diff --git a/BioC_XML/4746701_v0.xml b/BioC_XML/4746701_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..c02b01e8e17606bb9d91dd5e9a2986184900e7c3 --- /dev/null +++ b/BioC_XML/4746701_v0.xml @@ -0,0 +1,11066 @@ + + + + PMC + 20201216 + pmc.key + + 4746701 + CC BY + no + 0 + 0 + + 10.1038/srep20261 + srep20261 + 4746701 + 27064360 + 20261 + This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ + surname:Jeong;given-names:Hanbin + surname:Sim;given-names:Hyo Jung + surname:Song;given-names:Eun Kyung + surname:Lee;given-names:Hakbong + surname:Ha;given-names:Sung Chul + surname:Jun;given-names:Youngsoo + surname:Park;given-names:Tae Joo + surname:Lee;given-names:Changwook + TITLE + front + 6 + 2016 + 0 + Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway + + 0.9988518 + evidence + cleaner0 + 2023-07-26T11:52:54Z + DUMMY: + + Crystal structure + + + 0.9992508 + protein + cleaner0 + 2023-07-26T08:23:58Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:35Z + + SLR + + + + ABSTRACT + abstract + 81 + Terminally misfolded proteins are selectively recognized and cleared by the endoplasmic reticulum-associated degradation (ERAD) pathway. SEL1L, a component of the ERAD machinery, plays an important role in selecting and transporting ERAD substrates for degradation. We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). Strikingly, SEL1Lcent forms a homodimer with two-fold symmetry in a head-to-tail manner. Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery. + + 0.9992866 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.99754673 + evidence + cleaner0 + 2023-07-26T11:52:55Z + DUMMY: + + crystal structure + + + 0.9909797 + taxonomy_domain + cleaner0 + 2023-07-26T08:29:39Z + DUMMY: + + mouse + + + 0.9992374 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.9963008 + structure_element + cleaner0 + 2023-07-26T09:47:28Z + SO: + + central domain + + + 0.99568605 + structure_element + cleaner0 + 2023-07-26T08:25:29Z + SO: + + Sel1-Like Repeats + + + 0.9770133 + structure_element + cleaner0 + 2023-07-26T08:25:35Z + SO: + + SLR motifs 5 to 9 + + + 0.51770043 + structure_element + cleaner0 + 2023-07-26T08:23:44Z + SO: + + SEL1Lcent + + + 0.98432606 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.99863064 + oligomeric_state + cleaner0 + 2023-07-26T08:24:28Z + DUMMY: + + homodimer + + + 0.9969376 + protein_state + cleaner0 + 2023-07-26T08:24:19Z + DUMMY: + + head-to-tail + + + 0.996372 + structure_element + cleaner0 + 2023-07-26T08:25:41Z + SO: + + SLR motif 9 + + + 0.9987109 + oligomeric_state + cleaner0 + 2023-07-26T08:24:33Z + DUMMY: + + dimer + + + 0.917501 + protein_state + cleaner0 + 2023-07-26T09:03:04Z + DUMMY: + + domain-swapped + + + 0.9982927 + site + cleaner0 + 2023-07-26T12:02:50Z + SO: + + dimeric interface + + + 0.9991167 + protein_state + cleaner0 + 2023-07-26T08:24:13Z + DUMMY: + + full-length + + + 0.9992772 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T08:25:00Z + + self-oligomer + + + 0.526936 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.9985261 + taxonomy_domain + cleaner0 + 2023-07-26T08:25:51Z + DUMMY: + + mammalian + + + 0.99810743 + structure_element + cleaner0 + 2023-07-26T08:33:21Z + SO: + + SLR-C + + + 0.9958229 + structure_element + cleaner0 + 2023-07-26T08:33:26Z + SO: + + SLR motifs 10 and 11 + + + 0.9992606 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T09:51:11Z + + luminal loops + + + 0.9960486 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:35Z + + SLR + + + 0.9993025 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + + INTRO + paragraph + 1119 + Protein quality control in the endoplasmic reticulum (ER) is essential for maintenance of cellular homeostasis in eukaryotes and is implicated in many severe diseases. Terminally misfolded proteins in the lumen or membrane of the ER are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. The process is called ER-associated protein degradation (ERAD) and is conserved in all eukaryotes. Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. The components are differentially localized from the lumen and membrane of the ER to the cytosol, and have different functions in the ERAD process. Yeast ERAD components, which have been extensively characterized through genetic and biochemical studies, are comparable with mammalian ERAD components, sharing similar molecular functions and structural composition. + + 0.9985447 + taxonomy_domain + cleaner0 + 2023-07-26T08:25:56Z + DUMMY: + + eukaryotes + + + 0.9758303 + protein_state + cleaner0 + 2023-07-26T11:59:39Z + DUMMY: + + polyubiquitinated + + + 0.9492112 + complex_assembly + cleaner0 + 2023-07-26T12:05:11Z + GO: + + proteasome + + + 0.9905149 + protein_state + cleaner0 + 2023-07-26T11:59:44Z + DUMMY: + + conserved + + + 0.9946179 + taxonomy_domain + cleaner0 + 2023-07-26T08:25:57Z + DUMMY: + + eukaryotes + + + 0.9990409 + protein + cleaner0 + 2023-07-26T08:26:24Z + PR: + + HRD1 + + + 0.99905616 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.99904174 + protein + cleaner0 + 2023-07-26T08:26:29Z + PR: + + Hrd3p + + + 0.9990537 + protein + cleaner0 + 2023-07-26T08:26:37Z + PR: + + Derlin-1, -2, -3 + + + 0.99914646 + protein + cleaner0 + 2023-07-26T08:26:42Z + PR: + + Der1p + + + 0.99912745 + protein + cleaner0 + 2023-07-26T08:26:53Z + PR: + + HERP-1, -2 + + + 0.9991105 + protein + cleaner0 + 2023-07-26T08:26:59Z + PR: + + Usa1p + + + 0.99894625 + protein + cleaner0 + 2023-07-26T08:27:04Z + PR: + + OS9 + + + 0.99881977 + protein + cleaner0 + 2023-07-26T08:27:09Z + PR: + + Yos9 + + + 0.99912435 + protein + cleaner0 + 2023-07-26T08:27:13Z + PR: + + XTP-B + + + 0.9990497 + protein + cleaner0 + 2023-07-26T08:27:19Z + PR: + + Grp94 + + + 0.99891496 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:25Z + DUMMY: + + yeast + + + 0.99853647 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:32Z + DUMMY: + + metazoans + + + 0.99890244 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:26Z + DUMMY: + + Yeast + + + 0.9988557 + experimental_method + cleaner0 + 2023-07-26T12:13:45Z + MESH: + + genetic and biochemical studies + + + 0.9987692 + taxonomy_domain + cleaner0 + 2023-07-26T08:25:52Z + DUMMY: + + mammalian + + + + INTRO + paragraph + 2186 + The HRD1 E3 ubiquitin ligase, which is embedded in the ER membrane, is involved in translocating ERAD substrates across the ER membrane and catalyzing substrate ubiquitination via its cytosolic RING finger domain. SEL1L, the mammalian homolog of Hrd3p, associates with HRD1, mediates HRD1 interactions with the ER luminal lectin OS9, and recognizes substrates to be degraded. In particular, SEL1L is crucial for translocation of Class I major histocompatibility complex (MHC) heavy chains (HCs). Recent research based on the inducible Sel1l knockout mouse model highlights the physiological functions of SEL1L. SEL1L is required for ER homeostasis, which is essential for protein translation, pancreatic function, and cellular and organismal survival. However, despite the functional importance of SEL1L, the molecular structure of SEL1L has not been solved. Previous biochemical studies reveal that SEL1L is a type I transmembrane protein and has a large luminal domain comprising sets of repeated Sel1-like (SLR) motifs. The SLR motif is a structural motif that closely resembles the tetratricopeptide-repeat (TPR) motif, which is a protein-protein interaction module. Although there is evidence that the luminal domain of SEL1L is involved in substrate recognition or in forming complexes with chaperones, it is not known how the unique structure of the repeated SLR motifs contributes to the molecular function of the HRD1-SEL1L E3 ligase complex and affects ERAD at the molecular level. Furthermore, while repeated SLR motifs are commonly found in tandem arrays, the SLR motifs in SEL1L are, according to the primary structure prediction of SEL1L, interspersed among other sequences in the luminal domain and form three SLR domain clusters. Therefore, the way in which these unique structural features of SEL1L are related to its critical function in ERAD remains to be elucidated. + + 0.99861705 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + 0.9934304 + protein_type + cleaner0 + 2023-07-26T08:27:55Z + MESH: + + E3 ubiquitin ligase + + + 0.9971369 + structure_element + cleaner0 + 2023-07-26T08:28:08Z + SO: + + RING finger domain + + + 0.9992901 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.998187 + taxonomy_domain + cleaner0 + 2023-07-26T08:25:52Z + DUMMY: + + mammalian + + + 0.99932337 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + 0.998701 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + 0.9979652 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + 0.9981773 + protein_type + cleaner0 + 2023-07-26T12:25:11Z + MESH: + + lectin + + + 0.9990397 + protein + cleaner0 + 2023-07-26T08:27:05Z + PR: + + OS9 + + + 0.9991423 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + complex_assembly + GO: + cleaner0 + 2023-07-26T08:28:59Z + + Class I major histocompatibility complex + + + 0.9961902 + complex_assembly + cleaner0 + 2023-07-26T12:05:21Z + GO: + + MHC + + + 0.703586 + protein_type + cleaner0 + 2023-07-26T08:29:20Z + MESH: + + heavy chains + + + 0.958494 + protein_type + cleaner0 + 2023-07-26T08:29:31Z + MESH: + + HCs + + + 0.5361406 + gene + cleaner0 + 2023-07-26T08:30:21Z + GENE: + + Sel1l + + + experimental_method + MESH: + cleaner0 + 2023-07-26T08:30:54Z + + knockout mouse + + + 0.99895835 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.99898237 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.99911755 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.5043668 + evidence + cleaner0 + 2023-07-26T11:53:04Z + DUMMY: + + structure + + + 0.9992186 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.9939858 + experimental_method + cleaner0 + 2023-07-26T12:13:52Z + MESH: + + biochemical studies + + + 0.99927205 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.9878181 + protein_type + cleaner0 + 2023-07-26T08:31:16Z + MESH: + + type I transmembrane protein + + + 0.992851 + structure_element + cleaner0 + 2023-07-26T08:31:23Z + SO: + + luminal domain + + + 0.95882595 + structure_element + cleaner0 + 2023-07-26T08:31:31Z + SO: + + repeated Sel1-like + + + 0.98645127 + structure_element + cleaner0 + 2023-07-26T08:31:35Z + SO: + + SLR + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:35Z + + SLR + + + 0.9975922 + structure_element + cleaner0 + 2023-07-26T09:44:59Z + SO: + + tetratricopeptide-repeat + + + 0.99426264 + structure_element + cleaner0 + 2023-07-26T09:43:43Z + SO: + + TPR + + + 0.99419355 + structure_element + cleaner0 + 2023-07-26T08:31:24Z + SO: + + luminal domain + + + 0.9992374 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.82589394 + protein_type + cleaner0 + 2023-07-26T11:53:10Z + MESH: + + chaperones + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:35Z + + SLR + + + 0.9983583 + complex_assembly + cleaner0 + 2023-07-26T08:32:42Z + GO: + + HRD1-SEL1L + + + 0.78861254 + protein_type + cleaner0 + 2023-07-26T08:32:48Z + MESH: + + E3 ligase + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:35Z + + SLR + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:35Z + + SLR + + + 0.99923146 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.9991757 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.99028516 + structure_element + cleaner0 + 2023-07-26T08:31:24Z + SO: + + luminal domain + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:35Z + + SLR + + + 0.9992945 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + + INTRO + paragraph + 4073 + To clearly understand the biochemical role of the SLR domains of SEL1L in ERAD, we determined the crystal structure of the central SLR domain of SEL1L. We found that the central domain of SEL1L, comprising SLR motifs 5 through 9 (SEL1Lcent), forms a tight dimer with two-fold symmetry due to domain swapping of the SLR motif 9. We also found that SLR-C, consisting of SLR motifs 10 and 11, directly interacts with the N-terminus luminal loop of HRD1. Based on these observations, we propose a model wherein the SLR domains of SEL1L contribute to the formation of stable oligomers of the ERAD translocation machinery, which is indispensable for ERAD. + + 0.9992932 + structure_element + cleaner0 + 2023-07-26T08:31:35Z + SO: + + SLR + + + 0.99931407 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.9978889 + evidence + cleaner0 + 2023-07-26T11:52:55Z + DUMMY: + + crystal structure + + + 0.99859124 + structure_element + cleaner0 + 2023-07-26T08:31:35Z + SO: + + SLR + + + 0.9993007 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.9929137 + structure_element + cleaner0 + 2023-07-26T09:47:38Z + SO: + + central domain + + + 0.99927694 + protein + cleaner0 + 2023-07-26T08:23:59Z + PR: + + SEL1L + + + 0.8446561 + structure_element + cleaner0 + 2023-07-26T09:47:43Z + SO: + + SLR motifs 5 through 9 + + + 0.9971794 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.9976841 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.9741096 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.8348906 + structure_element + cleaner0 + 2023-07-26T08:33:22Z + SO: + + SLR-C + + + 0.9868504 + structure_element + cleaner0 + 2023-07-26T08:33:26Z + SO: + + SLR motifs 10 and 11 + + + 0.99824077 + structure_element + cleaner0 + 2023-07-26T09:47:48Z + SO: + + luminal loop + + + 0.9978137 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + 0.9992292 + structure_element + cleaner0 + 2023-07-26T08:31:36Z + SO: + + SLR + + + 0.9993494 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.9854754 + protein_state + cleaner0 + 2023-07-26T11:59:55Z + DUMMY: + + stable + + + 0.99838114 + oligomeric_state + cleaner0 + 2023-07-26T11:55:49Z + DUMMY: + + oligomers + + + + RESULTS + title_1 + 4723 + Results + + + RESULTS + title_2 + 4731 + Structure Determination of SEL1Lcent + + 0.99846506 + experimental_method + cleaner0 + 2023-07-26T12:13:58Z + MESH: + + Structure Determination + + + 0.998895 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + + RESULTS + paragraph + 4768 + The Mus musculus SEL1L protein contains 790 amino acids and has 17% sequence identity to its yeast homolog, Hrd3p. Mouse SEL1L contains a fibronectin type II domain at the N-terminus, followed by 11 SLR motifs and a single transmembrane domain at the C-terminus (Fig. 1A). The 11 SLR motifs are located in the ER lumen and account for more than two thirds of the mass of full-length SEL1L. The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). We first tried to prepare the full-length mouse SEL1L protein, excluding the transmembrane domain at the C-terminus (residues 735–755), by expression in bacteria. However, the full-length SEL1L protein aggregated in solution and produced no soluble protein. To identify a soluble form of SEL1L, we generated serial truncation constructs of SEL1L based on the predicted SLR motifs and highly conserved regions across several different species. Both SLR-N (residues 194–343) and SLR-C (residues 639–719) alone could be solubilized with an MBP tag at the N-terminus, but appeared to be polydisperse when analyzed by size-exclusion chromatography. However, the central region of SEL1L, comprising residues 337–554, was soluble and homogenous in size, as determined by size-exclusion chromatography. To define compact domain boundaries for the central region of SEL1L, we digested the protein with trypsin and analyzed the proteolysis products by SDS-PAGE and N-terminal sequencing. The results of this preliminary biochemical analysis suggested that SEL1L residues 348–533 (SEL1Lcent) would be amenable to structural analysis (Fig. 1A). + + 0.99817586 + species + cleaner0 + 2023-07-26T08:34:14Z + MESH: + + Mus musculus + + + 0.99881727 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.9986405 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:26Z + DUMMY: + + yeast + + + 0.9986916 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + 0.99675995 + taxonomy_domain + cleaner0 + 2023-07-26T08:29:39Z + DUMMY: + + Mouse + + + 0.9990717 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.9988529 + structure_element + cleaner0 + 2023-07-26T09:47:57Z + SO: + + fibronectin type II domain + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + structure_element + SO: + cleaner0 + 2023-07-26T08:38:12Z + + transmembrane domain + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9991053 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.99896204 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9866763 + structure_element + cleaner0 + 2023-07-26T08:35:21Z + SO: + + linker sequences + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9888961 + structure_element + cleaner0 + 2023-07-26T08:35:33Z + SO: + + SLR-N + + + 0.9852956 + structure_element + cleaner0 + 2023-07-26T08:35:37Z + SO: + + SLR motifs 1 to 4 + + + 0.99116176 + structure_element + cleaner0 + 2023-07-26T08:35:41Z + SO: + + SLR-M + + + 0.93657416 + structure_element + cleaner0 + 2023-07-26T08:25:36Z + SO: + + SLR motifs 5 to 9 + + + 0.9188196 + structure_element + cleaner0 + 2023-07-26T08:33:22Z + SO: + + SLR-C + + + 0.98369825 + structure_element + cleaner0 + 2023-07-26T09:48:02Z + SO: + + SLR motifs 10 to 11 + + + 0.99843454 + experimental_method + cleaner0 + 2023-07-26T12:14:02Z + MESH: + + Sequence alignment + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.8135656 + structure_element + cleaner0 + 2023-07-26T08:35:28Z + SO: + + linker sequence + + + 0.9977494 + residue_range + cleaner0 + 2023-07-26T08:36:20Z + DUMMY: + + 336–345 + + + 0.99132603 + structure_element + cleaner0 + 2023-07-26T08:35:33Z + SO: + + SLR-N + + + 0.9862742 + structure_element + cleaner0 + 2023-07-26T08:35:41Z + SO: + + SLR-M + + + 0.95723855 + structure_element + cleaner0 + 2023-07-26T08:35:28Z + SO: + + linker sequence + + + 0.9976837 + residue_range + cleaner0 + 2023-07-26T08:36:26Z + DUMMY: + + 528–635 + + + 0.98249346 + structure_element + cleaner0 + 2023-07-26T08:35:41Z + SO: + + SLR-M + + + 0.9578497 + structure_element + cleaner0 + 2023-07-26T08:33:22Z + SO: + + SLR-C + + + 0.99900556 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.9963148 + taxonomy_domain + cleaner0 + 2023-07-26T08:29:39Z + DUMMY: + + mouse + + + 0.9991549 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.99889475 + structure_element + cleaner0 + 2023-07-26T08:38:11Z + SO: + + transmembrane domain + + + 0.9977551 + residue_range + cleaner0 + 2023-07-26T08:36:22Z + DUMMY: + + 735–755 + + + 0.9981437 + experimental_method + cleaner0 + 2023-07-26T12:14:12Z + MESH: + + expression in bacteria + + + 0.9991067 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.99904925 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.9991609 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.9660775 + experimental_method + cleaner0 + 2023-07-26T12:14:18Z + MESH: + + serial truncation constructs + + + 0.99915016 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9989929 + protein_state + cleaner0 + 2023-07-26T08:36:14Z + DUMMY: + + highly conserved + + + 0.88397986 + structure_element + cleaner0 + 2023-07-26T08:35:33Z + SO: + + SLR-N + + + 0.997901 + residue_range + cleaner0 + 2023-07-26T08:36:28Z + DUMMY: + + 194–343 + + + 0.83972365 + structure_element + cleaner0 + 2023-07-26T08:33:22Z + SO: + + SLR-C + + + 0.9978097 + residue_range + cleaner0 + 2023-07-26T08:36:31Z + DUMMY: + + 639–719 + + + experimental_method + MESH: + cleaner0 + 2023-07-26T08:38:02Z + + MBP tag at the N-terminus + + + 0.998687 + experimental_method + cleaner0 + 2023-07-26T12:14:24Z + MESH: + + size-exclusion chromatography + + + structure_element + SO: + cleaner0 + 2023-07-26T08:36:53Z + + central region + + + 0.9990677 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.9978606 + residue_range + cleaner0 + 2023-07-26T08:36:34Z + DUMMY: + + 337–554 + + + 0.9987895 + experimental_method + cleaner0 + 2023-07-26T12:14:29Z + MESH: + + size-exclusion chromatography + + + structure_element + SO: + cleaner0 + 2023-07-26T08:36:54Z + + central region + + + 0.9991781 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + experimental_method + MESH: + cleaner0 + 2023-07-26T08:37:23Z + + digested the protein with trypsin + + + 0.9988027 + experimental_method + cleaner0 + 2023-07-26T12:14:37Z + MESH: + + SDS-PAGE + + + 0.99544513 + experimental_method + cleaner0 + 2023-07-26T12:14:41Z + MESH: + + N-terminal sequencing + + + 0.99861073 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.99793893 + residue_range + cleaner0 + 2023-07-26T08:38:17Z + DUMMY: + + 348–533 + + + 0.91120136 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + experimental_method + MESH: + cleaner0 + 2023-07-26T08:37:44Z + + structural analysis + + + + RESULTS + paragraph + 6731 + Crystals of SEL1Lcent grew in space group P21 with four copies of SEL1Lcent (a total of 82 kDa) in the asymmetric unit. The structure was determined by the single-wavelength anomalous diffraction (SAD) method using selenium as the anomalous scatterer (Table 1 and Methods). The assignment of residues during model building was aided by the selenium atom positions, and the structure was refined with native data to 2.6 Å resolution with Rwork/Rfree values of 20.7/27.7%. Statistics for data collection and refinement are presented in Table 1. + + 0.99207085 + evidence + cleaner0 + 2023-07-26T11:53:16Z + DUMMY: + + Crystals + + + 0.9989442 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.99910873 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.9972408 + evidence + cleaner0 + 2023-07-26T11:53:20Z + DUMMY: + + structure + + + 0.99886435 + experimental_method + cleaner0 + 2023-07-26T12:14:47Z + MESH: + + single-wavelength anomalous diffraction + + + 0.99847096 + experimental_method + cleaner0 + 2023-07-26T12:14:50Z + MESH: + + SAD + + + 0.9987256 + chemical + cleaner0 + 2023-07-26T08:38:44Z + CHEBI: + + selenium + + + 0.99861145 + chemical + cleaner0 + 2023-07-26T08:38:44Z + CHEBI: + + selenium + + + 0.9973163 + evidence + cleaner0 + 2023-07-26T11:53:28Z + DUMMY: + + structure + + + 0.76061416 + evidence + cleaner0 + 2023-07-26T11:53:31Z + DUMMY: + + Rwork/Rfree + + + + RESULTS + title_2 + 7279 + Overall Structure of SEL1Lcent + + 0.99723697 + evidence + cleaner0 + 2023-07-26T11:53:36Z + DUMMY: + + Structure + + + 0.998892 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + + RESULTS + paragraph + 7310 + The mouse SEL1Lcent crystallized as a homodimer, and there were two homodimers in the crystal asymmetric unit (Fig. 1B,C, Supplementary Fig. 1). The two SEL1Lcent molecules dimerize in a head-to-tail manner through a two-fold symmetry interface resulting in a cosmos-like shaped structure (Fig. 1B). The resulting structure resembles the yin-yang symbol with overall dimensions of 60 × 60 × 25 Å, where a SEL1Lcent monomer corresponds to half the symbol. The dimer formation buries a surface area of 1670 Å2 for each monomer, and no significant differences between the protomers were displayed (final root mean square deviation (RMSD) of 0.6 Å for all Cα atoms). Each protomer is composed of ten α-helices, which form the five SLRs, resulting in an elongated curved structure, confirming the primary structure prediction (Fig. 1D). + + 0.99545884 + taxonomy_domain + cleaner0 + 2023-07-26T08:29:39Z + DUMMY: + + mouse + + + 0.99923635 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.9950051 + experimental_method + cleaner0 + 2023-07-26T12:14:55Z + MESH: + + crystallized + + + 0.9987419 + oligomeric_state + cleaner0 + 2023-07-26T08:24:28Z + DUMMY: + + homodimer + + + 0.9987446 + oligomeric_state + cleaner0 + 2023-07-26T08:39:18Z + DUMMY: + + homodimers + + + 0.9991404 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.9984725 + oligomeric_state + cleaner0 + 2023-07-26T08:38:55Z + DUMMY: + + dimerize + + + 0.9923738 + protein_state + cleaner0 + 2023-07-26T08:24:20Z + DUMMY: + + head-to-tail + + + 0.9871208 + site + cleaner0 + 2023-07-26T12:03:32Z + SO: + + two-fold symmetry interface + + + 0.89937943 + evidence + cleaner0 + 2023-07-26T11:54:22Z + DUMMY: + + structure + + + 0.99790156 + evidence + cleaner0 + 2023-07-26T11:54:25Z + DUMMY: + + structure + + + 0.9990891 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.9988864 + oligomeric_state + cleaner0 + 2023-07-26T08:39:01Z + DUMMY: + + monomer + + + 0.99883777 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.99886155 + oligomeric_state + cleaner0 + 2023-07-26T08:39:02Z + DUMMY: + + monomer + + + 0.99703 + oligomeric_state + cleaner0 + 2023-07-26T08:39:12Z + DUMMY: + + protomers + + + 0.99834615 + evidence + cleaner0 + 2023-07-26T11:54:29Z + DUMMY: + + root mean square deviation + + + 0.9983158 + evidence + cleaner0 + 2023-07-26T08:41:28Z + DUMMY: + + RMSD + + + 0.99887854 + oligomeric_state + cleaner0 + 2023-07-26T08:39:06Z + DUMMY: + + protomer + + + 0.998384 + structure_element + cleaner0 + 2023-07-26T09:48:10Z + SO: + + α-helices + + + 0.9965168 + structure_element + cleaner0 + 2023-07-26T08:41:13Z + SO: + + SLRs + + + + RESULTS + paragraph + 8163 + The α-helices subdivide the structure into five pairs (A and B) as shown in a number of TPRs and SLRs. Helices A and B are 14 and 13 residues long, respectively, and the two helices are connected by a short turn and loop (Fig. 1D). In addition, a longer loop, consisting of approximately eight amino acids, is inserted between helix B of one SLR and helix A of the next SLR. This arrangement is a unique feature for SLRs among the major classes of repeats containing an α-solenoid. Starting from its N-terminus, the α-solenoid of SEL1L extends across a semi-circle in a right-handed superhelix fashion along the rotation axis of the yin-yang circle. However, the last helix, 9B, at the C-terminus adopts a different conformation, lying parallel to the long axis of helix 9A instead of forming an antiparallel SLR. This unique conformation of helix 9B most likely contributes to formation of the dimer structure of SEL1Lcent, as detailed below. With the exception of the last SLR, the four α-helix pairs possess similar conformations, with RMSD values of 0.7 Å for all Cα atoms. Although the sequence similarity for the pairwise alignments varies between 25% and 35%, all the residues present in the SLR motifs are conserved among the five pairs. The SLR domain of SLR-M ends at residue 524, and C-terminal amino acids 525–533 of the protein are not visible in the electron density map, suggesting that this region is highly flexible. + + 0.9993 + structure_element + cleaner0 + 2023-07-26T09:48:18Z + SO: + + α-helices + + + 0.8356808 + evidence + cleaner0 + 2023-07-26T11:54:34Z + DUMMY: + + structure + + + 0.99865365 + structure_element + cleaner0 + 2023-07-26T09:48:21Z + SO: + + A + + + 0.99602926 + structure_element + cleaner0 + 2023-07-26T09:48:24Z + SO: + + B + + + 0.99529266 + structure_element + cleaner0 + 2023-07-26T08:40:43Z + SO: + + TPRs + + + 0.93863434 + structure_element + cleaner0 + 2023-07-26T08:41:12Z + SO: + + SLRs + + + 0.9174499 + structure_element + cleaner0 + 2023-07-26T08:40:37Z + SO: + + Helices A and B + + + 0.964784 + structure_element + cleaner0 + 2023-07-26T09:48:28Z + SO: + + helices + + + 0.9637494 + structure_element + cleaner0 + 2023-07-26T09:48:34Z + SO: + + turn + + + 0.9960223 + structure_element + cleaner0 + 2023-07-26T09:48:36Z + SO: + + loop + + + 0.9986534 + structure_element + cleaner0 + 2023-07-26T09:48:39Z + SO: + + loop + + + 0.98835695 + structure_element + cleaner0 + 2023-07-26T08:40:26Z + SO: + + helix B + + + 0.99341077 + structure_element + cleaner0 + 2023-07-26T08:31:36Z + SO: + + SLR + + + 0.99514014 + structure_element + cleaner0 + 2023-07-26T08:40:32Z + SO: + + helix A + + + 0.9932869 + structure_element + cleaner0 + 2023-07-26T08:31:36Z + SO: + + SLR + + + 0.9989489 + structure_element + cleaner0 + 2023-07-26T08:41:13Z + SO: + + SLRs + + + 0.99916095 + structure_element + cleaner0 + 2023-07-26T09:48:43Z + SO: + + α-solenoid + + + 0.9992831 + structure_element + cleaner0 + 2023-07-26T09:48:46Z + SO: + + α-solenoid + + + 0.99934524 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.86892396 + structure_element + cleaner0 + 2023-07-26T08:40:53Z + SO: + + yin-yang circle + + + 0.9986014 + structure_element + cleaner0 + 2023-07-26T08:41:59Z + SO: + + 9B + + + structure_element + SO: + cleaner0 + 2023-07-26T08:40:19Z + + helix 9A + + + 0.8358868 + structure_element + cleaner0 + 2023-07-26T08:31:36Z + SO: + + SLR + + + structure_element + SO: + cleaner0 + 2023-07-26T08:41:50Z + + helix 9B + + + 0.99878865 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.9993019 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.92849016 + structure_element + cleaner0 + 2023-07-26T08:31:36Z + SO: + + SLR + + + 0.99918014 + structure_element + cleaner0 + 2023-07-26T09:48:59Z + SO: + + α-helix + + + 0.9987 + evidence + cleaner0 + 2023-07-26T08:41:27Z + DUMMY: + + RMSD + + + 0.99687785 + experimental_method + cleaner0 + 2023-07-26T12:15:12Z + MESH: + + pairwise alignments + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9889858 + protein_state + cleaner0 + 2023-07-26T08:42:36Z + DUMMY: + + conserved + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + structure_element + SO: + cleaner0 + 2023-07-26T08:35:41Z + + SLR-M + + + 0.9716945 + residue_number + cleaner0 + 2023-07-26T08:42:28Z + DUMMY: + + 524 + + + 0.9968249 + residue_range + cleaner0 + 2023-07-26T08:42:24Z + DUMMY: + + 525–533 + + + 0.99862355 + evidence + cleaner0 + 2023-07-26T08:42:46Z + DUMMY: + + electron density map + + + 0.99881995 + protein_state + cleaner0 + 2023-07-26T08:42:41Z + DUMMY: + + highly flexible + + + + RESULTS + paragraph + 9617 + Since no information regarding dimer formation by SEL1L through its SLR motifs is available, we tested whether the SEL1Lcent dimer shown in our crystal structure is a biological unit. First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. We detected bands at the mass of a dimer for both SEL1Lcent and SEL1Llong when cross-linked with low concentrations of GA (0.005%) or DMS (0.3 mM) (Supplementary Fig. 2A,B). Next, we conducted analytical ultracentrifugation of SEL1Lcent. Consistent with the cross-linking data, analytical ultracentrifugation revealed that the molecular weight of SEL1Lcent corresponds to a dimer (Supplementary Fig. 2C). Taken together, these data indicate that some kind of dimer is formed in solution. + + 0.9988261 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.9988477 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9933989 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.99881846 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.99609435 + evidence + cleaner0 + 2023-07-26T11:52:55Z + DUMMY: + + crystal structure + + + 0.9954019 + experimental_method + cleaner0 + 2023-07-26T12:15:19Z + MESH: + + cross-linked + + + 0.99408615 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.9866093 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.99746454 + mutant + cleaner0 + 2023-07-26T08:43:07Z + MESH: + + SEL1Llong + + + 0.99769783 + residue_range + cleaner0 + 2023-07-26T08:43:15Z + DUMMY: + + 337–554 + + + 0.9990128 + chemical + cleaner0 + 2023-07-26T08:43:22Z + CHEBI: + + glutaraldehyde + + + 0.9991844 + chemical + cleaner0 + 2023-07-26T08:43:26Z + CHEBI: + + GA + + + 0.9989731 + chemical + cleaner0 + 2023-07-26T08:43:31Z + CHEBI: + + dimethyl suberimidate + + + 0.99916816 + chemical + cleaner0 + 2023-07-26T08:43:36Z + CHEBI: + + DMS + + + 0.9988912 + experimental_method + cleaner0 + 2023-07-26T12:15:46Z + MESH: + + SDS-PAGE + + + 0.998814 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.99484646 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.9665328 + mutant + cleaner0 + 2023-07-26T08:43:07Z + MESH: + + SEL1Llong + + + 0.99625254 + experimental_method + cleaner0 + 2023-07-26T12:15:28Z + MESH: + + cross-linked + + + 0.9990452 + chemical + cleaner0 + 2023-07-26T08:43:27Z + CHEBI: + + GA + + + 0.9989182 + chemical + cleaner0 + 2023-07-26T08:43:37Z + CHEBI: + + DMS + + + 0.9989053 + experimental_method + cleaner0 + 2023-07-26T12:15:53Z + MESH: + + analytical ultracentrifugation + + + 0.99510103 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.99864703 + experimental_method + cleaner0 + 2023-07-26T12:16:01Z + MESH: + + cross-linking + + + 0.9988791 + experimental_method + cleaner0 + 2023-07-26T12:16:06Z + MESH: + + analytical ultracentrifugation + + + evidence + DUMMY: + cleaner0 + 2023-07-26T12:16:18Z + + molecular weight + + + 0.9970095 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.99879503 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.99886096 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + + RESULTS + title_2 + 10509 + Dimer Interface of SEL1Lcent + + 0.99903053 + site + cleaner0 + 2023-07-26T08:43:53Z + SO: + + Dimer Interface + + + 0.99924254 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + + RESULTS + paragraph + 10538 + In contrast to a previously described SLR motif containing proteins that exist as monomers in solution, SEL1Lcent forms an intimate two-fold homotypic dimer interface (Figs 1B and 2A). The concave surface of each SEL1L domain comprising helix 5A to 9A encircles its dimer counterpart in an interlocking clasp-like arrangement. However, no interactions were seen between the two-fold-related protomers through the concave inner surfaces themselves. Rather, the unique structure of SLR motif 9, consisting of two parallel helices (9A and 9B), is located in the space generated by the concave surface and provides an extensive dimerization interface between the two-fold-related molecules (Fig. 2A). Helix 9B from one protomer inserts into the empty space surrounded by the concave region in the other monomer, forming a domain-swapped conformation. + + protein_type + MESH: + cleaner0 + 2023-07-26T08:45:41Z + + SLR motif containing proteins + + + 0.99890316 + oligomeric_state + cleaner0 + 2023-07-26T11:55:54Z + DUMMY: + + monomers + + + 0.998408 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + site + SO: + cleaner0 + 2023-07-26T08:46:09Z + + two-fold homotypic dimer interface + + + 0.98965824 + site + cleaner0 + 2023-07-26T08:46:22Z + SO: + + concave surface + + + 0.9853652 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:44:27Z + + helix 5A to 9A + + + 0.9988918 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.9983084 + oligomeric_state + cleaner0 + 2023-07-26T08:39:13Z + DUMMY: + + protomers + + + 0.9283633 + site + cleaner0 + 2023-07-26T12:03:37Z + SO: + + concave inner surfaces + + + 0.99800134 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.9989574 + structure_element + cleaner0 + 2023-07-26T08:44:43Z + SO: + + 9A + + + 0.99379176 + structure_element + cleaner0 + 2023-07-26T08:44:45Z + SO: + + 9B + + + 0.9734504 + site + cleaner0 + 2023-07-26T08:46:23Z + SO: + + concave surface + + + 0.9988966 + site + cleaner0 + 2023-07-26T08:58:10Z + SO: + + dimerization interface + + + structure_element + SO: + cleaner0 + 2023-07-26T08:41:50Z + + Helix 9B + + + 0.99881697 + oligomeric_state + cleaner0 + 2023-07-26T08:39:07Z + DUMMY: + + protomer + + + 0.963019 + site + cleaner0 + 2023-07-26T12:03:42Z + SO: + + concave region + + + 0.99887115 + oligomeric_state + cleaner0 + 2023-07-26T08:39:02Z + DUMMY: + + monomer + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T09:03:04Z + + domain-swapped + + + + RESULTS + paragraph + 11385 + Three major contact interfaces are involved in the interactions, and all interfaces are symmetrically related between the dimer subunits (Fig. 2A). Structure-based sequence alignment of 135 SEL1L phylogenetic sequences using a ConSurf server revealed that the surface residues in the dimer interfaces were highly conserved among the SEL1L orthologs (Fig. 1E). First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR α-helices (5B, 6B, 7B, and 8B) from its counterpart. In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. The side chain of Gln 523 forms an H-bond to the side chain of Asp 480 on the two-fold-related protomer (Fig. 2A, Interface 1 detail). Second, the residues from helix 9A interact with the residues from helix 5A of its counterpart in a head-to-tail orientation. In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). Third, the helix 9B from each protomer is involved in the dimer interaction by forming a two-fold antiparallel symmetry. In particular, the side chains of hydrophobic residues, including Phe 518, Leu 521, and Met 524, are directed toward each other, where they make both inter- and intramolecular contacts (Fig. 2A, Interface 3 detail). + + 0.998649 + site + cleaner0 + 2023-07-26T12:03:48Z + SO: + + contact interfaces + + + 0.9989027 + site + cleaner0 + 2023-07-26T12:03:55Z + SO: + + interfaces + + + 0.9985661 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.9989074 + experimental_method + cleaner0 + 2023-07-26T12:16:44Z + MESH: + + Structure-based sequence alignment + + + 0.9986141 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.76681197 + experimental_method + cleaner0 + 2023-07-26T12:16:49Z + MESH: + + ConSurf server + + + 0.9986466 + site + cleaner0 + 2023-07-26T08:47:05Z + SO: + + dimer interfaces + + + 0.9988012 + protein_state + cleaner0 + 2023-07-26T08:47:52Z + DUMMY: + + highly conserved + + + 0.921708 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.9958381 + structure_element + cleaner0 + 2023-07-26T08:41:50Z + SO: + + helix 9B + + + 0.9777349 + structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + 0.9805442 + site + cleaner0 + 2023-07-26T12:04:23Z + SO: + + inner groove + + + 0.99077106 + structure_element + cleaner0 + 2023-07-26T08:31:36Z + SO: + + SLR + + + 0.9991037 + structure_element + cleaner0 + 2023-07-26T08:47:40Z + SO: + + α-helices + + + 0.9984108 + structure_element + cleaner0 + 2023-07-26T08:47:43Z + SO: + + 5B + + + 0.9963392 + structure_element + cleaner0 + 2023-07-26T08:47:45Z + SO: + + 6B + + + 0.9923379 + structure_element + cleaner0 + 2023-07-26T08:47:48Z + SO: + + 7B + + + 0.99450463 + structure_element + cleaner0 + 2023-07-26T08:47:50Z + SO: + + 8B + + + 0.99847454 + site + cleaner0 + 2023-07-26T12:04:30Z + SO: + + interface + + + 0.9957429 + residue_name_number + cleaner0 + 2023-07-26T08:48:11Z + DUMMY: + + Leu 516 + + + 0.9974791 + residue_name_number + cleaner0 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structure_element + cleaner0 + 2023-07-26T08:23:45Z + SO: + + SEL1Lcent + + + site + SO: + cleaner0 + 2023-07-26T08:51:38Z + + Interface 1 + + + 0.9971962 + bond_interaction + cleaner0 + 2023-07-26T08:50:40Z + MESH: + + hydrophobic interactions + + + 0.9972205 + residue_name_number + cleaner0 + 2023-07-26T08:48:16Z + DUMMY: + + Tyr 519 + + + 0.9973451 + residue_name_number + cleaner0 + 2023-07-26T08:48:47Z + DUMMY: + + Ile 515 + + + 0.99737597 + bond_interaction + cleaner0 + 2023-07-26T08:50:46Z + MESH: + + H-bonds + + + 0.9988205 + protein_state + cleaner0 + 2023-07-26T12:00:49Z + DUMMY: + + conserved + + + 0.9975953 + residue_name_number + cleaner0 + 2023-07-26T08:48:54Z + DUMMY: + + Gln 377 + + + 0.99773026 + residue_name_number + cleaner0 + 2023-07-26T08:48:59Z + DUMMY: + + His 381 + + + 0.9872621 + structure_element + cleaner0 + 2023-07-26T08:48:03Z + SO: + + helix 5B + + + 0.9984987 + oligomeric_state + cleaner0 + 2023-07-26T08:39:07Z + DUMMY: + + protomer + + + 0.9967507 + residue_name_number + cleaner0 + 2023-07-26T08:49:04Z + DUMMY: + + Gln 523 + + + 0.99719113 + bond_interaction + cleaner0 + 2023-07-26T08:50:51Z + MESH: + + H-bond + + + 0.99751633 + residue_name_number + cleaner0 + 2023-07-26T08:49:10Z + DUMMY: + + Asp 480 + + + 0.99792105 + oligomeric_state + cleaner0 + 2023-07-26T08:39:07Z + DUMMY: + + protomer + + + site + SO: + cleaner0 + 2023-07-26T08:51:38Z + + Interface 1 + + + 0.9912703 + structure_element + cleaner0 + 2023-07-26T08:40:20Z + SO: + + helix 9A + + + 0.99822116 + structure_element + cleaner0 + 2023-07-26T09:49:09Z + SO: + + helix 5A + + + 0.9975549 + protein_state + cleaner0 + 2023-07-26T08:24:20Z + DUMMY: + + head-to-tail + + + 0.99807405 + site + cleaner0 + 2023-07-26T12:04:35Z + SO: + + interface + + + 0.99662375 + structure_element + cleaner0 + 2023-07-26T08:40:20Z + SO: + + helix 9A + + + 0.99731815 + residue_name_number + cleaner0 + 2023-07-26T08:49:17Z + DUMMY: + + Leu 503 + + + 0.9975286 + residue_name_number + cleaner0 + 2023-07-26T08:49:21Z + DUMMY: + + Tyr 499 + + + 0.99735534 + residue_name_number + cleaner0 + 2023-07-26T08:49:27Z + DUMMY: + + Lys 500 + + + 0.993017 + bond_interaction + cleaner0 + 2023-07-26T08:50:55Z + MESH: + + van der Waals contacts + + + 0.99887514 + structure_element + cleaner0 + 2023-07-26T09:49:09Z + SO: + + helix 5A + + + 0.9965479 + residue_name_number + cleaner0 + 2023-07-26T08:49:32Z + DUMMY: + + Tyr 360 + + + 0.9968129 + residue_name_number + cleaner0 + 2023-07-26T08:49:37Z + DUMMY: + + Leu 356 + + + 0.99606204 + residue_name_number + cleaner0 + 2023-07-26T08:49:42Z + DUMMY: + + Tyr 359 + + + 0.99658686 + residue_name_number + cleaner0 + 2023-07-26T08:49:46Z + DUMMY: + + Leu 363 + + + 0.9970648 + bond_interaction + cleaner0 + 2023-07-26T08:50:40Z + MESH: + + hydrophobic interactions + + + 0.99649847 + residue_name_number + cleaner0 + 2023-07-26T08:49:51Z + DUMMY: + + Asn 507 + + + 0.9968495 + residue_name_number + cleaner0 + 2023-07-26T08:49:56Z + DUMMY: + + Ser 510 + + 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+ residue_name_number + cleaner0 + 2023-07-26T08:50:06Z + DUMMY: + + Phe 518 + + + 0.9968066 + residue_name_number + cleaner0 + 2023-07-26T08:50:10Z + DUMMY: + + Leu 521 + + + 0.9973854 + residue_name_number + cleaner0 + 2023-07-26T08:50:15Z + DUMMY: + + Met 524 + + + site + SO: + cleaner0 + 2023-07-26T08:53:07Z + + Interface 3 + + + + RESULTS + paragraph + 13524 + To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L348–497) lacking SLR motif 9 (helix 9A and 9B) from SEL1Lcent for comparative analysis. The deletion mutant and the wild-type SEL1Lcent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1Lcent (Supplementary Fig. 3). However, the mutant behaved as a monomer in size-exclusion chromatography and analytical ultracentrifugation experiments (Fig. 2B, Supplementary Fig. 2C). Additionally, to further validate the key residues involved in dimer formation, we generated a triple point mutant (Interface 1, I515A, L516A, and Y519A) of the hydrophobic residues that are involved in dimerization. The triple point mutant eluted at the monomer position upon size-exclusion chromatography, while the negative control point mutant (Q460A) eluted at the same position as the wild-type. Notably, a single-residue mutation (L521A in interface 3) abolished the dimerization of SEL1Lcent (Fig. 2B). Leu 521 is located in the dimerization center of the antiparallel 9B helices in the SEL1Lcent dimer. + + 0.99796003 + evidence + cleaner0 + 2023-07-26T11:52:55Z + DUMMY: + + crystal structure + + + 0.99194884 + protein_state + cleaner0 + 2023-07-26T08:53:27Z + DUMMY: + + deletion mutant + + + mutant + MESH: + cleaner0 + 2023-07-26T09:04:56Z + + SEL1L348–497 + + + 0.99806386 + protein_state + cleaner0 + 2023-07-26T12:00:55Z + DUMMY: + + lacking + + + 0.9974417 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + structure_element + SO: + cleaner0 + 2023-07-26T08:40:20Z + + helix 9A + + + 0.99706894 + structure_element + cleaner0 + 2023-07-26T08:51:24Z + SO: + + 9B + + + 0.9993037 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.97612184 + protein_state + cleaner0 + 2023-07-26T08:53:26Z + DUMMY: + + deletion mutant + + + 0.9989631 + protein_state + cleaner0 + 2023-07-26T08:53:20Z + DUMMY: + + wild-type + + + 0.9991844 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + evidence + DUMMY: + cleaner0 + 2023-07-26T08:53:45Z + + spectra + + + 0.9971682 + experimental_method + cleaner0 + 2023-07-26T12:16:53Z + MESH: + + CD spectroscopy + + + 0.9705612 + experimental_method + cleaner0 + 2023-07-26T12:16:58Z + MESH: + + deletion + + + 0.9956174 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.99916804 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.9989379 + protein_state + cleaner0 + 2023-07-26T12:00:59Z + DUMMY: + + mutant + + + 0.9988734 + oligomeric_state + cleaner0 + 2023-07-26T08:39:02Z + DUMMY: + + monomer + + + 0.9987341 + experimental_method + cleaner0 + 2023-07-26T12:17:01Z + MESH: + + size-exclusion chromatography + + + 0.9985672 + experimental_method + cleaner0 + 2023-07-26T12:17:04Z + MESH: + + analytical ultracentrifugation + + + 0.9988134 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: 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cleaner0 + 2023-07-26T08:53:21Z + DUMMY: + + wild-type + + + experimental_method + MESH: + cleaner0 + 2023-07-26T08:52:45Z + + single-residue mutation + + + 0.99892384 + mutant + cleaner0 + 2023-07-26T08:52:49Z + MESH: + + L521A + + + 0.9969276 + site + cleaner0 + 2023-07-26T08:53:06Z + SO: + + interface 3 + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T08:54:10Z + + abolished the dimerization + + + 0.99925107 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.9963031 + residue_name_number + cleaner0 + 2023-07-26T08:50:11Z + DUMMY: + + Leu 521 + + + 0.99869436 + site + cleaner0 + 2023-07-26T11:58:04Z + SO: + + dimerization center + + + 0.9960935 + structure_element + cleaner0 + 2023-07-26T09:49:23Z + SO: + + 9B helices + + + 0.9992654 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.998847 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + + RESULTS + paragraph + 14728 + Taken together, these structural and biochemical data demonstrate that SEL1Lcent exists as a dimer in solution and that SLR motif 9 in SEL1Lcent plays an important role in generating a two-fold dimerization interface. + + 0.99832624 + evidence + cleaner0 + 2023-07-26T11:54:41Z + DUMMY: + + structural and biochemical data + + + 0.9992519 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.99875975 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.99915147 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.9993118 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.99687177 + site + cleaner0 + 2023-07-26T08:58:10Z + SO: + + dimerization interface + + + + RESULTS + title_2 + 14946 + The Two Glycine Residues (G512 and G513) Create a Hinge for Domain Swapping of SLR Motif 9 + + 0.9976674 + residue_name + cleaner0 + 2023-07-26T12:27:43Z + SO: + + Glycine + + + 0.9995813 + residue_name_number + cleaner0 + 2023-07-26T08:58:15Z + DUMMY: + + G512 + + + 0.9995747 + residue_name_number + cleaner0 + 2023-07-26T08:58:21Z + DUMMY: + + G513 + + + 0.9722741 + structure_element + cleaner0 + 2023-07-26T09:00:42Z + SO: + + Hinge + + + structure_element + SO: + cleaner0 + 2023-07-26T08:25:42Z + + SLR Motif 9 + + + + RESULTS + paragraph + 15037 + SLRs of mouse SEL1L were predicted using the TPRpred server. Based on the prediction, full-length SEL1L contains a total of 11 SLR motifs, and our construct corresponds to SLR motifs 5 through 9. Although amino acid sequences from helix 9A and 9B correctly aligned with the regular SLR repeats and corresponded to SLR motif 9 (Fig. 3A), the structural arrangement of the two helices deviated from the common structure for the SLR motif. According to our crystal structure, the central axis of helix 9B is almost parallel to that of helix 9A (Fig. 3B). However, this unusual conformation of SLR motif 9 seems to be essential for dimer formation, as described earlier. For this structural geometry, two adjacent residues, Gly 512 and Gly 513, in SEL1L confer flexibility at this position by adopting main-chain dihedral angles that are disallowed for non-glycine residues. The phi and psi dihedrals are 100° and 20° for Gly 512, and 110° and −20° for Gly 513, respectively (Fig. 3C). Gly 513 is conserved among other SLR motifs in the SEL1Lcent, but Gly 512 is present only in the SLR motif 9 of SEL1Lcent (Fig. 3A). Thus, the Gly-Gly residues generate an unusual sharp bend at the C-terminal SLR motif 9. The involvement of a glycine residue in forming a hinge for domain swapping has been reported previously. The significance of Gly 513 is further highlighted by its absolute conservation among different species, including the budding yeast homolog Hrd3p. + + 0.99879164 + structure_element + cleaner0 + 2023-07-26T08:41:13Z + SO: + + SLRs + + + 0.99712 + taxonomy_domain + cleaner0 + 2023-07-26T08:29:39Z + DUMMY: + + mouse + + + 0.9992617 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.99805737 + experimental_method + cleaner0 + 2023-07-26T12:17:13Z + MESH: + + TPRpred server + + + 0.99909896 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.9992016 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9956316 + structure_element + cleaner0 + 2023-07-26T09:49:28Z + SO: + + SLR motifs 5 through 9 + + + structure_element + SO: + cleaner0 + 2023-07-26T08:40:20Z + + helix 9A + + + 0.9945779 + structure_element + cleaner0 + 2023-07-26T08:59:33Z + SO: + + 9B + + + 0.9980234 + structure_element + cleaner0 + 2023-07-26T08:59:40Z + SO: + + SLR repeats + + + 0.9980931 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.9152161 + structure_element + cleaner0 + 2023-07-26T09:49:33Z + SO: + + helices + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9985614 + evidence + cleaner0 + 2023-07-26T11:52:55Z + DUMMY: + + crystal structure + + + structure_element + SO: + cleaner0 + 2023-07-26T08:41:50Z + + helix 9B + + + structure_element + SO: + cleaner0 + 2023-07-26T08:40:20Z + + helix 9A + + + 0.9983694 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.99886876 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.9963238 + residue_name_number + cleaner0 + 2023-07-26T09:34:15Z + DUMMY: + + Gly 512 + + + 0.9969952 + residue_name_number + cleaner0 + 2023-07-26T09:34:18Z + DUMMY: + + Gly 513 + + + 0.99919623 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.99500006 + residue_name_number + cleaner0 + 2023-07-26T09:34:15Z + DUMMY: + + Gly 512 + + + 0.9959071 + residue_name_number + cleaner0 + 2023-07-26T09:34:18Z + DUMMY: + + Gly 513 + + + 0.9964285 + residue_name_number + cleaner0 + 2023-07-26T09:34:18Z + DUMMY: + + Gly 513 + + + 0.9982552 + protein_state + cleaner0 + 2023-07-26T09:00:04Z + DUMMY: + + conserved + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.99914706 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.99679244 + residue_name_number + cleaner0 + 2023-07-26T09:34:15Z + DUMMY: + + Gly 512 + + + 0.9982619 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.9991812 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + structure_element + SO: + cleaner0 + 2023-07-26T09:01:23Z + + Gly-Gly + + + 0.99818 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.9979327 + residue_name + cleaner0 + 2023-07-26T09:00:37Z + SO: + + glycine + + + 0.9945412 + structure_element + cleaner0 + 2023-07-26T09:00:43Z + SO: + + hinge + + + 0.9972619 + residue_name_number + cleaner0 + 2023-07-26T09:34:18Z + DUMMY: + + Gly 513 + + + 0.98326504 + protein_state + cleaner0 + 2023-07-26T09:01:33Z + DUMMY: + + absolute conservation + + + 0.9730308 + taxonomy_domain + cleaner0 + 2023-07-26T09:00:54Z + DUMMY: + + budding yeast + + + 0.99933845 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + + RESULTS + paragraph + 16501 + To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). This means that the effect of the mutation is mainly to generate a more restricted geometry at the hinge region. G512A or G513A alone showed no differences from wild-type in terms of the size-exclusion chromatography elution profile (Fig. 3D), suggesting that the restriction for single glycine flexibility would not be enough to break the swapped structure of helix 9B. However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely. The G512K/G513K double mutant eluted at the monomer position in size-exclusion chromatography (Fig. 3D). A previous study shows that induction of steric hindrance by mutation destabilizes the dimerization interface of a different protein, ClC transporter. + + 0.9956256 + residue_name_number + cleaner0 + 2023-07-26T09:34:15Z + DUMMY: + + Gly 512 + + + 0.9962336 + residue_name_number + cleaner0 + 2023-07-26T09:34:18Z + DUMMY: + + Gly 513 + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9958221 + experimental_method + cleaner0 + 2023-07-26T12:17:27Z + MESH: + + point mutation + + + 0.8371418 + mutant + cleaner0 + 2023-07-26T09:01:49Z + MESH: + + Gly to Ala + + + 0.99523824 + residue_name_number + cleaner0 + 2023-07-26T09:34:15Z + DUMMY: + + Gly 512 + + + 0.9954579 + residue_name_number + cleaner0 + 2023-07-26T09:34:19Z + DUMMY: + + Gly 513 + + + 0.9959911 + structure_element + cleaner0 + 2023-07-26T08:41:50Z + SO: + + helix 9B + + + 0.994381 + oligomeric_state + cleaner0 + 2023-07-26T08:39:07Z + DUMMY: + + protomer + + + 0.9974095 + residue_name + cleaner0 + 2023-07-26T09:01:59Z + SO: + + alanine + + + 0.9810831 + experimental_method + cleaner0 + 2023-07-26T12:17:36Z + MESH: + + mutation + + + 0.9816042 + experimental_method + cleaner0 + 2023-07-26T12:17:38Z + MESH: + + mutation + + + structure_element + SO: + cleaner0 + 2023-07-26T09:00:43Z + + hinge + + + 0.9990533 + mutant + cleaner0 + 2023-07-26T09:02:13Z + MESH: + + G512A + + + 0.99901223 + mutant + cleaner0 + 2023-07-26T09:02:17Z + MESH: + + G513A + + + 0.9987969 + protein_state + cleaner0 + 2023-07-26T08:53:21Z + DUMMY: + + wild-type + + + 0.99452174 + experimental_method + cleaner0 + 2023-07-26T12:17:41Z + MESH: + + size-exclusion chromatography + + + 0.9920854 + residue_name + cleaner0 + 2023-07-26T09:02:26Z + SO: + + glycine + + + 0.99700415 + structure_element + cleaner0 + 2023-07-26T08:41:50Z + SO: + + helix 9B + + + 0.89237595 + protein_state + cleaner0 + 2023-07-26T09:02:33Z + DUMMY: + + double mutant + + + 0.9990127 + mutant + cleaner0 + 2023-07-26T09:02:13Z + MESH: + + G512A + + + 0.9969656 + mutant + cleaner0 + 2023-07-26T09:02:18Z + MESH: + + G513A + + + 0.998868 + protein_state + cleaner0 + 2023-07-26T08:53:21Z + DUMMY: + + wild-type + + + 0.9976515 + experimental_method + cleaner0 + 2023-07-26T12:17:44Z + MESH: + + mutation + + + 0.9992169 + structure_element + cleaner0 + 2023-07-26T09:00:43Z + SO: + + hinge + + + 0.9940084 + structure_element + cleaner0 + 2023-07-26T08:40:20Z + SO: + + helix 9A + + + 0.89214116 + structure_element + cleaner0 + 2023-07-26T09:49:44Z + SO: + + 9B + + + 0.9929811 + structure_element + cleaner0 + 2023-07-26T08:41:50Z + SO: + + helix 9B + + + 0.9993969 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.98809755 + experimental_method + cleaner0 + 2023-07-26T12:17:51Z + MESH: + + mutated to + + + 0.99598 + residue_name + cleaner0 + 2023-07-26T09:02:28Z + SO: + + lysine + + + 0.9989839 + mutant + cleaner0 + 2023-07-26T09:02:42Z + MESH: + + G512K + + + 0.99544466 + mutant + cleaner0 + 2023-07-26T09:02:47Z + MESH: + + G513K + + + 0.9972485 + protein_state + cleaner0 + 2023-07-26T09:02:37Z + DUMMY: + + mutant + + + 0.9987363 + structure_element + cleaner0 + 2023-07-26T09:00:43Z + SO: + + hinge + + + 0.9963349 + oligomeric_state + cleaner0 + 2023-07-26T08:39:07Z + DUMMY: + + protomer + + + 0.99938715 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.9994 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.9989249 + mutant + cleaner0 + 2023-07-26T09:02:43Z + MESH: + + G512K + + + 0.99053127 + mutant + cleaner0 + 2023-07-26T09:02:47Z + MESH: + + G513K + + + 0.93179905 + protein_state + cleaner0 + 2023-07-26T09:02:33Z + DUMMY: + + double mutant + + + 0.9976248 + oligomeric_state + cleaner0 + 2023-07-26T08:39:02Z + DUMMY: + + monomer + + + 0.9979068 + experimental_method + cleaner0 + 2023-07-26T12:17:55Z + MESH: + + size-exclusion chromatography + + + 0.5608726 + experimental_method + cleaner0 + 2023-07-26T12:17:59Z + MESH: + + mutation + + + 0.9989898 + site + cleaner0 + 2023-07-26T08:58:10Z + SO: + + dimerization interface + + + 0.98787004 + protein_type + cleaner0 + 2023-07-26T12:18:03Z + MESH: + + ClC transporter + + + + RESULTS + paragraph + 18214 + Collectively, these data suggest that the Gly 512 and Gly 513 at the connection between helix 9A and 9B play a crucial role in forming the domain-swapped conformation that enables dimer formation. + + 0.9961103 + residue_name_number + cleaner0 + 2023-07-26T09:34:15Z + DUMMY: + + Gly 512 + + + 0.9966997 + residue_name_number + cleaner0 + 2023-07-26T09:34:19Z + DUMMY: + + Gly 513 + + + 0.99120015 + structure_element + cleaner0 + 2023-07-26T08:40:20Z + SO: + + helix 9A + + + 0.5932326 + structure_element + cleaner0 + 2023-07-26T09:49:50Z + SO: + + 9B + + + 0.99841547 + protein_state + cleaner0 + 2023-07-26T09:03:04Z + DUMMY: + + domain-swapped + + + 0.9987753 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + + RESULTS + title_2 + 18411 + SEL1L Forms Self-oligomers through SEL1Lcent domain in vivo + + 0.9990978 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T09:06:04Z + + Self-oligomers + + + 0.99933666 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + + RESULTS + paragraph + 18471 + Next, we examined if SEL1L also forms self-oligomers in vivo using HEK293T cells. We generated full-length SEL1L-HA and SEL1L-FLAG fusion constructs and co-transfected the constructs into HEK293T cells. A co-immunoprecipitation assay using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody showed that full-length SEL1L forms self-oligomers in vivo (Fig. 4A). To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. Co-immunoprecipitation analysis showed that the SEL1Lcent was sufficient to physically interact with the full-length SEL1L, while SEL1L348–497 failed to do so (Fig. 4A). Interestingly, however, the expression level of SEL1L348–497 was consistently lower than that of SEL1Lcent (Fig. 4A,B). Semi-quantitative RT-PCR revealed no significant difference in transcriptional levels of the two constructs (data not shown). We speculated that SEL1L348–497 could be secreted while the SEL1Lcent is retained in the ER by association with the endogenous ERAD complex. Indeed, immunoprecipitation followed by western blot analysis using the culture medium detected secreted SEL1L348–497 fragment, but not SEL1Lcent (Fig. 4B). We next examined if the reason why SEL1L348–497 failed to bind to the full-length SEL1L may be because of the lower level of SEL1L348–497 in the ER lumen compared to SEL1Lcent fragment. In order to retain two SEL1L fragments in the ER lumen, we added KDEL ER retention sequence to the C-terminus of both fragments. Indeed, the addition of KDEL peptide increased the level of SEL1L348–497 in the ER lumen (Fig. 4D,E) and the immunostaining analysis showed both constructs were well localized to the ER (Fig. 4C). We further analyzed whether SEL1Lcent may competitively inhibit the self-oligomerization of SEL1L in vivo. To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. Co-immunoprecipitation assay revealed that wild-type SEL1Lcent-KDEL, indeed, competitively disrupted the self-association of the full-length SEL1L (Fig. 4E). In contrast, SEL1L348–497-KDEL and the single-residue mutation L521A in SEL1Lcent did not competitively inhibit the self-association of full-length SEL1L (Fig. 4E,F). These data suggest that the SEL1L forms self-oligomers and the oligomerization is mediated by the SEL1Lcent domain in vivo. + + 0.99905807 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T09:06:04Z + + self-oligomers + + + 0.9990368 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.99192 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:03:35Z + + HA + + + 0.9596383 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.60219574 + experimental_method + cleaner0 + 2023-07-26T09:03:44Z + MESH: + + FLAG + + + experimental_method + MESH: + cleaner0 + 2023-07-26T12:18:38Z + + fusion constructs + + + 0.9982791 + experimental_method + cleaner0 + 2023-07-26T12:18:41Z + MESH: + + co-transfected + + + 0.9990059 + experimental_method + cleaner0 + 2023-07-26T12:18:44Z + MESH: + + co-immunoprecipitation assay + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:03:45Z + + FLAG + + + 0.9982916 + experimental_method + cleaner0 + 2023-07-26T12:18:49Z + MESH: + + Western blot + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:09:28Z + + HA + + + 0.999084 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.9989919 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T09:06:04Z + + self-oligomers + + + 0.8052421 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.9991011 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.9988022 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.8979191 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + structure_element + SO: + cleaner0 + 2023-07-26T09:04:35Z + + SLR motif 9 + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:04:44Z + + deletion + + + 0.79511404 + mutant + cleaner0 + 2023-07-26T09:04:55Z + MESH: + + SEL1L348–497 + + + 0.88660175 + experimental_method + cleaner0 + 2023-07-26T12:18:55Z + MESH: + + fused to + + + 0.9970625 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.9947206 + structure_element + cleaner0 + 2023-07-26T09:05:43Z + SO: + + signal peptides + + + 0.9989898 + experimental_method + cleaner0 + 2023-07-26T12:18:58Z + MESH: + + Co-immunoprecipitation analysis + + + 0.6223402 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.9991303 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.9983606 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.8710802 + mutant + cleaner0 + 2023-07-26T09:04:56Z + MESH: + + SEL1L348–497 + + + 0.8748493 + mutant + cleaner0 + 2023-07-26T09:04:56Z + MESH: + + SEL1L348–497 + + + 0.5263394 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.9990075 + experimental_method + cleaner0 + 2023-07-26T12:19:05Z + MESH: + + Semi-quantitative RT-PCR + + + mutant + MESH: + cleaner0 + 2023-07-26T09:04:56Z + + SEL1L348–497 + + + 0.91881275 + structure_element + cleaner0 + 2023-07-26T08:23:46Z + SO: + + SEL1Lcent + + + 0.9988921 + experimental_method + cleaner0 + 2023-07-26T12:19:09Z + MESH: + + immunoprecipitation + + + 0.9573611 + experimental_method + cleaner0 + 2023-07-26T12:19:12Z + MESH: + + western blot + + + mutant + MESH: + cleaner0 + 2023-07-26T09:04:56Z + + SEL1L348–497 + + + 0.9566656 + structure_element + cleaner0 + 2023-07-26T08:23:47Z + SO: + + SEL1Lcent + + + 0.8454278 + mutant + cleaner0 + 2023-07-26T09:04:56Z + MESH: + + SEL1L348–497 + + + 0.9990664 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.99819946 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + mutant + MESH: + cleaner0 + 2023-07-26T09:04:56Z + + SEL1L348–497 + + + 0.86057234 + structure_element + cleaner0 + 2023-07-26T08:23:47Z + SO: + + SEL1Lcent + + + 0.87872255 + protein + cleaner0 + 2023-07-26T08:24:00Z + PR: + + SEL1L + + + 0.7766495 + structure_element + cleaner0 + 2023-07-26T12:19:53Z + SO: + + KDEL + + + 0.9066809 + structure_element + cleaner0 + 2023-07-26T09:50:01Z + SO: + + ER retention sequence + + + structure_element + SO: + cleaner0 + 2023-07-26T12:20:10Z + + KDEL + + + mutant + MESH: + cleaner0 + 2023-07-26T09:04:56Z + + SEL1L348–497 + + + 0.9971819 + experimental_method + cleaner0 + 2023-07-26T12:19:16Z + MESH: + + immunostaining + + + 0.7080341 + structure_element + cleaner0 + 2023-07-26T08:23:47Z + SO: + + SEL1Lcent + + + 0.99902713 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.99842024 + experimental_method + cleaner0 + 2023-07-26T12:19:20Z + MESH: + + co-transfected + + + 0.9777483 + protein_state + cleaner0 + 2023-07-26T12:01:08Z + DUMMY: + + tagged + + + 0.9990776 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.9985807 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.99638003 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.4470757 + experimental_method + cleaner0 + 2023-07-26T09:09:27Z + MESH: + + HA + + + 0.98915076 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:03:45Z + + FLAG + + + 0.89780843 + experimental_method + cleaner0 + 2023-07-26T12:19:26Z + MESH: + + increasing doses + + + mutant + MESH: + cleaner0 + 2023-07-26T09:07:32Z + + SEL1Lcent-KDEL + + + mutant + MESH: + cleaner0 + 2023-07-26T09:08:07Z + + SEL1L348–497-KDEL + + + mutant + MESH: + cleaner0 + 2023-07-26T09:09:00Z + + SEL1Lcent (L521A)-KDEL + + + 0.9989825 + experimental_method + cleaner0 + 2023-07-26T12:19:29Z + MESH: + + Co-immunoprecipitation assay + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T08:53:21Z + + wild-type + + + mutant + MESH: + cleaner0 + 2023-07-26T09:07:33Z + + SEL1Lcent-KDEL + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T08:24:14Z + + full-length + + + mutant + MESH: + cleaner0 + 2023-07-26T09:08:08Z + + SEL1L348–497-KDEL + + + mutant + MESH: + cleaner0 + 2023-07-26T08:52:49Z + + L521A + + + structure_element + SO: + cleaner0 + 2023-07-26T08:23:47Z + + SEL1Lcent + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T08:24:14Z + + full-length + + + protein + PR: + cleaner0 + 2023-07-26T08:24:01Z + + SEL1L + + + protein + PR: + cleaner0 + 2023-07-26T08:24:01Z + + SEL1L + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T09:06:04Z + + self-oligomers + + + structure_element + SO: + cleaner0 + 2023-07-26T08:23:47Z + + SEL1Lcent + + + + RESULTS + title_2 + 21109 + Structural Comparison of SEL1L SLRs with TPRs or SLRs of Other Proteins + + 0.99825406 + experimental_method + cleaner0 + 2023-07-26T12:20:20Z + MESH: + + Structural Comparison + + + 0.99931073 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.99896467 + structure_element + cleaner0 + 2023-07-26T08:41:13Z + SO: + + SLRs + + + 0.99888355 + structure_element + cleaner0 + 2023-07-26T08:40:43Z + SO: + + TPRs + + + 0.99914634 + structure_element + cleaner0 + 2023-07-26T08:41:13Z + SO: + + SLRs + + + + RESULTS + paragraph + 21181 + Previous studies reveal that TPRs and SLRs have similar consensus sequences, suggesting that their three-dimensional structures are also similar. The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar. This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). The differences may result from the differing numbers of residues in the loops and differences in antiparallel helix packing. Moreover, there are conserved disulfide bonds in the SLR motifs of HcpC and HcpB, but no such bonds are observed in SEL1Lcent. These factors contribute to the differences in the overall conformation of the SLR motifs in SEL1L and other SLR or TPR motif-containing proteins. + + 0.99915004 + structure_element + cleaner0 + 2023-07-26T08:40:43Z + SO: + + TPRs + + + 0.9991611 + structure_element + cleaner0 + 2023-07-26T08:41:13Z + SO: + + SLRs + + + 0.9988194 + experimental_method + cleaner0 + 2023-07-26T12:20:23Z + MESH: + + superposition + + + 0.9993436 + structure_element + cleaner0 + 2023-07-26T08:40:43Z + SO: + + TPRs + + + 0.9990426 + protein + cleaner0 + 2023-07-26T09:10:13Z + PR: + + Cdc23 + + + 0.9972758 + species + cleaner0 + 2023-07-26T09:10:05Z + MESH: + + S. pombe + + + protein + PR: + cleaner0 + 2023-07-26T09:10:01Z + + cell division cycle 23 + + + 0.9991893 + structure_element + cleaner0 + 2023-07-26T08:41:13Z + SO: + + SLRs + + + 0.9991386 + protein + cleaner0 + 2023-07-26T09:10:22Z + PR: + + HcpC + + + 0.9924793 + protein + cleaner0 + 2023-07-26T09:10:29Z + PR: + + Helicobacter Cysteine-rich Protein C + + + 0.9976896 + evidence + cleaner0 + 2023-07-26T11:54:48Z + DUMMY: + + RMSDs + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9987198 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9984101 + structure_element + cleaner0 + 2023-07-26T08:23:47Z + SO: + + SEL1Lcent + + + 0.9481077 + experimental_method + cleaner0 + 2023-07-26T12:20:34Z + MESH: + + structural alignment + + + 0.99925023 + structure_element + cleaner0 + 2023-07-26T08:40:43Z + SO: + + TPRs + + + 0.99756396 + evidence + cleaner0 + 2023-07-26T08:41:28Z + DUMMY: + + RMSD + + + protein + PR: + cleaner0 + 2023-07-26T09:11:47Z + + Cdc23 + + + 0.9993161 + structure_element + cleaner0 + 2023-07-26T08:41:13Z + SO: + + SLRs + + + 0.9979068 + evidence + cleaner0 + 2023-07-26T08:41:28Z + DUMMY: + + RMSD + + + 0.99843234 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.9988809 + experimental_method + cleaner0 + 2023-07-26T12:20:37Z + MESH: + + superimposing + + + 0.9978993 + evidence + cleaner0 + 2023-07-26T11:54:52Z + DUMMY: + + structure + + + 0.96890366 + structure_element + cleaner0 + 2023-07-26T08:25:36Z + SO: + + SLR motifs 5 to 9 + + + 0.9973239 + structure_element + cleaner0 + 2023-07-26T08:23:47Z + SO: + + SEL1Lcent + + + protein + PR: + cleaner0 + 2023-07-26T09:12:02Z + + Cdc23 + + + 0.9991424 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.99682415 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.9979621 + evidence + cleaner0 + 2023-07-26T11:54:56Z + DUMMY: + + structures + + + 0.969845 + structure_element + cleaner0 + 2023-07-26T08:25:36Z + SO: + + SLR motifs 5 to 9 + + + 0.9980794 + structure_element + cleaner0 + 2023-07-26T08:23:47Z + SO: + + SEL1Lcent + + + 0.9989575 + protein + cleaner0 + 2023-07-26T09:10:14Z + PR: + + Cdc23 + + + 0.9989826 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.99841034 + evidence + cleaner0 + 2023-07-26T08:41:28Z + DUMMY: + + RMSD + + + 0.9975805 + structure_element + cleaner0 + 2023-07-26T09:50:22Z + SO: + + loops + + + structure_element + SO: + cleaner0 + 2023-07-26T09:50:44Z + + antiparallel helix + + + 0.99637115 + protein_state + cleaner0 + 2023-07-26T09:12:07Z + DUMMY: + + conserved + + + 0.98580664 + ptm + cleaner0 + 2023-07-26T09:12:10Z + MESH: + + disulfide bonds + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9988392 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.99880195 + protein + cleaner0 + 2023-07-26T09:42:10Z + PR: + + HcpB + + + 0.99857056 + structure_element + cleaner0 + 2023-07-26T08:23:47Z + SO: + + SEL1Lcent + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:36Z + + SLR + + + 0.9981993 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + protein_type + MESH: + cleaner0 + 2023-07-26T09:12:43Z + + SLR or TPR motif-containing proteins + + + + RESULTS + paragraph + 22532 + Another major difference in the structure of SLR motifs between SEL1L and HcpC is the oligomeric state of proteins. The TPR motif is involved in the dimerization of proteins such as Cdc23, Cdc16, and Cdc27. In particular, the N-terminal domain of Cdc23 (Cdc23N-term) has a TPR-motif organization similar to that of the SLR motif in SEL1Lcent. The seven TPR motifs of Cdc23N-term are assembled into a superhelical structure, generating a hollow surface and encircling its dimer counterpart in an interlocking clasp-like arrangement (Fig. 5C). The TPR motif 1 (TPR1) of each Cdc23N-term subunit is located in the hollow surface of the counter subunit and interacts with residues lining the inner groove TPR α-helices, generating two-fold symmetry homotype interactions. However, in this structure, a conformational change in the TPR motif itself is not observed. + + 0.99250495 + evidence + cleaner0 + 2023-07-26T09:44:28Z + DUMMY: + + structure + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.99894506 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.99799466 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + structure_element + SO: + cleaner0 + 2023-07-26T09:43:43Z + + TPR + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T11:57:09Z + + dimerization + + + 0.99848664 + protein + cleaner0 + 2023-07-26T09:10:14Z + PR: + + Cdc23 + + + 0.99822587 + protein + cleaner0 + 2023-07-26T09:13:06Z + PR: + + Cdc16 + + + 0.9979845 + protein + cleaner0 + 2023-07-26T09:13:11Z + PR: + + Cdc27 + + + 0.9977392 + protein + cleaner0 + 2023-07-26T09:10:14Z + PR: + + Cdc23 + + + protein + PR: + cleaner0 + 2023-07-26T09:13:28Z + + Cdc23 + + + structure_element + SO: + cleaner0 + 2023-07-26T09:43:43Z + + TPR + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.9988959 + structure_element + cleaner0 + 2023-07-26T08:23:47Z + SO: + + SEL1Lcent + + + structure_element + SO: + cleaner0 + 2023-07-26T09:43:43Z + + TPR + + + protein + PR: + cleaner0 + 2023-07-26T09:42:30Z + + Cdc23 + + + 0.9910953 + structure_element + cleaner0 + 2023-07-26T09:51:00Z + SO: + + superhelical structure + + + 0.99880505 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.999269 + structure_element + cleaner0 + 2023-07-26T09:13:49Z + SO: + + TPR motif 1 + + + 0.99945503 + structure_element + cleaner0 + 2023-07-26T09:13:53Z + SO: + + TPR1 + + + protein + PR: + cleaner0 + 2023-07-26T09:42:46Z + + Cdc23 + + + site + SO: + cleaner0 + 2023-07-26T12:04:24Z + + inner groove + + + 0.9081493 + structure_element + cleaner0 + 2023-07-26T09:43:42Z + SO: + + TPR + + + 0.9844491 + structure_element + cleaner0 + 2023-07-26T09:44:22Z + SO: + + α-helices + + + 0.9981635 + evidence + cleaner0 + 2023-07-26T09:44:26Z + DUMMY: + + structure + + + structure_element + SO: + cleaner0 + 2023-07-26T09:43:43Z + + TPR + + + + RESULTS + paragraph + 23397 + Self-association of HcpC has not been reported, and there is no domain-swapped structure in the SLR motifs of HcpC, in contrast to that observed in SEL1Lcent. Although SEL1L contains a number of SLR motifs comparable to HcpC, the SLR motifs in SEL1L are interrupted by other sequences, making three SLR motif clusters (Fig. 1A). The interrupted SLR motifs may be required for dimerization of SEL1Lcent, as five SLR motifs are more than enough to form the semicircle of the yin-yang symbol (Fig. 1B). Helix 5A from SLR motif 5 meets helix 9A from SLR motif 9 of the counterpart SEL1L. If the SLR motifs 5 to 9 were not isolated from other SLR motifs, steric hindrance could interfere with dimerization of SEL1L. This is one of the biggest differences from TPRs in Cdc23 and from the SLRs in HcpC, where the motifs exist in tandem. TPR and SLR motifs are generally involved in protein-protein interaction modules, and the sequences between the SLR motifs of SEL1L might actually facilitate the self-association of this protein. + + 0.99859196 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.9856379 + protein_state + cleaner0 + 2023-07-26T09:03:04Z + DUMMY: + + domain-swapped + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.9986305 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.9993549 + structure_element + cleaner0 + 2023-07-26T08:23:47Z + SO: + + SEL1Lcent + + + 0.9992762 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.9985732 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.9992106 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T11:57:09Z + + dimerization + + + 0.9993672 + structure_element + cleaner0 + 2023-07-26T08:23:47Z + SO: + + SEL1Lcent + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + structure_element + SO: + cleaner0 + 2023-07-26T09:15:01Z + + semicircle of the yin-yang + + + 0.9988102 + structure_element + cleaner0 + 2023-07-26T09:49:09Z + SO: + + Helix 5A + + + 0.9962883 + structure_element + cleaner0 + 2023-07-26T09:15:11Z + SO: + + SLR motif 5 + + + 0.99878836 + structure_element + cleaner0 + 2023-07-26T08:40:20Z + SO: + + helix 9A + + + 0.9975462 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.9993511 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.98970985 + structure_element + cleaner0 + 2023-07-26T08:25:36Z + SO: + + SLR motifs 5 to 9 + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T11:57:09Z + + dimerization + + + 0.9993968 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.9991345 + structure_element + cleaner0 + 2023-07-26T08:40:43Z + SO: + + TPRs + + + 0.9992237 + protein + cleaner0 + 2023-07-26T09:10:14Z + PR: + + Cdc23 + + + 0.9994486 + structure_element + cleaner0 + 2023-07-26T08:41:13Z + SO: + + SLRs + + + 0.9989011 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.9992543 + structure_element + cleaner0 + 2023-07-26T09:43:43Z + SO: + + TPR + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.99938834 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + + RESULTS + title_2 + 24423 + SLR-C of SEL1L Binds HRD1 N-terminus Luminal Loop + + 0.99893826 + structure_element + cleaner0 + 2023-07-26T08:33:22Z + SO: + + SLR-C + + + 0.9991726 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.7173637 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + 0.9890427 + structure_element + cleaner0 + 2023-07-26T09:47:49Z + SO: + + Luminal Loop + + + + RESULTS + paragraph + 24473 + Based on the structural data presented herein, a possible arrangement of membrane-associated ERAD components in mammals, highlighting the molecular functions of SLR domains in SEL1L, is shown in Fig. 6C. We suggest that the middle SLR domains are involved in the dimerization of SEL1L based on the crystal structure and biochemical data. SLR-C, which contains SLR motifs 10 to 11, might be involved in the interaction with HRD1. Indirect evidence from a previous yeast study shows that the circumscribed region of C-terminal Hrd3p, specifically residues 664–695, forms contacts with the Hrd1 luminal loops. The Hrd3p residues 664–695 correspond to mouse SEL1L residues 696–727, which include the entire helix 11B (residue 697–709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4). This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). To address this hypothesis, we prepared constructs encoding mouse HRD1 luminal fragments fused to GST as shown in Fig. 6A, and tested their ability to bind certain SLR motifs in SEL1L. The fusion proteins were immobilized on glutathione-Sepharose beads and probed for binding to SLR-N, SLR-M, SLR-C, and monomer form of SLR-M (SLR-ML521A). Figure 6B shows that the SLR-C, consisting of SLR motifs 10 and 11, exclusively interacts with N-terminal luminal loop (residues 21–42) of HRD1. + + 0.99301314 + evidence + cleaner0 + 2023-07-26T11:55:02Z + DUMMY: + + structural data + + + 0.99872905 + taxonomy_domain + cleaner0 + 2023-07-26T11:55:33Z + DUMMY: + + mammals + + + 0.99515957 + structure_element + cleaner0 + 2023-07-26T08:31:37Z + SO: + + SLR + + + 0.99928576 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T11:57:09Z + + dimerization + + + 0.99938464 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.99822503 + evidence + cleaner0 + 2023-07-26T11:52:55Z + DUMMY: + + crystal structure + + + structure_element + SO: + cleaner0 + 2023-07-26T08:33:22Z + + SLR-C + + + 0.9579142 + structure_element + cleaner0 + 2023-07-26T09:51:05Z + SO: + + SLR motifs 10 to 11 + + + 0.50802 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + 0.9944859 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:26Z + DUMMY: + + yeast + + + 0.9987024 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + 0.9977992 + residue_range + cleaner0 + 2023-07-26T09:16:48Z + DUMMY: + + 664–695 + + + 0.92809564 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + Hrd1 + + + 0.99864084 + structure_element + cleaner0 + 2023-07-26T09:51:10Z + SO: + + luminal loops + + + 0.99743974 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + 0.99779993 + residue_range + cleaner0 + 2023-07-26T09:16:51Z + DUMMY: + + 664–695 + + + 0.9900275 + taxonomy_domain + cleaner0 + 2023-07-26T08:29:39Z + DUMMY: + + mouse + + + 0.9992859 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.9977267 + residue_range + cleaner0 + 2023-07-26T09:16:53Z + DUMMY: + + 696–727 + + + 0.9986229 + structure_element + cleaner0 + 2023-07-26T09:17:17Z + SO: + + helix 11B + + + 0.99773 + residue_range + cleaner0 + 2023-07-26T09:16:56Z + DUMMY: + + 697–709 + + + 0.99542075 + structure_element + cleaner0 + 2023-07-26T09:17:14Z + SO: + + SLR motif 11 + + + 0.99896866 + protein_state + cleaner0 + 2023-07-26T09:16:58Z + DUMMY: + + well-conserved + + + 0.9705908 + structure_element + cleaner0 + 2023-07-26T09:17:21Z + SO: + + SLR motif 10 to 11 + + + 0.9988179 + experimental_method + cleaner0 + 2023-07-26T12:20:42Z + MESH: + + structure-guided SLR motif alignment + + + 0.82161427 + experimental_method + cleaner0 + 2023-07-26T12:20:46Z + MESH: + + structure study + + + 0.7311754 + protein_state + cleaner0 + 2023-07-26T12:01:37Z + DUMMY: + + sequence conservation + + + 0.99881953 + taxonomy_domain + cleaner0 + 2023-07-26T08:25:52Z + DUMMY: + + mammalian + + + 0.99931455 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.9988205 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:26Z + DUMMY: + + yeast + + + 0.95084345 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + 0.98013896 + structure_element + cleaner0 + 2023-07-26T09:17:31Z + SO: + + SLR motifs 10 to 11 + + + 0.73068887 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + 0.9921606 + protein + cleaner0 + 2023-07-26T09:20:05Z + PR: + + Hrd1p + + + 0.99814737 + taxonomy_domain + cleaner0 + 2023-07-26T08:29:39Z + DUMMY: + + mouse + + + 0.9586618 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:17:58Z + + fused to GST + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.99931836 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.95016915 + structure_element + cleaner0 + 2023-07-26T08:35:33Z + SO: + + SLR-N + + + 0.95825976 + structure_element + cleaner0 + 2023-07-26T08:35:41Z + SO: + + SLR-M + + + 0.98809576 + structure_element + cleaner0 + 2023-07-26T08:33:22Z + SO: + + SLR-C + + + 0.99815935 + oligomeric_state + cleaner0 + 2023-07-26T08:39:02Z + DUMMY: + + monomer + + + 0.98904943 + structure_element + cleaner0 + 2023-07-26T08:35:41Z + SO: + + SLR-M + + + 0.9985962 + mutant + cleaner0 + 2023-07-26T09:18:25Z + MESH: + + SLR-ML521A + + + structure_element + SO: + cleaner0 + 2023-07-26T08:33:22Z + + SLR-C + + + 0.9908142 + structure_element + cleaner0 + 2023-07-26T08:33:26Z + SO: + + SLR motifs 10 and 11 + + + 0.99880993 + structure_element + cleaner0 + 2023-07-26T09:47:49Z + SO: + + luminal loop + + + 0.9976925 + residue_range + cleaner0 + 2023-07-26T09:18:30Z + DUMMY: + + 21–42 + + + 0.591227 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + + RESULTS + paragraph + 26172 + The molecular functions of SLR-N are unclear. One possibility is that SLR-N contributes to substrate recognition of proteins to be degraded because there are a couple of putative glycosylation sites within the SLR-N domain (Fig. 1A). SEL1Lcent contains a putative N-glycosylation site, Asn 427, which is highly conserved among different species and structurally exposed to the surface of the SEL1L dimer according to the crystal structure (Fig. 6C). + + 0.9861813 + structure_element + cleaner0 + 2023-07-26T08:35:33Z + SO: + + SLR-N + + + 0.7683429 + structure_element + cleaner0 + 2023-07-26T08:35:33Z + SO: + + SLR-N + + + site + SO: + cleaner0 + 2023-07-26T09:19:34Z + + glycosylation sites + + + structure_element + SO: + cleaner0 + 2023-07-26T08:35:33Z + + SLR-N + + + 0.99923563 + structure_element + cleaner0 + 2023-07-26T08:23:47Z + SO: + + SEL1Lcent + + + site + SO: + cleaner0 + 2023-07-26T09:19:45Z + + N-glycosylation site + + + 0.9950211 + residue_name_number + cleaner0 + 2023-07-26T09:19:48Z + DUMMY: + + Asn 427 + + + 0.9987097 + protein_state + cleaner0 + 2023-07-26T09:19:51Z + DUMMY: + + highly conserved + + + 0.9993482 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.9988103 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.99884605 + evidence + cleaner0 + 2023-07-26T11:52:55Z + DUMMY: + + crystal structure + + + + DISCUSS + title_1 + 26622 + Discussion + + + DISCUSS + paragraph + 26633 + Many reports demonstrate that membrane-bound ERAD machinery proteins in yeast, such as Hrd1p, Der1p, and Usa1p, are involved in oligomerization of ERAD components. The Hrd1p complex forms dimers upon sucrose gradient sedimentation and size-exclusion chromatography. Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. Considering that the functional and structural composition of ERAD components are conserved in both yeast and mammals, we propose that the mammalian ERAD components also form self-associating oligomers. This hypothesis is supported by cross-linking data suggesting that human HRD1 forms a homodimer. Consistent with the previous data, our crystal structure and biochemical data demonstrate that mouse SEL1Lcent exists as a homodimer in the ER lumen via domain swapping of SLR motif 9. We need to further test whether there are contacts involved in dimer formation in SEL1L in addition to those in the SLR-M region. + + 0.9989015 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:26Z + DUMMY: + + yeast + + + 0.9981091 + protein + cleaner0 + 2023-07-26T09:20:04Z + PR: + + Hrd1p + + + 0.99841964 + protein + cleaner0 + 2023-07-26T08:26:42Z + PR: + + Der1p + + + 0.99871147 + protein + cleaner0 + 2023-07-26T08:27:00Z + PR: + + Usa1p + + + 0.99583495 + protein + cleaner0 + 2023-07-26T09:20:05Z + PR: + + Hrd1p + + + 0.9987563 + oligomeric_state + cleaner0 + 2023-07-26T11:56:32Z + DUMMY: + + dimers + + + 0.9988889 + experimental_method + cleaner0 + 2023-07-26T12:21:08Z + MESH: + + sucrose gradient sedimentation + + + 0.99879324 + experimental_method + cleaner0 + 2023-07-26T12:21:11Z + MESH: + + size-exclusion chromatography + + + 0.9873705 + protein_state + cleaner0 + 2023-07-26T09:20:21Z + DUMMY: + + HA-epitope-tagged + + + 0.9904461 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + 0.97846574 + protein + cleaner0 + 2023-07-26T09:20:05Z + PR: + + Hrd1p + + + 0.99916005 + protein_state + cleaner0 + 2023-07-26T09:20:24Z + DUMMY: + + unmodified + + + 0.99035174 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + 0.9786226 + protein + cleaner0 + 2023-07-26T09:20:05Z + PR: + + Hrd1p + + + 0.98326415 + protein + cleaner0 + 2023-07-26T09:20:05Z + PR: + + Hrd1p + + + 0.986401 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + 0.99876297 + oligomeric_state + cleaner0 + 2023-07-26T08:39:18Z + DUMMY: + + homodimers + + + 0.97608227 + complex_assembly + cleaner0 + 2023-07-26T09:24:15Z + GO: + + Hrd + + + 0.9987778 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:26Z + DUMMY: + + yeast + + + 0.99861693 + taxonomy_domain + cleaner0 + 2023-07-26T11:55:38Z + DUMMY: + + mammals + + + 0.9984384 + taxonomy_domain + cleaner0 + 2023-07-26T08:25:52Z + DUMMY: + + mammalian + + + 0.99883693 + oligomeric_state + cleaner0 + 2023-07-26T11:56:36Z + DUMMY: + + oligomers + + + 0.7436882 + experimental_method + cleaner0 + 2023-07-26T12:21:15Z + MESH: + + cross-linking data + + + 0.99834514 + species + cleaner0 + 2023-07-26T12:25:26Z + MESH: + + human + + + 0.997649 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + 0.9987276 + oligomeric_state + cleaner0 + 2023-07-26T08:24:28Z + DUMMY: + + homodimer + + + 0.9978006 + evidence + cleaner0 + 2023-07-26T11:52:55Z + DUMMY: + + crystal structure + + + 0.92263424 + evidence + cleaner0 + 2023-07-26T11:55:09Z + DUMMY: + + biochemical data + + + 0.995531 + taxonomy_domain + cleaner0 + 2023-07-26T08:29:39Z + DUMMY: + + mouse + + + 0.99918634 + structure_element + cleaner0 + 2023-07-26T08:23:47Z + SO: + + SEL1Lcent + + + 0.9987508 + oligomeric_state + cleaner0 + 2023-07-26T08:24:28Z + DUMMY: + + homodimer + + + 0.99854213 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.9988674 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.99924845 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:35:41Z + + SLR-M + + + + DISCUSS + paragraph + 27746 + In yeast, Usa1p acts as a scaffold for Hrd1p and Der1p, in which the N-terminus of Usa1p interacts with the C-terminal 34 amino acids of Hrd1p in the cytosol to induce oligomerization of Hrd1p, which is essential for its activity. However, metazoans lack a clear Usa1p homolog. Although mammalian HERP has sequences and domains that are conserved in Usa1p, the molecular function of HERP is not clearly related to that of Usa1p. Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. Assuming that the correct oligomerization of ERAD components may be critical for their function, we hypothesize that homodimer formation of SEL1L in the ER lumen may stabilize oligomerization of the HRD complex, given that SEL1L forms a stoichiometric complex with HRD1. This is further supported by our data showing that the SLR-C of SEL1L directly interacts with the luminal fragment of HRD1 in the ER lumen. + + 0.99887055 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:26Z + DUMMY: + + yeast + + + 0.99914896 + protein + cleaner0 + 2023-07-26T08:27:00Z + PR: + + Usa1p + + + 0.99887496 + protein + cleaner0 + 2023-07-26T09:20:05Z + PR: + + Hrd1p + + + 0.99909854 + protein + cleaner0 + 2023-07-26T08:26:42Z + PR: + + Der1p + + + 0.9991042 + protein + cleaner0 + 2023-07-26T08:27:00Z + PR: + + Usa1p + + + 0.9988599 + protein + cleaner0 + 2023-07-26T09:20:05Z + PR: + + Hrd1p + + + 0.9987087 + protein + cleaner0 + 2023-07-26T09:20:05Z + PR: + + Hrd1p + + + 0.99869114 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:33Z + DUMMY: + + metazoans + + + 0.9989818 + protein + cleaner0 + 2023-07-26T08:27:00Z + PR: + + Usa1p + + + 0.9985386 + taxonomy_domain + cleaner0 + 2023-07-26T08:25:52Z + DUMMY: + + mammalian + + + 0.9656231 + protein_type + cleaner0 + 2023-07-26T09:46:58Z + MESH: + + HERP + + + 0.8323746 + protein_state + cleaner0 + 2023-07-26T12:01:47Z + DUMMY: + + conserved in + + + 0.99918956 + protein + cleaner0 + 2023-07-26T08:27:00Z + PR: + + Usa1p + + + 0.98418415 + protein_type + cleaner0 + 2023-07-26T09:46:59Z + MESH: + + HERP + + + 0.99921775 + protein + cleaner0 + 2023-07-26T08:27:00Z + PR: + + Usa1p + + + 0.6903281 + protein_state + cleaner0 + 2023-07-26T09:21:44Z + DUMMY: + + transiently expressed + + + 0.9989593 + complex_assembly + cleaner0 + 2023-07-26T08:32:43Z + GO: + + HRD1-SEL1L + + + 0.8359795 + protein_type + cleaner0 + 2023-07-26T12:25:19Z + MESH: + + lectins + + + 0.9986066 + protein + cleaner0 + 2023-07-26T08:27:05Z + PR: + + OS9 + + + 0.997894 + protein + cleaner0 + 2023-07-26T08:27:14Z + PR: + + XTP-B + + + protein + PR: + cleaner0 + 2023-07-26T09:22:22Z + + α-antitrypsin null Hong-Kong + + + 0.98373795 + protein + cleaner0 + 2023-07-26T09:22:35Z + PR: + + NHK + + + 0.998617 + mutant + cleaner0 + 2023-07-26T12:27:17Z + MESH: + + NHK-QQQ + + + 0.99814117 + protein_state + cleaner0 + 2023-07-26T12:01:57Z + DUMMY: + + lacks + + + 0.99818313 + site + cleaner0 + 2023-07-26T09:21:19Z + SO: + + N-glycosylation sites + + + 0.9988011 + oligomeric_state + cleaner0 + 2023-07-26T08:24:28Z + DUMMY: + + homodimer + + + 0.99926955 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.99525696 + complex_assembly + cleaner0 + 2023-07-26T09:24:15Z + GO: + + HRD + + + 0.99923074 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T12:27:01Z + + complex with + + + 0.9989691 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + 0.99814767 + structure_element + cleaner0 + 2023-07-26T08:33:22Z + SO: + + SLR-C + + + 0.9991585 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.9979825 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + + DISCUSS + paragraph + 28919 + Although the organization of membrane-bound HRD complex components may be very similar between metazoans and yeast, the molecular details of interactions between the components may not necessarily be conserved. In yeast, it is unclear whether self-association of Hrd3p is due to SLR motifs because the sequence of Hrd3p does not align precisely with the SLR motifs in SEL1L. Furthermore, we are uncertain whether self-association of Hrd3p contributes to formation of the active form of the Hrd1p complex. Recently, a truncated version of Yos9 was shown to form a dimer in the ER lumen and to contribute to the dimeric state of the Hrd1p complex. This interaction seems to be weak because direct Yos9-Yos9 interactions were not detected in immunoprecipitation experiments from yeast cell extracts containing different epitope-tagged variants of Yos9. However, the dimerization of Yos9 could provide a higher stability for the Hrd1p complex oligomer. Likewise, the dimerization of SEL1L might provide stability for the mammalian HRD oligomer complex. Further cell biological studies are required to clarify whether SEL1L (Hrd3p) dimerization could be cooperative with the oligomerization of the HRD complex. + + 0.7623892 + complex_assembly + cleaner0 + 2023-07-26T09:24:15Z + GO: + + HRD + + + 0.9986921 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:33Z + DUMMY: + + metazoans + + + 0.998847 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:26Z + DUMMY: + + yeast + + + 0.9988938 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:26Z + DUMMY: + + yeast + + + 0.9938378 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.9938076 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.99895895 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.98880565 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + 0.9991412 + protein_state + cleaner0 + 2023-07-26T12:02:08Z + DUMMY: + + active + + + 0.79716533 + protein + cleaner0 + 2023-07-26T09:20:05Z + PR: + + Hrd1p + + + 0.9986615 + protein_state + cleaner0 + 2023-07-26T12:02:11Z + DUMMY: + + truncated + + + 0.9987722 + protein + cleaner0 + 2023-07-26T08:27:09Z + PR: + + Yos9 + + + 0.99889237 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.9989335 + oligomeric_state + cleaner0 + 2023-07-26T11:56:43Z + DUMMY: + + dimeric + + + 0.72566545 + protein + cleaner0 + 2023-07-26T09:20:05Z + PR: + + Hrd1p + + + 0.91094744 + protein + cleaner0 + 2023-07-26T08:27:09Z + PR: + + Yos9 + + + 0.65282786 + protein + cleaner0 + 2023-07-26T08:27:09Z + PR: + + Yos9 + + + 0.98758864 + experimental_method + cleaner0 + 2023-07-26T12:21:23Z + MESH: + + immunoprecipitation experiments + + + 0.9987257 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:26Z + DUMMY: + + yeast + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T09:23:32Z + + epitope-tagged + + + 0.9984559 + protein + cleaner0 + 2023-07-26T08:27:09Z + PR: + + Yos9 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T11:57:09Z + + dimerization + + + 0.99885285 + protein + cleaner0 + 2023-07-26T08:27:09Z + PR: + + Yos9 + + + 0.68382525 + protein + cleaner0 + 2023-07-26T09:20:05Z + PR: + + Hrd1p + + + 0.99890447 + oligomeric_state + cleaner0 + 2023-07-26T11:57:18Z + DUMMY: + + oligomer + + + 0.99372673 + oligomeric_state + cleaner0 + 2023-07-26T11:57:08Z + DUMMY: + + dimerization + + + 0.9992169 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.9986565 + taxonomy_domain + cleaner0 + 2023-07-26T08:25:52Z + DUMMY: + + mammalian + + + 0.9694277 + complex_assembly + cleaner0 + 2023-07-26T09:24:15Z + GO: + + HRD + + + 0.99869686 + oligomeric_state + cleaner0 + 2023-07-26T11:57:18Z + DUMMY: + + oligomer + + + 0.9991221 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.9679804 + protein + cleaner0 + 2023-07-26T08:26:30Z + PR: + + Hrd3p + + + 0.93076116 + oligomeric_state + cleaner0 + 2023-07-26T11:57:09Z + DUMMY: + + dimerization + + + 0.5775222 + complex_assembly + cleaner0 + 2023-07-26T09:24:15Z + GO: + + HRD + + + + DISCUSS + paragraph + 30125 + Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans. These findings should provide a foundation for molecular-level studies to understand the membrane-associated HRD complex assembly in ERAD. + + 0.6018751 + complex_assembly + cleaner0 + 2023-07-26T09:24:14Z + GO: + + HRD + + + 0.9982066 + oligomeric_state + cleaner0 + 2023-07-26T11:57:23Z + DUMMY: + + oligomers + + + 0.9993987 + protein + cleaner0 + 2023-07-26T08:24:01Z + PR: + + SEL1L + + + 0.998985 + protein_state + cleaner0 + 2023-07-26T12:02:15Z + DUMMY: + + active + + + 0.58802515 + complex_assembly + cleaner0 + 2023-07-26T09:24:15Z + GO: + + HRD + + + 0.9988446 + protein_state + cleaner0 + 2023-07-26T12:02:21Z + DUMMY: + + absence of + + + 0.99927646 + protein + cleaner0 + 2023-07-26T08:27:00Z + PR: + + Usa1p + + + 0.9982326 + taxonomy_domain + cleaner0 + 2023-07-26T08:27:33Z + DUMMY: + + metazoans + + + 0.62106395 + complex_assembly + cleaner0 + 2023-07-26T09:24:15Z + GO: + + HRD + + + + METHODS + title_1 + 30539 + Methods + + + METHODS + title_2 + 30547 + Protein Production + + + METHODS + paragraph + 30566 + The expression and purification of SEL1L was performed as described previously. + + + METHODS + title_2 + 30646 + Crystallization and SAD Structure Determination of SEL1Lcent + + + METHODS + paragraph + 30707 + Crystals were grown using the hanging-drop vapor diffusion method at 4 °C. For crystallization of the M. musculus SEL1Lcent, 1 μl of protein solution (in 25 mM Tris-HCl, 150 mM NaCl, and 5 mM DTT, pH 7.5) was equilibrated with 1 μl of well solution (30% isopropanol, 100 mM NaCl, 100 mM Tris, 5 mM DTT, and 20 mM phenol, pH 8.5). The crystals, which appeared after 4 days, contain two SEL1Lcent dimers in the asymmetric unit (space group P21, a = 29.13, b = 110.52, c = 109.81 Å, α = 90.00, β = 90.61, γ = 90.00, 44% solvent). For X-ray diffraction experiments, crystals were transferred to well solution plus paraffin-oil, then flash frozen in liquid nitrogen. + + + METHODS + paragraph + 31422 + SAD data were collected with a Se-Met crystal at beamline 7A of the Pohang Accelerator Laboratory (PAL) and processed using HKL2000 software. Native data (2.6 Å resolution) were collected from a single frozen crystal at the same beamline of PAL and were integrated and scaled as described above. The SAD data analysis was performed using Phenix software using data between 50 and 2.9 Å resolution. Phenix identified 31 of the 32 selenium sites and refined these to give a mean f.o.m. = 0.472. Electron density modification, including non-crystallographic symmetry (NCS) averaging, using the RESOLVE software yielded an initial electron density map of excellent quality. Model building and refinement were carried out with the Coot and Phenix programs, respectively. The final model was refined to an R factor of 20.7% (Rfree = 27.7%) for native data between 30 and 2.6 Å resolution (Table 1). The final model consisted of 5402 protein atoms and 47 water molecules. There were no outliers in a Ramachandran plot of the final model. The model contained four copies of SEL1Lcent (residues 348–533) in the asymmetric unit. Of these, the following residues were not modeled due to weak electron densities: SEL1Lcent residues 348–351, 420, 421, and 525–533 in the first copy; residues 348–351 and 525–533 in the second and third copies; and residues 348–352 and 525–533 in the fourth copy. The X-ray data and refinement statistics are summarized in Table 1. + + + METHODS + title_2 + 32905 + Cell Culture and Plasmids Construction + + + METHODS + paragraph + 32944 + HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS. The mouse Sel1L gene was cloned into pCS108 and the 3 × HA or 3 × FLAG tag was fused to the C-terminus of SEL1L. The signal peptide from Xenopus Sel1L was cloned into pCS108 and the mouse SEL1Lcent domain, SEL1L (348-497) fragments, and SEL1Lcent (L521A) were fused to the C-terminus of the signal peptide. Then, a 3 × HA or a 3 × FLAG tag was fused to the C-terminus of the constructs. For the ER retention signal, the KDEL sequences were added to the C-terminus of the fragments. The plasmids were transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s manual. + + + METHODS + title_2 + 33634 + Western Blot Analysis and Immunostaining + + + METHODS + paragraph + 33675 + For western blot analysis, HEK293T cells were transfected with the indicated construct and harvested after washing in PBS. The cells were homogenized in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 5% glycerol), supplemented with protease and phosphatase inhibitor cocktails. Homogenates were cleared by centrifugation at 13,200 rpm for 15 minutes at 4 °C. The lysates were subsequently used for either co-immunoprecipitation experiment or western blot analysis. For the western blot analysis, the samples were run onto 6–12% polyacrylamide gel. Blots were blocked in 5% TBS + 0.05% Tween 20 and incubated with anti-DDDD-K (Abcam) or anti-HA (Roche) antibodies. Proteins were visualized using HRP-conjugated secondary antibodies (1:4000) and SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Dura Extended Duration Substrate (Thermo) and exposed to ChemiDoc MP (Bio-Rad). + + + METHODS + paragraph + 34604 + For immunostaining, the cells were fixed in 4% formaldehyde and incubated with the indicated antibodies. The coverslips were incubated in blocking solution (10% FBS + 2% DMSO in TBS + 0.1% Triton X-100) at room temperature for 30 minutes to block non-specific binding. Fluorescent labeling was performed using Alexa Fluor 555 or 488-conjugated secondary antibodies and nuclei were stained with DAPI. The samples were mounted and confocal images were obtained using a Zeiss LSM700. + + + METHODS + title_2 + 35095 + GST Pull-down Assay + + + METHODS + paragraph + 35115 + For pull-down experiments, 400 μg of HRD1 luminal fragment GST-fusion proteins were incubated with 5 μl of a 50% (v/v) slurry of glutathione sepharose 4B beads (GE Healthcare) for 50 min at 4 °C. Beads were washed twice with buffer A (150 mM NaCl, 25 mM sodium phosphate pH 7.5, 5 mM DTT), and then mixed with 100 μg of MBP-SEL1L protein (SLR-N, SLR-M, SLR-C, and SLR-ML521A) in buffer A, in a total assay volume of 500 μl. The assay mix was incubated at 4 °C for 15 minutes, and beads were washed twice with 500 μl buffer A. Proteins were eluted with SDS sample buffer, and analyzed by SDS-PAGE. + + + METHODS + title_1 + 35743 + Additional Information + + + METHODS + paragraph + 35766 + Accession Numbers: The coordinates and structure factors have been deposited in the Protein Data Bank with the accession code of 5B26. + + + METHODS + paragraph + 35901 + How to cite this article: Jeong, H. et al. Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway. Sci. Rep. 6, 20261; doi: 10.1038/srep20261 (2016). + + + SUPPL + title_1 + 36077 + Supplementary Material + + + 333 + 366 + surname:Chiti;given-names:F. + surname:Dobson;given-names:C. M. + 16756495 + REF + Annu Rev Biochem + ref + 75 + 2006 + 36100 + Protein misfolding, functional amyloid, and human disease + + + 1163 + 1167 + surname:Brodsky;given-names:J. L. + 23217703 + REF + Cell + ref + 151 + 2012 + 36158 + Cleaning up: ER-associated degradation to the rescue + + + 93 + 105 + surname:Christianson;given-names:J. C. + 22119785 + REF + Nat Cell Biol + ref + 14 + 2012 + 36211 + Defining human ERAD networks through an integrative mapping strategy + + + 1086 + 1090 + surname:Smith;given-names:M. H. + surname:Ploegh;given-names:H. L. + surname:Weissman;given-names:J. S. + 22116878 + REF + Science + ref + 334 + 2011 + 36280 + Road to ruin: targeting proteins for degradation in the endoplasmic reticulum + + + 361 + 373 + surname:Carvalho;given-names:P. + surname:Goder;given-names:V. + surname:Rapoport;given-names:T. A. + 16873066 + REF + Cell + ref + 126 + 2006 + 36358 + Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins + + + 272 + 282 + surname:Christianson;given-names:J. C. + surname:Shaler;given-names:T. A. + surname:Tyler;given-names:R. E. + surname:Kopito;given-names:R. R. + 18264092 + REF + Nat Cell Biol + ref + 10 + 2008 + 36456 + OS-9 and GRP94 deliver mutant α1-antitrypsin to the Hrd1–SEL1L ubiquitin ligase complex for ERAD + + + 349 + 359 + surname:Denic;given-names:V. + surname:Quan;given-names:E. M. + surname:Weissman;given-names:J. S. + 16873065 + REF + Cell + ref + 126 + 2006 + 36557 + A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation + + + 1827 + 1835 + surname:Gauss;given-names:R. + surname:Sommer;given-names:T. + surname:Jarosch;given-names:E. + 16619026 + REF + EMBO J + ref + 25 + 2006 + 36655 + The Hrd1p ligase complex forms a linchpin between ER-lumenal substrate selection and Cdc48p recruitment + + + 453 + 460 + surname:Hirsch;given-names:C. + surname:Gauss;given-names:R. + surname:Horn;given-names:S. C. + surname:Neuber;given-names:O. + surname:Sommer;given-names:T. + 19325625 + REF + Nature + ref + 458 + 2009 + 36759 + The ubiquitylation machinery of the endoplasmic reticulum + + + 69 + 82 + surname:Gardner;given-names:R. G. + 11018054 + REF + J Cell Biol + ref + 151 + 2000 + 36817 + Endoplasmic reticulum degradation requires lumen to cytosol signaling transmembrane control of Hrd1p by Hrd3p + + + 223 + 235 + surname:Bernasconi;given-names:R. + surname:Galli;given-names:C. + surname:Calanca;given-names:V. + surname:Nakajima;given-names:T. + surname:Molinari;given-names:M. + 20100910 + REF + J Cell Biol + ref + 188 + 2010 + 36927 + Stringent requirement for HRD1, SEL1L, and OS-9/XTP3-B for disposal of ERAD-LS substrates + + + 849 + 854 + surname:Gauss;given-names:R. + surname:Jarosch;given-names:E. + surname:Sommer;given-names:T. + surname:Hirsch;given-names:C. + 16845381 + REF + Nat Cell Biol + ref + 8 + 2006 + 37017 + A complex of Yos9p and the HRD ligase integrates endoplasmic reticulum quality control into the degradation machinery + + + 14296 + 14301 + surname:Lilley;given-names:B. N. + surname:Ploegh;given-names:H. L. + 16186509 + REF + Proc Natl Acad Sci USA + ref + 102 + 2005 + 37135 + Multiprotein complexes that link dislocation, ubiquitination, and extraction of misfolded proteins from the endoplasmic reticulum membrane + + + 12325 + 12330 + surname:Mueller;given-names:B. + surname:Klemm;given-names:E. J. + surname:Spooner;given-names:E. + surname:Claessen;given-names:J. H. + surname:Ploegh;given-names:H. L. + 18711132 + REF + Proc Natl Acad Sci USA + ref + 105 + 2008 + 37274 + SEL1L nucleates a protein complex required for dislocation of misfolded glycoproteins + + + 261 + 270 + surname:Mueller;given-names:B. + surname:Lilley;given-names:B. N. + surname:Ploegh;given-names:H. L. + 17043138 + REF + J Cell Biol + ref + 175 + 2006 + 37360 + SEL1L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER + + + 458 + 470 + surname:Sha;given-names:H. + 25066055 + REF + Cell Metab + ref + 20 + 2014 + 37454 + The ER-associated degradation adaptor protein Sel1L regulates LPL secretion and lipid metabolism + + + 582 + 591 + surname:Sun;given-names:S. + REF + Proc Natl Acad Sci USA + ref + 111 + 2014 + 37551 + Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival + + + 20 + 31 + surname:Mittl;given-names:P. R. + surname:Schneider-Brachert;given-names:W. + 16870393 + REF + Cell Signal + ref + 19 + 2007 + 37682 + Sel1-like repeat proteins in signal transduction + + + 1192 + 1199 + surname:Das;given-names:A. K. + surname:Cohen;given-names:P. W. + surname:Barford;given-names:D. + 9482716 + REF + EMBO J + ref + 17 + 1998 + 37731 + The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions + + + 829 + 841 + surname:Lüthy;given-names:L. + surname:Grütter;given-names:M. G. + surname:Mittl;given-names:P. R. + 15223324 + REF + J Mol Biol + ref + 340 + 2004 + 37863 + The crystal structure of Helicobacter cysteine-rich protein C at 2.0 Å resolution: similar peptide-binding sites in TPR and SEL1-like repeat proteins + + + 10187 + 10193 + surname:Lüthy;given-names:L. + surname:Grütter;given-names:M. G. + surname:Mittl;given-names:P. R. + 11777911 + REF + J Biol Chem + ref + 277 + 2002 + 38016 + The Crystal Structure of Helicobacter pyloriCysteine-rich Protein B Reveals a Novel Fold for a Penicillin-binding Protein + + + 299 + 302 + surname:Landau;given-names:M. + REF + Nucleic Acids Res + ref + 33 + 2005 + 38138 + ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures + + + 2 + surname:Karpenahalli;given-names:M. R. + surname:Lupas;given-names:A. N. + surname:Söding;given-names:J. + 17199898 + REF + BMC Bioinformatics + ref + 8 + 2007 + 38237 + TPRpred: a tool for prediction of TPR-, PPR-and SEL1-like repeats from protein sequences + + + 739 + 748 + surname:Ramoni;given-names:R. + 11931632 + REF + Biochem J + ref + 365 + 2002 + 38326 + Control of domain swapping in bovine odorant-binding protein + + + 844 + 847 + surname:Robertson;given-names:J. L. + surname:Kolmakova-Partensky;given-names:L. + surname:Miller;given-names:C. + 21048711 + REF + Nature + ref + 468 + 2010 + 38387 + Design, function and structure of a monomeric ClC transporter + + + 4236 + 4248 + surname:Zhang;given-names:Z. + 23583778 + REF + J Mol Biol + ref + 425 + 2013 + 38449 + The four canonical TPR subunits of human APC/C form related homo-dimeric structures and stack in parallel to form a TPR suprahelix + + + 140 + 151 + surname:Bernardi;given-names:K. M. + 19864457 + REF + Mol Biol Cell + ref + 21 + 2010 + 38580 + The E3 ubiquitin ligases Hrd1 and gp78 bind to and promote cholera toxin retro-translocation + + + 5151 + 5156 + surname:Kny;given-names:M. + surname:Standera;given-names:S. + surname:Hartmann-Petersen;given-names:R. + surname:Kloetzel;given-names:P.-M. + surname:Seeger;given-names:M. + 21149444 + REF + J Biol Chem + ref + 286 + 2011 + 38673 + Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like domain-dependent manner + + + 77 + 86 + surname:Mehnert;given-names:M. + surname:Sommer;given-names:T. + surname:Jarosch;given-names:E. + 24292014 + REF + Nat Cell Biol + ref + 16 + 2014 + 38761 + Der1 promotes movement of misfolded proteins through the endoplasmic reticulum membrane + + + 782 + 793 + surname:Horn;given-names:S. C. + 20005842 + REF + Mol Cell + ref + 36 + 2009 + 38849 + Usa1 functions as a scaffold of the HRD-ubiquitin ligase + + + 4444 + 4454 + surname:Huang;given-names:C.-H. + surname:Chu;given-names:Y.-R. + surname:Ye;given-names:Y. + surname:Chen;given-names:X. + 24366871 + REF + J Biol Chem + ref + 289 + 2014 + 38906 + Role of HERP and a HERP-related Protein in HRD1-dependent Protein Degradation at the Endoplasmic Reticulum + + + 579 + 591 + surname:Carvalho;given-names:P. + surname:Stanley;given-names:A. M. + surname:Rapoport;given-names:T. A. + 21074049 + REF + Cell + ref + 143 + 2010 + 39013 + Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p + + + 16929 + 16939 + surname:Iida;given-names:Y. + 21454652 + REF + J Biol Chem + ref + 286 + 2011 + 39096 + SEL1L protein critically determines the stability of the HRD1-SEL1L endoplasmic reticulum-associated degradation (ERAD) complex to optimize the degradation kinetics of ERAD substrates + + + 8633 + 8640 + surname:Hanna;given-names:J. + 22262864 + REF + J Biol Chem + ref + 287 + 2012 + 39280 + Structural and biochemical basis of Yos9 protein dimerization and possible contribution to self-association of 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation ubiquitin-ligase complex + + + 1624 + 1627 + surname:Jeong;given-names:H. + surname:Lee;given-names:H. + surname:Lee;given-names:C. + REF + Acta Crystallogr F + ref + 70 + 2014 + 39476 + Crystallization and preliminary X-ray diffraction analysis of the Sel1-like repeats of SEL1L + + + 307 + 326 + surname:Otwinowski;given-names:Z. + surname:Minor;given-names:W. + REF + Method Enzymol + ref + 276 + 1997 + 39569 + Processing of X-ray diffraction data collected in oscillation mode + + + 213 + 221 + surname:Adams;given-names:P. D. + 20124702 + REF + Acta Crystallogr D + ref + 66 + 2010 + 39636 + PHENIX: a comprehensive Python-based system for macromolecular structure solution + + + 849 + 861 + surname:Terwilliger;given-names:T. C. + surname:Berendzen;given-names:J. + 10089316 + REF + Acta Crystallogr D + ref + 55 + 1999 + 39718 + Automated MAD and MIR structure solution + + + 486 + 501 + surname:Emsley;given-names:P. + surname:Lohkamp;given-names:B. + surname:Scott;given-names:W. G. + surname:Cowtan;given-names:K. + 20383002 + REF + Acta Crystallogr D + ref + 66 + 2010 + 39759 + Features and development of Coot + + + SUPPL + footnote + 39792 + Author Contributions H.J. and H.L. performed cloning, expression, and protein purification. H.J., S.H. and C.L. crystallized, collected X-ray data, and solved the protein structure. H.J., H.S., E.S. and Y.J performed biochemical experiments. H.J., T.P. and C.L. wrote the manuscript. T.P. and C.L. designed and supervised the project. All the authors discussed the results, commented on the manuscript, and approved the manuscript. + + + srep20261-f1.jpg + f1 + FIG + fig_title_caption + 40224 + Crystal Structure of SEL1Lcent. + + 0.9985441 + evidence + cleaner0 + 2023-07-26T11:52:56Z + DUMMY: + + Crystal Structure + + + 0.9989202 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + + srep20261-f1.jpg + f1 + FIG + fig_caption + 40256 + (A) The diagram shows the domain structure of Mus musculus SEL1L, as defined by proteolytic mapping and sequence/structure analysis. The 11 SLR motifs were divided into three groups (SLR-N, SLR-M, and SLR-C) due to the presence of linker sequences that are not predicted SLR motifs. Putative N-glycosylation sites are indicated by black triangles. We determined the crystal structure of the SLR-M, residues 348–533. (B) Ribbon diagram of the biological unit of the SEL1Lcent, viewed along the two-fold NCS axis. The crystal structure was determined by SAD phasing using selenium as the anomalous scatterer and refined to 2.6 Å resolution (Table 1). (C) SEL1Lcent ribbon diagram rotated 90° around a horizontal axis relative to (B). (D) One protomer of the SEL1Lcent dimer. This view is rotated about 90° anticlockwise from the bottom copy in (B), along the two-fold NCS axis. Starting from the N-terminus, SEL1Lcent has five SLR motifs comprising ten α helices. Each SLR motif (from 5 to 9) is indicated in a different color. (E) Evolutionary conservation of surface residues in SEL1Lcent, calculated using ConSurf, from a structure-based alignment of 135 SEL1L sequences. The surface is colored from red (high) to white (poor) according to the degree of conservation in the SEL1L phylogenetic orthologs. The ribbon diagram of the counterpart protomer is drawn to show the orientation of the SEL1Lcent dimer. + + 0.99830663 + species + cleaner0 + 2023-07-26T08:34:15Z + MESH: + + Mus musculus + + + 0.99923646 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.9986738 + experimental_method + cleaner0 + 2023-07-26T12:21:30Z + MESH: + + proteolytic mapping + + + 0.9985619 + experimental_method + cleaner0 + 2023-07-26T12:21:35Z + MESH: + + sequence/structure analysis + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.99073833 + structure_element + cleaner0 + 2023-07-26T08:35:33Z + SO: + + SLR-N + + + 0.9909288 + structure_element + cleaner0 + 2023-07-26T08:35:42Z + SO: + + SLR-M + + + 0.9904032 + structure_element + cleaner0 + 2023-07-26T08:33:22Z + SO: + + SLR-C + + + 0.997459 + structure_element + cleaner0 + 2023-07-26T08:35:21Z + SO: + + linker sequences + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.99653506 + site + cleaner0 + 2023-07-26T09:30:56Z + SO: + + N-glycosylation sites + + + 0.99770147 + evidence + cleaner0 + 2023-07-26T11:52:56Z + DUMMY: + + crystal structure + + + 0.8388017 + structure_element + cleaner0 + 2023-07-26T08:35:42Z + SO: + + SLR-M + + + 0.99781173 + residue_range + cleaner0 + 2023-07-26T09:31:05Z + DUMMY: + + 348–533 + + + 0.9988219 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.99648273 + evidence + cleaner0 + 2023-07-26T11:52:56Z + DUMMY: + + crystal structure + + + 0.99892163 + experimental_method + cleaner0 + 2023-07-26T12:21:44Z + MESH: + + SAD phasing + + + 0.99900913 + chemical + cleaner0 + 2023-07-26T08:38:45Z + CHEBI: + + selenium + + + 0.9987111 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.9989466 + oligomeric_state + cleaner0 + 2023-07-26T08:39:07Z + DUMMY: + + protomer + + + 0.99757284 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.9988978 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.9988696 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.9988542 + structure_element + cleaner0 + 2023-07-26T09:31:20Z + SO: + + α helices + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.99901414 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.998268 + experimental_method + cleaner0 + 2023-07-26T12:21:50Z + MESH: + + ConSurf + + + 0.9988711 + experimental_method + cleaner0 + 2023-07-26T12:21:54Z + MESH: + + structure-based alignment + + + 0.9931625 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.99388146 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.99892056 + oligomeric_state + cleaner0 + 2023-07-26T08:39:07Z + DUMMY: + + protomer + + + 0.99843997 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.998904 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + + srep20261-f2.jpg + f2 + FIG + fig_title_caption + 41671 + Dimer Interface of SEL1Lcent. + + 0.99903095 + site + cleaner0 + 2023-07-26T08:43:54Z + SO: + + Dimer Interface + + + 0.9991755 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + + srep20261-f2.jpg + f2 + FIG + fig_caption + 41701 + (A) The diagram on the left shows the SEL1Lcent dimer viewed along the two-fold symmetry axis. Three distinct contact regions are indicated with labeled boxes. The close-up view on the right shows the residues of SEL1Lcent that contribute to dimer formation via the three contact interfaces. Oxygen and nitrogen atoms are shown as red and blue, respectively. The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins. The standard molecular masses for the SEC experiments (top) were obtained from the following proteins: aldolase, 158 kDa; cobalbumin, 75 kDa; ovalbumin, 44 kDa; and carbonic anhydrase, 29 kDa. Chromatography was performed on a Superdex 200 column with a buffer containing 25 mM Tris, 150 mM NaCl, and 5 mM DTT (pH 7.5). The elution fractions, indicated by the gray shading, were run on SDS-PAGE and are shown below the gel-filtration elution profile. The schematic diagrams representing the protein constructs used in the SEC are shown on the left of each SDS-PAGE profile. + + 0.99904746 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.9988695 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.9326523 + site + cleaner0 + 2023-07-26T12:04:53Z + SO: + + contact regions + + + 0.99910814 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.9988939 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.9985516 + site + cleaner0 + 2023-07-26T12:04:58Z + SO: + + contact interfaces + + + 0.99579656 + bond_interaction + cleaner0 + 2023-07-26T09:31:54Z + MESH: + + hydrogen bonds + + + 0.9982621 + oligomeric_state + cleaner0 + 2023-07-26T08:39:13Z + DUMMY: + + protomers + + + 0.9991347 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.9989516 + experimental_method + cleaner0 + 2023-07-26T12:22:05Z + MESH: + + Size-exclusion chromatography + + + 0.9987413 + experimental_method + cleaner0 + 2023-07-26T12:22:10Z + MESH: + + SEC + + + 0.9990842 + protein_state + cleaner0 + 2023-07-26T08:53:21Z + DUMMY: + + wild-type + + + site + SO: + cleaner0 + 2023-07-26T09:32:46Z + + dimeric interface + + + 0.9965245 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.9806871 + protein_state + cleaner0 + 2023-07-26T12:02:31Z + DUMMY: + + mutants + + + 0.99843615 + experimental_method + cleaner0 + 2023-07-26T12:22:16Z + MESH: + + SEC + + + 0.99640155 + experimental_method + cleaner0 + 2023-07-26T12:22:19Z + MESH: + + SDS-PAGE + + + evidence + DUMMY: + cleaner0 + 2023-07-26T12:22:35Z + + gel-filtration elution profile + + + 0.9978103 + experimental_method + cleaner0 + 2023-07-26T12:22:58Z + MESH: + + SEC + + + 0.9939421 + experimental_method + cleaner0 + 2023-07-26T12:23:01Z + MESH: + + SDS-PAGE + + + + srep20261-f3.jpg + f3 + FIG + fig_title_caption + 42903 + Domain Swapping for Dimerization of SEL1Lcent. + + 0.99684453 + oligomeric_state + cleaner0 + 2023-07-26T11:57:09Z + DUMMY: + + Dimerization + + + 0.9990489 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + + srep20261-f3.jpg + f3 + FIG + fig_caption + 42950 + (A) Sequence alignment of the SLR motifs in SEL1L. The 11 SLR motifs were aligned based on the present crystal structure of SEL1Lcent. The sequences of SEL1Lcent included in the crystal structure are highlighted by the blue box. The secondary structure elements are indicated above the sequences, with helices depicted as cylinders. Residues that are conserved in at least 7 out of 11 sequences are red. The GG sequence in SLR motif 9, which creates the hinge for domain swapping (see text), is shaded yellow. Stars below the sequences indicate the specific residues that commonly appear in SLRs. (B) Structure alignment of five SLR motifs in SEL1Lcent is shown to highlight the unusual geometry of SLR motif 9. Each SLR motif is shown in a different color. The arrow indicates the direction of the helical axes. In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an α-hairpin structure. (C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer. Oxygen and nitrogen atoms are colored red and blue, respectively. The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. Size-exclusion chromatography was conducted as described in Fig. 2B. The standard molecular masses are shown at the top as in Fig. 2B. + + 0.9987904 + experimental_method + cleaner0 + 2023-07-26T12:23:20Z + MESH: + + Sequence alignment + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.9992772 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.9936063 + experimental_method + cleaner0 + 2023-07-26T12:23:26Z + MESH: + + aligned + + + 0.99686766 + evidence + cleaner0 + 2023-07-26T11:52:56Z + DUMMY: + + crystal structure + + + 0.9993211 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.9993175 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.9664356 + evidence + cleaner0 + 2023-07-26T11:52:56Z + DUMMY: + + crystal structure + + + 0.70380676 + structure_element + cleaner0 + 2023-07-26T09:51:23Z + SO: + + helices + + + 0.5623606 + structure_element + cleaner0 + 2023-07-26T09:33:39Z + SO: + + GG + + + 0.9946766 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.99734586 + structure_element + cleaner0 + 2023-07-26T09:00:43Z + SO: + + hinge + + + 0.99871385 + structure_element + cleaner0 + 2023-07-26T08:41:13Z + SO: + + SLRs + + + 0.9989205 + experimental_method + cleaner0 + 2023-07-26T12:23:31Z + MESH: + + Structure alignment + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.9992717 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.9945833 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.99119973 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.9149917 + structure_element + cleaner0 + 2023-07-26T09:51:26Z + SO: + + helices + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.9991172 + structure_element + cleaner0 + 2023-07-26T09:51:29Z + SO: + + α-hairpin + + + 0.9924176 + residue_name_number + cleaner0 + 2023-07-26T09:34:14Z + DUMMY: + + Gly 512 + + + 0.995074 + residue_name_number + cleaner0 + 2023-07-26T09:34:18Z + DUMMY: + + Gly 513 + + + structure_element + SO: + cleaner0 + 2023-07-26T08:41:50Z + + helix 9B + + + 0.9989567 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + 0.9913488 + residue_name_number + cleaner0 + 2023-07-26T09:34:15Z + DUMMY: + + Gly 512 + + + 0.9942075 + residue_name_number + cleaner0 + 2023-07-26T09:34:19Z + DUMMY: + + Gly 513 + + + 0.99725544 + experimental_method + cleaner0 + 2023-07-26T12:23:38Z + MESH: + + point mutations + + + 0.9937947 + residue_name_number + cleaner0 + 2023-07-26T09:34:15Z + DUMMY: + + Gly 512 + + + 0.99478316 + residue_name_number + cleaner0 + 2023-07-26T09:34:19Z + DUMMY: + + Gly 513 + + + 0.9989575 + structure_element + cleaner0 + 2023-07-26T09:00:43Z + SO: + + hinge + + + 0.99069184 + structure_element + cleaner0 + 2023-07-26T08:25:42Z + SO: + + SLR motif 9 + + + 0.9985311 + mutant + cleaner0 + 2023-07-26T09:02:13Z + MESH: + + G512A + + + 0.9986308 + mutant + cleaner0 + 2023-07-26T09:02:18Z + MESH: + + G513A + + + 0.9983796 + mutant + cleaner0 + 2023-07-26T09:02:13Z + MESH: + + G512A + + + 0.99881685 + mutant + cleaner0 + 2023-07-26T09:02:18Z + MESH: + + G513A + + + 0.9988588 + mutant + cleaner0 + 2023-07-26T09:02:43Z + MESH: + + G512K + + + 0.99901724 + mutant + cleaner0 + 2023-07-26T09:02:48Z + MESH: + + G513K + + + 0.9989951 + experimental_method + cleaner0 + 2023-07-26T12:23:43Z + MESH: + + Size-exclusion chromatography + + + + srep20261-f4.jpg + f4 + FIG + fig_title_caption + 44504 + SEL1L forms self-oligomer mediated by the SEL1Lcent domain in vivo. + + 0.9991986 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.52479684 + oligomeric_state + cleaner0 + 2023-07-26T11:59:16Z + DUMMY: + + self-oligomer + + + 0.99886084 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + + srep20261-f4.jpg + f4 + FIG + fig_caption + 44572 + (A) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. The full-length SEL1L-FLAG was co-immunoprecipitated with the full-length SEL1L-HA. Also, SEL1Lcent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively. The SEL1L348–497 fragment was secreted to the culture media but the SEL1Lcent was retained in the ER. (C) SEL1Lcent-FLAG-KDEL and SEL1L348–497-FLAG-KDEL localized to the ER. The nuclei were stained with DAPI in blue. The ER was visualized with the anti-calnexin antibody in green. The SEL1L fragments were stained in red. (D) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-HA antibody followed by Western blot analysis using an anti-FLAG antibody. The full-length SEL1L forms self-oligomers and the SEL1Lcent-FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA. The red asterisk indicates the expected signal for SEL1L348–497-FLAG-KDEL. SEL1L348–497-FLAG-KDEL did not co-immunoprecipitate with full-length SEL1L-HA. The white asterisks indicate non-specific bands. (E) SEL1Lcent-HA-KDEL competitively inhibited self-oligomerization of full-length SEL1L. The indicated plasmid constructs were transfected and immunoprecipitation assay was performed using an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. The red rectangle indicates competitively inhibited SEL1L self-oligomer formation by the increasing doses of SEL1Lcent-HA-KDEL. (F) L521A point mutant in SEL1Lcent did not inhibit the self-association of SEL1L. + + 0.9971973 + experimental_method + cleaner0 + 2023-07-26T12:23:51Z + MESH: + + immunoprecipitated + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:03:45Z + + FLAG + + + 0.9885245 + experimental_method + cleaner0 + 2023-07-26T12:23:55Z + MESH: + + western blot + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:09:28Z + + HA + + + 0.99912053 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.9982527 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:03:45Z + + FLAG + + + 0.9940212 + experimental_method + cleaner0 + 2023-07-26T12:23:59Z + MESH: + + co-immunoprecipitated + + + 0.9991062 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.99856913 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.61573565 + experimental_method + cleaner0 + 2023-07-26T09:09:28Z + MESH: + + HA + + + 0.997417 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.9880214 + experimental_method + cleaner0 + 2023-07-26T12:24:02Z + MESH: + + co-immunoprecipitated + + + 0.99909383 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.9989844 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:25:42Z + + SLR motif 9 + + + 0.42224488 + experimental_method + cleaner0 + 2023-07-26T12:24:05Z + MESH: + + deletion + + + 0.99481857 + experimental_method + cleaner0 + 2023-07-26T12:24:08Z + MESH: + + western blot + + + 0.9980458 + experimental_method + cleaner0 + 2023-07-26T12:24:12Z + MESH: + + immunoprecipitation + + + mutant + MESH: + cleaner0 + 2023-07-26T09:04:56Z + + SEL1L348–497 + + + 0.99846137 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + mutant + MESH: + cleaner0 + 2023-07-26T09:35:10Z + + SEL1Lcent-FLAG-KDEL + + + mutant + MESH: + cleaner0 + 2023-07-26T09:35:44Z + + SEL1L348–497-FLAG-KDEL + + + 0.3403473 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.99533045 + experimental_method + cleaner0 + 2023-07-26T12:24:17Z + MESH: + + immunoprecipitated + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:09:28Z + + HA + + + 0.9932866 + experimental_method + cleaner0 + 2023-07-26T12:24:20Z + MESH: + + Western blot + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:03:45Z + + FLAG + + + 0.99895054 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.99914587 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T09:06:04Z + + self-oligomers + + + mutant + MESH: + cleaner0 + 2023-07-26T09:36:31Z + + SEL1Lcent-FLAG-KDEL + + + 0.9982154 + experimental_method + cleaner0 + 2023-07-26T12:24:24Z + MESH: + + co-immunoprecipitated + + + 0.9989381 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.99822336 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.70323896 + experimental_method + cleaner0 + 2023-07-26T09:09:28Z + MESH: + + HA + + + 0.8285012 + mutant + cleaner0 + 2023-07-26T09:35:45Z + MESH: + + SEL1L348–497-FLAG-KDEL + + + mutant + MESH: + cleaner0 + 2023-07-26T09:35:45Z + + SEL1L348–497-FLAG-KDEL + + + 0.9989348 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.99864477 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.5467928 + experimental_method + cleaner0 + 2023-07-26T09:09:28Z + MESH: + + HA + + + mutant + MESH: + cleaner0 + 2023-07-26T09:36:58Z + + SEL1Lcent-HA-KDEL + + + 0.9990277 + protein_state + cleaner0 + 2023-07-26T08:24:14Z + DUMMY: + + full-length + + + 0.99917597 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.99865973 + experimental_method + cleaner0 + 2023-07-26T12:24:28Z + MESH: + + immunoprecipitation assay + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:03:45Z + + FLAG + + + 0.95454943 + experimental_method + cleaner0 + 2023-07-26T12:24:30Z + MESH: + + western blot + + + experimental_method + MESH: + cleaner0 + 2023-07-26T09:09:28Z + + HA + + + 0.9990332 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T11:57:18Z + + oligomer + + + mutant + MESH: + cleaner0 + 2023-07-26T09:36:59Z + + SEL1Lcent-HA-KDEL + + + 0.9991394 + mutant + cleaner0 + 2023-07-26T08:52:49Z + MESH: + + L521A + + + 0.9413898 + protein_state + cleaner0 + 2023-07-26T12:02:37Z + DUMMY: + + point mutant + + + 0.9989951 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + 0.9991678 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + + srep20261-f5.jpg + f5 + FIG + fig_title_caption + 46507 + Comparison of SLR in SEL1L with TPR or Other SLR-Containing Proteins. + + 0.9993629 + structure_element + cleaner0 + 2023-07-26T08:31:37Z + SO: + + SLR + + + 0.9993555 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.9819749 + structure_element + cleaner0 + 2023-07-26T09:43:43Z + SO: + + TPR + + + protein_type + MESH: + cleaner0 + 2023-07-26T09:37:53Z + + SLR-Containing Proteins + + + + srep20261-f5.jpg + f5 + FIG + fig_caption + 46577 + (A) Ribbon diagram showing superimposition of an isolated TPR motif from Cdc23 and an SLR motif from SEL1Lcent (left), and SLR motifs in HcpC and SEL1Lcent (right). The SEL1L, Cdc23, and HcpC are colored magenta, green and cyan, respectively. Black arrows indicate the helical axes. The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. Both SEL1Lcent schematics are identically oriented for comparison. The Cα atoms of the residues in each α-solenoid domain are superimposed with a root-mean-squared deviation of 3.3 Å for Cdc23 and SEL1Lcent (left), and 2.5 Å for HcpC and SEL1Lcent (right). SEL1Lcent, Cdc23, and HcpC are colored as in (A). (C) Ribbon diagram showing the overall structure of Cdc23N-term (left) and SEL1Lcent (right) to compare their similarities regarding dimer formation through domain swapping. The view is along the two-fold axis. + + 0.9983388 + experimental_method + cleaner0 + 2023-07-26T12:24:35Z + MESH: + + superimposition + + + structure_element + SO: + cleaner0 + 2023-07-26T09:43:43Z + + TPR + + + 0.99884206 + protein + cleaner0 + 2023-07-26T09:10:14Z + PR: + + Cdc23 + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.9989537 + structure_element + cleaner0 + 2023-07-26T08:23:48Z + SO: + + SEL1Lcent + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:37Z + + SLR + + + 0.9991535 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.9990169 + structure_element + cleaner0 + 2023-07-26T08:23:49Z + SO: + + SEL1Lcent + + + 0.9981001 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.9981516 + protein + cleaner0 + 2023-07-26T09:10:14Z + PR: + + Cdc23 + + + 0.9990277 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.98901033 + ptm + cleaner0 + 2023-07-26T09:12:11Z + MESH: + + disulfide bonds + + + 0.9985221 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.9974023 + residue_name + cleaner0 + 2023-07-26T12:27:50Z + SO: + + Cys + + + 0.9873022 + ptm + cleaner0 + 2023-07-26T12:27:58Z + MESH: + + disulfide bonding + + + 0.99798596 + experimental_method + cleaner0 + 2023-07-26T12:24:38Z + MESH: + + superimposition + + + 0.99736744 + protein + cleaner0 + 2023-07-26T09:10:14Z + PR: + + Cdc23 + + + 0.9981723 + structure_element + cleaner0 + 2023-07-26T08:23:49Z + SO: + + SEL1Lcent + + + 0.9984665 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.99831355 + structure_element + cleaner0 + 2023-07-26T08:23:49Z + SO: + + SEL1Lcent + + + 0.9990736 + structure_element + cleaner0 + 2023-07-26T09:38:15Z + SO: + + α-solenoid domain + + + 0.99877936 + structure_element + cleaner0 + 2023-07-26T08:23:49Z + SO: + + SEL1Lcent + + + 0.99905175 + structure_element + cleaner0 + 2023-07-26T09:38:15Z + SO: + + α-solenoid domain + + + 0.9977223 + experimental_method + cleaner0 + 2023-07-26T12:24:43Z + MESH: + + superimposed + + + 0.9984053 + evidence + cleaner0 + 2023-07-26T09:38:21Z + DUMMY: + + root-mean-squared deviation + + + 0.9972836 + protein + cleaner0 + 2023-07-26T09:10:14Z + PR: + + Cdc23 + + + 0.99782985 + structure_element + cleaner0 + 2023-07-26T08:23:49Z + SO: + + SEL1Lcent + + + 0.9985839 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.9982064 + structure_element + cleaner0 + 2023-07-26T08:23:49Z + SO: + + SEL1Lcent + + + 0.99795073 + structure_element + cleaner0 + 2023-07-26T08:23:49Z + SO: + + SEL1Lcent + + + 0.99767953 + protein + cleaner0 + 2023-07-26T09:10:14Z + PR: + + Cdc23 + + + 0.99890506 + protein + cleaner0 + 2023-07-26T09:10:23Z + PR: + + HcpC + + + 0.7976578 + evidence + cleaner0 + 2023-07-26T11:55:17Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-26T12:27:35Z + + Cdc23 + + + 0.99864715 + structure_element + cleaner0 + 2023-07-26T08:23:49Z + SO: + + SEL1Lcent + + + 0.9988312 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + + srep20261-f6.jpg + f6 + FIG + fig_title_caption + 47690 + The Role of SLR-C in ERAD machinery and Model for the Organization of Proteins in Membrane-Associated ERAD Components. + + 0.9980087 + structure_element + cleaner0 + 2023-07-26T08:33:22Z + SO: + + SLR-C + + + + srep20261-f6.jpg + f6 + FIG + fig_caption + 47809 + (A) Schematic diagram shows three HRD1 fragment constructs used in the GST pull-down experiment. (B) Pull-down experiments to examine the interactions between HRD luminal loops and certain SLR motifs of SEL1L. Fragments of the luminal loop of HRD1 fused to GST were immobilized on glutathione sepharose beads and incubated with purified three clusters of SLR motifs and monomer form of SLR-M (SLR-ML521A, right panel) in SEL1L. Proteins were analyzed by 12% SDS-PAGE and Coomassie blue staining. (C) Schematic representation of the organization of metazoan ERAD components in the ER membrane. The 11 SLR motifs of SEL1L were expressed with red cylinders and grouped into three parts (SLR-N, SLR-M, and SLR-C) based on the sequence alignment across the motifs and the crystal structure presented herein. We hypothesized that the interrupted SLR motifs of SEL1L have distinct functions such that the SLR-M is important for dimer formation of the protein, and SLR-C is involved in the interaction with HRD1 in the ER lumen. The surface representation of SEL1Lcent is placed in the same orientation as that shown in the schematic model to show that the putative N-glycosylation site, residue N427 (indicated in yellow), is exposed on the surface of the protein. The yellow arrow indicates self-association among the respective components. + + 0.9831434 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + 0.99888813 + experimental_method + cleaner0 + 2023-07-26T12:24:48Z + MESH: + + GST pull-down + + + 0.98592913 + experimental_method + cleaner0 + 2023-07-26T12:24:53Z + MESH: + + Pull-down experiments + + + 0.99947685 + complex_assembly + cleaner0 + 2023-07-26T09:24:15Z + GO: + + HRD + + + 0.9987311 + structure_element + cleaner0 + 2023-07-26T09:51:11Z + SO: + + luminal loops + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:38Z + + SLR + + + 0.9991185 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.9990668 + structure_element + cleaner0 + 2023-07-26T09:47:49Z + SO: + + luminal loop + + + 0.73801214 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + 0.42447874 + chemical + cleaner0 + 2023-07-26T09:39:31Z + CHEBI: + + GST + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:38Z + + SLR + + + 0.9988919 + oligomeric_state + cleaner0 + 2023-07-26T08:39:02Z + DUMMY: + + monomer + + + 0.82218844 + structure_element + cleaner0 + 2023-07-26T08:35:42Z + SO: + + SLR-M + + + 0.9988654 + mutant + cleaner0 + 2023-07-26T09:18:26Z + MESH: + + SLR-ML521A + + + 0.99908733 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + 0.9981499 + experimental_method + cleaner0 + 2023-07-26T12:25:00Z + MESH: + + SDS-PAGE + + + 0.9984635 + taxonomy_domain + cleaner0 + 2023-07-26T11:55:43Z + DUMMY: + + metazoan + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:38Z + + SLR + + + 0.99918073 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:35:34Z + + SLR-N + + + structure_element + SO: + cleaner0 + 2023-07-26T08:35:42Z + + SLR-M + + + 0.7746102 + structure_element + cleaner0 + 2023-07-26T08:33:22Z + SO: + + SLR-C + + + 0.9975531 + experimental_method + cleaner0 + 2023-07-26T12:25:04Z + MESH: + + sequence alignment + + + 0.9978694 + evidence + cleaner0 + 2023-07-26T11:52:56Z + DUMMY: + + crystal structure + + + structure_element + SO: + cleaner0 + 2023-07-26T08:31:38Z + + SLR + + + 0.9992436 + protein + cleaner0 + 2023-07-26T08:24:02Z + PR: + + SEL1L + + + structure_element + SO: + cleaner0 + 2023-07-26T08:35:42Z + + SLR-M + + + 0.9989221 + oligomeric_state + cleaner0 + 2023-07-26T08:24:34Z + DUMMY: + + dimer + + + structure_element + SO: + cleaner0 + 2023-07-26T08:33:22Z + + SLR-C + + + 0.9812152 + protein + cleaner0 + 2023-07-26T08:26:25Z + PR: + + HRD1 + + + 0.9988286 + structure_element + cleaner0 + 2023-07-26T08:23:49Z + SO: + + SEL1Lcent + + + 0.9985453 + site + cleaner0 + 2023-07-26T12:05:05Z + SO: + + N-glycosylation site + + + 0.99954575 + residue_name_number + cleaner0 + 2023-07-26T09:40:08Z + DUMMY: + + N427 + + + + t1.xml + t1 + TABLE + table_title_caption + 49144 + Data Collection and Refinement Statistics. + + + t1.xml + t1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/></colgroup><tbody valign="top"><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">SEL1L<sup>cent</sup></td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Data set:</td><td align="center" valign="top" charoff="50">Native</td><td align="center" valign="top" charoff="50">Se-SAD</td></tr><tr><td align="left" valign="top" charoff="50">PDB accession #:</td><td align="center" valign="top" charoff="50">5B26</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">X-ray source</td><td align="center" valign="top" charoff="50">Beamline 7A, PAL</td><td align="center" valign="top" charoff="50">Beamline 7A, PAL</td></tr><tr><td align="left" valign="top" charoff="50">Temperature (K)</td><td align="center" valign="top" charoff="50">100</td><td align="center" valign="top" charoff="50">100</td></tr><tr><td align="left" valign="top" charoff="50">Space group:</td><td align="center" valign="top" charoff="50">P2<sub>1</sub></td><td align="center" valign="top" charoff="50">P2<sub>1</sub></td></tr><tr><td align="left" valign="top" charoff="50">Cell parameters a, b, c (Å)</td><td align="center" valign="top" charoff="50">29.13, 110.52, 109.81</td><td align="center" valign="top" charoff="50">29.51, 110.49, 109.81</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">90.00, 90.61, 90.00</td><td align="center" valign="top" charoff="50">90.00, 90.74, 90.00</td></tr><tr><td colspan="3" align="left" valign="top" charoff="50"><bold>Data processing</bold></td></tr><tr><td align="left" valign="top" charoff="50">Wavelength (Å)</td><td align="center" valign="top" charoff="50">1.00000</td><td align="center" valign="top" charoff="50">0.97923</td></tr><tr><td align="left" valign="top" charoff="50">Resolution (Å)</td><td align="center" valign="top" charoff="50">50-2.60</td><td align="center" valign="top" charoff="50">50–2.90</td></tr><tr><td align="left" valign="top" charoff="50">R<sub>merge</sub> (%)<xref ref-type="fn" rid="t1-fn1">a</xref></td><td align="center" valign="top" charoff="50">6.1 (38.7)<xref ref-type="fn" rid="t1-fn1">*</xref></td><td align="center" valign="top" charoff="50">9.4 (40.6)</td></tr><tr><td align="left" valign="top" charoff="50">I/σ</td><td align="center" valign="top" charoff="50">29.4 (4.6)</td><td align="center" valign="top" charoff="50">21.0 (3.3)</td></tr><tr><td align="left" valign="top" charoff="50">Completeness (%)</td><td align="center" valign="top" charoff="50">99.5 (99.3)</td><td align="center" valign="top" charoff="50">99.9 (100.0)</td></tr><tr><td align="left" valign="top" charoff="50">Redundancy</td><td align="center" valign="top" charoff="50">4.1 (4.1)</td><td align="center" valign="top" charoff="50">3.8 (3.8)</td></tr><tr><td align="left" valign="top" charoff="50">Measured reflections</td><td align="center" valign="top" charoff="50">88070</td><td align="center" valign="top" charoff="50">116951</td></tr><tr><td align="left" valign="top" charoff="50">Unique reflections</td><td align="center" valign="top" charoff="50">21479</td><td align="center" valign="top" charoff="50">30823</td></tr><tr><td colspan="3" align="left" valign="top" charoff="50"><bold>Refinement statistics</bold></td></tr><tr><td align="left" valign="top" charoff="50">Data range (Å)</td><td align="center" valign="top" charoff="50">30-2.60</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Reflections</td><td align="center" valign="top" charoff="50">21446</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Nonhydrogen atoms</td><td align="center" valign="top" charoff="50">5402</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Water molecules</td><td align="center" valign="top" charoff="50">47</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">R.m.s. ∆ bonds (Å)<xref ref-type="fn" rid="t1-fn2">b</xref></td><td align="center" valign="top" charoff="50">0.010</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">R.m.s. ∆ angles (°)<xref ref-type="fn" rid="t1-fn2">b</xref></td><td align="center" valign="top" charoff="50">1.365</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">R-factor (%)<xref ref-type="fn" rid="t1-fn3">c</xref></td><td align="center" valign="top" charoff="50">20.7</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">R<sub>free</sub> (%)<xref ref-type="fn" rid="t1-fn3">c</xref><sup>,<xref ref-type="fn" rid="t1-fn4">d</xref></sup></td><td align="center" valign="top" charoff="50">27.7</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Ramachandran plot, residues in</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Most favored regions (%)</td><td align="center" valign="top" charoff="50">92.8</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Additional allowed regions (%)</td><td align="center" valign="top" charoff="50">6.5</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Generously allowed regions (%)</td><td align="center" valign="top" charoff="50">0.7</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Disallowed regions (%)</td><td align="center" valign="top" charoff="50">0.0</td><td align="center" valign="top" charoff="50"> </td></tr></tbody></table> + + 49187 +   SEL1Lcent   Data set: Native Se-SAD PDB accession #: 5B26   X-ray source Beamline 7A, PAL Beamline 7A, PAL Temperature (K) 100 100 Space group: P21 P21 Cell parameters a, b, c (Å) 29.13, 110.52, 109.81 29.51, 110.49, 109.81   90.00, 90.61, 90.00 90.00, 90.74, 90.00 Data processing Wavelength (Å) 1.00000 0.97923 Resolution (Å) 50-2.60 50–2.90 Rmerge (%)a 6.1 (38.7)* 9.4 (40.6) I/σ 29.4 (4.6) 21.0 (3.3) Completeness (%) 99.5 (99.3) 99.9 (100.0) Redundancy 4.1 (4.1) 3.8 (3.8) Measured reflections 88070 116951 Unique reflections 21479 30823 Refinement statistics Data range (Å) 30-2.60   Reflections 21446   Nonhydrogen atoms 5402   Water molecules 47   R.m.s. ∆ bonds (Å)b 0.010   R.m.s. ∆ angles (°)b 1.365   R-factor (%)c 20.7   Rfree (%)c,d 27.7   Ramachandran plot, residues in     Most favored regions (%) 92.8   Additional allowed regions (%) 6.5   Generously allowed regions (%) 0.7   Disallowed regions (%) 0.0   + + + t1.xml + t1 + TABLE + table_footnote + 50207 + *Highest resolution shell is shown in parenthesis. + + + t1.xml + t1 + TABLE + table_footnote + 50258 + aRmerge = 100 × ∑h∑i | Ii(h) − <I(h) >|/∑h <I(h)>, where Ii(h) is the ith measurement and <I(h)> is the weighted mean of all measurement of I(h) for Miller indices h. + + + t1.xml + t1 + TABLE + table_footnote + 50446 + bRoot-mean-squared deviation (r.m.s. ∆) from target geometries. + + + t1.xml + t1 + TABLE + table_footnote + 50512 + cR-factor = 100 × ∑|FP – FP(calc)|/∑ FP. + + + t1.xml + t1 + TABLE + table_footnote + 50572 + dRfree was calculated with 5% of the data. + + + diff --git a/BioC_XML/4772114_v0.xml b/BioC_XML/4772114_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..9290164e2817850f6edec1adb9c536917ee5732e --- /dev/null +++ b/BioC_XML/4772114_v0.xml @@ -0,0 +1,8017 @@ + + + + PMC + 20201220 + pmc.key + + 4772114 + CC BY + no + 0 + 0 + + 10.1038/srep22324 + srep22324 + 4772114 + 26927947 + 22324 + This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ + surname:Yokogawa;given-names:Mariko + surname:Tsushima;given-names:Takashi + surname:Noda;given-names:Nobuo N. + surname:Kumeta;given-names:Hiroyuki + surname:Enokizono;given-names:Yoshiaki + surname:Yamashita;given-names:Kazuo + surname:Standley;given-names:Daron M. + surname:Takeuchi;given-names:Osamu + surname:Akira;given-names:Shizuo + surname:Inagaki;given-names:Fuyuhiko + TITLE + front + 6 + 2016 + 0 + Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions + + 0.99706316 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + + ABSTRACT + abstract + 101 + Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions. + + 0.9963355 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + 0.9934494 + protein_type + cleaner0 + 2023-07-06T12:55:04Z + MESH: + + RNase + + + chemical + CHEBI: + cleaner0 + 2023-07-06T12:55:18Z + + mRNAs + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:16Z + + IL-6 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:52Z + + IL-12p40 + + + 0.99686384 + evidence + cleaner0 + 2023-07-06T13:52:13Z + DUMMY: + + structures + + + 0.9966231 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + 0.99424475 + species + cleaner0 + 2023-07-06T12:55:35Z + MESH: + + Mus musculus + + + 0.95943165 + structure_element + cleaner0 + 2023-07-06T12:55:54Z + SO: + + N-terminal domain + + + 0.9985343 + structure_element + cleaner0 + 2023-07-06T12:55:57Z + SO: + + NTD + + + 0.9938652 + structure_element + cleaner0 + 2023-07-06T12:55:45Z + SO: + + PilT N-terminus like + + + 0.9969989 + structure_element + cleaner0 + 2023-07-06T12:55:49Z + SO: + + PIN + + + 0.9811925 + structure_element + cleaner0 + 2023-07-06T12:57:31Z + SO: + + zinc finger + + + 0.5748093 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.9770051 + structure_element + cleaner0 + 2023-07-06T12:57:38Z + SO: + + C-terminal domain + + + 0.9980526 + structure_element + cleaner0 + 2023-07-06T12:57:42Z + SO: + + CTD + + + 0.9985806 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + site + SO: + cleaner0 + 2023-07-06T12:58:08Z + + catalytic center + + + 0.99840075 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99846005 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.99839157 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:03:21Z + + head-to-tail + + + 0.9782215 + oligomeric_state + cleaner0 + 2023-07-06T13:11:19Z + DUMMY: + + oligomer + + + 0.99730396 + site + cleaner0 + 2023-07-06T13:42:58Z + SO: + + dimer interface + + + 0.9981868 + site + cleaner0 + 2023-07-06T14:02:46Z + SO: + + NTD binding site + + + 0.81668115 + experimental_method + cleaner0 + 2023-07-06T14:12:00Z + MESH: + + mutations + + + 0.6376089 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:58Z + + NTD + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.99546367 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.9505898 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.29619977 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.860189 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.8859175 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + + INTRO + paragraph + 1084 + The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. Since aberrant activation of this system leads to auto immune disorders, it must be tightly regulated. Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. Regnase-1 accelerates target mRNA degradation via their 3′-terminal untranslated region (3′UTR), and also degrades its own mRNA. + + 0.9920222 + protein_type + cleaner0 + 2023-07-06T12:59:53Z + MESH: + + pattern-recognition receptors + + + 0.98915255 + protein_type + cleaner0 + 2023-07-06T12:59:56Z + MESH: + + PRRs + + + 0.94873893 + protein_type + cleaner0 + 2023-07-06T12:59:59Z + MESH: + + Toll-like receptors + + + 0.9913421 + protein_type + cleaner0 + 2023-07-06T13:00:04Z + MESH: + + TLRs + + + 0.9883321 + protein_type + cleaner0 + 2023-07-06T13:00:05Z + MESH: + + TLRs + + + 0.9960312 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + 0.99511456 + protein + cleaner0 + 2023-07-06T12:59:45Z + PR: + + Zc3h12a + + + 0.9936785 + protein + cleaner0 + 2023-07-06T12:59:50Z + PR: + + MCPIP1 + + + 0.9958835 + protein_type + cleaner0 + 2023-07-06T12:55:05Z + MESH: + + RNase + + + 0.9770538 + chemical + cleaner0 + 2023-07-06T13:51:16Z + CHEBI: + + lipopolysaccharides + + + chemical + CHEBI: + cleaner0 + 2023-07-06T12:55:19Z + + mRNAs + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:59:01Z + + (IL)-6 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:59:18Z + + IL-1β + + + protein_type + MESH: + cleaner0 + 2023-07-06T13:00:54Z + + IL-2 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:53Z + + IL-12p40 + + + 0.99513847 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:25Z + + mRNA + + + 0.9653266 + structure_element + cleaner0 + 2023-07-06T13:00:11Z + SO: + + 3′-terminal untranslated region + + + 0.9756991 + structure_element + cleaner0 + 2023-07-06T13:07:06Z + SO: + + 3′UTR + + + 0.91452265 + chemical + cleaner0 + 2023-07-06T13:00:26Z + CHEBI: + + mRNA + + + + INTRO + paragraph + 1904 + Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. Recently, the crystal structure of the Regnase-1 PIN domain derived from Homo sapiens was reported. The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity. Several CCCH-type ZF motifs in RNA-binding proteins have been reported to directly bind RNA. In addition, Regnase-1 has been predicted to possess other domains in the N- and C- terminal regions. However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved. + + 0.99689364 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + 0.9889549 + protein_type + cleaner0 + 2023-07-06T13:51:03Z + MESH: + + Regnase family + + + 0.99654186 + structure_element + cleaner0 + 2023-07-06T14:05:32Z + SO: + + PilT N-terminus like + + + 0.99797446 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9965479 + structure_element + cleaner0 + 2023-07-06T14:05:37Z + SO: + + CCCH-type zinc–finger + + + 0.8693028 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.997024 + protein_state + cleaner0 + 2023-07-06T14:09:26Z + DUMMY: + + conserved + + + 0.95484096 + protein_type + cleaner0 + 2023-07-06T13:51:07Z + MESH: + + Regnase family members + + + 0.99717337 + evidence + cleaner0 + 2023-07-06T13:52:18Z + DUMMY: + + crystal structure + + + 0.99680424 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + 0.99518687 + species + cleaner0 + 2023-07-06T13:01:57Z + MESH: + + Homo sapiens + + + 0.99719834 + evidence + cleaner0 + 2023-07-06T13:52:23Z + DUMMY: + + structure + + + 0.98988986 + residue_name + cleaner0 + 2023-07-06T14:00:50Z + SO: + + Asp + + + 0.9951724 + site + cleaner0 + 2023-07-06T13:10:22Z + SO: + + catalytic center + + + 0.97751343 + chemical + cleaner0 + 2023-07-06T13:51:20Z + CHEBI: + + Mg2+ + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.9969994 + structure_element + cleaner0 + 2023-07-06T14:05:40Z + SO: + + CCCH-type ZF motifs + + + 0.9912995 + protein_type + cleaner0 + 2023-07-06T13:51:10Z + MESH: + + RNA-binding proteins + + + 0.9979538 + chemical + cleaner0 + 2023-07-06T13:06:21Z + CHEBI: + + RNA + + + 0.99688846 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + 0.8052354 + structure_element + cleaner0 + 2023-07-06T14:05:45Z + SO: + + N- and C- terminal regions + + + 0.8256673 + evidence + cleaner0 + 2023-07-06T13:52:27Z + DUMMY: + + structure + + + structure_element + SO: + cleaner0 + 2023-07-06T12:57:35Z + + ZF + + + 0.9932221 + structure_element + cleaner0 + 2023-07-06T14:05:49Z + SO: + + N-terminal domain + + + 0.99760604 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.9944104 + structure_element + cleaner0 + 2023-07-06T14:05:52Z + SO: + + C-terminal domain + + + 0.9967349 + structure_element + cleaner0 + 2023-07-06T12:57:43Z + SO: + + CTD + + + 0.9967521 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + + INTRO + paragraph + 2729 + Here, we performed structural and functional analyses of individual domains of Regnase-1 derived from Mus musculus in order to understand the catalytic activity in vitro. Our data revealed that the catalytic activity of Regnase-1 is regulated through both intra and intermolecular domain interactions in vitro. The NTD plays a crucial role in efficient cleavage of target mRNA, through intramolecular NTD-PIN interactions. Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA. Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. + + 0.9942131 + experimental_method + cleaner0 + 2023-07-06T14:12:08Z + MESH: + + structural and functional analyses + + + 0.9970216 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + 0.9943695 + species + cleaner0 + 2023-07-06T12:55:36Z + MESH: + + Mus musculus + + + 0.99688476 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + 0.99837047 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99365187 + chemical + cleaner0 + 2023-07-06T13:00:26Z + CHEBI: + + mRNA + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:58Z + + NTD + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + 0.99676126 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + 0.9967153 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + 0.9953933 + chemical + cleaner0 + 2023-07-06T13:00:26Z + CHEBI: + + mRNA + + + 0.9967844 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + 0.99598 + chemical + cleaner0 + 2023-07-06T13:00:26Z + CHEBI: + + mRNA + + + 0.9966903 + protein_state + cleaner0 + 2023-07-06T14:09:32Z + DUMMY: + + NTD-activated + + + 0.9873679 + protein_state + cleaner0 + 2023-07-06T14:09:41Z + DUMMY: + + functional + + + 0.97070014 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9956506 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + 0.9979144 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:06:21Z + + RNA + + + 0.9399824 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9955165 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + + RESULTS + title_1 + 3441 + Results + + + RESULTS + title_2 + 3449 + Domain structures of Regnase-1 + + 0.7206502 + evidence + cleaner0 + 2023-07-06T13:52:32Z + DUMMY: + + structures + + + 0.99688196 + protein + cleaner0 + 2023-07-06T12:54:59Z + PR: + + Regnase-1 + + + + RESULTS + paragraph + 3480 + We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. This suggests that the PIN and ZF domains exist independently without interacting with each other. The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e). The NTD and CTD are both composed of three α helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server. + + 0.99682 + protein + cleaner0 + 2023-07-06T13:50:57Z + PR: + + Rengase-1 + + + 0.9952018 + species + cleaner0 + 2023-07-06T12:55:36Z + MESH: + + Mus musculus + + + 0.8869559 + experimental_method + cleaner0 + 2023-07-06T14:12:12Z + MESH: + + solved + + + 0.99640036 + evidence + cleaner0 + 2023-07-06T13:52:36Z + DUMMY: + + structures + + + 0.9984811 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.9984528 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9985355 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.998519 + structure_element + cleaner0 + 2023-07-06T12:57:43Z + SO: + + CTD + + + 0.9959334 + experimental_method + cleaner0 + 2023-07-06T13:25:25Z + MESH: + + X-ray crystallography + + + 0.99162745 + experimental_method + cleaner0 + 2023-07-06T13:24:17Z + MESH: + + NMR + + + 0.99591327 + experimental_method + cleaner0 + 2023-07-06T13:25:25Z + MESH: + + X-ray crystallography + + + 0.99827635 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99820614 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.9959991 + evidence + cleaner0 + 2023-07-06T13:52:40Z + DUMMY: + + electron density + + + 0.99839526 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9966171 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.99525744 + species + cleaner0 + 2023-07-06T13:01:57Z + MESH: + + Homo sapiens + + + 0.9983779 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9983346 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.9134344 + evidence + cleaner0 + 2023-07-06T13:52:44Z + DUMMY: + + structures + + + 0.99859136 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.9986003 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.99869186 + structure_element + cleaner0 + 2023-07-06T12:57:43Z + SO: + + CTD + + + 0.9929824 + experimental_method + cleaner0 + 2023-07-06T13:24:17Z + MESH: + + NMR + + + 0.99878925 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.9986803 + structure_element + cleaner0 + 2023-07-06T12:57:43Z + SO: + + CTD + + + structure_element + SO: + cleaner0 + 2023-07-06T14:06:11Z + + α helices + + + protein + PR: + cleaner0 + 2023-07-06T13:04:43Z + + ubiquitin conjugating enzyme E2 K + + + protein + PR: + cleaner0 + 2023-07-06T13:05:07Z + + ubiquitin associated protein 1 + + + 0.9484439 + experimental_method + cleaner0 + 2023-07-06T14:12:17Z + MESH: + + Dali server + + + + RESULTS + title_2 + 4303 + Contribution of each domain of Regnase-1 to the mRNA binding activity + + 0.99704736 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + + RESULTS + paragraph + 4373 + Although the PIN domain is responsible for the catalytic activity of Regnase-1, the roles of the other domains are largely unknown. First, we evaluated a role of the NTD and ZF domains for mRNA binding by an in vitro gel shift assay (Fig. 1f). Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. Based on the decrease in the free RNA fluorescence band, we evaluated the contribution of each domain of Regnase-1 to RNA binding. While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1). Direct binding of the ZF domain and RNA were confirmed by NMR spectral changes. The fitting of the titration curve of Y314 resulted in an apparent dissociation constant (Kd) of 10 ± 1.1 μM (Supplementary Fig. 2). These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA. + + 0.99853945 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99647266 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.9984365 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99807453 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + 0.89889586 + experimental_method + cleaner0 + 2023-07-06T14:12:24Z + MESH: + + in vitro gel shift assay + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:52:00Z + + Fluorescently 5′-labeled + + + 0.9976273 + chemical + cleaner0 + 2023-07-06T13:06:21Z + CHEBI: + + RNA + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:16Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + structure_element + SO: + cleaner0 + 2023-07-06T13:07:07Z + + 3′UTR + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:53:06Z + + inactive + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:19:22Z + + mutant + + + 0.99841 + mutant + cleaner0 + 2023-07-06T13:07:42Z + MESH: + + D226N + + + 0.998334 + mutant + cleaner0 + 2023-07-06T13:07:46Z + MESH: + + D244N + + + 0.9962869 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + mutant + MESH: + cleaner0 + 2023-07-06T13:08:09Z + + DDNN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:08:20Z + + mutant + + + 0.9952104 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + evidence + DUMMY: + cleaner0 + 2023-07-06T14:10:14Z + + fluorescence + + + 0.7604997 + protein_state + cleaner0 + 2023-07-06T14:09:52Z + DUMMY: + + free + + + 0.99726033 + chemical + cleaner0 + 2023-07-06T13:06:21Z + CHEBI: + + RNA + + + 0.99568486 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.8467543 + protein_state + cleaner0 + 2023-07-06T14:10:23Z + DUMMY: + + bound to + + + 0.9962359 + chemical + cleaner0 + 2023-07-06T13:06:21Z + CHEBI: + + RNA + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:06:21Z + + RNA + + + 0.99588823 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:06:21Z + + RNA + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:06:21Z + + RNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:06:08Z + + presence of + + + 0.9979705 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:50:31Z + + presence of + + + 0.99823594 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.9982657 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.9935913 + chemical + cleaner0 + 2023-07-06T13:06:21Z + CHEBI: + + RNA + + + 0.9553847 + experimental_method + cleaner0 + 2023-07-06T13:24:17Z + MESH: + + NMR + + + 0.95748454 + evidence + cleaner0 + 2023-07-06T13:53:11Z + DUMMY: + + spectral changes + + + 0.8526306 + evidence + cleaner0 + 2023-07-06T13:53:14Z + DUMMY: + + titration curve + + + 0.99872357 + residue_name_number + cleaner0 + 2023-07-06T13:58:23Z + DUMMY: + + Y314 + + + 0.98858047 + evidence + cleaner0 + 2023-07-06T13:53:16Z + DUMMY: + + dissociation constant + + + 0.98417723 + evidence + cleaner0 + 2023-07-06T13:28:19Z + DUMMY: + + Kd + + + 0.99861693 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9984654 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:06:21Z + + RNA + + + 0.9986304 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.9904041 + chemical + cleaner0 + 2023-07-06T13:06:21Z + CHEBI: + + RNA + + + + RESULTS + title_2 + 5661 + Contribution of each domain of Regnase-1 to RNase activity + + 0.9970178 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + + RESULTS + paragraph + 5720 + In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). Regnase-1 constructs consisting of NTD-PIN-ZF completely cleaved the target mRNA and generated the cleaved products. The apparent half-life (T1/2) of the RNase activity was about 20 minutes. Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2 ~ 70 minutes), while those lacking the NTD did not generate cleaved products (T1/2 > 90 minutes). It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. Taken together with the results in the previous section, we conclude that the NTD is crucial for the RNase activity of Regnase-1 in vitro, although it does not contribute to the direct mRNA binding. + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.99303466 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.9693205 + experimental_method + cleaner0 + 2023-07-06T14:12:28Z + MESH: + + in vitro cleavage assay + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:52:00Z + + fluorescently 5′-labeled + + + 0.99763703 + chemical + cleaner0 + 2023-07-06T13:06:21Z + CHEBI: + + RNA + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:16Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + structure_element + SO: + cleaner0 + 2023-07-06T13:07:07Z + + 3′UTR + + + 0.91905814 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.8367156 + mutant + cleaner0 + 2023-07-06T13:59:49Z + MESH: + + NTD-PIN-ZF + + + 0.7324506 + chemical + cleaner0 + 2023-07-06T13:00:26Z + CHEBI: + + mRNA + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.9866276 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.98778534 + protein_state + cleaner0 + 2023-07-06T14:10:28Z + DUMMY: + + lacking + + + structure_element + SO: + cleaner0 + 2023-07-06T12:57:35Z + + ZF + + + 0.9885648 + protein_state + cleaner0 + 2023-07-06T14:10:31Z + DUMMY: + + lacking + + + 0.9975284 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.9904258 + mutant + cleaner0 + 2023-07-06T13:59:52Z + MESH: + + NTD-PIN(DDNN)-ZF + + + 0.99837947 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.88263583 + protein_state + cleaner0 + 2023-07-06T14:10:34Z + DUMMY: + + lacks + + + site + SO: + cleaner0 + 2023-07-06T14:03:13Z + + catalytic residues + + + 0.99856585 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + site + SO: + cleaner0 + 2023-07-06T13:10:21Z + + catalytic center + + + 0.99861443 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99850696 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.9938163 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + + RESULTS + title_2 + 6810 + Dimer formation of the PIN domains + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-06T13:11:23Z + + Dimer + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + + RESULTS + paragraph + 6845 + During purification by gel filtration, the PIN domain exhibited extremely asymmetric elution peaks in a concentration dependent manner (Fig. 2a). By comparison with the elution volume of standard marker proteins, the PIN domain was assumed to be in equilibrium between a monomer and a dimer in solution at concentrations in the 20–200 μM range. The crystal structure of the PIN domain has been determined in three distinct crystal forms with a space group of P3121 (form I in this study and PDB ID 3V33), P3221 (form II in this study), and P41 (PDB ID 3V32 and 3V34), respectively. We found that the PIN domain formed a head-to-tail oligomer that was commonly observed in all three crystal forms in spite of the different crystallization conditions (Supplementary Fig. 3). Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. On the other hand, single mutations of side chains involved in the PIN–PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b). Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4). These results indicate that the PIN domain forms a head-to-tail oligomer in solution similar to the crystal structure. Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). The side chains of these residues point away from the catalytic center on the same molecule (Fig. 2b). Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro. + + 0.49429753 + experimental_method + cleaner0 + 2023-07-06T14:12:40Z + MESH: + + purification + + + 0.98449016 + experimental_method + cleaner0 + 2023-07-06T14:12:42Z + MESH: + + gel filtration + + + 0.9975926 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + experimental_method + MESH: + cleaner0 + 2023-07-06T13:53:47Z + + comparison with the elution volume of standard marker proteins + + + 0.9975043 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9934463 + oligomeric_state + cleaner0 + 2023-07-06T13:11:28Z + DUMMY: + + monomer + + + 0.99505746 + oligomeric_state + cleaner0 + 2023-07-06T13:11:22Z + DUMMY: + + dimer + + + 0.9972782 + evidence + cleaner0 + 2023-07-06T13:53:51Z + DUMMY: + + crystal structure + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + 0.9903691 + evidence + cleaner0 + 2023-07-06T13:53:56Z + DUMMY: + + crystal forms + + + 0.99769187 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.7567082 + protein_state + cleaner0 + 2023-07-06T13:11:11Z + DUMMY: + + head-to-tail + + + 0.61930215 + oligomeric_state + cleaner0 + 2023-07-06T13:11:19Z + DUMMY: + + oligomer + + + 0.99009776 + evidence + cleaner0 + 2023-07-06T13:53:59Z + DUMMY: + + crystal forms + + + 0.97776425 + experimental_method + cleaner0 + 2023-07-06T14:12:48Z + MESH: + + Mutation + + + 0.9985078 + residue_name_number + cleaner0 + 2023-07-06T13:58:29Z + DUMMY: + + Arg215 + + + site + SO: + cleaner0 + 2023-07-06T14:01:25Z + + oligomeric surface + + + 0.9858667 + residue_name + cleaner0 + 2023-07-06T14:00:56Z + SO: + + Glu + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-06T13:11:29Z + + monomer + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-06T13:11:23Z + + dimer + + + 0.99677914 + protein_state + cleaner0 + 2023-07-06T13:10:58Z + DUMMY: + + wild type + + + 0.9691986 + experimental_method + cleaner0 + 2023-07-06T14:12:51Z + MESH: + + single mutations + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-06T13:11:29Z + + monomer + + + 0.95763874 + experimental_method + cleaner0 + 2023-07-06T14:12:58Z + MESH: + + gel filtration + + + 0.9966627 + protein_state + cleaner0 + 2023-07-06T13:10:58Z + DUMMY: + + Wild type + + + 0.81764865 + oligomeric_state + cleaner0 + 2023-07-06T13:54:29Z + DUMMY: + + monomeric + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + 0.9986314 + mutant + cleaner0 + 2023-07-06T13:11:34Z + MESH: + + P212A + + + 0.9985985 + mutant + cleaner0 + 2023-07-06T13:11:39Z + MESH: + + D278R + + + 0.99153614 + experimental_method + cleaner0 + 2023-07-06T13:24:17Z + MESH: + + NMR + + + 0.9876031 + evidence + cleaner0 + 2023-07-06T13:54:02Z + DUMMY: + + spectra + + + 0.996472 + site + cleaner0 + 2023-07-06T13:42:59Z + SO: + + dimer interface + + + 0.99663275 + protein_state + cleaner0 + 2023-07-06T13:10:58Z + DUMMY: + + wild type + + + 0.9971969 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.77656555 + oligomeric_state + cleaner0 + 2023-07-06T13:54:30Z + DUMMY: + + monomeric + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + 0.9976539 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.8635138 + protein_state + cleaner0 + 2023-07-06T13:11:11Z + DUMMY: + + head-to-tail + + + 0.95340234 + oligomeric_state + cleaner0 + 2023-07-06T13:11:18Z + DUMMY: + + oligomer + + + 0.9970512 + evidence + cleaner0 + 2023-07-06T13:54:06Z + DUMMY: + + crystal structure + + + 0.6994014 + oligomeric_state + cleaner0 + 2023-07-06T13:54:30Z + DUMMY: + + monomeric + + + 0.3403942 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + 0.99864393 + mutant + cleaner0 + 2023-07-06T13:11:35Z + MESH: + + P212A + + + 0.99861157 + mutant + cleaner0 + 2023-07-06T13:11:44Z + MESH: + + R214A + + + 0.99861526 + mutant + cleaner0 + 2023-07-06T13:11:40Z + MESH: + + D278R + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:16Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + 0.77138484 + site + cleaner0 + 2023-07-06T13:10:22Z + SO: + + catalytic center + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:11:11Z + + head-to-tail + + + 0.487095 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99777055 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.9731679 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + + RESULTS + title_2 + 8692 + Domain-domain interaction between the NTD and the PIN domain + + 0.9979849 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99878246 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + + RESULTS + paragraph + 8753 + While the NTD does not contribute to RNA binding (Fig. 1f,g, and Supplementary Fig. 1), it increases the RNase activity of Regnase-1 (Fig. 1h). In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR. We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). These results clearly indicate a direct interaction between the PIN domain and the NTD. Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a). Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can “terminate” PIN-PIN oligomerization (Fig. 2b). An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c). + + 0.9982394 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:06:21Z + + RNA + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.99550647 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.64479476 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.9949989 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.9981713 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99805367 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.984757 + experimental_method + cleaner0 + 2023-07-06T13:24:17Z + MESH: + + NMR + + + 0.9969028 + protein_state + cleaner0 + 2023-07-06T14:10:39Z + DUMMY: + + catalytically inactive + + + 0.55651623 + oligomeric_state + cleaner0 + 2023-07-06T13:54:30Z + DUMMY: + + monomeric + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:13:23Z + + mutant + + + 0.9971111 + mutant + cleaner0 + 2023-07-06T13:08:10Z + MESH: + + DDNN + + + 0.99854714 + mutant + cleaner0 + 2023-07-06T13:11:40Z + MESH: + + D278R + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-06T13:11:23Z + + dimer + + + 0.99785584 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.6698075 + experimental_method + cleaner0 + 2023-07-06T13:24:17Z + MESH: + + NMR + + + 0.99814427 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99865305 + residue_name_number + cleaner0 + 2023-07-06T13:58:33Z + DUMMY: + + V177 + + + residue_range + DUMMY: + cleaner0 + 2023-07-06T13:13:50Z + + F210-T211 + + + 0.9985494 + residue_name_number + cleaner0 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2023-07-06T13:58:41Z + DUMMY: + + D53 + + + 0.99868435 + residue_name_number + cleaner0 + 2023-07-06T13:58:44Z + DUMMY: + + F55 + + + 0.998694 + residue_name_number + cleaner0 + 2023-07-06T13:58:46Z + DUMMY: + + K57 + + + residue_range + DUMMY: + cleaner0 + 2023-07-06T13:15:18Z + + Y60-S61 + + + 0.9986437 + residue_name_number + cleaner0 + 2023-07-06T13:58:50Z + DUMMY: + + V68 + + + residue_range + DUMMY: + cleaner0 + 2023-07-06T13:15:36Z + + T80-G83 + + + 0.9986634 + residue_name_number + cleaner0 + 2023-07-06T13:58:53Z + DUMMY: + + L85 + + + 0.9986873 + residue_name_number + cleaner0 + 2023-07-06T13:58:56Z + DUMMY: + + G89 + + + 0.9984981 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99816114 + residue_name_number + cleaner0 + 2023-07-06T13:58:59Z + DUMMY: + + N79 + + + 0.9981133 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9982187 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.93074256 + evidence + cleaner0 + 2023-07-06T13:54:52Z + DUMMY: + + titration curve + + + evidence + DUMMY: + cleaner0 + 2023-07-06T13:55:10Z + + chemical shift changes + + + 0.9986505 + residue_name_number + cleaner0 + 2023-07-06T13:59:02Z + DUMMY: + + L58 + + + 0.99329054 + evidence + cleaner0 + 2023-07-06T13:28:19Z + DUMMY: + + Kd + + + 0.99828786 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.96001387 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9982845 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99725014 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9599482 + structure_element + cleaner0 + 2023-07-06T14:06:22Z + SO: + + linker + + + 0.98603517 + evidence + cleaner0 + 2023-07-06T13:55:14Z + DUMMY: + + binding affinity + + + 0.6444536 + protein_state + cleaner0 + 2023-07-06T14:10:42Z + DUMMY: + + native + + + 0.9977081 + site + cleaner0 + 2023-07-06T14:03:19Z + SO: + + PIN/NTD interface + + + 0.97318345 + structure_element + cleaner0 + 2023-07-06T14:06:27Z + SO: + + helix + + + 0.9988331 + residue_name_number + cleaner0 + 2023-07-06T13:59:06Z + DUMMY: + + D225 + + + 0.9988852 + residue_name_number + cleaner0 + 2023-07-06T13:59:09Z + DUMMY: + + D226 + + + 0.9984113 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9964691 + site + cleaner0 + 2023-07-06T14:03:23Z + SO: + + binding site + + + 0.9971938 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99723375 + site + cleaner0 + 2023-07-06T13:44:44Z + SO: + + PIN-PIN dimer interface + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:58Z + + NTD + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + 0.68101674 + experimental_method + cleaner0 + 2023-07-06T14:13:33Z + MESH: + + in silico docking + + + 0.9981969 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99754286 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9163096 + evidence + cleaner0 + 2023-07-06T13:55:17Z + DUMMY: + + chemical shift restraints + + + 0.9760098 + experimental_method + cleaner0 + 2023-07-06T13:24:17Z + MESH: + + NMR + + + + RESULTS + title_2 + 10692 + Residues critical for Regnase-1 RNase activity + + 0.9926675 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + + RESULTS + paragraph + 10739 + To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs. The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). In contrast, R214 is important for oligomerization of the PIN domain and the “secondary” chain’s residue R214 is also positioned near the “primary” chain’s active site within the dimer interface. It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains. + + 0.9933803 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.9982642 + site + cleaner0 + 2023-07-06T13:17:44Z + SO: + + catalytic site + + + 0.5471427 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.85306406 + oligomeric_state + cleaner0 + 2023-07-06T13:11:19Z + DUMMY: + + oligomer + + + 0.95547485 + experimental_method + cleaner0 + 2023-07-06T14:13:37Z + MESH: + + mutated to + + + 0.98409975 + residue_name + cleaner0 + 2023-07-06T14:01:44Z + SO: + + alanine + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.99340606 + experimental_method + cleaner0 + 2023-07-06T14:13:41Z + MESH: + + gel filtration assays + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + 0.99769235 + mutant + cleaner0 + 2023-07-06T13:11:44Z + MESH: + + R214A + + + 0.9367153 + oligomeric_state + cleaner0 + 2023-07-06T14:02:14Z + DUMMY: + + dimers + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + 0.9975062 + mutant + cleaner0 + 2023-07-06T13:11:44Z + MESH: + + R214A + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-06T13:11:23Z + + dimer + + + 0.99367803 + mutant + cleaner0 + 2023-07-06T13:21:17Z + MESH: + + W182A + + + 0.9938613 + mutant + cleaner0 + 2023-07-06T13:59:57Z + MESH: + + R183A + + + 0.9961654 + mutant + cleaner0 + 2023-07-06T13:11:44Z + MESH: + + R214A + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:16Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + protein + PR: + cleaner0 + 2023-07-06T12:55:00Z + + Regnase-1 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + 0.9929792 + mutant + cleaner0 + 2023-07-06T13:21:22Z + MESH: + + K184A + + + 0.9969901 + mutant + cleaner0 + 2023-07-06T14:00:01Z + MESH: + + R215A + + + 0.996974 + mutant + cleaner0 + 2023-07-06T13:21:32Z + MESH: + + R220A + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + chemical + CHEBI: + cleaner0 + 2023-07-06T12:55:19Z + + mRNAs + + + 0.9987538 + residue_name_number + cleaner0 + 2023-07-06T13:46:04Z + DUMMY: + + K219 + + + 0.99883634 + residue_name_number + cleaner0 + 2023-07-06T13:46:09Z + DUMMY: + + R247 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:16Z + + IL-6 + + + 0.7909548 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + 0.99872726 + residue_name_number + cleaner0 + 2023-07-06T13:46:03Z + DUMMY: + + K219 + + + 0.9987649 + residue_name_number + cleaner0 + 2023-07-06T13:46:09Z + DUMMY: + + R247 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:16Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + 0.73296374 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + 0.998792 + residue_name_number + cleaner0 + 2023-07-06T13:46:13Z + DUMMY: + + K152 + + + 0.99888474 + residue_name_number + cleaner0 + 2023-07-06T13:46:17Z + DUMMY: + + R158 + + + 0.9988695 + residue_name_number + cleaner0 + 2023-07-06T13:46:22Z + DUMMY: + + R188 + + + 0.9988493 + residue_name_number + cleaner0 + 2023-07-06T13:46:26Z + DUMMY: + + R200 + + + 0.99886715 + residue_name_number + cleaner0 + 2023-07-06T13:46:30Z + DUMMY: + + K204 + + + 0.9988532 + residue_name_number + cleaner0 + 2023-07-06T13:46:35Z + DUMMY: + + K206 + + + 0.99883693 + residue_name_number + cleaner0 + 2023-07-06T13:46:43Z + DUMMY: + + K257 + + + 0.99881834 + residue_name_number + cleaner0 + 2023-07-06T13:46:49Z + DUMMY: + + R258 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.99880743 + residue_name_number + cleaner0 + 2023-07-06T13:46:54Z + DUMMY: + + W182 + + + 0.9988092 + residue_name_number + cleaner0 + 2023-07-06T13:46:59Z + DUMMY: + + R183 + + + 0.7364476 + oligomeric_state + cleaner0 + 2023-07-06T13:54:30Z + DUMMY: + + monomeric + + + 0.41677222 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9933302 + evidence + cleaner0 + 2023-07-06T13:55:23Z + DUMMY: + + structure + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + site + SO: + cleaner0 + 2023-07-06T13:17:44Z + + catalytic site + + + 0.9987658 + residue_name_number + cleaner0 + 2023-07-06T13:47:03Z + DUMMY: + + K184 + + + 0.99793285 + site + cleaner0 + 2023-07-06T14:03:30Z + SO: + + active site + + + 0.71547395 + evidence + cleaner0 + 2023-07-06T13:55:28Z + DUMMY: + + structure + + + 0.998803 + residue_name_number + cleaner0 + 2023-07-06T13:47:04Z + DUMMY: + + K184 + + + 0.6941805 + protein_state 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Regnase-1 + + + 0.6059957 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + + RESULTS + title_2 + 12527 + Molecular mechanism of target mRNA cleavage by the PIN dimer + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + 0.8507944 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.98532355 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + + RESULTS + paragraph + 12588 + Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. In order to test this hypothesis, we performed in vitro cleavage assays using combinations of Regnase-1 mutants that had no or decreased RNase activities by themselves (Fig. 5). One group consisted of catalytically active PIN domains with mutation of basic residues found in the previous section to confer decreased RNase activity (Fig. 4). These were paired with a DDNN mutant that had no RNase activity by itself. When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). Rescue of K184A and R214A by the DDNN mutant was also confirmed by a significant increase in the cleaved products. This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize. According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. Taken together, the rescue experiments above support the proposed model in which the head-to-tail dimer is functional in vitro. + + 0.9658383 + experimental_method + cleaner0 + 2023-07-06T14:13:46Z + MESH: + + mutational experiments + + + 0.99637294 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + 0.973656 + protein_state + cleaner0 + 2023-07-06T14:06:42Z + DUMMY: + + secondary + + + 0.5243988 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.8478697 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.99665636 + site + cleaner0 + 2023-07-06T14:03:46Z + SO: + + RNA binding residues + + + 0.99816585 + site + cleaner0 + 2023-07-06T14:03:50Z + SO: + + active site + + + 0.9636926 + protein_state + cleaner0 + 2023-07-06T14:06:52Z + DUMMY: + + primary + + + 0.54169255 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9962474 + protein_state + cleaner0 + 2023-07-06T14:10:48Z + DUMMY: + + 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DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + 0.9852212 + mutant + cleaner0 + 2023-07-06T13:21:17Z + MESH: + + W182A + + + 0.9861892 + mutant + cleaner0 + 2023-07-06T13:21:22Z + MESH: + + K184A + + + 0.96760845 + mutant + cleaner0 + 2023-07-06T13:11:45Z + MESH: + + R214A + + + 0.96821 + mutant + cleaner0 + 2023-07-06T13:21:31Z + MESH: + + R220A + + + mutant + MESH: + cleaner0 + 2023-07-06T13:08:10Z + + DDNN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:19:22Z + + mutant + + + evidence + DUMMY: + cleaner0 + 2023-07-06T13:42:11Z + + fluorescence intensity + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:49:26Z + + uncleaved + + + protein + PR: + cleaner0 + 2023-07-06T12:55:00Z + + Regnase-1 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + 0.9902086 + mutant + cleaner0 + 2023-07-06T13:21:22Z + MESH: + + K184A + + + 0.9869365 + mutant + cleaner0 + 2023-07-06T13:11:45Z + MESH: + + R214A + + + mutant + MESH: + cleaner0 + 2023-07-06T13:08:10Z + + DDNN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:19:22Z + + mutant + + + 0.9826202 + mutant + cleaner0 + 2023-07-06T13:21:22Z + MESH: + + K184A + + + 0.98075163 + mutant + cleaner0 + 2023-07-06T13:11:45Z + MESH: + + R214A + + + mutant + MESH: + cleaner0 + 2023-07-06T13:08:10Z + + DDNN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:19:22Z + + mutant + + + 0.9985215 + residue_name_number + cleaner0 + 2023-07-06T13:47:04Z + DUMMY: + + K184 + + + 0.99844944 + residue_name_number + cleaner0 + 2023-07-06T13:47:11Z + DUMMY: + + R214 + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:22:41Z + + primary + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + site + SO: + cleaner0 + 2023-07-06T13:10:22Z + + catalytic center + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:23:10Z + + secondary + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + site + SO: + cleaner0 + 2023-07-06T13:10:22Z + + catalytic center + + + 0.8445035 + protein_state + cleaner0 + 2023-07-06T13:22:53Z + DUMMY: + + primary + + + 0.7418916 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9984372 + residue_name_number + cleaner0 + 2023-07-06T13:47:11Z + DUMMY: + + R214 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-06T13:11:23Z + + dimer + + + 0.96471846 + mutant + cleaner0 + 2023-07-06T13:11:45Z + MESH: + + R214A + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:23:31Z + + secondary + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + 0.996743 + mutant + cleaner0 + 2023-07-06T13:11:45Z + MESH: + + R214A + + + 0.9974546 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99503005 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + 0.8482282 + mutant + cleaner0 + 2023-07-06T13:08:10Z + MESH: + + DDNN + + + 0.99564797 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:23:48Z + + secondary + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:50Z + + PIN + + + 0.98570395 + protein_state + cleaner0 + 2023-07-06T13:11:11Z + DUMMY: + + head-to-tail + + + 0.99637955 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + + DISCUSS + title_1 + 14859 + Discussion + + + DISCUSS + paragraph + 14870 + We determined the individual domain structures of Regnase-1 by NMR and X-ray crystallography. Although the function of the CTD remains elusive, we revealed the functions of the NTD, PIN, and ZF domains. A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. These results indicate that Regnase-1 directly binds to RNA and precipitates under such experimental conditions. Due to this limitation, it is difficult to perform further structural analyses of mRNA-Regnase-1 complexes by X-ray crystallography or NMR. + + 0.97923684 + evidence + cleaner0 + 2023-07-06T13:55:34Z + DUMMY: + + structures + + + 0.99567956 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.9941907 + experimental_method + cleaner0 + 2023-07-06T13:24:16Z + MESH: + + NMR + + + 0.99620944 + experimental_method + cleaner0 + 2023-07-06T13:25:25Z + MESH: + + X-ray crystallography + + + 0.9986823 + structure_element + cleaner0 + 2023-07-06T12:57:43Z + SO: + + CTD + + + 0.9985928 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.9983541 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99825305 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.98885316 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.9979913 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9980254 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.99311554 + species + cleaner0 + 2023-07-06T12:55:36Z + MESH: + + Mus musculus + + + 0.97050124 + experimental_method + cleaner0 + 2023-07-06T14:14:04Z + MESH: + + crystallized + + + 0.99573576 + evidence + cleaner0 + 2023-07-06T13:55:38Z + DUMMY: + + electron density + + + 0.9982503 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.99840987 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.9916284 + protein_state + cleaner0 + 2023-07-06T14:11:16Z + DUMMY: + + highly mobile + + + 0.9928031 + protein_state + cleaner0 + 2023-07-06T13:49:31Z + DUMMY: + + absence of + + + 0.9949568 + chemical + cleaner0 + 2023-07-06T13:00:26Z + CHEBI: + + mRNA + + + 0.9919566 + experimental_method + cleaner0 + 2023-07-06T13:24:17Z + MESH: + + NMR + + + 0.9981407 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.98258847 + protein_type + cleaner0 + 2023-07-06T12:54:16Z + MESH: + + IL-6 + + + 0.93520975 + chemical + cleaner0 + 2023-07-06T13:00:26Z + CHEBI: + + mRNA + + + 0.99338335 + evidence + cleaner0 + 2023-07-06T13:28:19Z + DUMMY: + + Kd + + + 0.9251319 + experimental_method + cleaner0 + 2023-07-06T14:14:07Z + MESH: + + in vitro gel shift assay + + + 0.99186295 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.9976367 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + 0.9452816 + chemical + cleaner0 + 2023-07-06T13:06:22Z + CHEBI: + + RNA + + + 0.9949519 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.9977245 + chemical + cleaner0 + 2023-07-06T13:06:22Z + CHEBI: + + RNA + + + experimental_method + MESH: + cleaner0 + 2023-07-06T13:25:57Z + + structural analyses + + + complex_assembly + GO: + cleaner0 + 2023-07-06T13:25:13Z + + mRNA-Regnase-1 + + + 0.996264 + experimental_method + cleaner0 + 2023-07-06T13:25:24Z + MESH: + + X-ray crystallography + + + 0.99421906 + experimental_method + cleaner0 + 2023-07-06T13:24:17Z + MESH: + + NMR + + + + DISCUSS + paragraph + 15945 + The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms. Rao and co-workers previously argued that PIN dimerization is likely to be a crystallographic artifact with no physiological significance, since monomers were dominant in their analytical ultra-centrifugation experiments. In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. This inconsistency might be due to difference in the analytical methods and/or protein concentrations used in each experiment, since the oligomer formation of PIN was dependent on the protein concentration in our study. + + 0.997193 + evidence + cleaner0 + 2023-07-06T13:55:41Z + DUMMY: + + crystal structure + + + 0.9956177 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.9980925 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9952601 + species + cleaner0 + 2023-07-06T13:01:57Z + MESH: + + Homo sapiens + + + 0.9953058 + species + cleaner0 + 2023-07-06T12:55:36Z + MESH: + + Mus musculus + + + 0.9115232 + evidence + cleaner0 + 2023-07-06T13:55:45Z + DUMMY: + + RMSD + + + 0.90184754 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.98497796 + residue_range + cleaner0 + 2023-07-06T13:47:29Z + DUMMY: + + 134–295 + + + 0.9944193 + taxonomy_domain + cleaner0 + 2023-07-06T13:26:14Z + DUMMY: + + mouse + + + 0.9957675 + species + cleaner0 + 2023-07-06T13:26:21Z + MESH: + + human + + + 0.99777824 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.97708714 + protein_state + cleaner0 + 2023-07-06T13:11:11Z + DUMMY: + + head-to-tail + + + 0.98037237 + oligomeric_state + cleaner0 + 2023-07-06T14:02:19Z + DUMMY: + + oligomers + + + 0.99634933 + evidence + cleaner0 + 2023-07-06T13:55:48Z + DUMMY: + + crystal forms + + + 0.92157084 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.98985136 + oligomeric_state + cleaner0 + 2023-07-06T14:02:25Z + DUMMY: + + monomers + + + 0.9923066 + experimental_method + cleaner0 + 2023-07-06T14:14:12Z + MESH: + + analytical ultra-centrifugation + + + 0.9894432 + experimental_method + cleaner0 + 2023-07-06T14:14:16Z + MESH: + + gel filtration + + + 0.98712045 + experimental_method + cleaner0 + 2023-07-06T14:14:19Z + MESH: + + mutational analyses + + + 0.98363185 + experimental_method + cleaner0 + 2023-07-06T13:24:17Z + MESH: + + NMR + + + 0.49351272 + evidence + cleaner0 + 2023-07-06T13:55:50Z + DUMMY: + + spectra + + + 0.99800354 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99093944 + protein_state + cleaner0 + 2023-07-06T13:11:11Z + DUMMY: + + head-to-tail + + + 0.9965288 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + 0.997293 + evidence + cleaner0 + 2023-07-06T13:55:52Z + DUMMY: + + crystal structure + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-06T13:11:19Z + + oligomer + + + 0.67573106 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + + DISCUSS + paragraph + 17024 + Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well. Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c). Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro. Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). Moreover, our structure-based mutational analyses showed these two Regnase-1 specific basic regions were essential for target mRNA cleavage in vitro. + + 0.97857404 + experimental_method + cleaner0 + 2023-07-06T14:14:26Z + MESH: + + Single mutations + + + 0.8207907 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.8227865 + oligomeric_state + cleaner0 + 2023-07-06T14:02:28Z + DUMMY: + + monomerized + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.850628 + experimental_method + cleaner0 + 2023-07-06T13:24:17Z + MESH: + + NMR + + + 0.7450116 + evidence + cleaner0 + 2023-07-06T13:55:58Z + DUMMY: + + spectra + + + 0.5644939 + oligomeric_state + cleaner0 + 2023-07-06T13:54:30Z + DUMMY: + + monomeric + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + 0.6800348 + oligomeric_state + cleaner0 + 2023-07-06T13:54:30Z + DUMMY: + + monomeric + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + structure_element + SO: + cleaner0 + 2023-07-06T13:48:07Z + + PIN + + + structure_element + SO: + cleaner0 + 2023-07-06T13:48:15Z + + PIN + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-06T13:11:23Z + + dimer + + + 0.7938333 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.99724627 + evidence + cleaner0 + 2023-07-06T13:56:01Z + DUMMY: + + crystal structure + + + 0.72638947 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99680114 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + protein + PR: + cleaner0 + 2023-07-06T12:55:00Z + + Regnase-1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T14:07:47Z + + primary + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T14:07:58Z + + secondary + + + 0.72285646 + structure_element + cleaner0 + 2023-07-06T14:08:01Z + SO: + + PINs + + + 0.9981303 + site + cleaner0 + 2023-07-06T13:17:44Z + SO: + + catalytic site + + + 0.7775953 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99617386 + experimental_method + cleaner0 + 2023-07-06T14:14:29Z + MESH: + + structure-based mutational analyses + + + protein + PR: + cleaner0 + 2023-07-06T12:55:00Z + + Regnase-1 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + + DISCUSS + paragraph + 17888 + The cleavage assay also showed that the NTD is crucial for efficient mRNA cleavage. Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a). Taken together, this suggests that the NTD and the PIN domain compete for a common binding site. The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+. In this context, it is interesting that, in response to TCR stimulation, Malt1 cleaves Regnase-1 at R111 to control immune responses in vivo. This result is consistent with a model in which the NTD acts as an enhancer, and cleavage of the linker lowers enzymatic activity dramatically. + + 0.9923765 + experimental_method + cleaner0 + 2023-07-06T14:14:33Z + MESH: + + cleavage assay + + + 0.998659 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.6335911 + chemical + cleaner0 + 2023-07-06T13:00:26Z + CHEBI: + + mRNA + + + 0.9985391 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.9485144 + site + cleaner0 + 2023-07-06T14:04:02Z + SO: + + oligomeric surface + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T14:08:46Z + + primary + + + 0.99635243 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.98555815 + structure_element + cleaner0 + 2023-07-06T14:08:05Z + SO: + + helix + + + 0.7927509 + site + cleaner0 + 2023-07-06T14:04:06Z + SO: + + catalytic residues + + + 0.9986872 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99848086 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.96827227 + site + cleaner0 + 2023-07-06T14:04:27Z + SO: + + common binding site + + + 0.8903143 + evidence + cleaner0 + 2023-07-06T13:56:04Z + DUMMY: + + affinity + + + 0.9919689 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + evidence + DUMMY: + cleaner0 + 2023-07-06T13:28:19Z + + Kd + + + 0.81601435 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.6422894 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + evidence + DUMMY: + cleaner0 + 2023-07-06T13:28:19Z + + Kd + + + 0.97889966 + residue_range + cleaner0 + 2023-07-06T14:00:35Z + DUMMY: + + 90–133 + + + 0.99872094 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T14:08:33Z + + primary + + + 0.99739 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99738455 + protein_state + cleaner0 + 2023-07-06T14:11:22Z + DUMMY: + + full-length + + + 0.9968293 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.95738995 + experimental_method + cleaner0 + 2023-07-06T14:14:36Z + MESH: + + docking and molecular dynamics simulations + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:58Z + + NTD + + + 0.6960801 + site + cleaner0 + 2023-07-06T14:04:33Z + SO: + + catalytic residues + + + 0.99850863 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99502134 + protein_state + cleaner0 + 2023-07-06T14:11:27Z + DUMMY: + + active + + + 0.99603933 + chemical + cleaner0 + 2023-07-06T13:52:07Z + CHEBI: + + Mg2+ + + + 0.998787 + protein + cleaner0 + 2023-07-06T13:29:05Z + PR: + + Malt1 + + + 0.99686813 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.99852484 + residue_name_number + cleaner0 + 2023-07-06T13:59:16Z + DUMMY: + + R111 + + + 0.99869967 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.998374 + structure_element + cleaner0 + 2023-07-06T14:08:09Z + SO: + + linker + + + + DISCUSS + paragraph + 19145 + Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. We incorporated information from the cleavage site of IL-6 mRNA in vitro is indicated by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 7a,b). The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1. + + 0.9916974 + experimental_method + cleaner0 + 2023-07-06T14:14:41Z + MESH: + + structural and functional analyses + + + 0.99671763 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.99478006 + experimental_method + cleaner0 + 2023-07-06T14:14:44Z + MESH: + + docking simulations + + + 0.64951575 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.7890433 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9949609 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + 0.9901457 + protein_type + cleaner0 + 2023-07-06T12:54:16Z + MESH: + + IL-6 + + + 0.976857 + chemical + cleaner0 + 2023-07-06T13:00:26Z + CHEBI: + + mRNA + + + 0.98416615 + site + cleaner0 + 2023-07-06T14:04:37Z + SO: + + cleavage site + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:16Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:26Z + + mRNA + + + 0.97995067 + experimental_method + cleaner0 + 2023-07-06T14:14:48Z + MESH: + + polyacrylamide gel electrophoresis + + + 0.90254337 + experimental_method + cleaner0 + 2023-07-06T14:14:51Z + MESH: + + docking + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:06:22Z + + RNA + + + protein_type + MESH: + cleaner0 + 2023-07-06T13:30:27Z + + RNA-binding proteins + + + 0.74459183 + structure_element + cleaner0 + 2023-07-06T14:09:02Z + SO: + + loop + + + 0.99668485 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + + DISCUSS + paragraph + 19757 + The overall model of regulation of Regnase-1 RNase activity through domain-domain interactions in vitro is summarized in Fig. 6. In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration. A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). While further investigations on the domain-domain interactions of Regnase-1 in vivo are necessary, these intramolecular and intermolecular domain interactions of Regnase-1 appear to structurally constrain Regnase-1activity, which, in turn, enables tight regulation of immune responses. + + 0.99697834 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:05Z + + RNase + + + 0.994583 + protein_state + cleaner0 + 2023-07-06T13:49:31Z + DUMMY: + + absence of + + + 0.9962379 + chemical + cleaner0 + 2023-07-06T13:00:26Z + CHEBI: + + mRNA + + + 0.9986432 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.9353172 + protein_state + cleaner0 + 2023-07-06T13:11:11Z + DUMMY: + + head-to-tail + + + 0.99412054 + oligomeric_state + cleaner0 + 2023-07-06T14:02:33Z + DUMMY: + + oligomers + + + 0.9860203 + protein_state + cleaner0 + 2023-07-06T14:11:32Z + DUMMY: + + fully active + + + 0.9961766 + site + cleaner0 + 2023-07-06T13:10:22Z + SO: + + catalytic center + + + 0.9985299 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-06T13:11:19Z + + oligomer + + + 0.9985869 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:11:11Z + + head-to-tail + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-06T13:11:19Z + + oligomer + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:48:38Z + + primary + + + 0.95147157 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.89128834 + protein_state + cleaner0 + 2023-07-06T14:11:38Z + DUMMY: + + functional + + + 0.9970004 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + 0.9982644 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:48:49Z + + secondary + + + 0.9390025 + structure_element + cleaner0 + 2023-07-06T12:55:50Z + SO: + + PIN + + + 0.99683267 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.99686545 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein + PR: + cleaner0 + 2023-07-06T13:31:15Z + + Regnase-1 + + + + METHODS + title_1 + 20555 + Methods + + + METHODS + title_2 + 20563 + Protein expression and purification + + + METHODS + paragraph + 20599 + The DNA fragment encoding Regnase-1 derived from Mus musculus was cloned into pGEX6p vector (GE Healthcare). All the mutants were generated by PCR-mediated site-directed mutagenesis and confirmed by the DNA sequence analyses. As a catalytically deficient mutant, both Asp226 and Asp244 at the catalytic center of PIN were mutated to Asn, which is referred to as DDNN mutant. Regnase-1 was expressed at 16 °C using the Escherichia coli RosettaTM(DE3)pLysS strain. After purification with a GST-affinity resin, an N-terminal GST tag was digested by HRV-3 C protease. NTD was further purified by gel filtration chromatography using a HiLoad 16/60 Superdex 75 pg (GE Healthcare). The other domains were further purified by cation exchange chromatography using Resource S (GE Healthcare), followed by gel filtration chromatography using a HiLoad 16/60 Superdex 75 pg (GE Healthcare). Uniformly 15N or 13C, 15N-double labeled proteins for NMR experiments were prepared by growing E. coli host in M9 minimal medium containing 15NH4Cl, unlabeled glucose and 15N CELTONE® Base Powder (CIL) or 15NH4Cl, 13C6-glucose, and13C, 15N CELTONE® Base Powder (CIL), respectively. + + + METHODS + title_2 + 21771 + X-ray crystallography + + + METHODS + paragraph + 21793 + Crystallization was performed using the sitting drop vapor diffusion method at 20 °C and two crystal forms (I and II) were obtained. In the case of form I crystals, drops (0.5 μl) of 6 mg/ml selenomethionine-labeled Regnase-1 PIN-ZF (residues 134–339 derived from Mus musculus) in 20 mM HEPES-NaOH (pH 6.8), 200 mM NaCl and 5 mM DTT were mixed with reservoir solution consisting of 1 M (NH4)2HPO4, 200 mM NaCl and 100 mM sodium citrate (pH 5.5) whereas in the case of form II crystals, drops (0.5 μl) of 6 mg/ml native Regnase-1 PIN-ZF (residues 134–339) in 20 mM HEPES-NaOH (pH 6.8), 200 mM NaCl and 5 mM DTT were mixed with reservoir solution consisting of 1.7 M NaCl and 100 mM HEPES-NaOH (pH 7.0). Diffraction data were collected at a Photon Factory Advanced Ring beamline NE3A (form I) or at a SPring-8 beamline BL41XU (form II), and were processed with HKL2000. The structure of the form I crystal was determined by the multiple anomalous dispersion (MAD) method. Nine Se sites were found using the program SOLVE; however, the electron density obtained by MAD phases calculated using SOLVE was not good enough to build a model even after density modification using the program RESOLVE. Then the program CNS was used to find additional three Se sites and calculate MAD phases using 12 Se sites. The electron density after density modification using CNS was good enough to build a model. Structure of the form II crystal was determined by the molecular replacement method using CNS and using the structure of the form I crystal as a search model. For all structures, further model building was performed manually with COOT, and TLS and restrained refinement using isotropic individual B factors was performed with REFMAC5 in the CCP4 program suite. Crystallographic parameters are summarized in Supplementary Table 1. + + + METHODS + title_2 + 23654 + NMR measurements + + + METHODS + paragraph + 23671 + All NMR experiments were carried out at 298 K on Inova 500-MHz, 600-MHz, and 800-MHz spectrometer (Agilent). The NMR data were processed using the NMRPipe, the Olivia (fermi.pharm.hokudai.ac.jp/olivia/), and the Sparky program (Sparky3, University of California, San Francisco). + + + METHODS + paragraph + 23952 + For structure calculation, NOE distance restraints were obtained from 3D 15N-NOESY-HSQC (100 ms mixing time for the NTD, 75 ms mixing time for the ZF domain and the CTD) and 13C-NOESY-HSQC spectra (100 ms mixing time for the NTD, 75 ms mixing time for the ZF domain and the CTD). The NMR structures were determined using the CANDID/CYANA2.1. Dihedral restraints were derived from backbone chemical shifts using TALOS. + + + METHODS + paragraph + 24378 + For the domain-domain interaction analyses between the NTD and the PIN domain, 1H-15N HSQC spectra of uniformly 15N-labeled proteins in the concentration of 100 μM were obtained in the presence of 3 or 6 molar equivalents of unlabeled proteins. + + + METHODS + title_2 + 24626 + Preparation of RNAs + + + METHODS + paragraph + 24646 + The fluorescently labeled RNAs at the 5′-end by 6-FAM were purchased from SIGMA-ALDORICH. The RNA sequences used in this study were shown below. + + + METHODS + paragraph + 24793 + IL-6 mRNA 3′UTR (82–106): 5′-UGUUGUUCUCUACGAAGAACUGACA-3′ (25 nts) + + + METHODS + paragraph + 24868 + Regnase-1 mRNA 3′UTR (191–211): 5′- CUGUUGAUACACAUUGUAUCU-3′ (21 nts) + + + METHODS + title_2 + 24946 + Electrophoretic mobility shift assay + + + METHODS + paragraph + 24983 + Catalytically deficient Regnase-1 proteins, containing DDNN mutations, and 5′-terminally 6-FAM labeled RNAs were incubated in the RNA-binding buffer (20 mM HEPES-NaOH (pH 6.8), 150 mM NaCl, 1 mM DTT, 10% glycerol (v/v), and 0.1% NP-40 (v/v)) at 4 °C for 30 minutes, then analyzed by non-denaturing polyacrylamide gel electrophoresis. The electrophoreses were performed at 4 °C using the 7.5% polyacrylamide (w/v) gel (monomer : bis = 29 : 1) in the electrophoresis buffer (25 mM Tris-HCl (pH 7.5) and 200 mM glycine). The fluorescence of 6-FAM labeled RNA was directly detected at the excitation wavelength of 460 nm with a fluorescence filter (Y515-Di) using a fluoroimaging analyzer (LAS-4000 (FUJIFILM)). The fluorescence intensity of each sample was quantified using ImageJ software. + + + METHODS + title_2 + 25797 + In vitro RNA cleavage assay + + + METHODS + paragraph + 25825 + Regnase-1 (2 μM) and 5′-terminally 6-FAM labeled RNA (1 μM) were incubated in the RNA-cleavage buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, and 1 mM DTT) at 37 °C. For the assay using combinations of Regnase-1 mutants, equimolar amounts of Regnase-1 mutants (2 μM each) were mixed with fluorescently labeled RNA (1 μM). After incubation for 30–120 minutes, the reaction was stopped by the addition of 1.5-fold volume of denaturing buffer containing 8 M urea and 100 mM EDTA, and samples were boiled. The electrophoreses were performed at room temperature using the 8 M urea containing denaturing gel with 20% polyacrylamide (w/v) (monomer : bis = 19 : 1) in 0.5 × TBE as the electrophoresis buffer. + + + METHODS + title_2 + 26581 + Docking calculations + + + METHODS + paragraph + 26602 + For docking NTD to PIN, OSCAR-star was first used to rebuild sidechains in the head-to-tail PIN dimer. Docking was carried out by surFit (http://sysimm.ifrec.osaka-u.ac.jp/docking/main/) with restraints obtained from NMR data (Fig. 3a,b) as follows. NTD: R56, L58, G59, V86, K87, H88; PIN: V177, F210, T211, R214, F228, I229, V230, K231, L232, F234, D235, S236. Top-scoring model was selected. + + + METHODS + paragraph + 26996 + For docking IL-6 mRNA 3′UTR to the PIN dimer, each domain of the PIN dimer structure was superimposed onto the PIN dimer of the human X-ray structure (PDB ID: 3V34) in order to graft both water molecules and Mg2+ ions to the mouse model. Each IL-6 representative structure was submitted to the HADDOCK 2.0 server, for total of 10 independent jobs. In order to be consistent with the cleavage assay, active residues consisted of all nucleotides in RNA, Mg2+ and W182, R183, K184, R188, R214, R215, K219, R220, and R247 in the protein. Docked models were selected based on the following criteria: one heavy atom within 7, 8, or 9th nucleotide from the 5′ end was <5 Å from the Mg2+ ion on the primary PIN. Further classification was done manually in order to divide the selected models into two clusters. + + + METHODS + title_1 + 27806 + Additional Information + + + METHODS + paragraph + 27829 + Accession codes: The crystal structure of the Regnase-1 PIN domain has been deposited in the Protein Data Bank (accession codes: 5H9V (Form I) and 5H9W (Form II)). The chemical shift assignments of the NTD, the ZF domain, and the CTD have been deposited at Biological Magnetic Resonance Bank (accession codes: 25718, 25719, and 25720, respectively), and the coordinates for the ensemble have been deposited in the Protein Data Bank (accession codes: 2N5J, 2N5K, and 2N5L, respectively). + + + METHODS + paragraph + 28317 + How to cite this article: Yokogawa, M. et al. Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions. Sci. Rep. 6, 22324; doi: 10.1038/srep22324 (2016). + + + SUPPL + title_1 + 28516 + Supplementary Material + + + 783 + 801 + surname:Akira;given-names:S. + surname:Uematsu;given-names:S. + surname:Takeuchi;given-names:O. + 16497588 + REF + Cell, + ref + 124 + 2006 + 28539 + Pathogen recognition and innate immunity. + + + 819 + 26 + surname:Medzhitov;given-names:R. + 17943118 + REF + Nature + ref + 449 + 2007 + 28581 + Recognition of microorganisms and activation of the immune response + + + 353 + 89 + surname:Beutler;given-names:B. + 16551253 + REF + Annu Rev Immunol + ref + 24 + 2006 + 28649 + Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large + + + 1185 + 90 + surname:Matsushita;given-names:K. + 19322177 + REF + Nature + ref + 458 + 2009 + 28737 + Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay + + + 7386 + 99 + surname:Mizgalska;given-names:D. + 19909337 + REF + FEBS J + ref + 276 + 2009 + 28825 + Interleukin-1-inducible MCPIP protein has structural and functional properties of RNase and participates in degradation of IL-1beta mRNA + + + e49841 + surname:Li;given-names:M. + 23185455 + REF + PLoS One, + ref + 7 + 2012 + 28962 + MCPIP1 down-regulates IL-2 expression through an ARE-independent pathway + + + 708 + 13 + surname:Uehata;given-names:T. + surname:Akira;given-names:S. + 23500036 + REF + Biochim Biophys Acta + ref + 1829 + 2013 + 29035 + mRNA degradation by the endoribonuclease Regnase-1/ZC3H12a/MCPIP-1 + + + 1167 + 75 + surname:Iwasaki;given-names:H. + 22037600 + REF + Nat Immunol + ref + 12 + 2011 + 29102 + The IκB kinase complex regulates the stability of cytokine-encoding mRNA induced by TLR-IL-1R by controlling degradation of regnase-1 + + + 6337 + 46 + surname:Liang;given-names:J. + 18178554 + REF + J Biol Chem + ref + 283 + 2008 + 29240 + A novel CCCH-zinc finger protein family regulates proinflammatory activation of macrophages + + + 903 + 10 + surname:Xu;given-names:J. + surname:Fu;given-names:S. + surname:Peng;given-names:W. + surname:Rao;given-names:Z. + 23132255 + REF + Protein Cell + ref + 3 + 2012 + 29332 + MCP-1-induced protein-1, an immune regulator + + + 6957 + 65 + surname:Xu;given-names:J. + 22561375 + REF + Nucleic Acids Res + ref + 40 + 2012 + 29377 + Structural study of MCPIP1 N-terminal conserved domain reveals a PIN-like RNase + + + 685 + 93 + surname:Hake;given-names:L. E. + surname:Mendez;given-names:R. + surname:Richter;given-names:J. D. + 9447964 + REF + Mol Cell Biol + ref + 18 + 1998 + 29457 + Specificity of RNA binding by CPEB: requirement for RNA recognition motifs and a novel zinc finger + + + 9606 + 13 + surname:Lai;given-names:W. S. + surname:Kennington;given-names:E. A. + surname:Blackshear;given-names:P. J. + 11782475 + REF + J Biol Chem + ref + 277 + 2002 + 29556 + Interactions of CCCH zinc finger proteins with mRNA: non-binding tristetraprolin mutants exert an inhibitory effect on degradation of AU-rich element-containing mRNAs + + + 257 + 64 + surname:Hudson;given-names:B. P. + surname:Martinez-Yamout;given-names:M. A. + surname:Dyson;given-names:H. J. + surname:Wright;given-names:P. E. + 14981510 + REF + Nat Struct Mol Biol + ref + 11 + 2004 + 29723 + Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d + + + 367 + 73 + surname:Hall;given-names:T. M. + 15963892 + REF + Curr Opin Struct Biol + ref + 15 + 2005 + 29799 + Multiple modes of RNA recognition by zinc finger proteins + + + 1177 + 85 + surname:Zhou;given-names:L. + 16574901 + REF + Circ Res + ref + 98 + 2006 + 29857 + Monocyte chemoattractant protein-1 induces a novel transcription factor that causes cardiac myocyte apoptosis and ventricular dysfunction + + + 2959 + 73 + surname:Liang;given-names:J. + 21115689 + REF + J Exp Med + ref + 207 + 2010 + 29995 + MCP-induced protein 1 deubiquitinates TRAF proteins and negatively regulates JNK and NF-kappaB signaling + + + W545 + 9 + surname:Holm;given-names:L. + surname:Rosenström;given-names:P. + 20457744 + REF + Nucleic Acids Res + ref + 38 + 2010 + 30100 + Dali server: conservation mapping in 3D + + + 1036 + 49 + surname:Uehata;given-names:T. + 23706741 + REF + Cell + ref + 153 + 2013 + 30140 + Malt1-induced cleavage of regnase-1 in CD4(+) helper T cells regulates immune activation + + + 307 + 326 + surname:Otwinowski;given-names:Z. + surname:Minor;given-names:W. + REF + Methods Enzymol + ref + 276 + 1997 + 30229 + Processing of X-ray Diffraction Data Collected in Oscillation Mode + + + 849 + 61 + surname:Terwilliger;given-names:T. C. + surname:Berendzen;given-names:J. + 10089316 + REF + Acta Crystallogr D Biol Crystallogr + ref + 55 + 1999 + 30296 + Automated MAD and MIR structure solution + + + 965 + 72 + surname:Terwilliger;given-names:T. C. + 10944333 + REF + Acta Crystallogr D Biol Crystallogr + ref + 56 + 2000 + 30337 + Maximum-likelihood density modification + + + 905 + 21 + surname:Brünger;given-names:A. T. + 9757107 + REF + Acta Crystallogr D Biol Crystallogr + ref + 54 + 1998 + 30377 + Crystallography & NMR system: A new software suite for macromolecular structure determination + + + 486 + 501 + surname:Emsley;given-names:P. + surname:Lohkamp;given-names:B. + surname:Scott;given-names:W. G. + surname:Cowtan;given-names:K. + 20383002 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 30471 + Features and development of Coot + + + 240 + 55 + surname:Murshudov;given-names:G. N. + surname:Vagin;given-names:A. A. + surname:Dodson;given-names:E. J. + 15299926 + REF + Acta Crystallogr D Biol Crystallogr + ref + 53 + 1997 + 30504 + Refinement of macromolecular structures by the maximum-likelihood method + + + 235 + 42 + surname:Winn;given-names:M. D. + 21460441 + REF + Acta Crystallogr D Biol Crystallogr + ref + 67 + 2011 + 30577 + Overview of the CCP4 suite and current developments + + + 277 + 93 + surname:Delaglio;given-names:F. + 8520220 + REF + J Biomol NMR + ref + 6 + 1995 + 30629 + NMRPipe: a multidimensional spectral processing system based on UNIX pipes + + + 353 + 78 + surname:Guntert;given-names:P. + 15318003 + REF + Methods Mol Biol + ref + 278 + 2004 + 30704 + Automated NMR structure calculation with CYANA + + + 289 + 302 + surname:Cornilescu;given-names:G. + surname:Delaglio;given-names:F. + surname:Bax;given-names:A. + 10212987 + REF + J Biomol NMR + ref + 13 + 1999 + 30751 + Protein backbone angle restraints from searching a database for chemical shift and sequence homology + + + 2913 + 4 + surname:Liang;given-names:S. + surname:Zheng;given-names:D. + surname:Zhang;given-names:C. + surname:Standley;given-names:D. M. + 21873640 + REF + Bioinformatics + ref + 27 + 2011 + 30852 + Fast and accurate prediction of protein side-chain conformations + + + SUPPL + footnote + 30917 + Author Contributions F.I. supervised the overall project. M.Y., T.T., Y.E., D.M.S., O.T., S.A. and F.I. designed the research; M.Y. and T.T. performed the research; M.Y., T.T., N.N.N., H.K., K.Y., D.M.S. and F.I. analyzed the data; and M.Y., N.N.N., H.K., K.Y., D.M.S. and F.I. wrote the paper. All authors reviewed the manuscript. + + + srep22324-f1.jpg + f1 + FIG + fig_title_caption + 31249 + Structural and functional analyses of Regnase-1. + + 0.99234915 + experimental_method + cleaner0 + 2023-07-06T14:15:01Z + MESH: + + Structural and functional analyses + + + 0.9971781 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + + srep22324-f1.jpg + f1 + FIG + fig_caption + 31298 + (a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain. Catalytic Asp residues were shown in sticks. (d) Solution structure of the ZF domain. Three Cys residues and one His residue responsible for Zn2+-binding were shown in sticks. (e) Solution structure of the CTD. All the structures were colored in rainbow from N-terminus (blue) to C-terminus (red). (f) In vitro gel shift binding assay between Regnase-1 and IL-6 mRNA. Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1. (g) Binding of Regnase-1 and IL-6 mRNA was plotted. The percentage of the bound IL-6 was calculated based on the fluorescence intensities of the free IL-6 quantified in (f). (h) In vitro cleavage assay of Regnase-1 to IL-6 mRNA. Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1. + + 0.9957292 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + evidence + DUMMY: + cleaner0 + 2023-07-06T13:56:33Z + + Solution structure + + + 0.99858487 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.9967145 + evidence + cleaner0 + 2023-07-06T13:56:15Z + DUMMY: + + Crystal structure + + + 0.9985958 + structure_element + cleaner0 + 2023-07-06T12:55:51Z + SO: + + PIN + + + 0.7488216 + protein_state + cleaner0 + 2023-07-06T14:11:44Z + DUMMY: + + Catalytic + + + 0.98785895 + residue_name + cleaner0 + 2023-07-06T14:01:52Z + SO: + + Asp + + + evidence + DUMMY: + cleaner0 + 2023-07-06T13:56:33Z + + Solution structure + + + 0.99852425 + structure_element + cleaner0 + 2023-07-06T12:57:35Z + SO: + + ZF + + + 0.9902659 + residue_name + cleaner0 + 2023-07-06T14:01:55Z + SO: + + Cys + + + 0.9896445 + residue_name + cleaner0 + 2023-07-06T14:01:57Z + SO: + + His + + + evidence + DUMMY: + cleaner0 + 2023-07-06T13:56:33Z + + Solution structure + + + 0.99867463 + structure_element + cleaner0 + 2023-07-06T12:57:43Z + SO: + + CTD + + + 0.96567667 + evidence + cleaner0 + 2023-07-06T13:56:53Z + DUMMY: + + structures + + + 0.89467937 + experimental_method + cleaner0 + 2023-07-06T14:15:05Z + MESH: + + In vitro gel shift binding assay + + + 0.9954107 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:16Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:27Z + + mRNA + + + evidence + DUMMY: + cleaner0 + 2023-07-06T13:42:11Z + + Fluorescence intensity + + + 0.7841645 + protein_state + cleaner0 + 2023-07-06T14:11:48Z + DUMMY: + + free + + + 0.9748128 + protein_type + cleaner0 + 2023-07-06T12:54:16Z + MESH: + + IL-6 + + + 0.99419165 + protein_state + cleaner0 + 2023-07-06T13:49:31Z + DUMMY: + + absence of + + + 0.99360347 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.9940892 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:17Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:27Z + + mRNA + + + 0.9351158 + protein_type + cleaner0 + 2023-07-06T12:54:17Z + MESH: + + IL-6 + + + evidence + DUMMY: + cleaner0 + 2023-07-06T13:42:42Z + + fluorescence intensities + + + 0.9395353 + protein_type + cleaner0 + 2023-07-06T12:54:17Z + MESH: + + IL-6 + + + 0.9077164 + experimental_method + cleaner0 + 2023-07-06T14:15:08Z + MESH: + + In vitro cleavage assay + + + 0.99444556 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:17Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:27Z + + mRNA + + + evidence + DUMMY: + cleaner0 + 2023-07-06T13:42:11Z + + Fluorescence intensity + + + 0.96524453 + protein_state + cleaner0 + 2023-07-06T13:49:26Z + DUMMY: + + uncleaved + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:17Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:27Z + + mRNA + + + 0.9879551 + protein_state + cleaner0 + 2023-07-06T13:49:30Z + DUMMY: + + absence of + + + 0.9940297 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + + srep22324-f2.jpg + f2 + FIG + fig_title_caption + 32262 + Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1. + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:11:11Z + + Head-to-tail + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-06T13:11:19Z + + oligomer + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:51Z + + PIN + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:06Z + + RNase + + + 0.9969178 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + + srep22324-f2.jpg + f2 + FIG + fig_caption + 32360 + (a) Gel filtration analyses of the PIN domain. Elution volumes of the standard marker proteins were indicated by arrows at the upper part. (b) Dimer structure of the PIN domain. Two PIN molecules in the crystal were colored white and green, respectively. Catalytic residues and mutated residues were shown in sticks. Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed. + + 0.9257605 + experimental_method + cleaner0 + 2023-07-06T14:15:13Z + MESH: + + Gel filtration analyses + + + 0.9976031 + structure_element + cleaner0 + 2023-07-06T12:55:51Z + SO: + + PIN + + + 0.9937178 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + Dimer + + + 0.948103 + evidence + cleaner0 + 2023-07-06T13:57:00Z + DUMMY: + + structure + + + 0.99757046 + structure_element + cleaner0 + 2023-07-06T12:55:51Z + SO: + + PIN + + + 0.52628136 + structure_element + cleaner0 + 2023-07-06T12:55:51Z + SO: + + PIN + + + 0.99690825 + evidence + cleaner0 + 2023-07-06T13:57:03Z + DUMMY: + + crystal + + + 0.79562724 + site + cleaner0 + 2023-07-06T14:04:43Z + SO: + + Catalytic residues + + + 0.99900573 + residue_name_number + cleaner0 + 2023-07-06T13:59:22Z + DUMMY: + + R215 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:06Z + + RNase + + + 0.96012145 + oligomeric_state + cleaner0 + 2023-07-06T13:54:30Z + DUMMY: + + monomeric + + + 0.5150391 + protein_state + cleaner0 + 2023-07-06T13:20:01Z + DUMMY: + + mutants + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:17Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:27Z + + mRNA + + + + srep22324-f3.jpg + f3 + FIG + fig_title_caption + 32894 + Domain-domain interaction between the NTD and the PIN domain. + + 0.99806803 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99878544 + structure_element + cleaner0 + 2023-07-06T12:55:51Z + SO: + + PIN + + + + srep22324-f3.jpg + f3 + FIG + fig_caption + 32956 + (a) NMR analyses of the NTD-binding to the PIN domain. The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red on the surface and ribbon structure of the PIN domain (right). Pro and the residues without analysis were colored black and gray, respectively. (b) NMR analyses of the PIN-binding to the NTD. The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red, yellow, or green on the surface and ribbon structure of the NTD. S62 was colored gray and excluded from the analysis, due to low signal intensity. (c) Docking model of the NTD and the PIN domain. The NTD and the PIN domain are shown in cyan and white, respectively. Residues in close proximity (<5 Å) to each other in the docking structure were colored yellow. Catalytic residues of the PIN domain are shown in sticks, and the residues that exhibited significant chemical shift changes in (a,b) were labeled. + + experimental_method + MESH: + cleaner0 + 2023-07-06T13:57:49Z + + NMR analyses + + + 0.390678 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:51Z + + PIN + + + 0.51126844 + experimental_method + cleaner0 + 2023-07-06T14:15:18Z + MESH: + + overlaid + + + 0.6816016 + evidence + cleaner0 + 2023-07-06T13:57:09Z + DUMMY: + + spectra + + + 0.99837154 + structure_element + cleaner0 + 2023-07-06T12:55:51Z + SO: + + PIN + + + 0.9659381 + residue_name + cleaner0 + 2023-07-06T14:02:05Z + SO: + + Pro + + + experimental_method + MESH: + cleaner0 + 2023-07-06T13:57:50Z + + NMR analyses + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:51Z + + PIN + + + 0.9985783 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + evidence + DUMMY: + cleaner0 + 2023-07-06T14:05:19Z + + significant chemical shift changes + + + experimental_method + MESH: + cleaner0 + 2023-07-06T13:57:27Z + + overlaid + + + 0.6049998 + evidence + cleaner0 + 2023-07-06T13:57:14Z + DUMMY: + + spectra + + + 0.99854714 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99872404 + residue_name_number + cleaner0 + 2023-07-06T13:59:27Z + DUMMY: + + S62 + + + 0.99856657 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.9984694 + structure_element + cleaner0 + 2023-07-06T12:55:51Z + SO: + + PIN + + + 0.99852055 + structure_element + cleaner0 + 2023-07-06T12:55:58Z + SO: + + NTD + + + 0.99842215 + structure_element + cleaner0 + 2023-07-06T12:55:51Z + SO: + + PIN + + + 0.87693775 + evidence + cleaner0 + 2023-07-06T13:58:03Z + DUMMY: + + docking structure + + + 0.68959475 + site + cleaner0 + 2023-07-06T14:04:47Z + SO: + + Catalytic residues + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:51Z + + PIN + + + evidence + DUMMY: + cleaner0 + 2023-07-06T14:05:03Z + + significant chemical shift changes + + + + srep22324-f4.jpg + f4 + FIG + fig_title_caption + 33942 + Critical residues in the PIN domain for the RNase activity of Regnase-1. + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:51Z + + PIN + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:06Z + + RNase + + + 0.9968206 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + + srep22324-f4.jpg + f4 + FIG + fig_caption + 34015 + (a) In vitro cleavage assay of basic residue mutants for IL-6 mRNA. The results indicate mean ± SD of four independent experiments. (b) +In vitro cleavage assay of basic residue mutants for Regnase-1 mRNA. The results indicate mean ± SD of three independent experiments. The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure. Mutated basic residues were shown in sticks and those with significantly reduced RNase activities were colored red or yellow. + + 0.9900084 + experimental_method + cleaner0 + 2023-07-06T14:15:23Z + MESH: + + In vitro cleavage assay + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:17Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:27Z + + mRNA + + + 0.9858886 + experimental_method + cleaner0 + 2023-07-06T14:15:26Z + MESH: + + In vitro cleavage assay + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + 0.59535795 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + 0.65554714 + chemical + cleaner0 + 2023-07-06T13:00:27Z + CHEBI: + + mRNA + + + evidence + DUMMY: + cleaner0 + 2023-07-06T13:42:11Z + + fluorescence intensity + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:49:27Z + + uncleaved + + + 0.997189 + chemical + cleaner0 + 2023-07-06T13:00:27Z + CHEBI: + + mRNA + + + 0.89147466 + structure_element + cleaner0 + 2023-07-06T12:55:51Z + SO: + + PIN + + + 0.9949576 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + 0.99110913 + evidence + cleaner0 + 2023-07-06T13:58:10Z + DUMMY: + + structure + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:06Z + + RNase + + + + srep22324-f5.jpg + f5 + FIG + fig_title_caption + 34541 + Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity. + + 0.9645019 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:20:01Z + + mutants + + + 0.9961874 + mutant + cleaner0 + 2023-07-06T13:08:10Z + MESH: + + DDNN + + + 0.6436507 + protein_state + cleaner0 + 2023-07-06T13:19:23Z + DUMMY: + + mutant + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:06Z + + RNase + + + + srep22324-f5.jpg + f5 + FIG + fig_caption + 34666 + (a) Cartoon representation of the concept of the experiment. (b) In vitro cleavage assay of Regnase-1 for IL-6 mRNA. (c) In vitro cleavage assay of Regnase-1 for Regnase-1 mRNA. The results indicate mean ± SD of three independent experiments. The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer. The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN. The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN. + + 0.98775005 + experimental_method + cleaner0 + 2023-07-06T14:15:32Z + MESH: + + In vitro cleavage assay + + + 0.99594265 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:54:17Z + + IL-6 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:27Z + + mRNA + + + 0.98873675 + experimental_method + cleaner0 + 2023-07-06T14:15:35Z + MESH: + + In vitro cleavage assay + + + 0.9957145 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + protein + PR: + cleaner0 + 2023-07-06T12:55:00Z + + Regnase-1 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T13:00:27Z + + mRNA + + + evidence + DUMMY: + cleaner0 + 2023-07-06T13:42:11Z + + fluorescence intensity + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:49:27Z + + uncleaved + + + 0.99734384 + chemical + cleaner0 + 2023-07-06T13:00:27Z + CHEBI: + + mRNA + + + 0.9837165 + structure_element + cleaner0 + 2023-07-06T12:55:51Z + SO: + + PIN + + + 0.9794327 + oligomeric_state + cleaner0 + 2023-07-06T13:11:23Z + DUMMY: + + dimer + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:06Z + + RNase + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:50:30Z + + presence of + + + mutant + MESH: + cleaner0 + 2023-07-06T13:08:10Z + + DDNN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:19:23Z + + mutant + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:51Z + + PIN + + + protein_type + MESH: + cleaner0 + 2023-07-06T12:55:06Z + + RNase + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:50:31Z + + presence of + + + mutant + MESH: + cleaner0 + 2023-07-06T13:08:10Z + + DDNN + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T13:19:23Z + + mutant + + + structure_element + SO: + cleaner0 + 2023-07-06T12:55:51Z + + PIN + + + + srep22324-f6.jpg + f6 + FIG + fig_title_caption + 35284 + Schematic representation of regulation of the Regnase-1 catalytic activity through the domain-domain interactions. + + 0.99573964 + protein + cleaner0 + 2023-07-06T12:55:00Z + PR: + + Regnase-1 + + + + diff --git a/BioC_XML/4773095_v0.xml b/BioC_XML/4773095_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..65eacbfa591ddc5c7164ab3e34b48c09606af28b --- /dev/null +++ b/BioC_XML/4773095_v0.xml @@ -0,0 +1,13760 @@ + + + + PMC + 20201223 + pmc.key + + 4773095 + CC BY + no + 0 + 0 + + Structure of the Lantibiotic Resistance Response Regulator + 10.1371/journal.pone.0149903 + 4773095 + 26930060 + PONE-D-15-49972 + e0149903 + 3 + This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + surname:Khosa;given-names:Sakshi + surname:Hoeppner;given-names:Astrid + surname:Gohlke;given-names:Holger + surname:Schmitt;given-names:Lutz + surname:Smits;given-names:Sander H. J. + surname:Cascales;given-names:Eric + All relevant data are within the paper and its Supporting Information files. + TITLE + Data Availability + front + 11 + 2016 + 0 + Structure of the Response Regulator NsrR from Streptococcus agalactiae, Which Is Involved in Lantibiotic Resistance + + 0.9945463 + evidence + cleaner0 + 2023-07-27T14:51:22Z + DUMMY: + + Structure + + + 0.998559 + protein_type + cleaner0 + 2023-07-27T12:47:37Z + MESH: + + Response Regulator + + + 0.9993967 + protein + cleaner0 + 2023-07-27T12:47:43Z + PR: + + NsrR + + + 0.9984838 + species + cleaner0 + 2023-07-27T12:47:50Z + MESH: + + Streptococcus agalactiae + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:52:04Z + + Lantibiotic + + + + ABSTRACT + abstract + 116 + Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria. Interestingly, several clinically relevant and human pathogenic strains are inherently resistant towards lantibiotics. The expression of the genes responsible for lantibiotic resistance is regulated by a specific two-component system consisting of a histidine kinase and a response regulator. Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. The C-terminal domain exhibits a fold that classifies NsrR as a member of the OmpR/PhoB subfamily of regulators. Amino acids involved in phosphorylation, dimerization, and DNA-binding were identified and demonstrated to be conserved in lantibiotic resistance regulators. Finally, a model of the full-length NsrR in the active and inactive state provides insights into protein dimerization and DNA-binding. + + 0.99717027 + chemical + cleaner0 + 2023-07-27T12:48:04Z + CHEBI: + + Lantibiotics + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:48:28Z + + antimicrobial peptides + + + 0.97839725 + taxonomy_domain + cleaner0 + 2023-07-27T12:48:34Z + DUMMY: + + Gram-positive bacteria + + + 0.9982835 + species + cleaner0 + 2023-07-27T12:48:39Z + MESH: + + human + + + 0.99746597 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + lantibiotics + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:52:04Z + + lantibiotic + + + complex_assembly + GO: + cleaner0 + 2023-07-27T12:57:48Z + + two-component system + + + 0.9987494 + protein_type + cleaner0 + 2023-07-27T12:48:45Z + MESH: + + histidine kinase + + + 0.99863535 + protein_type + cleaner0 + 2023-07-27T12:47:38Z + MESH: + + response regulator + + + 0.998608 + protein_type + cleaner0 + 2023-07-27T12:47:38Z + MESH: + + response regulator + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:52:04Z + + lantibiotic + + + 0.9994099 + protein + cleaner0 + 2023-07-27T12:47:43Z + PR: + + NsrR + + + 0.9986101 + species + cleaner0 + 2023-07-27T12:47:51Z + MESH: + + Streptococcus agalactiae + + + 0.99849606 + evidence + cleaner0 + 2023-07-27T14:51:26Z + DUMMY: + + crystal structures + + + 0.9992875 + structure_element + cleaner0 + 2023-07-27T12:48:55Z + SO: + + receiver domain + + + 0.9989768 + structure_element + cleaner0 + 2023-07-27T12:49:01Z + SO: + + DNA-binding effector domain + + + 0.9987072 + structure_element + cleaner0 + 2023-07-27T12:49:11Z + SO: + + C-terminal domain + + + 0.9993938 + protein + cleaner0 + 2023-07-27T12:47:43Z + PR: + + NsrR + + + protein_type + MESH: + cleaner0 + 2023-07-27T12:49:40Z + + OmpR/PhoB subfamily + + + 0.8666049 + ptm + cleaner0 + 2023-07-27T12:50:11Z + MESH: + + phosphorylation + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:49:57Z + + DNA + + + 0.9957343 + protein_state + cleaner0 + 2023-07-27T14:23:03Z + DUMMY: + + conserved + + + 0.99867386 + protein_type + cleaner0 + 2023-07-27T12:49:44Z + MESH: + + lantibiotic resistance regulators + + + 0.99908096 + protein_state + cleaner0 + 2023-07-27T12:50:18Z + DUMMY: + + full-length + + + 0.9994062 + protein + cleaner0 + 2023-07-27T12:47:43Z + PR: + + NsrR + + + 0.99904615 + protein_state + cleaner0 + 2023-07-27T12:50:24Z + DUMMY: + + active + + + 0.99918455 + protein_state + cleaner0 + 2023-07-27T12:50:30Z + DUMMY: + + inactive + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:49:58Z + + DNA + + + + INTRO + title_1 + 1120 + Introduction + + + INTRO + paragraph + 1133 + The dramatic rise in antibiotic resistance has posed a major threat to the treatment of infectious diseases. This has led to the search for novel antibiotics that can be used as pharmaceuticals against human pathogenic bacteria. One of the potential antibiotic alternatives are lantibiotics. Lantibiotics are small antimicrobial peptides (30–50 amino acids in length), which are produced by several Gram-positive bacterial strains. They are post-translationally modified and contain specific lanthionine/methyl-lanthionine rings, which are crucial for their high antimicrobial activity. Lantibiotics are for example highly effective against various Gram-positive, human pathogenic bacteria including Streptococcus pneumoniae and several methicillin-resistant Staphylococcus aureus (MRSA) strains. The high potency of lantibiotics for medical usage has already been noticed, and several lantibiotics are already included in clinical trials. Their high potency is highlighted by the fact that, although being extensively used in food industry, resistance has not been described so far. Nisin is the most prominent member of the lantibiotic family and is able to inhibit cell growth, penetrates the membranes of various Gram-positive bacteria, and is characterized by five specific (methyl-)lanthionine rings, which are crucial for stability and activity in the nanomolar range. Thus, the lantibiotic producer strains have an inbuilt self-protection mechanism (immunity) to prevent cell death caused due to the action of its cognate lantibiotic. This immunity system consists of a membrane–associated lipoprotein (usually referred to as LanI) and/or an ABC transporter (termed as LanFEG and comprising three subunits). Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. Examples for LanFEG are NisI and NisFEG of the nisin system, SpaI and SpaFEG conferring immunity towards subtilin, and PepI constituting the immunity system of Pep5 producing strains. Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024. + + 0.9987451 + species + cleaner0 + 2023-07-27T12:48:40Z + MESH: + + human + + + 0.9874534 + taxonomy_domain + cleaner0 + 2023-07-27T12:51:04Z + DUMMY: + + bacteria + + + 0.9956435 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + lantibiotics + + + 0.99147624 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + Lantibiotics + + + 0.9319806 + chemical + cleaner0 + 2023-07-27T12:48:29Z + CHEBI: + + antimicrobial peptides + + + 0.9792487 + taxonomy_domain + cleaner0 + 2023-07-27T14:20:56Z + DUMMY: + + Gram-positive bacterial + + + 0.9981494 + chemical + cleaner0 + 2023-07-27T12:52:10Z + CHEBI: + + lanthionine + + + 0.9968582 + chemical + cleaner0 + 2023-07-27T12:52:25Z + CHEBI: + + methyl-lanthionine + + + 0.99280035 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + Lantibiotics + + + 0.9802711 + taxonomy_domain + cleaner0 + 2023-07-27T14:20:59Z + DUMMY: + + Gram-positive + + + 0.997926 + species + cleaner0 + 2023-07-27T12:48:40Z + MESH: + + human + + + 0.6351519 + taxonomy_domain + cleaner0 + 2023-07-27T12:51:05Z + DUMMY: + + bacteria + + + 0.9978671 + species + cleaner0 + 2023-07-27T14:26:51Z + MESH: + + Streptococcus pneumoniae + + + 0.95391035 + species + cleaner0 + 2023-07-27T14:26:59Z + MESH: + + methicillin-resistant Staphylococcus aureus + + + 0.9970284 + species + cleaner0 + 2023-07-27T14:27:03Z + MESH: + + MRSA + + + 0.9952632 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + lantibiotics + + + 0.9958437 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + lantibiotics + + + 0.9949949 + chemical + cleaner0 + 2023-07-27T12:51:59Z + CHEBI: + + Nisin + + + 0.9946043 + chemical + cleaner0 + 2023-07-27T12:52:04Z + CHEBI: + + lantibiotic + + + 0.9794172 + taxonomy_domain + cleaner0 + 2023-07-27T12:48:34Z + DUMMY: + + Gram-positive bacteria + + + 0.9782201 + chemical + cleaner0 + 2023-07-27T12:52:04Z + CHEBI: + + lantibiotic + + + 0.9982658 + chemical + cleaner0 + 2023-07-27T12:52:04Z + CHEBI: + + lantibiotic + + + 0.92543674 + protein_type + cleaner0 + 2023-07-27T12:51:28Z + MESH: + + membrane–associated lipoprotein + + + 0.9788287 + protein_type + cleaner0 + 2023-07-27T12:54:45Z + MESH: + + LanI + + + 0.9981523 + protein_type + cleaner0 + 2023-07-27T12:51:33Z + MESH: + + ABC transporter + + + 0.9421355 + protein_type + cleaner0 + 2023-07-27T12:54:56Z + MESH: + + LanFEG + + + 0.9975822 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + lantibiotics + + + 0.9969531 + chemical + cleaner0 + 2023-07-27T12:52:55Z + CHEBI: + + Pep5 + + + 0.9987821 + chemical + cleaner0 + 2023-07-27T12:53:00Z + CHEBI: + + epicidin + + + 0.99894005 + chemical + cleaner0 + 2023-07-27T12:53:05Z + CHEBI: + + epilancin + + + 0.9979113 + chemical + cleaner0 + 2023-07-27T12:53:10Z + CHEBI: + + lactocin S + + + 0.9869429 + protein_type 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2023-07-27T12:54:25Z + PR: + + SpaI + + + 0.99916637 + protein + cleaner0 + 2023-07-27T12:54:30Z + PR: + + SpaFEG + + + 0.99042773 + chemical + cleaner0 + 2023-07-27T12:53:31Z + CHEBI: + + subtilin + + + 0.9991824 + protein + cleaner0 + 2023-07-27T12:55:05Z + PR: + + PepI + + + 0.9694549 + chemical + cleaner0 + 2023-07-27T12:52:56Z + CHEBI: + + Pep5 + + + 0.6991327 + evidence + cleaner0 + 2023-07-27T14:51:33Z + DUMMY: + + Structural data + + + 0.8691757 + protein_type + cleaner0 + 2023-07-27T14:58:00Z + MESH: + + immunity proteins + + + 0.99902976 + protein + cleaner0 + 2023-07-27T12:54:13Z + PR: + + NisI + + + 0.9983518 + species + cleaner0 + 2023-07-27T12:55:14Z + MESH: + + Lactococcus lactis + + + 0.9991961 + protein + cleaner0 + 2023-07-27T12:54:26Z + PR: + + SpaI + + + 0.99803007 + species + cleaner0 + 2023-07-27T12:55:19Z + MESH: + + Bacillus subtilis + + + 0.99903035 + protein + cleaner0 + 2023-07-27T12:55:24Z + PR: + + MlbQ + + + 0.9953537 + chemical + cleaner0 + 2023-07-27T12:52:04Z + CHEBI: + + lantibiotic + + + 0.9741191 + chemical + cleaner0 + 2023-07-27T12:55:31Z + CHEBI: + + NAI-107 + + + 0.7365977 + species + cleaner0 + 2023-07-27T12:55:37Z + MESH: + + Microbispora ATCC PTA-5024 + + + + INTRO + paragraph + 3545 + Recently, gene clusters were identified in certain clinically relevant human pathogenic strains such as Streptococcus agalactiae, S. aureus, and others that confer inherent resistance against specific lantibiotics such as nisin and resemble the genetic architecture of the lantibiotic immunity genes found in the producing strains. Within these resistance operons, genes encoding for a membrane-associated protease and an ABC transporter were identified. Expression of these proteins provides resistance against lantibiotics. Recently, the structure of SaNSR from S. agalactiae was solved which provides resistance against nisin by a protease activity. Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. Bacteria have the ability to sense and survive various environmental stimuli through adaptive responses, which are regulated by TCSs. These processes include drug resistance, quorum-sensing, phosphate uptake, sporulation, and osmoregulation. The absence of TCSs within mammals makes them unique targets for novel antimicrobial drugs. + + 0.99886096 + species + cleaner0 + 2023-07-27T12:48:40Z + MESH: + + human + + + 0.9984276 + species + cleaner0 + 2023-07-27T12:47:51Z + MESH: + + Streptococcus agalactiae + + + 0.9982524 + species + cleaner0 + 2023-07-27T12:55:50Z + MESH: + + S. aureus + + + 0.99864274 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + lantibiotics + + + 0.9991204 + chemical + cleaner0 + 2023-07-27T12:52:00Z + CHEBI: + + nisin + + + 0.9943542 + protein_type + cleaner0 + 2023-07-27T12:56:19Z + MESH: + + membrane-associated protease + + + 0.99774075 + protein_type + cleaner0 + 2023-07-27T12:51:33Z + MESH: + + ABC transporter + + + 0.99697626 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + lantibiotics + + + 0.99493057 + evidence + cleaner0 + 2023-07-27T14:51:39Z + DUMMY: + + structure + + + 0.99870455 + protein + cleaner0 + 2023-07-27T12:56:31Z + PR: + + SaNSR + + + 0.9984689 + species + cleaner0 + 2023-07-27T12:56:38Z + MESH: + + S. agalactiae + + + 0.9989241 + chemical + cleaner0 + 2023-07-27T12:52:00Z + CHEBI: + + nisin + + + complex_assembly + GO: + cleaner0 + 2023-07-27T12:57:48Z + + two-component system + + + 0.7977292 + complex_assembly + cleaner0 + 2023-07-27T12:57:55Z + GO: + + TCS + + + 0.532767 + chemical + cleaner0 + 2023-07-27T12:52:04Z + CHEBI: + + lantibiotic + + + protein_type + MESH: + cleaner0 + 2023-07-27T12:48:45Z + + histidine kinase + + + 0.99778545 + protein_type + cleaner0 + 2023-07-27T12:58:38Z + MESH: + + HK + + + 0.9968966 + protein_type + cleaner0 + 2023-07-27T12:47:38Z + MESH: + + response regulator + + + 0.9954798 + protein_type + cleaner0 + 2023-07-27T12:58:45Z + MESH: + + RR + + + 0.998307 + protein_type + cleaner0 + 2023-07-27T12:58:39Z + MESH: + + HK + + + 0.9990539 + chemical + cleaner0 + 2023-07-27T12:52:04Z + CHEBI: + + lantibiotic + + + 0.6605025 + ptm + cleaner0 + 2023-07-27T12:58:52Z + MESH: + + auto-phosphorylates + + + 0.9977049 + protein_state + cleaner0 + 2023-07-27T12:59:07Z + DUMMY: + + conserved + + + 0.99674785 + residue_name + cleaner0 + 2023-07-27T14:08:48Z + SO: + + histidine + + + 0.99574405 + protein_type + cleaner0 + 2023-07-27T12:58:46Z + MESH: + + RR + + + 0.99509084 + protein_type + cleaner0 + 2023-07-27T12:58:46Z + MESH: + + RR + + + 0.99866474 + taxonomy_domain + cleaner0 + 2023-07-27T12:51:05Z + DUMMY: + + Bacteria + + + 0.965592 + complex_assembly + cleaner0 + 2023-07-27T12:58:13Z + GO: + + TCSs + + + 0.99651206 + protein_state + cleaner0 + 2023-07-27T12:59:03Z + DUMMY: + + absence of + + + 0.9872185 + complex_assembly + cleaner0 + 2023-07-27T12:58:14Z + GO: + + TCSs + + + 0.9981237 + taxonomy_domain + cleaner0 + 2023-07-27T12:58:58Z + DUMMY: + + mammals + + + + INTRO + paragraph + 5141 + The expression of the lantibiotic-resistance genes via TCS is generally regulated by microorganism-specific lantibiotics, which act via external stimuli. Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples. Interestingly, the histidine kinase contains two-transmembrane helices but lacks an extracellular sensory domain, and are therefore known as ‘intramembrane-sensing’ histidine kinases. It has been suggested that in addition to conferring general resistance against lantibiotics, the BceAB-type transporters assist in signalling as via the presence of a large extracellular domain within the transmembrane segment indicated by experimental evidence from various systems. + + 0.668119 + chemical + cleaner0 + 2023-07-27T12:52:04Z + CHEBI: + + lantibiotic + + + 0.35961458 + complex_assembly + cleaner0 + 2023-07-27T12:57:55Z + GO: + + TCS + + + 0.9985266 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + lantibiotics + + + 0.5892956 + complex_assembly + cleaner0 + 2023-07-27T12:57:55Z + GO: + + TCS + + + 0.9971806 + protein + cleaner0 + 2023-07-27T12:59:44Z + PR: + + BraRS + + + 0.99837446 + species + cleaner0 + 2023-07-27T12:55:51Z + MESH: + + S. aureus + + + 0.99902904 + chemical + cleaner0 + 2023-07-27T14:27:20Z + CHEBI: + + bacitracin + + + 0.9989477 + chemical + cleaner0 + 2023-07-27T12:52:00Z + CHEBI: + + nisin + + + 0.9878618 + chemical + cleaner0 + 2023-07-27T13:01:00Z + CHEBI: + + nukacin-ISK-1 + + + 0.9742468 + protein + cleaner0 + 2023-07-27T12:59:50Z + PR: + + BceRS + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-27T13:00:06Z + + Bacillus spp + + + 0.9970746 + chemical + cleaner0 + 2023-07-27T14:27:24Z + CHEBI: + + actagardine + + + 0.99615234 + chemical + cleaner0 + 2023-07-27T14:27:27Z + CHEBI: + + mersacidin + + + 0.99828523 + protein + cleaner0 + 2023-07-27T13:00:14Z + PR: + + LcrRS + + + 0.9982466 + species + cleaner0 + 2023-07-27T14:27:08Z + MESH: + + Streptococcus mutans + + + 0.9896021 + chemical + cleaner0 + 2023-07-27T13:01:00Z + CHEBI: + + nukacin-ISK-1 + + + 0.9980278 + chemical + cleaner0 + 2023-07-27T13:01:06Z + CHEBI: + + lacticin 481 + + + 0.998494 + protein + cleaner0 + 2023-07-27T13:00:20Z + PR: + + LisRK + + + 0.998415 + species + cleaner0 + 2023-07-27T13:00:31Z + MESH: + + Listeria monocytogenes + + + 0.99782085 + chemical + cleaner0 + 2023-07-27T12:52:00Z + CHEBI: + + nisin + + + 0.9961661 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + lantibiotics + + + 0.6338489 + complex_assembly + cleaner0 + 2023-07-27T12:57:55Z + GO: + + TCS + + + 0.7201387 + protein + cleaner0 + 2023-07-27T13:00:25Z + PR: + + CprRK + + + 0.99792933 + species + cleaner0 + 2023-07-27T13:00:37Z + MESH: + + Clostridium difficile + + + gene + GENE: + cleaner0 + 2023-07-27T14:09:07Z + + cpr + + + 0.9980063 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + lantibiotics + + + 0.99929786 + chemical + cleaner0 + 2023-07-27T12:52:00Z + CHEBI: + + nisin + + + 0.9993292 + chemical + cleaner0 + 2023-07-27T12:53:40Z + CHEBI: + + gallidermin + + + 0.99928635 + chemical + cleaner0 + 2023-07-27T12:53:31Z + CHEBI: + + subtilin + + + 0.99885315 + chemical + cleaner0 + 2023-07-27T13:00:43Z + CHEBI: + + mutacin 1140 + + + 0.9988773 + protein_type + cleaner0 + 2023-07-27T12:48:45Z + MESH: + + histidine kinase + + + 0.9979497 + structure_element + cleaner0 + 2023-07-27T14:46:05Z + SO: + + two-transmembrane helices + + + 0.998926 + structure_element + cleaner0 + 2023-07-27T14:46:10Z + SO: + + extracellular sensory domain + + + protein_type + MESH: + cleaner0 + 2023-07-27T14:58:28Z + + ‘intramembrane-sensing’ histidine kinases + + + 0.9964479 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + lantibiotics + + + 0.9987182 + protein_type + cleaner0 + 2023-07-27T13:01:28Z + MESH: + + BceAB-type transporters + + + 0.9970465 + structure_element + cleaner0 + 2023-07-27T14:46:13Z + SO: + + extracellular domain + + + 0.9991809 + structure_element + cleaner0 + 2023-07-27T14:46:16Z + SO: + + transmembrane segment + + + + INTRO + paragraph + 6383 + The recently discovered nsr gene cluster of the human pathogen S. agalactiae encodes for the resistance protein NSR and the ABC transporter NsrFP, both conferring resistance against nisin. Homologous operons have been identified in various human pathogenic strains such as Staphylococcus epidermis and Streptococcus ictaluri based on the high sequence identity of NSR and NsrFP. In this gene cluster, the TCS NsrRK is responsible for the expression of the nsr and nsrFP genes. The similarity of the TCS within all the described nisin resistance operons suggests an expression specifically induced by nisin. Thus, NsrRK might be a useful target to combat inherently pathogenic lantibiotic-resistant strains. + + gene + GENE: + cleaner0 + 2023-07-27T13:02:41Z + + nsr + + + 0.9989058 + species + cleaner0 + 2023-07-27T12:48:40Z + MESH: + + human + + + 0.99860716 + species + cleaner0 + 2023-07-27T12:56:39Z + MESH: + + S. agalactiae + + + 0.99846685 + protein_type + cleaner0 + 2023-07-27T14:58:33Z + MESH: + + resistance protein + + + 0.99936527 + protein + cleaner0 + 2023-07-27T15:00:30Z + PR: + + NSR + + + 0.997722 + protein_type + cleaner0 + 2023-07-27T12:51:33Z + MESH: + + ABC transporter + + + 0.9993332 + protein + cleaner0 + 2023-07-27T15:00:43Z + PR: + + NsrFP + + + 0.99881834 + chemical + cleaner0 + 2023-07-27T12:52:00Z + CHEBI: + + nisin + + + 0.9989549 + species + cleaner0 + 2023-07-27T12:48:40Z + MESH: + + human + + + 0.99869466 + species + cleaner0 + 2023-07-27T13:03:08Z + MESH: + + Staphylococcus epidermis + + + 0.9987157 + species + cleaner0 + 2023-07-27T13:03:14Z + MESH: + + Streptococcus ictaluri + + + 0.9989485 + protein + cleaner0 + 2023-07-27T15:00:26Z + PR: + + NSR + + + 0.99765635 + protein + cleaner0 + 2023-07-27T15:00:35Z + PR: + + NsrFP + + + 0.97538346 + complex_assembly + cleaner0 + 2023-07-27T12:57:55Z + GO: + + TCS + + + 0.9808241 + protein + cleaner0 + 2023-07-27T14:10:58Z + PR: + + NsrRK + + + 0.9584849 + gene + cleaner0 + 2023-07-27T13:02:41Z + GENE: + + nsr + + + 0.902056 + gene + cleaner0 + 2023-07-27T13:02:49Z + GENE: + + nsrFP + + + 0.8232556 + complex_assembly + cleaner0 + 2023-07-27T12:57:55Z + GO: + + TCS + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:52:00Z + + nisin + + + 0.9988973 + chemical + cleaner0 + 2023-07-27T12:52:00Z + CHEBI: + + nisin + + + 0.9986237 + protein + cleaner0 + 2023-07-27T14:10:59Z + PR: + + NsrRK + + + 0.9980398 + chemical + cleaner0 + 2023-07-27T12:52:04Z + CHEBI: + + lantibiotic + + + + INTRO + paragraph + 7090 + Generally, RRs consist of two distinct structural domains, a receiver domain (RD) and an effector domain (ED), that are separated from each other by a flexible linker. RDs contain a highly conserved aspartate residue, which acts as a phosphoryl acceptor that becomes phosphorylated by the kinase domain of the histidine kinase upon reception of an external signal. The ED is thereby activated and binds to the designated promoters, thus initiating transcription of the target genes. + + 0.94559 + protein_type + cleaner0 + 2023-07-27T13:02:15Z + MESH: + + RRs + + + 0.99930763 + structure_element + cleaner0 + 2023-07-27T12:48:55Z + SO: + + receiver domain + + + 0.99952686 + structure_element + cleaner0 + 2023-07-27T13:01:57Z + SO: + + RD + + + 0.9992855 + structure_element + cleaner0 + 2023-07-27T13:02:02Z + SO: + + effector domain + + + 0.9994966 + structure_element + cleaner0 + 2023-07-27T13:02:08Z + SO: + + ED + + + 0.38903397 + protein_state + cleaner0 + 2023-07-27T14:46:31Z + DUMMY: + + flexible + + + 0.6832957 + structure_element + cleaner0 + 2023-07-27T14:46:36Z + SO: + + linker + + + 0.99942696 + structure_element + cleaner0 + 2023-07-27T13:34:52Z + SO: + + RDs + + + 0.9986931 + protein_state + cleaner0 + 2023-07-27T14:23:30Z + DUMMY: + + highly conserved + + + 0.9979576 + residue_name + cleaner0 + 2023-07-27T13:26:45Z + SO: + + aspartate + + + 0.5846595 + protein_state + cleaner0 + 2023-07-27T14:22:49Z + DUMMY: + + phosphorylated + + + 0.9991957 + structure_element + cleaner0 + 2023-07-27T14:46:40Z + SO: + + kinase domain + + + 0.99852765 + protein_type + cleaner0 + 2023-07-27T12:48:45Z + MESH: + + histidine kinase + + + 0.9995159 + structure_element + cleaner0 + 2023-07-27T13:02:08Z + SO: + + ED + + + + INTRO + paragraph + 7573 + The RRs are classified into different subfamilies depending on the three-dimensional structure of their EDs. The OmpR/PhoB subfamily is the largest subgroup of RRs and comprises approximately 40% of all response regulators in bacteria. All their members are characterized by a winged helix-turn-helix (wHTH) motif. Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). The various structures of RRs reveal that in addition to being in either “inactive” or “active” state, the RRs can also exist in two distinct conformations: “open” and “closed”. MtrA and PrrA exhibit a very compact, closed structure with the DNA-binding sequence, called recognition helix, of the ED being inaccessible to DNA. The structures of DrrD and DrrB exist in an open conformation, here the recognition helix is fully exposed, suggesting that RRs are flexible in solution and can adopt multiple conformations. + + 0.99907553 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.9992859 + structure_element + cleaner0 + 2023-07-27T14:46:45Z + SO: + + EDs + + + 0.8960476 + protein_type + cleaner0 + 2023-07-27T13:03:41Z + MESH: + + OmpR/PhoB subfamily + + + 0.99917954 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.99859655 + protein_type + cleaner0 + 2023-07-27T13:03:36Z + MESH: + + response regulators + + + 0.9985084 + taxonomy_domain + cleaner0 + 2023-07-27T12:51:05Z + DUMMY: + + bacteria + + + 0.99917966 + structure_element + cleaner0 + 2023-07-27T13:04:08Z + SO: + + winged helix-turn-helix + + + 0.99807054 + structure_element + cleaner0 + 2023-07-27T13:04:13Z + SO: + + wHTH + + + 0.9970993 + evidence + cleaner0 + 2023-07-27T14:51:45Z + DUMMY: + + structures + + + 0.9978765 + evidence + cleaner0 + 2023-07-27T14:51:53Z + DUMMY: + + structures + + + 0.9991668 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.9771588 + protein_type + cleaner0 + 2023-07-27T13:03:56Z + MESH: + + OmpR/PhoB-type RRs + + + 0.999338 + protein + cleaner0 + 2023-07-27T13:04:18Z + PR: + + RegX3 + + + 0.99928516 + protein + cleaner0 + 2023-07-27T13:04:25Z + PR: + + MtrA + + + 0.99929607 + protein + cleaner0 + 2023-07-27T13:04:30Z + PR: + + PrrA + + + 0.99932086 + protein + cleaner0 + 2023-07-27T13:04:35Z + PR: + + PhoP + + + 0.99864024 + species + cleaner0 + 2023-07-27T13:04:58Z + MESH: + + Mycobacterium tuberculosis + + + 0.99932957 + protein + cleaner0 + 2023-07-27T13:04:42Z + PR: + + DrrB + + + 0.99933076 + protein + cleaner0 + 2023-07-27T13:04:47Z + PR: + + DrrD + + + 0.99869025 + species + cleaner0 + 2023-07-27T13:05:04Z + MESH: + + Thermotoga maritima + + + 0.9992974 + protein + cleaner0 + 2023-07-27T13:04:52Z + PR: + + KdpE + + + 0.9986049 + species + cleaner0 + 2023-07-27T13:05:09Z + MESH: + + Escherichia coli + + + 0.99784315 + evidence + cleaner0 + 2023-07-27T14:51:49Z + DUMMY: + + structures + + + 0.9987684 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.99910814 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.99913764 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.9983626 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.9992224 + protein_state + cleaner0 + 2023-07-27T13:05:18Z + DUMMY: + + open + + + 0.9992557 + protein_state + cleaner0 + 2023-07-27T13:05:24Z + DUMMY: + + closed + + + 0.99927944 + protein + cleaner0 + 2023-07-27T13:04:25Z + PR: + + MtrA + + + 0.99929 + protein + cleaner0 + 2023-07-27T13:04:30Z + PR: + + PrrA + + + 0.7214432 + protein_state + cleaner0 + 2023-07-27T14:23:34Z + DUMMY: + + very compact + + + 0.999193 + protein_state + cleaner0 + 2023-07-27T13:05:25Z + DUMMY: + + closed + + + 0.99700975 + evidence + cleaner0 + 2023-07-27T14:51:57Z + DUMMY: + + structure + + + structure_element + SO: + cleaner0 + 2023-07-27T13:06:26Z + + DNA-binding sequence + + + 0.9984138 + structure_element + cleaner0 + 2023-07-27T13:06:31Z + SO: + + recognition helix + + + 0.9992761 + structure_element + cleaner0 + 2023-07-27T13:02:08Z + SO: + + ED + + + 0.94537944 + chemical + cleaner0 + 2023-07-27T12:49:58Z + CHEBI: + + DNA + + + 0.997687 + evidence + cleaner0 + 2023-07-27T14:52:00Z + DUMMY: + + structures + + + 0.9993082 + protein + cleaner0 + 2023-07-27T13:04:48Z + PR: + + DrrD + + + 0.999303 + protein + cleaner0 + 2023-07-27T13:04:43Z + PR: + + DrrB + + + 0.9992262 + protein_state + cleaner0 + 2023-07-27T13:05:19Z + DUMMY: + + open + + + 0.99932104 + structure_element + cleaner0 + 2023-07-27T13:06:32Z + SO: + + recognition helix + + + 0.99866134 + protein_state + cleaner0 + 2023-07-27T14:23:38Z + DUMMY: + + fully exposed + + + 0.9987412 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.67741996 + protein_state + cleaner0 + 2023-07-27T14:23:43Z + DUMMY: + + flexible + + + + INTRO + paragraph + 8813 + Here, we describe the crystal structures of the N-terminal RD and the C-terminal ED of the lantibiotic resistance-associated RR NsrR from S. agalactiae. NsrR is part of the nisin resistance operon. The expression of the genes of this operon is induced by a TCS consisting of the HK NsrK and the RR NsrR. Based on the crystal structures of both the domains, modeling was employed to shed light on the putative DNA-bound state of full-length NsrR. + + 0.9987211 + evidence + cleaner0 + 2023-07-27T14:52:03Z + DUMMY: + + crystal structures + + + 0.9995086 + structure_element + cleaner0 + 2023-07-27T13:01:57Z + SO: + + RD + + + 0.9994874 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + protein_type + MESH: + cleaner0 + 2023-07-27T14:24:11Z + + lantibiotic resistance-associated RR + + + 0.9993624 + protein + cleaner0 + 2023-07-27T12:47:43Z + PR: + + NsrR + + + 0.9983449 + species + cleaner0 + 2023-07-27T12:56:39Z + MESH: + + S. agalactiae + + + 0.59359527 + protein + cleaner0 + 2023-07-27T12:47:43Z + PR: + + NsrR + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:52:00Z + + nisin + + + 0.7663454 + complex_assembly + cleaner0 + 2023-07-27T12:57:55Z + GO: + + TCS + + + 0.9973092 + protein_type + cleaner0 + 2023-07-27T12:58:39Z + MESH: + + HK + + + 0.9976102 + protein + cleaner0 + 2023-07-27T15:00:50Z + PR: + + NsrK + + + 0.9794317 + protein_type + cleaner0 + 2023-07-27T12:58:46Z + MESH: + + RR + + + 0.9993907 + protein + cleaner0 + 2023-07-27T12:47:43Z + PR: + + NsrR + + + 0.998714 + evidence + cleaner0 + 2023-07-27T14:52:06Z + DUMMY: + + crystal structures + + + 0.9989874 + protein_state + cleaner0 + 2023-07-27T13:06:02Z + DUMMY: + + DNA-bound + + + 0.9991117 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.99933654 + protein + cleaner0 + 2023-07-27T12:47:43Z + PR: + + NsrR + + + + METHODS + title_1 + 9259 + Materials and Methods + + + METHODS + title_2 + 9281 + Cloning, expression and purification NsrR + + + METHODS + paragraph + 9323 + NsrR was constructed, expressed, and purified as described previously. In brief, the nsrR gene (accession no. HG939456.1) from S. agalactiae COH1 was ligated into the expression vector pET24a allowing expression in E. coli with a His6-tag introduced at the C-terminus. The resulting plasmid pET24a-NsrR was transformed into E. coli BL21 (DE3) for expression. A single transformed colony was inoculated into 20 ml LB media containing 30 μg/ml kanamycin. The culture was grown for 14 h at 310 K with shaking at 200 rpm. 4 l LB media with 30 μg/ml kanamycin were inoculated with the overnight culture at an OD600 of 0.05 and grown at 310 K with shaking at 170 rpm until an OD600 of 0.3 was reached. Subsequently, temperature was lowered to 291 K, and cells were further grown until an OD600 of 0.8 was reached before inducing the expression by addition of 1 mM IPTG. Cells were further grown for 15 h and harvested by centrifugation at 8000 rpm for 20 min at 277 K. The harvested cell pellet was re-suspended in 10 ml of buffer A (50 mM Tris pH 8.0, 50 mM NaCl, 2 mM PMSF and 10% (v/v) glycerol) and 10 mg of DNase (Deoxyribonuclease I from bovine pancreas, Sigma Aldrich) was added. Cells were lysed using a cell disruptor (Constant Cell Disruption Systems, United Kingdom) at 2.6 × 105 kPa. The lysate was centrifuged at 42000 rpm for 60 min using a Ti60 rotor to remove non-lysed cells and cell debris. + + + METHODS + paragraph + 10729 + 20 mM imidazole was added to the cleared lysate prior to applying it onto a Ni2+ loaded Hi-Trap HP Chelating column (GE Healthcare) pre-equilibrated with buffer B (20 mM Tris pH 8.0, 250 mM NaCl and 20 mM imidazole, 2 mM PMSF). The column was washed with six column volumes of buffer B. Protein was eluted with a linear gradient of imidazole from 20 mM to 400 mM in buffer B. The fractions containing NsrR were pooled and concentrated up to 8 mg/ml in an Amicon centrifugal filter concentrator with a 10 kDa cut-off membrane (Millipore). The concentrated protein was further purified by size exclusion chromatography using a Superdex 200 GL 10/300 column (GE Healthcare), equilibrated with buffer C (25 mM Tris pH 9.0, 50 mM NaCl, 2 mM PMSF). The eluted protein fractions were pooled and concentrated to 11 mg/ml as described above. The purity of the protein was analyzed with 15% SDS-PAGE using colloidal Coomassie blue staining. + + + METHODS + title_2 + 11660 + Crystallization of NsrR + + + METHODS + paragraph + 11684 + Crystals were obtained by using 1 μl of protein solution (concentration of 6.0 mg/ml) mixed with 1 μl of reservoir solution using the hanging-drop vapor diffusion method at 285 K. The reservoir solution contained PEG 20000 (11, 13, 15, 17 and 21% (w/v)) and 0.1 M MES pH (6.0, 6.5, 7.0 and 7.5). Crystals were obtained after three weeks and grew to their maximum dimensions within one month. Two different crystal forms, rectangular plate-shaped crystals and thin plates, were observed in the same drop. Both crystals forms were transferred into a buffer containing the reservoir solution plus 30% (v/v) ethylene glycol for 5 min prior to flash cooling using liquid nitrogen. For phasing, 20 mM tetra-chloro platinate IV (Hampton Research) was added to the crystallization drop, and the rectangular plate-shaped crystals were soaked for 30 min. The crystals with no obvious optical damage were harvested and flash-cooled in liquid nitrogen following the procedure above. + + + METHODS + title_2 + 12657 + Data collection + + + METHODS + paragraph + 12673 + Initially crystals were screened for quality at beamline P13 (DESY, EMBL Hamburg). All X-ray diffraction data were collected at beamline ID23eh1 of the European Synchrotron Radiation Facility (ESRF), Grenoble. All data sets were processed and scaled using XDS and XSCALE software package. Data sets from both native crystal forms were collected at 100 K. A single-wavelength anomalous dispersion (SAD) dataset from a single heavy-atom derivatized crystal (rectangular plate-shaped crystal) was collected at 1.0714 Å at 100 K. Diffraction data up to 1.7 Å was used for heavy atom localization and subsequent phasing. + + + METHODS + title_2 + 13291 + Structure determination of NsrR + + + METHODS + paragraph + 13323 + The structure of the thin plate-shaped crystals was solved by molecular replacement using the structure of the receiver domain of PhoB (PDB entry: 1B00) as a model to phase the native data set at 1.8 Å resolution. The model generated was refined manually in COOT followed by iterative cycles of refinement using the program phenix.refine. Manual adjustments between the refinement cycles were performed with the program COOT and Ramachandran validation was done using MolProbity. + + + METHODS + paragraph + 13804 + The SAD dataset of the rectangular plate-shaped crystal was used for phasing via the Auto-Rickshaw server. The initial model was further built and refined manually using COOT and phenix.refine from the Phenix package with iterative cycles of refinement. This model was used to phase the native data set of the rectangular plate-shaped crystals at a resolution of 1.6 Å. + + + METHODS + paragraph + 14175 + Data collection and refinement statistics are listed in Table 1 and all images of the models were prepared using PyMOL. + + + pone.0149903.t001.xml + pone.0149903.t001 + TABLE + table_title_caption + 14295 + Data collection, phasing, and refinement statistics for the receiver and effector domains of NsrR. + + + pone.0149903.t001.xml + pone.0149903.t001 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><thead><tr><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1">NsrR-RD (native)</th><th align="center" rowspan="1" colspan="1">NsrR-ED (native)</th><th align="center" rowspan="1" colspan="1">NsrR-ED (SAD dataset)</th></tr></thead><tbody><tr><td align="justify" rowspan="1" colspan="1"><bold>Data collection</bold></td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">Space group</td><td align="justify" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2</td><td align="justify" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2</td><td align="justify" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2</td></tr><tr><td align="justify" rowspan="1" colspan="1"><italic>Cell dimensions</italic></td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    a, b, c (Å)</td><td align="justify" rowspan="1" colspan="1">57.0 107.1 39.4</td><td align="justify" rowspan="1" colspan="1">56.3 60.4 56.8</td><td align="justify" rowspan="1" colspan="1">56.3 60.6 56.7</td></tr><tr><td align="justify" rowspan="1" colspan="1">    α, β, γ (°)</td><td align="justify" rowspan="1" colspan="1">90.0 90.0 90.0</td><td align="justify" rowspan="1" colspan="1">90.0 90.0 90.0</td><td align="justify" rowspan="1" colspan="1">90.0 90.0 90.0</td></tr><tr><td align="justify" rowspan="1" colspan="1">Wavelength (λ)</td><td align="justify" rowspan="1" colspan="1">1.0688</td><td align="justify" rowspan="1" colspan="1">0.9677</td><td align="justify" rowspan="1" colspan="1">1.0714</td></tr><tr><td align="justify" rowspan="1" colspan="1">Resolution (Å)</td><td align="justify" rowspan="1" colspan="1">39.48–1.80 (1.86–1.80)</td><td align="justify" rowspan="1" colspan="1">56.85–1.60 (1.65–1.60)</td><td align="justify" rowspan="1" colspan="1">100.00–1.70 (1.75–1.70)</td></tr><tr><td align="justify" rowspan="1" colspan="1">R<sub>merge</sub> +<xref ref-type="table-fn" rid="t001fn002"><sup>a</sup></xref></td><td align="justify" rowspan="1" colspan="1">3.4 (33.3)</td><td align="justify" rowspan="1" colspan="1">4.8 (30.5)</td><td align="justify" rowspan="1" colspan="1">6.8 (97.0)</td></tr><tr><td align="justify" rowspan="1" colspan="1">I /σ(I)</td><td align="justify" rowspan="1" colspan="1">26.2 (5.1)</td><td align="justify" rowspan="1" colspan="1">18.2 (4.6)</td><td align="justify" rowspan="1" colspan="1">21.6 (1.7)</td></tr><tr><td align="justify" rowspan="1" colspan="1">Completeness (%)</td><td align="justify" rowspan="1" colspan="1">98.8 (98.8)</td><td align="justify" rowspan="1" colspan="1">99.5 (99.7)</td><td align="justify" rowspan="1" colspan="1">98.8 (90.2)</td></tr><tr><td align="justify" rowspan="1" colspan="1">Redundancy</td><td align="justify" rowspan="1" colspan="1">4.8 (4.8)</td><td align="justify" rowspan="1" colspan="1">4.8 (4.9)</td><td align="justify" rowspan="1" colspan="1">11.7 (6.5)</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Structure Refinement</bold></td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">Resolution (Å)</td><td align="justify" rowspan="1" colspan="1">39.48–1.80 (1.86–1.80)</td><td align="justify" rowspan="1" colspan="1">56.85–1.60 (1.65–1.60)</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">No. of reflections</td><td align="justify" rowspan="1" colspan="1">109201 (10602)</td><td align="justify" rowspan="1" colspan="1">124810 (12438)</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">CC1/2</td><td align="left" rowspan="1" colspan="1">0.999 (0.924)</td><td align="left" rowspan="1" colspan="1">0.999 (0.923)</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">R<sub>work</sub> +<sup>b</sup> / R<sub>free</sub> +<xref ref-type="table-fn" rid="t001fn003"><sup>b</sup></xref></td><td align="justify" rowspan="1" colspan="1">0.17 (0.20)/ 0.22 (0.27)</td><td align="justify" rowspan="1" colspan="1">0.18 (0.22)/ 0.22 (0.27)</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1"><italic>No</italic>. <italic>of atoms</italic></td><td align="justify" rowspan="1" colspan="1">2027</td><td align="justify" rowspan="1" colspan="1">1843</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Macromolecules</td><td align="justify" rowspan="1" colspan="1">1894</td><td align="justify" rowspan="1" colspan="1">1580</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Ligand/ion</td><td align="justify" rowspan="1" colspan="1">20</td><td align="justify" rowspan="1" colspan="1">8</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Water</td><td align="justify" rowspan="1" colspan="1">113</td><td align="justify" rowspan="1" colspan="1">255</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1"><italic>B-factors (Å</italic><sup><italic>2</italic></sup><italic>)</italic></td><td align="justify" rowspan="1" colspan="1">28.3</td><td align="justify" rowspan="1" colspan="1">21.7</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Macromolecules</td><td align="justify" rowspan="1" colspan="1">27.7</td><td align="justify" rowspan="1" colspan="1">20.2</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Ligand/ion</td><td align="justify" rowspan="1" colspan="1">34.0</td><td align="justify" rowspan="1" colspan="1">38.6</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Solvent</td><td align="justify" rowspan="1" colspan="1">36.9</td><td align="justify" rowspan="1" colspan="1">30.4</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1"><italic>R</italic>.<italic>m</italic>.<italic>s</italic>. <italic>deviations</italic></td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Bond lengths (Å)</td><td align="justify" rowspan="1" colspan="1">0.007</td><td align="justify" rowspan="1" colspan="1">0.008</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Bond angles (°)</td><td align="justify" rowspan="1" colspan="1">1.11</td><td align="justify" rowspan="1" colspan="1">1.18</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1"><italic>Ramachandran plot (%)</italic></td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Favored</td><td align="justify" rowspan="1" colspan="1">99.0</td><td align="justify" rowspan="1" colspan="1">97.0</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Allowed</td><td align="justify" rowspan="1" colspan="1">1.0</td><td align="justify" rowspan="1" colspan="1">2.48</td><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">    Outliers</td><td align="justify" rowspan="1" colspan="1">0.0</td><td align="justify" rowspan="1" colspan="1">0.52</td><td align="justify" rowspan="1" colspan="1"/></tr></tbody></table> + + 14394 + NsrR-RD (native) NsrR-ED (native) NsrR-ED (SAD dataset) Data collection Space group P 21 21 2 P 21 21 2 P 21 21 2 Cell dimensions     a, b, c (Å) 57.0 107.1 39.4 56.3 60.4 56.8 56.3 60.6 56.7     α, β, γ (°) 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 Wavelength (λ) 1.0688 0.9677 1.0714 Resolution (Å) 39.48–1.80 (1.86–1.80) 56.85–1.60 (1.65–1.60) 100.00–1.70 (1.75–1.70) Rmergea 3.4 (33.3) 4.8 (30.5) 6.8 (97.0) I /σ(I) 26.2 (5.1) 18.2 (4.6) 21.6 (1.7) Completeness (%) 98.8 (98.8) 99.5 (99.7) 98.8 (90.2) Redundancy 4.8 (4.8) 4.8 (4.9) 11.7 (6.5) Structure Refinement Resolution (Å) 39.48–1.80 (1.86–1.80) 56.85–1.60 (1.65–1.60) No. of reflections 109201 (10602) 124810 (12438) CC1/2 0.999 (0.924) 0.999 (0.923) Rworkb / Rfreeb 0.17 (0.20)/ 0.22 (0.27) 0.18 (0.22)/ 0.22 (0.27) No. of atoms 2027 1843     Macromolecules 1894 1580     Ligand/ion 20 8     Water 113 255 B-factors (Å2) 28.3 21.7     Macromolecules 27.7 20.2     Ligand/ion 34.0 38.6     Solvent 36.9 30.4 R.m.s. deviations     Bond lengths (Å) 0.007 0.008     Bond angles (°) 1.11 1.18 Ramachandran plot (%)     Favored 99.0 97.0     Allowed 1.0 2.48     Outliers 0.0 0.52 + + + pone.0149903.t001.xml + pone.0149903.t001 + TABLE + table_footnote + 15717 + Values in parentheses are for the highest resolution shell. + + + pone.0149903.t001.xml + pone.0149903.t001 + TABLE + table_footnote + 15777 + a Rmerge is defined as Rsym = ∑hkl∑i|Ii(hkl) − ⟨I(hkl)⟩|/∑hkl∑iIi(hkl) and + + + pone.0149903.t001.xml + pone.0149903.t001 + TABLE + table_footnote + 15866 + b RF as Rf = ∑hkl‖Fobs|−|Fcalc‖/∑hkl|Fobs| + + + METHODS + title_2 + 15919 + Accession numbers + + + METHODS + paragraph + 15937 + Coordinates and structure factors have been deposited in the PDB with accession numbers 5DCL (NsrR-RD) and 5DCM (NsrR-ED). + + + RESULTS + title_1 + 16060 + Results and Discussion + + + RESULTS + paragraph + 16083 + NsrR was expressed and purified as described, resulting in a homogenous protein as observed by size exclusion chromatography (Fig 1A), with a yield of 2 mg per liter of cell culture. By calibrating the column with proteins of known molecular weight the NsrR full length protein elutes as a dimer. The purified NsrR protein has a theoretical molecular mass of 27.7 kDa and was >98% pure as assessed by SDS-PAGE (Fig 1B, indicated by *). Surprisingly, over time NsrR degraded into two distinct fragments as visible on SDS-PAGE analysis using the same purified protein sample after one week (Fig 1C, indicated by ** and ***, respectively). This was also observed by size exclusion chromatography where a peak at an elution time of 18 min appeared (Fig 1A). Both bands were subjected to mass spectrometry analysis. The analysis revealed that the larger fragment (**) represents the N-terminal receiver domain (residues 1–119; referred to as NsrR-RD) whereas the smaller fragment (***) contained the C-terminal DNA-binding effector domain of NsrR (residues 129–243 including 21 amino acids derived from the expression tag; referred to as NsrR-ED) (Fig 1C). Residues 120–128 form the linker connecting the RD and ED. Such a cleavage of the full-length RR into two specific domains is not unusual and has been previously reported for other RRs as well. Mass spectrometry analysis did not reveal the presence of any specific protease in the purified NsrR sample. Furthermore, addition of a protease inhibitor, such as PMSF (Phenylmethylsulfonyl fluoride) and AEBSF {4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride}, even at high concentrations, did not inhibit proteolysis (data not shown). + + 0.99927205 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.98291045 + experimental_method + cleaner0 + 2023-07-27T14:35:16Z + MESH: + + expressed and purified + + + 0.99884963 + experimental_method + cleaner0 + 2023-07-27T14:35:20Z + MESH: + + size exclusion chromatography + + + 0.99928325 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9991232 + protein_state + cleaner0 + 2023-07-27T14:28:34Z + DUMMY: + + full length + + + 0.9988662 + oligomeric_state + cleaner0 + 2023-07-27T13:37:59Z + DUMMY: + + dimer + + + 0.99927896 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + evidence + DUMMY: + cleaner0 + 2023-07-27T13:07:27Z + + molecular mass + + + 0.9988087 + experimental_method + cleaner0 + 2023-07-27T14:36:27Z + MESH: + + SDS-PAGE + + + 0.99922633 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99886465 + experimental_method + cleaner0 + 2023-07-27T14:35:24Z + MESH: + + SDS-PAGE + + + 0.9988444 + experimental_method + cleaner0 + 2023-07-27T14:35:28Z + MESH: + + size exclusion chromatography + + + experimental_method + MESH: + cleaner0 + 2023-07-27T14:35:48Z + + mass spectrometry analysis + + + 0.9993094 + structure_element + cleaner0 + 2023-07-27T12:48:55Z + SO: + + receiver domain + + + 0.99736327 + residue_range + cleaner0 + 2023-07-27T14:08:24Z + DUMMY: + + 1–119 + + + 0.9771807 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9815507 + structure_element + cleaner0 + 2023-07-27T13:01:57Z + SO: + + RD + + + 0.9990264 + structure_element + cleaner0 + 2023-07-27T12:49:02Z + SO: + + DNA-binding effector domain + + + 0.99936086 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99765706 + residue_range + cleaner0 + 2023-07-27T14:08:27Z + DUMMY: + + 129–243 + + + 0.9743844 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.7150254 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.9978265 + residue_range + cleaner0 + 2023-07-27T14:08:31Z + DUMMY: + + 120–128 + + + 0.9986193 + structure_element + cleaner0 + 2023-07-27T14:46:51Z + SO: + + linker + + + 0.9995346 + structure_element + cleaner0 + 2023-07-27T13:01:57Z + SO: + + RD + + + 0.9995073 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.9991009 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.98067904 + protein_type + cleaner0 + 2023-07-27T12:58:46Z + MESH: + + RR + + + 0.99906784 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + experimental_method + MESH: + cleaner0 + 2023-07-27T14:36:21Z + + Mass spectrometry analysis + + + 0.99928313 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99845946 + chemical + cleaner0 + 2023-07-27T13:08:05Z + CHEBI: + + PMSF + + + 0.9973788 + chemical + cleaner0 + 2023-07-27T13:08:10Z + CHEBI: + + Phenylmethylsulfonyl fluoride + + + 0.99913484 + chemical + cleaner0 + 2023-07-27T13:08:14Z + CHEBI: + + AEBSF + + + 0.9984695 + chemical + cleaner0 + 2023-07-27T13:08:19Z + CHEBI: + + 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride + + + + pone.0149903.g001.jpg + pone.0149903.g001 + FIG + fig_title_caption + 17780 + Purification of NsrR and SDS PAGE analysis of purified NsrR directly and one week after purification. + + experimental_method + MESH: + cleaner0 + 2023-07-27T14:36:54Z + + Purification + + + 0.99923396 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9983078 + experimental_method + cleaner0 + 2023-07-27T14:36:42Z + MESH: + + SDS PAGE + + + 0.9992512 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + + pone.0149903.g001.jpg + pone.0149903.g001 + FIG + fig_caption + 17882 + (a) Elution profile of size-exclusion chromatography step of NsrR. The y-axis represents the UV absorption of the protein at 280 nm, while the x-axis represents the elution volume. a, b, c refer to the protein standards dextran blue (2,000 kDa), BSA (67 kDa), and lysozyme (14.3 kDa), respectively. The bold line represents the chromatogram of freshly purified NsrR while the dashed line shows the chromatogram of the same NsrR protein after one week. (b) Freshly purified NsrR protein, and (c) NsrR protein after one week. Lanes: M represents the PAGE Ruler Unstained Ladder; 1: NsrR after a two-step purification; 2: NsrR one week after purification. * corresponds to full-length NsrR protein at 27 kDa, while ** and *** correspond to the NsrR-RD and NsrR-ED domain at around 13 kDa, respectively. + + 0.624071 + evidence + cleaner0 + 2023-07-27T13:09:02Z + DUMMY: + + Elution profile + + + 0.9987503 + experimental_method + cleaner0 + 2023-07-27T14:36:58Z + MESH: + + size-exclusion chromatography + + + 0.99920565 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9852395 + evidence + cleaner0 + 2023-07-27T13:08:56Z + DUMMY: + + chromatogram + + + 0.99915934 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9861688 + evidence + cleaner0 + 2023-07-27T13:08:55Z + DUMMY: + + chromatogram + + + 0.9992047 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99925953 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99921334 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99917966 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99925095 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9990738 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.99925727 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99833435 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99890995 + structure_element + cleaner0 + 2023-07-27T13:01:57Z + SO: + + RD + + + 0.99834394 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9976876 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + + RESULTS + paragraph + 18682 + Since formation of the crystals took around one month, it is not surprising that this cleavage also occurred in the crystallization drop. NsrR was crystallized yielding two crystal forms, which were distinguishable by visual inspection. Initially, we tried to solve the structure of NsrR by molecular replacement, which was not successful. Therefore, we tried heavy atom phasing using a platinum compound. This succeeded for the rectangular plate-shaped crystals. After the structure was solved, it became evident that these crystals contained two monomers of the ED of NsrR in the asymmetric unit. + + 0.96927536 + evidence + cleaner0 + 2023-07-27T14:52:11Z + DUMMY: + + crystals + + + 0.9993742 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99806994 + experimental_method + cleaner0 + 2023-07-27T14:37:27Z + MESH: + + crystallized + + + 0.98949033 + evidence + cleaner0 + 2023-07-27T14:52:14Z + DUMMY: + + structure + + + 0.99938893 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.998575 + experimental_method + cleaner0 + 2023-07-27T14:37:31Z + MESH: + + molecular replacement + + + 0.99857587 + experimental_method + cleaner0 + 2023-07-27T14:37:34Z + MESH: + + heavy atom phasing + + + 0.8848682 + chemical + cleaner0 + 2023-07-27T14:27:32Z + CHEBI: + + platinum + + + 0.86987334 + evidence + cleaner0 + 2023-07-27T14:52:17Z + DUMMY: + + crystals + + + 0.9626586 + evidence + cleaner0 + 2023-07-27T14:52:19Z + DUMMY: + + structure + + + 0.9928011 + evidence + cleaner0 + 2023-07-27T14:52:22Z + DUMMY: + + crystals + + + 0.99839395 + oligomeric_state + cleaner0 + 2023-07-27T13:18:37Z + DUMMY: + + monomers + + + 0.99861085 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.99940157 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + + RESULTS + paragraph + 19281 + We also tried to solve the structure of the thin plate-shaped crystals with this template, but the resulting model generated was not sufficient. Therefore, we thought that these crystals contained the N-terminal domain of NsrR and successfully phased this dataset using molecular replacement with the N-terminal domain of PhoB (PDB code: 1B00; as a template. This approach revealed that this crystal form indeed contained two monomers of the RD of NsrR in the asymmetric unit. Since both crystals forms were obtained in the same drop it is not surprising that, when dissolving several crystals and performing subsequent mass-spectrometry to identify the protein in the crystals, it yielded peptide fragments throughout the NsrR sequence. + + 0.9663912 + evidence + cleaner0 + 2023-07-27T14:52:26Z + DUMMY: + + structure + + + 0.98445845 + evidence + cleaner0 + 2023-07-27T14:52:29Z + DUMMY: + + crystals + + + 0.9924428 + evidence + cleaner0 + 2023-07-27T14:52:32Z + DUMMY: + + crystals + + + 0.99763584 + structure_element + cleaner0 + 2023-07-27T14:47:24Z + SO: + + N-terminal domain + + + 0.9993974 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9987139 + experimental_method + cleaner0 + 2023-07-27T14:37:48Z + MESH: + + molecular replacement + + + 0.9982074 + structure_element + cleaner0 + 2023-07-27T14:47:26Z + SO: + + N-terminal domain + + + 0.9993304 + protein + cleaner0 + 2023-07-27T13:28:31Z + PR: + + PhoB + + + 0.99808645 + oligomeric_state + cleaner0 + 2023-07-27T13:18:37Z + DUMMY: + + monomers + + + 0.99933535 + structure_element + cleaner0 + 2023-07-27T13:01:57Z + SO: + + RD + + + 0.99941313 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.56550413 + evidence + cleaner0 + 2023-07-27T14:52:35Z + DUMMY: + + crystals + + + 0.99880904 + experimental_method + cleaner0 + 2023-07-27T14:38:11Z + MESH: + + mass-spectrometry + + + 0.7979644 + evidence + cleaner0 + 2023-07-27T14:52:38Z + DUMMY: + + crystals + + + 0.9993994 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + + RESULTS + paragraph + 20019 + In summary, the two crystal forms contained one of the two domains, respectively, such that both domains were successfully crystallized. We determined the crystal structures of NsrR-RD and NsrR-ED separately. However, a part of the linker region (residues 120–128; 120RRSQQFIQQ128; underlined are the amino acid residues not visible in either domain) could not be traced in the electron density. + + 0.97675586 + evidence + cleaner0 + 2023-07-27T14:52:41Z + DUMMY: + + crystal forms + + + 0.9501939 + experimental_method + cleaner0 + 2023-07-27T14:38:26Z + MESH: + + crystallized + + + 0.99843526 + evidence + cleaner0 + 2023-07-27T14:52:44Z + DUMMY: + + crystal structures + + + protein + PR: + cleaner0 + 2023-07-27T12:47:44Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:57Z + + RD + + + protein + PR: + cleaner0 + 2023-07-27T12:47:44Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:02:09Z + + ED + + + 0.9992579 + structure_element + cleaner0 + 2023-07-27T13:52:42Z + SO: + + linker region + + + 0.9973113 + residue_range + cleaner0 + 2023-07-27T14:08:37Z + DUMMY: + + 120–128 + + + 0.86698794 + structure_element + cleaner0 + 2023-07-27T14:08:40Z + SO: + + 120RRSQQFIQQ128 + + + 0.9987925 + evidence + cleaner0 + 2023-07-27T14:52:46Z + DUMMY: + + electron density + + + + RESULTS + title_2 + 20417 + Overall structure of the N-terminal NsrR receiver domain (NsrR-RD) + + 0.99032 + evidence + cleaner0 + 2023-07-27T14:52:52Z + DUMMY: + + structure + + + 0.9990251 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99905497 + structure_element + cleaner0 + 2023-07-27T12:48:55Z + SO: + + receiver domain + + + protein + PR: + cleaner0 + 2023-07-27T12:47:44Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:57Z + + RD + + + + RESULTS + paragraph + 20484 + The structure of the NsrR-RD was determined at a resolution of 1.8 Å (Table 1). The Rwork and Rfree values after refinement were 0.17 and 0.22, respectively. Ramachandran validation revealed that all residues (100%, 236 amino acids) were in the preferred or allowed regions. The structure contained many ethylene glycol molecules arising from the cryo-protecting procedure. Data collection and refinement statistics are listed in Table 1. + + 0.99849343 + evidence + cleaner0 + 2023-07-27T14:52:56Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-27T12:47:44Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:57Z + + RD + + + 0.9982931 + evidence + cleaner0 + 2023-07-27T14:52:59Z + DUMMY: + + Rwork + + + 0.99802566 + evidence + cleaner0 + 2023-07-27T14:53:03Z + DUMMY: + + Rfree + + + 0.97388566 + evidence + cleaner0 + 2023-07-27T13:41:47Z + DUMMY: + + Ramachandran validation + + + 0.99851376 + evidence + cleaner0 + 2023-07-27T14:53:06Z + DUMMY: + + structure + + + 0.6619525 + chemical + cleaner0 + 2023-07-27T14:27:38Z + CHEBI: + + ethylene glycol + + + + RESULTS + paragraph + 20924 + The asymmetric unit contains two copies of NsrR-RD. Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. For Asn85, Asp86, and Glu87 of chain A, poor electron density was observed for the side chains and, thus, these side chains were deleted during refinement and are not present in the final structure. Since the two monomers of NsrR-RD were virtually identical (rmsd of 0.6 Å over 116 Cα atoms for the two monomers). Therefore, the overall structure is described for monomer A only. + + protein + PR: + cleaner0 + 2023-07-27T12:47:44Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:57Z + + RD + + + 0.9993507 + structure_element + cleaner0 + 2023-07-27T12:48:55Z + SO: + + receiver domain + + + 0.99704796 + residue_range + cleaner0 + 2023-07-27T13:18:20Z + DUMMY: + + Met1-Leu119 + + + 0.9967788 + residue_range + cleaner0 + 2023-07-27T13:18:24Z + DUMMY: + + Asn4 to Arg121 + + + 0.9990872 + structure_element + cleaner0 + 2023-07-27T13:43:00Z + SO: + + chain A + + + 0.9973508 + residue_name_number + cleaner0 + 2023-07-27T13:19:04Z + DUMMY: + + Arg120 + + + 0.9985317 + residue_name_number + cleaner0 + 2023-07-27T13:19:09Z + DUMMY: + + Arg121 + + + 0.9991097 + structure_element + cleaner0 + 2023-07-27T14:47:31Z + SO: + + linker + + + 0.99671906 + residue_range + cleaner0 + 2023-07-27T13:18:27Z + DUMMY: + + Gln5 to Ser122 + + + 0.99902666 + structure_element + cleaner0 + 2023-07-27T13:43:05Z + SO: + + chain B + + + residue_range + DUMMY: + cleaner0 + 2023-07-27T13:18:09Z + + Arg120 until Ser122 + + + 0.9988379 + structure_element + cleaner0 + 2023-07-27T14:47:36Z + SO: + + linker + + + 0.9987049 + evidence + cleaner0 + 2023-07-27T14:53:10Z + DUMMY: + + electron density + + + protein + PR: + cleaner0 + 2023-07-27T12:47:44Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:57Z + + RD + + + 0.99953294 + residue_name_number + cleaner0 + 2023-07-27T13:19:25Z + DUMMY: + + Asn85 + + + 0.9995608 + residue_name_number + cleaner0 + 2023-07-27T13:19:29Z + DUMMY: + + Asp86 + + + 0.9995659 + residue_name_number + cleaner0 + 2023-07-27T13:19:33Z + DUMMY: + + Glu87 + + + 0.99893194 + structure_element + cleaner0 + 2023-07-27T13:43:00Z + SO: + + chain A + + + 0.99816 + evidence + cleaner0 + 2023-07-27T14:53:12Z + DUMMY: + + electron density + + + 0.997624 + evidence + cleaner0 + 2023-07-27T14:53:16Z + DUMMY: + + structure + + + 0.9985448 + oligomeric_state + cleaner0 + 2023-07-27T13:18:36Z + DUMMY: + + monomers + + + protein + PR: + cleaner0 + 2023-07-27T12:47:44Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + 0.99848664 + evidence + cleaner0 + 2023-07-27T13:18:48Z + DUMMY: + + rmsd + + + 0.9985921 + oligomeric_state + cleaner0 + 2023-07-27T13:18:37Z + DUMMY: + + monomers + + + 0.99662185 + evidence + cleaner0 + 2023-07-27T14:53:18Z + DUMMY: + + structure + + + 0.99799687 + oligomeric_state + cleaner0 + 2023-07-27T13:18:42Z + DUMMY: + + monomer + + + 0.9981139 + structure_element + cleaner0 + 2023-07-27T14:47:41Z + SO: + + A + + + + RESULTS + paragraph + 21674 + NsrR-RD structurally adopts a αβ doubly-wound fold previously observed in OmpR/PhoB type regulators. Five β-strands (β1-β5) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two α-helices (α1 and α5) on one and three helices (α2, α3, α4) on the other side (Fig 2). The NsrR-RD structure shows a β1-α1-β2-α2-β3-α3-β4-α4-β5-α5 topology as also observed for other RRs. + + protein + PR: + cleaner0 + 2023-07-27T12:47:44Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + 0.9978269 + structure_element + cleaner0 + 2023-07-27T14:47:46Z + SO: + + αβ doubly-wound fold + + + protein_type + MESH: + cleaner0 + 2023-07-27T13:19:55Z + + OmpR/PhoB type regulators + + + 0.92422724 + structure_element + cleaner0 + 2023-07-27T13:44:03Z + SO: + + β-strands + + + 0.99796885 + structure_element + cleaner0 + 2023-07-27T13:23:38Z + SO: + + β1-β5 + + + 0.9941896 + evidence + cleaner0 + 2023-07-27T14:53:21Z + DUMMY: + + structure + + + 0.9989569 + structure_element + cleaner0 + 2023-07-27T13:43:54Z + SO: + + α-helices + + + 0.9994605 + structure_element + cleaner0 + 2023-07-27T13:23:14Z + SO: + + α1 + + + 0.9994593 + structure_element + cleaner0 + 2023-07-27T13:23:19Z + SO: + + α5 + + + 0.9925608 + structure_element + cleaner0 + 2023-07-27T14:48:06Z + SO: + + helices + + + 0.9994474 + structure_element + cleaner0 + 2023-07-27T13:23:25Z + SO: + + α2 + + + 0.9994404 + structure_element + cleaner0 + 2023-07-27T13:23:30Z + SO: + + α3 + + + 0.99948514 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + protein + PR: + cleaner0 + 2023-07-27T12:47:44Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + 0.9972779 + evidence + cleaner0 + 2023-07-27T14:53:24Z + DUMMY: + + structure + + + structure_element + SO: + cleaner0 + 2023-07-27T13:23:09Z + + β1-α1-β2-α2-β3-α3-β4-α4-β5-α5 + + + 0.9992448 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + + pone.0149903.g002.jpg + pone.0149903.g002 + FIG + fig_title_caption + 22171 + Structure of NsrR-RD. + + 0.9948383 + evidence + cleaner0 + 2023-07-27T14:53:30Z + DUMMY: + + Structure + + + protein + PR: + cleaner0 + 2023-07-27T12:47:44Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + + pone.0149903.g002.jpg + pone.0149903.g002 + FIG + fig_caption + 22193 + Cartoon representation of the helices (α1 – α5) and β-sheets (β1 - β5). Structural areas with the highest variations to the receiver domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1B00) are marked in separate boxes. + + 0.9325321 + structure_element + cleaner0 + 2023-07-27T14:48:12Z + SO: + + helices + + + 0.99635005 + structure_element + cleaner0 + 2023-07-27T13:24:02Z + SO: + + α1 – α5 + + + 0.9991474 + structure_element + cleaner0 + 2023-07-27T13:43:49Z + SO: + + β-sheets + + + 0.9984813 + structure_element + cleaner0 + 2023-07-27T14:48:22Z + SO: + + β1 - β5 + + + 0.99939233 + structure_element + cleaner0 + 2023-07-27T14:48:25Z + SO: + + receiver domains + + + 0.99928397 + protein + cleaner0 + 2023-07-27T13:04:43Z + PR: + + DrrB + + + 0.99928206 + protein + cleaner0 + 2023-07-27T13:04:25Z + PR: + + MtrA + + + 0.99928015 + protein + cleaner0 + 2023-07-27T13:28:31Z + PR: + + PhoB + + + + RESULTS + title_2 + 22444 + Comparison with structures of other receiver domains + + experimental_method + MESH: + cleaner0 + 2023-07-27T14:53:43Z + + Comparison + + + 0.9972778 + evidence + cleaner0 + 2023-07-27T14:53:33Z + DUMMY: + + structures + + + 0.99821997 + structure_element + cleaner0 + 2023-07-27T14:48:30Z + SO: + + receiver domains + + + + RESULTS + paragraph + 22497 + NsrR belongs to the OmpR/PhoB family of RRs. The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. Superimposition of the structures revealed that helix α4 is slightly rotated outward in NsrR-RD (Fig 2). In receiver domains of response regulators, helix α4 has been shown to be a crucial part of the dimerization interface. Furthermore, helix α4 in NsrR is shorter than in other RRs. The first helical turn is unwound and adopts an unstructured region (see Fig 2). A slightly outward rotation or unwinding of helix α4 has been observed in the structures of other RD of regulators. For example, the structure of BaeR and RegX3 displayed a completely unwound helix α4. In the structure of DrrD, helix α4 is only partially displaced. In the receiver domain of NsrR, helix α4 is also partially displaced but in a different direction (S1 Fig). Inspection of the crystal contacts revealed no major interactions in this region that could have influenced the orientation of helix α4. Furthermore, NsrR is crystallized as a monomer, and investigation of the symmetry-related molecules did not reveal a functional dimer within the crystal. This could explain the flexibility and thereby the different orientation of helix α4 in NsrR. + + 0.9992686 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.92631733 + protein_type + cleaner0 + 2023-07-27T14:29:15Z + MESH: + + OmpR/PhoB family + + + 0.9988243 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.9993143 + structure_element + cleaner0 + 2023-07-27T12:48:55Z + SO: + + receiver domain + + + 0.99924767 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99867636 + experimental_method + cleaner0 + 2023-07-27T14:38:37Z + MESH: + + superimposed + + + 0.9992309 + structure_element + cleaner0 + 2023-07-27T14:48:34Z + SO: + + receiver domains + + + 0.9120915 + protein_type + cleaner0 + 2023-07-27T14:29:23Z + MESH: + + OmpR/PhoB family + + + 0.9993724 + protein + cleaner0 + 2023-07-27T13:04:43Z + PR: + + DrrB + + + 0.9992994 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9993486 + protein + cleaner0 + 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structure_element + cleaner0 + 2023-07-27T14:48:38Z + SO: + + receiver domains + + + 0.99828744 + protein_type + cleaner0 + 2023-07-27T13:03:37Z + MESH: + + response regulators + + + 0.99895096 + structure_element + cleaner0 + 2023-07-27T13:24:12Z + SO: + + helix + + + 0.99940157 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + 0.99892545 + site + cleaner0 + 2023-07-27T14:33:02Z + SO: + + dimerization interface + + + 0.9989354 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.99936026 + structure_element + cleaner0 + 2023-07-27T13:20:46Z + SO: + + α4 + + + 0.9992988 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99918586 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.9848738 + structure_element + cleaner0 + 2023-07-27T14:48:48Z + SO: + + first helical turn + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T14:28:59Z + + unwound + + + 0.9173802 + protein_state + cleaner0 + 2023-07-27T14:28:41Z + DUMMY: + + unstructured + + + 0.998276 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.9991788 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + 0.99831796 + evidence + cleaner0 + 2023-07-27T14:53:56Z + DUMMY: + + structures + + + 0.99908435 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9976528 + evidence + cleaner0 + 2023-07-27T14:54:01Z + DUMMY: + + structure + + + 0.99934536 + protein + cleaner0 + 2023-07-27T15:00:57Z + PR: + + BaeR + + + 0.9994049 + protein + cleaner0 + 2023-07-27T13:04:20Z + PR: + + RegX3 + + + 0.75553304 + protein_state + cleaner0 + 2023-07-27T14:29:02Z + DUMMY: + + unwound + + + 0.9991953 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.9988997 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + 0.99758387 + evidence + cleaner0 + 2023-07-27T14:53:59Z + DUMMY: + + structure + + + 0.99940825 + protein + cleaner0 + 2023-07-27T13:04:48Z + PR: + + DrrD + + + 0.99679846 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.99936146 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + 0.9991323 + structure_element + cleaner0 + 2023-07-27T12:48:55Z + SO: + + receiver domain + + + 0.99933666 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9980818 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.9993893 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + 0.99822336 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.9993075 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + 0.99936146 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9984592 + experimental_method + cleaner0 + 2023-07-27T14:39:05Z + MESH: + + crystallized + + + 0.9988293 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + 0.998806 + oligomeric_state + cleaner0 + 2023-07-27T13:38:00Z + DUMMY: + + dimer + + + 0.85652447 + evidence + cleaner0 + 2023-07-27T14:54:04Z + DUMMY: + + crystal + + + 0.9982503 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.9992778 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + 0.99934965 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + + pone.0149903.t002.xml + pone.0149903.t002 + TABLE + table_title_caption + 24009 + The structures of the RD and ED domains of NsrR aligned to other response regulators. + + 0.9971283 + evidence + cleaner0 + 2023-07-27T14:54:10Z + DUMMY: + + structures + + + 0.9994692 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9994423 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.99930155 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.6662647 + experimental_method + cleaner0 + 2023-07-27T14:39:08Z + MESH: + + aligned + + + 0.9969348 + protein_type + cleaner0 + 2023-07-27T13:03:37Z + MESH: + + response regulators + + + + pone.0149903.t002.xml + pone.0149903.t002 + TABLE + table_caption + 24095 + The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence. + + 0.9984945 + evidence + cleaner0 + 2023-07-27T13:18:49Z + DUMMY: + + rmsd + + + 0.9987993 + experimental_method + cleaner0 + 2023-07-27T14:39:12Z + MESH: + + superimpositions + + + 0.9979717 + evidence + cleaner0 + 2023-07-27T14:54:13Z + DUMMY: + + structures + + + protein + PR: + cleaner0 + 2023-07-27T12:47:44Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + protein + PR: + cleaner0 + 2023-07-27T12:47:44Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:02:09Z + + ED + + + 0.9972584 + evidence + cleaner0 + 2023-07-27T14:54:16Z + DUMMY: + + structures + + + protein_type + MESH: + cleaner0 + 2023-07-27T13:03:42Z + + OmpR/PhoB subfamily + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:50:19Z + + full-length + + + + pone.0149903.t002.xml + pone.0149903.t002 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><thead><tr><th align="left" rowspan="1" colspan="1"><italic>Protein</italic></th><th align="left" rowspan="1" colspan="1"><italic>PDB</italic></th><th align="left" rowspan="1" colspan="1"><italic>Z-score</italic></th><th align="left" rowspan="1" colspan="1"><italic>RMSD (Å)</italic></th><th align="left" rowspan="1" colspan="1"><italic>Number of residues (total number of residues)</italic></th><th align="left" rowspan="1" colspan="1"><italic>Seq</italic>. <italic>ID (%)*</italic></th><th align="left" rowspan="1" colspan="1"><italic>Reference</italic></th></tr></thead><tbody><tr><td align="center" colspan="7" rowspan="1"><bold>Receiver domain</bold></td></tr><tr><td align="justify" rowspan="1" colspan="1">KdpE</td><td align="justify" rowspan="1" colspan="1">4KNY</td><td align="justify" rowspan="1" colspan="1">18.8</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">117 (222)</td><td align="left" rowspan="1" colspan="1">28</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref042" ref-type="bibr">42</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">YycF</td><td align="justify" rowspan="1" colspan="1">2ZWM</td><td align="justify" rowspan="1" colspan="1">18.3</td><td align="justify" rowspan="1" colspan="1">1.7</td><td align="justify" rowspan="1" colspan="1">115 (120)</td><td align="left" rowspan="1" colspan="1">35</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref053" ref-type="bibr">53</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">YycF</td><td align="justify" rowspan="1" colspan="1">3F6P</td><td align="justify" rowspan="1" colspan="1">18.1</td><td align="justify" rowspan="1" colspan="1">1.7</td><td align="justify" rowspan="1" colspan="1">114 (120)</td><td align="left" rowspan="1" colspan="1">35</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref081" ref-type="bibr">81</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">DivK</td><td align="justify" rowspan="1" colspan="1">1M5T</td><td align="justify" rowspan="1" colspan="1">18.1</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">116 (123)</td><td align="left" rowspan="1" colspan="1">27</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref065" ref-type="bibr">65</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">KdpE</td><td align="justify" rowspan="1" colspan="1">1ZH2</td><td align="justify" rowspan="1" colspan="1">18.0</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">115 (120)</td><td align="left" rowspan="1" colspan="1">28</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref074" ref-type="bibr">74</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">PhoB</td><td align="justify" rowspan="1" colspan="1">1B00</td><td align="justify" rowspan="1" colspan="1">17.0</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">113 (122)</td><td align="left" rowspan="1" colspan="1">30</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref047" ref-type="bibr">47</xref></td></tr><tr><td align="center" colspan="7" rowspan="1"><bold>Effector domain</bold></td></tr><tr><td align="justify" rowspan="1" colspan="1">PhoB</td><td align="justify" rowspan="1" colspan="1">1GXQ</td><td align="justify" rowspan="1" colspan="1">13.7</td><td align="justify" rowspan="1" colspan="1">1.7</td><td align="justify" rowspan="1" colspan="1">92 (105)</td><td align="left" rowspan="1" colspan="1">30</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref054" ref-type="bibr">54</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">PhoP</td><td align="justify" rowspan="1" colspan="1">2PMU</td><td align="justify" rowspan="1" colspan="1">13.4</td><td align="justify" rowspan="1" colspan="1">1.7</td><td align="justify" rowspan="1" colspan="1">87 (93)</td><td align="left" rowspan="1" colspan="1">30</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref082" ref-type="bibr">82</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">PhoB</td><td align="justify" rowspan="1" colspan="1">2Z33</td><td align="justify" rowspan="1" colspan="1">13.3</td><td align="justify" rowspan="1" colspan="1">1.8</td><td align="justify" rowspan="1" colspan="1">92 (104)</td><td align="left" rowspan="1" colspan="1">30</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref083" ref-type="bibr">83</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">PhoB (DNA bound)</td><td align="justify" rowspan="1" colspan="1">1GXP</td><td align="justify" rowspan="1" colspan="1">13.3</td><td align="justify" rowspan="1" colspan="1">2.0</td><td align="justify" rowspan="1" colspan="1">92 (101)</td><td align="left" rowspan="1" colspan="1">30</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref054" ref-type="bibr">54</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">SaeR</td><td align="justify" rowspan="1" colspan="1">4IXA</td><td align="justify" rowspan="1" colspan="1">13.0</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">94 (102)</td><td align="left" rowspan="1" colspan="1">29</td><td align="justify" rowspan="1" colspan="1">Not available</td></tr><tr><td align="justify" rowspan="1" colspan="1">RstA</td><td align="justify" rowspan="1" colspan="1">4NHJ</td><td align="justify" rowspan="1" colspan="1">11.8</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">85 (101)</td><td align="left" rowspan="1" colspan="1">29</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref084" ref-type="bibr">84</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">KdpE</td><td align="justify" rowspan="1" colspan="1">4KNY</td><td align="justify" rowspan="1" colspan="1">11.5</td><td align="justify" rowspan="1" colspan="1">2.6</td><td align="justify" rowspan="1" colspan="1">86 (222)</td><td align="left" rowspan="1" colspan="1">28</td><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref042" ref-type="bibr">42</xref></td></tr><tr><td align="center" colspan="7" rowspan="1"><bold>Full-length Response Regulators</bold></td></tr><tr><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"><italic>PDB code</italic></td><td align="center" rowspan="1" colspan="1"><italic>N-terminal rmsd (Å)</italic></td><td align="center" rowspan="1" colspan="1"><italic>C-terminal rmsd (Å)</italic></td><td align="center" rowspan="1" colspan="1"><italic>DNA bound</italic></td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"><italic>Reference</italic></td></tr><tr><td align="justify" rowspan="1" colspan="1">DrrB</td><td align="justify" rowspan="1" colspan="1">1P2F</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">2.3</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref040" ref-type="bibr">40</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">DrrD</td><td align="justify" rowspan="1" colspan="1">1KGS</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref041" ref-type="bibr">41</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">KdpE</td><td align="justify" rowspan="1" colspan="1">4KNY</td><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">2.6</td><td align="justify" rowspan="1" colspan="1">Yes</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref042" ref-type="bibr">42</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">MtrA</td><td align="justify" rowspan="1" colspan="1">2GWR</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">2.0</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref037" ref-type="bibr">37</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">PrrA</td><td align="justify" rowspan="1" colspan="1">1YS6</td><td align="justify" rowspan="1" colspan="1">2.0</td><td align="justify" rowspan="1" colspan="1">2.2</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref038" ref-type="bibr">38</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">RegX3</td><td align="justify" rowspan="1" colspan="1">2OQR</td><td align="justify" rowspan="1" colspan="1">2.3</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref036" ref-type="bibr">36</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">BaeR</td><td align="justify" rowspan="1" colspan="1">4B09</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">2.1</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref057" ref-type="bibr">57</xref></td></tr><tr><td align="justify" rowspan="1" colspan="1">VraR</td><td align="justify" rowspan="1" colspan="1">4GVP</td><td align="justify" rowspan="1" colspan="1">2.3</td><td align="justify" rowspan="1" colspan="1">2.6</td><td align="justify" rowspan="1" colspan="1">No</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"><xref rid="pone.0149903.ref085" ref-type="bibr">85</xref></td></tr></tbody></table> + + 24322 + Protein PDB Z-score RMSD (Å) Number of residues (total number of residues) Seq. ID (%)* Reference Receiver domain KdpE 4KNY 18.8 1.9 117 (222) 28 YycF 2ZWM 18.3 1.7 115 (120) 35 YycF 3F6P 18.1 1.7 114 (120) 35 DivK 1M5T 18.1 1.9 116 (123) 27 KdpE 1ZH2 18.0 1.9 115 (120) 28 PhoB 1B00 17.0 1.9 113 (122) 30 Effector domain PhoB 1GXQ 13.7 1.7 92 (105) 30 PhoP 2PMU 13.4 1.7 87 (93) 30 PhoB 2Z33 13.3 1.8 92 (104) 30 PhoB (DNA bound) 1GXP 13.3 2.0 92 (101) 30 SaeR 4IXA 13.0 2.1 94 (102) 29 Not available RstA 4NHJ 11.8 1.9 85 (101) 29 KdpE 4KNY 11.5 2.6 86 (222) 28 Full-length Response Regulators PDB code N-terminal rmsd (Å) C-terminal rmsd (Å) DNA bound Reference DrrB 1P2F 2.1 2.3 No DrrD 1KGS 2.1 1.9 No KdpE 4KNY 1.9 2.6 Yes MtrA 2GWR 2.1 2.0 No PrrA 1YS6 2.0 2.2 No RegX3 2OQR 2.3 2.1 No BaeR 4B09 2.1 2.1 No VraR 4GVP 2.3 2.6 No + + + RESULTS + paragraph + 25243 + Based on the Dali server, the NsrR-RD domain is structurally closely related to KdpE (PDB code: 4KNY) from E. coli, displaying a sequence identity of 28%. This structural homology is also reflected by the low rmsd of 1.9 Å over 117 Cα atoms after superimposition of the receiver domains of NsrR and KdpE (Table 2). Furthermore, the orientation of the helix α4 in NsrR is close to that present in KdpE (S1 Fig). + + 0.9987018 + experimental_method + cleaner0 + 2023-07-27T14:39:20Z + MESH: + + Dali server + + + 0.99931526 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.86894065 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9992918 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.99862313 + species + cleaner0 + 2023-07-27T13:21:52Z + MESH: + + E. coli + + + 0.9976701 + evidence + cleaner0 + 2023-07-27T13:18:49Z + DUMMY: + + rmsd + + + 0.99874544 + experimental_method + cleaner0 + 2023-07-27T14:39:23Z + MESH: + + superimposition + + + 0.99932015 + structure_element + cleaner0 + 2023-07-27T14:48:54Z + SO: + + receiver domains + + + 0.9993705 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99928564 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.99937433 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.99948055 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + 0.99938893 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99926966 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + + RESULTS + title_2 + 25662 + Active site residues and dimerization + + site + SO: + cleaner0 + 2023-07-27T13:26:23Z + + Active site + + + + RESULTS + paragraph + 25700 + All RRs contain a highly conserved aspartate residue in the active site (Fig 3; shown in red). Phosphorylation of this aspartate residue induces a conformational change leading to the activation of the effector domain that binds DNA and regulates the transcription of target genes. This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). + + 0.9992574 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.99878275 + protein_state + cleaner0 + 2023-07-27T13:26:48Z + DUMMY: + + highly conserved + + + 0.99776244 + residue_name + cleaner0 + 2023-07-27T13:26:44Z + SO: + + aspartate + + + 0.99911153 + site + cleaner0 + 2023-07-27T13:26:22Z + SO: + + active site + + + 0.9962476 + ptm + cleaner0 + 2023-07-27T12:50:12Z + MESH: + + Phosphorylation + + + 0.99746764 + residue_name + cleaner0 + 2023-07-27T13:26:45Z + SO: + + aspartate + + + 0.998433 + structure_element + cleaner0 + 2023-07-27T13:02:03Z + SO: + + effector domain + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:49:58Z + + DNA + + + 0.9949208 + ptm + cleaner0 + 2023-07-27T12:50:12Z + MESH: + + phosphorylation + + + 0.99892527 + protein_state + cleaner0 + 2023-07-27T13:26:50Z + DUMMY: + + conserved + + + 0.99866056 + protein_type + cleaner0 + 2023-07-27T13:03:37Z + MESH: + + response regulators + + + protein_type + MESH: + cleaner0 + 2023-07-27T13:26:11Z + + lantibiotic resistance-associated RRs + + + 0.9993311 + protein + cleaner0 + 2023-07-27T15:01:01Z + PR: + + BraR + + + 0.9983806 + species + cleaner0 + 2023-07-27T13:24:30Z + MESH: + + L. monocytogenes + + + 0.9992518 + protein + cleaner0 + 2023-07-27T13:25:32Z + PR: + + BceR + + + 0.9978348 + species + cleaner0 + 2023-07-27T12:55:20Z + MESH: + + Bacillus subtilis + + + 0.999303 + protein + cleaner0 + 2023-07-27T13:25:27Z + PR: + + CprR + + + 0.99798745 + species + cleaner0 + 2023-07-27T13:24:37Z + MESH: + + C. difficile + + + 0.9993249 + protein + cleaner0 + 2023-07-27T13:25:21Z + PR: + + GraR + + + 0.9983581 + species + cleaner0 + 2023-07-27T12:55:51Z + MESH: + + S. aureus + + + 0.9993357 + protein + cleaner0 + 2023-07-27T13:25:16Z + PR: + + LcrR + + + 0.9983671 + species + cleaner0 + 2023-07-27T13:24:43Z + MESH: + + S. mutans + + + 0.999337 + protein + cleaner0 + 2023-07-27T13:25:01Z + PR: + + LisR + + + 0.9993486 + protein + cleaner0 + 2023-07-27T13:24:55Z + PR: + + VirR + + + 0.9984111 + species + cleaner0 + 2023-07-27T13:24:31Z + MESH: + + L. monocytogenes + + + + pone.0149903.g003.jpg + pone.0149903.g003 + FIG + fig_title_caption + 26299 + Sequence alignment of NsrR protein with other response regulators. + + 0.9984927 + experimental_method + cleaner0 + 2023-07-27T14:39:33Z + MESH: + + Sequence alignment + + + 0.99662125 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9976491 + protein_type + cleaner0 + 2023-07-27T13:03:37Z + MESH: + + response regulators + + + + pone.0149903.g003.jpg + pone.0149903.g003 + FIG + fig_caption + 26366 + A sequence alignment of NsrR with RRs belonging to the OmpR/PhoB subfamily (marked in grey) and RRs involved in lantibiotic resistance (black) is shown. The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. The linker region of the known structures is underlined within the sequence. + + 0.99870265 + experimental_method + cleaner0 + 2023-07-27T14:39:36Z + MESH: + + sequence alignment + + + 0.9993049 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99912876 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + protein_type + MESH: + cleaner0 + 2023-07-27T13:03:42Z + + OmpR/PhoB subfamily + + + 0.99905664 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:52:04Z + + lantibiotic + + + 0.9988451 + site + cleaner0 + 2023-07-27T13:26:23Z + SO: + + active site + + + 0.9969194 + residue_name + cleaner0 + 2023-07-27T13:26:45Z + SO: + + aspartate + + + 0.99909055 + site + cleaner0 + 2023-07-27T14:33:08Z + SO: + + acidic pocket + + + 0.99875534 + site + cleaner0 + 2023-07-27T13:29:10Z + SO: + + switch residues + + + 0.99890363 + protein_state + cleaner0 + 2023-07-27T13:27:17Z + DUMMY: + + conserved + + + 0.99776745 + residue_name + cleaner0 + 2023-07-27T13:27:21Z + SO: + + lysine + + + 0.9988321 + protein_state + cleaner0 + 2023-07-27T13:27:15Z + DUMMY: + + highly conserved + + + 0.9983248 + structure_element + cleaner0 + 2023-07-27T13:52:42Z + SO: + + linker region + + + 0.9989053 + site + cleaner0 + 2023-07-27T13:52:10Z + SO: + + dimer interface + + + 0.99926007 + structure_element + cleaner0 + 2023-07-27T12:48:56Z + SO: + + receiver domain + + + 0.99575883 + chemical + cleaner0 + 2023-07-27T12:49:58Z + CHEBI: + + DNA + + + 0.9976224 + structure_element + cleaner0 + 2023-07-27T13:52:42Z + SO: + + linker region + + + 0.6837149 + evidence + cleaner0 + 2023-07-27T14:54:20Z + DUMMY: + + structures + + + + RESULTS + paragraph + 27143 + The putative phosphorylation site of NsrR is Asp55, which is localized at the end of strand β3 (Fig 3, shown in red; Fig 4) and lies within an acidic environment composed of the side chains of Glu12 and Asp13 (Fig 3, highlighted in pink). This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. In NsrR, Glu12, Asp13, and Asp55 are in close proximity of a highly conserved Lys104 residue (highlighted in green in Fig 3). + + 0.9982375 + site + cleaner0 + 2023-07-27T13:27:36Z + SO: + + phosphorylation site + + + 0.9994178 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9995726 + residue_name_number + cleaner0 + 2023-07-27T13:27:43Z + DUMMY: + + Asp55 + + + 0.9946991 + structure_element + cleaner0 + 2023-07-27T14:49:00Z + SO: + + strand + + + 0.99752575 + structure_element + cleaner0 + 2023-07-27T13:27:50Z + SO: + + β3 + + + 0.999574 + residue_name_number + cleaner0 + 2023-07-27T13:28:01Z + DUMMY: + + Glu12 + + + 0.99957496 + residue_name_number + cleaner0 + 2023-07-27T13:28:07Z + DUMMY: + + Asp13 + + + 0.99850416 + site + cleaner0 + 2023-07-27T14:33:14Z + SO: + + pocket + + + 0.645693 + protein_state + cleaner0 + 2023-07-27T14:30:06Z + DUMMY: + + acidic + + + 0.9391881 + site + cleaner0 + 2023-07-27T13:26:23Z + SO: + + active site + + + 0.9979899 + evidence + cleaner0 + 2023-07-27T14:54:24Z + DUMMY: + + structures + + + 0.9990188 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.99903286 + protein + cleaner0 + 2023-07-27T13:28:31Z + PR: + + PhoB + + + 0.9978824 + species + cleaner0 + 2023-07-27T13:21:53Z + MESH: + + E. coli + + + 0.9987301 + protein + cleaner0 + 2023-07-27T13:04:36Z + PR: + + PhoP + + + 0.99839306 + species + cleaner0 + 2023-07-27T13:28:24Z + MESH: + + M. tuberculosis + + + 0.9990707 + protein + cleaner0 + 2023-07-27T13:28:37Z + PR: + + DivK + + + 0.9986853 + species + cleaner0 + 2023-07-27T13:28:43Z + MESH: + + Caulobacter crescentus + + + 0.99941945 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9995726 + residue_name_number + cleaner0 + 2023-07-27T13:28:02Z + DUMMY: + + Glu12 + + + 0.999577 + residue_name_number + cleaner0 + 2023-07-27T13:28:07Z + DUMMY: + + Asp13 + + + 0.9995803 + residue_name_number + cleaner0 + 2023-07-27T13:27:44Z + DUMMY: + + Asp55 + + + 0.99885833 + protein_state + cleaner0 + 2023-07-27T13:28:51Z + DUMMY: + + highly conserved + + + 0.9995635 + residue_name_number + cleaner0 + 2023-07-27T13:28:55Z + DUMMY: + + Lys104 + + + + pone.0149903.g004.jpg + pone.0149903.g004 + FIG + fig_title_caption + 27691 + Location of the highly conserved Asp55 and inactive state conformation of the key switch residues, Ser82 and Phe101 in NsrR-RD. + + 0.99882954 + protein_state + cleaner0 + 2023-07-27T13:29:02Z + DUMMY: + + highly conserved + + + 0.999592 + residue_name_number + cleaner0 + 2023-07-27T13:27:44Z + DUMMY: + + Asp55 + + + 0.9993012 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.9986496 + site + cleaner0 + 2023-07-27T13:29:09Z + SO: + + switch residues + + + 0.9995976 + residue_name_number + cleaner0 + 2023-07-27T13:29:15Z + DUMMY: + + Ser82 + + + 0.9996113 + residue_name_number + cleaner0 + 2023-07-27T13:29:19Z + DUMMY: + + Phe101 + + + 0.49365017 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + + pone.0149903.g004.jpg + pone.0149903.g004 + FIG + fig_caption + 27819 + NsrR (represented in yellow) displays a geometry representing the inactive state as deduced from the inactive state structure of PhoB (shown in brown, PDB code 1B00) (a). The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). + + 0.9993332 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.99925977 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.999292 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.9965479 + evidence + cleaner0 + 2023-07-27T14:54:29Z + DUMMY: + + structure + + + 0.999335 + protein + cleaner0 + 2023-07-27T13:28:32Z + PR: + + PhoB + + + 0.99931276 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.99935955 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + 0.9992592 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.9966697 + evidence + cleaner0 + 2023-07-27T14:54:31Z + DUMMY: + + structure + + + 0.99936277 + protein + cleaner0 + 2023-07-27T13:28:32Z + PR: + + PhoB + + + 0.9986987 + site + cleaner0 + 2023-07-27T13:29:10Z + SO: + + switch residues + + + 0.9996013 + residue_name_number + cleaner0 + 2023-07-27T13:29:15Z + DUMMY: + + Ser82 + + + 0.9996087 + residue_name_number + cleaner0 + 2023-07-27T13:29:21Z + DUMMY: + + Phe101 + + + 0.99907035 + site + cleaner0 + 2023-07-27T13:26:23Z + SO: + + active site + + + 0.9995894 + residue_name_number + cleaner0 + 2023-07-27T13:27:44Z + DUMMY: + + Asp55 + + + 0.9993617 + protein + cleaner0 + 2023-07-27T12:47:44Z + PR: + + NsrR + + + + RESULTS + paragraph + 28258 + A divalent metal ion is usually bound in this acidic environment and is essential for phosphorylation and de-phosphorylation of RRs. In some RRs like CheY, Mg2+ is observed in the structure, bound near the phosphorylation site. In the KdpE regulator from E. coli that is involved in osmoregulation, a divalent calcium ion is present. However, the structure of NsrR-RD did not contain any divalent ion. Instead, a water molecule is present, which interacts with Glu12 of the acidic pocket, Lys104, and another water molecule in the vicinity. + + 0.9805444 + ptm + cleaner0 + 2023-07-27T12:50:12Z + MESH: + + phosphorylation + + + 0.8213034 + ptm + cleaner0 + 2023-07-27T13:30:09Z + MESH: + + de-phosphorylation + + + 0.99926823 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.99929166 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.998963 + protein + cleaner0 + 2023-07-27T13:30:25Z + PR: + + CheY + + + 0.9990341 + chemical + cleaner0 + 2023-07-27T13:30:17Z + CHEBI: + + Mg2+ + + + 0.9984463 + evidence + cleaner0 + 2023-07-27T14:54:35Z + DUMMY: + + structure + + + 0.9743113 + protein_state + cleaner0 + 2023-07-27T14:30:17Z + DUMMY: + + bound + + + 0.9988899 + site + cleaner0 + 2023-07-27T13:27:37Z + SO: + + phosphorylation site + + + protein + PR: + cleaner0 + 2023-07-27T13:04:53Z + + KdpE + + + protein_type + MESH: + cleaner0 + 2023-07-27T13:30:53Z + + regulator + + + 0.9986133 + species + cleaner0 + 2023-07-27T13:21:53Z + MESH: + + E. coli + + + 0.99884295 + chemical + cleaner0 + 2023-07-27T14:28:05Z + CHEBI: + + calcium + + + 0.9983334 + evidence + cleaner0 + 2023-07-27T14:54:38Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + 0.9990127 + chemical + cleaner0 + 2023-07-27T14:28:09Z + CHEBI: + + water + + + 0.99956554 + residue_name_number + cleaner0 + 2023-07-27T13:28:02Z + DUMMY: + + Glu12 + + + 0.9985113 + site + cleaner0 + 2023-07-27T14:33:19Z + SO: + + acidic pocket + + + 0.9995339 + residue_name_number + cleaner0 + 2023-07-27T13:28:56Z + DUMMY: + + Lys104 + + + 0.99894077 + chemical + cleaner0 + 2023-07-27T14:28:14Z + CHEBI: + + water + + + + RESULTS + paragraph + 28799 + Within the β4-α4 loop and in β5 of the RD of RRs, specific amino acids are crucial for signal transduction from the RD to the ED via conformational changes that are a consequence of phosphorylation of the RD. These amino acids are Ser/Thr and Phe/Tyr located at the end of β4 and before β5, respectively, and designated as “signature switch residues”. As seen in the alignment (Fig 3, highlighted in blue), these signature residues (Ser/Thr and Phe/Tyr) are highly conserved in the lantibiotic resistance-associated RRs. The orientation of the side chains of these residues determines whether the RD is in an active or inactive state. In the inactive state, the phenylalanine or tyrosine residue faces away from the active site, and the corresponding serine or threonine residue adopts an outward-facing conformation as well (Fig 4A). In contrast, the switch residues face towards the active site in the active state conformation (Fig 4B). + + 0.9991443 + structure_element + cleaner0 + 2023-07-27T13:31:13Z + SO: + + β4-α4 loop + + + 0.99936444 + structure_element + cleaner0 + 2023-07-27T13:31:29Z + SO: + + β5 + + + 0.99957186 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9990752 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.99949634 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9982326 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.9962458 + ptm + cleaner0 + 2023-07-27T12:50:12Z + MESH: + + phosphorylation + + + 0.99957305 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9958106 + residue_name + cleaner0 + 2023-07-27T13:31:39Z + SO: + + Ser + + + 0.99469405 + residue_name + cleaner0 + 2023-07-27T13:31:44Z + SO: + + Thr + + + 0.9963182 + residue_name + cleaner0 + 2023-07-27T13:31:48Z + SO: + + Phe + + + 0.9953441 + residue_name + cleaner0 + 2023-07-27T13:31:53Z + SO: + + Tyr + + + 0.9993119 + structure_element + cleaner0 + 2023-07-27T13:31:33Z + SO: + + β4 + + + 0.99923587 + structure_element + cleaner0 + 2023-07-27T13:31:28Z + SO: + + β5 + + + 0.9972958 + site + cleaner0 + 2023-07-27T13:32:48Z + SO: + + signature switch residues + + + 0.99501884 + experimental_method + cleaner0 + 2023-07-27T14:39:45Z + MESH: + + alignment + + + 0.99686414 + residue_name + cleaner0 + 2023-07-27T13:31:40Z + SO: + + Ser + + + 0.9956923 + residue_name + cleaner0 + 2023-07-27T13:31:45Z + SO: + + Thr + + + 0.9970174 + residue_name + cleaner0 + 2023-07-27T13:31:49Z + SO: + + Phe + + + 0.99610436 + residue_name + cleaner0 + 2023-07-27T13:31:53Z + SO: + + Tyr + + + 0.99895 + protein_state + cleaner0 + 2023-07-27T14:30:22Z + DUMMY: + + highly conserved + + + 0.99675304 + protein_type + cleaner0 + 2023-07-27T13:32:10Z + MESH: + + lantibiotic resistance-associated RRs + + + 0.99955064 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.99911636 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.99923444 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.99929166 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.9975184 + residue_name + cleaner0 + 2023-07-27T13:32:23Z + SO: + + phenylalanine + + + 0.9971637 + residue_name + cleaner0 + 2023-07-27T13:32:32Z + SO: + + tyrosine + + + 0.99902064 + site + cleaner0 + 2023-07-27T13:26:23Z + SO: + + active site + + + 0.9974087 + residue_name + cleaner0 + 2023-07-27T13:32:18Z + SO: + + serine + + + 0.9970175 + residue_name + cleaner0 + 2023-07-27T13:32:27Z + SO: + + threonine + + + 0.99904776 + protein_state + cleaner0 + 2023-07-27T14:30:26Z + DUMMY: + + outward-facing + + + 0.9982003 + site + cleaner0 + 2023-07-27T13:29:10Z + SO: + + switch residues + + + 0.99894565 + site + cleaner0 + 2023-07-27T13:26:23Z + SO: + + active site + + + 0.99922574 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + + RESULTS + paragraph + 29755 + By sequence alignment with other lantibiotic resistance-associated RRs, these “signature switch residues” are identified as Ser82 and Phe101 in NsrR (see above). Although some RRs such as KdpE, BraR, BceR, GraR, and VirR contain a serine residue as the first switch residue, the others possess a threonine instead. Furthermore, the second switch residue is mostly a tyrosine, with NsrR, BraR, and BceR being the only exceptions containing a phenylalanine at that position. A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). + + 0.9987155 + experimental_method + cleaner0 + 2023-07-27T14:39:54Z + MESH: + + sequence alignment + + + 0.9867547 + protein_type + cleaner0 + 2023-07-27T13:32:11Z + MESH: + + lantibiotic resistance-associated RRs + + + 0.9982168 + site + cleaner0 + 2023-07-27T13:32:49Z + SO: + + signature switch residues + + + 0.9995956 + residue_name_number + cleaner0 + 2023-07-27T13:29:15Z + DUMMY: + + Ser82 + + + 0.9995952 + residue_name_number + cleaner0 + 2023-07-27T13:29:21Z + DUMMY: + + Phe101 + + + 0.9993794 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.9990345 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.9992231 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.99924433 + protein + cleaner0 + 2023-07-27T15:01:06Z + PR: + + BraR + + + 0.99924636 + protein + cleaner0 + 2023-07-27T13:25:33Z + PR: + + BceR + + + 0.999281 + protein + cleaner0 + 2023-07-27T13:25:22Z + PR: + + GraR + + + 0.9993087 + protein + cleaner0 + 2023-07-27T13:24:56Z + PR: + + VirR + + + 0.99800855 + residue_name + cleaner0 + 2023-07-27T13:32:19Z + SO: + + serine + + + 0.9939422 + site + cleaner0 + 2023-07-27T13:33:54Z + SO: + + first switch residue + + + 0.9978789 + residue_name + cleaner0 + 2023-07-27T13:32:28Z + SO: + + threonine + + + 0.9968428 + site + cleaner0 + 2023-07-27T13:33:59Z + SO: + + second switch residue + + + 0.99760485 + residue_name + cleaner0 + 2023-07-27T13:32:33Z + SO: + + tyrosine + + + 0.9993279 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99930656 + protein + cleaner0 + 2023-07-27T15:01:10Z + PR: + + BraR + + + 0.99928623 + protein + cleaner0 + 2023-07-27T13:25:33Z + PR: + + BceR + + + 0.99770194 + residue_name + cleaner0 + 2023-07-27T13:32:23Z + SO: + + phenylalanine + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + 0.9980069 + evidence + cleaner0 + 2023-07-27T14:54:45Z + DUMMY: + + structure + + + 0.9976042 + evidence + cleaner0 + 2023-07-27T14:54:50Z + DUMMY: + + structures + + + 0.9993369 + protein + cleaner0 + 2023-07-27T13:28:32Z + PR: + + PhoB + + + 0.9991748 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.99925095 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.9996037 + residue_name_number + cleaner0 + 2023-07-27T13:29:15Z + DUMMY: + + Ser82 + + + 0.99407935 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + 0.998924 + site + cleaner0 + 2023-07-27T13:26:23Z + SO: + + active site + + + 0.9995777 + residue_name_number + cleaner0 + 2023-07-27T13:27:44Z + DUMMY: + + Asp55 + + + 0.99959034 + residue_name_number + cleaner0 + 2023-07-27T13:29:21Z + DUMMY: + + Phe101 + + + 0.99907637 + protein_state + cleaner0 + 2023-07-27T14:30:33Z + DUMMY: + + outward + + + 0.999186 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.9952152 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + + RESULTS + paragraph + 30568 + As mentioned above, RRs contain a phosphorylation-activated switch and normally exist in equilibrium between the active and inactive conformations. Phosphorylation shifts the equilibrium towards the active conformation and induces the formation of rotationally symmetric dimers on the α4-β5-α5 interface of RDs. It has been suggested that dimerization is crucial for DNA-binding of RRs of the OmpR/PhoB subfamily. + + 0.9986951 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + 0.90515757 + protein_state + cleaner0 + 2023-07-27T13:34:19Z + DUMMY: + + phosphorylation-activated + + + 0.9873713 + site + cleaner0 + 2023-07-27T14:33:36Z + SO: + + switch + + + 0.9990515 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.9990963 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.99587303 + ptm + cleaner0 + 2023-07-27T12:50:12Z + MESH: + + Phosphorylation + + + 0.99908197 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.9985526 + oligomeric_state + cleaner0 + 2023-07-27T13:37:55Z + DUMMY: + + dimers + + + 0.9987912 + site + cleaner0 + 2023-07-27T13:34:35Z + SO: + + α4-β5-α5 interface + + + 0.6789144 + structure_element + cleaner0 + 2023-07-27T13:34:51Z + SO: + + RDs + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:49:58Z + + DNA + + + 0.99902534 + protein_type + cleaner0 + 2023-07-27T13:02:16Z + MESH: + + RRs + + + protein_type + MESH: + cleaner0 + 2023-07-27T13:03:42Z + + OmpR/PhoB subfamily + + + + RESULTS + paragraph + 30993 + The RD domain of NsrR was crystallized with two separate monomers in the asymmetric unit. Therefore, we performed a DALI search and focused on RD domains that were structurally determined as functional dimers. In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. + + 0.9994293 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.99937123 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99852055 + experimental_method + cleaner0 + 2023-07-27T14:40:01Z + MESH: + + crystallized + + + 0.998896 + oligomeric_state + cleaner0 + 2023-07-27T13:18:37Z + DUMMY: + + monomers + + + 0.998592 + experimental_method + cleaner0 + 2023-07-27T14:40:04Z + MESH: + + DALI search + + + 0.9994253 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.57029694 + protein_state + cleaner0 + 2023-07-27T14:30:38Z + DUMMY: + + functional + + + 0.99879634 + oligomeric_state + cleaner0 + 2023-07-27T13:37:55Z + DUMMY: + + dimers + + + 0.9987651 + oligomeric_state + cleaner0 + 2023-07-27T13:38:00Z + DUMMY: + + dimer + + + 0.9991476 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.9984451 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9985321 + species + cleaner0 + 2023-07-27T13:21:53Z + MESH: + + E. coli + + + 0.99824125 + evidence + cleaner0 + 2023-07-27T13:34:58Z + DUMMY: + + Z-score + + + 0.9985576 + evidence + cleaner0 + 2023-07-27T13:18:49Z + DUMMY: + + rmsd + + + 0.99799037 + evidence + cleaner0 + 2023-07-27T14:54:55Z + DUMMY: + + structure + + + 0.9678832 + protein_state + cleaner0 + 2023-07-27T14:30:42Z + DUMMY: + + functional + + + 0.99880075 + oligomeric_state + cleaner0 + 2023-07-27T13:38:00Z + DUMMY: + + dimer + + + 0.9994287 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9983266 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.99853164 + species + cleaner0 + 2023-07-27T13:21:53Z + MESH: + + E. coli + + + 0.58302724 + evidence + cleaner0 + 2023-07-27T14:54:57Z + DUMMY: + + structures + + + + RESULTS + paragraph + 31471 + We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). Therefore, helix α4 and the N-terminal loop were shifted to the position of KdpE by primarily modifying backbone torsion angles in the region immediately C-terminal to helix α4. Afterwards, helix α4 and the adjacent loops were energy minimized with the MAB force field as implemented in the program Moloc; all other atoms of NsrR-RD were kept fixed. The result is highlighted in S2B Fig. The energy minimized structure of NsrR-RD was then superimposed on the dimeric structure of KdpE. + + 0.99855644 + experimental_method + cleaner0 + 2023-07-27T14:40:15Z + MESH: + + aligned + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + 0.99868745 + oligomeric_state + cleaner0 + 2023-07-27T13:18:37Z + DUMMY: + + monomers + + + 0.9988589 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9914846 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9993125 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.9994419 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + 0.9982222 + evidence + cleaner0 + 2023-07-27T14:55:02Z + DUMMY: + + structures + + + 0.97735256 + structure_element + cleaner0 + 2023-07-27T13:34:52Z + SO: + + RDs + + + 0.99917585 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.99945635 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + 0.9992138 + structure_element + cleaner0 + 2023-07-27T14:49:05Z + SO: + + loop + + + 0.99885714 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + 0.99884015 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + 0.9991743 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.9994629 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + 0.9989548 + structure_element + cleaner0 + 2023-07-27T14:49:09Z + SO: + + loop + + + 0.9748784 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9989729 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.9994205 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + 0.9989839 + structure_element + cleaner0 + 2023-07-27T13:24:13Z + SO: + + helix + + + 0.9994455 + structure_element + cleaner0 + 2023-07-27T13:20:47Z + SO: + + α4 + + + 0.99017185 + structure_element + cleaner0 + 2023-07-27T14:49:12Z + SO: + + loops + + + experimental_method + MESH: + cleaner0 + 2023-07-27T14:40:43Z + + energy minimized with the MAB force field + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + 0.90363157 + protein_state + cleaner0 + 2023-07-27T14:41:01Z + DUMMY: + + energy minimized + + + 0.9972216 + evidence + cleaner0 + 2023-07-27T14:55:06Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + 0.99862397 + experimental_method + cleaner0 + 2023-07-27T14:41:04Z + MESH: + + superimposed + + + 0.9988412 + oligomeric_state + cleaner0 + 2023-07-27T13:37:49Z + DUMMY: + + dimeric + + + 0.99755234 + evidence + cleaner0 + 2023-07-27T14:55:09Z + DUMMY: + + structure + + + 0.9974003 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + + RESULTS + paragraph + 32240 + The putative functional dimer of NsrR-RD is depicted in Fig 5. The dimeric interface is formed by α4-β5-α5 of RD (Fig 5A), as previously observed in other RRs. In KdpE, a network of salt bridges and other electrostatic interactions stabilize the interface within a single monomer as well as between the monomers. Majority of these interactions involve residues that are highly conserved within the OmpR/PhoB subfamily of RRs. In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). Structurally, a similar set of residues is also found in NsrR: Leu94 (α4), Val110 (α5) and Ala113 (α5), respectively (depicted as spheres in Fig 5B), which are conserved to some extent on sequence level (highlighted in yellow; Fig 3). + + 0.99889624 + oligomeric_state + cleaner0 + 2023-07-27T13:38:00Z + DUMMY: + + dimer + + + 0.51494896 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.630312 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9989375 + site + cleaner0 + 2023-07-27T13:37:42Z + SO: + + dimeric interface + + + 0.9579359 + structure_element + cleaner0 + 2023-07-27T13:35:58Z + SO: + + α4-β5-α5 + + + 0.99956626 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9991442 + protein_type + cleaner0 + 2023-07-27T13:02:17Z + MESH: + + RRs + + + 0.95380193 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9955318 + bond_interaction + cleaner0 + 2023-07-27T13:37:04Z + MESH: + + salt bridges + + + 0.9959799 + bond_interaction + cleaner0 + 2023-07-27T13:37:08Z + MESH: + + electrostatic interactions + + + 0.99902105 + site + cleaner0 + 2023-07-27T14:33:42Z + SO: + + interface + + + 0.99887425 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + 0.9986094 + oligomeric_state + cleaner0 + 2023-07-27T13:18:37Z + DUMMY: + + monomers + + + 0.99877894 + protein_state + cleaner0 + 2023-07-27T14:30:45Z + DUMMY: + + highly conserved + + + protein_type + MESH: + cleaner0 + 2023-07-27T13:03:42Z + + OmpR/PhoB subfamily + + + 0.99906415 + protein_type + cleaner0 + 2023-07-27T13:02:17Z + MESH: + + RRs + + + 0.9990164 + site + cleaner0 + 2023-07-27T13:37:00Z + SO: + + dimeric interface + + + 0.55356544 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9988632 + site + cleaner0 + 2023-07-27T13:36:57Z + SO: + + hydrophobic patch + + + 0.9995628 + residue_name_number + cleaner0 + 2023-07-27T13:36:13Z + DUMMY: + + Ile88 + + + 0.99930334 + structure_element + cleaner0 + 2023-07-27T13:20:48Z + SO: + + α4 + + + 0.9995664 + residue_name_number + cleaner0 + 2023-07-27T13:36:18Z + DUMMY: + + Leu91 + + + 0.9991937 + structure_element + cleaner0 + 2023-07-27T13:20:48Z + SO: + + α4 + + + 0.9995665 + residue_name_number + cleaner0 + 2023-07-27T13:36:23Z + DUMMY: + + Ala110 + + + 0.9991861 + structure_element + cleaner0 + 2023-07-27T13:23:20Z + SO: + + α5 + + + 0.99957925 + residue_name_number + cleaner0 + 2023-07-27T13:36:28Z + DUMMY: + + Val114 + + + 0.9991819 + structure_element + cleaner0 + 2023-07-27T13:23:20Z + SO: + + α5 + + + 0.9992605 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.9995808 + residue_name_number + cleaner0 + 2023-07-27T13:36:34Z + DUMMY: + + Leu94 + + + 0.9992924 + structure_element + cleaner0 + 2023-07-27T13:20:48Z + SO: + + α4 + + + 0.9995852 + residue_name_number + cleaner0 + 2023-07-27T13:36:39Z + DUMMY: + + Val110 + + + 0.99926513 + structure_element + cleaner0 + 2023-07-27T13:23:20Z + SO: + + α5 + + + 0.9995722 + residue_name_number + cleaner0 + 2023-07-27T13:36:43Z + DUMMY: + + Ala113 + + + 0.99918777 + structure_element + cleaner0 + 2023-07-27T13:23:20Z + SO: + + α5 + + + 0.85403407 + protein_state + cleaner0 + 2023-07-27T13:36:48Z + DUMMY: + + conserved + + + + pone.0149903.g005.jpg + pone.0149903.g005 + FIG + fig_title_caption + 33094 + Functional dimer orientation of the RDs of NsrR. + + 0.99872226 + oligomeric_state + cleaner0 + 2023-07-27T13:37:59Z + DUMMY: + + dimer + + + 0.9926811 + structure_element + cleaner0 + 2023-07-27T13:34:52Z + SO: + + RDs + + + 0.99941933 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + + pone.0149903.g005.jpg + pone.0149903.g005 + FIG + fig_caption + 33143 + Dimeric structure of the RD of NsrR aligned to the structure of KdpE (PDB code 1ZH2, not shown). (a) The two monomers of NsrR as functional dimers are represented in a cartoon representation displayed in cyan and yellow colors. (b) Zoom-in of the dimeric interface mediated by α4-β5-α5. The monomer-monomer interactions are facilitated by hydrophobic residues (displayed as spheres), inter- and intra-domain interactions (displayed as sticks). The layout is adopted from. + + 0.9987577 + oligomeric_state + cleaner0 + 2023-07-27T13:37:49Z + DUMMY: + + Dimeric + + + 0.992486 + evidence + cleaner0 + 2023-07-27T14:55:17Z + DUMMY: + + structure + + + 0.9995028 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9994117 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.9930173 + evidence + cleaner0 + 2023-07-27T14:55:20Z + DUMMY: + + structure + + + 0.99721897 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.99883336 + oligomeric_state + cleaner0 + 2023-07-27T13:18:37Z + DUMMY: + + monomers + + + 0.9994098 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99882084 + oligomeric_state + cleaner0 + 2023-07-27T13:37:54Z + DUMMY: + + dimers + + + 0.99887955 + site + cleaner0 + 2023-07-27T13:37:41Z + SO: + + dimeric interface + + + structure_element + SO: + cleaner0 + 2023-07-27T13:37:36Z + + α4-β5-α5 + + + 0.9766981 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + 0.95569986 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + + RESULTS + paragraph + 33626 + Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). Some of these interactions can also be identified in the dimeric model of NsrR-RD. Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). Asp99 (α4–β5 loop; Fig 3, shown in cyan) points toward the side chain of Arg121. This interaction is also observed in KdpE (Asp96 (α4–β5 loop) and Arg118 (α5)). In KdpE, Arg111 is additionally stabilized by another intra-molecular salt bridge with Glu107 (α5). Interestingly, in NsrR-RD this amino acid corresponds to Val110 (highlighted in yellow in Fig 3). As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan). Interestingly, whenever an arginine is present at this position (Arg111 in KdpE), a glutamate (Glu107 in KdpE) is present as well, presumably stabilizing the arginine side chain. However, when a lysine is present at this position, the glutamate is exchanged to a hydrophobic residue contributing to the hydrophobic patch described above. Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. + + 0.99631524 + bond_interaction + cleaner0 + 2023-07-27T13:37:09Z + MESH: + + electrostatic interactions + + + 0.9952254 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + 0.98631454 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + 0.99910814 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.99952424 + residue_name_number + cleaner0 + 2023-07-27T13:38:33Z + DUMMY: + + Asp97 + + + 0.9993235 + structure_element + cleaner0 + 2023-07-27T13:31:29Z + SO: + + β5 + + + 0.99950457 + residue_name_number + cleaner0 + 2023-07-27T13:38:39Z + DUMMY: + + Arg111 + + + 0.99937016 + structure_element + cleaner0 + 2023-07-27T13:23:20Z + SO: + + α5 + + + 0.9995074 + residue_name_number + cleaner0 + 2023-07-27T13:38:43Z + DUMMY: + + Asp96 + + + 0.9988633 + structure_element + cleaner0 + 2023-07-27T13:38:27Z + SO: + + α4–β5 loop + + + 0.9994844 + residue_name_number + cleaner0 + 2023-07-27T13:38:49Z + DUMMY: + + Arg118 + + + 0.9993494 + structure_element + cleaner0 + 2023-07-27T13:23:20Z + SO: + + α5 + + + 0.9995092 + residue_name_number + cleaner0 + 2023-07-27T13:38:54Z + DUMMY: + + Asp92 + + + 0.99934167 + structure_element + cleaner0 + 2023-07-27T13:20:48Z + SO: + + α4 + + + 0.9995054 + residue_name_number + cleaner0 + 2023-07-27T13:38:59Z + DUMMY: + + Arg113 + + + 0.9993774 + structure_element + cleaner0 + 2023-07-27T13:23:20Z + SO: + + α5 + + + 0.99893016 + oligomeric_state + cleaner0 + 2023-07-27T13:37:50Z + DUMMY: + + dimeric + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + 0.99952006 + residue_name_number + cleaner0 + 2023-07-27T13:39:13Z + DUMMY: + + Asp100 + + + 0.9993284 + structure_element + cleaner0 + 2023-07-27T13:31:29Z + SO: + + β5 + + + 0.9995098 + residue_name_number + cleaner0 + 2023-07-27T13:39:18Z + DUMMY: + + Lys114 + + + 0.99942315 + structure_element + cleaner0 + 2023-07-27T13:23:20Z + SO: + + α5 + + + 0.9988727 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + 0.9995421 + residue_name_number + cleaner0 + 2023-07-27T13:39:23Z + DUMMY: + + Asn95 + + + 0.9994081 + structure_element + cleaner0 + 2023-07-27T13:20:48Z + SO: + + α4 + + + 0.9987913 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + 0.9995415 + residue_name_number + cleaner0 + 2023-07-27T13:39:29Z + DUMMY: + + Thr116 + + + 0.9993672 + structure_element + cleaner0 + 2023-07-27T13:23:20Z + SO: + + α5 + + + 0.99874914 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + 0.9995491 + residue_name_number + cleaner0 + 2023-07-27T13:39:34Z + DUMMY: + + Asp99 + + + 0.9990548 + structure_element + cleaner0 + 2023-07-27T13:38:28Z + SO: + + α4–β5 loop + + + 0.9995134 + residue_name_number + cleaner0 + 2023-07-27T13:19:10Z + DUMMY: + + Arg121 + + + 0.9983334 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.99952364 + residue_name_number + cleaner0 + 2023-07-27T13:38:44Z + DUMMY: + + Asp96 + + + 0.998955 + structure_element + cleaner0 + 2023-07-27T13:38:28Z + SO: + + α4–β5 loop + + + 0.99951935 + residue_name_number + cleaner0 + 2023-07-27T13:38:49Z + DUMMY: + + Arg118 + + + 0.9993787 + structure_element + cleaner0 + 2023-07-27T13:23:20Z + SO: + + α5 + + + 0.9982463 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9995041 + residue_name_number + cleaner0 + 2023-07-27T13:38:39Z + DUMMY: + + Arg111 + + + 0.9941443 + bond_interaction + cleaner0 + 2023-07-27T13:40:59Z + MESH: + + salt bridge + + + 0.9995147 + residue_name_number + cleaner0 + 2023-07-27T13:40:07Z + DUMMY: + + Glu107 + + + 0.9993493 + structure_element + cleaner0 + 2023-07-27T13:23:20Z + SO: + + α5 + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:01:58Z + + RD + + + 0.9995579 + residue_name_number + cleaner0 + 2023-07-27T13:36:40Z + DUMMY: + + Val110 + + + 0.9959351 + experimental_method + cleaner0 + 2023-07-27T14:41:12Z + MESH: + + alignment + + + 0.99778134 + residue_name + cleaner0 + 2023-07-27T13:40:32Z + SO: + + arginine + + + 0.999509 + residue_name_number + cleaner0 + 2023-07-27T13:38:39Z + DUMMY: + + Arg111 + + + 0.99797183 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9978527 + residue_name + cleaner0 + 2023-07-27T13:40:32Z + SO: + + arginine + + + 0.99764544 + residue_name + cleaner0 + 2023-07-27T13:27:21Z + SO: + + lysine + + + 0.99949944 + residue_name_number + cleaner0 + 2023-07-27T13:39:18Z + DUMMY: + + Lys114 + + + 0.99928856 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.9987174 + protein_type + cleaner0 + 2023-07-27T13:02:17Z + MESH: + + RRs + + + 0.95484114 + experimental_method + cleaner0 + 2023-07-27T14:41:18Z + MESH: + + alignment + + + 0.9974968 + residue_name + cleaner0 + 2023-07-27T13:40:32Z + SO: + + arginine + + + 0.99949026 + residue_name_number + cleaner0 + 2023-07-27T13:38:39Z + DUMMY: + + Arg111 + + + 0.9955577 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9974166 + residue_name + cleaner0 + 2023-07-27T13:40:38Z + SO: + + glutamate + + + 0.9995074 + residue_name_number + cleaner0 + 2023-07-27T13:40:08Z + DUMMY: + + Glu107 + + + 0.99625236 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.997759 + residue_name + cleaner0 + 2023-07-27T13:40:33Z + SO: + + arginine + + + 0.99775827 + residue_name + cleaner0 + 2023-07-27T13:27:21Z + SO: + + lysine + + + 0.99813676 + residue_name + cleaner0 + 2023-07-27T13:40:39Z + SO: + + glutamate + + + 0.99848545 + site + cleaner0 + 2023-07-27T14:33:53Z + SO: + + hydrophobic patch + + + 0.9991761 + protein + cleaner0 + 2023-07-27T13:28:32Z + PR: + + PhoB + + + 0.99839574 + species + cleaner0 + 2023-07-27T13:21:53Z + MESH: + + E. coli + + + 0.999171 + protein + cleaner0 + 2023-07-27T13:04:37Z + PR: + + PhoP + + + 0.99854344 + species + cleaner0 + 2023-07-27T14:27:14Z + MESH: + + B. subtilis + + + 0.99686015 + experimental_method + cleaner0 + 2023-07-27T14:41:21Z + MESH: + + mutating + + + 0.99954164 + residue_name_number + cleaner0 + 2023-07-27T13:39:13Z + DUMMY: + + Asp100 + + + 0.9995635 + residue_name_number + cleaner0 + 2023-07-27T13:36:40Z + DUMMY: + + Val110 + + + 0.99952424 + residue_name_number + cleaner0 + 2023-07-27T13:39:18Z + DUMMY: + + Lys114 + + + 0.99932265 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99883074 + oligomeric_state + cleaner0 + 2023-07-27T14:32:29Z + DUMMY: + + monomeric + + + 0.99803215 + protein_type + cleaner0 + 2023-07-27T12:47:38Z + MESH: + + response regulator + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T14:42:08Z + + lost the ability to dimerize + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:49:58Z + + DNA + + + + RESULTS + title_2 + 35450 + Overall Structure of C-terminal DNA-binding effector domain of NsrR + + 0.9876565 + evidence + cleaner0 + 2023-07-27T14:55:28Z + DUMMY: + + Structure + + + 0.9935832 + structure_element + cleaner0 + 2023-07-27T12:49:02Z + SO: + + DNA-binding effector domain + + + 0.99934393 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + + RESULTS + paragraph + 35518 + The structure of NsrR-ED from S. agalactiae was determined using experimental phases from a single-wavelength anomalous dispersion dataset from the rectangular plate-shaped crystal derivatized with platinum at a resolution of 1.6 Å in space group P21212. The Rwork and Rfree values after refinement were 0.18 and 0.22, respectively. Ramachandran validation was done using MolProbity. Almost all residues (99.48%, 193 amino acids) were in the preferred or allowed regions, while 0.52% (1 amino acid) were localized in the disallowed region. The latter is Glu128 (last residue of the linker region) of chain B that is involved in crystal contacts and, therefore, likely adopts an unfavorable conformation. The structure contained a few ethylene glycol molecules introduced by the cryo-protecting procedure. The data collection and refinement statistics are listed in Table 1. + + 0.99821424 + evidence + cleaner0 + 2023-07-27T14:55:31Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:02:09Z + + ED + + + 0.9984174 + species + cleaner0 + 2023-07-27T12:56:39Z + MESH: + + S. agalactiae + + + 0.9618526 + experimental_method + cleaner0 + 2023-07-27T14:42:34Z + MESH: + + single-wavelength anomalous dispersion dataset + + + 0.7794421 + chemical + cleaner0 + 2023-07-27T13:42:13Z + CHEBI: + + platinum + + + 0.9981863 + evidence + cleaner0 + 2023-07-27T14:55:34Z + DUMMY: + + Rwork + + + 0.9977349 + evidence + cleaner0 + 2023-07-27T14:55:37Z + DUMMY: + + Rfree + + + evidence + DUMMY: + cleaner0 + 2023-07-27T13:41:43Z + + Ramachandran validation + + + 0.9995865 + residue_name_number + cleaner0 + 2023-07-27T13:42:01Z + DUMMY: + + Glu128 + + + 0.99935395 + structure_element + cleaner0 + 2023-07-27T13:52:42Z + SO: + + linker region + + + 0.9993657 + structure_element + cleaner0 + 2023-07-27T13:43:05Z + SO: + + chain B + + + 0.9984049 + evidence + cleaner0 + 2023-07-27T14:55:41Z + DUMMY: + + structure + + + 0.9827248 + chemical + cleaner0 + 2023-07-27T13:42:16Z + CHEBI: + + ethylene glycol + + + + RESULTS + paragraph + 36393 + The C-terminal effector DNA-binding domain of NsrR is about 13 kDa in size and consists of residues 129–243 (including 21 amino acid residues of the expression tag). Monomer A contains residue 129–224 and monomer B contain residues 128–225. For Asp147 of chain A and Glu174 of chain B, poor electron density was observed for the side chains and, thus, these side chains were removed during refinement. The asymmetric unit contains two copies of NsrR-ED related by two-fold rotational symmetry. An overlay revealed that both monomers display high similarity in their overall structure with an rmsd of 0.5 Å over 95 Cα atoms. We therefore describe for the overall structure only monomer A. + + 0.9993509 + structure_element + cleaner0 + 2023-07-27T13:42:27Z + SO: + + effector DNA-binding domain + + + 0.99940264 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.9978315 + residue_range + cleaner0 + 2023-07-27T13:42:37Z + DUMMY: + + 129–243 + + + 0.99845517 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + Monomer + + + 0.99803346 + structure_element + cleaner0 + 2023-07-27T13:42:49Z + SO: + + A + + + 0.99786425 + residue_range + cleaner0 + 2023-07-27T13:42:44Z + DUMMY: + + 129–224 + + + 0.9984761 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + 0.9977214 + structure_element + cleaner0 + 2023-07-27T13:42:53Z + SO: + + B + + + 0.9979051 + residue_range + cleaner0 + 2023-07-27T13:42:47Z + DUMMY: + + 128–225 + + + 0.99954945 + residue_name_number + cleaner0 + 2023-07-27T14:07:39Z + DUMMY: + + Asp147 + + + 0.99925065 + structure_element + cleaner0 + 2023-07-27T13:42:59Z + SO: + + chain A + + + 0.99954873 + residue_name_number + cleaner0 + 2023-07-27T14:08:04Z + DUMMY: + + Glu174 + + + 0.99925137 + structure_element + cleaner0 + 2023-07-27T13:43:04Z + SO: + + chain B + + + 0.9981958 + evidence + cleaner0 + 2023-07-27T14:55:44Z + DUMMY: + + electron density + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:02:09Z + + ED + + + 0.99870884 + experimental_method + cleaner0 + 2023-07-27T14:42:54Z + MESH: + + overlay + + + 0.9979359 + oligomeric_state + cleaner0 + 2023-07-27T13:18:37Z + DUMMY: + + monomers + + + 0.9912305 + evidence + cleaner0 + 2023-07-27T14:55:49Z + DUMMY: + + structure + + + 0.9986547 + evidence + cleaner0 + 2023-07-27T13:18:49Z + DUMMY: + + rmsd + + + 0.9728925 + evidence + cleaner0 + 2023-07-27T14:55:52Z + DUMMY: + + structure + + + 0.9981357 + oligomeric_state + cleaner0 + 2023-07-27T13:18:43Z + DUMMY: + + monomer + + + 0.9901218 + structure_element + cleaner0 + 2023-07-27T13:43:14Z + SO: + + A + + + + RESULTS + paragraph + 37089 + The ED domain of NsrR consists of six β-strands and three α-helices in a β6-β7-β8-β9-α6-α7-α8-β10-β11 topology (the secondary structure elements are counted in continuation of those of the RD). The effector domain starts with a 4-stranded antiparallel β-sheet, followed by three α-helices and eventually ends in a C-terminal β-hairpin (Fig 6). The two β-sheets sandwich the three α-helices. + + 0.99951637 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.9992681 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.9784997 + structure_element + cleaner0 + 2023-07-27T13:44:02Z + SO: + + β-strands + + + 0.99849993 + structure_element + cleaner0 + 2023-07-27T13:43:54Z + SO: + + α-helices + + + structure_element + SO: + cleaner0 + 2023-07-27T13:43:36Z + + β6-β7-β8-β9-α6-α7-α8-β10-β11 + + + 0.9995215 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9992093 + structure_element + cleaner0 + 2023-07-27T13:02:03Z + SO: + + effector domain + + + 0.99837077 + structure_element + cleaner0 + 2023-07-27T13:44:07Z + SO: + + 4-stranded antiparallel β-sheet + + + 0.9986458 + structure_element + cleaner0 + 2023-07-27T13:43:54Z + SO: + + α-helices + + + 0.99908113 + structure_element + cleaner0 + 2023-07-27T13:43:43Z + SO: + + β-hairpin + + + 0.998061 + structure_element + cleaner0 + 2023-07-27T13:43:47Z + SO: + + β-sheets + + + 0.99907786 + structure_element + cleaner0 + 2023-07-27T13:43:53Z + SO: + + α-helices + + + + pone.0149903.g006.jpg + pone.0149903.g006 + FIG + fig_title_caption + 37534 + Structure of the C-terminal effector domain of NsrR. + + 0.99674857 + evidence + cleaner0 + 2023-07-27T14:55:57Z + DUMMY: + + Structure + + + 0.9992944 + structure_element + cleaner0 + 2023-07-27T13:02:03Z + SO: + + effector domain + + + 0.99928445 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + + pone.0149903.g006.jpg + pone.0149903.g006 + FIG + fig_caption + 37587 + Cartoon representation of the C-terminal effector domain of NsrR (green; recognition helix in cyan). The structural areas with the highest variations compared to the effector domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1GXQ) are marked. The transactivation loop of MtrA is missing in the structure, therefore, the two termini are connected by a dashed line. + + 0.99799705 + structure_element + cleaner0 + 2023-07-27T13:02:03Z + SO: + + effector domain + + + 0.99933964 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.999251 + structure_element + cleaner0 + 2023-07-27T13:06:32Z + SO: + + recognition helix + + + 0.9877807 + structure_element + cleaner0 + 2023-07-27T14:49:18Z + SO: + + effector domains + + + 0.9993525 + protein + cleaner0 + 2023-07-27T13:04:43Z + PR: + + DrrB + + + 0.99932384 + protein + cleaner0 + 2023-07-27T13:04:26Z + PR: + + MtrA + + + 0.9993337 + protein + cleaner0 + 2023-07-27T13:28:32Z + PR: + + PhoB + + + 0.99938977 + structure_element + cleaner0 + 2023-07-27T13:46:56Z + SO: + + transactivation loop + + + 0.99929297 + protein + cleaner0 + 2023-07-27T13:04:26Z + PR: + + MtrA + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T14:32:10Z + + missing + + + 0.99806935 + evidence + cleaner0 + 2023-07-27T14:56:08Z + DUMMY: + + structure + + + + RESULTS + paragraph + 37966 + The characteristic feature of the OmpR/PhoB subfamily of RRs is a winged helix-turn-helix (wHTH) fold that is adopted by the α7-loop-α8 segment in full-length and single effector domain structures of RRs of this subfamily. The structure of NsrR-ED also contains such a wHTH motif built up by helices α7 and α8 (Fig 6). The second helix of the wHTH motif is important for DNA-binding and, therefore, is termed “recognition helix” (shown in cyan in Fig 6). Furthermore, a helix within the HTH motif, named “positioning helix”, is important for proper orientation and positioning of the loop between these two helices and is referred to as “transactivation loop” (also called α-loop; Fig 6). In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). The 16-residue long, solvent-exposed recognition helix α8 of NsrR-ED contains four positively charged residues that can potentially interact with DNA. These are Arg198, Arg200, Lys201, and Lys202. When comparing the sequence of NsrR with PhoB, KdpE, and MtrA, the alignment (Fig 3, colored in blue) emphasizes the variations at these positions, except for Arg200, which is conserved throughout the lantibiotic resistance RRs. Additionally, Lys202 is also highly conserved throughout the family of RRs except PhoB, clearly reflecting differences in the sequences of DNA to be bound. + + protein_type + MESH: + cleaner0 + 2023-07-27T13:03:42Z + + OmpR/PhoB subfamily + + + 0.99827945 + protein_type + cleaner0 + 2023-07-27T13:02:17Z + MESH: + + RRs + + + 0.9990485 + structure_element + cleaner0 + 2023-07-27T13:04:09Z + SO: + + winged helix-turn-helix + + + 0.99674976 + structure_element + cleaner0 + 2023-07-27T13:04:14Z + SO: + + wHTH + + + 0.9984329 + structure_element + cleaner0 + 2023-07-27T13:46:34Z + SO: + + α7-loop-α8 segment + + + 0.999053 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + structure_element + SO: + cleaner0 + 2023-07-27T13:02:03Z + + effector domain + + + 0.99596786 + evidence + cleaner0 + 2023-07-27T14:56:16Z + DUMMY: + + structures + + + 0.99909353 + protein_type + cleaner0 + 2023-07-27T13:02:17Z + MESH: + + RRs + + + 0.9975446 + evidence + cleaner0 + 2023-07-27T14:56:12Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:02:09Z + + ED + + + structure_element + SO: + cleaner0 + 2023-07-27T13:04:14Z + + wHTH + + + 0.98942417 + structure_element + cleaner0 + 2023-07-27T14:49:22Z + SO: + + helices + + + 0.9994035 + structure_element + cleaner0 + 2023-07-27T13:45:32Z + SO: + + α7 + + + 0.99931467 + structure_element + cleaner0 + 2023-07-27T13:47:07Z + SO: + + α8 + + + structure_element + SO: + cleaner0 + 2023-07-27T13:46:02Z + + helix + + + structure_element + SO: + cleaner0 + 2023-07-27T13:04:14Z + + wHTH + + + 0.8894096 + chemical + cleaner0 + 2023-07-27T12:49:58Z + CHEBI: + + DNA + + + 0.9991862 + structure_element + cleaner0 + 2023-07-27T13:06:32Z + SO: + + recognition helix + + + 0.92855954 + structure_element + cleaner0 + 2023-07-27T13:24:14Z + SO: + + helix + + + structure_element + SO: + cleaner0 + 2023-07-27T13:46:19Z + + HTH + + + 0.9990045 + structure_element + cleaner0 + 2023-07-27T13:46:49Z + SO: + + positioning helix + + + 0.99816555 + structure_element + cleaner0 + 2023-07-27T14:49:26Z + SO: + + loop + + + 0.9992594 + structure_element + cleaner0 + 2023-07-27T13:46:55Z + SO: + + transactivation loop + + + 0.9993288 + structure_element + cleaner0 + 2023-07-27T14:49:30Z + SO: + + α-loop + + + 0.99809307 + evidence + cleaner0 + 2023-07-27T14:56:19Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:02:09Z + + ED + + + 0.99365133 + structure_element + cleaner0 + 2023-07-27T13:24:14Z + SO: + + helix + + + 0.99941874 + structure_element + cleaner0 + 2023-07-27T13:47:06Z + SO: + + α8 + + + 0.99918497 + structure_element + cleaner0 + 2023-07-27T13:06:32Z + SO: + + recognition helix + + + 0.9993969 + structure_element + cleaner0 + 2023-07-27T13:45:33Z + SO: + + α7 + + + 0.99906194 + structure_element + cleaner0 + 2023-07-27T13:46:50Z + SO: + + positioning helix + + + 0.84965134 + structure_element + cleaner0 + 2023-07-27T14:49:36Z + SO: + + loop region + + + 0.9968534 + structure_element + cleaner0 + 2023-07-27T13:47:42Z + SO: + + α7-α8 + + + 0.9992547 + structure_element + cleaner0 + 2023-07-27T13:46:56Z + SO: + + transactivation loop + + + 0.9991375 + protein_type + cleaner0 + 2023-07-27T13:02:17Z + MESH: + + RRs + + + 0.95358604 + protein_state + cleaner0 + 2023-07-27T14:31:06Z + DUMMY: + + solvent-exposed + + + 0.99926454 + structure_element + cleaner0 + 2023-07-27T13:06:32Z + SO: + + recognition helix + + + 0.9992513 + structure_element + cleaner0 + 2023-07-27T13:47:07Z + SO: + + α8 + + + 0.58672327 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:02:09Z + + ED + + + 0.97925687 + chemical + cleaner0 + 2023-07-27T12:49:58Z + CHEBI: + + DNA + + + 0.99947995 + residue_name_number + cleaner0 + 2023-07-27T13:48:03Z + DUMMY: + + Arg198 + + + 0.999479 + residue_name_number + cleaner0 + 2023-07-27T13:48:08Z + DUMMY: + + Arg200 + + + 0.99947435 + residue_name_number + cleaner0 + 2023-07-27T13:48:12Z + DUMMY: + + Lys201 + + + 0.9994831 + residue_name_number + cleaner0 + 2023-07-27T13:48:17Z + DUMMY: + + Lys202 + + + 0.99930596 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.9993229 + protein + cleaner0 + 2023-07-27T13:28:32Z + PR: + + PhoB + + + 0.99927527 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9993191 + protein + cleaner0 + 2023-07-27T13:04:26Z + PR: + + MtrA + + + 0.9937802 + experimental_method + cleaner0 + 2023-07-27T14:42:59Z + MESH: + + alignment + + + 0.9994955 + residue_name_number + cleaner0 + 2023-07-27T13:48:09Z + DUMMY: + + Arg200 + + + 0.9980679 + protein_state + cleaner0 + 2023-07-27T13:48:38Z + DUMMY: + + conserved + + + 0.99790066 + protein_type + cleaner0 + 2023-07-27T13:48:33Z + MESH: + + lantibiotic resistance RRs + + + 0.9994874 + residue_name_number + cleaner0 + 2023-07-27T13:48:18Z + DUMMY: + + Lys202 + + + 0.99876195 + protein_state + cleaner0 + 2023-07-27T13:48:41Z + DUMMY: + + highly conserved + + + 0.99916935 + protein_type + cleaner0 + 2023-07-27T13:02:17Z + MESH: + + RRs + + + 0.9992446 + protein + cleaner0 + 2023-07-27T13:28:32Z + PR: + + PhoB + + + 0.96336824 + chemical + cleaner0 + 2023-07-27T12:49:58Z + CHEBI: + + DNA + + + + RESULTS + title_2 + 39484 + Comparison with structures of other effector domains + + 0.9963588 + evidence + cleaner0 + 2023-07-27T14:56:24Z + DUMMY: + + structures + + + 0.7678622 + structure_element + cleaner0 + 2023-07-27T14:49:40Z + SO: + + effector domains + + + + RESULTS + paragraph + 39537 + We performed a DALI search to identify structurally related proteins to NsrR-ED. Here the structure of the effector domain of PhoB from E. coli (PDB code: 1GXQ) (Z-score of 13.7) is structurally the most closely related. Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182–189) is also present in the structure of NsrR-ED that connects helices α7 and α8. The rmsd between the three helices of the effector domain (including the two helices forming the wHTH motif) of PhoB and NsrR-ED is 1.6 Å over 47 Cα atoms, clearly indicating that NsrR belongs to the OmpR/PhoB family of RRs. + + 0.9987474 + experimental_method + cleaner0 + 2023-07-27T14:43:29Z + MESH: + + DALI search + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:02:09Z + + ED + + + 0.99845695 + evidence + cleaner0 + 2023-07-27T14:56:27Z + DUMMY: + + structure + + + 0.9990599 + structure_element + cleaner0 + 2023-07-27T13:02:03Z + SO: + + effector domain + + + 0.99923027 + protein + cleaner0 + 2023-07-27T13:28:32Z + PR: + + PhoB + + + 0.99851304 + species + cleaner0 + 2023-07-27T13:21:53Z + MESH: + + E. coli + + + 0.99834114 + evidence + cleaner0 + 2023-07-27T13:34:58Z + DUMMY: + + Z-score + + + 0.9986601 + protein + cleaner0 + 2023-07-27T13:28:32Z + PR: + + PhoB + + + 0.9989355 + structure_element + cleaner0 + 2023-07-27T13:02:03Z + SO: + + effector domain + + + 0.96873546 + structure_element + cleaner0 + 2023-07-27T14:49:49Z + SO: + + loop + + + 0.9973235 + residue_range + cleaner0 + 2023-07-27T13:49:51Z + DUMMY: + + 182–189 + + + 0.99845123 + evidence + cleaner0 + 2023-07-27T14:56:30Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:02:09Z + + ED + + + 0.9939365 + structure_element + cleaner0 + 2023-07-27T14:49:58Z + SO: + + helices + + + 0.99922574 + structure_element + cleaner0 + 2023-07-27T13:45:33Z + SO: + + α7 + + + 0.9988863 + structure_element + cleaner0 + 2023-07-27T13:47:07Z + SO: + + α8 + + + 0.99877197 + evidence + cleaner0 + 2023-07-27T13:18:49Z + DUMMY: + + rmsd + + + 0.99845666 + structure_element + cleaner0 + 2023-07-27T13:02:03Z + SO: + + effector domain + + + 0.6872202 + structure_element + cleaner0 + 2023-07-27T14:49:54Z + SO: + + helices + + + structure_element + SO: + cleaner0 + 2023-07-27T13:04:14Z + + wHTH + + + 0.99910176 + protein + cleaner0 + 2023-07-27T13:28:32Z + PR: + + PhoB + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:02:09Z + + ED + + + 0.99917275 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + protein_type + MESH: + cleaner0 + 2023-07-27T13:49:46Z + + OmpR/PhoB family of RRs + + + + RESULTS + paragraph + 40153 + Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. Overall, the structures are very similar with rmsd’s ranging from 1.7 to 2.6 Å (Table 2). The highest variations (Fig 6) are visible in in the loop regions α7-α8, which corresponds to the transactivation loop. Interestingly, this region also shows low sequence conservation (Fig 3). In many RRs this transactivation loop along with the recognition helix α8, form inter-domain contacts in the inactive state and are only exposed upon activation of the RRs via a conformational change where the N- and C-terminal domains move away from each other. + + 0.9987494 + experimental_method + cleaner0 + 2023-07-27T14:43:43Z + MESH: + + superimposed + + + 0.9971052 + structure_element + cleaner0 + 2023-07-27T13:02:03Z + SO: + + effector domain + + + 0.99924135 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + structure_element + SO: + cleaner0 + 2023-07-27T13:02:09Z + + ED + + + 0.9866476 + structure_element + cleaner0 + 2023-07-27T14:50:01Z + SO: + + effector domains + + + protein_type + MESH: + cleaner0 + 2023-07-27T13:50:19Z + + OmpR/PhoB family + + + 0.99935406 + protein + cleaner0 + 2023-07-27T13:04:43Z + PR: + + DrrB + + + 0.9993337 + protein + cleaner0 + 2023-07-27T13:04:26Z + PR: + + MtrA + + + 0.9747043 + structure_element + cleaner0 + 2023-07-27T13:02:03Z + SO: + + effector domain + + + 0.99701023 + evidence + cleaner0 + 2023-07-27T14:56:48Z + DUMMY: + + structure + + + 0.9992217 + protein + cleaner0 + 2023-07-27T13:28:32Z + PR: + + PhoB + + + 0.9985159 + species + cleaner0 + 2023-07-27T13:21:53Z + MESH: + + E. coli + + + 0.9981047 + evidence + cleaner0 + 2023-07-27T14:56:50Z + DUMMY: + + structures + + + 0.9984321 + evidence + cleaner0 + 2023-07-27T13:18:49Z + DUMMY: + + rmsd + + + 0.9979315 + structure_element + cleaner0 + 2023-07-27T14:50:08Z + SO: + + loop + + + 0.9983066 + structure_element + cleaner0 + 2023-07-27T13:50:38Z + SO: + + α7-α8 + + + 0.99934065 + structure_element + cleaner0 + 2023-07-27T13:46:56Z + SO: + + transactivation loop + + + 0.9992601 + protein_type + cleaner0 + 2023-07-27T13:02:17Z + MESH: + + RRs + + + 0.9994088 + structure_element + cleaner0 + 2023-07-27T13:46:56Z + SO: + + transactivation loop + + + 0.9993139 + structure_element + cleaner0 + 2023-07-27T13:06:32Z + SO: + + recognition helix + + + 0.99946314 + structure_element + cleaner0 + 2023-07-27T13:47:07Z + SO: + + α8 + + + 0.9992349 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.9992236 + protein_type + cleaner0 + 2023-07-27T13:02:17Z + MESH: + + RRs + + + + RESULTS + title_2 + 40956 + Linker region + + 0.99827135 + structure_element + cleaner0 + 2023-07-27T13:52:42Z + SO: + + Linker region + + + + RESULTS + paragraph + 40970 + The linkers that connect the RDs and EDs in response regulators are highly variable with respect to both length and sequence. The exact boundaries of these linkers are difficult to predict from sequence alignments in the absence of structural information of the distinct RR. Linker lengths in OmpR/PhoB proteins of unknown structure have been estimated by comparing the number of residues between conserved landmark residues in the regulatory and effector domains to those from structurally characterized family members. Such analysis has indicated that linker lengths vary from 5 to 20 residues. Similar to the OmpR/PhoB family, the lantibiotic resistance-associated family of response regulators also displays diverse linker regions, which are recognized in sequence alignments by the introduction of gaps (Fig 3). Interestingly, two arginine residues (Arg120 and Arg121 in NsrR; Fig 3, shown in purple) at the end of the RDs seem to be strictly conserved throughout the family of response regulators in both the OmpR/PhoB and lantibiotic resistance-associated RRs, indicating a conserved similarity. As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of α5 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue. This aspartate residue is identified in NsrR as Asp99 (see above). Arginine 121 of NsrR points towards this Asp99 residue however, the distance for a salt bridge interaction is too large. + + 0.99914396 + structure_element + cleaner0 + 2023-07-27T14:50:18Z + SO: + + linkers + + + 0.9995012 + structure_element + cleaner0 + 2023-07-27T13:34:52Z + SO: + + RDs + + + 0.99951756 + structure_element + cleaner0 + 2023-07-27T14:50:23Z + SO: + + EDs + + + 0.998411 + protein_type + cleaner0 + 2023-07-27T13:03:37Z + MESH: + + response regulators + + + 0.99304634 + protein_state + cleaner0 + 2023-07-27T14:31:40Z + DUMMY: + + highly variable + + + 0.9991146 + structure_element + cleaner0 + 2023-07-27T14:50:26Z + SO: + + linkers + + + 0.99842864 + experimental_method + cleaner0 + 2023-07-27T14:43:57Z + MESH: + + sequence alignments + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:59:04Z + + absence of + + + 0.9994678 + protein_type + cleaner0 + 2023-07-27T12:58:46Z + MESH: + + RR + + + 0.9903804 + structure_element + cleaner0 + 2023-07-27T14:50:30Z + SO: + + Linker + + + 0.9952068 + protein_type + cleaner0 + 2023-07-27T13:50:54Z + MESH: + + OmpR/PhoB proteins + + + 0.99726564 + structure_element + cleaner0 + 2023-07-27T14:50:37Z + SO: + + regulatory and effector domains + + + 0.9916016 + protein_type + cleaner0 + 2023-07-27T13:51:15Z + MESH: + + OmpR/PhoB family + + + 0.9810604 + protein_type + cleaner0 + 2023-07-27T13:51:07Z + MESH: + + lantibiotic resistance-associated family of response regulators + + + 0.9988881 + structure_element + cleaner0 + 2023-07-27T14:50:45Z + SO: + + linker regions + + + 0.9986211 + experimental_method + cleaner0 + 2023-07-27T14:43:59Z + MESH: + + sequence alignments + + + 0.99692875 + residue_name + cleaner0 + 2023-07-27T13:40:33Z + SO: + + arginine + + + 0.9995346 + residue_name_number + cleaner0 + 2023-07-27T13:19:05Z + DUMMY: + + Arg120 + + + 0.99953735 + residue_name_number + cleaner0 + 2023-07-27T13:19:10Z + DUMMY: + + Arg121 + + + 0.99940956 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99954176 + structure_element + cleaner0 + 2023-07-27T13:34:52Z + SO: + + RDs + + + 0.9988153 + protein_state + cleaner0 + 2023-07-27T14:31:45Z + DUMMY: + + strictly conserved + + + 0.9983387 + protein_type + cleaner0 + 2023-07-27T13:03:37Z + MESH: + + response regulators + + + protein_type + MESH: + cleaner0 + 2023-07-27T13:51:41Z + + OmpR/PhoB and lantibiotic resistance-associated RRs + + + 0.98540246 + protein_state + cleaner0 + 2023-07-27T14:31:49Z + DUMMY: + + conserved + + + 0.99819034 + evidence + cleaner0 + 2023-07-27T14:56:55Z + DUMMY: + + structures + + + 0.99942976 + protein + cleaner0 + 2023-07-27T13:04:26Z + PR: + + MtrA + + + 0.99939 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9974917 + residue_name + cleaner0 + 2023-07-27T13:40:33Z + SO: + + arginine + + + 0.9993438 + structure_element + cleaner0 + 2023-07-27T13:23:20Z + SO: + + α5 + + + 0.9989869 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.9989382 + site + cleaner0 + 2023-07-27T13:52:09Z + SO: + + dimer interface + + + 0.9995241 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.99504364 + bond_interaction + cleaner0 + 2023-07-27T13:41:00Z + MESH: + + salt bridge + + + 0.99703443 + residue_name + cleaner0 + 2023-07-27T13:26:45Z + SO: + + aspartate + + + 0.9972184 + residue_name + cleaner0 + 2023-07-27T13:26:45Z + SO: + + aspartate + + + 0.99941874 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.999471 + residue_name_number + cleaner0 + 2023-07-27T13:39:35Z + DUMMY: + + Asp99 + + + 0.9929842 + residue_name_number + cleaner0 + 2023-07-27T13:51:51Z + DUMMY: + + Arginine 121 + + + 0.9994128 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99952173 + residue_name_number + cleaner0 + 2023-07-27T13:39:35Z + DUMMY: + + Asp99 + + + 0.99290204 + bond_interaction + cleaner0 + 2023-07-27T13:41:00Z + MESH: + + salt bridge + + + + RESULTS + paragraph + 42477 + Although we aimed at crystallizing full-length NsrR, this endeavor failed due to proteolytic cleavage within the linker region during the time period of crystallization. Nonetheless, the structures of NsrR-RD and NsrR-ED together provide the required structural knowledge to predict the linker region that joins the receiver and effector domains. The linker region of NsrR consists of approximately nine residues (Fig 3), comprising 120RRSQQFIQQ128 (underlined residues are neither present in the structure of RD nor in ED of NsrR) and contains two positively charged amino acids. + + 0.99736965 + experimental_method + cleaner0 + 2023-07-27T14:44:04Z + MESH: + + crystallizing + + + 0.99897003 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.99939334 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.9992441 + structure_element + cleaner0 + 2023-07-27T13:52:42Z + SO: + + linker region + + + 0.9977715 + experimental_method + cleaner0 + 2023-07-27T14:44:12Z + MESH: + + crystallization + + + 0.9983114 + evidence + cleaner0 + 2023-07-27T14:57:00Z + DUMMY: + + structures + + + protein + PR: + cleaner0 + 2023-07-27T12:47:45Z + + NsrR + + + 0.50491655 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.49194387 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.5739222 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.9992206 + structure_element + cleaner0 + 2023-07-27T13:52:41Z + SO: + + linker region + + + 0.9984927 + structure_element + cleaner0 + 2023-07-27T14:50:52Z + SO: + + receiver and effector domains + + + 0.9992281 + structure_element + cleaner0 + 2023-07-27T13:52:42Z + SO: + + linker region + + + 0.9993925 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.91002107 + structure_element + cleaner0 + 2023-07-27T13:52:45Z + SO: + + 120RRSQQFIQQ128 + + + 0.9981325 + evidence + cleaner0 + 2023-07-27T14:57:02Z + DUMMY: + + structure + + + 0.9994654 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9994773 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.9993216 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + + RESULTS + title_2 + 43058 + DNA-binding mode of NsrR using a full-length model + + 0.97402394 + chemical + cleaner0 + 2023-07-27T12:49:58Z + CHEBI: + + DNA + + + 0.9993411 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99909955 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + + RESULTS + paragraph + 43109 + Since the structures of both domains of NsrR were determined, we used this structural information together with the available crystal structures of related proteins to create a model of the full-length NsrR in its active and inactive state. + + 0.9957717 + evidence + cleaner0 + 2023-07-27T14:57:08Z + DUMMY: + + structures + + + 0.9993992 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.92059565 + evidence + cleaner0 + 2023-07-27T14:57:11Z + DUMMY: + + structural information + + + 0.9983585 + evidence + cleaner0 + 2023-07-27T14:57:14Z + DUMMY: + + crystal structures + + + 0.99907106 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.99939525 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99906856 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.99910116 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + + RESULTS + paragraph + 43350 + To achieve this, we first carefully analyzed the outcome of the Dali search for each domain and identified structurally highly similar proteins (based on Z-scores and rmsd values) and choose the full-length structures previously reported. This resulted in a list of possible templates for modeling the full-length structure of NsrR (Table 2). In solution, RRs exist in equilibrium between the active and inactive state, which is shifted towards the active state upon phosphorylation of the ED. This results in oligomerization of the RR and a higher affinity towards DNA. + + 0.9985951 + experimental_method + cleaner0 + 2023-07-27T14:44:17Z + MESH: + + Dali search + + + 0.99855995 + evidence + cleaner0 + 2023-07-27T14:57:27Z + DUMMY: + + Z-scores + + + 0.9982128 + evidence + cleaner0 + 2023-07-27T13:18:49Z + DUMMY: + + rmsd + + + 0.9991496 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.99840325 + evidence + cleaner0 + 2023-07-27T14:57:17Z + DUMMY: + + structures + + + 0.9990954 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.99808913 + evidence + cleaner0 + 2023-07-27T14:57:32Z + DUMMY: + + structure + + + 0.9993893 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99833775 + protein_type + cleaner0 + 2023-07-27T13:02:17Z + MESH: + + RRs + + + 0.9991147 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.99910897 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.99906796 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.99414575 + ptm + cleaner0 + 2023-07-27T12:50:12Z + MESH: + + phosphorylation + + + 0.999181 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.9882895 + protein_type + cleaner0 + 2023-07-27T12:58:46Z + MESH: + + RR + + + 0.9954269 + chemical + cleaner0 + 2023-07-27T12:49:58Z + CHEBI: + + DNA + + + + RESULTS + paragraph + 43921 + Based on the above-mentioned criteria, the structure of MtrA from M. tuberculosis, crystallized in an inactive and non-phosphorylated state, seemed best suited for modeling purposes. Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure. This mimics the closed inactive conformation of NsrR (Fig 7A; the missing linker is represented as dotted line). + + 0.9976841 + evidence + cleaner0 + 2023-07-27T14:57:20Z + DUMMY: + + structure + + + 0.99903095 + protein + cleaner0 + 2023-07-27T13:04:26Z + PR: + + MtrA + + + 0.9985315 + species + cleaner0 + 2023-07-27T13:28:25Z + MESH: + + M. tuberculosis + + + 0.99837327 + experimental_method + cleaner0 + 2023-07-27T14:44:21Z + MESH: + + crystallized + + + 0.9992687 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.99901205 + protein_state + cleaner0 + 2023-07-27T14:31:56Z + DUMMY: + + non-phosphorylated + + + 0.9976125 + structure_element + cleaner0 + 2023-07-27T13:53:09Z + SO: + + linker + + + 0.9992268 + protein + cleaner0 + 2023-07-27T13:04:26Z + PR: + + MtrA + + + 0.9992353 + structure_element + cleaner0 + 2023-07-27T13:53:04Z + SO: + + linker + + + 0.9994473 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99869484 + experimental_method + cleaner0 + 2023-07-27T14:44:24Z + MESH: + + aligned + + + 0.99834764 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99888676 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.99809986 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.96828985 + protein + cleaner0 + 2023-07-27T13:04:26Z + PR: + + MtrA + + + 0.99627304 + evidence + cleaner0 + 2023-07-27T14:57:22Z + DUMMY: + + structure + + + 0.99923456 + protein_state + cleaner0 + 2023-07-27T13:05:25Z + DUMMY: + + closed + + + 0.9991221 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.9994073 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.9783392 + protein_state + cleaner0 + 2023-07-27T14:32:09Z + DUMMY: + + missing + + + 0.9979387 + structure_element + cleaner0 + 2023-07-27T13:53:06Z + SO: + + linker + + + + pone.0149903.g007.jpg + pone.0149903.g007 + FIG + fig_title_caption + 44441 + Model of full-length NsrR in its inactive state and active state. + + 0.9991148 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.99936193 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.9992078 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.9992238 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + + pone.0149903.g007.jpg + pone.0149903.g007 + FIG + fig_caption + 44507 + The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. The missing linker is represented by a dotted line. (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. + + 0.99949074 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.99939835 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99950325 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.9961858 + structure_element + cleaner0 + 2023-07-27T13:06:32Z + SO: + + recognition helix + + + 0.99931276 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + Inactive + + + 0.9993789 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.9916055 + experimental_method + cleaner0 + 2023-07-27T14:44:36Z + MESH: + + aligned + + + 0.9903324 + evidence + cleaner0 + 2023-07-27T14:57:37Z + DUMMY: + + structure + + + 0.99943084 + protein + cleaner0 + 2023-07-27T13:04:26Z + PR: + + MtrA + + + 0.99928015 + protein_state + cleaner0 + 2023-07-27T13:05:25Z + DUMMY: + + closed + + + 0.99893826 + protein_state + cleaner0 + 2023-07-27T12:50:31Z + DUMMY: + + inactive + + + 0.9991382 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.9993907 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.999585 + residue_name_number + cleaner0 + 2023-07-27T13:29:21Z + DUMMY: + + Phe101 + + + 0.99958366 + residue_name_number + cleaner0 + 2023-07-27T14:08:18Z + DUMMY: + + Asp187 + + + 0.9992489 + protein_state + cleaner0 + 2023-07-27T13:05:25Z + DUMMY: + + closed + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T14:32:10Z + + missing + + + 0.9982326 + structure_element + cleaner0 + 2023-07-27T14:51:00Z + SO: + + linker + + + 0.99928844 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + Active + + + 0.9992003 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.9993948 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99929166 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.99759716 + experimental_method + cleaner0 + 2023-07-27T14:44:41Z + MESH: + + alignment + + + 0.9993851 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99301624 + evidence + cleaner0 + 2023-07-27T14:57:39Z + DUMMY: + + structure + + + 0.9988744 + protein_state + cleaner0 + 2023-07-27T13:53:38Z + DUMMY: + + DNA bound + + + 0.9658328 + evidence + cleaner0 + 2023-07-27T14:57:42Z + DUMMY: + + structure + + + 0.8901941 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:50:25Z + + active + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T13:05:19Z + + open + + + 0.9993364 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.9990963 + structure_element + cleaner0 + 2023-07-27T13:06:32Z + SO: + + recognition helix + + + + RESULTS + paragraph + 45268 + In MtrA, the two domains interact via the α4-β5-α5 interface of the receiver domain and the end of α7, α7-α8 loop and α8 of the effector domain. Both interfaces have been shown to form functionally important contact areas in the active state within members of the OmpR/PhoB subfamily. In our model of full-length NsrR, a similar orientation between the domains is observed, contributing to the inter-domain interactions. The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation. In the model of NsrR, similar amino acids are present, Phe101 (switch residue) and Asp188 (Fig 3, represented by orange boxes) forming a likewise similar network of interaction. + + 0.9994369 + protein + cleaner0 + 2023-07-27T13:04:26Z + PR: + + MtrA + + + 0.9988864 + site + cleaner0 + 2023-07-27T13:34:36Z + SO: + + α4-β5-α5 interface + + + 0.99934936 + structure_element + cleaner0 + 2023-07-27T12:48:56Z + SO: + + receiver domain + + + 0.9994467 + structure_element + cleaner0 + 2023-07-27T13:45:33Z + SO: + + α7 + + + 0.99927455 + structure_element + cleaner0 + 2023-07-27T13:54:04Z + SO: + + α7-α8 loop + + + 0.9993687 + structure_element + cleaner0 + 2023-07-27T13:47:07Z + SO: + + α8 + + + 0.9989896 + structure_element + cleaner0 + 2023-07-27T13:02:03Z + SO: + + effector domain + + + 0.99842715 + site + cleaner0 + 2023-07-27T14:34:06Z + SO: + + interfaces + + + 0.9991906 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + protein_type + MESH: + cleaner0 + 2023-07-27T13:03:42Z + + OmpR/PhoB subfamily + + + 0.9991438 + protein_state + cleaner0 + 2023-07-27T12:50:19Z + DUMMY: + + full-length + + + 0.99945253 + protein + cleaner0 + 2023-07-27T12:47:45Z + PR: + + NsrR + + + 0.99926037 + protein_state + cleaner0 + 2023-07-27T12:50:32Z + DUMMY: + + inactive + + + 0.9994436 + protein + cleaner0 + 2023-07-27T13:04:26Z + PR: + + MtrA + + + 0.9995383 + residue_name_number + cleaner0 + 2023-07-27T13:54:29Z + DUMMY: + + Tyr102 + + + 0.99907994 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.9995233 + residue_name_number + cleaner0 + 2023-07-27T13:54:43Z + DUMMY: + + Asp56 + + + 0.9995345 + residue_name_number + cleaner0 + 2023-07-27T13:54:30Z + DUMMY: + + Tyr102 + + + 0.99953544 + residue_name_number + cleaner0 + 2023-07-27T13:54:37Z + DUMMY: + + Asp190 + + + 0.9995652 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.999433 + protein + cleaner0 + 2023-07-27T13:04:26Z + PR: + + MtrA + + + 0.9992292 + protein_state + cleaner0 + 2023-07-27T13:05:25Z + DUMMY: + + closed + + + 0.9994388 + protein + cleaner0 + 2023-07-27T12:47:46Z + PR: + + NsrR + + + 0.99956363 + residue_name_number + cleaner0 + 2023-07-27T13:29:21Z + DUMMY: + + Phe101 + + + 0.9895373 + site + cleaner0 + 2023-07-27T14:34:51Z + SO: + + switch residue + + + 0.9995402 + residue_name_number + cleaner0 + 2023-07-27T13:54:50Z + DUMMY: + + Asp188 + + + + RESULTS + paragraph + 46155 + Next, we were interested in the active conformation of the NsrR protein adopting an active “open” conformation in the dimeric state. We compared and aligned the NsrR-RD and ED on the dimeric structure of KdpE that was solved in the DNA-bound state (Fig 7B). + + 0.9992353 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.99932516 + protein + cleaner0 + 2023-07-27T12:47:46Z + PR: + + NsrR + + + 0.9992791 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.99926335 + protein_state + cleaner0 + 2023-07-27T13:05:19Z + DUMMY: + + open + + + 0.998841 + oligomeric_state + cleaner0 + 2023-07-27T13:37:50Z + DUMMY: + + dimeric + + + 0.9955654 + experimental_method + cleaner0 + 2023-07-27T14:44:48Z + MESH: + + compared and aligned + + + 0.998359 + protein + cleaner0 + 2023-07-27T12:47:46Z + PR: + + NsrR + + + 0.9993024 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.99938285 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.99880266 + oligomeric_state + cleaner0 + 2023-07-27T13:37:50Z + DUMMY: + + dimeric + + + 0.9975605 + evidence + cleaner0 + 2023-07-27T14:57:45Z + DUMMY: + + structure + + + 0.998285 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9948185 + experimental_method + cleaner0 + 2023-07-27T14:44:52Z + MESH: + + solved + + + 0.9988813 + protein_state + cleaner0 + 2023-07-27T13:06:03Z + DUMMY: + + DNA-bound + + + + RESULTS + paragraph + 46417 + Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer. We superimposed the ED of NsrR with the DNA-binding domain of KdpE resulting in a reasonably well-aligned structure (rmsd of 2.6Å over 86 Cα atoms; Table 2). As a result, a highly positive groove is created by the two ED domains of NsrR which likely represents the DNA binding site as observed in KdpE. A prediction of the putative promoter sequence that NsrR binds via the BPROM online server was performed (S3 Fig). A promoter region was identified upstream of the nsr operon. However, the regulation of the predicted promoter and the DNA binding by NsrR has to be confirmed. + + 0.99928784 + structure_element + cleaner0 + 2023-07-27T13:52:42Z + SO: + + linker region + + + 0.9978363 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.99921405 + protein + cleaner0 + 2023-07-27T12:47:46Z + PR: + + NsrR + + + 0.9989531 + protein_state + cleaner0 + 2023-07-27T13:06:03Z + DUMMY: + + DNA-bound + + + 0.99952734 + structure_element + cleaner0 + 2023-07-27T13:01:58Z + SO: + + RD + + + 0.9995184 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.9993623 + protein + cleaner0 + 2023-07-27T12:47:46Z + PR: + + NsrR + + + 0.9984419 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.9992557 + protein_state + cleaner0 + 2023-07-27T12:50:25Z + DUMMY: + + active + + + 0.9987226 + oligomeric_state + cleaner0 + 2023-07-27T13:38:00Z + DUMMY: + + dimer + + + 0.9987375 + experimental_method + cleaner0 + 2023-07-27T14:44:57Z + MESH: + + superimposed + + + 0.9994961 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.9992569 + protein + cleaner0 + 2023-07-27T12:47:46Z + PR: + + NsrR + + + 0.9989898 + structure_element + cleaner0 + 2023-07-27T13:55:17Z + SO: + + DNA-binding domain + + + 0.9977089 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.99800843 + evidence + cleaner0 + 2023-07-27T14:57:51Z + DUMMY: + + structure + + + 0.998494 + evidence + cleaner0 + 2023-07-27T13:18:49Z + DUMMY: + + rmsd + + + 0.9923591 + site + cleaner0 + 2023-07-27T14:34:55Z + SO: + + highly positive groove + + + 0.99947745 + structure_element + cleaner0 + 2023-07-27T13:02:09Z + SO: + + ED + + + 0.9993467 + protein + cleaner0 + 2023-07-27T12:47:46Z + PR: + + NsrR + + + 0.9987176 + site + cleaner0 + 2023-07-27T13:55:37Z + SO: + + DNA binding site + + + 0.9975803 + protein + cleaner0 + 2023-07-27T13:04:53Z + PR: + + KdpE + + + 0.99932516 + protein + cleaner0 + 2023-07-27T12:47:46Z + PR: + + NsrR + + + gene + GENE: + cleaner0 + 2023-07-27T13:02:41Z + + nsr + + + chemical + CHEBI: + cleaner0 + 2023-07-27T12:49:59Z + + DNA + + + 0.99938023 + protein + cleaner0 + 2023-07-27T12:47:46Z + PR: + + NsrR + + + + RESULTS + title_2 + 47199 + Conclusion + + + RESULTS + paragraph + 47210 + In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. The regulation of the expression of these genes is mediated by two-component systems. The structure of the response regulator NsrR from S. agalactiae presented in this study is the first structural information available for the subgroup of lantibiotic resistance-associated RRs. + + 0.9978606 + taxonomy_domain + cleaner0 + 2023-07-27T12:51:05Z + DUMMY: + + bacteria + + + 0.9981645 + species + cleaner0 + 2023-07-27T12:56:39Z + MESH: + + S. agalactiae + + + 0.9976611 + species + cleaner0 + 2023-07-27T12:55:51Z + MESH: + + S. aureus + + + 0.9971164 + species + cleaner0 + 2023-07-27T13:24:38Z + MESH: + + C. difficile + + + 0.9989114 + chemical + cleaner0 + 2023-07-27T12:52:05Z + CHEBI: + + lantibiotic + + + 0.99796283 + chemical + cleaner0 + 2023-07-27T12:48:05Z + CHEBI: + + lantibiotics + + + 0.9993581 + chemical + cleaner0 + 2023-07-27T12:52:00Z + CHEBI: + + nisin + + + 0.9985107 + chemical + cleaner0 + 2023-07-27T14:28:19Z + CHEBI: + + nukacin ISK-1 + + + 0.99897635 + chemical + cleaner0 + 2023-07-27T13:01:07Z + CHEBI: + + lacticin 481 + + + 0.9987948 + chemical + cleaner0 + 2023-07-27T12:53:41Z + CHEBI: + + gallidermin + + + 0.9993537 + chemical + cleaner0 + 2023-07-27T14:28:23Z + CHEBI: + + actagardine + + + 0.9993405 + chemical + cleaner0 + 2023-07-27T14:28:26Z + CHEBI: + + mersacidin + + + 0.99779874 + evidence + cleaner0 + 2023-07-27T14:57:54Z + DUMMY: + + structure + + + 0.9980495 + protein_type + cleaner0 + 2023-07-27T12:47:38Z + MESH: + + response regulator + + + 0.99939275 + protein + cleaner0 + 2023-07-27T12:47:46Z + PR: + + NsrR + + + 0.9983414 + species + cleaner0 + 2023-07-27T12:56:39Z + MESH: + + S. agalactiae + + + protein_type + MESH: + cleaner0 + 2023-07-27T13:32:11Z + + lantibiotic resistance-associated RRs + + + + SUPPL + title_1 + 47771 + Supporting Information + + + REF + title + 47794 + References + + + 95 + 105 + surname:Cotter;given-names:PD + surname:Ross;given-names:RP + surname:Hill;given-names:C + 10.1038/nrmicro2937 + 23268227 + REF + Nature Reviews Microbiology + ref + 11 + 2012 + 47805 + Bacteriocins—a viable alternative to antibiotics? + + + 385 + 392 + surname:Bierbaum;given-names:G + surname:Szekat;given-names:C + surname:Josten;given-names:M + surname:Heidrich;given-names:C + surname:Kempter;given-names:C + surname:Jung;given-names:G + 8593044 + REF + Applied and environmental microbiology + ref + 62 + 1996 + 47857 + Engineering of a novel thioether bridge and role of modified residues in the lantibiotic Pep5 + + + 41 + 79 + surname:Sahl;given-names:H-G + surname:Bierbaum;given-names:G + REF + Annual Reviews in Microbiology + ref + 52 + 1998 + 47951 + Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from gram-positive bacteria + + + 61 + 75 + surname:Cotter;given-names:PD + surname:Hill;given-names:C + surname:Ross;given-names:RP + 15638769 + REF + Current Protein and Peptide Science + ref + 6 + 2005 + 48062 + Bacterial lantibiotics: strategies to improve therapeutic potential + + + 51 + 62 + surname:Dischinger;given-names:J + surname:Basi;given-names:Chipalu S + surname:Bierbaum;given-names:G + 10.1016/j.ijmm.2013.09.003 + 24210177 + REF + International Journal of Medical Microbiology + ref + 304 + 2014 + 48130 + Lantibiotics: promising candidates for future applications in health care + + + 22 + 25 + surname:Boakes;given-names:S + surname:Wadman;given-names:S + REF + Innov Pharm Technol + ref + 27 + 2008 + 48204 + The therapeutic potential of lantibiotics + + + 752 + 763 + surname:Alkhatib;given-names:Z + surname:Lagedroste;given-names:M + surname:Zaschke;given-names:J + surname:Wagner;given-names:M + surname:Abts;given-names:A + surname:Fey;given-names:I + 10.1002/mbo3.205 + 25176038 + REF + MicrobiologyOpen + ref + 3 + 2014 + 48246 + The C‐terminus of nisin is important for the ABC transporter NisFEG to confer immunity in Lactococcus lactis + + + 1772 + 1779 + surname:Wiedemann;given-names:I + surname:Breukink;given-names:E + surname:Van Kraaij;given-names:C + surname:Kuipers;given-names:OP + surname:Bierbaum;given-names:G + surname:De Kruijff;given-names:B + 11038353 + REF + Journal of Biological Chemistry + ref + 276 + 2001 + 48357 + Specific Binding of Nisin to the Peptidoglycan Precursor Lipid II Combines Pore Formation and Inhibition of Cell Wall Biosynthesis for Potent Antibiotic Activity + + + 814 + 825 + surname:Engelke;given-names:G + surname:Gutowski-Eckel;given-names:Z + surname:Kiesau;given-names:P + surname:Siegers;given-names:K + surname:Hammelmann;given-names:M + surname:Entian;given-names:K + 8161176 + REF + Applied and environmental microbiology + ref + 60 + 1994 + 48519 + Regulation of nisin biosynthesis and immunity in Lactococcus lactis 6F3 + + + 281 + 291 + surname:Kuipers;given-names:OP + surname:Beerthuyzen;given-names:MM + surname:Siezen;given-names:RJ + surname:VOS;given-names:WM + 7689965 + REF + European Journal of Biochemistry + ref + 216 + 1993 + 48591 + Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis + + + 106 + 113 + surname:Guder;given-names:A + surname:Schmitter;given-names:T + surname:Wiedemann;given-names:I + surname:Sahl H-;given-names:G + surname:Bierbaum;given-names:G + 11772616 + REF + Applied and environmental microbiology + ref + 68 + 2002 + 48667 + Role of the single regulator MrsR1 and the two-component system MrsR2/K2 in the regulation of mersacidin production and immunity + + + 145 + 154 + surname:Alkhatib;given-names:Z + surname:Abts;given-names:A + surname:Mavaro;given-names:A + surname:Schmitt;given-names:L + surname:Smits;given-names:SH + 10.1016/j.jbiotec.2012.01.032 + 22329892 + REF + Journal of biotechnology + ref + 159 + 2012 + 48796 + Lantibiotics: how do producers become self-protected? + + + 633 + 684 + surname:Chatterjee;given-names:C + surname:Paul;given-names:M + surname:Xie;given-names:L + surname:van der Donk;given-names:WA + 15700960 + REF + Chemical Reviews + ref + 105 + 2005 + 48850 + Biosynthesis and mode of action of lantibiotics + + + 151 + 159 + surname:Saris;given-names:PEJ + surname:Immonen;given-names:T + surname:Reis;given-names:M + surname:Sahl;given-names:H-G + 8775975 + REF + Antonie van Leeuwenhoek + ref + 69 + 1996 + 48898 + Immunity to lantibiotics + + + surname:Wolter;given-names:AC + surname:Duchardt-Ferner;given-names:E + surname:Nasiri;given-names:AH + surname:Hantke;given-names:K + surname:Wunderlich;given-names:CH + surname:Kreutz;given-names:C + REF + Biomol NMR Assign + ref + 2015 + 48923 + NMR resonance assignments for the class II GTP binding RNA aptamer in complex with GTP + + + 35286 + 35298 + surname:Christ;given-names:NA + surname:Bochmann;given-names:S + surname:Gottstein;given-names:D + surname:Duchardt-Ferner;given-names:E + surname:Hellmich;given-names:UA + surname:Dusterhus;given-names:S + 10.1074/jbc.M112.401620 + 22904324 + REF + J Biol Chem + ref + 287 + 2012 + 49010 + The First structure of a lantibiotic immunity protein, SpaI from Bacillus subtilis, reveals a novel fold + + + surname:Pozzi;given-names:R + surname:Coles;given-names:M + surname:Linke;given-names:D + surname:Kulik;given-names:A + surname:Nega;given-names:M + surname:Wohlleben;given-names:W + REF + Environ Microbiol + ref + 2015 + 49115 + Distinct mechanisms contribute to immunity in the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024 + + + 1543 + 1549 + surname:Khosa;given-names:S + surname:Alkhatib;given-names:Z + surname:Smits;given-names:SH + 10.1515/hsz-2013-0167 + 23893686 + REF + Biological chemistry + ref + 394 + 2013 + 49228 + NSR from Streptococcus agalactiae confers resistance against nisin and is encoded by a conserved nsr operon + + + 4751 + 4755 + surname:Kawada-Matsuo;given-names:M + surname:Oogai;given-names:Y + surname:Zendo;given-names:T + surname:Nagao;given-names:J + surname:Shibata;given-names:Y + surname:Yamashita;given-names:Y + 10.1128/AEM.00780-13 + 23709506 + REF + Applied and Environmental Microbiology + ref + 79 + 2013 + 49336 + Involvement of the Novel Two-Component NsrRS and LcrRS Systems in Distinct Resistance Pathways against Nisin A and Nukacin ISK-1 in Streptococcus mutans + + + 1047 + 1058 + surname:Falord;given-names:M + surname:Karimova;given-names:G + surname:Hiron;given-names:A + surname:Msadek;given-names:T + 10.1128/AAC.05054-11 + 22123691 + REF + Antimicrobial agents and chemotherapy + ref + 56 + 2012 + 49489 + GraXSR proteins interact with the VraFG ABC transporter to form a five-component system required for cationic antimicrobial peptide sensing and resistance in Staphylococcus aureus + + + 18679 + surname:Khosa;given-names:S + surname:Frieg;given-names:B + surname:Mulnaes;given-names:D + surname:Kleinschrodt;given-names:D + surname:Hoeppner;given-names:A + surname:Gohlke;given-names:H + 10.1038/srep18679 + 26727488 + REF + Sci Rep + ref + 6 + 2016 + 49669 + Structural basis of lantibiotic recognition by the nisin resistance protein from Streptococcus agalactiae + + + 171 + 191 + surname:Draper;given-names:LA + surname:Cotter;given-names:PD + surname:Hill;given-names:C + surname:Ross;given-names:RP + 10.1128/MMBR.00051-14 + 25787977 + REF + Microbiology and Molecular Biology Reviews + ref + 79 + 2015 + 49775 + Lantibiotic Resistance + + + 895 + 904 + surname:Kleerebezem;given-names:M + surname:Quadri;given-names:LE + surname:Kuipers;given-names:OP + surname:De Vos;given-names:WM + 9219998 + REF + Molecular microbiology + ref + 24 + 1997 + 49798 + Quorum sensing by peptide pheromones and two-component signal-transduction systems in Gram-positive bacteria + + + 183 + 215 + surname:Stock;given-names:AM + surname:Robinson;given-names:VL + surname:Goudreau;given-names:PN + 10966457 + REF + Annual review of biochemistry + ref + 69 + 2000 + 49907 + Two-component signal transduction + + + 1529 + 1536 + surname:Barrett;given-names:JF + surname:Hoch;given-names:JA + 9660978 + REF + Antimicrobial agents and chemotherapy + ref + 42 + 1998 + 49941 + Two-component signal transduction as a target for microbial anti-infective therapy + + + 602 + 622 + surname:Hiron;given-names:A + surname:Falord;given-names:M + surname:Valle;given-names:J + surname:Débarbouillé;given-names:M + surname:Msadek;given-names:T + 10.1111/j.1365-2958.2011.07735.x + 21696458 + REF + Molecular microbiology + ref + 81 + 2011 + 50024 + Bacitracin and nisin resistance in Staphylococcus aureus: a novel pathway involving the BraS/BraR two-component system (SA2417/SA2418) and both the BraD/BraE and VraD/VraE ABC transporters + + + 1135 + 1144 + surname:Ohki;given-names:R + surname:Giyanto + surname:Tateno;given-names:K + surname:Masuyama;given-names:W + surname:Moriya;given-names:S + surname:Kobayashi;given-names:K + 12890034 + REF + Molecular Microbiology + ref + 49 + 2003 + 50213 + The BceRS two-component regulatory system induces expression of the bacitracin transporter, BceAB, in Bacillus subtilis + + + 6840 + 6843 + surname:Cotter;given-names:PD + surname:Emerson;given-names:N + surname:Gahan;given-names:CG + surname:Hill;given-names:C + 10542190 + REF + Journal of bacteriology + ref + 181 + 1999 + 50333 + Identification and disruption of lisRK, a genetic locus encoding a two-component signal transduction system involved in stress tolerance and virulence in Listeria monocytogenes + + + 3851 + 3862 + surname:Dintner;given-names:S + surname:Staroń;given-names:A + surname:Berchtold;given-names:E + surname:Petri;given-names:T + surname:Mascher;given-names:T + surname:Gebhard;given-names:S + 10.1128/JB.05175-11 + 21665979 + REF + Journal of bacteriology + ref + 193 + 2011 + 50510 + Coevolution of ABC transporters and two-component regulatory systems as resistance modules against antimicrobial peptides in Firmicutes bacteria + + + 133 + 144 + surname:Mascher;given-names:T + 17064367 + REF + FEMS microbiology letters + ref + 264 + 2006 + 50655 + Intramembrane-sensing histidine kinases: a new family of cell envelope stress sensors in Firmicutes bacteria + + + 768 + 785 + surname:Rietkötter;given-names:E + surname:Hoyer;given-names:D + surname:Mascher;given-names:T + 10.1111/j.1365-2958.2008.06194.x + 18394148 + REF + Molecular microbiology + ref + 68 + 2008 + 50764 + Bacitracin sensing in Bacillus subtilis + + + 515 + 525 + surname:Staroń;given-names:A + surname:Finkeisen;given-names:DE + surname:Mascher;given-names:T + 10.1128/AAC.00352-10 + 21078927 + REF + Antimicrobial agents and chemotherapy + ref + 55 + 2011 + 50804 + Peptide antibiotic sensing and detoxification modules of Bacillus subtilis + + + 3895 + 3906 + surname:Ouyang;given-names:J + surname:Tian;given-names:X-L + surname:Versey;given-names:J + surname:Wishart;given-names:A + surname:Li;given-names:Y-H + 10.1128/AAC.01802-09 + 20606066 + REF + Antimicrobial agents and chemotherapy + ref + 54 + 2010 + 50879 + The BceABRS four-component system regulates the bacitracin-induced cell envelope stress response in Streptococcus mutans + + + 225 + 234 + surname:Gao;given-names:R + surname:Mack;given-names:TR + surname:Stock;given-names:AM + 17433693 + REF + Trends in biochemical sciences + ref + 32 + 2007 + 51000 + Bacterial response regulators: versatile regulatory strategies from common domains + + + 301 + 312 + surname:Martínez-Hackert;given-names:E + surname:Stock;given-names:AM + 9199401 + REF + Journal of molecular biology + ref + 269 + 1997 + 51083 + Structural relationships in the OmpR family of winged-helix transcription factors + + + 37717 + 37729 + surname:King-Scott;given-names:J + surname:Nowak;given-names:E + surname:Mylonas;given-names:E + surname:Panjikar;given-names:S + surname:Roessle;given-names:M + surname:Svergun;given-names:DI + 17942407 + REF + Journal of Biological Chemistry + ref + 282 + 2007 + 51165 + The structure of a full-length response regulator from Mycobacterium tuberculosis in a stabilized three-dimensional domain-swapped, activated state + + + 6733 + 6743 + surname:Friedland;given-names:N + surname:Mack;given-names:TR + surname:Yu;given-names:M + surname:Hung;given-names:L-W + surname:Terwilliger;given-names:TC + surname:Waldo;given-names:GS + 17511470 + REF + Biochemistry + ref + 46 + 2007 + 51313 + Domain orientation in the inactive response regulator Mycobacterium tuberculosis MtrA provides a barrier to activation + + + 9659 + 9666 + surname:Nowak;given-names:E + surname:Panjikar;given-names:S + surname:Konarev;given-names:P + surname:Svergun;given-names:DI + surname:Tucker;given-names:PA + 16434396 + REF + Journal of Biological Chemistry + ref + 281 + 2006 + 51432 + The structural basis of signal transduction for the response regulator PrrA from Mycobacterium tuberculosis + + + 5948 + 5957 + surname:Menon;given-names:S + surname:Wang;given-names:S + 10.1021/bi2005575 + 21634789 + REF + Biochemistry + ref + 50 + 2011 + 51540 + Structure of the response regulator PhoP from Mycobacterium tuberculosis reveals a dimer through the receiver domain + + + 4186 + 4194 + surname:Robinson;given-names:VL + surname:Wu;given-names:T + surname:Stock;given-names:AM + 12837793 + REF + Journal of bacteriology + ref + 185 + 2003 + 51657 + Structural analysis of the domain interface in DrrB, a response regulator of the OmpR/PhoB subfamily + + + 153 + 164 + surname:Buckler;given-names:DR + surname:Zhou;given-names:Y + surname:Stock;given-names:AM + 11839301 + REF + Structure + ref + 10 + 2002 + 51758 + Evidence of intradomain and interdomain flexibility in an OmpR/PhoB homolog from Thermotoga maritima + + + surname:Narayanan;given-names:A + surname:Kumar;given-names:S + surname:Evrard;given-names:AN + surname:Paul;given-names:LN + surname:Yernool;given-names:DA + REF + Nat Commun + ref + 5 + 2014 + 51859 + An asymmetric heterodomain interface stabilizes a response regulator–DNA complex + + + 1322 + 1326 + surname:Khosa;given-names:S + surname:Hoeppner;given-names:A + surname:Kleinschrodt;given-names:D + surname:Smits;given-names:SHJ + REF + Acta Crystallographica Section F + ref + 71 + 2015 + 51942 + Overexpression, purification and crystallization of the response regulator NsrR involved in nisin resistance + + + surname:Dyballa;given-names:N + surname:Metzger;given-names:S + REF + Journal of visualized experiments: JoVE + ref + 2009 + 52051 + Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels + + + 227 + 238 + surname:Nurizzo;given-names:D + surname:Mairs;given-names:T + surname:Guijarro;given-names:M + surname:Rey;given-names:V + surname:Meyer;given-names:J + surname:Fajardo;given-names:P + 16645249 + REF + Journal of synchrotron radiation + ref + 13 + 2006 + 52141 + The ID23-1 structural biology beamline at the ESRF + + + 125 + 132 + surname:Kabsch;given-names:W + 10.1107/S0907444909047337 + 20124692 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 52192 + Xds + + + 675 + 687 + surname:Solà;given-names:M + surname:Gomis-RuÈth;given-names:FX + surname:Serrano;given-names:L + surname:González;given-names:A + surname:Coll;given-names:M + 9878437 + REF + Journal of molecular biology + ref + 285 + 1999 + 52196 + Three-dimensional crystal structure of the transcription factor PhoB receiver domain + + + 213 + 221 + surname:Adams;given-names:PD + surname:Afonine;given-names:PV + surname:Bunkóczi;given-names:G + surname:Chen;given-names:VB + surname:Davis;given-names:IW + surname:Echols;given-names:N + 10.1107/S0907444909052925 + 20124702 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 52281 + PHENIX: a comprehensive Python-based system for macromolecular structure solution + + + 486 + 501 + surname:Emsley;given-names:P + surname:Lohkamp;given-names:B + surname:Scott;given-names:WG + surname:Cowtan;given-names:K + 20383002 + REF + Acta Crystallographica Section D: Biological Crystallography + ref + 66 + 2010 + 52363 + Features and development of Coot + + + 12 + 21 + surname:Chen;given-names:VB + surname:Arendall;given-names:WB;suffix:3rd + surname:Headd;given-names:JJ + surname:Keedy;given-names:DA + surname:Immormino;given-names:RM + surname:Kapral;given-names:GJ + 10.1107/S0907444909042073 + 20057044 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 52396 + MolProbity: all-atom structure validation for macromolecular crystallography + + + 1089 + 1097 + surname:Panjikar;given-names:S + surname:Parthasarathy;given-names:V + surname:Lamzin;given-names:VS + surname:Weiss;given-names:MS + surname:Tucker;given-names:PA + 10.1107/S0907444909029643 + 19770506 + REF + Acta Crystallogr D Biol Crystallogr + ref + 65 + 2009 + 52473 + On the combination of molecular replacement and single-wavelength anomalous diffraction phasing for automated structure determination + + + REF + ref + 52607 + Delano WL (2002) The PyMOL molecular graphics system. + + + 3434 + 3438 + surname:Okajima;given-names:T + surname:Doi;given-names:A + surname:Okada;given-names:A + surname:Gotoh;given-names:Y + surname:Tanizawa;given-names:K + surname:Utsumi;given-names:R + 10.1016/j.febslet.2008.09.007 + 18789936 + REF + FEBS letters + ref + 582 + 2008 + 52661 + Response regulator YycF essential for bacterial growth: X-ray crystal structure of the DNA-binding domain and its PhoB-like DNA recognition motif + + + 701 + 713 + surname:Blanco;given-names:AG + surname:Sola;given-names:M + surname:Gomis-Rüth;given-names:FX + surname:Coll;given-names:M + 12015152 + REF + Structure + ref + 10 + 2002 + 52807 + Tandem DNA Recognition by PhoB, a Two-Component Signal Transduction Transcriptional Activator + + + 11 + 26 + surname:Toro-Roman;given-names:A + surname:Mack;given-names:TR + surname:Stock;given-names:AM + 15876365 + REF + Journal of molecular biology + ref + 349 + 2005 + 52901 + Structural analysis and solution studies of the activated regulatory domain of the response regulator ArcA: a symmetric dimer mediated by the α4-β5-α5 face + + + 1353 + 1363 + surname:Bachhawat;given-names:P + surname:Swapna;given-names:G + surname:Montelione;given-names:GT + surname:Stock;given-names:AM + 16154092 + REF + Structure + ref + 13 + 2005 + 53068 + Mechanism of activation for transcription factor PhoB suggested by different modes of dimerization in the inactive and active states + + + 1287 + 1293 + surname:Choudhury;given-names:HG + surname:Beis;given-names:K + 10.1002/pro.2311 + 23868292 + REF + Protein Science + ref + 22 + 2013 + 53201 + The dimeric form of the unphosphorylated response regulator BaeR + + + W545 + W549 + surname:Holm;given-names:L + surname:Rosenström;given-names:P + 10.1093/nar/gkq366 + 20457744 + REF + Nucleic acids research + ref + 38 + 2010 + 53266 + Dali server: conservation mapping in 3D + + + 403 + 410 + surname:Altschul;given-names:SF + surname:Gish;given-names:W + surname:Miller;given-names:W + surname:Myers;given-names:EW + surname:Lipman;given-names:DJ + 2231712 + REF + Journal of molecular biology + ref + 215 + 1990 + 53306 + Basic local alignment search tool + + + 2621 + 2631 + surname:Suárez;given-names:JM + surname:Edwards;given-names:AN + surname:McBride;given-names:SM + 10.1128/JB.00166-13 + 23543720 + REF + Journal of bacteriology + ref + 195 + 2013 + 53340 + The Clostridium difficile cpr locus is regulated by a noncontiguous two-component system in response to type A and B lantibiotics + + + 167 + 176 + surname:McBride;given-names:SM + surname:Sonenshein;given-names:AL + 10.1128/IAI.00731-10 + 20974818 + REF + Infection and immunity + ref + 79 + 2011 + 53470 + Identification of a genetic locus responsible for antimicrobial peptide resistance in Clostridium difficile + + + 45 + 53 + surname:Neoh;given-names:H-m + surname:Cui;given-names:L + surname:Yuzawa;given-names:H + surname:Takeuchi;given-names:F + surname:Matsuo;given-names:M + surname:Hiramatsu;given-names:K + 17954695 + REF + Antimicrobial agents and chemotherapy + ref + 52 + 2008 + 53578 + Mutated response regulator graR is responsible for phenotypic conversion of Staphylococcus aureus from heterogeneous vancomycin-intermediate resistance to vancomycin-intermediate resistance + + + 2679 + 2689 + surname:Meehl;given-names:M + surname:Herbert;given-names:S + surname:Götz;given-names:F + surname:Cheung;given-names:A + 17502406 + REF + Antimicrobial agents and chemotherapy + ref + 51 + 2007 + 53768 + Interaction of the GraRS two-component system with the VraFG ABC transporter to support vancomycin-intermediate resistance in Staphylococcus aureus + + + 1367 + 1380 + surname:Mandin;given-names:P + surname:Fsihi;given-names:H + surname:Dussurget;given-names:O + surname:Vergassola;given-names:M + surname:Milohanic;given-names:E + surname:Toledo;given-names:Arana A + 16102006 + REF + Molecular microbiology + ref + 57 + 2005 + 53916 + VirR, a response regulator critical for Listeria monocytogenes virulence + + + 42003 + 42010 + surname:Guillet;given-names:V + surname:Ohta;given-names:N + surname:Cabantous;given-names:S + surname:Newton;given-names:A + surname:Samama;given-names:J-P + 12176983 + REF + Journal of Biological Chemistry + ref + 277 + 2002 + 53989 + Crystallographic and biochemical studies of DivK reveal novel features of an essential response regulator in Caulobacter crescentus + + + 41 + 46 + surname:Lukat;given-names:GS + surname:Stock;given-names:JB + 8381790 + REF + Journal of cellular biochemistry + ref + 51 + 1993 + 54121 + Response regulation in bacterial chemotaxis + + + 31567 + 31572 + surname:McCleary;given-names:WR + surname:Stock;given-names:JB + 7989325 + REF + Journal of Biological Chemistry + ref + 269 + 1994 + 54165 + Acetyl phosphate and the activation of two-component response regulators + + + 13375 + 13380 + surname:Stock;given-names:AM + surname:Martinez-Hackert;given-names:E + surname:Rasmussen;given-names:BF + surname:West;given-names:AH + surname:Stock;given-names:JB + surname:Ringe;given-names:D + 8257674 + REF + Biochemistry + ref + 32 + 1993 + 54238 + Structure of the magnesium-bound form of CheY and mechanism of phosphoryl transfer in bacterial chemotaxis + + + 116 + 128 + surname:Bellsolell;given-names:Ls + surname:Cronet;given-names:P + surname:Majolero;given-names:M + surname:Serrano;given-names:L + surname:Coll;given-names:M + 8632450 + REF + Journal of molecular biology + ref + 257 + 1996 + 54345 + The three-dimensional structure of two mutants of the signal transduction protein CheY suggest its molecular activation mechanism + + + 369 + 376 + surname:West;given-names:AH + surname:Stock;given-names:AM + 11406410 + REF + Trends in biochemical sciences + ref + 26 + 2001 + 54475 + Histidine kinases and response regulator proteins in two-component signaling systems + + + 5000 + 5006 + surname:Zhu;given-names:X + surname:Rebello;given-names:J + surname:Matsumura;given-names:P + surname:Volz;given-names:K + 9030562 + REF + Journal of Biological Chemistry + ref + 272 + 1997 + 54560 + Crystal Structures of CheY Mutants Y106W and T87I/Y106W CheY Activation Correlates with Movement of Residue 106 + + + 3563 + 3569 + surname:Appleby;given-names:JL + surname:Bourret;given-names:RB + 9657998 + REF + Journal of bacteriology + ref + 180 + 1998 + 54672 + Proposed signal transduction role for conserved CheY residue Thr87, a member of the response regulator active-site quintet + + + 149 + 165 + surname:Gardino;given-names:AK + surname:Kern;given-names:D + 17609130 + REF + Methods in enzymology + ref + 423 + 2007 + 54795 + Functional Dynamics of Response Regulators Using NMR Relaxation Techniques + + + 3077 + 3088 + surname:Toro;given-names:Roman A + surname:Wu;given-names:T + surname:Stock;given-names:AM + 16322582 + REF + Protein science + ref + 14 + 2005 + 54870 + A common dimerization interface in bacterial response regulators KdpE and TorR + + + 251 + 268 + surname:Gerber;given-names:PR + surname:Müller;given-names:K + 7561977 + REF + Journal of computer-aided molecular design + ref + 9 + 1995 + 54949 + MAB, a generally applicable molecular force field for structure modelling in medicinal chemistry + + + 349 + 364 + surname:Mack;given-names:TR + surname:Gao;given-names:R + surname:Stock;given-names:AM + 10.1016/j.jmb.2009.04.014 + 19371748 + REF + Journal of molecular biology + ref + 389 + 2009 + 55046 + Probing the roles of the two different dimers mediated by the receiver domain of the response regulator PhoB + + + 262 + 273 + surname:Chen;given-names:Y + surname:Birck;given-names:C + surname:Samama J-;given-names:P + surname:Hulett;given-names:FM + 12486063 + REF + Journal of bacteriology + ref + 185 + 2003 + 55155 + Residue R113 is essential for PhoP dimerization and function: a residue buried in the asymmetric PhoP dimer interface determined in the PhoPN three-dimensional crystal structure + + + 109 + 124 + surname:Martínez-Hackert;given-names:E + surname:Stock;given-names:AM + 9016718 + REF + Structure + ref + 5 + 1997 + 55333 + The DNA-binding domain of OmpR: crystal structures of a winged helix transcription factor + + + 341 + 348 + surname:Hasegawa;given-names:H + surname:Holm;given-names:L + 10.1016/j.sbi.2009.04.003 + 19481444 + REF + Current opinion in structural biology + ref + 19 + 2009 + 55423 + Advances and pitfalls of protein structural alignment + + + 61 + 78 + surname:Solovyev;given-names:V + surname:Salamov;given-names:A + REF + Metagenomics and its applications in agriculture, biomedicine and environmental studies + ref + 2011 + 55477 + Automatic annotation of microbial genomes and metagenomic sequences + + + 719 + 722 + surname:Zhao;given-names:H + surname:Heroux;given-names:A + surname:Sequeira;given-names:RD + surname:Tang;given-names:L + 10.1107/S1744309109022696 + 19574649 + REF + Structural Biology and Crystallization Communications + ref + 65 + 2009 + 55545 + Preliminary crystallographic studies of the regulatory domain of response regulator YycF from an essential two-component signal transduction system. Acta Crystallographica Section F + + + 14751 + 14761 + surname:Wang;given-names:S + surname:Engohang-Ndong;given-names:J + surname:Smith;given-names:I + 18052041 + REF + Biochemistry + ref + 46 + 2007 + 55727 + Structure of the DNA-binding domain of the response regulator PhoP from Mycobacterium tuberculosis + + + 1970 + 1983 + surname:Yamane;given-names:T + surname:Okamura;given-names:H + surname:Ikeguchi;given-names:M + surname:Nishimura;given-names:Y + surname:Kidera;given-names:A + REF + Proteins: Structure, Function, and Bioinformatics + ref + 71 + 2008 + 55826 + Water-mediated interactions between DNA and PhoB DNA-binding/transactivation domain: NMR-restrained molecular dynamics in explicit water environment + + + gku572 + surname:Li;given-names:Y-C + surname:Chang;given-names:C-k + surname:Chang;given-names:C-F + surname:Cheng;given-names:Y-H + surname:Fang;given-names:P-J + surname:Yu;given-names:T + REF + Nucleic acids research + ref + 2014 + 55975 + Structural dynamics of the two-component response regulator RstA in recognition of promoter DNA element + + + 8525 + 8530 + surname:Leonard;given-names:PG + surname:Golemi-Kotra;given-names:D + surname:Stock;given-names:AM + REF + Proceedings of the National Academy of Sciences + ref + 110 + 2013 + 56079 + Phosphorylation-dependent conformational changes and domain rearrangements in Staphylococcus aureus VraR activation + + + diff --git a/BioC_XML/4774019_v0.xml b/BioC_XML/4774019_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..26298418e99a4207e6c4fe3a288525dfbf200de0 --- /dev/null +++ b/BioC_XML/4774019_v0.xml @@ -0,0 +1,6132 @@ + + + + PMC + 20201215 + pmc.key + + 4774019 + CC BY + no + 0 + 0 + + 10.1186/s12915-016-0236-7 + 4774019 + 26934976 + 236 + 14 + Innate immunity IRG proteins GTPase Dynamin superfamily Dimerization Oligomerization + +Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. + surname:Schulte;given-names:Kathrin + surname:Pawlowski;given-names:Nikolaus + surname:Faelber;given-names:Katja + surname:Fröhlich;given-names:Chris + surname:Howard;given-names:Jonathan + surname:Daumke;given-names:Oliver + surname:Howard;given-names:Jonathan + surname:Daumke;given-names:Oliver + TITLE + Keywords + front + 14 + 2016 + 0 + The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion + + 0.99856925 + protein_type + cleaner0 + 2023-07-27T10:33:20Z + MESH: + + immunity-related GTPase + + + 0.99922526 + protein + cleaner0 + 2023-07-27T10:33:25Z + PR: + + Irga6 + + + 0.99804074 + oligomeric_state + cleaner0 + 2023-07-27T10:33:31Z + DUMMY: + + dimerizes + + + 0.998421 + protein_state + cleaner0 + 2023-07-27T10:33:35Z + DUMMY: + + parallel head-to-head + + + + ABSTRACT + abstract_title_1 + 79 + Background + + + ABSTRACT + abstract + 90 + The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens. Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization. + + 0.99871874 + protein_type + cleaner0 + 2023-07-27T10:37:44Z + MESH: + + immunity-related GTPases + + + 0.99926287 + protein_type + cleaner0 + 2023-07-27T10:33:42Z + MESH: + + IRGs + + + 0.9991574 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9987838 + protein_type + cleaner0 + 2023-07-27T10:33:48Z + MESH: + + dynamin-like protein + + + 0.99836946 + species + cleaner0 + 2023-07-27T10:33:54Z + MESH: + + Toxoplasma gondii + + + 0.99855673 + experimental_method + cleaner0 + 2023-07-27T10:34:13Z + MESH: + + biochemical analysis + + + 0.9992793 + structure_element + cleaner0 + 2023-07-27T10:34:08Z + SO: + + GTPase domains + + + 0.99919015 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.99237704 + oligomeric_state + cleaner0 + 2023-07-27T12:30:12Z + DUMMY: + + dimerize + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:29:53Z + + antiparallel + + + + ABSTRACT + abstract_title_1 + 526 + Results + + + ABSTRACT + abstract + 534 + We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion. The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule. The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. Further dimerization contacts involve switch II, the G4 helix and the trans stabilizing loop. + + experimental_method + MESH: + cleaner0 + 2023-07-27T12:41:13Z + + determined + + + 0.9973475 + evidence + cleaner0 + 2023-07-27T12:40:59Z + DUMMY: + + crystal structure + + + 0.9970346 + protein_state + cleaner0 + 2023-07-27T10:34:19Z + DUMMY: + + oligomerization-impaired + + + 0.99841 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9990128 + protein_state + cleaner0 + 2023-07-27T10:34:25Z + DUMMY: + + mutant + + + 0.99903274 + protein_state + cleaner0 + 2023-07-27T10:34:29Z + DUMMY: + + bound to + + + chemical + CHEBI: + cleaner0 + 2023-07-27T10:35:02Z + + GTP + + + 0.99806577 + evidence + cleaner0 + 2023-07-27T12:41:19Z + DUMMY: + + structure + + + 0.99867094 + structure_element + cleaner0 + 2023-07-27T10:34:08Z + SO: + + GTPase domains + + + 0.99238014 + oligomeric_state + cleaner0 + 2023-07-27T12:30:12Z + DUMMY: + + dimerize + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:09:49Z + + parallel + + + 0.9541489 + chemical + cleaner0 + 2023-07-27T12:37:37Z + CHEBI: + + nucleotides + + + 0.9987698 + site + cleaner0 + 2023-07-27T12:37:40Z + SO: + + interface + + + site + SO: + cleaner0 + 2023-07-27T10:35:34Z + + switch I + + + 0.9991636 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.99857926 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.9832497 + protein_state + cleaner0 + 2023-07-27T10:35:43Z + DUMMY: + + catalytic + + + 0.83116907 + residue_name_number + cleaner0 + 2023-07-27T10:35:49Z + DUMMY: + + glutamate 106 + + + 0.9981953 + site + cleaner0 + 2023-07-27T10:35:35Z + SO: + + switch I + + + 0.99897873 + site + cleaner0 + 2023-07-27T12:37:45Z + SO: + + active site + + + 0.9981766 + site + cleaner0 + 2023-07-27T10:35:38Z + SO: + + switch II + + + 0.9988177 + structure_element + cleaner0 + 2023-07-27T10:50:58Z + SO: + + G4 helix + + + structure_element + SO: + cleaner0 + 2023-07-27T10:44:28Z + + trans stabilizing loop + + + + ABSTRACT + abstract_title_1 + 1189 + Conclusions + + + ABSTRACT + abstract + 1201 + The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. This study contributes important insights into the assembly and catalytic mechanisms of IRG proteins as prerequisite to understand their anti-microbial action. + + 0.99903893 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9976412 + evidence + cleaner0 + 2023-07-27T12:41:35Z + DUMMY: + + structure + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:09:49Z + + parallel + + + structure_element + SO: + cleaner0 + 2023-07-27T10:36:19Z + + GTPase domain + + + 0.9474066 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.99884295 + protein_type + cleaner0 + 2023-07-27T10:36:29Z + MESH: + + dynamin + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:36:51Z + + septin + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:37:26Z + + IRG + + + + ABSTRACT + abstract_title_1 + 1508 + Electronic supplementary material + + + ABSTRACT + abstract + 1542 + The online version of this article (doi:10.1186/s12915-016-0236-7) contains supplementary material, which is available to authorized users. + + + INTRO + title_1 + 1682 + Background + + + INTRO + paragraph + 1693 + Immunity-related GTPases (IRGs) comprise a family of dynamin-related cell-autonomous resistance proteins targeting intracellular pathogens, such as Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Trypanosoma cruzi, and Toxoplasma gondii. In mice, the 23 IRG members are induced by interferons, whereas the single human homologue is constitutively expressed in some tissues, especially in testis. In non-infected cells, most IRGs are largely cytosolic. However, members of a small sub-family with regulatory function associate with specific intracellular membranes, with one member favoring the endoplasmic reticulum and others the Golgi membrane and the endolysosomal system. Infection by certain intracellular pathogens initiates the redistribution of several effector members to the parasitophorous vacuole, followed by its disruption. In this way, IRGs contribute to the release of the pathogen into the cytoplasm and its subsequent destruction. + + 0.9982124 + protein_type + cleaner0 + 2023-07-27T10:37:43Z + MESH: + + Immunity-related GTPases + + + 0.9992719 + protein_type + cleaner0 + 2023-07-27T10:33:43Z + MESH: + + IRGs + + + 0.9983795 + protein_type + cleaner0 + 2023-07-27T10:37:51Z + MESH: + + dynamin-related cell-autonomous resistance proteins + + + 0.997863 + species + cleaner0 + 2023-07-27T10:38:06Z + MESH: + + Mycobacterium tuberculosis + + + 0.99792004 + species + cleaner0 + 2023-07-27T10:38:11Z + MESH: + + Mycobacterium avium + + + 0.99799746 + species + cleaner0 + 2023-07-27T10:38:15Z + MESH: + + Listeria monocytogenes + + + 0.99820083 + species + cleaner0 + 2023-07-27T10:38:18Z + MESH: + + Trypanosoma cruzi + + + 0.9983006 + species + cleaner0 + 2023-07-27T10:33:54Z + MESH: + + Toxoplasma gondii + + + 0.7319738 + taxonomy_domain + cleaner0 + 2023-07-27T10:38:34Z + DUMMY: + + mice + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:37:26Z + + IRG + + + 0.77819955 + protein_type + cleaner0 + 2023-07-27T10:38:00Z + MESH: + + interferons + + + 0.9984085 + species + cleaner0 + 2023-07-27T10:38:38Z + MESH: + + human + + + 0.99926263 + protein_type + cleaner0 + 2023-07-27T10:33:43Z + MESH: + + IRGs + + + 0.99927527 + protein_type + cleaner0 + 2023-07-27T10:33:43Z + MESH: + + IRGs + + + + INTRO + paragraph + 2667 + Irga6, one of the effector IRG proteins, localizes to the intact parasitophorous vacuole membrane (PVM) and, after disruption of the PVM, is found associated with vesicular accumulations, presumably derived from the PVM. A myristoylation site at Gly2 is necessary for the recruitment to the PVM but not for the weak constitutive binding to the ER membrane. An internally oriented antibody epitope on helix A between positions 20 and 24 was demonstrated to be accessible in the GTP-, but not in the GDP-bound state. This indicates large-scale structural changes upon GTP binding that probably include exposure of the myristoyl group, enhancing binding to the PVM. Biochemical studies indicated that Irga6 hydrolyses GTP in a cooperative manner and forms GTP-dependent oligomers in vitro and in vivo. + + 0.9990559 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:37:26Z + + IRG + + + 0.9987713 + site + cleaner0 + 2023-07-27T10:39:19Z + SO: + + myristoylation site + + + 0.9995802 + residue_name_number + cleaner0 + 2023-07-27T10:39:25Z + DUMMY: + + Gly2 + + + 0.9992757 + structure_element + cleaner0 + 2023-07-27T12:33:53Z + SO: + + helix A + + + residue_range + DUMMY: + cleaner0 + 2023-07-27T10:39:50Z + + 20 and 24 + + + 0.9980969 + protein_state + cleaner0 + 2023-07-27T10:40:06Z + DUMMY: + + GTP-, + + + 0.9988313 + protein_state + cleaner0 + 2023-07-27T10:40:09Z + DUMMY: + + GDP-bound + + + 0.9982768 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.9988266 + experimental_method + cleaner0 + 2023-07-27T10:40:21Z + MESH: + + Biochemical studies + + + 0.9991855 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9988405 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.99385804 + protein_state + cleaner0 + 2023-07-27T10:40:16Z + DUMMY: + + GTP-dependent + + + 0.9974528 + oligomeric_state + cleaner0 + 2023-07-27T10:40:32Z + DUMMY: + + oligomers + + + + INTRO + paragraph + 3466 + Crystal structures of Irga6 in various nucleotide-loaded states revealed the basic architecture of IRG proteins, including a GTPase domain and a composite helical domain. These studies additionally showed a dimerization interface in the nucleotide-free protein as well as in all nucleotide-bound states. It involves a GTPase domain surface, which is located at the opposite side of the nucleotide, and an interface in the helical domain, with a water-filled gap between the two contact surfaces. Mutagenesis of the contact surfaces suggests that this "backside" interface is not required for GTP-dependent oligomerization or cooperative hydrolysis, despite an earlier suggestion to the contrary. + + 0.99883634 + evidence + cleaner0 + 2023-07-27T12:41:41Z + DUMMY: + + Crystal structures + + + 0.99927765 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9988356 + protein_state + cleaner0 + 2023-07-27T10:41:38Z + DUMMY: + + nucleotide-loaded + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:37:26Z + + IRG + + + 0.998955 + structure_element + cleaner0 + 2023-07-27T10:36:20Z + SO: + + GTPase domain + + + structure_element + SO: + cleaner0 + 2023-07-27T10:41:04Z + + composite helical domain + + + 0.9988985 + site + cleaner0 + 2023-07-27T10:41:23Z + SO: + + dimerization interface + + + 0.99874526 + protein_state + cleaner0 + 2023-07-27T10:41:29Z + DUMMY: + + nucleotide-free + + + 0.9989639 + protein_state + cleaner0 + 2023-07-27T10:41:33Z + DUMMY: + + nucleotide-bound + + + 0.99890107 + site + cleaner0 + 2023-07-27T10:41:20Z + SO: + + GTPase domain surface + + + 0.9985129 + site + cleaner0 + 2023-07-27T12:37:52Z + SO: + + interface + + + 0.99901116 + structure_element + cleaner0 + 2023-07-27T10:41:51Z + SO: + + helical domain + + + 0.992031 + chemical + cleaner0 + 2023-07-27T12:40:24Z + CHEBI: + + water + + + 0.9884542 + site + cleaner0 + 2023-07-27T12:37:57Z + SO: + + contact surfaces + + + 0.9971976 + experimental_method + cleaner0 + 2023-07-27T12:43:20Z + MESH: + + Mutagenesis + + + 0.99702406 + site + cleaner0 + 2023-07-27T12:38:00Z + SO: + + contact surfaces + + + 0.99781394 + site + cleaner0 + 2023-07-27T12:38:10Z + SO: + + backside + + + 0.98933053 + site + cleaner0 + 2023-07-27T12:38:13Z + SO: + + interface + + + 0.99357283 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + + INTRO + paragraph + 4162 + Extensive biochemical studies suggested that GTP-induced oligomerization of Irga6 requires an interface in the GTPase domain across the nucleotide-binding site. Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). For several of these proteins, formation of the G interface was shown to trigger GTP hydrolysis by inducing rearrangements of catalytic residues in cis. In dynamin, the G interface includes residues in the phosphate binding loop, the two switch regions, the 'trans stabilizing loop' and the 'G4 loop'. For Irga6, it was demonstrated that besides residues in the switch I and switch II regions, the 3'-OH group of the ribose participates in this interface. Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6. However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. This structure was obtained by soaking GMPPNP in nucleotide-free crystals of Irga6, an approach which may have interfered with nucleotide-induced domain rearrangements. + + 0.99814224 + experimental_method + cleaner0 + 2023-07-27T12:43:23Z + MESH: + + biochemical studies + + + 0.99528426 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.9993851 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9989949 + site + cleaner0 + 2023-07-27T12:38:17Z + SO: + + interface + + + 0.9989977 + structure_element + cleaner0 + 2023-07-27T10:36:20Z + SO: + + GTPase domain + + + 0.9990971 + site + cleaner0 + 2023-07-27T12:38:21Z + SO: + + nucleotide-binding site + + + 0.998345 + experimental_method + cleaner0 + 2023-07-27T12:43:26Z + MESH: + + structural studies + + + 0.9990305 + site + cleaner0 + 2023-07-27T10:42:59Z + SO: + + G interface + + + protein_type + MESH: + cleaner0 + 2023-07-27T12:26:37Z + + dynamin + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:42:32Z + + dynamin + + + 0.9992785 + protein + cleaner0 + 2023-07-27T10:42:39Z + PR: + + MxA + + + 0.9988429 + protein + cleaner0 + 2023-07-27T10:42:42Z + PR: + + guanylate binding protein-1 + + + 0.99911195 + protein + cleaner0 + 2023-07-27T10:42:47Z + PR: + + GBP-1 + + + 0.9993549 + protein_type + cleaner0 + 2023-07-27T12:13:53Z + MESH: + + atlastin + + + 0.99868435 + taxonomy_domain + cleaner0 + 2023-07-27T10:42:55Z + DUMMY: + + bacterial + + + 0.99672365 + protein_type + cleaner0 + 2023-07-27T10:44:43Z + MESH: + + dynamin-like proteins + + + 0.9980044 + protein_type + cleaner0 + 2023-07-27T10:44:54Z + MESH: + + BDLP + + + 0.99910605 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.9984066 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.9871726 + protein_type + cleaner0 + 2023-07-27T10:36:30Z + MESH: + + dynamin + + + 0.9990924 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.998894 + structure_element + cleaner0 + 2023-07-27T10:44:20Z + SO: + + phosphate binding loop + + + 0.99662805 + site + cleaner0 + 2023-07-27T12:38:44Z + SO: + + switch regions + + + 0.99888474 + structure_element + cleaner0 + 2023-07-27T10:44:27Z + SO: + + trans stabilizing loop + + + 0.9989444 + structure_element + cleaner0 + 2023-07-27T10:44:31Z + SO: + + G4 loop + + + 0.9993806 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9983735 + site + cleaner0 + 2023-07-27T10:35:35Z + SO: + + switch I + + + site + SO: + cleaner0 + 2023-07-27T10:35:39Z + + switch II + + + 0.99814296 + site + cleaner0 + 2023-07-27T12:38:48Z + SO: + + interface + + + 0.65104276 + protein_type + cleaner0 + 2023-07-27T12:27:47Z + MESH: + + signal recognition particle GTPase + + + 0.8848597 + protein_type + cleaner0 + 2023-07-27T10:45:16Z + MESH: + + receptor + + + 0.9990214 + protein + cleaner0 + 2023-07-27T10:45:07Z + PR: + + FfH + + + 0.99926084 + protein + cleaner0 + 2023-07-27T10:45:11Z + PR: + + FtsY + + + 0.99863774 + taxonomy_domain + cleaner0 + 2023-07-27T10:44:50Z + DUMMY: + + bacteria + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-27T12:30:12Z + + dimerize + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:10:00Z + + anti-parallel + + + 0.9983564 + protein_type + cleaner0 + 2023-07-27T10:45:14Z + MESH: + + GTPase + + + 0.99937195 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.99760354 + evidence + cleaner0 + 2023-07-27T12:41:55Z + DUMMY: + + crystal structure + + + 0.9993901 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.98288035 + protein_state + cleaner0 + 2023-07-27T10:44:05Z + DUMMY: + + presence of + + + 0.99829143 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.99908435 + chemical + cleaner0 + 2023-07-27T10:43:21Z + CHEBI: + + 5'-guanylyl imidodiphosphate + + + 0.99934644 + chemical + cleaner0 + 2023-07-27T10:43:26Z + CHEBI: + + GMPPNP + + + 0.9992712 + protein_state + cleaner0 + 2023-07-27T10:44:00Z + DUMMY: + + apo + + + 0.99909383 + protein_state + cleaner0 + 2023-07-27T10:44:10Z + DUMMY: + + GDP-bound + + + 0.99903345 + site + cleaner0 + 2023-07-27T10:43:18Z + SO: + + dimer interface + + + 0.99882746 + structure_element + cleaner0 + 2023-07-27T10:36:20Z + SO: + + GTPase domain + + + 0.99785787 + evidence + cleaner0 + 2023-07-27T12:41:57Z + DUMMY: + + structure + + + 0.9982498 + experimental_method + cleaner0 + 2023-07-27T12:43:33Z + MESH: + + soaking + + + 0.99927694 + chemical + cleaner0 + 2023-07-27T10:43:26Z + CHEBI: + + GMPPNP + + + 0.99860454 + protein_state + cleaner0 + 2023-07-27T10:41:30Z + DUMMY: + + nucleotide-free + + + 0.9980411 + evidence + cleaner0 + 2023-07-27T12:42:00Z + DUMMY: + + crystals + + + 0.99938655 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + + INTRO + paragraph + 5722 + To clarify the dimerization mode via the G interface, we determined the GMPPNP-bound crystal structure of a non-oligomerizing Irga6 variant. The structure revealed that Irga6 can dimerize via the G interface in a parallel head-to-head fashion. This dimerization mode explains previously published biochemical data, and shows in particular how the 3'-OH group of the ribose participates in the assembly. Our data suggest that a parallel dimerization mode may be a unifying feature in all dynamin and septin superfamily proteins. + + 0.9990747 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.9935353 + experimental_method + cleaner0 + 2023-07-27T12:43:37Z + MESH: + + determined + + + 0.9989519 + protein_state + cleaner0 + 2023-07-27T10:45:26Z + DUMMY: + + GMPPNP-bound + + + 0.99767303 + evidence + cleaner0 + 2023-07-27T12:41:50Z + DUMMY: + + crystal structure + + + 0.9989877 + protein_state + cleaner0 + 2023-07-27T10:45:32Z + DUMMY: + + non-oligomerizing + + + 0.97425914 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9017479 + protein_state + cleaner0 + 2023-07-27T12:30:56Z + DUMMY: + + variant + + + 0.998187 + evidence + cleaner0 + 2023-07-27T12:42:03Z + DUMMY: + + structure + + + 0.9992094 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9518729 + oligomeric_state + cleaner0 + 2023-07-27T12:30:12Z + DUMMY: + + dimerize + + + 0.99904406 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.9986 + protein_state + cleaner0 + 2023-07-27T10:33:35Z + DUMMY: + + parallel head-to-head + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:09:49Z + + parallel + + + 0.99881124 + protein_type + cleaner0 + 2023-07-27T10:36:30Z + MESH: + + dynamin + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:36:52Z + + septin + + + + RESULTS + title_1 + 6250 + Results + + + RESULTS + paragraph + 6258 + Previous results indicated that Irga6 mutations in a loosely defined surface region (the "secondary patch"), which is distant from the G-interface and only slightly overlapping with the backside interface (see below), individually reduced GTP-dependent oligomerization. A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. Crystals diffracted to 3.2 Å resolution and displayed one exceptionally long unit cell axis of 1289 Å (Additional file 1: Table S1). The structure was solved by molecular replacement and refined to Rwork/Rfree of 29.7 %/31.7 % (Additional file 1: Table S2). The asymmetric unit contained seven Irga6 molecules that were arranged in a helical pattern along the long cell axis (Additional file 1: Figure S2). + + 0.99924433 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.7618282 + experimental_method + cleaner0 + 2023-07-27T12:43:45Z + MESH: + + mutations + + + 0.72960615 + site + cleaner0 + 2023-07-27T12:38:53Z + SO: + + surface region + + + 0.98579633 + site + cleaner0 + 2023-07-27T10:48:51Z + SO: + + secondary patch + + + 0.99895334 + site + cleaner0 + 2023-07-27T12:38:58Z + SO: + + G-interface + + + 0.9989779 + site + cleaner0 + 2023-07-27T10:46:44Z + SO: + + backside interface + + + 0.99561846 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + experimental_method + MESH: + cleaner0 + 2023-07-27T10:46:21Z + + combination of four of these mutations + + + 0.9989084 + mutant + cleaner0 + 2023-07-27T10:46:24Z + MESH: + + R31E + + + 0.99891293 + mutant + cleaner0 + 2023-07-27T10:46:29Z + MESH: + + K32E + + + 0.9988226 + mutant + cleaner0 + 2023-07-27T10:46:33Z + MESH: + + K176E + + + 0.99895966 + mutant + cleaner0 + 2023-07-27T10:46:37Z + MESH: + + K246E + + + 0.530567 + protein_state + cleaner0 + 2023-07-27T12:31:20Z + DUMMY: + + eliminated + + + 0.96393704 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.9978302 + experimental_method + cleaner0 + 2023-07-27T12:43:50Z + MESH: + + crystallization + + + 0.9992912 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9966885 + protein_state + cleaner0 + 2023-07-27T10:44:05Z + DUMMY: + + presence of + + + 0.99935204 + chemical + cleaner0 + 2023-07-27T10:43:26Z + CHEBI: + + GMPPNP + + + 0.9900343 + evidence + cleaner0 + 2023-07-27T12:42:08Z + DUMMY: + + Crystals + + + 0.98809695 + evidence + cleaner0 + 2023-07-27T12:42:11Z + DUMMY: + + structure + + + 0.9985469 + experimental_method + cleaner0 + 2023-07-27T12:43:54Z + MESH: + + molecular replacement + + + 0.9984352 + evidence + cleaner0 + 2023-07-27T12:42:19Z + DUMMY: + + Rwork + + + 0.7509895 + evidence + cleaner0 + 2023-07-27T12:42:21Z + DUMMY: + + Rfree + + + 0.99925333 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + + 12915_2016_236_Fig1_HTML.jpg + Fig1 + FIG + fig_caption + 7150 + Structure of the Irga6 dimer. a Schematic view of the domain architecture of mouse Irga6. The first and last amino acids of each domain are indicated. b Ribbon-type representation of the Irga6 dimer. In the left molecule, domains are colored according to the domain architecture, the right molecule is colored in grey. The nucleotide and Mg2+ ion (green) are shown in sphere representation. The GTPase domain dimer is boxed. The dotted line indicates a 2-fold axis. Secondary structure was numbered according to ref.. c +Top view on the GTPase domain dimer. d Magnification of the contact sites. Dotted lines indicate interactions. e Superposition of different switch I conformations in the asymmetric unit; the same colors as in Additional file 1: Figure S2 are used for the switch I regions of the individual subunits. Switch I residues of subunit A (yellow) involved in ribose binding are labelled and shown in stick representation. Irga6 immunity-related GTPase 6 + + evidence + DUMMY: + cleaner0 + 2023-07-27T12:44:12Z + + Structure + + + 0.99921584 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9988256 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.9914909 + taxonomy_domain + cleaner0 + 2023-07-27T12:37:17Z + DUMMY: + + mouse + + + 0.9991703 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.999255 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.99884343 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.9989561 + chemical + cleaner0 + 2023-07-27T12:40:29Z + CHEBI: + + Mg2+ + + + 0.98754936 + structure_element + cleaner0 + 2023-07-27T10:36:20Z + SO: + + GTPase domain + + + 0.998869 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.9723335 + structure_element + cleaner0 + 2023-07-27T10:36:20Z + SO: + + GTPase domain + + + 0.99883145 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.9263503 + site + cleaner0 + 2023-07-27T12:39:04Z + SO: + + contact sites + + + 0.9949509 + experimental_method + cleaner0 + 2023-07-27T12:44:02Z + MESH: + + Superposition + + + 0.9954356 + site + cleaner0 + 2023-07-27T10:35:35Z + SO: + + switch I + + + site + SO: + cleaner0 + 2023-07-27T10:35:35Z + + switch I + + + 0.99632245 + site + cleaner0 + 2023-07-27T10:35:35Z + SO: + + Switch I + + + 0.99873453 + structure_element + cleaner0 + 2023-07-27T12:34:00Z + SO: + + A + + + 0.999233 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.99642575 + protein + cleaner0 + 2023-07-27T10:47:22Z + PR: + + immunity-related GTPase 6 + + + + RESULTS + paragraph + 8117 + Like other dynamin superfamily members, the GTPase domain of Irga6 comprises a canonical GTPase domain fold, with a central β-sheet surrounded by helices on both sides (Fig. 1a-c). The helical domain is a bipartite structure composed of helices αA-C at the N-terminus and helix αF-L at the C-terminus of the GTPase domain. Overall, the seven molecules in the asymmetric unit are very similar to each other, with root mean square deviations (rmsd) ranging from 0.32 – 0.45 Å over all Cα atoms. The structures of the seven molecules also agree well with the previously determined structure of native GMPPNP-bound Irga6 (PDB: 1TQ6; rmsd of 1.00-1.13 Å over all Cα atoms). + + protein_type + MESH: + cleaner0 + 2023-07-27T10:36:30Z + + dynamin + + + 0.99935675 + structure_element + cleaner0 + 2023-07-27T10:36:20Z + SO: + + GTPase domain + + + 0.9993537 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + structure_element + SO: + cleaner0 + 2023-07-27T10:36:20Z + + GTPase domain + + + 0.99903613 + structure_element + cleaner0 + 2023-07-27T12:34:04Z + SO: + + β-sheet + + + 0.99515283 + structure_element + cleaner0 + 2023-07-27T12:34:08Z + SO: + + helices + + + 0.9993558 + structure_element + cleaner0 + 2023-07-27T10:41:52Z + SO: + + helical domain + + + 0.9986992 + structure_element + cleaner0 + 2023-07-27T12:34:10Z + SO: + + helices + + + 0.999157 + structure_element + cleaner0 + 2023-07-27T12:34:13Z + SO: + + αA-C + + + 0.99909174 + structure_element + cleaner0 + 2023-07-27T12:34:15Z + SO: + + helix + + + 0.9992301 + structure_element + cleaner0 + 2023-07-27T12:34:18Z + SO: + + αF-L + + + 0.99927294 + structure_element + cleaner0 + 2023-07-27T10:36:20Z + SO: + + GTPase domain + + + 0.9981309 + evidence + cleaner0 + 2023-07-27T10:48:13Z + DUMMY: + + root mean square deviations + + + 0.9978098 + evidence + cleaner0 + 2023-07-27T10:48:17Z + DUMMY: + + rmsd + + + 0.998288 + evidence + cleaner0 + 2023-07-27T12:42:29Z + DUMMY: + + structures + + + 0.99807537 + evidence + cleaner0 + 2023-07-27T12:42:34Z + DUMMY: + + structure + + + 0.99919444 + protein_state + cleaner0 + 2023-07-27T10:48:08Z + DUMMY: + + native + + + 0.9989428 + protein_state + cleaner0 + 2023-07-27T10:45:26Z + DUMMY: + + GMPPNP-bound + + + 0.999371 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.99834836 + evidence + cleaner0 + 2023-07-27T10:48:17Z + DUMMY: + + rmsd + + + + RESULTS + paragraph + 8805 + The seven Irga6 molecules in the asymmetric unit form various higher order contacts in the crystals. Within the asymmetric unit, six molecules dimerize via the symmetric backside dimer interface (buried surface area 930 Å2), and the remaining seventh molecule forms the same type of interaction with its symmetry mate of the adjacent asymmetric unit (Additional file 1: Figure S2a, b, Figure S3). This indicates that the introduced mutations in the secondary patch, from which only Lys176 is part of the backside interface, do, in fact, not prevent this interaction. + + 0.9991252 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.98424673 + evidence + cleaner0 + 2023-07-27T12:42:37Z + DUMMY: + + crystals + + + 0.99772984 + oligomeric_state + cleaner0 + 2023-07-27T12:30:12Z + DUMMY: + + dimerize + + + 0.96250945 + site + cleaner0 + 2023-07-27T10:48:42Z + SO: + + backside dimer interface + + + 0.9550488 + experimental_method + cleaner0 + 2023-07-27T12:44:31Z + MESH: + + mutations + + + 0.9822448 + site + cleaner0 + 2023-07-27T10:48:50Z + SO: + + secondary patch + + + 0.9995739 + residue_name_number + cleaner0 + 2023-07-27T10:48:33Z + DUMMY: + + Lys176 + + + 0.9989483 + site + cleaner0 + 2023-07-27T10:46:44Z + SO: + + backside interface + + + + RESULTS + paragraph + 9373 + Another assembly interface with a buried surface area of 450 Å2, which we call the “tertiary patch”, was formed via two interaction sites in the helical domains (Additional file 1: Figure S2c, d, S3). In this interface, helices αK from two adjacent molecules form a hydrogen bonding network involving residues 373-376. Furthermore, two adjacent helices αA form hydrophobic contacts. It was previously shown that the double mutation L372R/A373R did not prevent GTP-induced assembly, so there is currently no evidence supporting an involvement of this interface in higher-order oligomerization. + + 0.99892914 + site + cleaner0 + 2023-07-27T12:39:10Z + SO: + + assembly interface + + + 0.99890065 + site + cleaner0 + 2023-07-27T10:48:59Z + SO: + + tertiary patch + + + 0.99861753 + site + cleaner0 + 2023-07-27T12:39:15Z + SO: + + interaction sites + + + 0.9989256 + structure_element + cleaner0 + 2023-07-27T12:34:22Z + SO: + + helical domains + + + 0.99846673 + site + cleaner0 + 2023-07-27T12:39:18Z + SO: + + interface + + + 0.999212 + structure_element + cleaner0 + 2023-07-27T12:34:25Z + SO: + + helices + + + 0.9995142 + structure_element + cleaner0 + 2023-07-27T12:34:28Z + SO: + + αK + + + 0.99816227 + bond_interaction + cleaner0 + 2023-07-27T10:49:12Z + MESH: + + hydrogen bonding network + + + 0.99330187 + residue_range + cleaner0 + 2023-07-27T10:49:40Z + DUMMY: + + 373-376 + + + 0.9990397 + structure_element + cleaner0 + 2023-07-27T12:34:30Z + SO: + + helices + + + 0.9995098 + structure_element + cleaner0 + 2023-07-27T12:34:33Z + SO: + + αA + + + 0.99618495 + bond_interaction + cleaner0 + 2023-07-27T10:49:17Z + MESH: + + hydrophobic contacts + + + 0.9097497 + protein_state + cleaner0 + 2023-07-27T10:49:30Z + DUMMY: + + double mutation + + + 0.9991792 + mutant + cleaner0 + 2023-07-27T10:49:33Z + MESH: + + L372R + + + 0.999008 + mutant + cleaner0 + 2023-07-27T10:49:37Z + MESH: + + A373R + + + 0.9986527 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.998389 + site + cleaner0 + 2023-07-27T12:39:24Z + SO: + + interface + + + + RESULTS + paragraph + 9974 + Strikingly, molecule A of one asymmetric unit assembled with an equivalent molecule of the adjacent asymmetric unit via the G-interface in a symmetric parallel fashion via a 470 Å2 interface. This assembly results in a butterfly-shaped Irga6 dimer in which the helical domains protrude in parallel orientations (Fig. 1b, Additional file 1: Figure S3). In contrast, the other six molecules in the asymmetric unit do not assemble via the G interface. + + 0.76502633 + structure_element + cleaner0 + 2023-07-27T10:49:47Z + SO: + + A + + + 0.9990626 + site + cleaner0 + 2023-07-27T12:39:30Z + SO: + + G-interface + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:09:49Z + + parallel + + + 0.99863386 + protein_state + cleaner0 + 2023-07-27T10:49:48Z + DUMMY: + + butterfly-shaped + + + 0.99940443 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.99864584 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.9984453 + structure_element + cleaner0 + 2023-07-27T12:34:39Z + SO: + + helical domains + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:09:49Z + + parallel + + + 0.99906284 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + + RESULTS + paragraph + 10425 + The G interface in molecule A can be subdivided into three distinct contact sites (Fig. 1c, d). Contact site I is formed between R159 and K161 in the trans stabilizing loops, and S132 in the switch II regions of the opposing molecules. Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. In contact site III, G103 of switch I interacts via its main chain nitrogen with the exocyclic 2’-OH and 3’-OH groups of the opposing ribose in trans, whereas the two opposing exocyclic 3’-OH group of the ribose form hydrogen bonds with each other. Via the ribose contact, switch I is pulled towards the opposing nucleotide (Fig. 1e). In turn, E106 of switch I reorients towards the nucleotide and now participates in the coordination of the Mg2+ ion (Fig. 1e, Additional file 1: Figure S4). E106 was previously shown to be essential for catalysis, and the observed interactions in contact site III explain how dimerization via the ribose is directly coupled to the activation of GTP hydrolysis. + + 0.9990487 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.9987433 + site + cleaner0 + 2023-07-27T12:39:36Z + SO: + + contact sites + + + 0.99637073 + site + cleaner0 + 2023-07-27T10:51:05Z + SO: + + Contact site I + + + 0.9995734 + residue_name_number + cleaner0 + 2023-07-27T10:51:14Z + DUMMY: + + R159 + + + 0.9995622 + residue_name_number + cleaner0 + 2023-07-27T10:51:19Z + DUMMY: + + K161 + + + structure_element + SO: + cleaner0 + 2023-07-27T12:31:43Z + + trans stabilizing loops + + + 0.999587 + residue_name_number + cleaner0 + 2023-07-27T10:51:25Z + DUMMY: + + S132 + + + 0.9942776 + site + cleaner0 + 2023-07-27T10:35:39Z + SO: + + switch II + + + 0.9976573 + site + cleaner0 + 2023-07-27T10:51:10Z + SO: + + Contact site II + + + 0.99633247 + bond_interaction + cleaner0 + 2023-07-27T10:51:30Z + MESH: + + polar and hydrophobic interactions + + + 0.998207 + site + cleaner0 + 2023-07-27T10:35:35Z + SO: + + switch I + + + 0.9996013 + residue_name_number + cleaner0 + 2023-07-27T10:51:34Z + DUMMY: + + V104 + + + 0.9996019 + residue_name_number + cleaner0 + 2023-07-27T10:51:38Z + DUMMY: + + V107 + + + 0.7647327 + structure_element + cleaner0 + 2023-07-27T12:34:43Z + SO: + + helix + + + 0.99911284 + structure_element + cleaner0 + 2023-07-27T10:50:53Z + SO: + + guanine specificity motif + + + 0.9983238 + structure_element + cleaner0 + 2023-07-27T10:50:57Z + SO: + + G4 helix + + + 0.99954104 + residue_name_number + cleaner0 + 2023-07-27T10:51:42Z + DUMMY: + + K184 + + + 0.99954873 + residue_name_number + cleaner0 + 2023-07-27T10:51:47Z + DUMMY: + + S187 + + + structure_element + SO: + cleaner0 + 2023-07-27T12:27:32Z + + trans stabilizing loop + + + 0.9995708 + residue_name_number + cleaner0 + 2023-07-27T10:51:53Z + DUMMY: + + T158 + + + 0.99916863 + structure_element + cleaner0 + 2023-07-27T10:36:20Z + SO: + + GTPase domain + + + 0.99693394 + site + cleaner0 + 2023-07-27T10:52:29Z + SO: + + contact site III + + + 0.9996068 + residue_name_number + cleaner0 + 2023-07-27T10:52:22Z + DUMMY: + + G103 + + + 0.997262 + site + cleaner0 + 2023-07-27T10:35:35Z + SO: + + switch I + + + 0.99728584 + chemical + cleaner0 + 2023-07-27T12:32:30Z + CHEBI: + + ribose + + + 0.9979128 + chemical + cleaner0 + 2023-07-27T12:32:33Z + CHEBI: + + ribose + + + 0.99671197 + bond_interaction + cleaner0 + 2023-07-27T10:52:41Z + MESH: + + hydrogen bonds + + + 0.97772825 + chemical + cleaner0 + 2023-07-27T12:32:37Z + CHEBI: + + ribose + + + 0.9978828 + site + cleaner0 + 2023-07-27T10:35:35Z + SO: + + switch I + + + 0.85826856 + chemical + cleaner0 + 2023-07-27T12:32:41Z + CHEBI: + + nucleotide + + + 0.9996145 + residue_name_number + cleaner0 + 2023-07-27T10:52:35Z + DUMMY: + + E106 + + + 0.997312 + site + cleaner0 + 2023-07-27T10:35:35Z + SO: + + switch I + + + 0.88709295 + chemical + cleaner0 + 2023-07-27T12:32:44Z + CHEBI: + + nucleotide + + + bond_interaction + MESH: + cleaner0 + 2023-07-27T10:52:59Z + + coordination of + + + 0.9984646 + chemical + cleaner0 + 2023-07-27T10:52:25Z + CHEBI: + + Mg2+ + + + 0.999617 + residue_name_number + cleaner0 + 2023-07-27T10:52:35Z + DUMMY: + + E106 + + + 0.99557066 + site + cleaner0 + 2023-07-27T10:52:29Z + SO: + + contact site III + + + 0.99209434 + chemical + cleaner0 + 2023-07-27T12:32:53Z + CHEBI: + + ribose + + + 0.9988549 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + + RESULTS + paragraph + 11606 + The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). Residues E77, K162, and D164 appear to orient the trans stabilizing loop which is involved in interface formation in contact site II. In the earlier model of an anti-parallel G interface, it was not possible to position the side chain of R159 to avoid steric conflict. In the present structure, the side-chain of R159 projects laterally along the G interface and, therefore, does not cause a steric conflict. + + 0.999061 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.9994791 + residue_name_number + cleaner0 + 2023-07-27T12:08:18Z + DUMMY: + + E77 + + + 0.9994943 + residue_name_number + cleaner0 + 2023-07-27T10:52:22Z + DUMMY: + + G103 + + + 0.9994764 + residue_name_number + cleaner0 + 2023-07-27T10:52:35Z + DUMMY: + + E106 + + + 0.99947697 + residue_name_number + cleaner0 + 2023-07-27T10:51:25Z + DUMMY: + + S132 + + + 0.9993932 + residue_name_number + cleaner0 + 2023-07-27T10:51:15Z + DUMMY: + + R159 + + + 0.9993832 + residue_name_number + cleaner0 + 2023-07-27T10:51:20Z + DUMMY: + + K161 + + + 0.9993795 + residue_name_number + cleaner0 + 2023-07-27T12:08:37Z + DUMMY: + + K162 + + + 0.99940157 + residue_name_number + cleaner0 + 2023-07-27T12:08:41Z + DUMMY: + + D164 + + + 0.999419 + residue_name_number + cleaner0 + 2023-07-27T12:08:45Z + DUMMY: + + N191 + + + 0.999411 + residue_name_number + cleaner0 + 2023-07-27T12:08:50Z + DUMMY: + + K196 + + + 0.9982338 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.9994584 + residue_name_number + cleaner0 + 2023-07-27T10:52:22Z + DUMMY: + + G103 + + + 0.999456 + residue_name_number + cleaner0 + 2023-07-27T10:51:25Z + DUMMY: + + S132 + + + 0.9993513 + residue_name_number + cleaner0 + 2023-07-27T10:51:15Z + DUMMY: + + R159 + + + 0.99934167 + residue_name_number + cleaner0 + 2023-07-27T10:51:20Z + DUMMY: + + K161 + + + 0.99899274 + site + cleaner0 + 2023-07-27T12:39:43Z + SO: + + interface + + + 0.9994425 + residue_name_number + cleaner0 + 2023-07-27T12:08:18Z + DUMMY: + + E77 + + + 0.99947435 + residue_name_number + cleaner0 + 2023-07-27T10:52:35Z + DUMMY: + + E106 + + + 0.99937785 + residue_name_number + cleaner0 + 2023-07-27T12:08:37Z + DUMMY: + + K162 + + + 0.99940526 + residue_name_number + cleaner0 + 2023-07-27T12:08:41Z + DUMMY: + + D164 + + + 0.99940145 + residue_name_number + cleaner0 + 2023-07-27T12:08:46Z + DUMMY: + + N191 + + + 0.9994404 + residue_name_number + cleaner0 + 2023-07-27T12:08:18Z + DUMMY: + + E77 + + + 0.9993716 + residue_name_number + cleaner0 + 2023-07-27T12:08:37Z + DUMMY: + + K162 + + + 0.9993992 + residue_name_number + cleaner0 + 2023-07-27T12:08:41Z + DUMMY: + + D164 + + + structure_element + SO: + cleaner0 + 2023-07-27T12:27:32Z + + trans stabilizing loop + + + 0.9987205 + site + cleaner0 + 2023-07-27T12:39:48Z + SO: + + interface + + + 0.99573773 + site + cleaner0 + 2023-07-27T10:51:10Z + SO: + + contact site II + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:08:08Z + + anti-parallel + + + site + SO: + cleaner0 + 2023-07-27T10:43:00Z + + G interface + + + 0.9994363 + residue_name_number + cleaner0 + 2023-07-27T10:51:15Z + DUMMY: + + R159 + + + 0.9985574 + evidence + cleaner0 + 2023-07-27T12:42:42Z + DUMMY: + + structure + + + 0.9994172 + residue_name_number + cleaner0 + 2023-07-27T10:51:15Z + DUMMY: + + R159 + + + 0.999063 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + + 12915_2016_236_Fig2_HTML.jpg + Fig2 + FIG + fig_caption + 12414 + A conserved dimerization mode via the G interface in dynamin and septin GTPases. The overall architecture of the parallel GTPase domain dimer of Irga6 is related to that of other dynamin and septin superfamily proteins. The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 +--bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. The GTPase domains of the left molecules are shown in orange, helical domains or extensions in blue. Nucleotide, Mg2+ (green) and AlF4 +- are shown in sphere representation, the buried interface sizes per molecule are indicated on the right. Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 + + 0.9989195 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.998784 + protein_type + cleaner0 + 2023-07-27T10:36:30Z + MESH: + + dynamin + + + 0.99875796 + protein_type + cleaner0 + 2023-07-27T12:10:14Z + MESH: + + septin GTPases + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:09:49Z + + parallel + + + structure_element + SO: + cleaner0 + 2023-07-27T10:36:20Z + + GTPase domain + + + 0.9963877 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.99923456 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9982521 + protein_type + cleaner0 + 2023-07-27T10:36:30Z + MESH: + + dynamin + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:36:52Z + + septin + + + 0.99592704 + evidence + cleaner0 + 2023-07-27T12:42:47Z + DUMMY: + + structures + + + 0.9990865 + structure_element + cleaner0 + 2023-07-27T10:34:08Z + SO: + + GTPase domains + + + 0.9987429 + protein_state + cleaner0 + 2023-07-27T10:45:26Z + DUMMY: + + GMPPNP-bound + + + 0.99917185 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.99885464 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.99870783 + protein_state + cleaner0 + 2023-07-27T12:33:13Z + DUMMY: + + GDP-AlF4 +--bound + + + 0.9958458 + protein + cleaner0 + 2023-07-27T12:10:59Z + PR: + + dynamin 1 + + + structure_element + SO: + cleaner0 + 2023-07-27T12:36:38Z + + GTPase-minimal + + + 0.9989162 + protein_state + cleaner0 + 2023-07-27T10:44:10Z + DUMMY: + + GDP-bound + + + 0.99906284 + protein + cleaner0 + 2023-07-27T12:11:08Z + PR: + + atlastin 1 + + + 0.9988581 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.99890757 + protein_state + cleaner0 + 2023-07-27T12:33:17Z + DUMMY: + + GDP-AlF3- bound + + + 0.99915755 + protein + cleaner0 + 2023-07-27T12:28:42Z + PR: + + GBP1 + + + 0.99882436 + structure_element + cleaner0 + 2023-07-27T10:36:20Z + SO: + + GTPase domain + + + 0.9982911 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.9982279 + protein_type + cleaner0 + 2023-07-27T10:44:54Z + MESH: + + BDLP + + + 0.9988255 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.9990673 + protein_state + cleaner0 + 2023-07-27T10:34:29Z + DUMMY: + + bound to + + + 0.9962359 + chemical + cleaner0 + 2023-07-27T12:40:34Z + CHEBI: + + GDP + + + 0.99897075 + protein_state + cleaner0 + 2023-07-27T12:11:28Z + DUMMY: + + GTP-bound + + + 0.9991867 + protein + cleaner0 + 2023-07-27T12:12:18Z + PR: + + GIMAP2 + + + 0.998862 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.9988321 + structure_element + cleaner0 + 2023-07-27T10:34:08Z + SO: + + GTPase domains + + + 0.9896811 + structure_element + cleaner0 + 2023-07-27T12:36:44Z + SO: + + helical domains + + + 0.9989772 + chemical + cleaner0 + 2023-07-27T12:11:32Z + CHEBI: + + Mg2+ + + + 0.99882233 + chemical + cleaner0 + 2023-07-27T12:11:37Z + CHEBI: + + AlF4 +- + + + 0.99710995 + site + cleaner0 + 2023-07-27T12:39:54Z + SO: + + interface + + + 0.99926573 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.998515 + protein + cleaner0 + 2023-07-27T10:47:22Z + PR: + + immunity-related GTPase 6 + + + 0.99926454 + chemical + cleaner0 + 2023-07-27T10:43:26Z + CHEBI: + + GMPPNP + + + 0.99905527 + chemical + cleaner0 + 2023-07-27T12:40:39Z + CHEBI: + + 5'-guanylyl imidodiphosphate + + + 0.9871029 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.99913836 + chemical + cleaner0 + 2023-07-27T12:40:43Z + CHEBI: + + guanosine-triphosphate + + + 0.99609816 + protein_type + cleaner0 + 2023-07-27T10:44:54Z + MESH: + + BDLP + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-27T10:42:55Z + + bacterial + + + protein_type + MESH: + cleaner0 + 2023-07-27T12:12:13Z + + dynamin like protein + + + 0.99891984 + protein + cleaner0 + 2023-07-27T12:12:18Z + PR: + + GIMAP2 + + + 0.99787754 + protein + cleaner0 + 2023-07-27T12:12:25Z + PR: + + GTPase of immunity associated protein 2 + + + + RESULTS + paragraph + 13467 + The buried surface area per molecule (BSA) of the G interface in Irga6 is relatively small (470 Å2) compared to that of other dynamin superfamily members, such as dynamin (BSA: 1400 Å2), atlastin (BSA: 820 Å2), GBP-1 (BSA: 2060 Å2), BDLP (BSA: 2300 Å2) or the septin-related GTPase of immunity associated protein 2 (GIMAP2) (BSA: 590 Å2) (Fig. 2). However, the relative orientations of the GTPase domains in these dimers are strikingly similar, and the same elements, such as switch I, switch II, the trans activating and G4 loops are involved in the parallel dimerization mode in all of these GTPase families. + + 0.9989669 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.99922585 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:36:30Z + + dynamin + + + 0.99929154 + protein_type + cleaner0 + 2023-07-27T10:36:30Z + MESH: + + dynamin + + + 0.99930716 + protein_type + cleaner0 + 2023-07-27T12:13:53Z + MESH: + + atlastin + + + 0.99926883 + protein + cleaner0 + 2023-07-27T10:42:47Z + PR: + + GBP-1 + + + 0.9990445 + protein_type + cleaner0 + 2023-07-27T10:44:54Z + MESH: + + BDLP + + + 0.99843323 + protein + cleaner0 + 2023-07-27T12:12:49Z + PR: + + septin-related GTPase of immunity associated protein 2 + + + 0.9993419 + protein + cleaner0 + 2023-07-27T12:12:18Z + PR: + + GIMAP2 + + + 0.99648523 + structure_element + cleaner0 + 2023-07-27T10:34:08Z + SO: + + GTPase domains + + + 0.9986093 + oligomeric_state + cleaner0 + 2023-07-27T12:30:22Z + DUMMY: + + dimers + + + 0.9986375 + site + cleaner0 + 2023-07-27T10:35:35Z + SO: + + switch I + + + 0.9985064 + site + cleaner0 + 2023-07-27T10:35:39Z + SO: + + switch II + + + 0.9989953 + structure_element + cleaner0 + 2023-07-27T12:36:52Z + SO: + + trans activating and G4 loops + + + 0.89991593 + protein_state + cleaner0 + 2023-07-27T12:09:49Z + DUMMY: + + parallel + + + protein_type + MESH: + cleaner0 + 2023-07-27T12:28:06Z + + GTPase + + + + DISCUSS + title_1 + 14085 + Discussion + + + DISCUSS + paragraph + 14096 + IRG proteins are crucial mediators of the innate immune response in mice against a specific subset of intracellular pathogens, all of which enter the cell to form a membrane-bounded vacuole without engagement of the phagocytic machinery. As members of the dynamin superfamily, IRGs oligomerize at cellular membranes in response to GTP binding. Oligomerization and oligomerization-induced GTP hydrolysis are thought to induce membrane remodeling events ultimately leading to disruption of the PVM. Recent structural and mechanistic analyses have begun to unravel the molecular basis for the membrane-remodeling activity and mechano-chemical function of some members (reviewed in). For example, for dynamin and atlastin, it was shown that GTP binding and/or hydrolysis leads to dimerization of the GTPase domains and to the reorientation of the adjacent helical domains. The resulting domain movement was suggested to act as a “power stroke” during membrane remodeling events. However, for other dynamin superfamily members such as IRGs, the molecular basis for GTP hydrolysis and the exact role of the mechano-chemical function are still unclear. + + protein_type + MESH: + cleaner0 + 2023-07-27T10:37:26Z + + IRG + + + 0.99626875 + taxonomy_domain + cleaner0 + 2023-07-27T10:38:34Z + DUMMY: + + mice + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:36:30Z + + dynamin + + + 0.99929845 + protein_type + cleaner0 + 2023-07-27T10:33:43Z + MESH: + + IRGs + + + 0.99870706 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.9958644 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.9985744 + experimental_method + cleaner0 + 2023-07-27T12:44:55Z + MESH: + + structural and mechanistic analyses + + + 0.97879887 + protein_type + cleaner0 + 2023-07-27T10:36:30Z + MESH: + + dynamin + + + 0.99768 + protein_type + cleaner0 + 2023-07-27T12:13:52Z + MESH: + + atlastin + + + 0.9983102 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.9991524 + structure_element + cleaner0 + 2023-07-27T10:34:08Z + SO: + + GTPase domains + + + 0.99791276 + structure_element + cleaner0 + 2023-07-27T12:36:56Z + SO: + + helical domains + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:36:30Z + + dynamin + + + 0.99784553 + protein_type + cleaner0 + 2023-07-27T10:33:43Z + MESH: + + IRGs + + + 0.9967393 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + + DISCUSS + paragraph + 15246 + Our structural analysis of an oligomerization- and GTPase-defective Irga6 mutant indicates that Irga6 dimerizes via the G interface in a parallel orientation. Only one of the seven Irga6 molecules in the asymmetric unit formed this contact pointing to a low affinity interaction via the G interface, which is in agreement with its small size. In the crystals, dimerization via the G interface is promoted by the high protein concentrations which may mimic a situation when Irga6 oligomerizes on a membrane surface. Such a low affinity interaction mode may allow reversibility of oligomerization following GTP hydrolysis. Similar low affinity G interface interactions were reported for dynamin and MxA. + + 0.9986919 + experimental_method + cleaner0 + 2023-07-27T12:44:58Z + MESH: + + structural analysis + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T12:14:27Z + + oligomerization- and GTPase-defective + + + 0.99571157 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.99869686 + protein_state + cleaner0 + 2023-07-27T10:34:25Z + DUMMY: + + mutant + + + 0.99924856 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.99752396 + oligomeric_state + cleaner0 + 2023-07-27T10:33:31Z + DUMMY: + + dimerizes + + + 0.9991144 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.79277337 + protein_state + cleaner0 + 2023-07-27T12:09:49Z + DUMMY: + + parallel + + + 0.9992861 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9991021 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.99705374 + evidence + cleaner0 + 2023-07-27T12:42:53Z + DUMMY: + + crystals + + + 0.99911535 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.99934715 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9984047 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.9991163 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.9990403 + protein_type + cleaner0 + 2023-07-27T10:36:30Z + MESH: + + dynamin + + + 0.9990915 + protein + cleaner0 + 2023-07-27T10:42:39Z + PR: + + MxA + + + + DISCUSS + paragraph + 15948 + The dimerization mode is strikingly different from the previously proposed anti-parallel model that was based on the crystal structure of the signal recognition particle GTPase, SRP54 and its homologous receptor. However, the G dimer interface is reminiscent of the GTPase domain dimers observed for several other dynamin superfamily members, such as dynamin, GBP1, atlastin, and BDLP. It was recently shown that septin and septin-related GTPases, such as the Tocs GTPases or GTPases of immunity related proteins (GIMAPs), also employ a GTP-dependent parallel dimerization mode. Based on phylogenetic and structural analysis, these observations suggest that dynamin and septin superfamilies are derived from a common ancestral membrane-associated GTPase that featured a GTP-dependent parallel dimerization mode. Importantly, our analysis indicates that IRGs are not outliers, but bona-fide representatives of the dynamin superfamily. + + 0.90689725 + protein_state + cleaner0 + 2023-07-27T12:10:00Z + DUMMY: + + anti-parallel + + + 0.99784887 + evidence + cleaner0 + 2023-07-27T12:42:57Z + DUMMY: + + crystal structure + + + protein_type + MESH: + cleaner0 + 2023-07-27T12:29:15Z + + signal recognition particle GTPase + + + 0.99929106 + protein + cleaner0 + 2023-07-27T12:29:19Z + PR: + + SRP54 + + + 0.9989607 + site + cleaner0 + 2023-07-27T12:14:51Z + SO: + + G dimer interface + + + structure_element + SO: + cleaner0 + 2023-07-27T12:15:22Z + + GTPase domain + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-27T12:15:33Z + + dimers + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:36:30Z + + dynamin + + + 0.9990158 + protein_type + cleaner0 + 2023-07-27T10:36:30Z + MESH: + + dynamin + + + 0.9991697 + protein + cleaner0 + 2023-07-27T12:29:23Z + PR: + + GBP1 + + + 0.9991943 + protein_type + cleaner0 + 2023-07-27T12:13:53Z + MESH: + + atlastin + + + 0.9991836 + protein_type + cleaner0 + 2023-07-27T10:44:54Z + MESH: + + BDLP + + + 0.9989994 + protein_type + cleaner0 + 2023-07-27T10:36:52Z + MESH: + + septin + + + 0.99870986 + protein_type + cleaner0 + 2023-07-27T12:15:51Z + MESH: + + septin-related GTPases + + + 0.99816024 + protein_type + cleaner0 + 2023-07-27T12:28:22Z + MESH: + + Tocs GTPases + + + 0.9955791 + protein_type + cleaner0 + 2023-07-27T12:15:54Z + MESH: + + GTPases of immunity related proteins + + + 0.9981299 + protein_type + cleaner0 + 2023-07-27T12:15:58Z + MESH: + + GIMAPs + + + 0.9936714 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.9250834 + protein_state + cleaner0 + 2023-07-27T12:09:50Z + DUMMY: + + parallel + + + 0.9989378 + experimental_method + cleaner0 + 2023-07-27T12:45:04Z + MESH: + + phylogenetic and structural analysis + + + 0.99865425 + protein_type + cleaner0 + 2023-07-27T10:36:30Z + MESH: + + dynamin + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:36:52Z + + septin + + + 0.998357 + protein_type + cleaner0 + 2023-07-27T12:16:01Z + MESH: + + membrane-associated GTPase + + + 0.9872011 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.71070504 + protein_state + cleaner0 + 2023-07-27T12:09:50Z + DUMMY: + + parallel + + + 0.99881625 + protein_type + cleaner0 + 2023-07-27T10:33:43Z + MESH: + + IRGs + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:36:30Z + + dynamin + + + + DISCUSS + paragraph + 16882 + Whereas the overall dimerization mode is similar in septin and dynamin GTPases, family-specific differences in the G interface and the oligomerization interfaces exist. For example, the involvement of the 2’ and 3’-OH groups of the ribose in the dimerization interface of Irga6 has not been observed for other dynamin and septin superfamily members. The surface-exposed location of the ribose in the Irga6 structure, with a wide-open nucleotide-binding pocket, facilitates its engagement in the dimerization interface. This contact, in turn, appears to activate GTP hydrolysis by inducing rearrangements in switch I and the positioning of the catalytic E106. During dimerization of GBP1, an arginine finger from the P loop reorients towards the nucleotide in cis to trigger GTP hydrolysis. In dynamin, the corresponding serine residue coordinates a sodium ion that is crucial for GTP hydrolysis. Irga6 bears Gly79 at this position, which in the dimerizing molecule A appears to approach the bridging imido group of GMPPNP via a main chain hydrogen bond. Higher resolution structures of the Irga6 dimer in the presence of a transition state analogue are required to show whether Gly79 directly participates in GTP hydrolysis or whether it may also position a catalytic cation. + + 0.99877435 + protein_type + cleaner0 + 2023-07-27T10:36:52Z + MESH: + + septin + + + 0.9981336 + protein_type + cleaner0 + 2023-07-27T12:16:25Z + MESH: + + dynamin GTPases + + + 0.9990227 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.9990978 + site + cleaner0 + 2023-07-27T12:39:59Z + SO: + + oligomerization interfaces + + + 0.99857116 + chemical + cleaner0 + 2023-07-27T12:40:48Z + CHEBI: + + ribose + + + 0.9990822 + site + cleaner0 + 2023-07-27T10:41:24Z + SO: + + dimerization interface + + + 0.99931896 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.99840385 + protein_type + cleaner0 + 2023-07-27T10:36:30Z + MESH: + + dynamin + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:36:52Z + + septin + + + 0.9993469 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9976046 + evidence + cleaner0 + 2023-07-27T12:43:05Z + DUMMY: + + structure + + + 0.95810074 + protein_state + cleaner0 + 2023-07-27T12:33:29Z + DUMMY: + + wide-open + + + 0.9990585 + site + cleaner0 + 2023-07-27T12:40:04Z + SO: + + nucleotide-binding pocket + + + 0.9991007 + site + cleaner0 + 2023-07-27T10:41:24Z + SO: + + dimerization interface + + + 0.9983584 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.9976095 + site + cleaner0 + 2023-07-27T10:35:35Z + SO: + + switch I + + + 0.9610252 + protein_state + cleaner0 + 2023-07-27T12:17:07Z + DUMMY: + + catalytic + + + 0.9995932 + residue_name_number + cleaner0 + 2023-07-27T10:52:35Z + DUMMY: + + E106 + + + 0.9994085 + protein + cleaner0 + 2023-07-27T12:29:27Z + PR: + + GBP1 + + + 0.9987834 + structure_element + cleaner0 + 2023-07-27T12:17:24Z + SO: + + arginine finger + + + 0.99936163 + structure_element + cleaner0 + 2023-07-27T12:17:30Z + SO: + + P loop + + + 0.9880989 + chemical + cleaner0 + 2023-07-27T12:40:53Z + CHEBI: + + nucleotide + + + 0.9986308 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.99870455 + protein_type + cleaner0 + 2023-07-27T10:36:30Z + MESH: + + dynamin + + + 0.99738985 + residue_name + cleaner0 + 2023-07-27T12:17:18Z + SO: + + serine + + + 0.97945315 + chemical + cleaner0 + 2023-07-27T12:17:15Z + CHEBI: + + sodium + + + 0.99860805 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + 0.99933213 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9995295 + residue_name_number + cleaner0 + 2023-07-27T12:16:51Z + DUMMY: + + Gly79 + + + 0.9914948 + oligomeric_state + cleaner0 + 2023-07-27T12:30:30Z + DUMMY: + + dimerizing + + + 0.6915696 + structure_element + cleaner0 + 2023-07-27T12:37:02Z + SO: + + A + + + 0.99933606 + chemical + cleaner0 + 2023-07-27T10:43:26Z + CHEBI: + + GMPPNP + + + 0.9966464 + bond_interaction + cleaner0 + 2023-07-27T12:16:45Z + MESH: + + hydrogen bond + + + 0.99851006 + evidence + cleaner0 + 2023-07-27T12:43:10Z + DUMMY: + + structures + + + 0.99937075 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9987482 + oligomeric_state + cleaner0 + 2023-07-27T10:37:33Z + DUMMY: + + dimer + + + 0.8419397 + protein_state + cleaner0 + 2023-07-27T10:44:05Z + DUMMY: + + presence of + + + 0.99955744 + residue_name_number + cleaner0 + 2023-07-27T12:16:52Z + DUMMY: + + Gly79 + + + 0.99833554 + chemical + cleaner0 + 2023-07-27T10:35:02Z + CHEBI: + + GTP + + + + DISCUSS + paragraph + 18162 + In dynamin, further assembly sites are provided by the helical domains which assemble in a criss-cross fashion to form a helical filament. In dynamin-related Eps15 homology domain containing proteins (EHDs), a second assembly interface is present in the GTPase domain. For Irga6, additional interfaces in the helical domain are presumably involved in oligomerization, such as the secondary patch residues whose mutation prevented oligomerization in the crystallized mutant. Further structural studies, especially electron microscopy analysis of the Irga6 oligomers, are required to clarify the assembly mode via the helical domains and to show how these interfaces cooperate with the G interface to mediate the regulated assembly on a membrane surface. Notably, we did not observe major rearrangements of the helical domain versus the GTPase domain in the Irga6 molecules that dimerized via the G interface. In a manner similar to BDLP, such large-scale conformational changes may be induced by membrane binding. Our structural analysis and the identification of the G-interface paves the way for determining the specific assembly of Irga6 into a membrane-associated scaffold as the prerequisite to understand its action as an anti-parasitic machine. + + 0.8942208 + protein_type + cleaner0 + 2023-07-27T10:36:30Z + MESH: + + dynamin + + + 0.99581313 + site + cleaner0 + 2023-07-27T12:17:56Z + SO: + + assembly sites + + + 0.99914 + structure_element + cleaner0 + 2023-07-27T12:18:05Z + SO: + + helical domains + + + 0.9960346 + structure_element + cleaner0 + 2023-07-27T12:18:07Z + SO: + + helical filament + + + 0.99539596 + protein_type + cleaner0 + 2023-07-27T12:17:42Z + MESH: + + dynamin-related Eps15 homology domain containing proteins + + + 0.99789655 + protein_type + cleaner0 + 2023-07-27T12:17:45Z + MESH: + + EHDs + + + 0.998976 + site + cleaner0 + 2023-07-27T12:17:58Z + SO: + + second assembly interface + + + 0.9990665 + structure_element + cleaner0 + 2023-07-27T10:36:20Z + SO: + + GTPase domain + + + 0.99937326 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9970872 + site + cleaner0 + 2023-07-27T12:40:07Z + SO: + + interfaces + + + 0.99917567 + structure_element + cleaner0 + 2023-07-27T10:41:52Z + SO: + + helical domain + + + 0.99605507 + site + cleaner0 + 2023-07-27T10:48:51Z + SO: + + secondary patch + + + 0.9845604 + experimental_method + cleaner0 + 2023-07-27T12:45:15Z + MESH: + + mutation + + + 0.60325915 + evidence + cleaner0 + 2023-07-27T12:43:14Z + DUMMY: + + crystallized + + + 0.53057414 + protein_state + cleaner0 + 2023-07-27T10:34:25Z + DUMMY: + + mutant + + + 0.9892376 + experimental_method + cleaner0 + 2023-07-27T12:45:18Z + MESH: + + structural studies + + + experimental_method + MESH: + cleaner0 + 2023-07-27T12:45:38Z + + electron microscopy analysis + + + 0.99935025 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.9985233 + oligomeric_state + cleaner0 + 2023-07-27T10:40:32Z + DUMMY: + + oligomers + + + 0.9989483 + structure_element + cleaner0 + 2023-07-27T12:37:06Z + SO: + + helical domains + + + 0.99807835 + site + cleaner0 + 2023-07-27T12:40:10Z + SO: + + interfaces + + + 0.99912065 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.99904406 + structure_element + cleaner0 + 2023-07-27T10:41:52Z + SO: + + helical domain + + + 0.99897075 + structure_element + cleaner0 + 2023-07-27T10:36:20Z + SO: + + GTPase domain + + + 0.9993631 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + 0.99770314 + protein_state + cleaner0 + 2023-07-27T12:30:44Z + DUMMY: + + dimerized + + + 0.9990767 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.6128288 + protein_type + cleaner0 + 2023-07-27T10:44:54Z + MESH: + + BDLP + + + 0.995021 + experimental_method + cleaner0 + 2023-07-27T12:45:44Z + MESH: + + structural analysis + + + 0.999104 + site + cleaner0 + 2023-07-27T12:39:31Z + SO: + + G-interface + + + 0.99935454 + protein + cleaner0 + 2023-07-27T10:33:26Z + PR: + + Irga6 + + + + METHODS + title_1 + 19413 + Methods + + + METHODS + title_2 + 19421 + Protein expression and purification + + + METHODS + paragraph + 19457 + Selenomethionine-substituted Mus musculus Irga6R31E, K32E, K176E, K246E was expressed as a GST-fusion from the vector pGEX-4T-2 in BL21 Rosetta2(DE3) cells according to reference. Protein was purified as previously described and the protein stored in small aliquots at a concentration of 118 mg/mL in 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 2 mM DTT. + + + METHODS + title_2 + 19804 + Biochemical analyses + + + METHODS + paragraph + 19825 + Oligomerization and GTPase assays for the Irga6R31E, K32E, K176E, K246E mutant were carried out as described in. + + + METHODS + title_2 + 19938 + Protein crystallization + + + METHODS + paragraph + 19962 + The protein was gently thawed on ice and diluted to a final concentration of 10 mg/mL with buffer containing 20 mM Tris-HCl, pH 7.5, 8 mM MgCl2, 3 mM DTT. GMPPNP was added to a final concentration of 2 mM. Crystallization was carried out in a 96 well format using the sitting drop vapor diffusion method. The reservoir contained 100 mM HEPES-NaOH pH 7.0, 9 % PEG4000, 6 % isopropanol. The sitting drop was set up using an Art Robbins Gryphon system and consisted of 200 nL protein solution and 200 nL reservoir solution. + + + METHODS + paragraph + 20483 + For cryo-protection, crystals were transferred into a cryo solution containing 33 % PEG4000, 3 % isopropanol, 50 mM HEPES pH 7.0, 4 mM MgCl2, 2 mM DTT, and 2 mM GMPPNP at 4 °C for at least 5 sec. Crystals were screened for diffraction at beamline BL 14.1 at BESSY II, Berlin, Germany. + + + METHODS + title_2 + 20769 + Data collection + + + METHODS + paragraph + 20785 + All data were recorded at beamline P11 at PETRA III, DESY Hamburg, Germany using a PILATUS 6 M detector. To achieve spot separation along the long cell axis, three data sets were collected with a φ increment of 0.05/0.1° at a temperature of 100 K using detector distances between 1300 and 598.5 mm (Additional file 1: Table S1). The wavelength was 0.972/0.979 Å. Calculation of an optimal data collection strategy was done with the Mosflm software. The high- and low-resolution datasets were processed and merged using the XDS program suite. + + + METHODS + title_2 + 21330 + Structure solution and refinement + + + METHODS + paragraph + 21364 + Structure solution was done by molecular replacement with Phaser employing the structure of Irga6 without nucleotide [PDB: 1TQ2] as search model. Atomic model building was done by Coot. Iterative refinement was done using Phenix at a maximum resolution of 3.2 Å. For the refinement strategy, a seven-fold non-crystallographic symmetry as well as one molecule of Irga6 [PDB: 1TQ4] as high resolution reference structure was chosen. Five percent of the measured X-ray intensities were set aside from the refinement as cross-validation. Methionine sites in the protein were confirmed by the anomalous signal of the selenium atoms. Protein superposition was done with lsqkab and the PyMol Molecular Graphics System, Version 1.3 Schrödinger, LLC. Figures were prepared using the PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC. Evaluation of atom contacts and geometry of the atomic model was done by the Molprobity server. Interface sizes were calculated by the PISA server. + + + METHODS + title_2 + 22354 + Accession numbers + + + METHODS + paragraph + 22372 + The Irga6 coordinates were submitted to the Protein Data Bank (pdb) database with accession code 5fph. http://www.rcsb.org/pdb/explore/explore.do?structureId=5fph. + + + CONCL + title_1 + 22536 + Conclusions + + + CONCL + paragraph + 22548 + Our study indicates that Irg proteins dimerize via the G interface in a parallel head-to-head fashion thereby facilitating GTPase activation. These findings contribute to a molecular understanding of the anti-parasitic action of the Irg protein family and suggest that Irgs are bona-fide members of the dynamin superfamily. + + protein_type + MESH: + cleaner0 + 2023-07-27T10:37:26Z + + Irg + + + 0.9966157 + oligomeric_state + cleaner0 + 2023-07-27T12:30:12Z + DUMMY: + + dimerize + + + 0.9990368 + site + cleaner0 + 2023-07-27T10:43:00Z + SO: + + G interface + + + 0.9982672 + protein_state + cleaner0 + 2023-07-27T10:33:36Z + DUMMY: + + parallel head-to-head + + + 0.82288593 + protein_type + cleaner0 + 2023-07-27T12:28:26Z + MESH: + + GTPase + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:37:26Z + + Irg + + + 0.999027 + protein_type + cleaner0 + 2023-07-27T10:33:43Z + MESH: + + Irgs + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:36:30Z + + dynamin + + + + CONCL + title_1 + 22872 + Additional file + + + ABBR + title + 22888 + Abbreviations + + + ABBR + paragraph + 22902 + BDLP + + + ABBR + paragraph + 22907 + Bacterial dynamin like protein + + + ABBR + paragraph + 22938 + EHD2 + + + ABBR + paragraph + 22943 + Eps15 homology domain containing protein 2 + + + ABBR + paragraph + 22986 + GBP + + + ABBR + paragraph + 22990 + Guanylate-binding protein + + + ABBR + paragraph + 23016 + GDP + + + ABBR + paragraph + 23020 + Guanosine-diphosphate + + + ABBR + paragraph + 23042 + GIMAP2 + + + ABBR + paragraph + 23049 + GTPase of immunity associated protein 2 + + + ABBR + paragraph + 23089 + GMPPNP + + + ABBR + paragraph + 23096 + 5'-guanylyl imidodiphosphate + + + ABBR + paragraph + 23125 + GTP + + + ABBR + paragraph + 23129 + Guanosine-triphosphate + + + ABBR + paragraph + 23152 + IRG + + + ABBR + paragraph + 23156 + Immunity-related GTPase + + + ABBR + paragraph + 23180 + Irga6 + + + ABBR + paragraph + 23186 + Immunity-related GTPase 6 + + + ABBR + paragraph + 23212 + MxA + + + ABBR + paragraph + 23216 + Myxovirus resistance protein A + + + ABBR + paragraph + 23247 + PVM + + + ABBR + paragraph + 23251 + Parasitophorous vacuolar membrane + + + ABBR + footnote + 23285 + Competing interests + + + ABBR + footnote + 23305 + The authors declare that they have no competing interests. + + + ABBR + footnote + 23364 + Authors’ contributions + + + ABBR + footnote + 23389 + All authors planned the experimental design. NP cloned, characterized, and purified the Irga6 construct and found initial crystallization conditions. KS and CF optimized the crystallization condition and found suitable cryo conditions. KS and KF collected data, KF solved, and KS and KF refined the structure. KS, NP, CF, JCH, and OD wrote the manuscript. All authors read and approved the final manuscript. + + + REF + title + 23797 + References + + + 654 + 5645 + 9 + surname:MacMicking;given-names:JD + surname:Taylor;given-names:GA + surname:McKinney;given-names:JD + 10.1126/science.1088063 + 14576437 + REF + Science + ref + 302 + 2003 + 23808 + Immune control of tuberculosis by IFN-gamma-inducible LRG-47 + + + 1163 + 2 + 8 + surname:Feng;given-names:CG + surname:Collazo-Custodio;given-names:CM + surname:Eckhaus;given-names:M + surname:Hieny;given-names:S + surname:Belkaid;given-names:Y + surname:Elkins;given-names:K + 10.4049/jimmunol.172.2.1163 + 14707092 + REF + J Immunol + ref + 172 + 2004 + 23869 + Mice deficient in LRG-47 display increased susceptibility to mycobacterial infection associated with the induction of lymphopenia + + + 181 + 2 + 8 + surname:Collazo;given-names:CM + surname:Yap;given-names:GS + surname:Sempowski;given-names:GD + surname:Lusby;given-names:KC + surname:Tessarollo;given-names:L + surname:Vande Woude;given-names:GF + 10.1084/jem.194.2.181 + 11457893 + REF + J Exp Med + ref + 194 + 2001 + 23999 + Inactivation of LRG-47 and IRG-47 reveals a family of interferon gamma-inducible genes with essential, pathogen-specific roles in resistance to infection + + + 8165 + 12 + 72 + surname:Santiago;given-names:HC + surname:Feng;given-names:CG + surname:Bafica;given-names:A + surname:Roffe;given-names:E + surname:Arantes;given-names:RM + surname:Cheever;given-names:A + 10.4049/jimmunol.175.12.8165 + 16339555 + REF + J Immunol + ref + 175 + 2005 + 24153 + Mice deficient in LRG-47 display enhanced susceptibility to Trypanosoma cruzi infection associated with defective hemopoiesis and intracellular control of parasite growth + + + 751 + 2 + 5 + surname:Taylor;given-names:GA + surname:Collazo;given-names:CM + surname:Yap;given-names:GS + surname:Nguyen;given-names:K + surname:Gregorio;given-names:TA + surname:Taylor;given-names:LS + 10.1073/pnas.97.2.751 + 10639151 + REF + Proc Natl Acad Sci U S A + ref + 97 + 2000 + 24324 + Pathogen-specific loss of host resistance in mice lacking the IFN-gamma-inducible gene IGTP + + + 3278 + 6 + 86 + surname:Butcher;given-names:BA + surname:Greene;given-names:RI + surname:Henry;given-names:SC + surname:Annecharico;given-names:KL + surname:Weinberg;given-names:JB + surname:Denkers;given-names:EY + 10.1128/IAI.73.6.3278-3286.2005 + 15908352 + REF + Infect Immun + ref + 73 + 2005 + 24416 + p47 GTPases regulate Toxoplasma gondii survival in activated macrophages + + + e24 + 3 + surname:Martens;given-names:S + surname:Parvanova;given-names:I + surname:Zerrahn;given-names:J + surname:Griffiths;given-names:G + surname:Schell;given-names:G + surname:Reichmann;given-names:G + 10.1371/journal.ppat.0010024 + 16304607 + REF + PLoS Pathog + ref + 1 + 2005 + 24489 + Disruption of Toxoplasma gondii parasitophorous vacuoles by the mouse p47-resistance GTPases + + + 877 + 5 + 85 + surname:Henry;given-names:SC + surname:Daniell;given-names:XG + surname:Burroughs;given-names:AR + surname:Indaram;given-names:M + surname:Howell;given-names:DN + surname:Coers;given-names:J + 10.1189/jlb.1008599 + 19176402 + REF + J Leukoc Biol + ref + 85 + 2009 + 24582 + Balance of Irgm protein activities determines IFN-gamma-induced host defense + + + 100 + 2 + 9 + surname:Taylor;given-names:GA + surname:Feng;given-names:CG + surname:Sher;given-names:A + 10.1038/nri1270 + 15040583 + REF + Nat Rev Immunol + ref + 4 + 2004 + 24659 + p47 GTPases: regulators of immunity to intracellular pathogens + + + 559 + 89 + surname:Martens;given-names:S + surname:Howard;given-names:J + 10.1146/annurev.cellbio.22.010305.104619 + 16824009 + REF + Annu Rev Cell Dev Biol. + ref + 22 + 2006 + 24722 + The interferon-inducible GTPases + + + 43 + 1-2 + 54 + surname:Hunn;given-names:JP + surname:Feng;given-names:CG + surname:Sher;given-names:A + surname:Howard;given-names:JC + 10.1007/s00335-010-9293-3 + 21052678 + REF + Mamm Genome + ref + 22 + 2011 + 24755 + The immunity-related GTPases in mammals: a fast-evolving cell-autonomous resistance system against intracellular pathogens + + + e1000403 + 3 + surname:Bekpen;given-names:C + surname:Marques-Bonet;given-names:T + surname:Alkan;given-names:C + surname:Antonacci;given-names:F + surname:Leogrande;given-names:MB + surname:Ventura;given-names:M + 10.1371/journal.pgen.1000403 + 19266026 + REF + PLoS Genet + ref + 5 + 2009 + 24878 + Death and resurrection of the human IRGM gene + + + 10639 + 16 + 45 + surname:Taylor;given-names:GA + surname:Stauber;given-names:R + surname:Rulong;given-names:S + surname:Hudson;given-names:E + surname:Pei;given-names:V + surname:Pavlakis;given-names:GN + 10.1074/jbc.272.16.10639 + 9099712 + REF + J Biol Chem + ref + 272 + 1997 + 24924 + The inducibly expressed GTPase localizes to the endoplasmic reticulum, independently of GTP binding + + + 2594 + 4 + 606 + surname:Martens;given-names:S + surname:Sabel;given-names:K + surname:Lange;given-names:R + surname:Uthaiah;given-names:R + surname:Wolf;given-names:E + surname:Howard;given-names:JC + 10.4049/jimmunol.173.4.2594 + 15294976 + REF + J Immunol + ref + 173 + 2004 + 25024 + Mechanisms regulating the positioning of mouse p47 resistance GTPases LRG-47 and IIGP1 on cellular membranes: retargeting to plasma membrane induced by phagocytosis + + + e1000288 + 2 + surname:Zhao;given-names:YO + surname:Khaminets;given-names:A + surname:Hunn;given-names:JP + surname:Howard;given-names:JC + 10.1371/journal.ppat.1000288 + 19197351 + REF + PLoS Pathog + ref + 5 + 2009 + 25189 + Disruption of the Toxoplasma gondii parasitophorous vacuole by IFNgamma-inducible immunity-related GTPases (IRG proteins) triggers necrotic cell death + + + 939 + 7 + 61 + surname:Khaminets;given-names:A + surname:Hunn;given-names:JP + surname:Konen-Waisman;given-names:S + surname:Zhao;given-names:YO + surname:Preukschat;given-names:D + surname:Coers;given-names:J + 10.1111/j.1462-5822.2010.01443.x + 20109161 + REF + Cell Microbiol + ref + 12 + 2010 + 25340 + Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole + + + 517 + 5 + 8 + surname:Zhao;given-names:Y + surname:Yap;given-names:GS + 10.1016/j.chom.2014.05.002 + 24832444 + REF + Cell Host Microbe + ref + 15 + 2014 + 25438 + Toxoplasma's arms race with the host interferon response: a menage a trois of ROPs + + + 2063 + 9 + 71 + surname:Ling;given-names:YM + surname:Shaw;given-names:MH + surname:Ayala;given-names:C + surname:Coppens;given-names:I + surname:Taylor;given-names:GA + surname:Ferguson;given-names:DJ + 10.1084/jem.20061318 + 16940170 + REF + J Exp Med + ref + 203 + 2006 + 25521 + Vacuolar and plasma membrane stripping and autophagic elimination of Toxoplasma gondii in primed effector macrophages + + + 4883 + 11 + 94 + surname:Melzer;given-names:T + surname:Duffy;given-names:A + surname:Weiss;given-names:LM + surname:Halonen;given-names:SK + 10.1128/IAI.01288-07 + 18765738 + REF + Infect Immun + ref + 76 + 2008 + 25639 + The gamma interferon (IFN-gamma)-inducible GTP-binding protein IGTP is necessary for toxoplasma vacuolar disruption and induces parasite egression in IFN-gamma-stimulated astrocytes + + + 32143 + 46 + 51 + surname:Papic;given-names:N + surname:Hunn;given-names:JP + surname:Pawlowski;given-names:N + surname:Zerrahn;given-names:J + surname:Howard;given-names:JC + 10.1074/jbc.M804846200 + 18784077 + REF + J Biol Chem + ref + 283 + 2008 + 25821 + Inactive and active states of the interferon-inducible resistance GTPase, Irga6, in vivo + + + 2495 + 19 + 509 + surname:Hunn;given-names:JP + surname:Koenen-Waisman;given-names:S + surname:Papic;given-names:N + surname:Schroeder;given-names:N + surname:Pawlowski;given-names:N + surname:Lange;given-names:R + 10.1038/emboj.2008.176 + 18772884 + REF + EMBO J + ref + 27 + 2008 + 25910 + Regulatory interactions between IRG resistance GTPases in the cellular response to Toxoplasma gondii + + + 29336 + 31 + 43 + surname:Uthaiah;given-names:RC + surname:Praefcke;given-names:GJ + surname:Howard;given-names:JC + surname:Herrmann;given-names:C + 10.1074/jbc.M211973200 + 12732635 + REF + J Biol Chem + ref + 278 + 2003 + 26011 + IIGP1, an interferon-gamma-inducible 47-kDa GTPase of the mouse, showing cooperative enzymatic activity and GTP-dependent multimerization + + + 727 + 5 + 39 + surname:Ghosh;given-names:A + surname:Uthaiah;given-names:R + surname:Howard;given-names:J + surname:Herrmann;given-names:C + surname:Wolf;given-names:E + 10.1016/j.molcel.2004.07.017 + 15350217 + REF + Mol Cell + ref + 15 + 2004 + 26149 + Crystal structure of IIGP1: a paradigm for interferon-inducible p47 resistance GTPases + + + 7 + surname:Pawlowski;given-names:N + surname:Khaminets;given-names:A + surname:Hunn;given-names:JP + surname:Papic;given-names:N + surname:Schmidt;given-names:A + surname:Uthaiah;given-names:RC + 10.1186/1741-7007-9-7 + 21276251 + REF + BMC Biol. + ref + 9 + 2011 + 26236 + The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii + + + 209 + 1 + 22 + surname:Chappie;given-names:JS + surname:Mears;given-names:JA + surname:Fang;given-names:S + surname:Leonard;given-names:M + surname:Schmid;given-names:SL + surname:Milligan;given-names:RA + 10.1016/j.cell.2011.09.003 + 21962517 + REF + Cell + ref + 147 + 2011 + 26361 + A pseudoatomic model of the dynamin polymer identifies a hydrolysis-dependent powerstroke + + + 435 + 7297 + 40 + surname:Chappie;given-names:JS + surname:Acharya;given-names:S + surname:Leonard;given-names:M + surname:Schmid;given-names:SL + surname:Dyda;given-names:F + 10.1038/nature09032 + 20428113 + REF + Nature + ref + 465 + 2010 + 26451 + G domain dimerization controls dynamin's assembly-stimulated GTPase activity + + + 1433 + 10 + 45 + surname:Rennie;given-names:ML + surname:McKelvie;given-names:SA + surname:Bulloch;given-names:EM + surname:Kingston;given-names:RL + 10.1016/j.str.2014.08.015 + 25295396 + REF + Structure + ref + 22 + 2014 + 26528 + Transient dimerization of human MxA promotes GTP hydrolysis, resulting in a mechanical power stroke + + + 12779 + 20 + 92 + surname:Dick;given-names:A + surname:Graf;given-names:L + surname:Olal;given-names:D + surname:von der Malsburg;given-names:A + surname:Gao;given-names:S + surname:Kochs;given-names:G + 10.1074/jbc.M115.650325 + 25829498 + REF + J Biol Chem + ref + 290 + 2015 + 26628 + Role of nucleotide binding and GTPase domain dimerization in dynamin-like myxovirus resistance protein A for GTPase activation and antiviral activity + + + 101 + 7080 + 4 + surname:Ghosh;given-names:A + surname:Praefcke;given-names:GJ + surname:Renault;given-names:L + surname:Wittinghofer;given-names:A + surname:Herrmann;given-names:C + 10.1038/nature04510 + 16511497 + REF + Nature + ref + 440 + 2006 + 26778 + How guanylate-binding proteins achieve assembly-stimulated processive cleavage of GTP to GMP + + + 2216 + 6 + 21 + surname:Byrnes;given-names:LJ + surname:Sondermann;given-names:H + 10.1073/pnas.1012792108 + 21220294 + REF + Proc Natl Acad Sci U S A + ref + 108 + 2011 + 26871 + Structural basis for the nucleotide-dependent dimerization of the large G protein atlastin-1/SPG3A + + + 3976 + 10 + 81 + surname:Bian;given-names:X + surname:Klemm;given-names:RW + surname:Liu;given-names:TY + surname:Zhang;given-names:M + surname:Sun;given-names:S + surname:Sui;given-names:XW + 10.1073/pnas.1101643108 + 21368113 + REF + Proc Natl Acad Sci U S A + ref + 108 + 2011 + 26970 + Structures of the atlastin GTPase provide insight into homotypic fusion of endoplasmic reticulum membranes + + + 766 + 7120 + 9 + surname:Low;given-names:HH + surname:Lowe;given-names:J + 10.1038/nature05312 + 17122778 + REF + Nature + ref + 444 + 2006 + 27077 + A bacterial dynamin-like protein + + + 1342 + 7 + 52 + surname:Low;given-names:HH + surname:Sachse;given-names:C + surname:Amos;given-names:LA + surname:Lowe;given-names:J + 10.1016/j.cell.2009.11.003 + 20064379 + REF + Cell + ref + 139 + 2009 + 27110 + Structure of a bacterial dynamin-like protein lipid tube provides a mechanism for assembly and membrane curving + + + 215 + 6971 + 21 + surname:Egea;given-names:PF + surname:Shan;given-names:SO + surname:Napetschnig;given-names:J + surname:Savage;given-names:DF + surname:Walter;given-names:P + surname:Stroud;given-names:RM + 10.1038/nature02250 + 14724630 + REF + Nature + ref + 427 + 2004 + 27222 + Substrate twinning activates the signal recognition particle and its receptor + + + 411 + 43 + surname:Faelber;given-names:K + surname:Gao;given-names:S + surname:Held;given-names:M + surname:Posor;given-names:Y + surname:Haucke;given-names:V + surname:Noe;given-names:F + 10.1016/B978-0-12-386931-9.00015-5 + 23663977 + REF + Prog Mol Biol Transl Sci. + ref + 117 + 2013 + 27300 + Oligomerization of dynamin superfamily proteins in health and disease + + + 311 + 7160 + 5 + surname:Sirajuddin;given-names:M + surname:Farkasovsky;given-names:M + surname:Hauer;given-names:F + surname:Kuhlmann;given-names:D + surname:Macara;given-names:IG + surname:Weyand;given-names:M + 10.1038/nature06052 + 17637674 + REF + Nature + ref + 449 + 2007 + 27370 + Structural insight into filament formation by mammalian septins + + + 95 + 2 + 100 + surname:Sun;given-names:YJ + surname:Forouhar;given-names:F + surname:Li Hm;given-names:HM + surname:Tu;given-names:SL + surname:Yeh;given-names:YH + surname:Kao;given-names:S + 10.1038/nsb744 + 11753431 + REF + Nat Struct Biol + ref + 9 + 2002 + 27434 + Crystal structure of pea Toc34, a novel GTPase of the chloroplast protein translocon + + + 20299 + 47 + 304 + surname:Schwefel;given-names:D + surname:Frohlich;given-names:C + surname:Eichhorst;given-names:J + surname:Wiesner;given-names:B + surname:Behlke;given-names:J + surname:Aravind;given-names:L + 10.1073/pnas.1010322107 + 21059949 + REF + Proc Natl Acad Sci U S A + ref + 107 + 2010 + 27519 + Structural basis of oligomerization in septin-like GTPase of immunity-associated protein 2 (GIMAP2) + + + 923 + 7164 + 7 + surname:Daumke;given-names:O + surname:Lundmark;given-names:R + surname:Vallis;given-names:Y + surname:Martens;given-names:S + surname:Butler;given-names:PJ + surname:McMahon;given-names:HT + 10.1038/nature06173 + 17914359 + REF + Nature + ref + 449 + 2007 + 27619 + Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling + + + 105 + 1 + 24 + surname:Van Duyne;given-names:GD + surname:Standaert;given-names:RF + surname:Karplus;given-names:PA + surname:Schreiber;given-names:SL + surname:Clardy;given-names:J + 10.1006/jmbi.1993.1012 + 7678431 + REF + J Mol Biol + ref + 229 + 1993 + 27710 + Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin + + + 48 + 57 + surname:Leslie;given-names:AG + 10.1107/S0907444905039107 + 16369093 + REF + Acta Crystallogr D Biol Crystallogr. + ref + 62 + 2006 + 27797 + The integration of macromolecular diffraction data + + + 125 + 32 + surname:Kabsch;given-names:W + 10.1107/S0907444909047337 + 20124692 + REF + Acta Crystallogr D Biol Crystallogr. + ref + 66 + 2010 + 27848 + XDS + + + 658 + 74 + surname:McCoy;given-names:AJ + surname:Grosse-Kunstleve;given-names:RW + surname:Adams;given-names:PD + surname:Winn;given-names:MD + surname:Storoni;given-names:LC + surname:Read;given-names:RJ + 10.1107/S0021889807021206 + 19461840 + REF + J Appl Crystallogr. + ref + 40 + 2007 + 27852 + Phaser crystallographic software + + + 486 + Pt 4 + 501 + surname:Emsley;given-names:P + surname:Lohkamp;given-names:B + surname:Scott;given-names:WG + surname:Cowtan;given-names:K + 10.1107/S0907444910007493 + 20383002 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 27885 + Features and development of Coot + + + 213 + 21 + surname:Adams;given-names:PD + surname:Afonine;given-names:PV + surname:Bunkoczi;given-names:G + surname:Chen;given-names:VB + surname:Davis;given-names:IW + surname:Echols;given-names:N + REF + Acta Cryst. + ref + D66 + 2010 + 27918 + PHENIX: a comprehensive Python-based system for macromolecular structure solution + + + 366 + 96 + surname:Brunger;given-names:AT + 10.1016/S0076-6879(97)77021-6 + 18488318 + REF + Methods Enzymol. + ref + 277 + 1997 + 28000 + Free R, value: cross-validation in crystallography + + + 922 + 3 + surname:Kabsch;given-names:W + 10.1107/S0567739476001873 + REF + Acta Cryst. + ref + A32 + 1976 + 28051 + A solution for the best rotation to relate two sets of vectors + + + 12 + Pt 1 + 21 + surname:Chen;given-names:VB + surname:Arendall;given-names:WB;suffix:3rd + surname:Headd;given-names:JJ + surname:Keedy;given-names:DA + surname:Immormino;given-names:RM + surname:Kapral;given-names:GJ + 10.1107/S0907444909042073 + 20057044 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 28114 + MolProbity: all-atom structure validation for macromolecular crystallography + + + 774 + 97 + surname:Krissinel;given-names:E + surname:Henrick;given-names:K + 10.1016/j.jmb.2007.05.022 + 17681537 + REF + J Mol Biol. + ref + 372 + 2007 + 28191 + Inference of macromolecular assemblies from crystalline state + + + diff --git a/BioC_XML/4781976_v0.xml b/BioC_XML/4781976_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..ebcf947d8979183738bccd01a4b45349321d7231 --- /dev/null +++ b/BioC_XML/4781976_v0.xml @@ -0,0 +1,1310 @@ + + + + PMC + 20201222 + pmc.key + + 4781976 + CC BY + no + 0 + 0 + + 10.1016/j.dib.2016.02.042 + 4781976 + 26977434 + S2352-3409(16)30064-6 + 344 + Tom1, GAT domain, Tollip, Ubiquitin, nuclear magnetic resonance + This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). + 348 + surname:Xiao;given-names:Shuyan + surname:Ellena;given-names:Jeffrey F. + surname:Armstrong;given-names:Geoffrey S. + surname:Capelluto;given-names:Daniel G.S. + TITLE + Keywords + front + 7 + 2016 + 0 + Structure of the GAT domain of the endosomal adapter protein Tom1 + + 0.9991358 + evidence + cleaner0 + 2023-07-20T14:57:22Z + DUMMY: + + Structure + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:58Z + + GAT + + + 0.9416477 + protein_type + cleaner0 + 2023-07-20T14:56:57Z + MESH: + + adapter protein + + + 0.9997857 + protein + cleaner0 + 2023-07-20T14:57:11Z + PR: + + Tom1 + + + + ABSTRACT + abstract + 66 + Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments. The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD). In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. The estimated protein structure exhibits a bundle of three helical elements. We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states. + + 0.8085951 + protein_type + cleaner0 + 2023-07-20T14:56:52Z + MESH: + + cell-surface receptor + + + 0.99906075 + protein_type + cleaner0 + 2023-07-20T14:57:01Z + MESH: + + adapter proteins + + + 0.99983084 + protein + cleaner0 + 2023-07-20T14:57:11Z + PR: + + Tom1 + + + 0.99978274 + protein + cleaner0 + 2023-07-20T14:57:16Z + PR: + + Tollip + + + 0.96031576 + ptm + cleaner0 + 2023-07-20T14:59:03Z + MESH: + + ubiquitinated + + + 0.9998591 + protein + cleaner0 + 2023-07-20T14:57:11Z + PR: + + Tom1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:58Z + + GAT + + + 0.99981624 + protein + cleaner0 + 2023-07-20T14:57:16Z + PR: + + Tollip + + + 0.999658 + structure_element + cleaner0 + 2023-07-20T14:57:32Z + SO: + + Tom1-binding domain + + + 0.9997849 + structure_element + cleaner0 + 2023-07-20T14:57:36Z + SO: + + TBD + + + 0.9995477 + experimental_method + cleaner0 + 2023-07-20T15:00:01Z + MESH: + + solution NMR + + + 0.9994789 + evidence + cleaner0 + 2023-07-20T14:57:22Z + DUMMY: + + structure + + + 0.9998665 + protein + cleaner0 + 2023-07-20T14:57:11Z + PR: + + Tom1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:58Z + + GAT + + + 0.999076 + evidence + cleaner0 + 2023-07-20T14:57:22Z + DUMMY: + + structure + + + 0.917164 + experimental_method + cleaner0 + 2023-07-20T15:06:26Z + MESH: + + compare + + + 0.9998603 + protein + cleaner0 + 2023-07-20T14:57:11Z + PR: + + Tom1 + + + 0.99962676 + structure_element + cleaner0 + 2023-07-20T14:57:58Z + SO: + + GAT + + + 0.9995364 + evidence + cleaner0 + 2023-07-20T14:57:22Z + DUMMY: + + structure + + + 0.9989391 + evidence + cleaner0 + 2023-07-20T15:06:36Z + DUMMY: + + structures + + + 0.9998318 + protein + cleaner0 + 2023-07-20T14:57:16Z + PR: + + Tollip + + + 0.9995357 + protein_state + cleaner0 + 2023-07-20T14:58:41Z + DUMMY: + + TBD- + + + 0.9995535 + protein_state + cleaner0 + 2023-07-20T14:58:47Z + DUMMY: + + ubiquitin-bound + + + + TABLE + title_1 + 730 + Specifications table + + + t0010.xml + t0010 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><tbody><tr><td>Subject area</td><td><italic>Biology</italic></td></tr><tr><td>More specific subject area</td><td><italic>Structural biology</italic></td></tr><tr><td>Type of data</td><td><italic>Table, text file, graph, figures</italic></td></tr><tr><td>How data was acquired</td><td><italic>Circular dichroism and NMR. NMR data was recorded using a Bruker 800 MHz</italic></td></tr><tr><td>Data format</td><td><italic>PDB format text file. Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol</italic></td></tr><tr><td>Experimental factors</td><td><italic>Recombinant human Tom1 GAT domain was purified to homogeneity before use</italic></td></tr><tr><td>Experimental features</td><td><italic>Solution structure of Tom1 GAT was determined from NMR chemical shift data</italic></td></tr><tr><td>Data source location</td><td><italic>Virginia and Colorado, United States.</italic></td></tr><tr><td>Data accessibility</td><td><italic>Data is available within this article. Tom1 GAT structural data is publicly available in the RCSB Protein Data Bank (http://www.rscb.org/) under the accession number PDB: 2n9d</italic></td></tr></tbody></table> + + 751 + Table + + + t0010.xml + t0010 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><tbody><tr><td>Subject area</td><td><italic>Biology</italic></td></tr><tr><td>More specific subject area</td><td><italic>Structural biology</italic></td></tr><tr><td>Type of data</td><td><italic>Table, text file, graph, figures</italic></td></tr><tr><td>How data was acquired</td><td><italic>Circular dichroism and NMR. NMR data was recorded using a Bruker 800 MHz</italic></td></tr><tr><td>Data format</td><td><italic>PDB format text file. Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol</italic></td></tr><tr><td>Experimental factors</td><td><italic>Recombinant human Tom1 GAT domain was purified to homogeneity before use</italic></td></tr><tr><td>Experimental features</td><td><italic>Solution structure of Tom1 GAT was determined from NMR chemical shift data</italic></td></tr><tr><td>Data source location</td><td><italic>Virginia and Colorado, United States.</italic></td></tr><tr><td>Data accessibility</td><td><italic>Data is available within this article. Tom1 GAT structural data is publicly available in the RCSB Protein Data Bank (http://www.rscb.org/) under the accession number PDB: 2n9d</italic></td></tr></tbody></table> + + 760 + Subject area Biology More specific subject area Structural biology Type of data Table, text file, graph, figures How data was acquired Circular dichroism and NMR. NMR data was recorded using a Bruker 800 MHz Data format PDB format text file. Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States. Data accessibility Data is available within this article. Tom1 GAT structural data is publicly available in the RCSB Protein Data Bank (http://www.rscb.org/) under the accession number PDB: 2n9d + + 0.99951756 + experimental_method + cleaner0 + 2023-07-20T14:59:18Z + MESH: + + Circular dichroism + + + 0.9996252 + experimental_method + cleaner0 + 2023-07-20T14:59:32Z + MESH: + + NMR + + + 0.99964654 + experimental_method + cleaner0 + 2023-07-20T14:59:32Z + MESH: + + NMR + + + 0.9992082 + experimental_method + cleaner0 + 2023-07-20T15:06:30Z + MESH: + + CS-Rosetta + + + 0.9445149 + experimental_method + cleaner0 + 2023-07-20T14:59:43Z + MESH: + + Protein Structure Validation Server + + + experimental_method + MESH: + cleaner0 + 2023-07-20T14:59:52Z + + PSVS + + + 0.97021425 + experimental_method + cleaner0 + 2023-07-20T15:00:05Z + MESH: + + NMRPipe + + + 0.9534222 + experimental_method + cleaner0 + 2023-07-20T15:00:07Z + MESH: + + NMRDraw + + + 0.99847716 + species + cleaner0 + 2023-07-20T15:00:38Z + MESH: + + human + + + 0.9998481 + protein + cleaner0 + 2023-07-20T14:57:11Z + PR: + + Tom1 + + + 0.5058204 + structure_element + cleaner0 + 2023-07-20T14:57:58Z + SO: + + GAT + + + 0.99844825 + evidence + cleaner0 + 2023-07-20T15:00:14Z + DUMMY: + + Solution structure + + + 0.99984014 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + 0.98831964 + structure_element + cleaner0 + 2023-07-20T14:57:58Z + SO: + + GAT + + + 0.9996598 + experimental_method + cleaner0 + 2023-07-20T14:59:32Z + MESH: + + NMR + + + evidence + DUMMY: + cleaner0 + 2023-07-20T15:00:28Z + + chemical shift + + + protein + PR: + cleaner0 + 2023-07-20T14:57:12Z + + Tom1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:58Z + + GAT + + + + TABLE + title_1 + 1563 + Value of the data + + + TABLE + paragraph + 1581 + The Tom1 GAT domain solution structure will provide additional tools for modulating its biological function. + + 0.99983656 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:58Z + + GAT + + + 0.9995917 + evidence + cleaner0 + 2023-07-20T15:00:14Z + DUMMY: + + solution structure + + + + TABLE + paragraph + 1690 + Tom1 GAT can adopt distinct conformations upon ligand binding. + + 0.999843 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + 0.9992766 + structure_element + cleaner0 + 2023-07-20T14:57:58Z + SO: + + GAT + + + + TABLE + paragraph + 1753 + A conformational response of the Tom1 GAT domain upon Tollip TBD binding can serve as an example to explain mutually exclusive ligand binding events. + + 0.99981683 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:58Z + + GAT + + + 0.9990669 + protein + cleaner0 + 2023-07-20T14:57:16Z + PR: + + Tollip + + + 0.99973947 + structure_element + cleaner0 + 2023-07-20T14:57:36Z + SO: + + TBD + + + + TABLE + title_1 + 1903 + Data + + + TABLE + paragraph + 1908 + Analysis of the far-UV circular dichroism (CD) spectrum of the Tom 1 GAT domain (Fig. 1) predicts 58.7% α-helix, 3% β-strand, 15.5% turn, and 22.8% disordered regions. The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). Unlike ubiquitin binding, data suggest that conformational changes of the Tom1 GAT α-helices 1 and 2 occur upon Tollip TBD binding (Fig. 3A,B). + + 0.99765813 + experimental_method + cleaner0 + 2023-07-20T15:01:25Z + MESH: + + far-UV circular dichroism + + + 0.97389156 + experimental_method + cleaner0 + 2023-07-20T15:01:22Z + MESH: + + CD + + + 0.9930906 + evidence + cleaner0 + 2023-07-20T15:01:19Z + DUMMY: + + spectrum + + + 0.99947125 + protein + cleaner0 + 2023-07-20T15:06:22Z + PR: + + Tom 1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:58Z + + GAT + + + 0.999609 + structure_element + cleaner0 + 2023-07-20T15:01:58Z + SO: + + α-helix + + + 0.9994879 + structure_element + cleaner0 + 2023-07-20T15:01:59Z + SO: + + β-strand + + + 0.99985206 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + 0.7947963 + structure_element + cleaner0 + 2023-07-20T14:57:58Z + SO: + + GAT + + + 0.9988989 + evidence + cleaner0 + 2023-07-20T15:06:41Z + DUMMY: + + structural restraints + + + 0.9295965 + evidence + cleaner0 + 2023-07-20T15:06:45Z + DUMMY: + + structures + + + 0.9993277 + evidence + cleaner0 + 2023-07-20T15:01:33Z + DUMMY: + + root mean square deviation + + + 0.9996222 + evidence + cleaner0 + 2023-07-20T15:01:36Z + DUMMY: + + RMSD + + + 0.9990892 + residue_range + cleaner0 + 2023-07-20T15:01:42Z + DUMMY: + + Q216-E240 + + + 0.9997021 + structure_element + cleaner0 + 2023-07-20T15:01:49Z + SO: + + α-helix 1 + + + 0.9991074 + residue_range + cleaner0 + 2023-07-20T15:01:44Z + DUMMY: + + P248-Q274 + + + 0.9997027 + structure_element + cleaner0 + 2023-07-20T15:01:51Z + SO: + + α-helix 2 + + + 0.99909496 + residue_range + cleaner0 + 2023-07-20T15:01:46Z + DUMMY: + + E278-T306 + + + 0.99970627 + structure_element + cleaner0 + 2023-07-20T15:01:53Z + SO: + + α-helix 3 + + + 0.99831545 + chemical + cleaner0 + 2023-07-20T15:07:05Z + CHEBI: + + ubiquitin + + + 0.99984026 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + 0.99932325 + structure_element + cleaner0 + 2023-07-20T14:57:58Z + SO: + + GAT + + + 0.9996645 + structure_element + cleaner0 + 2023-07-20T15:01:56Z + SO: + + α-helices 1 and 2 + + + 0.9998336 + protein + cleaner0 + 2023-07-20T14:57:16Z + PR: + + Tollip + + + 0.9960354 + structure_element + cleaner0 + 2023-07-20T14:57:36Z + SO: + + TBD + + + + METHODS + title_1 + 2559 + Experimental design, materials, and methods + + + METHODS + title_2 + 2603 + Protein expression and purification + + + METHODS + paragraph + 2639 + Human Tom1 GAT (residues 215–309) cDNA was cloned into both pGEX6P1 and pET28a vectors, and expressed as GST-tagged and His-tagged fusion proteins, respectively, using Escherichia coli [Rosetta (DE3) strain]. The 13C, 15N-labeled Tom1 GAT domain was expressed and purified as described previously. + + + METHODS + title_2 + 2939 + Circular dichroism + + + METHODS + paragraph + 2958 + Far-UV CD spectra of the His-Tom1 GAT domain were collected on a Jasco J-815 spectropolarimeter using a 1 mm path length quartz cell at room temperature. The protein (10 μM) was solubilized in 5 mM Tris–HCl (pH 7) and 100 mM KF. Spectra were obtained from five accumulated scans from 190 to 260 nm using a bandwidth of 1 nm and a response time of 1 s at a scan speed of 20 nm/min. Buffer backgrounds were employed to subtract the protein spectra. Data was processed using the Dichroweb server and the CONTIN algorithm (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml). + + + METHODS + title_2 + 3539 + NMR structure determination + + + METHODS + paragraph + 3567 + NMR experiments were performed using 1 mM 13C, 15N-labeled Tom1 GAT domain in a buffer containing 20 mM d11-TrisHCl (pH 7), 50 mM KCl, 1 mM d18-DTT, and 1 mM NaN3. NMR spectra were recorded at 25 °C on a Bruker 800-MHz spectrometer (University of Virginia). The individual structure of Tom1 GAT was generated using CS-Rosetta (https://csrosetta.bmrb.wisc.edu/csrosetta). Chemical shift information (BMRB #26574) was used to obtain the structure calculation. The Rosetta calculations yielded 3000 structures of Tom1 GAT. From these, ten structures were selected based on their score and RMSDs, and converted to Protein Data Bank (PDB) format. NMR structural statistics for the ten lowest energy conformers of Tom1 GAT was generated using the Protein Structure Validation Suite. By using MolProbity, the Ramachandran analysis of the ten superimposed Tom1 GAT structures identified that 100% of the residues were in the most favored regions and there were no Ramachandran outliers in the allowed and disallowed regions. Protein structure images were obtained using PyMol (http://www.pymol.org). The structures of the ubiquitin- and Tollip TBD-bound states of the Tom1 GAT domain were obtained from data reported in Refs. and. + + + REF + title + 4797 + References + + + 1910 + 1920 + surname:Xiao;given-names:S. + surname:Brannon;given-names:M.K. + surname:Zhao;given-names:X. + surname:Fread;given-names:K.I. + surname:Ellena;given-names:J.F. + surname:Bushweller;given-names:J.H. + surname:Finkielstein;given-names:C.V. + surname:Armstrong;given-names:G.S. + surname:Capelluto;given-names:D.G. + REF + Structure + ref + 23 + 2015 + 4808 + Tom1 modulates binding of Tollip to phosphatidylinositol 3-phosphate via a coupled folding and binding mechanism + + + 5385 + 5391 + surname:Akutsu;given-names:M. + surname:Kawasaki;given-names:M. + surname:Katoh;given-names:Y. + surname:Shiba;given-names:T. + surname:Yamaguchi;given-names:Y. + surname:Kato;given-names:R. + surname:Kato;given-names:K. + surname:Nakayama;given-names:K. + surname:Wakatsuki;given-names:S. + 16199040 + REF + FEBS Lett. + ref + 579 + 2005 + 4921 + Structural basis for recognition of ubiquitinated cargo by Tom1-GAT domain + + + SUPPL + title_1 + 4996 + Supplementary material + + + SUPPL + footnote + 5019 + Supplementary data associated with this article can be found in the online version at doi:10.1016/j.dib.2016.02.042. + + + gr1.jpg + f0005 + FIG + fig_caption + 5136 + Representative far-UV CD spectrum of the His-Tom1 GAT domain. + + 0.99858 + experimental_method + cleaner0 + 2023-07-20T15:03:56Z + MESH: + + far-UV CD + + + 0.71714723 + evidence + cleaner0 + 2023-07-20T15:06:50Z + DUMMY: + + spectrum + + + 0.8865229 + experimental_method + cleaner0 + 2023-07-20T15:04:25Z + MESH: + + His- + + + 0.9922563 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:59Z + + GAT + + + + gr1.jpg + f0005 + FIG + fig + 5198 + Fig. 1. + + + gr2.jpg + f0010 + FIG + fig_caption + 5206 + (A) Stereo view displaying the best-fit backbone superposition of the refined structures for the Tom1 GAT domain. Helices are shown in orange, whereas loops are colored in green. (B) Ribbon illustration of the Tom1 GAT domain. + + 0.9992467 + experimental_method + cleaner0 + 2023-07-20T15:04:41Z + MESH: + + backbone superposition + + + 0.9941738 + evidence + cleaner0 + 2023-07-20T15:06:54Z + DUMMY: + + structures + + + 0.9998486 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:59Z + + GAT + + + 0.99984694 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:59Z + + GAT + + + + gr2.jpg + f0010 + FIG + fig + 5433 + Fig. 2. + + + gr3.jpg + f0015 + FIG + fig_caption + 5441 + (A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green). + + 0.9534955 + experimental_method + cleaner0 + 2023-07-20T15:05:10Z + MESH: + + superimposed structures + + + 0.99984646 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:59Z + + GAT + + + 0.99965954 + protein_state + cleaner0 + 2023-07-20T15:05:18Z + DUMMY: + + free + + + 0.9996728 + protein + cleaner0 + 2023-07-20T14:57:16Z + PR: + + Tollip + + + 0.99953216 + protein_state + cleaner0 + 2023-07-20T15:05:15Z + DUMMY: + + TBD-bound + + + 0.9632287 + experimental_method + cleaner0 + 2023-07-20T15:05:12Z + MESH: + + superimposed structures + + + 0.9998419 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:59Z + + GAT + + + 0.9995443 + protein_state + cleaner0 + 2023-07-20T15:05:16Z + DUMMY: + + Ub-bound + + + + gr3.jpg + f0015 + FIG + fig + 5697 + Fig. 3. + + + t0005.xml + t0005 + TABLE + table_caption + 5705 + NMR and refinement statistics for the Tom1 GAT domain. NMR structural statistics for lowest energy conformers of Tom1 GAT using PSVS. + + 0.99963295 + experimental_method + cleaner0 + 2023-07-20T14:59:32Z + MESH: + + NMR + + + 0.9961254 + evidence + cleaner0 + 2023-07-20T15:05:29Z + DUMMY: + + refinement statistics + + + 0.9998447 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:59Z + + GAT + + + 0.999608 + experimental_method + cleaner0 + 2023-07-20T14:59:32Z + MESH: + + NMR + + + 0.9994303 + evidence + cleaner0 + 2023-07-20T15:05:33Z + DUMMY: + + structural statistics + + + 0.99985063 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + 0.9986708 + structure_element + cleaner0 + 2023-07-20T14:57:59Z + SO: + + GAT + + + 0.9996277 + experimental_method + cleaner0 + 2023-07-20T15:05:31Z + MESH: + + PSVS + + + + t0005.xml + t0005 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><thead><tr><th/><th><bold>Tom1 GAT</bold></th></tr></thead><tbody><tr><td><bold>NMR distance and dihedral constraints</bold></td><td/></tr><tr><td> Dihedral angle restraints total</td><td>178</td></tr><tr><td><italic> ϕ</italic></td><td>89</td></tr><tr><td><italic> ψ</italic></td><td>89</td></tr><tr><td><bold>Structure statistics</bold></td><td/></tr><tr><td> Dihedral angle constraints (deg)</td><td>8.8±0.2</td></tr><tr><td> Max. dihedral angle violation (deg)</td><td>111±3</td></tr><tr><td>Deviations from idealized geometry</td><td/></tr><tr><td> Bond lengths (Å)</td><td>0.011</td></tr><tr><td> Bond angles (deg)</td><td>0.7</td></tr><tr><td>Average pairwise r.m.s. deviation (Å)<xref rid="tbl1fna" ref-type="table-fn">a</xref></td><td/></tr><tr><td> Protein</td><td/></tr><tr><td> Heavy</td><td>1.3</td></tr><tr><td> Backbone</td><td>0.9</td></tr></tbody></table> + + 5839 + Table 1. + + + t0005.xml + t0005 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><thead><tr><th/><th><bold>Tom1 GAT</bold></th></tr></thead><tbody><tr><td><bold>NMR distance and dihedral constraints</bold></td><td/></tr><tr><td> Dihedral angle restraints total</td><td>178</td></tr><tr><td><italic> ϕ</italic></td><td>89</td></tr><tr><td><italic> ψ</italic></td><td>89</td></tr><tr><td><bold>Structure statistics</bold></td><td/></tr><tr><td> Dihedral angle constraints (deg)</td><td>8.8±0.2</td></tr><tr><td> Max. dihedral angle violation (deg)</td><td>111±3</td></tr><tr><td>Deviations from idealized geometry</td><td/></tr><tr><td> Bond lengths (Å)</td><td>0.011</td></tr><tr><td> Bond angles (deg)</td><td>0.7</td></tr><tr><td>Average pairwise r.m.s. deviation (Å)<xref rid="tbl1fna" ref-type="table-fn">a</xref></td><td/></tr><tr><td> Protein</td><td/></tr><tr><td> Heavy</td><td>1.3</td></tr><tr><td> Backbone</td><td>0.9</td></tr></tbody></table> + + 5851 + Tom1 GAT NMR distance and dihedral constraints  Dihedral angle restraints total 178  ϕ 89  ψ 89 Structure statistics  Dihedral angle constraints (deg) 8.8±0.2  Max. dihedral angle violation (deg) 111±3 Deviations from idealized geometry  Bond lengths (Å) 0.011  Bond angles (deg) 0.7 Average pairwise r.m.s. deviation (Å)a  Protein  Heavy 1.3  Backbone 0.9 + + + t0005.xml + t0005 + TABLE + table_footnote + 6261 + Pairwise backbone and heavy-atom r.m.s. deviations were obtained by superimposing residues 215–309 of Tom1 GAT among 10 lowest energy refined structures. + + 0.9994108 + evidence + cleaner0 + 2023-07-20T15:05:50Z + DUMMY: + + r.m.s. deviations + + + 0.99964523 + experimental_method + cleaner0 + 2023-07-20T15:05:52Z + MESH: + + superimposing + + + 0.9990513 + residue_range + cleaner0 + 2023-07-20T15:05:56Z + DUMMY: + + 215–309 + + + 0.99986255 + protein + cleaner0 + 2023-07-20T14:57:12Z + PR: + + Tom1 + + + structure_element + SO: + cleaner0 + 2023-07-20T14:57:59Z + + GAT + + + 0.9991259 + evidence + cleaner0 + 2023-07-20T15:06:59Z + DUMMY: + + structures + + + + diff --git a/BioC_XML/4784909_v0.xml b/BioC_XML/4784909_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..fed6b2af0181ce075e8420ce664ca6d99d306be8 --- /dev/null +++ b/BioC_XML/4784909_v0.xml @@ -0,0 +1,8679 @@ + + + + PMC + 20140719 + pmc.key + + 4784909 + CC BY + no + 0 + 0 + + The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle + 10.1371/journal.pbio.1002399 + 4784909 + 26959993 + PBIOLOGY-D-15-02496 + e1002399 + 3 + This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. + surname:Erbilgin;given-names:Onur + surname:Sutter;given-names:Markus + surname:Kerfeld;given-names:Cheryl A. + surname:Petsko;given-names:Gregory A. + PDB files are available from the Protein Data Bank under accession codes 5CUO and 5CUP. + TITLE + Data Availability + front + 14 + 2016 + 0 + The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:19:42Z + + Coenzyme A + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:15:03Z + + Bacterial + + + + ABSTRACT + abstract + 70 + Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:15:03Z + + Bacterial + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:39Z + + Microcompartments + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:46Z + + BMCs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:17:56Z + + catabolic + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:46Z + + BMCs + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:18:05Z + + metabolosomes + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:19:34Z + + aldehyde + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:12Z + + phosphotransacylase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:19Z + + PTAC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:19:42Z + + Coenzyme A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:19:53Z + + acyl phosphate + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:19Z + + PTAC + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:18:05Z + + metabolosomes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:27Z + + PduL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:19Z + + PTAC + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:21:12Z + + fermentative bacteria + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:20Z + + Pta + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:27Z + + PduL + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:21:32Z + + crystal structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:21:40Z + + with bound substrates + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:22:32Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:22:46Z + + fold + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:21:59Z + + that + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:20Z + + Pta + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:25:04Z + + dimetal active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:22:56Z + + housekeeping + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:19Z + + PTAC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:27Z + + PduL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:20Z + + Pta + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:27Z + + PduL + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:23:00Z + + structure + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + metabolosome + + + + ABSTRACT + abstract + 1219 + This study describes the structure of a novel phosphotransacylase enzyme that facilitates the recycling of the essential cofactor acetyl-CoA within a bacterial organelle and discusses the properties of the enzyme's active site and how it is packaged into the organelle. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:23:53Z + + structure + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:12Z + + phosphotransacylase + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:24:05Z + + acetyl-CoA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:15:03Z + + bacterial + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:14Z + + active site + + + + ABSTRACT + abstract_title_1 + 1489 + Author Summary + + + ABSTRACT + abstract + 1504 + In metabolism, molecules with “high-energy” bonds (e.g., ATP and Acetyl~CoA) are critical for both catabolic and anabolic processes. Accordingly, the retention of these bonds during biochemical transformations is incredibly important. The phosphotransacylase (Pta) enzyme catalyzes the conversion between acyl-CoA and acyl-phosphate. This reaction directly links an acyl-CoA with ATP generation via substrate-level phosphorylation, producing short-chain fatty acids (e.g., acetate), and also provides a path for short-chain fatty acids to enter central metabolism. Due to this key function, Pta is conserved across the bacterial kingdom. Recently, a new type of phosphotransacylase was described that shares no evolutionary relation to Pta. This enzyme, PduL, is exclusively associated with organelles called bacterial microcompartments, which are used to catabolize various compounds. Not only does PduL facilitate substrate level phosphorylation, but it also is critical for cofactor recycling within, and product efflux from, the organelle. We solved the structure of this convergent phosphotransacylase and show that it is completely structurally different from Pta, including its active site architecture. We also discuss features of the protein important to its packaging in the organelle. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:10Z + + ATP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:17Z + + Acetyl~CoA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:12Z + + phosphotransacylase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:28Z + + acyl-CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:37Z + + acyl-phosphate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:28Z + + acyl-CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:10Z + + ATP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:53Z + + short-chain fatty acids + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:29:00Z + + acetate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:53Z + + short-chain fatty acids + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:29:05Z + + conserved + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:29:09Z + + bacterial kingdom + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:12Z + + phosphotransacylase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:27Z + + PduL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:29:19Z + + exclusively + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:15:03Z + + bacterial + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:39Z + + microcompartments + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:29:26Z + + solved + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:29:29Z + + structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:29:33Z + + convergent + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:12Z + + phosphotransacylase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:14Z + + active site + + + + INTRO + title_1 + 2804 + Introduction + + + INTRO + paragraph + 2817 + Bacterial Microcompartments (BMCs) are organelles that encapsulate enzymes for sequential biochemical reactions within a protein shell. The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell. The facets of the shell are composed primarily of hexamers that are typically perforated by pores lined with highly conserved, polar residues that presumably function as the conduits for metabolites into and out of the shell. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:15:03Z + + Bacterial + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:39Z + + Microcompartments + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:46Z + + BMCs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:29Z + + shell + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:30Z + + shell + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:31:44Z + + hexagonal + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:48Z + + BMC-H + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:52Z + + BMC-T + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:31:54Z + + pentagonal + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:57Z + + BMC-P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:32:00Z + + polyhedral + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:30Z + + shell + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:30Z + + shell + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:32:06Z + + hexamers + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:32:12Z + + pores + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:32:18Z + + highly conserved + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:32:21Z + + polar + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:32:25Z + + residues + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:30Z + + shell + + + + INTRO + paragraph + 3360 + The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1). More recently, bioinformatic studies have demonstrated the widespread distribution of BMCs among diverse bacterial phyla and grouped them into 23 different functional types. The reactions carried out in the majority of catabolic BMCs (also known as metabolosomes) fit a generalized biochemical paradigm for the oxidation of aldehydes (Fig 1). This involves a BMC-encapsulated signature enzyme that generates a toxic and/or volatile aldehyde that the BMC shell sequesters from the cytosol. The aldehyde is subsequently converted into an acyl-CoA by aldehyde dehydrogenase, which uses NAD+ and CoA as cofactors. These two cofactors are relatively large, and their diffusion across the protein shell is thought to be restricted, necessitating their regeneration within the BMC lumen. NAD+ is recycled via alcohol dehydrogenase, and CoA is recycled via phosphotransacetylase (PTAC) (Fig 1). The final product of the BMC, an acyl-phosphate, can then be used to generate ATP via acyl kinase, or revert back to acyl-CoA by Pta for biosynthesis. Collectively, the aldehyde and alcohol dehydrogenases, as well as the PTAC, constitute the common metabolosome core. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:35:57Z + + vitamin B12-dependent propanediol-utilizing (PDU) BMC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:17:56Z + + catabolic + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:46Z + + BMCs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:36:05Z + + ethanolamine + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:36:08Z + + choline + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:54:19Z + + fucose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:36:10Z + + rhamnose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:36:13Z + + ethanol + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:19:34Z + + aldehyde + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:36:19Z + + bioinformatic studies + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:46Z + + BMCs + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:36:22Z + + bacterial phyla + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:17:56Z + + catabolic + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:46Z + + BMCs + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:18:05Z + + metabolosomes + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:36:31Z + + aldehydes + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:00Z + + BMC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:19:34Z + + aldehyde + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:00Z + + BMC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:30Z + + shell + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:19:34Z + + aldehyde + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:28Z + + acyl-CoA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:38:03Z + + aldehyde dehydrogenase + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:38:07Z + + NAD+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:38:10Z + + CoA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:38:16Z + + protein shell + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:00Z + + BMC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:38:21Z + + NAD+ + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:38:28Z + + alcohol dehydrogenase + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:38:32Z + + CoA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:38:41Z + + phosphotransacetylase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:00Z + + BMC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:37Z + + acyl-phosphate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:10Z + + ATP + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:38:57Z + + acyl kinase + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:28Z + + acyl-CoA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:39:04Z + + aldehyde and alcohol dehydrogenases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + metabolosome + + + + pbio.1002399.g001.jpg + pbio.1002399.g001 + FIG + fig_title_caption + 4825 + General biochemical model of aldehyde-degrading BMCs (metabolosomes) illustrating the common metabolosome core enzymes and reactions. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:39:32Z + + aldehyde-degrading + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:46Z + + BMCs + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:18:05Z + + metabolosomes + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + metabolosome + + + + pbio.1002399.g001.jpg + pbio.1002399.g001 + FIG + fig_caption + 4959 + Substrates and cofactors involving the PTAC reaction are shown in red; other substrates and enzymes are shown in black, and other cofactors are shown in gray. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + + pbio.1002399.t001.xml + pbio.1002399.t001 + TABLE + table_title_caption + 5118 + Characterized and predicted catabolic BMC (metabolosome) types that represent the aldehyde-degrading paradigm (for definition of types see Kerfeld and Erbilgin). + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:17:56Z + + catabolic + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:00Z + + BMC + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + metabolosome + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:19:34Z + + aldehyde + + + + pbio.1002399.t001.xml + pbio.1002399.t001 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><thead><tr><th align="justify" rowspan="1" colspan="1">Name</th><th align="justify" rowspan="1" colspan="1">PTAC Type</th><th align="justify" rowspan="1" colspan="1">Sequestered Aldehyde</th></tr></thead><tbody><tr><td align="justify" rowspan="1" colspan="1">PDU<xref ref-type="table-fn" rid="t001fn001">*</xref> +</td><td align="justify" rowspan="1" colspan="1">PduL</td><td align="justify" rowspan="1" colspan="1">propionaldehyde</td></tr><tr><td align="justify" rowspan="1" colspan="1">EUT1</td><td align="justify" rowspan="1" colspan="1">PTA_PTB</td><td align="justify" rowspan="1" colspan="1">acetaldehyde</td></tr><tr><td align="justify" rowspan="1" colspan="1">EUT2</td><td align="justify" rowspan="1" colspan="1">PduL</td><td align="justify" rowspan="1" colspan="1">acetaldehyde</td></tr><tr><td align="justify" rowspan="1" colspan="1">ETU</td><td align="justify" rowspan="1" colspan="1">None</td><td align="justify" rowspan="1" colspan="1">acetaldehyde</td></tr><tr><td align="justify" rowspan="1" colspan="1">GRM1/CUT</td><td align="justify" rowspan="1" colspan="1">PduL</td><td align="justify" rowspan="1" colspan="1">acetaldehyde</td></tr><tr><td align="justify" rowspan="1" colspan="1">GRM2</td><td align="justify" rowspan="1" colspan="1">PduL</td><td align="justify" rowspan="1" colspan="1">acetaldehyde</td></tr><tr><td align="justify" rowspan="1" colspan="1">GRM3<xref ref-type="table-fn" rid="t001fn001">*</xref>,4</td><td align="justify" rowspan="1" colspan="1">PduL</td><td align="justify" rowspan="1" colspan="1">propionaldehyde</td></tr><tr><td align="justify" rowspan="1" colspan="1">GRM5/GRP</td><td align="justify" rowspan="1" colspan="1">PduL</td><td align="justify" rowspan="1" colspan="1">propionaldehyde</td></tr><tr><td align="justify" rowspan="1" colspan="1">PVM<xref ref-type="table-fn" rid="t001fn001">*</xref> +</td><td align="justify" rowspan="1" colspan="1">PduL</td><td align="justify" rowspan="1" colspan="1">lactaldehyde</td></tr><tr><td align="justify" rowspan="1" colspan="1">RMM1,2</td><td align="justify" rowspan="1" colspan="1">None</td><td align="justify" rowspan="1" colspan="1">unknown</td></tr><tr><td align="justify" rowspan="1" colspan="1">SPU</td><td align="justify" rowspan="1" colspan="1">PduL</td><td align="justify" rowspan="1" colspan="1">unknown</td></tr></tbody></table> + + 5280 + Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:19:34Z + + Aldehyde + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:41:59Z + + PDU + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:42:02Z + + propionaldehyde + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:42:09Z + + EUT1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:42:12Z + + PTA_PTB + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:42:15Z + + acetaldehyde + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:42:18Z + + EUT2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:42:23Z + + acetaldehyde + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:42:26Z + + ETU + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:42:28Z + + acetaldehyde + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:42:31Z + + GRM1/CUT + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:42:35Z + + acetaldehyde + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:42:38Z + + GRM2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:42:41Z + + acetaldehyde + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:42:44Z + + GRM3*,4 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:42:47Z + + propionaldehyde + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:42:49Z + + GRM5/GRP + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:42:52Z + + propionaldehyde + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:42:55Z + + PVM + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:42:58Z + + lactaldehyde + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:43:01Z + + RMM1,2 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:43:04Z + + SPU + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + + pbio.1002399.t001.xml + pbio.1002399.t001 + TABLE + table_footnote + 5607 + * PduL from these functional types of metabolosomes were purified in this study. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:18:05Z + + metabolosomes + + + + INTRO + paragraph + 5688 + The activities of core enzymes are not confined to BMC-associated functions: aldehyde and alcohol dehydrogenases are utilized in diverse metabolic reactions, and PTAC catalyzes a key biochemical reaction in the process of obtaining energy during fermentation. The concerted functioning of a PTAC and an acetate kinase (Ack) is crucial for ATP generation in the fermentation of pyruvate to acetate (see Reactions 1 and 2). Both enzymes are, however, not restricted to fermentative organisms. They can also work in the reverse direction to activate acetate to the CoA-thioester. This occurs, for example, during acetoclastic methanogenesis in the archaeal Methanosarcina species. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:00Z + + BMC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:45:57Z + + aldehyde and alcohol dehydrogenases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:45:54Z + + acetate kinase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:46:04Z + + Ack + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:10Z + + ATP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:46:13Z + + pyruvate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:29:00Z + + acetate + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:45:44Z + + fermentative organisms + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:29:00Z + + acetate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:46:23Z + + CoA-thioester + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:45:22Z + + archaeal + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:45:08Z + + Methanosarcina species + + + + INTRO + paragraph + 6366 + Reaction 1: acetyl-S-CoA + Pi ←→ acetyl phosphate + CoA-SH (PTAC) + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:46:56Z + + acetyl-S-CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:47:02Z + + Pi + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:47:09Z + + acetyl phosphate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:47:19Z + + CoA-SH + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + + INTRO + paragraph + 6437 + Reaction 2: acetyl phosphate + ADP ←→ acetate + ATP (Ack) + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:47:10Z + + acetyl phosphate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:47:55Z + + ADP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:29:01Z + + acetate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:10Z + + ATP + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:46:04Z + + Ack + + + + INTRO + paragraph + 6500 + The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515). Pta has been extensively characterized due to its key role in fermentation. More recently, a second type of PTAC without any sequence homology to Pta was identified. This protein, PduL (Pfam domain PF06130), was shown to catalyze the conversion of propionyl-CoA to propionyl-phosphate and is associated with a BMC involved in propanediol utilization, the PDU BMC. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:50:42Z + + eukaryotes + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:50:50Z + + archaea + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:51:00Z + + bacteria + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:15:03Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:51:05Z + + PTA_PTB phosphotransacylase + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:51:16Z + + PF01515 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:51:25Z + + PF06130 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:51:34Z + + propionyl-CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:51:42Z + + propionyl-phosphate + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:00Z + + BMC + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:51:51Z + + PDU BMC + + + + INTRO + paragraph + 7136 + Both pduL and pta genes can be found in genetic loci of functionally distinct BMCs, although the PduL type is much more prevalent, being found in all but one type of metabolosome locus: EUT1 (Table 1). Furthermore, in the Integrated Microbial Genomes Database, 91% of genomes that encode PF06130 also encode genes for shell proteins. As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. Another distinctive feature of BMC-associated PduL homologs is an N-terminal encapsulation peptide (EP) that is thought to “target” proteins for encapsulation by the BMC shell. EPs are frequently found on BMC-associated proteins and have been shown to interact with shell proteins. EPs have also been observed to cause proteins to aggregate, and this has recently been suggested to be functionally relevant as an initial step in metabolosome assembly, in which a multifunctional protein core is formed, around which the shell assembles. + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-21T15:54:31Z + + pduL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-21T15:54:49Z + + pta + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:46Z + + BMCs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-21T17:55:23Z + + metabolosome locus + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-21T17:55:27Z + + EUT1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:51:25Z + + PF06130 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:55:52Z + + aldehyde-oxidizing + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:18:05Z + + metabolosomes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:24:05Z + + acetyl-CoA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:51:51Z + + PDU BMC + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:56:10Z + + Salmonella enterica + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:51:34Z + + propionyl-CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:24:05Z + + acetyl-CoA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:46Z + + BMCs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:56:18Z + + CoA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:56:22Z + + BMC-associated + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:32Z + + encapsulation peptide + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:30Z + + shell + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:54Z + + EPs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:56:58Z + + BMC-associated proteins + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:54Z + + EPs + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + metabolosome + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:30Z + + shell + + + + INTRO + paragraph + 8478 + Of the three common metabolosome core enzymes, crystal structures are available for both the alcohol and aldehyde dehydrogenases. In contrast, the structure of PduL, the PTAC found in the vast majority of catabolic BMCs, has not been determined. This is a major gap in our understanding of metabolosome-encapsulated biochemistry and cofactor recycling. Structural information will be essential to working out how the core enzymes and their cofactors assemble and organize within the organelle lumen to enhance catalysis. Moreover, it will be useful for guiding efforts to engineer novel BMC cores for biotechnological applications. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + metabolosome + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:21:32Z + + crystal structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:58:16Z + + alcohol and aldehyde dehydrogenases + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:58:19Z + + structure + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:17:56Z + + catabolic + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:47Z + + BMCs + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + metabolosome + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + + INTRO + paragraph + 9110 + The primary structure of PduL homologs is subdivided into two PF06130 domains, each roughly 80 residues in length. No available protein structures contain the PF06130 domain, and homology searches using the primary structure of PduL do not return any significant results that would allow prediction of the structure. Moreover, the evident novelty of PduL makes its structure interesting in the context of convergent evolution of PTAC function; to-date, only the Pta active site and catalytic mechanism is known. Here we report high-resolution crystal structures of a PduL-type PTAC in both CoA- and phosphate-bound forms, completing our understanding of the structural basis of catalysis by the metabolosome common core enzymes. We propose a catalytic mechanism analogous but yet distinct from the ubiquitous Pta enzyme, highlighting the functional convergence of two enzymes with completely different structures and metal requirements. We also investigate the quaternary structures of three different PduL homologs and situate our findings in the context of organelle biogenesis in functionally diverse BMCs. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:51:25Z + + PF06130 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:00:07Z + + 80 residues in length + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:51:25Z + + PF06130 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:00:18Z + + homology searches + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:00:22Z + + structure + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:00:25Z + + structure + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:14Z + + active site + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:21:32Z + + crystal structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:00:29Z + + PduL-type PTAC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:48:27Z + + CoA- + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:47:26Z + + phosphate-bound + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + metabolosome + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:47Z + + BMCs + + + + RESULTS + title_1 + 10220 + Results + + + RESULTS + title_2 + 10228 + Structure Determination of PduL + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:00:48Z + + Structure Determination + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + + RESULTS + paragraph + 10260 + We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). While purifying full-length sPduL, we observed a tendency to aggregation as described previously, with a large fraction of the expressed protein found in the insoluble fraction in a white, cake-like pellet. Remarkably, after removing the N-terminal putative EP (27 amino acids), most of the sPduLΔEP protein was in the soluble fraction upon cell lysis. Similar differences in solubility were observed for pPduL and rPduL when comparing EP-truncated forms to the full-length protein, but none were quite as dramatic as for sPduL. We confirmed that all homologs were active (S1a and S1b Fig). Among these, we were only able to obtain diffraction-quality crystals of rPduL after removing the N-terminal putative EP (33 amino acids, also see Fig 2a) (rPduLΔEP). Truncated rPduLΔEP had comparable enzymatic activity to the full-length enzyme (S1a Fig). + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:03:27Z + + cloned, expressed, and purified + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:47Z + + BMCs + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-21T16:03:31Z + + pdu locus + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:03:38Z + + S. enterica Typhimurium LT2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:03:44Z + + sPduL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-21T16:03:48Z + + pvm locus + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:03:54Z + + Planctomyces limnophilus + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:00Z + + pPduL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-21T16:04:05Z + + grm3 locus + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:04:10Z + + Rhodopseudomonas palustris BisB18 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:17Z + + rPduL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:04:25Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:03:44Z + + sPduL + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:04:30Z + + removing + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:04:37Z + + 27 amino acids + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:04:44Z + + sPduLΔEP + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:00Z + + pPduL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:17Z + + rPduL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:04:54Z + + EP-truncated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:04:25Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:03:44Z + + sPduL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:05:01Z + + active + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:05:06Z + + diffraction-quality crystals + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:17Z + + rPduL + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:05:09Z + + removing + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:05:12Z + + 33 amino acids + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:05:18Z + + rPduLΔEP + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:05:26Z + + Truncated + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:05:18Z + + rPduLΔEP + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:04:25Z + + full-length + + + + pbio.1002399.g002.jpg + pbio.1002399.g002 + FIG + fig_title_caption + 11449 + Structural overview of R. palustris PduL from the grm3 locus. + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:06:40Z + + R. palustris + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-21T16:06:44Z + + grm3 locus + + + + pbio.1002399.g002.jpg + pbio.1002399.g002 + FIG + fig_caption + 11511 + (a) Primary and secondary structure of rPduL (tubes represent α-helices, arrows β-sheets and dashed line residues disordered in the structure. Blocks of ten residues are shaded alternatively black/dark gray. The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). Metal coordination residues are highlighted in light blue and CoA contacting residues in magenta, residues contacting the CoA of the other chain are also outlined. (b) Cartoon representation of the structure colored by domains and including secondary structure numbering. The N-and C-termini are in close proximity. Coenzyme A is shown in magenta sticks and Zinc (grey) as spheres. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:17Z + + rPduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:08:37Z + + α-helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:08:40Z + + β-sheets + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:08:43Z + + structure + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:08:47Z + + first 33 amino acids + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:09:10Z + + EP + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:09:24Z + + alpha helix + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:09:35Z + + α0 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:05:26Z + + truncated + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:05:18Z + + rPduLΔEP + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:09:39Z + + crystallized + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:09:42Z + + M + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:09:44Z + + G + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:09:47Z + + V + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:09:51Z + + domain 1 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:09:53Z + + D36-N46 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:09:56Z + + Q155-C224 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:10:00Z + + loop insertion + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:10:02Z + + G61-E81 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:10:05Z + + domain 2 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:10:07Z + + R47-F60 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:10:10Z + + E82-A154 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:10:12Z + + Metal coordination residues + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:10:16Z + + CoA contacting residues + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:10:18Z + + CoA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:54:54Z + + structure + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:54:57Z + + structure + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:19:43Z + + Coenzyme A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:10:26Z + + Zinc + + + + RESULTS + paragraph + 12438 + We collected a native dataset from rPduLΔEP crystals diffracting to a resolution of 1.54 Å (Table 2). Using a mercury-derivative crystal form diffracting to 1.99 Å (Table 2), we obtained high quality electron density for model building and used the initial model to refine against the native data to Rwork/Rfree values of 18.9/22.1%. There are two PduL molecules in the asymmetric unit of the P212121 unit cell. We were able to fit all of the primary structure of PduLΔEP into the electron density with the exception of three amino acids at the N-terminus and two amino acids at the C-terminus (Fig 2a); the model is of excellent quality (Table 2). A CoA cofactor as well as two metal ions are clearly resolved in the density (for omit maps of CoA see S2 Fig). + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:33:57Z + + collected a native dataset + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:05:18Z + + rPduLΔEP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:33:53Z + + crystals + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:33:59Z + + mercury-derivative crystal + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:34:03Z + + electron density + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:34:38Z + + Rwork + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:34:52Z + + Rfree + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:35:27Z + + PduLΔEP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:35:31Z + + electron density + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:35:42Z + + CoA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:35:52Z + + density + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:35:54Z + + omit maps + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:35:57Z + + CoA + + + + pbio.1002399.t002.xml + pbio.1002399.t002 + TABLE + table_title_caption + 13207 + Data collection and refinement statistics + + + pbio.1002399.t002.xml + pbio.1002399.t002 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><thead><tr><th align="justify" rowspan="1" colspan="1"/><th align="justify" rowspan="1" colspan="1">PduL native</th><th align="left" rowspan="1" colspan="1">PduL mercury derivative</th><th align="left" rowspan="1" colspan="1">PduL phosphate soaked</th></tr></thead><tbody><tr><td align="justify" rowspan="1" colspan="1"> +<bold>Data collection</bold> +</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">Space group</td><td align="justify" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2<sub>1</sub> +</td><td align="justify" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2<sub>1</sub> +</td><td align="justify" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2<sub>1</sub> +</td></tr><tr><td align="justify" rowspan="1" colspan="1">Cell dimensions</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1"> +<italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td align="justify" rowspan="1" colspan="1">57.7, 56.4, 150.4</td><td align="justify" rowspan="1" colspan="1">55.6, 57.7, 150.2</td><td align="justify" rowspan="1" colspan="1">57.1, 58.8, 136.7</td></tr><tr><td align="justify" rowspan="1" colspan="1"> +<italic>α</italic>, <italic>β</italic>, <italic>γ</italic> (°)</td><td align="justify" rowspan="1" colspan="1">90, 90, 90</td><td align="justify" rowspan="1" colspan="1">90, 90, 90</td><td align="justify" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="justify" rowspan="1" colspan="1">Resolution (Å)</td><td align="justify" rowspan="1" colspan="1">31.4 − 1.54 (1.60 − 1.54)<xref ref-type="table-fn" rid="t002fn001">*</xref> +</td><td align="justify" rowspan="1" colspan="1">35.3 − 1.99 (2.07 − 1.99)</td><td align="justify" rowspan="1" colspan="1">39.2 − 2.10 (2.21 − 2.10)</td></tr><tr><td align="justify" rowspan="1" colspan="1"> +<italic>R</italic> +<sub>merge</sub> +</td><td align="justify" rowspan="1" colspan="1">0.169 (1.223)</td><td align="justify" rowspan="1" colspan="1">0.084 (0.299)</td><td align="justify" rowspan="1" colspan="1">0.122 (0.856)</td></tr><tr><td align="justify" rowspan="1" colspan="1">I/σ(I)</td><td align="justify" rowspan="1" colspan="1">12.9 (1.7)</td><td align="justify" rowspan="1" colspan="1">22.1 (7.1)</td><td align="justify" rowspan="1" colspan="1">12.6 (2.0)</td></tr><tr><td align="justify" rowspan="1" colspan="1">Completeness (%)</td><td align="justify" rowspan="1" colspan="1">99.4 (94.4)</td><td align="justify" rowspan="1" colspan="1">99.3 (93.3)</td><td align="justify" rowspan="1" colspan="1">100 (99.9)</td></tr><tr><td align="justify" rowspan="1" colspan="1">Redundancy</td><td align="justify" rowspan="1" colspan="1">13.9 (12.1)</td><td align="justify" rowspan="1" colspan="1">7.2 (7.0)</td><td align="justify" rowspan="1" colspan="1">6.5 (6.1)</td></tr><tr><td align="justify" rowspan="1" colspan="1"> +<bold>Refinement</bold> +</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">Resolution (Å)</td><td align="justify" rowspan="1" colspan="1">31.4 − 1.54 (1.60 − 1.54)<xref ref-type="table-fn" rid="t002fn001">*</xref> +</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">39.2 − 2.10 (2.18 − 2.1)</td></tr><tr><td align="justify" rowspan="1" colspan="1">No. reflections</td><td align="justify" rowspan="1" colspan="1">72,698</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">27,554</td></tr><tr><td align="justify" rowspan="1" colspan="1"> +<italic>R</italic> +<sub>work/</sub> +<italic>R</italic> +<sub>free</sub> (%)</td><td align="justify" rowspan="1" colspan="1">18.9 (30.7) / 22.1 (34.7)</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">17.5 (24.2) / 22.6 (30.0)</td></tr><tr><td align="justify" rowspan="1" colspan="1">No. atoms</td><td align="justify" rowspan="1" colspan="1">3,453</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">3,127</td></tr><tr><td align="justify" rowspan="1" colspan="1">Protein</td><td align="justify" rowspan="1" colspan="1">2,841</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">2,838</td></tr><tr><td align="justify" rowspan="1" colspan="1">Ligand/ion</td><td align="justify" rowspan="1" colspan="1">100</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">24</td></tr><tr><td align="justify" rowspan="1" colspan="1">Water</td><td align="justify" rowspan="1" colspan="1">512</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">265</td></tr><tr><td align="justify" rowspan="1" colspan="1">B-factors</td><td align="justify" rowspan="1" colspan="1">22.8</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">34.7</td></tr><tr><td align="justify" rowspan="1" colspan="1">Protein</td><td align="justify" rowspan="1" colspan="1">21.5</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">24.3</td></tr><tr><td align="justify" rowspan="1" colspan="1">Ligand/ion</td><td align="justify" rowspan="1" colspan="1">21.9</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">40.6</td></tr><tr><td align="justify" rowspan="1" colspan="1">Water</td><td align="justify" rowspan="1" colspan="1">30.3</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">37.9</td></tr><tr><td align="justify" rowspan="1" colspan="1">R.m.s deviations</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">Bond lengths (Å)</td><td align="justify" rowspan="1" colspan="1">0.006</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">0.013</td></tr><tr><td align="justify" rowspan="1" colspan="1">Bond angles (°)</td><td align="justify" rowspan="1" colspan="1">1.26</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">1.30</td></tr><tr><td align="justify" rowspan="1" colspan="1">Ramachandran Plot</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">favored (%)</td><td align="justify" rowspan="1" colspan="1">99</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">99</td></tr><tr><td align="justify" rowspan="1" colspan="1">allowed (%)</td><td align="justify" rowspan="1" colspan="1">1</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">1</td></tr><tr><td align="justify" rowspan="1" colspan="1">disallowed (%)</td><td align="justify" rowspan="1" colspan="1">0</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">0</td></tr></tbody></table> + + 13249 + PduL native PduL mercury derivative PduL phosphate soaked Data collection Space group P 21 21 21 P 21 21 21 P 21 21 21 Cell dimensions a, b, c (Å) 57.7, 56.4, 150.4 55.6, 57.7, 150.2 57.1, 58.8, 136.7 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) 31.4 − 1.54 (1.60 − 1.54)* 35.3 − 1.99 (2.07 − 1.99) 39.2 − 2.10 (2.21 − 2.10) Rmerge 0.169 (1.223) 0.084 (0.299) 0.122 (0.856) I/σ(I) 12.9 (1.7) 22.1 (7.1) 12.6 (2.0) Completeness (%) 99.4 (94.4) 99.3 (93.3) 100 (99.9) Redundancy 13.9 (12.1) 7.2 (7.0) 6.5 (6.1) Refinement Resolution (Å) 31.4 − 1.54 (1.60 − 1.54)* 39.2 − 2.10 (2.18 − 2.1) No. reflections 72,698 27,554 Rwork/Rfree (%) 18.9 (30.7) / 22.1 (34.7) 17.5 (24.2) / 22.6 (30.0) No. atoms 3,453 3,127 Protein 2,841 2,838 Ligand/ion 100 24 Water 512 265 B-factors 22.8 34.7 Protein 21.5 24.3 Ligand/ion 21.9 40.6 Water 30.3 37.9 R.m.s deviations Bond lengths (Å) 0.006 0.013 Bond angles (°) 1.26 1.30 Ramachandran Plot favored (%) 99 99 allowed (%) 1 1 disallowed (%) 0 0 + + + pbio.1002399.t002.xml + pbio.1002399.t002 + TABLE + table_footnote + 14364 + *Highest resolution shell is shown in parentheses. + + + RESULTS + paragraph + 14415 + Structurally, PduL consists of two domains (Fig 2, blue/red), each a beta-barrel that is capped on both ends by short α-helices. β-Barrel 1 consists of the N-terminal β strand and β strands from the C-terminal half of the polypeptide chain (β1, β10-β14; residues 37–46 and 155–224). β-Barrel 2 consists mainly of the central segment of primary structure (β2, β5–β9; residues 47–60 and 82–154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (β3-β4, residues 61–81). This β-sheet is involved in contacts between the two domains and forms a lid over the active site. Residues in this region (Gln42, Pro43, Gly44), covering the active site, are strongly conserved (Fig 3). This structural arrangement is completely different from the functionally related Pta, which is composed of two domains, each consisting of a central flat beta sheet with alpha-helices on the top and bottom. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:38:19Z + + domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:38:22Z + + beta-barrel + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:38:24Z + + α-helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:38:31Z + + β-Barrel 1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:38:34Z + + β strand + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:38:37Z + + β strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:38:40Z + + C-terminal half + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:38:43Z + + β1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:38:45Z + + β10-β14 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:38:48Z + + 37–46 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:38:51Z + + 155–224 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:38:57Z + + β-Barrel 2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:39:02Z + + β2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:39:05Z + + β5–β9 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:39:07Z + + 47–60 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:55:31Z + + 82–154 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:39:14Z + + short two-strand beta sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:39:16Z + + β3-β4 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:39:19Z + + 61–81 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:39:22Z + + β-sheet + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:14Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:39:34Z + + Gln42 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:39:41Z + + Pro43 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:39:47Z + + Gly44 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:14Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:54:31Z + + strongly conserved + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:39:52Z + + domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:39:54Z + + beta sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:39:57Z + + alpha-helices + + + + pbio.1002399.g003.jpg + pbio.1002399.g003 + FIG + fig_title_caption + 15354 + Primary structure conservation of the PduL protein family. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + + pbio.1002399.g003.jpg + pbio.1002399.g003 + FIG + fig_caption + 15413 + Sequence logo calculated from the multiple sequence alignment of PduL homologs (see Materials and Methods), but not including putative EP sequences. Residues 100% conserved across all PduL homologs in our dataset are noted with an asterisk, and residues conserved in over 90% of sequences are noted with a colon. The sequences aligning to the PF06130 domain (determined by BLAST) are highlighted in red and blue. The position numbers shown correspond to the residue numbering of rPduL; note that some positions in the logo represent gaps in the rPduL sequence. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:40:47Z + + multiple sequence alignment + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:28Z + + PduL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:40:54Z + + not including + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:51:25Z + + PF06130 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:17Z + + rPduL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:17Z + + rPduL + + + + RESULTS + paragraph + 15974 + There are two PduL molecules in the asymmetric unit forming a butterfly-shaped dimer (Fig 4c). Consistent with this, results from size exclusion chromatography of rPduLΔEP suggest that it is a dimer in solution (Fig 5e). The interface between the two chains buries 882 Å2 per monomer and is mainly formed by α-helices 2 and 4 and parts of β-sheets 12 and 14, as well as a π–π stacking of the adenine moiety of CoA with Phe116 of the adjacent chain (Fig 4c). The folds of the two chains in the asymmetric unit are very similar, superimposing with a rmsd of 0.16 Å over 2,306 aligned atom pairs. The peripheral helices and the short antiparallel β3–4 sheet mediate most of the crystal contacts. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:17Z + + butterfly-shaped + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:24Z + + dimer + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:48:56Z + + size exclusion chromatography + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:05:18Z + + rPduLΔEP + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:24Z + + dimer + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:42:34Z + + interface + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:40Z + + monomer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:42:44Z + + α-helices 2 and 4 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:42:47Z + + β-sheets 12 and 14 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:42:58Z + + adenine + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:43:00Z + + CoA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:43:09Z + + Phe116 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:44:40Z + + superimposing + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:44:59Z + + rmsd + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:45:10Z + + helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:45:33Z + + short antiparallel β3–4 sheet + + + + pbio.1002399.g004.jpg + pbio.1002399.g004 + FIG + fig_title_caption + 16683 + Details of active site, dimeric assembly, and sequence conservation of PduL. + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:14Z + + active site + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:45:56Z + + dimeric + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + + pbio.1002399.g004.jpg + pbio.1002399.g004 + FIG + fig_caption + 16760 + (a,b) Proposed active site of PduL with relevant residues shown as sticks in atom coloring (nitrogen blue, oxygen red, sulfur yellow), zinc as grey colored spheres and coordinating ordered water molecules in red. Distances between atom centers are indicated in Å. (a) Coenzyme A containing, (b) phosphate-bound structure. (c) View of the dimer in the asymmetric unit from the side, domains 1 and 2 colored as in Fig 2 and the two chains differentiated by blue/red versus slate/firebrick. The bottom panel shows a top view down the 2-fold axis as indicated by the arrow in the top panel. The asterisk and double arrow marks the location of the π–π interaction between F116 and the CoA base of the other dimer chain. (d) Surface representation of the structure with indicated conservation (red: high, white: intermediate, yellow: low). + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:14Z + + active site + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:47:01Z + + nitrogen + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:47:04Z + + oxygen + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:47:06Z + + sulfur + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:10:27Z + + zinc + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:47:14Z + + water + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:19:43Z + + Coenzyme A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:47:26Z + + phosphate-bound + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:47:31Z + + structure + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:24Z + + dimer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:47:37Z + + domains 1 and 2 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:28Z + + π–π interaction + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:47:43Z + + F116 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:47:53Z + + CoA + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:24Z + + dimer + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:47:59Z + + structure + + + + pbio.1002399.g005.jpg + pbio.1002399.g005 + FIG + fig_title_caption + 17599 + Size exclusion chromatography of PduL homologs. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:48:56Z + + Size exclusion chromatography + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + + pbio.1002399.g005.jpg + pbio.1002399.g005 + FIG + fig_caption + 17647 + (a)–(c): Chromatograms of sPduL (a), rPduL (b), and pPduL (c) with (orange) or without (blue) the predicted EP, post-nickel affinity purification, applied over a preparative size exclusion column (see Materials and Methods). (d)–(f): Chromatograms of sPduL (d), rPduL (e), and pPduL (f) post-preparative size exclusion chromatography with different size fractions separated, applied over an analytical size exclusion column (see Materials and Methods). All chromatograms are cropped to show only the linear range of separation based on standard runs, shown in black squares with a dashed linear trend line. All y-axes are arbitrary absorbance units except the right-hand axes for panels (a) and (d), which is the log10(molecular weight) of the standards. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:49:50Z + + Chromatograms + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:03:44Z + + sPduL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:17Z + + rPduL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:01Z + + pPduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:49:53Z + + nickel affinity purification + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:49:48Z + + Chromatograms + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:03:44Z + + sPduL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:17Z + + rPduL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:01Z + + pPduL + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:48:56Z + + size exclusion chromatography + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:49:45Z + + chromatograms + + + + RESULTS + title_2 + 18406 + Active Site Properties + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:14Z + + Active Site + + + + RESULTS + paragraph + 18429 + CoA and the metal ions bind between the two domains, presumably in the active site (Figs 2b and 4a). To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn). There was a large signal at the zinc edge, and we tested for the presence of zinc by collecting full data sets before and after the Zn K-edge (1.2861 and 1.2822 Å, respectively). The large differences between the anomalous signals confirm the presence of zinc at both metal sites (S3 Fig). + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:51:21Z + + CoA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:14Z + + active site + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:51:26Z + + X-ray fluorescence scan + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:51:29Z + + crystals + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:51:32Z + + Mn + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:51:34Z + + Fe + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:51:37Z + + Co + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:51:40Z + + Ni + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:51:42Z + + Cu + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:51:45Z + + Zn + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:10:27Z + + zinc + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:51:47Z + + collecting full data sets before and after the Zn K-edge (1.2861 and 1.2822 Å + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:10:27Z + + zinc + + + + RESULTS + paragraph + 18992 + The first zinc ion (Zn1) is in a tetrahedral coordination state with His48, His50, Glu109, and the CoA sulfur (Fig 4a). The second (Zn2) is in octahedral coordination by three conserved histidine residues (His157, His159 and His204) as well as three water molecules (Fig 4a). The nitrogen atom coordinating the zinc is the Nε in each histidine residue, as is typical for this interaction. When the crystals were soaked in a sodium phosphate solution for 2 d prior to data collection, the CoA dissociates, and density for a phosphate molecule is visible at the active site (Table 2, Fig 4b). The phosphate-bound structure aligns well with the CoA-bound structure (0.43 Å rmsd over 2,361 atoms for the monomer, 0.83 Å over 5,259 aligned atoms for the dimer). The phosphate contacts both zinc atoms (Fig 4b) and replaces the coordination by CoA at Zn1; the coordination for Zn2 changes from octahedral with three bound waters to tetrahedral with a phosphate ion as one of the ligands (Fig 4b). Conserved Arg103 seems to be involved in maintaining the phosphate in that position. The two zinc atoms are slightly closer together in the phosphate-bound form (5.8 Å vs 6.3 Å), possibly due to the bridging effect of the phosphate. An additional phosphate molecule is bound at a crystal contact interface, perhaps accounting for the 14 Å shorter c-axis in the phosphate-bound crystal form (Table 2). + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:10:27Z + + zinc + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:55:24Z + + Zn1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:55:33Z + + His48 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:55:39Z + + His50 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:55:45Z + + Glu109 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:55:48Z + + CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:55:51Z + + sulfur + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:56:02Z + + Zn2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:56:06Z + + conserved + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:56:11Z + + histidine + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:56:17Z + + His157 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:56:23Z + + His159 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:56:29Z + + His204 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:47:14Z + + water + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:10:27Z + + zinc + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T16:56:11Z + + histidine + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:56:39Z + + crystals were soaked + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:56:43Z + + sodium phosphate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:56:45Z + + CoA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:56:48Z + + density + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:56:51Z + + phosphate + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:14Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:47:26Z + + phosphate-bound + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:56:57Z + + structure + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:56:59Z + + aligns + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:57:04Z + + CoA-bound + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:57:07Z + + structure + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:45:02Z + + rmsd + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:40Z + + monomer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:24Z + + dimer + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:57:13Z + + phosphate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:10:27Z + + zinc + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:57:20Z + + CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:55:24Z + + Zn1 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:56:02Z + + Zn2 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:54:23Z + + waters + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:57:30Z + + phosphate + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:57:33Z + + Conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:57:39Z + + Arg103 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:54:27Z + + phosphate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:10:27Z + + zinc + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:47:26Z + + phosphate-bound + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:57:48Z + + phosphate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T16:57:51Z + + phosphate + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:47:26Z + + phosphate-bound + + + + RESULTS + title_2 + 20390 + Oligomeric States of PduL Orthologs Are Influenced by the EP + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + + RESULTS + paragraph + 20451 + Interestingly, some of the residues important for dimerization of rPduL, particularly Phe116, are poorly conserved across PduL homologs associated with functionally diverse BMCs (Figs 4c and 3), suggesting that they may have alternative oligomeric states. We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, ΔEP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1). Therefore, they are packaged with different signature enzymes and different ancillary proteins. It has been proposed that the catabolic BMCs may assemble in a core-first manner, with the luminal enzymes (signature enzyme, aldehyde, and alcohol dehydrogenases and the BMC PTAC) forming an initial bolus, or prometabolosome, around which a shell assembles. Given the diversity of signature enzymes (Table 1), it is plausible that PduL orthologs may adopt different oligomeric states that reflect the differences in the proteins being packaged with them in the organelle lumen. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:17Z + + rPduL + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:43:09Z + + Phe116 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:00:04Z + + poorly conserved + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:47Z + + BMCs + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:48:56Z + + size exclusion chromatography + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:04:25Z + + full-length + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:00:50Z + + lacking + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:00:55Z + + ΔEP + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:03:44Z + + sPduL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:17Z + + rPduL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:01Z + + pPduL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:47Z + + BMCs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:17:56Z + + catabolic + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:47Z + + BMCs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:01:05Z + + aldehyde, and alcohol dehydrogenases + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:30Z + + shell + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + + RESULTS + paragraph + 21521 + We found that not only did the different orthologs appear to assemble into different oligomeric states, but that quaternary structure was dependent on whether or not the EP was present. Full-length sPduL was unstable in solution—precipitating over time—and eluted throughout the entire volume of a size exclusion column, indicating it was nonspecifically aggregating. However, when the putative EP (residues 1–27) was removed (sPduL ΔEP), the truncated protein was stable and eluted as a single peak (Fig 5a) consistent with the size of a monomer (Fig 5d, blue curve). In contrast, both full-length rPduL and pPduL appeared to exist in two distinct oligomeric states (Fig 5b and 5c respectively, orange curves), one form of the approximate size of a dimer and the second, a higher molecular weight oligomer (~150 kDa). Upon deletion of the putative EP (residues 1–47 for rPduL, and 1–20 for pPduL), there was a distinct change in the elution profiles (Fig 5b and 5c respectively, blue curves). pPduLΔEP eluted as two smaller forms, possibly corresponding to a trimer and a monomer. In contrast, rPduLΔEP eluted as one smaller oligomer, possibly a dimer. We also analyzed purified rPduL and rPduLΔEP by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) for a complementary approach to assessing oligomeric state. SEC-MALS analysis of rPdulΔEP is consistent with a dimer (as observed in the crystal structure) with a weighted average (Mw) and number average (Mn) of the molar mass of 58.4 kDa +/− 11.2% and 58.8 kDa +/− 10.9%, respectively (S4a Fig). rPduL full length runs as Mw = 140.3 kDa +/− 1.2% and Mn = 140.5 kDa +/− 1.2%. This corresponds to an oligomeric state of six subunits (calculated molecular weight of 144 kDa). Collectively, these data strongly suggest that the N-terminal EP of PduL plays a role in defining the quaternary structure of the protein. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:04:25Z + + Full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:03:44Z + + sPduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:04:30Z + + 1–27 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:04:33Z + + removed + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:04:36Z + + sPduL ΔEP + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:05:26Z + + truncated + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:40Z + + monomer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:04:26Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:17Z + + rPduL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:01Z + + pPduL + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:24Z + + dimer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:04:51Z + + oligomer + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:04:54Z + + deletion + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:04:57Z + + 1–47 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:18Z + + rPduL + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:05:01Z + + 1–20 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:01Z + + pPduL + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:55:36Z + + pPduLΔEP + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:05:08Z + + trimer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:40Z + + monomer + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:05:18Z + + rPduLΔEP + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:24Z + + dimer + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:18Z + + rPduL + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:05:18Z + + rPduLΔEP + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:48:56Z + + size exclusion chromatography + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:05:33Z + + multiangle light scattering + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:05:57Z + + SEC-MALS + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:05:57Z + + SEC-MALS + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T16:05:18Z + + rPdulΔEP + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:42:24Z + + dimer + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:55:02Z + + crystal structure + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:06:36Z + + weighted average (Mw) and number average (Mn) of the molar mass + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:18Z + + rPduL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:06:45Z + + full length + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:07:03Z + + Mw + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:07:17Z + + Mn + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:07:22Z + + six subunits + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:07:48Z + + molecular weight + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + + DISCUSS + title_1 + 23441 + Discussion + + + DISCUSS + paragraph + 23452 + The hallmark attribute of an organelle is that it serves as a discrete subcellular compartment functioning as an isolated microenvironment distinct from the cytosol. In order to create and preserve this microenvironment, the defining barrier (i.e., lipid bilayer membrane or microcompartment shell) must be selectively permeable. The BMC shell not only sequesters specific enzymes but also their cofactors, thereby establishing a private cofactor pool dedicated to the encapsulated reactions. In catabolic BMCs, CoA and NAD+ must be continually recycled within the organelle (Fig 1). Homologs of the predominant cofactor utilizer (aldehyde dehydrogenase) and NAD+ regenerator (alcohol dehydrogenase) have been structurally characterized, but until now structural information was lacking for PduL, which recycles CoA in the organelle lumen. Curiously, while the housekeeping Pta could provide this function, and indeed does so in the case of one type of ethanolamine-utilizing (EUT) BMC, the evolutionarily unrelated PduL fulfills this function for the majority of metabolosomes using a novel structure and active site for convergent evolution of function. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:31:30Z + + shell + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:17:56Z + + catabolic + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:47Z + + BMCs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:11:06Z + + CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:11:09Z + + NAD+ + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:38:03Z + + aldehyde dehydrogenase + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:11:20Z + + NAD+ + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:38:28Z + + alcohol dehydrogenase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:11:30Z + + CoA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:22:56Z + + housekeeping + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T17:11:36Z + + ethanolamine-utilizing (EUT) BMC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:18:05Z + + metabolosomes + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:11:39Z + + structure + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + + DISCUSS + title_2 + 24608 + The Tertiary Structure of PduL Is Formed by Discontinuous Segments of Primary Structure + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + + DISCUSS + paragraph + 24696 + The structure of PduL consists of two β-barrel domains capped by short alpha helical segments (Fig 2b). The two domains are structurally very similar (superimposing with a rmsd of 1.34 Å (over 123 out of 320/348 aligned backbone atoms, S5a Fig). However, the amino acid sequences of the two domains are only 16% identical (mainly the RHxH motif, β2 and β10), and 34% similar. Our structure reveals that the two assigned PF06130 domains (Fig 3) do not form structurally discrete units; this reduces the apparent sequence conservation at the level of primary structure. One strand of the domain 1 beta barrel (shown in blue in Fig 2) is contributed by the N-terminus, while the rest of the domain is formed by the residues from the C-terminal half of the protein. When aligned by structure, the β1 strand of the first domain (Fig 2a and 2b, blue) corresponds to the final strand of the second domain (β9), effectively making the domains continuous if the first strand was transplanted to the C-terminus. Refined domain assignment based on our structure should be able to predict domains of PF06130 homologs much more accurately. The closest structural homolog of the PduL barrel domain is a subdomain of a multienzyme complex, the alpha subunit of ethylbenzene dehydrogenase (S5b Fig, rmsd of 2.26 Å over 226 aligned atoms consisting of one beta barrel and one capping helix). In contrast to PduL, there is only one barrel present in ethylbenzene dehydrogenase, and there is no comparable active site arrangement. The PduL signature primary structure, two PF06130 domains, occurs in some multidomain proteins, most of them annotated as Acks, suggesting that PduL may also replace Pta in variants of the phosphotransacetylase-Ack pathway. These PduL homologs lack EPs, and their fusion to Ack may have evolved as a way to facilitate substrate channeling between the two enzymes. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:15:35Z + + structure + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:15:33Z + + two β-barrel domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:15:37Z + + short alpha helical segments + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:15:40Z + + superimposing + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:45:02Z + + rmsd + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:15:45Z + + RHxH motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:15:48Z + + β2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:15:51Z + + β10 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:15:53Z + + structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:51:25Z + + PF06130 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:15:56Z + + strand + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:00Z + + domain 1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:02Z + + beta barrel + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:05Z + + C-terminal half + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:16:08Z + + protein + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:16:11Z + + aligned + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:16:13Z + + structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:17Z + + β1 strand + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:19Z + + first domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:21Z + + final strand + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:24Z + + second domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:26Z + + β9 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:16:34Z + + structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:51:25Z + + PF06130 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:37Z + + barrel domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:40Z + + alpha subunit + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:16:43Z + + ethylbenzene dehydrogenase + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:45:02Z + + rmsd + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:47Z + + beta barrel + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:49Z + + capping helix + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:16:51Z + + barrel + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:16:54Z + + ethylbenzene dehydrogenase + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:51:25Z + + PF06130 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:16:58Z + + Acks + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:38:41Z + + phosphotransacetylase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:46:04Z + + Ack + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:17:06Z + + lack + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:54Z + + EPs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:17:30Z + + their + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:46:04Z + + Ack + + + + DISCUSS + title_2 + 26587 + Implications for Metabolosome Core Assembly + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + Metabolosome + + + + DISCUSS + paragraph + 26631 + For BMC-encapsulated proteins to properly function together, they must be targeted to the lumen and assemble into an organization that facilitates substrate/product channeling among the different catalytic sites of the signature and core enzymes. The N-terminal extension on PduL homologs may serve both of these functions. The extension shares many features with previously characterized EPs: it is present only in homologs associated with BMC loci, and it is predicted to form an amphipathic α-helix. Moreover, its removal affects the oligomeric state of the protein. EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP. sPduL has also previously been reported to localize to inclusion bodies when overexpressed; we show here that this is dependent on the presence of the EP. This propensity of the EP to cause proteins to form complexes (Fig 5) might not be a coincidence, but could be a necessary step in the assembly of BMCs. Structured aggregation of the core enzymes has been proposed to be the initial step in metabolosome assembly and is known to be the first step of β-carboxysome biogenesis, where the core enzyme Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) is aggregated by the CcmM protein. Likewise, CsoS2, a protein in the α-carboxysome core, also aggregates when purified and is proposed to facilitate the nucleation and encapsulation of RuBisCO molecules in the lumen of the organelle. Coupled with protein–protein interactions with other luminal components, the aggregation of these enzymes could lead to a densely packed organelle core. This role for EPs in BMC assembly is in addition to their interaction with shell proteins. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:55:12Z + + catalytic sites + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:20:42Z + + N-terminal extension + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:20:45Z + + The extension + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:54Z + + EPs + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-21T17:20:53Z + + BMC loci + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:20:48Z + + amphipathic + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:20:51Z + + α-helix + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:20:56Z + + removal + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:04:26Z + + full-length + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:54:36Z + + propanediol dehydratase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:54:40Z + + ethanolamine ammonia-lyase + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T17:21:18Z + + PDU + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T17:21:49Z + + EUT BMCs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:03:44Z + + sPduL + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:21:53Z + + overexpressed + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:47Z + + BMCs + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + metabolosome + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:22:01Z + + Ribulose Bisphosphate Carboxylase/Oxygenase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:22:03Z + + RuBisCO + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:22:05Z + + CcmM + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:22:08Z + + CsoS2 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T17:22:10Z + + α-carboxysome + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:22:13Z + + RuBisCO + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:54Z + + EPs + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + + DISCUSS + paragraph + 28535 + Moreover, the PduL crystal structures offer a clue as to how required cofactors enter the BMC lumen during assembly. Free CoA and NAD+/H could potentially be bound to the enzymes as the core assembles and is encapsulated. However, this raises an issue of stoichiometry: if the ratio of cofactors to core enzymes is too low, then the sequestered metabolism would proceed at suboptimal rates. Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the “ground state” of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:21:32Z + + crystal structures + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:23:45Z + + CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:24:02Z + + NAD+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:24:11Z + + H + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:24:15Z + + crystals + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:24:18Z + + CoA + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:24:22Z + + Escherichia coli + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:57:04Z + + CoA-bound + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T17:24:35Z + + CoA:PduL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + metabolosome + + + + DISCUSS + title_2 + 29200 + Active Site Identification and Structural Insights into Catalysis + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + Active Site + + + + DISCUSS + paragraph + 29266 + The active site of PduL is formed at the interface of the two structural domains (Fig 2b). As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211). All of the metal-coordinating residues (Fig 2a) are absolutely conserved, implicating them in catalysis or the correct spatial orientation of the substrates. Arg103, which contacts the phosphate (Fig 4b), is present in all PduL homologs. The close resemblance between the structures binding CoA and phosphate likely indicates that no large changes in protein conformation are involved in catalysis, and that our crystal structures are representative of the active form. The native substrate for the forward reaction of rPduL and pPduL, propionyl-CoA, most likely binds to the enzyme in the same way at the observed nucleotide and pantothenic acid moiety, but the propionyl group in the CoA-thioester might point in a different direction. There is a pocket nearby the active site between the well-conserved residues Ser45 and Ala154, which could accommodate the propionyl group (S6 Fig). A homology model of sPduL indicates that the residues making up this pocket and the surrounding active site region are identical to that of rPduL, which is not surprising, because these two homologs presumably have the same propionyl-CoA substrate. The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. The catalytic mechanism of Pta involves the abstraction of a thiol hydrogen by an aspartate residue, resulting in the nucleophilic attack of thiolate upon the carbonyl carbon of acetyl-phosphate, oriented by an arginine and stabilized by a serine —there are no metals involved. In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions. These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site. + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:33:23Z + + interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:33:26Z + + domains + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:32:18Z + + highly conserved + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:33:36Z + + CoA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:33:41Z + + Ser45 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:33:47Z + + Lys70 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:33:52Z + + Arg97 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:33:57Z + + Leu99 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:56:29Z + + His204 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:34:07Z + + Asn211 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:34:11Z + + metal-coordinating residues + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:34:13Z + + absolutely conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:57:39Z + + Arg103 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:34:20Z + + phosphate + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:34:23Z + + CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:34:26Z + + phosphate + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:21:32Z + + crystal structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:05:01Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:18Z + + rPduL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:01Z + + pPduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:51:34Z + + propionyl-CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:34:42Z + + nucleotide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:34:45Z + + pantothenic acid + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:46:23Z + + CoA-thioester + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:34:54Z + + pocket + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:34:56Z + + well-conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:33:41Z + + Ser45 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:35:05Z + + Ala154 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:35:13Z + + homology model + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:03:44Z + + sPduL + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:35:15Z + + pocket + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:18Z + + rPduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:51:34Z + + propionyl-CoA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:35:22Z + + homology model + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:01Z + + pPduL + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:35:29Z + + pocket + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:35:36Z + + Gln77 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:18Z + + rPduL + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:35:40Z + + tyrosine + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:35:45Z + + Tyr77 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:01Z + + pPduL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:01Z + + pPduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:35:49Z + + lactyl-CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:51:34Z + + propionyl-CoA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:36:02Z + + aromatic + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:36:04Z + + residue + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-21T17:36:09Z + + pvm locus + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:54:45Z + + PduLs + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-21T17:36:12Z + + pvm loci + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:35:36Z + + Gln77 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:36:17Z + + Tyr + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:36:20Z + + Phe + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:36:22Z + + Gln + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:36:24Z + + Glu + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:54:49Z + + PduLs + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:36:40Z + + acetyl- + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:51:34Z + + propionyl-CoA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:36:43Z + + comparison + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:36:58Z + + aspartate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:37:22Z + + acetyl-phosphate + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:37:25Z + + arginine + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:37:28Z + + serine + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T16:04:18Z + + rPduL + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:37:30Z + + structure + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:37:33Z + + aspartate + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:37:35Z + + well-conserved + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:37:38Z + + glutamate + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:28Z + + coordinating + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:37:40Z + + acidic + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:37:42Z + + residue + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:37:45Z + + dimetal active site + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:37:50Z + + phosphate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:28Z + + acyl-CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:37Z + + acyl-phosphate + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:38:26Z + + phosphatases + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:38:34Z + + dimetal active site + + + + DISCUSS + paragraph + 32659 + Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. In the phosphate-bound crystal structure, Ser127 and Arg103 appear to position the phosphate (Fig 4b). Alternatively, Arg103 might act as a base to render the phosphate more nucleophilic. The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:40:34Z + + structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:40:37Z + + Conserved + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:40:42Z + + Ser127 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:57:39Z + + Arg103 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:40:51Z + + Arg194 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:40:56Z + + Gln107 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:55:45Z + + Gln74 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:41:44Z + + Gln + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:41:58Z + + Glu77 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:47:26Z + + phosphate-bound + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:55:07Z + + crystal structure + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:40:42Z + + Ser127 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:57:39Z + + Arg103 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:42:07Z + + phosphate + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:57:40Z + + Arg103 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:42:11Z + + phosphate + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:55:49Z + + Gln74 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:42:32Z + + Gln + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:42:45Z + + Glu77 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:40:51Z + + Arg194 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:43:09Z + + CoA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:47:26Z + + phosphate-bound + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:21:32Z + + crystal structures + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:28Z + + hydrogen bonding + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:37Z + + acyl-phosphate + + + + DISCUSS + paragraph + 33425 + The free CoA-bound form is presumably poised for attack upon an acyl-phosphate, indicating that the enzyme initially binds CoA as opposed to acyl-phosphate. This hypothesis is strengthened by the fact that the CoA-bound crystals were obtained without added CoA, indicating that the protein bound CoA from the E. coli expression strain and retained it throughout purification and crystallization. The phosphate-bound structure indicates that in the opposite reaction direction phosphate is bound first, and then an acyl-CoA enters. The two high-resolution crystal structures presented here will serve as the foundation for mechanistic studies on this noncanonical PTAC enzyme to determine how the dimetal active site functions to catalyze both forward and reverse reactions. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:57:04Z + + CoA-bound + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:37Z + + acyl-phosphate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:44:53Z + + CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:37Z + + acyl-phosphate + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:57:04Z + + CoA-bound + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:45:00Z + + crystals + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:45:02Z + + CoA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:45:07Z + + bound + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:45:09Z + + CoA + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:45:12Z + + E. coli + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T16:47:26Z + + phosphate-bound + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:45:16Z + + structure + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T17:45:19Z + + phosphate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:28:28Z + + acyl-CoA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:21:32Z + + crystal structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:45:25Z + + dimetal active site + + + + DISCUSS + title_2 + 34199 + Functional, but Not Structural, Convergence of PduL and Pta + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:29Z + + PduL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + + DISCUSS + paragraph + 34259 + PduL and Pta are mechanistically and structurally distinct enzymes that catalyze the same reaction, a prime example of evolutionary convergence upon a function. There are several examples of such functional convergence of enzymes, although typically the enzymes have independently evolved similar, or even identical active sites; for example, the carbonic anhydrase family. However, apparently less frequent is functional convergence that is supported by distinctly different active sites and accordingly catalytic mechanism, as revealed by comparison of the structures of Pta and PduL. One well-studied example of this is the β-lactamase family of enzymes, in which the active site of Class A and Class C enzymes involve serine-based catalysis, but Class B enzymes are metalloproteins. This is not surprising, as β-lactamases are not so widespread among bacteria and therefore would be expected to have evolved independently several times as a defense mechanism against β-lactam antibiotics. However, nearly all bacteria encode Pta, and it is not immediately clear why the Pta/PduL functional convergence should have evolved: it would seem to be evolutionarily more resourceful for the Pta-encoding gene to be duplicated and repurposed for BMCs, as is apparently the case in one type of BMC—EUT1 (Table 1). There could be some intrinsic biochemical difference between the two enzymes that renders PduL a more attractive candidate for encapsulation in a BMC—for example, PduL might be more amenable to tight packaging, or is better suited for the chemical microenvironment formed within the lumen of the BMC, which can be quite different from the cytosol. Further biochemical comparison between the two PTACs will likely yield exciting results that could answer this evolutionary question. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:30Z + + PduL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:55:17Z + + active sites + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:47:24Z + + carbonic anhydrase + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T17:56:41Z + + active sites + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:30Z + + PduL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:47:26Z + + β-lactamase + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:24:15Z + + active site + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:47:28Z + + metalloproteins + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:47:31Z + + β-lactamases + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:51:00Z + + bacteria + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:51:01Z + + bacteria + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:30Z + + PduL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-21T17:47:39Z + + Pta-encoding gene + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:47Z + + BMCs + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T17:47:42Z + + BMC—EUT1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:30Z + + PduL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:30Z + + PduL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T17:47:50Z + + PTACs + + + + DISCUSS + title_2 + 36058 + Implications + + + DISCUSS + paragraph + 36071 + BMCs are now known to be widespread among the bacteria and are involved in critical segments of both autotrophic and heterotrophic biochemical pathways that confer to the host organism a competitive (metabolic) advantage in select niches. As one of the three common metabolosome core enzymes, the structure of PduL provides a key missing piece to our structural picture of the shared core biochemistry (Fig 1) of functionally diverse catabolic BMCs. We have observed the oligomeric state differences of PduL to correlate with the presence of an EP, providing new insight into the function of this sequence extension in BMC assembly. Moreover, our results suggest a means for Coenzyme A incorporation during metabolosome biogenesis. A detailed understanding of the underlying principles governing the assembly and internal structural organization of BMCs is a requisite for synthetic biologists to design custom nanoreactors that use BMC architectures as a template. Furthermore, given the growing number of metabolosomes implicated in pathogenesis, the PduL structure will be useful in the development of therapeutics. It is gradually being realized that the metabolic capabilities of a pathogen are also important for virulence, along with the more traditionally cited factors like secretion systems and effector proteins. The fact that PduL is confined almost exclusively to metabolosomes can be used to develop an inhibitor that blocks only PduL and not Pta as a way to selectively disrupt BMC-based metabolism, while not affecting most commensal organisms that require PTAC activity. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:47Z + + BMCs + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:51:01Z + + bacteria + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + metabolosome + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:49:22Z + + structure + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:30Z + + PduL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T15:17:56Z + + catabolic + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:47Z + + BMCs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:30Z + + PduL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T15:56:46Z + + EP + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T15:19:43Z + + Coenzyme A + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:39:38Z + + metabolosome + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:17:47Z + + BMCs + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:18:05Z + + metabolosomes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:30Z + + PduL + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T17:49:24Z + + structure + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:30Z + + PduL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:18:05Z + + metabolosomes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:20:30Z + + PduL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:21:21Z + + Pta + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T15:37:01Z + + BMC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T15:19:20Z + + PTAC + + + + METHODS + title_1 + 37659 + Materials and Methods + + + METHODS + title_2 + 37681 + Molecular Cloning + + + METHODS + paragraph + 37699 + Genes for PduL homologs with and without the EP were amplified via PCR using the primers listed in S1 Table. sPduL was amplified using S. enterica Typhimurium LT2 genomic DNA, and pPduL and rPduL sequences were codon optimized and synthesized by GenScript with the 6xHis tag. All 5’ primers included EcoRI and BglII restriction sites, and all 3’ primers included a BamHI restriction site to facilitate cloning using the BglBricks strategy. 5’ primers also included the sequence TTTAAGAAGGAGATATACCATG downstream of the restriction sites, serving as a strong ribosome binding site. The 6x polyhistidine tag sequence was added to the 3’ end of the gene using the BglBricks strategy and was subcloned into the pETBb3 vector, a pET21b-based vector modified to be BglBricks compatible. + + + METHODS + title_2 + 38488 + Protein Purification, Size Exclusion Chromatography, and Protein Crystallization + + + METHODS + paragraph + 38569 + E. coli BL21(DE3) expression strains containing the relevant PduL construct in the pETBb3 vector were grown overnight at 37°C in standard LB medium and then used to inoculate 1L of standard LB medium in 2.8 L Fernbach flasks at a 1:100 dilution, which were then incubated at 37°C shaking at 150 rpm, until the culture reached an OD600 of 0.8–1.0, at which point cultures were induced with 200 μM IPTG (isopropylthio-β-D-galactoside) and incubated at 20°C for 18 h, shaking at 150 rpm. Cells were centrifuged at 5,000 xg for 15 min, and cell pellets were frozen at –20°C. + + + METHODS + paragraph + 39151 + For protein purifications, cell pellets from 1–3 L cultures were resuspended in 20–30 ml buffer A (50 mM Tris-HCl pH 7.4, 300 mM NaCl) and lysed using a French pressure cell at 20,000 lb/in2. The resulting cell lysate was centrifuged at 15,000 xg. 30 mM imidazole was added to the supernatant that was then applied to a 5 mL HisTrap column (GE Healthcare Bio-Sciences, Pittsburgh, PA). Protein was eluted off the column using a gradient of buffer A from 0 mM to 500 mM imidazole over 20 column volumes. Fractions corresponding to PduL were pooled and concentrated using Amicon Ultra Centrifugal filters (EMD Millipore, Billerica, MA) to a volume of no more than 2.5 mL. The protein sample was then applied to a HiLoad 26/60 Superdex 200 preparative size exclusion column (GE Healthcare Bio-Sciences, Pittsburgh, PA) and eluted with buffer B (20 mM Tris pH 7.4, 50 mM NaCl). Where applicable, fractions corresponding to different oligomeric states were pooled separately, leaving one or two fractions in between to prevent cross contamination. Pooled fractions were concentrated to 1–20 mg/mL protein as determined by the Bradford method prior to applying on a Superdex 200 10/300 GL analytical size exclusion column (GE Healthcare Bio-Sciences, Pittsburgh, PA). Size standards used were Thyroglobulin 670 kDa, γ-globulin 158 kDa, Ovalbumin 44 kDa, and Myoglobin 17 kDa. For light scattering, the proteins were measured in a Protein Solutions Dynapro dynamic light scattering instrument with an acquisition time of 5 s, averaging 10 acquisitions at a constant temperature of 25°C. The radii were calculated assuming a globular particle shape. + + + METHODS + paragraph + 40801 + Size exclusion chromatography coupled with SEC-MALS was performed on full-length rPduL and rPduL-ΔEP similar to Luzi et al. 2015. A Wyatt DAWN Heleos-II 18-angle light scattering instrument was used in tandem with a GE AKTA pure FPLC with built in UV detector, and a Wyatt Optilab T-Rex refractive index detector. Detector 16 of the DAWN Heleos-II was replaced with a Wyatt Dynapro Nanostar QELS detector for dynamic light scattering. A GE Superdex S200 10/300 GL column was used, with 125–100 μl of protein sample at 1 mg/ml concentration injected, and the column run at 0.5 ml/min in 20 mM Tris, 50 mM NaCl, pH 7.4. + + + METHODS + paragraph + 41424 + Each detector of the DAWN-Heleos-II was plotted with the Zimm model in the Wyatt ASTRA software to calculate the molar mass. The molar mass was measured at each collected data point across the peaks at ~1 point per 8 μl eluent. Both the Mw and Mn of the molar mass calculations, as well as percent deviations, were also determined using Wyatt software program ASTRA. + + + METHODS + paragraph + 41792 + For preparing protein for crystallography, expression cells were grown as above, except were induced with 50 μM IPTG. Harvested cells were resuspended in buffer B and lysed using a French Press. Cleared lysate was applied on a 5 ml HisTrap HP column (GE Healthcare) and washed with buffer A containing 20 mM imidazole. Pdul-His was eluted with 2 CV buffer B containing 300 mM imidazole, concentrated and then applied on a HiLoad 26/60 Superdex 200 (GE Healthcare) column equilibrated in buffer B for final cleanup. Protein was then concentrated to 20–30 mg/ml for crystallization. Crystals were obtained from sitting drop experiments at 22°C, mixing 3 μl of protein solution with 3 μl of reservoir solution containing 39%–35% MPD. Crystals were flash frozen in liquid nitrogen after being adding 5 μl of a reservoir solution. For heavy atom derivatives, 0.2 μl of 100 mM Thiomerosal (Hampton Research) was added to the crystallization drop 36 h prior to freezing. For phosphate soaks, 5 μl reservoir and 1.5 μl 200 mM sodium phosphate solution (pH 7.0) were added 2 d prior to flash freezing. + + + METHODS + title_2 + 42897 + PTAC Activity Assay + + + METHODS + paragraph + 42917 + Enzyme reactions were performed in a 2 mL cuvette containing 50 mM Tris-HCl pH 7.5, 0.2 mM 5,5'-dithiobis-2-nitrobenzoic acid (DTNB; Ellman’s reagent), 0.1 mM acyl-CoA, and 0.5 μg purified PTAC, unless otherwise noted. To initiate the reaction, 5 mM NaH2PO4 was added, the cuvette was inverted to mix, and the absorbance at 412 nm was measured every 2 s over the course of four minutes in a Nanodrop 2000c, in the cuvette holder. 14,150 M-1cm-1 was used as the extinction coefficient of DTNB to determine the specific activity. + + + METHODS + title_2 + 43448 + PduL Sequence Analysis + + + METHODS + paragraph + 43471 + A multiple sequence alignment of 228 PduL sequences associated with BMCs and 20 PduL sequences not associated with BMCs was constructed using MUSCLE. PduL sequences associated with BMCs were determined from Dataset S1 of Reference, and those not associated with BMCs were determined by searching for genomes that encoded PF06130 but not PF03319 nor PF00936 in the IMG database. The multiple sequence alignment was visualized in Jalview, and the nonconserved N- and C-terminal amino acids were deleted. This trimmed alignment was used to build the sequence logo using WebLogo. + + + METHODS + title_2 + 44047 + Diffraction Data Collection, Structure Determination and Visualization + + + METHODS + paragraph + 44118 + Diffraction data were collected at the Advanced Light Source at Lawrence Berkeley National Laboratory beamline 5.0.2 (100 K, 1.0000 Å wavelength for native data, 1.0093 Å for mercury derivative, 1.2861 Å for Zn pre-edge and 1.2822 Å for Zn peak). Diffraction data were integrated with XDS and scaled with SCALA (CCP4). The structure of PduL was solved using phenix.autosol, which found 11 heavy atom sites and produced density suitable for automatic model building. The model was refined with phenix.refine, with refinement alternating with model building using 2Fo-Fc and Fo-Fc maps visualized in COOT. Statistics for diffraction data collection, structure determination and refinement are summarized in Table 2. Figures were prepared using pymol (www.pymol.org) and Raster3D. + + + METHODS + title_2 + 44900 + Homology Modeling + + + METHODS + paragraph + 44918 + Models of S. enterica Typhimurium LT2 and P. limnophilus PduL were generated with Modeller using the align2d and model-default scripts. + + + SUPPL + title_1 + 45054 + Supporting Information + + + ABBR + title + 45077 + Abbreviations + + + ABBR + paragraph + 45091 + Ack + + + ABBR + paragraph + 45095 + acetate kinase + + + ABBR + paragraph + 45110 + BMC + + + ABBR + paragraph + 45114 + Bacterial Microcompartment + + + ABBR + paragraph + 45141 + EP + + + ABBR + paragraph + 45144 + encapsulation peptide + + + ABBR + paragraph + 45166 + EUT + + + ABBR + paragraph + 45170 + ethanolamine-utilizing + + + ABBR + paragraph + 45193 + Mn + + + ABBR + paragraph + 45196 + number average + + + ABBR + paragraph + 45211 + Mw + + + ABBR + paragraph + 45214 + weighted average + + + ABBR + paragraph + 45231 + PDU + + + ABBR + paragraph + 45235 + propanediol-utilizing + + + ABBR + paragraph + 45257 + Pta + + + ABBR + paragraph + 45261 + phosphotransacylase + + + ABBR + paragraph + 45281 + PTAC + + + ABBR + paragraph + 45286 + phosphotransacylase + + + ABBR + paragraph + 45306 + RuBisCO + + + ABBR + paragraph + 45314 + Ribulose Bisphosphate Carboxylase/Oxygenase + + + ABBR + paragraph + 45358 + SEC-MALS + + + ABBR + paragraph + 45367 + multiangle light scattering + + + REF + title + 45395 + References + + + 22 + 1 + 34 + surname:Kerfeld;given-names:CA + surname:Erbilgin;given-names:O + 10.1016/j.tim.2014.10.003 + 25455419 + REF + Trends in microbiology + ref + 23 + 2015 + 45406 + Bacterial microcompartments and the modular construction of microbial metabolism + + + e1003898 + 10 + surname:Axen;given-names:SD + surname:Erbilgin;given-names:O + surname:Kerfeld;given-names:CA + 10.1371/journal.pcbi.1003898 + 25340524 + REF + PLoS Comput Biol + ref + 10 + 2014 + 45487 + A taxonomy of bacterial microcompartment loci constructed by a novel scoring method + + + surname:Liu;given-names:Y + surname:Jorda;given-names:J + surname:Yeates;given-names:TO + surname:Bobik;given-names:TA + 10.1128/JB.00056-15 + 25962918 + REF + Journal of bacteriology + ref + 2015 + 45571 + The PduL phosphotransacylase is used to recycle coenzyme A within the Pdu microcompartment + + + 1589 + 5 + 96 + surname:Liu;given-names:Y + surname:Leal;given-names:NA + surname:Sampson;given-names:EM + surname:Johnson;given-names:CLV + surname:Havemann;given-names:GD + surname:Bobik;given-names:Ta + 10.1128/JB.01151-06 + 17158662 + REF + Journal of bacteriology + ref + 189 + 2007 + 45662 + PduL is an Evolutionarily Distinct Phosphotransacylase Involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar typhimurium LT2 + + + 936 + 8 + surname:Kerfeld;given-names:CA + surname:Sawaya;given-names:MR + surname:Tanaka;given-names:S + surname:Nguyen;given-names:CV + surname:Phillips;given-names:M + surname:Beeby;given-names:M + 10.1126/science.1113397 + REF + Science (New York, NY) + ref + 309 + 2005 + 45818 + Protein structures forming the shell of primitive bacterial organelles + + + 1642 + 12 + 52 + surname:Pang;given-names:A + surname:Liang;given-names:M + surname:Prentice;given-names:MB + surname:Pickersgill;given-names:RW + 10.1107/S0907444912039315 + 23151629 + REF + Acta Crystallographica Section D Biological Crystallography + ref + 68 + 2012 + 45889 + Substrate channels revealed in the trimeric Lactobacillus reuteri bacterial microcompartment shell protein PduB + + + 5967 + 19 + 75 + surname:Bobik;given-names:TA + surname:Havemann;given-names:GD + surname:Busch;given-names:RJ + surname:Williams;given-names:DS + surname:Aldrich;given-names:HC + 10498708 + REF + Journal of bacteriology + ref + 181 + 1999 + 46001 + The Propanediol Utilization (pdu) Operon of Salmonella enterica serovar Typhimurium LT2 Includes Genes Necessary for Formation of Polyhedral Organelles Involved in coenzyme B12-Dependent 1, 2-Propanediol Degradation + + + 1000118 + 4 + surname:Abdul-Rahman;given-names:F + surname:Petit;given-names:E + surname:Blanchard;given-names:JL + 10.4172/2329-9002.1000118 + REF + Phylogenetics and Evolutionary Biology + ref + 1 + 2013 + 46217 + The Distribution of Polyhedral Bacterial Microcompartments Suggests Frequent Horizontal Transfer and Operon Reassembly + + + 179 + 2 + 95 + surname:Jorda;given-names:J + surname:Lopez;given-names:D + surname:Wheatley;given-names:NM + surname:Yeates;given-names:TO + 10.1002/pro.2196 + 23188745 + REF + Protein Science + ref + 22 + 2013 + 46336 + Using comparative genomics to uncover new kinds of protein-based metabolic organelles in bacteria + + + 3855 + 9 + 63 + surname:Roof;given-names:DM + surname:Roth;given-names:JR + 3045078 + REF + Journal of bacteriology + ref + 170 + 1988 + 46434 + Ethanolamine utilization in Salmonella typhimurium + + + 353 + 5 + 61 + surname:Leal;given-names:NA + surname:Havemann;given-names:GD + surname:Bobik;given-names:TA + 10.1007/s00203-003-0601-0 + 14504694 + REF + Archives of microbiology + ref + 180 + 2003 + 46486 + PduP is a coenzyme-a-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2 + + + 2864 + 12 + 79 + surname:Huseby;given-names:DL + surname:Roth;given-names:JR + 10.1128/JB.02179-12 + 23585538 + REF + Journal of bacteriology + ref + 195 + 2013 + 46686 + Evidence that a metabolic microcompartment contains and recycles private cofactor pools + + + e47144 + 10 + e + surname:Cheng;given-names:S + surname:Fan;given-names:C + surname:Sinha;given-names:S + surname:Bobik;given-names:TA + 10.1371/journal.pone.0047144 + 23077559 + REF + PloS ONE + ref + 7 + 2012 + 46774 + The PduQ enzyme is an alcohol dehydrogenase used to recycle NAD+ internally within the Pdu microcompartment of Salmonella enterica + + + surname:White;given-names:D + REF + The Physiology and Biochemistry of Prokaryotes + ref + 2000 + 46905 + + + 18392 + 31 + 6 + surname:Lundie;given-names:LL;suffix:Jr. + surname:Ferry;given-names:JG + 2808380 + REF + J Biol Chem + ref + 264 + 1989 + 46906 + Activation of acetate by Methanosarcina thermophila. Purification and characterization of phosphotransacetylase + + + 25 + 1 + 35 + surname:Ferry;given-names:JG + 9233537 + REF + Biofactors + ref + 6 + 1997 + 47018 + Enzymology of the fermentation of acetate to methane by Methanosarcina thermophila + + + 387 + 4 + 97 + surname:Tielens;given-names:AG + surname:van Grinsven;given-names:KW + surname:Henze;given-names:K + surname:van Hellemond;given-names:JJ + surname:Martin;given-names:W + 10.1016/j.ijpara.2009.12.006 + 20085767 + REF + Int J Parasitol + ref + 40 + 2010 + 47101 + Acetate formation in the energy metabolism of parasitic helminths and protists + + + D115 + Database issue + 22 + surname:Markowitz;given-names:VM + surname:Chen;given-names:IMA + surname:Palaniappan;given-names:K + surname:Chu;given-names:K + surname:Szeto;given-names:E + surname:Grechkin;given-names:Y + 10.1093/nar/gkr1044 + 22194640 + REF + Nucleic acids research + ref + 40 + 2012 + 47180 + IMG: the Integrated Microbial Genomes database and comparative analysis system + + + D222 + Database issue + 30 + surname:Finn;given-names:RD + surname:Bateman;given-names:A + surname:Clements;given-names:J + surname:Coggill;given-names:P + surname:Eberhardt;given-names:RY + surname:Eddy;given-names:SR + 10.1093/nar/gkt1223 + 24288371 + REF + Nucleic acids research + ref + 42 + 2014 + 47259 + Pfam: the protein families database + + + D290 + Database issue + 301 + surname:Punta;given-names:M + surname:Coggill;given-names:PC + surname:Eberhardt;given-names:RY + surname:Mistry;given-names:J + surname:Tate;given-names:J + surname:Boursnell;given-names:C + 10.1093/nar/gkr1065 + 22127870 + REF + Nucleic acids research + ref + 40 + 2012 + 47295 + The Pfam protein families database + + + 527 + 2 + 34 + surname:Stadtman;given-names:ER + 12980995 + REF + J Biol Chem + ref + 196 + 1952 + 47330 + The purification and properties of phosphotransacetylase + + + 114 + 1 + 25 + surname:Rado;given-names:TA + surname:Hoch;given-names:JA + 4201530 + REF + Biochim Biophys Acta + ref + 321 + 1973 + 47387 + Phosphotransacetylase from Bacillus subtilis: purification and physiological studies + + + 1657 + 11 + 64 + surname:Smith;given-names:JE + surname:Ng;given-names:WS + 4263885 + REF + Can J Microbiol + ref + 18 + 1972 + 47472 + Fluorometric determination of glycolytic intermediates and adenylates during sequential changes in replacement culture of Aspergillus niger + + + e1039755 + 3 + surname:Aussignargues;given-names:C + surname:Paasch;given-names:BC + surname:Gonzales-Esquer;given-names:R + surname:Erbilgin;given-names:O + surname:Kerfeld;given-names:CA + 26478774 + REF + Communicative & Integrative Biology + ref + 8 + 2015 + 47612 + Bacterial Microcompartment Assembly: The Key Role of Encapsulation Peptides + + + 14995 + 37 + 5000 + surname:Fan;given-names:C + surname:Cheng;given-names:S + surname:Sinha;given-names:S + surname:Bobik;given-names:Ta + 10.1073/pnas.1207516109 + 22927404 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 109 + 2012 + 47688 + Interactions between the termini of lumen enzymes and shell proteins mediate enzyme encapsulation into bacterial microcompartments + + + 17729 + 21 + 36 + surname:Kinney;given-names:JN + surname:Salmeen;given-names:A + surname:Cai;given-names:F + surname:Kerfeld;given-names:CA + 10.1074/jbc.M112.355305 + 22461622 + REF + The Journal of biological chemistry + ref + 287 + 2012 + 47819 + Elucidating the essential role of the conserved carboxysomal protein CcmN reveals a common feature of bacterial microcompartment assembly + + + 455 + 3 + 62 + surname:Tobimatsu;given-names:T + surname:Kawata;given-names:M + surname:Toraya;given-names:T + surname:Obimatsu;given-names:TT + surname:Awata;given-names:MK + surname:Oraya;given-names:TT + 15784971 + REF + Bioscience, Biotechnology, and Biochemistry + ref + 69 + 2005 + 47957 + The N-Terminal Regions of β and γ Subunits Lower the Solubility of Adenosylcobalamin-Dependent Diol Dehydratase + + + 26484 + 34 + 93 + surname:Shibata;given-names:N + surname:Tamagaki;given-names:H + surname:Hieda;given-names:N + surname:Akita;given-names:K + surname:Komori;given-names:H + surname:Shomura;given-names:Y + 10.1074/jbc.M110.125112 + 20519496 + REF + The Journal of biological chemistry + ref + 285 + 2010 + 48076 + Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates + + + 273 + 2 + 9 + surname:Frank;given-names:S + surname:Lawrence;given-names:AD + surname:Prentice;given-names:MB + surname:Warren;given-names:MJ + 10.1016/j.jbiotec.2012.09.002 + 22982517 + REF + Journal of biotechnology + ref + 163 + 2013 + 48176 + Bacterial microcompartments moving into a synthetic biological world + + + surname:Gonzalez-Esquer;given-names:CR + surname:Shubitowski;given-names:TB + surname:Kerfeld;given-names:CA + REF + Plant Cell + ref + 48245 + Streamlined construction of the cyanobacterial CO2-fixing organelle via protein domain fusions + + + 1143 + 3 + 54 + surname:Lawrence;given-names:SH + surname:Luther;given-names:KB + surname:Schindelin;given-names:H + surname:Ferry;given-names:JG + 10.1128/JB.188.3.1143-1154.2006 + 16428418 + REF + J Bacteriol + ref + 188 + 2006 + 48340 + Structural and functional studies suggest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophila + + + 2193 + 7 + 205 + surname:Erbilgin;given-names:O + surname:McDonald;given-names:KL + surname:Kerfeld;given-names:Ca + 10.1128/AEM.03887-13 + 24487526 + REF + Applied and environmental microbiology + ref + 80 + 2014 + 48466 + Characterization of a planctomycetal organelle: a novel bacterial microcompartment for the aerobic degradation of plant saccharides + + + 57 + 1 + 63 + surname:Chakrabarti;given-names:P + 2290835 + REF + Protein engineering + ref + 4 + 1990 + 48598 + Geometry of interaction of metal ions with histidine residues in protein structures + + + 2392 + 14 + 9 + surname:Liu;given-names:Y + surname:Jorda;given-names:J + surname:Yeates;given-names:TO + surname:Bobik;given-names:TA + 10.1128/JB.00056-15 + 25962918 + REF + J Bacteriol + ref + 197 + 2015 + 48682 + The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment + + + 1377 + 9 + 88 + surname:Kloer;given-names:DP + surname:Hagel;given-names:C + surname:Heider;given-names:J + surname:Schulz;given-names:GE + 10.1016/j.str.2006.07.001 + 16962969 + REF + Structure + ref + 14 + 2006 + 48773 + Crystal structure of ethylbenzene dehydrogenase from Aromatoleum aromaticum + + + 1141 + 2 + 71 + surname:Cai;given-names:F + surname:Dou;given-names:Z + surname:Bernstein;given-names:SL + surname:Leverenz;given-names:R + surname:Williams;given-names:EB + surname:Heinhorst;given-names:S + 10.3390/life5021141 + 25826651 + REF + Life (Basel) + ref + 5 + 2015 + 48849 + Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component + + + 1131 + 5 + 40 + surname:Cameron;given-names:JC + surname:Wilson;given-names:SC + surname:Bernstein;given-names:SL + surname:Kerfeld;given-names:CA + 10.1016/j.cell.2013.10.044 + 24267892 + REF + Cell + ref + 155 + 2013 + 48973 + Biogenesis of a Bacterial Organelle: The Carboxysome Assembly Pathway + + + 140130103032007 + surname:Lawrence;given-names:AD + surname:Frank;given-names:S + surname:Newnham;given-names:S + surname:Lee;given-names:MJ + surname:Brown;given-names:IR + surname:Xue;given-names:W-F + 10.1021/sb4001118 + REF + ACS Synthetic Biology + ref + 2014 + 49043 + Solution structure of a bacterial microcompartment targeting peptide and its application in the construction of an ethanol bioreactor + + + 3252 + 8 + 78 + surname:Cleland;given-names:WW + surname:Hengge;given-names:AC + 10.1021/cr050287o + 16895327 + REF + Chem Rev + ref + 106 + 2006 + 49177 + Enzymatic mechanisms of phosphate and sulfate transfer + + + 1407 + 7 + 18 + surname:Kimber;given-names:MS + surname:Pai;given-names:EF + 10.1093/emboj/19.7.1407 + 10747009 + REF + EMBO J + ref + 19 + 2000 + 49232 + The active site architecture of Pisum sativum beta-carbonic anhydrase is a mirror image of that of alpha-carbonic anhydrases + + + 48615 + 52 + 8 + surname:Tripp;given-names:BC + surname:Smith;given-names:K + surname:Ferry;given-names:JG + 10.1074/jbc.R100045200 + 11696553 + REF + J Biol Chem + ref + 276 + 2001 + 49357 + Carbonic anhydrase: new insights for an ancient enzyme + + + 4914 + 20 + 21 + surname:Carfi;given-names:A + surname:Pares;given-names:S + surname:Duee;given-names:E + surname:Galleni;given-names:M + surname:Duez;given-names:C + surname:Frere;given-names:JM + 7588620 + REF + EMBO J + ref + 14 + 1995 + 49412 + The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold + + + 160 + 1 + 201 + surname:Drawz;given-names:SM + surname:Bonomo;given-names:RA + 10.1128/CMR.00037-09 + 20065329 + REF + Clin Microbiol Rev + ref + 23 + 2010 + 49519 + Three decades of beta-lactamase inhibitors + + + 2455 + 6 + 60 + surname:Peña;given-names:KL + surname:Castel;given-names:SE + surname:de Araujo;given-names:C + surname:Espie;given-names:GS + surname:Kimber;given-names:MS + 10.1073/pnas.0910866107 + 20133749 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 107 + 2010 + 49562 + Structural basis of the oxidative activation of the carboxysomal gamma-carbonic anhydrase, CcmM + + + e76127 + 9 + e + surname:Chen;given-names:AH + surname:Robinson-Mosher;given-names:A + surname:Savage;given-names:DF + surname:Silver;given-names:Pa + surname:Polka;given-names:JK + 10.1371/journal.pone.0076127 + 24023971 + REF + PloS ONE + ref + 8 + 2013 + 49658 + The bacterial carbon-fixing organelle is formed by shell envelopment of preassembled cargo + + + 4105 + 10 + 21 + surname:Harvey;given-names:PC + surname:Watson;given-names:M + surname:Hulme;given-names:S + surname:Jones;given-names:Ma + surname:Lovell;given-names:M + surname:Berchieri;given-names:A + 10.1128/IAI.01390-10 + 21768276 + REF + Infection and immunity + ref + 79 + 2011 + 49749 + Salmonella enterica typhimurium colonizing the lumen of the chicken intestine grows slowly and upregulates a unique set of virulence and metabolism genes + + + 556 + 2 + 68 + surname:Joseph;given-names:B + surname:Przybilla;given-names:K + surname:Stu;given-names:C + surname:Schauer;given-names:K + surname:Fuchs;given-names:TM + surname:Goebel;given-names:W + 16385046 + REF + Journal of bacteriology + ref + 188 + 2006 + 49904 + Identification of Listeria monocytogenes Genes Contributing to Intracellular Replication by Expression Profiling and Mutant Screening + + + 1 + 3 + 10 + surname:Kendall;given-names:MM + surname:Gruber;given-names:CC + surname:Parker;given-names:CT + surname:Sperandio;given-names:V + 10.1128/mBio.00050-12 + REF + mBio + ref + 3 + 2012 + 50038 + Ethanolamine controls expression of genes encoding components involved in interkingdom signaling and virulence in enterohemorrhagic Escherichia coli O157:H7 + + + 1207 + 20 + surname:Klumpp;given-names:J + surname:Fuchs;given-names:TM + REF + Microbiology + ref + 2 + 2007 + 50195 + Identification of novel genes in genomic islands that contribute to Salmonella typhimurium replication in macrophages + + + 2634 + 5 + 7 + surname:Maadani;given-names:A + surname:Fox;given-names:KA + surname:Mylonakis;given-names:E + surname:Garsin;given-names:DA + 10.1128/IAI.01372-06 + 17307944 + REF + Infection and immunity + ref + 75 + 2007 + 50313 + Enterococcus faecalis mutations affecting virulence in the Caenorhabditis elegans model host + + + 239 + 6 + 46 + surname:Mobley;given-names:H + REF + Microbe + ref + 10 + 2015 + 50407 + Redefining Virulence of Bacterial Pathogens + + + 248 + 54 + surname:Bradford;given-names:MM + 942051 + REF + Anal Biochem + ref + 72 + 1976 + 50451 + A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding + + + 45 + 2 + 52 + surname:Luzi;given-names:S + surname:Kondo;given-names:Y + surname:Bernard;given-names:E + surname:Stadler;given-names:LK + surname:Vaysburd;given-names:M + surname:Winter;given-names:G + 10.1093/protein/gzu055 + 25614525 + REF + Protein Eng Des Sel + ref + 28 + 2015 + 50583 + Subunit disassembly and inhibition of TNFalpha by a semi-synthetic bicyclic peptide + + + 1792 + 5 + 7 + surname:Edgar;given-names:RC + 10.1093/nar/gkh340 + 15034147 + REF + Nucleic acids research + ref + 32 + 2004 + 50667 + MUSCLE: multiple sequence alignment with high accuracy and high throughput + + + 1189 + 9 + 91 + surname:Waterhouse;given-names:AM + surname:Procter;given-names:JB + surname:Martin;given-names:DMa + surname:Clamp;given-names:M + surname:Barton;given-names:GJ + 10.1093/bioinformatics/btp033 + REF + Bioinformatics (Oxford, England) + ref + 25 + 2009 + 50742 + Jalview Version 2—a multiple sequence alignment editor and analysis workbench + + + 1188 + 6 + 90 + surname:Crooks;given-names:GE + surname:Hon;given-names:G + surname:Chandonia;given-names:JM + surname:Brenner;given-names:SE + 10.1101/gr.849004 + 15173120 + REF + Genome Res + ref + 14 + 2004 + 50822 + WebLogo: a sequence logo generator + + + 125 + Pt 2 + 32 + surname:Kabsch;given-names:W + 10.1107/S0907444909047337 + 20124692 + REF + Acta crystallographica Section D, Biological crystallography + ref + 66 + 2010 + 50857 + XDS + + + 235 + Pt 4 + 42 + surname:Winn;given-names:MD + surname:Ballard;given-names:CC + surname:Cowtan;given-names:KD + surname:Dodson;given-names:EJ + surname:Emsley;given-names:P + surname:Evans;given-names:PR + 10.1107/S0907444910045749 + 21460441 + REF + Acta crystallographica Section D, Biological crystallography + ref + 67 + 2011 + 50861 + Overview of the CCP4 suite and current developments + + + 352 + 67 + surname:Afonine;given-names:PV + surname:Grosse-Kunstleve;given-names:RW + surname:Echols;given-names:N + surname:Headd;given-names:JJ + surname:Moriarty;given-names:NW + surname:Mustyakimov;given-names:M + 10.1107/S0907444912001308 + 22505256 + REF + Acta Crystallogr D + ref + 68 + 2012 + 50913 + Towards automated crystallographic structure refinement with phenix.refine + + + 2126 + Pt 12 Pt 1 + 32 + surname:Emsley;given-names:P + surname:Cowtan;given-names:K + 10.1107/S0907444904019158 + 15572765 + REF + Acta crystallographica Section D, Biological crystallography + ref + 60 + 2004 + 50988 + Coot: model-building tools for molecular graphics + + + 505 + 24 + surname:Merritt;given-names:EA + surname:Bacon;given-names:DJ + 18488322 + REF + Methods in enzymology + ref + 277 + 1997 + 51038 + Raster3D: photorealistic molecular graphics + + + 291 + 325 + surname:Marti-Renom;given-names:MA + surname:Stuart;given-names:AC + surname:Fiser;given-names:A + surname:Sanchez;given-names:R + surname:Melo;given-names:F + surname:Sali;given-names:A + 10.1146/annurev.biophys.29.1.291 + 10940251 + REF + Annu Rev Biophys Biomol Struct + ref + 29 + 2000 + 51082 + Comparative protein structure modeling of genes and genomes + + + diff --git a/BioC_XML/4786784_v0.xml b/BioC_XML/4786784_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..92a3c2c022824db5b1d8abfb8b5e8c609b59d08f --- /dev/null +++ b/BioC_XML/4786784_v0.xml @@ -0,0 +1,13915 @@ + + + + PMC + 20140719 + pmc.key + + 4786784 + CC BY + no + 0 + 0 + + 10.1038/ncomms10950 + ncomms10950 + 4786784 + 26952537 + 10950 + This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ + surname:Agrawal;given-names:Anant A. + surname:Salsi;given-names:Enea + surname:Chatrikhi;given-names:Rakesh + surname:Henderson;given-names:Steven + surname:Jenkins;given-names:Jermaine L. + surname:Green;given-names:Michael R. + surname:Ermolenko;given-names:Dmitri N. + surname:Kielkopf;given-names:Clara L. + TITLE + front + 7 + 2016 + 0 + An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:25Z + + extended + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:09:24Z + + U2AF65–RNA-binding domain + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:09:33Z + + 3′ splice site + + + + ABSTRACT + abstract + 79 + How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:12:17Z + + pre-mRNA splicing factor + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:12:38Z + + polypyrimidine + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:12:46Z + + Py + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:12:54Z + + 3′ splice sites + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:18:14Z + + human + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:52:31Z + + determined four structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:25Z + + extended + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:09:24Z + + U2AF65–RNA-binding domain + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:43:11Z + + Py-tract oligonucleotides + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:47Z + + structures + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:13:51Z + + RNA binding and splicing assays + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:13:56Z + + inter-domain residues + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:39:02Z + + contiguous + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:49Z + + nucleotide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:09Z + + Py tract + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:21Z + + linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:29Z + + RNA recognition motifs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:17:00Z + + RRM extensions + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:17:09Z + + 3′ terminus + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:17:33Z + + third + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:17:19Z + + Single-molecule FRET + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:25:08Z + + Py-tract + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:21:03Z + + splice site + + + + ABSTRACT + abstract + 1090 + The pre-mRNA splicing factor U2AF65 recognizes 3′ splice sites in human gene transcripts, but the details are not fully understood. Here, the authors report U2AF65 structures and single molecule FRET that reveal mechanistic insights into splice site recognition. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:12:17Z + + pre-mRNA splicing factor + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:12:54Z + + 3′ splice sites + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:18:14Z + + human + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:18:19Z + + structures + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:18:27Z + + single molecule FRET + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:21:03Z + + splice site + + + + INTRO + paragraph + 1356 + The differential skipping or inclusion of alternatively spliced pre-mRNA regions is a major source of diversity for nearly all human gene transcripts. The splice sites are marked by relatively short consensus sequences and are regulated by additional pre-mRNA motifs (reviewed in ref.). At the 3′ splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3′ splice site junction. Disease-causing mutations often compromise pre-mRNA splicing (reviewed in refs), yet a priori predictions of splice sites and the consequences of their mutations are challenged by the brevity and degeneracy of known splice site sequences. High-resolution structures of intact splicing factor–RNA complexes would offer key insights regarding the juxtaposition of the distinct splice site consensus sequences and their relationship to disease-causing point mutations. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T13:49:28Z + + pre-mRNA regions + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:18:14Z + + human + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:19:52Z + + splice sites + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:20:09Z + + short consensus sequences + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:20:13Z + + pre-mRNA motifs + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:09:33Z + + 3′ splice site + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:20:18Z + + polypyrimidine (Py) tract + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:20:24Z + + U + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:20:27Z + + C + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:20:05Z + + branch point sequence + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:24:05Z + + BPS + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:20:35Z + + AG-dinucleotide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:09:33Z + + 3′ splice site + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:50:01Z + + pre-mRNA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:19:52Z + + splice sites + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:21:03Z + + splice site + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:20:45Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:30:59Z + + intact + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:20:56Z + + splicing factor–RNA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:21:03Z + + splice site + + + + INTRO + paragraph + 2410 + The early-stage pre-mRNA splicing factor U2AF65 is essential for viability in vertebrates and other model organisms (for example, ref.). A tightly controlled assembly among U2AF65, the pre-mRNA, and partner proteins sequentially identifies the 3′ splice site and promotes association of the spliceosome, which ultimately accomplishes the task of splicing. Initially U2AF65 recognizes the Py-tract splice site signal. In turn, the ternary complex of U2AF65 with SF1 and U2AF35 identifies the surrounding BPS and 3′ splice site junctions. Subsequently U2AF65 recruits the U2 small nuclear ribonucleoprotein particle (snRNP) and ultimately dissociates from the active spliceosome. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:12:17Z + + pre-mRNA splicing factor + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:24:20Z + + vertebrates + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:24:24Z + + assembly + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:24:29Z + + pre-mRNA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:09:33Z + + 3′ splice site + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:26:09Z + + spliceosome + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:25:06Z + + Py-tract + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:21:03Z + + splice site + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:26:18Z + + ternary complex + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:25:48Z + + SF1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:25:51Z + + U2AF35 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:24:06Z + + BPS + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:09:33Z + + 3′ splice site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:25:43Z + + U2 small nuclear ribonucleoprotein particle + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:25:55Z + + snRNP + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:26:02Z + + active + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:26:09Z + + spliceosome + + + + INTRO + paragraph + 3092 + Biochemical characterizations of U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract (Fig. 1a). Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. As such, the molecular mechanisms for Py-tract recognition by the intact U2AF65–RNA-binding domain remained unknown. Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs. We use single-molecule Förster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65–RNA-binding domain during Py-tract recognition. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:29:00Z + + Biochemical characterizations + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:29Z + + RNA recognition motifs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:14Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:09Z + + Py tract + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:39Z + + crystal structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:47Z + + core + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:14Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:57Z + + shortened + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:13Z + + dU2AF651,2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:16:43Z + + NMR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:16:52Z + + structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:08Z + + side-by-side + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:35:25Z + + minimal + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:14Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:48:49Z + + linker + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:48:19Z + + natural length + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:13Z + + dU2AF651,2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:39Z + + crystal structures + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-06-15T11:52:42Z + + RNA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:25:08Z + + Py-tract + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:30:59Z + + intact + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:09:24Z + + U2AF65–RNA-binding domain + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:10Z + + X-ray crystallography + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:13Z + + biochemical studies + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:25:08Z + + Py-tract + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:47Z + + core + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:17Z + + single-molecule Förster resonance energy transfer + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:24Z + + smFRET + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:31:30Z + + conformational dynamics + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:25Z + + extended + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:09:24Z + + U2AF65–RNA-binding domain + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:25:08Z + + Py-tract + + + + RESULTS + title_1 + 4200 + Results + + + RESULTS + title_2 + 4208 + Cognate U2AF65–Py-tract recognition requires RRM extensions + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:25:08Z + + Py-tract + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:17:00Z + + RRM extensions + + + + RESULTS + paragraph + 4270 + The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). Historically, this difference was attributed to the U2AF65 arginine–serine rich domain, which contacts pre-mRNA–U2 snRNA duplexes outside of the Py tract. We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141–342; Fig. 1a). In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h). + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:10Z + + RNA affinity + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:35:25Z + + minimal + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:47Z + + core + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:14Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:48:54Z + + folds + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:22Z + + 148–336 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:30Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:34:43Z + + arginine–serine rich domain + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:34:50Z + + pre-mRNA–U2 snRNA duplexes + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:09Z + + Py tract + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:35:03Z + + RNA-binding affinity + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:14Z + + addition of seven and six residues + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:35:25Z + + minimal + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:14Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:39Z + + U2AF651,2L + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:35:47Z + + 141–342 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:50Z + + fluorescence anisotropy assay + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:09Z + + Py tract + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:21:04Z + + splice site + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:35:56Z + + adenovirus major late promoter + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:15Z + + AdML + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:10Z + + RNA affinity + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:39Z + + U2AF651,2L + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:30Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:39Z + + U2AF651,2L + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:30Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:26Z + + U2AF65 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:36:28Z + + sequence specificity + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:36:32Z + + U-rich stretches + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:36:40Z + + 5′-region + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:09Z + + Py tract + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:36:45Z + + C-rich regions + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:36:52Z + + 3′-region + + + + RESULTS + title_2 + 5425 + U2AF65-bound Py tract comprises nine contiguous nucleotides + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:37:53Z + + U2AF65-bound + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:09Z + + Py tract + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:39:00Z + + contiguous + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:50:55Z + + nucleotides + + + + RESULTS + paragraph + 5485 + To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1). By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. The protein and oligonucleotide conformations are nearly identical among the four new U2AF651,2L structures (Supplementary Fig. 2a). The U2AF651,2L RRM1 and RRM2 associate with the Py tract in a parallel, side-by-side arrangement (shown for representative structure iv in Fig. 2b,c; Supplementary Movie 1). An extended conformation of the U2AF65 inter-RRM linker traverses across the α-helical surface of RRM1 and the central β-strands of RRM2 and is well defined in the electron density (Fig. 2b). The extensions at the N terminus of RRM1 and C terminus of RRM2 adopt well-ordered α-helices. Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide. We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:39:03Z + + contiguous + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:09Z + + Py tract + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:42:55Z + + determined + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:39Z + + crystal structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:39Z + + U2AF651,2L + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:43:11Z + + Py-tract oligonucleotides + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:43:16Z + + sequential boot strapping + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:43:21Z + + oligonucleotide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:43:27Z + + Br-dU + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:44:02Z + + rU + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:44:11Z + + dU + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:44:18Z + + rC + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:39Z + + U2AF651,2L + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:39:03Z + + contiguous + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:00Z + + Py tracts + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:44:37Z + + oligonucleotide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:39Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:44:41Z + + structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:39Z + + U2AF651,2L + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:14Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:09Z + + Py tract + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:01Z + + parallel + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:08Z + + side-by-side + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:14Z + + extended conformation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:45:21Z + + α-helical surface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:14Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:45:33Z + + β-strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:40Z + + electron density + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:45:45Z + + extensions + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:14Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:45:51Z + + α-helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:45:59Z + + extensions + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:39Z + + U2AF651,2L + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:46:10Z + + bound + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:46:07Z + + oligonucleotide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:39Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:46:20Z + + structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:13Z + + dU2AF651,2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:46:29Z + + crystal structure + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:17:19Z + + NMR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:17:27Z + + structure + + + + RESULTS + paragraph + 6853 + The discovery of nine U2AF65-binding sites for contiguous Py-tract nucleotides was unexpected. Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures. Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3′-terminal nucleotide (Fig. 2c; Supplementary Movie 1). The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. Qualitatively, a subset of the U2AF651,2L-nucleotide-binding sites (sites 1–3 and 7–9) share similar locations to those of the dU2AF651,2 structures (Supplementary Figs 2c,d and 3). Yet, only the U2AF651,2L interactions at sites 1 and 7 are nearly identical to those of the dU2AF651,2 structures (Supplementary Fig. 3a,f). In striking departures from prior partial views, the U2AF651,2L structures reveal three unanticipated nucleotide-binding sites at the centre of the Py tract, as well as numerous new interactions that underlie cognate recognition of the Py tract (Fig. 3a–h). + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:50:02Z + + U2AF65-binding sites + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:39:03Z + + contiguous + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:50:08Z + + Py-tract nucleotides + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:13Z + + dU2AF651,2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:50:12Z + + structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:35:25Z + + minimal + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:50:22Z + + nucleotides + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:50:25Z + + structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:50:33Z + + RRM2 extension + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:53:30Z + + nucleotide-binding sites + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:50:53Z + + RRM1/RRM2 junction + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:51:02Z + + RRM1 extension + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:39Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:51:33Z + + structures + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:51:37Z + + ribose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:51:40Z + + r + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:51:45Z + + nucleotides + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:51:50Z + + binding sites + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:02Z + + seventh + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:08Z + + eighth + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:52:16Z + + deoxy-(d)U + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:31Z + + RNA-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:36Z + + structure + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:52:45Z + + U2AF651,2L-nucleotide-binding sites + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:52:50Z + + sites 1–3 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:52:54Z + + 7–9 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:58Z + + structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-06-15T11:53:56Z + + U2AF651,2L + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:53:06Z + + sites 1 and 7 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:53:10Z + + structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:53:18Z + + structures + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:53:30Z + + nucleotide-binding sites + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:09Z + + Py tract + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:09Z + + Py tract + + + + RESULTS + title_2 + 8134 + U2AF65 inter-RRM linker interacts with the Py tract + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:09Z + + Py tract + + + + RESULTS + paragraph + 8186 + The U2AF651,2L RRM2, the inter-RRM linker and RRM1 concomitantly recognize the three central nucleotides of the Py tract, which are likely to coordinate the conformational arrangement of these disparate portions of the protein. Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d). The backbone amide of the linker V254 and the carbonyl of T252 engage in hydrogen bonds with the rU5-O4 and -N3H atoms. In the C-terminal β-strand of RRM1, the side chains of K225 and R227 donate additional hydrogen bonds to the rU5-O2 lone pair electrons. The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c). Otherwise, the rU4 nucleotide packs against F304 in the signature ribonucleoprotein consensus motif (RNP)-2 of RRM2. + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:57:34Z + + nucleotides + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:09Z + + Py tract + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:58:12Z + + C-terminal region + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:58:17Z + + binding site + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:25Z + + fifth + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:58:36Z + + RRM2 surface + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:58:44Z + + RRM1/RRM2 interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:58:49Z + + linker + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:56Z + + V254 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:03Z + + T252 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bonds + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:22Z + + rU5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:59:34Z + + β-strand + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:40Z + + K225 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:47Z + + R227 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bonds + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:22Z + + rU5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:58:12Z + + C-terminal region + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:00:00Z + + rU4-binding site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:56Z + + V254 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:00:11Z + + D256 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:00:19Z + + K260 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:00:39Z + + rU4 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:00:39Z + + rU4 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:01:06Z + + F304 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:01:15Z + + ribonucleoprotein consensus motif (RNP)-2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + + RESULTS + paragraph + 9217 + At the opposite side of the central fifth nucleotide, the sixth rU6 nucleotide is located at the inter-RRM1/RRM2 interface (Fig. 3e; Supplementary Movie 1). This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the β2/β3 loops of RRM1 and RRM2. The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain. + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:25Z + + fifth + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:03:25Z + + sixth + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:03:32Z + + rU6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:03:40Z + + inter-RRM1/RRM2 interface + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:03:46Z + + linker + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:03:32Z + + rU6 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:06:59Z + + uracil + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:03:55Z + + β2/β3 loops + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:03:32Z + + rU6 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:03:59Z + + solvent exposed + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:03:32Z + + rU6 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bonds + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T17:04:09Z + + water + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:04:19Z + + N196 + + + + RESULTS + paragraph + 9716 + We tested the contribution of the U2AF651,2L interactions with the new central nucleotide to Py-tract affinity (Fig. 3i; Supplementary Fig. 4a,b). Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. The energetic penalties due to these mutations (ΔΔG 0.8–0.9 kcal mol−1) are consistent with the loss of each hydrogen bond with the rU5 base and support the relevance of the central nucleotide interactions observed in the U2AF651,2L structures. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T17:06:01Z + + tested the contribution + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-06-15T11:54:58Z + + U2AF651,2L + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:06:18Z + + Py-tract affinity + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T17:06:23Z + + Mutagenesis + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:56Z + + V254 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:06:33Z + + proline + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:47Z + + R227 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:06:44Z + + alanine + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bond + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:25Z + + fifth + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:07:00Z + + uracil + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:07:08Z + + affinities + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:15Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:07:36Z + + ΔΔG + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:22Z + + rU5 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:07:50Z + + structures + + + + RESULTS + title_2 + 10389 + U2AF65 RRM extensions interact with the Py tract + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:17:00Z + + RRM extensions + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + + RESULTS + paragraph + 10438 + The N- and C-terminal extensions of the U2AF65 RRM1 and RRM2 directly contact the bound Py tract. Rather than interacting with a new 5′-terminal nucleotide as we had hypothesized, the C-terminal α-helix of RRM2 instead folds across one surface of rU3 in the third binding site (Fig. 3b). There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal β-strand of RRM2. Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b). + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:15:47Z + + N- and C-terminal extensions + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:15:55Z + + bound + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:16:11Z + + α-helix + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:16:19Z + + rU3 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:16:25Z + + third binding site + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + salt bridge + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:16:42Z + + K340 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:17:02Z + + G338 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + stacking + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:17:02Z + + G338 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:16:19Z + + rU3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:50:33Z + + RRM2 extension + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:17:33Z + + third + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:50Z + + nucleotide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:17:58Z + + rU2 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:51Z + + nucleotide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:50:21Z + + second binding site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:48:59Z + + β-strand + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:48:24Z + + U2AF651,2L-bound + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:17:58Z + + rU2 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bonds + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:18:12Z + + K329 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:18:12Z + + K329 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:18:20Z + + structure + + + + RESULTS + paragraph + 11331 + At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). The Q147 residue participates in hydrogen bonds with the -N3H of the eighth uracil and -O2 of the ninth pyrimidine. The adjacent R146 guanidinium group donates hydrogen bonds to the 3′-terminal ribose-O2′ and O3′ atoms, where it could form a salt bridge with a phospho-diester group in the context of a longer pre-mRNA. Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). We compare U2AF65 interactions with uracil relative to cytosine pyrimidines at the ninth binding site in Fig. 3g,h and the Supplementary Discussion. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:20:40Z + + α-helical extension + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:20:46Z + + Q147 + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:08Z + + eighth + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:21:03Z + + ninth + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T17:21:09Z + + nucleotides + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:17:10Z + + 3′ terminus + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:20:46Z + + Q147 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bonds + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:08Z + + eighth + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:07:00Z + + uracil + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:21:03Z + + ninth + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T17:21:27Z + + pyrimidine + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:21:35Z + + R146 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bonds + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T17:21:43Z + + ribose + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + salt bridge + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T17:22:08Z + + pre-mRNA + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bond + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:21:03Z + + ninth + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T17:21:27Z + + pyrimidine + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:22:35Z + + ΔΔG + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T17:22:44Z + + mutation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:20:46Z + + Q147 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:06:44Z + + alanine + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:53:08Z + + U2AF651,2L affinity + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T17:22:55Z + + compare + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:07:00Z + + uracil + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:23:03Z + + cytosine + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T17:23:11Z + + pyrimidines + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:23:24Z + + ninth binding site + + + + RESULTS + title_2 + 12221 + Versatile primary sequence of the U2AF65 inter-RRM linker + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + + RESULTS + paragraph + 12279 + The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. Altogether, the U2AF65 inter-RRM linker residues (R228–K260) bury 2,800 Å2 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900 Å2 for a typical protein–protein complex. The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two α-helices of the core RRM1. The adjacent V249 and V250 are notable for their respective interactions that connect RRM1 and RRM2 at this distal interface from the RNA-binding site (Fig. 4a, top). A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). Few direct contacts are made between the remaining residues of the linker and the U2AF65 RRM2; instead, the C-terminal conformation of the linker appears primarily RNA mediated (Fig. 3c,d). + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:09:23Z + + structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:09:29Z + + extensive interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:09:52Z + + α-helix + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:09:56Z + + β2/β3 strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:10:00Z + + α-helical extension + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:10:07Z + + R228–K260 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:10:11Z + + holo-protein + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:10:14Z + + cognate interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:10:19Z + + linker + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:10:31Z + + P229 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:47Z + + core + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:10:35Z + + β-strand + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:10:39Z + + kink + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + intra-molecular stacking + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:10:52Z + + R228 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:11:02Z + + Y232 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:11:12Z + + P234 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:11:24Z + + second kink + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:11:35Z + + P236 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:11:45Z + + L235 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:11:53Z + + M238 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:12:01Z + + α-helical RRM1 extension + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:47Z + + core + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:12:06Z + + α2-helix + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:58:44Z + + RRM1/RRM2 interface + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:12:17Z + + RNA-binding site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:12:36Z + + linker + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:12:46Z + + V244 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:12:54Z + + V246 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:13:01Z + + hydrophobic pocket + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:13:05Z + + α-helices + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:47Z + + core + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:21:39Z + + V249 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:21:47Z + + V250 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:13:49Z + + interface + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:12:17Z + + RNA-binding site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:13:13Z + + third kink + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + stacks + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:13:25Z + + P247 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:13:32Z + + G248 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:13:39Z + + Y245 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:58:13Z + + C-terminal region + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:14:02Z + + linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:24Z + + RRM2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:14:19Z + + bound + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:14:24Z + + RNA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:14:54Z + + RNA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:56Z + + V254 + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:25Z + + fifth + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:07:00Z + + uracil + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrophobic contacts + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:15:11Z + + β-sheet surface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:15:15Z + + RNP1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:01:06Z + + F304 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + stacks + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:54:34Z + + fourth + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T17:07:00Z + + uracil + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:15:29Z + + linker + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:15:34Z + + linker + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:15:37Z + + RNA + + + + RESULTS + paragraph + 14100 + We investigated whether the observed contacts between the RRMs and linker were critical for RNA binding by structure-guided mutagenesis (Fig. 4b). We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d–h). We introduced glycine substitutions to maximally reduce the buried surface area without directly interfering with its hydrogen bonds between backbone atoms and the base. First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). However, the resulting decrease in the AdML RNA affinity of the U2AF651,2L-3Gly mutant relative to wild-type protein was not significant (Fig. 4b). In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. A more conservative substitution of these five residues (251–255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:20:33Z + + linker + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:20:36Z + + structure-guided mutagenesis + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:20:40Z + + titrated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:48:29Z + + mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:20:44Z + + fluorescein + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:20:57Z + + Py-tract RNA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:21:02Z + + fluorescence anisotropy changes + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:21:07Z + + equilibrium affinities + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:21:18Z + + glycine + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:21:24Z + + substitutions + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bonds + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:21:32Z + + replaced + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:21:39Z + + V249 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:21:47Z + + V250 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:58:44Z + + RRM1/RRM2 interface + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:56Z + + V254 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:21:59Z + + bound + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:22:18Z + + RNA + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:21:18Z + + glycine + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:22:23Z + + 3Gly + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:10Z + + RNA affinity + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:26:32Z + + U2AF651,2L-3Gly + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:48:33Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:22:39Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T09:22:43Z + + protein + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:22:47Z + + replaced + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:22:50Z + + linker residues + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:22:56Z + + S251 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:04Z + + T252 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:23:06Z + + V253 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:56Z + + V254 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:23:21Z + + P255 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:24:25Z + + fifth nucleotide-binding site + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:24:41Z + + glycines + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:24:46Z + + 5Gly + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:10Z + + RNA affinity + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:26:35Z + + U2AF651,2L-5Gly + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:48:37Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:22:39Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T09:24:53Z + + protein + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:24:56Z + + conservative substitution + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:25:02Z + + 251–255 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bonds + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:25:09Z + + STVVP>NLALA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:25:13Z + + inter-RRM sequence + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:25:17Z + + affinity + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T11:57:13Z + + linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T11:57:22Z + + RRM + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:52:37Z + + substituted + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:21:18Z + + glycine + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:25:36Z + + M144 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:11:45Z + + L235 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:11:53Z + + M238 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:12:46Z + + V244 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:12:54Z + + V246 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:21:39Z + + V249 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:21:47Z + + V250 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:22:56Z + + S251 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:04Z + + T252 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:23:06Z + + V253 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:56Z + + V254 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:23:21Z + + P255 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:26:06Z + + 12Gly + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:52:41Z + + 12 concurrent mutations + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:10Z + + RNA affinity + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:26:29Z + + U2AF651,2L-12Gly + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:48:40Z + + variant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:26:40Z + + unmodified + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T09:26:43Z + + protein + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:54Z + + V254P + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:23Z + + rU5 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bond + + + + RESULTS + paragraph + 15968 + To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). We found that the affinity of dU2AF651,2L for the AdML RNA was significantly reduced relative to U2AF651,2L (four-fold, Figs 1b and 4b; Supplementary Fig. 4i). Yet, it is well known that the linker deletion in the context of the minimal RRM1–RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j). The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. However, stretching of the truncated dU2AF651,2L linker to connect the RRM termini is expected to disrupt its nucleotide interactions. Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:17:00Z + + RRM extensions + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:30:55Z + + constructed + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T12:57:18Z + + linker deletion + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:31:18Z + + 20-residues + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:25Z + + extended + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:31:24Z + + RNA-binding domain + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:54:50Z + + dU2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:53:36Z + + affinity + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:54:50Z + + dU2AF651,2L + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:05Z + + RNA + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:31:39Z + + linker deletion + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:35:25Z + + minimal + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:53:39Z + + RNA affinities + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:32:13Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:32:17Z + + extended conformation + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:32:20Z + + truncated + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:48Z + + R227 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:32:31Z + + H259 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:53:43Z + + RNA affinities + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:32:36Z + + dual + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:32:46Z + + individual + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:32:51Z + + truncated + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:54:50Z + + dU2AF651,2L + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:32:54Z + + linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:49:26Z + + RRM termini + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:33:05Z + + deletion + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:51:02Z + + RRM1 extension + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:57Z + + shortened + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:33:24Z + + linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:33:27Z + + kinked turn + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:10:31Z + + P229 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:53:46Z + + RNA affinities + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:54:50Z + + dU2AF651,2L + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + + RESULTS + paragraph + 17374 + To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d). Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the ΔΔG's for the single mutations. This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. Altogether, we conclude that the conformation of the U2AF65 inter-RRM linker is key for recognizing RNA and is positioned by the RRM extension but otherwise relatively independent of the side chain composition. The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:17:00Z + + RRM extensions + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:38Z + + Q147A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:54Z + + V254P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:46Z + + R227A + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:35:53Z + + mutation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:35:56Z + + U2AF651,2L-3Mut + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:38Z + + Q147A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:54Z + + V254P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:46Z + + R227A + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:36:10Z + + mutation + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:10Z + + RNA affinity + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:36:13Z + + U2AF651,2L-3Mut + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:22:38Z + + ΔΔG + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:36:16Z + + linearly distant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:36:22Z + + regions + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:20:47Z + + Q147 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:51:02Z + + RRM1 extension + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:48Z + + R227 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:56Z + + V254 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:36:37Z + + linker regions + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:36:41Z + + fifth nucleotide site + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:09Z + + RNA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:49:30Z + + RRM extension + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:38Z + + Q147A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:54Z + + V254P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:46Z + + R227A + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:36:58Z + + triple mutation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:27Z + + U2AF65 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:37:02Z + + linker deletion + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:37:06Z + + linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:37:10Z + + N- and C-terminal extensions + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:47Z + + core + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + + RESULTS + title_2 + 18570 + Importance of U2AF65–RNA contacts for pre-mRNA splicing + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-06-15T12:02:00Z + + U2AF65–RNA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:51:29Z + + pre-mRNA + + + + RESULTS + paragraph + 18628 + We proceeded to test the importance of new U2AF65–Py-tract interactions for splicing of a model pre-mRNA substrate in a human cell line (Fig. 5; Supplementary Fig. 5). As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a). When transfected into HEK293T cells containing only endogenous U2AF65, the PY splice site is used and the remaining transcript remains unspliced. When co-transfected with an expression plasmid for wild-type U2AF65, use of the py splice site significantly increases (by more than five-fold) and as documented converts a fraction of the unspliced to spliced transcript. The strong PY splice site is insensitive to added U2AF65, suggesting that endogenous U2AF65 levels are sufficient to saturate this site (Supplementary Fig. 5b). We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak ‘py' splice site relative to wild-type U2AF65 (Fig. 5b,c). We conclude that the Py-tract interactions with these residues of the U2AF65 inter-RRM linker and RRM extensions are important for splicing as well as for binding a representative of the major U2-class of splice sites. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-06-15T12:02:17Z + + U2AF65–Py-tract + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:41:22Z + + pre-mRNA + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:18:14Z + + human + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:41:29Z + + minigene splicing reporter + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:41:40Z + + pyPY + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:42:43Z + + py + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:49:35Z + + U-rich + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:42:31Z + + PY + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:42:15Z + + polypyrimidine tracts + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:19:52Z + + splice sites + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:43:26Z + + transfected + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:42:07Z + + endogenous + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:50:25Z + + PY splice site + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:43:43Z + + co-transfected + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:43:46Z + + expression plasmid + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:22:39Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:43:57Z + + py splice site + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:44:08Z + + PY splice site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:42:07Z + + endogenous + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:44:12Z + + triple mutation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:54Z + + V254P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:46Z + + R227A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:38Z + + Q147A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:30Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:55:01Z + + U2AF65-3Mut + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:44:34Z + + Co-transfection + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:55:01Z + + U2AF65-3Mut + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:41:40Z + + pyPY + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:50:29Z + + ‘py' splice site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:22:39Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-06-15T11:59:05Z + + Py-tract + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:17:00Z + + RRM extensions + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:45:08Z + + major U2-class of splice sites + + + + RESULTS + title_2 + 20165 + Sparse inter-RRM contacts underlie apo-U2AF65 dynamics + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:53:02Z + + inter-RRM + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:45:31Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + + RESULTS + paragraph + 20220 + The direct interface between U2AF651,2L RRM1 and RRM2 is minor, burying 265 Å2 of solvent accessible surface area compared with 570 Å2 on average for a crystal packing interface. A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). This minor U2AF65 RRM1/RRM2 interface, coupled with the versatile sequence of the inter-RRM linker, highlighted the potential role for inter-RRM conformational dynamics in U2AF65-splice site recognition. Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution. + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T09:55:15Z + + interface + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:53:02Z + + inter-RRM + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:17Z + + hydrogen bonds + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:55:24Z + + N155 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:55:27Z + + K292 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:55:31Z + + N155 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:55:35Z + + D272 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:55:38Z + + G221 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:55:41Z + + D273 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:58:44Z + + RRM1/RRM2 interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:53:02Z + + inter-RRM + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:53:17Z + + U2AF65 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:52:03Z + + Paramagnetic resonance enhancement + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:42:21Z + + PRE + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:42:12Z + + back-to-back + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:20Z + + closed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:45:31Z + + apo + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:12Z + + open + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:31Z + + RNA-bound + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:53:02Z + + inter-RRM + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:56:33Z + + small-angle X-ray scattering + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:56:36Z + + SAXS + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:35:25Z + + minimal + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:45Z + + highly diverse continuum of conformations + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:05:45Z + + absence of + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:13Z + + RNA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:20Z + + closed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:12Z + + open + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:57:01Z + + structure + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T09:57:04Z + + X-ray crystallography + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:24Z + + smFRET + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T12:50:22Z + + probability distribution functions + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:53:03Z + + inter-RRM + + + + RESULTS + paragraph + 21568 + The inter-RRM dynamics of U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2 (Fig. 6a,b, Methods). The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). The FRET efficiencies of either of these structurally characterized conformations also are expected to be significantly greater than elongated U2AF65 conformations that lack inter-RRM contacts. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:53:03Z + + inter-RRM + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T12:56:16Z + + FRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:56:19Z + + fluorophores + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:56:22Z + + cysteine + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T12:57:03Z + + mutations + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:57:31Z + + fluorophore + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T12:56:30Z + + A181C + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T12:56:33Z + + Q324C + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T12:58:07Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:20Z + + closed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:45:31Z + + apo + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T12:58:12Z + + RRM/RNA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:58:16Z + + inter-RRM interfaces + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T12:58:20Z + + U2AF651,2L–Py tract + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:20Z + + closed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:45:31Z + + apo + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T12:58:28Z + + Förster radius (Ro) + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T12:59:01Z + + FRET efficiencies + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:20Z + + closed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:45:31Z + + apo + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:12Z + + open + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:31Z + + RNA-bound + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T12:59:05Z + + structures + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T12:59:01Z + + FRET efficiencies + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T12:59:16Z + + elongated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T12:59:19Z + + lack + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T12:02:44Z + + RRM + + + + RESULTS + paragraph + 22670 + Double-cysteine variant of U2AF651,2 was modified with equimolar amount of Cy3 and Cy5. Only traces that showed single photobleaching events for both donor and acceptor dyes and anti-correlated changes in acceptor and donor fluorescence were included in smFRET data analysis. Hence, molecules that were conjugated to two donor or two acceptor fluorophores were excluded from analysis. + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-21T13:00:04Z + + cysteine + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:00:14Z + + variant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:00:43Z + + modified + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:24Z + + smFRET + + + + RESULTS + paragraph + 23055 + We first characterized the conformational dynamics spectrum of U2AF65 in the absence of RNA (Fig. 6c,d; Supplementary Fig. 7a,b). The double-labelled U2AF651,2LFRET(Cy3/Cy5) protein was tethered to a slide via biotin-NTA/Ni+2 resin. Virtually no fluorescent molecules were detected in the absence of biotin-NTA/Ni+2, which demonstrates the absence of detectable non-specific binding of U2AF651,2LFRET to the slide. The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4 (Fig. 6d). Approximately 40% of the smFRET traces showed apparent transitions between multiple FRET values (for example, Fig. 6c). Despite the large width of the FRET-distribution histogram, the majority (80%) of traces that showed fluctuations sampled only two distinct FRET states (for example, Supplementary Fig. 7a). Approximately 70% of observed fluctuations were interchanges between the ∼0.65 and ∼0.45 FRET values (Supplementary Fig. 7b). We cannot exclude a possibility that tethering of U2AF651,2LFRET(Cy3/Cy5) to the microscope slide introduces structural heterogeneity into the protein and, thus, contributes to the breadth of the FRET distribution histogram. However, the presence of repetitive fluctuations between particular FRET values supports the hypothesis that RNA-free U2AF65 samples several distinct conformations. This result is consistent with the broad ensembles of extended solution conformations that best fit the SAXS data collected for U2AF651,2 as well as for a longer construct (residues 136–347). We conclude that weak contacts between the U2AF65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:05:45Z + + absence of + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:18Z + + RNA + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:09:40Z + + tethered + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:52:39Z + + biotin-NTA/Ni+2 resin + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:05:45Z + + absence of + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:40:49Z + + biotin-NTA/Ni+2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:05:45Z + + absence of + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:53:50Z + + FRET distribution histogram + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:05:45Z + + absence of + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:06:07Z + + ligand + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:03:17Z + + FRET efficiency + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:24Z + + smFRET + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:03:39Z + + FRET values + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:05:05Z + + FRET-distribution histogram + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-22T10:41:05Z + + FRET states + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:03:41Z + + FRET values + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:53:57Z + + FRET distribution histogram + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:03:41Z + + FRET values + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:05:13Z + + RNA-free + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:26Z + + extended + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:05:26Z + + SAXS + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:05:32Z + + 136–347 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:05:45Z + + absence of + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:05:36Z + + RNA + + + + RESULTS + title_2 + 24827 + U2AF65 conformational selection and induced fit by bound RNA + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:06:39Z + + bound + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:06:42Z + + RNA + + + + RESULTS + paragraph + 24888 + We next used smFRET to probe the conformational selection of distinct inter-RRM arrangements following association of U2AF65 with the AdML Py-tract prototype. Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f). To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5). + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:24Z + + smFRET + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:53:03Z + + inter-RRM + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:25:08Z + + Py-tract + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:22Z + + RNA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:09:40Z + + tethered + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:03:20Z + + FRET efficiency + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:09:11Z + + FRET state + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:10:54Z + + RNA-free + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:09:58Z + + tethering + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:10:01Z + + distribution of FRET values + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:10:12Z + + reversed the immobilization scheme + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:09:40Z + + tethered + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:26Z + + RNA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:30Z + + biotinylated oligonucleotide DNA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:52:46Z + + added + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:05:45Z + + absence of + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:10:34Z + + biotin-NTA resin + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:17Z + + FRET value + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:31Z + + RNA-bound + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:28Z + + untethered + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:09:40Z + + tethered + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + + RESULTS + paragraph + 25927 + We examined the effect on U2AF651,2L conformations of purine interruptions that often occur in relatively degenerate human Py tracts. We introduced an rArA purine dinucleotide within a variant of the AdML Py tract (detailed in Methods). Insertion of adenine nucleotides decreased binding affinity of U2AF65 to RNA by approximately five-fold. Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent ∼0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract. Therefore, RRM1-to-RRM2 distance remains similar regardless of whether U2AF65 is bound to interrupted or continuous Py tract. + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:13:55Z + + purine interruptions + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:18:14Z + + human + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:14:03Z + + Py tracts + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:14:07Z + + introduced + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:14:35Z + + rArA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:14:51Z + + purine dinucleotide + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:15:44Z + + Insertion + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:16:30Z + + adenine nucleotides + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:16:01Z + + binding affinity + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:16:26Z + + RNA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:16:16Z + + rArA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:34Z + + RNA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:16:21Z + + slide-tethered + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:19Z + + FRET value + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:15Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:28Z + + U2AF65 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + + RESULTS + paragraph + 26644 + The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the α-carbon atoms of the respective residues in the crystal structures of U2AF651,2L bound to Py-tract oligonucleotides. It should be noted that inferring distances from FRET values is prone to significant error because of uncertainties in the determination of fluorophore orientation factor κ2 and Förster radius R0, the parameters used in distance calculations. Nevertheless, the predominant 0.45 FRET state in the presence of RNA agrees with the Py-tract-bound crystal structure of U2AF651,2L. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:18:15Z + + inter-fluorophore distances + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:09:17Z + + FRET state + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:39Z + + crystal structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:43:11Z + + Py-tract oligonucleotides + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:03:41Z + + FRET values + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-22T10:41:32Z + + FRET state + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:18:27Z + + RNA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:18:11Z + + Py-tract-bound + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:46:29Z + + crystal structure + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:40Z + + U2AF651,2L + + + + RESULTS + paragraph + 27257 + Importantly, the majority of traces (∼70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a ∼0.45 FRET value (for example, Fig. 6g). The remaining ∼30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values. The majority of traces that show fluctuations began at high (0.65–0.8) FRET value and transitioned to a ∼0.45 FRET value (Supplementary Fig. 7c–g). Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to ∼0.7–0.8 and ∼0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state. Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:22:56Z + + RNA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:19Z + + FRET value + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:22:59Z + + RNA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:03:41Z + + FRET values + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:19Z + + FRET value + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:19Z + + FRET value + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:21:47Z + + Hidden Markov modelling analysis + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:24Z + + smFRET + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:31Z + + RNA-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:03:41Z + + FRET values + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:09:17Z + + FRET state + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:03:42Z + + FRET values + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:23:11Z + + RNA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:41:47Z + + RNA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:23:35Z + + compact + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:19Z + + FRET value + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:08Z + + side-by-side + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:53:03Z + + inter-RRM + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:39Z + + crystal structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:24Z + + smFRET + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:25:09Z + + Py-tract + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:23:45Z + + pre-configured + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:08Z + + side-by-side + + + + DISCUSS + title_1 + 28824 + Discussion + + + DISCUSS + paragraph + 28835 + The U2AF65 structures and analyses presented here represent a successful step towards defining a molecular map of the 3′ splice site. Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65–Py-tract recognition. Truncation of U2AF65 to the core RRM1–RRM2 region reduces its RNA affinity by 100-fold. Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold. Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition. The implications of this finding for U2AF65 conservation and Py-tract recognition are detailed in the Supplementary Discussion. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:26:55Z + + structures + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:26:57Z + + analyses + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:09:33Z + + 3′ splice site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:16Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:51:02Z + + RRM1 extension + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:53:17Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-07-21T09:20:50Z + + Py-tract + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:27:17Z + + Truncation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:47Z + + core + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:49:42Z + + RRM1–RRM2 region + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:10Z + + RNA affinity + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:27:24Z + + deletion + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:27:31Z + + 20 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T13:27:50Z + + inter-RRM linker residues + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:53:17Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:42:03Z + + RNA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:27:55Z + + longer + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:17:00Z + + RRM extensions + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T13:27:59Z + + linker + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:42:13Z + + RNA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:28:06Z + + triple mutation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:54Z + + V254P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:38Z + + Q147A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:46Z + + R227A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T13:28:19Z + + N- and C-terminal extensions + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:54:02Z + + RNA binding + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:16Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:05Z + + inter-RRM linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T13:28:26Z + + N-and C-terminal extensions + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:28:47Z + + Py-tract + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:25:09Z + + Py-tract + + + + DISCUSS + paragraph + 30033 + Recently, high-throughput sequencing studies have shown that somatic mutations in pre-mRNA splicing factors occur in the majority of patients with myelodysplastic syndrome (MDS). MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)—possibly due to the selective versus nearly universal requirements of these factors for splicing. A confirmed somatic mutation of U2AF65 in patients with MDS, L187V, is located on a solvent-exposed surface of RRM1 that is distinct from the RNA interface (Fig. 7a). This L187 surface is oriented towards the N terminus of the U2AF651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer. Likewise, an unconfirmed M144I mutation reported by the same group corresponds to the N-terminal residue of U2AF651,2L, which is separated by only ∼20 residues from the U2AF35-binding site. As such, we suggest that the MDS-relevant U2AF65 mutations contribute to MDS progression indirectly, by destabilizing a relevant conformation of the conjoined U2AF35 subunit rather than affecting U2AF65 functions in RNA binding or spliceosome recruitment per se. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:31:12Z + + high-throughput sequencing studies + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:31:15Z + + pre-mRNA splicing factors + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:31:19Z + + small + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:31:27Z + + U2AF subunit + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:31:33Z + + U2AF35 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:31:30Z + + U2AF1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:31:38Z + + large + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:31:41Z + + U2AF2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:31:46Z + + L187V + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T13:31:55Z + + solvent-exposed surface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:16Z + + RRM1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T13:31:58Z + + RNA interface + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:31:50Z + + L187 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T13:32:03Z + + U2AF35-binding site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:30Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:32:08Z + + U2AF + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:55:53Z + + heterodimer + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:32:11Z + + M144I + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T13:32:14Z + + U2AF35-binding site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T14:50:38Z + + U2AF35 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:42:28Z + + RNA + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:26:09Z + + spliceosome + + + + DISCUSS + paragraph + 31277 + Our smFRET results agree with prior NMR/PRE evidence for multi-domain conformational selection as one mechanistic basis for U2AF65–RNA association (Fig. 7b). The ‘induced fit' versus ‘conformational selection' models are the prevailing views of the mechanisms underlying bio-molecular interactions (reviewed in ref.). In the former, ligand binding promotes a subsequent conformational change in the protein, whereas in the latter, the ligand selects a protein conformation from a pre-existing ensemble and thereby shifts the population towards that state. An ∼0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide. The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:24Z + + smFRET + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:41:44Z + + NMR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:42:21Z + + PRE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:53:17Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:42:40Z + + RNA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:19Z + + FRET value + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:39Z + + crystal structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:16Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:08Z + + side-by-side + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T13:41:59Z + + Py-tract oligonucleotide + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:03:42Z + + FRET values + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:28Z + + untethered + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:35Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:20Z + + closed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:42:12Z + + back-to-back + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:52:50Z + + NMR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:42:21Z + + PRE + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:26Z + + extended + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T12:03:37Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T12:03:45Z + + RRM2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:42:32Z + + protein + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:39Z + + RNA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:42:29Z + + single + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:20Z + + FRET value + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:53:18Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:42:50Z + + RNA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:05:45Z + + absence of + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:44Z + + RNA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:42:54Z + + pre-configured + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:53:03Z + + inter-RRM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:20Z + + FRET value + + + + DISCUSS + paragraph + 32792 + Notably, our smFRET results reveal that U2AF65–Py-tract recognition can be characterized by an ‘extended conformational selection' model (Fig. 7b). In this recent model for macromolecular interactions, the pure ‘conformational selection' and ‘induced fit' scenarios represent the limits of a mechanistic spectrum and may compete or occur sequentially. Examples of ‘extended conformational selection' during ligand binding have been characterized for a growing number of macromolecules (for example, adenylate kinase, LAO-binding protein, poly-ubiquitin, maltose-binding protein and the preQ1 riboswitch, among others). Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein. Similar interdisciplinary structural approaches are likely to illuminate whether similar mechanistic bases for RNA binding are widespread among other members of the vast multi-RRM family. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:24Z + + smFRET + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:53:18Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:43:04Z + + Py-tract + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:26Z + + extended + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:45:13Z + + adenylate kinase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:45:16Z + + LAO-binding protein + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:45:19Z + + poly-ubiquitin + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:45:21Z + + maltose-binding protein + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:45:24Z + + preQ1 riboswitch + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:24Z + + smFRET + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:36Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:43Z + + Cy5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:48Z + + RNA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:20Z + + FRET value + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:20Z + + FRET value + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:20Z + + closed + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:45:37Z + + NMR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:42:21Z + + PRE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:50:34Z + + RNA-binding surface + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:45:31Z + + single + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:49:47Z + + RRM + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:08Z + + side-by-side + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:31Z + + RNA-bound + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:46:29Z + + crystal structure + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:31:24Z + + smFRET + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:45:40Z + + NMR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:42:21Z + + PRE + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:53:03Z + + inter-RRM + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:45:45Z + + SAXS + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:45:31Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:45:48Z + + protein + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:49:51Z + + RRM + + + + DISCUSS + paragraph + 34345 + The finding that U2AF65 recognizes a nine base pair Py tract contributes to an elusive ‘code' for predicting splicing patterns from primary sequences in the post-genomic era (reviewed in ref.). Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. Further research will be needed to understand the roles of SF1 and U2AF35 subunits in the conformational equilibria underlying U2AF65 association with Py tracts. Moreover, structural differences among U2AF65 homologues and paralogues may regulate splice site selection. Ultimately, these guidelines will assist the identification of 3′ splice sites and the relationship of disease-causing mutations to penalties for U2AF65 association. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:54:06Z + + RNA affinities + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:29Z + + U2AF65 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:48:06Z + + structures + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:48:09Z + + penalties + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:48:11Z + + structure-guided mutations + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:48:16Z + + RNA binding and splicing assays + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:26Z + + extended + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T13:47:43Z + + inter-RRM regions + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:48:20Z + + structures + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:25:09Z + + Py-tract + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:30Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:30Z + + U2AF65 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:48:25Z + + SF1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T13:48:29Z + + U2AF35 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:30Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:53Z + + Py tracts + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:30Z + + U2AF65 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:21:04Z + + splice site + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:12:54Z + + 3′ splice sites + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:30Z + + U2AF65 + + + + METHODS + title_1 + 35392 + Methods + + + METHODS + title_2 + 35400 + Protein expression and purification + + + METHODS + paragraph + 35436 + For crystallization and RNA-binding experiments, human U2AF651,2L (residues 141–342 of NCBI RefSeq NP_009210) was expressed in Escherichia coli strain BL21 Rosetta-2 as a GST-fusion protein in the vector pGEX6P-2 and purified by glutathione affinity, followed by anion exchange and gel filtration chromatography. The GST-tagged protein was bound to a GSTrap column (GE Healthcare) in 1 M NaCl, 25 mM HEPES, pH 7.4 and eluted using 150 mM NaCl, 100 mM Tris, pH 8 containing 10 mM glutathione. The GST tag was cleaved from the protein by treatment with PreScission Protease during dialysis against a buffer containing 100 mM NaCl, 25 mM HEPES, pH 8, 5% (v/v) glycerol, 5 mM DTT, 0.25 mM EDTA and 0.1 mM PMSF. Cleaved GST was separated from the U2AF651,2L by subtractive glutathione affinity chromatography in 100 mM NaCl, 25 mM Tris, pH 8, 0.2 mM TCEP followed by subtractive anion-exchange chromatography with a HiTrap Q column (GE Healthcare). The final purification step was size-exclusion chromatography on a Superdex-75 prep-grade column (GE Healthcare) that had been previously equilibrated with 100 mM NaCl, 15 mM HEPES, pH 6.8, 0.2 mM tris(2-carboxy-ethyl)phosphine (TCEP). The purified U2AF651,2L was concentrated using a Vivaspin 15 R (Sartorius) centrifugal concentrator with 10 kDa MWCO, and the protein concentration was estimated using the calculated extinction coefficient of 8,940 M−1cm−1 and absorbance at 280 nm. Shorter constructs (U2AF651,2, residues 148–336; dU2AF651,2, residues 148–237, 258–336; dU2AF651,2L, residues 141–237, 258–342) (Fig. 1a) and individual U2AF651,2L Q147A, R227A, V254P mutants used for RNA-binding experiments were purified similarly. + + + METHODS + paragraph + 37171 + For comparative RNA-binding experiments, full-length human U2AF65 (residues 1–475) and the U2AF35-UHM (U2AF homology motif; residues 43–146, NCBI RefSeq NP_006749) initially were expressed and purified separately as GST fusion proteins. Following GST cleavage and ion-exchange chromatography (SP-HiTrap and Q-HiTrap, respectively), U2AF65 was combined with slight excess U2AF35-UHM (in stoichiometric ratio of 1:1.2) and dialysed overnight. The final U2AF heterodimer was purified by size-exclusion chromatography using a Superdex-200 prep-grade column (GE Healthcare) pre-equilibrated with 150 mM NaCl, 25 mM HEPES, pH 6.8, 0.2 mM TCEP. Representative purified U2AF651,2L and U2AF65–U2AF35-UHM proteins are shown in Supplementary Fig. 1a. + + + METHODS + title_2 + 37923 + Oligonucleotide preparation + + + METHODS + paragraph + 37951 + High-performance liquid chromatography-purified oligonucleotides (sequences shown in Supplementary Fig. 2a) were purchased for crystallization (Integrated DNA Technologies, Inc.). The lyophilized oligonucleotides were diluted in gel filtration buffer for crystallization experiments. The 5′-fluorescein (Fl)-labelled RNAs (AdML: 5′-Fl-CCCUUUUUUUUCC-3′, Py tract of the AdML splicing substrate; 5′-4rU: 5′-Fl-CCUUUUCCCCCCC-3′; 3′-4rU: 5′-Fl-CCCCCCCUUUUCC-3′) for RNA-binding experiments (Dharmacon Research, Inc., Thermo Scientific) was deprotected according to the manufacturer's protocol, vacuum dried and resuspended in nuclease-free water. RNA and RNA–DNA concentrations were calculated using the calculated molar extinction coefficients and absorbance at 260 nm. + + + METHODS + title_2 + 38740 + Fluorescence anisotropy RNA-binding experiments + + + METHODS + paragraph + 38788 + For RNA-binding experiments, purified proteins and RNA were diluted separately >100-fold in binding buffer (100 mM NaCl, 15 mM HEPES, pH 6.8, 0.2 mM TCEP, 0.1 U μl−1 Superase-In (Ambion Life Technologies)). The final RNA concentration in the cuvette was 30 nM. Volume changes during addition of the protein were <10% to minimize dilution effects. The fluorescence anisotropy changes during titration were measured using a FluoroMax-3 spectrophotometer temperature controlled by a circulating water bath at 23 °C. Samples were excited at 490 nm and emission intensities recorded at 520 nm with a slit width of 5 nm. The titrations were repeated three times in succession. Each titration was fit with Graphpad Prism v4.0 to obtain the apparent equilibrium dissociation constant (KD). The apparent equilibrium affinities (KA) are the reciprocal of the KD. The average KD's or KA's and s.e.m. among the three replicates were calculated using Excel and are reported in Figs 3 and 4; Supplementary Figs 1 and 4. The P values from a two-tailed unpaired t-test were calculated using Graphpad Prism v4.0. + + + METHODS + title_2 + 39908 + Transfection, immunoblotting and RT-PCR analyses + + + METHODS + paragraph + 39957 + For transfection experiments, the full-length human U2AF65 cDNA in pCMV6-XL5 (Origene Tech. Inc., clone ID BC008740) was used (WT U2AF65) and in parallel mutated to encode the Q147A/R227A/V254P triple-mutant protein (Mut U2AF65). The pyPY minigene was a gift from M. Carmo-Fonseca (University of Lisbon, Portugal). HEK293T cells (kindly provided by Dr Lata Balakrishnan, originally purchased from ATCC, cat. no. CRL3216) were seeded into 12-well plates (2–4 × 105 cells per well) and grown as monolayers in MEM (Gibco Life Technologies) supplemented with 10% (v/v) of heat-inactivated fetal bovine serum, 1% (v/v) L-glutamine and 1% (v/v) penicillin–streptomycin. After 1 day, the cells were transiently transfected with either 0.5 μg of pyPY plasmid or a mixture of 0.5 μg of U2AF65 variant and 0.5 μg of pyPY plasmid per well using appropriately adjusted Lipofectamine 2000 (Invitrogen Life Technologies) ratio according to the manufacturer's instructions. + + + METHODS + paragraph + 40932 + For immunoblots of WT U2AF65 and Mut U2AF65 expression levels (Supplementary Fig. 5a), transfected or control cells were lysed in radioimmunoprecipitation assay buffer with proteinase and kinase inhibitors. Total protein (20 μg) was separated by SDS–PAGE, and transferred onto polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA, USA) and immunoblotted using mouse monoclonal antibodies directed against U2AF65 (ref.) (MC3, cat. no. U4758 Sigma-Aldrich at 1:500 dilution) or as a control for comparison, GAPDH (glyceraldehyde-3-phosphate dehydrogenase; monoclonal clone 71.1, cat. no. G8795 Sigma-Aldrich at 1:5,000 dilution). Immunoblots were developed using anti-mouse horseradish peroxidase-conjugates (cat. no. U4758 Sigma-Aldrich, Co. at 1:2,500 or 1:10,000 dilutions for GAPDH and U2AF65, respectively) and detected using SuperSignal WestPico chemi-luminescent substrate (Pierce Thermo Scientific Inc.). Blots were imaged using a IS4000MM system (Carestream, Rochester, NY, USA). For size analysis, fluorescent images of the BioRad Precision Plus Dual Color Standards were overlaid directly. + + + METHODS + paragraph + 42051 + For reverse transcription PCR (RT-PCR), the total RNA was isolated 2 days post transfection using the Cells-to-cDNA II kit (Ambion Life Technologies). The RT-PCR reaction comprised 35 cycles (94 °C per 60 s—60 °C per 50 s—72 °C per 60 s) with forward (5′-TGAGGGGAGGTGAATGAGGAG-3′) and reverse (5′-TCCACTGGAAAGACCGCGAAG-3′) primers for the pyPY product or forward (5′-CATGTTCGTCATGGGTGTGAACCA-3′) and reverse (5′-ATGGCATGGACTGTGGTCATGAGT-3′) primers for a GAPDH control. The RT-PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. The percentages of splice site use were calculated from the background corrected intensities I using the formula 100% × I(py)/[I(py)+I(PY)+I(unspliced)] for py spliced (Fig. 5b,c) or 100% × I(PY)/[I(py)+I(PY)+I(unspliced)] for PY spliced (Supplementary Fig. 5b). The band intensities of four independent biological replicates were measured using ImageQuant software. + + + METHODS + title_2 + 43029 + Crystallization, data collection and structure determination + + + METHODS + paragraph + 43090 + Before crystallization, the purified U2AF651,2L and given oligonucleotide were mixed to achieve respective final concentrations of 1.0 and 1.1 mM and incubated on ice for 20–30 min. For each oligonucleotide, sparse matrix screens of the Jancarik and Kim Crystal Screen(in hanging drop format; Hampton Research, Corp.) and JCSG-Plus (in sitting drop format; Molecular Dimensions) were used to identify initial crystallization conditions, which were obtained from the latter screen and further optimized in hanging drop format. In optimized crystallization experiments, a mixture of sample and reservoir solution (1.2:1 μl) was equilibrated against 700 μl reservoir solution at 4 °C. + + + METHODS + paragraph + 43787 + The oligonucleotide sequences were optimized and the structures were determined as follows: in addition to the previously characterized dU2AF651,2-binding sites for seven nucleotides, the new terminal residues of the U2AF651,2L construct were presumed to contact an additional nucleotide and the crystal packing of a central nucleotide between the RRM1/RRM2 of dU2AF651,2 was presumed to represent one nucleotide. Also considering the known proclivity for deoxy(d)U to co-crystallize with dU2AF651,2 (ref.) and for 5-bromo-dU (5BrdU) to bind a given site of dU2AF651,2 (ref.), we initially designed two 9-mer oligonucleotides (5′-ribose (r)UrUrUrUrU(5BrdU)dUrUrU and 5′-rUrUrUdUdU(5BrdU)dUrUrU) and screened for co-crystallization with U2AF651,2L. The former oligonucleotide failed to produce crystals in these screens. The latter oligonucleotide comprising central dU nucleotides produced diffracting crystals, which were frozen directly from a reservoir comprising 100 mM phosphate–citrate buffer pH 4.2, 40% Peg 300. The structure determined by molecular replacement using Phenix with a data set collected at beamline (BL) 12-2 of the Stanford Synchrotron Radiation Lightsource (SSRL; Menlo Park, CA, USA) (Table 1). The search models comprising each of the individual RRMs bound to two nucleotides were derived from the dU2AF651,2 structure (PDB ID 2G4B) (translation function Z-score equivalent 12.9, log-likelihood gain 528). For comparison, searches with the NMR structure (PDB ID 2YH1) as a search model failed to find a solution. The initial structure revealed a greater number of central nucleotide-binding sites than expected. The oligonucleotide binding register had slipped to place the BrdU in the preferred site, leave the 5′ terminal-binding sites empty, and the terminal nucleotide unbound and disordered. Subsequent oligonucleotides were designed to place BrdU in the preferred site, fill the unoccupied 5′ terminal sites, capture rU at the central sites, and compare rC at the terminal site. + + + METHODS + paragraph + 45810 + The U2AF651,2L protein co-crystals with oligonucleotide 5′-phosphorylated (P)-rUrUdUdUrUdU(BrdU)dU were obtained using a reservoir of 200 mM LiCl, 100 mM sodium citrate pH 4.0, 8% (w/v) polyethylene glycol (PEG) 6,000, 10% (v/v) PEG 300, 10% (v/v) dioxane with 0.1 μl of N,N-bis[3-(D-gluconamido)propyl]deoxy-cholamide (deoxy-BigCHAP) (14 mM) added to the hanging drop and cryoprotected by sequential layering with reservoir solution supplemented with increasing PEG 300 to a final concentration of 26%. Co-crystals with either 5′-(P)rUrUdUrUrU(BrdU)dUdU or 5′-(P)rUrUrUdUrUrU(BrdU)dUrC were obtained from 1 M succinate, 100 mM HEPES, pH 7.0, 1–3% (w/v) PEG monomethylether 2,000. The former was cryoprotected by coating with a 1:1 (v/v) mixture of silicon oil and Paratone-N and the latter by sequential transfer to 21% (v/v) glycerol. Data sets for flash-cooled crystals were collected at 100 K using remote access to SSRL BL12-2. Structures were determined by molecular replacement using the initial U2AF651,2L/rUrUrUdUdU(BrdU)dUrUrU structure as a search model. Consistent sets of free-R reflections were maintained (6% of the total reflections). Models were built using COOT and refined with PHENIX. No non-glycine/non-proline residues were found in the disallowed regions of the Ramachandran plots. Clash scores and Molprobity scores calculated using the program Molprobity were above average. Structure illustrations were prepared using PYMOL. Crystallographic data and refinement statistics are given in Table 1. + + + METHODS + title_2 + 47354 + Sample preparation for single-molecule FRET + + + METHODS + paragraph + 47398 + The U2AF651,2LFRET construct used for smFRET comprises the six histidine and T7 tags from the pET28a vector (Merck), a GGGS linker and U2AF65 residues 113–343. The single cysteine of human U2AF65 was replaced by alanine (C305A), which is a natural amino-acid variation among U2AF65 homologues. Single A181C and Q324C mutations were introduced in each RRM for fluorophore attachment at residues that were carefully selected to meet experimental criteria described in the Results. The U2AF651,2LFRET was purified by the same method as described above for U2AF651,2L protein and binds RNA with similar affinity as U2AF651,2L (Supplementary Fig. 6a). Before labelling, the purified U2AF651,2LFRET protein was incubated with 10 mM DTT on ice for 30 min and then buffer exchanged into Labelling Buffer (100 mM NaCl, 25 mM HEPES pH 7.0, 5 mM EDTA, 0.5 mM tris(2-carboxy-ethyl)phosphine (TCEP)) using Zeba Spin Desalting Columns 7K MWCO (Pierce, ThermoFisher Scientific). To initiate the labelling reaction, 4 μl each of cyanine (Cy)3-Maleimide and Cy5-Maleimide (Combinix, Inc.) stock solutions (10 mM in DMSO) were pre-mixed (total volume 8 μl) and then added to 200 μl of 20 μM protein (final 20:1 molar ratio of dye:protein). The labelling reaction was incubated at room temperature in the dark for 2 h and then quenched by the addition of 10 mM DTT. The labelled protein was separated from excess dye using a Zeba Spin Desalting Column followed by size exclusion chromatography using a pre-packed Superdex-75 10/300 GL (GE Healthcare) column in Labelling Buffer. Our previous experience of conjugating cysteines with maleimide derivatives of fluorophores and suggests that nonspecific modification of aminogroups of proteins with fluorescent dyes under the employed experimental conditions is negligible. Consistent with specific labelling of A181C and Q324C, the labelling efficiencies were ∼60% each for Cy3 and Cy5 as estimated using the dye extinction coefficients (ɛCy3=150,000 M−1 cm−1 at 550 nm, ɛCy5=170,000 M−1 cm−1 at 650 nm) and the calculated extinction coefficient of the U2AF651,2LFRET protein (ɛprot=8,940 M−1 cm−1 at 280 nm), and correcting for the absorbance (A) of the dyes at 280 nm (GE Healthcare, Amersham CyDye Maleimide product booklet): + + + METHODS + paragraph + 49725 + For smFRET experiments with a ‘strong', homogeneous Py tract, we used the prototypical AdML sequence (5′-CCUUUUUUUUCC-3′). To investigate the inter-RRM separation in the presence of a ‘weak' Py tract interrupted by purines, we compared the U2AF651,2L affinity for a purine-interrupted Py tract comprising an rUrUrUrUrU tract that is expected to bind U2AF65 RRM2/inter-RRM linker, a central rArA and an rUrUrUrCrC tract that is expected to bind RRM1. The tandem purines represent a compromise between significant inhibition of U2AF65 binding by longer A interruptions and an approximately five-fold penalty for the rArA mutation in the AdML Py tract (Supplementary Fig. 6b,c). To maintain avidity and provide flanking phosphoryl groups in case of inter-RRM adjustment, we included the 5′-C and 3′-A of parent AdML sequence, which are respective low-affinity nucleotides for binding RRM2 and RRM1 (ref.), in the final rArA-interrupted RNA oligonucleotide (5′-rCrUrUrUrUrUrArArUrUrUrCrCrA-3′). + + + METHODS + paragraph + 50732 + For the reversed immobilization of RNA via a complementary biotinyl-DNA primer experiment, the AdML Py-tract RNA was extended to include the DNA counterpart of downstream AdML intron/exon sequences that were complementary to the biotinyl-DNA primer. To increase separation from the slide surface, a hexaethylene glycol linker (18PEG) was inserted between the AdML Py-tract RNA and the tethered DNA duplex. The tethered oligonucleotide sequences included: 5′-rCrCrUrUrUrUrUrUrUrUrCrC/18PEG/dAdCdAdGdCdTdCdGdCdG-dGdTdTdGdAdGdGdAdCdAdA-3′ annealed to 5′-biotinyl-dTdTdGdTdCdCdTdCdAdA-dCdCdGdCdGdAdGdCdTdGdT-3' (purchased with high-performance liquid chromatography purification from Integrated DNA Technologies). + + + METHODS + title_2 + 51448 + Single-molecule FRET data acquisition and analysis + + + METHODS + paragraph + 51499 + The smFRET measurements were carried out at room temperature in 50 mM HEPES, pH 7.4, 100 mM NaCl. The imaging buffer also contained an oxygen-scavenging system (0.8 mg ml−1 glucose oxidase, 0.625% glucose, 0.02 mg ml−1 catalase), 1.5 mM Trolox (used to eliminate Cy5 blinking) and 6 mM β-mercaptoethanol. The sample chamber was assembled from quartz microscope slides and glass cover slips coated with a mixture of m-PEG and biotin-PEG and pre-treated with neutravidin (0.2 mg ml−1). Surface tethering of doubly labelled U2AF651,2LFRET(Cy3/Cy5) via its His-tag (Fig. 6c–f,i,j; Supplementary Fig. 7a,b) was achieved by pre-incubating the sample chamber with 50 nM biotinyl-NTA resin (Biotin-X NTA, Biotium), pre-loaded with three-fold excess NiSO4) for 20 min before addition of 5 nM U2AF651,2LFRET(Cy3/Cy5). After 10 min, unbound sample was removed by washing the sample chamber with imaging buffer. The AdML RNA ligand was added to the imaging buffer at a concentration of 5 μM (100-fold higher than the measured KD value), whereas the rArA-interrupted RNA was added at a concentration of 10 μM. Alternatively, to detect binding of doubly labelled U2AF651,2LFRET(Cy3/Cy5) to surface-tethered RNA ligand (Fig. 6g,h Supplementary Fig. 7c–g), 10 nM AdML RNA (pre-annealed to biotinyl-DNA primer) was incubated in the neutravidin-treated sample chamber for 20 min, and 1 nM U2AF651,2LFRET(Cy3/Cy5) was then added to the imaging buffer. + + + METHODS + paragraph + 52983 + Single-molecule FRET measurements were taken as previously described. An Olympus IX71 inverted microscope, equipped with a UPlanApo 60x/1.20w objective lens, a 532 nm laser (Spectra-Physics) for excitation of Cy3 dyes, and a 642 nm laser (Spectra-Physics) for excitation of Cy5 dyes was used. Total internal reflection (TIR) was obtained by a quartz prism (ESKMA Optics). Fluorescence emission was split into Cy3 and Cy5 fluorescence using a dual view imaging system DV2 (Photometrics) equipped with a 630 nm dichroic mirror and recorded via an Andor iXon+ EMCCD camera. Movies were recorded using the Single software (downloaded from Prof. Taekjip Ha's laboratory website at the University of Illinois at Urbana-Champaign, physics.illinois.edu/cplc/software), with the exposure time set at 100 ms. We typically took up to five 5-minute-long movies while imaging different sections of the slide for each sample. Before each measurement, we checked for non-specific binding by adding doubly-labeled U2Fret to the slide in the absence of neutravidin and imaging the slide. Non-specific binding was virtually absent. + + + METHODS + paragraph + 54105 + Collected data sets were processed with IDL and Matlab softwares, using scripts downloaded from a freely available source: physics.illinois.edu/cplc/software. Apparent FRET efficiencies (Eapp) were calculated from the emission intensities of donor (ICy3) and acceptor (ICy5) as follows: Eapp=ICy5/(ICy5+ICy3). The FRET distribution histograms were built from traces that showed single-step photobleaching in both Cy3 and Cy5 signals using a Matlab script generously provided by Prof. Peter Cornish (University of Missouri, Columbia). Anti-correlated changes in donor and acceptor intensities with constant sum of intensities indicated the presence of an energy transfer in single molecules labelled with one donor and one acceptor dye. All histograms were smoothed with a five-point window and plotted using Origin software (Origin Lab Co). Idealization of FRET trajectories was done using the hidden Markov model algorithms via HaMMy software (http://bio.physics.illinois.edu/HaMMy.asp). Transition density plots were generated from transitions detected in idealized FRET trajectories obtained by HaMMy fit of raw FRET traces via Matlab. Frequency of transitions from starting FRET efficiency value (x-axis) to ending FRET efficiency value (y-axis) was represented by a heat map. The range of FRET efficiencies from 0 to 1 was separated in 200 bins. The resulting heat map was normalized to the most populated bin in the plot; the lower- and upper-bound thresholds were set to 20% and 100% of the most populated bin, respectively. + + + METHODS + paragraph + 55637 + The surface contour plots were generated as follows: the individual single-molecule FRET traces (for example, Fig. 6g of the main text and Supplementary Fig. 7e,f) were post synchronized at the first time point showing non-zero (>0.15) FRET efficiency, corresponding to binding. The time range (x-axis, 0–10 s) was separated into 100 bins. The FRET efficiency range (y-axis, 0–1 FRET) was separated into 100 bins. A heat map is used to represent the frequency of sampling of each FRET state over time; frequency in each bin was normalized to the most populated bin in the plot with lower- and upper-bound thresholds set at 10% and 80% of the most populated bin, respectively. + + + METHODS + title_1 + 56319 + Additional information + + + METHODS + paragraph + 56342 + Accession codes: Coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5EV1, 5EV2, 5EV3 and 5EV4 for respective U2AF651,2L-oligonucleotide structures (i)–(iv). + + + METHODS + paragraph + 56550 + How to cite this article: Agrawal, A. A. et al. An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal. Nat. Commun. 7:10950 doi: 10.1038/ncomms10950 (2016). + + + SUPPL + title_1 + 56732 + Supplementary Material + + + 470 + 476 + surname:Wang;given-names:E. T. + 18978772 + REF + Nature + ref + 456 + 2008 + 56755 + Alternative isoform regulation in human tissue transcriptomes + + + 525 + 560 + surname:Burge;given-names:C. B. + surname:Tuschl;given-names:T. + surname:Sharp;given-names:P. A. + REF + The RNA World + ref + 1999 + 56817 + + + 472 + 482 + surname:Singh;given-names:R. K. + surname:Cooper;given-names:T. A. + 22819011 + REF + Trends Mol. Med. + ref + 18 + 2012 + 56818 + Pre-mRNA splicing in disease and therapeutics + + + 1540 + 1549 + surname:Scott;given-names:L. M. + surname:Rebel;given-names:V. I. + 24052622 + REF + J. Natl Cancer Inst. + ref + 105 + 2013 + 56864 + Acquired mutations that affect pre-mRNA splicing in hematologic malignancies and solid tumors + + + 135 + 140 + surname:Golling;given-names:G. + 12006978 + REF + Nat. Genet. + ref + 31 + 2002 + 56958 + Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development + + + 207 + 219 + surname:Ruskin;given-names:B. + surname:Zamore;given-names:P. D. + surname:Green;given-names:M. R. + 2963698 + REF + Cell + ref + 52 + 1988 + 57063 + A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly + + + 781 + 787 + surname:Berglund;given-names:J. A. + surname:Chua;given-names:K. + surname:Abovich;given-names:N. + surname:Reed;given-names:R. + surname:Rosbash;given-names:M. + 9182766 + REF + Cell + ref + 89 + 1997 + 57142 + The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC + + + 858 + 867 + surname:Berglund;given-names:J. A. + surname:Abovich;given-names:N. + surname:Rosbash;given-names:M. + 9512519 + REF + Genes Dev. + ref + 12 + 1998 + 57236 + A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition + + + 832 + 835 + surname:Wu;given-names:S. + surname:Romfo;given-names:C. M. + surname:Nilsen;given-names:T. W. + surname:Green;given-names:M. R. + 10617206 + REF + Nature + ref + 402 + 1999 + 57333 + Functional recognition of the 3' splice site AG by the splicing factor U2AF35 + + + 835 + 838 + surname:Zorio;given-names:D. A. + surname:Blumenthal;given-names:T. + 10617207 + REF + Nature + ref + 402 + 1999 + 57411 + Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans + + + 838 + 841 + surname:Merendino;given-names:L. + surname:Guth;given-names:S. + surname:Bilbao;given-names:D. + surname:Martinez;given-names:C. + surname:Valcarcel;given-names:J. + 10617208 + REF + Nature + ref + 402 + 1999 + 57488 + Inhibition of msl-2 splicing by Sex-lethal reveals interaction between U2AF35 and the 3' splice site AG + + + 1173 + 1176 + surname:Singh;given-names:R. + surname:Valcarcel;given-names:J. + surname:Green;given-names:M. R. + 7761834 + REF + Science + ref + 268 + 1995 + 57592 + Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins + + + 609 + 614 + surname:Zamore;given-names:P. D. + surname:Patton;given-names:J. G. + surname:Green;given-names:M. R. + 1538748 + REF + Nature + ref + 355 + 1992 + 57696 + Cloning and domain structure of the mammalian splicing factor U2AF + + + 3859 + 3873 + surname:Jenkins;given-names:J. L. + surname:Agrawal;given-names:A. A. + surname:Gupta;given-names:A. + surname:Green;given-names:M. R. + surname:Kielkopf;given-names:C. L. + 23376934 + REF + Nucleic Acids Res. + ref + 41 + 2013 + 57763 + U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs + + + 49 + 59 + surname:Sickmier;given-names:E. A. + 16818232 + REF + Mol. Cell + ref + 23 + 2006 + 57894 + Structural basis of polypyrimidine tract recognition by the essential pre-mRNA splicing factor, U2AF65 + + + 408 + 411 + surname:Mackereth;given-names:C. D. + 21753750 + REF + Nature + ref + 475 + 2011 + 57997 + Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF + + + 50572 + 50577 + surname:Kent;given-names:O. A. + surname:Reayi;given-names:A. + surname:Foong;given-names:L. + surname:Chilibeck;given-names:K. A. + surname:MacMillan;given-names:A. M. + 14506271 + REF + J. Biol. Chem. + ref + 278 + 2003 + 58082 + Structuring of the 3' splice site by U2AF65 + + + 1706 + 1709 + surname:Valcarcel;given-names:J. + surname:Gaur;given-names:R. K. + surname:Singh;given-names:R. + surname:Green;given-names:M. R. + 8781232 + REF + Science + ref + 273 + 1996 + 58126 + Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA + + + 363 + 373 + surname:Shen;given-names:H. + surname:Green;given-names:M. R. + 15525510 + REF + Mol. Cell + ref + 16 + 2004 + 58229 + A pathway of sequential arginine-serine-rich domain-splicing signal interactions during mammalian spliceosome assembly + + + 17420 + 17425 + surname:Agrawal;given-names:A. A. + surname:McLaughlin;given-names:K. J. + surname:Jenkins;given-names:J. L. + surname:Kielkopf;given-names:C. L. + 25422459 + REF + Proc. Natl Acad. Sci. USA + ref + 111 + 2014 + 58348 + Structure-guided U2AF65 variant improves recognition and splicing of a defective pre-mRNA + + + 133 + 180 + surname:Janin;given-names:J. + surname:Bahadur;given-names:R. P. + surname:Chakrabarti;given-names:P. + 18812015 + REF + Q. Rev. Biophys. + ref + 41 + 2008 + 58438 + Protein-protein interaction and quaternary structure + + + 33641 + 33649 + surname:Jenkins;given-names:J. L. + surname:Shen;given-names:H. + surname:Green;given-names:M. R. + surname:Kielkopf;given-names:C. L. + 18842594 + REF + J. Biol. Chem. + ref + 283 + 2008 + 58491 + Solution conformation and thermodynamic characteristics of RNA binding by the splicing factor U2AF65 + + + 4523 + 4534 + surname:Ito;given-names:T. + surname:Muto;given-names:Y. + surname:Green;given-names:M. R. + surname:Yokoyama;given-names:S. + 10449418 + REF + EMBO J. + ref + 18 + 1999 + 58592 + Solution structures of the first and second RNA-binding domains of human U2 small nuclear ribonucleoprotein particle auxiliary factor (U2AF65) + + + 8183 + 8190 + surname:Pacheco;given-names:T. R. + surname:Coelho;given-names:M. B. + surname:Desterro;given-names:J. M. + surname:Mollet;given-names:I. + surname:Carmo-Fonseca;given-names:M. + 16940179 + REF + Mol. Cell. Biol. + ref + 26 + 2006 + 58735 + In vivo requirement of the small subunit of U2AF for recognition of a weak 3' splice site + + + 5223 + 5225 + surname:Jenkins;given-names:J. L. + surname:Laird;given-names:K. M. + surname:Kielkopf;given-names:C. L. + 22702716 + REF + Biochemistry + ref + 51 + 2012 + 58825 + A broad range of conformations contribute to the solution ensemble of the essential splicing factor U2AF65 + + + 7068 + 7076 + surname:Huang;given-names:J. R. + 24734879 + REF + J. Am. Chem. Soc. + ref + 136 + 2014 + 58932 + Transient electrostatic interactions dominate the conformational equilibrium sampled by multidomain splicing factor U2AF65: a combined NMR and SAXS study + + + 211 + 231 + surname:Dietrich;given-names:A. + surname:Buschmann;given-names:V. + surname:Muller;given-names:C. + surname:Sauer;given-names:M. + 11999691 + REF + J. Biotechnol. + ref + 82 + 2002 + 59086 + Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single molecule data + + + 507 + 516 + surname:Roy;given-names:R. + surname:Hohng;given-names:S. + surname:Ha;given-names:T. + 18511918 + REF + Nat. Methods + ref + 5 + 2008 + 59256 + A practical guide to single-molecule FRET + + + 241 + 247 + surname:Haferlach;given-names:T. + 24220272 + REF + Leukemia + ref + 28 + 2014 + 59298 + Landscape of genetic lesions in 944 patients with myelodysplastic syndromes + + + 64 + 69 + surname:Yoshida;given-names:K. + 21909114 + REF + Nature + ref + 478 + 2011 + 59374 + Frequent pathway mutations of splicing machinery in myelodysplasia + + + 595 + 605 + surname:Kielkopf;given-names:C. L. + surname:Rodionova;given-names:N. A. + surname:Green;given-names:M. R. + surname:Burley;given-names:S. K. + 11551507 + REF + Cell + ref + 106 + 2001 + 59441 + A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer + + + 539 + 546 + surname:Csermely;given-names:P. + surname:Palotai;given-names:R. + surname:Nussinov;given-names:R. + 20541943 + REF + Trends Biochem. Sci. + ref + 35 + 2010 + 59542 + Induced fit, conformational selection and independent dynamic segments: an extended view of binding events + + + 13737 + 13741 + surname:Hammes;given-names:G. G. + surname:Chang;given-names:Y. C. + surname:Oas;given-names:T. G. + 19666553 + REF + Proc. Natl Acad. Sci. USA + ref + 106 + 2009 + 59649 + Conformational selection or induced fit: a flux description of reaction mechanism + + + 18055 + 18060 + surname:Hanson;given-names:J. A. + 17989222 + REF + Proc. Natl Acad. Sci. USA + ref + 104 + 2007 + 59731 + Illuminating the mechanistic roles of enzyme conformational dynamics + + + 838 + 844 + surname:Henzler-Wildman;given-names:K. A. + 18026086 + REF + Nature + ref + 450 + 2007 + 59800 + Intrinsic motions along an enzymatic reaction trajectory + + + e1002054 + surname:Silva;given-names:D. A. + surname:Bowman;given-names:G. R. + surname:Sosa-Peinado;given-names:A. + surname:Huang;given-names:X. + 21637799 + REF + PLoS Comput. Biol. + ref + 7 + 2011 + 59857 + A role for both conformational selection and induced fit in ligand binding by the LAO protein + + + 19346 + 19351 + surname:Wlodarski;given-names:T. + surname:Zagrovic;given-names:B. + 19887638 + REF + Proc. Natl Acad. Sci. USA + ref + 106 + 2009 + 59951 + Conformational selection and induced fit mechanism underlie specificity in noncovalent interactions with ubiquitin + + + 313 + 318 + surname:Kim;given-names:E. + 23502425 + REF + Nat. Chem. Biol. + ref + 9 + 2013 + 60066 + A single-molecule dissection of ligand binding to a protein with intrinsic dynamics + + + 14075 + 14083 + surname:Suddala;given-names:K. C. + surname:Wang;given-names:J. + surname:Hou;given-names:Q. + surname:Walter;given-names:N. G. + 26471732 + REF + J. Am. Chem. Soc. + ref + 137 + 2015 + 60150 + Mg(2+) shifts ligand-mediated folding of a riboswitch from induced-fit to conformational selection + + + 802 + 813 + surname:Wang;given-names:Z. + surname:Burge;given-names:C. B. + 18369186 + REF + RNA + ref + 14 + 2008 + 60249 + Splicing regulation: from a parts list of regulatory elements to an integrated splicing code + + + e13 + surname:Cavaluzzi;given-names:M. J. + surname:Borer;given-names:P. N. + 14722228 + REF + Nucleic Acids Res. + ref + 32 + 2004 + 60342 + Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA + + + 975 + 987 + surname:Gama-Carvalho;given-names:M. + 9166400 + REF + J. Cell Biol. + ref + 137 + 1997 + 60435 + Targeting of U2AF65 to sites of active splicing in the nucleus + + + 409 + 411 + surname:Jancarik;given-names:J. + surname:Kim;given-names:S.-H. + REF + J. Appl. Cryst. + ref + 24 + 1991 + 60498 + Sparse matrix sampling: a screening method for crystallization of proteins + + + 457 + 459 + surname:Sickmier;given-names:E. A. + surname:Frato;given-names:K. E. + surname:Kielkopf;given-names:C. L. + REF + Acta Crystallogr. + ref + F62 + 2006 + 60573 + Crystallization and preliminary X-ray analysis of U2AF65 variant in complex with a polypyrimidine tract analogue by use of protein engineering + + + 213 + 221 + surname:Adams;given-names:P. D. + REF + Acta Crystallogr. + ref + D66 + 2010 + 60716 + PHENIX: a comprehensive Python-based system for macromolecular structure solution + + + 2126 + 2132 + surname:Emsley;given-names:P. + surname:Cowtan;given-names:K. + REF + Acta Crystallogr. + ref + D60 + 2004 + 60798 + Coot: model-building tools for molecular graphics + + + W615 + W619 + surname:Davis;given-names:I. W. + surname:Murray;given-names:L. W. + surname:Richardson;given-names:J. S. + surname:Richardson;given-names:D. C. + 15215462 + REF + Nucleic Acids Res. + ref + 32 + 2004 + 60848 + MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes + + + surname:DeLano;given-names:W. L. + REF + The PyMOL Molecular Graphics System, Version 1.8. + ref + 2015 + 60949 + + + 15060 + 15065 + surname:Salsi;given-names:E. + surname:Farah;given-names:E. + surname:Dann;given-names:J. + surname:Ermolenko;given-names:D. N. + 25288752 + REF + Proc. Natl Acad. Sci. USA + ref + 111 + 2014 + 60950 + Following movement of domain IV of elongation factor G during ribosomal translocation + + + 1941 + 1951 + surname:McKinney;given-names:S. A. + surname:Joo;given-names:C. + surname:Ha;given-names:T. + 16766620 + REF + Biophys. J. + ref + 91 + 2006 + 61036 + Analysis of single-molecule FRET trajectories using hidden Markov modeling + + + SUPPL + footnote + 61111 + Author contributions A.A.A. performed crystallization, refinement, molecular biology and most RNA-binding experiments. E.S. performed smFRET experiments. R.C. labelled protein and S.H. completed a subset of RNA-binding experiments. C.L.K. cryoprotected crystals, collected crystallographic data and built structures. J.L.J. performed molecular replacement and completed structure refinements. M.R.G. and C.L.K. conceived the study. C.L.K. and D.N.E. designed the experiments. C.L.K., D.N.E. and E.S. wrote the paper with input from J.L.J. and A.A.A. + + + ncomms10950-f1.jpg + f1 + FIG + fig_title_caption + 61661 + The intact U2AF65 RRM1/RRM2-containing domain and flanking residues are required for binding contiguous Py tracts. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:30:59Z + + intact + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:16Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:39:03Z + + contiguous + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:51:58Z + + Py tracts + + + + ncomms10950-f1.jpg + f1 + FIG + fig_caption + 61776 + (a) Domain organization of full-length (fl) U2AF65 and constructs used for RNA binding and structural experiments. The N- and C-terminal residue numbers are indicated. An internal deletion (d, Δ) of residues 238–257 removes a portion of the inter-RRM linker from the dU2AF651,2 and dU2AF651,2L constructs. (b) Comparison of the apparent equilibrium affinities of various U2AF65 constructs for binding the prototypical AdML Py tract (5′-CCCUUUUUUUUCC-3′). The flU2AF65 protein includes a heterodimerization domain of the U2AF35 subunit to promote solubility and folding. The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t-tests with Welch's correction are indicated as follows: **P<0.01; NS, not significant, P>0.05. The purified protein and average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 1. RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:34:30Z + + full-length + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:03:48Z + + fl + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:43:52Z + + RNA + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:03:59Z + + d + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:04:03Z + + Δ + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:04:06Z + + 238–257 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:06Z + + inter-RRM linker + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:54:50Z + + dU2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:04:12Z + + equilibrium affinities + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:04:30Z + + 5′-CCCUUUUUUUUCC-3′ + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T14:05:17Z + + flU2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:49:57Z + + heterodimerization domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T14:50:42Z + + U2AF35 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:05:26Z + + equilibrium dissociation constants + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:05:32Z + + KD + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T14:05:17Z + + flU2AF65 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:54:12Z + + RNA sequence specificities + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T14:05:17Z + + flU2AF65 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:41Z + + U2AF651,2L + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:50:00Z + + C-rich + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:52:02Z + + Py tracts + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:05:32Z + + KD + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:04:49Z + + 5′-CCUUUUCCCCCCC-3′ + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T14:05:17Z + + flU2AF65 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:05:32Z + + KD + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:05:09Z + + 5′-CCCCCCCUUUUCC-3′ + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T14:05:17Z + + flU2AF65 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:14:07Z + + average apparent equilibrium affinity + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:14:16Z + + KA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:14:18Z + + average fitted fluorescence anisotropy RNA-binding curves + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:50:04Z + + RRM + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:14:23Z + + RNA recognition motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:14:39Z + + RS + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:14:27Z + + arginine-serine rich + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:14:43Z + + UHM + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:14:45Z + + U2AF homology motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:14:48Z + + ULM + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:14:50Z + + U2AF ligand motif + + + + ncomms10950-f2.jpg + f2 + FIG + fig_title_caption + 63443 + Structures of U2AF651,2L recognizing a contiguous Py tract. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:15:10Z + + Structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:39:03Z + + contiguous + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + + ncomms10950-f2.jpg + f2 + FIG + fig_caption + 63503 + (a) Alignment of oligonucleotide sequences that were co-crystallized in the indicated U2AF651,2L structures. The regions of RRM1, RRM2 and linker contacts are indicated above by respective black and blue arrows from N- to C-terminus. For clarity, we consistently number the U2AF651,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty. The prior dU2AF651,2 nucleotide-binding sites are given in parentheses (site 4' interacts with dU2AF65 RRM1 and RRM2 by crystallographic symmetry). Italics, disordered in the structure. (b) Stereo views of a ‘kicked' 2|Fo|−|Fc| electron density map contoured at 1σ for the inter-RRM linker, N- and C-terminal residues (blue) or bound oligonucleotide of a representative U2AF651,2L structure (structure iv, bound to 5′-(P)rUrUrUdUrUrU(BrdU)dUrC) (magenta). (c) Cartoon diagram of this structure. Crystallographic statistics are given in Table 1 and the overall conformations of U2AF651,2L and prior dU2AF651,2/U2AF651,2 structures are compared in Supplementary Fig. 2. BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:52:55Z + + Alignment + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:52:07Z + + oligonucleotide + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:52:58Z + + co-crystallized + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:17:15Z + + structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:16Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:50:09Z + + linker + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:53:30Z + + nucleotide-binding sites + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:17:22Z + + co-crystallized + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:17:35Z + + oligonucleotide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:17:43Z + + nucleotides + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:17:47Z + + first binding site + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:53:30Z + + nucleotide-binding sites + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:55:13Z + + dU2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:16Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:17:53Z + + 2|Fo|−|Fc| electron density map + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:06Z + + inter-RRM linker + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:52:14Z + + oligonucleotide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:18:17Z + + structures + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:18:20Z + + BrdU + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:18:22Z + + 5-bromo-deoxy-uridine + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:18:26Z + + d + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:18:28Z + + deoxy-ribose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:18:31Z + + P- + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:18:37Z + + 5′-phosphorylation + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:18:40Z + + r + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:18:43Z + + ribose + + + + ncomms10950-f3.jpg + f3 + FIG + fig_title_caption + 64725 + Representative views of the U2AF651,2L interactions with each new nucleotide of the bound Py tract. + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:16:51Z + + nucleotide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:19:16Z + + bound + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + + ncomms10950-f3.jpg + f3 + FIG + fig_caption + 64825 + New residues of the U2AF651,2L structures are coloured a darker shade of blue, apart from residues that were tested by site-directed mutagenesis, which are coloured yellow. The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. (i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t-tests with Welch's correction are indicated as follows: **P<0.01; *P<0.05; NS, not significant, P>0.05. The average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4a–c. + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:21:31Z + + structures + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:21:34Z + + site-directed mutagenesis + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:53:30Z + + nucleotide-binding sites + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:21:50Z + + structure + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:21:47Z + + first and seventh U2AF651,2L-binding sites + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T14:21:57Z + + dU2AF651,2–RNA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:00Z + + structure + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:03Z + + structures + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:22:09Z + + ninth site + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:05Z + + structure + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:22:12Z + + ribose nucleotide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:17:58Z + + rU2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:16:19Z + + rU3 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:00:39Z + + rU4 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:23Z + + rU5 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T17:03:32Z + + rU6 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:28Z + + dU8 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:32Z + + dU9 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:35Z + + rC9 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:39Z + + equilibrium affinities + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:23:12Z + + KA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:42Z + + wild type + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:45Z + + mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:44:35Z + + 5′-CCCUUUUUUUUCC-3′ + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:49Z + + equilibrium dissociation constants + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:05:32Z + + KD + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:52Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:54Z + + wild type + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:22:57Z + + WT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:46Z + + R227A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:54Z + + V254P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:39Z + + Q147A + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:23:09Z + + KA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:23:17Z + + RNA-binding curves + + + + ncomms10950-f4.jpg + f4 + FIG + fig_title_caption + 66426 + The U2AF65 linker/RRM and inter-RRM interactions. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T12:04:16Z + + linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T12:04:25Z + + RRM + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T12:04:36Z + + RRM + + + + ncomms10950-f4.jpg + f4 + FIG + fig_caption + 66476 + (a) Contacts of the U2AF65 inter-RRM linker with the RRMs. A semi-transparent space-filling surface is shown for the RRM1 (green) and RRM2 (light blue). Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. Other linker residues are coloured either dark blue for new residues in the U2AF651,2L structure or light blue for the remaining inter-RRM residues. The central panel shows an overall view with stick diagrams for mutated residues; boxed regions are expanded to show the C-terminal (bottom left) and central linker regions (top) at the inter-RRM interfaces, and N-terminal linker region contacts with RRM1 (bottom right). (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t-tests with Welch's correction are indicated as follows: **P<0.01; *P<0.05; NS, not significant, P>0.05. The fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4d–j. (c) Close view of the U2AF65 RRM1/RRM2 interface following a two-fold rotation about the x-axis relative to a. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:30:06Z + + inter-RRM linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:16Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:25Z + + RRM2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:21:39Z + + V249 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:21:47Z + + V250 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:56Z + + V254 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:27:47Z + + mutated + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:27:53Z + + V249G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:27:59Z + + V250G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:06Z + + V254G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:08Z + + 3Gly mutant + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:22:56Z + + S251 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:59:04Z + + T252 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:23:07Z + + V253 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:23:21Z + + P255 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:58:56Z + + V254 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:25Z + + mutated + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:33Z + + S251G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:40Z + + T252G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:47Z + + V253G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:06Z + + V254G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:57Z + + P255G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:29:00Z + + 5Gly mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:29:07Z + + S251N + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:29:15Z + + T252L + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:29:23Z + + V253A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:29:30Z + + V254L + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:29:36Z + + P255A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:29:39Z + + NLALA mutant + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:25:36Z + + M144 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:11:45Z + + L235 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:11:53Z + + M238 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:12:46Z + + V244 + + 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2023-03-21T14:30:34Z + + M238G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:30:42Z + + V244G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:30:49Z + + V246G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:27:53Z + + V249G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:27:59Z + + V250G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:33Z + + S251G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:40Z + + T252G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:47Z + + V253G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:06Z + + V254G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:28:57Z + + P255G + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:31:13Z + + 12Gly mutant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:31:16Z + + linker + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T12:53:03Z + + inter-RRM + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:31:24Z + + central linker regions + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:31:27Z + + inter-RRM interfaces + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:16Z + + RRM1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:31:33Z + + equilibrium affinities + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:31:36Z + + KA + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:44:52Z + + 5′-CCCUUUUUUUUCC-3′ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:22:39Z + + wild-type + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:31:47Z + + 3Gly + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:31:49Z + + 5Gly + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:31:51Z + + NLALA + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:31:54Z + + 12Gly + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:31:56Z + + linker deletions + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:14Z + + dU2AF651,2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:35:26Z + + minimal + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:50:16Z + + RRM1–RRM2 region + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:32:13Z + + 148–237 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:32:16Z + + 258–336 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:54:50Z + + dU2AF651,2L + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:32:18Z + + 141–237 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:32:21Z + + 258–342 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:32:11Z + + equilibrium dissociation constants + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:05:32Z + + KD + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:32:26Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:32:29Z + + wild type + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:32:31Z + + WT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:32:38Z + + 3Gly + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:32:41Z + + 5Gly + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:32:44Z + + 12Gly + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:32:47Z + + NLALA + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:54:50Z + + dU2AF651,2L + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:15Z + + dU2AF651,2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:32:51Z + + 3Mut + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:32:54Z + + KA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:32:57Z + + RNA-binding curves + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:58:45Z + + RRM1/RRM2 interface + + + + ncomms10950-f5.jpg + f5 + FIG + fig_title_caption + 68622 + U2AF65 inter-domain residues are important for splicing a representative pre-mRNA substrate in human cells. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:33:34Z + + pre-mRNA + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:18:14Z + + human + + + + ncomms10950-f5.jpg + f5 + FIG + fig_caption + 68730 + (a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset). (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). The average percentages and s.d.'s are given among four independent biological replicates. ****P<0.0001 for two-tailed unpaired t-test with Welch's correction. Protein overexpression and qRT-PCR results are shown in Supplementary Fig. 5. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:41:41Z + + pyPY + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:19:52Z + + splice sites + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:42:43Z + + py + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:42:32Z + + PY + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:00Z + + RT-PCR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:41:41Z + + pyPY + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:12Z + + co-transfected + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:41:41Z + + pyPY + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:22:39Z + + wild-type + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:36:19Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:36:23Z + + mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:25Z + + 3Mut + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:39Z + + Q147A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:46Z + + R227A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:36:54Z + + V254P + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:42:43Z + + py + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:37:02Z + + mRNA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T09:41:41Z + + pyPY + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:37:07Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:37:09Z + + 3Mut + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:35:37Z + + Protein overexpression + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:35:54Z + + qRT-PCR + + + + ncomms10950-f6.jpg + f6 + FIG + fig_title_caption + 69621 + RNA binding stabilizes the side-by-side conformation of U2AF65 RRMs. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:08Z + + side-by-side + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + + ncomms10950-f6.jpg + f6 + FIG + fig_caption + 69690 + (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. The U2AF651,2LFRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site. (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). In g and h, the immobilization protocol was reversed. The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1 nM) was added to AdML RNA–polyethylene-glycol-linker–DNA oligonucleotide (10 nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line). Typical single-molecule FRET traces (c,e,g,i) show fluorescence intensities from Cy3 (green) and Cy5 (red) and the calculated apparent FRET efficiency (blue). Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). N is the number of single-molecule traces compiled for each histogram. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:40:56Z + + FRET + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:16Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:26Z + + RRM2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:46:29Z + + crystal structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:08Z + + side-by-side + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:41:02Z + + Py-tract oligonucleotide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:20Z + + closed + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:53:03Z + + NMR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:42:22Z + + PRE + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:30:38Z + + U2AF651,2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:26Z + + RRM2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:33Z + + U2AF651,2LFRET + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:55:18Z + + A181C + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:55:21Z + + Q324C + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:36Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:44Z + + Cy5 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:41:11Z + + fluorophores + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:34Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:36Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:44Z + + Cy5 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:46:00Z + + biotin-NTA/Ni+2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:05:45Z + + absence of + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:42:22Z + + ligands + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:41:16Z + + Py-tract RNA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:45:20Z + + 5′-CCUUUUUUUUCC-3′ + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-22T10:58:03Z + + adenosine + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:52:19Z + + RNA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:45:50Z + + 5′-CUUUUUAAUUUCCA-3′ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:28Z + + untethered + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:34Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:36Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:44Z + + Cy5 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:42:32Z + + RNA–polyethylene-glycol-linker–DNA oligonucleotide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:42:36Z + + biotinyl-DNA oligonucleotide + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:42:40Z + + single-molecule FRET + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:36Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:44Z + + Cy5 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:54:17Z + + calculated apparent FRET efficiency + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:28Z + + untethered + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:31Z + + RNA-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:34Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:36Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:44Z + + Cy5 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:54:21Z + + Histograms + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:54:28Z + + distribution of FRET values + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:42:49Z + + RNA-free + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:42:55Z + + slide-tethered + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:34Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:36Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:44Z + + Cy5 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:31Z + + RNA-bound + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:43:03Z + + slide-tethered + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:34Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:36Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:44Z + + Cy5 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T16:36:16Z + + AdML + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:31Z + + RNA-bound + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:28Z + + untethered + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:34Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:36Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:44Z + + Cy5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:52:31Z + + RNA-bound + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:43:15Z + + slide-tethered + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:04:34Z + + U2AF651,2LFRET + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:36Z + + Cy3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T12:58:44Z + + Cy5 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:04:09Z + + traces + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-22T10:46:20Z + + histogram + + + + ncomms10950-f7.jpg + f7 + FIG + fig_title_caption + 71544 + Schematic models of U2AF65 recognizing the Py tract. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T16:14:10Z + + Py tract + + + + ncomms10950-f7.jpg + f7 + FIG + fig_caption + 71597 + (a) Diagram of the U2AF65, SF1 and U2AF35 splicing factors bound to the consensus elements of the 3′ splice site. A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3′ splice site are unknown. MDS-relevant mutated residues of U2AF65 are shown as yellow spheres (L187 and M144). (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. Alternatively, a conformation of U2AF65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF65 population towards the ∼0.45 FRET value. RRM1, green; RRM2, pale blue; RRM extensions/linker, blue. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T14:50:48Z + + SF1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-21T14:50:51Z + + U2AF35 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:09:33Z + + 3′ splice site + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:13:37Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:46:27Z + + nucleotides + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:20:05Z + + branch point sequence + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:24:06Z + + BPS + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:46:43Z + + AG dinucleotide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:09:33Z + + 3′ splice site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:46:35Z + + L187 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:25:36Z + + M144 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:52:25Z + + Py-tract RNA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T14:46:41Z + + high FRET + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:20Z + + closed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:42:12Z + + back-to-back + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:45:31Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T13:42:22Z + + PRE + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T14:46:49Z + + NMR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:20Z + + FRET value + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:12Z + + open + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:08Z + + side-by-side + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:16:37Z + + RRMs + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:35:42Z + + U2AF651,2L + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:29:39Z + + crystal structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:20Z + + FRET value + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-21T14:47:02Z + + RNA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:46:56Z + + RNA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T09:56:12Z + + open + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:45:08Z + + side-by-side + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:12:31Z + + U2AF65 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-21T13:11:20Z + + FRET value + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:16Z + + RRM1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:29:26Z + + RRM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:17:00Z + + RRM extensions + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-21T14:46:59Z + + linker + + + + t1.xml + t1 + TABLE + table_title_caption + 72689 + Crystallographic data and refinement statistics*. + + + t1.xml + t1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"><bold>Structure</bold></th><th align="center" valign="top" charoff="50"><bold>U2AF</bold><sup><bold>65</bold></sup><bold>1,2L with rUrUrUdUdU(BrdU)dUrUrU</bold></th><th align="center" valign="top" charoff="50"><bold>U2AF</bold><sup><bold>65</bold></sup><bold>1,2L with (P)rUrUdUdUrUdU(BrdU)dU</bold></th><th align="center" valign="top" charoff="50"><bold>U2AF</bold><sup><bold>65</bold></sup><bold>1,2L with (P)rUrUdUrUrU(BrdU)dUdU</bold></th><th align="center" valign="top" charoff="50"><bold>U2AF</bold><sup><bold>65</bold></sup><bold>1,2L with (P)rUrUrUdUrUrU(BrdU)dUrC</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50"><italic>Data collection</italic></td><td align="center" valign="top" charoff="50">(i)</td><td align="center" valign="top" charoff="50">(ii)</td><td align="center" valign="top" charoff="50">(iii)</td><td align="center" valign="top" charoff="50">(iv)</td></tr><tr><td align="left" valign="top" charoff="50">Space group</td><td align="center" valign="top" charoff="50">C222<sub>1</sub></td><td align="center" valign="top" charoff="50">C222<sub>1</sub></td><td align="center" valign="top" charoff="50">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="center" valign="top" charoff="50">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td></tr><tr><td align="left" valign="top" charoff="50">Unit cell (Å) <italic>a,b,c</italic></td><td align="center" valign="top" charoff="50">62.1, 114.2, 59.4</td><td align="center" valign="top" charoff="50">61.9, 115.1, 59.5</td><td align="center" valign="top" charoff="50">43.4, 62.2, 77.4</td><td align="center" valign="top" charoff="50">43.5, 63.4, 77.7</td></tr><tr><td align="left" valign="top" charoff="50">Resolution limits (Å)</td><td align="center" valign="top" charoff="50">32.46–2.04</td><td align="center" valign="top" charoff="50">32.57–1.86</td><td align="center" valign="top" charoff="50">38.71–1.50</td><td align="center" valign="top" charoff="50">38.83–1.57</td></tr><tr><td align="left" valign="top" charoff="50">Completeness (%)</td><td align="center" valign="top" charoff="50">95.5 (78.3)</td><td align="center" valign="top" charoff="50">98.7 (95.9)</td><td align="center" valign="top" charoff="50">98.2 (69.8)</td><td align="center" valign="top" charoff="50">98.3 (71.7)</td></tr><tr><td align="left" valign="top" charoff="50">Redundancy</td><td align="center" valign="top" charoff="50">4.6 (4.1)</td><td align="center" valign="top" charoff="50">4.3 (4.2)</td><td align="center" valign="top" charoff="50">6.1 (3.0)</td><td align="center" valign="top" charoff="50">6.2 (3.1)</td></tr><tr><td align="left" valign="top" charoff="50"><italic>I</italic>/<italic>σ</italic>(<italic>I</italic>)</td><td align="center" valign="top" charoff="50">21.2 (4.2)</td><td align="center" valign="top" charoff="50">24.6 (4.6)</td><td align="center" valign="top" charoff="50">38.0 (6.5)</td><td align="center" valign="top" charoff="50">42.9 (6.9)</td></tr><tr><td align="left" valign="top" charoff="50"><italic>R</italic><sub>sym</sub> (%)</td><td align="center" valign="top" charoff="50">3.9 (32.1)</td><td align="center" valign="top" charoff="50">3.9 (30.3)</td><td align="center" valign="top" charoff="50">2.4 (14.8)</td><td align="center" valign="top" charoff="50">2.2 (14.9)</td></tr><tr><td colspan="5" align="center" valign="top" charoff="50"><italic>Refinement</italic></td></tr><tr><td align="left" valign="top" charoff="50"> No. reflections (work/test)</td><td align="center" valign="top" charoff="50">12,124/1,055</td><td align="center" valign="top" charoff="50">17,870/1,456</td><td align="center" valign="top" charoff="50">31,802/1,996</td><td align="center" valign="top" charoff="50">28,162/2,000</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub> (%)</td><td align="center" valign="top" charoff="50">17.3/22.8</td><td align="center" valign="top" charoff="50">15.1/18.8</td><td align="center" valign="top" charoff="50">15.3/18.6</td><td align="center" valign="top" charoff="50">15.4/17.6</td></tr><tr><td colspan="5" align="center" valign="top" charoff="50"><italic>No. atoms</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Protein</td><td align="center" valign="top" charoff="50">2,982</td><td align="center" valign="top" charoff="50">3,052</td><td align="center" valign="top" charoff="50">2,986</td><td align="center" valign="top" charoff="50">2,978</td></tr><tr><td align="left" valign="top" charoff="50"> Oligonucleotide</td><td align="char" valign="top" char="." charoff="50">214</td><td align="char" valign="top" char="." charoff="50">209</td><td align="char" valign="top" char="." charoff="50">198</td><td align="char" valign="top" char="." charoff="50">255</td></tr><tr><td align="left" valign="top" charoff="50"> Water</td><td align="char" valign="top" char="." charoff="50">118</td><td align="char" valign="top" char="." charoff="50">203</td><td align="char" valign="top" char="." charoff="50">263</td><td align="char" valign="top" char="." charoff="50">177</td></tr><tr><td colspan="5" align="center" valign="top" charoff="50"><italic>Bond r.m.s.d.</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Bond lengths (Å)</td><td align="char" valign="top" char="." charoff="50">0.013</td><td align="char" valign="top" char="." charoff="50">0.010</td><td align="char" valign="top" char="." charoff="50">0.008</td><td align="char" valign="top" char="." charoff="50">0.009</td></tr><tr><td align="left" valign="top" charoff="50"> Bond angles (°)</td><td align="char" valign="top" char="." charoff="50">1.32</td><td align="char" valign="top" char="." charoff="50">1.1</td><td align="char" valign="top" char="." charoff="50">1.05</td><td align="char" valign="top" char="." charoff="50">1.05</td></tr><tr><td colspan="5" align="center" valign="top" charoff="50"><italic>&lt;B&gt; factors (Å<sup>2</sup>)</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Protein</td><td align="char" valign="top" char="." charoff="50">46.4</td><td align="char" valign="top" char="." charoff="50">27.4</td><td align="char" valign="top" char="." charoff="50">26.3</td><td align="char" valign="top" char="." charoff="50">26.7</td></tr><tr><td align="left" valign="top" charoff="50"> Oligonucleotide</td><td align="char" valign="top" char="." charoff="50">61.8</td><td align="char" valign="top" char="." charoff="50">35.2</td><td align="char" valign="top" char="." charoff="50">24.5</td><td align="char" valign="top" char="." charoff="50">30.5</td></tr><tr><td align="left" valign="top" charoff="50"> Water</td><td align="char" valign="top" char="." charoff="50">45.2</td><td align="char" valign="top" char="." charoff="50">35.2</td><td align="char" valign="top" char="." charoff="50">30.7</td><td align="char" valign="top" char="." charoff="50">29.8</td></tr></tbody></table> + + 72739 + Structure U2AF651,2L with rUrUrUdUdU(BrdU)dUrUrU U2AF651,2L with (P)rUrUdUdUrUdU(BrdU)dU U2AF651,2L with (P)rUrUdUrUrU(BrdU)dUdU U2AF651,2L with (P)rUrUrUdUrUrU(BrdU)dUrC Data collection (i) (ii) (iii) (iv) Space group C2221 C2221 P212121 P212121 Unit cell (Å) a,b,c 62.1, 114.2, 59.4 61.9, 115.1, 59.5 43.4, 62.2, 77.4 43.5, 63.4, 77.7 Resolution limits (Å) 32.46–2.04 32.57–1.86 38.71–1.50 38.83–1.57 Completeness (%) 95.5 (78.3) 98.7 (95.9) 98.2 (69.8) 98.3 (71.7) Redundancy 4.6 (4.1) 4.3 (4.2) 6.1 (3.0) 6.2 (3.1) I/σ(I) 21.2 (4.2) 24.6 (4.6) 38.0 (6.5) 42.9 (6.9) Rsym (%) 3.9 (32.1) 3.9 (30.3) 2.4 (14.8) 2.2 (14.9) Refinement  No. reflections (work/test) 12,124/1,055 17,870/1,456 31,802/1,996 28,162/2,000  Rwork/Rfree (%) 17.3/22.8 15.1/18.8 15.3/18.6 15.4/17.6 No. atoms  Protein 2,982 3,052 2,986 2,978  Oligonucleotide 214 209 198 255  Water 118 203 263 177 Bond r.m.s.d.  Bond lengths (Å) 0.013 0.010 0.008 0.009  Bond angles (°) 1.32 1.1 1.05 1.05 <B> factors (Å2)  Protein 46.4 27.4 26.3 26.7  Oligonucleotide 61.8 35.2 24.5 30.5  Water 45.2 35.2 30.7 29.8 + + + t1.xml + t1 + TABLE + table_footnote + 73893 + All available crystallographic data was used for refinement. + + + t1.xml + t1 + TABLE + table_footnote + 73954 + *A single crystal was used for each structure. Values from the highest resolution shell are given in parentheses: 2.15–2.04; 1.90–1.86; 1.53–1.50; 1.61–1.57. + + + diff --git a/BioC_XML/4792962_v0.xml b/BioC_XML/4792962_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..bf7e12269749472745587a899796a00d24378d16 --- /dev/null +++ b/BioC_XML/4792962_v0.xml @@ -0,0 +1,11431 @@ + + + + PMC + 20140719 + pmc.key + + 4792962 + CC BY + no + 0 + 0 + + 10.1038/ncomms10900 + ncomms10900 + 4792962 + 26964885 + 10900 + This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ + surname:Huber;given-names:Eva M. + surname:Heinemeyer;given-names:Wolfgang + surname:Li;given-names:Xia + surname:Arendt;given-names:Cassandra S. + surname:Hochstrasser;given-names:Mark + surname:Groll;given-names:Michael + TITLE + front + 7 + 2016 + 0 + A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:54:48Z + + autocatalytic activation + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T11:54:41Z + + 20S proteasome + + + + ABSTRACT + abstract + 87 + Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T11:54:41Z + + 20S proteasome + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:28Z + + active-site + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:33Z + + threonines + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-22T10:33:38Z + + autocatalytically + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:59:38Z + + strict conservation + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:41Z + + threonine + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T12:00:07Z + + mutagenesis + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T12:00:09Z + + X-ray crystallography + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T12:00:12Z + + biochemical assays + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:18Z + + Lys33 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:37Z + + Asp17 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T12:00:53Z + + catalytic triad + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T12:00:57Z + + Substitution + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:10Z + + Cys + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:07Z + + inactivates + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:22Z + + proteasome + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T12:01:44Z + + Thr1Ser + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:50Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:59Z + + active + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:02:10Z + + wild type + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:04:54Z + + Ser1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T12:02:47Z + + propeptide autolysis + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:22Z + + proteasome + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T12:02:54Z + + threonine protease + + + + ABSTRACT + abstract + 1120 + The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear. Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:22Z + + proteasome + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T12:02:54Z + + threonine protease + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:22Z + + proteasome + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:13Z + + active site + + + + INTRO + paragraph + 1415 + The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells. Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. While the inactive α subunits build the two outer rings, the β subunits form the inner rings. Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:13:51Z + + 20S proteasome core particle + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:14:09Z + + non-lysosomal protease + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:14:15Z + + eukaryotic + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:15:20Z + + α + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:15:27Z + + β subunits + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:15:38Z + + heptameric + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:15:45Z + + rings + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:15:49Z + + hollow cylinder + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:15:56Z + + inactive + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:05Z + + α subunits + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:15:45Z + + rings + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:15:27Z + + β subunits + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:15:45Z + + rings + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:15:27Z + + β subunits + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:42Z + + β1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:51Z + + β2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:17:20Z + + proteolytic active centres + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:17:44Z + + prosegments + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:17:55Z + + autocatalytically removed + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:18:08Z + + active site residue + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:18:29Z + + Gly(-1) + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:59Z + + active + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + + INTRO + paragraph + 2246 + Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds. Nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of the scissile peptide bond creates a first cleavage product and a covalent acyl-enzyme intermediate. Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment. The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å). In principle it could function as the general base during both autocatalytic removal of the propeptide and protein substrate cleavage. Here we provide experimental evidences for this distinct view of the proteasome active-site mechanism. Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays. + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:22:13Z + + substrate-binding channel + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:59Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:15:27Z + + β subunits + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:02:57Z + + active sites + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:05:01Z + + Thr1 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:22:47Z + + complex + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:07Z + + water + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:23:11Z + + enzyme + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:14Z + + peptide + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:23:19Z + + N-terminal nucleophilic (Ntn) hydrolases + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:23:26Z + + free + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:05:07Z + + Thr1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:23:35Z + + autocatalytic precursor processing + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:23:43Z + + immature + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:03:01Z + + active sites + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:05:10Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:23:59Z + + Gly(-1) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen-bonding + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:05:14Z + + Thr1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:24:40Z + + autocatalytic removal + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:28Z + + active-site + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:24:46Z + + biochemical and structural analyses + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:13Z + + active site + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:54:48Z + + autocatalytic activation + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:03Z + + Thr + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:10Z + + Cys + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:17Z + + Ser + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:25:21Z + + X-ray crystallography + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:25:25Z + + activity and inhibition assays + + + + RESULTS + title_1 + 4066 + Results + + + RESULTS + title_2 + 4074 + Inactivation of proteasome subunits by T1A mutations + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:25:52Z + + subunits + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:26:07Z + + T1A + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:26:13Z + + mutations + + + + RESULTS + paragraph + 4127 + Proteasome-mediated degradation of cell-cycle regulators and potentially toxic misfolded proteins is required for the viability of eukaryotic cells. Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). These results indicate that the β1 and β2 proteolytic activities are not essential for cell survival. By contrast, the T1A mutation in subunit β5 has been reported to be lethal or nearly so. Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + Proteasome + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:14:15Z + + eukaryotic + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:13Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:28:44Z + + mutation to + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:28:50Z + + Ala + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:03:19Z + + active sites + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:58Z + + Yeast + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:08Z + + β1-T1A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:16Z + + β2-T1A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:29:45Z + + catalytic + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:15:27Z + + β subunits + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:29:56Z + + disabled + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:05Z + + carry remnants of + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:42Z + + β1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:51Z + + β2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:26:07Z + + T1A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:30:17Z + + β5-T1A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:30:31Z + + deleted but expressed separately + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:39Z + + trans + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:30:17Z + + β5-T1A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:39Z + + trans + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:30:46Z + + crystallographic analysis + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:30:17Z + + β5-T1A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:39Z + + trans + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:50Z + + mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:30:50Z + + mutation + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:31:01Z + + catalytic active site + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:31:13Z + + trans-expressed + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:31:19Z + + not bound + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:31:22Z + + substrate-binding channel + + + + RESULTS + paragraph + 5343 + The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy. Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:30:17Z + + β5-T1A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:50Z + + mutant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:33:19Z + + cis + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:33:23Z + + Sequencing of the plasmids + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:58Z + + yeast + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:33:27Z + + site-directed mutagenesis + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:30:17Z + + β5-T1A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:50Z + + mutant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:33:19Z + + cis + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:33:38Z + + K81R + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:33:31Z + + This single amino-acid exchange + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:03:23Z + + interface + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:33:46Z + + α4 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:33:53Z + + β4 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:34:12Z + + β5-T1A-K81R + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:50Z + + mutant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:34:21Z + + crystal structure + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:58Z + + yeast + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:34:28Z + + yCP + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:30:17Z + + β5-T1A + + + + RESULTS + title_2 + 6407 + Propeptide conformation and triggering of autolysis + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + Propeptide + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:34:49Z + + autolysis + + + + RESULTS + paragraph + 6459 + In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a). However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1. Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b). Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant. Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. Nonetheless, the carbonyl carbon of Gly(-1) would be ideally placed for nucleophilic attack by Thr1Oγ (Fig. 1c and Supplementary Fig. 2c,d). As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:39:00Z + + autocatalytically cleaved + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:39:22Z + + mature + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:39:26Z + + β-subunit domains + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:42Z + + β1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:39:13Z + + (-2) + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:39:36Z + + S1 specificity pocket + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:39:40Z + + substrate-binding channel + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:39:44Z + + 45 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:40:10Z + + prosegment + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:40:16Z + + antiparallel β-sheet + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:41:13Z + + Gly(-1) + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:40:21Z + + γ-turn conformation + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:40:35Z + + Leu(-2) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:40:44Z + + Thr1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:08Z + + β1-T1A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:50Z + + mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:40:54Z + + crystallographically + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:40:57Z + + structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:16Z + + β2-T1A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:41:04Z + + β1-T1A-β2-T1A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:43Z + + β1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:41:10Z + + Gly(-1) + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:41:19Z + + sharp turn + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:41:36Z + + prosegment cleavage + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:41:47Z + + γ-turn conformation + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:41:55Z + + Gly(-1) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:51Z + + β2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:41:59Z + + Thr(-2) + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:05Z + + S1 pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:42:10Z + + Leu(-2) + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:43Z + + β1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:51Z + + β2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:05Z + + S1 pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:42:17Z + + Gly45 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:42:20Z + + Thr(-2) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:18:31Z + + Gly(-1) + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bonding + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:42:31Z + + Thr1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:34:12Z + + β5-T1A-K81R + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:42:36Z + + Gly(-1) + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:40Z + + histidine + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:42:45Z + + (-2) + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:49Z + + S2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:05Z + + S1 pocket + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:55Z + + antiparallel β-sheet + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:42:59Z + + Gly(-1) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:43:02Z + + Thr1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:33:38Z + + K81R + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:43:42Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:43:45Z + + Arg81 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:05Z + + S1 pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:53:10Z + + Met45 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:43:57Z + + His(-2) + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:44:04Z + + S1 site + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:44:22Z + + autolysis + + + + RESULTS + paragraph + 8870 + Processing of β-subunit precursors requires deprotonation of Thr1OH; however, the general base initiating autolysis is unknown. Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c). Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base. + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:46:37Z + + Thr1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:46:40Z + + autolysis + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:14:15Z + + eukaryotic + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:46:46Z + + His + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:46:49Z + + (-2) + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:46:53Z + + histidine + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:47:01Z + + catalytic triads + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:47:09Z + + serine proteases + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:47:13Z + + His(-2) + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:47:16Z + + exchanging it for + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:47:19Z + + Asn + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:47:22Z + + Lys + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:47:25Z + + Phe + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:28:50Z + + Ala + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:58Z + + yeast + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:47:35Z + + H(-2)N + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:47:41Z + + H(-2)F + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:47:35Z + + H(-2)N + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:47:41Z + + H(-2)F + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:47:50Z + + yCPs + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:47:55Z + + Structural analyses + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:47:50Z + + yCPs + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:48:03Z + + 2FO–FC electron densities + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:48:06Z + + Gly(-1) + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:48:10Z + + Phe + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:48:13Z + + Lys(-2) + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:28:50Z + + Ala + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:48:18Z + + Asn(-2) + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:48:25Z + + processed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:48:31Z + + unprocessed + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:48:36Z + + autolysis + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:48:40Z + + (-2) + + + + RESULTS + paragraph + 10064 + Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. As expected from β5-T1A mutants, the yeasts show severe growth phenotypes, with minor variations (Supplementary Fig. 4a and Table 1). We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1). For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h). This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site. Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:52:19Z + + (-2) + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:52:22Z + + creating mutants that combine + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:26:07Z + + T1A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:33:38Z + + K81R + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:52:26Z + + mutation(s) + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:52:32Z + + H(-2)L + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:52:40Z + + H(-2)T + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:52:48Z + + H(-2)A + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:52:51Z + + substitutions + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:52:55Z + + Leu(-2) + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:58Z + + yeast + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:43Z + + β1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:53:01Z + + Thr(-2) + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:51Z + + β2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:53:03Z + + Ala(-2) + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:59Z + + β5 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:05Z + + S1 pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:53:10Z + + Met45 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:30:17Z + + β5-T1A + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:06:35Z + + yeasts + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:53:26Z + + crystal structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:53:40Z + + β5-H(-2)L-T1A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:53:52Z + + β5-H(-2)T-T1A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:54:05Z + + β5-H(-2)A-T1A-K81R + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:54:05Z + + β5-H(-2)A-T1A-K81R + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:54:15Z + + Gly(-1) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:54:17Z + + Ala(-2) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:54:23Z + + Ala(-2) + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:54:38Z + + substrate-binding channel + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:17:44Z + + prosegments + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:53:40Z + + β5-H(-2)L-T1A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:53:52Z + + β5-H(-2)T-T1A + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:54:57Z + + 2FO–FC electron-density maps + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:05Z + + His(-2) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:16Z + + Leu(-2) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:23Z + + Thr(-2) + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:39:36Z + + S1 specificity pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:53:10Z + + Met45 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:38Z + + His(-2) + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:44:04Z + + S1 site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:45Z + + Gly(-1) + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:52Z + + wild-type + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:59Z + + WT + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:05Z + + Thr1 + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:09Z + + (-2) + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:05Z + + S1 pocket + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:56:12Z + + antiparallel β-sheet + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:01:52Z + + autolysis + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + + RESULTS + paragraph + 12011 + Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable. Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k). These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:34:21Z + + crystal structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:58:43Z + + chimeric + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:34:28Z + + yCP + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:58Z + + yeast + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:43Z + + β1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:58:40Z + + replaced by + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:58:56Z + + counterpart + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:59:04Z + + 2FO–FC electron density + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:43Z + + β1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:59:10Z + + Thr(-2) + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:51Z + + β2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:59:13Z + + Leu(-2) + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:43Z + + β1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:59:16Z + + not conserved + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:59:20Z + + created + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:59:23Z + + β2-T(-2)V + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:16Z + + β2-T1A + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:53:26Z + + crystal structures + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:59:33Z + + Thr(-2) + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bonds + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:18:31Z + + Gly(-1) + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:53:52Z + + β5-H(-2)T-T1A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:59:50Z + + exchange + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:59:53Z + + Thr(-2) + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:59:56Z + + Val + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:51Z + + β2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:00:04Z + + 2FO–FC electron-density map + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:51Z + + β2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:16Z + + β2-T1A + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:00:10Z + + Val(-2) + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:44:04Z + + S1 site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:00:15Z + + Gly(-1) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:00:21Z + + active-site residues + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:00:19Z + + Gly(-1) + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + + RESULTS + title_2 + 13352 + The active site of the proteasome + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + + RESULTS + paragraph + 13386 + Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis. However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. Owing to its likely protonation at neutral pH, however, it was assumed not to act as the general base. A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base. Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a). This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a). Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b). Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:08:11Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:07:50Z + + Thr1 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:07Z + + water + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:23:43Z + + immature + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:02:07Z + + particle + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:08:28Z + + Thr1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:08:32Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:08:35Z + + Lys33 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen-bonding + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:08:41Z + + Thr1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:08:44Z + + catalytically active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:15:27Z + + β subunits + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:08:55Z + + catalytic tetrad + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:08:58Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:09:01Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:09:04Z + + Lys33 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:09:07Z + + Asp17 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:07Z + + water + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:34:28Z + + yCP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:09:15Z + + X-ray structures + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:09:22Z + + Mutation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:28:50Z + + Ala + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:06:59Z + + structural and biochemical analyses + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:01:56Z + + propeptide cleavage + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:06:15Z + + β5-K33A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:34:12Z + + β5-T1A-K81R + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:58Z + + yeast + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:11:34Z + + L(-49)S + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:11:40Z + + β5-K33A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:11:44Z + + Structural comparison + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:11:47Z + + β5-L(-49)S-K33A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:34:12Z + + β5-T1A-K81R + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:03:28Z + + active sites + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:11:52Z + + mutation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:28:50Z + + Ala + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:28Z + + active-site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:11:58Z + + β5-K33A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:12:02Z + + expressed separately + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:39Z + + trans + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:34:21Z + + crystal structure + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:12:27Z + + β5-K33A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:39Z + + trans + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:12:30Z + + in complex with + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:36Z + + carfilzomib + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:03:32Z + + active sites + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:12:42Z + + acetylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:12:53Z + + β5-K33A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:39Z + + trans + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:49:49Z + + apo + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:34:21Z + + crystal structure + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:12:58Z + + Lys33 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:13:01Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:13:04Z + + Thr1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:34:21Z + + crystal structure + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:13:07Z + + β5-K33A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:39Z + + trans + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:13:10Z + + without inhibitor + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:13:15Z + + Thr1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + coordinates + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:13:47Z + + water + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:07Z + + water + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bonds + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:14:01Z + + Arg19 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:14:05Z + + Asp17 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:14:08Z + + Lys33 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:59Z + + WT + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:14:11Z + + structure + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:14:15Z + + autolysis + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:14:18Z + + Conservative substitution + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:14:21Z + + Arg + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:14:24Z + + autolysis + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:59Z + + yeast + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:28Z + + Thr1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:59Z + + WT + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:47:50Z + + yCPs + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:14:35Z + + Arg33 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:37Z + + Asp17 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + + RESULTS + paragraph + 16924 + The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:20:48Z + + conservative mutation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:38Z + + Asp17 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:20:51Z + + Asn + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:34:28Z + + yCP + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:20:57Z + + L(-49)S + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:21:00Z + + β5-D17N + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:34:28Z + + yCP + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:21:04Z + + crystallographic analysis + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:21:33Z + + partially processed + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:36Z + + carfilzomib + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:43Z + + β1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:51Z + + β2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:59Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:33:19Z + + cis + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:21:47Z + + expression + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:39Z + + trans + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:21:51Z + + isolation + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:21:54Z + + crystallization + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:22:01Z + + D17N + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:22:05Z + + β5-D17N + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:39Z + + trans + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:22:10Z + + N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:22:17Z + + Suc-LLVY-AMC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:22:21Z + + carboxybenzyl-Gly-Gly-Leu-para-nitroanilide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:22:27Z + + Z-GGL-pNA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:38Z + + Asp17 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:39:22Z + + mature + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:22:33Z + + β5-D17N + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:39Z + + trans + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:34:28Z + + yCP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:34:21Z + + crystal structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:59Z + + WT + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:34:28Z + + yCP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:22:36Z + + co-crystal structure + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:35:36Z + + α′, β′ epoxyketone + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:36Z + + carfilzomib + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:34:21Z + + crystal structure + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:22:44Z + + β5-D17N + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:33:19Z + + cis + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:22:47Z + + in complex with + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:36Z + + carfilzomib + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:22:53Z + + Autolysis + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:22:56Z + + β5-D17N + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:05:19Z + + Asn17 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:23:02Z + + E17A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:06:41Z + + β-subunit + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:23:10Z + + archaeon + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:23:15Z + + Thermoplasma acidophilum + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:23:18Z + + autolysis + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:23:22Z + + X-ray data + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:23:25Z + + β5-D17N + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:23:27Z + + expressed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:33:19Z + + cis + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:39Z + + trans + + + + RESULTS + paragraph + 19094 + On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T14:24:38Z + + CPs + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T12:00:53Z + + catalytic triad + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:24:46Z + + Asp + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:24:52Z + + Glu17 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:24:56Z + + autocatalytic precursor processing + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:07Z + + water + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:39:22Z + + mature + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:59Z + + WT + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:25:17Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:25:02Z + + Thr1 + + + + RESULTS + paragraph + 19489 + To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. The hydrogen bonds involving Ser169OH are intact and may account for residual substrate turnover. Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis. These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis. + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:28Z + + active-site + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:28:38Z + + exchanged the conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:30:47Z + + Asp166 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:28:43Z + + Asn + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:59Z + + yeast + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:28:46Z + + Asp166 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen-bonded + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:28:49Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:28:52Z + + Ser129 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:28:55Z + + Ser169 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:28:57Z + + β5-D166N + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:33:19Z + + cis + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:59Z + + yeast + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:29:12Z + + X-ray data + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:29:15Z + + β5-D166N + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:29:18Z + + Ser129 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:29:24Z + + Asn166 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:07Z + + water + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:29:27Z + + bound to + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:29:29Z + + Ser129 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:29:32Z + + Thr1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:29:35Z + + precursor processing + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bonds + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:29:40Z + + Ser169 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:29:43Z + + Soaking + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:29:46Z + + β5-D166N + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:29:49Z + + crystals + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:36Z + + carfilzomib + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:29:55Z + + MG132 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T14:30:07Z + + carfilzomib complex + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:30:09Z + + structure + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:30:12Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:30:14Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:31:06Z + + Ser129 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:59Z + + WT + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:30:25Z + + MG132-bound state + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:30:28Z + + Thr1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:30:30Z + + unmodified + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:31:06Z + + Ser129 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen-bonded + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:07Z + + water + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:05:25Z + + Asn166 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:30:40Z + + Asn + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:30:47Z + + Asp166 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:28:50Z + + Ala + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:30:55Z + + autolysis + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:30:47Z + + Asp166 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:31:06Z + + Ser129 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:31:00Z + + Thr1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:30:58Z + + autolysis + + + + RESULTS + title_2 + 21052 + Substitution of the active-site Thr1 by Cys + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:31:37Z + + Substitution + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:28Z + + active-site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:10Z + + Cys + + + + RESULTS + paragraph + 21096 + Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure. In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. Owing to the unequal positions of the two β5 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:33:59Z + + Mutation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:10Z + + Cys + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T11:54:41Z + + 20S proteasome + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:23:10Z + + archaeon + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:33:51Z + + T. acidophilum + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:59Z + + yeast + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:33:56Z + + mutation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:34:09Z + + X-ray structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:34:34Z + + cleaved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:34Z + + propeptide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:34:38Z + + still bound + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:34:41Z + + substrate-binding channel + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:34:44Z + + His(-2) + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:34:47Z + + S2 pocket + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:34:12Z + + β5-T1A-K81R + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:34:57Z + + T1C + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:35:00Z + + antiparallel β-sheet + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:29:55Z + + MG132 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:35:05Z + + T1C + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:35:12Z + + autolysis + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:35:16Z + + crystallization + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:00Z + + β5 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + + RESULTS + paragraph + 22121 + Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a). In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74° with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b). This presumably results from the larger radius of the sulfur atom compared with oxygen. Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. Together, these observations suggest that efficient peptide-bond hydrolysis requires that Lys33NH2 hydrogen bonds to the active site nucleophile. + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:39:29Z + + propeptide hydrolysis + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:40:08Z + + β5-T1C + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:40:12Z + + catalytically inactive + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:40:15Z + + soaking crystals + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:40:22Z + + bortezomib + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:36Z + + carfilzomib + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:43Z + + β1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:51Z + + β2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:03:37Z + + active sites + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:40:26Z + + β5-T1C + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:40:29Z + + proteolytic centres + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:40:31Z + + unmodified + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:40:34Z + + cleaved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:40:41Z + + structural data + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:40:44Z + + Cys1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:59Z + + WT + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T14:24:38Z + + CPs + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:40:55Z + + Cys1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:00:04Z + + 2FO–FC electron-density map + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:40:58Z + + T1C + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:05:31Z + + Lys33 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:41:11Z + + disordered + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:05:35Z + + Lys33 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bonds + + + + RESULTS + title_2 + 23124 + The benefit of Thr over Ser as the active-site nucleophile + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:04Z + + Thr + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:17Z + + Ser + + + + RESULTS + paragraph + 23183 + All proteasomes strictly employ threonine as the active-site residue instead of serine. To investigate the reason for this singularity, we analysed a β5-T1S mutant, which is viable but suffers from growth defects (Fig. 4a and Table 1). Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). By contrast, turnover of the substrate Z-GGL-pNA, used to monitor ChT-L activity in situ but in a less quantitative fashion, is not detectably impaired (Supplementary Fig. 9a). Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:02:12Z + + proteasomes + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:04:29Z + + strictly employ + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:45:18Z + + threonine + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:03:40Z + + active-site residue + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:45:23Z + + serine + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:45:27Z + + β5-T1S + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:45:30Z + + Activity assays + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:01Z + + β5 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:22:17Z + + Suc-LLVY-AMC + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:45:37Z + + T1S + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:59Z + + WT + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:02:16Z + + proteasomes + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:22:27Z + + Z-GGL-pNA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:34:21Z + + Crystal structure + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:45:42Z + + β5-T1S + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:45:45Z + + precursor processing + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T14:46:11Z + + ligand-complex + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:46:17Z + + structures + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:40:22Z + + bortezomib + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:37Z + + carfilzomib + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:46:22Z + + Ser1 + + + + RESULTS + paragraph + 24059 + However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g). Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. This renders it unavailable for direct nucleophilic attack onto incoming substrates and first requires its reorientation, which is expected to delay substrate turnover. Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1. The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). Consequently, the hydroxyl group of Thr1 requires no reorientation before substrate cleavage and is thus more catalytically efficient than Ser1. In agreement, at an elevated growing temperature of 37 °C the T1S mutant is unable to grow (Fig. 4a). In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein. Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased. Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.). + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:49:49Z + + apo + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:34:21Z + + crystal structure + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:49:55Z + + Ser1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:49:59Z + + substrate-binding channel + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:05:42Z + + Thr1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:59Z + + WT + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:50:04Z + + structures + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:50:06Z + + Ser1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:50:16Z + + Ser1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen-bonded + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:50:25Z + + Lys33 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:50:30Z + + active-site residue + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrophobic interactions + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:50:40Z + + Thr3 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:50:38Z + + Ala46 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:50:47Z + + Ser1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:50:50Z + + T1S + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:40:22Z + + bortezomib + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:37Z + + carfilzomib + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T14:51:08Z + + inhibitor complex + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:51:13Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:59Z + + WT + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:34:28Z + + yCP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:51:16Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:51:19Z + + with the same inhibitors + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:51:23Z + + affinity + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:37Z + + carfilzomib + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:51:30Z + + substrate-binding channel + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:40:22Z + + bortezomib + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:51:34Z + + mean residence time + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:37Z + + carfilzomib + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:51:37Z + + Ser1 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:51:43Z + + threonine + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-22T10:36:40Z + + proteasomes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:52:12Z + + proteasome-like proteases + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T14:52:15Z + + HslV + + + + DISCUSS + title_1 + 25916 + Discussion + + + DISCUSS + paragraph + 25927 + The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized. The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex. In addition, they prevent irreversible inactivation of the Thr1 N terminus by N-acetylation. By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated. However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects. These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T11:54:41Z + + 20S proteasome + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:53:53Z + + non-lysosomal protease + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:14:15Z + + eukaryotic + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T16:06:46Z + + β-subunit + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:01Z + + β5 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:54:03Z + + N-acetylation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:17:44Z + + prosegments + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:15:27Z + + β subunits + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:54:13Z + + archaeal + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:54:17Z + + heterologously expressed + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:54:25Z + + Escherichia coli + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:54:32Z + + eukaryotes + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:43Z + + β1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:51Z + + β2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:54:37Z + + removal of + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:01Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:40:10Z + + prosegment + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:01Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:01Z + + β5 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:14:15Z + + eukaryotic + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + + DISCUSS + paragraph + 26801 + Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide. Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis. In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1Oγ due to the rotational freedom and flexibility of the acetyl group. The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2). + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:56:42Z + + atomic structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:30:17Z + + β5-T1A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:01Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:56:46Z + + (-2) + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-22T10:37:05Z + + Gly(-1) + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:56:49Z + + structures + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:56:52Z + + Thr1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:56:58Z + + tight turn + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:40:10Z + + prosegment + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:43Z + + β1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:57:01Z + + Gly(-1) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:57:05Z + + autolysis + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:57:09Z + + N-acetylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:57:13Z + + Thr1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:57:17Z + + substrate-binding channel + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:57:20Z + + Gly(-1) + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:57:22Z + + (-2) + + + + DISCUSS + paragraph + 27707 + Autolytic activation of the CP constitutes one of the final steps of proteasome biogenesis, but the trigger for propeptide cleavage had remained enigmatic. On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture. We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase. Thus, specific protein surroundings can significantly alter the chemical properties of amino acids such as Lys to function as an acid–base catalyst. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:00Z + + CP + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T14:59:25Z + + propeptide cleavage + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T14:59:29Z + + CP:ligand + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:59:33Z + + active-site architecture + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T12:00:54Z + + catalytic triad + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:01Z + + CP + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T14:59:41Z + + Asp + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:24:52Z + + Glu17 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:14:15Z + + eukaryotic + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:59:54Z + + bacterial + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:54:13Z + + archaeal + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:59:59Z + + Lys33 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:00:23Z + + autocatalytic removal + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:00:28Z + + Asp17 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:00:31Z + + Lys33 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:00:43Z + + Lys33 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:00:46Z + + Asp17 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:00:48Z + + Thr–Lys–Asp triad + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:00:52Z + + L-asparaginase + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:00:55Z + + Lys + + + + DISCUSS + paragraph + 28792 + In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+. The resulting uncharged Thr1NH2 is hydrogen-bridged to the C3-OH group. In agreement, acetylation of the Thr1 N terminus irreversibly blocks hydrolytic activity, and binding of substrates is prevented for steric reasons. By acting as a proton donor during catalysis, the Thr1 N terminus may also favour cleavage of substrate peptide bonds (Fig. 3d). In all proteases, collapse of the tetrahedral transition state results in selective breakage of the substrate amide bond, while the covalent interaction between the substrate and the enzyme persists. Cleavage of the scissile peptide bond requires protonation of the emerging free amine, and in the proteasome, the Thr1 amine group is likely to assume this function. Analogously, Thr1NH3+ might promote the bivalent reaction mode of epoxyketone inhibitors by protonating the epoxide moiety to create a positively charged trivalent oxygen atom that is subsequently nucleophilically attacked by Thr1NH2. + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:18:47Z + + Thr1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bonds + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:18:59Z + + endoproteolytic cleavage + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:19:02Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bonds + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T15:19:22Z + + fellutamide B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T15:19:25Z + + α-ketoamides + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T15:19:27Z + + homobelactosin C + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T15:19:30Z + + salinosporamide A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T15:19:38Z + + omuralide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:19:48Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:19:51Z + + Thr1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen-bridged + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:19:57Z + + acetylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:20:13Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:20:16Z + + Thr1 + + + + DISCUSS + paragraph + 30357 + During autolysis the Thr1 N terminus is engaged in a hydroxyoxazolidine ring intermediate (Fig. 3d), which is unstable and short-lived. Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+. The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state. Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2). This renders the N terminus less suitable to stabilize substrates and to protonate the first cleavage product during catalysis, although it favours its ability to act as a nucleophile. This interpretation agrees with the strongly reduced catalytic activity of the β5-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other. Hence, the proteasome can be viewed as having a second triad that is essential for efficient proteolysis. While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:02:02Z + + autolysis + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:22:46Z + + aspartic acid 166 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:22:49Z + + Ser129 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:22:52Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:31:06Z + + Ser129 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:30:47Z + + Asp166 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:22:56Z + + Thr1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:23:01Z + + mutation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:23:03Z + + D166A + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:23:06Z + + autolysis + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:54:13Z + + archaeal + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:01Z + + CP + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:23:09Z + + exchange + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:23:12Z + + D166N + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:59Z + + yeast + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:01Z + + CP + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:23:15Z + + mutation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:23:17Z + + D166N + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:23:19Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:23:22Z + + Thr1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:23:25Z + + β5-D166N + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:37Z + + carfilzomib + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:03:45Z + + second triad + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:23:29Z + + Lys33 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:23:32Z + + Asp17 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:23:34Z + + Ser129 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:23:37Z + + Asp166 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + + DISCUSS + paragraph + 31717 + In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. Consequently, efficient substrate turnover or covalent modification by ligands is prevented. However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time. While only one single turnover is necessary for autolysis, continuous enzymatic activity is required for significant and detectable substrate hydrolysis. Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:38Z + + Asp17 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T12:00:54Z + + catalytic triad + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:25:23Z + + crystallographic data + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:25:26Z + + proteolytically inactive + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:25:29Z + + β5-T1C + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:25:35Z + + Lys33 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:05:49Z + + Cys1 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:10Z + + Cys + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:25:38Z + + cleaved + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:25:41Z + + autolysis + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:25:45Z + + Ntn hydrolase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:25:47Z + + penicillin acylase + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:25:50Z + + substitution + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:29:45Z + + catalytic + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:17Z + + Ser + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:10Z + + Cys + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:25:58Z + + inactivates + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:26:01Z + + enzyme + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:26:03Z + + precursor processing + + + + DISCUSS + paragraph + 32443 + To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC. We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:14:01Z + + CP + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:30:21Z + + threonine + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:30:24Z + + active-site residue + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:30:27Z + + β5-T1S + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:34:28Z + + yCP + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:30:34Z + + biochemically and structurally + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:30:37Z + + Activity assays + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:30:39Z + + β5-T1S + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:22:17Z + + Suc-LLVY-AMC + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:30:43Z + + β5-T1S + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T13:34:28Z + + yCP + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:40:22Z + + bortezomib + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:37Z + + carfilzomib + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:30:47Z + + Structural analyses + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:30:50Z + + T1S + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:30:54Z + + strict use of + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:04Z + + Thr + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:02:21Z + + proteasomes + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrophobic interactions + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:31:05Z + + Ala46 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:31:12Z + + Thr3 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:31:16Z + + proteolytically active + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:23Z + + proteasome + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:31:19Z + + archaea + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:59Z + + yeast + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:31:25Z + + mammals + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:04Z + + Thr + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:31:32Z + + Ile + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:31:35Z + + 3 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:32:02Z + + Ser1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T15:31:55Z + + enzyme + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:32:06Z + + threonine aspartase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T15:32:09Z + + Taspase1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:32:12Z + + mutation + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:29Z + + active-site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:32:20Z + + Thr234 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:17Z + + Ser + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:32:23Z + + Thr234 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:32:26Z + + serine + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:51Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:02:01Z + + active + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:32:32Z + + threonine + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:24Z + + proteasome + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:32:35Z + + threonine + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:24Z + + proteasome + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:32:38Z + + conservation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:02:25Z + + proteasomes + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:32:41Z + + bacteria + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:54:32Z + + eukaryotes + + + + METHODS + title_1 + 34241 + Methods + + + METHODS + title_2 + 34249 + Yeast mutagenesis + + + METHODS + paragraph + 34267 + Site-directed mutagenesis was performed by standard techniques using oligonucleotides listed in Supplementary Table 2. The pre2/doa3 (β5) mutant alleles in the centromeric, TRP1- or LEU2-marked shuttle vectors YCplac22 and pRS315, respectively, were verified by sequencing and subsequently introduced into the yeast strains MHY784 (ref.) or YWH20 (ref.), which express WT PRE2 from a URA3-marked plasmid. Counter-selection against the URA3 marker with 5-fluoroorotic acid yielded strains expressing only the mutant forms of β5. + + + METHODS + paragraph + 34801 + The strain producing a processed β5-T1A variant and the β5 propeptide in trans is a derivative of YWH212 (ref.). It carries an additional deletion of the NAT1 gene to avoid N-acetylation of Ala1; this strain exhibits extremely slow growth rates and served for crystallographic analysis only. All strains used in this study are listed in Supplementary Table 3. + + + METHODS + title_2 + 35167 + Purification of yeast proteasomes + + + METHODS + paragraph + 35201 + Yeast strains were grown in 18-l cultures at 30 °C in YPD into early stationary phase, and the yCPs were purified according to published procedures. In brief, 120 g yeast cells were solubilized in 150 ml of 50 mM KH2PO4/K2HPO4 buffer (pH 7.5) and disrupted with a French press. Cell debris were removed by centrifugation for 30 min at 21,000 r.p.m. (4 °C). The resulting supernatant was filtered and ammonium sulfate (saturated solution) was added to a final concentration of 30% (v/v). This solution was loaded on a Phenyl Sepharose 6 Fast Flow column (GE Healthcare) pre-equilibrated with 1 M ammonium sulfate in 20 mM KH2PO4/K2HPO4 (pH 7.5). CPs were eluted by applying a linear gradient from 1 to 0 M ammonium sulfate. Proteasome-containing fractions were pooled and loaded onto a hydroxyapatite column (Bio-Rad) equilibrated with 20 mM KH2PO4/K2HPO4 (pH 7.5). Elution of the CPs was achieved by increasing the phosphate buffer concentration from 20 to 500 mM. Anion-exchange chromatogaphy (Resource Q column (GE Healthcare), elution gradient from 0 to 500 mM sodium chloride in 20 mM Tris-HCl (pH 7.5)) and subsequent size-exclusion chromatography (Superose 6 10/300 GL (GE Healthcare), 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl) resulted in pure CPs for crystallization and activity assays. + + + METHODS + title_2 + 36530 + Fluorescence-based activity assay + + + METHODS + paragraph + 36564 + ChT-L (β5) activity of CPs was monitored by fluorescence spectroscopy using the model substrate Suc-LLVY-AMC. Purified yCPs (66 nM in 100 mM Tris-HCl, pH 7.5) were incubated with 300 μM substrate for 1 h at room temperature or 37 °C. The reactions were stopped by diluting samples 1:10 in 20 mM Tris-HCl, pH 7.5. AMC fluorophores released by proteasomal activity were measured in triplicate with a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) at λexc=360 nm and λem=460 nm. + + + METHODS + title_2 + 37088 + Inhibition assays + + + METHODS + paragraph + 37106 + Purified yCPs were mixed with dimethylsulfoxide as a control or serial dilutions of inhibitor and incubated for 45 min at room temperature. A final concentration of yCP of 66 nM was used. After addition of the peptide substrate Suc-LLVY-AMC to a final concentration of 200 μM and incubation for 1 h at room temperature, the reaction was stopped by diluting the samples 1:10 in 20 mM Tris-HCl, pH 7.5. AMC fluorophores released by residual proteasomal activity were measured in triplicate at λexc=360 nm and λem=460 nm. Relative fluorescence units were normalized to the dimethylsulfoxide-treated control. The calculated residual activities were plotted against the logarithm of the applied inhibitor concentration and fitted with GraphPad Prism 5. The IC50 value, the ligand concentration that leads to 50% inhibition of the enzymatic activity, was deduced from the fitted data. + + + METHODS + title_2 + 38002 + Crystallization and structure determination + + + METHODS + paragraph + 38046 + Mutant yCPs were crystallized as previously described for WT 20S proteasomes. Crystals were grown at 20 °C using the hanging drop vapour diffusion method. Drops contained a 1:1 mixture of protein (40 mg ml−1) and reservoir solution (25 mM magnesium acetate, 100 mM 2-(N-morpholino)ethanesulfonic acid (MES; pH 6.8) and 9–13% (v/v) 2-methyl-2,4-pentanediol (MPD)). Crystals were cryoprotected by addition of 5 μl cryobuffer (20 mM magnesium acetate, 100 mM MES, pH 6.8, and 30% (v/v) MPD). Inhibitor complex structures were obtained by incubating crystals in 5 μl cryobuffer supplemented with bortezomib or carfilzomib at a final concentration of 1.5 mM for at least 8 h. + + + METHODS + paragraph + 38746 + Diffraction data were collected at the beamline X06SA at the Paul Scherrer Institute, SLS, Villigen, Switzerland (λ=1.0 Å). Evaluation of reflection intensities and data reduction were performed with the programme package XDS. Molecular replacement was carried out with the coordinates of the yeast 20S proteasome (PDB entry code: 5CZ4) by rigid body refinements (REFMAC5; ref.). MAIN and COOT were used to build models. TLS (Translation/Libration/Screw) refinements finally yielded excellent Rwork and Rfree, as well as root mean squared deviation bond and angle values. The coordinates, proven to have good stereochemistry from the Ramachandran plots, were deposited in the RCSB Protein Data Bank (Supplementary Table 1). + + + METHODS + paragraph + 39475 + The coordinates for the yeast 20S proteasome deposited under the entry code 1RYP do not represent the WT yCP but the double-mutant β5-K33R β1-T1A. At the time of deposition (in 1997), these data were the best available on the yCP. As yCP structure determination has become routine today, and structure refinement procedures have significantly improved, we here provide coordinates for the WT yCP at 2.3 Å resolution (PDB entry code: 5CZ4). Furthermore, the structures of most mutant yCPs described in this work were determined in their apo and ligand-bound states. For mutants with proteolytically inactive β5 subunits, the best crystallographic data obtained are given. For ligands or propeptide segments that were only partially defined in the 2FO–FC electron-density map the occupancy was reduced (for details see Supplementary Table 1). + + + METHODS + title_1 + 40325 + Additional information + + + METHODS + paragraph + 40348 + Accession codes: Coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (for PDB entry codes see Supplementary Table 1). + + + METHODS + paragraph + 40506 + How to cite this article: Huber, E. M. et al. A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome. Nat. Commun. 7:10900 doi: 10.1038/ncomms10900 (2016). + + + SUPPL + title_1 + 40694 + Supplementary Material + + + 961 + 972 + surname:Chen;given-names:P. + surname:Hochstrasser;given-names:M. + 8808631 + REF + Cell + ref + 86 + 1996 + 40717 + Autocatalytic subunit processing couples active site formation in the 20S proteasome to completion of assembly + + + 463 + 471 + surname:Groll;given-names:M. + 9087403 + REF + Nature + ref + 386 + 1997 + 40828 + Structure of 20S proteasome from yeast at 2.4 Å resolution + + + 289 + 293 + surname:Krüger;given-names:E. + surname:Kloetzel;given-names:P. M. + surname:Enenkel;given-names:C. + 11295488 + REF + Biochimie + ref + 83 + 2001 + 40890 + 20S proteasome biogenesis + + + 10976 + 10983 + surname:Groll;given-names:M. + 10500111 + REF + Proc. Natl Acad. Sci. USA + ref + 96 + 1999 + 40916 + The catalytic sites of 20S proteasomes and their role in subunit maturation: a mutational and crystallographic study + + + 1187 + 1191 + surname:Ditzel;given-names:L. + 9642094 + REF + J. Mol. Biol. + ref + 279 + 1998 + 41033 + Conformational constraints for protein self-cleavage in the proteasome + + + 727 + 738 + surname:Huber;given-names:E. M. + 22341445 + REF + Cell + ref + 148 + 2012 + 41104 + Immuno- and constitutive proteasome crystal structures reveal differences in substrate and inhibitor specificity + + + 7835 + 7842 + surname:Huber;given-names:E. M. + 26020686 + REF + J. Am. Chem. Soc. + ref + 137 + 2015 + 41217 + Systematic analyses of substrate preferences of 20S proteasomes using peptidic epoxyketone inhibitors + + + 1509 + 1536 + surname:Marques;given-names:A. J. + surname:Palanimurugan;given-names:R. + surname:Matias;given-names:A. C. + surname:Ramos;given-names:P. C. + surname:Dohmen;given-names:R. J. + 19265443 + REF + Chem. Rev. + ref + 109 + 2009 + 41319 + Catalytic mechanism and assembly of the proteasome + + + 533 + 539 + surname:Löwe;given-names:J. + 7725097 + REF + Science + ref + 268 + 1995 + 41370 + Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution + + + 416 + 419 + surname:Brannigan;given-names:J. A. + 7477383 + REF + Nature + ref + 378 + 1995 + 41466 + A protein catalytic framework with an N-terminal nucleophile is capable of self-activation + + + 579 + 582 + surname:Seemüller;given-names:E. + 7725107 + REF + Science + ref + 268 + 1995 + 41557 + Proteasome from Thermoplasma acidophilum: a threonine protease + + + 25637 + 25646 + surname:Dick;given-names:T. P. + 9748229 + REF + J. Biol. Chem. + ref + 273 + 1998 + 41620 + Contribution of proteasomal beta-subunits to the cleavage of peptide substrates analyzed with yeast mutants + + + 25200 + 25209 + surname:Heinemeyer;given-names:W. + surname:Fischer;given-names:M. + surname:Krimmer;given-names:T. + surname:Stachon;given-names:U. + surname:Wolf;given-names:D. H. + 9312134 + REF + J. Biol. Chem. + ref + 272 + 1997 + 41728 + The active sites of the eukaryotic 20S proteasome and their involvement in subunit precursor processing + + + 7156 + 7161 + surname:Arendt;given-names:C. S. + surname:Hochstrasser;given-names:M. + 9207060 + REF + Proc. Natl Acad. Sci. USA + ref + 94 + 1997 + 41832 + Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation + + + 997 + 1013 + surname:Jäger;given-names:S. + surname:Groll;given-names:M. + surname:Huber;given-names:R. + surname:Wolf;given-names:D. H. + surname:Heinemeyer;given-names:W. + 10452902 + REF + J. Mol. Biol. + ref + 291 + 1999 + 41953 + Proteasome beta-type subunits: unequal roles of propeptides in core particle maturation and a hierarchy of active site function + + + 3575 + 3585 + surname:Arendt;given-names:C. S. + surname:Hochstrasser;given-names:M. + 10393174 + REF + EMBO J. + ref + 18 + 1999 + 42081 + Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly + + + 2161 + 2172 + surname:Polgar;given-names:L. + 16003488 + REF + Cell. Mol. Life Sci. + ref + 62 + 2005 + 42226 + The catalytic triad of serine peptidases + + + 1991 + 2003 + surname:Li;given-names:X. + surname:Li;given-names:Y. + surname:Arendt;given-names:C. S. + surname:Hochstrasser;given-names:M. + 26627836 + REF + J. Biol. Chem. + ref + 291 + 2015 + 42267 + Distinct elements in the proteasomal beta5 subunit propeptide required for autocatalytic processing and proteasome assembly + + + 6887 + 6898 + surname:Schmidtke;given-names:G. + 9003765 + REF + EMBO J. + ref + 15 + 1996 + 42391 + Analysis of mammalian 20S proteasome biogenesis: the maturation of beta-subunits is an ordered two-step mechanism involving autocatalysis + + + 417 + 420 + surname:Wlodawer;given-names:A. + 7663937 + REF + Structure + ref + 3 + 1995 + 42529 + Proteasome: a complex protease with a new fold and a distinct mechanism + + + 468 + 471 + surname:Seemüller;given-names:E. + surname:Lupas;given-names:A. + surname:Baumeister;given-names:W. + 8684489 + REF + Nature + ref + 382 + 1996 + 42601 + Autocatalytic processing of the 20S proteasome + + + 451 + 456 + surname:Groll;given-names:M. + surname:Berkers;given-names:C. R. + surname:Ploegh;given-names:H. L. + surname:Ovaa;given-names:H. + 16531229 + REF + Structure + ref + 14 + 2006 + 42648 + Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome + + + 407 + 417 + surname:Huber;given-names:E. M. + surname:Heinemeyer;given-names:W. + surname:Groll;given-names:M. + 25599643 + REF + Structure + ref + 23 + 2015 + 42765 + Bortezomib-resistant mutant proteasomes: structural and biochemical evaluation with carfilzomib and ONX 0914 + + + 6070 + 6074 + surname:Bochtler;given-names:M. + surname:Ditzel;given-names:L. + surname:Groll;given-names:M. + surname:Huber;given-names:R. + 9177170 + REF + Proc. Natl Acad. Sci. USA + ref + 94 + 1997 + 42874 + Crystal structure of heat shock locus V (HslV) from Escherichia coli + + + 765 + 770 + surname:Zwickl;given-names:P. + surname:Kleinz;given-names:J. + surname:Baumeister;given-names:W. + 7634086 + REF + Nat. Struct. Biol. + ref + 1 + 1994 + 42943 + Critical elements in proteasome assembly + + + 75 + 83 + surname:Groll;given-names:M. + surname:Brandstetter;given-names:H. + surname:Bartunik;given-names:H. + surname:Bourenkow;given-names:G. + surname:Huber;given-names:R. + 12614609 + REF + J. Mol. Biol. + ref + 327 + 2003 + 42984 + Investigations on the maturation and regulation of archaebacterial proteasomes + + + 84 + 92 + surname:Lubkowski;given-names:J. + surname:Dauter;given-names:M. + surname:Aghaiypour;given-names:K. + surname:Wlodawer;given-names:A. + surname:Dauter;given-names:Z. + 12499544 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 59 + 2003 + 43063 + Atomic resolution structure of Erwinia chrysanthemi L-asparaginase + + + 622 + 629 + surname:Gutteridge;given-names:A. + surname:Thornton;given-names:J. M. + 16214343 + REF + Trends Biochem. Sci. + ref + 30 + 2005 + 43130 + Understanding nature's catalytic toolkit + + + 501 + 512 + surname:Hines;given-names:J. + surname:Groll;given-names:M. + surname:Fahnestock;given-names:M. + surname:Crews;given-names:C. M. + 18482702 + REF + Chem. Biol. + ref + 15 + 2008 + 43171 + Proteasome inhibition by fellutamide B induces nerve growth factor synthesis + + + 1679 + 1683 + surname:Stein;given-names:M. L. + REF + Angew. Chem. Int. Ed. + ref + 53 + 2014 + 43248 + Systematic comparison of peptidic proteasome inhibitors highlights the alpha-ketoamide electrophile as an auspicious reversible lead motif + + + 4576 + 4579 + surname:Groll;given-names:M. + surname:Larionov;given-names:O. V. + surname:Huber;given-names:R. + surname:de Meijere;given-names:A. + 16537370 + REF + Proc. Natl Acad. Sci. USA + ref + 103 + 2006 + 43387 + Inhibitor-binding mode of homobelactosin C to proteasomes: new insights into class I MHC ligand generation + + + 5136 + 5141 + surname:Groll;given-names:M. + surname:Huber;given-names:R. + surname:Potts;given-names:B. C. + 16608349 + REF + J. Am. Chem. Soc. + ref + 128 + 2006 + 43494 + Crystal structures of Salinosporamide A (NPI-0052) and B (NPI-0047) in complex with the 20S proteasome reveal important consequences of beta-lactone ring opening and a mechanism for irreversible binding + + + 6270 + 6276 + surname:Choi;given-names:K. S. + surname:Kim;given-names:J. A. + surname:Kang;given-names:H. S. + 1400178 + REF + J. Bacteriol. + ref + 174 + 1992 + 43697 + Effects of site-directed mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC 11105 + + + 1443 + 1452 + surname:Khan;given-names:J. A. + surname:Dunn;given-names:B. M. + surname:Tong;given-names:L. + 16216576 + REF + Structure + ref + 13 + 2005 + 43818 + Crystal structure of human Taspase1, a crucial protease regulating the function of MLL + + + 14831 + 14837 + surname:Kisselev;given-names:A. F. + surname:Songyang;given-names:Z. + surname:Goldberg;given-names:A. L. + 10809725 + REF + J. Biol. Chem. + ref + 275 + 2000 + 43905 + Why does threonine, and not serine, function as the active site nucleophile in proteasomes? + + + 373 + 390 + surname:Gallastegui;given-names:N. + surname:Groll;given-names:M. + 22350899 + REF + Methods Mol. Biol. + ref + 832 + 2012 + 43997 + Analysing properties of proteasome inhibitors using kinetic and X-ray crystallographic studies + + + 329 + 336 + surname:Groll;given-names:M. + surname:Huber;given-names:R. + 16275340 + REF + Methods Enzymol. + ref + 398 + 2005 + 44092 + Purification, crystallization, and X-ray analysis of the yeast 20S proteasome + + + 125 + 132 + surname:Kabsch;given-names:W. + 20124692 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 44170 + XDS + + + 2184 + 2195 + surname:Vagin;given-names:A. A. + 15572771 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 60 + 2004 + 44174 + REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use + + + 1342 + 1357 + surname:Turk;given-names:D. + 23897458 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 69 + 2013 + 44262 + MAIN software for density averaging, model building, structure refinement and validation + + + 486 + 501 + surname:Emsley;given-names:P. + surname:Lohkamp;given-names:B. + surname:Scott;given-names:W. G. + surname:Cowtan;given-names:K. + 20383002 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 44351 + Features and development of Coot + + + SUPPL + footnote + 44384 + Author contributions E.M.H., W.H., X.L., C.S.A. and M.H. created yeast mutants; E.M.H. and W.H. performed activity and growth assays; E.M.H. and M.G. collected and analysed X-ray data; E.M.H., M.H. and M.G. wrote the manuscript. + + + ncomms10900-f1.jpg + f1 + FIG + fig_title_caption + 44613 + Conformation of proteasomal propeptides. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + + ncomms10900-f1.jpg + f1 + FIG + fig_caption + 44654 + (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed. The major determinant of the S1 specificity pocket, residue 45, is depicted. Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). The black arrow indicates the attack of Thr1Oγ onto the carbonyl carbon atom of Gly(-1). (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. Thr(-2)OH is hydrogen-bonded to Gly(-1)O (∼2.8 Å; black dashed line). The major determinant of the S1 specificity pocket, residue 45, is depicted. (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. Nonetheless, in all mutants the carbonyl carbon atom of Gly(-1) is ideally placed for the nucleophilic attack by Thr1Oγ. The hydrogen bond between Thr(-2)OH and Gly(-1)O (∼2.8 Å) is indicated by a black dashed line. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:40:12Z + + Structural superposition + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:08Z + + β1-T1A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:04:34Z + + matured + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:43Z + + β1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:29Z + + active-site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:40:22Z + + (-5) to (-1) + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:08Z + + β1-T1A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:39:36Z + + S1 specificity pocket + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:40:29Z + + 45 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:40:33Z + + Gly(-1) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:40:38Z + + Ala1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:40:40Z + + G(-1) + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:48:25Z + + processed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:29Z + + active-site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:29Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:40:50Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:40:52Z + + Gly(-1) + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:40:55Z + + Structural superposition + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:08Z + + β1-T1A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:16Z + + β2-T1A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:41:01Z + + Ala1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:41:03Z + + Gly(-1) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:26Z + + Thr(-2) + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen-bonded + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:18:31Z + + Gly(-1) + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:39:36Z + + S1 specificity pocket + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:41:17Z + + 45 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:41:20Z + + Structural superposition + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:08Z + + β1-T1A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:16Z + + β2-T1A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:34:12Z + + β5-T1A-K81R + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:41:25Z + + (-2) + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:43Z + + β1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:51Z + + β2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:17:45Z + + prosegments + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:05Z + + S1 pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:41:30Z + + His(-2) + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:01Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:41:34Z + + S2 pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:41:37Z + + Gly(-1) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:41:40Z + + Thr1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:55:26Z + + Thr(-2) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:18:31Z + + Gly(-1) + + + + ncomms10900-f2.jpg + f2 + FIG + fig_title_caption + 46013 + Mutations of residue (-2) and their influence on propeptide conformation and autolysis. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:42:07Z + + Mutations + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:42:09Z + + (-2) + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:42:11Z + + autolysis + + + + ncomms10900-f2.jpg + f2 + FIG + fig_caption + 46101 + (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. The (-2) residues of both prosegments point into the S1 pocket. (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions. (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). For all panels stereo views are provided in Supplementary Fig. 4g–j. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:44:34Z + + Structural superposition + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:08Z + + β1-T1A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:53:40Z + + β5-H(-2)L-T1A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:44:40Z + + (-2) + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:17:45Z + + prosegments + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:05Z + + S1 pocket + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:44:43Z + + Structural superposition + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:01Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:18Z + + propeptides + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:53:40Z + + β5-H(-2)L-T1A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:53:53Z + + β5-H(-2)T-T1A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:06:20Z + + β5-(H-2)A-T1A-K81R + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:34:12Z + + β5-T1A-K81R + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:02:30Z + + proteasomes + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:44:51Z + + (-2) to (-4) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:44:53Z + + Gly(-1) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:44:56Z + + Ala1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:45:00Z + + structures + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:45:02Z + + Structural superposition + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:16Z + + β2-T1A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:53:53Z + + β5-H(-2)T-T1A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:45:10Z + + (-2) + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:17:45Z + + prosegments + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:05Z + + S1 pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:45:18Z + + Thr(-2) + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:52Z + + β2 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bridge + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:18:31Z + + Gly(-1) + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:45:27Z + + Structural superposition + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T16:04:38Z + + matured + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:16:52Z + + β2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:16Z + + β2-T1A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:06:24Z + + β2-T(-2)V + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:45:33Z + + Val(-2) + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:05Z + + S1 pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:45:40Z + + Gly(-1) + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:45:43Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:45:45Z + + Gly(-1) + + + + ncomms10900-f3.jpg + f3 + FIG + fig_title_caption + 47199 + Architecture and proposed reaction mechanism of the proteasomal active site. + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + + ncomms10900-f3.jpg + f3 + FIG + fig_caption + 47276 + (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ. The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located. Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place. (d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome. The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red. Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state. The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle. Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH. Next, Thr1NH2 polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate. On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures). Substrate processing starts with nucleophilic attack of the carbonyl carbon atom of the scissile peptide bond. The charged Thr1 N terminus may engage in the orientation of the amide moiety and donate a proton to the emerging N terminus of the C-terminal cleavage product. The resulting deprotonated Thr1NH2 finally activates a water molecule for hydrolysis of the acyl-enzyme. + + site + SO: + melaniev@ebi.ac.uk + 2023-06-15T10:46:14Z + + Hydrogen-bonding network + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:39:22Z + + mature + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:01Z + + β5 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:49:06Z + + Thr1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen-bonded + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:49:13Z + + Lys33 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:49:18Z + + Asp17 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:30Z + + Thr1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bonds + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:49:23Z + + Ser129 + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:49:27Z + + 168 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:49:29Z + + Ser169 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:49:32Z + + Asp166 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:49:36Z + + active-site residues + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bonding + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:49:41Z + + strictly conserved + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:49:44Z + + proteolytic centre + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:49:47Z + + superposition + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:15:27Z + + β subunits + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:49:50Z + + Structural superposition + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:01Z + + β5 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:49:55Z + + β5-K33A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:30:26Z + + pp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:30:39Z + + trans + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:07Z + + water + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-22T10:38:25Z + + Lys33 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:19Z + + Lys33 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:07Z + + water + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrogen bonds + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:50:05Z + + Arg19 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:50:07Z + + Asp17 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:50:10Z + + Thr1 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:07Z + + water + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:30Z + + Thr1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:50:15Z + + active-site residues + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:50:18Z + + autocatalytic precursor processing + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:25Z + + proteasome + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:29Z + + active-site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:30Z + + Thr1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:50:28Z + + Autolysis + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:50:31Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:50:33Z + + Lys33 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:50:36Z + + strictly conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:50:39Z + + Gly47 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:30Z + + Thr1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:50:43Z + + Asp166 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:50:45Z + + Ser129 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:50:47Z + + Thr1 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:07Z + + water + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:29Z + + active-site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:30Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:30Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:50:52Z + + Thr1 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T13:23:07Z + + water + + + + ncomms10900-f4.jpg + f4 + FIG + fig_title_caption + 49537 + The proteasome favours threonine as the active-site nucleophile. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:25Z + + proteasome + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:51:29Z + + threonine + + + + ncomms10900-f4.jpg + f4 + FIG + fig_caption + 49602 + (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. Relative fluorescence units were measured in triplicate after 1 h of incubation at room temperature and are given as mean values. S.d.'s are indicated by error bars. (c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment. The prosegment is cleaved but still bound in the substrate-binding channel. Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel. (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. The SH group is rotated by 74° compared with the OH group. (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. The 2FO–FC electron-density map for Ser1 (blue mesh contoured at 1σ) is illustrated. (h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ. Ser1 lacks this stabilization and is therefore rotated by 60°. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:54:38Z + + Growth tests by serial dilution + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:01Z + + β5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:28:59Z + + yeast + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:59:29Z + + active-site + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:04:43Z + + mutants + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:02:34Z + + proteasomes + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:01Z + + β5 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:22:17Z + + Suc-LLVY-AMC + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:00:04Z + + 2FO–FC electron-density map + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:54:57Z + + β5-T1C + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:40:10Z + + prosegment + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:55:04Z + + cleaved + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:55:02Z + + still bound + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:55:07Z + + substrate-binding channel + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:55:11Z + + His(-2) + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:42:05Z + + S1 pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:53:10Z + + Met45 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:34:12Z + + β5-T1A-K81R + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:55:18Z + + Structural superposition + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:34:12Z + + β5-T1A-K81R + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:55:21Z + + β5-T1C + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:02Z + + β5 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:55:25Z + + Structural superposition + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:55:28Z + + β5-T1C + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:35Z + + propeptide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T13:29:08Z + + β1-T1A + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:14Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:02Z + + β5 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:12:15Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:55:36Z + + in complex with + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T12:01:25Z + + proteasome + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:29:55Z + + MG132 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T15:55:41Z + + inhibitor + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:57:19Z + + propeptides + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:55:45Z + + substrate-binding channel + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:55:48Z + + Structural superposition + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:02Z + + β5 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:55:50Z + + β5-T1C + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:03:50Z + + active sites + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:30Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:55:55Z + + Cys1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:04:48Z + + Structural superposition + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:02Z + + β5 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T16:06:30Z + + β5-T1S + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T16:03:54Z + + active sites + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:30Z + + Thr1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:56:02Z + + Ser1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T14:00:04Z + + 2FO–FC electron-density map + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:56:09Z + + Ser1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:30Z + + Thr1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:23:04Z + + hydrophobic interactions + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:56:18Z + + Ala46 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:56:21Z + + Thr3 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:56:24Z + + Ser1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:56:44Z + + lacks + + + + ncomms10900-f5.jpg + f5 + FIG + fig_title_caption + 51469 + Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:57:14Z + + β5-T1S + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:02:39Z + + proteasomes + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:40:22Z + + bortezomib + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:37Z + + carfilzomib + + + + ncomms10900-f5.jpg + f5 + FIG + fig_caption + 51550 + Inhibition assays (left panel). Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. IC50 values were determined in triplicate; s.d.'s are indicated by error bars. Note that IC50 values depend on time and enzyme concentration. Proteasomes (final concentration: 66 nM) were incubated with inhibitor for 45 min before substrate addition (final concentration: 200 μM). Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ. The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:58:48Z + + Inhibition assays + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:29:00Z + + yeast + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:02:43Z + + proteasomes + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T13:17:02Z + + β5 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:40:22Z + + bortezomib + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:37Z + + carfilzomib + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:22:17Z + + Suc-LLVY-AMC + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:58:51Z + + IC50 values + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:58:55Z + + IC50 values + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:02:47Z + + Proteasomes + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:58:57Z + + Structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:59:00Z + + β5-T1S + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:01:52Z + + mutant + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T15:59:04Z + + complex with both ligands + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:59:08Z + + Ser1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:54:57Z + + 2FO–FC electron-density maps + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:59:11Z + + Ser1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T15:59:21Z + + P1 site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:59:18Z + + Leu1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T13:56:00Z + + WT + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T16:02:52Z + + proteasome:inhibitor complex + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T15:59:26Z + + structures + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:30Z + + Thr1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:59:33Z + + superimposed + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T15:59:35Z + + mutation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T12:00:30Z + + Thr1 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T13:25:17Z + + Ser + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:40:22Z + + bortezomib + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T14:12:37Z + + carfilzomib + + + + t1.xml + t1 + TABLE + table_title_caption + 52522 + Growth phenotypes and status of autolysis and catalysis of mutants. + + + t1.xml + t1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"><bold>Mutant</bold></th><th align="center" valign="top" charoff="50"><bold>Viability</bold></th><th align="center" valign="top" charoff="50"><bold>Temperature sensitivity</bold></th><th align="center" valign="top" charoff="50"><bold>Autolysis state of the mutant subunit</bold><xref ref-type="fn" rid="t1-fn2">*</xref></th><th align="center" valign="top" charoff="50"><bold>Activity of the mutant subunit</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">WT</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">+++</td></tr><tr><td align="left" valign="top" charoff="50">β1-T1A (ref.<xref ref-type="bibr" rid="b13">13</xref>)</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β2-T1A (ref.<xref ref-type="bibr" rid="b13">13</xref>)</td><td align="center" valign="top" charoff="50">+++</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β1-T1A β2-T1A (ref.<xref ref-type="bibr" rid="b13">13</xref>)</td><td align="center" valign="top" charoff="50">+++</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-T1A</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)A-T1A</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)T-T1A</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)L-T1A</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-T1A, pp <italic>trans</italic>, <italic>nat1</italic>Δ</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">pp <italic>trans</italic></td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-T1A-K81R</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)A-T1A-K81R</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)T-T1A-K81R</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)L-T1A-K81R</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)A</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">+++</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)K</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">+++</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)F</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++</td></tr><tr><td align="left" valign="top" charoff="50">β5-H(-2)N</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++</td></tr><tr><td align="left" valign="top" charoff="50">β5pp-β1 (ref. 18)</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">+/−</td></tr><tr><td align="left" valign="top" charoff="50">β2-T(-2)V</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-L(-49S)-K33A (ref.<xref ref-type="bibr" rid="b13">13</xref>)</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">−</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-K33A, pp <italic>trans</italic><xref ref-type="bibr" rid="b13">13</xref></td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">pp <italic>trans</italic></td><td align="center" valign="top" charoff="50">+/−</td></tr><tr><td align="left" valign="top" charoff="50">β5-F(-45)S-K33R (ref.<xref ref-type="bibr" rid="b13">13</xref>)</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">−</td></tr><tr><td align="left" valign="top" charoff="50">β5-D17N</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">ND<xref ref-type="fn" rid="t1-fn3">†</xref></td><td align="center" valign="top" charoff="50">ND<xref ref-type="fn" rid="t1-fn3">†</xref></td></tr><tr><td align="left" valign="top" charoff="50">β5-L(-49)S-D17N</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+/−</td><td align="center" valign="top" charoff="50">+/−</td></tr><tr><td align="left" valign="top" charoff="50">β5-D17N, pp <italic>trans</italic></td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">pp <italic>trans</italic></td><td align="center" valign="top" charoff="50">+/−</td></tr><tr><td align="left" valign="top" charoff="50">β5-D166N</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">+/−</td></tr><tr><td align="left" valign="top" charoff="50">β5-D166N, pp <italic>trans</italic></td><td align="center" valign="top" charoff="50">+++</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">pp <italic>trans</italic></td><td align="center" valign="top" charoff="50">+/−</td></tr><tr><td align="left" valign="top" charoff="50">β5-T1S</td><td align="center" valign="top" charoff="50">+++</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">++</td></tr><tr><td align="left" valign="top" charoff="50">β5-T1C</td><td align="center" valign="top" charoff="50">++</td><td align="center" valign="top" charoff="50">++++</td><td align="center" valign="top" charoff="50">+</td><td align="center" valign="top" charoff="50">−</td></tr></tbody></table> + + 52591 + Mutant Viability Temperature sensitivity Autolysis state of the mutant subunit* Activity of the mutant subunit WT ++++ + + +++ β1-T1A (ref.) ++++ + − − β2-T1A (ref.) +++ ++ − − β1-T1A β2-T1A (ref.) +++ ++ − − β5-T1A +/− ++++ − − β5-H(-2)A-T1A +/− ND − − β5-H(-2)T-T1A +/− ND − − β5-H(-2)L-T1A ++ ND − − β5-T1A, pp trans, nat1Δ +/− ++++ pp trans − β5-T1A-K81R + ++++ − − β5-H(-2)A-T1A-K81R +/− ND − − β5-H(-2)T-T1A-K81R +/− ND − − β5-H(-2)L-T1A-K81R ++ ND − − β5-H(-2)A ++++ ++ + +++ β5-H(-2)K ++++ ++ + +++ β5-H(-2)F ++++ +++ + ++ β5-H(-2)N ++++ +++ + ++ β5pp-β1 (ref. 18) ++++ + +/− +/− β2-T(-2)V ++++ + − − β5-L(-49S)-K33A (ref.) + ++++ − − β5-K33A, pp trans + ++++ pp trans +/− β5-F(-45)S-K33R (ref.) ++ ++++ + − β5-D17N +/− ++++ ND† ND† β5-L(-49)S-D17N + ++++ +/− +/− β5-D17N, pp trans + ++++ pp trans +/− β5-D166N ++ ++++ + +/− β5-D166N, pp trans +++ ++++ pp trans +/− β5-T1S +++ ++++ + ++ β5-T1C ++ ++++ + − + + + t1.xml + t1 + TABLE + table_footnote + 53689 + ND, not determined. + + + t1.xml + t1 + TABLE + table_footnote + 53709 + *The autolysis state was assessed by purification and crystallization of the mutant proteasomes. + + + t1.xml + t1 + TABLE + table_footnote + 53806 + †Purification of this mutant proteasome was not possible. + + + diff --git a/BioC_XML/4795551_v0.xml b/BioC_XML/4795551_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..32e1735b555ed5fcebd6068cfddb831ea7be50c1 --- /dev/null +++ b/BioC_XML/4795551_v0.xml @@ -0,0 +1,14855 @@ + + + + PMC + 20201215 + pmc.key + + 4795551 + CC BY + no + 0 + 0 + + Functional and Structural Characterization of Ectoine Synthase + 10.1371/journal.pone.0151285 + 4795551 + 26986827 + PONE-D-15-52796 + e0151285 + 3 + This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + surname:Widderich;given-names:Nils + surname:Kobus;given-names:Stefanie + surname:Höppner;given-names:Astrid + surname:Riclea;given-names:Ramona + surname:Seubert;given-names:Andreas + surname:Dickschat;given-names:Jeroen S. + surname:Heider;given-names:Johann + surname:Smits;given-names:Sander H. J. + surname:Bremer;given-names:Erhard + surname:Hofmann;given-names:Andreas + All relevant data are within the paper and its Supporting Information files. + TITLE + Data Availability + front + 11 + 2016 + 0 + Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily + + 0.99961436 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + Crystal Structure + + + 0.9995517 + protein_type + cleaner0 + 2023-07-20T10:07:27Z + MESH: + + Ectoine Synthase + + + protein_state + DUMMY: + cleaner0 + 2023-07-20T10:06:42Z + + Metal-Containing + + + 0.9991269 + protein_type + cleaner0 + 2023-07-20T10:07:20Z + MESH: + + Cupin Superfamily + + + + ABSTRACT + abstract + 107 + Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel. + + 0.9922638 + chemical + cleaner0 + 2023-07-20T10:07:51Z + CHEBI: + + Ectoine + + + 0.9993475 + taxonomy_domain + cleaner0 + 2023-07-20T10:09:01Z + DUMMY: + + Bacteria + + + 0.9993168 + taxonomy_domain + cleaner0 + 2023-07-20T10:09:07Z + DUMMY: + + Archaea + + + 0.9973253 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + Ectoine synthase + + + 0.9420252 + protein_type + cleaner0 + 2023-07-20T10:09:58Z + MESH: + + EctC + + + 0.9979506 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + ectoine + + + 0.999782 + chemical + cleaner0 + 2023-07-20T10:08:34Z + CHEBI: + + N-gamma-acetyl-L-2,4-diaminobutyric acid + + + 0.99200696 + chemical + cleaner0 + 2023-07-20T14:18:25Z + CHEBI: + + water + + + 0.99961984 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + 0.9996322 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.9996338 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-20T10:11:50Z + + marine bacterium + + + 0.9991011 + species + cleaner0 + 2023-07-20T10:06:48Z + MESH: + + Sphingopyxis alaskensis + + + 0.9982413 + species + cleaner0 + 2023-07-20T10:06:55Z + MESH: + + Sa + + + 0.99890184 + protein + cleaner0 + 2023-07-20T10:09:39Z + PR: + + EctC + + + 0.99962497 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + 0.9996493 + protein_state + cleaner0 + 2023-07-20T10:12:17Z + DUMMY: + + apo + + + 0.99915904 + experimental_method + cleaner0 + 2023-07-20T14:28:34Z + MESH: + + Structural analysis + + + species + MESH: + cleaner0 + 2023-07-20T10:10:28Z + + Sa + + + 0.50138265 + protein + cleaner0 + 2023-07-20T10:09:38Z + PR: + + EctC + + + 0.98862517 + protein_type + cleaner0 + 2023-07-20T10:07:21Z + MESH: + + cupin superfamily + + + 0.911944 + protein + cleaner0 + 2023-07-20T10:09:39Z + PR: + + EctC + + + 0.9992981 + oligomeric_state + cleaner0 + 2023-07-20T10:12:02Z + DUMMY: + + dimer + + + 0.99934924 + protein_state + cleaner0 + 2023-07-20T10:12:22Z + DUMMY: + + head-to-tail + + + 0.9996296 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + 0.9992674 + site + cleaner0 + 2023-07-20T14:38:53Z + SO: + + interface + + + 0.99930906 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:19Z + + hydrophobic interactions + + + 0.999515 + structure_element + cleaner0 + 2023-07-20T14:39:50Z + SO: + + beta-sheets + + + 0.99926466 + oligomeric_state + cleaner0 + 2023-07-20T10:12:08Z + DUMMY: + + monomer + + + 0.99964887 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.99949694 + experimental_method + cleaner0 + 2023-07-20T14:18:33Z + MESH: + + metal depletion and reconstitution experiments + + + 0.8798571 + protein + cleaner0 + 2023-07-20T10:09:39Z + PR: + + EctC + + + protein_state + DUMMY: + cleaner0 + 2023-07-20T10:11:01Z + + iron-dependent + + + 0.8113488 + protein + cleaner0 + 2023-07-20T10:09:39Z + PR: + + EctC + + + 0.99978054 + chemical + cleaner0 + 2023-07-20T10:08:36Z + CHEBI: + + N-gamma-acetyl-L-2,4-diaminobutyric acid + + + 0.99935395 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + ectoine + + + 0.9997851 + chemical + cleaner0 + 2023-07-20T14:19:09Z + CHEBI: + + N-alpha-acetyl-L-2,4-diaminobutyric acid + + + evidence + DUMMY: + cleaner0 + 2023-07-20T11:31:32Z + + catalytic efficiency + + + 0.99946934 + experimental_method + cleaner0 + 2023-07-20T14:18:52Z + MESH: + + Structure-guided site-directed mutagenesis + + + 0.99933404 + protein_state + cleaner0 + 2023-07-20T14:46:26Z + DUMMY: + + evolutionarily highly conserved + + + protein_type + MESH: + cleaner0 + 2023-07-20T10:11:19Z + + EctC protein family + + + 0.9996268 + site + cleaner0 + 2023-07-20T13:28:16Z + SO: + + iron-binding site + + + 0.77332145 + protein + cleaner0 + 2023-07-20T10:09:39Z + PR: + + EctC + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:00Z + + iron + + + 0.9996016 + site + cleaner0 + 2023-07-20T13:43:14Z + SO: + + active site + + + 0.9987109 + protein + cleaner0 + 2023-07-20T10:09:39Z + PR: + + EctC + + + 0.9990552 + structure_element + cleaner0 + 2023-07-20T13:33:43Z + SO: + + cupin barrel + + + + INTRO + title_1 + 2268 + Introduction + + + INTRO + paragraph + 2281 + Compatible solutes are exploited by members of all three domains of life as versatile cyto-protectants, in particular against cellular stress elicited by high osmolarity environments. They are especially useful for this latter purpose since their benign nature allows their amassing to exceedingly high cellular concentrations. As a result of compatible solute accumulation, dehydration of the cytoplasm of osmotically stressed cells is counteracted, and concomitantly, its solvent properties are optimized for the functioning of vital biochemical and physiological processes. + + + INTRO + paragraph + 2858 + Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes. Both marine and terrestrial microorganisms produce them widely in response to osmotic or temperature stress. Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1). The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers. This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. The remarkable function preserving effects of ectoines for macromolecules and cells, frequently also addressed as chemical chaperones, led to a substantial interest in exploiting these compounds for biotechnological purposes and medical applications. + + 0.99967897 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + Ectoine + + + 0.99959415 + chemical + cleaner0 + 2023-07-20T10:12:52Z + CHEBI: + + (S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid + + + 0.99977165 + chemical + cleaner0 + 2023-07-20T10:13:04Z + CHEBI: + + 5-hydroxyectoine + + + 0.99902165 + chemical + cleaner0 + 2023-07-20T10:13:09Z + CHEBI: + + (4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-20T10:19:43Z + + marine and terrestrial microorganisms + + + 0.99904186 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + ectoine + + + 0.9997767 + chemical + cleaner0 + 2023-07-20T10:13:13Z + CHEBI: + + L-aspartate-ß-semialdehyde + + + 0.99959826 + protein_type + cleaner0 + 2023-07-20T10:13:35Z + MESH: + + L-2,4-diaminobutyrate transaminase + + + 0.9996469 + protein_type + cleaner0 + 2023-07-20T10:16:02Z + MESH: + + EctB + + + 0.999636 + protein_type + cleaner0 + 2023-07-20T10:13:38Z + MESH: + + 2,4-diaminobutyrate acetyltransferase + + + 0.9996762 + protein_type + cleaner0 + 2023-07-20T10:16:26Z + MESH: + + EctA + + + protein_type + MESH: + cleaner0 + 2023-07-20T10:07:29Z + + ectoine synthase + + + 0.99961436 + protein_type + cleaner0 + 2023-07-20T10:16:38Z + MESH: + + EctC + + + 0.99946684 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + ectoine + + + 0.99977547 + chemical + cleaner0 + 2023-07-20T10:13:18Z + CHEBI: + + 5-hydroxyectoine + + + 0.5892295 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + ectoine + + + 0.99958915 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + ectoine + + + 0.99327844 + protein_type + cleaner0 + 2023-07-20T14:16:45Z + MESH: + + ectoine hydroxylase + + + 0.99969697 + protein_type + cleaner0 + 2023-07-20T10:16:49Z + MESH: + + EctD + + + 0.9819392 + protein_type + cleaner0 + 2023-07-20T10:16:54Z + MESH: + + non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily + + + 0.9726849 + chemical + cleaner0 + 2023-07-20T10:16:59Z + CHEBI: + + ectoines + + + + pone.0151285.g001.jpg + pone.0151285.g001 + FIG + fig_title_caption + 4127 + Biosynthetic routes for ectoine and 5-hydroxyectoine. + + 0.99978477 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + ectoine + + + 0.9997826 + chemical + cleaner0 + 2023-07-20T10:13:18Z + CHEBI: + + 5-hydroxyectoine + + + + pone.0151285.g001.jpg + pone.0151285.g001 + FIG + fig_caption + 4181 + Scheme of the ectoine and 5-hydroxyectoine biosynthetic pathway. + + 0.9997751 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + ectoine + + + 0.99978995 + chemical + cleaner0 + 2023-07-20T10:13:18Z + CHEBI: + + 5-hydroxyectoine + + + + INTRO + paragraph + 4246 + Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1). Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out. Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1). In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes. + + 0.9990746 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.9434349 + protein + cleaner0 + 2023-07-20T10:09:39Z + PR: + + EctC + + + 0.99730337 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + ectoine + + + 0.9768098 + experimental_method + cleaner0 + 2023-07-20T14:28:42Z + MESH: + + characterizations + + + 0.9996544 + protein_type + cleaner0 + 2023-07-20T10:17:22Z + MESH: + + ectoine synthases + + + 0.9770065 + taxonomy_domain + cleaner0 + 2023-07-20T10:17:57Z + DUMMY: + + extremophiles + + + 0.9993757 + species + cleaner0 + 2023-07-20T10:17:30Z + MESH: + + Halomonas elongata + + + 0.99943984 + species + cleaner0 + 2023-07-20T10:17:36Z + MESH: + + Methylomicrobium alcaliphilum + + + 0.9993987 + species + cleaner0 + 2023-07-20T10:17:42Z + MESH: + + Acidiphilium cryptum + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-20T10:18:19Z + + nitrifying archaeon + + + 0.99944043 + species + cleaner0 + 2023-07-20T10:17:47Z + MESH: + + Nitrosopumilus maritimus + + + 0.99977577 + chemical + cleaner0 + 2023-07-20T10:18:25Z + CHEBI: + + N-γ-acetyl-L-2,4-diaminobutyric acid + + + 0.9997686 + chemical + cleaner0 + 2023-07-20T10:18:34Z + CHEBI: + + N-γ-ADABA + + + 0.9994516 + protein_type + cleaner0 + 2023-07-20T10:18:29Z + MESH: + + 2,4-diaminobutyrate acetyltransferase + + + 0.9980604 + protein_type + cleaner0 + 2023-07-20T10:19:02Z + MESH: + + EctA + + + 0.99930656 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + ectoine + + + 0.9997781 + chemical + cleaner0 + 2023-07-20T14:19:14Z + CHEBI: + + water + + + 0.9992575 + protein + cleaner0 + 2023-07-20T10:09:39Z + PR: + + EctC + + + 0.9997859 + chemical + cleaner0 + 2023-07-20T10:18:43Z + CHEBI: + + 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate + + + 0.99978095 + chemical + cleaner0 + 2023-07-20T10:18:48Z + CHEBI: + + ADPC + + + 0.9978382 + chemical + cleaner0 + 2023-07-20T10:19:06Z + CHEBI: + + glutamine + + + 0.99942786 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + ectoine + + + 0.9993905 + chemical + cleaner0 + 2023-07-20T10:07:52Z + CHEBI: + + ectoine + + + + INTRO + paragraph + 5189 + Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa). We demonstrate here for the first time that the ectoine synthase is a metal-dependent enzyme, with iron as the most likely physiologically relevant co-factor. The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily. The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase. + + 0.99968475 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.7485945 + evidence + cleaner0 + 2023-07-20T14:10:36Z + DUMMY: + + structure + + + 0.8181996 + experimental_method + cleaner0 + 2023-07-20T14:28:46Z + MESH: + + biochemically characterized + + + 0.99963343 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-20T10:19:51Z + + marine bacterium + + + 0.9990964 + species + cleaner0 + 2023-07-20T10:06:50Z + MESH: + + Sphingopyxis alaskensis + + + 0.9987779 + species + cleaner0 + 2023-07-20T10:06:56Z + MESH: + + Sa + + + 0.9996583 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.99915826 + chemical + cleaner0 + 2023-07-20T11:22:00Z + CHEBI: + + iron + + + 0.9993026 + protein + cleaner0 + 2023-07-20T10:09:39Z + PR: + + EctC + + + 0.99935967 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + 0.99945956 + experimental_method + cleaner0 + 2023-07-20T14:28:50Z + MESH: + + structural analysis + + + 0.99953306 + protein_type + cleaner0 + 2023-07-20T10:07:21Z + MESH: + + cupin superfamily + + + 0.99961185 + evidence + cleaner0 + 2023-07-20T13:57:50Z + DUMMY: + + crystal structures + + + species + MESH: + cleaner0 + 2023-07-20T10:06:56Z + + Sa + + + 0.9697854 + protein + cleaner0 + 2023-07-20T10:09:39Z + PR: + + EctC + + + 0.9992135 + experimental_method + cleaner0 + 2023-07-20T14:28:53Z + MESH: + + site-directed mutagenesis + + + 0.9993755 + protein_state + cleaner0 + 2023-07-20T14:40:00Z + DUMMY: + + evolutionarily highly conserved + + + 0.9995388 + protein_type + cleaner0 + 2023-07-20T10:20:35Z + MESH: + + EctC protein + + + 0.9978783 + site + cleaner0 + 2023-07-20T14:39:00Z + SO: + + catalytic core + + + 0.9996662 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + + METHODS + title_1 + 6216 + Materials and Methods + + + METHODS + title_2 + 6238 + Chemicals + + + METHODS + paragraph + 6248 + Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] was a kind gift from bitop AG (Witten, Germany). Anhydrotetracycline (AHT), desthiobiotine and the strepavidin affinity matrix for the purification of Strep-tag II labeled proteins was purchased from IBA GmbH (Göttingen, Germany). Hydroxylamine and phenanthroline for the photometric determination of the iron-content of the recombinant (Sa)EctC proteins were purchased from Sigma-Aldrich (München, Germany). + + + METHODS + title_2 + 6729 + Synthesis of N-γ-acetyl-L-2,4-diaminobutyric acid and N-α-acetyl-L-2,4-diaminobutyric acid through hydrolysis of ectoine + + + METHODS + paragraph + 6858 + All chemicals used to synthesize the gamma and alpha forms of N-acetyl-l-2,4-diaminobutyric acid (ADABA) for EctC enzyme activity assays were purchased either from Sigma Aldrich (Steinheim, Germany), or Acros (Geel, Belgium). Alkaline hydrolysis of ectoine (284 mg, 2.0 mmol) was accomplished in aqueous KOH (50 mL, 0.1 M) for 20 h at 50°C. The reaction mixture was subsequently neutralized with perchloric acid (60% in water, 4 mL) and the precipitated potassium perchlorate was filtered off. Subsequently, the filtrate was concentrated under reduced pressure. Purification of the residue and separation of the formed compounds was then performed by repeated chromatography on a silica gel column (Merck silica gel 60) using a gradient of ethanol/25% ammonia/water 50:1:2–10:1:2 as eluent to yield pure N-γ-ADABA (192 mg, 1.20 mmol, 60%) and N-α-ADABA (32 mg, 0.20 mmol, 10%). The identity and purity of theses compounds was unequivocally established by thin-layer chromatography (TLC) and nuclear magnetic resonance (1H-NMR and 13C-NMR) spectroscopy (S1a and S1b Fig) as described on a Bruker AVIII-400 or DRX-500 NMR spectrometer. (i) Analytical data for N-γ-ADABA: TLC: Rf = 0.55 (ethanol/25% ammonia/water 7:1:2); 1H-NMR (400 MHz, D2O): δ = 3.71 (dd, 3J(H,H) = 7.6 Hz, 3J(H,H) = 5.6 Hz, 1H, CH), 3.41–3.24 (m, 2H, CH2), 2.15–2.01 (m, 2H, CH2), 1.99 (s, 3H, CH3) ppm; 13C-NMR (100 MHz, D2O): δ = 177.5 (CO), 177.0 (COOH), 55.3 (CH), 38.3 (CH2), 33.0 (CH2), 24.6 (CH3) ppm. (ii) Analytical data for N-α-ADABA: TLC: Rf = 0.38 (ethanol/25% ammonia/water 7:1:2); 1H-NMR (400 MHz, D2O): δ = 4.24 (dd, 3J(H,H) = 8.8 Hz, 3J(H,H) = 5.1 Hz, 1H, CH), 3.07–3.02 (m, 2H, CH2), 2.22–2.11 (m, 2H, CH2), 2.04 (s, 3H, CH3) ppm; 13C-NMR (100 MHz, D2O): δ = 180.1 (CO), 176.7 (COOH), 55.4 (CH), 39.6 (CH2), 32.5 (CH2), 24.7 (CH3) ppm. + + + METHODS + title_2 + 8711 + Bacterial strains, plasmids and media + + + METHODS + paragraph + 8749 + The nucleotide sequence of the ectC gene from S. alaskensis (genome accession number: NC_008048) was used as a template to obtain a codon-optimized ectC DNA sequence (Life Technologies, Darmstadt, Germany) for its expression in E. coli. The nucleotide sequence of the synthetic ectC gene was deposited in the NCBI database under accession number KR002036. The synthetic ectC gene was used to construct an expression plasmid (pNW12) that is based on the pASG-IBA3 vector (IBA GmbH, Göttingen, Germany). In plasmid pNW12, the ectC gene is fused at its 3’ end to a short open reading frame encoding a Strep-tag II affinity peptide (NWSHPQFEK). It is transcribed from the TetR-controlled tet promoter carried by the backbone of the pASG-IBA3 expression vector. De-repression of tet promoter activity can be triggered by adding the synthetic inducer AHT for the TetR repressor to the growth medium. The details of the construction of pNW12 have been reported. + + + METHODS + paragraph + 9707 + Plasmids carrying ectC genes were routinely maintained in the Escherichia coli strain DH5α (Invitrogen, Karlsruhe, Germany) on LB agar plates containing ampicillin (100 μg ml-1). Plasmid DNA was isolated by routine procedures. Minimal medium A (MMA) containing 0.5% (w/v) glucose as the carbon source, 0.5% (w/v) casamino acids, 1 mM MgSO4, and 3 mM thiamine was used to cultivate the E. coli strain BL21 carrying pNW12 for the overproduction of the (Sa)EctC protein and its mutant derivatives. No additional metal solution was added to the components of the original recipe of MMA. + + + METHODS + title_2 + 10295 + Site-directed mutagenesis of the ectC gene + + + METHODS + paragraph + 10338 + Variants of the codon-optimized ectC gene from S. alaskensis present on plasmid pNW12 were prepared by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, Waldbronn, Germany) with custom synthesized DNA primers purchased from Microsynth AG (Lindau, Germany). The DNA sequence of the entire coding region of each mutant ectC gene was determined by Eurofins MWG (Ebersberg, Germany) to ensure the presence of the desired mutation and the absence of unwanted alterations. Details on the genetic changes introduced into ectC genes are listed in Table 1. + + + pone.0151285.t001.xml + pone.0151285.t001 + TABLE + table_title_caption + 10933 + Conversion of N-γ-ADABA into ectoine by (Sa)EctC mutant derivatives and their iron-content. + + + pone.0151285.t001.xml + pone.0151285.t001 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><thead><tr><th align="center" rowspan="1" colspan="1">Mutation</th><th align="center" rowspan="1" colspan="1">Amino acid-substitution</th><th align="center" rowspan="1" colspan="1">Ectoine formed [mM]</th><th align="center" rowspan="1" colspan="1">Activity (%)</th><th align="center" rowspan="1" colspan="1">Iron content of the protein preparation (mol %)</th></tr></thead><tbody><tr><td align="center" rowspan="1" colspan="1">WT</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">9.33 ± 0.28</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">92.1 ± 3.4</td></tr><tr><td align="left" rowspan="1" colspan="1">TAT/GCT</td><td align="left" rowspan="1" colspan="1">Tyr-52/Ala</td><td align="center" rowspan="1" colspan="1">2.53 ± 0.18</td><td align="center" rowspan="1" colspan="1">27</td><td align="center" rowspan="1" colspan="1">19.8 ± 1.7</td></tr><tr><td align="center" rowspan="1" colspan="1">GAA/GCA</td><td align="left" rowspan="1" colspan="1">Glu-57/Ala</td><td align="center" rowspan="1" colspan="1">0.97 ± 0.16</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">4.3 ± 2.9</td></tr><tr><td align="center" rowspan="1" colspan="1">GAA/GAT</td><td align="left" rowspan="1" colspan="1">Glu-57/Asp</td><td align="center" rowspan="1" colspan="1">6.19 ± 0.42</td><td align="center" rowspan="1" colspan="1">66</td><td align="center" rowspan="1" colspan="1">68.7 ± 5.1</td></tr><tr><td align="left" rowspan="1" colspan="1">TAT/GCT</td><td align="left" rowspan="1" colspan="1">Tyr-85/Ala</td><td align="center" rowspan="1" colspan="1">2.15 ± 0.28</td><td align="center" rowspan="1" colspan="1">23</td><td align="center" rowspan="1" colspan="1">8.3 ± 2.3</td></tr><tr><td align="left" rowspan="1" colspan="1">TAT/TTT</td><td align="left" rowspan="1" colspan="1">Tyr-85/Phe</td><td align="center" rowspan="1" colspan="1">2.74± 0.50</td><td align="center" rowspan="1" colspan="1">29</td><td align="center" rowspan="1" colspan="1">9.4 ± 3.9</td></tr><tr><td align="left" rowspan="1" colspan="1">TAT/TGG</td><td align="left" rowspan="1" colspan="1">Tyr-85/Trp</td><td align="center" rowspan="1" colspan="1">0.95 ± 0.08</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">5.1 ± 1.7</td></tr><tr><td align="left" rowspan="1" colspan="1">CAT/GCT</td><td align="left" rowspan="1" colspan="1">His-93/Ala</td><td align="center" rowspan="1" colspan="1">0.72 ± 0.09</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">4.5 ± 0.8</td></tr><tr><td align="left" rowspan="1" colspan="1">CAT/AAT</td><td align="left" rowspan="1" colspan="1">His-93/Asn</td><td align="center" rowspan="1" colspan="1">2.14 ± 0.31</td><td align="center" rowspan="1" colspan="1">23</td><td align="center" rowspan="1" colspan="1">12.9 ± 2.6</td></tr><tr><td align="left" rowspan="1" colspan="1">TGG/GCG</td><td align="left" rowspan="1" colspan="1">Trp-21/Ala</td><td align="center" rowspan="1" colspan="1">2.41 ± 0.39</td><td align="center" rowspan="1" colspan="1">26</td><td align="center" rowspan="1" colspan="1">89.4 ± 4.5</td></tr><tr><td align="left" rowspan="1" colspan="1">ACG/GCC</td><td align="left" rowspan="1" colspan="1">Ser-23/Ala</td><td align="center" rowspan="1" colspan="1">1.98 ± 0.42</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">91.6 ± 2.8</td></tr><tr><td align="left" rowspan="1" colspan="1">ACC/GCC</td><td align="left" rowspan="1" colspan="1">Thr-40/Ala</td><td align="center" rowspan="1" colspan="1">1.12 ± 0.13</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">89.6 ± 2.2</td></tr><tr><td align="left" rowspan="1" colspan="1">TGT/GCT</td><td align="left" rowspan="1" colspan="1">Cys-105/Ala</td><td align="center" rowspan="1" colspan="1">0.96 ± 0.21</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">90.1 ± 1.6</td></tr><tr><td align="left" rowspan="1" colspan="1">TGT/TCT</td><td align="left" rowspan="1" colspan="1">Cys-105/Ser</td><td align="center" rowspan="1" colspan="1">7.81 ± 0.65</td><td align="center" rowspan="1" colspan="1">84</td><td align="center" rowspan="1" colspan="1">88.7 ± 3.1</td></tr><tr><td align="left" rowspan="1" colspan="1">TTT/GCT</td><td align="left" rowspan="1" colspan="1">Phe-107/Ala</td><td align="center" rowspan="1" colspan="1">4.77 ± 0.10</td><td align="center" rowspan="1" colspan="1">51</td><td align="center" rowspan="1" colspan="1">87.9 ± 2.2</td></tr><tr><td align="left" rowspan="1" colspan="1">TTT/TAT</td><td align="left" rowspan="1" colspan="1">Phe-107/Tyr</td><td align="center" rowspan="1" colspan="1">8.87 ± 0.62</td><td align="center" rowspan="1" colspan="1">95</td><td align="center" rowspan="1" colspan="1">90.9 ± 3.9</td></tr><tr><td align="left" rowspan="1" colspan="1">TTT/TGG</td><td align="left" rowspan="1" colspan="1">Phe-107/Trp</td><td align="center" rowspan="1" colspan="1">1.08 ± 0.27</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">72.6 ± 5.8</td></tr><tr><td align="left" rowspan="1" colspan="1">CAT/GCT</td><td align="left" rowspan="1" colspan="1">His-117/Ala</td><td align="center" rowspan="1" colspan="1">4.14 ± 0.27</td><td align="center" rowspan="1" colspan="1">44</td><td align="center" rowspan="1" colspan="1">82.9 ± 1.1</td></tr><tr><td align="left" rowspan="1" colspan="1">CAT/GCT</td><td align="left" rowspan="1" colspan="1">His-55/Ala</td><td align="center" rowspan="1" colspan="1">1.53 ± 0.19</td><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1">15.4 ± 4.3</td></tr><tr><td align="left" rowspan="1" colspan="1">GAA/GCA</td><td align="center" rowspan="1" colspan="1">Glu-115/Ala</td><td align="center" rowspan="1" colspan="1">1.92 ± 0.44</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">87.6 ± 4.4</td></tr><tr><td align="left" rowspan="1" colspan="1">GAA/GAT</td><td align="center" rowspan="1" colspan="1">Glu-115/Asp</td><td align="center" rowspan="1" colspan="1">7.15 ± 0.60</td><td align="center" rowspan="1" colspan="1">77</td><td align="center" rowspan="1" colspan="1">88.0 ± 3.2</td></tr><tr><td align="left" rowspan="1" colspan="1">CTG/GCG</td><td align="left" rowspan="1" colspan="1">Leu-87/Ala</td><td align="center" rowspan="1" colspan="1">5.81 ± 0.44</td><td align="center" rowspan="1" colspan="1">62</td><td align="center" rowspan="1" colspan="1">92.0 ± 1.3</td></tr><tr><td align="left" rowspan="1" colspan="1">GAT/GCT</td><td align="left" rowspan="1" colspan="1">Asp-91/Ala</td><td align="center" rowspan="1" colspan="1">7.48 ± 0.81</td><td align="center" rowspan="1" colspan="1">80</td><td align="center" rowspan="1" colspan="1">89.6 ± 2.2</td></tr><tr><td align="left" rowspan="1" colspan="1">GAT/GAA</td><td align="left" rowspan="1" colspan="1">Asp-91/Glu</td><td align="center" rowspan="1" colspan="1">9.13 ± 0.57</td><td align="center" rowspan="1" colspan="1">98</td><td align="center" rowspan="1" colspan="1">90.0 ± 1.9</td></tr><tr><td align="left" rowspan="1" colspan="1">ACC/GCC</td><td align="left" rowspan="1" colspan="1">Thr-41/Ala</td><td align="center" rowspan="1" colspan="1">8.84 ± 0.63</td><td align="center" rowspan="1" colspan="1">95</td><td align="center" rowspan="1" colspan="1">91.7 ± 1.9</td></tr><tr><td align="left" rowspan="1" colspan="1">CAT/GCT</td><td align="left" rowspan="1" colspan="1">His-51/Ala</td><td align="center" rowspan="1" colspan="1">8.94 ± 0.47</td><td align="center" rowspan="1" colspan="1">96</td><td align="center" rowspan="1" colspan="1">90.2 ± 2.6</td></tr></tbody></table> + + 11029 + Mutation Amino acid-substitution Ectoine formed [mM] Activity (%) Iron content of the protein preparation (mol %) WT - 9.33 ± 0.28 100 92.1 ± 3.4 TAT/GCT Tyr-52/Ala 2.53 ± 0.18 27 19.8 ± 1.7 GAA/GCA Glu-57/Ala 0.97 ± 0.16 10 4.3 ± 2.9 GAA/GAT Glu-57/Asp 6.19 ± 0.42 66 68.7 ± 5.1 TAT/GCT Tyr-85/Ala 2.15 ± 0.28 23 8.3 ± 2.3 TAT/TTT Tyr-85/Phe 2.74± 0.50 29 9.4 ± 3.9 TAT/TGG Tyr-85/Trp 0.95 ± 0.08 10 5.1 ± 1.7 CAT/GCT His-93/Ala 0.72 ± 0.09 8 4.5 ± 0.8 CAT/AAT His-93/Asn 2.14 ± 0.31 23 12.9 ± 2.6 TGG/GCG Trp-21/Ala 2.41 ± 0.39 26 89.4 ± 4.5 ACG/GCC Ser-23/Ala 1.98 ± 0.42 21 91.6 ± 2.8 ACC/GCC Thr-40/Ala 1.12 ± 0.13 12 89.6 ± 2.2 TGT/GCT Cys-105/Ala 0.96 ± 0.21 10 90.1 ± 1.6 TGT/TCT Cys-105/Ser 7.81 ± 0.65 84 88.7 ± 3.1 TTT/GCT Phe-107/Ala 4.77 ± 0.10 51 87.9 ± 2.2 TTT/TAT Phe-107/Tyr 8.87 ± 0.62 95 90.9 ± 3.9 TTT/TGG Phe-107/Trp 1.08 ± 0.27 12 72.6 ± 5.8 CAT/GCT His-117/Ala 4.14 ± 0.27 44 82.9 ± 1.1 CAT/GCT His-55/Ala 1.53 ± 0.19 16 15.4 ± 4.3 GAA/GCA Glu-115/Ala 1.92 ± 0.44 21 87.6 ± 4.4 GAA/GAT Glu-115/Asp 7.15 ± 0.60 77 88.0 ± 3.2 CTG/GCG Leu-87/Ala 5.81 ± 0.44 62 92.0 ± 1.3 GAT/GCT Asp-91/Ala 7.48 ± 0.81 80 89.6 ± 2.2 GAT/GAA Asp-91/Glu 9.13 ± 0.57 98 90.0 ± 1.9 ACC/GCC Thr-41/Ala 8.84 ± 0.63 95 91.7 ± 1.9 CAT/GCT His-51/Ala 8.94 ± 0.47 96 90.2 ± 2.6 + + + pone.0151285.t001.xml + pone.0151285.t001 + TABLE + table_footnote + 12408 + The conversion of N-γ-ADABA into ectoine by the (Sa)EctC protein and its mutant derivatives was monitored in a reaction that contained 10 mM N-γ-ADABA as the substrate, 1 mM FeSO4 and 5 μg of the EctC protein under study. The amount of ectoine formed was measured after 20 min of incubation of the enzyme-substrate mixture by HPLC analysis. The iron-content of the investigated protein preparations was determined photometrically; note that in comparison with data obtained via ICP-MS, the colorimetric assay overestimates somewhat the iron content of the (Sa)EctC protein preparations. + + + METHODS + title_2 + 13004 + Overproduction and purification of recombinant EctC proteins + + + METHODS + paragraph + 13065 + For the overproduction of the (Sa)EctC-Strep-tag II protein, an overnight culture of strain [BL21 (pNW12)] was prepared in MMA and used to inoculate 1 L of MMA (in a 2 L Erlenmeyer flask) to an OD578 of 0.05. The cells were grown on an aerial shaker (set to 180 rpm) at 37°C until the culture reached an OD578 of 0.5. At this time point, the growth temperature was lowered to 30°C and the speed of the shaker was reduced to 100 rpm. Growth of the culture was continued and when it reached an OD578 of 0.7, AHT was added to the growth medium at a final concentration of 0.2 mg ml-1 to boost expression of the recombinant ectC gene. After 2 h of further incubation of the culture, the E. coli cells were harvested by centrifugation and disrupted by passing them several times through a French Pressure cell; a cleared cell lysate was prepared by ultracentrifugation (100 000 g) at 4°C for 1 h. The supernatant of this cleared lysate was then passed through a column filled with 5 ml of Strep-Tactin Superflow material (IBA GmbH, Göttingen, Germany); the column had been equilibrated with a buffer containing 200 mM NaCl and 20 mM Tris-HCl (pH 8). The (Sa)EctC-Strep-tag II protein was eluted from the affinity matrix with three column volumes of the same buffer containing 2.5 mM desthiobiotin. The recombinant (Sa)EctC-Strep-tag II protein was then concentrated to either 5 mg ml-1 for enzymes assays or 10 mg ml-1 for crystallization trials with Vivaspin 6 columns (Satorius Stedim Biotech GmbH, Göttingen, Germany) in the same buffer as described above. Desthiobiotin was not removed by dialysis from these protein preparations. The purified and concentrated (Sa)EctC-Strep-tag II protein was either used immediately for enzymes assays or kept at 4°C since the flash-freezing of the protein with liquid nitrogen and its subsequent storage at -80°C resulted in a rapid inactivation of ectoine synthase activity. 25 Variants of the (Sa)EctC-Strep-tag II protein carrying singe amino acid substitutions (Table 1) were overproduced and purified using the same procedure employed for the isolation of the wild-type protein. These mutant proteins behaved like the wild-type (Sa)EctC-Strep-tag II protein during the overproduction and purification procedure. Protein concentrations were determined both with a Pierce BCA Protein Assay Kit (Thermo Scientific, Schwerte, Germany) using BSA as the standard protein and spectrophotometrically by using an extinction coefficient of 15 470 M-1 cm-1 for the (Sa)EctC-Strep-tag II protein at a wavelength of 280 nm. The purity and integrity of the isolated (Sa)EctC-Strep-tag II proteins was inspected by SDS-polyacrylamide (15%) gel electrophoresis (SDS-PAGE). Molecular mass marker proteins for SDS-PAGE were purchased from LifeTechnologies (Darmstadt, Germany). + + + METHODS + title_2 + 15873 + Ectoine synthase enzyme activity assays + + + METHODS + paragraph + 15913 + The ectoine synthase activity of the (Sa)EctC protein was determined by HPLC-based enzyme assays. The initial enzyme activity assays were performed in a 30 μl-reaction volume for 20 min at 20°C. The used standard buffer (20 mM Tris, pH 8.0) contained 150 mM NaCl, 1 mM FeCl2, and 10 mM N-γ-ADABA. To determine optimal enzyme assay conditions for the (Sa)EctC-Strep-tag II protein, assay parameters and buffer conditions (e.g., the salt-concentrations, temperature, pH) were individually changed. The finally optimized assay buffer for ectoine synthase activity of the (Sa)EctC protein contained 20 mM Tris (pH 8.5), 200 mM NaCl, 1 mM FeCl2 and 10 mM N-γ-ADABA. Activity assays were run for 20 min at 15°C. Usually, 10 μg of the purified (Sa)EctC protein were added to start the enzyme assay. To assess the kinetic parameters of the ectoine synthase, varying concentrations of the substrates were used in the optimized assay buffer with a constant amount (10 μg) of the (Sa)EctC protein. The concentration of the natural EctC substrate N-γ-ADABA was varied between 0 and 40 mM, whereas that of N-α-ADABA was varied between 0 and 200 mM in the enzyme assays. Enzyme reactions were stopped by adding 30 μl of acetonitrile (100%) to the reaction vessel. The samples were centrifuged (13000 rpm, at room temperature for 5 min) to remove denatured proteins; the supernatant was subsequently analyzed for the formation of ectoine by HPLC analysis. Usually, 5- to 10-μl samples were injected into the HPLC system and the reaction product ectoine was analytically detected on a GROM-SIL Amino-1PR column (125 x 4 mm with a particle size of 3μm; purchased from GROM, Rottenburg-Hailfingen, Germany). Synthesis of ectoine by the purified (Sa)EctC-Strep-tag II protein and its mutant derivatives was monitored using a Infinity 1260 Diode Array Detector (DAD) (Agilent, Waldbronn, Germany) integrated into an Agilent 1260 Infinity LC system (Agilent). The ectoine content of the samples was quantified using the OpenLAB software suite (Agilent). The data shown for each ectC mutant (Table 1) were derived from two independent (Sa)EctC preparations, and each (Sa)EctC protein solution was assayed three times for its enzyme activity. + + + METHODS + title_2 + 18155 + Metal depletion and reconstitution of the (Sa)EctC protein + + + METHODS + paragraph + 18214 + To assess the dependency of the ectoine synthase for its enzyme activity on iron and other metals, purified and concentrated (Sa)EctC protein preparations (10 μM) were treated with different concentrations of EDTA for 10 minutes. They were subsequently dialyzed to remove the EDTA and the remaining (Sa)EctC enzyme activity was analyzed. To determine metal ion specificity of the ectoine synthase, 500 μl of the (Sa)EctC protein (100 μM) were initially treated with 1 mM EDTA for 10 minutes to obtain apo-(Sa)EctC protein preparations and the EDTA was then removed by dialysis. Enzyme activity assays with 10 μM of such protein preparations were then performed in the presence of either stoichiometric (10 μM) or excess amounts (1 mM) of FeCl2, FeCl3, ZnCl2, CoCl2, NiCl2, CuCl2, and MnCl2 to monitor metal ion specificity of the ectoine synthase. Prior to initiation of the enzyme reaction (by addition of the substrate), the (Sa)EctC protein solution was incubated with the different indicated metal ions for 10 minutes. + + + METHODS + title_2 + 19242 + Determination of the oligomeric state of (Sa)EctC protein + + + METHODS + paragraph + 19300 + To determine the oligomeric state of the (Sa)EctC protein in solution, we used high-performance liquid chromatography coupled to multi-angle light scattering detection (HPLC-MALS). A Bio SEC-5 HPLC column (Agilent Technologies Deutschland GmbH, Böblingen, Germany) with a pore size of 300 Å was equilibrated with 20 mM Tris-HCl (pH 7.5), 200 mM NaCl for high-performance liquid chromatography analysis. For these experiments, an Agilent Technologies system connected to a triple-angle light scattering detector (miniDAWN TREOS, Wyatt Technology Europe GmbH, Dernbach, Germany) followed by a differential refractive index detection system (Optilab t-rEX, Wyatt Technology) was used. Typically, 100 μl of purified (Sa)EctC protein (2 mg ml-1) was loaded onto the Bio SEC-5 HPLC column and the obtained data were analyzed with the ASTRA software package (Wyatt Technology). + + + METHODS + title_2 + 20174 + Determination of metal content of recombinant (Sa)EctC protein by ICP-MS + + + METHODS + paragraph + 20247 + The elemental contents of P, Fe, Ni, Cu and Zn of the (Sa)EctC-Strep-Tag-II protein sample were determined by inductive-coupled plasma mass spectrometry (ICP-MS) using an Agilent 7900 ICP-MS system equipped with a HEN nebulizer and cooled scott spray chamber under standard operating conditions. The isotopes 31P, 56Fe, 57Fe, 58Ni, 60Ni, 62Ni, 63Cu, 65Cu, 64Zn, 66Zn, and 67Zn were measured under NoGas, He collision and H2 reaction mode conditions. Some isotopes are strongly interfered from the matrix (mainly 56Fe, 63Cu) in the NoGas mode and are therefore rejected. The (Sa)EctC protein samples and buffer blanks were diluted 100-fold with ultra pure water and spiked with 10 μg kg-1 Y as the internal standard. The calibration of the ICP-MS was performed in the concentration range between 0.1 to 100 μg kg-1 using a homemade P standard solution prepared from titrimetrically analyzed H3PO4 solution and from dilutions of a Merck ICP multi-element standard solution IV (Merck No. 111355, Darmstadt, Germany). + + + METHODS + title_2 + 21263 + Photometric determination of non-heme-iron in (Sa)EctC and its mutant derivatives + + + METHODS + paragraph + 21345 + To determine the iron content in our (Sa)EctC-Strep-Tag-II preparations photometrically, 10 nmol of the purified proteins were heated at 80°C for 10 min in 250 μl of a 1% HCl solution. The reaction assay was cooled down on ice and then centrifuged (13000 rpm, 10 min at room temperature). The supernatant was transferred to a new reaction tube, and 750 μl H2O, 50 μl of 10% hydroxylamine/HCl, and 250 μl of 0.1% phenanthroline were added to the reaction vessel. After 30 min of incubation at room temperature, the absorbance of the solution was measured at 512 nm. 5 to 40 nmol of ammonium iron(II) sulfate were used for calibration of the assay. + + + METHODS + title_2 + 21997 + Crystallization of the (Sa)EctC protein + + + METHODS + paragraph + 22037 + Several conditions under which the (Sa)EctC protein formed crystals were found by using commercial screens (Nextal, Qiagen, Hilden, Germany; Molecular Dimensions, Suffolk, UK) in 96-well sitting drop plates (Corning 3553) at 12°C. Homogeneous (Sa)EctC protein (0.1 μl from a solution of 11 mg protein ml-1) was mixed with 0.1 μl reservoir solution and equilibrated against 50 μl reservoir solution. The most promising condition was found with a solution containing 0.05 M calcium acetate, 0.1 M sodium acetate (pH 4.5), and 40% (v/v) 1,2-propanediol from the Nextal Core IV suite (Qiagen, Hilden, Germany). A second condition under which (Sa)EctC crystallized was identified in microbatch setups (1 μl + 1 μl drops) using 20% (w/v) PEG 6000, 0.9 M lithium chloride, and 0.1 M citric acid (pH 5) from the Nextal Core II suite (Qiagen, Hilden, Germany). These conditions were optimized by grid screens around the initial condition and/or after the addition of tert-butanol as an additive. Large crystals were obtained either without any additive or after the addition of tert-butanol to the (Sa)EctC protein solution 30 minutes before the drops were spotted. Crystals reached their maximum dimensions of about 50 × 50 × 70 μm3 after 3–10 weeks. The crystals were fished after overlaying the drop with 2 μl mineral oil and flash frozen in liquid nitrogen. To obtain heavy atom derivatized crystals, methylmercury(II) chloride was added (final concentration: 0.5 mM) to the crystals in their drop for 30 minutes before they were fished and flash frozen in liquid nitrogen. + + + METHODS + title_2 + 23618 + Data processing and structure determination + + + METHODS + paragraph + 23662 + Native data sets were collected from a single crystal of (Sa)EctC obtained from the various crystallization trials at the ERSF beamline ID23eh2 (Grenoble, France) at 100 K. These data sets were processed using the XDS package and scaled with XSCALE showing a maximum resolution of 1.2 Å. To obtain initial phases of (Sa)EctC, a mercury-derivatized crystal was used to collect a conservative dataset at 2.8 Å resolution. The data were processed and scaled as described above, before the program AUTORICKSHAW using single isomorphous replacement (SIRAS), was used to localize the Hg atom, phase and built an initial model of the (Sa)EctC protein. This initial model was used as a template for molecular replacement on the 2.0 Å dataset revealing four monomers in the asymmetric unit. Once the 2.0 Å structure was refined, a single monomer of this structure was used as a template for molecular replacement to phase the 1.2 Å resolution dataset using the PHENIX software. Model building and refinement were performed using COOT, Refmac5 and Phenix_refine. Data refinement statistics and model content are summarized in Table 1. + + + METHODS + title_2 + 24792 + PDB accession numbers + + + METHODS + paragraph + 24814 + The atomic coordinates and structural factors have been deposited into the Protein Data Bank (PDB) (Brookhaven, USA) under the following accession codes: 5BXX (for the “semi-closed” (Sa)EctC structure) and 5BY5 (for the “open” (Sa)EctC structure). + + + METHODS + title_2 + 25070 + Figure preparation of crystal structures + + + METHODS + paragraph + 25111 + Figures of the crystal structures of SaEctC were prepared using the PyMol software suite (www.pymol.org). + + + RESULTS + title_1 + 25217 + Results + + + RESULTS + title_2 + 25225 + Overproduction, purification and oligomeric state of the ectoine synthase in solution + + 0.9935509 + experimental_method + cleaner0 + 2023-07-20T14:28:59Z + MESH: + + Overproduction + + + 0.8892884 + experimental_method + cleaner0 + 2023-07-20T14:29:04Z + MESH: + + purification + + + 0.99961185 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + + RESULTS + paragraph + 25311 + We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product. We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography. The (Sa)EctC protein was overproduced and isolated with good yields (30–40 mg L-1 of culture) and purity (S2a Fig). Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution. High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig). This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa). Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus. + + 0.9994874 + experimental_method + cleaner0 + 2023-07-20T14:29:08Z + MESH: + + biochemical and structural studies + + + 0.9996302 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.99917936 + species + cleaner0 + 2023-07-20T10:21:23Z + MESH: + + S. alaskensis + + + species + MESH: + cleaner0 + 2023-07-20T10:22:10Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:40Z + + EctC + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-20T14:21:30Z + + marine ultra-microbacterium + + + 0.99960726 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + 0.99947584 + protein_type + cleaner0 + 2023-07-20T10:22:20Z + MESH: + + ectoine hydroxylase + + + 0.99979144 + protein_type + cleaner0 + 2023-07-20T10:22:39Z + MESH: + + EctD + + + 0.9992048 + protein_state + cleaner0 + 2023-07-20T13:57:28Z + DUMMY: + + in complex with + + + 0.99922276 + species + cleaner0 + 2023-07-20T10:21:24Z + MESH: + + S. alaskensis + + + 0.99774545 + gene + cleaner0 + 2023-07-20T10:22:58Z + GENE: + + ectC + + + 0.99888724 + species + cleaner0 + 2023-07-20T10:23:06Z + MESH: + + E. coli + + + 0.90680575 + experimental_method + cleaner0 + 2023-07-20T14:19:30Z + MESH: + + Strep-tag II affinity peptide + + + species + MESH: + cleaner0 + 2023-07-20T10:06:57Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:40Z + + EctC + + + experimental_method + MESH: + cleaner0 + 2023-07-20T10:23:30Z + + Strep-Tag-II + + + 0.9994743 + experimental_method + cleaner0 + 2023-07-20T14:29:28Z + MESH: + + affinity chromatography + + + 0.3394467 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:40Z + + EctC + + + 0.9861797 + experimental_method + cleaner0 + 2023-07-20T14:29:37Z + MESH: + + size-exclusion chromatography + + + 0.99960774 + experimental_method + cleaner0 + 2023-07-20T13:22:12Z + MESH: + + SEC + + + 0.4025306 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.99941814 + oligomeric_state + cleaner0 + 2023-07-20T14:22:55Z + DUMMY: + + dimers + + + 0.9995423 + experimental_method + cleaner0 + 2023-07-20T14:29:41Z + MESH: + + High performance liquid chromatography + + + 0.99823344 + experimental_method + cleaner0 + 2023-07-20T14:29:45Z + MESH: + + multi-angle light-scattering detection + + + 0.99953747 + experimental_method + cleaner0 + 2023-07-20T13:22:18Z + MESH: + + HPLC-MALS + + + 0.44877085 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.34720233 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.9993968 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + 0.99939466 + oligomeric_state + cleaner0 + 2023-07-20T10:12:09Z + DUMMY: + + monomer + + + 0.85244745 + experimental_method + cleaner0 + 2023-07-20T14:19:43Z + MESH: + + Strep-tag II affinity peptide + + + 0.9994011 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + 0.9995308 + protein_type + cleaner0 + 2023-07-20T10:24:20Z + MESH: + + EctC proteins + + + 0.9994009 + species + cleaner0 + 2023-07-20T10:21:29Z + MESH: + + H. elongata + + + 0.99942994 + species + cleaner0 + 2023-07-20T10:21:34Z + MESH: + + N. maritimus + + + + RESULTS + title_2 + 26746 + Biochemical properties of the ectoine synthase + + 0.9996504 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + + RESULTS + paragraph + 26793 + The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available. We used alkaline hydrolysis of ectoine and subsequent chromatography on silica gel columns to obtain N-γ-ADABA in chemically highly purified form (S1a Fig). This procedure also yielded the isomer of N-γ-ADABA, N-α-acetyl-L-2,4-diaminobutyric acid (N-α-ADABA) (S1b Fig). N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism. + + 0.5488887 + protein + cleaner0 + 2023-07-20T10:13:49Z + PR: + + EctA + + + 0.999634 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.9997744 + chemical + cleaner0 + 2023-07-20T10:24:44Z + CHEBI: + + N-γ-acetyl-L-2,4-diaminobutyric acid + + + 0.99975413 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.99936503 + chemical + cleaner0 + 2023-07-20T10:07:53Z + CHEBI: + + ectoine + + + 0.9997587 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.99974823 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.99976957 + chemical + cleaner0 + 2023-07-20T10:24:47Z + CHEBI: + + N-α-acetyl-L-2,4-diaminobutyric acid + + + 0.9997544 + chemical + cleaner0 + 2023-07-20T10:24:51Z + CHEBI: + + N-α-ADABA + + + 0.9997612 + chemical + cleaner0 + 2023-07-20T10:24:52Z + CHEBI: + + N-α-ADABA + + + 0.9994758 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.9994418 + taxonomy_domain + cleaner0 + 2023-07-20T11:32:08Z + DUMMY: + + microorganisms + + + 0.9997521 + chemical + cleaner0 + 2023-07-20T10:07:53Z + CHEBI: + + ectoine + + + + RESULTS + paragraph + 27398 + Using N-γ-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein. S. alaskensis, from which the studied ectoine synthase was originally derived, is a microorganism that is well-adapted to a life in permanently cold ocean waters. Consistent with the physicochemical attributes of this habitat, the (Sa)EctC protein was already enzymatically active at 5°C, had a temperature optimum of 15°C and was able to function over a broad range of temperatures (S3a Fig). It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0). + + 0.9997508 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.5292546 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + 0.6270969 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.99938875 + species + cleaner0 + 2023-07-20T10:21:24Z + MESH: + + S. alaskensis + + + 0.9996713 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.9974341 + taxonomy_domain + cleaner0 + 2023-07-20T14:21:36Z + DUMMY: + + microorganism + + + species + MESH: + cleaner0 + 2023-07-20T10:06:57Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.9985224 + protein_state + cleaner0 + 2023-07-20T14:46:40Z + DUMMY: + + enzymatically active + + + 0.6991387 + protein_state + cleaner0 + 2023-07-20T14:47:02Z + DUMMY: + + alkaline + + + 0.99969196 + protein_type + cleaner0 + 2023-07-20T14:17:02Z + MESH: + + ectoine synthases + + + 0.9490307 + protein_state + cleaner0 + 2023-07-20T14:47:07Z + DUMMY: + + halo-tolerant + + + 0.999429 + species + cleaner0 + 2023-07-20T10:21:30Z + MESH: + + H. elongata + + + 0.98819995 + taxonomy_domain + cleaner0 + 2023-07-20T14:21:42Z + DUMMY: + + alkaliphile + + + 0.99946517 + species + cleaner0 + 2023-07-20T10:25:07Z + MESH: + + M. alcaliphilum + + + 0.9690805 + taxonomy_domain + cleaner0 + 2023-07-20T14:21:45Z + DUMMY: + + acidophile + + + 0.99937564 + species + cleaner0 + 2023-07-20T10:17:43Z + MESH: + + Acidiphilium cryptum + + + 0.9995845 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.9994826 + species + cleaner0 + 2023-07-20T10:21:35Z + MESH: + + N. maritimus + + + 0.9332136 + protein_state + cleaner0 + 2023-07-20T14:47:11Z + DUMMY: + + neutral pH + + + + RESULTS + paragraph + 28279 + The salinity of the assay buffer had a significant influence on the maximal enzyme activity of the (Sa)EctC protein. An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity. The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively. (Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig). The stimulation of EctC enzyme activity by salts has previously also been observed for other ectoine synthases. + + species + MESH: + cleaner0 + 2023-07-20T10:06:57Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.99961096 + chemical + cleaner0 + 2023-07-20T10:26:08Z + CHEBI: + + NaCl + + + 0.9996014 + chemical + cleaner0 + 2023-07-20T10:26:14Z + CHEBI: + + KCl + + + 0.9996583 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + species + MESH: + cleaner0 + 2023-07-20T10:06:57Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.9995086 + chemical + cleaner0 + 2023-07-20T10:26:10Z + CHEBI: + + NaCl + + + 0.99956685 + chemical + cleaner0 + 2023-07-20T10:26:15Z + CHEBI: + + KCl + + + species + MESH: + cleaner0 + 2023-07-20T10:06:57Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.9996302 + chemical + cleaner0 + 2023-07-20T10:26:10Z + CHEBI: + + NaCl + + + 0.9996433 + chemical + cleaner0 + 2023-07-20T10:26:15Z + CHEBI: + + KCl + + + 0.9996387 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.99969184 + protein_type + cleaner0 + 2023-07-20T10:26:36Z + MESH: + + ectoine synthases + + + + RESULTS + title_2 + 28902 + The ectoine synthase is a metal-containing protein + + 0.99967754 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.985259 + protein_type + cleaner0 + 2023-07-20T14:17:21Z + MESH: + + metal-containing protein + + + + RESULTS + paragraph + 28953 + Considerations based on bioinformatics suggests that EctC belongs to the cupin superfamily. Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel. Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal). Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family. An abbreviated alignment of the amino acid sequence of EctC-type proteins is shown in Fig 2. + + 0.99895215 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.9994916 + protein_type + cleaner0 + 2023-07-20T10:07:22Z + MESH: + + cupin superfamily + + + 0.99914396 + chemical + cleaner0 + 2023-07-20T11:21:59Z + CHEBI: + + iron + + + 0.9990225 + chemical + cleaner0 + 2023-07-20T11:22:06Z + CHEBI: + + copper + + + 0.9989623 + chemical + cleaner0 + 2023-07-20T11:22:12Z + CHEBI: + + zinc + + + 0.9989072 + chemical + cleaner0 + 2023-07-20T11:22:20Z + CHEBI: + + manganese + + + 0.9986871 + chemical + cleaner0 + 2023-07-20T11:22:25Z + CHEBI: + + cobalt + + + 0.99882394 + chemical + cleaner0 + 2023-07-20T11:22:32Z + CHEBI: + + nickel + + + 0.99960715 + protein_type + cleaner0 + 2023-07-20T11:22:38Z + MESH: + + Cupins + + + protein_state + DUMMY: + cleaner0 + 2023-07-20T11:24:19Z + + conserved + + + structure_element + SO: + cleaner0 + 2023-07-20T11:23:06Z + + G(X)5HXH(X)3,4E(X)6G + + + structure_element + SO: + cleaner0 + 2023-07-20T11:23:28Z + + G(X)5PXG(X)2H(X)3N + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.9817178 + experimental_method + cleaner0 + 2023-07-20T14:30:15Z + MESH: + + alignment of the amino acid sequences + + + 0.9996746 + protein_type + cleaner0 + 2023-07-20T11:23:47Z + MESH: + + EctC-type proteins + + + 0.70793587 + structure_element + cleaner0 + 2023-07-20T11:23:52Z + SO: + + metal-binding motif + + + 0.9996796 + protein_type + cleaner0 + 2023-07-20T11:23:56Z + MESH: + + cupin-type proteins + + + 0.9992851 + protein_state + cleaner0 + 2023-07-20T11:24:00Z + DUMMY: + + not conserved + + + protein_type + MESH: + cleaner0 + 2023-07-20T11:24:39Z + + ectoine synthase protein family + + + experimental_method + MESH: + cleaner0 + 2023-07-20T14:30:36Z + + alignment of the amino acid sequence + + + 0.9996861 + protein_type + cleaner0 + 2023-07-20T11:24:52Z + MESH: + + EctC-type proteins + + + + pone.0151285.g002.jpg + pone.0151285.g002 + FIG + fig_title_caption + 29679 + Abbreviated alignment of EctC-type proteins. + + 0.97866905 + experimental_method + cleaner0 + 2023-07-20T14:30:40Z + MESH: + + alignment + + + 0.99968976 + protein_type + cleaner0 + 2023-07-20T11:24:53Z + MESH: + + EctC-type proteins + + + + pone.0151285.g002.jpg + pone.0151285.g002 + FIG + fig_caption + 29724 + The amino acid sequences of 20 selected EctC-type proteins are compared. Strictly conserved amino acid residues are shown in yellow. Dots shown above the (Sa)EctC protein sequence indicate residues likely to be involved in iron-binding (red), ligand-binding (green) and stabilization of the loop-architecture (blue). The conserved residue Tyr-52 with so-far undefined functions is indicated by a green dot circled in red. Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins. + + 0.99966794 + protein_type + cleaner0 + 2023-07-20T11:24:53Z + MESH: + + EctC-type proteins + + + 0.9993727 + protein_state + cleaner0 + 2023-07-20T11:25:19Z + DUMMY: + + Strictly conserved + + + 0.43636063 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + 0.32576406 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.99935764 + protein_state + cleaner0 + 2023-07-20T11:25:40Z + DUMMY: + + conserved + + + 0.99919754 + residue_name_number + cleaner0 + 2023-07-20T13:38:45Z + DUMMY: + + Tyr-52 + + + 0.99957323 + structure_element + cleaner0 + 2023-07-20T14:40:15Z + SO: + + α-helices + + + 0.999482 + structure_element + cleaner0 + 2023-07-20T14:40:19Z + SO: + + β-sheets + + + 0.6731305 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.9995988 + evidence + cleaner0 + 2023-07-20T11:25:35Z + DUMMY: + + crystal structure + + + 0.99965817 + protein_type + cleaner0 + 2023-07-20T11:24:53Z + MESH: + + EctC-type proteins + + + + RESULTS + paragraph + 30315 + Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS). For this analysis we used recombinant (Sa)EctC preparations from three independent protein overproduction and purification experiments. The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein). All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively). The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein. Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein. We note in this context, that the values obtained for the iron content of the (Sa)EctC proteins varied by approximately 10 to 20% between the two methods. The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree. + + 0.9835426 + structure_element + cleaner0 + 2023-07-20T11:25:52Z + SO: + + metal-binding motif + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.40864503 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.9995365 + experimental_method + cleaner0 + 2023-07-20T14:30:45Z + MESH: + + inductive-coupled plasma mass spectrometry + + + 0.9995499 + experimental_method + cleaner0 + 2023-07-20T13:55:27Z + MESH: + + ICP-MS + + + 0.2987374 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.99955463 + experimental_method + cleaner0 + 2023-07-20T13:55:27Z + MESH: + + ICP-MS + + + 0.9986418 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.99890316 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.41933072 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.99888176 + chemical + cleaner0 + 2023-07-20T11:22:14Z + CHEBI: + + zinc + + + 0.9990158 + chemical + cleaner0 + 2023-07-20T11:22:14Z + CHEBI: + + zinc + + + 0.99899393 + chemical + cleaner0 + 2023-07-20T11:22:07Z + CHEBI: + + copper + + + 0.9987733 + chemical + cleaner0 + 2023-07-20T11:22:33Z + CHEBI: + + nickel + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.9990601 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.37607437 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + experimental_method + MESH: + cleaner0 + 2023-07-20T11:27:02Z + + colorimetric method + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.9954098 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.40473375 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.99956316 + experimental_method + cleaner0 + 2023-07-20T13:55:27Z + MESH: + + ICP-MS + + + 0.8751861 + experimental_method + cleaner0 + 2023-07-20T11:27:02Z + MESH: + + colorimetric method + + + 0.9994973 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.9993145 + species + cleaner0 + 2023-07-20T10:21:24Z + MESH: + + S. alaskensis + + + 0.65454566 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.98509324 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.5177903 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.8369187 + experimental_method + cleaner0 + 2023-07-20T11:27:07Z + MESH: + + colorimetric assay + + + 0.9820753 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.45383352 + species + cleaner0 + 2023-07-20T10:06:57Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + + RESULTS + title_2 + 31796 + A metal cofactor is important for the catalytic activity of EctC + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.9996711 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + + RESULTS + paragraph + 31861 + The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily. To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity. The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a). + + 0.9994646 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.99937034 + protein_type + cleaner0 + 2023-07-20T10:07:22Z + MESH: + + cupin superfamily + + + 0.9995086 + experimental_method + cleaner0 + 2023-07-20T14:31:05Z + MESH: + + incubated + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + experimental_method + MESH: + cleaner0 + 2023-07-20T14:31:27Z + + with increasing concentrations + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.9996955 + chemical + cleaner0 + 2023-07-20T11:27:57Z + CHEBI: + + ethylene-diamine-tetraacetic-acid + + + 0.9996581 + chemical + cleaner0 + 2023-07-20T11:28:02Z + CHEBI: + + EDTA + + + 0.9993819 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.9996971 + chemical + cleaner0 + 2023-07-20T11:28:04Z + CHEBI: + + EDTA + + + 0.99951756 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.99938935 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.99970055 + chemical + cleaner0 + 2023-07-20T11:28:04Z + CHEBI: + + EDTA + + + + pone.0151285.g003.jpg + pone.0151285.g003 + FIG + fig_title_caption + 32488 + Dependency of the ectoine synthase activity on metals. + + 0.9996023 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + + pone.0151285.g003.jpg + pone.0151285.g003 + FIG + fig_caption + 32543 + (a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein. Metal depletion and reconstitution experiments with (b) stoichiometric and (c) excess amounts of metals. The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added. + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.99977547 + chemical + cleaner0 + 2023-07-20T11:28:04Z + CHEBI: + + EDTA + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.81504804 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.99951935 + experimental_method + cleaner0 + 2023-07-20T11:28:35Z + MESH: + + Metal depletion and reconstitution experiments + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.3710249 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.99964386 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.99955547 + experimental_method + cleaner0 + 2023-07-20T14:31:34Z + MESH: + + enzyme assays + + + 0.9997687 + chemical + cleaner0 + 2023-07-20T11:29:01Z + CHEBI: + + FeCl2 + + + + RESULTS + paragraph + 32944 + We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme. The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively. All other tested metals, including Fe3+, were unable to restore activity (Fig 3b). When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c). However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c). This is in line with literature data showing that cupin-type enzymes are often promiscuous with respect to the use of the catalytically important metal. + + 0.9988016 + protein_state + cleaner0 + 2023-07-20T14:47:17Z + DUMMY: + + inactivated + + + 0.99844754 + chemical + cleaner0 + 2023-07-20T11:28:04Z + CHEBI: + + EDTA + + + 0.9991221 + experimental_method + cleaner0 + 2023-07-20T14:31:38Z + MESH: + + dialysis + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.77772117 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.99972755 + chemical + cleaner0 + 2023-07-20T11:29:00Z + CHEBI: + + FeCl2 + + + 0.99939704 + experimental_method + cleaner0 + 2023-07-20T11:29:12Z + MESH: + + enzyme assay + + + 0.99973184 + chemical + cleaner0 + 2023-07-20T11:28:55Z + CHEBI: + + ZnCl2 + + + 0.99973637 + chemical + cleaner0 + 2023-07-20T11:29:05Z + CHEBI: + + CoCl2 + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.7600365 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.99975914 + chemical + cleaner0 + 2023-07-20T11:29:26Z + CHEBI: + + Fe3+ + + + 0.99914694 + experimental_method + cleaner0 + 2023-07-20T11:29:13Z + MESH: + + enzyme assay + + + 0.99975884 + chemical + cleaner0 + 2023-07-20T11:29:23Z + CHEBI: + + Fe2+ + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.8068256 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.99578047 + chemical + cleaner0 + 2023-07-20T11:28:04Z + CHEBI: + + EDTA + + + 0.9994541 + chemical + cleaner0 + 2023-07-20T11:22:14Z + CHEBI: + + zinc + + + 0.99934477 + chemical + cleaner0 + 2023-07-20T11:22:27Z + CHEBI: + + cobalt + + + 0.999358 + chemical + cleaner0 + 2023-07-20T11:22:33Z + CHEBI: + + nickel + + + 0.9994154 + chemical + cleaner0 + 2023-07-20T11:22:08Z + CHEBI: + + copper + + + 0.9994241 + chemical + cleaner0 + 2023-07-20T11:22:21Z + CHEBI: + + manganese + + + 0.99947345 + protein_type + cleaner0 + 2023-07-20T10:07:22Z + MESH: + + cupin superfamily + + + 0.99968255 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.999539 + protein_type + cleaner0 + 2023-07-20T14:17:27Z + MESH: + + cupin-type enzymes + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + + RESULTS + title_2 + 34093 + Kinetic parameters of EctC for N-γ-ADABA and N-α-ADABA + + 0.99944764 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.9997571 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.99975604 + chemical + cleaner0 + 2023-07-20T10:24:52Z + CHEBI: + + N-α-ADABA + + + + RESULTS + paragraph + 34156 + Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA. The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig). Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine. This was indeed the case. (Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig). Hence, N-α-ADABA is a newly recognized substrate for ectoine synthase. However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA. The Km dropped fife-fold from 4.9 ± 0.5 mM to 25.4 ± 2.9 mM, and the catalytic efficiency was reduced from 1.47 mM-1 s-1 to 0.02 mM-1 s-1, a 73-fold decrease. + + 0.9988838 + experimental_method + cleaner0 + 2023-07-20T11:30:39Z + MESH: + + activity assay + + + 0.99960184 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.9992654 + species + cleaner0 + 2023-07-20T10:21:24Z + MESH: + + S. alaskensis + + + 0.33515686 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + 0.6267819 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.9997531 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.9997481 + chemical + cleaner0 + 2023-07-20T10:24:53Z + CHEBI: + + N-α-ADABA + + + 0.9973105 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.9997524 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.9990388 + chemical + cleaner0 + 2023-07-20T10:07:53Z + CHEBI: + + ectoine + + + 0.9958072 + experimental_method + cleaner0 + 2023-07-20T11:30:32Z + MESH: + + Michaelis-Menten-kinetics + + + 0.99950933 + evidence + cleaner0 + 2023-07-20T11:30:04Z + DUMMY: + + Km + + + 0.99886847 + evidence + cleaner0 + 2023-07-20T11:31:42Z + DUMMY: + + vmax + + + 0.9994772 + evidence + cleaner0 + 2023-07-20T11:30:13Z + DUMMY: + + kcat + + + 0.9997486 + chemical + cleaner0 + 2023-07-20T10:24:53Z + CHEBI: + + N-α-ADABA + + + 0.9997503 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.99965304 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.77422124 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.9997444 + chemical + cleaner0 + 2023-07-20T10:24:53Z + CHEBI: + + N-α-ADABA + + + 0.9989826 + chemical + cleaner0 + 2023-07-20T10:07:53Z + CHEBI: + + ectoine + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.82548434 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + 0.9978008 + experimental_method + cleaner0 + 2023-07-20T11:30:34Z + MESH: + + Michaelis-Menten-kinetics + + + 0.9995671 + evidence + cleaner0 + 2023-07-20T11:30:04Z + DUMMY: + + Km + + + 0.99924266 + evidence + cleaner0 + 2023-07-20T11:31:43Z + DUMMY: + + vmax + + + 0.9994406 + evidence + cleaner0 + 2023-07-20T11:30:12Z + DUMMY: + + kcat + + + 0.99975175 + chemical + cleaner0 + 2023-07-20T10:24:53Z + CHEBI: + + N-α-ADABA + + + 0.9996559 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.99953246 + evidence + cleaner0 + 2023-07-20T14:10:42Z + DUMMY: + + affinity + + + 0.99958366 + evidence + cleaner0 + 2023-07-20T11:30:03Z + DUMMY: + + Km + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.736817 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + evidence + DUMMY: + cleaner0 + 2023-07-20T11:31:31Z + + catalytic efficiency + + + 0.99560183 + evidence + cleaner0 + 2023-07-20T11:31:19Z + DUMMY: + + kcat/Km + + + 0.9997519 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.99958986 + evidence + cleaner0 + 2023-07-20T11:30:04Z + DUMMY: + + Km + + + evidence + DUMMY: + cleaner0 + 2023-07-20T11:31:33Z + + catalytic efficiency + + + + RESULTS + paragraph + 35415 + Both N-γ-ADABA and N-α-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism. Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine. However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig). + + 0.99971217 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.9997239 + chemical + cleaner0 + 2023-07-20T10:24:53Z + CHEBI: + + N-α-ADABA + + + 0.99963677 + chemical + cleaner0 + 2023-07-20T10:07:53Z + CHEBI: + + ectoine + + + 0.99970275 + chemical + cleaner0 + 2023-07-20T10:24:53Z + CHEBI: + + N-α-ADABA + + + 0.99965507 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.99938774 + taxonomy_domain + cleaner0 + 2023-07-20T11:32:08Z + DUMMY: + + microorganisms + + + 0.999724 + chemical + cleaner0 + 2023-07-20T10:07:54Z + CHEBI: + + ectoine + + + 0.9992518 + taxonomy_domain + cleaner0 + 2023-07-20T11:32:07Z + DUMMY: + + microorganisms + + + 0.9993874 + evidence + cleaner0 + 2023-07-20T11:31:59Z + DUMMY: + + affinity + + + 0.9996618 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.99972785 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.99971116 + chemical + cleaner0 + 2023-07-20T10:24:53Z + CHEBI: + + N-α-ADABA + + + evidence + DUMMY: + cleaner0 + 2023-07-20T11:31:33Z + + catalytic efficiency + + + + RESULTS + title_2 + 36001 + Crystallization of the (Sa)EctC protein + + 0.9996445 + experimental_method + cleaner0 + 2023-07-20T14:31:43Z + MESH: + + Crystallization + + + 0.18065484 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + 0.49151045 + protein + cleaner0 + 2023-07-20T10:09:41Z + PR: + + EctC + + + + RESULTS + paragraph + 36041 + Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein. Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful. However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained. Crystal form A diffracted to 1.2 Å and had a unit cell of a = 72.71 b = 72.71 c = 52.33 Å and α = 90 β = 90 γ = 120° displaying a P3221 symmetry (S1 Table). Crystal form B diffracted to 2.0 Å and had a unit cell of a = 97.52 b = 43.96 c = 138.54 Å and α = 90 β = 101.5 γ = 120° and displayed a C2 symmetry (S1 Table). Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed. However, we were able to obtain crystals of form B that were derivatized with mercury and these diffracted up to 2.8 Å (S1 Table). This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table). Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table). + + 0.99962616 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + 0.99959636 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.99956125 + experimental_method + cleaner0 + 2023-07-20T14:31:46Z + MESH: + + crystallize + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:41Z + + EctC + + + 0.9958228 + evidence + cleaner0 + 2023-07-20T14:10:52Z + DUMMY: + + crystals + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.99944985 + protein_state + cleaner0 + 2023-07-20T14:47:22Z + DUMMY: + + in complex + + + 0.99971485 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.999548 + chemical + cleaner0 + 2023-07-20T10:07:54Z + CHEBI: + + ectoine + + + 0.9986279 + evidence + cleaner0 + 2023-07-20T14:11:01Z + DUMMY: + + crystal forms + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9994385 + protein_state + cleaner0 + 2023-07-20T13:34:29Z + DUMMY: + + absence of + + + 0.9996357 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.99954855 + experimental_method + cleaner0 + 2023-07-20T14:31:50Z + MESH: + + molecular replacement + + + 0.9473007 + evidence + cleaner0 + 2023-07-20T14:11:06Z + DUMMY: + + crystals + + + 0.9993457 + chemical + cleaner0 + 2023-07-20T11:33:32Z + CHEBI: + + mercury + + + 0.9893433 + evidence + cleaner0 + 2023-07-20T14:11:10Z + DUMMY: + + structural model + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9995866 + experimental_method + cleaner0 + 2023-07-20T14:31:53Z + MESH: + + molecular replacement + + + 0.9993136 + oligomeric_state + cleaner0 + 2023-07-20T13:33:48Z + DUMMY: + + monomers + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.99962366 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + 0.99959403 + evidence + cleaner0 + 2023-07-20T11:33:19Z + DUMMY: + + Rcryst + + + 0.99952066 + evidence + cleaner0 + 2023-07-20T11:33:24Z + DUMMY: + + Rfree + + + 0.9993229 + oligomeric_state + cleaner0 + 2023-07-20T10:12:09Z + DUMMY: + + monomer + + + 0.99949193 + evidence + cleaner0 + 2023-07-20T14:11:15Z + DUMMY: + + structure + + + 0.9995883 + experimental_method + cleaner0 + 2023-07-20T14:31:56Z + MESH: + + molecular replacement + + + 0.9995921 + evidence + cleaner0 + 2023-07-20T11:33:20Z + DUMMY: + + Rcryst + + + 0.99957305 + evidence + cleaner0 + 2023-07-20T11:33:24Z + DUMMY: + + Rfree + + + + RESULTS + title_2 + 37632 + Overall fold of the (Sa)EctC protein + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + + RESULTS + paragraph + 37669 + The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c). However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2). First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein. This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b). In this structure no metal co-factor was identified. The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation. Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2). We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure. Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure. + + 0.997881 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.9990163 + evidence + cleaner0 + 2023-07-20T14:11:19Z + DUMMY: + + structures + + + 0.99481285 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.99909353 + protein_type + cleaner0 + 2023-07-20T10:07:22Z + MESH: + + cupin superfamily + + + 0.9970457 + residue_range + cleaner0 + 2023-07-20T14:37:05Z + DUMMY: + + 137 amino acids + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.30935073 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.99948114 + evidence + cleaner0 + 2023-07-20T14:11:23Z + DUMMY: + + structure + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.421482 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.94030523 + residue_range + cleaner0 + 2023-07-20T14:11:35Z + DUMMY: + + Met-1 to Glu-115 + + + 0.99928635 + protein_state + cleaner0 + 2023-07-20T14:47:26Z + DUMMY: + + lacks + + + 0.96660024 + residue_range + cleaner0 + 2023-07-20T14:37:19Z + DUMMY: + + 22 amino acids + + + structure_element + SO: + cleaner0 + 2023-07-20T14:44:23Z + + carboxy-terminus + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.37216732 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.99869365 + evidence + cleaner0 + 2023-07-20T14:11:48Z + DUMMY: + + structure + + + 0.9996549 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.9987495 + structure_element + cleaner0 + 2023-07-20T14:40:25Z + SO: + + cupin barrels + + + 0.9996737 + protein_state + cleaner0 + 2023-07-20T11:35:51Z + DUMMY: + + open + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.45329663 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.99948573 + evidence + cleaner0 + 2023-07-20T14:11:53Z + DUMMY: + + structure + + + 0.9987312 + evidence + cleaner0 + 2023-07-20T14:11:57Z + DUMMY: + + structure + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.99953264 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.81937975 + experimental_method + cleaner0 + 2023-07-20T14:32:01Z + MESH: + + solved + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-20T14:24:15Z + + protomer + + + structure_element + SO: + cleaner0 + 2023-07-20T14:23:59Z + + A + + + 0.9478492 + residue_range + cleaner0 + 2023-07-20T14:11:44Z + DUMMY: + + Met-1 to Gly-121 + + + 0.99966216 + protein_state + cleaner0 + 2023-07-20T14:47:40Z + DUMMY: + + closed + + + 0.8067061 + protein_state + cleaner0 + 2023-07-20T14:47:43Z + DUMMY: + + lacks + + + 0.9836785 + residue_range + cleaner0 + 2023-07-20T14:37:24Z + DUMMY: + + 16 amino acid + + + structure_element + SO: + cleaner0 + 2023-07-20T14:44:23Z + + carboxy-terminus + + + 0.9969917 + residue_range + cleaner0 + 2023-07-20T14:37:29Z + DUMMY: + + 137 amino acids + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.99961877 + evidence + cleaner0 + 2023-07-20T11:35:41Z + DUMMY: + + crystal structure + + + 0.99947864 + protein_state + cleaner0 + 2023-07-20T11:35:58Z + DUMMY: + + fully closed + + + 0.99901295 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.9992604 + protein_state + cleaner0 + 2023-07-20T11:36:03Z + DUMMY: + + semi-closed + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.5132443 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.9994236 + evidence + cleaner0 + 2023-07-20T14:12:00Z + DUMMY: + + structure + + + 0.9989819 + oligomeric_state + cleaner0 + 2023-07-20T13:33:48Z + DUMMY: + + monomers + + + 0.8134865 + residue_range + cleaner0 + 2023-07-20T14:12:12Z + DUMMY: + + Met-1 to Glu-115 + + + 0.9996649 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.91006696 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.9993042 + evidence + cleaner0 + 2023-07-20T14:12:04Z + DUMMY: + + structure + + + + pone.0151285.g004.jpg + pone.0151285.g004 + FIG + fig_title_caption + 39144 + Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC. + + 0.99957496 + evidence + cleaner0 + 2023-07-20T14:12:18Z + DUMMY: + + structure + + + 0.999647 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.9995422 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.99960786 + evidence + cleaner0 + 2023-07-20T13:57:50Z + DUMMY: + + crystal structures + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + + pone.0151285.g004.jpg + pone.0151285.g004 + FIG + fig_caption + 39234 + (a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation. The β-strands are numbered β1-β11 and the helices α-I to α-II. (b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation. The entrance to the active site of the ectoine synthase is marked. (c) Overlay of the “semi-closed” and “open” (Sa)EctC structures. + + 0.9980724 + evidence + cleaner0 + 2023-07-20T14:12:21Z + DUMMY: + + structure + + + 0.99951744 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.54517436 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + 0.99927276 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.99968773 + structure_element + cleaner0 + 2023-07-20T14:40:31Z + SO: + + β-strands + + + 0.9995842 + structure_element + cleaner0 + 2023-07-20T13:54:20Z + SO: + + β1-β11 + + + 0.99951017 + structure_element + cleaner0 + 2023-07-20T14:40:34Z + SO: + + helices + + + 0.9993981 + structure_element + cleaner0 + 2023-07-20T14:40:37Z + SO: + + α-I to α-II + + + 0.99891806 + evidence + cleaner0 + 2023-07-20T14:12:24Z + DUMMY: + + structure + + + 0.99967027 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.5190078 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + 0.9992449 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.9996062 + site + cleaner0 + 2023-07-20T13:43:15Z + SO: + + active site + + + 0.9995431 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.9994759 + experimental_method + cleaner0 + 2023-07-20T14:32:08Z + MESH: + + Overlay + + + 0.9995149 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.9996666 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.5526954 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + 0.9992661 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.99936575 + evidence + cleaner0 + 2023-07-20T14:12:26Z + DUMMY: + + structures + + + + RESULTS + paragraph + 39782 + The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure. This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other. However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure. Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail. + + 0.9994179 + evidence + cleaner0 + 2023-07-20T14:12:41Z + DUMMY: + + structure + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9992894 + evidence + cleaner0 + 2023-07-20T14:12:44Z + DUMMY: + + crystals + + + structure_element + SO: + cleaner0 + 2023-07-20T14:44:23Z + + carboxy-terminus + + + 0.99950683 + structure_element + cleaner0 + 2023-07-20T13:33:43Z + SO: + + cupin barrel + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-20T13:23:23Z + + monomer + + + structure_element + SO: + cleaner0 + 2023-07-20T14:23:26Z + + A + + + 0.9995088 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.99846053 + evidence + cleaner0 + 2023-07-20T14:12:49Z + DUMMY: + + structure + + + 0.99302316 + evidence + cleaner0 + 2023-07-20T14:12:52Z + DUMMY: + + root mean square deviation + + + 0.9995968 + evidence + cleaner0 + 2023-07-20T13:17:42Z + DUMMY: + + RMSD + + + 0.9996617 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.9990269 + oligomeric_state + cleaner0 + 2023-07-20T13:33:48Z + DUMMY: + + monomers + + + 0.9995272 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.99902415 + oligomeric_state + cleaner0 + 2023-07-20T10:12:10Z + DUMMY: + + monomer + + + 0.99965227 + evidence + cleaner0 + 2023-07-20T13:17:42Z + DUMMY: + + RMSD + + + 0.9996704 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.9994696 + evidence + cleaner0 + 2023-07-20T14:12:57Z + DUMMY: + + structure + + + 0.9996313 + evidence + cleaner0 + 2023-07-20T14:13:00Z + DUMMY: + + structure + + + 0.9995301 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9996687 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.9995306 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + + RESULTS + paragraph + 40595 + The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a). The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. These two β-sheets pack against each other, forming a cup-shaped β-sandwich with a topology characteristic for the cupin-fold. Hence, (Sa)EctC adopts an overall bowl shape in which one side is opened towards the solvent (Fig 4a to 4c). In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a). The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). + + 0.9994899 + evidence + cleaner0 + 2023-07-20T14:13:04Z + DUMMY: + + structure + + + 0.99953014 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.32368964 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9989476 + structure_element + cleaner0 + 2023-07-20T14:40:40Z + SO: + + β-strands + + + 0.9996641 + structure_element + cleaner0 + 2023-07-20T13:54:20Z + SO: + + β1-β11 + + + 0.9994936 + structure_element + cleaner0 + 2023-07-20T14:40:43Z + SO: + + α-helices + + + 0.9996903 + structure_element + cleaner0 + 2023-07-20T14:40:47Z + SO: + + α-I + + + 0.9996945 + structure_element + cleaner0 + 2023-07-20T14:40:50Z + SO: + + α-II + + + 0.9995985 + structure_element + cleaner0 + 2023-07-20T14:40:53Z + SO: + + β-strands + + + 0.9996136 + structure_element + cleaner0 + 2023-07-20T14:40:57Z + SO: + + anti-parallel β-sheets + + + 0.9997389 + structure_element + cleaner0 + 2023-07-20T14:41:04Z + SO: + + β2 + + + 0.97667503 + structure_element + cleaner0 + 2023-07-20T14:41:07Z + SO: + + β3 + + + 0.99975175 + structure_element + cleaner0 + 2023-07-20T14:41:10Z + SO: + + β4 + + + 0.99974126 + structure_element + cleaner0 + 2023-07-20T13:26:56Z + SO: + + β11 + + + 0.9997428 + structure_element + cleaner0 + 2023-07-20T13:24:59Z + SO: + + β6 + + + 0.99973994 + structure_element + cleaner0 + 2023-07-20T14:41:14Z + SO: + + β9 + + + 0.9830336 + structure_element + cleaner0 + 2023-07-20T14:41:17Z + SO: + + three-stranded β-sheet + + + 0.99974567 + structure_element + cleaner0 + 2023-07-20T14:41:20Z + SO: + + β7 + + + 0.9997398 + structure_element + cleaner0 + 2023-07-20T14:41:23Z + SO: + + β8 + + + 0.99974865 + structure_element + cleaner0 + 2023-07-20T14:41:26Z + SO: + + β10 + + + 0.99933386 + structure_element + cleaner0 + 2023-07-20T14:41:29Z + SO: + + β-sheets + + + 0.9995074 + structure_element + cleaner0 + 2023-07-20T14:41:31Z + SO: + + cup-shaped β-sandwich + + + 0.9976778 + structure_element + cleaner0 + 2023-07-20T13:28:25Z + SO: + + cupin-fold + + + 0.4580153 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.999531 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.99948585 + evidence + cleaner0 + 2023-07-20T14:13:08Z + DUMMY: + + structure + + + 0.9988877 + structure_element + cleaner0 + 2023-07-20T14:41:37Z + SO: + + carboxy-terminal tail + + + 0.9995363 + evidence + cleaner0 + 2023-07-20T13:31:03Z + DUMMY: + + electron density + + + structure_element + SO: + cleaner0 + 2023-07-20T14:42:03Z + + small helix + + + 0.99971133 + structure_element + cleaner0 + 2023-07-20T14:42:06Z + SO: + + α-II + + + 0.9995864 + site + cleaner0 + 2023-07-20T13:43:15Z + SO: + + active site + + + 0.4139034 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9987648 + structure_element + cleaner0 + 2023-07-20T14:42:11Z + SO: + + α-II helix + + + 0.45860088 + protein_state + cleaner0 + 2023-07-20T14:42:26Z + DUMMY: + + unstructured + + + 0.99947053 + structure_element + cleaner0 + 2023-07-20T14:42:30Z + SO: + + loop + + + 0.99964976 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.99924123 + evidence + cleaner0 + 2023-07-20T14:13:29Z + DUMMY: + + structure + + + 0.9997776 + structure_element + cleaner0 + 2023-07-20T14:42:33Z + SO: + + β4 + + + 0.9997366 + structure_element + cleaner0 + 2023-07-20T13:24:59Z + SO: + + β6 + + + 0.99904567 + protein_state + cleaner0 + 2023-07-20T14:48:01Z + DUMMY: + + stable + + + 0.9996109 + structure_element + cleaner0 + 2023-07-20T13:27:53Z + SO: + + β-strand + + + 0.99972516 + structure_element + cleaner0 + 2023-07-20T13:27:47Z + SO: + + β5 + + + 0.99952763 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.5325583 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + + RESULTS + paragraph + 41663 + Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily. The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction. Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily. + + 0.9995205 + experimental_method + cleaner0 + 2023-07-20T14:32:14Z + MESH: + + Structural comparison analyses + + + 0.99941206 + experimental_method + cleaner0 + 2023-07-20T14:32:17Z + MESH: + + DALI server + + + 0.41508162 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + 0.8436447 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.99930793 + protein_type + cleaner0 + 2023-07-20T10:07:22Z + MESH: + + cupin superfamily + + + protein + PR: + cleaner0 + 2023-07-20T13:19:00Z + + Cupin 2 conserved barrel domain protein + + + protein + PR: + cleaner0 + 2023-07-20T13:18:40Z + + YP_751781.1 + + + 0.999388 + species + cleaner0 + 2023-07-20T13:19:06Z + MESH: + + Shewanella frigidimarina + + + 0.96734786 + evidence + cleaner0 + 2023-07-20T13:17:34Z + DUMMY: + + Z-score + + + 0.9995598 + evidence + cleaner0 + 2023-07-20T13:17:41Z + DUMMY: + + RMSD + + + 0.968627 + evidence + cleaner0 + 2023-07-20T14:13:35Z + DUMMY: + + structure + + + 0.99936956 + protein + cleaner0 + 2023-07-20T13:19:27Z + PR: + + manganese-containing cupin + + + 0.9408579 + protein + cleaner0 + 2023-07-20T13:19:41Z + PR: + + TM1459 + + + 0.99944925 + species + cleaner0 + 2023-07-20T13:19:59Z + MESH: + + Thermotoga maritima + + + 0.8786765 + evidence + cleaner0 + 2023-07-20T13:17:35Z + DUMMY: + + Z-score + + + 0.99958724 + evidence + cleaner0 + 2023-07-20T13:17:43Z + DUMMY: + + RMSD + + + 0.99950814 + protein_type + cleaner0 + 2023-07-20T13:20:07Z + MESH: + + cyclase + + + 0.9994784 + protein + cleaner0 + 2023-07-20T13:20:12Z + PR: + + RemF + + + 0.99944437 + species + cleaner0 + 2023-07-20T13:20:17Z + MESH: + + Streptomyces resistomycificus + + + evidence + DUMMY: + cleaner0 + 2023-07-20T13:17:35Z + + Z-score + + + 0.99956745 + evidence + cleaner0 + 2023-07-20T13:17:43Z + DUMMY: + + RMSD + + + protein + PR: + cleaner0 + 2023-07-20T13:20:48Z + + auxin-binding protein 1 + + + 0.99945617 + species + cleaner0 + 2023-07-20T13:20:53Z + MESH: + + Zea mays + + + evidence + DUMMY: + cleaner0 + 2023-07-20T13:17:35Z + + Z-score + + + 0.9995503 + evidence + cleaner0 + 2023-07-20T13:17:43Z + DUMMY: + + RMSD + + + 0.9280039 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.9996645 + protein_type + cleaner0 + 2023-07-20T13:21:02Z + MESH: + + polyketide cyclase + + + 0.9996055 + protein + cleaner0 + 2023-07-20T13:20:13Z + PR: + + RemF + + + 0.99893826 + protein_type + cleaner0 + 2023-07-20T14:17:35Z + MESH: + + cupin-related + + + 0.999348 + protein + cleaner0 + 2023-07-20T13:20:13Z + PR: + + RemF + + + 0.9995912 + protein_type + cleaner0 + 2023-07-20T13:21:25Z + MESH: + + aldos-2-ulose dehydratase + + + 0.906959 + protein_type + cleaner0 + 2023-07-20T13:21:27Z + MESH: + + isomerase + + + 0.9988444 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.9995653 + protein_type + cleaner0 + 2023-07-20T13:21:31Z + MESH: + + dehydratase + + + 0.999416 + protein_type + cleaner0 + 2023-07-20T10:07:22Z + MESH: + + cupin superfamily + + + + RESULTS + title_2 + 42925 + Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure + + 0.9020333 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.99927044 + site + cleaner0 + 2023-07-20T13:21:55Z + SO: + + dimer interface + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9996129 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + + RESULTS + paragraph + 43008 + Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution. The crystal structure of this protein reflects this quaternary arrangement. In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit. This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). The strong interactions between these β-strands rely primarily on backbone contacts. In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands. As calculated with PDBePISA, the surface area buried upon dimer formation is 1462 Å2, which is 20.5% of the total accessible surface of a monomer of this protein. Both values fall within the range for known functional dimers. + + 0.9996774 + experimental_method + cleaner0 + 2023-07-20T13:22:11Z + MESH: + + SEC + + + 0.99950904 + experimental_method + cleaner0 + 2023-07-20T13:22:16Z + MESH: + + HPLC-MALS + + + 0.99953663 + protein_type + cleaner0 + 2023-07-20T10:07:29Z + MESH: + + ectoine synthase + + + 0.9993809 + species + cleaner0 + 2023-07-20T10:21:24Z + MESH: + + S. alaskensis + + + 0.9993979 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + 0.99958277 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + 0.99948746 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.99957633 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + 0.29558715 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.999613 + experimental_method + cleaner0 + 2023-07-20T14:32:22Z + MESH: + + crystallized + + + 0.99940705 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + 0.99937505 + oligomeric_state + cleaner0 + 2023-07-20T14:23:00Z + DUMMY: + + dimers + + + 0.99941826 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + 0.999331 + oligomeric_state + cleaner0 + 2023-07-20T13:33:48Z + DUMMY: + + monomers + + + 0.99941456 + protein_state + cleaner0 + 2023-07-20T10:12:24Z + DUMMY: + + head-to-tail + + + 0.9994602 + structure_element + cleaner0 + 2023-07-20T14:42:40Z + SO: + + antiparallel β-strands + + + 0.9996492 + structure_element + cleaner0 + 2023-07-20T13:27:53Z + SO: + + β-strand + + + 0.9997371 + structure_element + cleaner0 + 2023-07-20T13:24:53Z + SO: + + β1 + + + 0.9195826 + structure_element + cleaner0 + 2023-07-20T13:23:50Z + SO: + + 1MIVRN5 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-20T13:23:23Z + + monomer + + + structure_element + SO: + cleaner0 + 2023-07-20T13:23:41Z + + A + + + 0.9996632 + structure_element + cleaner0 + 2023-07-20T13:27:53Z + SO: + + β-strand + + + 0.9997193 + structure_element + cleaner0 + 2023-07-20T14:42:44Z + SO: + + β8 + + + 0.99917704 + oligomeric_state + cleaner0 + 2023-07-20T10:12:10Z + DUMMY: + + monomer + + + 0.99017453 + structure_element + cleaner0 + 2023-07-20T13:23:46Z + SO: + + B + + + 0.9666013 + structure_element + cleaner0 + 2023-07-20T13:23:53Z + SO: + + 82GVMYAL87 + + + 0.99965686 + structure_element + cleaner0 + 2023-07-20T14:42:51Z + SO: + + β-strands + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:19Z + + hydrophobic interactions + + + 0.9992143 + oligomeric_state + cleaner0 + 2023-07-20T13:33:48Z + DUMMY: + + monomers + + + 0.99970716 + structure_element + cleaner0 + 2023-07-20T14:42:54Z + SO: + + loops + + + 0.99967855 + structure_element + cleaner0 + 2023-07-20T14:42:49Z + SO: + + β-strands + + + 0.56406224 + experimental_method + cleaner0 + 2023-07-20T14:32:26Z + MESH: + + PDBePISA + + + 0.99939084 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + 0.9993728 + oligomeric_state + cleaner0 + 2023-07-20T10:12:10Z + DUMMY: + + monomer + + + 0.9993892 + oligomeric_state + cleaner0 + 2023-07-20T14:23:04Z + DUMMY: + + dimers + + + + pone.0151285.g005.jpg + pone.0151285.g005 + FIG + fig_title_caption + 44113 + Crystal structure of (Sa)EctC. + + 0.99959344 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + Crystal structure + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + + pone.0151285.g005.jpg + pone.0151285.g005 + FIG + fig_caption + 44144 + (a) Top-view of the dimer of the (Sa)EctC protein. The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer. (c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6. + + 0.9994184 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9997701 + chemical + cleaner0 + 2023-07-20T14:19:48Z + CHEBI: + + water + + + 0.9994006 + oligomeric_state + cleaner0 + 2023-07-20T13:33:48Z + DUMMY: + + monomers + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9993993 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + 0.9994203 + site + cleaner0 + 2023-07-20T13:21:56Z + SO: + + dimer interface + + + 0.99967796 + structure_element + cleaner0 + 2023-07-20T14:42:58Z + SO: + + β-strands + + + 0.9993344 + oligomeric_state + cleaner0 + 2023-07-20T10:12:10Z + DUMMY: + + monomer + + + 0.9994918 + site + cleaner0 + 2023-07-20T13:21:56Z + SO: + + dimer interface + + + 0.99967194 + structure_element + cleaner0 + 2023-07-20T14:43:01Z + SO: + + beta-strand + + + 0.9997563 + structure_element + cleaner0 + 2023-07-20T13:24:52Z + SO: + + β1 + + + 0.9997464 + structure_element + cleaner0 + 2023-07-20T13:24:58Z + SO: + + β6 + + + + RESULTS + paragraph + 44533 + In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit. We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well. Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets. The crystallographic two-fold axis present within the crystal symmetry is located exactly in between the two monomers, resulting in a monomer within the asymmetric unit. Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure. Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5). + + 0.99965227 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.8590964 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.99935895 + evidence + cleaner0 + 2023-07-20T14:13:42Z + DUMMY: + + structure + + + 0.9992888 + oligomeric_state + cleaner0 + 2023-07-20T10:12:10Z + DUMMY: + + monomer + + + 0.54075 + experimental_method + cleaner0 + 2023-07-20T14:32:31Z + MESH: + + packing + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-20T10:12:10Z + + monomer + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-20T10:12:10Z + + monomer + + + 0.9993844 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + 0.7603926 + evidence + cleaner0 + 2023-07-20T14:13:46Z + DUMMY: + + crystal form + + + 0.99921775 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + 0.99952525 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.8735654 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9993697 + evidence + cleaner0 + 2023-07-20T14:13:49Z + DUMMY: + + structure + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-20T10:12:10Z + + monomer + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-20T10:12:10Z + + monomer + + + 0.9995718 + structure_element + cleaner0 + 2023-07-20T14:43:07Z + SO: + + β-sheets + + + 0.9992028 + oligomeric_state + cleaner0 + 2023-07-20T13:33:48Z + DUMMY: + + monomers + + + 0.9992398 + oligomeric_state + cleaner0 + 2023-07-20T10:12:10Z + DUMMY: + + monomer + + + 0.9993611 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + 0.9995227 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.99945027 + evidence + cleaner0 + 2023-07-20T14:13:52Z + DUMMY: + + structure + + + 0.6170136 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9996438 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.99921894 + evidence + cleaner0 + 2023-07-20T14:13:57Z + DUMMY: + + structure + + + 0.9993315 + experimental_method + cleaner0 + 2023-07-20T14:32:34Z + MESH: + + DALI search + + + 0.9978916 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.9994137 + oligomeric_state + cleaner0 + 2023-07-20T14:23:09Z + DUMMY: + + dimers + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-20T10:12:10Z + + monomer + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-20T10:12:10Z + + monomer + + + 0.9993254 + oligomeric_state + cleaner0 + 2023-07-20T10:12:03Z + DUMMY: + + dimer + + + + RESULTS + title_2 + 45634 + Structural rearrangements of the flexible (Sa)EctC carboxy-terminus + + 0.99960583 + protein_state + cleaner0 + 2023-07-20T14:48:07Z + DUMMY: + + flexible + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + 0.9870107 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + structure_element + SO: + cleaner0 + 2023-07-20T14:44:23Z + + carboxy-terminus + + + + RESULTS + paragraph + 45702 + The cupin core represents the structural framework of ectoine synthase (Figs 4 and 5). The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus. Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family. At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein. In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121. These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a). Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement. This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure. As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c). It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below). The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5. Therefore the sealing of the cupin fold, as described above, seem to have an indirect influence on the architecture of the postulated iron-binding site. + + 0.99961126 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.9996259 + evidence + cleaner0 + 2023-07-20T13:57:50Z + DUMMY: + + crystal structures + + + 0.61970377 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + structure_element + SO: + cleaner0 + 2023-07-20T14:44:23Z + + carboxy-terminus + + + structure_element + SO: + cleaner0 + 2023-07-20T13:50:49Z + + carboxy-terminal region + + + 0.99818975 + residue_range + cleaner0 + 2023-07-20T14:37:36Z + DUMMY: + + 137 amino acids + + + 0.5200333 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9994724 + protein_state + cleaner0 + 2023-07-20T14:48:11Z + DUMMY: + + highly conserved + + + 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2023-07-20T10:09:42Z + + EctC + + + 0.90157557 + structure_element + cleaner0 + 2023-07-20T14:43:25Z + SO: + + helix + + + 0.99969316 + structure_element + cleaner0 + 2023-07-20T14:43:28Z + SO: + + loop region + + + 0.9996677 + structure_element + cleaner0 + 2023-07-20T14:43:32Z + SO: + + β-strands + + + 0.99966717 + structure_element + cleaner0 + 2023-07-20T14:43:35Z + SO: + + β4 + + + 0.99880993 + structure_element + cleaner0 + 2023-07-20T13:24:59Z + SO: + + β6 + + + 0.99965495 + structure_element + cleaner0 + 2023-07-20T13:27:51Z + SO: + + β-strand + + + 0.9994319 + structure_element + cleaner0 + 2023-07-20T13:27:45Z + SO: + + β5 + + + 0.9923357 + structure_element + cleaner0 + 2023-07-20T14:43:38Z + SO: + + small C-terminal helix + + + 0.9948067 + protein_state + cleaner0 + 2023-07-20T14:48:22Z + DUMMY: + + absent + + + 0.99961144 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.42274258 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.99960667 + evidence + cleaner0 + 2023-07-20T14:14:06Z + DUMMY: + + structure + + + 0.9996436 + structure_element + cleaner0 + 2023-07-20T13:27:53Z + SO: + + β-strand + + + 0.99935025 + structure_element + cleaner0 + 2023-07-20T13:27:47Z + SO: + + β5 + + + 0.9994707 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.80144036 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9996332 + evidence + cleaner0 + 2023-07-20T14:14:09Z + DUMMY: + + structure + + + 0.9996664 + structure_element + cleaner0 + 2023-07-20T13:27:53Z + SO: + + β-strand + + + 0.99895096 + structure_element + cleaner0 + 2023-07-20T13:27:47Z + SO: + + β5 + + + 0.99931455 + residue_name_number + cleaner0 + 2023-07-20T13:31:18Z + DUMMY: + + His-93 + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.9986792 + residue_name + cleaner0 + 2023-07-20T13:31:09Z + SO: + + His + + + species + MESH: + cleaner0 + 2023-07-20T10:06:58Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.99929583 + evidence + cleaner0 + 2023-07-20T14:14:13Z + DUMMY: + + structures + + + 0.999655 + structure_element + cleaner0 + 2023-07-20T13:27:53Z + SO: + + β-strand + + + 0.9993672 + structure_element + cleaner0 + 2023-07-20T13:27:47Z + SO: + + β5 + + + 0.99949586 + structure_element + cleaner0 + 2023-07-20T14:43:43Z + SO: + + cupin fold + + + 0.9996346 + site + cleaner0 + 2023-07-20T13:28:15Z + SO: + + iron-binding site + + + + RESULTS + paragraph + 47466 + The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2). They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold. In the “semi-closed” structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c). In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus. A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures. The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11. Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein. + + 0.99903864 + residue_name_number + cleaner0 + 2023-07-20T13:28:44Z + DUMMY: + + Pro-109 + + + 0.99906427 + residue_name_number + cleaner0 + 2023-07-20T13:28:50Z + DUMMY: + + Pro-110 + + + 0.9996564 + structure_element + cleaner0 + 2023-07-20T13:27:53Z + SO: + + β-strand + + + structure_element + SO: + cleaner0 + 2023-07-20T13:29:38Z + + β11 + + + 0.9995168 + protein_state + cleaner0 + 2023-07-20T14:48:26Z + DUMMY: + + highly conserved + + + 0.999655 + protein_type + cleaner0 + 2023-07-20T11:24:54Z + MESH: + + EctC-type proteins + + + structure_element + SO: + cleaner0 + 2023-07-20T14:44:23Z + + carboxy-terminus + + + 0.99866015 + residue_range + cleaner0 + 2023-07-20T14:38:07Z + DUMMY: + + 27 amino acid residues + + + 0.7135056 + species + cleaner0 + 2023-07-20T10:06:58Z + MESH: + + Sa + + + 0.41343936 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.9989593 + structure_element + cleaner0 + 2023-07-20T14:43:47Z + SO: + + cupin fold + + + 0.999533 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.9993531 + evidence + cleaner0 + 2023-07-20T14:14:17Z + DUMMY: + + structure + + + 0.9986342 + structure_element + cleaner0 + 2023-07-20T13:33:43Z + SO: + + cupin barrel + + + 0.9996474 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.45168442 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.99943846 + evidence + cleaner0 + 2023-07-20T14:14:20Z + DUMMY: + + structure + + + 0.9989772 + residue_name + cleaner0 + 2023-07-20T13:29:05Z + SO: + + proline + + + 0.99952924 + evidence + cleaner0 + 2023-07-20T13:31:02Z + DUMMY: + + electron density + + + 0.9990189 + residue_name_number + cleaner0 + 2023-07-20T13:28:50Z + DUMMY: + + Pro-110 + + + 0.99954295 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + structure_element + SO: + cleaner0 + 2023-07-20T14:44:23Z + + carboxy-terminus + + + 0.9990298 + residue_name_number + cleaner0 + 2023-07-20T13:28:44Z + DUMMY: + + Pro-109 + + + 0.99891084 + residue_name_number + cleaner0 + 2023-07-20T13:30:39Z + DUMMY: + + His-55 + + + 0.9996402 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.99952775 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.4830879 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.99958783 + evidence + cleaner0 + 2023-07-20T14:14:24Z + DUMMY: + + structures + + + 0.9988208 + residue_name_number + cleaner0 + 2023-07-20T13:28:44Z + DUMMY: + + Pro-109 + + + 0.9977756 + residue_name_number + cleaner0 + 2023-07-20T13:30:41Z + DUMMY: + + His-55 + + + 0.997267 + protein_state + cleaner0 + 2023-07-20T14:48:31Z + DUMMY: + + stable + + + 0.99815995 + residue_name + cleaner0 + 2023-07-20T13:29:08Z + SO: + + proline + + + 0.9996521 + structure_element + cleaner0 + 2023-07-20T13:27:53Z + SO: + + β-strand + + + 0.9996872 + structure_element + cleaner0 + 2023-07-20T13:26:56Z + SO: + + β11 + + + 0.99806434 + residue_name + cleaner0 + 2023-07-20T13:29:10Z + SO: + + proline + + + 0.99933326 + structure_element + cleaner0 + 2023-07-20T13:50:49Z + SO: + + carboxy-terminal region + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.99878997 + residue_name_number + cleaner0 + 2023-07-20T13:30:41Z + DUMMY: + + His-55 + + + 0.99891657 + residue_name_number + cleaner0 + 2023-07-20T13:28:44Z + DUMMY: + + Pro-109 + + + + RESULTS + paragraph + 48657 + In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further. The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density. In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b). Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region. + + 0.9991196 + residue_name_number + cleaner0 + 2023-07-20T13:28:44Z + DUMMY: + + Pro-109 + + + 0.99898905 + residue_name_number + cleaner0 + 2023-07-20T13:30:41Z + DUMMY: + + His-55 + + + 0.9983686 + structure_element + cleaner0 + 2023-07-20T13:50:49Z + SO: + + carboxy-terminal region + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9990506 + residue_name_number + cleaner0 + 2023-07-20T13:31:31Z + DUMMY: + + Glu-115 + + + 0.998964 + residue_name_number + cleaner0 + 2023-07-20T13:30:41Z + DUMMY: + + His-55 + + + 0.9996947 + structure_element + cleaner0 + 2023-07-20T14:43:53Z + SO: + + small helix + + + structure_element + SO: + cleaner0 + 2023-07-20T14:44:22Z + + carboxy-terminus + + + 0.9988809 + residue_name_number + cleaner0 + 2023-07-20T13:31:32Z + DUMMY: + + Glu-115 + + + 0.9987626 + residue_name_number + cleaner0 + 2023-07-20T13:30:41Z + DUMMY: + + His-55 + + + 0.9995065 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.99943584 + evidence + cleaner0 + 2023-07-20T14:14:29Z + DUMMY: + + structure + + + 0.99632454 + protein_state + cleaner0 + 2023-07-20T14:48:36Z + DUMMY: + + partially extended + + + 0.9766502 + structure_element + cleaner0 + 2023-07-20T14:44:24Z + SO: + + carboxy-terminus + + + 0.9995966 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + 0.99962103 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.99952877 + evidence + cleaner0 + 2023-07-20T14:14:32Z + DUMMY: + + structure + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9989541 + residue_name_number + cleaner0 + 2023-07-20T13:31:32Z + DUMMY: + + Glu-115 + + + 0.9691086 + structure_element + cleaner0 + 2023-07-20T14:44:24Z + SO: + + carboxy-terminus + + + 0.99963653 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9996598 + evidence + cleaner0 + 2023-07-20T14:14:36Z + DUMMY: + + structure + + + 0.99958694 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + + pone.0151285.g006.jpg + pone.0151285.g006 + FIG + fig_title_caption + 49457 + Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus. + + 0.9996562 + site + cleaner0 + 2023-07-20T14:14:46Z + SO: + + metal-binding site + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9995962 + protein_state + cleaner0 + 2023-07-20T14:48:43Z + DUMMY: + + flexible + + + structure_element + SO: + cleaner0 + 2023-07-20T14:44:24Z + + carboxy-terminus + + + + pone.0151285.g006.jpg + pone.0151285.g006 + FIG + fig_caption + 49564 + (a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93. The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase. His-55 interacts with the double proline motif (Pro-109 and Pro-110). It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure. (b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein. + + 0.9997719 + chemical + cleaner0 + 2023-07-20T14:19:52Z + CHEBI: + + water + + + 0.99921626 + residue_name_number + cleaner0 + 2023-07-20T13:32:56Z + DUMMY: + + Glu-57 + + + 0.9992302 + residue_name_number + cleaner0 + 2023-07-20T13:33:02Z + DUMMY: + + Tyr-85 + + + 0.99922436 + residue_name_number + cleaner0 + 2023-07-20T13:31:19Z + DUMMY: + + His-93 + + + 0.9997919 + chemical + cleaner0 + 2023-07-20T14:19:55Z + CHEBI: + + water + + + 0.99941945 + chemical + cleaner0 + 2023-07-20T14:20:01Z + CHEBI: + + Fe2+ + + + 0.88510096 + site + cleaner0 + 2023-07-20T14:39:11Z + SO: + + active side + + + 0.99900013 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.9992514 + residue_name_number + cleaner0 + 2023-07-20T13:30:41Z + DUMMY: + + His-55 + + + 0.9992842 + structure_element + cleaner0 + 2023-07-20T13:32:18Z + SO: + + double proline motif + + + 0.99929523 + residue_name_number + cleaner0 + 2023-07-20T13:28:44Z + DUMMY: + + Pro-109 + + + 0.9992961 + residue_name_number + cleaner0 + 2023-07-20T13:28:50Z + DUMMY: + + Pro-110 + + + 0.99925417 + residue_name_number + cleaner0 + 2023-07-20T13:31:32Z + DUMMY: + + Glu-115 + + + 0.99933594 + protein_state + cleaner0 + 2023-07-20T14:48:45Z + DUMMY: + + flexible + + + structure_element + SO: + cleaner0 + 2023-07-20T14:44:24Z + + carboxy-terminus + + + 0.4762111 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + 0.5403825 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.9995139 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.46799707 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9995493 + evidence + cleaner0 + 2023-07-20T14:14:51Z + DUMMY: + + structure + + + 0.999553 + experimental_method + cleaner0 + 2023-07-20T14:32:39Z + MESH: + + overlay + + + 0.99967325 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.9995157 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.9994874 + evidence + cleaner0 + 2023-07-20T14:14:54Z + DUMMY: + + structure + + + 0.37436584 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + + RESULTS + title_2 + 50273 + The putative iron binding site of (Sa)EctC + + 0.99960923 + site + cleaner0 + 2023-07-20T13:33:21Z + SO: + + iron binding site + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + + RESULTS + paragraph + 50316 + In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel. Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy. Only the refinement of Fe2+ resulted in a visibly improved electron density, however with a low degree of occupancy. This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b). The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively. These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure. Since both the refinement and the distance did not clearly identify an iron molecule, we decided to conservatively place a water molecule at this position. The position of this water molecule is described in more detail below and is highlighted in Figs 5a and 5b and 6a and 6b as a sphere. Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2). + + 0.9995599 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.9994135 + evidence + cleaner0 + 2023-07-20T14:14:59Z + DUMMY: + + structure + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9990086 + oligomeric_state + cleaner0 + 2023-07-20T13:33:47Z + DUMMY: + + monomers + + + 0.99944574 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + 0.9995765 + structure_element + cleaner0 + 2023-07-20T13:33:41Z + SO: + + cupin barrel + + + 0.5870309 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.99962735 + chemical + cleaner0 + 2023-07-20T13:34:02Z + CHEBI: + + Fe2+ + + + 0.99964345 + chemical + cleaner0 + 2023-07-20T13:34:10Z + CHEBI: + + Zn2+ + + + 0.9991924 + evidence + cleaner0 + 2023-07-20T14:15:03Z + DUMMY: + + density + + + experimental_method + MESH: + cleaner0 + 2023-07-20T14:33:11Z + + refined occupancy + + + 0.999634 + chemical + cleaner0 + 2023-07-20T13:34:07Z + CHEBI: + + Fe2+ + + + 0.9995745 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + 0.9994524 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.99923414 + residue_name_number + cleaner0 + 2023-07-20T13:32:58Z + DUMMY: + + Glu-57 + + + 0.9992427 + residue_name_number + cleaner0 + 2023-07-20T13:33:03Z + DUMMY: + + Tyr-85 + + + 0.9992547 + residue_name_number + cleaner0 + 2023-07-20T13:31:19Z + DUMMY: + + His-93 + + + 0.9990397 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.9992247 + residue_name_number + cleaner0 + 2023-07-20T13:32:58Z + DUMMY: + + Glu-57 + + + 0.9992345 + residue_name_number + cleaner0 + 2023-07-20T13:33:03Z + DUMMY: + + Tyr-85 + + + 0.9992223 + residue_name_number + cleaner0 + 2023-07-20T13:31:19Z + DUMMY: + + His-93 + + + 0.99948376 + site + cleaner0 + 2023-07-20T13:34:21Z + SO: + + iron binding sites + + + 0.9988392 + protein_state + cleaner0 + 2023-07-20T13:34:28Z + DUMMY: + + absence of + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9996319 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + 0.99956316 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.99976 + chemical + cleaner0 + 2023-07-20T14:20:05Z + CHEBI: + + water + + + 0.9997688 + chemical + cleaner0 + 2023-07-20T14:20:10Z + CHEBI: + + water + + + 0.99977607 + chemical + cleaner0 + 2023-07-20T14:20:13Z + CHEBI: + + water + + + 0.99950516 + protein_state + cleaner0 + 2023-07-20T14:48:50Z + DUMMY: + + strictly conserved + + + 0.978961 + experimental_method + cleaner0 + 2023-07-20T14:33:16Z + MESH: + + alignment + + + 0.9155193 + protein_type + cleaner0 + 2023-07-20T13:34:49Z + MESH: + + EctC protein + + + 0.9996103 + protein_type + cleaner0 + 2023-07-20T11:24:54Z + MESH: + + EctC-type proteins + + + + RESULTS + paragraph + 51703 + In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure. However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure. In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above. Taken together, this observations indicate, that the architecture of the presumptive iron-binding site is pre-set for the binding of the catalytically important metal by the ectoine synthase. + + 0.9996517 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.9994394 + evidence + cleaner0 + 2023-07-20T14:15:12Z + DUMMY: + + structure + + + 0.33843443 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.99958664 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + 0.9984459 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.9995256 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.9993711 + evidence + cleaner0 + 2023-07-20T14:15:16Z + DUMMY: + + structure + + + 0.9995761 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + 0.99966013 + chemical + cleaner0 + 2023-07-20T14:20:17Z + CHEBI: + + water + + + 0.9975165 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.99964285 + chemical + cleaner0 + 2023-07-20T14:20:19Z + CHEBI: + + water + + + 0.99965155 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.47516298 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9995999 + evidence + cleaner0 + 2023-07-20T14:15:19Z + DUMMY: + + structure + + + 0.99967086 + experimental_method + cleaner0 + 2023-07-20T14:33:22Z + MESH: + + superimposition + + + 0.55314845 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + 0.9995959 + evidence + cleaner0 + 2023-07-20T13:57:50Z + DUMMY: + + crystal structures + + + 0.9993043 + residue_name_number + cleaner0 + 2023-07-20T13:32:58Z + DUMMY: + + Glu-57 + + + 0.99930924 + residue_name_number + cleaner0 + 2023-07-20T13:33:03Z + DUMMY: + + Tyr-85 + + + 0.9993059 + residue_name_number + cleaner0 + 2023-07-20T13:31:20Z + DUMMY: + + His-93 + + + 0.99863094 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.99952936 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.9990138 + evidence + cleaner0 + 2023-07-20T14:15:23Z + DUMMY: + + structure + + + 0.9994984 + protein_state + cleaner0 + 2023-07-20T13:38:06Z + DUMMY: + + iron-free + + + 0.99965525 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.99928147 + evidence + cleaner0 + 2023-07-20T14:15:26Z + DUMMY: + + structure + + + 0.9992802 + residue_name_number + cleaner0 + 2023-07-20T13:31:20Z + DUMMY: + + His-93 + + + 0.9995225 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.9994363 + evidence + cleaner0 + 2023-07-20T14:15:28Z + DUMMY: + + structure + + + 0.99964875 + structure_element + cleaner0 + 2023-07-20T13:27:53Z + SO: + + β-strand + + + 0.9996643 + structure_element + cleaner0 + 2023-07-20T13:27:47Z + SO: + + β5 + + + 0.9996266 + site + cleaner0 + 2023-07-20T13:28:17Z + SO: + + iron-binding site + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.99957365 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + + RESULTS + paragraph + 52742 + Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures. In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding. Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1). It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility. + + 0.9991734 + residue_name_number + cleaner0 + 2023-07-20T13:38:45Z + DUMMY: + + Tyr-52 + + + 0.9997297 + structure_element + cleaner0 + 2023-07-20T14:44:33Z + SO: + + loop + + + 0.9996465 + structure_element + cleaner0 + 2023-07-20T13:27:53Z + SO: + + β-strand + + + 0.9996942 + structure_element + cleaner0 + 2023-07-20T13:27:47Z + SO: + + β5 + + + 0.9996644 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.99950296 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.41048887 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.9991352 + evidence + cleaner0 + 2023-07-20T14:15:49Z + DUMMY: + + structures + + + 0.99953073 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.9989262 + evidence + cleaner0 + 2023-07-20T14:15:52Z + DUMMY: + + structure + + + 0.9991581 + residue_name_number + cleaner0 + 2023-07-20T13:38:45Z + DUMMY: + + Tyr-52 + + + 0.9957058 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.9991247 + residue_name_number + cleaner0 + 2023-07-20T13:38:43Z + DUMMY: + + Tyr-52 + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.99670815 + experimental_method + cleaner0 + 2023-07-20T14:33:29Z + MESH: + + substitution + + + 0.9991492 + residue_name + cleaner0 + 2023-07-20T13:38:55Z + SO: + + Ala + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.9722253 + protein_state + cleaner0 + 2023-07-20T13:40:44Z + DUMMY: + + mutant + + + 0.99968576 + experimental_method + cleaner0 + 2023-07-20T14:33:33Z + MESH: + + overlay + + + 0.9996631 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.9995298 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.42811933 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.9995607 + evidence + cleaner0 + 2023-07-20T13:57:50Z + DUMMY: + + crystal structures + + + 0.99912995 + residue_name_number + cleaner0 + 2023-07-20T13:38:45Z + DUMMY: + + Tyr-52 + + + 0.99530953 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.9992278 + residue_name_number + cleaner0 + 2023-07-20T13:32:58Z + DUMMY: + + Glu-57 + + + 0.99924177 + residue_name_number + cleaner0 + 2023-07-20T13:33:03Z + DUMMY: + + Tyr-85 + + + 0.9992725 + residue_name_number + cleaner0 + 2023-07-20T13:31:20Z + DUMMY: + + His-93 + + + 0.9991281 + residue_name_number + cleaner0 + 2023-07-20T13:38:45Z + DUMMY: + + Tyr-52 + + + 0.999544 + protein_state + cleaner0 + 2023-07-20T14:49:00Z + DUMMY: + + strictly conserved + + + 0.99956137 + experimental_method + cleaner0 + 2023-07-20T14:33:36Z + MESH: + + alignment + + + 0.99964917 + protein_type + cleaner0 + 2023-07-20T11:24:54Z + MESH: + + EctC-type proteins + + + 0.9996603 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.999398 + protein_state + cleaner0 + 2023-07-20T13:34:29Z + DUMMY: + + absence of + + + 0.9994852 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.7439358 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.99959594 + evidence + cleaner0 + 2023-07-20T13:57:50Z + DUMMY: + + crystal structures + + + 0.99913955 + residue_name_number + cleaner0 + 2023-07-20T13:38:45Z + DUMMY: + + Tyr-52 + + + + RESULTS + paragraph + 53965 + To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity. When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1). For some of the presumptive iron-coordinating residues, additional site-directed mutagenesis experiments were carried out. To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant. This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1). We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative. Collectively, these data suggest that the hydroxyl group of the Tyr-85 side chain is needed for the binding of the iron (Fig 6a). We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1). Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance. + + 0.9995678 + site + cleaner0 + 2023-07-20T13:33:22Z + SO: + + iron binding site + + + 0.99942875 + experimental_method + cleaner0 + 2023-07-20T13:39:48Z + MESH: + + structure-guided site-directed mutagenesis + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:42Z + + EctC + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.99915075 + residue_name_number + cleaner0 + 2023-07-20T13:32:58Z + DUMMY: + + Glu-57 + + + 0.9991446 + residue_name_number + cleaner0 + 2023-07-20T13:33:03Z + DUMMY: + + Tyr-85 + + + 0.99909955 + residue_name_number + cleaner0 + 2023-07-20T13:31:20Z + DUMMY: + + His-93 + + + 0.9344692 + site + cleaner0 + 2023-07-20T13:40:01Z + SO: + + mono-nuclear iron center + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.67269844 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + 0.9995788 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + 0.98794144 + experimental_method + cleaner0 + 2023-07-20T14:33:41Z + MESH: + + replaced + + + 0.99890435 + residue_name + cleaner0 + 2023-07-20T13:40:27Z + SO: + + Ala + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.5676748 + protein_state + cleaner0 + 2023-07-20T13:40:44Z + DUMMY: + + mutant + + + 0.99950314 + site + cleaner0 + 2023-07-20T13:40:16Z + SO: + + iron-coordinating residues + + + 0.99941367 + experimental_method + cleaner0 + 2023-07-20T13:40:21Z + MESH: + + site-directed mutagenesis + + + 0.99917907 + residue_name_number + cleaner0 + 2023-07-20T13:32:58Z + DUMMY: + + Glu-57 + + + 0.9856354 + residue_name + cleaner0 + 2023-07-20T13:40:30Z + SO: + + Asp + + + 0.8235472 + protein_state + cleaner0 + 2023-07-20T14:38:42Z + DUMMY: + + variant + + + 0.9981299 + protein_state + cleaner0 + 2023-07-20T13:40:44Z + DUMMY: + + mutant + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.9215003 + residue_name + cleaner0 + 2023-07-20T13:40:33Z + SO: + + Ala + + + 0.7259734 + experimental_method + cleaner0 + 2023-07-20T14:33:45Z + MESH: + + substitution + + + 0.6129925 + experimental_method + cleaner0 + 2023-07-20T14:33:48Z + MESH: + + replaced + + + 0.9991696 + residue_name_number + cleaner0 + 2023-07-20T13:33:03Z + DUMMY: + + Tyr-85 + + + 0.99924386 + residue_name + cleaner0 + 2023-07-20T13:40:36Z + SO: + + Phe + + + 0.99925524 + residue_name + cleaner0 + 2023-07-20T13:40:40Z + SO: + + Trp + + + 0.99584913 + protein_state + cleaner0 + 2023-07-20T13:40:43Z + DUMMY: + + mutant + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.99915314 + residue_name_number + cleaner0 + 2023-07-20T13:33:03Z + DUMMY: + + Tyr-85 + + + 0.99948275 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.9980252 + experimental_method + cleaner0 + 2023-07-20T14:33:51Z + MESH: + + replaced + + + 0.9995284 + site + cleaner0 + 2023-07-20T14:39:16Z + SO: + + iron-binding residue + + + 0.99909085 + residue_name_number + cleaner0 + 2023-07-20T13:31:20Z + DUMMY: + + His-93 + + + 0.99927825 + residue_name + cleaner0 + 2023-07-20T13:40:51Z + SO: + + Asn + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.58080834 + protein + cleaner0 + 2023-07-20T10:09:42Z + PR: + + EctC + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.9995037 + protein_state + cleaner0 + 2023-07-20T13:41:16Z + DUMMY: + + wild-type + + + 0.99956393 + site + cleaner0 + 2023-07-20T13:40:18Z + SO: + + iron-coordinating residues + + + 0.9991215 + residue_name_number + cleaner0 + 2023-07-20T13:32:58Z + DUMMY: + + Glu-57 + + + 0.99911183 + residue_name_number + cleaner0 + 2023-07-20T13:33:03Z + DUMMY: + + Tyr-85 + + + 0.9990993 + residue_name_number + cleaner0 + 2023-07-20T13:31:20Z + DUMMY: + + His-93 + + + 0.99946946 + chemical + cleaner0 + 2023-07-20T13:41:21Z + CHEBI: + + Fe2+ + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.68746006 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + + RESULTS + title_2 + 55597 + A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate + + 0.50598353 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.99949026 + evidence + cleaner0 + 2023-07-20T14:15:57Z + DUMMY: + + structure + + + 0.99971735 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + + RESULTS + paragraph + 55715 + Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1). However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement. This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement. + + 0.9995761 + experimental_method + cleaner0 + 2023-07-20T14:33:56Z + MESH: + + co-crystallization + + + 0.99930274 + experimental_method + cleaner0 + 2023-07-20T14:34:00Z + MESH: + + soaking experiments + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.99963343 + evidence + cleaner0 + 2023-07-20T13:57:50Z + DUMMY: + + crystal structures + + + 0.99975777 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.9989285 + chemical + cleaner0 + 2023-07-20T10:07:54Z + CHEBI: + + ectoine + + + 0.99956965 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.9995155 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.52894163 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.99962676 + evidence + cleaner0 + 2023-07-20T14:16:02Z + DUMMY: + + structure + + + 0.9972017 + structure_element + cleaner0 + 2023-07-20T13:43:34Z + SO: + + carboxy-terminal loop + + + 0.99951136 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + 0.99958414 + site + cleaner0 + 2023-07-20T13:43:15Z + SO: + + active site + + + 0.9973748 + experimental_method + cleaner0 + 2023-07-20T14:34:04Z + MESH: + + crystallographic refinement + + + 0.9996444 + protein_state + cleaner0 + 2023-07-20T11:35:52Z + DUMMY: + + open + + + 0.99958867 + evidence + cleaner0 + 2023-07-20T14:16:06Z + DUMMY: + + structure + + + 0.41580853 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.99953204 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + + RESULTS + paragraph + 56388 + We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched. This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization. Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments. We therefore stress that we cannot identify neither the true chemically nature of this compound, nor its precise origin. + + experimental_method + MESH: + cleaner0 + 2023-07-20T14:34:37Z + + purification + + + 0.99943024 + experimental_method + cleaner0 + 2023-07-20T14:34:41Z + MESH: + + crystallization + + + 0.999591 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.9980836 + species + cleaner0 + 2023-07-20T10:23:07Z + MESH: + + E. coli + + + 0.99773324 + experimental_method + cleaner0 + 2023-07-20T14:34:44Z + MESH: + + crystallization + + + 0.9997731 + chemical + cleaner0 + 2023-07-20T13:42:28Z + CHEBI: + + PEG + + + 0.99964523 + evidence + cleaner0 + 2023-07-20T14:16:14Z + DUMMY: + + density + + + 0.9997856 + chemical + cleaner0 + 2023-07-20T13:42:30Z + CHEBI: + + PEG + + + 0.99977535 + chemical + cleaner0 + 2023-07-20T13:42:30Z + CHEBI: + + PEG + + + 0.9997731 + chemical + cleaner0 + 2023-07-20T13:42:30Z + CHEBI: + + PEG + + + + RESULTS + paragraph + 57081 + Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density. However, to the best of our knowledge, hexane-1,6-diol is not part of the E. coli metabolome. Despite these notable limitations, we considered the serendipitously trapped compound as a mock ligand that might provide useful insights into the spatial positioning of the true EctC substrate and those residues that coordinate it within the ectoine synthase active site. We note that both N-γ-ADABA and hexane-1,6-diol are both C6-compounds and display similar length (Fig 7a). + + 0.9991968 + evidence + cleaner0 + 2023-07-20T13:42:46Z + DUMMY: + + electron density feature + + + 0.26774096 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + 0.70911974 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + 0.99976677 + chemical + cleaner0 + 2023-07-20T13:42:36Z + CHEBI: + + hexane-1,6-diol + + + 0.99958235 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + 0.99976915 + chemical + cleaner0 + 2023-07-20T13:42:37Z + CHEBI: + + hexane-1,6-diol + + + 0.99940926 + species + cleaner0 + 2023-07-20T10:23:07Z + MESH: + + E. coli + + + 0.99940443 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + 0.9996128 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.9996047 + site + cleaner0 + 2023-07-20T13:43:15Z + SO: + + active site + + + 0.9997796 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.9997713 + chemical + cleaner0 + 2023-07-20T13:42:37Z + CHEBI: + + hexane-1,6-diol + + + + pone.0151285.g007.jpg + pone.0151285.g007 + FIG + fig_title_caption + 57795 + A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure. + + 0.99954915 + site + cleaner0 + 2023-07-20T13:43:13Z + SO: + + active site + + + 0.9995071 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.9989201 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + 0.9980453 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + 0.9996127 + evidence + cleaner0 + 2023-07-20T11:35:43Z + DUMMY: + + crystal structure + + + + pone.0151285.g007.jpg + pone.0151285.g007 + FIG + fig_caption + 57909 + (a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds. (b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted. The amino acid side chains involved in iron-ligand binding are colored in blue and those involved in the binding of the chemically undefined ligand are colored in green using a ball and stick representation. The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange. The electron density was calculated as an omit map and contoured at 1.0 σ. + + 0.9995905 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + 0.99960107 + site + cleaner0 + 2023-07-20T13:43:15Z + SO: + + active site + + + 0.9995239 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + 0.99727964 + evidence + cleaner0 + 2023-07-20T14:16:21Z + DUMMY: + + structure + + + 0.5305522 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.9997797 + chemical + cleaner0 + 2023-07-20T13:42:37Z + CHEBI: + + hexane-1,6-diol + + + 0.9995757 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + 0.9997587 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.9996003 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.999565 + site + cleaner0 + 2023-07-20T14:39:22Z + SO: + + binding site + + + 0.9704798 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.9997806 + chemical + cleaner0 + 2023-07-20T13:42:37Z + CHEBI: + + hexane-1,6-diol + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.9990362 + protein_state + cleaner0 + 2023-07-20T14:49:06Z + DUMMY: + + flexible + + + 0.9996913 + structure_element + cleaner0 + 2023-07-20T13:43:33Z + SO: + + carboxy-terminal loop + + + 0.4624071 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.9996306 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + 0.99962413 + evidence + cleaner0 + 2023-07-20T13:44:12Z + DUMMY: + + omit map + + + + RESULTS + paragraph + 58667 + We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%. We also calculated an omit map and the electron density reappeared (Fig 7b). When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. Remarkably, all of these residues are highly conserved throughout the extended EctC protein family (Fig 2). + + 0.99945074 + experimental_method + cleaner0 + 2023-07-20T14:34:50Z + MESH: + + refined + + + 0.56919813 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.99954236 + evidence + cleaner0 + 2023-07-20T14:16:25Z + DUMMY: + + structure + + + 0.9994499 + evidence + cleaner0 + 2023-07-20T14:16:28Z + DUMMY: + + R- and Rfree-factor + + + 0.99959517 + evidence + cleaner0 + 2023-07-20T13:44:11Z + DUMMY: + + omit map + + + 0.9995711 + evidence + cleaner0 + 2023-07-20T13:31:04Z + DUMMY: + + electron density + + + 0.6303048 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.9996358 + protein_state + cleaner0 + 2023-07-20T14:49:10Z + DUMMY: + + bound + + + 0.99931145 + residue_name_number + cleaner0 + 2023-07-20T14:22:14Z + DUMMY: + + Trp-21 + + + 0.99931717 + residue_name_number + cleaner0 + 2023-07-20T14:22:17Z + DUMMY: + + Ser-23 + + + 0.9996448 + structure_element + cleaner0 + 2023-07-20T14:44:38Z + SO: + + β-sheet + + + 0.9996158 + structure_element + cleaner0 + 2023-07-20T14:44:42Z + SO: + + β3 + + + 0.9993132 + residue_name_number + cleaner0 + 2023-07-20T13:45:00Z + DUMMY: + + Thr-40 + + + 0.99966 + structure_element + cleaner0 + 2023-07-20T14:44:47Z + SO: + + β-sheet + + + 0.99950767 + structure_element + cleaner0 + 2023-07-20T14:44:50Z + SO: + + β4 + + + 0.9993027 + residue_name_number + cleaner0 + 2023-07-20T14:22:21Z + DUMMY: + + Cys-105 + + + 0.99930197 + residue_name_number + cleaner0 + 2023-07-20T13:45:34Z + DUMMY: + + Phe-107 + + + 0.99964255 + structure_element + cleaner0 + 2023-07-20T14:44:53Z + SO: + + β-sheet + + + 0.99967504 + structure_element + cleaner0 + 2023-07-20T13:26:56Z + SO: + + β11 + + + 0.99954045 + protein_state + cleaner0 + 2023-07-20T14:49:14Z + DUMMY: + + highly conserved + + + 0.8928305 + protein_type + cleaner0 + 2023-07-20T13:44:25Z + MESH: + + EctC protein + + + + RESULTS + title_2 + 59274 + Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase + + 0.9994589 + experimental_method + cleaner0 + 2023-07-20T14:35:01Z + MESH: + + Structure-guided site-directed mutagenesis + + + 0.98101175 + site + cleaner0 + 2023-07-20T14:39:26Z + SO: + + catalytic core + + + 0.99961734 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + + RESULTS + paragraph + 59363 + In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues. These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices. We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein. As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a). + + 0.9936033 + experimental_method + cleaner0 + 2023-07-20T14:35:05Z + MESH: + + alignment of the amino acid sequences + + + 0.99969447 + protein_type + cleaner0 + 2023-07-20T11:24:54Z + MESH: + + EctC-type proteins + + + 0.9992889 + protein_state + cleaner0 + 2023-07-20T14:49:26Z + DUMMY: + + strictly conserved + + + 0.99925977 + residue_name_number + cleaner0 + 2023-07-20T13:44:59Z + DUMMY: + + Thr-40 + + + 0.999253 + residue_name_number + cleaner0 + 2023-07-20T13:38:45Z + DUMMY: + + Tyr-52 + + + 0.99923754 + residue_name_number + cleaner0 + 2023-07-20T13:30:41Z + DUMMY: + + His-55 + + + 0.9992304 + residue_name_number + cleaner0 + 2023-07-20T13:32:58Z + DUMMY: + + Glu-57 + + + 0.9992234 + residue_name_number + cleaner0 + 2023-07-20T13:45:14Z + DUMMY: + + Gly-64 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-20T13:33:03Z + + Tyr-85 + + + 0.9920979 + residue_name_number + cleaner0 + 2023-07-20T13:45:24Z + DUMMY: + + Leu-87 + + + 0.9992099 + residue_name_number + cleaner0 + 2023-07-20T13:31:20Z + DUMMY: + + His-93 + + + 0.9992581 + residue_name_number + cleaner0 + 2023-07-20T13:45:32Z + DUMMY: + + Phe-107 + + + 0.9992681 + residue_name_number + cleaner0 + 2023-07-20T13:28:44Z + DUMMY: + + Pro-109 + + + 0.9992566 + residue_name_number + cleaner0 + 2023-07-20T13:45:41Z + DUMMY: + + Gly-113 + + + 0.99928117 + residue_name_number + cleaner0 + 2023-07-20T13:31:32Z + DUMMY: + + Glu-115 + + + 0.999279 + residue_name_number + cleaner0 + 2023-07-20T13:45:50Z + DUMMY: + + His-117 + + + 0.7303695 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.9992512 + residue_name_number + cleaner0 + 2023-07-20T13:45:15Z + DUMMY: + + Gly-64 + + + 0.99925476 + residue_name_number + cleaner0 + 2023-07-20T13:28:44Z + DUMMY: + + Pro-109 + + + 0.9992551 + residue_name_number + cleaner0 + 2023-07-20T13:45:42Z + DUMMY: + + Gly-113 + + + 0.9996702 + structure_element + cleaner0 + 2023-07-20T14:45:09Z + SO: + + β-strands + + + 0.99966365 + structure_element + cleaner0 + 2023-07-20T14:45:13Z + SO: + + α-helices + + + 0.99937916 + protein_state + cleaner0 + 2023-07-20T14:49:30Z + DUMMY: + + flexible + + + structure_element + SO: + cleaner0 + 2023-07-20T14:44:24Z + + carboxy-terminus + + + 0.3778841 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + 0.6953704 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + 0.999218 + residue_name_number + cleaner0 + 2023-07-20T13:32:58Z + DUMMY: + + Glu-57 + + + 0.9992469 + residue_name_number + cleaner0 + 2023-07-20T13:33:03Z + DUMMY: + + Tyr-85 + + + 0.99925154 + residue_name_number + cleaner0 + 2023-07-20T13:31:20Z + DUMMY: + + His-93 + + + 0.99963474 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + + RESULTS + paragraph + 60186 + In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1). Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations. We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min. In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1). + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.54778117 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + 0.9992084 + evidence + cleaner0 + 2023-07-20T14:16:35Z + DUMMY: + + structure + + + 0.99942106 + experimental_method + cleaner0 + 2023-07-20T13:46:32Z + MESH: + + structure-guided site-directed mutagenesis + + + 0.9990582 + protein_state + cleaner0 + 2023-07-20T13:40:44Z + DUMMY: + + mutant + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.43671474 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + 0.9973287 + species + cleaner0 + 2023-07-20T10:23:07Z + MESH: + + E. coli + + + 0.99090666 + experimental_method + cleaner0 + 2023-07-20T14:35:14Z + MESH: + + affinity chromatography + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.9626147 + experimental_method + cleaner0 + 2023-07-20T13:46:48Z + MESH: + + single time-point enzyme assay + + + 0.99954396 + protein_state + cleaner0 + 2023-07-20T13:41:17Z + DUMMY: + + wild-type + + + 0.44324213 + species + cleaner0 + 2023-07-20T10:06:59Z + MESH: + + Sa + + + 0.34864613 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + 0.9997834 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.9997422 + chemical + cleaner0 + 2023-07-20T10:07:54Z + CHEBI: + + ectoine + + + 0.99838936 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.9993874 + protein_state + cleaner0 + 2023-07-20T13:40:44Z + DUMMY: + + mutant + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.25436148 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + 0.9979036 + experimental_method + cleaner0 + 2023-07-20T11:27:08Z + MESH: + + colorimetric assay + + + + RESULTS + paragraph + 60967 + The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b). We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins. However, their catalytic activity was substantially impaired (Table 1). Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b). We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding. Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1). Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase. + + 0.9990735 + protein_state + cleaner0 + 2023-07-20T14:49:36Z + DUMMY: + + evolutionarily conserved + + + 0.9991827 + residue_name_number + cleaner0 + 2023-07-20T14:22:28Z + DUMMY: + + Trp-21 + + + 0.999167 + residue_name_number + cleaner0 + 2023-07-20T14:22:33Z + DUMMY: + + Ser-23 + + + 0.99915034 + residue_name_number + cleaner0 + 2023-07-20T13:45:00Z + DUMMY: + + Thr-40 + + + 0.9991319 + residue_name_number + cleaner0 + 2023-07-20T14:22:36Z + DUMMY: + + Cys-105 + + + 0.99915427 + residue_name_number + cleaner0 + 2023-07-20T13:45:34Z + DUMMY: + + Phe-107 + + + 0.9995274 + protein_state + cleaner0 + 2023-07-20T11:36:05Z + DUMMY: + + semi-closed + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.6939467 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + 0.99955386 + evidence + cleaner0 + 2023-07-20T14:16:38Z + DUMMY: + + structure + + + 0.9988753 + experimental_method + cleaner0 + 2023-07-20T14:35:21Z + MESH: + + replaced + + + 0.99910176 + residue_name + cleaner0 + 2023-07-20T13:48:04Z + SO: + + Ala + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.4964614 + protein_state + cleaner0 + 2023-07-20T13:40:44Z + DUMMY: + + mutant + + + 0.9991894 + residue_name_number + cleaner0 + 2023-07-20T13:45:00Z + DUMMY: + + Thr-40 + + + 0.99962234 + structure_element + cleaner0 + 2023-07-20T13:27:53Z + SO: + + β-strand + + + 0.9996573 + structure_element + cleaner0 + 2023-07-20T13:27:47Z + SO: + + β5 + + + 0.99946547 + structure_element + cleaner0 + 2023-07-20T13:33:43Z + SO: + + cupin barrel + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.62484354 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + 0.99685633 + experimental_method + cleaner0 + 2023-07-20T14:35:25Z + MESH: + + replaced + + + 0.99922866 + 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Ser-105 + + + 0.9995801 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + + RESULTS + paragraph + 62729 + We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1). Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1). This is different for the His-55/Ala substitution. The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b). Each of these residues is evolutionarily highly conserved. The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity. Indeed, the Glu-115/Ala and the His-55/Ala substitutions possessed only 21% and 16% activity of the wild-type protein, respectively (Table 1). The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1). We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity. Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase. + + 0.8126707 + experimental_method + cleaner0 + 2023-07-20T14:35:43Z + MESH: + + amino acid substitutions + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + mutant + MESH: + cleaner0 + 2023-07-20T13:49:54Z + + Tyr-52/Ala + + + 0.99514467 + mutant + cleaner0 + 2023-07-20T13:50:07Z + MESH: + + His-55/Ala + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + 0.4194266 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + species + MESH: + cleaner0 + 2023-07-20T10:06:59Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.99959433 + evidence + cleaner0 + 2023-07-20T13:57:50Z + DUMMY: + + crystal structures + + + 0.9994112 + experimental_method + cleaner0 + 2023-07-20T14:35:47Z + MESH: + + replacement + + + 0.99900836 + residue_name_number + cleaner0 + 2023-07-20T13:38:45Z + DUMMY: + + Tyr-52 + + + 0.9989157 + residue_name + cleaner0 + 2023-07-20T13:50:25Z + SO: + + Ala + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.9857507 + mutant + cleaner0 + 2023-07-20T13:50:08Z + MESH: + + His-55/Ala + + + 0.97617674 + structure_element + cleaner0 + 2023-07-20T13:50:48Z + SO: + + carboxy-terminal region + + + species + MESH: + cleaner0 + 2023-07-20T10:07:00Z + + Sa + + + 0.45931357 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + 0.9991133 + residue_name_number + cleaner0 + 2023-07-20T13:31:32Z + DUMMY: + + Glu-115 + + + 0.9989636 + residue_name_number + cleaner0 + 2023-07-20T13:30:41Z + DUMMY: + + His-55 + + + 0.999028 + residue_name_number + cleaner0 + 2023-07-20T13:30:41Z + DUMMY: + + His-55 + + + 0.99918747 + residue_name_number + cleaner0 + 2023-07-20T13:28:50Z + DUMMY: + + Pro-110 + + + 0.9993026 + protein_state + cleaner0 + 2023-07-20T14:49:58Z + DUMMY: + + evolutionarily highly conserved + + + 0.9575634 + experimental_method + cleaner0 + 2023-07-20T14:35:49Z + MESH: + + substitution + + + 0.99907035 + residue_name_number + cleaner0 + 2023-07-20T13:31:32Z + DUMMY: + + Glu-115 + + + 0.9990751 + residue_name_number + cleaner0 + 2023-07-20T13:30:41Z + DUMMY: + + His-55 + + + 0.99904245 + residue_name + cleaner0 + 2023-07-20T13:51:41Z + SO: + + Ala + + + 0.9991319 + site + cleaner0 + 2023-07-20T14:39:33Z + SO: + + interactive network + + + 0.96925527 + mutant + cleaner0 + 2023-07-20T13:51:08Z + MESH: + + Glu-115/Ala + + + 0.98248357 + mutant + cleaner0 + 2023-07-20T13:50:08Z + MESH: + + His-55/Ala + + + 0.9995592 + protein_state + cleaner0 + 2023-07-20T13:41:17Z + DUMMY: + + wild-type + + + 0.9973091 + mutant + cleaner0 + 2023-07-20T13:51:09Z + MESH: + + Glu-115/Ala + + + 0.9991985 + protein_state + cleaner0 + 2023-07-20T13:40:45Z + DUMMY: + + mutant + + + 0.9979682 + protein_state + cleaner0 + 2023-07-20T13:41:17Z + DUMMY: + + wild-type + + + 0.9442199 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:01Z + + iron + + + 0.9923347 + mutant + cleaner0 + 2023-07-20T13:50:08Z + MESH: + + His-55/Ala + + + 0.99822956 + protein_state + cleaner0 + 2023-07-20T13:41:17Z + DUMMY: + + wild-type + + + 0.9986185 + experimental_method + cleaner0 + 2023-07-20T14:35:54Z + MESH: + + replaced + + + 0.99916977 + residue_name_number + cleaner0 + 2023-07-20T13:31:32Z + DUMMY: + + Glu-115 + + + 0.99923563 + residue_name + cleaner0 + 2023-07-20T13:51:15Z + SO: + + Asp + + + species + MESH: + cleaner0 + 2023-07-20T10:07:00Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.99856275 + protein_state + cleaner0 + 2023-07-20T13:41:17Z + DUMMY: + + wild-type + + + 0.9908215 + chemical + cleaner0 + 2023-07-20T11:22:01Z + CHEBI: + + iron + + + 0.9920697 + protein_state + cleaner0 + 2023-07-20T13:41:17Z + DUMMY: + + wild-type + + + structure_element + SO: + cleaner0 + 2023-07-20T14:44:24Z + + carboxy-terminus + + + 0.476489 + species + cleaner0 + 2023-07-20T10:07:00Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.99950236 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + + RESULTS + paragraph + 64268 + Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family. The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1). Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1). We currently cannot comment on possible functional role Asp-91. However, Leu-87 is positioned at the end of one of the β-sheets that form the dimer interface (Fig 5c) and it might therefore possess a structural role. It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects. His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1). We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase. + + 0.999248 + residue_name_number + cleaner0 + 2023-07-20T13:45:25Z + DUMMY: + + Leu-87 + + + 0.9992301 + residue_name_number + cleaner0 + 2023-07-20T13:51:53Z + DUMMY: + + Asp-91 + + + 0.9995029 + protein_state + cleaner0 + 2023-07-20T14:50:02Z + DUMMY: + + highly conserved + + + 0.99949646 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.9994337 + experimental_method + cleaner0 + 2023-07-20T14:35:57Z + MESH: + + replacement + + + 0.9992523 + residue_name_number + cleaner0 + 2023-07-20T13:45:25Z + DUMMY: + + Leu-87 + + + 0.9991021 + residue_name + cleaner0 + 2023-07-20T13:52:17Z + SO: + + Ala + + + 0.9994435 + experimental_method + cleaner0 + 2023-07-20T14:36:02Z + MESH: + + replacement + + + 0.9992148 + residue_name_number + cleaner0 + 2023-07-20T13:51:54Z + DUMMY: + + Asp-91 + + + 0.99909496 + residue_name + cleaner0 + 2023-07-20T13:52:12Z + SO: + + Ala + + + 0.999158 + residue_name + cleaner0 + 2023-07-20T13:52:14Z + SO: + + Glu + + + species + MESH: + cleaner0 + 2023-07-20T10:07:00Z + + Sa + + + 0.5594292 + protein + cleaner0 + 2023-07-20T10:09:43Z + PR: + + EctC + + + 0.9992165 + residue_name_number + cleaner0 + 2023-07-20T13:51:54Z + DUMMY: + + Asp-91 + + + 0.9992547 + residue_name_number + cleaner0 + 2023-07-20T13:45:25Z + DUMMY: + + Leu-87 + + + 0.9996314 + structure_element + cleaner0 + 2023-07-20T14:45:18Z + SO: + + β-sheets + + + 0.9995923 + site + cleaner0 + 2023-07-20T13:21:56Z + SO: + + dimer interface + + + 0.9992736 + residue_name_number + cleaner0 + 2023-07-20T13:33:03Z + DUMMY: + + Tyr-85 + + + 0.9993905 + chemical + cleaner0 + 2023-07-20T11:22:02Z + CHEBI: + + iron + + + 0.63578343 + species + cleaner0 + 2023-07-20T10:07:00Z + MESH: + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:43Z + + EctC + + + 0.99964094 + site + cleaner0 + 2023-07-20T13:43:15Z + SO: + + active site + + + 0.9992296 + residue_name_number + cleaner0 + 2023-07-20T13:45:51Z + DUMMY: + + His-117 + + + 0.9994899 + protein_state + cleaner0 + 2023-07-20T14:50:05Z + DUMMY: + + strictly conserved + + + 0.9991002 + experimental_method + cleaner0 + 2023-07-20T14:36:05Z + MESH: + + substitution + + + 0.9991954 + residue_name + cleaner0 + 2023-07-20T14:22:07Z + SO: + + Ala + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:02Z + + iron + + + 0.9992239 + residue_name_number + cleaner0 + 2023-07-20T13:45:51Z + DUMMY: + + His-117 + + + species + MESH: + cleaner0 + 2023-07-20T10:07:00Z + + Sa + + + 0.632539 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.9993117 + evidence + cleaner0 + 2023-07-20T13:52:53Z + DUMMY: + + structure + + + 0.9996658 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + + RESULTS + paragraph + 65360 + As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues. Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1). This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content. + + experimental_method + MESH: + cleaner0 + 2023-07-20T14:36:39Z + + mutagenesis experiments + + + 0.93242997 + experimental_method + cleaner0 + 2023-07-20T14:36:13Z + MESH: + + substituted + + + 0.99923414 + residue_name_number + cleaner0 + 2023-07-20T13:53:06Z + DUMMY: + + Thr-41 + + + 0.9992416 + residue_name_number + cleaner0 + 2023-07-20T13:53:12Z + DUMMY: + + His-51 + + + 0.99938995 + protein_state + cleaner0 + 2023-07-20T14:50:08Z + DUMMY: + + not evolutionarily conserved + + + 0.9996739 + protein_type + cleaner0 + 2023-07-20T11:24:54Z + MESH: + + EctC-type proteins + + + 0.99900913 + residue_name + cleaner0 + 2023-07-20T13:53:02Z + SO: + + Ala + + + species + MESH: + cleaner0 + 2023-07-20T10:07:00Z + + Sa + + + 0.40038544 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.999278 + protein_state + cleaner0 + 2023-07-20T13:41:17Z + DUMMY: + + wild-type + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:02Z + + iron + + + 0.9995473 + protein_state + cleaner0 + 2023-07-20T13:41:17Z + DUMMY: + + wild-type + + + species + MESH: + cleaner0 + 2023-07-20T10:07:00Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:44Z + + EctC + + + chemical + CHEBI: + cleaner0 + 2023-07-20T11:22:02Z + + iron + + + + DISCUSS + title_1 + 65848 + Discussion + + + DISCUSS + paragraph + 65859 + The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily. The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins. In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different. As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins. + + 0.99896866 + evidence + cleaner0 + 2023-07-20T13:53:40Z + DUMMY: + + crystallographic data + + + 0.9955268 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.7606321 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.9992632 + taxonomy_domain + cleaner0 + 2023-07-20T13:54:44Z + DUMMY: + + microbial + + + 0.86524945 + chemical + cleaner0 + 2023-07-20T10:07:54Z + CHEBI: + + ectoine + + + 0.99958295 + protein_type + cleaner0 + 2023-07-20T10:07:22Z + MESH: + + cupin superfamily + + + 0.58500177 + species + cleaner0 + 2023-07-20T10:07:00Z + MESH: + + Sa + + + 0.8823065 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.9165128 + structure_element + cleaner0 + 2023-07-20T14:45:23Z + SO: + + β-strands + + + 0.9996851 + structure_element + cleaner0 + 2023-07-20T13:54:19Z + SO: + + β1-β11 + + + 0.9996598 + structure_element + cleaner0 + 2023-07-20T14:45:27Z + SO: + + α-helices + + + 0.99969345 + structure_element + cleaner0 + 2023-07-20T14:45:29Z + SO: + + α-I + + + 0.99968463 + structure_element + cleaner0 + 2023-07-20T14:45:32Z + SO: + + α-II + + + 0.99958915 + evidence + cleaner0 + 2023-07-20T13:57:50Z + DUMMY: + + crystal structures + + + 0.9995442 + protein_type + cleaner0 + 2023-07-20T11:22:39Z + MESH: + + cupins + + + 0.9996916 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.9996798 + protein_type + cleaner0 + 2023-07-20T14:17:41Z + MESH: + + polyketide cyclase + + + 0.99957806 + protein + cleaner0 + 2023-07-20T13:20:13Z + PR: + + RemF + + + 0.99837685 + protein_type + cleaner0 + 2023-07-20T14:17:54Z + MESH: + + cupin-related + + + 0.9934255 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.99956805 + protein + cleaner0 + 2023-07-20T13:20:13Z + PR: + + RemF + + + 0.99773055 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.9995147 + protein + cleaner0 + 2023-07-20T13:20:13Z + PR: + + RemF + + + 0.9996712 + protein_type + cleaner0 + 2023-07-20T11:24:54Z + MESH: + + EctC-type proteins + + + 0.9993017 + taxonomy_domain + cleaner0 + 2023-07-20T13:54:43Z + DUMMY: + + microbial + + + 0.999644 + protein_type + cleaner0 + 2023-07-20T13:54:39Z + MESH: + + RemF-like + + + + DISCUSS + paragraph + 66824 + The pro- and eukaryotic members of the cupin superfamily perform a variety of both enzymatic and non-enzymatic functions that are built upon a common structural scaffold. Most cupins contain transition state metals that can promote different types of chemical reactions. Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites. We report here for the first time that the ectoine synthase is a metal-dependent enzyme. ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction. However, as observed with other cupins, EctC is a somewhat promiscuous enzyme as far as the catalytically important metal is concerned when they are provided in large molar excess (Fig 3c). + + 0.9402873 + taxonomy_domain + cleaner0 + 2023-07-20T13:54:54Z + DUMMY: + + pro- + + + 0.6676487 + taxonomy_domain + cleaner0 + 2023-07-20T13:54:57Z + DUMMY: + + eukaryotic + + + 0.9990274 + protein_type + cleaner0 + 2023-07-20T10:07:22Z + MESH: + + cupin superfamily + + + 0.99962854 + protein_type + cleaner0 + 2023-07-20T11:22:39Z + MESH: + + cupins + + + 0.9995135 + protein_type + cleaner0 + 2023-07-20T14:18:01Z + MESH: + + cupin-related proteins + + + 0.99931794 + protein_type + cleaner0 + 2023-07-20T14:18:10Z + MESH: + + metallo-chaperones + + + 0.97990215 + protein_state + cleaner0 + 2023-07-20T14:50:18Z + DUMMY: + + bound + + + 0.98385483 + chemical + cleaner0 + 2023-07-20T13:55:09Z + CHEBI: + + metal + + + 0.99956536 + site + cleaner0 + 2023-07-20T14:39:38Z + SO: + + active sites + + + 0.9996103 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.9995976 + experimental_method + cleaner0 + 2023-07-20T13:55:26Z + MESH: + + ICP-MS + + + 0.99949604 + experimental_method + cleaner0 + 2023-07-20T13:55:31Z + MESH: + + metal-depletion and reconstitution experiments + + + 0.99902046 + chemical + cleaner0 + 2023-07-20T11:22:02Z + CHEBI: + + iron + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + 0.99965394 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.9996189 + protein_type + cleaner0 + 2023-07-20T11:22:39Z + MESH: + + cupins + + + 0.9996886 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + + DISCUSS + paragraph + 67707 + Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1). The architecture of the metal center of ectoine synthase seems to be subjected to considerable evolutionary constraints. The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2). + + chemical + CHEBI: + cleaner0 + 2023-07-20T13:55:10Z + + metal + + + species + MESH: + cleaner0 + 2023-07-20T10:07:00Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:44Z + + EctC + + + 0.99939793 + experimental_method + cleaner0 + 2023-07-20T13:55:45Z + MESH: + + structure-guided site-directed mutagenesis + + + 0.9995793 + site + cleaner0 + 2023-07-20T13:55:55Z + SO: + + iron-binding residues + + + 0.99940354 + site + cleaner0 + 2023-07-20T13:56:05Z + SO: + + metal center + + + 0.9993664 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.99930215 + residue_name_number + cleaner0 + 2023-07-20T13:32:58Z + DUMMY: + + Glu-57 + + + 0.9993043 + residue_name_number + cleaner0 + 2023-07-20T13:33:03Z + DUMMY: + + Tyr-85 + + + 0.99931043 + residue_name_number + cleaner0 + 2023-07-20T13:31:20Z + DUMMY: + + His-93 + + + 0.9994848 + protein_state + cleaner0 + 2023-07-20T13:56:17Z + DUMMY: + + strictly conserved + + + 0.9996911 + protein_type + cleaner0 + 2023-07-20T11:24:54Z + MESH: + + EctC-type proteins + + + 0.99945575 + taxonomy_domain + cleaner0 + 2023-07-20T14:21:50Z + DUMMY: + + bacterial + + + 0.99945897 + taxonomy_domain + cleaner0 + 2023-07-20T14:21:55Z + DUMMY: + + archaeal + + + + DISCUSS + paragraph + 68362 + We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig). Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules. Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route. + + 0.9997269 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.95461667 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.99970835 + chemical + cleaner0 + 2023-07-20T10:24:53Z + CHEBI: + + N-α-ADABA + + + 0.99932873 + chemical + cleaner0 + 2023-07-20T10:07:54Z + CHEBI: + + ectoine + + + evidence + DUMMY: + cleaner0 + 2023-07-20T11:31:33Z + + catalytic efficiency + + + 0.9995589 + site + cleaner0 + 2023-07-20T13:43:15Z + SO: + + active site + + + 0.99962765 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.99893576 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.99979264 + chemical + cleaner0 + 2023-07-20T10:18:49Z + CHEBI: + + ADPC + + + 0.9997565 + chemical + cleaner0 + 2023-07-20T13:56:30Z + CHEBI: + + glutamine + + + 0.9997123 + chemical + cleaner0 + 2023-07-20T10:24:53Z + CHEBI: + + N-α-ADABA + + + 0.99957216 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.99946934 + taxonomy_domain + cleaner0 + 2023-07-20T11:32:08Z + DUMMY: + + microorganisms + + + 0.99968433 + chemical + cleaner0 + 2023-07-20T10:07:54Z + CHEBI: + + ectoine + + + 0.9997078 + chemical + cleaner0 + 2023-07-20T10:24:53Z + CHEBI: + + N-α-ADABA + + + + DISCUSS + paragraph + 69193 + Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a). We assumed that its location and mode of binding gives, in all likelihood, clues as to the position of the true substrate N-γ-ADABA within the EctC active site. Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1). This set of data and the fact that the targeted residues are strongly conserved among EctC-type proteins (Fig 2) is consistent with their potential role in N-γ-ADABA binding or enzyme catalysis. We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b). + + 0.94073266 + chemical + cleaner0 + 2023-07-20T14:20:24Z + CHEBI: + + C6 + + + species + MESH: + cleaner0 + 2023-07-20T10:07:00Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:44Z + + EctC + + + 0.9991386 + evidence + cleaner0 + 2023-07-20T13:56:52Z + DUMMY: + + structure + + + protein_type + MESH: + cleaner0 + 2023-07-20T10:07:30Z + + ectoine synthase + + + 0.999745 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.9959065 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.99957526 + site + cleaner0 + 2023-07-20T13:43:15Z + SO: + + active site + + + 0.99945617 + experimental_method + cleaner0 + 2023-07-20T14:36:44Z + MESH: + + site-directed mutagenesis + + + species + MESH: + cleaner0 + 2023-07-20T10:07:00Z + + Sa + + + protein + PR: + cleaner0 + 2023-07-20T10:09:44Z + + EctC + + + 0.98933226 + protein_state + cleaner0 + 2023-07-20T13:41:17Z + DUMMY: + + wild-type + + + 0.9982116 + chemical + cleaner0 + 2023-07-20T11:22:02Z + CHEBI: + + iron + + + 0.9994867 + protein_state + cleaner0 + 2023-07-20T13:57:10Z + DUMMY: + + strongly conserved + + + 0.9996275 + protein_type + cleaner0 + 2023-07-20T11:24:54Z + MESH: + + EctC-type proteins + + + 0.9997193 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.999475 + evidence + cleaner0 + 2023-07-20T13:53:41Z + DUMMY: + + crystallographic data + + + 0.9749029 + experimental_method + cleaner0 + 2023-07-20T14:36:48Z + MESH: + + site-directed mutagenesis study + + + 0.99662185 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.99958104 + site + cleaner0 + 2023-07-20T13:43:15Z + SO: + + active site + + + + DISCUSS + paragraph + 70236 + The ectoine synthase from the cold-adapted marine bacterium S. alaskensis can be considered as a psychrophilic enzyme (S3a Fig), types of proteins with a considerable structural flexibility. This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine. Because microbial ectoine producers can colonize ecological niches with rather different physicochemical attributes, it seems promising to exploit this considerable biodiversity to identify EctC proteins with enhanced protein stability. It is hoped that these can be further employed to obtain EctC crystal structures with either the substrate or the reaction product. Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction. + + 0.99967456 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-20T10:19:51Z + + marine bacterium + + + 0.9993682 + species + cleaner0 + 2023-07-20T10:21:25Z + MESH: + + S. alaskensis + + + 0.9996017 + evidence + cleaner0 + 2023-07-20T13:57:50Z + DUMMY: + + crystal structures + + + 0.99951786 + protein_state + cleaner0 + 2023-07-20T14:50:23Z + DUMMY: + + full-length + + + 0.9908317 + species + cleaner0 + 2023-07-20T10:07:00Z + MESH: + + Sa + + + 0.9994854 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.99932766 + protein_state + cleaner0 + 2023-07-20T13:57:27Z + DUMMY: + + in complex with + + + 0.9996761 + chemical + cleaner0 + 2023-07-20T10:18:35Z + CHEBI: + + N-γ-ADABA + + + 0.85791844 + chemical + cleaner0 + 2023-07-20T10:07:54Z + CHEBI: + + ectoine + + + 0.99936575 + taxonomy_domain + cleaner0 + 2023-07-20T13:54:44Z + DUMMY: + + microbial + + + 0.9690008 + chemical + cleaner0 + 2023-07-20T10:07:54Z + CHEBI: + + ectoine + + + 0.99842644 + protein_type + cleaner0 + 2023-07-20T14:18:15Z + MESH: + + EctC proteins + + + 0.9996333 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + 0.99962413 + evidence + cleaner0 + 2023-07-20T13:57:49Z + DUMMY: + + crystal structures + + + 0.99965763 + protein_type + cleaner0 + 2023-07-20T10:07:30Z + MESH: + + ectoine synthase + + + 0.99684787 + protein_state + cleaner0 + 2023-07-20T14:50:28Z + DUMMY: + + metal dependent + + + 0.9996215 + evidence + cleaner0 + 2023-07-20T13:57:50Z + DUMMY: + + crystal structures + + + 0.99962616 + protein + cleaner0 + 2023-07-20T10:09:44Z + PR: + + EctC + + + + SUPPL + title_1 + 71175 + Supporting Information + + + REF + title + 71198 + References + + + 1 + 24 + surname:Yancey;given-names:PH + 15651637 + REF + Sci Prog + ref + 87 + 2004 + 71209 + Compatible and counteracting solutes: protecting cells from the Dead Sea to the deep sea + + + 319 + 330 + surname:Kempf;given-names:B + surname:Bremer;given-names:E + 9818351 + REF + Arch Microbiol + ref + 170 + 1998 + 71298 + Uptake and synthesis of compatible solutes as microbial stress responses to high osmolality environments + + + 121 + 147 + surname:Csonka;given-names:LN + 2651863 + REF + Microbiol Rev + ref + 53 + 1989 + 71403 + Physiological and genetic responses of bacteria to osmotic stress + + + 7309 + 7313 + surname:Burg;given-names:MB + surname:Ferraris;given-names:JD + 10.1074/jbc.R700042200 + 18256030 + REF + J Biol Chem + ref + 283 + 2008 + 71469 + Intracellular organic osmolytes: function and regulation + + + 743 + 754 + surname:Roeßler;given-names:M + surname:Müller;given-names:V + REF + Env Microbiol Rep + ref + 3 + 2001 + 71526 + Osmoadaptation in bacteria and archaea: common principles and differences + + + 13997 + 14002 + surname:Street;given-names:TO + surname:Bolen;given-names:DW + surname:Rose;given-names:GD + 16968772 + REF + Proc Natl Acad Sci U S A + ref + 103 + 2006 + 71600 + A molecular mechanism for osmolyte-induced protein stability + + + 143 + 148 + surname:Record;given-names:MT;suffix:Jr + surname:Courtenay;given-names:ES + surname:Cayley;given-names:DS + surname:Guttman;given-names:HJ + 9584618 + REF + Trends Biochem Sci + ref + 23 + 1998 + 71661 + Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water + + + 215 + 238 + surname:Wood;given-names:JM + 10.1146/annurev-micro-090110-102815 + 21663439 + REF + Annu Rev Microbiol + ref + 65 + 2011 + 71759 + Bacterial osmoregulation: a paradigm for the study of cellular homeostasis + + + 12596 + 12609 + surname:Cayley;given-names:S + surname:Record;given-names:MT;suffix:Jr + 14580206 + REF + Biochemistry + ref + 42 + 2003 + 71834 + Roles of cytoplasmic osmolytes, water, and crowding in the response of Escherichia coli to osmotic stress: biophysical basis of osmoprotection by glycine betaine + + + 135 + 139 + surname:Galinski;given-names:EA + surname:Pfeiffer;given-names:HP + surname:Trüper;given-names:HG + 3838936 + REF + Eur J Biochem + ref + 149 + 1985 + 71996 + 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira + + + 16014 + 16022 + surname:Inbar;given-names:L + surname:Lapidot;given-names:A + 2903148 + REF + J Biol Chem + ref + 263 + 1988 + 72149 + The structure and biosynthesis of new tetrahydropyrimidine derivatives in actinomycin D producer Streptomyces parvulus. Use of 13C- and 15N-labeled L-glutamate and 13C and 15N NMR spectroscopy + + + 782 + 801 + surname:Pastor;given-names:JM + surname:Salvador;given-names:M + surname:Argandona;given-names:M + surname:Bernal;given-names:V + surname:Reina-Bueno;given-names:M + surname:Csonka;given-names:LN + 10.1016/j.biotechadv.2010.06.005 + 20600783 + REF + Biotechnol Adv + ref + 28 + 2010 + 72342 + Ectoines in cell stress protection: uses and biotechnological production + + + e93809 + surname:Widderich;given-names:N + surname:Hoppner;given-names:A + surname:Pittelkow;given-names:M + surname:Heider;given-names:J + surname:Smits;given-names:SH + surname:Bremer;given-names:E + 10.1371/journal.pone.0093809 + 24714029 + REF + PLoS One + ref + 9 + 2014 + 72415 + Biochemical properties of ectoine hydroxylases from extremophiles and their wider taxonomic distribution among microorganisms + + + surname:Widderich;given-names:N + surname:Czech;given-names:L + surname:Elling;given-names:FJ + surname:Könneke;given-names:M + surname:Stöveken;given-names:N + surname:Pittelkow;given-names:M + 10.1111/1462-2920.13156 + REF + Env Microbiol + ref + 2016 + 72541 + Strangers in the archaeal world: osmostress-responsive biosynthesis of ectoine and hydroxyectoine by the marine thaumarchaeon Nitrosopumilus maritimus + + + 772 + 783 + surname:Kuhlmann;given-names:AU + surname:Bremer;given-names:E + 11823218 + REF + Appl Environ Microbiol + ref + 68 + 2002 + 72692 + Osmotically regulated synthesis of the compatible solute ectoine in Bacillus pasteurii and related Bacillus spp + + + 4560 + 4563 + surname:Kuhlmann;given-names:AU + surname:Bursy;given-names:J + surname:Gimpel;given-names:S + surname:Hoffmann;given-names:T + surname:Bremer;given-names:E + 10.1128/AEM.00492-08 + 18487398 + REF + Appl Environ Microbiol + ref + 74 + 2008 + 72804 + Synthesis of the compatible solute ectoine in Virgibacillus pantothenticus is triggered by high salinity and low growth temperature + + + 14 + surname:Vargas;given-names:C + surname:Argandona;given-names:M + surname:Reina-Bueno;given-names:M + surname:Rodriguez-Moya;given-names:J + surname:Fernandez-Aunion;given-names:C + surname:Nieto;given-names:JJ + 10.1186/1746-1448-4-14 + 18793408 + REF + Saline Systems + ref + 4 + 2008 + 72936 + Unravelling the adaptation responses to osmotic and temperature stress in Chromohalobacter salexigens, a bacterium with broad salinity tolerance + + + 594 + 651 + surname:Lo;given-names:CC + surname:Bonner;given-names:CA + surname:Xie;given-names:G + surname:D'Souza;given-names:M + surname:Jensen;given-names:RA + 10.1128/MMBR.00024-09 + 19946135 + REF + Microbiol Mol Biol Rev + ref + 73 + 2009 + 73081 + Cohesion group approach for evolutionary analysis of aspartokinase, an enzyme that feeds a branched network of many biochemical pathways + + + 4456 + 4468 + surname:Stöveken;given-names:N + surname:Pittelkow;given-names:M + surname:Sinner;given-names:T + surname:Jensen;given-names:RA + surname:Heider;given-names:J + surname:Bremer;given-names:E + 10.1128/JB.00345-11 + 21725014 + REF + J Bacteriol + ref + 193 + 2011 + 73218 + A specialized aspartokinase enhances the biosynthesis of the osmoprotectants ectoine and hydroxyectoine in Pseudomonas stutzeri A1501 + + + 91 + 99 + surname:Ono;given-names:H + surname:Sawada;given-names:K + surname:Khunajakr;given-names:N + surname:Tao;given-names:T + surname:Yamamoto;given-names:M + surname:Hiramoto;given-names:M + 9864317 + REF + J Bacteriol + ref + 181 + 1999 + 73352 + Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata + + + 157 + 162 + surname:Peters;given-names:P + surname:Galinski;given-names:EA + surname:Trüper;given-names:HG + REF + The biosyntheis of ectoine FEMS Microbiol Lett + ref + 71 + 1990 + 73487 + + + 150 + surname:Tanne;given-names:C + surname:Golovina;given-names:EA + surname:Hoekstra;given-names:FA + surname:Meffert;given-names:A + surname:Galinski;given-names:EA + 10.3389/fmicb.2014.00150 + 24772110 + REF + Front Microbiol + ref + 5 + 2014 + 73488 + Glass-forming property of hydroxyectoine is the cause of its superior function as a dessication protectant + + + 7286 + 7296 + surname:Bursy;given-names:J + surname:Kuhlmann;given-names:AU + surname:Pittelkow;given-names:M + surname:Hartmann;given-names:H + surname:Jebbar;given-names:M + surname:Pierik;given-names:AJ + 10.1128/AEM.00768-08 + 18849444 + REF + Appl Environ Microbiol + ref + 74 + 2008 + 73595 + Synthesis and uptake of the compatible solutes ectoine and 5-hydroxyectoine by Streptomyces coelicolor A3(2) in response to salt and heat stresses + + + 3774 + 3784 + surname:Garcia-Estepa;given-names:R + surname:Argandona;given-names:M + surname:Reina-Bueno;given-names:M + surname:Capote;given-names:N + surname:Iglesias-Guerra;given-names:F + surname:Nieto;given-names:JJ + 16707670 + REF + J Bacteriol + ref + 188 + 2006 + 73742 + The ectD gene, which is involved in the synthesis of the compatible solute hydroxyectoine, is essential for thermoprotection of the halophilic bacterium Chromohalobacter salexigens + + + 31147 + 31155 + surname:Bursy;given-names:J + surname:Pierik;given-names:AJ + surname:Pica;given-names:N + surname:Bremer;given-names:E + 17636255 + REF + J Biol Chem + ref + 282 + 2007 + 73923 + Osmotically induced synthesis of the compatible solute hydroxyectoine is mediated by an evolutionarily conserved ectoine hydroxylase + + + 586 + 600 + surname:Widderich;given-names:N + surname:Pittelkow;given-names:M + surname:Höppner;given-names:A + surname:Mulnaes;given-names:D + surname:Buckel;given-names:W + surname:Gohlke;given-names:H + 10.1016/j.jmb.2013.10.028 + 24184278 + REF + J Mol Biol + ref + 426 + 2014 + 74056 + Molecular dynamics simulations and structure-guided mutagenesis provide insight into the architecture of the catalytic core of the ectoine hydroxylase + + + 29570 + 29583 + surname:Höppner;given-names:A + surname:Widderich;given-names:N + surname:Lenders;given-names:M + surname:Bremer;given-names:E + surname:Smits;given-names:SHJ + 10.1074/jbc.M114.576769 + 25172507 + REF + J Biol Chem + ref + 289 + 2014 + 74207 + Crystal structure of the ectoine hydroxylase, a snapshot of the active site + + + 61 + 65 + surname:Lippert;given-names:K + surname:Galinski;given-names:EA + REF + Appl Microbial Biotechnol + ref + 37 + 1992 + 74283 + Enzyme stabilization by ectoine-type compatible solutes: protection against heating, freezing and drying + + + 6 + surname:Kurz;given-names:M + 10.1186/1746-1448-4-6 + 18522725 + REF + Saline Systems + ref + 4 + 2008 + 74388 + Compatible solute influence on nucleic acids: many questions but few answers + + + 191 + 198 + surname:Knapp;given-names:S + surname:Ladenstein;given-names:R + surname:Galinski;given-names:EA + 10484175 + REF + Extremophiles + ref + 3 + 1999 + 74465 + Extrinsic protein stabilization by the naturally occurring osmolytes beta-hydroxyectoine and betaine + + + 37 + 46 + surname:Harishchandra;given-names:RK + surname:Wulff;given-names:S + surname:Lentzen;given-names:G + surname:Neuhaus;given-names:T + surname:Galla;given-names:HJ + 10.1016/j.bpc.2010.02.007 + 20206435 + REF + Biophys Chem + ref + 150 + 2010 + 74566 + The effect of compatible solute ectoines on the structural organization of lipid monolayer and bilayer membranes + + + 326 + 333 + surname:Graf;given-names:R + surname:Anzali;given-names:S + surname:Buenger;given-names:J + surname:Pfluecker;given-names:F + surname:Driller;given-names:H + 10.1016/j.clindermatol.2008.01.002 + 18691511 + REF + Clin Dermatol + ref + 26 + 2008 + 74679 + The multifunctional role of ectoine as a natural cell protectant + + + 10 + 25 + surname:Kunte;given-names:HJ + surname:Lentzen;given-names:G + surname:Galinski;given-names:E + REF + Cur Biotechnol + ref + 3 + 2014 + 74744 + Industrial production of the cell protectant ectoine: protection, mechanisms, processes, and products + + + 1141 + 1149 + surname:Louis;given-names:P + surname:Galinski;given-names:EA + 9141677 + REF + Microbiology + ref + 143 + 1997 + 74846 + Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli + + + 91 + 99 + surname:Ono;given-names:H + surname:Sawada;given-names:K + surname:Khunajakr;given-names:N + surname:Tao;given-names:T + surname:Yamamoto;given-names:M + surname:Hiramoto;given-names:M + 9864317 + REF + J Bacteriol + ref + 181 + 1999 + 75004 + Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata + + + 113 + 122 + surname:Witt;given-names:EM + surname:Davies;given-names:NW + surname:Galinski;given-names:EA + 10.1007/s00253-011-3211-9 + 21468713 + REF + Appl Microbiol Biotechnol + ref + 91 + 2011 + 75139 + Unexpected property of ectoine synthase and its application for synthesis of the engineered compatible solute ADPC + + + 87 + 99 + surname:Moritz;given-names:KD + surname:Amendt;given-names:B + surname:Witt;given-names:EMHJ + surname:Galinski;given-names:EA + 10.1007/s00792-014-0687-0 + 25142158 + REF + Extremophiles + ref + 19 + 2015 + 75254 + The hydroxyectoine gene cluster of the non-halophilic acidophile Acidiphilium cryptum + + + 286 + 297 + surname:Reshetnikov;given-names:AS + surname:Khmelenina;given-names:VN + surname:Trotsenko;given-names:YA + 16283251 + REF + Arch Microbiol + ref + 184 + 2006 + 75340 + Characterization of the ectoine biosynthesis genes of haloalkalotolerant obligate methanotroph "Methylomicrobium alcaliphilum 20Z" + + + 129 + 136 + surname:Kunte;given-names:HJ + surname:Galinski;given-names:EA + surname:Trüper;given-names:GH + REF + J Microbiol Meth + ref + 17 + 1993 + 75471 + A modified FMOC-method for the detection of amino acid-type osmolytes and tetrahydropyrimidines (ectoines) + + + 2658 + 2676 + surname:Ting;given-names:L + surname:Williams;given-names:TJ + surname:Cowley;given-names:MJ + surname:Lauro;given-names:FM + surname:Guilhaus;given-names:M + surname:Raftery;given-names:MJ + 10.1111/j.1462-2920.2010.02235.x + 20482592 + REF + Environ Microbiol + ref + 12 + 2010 + 75578 + Cold adaptation in the marine bacterium, Sphingopyxis alaskensis, assessed using quantitative proteomics + + + 1027 + 1032 + surname:Kobus;given-names:S + surname:Widderich;given-names:N + surname:Hoeppner;given-names:A + surname:Bremer;given-names:E + surname:Smits;given-names:SHJ + REF + Acta Cryst + ref + F71 + 2015 + 75683 + Overproduction, crystallization and X-ray diffraction data analysis of ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis + + + surname:Miller;given-names:JH + REF + Experiments in molecular genetics + ref + 1972 + 75834 + + + 3899 + 3913 + surname:Lovenberg;given-names:W + surname:Buchanan;given-names:BB + surname:Rabinowitz;given-names:JC + 14086723 + REF + J Biol Chem + ref + 238 + 1963 + 75835 + Studies on the chemical nature of Clostridial ferredoxin + + + 125 + 132 + surname:Kabsch;given-names:W + 10.1107/S0907444909047337 + 20124692 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 75892 + XDS + + + 133 + 144 + surname:Kabsch;given-names:W + 10.1107/S0907444909047374 + 20124693 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 75896 + Integration, scaling, space-group assignment and post-refinement + + + 1089 + 1097 + surname:Panjikar;given-names:S + surname:Parthasarathy;given-names:V + surname:Lamzin;given-names:VS + surname:Weiss;given-names:MS + surname:Tucker;given-names:PA + 10.1107/S0907444909029643 + 19770506 + REF + Acta Crystallogr D Biol Crystallogr + ref + 65 + 2009 + 75961 + On the combination of molecular replacement and single-wavelength anomalous diffraction phasing for automated structure determination + + + 352 + 367 + surname:Afonine;given-names:PV + surname:Grosse-Kunstleve;given-names:RW + surname:Echols;given-names:N + surname:Headd;given-names:JJ + surname:Moriarty;given-names:NW + surname:Mustyakimov;given-names:M + 10.1107/S0907444912001308 + 22505256 + REF + Acta Crystallogr D Biol Crystallogr + ref + 68 + 2012 + 76095 + Towards automated crystallographic structure refinement with phenix.refine + + + 486 + 501 + surname:Emsley;given-names:P + surname:Lohkamp;given-names:B + surname:Scott;given-names:WG + surname:Cowtan;given-names:K + 10.1107/S0907444910007493 + 20383002 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 76170 + Features and development of Coot + + + 355 + 367 + surname:Murshudov;given-names:GN + surname:Skubak;given-names:P + surname:Lebedev;given-names:AA + surname:Pannu;given-names:NS + surname:Steiner;given-names:RA + surname:Nicholls;given-names:RA + 10.1107/S0907444911001314 + 21460454 + REF + Acta Crystallogr D Biol Crystallogr + ref + 67 + 2011 + 76203 + REFMAC5 for the refinement of macromolecular crystal structures + + + surname:Delano;given-names:WL + REF + The PyMol molecular graphics system + ref + 2002 + 76267 + + + 1973 + 1994 + surname:Schwibbert;given-names:K + surname:Marin-Sanguino;given-names:A + surname:Bagyan;given-names:I + surname:Heidrich;given-names:G + surname:Lentzen;given-names:G + surname:Seitz;given-names:H + 10.1111/j.1462-2920.2010.02336.x + 20849449 + REF + Environ Microbiol + ref + 13 + 2011 + 76268 + A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T + + + 740 + 746 + surname:Dunwell;given-names:JM + surname:Culham;given-names:A + surname:Carter;given-names:CE + surname:Sosa-Aguirre;given-names:CR + surname:Goodenough;given-names:PW + 11738598 + REF + Trends Biochem Sci + ref + 26 + 2001 + 76374 + Evolution of functional diversity in the cupin superfamily + + + e74477 + surname:Uberto;given-names:R + surname:Moomaw;given-names:EW + 10.1371/journal.pone.0074477 + 24040257 + REF + PLoS One + ref + 8 + 2013 + 76433 + Protein similarity networks reveal relationships among sequence, structure, and function within the Cupin superfamily + + + 7 + 17 + surname:Dunwell;given-names:JM + surname:Purvis;given-names:A + surname:Khuri;given-names:S + 14697267 + REF + Phytochemistry + ref + 65 + 2004 + 76551 + Cupins: the most functionally diverse protein superfamily? + + + e5736 + surname:Agarwal;given-names:G + surname:Rajavel;given-names:M + surname:Gopal;given-names:B + surname:Srinivasan;given-names:N + 10.1371/journal.pone.0005736 + 19478949 + REF + PLoS One + ref + 4 + 2009 + 76610 + Structure-based phylogeny as a diagnostic for functional characterization of proteins with a cupin fold + + + 2917 + 2921 + surname:Silvennoinen;given-names:L + surname:Sandalova;given-names:T + surname:Schneider;given-names:G + 10.1016/j.febslet.2009.07.061 + 19665022 + REF + FEBS Lett + ref + 583 + 2009 + 76714 + The polyketide cyclase RemF from Streptomyces resistomycificus contains an unusual octahedral zinc binding site + + + 5815 + 5828 + surname:Hajnal;given-names:I + surname:Lyskowski;given-names:A + surname:Hanefeld;given-names:U + surname:Gruber;given-names:K + surname:Schwab;given-names:H + surname:Steiner;given-names:K + 10.1111/febs.12501 + 23981508 + REF + FEBS J + ref + 280 + 2013 + 76826 + Biochemical and structural characterization of a novel bacterial manganese-dependent hydroxynitrile lyase + + + 193 + 201 + surname:Gopal;given-names:B + surname:Madan;given-names:LL + surname:Betz;given-names:SF + surname:Kossiakoff;given-names:AA + 15628860 + REF + Biochemistry + ref + 44 + 2005 + 76932 + The crystal structure of a quercetin 2,3-dioxygenase from Bacillus subtilis suggests modulation of enzyme activity by a change in the metal ion at the active site(s) + + + W545 + 549 + surname:Holm;given-names:L + surname:Rosenström;given-names:P + 10.1093/nar/gkq366 + 20457744 + REF + Nucleic Acids Res + ref + 38 + 2010 + 77098 + Dali server: conservation mapping in 3D + + + 611 + 614 + surname:Jaroszewski;given-names:L + surname:Schwarzenbacher;given-names:R + surname:von Delft;given-names:F + surname:McMullan;given-names:D + surname:Brinen;given-names:LS + surname:Canaves;given-names:JM + 15229893 + REF + Proteins + ref + 56 + 2004 + 77138 + Crystal structure of a novel manganese-containing cupin (TM1459) from Thermotoga maritima at 1.65 A resolution + + + 2877 + 2885 + surname:Woo;given-names:EJ + surname:Marshall;given-names:J + surname:Bauly;given-names:J + surname:Chen;given-names:JG + surname:Venis;given-names:M + surname:Napier;given-names:RM + 12065401 + REF + EMBO J + ref + 21 + 2002 + 77249 + Crystal structure of auxin-binding protein 1 in complex with auxin + + + 279 + 293 + surname:Claesson;given-names:M + surname:Lindqvist;given-names:Y + surname:Madrid;given-names:S + surname:Sandalova;given-names:T + surname:Fiskesund;given-names:R + surname:Yu;given-names:S + 10.1016/j.jmb.2012.02.001 + 22330145 + REF + J Mol Biol + ref + 417 + 2012 + 77316 + Crystal structure of bifunctional aldos-2-ulose dehydratase/isomerase from Phanerochaete chrysosporium with the reaction intermediate ascopyrone M + + + 774 + 797 + surname:Krissinel;given-names:E + surname:Henrick;given-names:K + 17681537 + REF + J Mol Biol + ref + 372 + 2007 + 77463 + Inference of macromolecular assemblies from crystalline state + + + 1091 + 1097 + surname:Link;given-names:H + surname:Fuhrer;given-names:T + surname:Gerosa;given-names:L + surname:Zamboni;given-names:N + surname:Sauer;given-names:U + REF + Nature Meth + ref + 12 + 2015 + 77525 + Real-time metabolome profiling of the metabolic switch between starvation and growth + + + 1138 + 1142 + surname:Tottey;given-names:S + surname:Waldron;given-names:KJ + surname:Firbank;given-names:SJ + surname:Reale;given-names:B + surname:Bessant;given-names:C + surname:Sato;given-names:K + 10.1038/nature07340 + 18948958 + REF + Nature + ref + 455 + 2008 + 77610 + Protein-folding location can regulate manganese-binding versus copper- or zinc-binding + + + 2505 + 2514 + surname:Fetzner;given-names:S + 10.1128/AEM.07651-11 + 22287012 + REF + Appl Environ Microbiol + ref + 78 + 2012 + 77697 + Ring-cleaving dioxygenases with a cupin fold + + + 3150 + 3164 + surname:Yoshida;given-names:H + surname:Yoshihara;given-names:A + surname:Teraoka;given-names:M + surname:Terami;given-names:Y + surname:Takata;given-names:G + surname:Izumori;given-names:K + 10.1111/febs.12850 + 24846739 + REF + FEBS J + ref + 281 + 2014 + 77742 + X-ray structure of a novel L-ribose isomerase acting on a non-natural sugar L-ribose as its ideal substrate + + + 1 + 7 + surname:Elias;given-names:M + surname:Wieczorek;given-names:G + surname:Rosenne;given-names:S + surname:Tawfik;given-names:DS + 10.1016/j.tibs.2013.11.001 + 24315123 + REF + Trends Biochem Sci + ref + 39 + 2014 + 77850 + The universality of enzymatic rate-temperature dependency + + + 25 + 42 + surname:Georlette;given-names:D + surname:Blaise;given-names:V + surname:Collins;given-names:T + surname:D'Amico;given-names:S + surname:Gratia;given-names:E + surname:Hoyoux;given-names:A + 14975528 + REF + FEMS Microbiol Rev + ref + 28 + 2004 + 77908 + Some like it cold: biocatalysis at low temperatures + + + 362 + 368 + surname:Ono;given-names:H + surname:Okuda;given-names:M + surname:Tongpim;given-names:S + surname:Imai;given-names:K + surname:Shinmyo;given-names:A + surname:Sakuda;given-names:S + REF + J Ferment Bioeng + ref + 85 + 1998 + 77960 + Accumulation of compatible solutes, ectoine and hydroxyectoine, in a moderate halophile, Halomonas elongata KS3, isolated from dry salty land in Thailand + + + diff --git a/BioC_XML/4802042_v0.xml b/BioC_XML/4802042_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..d310e80b44d0b980bb7493ad4a1261fe1bc71c91 --- /dev/null +++ b/BioC_XML/4802042_v0.xml @@ -0,0 +1,14246 @@ + + + + PMC + 20201215 + pmc.key + + 4802042 + CC BY + no + 0 + 0 + + 10.1038/ncomms10879 + ncomms10879 + 4802042 + 26988444 + 10879 + This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ + surname:Liu;given-names:Xin + surname:Zhang;given-names:Chen-Song + surname:Lu;given-names:Chang + surname:Lin;given-names:Sheng-Cai + surname:Wu;given-names:Jia-Wei + surname:Wang;given-names:Zhi-Xin + TITLE + front + 7 + 2016 + 0 + A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation + + 0.99966806 + protein_type + cleaner0 + 2023-07-11T20:07:53Z + MESH: + + JNK/p38-specific MAPK phosphatases + + + 0.99971896 + protein + cleaner0 + 2023-07-11T14:03:57Z + PR: + + JNK1 + + + + ABSTRACT + abstract + 111 + Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs). MAPK signalling efficiency and specificity is modulated by protein–protein interactions between individual MAPKs and the docking motifs in cognate binding partners. Two types of docking interactions have been identified: D-motif-mediated interaction and FXF-docking interaction. Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-Å resolution, providing high-resolution structural insight into the FXF-docking interaction. The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. + + 0.99968576 + protein_type + cleaner0 + 2023-07-11T14:02:48Z + MESH: + + Mitogen-activated protein kinases + + + 0.9995889 + protein_type + cleaner0 + 2023-07-11T14:03:00Z + MESH: + + MAPKs + + + 0.9994999 + protein_type + cleaner0 + 2023-07-11T14:03:07Z + MESH: + + protein kinases + + + 0.99964887 + protein_type + cleaner0 + 2023-07-11T14:03:13Z + MESH: + + MAPK phosphatases + + + 0.99966013 + protein_type + cleaner0 + 2023-07-11T14:03:19Z + MESH: + + MKPs + + + 0.99961084 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + 0.99963725 + protein_type + cleaner0 + 2023-07-11T14:03:01Z + MESH: + + MAPKs + + + 0.9891355 + structure_element + cleaner0 + 2023-07-11T14:06:52Z + SO: + + docking motifs + + + 0.99399155 + structure_element + cleaner0 + 2023-07-11T14:07:23Z + SO: + + D-motif + + + 0.99577826 + site + cleaner0 + 2023-07-11T19:44:49Z + SO: + + FXF-docking interaction + + + 0.99961877 + evidence + cleaner0 + 2023-07-12T14:40:02Z + DUMMY: + + crystal structure + + + 0.9997868 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.99941844 + protein_state + cleaner0 + 2023-07-12T14:47:24Z + DUMMY: + + bound to + + + 0.9996116 + structure_element + cleaner0 + 2023-07-11T20:18:43Z + SO: + + catalytic domain + + + 0.9998472 + protein + cleaner0 + 2023-07-11T14:04:12Z + PR: + + MKP7 + + + 0.9984511 + site + cleaner0 + 2023-07-11T19:44:50Z + SO: + + FXF-docking interaction + + + 0.94341755 + structure_element + cleaner0 + 2023-07-11T19:45:10Z + SO: + + 285FNFL288 segment + + + 0.9998379 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9994817 + site + cleaner0 + 2023-07-11T19:44:57Z + SO: + + hydrophobic site + + + 0.9998085 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + protein_type + MESH: + cleaner0 + 2023-07-11T14:04:43Z + + MAPK + + + 0.9996178 + structure_element + cleaner0 + 2023-07-11T20:18:49Z + SO: + + helix + + + 0.9984957 + structure_element + cleaner0 + 2023-07-11T20:18:52Z + SO: + + αG + + + 0.99952626 + experimental_method + cleaner0 + 2023-07-12T14:52:10Z + MESH: + + Biochemical studies + + + 0.999537 + protein_state + cleaner0 + 2023-07-12T14:47:28Z + DUMMY: + + highly conserved + + + 0.999419 + structure_element + cleaner0 + 2023-07-11T20:18:57Z + SO: + + structural motif + + + 0.99936134 + protein_type + cleaner0 + 2023-07-11T20:14:06Z + MESH: + + MKP family + + + 0.99933404 + protein_type + cleaner0 + 2023-07-11T20:14:34Z + MESH: + + MKP + + + 0.89675665 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + + ABSTRACT + abstract + 1110 + The important MAPK family of signalling proteins is controlled by MAPK phosphatases (MKPs). Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction. + + protein_type + MESH: + cleaner0 + 2023-07-11T20:14:29Z + + MAPK family + + + 0.99956846 + protein_type + cleaner0 + 2023-07-11T14:03:14Z + MESH: + + MAPK phosphatases + + + 0.99966645 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.99955636 + evidence + cleaner0 + 2023-07-12T14:40:06Z + DUMMY: + + structure + + + 0.99985623 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.99945796 + protein_state + cleaner0 + 2023-07-12T14:47:31Z + DUMMY: + + bound to + + + 0.99978846 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.9993968 + protein_state + cleaner0 + 2023-07-12T14:47:35Z + DUMMY: + + conserved + + + 0.9992163 + protein_type + cleaner0 + 2023-07-11T20:14:41Z + MESH: + + MKP + + + protein_type + MESH: + cleaner0 + 2023-07-11T14:04:43Z + + MAPK + + + + INTRO + paragraph + 1317 + The mitogen-activated protein kinases (MAPKs) are central components of the signal-transduction pathways, which mediate the cellular response to a variety of extracellular stimuli, ranging from growth factors to environmental stresses. The MAPK signalling pathways are evolutionally highly conserved. The basic assembly of MAPK pathways is a three-tier kinase module that establishes a sequential activation cascade: a MAPK kinase kinase activates a MAPK kinase, which in turn activates a MAPK. The three best-characterized MAPK signalling pathways are mediated by the kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. The ERK pathway is activated by various mitogens and phorbol esters, whereas the JNK and p38 pathways are stimulated mainly by environmental stress and inflammatory cytokines. The MAPKs are activated by MAPK kinases that phosphorylate the MAPKs at conserved threonine and tyrosine residues within their activation loop. After activation, each MAPK phosphorylates a distinct set of protein substrates, which act as the critical effectors that enable cells to mount the appropriate responses to varied stimuli. + + 0.9996603 + protein_type + cleaner0 + 2023-07-11T14:02:49Z + MESH: + + mitogen-activated protein kinases + + + 0.9995908 + protein_type + cleaner0 + 2023-07-11T14:03:01Z + MESH: + + MAPKs + + + 0.9994746 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + 0.99851507 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + protein_type + MESH: + cleaner0 + 2023-07-11T20:08:19Z + + kinase + + + 0.95697093 + protein_type + cleaner0 + 2023-07-11T14:05:44Z + MESH: + + MAPK kinase kinase + + + 0.99957675 + protein_type + cleaner0 + 2023-07-11T14:05:47Z + MESH: + + MAPK kinase + + + 0.9996008 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + 0.99944526 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + 0.9977592 + protein_type + cleaner0 + 2023-07-11T14:05:29Z + MESH: + + kinases + + + 0.9959944 + protein_type + cleaner0 + 2023-07-11T20:14:47Z + MESH: + + extracellular signal-regulated kinase + + + 0.99826115 + protein_type + cleaner0 + 2023-07-11T14:06:43Z + MESH: + + ERK + + + 0.989564 + protein_type + cleaner0 + 2023-07-11T14:06:25Z + MESH: + + c-Jun N-terminal kinase + + + 0.98444486 + protein_type + cleaner0 + 2023-07-11T14:06:29Z + MESH: + + JNK + + + 0.993874 + protein_type + cleaner0 + 2023-07-11T14:06:35Z + MESH: + + p38 + + + 0.9856688 + protein_type + cleaner0 + 2023-07-11T14:06:42Z + MESH: + + ERK + + + 0.9965958 + protein_type + cleaner0 + 2023-07-11T14:06:30Z + MESH: + + JNK + + + 0.9974468 + protein_type + cleaner0 + 2023-07-11T14:06:36Z + MESH: + + p38 + + + protein_type + MESH: + cleaner0 + 2023-07-11T20:08:29Z + + cytokines + + + 0.9995852 + protein_type + cleaner0 + 2023-07-11T14:03:01Z + MESH: + + MAPKs + + + 0.99966526 + protein_type + cleaner0 + 2023-07-11T20:14:51Z + MESH: + + MAPK kinases + + + 0.9996138 + protein_type + cleaner0 + 2023-07-11T14:03:01Z + MESH: + + MAPKs + + + 0.9992895 + protein_state + cleaner0 + 2023-07-12T14:48:09Z + DUMMY: + + conserved + + + 0.99927574 + residue_name + cleaner0 + 2023-07-11T20:32:43Z + SO: + + threonine + + + 0.9992637 + residue_name + cleaner0 + 2023-07-11T20:32:45Z + SO: + + tyrosine + + + 0.99943674 + structure_element + cleaner0 + 2023-07-11T20:19:03Z + SO: + + activation loop + + + 0.9995946 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + + INTRO + paragraph + 2487 + MAPKs lie at the bottom of conserved three-component phosphorylation cascades and utilize docking interactions to link module components and bind substrates. Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF). The best-studied docking interactions are those between MAP kinases and ‘D-motifs', which consists of two or more basic residues followed by a short linker and a cluster of hydrophobic residues. The D-motif-docking site (D-site) in MAPKs is situated in a noncatalytic region opposite of the kinase catalytic pocket and is comprised of a highly acidic patch and a hydrophobic groove. D-motifs are found in many MAPK-interacting proteins, including substrates, activating kinases and inactivating phosphatases, as well as scaffolding proteins. A second docking motif for MAPKs consists of two Phe residues separated by one residue (FXF-motif). This motif has been observed in several MAPK substrates. The FXF-motif-binding site of ERK2 has been mapped to a hydrophobic pocket formed between the P+1 site, αG helix and the MAPK insert. However, the generality and mechanism of the FXF-mediated interaction is unclear. + + 0.999374 + protein_type + cleaner0 + 2023-07-11T14:03:01Z + MESH: + + MAPKs + + + 0.7972738 + structure_element + cleaner0 + 2023-07-11T14:06:51Z + SO: + + docking motifs + + + 0.9995433 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + 0.99959016 + structure_element + cleaner0 + 2023-07-11T20:19:07Z + SO: + + kinase-interacting motif + + + 0.9996815 + structure_element + cleaner0 + 2023-07-11T14:07:22Z + SO: + + D-motif + + + 0.9996833 + structure_element + cleaner0 + 2023-07-11T14:06:57Z + SO: + + FXF-motif + + + 0.99970543 + structure_element + cleaner0 + 2023-07-11T14:07:02Z + SO: + + DEF motif + + + 0.99915403 + site + cleaner0 + 2023-07-12T14:33:02Z + SO: + + docking site + + + 0.72890854 + protein_type + cleaner0 + 2023-07-11T14:06:43Z + MESH: + + ERK + + + 0.9997389 + structure_element + cleaner0 + 2023-07-11T14:07:11Z + SO: + + FXF + + + 0.99966234 + protein_type + cleaner0 + 2023-07-11T20:14:55Z + MESH: + + MAP kinases + + + 0.99965477 + structure_element + cleaner0 + 2023-07-11T20:19:13Z + SO: + + D-motifs + + + 0.996764 + structure_element + cleaner0 + 2023-07-11T20:19:22Z + SO: + + short linker + + + 0.9980342 + site + cleaner0 + 2023-07-11T14:08:22Z + SO: + + D-motif-docking site + + + 0.999386 + site + cleaner0 + 2023-07-11T14:08:27Z + SO: + + D-site + + + 0.99951327 + protein_type + cleaner0 + 2023-07-11T14:03:01Z + MESH: + + MAPKs + + + site + SO: + cleaner0 + 2023-07-11T20:20:02Z + + noncatalytic region + + + 0.98805374 + protein_type + cleaner0 + 2023-07-11T20:15:02Z + MESH: + + kinase + + + 0.999212 + site + cleaner0 + 2023-07-12T14:33:15Z + SO: + + catalytic pocket + + + 0.99618626 + site + cleaner0 + 2023-07-12T14:33:19Z + SO: + + highly acidic patch + + + 0.99940896 + site + cleaner0 + 2023-07-12T14:33:24Z + SO: + + hydrophobic groove + + + 0.9996321 + structure_element + cleaner0 + 2023-07-11T20:20:08Z + SO: + + D-motifs + + + 0.9996097 + protein_type + cleaner0 + 2023-07-11T20:15:06Z + MESH: + + MAPK-interacting proteins + + + 0.98448354 + protein_type + cleaner0 + 2023-07-11T14:07:33Z + MESH: + + kinases + + + 0.9988788 + protein_type + cleaner0 + 2023-07-11T14:07:31Z + MESH: + + phosphatases + + + 0.9901715 + structure_element + cleaner0 + 2023-07-11T20:20:12Z + SO: + + second docking motif + + + 0.99957615 + protein_type + cleaner0 + 2023-07-11T14:03:01Z + MESH: + + MAPKs + + + 0.9990539 + residue_name + cleaner0 + 2023-07-11T19:59:38Z + SO: + + Phe + + + 0.999645 + structure_element + cleaner0 + 2023-07-11T14:06:58Z + SO: + + FXF-motif + + + 0.99953496 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + 0.9996225 + site + cleaner0 + 2023-07-11T14:07:52Z + SO: + + FXF-motif-binding site + + + 0.99954295 + protein + cleaner0 + 2023-07-11T14:16:33Z + PR: + + ERK2 + + + 0.9996133 + site + cleaner0 + 2023-07-12T14:33:29Z + SO: + + hydrophobic pocket + + + 0.99959314 + site + cleaner0 + 2023-07-12T14:33:32Z + SO: + + P+1 site + + + 0.99970084 + structure_element + cleaner0 + 2023-07-11T20:20:16Z + SO: + + αG helix + + + 0.8454261 + structure_element + cleaner0 + 2023-07-11T20:20:20Z + SO: + + MAPK insert + + + 0.99787986 + structure_element + cleaner0 + 2023-07-11T14:07:12Z + SO: + + FXF + + + + INTRO + paragraph + 3754 + The physiological outcome of MAPK signalling depends on both the magnitude and the duration of kinase activation. Downregulation of MAPK activity can be achieved through direct dephosphorylation of the phospho-threonine and/or tyrosine residues by various serine/threonine phosphatases, tyrosine phosphatases and dual-specificity phosphatases (DUSPs) termed MKPs. MKPs constitute a group of DUSPs that are characterized by their ability to dephosphorylate both phosphotyrosine and phosphoserine/phospho-threonine residues within a substrate. Dysregulated expression of MKPs has been associated with pathogenesis of various diseases, and understanding their precise recognition mechanism presents an important challenge and opportunity for drug development. + + 0.9996532 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + 0.9996407 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-07-21T14:00:06Z + + phospho-threonine and/or tyrosine + + + 0.9996766 + protein_type + cleaner0 + 2023-07-11T14:08:37Z + MESH: + + serine/threonine phosphatases + + + 0.9996767 + protein_type + cleaner0 + 2023-07-11T14:08:40Z + MESH: + + tyrosine phosphatases + + + 0.99968606 + protein_type + cleaner0 + 2023-07-11T14:08:45Z + MESH: + + dual-specificity phosphatases + + + 0.99966526 + protein_type + cleaner0 + 2023-07-11T14:08:51Z + MESH: + + DUSPs + + + 0.9996773 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.99967456 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.99966264 + protein_type + cleaner0 + 2023-07-11T14:08:52Z + MESH: + + DUSPs + + + 0.57390255 + residue_name + melaniev@ebi.ac.uk + 2023-07-21T14:00:06Z + SO: + + phosphotyrosine + + + 0.53731674 + residue_name + melaniev@ebi.ac.uk + 2023-07-21T14:00:06Z + SO: + + phosphoserine + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-07-21T14:00:06Z + + phospho-threonine + + + 0.9996741 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + + INTRO + paragraph + 4511 + Here, we present the crystal structure of JNK1 in complex with the catalytic domain of MKP7. This structure reveals the molecular mechanism underlying the docking interaction between MKP7 and JNK1. In the JNK1–MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix α5 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction. Biochemical and modelling studies further demonstrate that the molecular interactions mediate this key element for substrate recognition are highly conserved among all MKP-family members. Thus, our study reveals a hitherto unrecognized interaction mode for encoding complex target specificity among MAPK isoforms. + + 0.9996257 + evidence + cleaner0 + 2023-07-12T14:40:11Z + DUMMY: + + crystal structure + + + 0.99982554 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.99903136 + protein_state + cleaner0 + 2023-07-12T14:48:29Z + DUMMY: + + in complex with + + + 0.99969137 + structure_element + cleaner0 + 2023-07-11T20:20:25Z + SO: + + catalytic domain + + + 0.99986076 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9979746 + evidence + cleaner0 + 2023-07-12T14:40:26Z + DUMMY: + + structure + + + 0.9998572 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.99982244 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.9996434 + complex_assembly + cleaner0 + 2023-07-11T14:13:00Z + GO: + + JNK1–MKP7 + + + 0.9996366 + structure_element + cleaner0 + 2023-07-11T20:20:28Z + SO: + + hydrophobic motif + + + 0.9920007 + structure_element + cleaner0 + 2023-07-11T20:20:31Z + SO: + + 285FNFL288 + + + 0.9997838 + structure_element + cleaner0 + 2023-07-11T20:20:34Z + SO: + + helix + + + 0.9869927 + structure_element + cleaner0 + 2023-07-11T20:20:38Z + SO: + + α5 + + + 0.9998592 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.99969554 + structure_element + cleaner0 + 2023-07-11T20:20:41Z + SO: + + catalytic domain + + + 0.99961805 + site + cleaner0 + 2023-07-11T14:07:53Z + SO: + + FXF-motif-binding site + + + 0.9998104 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.9645349 + site + cleaner0 + 2023-07-12T14:33:42Z + SO: + + FXF-type docking interaction + + + 0.9995483 + experimental_method + cleaner0 + 2023-07-12T14:52:15Z + MESH: + + Biochemical and modelling studies + + + 0.99965215 + protein_type + cleaner0 + 2023-07-11T20:15:11Z + MESH: + + MKP-family members + + + 0.8800726 + protein_type + cleaner0 + 2023-07-11T20:15:14Z + MESH: + + MAPK isoforms + + + + RESULTS + title_1 + 5277 + Results + + + RESULTS + title_2 + 5285 + Interaction of JNK1 with the MKP7 catalytic domain + + 0.9998319 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.99986243 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9988241 + structure_element + cleaner0 + 2023-07-11T20:20:48Z + SO: + + catalytic domain + + + + RESULTS + paragraph + 5336 + DUSPs belong to the protein-tyrosine phosphatases (PTPase) superfamily, which is defined by the PTPase-signature motif CXXGXXR. MKPs represent a distinct subfamily within a larger group of DUSPs. In mammalian cells, the MKP subfamily includes 10 distinct catalytically active MKPs. All MKPs contain a highly conserved C-terminal catalytic domain (CD) and an N-terminal kinase-binding domain (KBD). The KBD is homologous to the rhodanese family and contains an intervening cluster of basic amino acids, which has been suggested to be important for interacting with the target MAPKs. On the basis of sequence similarity, substrate specificity and predominant subcellular localization, the MKP family can be further divided into three groups (Fig. 1). Biochemical and structural studies have revealed that the KBD of MKPs is critical for MKP3 docking to ERK2, and MKP5 binding to p38α, although their binding mechanisms are completely different. However, it is unknown if other MAPKs can interact with the KBD of their cognate phosphatases in the same manner as observed for recognition of ERK2 and p38α by their MKPs, or whether they recognize distinct docking motifs of MKPs. + + 0.99967456 + protein_type + cleaner0 + 2023-07-11T14:08:52Z + MESH: + + DUSPs + + + 0.99966884 + protein_type + cleaner0 + 2023-07-11T14:13:58Z + MESH: + + protein-tyrosine phosphatases + + + 0.9996518 + protein_type + cleaner0 + 2023-07-11T14:14:03Z + MESH: + + PTPase + + + 0.6538119 + protein_type + cleaner0 + 2023-07-11T14:14:03Z + MESH: + + PTPase + + + 0.9993011 + structure_element + cleaner0 + 2023-07-11T20:20:59Z + SO: + + CXXGXXR + + + 0.9996785 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.99966824 + protein_type + cleaner0 + 2023-07-11T14:08:52Z + MESH: + + DUSPs + + + 0.9990175 + taxonomy_domain + cleaner0 + 2023-07-11T14:14:09Z + DUMMY: + + mammalian + + + 0.9919881 + protein_type + cleaner0 + 2023-07-11T20:15:19Z + MESH: + + MKP subfamily + + + 0.99940425 + protein_state + cleaner0 + 2023-07-12T14:48:43Z + DUMMY: + + catalytically active + + + 0.99965084 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.99967396 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.9995341 + protein_state + cleaner0 + 2023-07-12T14:48:46Z + DUMMY: + + highly conserved + + + 0.99970675 + structure_element + cleaner0 + 2023-07-11T14:16:05Z + SO: + + catalytic domain + + + 0.9998092 + structure_element + cleaner0 + 2023-07-11T14:16:09Z + SO: + + CD + + + 0.9996517 + structure_element + cleaner0 + 2023-07-11T14:14:16Z + SO: + + kinase-binding domain + + + 0.9997887 + structure_element + cleaner0 + 2023-07-11T14:14:21Z + SO: + + KBD + + + 0.99979013 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9995134 + protein_type + cleaner0 + 2023-07-11T20:15:22Z + MESH: + + rhodanese family + + + 0.9996666 + protein_type + cleaner0 + 2023-07-11T14:03:01Z + MESH: + + MAPKs + + + 0.9992954 + protein_type + cleaner0 + 2023-07-11T20:14:07Z + MESH: + + MKP family + + + 0.9995157 + experimental_method + cleaner0 + 2023-07-12T14:52:20Z + MESH: + + Biochemical and structural studies + + + 0.99979573 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9996674 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.9998385 + protein + cleaner0 + 2023-07-11T14:16:26Z + PR: + + MKP3 + + + 0.99919564 + protein + cleaner0 + 2023-07-11T14:16:33Z + PR: + + ERK2 + + + 0.9998448 + protein + cleaner0 + 2023-07-11T14:16:41Z + PR: + + MKP5 + + + 0.99958605 + protein + cleaner0 + 2023-07-11T14:16:52Z + PR: + + p38α + + + 0.9996512 + protein_type + cleaner0 + 2023-07-11T14:03:01Z + MESH: + + MAPKs + + + 0.9997993 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.99967206 + protein_type + cleaner0 + 2023-07-11T20:15:28Z + MESH: + + phosphatases + + + 0.99907947 + protein + cleaner0 + 2023-07-11T14:16:33Z + PR: + + ERK2 + + + 0.99940205 + protein + cleaner0 + 2023-07-11T14:16:53Z + PR: + + p38α + + + 0.99967 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.99714094 + structure_element + cleaner0 + 2023-07-11T14:06:52Z + SO: + + docking motifs + + + 0.99968064 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + + RESULTS + paragraph + 6519 + MKP7, the biggest molecule in the MKP family, selectively inactivates JNK and p38 following stress activation. In addition to the CD and KBD, MKP7 has a long C-terminal region that contains both nuclear localization and export sequences by which MKP7 shuttles between the nucleus and the cytoplasm (Fig. 2a). To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). Figure 2b shows the variation of initial rates of the MKP7ΔC304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid. Thus, the kinetic data were analysed using the general initial velocity equation, taking substrate depletion into account: + + 0.99967444 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9996246 + protein_type + cleaner0 + 2023-07-11T20:14:07Z + MESH: + + MKP family + + + 0.8621272 + protein_type + cleaner0 + 2023-07-11T14:06:30Z + MESH: + + JNK + + + 0.9341276 + protein_type + cleaner0 + 2023-07-11T14:06:36Z + MESH: + + p38 + + + 0.9998054 + structure_element + cleaner0 + 2023-07-11T14:16:10Z + SO: + + CD + + + 0.99979025 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9998404 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.999457 + structure_element + cleaner0 + 2023-07-11T20:21:18Z + SO: + + C-terminal region + + + 0.9998524 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9995566 + structure_element + cleaner0 + 2023-07-11T20:21:21Z + SO: + + N-terminal domain + + + 0.9998579 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9997814 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + ptm + MESH: + cleaner0 + 2023-07-11T14:18:30Z + + dephosphorylation + + + 0.6796221 + evidence + cleaner0 + 2023-07-12T14:41:01Z + DUMMY: + + kinetic + + + 0.7138995 + experimental_method + cleaner0 + 2023-07-12T14:52:37Z + MESH: + + truncation + + + 0.99983287 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9971969 + mutant + cleaner0 + 2023-07-11T14:17:22Z + MESH: + + MKP7ΔC304 + + + 0.99902564 + residue_range + cleaner0 + 2023-07-12T15:00:47Z + DUMMY: + + 5–303 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:10Z + + CD + + + 0.9990709 + residue_range + cleaner0 + 2023-07-12T15:00:50Z + DUMMY: + + 156–301 + + + 0.99956316 + protein_state + cleaner0 + 2023-07-11T20:03:11Z + DUMMY: + + phosphorylated + + + 0.9997745 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T14:18:06Z + + p + + + protein + PR: + cleaner0 + 2023-07-11T14:17:56Z + + JNK1 + + + 0.8938303 + evidence + cleaner0 + 2023-07-12T14:41:12Z + DUMMY: + + variation of initial rates + + + 0.99916244 + mutant + cleaner0 + 2023-07-11T14:17:23Z + MESH: + + MKP7ΔC304 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:10Z + + CD + + + 0.99105 + protein_state + cleaner0 + 2023-07-12T14:48:56Z + DUMMY: + + phospho + + + 0.9994254 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.99984777 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:01:02Z + + p + + + protein + PR: + cleaner0 + 2023-07-11T19:46:53Z + + JNK1 + + + evidence + DUMMY: + cleaner0 + 2023-07-12T14:41:37Z + + kinetic data + + + 0.98305637 + evidence + cleaner0 + 2023-07-12T14:41:39Z + DUMMY: + + initial velocity equation + + + + RESULTS + paragraph + 7572 + The kcat and Km of the MKP7-CD (0.028 s−1 and 0.26 μM) so determined were nearly identical to those of MKP7ΔC304 (0.029 s−1 and 0.27 μM), indicating that the MKP7-KBD has no effect on enzyme catalysis. + + 0.9995908 + evidence + cleaner0 + 2023-07-12T14:41:43Z + DUMMY: + + kcat + + + 0.99959475 + evidence + cleaner0 + 2023-07-12T14:41:46Z + DUMMY: + + Km + + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:10Z + + CD + + + 0.99417454 + mutant + cleaner0 + 2023-07-11T14:17:23Z + MESH: + + MKP7ΔC304 + + + 0.6997105 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:14:22Z + + KBD + + + + RESULTS + paragraph + 7789 + We next examined the interaction of JNK1 with the CD and KBD of MKP7 by gel filtration analysis. When 3 molar equivalents of CD were mixed with 1 molar equivalent of JNK1, a significant amount of CD co-migrated with JNK1 to earlier fractions, and the excess amount of CD was eluted from the size exclusion column as a monomer, indicating stable complex formation (Fig. 2c). In contrast, no KBD–JNK1 complex was detected when 3 molar equivalents of KBD were mixed with 1 molar equivalent of JNK1. To further confirm the JNK1–MKP7-CD interaction, we performed a pull-down assay using the purified proteins. As shown in Fig. 2d, the CD of MKP7 can be pulled down by JNK1, while the KBD failed to bind to the counterpart protein. Taken together, our data indicate that the CD of MKP7, but not the KBD domain, is responsible for JNK substrate-binding and enzymatic specificity. + + 0.999777 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.99978906 + structure_element + cleaner0 + 2023-07-11T14:16:10Z + SO: + + CD + + + 0.9997913 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9998486 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9995236 + experimental_method + cleaner0 + 2023-07-12T14:52:42Z + MESH: + + gel filtration analysis + + + 0.9997826 + structure_element + cleaner0 + 2023-07-11T14:16:10Z + SO: + + CD + + + 0.999775 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.9997471 + structure_element + cleaner0 + 2023-07-11T14:16:10Z + SO: + + CD + + + 0.99950635 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.99974126 + structure_element + cleaner0 + 2023-07-11T14:16:10Z + SO: + + CD + + + 0.99936455 + oligomeric_state + cleaner0 + 2023-07-12T15:00:56Z + DUMMY: + + monomer + + + 0.995395 + complex_assembly + cleaner0 + 2023-07-11T20:33:13Z + GO: + + KBD–JNK1 + + + 0.81012785 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.99950385 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + complex_assembly + GO: + cleaner0 + 2023-07-11T19:51:19Z + + JNK1–MKP7-CD + + + 0.999572 + experimental_method + cleaner0 + 2023-07-12T14:52:53Z + MESH: + + pull-down assay + + + 0.9997962 + structure_element + cleaner0 + 2023-07-11T14:16:10Z + SO: + + CD + + + 0.999845 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9998209 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.9997838 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.99978584 + structure_element + cleaner0 + 2023-07-11T14:16:10Z + SO: + + CD + + + 0.99984205 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9997806 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.99772984 + protein_type + cleaner0 + 2023-07-11T14:06:30Z + MESH: + + JNK + + + + RESULTS + title_2 + 8674 + Crystal structure of JNK1 in complex with the MKP7-CD + + 0.99959636 + evidence + cleaner0 + 2023-07-12T14:41:51Z + DUMMY: + + Crystal structure + + + 0.99980146 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.99925405 + protein_state + cleaner0 + 2023-07-12T14:49:01Z + DUMMY: + + in complex with + + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:10Z + + CD + + + + RESULTS + paragraph + 8728 + To understand the molecular basis of JNK1 recognition by MKP7, we determined the crystal structure of unphosphorylated JNK1 in complex with the MKP7-CD (Fig. 3a, Supplementary Fig. 1a and Table 1). In the complex, JNK1 has its characteristic bilobal structure comprising an N-terminal lobe rich in β-sheet and a C-terminal lobe that is mostly α-helical. The overall folding of MKP7-CD is typical of DUSPs, with a central twisted five-stranded β-sheet surrounded by six α-helices. One side of the β-sheet is covered with two α-helices and the other is covered with four α-helices (Fig. 3b). The catalytic domain of MKP7 interacts with JNK1 through a contiguous surface area that is remote from the active site. MKP7-CD is positioned onto the JNK1 molecule so that the active site of the phosphatase faces towards the activation segment. In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). The most striking difference is that helix α0 and loop α0–β1 of VHR are absent in MKP7-CD. Another region that cannot be aligned with VHR is found in loop β3–β4. This loop is shortened by nine residues in MKP7-CD compared with that in VHR. Since helix α0 and the following loop α0–β1 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7. + + 0.9998361 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.999859 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9996276 + evidence + cleaner0 + 2023-07-12T14:41:54Z + DUMMY: + + crystal structure + + + 0.999602 + protein_state + cleaner0 + 2023-07-12T14:49:04Z + DUMMY: + + unphosphorylated + + + 0.999816 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.9991393 + protein_state + cleaner0 + 2023-07-12T14:49:08Z + DUMMY: + + in complex with + + + 0.93712324 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9997706 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.9554019 + structure_element + cleaner0 + 2023-07-11T20:21:29Z + SO: + + N-terminal lobe + + + 0.99935323 + structure_element + cleaner0 + 2023-07-11T20:21:32Z + SO: + + β-sheet + + + 0.98967075 + structure_element + cleaner0 + 2023-07-11T20:21:36Z + SO: + + C-terminal lobe + + + 0.99887353 + structure_element + cleaner0 + 2023-07-11T20:21:39Z + SO: + + α-helical + + + 0.7400553 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.99963737 + protein_type + cleaner0 + 2023-07-11T14:08:52Z + MESH: + + DUSPs + + + 0.9988386 + structure_element + cleaner0 + 2023-07-11T20:21:43Z + SO: + + twisted five-stranded β-sheet + + + 0.99922544 + structure_element + cleaner0 + 2023-07-11T20:21:46Z + SO: + + α-helices + + + 0.99953717 + structure_element + cleaner0 + 2023-07-11T20:21:49Z + SO: + + β-sheet + + + 0.9994037 + structure_element + cleaner0 + 2023-07-11T20:21:52Z + SO: + + α-helices + + + 0.9919618 + structure_element + cleaner0 + 2023-07-11T20:21:55Z + SO: + + α-helices 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structure_element + cleaner0 + 2023-07-11T20:22:10Z + SO: + + loop + + + 0.99964577 + structure_element + cleaner0 + 2023-07-11T20:22:13Z + SO: + + α0–β1 + + + 0.9995042 + protein + cleaner0 + 2023-07-11T14:22:09Z + PR: + + VHR + + + 0.6807224 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.99950767 + protein + cleaner0 + 2023-07-11T14:22:09Z + PR: + + VHR + + + 0.99970764 + structure_element + cleaner0 + 2023-07-11T20:22:17Z + SO: + + loop + + + 0.9996819 + structure_element + cleaner0 + 2023-07-11T20:22:19Z + SO: + + β3–β4 + + + 0.9990049 + structure_element + cleaner0 + 2023-07-11T20:22:22Z + SO: + + loop + + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.99971765 + protein + cleaner0 + 2023-07-11T14:22:09Z + PR: + + VHR + + + 0.9990748 + structure_element + cleaner0 + 2023-07-11T20:22:25Z + SO: + + helix + + + 0.999754 + structure_element + cleaner0 + 2023-07-11T20:22:27Z + SO: + + α0 + + + 0.9996567 + structure_element + cleaner0 + 2023-07-11T20:22:30Z + SO: + + loop + + + 0.99963564 + structure_element + cleaner0 + 2023-07-11T20:22:32Z + SO: + + α0–β1 + + + 0.95910877 + site + cleaner0 + 2023-07-12T14:34:29Z + SO: + + substrate-recognition motif + + + 0.9991061 + protein + cleaner0 + 2023-07-11T14:22:08Z + PR: + + VHR + + + 0.9996191 + protein_type + cleaner0 + 2023-07-11T14:21:30Z + MESH: + + phosphatases + + + 0.99985707 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + + RESULTS + paragraph + 10297 + The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs. The catalytic residue, Cys244, is located just after strand β5 and optimally positioned for nucleophilic attack. Asp213 in MKP7 also adopts a position similar to that of Asp92 in VHR (Supplementary Fig. 1c), indicating that Asp213 is likely to function as the general acid in MKP7. We also observed the binding of a chloride ion in the active site of MKP7-CD. It is located 3.36 Å from the Cys244 side chain and makes electrostatic interactions with the dipole moment of helix α3 and with several main-chain amide groups. The side chain of strictly conserved Arg250 is oriented towards the negatively charged chloride, similar to the canonical phosphate-coordinating conformation. Thus this chloride ion is a mimic for the phosphate group of the substrate, as revealed by a comparison with the structure of PTP1B in complex with phosphotyrosine (Supplementary Fig. 1d). Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). The difference in the polarity/hydrophobicity of the surface may also point to the origin of the differences in the substrate-recognition mechanism for these two phosphatases (Supplementary Fig. 1e,f). + + 0.99956113 + site + cleaner0 + 2023-07-12T14:34:34Z + SO: + + active site + + + 0.9998584 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9995509 + structure_element + cleaner0 + 2023-07-11T20:22:35Z + SO: + + phosphate-binding loop + + + 0.9997042 + structure_element + cleaner0 + 2023-07-11T15:41:47Z + SO: + + P-loop + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-11T14:23:54Z + + Cys244 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-11T14:24:06Z + + Leu245 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-11T14:24:28Z + + Ala246 + + + 0.4037717 + residue_name_number + cleaner0 + 2023-07-11T14:24:35Z + DUMMY: + + Gly247 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-11T14:24:57Z + + Ile248 + + + residue_name_number + DUMMY: + 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0.99766505 + evidence + cleaner0 + 2023-07-12T14:42:09Z + DUMMY: + + structure + + + 0.9998487 + protein + cleaner0 + 2023-07-11T15:49:21Z + PR: + + PTP1B + + + 0.99920243 + protein_state + cleaner0 + 2023-07-12T14:49:22Z + DUMMY: + + in complex with + + + 0.9996356 + residue_name + melaniev@ebi.ac.uk + 2023-07-21T14:00:06Z + SO: + + phosphotyrosine + + + 0.99006784 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9998066 + protein + cleaner0 + 2023-07-11T14:22:09Z + PR: + + VHR + + + 0.99970084 + structure_element + cleaner0 + 2023-07-11T15:41:47Z + SO: + + P-loop + + + 0.9998542 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9996877 + protein + cleaner0 + 2023-07-11T14:22:09Z + PR: + + VHR + + + 0.56615037 + residue_name_number + cleaner0 + 2023-07-11T14:28:39Z + DUMMY: + + Cys124 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-11T14:29:03Z + + Arg125 + + + 0.7130929 + residue_name_number + cleaner0 + 2023-07-11T14:30:49Z + DUMMY: + + Glu126 + + + 0.71502244 + residue_name_number + cleaner0 + 2023-07-11T14:30:22Z + DUMMY: + + Gly127 + + + 0.98875487 + residue_name_number + cleaner0 + 2023-07-11T14:30:29Z + DUMMY: + + Tyr128 + + + 0.99159086 + residue_name_number + cleaner0 + 2023-07-11T14:30:38Z + DUMMY: + + Gly129 + + + 0.99976474 + residue_name_number + cleaner0 + 2023-07-11T14:30:43Z + DUMMY: + + Arg130 + + + 0.999539 + protein_type + cleaner0 + 2023-07-11T14:30:55Z + MESH: + + phosphatases + + + + RESULTS + paragraph + 11945 + In the complex, MKP7-CD and JNK1 form extensive protein–protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e). As a result, the buried solvent-accessible surface area is ∼1,315 Å. In the C-terminal domain, JNK1 has an insertion after the helix αG. This insertion consists of two helices (α1L14 and α2L14) that are common to all members of the MAPK family. The interactive surface in JNK1, formed by the helices αG and α2L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d). The MKP7-docking region includes two helices, α4 and α5, and the general acid loop. The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. The second interactive area involves the α4 helix of MKP7 and charged/polar residues of JNK1 (Fig. 3e). The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9997383 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.9009522 + structure_element + cleaner0 + 2023-07-11T20:23:02Z + SO: + + C-terminal helices + + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9996624 + structure_element + cleaner0 + 2023-07-11T20:23:08Z + SO: + + C-lobe + + + 0.99978286 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.99970305 + structure_element + cleaner0 + 2023-07-11T20:23:11Z + SO: + + C-terminal domain + + + 0.9997002 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.99934155 + 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residue_name_number + cleaner0 + 2023-07-11T15:42:56Z + DUMMY: + + Ile197 + + + 0.9999 + residue_name_number + cleaner0 + 2023-07-11T15:43:02Z + DUMMY: + + Leu198 + + + 0.9999014 + residue_name_number + cleaner0 + 2023-07-11T15:43:08Z + DUMMY: + + Ile231 + + + 0.99990284 + residue_name_number + cleaner0 + 2023-07-11T15:42:41Z + DUMMY: + + Trp234 + + + 0.99990404 + residue_name_number + cleaner0 + 2023-07-11T15:43:16Z + DUMMY: + + Val256 + + + 0.99990463 + residue_name_number + cleaner0 + 2023-07-11T15:43:21Z + DUMMY: + + Tyr259 + + + 0.9999045 + residue_name_number + cleaner0 + 2023-07-11T15:43:26Z + DUMMY: + + Val260 + + + 0.9999076 + residue_name_number + cleaner0 + 2023-07-11T15:43:33Z + DUMMY: + + His230 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:06Z + + hydrogen bonds + + + 0.9999056 + residue_name_number + cleaner0 + 2023-07-11T15:43:38Z + DUMMY: + + Ser282 + + + 0.99990463 + residue_name_number + cleaner0 + 2023-07-11T15:43:44Z + DUMMY: + + Asn286 + + 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2023-07-11T20:24:17Z + SO: + + α4 helix + + + 0.9998319 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.99964833 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.99990463 + residue_name_number + cleaner0 + 2023-07-11T15:44:17Z + DUMMY: + + Asp268 + + + 0.99982387 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:06Z + + salt bridge + + + 0.99990475 + residue_name_number + cleaner0 + 2023-07-11T15:44:33Z + DUMMY: + + Arg263 + + + 0.99966395 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.9999026 + residue_name_number + cleaner0 + 2023-07-11T15:44:38Z + DUMMY: + + Lys275 + + + 0.9998275 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:06Z + + hydrogen bond + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:06Z + + salt bridge + + + 0.9999002 + residue_name_number + cleaner0 + 2023-07-11T15:44:22Z + DUMMY: + + Thr228 + + + 0.99990034 + residue_name_number + cleaner0 + 2023-07-11T15:44:27Z + DUMMY: + + Asp229 + + + 0.9996506 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + + RESULTS + title_2 + 13355 + Mutational analysis of the JNK1–MKP7 docking interface + + 0.9995778 + experimental_method + cleaner0 + 2023-07-12T14:53:09Z + MESH: + + Mutational analysis + + + site + SO: + cleaner0 + 2023-07-11T15:45:03Z + + JNK1–MKP7 docking interface + + + + RESULTS + paragraph + 13412 + To assess the importance of the aforementioned interactions, we generated a series of point mutations on the MKP7-CD and examined their effect on the MKP7-catalysed JNK1 dephosphorylation (Fig. 4a). When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold. In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP. Mutation of Leu288 markedly reduced its solubility when expressed in Escherichia coli, resulting in the insoluble aggregation of the mutant protein. Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b). Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a). We also generated a series of point mutations in the JNK1 and assessed the effect on JNK1 binding using the GST pull-down assay (Fig. 4c). Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. To determine whether the deficiencies in their abilities to bind partner proteins or carry out catalytic function are owing to misfolding of the purified mutant proteins, we also examined the folding properties of the JNK1 and MKP7 mutants with circular dichroism. The spectra of these mutants are similar to the wild-type proteins, indicating that these mutants fold as well as the wild-type proteins (Fig. 4d,e). Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1. + + experimental_method + MESH: + cleaner0 + 2023-07-12T14:53:26Z + + point mutations + + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + 0.58665764 + structure_element + cleaner0 + 2023-07-11T14:16:11Z + SO: + + CD + + + 0.99985516 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9996363 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T13:57:01Z + + dephosphorylation + + + 0.99989676 + residue_name_number + cleaner0 + 2023-07-11T15:42:48Z + DUMMY: + + Phe285 + + + 0.99989974 + residue_name_number + cleaner0 + 2023-07-11T15:47:33Z + DUMMY: + + Phe287 + + + 0.9997677 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residue_name_number + cleaner0 + 2023-07-11T15:42:48Z + DUMMY: + + Phe285 + + + 0.9884856 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.7853465 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + complex_assembly + GO: + cleaner0 + 2023-07-12T14:42:42Z + + F285D–JNK1 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9996942 + mutant + cleaner0 + 2023-07-11T15:53:12Z + MESH: + + F285D + + + 0.999648 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + 0.99958867 + experimental_method + cleaner0 + 2023-07-12T14:54:46Z + MESH: + + mutation + + + 0.99990153 + residue_name_number + cleaner0 + 2023-07-11T15:47:32Z + DUMMY: + + Phe287 + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:02:20Z + + p + + + protein + PR: + cleaner0 + 2023-07-11T19:47:26Z + + JNK1 + + + 0.9948709 + evidence + cleaner0 + 2023-07-12T14:42:18Z + DUMMY: + + affinity + + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.99906033 + protein + cleaner0 + 2023-07-11T14:03:58Z + PR: + + JNK1 + + + experimental_method + MESH: + cleaner0 + 2023-07-12T14:55:11Z + + point mutations + + + 0.99979895 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.99973184 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.9995672 + experimental_method + cleaner0 + 2023-07-12T14:55:19Z + MESH: + + GST pull-down assay + + + 0.99960667 + experimental_method + cleaner0 + 2023-07-12T14:55:23Z + MESH: + + Substitution + + + 0.9998933 + residue_name_number + cleaner0 + 2023-07-11T15:44:28Z + DUMMY: + + Asp229 + + + 0.9998952 + residue_name_number + cleaner0 + 2023-07-11T15:42:41Z + DUMMY: + + Trp234 + + + 0.99989414 + residue_name_number + cleaner0 + 2023-07-11T15:43:53Z + DUMMY: + + Thr255 + + + 0.9998956 + residue_name_number + cleaner0 + 2023-07-11T15:43:17Z + DUMMY: + + Val256 + + + 0.9998952 + residue_name_number + cleaner0 + 2023-07-11T15:43:22Z + DUMMY: + + Tyr259 + + + 0.99989724 + residue_name_number + cleaner0 + 2023-07-11T15:43:27Z + DUMMY: + + Val260 + + + 0.9995673 + evidence + cleaner0 + 2023-07-12T14:42:49Z + DUMMY: + + binding affinity + + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + 0.5284776 + structure_element + cleaner0 + 2023-07-11T14:16:11Z + SO: + + CD + + + 0.99918693 + protein_type + cleaner0 + 2023-07-11T14:06:30Z + MESH: + + JNK + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T15:48:15Z + + mutant + + + 0.9997172 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.99974996 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.99952084 + experimental_method + cleaner0 + 2023-07-12T14:46:19Z + MESH: + + circular dichroism + + + 0.9995652 + evidence + cleaner0 + 2023-07-12T14:42:52Z + DUMMY: + + spectra + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.99955815 + protein_state + cleaner0 + 2023-07-11T15:48:09Z + DUMMY: + + wild-type + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.9995454 + protein_state + cleaner0 + 2023-07-11T15:48:08Z + DUMMY: + + wild-type + + + 0.9967828 + evidence + cleaner0 + 2023-07-12T14:42:55Z + DUMMY: + + crystallographic model + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:06Z + + hydrophobic contacts + + + 0.999858 + protein + cleaner0 + 2023-07-11T14:04:13Z + PR: + + MKP7 + + + 0.9994093 + structure_element + cleaner0 + 2023-07-11T20:20:49Z + SO: + + catalytic domain + + + 0.999542 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.9999 + residue_name_number + cleaner0 + 2023-07-11T15:42:48Z + DUMMY: + + Phe285 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + 0.7495031 + structure_element + cleaner0 + 2023-07-11T14:16:11Z + SO: + + CD + + + 0.9997335 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + + RESULTS + paragraph + 15610 + It has previously been reported that several cytosolic and inducible nuclear MKPs undergo catalytic activation upon interaction with the MAPK substrates. This allosteric activation of MKP3 has been well-documented in vitro using pNPP, a small-molecule phosphotyrosine analogue of its normal substrate. We then assayed pNPPase activities of MKP7ΔC304 and MKP7-CD in the presence of JNK1. Incubation of MKP7 with JNK1 did not markedly stimulate the phosphatase activity, which is consistent with previous results that MKP7 solely possesses the intrinsic activity (Supplementary Fig. 2b). The small pNPP molecule binds directly at the enzyme active site and can be used to probe the reaction mechanism of protein phosphatases. We therefore examined the effects of the MKP7-CD mutants on their pNPPase activities. As shown in Fig. 4f, all the mutants, except F287D/A, showed little or no activity change compared with the wild-type MKP7-CD. In the JNK1/MKP7-CD complex structure, Phe287 of MKP7 does not make contacts with JNK1 substrate. It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). + + 0.99966466 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.99960107 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + 0.99984586 + protein + cleaner0 + 2023-07-11T14:16:27Z + PR: + + MKP3 + + + 0.99979967 + chemical + cleaner0 + 2023-07-12T15:01:52Z + CHEBI: + + pNPP + + + 0.9996563 + residue_name + melaniev@ebi.ac.uk + 2023-07-21T14:00:06Z + SO: + + phosphotyrosine + + + 0.9685103 + protein_type + cleaner0 + 2023-07-11T20:16:18Z + MESH: + + pNPPase + + + 0.9973181 + mutant + cleaner0 + 2023-07-11T14:17:23Z + MESH: + + MKP7ΔC304 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:13Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9957655 + protein_state + cleaner0 + 2023-07-12T14:49:29Z + DUMMY: + + presence of + + + 0.9997247 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.98657924 + experimental_method + cleaner0 + 2023-07-12T14:55:33Z + MESH: + + Incubation + + + 0.99985695 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9997564 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.99723995 + protein_type + cleaner0 + 2023-07-11T20:16:22Z + MESH: + + phosphatase + + + 0.9998536 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9998017 + chemical + cleaner0 + 2023-07-12T15:01:55Z + CHEBI: + + pNPP + + + 0.9995545 + site + cleaner0 + 2023-07-12T14:36:41Z + SO: + + active site + + + 0.99950284 + protein_type + cleaner0 + 2023-07-11T20:16:25Z + MESH: + + protein phosphatases + + + protein + PR: + cleaner0 + 2023-07-11T14:04:14Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9039243 + protein_state + cleaner0 + 2023-07-11T20:04:10Z + DUMMY: + + mutants + + + 0.99861705 + protein_type + cleaner0 + 2023-07-11T20:16:29Z + MESH: + + pNPPase + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.9993043 + mutant + cleaner0 + 2023-07-11T20:02:34Z + MESH: + + F287D/A + + + 0.9995575 + protein_state + cleaner0 + 2023-07-11T15:48:09Z + DUMMY: + + wild-type + + + protein + PR: + cleaner0 + 2023-07-11T14:04:14Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.99967325 + complex_assembly + cleaner0 + 2023-07-11T20:33:18Z + GO: + + JNK1/MKP7-CD + + + 0.9995315 + evidence + cleaner0 + 2023-07-12T14:43:01Z + DUMMY: + + structure + + + 0.999907 + residue_name_number + cleaner0 + 2023-07-11T15:47:33Z + DUMMY: + + Phe287 + + + 0.9998541 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9997867 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.9994991 + site + cleaner0 + 2023-07-12T14:36:47Z + SO: + + pocket + + + 0.9997178 + structure_element + cleaner0 + 2023-07-11T15:41:48Z + SO: + + P-loop + + + 0.99967957 + structure_element + cleaner0 + 2023-07-11T20:24:31Z + SO: + + general acid loop + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:06Z + + hydrophobic contacts + + + 0.9999043 + residue_name_number + cleaner0 + 2023-07-11T14:25:16Z + DUMMY: + + Arg250 + + + 0.99990535 + residue_name_number + cleaner0 + 2023-07-11T20:31:42Z + DUMMY: + + Glu217 + + + 0.99990547 + residue_name_number + cleaner0 + 2023-07-11T20:31:44Z + DUMMY: + + Ile219 + + + 0.9999083 + residue_name_number + cleaner0 + 2023-07-11T15:47:33Z + DUMMY: + + Phe287 + + + 0.999853 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.99966335 + protein_type + cleaner0 + 2023-07-11T20:16:32Z + MESH: + + PTPs + + + 0.99990714 + residue_name_number + cleaner0 + 2023-07-11T15:49:12Z + DUMMY: + + Gln266 + + + 0.9997998 + protein + cleaner0 + 2023-07-11T15:49:20Z + PR: + + PTP1B + + + 0.9814004 + protein + cleaner0 + 2023-07-11T14:22:09Z + PR: + + VHR + + + 0.9999057 + residue_name_number + cleaner0 + 2023-07-11T15:49:26Z + DUMMY: + + Phe166 + + + 0.99110174 + protein + cleaner0 + 2023-07-11T14:22:09Z + PR: + + VHR + + + 0.9995959 + site + cleaner0 + 2023-07-12T14:36:52Z + SO: + + active-site residues + + + 0.9998474 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + + RESULTS + paragraph + 17123 + Kinase-associated phosphatase (KAP), a member of the DUSP family, plays a crucial role in cell cycle regulation by dephosphorylating the pThr160 residue of CDK2 (cyclin-dependent kinase 2). The crystal structure of the CDK2/KAP complex has been determined at 3.0 Å (Fig. 5a). The interface between these two proteins consists of three discontinuous contact regions. Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the αG helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b). There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2. Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). The substitution of the two hydrophobic residues with charged/polar residues (F285I/N286K) seriously disrupts the hydrophobic interaction required for MKP7 binding on JNK1 (Fig. 4a). In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK. These data indicated that a unique hydrophobic pocket formed between the MAPK insert and αG helix plays a major role in the substrate recognition by MKPs. + + 0.8595056 + protein + cleaner0 + 2023-07-11T15:50:57Z + PR: + + Kinase-associated phosphatase + + + 0.8032938 + protein + cleaner0 + 2023-07-11T15:50:48Z + PR: + + KAP + + + 0.9996263 + protein_type + cleaner0 + 2023-07-11T20:16:37Z + MESH: + + DUSP family + + + 0.9998877 + ptm + cleaner0 + 2023-07-11T20:30:37Z + MESH: + + pThr160 + + + 0.99959224 + protein + cleaner0 + 2023-07-11T15:51:11Z + PR: + + CDK2 + + + 0.84290755 + protein + cleaner0 + 2023-07-11T15:51:07Z + PR: + + cyclin-dependent kinase 2 + + + 0.9995709 + evidence + cleaner0 + 2023-07-12T14:43:16Z + DUMMY: + + crystal structure + + + 0.9996457 + complex_assembly + cleaner0 + 2023-07-11T20:33:22Z + GO: + + CDK2/KAP + + + 0.99838305 + site + cleaner0 + 2023-07-12T14:36:57Z + SO: + + interface + + + 0.9886779 + protein + cleaner0 + 2023-07-11T15:50:49Z + PR: + + KAP + + 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CDK2 + + + 0.99989843 + residue_name_number + cleaner0 + 2023-07-11T20:32:01Z + DUMMY: + + Lys184 + + + 0.9994535 + protein + cleaner0 + 2023-07-11T15:50:49Z + PR: + + KAP + + + 0.9998981 + residue_name_number + cleaner0 + 2023-07-11T20:32:04Z + DUMMY: + + Asp235 + + + 0.9997507 + protein + cleaner0 + 2023-07-11T15:51:12Z + PR: + + CDK2 + + + 0.99959075 + experimental_method + cleaner0 + 2023-07-12T14:56:07Z + MESH: + + Structural analysis + + + 0.99918985 + experimental_method + cleaner0 + 2023-07-12T14:56:09Z + MESH: + + sequence alignment + + + protein + PR: + cleaner0 + 2023-07-11T14:04:14Z + + MKP7 + + + 0.62528604 + structure_element + cleaner0 + 2023-07-11T14:16:11Z + SO: + + CD + + + 0.9986525 + protein + cleaner0 + 2023-07-11T15:50:49Z + PR: + + KAP + + + 0.9995457 + site + cleaner0 + 2023-07-12T14:37:26Z + SO: + + substrate-binding region + + + 0.99974567 + structure_element + cleaner0 + 2023-07-11T20:25:09Z + SO: + + FNFL + + + 0.99516207 + protein + cleaner0 + 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2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.9998976 + residue_name_number + cleaner0 + 2023-07-11T20:32:07Z + DUMMY: + + Glu208 + + + 0.9998958 + residue_name_number + cleaner0 + 2023-07-11T20:32:10Z + DUMMY: + + Asp235 + + + 0.9998272 + protein + cleaner0 + 2023-07-11T15:51:12Z + PR: + + CDK2 + + + 0.96913147 + protein + cleaner0 + 2023-07-11T15:51:12Z + PR: + + CDK2 + + + 0.99889547 + site + cleaner0 + 2023-07-12T14:37:30Z + SO: + + interactive surface + + + 0.9995783 + protein_type + cleaner0 + 2023-07-11T14:06:30Z + MESH: + + JNK + + + 0.99939847 + site + cleaner0 + 2023-07-12T14:37:35Z + SO: + + hydrophobic pocket + + + 0.9705114 + structure_element + cleaner0 + 2023-07-11T20:25:17Z + SO: + + MAPK insert + + + 0.99964076 + structure_element + cleaner0 + 2023-07-11T20:25:19Z + SO: + + αG helix + + + 0.999629 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + + RESULTS + title_2 + 18683 + F-site interaction is crucial for JNK1 inactivation in vivo + + 0.9995083 + site + cleaner0 + 2023-07-12T14:37:38Z + SO: + + F-site + + + 0.9998393 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + + RESULTS + paragraph + 18743 + JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol). To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. As shown in Fig. 6a–c, immunobloting showed similar expression levels for the different MKP7 constructs in all the cells. Overexpressed full-length MKP7, MKP7ΔC304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells. Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. We next tested in vivo interactions between JNK1 mutants and full-length MKP7 by coimmunoprecipitation experiments under unstimulated conditions. When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo. In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a). + + 0.9604673 + protein_type + cleaner0 + 2023-07-11T14:06:30Z + MESH: + + JNK + + + 0.9985983 + chemical + cleaner0 + 2023-07-12T15:02:01Z + CHEBI: + + anisomycin + + + 0.99502426 + chemical + cleaner0 + 2023-07-12T15:02:05Z + CHEBI: + + H2O2 + + + 0.98596144 + chemical + cleaner0 + 2023-07-11T15:51:53Z + CHEBI: + + sorbitol + + + 0.9941076 + chemical + cleaner0 + 2023-07-11T15:51:59Z + CHEBI: + + etoposide + + + 0.9951277 + chemical + cleaner0 + 2023-07-11T15:52:04Z + CHEBI: + + cisplatin + + + 0.9956655 + chemical + cleaner0 + 2023-07-11T15:52:09Z + CHEBI: + + taxol + + + 0.9998487 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.6975378 + protein_type + cleaner0 + 2023-07-11T14:06:30Z + MESH: + + JNK + + + 0.9971235 + protein_state + cleaner0 + 2023-07-12T14:49:37Z + DUMMY: + + HA-tagged + + + 0.99959403 + protein_state + cleaner0 + 2023-07-11T15:54:25Z + DUMMY: + + full-length + + + 0.9998418 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.99450654 + mutant + cleaner0 + 2023-07-11T14:17:23Z + MESH: + + MKP7ΔC304 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:14Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9996773 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.94985515 + chemical + cleaner0 + 2023-07-11T15:52:00Z + CHEBI: + + etoposide + + + 0.99964476 + experimental_method + cleaner0 + 2023-07-12T14:56:29Z + MESH: + + immunobloting + + + 0.9997931 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.99441683 + experimental_method + cleaner0 + 2023-07-12T14:56:32Z + MESH: + 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mutant + cleaner0 + 2023-07-11T15:53:11Z + MESH: + + F285D + + + 0.9996712 + mutant + cleaner0 + 2023-07-11T15:53:17Z + MESH: + + F287D + + + 0.99967074 + mutant + cleaner0 + 2023-07-11T15:53:45Z + MESH: + + L288D + + + 0.999846 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9996885 + mutant + cleaner0 + 2023-07-11T15:53:33Z + MESH: + + D268A + + + 0.99969137 + mutant + cleaner0 + 2023-07-11T15:53:39Z + MESH: + + N286A + + + 0.99719185 + protein_state + cleaner0 + 2023-07-11T20:04:10Z + DUMMY: + + mutants + + + 0.9376806 + protein_type + cleaner0 + 2023-07-11T14:06:30Z + MESH: + + JNK + + + 0.9992361 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.999608 + protein_state + cleaner0 + 2023-07-11T15:54:24Z + DUMMY: + + full-length + + + 0.99984133 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.99945056 + experimental_method + cleaner0 + 2023-07-12T14:56:41Z + MESH: + + coimmunoprecipitation experiments + + + 0.9995473 + experimental_method + cleaner0 + 2023-07-12T14:56:44Z + MESH: + + co-expressed + + + 0.9995973 + protein_state + cleaner0 + 2023-07-11T15:48:09Z + DUMMY: + + wild-type + + + 0.9994735 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.9990482 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9998165 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9996216 + protein_state + cleaner0 + 2023-07-11T20:03:16Z + DUMMY: + + dephosphorylated + + + 0.99959725 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.9960538 + experimental_method + cleaner0 + 2023-07-12T14:56:47Z + MESH: + + in vitro pull-down + + + 0.9902006 + protein_state + cleaner0 + 2023-07-11T20:04:10Z + DUMMY: + + mutants + + + 0.99968827 + mutant + cleaner0 + 2023-07-11T15:53:51Z + MESH: + + D229A + + + 0.99967825 + mutant + cleaner0 + 2023-07-11T15:53:59Z + MESH: + + W234D + + + 0.9996753 + mutant 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To examine whether the inhibition of JNK activity by MKP7 would provide protections against the apoptosis, we analysed the rate of apoptosis in ultraviolet-irradiated cells transfected with MKP7 (wild type or mutants) by flow cytometry. The results showed similar apoptotic rates between cells transfected with blank vector or with MKP7 (wild type or mutants) under unstimulated conditions (Supplementary Fig. 3b), while ultraviolet-irradiation significantly increased apoptotic rate in cells transfected with blank vector (Fig. 6e). Expressions of wild-type MKP7, MKP7ΔC304 and MKP7-CD significantly decreased the proportion of apoptotic cells after ultraviolet treatment. Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did. In contrast, cells transfected with the MKP7 FXF-motif mutants (F285D, F287D and L288D) showed little protective effect after ultraviolet treatment and similar levels of apoptosis rates were detected to cells transfected with control vectors (Fig. 6e,f). Taken together, our results suggested that FXF-motif-mediated, rather than KBD-mediated, interaction is essential for MKP7 to block ultraviolet-induced apoptosis. + + 0.966076 + protein_type + cleaner0 + 2023-07-11T14:06:30Z + MESH: + + JNK + + + 0.98420215 + protein_type + cleaner0 + 2023-07-11T14:06:30Z + MESH: + + JNK + + + 0.9612107 + protein_type + cleaner0 + 2023-07-11T14:06:30Z + MESH: + + JNK + + + 0.99984944 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.99985313 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9995705 + protein_state + cleaner0 + 2023-07-11T15:54:35Z + DUMMY: + + wild type + + + 0.9624378 + protein_state + cleaner0 + 2023-07-11T20:04:10Z + DUMMY: + + mutants + + + 0.9994309 + experimental_method + cleaner0 + 2023-07-12T14:56:52Z + MESH: + + flow cytometry + + + 0.9998561 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.99956214 + protein_state + cleaner0 + 2023-07-11T15:54:35Z + DUMMY: + + wild type + + + 0.9825302 + protein_state + cleaner0 + 2023-07-11T20:04:09Z + DUMMY: + + mutants + + + 0.55191964 + experimental_method + cleaner0 + 2023-07-12T14:56:57Z + MESH: + + Expressions + + + 0.9995586 + protein_state + cleaner0 + 2023-07-11T15:48:09Z + DUMMY: + + wild-type + + + 0.9998543 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9996325 + mutant + cleaner0 + 2023-07-11T14:17:23Z + MESH: + + MKP7ΔC304 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:14Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.79603076 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:14:22Z + + KBD + + + 0.99613756 + protein_state + cleaner0 + 2023-07-11T20:04:10Z + DUMMY: + + mutants + + + 0.9996768 + mutant + cleaner0 + 2023-07-11T15:52:50Z + MESH: + + R56A + + + 0.78404593 + mutant + cleaner0 + 2023-07-11T15:52:54Z + MESH: + + R57A + + + 0.9996897 + mutant + cleaner0 + 2023-07-11T15:53:00Z + MESH: + + V63A + + + 0.9893594 + mutant + cleaner0 + 2023-07-11T15:53:05Z + MESH: + + I65A + + + 0.99985623 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9995667 + protein_state + cleaner0 + 2023-07-11T15:54:35Z + DUMMY: + + wild type + + + 0.99985516 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9996304 + structure_element + cleaner0 + 2023-07-11T14:06:58Z + SO: + + FXF-motif + + + 0.99723876 + protein_state + cleaner0 + 2023-07-11T20:04:10Z + DUMMY: + + mutants + + + 0.99966097 + mutant + cleaner0 + 2023-07-11T15:53:12Z + MESH: + + F285D + + + 0.9996561 + mutant + cleaner0 + 2023-07-11T15:53:18Z + MESH: + + F287D + + + 0.9996691 + mutant + cleaner0 + 2023-07-11T15:53:46Z + MESH: + + L288D + + + 0.99962074 + structure_element + cleaner0 + 2023-07-11T14:06:58Z + SO: + + FXF-motif + + + 0.99956053 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9998573 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + + RESULTS + title_2 + 22013 + A similar docking mechanism for JNK1 recognition by MKP5 + + 0.9998292 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.9998512 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + + RESULTS + paragraph + 22070 + MKP5 belongs to the same subfamily as MKP7. MKP5 is unique among the MKPs in possessing an additional domain of unknown function at the N-terminus (Fig. 7a). The KBD of MKP5 interacts with the D-site of p38α to mediate the enzyme–substrate interaction. Deletion of the KBD in MKP5 leads to a 280-fold increase in Km for p38α substrate. In contrast to p38α substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b). The substrate specificity constant kcat /Km value for MKP5-CD was calculated as 1.0 × 105 M−1 s−1, which is very close to that of MKP7-CD (1.07 × 105 M−1 s−1). The crystal structure of human MKP5-CD has been determined. Comparisons between catalytic domains structures of MKP5 and MKP7 reveal that the overall folds of the two proteins are highly similar, with only a few regions exhibiting small deviations (r.m.s.d. of 0.79 Å; Fig. 7c). + + 0.99984145 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.999816 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.999853 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.9996393 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.9997284 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9998511 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.99933696 + site + cleaner0 + 2023-07-11T14:08:28Z + SO: + + D-site + + + 0.9997428 + protein + cleaner0 + 2023-07-11T14:16:53Z + PR: + + p38α + + + 0.995196 + experimental_method + cleaner0 + 2023-07-12T14:57:09Z + MESH: + + Deletion of + + + 0.99958664 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9998568 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.9995534 + evidence + cleaner0 + 2023-07-12T14:43:22Z + DUMMY: + + Km + + + 0.99974126 + protein + cleaner0 + 2023-07-11T14:16:53Z + PR: + + p38α + + + 0.99971825 + protein + cleaner0 + 2023-07-11T14:16:53Z + PR: + + p38α + + + 0.94834405 + experimental_method + cleaner0 + 2023-07-12T14:57:11Z + MESH: + + deletion of + + + 0.9998579 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.7168404 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9996953 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.9997081 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.99985504 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.99968207 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.9992036 + evidence + cleaner0 + 2023-07-12T14:43:26Z + DUMMY: + + substrate specificity constant + + + 0.9992015 + evidence + cleaner0 + 2023-07-12T14:43:29Z + DUMMY: + + kcat /Km + + + 0.9990727 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + protein + PR: + cleaner0 + 2023-07-11T14:04:14Z + + MKP7 + + + 0.5896785 + structure_element + cleaner0 + 2023-07-11T14:16:11Z + SO: + + CD + + + 0.99961245 + evidence + cleaner0 + 2023-07-12T14:43:32Z + DUMMY: + + crystal structure + + + 0.99919623 + species + cleaner0 + 2023-07-11T20:32:27Z + MESH: + + human + + + 0.9881953 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9996147 + structure_element + cleaner0 + 2023-07-11T20:25:29Z + SO: + + catalytic domains + + + 0.99957293 + evidence + cleaner0 + 2023-07-12T14:43:36Z + DUMMY: + + structures + + + 0.99985886 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.9998543 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9995599 + evidence + cleaner0 + 2023-07-12T14:43:39Z + DUMMY: + + r.m.s.d. + + + + RESULTS + paragraph + 23094 + Given the distinct interaction mode revealed by the crystal structure of JNK1–MKP7-CD, one obvious question is whether this is a general mechanism used by all members of the JNK-specific MKPs. To address this issue, we first examined the docking ability of JNK1 to the KBD and CD of MKP5 using gel filtration analysis and pull-down assays. It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d). Pull-down assays also confirmed the protein–protein interactions observed above. The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs. To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity. As shown in Fig. 7f, the T432A and L449F MKP5 mutant showed little or no difference in phosphatase activity, whereas the other mutants showed reduced specific activities of MKP5. As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a). Taken together, our results suggest that MKP5 binds JNK1 in a docking mode similar to that in the JNK1–MKP7 complex, and the detailed interaction model can be generated using molecular dynamics simulation based on the structure of JNK1–MKP7-CD complex (Supplementary Fig. 4b,c). In this model, the MKP5-CD adopts a conformation nearly identical to that in its unbound form, suggesting that the conformation of the catalytic domain undergoes little change, if any at all, upon JNK1 binding. In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1–MKP7-CD complex (Supplementary Fig. 4d). Despite the strong similarities between JNK1–MKP5-CD and JNK1–MKP7-CD, however, there are differences. The JNK1–MKP7-CD interaction is better and more extensive. Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1. In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). + + 0.9996052 + evidence + cleaner0 + 2023-07-12T14:43:43Z + DUMMY: + + crystal structure + + + 0.99971896 + complex_assembly + cleaner0 + 2023-07-11T15:55:37Z + GO: + + JNK1–MKP7-CD + + + 0.9985678 + protein_type + cleaner0 + 2023-07-11T19:54:00Z + MESH: + + JNK-specific MKPs + + + 0.9996025 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.99980146 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9997985 + structure_element + cleaner0 + 2023-07-11T14:16:11Z + SO: + + CD + + + 0.9998498 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.9994723 + experimental_method + cleaner0 + 2023-07-12T14:57:21Z + MESH: + + gel filtration analysis + + + 0.9995785 + experimental_method + cleaner0 + 2023-07-12T14:57:24Z + MESH: + + pull-down assays + + + 0.99940306 + experimental_method + cleaner0 + 2023-07-12T14:57:27Z + MESH: + + gel filtration experiments + + + 0.99954104 + protein + cleaner0 + 2023-07-11T14:03:59Z + PR: + + JNK1 + + + 0.9941077 + protein_state + cleaner0 + 2023-07-12T14:49:42Z + DUMMY: + + stable + + + 0.99865675 + oligomeric_state + cleaner0 + 2023-07-12T15:01:02Z + DUMMY: + + heterodimer + + + 0.99686253 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9997793 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9995878 + experimental_method + cleaner0 + 2023-07-12T14:57:30Z + MESH: + + Pull-down assays + + + 0.9997058 + structure_element + cleaner0 + 2023-07-11T20:20:49Z + SO: + + catalytic domain + + + 0.9998578 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.99981016 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9997764 + protein + 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2023-07-11T15:46:01Z + + Leu288 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-11T20:04:57Z + + Asn379 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-11T20:05:07Z + + Met431 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-11T20:05:18Z + + Met452 + + + protein + PR: + cleaner0 + 2023-07-11T14:16:42Z + + MKP5 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:06Z + + hydrophobic interactions + + + protein + PR: + cleaner0 + 2023-07-11T14:16:42Z + + MKP5 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + protein + PR: + cleaner0 + 2023-07-11T14:04:00Z + + JNK1 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:00Z + + JNK1 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:14Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + protein + PR: + cleaner0 + 2023-07-11T14:16:42Z + + MKP5 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + protein + PR: + cleaner0 + 2023-07-11T14:16:42Z + + MKP5 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:05:56Z + + p + + + protein + PR: + cleaner0 + 2023-07-11T20:06:05Z + + JNK1 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:14Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + + DISCUSS + title_1 + 26278 + Discussion + + + DISCUSS + paragraph + 26289 + The MAPKs p38, ERK and JNK, are central to evolutionarily conserved signalling pathways that are present in all eukaryotic cells. Each MAPK cascade is activated in response to a diverse array of extracellular signals and culminates in the dual-phosphorylation of a threonine and a tyrosine residue in the MAPK-activation loop. The propagation of MAPK signals is attenuated through the actions of the MKPs. Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the ‘kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL). Crystal structures of ERK2 bound with the D-motif sequences derived from MKP3 and HePTP have been reported. These structures revealed that linear docking motifs in interacting proteins bind to a common docking site on MAPKs outside the kinase active site. The particular amino acids and their spacing within D-motif sequences and amino acid composition of the docking sites on MAPKs appear to determine the specificity of D-motifs for individual MAPKs. + + 0.9995801 + protein_type + cleaner0 + 2023-07-11T14:03:02Z + MESH: + + MAPKs + + + 0.9994368 + protein_type + cleaner0 + 2023-07-11T14:06:36Z + MESH: + + p38 + + + 0.99916995 + protein_type + cleaner0 + 2023-07-11T14:06:43Z + MESH: + + ERK + + + 0.99858177 + protein_type + cleaner0 + 2023-07-11T14:06:31Z + MESH: + + JNK + + + 0.99923587 + taxonomy_domain + cleaner0 + 2023-07-11T20:32:36Z + DUMMY: + + eukaryotic + + + 0.99952877 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + 0.97636795 + ptm + cleaner0 + 2023-07-12T15:04:02Z + MESH: + + dual-phosphorylation + + + 0.9991327 + residue_name + cleaner0 + 2023-07-11T20:32:50Z + SO: + + threonine + + + 0.99905163 + residue_name + cleaner0 + 2023-07-11T20:33:01Z + SO: + + tyrosine + + + 0.9992236 + structure_element + cleaner0 + 2023-07-11T19:48:38Z + SO: + + MAPK-activation loop + + + 0.9995788 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + 0.99965894 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.99959964 + protein_type + cleaner0 + 2023-07-11T14:03:02Z + MESH: + + MAPKs + + + 0.9996265 + protein_type + cleaner0 + 2023-07-11T20:16:52Z + MESH: + + phosphatases + + + 0.9995668 + structure_element + cleaner0 + 2023-07-11T19:48:41Z + SO: + + kinase-interaction motif + + + 0.9996681 + structure_element + cleaner0 + 2023-07-11T14:07:23Z + SO: + + D-motif + + + 0.99964 + protein_type + cleaner0 + 2023-07-11T14:08:52Z + MESH: + + DUSPs + + + 0.99963486 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.9996373 + protein_type + cleaner0 + 2023-07-11T14:08:41Z + MESH: + + tyrosine phosphatases + + + 0.9723622 + protein + cleaner0 + 2023-07-11T19:48:17Z + PR: + + HePTP + + + 0.73130083 + protein + cleaner0 + 2023-07-11T19:48:22Z + PR: + + STEP + + + 0.998229 + protein + cleaner0 + 2023-07-11T19:48:27Z + PR: + + PTP-SL + + + 0.9996085 + evidence + cleaner0 + 2023-07-12T14:44:03Z + DUMMY: + + Crystal structures + + + 0.9965006 + protein + cleaner0 + 2023-07-11T14:16:33Z + PR: + + ERK2 + + + 0.99931073 + protein_state + cleaner0 + 2023-07-12T14:49:55Z + DUMMY: + + bound with + + + 0.9996767 + structure_element + cleaner0 + 2023-07-11T14:07:23Z + SO: + + D-motif + + + 0.9997563 + protein + cleaner0 + 2023-07-11T14:16:27Z + PR: + + MKP3 + + + 0.9996892 + protein + cleaner0 + 2023-07-11T19:48:17Z + PR: + + HePTP + + + 0.998662 + evidence + cleaner0 + 2023-07-12T14:44:06Z + DUMMY: + + structures + + + 0.84627134 + structure_element + cleaner0 + 2023-07-11T19:48:06Z + SO: + + linear docking motifs + + + 0.9994099 + site + cleaner0 + 2023-07-12T14:37:56Z + SO: + + docking site + + + 0.9995753 + protein_type + cleaner0 + 2023-07-11T14:03:02Z + MESH: + + MAPKs + + + 0.9915377 + protein_type + cleaner0 + 2023-07-11T20:06:36Z + MESH: + + kinase + + + 0.980252 + site + cleaner0 + 2023-07-12T14:37:58Z + SO: + + active site + + + 0.9995621 + structure_element + cleaner0 + 2023-07-11T14:07:23Z + SO: + + D-motif + + + 0.9995978 + site + cleaner0 + 2023-07-12T14:38:01Z + SO: + + docking sites + + + 0.99960834 + protein_type + cleaner0 + 2023-07-11T14:03:02Z + MESH: + + MAPKs + + + 0.99966985 + structure_element + cleaner0 + 2023-07-11T20:25:35Z + SO: + + D-motifs + + + 0.9996258 + protein_type + cleaner0 + 2023-07-11T14:03:02Z + MESH: + + MAPKs + + + + DISCUSS + paragraph + 27390 + Recently, the crystal structure of a complex between the KBD of MKP5 and p38α has been obtained. This complex has revealed a distinct interaction mode for MKP5. The KBD of MKP5 binds to p38α in the opposite polypeptide direction compared with how the D-motif of MKP3 binds to ERK2. In contrast to the canonical D-motif-binding mode, separate helices, α2 and α3′, in the KBD of MKP5 engage the p38α-docking site. Further structural and biochemical studies indicate that KBD of MKP7 may interact with p38α in a similar manner to that of MKP5. In contrast to MKP5, removal of the KBD domain from MKP7 does not drastically affect enzyme catalysis, and the kinetic parameters of MKP7-CD for p38α substrate are very similar to those for JNK1 substrate. Taken together, these results suggest that MKP7 utilizes a bipartite recognition mechanism to achieve the efficiency and fidelity of p38α signalling. The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. + + 0.9996283 + evidence + cleaner0 + 2023-07-12T14:44:09Z + DUMMY: + + crystal structure + + + 0.99979156 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9998586 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.9997658 + protein + cleaner0 + 2023-07-11T14:16:53Z + PR: + + p38α + + + 0.9998615 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.9997876 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.99985623 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.99980146 + protein + cleaner0 + 2023-07-11T14:16:53Z + PR: + + p38α + + + 0.9996622 + structure_element + cleaner0 + 2023-07-11T14:07:23Z + SO: + + D-motif + + + 0.9998518 + protein + cleaner0 + 2023-07-11T14:16:27Z + PR: + + MKP3 + + + 0.99966013 + protein + cleaner0 + 2023-07-11T14:16:33Z + PR: + + ERK2 + + + 0.94805497 + site + cleaner0 + 2023-07-11T19:48:51Z + SO: + + D-motif-binding mode + + + 0.9082678 + structure_element + cleaner0 + 2023-07-11T20:25:43Z + SO: + + helices + + + 0.9998097 + structure_element + cleaner0 + 2023-07-11T20:25:46Z + SO: + + α2 + + + 0.99963236 + structure_element + cleaner0 + 2023-07-11T20:25:48Z + SO: + + α3′ + + + 0.99978834 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.99986005 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + site + SO: + cleaner0 + 2023-07-11T19:49:31Z + + p38α-docking site + + + 0.9994786 + experimental_method + cleaner0 + 2023-07-12T14:57:53Z + MESH: + + structural and biochemical studies + + + 0.99978584 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9998585 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.99980944 + protein + cleaner0 + 2023-07-11T14:16:53Z + PR: + + p38α + + + 0.99985754 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.9998597 + protein + cleaner0 + 2023-07-11T14:16:42Z + PR: + + MKP5 + + + 0.9972884 + experimental_method + cleaner0 + 2023-07-12T14:57:59Z + MESH: + + removal of + + + 0.9997774 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9998616 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9995437 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9997954 + protein + cleaner0 + 2023-07-11T14:16:53Z + PR: + + p38α + + + 0.99980885 + protein + cleaner0 + 2023-07-11T14:04:00Z + PR: + + JNK1 + + + 0.99985254 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9997168 + protein + cleaner0 + 2023-07-11T14:16:53Z + PR: + + p38α + + + 0.9990908 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.92762035 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.99951345 + site + cleaner0 + 2023-07-11T14:08:28Z + SO: + + D-site + + + 0.99981517 + protein + cleaner0 + 2023-07-11T14:16:53Z + PR: + + p38α + + + 0.9996432 + site + cleaner0 + 2023-07-12T14:38:06Z + SO: + + catalytic pocket + + + 0.999741 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9997526 + protein + cleaner0 + 2023-07-11T14:16:53Z + PR: + + p38α + + + 0.99972093 + structure_element + cleaner0 + 2023-07-11T20:26:06Z + SO: + + activation loop + + + 0.9998603 + protein + cleaner0 + 2023-07-11T14:04:14Z + PR: + + MKP7 + + + 0.9996278 + site + cleaner0 + 2023-07-12T14:38:09Z + SO: + + active site + + + 0.9998229 + protein + cleaner0 + 2023-07-11T14:16:53Z + PR: + + p38α + + + + DISCUSS + paragraph + 28756 + In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). MKP3 is highly specific in dephosphorylating and inactivating ERK2, and the phosphatase activity of the MKP3-catalysed pNPP reaction can be markedly increased in the presence of ERK2 (refs). Sequence alignment of all MKPs reveals a high degree of conservation of residues surrounding the interacting region observed in JNK1–MKP7-CD complex (Supplementary Fig. 5). Therefore, it is tempting to speculate that the catalytic domain of MKP3 may bind to ERK2 in a manner analogous to the way by which MKP7-CD binds to JNK1. A comprehensive examination of the molecular basis of the specific ERK2 recognition by MKP3 is underway. The ongoing work demonstrates that although the overall interaction modes are similar between the JNK1–MKP7-CD and ERK2–MKP3-CD complexes, the ERK2–MKP3-CD interaction is less extensive and helix α4 from MKP3-CD does not interact directly with ERK2. The FXF-motif-mediated interaction is critical for both pERK2 inactivation and ERK2-induced MKP3 activation (manuscript in preparation). + + 0.99955744 + site + cleaner0 + 2023-07-11T14:08:28Z + SO: + + D-site + + + 0.99954635 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + 0.99971265 + protein + cleaner0 + 2023-07-11T14:16:33Z + PR: + + ERK2 + + + 0.99957544 + site + cleaner0 + 2023-07-12T14:38:17Z + SO: + + second binding site + + + 0.99919015 + protein_type + cleaner0 + 2023-07-11T20:17:00Z + MESH: + + phosphatases + + + 0.99955225 + site + cleaner0 + 2023-07-11T14:07:53Z + SO: + + FXF-motif-binding site + + + 0.99956304 + site + cleaner0 + 2023-07-12T14:38:21Z + SO: + + F-site + + + 0.99965453 + protein_state + cleaner0 + 2023-07-12T14:50:01Z + DUMMY: + + active + + + 0.9998123 + protein + cleaner0 + 2023-07-11T14:16:33Z + PR: + + ERK2 + + + 0.746325 + structure_element + cleaner0 + 2023-07-11T14:07:23Z + SO: + + D-motif + + + 0.9995926 + protein_type + cleaner0 + 2023-07-11T14:03:02Z + MESH: + + MAPKs + + + 0.9996002 + site + cleaner0 + 2023-07-11T19:44:58Z + SO: + + hydrophobic site + + + 0.8181511 + evidence + cleaner0 + 2023-07-12T14:44:20Z + DUMMY: + + changes in deuterium exchange profiles + + + 0.8763486 + structure_element + cleaner0 + 2023-07-11T20:26:10Z + SO: + + MAPK insertion + + + 0.99932504 + structure_element + cleaner0 + 2023-07-11T20:26:13Z + SO: + + helix + + + 0.9997248 + structure_element + cleaner0 + 2023-07-11T20:26:16Z + SO: + + αG + + + 0.99982846 + protein + cleaner0 + 2023-07-11T14:04:00Z + PR: + + JNK1 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:14Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.99978536 + protein + cleaner0 + 2023-07-11T14:16:33Z + PR: + + ERK2 + + + 0.99960566 + site + cleaner0 + 2023-07-12T14:38:25Z + SO: + + DEF-site + + + 0.9997335 + protein + cleaner0 + 2023-07-11T14:16:34Z + PR: + + ERK2 + + + 0.9998536 + protein + cleaner0 + 2023-07-11T14:16:27Z + PR: + + MKP3 + + + 0.99983525 + protein + cleaner0 + 2023-07-11T14:16:34Z + PR: + + ERK2 + + + 0.9998573 + protein + cleaner0 + 2023-07-11T14:16:27Z + PR: + + MKP3 + + + 0.9996147 + chemical + cleaner0 + 2023-07-12T15:02:14Z + CHEBI: + + pNPP + + + 0.999213 + protein_state + cleaner0 + 2023-07-12T14:50:14Z + DUMMY: + + presence of + + + 0.9997465 + protein + cleaner0 + 2023-07-11T14:16:34Z + PR: + + ERK2 + + + 0.9995199 + experimental_method + cleaner0 + 2023-07-12T14:58:02Z + MESH: + + Sequence alignment + + + 0.99966586 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.99782705 + site + cleaner0 + 2023-07-12T14:38:30Z + SO: + + interacting region + + + 0.9997303 + complex_assembly + cleaner0 + 2023-07-11T19:51:18Z + GO: + + JNK1–MKP7-CD + + + 0.9996654 + structure_element + cleaner0 + 2023-07-11T20:20:49Z + SO: + + catalytic domain + + + 0.9998479 + protein + cleaner0 + 2023-07-11T14:16:27Z + PR: + + MKP3 + + + 0.9998105 + protein + cleaner0 + 2023-07-11T14:16:34Z + PR: + + ERK2 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:14Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:11Z + + CD + + + 0.9997949 + protein + cleaner0 + 2023-07-11T14:04:00Z + PR: + + JNK1 + + + 0.9998253 + protein + cleaner0 + 2023-07-11T14:16:34Z + PR: + + ERK2 + + + 0.9998517 + protein + cleaner0 + 2023-07-11T14:16:27Z + PR: + + MKP3 + + + 0.9997066 + complex_assembly + cleaner0 + 2023-07-11T19:51:19Z + GO: + + JNK1–MKP7-CD + + + 0.9997077 + complex_assembly + cleaner0 + 2023-07-11T19:51:53Z + GO: + + ERK2–MKP3-CD + + + 0.9992817 + complex_assembly + cleaner0 + 2023-07-11T19:51:54Z + GO: + + ERK2–MKP3-CD + + + 0.99969053 + structure_element + cleaner0 + 2023-07-11T20:26:21Z + SO: + + helix + + + 0.9997199 + structure_element + cleaner0 + 2023-07-11T20:26:23Z + SO: + + α4 + + + protein + PR: + cleaner0 + 2023-07-11T14:16:27Z + + MKP3 + + + 0.947457 + structure_element + cleaner0 + 2023-07-11T14:16:11Z + SO: + + CD + + + 0.9998349 + protein + cleaner0 + 2023-07-11T14:16:34Z + PR: + + ERK2 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:06:58Z + + FXF-motif + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:07:12Z + + p + + + protein + PR: + cleaner0 + 2023-07-11T19:54:59Z + + ERK2 + + + 0.9995442 + protein + cleaner0 + 2023-07-11T14:16:34Z + PR: + + ERK2 + + + 0.9998511 + protein + cleaner0 + 2023-07-11T14:16:27Z + PR: + + MKP3 + + + + DISCUSS + paragraph + 30429 + In summary, we have resolved the structure of JNK1 in complex with the catalytic domain of MKP7. This structure reveals an FXF-docking interaction mode between MAPK and MKP. Results from biochemical characterization of the Phe285 and Phe287 MKP7 mutants combined with structural information support the conclusion that the two Phe residues serve different roles in the catalytic reaction. Phe285 is essential for JNK1 substrate binding, whereas Phe287 plays a role for the precise alignment of active-site residues, which are important for transition-state stabilization. This newly identified FXF-type motif is present in all MKPs, except that the residue at the first position in MKP5 is an equivalent hydrophobic leucine residue (see also Fig. 7f,g), suggesting that these two Phe residues would play a similar role in facilitating substrate recognition and catalysis, respectively. An important feature of MKP–JNK1 interactions is that MKP7 or MKP5 only interact with the F-site of JNK1. One possible explanation is that JNK1 needs to use the D-site to interact with JIP-1, a scaffold protein for JNK signalling. The N-terminal JNK-binding domain of JIP-1 interacts with the D-site on JNK and this interaction is required for JIP-1-mediated enhancement of JNK activation. In addition, JIP-1 can also associate with MKP7 via the C-terminal region of MKP7 (ref.). When MKP7 is bound to JIP-1, it reduces JNK activation, leading to reduced phosphorylation of the JNK target c-Jun. Thus, our biochemical and structural data allow us to present a model for the JNK1–JIP-1–MKP7 ternary complex and provide an important insight into the assembly and function of JNK signalling modules (Supplementary Fig. 6). + + 0.99756986 + evidence + cleaner0 + 2023-07-12T14:44:22Z + DUMMY: + + structure + + + 0.9997992 + protein + cleaner0 + 2023-07-11T14:04:00Z + PR: + + JNK1 + + + 0.99904174 + protein_state + cleaner0 + 2023-07-12T14:50:31Z + DUMMY: + + in complex with + + + 0.9996628 + structure_element + cleaner0 + 2023-07-11T20:20:49Z + SO: + + catalytic domain + + + 0.99985576 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + 0.9973513 + evidence + cleaner0 + 2023-07-12T14:44:25Z + DUMMY: + + structure + + + 0.99884284 + site + cleaner0 + 2023-07-12T14:38:36Z + SO: + + FXF-docking interaction mode + + + 0.999608 + protein_type + cleaner0 + 2023-07-11T14:04:43Z + MESH: + + MAPK + + + 0.9996295 + protein_type + cleaner0 + 2023-07-11T20:17:04Z + MESH: + + MKP + + + 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D-site + + + 0.9997222 + protein + cleaner0 + 2023-07-11T19:55:47Z + PR: + + JIP-1 + + + 0.9912742 + protein_type + cleaner0 + 2023-07-11T14:06:31Z + MESH: + + JNK + + + 0.99856734 + structure_element + cleaner0 + 2023-07-11T20:26:33Z + SO: + + JNK-binding domain + + + 0.99969196 + protein + cleaner0 + 2023-07-11T19:55:47Z + PR: + + JIP-1 + + + 0.99909693 + site + cleaner0 + 2023-07-11T14:08:28Z + SO: + + D-site + + + 0.6355919 + protein_type + cleaner0 + 2023-07-11T14:06:31Z + MESH: + + JNK + + + 0.9996818 + protein + cleaner0 + 2023-07-11T19:55:47Z + PR: + + JIP-1 + + + 0.91881114 + protein_type + cleaner0 + 2023-07-11T14:06:31Z + MESH: + + JNK + + + 0.99969906 + protein + cleaner0 + 2023-07-11T19:55:47Z + PR: + + JIP-1 + + + 0.99985313 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + 0.9994958 + structure_element + cleaner0 + 2023-07-11T20:26:36Z + SO: + + C-terminal region + + + 0.9998529 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + 0.9998578 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + 0.9994645 + protein_state + cleaner0 + 2023-07-12T14:50:35Z + DUMMY: + + bound to + + + 0.99965245 + protein + cleaner0 + 2023-07-11T19:55:47Z + PR: + + JIP-1 + + + 0.52655923 + protein_type + cleaner0 + 2023-07-11T14:06:31Z + MESH: + + JNK + + + protein_type + MESH: + cleaner0 + 2023-07-11T14:06:31Z + + JNK + + + 0.9402229 + protein_type + cleaner0 + 2023-07-11T20:18:37Z + MESH: + + c-Jun + + + 0.9990047 + evidence + cleaner0 + 2023-07-12T14:44:44Z + DUMMY: + + biochemical and structural data + + + 0.99968415 + complex_assembly + cleaner0 + 2023-07-11T19:55:38Z + GO: + + JNK1–JIP-1–MKP7 + + + 0.9929068 + protein_type + cleaner0 + 2023-07-11T14:06:31Z + MESH: + + JNK + + + + METHODS + title_1 + 32141 + Methods + + + METHODS + title_2 + 32149 + Protein preparation + + + METHODS + paragraph + 32169 + The cDNAs of human MKP7 and MKP5 were kindly provided by Dr Mathijs Baens (University of Leuven) and Dr Eisuke Nishida (Kyoto University), respectively. The cDNAs of human ASK1, MKK4, MKK7 and JNK1 were kindly provided by Dr Zhenguo Wu (Hong Kong University of Science and Technology). The catalytic domains of MKP7 (MKP7-CD, residues 156–301) and MKP5 (MKP5-CD, 320–467) and the full-length MKP5 were cloned into the pET15b vector, resulting in the N-terminal His-fusion proteins. The KBD domains of MKP7 (MKP7-KBD, 5–138) and MKP5 (MKP5-KBD, 139–287), and the C-terminal truncation of MKP7 (MKP7ΔC304, 5–303) were cloned into pET21b vector for generation of C-terminal His-tagged proteins. The human full-length JNK1, MKK4, MKK7 and the kinase domain of ASK1 (659–951) were cloned into pGEX4T-2, pET15b and/or pET21b vectors to produce a GST- or His-tagged protein. Mutations of MKP7-CD, MKP5-CD and JNK1 were generated by overlap PCR procedure. All constructs were verified by DNA sequencing. All proteins, overexpressed in BL21(DE3) cells at 20 °C, were first purified over Ni-NTA (Qiagen) or GS4B (GE Healthcare) columns, and then by ion exchange and gel filtration chromatography (Source-15Q/15S and Superdex-200, GE Healthcare) at 4 °C. The double-phosphorylated JNK1 (phospho-JNK1) was generated by mixing JNK1 with upstream kinases MKK4, MKK7 and ASK1 in buffer containing 10 mM MgCl2 and 2 mM ATP, and further purified by gel filtration chromatography (Superdex-200, GE Healthcare) at 4 °C (ref.). Proteins were stored at −80 °C, and stocks for phosphatase assays were supplemented with glycerol to a final concentration of 20% (v/v). Protein concentrations were determined spectrophotometrically using theoretical molar extinction coefficients at 280 nm (ref.). + + + METHODS + title_2 + 33975 + Crystallography + + + METHODS + paragraph + 33991 + The mixture of unphosphorylated JNK1 and MKP7-CD at 1:1 molar ratio was subjected to crystallization trials. Crystals were grown by the vapor-diffusion technique in hanging drops, and the drops were prepared by mixing equal volumes of protein with reservoir solution containing 0.1 M HEPES, pH 7.0, 14% PEG3350, 0.2 M MgCl2, 6% 1,6-Hexanediol and 0.005 M EDTA at 21 °C. Crystals were cryo-protected in reservoir solutions supplemented with 10% glycerol and then flash frozen in liquid nitrogen. The diffraction data sets were collected at beamline 17U at Shanghai Synchrotron Radiation Facility and processed with the HKL2000 package. The crystals belong to space group P1 and comprise eight molecules per asymmetric unit (four complexes). Structure was solved by molecular replacement using Phaser with JNK1 (PDB 1UKH) and MKP5-CD (PDB 1ZZW) as the search models. Standard refinement was performed with programs PHENIX and Coot. The crystal structure of unphosphorylated JNK1 in complex with the catalytic domain of MKP7 was refined to 2.4 Å resolution. Initial structural refinement was performed with NCS restraints, and after several rounds the restraints were removed from the calculations. The final Rwork and Rfree were 21.7 and 23.9%, respectively. The crystallographic asymmetric unit contains four JNK1–MKP7-CD complexes. The four complexes are nearly identical with an r.m.s.d.<1 Å for any complex pair in the asymmetric unit. Ramachandran analysis was carried out using PROCHECK. Additional density at the active site of MKP7-CD was attributed to a chloride ion incorporated as a crystallizing agent, similar to those observed in the structures of MKP3-CD and MKP5-CD (refs). The data collection and refinement statistics are summarized in Table 1. All structural representations in this paper were prepared with PYMOL (http://www.pymol.org). + + + METHODS + title_2 + 35863 + Phosphatase assays + + + METHODS + paragraph + 35882 + The activities of MKP7 and MKP5 was assayed using phospho-JNK1 as substrate in the coupled enzyme system containing 50 mM MOPS, pH 7.0, 100 mM NaCl, 0.1 mM EDTA, 50 μM MESG and 0.1 mg ml−1 PNPase. This coupled assay uses PNPase and its chromogenic substrate MESG to monitor the production of inorganic phosphate. The reactions were initiated by addition of 0.4 μM MKP7-CD and MKP7ΔC304 (or 0.1 μM MKP5 full-length and 0.15 μM MKP5-CD) for substrate phospho-JNK1. All experiments were carried out at 25 °C in 1.8 ml reaction mixtures, and the continuous absorbance changes were recorded with a PerkinElmer LAMBDA 45 spectrophotometer equipped with a magnetic stirrer in the cuvette holder. The quantification of inorganic phosphate produced was monitored at 360 nm with the extinction coefficient of 11,200 M−1 cm−1 (ref.). The initial rates were determined from the linear slope of the progress curves obtained. The activity of MKP7-CD or MKP5-CD mutants were assayed using pNPP or phospho-JNK1 as substrate. The phospho-JNK1 assay was performed as the same procedure mentioned above, and in the presence of wild type (as a control) or indicated mutants, and equal concentrations of phospho-JNK1. The pNPP assay was performed in the reaction mixture containg 50 mM MOPS, pH 7.0, 100 mM NaCl, 0.1 mM EDTA and 20 mM pNPP. The amount of the product p-nitrophenol was determined from the absorbance at 405 nm using a molar extinction coefficient of 18,000 M−1 cm−1 (ref.). + + + METHODS + title_2 + 37409 + Assays for protein–protein interaction + + + METHODS + paragraph + 37450 + The interactions of JNK1 with the CD and KBD domains of MKP7 and MKP5 were examined by gel filtration analyses using a Superdex-200 10/300 column on an ÄKTA FPLC (GE Healthcare). The column was equilibrated with a buffer containing 10 mM HEPES, pH 7.5, 150 mM NaCl and 2 mM dithiothreitol, and calibrated with molecular mass standards. Samples of individual proteins and indicated mixtures (500 μl each) were loaded to the Superdex-200 column and then eluted at a flow rate of 0.5 ml min−1. Fractions of 0.5 ml each were collected, and aliquots of relevant fractions were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed Coomassie Blue staining. + + + METHODS + paragraph + 38133 + The interactions between various JNK1 mutants and MKP7-CD or MKP5-CD were assessed by GST-mediated pull-down assays at 4 °C. First, 0.5 ml GST-JNK1 proteins (6 μM) were loaded to 0.2 ml GS4B resin. The excess unbound JNK1 or other contaminants were removed by washing the column 5 times, each with 1.0 ml buffer containing 25 mM Tris-HCl, pH 8.0, 150 mM NaCl and 2 mM dithiothreitol. Then, 0.5 ml MKP7-CD or MKP5-CD (20 μM) was allowed to flow through the JNK1-bound column. After extensive washing, the bound proteins were eluted with 0.5 ml reduced glutathione (10 mM). The interactions of JNK1 with the CD and KBD domains of MKP7 and MKP5 were also examined by GST-mediated pull-down assays. The GST protein alone was used as a control. Aliquots of all eluates were subjected to SDS-PAGE, and proteins were visualized by Coomassie Blue staining. The uncropped gels are shown in Supplementary Fig. 7. + + + METHODS + title_2 + 39063 + Building JNK and MKP5 interaction model + + + METHODS + paragraph + 39103 + The model of catalytic domain of MKP5 bound to JNK was constructed by superimposition of previous deposited structures of MKP5-CD (PDB 1ZZW) to the corresponding domains in the crystal structure of JNK1–MKP7-CD. The fractured loops in deposited structures were computationally generated using Modeller. The program CHARMM22 (ref.) was then used to add hydrogen atoms, N- and C-terminal patches to the model. The model was then subjected to restrained energy minimization to optimize bonds and remove any nonbonded steric clashes. Refinement of the modelled complex was performed using NAMD2.9 package at 1 atm pressure and 300 K. The generated complex structure was solvated and neutralized in a box with TIP3P water at a minimum of 13 Å between the model and the wall of the box. The simulation was first set up with 1 fs time step under periodic boundary conditions. The particle mesh Ewald method was applied to model the electrostatics and the van der Waals interactions cutoff was set at 12 Å. The system was restrained for 5 ps minimization and 5 ps simulation, and followed by removing all the restraints and performing a minimization of 10 ps and an equilibration of 10 ns. Simulations were viewed using VMD. + + + METHODS + title_2 + 40342 + Circular dichroism spectra + + + METHODS + paragraph + 40369 + The experiments were performed on a Chirascan-plus circular dichroism Spectrometer (Applied Photophysics, Surrey, UK) using 0.1 mm quartz cuvette. The protein sample were analysed at a concentration of 0.5 mg ml−1. Data were collected over a wavelength range from 260 to 190 nm with 1 nm intervals at room temperature, three scans were averaged, and the baseline spetrum of solution buffer containing 10 mM HEPES (pH 7.5) and 150 mM NaCl was subtracted. + + + METHODS + title_2 + 40839 + Cell culture and transfections + + + METHODS + paragraph + 40870 + pcDNA3.3-Myc-MKP7, pCMV5-3HA-JNK1 and pBOBI-HA-MKP7 were generated with standard molecular techniques. Mutants with amino acid substitution and truncation constructs were generated through PCR-based site-directed mutagenesis method using Pfu polymerase (Stratagene). The authenticities of all constructs were confirmed by sequencing (Invitrogen, China). HEK293T and HeLa cells (ATCC) were maintained in DMEM supplemented with 10% fetal bovine serum, 100 IU penicillin, 100 mg ml−1 streptomycin at 37 °C in a humidified incubator containing 5% CO2. Polyethylenimine (Polysciences, #23966) at a final concentration of 10 μM was used to transfect HEK293T cells. Total DNA for each plate was adjusted to the same amount by adding relevant blank vector. Lentiviruses for infection were packaged in HEK293T cells after transfection using Lipofectamine 2000 (Invitrogen, 11668-027). At 30 h post transfection, medium was collected for further infection. + + + METHODS + title_2 + 41833 + Coimmunoprecipitation and immunoblotting + + + METHODS + paragraph + 41874 + Cells were lysed in a lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 2 mM Na3VO4, 25 mM NaF, 1 mM phenylmethanesulfonyl fluoride, 1 μg ml−1 leupeptin and 1 μg ml−1 aprotinin. Cell lysates were incubated with respective antibodies overnight at 4 °C. Protein aggregates resulting from the overnight incubation were removed by centrifugation, and protein A/G beads (Santa-Cruz Biotechnology, Dallas, TX, USA) were then added into the lysates and incubated for another 3 h. After spinning and washing for three times with the lysis buffer, the beads were mixed with 2 × SDS sample buffer, boiled and subjected to 15% SDS/PAGE. The samples were transferred to PVDF membranes (Millipore), and immunoblotted with indicated antibodies. Levels of total proteins and the levels of phosphorylation of proteins were analysed on separate gels. The uncropped blots are shown in Supplementary Fig. 7. + + + METHODS + title_2 + 42830 + Antibodies and drugs + + + METHODS + paragraph + 42851 + Antibodies used in this study: mouse anti-HA (1:100 for immunoprecipitation; F-7) and anti-JNK1 (1:1,000 for immunoblotting; F-3) antibodies were purchased from Santa-Cruz Biotechnology. Anti-c-Myc Agarose Affinity Gel antibody produced in rabbit (1:200 for immunoprecipitation; A7470) was purchased from Sigma. Rabbit anti-HA-tag (1:1,000 for immunoblotting; #3724), anti-phospho-JNK-T183/Y185 (1:1,000 for immunoblotting; #4668) and mouse anti-Myc-tag (1:1,000 for immunoblotting; #2276) antibodies were purchased from Cell Signaling Technology. Etoposide (E1383) was purchased from Sigma. + + + METHODS + title_2 + 43443 + Apoptosis assay + + + METHODS + paragraph + 43459 + HeLa cells were infected with lentiviruses expressing MKP7 or its mutants. At 36 h post infection, cells were irradiated with 25 J m−2 ultraviolet light and collected at 6 h after irradiation. Cells were then stained with the Annexin-V-APC/PI double-staining solution (BD Biosciences) and analysed with a flow cytometer (BD LSRFortessa). The percentages of apoptotic cells were quantified with FlowJo 7.6.1 software. + + + METHODS + title_1 + 43886 + Additional information + + + METHODS + paragraph + 43909 + Accession codes: The coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 4YR8 for the JNK1–MKP7-CD structure. + + + METHODS + paragraph + 44069 + How to cite this article: Liu, X. et al. A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation. Nat. Commun. 7:10879 doi: 10.1038/ncomms10879 (2016). + + + SUPPL + title_1 + 44276 + Supplementary Material + + + 1150 + 1160 + surname:Avruch;given-names:J. + 17229475 + REF + Biochim. Biophys. Acta + ref + 1773 + 2007 + 44299 + MAP kinase pathways: the first twenty years + + + 50 + 83 + surname:Cargnello;given-names:M. + surname:Roux;given-names:P. P. + 21372320 + REF + Microbiol. Mol. Biol. Rev. + ref + 75 + 2011 + 44343 + Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases + + + 3100 + 3112 + surname:Raman;given-names:M. + surname:Chen;given-names:W. + surname:Cobb;given-names:M. H. + 17496909 + REF + Oncogene + ref + 26 + 2007 + 44437 + Differential regulation and properties of MAPKs + + + 827 + 837 + surname:Kolch;given-names:W. + 16227978 + REF + Nat. Rev. Mol. Cell Biol. + ref + 6 + 2005 + 44485 + Coordinating ERK/MAPK signalling through scaffolds and inhibitors + + + 142 + 149 + surname:Weston;given-names:C. R. + surname:Davis;given-names:R. J. + 17303404 + REF + Curr. Opin. Cell Biol. + ref + 19 + 2007 + 44551 + The JNK signal transduction pathway + + + 537 + 549 + surname:Wagner;given-names:E. F. + surname:Nebreda;given-names:A. R. + 19629069 + REF + Nat. Rev. Cancer + ref + 9 + 2009 + 44587 + Signal integration by JNK and p38 MAPK pathways in cancer development + + + 837 + 841 + surname:Bardwell;given-names:L. + 17052210 + REF + Biochem. Soc. Trans. + ref + 34 + 2006 + 44657 + Mechanisms of MAPK signalling specificity + + + 48 + 55 + surname:Akella;given-names:R. + surname:Moon;given-names:T. M. + surname:Goldsmith;given-names:E. J. + 18068683 + REF + Biochim. Biophys. Acta + ref + 1784 + 2008 + 44699 + Unique MAP kinase binding sites + + + 163 + 175 + surname:Jacobs;given-names:D. + surname:Glossip;given-names:D. + surname:Xing;given-names:H. + surname:Muslin;given-names:A. J. + surname:Kornfeld;given-names:K. + 9925641 + REF + Genes Dev. + ref + 13 + 1999 + 44731 + Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase + + + 27256 + 27265 + surname:Fantz;given-names:D. A. + surname:Jacobs;given-names:D. + surname:Glossip;given-names:D. + surname:Kornfeld;given-names:K. + 11371562 + REF + J. Biol. Chem. + ref + 276 + 2001 + 44842 + Docking sites on substrate proteins direct extracellular signal-regulated kinase to phosphorylate specific residues + + + 965 + 973 + surname:Galanis;given-names:A. + surname:Yang;given-names:S. H. + surname:Sharrocks;given-names:A. D. + 11029469 + REF + J. Biol. Chem. + ref + 276 + 2001 + 44958 + Selective targeting of MAPKs to the ETS domain transcription factor SAP-1 + + + 16609 + 16617 + surname:MacKenzie;given-names:S. J. + surname:Baillie;given-names:G. S. + surname:McPhee;given-names:I. + surname:Bolger;given-names:G. B. + surname:Houslay;given-names:M. D. + REF + J. Biol. Chem. + ref + 275 + 2000 + 45032 + ERK2 mitogen-activated protein kinase binding, phosphorylation, and regulation of the PDE4D cAMP-specific phosphodiesterases. The involvement of COOH-terminal docking sites and NH2-terminal UCR regions + + + 556 + 564 + surname:Murphy;given-names:L. O. + surname:Smith;given-names:S. + surname:Chen;given-names:R. H. + surname:Fingar;given-names:D. C. + surname:Blenis;given-names:J. + 12134156 + REF + Nat. Cell Biol. + ref + 4 + 2002 + 45234 + Molecular interpretation of ERK signal duration by immediate early gene products + + + 43 + 55 + surname:Lee;given-names:T. + 15068802 + REF + Mol. Cell + ref + 14 + 2004 + 45315 + Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry + + + 1227 + 1237 + surname:Kondoh;given-names:K. + surname:Nishida;given-names:E. + REF + Biochim. Biophys. Acta + ref + 1773 + 2007 + 45405 + Regulation of MAP kinases by MAP kinase phosphatases + + + 3203 + 3213 + surname:Owens;given-names:D. M. + surname:Keyse;given-names:S. M. + 17496916 + REF + Oncogene + ref + 26 + 2007 + 45458 + Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases + + + 475 + 489 + surname:Patterson;given-names:K. I. + surname:Brummer;given-names:T. + surname:O'Brien;given-names:P. M. + surname:Daly;given-names:R. J. + 19228121 + REF + Biochem. J. + ref + 418 + 2009 + 45548 + Dual-specificity phosphatases: critical regulators with diverse cellular targets + + + 391 + 403 + surname:Jeffrey;given-names:K. L. + surname:Camps;given-names:M. + surname:Rommel;given-names:C. + surname:Mackay;given-names:C. R. + 17473844 + REF + Nat. Rev. Drug Discov. + ref + 6 + 2007 + 45629 + Targeting dual-specificity phosphatases: manipulating MAP kinase signalling and immune responses + + + 253 + 261 + surname:Keyse;given-names:S. M. + 18330678 + REF + Cancer Metastasis Rev. + ref + 27 + 2008 + 45726 + Dual-specificity MAP kinase phosphatases (MKPs) and cancer + + + 833 + 846 + surname:Tonks;given-names:N. K. + 17057753 + REF + Nat. Rev. Mol. Cell Biol. + ref + 7 + 2006 + 45785 + Protein tyrosine phosphatases: from genes, to function, to disease + + + 4607 + 4615 + surname:Dickinson;given-names:R. J. + surname:Keyse;given-names:S. M. + 17093265 + REF + J. Cell Sci. + ref + 119 + 2006 + 45852 + Diverse physiological functions for dual-specificity MAP kinase phosphatases + + + 5326 + 5331 + surname:Liu;given-names:S. + surname:Sun;given-names:J. P. + surname:Zhou;given-names:B. + surname:Zhang;given-names:Z. Y. + 16567630 + REF + Proc. Natl Acad. Sci. USA + ref + 103 + 2006 + 45929 + Structural basis of docking interactions between ERK2 and MAP kinase phosphatase 3 + + + ra88 + surname:Zhang;given-names:Y. Y. + surname:Wu;given-names:J. W. + surname:Wang;given-names:Z. X. + 22375048 + REF + Sci. Signal. + ref + 4 + 2011 + 46012 + A distinct interaction mode revealed by the crystal structure of the kinase p38alpha with the MAPK binding domain of the phosphatase MKP5 + + + 26629 + 26639 + surname:Tanoue;given-names:T. + surname:Yamamoto;given-names:T. + surname:Maeda;given-names:R. + surname:Nishida;given-names:E. + 11359773 + REF + J. Biol. Chem. + ref + 276 + 2001 + 46150 + A Novel MAPK phosphatase MKP-7 acts preferentially on JNK/SAPK and p38 alpha and beta MAPKs + + + 1328 + 1331 + surname:Yuvaniyama;given-names:J. + surname:Denu;given-names:J. M. + surname:Dixon;given-names:J. E. + surname:Saper;given-names:M. A. + 8650541 + REF + Science + ref + 272 + 1996 + 46242 + Crystal structure of the dual specificity protein phosphatase VHR + + + 1 + 52 + surname:Zhang;given-names:Z. Y. + 9543627 + REF + Crit. Rev. Biochem. Mol. Biol. + ref + 33 + 1998 + 46308 + Protein-tyrosine phosphatases: biological function, structural characteristics, and mechanism of catalysis + + + 3009 + 3017 + surname:Schumacher;given-names:M. A. + surname:Todd;given-names:J. L. + surname:Rice;given-names:A. E. + surname:Tanner;given-names:K. G. + surname:Denu;given-names:J. M. + 11863439 + REF + Biochemistry + ref + 41 + 2002 + 46415 + Structural basis for the recognition of a bisphosphorylated MAP kinase peptide by human VHR protein Phosphatase + + + 769 + 779 + surname:Farooq;given-names:A. + surname:Zhou;given-names:M. M. + 15115656 + REF + Cell. Signal. + ref + 16 + 2004 + 46527 + Structure and regulation of MAPK phosphatases + + + 13420 + 13425 + surname:Puius;given-names:Y. A. + 9391040 + REF + Proc. Natl Acad. Sci. USA + ref + 94 + 1997 + 46573 + Identification of a second aryl phosphate-binding site in protein-tyrosine phosphatase 1B: a paradigm for inhibitor design + + + 1262 + 1265 + surname:Camps;given-names:M. + 9596579 + REF + Science + ref + 280 + 1998 + 46696 + Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase + + + 35526 + 35534 + surname:Zhou;given-names:B. + surname:Zhang;given-names:Z. Y. + 10585426 + REF + J. Biol. Chem. + ref + 274 + 1999 + 46783 + Mechanism of mitogen-activated protein kinase phosphatase-3 activation by ERK2 + + + 5484 + 5492 + surname:Zhao;given-names:Y. + surname:Wu;given-names:L. + surname:Noh;given-names:S. J. + surname:Guan;given-names:K. L. + surname:Zhang;given-names:Z. Y. + 9488671 + REF + J. Biol. Chem. + ref + 273 + 1998 + 46862 + Altering the nucleophile specificity of a protein-tyrosine phosphatase-catalyzed reaction. Probing the function of the invariant glutamine residues + + + 352 + 363 + surname:Barr;given-names:A. J. + 19167335 + REF + Cell + ref + 136 + 2009 + 47010 + Large-scale structural analysis of the classical human protein tyrosine phosphatome + + + 693 + 699 + surname:Kim;given-names:S. J. + surname:Ryu;given-names:S. E. + 23261054 + REF + BMB Rep. + ref + 45 + 2012 + 47094 + Structure and catalytic mechanism of human protein tyrosine phosphatome + + + 15874 + 15883 + surname:Brandao;given-names:T. A. + surname:Hengge;given-names:A. C. + surname:Johnson;given-names:S. J. + 20236928 + REF + J. Biol. Chem. + ref + 285 + 2010 + 47166 + Insights into the reaction of protein-tyrosine phosphatase 1B: crystal structures for transition state analogs of both catalytic steps + + + 615 + 626 + surname:Song;given-names:H. + 11463386 + REF + Mol. Cell + ref + 7 + 2001 + 47301 + Phosphoprotein-protein interactions revealed by the crystal structure of kinase-associated phosphatase in complex with phosphoCDK2 + + + 283 + 295 + surname:Karin;given-names:M. + surname:Gallagher;given-names:E. + 16036612 + REF + IUBMB Life + ref + 57 + 2005 + 47432 + From JNK to pay dirt: jun kinases, their biochemistry, physiology and clinical importance + + + 4631 + 4636 + surname:Seimiya;given-names:H. + surname:Mashima;given-names:T. + surname:Toho;given-names:M. + surname:Tsuruo;given-names:T. + 9020192 + REF + J. Biol. Chem. + ref + 272 + 1997 + 47522 + c-Jun NH2-terminal kinase-mediated activation of interleukin-1beta converting enzyme/CED-3-like protease during anticancer drug-induced apoptosis + + + 5142 + 5152 + surname:Sanchez-Perez;given-names:I. + surname:Martinez-Gomariz;given-names:M. + surname:Williams;given-names:D. + surname:Keyse;given-names:S. M. + surname:Perona;given-names:R. + 11064451 + REF + Oncogene + ref + 19 + 2000 + 47668 + CL100/MKP-1 modulates JNK activation and apoptosis in response to cisplatin + + + 6245 + 6251 + surname:Dhanasekaran;given-names:D. N. + surname:Reddy;given-names:E. P. + 18931691 + REF + Oncogene + ref + 27 + 2008 + 47744 + JNK signaling in apoptosis + + + 3014 + 3019 + surname:Franklin;given-names:C. C. + surname:Srikanth;given-names:S. + surname:Kraft;given-names:A. S. + 9501207 + REF + Proc. Natl Acad. Sci. USA + ref + 95 + 1998 + 47771 + Conditional expression of mitogen-activated protein kinase phosphatase-1, MKP-1, is cytoprotective against UV-induced apoptosis + + + 16917 + 16923 + surname:Franklin;given-names:C. C. + surname:Kraft;given-names:A. S. + 9202001 + REF + J. Biol. Chem. + ref + 272 + 1997 + 47899 + Conditional expression of the mitogen-activated protein kinase (MAPK) phosphatase MKP-1 preferentially inhibits p38 MAPK and stress-activated protein kinase in U937 cells + + + G761 + G770 + surname:Ray;given-names:R. M. + surname:Jin;given-names:S. + surname:Bavaria;given-names:M. N. + surname:Johnson;given-names:L. R. + 21350193 + REF + Am. J. Physiol. Gastrointest. Liver Physiol. + ref + 300 + 2011 + 48070 + Regulation of JNK activity in the apoptotic response of intestinal epithelial cells + + + 19949 + 19956 + surname:Tanoue;given-names:T. + surname:Moriguchi;given-names:T. + surname:Nishida;given-names:E. + 10391943 + REF + J. Biol. Chem. + ref + 274 + 1999 + 48154 + Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5 + + + 946 + 955 + surname:Jeong;given-names:D. G. + 16806267 + REF + J. Mol. Biol. + ref + 360 + 2006 + 48240 + Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase + + + 193 + 201 + surname:Munoz;given-names:J. J. + surname:Tarrega;given-names:C. + surname:Blanco-Aparicio;given-names:C. + surname:Pulido;given-names:R. + 12583813 + REF + Biochem. J. + ref + 372 + 2003 + 48375 + Differential interaction of the tyrosine phosphatases PTP-SL, STEP and HePTP with the mitogen-activated protein kinases ERK1/2 and p38alpha is determined by a kinase specificity sequence and influenced by reducing agents + + + 7337 + 7350 + surname:Pulido;given-names:R. + surname:Zuniga;given-names:A. + surname:Ullrich;given-names:A. + 9857190 + REF + EMBO J. + ref + 17 + 1998 + 48596 + PTP-SL and STEP protein tyrosine phosphatases regulate the activation of the extracellular signal-regulated kinases ERK1 and ERK2 by association through a kinase interaction motif + + + 1011 + 1019 + surname:Zhou;given-names:T. + surname:Sun;given-names:L. + surname:Humphreys;given-names:J. + surname:Goldsmith;given-names:E. J. + 16765894 + REF + Structure + ref + 14 + 2006 + 48776 + Docking interactions induce exposure of activation loop in the MAP kinase ERK2 + + + 859 + 869 + surname:Canagarajah;given-names:B. J. + surname:Khokhlatchev;given-names:A. + surname:Cobb;given-names:M. H. + surname:Goldsmith;given-names:E. J. + 9298898 + REF + Cell + ref + 90 + 1997 + 48855 + Activation mechanism of the MAP kinase ERK2 by dual phosphorylation + + + 693 + 696 + surname:Dickens;given-names:M. + 9235893 + REF + Science + ref + 277 + 1997 + 48923 + A cytoplasmic inhibitor of the JNK signal transduction pathway + + + 11843 + 11852 + surname:Mooney;given-names:L. M. + surname:Whitmarsh;given-names:A. J. + 14699111 + REF + J. Biol. Chem. + ref + 279 + 2004 + 48986 + Docking interactions in the c-Jun N-terminal kinase pathway + + + 2185 + 2195 + surname:Heo;given-names:Y. S. + 15141161 + REF + EMBO J. + ref + 23 + 2004 + 49046 + Structural basis for the selective inhibition of JNK1 by the scaffolding protein JIP1 and SP600125 + + + 1671 + 1674 + surname:Whitmarsh;given-names:A. J. + surname:Cavanagh;given-names:J. + surname:Tournier;given-names:C. + surname:Yasuda;given-names:J. + surname:Davis;given-names:R. J. + 9733513 + REF + Science + ref + 281 + 1998 + 49145 + A mammalian scaffold complex that selectively mediates MAP kinase activation + + + 10731 + 10736 + surname:Willoughby;given-names:E. A. + surname:Perkins;given-names:G. R. + surname:Collins;given-names:M. K. + surname:Whitmarsh;given-names:A. J. + 12524447 + REF + J. Biol. Chem. + ref + 278 + 2003 + 49222 + The JNK-interacting protein-1 scaffold protein targets MAPK phosphatase-7 to dephosphorylate JNK + + + 87 + 99 + surname:Owen;given-names:G. R. + surname:Achilonu;given-names:I. + surname:Dirr;given-names:H. W. + 23147205 + REF + Protein Expr. Purif. + ref + 87 + 2013 + 49319 + High yield purification of JNK1beta1 and activation by in vitro reconstitution of the MEKK1-->MKK4-->JNK MAPK phosphorylation cascade + + + 319 + 326 + surname:Gill;given-names:S. C. + surname:von Hippel;given-names:P. H. + 2610349 + REF + Anal. Biochem. + ref + 182 + 1989 + 49453 + Calculation of protein extinction coefficients from amino acid sequence data + + + 307 + 326 + surname:Otwinowski;given-names:Z. + surname:Minor;given-names:W. + REF + Macromol. Crystallogr. A + ref + 276 + 1997 + 49530 + Processing of X-ray diffraction data collected in oscillation mode + + + 658 + 674 + surname:McCoy;given-names:A. J. + 19461840 + REF + J. Appl. Crystallogr. + ref + 40 + 2007 + 49597 + Phaser crystallographic software + + + 1948 + 1954 + surname:Adams;given-names:P. D. + 12393927 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 58 + 2002 + 49630 + PHENIX: building new software for automated crystallographic structure determination + + + 2126 + 2132 + surname:Emsley;given-names:P. + surname:Cowtan;given-names:K. + 15572765 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 60 + 2004 + 49715 + Coot: model-building tools for molecular graphics + + + 283 + 291 + surname:Laskowski;given-names:R. A. + surname:Macarthur;given-names:M. W. + surname:Moss;given-names:D. S. + surname:Thornton;given-names:J. M. + REF + J. Appl. Crystallogr. + ref + 26 + 1993 + 49765 + Procheck—a program to check the stereochemical quality of protein structures + + + 174 + 181 + surname:Stewart;given-names:A. E. + surname:Dowd;given-names:S. + surname:Keyse;given-names:S. M. + surname:McDonald;given-names:N. Q. + 10048930 + REF + Nat. Struct. Biol. + ref + 6 + 1999 + 49844 + Crystal structure of the MAPK phosphatase Pyst1 catalytic domain and implications for regulated activation + + + 880 + 886 + surname:Tao;given-names:X. + surname:Tong;given-names:L. + 17400920 + REF + Protein Sci. + ref + 16 + 2007 + 49951 + Crystal structure of the MAP kinase binding domain and the catalytic domain of human MKP5 + + + 4884 + 4887 + surname:Webb;given-names:M. R. + 1534409 + REF + Proc. Natl Acad. Sci. USA + ref + 89 + 1992 + 50041 + A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems + + + 348 + 355 + surname:Sergienko;given-names:E. A. + surname:Srivastava;given-names:D. K. + 7810877 + REF + Anal. Biochem. + ref + 221 + 1994 + 50170 + A continuous spectrophotometric method for the determination of glycogen phosphorylase-catalyzed reaction in the direction of glycogen synthesis + + + Unit 5.6 + surname:Eswar;given-names:N. + 18428767 + REF + Curr. Protoc. Bioinformatics + ref + 5 + 2006 + 50315 + Comparative protein structure modeling using Modeller + + + 3586 + 3616 + surname:MacKerell;given-names:A. D. + 24889800 + REF + J. Phys. Chem. B + ref + 102 + 1998 + 50369 + All-atom empirical potential for molecular modeling and dynamics studies of proteins + + + 1781 + 1802 + surname:Phillips;given-names:J. C. + 16222654 + REF + J. Comput. Chem. + ref + 26 + 2005 + 50454 + Scalable molecular dynamics with NAMD + + + 33 + 38 + surname:Humphrey;given-names:W. + surname:Dalke;given-names:A. + surname:Schulten;given-names:K. + 8744570 + REF + J. Mol. Graph. + ref + 14 + 1996 + 50492 + VMD: visual molecular dynamics + + + SUPPL + footnote + 50523 + Author contributions X.L. and C.-S.Z. performed the experiments and analysed the data. C.L. performed the MD simulation. Z.-X.W., J.-W.W. and S.-C.L. conceived and designed the experiments, and wrote the manuscript. + + + ncomms10879-f1.jpg + f1 + FIG + fig_title_caption + 50739 + Domain structures of ten human MKPs and the atypical VHR. + + 0.9988306 + evidence + cleaner0 + 2023-07-12T14:44:49Z + DUMMY: + + structures + + + 0.999137 + species + cleaner0 + 2023-07-11T20:32:28Z + MESH: + + human + + + 0.9996724 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.9510513 + protein + cleaner0 + 2023-07-11T14:22:09Z + PR: + + VHR + + + + ncomms10879-f1.jpg + f1 + FIG + fig_caption + 50797 + On the basis of sequence similarity, protein structure, substrate specificity and subcellular localization, the ten members of MKP family can be divided into three groups. The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs. The second subfamily contains MKP3, MKP4 and MKPX, which are cytoplasmic ERK-specific MKPs. The third subfamily comprises MKP5, MKP7 and hVH5, which were located in both nucleus and cytoplasm, and selectively inactivate JNK and p38. All MKPs contain both the CD and KBD domains, whereas VHR, an atypical MKP, only contains a highly conserved catalytic domain. In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs. NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines. + + 0.6954108 + evidence + cleaner0 + 2023-07-12T14:44:52Z + DUMMY: + + structure + + + 0.9993874 + protein_type + cleaner0 + 2023-07-11T20:14:08Z + MESH: + + MKP family + + + 0.99983966 + protein + cleaner0 + 2023-07-11T19:57:04Z + PR: + + MKP1 + + + 0.9998387 + protein + cleaner0 + 2023-07-11T19:57:06Z + PR: + + MKP2 + + + 0.99979764 + protein + cleaner0 + 2023-07-11T19:57:09Z + PR: + + PAC1 + + + 0.9997702 + protein + cleaner0 + 2023-07-11T19:57:11Z + PR: + + hVH3 + + + 0.99476105 + protein_state + cleaner0 + 2023-07-12T14:50:43Z + DUMMY: + + inducible + + + protein_type + MESH: + cleaner0 + 2023-07-12T14:50:59Z + + nuclear phosphatases + + + 0.9887932 + protein_type + cleaner0 + 2023-07-11T14:06:43Z + MESH: + + ERK + + + 0.69141513 + protein_type + cleaner0 + 2023-07-11T14:06:31Z + MESH: + + JNK + + + 0.91759735 + protein_type + cleaner0 + 2023-07-11T14:06:37Z + MESH: + + p38 + + + 0.99923766 + protein_type + cleaner0 + 2023-07-11T14:03:02Z + MESH: + + MAPKs + + + 0.99983656 + protein + cleaner0 + 2023-07-11T14:16:27Z + PR: + + MKP3 + + + 0.9998363 + protein + cleaner0 + 2023-07-11T19:57:16Z + PR: + + MKP4 + + + 0.9997894 + protein + cleaner0 + 2023-07-11T19:57:18Z + PR: + + MKPX + + + 0.9996675 + protein_type + cleaner0 + 2023-07-11T18:52:27Z + MESH: + + ERK-specific MKPs + + + 0.99985766 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + 0.9998541 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + 0.99983954 + protein + cleaner0 + 2023-07-11T19:57:27Z + PR: + + hVH5 + + + 0.81356543 + protein_type + cleaner0 + 2023-07-11T14:06:31Z + MESH: + + JNK + + + 0.81705546 + protein_type + cleaner0 + 2023-07-11T14:06:37Z + MESH: + + p38 + + + 0.99963415 + protein_type + cleaner0 + 2023-07-11T14:03:20Z + MESH: + + MKPs + + + 0.9998186 + structure_element + cleaner0 + 2023-07-11T14:16:12Z + SO: + + CD + + + 0.9998085 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.99979275 + protein + cleaner0 + 2023-07-11T14:22:09Z + PR: + + VHR + + + 0.9995327 + protein_type + cleaner0 + 2023-07-11T20:17:18Z + MESH: + + MKP + + + 0.9995488 + protein_state + cleaner0 + 2023-07-12T14:51:29Z + DUMMY: + + highly conserved + + + 0.9996837 + structure_element + cleaner0 + 2023-07-11T20:20:49Z + SO: + + catalytic domain + + + 0.99982965 + structure_element + cleaner0 + 2023-07-11T14:16:12Z + SO: + + CD + + + 0.99981815 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9998565 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + 0.9995472 + structure_element + cleaner0 + 2023-07-11T20:26:41Z + SO: + + C-terminal region + + + 0.9997634 + structure_element + cleaner0 + 2023-07-11T19:57:33Z + SO: + + NES + + + 0.99974984 + structure_element + cleaner0 + 2023-07-11T19:57:36Z + SO: + + NLS + + + 0.9996542 + structure_element + cleaner0 + 2023-07-11T20:26:46Z + SO: + + PEST motifs + + + 0.9998586 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + 0.9992887 + protein_type + cleaner0 + 2023-07-11T14:03:02Z + MESH: + + MAPKs + + + 0.9963917 + structure_element + cleaner0 + 2023-07-11T19:57:40Z + SO: + + NES + + + structure_element + SO: + cleaner0 + 2023-07-11T19:57:55Z + + nuclear export signal + + + 0.9986312 + structure_element + cleaner0 + 2023-07-11T19:57:38Z + SO: + + NLS + + + 0.90655786 + structure_element + cleaner0 + 2023-07-11T19:57:57Z + SO: + + nuclear localization signal + + + 0.99957305 + structure_element + cleaner0 + 2023-07-11T19:57:59Z + SO: + + PEST + + + 0.94908106 + structure_element + cleaner0 + 2023-07-11T19:58:01Z + SO: + + C-terminal sequence rich + + + 0.9986249 + residue_name + cleaner0 + 2023-07-11T18:52:32Z + SO: + + prolines + + + 0.99889416 + residue_name + cleaner0 + 2023-07-11T18:52:34Z + SO: + + glutamates + + + 0.9987664 + residue_name + cleaner0 + 2023-07-11T18:52:37Z + SO: + + serines + + + 0.99859756 + residue_name + cleaner0 + 2023-07-11T18:52:39Z + SO: + + threonines + + + + ncomms10879-f2.jpg + f2 + FIG + fig_title_caption + 51826 + MKP7-CD is crucial for JNK1 binding and enzyme catalysis. + + 0.9993863 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.9998307 + protein + cleaner0 + 2023-07-11T14:04:01Z + PR: + + JNK1 + + + + ncomms10879-f2.jpg + f2 + FIG + fig_caption + 51884 + (a) Domain organization of human MKP7 and JNK1. The KBD and CD of MKP7 are shown in green and blue, and the N-lobe and C-lobe of JNK1 are coloured in lemon and yellow, respectively. The key structural elements are indicated. The colour scheme is the same in the following figures unless indicated otherwise. (b) Plots of initial velocity of the MKP7-catalysed reaction versus phospho-JNK1 concentration. The solid lines are best-fitting results according to equation (1). Each experiment was performed in replicate for at least three times. The error bars represent s.e.m. (c) Gel filtration analysis for interaction of JNK1 with MKP7-CD and MKP7-KBD. (d) GST-mediated pull-down assay for interaction of JNK1 with MKP7-CD and MKP7-KBD. The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. The protein amounts of MKP7 used are shown at the bottom. + + 0.9990736 + species + cleaner0 + 2023-07-11T20:32:28Z + MESH: + + human + + + 0.9998344 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + 0.99976367 + protein + cleaner0 + 2023-07-11T14:04:01Z + PR: + + JNK1 + + + 0.9998006 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.9997886 + structure_element + cleaner0 + 2023-07-11T14:16:12Z + SO: + + CD + + + 0.99983644 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + 0.99769574 + structure_element + cleaner0 + 2023-07-11T20:26:51Z + SO: + + N-lobe + + + 0.9928906 + structure_element + cleaner0 + 2023-07-11T20:26:54Z + SO: + + C-lobe + + + 0.99968266 + protein + cleaner0 + 2023-07-11T14:04:01Z + PR: + + JNK1 + + + evidence + DUMMY: + cleaner0 + 2023-07-12T14:45:13Z + + Plots of initial velocity + + + 0.9997818 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + 0.7825839 + ptm + cleaner0 + 2023-07-12T15:02:31Z + MESH: + + phospho + + + 0.9747847 + protein + cleaner0 + 2023-07-11T14:04:01Z + PR: + + JNK1 + + + 0.99949336 + experimental_method + cleaner0 + 2023-07-12T14:58:18Z + MESH: + + Gel filtration analysis + + + 0.9996594 + protein + cleaner0 + 2023-07-11T14:04:01Z + PR: + + JNK1 + + + 0.99578506 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + protein + PR: + cleaner0 + 2023-07-11T14:04:15Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:14:22Z + + KBD + + + 0.9995559 + experimental_method + cleaner0 + 2023-07-12T14:58:21Z + MESH: + + GST-mediated pull-down assay + + + 0.9995567 + protein + cleaner0 + 2023-07-11T14:04:01Z + PR: + + JNK1 + + + 0.9985934 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + protein + PR: + cleaner0 + 2023-07-11T14:04:15Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:14:22Z + + KBD + + + 0.9938221 + evidence + cleaner0 + 2023-07-12T14:45:20Z + DUMMY: + + affinities + + + 0.984475 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + protein + PR: + cleaner0 + 2023-07-11T14:04:15Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:14:22Z + + KBD + + + 0.9995722 + protein + cleaner0 + 2023-07-11T14:04:01Z + PR: + + JNK1 + + + 0.999506 + evidence + cleaner0 + 2023-07-12T14:45:23Z + DUMMY: + + affinity + + + 0.9986137 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.99982244 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + 0.9996997 + protein + cleaner0 + 2023-07-11T14:04:01Z + PR: + + JNK1 + + + 0.9995669 + experimental_method + cleaner0 + 2023-07-12T14:58:25Z + MESH: + + GST pull-down assays + + + 0.9998379 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + + ncomms10879-f3.jpg + f3 + FIG + fig_title_caption + 52894 + Structure of JNK1 in complex with MKP7-CD. + + 0.99948704 + evidence + cleaner0 + 2023-07-12T14:45:27Z + DUMMY: + + Structure + + + 0.9998179 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.9992642 + protein_state + cleaner0 + 2023-07-12T14:51:34Z + DUMMY: + + in complex with + + + protein + PR: + cleaner0 + 2023-07-11T14:04:15Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + + ncomms10879-f3.jpg + f3 + FIG + fig_caption + 52937 + (a) Ribbon diagram of JNK1–MKP7-CD complex in two views related by a 45° rotation around a vertical axis. (b) Structure of MKP7-CD with its active site highlight in cyan. The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). The JNK1 is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). (e) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. MKP7-CD is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). Blue dashed lines represent polar interactions. (f) The 2Fo−Fc omit map (contoured at 1.5σ) clearly shows electron density for the 285FNFL288 segment of MKP7-CD. + + 0.99973774 + complex_assembly + cleaner0 + 2023-07-11T19:51:19Z + GO: + + JNK1–MKP7-CD + + + 0.95589375 + evidence + cleaner0 + 2023-07-12T14:45:30Z + DUMMY: + + Structure + + + 0.9310647 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.9996388 + site + cleaner0 + 2023-07-12T14:38:56Z + SO: + + active site + + + 0.9995763 + evidence + cleaner0 + 2023-07-12T14:45:33Z + DUMMY: + + 2Fo−Fc omit map + + + 0.99972206 + structure_element + cleaner0 + 2023-07-11T15:41:48Z + SO: + + P-loop + + + protein + PR: + cleaner0 + 2023-07-11T14:04:15Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.9885592 + evidence + cleaner0 + 2023-07-12T14:45:36Z + DUMMY: + + Structure + + + 0.99786264 + protein + cleaner0 + 2023-07-11T14:22:09Z + PR: + + VHR + + + 0.99964476 + site + cleaner0 + 2023-07-12T14:38:59Z + SO: + + active site + + + 0.9995858 + site + cleaner0 + 2023-07-12T14:39:02Z + SO: + + JNK1–MKP7 interface + + + 0.9985259 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:15Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.9991696 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.9993895 + site + cleaner0 + 2023-07-12T14:39:07Z + SO: + + Interaction networks + + + 0.9997509 + structure_element + cleaner0 + 2023-07-11T20:26:59Z + SO: + + helices + + + 0.9997986 + structure_element + cleaner0 + 2023-07-11T20:27:02Z + SO: + + α4 + + + 0.9997985 + structure_element + cleaner0 + 2023-07-11T20:27:04Z + SO: + + α5 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:15Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.9997863 + structure_element + cleaner0 + 2023-07-11T20:27:07Z + SO: + + αG + + + 0.9997346 + structure_element + cleaner0 + 2023-07-11T20:27:09Z + SO: + + α2L14 + + + 0.99909556 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:15Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:06Z + + polar interactions + + + 0.9995697 + evidence + cleaner0 + 2023-07-12T14:45:39Z + DUMMY: + + 2Fo−Fc omit map + + + 0.99958444 + evidence + cleaner0 + 2023-07-12T14:45:42Z + DUMMY: + + electron density + + + structure_element + SO: + cleaner0 + 2023-07-11T19:45:11Z + + 285FNFL288 segment + + + protein + PR: + cleaner0 + 2023-07-11T14:04:15Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + + ncomms10879-f4.jpg + f4 + FIG + fig_title_caption + 53913 + Mutational analysis on interactions between MKP7-CD and JNK1. + + 0.99958706 + experimental_method + cleaner0 + 2023-07-12T14:58:42Z + MESH: + + Mutational analysis + + + 0.9992169 + protein + cleaner0 + 2023-07-11T14:04:15Z + PR: + + MKP7 + + + 0.58408344 + structure_element + cleaner0 + 2023-07-11T14:16:12Z + SO: + + CD + + + 0.99970716 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + + ncomms10879-f4.jpg + f4 + FIG + fig_caption + 53975 + (a) Effects of mutations in MKP7-CD on the JNK1 dephosphorylation (mean±s.e.m., n=3). Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. (b) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D. Mutant F285D and JNK1 were eluted as monomers, with the molecular masses of ∼17 and 44 kDa, respectively. However, in contrast to the wild-type MKP7-CD, mutant F285D did not co-migrate with JNK1. (c) Pull-down assays of MKP7-CD by GST-tagged JNK1 mutants. The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. The protein amounts of MKP7-CD used are shown at the bottom. (d) Circular dichroism spectra for MKP7-CD wild type and mutants. Measurements were averaged for three scans. (e) Circular dichroism spectra for JNK1 wild type and mutants. Measurements were averaged for three scans. (f) Effects of mutations in MKP7-CD on the pNPP hydrolysis reaction (mean±s.e.m., n=3). + + protein + PR: + cleaner0 + 2023-07-11T14:04:16Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.99977106 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T14:01:33Z + + dephosphorylation + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:06Z + + hydrophobic and hydrophilic contacts + + + 0.9995289 + experimental_method + cleaner0 + 2023-07-12T14:58:45Z + MESH: + + Gel filtration analysis + + + 0.9996489 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:16Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.9994962 + protein_state + cleaner0 + 2023-07-11T15:48:15Z + DUMMY: + + mutant + + + 0.99963117 + mutant + cleaner0 + 2023-07-11T15:53:12Z + MESH: + + F285D + + + 0.9994444 + protein_state + cleaner0 + 2023-07-11T15:48:15Z + DUMMY: + + Mutant + + + 0.99965394 + mutant + cleaner0 + 2023-07-11T15:53:12Z + MESH: + + F285D + + + 0.9996574 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.9994036 + oligomeric_state + cleaner0 + 2023-07-12T15:01:07Z + DUMMY: + + monomers + + + 0.9995597 + protein_state + cleaner0 + 2023-07-11T15:48:09Z + DUMMY: + + wild-type + + + protein + PR: + cleaner0 + 2023-07-11T14:04:16Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.99948275 + protein_state + cleaner0 + 2023-07-11T15:48:15Z + DUMMY: + + mutant + + + 0.99964154 + mutant + cleaner0 + 2023-07-11T15:53:12Z + MESH: + + F285D + + + 0.9996581 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.99957776 + experimental_method + cleaner0 + 2023-07-12T14:58:48Z + MESH: + + Pull-down assays + + + protein + PR: + cleaner0 + 2023-07-11T14:04:16Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.9913747 + protein_state + cleaner0 + 2023-07-12T14:51:39Z + DUMMY: + + GST-tagged + + + 0.9995147 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.96717983 + evidence + cleaner0 + 2023-07-12T14:45:47Z + DUMMY: + + affinities + + + protein + PR: + cleaner0 + 2023-07-11T14:04:16Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.9995888 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.9993345 + evidence + cleaner0 + 2023-07-12T14:45:51Z + DUMMY: + + affinity + + + 0.9995756 + protein_state + cleaner0 + 2023-07-11T15:48:09Z + DUMMY: + + wild-type + + + 0.99977297 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + protein + PR: + cleaner0 + 2023-07-11T14:04:16Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.99935895 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.99956286 + experimental_method + cleaner0 + 2023-07-12T14:58:51Z + MESH: + + GST pull-down assays + + + protein + PR: + cleaner0 + 2023-07-11T14:04:16Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + experimental_method + MESH: + cleaner0 + 2023-07-12T14:46:18Z + + Circular dichroism + + + evidence + DUMMY: + cleaner0 + 2023-07-12T14:46:27Z + + spectra + + + protein + PR: + cleaner0 + 2023-07-11T14:04:16Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.9995651 + protein_state + cleaner0 + 2023-07-11T15:54:35Z + DUMMY: + + wild type + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + experimental_method + MESH: + cleaner0 + 2023-07-12T14:46:19Z + + Circular dichroism + + + evidence + DUMMY: + cleaner0 + 2023-07-12T14:46:44Z + + spectra + + + 0.99968576 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.99957126 + protein_state + cleaner0 + 2023-07-11T15:54:35Z + DUMMY: + + wild type + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.59046876 + experimental_method + cleaner0 + 2023-07-12T14:58:55Z + MESH: + + mutations + + + protein + PR: + cleaner0 + 2023-07-11T14:04:16Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.99979097 + chemical + cleaner0 + 2023-07-12T15:02:37Z + CHEBI: + + pNPP + + + + ncomms10879-f5.jpg + f5 + FIG + fig_title_caption + 55099 + Comparison of CDK2-KAP and JNK1–MKP7-CD. + + 0.8294056 + complex_assembly + cleaner0 + 2023-07-11T20:33:53Z + GO: + + CDK2-KAP + + + 0.9901239 + complex_assembly + cleaner0 + 2023-07-11T19:51:19Z + GO: + + JNK1–MKP7-CD + + + + ncomms10879-f5.jpg + f5 + FIG + fig_caption + 55142 + (a) Superposition of the complex structures of CDK2-KAP (PDB 1FQ1) and JNK1–MKP7-CD. The N-lobe and C-lobe of CDK2 are coloured in grey and pink, respectively, and KAP is coloured in green. The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I). Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II). (b) Interactions networks at the auxiliary region II mainly involving helix α7 from KAP and the αG helix and following L14 loop of CDK2. The orientation of the panel is almost identical to those of Fig. 3d. The CDK2 is shown in surface representation coloured according to the electrostatic potential (positive, blue; negative, red). Residues of KAP and CDK2 are highlighted as green and red sticks, respectively. Blue dashed lines represent polar interactions. One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. Residues of MKP7-CD involved in JNK1 recognition are indicated by cyan asterisks, and the conserved FXF-motif is highlighted in cyan. The secondary structure assignments of MKP7-CD and KAP are shown above and below each sequence. (d) Sequence alignment of the F-site regions on MAPKs. Residues of JNK1 involved in recognition of MKP7 are indicated by orange asterisks, and those forming the F-site are highlighted in yellow. + + 0.99969125 + experimental_method + cleaner0 + 2023-07-12T14:58:59Z + MESH: + + Superposition + + + 0.99635834 + evidence + cleaner0 + 2023-07-12T14:46:51Z + DUMMY: + + structures + + + 0.9996756 + complex_assembly + cleaner0 + 2023-07-11T20:33:59Z + GO: + + CDK2-KAP + + + 0.9996969 + complex_assembly + cleaner0 + 2023-07-11T19:51:19Z + GO: + + JNK1–MKP7-CD + + + 0.9981871 + structure_element + cleaner0 + 2023-07-11T20:27:15Z + SO: + + N-lobe + + + 0.996067 + structure_element + cleaner0 + 2023-07-11T20:27:18Z + SO: + + C-lobe + + + 0.9996972 + protein + cleaner0 + 2023-07-11T15:51:12Z + PR: + + CDK2 + + + 0.9975011 + protein + cleaner0 + 2023-07-11T15:50:50Z + PR: + + KAP + + + 0.8763778 + site + cleaner0 + 2023-07-12T14:39:12Z + SO: + + contact regions + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:06Z + + hydrogen 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protein + PR: + cleaner0 + 2023-07-11T14:04:16Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.9996482 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.9995695 + protein_state + cleaner0 + 2023-07-12T14:51:48Z + DUMMY: + + conserved + + + 0.9996901 + structure_element + cleaner0 + 2023-07-11T14:06:58Z + SO: + + FXF-motif + + + protein + PR: + cleaner0 + 2023-07-11T14:04:16Z + + MKP7 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.99891937 + protein + cleaner0 + 2023-07-11T15:50:50Z + PR: + + KAP + + + 0.99956197 + experimental_method + cleaner0 + 2023-07-12T14:59:06Z + MESH: + + Sequence alignment + + + structure_element + SO: + cleaner0 + 2023-07-11T20:28:31Z + + F-site regions + + + 0.9995608 + protein_type + cleaner0 + 2023-07-11T14:03:02Z + MESH: + + MAPKs + + + 0.9998103 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.99984515 + protein + cleaner0 + 2023-07-11T14:04:16Z + PR: + + MKP7 + + + 0.99884516 + site + cleaner0 + 2023-07-12T14:39:31Z + SO: + + F-site + + + + ncomms10879-f6.jpg + f6 + FIG + fig_title_caption + 56864 + FXF-motif is critical for controlling the phosphorylation of JNK and ultraviolet-induced apoptosis. + + 0.99970436 + structure_element + cleaner0 + 2023-07-11T14:06:58Z + SO: + + FXF-motif + + + 0.9987393 + ptm + cleaner0 + 2023-07-12T15:04:07Z + MESH: + + phosphorylation + + + 0.8759518 + protein_type + cleaner0 + 2023-07-11T14:06:31Z + MESH: + + JNK + + + + ncomms10879-f6.jpg + f6 + FIG + fig_caption + 56964 + (a–c) FXF-motif is essential for the dephosphorylation of JNK by MKP7. HEK293T cells were infected with lentiviruses expressing MKP7 and its mutants (1.0 μg). After 36 h infection, cells were untreated in a, stimulated with 30 μM etoposide for 3 h in b or irradiated with 25 J m−2 ultraviolet light at 30 min before lysis in c. Whole-cell extracts were then immunoblotted with antibody indicated. Shown is a typical immunoblot for phosphorylated JNK from three independent experiments. (d) F-site is required for JNK1 to interact with MKP7. HEK293T cells were co-transfected with MKP7 full-length (1.0 μg) and JNK1 (wild type or mutants as indicated, 1.0 μg). At 16 h post transfection, cells were lysed. Whole-cell extracts were then immunoprecipitated with antibody against Myc for MKP7; immunobloting was carried out with antibodies indicated. IP, immunoprecipitation; TCL, total cell lysate. Shown is a typical result from three independent experiments. (e) Effect of MKP7 (wild type or mutants) expression on ultraviolet-induced apoptosis. HeLa cells were infected with lentiviruses expressing MKP7 full-length and its mutants. At 36 h post infection, cells were irradiated with 25 J m−2 ultraviolet light and collected at 6 h after irradiation. Cells were then subjected to flow cytometry analysis. Apoptotic cells were determined by Annexin-V-APC/PI staining. The results using Annexin-V stain for membrane phosphatidylserine eversion, combined with propidium iodide (PI) uptake to evaluate cells whose membranes had been compromised. Staining with both Annexin-V and PI indicate apoptosis (upper right quadrant). The values shown in the lower left, and upper right quadrants of each panel represent the percentage of viable, and apoptotic cells, respectively. All results are representative of three independent experiments. (f) Statistical analysis of apoptotic cells (mean±s.e.m., n=3), *P<0.05, ***P<0.001 (ANOVA followed by Tukey's test). NS, not significant. + + 0.99860597 + structure_element + cleaner0 + 2023-07-11T14:06:58Z + SO: + + FXF-motif + + + 0.9997149 + protein_type + cleaner0 + 2023-07-11T14:06:31Z + MESH: + + JNK + + + 0.9998548 + protein + cleaner0 + 2023-07-11T14:04:16Z + PR: + + MKP7 + + + 0.99673367 + taxonomy_domain + cleaner0 + 2023-07-12T14:59:33Z + DUMMY: + + lentiviruses + + + 0.99986315 + protein + cleaner0 + 2023-07-11T14:04:16Z + PR: + + MKP7 + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.75931406 + chemical + cleaner0 + 2023-07-11T15:52:00Z + CHEBI: + + etoposide + + + 0.99608105 + protein_state + cleaner0 + 2023-07-11T20:03:12Z + DUMMY: + + phosphorylated + + + 0.9987192 + protein_type + cleaner0 + 2023-07-11T14:06:31Z + MESH: + + JNK + + + 0.8712673 + site + cleaner0 + 2023-07-12T14:39:37Z + SO: + + F-site + + + 0.9998406 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.99986076 + protein + cleaner0 + 2023-07-11T14:04:16Z + PR: + + MKP7 + + + 0.9993498 + experimental_method + cleaner0 + 2023-07-12T14:59:46Z + MESH: + + co-transfected + + + 0.9998604 + protein + cleaner0 + 2023-07-11T14:04:16Z + PR: + + MKP7 + + + 0.99951607 + protein_state + cleaner0 + 2023-07-11T15:54:25Z + DUMMY: + + full-length + + + 0.9998323 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.9995818 + protein_state + cleaner0 + 2023-07-11T15:54:35Z + DUMMY: + + wild type + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.9974833 + experimental_method + cleaner0 + 2023-07-12T14:59:49Z + MESH: + + immunoprecipitated + + + 0.9998566 + protein + cleaner0 + 2023-07-11T14:04:16Z + PR: + + MKP7 + + + 0.99774736 + experimental_method + cleaner0 + 2023-07-12T14:59:53Z + MESH: + + IP + + + 0.99863476 + experimental_method + cleaner0 + 2023-07-12T14:59:56Z + MESH: + + immunoprecipitation + + + 0.9998597 + protein + cleaner0 + 2023-07-11T14:04:16Z + PR: + + MKP7 + + + 0.999576 + protein_state + cleaner0 + 2023-07-11T15:54:35Z + DUMMY: + + wild type + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.9986125 + taxonomy_domain + cleaner0 + 2023-07-12T15:00:10Z + DUMMY: + + lentiviruses + + + 0.9998605 + protein + cleaner0 + 2023-07-11T14:04:16Z + PR: + + MKP7 + + + 0.9995164 + protein_state + cleaner0 + 2023-07-11T15:54:25Z + DUMMY: + + full-length + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T20:04:10Z + + mutants + + + 0.99808496 + experimental_method + cleaner0 + 2023-07-12T15:00:15Z + MESH: + + flow cytometry + + + 0.9895574 + chemical + cleaner0 + 2023-07-12T15:03:07Z + CHEBI: + + Annexin-V-APC + + + 0.9218436 + chemical + cleaner0 + 2023-07-12T15:03:10Z + CHEBI: + + PI + + + 0.99781734 + chemical + cleaner0 + 2023-07-12T15:03:28Z + CHEBI: + + Annexin-V + + + 0.9977014 + chemical + cleaner0 + 2023-07-12T15:03:12Z + CHEBI: + + propidium iodide + + + 0.9991844 + chemical + cleaner0 + 2023-07-12T15:03:16Z + CHEBI: + + PI + + + 0.9990835 + chemical + cleaner0 + 2023-07-12T15:03:31Z + CHEBI: + + Annexin-V + + + 0.9988003 + chemical + cleaner0 + 2023-07-12T15:03:49Z + CHEBI: + + PI + + + evidence + DUMMY: + cleaner0 + 2023-07-11T20:13:34Z + + *P + + + evidence + DUMMY: + cleaner0 + 2023-07-11T20:13:41Z + + ***P + + + 0.99305326 + experimental_method + cleaner0 + 2023-07-12T15:00:17Z + MESH: + + ANOVA + + + experimental_method + MESH: + cleaner0 + 2023-07-11T20:13:19Z + + Tukey's test + + + + ncomms10879-f7.jpg + f7 + FIG + fig_title_caption + 58975 + MKP5-CD is crucial for JNK1 binding and enzyme catalysis. + + 0.99952304 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.99982834 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + + ncomms10879-f7.jpg + f7 + FIG + fig_caption + 59033 + (a) Domain organization of human MKP5. The KBD and CD of MKP5 are shown in brown and grey, respectively. (b) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration. The solid lines are best-fitting results according to the Michaelis–Menten equation with Km and kcat values indicated. Each experiment was performed in replicate for at least three times. The error bars represent s.e.m. (c) Structural comparison of the JNK-interacting residues on MKP5-CD (PDB 1ZZW) and MKP7-CD. The corresponding residues on MKP5 are depicted as orange sticks, and MKP5 residues numbers are in parentheses. (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. The panels are arranged the same as in Fig. 2d. (f) Effects of mutations in MKP5-CD on the JNK1 dephosphorylation (mean±s.e.m., n=3). (g) Effects of mutations in MKP5-CD on the pNPP hydrolysis reaction (mean±s.e.m., n=3). (h) Pull-down assays of MKP5-CD by GST-tagged JNK1 mutants. The panels are arranged the same as in Fig. 4c. + + 0.99896634 + species + cleaner0 + 2023-07-11T20:32:28Z + MESH: + + human + + + 0.9998555 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + 0.9997814 + structure_element + cleaner0 + 2023-07-11T14:14:22Z + SO: + + KBD + + + 0.999783 + structure_element + cleaner0 + 2023-07-11T14:16:12Z + SO: + + CD + + + 0.9998547 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + evidence + DUMMY: + cleaner0 + 2023-07-12T14:47:11Z + + Plots of initial velocity + + + 0.9998386 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + 0.61602974 + protein_state + cleaner0 + 2023-07-12T14:51:53Z + DUMMY: + + phospho + + + 0.99366367 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.99945515 + evidence + cleaner0 + 2023-07-12T14:47:14Z + DUMMY: + + Km + + + 0.9994019 + evidence + cleaner0 + 2023-07-12T14:47:17Z + DUMMY: + + kcat + + + 0.999575 + experimental_method + cleaner0 + 2023-07-12T15:00:23Z + MESH: + + Structural comparison + + + 0.9991039 + site + cleaner0 + 2023-07-12T14:39:54Z + SO: + + JNK-interacting residues + + + 0.99977833 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + 0.62858474 + structure_element + cleaner0 + 2023-07-11T14:16:12Z + SO: + + CD + + + protein + PR: + cleaner0 + 2023-07-11T14:04:16Z + + MKP7 + + + 0.46788436 + structure_element + cleaner0 + 2023-07-11T14:16:12Z + SO: + + CD + + + 0.99983907 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + 0.9998492 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + 0.9995819 + experimental_method + cleaner0 + 2023-07-12T15:00:26Z + MESH: + + Gel filtration analysis + + + 0.9997434 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.99965286 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.9639164 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:14:22Z + + KBD + + + 0.9995555 + experimental_method + cleaner0 + 2023-07-12T15:00:30Z + MESH: + + GST-mediated pull-down assays + + + 0.99949336 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.9991891 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + 0.49584374 + structure_element + cleaner0 + 2023-07-11T14:16:12Z + SO: + + CD + + + protein + PR: + cleaner0 + 2023-07-11T14:16:43Z + + MKP5 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:14:22Z + + KBD + + + 0.6695175 + experimental_method + cleaner0 + 2023-07-12T15:00:33Z + MESH: + + mutations + + + 0.9997912 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + 0.6887164 + structure_element + cleaner0 + 2023-07-11T14:16:12Z + SO: + + CD + + + 0.9993088 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.8962566 + experimental_method + cleaner0 + 2023-07-12T15:00:36Z + MESH: + + mutations + + + 0.9997826 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + 0.8319109 + structure_element + cleaner0 + 2023-07-11T14:16:12Z + SO: + + CD + + + 0.9997385 + chemical + cleaner0 + 2023-07-12T15:03:54Z + CHEBI: + + pNPP + + + 0.9995843 + experimental_method + cleaner0 + 2023-07-12T15:00:39Z + MESH: + + Pull-down assays + + + 0.9994916 + protein + cleaner0 + 2023-07-11T14:16:43Z + PR: + + MKP5 + + + structure_element + SO: + cleaner0 + 2023-07-11T14:16:12Z + + CD + + + 0.99779916 + protein_state + cleaner0 + 2023-07-12T14:51:56Z + DUMMY: + + GST-tagged + + + 0.99959534 + protein + cleaner0 + 2023-07-11T14:04:02Z + PR: + + JNK1 + + + 0.99732274 + protein_state + cleaner0 + 2023-07-11T20:04:10Z + DUMMY: + + mutants + + + + t1.xml + t1 + TABLE + table_title_caption + 60157 + Data collection and refinement statistics. + + + t1.xml + t1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th align="center" valign="top" charoff="50"><bold>JNK1–MKP7-CD</bold></th></tr></thead><tbody valign="top"><tr><td colspan="2" align="left" valign="top" charoff="50"><italic>Data collection</italic><xref ref-type="fn" rid="t1-fn1">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> Space group</td><td align="center" valign="top" charoff="50"><italic>P</italic>1</td></tr><tr><td colspan="2" align="left" valign="top" charoff="50"> Cell dimensions</td></tr><tr><td align="left" valign="top" charoff="50">  <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td align="center" valign="top" charoff="50">58.1, 74.8, 134.8</td></tr><tr><td align="left" valign="top" charoff="50">  α, β, γ (°)</td><td align="center" valign="top" charoff="50">76.9, 84.3, 67.4</td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="char" valign="top" char="(" charoff="50">40.00–2.40 (2.49–2.40)<xref ref-type="fn" rid="t1-fn2">†</xref></td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>merge</sub></td><td align="char" valign="top" char="(" charoff="50">6.4 (60.9)</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>I</italic>/σ<italic>I</italic></td><td align="char" valign="top" char="(" charoff="50">14.1 (1.9)</td></tr><tr><td align="left" valign="top" charoff="50"> Completeness (%)</td><td align="char" valign="top" char="(" charoff="50">99.6 (99.9)</td></tr><tr><td align="left" valign="top" charoff="50"> Redundancy</td><td align="char" valign="top" char="(" charoff="50">3.5 (3.4)</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="2" align="left" valign="top" charoff="50"><italic>Refinement</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="char" valign="top" char="(" charoff="50">38.02–2.40 (2.43–2.40)</td></tr><tr><td align="left" valign="top" charoff="50"> No. of reflections</td><td align="center" valign="top" charoff="50">66889</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub></td><td align="center" valign="top" charoff="50">21.7/23.9</td></tr><tr><td colspan="2" align="left" valign="top" charoff="50"> No. atoms</td></tr><tr><td align="left" valign="top" charoff="50">  Protein</td><td align="center" valign="top" charoff="50">14637</td></tr><tr><td align="left" valign="top" charoff="50">  Ligand/ion</td><td align="center" valign="top" charoff="50">4</td></tr><tr><td align="left" valign="top" charoff="50">  Water</td><td align="center" valign="top" charoff="50">457</td></tr><tr><td colspan="2" align="left" valign="top" charoff="50"> <italic>B</italic>-factors</td></tr><tr><td align="left" valign="top" charoff="50">  Protein</td><td align="center" valign="top" charoff="50">47.04</td></tr><tr><td align="left" valign="top" charoff="50">  Ligand/ion</td><td align="center" valign="top" charoff="50">30</td></tr><tr><td align="left" valign="top" charoff="50">  Water</td><td align="center" valign="top" charoff="50">37.28</td></tr><tr><td colspan="2" align="left" valign="top" charoff="50"> R.m.s.d.</td></tr><tr><td align="left" valign="top" charoff="50">  Bond lengths (Å)</td><td align="center" valign="top" charoff="50">0.015</td></tr><tr><td align="left" valign="top" charoff="50">  Bond angles (°)</td><td align="center" valign="top" charoff="50">1.564</td></tr></tbody></table> + + 60200 +   JNK1–MKP7-CD Data collection*  Space group P1  Cell dimensions   a, b, c (Å) 58.1, 74.8, 134.8   α, β, γ (°) 76.9, 84.3, 67.4  Resolution (Å) 40.00–2.40 (2.49–2.40)†  Rmerge 6.4 (60.9)  I/σI 14.1 (1.9)  Completeness (%) 99.6 (99.9)  Redundancy 3.5 (3.4)     Refinement  Resolution (Å) 38.02–2.40 (2.43–2.40)  No. of reflections 66889  Rwork/Rfree 21.7/23.9  No. atoms   Protein 14637   Ligand/ion 4   Water 457  B-factors   Protein 47.04   Ligand/ion 30   Water 37.28  R.m.s.d.   Bond lengths (Å) 0.015   Bond angles (°) 1.564 + + + t1.xml + t1 + TABLE + table_footnote + 60875 + *The data set was collected from a single crystal. + + + t1.xml + t1 + TABLE + table_footnote + 60926 + †Values in parentheses are for the highest resolution shell. + + + diff --git a/BioC_XML/4802085_v0.xml b/BioC_XML/4802085_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..598cab69e5285bdee3fec8f841fc70d77cc558a8 --- /dev/null +++ b/BioC_XML/4802085_v0.xml @@ -0,0 +1,10272 @@ + + + + PMC + 20201217 + pmc.key + + 4802085 + CC BY + no + 0 + 0 + + 10.1038/ncomms11030 + ncomms11030 + 4802085 + 26988023 + 11030 + This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ + surname:Kabe;given-names:Yasuaki + surname:Nakane;given-names:Takanori + surname:Yamamoto;given-names:Ayumi + surname:Uchida;given-names:Takeshi + surname:Iwata;given-names:So + surname:Yamaguchi;given-names:Yuki + surname:Krayukhina;given-names:Elena + surname:Noda;given-names:Masanori + surname:Handa;given-names:Hiroshi + surname:Ishimori;given-names:Koichiro + surname:Uchiyama;given-names:Susumu + surname:Kobayashi;given-names:Takuya + surname:Koike;given-names:Ikko + surname:Suematsu;given-names:Makoto + surname:Yamamoto;given-names:Tatsuya + surname:Sugiura;given-names:Yuki + surname:Harada;given-names:Erisa + surname:Sugase;given-names:Kenji + surname:Shimamura;given-names:Tatsuro + surname:Ohmura;given-names:Mitsuyo + surname:Muraoka;given-names:Kazumi + TITLE + front + 7 + 2016 + 0 + Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance + + 0.997191 + chemical + cleaner0 + 2023-07-11T10:02:49Z + CHEBI: + + Haem + + + 0.9126601 + oligomeric_state + cleaner0 + 2023-07-11T12:47:35Z + DUMMY: + + dimerization + + + 0.99922025 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.8080058 + protein + cleaner0 + 2023-07-11T10:02:38Z + PR: + + Sigma-2 + + + + ABSTRACT + abstract + 108 + Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem–haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer. + + 0.99617916 + protein + cleaner0 + 2023-07-11T10:03:09Z + PR: + + Progesterone-receptor membrane component 1 + + + 0.9996635 + protein + cleaner0 + 2023-07-11T10:03:14Z + PR: + + PGRMC1 + + + 0.9904061 + protein + cleaner0 + 2023-07-11T10:03:25Z + PR: + + Sigma-2 receptor + + + 0.99575883 + protein_type + cleaner0 + 2023-07-11T10:03:52Z + MESH: + + haem-containing protein + + + 0.99715096 + protein_type + cleaner0 + 2023-07-11T10:03:47Z + MESH: + + epidermal growth factor receptor + + + 0.9765944 + protein_type + cleaner0 + 2023-07-11T10:03:58Z + MESH: + + EGFR + + + 0.9995735 + protein_type + cleaner0 + 2023-07-11T10:04:04Z + MESH: + + cytochromes P450 + + + 0.99915475 + experimental_method + cleaner0 + 2023-07-11T13:20:56Z + MESH: + + crystallographic analyses + + + 0.99984443 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.99951756 + structure_element + cleaner0 + 2023-07-11T10:04:20Z + SO: + + cytosolic domain + + + 0.99889094 + protein_state + cleaner0 + 2023-07-11T13:41:08Z + DUMMY: + + stable + + + 0.99923956 + oligomeric_state + cleaner0 + 2023-07-11T10:05:41Z + DUMMY: + + dimer + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + stacking interactions + + + 0.99943525 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + 0.9986947 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + 0.9945403 + chemical + cleaner0 + 2023-07-11T10:04:43Z + CHEBI: + + iron + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + five-coordinated by + + + 0.9998828 + residue_name_number + cleaner0 + 2023-07-11T10:15:12Z + DUMMY: + + Tyr113 + + + site + SO: + cleaner0 + 2023-07-11T13:45:29Z + + surface + + + 0.9977743 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9994272 + chemical + cleaner0 + 2023-07-11T10:03:34Z + CHEBI: + + Carbon monoxide + + + 0.9997272 + chemical + cleaner0 + 2023-07-11T10:03:39Z + CHEBI: + + CO + + + 0.9998229 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9994967 + site + cleaner0 + 2023-07-11T13:45:40Z + SO: + + sixth coordination site + + + 0.99301106 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + 0.97565484 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + Haem + + + 0.99980956 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.97255594 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.9996189 + protein_type + cleaner0 + 2023-07-11T10:04:05Z + MESH: + + cytochromes P450 + + + 0.99971694 + chemical + cleaner0 + 2023-07-11T10:03:40Z + CHEBI: + + CO + + + 0.97554827 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + haem–haem stacking + + + 0.999215 + taxonomy_domain + cleaner0 + 2023-07-11T10:04:12Z + DUMMY: + + eukaryotes + + + + ABSTRACT + abstract + 1195 + PGRMC1 binds to EGFR and cytochromes P450, and is known to be involved in cancer proliferation and in drug resistance. Here, the authors determine the structure of the cytosolic domain of PGRMC1, which forms a dimer via haem–haem stacking, and propose how this interaction could be involved in its function. + + 0.99971217 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.51908803 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.9995978 + protein_type + cleaner0 + 2023-07-11T10:04:05Z + MESH: + + cytochromes P450 + + + 0.99931693 + evidence + cleaner0 + 2023-07-11T13:31:08Z + DUMMY: + + structure + + + 0.99953073 + structure_element + cleaner0 + 2023-07-11T10:04:21Z + SO: + + cytosolic domain + + + 0.99980575 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.9992767 + oligomeric_state + cleaner0 + 2023-07-11T10:05:41Z + DUMMY: + + dimer + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + haem–haem stacking + + + + INTRO + paragraph + 1506 + Much attention has been paid to the roles of haem-iron in cancer development. Increased dietary intake of haem is a risk factor for several types of cancer. Previous studies showed that deprivation of iron or haem suppresses tumourigenesis. On the other hand, carbon monoxide (CO), the gaseous mediator generated by oxidative degradation of haem via haem oxygenase (HO), inhibits tumour growth. Thus, a tenuous balance between free haem and CO plays key roles in cancer development and chemoresistance, although the underlying mechanisms are not fully understood. + + 0.999345 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + 0.924516 + chemical + cleaner0 + 2023-07-11T10:04:43Z + CHEBI: + + iron + + + 0.99958843 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + 0.98989916 + protein_state + cleaner0 + 2023-07-11T13:41:13Z + DUMMY: + + deprivation of + + + 0.99878734 + chemical + cleaner0 + 2023-07-11T10:04:42Z + CHEBI: + + iron + + + 0.99934775 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + 0.9995382 + chemical + cleaner0 + 2023-07-11T10:03:34Z + CHEBI: + + carbon monoxide + + + 0.9997185 + chemical + cleaner0 + 2023-07-11T10:03:40Z + CHEBI: + + CO + + + 0.99952734 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + 0.9991763 + protein_type + cleaner0 + 2023-07-11T10:04:34Z + MESH: + + haem oxygenase + + + 0.9995011 + protein_type + cleaner0 + 2023-07-11T10:04:48Z + MESH: + + HO + + + 0.9997608 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + 0.9997502 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + + INTRO + paragraph + 2070 + To gain insight into the underlying mechanisms, we took chemical biological approaches using affinity nanobeads carrying haem and identified progesterone-receptor membrane component 1 (PGRMC1) as a haem-binding protein from mouse liver extracts (Supplementary Fig. 1). PGRMC1 is a member of the membrane-associated progesterone receptor (MAPR) family with a cytochrome b5-like haem-binding region, and is known to be highly expressed in various types of cancers. PGRMC1 is anchored to the cell membrane through the N-terminal transmembrane helix and interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 (ref). While PGRMC1 is implicated in cell proliferation and cholesterol biosynthesis, the structural basis on which PGRMC1 exerts its function remains largely unknown. + + 0.99945414 + experimental_method + cleaner0 + 2023-07-11T13:21:03Z + MESH: + + affinity nanobeads + + + 0.9996358 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + 0.99765295 + protein + cleaner0 + 2023-07-11T10:03:10Z + PR: + + progesterone-receptor membrane component 1 + + + 0.9997446 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.52183425 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + 0.99361974 + taxonomy_domain + cleaner0 + 2023-07-11T10:05:12Z + DUMMY: + + mouse + + + 0.99970585 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.9996416 + protein_type + cleaner0 + 2023-07-11T10:05:21Z + MESH: + + membrane-associated progesterone receptor + + + 0.99951077 + protein_type + cleaner0 + 2023-07-11T10:05:26Z + MESH: + + MAPR + + + 0.89973205 + structure_element + cleaner0 + 2023-07-11T13:31:39Z + SO: + + cytochrome b5-like + + + 0.9463763 + site + cleaner0 + 2023-07-11T10:07:42Z + SO: + + haem-binding region + + + 0.64385563 + protein_state + cleaner0 + 2023-07-11T13:41:29Z + DUMMY: + + highly expressed + + + 0.9997782 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.99967134 + structure_element + cleaner0 + 2023-07-11T13:39:58Z + SO: + + transmembrane helix + + + 0.99742174 + protein_type + cleaner0 + 2023-07-11T10:03:48Z + MESH: + + epidermal growth factor receptor + + + 0.94433254 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.99960387 + protein_type + cleaner0 + 2023-07-11T10:04:06Z + MESH: + + cytochromes P450 + + + 0.9997558 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.99980503 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + + INTRO + paragraph + 2864 + Here we show that PGRMC1 exhibits a unique haem-dependent dimerization. The dimer binds to EGFR and cytochromes P450 to enhance tumour cell proliferation and chemoresistance. The dimer is dissociated to monomers by physiological levels of CO, suggesting that PGRMC1 serves as a CO-sensitive molecular switch regulating cancer cell proliferation. + + 0.99980026 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.42081538 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9994338 + oligomeric_state + cleaner0 + 2023-07-11T10:05:40Z + DUMMY: + + dimer + + + 0.9223955 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.9995984 + protein_type + cleaner0 + 2023-07-11T10:04:06Z + MESH: + + cytochromes P450 + + + 0.9994241 + oligomeric_state + cleaner0 + 2023-07-11T10:05:41Z + DUMMY: + + dimer + + + 0.99941826 + oligomeric_state + cleaner0 + 2023-07-11T10:10:59Z + DUMMY: + + monomers + + + 0.9996674 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + 0.99979967 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.99934083 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + + RESULTS + title_1 + 3210 + Results + + + RESULTS + title_2 + 3218 + X-ray crystal structure of PGRMC1 + + 0.9995533 + evidence + cleaner0 + 2023-07-11T13:31:48Z + DUMMY: + + X-ray crystal structure + + + 0.9998611 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + + RESULTS + paragraph + 3252 + We solved the crystal structure of the haem-bound PGRMC1 cytosolic domain (a.a.72–195) at 1.95 Å resolution (Supplementary Fig. 2). In the presence of haem, PGRMC1 forms a dimeric structure largely through hydrophobic interactions between the haem moieties of two monomers (Fig. 1a, Table 1 and Supplementary Fig. 3; a stereo-structural image is shown in Supplementary Fig 4). While the overall fold of PGRMC1 is similar to that of canonical cytochrome b5, their haem irons are coordinated differently. In cytochrome b5, the haem iron is six-coordinated by two axial histidine residues. These histidines are missing in PGRMC1, and the haem iron is five-coordinated by Tyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3). A homologous helix that holds haem in cytochrome b5 is longer, shifts away from haem, and does not form a coordinate bond in PGRMC1 (Fig. 1c). Consequently, the five-coordinated haem of PGRMC1 has an open surface that allows its dimerization through hydrophobic haem–haem stacking. Contrary to our finding, Kaluka et al. recently reported that Tyr164 of PGRMC1 is the axial ligand of haem because mutation of this residue impairs haem binding. Our structural data revealed that Tyr164 and a few other residues such as Tyr107 and Lys163 are in fact hydrogen-bonded to haem propionates. This is consistent with observations by Min et al. that Tyr 107 and Tyr113 of PGRMC1 are involved in binding with haem. These amino acid residues are conserved among MAPR family members (Supplementary Fig. 5a), suggesting that these proteins share the ability to exhibit haem-dependent dimerization. + + 0.9960192 + experimental_method + cleaner0 + 2023-07-11T13:21:11Z + MESH: + + solved + + + 0.9996319 + evidence + cleaner0 + 2023-07-11T13:31:51Z + DUMMY: + + crystal structure + + + 0.99956006 + protein_state + cleaner0 + 2023-07-11T10:05:56Z + DUMMY: + + haem-bound + + + 0.99983776 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.88886553 + structure_element + cleaner0 + 2023-07-11T10:04:21Z + SO: + + cytosolic domain + + + 0.9990271 + residue_range + cleaner0 + 2023-07-11T13:08:47Z + DUMMY: + + 72–195 + + + 0.99936044 + protein_state + cleaner0 + 2023-07-11T10:14:47Z + DUMMY: + + presence of + + + 0.9987789 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + 0.99973947 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.99914265 + oligomeric_state + cleaner0 + 2023-07-11T12:47:44Z + DUMMY: + + dimeric + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + hydrophobic interactions + + + 0.9972965 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + 0.99922407 + oligomeric_state + cleaner0 + 2023-07-11T10:10:59Z + DUMMY: + + monomers + + + 0.9997614 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.97160864 + protein_type + cleaner0 + 2023-07-11T10:06:30Z + MESH: + + cytochrome b5 + + + 0.94981354 + chemical + cleaner0 + 2023-07-11T10:02:50Z + CHEBI: + + haem + + + protein_type + MESH: + cleaner0 + 2023-07-11T10:06:30Z + + cytochrome b5 + + + 0.98066837 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:04:43Z + + iron + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + six-coordinated by + + + 0.99924004 + residue_name + cleaner0 + 2023-07-11T13:08:30Z + SO: + + histidine + + + 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MESH: + + cytochrome b5 + + + 0.7402097 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + 0.9997911 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.9992798 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + 0.9997787 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.9903881 + site + cleaner0 + 2023-07-11T13:45:45Z + SO: + + surface + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + hydrophobic haem–haem stacking + + + 0.99989355 + residue_name_number + cleaner0 + 2023-07-11T10:15:26Z + DUMMY: + + Tyr164 + + + 0.9997899 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.9974502 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + 0.9995301 + experimental_method + cleaner0 + 2023-07-11T13:21:35Z + MESH: + + mutation + + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:02:51Z + + haem + + + 0.99961376 + evidence + cleaner0 + 2023-07-11T13:31:55Z + DUMMY: + + structural data + + + 0.9998939 + residue_name_number + cleaner0 + 2023-07-11T10:15:26Z + DUMMY: + + Tyr164 + + + 0.99989355 + residue_name_number + cleaner0 + 2023-07-11T10:15:17Z + DUMMY: + + Tyr107 + + + 0.99989295 + residue_name_number + cleaner0 + 2023-07-11T10:15:21Z + DUMMY: + + Lys163 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + hydrogen-bonded + + + 0.99904174 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-11T13:11:18Z + + Tyr 107 + + + 0.9998927 + residue_name_number + cleaner0 + 2023-07-11T10:15:12Z + DUMMY: + + Tyr113 + + + 0.999806 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.9996282 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + 0.9994444 + protein_state + cleaner0 + 2023-07-11T13:42:00Z + DUMMY: + + conserved + + + protein_type + MESH: + cleaner0 + 2023-07-11T10:05:27Z + + MAPR + + + 0.47200882 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + + RESULTS + title_2 + 4868 + PGRMC1 exhibits haem-dependent dimerization in solution + + 0.9998222 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.99322426 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + + RESULTS + paragraph + 4924 + In the PGRMC1 crystal, two different types of crystal contacts (chain A–A″ and A–B) were observed in addition to the haem-mediated dimer (chain A–A′) (Supplementary Figs 3 and 6a). To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7). NMR signals from some amino acid residues of PGRMC1 disappeared due to the paramagnetic relaxation effect of haem (Supplementary Figs 6b); these residues were located in the haem-binding region. When chemical shifts of apo- and haem-bound forms of PGMRC1 were compared, some amino acid residues close to those which disappeared because of the paramagnetic relaxation effect of haem exhibit notable chemical shifts (Supplementary Fig. 6a,b; dark yellow). However, at the interfaces of the other possible dimeric structures (Supplementary Fig. 6a, chain A–A″; cyan and chain A–B; violet), no significant difference was observed. Furthermore, free energy of dissociation predicted by PISA suggested that the haem-mediated dimer is stable in solution while the other potential interactions are not. We also attempted to predict the secondary structure of PGRMC1 through NMR data by calculating with TALOS+ program (Supplementary Fig. 8); the prediction suggested that the overall secondary structure is comparable between apo- and haem-bound forms of PGRMC1 in solution. + + 0.9998647 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.9995252 + evidence + cleaner0 + 2023-07-11T13:32:01Z + DUMMY: + + crystal + + + 0.62014765 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + 0.99932265 + oligomeric_state + cleaner0 + 2023-07-11T10:05:41Z + DUMMY: + + dimer + + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:02:51Z + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9998646 + protein + cleaner0 + 2023-07-11T10:03:15Z + PR: + + PGRMC1 + + + 0.9995944 + evidence + cleaner0 + 2023-07-11T13:32:06Z + DUMMY: + + structure + + + 0.99966764 + protein_state + cleaner0 + 2023-07-11T10:07:57Z + DUMMY: + + apo + + + 0.9995765 + protein_state + cleaner0 + 2023-07-11T10:05:57Z + DUMMY: + + haem-bound + + + 0.9998635 + protein + cleaner0 + 2023-07-11T12:49:55Z + PR: + + PGMRC1 + + + 0.9964247 + experimental_method + cleaner0 + 2023-07-11T13:21:40Z + MESH: + + two-dimensional nuclear magnetic resonance + + + 0.9994337 + experimental_method + cleaner0 + 2023-07-11T13:21:43Z + MESH: + + NMR + + + 0.99752617 + experimental_method + cleaner0 + 2023-07-11T13:21:47Z + MESH: + + heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy + + + 0.99956316 + experimental_method + cleaner0 + 2023-07-11T13:21:54Z + MESH: + + NMR + + + 0.99986446 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.9993055 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + 0.9991949 + site + cleaner0 + 2023-07-11T10:07:41Z + SO: + + haem-binding region + + + 0.89633644 + evidence + cleaner0 + 2023-07-11T13:32:10Z + DUMMY: + + chemical shifts + + + 0.99966455 + protein_state + cleaner0 + 2023-07-11T10:07:57Z + DUMMY: + + apo + + + 0.99956113 + protein_state + cleaner0 + 2023-07-11T10:05:57Z + DUMMY: + + haem-bound + + + 0.9998461 + protein + cleaner0 + 2023-07-11T12:50:03Z + PR: + + PGMRC1 + + + 0.9983656 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + 0.99826884 + site + cleaner0 + 2023-07-11T13:45:51Z + SO: + + interfaces + + + 0.9981969 + oligomeric_state + cleaner0 + 2023-07-11T12:47:45Z + DUMMY: + + dimeric + + + 0.67957354 + evidence + cleaner0 + 2023-07-11T13:32:13Z + DUMMY: + + structures + + + 0.99630755 + evidence + cleaner0 + 2023-07-11T13:32:18Z + DUMMY: + + free energy of dissociation + + + 0.9241478 + experimental_method + cleaner0 + 2023-07-11T13:22:00Z + MESH: + + PISA + + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:02:51Z + + haem + + + 0.99919623 + oligomeric_state + cleaner0 + 2023-07-11T10:05:41Z + DUMMY: + + dimer + + + 0.9904857 + protein_state + cleaner0 + 2023-07-11T13:42:05Z + DUMMY: + + stable + + + 0.9998642 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.999435 + experimental_method + cleaner0 + 2023-07-11T13:22:07Z + MESH: + + NMR + + + 0.99423486 + experimental_method + cleaner0 + 2023-07-11T13:22:12Z + MESH: + + TALOS+ program + + + 0.99967337 + protein_state + cleaner0 + 2023-07-11T10:07:56Z + DUMMY: + + apo + + + 0.9995765 + protein_state + cleaner0 + 2023-07-11T10:05:57Z + DUMMY: + + haem-bound + + + 0.9998648 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + + RESULTS + paragraph + 6494 + We analysed the haem-dependent dimerization of the PGRMC1 cytosolic domain (a.a.44–195) in solution (Fig. 2 and Table 2). Mass spectrometry (MS) analyses under non-denaturing condition demonstrated that the apo-monomer PGRMC1 resulted in dimerization by binding with haem (Fig. 2a). It should be noted that a disulfide bond between two Cys129 residues is observed in the crystal of PGRMC1 (Fig. 1a), while Cys129 is not conserved among the MAPR family proteins (Supplementary Fig. 5a). This observation led us to examine whether or not the disulfide bond contributes to PGRMC1 dimerization. MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). Supporting this, MS analyses under denaturing conditions showed that haem-mediated PGRMC1 dimer is completely dissociated into monomer, indicating that dimerization of this kind is not mediated by any covalent bond such as disulfide bond (Supplementary Fig. 9). + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:02:51Z + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9998629 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.9885373 + structure_element + cleaner0 + 2023-07-11T10:04:21Z + SO: + + cytosolic domain + + + 0.9990666 + residue_range + cleaner0 + 2023-07-11T13:08:52Z + DUMMY: + + 44–195 + + + 0.9994917 + experimental_method + cleaner0 + 2023-07-11T13:22:17Z + MESH: + + Mass spectrometry + + + 0.9996383 + experimental_method + cleaner0 + 2023-07-11T13:22:20Z + MESH: + + MS + + + 0.8378052 + experimental_method + cleaner0 + 2023-07-11T13:22:22Z + MESH: + + non-denaturing condition + + + 0.9996264 + protein_state + cleaner0 + 2023-07-11T10:07:57Z + DUMMY: + + apo + + + 0.99931693 + oligomeric_state + cleaner0 + 2023-07-11T10:08:32Z + DUMMY: + + monomer + + + 0.9998591 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99948895 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + 0.9962624 + ptm + cleaner0 + 2023-07-11T10:08:12Z + MESH: + + disulfide bond + + + 0.9998779 + residue_name_number + cleaner0 + 2023-07-11T10:08:21Z + DUMMY: + + Cys129 + + + 0.99955493 + evidence + cleaner0 + 2023-07-11T13:32:23Z + DUMMY: + + crystal + + + 0.9998611 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.99987674 + residue_name_number + cleaner0 + 2023-07-11T10:08:20Z + DUMMY: + + Cys129 + + + 0.9994192 + protein_state + cleaner0 + 2023-07-11T13:42:10Z + DUMMY: + + not conserved + + + protein_type + MESH: + cleaner0 + 2023-07-11T10:05:27Z + + MAPR + + + 0.9960234 + ptm + cleaner0 + 2023-07-11T10:08:11Z + MESH: + + disulfide bond + + + 0.9998549 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99966466 + experimental_method + cleaner0 + 2023-07-11T13:22:34Z + MESH: + + MS + + + 0.81779677 + experimental_method + cleaner0 + 2023-07-11T13:22:38Z + MESH: + + non-denaturing conditions + + + 0.9996855 + mutant + cleaner0 + 2023-07-11T12:59:13Z + MESH: + + Cys129Ser + + + 0.999521 + mutant + cleaner0 + 2023-07-11T12:59:18Z + MESH: + + C129S + + + 0.99784625 + protein_state + cleaner0 + 2023-07-11T10:13:08Z + DUMMY: + + mutant + + + 0.99925834 + protein_state + cleaner0 + 2023-07-11T12:48:12Z + DUMMY: + + dimerized + + + 0.998912 + protein_state + cleaner0 + 2023-07-11T10:14:47Z + DUMMY: + + presence of + + + 0.9997181 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:02:51Z + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99986136 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.9959481 + ptm + cleaner0 + 2023-07-11T10:08:12Z + MESH: + + disulfide bond + + + 0.9998728 + residue_name_number + cleaner0 + 2023-07-11T10:08:21Z + DUMMY: + + Cys129 + + + 0.99966216 + experimental_method + cleaner0 + 2023-07-11T13:22:42Z + MESH: + + MS + + + experimental_method + MESH: + cleaner0 + 2023-07-11T13:22:58Z + + denaturing conditions + + + 0.7049826 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + 0.99985874 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.99940586 + oligomeric_state + cleaner0 + 2023-07-11T10:05:41Z + DUMMY: + + dimer + + + 0.9994506 + oligomeric_state + cleaner0 + 2023-07-11T10:08:33Z + DUMMY: + + monomer + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99528134 + ptm + cleaner0 + 2023-07-11T10:08:12Z + MESH: + + disulfide bond + + + + RESULTS + paragraph + 7612 + We also analysed the haem-dependent dimerization of PGRMC1 by diffusion-ordered NMR spectroscopy (DOSY) analyses (Table 2, Supplementary Fig. 10). The results suggested that the hydrodynamic radius of haem-bound PGRMC1 is larger than that of apo-PGRMC1. To further evaluate changes in molecular weights in dimerization of PGRMC1, sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis was carried out. Whereas the wild-type (wt) apo-PGRMC1 appeared at a 1.9 S peak as monomer, the haem-binding PGRMC1 was converted into dimer at a 3.1 S peak (Fig. 2b). Similarly, the C129S mutant of PGRMC1 converted from monomer to dimer by binding to haem (Fig. 2b). SV-AUC analyses also allowed us to examine the stability of haem/PGRMC1 dimer. To this end, we used different concentrations (3.5–147 μmol l−1) of haem-bound PGRMC1 protein (a.a. 72–195), which were identical to that used in the crystallographic analysis. The sedimentation coefficients calculated on the basis of the crystal structure were 1.71 S for monomer and 2.56 S for dimer (Supplementary Fig. 11, upper panel). The results showed that the PGRMC1 dimer is not dissociated into monomer at all concentrations examined (Supplementary Fig. 11, lower panel), suggesting that the Kd value of haem-mediated dimer of PGRMC1 is under 3.5 μmol l−1. A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20 μmol l−1) (ref.) and plexin A2 receptor (dimerization Kd higher than 300 μmol l−1) (ref.). The current analytical data confirmed that apo-PGRMC1 monomer converts into dimer by binding to haem in solution (Table 2). + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:02:51Z + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9998628 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.99955225 + experimental_method + cleaner0 + 2023-07-11T13:23:05Z + MESH: + + diffusion-ordered NMR spectroscopy + + + 0.99960417 + experimental_method + cleaner0 + 2023-07-11T13:23:08Z + MESH: + + DOSY + + + 0.9094112 + evidence + cleaner0 + 2023-07-11T13:32:42Z + DUMMY: + + hydrodynamic radius + + + 0.99956554 + protein_state + cleaner0 + 2023-07-11T10:05:57Z + DUMMY: + + haem-bound + + + 0.99985063 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.9996785 + protein_state + cleaner0 + 2023-07-11T10:07:58Z + DUMMY: + + apo + + + 0.99985325 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9998604 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.9995748 + experimental_method + cleaner0 + 2023-07-11T13:23:11Z + MESH: + + sedimentation velocity analytical ultracentrifugation + + + 0.9995594 + experimental_method + cleaner0 + 2023-07-11T13:23:14Z + MESH: + + SV-AUC + + + 0.9995815 + protein_state + cleaner0 + 2023-07-11T10:08:53Z + DUMMY: + + wild-type + + + 0.99965787 + protein_state + cleaner0 + 2023-07-11T10:08:58Z + DUMMY: + + wt + + + 0.99967706 + protein_state + cleaner0 + 2023-07-11T10:07:58Z + DUMMY: + + apo + + + 0.99985373 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.9994248 + oligomeric_state + cleaner0 + 2023-07-11T10:08:33Z + DUMMY: + + monomer + + + chemical + CHEBI: + cleaner0 + 2023-07-11T13:42:34Z + + haem + + + 0.9998572 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.9993969 + oligomeric_state + cleaner0 + 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2023-07-11T10:02:51Z + + haem + + + 0.99942833 + oligomeric_state + cleaner0 + 2023-07-11T10:05:41Z + DUMMY: + + dimer + + + 0.99985766 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.9998603 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.9994603 + oligomeric_state + cleaner0 + 2023-07-11T10:05:41Z + DUMMY: + + dimer + + + 0.99946505 + oligomeric_state + cleaner0 + 2023-07-11T12:48:34Z + DUMMY: + + dimers + + + 0.99969673 + structure_element + cleaner0 + 2023-07-11T13:40:42Z + SO: + + extracellular domain + + + 0.9881788 + protein_type + cleaner0 + 2023-07-11T13:34:38Z + MESH: + + membrane proteins + + + protein + PR: + cleaner0 + 2023-07-11T12:51:31Z + + Toll like receptor 9 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99925154 + evidence + cleaner0 + 2023-07-11T10:09:12Z + DUMMY: + + Kd + + + 0.70314854 + protein + cleaner0 + 2023-07-11T12:51:34Z + PR: + + plexin A2 receptor + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9986761 + evidence + cleaner0 + 2023-07-11T10:09:13Z + DUMMY: + + Kd + + + 0.99967265 + protein_state + cleaner0 + 2023-07-11T10:07:58Z + DUMMY: + + apo + + + 0.9998605 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.9994692 + oligomeric_state + cleaner0 + 2023-07-11T10:08:33Z + DUMMY: + + monomer + + + 0.9994217 + oligomeric_state + cleaner0 + 2023-07-11T10:05:41Z + DUMMY: + + dimer + + + 0.9997222 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + + RESULTS + paragraph + 9356 + We also showed by haem titration experiments that haem binding to PGRMC1 was of low affinity with a Kd value of 50 nmol l−1; this is comparable with that of iron regulatory protein 2, which is known to be regulated by intracellular levels of haem (Fig. 2c and Supplementary Table 1). These results raised the possibility that the function of PGRMC1 is regulated by intracellular haem concentrations. + + 0.99956113 + experimental_method + cleaner0 + 2023-07-11T13:23:26Z + MESH: + + haem titration experiments + + + 0.99978155 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + 0.99982446 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.99956983 + evidence + cleaner0 + 2023-07-11T10:09:13Z + DUMMY: + + Kd + + + 0.97503453 + protein + cleaner0 + 2023-07-11T13:35:18Z + PR: + + iron regulatory protein 2 + + + 0.9997582 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + 0.9998233 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.99977595 + chemical + cleaner0 + 2023-07-11T10:02:51Z + CHEBI: + + haem + + + + RESULTS + title_2 + 9762 + CO inhibits haem-dependent dimerization of PGRMC1 + + 0.99964213 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + 0.9986714 + chemical + cleaner0 + 2023-07-11T10:02:52Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99985695 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + + RESULTS + paragraph + 9812 + Crystallographic analyses revealed that Tyr113 of PGRMC1 is an axial ligand for haem and contributes to haem-dependent dimerization (Fig. 1a). Analysis of UV-visible spectra revealed that the heme of PGRMC1 is reducible from ferric to ferrous state, thus allowing CO binding (Fig. 3a). Furthermore, the UV-visible spectrum of the wild type PGRMC1 was the same as that of the C129S mutant of PGRMC1, and the R/Z ratio determined by the intensities between the Soret band (394 nm) peak and the 274-nm peak showed that these proteins were fully loaded with haem (Supplementary Fig. 12). Analysis of the ferric form of PGRMC1 using resonance Raman spectroscopy (Supplementary Fig. 13) showed that the relative intensity of oxidation and spin state marker bands (ν4 and ν3) is close to 1.0, which is consistent with it being a haem protein with a proximal Tyr coordination. A specific Raman shift peaking at vFe–CO=500 cm−1 demonstrated that the CO-bound haem of PGRMC1 is six-coordinated (Supplementary Fig. 13). + + 0.9995625 + experimental_method + cleaner0 + 2023-07-11T13:23:34Z + MESH: + + Crystallographic analyses + + + 0.9998559 + residue_name_number + cleaner0 + 2023-07-11T10:15:12Z + DUMMY: + + Tyr113 + + + 0.99985945 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.99927706 + chemical + cleaner0 + 2023-07-11T10:02:52Z + CHEBI: + + haem + + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:02:52Z + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9985645 + evidence + cleaner0 + 2023-07-11T13:33:04Z + DUMMY: + + UV-visible spectra + + + 0.87144 + chemical + cleaner0 + 2023-07-11T13:11:41Z + CHEBI: + + heme + + + 0.9998511 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.99957365 + protein_state + cleaner0 + 2023-07-11T10:10:20Z + DUMMY: + + ferric + + + 0.9995223 + protein_state + cleaner0 + 2023-07-11T10:10:24Z + DUMMY: + + ferrous + + + 0.99956876 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + 0.9964068 + evidence + cleaner0 + 2023-07-11T13:33:07Z + DUMMY: + + UV-visible spectrum + + + 0.9996103 + protein_state + cleaner0 + 2023-07-11T13:42:39Z + DUMMY: + + wild type + + + 0.99985707 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.9997043 + mutant + cleaner0 + 2023-07-11T12:59:19Z + MESH: + + C129S + + + 0.998828 + protein_state + cleaner0 + 2023-07-11T10:13:08Z + DUMMY: + + mutant + + + 0.9998555 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.99939615 + evidence + cleaner0 + 2023-07-11T13:33:10Z + DUMMY: + + R/Z ratio + + + 0.9772954 + protein_state + cleaner0 + 2023-07-11T13:42:51Z + DUMMY: + + fully loaded with + + + 0.9994116 + chemical + cleaner0 + 2023-07-11T10:02:52Z + CHEBI: + + haem + + + 0.99962366 + protein_state + cleaner0 + 2023-07-11T10:10:19Z + DUMMY: + + ferric + + + 0.9998579 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + 0.9995656 + experimental_method + cleaner0 + 2023-07-11T13:23:40Z + MESH: + + resonance Raman spectroscopy + + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:02:52Z + + haem + + + 0.9990225 + residue_name + cleaner0 + 2023-07-11T13:08:40Z + SO: + + Tyr + + + 0.99756503 + evidence + cleaner0 + 2023-07-11T13:33:22Z + DUMMY: + + Raman shift + + + 0.9995175 + protein_state + cleaner0 + 2023-07-11T10:10:07Z + DUMMY: + + CO-bound + + + 0.9991033 + chemical + cleaner0 + 2023-07-11T10:02:52Z + CHEBI: + + haem + + + 0.9998518 + protein + cleaner0 + 2023-07-11T10:03:16Z + PR: + + PGRMC1 + + + + RESULTS + paragraph + 10831 + Since PGRMC1 dimerization involves the open surface of haem on the opposite side of the axial Tyr113, no space for CO binding is available in the dimeric structure (Fig. 3b). This prompted us to ask if CO binding to haem causes dissociation of the PGRMC1 dimer. Analysis by gel filtration chromatography revealed that the relative molecular sizes of the wild-type and the C129S mutant of PGRMC1 are increased by adding haem to apo-PGRMC1 regardless of the oxidation state of the iron (Fig. 3c), which is in agreement with the results in Table 1. CO application to ferrous PGRMC1 abolished the haem-dependent increase in its molecular size. Under this reducing condition in the presence of dithionite, analyses of UV-visible spectra indicated that CO-binding with haem-PGRMC1 is stable, showing only 20% reduction of the absorbance at 412 nm within 2 h (Supplementary Fig. 14). Furthermore, the Tyr113Phe (Y113F) mutant of PGRMC1 was not responsive to haem. These results suggest that CO favours the six-coordinate form of haem and interferes with the haem-mediated dimerization of PGRMC1. To examine the inhibitory effects of CO on haem-mediated PGRMC1 dimerization, SV-AUC analysis was carried out. The peak corresponding to the haem/PGRMC1 dimer was detected under reducing conditions in the presence of dithionite (Supplementary Fig. 15, middle panel). Under these circumstances, CO application induced dissociation of the haem-mediated dimers of PGRMC1 to generate a peak of monomers (Supplementary Fig. 15, lower panel). These observations raised the transition model for structural regulation of PGRMC1 in response to haem (Fig. 3d). As mentioned above, apo-PGRMC1 exists as monomer. By binding with haem (binding Kd=50 nmol l−1), PGRMC1 forms a stable dimer (dimerization Kd<<3.5 μmol l−1) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer. CO induces the dissociation of the haem-mediated dimer of PGRMC1 by interfering with the haem-stacking interface via formation of the six-coordinated CO-haem-PGRMC1 complex. Such a dynamic structural regulation led us to further examine the regulation of PGRMC1 functions in cancer cells. + + 0.9998406 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + site + SO: + cleaner0 + 2023-07-11T13:46:10Z + + surface + + + 0.9996704 + chemical + cleaner0 + 2023-07-11T10:02:52Z + CHEBI: + + haem + + + 0.9998692 + residue_name_number + cleaner0 + 2023-07-11T10:15:12Z + DUMMY: + + Tyr113 + + + 0.9994343 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + 0.99941576 + oligomeric_state + cleaner0 + 2023-07-11T12:47:45Z + DUMMY: + + dimeric + + + 0.9993579 + evidence + cleaner0 + 2023-07-11T13:33:43Z + DUMMY: + + structure + + + 0.99627966 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + 0.9282887 + chemical + cleaner0 + 2023-07-11T10:02:52Z + CHEBI: + + haem + + + 0.99985695 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 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2023-07-11T10:08:33Z + DUMMY: + + monomer + + + 0.9996947 + chemical + cleaner0 + 2023-07-11T10:02:52Z + CHEBI: + + haem + + + 0.99718386 + evidence + cleaner0 + 2023-07-11T10:09:13Z + DUMMY: + + Kd + + + 0.9998217 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.99934345 + protein_state + cleaner0 + 2023-07-11T13:42:59Z + DUMMY: + + stable + + + 0.9994431 + oligomeric_state + cleaner0 + 2023-07-11T10:05:41Z + DUMMY: + + dimer + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99946743 + evidence + cleaner0 + 2023-07-11T10:09:13Z + DUMMY: + + Kd + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + stacking + + + 0.94421965 + site + cleaner0 + 2023-07-11T13:46:14Z + SO: + + surfaces + + + 0.99966574 + chemical + cleaner0 + 2023-07-11T10:02:52Z + CHEBI: + + haem + + + 0.9993855 + oligomeric_state + cleaner0 + 2023-07-11T10:08:33Z + DUMMY: + + monomer + + + 0.9994061 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:02:52Z + + haem + + + 0.99938047 + oligomeric_state + cleaner0 + 2023-07-11T10:05:41Z + DUMMY: + + dimer + + + 0.9998516 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9996438 + site + cleaner0 + 2023-07-11T13:46:17Z + SO: + + haem-stacking interface + + + 0.99660575 + complex_assembly + cleaner0 + 2023-07-11T10:11:25Z + GO: + + CO-haem-PGRMC1 + + + 0.9998492 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + + RESULTS + title_2 + 13032 + PGRMC1 dimerization is required for binding to EGFR + + 0.9998254 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9931778 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + + RESULTS + paragraph + 13084 + Because PGRMC1 is known to interact with EGFR and to accelerate tumour progression, we examined the effect of haem-dependent dimerization of PGRMC1 on its interaction with EGFR by using purified proteins. As shown in Fig. 4a, the cytosolic domain of wild-type PGRMC1, but not the Y113F mutant, interacted with purified EGFR in a haem-dependent manner. This interaction was disrupted by the ruthenium-based CO-releasing molecule, CORM3, but not by RuCl3 as a control reagent (Fig. 4b). We further analysed the intracellular interaction between PGRMC1 and EGFR. FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c). The C129S mutant of PGRMC1 also interacted with endogenous PGRMC1 and EGFR (Supplementary Fig. 16). Whereas FLAG-tagged wild-type PGRMC1 interacted with endogenous PGRMC1 and EGFR, the Y113F mutant did not. We also examined the effect of succinylacetone (SA), an inhibitor of haem biosynthesis (Fig. 4d). As expected, SA significantly reduced PGRMC1 dimerization and its interaction with EGFR (Fig. 4e), indicating that haem-mediated dimerization of PGMRC1 is critical for its binding to EGFR. + + 0.999824 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9776495 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.9987263 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99984765 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9926104 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.99920774 + structure_element + cleaner0 + 2023-07-11T10:04:21Z + SO: + + cytosolic domain + + + 0.9995993 + protein_state + cleaner0 + 2023-07-11T10:08:53Z + DUMMY: + + wild-type + + + 0.9998559 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9997048 + mutant + cleaner0 + 2023-07-11T10:13:03Z + MESH: + 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2023-07-11T13:04:15Z + + endogenous + + + 0.9998481 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9986162 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.99928635 + protein_state + cleaner0 + 2023-07-11T13:43:17Z + DUMMY: + + FLAG-tagged + + + 0.9995968 + protein_state + cleaner0 + 2023-07-11T10:08:54Z + DUMMY: + + wild-type + + + 0.99984515 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.5547055 + protein_state + cleaner0 + 2023-07-11T13:04:15Z + DUMMY: + + endogenous + + + 0.99985075 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.99929786 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.9996997 + mutant + cleaner0 + 2023-07-11T10:13:03Z + MESH: + + Y113F + + + 0.9983523 + protein_state + cleaner0 + 2023-07-11T10:13:08Z + DUMMY: + + mutant + + + 0.9997886 + chemical + cleaner0 + 2023-07-11T13:12:20Z + CHEBI: + + succinylacetone + + + 0.99974924 + chemical + cleaner0 + 2023-07-11T13:12:24Z + CHEBI: + + SA + + + 0.9991365 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + 0.9997638 + chemical + cleaner0 + 2023-07-11T13:12:25Z + CHEBI: + + SA + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T13:26:59Z + + reduced + + + 0.9998474 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99785066 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.9987841 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.999846 + protein + cleaner0 + 2023-07-11T12:50:03Z + PR: + + PGMRC1 + + + 0.9963064 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + + RESULTS + title_2 + 14383 + PGRMC1 dimer facilitates EGFR-mediated cancer growth + + 0.9998097 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.99929833 + oligomeric_state + cleaner0 + 2023-07-11T10:05:41Z + DUMMY: + + dimer + + + 0.9466698 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + + RESULTS + paragraph + 14436 + Next, we investigated the functional significance of PGRMC1 dimerization in EGFR signaling. EGF-induced phosphorylations of EGFR and its downstream targets AKT and ERK were decreased by PGRMC1 knockdown (PGRMC1-KD) (Fig. 4f). Similarly, EGFR signaling was suppressed by treatment of HCT116 cells with SA (Fig. 4g) or CORM3 (Fig. 4h). These results suggested that haem-mediated dimerization of PGRMC1 is critical for EGFR signaling. + + 0.99984276 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.8777582 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.9991768 + protein_type + cleaner0 + 2023-07-11T13:13:39Z + MESH: + + EGF + + + ptm + MESH: + cleaner0 + 2023-07-11T13:13:01Z + + phosphorylations + + + 0.9652565 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.5629122 + protein_type + cleaner0 + 2023-07-11T13:35:28Z + MESH: + + AKT + + + 0.51487744 + protein_type + cleaner0 + 2023-07-11T13:35:32Z + MESH: + + ERK + + + 0.9859196 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.5487161 + protein_state + cleaner0 + 2023-07-11T13:43:26Z + DUMMY: + + knockdown + + + mutant + MESH: + cleaner0 + 2023-07-11T10:12:32Z + + PGRMC1-KD + + + 0.555868 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.9997993 + chemical + cleaner0 + 2023-07-11T13:12:25Z + CHEBI: + + SA + + + 0.998782 + chemical + cleaner0 + 2023-07-11T13:12:01Z + CHEBI: + + CORM3 + + + 0.999782 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99984777 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.91323197 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + + RESULTS + paragraph + 14868 + To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b). Chemosensitivity enhancement by two different shRNAs to PGRMC1 was seen also in HCT116 cells and human hepatoma HuH7 cells (Supplementary Fig. 17). Furthermore, PGRMC1-KD inhibited spheroid formation of HCT116 cells in culture, and this inhibition was reversed by co-expression of wild-type PGRMC1 but not of the Y113F mutant (Fig. 5c and Supplementary Fig. 18). Thus, PGRMC1 dimerization is important for cancer cell proliferation and chemoresistance. + + 0.5924544 + protein_state + cleaner0 + 2023-07-11T12:48:13Z + DUMMY: + + dimerized + + + 0.99986446 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.8416364 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.99874413 + experimental_method + cleaner0 + 2023-07-11T13:24:15Z + MESH: + + knockdown-rescue experiments + + + 0.999378 + protein_state + cleaner0 + 2023-07-11T13:43:31Z + DUMMY: + + FLAG-tagged + + + 0.99957436 + protein_state + cleaner0 + 2023-07-11T10:08:54Z + DUMMY: + + wild-type + + + 0.99964285 + mutant + cleaner0 + 2023-07-11T10:13:03Z + MESH: + + Y113F + + + 0.9990972 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.72546744 + experimental_method + cleaner0 + 2023-07-11T13:24:24Z + MESH: + + expression vectors + + + 0.9838925 + experimental_method + cleaner0 + 2023-07-11T13:24:27Z + MESH: + + silent mutations + + + 0.8342758 + experimental_method + cleaner0 + 2023-07-11T13:24:31Z + MESH: + + introduced + + + 0.87169033 + chemical + cleaner0 + 2023-07-11T13:18:31Z + CHEBI: + + shRNA + + + 0.998572 + experimental_method + cleaner0 + 2023-07-11T13:24:34Z + MESH: + + knocking down + + + 0.99981123 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.7923005 + mutant + cleaner0 + 2023-07-11T10:12:33Z + MESH: + + PGRMC1-KD + + + 0.98196614 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.99952066 + chemical + cleaner0 + 2023-07-11T13:13:58Z + CHEBI: + + erlotinib + + + 0.9991309 + experimental_method + cleaner0 + 2023-07-11T13:24:38Z + MESH: + + co-expression + + + 0.9993799 + protein_state + cleaner0 + 2023-07-11T13:19:51Z + DUMMY: + + shRNA-resistant + + + 0.99959135 + protein_state + cleaner0 + 2023-07-11T10:08:54Z + DUMMY: + + wild-type + + + 0.99985266 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9996898 + mutant + cleaner0 + 2023-07-11T10:13:02Z + MESH: + + Y113F + + + 0.99927086 + protein_state + cleaner0 + 2023-07-11T10:13:07Z + DUMMY: + + mutant + + + 0.63183546 + chemical + cleaner0 + 2023-07-11T13:24:49Z + CHEBI: + + shRNAs + + + 0.9998554 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + species + MESH: + cleaner0 + 2023-07-11T10:15:41Z + + human + + + 0.8441641 + mutant + cleaner0 + 2023-07-11T10:12:33Z + MESH: + + PGRMC1-KD + + + 0.999172 + experimental_method + cleaner0 + 2023-07-11T13:25:09Z + MESH: + + co-expression + + + 0.9996086 + protein_state + cleaner0 + 2023-07-11T10:08:54Z + DUMMY: + + wild-type + + + 0.9998555 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9996973 + mutant + cleaner0 + 2023-07-11T10:13:03Z + MESH: + + Y113F + + + 0.99917185 + protein_state + cleaner0 + 2023-07-11T10:13:08Z + DUMMY: + + mutant + + + 0.99985874 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + + RESULTS + paragraph + 15927 + We examined the role of PGRMC1 in metastatic progression by xenograft transplantation assays using super-immunodeficient NOD/scid/γnull (NOG) mice. Ten days after intra-splenic implantation of HCT116 cells that were genetically tagged with a fluorescent protein Venus, the group implanted with PGRMC1-KD cells showed a significant decrease of liver metastasis in comparison with the control group (Fig. 5d). + + 0.9998597 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9993624 + experimental_method + cleaner0 + 2023-07-11T13:25:14Z + MESH: + + xenograft transplantation assays + + + 0.8666498 + experimental_method + cleaner0 + 2023-07-11T13:25:21Z + MESH: + + intra-splenic implantation + + + 0.81741047 + mutant + cleaner0 + 2023-07-11T10:12:33Z + MESH: + + PGRMC1-KD + + + + RESULTS + title_2 + 16339 + Interaction of PGRMC1 dimer with cytochromes P450 + + 0.9998543 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.99934536 + oligomeric_state + cleaner0 + 2023-07-11T10:05:42Z + DUMMY: + + dimer + + + 0.9991067 + protein_type + cleaner0 + 2023-07-11T10:04:06Z + MESH: + + cytochromes P450 + + + + RESULTS + paragraph + 16389 + Since PGRMC1 has been shown to interact with cytochromes P450 (ref), we investigated whether the haem-mediated dimerization of PGRMC1 is necessary for their interactions. Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b). Moreover, the interaction of PGRMC1 with CYP1A2 was blocked by CORM3 under reducing conditions (Fig. 6c), indicating that PGRMC1 dimerization is necessary for its interaction with cytochromes P450. Doxorubicin is an anti-cancer reagent that is metabolized into inactive doxorubicinol by CYP2D6 and CYP3A4 (Fig. 6d). PGRMC1-KD significantly suppressed the conversion of doxorubicin to doxorubicinol (Fig. 6d) and increased sensitivity to doxorubicin (Fig. 6e). Enhanced doxorubicin sensitivity was modestly but significantly induced by PGRMC1-KD. This effect was reversed by co-expression of the wild-type PGRMC1 but not of the Y113F mutant, suggesting that PGRMC1 enhances doxorubicin resistance of cancer cells by facilitating its degradation via cytochromes P450. To gain further insight into the interaction between PGRMC1 and cytochromes P450, surface plasmon resonance analyses were conducted using recombinant CYP51 and PGRMC1. This was based on a previous study showing that PGRMC1 binds to CYP51 and enhances cholesterol biosynthesis by CYP51 (refs). CYP51 interacted with PGRMC1 in a concentration-dependent manner in the presence of haem, but not in its absence (Supplementary Fig. 19), suggesting the requirement for the haem-dependent dimerization of PGRMC1. The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/Gαi1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant. + + 0.9998209 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9996052 + protein_type + cleaner0 + 2023-07-11T10:04:06Z + MESH: + + cytochromes P450 + + + 0.99583876 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99984634 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9998332 + protein + cleaner0 + 2023-07-11T12:51:49Z + PR: + + CYP1A2 + + + 0.99976724 + protein + cleaner0 + 2023-07-11T10:16:17Z + PR: + + CYP3A4 + + + 0.9742723 + protein_type + cleaner0 + 2023-07-11T10:06:30Z + MESH: + + cytochrome b5 + + + 0.9994921 + protein + cleaner0 + 2023-07-11T13:36:11Z + PR: + + 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protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9998517 + protein + cleaner0 + 2023-07-11T10:16:07Z + PR: + + CYP51 + + + 0.999843 + protein + cleaner0 + 2023-07-11T10:16:07Z + PR: + + CYP51 + + + 0.9998252 + protein + cleaner0 + 2023-07-11T10:16:07Z + PR: + + CYP51 + + + 0.9998311 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.99915564 + protein_state + cleaner0 + 2023-07-11T10:14:47Z + DUMMY: + + presence of + + + 0.9997129 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + 0.9995047 + protein_state + cleaner0 + 2023-07-11T10:14:52Z + DUMMY: + + absence + + + 0.7125231 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9998425 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9996069 + evidence + cleaner0 + 2023-07-11T10:09:13Z + DUMMY: + + Kd + + + 0.99983513 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9998565 + protein + cleaner0 + 2023-07-11T10:16:07Z + PR: + + CYP51 + + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:02:53Z + + haem + + + 0.99964935 + protein + cleaner0 + 2023-07-11T12:53:21Z + PR: + + cytochrome P450 reductase + + + 0.9992912 + protein + cleaner0 + 2023-07-11T12:52:33Z + PR: + + neuroglobin + + + 0.999418 + protein + cleaner0 + 2023-07-11T12:52:36Z + PR: + + Gαi1 + + + 0.9307969 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + 0.99983525 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9998615 + protein + cleaner0 + 2023-07-11T10:16:07Z + PR: + + CYP51 + + + + DISCUSS + title_1 + 18363 + Discussion + + + DISCUSS + paragraph + 18374 + In this study, we showed that PGRMC1 dimerizes by stacking interactions of haem molecules from each monomer. Recently, Lucas et al. reported that translationally-controlled tumour protein was dimerized by binding with haem, but its structural basis remains unclear. This is the report showing crystallographic evidence that indicates roles of the direct haem–haem stacking in haem-mediated dimerization in eukaryotes, although a few examples are known in bacteria. Sequence alignments show that haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in PGRMC1 are conserved among MAPR proteins (Supplementary Fig. 5). In the current study, the Y113 residue plays a crucial role for the haem-dependent dimerization of PGRMC1 and resultant regulation of cancer proliferation and chemoresistance (Figs 5c and 6e). Since the Y113 residue is involved in the putative consensus motif of phosphorylation by tyrosine kinases such as Abl and Lck, we investigated whether phosphorylated Y113 is present in HCT116 cells by ESI-MS analysis. It was, however, undetectable under current experimental conditions (Supplementary Fig. 20). Recently, Peluso et al. reported that PGRMC1 binds to PGRMC2, suggesting that MAPR family members may also undergo haem-mediated heterodimerization. + + 0.9998337 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9991786 + oligomeric_state + cleaner0 + 2023-07-11T12:48:43Z + DUMMY: + + dimerizes + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + stacking interactions + + + 0.9997578 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + 0.9993383 + oligomeric_state + cleaner0 + 2023-07-11T10:08:33Z + DUMMY: + + monomer + + + 0.99948996 + protein_type + cleaner0 + 2023-07-11T13:37:02Z + MESH: + + translationally-controlled tumour protein + + + 0.9988047 + protein_state + cleaner0 + 2023-07-11T12:48:13Z + DUMMY: + + dimerized + + + 0.9997185 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + haem–haem stacking + + + 0.8483521 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99942636 + taxonomy_domain + cleaner0 + 2023-07-11T10:04:13Z + DUMMY: + + eukaryotes + + + 0.99948066 + taxonomy_domain + cleaner0 + 2023-07-11T13:10:38Z + DUMMY: + + bacteria + + + 0.999531 + experimental_method + cleaner0 + 2023-07-11T13:25:39Z + MESH: + + Sequence alignments + + + 0.9995895 + site + cleaner0 + 2023-07-11T13:46:21Z + SO: + + haem-binding residues + + + 0.9998964 + residue_name_number + cleaner0 + 2023-07-11T10:15:11Z + DUMMY: + + Tyr113 + + + 0.9998957 + residue_name_number + cleaner0 + 2023-07-11T10:15:16Z + DUMMY: + + Tyr107 + + + 0.9998933 + residue_name_number + cleaner0 + 2023-07-11T10:15:20Z + DUMMY: + + Lys163 + + + 0.99989545 + residue_name_number + cleaner0 + 2023-07-11T10:15:25Z + DUMMY: + + Tyr164 + + + 0.99982834 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.9995223 + protein_state + cleaner0 + 2023-07-11T13:43:47Z + DUMMY: + + conserved + + + protein_type + MESH: + cleaner0 + 2023-07-11T10:05:27Z + + MAPR + + + 0.9998982 + residue_name_number + cleaner0 + 2023-07-11T10:15:32Z + DUMMY: + + Y113 + + + 0.9912539 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99984884 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.99989116 + residue_name_number + cleaner0 + 2023-07-11T10:15:33Z + DUMMY: + + Y113 + + + 0.9453771 + structure_element + cleaner0 + 2023-07-11T13:40:47Z + SO: + + consensus motif + + + 0.9983682 + ptm + cleaner0 + 2023-07-11T13:00:54Z + MESH: + + phosphorylation + + + 0.99956584 + protein_type + cleaner0 + 2023-07-11T13:37:08Z + MESH: + + tyrosine kinases + + + 0.9996031 + protein_type + cleaner0 + 2023-07-11T12:54:25Z + MESH: + + Abl + + + 0.9997259 + protein_type + cleaner0 + 2023-07-11T12:54:42Z + MESH: + + Lck + + + 0.9995066 + protein_state + cleaner0 + 2023-07-11T13:43:52Z + DUMMY: + + phosphorylated + + + 0.9998871 + residue_name_number + cleaner0 + 2023-07-11T10:15:33Z + DUMMY: + + Y113 + + + 0.99956065 + experimental_method + cleaner0 + 2023-07-11T13:25:52Z + MESH: + + ESI-MS + + + 0.9998331 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.999848 + protein + cleaner0 + 2023-07-11T12:55:24Z + PR: + + PGRMC2 + + + protein_type + MESH: + cleaner0 + 2023-07-11T10:05:27Z + + MAPR + + + 0.7666159 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + + DISCUSS + paragraph + 19650 + We showed that the haem-mediated dimer of PGRMC1 enables interaction with different subclasses of cytochromes P450 (CYP) (Fig. 6). While the effects of PGRMC1 on cholesterol synthesis mediated by CYP51 have been well documented in yeast and human cells, it has not been clear whether drug-metabolizing CYP activities are regulated by PGRMC1. Szczesna-Skorupa and Kemper reported that PGRMC1 exhibited an inhibitory effect on CYP3A4 drug metabolizing activity by competitively binding with cytochrome P450 reductase (CPR) in HEK293 or HepG2 cells. On the other hand, Oda et al. reported that PGRMC1 had no effect to CYP2E1 and CYP3A4 activities in HepG2 cell. Several other groups showed that PGRMC1 enhanced chemoresistance in several cancer cells such as uterine sarcoma, breast cancer, endometrial tumour and ovarian cancer; however, no evidence of PGRMC1-dependent regulation of CYP activity was provided. Our results showed that PGRMC1 contributes to enhancement of the doxorubicin metabolism, which is mediated by CYP2D6 or CYP3A4 in human colon cancer HCT116 cells (Fig. 6d). While the effects of structural diversity of CYP family proteins and interactions with different xenobiotic substrates should further be examined, the current results suggest that the interaction of drug-metabolizing CYPs with the haem-mediated dimer of PGRMC1 plays a crucial role in regulating their activities. + + 0.9624773 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + 0.9993843 + oligomeric_state + cleaner0 + 2023-07-11T10:05:42Z + DUMMY: + + dimer + + + 0.9998399 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.99937296 + protein_type + cleaner0 + 2023-07-11T10:04:06Z + MESH: + + cytochromes P450 + + + 0.99959105 + protein_type + cleaner0 + 2023-07-11T10:15:50Z + MESH: + + CYP + + + 0.9998441 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.99985826 + protein + cleaner0 + 2023-07-11T10:16:06Z + PR: + + CYP51 + + + 0.99940324 + taxonomy_domain + cleaner0 + 2023-07-11T13:10:46Z + DUMMY: + + yeast + + + 0.99935716 + species + cleaner0 + 2023-07-11T10:15:40Z + MESH: + + human + + + 0.9955434 + protein_type + cleaner0 + 2023-07-11T10:15:51Z + MESH: + + CYP + + + 0.9998425 + protein + cleaner0 + 2023-07-11T10:03:17Z + PR: + + PGRMC1 + + + 0.99982494 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + 0.99979335 + protein + cleaner0 + 2023-07-11T10:16:17Z + PR: + + CYP3A4 + + + protein + PR: + cleaner0 + 2023-07-11T12:56:02Z + + cytochrome P450 reductase + + + 0.9995902 + protein + cleaner0 + 2023-07-11T12:56:31Z + PR: + + CPR + + + 0.99982554 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + 0.9998056 + protein + cleaner0 + 2023-07-11T10:16:11Z + PR: + + CYP2E1 + + + 0.99980384 + protein + cleaner0 + 2023-07-11T10:16:16Z + PR: + + CYP3A4 + + + 0.9998271 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + 0.99979013 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + protein_type + MESH: + cleaner0 + 2023-07-11T10:15:51Z + + CYP + + + 0.9998399 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + 0.9997013 + chemical + cleaner0 + 2023-07-11T10:14:40Z + CHEBI: + + doxorubicin + + + 0.9997967 + protein + cleaner0 + 2023-07-11T12:52:10Z + PR: + + CYP2D6 + + + 0.99980694 + protein + cleaner0 + 2023-07-11T10:16:17Z + PR: + + CYP3A4 + + + 0.9991543 + species + cleaner0 + 2023-07-11T10:15:41Z + MESH: + + human + + + 0.9914016 + protein_type + cleaner0 + 2023-07-11T10:15:51Z + MESH: + + CYP + + + 0.99950373 + protein_type + cleaner0 + 2023-07-11T13:37:15Z + MESH: + + CYPs + + + 0.7879104 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + 0.9994192 + oligomeric_state + cleaner0 + 2023-07-11T10:05:42Z + DUMMY: + + dimer + + + 0.9998374 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + + DISCUSS + paragraph + 21046 + We showed that haem-mediated dimerization of PGRMC1 enhances proliferation and chemoresistance of cancer cells through binding to and regulating EGFR and cytochromes P450 (illustrated in Fig. 7). Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration. Considering microenvironments in and around malignant tumours, the haem concentration in cancer cells is likely to be elevated through multiple mechanisms, such as (i) an increased intake of haem, (ii) mutation of enzymes in TCA cycle (for example, fumarate hydratase) that increases the level of succinyl CoA, a substrate for haem biosynthesis and (iii) metastasis to haem-rich organs such as liver, brain and bone marrow. Moreover, exposure of cancer cells to stimuli such as hypoxia, radiation and chemotherapy causes cell damages and leads to protein degradation, resulting in increased levels of TCA cycle intermediates and in an enhanced haem biosynthesis. On the other hand, excessive haem induces HO-1, the enzyme that oxidatively degrades haem and generates CO. Thus, HO-1 induction in cancer cells may inhibit the haem-mediated dimerization of PGRMC1 through the production of CO and thereby suppress tumour progression. This idea is consistent with the observation that HO-1 induction or CO inhibits tumour growth. + + 0.9647786 + chemical + cleaner0 + 2023-07-11T10:02:53Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9998392 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + 0.5363099 + protein_type + cleaner0 + 2023-07-11T10:03:59Z + MESH: + + EGFR + + + 0.9995657 + protein_type + cleaner0 + 2023-07-11T10:04:06Z + MESH: + + cytochromes P450 + + + 0.9848647 + evidence + cleaner0 + 2023-07-11T13:34:00Z + DUMMY: + + haem-binding affinity + + + 0.99984384 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + 0.66475755 + protein_state + cleaner0 + 2023-07-11T13:44:06Z + DUMMY: + + constitutive + + + 0.9995326 + protein_type + cleaner0 + 2023-07-11T13:37:20Z + MESH: + + haem-binding proteins + + + 0.9998084 + protein + cleaner0 + 2023-07-11T12:56:47Z + PR: + + myoglobin + + + 0.99978274 + protein + cleaner0 + 2023-07-11T12:50:04Z + PR: + + PGMRC1 + + + 0.99966633 + protein_state + cleaner0 + 2023-07-11T10:07:58Z + DUMMY: + + apo + + + 0.999292 + oligomeric_state + cleaner0 + 2023-07-11T10:08:33Z + DUMMY: + + monomer + + + 0.999523 + protein_state + cleaner0 + 2023-07-11T10:05:57Z + DUMMY: + + haem-bound + + + 0.99920493 + oligomeric_state + cleaner0 + 2023-07-11T10:05:42Z + DUMMY: + + dimer + + + 0.99977523 + chemical + cleaner0 + 2023-07-11T10:02:54Z + CHEBI: + + haem + + + 0.9996656 + chemical + cleaner0 + 2023-07-11T10:02:54Z + CHEBI: + + haem + + + 0.9996393 + chemical + cleaner0 + 2023-07-11T10:02:54Z + CHEBI: + + haem + + + 0.99816644 + protein_type + cleaner0 + 2023-07-11T13:39:43Z + MESH: + + fumarate hydratase + + + 0.9997113 + chemical + cleaner0 + 2023-07-11T13:14:23Z + CHEBI: + + succinyl CoA + + + 0.99964297 + chemical + cleaner0 + 2023-07-11T10:02:54Z + CHEBI: + + haem + + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:02:54Z + + haem + + + 0.9997143 + chemical + cleaner0 + 2023-07-11T10:02:54Z + CHEBI: + + haem + + + 0.99975115 + chemical + cleaner0 + 2023-07-11T10:02:54Z + CHEBI: + + haem + + + 0.9821542 + protein + cleaner0 + 2023-07-11T12:56:52Z + PR: + + HO-1 + + + 0.99975115 + chemical + cleaner0 + 2023-07-11T10:02:54Z + CHEBI: + + haem + + + 0.99973446 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + 0.9974893 + protein + cleaner0 + 2023-07-11T12:56:53Z + PR: + + HO-1 + + + 0.94059193 + chemical + cleaner0 + 2023-07-11T10:02:54Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9998331 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + 0.9997408 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + 0.9963533 + protein + cleaner0 + 2023-07-11T12:56:53Z + PR: + + HO-1 + + + 0.9996458 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + + DISCUSS + paragraph + 22532 + Besides the regulatory roles of PGRMC1/Sigma-2 receptor in proliferation and chemoresistance in cancer cells (ref.), recent reports show that PGRMC1 is able to bind to amyloid beta oligomer to enhance its neurotoxicity. Furthermore, Sigma-2 ligand-binding is decreased in transgenic amyloid beta deposition model APP/PS1 female mice. These results suggest a possible involvement of PGRMC1 in Alzheimer's disease. The roles of haem-dependent dimerization of PGRMC1 in the functional regulation of its target proteins deserve further studies to find evidence that therapeutic interventions to interfere with the function of the dimer may control varied disease conditions. + + 0.9997824 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + protein + PR: + cleaner0 + 2023-07-11T12:57:31Z + + Sigma-2 + + + 0.999833 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + 0.9071528 + protein + cleaner0 + 2023-07-11T13:14:43Z + PR: + + amyloid beta + + + 0.6471054 + oligomeric_state + cleaner0 + 2023-07-11T12:48:52Z + DUMMY: + + oligomer + + + 0.98613906 + protein + cleaner0 + 2023-07-11T12:57:36Z + PR: + + Sigma-2 + + + 0.9998436 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + 0.99944144 + chemical + cleaner0 + 2023-07-11T10:02:54Z + CHEBI: + + haem + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.99983823 + protein + cleaner0 + 2023-07-11T10:03:18Z + PR: + + PGRMC1 + + + 0.9993774 + oligomeric_state + cleaner0 + 2023-07-11T10:05:42Z + DUMMY: + + dimer + + + + METHODS + title_1 + 23203 + Methods + + + METHODS + title_2 + 23211 + Materials + + + METHODS + paragraph + 23221 + Recombinant EGF, CYP1A2 and CYP3A4 proteins were purchased from Sigma. Erlotinib was purchased from Cayman. Doxorubicin was purchased from Wako. Anti-FLAG (M2) antibody, FLAG peptide and anti-FLAG antibody-conjugated agarose were purchased from Sigma. Haemin and protoporphyrin-IX (PP-IX) were purchased from Porphyrin Science. + + + METHODS + title_2 + 23549 + Plasmid constructions + + + METHODS + paragraph + 23571 + Human PGRMC1 cDNA was cloned from the cDNA library of HuH7 cells. The PGMRC1 (a.a.44–195 for in vitro studies and a.a.72–195 for crystallographic analyses and SV-AUC) cDNA fragment was amplified with PCR, digested with Bam HI and Sal I and then ligated into pGEX6P-1 (GE Healthcare). For NMR analysis, the PGRMC1 (a.a.44–195) cDNA fragment was amplified with PCR (with primers containing the factor Xa site) and ligated into the Bam HI and Sal I sites of pGEX6P-1. The full-length PGRMC1 cDNA fragment containing resistant sequences for shRNA was generated by using the primers (Supplementary Methods), and ligated into the Eco RI and Bam HI sites of the C-terminus of the 3xFLAG-tagged expression vector p3xFLAG CMV14 (Sigma). + + + METHODS + title_2 + 24305 + Preparations of recombinant proteins + + + METHODS + paragraph + 24342 + pGEX-PGRMC1 wt, Y113F or C129S mutant expression vectors were transformed into BL21 (DE3), and the bacteria were incubated in LB with ampicillin at 37 °C until OD600 reached at 0.8. Protein expression was induced by 1 mmol l−1 isopropyl-β-thiogalactopyranoside for 4 h at 37 °C. Cell pellets were resuspended in the buffer containing 20 mmol l−1 Tris-HCl (pH 7.5), 100 mmol l−1 NaCl and 0.1% Tween 20, sonicated twice for 5 min at 4 °C and centrifuged at 20,000 × g for 30 min. The supernatant was incubated with glutathione Sepharose 4B (GE Healthcare) for 1 h at 4 °C. The resin was then washed five times with the same buffer, and the GST tag was cleared by addition of Precision Protease (GE Healthcare) and further incubation for 16 h at 4 °C. The apo-PGRMC1 was prepared by eliminating the bacterial holo-PGRMC1 with size-exclusion chromatography (Superdex 200; GE Healthcare). Haem-bound PGRMC1 were prepared by treatment with 100 μmol l−1 haemin and purified by size-exclusion chromatography. The PGRMC1 protein treated with Precision Protease to cleave the GST-tag contained additional amino acid residues (GPLGSEF) derived from the restriction site and the protease site for Precision Protease at the N-terminal region of PGRMC1. + + + METHODS + paragraph + 25634 + Isotope-labelled PGRMC1 proteins for NMR analyses were prepared by growing cells (BL21 (DE3)) in minimal M9 media in H2O or 99.9% 2H2O, including ampicillin, metals, vitamins, 15N-ammonium chloride and 13C or 12C glucose as sources of nitrogen and carbon, respectively. These procedures were followed by addition of 1 mmol l−1 isopropyl-β-thiogalactopyranoside for 40 h at 20 °C. Protein purification was performed as mentioned above. The GST tag was cleaved with Factor Xa (GE Healthcare). The proteins were treated with Factor Xa to cleave the GST tag at the direct site of N-terminal region of PGRCM1 (a.a.44–195). + + + METHODS + title_2 + 26266 + X-ray crystallography + + + METHODS + paragraph + 26288 + PGRMC1 (a.a.72–195) crystals were grown at 20 °C using hanging-drop vapour diffusion by mixing equal volumes of protein solution and reservoir solution containing 100 mmol l−1 sodium cacodylate (pH 6.5) and 1.26–1.45 mol l−1 ammonium sulphate. Brown crystals reached maximum size in three weeks. The crystals were soaked in reservoir solution containing 30% trehalose and then flash-frozen in liquid nitrogen. The X-ray diffraction data for PGRMC1 crystals were collected at SPring-8 BL41XU and processed with XDS. The initial phase was obtained by single-wavelength anomalous dispersion, using a dataset collected at 1.73 Å with PHENIX AutoSol. Manual modeling and refinement were performed with COOT and phenix.refine. The deposited model was refined to a resolution of 1.95 Å. In this model, 95.4% of the residues were in favoured regions of Ramachandran plot, and all the others were in allowed regions. Data collection and refinement statistics are shown in Table 1. Molecular figures were created by PyMOL (Schrodinger, LLC. The PyMOL Molecular Graphics System, Version 1.5.0.3). + + + METHODS + title_2 + 27399 + Mass spectrometry analyses + + + METHODS + paragraph + 27426 + The purified PGRMC1 (a.a.44–195) proteins, the wild-type (apo and haem) and the C129S mutant (apo and haem), which included additional amino acid residues (GPLGSEF), were buffer-exchanged into 100 mmol l−1 ammonium acetate, pH 7.5, by passing the proteins through a Bio-Spin 6 column (Bio-Rad). The buffer-exchanged PGRMC1 wild-type (apo and haem) and PGMRC1 C129S mutants were immediately analysed by nanoflow electrospray ionization MS using gold-coated glass capillaries made in house. In the case of ESI-MS analyses under denaturing conditions, buffer-exchanged proteins were denatured before ESI-MS analyses by adding aliquots of formic acid at final concentration of 30%). Spectra were recorded on a SYNAPT G2 HDMS mass spectrometer (Waters, Manchester, UK) in positive ionization mode at 1.20 kV with a 120 V sampling cone voltage. The spectra were calibrated using 1 mg ml−1 caesium iodide and analysed with Mass Lynx software (Waters). + + + METHODS + title_2 + 28389 + SV-AUC analyses + + + METHODS + paragraph + 28405 + SV-AUC experiments were performed in a ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter) equipped with 4-hole An60Ti rotors at 20 °C using Beckman Coulter 12-mm double-sector aluminium centerpieces and sapphire windows. Recombinant PGRMC1 proteins were diluted with the buffer (20 mmol l−1 Tris-HCl (pH 7.5) and 100 mmol l−1 NaCl) at the indicated concentration. Scanning was performed as quickly as possible at 262,080 g at 6.5 cm (60,000 rpm), between 6.0 and 7.2 cm from the axis of rotation with a radial increment of 30 μm using an absorbance optical system. The sedimentation coefficient distributions were obtained using the c(s) method of SEDFIT. The partial specific volume, buffer density and viscosity were calculated using the program SEDNTERP 1.09 and were 0.7216, cm3 g−1, 1.00293, g ml−1, and 1.017 cP, respectively. The sedimentation coefficient of PGRMC1 (a.a.72–195) was calculated with the UltraScan Solution Modeler (US-SOMO) suite using the crystal structure determined in this study. To analyse the effect of CO, protein samples were prepared in a deaerated solution and treated with dithionite at 5 mmol l−1 and/or CO gas. + + + METHODS + title_2 + 29607 + DOSY analysis + + + METHODS + paragraph + 29621 + Diffusion-ordered 2D NMR spectroscopy (DOSY) was used to investigate the oligomerization state of PGRMC1 (a.a.44–195) induced by haem binding. Apo- or haem-bound PGRMC1 and reference proteins (including hen egg lysozyme, ovalbumin and bovine serum albumin (BSA)) dissolved in 50 mmol l−1 phosphate buffer (pH 7.0) containing 5% D2O were measured at 25 °C. The protein concentrations were 0.15–0.2 mmol l−1. DOSY spectra were measured using the stimulated echo sequence with a longitudinal-eddy-current delay, and diffusion coefficients were calculated from signals in the aliphatic regions using the software TOPSPIN (Bruker). The signal intensities fit the Stejskal-Tanner equation: + + + METHODS + paragraph + 30324 + where I represents the signal intensity when gradient pulses of length δ are applied at strength g, varying from 2 to 95% of the full gradient strength (55 G cm−1). The diffusion coefficient D25 is estimated by curve fitting with the term I(0), which corresponds to the signal intensity at a gradient strength of 0. The term γ represents the gyromagnetic ratio, and Δ represents the delay between two sets of gradients responsible for the stimulated echo. In this study, δ was set to 8 or 10 ms, and Δ was set to 40 ms. The hydrodynamic radii of the proteins were estimated on the basis of the Stokes–Einstein equation as follows: + + + METHODS + paragraph + 30975 + where T is the absolute temperature, r is the hydrodynamic radius of the spherical molecule, η is the viscosity of the solvent, and κB is the Boltzmann constant. The molecular weights (MWs) of apo- and haem-bound PGRMC1 proteins were estimated from a relationship between r and MWs. MWs of apo and haem-bound PGRMC1 proteins were obtained from the linear-fitting of measured r values for the reference proteins with known MWs according to the following equation: MW=1.2864 (r3)+8.0411. + + + METHODS + title_2 + 31467 + UV-visible absorption spectrometry and haem titration analysis + + + METHODS + paragraph + 31530 + UV-visible absorption spectra of the protein were recorded with a V-660 (Jasco) spectrophotometer at room temperature. Haem binding was tracked by difference spectroscopy in the Soret region of the UV-visible spectrum. Successive aliquots of 0.5 mmol l−1 haemin in N,N-dimethylformamide were added to both the sample cuvette, which contained 10 μmol l−1 apo-PGRMC1 (a.a.44–195), and the reference cuvette. Spectra were recorded 3 min after the addition of each haem aliquot. The absorbance difference at 400 nm was plotted as a function of haem concentration, and the dissociation constant (Kd) was calculated using a quadratic binding equation. + + + METHODS + title_2 + 32195 + Gel filtration chromatography + + + METHODS + paragraph + 32225 + Recombinant PGRMC1 (a.a.44–195) (10 μg) wt, Y113F or C129S mutant, treated with 5 mmol l−1 sodium dithionite and/or CO gas or left untreated, was separated on a Superdex 200 column equilibrated in buffer containing 20 mmol l−1 Tris-HCl (pH 7.5) and 100 mmol l−1 NaCl using a SMART system (GE Healthcare). To prepare the reducing conditions for ferrous haem proteins, the running buffer was deaerated by boiling and saturating it with argon gas according to modified versions of previously reported methods. Namely, immediately after adding dithionite to give a final concentration of 5 mmol l−1, the buffer was equilibrated into the column. The SMART system was sealed with gas-tight taping to maintain anaerobic conditions. Separations of proteins were completed within 1 h. Protein samples were also prepared in the deaerated solution and treated with dithionite at 5 mmol l−1 and/or CO gas, right before being injected into the column. Fractions were then subjected to SDS-PAGE under ambient conditions and visualized by silver staining. The size of proteins was estimated using molecular mass markers (thyroglobin, 669 kDa; catalase, 232 kDa; aldolase, 150 kDa; bovine albumin, 66 kDa and β-amylase, 20 kDa). Results showing that the molecular size of PGRMC1 became smaller in CO-treated conditions (Fig. 3) were collected ∼60 min after the start of experiments. The stability of CO-binding to PGRMC1 was examined with UV-visible absorption spectra to chase temporal alterations for 2 h, as shown in Supplementary Fig. 14. + + + METHODS + title_2 + 33806 + In vitro binding assays + + + METHODS + paragraph + 33830 + For in vitro binding assays, EGFR protein was obtained from ENZO (BML-SE116) as full length protein isolated from human A431 cells. Human CYP1A2, CYP3A4 proteins purified as a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase were obtained from Sigma (C1561 and C4982, respectively). Proteins for human CYP1A2, CYP3A4 or EGFR (1 μg) were incubated with 10 μg of FLAG-PGRMC1 (a.a.44–195) treated with or without 50 μmol l−1 haemin in 500 μl of binding buffer containing 20 mmol l−1 HEPES-NaOH (pH 7.9), 100 mmol l−1 NaCl, 0.2 mmol l−1 EDTA, 10% glycerol and 0.1% NP40 for 60 min at room temperature. 5 mmol l−1 sodium dithionite was added to produce the reducing conditions specified in the aforementioned methods, and the effects of CORM3 or RuCl3 at 10 μmol l−1 were examined. Then, 10 μl of equilibrated anti-FLAG (M2) agarose was added to the mixture, which was then incubated for 60 min at room temperature. Bound proteins were washed three times with 200 μl of binding buffer and eluted with 10 μl of 2 μg ml−1 FLAG peptide. The eluates were subjected to SDS-PAGE and visualized by Western blotting using antibodies against CYP1A2, CYP3A4 (Santa Cruz: sc-30085 and sc-53850, respectively), FLAG and EGFR (Cell signaling: #2232S). + + + METHODS + title_2 + 35162 + Cell culture analyses + + + METHODS + paragraph + 35184 + The human colon cancer cell line HCT116 and human hepatoma cell line HuH7 were maintained in DMEM medium containing 10% FCS. To generate a stable PGRMC1 knockdown cell line, lentivirus vectors encoding a control or PGRMC1 targeting shRNA sequence were transfected into 293FT cells. The lentivirus was prepared according to the manufacturer's instructions (Invitrogen). HCT116 and HuH7 cells were infected with the lentivirus, and a stable cell line was selected by maintaining the cells in medium containing 10 μg ml−1 blasticidin (Invitrogen) for 1 week. + + + METHODS + paragraph + 35747 + For co-immunoprecipitation assay, the expression vector of FLAG-PGRMC1 or an empty vector into HCT116 by using a transfection reagent Lipofectamine 2000 (Invitrogen). Cells were incubated with or without 250 μ mol l−1 succinylacetone (SA) for 48 h, and the cells were then lysed with NP40 lysis buffer (20 mmol l−1 Tris-HCl (pH 7.5), 150 mmol l−1 NaCl, 1% NP40). The lysates were incubated with 10 μl of equilibrated anti-FLAG (M2) agarose for 60 min at room temperature. Bound proteins were washed three times, and were subjected to SDS-PAGE and visualized by Western blotting using antibodies against PGRMC1 (NOVUS: NBP1–83220) and EGFR. + + + METHODS + paragraph + 36413 + For analysis of EGFR signaling, cells were incubated overnight with serum-deprived medium, and then 100 ng ml−1 EGF was added for 5 min. Cells were lysed with RIPA buffer, and the lysates were subjected to SDS-PAGE and visualized by Western blotting using antibodies against PGRMC1, EGFR, phospho-Y1068 EGFR (Cell signaling: #2234S), AKT (Cell signaling: #9272S), phospho-S473AKT (Cell signaling: #4060S), ERK (Cell signaling: #4695S) and phospho-T185 Y187 ERK (Invitrogen: 44680 G). + + + METHODS + paragraph + 36907 + To analyse proliferation of HCT116 cells, Lipofectamine 2000 (Invitrogen) was used to transfect the shRNA-resistant expression vector of FLAG-PGRMC1 or an empty vector into HCT116 control or PGRMC1-knockdown cells. After 24 h, the cells were seeded and incubated for 12 h on a 96-well plate, after which erlotinib or doxorubicin was added for 24 h. Cell viability was determined by using an MTT assay kit (Millipore) according to the manufacturer's instructions. + + + METHODS + paragraph + 37376 + For analysis of spheroid formation of HCT116 cells, the shRNA-resistant expression vector of FLAG-PGRMC1 or an empty vector was transfected as described above. After 24 h, cells were seeded at 1 × 104 cells per well onto a 96-well spheroid culture plate (NanoCulture plate with a microsquare pattern, SCIVAX Corp.) and incubated for three days. The size of individual spheroid was determined by measuring their optical areas using Image-J and by calculating the apparent radius (r) to estimate their apparent volume (v) according to the following formula: v=4/3 × πr3. + + + METHODS + title_2 + 37950 + Measurements of intracellular haem concentrations + + + METHODS + paragraph + 38000 + To measure protohaem (haem b) concentrations, LC-UV and LC-MS analyses were performed for quantification and molecular identification, respectively. Briefly, HCT116 cells (1 × 107 cells) were treated with vehicle or 250 μmol l−1 SA for 48 h. After centrifugation, haem b was extracted from cell pellets twice by adding acetone containing 30% formate, followed by a 5 min sonication and centrifugation. The supernatant was collected, and the solvent was evaporated. The dried residues were re-dissolved in acetonitrile containing 0.2% formate and subjected to a LCMS-8030 system equipped with photodiode array (PDA) detector (SPD-20A) (Shimadzu Corporation, Kyoto, Japan). Haem b was detected by monitoring the absorption at 400 nm. Its identity was confirmed by simultaneous mass spectrometric analysis at m/z 616. + + + METHODS + title_2 + 38829 + Analyses of doxorubicin metabolism + + + METHODS + paragraph + 38864 + HCT116 cells (5 × 106 cells/10 cm dish) were cultured for 2 days, after which the cells were cultured in the presence of 0.3 μmol l−1 doxorubicin overnight. The cells were lysed with methanol containing internal standard compounds, and then water-soluble fraction was separated by liquid-liquid extraction (chloroform: methanol: water=1: 2: 1). The amounts of doxorubicin and its metabolites were quantified using LC-MS/MS. Briefly, a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) ion source (LCMS-8030; Shimadzu Corporation) was used in the positive-ESI and multiple reaction monitoring modes. The samples were resolved on an ACQUITY UPLC BEH C18 column (100 × 2.1 mm i.d., 1.7 μm particle) using water and acetonitrile as mobile phases A and B, respectively, at a flow rate of 0.15 ml min−1 and a column temperature of 40 °C. Ion transitions from m/z 544 to m/z 130 and from m/z 546 to m/z 399 for doxorubicin and doxorubicinol, respectively, were monitored for their quantification. + + + METHODS + title_2 + 39912 + Xenograft implantation of HCT116 cells + + + METHODS + paragraph + 39951 + All the protocols for animal experiments in this study were approved by the Experimental Animal Committee of Keio University School of Medicine (the approved number; 08037-(7)). A model of liver metastases of human colon cancer was prepared according to our previous methods with minor modifications. Briefly, HCT116 cells transfected with the cDNA of Venus (1 × 106 cells/mice), a highly sensitive fluorescent protein on tissue slice sections, were transplanted into the spleens of 10-week-old male NOG mice. Ten days after transplantation, the animals were anesthetized with sevoflurane (Maruishi Pharmaceutical), and their livers were removed, embedded in super-cryoembedding medium and frozen quickly in liquid nitrogen. The frozen tissues were sliced with a cryostat (Leica CM1900) at a thickness of 5 μm, and haematoxylin-eosin staining was performed. Fluorescent microscopy (Keyence: BIOREVO, BZ-9000) was used to observe Venus fluorescence. Percentages of cross-sectional areas showing metastatic tumours were calculated by Image-J as previously described. + + + METHODS + title_1 + 41020 + Additional information + + + METHODS + paragraph + 41043 + Accession code: Structural information on PGRMC1 is available from the Protein Data Bank under accession code 4X8Y. + + + METHODS + paragraph + 41159 + How to cite this article: Kabe, Y. et al. Haem-dependent dimerization of PGRMC1/sigma-2 receptor facilitates cancer proliferation and chemoresistance. Nat. Commun. 7:11030 doi: 10.1038/ncomms11030 (2016). + + + SUPPL + title_1 + 41364 + Supplementary Material + + + S79 + S86 + surname:Kowdley;given-names:K. V. + 15508107 + REF + Gastroenterology + ref + 127 + 2004 + 41387 + Iron, hemochromatosis, and hepatocellular carcinoma + + + 2744 + 2750 + surname:Jakszyn;given-names:P. + REF + Int. J. Cancer. + ref + 133 + 2013 + 41439 + Meat and heme iron intake and esophageal adenocarcinoma in the European prospective investigation into cancer and nutrition study + + + 177 + 184 + surname:Bastide;given-names:N. M. + surname:Pierre;given-names:F. H. + surname:Corpet;given-names:D. E. + 21209396 + REF + Cancer Prev. Res. (Phila.) + ref + 4 + 2011 + 41569 + Heme iron from meat and risk of colorectal cancer: a meta-analysis and a review of the mechanisms involved + + + 180 + 193 + surname:Shen;given-names:J. + 24685134 + REF + Cell Rep. + ref + 7 + 2014 + 41676 + Iron metabolism regulates p53 signaling through direct heme-p53 interaction and modulation of p53 localization, stability and function + + + e63402 + surname:Hooda;given-names:J. + 23704904 + REF + PLoS ONE + ref + 8 + 2013 + 41811 + Enhanced heme function and mitochondrial respiration promote the progression of lung cancer cells + + + 7009 + 7021 + surname:Wegiel;given-names:B. + 24121491 + REF + Cancer Res. + ref + 73 + 2013 + 41909 + Carbon monoxide expedites metabolic exhaustion to inhibit tumor growth + + + 3480 + surname:Yamamoto;given-names:T. + 24633012 + REF + Nat. Commun. + ref + 5 + 2014 + 41980 + Reduced methylation of PFKFB3 in cancer cells shunts glucose towards the pentose phosphate pathway + + + 66 + 85 + surname:Sakamoto;given-names:S. + surname:Kabe;given-names:Y. + surname:Hatakeyama;given-names:M. + surname:Yamaguchi;given-names:Y. + surname:Handa;given-names:H. + 19243077 + REF + Chem. Rec. + ref + 9 + 2009 + 42079 + Development and application of high-performance affinity beads: toward chemical biology and drug discovery + + + RESEARCH0068 + surname:Mifsud;given-names:W. + surname:Bateman;given-names:A. + 12537557 + REF + Genome. Biol. + ref + 3 + 2002 + 42186 + Membrane-bound progesterone receptors contain a cytochrome b5-like ligand-binding domain + + + 509 + 513 + surname:Neubauer;given-names:H. + 23758160 + REF + Climacteric + ref + 16 + 2013 + 42275 + Possible role of PGRMC1 in breast cancer development + + + 113 + surname:Craven;given-names:R. J. + 19090968 + REF + Breast Cancer Res. + ref + 10 + 2008 + 42328 + PGRMC1: a new biomarker for the estrogen receptor in breast cancer + + + 1592 + 1599 + surname:Peluso;given-names:J. J. + surname:Liu;given-names:X. + surname:Saunders;given-names:M. M. + surname:Claffey;given-names:K. P. + surname:Phoenix;given-names:K. + 18319313 + REF + J. Clin. Endocrinol. Metab. + ref + 93 + 2008 + 42395 + Regulation of ovarian cancer cell viability and sensitivity to cisplatin by progesterone receptor membrane component-1 + + + 434 + 442 + surname:Friel;given-names:A. M. + 25304370 + REF + Cancer Lett. + ref + 356 + 2015 + 42514 + Progesterone receptor membrane component 1 deficiency attenuates growth while promoting chemosensitivity of human endometrial xenograft tumors + + + 914 + 933 + surname:Nie;given-names:A. Y. + 16921489 + REF + Mol. Carcinog. + ref + 45 + 2006 + 42657 + Predictive toxicogenomics approaches reveal underlying molecular mechanisms of nongenotoxic carcinogenicity + + + E1 + E9 + surname:Mir;given-names:S. U. + surname:Ahmed;given-names:I. S. + surname:Arnold;given-names:S. + surname:Craven;given-names:R. J. + 21918976 + REF + Int. J. Cancer + ref + 131 + 2012 + 42765 + Elevated progesterone receptor membrane component 1/sigma-2 receptor levels in lung tumors and plasma from lung cancer patients + + + S152 + S156 + surname:Hornick;given-names:J. R. + surname:Spitzer;given-names:D. + surname:Goedegebuure;given-names:P. + surname:Mach;given-names:R. H. + surname:Hawkins;given-names:W. G. + 22763259 + REF + Surgery + ref + 152 + 2012 + 42893 + Therapeutic targeting of pancreatic cancer utilizing sigma-2 ligands + + + 142 + 146 + surname:Crudden;given-names:G. + surname:Loesel;given-names:R. + surname:Craven;given-names:R. J. + 15970648 + REF + Tumour Biol. + ref + 26 + 2005 + 42962 + Overexpression of the cytochrome p450 activator hpr6 (heme-1 domain protein/human progesterone receptor) in tumors + + + 24775 + 24782 + surname:Ahmed;given-names:I. S. + surname:Rohe;given-names:H. J. + surname:Twist;given-names:K. E. + surname:Craven;given-names:R. J. + 20538600 + REF + J. Biol. Chem. + ref + 285 + 2010 + 43077 + Pgrmc1 (progesterone receptor membrane component 1) associates with epidermal growth factor receptor and regulates erlotinib sensitivity + + + 143 + 149 + surname:Hughes;given-names:A. L. + 17276356 + REF + Cell Metab. + ref + 5 + 2007 + 43214 + Dap1/PGRMC1 binds and regulates cytochrome P450 enzymes + + + 564 + 573 + surname:Ahmed;given-names:I. S. + surname:Rohe;given-names:H. J. + surname:Twist;given-names:K. E. + surname:Mattingly;given-names:M. N. + surname:Craven;given-names:R. J. + 20164297 + REF + J. Pharmacol. Exp. Ther. + ref + 333 + 2010 + 43270 + Progesterone receptor membrane component 1 (Pgrmc1): a heme-1 domain protein that promotes tumorigenesis and is inhibited by a small molecule + + + 361 + 370 + surname:Ahmed;given-names:I. S. + surname:Chamberlain;given-names:C. + surname:Craven;given-names:R. J. + 22292588 + REF + Expert Opin. Drug. Metab. Toxicol. + ref + 8 + 2012 + 43412 + S2R(Pgrmc1): the cytochrome-related sigma-2 receptor that regulates lipid and drug metabolism and hormone signaling + + + 1638 + 1647 + surname:Kaluka;given-names:D. + surname:Batabyal;given-names:D. + surname:Chiang;given-names:B. Y. + surname:Poulos;given-names:T. L. + surname:Yeh;given-names:S. R. + 25675345 + REF + Biochemistry + ref + 54 + 2015 + 43528 + Spectroscopic and mutagenesis studies of human PGRMC1 + + + 5832 + 5843 + surname:Min;given-names:L. + 16279947 + REF + FEBS J. + ref + 272 + 2005 + 43582 + Molecular identification of adrenal inner zone antigen as a heme-binding protein + + + 774 + 797 + surname:Krissinel;given-names:E. + surname:Henrick;given-names:K. + 17681537 + REF + J. Mol. Biol. + ref + 372 + 2007 + 43663 + Inference of macromolecular assemblies from crystalline state + + + 213 + 223 + surname:Shen;given-names:Y. + surname:Delaglio;given-names:F. + surname:Cornilescu;given-names:G. + surname:Bax;given-names:A. + 19548092 + REF + J. Biomol. NMR. + ref + 44 + 2009 + 43725 + TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts + + + 702 + 705 + surname:Ohto;given-names:U. + 25686612 + REF + Nature + ref + 520 + 2015 + 43821 + Structural basis of CpG and inhibitory DNA recognition by Toll-like receptor 9 + + + 1123 + 1127 + surname:Nogi;given-names:T. + 20881961 + REF + Nature + ref + 467 + 2010 + 43900 + Structural basis for semaphorin signalling through the plexin receptor + + + 171 + 181 + surname:Ishikawa;given-names:H. + 16039587 + REF + Mol. Cell. + ref + 19 + 2005 + 43971 + Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2 + + + 3733 + 3743 + surname:Liu;given-names:Y. + 10090762 + REF + Biochemistry + ref + 38 + 1999 + 44064 + Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase + + + 1895 + 1904 + surname:Kubo;given-names:A. + 21479793 + REF + Anal. Bioanal. Chem. + ref + 400 + 2011 + 44178 + Semi-quantitative analyses of metabolic systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG mice + + + 973 + 985 + surname:Bao;given-names:Y. + 23741060 + REF + Mol. Cancer Res. + ref + 11 + 2013 + 44313 + Energy management by enhanced glycolysis in G1-phase in human colon cancer cells in vitro and in vivo + + + 3232 + 3238 + surname:Quintieri;given-names:L. + 10866316 + REF + Cancer Res. + ref + 60 + 2000 + 44415 + In vivo antitumor activity and host toxicity of methoxymorpholinyl doxorubicin: role of cytochrome P450 3A + + + 207 + 212 + surname:McFadyen;given-names:M. C. + REF + Biochem. Pharmacol. + ref + 62 + 2001 + 44522 + Cytochrome P450 CYP1B1 protein expression: a novel mechanism of anticancer drug resistance + + + 340 + 350 + surname:Szczesna-Skorupa;given-names:E. + surname:Kemper;given-names:B. + 21081644 + REF + Mol. Pharmacol. + ref + 79 + 2011 + 44613 + Progesterone receptor membrane component 1 inhibits the activity of drug-metabolizing cytochromes P450 and binds to cytochrome P450 reductase + + + 729 + 737 + surname:Higashimoto;given-names:Y. + 15516695 + REF + J. Biol. Chem. + ref + 280 + 2005 + 44755 + Involvement of NADPH in the interaction between heme oxygenase-1 and cytochrome P450 reductase + + + 36505 + 36512 + surname:Wakasugi;given-names:K. + surname:Nakano;given-names:T. + surname:Morishima;given-names:I. + 12860983 + REF + J. Biol. Chem. + ref + 278 + 2003 + 44850 + Oxidized human neuroglobin acts as a heterotrimeric Galpha protein guanine nucleotide dissociation inhibitor + + + e112823 + surname:Lucas;given-names:A. T. + 25396429 + REF + PLoS ONE + ref + 9 + 2014 + 44959 + Ligand binding reveals a role for heme in translationally-controlled tumor protein dimerization + + + 28649 + 28659 + surname:Watanabe;given-names:M. + 18667422 + REF + J. Biol. Chem. + ref + 283 + 2008 + 45055 + Structural basis for multimeric heme complexation through a specific protein-heme interaction: the case of the third neat domain of IsdH from Staphylococcus aureus + + + 16 + 36 + surname:Cahill;given-names:M. A. + 17583495 + REF + J. Steroid Biochem. Mol. Biol. + ref + 105 + 2007 + 45219 + Progesterone receptor membrane component 1: an integrative review + + + 104 + surname:Peluso;given-names:J. J. + surname:Griffin;given-names:D. + surname:Liu;given-names:X. + surname:Horne;given-names:M. + 25253729 + REF + Biol. Reprod. + ref + 91 + 2014 + 45285 + Progesterone receptor membrane component-1 (PGRMC1) and PGRMC-2 interact to suppress entry into the cell cycle in spontaneously immortalized rat granulosa cells + + + 36543 + 36551 + surname:Craven;given-names:R. J. + surname:Mallory;given-names:J. C. + surname:Hand;given-names:R. A. + 17954932 + REF + J. Biol. Chem. + ref + 282 + 2007 + 45446 + Regulation of iron homeostasis mediated by the heme-binding protein Dap1 (damage resistance protein 1) via the P450 protein Erg11/Cyp51 + + + 2057 + 2065 + surname:Oda;given-names:S. + surname:Nakajima;given-names:M. + surname:Toyoda;given-names:Y. + surname:Fukami;given-names:T. + surname:Yokoi;given-names:T. + 21825115 + REF + Drug Metab. Dispos. + ref + 39 + 2011 + 45582 + Progesterone receptor membrane component 1 modulates human cytochrome P450 activities in an isoform-dependent manner + + + 2395 + 2409 + surname:Lin;given-names:S. T. + 25596698 + REF + Cell. Mol. Life Sci. + ref + 72 + 2015 + 45699 + PGRMC1 contributes to doxorubicin-induced chemoresistance in MES-SA uterine sarcoma + + + 273 + 279 + surname:Wendler;given-names:A. + surname:Keller;given-names:D. + surname:Albrecht;given-names:C. + surname:Peluso;given-names:J. J. + surname:Wehling;given-names:M. + 21109987 + REF + Oncol. Rep. + ref + 25 + 2011 + 45783 + Involvement of let-7/miR-98 microRNAs in the regulation of progesterone receptor membrane component 1 expression in ovarian cancer cells + + + 577 + 582 + surname:Liu;given-names:N. + surname:Zhou;given-names:C. + surname:Zhao;given-names:J. + surname:Chen;given-names:Y. + 22812695 + REF + Cancer Invest. + ref + 30 + 2012 + 45920 + Reversal of paclitaxel resistance in epithelial ovarian carcinoma cells by a MUC1 aptamer-let-7i chimera + + + 157 + 192 + surname:Kajimura;given-names:M. + surname:Fukuda;given-names:R. + surname:Bateman;given-names:R. M. + surname:Yamamoto;given-names:T. + surname:Suematsu;given-names:M. + 19939208 + REF + Antioxid. Redox. Signal. + ref + 13 + 2010 + 46025 + Interactions of multiple gas-transducing systems: hallmarks and uncertainties of CO, NO, and H2S gas biology + + + 1227 + 1240 + surname:Kyokane;given-names:T. + 11266386 + REF + Gastroenterology + ref + 120 + 2001 + 46134 + Carbon monoxide from heme catabolism protects against hepatobiliary dysfunction in endotoxin-treated rat liver + + + 3 + 6 + surname:Suematsu;given-names:M. + surname:Ishimura;given-names:Y. + 10613719 + REF + Hepatology + ref + 31 + 2000 + 46245 + The heme oxygenase-carbon monoxide system: a regulator of hepatobiliary function + + + 1459 + 1467 + surname:Chen;given-names:C. + surname:Paw;given-names:B. H. + 22285816 + REF + Biochim. Biophys. Acta. + ref + 1823 + 2012 + 46326 + Cellular and mitochondrial iron homeostasis in vertebrates + + + 225 + 228 + surname:Frezza;given-names:C. + 21849978 + REF + Nature + ref + 477 + 2011 + 46385 + Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase + + + 55 + 62 + surname:Lee;given-names:W. Y. + 24211270 + REF + Toxicol. Appl. Pharmacol. + ref + 274 + 2014 + 46470 + The induction of heme oxygenase-1 suppresses heat shock protein 90 and the proliferation of human breast cancer cells through its byproduct carbon monoxide + + + 380 + surname:Xu;given-names:J. + 21730960 + REF + Nat. Commun. + ref + 2 + 2011 + 46626 + Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site + + + e111899 + surname:Izzo;given-names:N. J. + 25390692 + REF + PLoS ONE + ref + 9 + 2014 + 46717 + Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers II: Sigma-2/PGRMC1 receptors mediate Abeta 42 oligomer binding and synaptotoxicity + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:48:52Z + + oligomer + + + + 302 + 311 + surname:Qin;given-names:Y. + 25576906 + REF + J. Steroid Biochem. Mol. Biol. + ref + 154 + 2015 + 46863 + Progesterone attenuates Abeta25-35-induced neuronal toxicity via JNK inactivation and progesterone receptor membrane component 1-dependent inhibition of mitochondrial apoptotic pathway + + + 439 + 445 + surname:Sahlholm;given-names:K. + surname:Liao;given-names:F. + surname:Holtzman;given-names:D. M. + surname:Xu;given-names:J. + surname:Mach;given-names:R. H. + 25796326 + REF + Biochem. Biophys. Res. Commun. + ref + 460 + 2015 + 47048 + Sigma-2 receptor binding is decreased in female, but not male, APP/PS1 mice + + + 125 + 132 + surname:Kabsch;given-names:W. + 20124692 + REF + Acta. Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 47124 + XDS + + + 582 + 601 + surname:Terwilliger;given-names:T. C. + 19465773 + REF + Acta. Crystallogr. D Biol. Crystallogr. + ref + 65 + 2009 + 47128 + Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard + + + 2126 + 2132 + surname:Emsley;given-names:P. + surname:Cowtan;given-names:K. + 15572765 + REF + Acta. Crystallogr. D Biol. Crystallogr. + ref + 60 + 2004 + 47233 + Coot: model-building tools for molecular graphics + + + 213 + 221 + surname:Adams;given-names:P. D. + 20124702 + REF + Acta. Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 47283 + PHENIX: a comprehensive Python-based system for macromolecular structure solution + + + 1606 + 1619 + surname:Schuck;given-names:P. + 10692345 + REF + Biophys. J. + ref + 78 + 2000 + 47365 + Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling + + + 423 + 435 + surname:Brookes;given-names:E. + surname:Demeler;given-names:B. + surname:Rosano;given-names:C. + surname:Rocco;given-names:M. + 19234696 + REF + Eur. Biophys. J. + ref + 39 + 2010 + 47483 + The implementation of SOMO (SOlution MOdeller) in the UltraScan analytical ultracentrifugation data analysis suite: enhanced capabilities allow the reliable hydrodynamic modeling of virtually any kind of biomacromolecule + + + 260 + 264 + surname:Wu;given-names:D. H. + surname:Chen;given-names:A. D. + surname:Johnson;given-names:C. S. + REF + J. Magn. Reson. A. + ref + 115 + 1995 + 47704 + An improved diffusion-ordered spectroscopy experiment incorporating bipolar-gradient pulses + + + 48 + 55 + surname:Esturau;given-names:N. + 11700080 + REF + J. Magn. Reson. + ref + 153 + 2001 + 47796 + The use of sample rotation for minimizing convection effects in self-diffusion NMR measurements + + + 2431 + 2437 + surname:Suematsu;given-names:M. + 7593631 + REF + J. Clin. Invest. + ref + 96 + 1995 + 47892 + Carbon monoxide: an endogenous modulator of sinusoidal tone in the perfused rat liver + + + 604 + 612 + surname:Goda;given-names:N. + 9449694 + REF + J. Clin. Invest. + ref + 101 + 1998 + 47978 + Distribution of heme oxygenase isoforms in rat liver. Topographic basis for carbon monoxide-mediated microvascular relaxation + + + 87 + 90 + surname:Nagai;given-names:T. + 11753368 + REF + Nat. Biotechnol. + ref + 20 + 2002 + 48104 + A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications + + + SUPPL + footnote + 48212 + Author contributions Y.K., T.K. and M.S. conceived the project; Y.K., T.N. and M.S. designed experiments and wrote the manuscript; Y.K., T.N. and T.S. performed X-ray crystallography. I.K., T.Y. and Y.S. performed cell biology experiments. E.H. and K.S. contributed to NMR experiments. E.K., M.N. and S.U. performed ESI-MS and SV-AUC analyses. M.O. and K.M. performed xenograft experiments in vivo. A.Y., T.U. and K.I. contributed to Raman spectrometric analyses. Y.Y., H.H. and S.I. contributed to consultations based on their expertise. + + + ncomms11030-f1.jpg + f1 + FIG + fig_title_caption + 48751 + X-ray crystal structure of PGRMC1. + + 0.9995518 + evidence + cleaner0 + 2023-07-11T13:34:05Z + DUMMY: + + X-ray crystal structure + + + 0.99986196 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + + ncomms11030-f1.jpg + f1 + FIG + fig_caption + 48786 + (a) Structure of the PGRMC1 dimer formed through stacked haems. Two PGRMC1 subunits (blue and green ribbons) dimerize via stacking of the haem molecules. (b) Haem coordination of PGRMC1 with Tyr113. Comparison of PGRMC1 (blue) and cytochrome b5 (yellow, ID: 3NER). (c) PGRMC1 has a longer helix (a.a.147–163), which is shifted away from the haem (arrow). + + 0.99985814 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.9994522 + oligomeric_state + cleaner0 + 2023-07-11T10:05:42Z + DUMMY: + + dimer + + + 0.99647886 + chemical + cleaner0 + 2023-07-11T13:14:55Z + CHEBI: + + haems + + + 0.9998598 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.9207685 + structure_element + cleaner0 + 2023-07-11T12:49:23Z + SO: + + subunits + + + 0.9993693 + oligomeric_state + cleaner0 + 2023-07-11T12:49:45Z + DUMMY: + + dimerize + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + stacking + + + 0.9973411 + chemical + cleaner0 + 2023-07-11T10:02:55Z + CHEBI: + + haem + + + chemical + CHEBI: + cleaner0 + 2023-07-11T10:02:56Z + + Haem + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + coordination + + + 0.9998574 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.99988806 + residue_name_number + cleaner0 + 2023-07-11T10:15:12Z + DUMMY: + + Tyr113 + + + 0.9998584 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.8310499 + protein_type + cleaner0 + 2023-07-11T10:06:31Z + MESH: + + cytochrome b5 + + + 0.9998435 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.9907663 + structure_element + cleaner0 + 2023-07-11T13:40:53Z + SO: + + helix + + + 0.9971724 + residue_range + cleaner0 + 2023-07-11T13:09:02Z + DUMMY: + + 147–163 + + + 0.995802 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + + ncomms11030-f2.jpg + f2 + FIG + fig_title_caption + 49143 + PGRCM1 is dimerized by binding with haem. + + 0.9996878 + protein + cleaner0 + 2023-07-11T12:57:43Z + PR: + + PGRCM1 + + + 0.9985904 + protein_state + cleaner0 + 2023-07-11T12:48:13Z + DUMMY: + + dimerized + + + 0.99976355 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + + ncomms11030-f2.jpg + f2 + FIG + fig_caption + 49185 + (a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in the presence or absence of haem under non-denaturing condition. Both proteins had identical lengths (a.a.44–195). Data of experimental mass show mean±s.d. (b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44–195) in the presence or absence of haem. SV-AUC experiments were performed with 1.5 mg ml−1 of PGRMC1 proteins. The major peak with sedimentation coefficient S20,w of 1.9∼2.0 S (monomer) or 3.1 S (dimer) was detected. (c) Difference absorption spectra of PGRMC1 (a.a.44–195) titrated with haem (left panel). The titration curve of haem to PGRMC1 (right panel). The absorbance difference at 400 nm is plotted against the haem concentration. + + 0.9995624 + experimental_method + cleaner0 + 2023-07-11T13:26:08Z + MESH: + + Mass spectrometric + + + 0.99958533 + protein_state + cleaner0 + 2023-07-11T10:08:54Z + DUMMY: + + wild-type + + + 0.99963 + protein_state + cleaner0 + 2023-07-11T10:08:59Z + DUMMY: + + wt + + + 0.999826 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.9997073 + mutant + cleaner0 + 2023-07-11T12:59:19Z + MESH: + + C129S + + + 0.99792045 + protein_state + cleaner0 + 2023-07-11T10:13:08Z + DUMMY: + + mutant + + + 0.934071 + protein_state + cleaner0 + 2023-07-11T13:44:13Z + DUMMY: + + presence + + + 0.84403944 + protein_state + cleaner0 + 2023-07-11T10:38:39Z + DUMMY: + + absence of + + + 0.99974006 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.9989166 + residue_range + cleaner0 + 2023-07-11T13:09:07Z + DUMMY: + + 44–195 + + + 0.9995753 + experimental_method + cleaner0 + 2023-07-11T13:26:11Z + MESH: + + SV-AUC + + + 0.99962234 + protein_state + cleaner0 + 2023-07-11T10:08:59Z + DUMMY: + + wt + + + 0.9998266 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.9997187 + mutant + cleaner0 + 2023-07-11T12:59:19Z + MESH: + + C129S + + + 0.99721175 + protein_state + cleaner0 + 2023-07-11T10:13:08Z + DUMMY: + + mutant + + + 0.99878955 + residue_range + cleaner0 + 2023-07-11T13:09:10Z + DUMMY: + + 44–195 + + + 0.8652999 + protein_state + cleaner0 + 2023-07-11T13:44:17Z + DUMMY: + + presence + + + 0.87766445 + protein_state + cleaner0 + 2023-07-11T10:38:40Z + DUMMY: + + absence of + + + 0.9997454 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.99957746 + experimental_method + cleaner0 + 2023-07-11T13:26:14Z + MESH: + + SV-AUC + + + 0.51118416 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.99951327 + evidence + cleaner0 + 2023-07-11T13:34:10Z + DUMMY: + + sedimentation coefficient + + + 0.9979966 + evidence + cleaner0 + 2023-07-11T13:34:13Z + DUMMY: + + S20,w + + + 0.99940455 + oligomeric_state + cleaner0 + 2023-07-11T10:08:33Z + DUMMY: + + monomer + + + 0.9993735 + oligomeric_state + cleaner0 + 2023-07-11T10:05:42Z + DUMMY: + + dimer + + + 0.99936885 + evidence + cleaner0 + 2023-07-11T13:34:16Z + DUMMY: + + Difference absorption spectra + + + 0.99980205 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.99883443 + residue_range + cleaner0 + 2023-07-11T13:09:14Z + DUMMY: + + 44–195 + + + 0.8623522 + experimental_method + cleaner0 + 2023-07-11T13:26:17Z + MESH: + + titrated with + + + 0.99974126 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.99945676 + evidence + cleaner0 + 2023-07-11T13:34:19Z + DUMMY: + + titration curve + + + 0.9996939 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.9998246 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.99908423 + evidence + cleaner0 + 2023-07-11T13:34:23Z + DUMMY: + + absorbance difference + + + 0.9995659 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + + ncomms11030-f3.jpg + f3 + FIG + fig_title_caption + 49947 + Carbon monoxide inhibits haem-dependent PGRMC1 dimerization. + + 0.9996653 + chemical + cleaner0 + 2023-07-11T10:03:35Z + CHEBI: + + Carbon monoxide + + + 0.9952827 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.99984586 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + + ncomms11030-f3.jpg + f3 + FIG + fig_caption + 50008 + (a) UV-visible absorption spectra of PGRMC1 (a.a.44–195). Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). (b) Close-up view of haem stacking. Spheres are drawn with van der Waals radii. (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. + + 0.99774075 + evidence + cleaner0 + 2023-07-11T13:34:29Z + DUMMY: + + UV-visible absorption spectra + + + 0.99984527 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.99811864 + residue_range + cleaner0 + 2023-07-11T13:09:25Z + DUMMY: + + 44–195 + + + 0.99841833 + protein_state + cleaner0 + 2023-07-11T10:14:47Z + DUMMY: + + presence of + + + 0.999592 + protein_state + cleaner0 + 2023-07-11T13:26:54Z + DUMMY: + + oxidized + + + 0.9996444 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.99959916 + protein_state + cleaner0 + 2023-07-11T10:10:20Z + DUMMY: + + ferric + + + 0.99959713 + protein_state + cleaner0 + 2023-07-11T13:26:59Z + DUMMY: + + reduced + + + 0.999368 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.9995776 + protein_state + cleaner0 + 2023-07-11T10:10:25Z + DUMMY: + + ferrous + + + 0.99956197 + protein_state + cleaner0 + 2023-07-11T13:26:59Z + DUMMY: + + reduced + + + 0.9990509 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.99953175 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + 0.99938774 + protein_state + cleaner0 + 2023-07-11T10:10:25Z + DUMMY: + + ferrous + + + 0.9992841 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + haem stacking + + + 0.9995644 + experimental_method + cleaner0 + 2023-07-11T13:26:37Z + MESH: + + Gel-filtration chromatography + + + 0.99983823 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.99805635 + residue_range + cleaner0 + 2023-07-11T13:09:29Z + DUMMY: + + 44–195 + + + 0.9995602 + protein_state + cleaner0 + 2023-07-11T10:08:54Z + DUMMY: + + wild-type + + + 0.9996201 + protein_state + cleaner0 + 2023-07-11T10:08:59Z + DUMMY: + + wt + + + 0.99970573 + mutant + cleaner0 + 2023-07-11T10:13:03Z + MESH: + + Y113F + + + 0.99971074 + mutant + cleaner0 + 2023-07-11T12:59:19Z + MESH: + + C129S + + + 0.9995121 + protein_state + cleaner0 + 2023-07-11T10:13:08Z + DUMMY: + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-07-21T13:48:17Z + + presence + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T10:38:40Z + + absence of + + + 0.9996362 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.99978393 + chemical + cleaner0 + 2023-07-11T13:11:49Z + CHEBI: + + dithionite + + + 0.9997497 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + 0.99985516 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.9996835 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.99974173 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + + ncomms11030-f4.jpg + f4 + FIG + fig_title_caption + 50577 + Haem-dependent dimerization of PGRMC1 is necessary for tumour proliferation mediated by EGFR signalling. + + 0.73282146 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + Haem + + + 0.8711033 + oligomeric_state + cleaner0 + 2023-07-11T12:47:36Z + DUMMY: + + dimerization + + + 0.9998617 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.50743526 + protein_type + cleaner0 + 2023-07-11T10:04:00Z + MESH: + + EGFR + + + + ncomms11030-f4.jpg + f4 + FIG + fig_caption + 50682 + (a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. Input and bound proteins were detected by Western blotting. (b) In vitro binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt (a.a.44–195) and purified EGFR with or without treatment of RuCl3 and CORM3. (c) FLAG-PGRMC1 wt or Y113F (full length) was over-expressed in HCT116 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads. Co-immunoprecipitated proteins (FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody. (d) HCT116 cells were treated with or without 250 μmol l−1 of succinylacetone (SA) for 48 h. The intracellular haem was extracted and quantified by reverse-phase HPLC. The data represent mean±s.d. of four separate experiments. **P<0.01 using unpaired Student's t-test. (e) Co-immunoprecipitation assay was performed as in (c) with or without SA treatment in HCT116 cells. (f) HCT116 cells expressing control shRNA or those knocking down PGRMC1 (PGRMC1-KD) were treated with EGF or left untreated, and components of the EGFR signaling pathway were detected by Western blotting. (g,h) HCT116 cells were treated with or without EGF, SA, RuCl3 and CORM3 as indicated, and components of the EGFR signaling pathway were detected by Western blotting. + + 0.65032303 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.99956894 + protein_state + cleaner0 + 2023-07-11T10:08:54Z + DUMMY: + + wild-type + + + 0.9996101 + protein_state + cleaner0 + 2023-07-11T10:08:59Z + DUMMY: + + wt + + + 0.9996736 + mutant + cleaner0 + 2023-07-11T10:13:03Z + MESH: + + Y113F + + + 0.99750805 + protein_state + cleaner0 + 2023-07-11T10:13:08Z + DUMMY: + + mutant + + + 0.99786013 + residue_range + cleaner0 + 2023-07-11T13:09:35Z + DUMMY: + + 44–195 + + + 0.99967265 + protein_state + cleaner0 + 2023-07-11T10:07:58Z + DUMMY: + + apo + + + 0.99957365 + protein_state + cleaner0 + 2023-07-11T10:05:57Z + DUMMY: + + haem-bound + + + 0.9995159 + experimental_method + cleaner0 + 2023-07-11T13:27:07Z + MESH: + + incubated + + + 0.99928683 + protein_type + cleaner0 + 2023-07-11T10:04:00Z + MESH: + + EGFR + + + 0.9989841 + experimental_method + cleaner0 + 2023-07-11T13:24:07Z + MESH: + + co-immunoprecipitated + + + 0.9990468 + experimental_method + cleaner0 + 2023-07-11T13:27:20Z + MESH: + + Western blotting + + + 0.99958646 + experimental_method + cleaner0 + 2023-07-11T13:27:25Z + MESH: + + In vitro binding assay + + + 0.9995735 + protein_state + cleaner0 + 2023-07-11T10:05:57Z + DUMMY: + + haem-bound + + + 0.67137414 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.99956673 + protein_state + cleaner0 + 2023-07-11T10:08:59Z + DUMMY: + + wt + + + 0.99717873 + residue_range + cleaner0 + 2023-07-11T13:09:40Z + DUMMY: + + 44–195 + + + 0.9992643 + protein_type + cleaner0 + 2023-07-11T10:04:00Z + MESH: + + EGFR + + + 0.99970835 + chemical + cleaner0 + 2023-07-11T13:12:08Z + CHEBI: + + RuCl3 + + + 0.99970764 + chemical + cleaner0 + 2023-07-11T13:12:02Z + CHEBI: + + CORM3 + + + 0.7659941 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.9996171 + protein_state + cleaner0 + 2023-07-11T10:08:59Z + DUMMY: + + wt + + + 0.9996636 + mutant + cleaner0 + 2023-07-11T10:13:03Z + MESH: + + Y113F + + + 0.99956185 + protein_state + cleaner0 + 2023-07-11T13:44:24Z + DUMMY: + + full length + + + 0.9995451 + experimental_method + cleaner0 + 2023-07-11T13:27:37Z + MESH: + + over-expressed + + + 0.9994822 + experimental_method + cleaner0 + 2023-07-11T13:27:40Z + MESH: + + immunoprecipitated + + + experimental_method + MESH: + cleaner0 + 2023-07-11T13:24:07Z + + Co-immunoprecipitated + + + 0.79199886 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T13:04:15Z + + endogenous + + + 0.9998264 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.9994973 + protein_type + cleaner0 + 2023-07-11T10:04:00Z + MESH: + + EGFR + + + 0.9994073 + experimental_method + cleaner0 + 2023-07-11T13:27:45Z + MESH: + + Western blotting + + + 0.99955744 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + protein_type + MESH: + cleaner0 + 2023-07-11T10:04:00Z + + EGFR + + + 0.9997801 + chemical + cleaner0 + 2023-07-11T13:15:02Z + CHEBI: + + succinylacetone + + + 0.99979025 + chemical + cleaner0 + 2023-07-11T13:12:25Z + CHEBI: + + SA + + + 0.9998016 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.9995403 + experimental_method + cleaner0 + 2023-07-11T13:27:48Z + MESH: + + reverse-phase HPLC + + + 0.9226739 + experimental_method + cleaner0 + 2023-07-11T13:27:52Z + MESH: + + Student's t-test + + + 0.9995975 + experimental_method + cleaner0 + 2023-07-11T13:28:26Z + MESH: + + Co-immunoprecipitation assay + + + 0.99978226 + chemical + cleaner0 + 2023-07-11T13:12:25Z + CHEBI: + + SA + + + 0.43960875 + chemical + cleaner0 + 2023-07-11T13:18:31Z + CHEBI: + + shRNA + + + experimental_method + MESH: + cleaner0 + 2023-07-11T13:28:19Z + + knocking down + + + 0.99907625 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.9763978 + mutant + cleaner0 + 2023-07-11T10:12:33Z + MESH: + + PGRMC1-KD + + + 0.9996431 + protein_type + cleaner0 + 2023-07-11T13:15:17Z + MESH: + + EGF + + + 0.8269775 + protein_type + cleaner0 + 2023-07-11T10:04:00Z + MESH: + + EGFR + + + 0.999485 + experimental_method + cleaner0 + 2023-07-11T13:28:28Z + MESH: + + Western blotting + + + 0.999752 + protein_type + cleaner0 + 2023-07-11T13:15:52Z + MESH: + + EGF + + + 0.9998056 + chemical + cleaner0 + 2023-07-11T13:12:25Z + CHEBI: + + SA + + + 0.99973816 + chemical + cleaner0 + 2023-07-11T13:12:08Z + CHEBI: + + RuCl3 + + + 0.9997198 + chemical + cleaner0 + 2023-07-11T13:12:02Z + CHEBI: + + CORM3 + + + 0.5486206 + protein_type + cleaner0 + 2023-07-11T10:04:00Z + MESH: + + EGFR + + + 0.9995396 + experimental_method + cleaner0 + 2023-07-11T13:28:31Z + MESH: + + Western blotting + + + + ncomms11030-f5.jpg + f5 + FIG + fig_title_caption + 52163 + Haem-dependent dimerization of PGRMC1 accelerates tumour growth through the EGFR signaling pathway. + + 0.48312646 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + Haem + + + 0.98892015 + oligomeric_state + cleaner0 + 2023-07-11T12:47:36Z + DUMMY: + + dimerization + + + 0.9998585 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.83838964 + protein_type + cleaner0 + 2023-07-11T10:04:00Z + MESH: + + EGFR + + + + ncomms11030-f5.jpg + f5 + FIG + fig_caption + 52263 + (a) Nucleotide sequences of PGRMC1 targeted by shRNA and of the shRNA-resistant full length PGRMC1 expression vector. Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). (b) Erlotinib was added to HCT116 (control) cells, PGRMC1-KD cells or PGRMC1-KD cells expressing shRNA-resistant PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. The data represent mean±s.d. of four separate experiments. *P<0.01 using ANOVA with Fischer's LSD test. (c) Spheroid formation in control and PGRMC1-KD HCT116 cells. Pictures indicate representative micrographs of spheroids formed under each condition. Spheroid size were measured and compared among groups. The graph represents mean±s.e. of each spheroid size. *P<0.01 using ANOVA with Fischer's LSD test. Scale bar: 0.1 mm. (d) Tumour-bearing livers of NOG mice at 10 days after intrasplenic injection of HCT116 (control) or PGRMC1-KD cells. Percentages of cross-sectional areas showing metastatic tumours were calculated. Data represent mean±s.d. of 10 separate experiments. *P<0.05 using unpaired Student's t-test. Scale bar: 5 mm. + + 0.9998652 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.92046005 + chemical + cleaner0 + 2023-07-11T13:18:30Z + CHEBI: + + shRNA + + + 0.9994595 + protein_state + cleaner0 + 2023-07-11T13:19:52Z + DUMMY: + + shRNA-resistant + + + 0.99961257 + protein_state + cleaner0 + 2023-07-11T13:44:29Z + DUMMY: + + full length + + + 0.99985754 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + mutant + MESH: + cleaner0 + 2023-07-11T13:19:25Z + + PGRMC1-knockdown + + + mutant + MESH: + cleaner0 + 2023-07-11T10:12:33Z + + PGRMC1-KD + + + 0.99932355 + experimental_method + cleaner0 + 2023-07-11T13:28:38Z + MESH: + + transiently transfected + + + 0.99941665 + protein_state + cleaner0 + 2023-07-11T13:19:52Z + DUMMY: + + shRNA-resistant + + + 0.9393594 + experimental_method + cleaner0 + 2023-07-11T13:28:41Z + MESH: + + expression vector + + + 0.9995915 + protein_state + cleaner0 + 2023-07-11T10:08:54Z + DUMMY: + + wild-type + + + 0.999858 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.99938774 + protein_state + cleaner0 + 2023-07-11T10:08:59Z + DUMMY: + + wt + + + 0.9996948 + mutant + cleaner0 + 2023-07-11T10:13:03Z + MESH: + + Y113F + + + 0.9992107 + protein_state + cleaner0 + 2023-07-11T10:13:08Z + DUMMY: + + mutant + + + 0.9996978 + mutant + cleaner0 + 2023-07-11T10:13:03Z + MESH: + + Y113F + + + 0.99971753 + chemical + cleaner0 + 2023-07-11T13:13:59Z + CHEBI: + + Erlotinib + + + mutant + MESH: + cleaner0 + 2023-07-11T10:12:33Z + + PGRMC1-KD + + + mutant + MESH: + cleaner0 + 2023-07-11T10:12:33Z + + PGRMC1-KD + + + 0.99945515 + protein_state + cleaner0 + 2023-07-11T13:19:52Z + DUMMY: + + shRNA-resistant + + + 0.99985015 + protein + cleaner0 + 2023-07-11T10:03:19Z + PR: + + PGRMC1 + + + 0.99955696 + protein_state + cleaner0 + 2023-07-11T10:08:59Z + DUMMY: + + wt + + + 0.999684 + mutant + cleaner0 + 2023-07-11T10:13:03Z + MESH: + + Y113F + + + 0.9995192 + experimental_method + cleaner0 + 2023-07-11T13:28:44Z + MESH: + + MTT assay + + + evidence + DUMMY: + cleaner0 + 2023-07-11T13:29:22Z + + *P + + + 0.992438 + experimental_method + cleaner0 + 2023-07-11T13:28:47Z + MESH: + + ANOVA + + + experimental_method + MESH: + cleaner0 + 2023-07-11T13:20:47Z + + Fischer's LSD test + + + mutant + MESH: + cleaner0 + 2023-07-11T10:12:33Z + + PGRMC1-KD + + + evidence + DUMMY: + cleaner0 + 2023-07-11T13:29:13Z + + *P + + + 0.99427176 + experimental_method + cleaner0 + 2023-07-11T13:28:51Z + MESH: + + ANOVA + + + 0.893418 + experimental_method + cleaner0 + 2023-07-11T10:40:24Z + MESH: + + Fischer's LSD test + + + 0.8566323 + experimental_method + cleaner0 + 2023-07-11T13:28:55Z + MESH: + + intrasplenic injection + + + mutant + MESH: + cleaner0 + 2023-07-11T10:12:33Z + + PGRMC1-KD + + + evidence + DUMMY: + cleaner0 + 2023-07-11T13:29:32Z + + *P + + + 0.98830795 + experimental_method + cleaner0 + 2023-07-11T13:28:58Z + MESH: + + Student's t-test + + + + ncomms11030-f6.jpg + f6 + FIG + fig_title_caption + 53478 + Haem-dependent PGRMC1 dimerization enhances tumour chemoresistance through interaction with cytochromes P450. + + 0.79347175 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + Haem + + + 0.9998203 + protein + cleaner0 + 2023-07-11T10:03:20Z + PR: + + PGRMC1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:36Z + + dimerization + + + 0.9994552 + protein_type + cleaner0 + 2023-07-11T10:04:06Z + MESH: + + cytochromes P450 + + + + ncomms11030-f6.jpg + f6 + FIG + fig_caption + 53588 + (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. Input and bound proteins were detected by Western blotting. (c) Binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt and CYP1A2 with or without RuCl3 and CORM3. (d) Schematic illustration of doxorubicin metabolism is shown on the left. Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD), and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS. Data represent mean±s.d. of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. Data represent mean±s.d. of four separate experiments. *P<0.01 using ANOVA with Fischer's LSD test. + + 0.9981668 + protein + cleaner0 + 2023-07-11T10:03:20Z + PR: + + PGRMC1 + + + 0.99958235 + protein_state + cleaner0 + 2023-07-11T10:08:54Z + DUMMY: + + wild-type + + + 0.99963427 + protein_state + cleaner0 + 2023-07-11T10:08:59Z + DUMMY: + + wt + + + 0.9996884 + mutant + cleaner0 + 2023-07-11T10:13:03Z + MESH: + + Y113F + + + 0.99812263 + protein_state + cleaner0 + 2023-07-11T10:13:08Z + DUMMY: + + mutant + + + 0.99884444 + residue_range + cleaner0 + 2023-07-11T13:09:45Z + DUMMY: + + 44–195 + + + 0.99968255 + protein_state + cleaner0 + 2023-07-11T10:07:58Z + DUMMY: + + apo + + + 0.99957067 + protein_state + cleaner0 + 2023-07-11T10:05:57Z + DUMMY: + + haem-bound + + + 0.9995882 + experimental_method + cleaner0 + 2023-07-11T13:29:39Z + MESH: + + incubated + + + 0.9998394 + protein + cleaner0 + 2023-07-11T12:51:50Z + PR: + + CYP1A2 + + + 0.9997769 + protein + cleaner0 + 2023-07-11T10:16:17Z + PR: + + CYP3A4 + + + 0.9995183 + experimental_method + cleaner0 + 2023-07-11T13:29:43Z + MESH: + + immunoprecipitated + + + 0.9991245 + experimental_method + cleaner0 + 2023-07-11T13:29:47Z + MESH: + + Western blotting + + + 0.9995997 + experimental_method + cleaner0 + 2023-07-11T13:29:49Z + MESH: + + Binding assay + + + 0.99957514 + protein_state + cleaner0 + 2023-07-11T10:05:57Z + DUMMY: + + haem-bound + + + 0.970778 + protein + cleaner0 + 2023-07-11T10:03:20Z + PR: + + PGRMC1 + + + 0.9996581 + protein_state + cleaner0 + 2023-07-11T10:08:59Z + DUMMY: + + wt + + + 0.9998621 + protein + cleaner0 + 2023-07-11T12:51:50Z + PR: + + CYP1A2 + + + 0.9996877 + chemical + cleaner0 + 2023-07-11T13:12:08Z + CHEBI: + + RuCl3 + + + 0.9996524 + chemical + cleaner0 + 2023-07-11T13:12:02Z + CHEBI: + + CORM3 + + + 0.9995505 + chemical + cleaner0 + 2023-07-11T10:14:40Z + CHEBI: + + doxorubicin + + + 0.999668 + chemical + cleaner0 + 2023-07-11T10:14:40Z + CHEBI: + + Doxorubicin + + + 0.9968923 + experimental_method + cleaner0 + 2023-07-11T13:29:53Z + MESH: + + incubated + + + 0.3211297 + chemical + cleaner0 + 2023-07-11T13:18:31Z + CHEBI: + + shRNA + + + 0.5181302 + chemical + cleaner0 + 2023-07-11T13:47:18Z + CHEBI: + + shPGRMC1 + + + mutant + MESH: + cleaner0 + 2023-07-11T10:12:33Z + + PGRMC1-KD + + + 0.999647 + chemical + cleaner0 + 2023-07-11T10:14:33Z + CHEBI: + + doxorubicinol + + + 0.9995933 + chemical + cleaner0 + 2023-07-11T10:14:40Z + CHEBI: + + doxorubicin + + + 0.99955636 + experimental_method + cleaner0 + 2023-07-11T13:29:56Z + MESH: + + LC-MS + + + evidence + DUMMY: + cleaner0 + 2023-07-11T13:30:30Z + + *P + + + 0.90229946 + experimental_method + cleaner0 + 2023-07-11T13:30:35Z + MESH: + + Student's t-test + + + 0.99974185 + chemical + cleaner0 + 2023-07-11T10:14:40Z + CHEBI: + + doxorubicin + + + mutant + MESH: + cleaner0 + 2023-07-11T10:12:33Z + + PGRMC1-KD + + + mutant + MESH: + cleaner0 + 2023-07-11T10:12:33Z + + PGRMC1-KD + + + 0.999475 + protein_state + cleaner0 + 2023-07-11T13:19:52Z + DUMMY: + + shRNA-resistant + + + 0.99956846 + protein_state + cleaner0 + 2023-07-11T13:44:35Z + DUMMY: + + full-length + + + 0.9998354 + protein + cleaner0 + 2023-07-11T10:03:20Z + PR: + + PGRMC1 + + + 0.99963653 + protein_state + cleaner0 + 2023-07-11T10:08:59Z + DUMMY: + + wt + + + 0.9996737 + mutant + cleaner0 + 2023-07-11T10:13:03Z + MESH: + + Y113F + + + 0.9995337 + experimental_method + cleaner0 + 2023-07-11T13:30:48Z + MESH: + + MTT assay + + + evidence + DUMMY: + cleaner0 + 2023-07-11T13:30:44Z + + *P + + + 0.99897254 + experimental_method + cleaner0 + 2023-07-11T13:30:51Z + MESH: + + ANOVA + + + 0.9984215 + experimental_method + cleaner0 + 2023-07-11T13:30:54Z + MESH: + + Fischer's LSD test + + + + ncomms11030-f7.jpg + f7 + FIG + fig_title_caption + 54670 + Schematic diagram for the regulation of PGRMC1 functions. + + 0.9998503 + protein + cleaner0 + 2023-07-11T10:03:20Z + PR: + + PGRMC1 + + + + ncomms11030-f7.jpg + f7 + FIG + fig_caption + 54728 + Apo-PGRMC1 exists as an inactive monomer. On binding to haem, PGRMC1 forms a dimer through stacking interactions between the haem moieties, which enables PGRMC1 to interact with EGFR and cytochromes P450, leading to an enhanced proliferation and chemoresistance of cancer cells. CO interferes with the stacking interactions of the haems and thereby inhibits PGRMC1 functions. + + 0.99966824 + protein_state + cleaner0 + 2023-07-11T10:07:58Z + DUMMY: + + Apo + + + 0.99981254 + protein + cleaner0 + 2023-07-11T10:03:20Z + PR: + + PGRMC1 + + + 0.99964905 + protein_state + cleaner0 + 2023-07-11T13:44:40Z + DUMMY: + + inactive + + + 0.9993481 + oligomeric_state + cleaner0 + 2023-07-11T10:08:33Z + DUMMY: + + monomer + + + 0.98189497 + protein_state + cleaner0 + 2023-07-11T13:44:45Z + DUMMY: + + binding to + + + 0.9996182 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.9998266 + protein + cleaner0 + 2023-07-11T10:03:20Z + PR: + + PGRMC1 + + + 0.9993005 + oligomeric_state + cleaner0 + 2023-07-11T10:05:42Z + DUMMY: + + dimer + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + stacking interactions + + + 0.99957377 + chemical + cleaner0 + 2023-07-11T10:02:56Z + CHEBI: + + haem + + + 0.99981076 + protein + cleaner0 + 2023-07-11T10:03:20Z + PR: + + PGRMC1 + + + 0.8467635 + protein_type + cleaner0 + 2023-07-11T10:04:00Z + MESH: + + EGFR + + + 0.9995526 + protein_type + cleaner0 + 2023-07-11T10:04:06Z + MESH: + + cytochromes P450 + + + 0.9997317 + chemical + cleaner0 + 2023-07-11T10:03:41Z + CHEBI: + + CO + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:55Z + + stacking interactions + + + 0.99605465 + chemical + cleaner0 + 2023-07-11T13:19:39Z + CHEBI: + + haems + + + 0.99982077 + protein + cleaner0 + 2023-07-11T10:03:20Z + PR: + + PGRMC1 + + + + t1.xml + t1 + TABLE + table_title_caption + 55104 + Data collection and refinement statistics. + + + t1.xml + t1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th align="center" valign="top" charoff="50"><bold>Native</bold></th><th align="center" valign="top" charoff="50"><bold>Phasing</bold></th></tr></thead><tbody valign="top"><tr><td colspan="3" align="center" valign="top" charoff="50"><italic>Data collection</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Space group</td><td align="center" valign="top" charoff="50"><italic>I</italic>4<sub>1</sub>22</td><td align="center" valign="top" charoff="50"><italic>I</italic>4<sub>1</sub>22</td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"> Cell dimensions</td></tr><tr><td align="left" valign="top" charoff="50">  <italic>a, b, c</italic> (Å)</td><td align="center" valign="top" charoff="50">167.23, 167.23, 63.46</td><td align="center" valign="top" charoff="50">168.11, 168.11, 63.65</td></tr><tr><td align="left" valign="top" charoff="50">  <italic>α, β, γ</italic> (°)</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td></tr><tr><td align="left" valign="top" charoff="50"> Wavelength (Å)</td><td align="center" valign="top" charoff="50">1.000</td><td align="center" valign="top" charoff="50">1.738</td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="center" valign="top" charoff="50">20.0–1.95 (2.00–1.95)</td><td align="center" valign="top" charoff="50">20.0–2.50 (2.56–2.50)</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>meas</sub></td><td align="center" valign="top" charoff="50">0.067 (1.168)</td><td align="center" valign="top" charoff="50">0.010 (0.850)</td></tr><tr><td align="left" valign="top" charoff="50"> I/σI</td><td align="center" valign="top" charoff="50">22.83 (2.39)</td><td align="center" valign="top" charoff="50">22.43 (4.54)</td></tr><tr><td align="left" valign="top" charoff="50"> Completeness (%)</td><td align="center" valign="top" charoff="50">97.8 (99.0)</td><td align="center" valign="top" charoff="50">99.1 (97.6)</td></tr><tr><td align="left" valign="top" charoff="50"> Multiplicity</td><td align="center" valign="top" charoff="50">11.2 (13.8)</td><td align="center" valign="top" charoff="50">14.9 (15.2)</td></tr><tr><td align="left" valign="top" charoff="50"> CC1/2</td><td align="center" valign="top" charoff="50">100 (81.8)</td><td align="center" valign="top" charoff="50">99.9 (93.5)</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"><italic>Refinement</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="center" valign="top" charoff="50">19.72–1.95</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"> Number of reflections</td><td align="center" valign="top" charoff="50">32,298 (2,384)</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub></td><td align="center" valign="top" charoff="50">0.1834/0.2123</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"> Number of atoms</td></tr><tr><td align="left" valign="top" charoff="50">  Protein</td><td align="center" valign="top" charoff="50">1,776</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  Ligand/ion</td><td align="center" valign="top" charoff="50">86</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  Water</td><td align="center" valign="top" charoff="50">109</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"> B-factors</td></tr><tr><td align="left" valign="top" charoff="50">  Protein</td><td align="center" valign="top" charoff="50">54.6</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  Ligand/ion</td><td align="center" valign="top" charoff="50">42.9</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  Water</td><td align="center" valign="top" charoff="50">46.6</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"> R.M.S deviations</td></tr><tr><td align="left" valign="top" charoff="50">  Bond lengths (Å)</td><td align="center" valign="top" charoff="50">0.008</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  Bond angles (°)</td><td align="center" valign="top" charoff="50">1.164</td><td align="center" valign="top" charoff="50"> </td></tr></tbody></table> + + 55147 +   Native Phasing Data collection  Space group I4122 I4122  Cell dimensions   a, b, c (Å) 167.23, 167.23, 63.46 168.11, 168.11, 63.65   α, β, γ (°) 90, 90, 90 90, 90, 90  Wavelength (Å) 1.000 1.738  Resolution (Å) 20.0–1.95 (2.00–1.95) 20.0–2.50 (2.56–2.50)  Rmeas 0.067 (1.168) 0.010 (0.850)  I/σI 22.83 (2.39) 22.43 (4.54)  Completeness (%) 97.8 (99.0) 99.1 (97.6)  Multiplicity 11.2 (13.8) 14.9 (15.2)  CC1/2 100 (81.8) 99.9 (93.5)       Refinement  Resolution (Å) 19.72–1.95    Number of reflections 32,298 (2,384)    Rwork/Rfree 0.1834/0.2123    Number of atoms   Protein 1,776     Ligand/ion 86     Water 109    B-factors   Protein 54.6     Ligand/ion 42.9     Water 46.6    R.M.S deviations   Bond lengths (Å) 0.008     Bond angles (°) 1.164   + + + t1.xml + t1 + TABLE + table_footnote + 56062 + *Highest resolution shell is shown in parenthesis. + + + t2.xml + t2 + TABLE + table_title_caption + 56113 + PGRMC1 proteins exhibit haem-dependent dimerization in solution. + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-11T12:47:37Z + + dimerization + + + + t2.xml + t2 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th colspan="2" align="center" valign="top" charoff="50"><bold>Apo form</bold><hr/></th><th colspan="2" align="center" valign="top" charoff="50"><bold>Haem-bound form</bold><hr/></th></tr><tr><th align="left" valign="top" charoff="50"> </th><th align="center" valign="top" charoff="50"> </th><th align="center" valign="top" charoff="50">Mass (Da)</th><th align="center" valign="top" charoff="50"> </th><th align="center" valign="top" charoff="50">Mass (Da)</th></tr></thead><tbody valign="top"><tr><td colspan="5" align="center" valign="top" charoff="50"><bold>a</bold> +<italic>PGRMC1 wt (a.a.44–195)</italic></td></tr><tr><td align="left" valign="top" charoff="50"> ESI-MS</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">17,844.14</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">36,920.19</td></tr><tr><td align="left" valign="top" charoff="50"> Theoretical</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">17,843.65</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">36,918.06</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">Hydrodynamic radius 10<sup>−9</sup> (m)</td><td align="center" valign="top" charoff="50">MW (kDa)</td><td align="center" valign="top" charoff="50">Hydrodynamic radius 10<sup>−9</sup> (m)</td><td align="center" valign="top" charoff="50">MW (kDa)</td></tr><tr><td align="left" valign="top" charoff="50"> DOSY</td><td align="center" valign="top" charoff="50">2.04–2.15</td><td align="center" valign="top" charoff="50">20</td><td align="center" valign="top" charoff="50">2.94–3.02</td><td align="center" valign="top" charoff="50">42</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"><italic>S</italic><sub>20,<italic>w</italic></sub> (S)</td><td align="center" valign="top" charoff="50">MW (kDa)</td><td align="center" valign="top" charoff="50"><italic>S</italic><sub>20,<italic>w</italic></sub> (S)</td><td align="center" valign="top" charoff="50">MW (kDa)</td></tr><tr><td align="left" valign="top" charoff="50"> SV-AUC</td><td align="center" valign="top" charoff="50">1.9</td><td align="center" valign="top" charoff="50">17.6</td><td align="center" valign="top" charoff="50">3.1</td><td align="center" valign="top" charoff="50">35.5</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td> </td><td> </td><td> </td><td> </td></tr><tr><td colspan="5" align="center" valign="top" charoff="50"><bold>b</bold> +<italic>PGRMC1 C129S (a.a.44–195)</italic></td></tr><tr><td align="left" valign="top" charoff="50"> ESI-MS</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">17,827.91</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">36,887.07</td></tr><tr><td align="left" valign="top" charoff="50"> Theoretical</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">17,827.59</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">36,885.6</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"><italic>S</italic><sub>20,<italic>w</italic></sub> (S)</td><td align="center" valign="top" charoff="50">MW (kDa)</td><td align="center" valign="top" charoff="50"><italic>S</italic><sub>20,<italic>w</italic></sub> (S)</td><td align="center" valign="top" charoff="50">MW (kDa)</td></tr><tr><td align="left" valign="top" charoff="50"> SV-AUC</td><td align="center" valign="top" charoff="50">2.0</td><td align="center" valign="top" charoff="50">18.1</td><td align="center" valign="top" charoff="50">3.1</td><td align="center" valign="top" charoff="50">35.8</td></tr></tbody></table> + + 56178 +   Apo form Haem-bound form     Mass (Da)   Mass (Da) aPGRMC1 wt (a.a.44–195)  ESI-MS — 17,844.14 — 36,920.19  Theoretical   17,843.65   36,918.06   Hydrodynamic radius 10−9 (m) MW (kDa) Hydrodynamic radius 10−9 (m) MW (kDa)  DOSY 2.04–2.15 20 2.94–3.02 42   S20,w (S) MW (kDa) S20,w (S) MW (kDa)  SV-AUC 1.9 17.6 3.1 35.5           bPGRMC1 C129S (a.a.44–195)  ESI-MS — 17,827.91 — 36,887.07  Theoretical   17,827.59   36,885.6   S20,w (S) MW (kDa) S20,w (S) MW (kDa)  SV-AUC 2.0 18.1 3.1 35.8 + + mutant + MESH: + cleaner0 + 2023-07-11T12:59:19Z + + C129S + + + + t2.xml + t2 + TABLE + table_footnote + 56752 + Differences in molecular weights of the wild-type (wt; a) and the C129S mutant (b) PGRMC1 proteins in the absence (apo form) or the presence of haem (haem-bound form). The protein sizes of the wt and C129S PGRMC1 cytosolic domains (a.a.44–195) in the presence or absence of haem were estimated by ESI-MS, DOSY and SV-AUC. + + mutant + MESH: + cleaner0 + 2023-07-11T12:59:19Z + + C129S + + + mutant + MESH: + cleaner0 + 2023-07-11T12:59:19Z + + C129S + + + + diff --git a/BioC_XML/4820378_v0.xml b/BioC_XML/4820378_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..520e24e364b8c27a2c438be8a2ca2aebacc456db --- /dev/null +++ b/BioC_XML/4820378_v0.xml @@ -0,0 +1,7487 @@ + + + + PMC + 20201223 + pmc.key + + 4820378 + CC BY-NC + no + 0 + 0 + + 10.1126/sciadv.1501397 + 4820378 + 27051866 + 1501397 + e1501397 + 3 + biomolecules nucleotides tRNA 3′-5′ addition reverse polymerization TLP crystal structure + This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited. + surname:Kimura;given-names:Shoko + surname:Suzuki;given-names:Tateki + surname:Chen;given-names:Meirong + surname:Kato;given-names:Koji + surname:Yu;given-names:Jian + surname:Nakamura;given-names:Akiyoshi + surname:Tanaka;given-names:Isao + surname:Yao;given-names:Min + surname:Tanaka;given-names:Isao + TITLE + Keywords + front + 2 + 2016 + 0 + Template-dependent nucleotide addition in the reverse (3′-5′) direction by Thg1-like protein + + 0.99769545 + protein_type + cleaner0 + 2023-07-27T08:36:51Z + MESH: + + Thg1-like protein + + + + ABSTRACT + abstract + 97 + Structures of Thg1-like proteins provide insight into the template-dependent nucleotide addition in the reverse (3′-5′) direction. + + 0.9979704 + evidence + cleaner0 + 2023-07-27T10:24:50Z + DUMMY: + + Structures + + + 0.99865925 + protein_type + cleaner0 + 2023-07-27T08:38:16Z + MESH: + + Thg1-like proteins + + + + ABSTRACT + abstract + 232 + Thg1-like protein (TLP) catalyzes the addition of a nucleotide to the 5′-end of truncated transfer RNA (tRNA) species in a Watson-Crick template–dependent manner. The reaction proceeds in two steps: the activation of the 5′-end by adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleotide addition. Structural analyses of the TLP and its reaction intermediates have revealed the atomic detail of the template-dependent elongation reaction in the 3′-5′ direction. The enzyme creates two substrate binding sites for the first- and second-step reactions in the vicinity of one reaction center consisting of two Mg2+ ions, and the two reactions are executed at the same reaction center in a stepwise fashion. When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3′-OH of the incoming nucleotide and the 5′-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. That the 3′-5′ elongation enzyme performs this elaborate two-step reaction in one catalytic center suggests that these two reactions have been inseparable throughout the process of protein evolution. Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNAHis-specific G−1 addition enzyme. Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. Furthermore, mutational analyses show that tRNAHis is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode. + + 0.9819144 + protein_type + cleaner0 + 2023-07-27T08:36:52Z + MESH: + + Thg1-like protein + + + 0.99417204 + protein_type + cleaner0 + 2023-07-27T08:37:27Z + MESH: + + TLP + + + 0.9968427 + chemical + cleaner0 + 2023-07-27T08:38:38Z + CHEBI: + + transfer RNA + + + 0.9986614 + chemical + cleaner0 + 2023-07-27T08:39:12Z + CHEBI: + + tRNA + + + 0.9991456 + chemical + cleaner0 + 2023-07-27T08:39:21Z + CHEBI: + + adenosine 5′-triphosphate + + + 0.99925095 + chemical + cleaner0 + 2023-07-27T08:39:26Z + CHEBI: + + ATP + + + 0.99916315 + chemical + cleaner0 + 2023-07-27T08:39:30Z + CHEBI: + + guanosine 5′-triphosphate + + + 0.99925977 + chemical + cleaner0 + 2023-07-27T08:39:34Z + CHEBI: + + GTP + + + 0.9986971 + experimental_method + cleaner0 + 2023-07-27T10:16:01Z + MESH: + + Structural analyses + + + 0.962661 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.99868774 + site + cleaner0 + 2023-07-27T10:03:50Z + SO: + + substrate binding sites + + + site + SO: + cleaner0 + 2023-07-27T10:02:25Z + + reaction center + + + 0.99890465 + chemical + cleaner0 + 2023-07-27T10:07:03Z + CHEBI: + + Mg2+ + + + site + SO: + cleaner0 + 2023-07-27T10:02:25Z + + reaction center + + + 0.9965724 + chemical + cleaner0 + 2023-07-27T10:07:09Z + CHEBI: + + nucleotide + + + 0.9980839 + protein_state + cleaner0 + 2023-07-27T08:40:17Z + DUMMY: + + bound to + + + 0.99871296 + site + cleaner0 + 2023-07-27T10:05:45Z + SO: + + second binding site + + + bond_interaction + MESH: + cleaner0 + 2023-07-27T08:37:54Z + + Watson-Crick hydrogen bonds + + + 0.8504399 + chemical + cleaner0 + 2023-07-27T09:32:05Z + CHEBI: + + 5′-triphosphate + + + 0.9985703 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.93671775 + site + cleaner0 + 2023-07-27T10:02:25Z + SO: + + reaction center + + + 0.9894528 + protein_type + cleaner0 + 2023-07-27T10:06:42Z + MESH: + + 3′-5′ elongation enzyme + + + site + SO: + cleaner0 + 2023-07-27T10:05:27Z + + catalytic center + + + 0.99867994 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.99912554 + protein + cleaner0 + 2023-07-27T08:38:02Z + PR: + + Thg1 + + + 0.99841225 + oligomeric_state + cleaner0 + 2023-07-27T08:39:47Z + DUMMY: + + tetrameric + + + 0.65315086 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.99869436 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.9993358 + protein + cleaner0 + 2023-07-27T08:38:02Z + PR: + + Thg1 + + + protein_type + MESH: + cleaner0 + 2023-07-27T08:40:09Z + + tRNAHis-specific G−1 addition enzyme + + + 0.9974462 + chemical + cleaner0 + 2023-07-27T08:40:24Z + CHEBI: + + tRNAHis + + + 0.99938846 + protein + cleaner0 + 2023-07-27T08:38:02Z + PR: + + Thg1 + + + 0.9986511 + oligomeric_state + cleaner0 + 2023-07-27T08:39:42Z + DUMMY: + + tetramer + + + 0.9982622 + structure_element + cleaner0 + 2023-07-27T10:31:16Z + SO: + + subunits + + + 0.99881375 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.9990613 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.99875957 + site + cleaner0 + 2023-07-27T09:50:09Z + SO: + + dimer interface + + + structure_element + SO: + cleaner0 + 2023-07-27T10:22:09Z + + accepter stem + + + 0.9910454 + protein_state + cleaner0 + 2023-07-27T10:29:14Z + DUMMY: + + flexible + + + 0.9992483 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + 0.99854076 + experimental_method + cleaner0 + 2023-07-27T10:16:05Z + MESH: + + mutational analyses + + + 0.99759763 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.99900013 + protein_state + cleaner0 + 2023-07-27T08:40:16Z + DUMMY: + + bound to + + + 0.9980719 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.99925965 + protein + cleaner0 + 2023-07-27T08:38:02Z + PR: + + Thg1 + + + 0.99786204 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + + INTRO + title_1 + 1972 + INTRODUCTION + + + INTRO + paragraph + 1985 + All polynucleotide chain elongation reactions, whether with DNA or RNA, proceed in the 5′-3′ direction. This reaction involves the nucleophilic attack of a 3′-OH of the terminal nucleotide in the elongating chain on the α-phosphate of an incoming nucleotide. The energy in the high-energy bond of the incoming nucleotide is used for its addition [termed tail polymerization ]. This elongation reaction of DNA/RNA chains is in clear contrast to the elongation of protein chains in which the high energy of the incoming aminoacyl-tRNA is not used for its own addition but for the addition of the next monomer (termed head polymerization). However, recent studies have shown that the Thg1/Thg1-like protein (TLP) family of proteins extends tRNA nucleotide chains in the reverse (3′-5′) direction. In this case, the 5′-end of tRNA is first activated using adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleophilic attack of a 3′-OH on the incoming nucleotide [nucleoside 5′-triphosphate (NTP)] to yield pppN-tRNA. Thus, the energy in the triphosphate bond of the incoming nucleotide is not used for its own addition but is reserved for subsequent polymerization (that is, head polymerization) (Fig. 1). + + 0.9901839 + chemical + cleaner0 + 2023-07-27T10:07:14Z + CHEBI: + + DNA + + + 0.99599516 + chemical + cleaner0 + 2023-07-27T10:07:31Z + CHEBI: + + RNA + + + 0.9835027 + chemical + cleaner0 + 2023-07-27T09:08:06Z + CHEBI: + + phosphate + + + 0.9404228 + chemical + cleaner0 + 2023-07-27T10:07:15Z + CHEBI: + + DNA + + + 0.9858251 + chemical + cleaner0 + 2023-07-27T10:07:32Z + CHEBI: + + RNA + + + 0.99878734 + chemical + cleaner0 + 2023-07-27T09:07:59Z + CHEBI: + + aminoacyl-tRNA + + + 0.9985611 + oligomeric_state + cleaner0 + 2023-07-27T09:07:37Z + DUMMY: + + monomer + + + protein + PR: + cleaner0 + 2023-07-27T08:38:03Z + + Thg1 + + + protein_type + MESH: + cleaner0 + 2023-07-27T08:36:52Z + + Thg1-like protein + + + 0.99842346 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.99820364 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.99828124 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.99920386 + chemical + cleaner0 + 2023-07-27T08:39:21Z + CHEBI: + + adenosine 5′-triphosphate + + + 0.9993376 + chemical + cleaner0 + 2023-07-27T08:39:26Z + CHEBI: + + ATP + + + 0.9992138 + chemical + cleaner0 + 2023-07-27T08:39:30Z + CHEBI: + + guanosine 5′-triphosphate + + + 0.99931574 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.999152 + chemical + cleaner0 + 2023-07-27T09:08:33Z + CHEBI: + + nucleoside 5′-triphosphate + + + 0.9992663 + chemical + cleaner0 + 2023-07-27T09:08:37Z + CHEBI: + + NTP + + + 0.76323205 + chemical + cleaner0 + 2023-07-27T09:08:17Z + CHEBI: + + pppN-tRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:24:41Z + + triphosphate + + + + 1501397-F1.jpg + F1 + FIG + fig_title_caption + 3237 + Reaction schemes of 3′-5′ and 5′-3′ elongation. + + + 1501397-F1.jpg + F1 + FIG + fig_caption + 3293 + Top: Reaction scheme of 3′-5′ elongation by Thg1/TLP family proteins. Bottom: Reaction scheme of 5′-3′ elongation by DNA/RNA polymerases. In 3′-5′ elongation by Thg1/TLP family proteins, the 5′-monophosphate of the tRNA is first activated by ATP/GTP, followed by the actual elongation reaction. The energy of the incoming nucleotide is not used for its own addition but is reserved for the subsequent addition (head polymerization). In 5′-3′ elongation by DNA/RNA polymerases, the energy of the incoming nucleotide is used for its own addition (tail polymerization). + + protein + PR: + cleaner0 + 2023-07-27T08:38:03Z + + Thg1 + + + protein_type + MESH: + cleaner0 + 2023-07-27T08:37:28Z + + TLP + + + 0.99611807 + protein_type + cleaner0 + 2023-07-27T09:09:53Z + MESH: + + DNA/RNA polymerases + + + protein + PR: + cleaner0 + 2023-07-27T08:38:03Z + + Thg1 + + + protein_type + MESH: + cleaner0 + 2023-07-27T08:37:28Z + + TLP + + + chemical + CHEBI: + cleaner0 + 2023-07-27T10:08:10Z + + 5′-monophosphate + + + 0.99824333 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9993061 + chemical + cleaner0 + 2023-07-27T08:39:26Z + CHEBI: + + ATP + + + 0.9993092 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.996169 + protein_type + cleaner0 + 2023-07-27T09:09:53Z + MESH: + + DNA/RNA polymerases + + + + INTRO + paragraph + 3878 + The best-characterized member of this family of proteins is eukaryotic Thg1 (tRNAHis guanylyltransferase), which catalyzes the nontemplated addition of a guanylate to the 5′-end of immature tRNAHis. This guanosine at position −1 (G−1) of tRNAHis is a critical identity element for recognition by the histidyl-tRNA synthase. Therefore, Thg1 is essential to the fidelity of protein synthesis in eukaryotes. However, Thg1 homologs or TLPs are found in organisms in which G−1 is genetically encoded, and thus, posttranscriptional modification is not required. This finding suggests that TLPs may have potential functions other than tRNAHis maturation. TLPs have been shown to catalyze 5′-end nucleotide addition to truncated tRNA species in vitro in a Watson-Crick template–dependent manner. This function of TLPs is not limited to tRNAHis but occurs efficiently with other tRNAs. Furthermore, the yeast homolog (Thg1p) has been shown to interact with the replication origin recognition complex for DNA replication, and the plant homolog (ICA1) was identified as a protein affecting the capacity to repair DNA damage. These observations suggest that TLPs may have more general functions in DNA/RNA repair. + + 0.99877626 + taxonomy_domain + cleaner0 + 2023-07-27T09:10:13Z + DUMMY: + + eukaryotic + + + 0.9991749 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.99857557 + protein_type + cleaner0 + 2023-07-27T09:10:07Z + MESH: + + tRNAHis guanylyltransferase + + + 0.97453237 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.51417506 + chemical + cleaner0 + 2023-07-27T09:52:51Z + CHEBI: + + guanosine + + + 0.97779673 + residue_number + cleaner0 + 2023-07-27T10:14:27Z + DUMMY: + + −1 + + + 0.93238014 + residue_name_number + cleaner0 + 2023-07-27T09:53:10Z + DUMMY: + + G−1 + + + 0.96735996 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.99853724 + protein_type + cleaner0 + 2023-07-27T09:10:27Z + MESH: + + histidyl-tRNA synthase + + + 0.9990308 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.9986656 + taxonomy_domain + cleaner0 + 2023-07-27T09:12:12Z + DUMMY: + + eukaryotes + + + 0.9842792 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.9990934 + protein_type + cleaner0 + 2023-07-27T09:10:32Z + MESH: + + TLPs + + + 0.42650872 + residue_name_number + cleaner0 + 2023-07-27T09:53:21Z + DUMMY: + + G−1 + + + 0.9991235 + protein_type + cleaner0 + 2023-07-27T09:10:32Z + MESH: + + TLPs + + + 0.9778497 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.9990957 + protein_type + cleaner0 + 2023-07-27T09:10:32Z + MESH: + + TLPs + + + 0.99528116 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9990841 + protein_type + cleaner0 + 2023-07-27T09:10:32Z + MESH: + + TLPs + + + 0.98222923 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.9904108 + chemical + cleaner0 + 2023-07-27T10:10:42Z + CHEBI: + + tRNAs + + + 0.99877506 + taxonomy_domain + cleaner0 + 2023-07-27T09:12:04Z + DUMMY: + + yeast + + + 0.99906677 + protein + cleaner0 + 2023-07-27T10:14:00Z + PR: + + Thg1p + + + chemical + CHEBI: + cleaner0 + 2023-07-27T10:07:15Z + + DNA + + + 0.99885345 + taxonomy_domain + cleaner0 + 2023-07-27T09:11:59Z + DUMMY: + + plant + + + 0.9990459 + protein + cleaner0 + 2023-07-27T10:14:08Z + PR: + + ICA1 + + + chemical + CHEBI: + cleaner0 + 2023-07-27T10:07:15Z + + DNA + + + 0.99915147 + protein_type + cleaner0 + 2023-07-27T09:10:32Z + MESH: + + TLPs + + + chemical + CHEBI: + cleaner0 + 2023-07-27T10:07:15Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-27T10:07:32Z + + RNA + + + + INTRO + paragraph + 5092 + The 3′-5′ addition reaction catalyzed by Thg1 occurs through three reaction steps. In the first step, the 5′-monophosphorylated tRNAHis, which is cleaved by ribonuclease P from pre-tRNAHis, is activated by ATP, creating a 5′-adenylylated tRNAHis intermediate. In the second step, the 3′-OH of the incoming GTP attacks the activated intermediate, yielding pppG−1-tRNAHis. Finally, the pyrophosphate is removed, and mature pG−1-tRNAHis is created. The crystal structure of human Thg1 (HsThg1) shows that its catalytic core shares structural homology with canonical 5′-3′ nucleotide polymerases, such as T7 DNA/RNA polymerases. This finding suggests that 3′-5′ elongation enzymes are related to 5′-3′ polymerases and raises important questions on why 5′-3′ polymerases predominate in nature. The crystal structure of TLP from Bacillus thuringiensis shows that it shares a similar tetrameric assembly and active-site architecture with HsThg1. Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase. However, in this structural analysis, the 5′-end of tRNAHis was not activated and the second substrate (GTP) was not bound. Thus, a detailed reaction mechanism remains unknown. + + 0.9993753 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.9984634 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.9819302 + protein_type + cleaner0 + 2023-07-27T09:54:03Z + MESH: + + ribonuclease P + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:54:24Z + + pre-tRNAHis + + + 0.9991885 + chemical + cleaner0 + 2023-07-27T08:39:26Z + CHEBI: + + ATP + + + 0.99659437 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.99922943 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:54:56Z + + pppG−1-tRNAHis + + + 0.9992478 + chemical + cleaner0 + 2023-07-27T09:55:05Z + CHEBI: + + pyrophosphate + + + 0.99007195 + chemical + cleaner0 + 2023-07-27T09:55:25Z + CHEBI: + + pG−1-tRNAHis + + + 0.99864215 + evidence + cleaner0 + 2023-07-27T10:24:58Z + DUMMY: + + crystal structure + + + 0.99866784 + species + cleaner0 + 2023-07-27T09:11:53Z + MESH: + + human + + + 0.99936336 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.9993332 + protein + cleaner0 + 2023-07-27T09:11:18Z + PR: + + HsThg1 + + + 0.93159294 + site + cleaner0 + 2023-07-27T10:02:20Z + SO: + + catalytic core + + + 0.99825877 + protein_type + cleaner0 + 2023-07-27T10:06:46Z + MESH: + + 5′-3′ nucleotide polymerases + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:06:22Z + + T7 DNA/RNA polymerases + + + 0.99781764 + protein_type + cleaner0 + 2023-07-27T09:11:09Z + MESH: + + 3′-5′ elongation enzymes + + + 0.99836665 + protein_type + cleaner0 + 2023-07-27T09:11:02Z + MESH: + + 5′-3′ polymerases + + + 0.998312 + protein_type + cleaner0 + 2023-07-27T09:11:02Z + MESH: + + 5′-3′ polymerases + + + 0.99878764 + evidence + cleaner0 + 2023-07-27T10:25:01Z + DUMMY: + + crystal structure + + + 0.48748377 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.9984957 + species + cleaner0 + 2023-07-27T09:11:46Z + MESH: + + Bacillus thuringiensis + + + 0.9980994 + oligomeric_state + cleaner0 + 2023-07-27T08:39:47Z + DUMMY: + + tetrameric + + + 0.9979635 + site + cleaner0 + 2023-07-27T10:08:44Z + SO: + + active-site + + + 0.9993067 + protein + cleaner0 + 2023-07-27T09:11:19Z + PR: + + HsThg1 + + + 0.9980274 + evidence + cleaner0 + 2023-07-27T10:25:03Z + DUMMY: + + structure + + + 0.99843144 + species + cleaner0 + 2023-07-27T09:11:35Z + MESH: + + Candida albicans + + + 0.9993672 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.99935013 + protein + cleaner0 + 2023-07-27T09:11:40Z + PR: + + CaThg1 + + + 0.99900305 + protein_state + cleaner0 + 2023-07-27T09:15:20Z + DUMMY: + + complexed with + + + 0.9984999 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.9954639 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.99536276 + site + cleaner0 + 2023-07-27T10:02:25Z + SO: + + reaction center + + + 0.998207 + protein_type + cleaner0 + 2023-07-27T10:06:51Z + MESH: + + DNA/RNA polymerase + + + 0.99610376 + experimental_method + cleaner0 + 2023-07-27T10:16:10Z + MESH: + + structural analysis + + + 0.99906045 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.99919957 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.998389 + protein_state + cleaner0 + 2023-07-27T10:29:29Z + DUMMY: + + not bound + + + + INTRO + paragraph + 6452 + Here, we successfully solved the structure of TLP from the methanogenic archaeon Methanosarcina acetivorans (MaTLP) in complex with ppptRNAPheΔ1, which mimics the activated intermediate of the repair substrate. Although TLP and Thg1 have similar tetrameric organization, the mode of tRNA binding is different in TLP. Furthermore, we obtained the structure in which the GTP analog (GDPNP) was inserted into this complex to form a Watson-Crick base pair with C72 at the 3′-end region of the tRNA. On the basis of these structures, we discuss the reaction mechanism of template-dependent reverse (3′-5′) polymerization in comparison with canonical 5′-3′ polymerization. + + 0.9828011 + experimental_method + cleaner0 + 2023-07-27T10:16:16Z + MESH: + + solved + + + 0.9475209 + evidence + cleaner0 + 2023-07-27T10:25:08Z + DUMMY: + + structure + + + 0.96883136 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.9628024 + taxonomy_domain + cleaner0 + 2023-07-27T09:13:32Z + DUMMY: + + methanogenic archaeon + + + 0.99863946 + species + cleaner0 + 2023-07-27T09:13:04Z + MESH: + + Methanosarcina acetivorans + + + 0.99919146 + protein + cleaner0 + 2023-07-27T09:13:09Z + PR: + + MaTLP + + + 0.99593526 + protein_state + cleaner0 + 2023-07-27T09:12:25Z + DUMMY: + + in complex with + + + 0.9728952 + chemical + cleaner0 + 2023-07-27T09:13:26Z + CHEBI: + + ppptRNAPheΔ1 + + + 0.9950616 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.9991167 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.9980457 + oligomeric_state + cleaner0 + 2023-07-27T08:39:47Z + DUMMY: + + tetrameric + + + 0.99848324 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.8676416 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.9974669 + evidence + cleaner0 + 2023-07-27T10:25:11Z + DUMMY: + + structure + + + 0.99928975 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.9993855 + chemical + cleaner0 + 2023-07-27T09:21:34Z + CHEBI: + + GDPNP + + + bond_interaction + MESH: + cleaner0 + 2023-07-27T09:12:49Z + + Watson-Crick base pair + + + 0.9993098 + residue_name_number + cleaner0 + 2023-07-27T09:17:23Z + DUMMY: + + C72 + + + 0.99910235 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.997968 + evidence + cleaner0 + 2023-07-27T10:25:15Z + DUMMY: + + structures + + + + RESULTS + title_1 + 7130 + RESULTS + + + RESULTS + title_2 + 7138 + Anticodon-independent binding of ppptRNAPheΔ1 to MaTLP + + 0.9988223 + chemical + cleaner0 + 2023-07-27T09:13:27Z + CHEBI: + + ppptRNAPheΔ1 + + + 0.9930177 + protein + cleaner0 + 2023-07-27T09:13:09Z + PR: + + MaTLP + + + + RESULTS + paragraph + 7197 + Previous biochemical experiments have suggested that ppptRNAPheΔ1, in which the 5′-end of tRNAPhe was triphosphorylated and G1 was deleted, can be an efficient substrate for the repair reaction (guanylyl transfer) of Thg1/TLP. Therefore, we prepared a crystal of MaTLP complexed with ppptRNAPheΔ1 and solved its structure to study the template-directed 3′-5′ elongation reaction by TLP (fig. S1). The crystal contained a dimer of TLP (A and B molecules) and one tRNA in an asymmetric unit. Two dimers in the crystal further assembled as a dimer of dimers by the crystallographic twofold axis (Fig. 2). This tetrameric structure and 4:2 stoichiometry of the TLP-tRNA complex are the same as those of the CaThg1-tRNA complex. However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. Thus, consistent with the notion that MaTLP is an anticodon-independent repair enzyme, the anticodon was not recognized in the MaTLP-tRNA complex, whereas the binding mode of CaThg1 is for the G−1 addition reaction, therefore the His anticodon has to be recognized (see “Dual binding mode for tRNA repair”). + + experimental_method + MESH: + cleaner0 + 2023-07-27T10:16:49Z + + biochemical experiments + + + 0.99851924 + chemical + cleaner0 + 2023-07-27T09:13:27Z + CHEBI: + + ppptRNAPheΔ1 + + + 0.9988738 + chemical + cleaner0 + 2023-07-27T09:27:24Z + CHEBI: + + tRNAPhe + + + 0.30236262 + residue_name_number + cleaner0 + 2023-07-27T10:15:20Z + DUMMY: + + G1 + + + 0.9096241 + experimental_method + cleaner0 + 2023-07-27T10:17:02Z + MESH: + + deleted + + + 0.9993913 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.99921095 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.9407621 + evidence + cleaner0 + 2023-07-27T10:25:26Z + DUMMY: + + crystal + + + 0.9993493 + protein + cleaner0 + 2023-07-27T09:13:09Z + PR: + + MaTLP + + + 0.99859333 + protein_state + cleaner0 + 2023-07-27T09:15:19Z + DUMMY: + + complexed with + + + 0.9969944 + chemical + cleaner0 + 2023-07-27T09:13:27Z + CHEBI: + + ppptRNAPheΔ1 + + + 0.8221576 + experimental_method + cleaner0 + 2023-07-27T10:17:05Z + MESH: + + solved + + + 0.9791672 + evidence + cleaner0 + 2023-07-27T10:25:33Z + DUMMY: + + structure + + + 0.99778384 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.9974795 + evidence + cleaner0 + 2023-07-27T10:25:57Z + DUMMY: + + crystal + + + 0.99883157 + oligomeric_state + cleaner0 + 2023-07-27T09:14:51Z + DUMMY: + + dimer + + + 0.9836731 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-28T13:58:56Z + + A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-28T13:58:56Z + + B + + + 0.9989492 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9988199 + oligomeric_state + cleaner0 + 2023-07-27T09:14:45Z + DUMMY: + + dimers + + + 0.9564452 + evidence + cleaner0 + 2023-07-27T10:25:49Z + DUMMY: + + crystal + + + 0.998803 + oligomeric_state + cleaner0 + 2023-07-27T09:14:51Z + DUMMY: + + dimer + + + 0.998838 + oligomeric_state + cleaner0 + 2023-07-27T09:14:46Z + DUMMY: + + dimers + + + 0.9986577 + oligomeric_state + cleaner0 + 2023-07-27T08:39:47Z + DUMMY: + + tetrameric + + + 0.99766195 + evidence + cleaner0 + 2023-07-27T10:25:52Z + DUMMY: + + structure + + + 0.99917907 + complex_assembly + cleaner0 + 2023-07-27T09:15:08Z + GO: + + TLP-tRNA + + + complex_assembly + GO: + cleaner0 + 2023-07-27T09:14:11Z + + CaThg1-tRNA + + + 0.9992673 + structure_element + cleaner0 + 2023-07-27T10:31:25Z + SO: + + AB + + + 0.9992729 + structure_element + cleaner0 + 2023-07-27T10:31:30Z + SO: + + CD + + + 0.99889463 + oligomeric_state + cleaner0 + 2023-07-27T09:14:46Z + DUMMY: + + dimers + + + 0.9985355 + oligomeric_state + cleaner0 + 2023-07-27T08:39:47Z + DUMMY: + + tetrameric + + + 0.99947053 + protein + cleaner0 + 2023-07-27T09:11:41Z + PR: + + CaThg1 + + + 0.9041023 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + 0.9974681 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.9992331 + structure_element + cleaner0 + 2023-07-27T10:31:25Z + SO: + + AB + + + 0.99891055 + oligomeric_state + cleaner0 + 2023-07-27T09:14:50Z + DUMMY: + + dimer + + + 0.9992483 + structure_element + cleaner0 + 2023-07-27T10:31:30Z + SO: + + CD + + + 0.99889743 + oligomeric_state + cleaner0 + 2023-07-27T09:14:51Z + DUMMY: + + dimer + + + 0.9977823 + oligomeric_state + cleaner0 + 2023-07-27T08:39:47Z + DUMMY: + + tetrameric + + + 0.9994438 + protein + cleaner0 + 2023-07-27T09:13:09Z + PR: + + MaTLP + + + 0.9987863 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.99582255 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + structure_element + SO: + cleaner0 + 2023-07-27T10:22:09Z + + accepter stem + + + 0.9753643 + structure_element + cleaner0 + 2023-07-27T10:23:13Z + SO: + + elbow region + + + 0.99939775 + protein + cleaner0 + 2023-07-27T09:13:09Z + PR: + + MaTLP + + + protein_type + MESH: + cleaner0 + 2023-07-27T10:17:42Z + + anticodon-independent repair enzyme + + + 0.99917156 + complex_assembly + cleaner0 + 2023-07-27T09:14:38Z + GO: + + MaTLP-tRNA + + + 0.9994748 + protein + cleaner0 + 2023-07-27T09:11:41Z + PR: + + CaThg1 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-27T10:18:02Z + + G−1 + + + 0.8723751 + residue_name + cleaner0 + 2023-07-27T09:29:31Z + SO: + + His + + + 0.95955354 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + + 1501397-F2.jpg + F2 + FIG + fig_title_caption + 8563 + Structure of the MaTLP complex with ppptRNAPheΔ1. + + 0.99634486 + evidence + cleaner0 + 2023-07-27T10:26:01Z + DUMMY: + + Structure + + + 0.92571753 + protein + cleaner0 + 2023-07-27T09:13:09Z + PR: + + MaTLP + + + 0.98812664 + protein_state + cleaner0 + 2023-07-27T09:16:07Z + DUMMY: + + complex with + + + 0.99362344 + chemical + cleaner0 + 2023-07-27T09:13:27Z + CHEBI: + + ppptRNAPheΔ1 + + + + 1501397-F2.jpg + F2 + FIG + fig_caption + 8617 + Left: One molecule of the tRNA substrate (ppptRNAPheΔ1) is bound to the MaTLP dimer. The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers). Right: Left figure rotated by 90o. The CD dimer is omitted for clarity. The accepter stem of the tRNA is recognized by molecule A (yellow), and the elbow region by molecule B (blue). Residues important for binding are depicted in stick form. The β-hairpin region of molecule B is shown in red. + + 0.99868494 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9433621 + chemical + cleaner0 + 2023-07-27T09:13:27Z + CHEBI: + + ppptRNAPheΔ1 + + + 0.9990138 + protein_state + cleaner0 + 2023-07-27T08:40:17Z + DUMMY: + + bound to + + + 0.9982882 + protein + cleaner0 + 2023-07-27T09:13:10Z + PR: + + MaTLP + + + 0.9988288 + oligomeric_state + cleaner0 + 2023-07-27T09:14:51Z + DUMMY: + + dimer + + + 0.99934846 + structure_element + cleaner0 + 2023-07-27T10:31:25Z + SO: + + AB + + + 0.99937457 + structure_element + cleaner0 + 2023-07-27T10:31:30Z + SO: + + CD + + + 0.9989034 + oligomeric_state + cleaner0 + 2023-07-27T09:14:46Z + DUMMY: + + dimers + + + 0.96084297 + oligomeric_state + cleaner0 + 2023-07-27T10:13:52Z + DUMMY: + + dimerized + + + 0.99878424 + oligomeric_state + cleaner0 + 2023-07-27T08:39:47Z + DUMMY: + + tetrameric + + + 0.8921645 + evidence + cleaner0 + 2023-07-27T10:26:21Z + DUMMY: + + structure + + + 0.9987697 + oligomeric_state + cleaner0 + 2023-07-27T09:14:51Z + DUMMY: + + dimer + + + 0.9981351 + oligomeric_state + cleaner0 + 2023-07-27T09:14:46Z + DUMMY: + + dimers + + + 0.99936455 + structure_element + cleaner0 + 2023-07-27T10:31:30Z + SO: + + CD + + + 0.9989243 + oligomeric_state + cleaner0 + 2023-07-27T09:14:51Z + DUMMY: + + dimer + + + 0.99393797 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + 0.9991868 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.91121864 + structure_element + cleaner0 + 2023-07-27T10:23:13Z + SO: + + elbow region + + + 0.99934506 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + + RESULTS + paragraph + 9133 + The elbow region of the tRNA substrate was recognized by the β-hairpin of molecule B of MaTLP. The N atoms in the side chain of R215 in the β-hairpin region of MaTLP were hydrogen-bonded to the phosphate groups of U55 and G57. The O atom on the S213 side chain was also hydrogen-bonded to the phosphate moiety of G57 of the tRNA (Fig. 2). This β-hairpin region was disordered in the crystal structure of the CaThg1-tRNA complex. + + 0.9842645 + structure_element + cleaner0 + 2023-07-27T10:23:13Z + SO: + + elbow region + + + 0.998276 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9993427 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + 0.9993599 + protein + cleaner0 + 2023-07-27T09:13:10Z + PR: + + MaTLP + + + 0.99948394 + residue_name_number + cleaner0 + 2023-07-27T09:16:27Z + DUMMY: + + R215 + + + structure_element + SO: + cleaner0 + 2023-07-27T10:20:20Z + + β-hairpin + + + 0.99937385 + protein + cleaner0 + 2023-07-27T09:13:10Z + PR: + + MaTLP + + + 0.99709296 + bond_interaction + cleaner0 + 2023-07-27T09:19:48Z + MESH: + + hydrogen-bonded + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:08:07Z + + phosphate + + + 0.99939704 + residue_name_number + cleaner0 + 2023-07-27T09:16:32Z + DUMMY: + + U55 + + + 0.99942434 + residue_name_number + cleaner0 + 2023-07-27T09:16:36Z + DUMMY: + + G57 + + + 0.9994429 + residue_name_number + cleaner0 + 2023-07-27T09:16:42Z + DUMMY: + + S213 + + + 0.9968627 + bond_interaction + cleaner0 + 2023-07-27T09:19:48Z + MESH: + + hydrogen-bonded + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:08:07Z + + phosphate + + + 0.9994149 + residue_name_number + cleaner0 + 2023-07-27T09:16:37Z + DUMMY: + + G57 + + + 0.99904877 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + structure_element + SO: + cleaner0 + 2023-07-27T10:20:20Z + + β-hairpin + + + 0.73507226 + protein_state + cleaner0 + 2023-07-27T10:26:27Z + DUMMY: + + disordered + + + 0.99873954 + evidence + cleaner0 + 2023-07-27T10:26:24Z + DUMMY: + + crystal structure + + + 0.99900657 + complex_assembly + cleaner0 + 2023-07-27T09:14:11Z + GO: + + CaThg1-tRNA + + + + RESULTS + paragraph + 9571 + The accepter stem of the tRNA substrate was recognized by molecule A of MaTLP. The N7 atom of G2 at the 5′-end was hydrogen-bonded to the N atom of the R136 side chain, whereas the α-phosphate was bonded to the N137 side chain (Fig. 2). R136 was also hydrogen-bonded to the base of C72 (the Watson-Crick bond partner of ΔG1). The triphosphate moiety at the 5′-end of the tRNA was bonded to the D21-K26 region. These phosphates were also coordinated to two metal ions, presumably Mg2+ (Mg2+A and Mg2+B) because they were observed at the same positions (figs. S3 and S4) previously identified by CaThg1 and HsThg1 structures. These ions were in turn coordinated by the O atoms of the side chains of D21 and D69 and the main-chain O of G22 (fig. S3A). Mutation of D29 and D76 in HsThg1 (corresponding to D21 and D69 of MaTLP) has been shown to markedly decrease G−1 addition activity. + + 0.99863183 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + 0.9986902 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.99942315 + protein + cleaner0 + 2023-07-27T09:13:10Z + PR: + + MaTLP + + + 0.9985697 + residue_name_number + cleaner0 + 2023-07-27T09:17:01Z + DUMMY: + + G2 + + + 0.99695307 + bond_interaction + cleaner0 + 2023-07-27T09:19:48Z + MESH: + + hydrogen-bonded + + + 0.999418 + residue_name_number + cleaner0 + 2023-07-27T09:17:06Z + DUMMY: + + R136 + + + 0.98731846 + chemical + cleaner0 + 2023-07-27T09:08:07Z + CHEBI: + + phosphate + + + 0.999443 + residue_name_number + cleaner0 + 2023-07-27T09:17:10Z + DUMMY: + + N137 + + + 0.99947983 + residue_name_number + cleaner0 + 2023-07-27T09:17:06Z + DUMMY: + + R136 + + + 0.99682266 + bond_interaction + cleaner0 + 2023-07-27T09:19:48Z + MESH: + + hydrogen-bonded + + + 0.9994659 + residue_name_number + cleaner0 + 2023-07-27T09:17:23Z + DUMMY: + + C72 + + + 0.99681926 + chemical + cleaner0 + 2023-07-27T09:24:41Z + CHEBI: + + triphosphate + + + 0.9985638 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + residue_range + DUMMY: + cleaner0 + 2023-07-27T09:17:52Z + + D21-K26 + + + 0.88514954 + chemical + cleaner0 + 2023-07-27T10:10:55Z + CHEBI: + + phosphates + + + 0.9750867 + bond_interaction + cleaner0 + 2023-07-27T09:19:44Z + MESH: + + coordinated to + + + 0.9988883 + chemical + cleaner0 + 2023-07-27T09:18:00Z + CHEBI: + + Mg2+ + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:18:14Z + + Mg2+ + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:18:47Z + + Mg2+ + + + 0.99945253 + protein + cleaner0 + 2023-07-27T09:11:41Z + PR: + + CaThg1 + + + 0.9994295 + protein + cleaner0 + 2023-07-27T09:11:19Z + PR: + + HsThg1 + + + 0.9982023 + evidence + cleaner0 + 2023-07-27T10:26:31Z + DUMMY: + + structures + + + bond_interaction + MESH: + cleaner0 + 2023-07-27T09:20:10Z + + coordinated by + + + 0.99948525 + residue_name_number + cleaner0 + 2023-07-27T09:19:11Z + DUMMY: + + D21 + + + 0.9995098 + residue_name_number + cleaner0 + 2023-07-27T09:19:15Z + DUMMY: + + D69 + + + 0.9994955 + residue_name_number + cleaner0 + 2023-07-27T09:19:19Z + DUMMY: + + G22 + + + 0.9975606 + experimental_method + cleaner0 + 2023-07-27T10:18:16Z + MESH: + + Mutation + + + 0.9994524 + residue_name_number + cleaner0 + 2023-07-27T09:19:24Z + DUMMY: + + D29 + + + 0.9994642 + residue_name_number + cleaner0 + 2023-07-27T09:19:29Z + DUMMY: + + D76 + + + 0.99943143 + protein + cleaner0 + 2023-07-27T09:11:19Z + PR: + + HsThg1 + + + 0.9994531 + residue_name_number + cleaner0 + 2023-07-27T09:19:11Z + DUMMY: + + D21 + + + 0.99944884 + residue_name_number + cleaner0 + 2023-07-27T09:19:15Z + DUMMY: + + D69 + + + 0.99944633 + protein + cleaner0 + 2023-07-27T09:13:10Z + PR: + + MaTLP + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-27T09:56:58Z + + G−1 + + + + RESULTS + title_2 + 10461 + Template-dependent binding of the GTP analog to the MaTLP-ppptRNAPheΔ1 complex + + 0.99917847 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.998625 + complex_assembly + cleaner0 + 2023-07-27T09:20:21Z + GO: + + MaTLP-ppptRNAPheΔ1 + + + + RESULTS + paragraph + 10544 + Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme. These features are consistent with the fact that this elongation reaction is template-dependent. The 5′-end (position 2) of the tRNA moved significantly (Fig. 3C) due to the insertion of GDPNP. Surprisingly, the 5′-triphosphate moiety after movement occupied the GTP/ATP triphosphate position during the activation step (Fig. 3D). Together with the observation that the 3′-OH of the incoming GTP analog was within coordination distance (2.8 Å) to Mg2+A (fig. S3B) and was able to execute a nucleophilic attack on the α-phosphate of the 5′-end, this structure indicates that the elongation reaction (second reaction) takes place at the same reaction center where the activation reaction (first reaction) occurs. + + 0.9968804 + evidence + cleaner0 + 2023-07-27T10:26:35Z + DUMMY: + + structure + + + 0.9987251 + protein + cleaner0 + 2023-07-27T09:13:10Z + PR: + + MaTLP + + + 0.99861515 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.5469938 + chemical + cleaner0 + 2023-07-27T09:13:27Z + CHEBI: + + ppptRNAPheΔ1 + + + 0.9993401 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.99941456 + chemical + cleaner0 + 2023-07-27T09:21:34Z + CHEBI: + + GDPNP + + + 0.998014 + experimental_method + cleaner0 + 2023-07-27T10:18:28Z + MESH: + + soaking + + + 0.99915266 + complex_assembly + cleaner0 + 2023-07-27T09:20:22Z + GO: + + MaTLP-ppptRNAPheΔ1 + + + 0.97418684 + evidence + cleaner0 + 2023-07-27T10:26:38Z + DUMMY: + + crystal + + + 0.9993868 + chemical + cleaner0 + 2023-07-27T09:21:34Z + CHEBI: + + GDPNP + + + 0.9970028 + evidence + cleaner0 + 2023-07-27T10:26:48Z + DUMMY: + + structure + + + 0.8970383 + chemical + cleaner0 + 2023-07-27T10:11:00Z + CHEBI: + + guanine + + + 0.9992805 + chemical + cleaner0 + 2023-07-27T09:21:34Z + CHEBI: + + GDPNP + + + 0.9721004 + bond_interaction + cleaner0 + 2023-07-27T08:37:55Z + MESH: + + Watson-Crick hydrogen bonds + + + 0.99924314 + residue_name_number + cleaner0 + 2023-07-27T09:17:23Z + DUMMY: + + C72 + + + 0.99525213 + bond_interaction + cleaner0 + 2023-07-27T09:21:16Z + MESH: + + base-stacking interactions + + + 0.9992003 + residue_name_number + cleaner0 + 2023-07-27T09:17:01Z + DUMMY: + + G2 + + + 0.9982686 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9052167 + chemical + cleaner0 + 2023-07-27T10:11:03Z + CHEBI: + + guanine + + + 0.9985051 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.99938846 + chemical + cleaner0 + 2023-07-27T09:21:34Z + CHEBI: + + GDPNP + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:32:05Z + + 5′-triphosphate + + + 0.9993123 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.99930036 + chemical + cleaner0 + 2023-07-27T08:39:26Z + CHEBI: + + ATP + + + 0.99679154 + chemical + cleaner0 + 2023-07-27T09:24:41Z + CHEBI: + + triphosphate + + + 0.99933904 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:20:58Z + + Mg2+ + + + 0.9886169 + chemical + cleaner0 + 2023-07-27T09:08:07Z + CHEBI: + + phosphate + + + 0.997259 + evidence + cleaner0 + 2023-07-27T10:26:51Z + DUMMY: + + structure + + + site + SO: + cleaner0 + 2023-07-27T10:02:25Z + + reaction center + + + + 1501397-F3.jpg + F3 + FIG + fig_title_caption + 11780 + Structural change of the tRNA (ppptRNAPheΔ1). + + 0.99411327 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9977137 + chemical + cleaner0 + 2023-07-27T09:13:27Z + CHEBI: + + ppptRNAPheΔ1 + + + + 1501397-F3.jpg + F3 + FIG + fig_caption + 11830 + Structural change of the tRNA (ppptRNAPheΔ1) accepter stem in MaTLP caused by insertion of GDPNP. (A) Structure before GDPNP binding. (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. + + 0.6159382 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.8077976 + chemical + cleaner0 + 2023-07-27T09:13:27Z + CHEBI: + + ppptRNAPheΔ1 + + + 0.9969453 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + 0.99880004 + protein + cleaner0 + 2023-07-27T09:13:10Z + PR: + + MaTLP + + + 0.9989936 + chemical + cleaner0 + 2023-07-27T09:21:33Z + CHEBI: + + GDPNP + + + 0.9961423 + evidence + cleaner0 + 2023-07-27T10:26:57Z + DUMMY: + + Structure + + + 0.9985405 + chemical + cleaner0 + 2023-07-27T09:21:34Z + CHEBI: + + GDPNP + + + 0.9974503 + evidence + cleaner0 + 2023-07-27T10:26:59Z + DUMMY: + + Structure + + + 0.9985354 + chemical + cleaner0 + 2023-07-27T09:21:34Z + CHEBI: + + GDPNP + + + 0.99718434 + experimental_method + cleaner0 + 2023-07-27T10:18:51Z + MESH: + + Superposition + + + 0.9865745 + evidence + cleaner0 + 2023-07-27T10:27:01Z + DUMMY: + + structures + + + 0.99856323 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.99900997 + chemical + cleaner0 + 2023-07-27T09:21:34Z + CHEBI: + + GDPNP + + + 0.99818677 + experimental_method + cleaner0 + 2023-07-27T10:18:55Z + MESH: + + Superposition + + + 0.99859303 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9986745 + chemical + cleaner0 + 2023-07-27T09:21:34Z + CHEBI: + + GDPNP + + + 0.99921167 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.5339933 + chemical + cleaner0 + 2023-07-27T09:24:40Z + CHEBI: + + triphosphate + + + + RESULTS + paragraph + 12306 + The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). All of these residues are well conserved (fig. S5), and mutation of corresponding residues in ScThg1 (R27, R93, K96, and R133) decreased the catalytic efficiency of G−1 addition. The triphosphate of the GDPNP was also bonded to the third Mg2+ (Mg2+C), which, unlike Mg2+A and Mg2+B, is not coordinated by the TLP molecule (fig. S3B). This triphosphate binding mode is the same as that for the second nucleotide binding site in Thg1. However, in previous analyses, the base moiety at the second site was either invisible or far beyond the reaction distance of the phosphate, and therefore, flipping of the base was expected to occur. + + 0.99445456 + chemical + cleaner0 + 2023-07-27T09:24:41Z + CHEBI: + + triphosphate + + + 0.9992803 + chemical + cleaner0 + 2023-07-27T09:21:34Z + CHEBI: + + GDPNP + + + 0.99835026 + site + cleaner0 + 2023-07-27T10:28:07Z + SO: + + interface + + + structure_element + SO: + cleaner0 + 2023-07-27T09:21:53Z + + A + + + structure_element + SO: + cleaner0 + 2023-07-27T09:22:01Z + + B + + + 0.9993624 + residue_name_number + cleaner0 + 2023-07-27T09:21:42Z + DUMMY: + + R19 + + + structure_element + SO: + cleaner0 + 2023-07-27T09:22:09Z + + A + + + 0.9993773 + residue_name_number + cleaner0 + 2023-07-27T09:22:50Z + DUMMY: + + R83 + + + structure_element + SO: + cleaner0 + 2023-07-27T09:22:18Z + + B + + + 0.9993542 + residue_name_number + cleaner0 + 2023-07-27T09:22:45Z + DUMMY: + + K86 + + + structure_element + SO: + cleaner0 + 2023-07-27T09:22:26Z + + B + + + 0.9993461 + residue_name_number + cleaner0 + 2023-07-27T09:22:55Z + DUMMY: + + R114 + + + structure_element + SO: + cleaner0 + 2023-07-27T09:22:41Z + + A + + + 0.99890375 + protein_state + cleaner0 + 2023-07-27T10:29:45Z + DUMMY: + + well conserved + + + 0.9977977 + experimental_method + cleaner0 + 2023-07-27T10:19:04Z + MESH: + + mutation + + + 0.99941695 + protein + cleaner0 + 2023-07-27T10:14:16Z + PR: + + ScThg1 + + + 0.99936813 + residue_name_number + cleaner0 + 2023-07-27T09:23:00Z + DUMMY: + + R27 + + + 0.9993944 + residue_name_number + cleaner0 + 2023-07-27T09:23:04Z + DUMMY: + + R93 + + + 0.9993637 + residue_name_number + cleaner0 + 2023-07-27T09:23:09Z + DUMMY: + + K96 + + + 0.99935395 + residue_name_number + cleaner0 + 2023-07-27T09:23:14Z + DUMMY: + + R133 + + + 0.9931157 + residue_name_number + cleaner0 + 2023-07-27T10:11:15Z + DUMMY: + + G−1 + + + 0.99587363 + chemical + cleaner0 + 2023-07-27T09:24:41Z + CHEBI: + + triphosphate + + + 0.9992716 + chemical + cleaner0 + 2023-07-27T09:21:34Z + CHEBI: + + GDPNP + + + 0.9977677 + chemical + cleaner0 + 2023-07-27T10:11:19Z + CHEBI: + + Mg2+ + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:23:34Z + + Mg2+ + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:24:00Z + + Mg2+ + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:24:23Z + + Mg2+ + + + bond_interaction + MESH: + cleaner0 + 2023-07-27T09:42:41Z + + coordinated by + + + 0.99687046 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:24:41Z + + triphosphate + + + 0.99195087 + site + cleaner0 + 2023-07-27T10:28:12Z + SO: + + second nucleotide binding site + + + 0.99942905 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.9525255 + site + cleaner0 + 2023-07-27T10:28:15Z + SO: + + second site + + + 0.90256834 + chemical + cleaner0 + 2023-07-27T09:08:07Z + CHEBI: + + phosphate + + + + RESULTS + title_2 + 13175 + tRNA binding and repair experiments of the β-hairpin mutants + + experimental_method + MESH: + cleaner0 + 2023-07-27T10:19:35Z + + tRNA binding and repair experiments + + + 0.99890774 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + 0.9695342 + protein_state + cleaner0 + 2023-07-27T10:29:48Z + DUMMY: + + mutants + + + + RESULTS + paragraph + 13239 + To confirm tRNA recognition by the β-hairpin, we created mutation variants with altered residues in the β-hairpin region. Then, tRNA binding and enzymatic activities were measured. β-Hairpin deletion variant delR198-R215 almost completely abolished the binding of tRNAPheΔ1 (fig. S6). Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). Experiments using the tRNAHisΔ1 substrate gave similar results (Fig. 4A). All these results are consistent with the crystal structure and suggest that the β-hairpin plays an important role in anticodon-independent binding of the tRNA substrate. Residues in the β-hairpin are not well conserved, except for R215 (fig. S5). Mutants R215A and R215A/S213A, in which the completely conserved R215 was changed to alanine, showed a moderate effect on the activity (27.3 and 16.3%, respectively). Thus, specific interactions with the conserved R215 and van der Waals contacts to residues in the β-hairpin would be important for tRNA recognition. + + chemical + CHEBI: + cleaner0 + 2023-07-27T08:39:13Z + + tRNA + + + 0.99931544 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + experimental_method + MESH: + cleaner0 + 2023-07-27T10:20:01Z + + created mutation variants + + + 0.9993222 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + experimental_method + MESH: + cleaner0 + 2023-07-27T10:20:48Z + + tRNA binding and enzymatic activities were measured + + + 0.96807307 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-Hairpin + + + 0.8194407 + protein_state + cleaner0 + 2023-07-27T10:29:51Z + DUMMY: + + deletion variant + + + 0.9983492 + mutant + cleaner0 + 2023-07-27T09:24:58Z + MESH: + + delR198-R215 + + + 0.9296625 + chemical + cleaner0 + 2023-07-27T09:25:56Z + CHEBI: + + tRNAPheΔ1 + + + 0.99831027 + mutant + cleaner0 + 2023-07-27T09:24:59Z + MESH: + + delR198-R215 + + + 0.99695975 + mutant + cleaner0 + 2023-07-27T09:25:09Z + MESH: + + delG202-E210 + + + 0.99902546 + protein_state + cleaner0 + 2023-07-27T09:26:30Z + DUMMY: + + wild type + + + 0.98946804 + experimental_method + cleaner0 + 2023-07-27T10:21:34Z + MESH: + + mutations + + + 0.99896884 + mutant + cleaner0 + 2023-07-27T09:25:13Z + MESH: + + N179A + + + 0.99896014 + mutant + cleaner0 + 2023-07-27T09:25:17Z + MESH: + + F174A + + + 0.9989961 + mutant + cleaner0 + 2023-07-27T09:25:14Z + MESH: + + N179A + + + 0.99903995 + mutant + cleaner0 + 2023-07-27T09:25:26Z + MESH: + + R188A + + + 0.99906284 + site + cleaner0 + 2023-07-27T10:28:19Z + SO: + + anticodon recognition site + + + 0.99927264 + complex_assembly + cleaner0 + 2023-07-27T09:25:39Z + GO: + + Thg1-tRNAHis + + + 0.5787097 + evidence + cleaner0 + 2023-07-27T10:27:07Z + DUMMY: + + structure + + + 0.97306955 + chemical + cleaner0 + 2023-07-27T10:11:23Z + CHEBI: + + tRNAHisΔ1 + + + 0.9987498 + evidence + cleaner0 + 2023-07-27T10:27:09Z + DUMMY: + + crystal structure + + + 0.99930507 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + 0.84993595 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9993302 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + 0.9987729 + protein_state + cleaner0 + 2023-07-27T10:29:54Z + DUMMY: + + not well conserved + + + 0.9993625 + residue_name_number + cleaner0 + 2023-07-27T09:16:27Z + DUMMY: + + R215 + + + 0.8830756 + protein_state + cleaner0 + 2023-07-27T10:29:59Z + DUMMY: + + Mutants + + + 0.9989693 + mutant + cleaner0 + 2023-07-27T09:26:15Z + MESH: + + R215A + + + 0.9989299 + mutant + cleaner0 + 2023-07-27T09:26:16Z + MESH: + + R215A + + + 0.9990251 + mutant + cleaner0 + 2023-07-27T09:26:23Z + MESH: + + S213A + + + 0.9988954 + protein_state + cleaner0 + 2023-07-27T10:30:03Z + DUMMY: + + completely conserved + + + 0.99940014 + residue_name_number + cleaner0 + 2023-07-27T09:16:27Z + DUMMY: + + R215 + + + 0.51008594 + experimental_method + cleaner0 + 2023-07-27T10:21:40Z + MESH: + + changed + + + 0.9953343 + residue_name + cleaner0 + 2023-07-27T09:58:38Z + SO: + + alanine + + + 0.99303716 + protein_state + cleaner0 + 2023-07-27T10:30:06Z + DUMMY: + + conserved + + + 0.99940646 + residue_name_number + cleaner0 + 2023-07-27T09:16:27Z + DUMMY: + + R215 + + + bond_interaction + MESH: + cleaner0 + 2023-07-27T09:58:17Z + + van der Waals contacts + + + 0.99931306 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + chemical + CHEBI: + cleaner0 + 2023-07-27T08:39:13Z + + tRNA + + + + 1501397-F4.jpg + F4 + FIG + fig_title_caption + 14513 + Mutational analysis of the β-hairpin and anticodon binding region. + + 0.9984823 + experimental_method + cleaner0 + 2023-07-27T10:21:45Z + MESH: + + Mutational analysis + + + 0.9993003 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + 0.9987523 + site + cleaner0 + 2023-07-27T10:28:24Z + SO: + + anticodon binding region + + + + 1501397-F4.jpg + F4 + FIG + fig_caption + 14583 + The rates of guanylylation by various mutants were measured. Error bars represent the SD of three independent experiments. (A) Guanylylation of ppptRNAPheΔ1 and ppptRNAHisΔ1 by various TLP mutants. The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. The activity to tRNAPheΔ1 is about 10% of ppptRNAPheΔ1. + + 0.99230766 + chemical + cleaner0 + 2023-07-27T09:13:27Z + CHEBI: + + ppptRNAPheΔ1 + + + 0.9945064 + chemical + cleaner0 + 2023-07-27T09:26:49Z + CHEBI: + + ppptRNAHisΔ1 + + + 0.96006143 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.75529 + protein_state + cleaner0 + 2023-07-27T10:30:10Z + DUMMY: + + mutants + + + 0.9910155 + chemical + cleaner0 + 2023-07-27T10:09:04Z + CHEBI: + + [α-32P]GTP + + + 0.9991336 + protein_state + cleaner0 + 2023-07-27T09:27:36Z + DUMMY: + + wild-type + + + 0.53344727 + protein + cleaner0 + 2023-07-27T09:13:10Z + PR: + + MaTLP + + + 0.9951326 + chemical + cleaner0 + 2023-07-27T09:13:27Z + CHEBI: + + ppptRNAPheΔ1 + + + 0.9966877 + chemical + cleaner0 + 2023-07-27T09:27:17Z + CHEBI: + + tRNAPheΔ1 + + + 0.99821585 + chemical + cleaner0 + 2023-07-27T09:27:24Z + CHEBI: + + tRNAPhe + + + 0.9968773 + chemical + cleaner0 + 2023-07-27T09:27:29Z + CHEBI: + + tRNAHisΔ−1 + + + 0.9707573 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.8338082 + protein_state + cleaner0 + 2023-07-27T10:30:12Z + DUMMY: + + mutants + + + 0.6254101 + chemical + cleaner0 + 2023-07-27T09:27:17Z + CHEBI: + + tRNAPheΔ1 + + + 0.9869407 + chemical + cleaner0 + 2023-07-27T09:13:27Z + CHEBI: + + ppptRNAPheΔ1 + + + + RESULTS + title_2 + 15033 + Termination of the elongation reaction by measuring the accepter stem + + structure_element + SO: + cleaner0 + 2023-07-27T10:22:09Z + + accepter stem + + + + RESULTS + paragraph + 15103 + TLPs catalyze the Watson-Crick template–dependent elongation or repair reaction for 5′-end truncated tRNAPhe substrates lacking G1 only (tRNAPheΔ1), or lacking both G1 and G2 (tRNAPheΔ1,2), whereas they do not show any activity with intact tRNAPhe (thus, repair is unnecessary). How TLP distinguishes between tRNAs that need 5′-end repair from ones that do not, or in other words, how the elongation reaction is properly terminated, remains unknown. The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. To confirm this speculation, we used computer graphics to examine whether the β-hairpin region was able to bind tRNA substrates with different accepter stem lengths when the 5′-end was properly placed in the reaction site. When the 5′-end was placed in the reaction site, the body of the tRNA molecule shifted in a manner dependent on the accepter stem length. The tRNA body also rotated because of the helical nature of the accepter stem (fig. S7). This model structure showed that the accepter stem of intact tRNAPhe was too long for the β-hairpin to recognize its elbow region, whereas tRNAPheΔ1 and tRNAPheΔ1,2 were recognized by the β-hairpin region (fig. S7), which is consistent with previous experiments. On the basis of these model structures, we concluded that the TLP molecule can properly terminate elongation by measuring the accepter stem length of tRNA substrates. + + 0.9963889 + protein_type + cleaner0 + 2023-07-27T09:10:32Z + MESH: + + TLPs + + + 0.9868966 + chemical + cleaner0 + 2023-07-27T09:27:24Z + CHEBI: + + tRNAPhe + + + 0.95910984 + residue_name_number + cleaner0 + 2023-07-27T10:15:25Z + DUMMY: + + G1 + + + 0.99755114 + chemical + cleaner0 + 2023-07-27T09:27:17Z + CHEBI: + + tRNAPheΔ1 + + + 0.96142435 + residue_name_number + cleaner0 + 2023-07-27T10:15:29Z + DUMMY: + + G1 + + + 0.9508771 + residue_name_number + cleaner0 + 2023-07-27T09:17:01Z + DUMMY: + + G2 + + + 0.99773103 + chemical + cleaner0 + 2023-07-27T09:28:04Z + CHEBI: + + tRNAPheΔ1,2 + + + 0.9463474 + chemical + cleaner0 + 2023-07-27T09:27:24Z + CHEBI: + + tRNAPhe + + + 0.98515874 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.99676067 + chemical + cleaner0 + 2023-07-27T10:10:42Z + CHEBI: + + tRNAs + + + 0.99747026 + evidence + cleaner0 + 2023-07-27T10:27:15Z + DUMMY: + + structure + + + 0.9989264 + complex_assembly + cleaner0 + 2023-07-27T09:20:22Z + GO: + + MaTLP-ppptRNAPheΔ1 + + + 0.9987936 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.9641728 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.9986859 + oligomeric_state + cleaner0 + 2023-07-27T09:14:51Z + DUMMY: + + dimer + + + 0.9985221 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.5924758 + structure_element + cleaner0 + 2023-07-27T10:23:12Z + SO: + + elbow region + + + 0.999376 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + 0.80603194 + structure_element + cleaner0 + 2023-07-27T10:31:36Z + SO: + + B + + + 0.90232784 + structure_element + cleaner0 + 2023-07-27T10:31:40Z + SO: + + A + + + 0.9393595 + protein_state + cleaner0 + 2023-07-27T10:30:18Z + DUMMY: + + flexible + + + 0.99939483 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + 0.99264103 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.86848617 + structure_element + cleaner0 + 2023-07-27T10:22:08Z + SO: + + accepter stem + + + experimental_method + MESH: + cleaner0 + 2023-07-27T10:24:01Z + + used computer graphics to examine + + + 0.9993806 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + 0.99490696 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.7337481 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + site + SO: + cleaner0 + 2023-07-27T10:23:06Z + + reaction site + + + 0.9975822 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + structure_element + SO: + cleaner0 + 2023-07-27T10:22:09Z + + accepter stem + + + 0.9953381 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9955665 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + 0.9954334 + evidence + cleaner0 + 2023-07-27T10:27:17Z + DUMMY: + + structure + + + 0.9975693 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + 0.967975 + chemical + cleaner0 + 2023-07-27T09:27:24Z + CHEBI: + + tRNAPhe + + + 0.99937016 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + 0.77153134 + structure_element + cleaner0 + 2023-07-27T10:23:13Z + SO: + + elbow region + + + 0.9978011 + chemical + cleaner0 + 2023-07-27T09:27:17Z + CHEBI: + + tRNAPheΔ1 + + + 0.9983471 + chemical + cleaner0 + 2023-07-27T09:28:10Z + CHEBI: + + tRNAPheΔ1,2 + + + 0.99938065 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + 0.99614185 + evidence + cleaner0 + 2023-07-27T10:27:19Z + DUMMY: + + structures + + + 0.9082034 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.98609275 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + 0.9883173 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + + RESULTS + title_2 + 16831 + Dual binding mode for tRNA repair + + 0.99911565 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + + RESULTS + paragraph + 16865 + The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. This difference in the tRNA binding modes is closely related to their enzymatic functions. The tRNAHis-specific G−1 addition enzyme Thg1 needs to recognize both the accepter stem and anticodon of tRNAHis. The tetrameric architecture of the Thg1 molecule allows it to access both regions located at the opposite side of the tRNA molecule [the AB dimer recognizes the accepter stem and CD dimer anticodon ]. In contrast, the binding mode of TLP corresponds to the anticodon-independent repair reactions of 5′-truncated general tRNAs. This binding mode is also suitable for the correct termination of the elongation or repair reaction by measuring the length of the accepter stem by the flexible β-hairpin. + + 0.99838936 + experimental_method + cleaner0 + 2023-07-27T10:24:07Z + MESH: + + structural analysis + + + 0.99898773 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.99926084 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.99870765 + oligomeric_state + cleaner0 + 2023-07-27T08:39:47Z + DUMMY: + + tetrameric + + + 0.9988476 + chemical + cleaner0 + 2023-07-27T10:10:42Z + CHEBI: + + tRNAs + + + 0.99926776 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.99909174 + protein_state + cleaner0 + 2023-07-27T08:40:17Z + DUMMY: + + bound to + + + 0.996358 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.99887127 + oligomeric_state + cleaner0 + 2023-07-27T08:39:42Z + DUMMY: + + tetramer + + + 0.999076 + protein_type + cleaner0 + 2023-07-27T08:37:28Z + MESH: + + TLP + + + 0.99910367 + protein_state + cleaner0 + 2023-07-27T08:40:17Z + DUMMY: + + bound to + + + 0.9971238 + chemical + cleaner0 + 2023-07-27T09:27:24Z + CHEBI: + + tRNAPhe + + + 0.99889284 + oligomeric_state + cleaner0 + 2023-07-27T09:14:51Z + DUMMY: + + dimer + + + chemical + CHEBI: + cleaner0 + 2023-07-27T08:39:13Z + + tRNA + + + protein_type + MESH: + cleaner0 + 2023-07-27T08:40:09Z + + tRNAHis-specific G−1 addition enzyme + + + 0.99932766 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.99702764 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + 0.59451216 + structure_element + cleaner0 + 2023-07-27T10:32:03Z + SO: + + anticodon + + + 0.9981139 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.9986755 + oligomeric_state + cleaner0 + 2023-07-27T08:39:47Z + DUMMY: + + tetrameric + + + 0.99936885 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.998466 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.99264705 + structure_element + cleaner0 + 2023-07-27T10:31:25Z + SO: + + AB + + + 0.99889743 + oligomeric_state + cleaner0 + 2023-07-27T09:14:51Z + DUMMY: + + dimer + + + 0.9975226 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + 0.99948126 + structure_element + cleaner0 + 2023-07-27T10:31:30Z + SO: + + CD + + + 0.99879384 + oligomeric_state + cleaner0 + 2023-07-27T09:14:51Z + DUMMY: + + dimer + + + 0.9991611 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.9989042 + chemical + cleaner0 + 2023-07-27T10:10:42Z + CHEBI: + + tRNAs + + + 0.9978206 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + 0.9984469 + protein_state + cleaner0 + 2023-07-27T10:30:26Z + DUMMY: + + flexible + + + 0.99937433 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + + RESULTS + paragraph + 17811 + Because tRNAHis requires an extra guanosine (G−1) at the 5′-end, the repair enzyme has to extend the 5′-end by one more nucleotide than other tRNAs. TLP has been shown to confer such catalytic activity on tRNAHisΔ−1 (Fig. 4B). Here, we showed that the TLP mutants, wherein the β-hairpin is truncated and tRNAPheΔ1 binding ability is lost, can still bind to tRNAPhe (GUG) whose anticodon is changed to that for His (fig. S6, C, H, and I). Also, the intact tRNAPhe, which is not recognized by TLP (Fig. 4B and fig. S6E), can be recognized when its anticodon is changed to that for His (fig. S6D). Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHisΔ−1 (Fig. 4B). All these experimental results indicate that TLP recognizes and binds tRNAs carrying the His anticodon in the same way that Thg1 recognizes tRNAHis. Thus, we concluded that TLP has two tRNA binding modes that are selectively used, depending on both the length of the accepter stem and the anticodon. The elongation or repair reaction normally terminates when the 5′-end reaches position 1, but when the His anticodon is present, TLP binds the tRNA in the second mode by recognizing the anticodon to execute the G−1 addition reaction. By having two different binding modes, TLP can manage this special feature of tRNAHis. + + 0.89148074 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.9935821 + chemical + cleaner0 + 2023-07-27T10:11:27Z + CHEBI: + + guanosine + + + 0.9969999 + residue_name_number + cleaner0 + 2023-07-27T10:11:38Z + DUMMY: + + G−1 + + + 0.99664366 + chemical + cleaner0 + 2023-07-27T10:10:42Z + CHEBI: + + tRNAs + + + 0.8903893 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.9946516 + chemical + cleaner0 + 2023-07-27T09:27:30Z + CHEBI: + + tRNAHisΔ−1 + + + 0.93582135 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.4843938 + protein_state + cleaner0 + 2023-07-27T10:30:30Z + DUMMY: + + mutants + + + 0.9993315 + structure_element + cleaner0 + 2023-07-27T10:20:20Z + SO: + + β-hairpin + + + 0.98948985 + protein_state + cleaner0 + 2023-07-27T10:30:35Z + DUMMY: + + truncated + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:27:17Z + + tRNAPheΔ1 + + + 0.99827754 + chemical + cleaner0 + 2023-07-27T09:27:24Z + CHEBI: + + tRNAPhe + + + 0.9803373 + chemical + cleaner0 + 2023-07-27T09:29:20Z + CHEBI: + + GUG + + + 0.9556936 + residue_name + cleaner0 + 2023-07-27T09:29:30Z + SO: + + His + + + 0.99863344 + chemical + cleaner0 + 2023-07-27T09:27:24Z + CHEBI: + + tRNAPhe + + + 0.99575424 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.9732659 + residue_name + cleaner0 + 2023-07-27T09:29:31Z + SO: + + His + + + 0.9255783 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.60005647 + protein_state + cleaner0 + 2023-07-27T10:30:38Z + DUMMY: + + variant + + + 0.9991592 + mutant + cleaner0 + 2023-07-27T09:25:18Z + MESH: + + F174A + + + 0.99910396 + mutant + cleaner0 + 2023-07-27T09:25:14Z + MESH: + + N179A + + + 0.99916065 + mutant + cleaner0 + 2023-07-27T09:25:26Z + MESH: + + R188A + + + 0.9990769 + site + cleaner0 + 2023-07-27T10:28:28Z + SO: + + anticodon recognition site + + + 0.9992427 + complex_assembly + cleaner0 + 2023-07-27T09:25:39Z + GO: + + Thg1-tRNAHis + + + 0.8880766 + evidence + cleaner0 + 2023-07-27T10:27:28Z + DUMMY: + + structure + + + 0.99190134 + chemical + cleaner0 + 2023-07-27T09:27:30Z + CHEBI: + + tRNAHisΔ−1 + + + 0.997844 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.9966815 + chemical + cleaner0 + 2023-07-27T10:10:42Z + CHEBI: + + tRNAs + + + 0.95553994 + residue_name + cleaner0 + 2023-07-27T09:29:31Z + SO: + + His + + + 0.9989329 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + 0.5086558 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.98881125 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + chemical + CHEBI: + cleaner0 + 2023-07-27T08:39:13Z + + tRNA + + + 0.98896366 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + 0.523069 + structure_element + cleaner0 + 2023-07-27T10:32:08Z + SO: + + anticodon + + + 0.7900665 + residue_name + cleaner0 + 2023-07-27T09:29:31Z + SO: + + His + + + 0.99240595 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.99671626 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9921144 + residue_name_number + cleaner0 + 2023-07-27T10:11:48Z + DUMMY: + + G−1 + + + 0.99529797 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.8667127 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + + DISCUSS + title_1 + 19268 + DISCUSSION + + + DISCUSS + paragraph + 19279 + The Thg1/TLP family of proteins extends tRNA chains in the 3′-5′ direction. The reaction involves two steps. First, the 5′-phosphate is activated by GTP/ATP. Then, the activated phosphate is attacked by the incoming nucleotide, resulting in an extension by one nucleotide at the 5′-end. Here, we successfully solved for the first time the intermediate structures of the template-dependent 3′-5′ elongation complex of MaTLP. On the basis of these structures, we will discuss the 3′-5′ addition reaction compared with canonical 5′-3′ elongation by DNA/RNA polymerases. + + protein + PR: + cleaner0 + 2023-07-27T08:38:03Z + + Thg1 + + + protein_type + MESH: + cleaner0 + 2023-07-27T08:37:29Z + + TLP + + + 0.9985511 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:35:20Z + + 5′-phosphate + + + 0.9992725 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.999213 + chemical + cleaner0 + 2023-07-27T08:39:26Z + CHEBI: + + ATP + + + 0.99731773 + chemical + cleaner0 + 2023-07-27T09:08:07Z + CHEBI: + + phosphate + + + 0.98605746 + experimental_method + cleaner0 + 2023-07-27T10:24:29Z + MESH: + + solved + + + 0.9901758 + evidence + cleaner0 + 2023-07-27T10:27:31Z + DUMMY: + + structures + + + 0.9988562 + protein + cleaner0 + 2023-07-27T09:13:10Z + PR: + + MaTLP + + + 0.9983096 + evidence + cleaner0 + 2023-07-27T10:27:33Z + DUMMY: + + structures + + + 0.98613816 + protein_type + cleaner0 + 2023-07-27T09:09:54Z + MESH: + + DNA/RNA polymerases + + + + DISCUSS + paragraph + 19866 + Figure 5 is a schematic diagram of the 3′-5′ addition reaction of TLP. This enzyme has two triphosphate binding sites and one reaction center at the position overlapping these two binding sites (Fig. 5A). In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). The structure of the MaTLP-ppptRNAPheΔ1 complex, wherein β- and γ-phosphates coordinate with Mg2+A and Mg2+B, respectively (Figs. 3A and 5C′), may represent this activated intermediate. Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3′-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5′-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D). Then, the elongation reaction of step 2 occurs (Fig. 5E). Thus, the present structure shows that this 3′-5′ elongation enzyme utilizes a reaction center homologous to that of 5′-3′ elongation enzymes for both activation and elongation in a stepwise fashion. Although these two reactions are similar in chemistry, their substrate characteristics are very different. It should be noted that TLP has evolved to allow the occurrence of these two elaborate reaction steps within one reaction center. + + 0.525246 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.9989221 + site + cleaner0 + 2023-07-27T09:29:55Z + SO: + + triphosphate binding sites + + + 0.99723035 + site + cleaner0 + 2023-07-27T10:02:25Z + SO: + + reaction center + + + 0.99826884 + site + cleaner0 + 2023-07-27T10:28:34Z + SO: + + binding sites + + + 0.99893254 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.9986119 + chemical + cleaner0 + 2023-07-27T08:39:26Z + CHEBI: + + ATP + + + 0.99674594 + protein_state + cleaner0 + 2023-07-27T08:40:17Z + DUMMY: + + bound to + + + 0.99489546 + site + cleaner0 + 2023-07-27T09:38:12Z + SO: + + site 1 + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:35:20Z + + 5′-phosphate + + + 0.9989851 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:31:14Z + + Mg2+ + + + 0.99080896 + chemical + cleaner0 + 2023-07-27T09:08:07Z + CHEBI: + + phosphate + + + 0.99920815 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.99906045 + chemical + cleaner0 + 2023-07-27T08:39:26Z + CHEBI: + + ATP + + + 0.99654704 + evidence + cleaner0 + 2023-07-27T10:27:37Z + DUMMY: + + structure + + + 0.9989104 + complex_assembly + cleaner0 + 2023-07-27T09:20:22Z + GO: + + MaTLP-ppptRNAPheΔ1 + + + 0.6651541 + chemical + cleaner0 + 2023-07-27T10:10:56Z + CHEBI: + + phosphates + + + 0.9484136 + bond_interaction + cleaner0 + 2023-07-27T09:30:12Z + MESH: + + coordinate with + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:30:29Z + + Mg2+ + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:30:51Z + + Mg2+ + + + 0.9949398 + chemical + cleaner0 + 2023-07-27T10:12:09Z + CHEBI: + + nucleotide + + + 0.9928668 + site + cleaner0 + 2023-07-27T09:38:06Z + SO: + + site 2 + + + bond_interaction + MESH: + cleaner0 + 2023-07-27T09:12:50Z + + Watson-Crick base pair + + + 0.98880345 + chemical + cleaner0 + 2023-07-27T10:12:18Z + CHEBI: + + nucleotide + + + 0.9964449 + chemical + cleaner0 + 2023-07-27T10:12:23Z + CHEBI: + + nucleotide + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:31:45Z + + Mg2+ + + + 0.9326544 + chemical + cleaner0 + 2023-07-27T09:32:04Z + CHEBI: + + 5′-triphosphate + + + 0.9990356 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9961376 + site + cleaner0 + 2023-07-27T10:02:25Z + SO: + + reaction center + + + 0.9960509 + evidence + cleaner0 + 2023-07-27T10:27:40Z + DUMMY: + + structure + + + protein_type + MESH: + cleaner0 + 2023-07-27T09:32:31Z + + 3′-5′ elongation enzyme + + + 0.99522007 + site + cleaner0 + 2023-07-27T10:02:25Z + SO: + + reaction center + + + 0.9950315 + protein_type + cleaner0 + 2023-07-27T09:32:15Z + MESH: + + 5′-3′ elongation enzymes + + + 0.7864525 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.9921572 + site + cleaner0 + 2023-07-27T10:02:25Z + SO: + + reaction center + + + + 1501397-F5.jpg + F5 + FIG + fig_title_caption + 21310 + Schematic representation of the 3′-5′ elongation mechanism. + + + 1501397-F5.jpg + F5 + FIG + fig_caption + 21374 + (A) The reaction center overlapped with two triphosphate binding sites. A, B, and C (in green) represent binding sites for Mg2+A, Mg2+B, and Mg2+C. P (in blue) represents the phosphate binding sites; O− (in red) is the binding site for the deprotonated OH group. Important TLP residues for tRNA and Mg2+ binding are also shown. (B) Structure of the activation complex (corresponding to fig. S8). GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). (C′) Structure before the elongation reaction (corresponding to Fig. 3A). The 5′-triphosphate of the tRNA binds to the same site as for activation of the 5′-terminus of the tRNA in (B). (D) Structure of initiation of the elongation reaction (corresponding to Fig. 3B). The base of the incoming GTP forms a Watson-Crick hydrogen bond with the nucleotide at position 72 in the template chain and a base-stacking interaction with a neighboring base (G2). Movement of the 5′-terminal chain leaves the 5′-triphosphate of the tRNA in the same site as the activation step in (B). The 3′-OH of the incoming GTP is deprotonated by Mg2+A and attacks the α-phosphate to form a covalent bond. (E) After the elongation reaction, the triphosphate of the new nucleotide is placed on site 1, as in (C′), and is ready for the next reaction. + + site + SO: + cleaner0 + 2023-07-27T10:02:25Z + + reaction center + + + 0.9989655 + site + cleaner0 + 2023-07-27T09:29:56Z + SO: + + triphosphate binding sites + + + 0.99841654 + site + cleaner0 + 2023-07-27T10:28:39Z + SO: + + binding sites + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:38:53Z + + Mg2+ + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:39:06Z + + Mg2+ + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:39:21Z + + Mg2+ + + + 0.6204343 + site + cleaner0 + 2023-07-27T10:28:53Z + SO: + + P + + + 0.99869686 + site + cleaner0 + 2023-07-27T09:33:28Z + SO: + + phosphate binding sites + + + 0.9984143 + site + cleaner0 + 2023-07-27T10:29:02Z + SO: + + binding site + + + 0.77258825 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.99759597 + chemical + cleaner0 + 2023-07-27T08:39:13Z + CHEBI: + + tRNA + + + 0.9947387 + chemical + cleaner0 + 2023-07-27T10:12:28Z + CHEBI: + + Mg2+ + + + 0.9144296 + evidence + cleaner0 + 2023-07-27T10:27:45Z + DUMMY: + + Structure + + + 0.93158305 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.99546534 + chemical + cleaner0 + 2023-07-27T08:39:26Z + CHEBI: + + ATP + + + 0.97349215 + site + cleaner0 + 2023-07-27T09:33:56Z + SO: + + triphosphate binding site 1 + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:34:45Z + + 5′-phosphate + + + 0.9780151 + chemical + cleaner0 + 2023-07-27T09:08:07Z + CHEBI: + + phosphate + + + 0.998922 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.99861634 + chemical + cleaner0 + 2023-07-27T08:39:26Z + CHEBI: + + ATP + + + 0.99910563 + chemical + cleaner0 + 2023-07-27T09:34:16Z + CHEBI: + + PPi + + + 0.94754815 + chemical + cleaner0 + 2023-07-27T09:34:19Z + CHEBI: + + inorganic pyrophosphate + + + 0.96327895 + evidence + cleaner0 + 2023-07-27T10:27:48Z + DUMMY: + + structure + + + 0.9909759 + evidence + cleaner0 + 2023-07-27T10:27:50Z + DUMMY: + + structure + + + 0.98339015 + evidence + cleaner0 + 2023-07-27T10:27:53Z + DUMMY: + + Structure + + + 0.8053533 + chemical + cleaner0 + 2023-07-27T09:32:05Z + CHEBI: + + 5′-triphosphate + + + 0.99874806 + chemical + cleaner0 + 2023-07-27T08:39:14Z + CHEBI: + + tRNA + + + 0.9983541 + chemical + cleaner0 + 2023-07-27T08:39:14Z + CHEBI: + + tRNA + + + 0.9921504 + evidence + cleaner0 + 2023-07-27T10:27:55Z + DUMMY: + + Structure + + + 0.9979594 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + 0.993615 + bond_interaction + cleaner0 + 2023-07-27T10:00:48Z + MESH: + + Watson-Crick hydrogen bond + + + 0.91873455 + chemical + cleaner0 + 2023-07-27T10:12:49Z + CHEBI: + + nucleotide + + + 0.97934216 + residue_number + cleaner0 + 2023-07-27T10:15:14Z + DUMMY: + + 72 + + + 0.9953772 + bond_interaction + cleaner0 + 2023-07-27T10:00:56Z + MESH: + + base-stacking interaction + + + 0.98064464 + residue_name_number + cleaner0 + 2023-07-27T09:17:02Z + DUMMY: + + G2 + + + 0.934088 + chemical + cleaner0 + 2023-07-27T09:32:05Z + CHEBI: + + 5′-triphosphate + + + 0.9988329 + chemical + cleaner0 + 2023-07-27T08:39:14Z + CHEBI: + + tRNA + + + 0.99860734 + chemical + cleaner0 + 2023-07-27T08:39:35Z + CHEBI: + + GTP + + + chemical + CHEBI: + cleaner0 + 2023-07-27T10:13:05Z + + Mg2+ + + + 0.946844 + chemical + cleaner0 + 2023-07-27T09:08:07Z + CHEBI: + + phosphate + + + 0.99588984 + chemical + cleaner0 + 2023-07-27T09:24:41Z + CHEBI: + + triphosphate + + + 0.9973099 + chemical + cleaner0 + 2023-07-27T10:13:28Z + CHEBI: + + nucleotide + + + 0.9555763 + site + cleaner0 + 2023-07-27T09:38:12Z + SO: + + site 1 + + + + DISCUSS + paragraph + 22881 + Figure 6 compares the 3′-5′ and 5′-3′ elongation mechanisms, showing the symmetrical nature of both elongation reactions using a similar reaction center composed of Mg2+A and Mg2+B in the conserved catalytic core. In TLP, which carries out 3′-5′ elongation, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the T7 RNA polymerase, a representative 5′-3′ DNA/RNA polymerase, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. In these reactions, the roles of the two Mg ions are identical. Mg2+A activates the 3′-OH of the incoming nucleotide in TLP and the 3′-OH of the 3′-end of the RNA chain in T7 RNA polymerase. The role of Mg2+B is to position the 5′-triphosphate of the tRNA in TLP and the incoming nucleotide in T7 RNA polymerase. These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core. + + 0.9598534 + site + cleaner0 + 2023-07-27T10:02:25Z + SO: + + reaction center + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:40:35Z + + Mg2+ + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:40:50Z + + Mg2+ + + + 0.9991924 + protein_state + cleaner0 + 2023-07-27T10:01:21Z + DUMMY: + + conserved + + + 0.8309485 + site + cleaner0 + 2023-07-27T10:02:20Z + SO: + + catalytic core + + + 0.99529254 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.9892129 + chemical + cleaner0 + 2023-07-27T09:08:07Z + CHEBI: + + phosphate + + + 0.99833184 + chemical + cleaner0 + 2023-07-27T08:39:14Z + CHEBI: + + tRNA + + + protein + PR: + cleaner0 + 2023-07-27T09:41:57Z + + T7 RNA polymerase + + + protein_type + MESH: + cleaner0 + 2023-07-27T09:40:04Z + + 5′-3′ DNA/RNA polymerase + + + 0.9950848 + chemical + cleaner0 + 2023-07-27T10:07:32Z + CHEBI: + + RNA + + + 0.992826 + chemical + cleaner0 + 2023-07-27T09:08:07Z + CHEBI: + + phosphate + + + 0.9984529 + chemical + cleaner0 + 2023-07-27T10:13:31Z + CHEBI: + + Mg + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:40:20Z + + Mg2+ + + + 0.99487984 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.9904358 + chemical + cleaner0 + 2023-07-27T10:07:32Z + CHEBI: + + RNA + + + protein + PR: + cleaner0 + 2023-07-27T09:41:58Z + + T7 RNA polymerase + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:41:06Z + + Mg2+ + + + chemical + CHEBI: + cleaner0 + 2023-07-27T09:32:05Z + + 5′-triphosphate + + + 0.9985898 + chemical + cleaner0 + 2023-07-27T08:39:14Z + CHEBI: + + tRNA + + + 0.9954899 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + protein + PR: + cleaner0 + 2023-07-27T09:41:58Z + + T7 RNA polymerase + + + 0.9950562 + chemical + cleaner0 + 2023-07-27T10:13:38Z + CHEBI: + + Mg2+ + + + bond_interaction + MESH: + cleaner0 + 2023-07-27T09:42:41Z + + coordinated by + + + 0.9991054 + protein_state + cleaner0 + 2023-07-27T10:01:23Z + DUMMY: + + conserved + + + 0.9874692 + residue_name + cleaner0 + 2023-07-27T09:42:20Z + SO: + + Asp + + + 0.9995602 + residue_name_number + cleaner0 + 2023-07-27T09:19:11Z + DUMMY: + + D21 + + + 0.9995504 + residue_name_number + cleaner0 + 2023-07-27T09:19:15Z + DUMMY: + + D69 + + + 0.9883781 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.9991966 + protein_state + cleaner0 + 2023-07-27T10:01:25Z + DUMMY: + + conserved + + + 0.8672683 + site + cleaner0 + 2023-07-27T10:02:20Z + SO: + + catalytic core + + + + 1501397-F6.jpg + F6 + FIG + fig_title_caption + 23926 + Structures of template-dependent nucleotide elongation in the 3′-5′ and 5′-3′ directions. + + 0.99832505 + evidence + cleaner0 + 2023-07-27T10:28:01Z + DUMMY: + + Structures + + + + 1501397-F6.jpg + F6 + FIG + fig_caption + 24024 + Symmetrical relationship between 3′-5′ elongation by TLP (this study) (left) and 5′-3′ elongation by T7 RNA polymerase [Protein Data Bank (PDB) ID: 1S76] (right). Red arrows represent elongation directions. In the 3′-5′ elongation reaction, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the 5′-3′ elongation reaction, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. Green spheres represent Mg2+ ions. + + 0.9417723 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + protein + PR: + cleaner0 + 2023-07-27T09:41:58Z + + T7 RNA polymerase + + + 0.9881513 + chemical + cleaner0 + 2023-07-27T09:08:07Z + CHEBI: + + phosphate + + + 0.99836165 + chemical + cleaner0 + 2023-07-27T08:39:14Z + CHEBI: + + tRNA + + + 0.99744284 + chemical + cleaner0 + 2023-07-27T10:07:32Z + CHEBI: + + RNA + + + 0.9937757 + chemical + cleaner0 + 2023-07-27T09:08:07Z + CHEBI: + + phosphate + + + 0.99891865 + chemical + cleaner0 + 2023-07-27T10:13:43Z + CHEBI: + + Mg2+ + + + + DISCUSS + paragraph + 24622 + Because the chemical roles of tRNA and the incoming nucleotide are reversed in these two reactions, these two substrates are inserted into a similar reaction center from opposite directions (Fig. 6). In spite of this difference, their fundamental reaction scheme is conserved. However, from an energetic viewpoint, these two reactions are clearly different: Whereas the high energy of the incoming nucleotide is used for its own addition in DNA/RNA polymerases, the high energy of the incoming nucleotide is used for subsequent addition in TLP. For this reason, TLP requires a mechanism that activates the 5′-terminus of the tRNA during the initial step of the reaction. Our analysis showed that the initial activation and subsequent elongation reactions occur sequentially at one reaction center. In this case, the enzyme needs to create two substrate binding sites for two different reactions in the vicinities of one reaction center. TLP has successfully created such sites by utilizing a conformational change in the tRNA through Watson-Crick base pairing (Fig. 3). These structural features of the TLP molecule suggest that development of an activation reaction site is a prerequisite for developing the 3′-5′ elongation enzyme. This is clearly more difficult than developing the 5′-3′ elongation enzyme, wherein the activation reaction site is not necessary, and which may be the primary reason why the 5′-3′ elongation enzyme has been exclusively developed. + + 0.998965 + chemical + cleaner0 + 2023-07-27T08:39:14Z + CHEBI: + + tRNA + + + 0.9934815 + site + cleaner0 + 2023-07-27T10:02:25Z + SO: + + reaction center + + + 0.9966459 + protein_type + cleaner0 + 2023-07-27T09:09:54Z + MESH: + + DNA/RNA polymerases + + + 0.8994305 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.88565826 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.99891484 + chemical + cleaner0 + 2023-07-27T08:39:14Z + CHEBI: + + tRNA + + + 0.86371475 + site + cleaner0 + 2023-07-27T10:02:25Z + SO: + + reaction center + + + 0.99890584 + site + cleaner0 + 2023-07-27T10:03:49Z + SO: + + substrate binding sites + + + 0.9167209 + site + cleaner0 + 2023-07-27T10:02:25Z + SO: + + reaction center + + + 0.7830006 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.999042 + chemical + cleaner0 + 2023-07-27T08:39:14Z + CHEBI: + + tRNA + + + bond_interaction + MESH: + cleaner0 + 2023-07-27T09:43:12Z + + Watson-Crick base pairing + + + 0.5639086 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.99901265 + site + cleaner0 + 2023-07-27T10:03:43Z + SO: + + activation reaction site + + + 0.9269032 + protein_type + cleaner0 + 2023-07-27T10:06:57Z + MESH: + + 3′-5′ elongation enzyme + + + 0.98154014 + protein_type + cleaner0 + 2023-07-27T09:43:20Z + MESH: + + 5′-3′ elongation enzyme + + + 0.99903107 + site + cleaner0 + 2023-07-27T10:03:44Z + SO: + + activation reaction site + + + 0.94371027 + protein_type + cleaner0 + 2023-07-27T09:43:20Z + MESH: + + 5′-3′ elongation enzyme + + + + DISCUSS + paragraph + 26101 + Here, we established a structural basis for 3′-5′ nucleotide elongation and showed that TLP has evolved to acquire a two-step Watson-Crick template–dependent 3′-5′ elongation reaction using the catalytic center homologous to 5′-3′ elongation enzymes. The active site of this enzyme is created at the dimerization interface. The dimerization also endows this protein with the ability to measure the length of the accepter stem of the tRNA substrate, so that the enzyme can properly terminate the elongation reaction. Furthermore, the dual binding mode of this protein suggests that it has further evolved to cover G−1 addition of tRNAHis by additional dimerization (dimer of dimers). Thus, the present structural analysis is consistent with the scenario in which TLP began as a 5′-end repair enzyme and evolved into a tRNAHis-specific G−1 addition enzyme. The detailed molecular mechanism of the Thg1/TLP family established by our analysis will open up new perspectives in our understanding of 3′-5′ versus 5′-3′ polymerization and the molecular evolution of template-dependent polymerases. + + 0.586542 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + 0.9988463 + site + cleaner0 + 2023-07-27T10:05:33Z + SO: + + catalytic center + + + 0.99679774 + protein_type + cleaner0 + 2023-07-27T09:32:16Z + MESH: + + 5′-3′ elongation enzymes + + + 0.99908924 + site + cleaner0 + 2023-07-27T10:29:07Z + SO: + + active site + + + 0.99904776 + site + cleaner0 + 2023-07-27T10:03:33Z + SO: + + dimerization interface + + + 0.98878664 + structure_element + cleaner0 + 2023-07-27T10:22:09Z + SO: + + accepter stem + + + 0.9981711 + chemical + cleaner0 + 2023-07-27T08:39:14Z + CHEBI: + + tRNA + + + 0.98732823 + residue_name_number + cleaner0 + 2023-07-27T10:03:23Z + DUMMY: + + G−1 + + + 0.9960594 + chemical + cleaner0 + 2023-07-27T08:40:25Z + CHEBI: + + tRNAHis + + + 0.9988643 + oligomeric_state + cleaner0 + 2023-07-27T09:14:51Z + DUMMY: + + dimer + + + 0.9988193 + oligomeric_state + cleaner0 + 2023-07-27T09:14:46Z + DUMMY: + + dimers + + + 0.9983082 + experimental_method + cleaner0 + 2023-07-27T10:24:40Z + MESH: + + structural analysis + + + 0.6366878 + protein_type + cleaner0 + 2023-07-27T08:37:29Z + MESH: + + TLP + + + protein_type + MESH: + cleaner0 + 2023-07-27T08:40:09Z + + tRNAHis-specific G−1 addition enzyme + + + 0.99843353 + protein + cleaner0 + 2023-07-27T08:38:03Z + PR: + + Thg1 + + + protein_type + MESH: + cleaner0 + 2023-07-27T08:37:29Z + + TLP + + + 0.9947258 + protein_type + cleaner0 + 2023-07-27T09:43:52Z + MESH: + + template-dependent polymerases + + + + METHODS + title_1 + 27219 + MATERIALS AND METHODS + + + METHODS + title_2 + 27241 + Plasmid construction + + + METHODS + paragraph + 27262 + Genomic DNA from M. acetivorans NBRC100939 was obtained from the NITE Biological Resource Center. The MaTLP gene was amplified by polymerase chain reaction from genomic DNA. The DNA fragment encoding MaTLP was then cloned between the Nde I and Xho I restriction sites in a pET26b vector with a C-terminal His tag. In the MaTLP gene, the amber stop codon (UAG) at position 142 was translated as Pyl. To express the full-length MaTLP in Escherichia coli, the TAG codon was altered to TGG (encoding Trp) with the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) as previously described. The inserted sequence was verified by DNA sequencing. + + + METHODS + title_2 + 27915 + Preparation of MaTLP and mutants + + + METHODS + paragraph + 27948 + Plasmids were transformed into E. coli strain BL21 (DE3) pLysSRARE by electroporation, and cells were grown in LB medium containing kanamycin (25 μg/ml) and chloramphenicol (34 μg/ml) at 37°C until reaching an optical density at 600 nm (OD600) of 0.45. The cells were then induced by the addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 250 μM and shifted to 18°C for approximately 20 hours before harvest. The cells were harvested and resuspended in buffer A [50 mM Hepes-NaOH (pH 7.5), 1 M NaCl, 4 mM MgCl2, 10% glycerol, 0.5 mM β-mercaptoethanol, lysozyme (0.5 mg/ml), and deoxyibonuclease (0.1 mg/ml)]. After sonication and centrifugation, the His6-tagged protein was purified by immobilized metal-ion affinity chromatography using a HisTrap HP column (GE Healthcare). The sample was washed with 75 mM imidazole and eluted with a 75 to 400 mM imidazole gradient in buffer B [50 mM tris-HCl (pH 7.5), 500 mM NaCl, 4 mM MgCl2, 20% glycerol, and 0.5 mM β-mercaptoethanol]. Then, the collected fractions were diluted in 300 mM NaCl with buffer C [25 mM tris-HCl (pH 7.5), 10% glycerol, 5 mM MgCl2, and 1 mM dithiothreitol (DTT)] and further purified on a HiTrap Heparin HP column (GE Healthcare) by elution with a 300 to 1000 mM NaCl gradient in buffer C. Finally, the protein was loaded onto a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) equilibrated with buffer D [20 mM Hepes-NaOH (pH 7.5), 500 mM NaCl, 5 mM MgCl2, 10% glycerol, and 1 mM DTT]. The protein was concentrated to 3.9 mg/ml by ultrafiltration. All MaTLP mutants were constructed with the QuikChange Site-Directed Mutagenesis Kit. MaTLP mutants were purified by a HisTrap HP column for RNA binding assay and further purified by a HiLoad 16/60 Superdex 200 prep grade column for 3′-5′ nucleotide addition assay. + + + METHODS + title_2 + 29779 + Preparation of tRNA and its mutants + + + METHODS + paragraph + 29815 + tRNA transcripts derived from yeast tRNAPhe and tRNAHis were prepared using T7 RNA polymerase as previously described. ppptRNA transcripts were prepared by excluding guanosine 5′-monophosphate (GMP) from the reaction mixture. Transcribed tRNAs were purified by a HiTrap DEAE FF column (GE Healthcare) as previously described. Pooled tRNAs were precipitated with isopropanol and dissolved in buffer E [20 mM Hepes-NaOH (pH 7.5), 100 mM NaCl, and 10 mM MgCl2]. + + chemical + CHEBI: + cleaner0 + 2023-07-27T10:10:42Z + + tRNAs + + + chemical + CHEBI: + cleaner0 + 2023-07-27T10:10:42Z + + tRNAs + + + + METHODS + title_2 + 30276 + Preparation of the MaTLP-ppptRNAPheΔ1 complex + + + METHODS + paragraph + 30326 + MaTLP and ppptRNAPheΔ1 (tRNAPhe with a triphosphorylated 5′-end and deleted G1) were mixed in a molar ratio of 1.7:1 and incubated for 30 min at room temperature. The mixture was then loaded onto a HiLoad 16/60 Superdex 200 prep grade column equilibrated with buffer F [20 mM Hepes-NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 10% glycerol, and 1 mM DTT]. Fractions containing the MaTLP-ppptRNAPheΔ1 complex were mixed with 1 mM spermine and concentrated to an OD280 of 16 by ultrafiltration. + + + METHODS + title_2 + 30822 + Crystallization and data collection + + + METHODS + paragraph + 30858 + All crystallization experiments were performed with the sitting-drop vapor diffusion method at 293 K. Initial crystals of MaTLP were obtained by mixing 1 μl of protein solution (3.9 mg/ml) with 1 μl of a reservoir solution containing 0.1 M Hepes-NaOH buffer (pH 7.5), 0.2 M magnesium chloride, and 30% polyethylene glycol 400 (PEG 400). MaTLP-GTP complex crystals were obtained by soaking the MaTLP crystals in the above reservoir solution supplemented with 1 mM GTP overnight. High-resolution crystals of MaTLP in apo form (MaTLP-apo) were obtained unexpectedly by mixing MaTLP with tRNAHis in 0.1 M sodium/potassium phosphate (pH 6.2) containing 2.5 M NaCl. Crystals of the MaTLP-ppptRNAPheΔ1 complex were obtained from a solution containing 0.2 M tripotassium citrate, 0.1 M tris (pH 8.0), 37% PEG3350, and 10 mM praseodymium (III) acetate. Crystals of the MaTLP-ppptRNAPheΔ1-GDPNP complex were obtained by soaking MaTLP-ppptRNAPheΔ1 complex crystals in a reservoir solution containing 0.2 M tripotassium citrate, 0.1 M tris (pH 8.0), 30% PEG3350, 5% glycerol, and 15 mM GDPNP overnight. Crystals of MaTLP-apo and MaTLP-GTP were cryoprotected with a reservoir solution containing 50% PEG400 before flash-cooling, whereas crystals of the MaTLP-ppptRNAPheΔ1-GDPNP and MaTLP-ppptRNAPheΔ1 complexes were flash-cooled without any cryoprotectant under a stream of liquid nitrogen at 100 K. X-ray diffraction data were collected from beamline BL41XU at SPring-8 (Hyogo, Japan) and beamlines BL5A and BL17A at Photon Factory (Ibaraki, Japan). All diffraction data were indexed, integrated, scaled, and merged using XDS. + + + METHODS + title_2 + 32495 + Structure determination and refinement + + + METHODS + paragraph + 32534 + The crystal structure of MaTLP-apo was determined by the molecular replacement (MR) method with Molrep, using the protomer structure of CaThg1 (PDB ID: 3WBZ) as a search model. The protomer structure of MaTLP-apo was then used as a search model to solve the structures of MaTLP-GTP. The crystal structure of the MaTLP-ppptRNAPheΔ1 complex was determined by the MR method with PHASER, using the protomer structures of MaTLP-apo and tRNAPhe from Saccharomyces cerevisiae (PDB ID: 1EHZ) as search models. The structure of the MaTLP-ppptRNAPheΔ1 complex was then used as a search model to solve the MaTLP-ppptRNAPheΔ1-GDPNP complex structure. Initial protein models were fitted manually using Coot, and tRNA models were automatically rebuilt by LAFIRE_NAFIT; these models were then refined using phenix.refine. The data collection and refinement statistics are summarized in Table 1. All structure figures were generated by PyMol. + + + T1.xml + T1 + TABLE + table_title_caption + 33473 + Summary of data collection and refinement statistics. + + + T1.xml + T1 + TABLE + table_caption + 33527 + Values in parentheses are for the highest-resolution shell. PF, Photon Factory; Rmsd, root-mean-square deviation. + + + T1.xml + T1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><col width="%" span="1"/><col width="%" span="1"/><col width="%" span="1"/><col width="%" span="1"/><col width="%" span="1"/><thead><tr><td valign="top" align="left" scope="col" rowspan="1" colspan="1"/><td valign="top" align="center" scope="col" rowspan="1" colspan="1"><bold><italic>Ma</italic>TLP-apo</bold></td><td valign="top" align="center" scope="col" rowspan="1" colspan="1"><bold><italic>Ma</italic>TLP-GTP</bold></td><td valign="top" align="center" scope="col" rowspan="1" colspan="1"><bold><italic>Ma</italic>TLP-ppptRNA<sup>Phe</sup>Δ<sub>1</sub></bold></td><td valign="top" align="center" scope="col" rowspan="1" colspan="1"><bold><italic>Ma</italic>TLP-ppptRNA<sup>Phe</sup>Δ<sub>1</sub>-GDPNP</bold></td></tr></thead><tbody><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">PDB ID</td><td valign="top" align="center" rowspan="1" colspan="1">5AXK</td><td valign="top" align="center" rowspan="1" colspan="1">5AXL</td><td valign="top" align="center" rowspan="1" colspan="1">5AXM</td><td valign="top" align="center" rowspan="1" colspan="1">5AXN</td></tr><tr><td colspan="5" valign="top" align="left" scope="row" rowspan="1"><bold>Data collection</bold></td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Beamline</td><td valign="top" align="center" rowspan="1" colspan="1">SPring-8 BL41XU</td><td valign="top" align="center" rowspan="1" colspan="1">SPring-8 BL41XU</td><td valign="top" align="center" rowspan="1" colspan="1">PF BL17A</td><td valign="top" align="center" rowspan="1" colspan="1">PF BL5A</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Space group</td><td valign="top" align="center" rowspan="1" colspan="1"><italic>C</italic>222<sub>1</sub></td><td valign="top" align="center" rowspan="1" colspan="1"><italic>C</italic>222<sub>1</sub></td><td valign="top" align="center" rowspan="1" colspan="1"><italic>I</italic>222</td><td valign="top" align="center" rowspan="1" colspan="1"><italic>I</italic>222</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Unit cell parameters <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td valign="top" align="center" rowspan="1" colspan="1">98.3, 120.5, 157.4</td><td valign="top" align="center" rowspan="1" colspan="1">103.1, 115.7, 144.9</td><td valign="top" align="center" rowspan="1" colspan="1">75.3, 127.6, 143.8</td><td valign="top" align="center" rowspan="1" colspan="1">82.3, 134.1, 147.4</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Wavelength (Å)</td><td valign="top" align="center" rowspan="1" colspan="1">0.9780</td><td valign="top" align="center" rowspan="1" colspan="1">1.0000</td><td valign="top" align="center" rowspan="1" colspan="1">0.97319</td><td valign="top" align="center" rowspan="1" colspan="1">1.0000</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Resolution range (Å)</td><td valign="top" align="center" rowspan="1" colspan="1">50.0–2.29 (2.43–2.29)</td><td valign="top" align="center" rowspan="1" colspan="1">50.0–2.99 (3.17–2.99)</td><td valign="top" align="center" rowspan="1" colspan="1">50.0–2.21 (2.34–2.21)</td><td valign="top" align="center" rowspan="1" colspan="1">50.0–2.70 (2.87–2.70)</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  <italic>R</italic><sub>meas</sub> (%)*</td><td valign="top" align="center" rowspan="1" colspan="1">8.9 (76.3)</td><td valign="top" align="center" rowspan="1" colspan="1">15.2 (90.0)</td><td valign="top" align="center" rowspan="1" colspan="1">9.7 (74.4)</td><td valign="top" align="center" rowspan="1" colspan="1">11.0 (87.2)</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  CC<sub>1/2</sub> (%)</td><td valign="top" align="center" rowspan="1" colspan="1">99.8 (80.4)</td><td valign="top" align="center" rowspan="1" colspan="1">99.5 (81.2)</td><td valign="top" align="center" rowspan="1" colspan="1">99.9 (83.6)</td><td valign="top" align="center" rowspan="1" colspan="1">99.9 (83.5)</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  〈<italic>I</italic>/σ(<italic>I</italic>)〉</td><td valign="top" align="center" rowspan="1" colspan="1">14.7 (2.8)</td><td valign="top" align="center" rowspan="1" colspan="1">12.0 (2.6)</td><td valign="top" align="center" rowspan="1" colspan="1">19.4 (3.2)</td><td valign="top" align="center" rowspan="1" colspan="1">16.9 (2.5)</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Completeness (%)</td><td valign="top" align="center" rowspan="1" colspan="1">98.3 (93.8)</td><td valign="top" align="center" rowspan="1" colspan="1">98.8 (93.4)</td><td valign="top" align="center" rowspan="1" colspan="1">99.7 (98.6)</td><td valign="top" align="center" rowspan="1" colspan="1">99.7 (99.3)</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Redundancy</td><td valign="top" align="center" rowspan="1" colspan="1">6.7 (6.6)</td><td valign="top" align="center" rowspan="1" colspan="1">7.2 (7.2)</td><td valign="top" align="center" rowspan="1" colspan="1">7.4 (7.3)</td><td valign="top" align="center" rowspan="1" colspan="1">8.1 (8.2)</td></tr><tr><td colspan="5" valign="top" align="left" scope="row" rowspan="1"><bold>Refinement</bold></td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  No. of reflections</td><td valign="top" align="center" rowspan="1" colspan="1">41,650</td><td valign="top" align="center" rowspan="1" colspan="1">17,581</td><td valign="top" align="center" rowspan="1" colspan="1">35,102</td><td valign="top" align="center" rowspan="1" colspan="1">22,669</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  <italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub> (%)<sup>†</sup></td><td valign="top" align="center" rowspan="1" colspan="1">20.6/24.0</td><td valign="top" align="center" rowspan="1" colspan="1">21.5/25.3</td><td valign="top" align="center" rowspan="1" colspan="1">21.6/24.3</td><td valign="top" align="center" rowspan="1" colspan="1">22.5/26.7</td></tr><tr><td colspan="5" valign="top" align="left" scope="row" rowspan="1">  No. of atoms</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Macromolecules</td><td valign="top" align="center" rowspan="1" colspan="1">3760</td><td valign="top" align="center" rowspan="1" colspan="1">3622</td><td valign="top" align="center" rowspan="1" colspan="1">5247</td><td valign="top" align="center" rowspan="1" colspan="1">5142</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Ligand/ion</td><td valign="top" align="center" rowspan="1" colspan="1">30</td><td valign="top" align="center" rowspan="1" colspan="1">68</td><td valign="top" align="center" rowspan="1" colspan="1">36</td><td valign="top" align="center" rowspan="1" colspan="1">101</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Water</td><td valign="top" align="center" rowspan="1" colspan="1">89</td><td valign="top" align="center" rowspan="1" colspan="1">8</td><td valign="top" align="center" rowspan="1" colspan="1">102</td><td valign="top" align="center" rowspan="1" colspan="1">16</td></tr><tr><td colspan="5" valign="top" align="left" scope="row" rowspan="1">  <italic>B</italic>-factors (Å<sup>2</sup>)</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Macromolecules</td><td valign="top" align="center" scope="col" rowspan="1" colspan="1">57.0</td><td valign="top" align="center" rowspan="1" colspan="1">68.4</td><td valign="top" align="center" rowspan="1" colspan="1">45.3</td><td valign="top" align="center" rowspan="1" colspan="1">57.3</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Ligand/ion</td><td valign="top" align="center" rowspan="1" colspan="1">60.5</td><td valign="top" align="center" rowspan="1" colspan="1">86.2</td><td valign="top" align="center" rowspan="1" colspan="1">46.6</td><td valign="top" align="center" rowspan="1" colspan="1">59.9</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Water</td><td valign="top" align="center" rowspan="1" colspan="1">49.0</td><td valign="top" align="center" rowspan="1" colspan="1">59.5</td><td valign="top" align="center" rowspan="1" colspan="1">33.0</td><td valign="top" align="center" rowspan="1" colspan="1">38.1</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Estimated coordinate error (Å)</td><td valign="top" align="center" rowspan="1" colspan="1">0.32</td><td valign="top" align="center" rowspan="1" colspan="1">0.48</td><td valign="top" align="center" rowspan="1" colspan="1">0.25</td><td valign="top" align="center" rowspan="1" colspan="1">0.41</td></tr><tr><td colspan="5" valign="top" align="left" scope="row" rowspan="1">  Rmsd from ideal</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Bond lengths (Å)</td><td valign="top" align="center" rowspan="1" colspan="1">0.009</td><td valign="top" align="center" rowspan="1" colspan="1">0.003</td><td valign="top" align="center" rowspan="1" colspan="1">0.003</td><td valign="top" align="center" rowspan="1" colspan="1">0.003</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Bond angles (°)</td><td valign="top" align="center" rowspan="1" colspan="1">1.11</td><td valign="top" align="center" rowspan="1" colspan="1">0.92</td><td valign="top" align="center" rowspan="1" colspan="1">0.72</td><td valign="top" align="center" rowspan="1" colspan="1">0.80</td></tr></tbody></table> + + 33641 + MaTLP-apo MaTLP-GTP MaTLP-ppptRNAPheΔ1 MaTLP-ppptRNAPheΔ1-GDPNP PDB ID 5AXK 5AXL 5AXM 5AXN Data collection   Beamline SPring-8 BL41XU SPring-8 BL41XU PF BL17A PF BL5A   Space group C2221 C2221 I222 I222   Unit cell parameters a, b, c (Å) 98.3, 120.5, 157.4 103.1, 115.7, 144.9 75.3, 127.6, 143.8 82.3, 134.1, 147.4   Wavelength (Å) 0.9780 1.0000 0.97319 1.0000   Resolution range (Å) 50.0–2.29 (2.43–2.29) 50.0–2.99 (3.17–2.99) 50.0–2.21 (2.34–2.21) 50.0–2.70 (2.87–2.70)   Rmeas (%)* 8.9 (76.3) 15.2 (90.0) 9.7 (74.4) 11.0 (87.2)   CC1/2 (%) 99.8 (80.4) 99.5 (81.2) 99.9 (83.6) 99.9 (83.5)   〈I/σ(I)〉 14.7 (2.8) 12.0 (2.6) 19.4 (3.2) 16.9 (2.5)   Completeness (%) 98.3 (93.8) 98.8 (93.4) 99.7 (98.6) 99.7 (99.3)   Redundancy 6.7 (6.6) 7.2 (7.2) 7.4 (7.3) 8.1 (8.2) Refinement   No. of reflections 41,650 17,581 35,102 22,669   Rwork/Rfree (%)† 20.6/24.0 21.5/25.3 21.6/24.3 22.5/26.7   No. of atoms     Macromolecules 3760 3622 5247 5142     Ligand/ion 30 68 36 101     Water 89 8 102 16   B-factors (Å2)     Macromolecules 57.0 68.4 45.3 57.3     Ligand/ion 60.5 86.2 46.6 59.9     Water 49.0 59.5 33.0 38.1   Estimated coordinate error (Å) 0.32 0.48 0.25 0.41   Rmsd from ideal     Bond lengths (Å) 0.009 0.003 0.003 0.003     Bond angles (°) 1.11 0.92 0.72 0.80 + + + T1.xml + T1 + TABLE + table_foot + 35061 + *Rmeas = Σhkl {N(hkl)/[N(hkl) − 1]}1/2 Σi | Ii(hkl) − 〈I(hkl)〉 |/Σhkl Σi +Ii(hkl), where 〈I(hkl)〉 and N(hkl) are the mean intensity of a set of equivalent reflections and the multiplicity, respectively. + + + T1.xml + T1 + TABLE + table_foot + 35279 + †Rwork += Σhkl ||Fobs| − |Fcalc||/Σhkl |Fobs|; Rfree was calculated for 5% randomly selected test sets that were not used in the refinement. + + + METHODS + title_2 + 35427 + Nucleotide addition assay + + + METHODS + paragraph + 35453 + Nucleotide addition assays were performed as previously described. A reaction mixture containing 25 mM Hepes-NaOH (pH 7.5), 400 mM NaCl, 10 mM MgCl2, 3 mM DTT, 5% glycerol, 0.1 μM [α-32P]GTP, 100 μM GTP, 1 μM MaTLP variants, and 10 μM tRNA transcript was incubated at 30°C for 2 hours. Then, the reaction was quenched with phenol/chloroform, and the supernatant was resolved on a 10% polyacrylamide gel containing 8 M urea. The radioactivity was visualized with a BAS-1800 II bioimaging analyzer (Fujifilm). + + + METHODS + title_2 + 35969 + tRNA binding assay + + + METHODS + paragraph + 35988 + A reaction mixture containing 34 μM MaTLP variants and 20 μM tRNA transcript was incubated in buffer F at room temperature for 30 min. Then, the mixture was loaded onto a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with the same buffer. + + + SUPPL + title_1 + 36242 + Supplementary Material + + + SUPPL + title + 36265 + SUPPLEMENTARY MATERIALS + + + REF + title + 36289 + REFERENCES AND NOTES + + + REF + ref + 36310 + B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Walter, Molecular Biology of the Cell (Garland Science, New York, ed. 5, 2008). + + + 333 + 344 + surname:Heinemann;given-names:I. U. + surname:Nakamura;given-names:A. + surname:O’Donoghue;given-names:P. + surname:Eiler;given-names:D. + surname:Söll;given-names:D. + 21890903 + REF + Nucleic Acids Res. + ref + 40 + 2012 + 36448 + tRNAHis-guanylyltransferase establishes tRNAHis identity + + + 886 + 899 + surname:Jackman;given-names:J. E. + surname:Gott;given-names:J. M. + surname:Gray;given-names:M. W. + 22456265 + REF + RNA + ref + 18 + 2012 + 36505 + Doing it in reverse: 3′-to-5′ polymerization by the Thg1 superfamily + + + 2889 + 2901 + surname:Gu;given-names:W. + surname:Jackman;given-names:J. E. + surname:Lohan;given-names:A. J. + surname:Gray;given-names:M. W. + surname:Phizicky;given-names:E. M. + 14633974 + REF + Genes Dev. + ref + 17 + 2003 + 36578 + tRNAHis maturation: An essential yeast protein catalyzes addition of a guanine nucleotide to the 5′ end of tRNAHis + + + 6475 + 6479 + surname:Cooley;given-names:L. + surname:Appel;given-names:B. + surname:Söll;given-names:D. + 6292903 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 79 + 1982 + 36695 + Post-transcriptional nucleotide addition is responsible for the formation of the 5′ terminus of histidine tRNA + + + 7855 + 7863 + surname:Himeno;given-names:H. + surname:Hasegawa;given-names:T. + surname:Ueda;given-names:T. + surname:Watanabe;given-names:K. + surname:Miura;given-names:K.-i. + surname:Shimizu;given-names:M. + 2678006 + REF + Nucleic Acids Res. + ref + 17 + 1989 + 36808 + Role of the extra G-C pair at the end of the acceptor stem of tRNAHb in aminoacylation + + + 5031 + 5037 + surname:Rudinger;given-names:J. + surname:Florentz;given-names:C. + surname:Giegé;given-names:R. + 7800496 + REF + Nucleic Acids Res. + ref + 22 + 1994 + 36895 + Histidylation by yeast HisRS of tRNA or tRNA-like structure relies on residues −1 and 73 but is dependent on the RNA context + + + 64 + 65 + surname:Rosen;given-names:A. E. + surname:Musier-Forsyth;given-names:K. + 14709061 + REF + J. Am. Chem. Soc. + ref + 126 + 2004 + 37022 + Recognition of G-1:C73 atomic groups by Escherichia coli histidyl-tRNA synthetase + + + 674 + 679 + surname:Abad;given-names:M. G. + surname:Rao;given-names:B. S. + surname:Jackman;given-names:J. E. + 20080734 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 107 + 2010 + 37104 + Template-dependent 3′–5′ nucleotide addition is a shared feature of tRNAHis guanylyltransferase enzymes from multiple domains of life + + + 3567 + 3572 + surname:Heinemann;given-names:I. U. + surname:Randau;given-names:L. + surname:Tomko;given-names:R. J.;suffix:Jr + surname:Söll;given-names:D. + 20650272 + REF + FEBS Lett. + ref + 584 + 2010 + 37244 + 3′–5′ tRNAHis guanylyltransferase in bacteria + + + 1833 + 1842 + surname:Rao;given-names:B. S. + surname:Maris;given-names:E. L. + surname:Jackman;given-names:J. E. + 21051361 + REF + Nucleic Acids Res. + ref + 39 + 2011 + 37296 + tRNA 5′-end repair activities of tRNAHis guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea + + + 832 + 835 + surname:Rice;given-names:T. S. + surname:Ding;given-names:M. + surname:Pederson;given-names:D. S. + surname:Heintz;given-names:N. H. + 15821142 + REF + Eukaryot. Cell + ref + 4 + 2005 + 37406 + The highly conserved tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex and is required for the G2/M phase transition in the yeast Saccharomyces cerevisiae + + protein + PR: + cleaner0 + 2023-07-27T10:14:01Z + + Thg1p + + + + e1005085 + surname:Zhu;given-names:W. + surname:Ausin;given-names:I. + surname:Alonso-Blanco;given-names:C. + surname:Balasubramanian;given-names:S. + surname:Seleznev;given-names:A. + surname:Méndez-Vigo;given-names:B. + surname:Picó;given-names:F. X. + surname:Sureshkumar;given-names:S. + surname:Sundaramoorthi;given-names:V. + surname:Bulach;given-names:D. + surname:Powell;given-names:D. + surname:Seemann;given-names:T. + 25951176 + REF + PLOS Genet. + ref + 11 + 2015 + 37591 + Natural variation identifies ICARUS1, a universal gene required for cell proliferation and growth at high temperatures in Arabidopsis thaliana + + + 22832 + 22836 + surname:Jahn;given-names:D. + surname:Pande;given-names:S. + 1660462 + REF + J. Biol. Chem. + ref + 266 + 1991 + 37734 + Histidine tRNA guanylyltransferase from Saccharomyces cerevisiae. II. Catalytic mechanism + + + 20305 + 20310 + surname:Hyde;given-names:S. J. + surname:Eckenroth;given-names:B. E. + surname:Smith;given-names:B. A. + surname:Eberley;given-names:W. A. + surname:Heintz;given-names:N. H. + surname:Jackman;given-names:J. E. + surname:Doublié;given-names:S. + 21059936 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 107 + 2010 + 37824 + tRNAHis guanylyltransferase (THG1), a unique 3′-5′ nucleotidyl transferase, shares unexpected structural homology with canonical 5′-3′ DNA polymerases + + + 251 + 258 + surname:Doublié;given-names:S. + surname:Tabor;given-names:S. + surname:Long;given-names:A. M. + surname:Richardson;given-names:C. C. + surname:Ellenberger;given-names:T. + 9440688 + REF + Nature + ref + 391 + 1998 + 37983 + Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution + + + 4101 + 4113 + surname:Jeruzalmi;given-names:D. + surname:Steitz;given-names:T. A. + 9670025 + REF + EMBO J. + ref + 17 + 1998 + 38068 + Structure of T7 RNA polymerase complexed to the transcriptional inhibitor T7 lysozyme + + + e67465 + surname:Hyde;given-names:S. J. + surname:Rao;given-names:B. S. + surname:Eckenroth;given-names:B. E. + surname:Jackman;given-names:J. E. + surname:Doublié;given-names:S. + 23844012 + REF + PLOS One + ref + 8 + 2013 + 38154 + Structural studies of a bacterial tRNAHis guanylyltransferase (Thg1)-like protein, with nucleotide in the activation and nucleotidyl transfer sites + + + 20970 + 20975 + surname:Nakamura;given-names:A. + surname:Nemoto;given-names:T. + surname:Heinemann;given-names:I. U. + surname:Yamashita;given-names:K. + surname:Sonoda;given-names:T. + surname:Komoda;given-names:K. + surname:Tanaka;given-names:I. + surname:Söll;given-names:D. + surname:Yao;given-names:M. + 24324136 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 110 + 2013 + 38302 + Structural basis of reverse nucleotide polymerization + + + 1007 + 1014 + surname:Jackman;given-names:J. E. + surname:Phizicky;given-names:E. M. + 16625026 + REF + RNA + ref + 12 + 2006 + 38356 + tRNAHis guanylyltransferase adds G-1 to the 5′ end of tRNAHis by recognition of the anticodon, one of several features unexpectedly shared with tRNA synthetases + + + 4817 + 4825 + surname:Jackman;given-names:J. E. + surname:Phizicky;given-names:E. M. + 18366186 + REF + Biochemistry + ref + 47 + 2008 + 38519 + Identification of critical residues for G-1 addition and substrate recognition by tRNAHis guanylyltransferase + + + 21103 + 21108 + surname:Heinemann;given-names:I. U. + surname:O’Donoghue;given-names:P. + surname:Madinger;given-names:C. + surname:Benner;given-names:J. + surname:Randau;given-names:L. + surname:Noren;given-names:C. J. + surname:Söll;given-names:D. + 19965368 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 106 + 2009 + 38629 + The appearance of pyrrolysine in tRNAHis guanylyltransferase by neutral evolution + + + 647 + 653 + surname:Easton;given-names:L. E. + surname:Shibata;given-names:Y. + surname:Lukavsky;given-names:P. J. + 20100812 + REF + RNA + ref + 16 + 2010 + 38711 + Rapid, nondenaturing RNA purification using weak anion-exchange fast performance liquid chromatography + + + 125 + 132 + surname:Kabsch;given-names:W. + 20124692 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 38814 + XDS + + + 22 + 25 + surname:Vagin;given-names:A. + surname:Teplyakov;given-names:A. + 20057045 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 38818 + Molecular replacement with MOLREP + + + 235 + 242 + surname:Winn;given-names:M. D. + surname:Ballard;given-names:C. C. + surname:McNicholas;given-names:S. J. + surname:Murshudov;given-names:G. N. + surname:Pannu;given-names:N. S. + surname:Potterton;given-names:E. A. + surname:Powell;given-names:H. R. + surname:Read;given-names:R. J. + surname:Vagin;given-names:A. + surname:Wilson;given-names:K. S. + surname:Cowtan;given-names:K. D. + surname:Dodson;given-names:E. J. + surname:Emsley;given-names:P. + surname:Evans;given-names:P. R. + surname:Keegan;given-names:R. M. + surname:Krissinel;given-names:E. B. + surname:Leslie;given-names:A. G. W. + surname:McCoy;given-names:A. + 21460441 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 67 + 2011 + 38852 + Overview of the CCP4 suite and current developments + + + 658 + 674 + surname:McCoy;given-names:A. J. + surname:Grosse-Kunstleve;given-names:R. W. + surname:Adams;given-names:P. D. + surname:Winn;given-names:M. D. + surname:Storoni;given-names:L. C. + surname:Read;given-names:R. J. + 19461840 + REF + J. Appl. Crystallogr. + ref + 40 + 2007 + 38904 + Phaser crystallographic software. + + + 1091 + 1105 + surname:Shi;given-names:H. + surname:Moore;given-names:P. B. + 10943889 + REF + RNA + ref + 6 + 2000 + 38938 + The crystal structure of yeast phenylalanine tRNA at 1.93 Å resolution: A classic structure revisited + + + 2126 + 2132 + surname:Emsley;given-names:P. + surname:Cowtan;given-names:K. + 15572765 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 60 + 2004 + 39041 + Coot: Model-building tools for molecular graphics + + + 1171 + 1179 + surname:Yamashita;given-names:K. + surname:Zhou;given-names:Y. + surname:Tanaka;given-names:I. + surname:Yao;given-names:M. + 23695261 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 69 + 2013 + 39091 + New model-fitting and model-completion programs for automated iterative nucleic acid refinement + + + 213 + 221 + surname:Adams;given-names:P. D. + surname:Afonine;given-names:P. V. + surname:McCoy;given-names:A. J. + surname:Moriarty;given-names:N. W. + surname:Oeffner;given-names:R. + surname:Read;given-names:R. J. + surname:Richardson;given-names:D. C. + surname:Richardson;given-names:J. S. + surname:Terwilliger;given-names:T. C. + surname:Zwart;given-names:P. H. + surname:Bunkóczi;given-names:G. + surname:Chen;given-names:V. B. + surname:Davis;given-names:I. W. + surname:Echols;given-names:N. + surname:Headd;given-names:J. J. + surname:Hung;given-names:L.-W. + surname:Kapral;given-names:G. J. + surname:Grosse-Kunstleve;given-names:R. W. + 20124702 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 39187 + PHENIX: A comprehensive Python-based system for macromolecular structure solution + + + REF + ref + 39269 + W. L. DeLano, The PyMOL Molecular Graphics System, Version 1.7.4 (Schrödinger, LLC, 2002). + + + diff --git a/BioC_XML/4822050_v0.xml b/BioC_XML/4822050_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..e2266b873ebcda0976cfff2954e71e08a79dc59c --- /dev/null +++ b/BioC_XML/4822050_v0.xml @@ -0,0 +1,14727 @@ + + + + PMC + 20201215 + pmc.key + + 4822050 + CC BY + no + 0 + 0 + + 10.1038/ncomms11197 + ncomms11197 + 4822050 + 27045799 + 11197 + This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ + surname:Fang;given-names:Jian + surname:Cheng;given-names:Jingdong + surname:Gong;given-names:Zhou + surname:Tang;given-names:Chun + surname:Wong;given-names:Jiemin + surname:Yang;given-names:Huirong + surname:Cao;given-names:Chunyang + surname:Xu;given-names:Yanhui + surname:Wang;given-names:Jiaolong + surname:Zhang;given-names:Qiao + surname:Liu;given-names:Mengjie + surname:Gong;given-names:Rui + surname:Wang;given-names:Ping + surname:Zhang;given-names:Xiaodan + surname:Feng;given-names:Yangyang + surname:Lan;given-names:Wenxian + TITLE + front + 7 + 2016 + 0 + Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:18Z + + Hemi-methylated DNA + + + 0.999231 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + 0.99887425 + protein + cleaner0 + 2023-07-25T14:58:00Z + PR: + + UHRF1 + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:08Z + + histone + + + + ABSTRACT + abstract + 95 + UHRF1 is an important epigenetic regulator for maintenance DNA methylation. UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD–PHD. The Spacer also facilitates UHRF1–DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. When TTD–PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1. + + 0.9992975 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:57Z + + methylation + + + 0.9993113 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.97070986 + chemical + cleaner0 + 2023-07-25T14:58:17Z + CHEBI: + + hemi-methylated DNA + + + 0.99714917 + chemical + cleaner0 + 2023-07-25T14:58:23Z + CHEBI: + + hm-DNA + + + 0.98988324 + ptm + cleaner0 + 2023-07-25T16:29:40Z + MESH: + + trimethylation + + + 0.995805 + protein_type + cleaner0 + 2023-07-25T15:05:08Z + MESH: + + histone + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:29:37Z + + H3 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-08-08T12:29:31Z + + K9 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:30:01Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:30:15Z + + K9me3 + + + 0.9994023 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.9992417 + protein_state + cleaner0 + 2023-07-25T14:59:19Z + DUMMY: + + closed + + + 0.99918896 + structure_element + cleaner0 + 2023-07-25T14:56:57Z + SO: + + C-terminal region + + + 0.9990657 + structure_element + cleaner0 + 2023-07-25T14:57:02Z + SO: + + Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:03Z + + binds to + + + 0.99929637 + structure_element + cleaner0 + 2023-07-25T14:57:11Z + SO: + + tandem Tudor domain + + + 0.99940634 + structure_element + cleaner0 + 2023-07-25T14:57:16Z + SO: + + TTD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:31:55Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:32:04Z + + K9me3 + + + 0.99883306 + structure_element + cleaner0 + 2023-07-25T14:57:27Z + SO: + + SET-and-RING-associated + + + 0.9949174 + structure_element + cleaner0 + 2023-07-25T14:57:33Z + SO: + + SRA + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:03Z + + binds to + + + 0.9913373 + structure_element + cleaner0 + 2023-07-25T14:57:42Z + SO: + + plant homeodomain + + + 0.99932337 + structure_element + cleaner0 + 2023-07-25T14:57:48Z + SO: + + PHD + + + 0.87803274 + site + cleaner0 + 2023-07-25T15:03:31Z + SO: + + H3R2 + + + 0.9954868 + chemical + cleaner0 + 2023-07-25T14:58:24Z + CHEBI: + + Hm-DNA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:30:55Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:31:41Z + + K9me3 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + 0.9989747 + structure_element + cleaner0 + 2023-07-25T14:57:03Z + SO: + + Spacer + + + complex_assembly + GO: + cleaner0 + 2023-07-25T16:03:29Z + + UHRF1–DNMT1 + + + evidence + DUMMY: + cleaner0 + 2023-07-25T14:56:50Z + + hm-DNA-binding affinity + + + 0.99947935 + structure_element + cleaner0 + 2023-07-25T14:57:34Z + SO: + + SRA + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:32:18Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:32:29Z + + K9me3 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:04:02Z + + SRA-Spacer + + + 0.9976597 + chemical + cleaner0 + 2023-07-25T14:58:24Z + CHEBI: + + hm-DNA + + + 0.99921703 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:32:46Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:32:56Z + + K9me3 + + + 0.99476165 + chemical + cleaner0 + 2023-07-25T14:58:24Z + CHEBI: + + hm-DNA + + + 0.9994437 + protein + cleaner0 + 2023-07-25T16:29:03Z + PR: + + URHF1 + + + + ABSTRACT + abstract + 1016 + UHRF1 is involved in the maintenance of DNA methylation, but the regulatory mechanism of this epigenetic regulator is unclear. Here, the authors show that it has a closed conformation and are able to make conclusions about the mechanism of recognition of epigenetic marks. + + 0.9987054 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:57Z + + methylation + + + 0.99924755 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + + INTRO + paragraph + 1290 + DNA methylation is an important epigenetic modification for gene repression, X-chromosome inactivation, genome imprinting and maintenance of genome stability. Mammalian DNA methylation is established by de novo DNA methyltransferases DNMT3A/3B, and DNA methylation patterns are maintained by maintenance DNA methyltransferase 1 (DNMT1) during DNA replication. Ubiquitin-like, containing PHD and RING fingers domains, 1 (UHRF1, also known as ICBP90 and NP95 in mouse) was shown to be essential for maintenance DNA methylation through recruiting DNMT1 to replication forks in S phase of the cell cycle. UHRF1 is essential for S phase entry and is involved in heterochromatin formation. UHRF1 also plays an important role in promoting proliferation and is shown to be upregulated in a number of cancers, suggesting that UHRF1 may serve as a potential drug target for therapeutic applications. + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:57Z + + methylation + + + 0.9980562 + taxonomy_domain + cleaner0 + 2023-07-25T15:00:03Z + DUMMY: + + Mammalian + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:57Z + + methylation + + + 0.9966591 + protein_type + cleaner0 + 2023-07-25T15:00:19Z + MESH: + + DNA methyltransferases + + + 0.9978175 + protein + cleaner0 + 2023-07-25T15:00:24Z + PR: + + DNMT3A/3B + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:57Z + + methylation + + + 0.9750573 + protein + cleaner0 + 2023-07-25T15:00:32Z + PR: + + DNA methyltransferase 1 + + + 0.99899286 + protein + cleaner0 + 2023-07-25T15:00:36Z + PR: + + DNMT1 + + + protein + PR: + cleaner0 + 2023-07-25T14:59:44Z + + Ubiquitin-like, containing PHD and RING fingers domains, 1 + + + 0.999151 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.9991623 + protein + cleaner0 + 2023-07-25T15:00:45Z + PR: + + ICBP90 + + + 0.9990305 + protein + cleaner0 + 2023-07-25T15:00:49Z + PR: + + NP95 + + + 0.99646413 + taxonomy_domain + cleaner0 + 2023-07-25T15:00:55Z + DUMMY: + + mouse + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:57Z + + methylation + + + 0.9990791 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + 0.99863005 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.998461 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.9986771 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + + INTRO + paragraph + 2180 + UHRF1 is a multi-domain containing protein connecting histone modification and DNA methylation. As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD. The SRA preferentially binds to hemi-methylated DNA (hm-DNA). Recent studies show that the SRA directly binds to replication focus targeting sequence (RFTS) of DNMT1 (RFTSDNMT1). A spacer region (Fig. 1a, designated Spacer hereafter) connecting the SRA and the RING is rich in basic residues and predicted to be unstructured for unknown function. Recent study shows that phosphatidylinostiol phosphate PI5P binds to the Spacer and induces a conformational change of UHRF1 to allow the TTD to recognize H3K9me3 (ref.). These studies indicate that UHRF1 connects dynamic regulation of DNA methylation and H3K9me3, which are positively correlated in human genome. However, how UHRF1 regulates the recognition of these two repressive epigenetic marks and recruits DNMT1 for chromatin localization remain largely unknown. + + 0.99919814 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:08Z + + histone + + + chemical + CHEBI: + cleaner0 + 2023-07-25T15:05:41Z + + DNA + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:51Z + + methylation + + + 0.9992995 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.99908644 + structure_element + cleaner0 + 2023-07-25T16:31:03Z + SO: + + ubiquitin-like domain + + + 0.99913794 + structure_element + cleaner0 + 2023-07-25T14:57:12Z + SO: + + tandem Tudor domain + + + 0.999468 + structure_element + cleaner0 + 2023-07-25T14:57:17Z + SO: + + TTD + + + 0.998676 + structure_element + cleaner0 + 2023-07-25T16:31:07Z + SO: + + TTDN + + + 0.9981198 + structure_element + cleaner0 + 2023-07-25T16:31:10Z + SO: + + TTDC + + + 0.9990694 + structure_element + cleaner0 + 2023-07-25T14:57:43Z + SO: + + plant homeodomain + + + 0.99924564 + structure_element + cleaner0 + 2023-07-25T14:57:49Z + SO: + + PHD + + + 0.9987116 + structure_element + cleaner0 + 2023-07-25T14:57:28Z + SO: + + SET-and-RING-associated + + + 0.9961398 + structure_element + cleaner0 + 2023-07-25T14:57:34Z + SO: + + SRA + + + 0.9305976 + structure_element + cleaner0 + 2023-07-25T15:01:22Z + SO: + + really interesting new gene + + + 0.9275266 + structure_element + cleaner0 + 2023-07-25T15:01:26Z + SO: + + RING + + + 0.9994217 + structure_element + cleaner0 + 2023-07-25T14:57:17Z + SO: + + TTD + + + 0.9994911 + structure_element + cleaner0 + 2023-07-25T14:57:49Z + SO: + + PHD + + + 0.9925216 + protein_type + cleaner0 + 2023-07-25T15:05:07Z + MESH: + + histone + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:33:20Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:33:29Z + + K9me3 + + + 0.8370888 + residue_name_number + cleaner0 + 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2023-07-25T15:02:17Z + SO: + + replication focus targeting sequence + + + 0.557462 + structure_element + cleaner0 + 2023-07-25T15:02:43Z + SO: + + RFTS + + + 0.9992735 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + 0.9992048 + protein + cleaner0 + 2023-07-25T16:02:12Z + PR: + + RFTSDNMT1 + + + 0.99836123 + structure_element + cleaner0 + 2023-07-25T15:01:57Z + SO: + + spacer region + + + 0.9973368 + structure_element + cleaner0 + 2023-07-25T14:57:03Z + SO: + + Spacer + + + 0.9995253 + structure_element + cleaner0 + 2023-07-25T14:57:34Z + SO: + + SRA + + + 0.9995208 + structure_element + cleaner0 + 2023-07-25T15:01:28Z + SO: + + RING + + + 0.91302955 + protein_state + cleaner0 + 2023-07-25T16:47:49Z + DUMMY: + + unstructured + + + 0.99880505 + chemical + cleaner0 + 2023-07-25T15:02:53Z + CHEBI: + + phosphatidylinostiol phosphate + + + 0.998345 + chemical + cleaner0 + 2023-07-25T15:03:00Z + CHEBI: + + PI5P + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + 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PR: + + DNMT1 + + + + INTRO + paragraph + 3589 + Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). Upon binding to hm-DNA, UHRF1 impairs the intramolecular interactions and promotes the H3K9me3 recognition by TTD–PHD, which may further enhance its genomic localization. As a result, UHRF1 is locked in the open conformation by the association of H3K9me3 by TTD–PHD, and thus SRA-Spacer either recognizes hm-DNA or recruits DNMT1 for DNA methylation. Therefore, UHRF1 may engage in a sophisticated regulation for its chromatin localization and recruitment of DNMT1 through a mechanism yet to be fully elucidated. Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by UHRF1. + + 0.99936396 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.9992605 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + structure_element + SO: + cleaner0 + 2023-07-25T14:57:03Z + + Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:03Z + + binds to + + + 0.99956805 + structure_element + cleaner0 + 2023-07-25T14:57:17Z + SO: + + TTD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:35:18Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:35:26Z + + K9me3 + + + 0.9994542 + structure_element + cleaner0 + 2023-07-25T14:57:34Z + SO: + + SRA + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.9995419 + structure_element + cleaner0 + 2023-07-25T14:57:49Z + SO: + + PHD + + + 0.79977167 + site + cleaner0 + 2023-07-25T15:03:30Z + SO: + + H3R2 + + + 0.99909306 + protein_state + cleaner0 + 2023-07-25T15:04:47Z + DUMMY: + + unmethylated + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:08Z + + histone + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:25Z + + H3 + + + 0.48252225 + residue_name_number + cleaner0 + 2023-07-25T15:06:49Z + DUMMY: + + R2 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:04:36Z + + binding to + + + 0.9958095 + chemical + cleaner0 + 2023-07-25T14:58:24Z + CHEBI: + + hm-DNA + + + 0.9993137 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:35:40Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:35:49Z + + K9me3 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + 0.9993806 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.9991967 + protein_state + cleaner0 + 2023-07-25T16:47:52Z + DUMMY: + + open + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:36:02Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:36:11Z + + K9me3 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + 0.9654761 + structure_element + cleaner0 + 2023-07-25T15:04:01Z + SO: + + SRA-Spacer + + + 0.99189776 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99909675 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T15:07:18Z + + DNA + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:57Z + + methylation + + + 0.99935514 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.99914134 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:36:27Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:36:35Z + + K9me3 + + + 0.9882726 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99934787 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + + RESULTS + title_1 + 4453 + Results + + + RESULTS + title_2 + 4461 + Hm-DNA facilitates histone H3K9me3 recognition by UHRF1 + + 0.9845609 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + Hm-DNA + + + 0.98075485 + protein_type + cleaner0 + 2023-07-25T15:05:08Z + MESH: + + histone + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:36:54Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:37:04Z + + K9me3 + + + 0.9989887 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + + RESULTS + paragraph + 4517 + To investigate how UHRF1 coordinates the recognition of H3K9me3 and hm-DNA, we purified recombinant UHRF1 (truncations and mutations) proteins from bacteria. We first performed an in vitro pull-down assay using biotinylated histone H3 peptides and hm-DNA (Supplementary Table 1). As shown in Fig. 1b, hm-DNA largely enhanced the interaction between full-length UHRF1 and unmethylated histone H3 (H3K9me0) or H3K9me3 peptide. Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a). In contrast, histone peptides showed no enhancement on the interaction between hm-DNA and UHRF1 (Fig. 1c). These results suggest that hm-DNA facilitates histone recognition by UHRF1. + + 0.9990995 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:37:24Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:37:32Z + + K9me3 + + + 0.9977552 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99919635 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.9987502 + experimental_method + cleaner0 + 2023-07-25T16:39:17Z + MESH: + + in vitro pull-down assay + + + 0.7566109 + protein_state + cleaner0 + 2023-07-25T16:30:09Z + DUMMY: + + biotinylated + + + 0.8355774 + protein_type + cleaner0 + 2023-07-25T15:05:08Z + MESH: + + histone + + + 0.8583309 + protein_type + cleaner0 + 2023-07-25T15:07:50Z + MESH: + + H3 + + + 0.9979067 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.9975527 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.999162 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.99920505 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.99908483 + protein_state + cleaner0 + 2023-07-25T15:04:48Z + DUMMY: + + unmethylated + + + 0.94932944 + protein_type + cleaner0 + 2023-07-25T15:05:08Z + MESH: + + histone + + + 0.8987189 + protein_type + cleaner0 + 2023-07-25T15:07:49Z + MESH: + + H3 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:37:47Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:37:57Z + + K9me0 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:38:09Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:38:18Z + + K9me3 + + + 0.99857134 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.9987547 + chemical + cleaner0 + 2023-07-25T15:08:28Z + CHEBI: + + um-DNA + + + 0.99885714 + protein_state + cleaner0 + 2023-07-25T15:04:48Z + DUMMY: + + unmethylated + + + 0.9731914 + chemical + cleaner0 + 2023-07-25T16:30:14Z + CHEBI: + + DNA + + + 0.99861336 + chemical + cleaner0 + 2023-07-25T15:08:22Z + CHEBI: + + fm-DNA + + + 0.9987992 + protein_state + cleaner0 + 2023-07-25T15:08:14Z + DUMMY: + + fully methylated + + + 0.9773302 + chemical + cleaner0 + 2023-07-25T16:30:18Z + CHEBI: + + DNA + + + 0.9990351 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:08Z + + histone + + + 0.9991819 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.99527615 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:08Z + + histone + + + 0.9965648 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.9990885 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.99166584 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:08Z + + histone + + + 0.99913424 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + + RESULTS + paragraph + 5416 + Our previous studies show that the PHD recognizes H3K9me0 and the TTD and the PHD together (TTD–PHD) coordinately recognize H3K9me3 (refs.). We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). The gel filtration analysis showed that UHRF1 is a monomer in solution (Supplementary Fig. 1b), indicating that the intramolecular (not intermolecular) interaction of UHRF1 regulates histone recognition. These results suggest that UHRF1 adopts an unfavourable conformation for histone H3 tails recognition, in which TTD–PHD might be blocked by other regions of UHRF1, and hm-DNA impairs this intramolecular interaction to facilitate its recognition of histone H3 tails. + + 0.99949586 + structure_element + cleaner0 + 2023-07-25T14:57:49Z + SO: + + PHD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:38:33Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:38:43Z + + K9me0 + + + 0.99951327 + structure_element + cleaner0 + 2023-07-25T14:57:17Z + SO: + + TTD + + + 0.9994549 + structure_element + cleaner0 + 2023-07-25T14:57:49Z + SO: + + PHD + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:38:56Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:39:10Z + + K9me3 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:16:09Z + + isolated + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + 0.99869025 + evidence + cleaner0 + 2023-07-25T15:10:17Z + DUMMY: + + binding affinity + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:39:22Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:39:31Z + + K9me3 + + + 0.9990852 + protein_state + cleaner0 + 2023-07-25T15:08:59Z + DUMMY: + + full-length + + + 0.99931014 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.9994672 + structure_element + cleaner0 + 2023-07-25T14:57:49Z + SO: + + PHD + + + 0.9987298 + evidence + cleaner0 + 2023-07-25T15:10:17Z + DUMMY: + + binding affinity + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:39:44Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:39:53Z + + K9me0 + + + 0.99909943 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.9993193 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.99865484 + experimental_method + cleaner0 + 2023-07-25T16:39:20Z + MESH: + + gel filtration analysis + + + 0.9992993 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.998701 + oligomeric_state + cleaner0 + 2023-07-25T16:39:02Z + DUMMY: + + monomer + + + 0.9992415 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:08Z + + histone + + + 0.99931514 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:08Z + + histone + + + 0.42043397 + protein_type + cleaner0 + 2023-07-25T15:07:50Z + MESH: + + H3 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + 0.9992786 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.9796707 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.69733983 + protein_type + cleaner0 + 2023-07-25T15:05:08Z + MESH: + + histone + + + 0.5546273 + protein_type + cleaner0 + 2023-07-25T15:07:50Z + MESH: + + H3 + + + + RESULTS + title_2 + 6321 + Intramolecular interaction within UHRF1 + + 0.9989747 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + + RESULTS + paragraph + 6361 + To test above hypothesis, we performed glutathione S-transferase (GST) pull-down assay using various truncations of UHRF1. Interestingly, the TTD directly bound to SRA-Spacer but not the SRA, suggesting that the Spacer (residues 587–674) is important for the intramolecular interaction (Fig. 2a). The isothermal titration calorimetry (ITC) measurements show that the TTD bound to the Spacer (but not the SRA) in a 1:1 stoichiometry with a binding affinity (KD) of 1.59 μM (Fig. 2b). The presence of the Spacer markedly impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). The results indicate that the Spacer directly binds to the TTD and inhibits its interaction with H3K9me3. + + experimental_method + MESH: + cleaner0 + 2023-07-25T16:36:22Z + + glutathione S-transferase (GST) pull-down assay + + + 0.8383519 + experimental_method + cleaner0 + 2023-07-25T16:39:24Z + MESH: + + truncations + + + 0.99936134 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.99953187 + structure_element + cleaner0 + 2023-07-25T14:57:17Z + SO: + + TTD + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:10:32Z + + bound to + + + structure_element + SO: + cleaner0 + 2023-07-25T15:04:02Z + + SRA-Spacer + + + 0.9419889 + structure_element + cleaner0 + 2023-07-25T14:57:34Z + SO: + + SRA + + + 0.99881434 + structure_element + cleaner0 + 2023-07-25T14:57:03Z + SO: + + Spacer + + + 0.9978974 + residue_range + cleaner0 + 2023-07-25T16:35:57Z + DUMMY: + + 587–674 + + + 0.99897915 + experimental_method + cleaner0 + 2023-07-25T16:36:28Z + MESH: + + isothermal titration calorimetry + + + 0.9986539 + experimental_method + cleaner0 + 2023-07-25T16:36:31Z + MESH: + + ITC + + + 0.99951494 + structure_element + cleaner0 + 2023-07-25T14:57:17Z + SO: + + TTD + + + 0.9987566 + protein_state + cleaner0 + 2023-07-25T15:10:31Z + DUMMY: + + bound to + + + 0.99883133 + structure_element + cleaner0 + 2023-07-25T14:57:03Z + SO: + + Spacer + + + 0.57471496 + structure_element + cleaner0 + 2023-07-25T14:57:34Z + SO: + + SRA + + + 0.9984056 + evidence + cleaner0 + 2023-07-25T15:10:16Z + DUMMY: + + binding affinity + + + 0.99863535 + evidence + cleaner0 + 2023-07-25T15:10:10Z + DUMMY: + + KD + + + 0.9984523 + protein_state + cleaner0 + 2023-07-25T15:10:24Z + DUMMY: + + presence of + + + 0.9855452 + structure_element + cleaner0 + 2023-07-25T14:57:03Z + SO: + + Spacer + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:20Z + + TTD–PHD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:40:09Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:40:20Z + + K9me3 + + + 0.9973073 + structure_element + cleaner0 + 2023-07-25T14:57:03Z + SO: + + Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.99943787 + structure_element + cleaner0 + 2023-07-25T14:57:17Z + SO: + + TTD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:40:32Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:40:42Z + + K9me3 + + + + RESULTS + paragraph + 7057 + The GST pull-down assay also shows that the PHD bound to the SRA, which was further confirmed by the ITC measurements (KD=26.7 μM; Fig. 2a,d). Compared with the PHD alone, PHD-SRA showed decreased binding affinity to H3K9me0 peptide by a factor of eight (Fig. 2e). Pre-incubation of the SRA also modestly impaired PHD–H3K9me0 interaction. These results indicate that the SRA directly binds to the PHD and inhibits its binding affinity to H3K9me0. Taken together, UHRF1 seems to adopt a closed form through intramolecular interactions (TTD–Spacer and PHD-SRA), which inhibit histone H3 tail recognition by UHRF1. + + 0.9989052 + experimental_method + cleaner0 + 2023-07-25T16:36:39Z + MESH: + + GST pull-down assay + + + 0.99954224 + structure_element + cleaner0 + 2023-07-25T14:57:49Z + SO: + + PHD + + + 0.9989557 + protein_state + cleaner0 + 2023-07-25T15:10:32Z + DUMMY: + + bound to + + + 0.98258084 + structure_element + cleaner0 + 2023-07-25T14:57:34Z + SO: + + SRA + + + 0.9976513 + experimental_method + cleaner0 + 2023-07-25T16:39:30Z + MESH: + + ITC + + + 0.99855214 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.9994531 + structure_element + cleaner0 + 2023-07-25T14:57:49Z + SO: + + PHD + + + 0.9981786 + protein_state + cleaner0 + 2023-07-25T16:47:57Z + DUMMY: + + alone + + + 0.94781685 + structure_element + cleaner0 + 2023-07-25T15:16:50Z + SO: + + PHD-SRA + + + 0.99865395 + evidence + cleaner0 + 2023-07-25T15:10:17Z + DUMMY: + + binding affinity + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:40:54Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:41:03Z + + K9me0 + + + 0.99731904 + experimental_method + cleaner0 + 2023-07-25T16:39:33Z + MESH: + + Pre-incubation + + + 0.48420557 + structure_element + cleaner0 + 2023-07-25T14:57:34Z + SO: + + SRA + + + 0.9934504 + structure_element + cleaner0 + 2023-07-25T14:57:49Z + SO: + + PHD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:41:14Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:41:25Z + + K9me0 + + + 0.9809187 + structure_element + cleaner0 + 2023-07-25T14:57:34Z + SO: + + SRA + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.9994642 + structure_element + cleaner0 + 2023-07-25T14:57:49Z + SO: + + PHD + + + 0.9982419 + evidence + cleaner0 + 2023-07-25T15:10:17Z + DUMMY: + + binding affinity + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:41:40Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:41:49Z + + K9me0 + + + 0.9993193 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + 0.9992454 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:20Z + + TTD–Spacer + + + structure_element + SO: + cleaner0 + 2023-07-25T15:16:51Z + + PHD-SRA + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:08Z + + histone + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:07:50Z + + H3 + + + 0.9993266 + protein + cleaner0 + 2023-07-25T14:58:01Z + PR: + + UHRF1 + + + + RESULTS + title_2 + 7676 + Overall structure of TTD–Spacer + + 0.9973246 + evidence + cleaner0 + 2023-07-25T16:44:33Z + DUMMY: + + structure + + + 0.99570864 + structure_element + cleaner0 + 2023-07-25T15:17:21Z + SO: + + TTD–Spacer + + + + RESULTS + paragraph + 7710 + To investigate the intramolecular interaction within UHRF1, we first mapped the minimal regions within the Spacer for the interaction with the TTD (Supplementary Fig. 2a). Internal deletions of the Spacer, including SpacerΔ660–664, SpacerΔ665–669, SpacerΔ670–674 and Spacer642–674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587–641 showed no detectable interaction. SpacerΔ642–651, SpacerΔ650–654 and SpacerΔ655–659 also decreased binding affinities, indicating that residues 642–674 are important for TTD–Spacer interaction. + + 0.99916494 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.99835926 + structure_element + cleaner0 + 2023-07-25T14:57:03Z + SO: + + Spacer + + + 0.9993787 + structure_element + cleaner0 + 2023-07-25T14:57:17Z + SO: + + TTD + + + 0.9698804 + experimental_method + cleaner0 + 2023-07-25T16:39:36Z + MESH: + + deletions + + + 0.99842167 + structure_element + cleaner0 + 2023-07-25T14:57:03Z + SO: + + Spacer + + + mutant + MESH: + cleaner0 + 2023-07-25T15:18:22Z + + SpacerΔ660–664 + + + mutant + MESH: + cleaner0 + 2023-07-25T15:18:42Z + + SpacerΔ665–669 + + + mutant + MESH: + cleaner0 + 2023-07-25T15:19:03Z + + SpacerΔ670–674 + + + mutant + MESH: + cleaner0 + 2023-07-25T15:19:23Z + + Spacer642–674 + + + 0.99854124 + protein_state + cleaner0 + 2023-07-25T15:10:32Z + DUMMY: + + bound to + + + 0.99894804 + structure_element + cleaner0 + 2023-07-25T14:57:17Z + SO: + + TTD + + + 0.9986196 + evidence + cleaner0 + 2023-07-25T16:44:38Z + DUMMY: + + binding affinities + + + 0.99589765 + structure_element + cleaner0 + 2023-07-25T14:57:03Z + SO: + + Spacer + + + mutant + MESH: + cleaner0 + 2023-07-25T15:21:58Z + + Spacer587–641 + + + mutant + MESH: + cleaner0 + 2023-07-25T15:19:44Z + + SpacerΔ642–651 + + + mutant + MESH: + cleaner0 + 2023-07-25T15:20:05Z + + SpacerΔ650–654 + + + mutant + MESH: + cleaner0 + 2023-07-25T15:20:29Z + + SpacerΔ655–659 + + + 0.9985037 + evidence + cleaner0 + 2023-07-25T16:44:42Z + DUMMY: + + binding affinities + + + 0.99734336 + residue_range + cleaner0 + 2023-07-25T15:20:40Z + DUMMY: + + 642–674 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + + RESULTS + paragraph + 8309 + We next determined the solution structure of the TTD (residues 134–285) bound to Spacer627–674 by conventional NMR techniques (Supplementary Table 3 and Supplementary Fig. 3a,b). In the complex structure, each Tudor domain adopts a ‘Royal' fold containing a characteristic five-stranded β-sheet and the two Tudor domains tightly pack against each other with a buried area of 573 Å2 (Fig. 3a). The TTD adopts similar fold to that in TTD–PHD–H3K9me3 complex structure (PDB: 4GY5) with a root-mean-square deviation of 1.09 Å for 128 Cα atoms, indicating that the Spacer does not result in obvious conformational change of the TTD (Fig. 3b). The Spacer (residues 643–655 were built in the model) adopts an extended conformation and binds to an acidic groove on the TTD (Fig. 3c). + + 0.9957992 + evidence + cleaner0 + 2023-07-25T16:44:45Z + DUMMY: + + solution structure + + + 0.9992454 + structure_element + cleaner0 + 2023-07-25T14:57:17Z + SO: + + TTD + + + 0.99801356 + residue_range + cleaner0 + 2023-07-25T15:20:43Z + DUMMY: + + 134–285 + + + 0.99913573 + protein_state + cleaner0 + 2023-07-25T15:10:32Z + DUMMY: + + bound to + + + 0.98736215 + residue_range + cleaner0 + 2023-07-25T15:24:25Z + DUMMY: + + Spacer627–674 + + + 0.9983102 + experimental_method + cleaner0 + 2023-07-25T16:39:42Z + MESH: + + NMR + + + 0.9735048 + evidence + cleaner0 + 2023-07-25T16:44:48Z + DUMMY: + + complex structure + + + 0.9985361 + structure_element + cleaner0 + 2023-07-25T16:31:14Z + SO: + + Tudor domain + + + structure_element + SO: + cleaner0 + 2023-07-25T16:31:40Z + + ‘Royal' fold + + + 0.9976389 + structure_element + cleaner0 + 2023-07-25T16:31:45Z + SO: + + five-stranded β-sheet + + + 0.99847436 + structure_element + cleaner0 + 2023-07-25T16:31:48Z + SO: + + Tudor domains + + + 0.99931407 + structure_element + cleaner0 + 2023-07-25T14:57:17Z + SO: + + TTD + + + 0.99921435 + complex_assembly + cleaner0 + 2023-07-25T15:22:28Z + GO: + + TTD–PHD–H3K9me3 + + + 0.7332863 + evidence + cleaner0 + 2023-07-25T16:44:50Z + DUMMY: + + structure + + + 0.9981439 + evidence + cleaner0 + 2023-07-25T16:44:53Z + DUMMY: + + root-mean-square deviation + + + 0.9938008 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.99923444 + structure_element + cleaner0 + 2023-07-25T14:57:17Z + SO: + + TTD + + + 0.9991209 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9979308 + residue_range + cleaner0 + 2023-07-25T15:24:36Z + DUMMY: + + 643–655 + + + 0.98440146 + protein_state + cleaner0 + 2023-07-25T16:48:08Z + DUMMY: + + extended conformation + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.9988172 + site + cleaner0 + 2023-07-25T16:51:32Z + SO: + + acidic groove + + + 0.9993272 + structure_element + cleaner0 + 2023-07-25T14:57:18Z + SO: + + TTD + + + + RESULTS + paragraph + 9105 + The TTD–Spacer interaction is mediated by a number of hydrogen bonds (Fig. 3d). The side chain of residue K648 forms hydrogen bonds with the carbonyl oxygen atom of D189 and side chain of D190 of the TTD. The side chain of residue R649 packs against an acidic surface mainly formed by residues D142 and E153. Residue S651 forms hydrogen bonds with the main chain of residues G236 and W238. The interaction is further supported by hydrogen bonds formed between residues K650, A652, G653 and G654 of the Spacer and residues N228, G236 and W238 of the TTD, respectively. + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + 0.99648 + bond_interaction + cleaner0 + 2023-07-25T15:24:58Z + MESH: + + hydrogen bonds + + + 0.9994708 + residue_name_number + cleaner0 + 2023-07-25T15:25:15Z + DUMMY: + + K648 + + + 0.99632293 + bond_interaction + cleaner0 + 2023-07-25T15:24:59Z + MESH: + + hydrogen bonds + + + 0.99950325 + residue_name_number + cleaner0 + 2023-07-25T15:25:05Z + DUMMY: + + D189 + + + 0.9995196 + residue_name_number + cleaner0 + 2023-07-25T15:25:10Z + DUMMY: + + D190 + + + 0.9994437 + structure_element + cleaner0 + 2023-07-25T14:57:18Z + SO: + + TTD + + + 0.99953413 + residue_name_number + cleaner0 + 2023-07-25T15:25:20Z + DUMMY: + + R649 + + + bond_interaction + MESH: + cleaner0 + 2023-07-25T15:25:44Z + + packs against + + + 0.9995499 + residue_name_number + cleaner0 + 2023-07-25T15:25:49Z + DUMMY: + + D142 + + + 0.9995509 + residue_name_number + cleaner0 + 2023-07-25T15:25:53Z + DUMMY: + + E153 + + + 0.9995384 + residue_name_number + cleaner0 + 2023-07-25T15:25:59Z + DUMMY: + + S651 + + + 0.9962274 + bond_interaction + cleaner0 + 2023-07-25T15:24:59Z + MESH: + + hydrogen bonds + + + 0.99953425 + residue_name_number + cleaner0 + 2023-07-25T15:26:05Z + DUMMY: + + G236 + + + 0.9995196 + residue_name_number + cleaner0 + 2023-07-25T15:26:11Z + DUMMY: + + W238 + + + 0.99586034 + bond_interaction + cleaner0 + 2023-07-25T15:24:59Z + MESH: + + hydrogen bonds + + + 0.99951375 + residue_name_number + cleaner0 + 2023-07-25T15:26:17Z + DUMMY: + + K650 + + + 0.99952185 + residue_name_number + cleaner0 + 2023-07-25T15:26:21Z + DUMMY: + + A652 + + + 0.99952614 + residue_name_number + cleaner0 + 2023-07-25T15:26:27Z + DUMMY: + + G653 + + + 0.9995352 + residue_name_number + cleaner0 + 2023-07-25T15:26:31Z + DUMMY: + + G654 + + + 0.9989109 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9995413 + residue_name_number + cleaner0 + 2023-07-25T15:26:36Z + DUMMY: + + N228 + + + 0.9995378 + residue_name_number + cleaner0 + 2023-07-25T15:26:06Z + DUMMY: + + G236 + + + 0.999521 + residue_name_number + cleaner0 + 2023-07-25T15:26:12Z + DUMMY: + + W238 + + + 0.99935097 + structure_element + cleaner0 + 2023-07-25T14:57:18Z + SO: + + TTD + + + + RESULTS + paragraph + 9675 + In support of above structural analyses, mutation D142A/E153A of the TTD abolished its interaction with the Spacer (Fig. 3e). Mutations K648D and S651D of the Spacer decreased their binding affinities to the TTD, and mutation R649A of the Spacer showed more significant decrease (∼13-fold) in the binding affinity (Fig. 3f). As negative control, mutations S639D and S666D of the Spacer showed little effect on the interaction. Interestingly, phosphorylation at residue S651 of UHRF1 was observed in previous mass-spectrometry analyses. Compared with the unmodified peptide of Spacer642–664, a phosphorylation at S651 markedly decreased the binding affinity to the TTD (Supplementary Fig. 2b), suggesting that the phosphorylation may regulate the intramolecular interaction within UHRF1. + + 0.99829507 + experimental_method + cleaner0 + 2023-07-25T16:39:46Z + MESH: + + structural analyses + + + 0.99609274 + experimental_method + cleaner0 + 2023-07-25T16:39:49Z + MESH: + + mutation + + + 0.99899954 + mutant + cleaner0 + 2023-07-25T15:26:52Z + MESH: + + D142A + + + 0.9990146 + mutant + cleaner0 + 2023-07-25T15:26:57Z + MESH: + + E153A + + + 0.9994748 + structure_element + cleaner0 + 2023-07-25T14:57:18Z + SO: + + TTD + + + 0.9990018 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.99218035 + experimental_method + cleaner0 + 2023-07-25T16:39:52Z + MESH: + + Mutations + + + 0.99906534 + mutant + cleaner0 + 2023-07-25T15:27:03Z + MESH: + + K648D + + + 0.9990694 + mutant + cleaner0 + 2023-07-25T15:27:08Z + MESH: + + S651D + + + 0.9989484 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9987575 + evidence + cleaner0 + 2023-07-25T16:44:57Z + DUMMY: + + binding affinities + + + 0.9994055 + structure_element + cleaner0 + 2023-07-25T14:57:18Z + SO: + + TTD + + + 0.99432313 + experimental_method + cleaner0 + 2023-07-25T16:39:55Z + MESH: + + mutation + + + 0.99906355 + mutant + cleaner0 + 2023-07-25T15:27:14Z + MESH: + + R649A + + + 0.9989303 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9987483 + evidence + cleaner0 + 2023-07-25T15:10:17Z + DUMMY: + + binding affinity + + + 0.98485273 + experimental_method + cleaner0 + 2023-07-25T16:39:57Z + MESH: + + mutations + + + 0.9991134 + mutant + cleaner0 + 2023-07-25T15:27:20Z + MESH: + + S639D + + + 0.99907804 + mutant + cleaner0 + 2023-07-25T15:27:26Z + MESH: + + S666D + + + 0.9987979 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.99615014 + ptm + cleaner0 + 2023-07-25T15:27:38Z + MESH: + + phosphorylation + + + 0.9994925 + residue_name_number + cleaner0 + 2023-07-25T15:25:59Z + DUMMY: + + S651 + + + 0.9993629 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.9987564 + experimental_method + cleaner0 + 2023-07-25T16:39:59Z + MESH: + + mass-spectrometry + + + 0.99917954 + protein_state + cleaner0 + 2023-07-25T15:27:58Z + DUMMY: + + unmodified + + + 0.96082616 + mutant + cleaner0 + 2023-07-25T16:38:03Z + MESH: + + Spacer642–664 + + + 0.99539644 + ptm + cleaner0 + 2023-07-25T15:27:39Z + MESH: + + phosphorylation + + + 0.99948895 + residue_name_number + cleaner0 + 2023-07-25T15:26:00Z + DUMMY: + + S651 + + + 0.9987745 + evidence + cleaner0 + 2023-07-25T15:10:17Z + DUMMY: + + binding affinity + + + 0.9994197 + structure_element + cleaner0 + 2023-07-25T14:57:18Z + SO: + + TTD + + + 0.9951279 + ptm + cleaner0 + 2023-07-25T15:27:39Z + MESH: + + phosphorylation + + + 0.9993599 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + + RESULTS + title_2 + 10466 + The spacer binds to the TTD by competing with the linker + + 0.99911445 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.99956805 + structure_element + cleaner0 + 2023-07-25T14:57:18Z + SO: + + TTD + + + 0.999511 + structure_element + cleaner0 + 2023-07-25T15:34:28Z + SO: + + linker + + + + RESULTS + paragraph + 10523 + Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286–306, designated Linker, Fig. 1a), and TTD–Linker interaction is essential for H3K9me3 recognition by TTD–PHD. Comparison of TTD–Spacer and TTD–PHD–H3K9me3 (PDB: 4GY5) structures indicates that the Spacer and the Linker bind to the TTD in a similar manner in the two complexes (Fig. 3b). In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. Similar intramolecular contacts (TTD–Linker and TTD–Spacer) were observed in the two structures (Fig. 3b,d and Supplementary Fig. 4a). Thus, the Spacer may disrupt the TTD–Linker interaction and inhibits the recognition of H3K9me3 by TTD–PHD. + + 0.99946636 + structure_element + cleaner0 + 2023-07-25T14:57:18Z + SO: + + TTD + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.96558005 + structure_element + cleaner0 + 2023-07-25T16:34:32Z + SO: + + linker region + + + 0.99939203 + structure_element + cleaner0 + 2023-07-25T14:57:18Z + SO: + + TTD + + + 0.9994456 + structure_element + cleaner0 + 2023-07-25T14:57:49Z + SO: + + PHD + + + 0.9974093 + residue_range + cleaner0 + 2023-07-25T16:38:11Z + DUMMY: + + 286–306 + + + 0.9985744 + structure_element + cleaner0 + 2023-07-25T15:34:28Z + SO: + + Linker + + + structure_element + SO: + cleaner0 + 2023-07-25T15:38:48Z + + TTD–Linker + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:42:09Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:42:19Z + + K9me3 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + 0.68116075 + experimental_method + cleaner0 + 2023-07-25T16:40:06Z + MESH: + + Comparison + + + 0.9833022 + structure_element + cleaner0 + 2023-07-25T15:17:21Z + SO: + + TTD–Spacer + + + 0.99882823 + complex_assembly + cleaner0 + 2023-07-25T15:22:29Z + GO: + + TTD–PHD–H3K9me3 + + + 0.9967913 + evidence + cleaner0 + 2023-07-25T16:45:05Z + DUMMY: + + structures + + + 0.9982766 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.99900836 + structure_element + cleaner0 + 2023-07-25T15:34:28Z + SO: + + Linker + + + 0.999508 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + 0.9989344 + complex_assembly + cleaner0 + 2023-07-25T15:22:29Z + GO: + + TTD–PHD–H3K9me3 + + + 0.9972197 + evidence + cleaner0 + 2023-07-25T16:45:08Z + DUMMY: + + structure + + + 0.99949956 + residue_name_number + cleaner0 + 2023-07-25T15:29:27Z + DUMMY: + + R295 + + + 0.99949014 + residue_name_number + cleaner0 + 2023-07-25T15:29:32Z + DUMMY: + + R296 + + + 0.9995214 + residue_name_number + cleaner0 + 2023-07-25T15:29:38Z + DUMMY: + + S298 + + + 0.998787 + structure_element + cleaner0 + 2023-07-25T15:34:29Z + SO: + + Linker + + + 0.9995097 + residue_name_number + cleaner0 + 2023-07-25T15:25:16Z + DUMMY: + + K648 + + + 0.9995159 + residue_name_number + cleaner0 + 2023-07-25T15:25:21Z + DUMMY: + + R649 + + + 0.9995147 + residue_name_number + cleaner0 + 2023-07-25T15:26:00Z + DUMMY: + + S651 + + + 0.99832743 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.98054665 + structure_element + cleaner0 + 2023-07-25T15:17:21Z + SO: + + TTD–Spacer + + + 0.9964238 + evidence + cleaner0 + 2023-07-25T16:45:10Z + DUMMY: + + structure + + + structure_element + SO: + cleaner0 + 2023-07-25T15:38:48Z + + TTD–Linker + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + 0.998024 + evidence + cleaner0 + 2023-07-25T16:45:12Z + DUMMY: + + structures + + + 0.9961163 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + structure_element + SO: + cleaner0 + 2023-07-25T15:38:48Z + + TTD–Linker + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:42:32Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:42:41Z + + K9me3 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + + RESULTS + paragraph + 11378 + To test this hypothesis, we first investigated the potential competition between the Linker and the Spacer for their interaction with the TTD. The ITC experiment shows that the Linker peptide (289–306) bound to the TTD with a binding affinity of 24.04 μM (Supplementary Fig. 4b), ∼15-fold lower than that of the Spacer peptide (KD=1.59 μM, Fig. 3e). The competitive ITC experiments show that TTD–Spacer binding affinity decreased by a factor of two in the presence of the Linker, whereas TTD–Linker interaction was abolished in the presence of the Spacer (Supplementary Fig. 4c). Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. Notably, although the Linker (in the context of TTD-PHD) impairs the TTD–Spacer interaction to some extent, the isolated Spacer could still bind to TTD–PHD with moderate binding affinity (KD=10.68 μM), supporting the existence of the intramolecular interaction within UHRF1. + + 0.99596643 + structure_element + cleaner0 + 2023-07-25T15:34:29Z + SO: + + Linker + + + 0.9945722 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.99871874 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + 0.99860686 + experimental_method + cleaner0 + 2023-07-25T16:40:11Z + MESH: + + ITC + + + 0.71808165 + structure_element + cleaner0 + 2023-07-25T15:34:29Z + SO: + + Linker + + + 0.99674845 + residue_range + cleaner0 + 2023-07-25T16:38:16Z + DUMMY: + + 289–306 + + + 0.9987063 + protein_state + cleaner0 + 2023-07-25T15:10:32Z + DUMMY: + + bound to + + + 0.9986609 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + 0.99879277 + evidence + cleaner0 + 2023-07-25T15:10:17Z + DUMMY: + + binding affinity + + + 0.64477235 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9950499 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.9985807 + experimental_method + cleaner0 + 2023-07-25T16:40:14Z + MESH: + + competitive ITC + + + evidence + DUMMY: + cleaner0 + 2023-07-25T15:30:27Z + + TTD–Spacer binding affinity + + + 0.9988144 + protein_state + cleaner0 + 2023-07-25T15:10:25Z + DUMMY: + + presence of + + + 0.9507958 + structure_element + cleaner0 + 2023-07-25T15:34:29Z + SO: + + Linker + + + structure_element + SO: + cleaner0 + 2023-07-25T15:38:48Z + + TTD–Linker + + + 0.9985734 + protein_state + cleaner0 + 2023-07-25T15:10:25Z + DUMMY: + + presence of + + + 0.941291 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + 0.9873067 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.9745458 + structure_element + cleaner0 + 2023-07-25T15:11:21Z + SO: + + TTD–PHD + + + 0.9986774 + evidence + cleaner0 + 2023-07-25T15:10:17Z + DUMMY: + + binding affinity + + + 0.9826287 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9945904 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.9967871 + experimental_method + cleaner0 + 2023-07-25T16:40:17Z + MESH: + + mutation + + + 0.9991032 + mutant + cleaner0 + 2023-07-25T15:37:56Z + MESH: + + R295D + + + 0.9991129 + mutant + cleaner0 + 2023-07-25T15:38:01Z + MESH: + + R296D + + + 0.99915695 + structure_element + cleaner0 + 2023-07-25T15:34:29Z + SO: + + Linker + + + structure_element + SO: + cleaner0 + 2023-07-25T15:38:48Z + + TTD–Linker + + + 0.98462725 + structure_element + cleaner0 + 2023-07-25T15:11:21Z + SO: + + TTD–PHD + + + 0.99872434 + evidence + cleaner0 + 2023-07-25T15:10:17Z + DUMMY: + + binding affinity + + + 0.9955218 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.95129967 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.99688077 + structure_element + cleaner0 + 2023-07-25T15:34:29Z + SO: + + Linker + + + 0.989062 + site + cleaner0 + 2023-07-25T16:51:36Z + SO: + + binding site + + + 0.9976774 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + 0.9987902 + structure_element + cleaner0 + 2023-07-25T15:34:29Z + SO: + + Linker + + + 0.9926465 + structure_element + cleaner0 + 2023-07-25T15:31:29Z + SO: + + TTD-PHD + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + 0.9983601 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.98772246 + structure_element + cleaner0 + 2023-07-25T15:11:21Z + SO: + + TTD–PHD + + + 0.9986695 + evidence + cleaner0 + 2023-07-25T15:10:17Z + DUMMY: + + binding affinity + + + 0.9967951 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.99931896 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + + RESULTS + paragraph + 12656 + To test whether TTD–Spacer association exists in the context of full-length UHRF1, we used various truncations of UHRF1 in the GST pull-down assay. As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. Moreover, GST-Linker showed very weak if not undetectable interaction with wild-type or deletions of UHRF1, suggesting that TTD–Linker interaction is much weaker than that of TTD–Spacer. Taken together, UHRF1 adopts a closed conformation, in which the Spacer binds to the TTD through competing with the Linker, and therefore inhibits H3K9me3 recognition by UHRF1. + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + 0.99904275 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.99914956 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.56910294 + experimental_method + cleaner0 + 2023-07-25T16:40:20Z + MESH: + + truncations + + + 0.9991579 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.9987602 + experimental_method + cleaner0 + 2023-07-25T16:40:24Z + MESH: + + GST pull-down assay + + + 0.99908614 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.9987546 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.9988024 + mutant + cleaner0 + 2023-07-25T15:32:20Z + MESH: + + UHRF1ΔSRA + + + 0.9920395 + protein_state + cleaner0 + 2023-07-25T16:48:12Z + DUMMY: + + GST-tagged + + + 0.99950266 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + 0.99916816 + structure_element + cleaner0 + 2023-07-25T15:34:28Z + SO: + + Linker + + + 0.9989662 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T16:48:55Z + + in-cis + + + 0.9990645 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.9991033 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.9989047 + mutant + cleaner0 + 2023-07-25T15:32:21Z + MESH: + + UHRF1ΔSRA + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T16:48:36Z + + in-trans + + + 0.99888223 + mutant + cleaner0 + 2023-07-25T15:32:49Z + MESH: + + UHRF1ΔTTD + + + 0.9990232 + protein_state + cleaner0 + 2023-07-25T15:10:33Z + DUMMY: + + bound to + + + 0.6337208 + experimental_method + cleaner0 + 2023-07-25T15:33:05Z + MESH: + + GST + + + 0.9788821 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + mutant + MESH: + cleaner0 + 2023-07-25T15:33:25Z + + UHRF1Δ627–674 + + + 0.99905694 + protein_state + cleaner0 + 2023-07-25T15:10:33Z + DUMMY: + + bound to + + + 0.72377217 + experimental_method + cleaner0 + 2023-07-25T15:33:34Z + MESH: + + GST + + + 0.76302904 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T16:48:54Z + + in-cis + + + 0.87800574 + structure_element + cleaner0 + 2023-07-25T15:17:21Z + SO: + + TTD–Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T16:48:37Z + + in-trans + + + 0.99933016 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.99834144 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9991 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.9991836 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.6764868 + experimental_method + cleaner0 + 2023-07-25T15:33:34Z + MESH: + + GST + + + 0.6613762 + structure_element + cleaner0 + 2023-07-25T15:34:29Z + SO: + + Linker + + + 0.9990788 + protein_state + cleaner0 + 2023-07-25T15:34:10Z + DUMMY: + + wild-type + + + 0.9992009 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:38:48Z + + TTD–Linker + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + 0.99930525 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.9992095 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + 0.9988832 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.99944085 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + 0.9992781 + structure_element + cleaner0 + 2023-07-25T15:34:29Z + SO: + + Linker + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T13:01:16Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T13:01:24Z + + K9me3 + + + 0.99924016 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + + RESULTS + title_2 + 13695 + The spacer inhibits H3K9me3 recognition by the isolated TTD + + 0.9306218 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + spacer + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:43:00Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:43:10Z + + K9me3 + + + 0.99938285 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + + RESULTS + paragraph + 13755 + Our previous study indicates that H3K9me3 binds to the TTD in different manner in TTD–PHD–H3K9me3 (ref.) and TTD-H3K9me3 (PDB: 2L3R) structures. Because the TTD is always associated with the PHD, whether the pattern of TTD–H3K9me3 interaction exists in vivo remains unknown. Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. As shown in Supplementary Fig. 4e, the Spacer inhibited TTD–H3K9me3 interaction, whereas its TTD-binding defective mutants of the Spacer or the SRA (a negative control) markedly decreased the inhibition. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:43:23Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:43:38Z + + K9me3 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.9995209 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + 0.99807155 + complex_assembly + cleaner0 + 2023-07-25T15:22:29Z + GO: + + TTD–PHD–H3K9me3 + + + 0.99896175 + complex_assembly + cleaner0 + 2023-07-25T15:35:15Z + GO: + + TTD-H3K9me3 + + + 0.9970138 + evidence + cleaner0 + 2023-07-25T16:45:20Z + DUMMY: + + structures + + + 0.99952006 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + 0.99954337 + structure_element + cleaner0 + 2023-07-25T14:57:49Z + SO: + + PHD + + + complex_assembly + GO: + cleaner0 + 2023-07-25T15:35:48Z + + TTD–H3K9me3 + + + 0.66193366 + experimental_method + cleaner0 + 2023-07-25T16:40:31Z + MESH: + + comparison + + + 0.9964395 + complex_assembly + cleaner0 + 2023-07-25T15:35:47Z + GO: + + TTD–H3K9me3 + + + 0.998569 + structure_element + cleaner0 + 2023-07-25T15:17:21Z + SO: + + TTD–Spacer + + + 0.99715745 + evidence + cleaner0 + 2023-07-25T16:45:22Z + DUMMY: + + structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:43:52Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:44:00Z + + K9me3 + + + 0.9979061 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.99949706 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + 0.99820197 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:44:19Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:44:28Z + + K9me3 + + + 0.9992218 + structure_element + cleaner0 + 2023-07-25T14:57:19Z + SO: + + TTD + + + 0.9980301 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + complex_assembly + GO: + cleaner0 + 2023-07-25T15:35:48Z + + TTD–H3K9me3 + + + 0.99777913 + protein_state + cleaner0 + 2023-07-25T15:36:16Z + DUMMY: + + TTD-binding defective + + + 0.92626643 + protein_state + cleaner0 + 2023-07-25T16:00:01Z + DUMMY: + + mutants + + + 0.9984737 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.554281 + structure_element + cleaner0 + 2023-07-25T14:57:34Z + SO: + + SRA + + + + RESULTS + paragraph + 14493 + We next tested whether such inhibition also occurs in the context of full-length UHRF1. Compared with full-length UHRF1, UHRF1Δ627–674 enhanced H3K9me3-binding affinity by a factor of four (Supplementary Fig. 4f). The restoration of H3K9me3-binding affinity is not dramatic because the PHD still binds to histone H3 in both proteins. To exclude this effect, we performed the assay using UHRF1D334A, which abolishes H3R2-binding affinity of the PHD. UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&Δ627–674 dramatically restored its H3K9me3-binding affinity (KD=8.69 μM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD. Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. + + 0.99906915 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.99927145 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.9990279 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.99917525 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.8828556 + mutant + cleaner0 + 2023-07-25T15:33:26Z + MESH: + + UHRF1Δ627–674 + + + 0.9981193 + evidence + cleaner0 + 2023-07-25T15:36:48Z + DUMMY: + + H3K9me3-binding affinity + + + 0.9981266 + evidence + cleaner0 + 2023-07-25T15:36:49Z + DUMMY: + + H3K9me3-binding affinity + + + 0.99945194 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.95636517 + protein_type + cleaner0 + 2023-07-25T15:05:08Z + MESH: + + histone + + + 0.80781966 + protein_type + cleaner0 + 2023-07-25T15:07:50Z + MESH: + + H3 + + + 0.9991373 + mutant + cleaner0 + 2023-07-25T15:37:36Z + MESH: + + UHRF1D334A + + + 0.6050428 + protein_state + cleaner0 + 2023-07-25T16:49:05Z + DUMMY: + + abolishes + + + 0.9379358 + evidence + cleaner0 + 2023-07-25T15:37:28Z + DUMMY: + + H3R2-binding affinity + + + 0.9994092 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.9990056 + mutant + cleaner0 + 2023-07-25T15:37:37Z + MESH: + + UHRF1D334A + + + 0.9981046 + evidence + cleaner0 + 2023-07-25T15:36:49Z + DUMMY: + + H3K9me3-binding affinity + + + 0.99837047 + mutant + cleaner0 + 2023-07-25T15:37:37Z + MESH: + + UHRF1D334A + + + 0.95115536 + mutant + cleaner0 + 2023-07-25T16:35:23Z + MESH: + + Δ627–674 + + + 0.9983492 + evidence + cleaner0 + 2023-07-25T15:36:49Z + DUMMY: + + H3K9me3-binding affinity + + + 0.99851674 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:44:46Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:44:54Z + + K9me3 + + + 0.999483 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.9982369 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9995005 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.99909306 + mutant + cleaner0 + 2023-07-25T15:37:56Z + MESH: + + R295D + + + 0.99889565 + mutant + cleaner0 + 2023-07-25T15:38:01Z + MESH: + + R296D + + + 0.9992405 + protein_state + cleaner0 + 2023-07-25T16:00:50Z + DUMMY: + + mutant + + + 0.99901754 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.9991984 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.9981595 + evidence + cleaner0 + 2023-07-25T15:10:17Z + DUMMY: + + binding affinity + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:45:16Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:45:10Z + + K9me3 + + + 0.99894714 + protein_state + cleaner0 + 2023-07-25T15:59:54Z + DUMMY: + + wild type + + + 0.9975069 + experimental_method + cleaner0 + 2023-07-25T16:40:37Z + MESH: + + mutation + + + 0.9991567 + mutant + cleaner0 + 2023-07-25T15:37:56Z + MESH: + + R295D + + + 0.9989969 + mutant + cleaner0 + 2023-07-25T15:38:01Z + MESH: + + R296D + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + 0.99926156 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.99921596 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + 0.9969368 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.9995086 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:45:35Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:45:44Z + + K9me3 + + + 0.99934036 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:38:48Z + + TTD–Linker + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:45:56Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:46:05Z + + K9me3 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:46:17Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:46:26Z + + K9me3 + + + 0.9995049 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + + RESULTS + title_2 + 15793 + TTD–PHD–H3K9me3 complex inhibits TTD–spacer interaction + + 0.99889505 + complex_assembly + cleaner0 + 2023-07-25T15:22:29Z + GO: + + TTD–PHD–H3K9me3 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–spacer + + + + RESULTS + paragraph + 15855 + Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). The results are also consistent with the previous observation that the interaction between TTD–PHD and the Spacer is much weaker (KD=10.68 μM, Fig. 3g) than that between TTD–PHD and H3K9me3 (KD=0.15 μM, Fig. 1d). These results suggest that once TTD–PHD binds to H3K9me3, UHRF1 will be locked by H3K9me3 and the Spacer is unlikely to fold back for the intramolecular interaction. + + 0.9957268 + experimental_method + cleaner0 + 2023-07-25T16:40:41Z + MESH: + + pre-incubation + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:46:39Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:46:48Z + + K9me3 + + + 0.9989543 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.99953127 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.9454766 + protein_state + cleaner0 + 2023-07-25T16:49:08Z + DUMMY: + + alone + + + 0.75461227 + structure_element + cleaner0 + 2023-07-25T15:11:21Z + SO: + + TTD–PHD + + + 0.77001154 + protein_state + cleaner0 + 2023-07-25T15:10:25Z + DUMMY: + + presence of + + + 0.99875605 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9114364 + structure_element + cleaner0 + 2023-07-25T15:11:21Z + SO: + + TTD–PHD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:47:02Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:47:17Z + + K9me3 + + + 0.9728093 + structure_element + cleaner0 + 2023-07-25T15:11:21Z + SO: + + TTD–PHD + + + 0.9988933 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9838965 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.80197483 + structure_element + cleaner0 + 2023-07-25T15:11:21Z + SO: + + TTD–PHD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:47:31Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:47:41Z + + K9me3 + + + 0.98408234 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.98543686 + structure_element + cleaner0 + 2023-07-25T15:11:21Z + SO: + + TTD–PHD + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:48:16Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:48:25Z + + K9me3 + + + 0.99848336 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:47:55Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:48:04Z + + K9me3 + + + 0.9985475 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + + RESULTS + title_2 + 16516 + Hm-DNA disrupts intramolecular interaction within UHRF1 + + 0.99885005 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + Hm-DNA + + + 0.9987213 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + + RESULTS + paragraph + 16572 + To investigate whether hm-DNA could open the closed conformation of UHRF1, we first measured the intramolecular interaction using UHRF1 truncations in the presence or absence of hm-DNA. The GST pull-down assays show that the PHD bound to the SRA and such interaction was impaired by the addition of hm-DNA (Fig. 4a). H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1ΔTTD and UHRF1ΔSpacer (Fig. 4b). The result indicates that hm-DNA disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. + + 0.9984625 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99766964 + protein_state + cleaner0 + 2023-07-25T16:49:12Z + DUMMY: + + open + + + 0.9992649 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + 0.9991647 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.9961184 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.28094894 + experimental_method + cleaner0 + 2023-07-25T16:40:48Z + MESH: + + truncations + + + 0.95577383 + protein_state + cleaner0 + 2023-07-25T16:49:16Z + DUMMY: + + presence + + + 0.99804974 + protein_state + cleaner0 + 2023-07-25T15:50:14Z + DUMMY: + + absence of + + + 0.99866885 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.9987156 + experimental_method + cleaner0 + 2023-07-25T15:44:48Z + MESH: + + GST pull-down assays + + + 0.9994842 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.9988121 + protein_state + cleaner0 + 2023-07-25T15:10:33Z + DUMMY: + + bound to + + + 0.99894243 + structure_element + cleaner0 + 2023-07-25T14:57:34Z + SO: + + SRA + + + 0.99869126 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99809307 + experimental_method + cleaner0 + 2023-07-25T15:44:56Z + MESH: + + H3 peptide pull-down assays + + + 0.998354 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99781346 + evidence + cleaner0 + 2023-07-25T15:45:08Z + DUMMY: + + H3K9me0-binding affinities + + + 0.9794041 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.385163 + experimental_method + cleaner0 + 2023-07-25T16:35:37Z + MESH: + + truncations + + + structure_element + SO: + cleaner0 + 2023-07-25T15:16:51Z + + PHD-SRA + + + structure_element + SO: + cleaner0 + 2023-07-25T15:16:51Z + + PHD-SRA + + + structure_element + SO: + cleaner0 + 2023-07-25T15:46:46Z + + TTD-PHD-SRA + + + structure_element + SO: + cleaner0 + 2023-07-25T15:47:05Z + + TTD-PHD-SRA-Spacer + + + 0.99860424 + mutant + cleaner0 + 2023-07-25T15:32:50Z + MESH: + + UHRF1ΔTTD + + + 0.9987993 + mutant + cleaner0 + 2023-07-25T15:47:15Z + MESH: + + UHRF1ΔSpacer + + + 0.99783105 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + structure_element + SO: + cleaner0 + 2023-07-25T15:47:39Z + + PHD–SRA + + + 0.9978884 + evidence + cleaner0 + 2023-07-25T15:47:57Z + DUMMY: + + H3K9me0-binding affinity + + + 0.9994618 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.99946874 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.99923813 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + + RESULTS + paragraph + 17280 + Moreover, the TTD or TTD–PHD bound to SRA–Spacer and the interaction was impaired by the addition of hm-DNA (Fig. 4c). The ITC measurements show that the presence of hm-DNA markedly impaired the interaction between the TTD and SRA–Spacer (Supplementary Fig. 5a). However, the TTD–Spacer interaction was not affected by the presence of the hm-DNA, indicating that hm-DNA displaces the Spacer from the TTD in a SRA-dependent manner (Supplementary Fig. 5b). + + 0.99950933 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + 0.9987109 + protein_state + cleaner0 + 2023-07-25T15:10:33Z + DUMMY: + + bound to + + + structure_element + SO: + cleaner0 + 2023-07-25T15:48:54Z + + SRA–Spacer + + + 0.99908704 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.9987098 + experimental_method + cleaner0 + 2023-07-25T16:40:53Z + MESH: + + ITC + + + 0.9988615 + protein_state + cleaner0 + 2023-07-25T15:10:25Z + DUMMY: + + presence of + + + 0.99899316 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99947697 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + structure_element + SO: + cleaner0 + 2023-07-25T15:48:55Z + + SRA–Spacer + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:10:25Z + + presence of + + + 0.99896955 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.9988248 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99763024 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9994855 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.8576784 + structure_element + cleaner0 + 2023-07-25T14:57:34Z + SO: + + SRA + + + + RESULTS + paragraph + 17743 + To investigate whether hm-DNA disrupts TTD–Spacer interaction in the context of full-length UHRF1, we monitored the conformational changes of UHRF1 using its histone-binding affinity as read-out. UHRF1D334A was used to exclude the effect of H3K9me0 recognition by the PHD. As expected, all D334A-containing mutants showed undetectable interaction with H3K9me0 (Fig. 4d). UHRF1D334A bound to H3K9me3 peptide in the presence of hm-DNA, but showed no interaction in the absence of hm-DNA, which is consistent with the ITC experiments (Supplementary Fig. 4f). In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. The results further support the conclusion that the Spacer binds to the TTD in the context of full-length UHRF1 and the intramolecular interactions are disrupted by hm-DNA. + + 0.9971008 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + 0.9991389 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.99922895 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + protein + PR: + cleaner0 + 2023-07-25T15:49:52Z + + UHRF1 + + + 0.9983196 + evidence + cleaner0 + 2023-07-25T15:49:57Z + DUMMY: + + histone-binding affinity + + + 0.9989108 + mutant + cleaner0 + 2023-07-25T15:37:37Z + MESH: + + UHRF1D334A + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:48:43Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:48:51Z + + K9me0 + + + 0.99943894 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.9989262 + mutant + cleaner0 + 2023-07-25T16:35:44Z + MESH: + + D334A + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T16:00:01Z + + mutants + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:49:04Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:49:12Z + + K9me0 + + + 0.99879444 + mutant + cleaner0 + 2023-07-25T15:37:37Z + MESH: + + UHRF1D334A + + + 0.9990998 + protein_state + cleaner0 + 2023-07-25T15:10:33Z + DUMMY: + + bound to + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:49:26Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:49:36Z + + K9me3 + + + 0.99886876 + protein_state + cleaner0 + 2023-07-25T15:10:25Z + DUMMY: + + presence of + + + 0.9983613 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99910927 + protein_state + cleaner0 + 2023-07-25T15:50:14Z + DUMMY: + + absence of + + + 0.997808 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99873966 + experimental_method + cleaner0 + 2023-07-25T16:40:58Z + MESH: + + ITC + + + 0.99884605 + mutant + cleaner0 + 2023-07-25T15:37:37Z + MESH: + + UHRF1D334A + + + 0.7490342 + mutant + cleaner0 + 2023-07-25T16:35:48Z + MESH: + + Δ627–674 + + + 0.96783024 + protein_state + cleaner0 + 2023-07-25T15:10:33Z + DUMMY: + + bound to + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:49:49Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:50:02Z + + K9me3 + + + 0.99908817 + protein_state + cleaner0 + 2023-07-25T15:50:13Z + DUMMY: + + absence of + + + 0.99811006 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.9919852 + experimental_method + cleaner0 + 2023-07-25T16:41:00Z + MESH: + + deletion + + + 0.99834204 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:50:15Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:50:23Z + + K9me3 + + + 0.99884856 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.99944717 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.9991366 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.9992418 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.9978318 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + + RESULTS + paragraph + 18682 + We next performed similar peptide pull-down assay using two mutants (N228C/G653C and R235C/G654C) generated on UHRF1D334A. Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. As negative controls, H3K9me3 recognition by UHRF1D334A or UHRF1D334A&Δ627–674 is not affected by DTT. + + 0.99872124 + experimental_method + cleaner0 + 2023-07-25T15:50:32Z + MESH: + + peptide pull-down assay + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T16:00:01Z + + mutants + + + 0.9989108 + mutant + cleaner0 + 2023-07-25T15:50:37Z + MESH: + + N228C + + + 0.9985165 + mutant + cleaner0 + 2023-07-25T15:50:42Z + MESH: + + G653C + + + 0.99888533 + mutant + cleaner0 + 2023-07-25T15:50:47Z + MESH: + + R235C + + + 0.9982223 + mutant + cleaner0 + 2023-07-25T15:50:53Z + MESH: + + G654C + + + 0.99885094 + mutant + cleaner0 + 2023-07-25T15:37:37Z + MESH: + + UHRF1D334A + + + 0.99957484 + residue_name_number + cleaner0 + 2023-07-25T15:26:37Z + DUMMY: + + N228 + + + 0.99957687 + residue_name_number + cleaner0 + 2023-07-25T15:51:05Z + DUMMY: + + R235 + + + 0.99939466 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.99954104 + residue_name_number + cleaner0 + 2023-07-25T15:26:28Z + DUMMY: + + G653 + + + 0.9995326 + residue_name_number + cleaner0 + 2023-07-25T15:26:32Z + DUMMY: + + G654 + + + 0.99916935 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9990664 + structure_element + cleaner0 + 2023-07-25T15:17:21Z + SO: + + TTD–Spacer + + + 0.9821614 + evidence + cleaner0 + 2023-07-25T16:45:30Z + DUMMY: + + structure + + + 0.99367803 + residue_name + cleaner0 + 2023-07-25T16:35:17Z + SO: + + Cysteine + + + 0.99936646 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.99905914 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9833516 + ptm + cleaner0 + 2023-07-25T15:51:59Z + MESH: + + disulphide bond + + + 0.99728125 + protein_state + cleaner0 + 2023-07-25T15:50:14Z + DUMMY: + + absence of + + + 0.9975418 + chemical + cleaner0 + 2023-07-25T15:51:45Z + CHEBI: + + dithiothreitol + + + 0.9974686 + chemical + cleaner0 + 2023-07-25T15:51:50Z + CHEBI: + + DTT + + + 0.9978681 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.9978291 + evidence + cleaner0 + 2023-07-25T15:51:32Z + DUMMY: + + H3K9me3-binding affinities + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T16:00:01Z + + mutants + + + 0.99578726 + protein_state + cleaner0 + 2023-07-25T15:10:25Z + DUMMY: + + presence of + + + 0.99850523 + chemical + cleaner0 + 2023-07-25T15:51:51Z + CHEBI: + + DTT + + + 0.99795115 + protein_state + cleaner0 + 2023-07-25T15:50:14Z + DUMMY: + + absence of + + + 0.9981852 + chemical + cleaner0 + 2023-07-25T15:51:51Z + CHEBI: + + DTT + + + 0.97308254 + ptm + cleaner0 + 2023-07-25T15:51:59Z + MESH: + + disulphide bond + + + 0.9947853 + protein_state + cleaner0 + 2023-07-25T15:50:14Z + DUMMY: + + absence of + + + 0.99812025 + chemical + cleaner0 + 2023-07-25T15:51:51Z + CHEBI: + + DTT + + + 0.9967267 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:50:40Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:50:48Z + + K9me3 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T13:01:42Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T13:01:50Z + + K9me3 + + + 0.99848133 + mutant + cleaner0 + 2023-07-25T15:37:37Z + MESH: + + UHRF1D334A + + + 0.99851066 + mutant + cleaner0 + 2023-07-25T15:37:37Z + MESH: + + UHRF1D334A + + + 0.9231658 + mutant + cleaner0 + 2023-07-25T16:35:52Z + MESH: + + Δ627–674 + + + 0.99822944 + chemical + cleaner0 + 2023-07-25T15:51:51Z + CHEBI: + + DTT + + + + RESULTS + paragraph + 19560 + The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). We have previously demonstrated that hm-DNA also disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. Taken together, hm-DNA disrupts the intramolecular interactions within UHRF1, and therefore facilitates the coordinate recognition of H3K9me3 by TTD–PHD. + + 0.9990444 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.99925977 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.9992508 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + 0.99818677 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.9995371 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:51:06Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:51:14Z + + K9me3 + + + 0.9987197 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.9989048 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9995277 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.9991288 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.9992617 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.73736984 + protein_type + cleaner0 + 2023-07-25T15:05:08Z + MESH: + + histone + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:51:25Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:51:33Z + + K9me3 + + + 0.9994729 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.9982949 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + 0.9982069 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + structure_element + SO: + cleaner0 + 2023-07-25T15:47:40Z + + PHD–SRA + + + 0.9877172 + evidence + cleaner0 + 2023-07-25T15:47:58Z + DUMMY: + + H3K9me0-binding affinity + + + 0.99947613 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.99953234 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.99911374 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.9984932 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99928147 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:51:45Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:51:54Z + + K9me3 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + + RESULTS + title_2 + 20269 + The spacer enhances hm-DNA-binding affinity of the SRA + + 0.82882965 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + spacer + + + evidence + DUMMY: + cleaner0 + 2023-07-25T14:56:50Z + + hm-DNA-binding affinity + + + 0.9169475 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + + RESULTS + paragraph + 20324 + To investigate how hm-DNA impairs TTD–Spacer interaction, we tested whether the Spacer is involved in hm-DNA recognition by the SRA, which is the only known domain for hm-DNA recognition within UHRF1. In the electrophoretic mobility-shift assay, SRA–Spacer showed higher hm-DNA-binding affinity than the SRA alone (Supplementary Fig. 6a). ITC measurements show that SRA–Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75 μM) than the SRA (KD=25.12 μM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a). In the fluorescence polarization (FP) measurements, SRA–Spacer, full-length UHRF1 and UHRF1ΔTTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not. In contrast, UHRF1ΔSRA abolished hm-DNA-binding affinity, indicating that the SRA is essential for hm-DNA recognition. Compared with full-length UHRF1, UHRF1Δ627–674 decreased the hm-DNA-binding affinity by a factor of 14 (Fig. 5b), further supporting that the Spacer plays an important role in hm-DNA recognition in the context of full-length UHRF1. In addition, hm-DNA-binding affinities of SRA or SRA–Spacer did not obviously vary upon the change of DNA lengths but did decrease with the increasing salt concentrations (Supplementary Fig. 6b,c and Supplementary Table 5). These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:25Z + + hm-DNA + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + 0.99807835 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:25Z + + hm-DNA + + + 0.81657904 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:25Z + + hm-DNA + + + 0.999308 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.9981696 + experimental_method + cleaner0 + 2023-07-25T16:41:07Z + MESH: + + electrophoretic mobility-shift assay + + + 0.987925 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.99830437 + evidence + cleaner0 + 2023-07-25T14:56:50Z + DUMMY: + + hm-DNA-binding affinity + + + 0.76229775 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + 0.98666745 + protein_state + cleaner0 + 2023-07-25T16:49:21Z + DUMMY: + + alone + + + 0.9979923 + experimental_method + cleaner0 + 2023-07-25T16:41:10Z + MESH: + + ITC + + + 0.9918042 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.9989643 + protein_state + cleaner0 + 2023-07-25T15:10:33Z + DUMMY: + + bound to + + + 0.92392224 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.9986793 + evidence + cleaner0 + 2023-07-25T15:10:17Z + DUMMY: + + binding affinity + + + 0.9982942 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.9393552 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + 0.9979615 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.94276625 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + 0.97806543 + protein_state + cleaner0 + 2023-07-25T16:49:23Z + DUMMY: + + alone + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:25Z + + hm-DNA + + + 0.997146 + experimental_method + cleaner0 + 2023-07-25T16:41:15Z + MESH: + + fluorescence polarization (FP + + + 0.9868372 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.99911547 + protein_state + cleaner0 + 2023-07-25T15:09:00Z + DUMMY: + + full-length + + + 0.99911135 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.9990601 + mutant + cleaner0 + 2023-07-25T15:32:50Z + MESH: + + UHRF1ΔTTD + + + 0.9985642 + evidence + cleaner0 + 2023-07-25T15:53:50Z + DUMMY: + + hm-DNA-binding affinities + + + 0.9991716 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:25Z + + hm-DNA + + + 0.9992442 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.99919313 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + 0.99904126 + mutant + cleaner0 + 2023-07-25T15:32:21Z + MESH: + + UHRF1ΔSRA + + + 0.99844027 + evidence + cleaner0 + 2023-07-25T14:56:50Z + DUMMY: + + hm-DNA-binding affinity + + + 0.99079424 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:25Z + + hm-DNA + + + 0.99907523 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + full-length + + + 0.99927646 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + mutant + MESH: + cleaner0 + 2023-07-25T15:33:26Z + + UHRF1Δ627–674 + + + 0.9985644 + evidence + cleaner0 + 2023-07-25T14:56:50Z + DUMMY: + + hm-DNA-binding affinity + + + 0.9980646 + structure_element + cleaner0 + 2023-07-25T14:57:04Z + SO: + + Spacer + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:25Z + + hm-DNA + + + 0.99915737 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + full-length + + + 0.999258 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.99851686 + evidence + cleaner0 + 2023-07-25T15:53:51Z + DUMMY: + + hm-DNA-binding affinities + + + 0.86080647 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + 0.9879043 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.99886537 + structure_element + cleaner0 + 2023-07-25T14:57:05Z + SO: + + Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.99944586 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:52:11Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:52:20Z + + K9me3 + + + 0.999356 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + 0.99925965 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:25Z + + hm-DNA + + + 0.47243175 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + 0.9991511 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:25Z + + hm-DNA + + + + RESULTS + paragraph + 21936 + We next mapped the minimal region of the Spacer for the enhancement of hm-DNA-binding affinity. SRA–Spacer-661 (residues 414–661) still maintained strong hm-DNA-binding affinity comparable to that of SRA–Spacer (residues 414–674), whereas SRA–Spacer-652 and SRA–Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642–661 are important for enhancing hm-DNA-binding affinity of the SRA. This minimal region largely overlaps with the Spacer region (643–655) essential for TTD interaction. We also determined the crystal structure of SRA–Spacer bound to hm-DNA at 3.15 Å resolution (Supplementary Table 6 and Supplementary Fig. 7a). The structure shows that the SRA binds to hm-DNA in a manner similar to that observed in the previously reported SRA-hm-DNA structures. Intriguingly, no electron density was observed for the Spacer. A possible explanation is that the Spacer facilitates SRA–hm-DNA interaction through nonspecific salt bridge contacts because DNA is rich in acidic groups and the Spacer is rich in basic residues (Supplementary Fig. 7b). The nonspecific interaction is consistent with the previous observation that UHRF1 has no DNA sequence selectivity besides hm-CpG dinucleotide. + + 0.9975426 + structure_element + cleaner0 + 2023-07-25T14:57:05Z + SO: + + Spacer + + + 0.9973096 + evidence + cleaner0 + 2023-07-25T14:56:50Z + DUMMY: + + hm-DNA-binding affinity + + + mutant + MESH: + cleaner0 + 2023-07-25T15:56:40Z + + SRA–Spacer-661 + + + 0.9965994 + residue_range + cleaner0 + 2023-07-25T16:38:23Z + DUMMY: + + 414–661 + + + 0.99742836 + evidence + cleaner0 + 2023-07-25T14:56:50Z + DUMMY: + + hm-DNA-binding affinity + + + 0.8045659 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.99663335 + residue_range + cleaner0 + 2023-07-25T16:38:30Z + DUMMY: + + 414–674 + + + mutant + MESH: + cleaner0 + 2023-07-25T15:57:14Z + + SRA–Spacer-652 + + + mutant + MESH: + cleaner0 + 2023-07-25T15:57:47Z + + SRA–Spacer-642 + + + 0.9978421 + evidence + cleaner0 + 2023-07-25T15:53:51Z + DUMMY: + + hm-DNA-binding affinities + + + 0.99661374 + residue_range + cleaner0 + 2023-07-25T16:38:34Z + DUMMY: + + 642–661 + + + 0.9960874 + evidence + cleaner0 + 2023-07-25T14:56:50Z + DUMMY: + + hm-DNA-binding affinity + + + 0.99648416 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + structure_element + SO: + cleaner0 + 2023-07-25T14:57:05Z + + Spacer + + + 0.99668616 + residue_range + cleaner0 + 2023-07-25T16:38:38Z + DUMMY: + + 643–655 + + + 0.7144268 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.998444 + evidence + cleaner0 + 2023-07-25T16:45:37Z + DUMMY: + + crystal structure + + + 0.9244888 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.99880683 + protein_state + cleaner0 + 2023-07-25T15:10:33Z + DUMMY: + + bound to + + + 0.99367905 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.997813 + evidence + cleaner0 + 2023-07-25T16:45:39Z + DUMMY: + + structure + + + 0.9913696 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + 0.8255575 + protein_state + cleaner0 + 2023-07-25T15:13:04Z + DUMMY: + + binds to + + + 0.9931057 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99895704 + complex_assembly + cleaner0 + 2023-07-25T15:58:37Z + GO: + + SRA-hm-DNA + + + 0.9978289 + evidence + cleaner0 + 2023-07-25T16:45:42Z + DUMMY: + + structures + + + 0.9962826 + evidence + cleaner0 + 2023-07-25T16:45:45Z + DUMMY: + + electron density + + + 0.9974815 + structure_element + cleaner0 + 2023-07-25T14:57:05Z + SO: + + Spacer + + + 0.9951716 + structure_element + cleaner0 + 2023-07-25T14:57:05Z + SO: + + Spacer + + + complex_assembly + GO: + cleaner0 + 2023-07-25T15:59:10Z + + SRA–hm-DNA + + + 0.9730364 + bond_interaction + cleaner0 + 2023-07-25T15:59:17Z + MESH: + + salt bridge + + + 0.94352984 + chemical + cleaner0 + 2023-07-25T16:30:22Z + CHEBI: + + DNA + + + 0.9973133 + structure_element + cleaner0 + 2023-07-25T14:57:05Z + SO: + + Spacer + + + 0.99935216 + protein + cleaner0 + 2023-07-25T14:58:02Z + PR: + + UHRF1 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T15:59:31Z + + DNA + + + 0.99895376 + chemical + cleaner0 + 2023-07-25T15:59:37Z + CHEBI: + + hm-CpG dinucleotide + + + + RESULTS + title_2 + 23199 + The spacer is important for PCH localization of UHRF1 + + 0.99830556 + structure_element + cleaner0 + 2023-07-25T14:57:05Z + SO: + + spacer + + + 0.9988042 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + + RESULTS + paragraph + 23253 + To investigate the role of the Spacer in the regulation of UHRF1 function, we transiently overexpressed GFP-tagged wild type or mutants of UHRF1 in NIH3T3 cells to determine their subcellular localization. For the NIH3T3 cells expressing wild-type UHRF1, most cells (∼74.6%) showed a focal pattern of protein that is co-localized with 4,6-diamidino-2-phenylindole (DAPI) foci (Fig. 5d), whereas the rest cells showed a diffuse nuclear staining pattern. The result is consistent with the previous studies that UHRF1 is mainly localized to highly methylated pericentromeric heterochromatin (PCH). In contrast, for the cells expressing UHRF1Δ627–674, a spacer deletion mutant with decreased hm-DNA-binding affinity (Fig. 5b), only ∼22.1% cells showed co-localization with DAPI. + + 0.9981256 + structure_element + cleaner0 + 2023-07-25T14:57:05Z + SO: + + Spacer + + + 0.9989207 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9978527 + experimental_method + cleaner0 + 2023-07-25T16:41:23Z + MESH: + + transiently overexpressed + + + 0.99685115 + protein_state + cleaner0 + 2023-07-25T15:59:46Z + DUMMY: + + GFP-tagged + + + 0.999069 + protein_state + cleaner0 + 2023-07-25T15:59:53Z + DUMMY: + + wild type + + + 0.9961945 + protein_state + cleaner0 + 2023-07-25T16:00:00Z + DUMMY: + + mutants + + + 0.9990792 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.99910134 + protein_state + cleaner0 + 2023-07-25T15:34:11Z + DUMMY: + + wild-type + + + 0.99903154 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9928932 + chemical + cleaner0 + 2023-07-25T16:00:05Z + CHEBI: + + 4,6-diamidino-2-phenylindole + + + 0.98459643 + chemical + cleaner0 + 2023-07-25T16:00:09Z + CHEBI: + + DAPI + + + 0.999064 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.96579576 + protein_state + cleaner0 + 2023-07-25T16:49:30Z + DUMMY: + + highly methylated + + + 0.9048956 + mutant + cleaner0 + 2023-07-25T15:33:26Z + MESH: + + UHRF1Δ627–674 + + + 0.981091 + protein_state + cleaner0 + 2023-07-25T16:00:39Z + DUMMY: + + spacer deletion mutant + + + 0.9975831 + evidence + cleaner0 + 2023-07-25T14:56:50Z + DUMMY: + + hm-DNA-binding affinity + + + 0.80328345 + chemical + cleaner0 + 2023-07-25T16:00:11Z + CHEBI: + + DAPI + + + + RESULTS + paragraph + 24035 + Previous reports have shown that the H3K9me3 recognition of UHRF1 also plays an important role in its heterochromatin localization. For example, UHRF1 mutant (within TTD domain) lacking H3K9me3-binding affinity largely reduces its co-localization with heterochromatin. Because manipulation of endogenous hm-DNA in cells is technically challenging, we used UHRF1ΔSRA (lacks hm-DNA-binding affinity but maintains closed conformation, Figs 3h and 5b) to test whether closed conformation of UHRF1 exists in vivo. In NIH3T3 cells, UHRF1ΔSRA largely decreased chromatin association (Fig. 5d). Only ∼4.8% cells expressing UHRF1ΔSRA showed an intermediate enrichment, but not characteristic focal pattern, at DAPI foci, whereas the majority of the cells showed a diffuse nuclear staining pattern. The results suggest that UHRF1ΔSRA adopts closed conformation so that H3K9me3 recognition by TTD–PHD is blocked by the intramolecular interaction, and support the regulatory role of the Spacer in PCH localization of UHRF1 in vivo. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:52:39Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:52:50Z + + K9me3 + + + 0.99911493 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.96556795 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9991499 + protein_state + cleaner0 + 2023-07-25T16:00:49Z + DUMMY: + + mutant + + + 0.9993081 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.9985348 + protein_state + cleaner0 + 2023-07-25T16:01:43Z + DUMMY: + + lacking + + + 0.9186321 + evidence + cleaner0 + 2023-07-25T15:36:49Z + DUMMY: + + H3K9me3-binding affinity + + + 0.95978385 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.9990237 + mutant + cleaner0 + 2023-07-25T15:32:21Z + MESH: + + UHRF1ΔSRA + + + 0.9810548 + protein_state + cleaner0 + 2023-07-25T16:01:46Z + DUMMY: + + lacks + + + 0.8604536 + evidence + cleaner0 + 2023-07-25T14:56:50Z + DUMMY: + + hm-DNA-binding affinity + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T14:59:20Z + + closed + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T14:59:20Z + + closed + + + 0.99917513 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9986072 + mutant + cleaner0 + 2023-07-25T15:32:21Z + MESH: + + UHRF1ΔSRA + + + 0.99880767 + mutant + cleaner0 + 2023-07-25T15:32:21Z + MESH: + + UHRF1ΔSRA + + + chemical + CHEBI: + cleaner0 + 2023-07-25T16:00:11Z + + DAPI + + + 0.9989556 + mutant + cleaner0 + 2023-07-25T15:32:21Z + MESH: + + UHRF1ΔSRA + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T14:59:20Z + + closed + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:53:06Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:53:14Z + + K9me3 + + + 0.80278236 + structure_element + cleaner0 + 2023-07-25T15:11:21Z + SO: + + TTD–PHD + + + 0.99870586 + structure_element + cleaner0 + 2023-07-25T14:57:05Z + SO: + + Spacer + + + 0.9991685 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + + RESULTS + title_2 + 25071 + The spacer facilitates UHRF1–DNMT1 interaction + + 0.99730814 + structure_element + cleaner0 + 2023-07-25T14:57:05Z + SO: + + spacer + + + complex_assembly + GO: + cleaner0 + 2023-07-25T16:03:29Z + + UHRF1–DNMT1 + + + + RESULTS + paragraph + 25120 + Previous studies show that UHRF1 recruits DNMT1 to hm-DNA for maintenance DNA methylation through the interaction between the SRA and RFTSDNMT1 (refs). We confirmed the direct interaction between RFTSDNMT1 and the SRA in a solution with low salt concentration (50 mM NaCl), but observed weak or undetectable interaction in a solution with higher salt concentrations (100 or 150 mM NaCl) (Supplementary Fig. 8a). Compared with the SRA, SRA–Spacer exhibited stronger interaction with RFTSDNMT1. In addition, RFTSDNMT1 bound to SRA–Spacer with a binding affinity of 7.09 μM, but showed no detectable interaction with the SRA (Supplementary Fig. 8b). Interestingly, the addition of hm-DNA abolished the interaction between RFTSDNMT1 and SRA–Spacer, suggesting that hm-DNA also regulates UHRF1–DNMT1 interaction (Supplementary Fig. 8c). These results indicate that the Spacer facilitates the interaction between RFTSDNMT1 and the SRA, and the interaction is impaired by the presence of hm-DNA. + + 0.9991511 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.99884087 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + 0.99011177 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-25T16:02:00Z + + DNA + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:57Z + + methylation + + + 0.9738806 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + 0.99923766 + protein + cleaner0 + 2023-07-25T16:02:12Z + PR: + + RFTSDNMT1 + + + 0.9991768 + protein + cleaner0 + 2023-07-25T16:02:12Z + PR: + + RFTSDNMT1 + + + 0.9504781 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + 0.99426115 + chemical + cleaner0 + 2023-07-25T16:02:18Z + CHEBI: + + NaCl + + + 0.9910999 + chemical + cleaner0 + 2023-07-25T16:02:19Z + CHEBI: + + NaCl + + + 0.990811 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + 0.9875321 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.9989967 + protein + cleaner0 + 2023-07-25T16:02:12Z + PR: + + RFTSDNMT1 + + + 0.9987704 + protein + cleaner0 + 2023-07-25T16:02:12Z + PR: + + RFTSDNMT1 + + + 0.99872077 + protein_state + cleaner0 + 2023-07-25T15:10:33Z + DUMMY: + + bound to + + + 0.8938754 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.99867624 + evidence + cleaner0 + 2023-07-25T15:10:18Z + DUMMY: + + binding affinity + + + 0.929079 + structure_element + cleaner0 + 2023-07-25T14:57:35Z + SO: + + SRA + + + 0.9985009 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + 0.99913883 + protein + cleaner0 + 2023-07-25T16:02:12Z + PR: + + RFTSDNMT1 + + + 0.9334615 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.99392587 + chemical + cleaner0 + 2023-07-25T14:58:25Z + CHEBI: + + hm-DNA + + + complex_assembly + GO: + cleaner0 + 2023-07-25T16:03:29Z + + UHRF1–DNMT1 + + + 0.9990073 + structure_element + cleaner0 + 2023-07-25T14:57:05Z + SO: + + Spacer + + + 0.99927133 + protein + cleaner0 + 2023-07-25T16:02:12Z + PR: + + RFTSDNMT1 + + + 0.8667852 + structure_element + cleaner0 + 2023-07-25T14:57:36Z + SO: + + SRA + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:10:25Z + + presence of + + + 0.9979096 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + + RESULTS + paragraph + 26124 + We next tested whether the UHRF1–DNMT1 interaction is regulated by the conformational change of UHRF1. Because the addition of hm-DNA disrupts the interaction between the SRA–Spacer and RFTSDNMT1, we used various truncations to mimic open and closed forms of UHRF1. In the absence of hm-DNA, only UHRF1ΔTTD bound to RFTSDNMT1, whereas full-length UHRF1, UHRF1ΔSRA and UHRF1Δ627–674 showed undetectable interaction (Fig. 5e). As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA–Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA. In support of above observations, the addition of large amount of RFTSDNMT1 impaired the interaction between UHRF1 and hm-DNA (Supplementary Fig. 8d), suggesting an existence of dynamic equilibrium between UHRF1–hm-DNA and UHRF1–DNMT1 complexes. + + complex_assembly + GO: + cleaner0 + 2023-07-25T16:03:29Z + + UHRF1–DNMT1 + + + 0.9991204 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.99723744 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.99853516 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.99903846 + protein + cleaner0 + 2023-07-25T16:02:12Z + PR: + + RFTSDNMT1 + + + 0.6827299 + experimental_method + cleaner0 + 2023-07-25T16:41:29Z + MESH: + + truncations + + + 0.99917966 + protein_state + cleaner0 + 2023-07-25T16:49:50Z + DUMMY: + + open + + + 0.99918646 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + 0.9992004 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.998818 + protein_state + cleaner0 + 2023-07-25T15:50:14Z + DUMMY: + + absence of + + + 0.9959512 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.9990483 + mutant + cleaner0 + 2023-07-25T15:32:50Z + MESH: + + UHRF1ΔTTD + + + 0.99897456 + protein_state + cleaner0 + 2023-07-25T15:10:33Z + DUMMY: + + bound to + + + 0.9410995 + protein + cleaner0 + 2023-07-25T16:02:12Z + PR: + + RFTSDNMT1 + + + 0.99909276 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + full-length + + + 0.9991623 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9990976 + mutant + cleaner0 + 2023-07-25T15:32:21Z + MESH: + + UHRF1ΔSRA + + + 0.8580041 + mutant + cleaner0 + 2023-07-25T15:33:27Z + MESH: + + UHRF1Δ627–674 + + + 0.79138756 + experimental_method + cleaner0 + 2023-07-25T16:41:33Z + MESH: + + deletion of + + + 0.99919194 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.9992625 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9991698 + protein_state + cleaner0 + 2023-07-25T16:49:53Z + DUMMY: + + open + + + 0.99846977 + protein + cleaner0 + 2023-07-25T16:02:12Z + PR: + + RFTSDNMT1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.99843246 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.9992735 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T16:50:11Z + + open + + + 0.9990318 + protein_state + cleaner0 + 2023-07-25T15:50:14Z + DUMMY: + + absence of + + + 0.9968054 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.5373807 + experimental_method + cleaner0 + 2023-07-25T16:41:39Z + MESH: + + addition + + + 0.8506688 + protein + cleaner0 + 2023-07-25T16:02:13Z + PR: + + RFTSDNMT1 + + + 0.9990897 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.99238986 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + complex_assembly + GO: + cleaner0 + 2023-07-25T16:04:33Z + + UHRF1–hm-DNA + + + 0.9837528 + complex_assembly + cleaner0 + 2023-07-25T16:03:29Z + GO: + + UHRF1–DNMT1 + + + + DISCUSS + title_1 + 27001 + Discussion + + + DISCUSS + paragraph + 27012 + According to the above results, we here proposed a working model for hm-DNA-mediated regulation of UHRF1 conformation (Fig. 5f). In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD. As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA. The interaction between UHRF1 and DNMT1 is also weak because the Spacer is unable to facilitate the intermolecular interaction. In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. The association of UHRF1 to the histone may facilitate the ubiquitination of histone tail (mediated by RING domain) for DNMT1 targeting. Moreover, through a mechanism yet to be fully elucidated, DNMT1 targets hm-DNA for maintenance DNA methylation, probably through interaction with the histone ubiquitylation and/or SRA-Spacer. This cartoon summarizes our findings in this study. The P(r) function obtained from small-angle X-ray scattering (SAXS) measurements of TTD–PHD–SRA–Spacer–hm-DNA complex showed a broader distribution than that of the TTD–PHD–SRA–Spacer alone, supporting the proposed model that UHRF1 adopts an open conformation in the presence of hm-DNA (Supplementary Fig. 8e). + + 0.991255 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.9993242 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9991345 + protein_state + cleaner0 + 2023-07-25T15:50:14Z + DUMMY: + + absence of + + + 0.99493146 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.99937004 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9992467 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + 0.99932206 + structure_element + cleaner0 + 2023-07-25T14:57:05Z + SO: + + Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.999443 + structure_element + 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DUMMY: + + P(r) function + + + 0.99899787 + experimental_method + cleaner0 + 2023-07-25T16:41:43Z + MESH: + + small-angle X-ray scattering + + + 0.9984654 + experimental_method + cleaner0 + 2023-07-25T16:41:45Z + MESH: + + SAXS + + + 0.9987902 + complex_assembly + cleaner0 + 2023-07-25T16:06:20Z + GO: + + TTD–PHD–SRA–Spacer–hm-DNA + + + 0.99797595 + complex_assembly + cleaner0 + 2023-07-25T16:06:09Z + GO: + + TTD–PHD–SRA–Spacer + + + 0.9993412 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9992655 + protein_state + cleaner0 + 2023-07-25T16:50:41Z + DUMMY: + + open + + + 0.99916625 + protein_state + cleaner0 + 2023-07-25T15:10:25Z + DUMMY: + + presence of + + + 0.99755687 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + + DISCUSS + paragraph + 28833 + Many questions need to be further clarified. We have tried crystallizing more than three sub-constructs with and without DNA across over 1,200 crystallization conditions but failed to determine the structure of TTD–PHD–SRA–Spacer in the absence or presence of hm-DNA. Getting these structures would greatly help for understanding the hm-DNA-mediated regulation of UHRF1. In addition, this regulatory process should be further characterized using advanced techniques, such as single molecular measurement. + + 0.9979419 + experimental_method + cleaner0 + 2023-07-25T16:41:49Z + MESH: + + crystallizing + + + 0.95115554 + protein_state + cleaner0 + 2023-07-25T16:30:30Z + DUMMY: + + with + + + 0.9905099 + protein_state + cleaner0 + 2023-07-25T16:30:32Z + DUMMY: + + without + + + 0.9972832 + chemical + cleaner0 + 2023-07-25T16:30:27Z + CHEBI: + + DNA + + + 0.99836916 + evidence + cleaner0 + 2023-07-25T16:45:56Z + DUMMY: + + structure + + + complex_assembly + GO: + cleaner0 + 2023-07-25T16:06:10Z + + TTD–PHD–SRA–Spacer + + + 0.9971886 + protein_state + cleaner0 + 2023-07-25T16:30:35Z + DUMMY: + + absence + + + 0.99712753 + protein_state + cleaner0 + 2023-07-25T15:10:25Z + DUMMY: + + presence of + + + 0.95390725 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.9982545 + evidence + cleaner0 + 2023-07-25T16:45:59Z + DUMMY: + + structures + + + 0.9149298 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.9990747 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9973283 + experimental_method + cleaner0 + 2023-07-25T16:41:59Z + MESH: + + single molecular measurement + + + + DISCUSS + paragraph + 29344 + Our previous studies show that phosphorylation at S639 within the Spacer disrupts interaction between UHRF1 and deubiquitylase USP7 and decreases UHRF1 stability in the M phase of the cell cycle. The Spacer was predicted to contain two nuclear localization signals, residues 581–600 and 648-670 (ref.). In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1. These findings together indicate that the Spacer plays a very important role in the dynamic regulation of UHRF1. When our manuscript was in preparation, Gelato et al. reported that binding of PI5P to the Spacer opens the closed conformation of UHRF1 and increases H3K9me3-binding affinity of the TTD. The result suggests that PI5P may facilitate the conformational change of UHRF1 induced by hm-DNA when UHRF1 is recruited to chromatin. In addition, mass-spectrometry analyses have identified several phosphorylation sites (S639, S651, S661) within the Spacer, suggesting that post-translational modification may add another layer of regulation of UHRF1 (refs). + + 0.9963503 + ptm + cleaner0 + 2023-07-25T15:27:39Z + MESH: + + phosphorylation + + + 0.9995302 + residue_name_number + cleaner0 + 2023-07-25T16:07:15Z + DUMMY: + + S639 + + + 0.9990552 + structure_element + cleaner0 + 2023-07-25T14:57:06Z + SO: + + Spacer + + + 0.9992318 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9987031 + protein_type + cleaner0 + 2023-07-25T16:06:41Z + MESH: + + deubiquitylase + + + 0.9992466 + protein + cleaner0 + 2023-07-25T16:06:36Z + PR: + + USP7 + + + 0.99921703 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9991099 + structure_element + cleaner0 + 2023-07-25T14:57:06Z + SO: + + Spacer + + + 0.9739017 + structure_element + cleaner0 + 2023-07-25T16:32:08Z + SO: + + nuclear localization signals + + + 0.9976179 + residue_range + cleaner0 + 2023-07-25T16:38:44Z + DUMMY: + + 581–600 + + + 0.99757415 + residue_range + cleaner0 + 2023-07-25T16:38:47Z + DUMMY: + + 648-670 + + + 0.99928963 + structure_element + cleaner0 + 2023-07-25T14:57:06Z + SO: + + Spacer + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.99951804 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.99922633 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + 0.99931645 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:55:10Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:55:18Z + + K9me3 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:26Z + + hm-DNA + + + 0.7548684 + structure_element + cleaner0 + 2023-07-25T14:57:36Z + SO: + + SRA + + + 0.58831096 + structure_element + cleaner0 + 2023-07-25T14:57:36Z + SO: + + SRA + + + 0.9992194 + protein + cleaner0 + 2023-07-25T16:02:13Z + PR: + + RFTSDNMT1 + + + 0.99913114 + structure_element + cleaner0 + 2023-07-25T14:57:06Z + SO: + + Spacer + + + 0.99928147 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9988167 + chemical + cleaner0 + 2023-07-25T15:03:00Z + CHEBI: + + PI5P + + + 0.9990702 + structure_element + cleaner0 + 2023-07-25T14:57:06Z + SO: + + Spacer + + + 0.99918395 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + 0.9993456 + protein + cleaner0 + 2023-07-25T14:58:03Z + PR: + + UHRF1 + + + 0.9901287 + evidence + cleaner0 + 2023-07-25T15:36:49Z + DUMMY: + + H3K9me3-binding affinity + + + 0.99946576 + structure_element + cleaner0 + 2023-07-25T14:57:20Z + SO: + + TTD + + + 0.9987204 + chemical + cleaner0 + 2023-07-25T15:03:00Z + CHEBI: + + PI5P + + + 0.9992894 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.7813921 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.9993051 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.99864024 + experimental_method + cleaner0 + 2023-07-25T16:42:02Z + MESH: + + mass-spectrometry + + + 0.96276474 + site + cleaner0 + 2023-07-25T16:07:07Z + SO: + + phosphorylation sites + + + 0.9995316 + residue_name_number + cleaner0 + 2023-07-25T16:07:14Z + DUMMY: + + S639 + + + 0.99952173 + residue_name_number + cleaner0 + 2023-07-25T15:26:00Z + DUMMY: + + S651 + + + 0.9995235 + residue_name_number + cleaner0 + 2023-07-25T16:07:21Z + DUMMY: + + S661 + + + 0.99915576 + structure_element + cleaner0 + 2023-07-25T14:57:06Z + SO: + + Spacer + + + 0.99927586 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + + DISCUSS + paragraph + 30560 + It has been well characterized that the SRA of UHRF1 preferentially recognizes hm-DNA through a base-flipping mechanism. Our study demonstrates that the Spacer markedly enhances the hm-DNA-binding affinity of the SRA and the deletion of the Spacer impairs heterochromatin localization of UHRF1, indicating that the Spacer is essential for recognition of hm-DNA in the context of full-length UHRF1. Interestingly, variant in methylation 1 (VIM1, a UHRF1 homologue in Arabidopsis) contains an equivalent spacer region, which was shown to be required for hm-DNA recognition by its SRA domain, suggesting a conserved regulatory mechanism in SRA domain-containing proteins. Intriguingly, UHRF2 (the only mammalian homologue of UHRF1) and UHRF1 show very high sequence similarities for all the domains but very low similarity for the Spacer (Supplementary Fig. 7c). Thus, although UHRF2 exhibits the histone- and hm-DNA-binding activities, the difference in the Spacer region may contribute to the functional differences between UHRF1 and UHRF2. This is also consistent with previous finding that UHRF2 is unable to replace UHRF1 to maintain the DNA methylation. + + 0.9994406 + structure_element + cleaner0 + 2023-07-25T14:57:36Z + SO: + + SRA + + + 0.9991404 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.9270676 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.9990067 + structure_element + cleaner0 + 2023-07-25T14:57:06Z + SO: + + Spacer + + + 0.99293834 + evidence + cleaner0 + 2023-07-25T14:56:50Z + DUMMY: + + hm-DNA-binding affinity + + + 0.99931526 + structure_element + cleaner0 + 2023-07-25T14:57:36Z + SO: + + SRA + + + 0.9863229 + experimental_method + cleaner0 + 2023-07-25T16:42:06Z + MESH: + + deletion of + + + 0.9961428 + structure_element + cleaner0 + 2023-07-25T14:57:06Z + SO: + + Spacer + + + 0.99889886 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.99849296 + structure_element + cleaner0 + 2023-07-25T14:57:06Z + SO: + + Spacer + + + 0.84966046 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.99905974 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + full-length + + + 0.9991301 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.9977554 + protein + cleaner0 + 2023-07-25T16:07:45Z + PR: + + variant in methylation 1 + + + 0.9988433 + protein + cleaner0 + 2023-07-25T16:07:49Z + PR: + + VIM1 + + + 0.9989505 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.9986185 + taxonomy_domain + cleaner0 + 2023-07-25T16:07:57Z + DUMMY: + + Arabidopsis + + + structure_element + SO: + cleaner0 + 2023-07-25T14:57:06Z + + spacer + + + 0.8714142 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.99954695 + structure_element + cleaner0 + 2023-07-25T14:57:36Z + SO: + + SRA + + + structure_element + SO: + cleaner0 + 2023-07-25T14:57:36Z + + SRA + + + 0.9990758 + protein + cleaner0 + 2023-07-25T16:08:25Z + PR: + + UHRF2 + + + 0.9986889 + taxonomy_domain + cleaner0 + 2023-07-25T15:00:05Z + DUMMY: + + mammalian + + + 0.99895585 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.99911803 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.99908423 + structure_element + cleaner0 + 2023-07-25T14:57:06Z + SO: + + Spacer + + + 0.9989396 + protein + cleaner0 + 2023-07-25T16:08:26Z + PR: + + UHRF2 + + + structure_element + SO: + cleaner0 + 2023-07-25T14:57:06Z + + Spacer + + + 0.99909437 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.9990441 + protein + cleaner0 + 2023-07-25T16:08:26Z + PR: + + UHRF2 + + + 0.99901295 + protein + cleaner0 + 2023-07-25T16:08:26Z + PR: + + UHRF2 + + + 0.9990362 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T16:09:23Z + + DNA + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:57Z + + methylation + + + + DISCUSS + paragraph + 31717 + One of the key questions in the field of DNA methylation is why UHRF1 contains modules recognizing two repressive epigenetic marks: H3K9me3 (by TTD–PHD) and hm-DNA (by the SRA). Previous studies show that chromatin localization of UHRF1 is dependent on hm-DNA, whereas other studies indicate that histone H3K9me3 recognition and hm-DNA association are both required for UHRF1-mediated maintenance DNA methylation. However, little is known about the crosstalk between these two epigenetic marks within UHRF1. In this study, we provide an explanation. As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61 μM) or H3K9me0 (KD=25.99 μM). We have shown that full-length UHRF1 and SRA–Spacer strongly bind to hm-DNA (0.35 and 0.49 μM, respectively) and the Spacer plays an important role in PCH localization (Fig. 5d). Therefore, genomic localization of UHRF1 is primarily determined by its recognition of hm-DNA, which allows UHRF1 to adopt an open form and promotes its histone tail recognition for proper genomic localization and function. As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. This explanation agrees nicely with previous observations and clarifies the importance of coordinate recognition of H3K9me3 and hm-DNA by UHRF1 for maintenance DNA methylation. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-08-08T12:55:32Z + + DNA + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:57Z + + methylation + + + 0.9992513 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:56:23Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:56:16Z + + K9me3 + + + 0.99834013 + structure_element + cleaner0 + 2023-07-25T15:11:21Z + SO: + + TTD–PHD + + + 0.99753475 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.97159886 + structure_element + cleaner0 + 2023-07-25T14:57:36Z + SO: + + SRA + + + 0.9992951 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.9839018 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.74091005 + protein_type + cleaner0 + 2023-07-25T15:05:08Z + MESH: + + histone + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:55:50Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:55:58Z + + K9me3 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:26Z + + hm-DNA + + + 0.999199 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T16:10:31Z + + DNA + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:57Z + + methylation + + + 0.99930465 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:56:37Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:56:46Z + + K9me3 + + + 0.99916047 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + full-length + + + 0.9992872 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.99787104 + protein_state + cleaner0 + 2023-07-25T15:04:48Z + DUMMY: + + unmethylated + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:56:59Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:57:08Z + + K9me3 + + + 0.992324 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:57:25Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:57:35Z + + K9me0 + + + 0.9930274 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.99916273 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + full-length + + + 0.9993338 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.99657935 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.9956055 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.99895966 + structure_element + cleaner0 + 2023-07-25T14:57:06Z + SO: + + Spacer + + + 0.99927944 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.9932789 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.9992743 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.9988243 + protein_state + cleaner0 + 2023-07-25T16:50:46Z + DUMMY: + + open + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:08Z + + histone + + + 0.99772996 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.9933903 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.9993279 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + 0.9992974 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.9956915 + structure_element + cleaner0 + 2023-07-25T15:11:21Z + SO: + + TTD–PHD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:57:54Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:58:04Z + + K9me3 + + + 0.966383 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.56240326 + protein_type + cleaner0 + 2023-07-25T15:07:50Z + MESH: + + H3 + + + 0.9991653 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T16:10:11Z + + DNA + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:57Z + + methylation + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:58:18Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:58:27Z + + K9me3 + + + 0.99602073 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.9992939 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:58Z + + methylation + + + + DISCUSS + paragraph + 33394 + UHRF1 is essential for maintenance DNA methylation through recruiting DNMT1 to DNA replication forks during S phase. This function is probably induced by a direct interaction between the SRA and RFTSDNMT1 (refs) or interaction between DNMT1 and ubiquitylation of histione tail. Recent study indicates that histone tail association of UHRF1 (by the PHD domain) is required for histone H3 ubiquitylation, which is dependent on ubiquitin ligase activity of the RING domain of UHRF1 (ref.). DNMT1 binds to ubiquitylated histone H3 and ubiquitylation is required for maintenance of DNA methylation in vivo. In this study, we found that both TTD and PHD are regulated by hm-DNA to recognize histone tail. Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation. + + 0.99912244 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T16:10:58Z + + DNA + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:58Z + + methylation + + + 0.9992249 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T16:11:06Z + + DNA + + + 0.99181515 + structure_element + cleaner0 + 2023-07-25T14:57:36Z + SO: + + SRA + + + 0.99929225 + protein + cleaner0 + 2023-07-25T16:02:13Z + PR: + + RFTSDNMT1 + + + 0.9993098 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + 0.9939644 + ptm + cleaner0 + 2023-07-25T16:11:21Z + MESH: + + ubiquitylation + + + protein_type + MESH: + cleaner0 + 2023-07-25T16:32:35Z + + histione + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:09Z + + histone + + + 0.9993056 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.99944574 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.8996436 + protein_type + cleaner0 + 2023-07-25T15:05:09Z + MESH: + + histone + + + 0.9478336 + protein_type + cleaner0 + 2023-07-25T15:07:50Z + MESH: + + H3 + + + 0.99182045 + ptm + cleaner0 + 2023-07-25T16:11:22Z + MESH: + + ubiquitylation + + + 0.97545373 + protein_type + cleaner0 + 2023-07-25T16:29:31Z + MESH: + + ubiquitin ligase + + + structure_element + SO: + cleaner0 + 2023-07-25T15:01:28Z + + RING + + + 0.99932575 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.9993137 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.9985134 + protein_state + cleaner0 + 2023-07-25T16:50:52Z + DUMMY: + + ubiquitylated + + + 0.9693887 + protein_type + cleaner0 + 2023-07-25T15:05:09Z + MESH: + + histone + + + 0.948022 + protein_type + cleaner0 + 2023-07-25T15:07:50Z + MESH: + + H3 + + + 0.9429411 + ptm + cleaner0 + 2023-07-25T16:11:22Z + MESH: + + ubiquitylation + + + chemical + CHEBI: + cleaner0 + 2023-07-25T16:11:16Z + + DNA + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:58Z + + methylation + + + 0.99955565 + structure_element + cleaner0 + 2023-07-25T14:57:21Z + SO: + + TTD + + + 0.99952936 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.995536 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:09Z + + histone + + + 0.99926776 + protein_state + cleaner0 + 2023-07-25T14:59:20Z + DUMMY: + + closed + + + 0.9993462 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.99938595 + protein + cleaner0 + 2023-07-25T16:29:17Z + PR: + + URHF1 + + + 0.9993699 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.99925655 + protein_state + cleaner0 + 2023-07-25T16:50:55Z + DUMMY: + + open + + + 0.99442863 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:09Z + + histone + + + ptm + MESH: + cleaner0 + 2023-07-25T16:11:22Z + + ubiquitylation + + + chemical + CHEBI: + cleaner0 + 2023-07-25T16:11:33Z + + DNA + + + ptm + MESH: + cleaner0 + 2023-07-25T15:05:58Z + + methylation + + + + DISCUSS + paragraph + 34313 + Moreover, structural analyses of DNMT1–DNA and SRA–DNA complexes also indicate that it is impossible for DNMT1 to methylate the hm-DNA that UHRF1 binds to because of steric hindrance. In our in vitro assays, we could detect interaction between SRA–Spacer and RFTSDNMT1, but not the interaction between full-length UHRF1 and RFTSDNMT1 (Supplementary Fig. 8a,b and Fig. 5e). The results suggest that UHRF1 adopts multiple conformations. Binding of UHRF1 to hm-DNA may serve as a switch for its recruitment of DNMT1. The S phase-dependent interaction between UHRF1 and DNMT1 (refs) suggest that DNMT1 may also undergo conformation changes so that RFTSDNMT1 binds to UHRF1 and the catalytic domain of DNMT1 binds to hm-DNA for reaction. + + 0.99893475 + experimental_method + cleaner0 + 2023-07-25T16:42:12Z + MESH: + + structural analyses + + + 0.9986643 + complex_assembly + cleaner0 + 2023-07-25T16:12:01Z + GO: + + DNMT1–DNA + + + 0.9991562 + complex_assembly + cleaner0 + 2023-07-25T16:12:07Z + GO: + + SRA–DNA + + + 0.99922836 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + 0.99494505 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.99907386 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.9988796 + experimental_method + cleaner0 + 2023-07-25T16:42:15Z + MESH: + + in vitro assays + + + 0.8854396 + structure_element + cleaner0 + 2023-07-25T15:48:55Z + SO: + + SRA–Spacer + + + 0.9989078 + protein + cleaner0 + 2023-07-25T16:02:13Z + PR: + + RFTSDNMT1 + + + 0.9990818 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + full-length + + + 0.99901927 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.9988921 + protein + cleaner0 + 2023-07-25T16:02:13Z + PR: + + RFTSDNMT1 + + + 0.99912745 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.99913675 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.99709946 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.99923027 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + 0.9991431 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.9992536 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + 0.9992206 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + 0.9991217 + protein + cleaner0 + 2023-07-25T16:02:13Z + PR: + + RFTSDNMT1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.99914336 + protein + cleaner0 + 2023-07-25T14:58:04Z + PR: + + UHRF1 + + + 0.9992223 + structure_element + cleaner0 + 2023-07-25T16:32:43Z + SO: + + catalytic domain + + + 0.99927205 + protein + cleaner0 + 2023-07-25T15:00:38Z + PR: + + DNMT1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:13:04Z + + binds to + + + 0.99804133 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + + METHODS + title_1 + 35052 + Methods + + + METHODS + title_2 + 35060 + Protein expression and purification + + + METHODS + paragraph + 35096 + The ubiquitin-like domain (residues 1–133), TTD (residues 134–285), PHD (residues 307–366), TTD–PHD (residues 134–366), SRA (residues 414–617), SRA–Spacer (residues 414–674), Spacer (residues 587–674), RING (residues 675–793), UHRF1 (residues 1–793) and other mutants or truncations of human UHRF1 were sub-cloned in a pGEX-6p-1 derivative vector. The truncated Spacer (residues 627–674) used for NMR analyses was inserted into modified pRSF-Duet-1 vector. All the proteins were expressed in E. coli strain BL21 (DE3) and purified as described previously. In brief, the transformants were grown at 37 °C in 2X YT medium and induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to 0.1 mM when the OD600 reached 0.6 and further incubated at 15 °C overnight. The cells were harvested and disrupted. After centrifugation, the supernatant of GST-tagged proteins was purified by GST affinity column (GE Healthcare) and the His-tagged truncated Spacer (residues 627–674) was purified by Nickel Nitrilotriacetic Acid affinity chromatography (GE Healthcare). The GST-tagged proteins used for GST pull-down experiment were eluted directly. The fusion proteins were digested with PreScission protease and further purified by ion exchange and gel filtration chromatography. The proteins were concentrated to 5–20 mg ml−1 for the following biochemical and structural analyses. + + + METHODS + paragraph + 36511 + To purify 15N- and 13C-labelled proteins, the transformants were grown in M9 medium containing 15N-labelled NH4Cl (1 g l−1) and 13C-labelled glucose (2 g l−1). The isotope-labelled TTD and truncated Spacer were purified as described above. + + + METHODS + title_2 + 36763 + Pull-down experiments + + + METHODS + paragraph + 36785 + For GST pull-down assays, 15 μg GST-tagged proteins were incubated with 40 μg recombinant proteins in 500 μl pull-down buffer (20 mM HEPES-NaOH, pH 8.0, 100 mM NaCl, 5% glycerol and 0.1% Triton X-100) for 1 h at 4 °C. Glutathione resins (GE Healthcare) were washed six times with pull-down buffer then mixed with the proteins for 1 h at 4 °C. After washed three times with pull-down buffer, the bound proteins were analysed by SDS–PAGE Coomassie blue staining. For competitive pull-down experiments: purified proteins were pre-incubated with hemi-methylated-DNA (12 bp, upper strand: 5′-GGGCCXGCAGGG-3′, X=5-methyldeoxycytosine) at the indicated molar ratios for 10 min at 4 °C. For salt concentration-dependent pull-down experiments, the pull-down buffer contains 50, 100 or 150 mM NaCl, respectively. + + + METHODS + paragraph + 37626 + For histone peptide or hm-DNA pull-down, 1 μg biotinylated histone H3 peptide (residues 1–21) or hm-DNA (12-bp, upper strand 5′-GAGGCXGCCTGC-3′ and lower strand 5′-biotin-GCAGGCGGCCTC-3′, X=5-methyldeoxycytosine) were incubated with 20 μg wild type or mutants of UHRF1 proteins in 500 μl pull-down buffer (20 mM HEPES-NaOH, pH 8.0, 100 mM NaCl, 5% glycerol and 0.1% Triton X-100) for 1 h at 4 °C. The proteins were pre-incubated with hm-DNA (12- bp, upper strand: 5′-GGGCCXGCAGGG-3′, X=5-methyldeoxycytosine) or indicated H3 peptide, or binding buffer as a control, at 1:2 molar ratios for 10 min at 4 °C. Then, 20 μl streptavidin beads were washed six times with pull-down buffer and incubated with the mixture for 1 h at 4 °C. The bound proteins were analysed as described above. The results are summarized in Supplementary Fig. 10. + + + METHODS + title_2 + 38508 + ITC measurements + + + METHODS + paragraph + 38525 + The binding affinity of protein/protein, protein/peptide or protein/DNA was measured by adding 0.05 mM protein in cell and titrated with 0.5 mM protein, peptide or hm-DNA (12 bp, upper strand: 5′-GGGCCXGCAGGG-3′, X=5-methyldeoxycytosine) in the syringe using iTC200 microcalorimeter (GE Healthcare) at 18 °C. For competition ITC experiments: the indicated proteins were pre-incubated with competitive peptide, protein or hm-DNA (at 1:2 molar ratio if not specified) for 10 min followed by ITC measurements as described above. Proteins, DNA and peptides were prepared within ITC buffer containing 10 mM HEPES, pH 8.0, 100 mM NaCl. The data were fitted by software Origin 7.0. All ITC results were summarized in Supplementary Table 2 and raw data were shown in Supplementary Fig. 9. + + + METHODS + title_2 + 39325 + NMR titration assay + + + METHODS + paragraph + 39345 + To determine the mole ratio of TTD versus Spacer peptide (residues 627–674) in the complex for NMR studies, NMR stepwise titration assay was performed at 20 °C in a PBS buffer supplemented with 0.01% NaN3, pH 7.4 and 10% D2O. The Spacer peptide was added into 15N-labelled TTD solution with an increasing molar ratio of TTD/Spacer as follows: 1:0.0, 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1.2 and 1:1.5. The 1H–15N heteronuclear single-quantum correlation (HSQC) spectra of the TTD were collected after each addition. + + + METHODS + title_2 + 39861 + NMR spectroscopy and analysis + + + METHODS + paragraph + 39891 + Two NMR samples were prepared in a mole ratio of 1:1.2 (TTD/Spacer). One is 0.7 mM uniformly 13C/15N-labelled TTD in complex with unlabelled Spacer peptide in NMR buffer (PBS buffer, 0.01% NaN3, pH 7.4 and 10% D2O). The other is 0.5 mM 15N-labelled Spacer peptide mixed with unlabelled TTD protein. All NMR experiments were performed at 20 °C on a Varian Unity Inova 600 NMR spectrometer equipped with a triple resonances cryoprobe and pulsed field gradients. The standard suite of experiments for assigning the 1H, 13C and 15N backbone, determining the side-chain chemical shifts of the TTD in complex with the Spacer peptide and collecting the Nuclear Overhauser effect (NOE)-based distance restraints were measured, including two-dimensional (2D) 13C-edited HSQC and 15N-edited HSQC; three-dimensional (3D) HNCA, HNCO, HN(CO)CA, HNCACB, CBCA(CO)NH, 15N-resolved HSQC-total correlation spectroscopy (TOCSY) and 13C-resolved HSQC-TOCSY in both aliphatic and aromatic regions; 15N-resolved HSQC-NOESY; 13C-resolved HSQC-NOESY for both aliphatic and aromatic resonances and 2D hbcbcgcdceheA and hbcbcgcdhdA spectra for the correlation of Cβ and Hδ or Hɛ in the aromatic ring that is used for aromatic proton assignment. The NMR signals of bound TTD were assigned according to the previously report. The proton NMR signals of the bound Spacer peptide were assigned by analysing the 2D 13C-filtered, 15N-filtered and J-resolved NOE spectroscopy (NOESY) and TOCSY spectra recorded for the 13C- and 15N-labelled protein with the unlabelled Spacer peptide and the 2D 1H–1H COSY, NOESY and TOCSY spectra recorded for the unlabelled free Spacer peptide, and 15N-edited HSQC, 3D 15N-resolved HSQC-TOCSY for the 15N-labelled Spacer in complex with the TTD protein in the NMR buffer described above, respectively. The intermolecular NOEs between the labelled protein and the unlabelled Spacer peptide were obtained by analysing the 3D 13C-F1-edited and 13C/15N-F3-filtered NOESY spectra. The spectra were processed with the NMRPipe programme and analysed using Sparky 3 (http://www.cgl.ucsf.edu/home/sparky/). + + + METHODS + title_2 + 42002 + Determining the NMR structure + + + METHODS + paragraph + 42032 + The calculations were performed using a standard simulated annealing protocol implemented in the XPLOR-2.29 programme (NIH version). The inter-proton distance restraints derived from the NOE intensities were grouped into three distance ranges, namely 1.8–2.9, 1.8–3.5and 1.8–6.0 Å, which corresponds to strong, medium and weak NOEs, respectively. The dihedral angles phi and psi were derived from the backbone chemical shifts (HN, HA, CO and CA) using the programme TALOS. The hydrogen-bond constraints were generated based on the observed NOE pattern between anti-β-sheets in the TTD domain, confirmed by H-D exchange experiments, and used in structural calculation. A total of ten iterations were performed (50 structures in the initial eight iterations). In total, 100 structures were computed during the last two iterations, and the 20 conformers with the lowest energy were used to represent the 3D structures. The conformers of these bundles (TTD in complex with the Spacer peptide) do not violate the following constraints: NOE >0.3 Å and dihedral angle >3o. The entire structure statistics were evaluated with PROCHECK and PROCHECK-NMR and are summarized in Supplementary Table 3. All of the structure figures were generated using the PyMOL and MOLMOL programmes. + + + METHODS + title_2 + 43317 + Electrophoretic mobility-shift assay + + + METHODS + paragraph + 43354 + A 6-carboxy-fluorescein (FAM)-labelled primer, 5′-CCATGCGCTGAC-3′, was annealed to a primer 5′-GTCAGXGCATGG-3′ (X=5-methyldeoxycytosine). The hemi-methylated double-strand DNA was used in both electrophoretic mobility-shift assay and FP assays. 50 nM FAM-hm-DNA (1 pmole per lane) was pre-incubated with indicated amount of proteins in reaction buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 8% glycerol and 1 mM DTT) for 20 min on ice. The samples were subjected to a 10% polyacrylamide gel electrophoresis and run in 0.5 × Tris-borate-EDTA buffer at 100 V for 1 h at 4 °C. The results were visualized on Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology Co., Ltd). + + + METHODS + title_2 + 44065 + FP measurements + + + METHODS + paragraph + 44081 + The 12-bp FAM-labelled hm-DNA (as described above) was incubated with increasing amount of indicated proteins for 20 min at 25 °C in reaction buffer containing 20 mM HEPES, pH 7.5, 175 mM NaCl, 8% glycerol and 1 mM DTT. FP measurements were performed at 25 °C on Synergy 4 Microplate Reader (BioTek). The 16-bp and 20-bp FAM-labelled hm-DNA (lower strand: 5′-GTGTCAGXGCATGGCC-3′ and 5′-CCGTGTCAGXGCATGGCCAT-3′, respectively. X=5-methyldeoxycytosine) were used in the FP experiment to test the effect of DNA length on the protein/DNA interaction. All experiments were performed in triplicate. The curves were fitted by GraphPad Prism 5. For salt concentration-dependent FP experiments, the reaction buffer contains 50 mM or 150 mM NaCl, respectively. + + + METHODS + title_2 + 44857 + Crystallization and data collection + + + METHODS + paragraph + 44893 + Crystals of SRA–Spacer in complex with an 18-bp hm-DNA (upper strand: 5′-CATCGTCCCTGCGGGCCC-3′, lower strand: 5′-GGGCCXGCAGGGACGATG-3′. X=5-methyldeoxycytosine) were grown at 18 °C using the hanging drop vapour diffusion method by mixing an equal volume of protein–DNA complex and crystallization buffer containing 12% PEG 3350, 45 mM citric acid/55 mM BIS-TRIS propane (pH 6.9). Protein and hm-DNA were mixed at the molar ratio of 1:1.5 and incubated for 0.5 h on ice before crystallization. Crystals were flash frozen in a cold nitrogen stream at −173 °C. All data sets were collected on beamline BL17U at the SSRF (Shanghai Synchrotron Radiation Facility, China). The data were processed using the programme HKL2000 (ref.). + + + METHODS + title_2 + 45646 + Structure determination + + + METHODS + paragraph + 45670 + The structure of SRA–Spacer–hm-DNA complex was determined by molecular replacement using structure of the SRA (PDB:3BI7) as a searching model. Rotation and translation function searches were performed with the programme PHASER. The model was manually built with COOT. All refinements were performed using the refinement module phenix.refine of PHENIX package. The model quality was checked with the PROCHECK programme and all structure figures were generated by PyMol. + + + METHODS + title_2 + 46143 + Cell culture, transient transfection and images capture + + + METHODS + paragraph + 46199 + NIH3T3 cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology. Wild type and mutants of UHRF1 were sub-cloned into pEGFP-C1 vector. Transient transfections of NIH3T3 cells were carried out using Lipofectamine 2000 (Invitrogen). The NIH3T3 cells were grown on glass coverslips and harvested in 36 h after transfection. The images were acquired and examined as previously described. Briefly, cells were fixed with 4% paraformaldehyde for 25 min, then washed with PBS three times. Coverslips were mounted with Antifade reagent containing DAPI (Molecular Probes) on slides and examined with a confocal microscopy. + + + METHODS + title_2 + 46839 + SAXS measurements + + + METHODS + paragraph + 46857 + SAXS measurements were performed with Anton Paar SAXSess mc2 instrument with linecolimation and charge-coupled-device detection. The X-ray wavelength was 1.5418 Å (CuKα), the sample to detector was 306.8 mm and the sample slit width was 10 mm. Each sample was prepared in 300 μl solution contained 150 mM NaCl, 10 mM HEPES, pH=8.0, 5 mM DTT and 5% glycerol. For the hm-DNA-bound form, the protein was pre-incubated with hm-DNA (12-bp, upper strand: 5′-GGGCCmCGCAGGG-3′, mC=5-methyldeoxycytosine) at 1:1.2 molar ratio for 10 min on ice. To correct for interparticle interference, the data of protein sample were collected twice and each time for 1 h. The solution containing no protein sample was also tested as background. + + + METHODS + paragraph + 47605 + The initial data were first processed using SAXSquant and the further analysis with ATSAS software. The SAXS data were only analysed these were collected in the first hour because there was no time effect on the samples. The radius of gyration Rg was estimated from primus. The distance distribution function P(r) was calculated in PCG package. The maximum particle dimension Dmax was estimated from the P(r) function as the r for which P(r)=0. + + + METHODS + title_1 + 48050 + Additional information + + + METHODS + paragraph + 48073 + Accession codes: The coordinate and structure factor for the TTD–Spacer complex structure have been deposited in the Protein Data Bank under accession code 5IAY. The chemical shift assignment of TTD–Spacer was deposited with BMRB ADIT-NMR online deposition system under the accession number 30019. + + + METHODS + paragraph + 48375 + How to cite this article: Fang, J. et al. Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition. Nat. Commun. 7:11197 doi: 10.1038/ncomms11197 (2016). + + + SUPPL + title_1 + 48567 + Supplementary Material + + + 245 + 254 + surname:Jaenisch;given-names:R. + surname:Bird;given-names:A. + 12610534 + REF + Nature Genet. + ref + 33 + 2003 + 48590 + Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals + + + 204 + 220 + surname:Smith;given-names:Z. D. + surname:Meissner;given-names:A. + 23400093 + REF + Nat. Rev. Genet. + ref + 14 + 2013 + 48694 + DNA methylation: roles in mammalian development + + + 204 + 220 + surname:Law;given-names:J. A. + surname:Jacobsen;given-names:S. E. + 20142834 + REF + Nat. Rev. Genet. + ref + 11 + 2010 + 48742 + Establishing, maintaining and modifying DNA methylation patterns in plants and animals + + + 81 + 98 + surname:Guibert;given-names:S. + surname:Forne;given-names:T. + surname:Weber;given-names:M. + 22122638 + REF + Epigenomics + ref + 1 + 2009 + 48829 + Dynamic regulation of DNA methylation during mammalian development + + + 6 + 21 + surname:Bird;given-names:A. + 11782440 + REF + Genes Dev. + ref + 16 + 2002 + 48896 + DNA methylation patterns and epigenetic memory + + + 5559 + 5563 + surname:Bestor;given-names:T. H. + surname:Ingram;given-names:V. M. + 6577443 + REF + Proc. Natl Acad. Sci. USA + ref + 80 + 1983 + 48943 + Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA + + + 247 + 257 + surname:Okano;given-names:M. + surname:Bell;given-names:D. W. + surname:Haber;given-names:D. A. + surname:Li;given-names:E. + 10555141 + REF + Cell + ref + 99 + 1999 + 49074 + DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development + + + 2395 + 2402 + surname:Bestor;given-names:T. H. + 11005794 + REF + Hum. Mol. Genet. + ref + 9 + 2000 + 49179 + The DNA methyltransferases of mammals + + + 1760 + 1764 + surname:Bostick;given-names:M. + 17673620 + REF + Science + ref + 317 + 2007 + 49217 + UHRF1 plays a role in maintaining DNA methylation in mammalian cells + + + 908 + 912 + surname:Sharif;given-names:J. + 17994007 + REF + Nature + ref + 450 + 2007 + 49286 + The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA + + + 909 + 914 + surname:Bonapace;given-names:I. M. + 12058012 + REF + J. Cell Biol. + ref + 157 + 2002 + 49377 + Np95 is regulated by E1A during mitotic reactivation of terminally differentiated cells and is essential for S phase entry + + + 7337 + 7345 + surname:Jeanblanc;given-names:M. + 16007129 + REF + Oncogene + ref + 24 + 2005 + 49500 + The retinoblastoma gene and its product are targeted by ICBP90: a key mechanism in the G1/S transition during the cell cycle + + + 705 + 717 + surname:Karagianni;given-names:P. + surname:Amazit;given-names:L. + surname:Qin;given-names:J. + surname:Wong;given-names:J. + 17967883 + REF + Mol. Cell. Biol. + ref + 28 + 2008 + 49625 + ICBP90, a novel methyl K9 H3 binding protein linking protein ubiquitination with heterochromatin formation + + + 419 + 434 + surname:Bronner;given-names:C. + 17658611 + REF + Pharmacol. Ther. + ref + 115 + 2007 + 49732 + The UHRF family: oncogenes that are drugable targets for cancer therapy in the near future? + + + 5621 + 5629 + surname:Jenkins;given-names:Y. + 16195352 + REF + Mol. Biol. Cell + ref + 16 + 2005 + 49824 + Critical role of the ubiquitin ligase activity of UHRF1, a nuclear RING finger protein, in tumor cell growth + + + 120 + 127 + surname:Mousli;given-names:M. + 12838312 + REF + Br. J. Cancer + ref + 89 + 2003 + 49933 + ICBP90 belongs to a new family of proteins with an expression that is deregulated in cancer cells + + + 217 + 222 + surname:Unoki;given-names:M. + 20517312 + REF + Br. J. Cancer + ref + 103 + 2010 + 50031 + UHRF1 is a novel diagnostic marker of lung cancer + + + 12950 + 12955 + surname:Arita;given-names:K. + 22837395 + REF + Proc. Natl Acad. Sci. USA + ref + 109 + 2012 + 50081 + Recognition of modification status on a histone H3 tail by linked histone reader modules of the epigenetic regulator UHRF1 + + + 1329 + 1339 + surname:Cheng;given-names:J. + 23161542 + REF + J. Biol. Chem. + ref + 288 + 2013 + 50204 + Structural insight into coordinated recognition of trimethylated histone H3 lysine 9 (H3K9me3) by the plant homeodomain (PHD) and tandem tudor domain (TTD) of UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) protein + + + 1374 + 1378 + surname:Hu;given-names:L. + surname:Li;given-names:Z. + surname:Wang;given-names:P. + surname:Lin;given-names:Y. + surname:Xu;given-names:Y. + 21808300 + REF + Cell Res. + ref + 21 + 2011 + 50437 + Crystal structure of PHD domain of UHRF1 and insights into recognition of unmodified histone H3 arginine residue 2 + + + 24300 + 24311 + surname:Nady;given-names:N. + 21489993 + REF + J. Biol. Chem. + ref + 286 + 2011 + 50552 + Recognition of multivalent histone states associated with heterochromatin by UHRF1 protein + + + 275 + 284 + surname:Rajakumara;given-names:E. + 21777816 + REF + Mol. Cell + ref + 43 + 2011 + 50643 + PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression + + + 1288 + 1298 + surname:Rothbart;given-names:S. B. + 23752590 + REF + Genes Dev. + ref + 27 + 2013 + 50750 + Multivalent histone engagement by the linked tandem Tudor and PHD domains of UHRF1 is required for the epigenetic inheritance of DNA methylation + + + 1379 + 1382 + surname:Wang;given-names:C. + 21808299 + REF + Cell Res. + ref + 21 + 2011 + 50895 + Structural basis for site-specific reading of unmodified R2 of histone H3 tail by UHRF1 PHD finger + + + 818 + 821 + surname:Arita;given-names:K. + surname:Ariyoshi;given-names:M. + surname:Tochio;given-names:H. + surname:Nakamura;given-names:Y. + surname:Shirakawa;given-names:M. + 18772891 + REF + Nature + ref + 455 + 2008 + 50994 + Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism + + + 822 + 825 + surname:Avvakumov;given-names:G. V. + 18772889 + REF + Nature + ref + 455 + 2008 + 51083 + Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1 + + + 826 + 829 + surname:Hashimoto;given-names:H. + 18772888 + REF + Nature + ref + 455 + 2008 + 51172 + The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix + + + 2187 + 2197 + surname:Achour;given-names:M. + 17934516 + REF + Oncogene + ref + 27 + 2008 + 51240 + The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression + + + 4106 + 4115 + surname:Bashtrykov;given-names:P. + surname:Jankevicius;given-names:G. + surname:Jurkowska;given-names:R. Z. + surname:Ragozin;given-names:S. + surname:Jeltsch;given-names:A. + 24368767 + REF + J. Biol. Chem. + ref + 289 + 2014 + 51367 + The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism + + + 379 + 386 + surname:Berkyurek;given-names:A. C. + 24253042 + REF + J. Biol. Chem. + ref + 289 + 2014 + 51499 + The DNA methyltransferase Dnmt1 directly interacts with the SET and RING finger-associated (SRA) domain of the multifunctional protein Uhrf1 to facilitate accession of the catalytic center to hemi-methylated DNA + + + 905 + 919 + surname:Gelato;given-names:K. A. + 24813945 + REF + Mol. Cell + ref + 54 + 2014 + 51711 + Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate + + + rs3 + surname:Rigbolt;given-names:K. T. + 21406692 + REF + Sci. Signal. + ref + 4 + 2011 + 51838 + System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation + + + 1098 + 1106 + surname:Papait;given-names:R. + 17182844 + REF + Mol. Biol. Cell + ref + 18 + 2007 + 51957 + Np95 is implicated in pericentromeric heterochromatin replication and in major satellite silencing + + + 1563 + surname:Liu;given-names:X. + 23463006 + REF + Nat. Commun. + ref + 4 + 2013 + 52056 + UHRF1 targets DNMT1 for DNA methylation through cooperative binding of hemi-methylated DNA and methylated H3K9 + + + 249 + 253 + surname:Nishiyama;given-names:A. + 24013172 + REF + Nature + ref + 502 + 2013 + 52167 + Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication + + + 911 + 929 + surname:Qin;given-names:W. + 26065575 + REF + Cell Res. + ref + 25 + 2015 + 52256 + DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination + + + 4828 + 4833 + surname:Ma;given-names:H. + 22411829 + REF + Proc. Natl Acad. Sci. USA + ref + 109 + 2012 + 52350 + M phase phosphorylation of the epigenetic regulator UHRF1 regulates its physical association with the deubiquitylase USP7 and stability + + + 121 + 128 + surname:Hopfner;given-names:R. + 10646863 + REF + Cancer Res. + ref + 60 + 2000 + 52486 + ICBP90, a novel human CCAAT binding protein, involved in the regulation of topoisomerase IIalpha expression + + + ra3 + surname:Olsen;given-names:J. V. + 20068231 + REF + Sci. Signal. + ref + 3 + 2010 + 52594 + Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis + + + 59 + 70 + surname:Chu;given-names:J. + 22072796 + REF + Mol. Biol. Cell + ref + 23 + 2012 + 52695 + UHRF1 phosphorylation by cyclin A2/cyclin-dependent kinase 2 is required for zebrafish embryogenesis + + + 267 + 277 + surname:Woo;given-names:H. R. + surname:Pontes;given-names:O. + surname:Pikaard;given-names:C. S. + surname:Richards;given-names:E. J. + 17242155 + REF + Genes Dev. + ref + 21 + 2007 + 52796 + VIM1, a methylcytosine-binding protein required for centromeric heterochromatinization + + + 2585 + 2593 + surname:Pichler;given-names:G. + 21598301 + REF + J. Cell. Biochem. + ref + 112 + 2011 + 52883 + Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways + + + 1723 + 1739 + surname:Zhang;given-names:J. + 22064703 + REF + Cell Res. + ref + 21 + 2011 + 52979 + S phase-dependent interaction with DNMT1 dictates the role of UHRF1 but not UHRF2 in DNA methylation maintenance + + + 911 + 929 + surname:Qin;given-names:W. H. + 26065575 + REF + Cell Res. + ref + 25 + 2015 + 53092 + DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination + + + 709 + 712 + surname:Song;given-names:J. + surname:Teplova;given-names:M. + surname:Ishibe-Murakami;given-names:S. + surname:Patel;given-names:D. J. + 22323818 + REF + Science + ref + 335 + 2012 + 53186 + Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation + + + 1036 + 1040 + surname:Song;given-names:J. + surname:Rechkoblit;given-names:O. + surname:Bestor;given-names:T. H. + surname:Patel;given-names:D. J. + 21163962 + REF + Science + ref + 331 + 2011 + 53271 + Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation + + + 22 + 34 + surname:Clore;given-names:G. M. + surname:Gronenborn;given-names:A. M. + 9470228 + REF + Trends Biotechnol. + ref + 16 + 1998 + 53367 + Determining the structures of large proteins and protein complexes by NMR + + + 11054 + 11055 + surname:Yamazaki;given-names:T. + surname:F.-K.;given-names:J. + surname:Kay;given-names:L. E. + REF + J. Am. Chem. Soc. + ref + 115 + 1993 + 53441 + Two-dimensional NMR experiments for correlating carbon-13. beta. and proton. delta./. epsilon. chemical shifts of aromatic residues in 13C-labeled proteins via scalar couplings + + + 277 + 293 + surname:Delaglio;given-names:F. + 8520220 + REF + J. Biomol. NMR. + ref + 6 + 1995 + 53618 + NMRPipe: a multidimensional spectral processing system based on UNIX pipes + + + 249 + 254 + surname:Kuszewski;given-names:J. + surname:Clore;given-names:G. M. + 11001840 + REF + J. Magn. Reson. + ref + 146 + 2000 + 53693 + Sources of and solutions to problems in the refinement of protein NMR structures against torsion angle potentials of mean force + + + 289 + 302 + surname:Cornilescu;given-names:G. + surname:Delaglio;given-names:F. + surname:Bax;given-names:A. + 10212987 + REF + J. Biomol. NMR + ref + 13 + 1999 + 53821 + Protein backbone angle restraints from searching a database for chemical shift and sequence homology + + + 283 + 291 + surname:Laskowski;given-names:R. A. + surname:MacArthur;given-names:M. W. + surname:Moss;given-names:D. S. + surname:Thornton;given-names:J. M. + REF + J. Appl. Crystallogr. + ref + 26 + 1993 + 53922 + PROCHECK: a program to check the stereochemical quality of protein structures + + + 477 + 486 + surname:Laskowski;given-names:R. A. + surname:Rullmannn;given-names:J. A. + surname:MacArthur;given-names:M. W. + surname:Kaptein;given-names:R. + surname:Thornton;given-names:J. M. + 9008363 + REF + J. Biomol. NMR + ref + 8 + 1996 + 54000 + AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR + + + surname:DeLano;given-names:W. L. + REF + ref + 2002 + 54093 + + + 51 + 55 + surname:Koradi;given-names:R. + surname:Billeter;given-names:M. + surname:Wuthrich;given-names:K. + 8744573 + REF + J. Mol. Graph. + ref + 14 + 1996 + 54094 + MOLMOL: a program for display and analysis of macromolecular structures + + + 307 + 326 + surname:Otwinowski;given-names:Z. + surname:Minor;given-names:W. + REF + Methods Enzymol. + ref + 276 + 1997 + 54166 + Processing of X-ray diffraction data collected in oscillation mode + + + 458 + 464 + surname:McCoy;given-names:A. J. + surname:Grosse-Kunstleve;given-names:R. W. + surname:Storoni;given-names:L. C. + surname:Read;given-names:R. J. + 15805601 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 61 + 2005 + 54233 + Likelihood-enhanced fast translation functions + + + 2126 + 2132 + surname:Emsley;given-names:P. + surname:Cowtan;given-names:K. + 15572765 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 60 + 2004 + 54280 + Coot: model-building tools for molecular graphics + + + 1948 + 1954 + surname:Adams;given-names:P. D. + 12393927 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 58 + 2002 + 54330 + PHENIX: building new software for automated crystallographic structure determination + + + SUPPL + footnote + 54415 + Author contributions J.F., H.Y., C.C. and Y.X. designed the experiments. J.F., J.C., M.L., R.G., X.Z. and Y.F. performed protein purification and crystallization of SRA-Spacer-DNA complex. J.F., J.C. and P.W. collected the data and determined the crystal structure. Jia.W. and W.L. performed the NMR titration assay. Jiaolong W. determined the NMR structure of TTD-Spacer complex. Q.Z. performed all the cell-based assays. J.F., M.L. and J.C. performed protein interaction assays. J.F. and Z.G. performed the SAXS measurements. C.T., W.L., C.C. and Jie.W. helped in the data analysis. J.F., H.Y. and Y.X. analysed the data and wrote the manuscript. Y.X. supervised the project. + + + ncomms11197-f1.jpg + f1 + FIG + fig_title_caption + 55093 + Hm-DNA facilities histione tails recognition by full-length UHRF1. + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:58:26Z + + Hm-DNA + + + protein_type + MESH: + cleaner0 + 2023-07-25T16:33:30Z + + histione + + + 0.9991283 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + full-length + + + 0.99892884 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + + ncomms11197-f1.jpg + f1 + FIG + fig_caption + 55160 + (a) Colour-coded domain structure of human UHRF1. The boundaries of the domains are indicated with the numbers representing the amino-acid positions. Note that the conserved motif (green background) of the Linker (residues 286–306) and the Spacer (residues 587–674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1. Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). The mixture was immobilized onto streptavidin Sepharose beads. The bound proteins were analysed in SDS–PAGE followed by Coomassie blue staining. Sequences of the peptides are indicated in Supplementary Table 1. (c) Histone peptides do not affect hm-DNA-binding affinity of UHRF1. Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). The estimated binding affinities (KD) are listed. The samples in the syringe (designated Sy hereafter) and cell are indicated. + + 0.9987324 + species + cleaner0 + 2023-07-25T16:39:11Z + MESH: + + human + + + 0.99917275 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.9983405 + protein_state + cleaner0 + 2023-07-25T16:51:01Z + DUMMY: + + conserved + + + 0.9991084 + structure_element + cleaner0 + 2023-07-25T15:34:29Z + SO: + + Linker + + + 0.9964264 + residue_range + cleaner0 + 2023-07-25T16:38:52Z + DUMMY: + + 286–306 + + + 0.99924755 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + 0.99628085 + residue_range + cleaner0 + 2023-07-25T16:38:56Z + DUMMY: + + 587–674 + + + 0.9995442 + structure_element + cleaner0 + 2023-07-25T14:57:22Z + SO: + + TTD + + + 0.9703517 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + Hm-DNA + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:09Z + + histone + + + 0.48119324 + protein_type + cleaner0 + 2023-07-25T15:07:50Z + MESH: + + H3 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T13:02:11Z + + H3 + + + 0.9991829 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.9990461 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + full-length + + + 0.99922454 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.87862474 + protein_state + cleaner0 + 2023-07-25T16:51:05Z + DUMMY: + + biotinylated + + + 0.91468906 + protein_type + cleaner0 + 2023-07-25T15:07:50Z + MESH: + + H3 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-08-08T13:03:21Z + + 1–21 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T13:02:23Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T13:02:42Z + + K9me3 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-08-08T13:03:32Z + + 1–21 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:50:14Z + + absence of + + + 0.99181557 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.99652356 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.96265155 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + 0.9976464 + experimental_method + cleaner0 + 2023-07-25T16:42:52Z + MESH: + + SDS–PAGE + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:09Z + + Histone + + + 0.85392904 + evidence + cleaner0 + 2023-07-25T14:56:51Z + DUMMY: + + hm-DNA-binding affinity + + + 0.99919266 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.9990065 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + Full-length + + + 0.9992055 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.8146759 + experimental_method + cleaner0 + 2023-07-25T16:42:55Z + MESH: + + incubated with + + + 0.57842284 + protein_state + cleaner0 + 2023-07-25T16:51:08Z + DUMMY: + + biotinylated + + + 0.99434835 + chemical + cleaner0 + 2023-07-25T14:58:26Z + CHEBI: + + hm-DNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:50:14Z + + absence of + + + 0.9817586 + protein_type + cleaner0 + 2023-07-25T15:07:50Z + MESH: + + H3 + + + residue_range + DUMMY: + cleaner0 + 2023-07-25T16:23:35Z + + 1–17 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:59:02Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:59:11Z + + K9me3 + + + residue_range + DUMMY: + cleaner0 + 2023-07-25T16:23:36Z + + 1–17 + + + 0.99561787 + experimental_method + cleaner0 + 2023-07-25T16:42:58Z + MESH: + + ITC + + + 0.96477485 + evidence + cleaner0 + 2023-07-25T16:46:04Z + DUMMY: + + enthalpy plots + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:59:33Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:59:26Z + + K9me3 + + + residue_range + DUMMY: + cleaner0 + 2023-07-25T16:23:36Z + + 1–17 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + 0.99911135 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + full-length + + + 0.9992053 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.9958145 + protein_type + cleaner0 + 2023-07-25T15:07:50Z + MESH: + + H3 + + + 0.6578081 + residue_range + cleaner0 + 2023-07-25T16:23:36Z + DUMMY: + + 1–17 + + + 0.99953353 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.99913436 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + full-length + + + 0.9992557 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.99829507 + evidence + cleaner0 + 2023-07-25T16:46:09Z + DUMMY: + + binding affinities + + + 0.99740285 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + + ncomms11197-f2.jpg + f2 + FIG + fig_title_caption + 56452 + Intramolecular interactions inhibit histone recognition by UHRF1. + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:09Z + + histone + + + 0.9990262 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + + ncomms11197-f2.jpg + f2 + FIG + fig_caption + 56518 + (a) GST pull-down assays for the intramolecular interactions. The isolated domains of UHRF1 were incubated with GST-tagged TTD or PHD immobilized on glutathione resin. The bound proteins were analysed by SDS–PAGE and Coomassie blue staining. (b,d) Superimposed ITC enthalpy plots for the intramolecular interactions of isolated UHRF1 domains. The estimated binding affinities (KD) were listed. ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). ND, not determined; Sy., syringe. + + 0.9989753 + experimental_method + cleaner0 + 2023-07-25T16:24:11Z + MESH: + + GST pull-down assays + + + 0.9977849 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.97791547 + experimental_method + cleaner0 + 2023-07-25T16:43:03Z + MESH: + + incubated + + + 0.9943831 + protein_state + cleaner0 + 2023-07-25T16:24:19Z + DUMMY: + + GST-tagged + + + 0.99942833 + structure_element + cleaner0 + 2023-07-25T14:57:22Z + SO: + + TTD + + + 0.9994708 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.9921228 + experimental_method + cleaner0 + 2023-07-25T16:43:06Z + MESH: + + SDS–PAGE + + + 0.9880918 + experimental_method + cleaner0 + 2023-07-25T16:43:09Z + MESH: + + ITC + + + 0.9660795 + evidence + cleaner0 + 2023-07-25T16:46:13Z + DUMMY: + + enthalpy plots + + + 0.9971623 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.9986558 + evidence + cleaner0 + 2023-07-25T16:46:22Z + DUMMY: + + binding affinities + + + 0.99723214 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.9923856 + experimental_method + cleaner0 + 2023-07-25T16:43:23Z + MESH: + + ITC + + + 0.981375 + evidence + cleaner0 + 2023-07-25T16:46:16Z + DUMMY: + + enthalpy plots + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:59:50Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T12:59:58Z + + K9me3 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + 0.99501556 + protein_state + cleaner0 + 2023-07-25T16:51:14Z + DUMMY: + + absence + + + 0.99276066 + protein_state + cleaner0 + 2023-07-25T15:10:25Z + DUMMY: + + presence of + + + 0.9653634 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + 0.9408276 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + 0.9922605 + experimental_method + cleaner0 + 2023-07-25T16:43:27Z + MESH: + + ITC + + + 0.98671687 + evidence + cleaner0 + 2023-07-25T16:46:18Z + DUMMY: + + enthalpy plots + + + 0.5302331 + protein_type + cleaner0 + 2023-07-25T15:07:50Z + MESH: + + H3 + + + structure_element + SO: + cleaner0 + 2023-07-25T15:47:40Z + + PHD–SRA + + + 0.9993455 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.99725914 + protein_state + cleaner0 + 2023-07-25T16:51:17Z + DUMMY: + + absence + + + 0.9969782 + protein_state + cleaner0 + 2023-07-25T15:10:25Z + DUMMY: + + presence of + + + 0.9905939 + structure_element + cleaner0 + 2023-07-25T14:57:37Z + SO: + + SRA + + + 0.99839264 + structure_element + cleaner0 + 2023-07-25T14:57:50Z + SO: + + PHD + + + 0.98487955 + structure_element + cleaner0 + 2023-07-25T14:57:37Z + SO: + + SRA + + + + ncomms11197-f3.jpg + f3 + FIG + fig_title_caption + 57271 + NMR structure of the TTD bound to the Spacer. + + 0.9986047 + experimental_method + cleaner0 + 2023-07-25T16:43:31Z + MESH: + + NMR + + + 0.998346 + evidence + cleaner0 + 2023-07-25T16:46:27Z + DUMMY: + + structure + + + 0.99939525 + structure_element + cleaner0 + 2023-07-25T14:57:22Z + SO: + + TTD + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:10:33Z + + bound to + + + 0.9795172 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + + ncomms11197-f3.jpg + f3 + FIG + fig_caption + 57317 + (a) Ribbon representation of TTD–Spacer structure. N- and C-termini of the Spacer are indicated. The TTD is coloured in green, and the Spacer is coloured in yellow. The colour scheme is used the all the structure figures. (b) Superimposition of TTD–Spacer and TTD–PHD–H3K9me3 (4GY5.PDB) structures shown in ribbon representations. The TTD is coloured in green and the Spacer in yellow in TTD–Spacer structure. TTD–PHD–H3K9me3 complex is coloured in grey, and the PHD and H3K9me3 are omitted for simplicity. Residues for the interactions are shown in stick representation. (c) Electrostatic potential surface representation of the TTD with the Spacer shown in ribbon representation. The critical residues on the Spacer for the interaction are shown in stick representation. (d) Close-up view of TTD–Spacer interaction. Critical residues for the interaction are shown in stick representation. Hydrogen bonds are indicated as dashed lines. (e–g) Superimposed ITC enthalpy plots for the interaction between the Spacer and the TTD (or TTD–PHD) with the estimated binding affinity (KD) indicated. Wild-type and mutant proteins for the measurements are indicated. (h) GST pull-down assays for the intramolecular interactions. The wild-type or indicated truncations of UHRF1 were incubated with GST-tagged TTD, Linker or Spacer. The mixtures were immobilized on glutathione resin. The bound proteins were analysed by SDS–PAGE and Coomassie blue staining. ND, not determined; Sy., syringe. + + 0.99809724 + structure_element + cleaner0 + 2023-07-25T15:17:21Z + SO: + + TTD–Spacer + + + 0.8751053 + evidence + cleaner0 + 2023-07-25T16:46:30Z + DUMMY: + + structure + + + 0.9955915 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + 0.99941146 + structure_element + cleaner0 + 2023-07-25T14:57:22Z + SO: + + TTD + + + 0.99854636 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + 0.988893 + experimental_method + cleaner0 + 2023-07-25T16:25:01Z + MESH: + + Superimposition + + + 0.9639978 + structure_element + cleaner0 + 2023-07-25T15:17:21Z + SO: + + TTD–Spacer + + + 0.99869716 + complex_assembly + cleaner0 + 2023-07-25T15:22:29Z + GO: + + TTD–PHD–H3K9me3 + + + 0.5059854 + evidence + cleaner0 + 2023-07-25T16:46:33Z + DUMMY: + + structures + + + 0.99940574 + structure_element + cleaner0 + 2023-07-25T14:57:22Z + SO: + + TTD + + + 0.99869233 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + 0.99854136 + structure_element + cleaner0 + 2023-07-25T15:17:21Z + SO: + + TTD–Spacer + + + 0.8394155 + evidence + cleaner0 + 2023-07-25T16:46:36Z + DUMMY: + + structure + + + 0.99902695 + complex_assembly + cleaner0 + 2023-07-25T15:22:29Z + GO: + + TTD–PHD–H3K9me3 + + + 0.9988858 + structure_element + cleaner0 + 2023-07-25T14:57:51Z + SO: + + PHD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T13:00:15Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T13:00:24Z + + K9me3 + + + 0.99935716 + structure_element + cleaner0 + 2023-07-25T14:57:22Z + SO: + + TTD + + + 0.9981571 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + 0.9972881 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + 0.9962218 + bond_interaction + cleaner0 + 2023-07-25T15:24:59Z + MESH: + + Hydrogen bonds + + + 0.99756014 + experimental_method + cleaner0 + 2023-07-25T16:43:38Z + MESH: + + ITC + + + 0.9872527 + evidence + cleaner0 + 2023-07-25T16:46:39Z + DUMMY: + + enthalpy plots + + + 0.9953733 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + 0.9989366 + structure_element + cleaner0 + 2023-07-25T14:57:22Z + SO: + + TTD + + + 0.9958517 + structure_element + cleaner0 + 2023-07-25T15:11:21Z + SO: + + TTD–PHD + + + 0.99859107 + evidence + cleaner0 + 2023-07-25T15:10:18Z + DUMMY: + + binding affinity + + + 0.9974821 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.9990232 + protein_state + cleaner0 + 2023-07-25T15:34:11Z + DUMMY: + + Wild-type + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T16:00:50Z + + mutant + + + 0.99896955 + experimental_method + cleaner0 + 2023-07-25T16:26:12Z + MESH: + + GST pull-down assays + + + 0.9991037 + protein_state + cleaner0 + 2023-07-25T15:34:11Z + DUMMY: + + wild-type + + + 0.99932194 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.9956762 + protein_state + cleaner0 + 2023-07-25T16:26:25Z + DUMMY: + + GST-tagged + + + 0.9994342 + structure_element + cleaner0 + 2023-07-25T14:57:22Z + SO: + + TTD + + + 0.99938715 + structure_element + cleaner0 + 2023-07-25T15:34:29Z + SO: + + Linker + + + 0.99925107 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + 0.99821776 + experimental_method + cleaner0 + 2023-07-25T16:43:43Z + MESH: + + SDS–PAGE + + + + ncomms11197-f4.jpg + f4 + FIG + fig_title_caption + 58820 + Hm-DNA impairs the intramolecular interaction of UHRF1 and facilitates its histone recognition. + + 0.9944139 + chemical + cleaner0 + 2023-07-25T14:58:27Z + CHEBI: + + Hm-DNA + + + 0.9990503 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + protein_type + MESH: + cleaner0 + 2023-07-25T15:05:09Z + + histone + + + + ncomms11197-f4.jpg + f4 + FIG + fig_caption + 58916 + (a) Hm-DNA impairs the intramolecular interaction of PHD–SRA. The SRA was incubated with GST-tagged PHD in the presence of increasing concentrations of hm-DNA and immobilized on glutathione resin. The bound proteins were analysed in SDS–PAGE and Coomassie blue staining (left) and quantified by band densitometry (right). Error bars, s.d. for triplicate experiments. (b) Purified fragments of UHRF1 were analysed by histone peptide (H3K9me0) pull-down assay as described in Fig. 1b. (c) Hm-DNA impairs the intramolecular interaction of TTD–Spacer. SRA–Spacer was incubated with GST-tagged TTD–PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining. Error bars, s.d. for triplicate experiments. (d) Histone peptide pull-down assay using UHRF1 mutants as indicated. The assays were performed in the presence (+DTT) or absence (−DTT) of 15 mM DTT. + + 0.9981992 + chemical + cleaner0 + 2023-07-25T14:58:27Z + CHEBI: + + Hm-DNA + + + structure_element + SO: + cleaner0 + 2023-07-25T15:47:40Z + + PHD–SRA + + + 0.99943036 + structure_element + cleaner0 + 2023-07-25T14:57:37Z + SO: + + SRA + + + 0.98517853 + experimental_method + cleaner0 + 2023-07-25T16:43:49Z + MESH: + + incubated + + + 0.9960082 + protein_state + cleaner0 + 2023-07-25T16:51:22Z + DUMMY: + + GST-tagged + + + 0.9994848 + structure_element + cleaner0 + 2023-07-25T14:57:51Z + SO: + + PHD + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:10:25Z + + presence of + + + 0.9988224 + chemical + cleaner0 + 2023-07-25T14:58:27Z + CHEBI: + + hm-DNA + + + 0.9975583 + experimental_method + cleaner0 + 2023-07-25T16:44:14Z + MESH: + + SDS–PAGE + + + experimental_method + MESH: + cleaner0 + 2023-07-25T16:44:05Z + + Coomassie blue staining + + + 0.97438693 + experimental_method + cleaner0 + 2023-07-25T16:44:09Z + MESH: + + band densitometry + + + 0.9990325 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.994632 + experimental_method + cleaner0 + 2023-07-25T16:27:22Z + MESH: + + histone peptide + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-08-08T13:00:40Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-08-08T13:00:52Z + + K9me0 + + + 0.99860966 + experimental_method + cleaner0 + 2023-07-25T16:27:25Z + MESH: + + pull-down assay + + + 0.9979406 + chemical + cleaner0 + 2023-07-25T14:58:27Z + CHEBI: + + Hm-DNA + + + structure_element + SO: + cleaner0 + 2023-07-25T15:17:21Z + + TTD–Spacer + + + structure_element + SO: + cleaner0 + 2023-07-25T15:48:56Z + + SRA–Spacer + + + 0.9862408 + experimental_method + cleaner0 + 2023-07-25T16:44:21Z + MESH: + + incubated + + + 0.9954321 + protein_state + cleaner0 + 2023-07-25T16:51:25Z + DUMMY: + + GST-tagged + + + structure_element + SO: + cleaner0 + 2023-07-25T15:11:21Z + + TTD–PHD + + + 0.9994454 + structure_element + cleaner0 + 2023-07-25T14:57:22Z + SO: + + TTD + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T15:10:25Z + + presence of + + + 0.99866724 + chemical + cleaner0 + 2023-07-25T14:58:27Z + CHEBI: + + hm-DNA + + + 0.9964418 + experimental_method + cleaner0 + 2023-07-25T16:44:24Z + MESH: + + pull-down experiment + + + 0.6687745 + experimental_method + cleaner0 + 2023-07-25T16:47:04Z + MESH: + + band densitometries + + + experimental_method + MESH: + cleaner0 + 2023-07-25T16:47:18Z + + Coomassie blue staining + + + 0.99877673 + experimental_method + cleaner0 + 2023-07-25T16:27:38Z + MESH: + + Histone peptide pull-down assay + + + 0.998818 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.9971113 + protein_state + cleaner0 + 2023-07-25T16:00:01Z + DUMMY: + + mutants + + + 0.9741964 + chemical + cleaner0 + 2023-07-25T15:51:51Z + CHEBI: + + DTT + + + chemical + CHEBI: + cleaner0 + 2023-07-25T15:51:51Z + + DTT + + + 0.9975981 + chemical + cleaner0 + 2023-07-25T15:51:51Z + CHEBI: + + DTT + + + + ncomms11197-f5.jpg + f5 + FIG + fig_title_caption + 59925 + The Spacer facilitates hm-DNA–SRA interaction and DNMT1–UHRF1 interaction. + + 0.99668473 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + 0.97400206 + chemical + cleaner0 + 2023-07-25T14:58:27Z + CHEBI: + + hm-DNA + + + 0.9543789 + structure_element + cleaner0 + 2023-07-25T14:57:37Z + SO: + + SRA + + + 0.90869576 + complex_assembly + cleaner0 + 2023-07-25T16:30:45Z + GO: + + DNMT1–UHRF1 + + + + ncomms11197-f5.jpg + f5 + FIG + fig_caption + 60004 + (a) Superimposed ITC enthalpy plots for hm-DNA-binding affinities of the SRA, the Spacer and SRA–Spacer. (b,c) Superimposed fluorescence polarization (FP) plots for hm-DNA-binding affinities of truncations or full-length UHRF1. The estimated binding affinities (KD) are listed above. (d) Subcellular localization of GFP-tagged wild-type or indicated mutants of UHRF1 in NIH3T3 cells. The percentages of cells showing co-localization with DAPI foci were counted from at least 100 cells and shown on the left of the corresponding representative confocal microscopy. The experiment was repeated three times in the same condition. Scale bar, 5 μm. (e) GST pull-down experiment for the interactions between wild-type or truncations of UHRF1 and RFTSDNMT1 as described in Fig. 2a. (f) Working model for hm-DNA-mediated conformational changes of UHRF1, as described in the Discussion. ND, not determined; Sy., syringe. + + 0.9923235 + experimental_method + cleaner0 + 2023-07-25T16:28:50Z + MESH: + + ITC + + + 0.9836543 + evidence + cleaner0 + 2023-07-25T16:47:28Z + DUMMY: + + enthalpy plots + + + 0.9968479 + evidence + cleaner0 + 2023-07-25T15:53:51Z + DUMMY: + + hm-DNA-binding affinities + + + 0.99944144 + structure_element + cleaner0 + 2023-07-25T14:57:37Z + SO: + + SRA + + + 0.9984194 + structure_element + cleaner0 + 2023-07-25T14:57:07Z + SO: + + Spacer + + + structure_element + SO: + cleaner0 + 2023-07-25T15:48:56Z + + SRA–Spacer + + + evidence + DUMMY: + cleaner0 + 2023-07-25T16:28:30Z + + fluorescence polarization (FP) plots + + + 0.99808145 + evidence + cleaner0 + 2023-07-25T15:53:51Z + DUMMY: + + hm-DNA-binding affinities + + + 0.99890304 + protein_state + cleaner0 + 2023-07-25T15:09:01Z + DUMMY: + + full-length + + + 0.99899775 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.9986728 + evidence + cleaner0 + 2023-07-25T16:47:34Z + DUMMY: + + binding affinities + + + 0.9979559 + evidence + cleaner0 + 2023-07-25T15:10:11Z + DUMMY: + + KD + + + 0.9150217 + protein_state + cleaner0 + 2023-07-25T15:59:47Z + DUMMY: + + GFP-tagged + + + 0.9991537 + protein_state + cleaner0 + 2023-07-25T15:34:11Z + DUMMY: + + wild-type + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T16:00:01Z + + mutants + + + 0.99895537 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T16:00:11Z + + DAPI + + + 0.9746206 + experimental_method + cleaner0 + 2023-07-25T16:28:45Z + MESH: + + confocal microscopy + + + 0.99787396 + experimental_method + cleaner0 + 2023-07-25T16:28:43Z + MESH: + + GST pull-down experiment + + + 0.9991854 + protein_state + cleaner0 + 2023-07-25T15:34:11Z + DUMMY: + + wild-type + + + 0.66345346 + experimental_method + cleaner0 + 2023-07-25T16:28:47Z + MESH: + + truncations + + + 0.99900025 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + 0.9981184 + protein + cleaner0 + 2023-07-25T16:02:13Z + PR: + + RFTSDNMT1 + + + 0.9931757 + chemical + cleaner0 + 2023-07-25T14:58:27Z + CHEBI: + + hm-DNA + + + 0.9990509 + protein + cleaner0 + 2023-07-25T14:58:05Z + PR: + + UHRF1 + + + + diff --git a/BioC_XML/4822561_v0.xml b/BioC_XML/4822561_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..b203f0fb609f0875cf52c07236c387135b9745ab --- /dev/null +++ b/BioC_XML/4822561_v0.xml @@ -0,0 +1,4015 @@ + + + + PMC + 20201216 + pmc.key + + 4822561 + CC BY + no + 0 + 0 + + Quinolone-stabilized cleavage complex of topoisomerase IV + ACSDAD + 10.1107/S2059798316001212 + S2059798316001212 + 4822561 + 27050128 + mn5108 + 488 + Pt 4 + Klebsiella pneumoniae cleavage complex quinolone levofloxacin topoisomerase IV DNA binding isomerase isomerase–DNA complex topoisomerases Gram-negative complexes X-ray crystallography protein–DNA–drug complexes + This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits +unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. + 496 + surname:Veselkov;given-names:Dennis A. + surname:Laponogov;given-names:Ivan + surname:Pan;given-names:Xiao-Su + surname:Selvarajah;given-names:Jogitha + surname:Skamrova;given-names:Galyna B. + surname:Branstrom;given-names:Arthur + surname:Narasimhan;given-names:Jana + surname:Prasad;given-names:Josyula V. N. Vara + surname:Fisher;given-names:L. Mark + surname:Sanderson;given-names:Mark R. + TITLE + front + 72 + 2016 + 0 + Structure of a quinolone-stabilized cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex + + 0.9950353 + evidence + cleaner0 + 2023-07-25T14:48:09Z + DUMMY: + + Structure + + + 0.83335567 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.9984047 + species + cleaner0 + 2023-07-25T13:55:59Z + MESH: + + Klebsiella pneumoniae + + + 0.99826175 + species + cleaner0 + 2023-07-25T13:56:04Z + MESH: + + Streptococcus pneumoniae + + + + ABSTRACT + abstract + 163 + Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared. For K. pneumoniae, this is the first high-resolution cleavage complex structure to be reported. + + 0.9985715 + evidence + cleaner0 + 2023-07-25T14:48:17Z + DUMMY: + + Crystal structures + + + 0.8690473 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.9693932 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:13Z + DUMMY: + + Gram-negative + + + 0.9980283 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.96933335 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:18Z + DUMMY: + + Gram-positive + + + 0.9981567 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.9947513 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:23Z + DUMMY: + + bacterial + + + 0.9992244 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.99786574 + species + cleaner0 + 2023-07-25T13:56:27Z + MESH: + + K. pneumoniae + + + 0.9451186 + evidence + cleaner0 + 2023-07-25T14:48:19Z + DUMMY: + + structure + + + + ABSTRACT + abstract + 522 + Klebsiella pneumoniae is a Gram-negative bacterium that is responsible for a range of common infections, including pulmonary pneumonia, bloodstream infections and meningitis. Certain strains of Klebsiella have become highly resistant to antibiotics. Despite the vast amount of research carried out on this class of bacteria, the molecular structure of its topoisomerase IV, a type II topoisomerase essential for catalysing chromosomal segregation, had remained unknown. In this paper, the structure of its DNA-cleavage complex is reported at 3.35 Å resolution. The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. This complex is compared with a similar complex from Streptococcus pneumoniae, which has recently been solved. + + 0.9980265 + species + cleaner0 + 2023-07-25T13:55:59Z + MESH: + + Klebsiella pneumoniae + + + 0.978526 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:41Z + DUMMY: + + Gram-negative bacterium + + + 0.8935523 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:53Z + DUMMY: + + Klebsiella + + + 0.9746953 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:49Z + DUMMY: + + bacteria + + + 0.9873208 + evidence + cleaner0 + 2023-07-25T14:48:23Z + DUMMY: + + structure + + + 0.81394833 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.99805975 + protein_type + cleaner0 + 2023-07-25T13:59:39Z + MESH: + + type II topoisomerase + + + 0.9964181 + evidence + cleaner0 + 2023-07-25T14:48:25Z + DUMMY: + + structure + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + protein + PR: + cleaner0 + 2023-07-25T14:03:41Z + + ParC + + + structure_element + SO: + cleaner0 + 2023-07-25T14:06:01Z + + breakage-reunion + + + protein + PR: + cleaner0 + 2023-07-25T14:03:46Z + + ParE + + + structure_element + SO: + cleaner0 + 2023-07-25T14:06:16Z + + TOPRIM + + + 0.99839836 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.60313827 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.99501544 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.9991844 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.9865645 + chemical + cleaner0 + 2023-07-25T14:01:12Z + CHEBI: + + fluoroquinolone + + + 0.998348 + species + cleaner0 + 2023-07-25T13:56:04Z + MESH: + + Streptococcus pneumoniae + + + + INTRO + title_1 + 1397 + Introduction   + + + INTRO + paragraph + 1414 + Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. K. pneumoniae is the most medically important species of the genus owing to its high resistance to antibiotics. Significant morbidity and mortality has been associated with an emerging, highly drug-resistant strain of K. pneumoniae characterized as producing the carbapenemase enzyme (KPC-producing bacteria; Nordmann et al., 2009). The best therapeutic approach to KPC-producing organisms has yet to be defined. However, common treatments (based on in vitro susceptibility testing) are the polymyxins, tigecycline and, less frequently, aminoglycoside antibiotics (Arnold et al., 2011). Another effective strategy involves the limited use of certain antimicrobials, specifically fluoroquinolones and cephalo­sporins (Gasink et al., 2009). Several new antibiotics are under development for KPC producers. These include combinations of existing β-lactam antibiotics with new β-lactamase inhibitors able to circumvent KPC resistance. Neoglycosides are novel aminoglycosides that have activity against KPC-producing bacteria that are also being developed (Chen et al., 2012). + + 0.7833224 + taxonomy_domain + cleaner0 + 2023-07-25T13:57:44Z + DUMMY: + + Klebsiella + + + 0.9896713 + taxonomy_domain + cleaner0 + 2023-07-25T13:57:24Z + DUMMY: + + Enterobacteriaceae + + + 0.95838034 + taxonomy_domain + cleaner0 + 2023-07-25T13:57:28Z + DUMMY: + + Gram-negative bacilli + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + 0.99668694 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.9967196 + species + cleaner0 + 2023-07-25T13:57:54Z + MESH: + + K. ozaenae + + + 0.99659187 + species + cleaner0 + 2023-07-25T13:57:58Z + MESH: + + K. rhinoscleromatis + + + 0.99684733 + species + cleaner0 + 2023-07-25T13:58:03Z + MESH: + + K. oxytoca + + + 0.99637413 + species + cleaner0 + 2023-07-25T13:58:07Z + MESH: + + K. planticola + + + 0.9967933 + species + cleaner0 + 2023-07-25T13:58:12Z + MESH: + + K. terrigena + + + 0.996679 + species + cleaner0 + 2023-07-25T13:58:16Z + MESH: + + K. ornithinolytica + + + 0.9955092 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.9969053 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.99565375 + protein_type + cleaner0 + 2023-07-25T14:01:33Z + MESH: + + carbapenemase + + + 0.9940204 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:49Z + DUMMY: + + bacteria + + + 0.9555781 + experimental_method + cleaner0 + 2023-07-25T14:52:53Z + MESH: + + in vitro susceptibility testing + + + 0.99856734 + chemical + cleaner0 + 2023-07-25T14:01:56Z + CHEBI: + + polymyxins + + + 0.9990127 + chemical + cleaner0 + 2023-07-25T14:02:00Z + CHEBI: + + tigecycline + + + 0.9477348 + chemical + cleaner0 + 2023-07-25T14:02:04Z + CHEBI: + + aminoglycoside + + + 0.9765737 + chemical + cleaner0 + 2023-07-25T14:42:26Z + CHEBI: + + fluoroquinolones + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:42:41Z + + cephalo­sporins + + + 0.9234915 + protein_type + cleaner0 + 2023-07-25T14:43:23Z + MESH: + + β-lactamase + + + 0.9992853 + chemical + cleaner0 + 2023-07-25T14:02:10Z + CHEBI: + + Neoglycosides + + + 0.99875367 + chemical + cleaner0 + 2023-07-25T14:02:18Z + CHEBI: + + aminoglycosides + + + 0.9785209 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:49Z + DUMMY: + + bacteria + + + + INTRO + paragraph + 2779 + Type II topoisomerase enzymes play important roles in prokaryotic and eukaryotic DNA replication, recombination and transcription (Drlica et al., 2008; Laponogov et al., 2013; Lee et al., 2013; Nitiss, 2009a ,b ; Schoeffler & Berger, 2008; Sissi & Palumbo, 2009; Vos et al., 2011; Wendorff et al., 2012; Wu et al., 2011, 2013). In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks. Both enzymes act via a double-strand DNA break involving a cleavage complex and are targets for quinolone antimicrobials that act by trapping these enzymes at the DNA-cleavage stage and preventing strand re-joining (Drlica et al., 2008). + + 0.95464545 + protein_type + cleaner0 + 2023-07-25T14:43:27Z + MESH: + + Type II topoisomerase enzymes + + + 0.99884963 + taxonomy_domain + cleaner0 + 2023-07-25T14:42:06Z + DUMMY: + + prokaryotic + + + 0.9986545 + taxonomy_domain + cleaner0 + 2023-07-25T14:42:09Z + DUMMY: + + eukaryotic + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + 0.99891686 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:49Z + DUMMY: + + bacteria + + + 0.73998654 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.9987166 + oligomeric_state + cleaner0 + 2023-07-25T14:24:22Z + DUMMY: + + tetramer + + + 0.9943597 + protein + cleaner0 + 2023-07-25T14:03:41Z + PR: + + ParC + + + 0.9907799 + protein + cleaner0 + 2023-07-25T14:03:45Z + PR: + + ParE + + + 0.86984783 + protein_type + cleaner0 + 2023-07-25T14:03:18Z + MESH: + + gyrase + + + 0.98574257 + complex_assembly + cleaner0 + 2023-07-25T14:04:09Z + GO: + + GyrA2GyrB2 + + + 0.99886 + oligomeric_state + cleaner0 + 2023-07-25T14:24:23Z + DUMMY: + + tetramer + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + + INTRO + paragraph + 3591 + Levofloxacin is a broad-spectrum third-generation fluoro­quinolone antibiotic. It is active against Gram-positive and Gram-negative bacteria and functions by inhibiting gyrase and topoisomerase IV (Drlica & Zhao, 1997; Laponogov et al., 2010). Acquiring a deep structural and functional understanding of the mode of action of fluoroquinolones (Tomašić & Mašič, 2014) and the development of new drugs targeted against topoisomerase IV and gyrase from a wide range of Gram-positive and Gram-negative pathogenic bacteria are highly active areas of current research directed at overcoming the vexed problem of drug resistance (Bax et al., 2010; Chan et al., 2015; Drlica et al., 2014; Mutsaev et al., 2014; Pommier, 2013; Srikannathasan et al., 2015). + + 0.9992699 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + Levofloxacin + + + 0.97983545 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:18Z + DUMMY: + + Gram-positive + + + 0.9393108 + taxonomy_domain + cleaner0 + 2023-07-25T13:58:45Z + DUMMY: + + Gram-negative bacteria + + + 0.79143083 + protein_type + cleaner0 + 2023-07-25T14:03:17Z + MESH: + + gyrase + + + 0.82725424 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.9948644 + chemical + cleaner0 + 2023-07-25T14:42:59Z + CHEBI: + + fluoroquinolones + + + 0.96102214 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.929206 + protein_type + cleaner0 + 2023-07-25T14:03:18Z + MESH: + + gyrase + + + 0.9806581 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:18Z + DUMMY: + + Gram-positive + + + 0.95993716 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:13Z + DUMMY: + + Gram-negative + + + 0.91757727 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:49Z + DUMMY: + + bacteria + + + + INTRO + paragraph + 4344 + Here, we report the first three-dimensional X-ray structure of a K. pneumoniae topoisomerase IV ParC/ParE cleavage complex with DNA stabilized by levofloxacin. The crystal structure provides structural information on topoisomerase IV from K. pneumoniae, a pathogen for which drug resistance is a serious concern. The structure of the ParC/ParE–DNA–levofloxacin binding site highlights the details of the cleavage-complex assembly that are essential for the rational design of Klebsiella topoisomerase inhibitors. + + 0.99835664 + evidence + cleaner0 + 2023-07-25T14:48:35Z + DUMMY: + + X-ray structure + + + 0.99824303 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.77934325 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.9941831 + complex_assembly + cleaner0 + 2023-07-25T14:06:40Z + GO: + + ParC/ParE + + + 0.99338806 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.9990916 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.9987276 + evidence + cleaner0 + 2023-07-25T14:48:37Z + DUMMY: + + crystal structure + + + 0.8437482 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.99829626 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.9978498 + evidence + cleaner0 + 2023-07-25T14:48:40Z + DUMMY: + + structure + + + complex_assembly + GO: + cleaner0 + 2023-07-25T14:06:41Z + + ParC/ParE + + + 0.8439887 + site + cleaner0 + 2023-07-25T14:07:30Z + SO: + + DNA–levofloxacin binding site + + + 0.7741324 + taxonomy_domain + cleaner0 + 2023-07-25T14:42:15Z + DUMMY: + + Klebsiella + + + 0.9223179 + protein_type + cleaner0 + 2023-07-25T14:43:32Z + MESH: + + topoisomerase + + + + METHODS + title_1 + 4861 + Materials and methods   + + + METHODS + title_2 + 4887 + Cloning, expression and purification of K. pneumoniae and Streptococcus pneumoniae ParC55/ParE30   + + + METHODS + paragraph + 4988 + Cloning, expression and purification protocols are described in detail in the Supporting Information. Table 1 ▸ contains the sequence information for all of the components of the complexes. Fig. 1 ▸ provides information about the protein and DNA constructs used in the experimental work. + + + METHODS + title_2 + 5280 + Preparation of the DNA oligomer   + + + METHODS + paragraph + 5316 + For the K. pneumoniae cleavage complex, two DNA oligomers (5′-CGTATTACGTTGTAT-3′ and 5′-GATCATACAACGTAATACG-3′) were synthesized by solid-phase phosphoramidite chemistry and doubly HPLC purified by Metabion, Munich, Germany. The DNA sequence was designed to make a complementary DNA 34-mer that contained the ‘pre-cut’ binding-site fragment: 5′-CGTATTACGTTGTAT↓GATCATACAACGTAATACG-3′ and 3′-GCATAATGCA­ACATACTAG↓TATGTTGCATTATGC-5′ (the cuts are shown by arrows; see Fig. 1 ▸ b). + + + METHODS + paragraph + 5822 + For the S. pneumoniae cleavage complex, two DNA oligomers (5′-CATGAATGACTATGCACG-3′ and 5′-CGTGCATAGTCATTCATG-3′) were synthesized by solid-phase phosphoramidite chemistry and doubly HPLC purified by Metabion, Munich, Germany. The DNA sequence corresponds to the E-site 18-mer, which was found to be a better DNA length for crystallization of the S. pneumoniae topo­isomerase IV cleavage complexes in order to give stable reproducible crystals (see Fig. 1 ▸ b). + + + METHODS + paragraph + 6295 + DNA stock solutions were made by mixing the required oligomers (at 1 mM in 20 mM Tris pH 7.5, 200 mM NaCl, 1 mM β-mercaptothanol, 0.05% NaN3) in equal volumes. For DNA annealing, the mixtures of complementary oligomers were heated to 98°C and then slowly cooled to 4°C over a 48 h period. + + + METHODS + title_2 + 6597 + Crystallization and data collection   + + + METHODS + paragraph + 6637 + Crystallization information for both the S. pneumoniae and the K. pneumoniae topoisomerase IV cleavage complexes is summarized in Table 2 ▸. Data-collection statistics and details are provided in Table 3 ▸. Structure-solution and refinement details are provided in Table 4 ▸. + + + METHODS + title_3 + 6919 + S. pneumoniae topoisomerase IV   + + + METHODS + paragraph + 6955 + Protein was mixed with DNA in a 1:1:1.2 molar ratio (ParC55:ParE30:18-mer E-site DNA) with an overall concentration of 4 mg ml−1. Levofloxacin and magnesium chloride were added to final concentrations of 2 and 10 mM, respectively. The mixture was pre-incubated at room temperature overnight. Initial crystallization screening was performed by sitting-drop vapour diffusion in a 96-well MRC crystallization plate (600 nl protein mixture + 400 nl reservoir solution) using a Mosquito robot (TTP Labtech; http://www.ttplabtech.com). The best crystals were obtained using capillary counter-diffusion against 50 mM sodium cacodylate pH 6.5, 2.5% Tacsimate (Hampton Research; McPherson & Cudney, 2006), 7% 2-propanol, 62.5 mM KCl, 7.5 mM MgCl2 at 304 K. The crystals were flash-cooled at 100 K in cryoprotectant buffer C [50 mM sodium cacodyl­ate pH 6.5, 2.5% Tacsimate, 62.5 mM KCl, 7.5 mM MgCl2, 1 mM β-mercapto­ethanol, 30%(v/v) MPD]. The best data set was collected on beamline I03 at Diamond Light Source at a wavelength of 0.9763 Å using an ADSC Quantum 315 detector. The data extended to 2.6 Å resolution anisotropically and were used in refinement with a maximum-likelihood target in the initial refinement cycles; they were deposited in the PDB without introducing a resolution cutoff. However, owing to the high R merge values in the outer shells, the final resolution is given as 2.9 Å and the statistics are reported according to this ‘trimmed’ resolution. The resolution cutoff was based on the rejection criteria R merge < 50% and I/σ(I) > 1.5 in the highest resolution shell. The data were integrated using HKL-2000 (Otwinowski & Minor, 1997). The space group was determined to be P3121, with unit-cell parameters a = b = 157.83, c = 211.15 Å. + + + METHODS + paragraph + 8761 + The structure was solved by molecular replacement using Phaser (McCoy et al., 2007) as implemented within the CCP4 suite (Winn et al., 2011) and our previously published topo­isomerase IV–levofloxacin structure (PDB entry 3k9f; Laponogov et al., 2010). Refinement was performed in PHENIX (Adams et al., 2002, 2010) with manual inspection and corrections performed in WinCoot (Emsley & Cowtan, 2004; Emsley et al., 2010).The structure was verified using WinCoot and PROCHECK (Laskowski et al., 1993). + + + METHODS + title_3 + 9264 + K. pneumoniae topoisomerase IV   + + + METHODS + paragraph + 9300 + ParC55/ParE30 protein stock in incubation buffer (at 4.5 mg ml−1) was mixed with the ‘pre-cut’ 34-mer DNA stock in a 1:1.2 protein:DNA molar ratio. High-concentration stocks of levofloxacin and MgCl2 were added to give final concentrations of 2 and 10 mM, respectively. The mixture was incubated overnight at room temperature. Initial crystallization screening was performed by sitting-drop vapour diffusion in 96-well MRC crystallization plates (600 nl protein mixture + 300 nl reservoir solution) using a Mosquito robot. When the optimal crystallization conditions had been established, conventional hanging-drop vapour diffusion in 24-well Linbro plates (4 µl protein mixture + 2 µl reservoir solution) was used to increase the crystal size. + + + METHODS + paragraph + 10067 + Crystals formed after ∼7–10 d at room temperature. The crystallization conditions varied slightly from batch to batch in the range 0.1 M Tris pH 7.5–8.0, 0–50 mM NaCl, 4–8% PEG 4000, 12–15% glycerol. + + + METHODS + paragraph + 10285 + It should be mentioned that several other DNA oligomers with the same binding-site sequence were tried for crystallization (i.e. 20-mer, ‘pre-cut’ 20-mer and 34-mer DNA sequences). However, these protein–DNA–drug complexes did not produce good-quality crystals for data collection. + + + METHODS + paragraph + 10575 + Crystals were tested in-house for diffraction quality using an Oxford Xcalibur Nova CCD diffractometer and were then transported for high-resolution data collection at Diamond Light Source (Harwell Science and Innovation Campus, Oxfordshire, England). The data were collected on beamline I03 (wavelength 0.9762 Å) using a Pilatus 6M-F detector (0.2° oscillation per image, 100 K nitrogen stream). The best crystals diffracted to ∼3.2 Å resolution. + + + METHODS + paragraph + 11034 + All data sets were integrated with MOSFLM (Leslie & Powell, 2007) and merged with SCALA (Evans, 2006) as implemented in CCP4 (Winn et al., 2011). The ParC55/ParE30–DNA–levofloxacin crystals belonged to space group P21, with unit-cell parameters a = 102.07, b = 161.53, c = 138.60 Å, α = 90.00, β = 94.22, γ = 90.00°. They contained two ParC/ParE–DNA heterodimers in the asymmetric unit. + + + METHODS + paragraph + 11438 + Several data sets were collected, some of which contained visible diffraction to 3.2 Å resolution, but owing to potential internal twinning and space-group ambiguity (most data sets could be integrated in space groups P21 and P212121) and the fact that the structure solution could be obtained in both space groups, careful selection of the integration ranges as well as appropriate data truncation were necessary. The best region of data was integrated to 3.35 Å (see Table 3 ▸ for statistics). The resolution cutoff was based on the rejection criteria R merge < 50% and I/σ(I) > 1.5 in the highest resolution shell. + + + METHODS + paragraph + 12065 + The structure was solved by the molecular-replacement method in Phaser (McCoy et al., 2007) using the levofloxacin–DNA cleavage complex of topoisomerase IV from S. pneumoniae as a search model (PDB entry 3rae; ∼41.8% sequence identity). Refinement was performed in PHENIX (Adams et al., 2002, 2010) using secondary-structure restraints derived by superposition of the K. pneumoniae ParC/ParE model with the previously solved complex of S. pneumoniae ParC/ParE. Rigid-body, positional and TLS refinements were performed. Levofloxacin molecules and magnesium ions were placed during the final stages of refinement based on missing electron density in the σA-weighted 2F obs − F calc and F obs − F calc maps. WinCoot (Emsley & Cowtan, 2004) was used for interactive model fitting. The structure was verified using WinCoot and PROCHECK (Laskowski et al., 1993). The resulting model had good geometry, with 87.8, 9.9 and 1.3% of residues in the favoured, allowed and generously allowed regions of the Ramachandran plot, respectively, and no more than 1% of residues in disallowed regions. The data-collection and final refinement statistics are given in Tables 3 ▸ and 4 ▸. Sequence alignment was performed in ClustalW (Larkin et al., 2007, McWilliam et al., 2013). Figures were prepared using PyMOL (DeLano, 2008), CHEMDRAW (Evans, 2014) and CorelDRAW (http://www.coreldraw.com). + + + RESULTS + title_1 + 13454 + Results and discussion   + + + RESULTS + paragraph + 13481 + We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. The X-ray crystal structure of the complex was determined to 3.35 Å resolution, revealing a closed ParC55 dimer flanked by two ParE30 monomers (Figs. 1 ▸, 2 ▸ and 3 ▸). The overall architecture of this complex is similar to that found for S. pneumoniae topoisomerase–DNA–drug complexes (Laponogov et al., 2009, 2010). Residues 6–30 of the N-terminal α-helix α1 of the ParC subunit again embrace the ParE subunit, ‘hugging’ the ParE subunits close to either side of the ParC dimer (Laponogov et al., 2010). Deletion of this ‘arm’ α1 results in loss of DNA-cleavage activity (Laponogov et al., 2007) and is clearly very important in complex stability (Fig. 3 ▸). This structural feature was absent in our original ParC55 structure (Laponogov et al., 2007; Sohi et al., 2008). The upper region of the topoisomerase complex consists of the E-subunit TOPRIM metal-binding domain formed of four parallel β-sheets and the surrounding α-helices. The C-subunit provides the WHD (winged-helix domain; a CAP-like structure; McKay & Steitz, 1981) and the ‘tower’ which form the U groove-shaped protein region into which the G-gate DNA binds with an induced U-shaped bend. The lower C-gate region (Fig. 3 ▸) consists of the same disposition of pairs of two long α-helices terminated by a spanning short α-helix forming a 30 Å wide DNA-accommodating cavity through which the T-gate DNA passes as found in the S. pneumoniae complex. Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013). + + 0.9987688 + experimental_method + cleaner0 + 2023-07-25T14:52:59Z + MESH: + + co-crystallized + + + 0.99840206 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.95960265 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + complex_assembly + GO: + cleaner0 + 2023-07-25T14:06:41Z + + ParC/ParE + + + structure_element + SO: + cleaner0 + 2023-07-25T14:06:01Z + + breakage-reunion + + + 0.9986889 + protein + cleaner0 + 2023-07-25T14:33:37Z + PR: + + ParC55 + + + 0.99739915 + residue_range + cleaner0 + 2023-07-25T14:23:07Z + DUMMY: + + 1–490 + + + 0.99886775 + protein + cleaner0 + 2023-07-25T14:03:46Z + PR: + + ParE + + + structure_element + SO: + cleaner0 + 2023-07-25T14:06:16Z + + TOPRIM + + + 0.9985896 + protein + cleaner0 + 2023-07-25T14:33:39Z + PR: + + ParE30 + + + 0.9976611 + residue_range + cleaner0 + 2023-07-25T14:23:04Z + DUMMY: + + 390–631 + + + 0.9976459 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.9927888 + site + cleaner0 + 2023-07-25T14:53:29Z + SO: + + E-site + + + 0.9991265 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.99823445 + evidence + cleaner0 + 2023-07-25T14:48:45Z + DUMMY: + + X-ray crystal structure + + + 0.9992544 + protein_state + cleaner0 + 2023-07-25T14:23:11Z + DUMMY: + + closed + + + 0.99875546 + protein + cleaner0 + 2023-07-25T14:33:44Z + PR: + + ParC55 + + + 0.998879 + oligomeric_state + cleaner0 + 2023-07-25T14:20:29Z + DUMMY: + + dimer + + + 0.9981425 + protein + cleaner0 + 2023-07-25T14:33:48Z + PR: + + ParE30 + + + 0.9988433 + oligomeric_state + cleaner0 + 2023-07-25T14:20:36Z + DUMMY: + + monomers + + + 0.9983236 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.9974201 + residue_range + cleaner0 + 2023-07-25T14:23:01Z + DUMMY: + + 6–30 + + + 0.99918294 + structure_element + cleaner0 + 2023-07-25T14:32:57Z + SO: + + α-helix + + + 0.9989114 + structure_element + cleaner0 + 2023-07-25T14:22:17Z + SO: + + α1 + + + 0.99914145 + protein + cleaner0 + 2023-07-25T14:03:41Z + PR: + + ParC + + + 0.99903774 + protein + cleaner0 + 2023-07-25T14:03:46Z + PR: + + ParE + + + 0.99867 + protein + cleaner0 + 2023-07-25T14:03:46Z + PR: + + ParE + + + 0.99911374 + protein + cleaner0 + 2023-07-25T14:03:41Z + PR: + + ParC + + + 0.99890816 + oligomeric_state + cleaner0 + 2023-07-25T14:20:30Z + DUMMY: + + dimer + + + 0.93558586 + experimental_method + cleaner0 + 2023-07-25T14:53:03Z + MESH: + + Deletion of + + + 0.9963787 + structure_element + cleaner0 + 2023-07-25T14:33:02Z + SO: + + arm + + + 0.9994838 + structure_element + cleaner0 + 2023-07-25T14:22:17Z + SO: + + α1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T14:37:19Z + + loss of DNA-cleavage activity + + + 0.9989447 + protein + cleaner0 + 2023-07-25T14:33:58Z + PR: + + ParC55 + + + 0.99683714 + evidence + cleaner0 + 2023-07-25T14:48:49Z + DUMMY: + + structure + + + 0.92049813 + protein_type + cleaner0 + 2023-07-25T14:38:12Z + MESH: + + topoisomerase + + + 0.99771005 + protein + cleaner0 + 2023-07-25T14:52:10Z + PR: + + E-subunit + + + 0.9520648 + structure_element + cleaner0 + 2023-07-25T14:22:55Z + SO: + + TOPRIM metal-binding domain + + + 0.99856985 + structure_element + cleaner0 + 2023-07-25T14:33:30Z + SO: + + parallel β-sheets + + + 0.9990757 + structure_element + cleaner0 + 2023-07-25T14:34:23Z + SO: + + α-helices + + + 0.99685293 + protein + cleaner0 + 2023-07-25T14:52:02Z + PR: + + C-subunit + + + 0.9993967 + structure_element + cleaner0 + 2023-07-25T14:38:27Z + SO: + + WHD + + + 0.99901634 + structure_element + cleaner0 + 2023-07-25T14:38:29Z + SO: + + winged-helix domain + + + 0.9520038 + structure_element + cleaner0 + 2023-07-25T14:38:31Z + SO: + + CAP-like structure + + + 0.97492325 + structure_element + cleaner0 + 2023-07-25T14:38:42Z + SO: + + tower + + + structure_element + SO: + cleaner0 + 2023-07-25T14:39:36Z + + U groove + + + structure_element + SO: + cleaner0 + 2023-07-25T14:21:46Z + + G-gate + + + 0.99712175 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + structure_element + SO: + cleaner0 + 2023-07-25T14:21:17Z + + C-gate + + + 0.97197616 + structure_element + cleaner0 + 2023-07-25T14:39:42Z + SO: + + long α-helices + + + 0.99347615 + structure_element + cleaner0 + 2023-07-25T14:39:44Z + SO: + + short α-helix + + + 0.990876 + site + cleaner0 + 2023-07-25T14:53:34Z + SO: + + DNA-accommodating cavity + + + structure_element + SO: + cleaner0 + 2023-07-25T14:52:31Z + + T-gate + + + 0.9967733 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.99772805 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + complex_assembly + GO: + cleaner0 + 2023-07-25T14:40:51Z + + topo­isomerases IV + + + 0.9982472 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.99819094 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.9854866 + protein_type + cleaner0 + 2023-07-25T14:43:37Z + MESH: + + topoisomerase + + + 0.99762684 + evidence + cleaner0 + 2023-07-25T14:48:52Z + DUMMY: + + structure + + + 0.98970866 + protein_state + cleaner0 + 2023-07-25T14:39:54Z + DUMMY: + + full complex + + + 0.9992118 + protein_state + cleaner0 + 2023-07-25T14:39:56Z + DUMMY: + + holoenzyme + + + structure_element + SO: + cleaner0 + 2023-07-25T14:40:24Z + + CTD β-pinwheel + + + 0.9982599 + structure_element + cleaner0 + 2023-07-25T14:40:28Z + SO: + + ATPase domain + + + 0.9992829 + protein_state + cleaner0 + 2023-07-25T14:39:59Z + DUMMY: + + open + + + + RESULTS + paragraph + 15603 + The G-gate DNA for the S. pneumoniae complex consists of an 18-base-pair E-site sequence (our designation for a DNA site which we first found from DNA-mapping studies; Leo et al., 2005; Arnoldi et al., 2013; Fig. 1 ▸). The crystallized complex was formed by turning over the topoisomerase tetramer in the presence of DNA and levofloxacin and crystallizing the product. In contrast, the K. pneumoniae complex was formed by co-crystallizing the topoisomerase tetramer complex in the presence of a 34-base-pair pre-cleaved DNA in the presence of levofloxacin. In both cases the DNA is bent into a U-form and bound snugly against the protein of the G-gate. We have been able to unambiguously read off the DNA sequences in the electron-density maps. + + 0.9945329 + structure_element + cleaner0 + 2023-07-25T14:21:46Z + SO: + + G-gate + + + 0.99850476 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.9983961 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.99768853 + site + cleaner0 + 2023-07-25T14:53:39Z + SO: + + E-site + + + 0.964101 + site + cleaner0 + 2023-07-25T14:23:52Z + SO: + + DNA site + + + 0.89909416 + experimental_method + cleaner0 + 2023-07-25T14:23:24Z + MESH: + + DNA-mapping studies + + + 0.48656628 + experimental_method + cleaner0 + 2023-07-25T14:23:30Z + MESH: + + crystallized + + + 0.99777657 + protein_type + cleaner0 + 2023-07-25T14:43:43Z + MESH: + + topoisomerase + + + 0.9984073 + oligomeric_state + cleaner0 + 2023-07-25T14:24:23Z + DUMMY: + + tetramer + + + 0.9606428 + protein_state + cleaner0 + 2023-07-25T14:23:38Z + DUMMY: + + presence of + + + 0.99778986 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.99881727 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.9870214 + experimental_method + cleaner0 + 2023-07-25T14:23:32Z + MESH: + + crystallizing + + + 0.99833447 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.998607 + experimental_method + cleaner0 + 2023-07-25T14:23:27Z + MESH: + + co-crystallizing + + + 0.9971288 + protein_type + cleaner0 + 2023-07-25T14:43:46Z + MESH: + + topoisomerase + + + 0.9982173 + oligomeric_state + cleaner0 + 2023-07-25T14:24:23Z + DUMMY: + + tetramer + + + 0.79218626 + protein_state + cleaner0 + 2023-07-25T14:23:38Z + DUMMY: + + presence of + + + 0.9968751 + protein_state + cleaner0 + 2023-07-25T14:23:43Z + DUMMY: + + pre-cleaved + + + 0.9985281 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.89513683 + protein_state + cleaner0 + 2023-07-25T14:23:38Z + DUMMY: + + presence of + + + 0.99858 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.9968882 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.99312526 + protein_state + cleaner0 + 2023-07-25T14:54:16Z + DUMMY: + + U-form + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T14:34:17Z + + bound + + + 0.997433 + structure_element + cleaner0 + 2023-07-25T14:21:46Z + SO: + + G-gate + + + 0.80029345 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.99893767 + evidence + cleaner0 + 2023-07-25T14:48:58Z + DUMMY: + + electron-density maps + + + + RESULTS + paragraph + 16350 + There is 41.6% sequence identity and 54.4% sequence homology between the ParE subunit of K. pneumoniae and that of S. pneumoniae. For the ParC subunits, the figures are 40.8 identity and 55.6% homology between the two organisms. The sequence alignment is given in Supplementary Fig. S1, with the key metal-binding residues and those which give rise to quinolone resistance highlighted. The binding of levofloxacin in the K. pneumoniae complex is shown in Figs. 2 ▸, 3 ▸ and 5 ▸ and is hemi-intercalated into the DNA and stacked against the DNA bases at the cleavage site (positions −1 and +1 of the four-base-pair staggered cut in the 34-mer DNA) which is similar to that found for the S. pneumoniae complex. Fig. 5 ▸ presents side-by-side views of the K. pneumoniae and S. pneumoniae active sites which shows that levofloxacin binds in a very similar manner in these two complexes with extensive π–π stacking interaction between the bases and the drug. The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. Interestingly, for S. pneumoniae we observe only one possible orientation of the C7 groups in both sub­units, while for K. pneumoniae we can see two: one with the same orientation as in S. pneumoniae and other rotated 180° away. They both exist within the same crystal in the two different dimers in the asymmetric unit. The side chains surrounding them in ParE are quite disordered and are more defined in K. pneumoniae (even though this complex is at lower resolution) than in S. pneumoniae. There are no direct hydrogen bonds from the drug to these residues (although it is possible that some are formed through water, which cannot be observed at this resolution). Obviously, the drug–ParE interaction in this region is less strong compared with PD 0305970 binding to the S. pneumoniae DNA complex, where PD 0305970 forms a hydrogen bond to ParE residue Asp475 and can form one to Asp474 if the bond rotates (Laponogov et al., 2010). This may explain why drug-resistance mutations for levofloxacin are more likely to form in the ParC subunits rather than in the ParE subunits. + + 0.9985201 + protein + cleaner0 + 2023-07-25T14:03:46Z + PR: + + ParE + + + 0.9981732 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.9981801 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.99809676 + protein + cleaner0 + 2023-07-25T14:03:41Z + PR: + + ParC + + + 0.9983183 + experimental_method + cleaner0 + 2023-07-25T14:53:08Z + MESH: + + sequence alignment + + + 0.9988264 + site + cleaner0 + 2023-07-25T14:53:46Z + SO: + + metal-binding residues + + + 0.9989184 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.9979357 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.96886516 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.81724125 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.9957226 + site + cleaner0 + 2023-07-25T14:53:50Z + SO: + + cleavage site + + + 0.91113573 + residue_number + cleaner0 + 2023-07-25T14:32:02Z + DUMMY: + + −1 + + + residue_number + DUMMY: + cleaner0 + 2023-07-25T14:32:43Z + + +1 + + + 0.97470677 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.9981618 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.9980726 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.9980286 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.9990308 + site + cleaner0 + 2023-07-25T14:53:54Z + SO: + + active sites + + + 0.99879223 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.993351 + bond_interaction + cleaner0 + 2023-07-25T14:26:29Z + MESH: + + π–π stacking interaction + + + 0.99655724 + chemical + cleaner0 + 2023-07-25T14:29:01Z + CHEBI: + + methylpiperazine + + + 0.9952042 + chemical + cleaner0 + 2023-07-25T14:43:04Z + CHEBI: + + quinolone + + + 0.99942684 + residue_name_number + cleaner0 + 2023-07-25T14:27:54Z + DUMMY: + + Glu474 + + + 0.9994332 + residue_name_number + cleaner0 + 2023-07-25T14:27:58Z + DUMMY: + + Glu475 + + + 0.9981634 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.9995127 + residue_name_number + cleaner0 + 2023-07-25T14:28:03Z + DUMMY: + + Gln460 + + + 0.9994655 + residue_name_number + cleaner0 + 2023-07-25T14:28:07Z + DUMMY: + + Glu461 + + + 0.9980914 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-25T14:27:07Z + + glutamate at 474 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-25T14:27:40Z + + glutamine at 460 + + + 0.99241626 + taxonomy_domain + cleaner0 + 2023-07-25T14:28:32Z + DUMMY: + + Klebsiella + + + 0.99809504 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.9971411 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.9979572 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.968922 + evidence + cleaner0 + 2023-07-25T14:49:04Z + DUMMY: + + crystal + + + 0.9987387 + oligomeric_state + cleaner0 + 2023-07-25T14:32:52Z + DUMMY: + + dimers + + + 0.9988141 + protein + cleaner0 + 2023-07-25T14:03:46Z + PR: + + ParE + + + 0.99787974 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.99789166 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.99686784 + bond_interaction + cleaner0 + 2023-07-25T14:28:41Z + MESH: + + hydrogen bonds + + + 0.9938929 + chemical + cleaner0 + 2023-07-25T14:29:27Z + CHEBI: + + water + + + 0.43409234 + protein + cleaner0 + 2023-07-25T14:03:46Z + PR: + + ParE + + + 0.998289 + chemical + cleaner0 + 2023-07-25T14:28:51Z + CHEBI: + + PD 0305970 + + + 0.9980553 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.98690504 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.9975598 + chemical + cleaner0 + 2023-07-25T14:28:52Z + CHEBI: + + PD 0305970 + + + 0.9970821 + bond_interaction + cleaner0 + 2023-07-25T14:28:44Z + MESH: + + hydrogen bond + + + 0.997598 + protein + cleaner0 + 2023-07-25T14:03:46Z + PR: + + ParE + + + 0.999468 + residue_name_number + cleaner0 + 2023-07-25T14:31:55Z + DUMMY: + + Asp475 + + + 0.9994392 + residue_name_number + cleaner0 + 2023-07-25T14:31:57Z + DUMMY: + + Asp474 + + + 0.99867827 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.99766207 + protein + cleaner0 + 2023-07-25T14:03:41Z + PR: + + ParC + + + protein + PR: + cleaner0 + 2023-07-25T14:03:46Z + + ParE + + + + RESULTS + paragraph + 18713 + For both complexes there is a Mg2+ ion bound to levofloxacin between the carbonyl group at position 4 of the quinolone and the carboxyl at position 6 (Figs. 2 ▸ and 5 ▸ and Supplementary Fig. 2 ▸). For S. pneumoniae topoisomerase IV, one of the O atoms of the carboxyl of Asp83 points towards the Mg2+ ion and is within hydrogen-bonding distance (5.04 Å) through an Mg2+-coordinated water. For K. pneumoniae both of the carboxyl O atoms are pointing towards the Mg2+ ion at distances of 4.86 and 4.23 Å. These residues are ordered in only one of the two dimers in the K. pneumoniae crystal (the one in which the C7 group is pointing towards the DNA away from ParE, although the conformations of these two groups on the drug are probably not correlated). + + 0.99876034 + chemical + cleaner0 + 2023-07-25T14:29:15Z + CHEBI: + + Mg2+ + + + 0.79460996 + protein_state + cleaner0 + 2023-07-25T14:43:16Z + DUMMY: + + bound to + + + 0.9990497 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.99841726 + chemical + cleaner0 + 2023-07-25T14:43:08Z + CHEBI: + + quinolone + + + 0.9985083 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.8904655 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.99946815 + residue_name_number + cleaner0 + 2023-07-25T14:31:06Z + DUMMY: + + Asp83 + + + 0.99855185 + chemical + cleaner0 + 2023-07-25T14:29:18Z + CHEBI: + + Mg2+ + + + 0.9971292 + bond_interaction + cleaner0 + 2023-07-25T14:31:00Z + MESH: + + hydrogen-bonding + + + 0.98301584 + chemical + cleaner0 + 2023-07-25T14:29:22Z + CHEBI: + + Mg2+ + + + 0.9988104 + chemical + cleaner0 + 2023-07-25T14:29:24Z + CHEBI: + + water + + + 0.99845415 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.99853647 + chemical + cleaner0 + 2023-07-25T14:29:20Z + CHEBI: + + Mg2+ + + + 0.9987025 + oligomeric_state + cleaner0 + 2023-07-25T14:32:52Z + DUMMY: + + dimers + + + 0.99833155 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.99787474 + evidence + cleaner0 + 2023-07-25T14:49:10Z + DUMMY: + + crystal + + + 0.9941613 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.6069613 + protein + cleaner0 + 2023-07-25T14:03:46Z + PR: + + ParE + + + + RESULTS + paragraph + 19479 + The topoisomerase IV ParE27-ParC55 fusion protein from K. pneumoniae was fully active in promoting levofloxacin-mediated cleavage of DNA (Fig. 6 ▸). In the absence of the drug and ATP, the protein converted supercoiled pBR322 into a ladder of bands corresponding to relaxed DNA. The inclusion of levofloxacin produced linear DNA in a dose-dependent and ATP-independent fashion. Similar behaviour was observed for the S. pneumoniae topo­isomerase IV ParE30-ParC55 fusion protein. The CC25 (the drug concentration that converted 25% of the supercoiled DNA substrate to a linear form) was 0.5 µM for the Klebsiella enzyme and 1 µM for the pneumococcal enzyme. Interestingly, K. pneumoniae strains are much more susceptible to levofloxacin than S. pneumoniae, with typical MIC values of 0.016 and 1 mg l−1, respectively (Odenholt & Cars, 2006), reflecting differences in multiple factors (in addition to binding affinity) that influence drug responses, including membrane, peptidoglycan structure, drug-uptake and efflux mechanisms. Moreover, although topoisomerase IV is primarily the target of levofloxacin in S. pneumoniae, it is likely to be gyrase in the Gram-negative K. pneumoniae. + + complex_assembly + GO: + cleaner0 + 2023-07-25T14:25:38Z + + topoisomerase IV + + + 0.9883413 + complex_assembly + cleaner0 + 2023-07-25T14:44:47Z + GO: + + ParE27-ParC55 + + + 0.99830294 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.99897254 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + 0.99761784 + protein_state + cleaner0 + 2023-07-25T14:54:21Z + DUMMY: + + absence of + + + 0.9889319 + chemical + cleaner0 + 2023-07-25T14:47:11Z + CHEBI: + + drug + + + 0.9983248 + chemical + cleaner0 + 2023-07-25T14:30:36Z + CHEBI: + + ATP + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + 0.9990933 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:30:36Z + + ATP + + + species + MESH: + cleaner0 + 2023-07-25T13:57:01Z + + S. pneumoniae + + + complex_assembly + GO: + cleaner0 + 2023-07-25T14:30:12Z + + topo­isomerase IV + + + 0.9783872 + complex_assembly + cleaner0 + 2023-07-25T14:44:51Z + GO: + + ParE30-ParC55 + + + 0.92020863 + evidence + cleaner0 + 2023-07-25T14:49:15Z + DUMMY: + + CC25 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + 0.9981312 + taxonomy_domain + cleaner0 + 2023-07-25T14:42:21Z + DUMMY: + + Klebsiella + + + 0.9982681 + taxonomy_domain + cleaner0 + 2023-07-25T14:31:24Z + DUMMY: + + pneumococcal + + + 0.9983346 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.9989604 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.9982436 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.99087524 + evidence + cleaner0 + 2023-07-25T14:49:19Z + DUMMY: + + binding affinity + + + 0.6844131 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.9989524 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.9983661 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.9983967 + protein_type + cleaner0 + 2023-07-25T14:03:18Z + MESH: + + gyrase + + + 0.86461306 + taxonomy_domain + cleaner0 + 2023-07-25T13:56:13Z + DUMMY: + + Gram-negative + + + 0.998044 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + + RESULTS + paragraph + 20679 + In summary, we have determined the first structure of a quinolone–DNA cleavage complex involving a type II topo­isomerase from K. pneumoniae. Given the current concerns about drug-resistant strains of Klebsiella, the structure reported here provides key information in understanding the action of currently used quinolones and should aid in the development of other topoisomerase-targeting therapeutics active against this major human pathogen. + + 0.9937563 + evidence + cleaner0 + 2023-07-25T14:49:30Z + DUMMY: + + structure + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:47:00Z + + quinolone + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + 0.99827665 + protein_type + cleaner0 + 2023-07-25T14:43:52Z + MESH: + + type II topo­isomerase + + + 0.99834776 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.8484614 + taxonomy_domain + cleaner0 + 2023-07-25T14:41:36Z + DUMMY: + + Klebsiella + + + 0.9980427 + evidence + cleaner0 + 2023-07-25T14:49:34Z + DUMMY: + + structure + + + 0.99839264 + chemical + cleaner0 + 2023-07-25T14:47:17Z + CHEBI: + + quinolones + + + 0.99797803 + protein_type + cleaner0 + 2023-07-25T14:43:57Z + MESH: + + topoisomerase + + + 0.9980672 + species + cleaner0 + 2023-07-25T14:31:36Z + MESH: + + human + + + + SUPPL + title_1 + 21127 + Supplementary Material + + + REF + title + 21150 + References + + + REF + ref + 21161 + Adams, P. D. et al. (2010). Acta Cryst. D66, 213–221. + + + REF + ref + 21217 + Adams, P. D., Grosse-Kunstleve, R. W., Hung, L.-W., Ioerger, T. R., McCoy, A. J., Moriarty, N. W., Read, R. J., Sacchettini, J. C., Sauter, N. K. & Terwilliger, T. C. (2002). Acta Cryst. D58, 1948–1954. + + + REF + ref + 21423 + Arnold, R. S., Thom, K. A., Sharma, S., Phillips, M., Kristie Johnson, J. & Morgan, D. J. (2011). South. Med. J. 104, 40–45. + + + REF + ref + 21550 + Arnoldi, E., Pan, X.-S. & Fisher, L. M. (2013). Nucleic Acids Res. 41, 9411–9423. + + + REF + ref + 21635 + Bax, B. D. et al. (2010). Nature (London), 466, 935–940. + + + REF + ref + 21694 + Chan, P. F. et al. (2015). Nature Commun. 6, 10048. + + + REF + ref + 21746 + Chen, L. F., Anderson, D. J. & Paterson, D. L. (2012). Infect. Drug Resist. 5, 133–141. + + + REF + ref + 21836 + DeLano, W. L. (2008). PyMOL. http://www.pymol.org. + + + REF + ref + 21887 + Drlica, K., Malik, M., Kerns, R. J. & Zhao, X. (2008). Antimicrob. Agents Chemother. 52, 385–392. + + + REF + ref + 21987 + Drlica, K., Mustaev, A., Towle, T. R., Luan, G., Kerns, R. J. & Berger, J. M. (2014). ACS Chem. Biol. 19, 2895–2904. + + + REF + ref + 22107 + Drlica, K. & Zhao, X. (1997). Microbiol. Mol. Biol. Rev. 61, 377–392. + + + REF + ref + 22179 + Emsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126–2132. + + + REF + ref + 22241 + Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486–501. + + + REF + ref + 22328 + Evans, P. (2006). Acta Cryst. D62, 72–82. + + + REF + ref + 22372 + Evans, D. A. (2014). Angew. Chem. Int. Ed. 53, 11140–11145. + + + REF + ref + 22434 + Gasink, L. B., Edelstein, P., Lautenbach, E., Synnestvedt, M. & Fishman, N. (2009). Infect. Control Hosp. Epidemiol. 30, 1180–1185. + + + REF + ref + 22568 + Laponogov, I., Pan, X.-S., Veselkov, D. A., McAuley, K. E., Fisher, L. M. & Sanderson, M. R. (2010). PLoS One, 5, e11338. + + + REF + ref + 22690 + Laponogov, I., Sohi, M. K., Veselkov, D. A., Pan, X.-S., Sawhney, R., Thompson, A. W., McAuley, K. E., Fisher, L. M. & Sanderson, M. R. (2009). Nature Struct. Mol. Biol. 16, 667–669. + + + REF + ref + 22875 + Laponogov, I., Veselkov, D. A., Crevel, I. M.-T., Pan, X.-S., Fisher, L. M. & Sanderson, M. R. (2013). Nucleic Acids Res. 41, 9911–9923. + + + REF + ref + 23014 + Laponogov, I., Veselkov, D. A., Sohi, M. K., Pan, X.-S., Achari, A., Yang, C., Ferrara, J. D., Fisher, L. M. & Sanderson, M. R. (2007). PLoS One, 2, e301. + + + REF + ref + 23169 + Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J. & Higgins, D. G. (2007). Bioinformatics, 23, 2947–2948. + + + REF + ref + 23402 + Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. (1993). J. Appl. Cryst. 26, 283–291. + + + REF + ref + 23507 + Lee, I., Dong, K. C. & Berger, J. M. (2013). Nucleic Acids Res. 41, 5444–5456. + + + REF + ref + 23588 + Leo, E., Gould, K. A., Pan, X.-S., Capranico, G., Sanderson, M. R., Palumbo, M. & Fisher, L. M. (2005). J. Biol. Chem. 280, 14252–14263. + + + REF + ref + 23727 + Leslie, A. G. W. & Powell, H. R. (2007). Evolving Methods for Macromolecular Crystallography, edited by R. Read & J. Sussman, pp. 41–51. Dordrecht: Springer. + + + REF + ref + 23887 + McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658–674. + + + REF + ref + 24021 + McKay, D. B. & Steitz, T. A. (1981). Nature (London), 290, 744–749. + + + REF + ref + 24091 + McPherson, A. & Cudney, B. (2006). J. Struct. Biol. 156, 387–406. + + + REF + ref + 24159 + McWilliam, H., Li, W., Uludag, M., Squizzato, S., Park, Y. M., Buso, N., Cowley, A. P. & Lopez, R. (2013). Nucleic Acids Res. 41, W597–W600. + + + REF + ref + 24302 + Mustaev, A., Malik, M., Zhao, X., Kurepina, N., Luan, G., Oppegard, L. M., Hiasa, H., Marks, K. R., Kerns, R. J., Berger, J. M. & Drlica, K. (2014). J. Biol. Chem. 289, 12300–12312. + + + REF + ref + 24486 + Nitiss, J. L. (2009a). Nature Rev. Cancer, 9, 327–337. + + + REF + ref + 24543 + Nitiss, J. L. (2009b). Nature Rev. Cancer, 9, 338–350. + + + REF + ref + 24600 + Nordmann, P., Cuzon, G. & Naas, T. (2009). Lancet Infect. Dis. 9, 228–236. + + + REF + ref + 24677 + Odenholt, I. & Cars, O. J. (2006). J. Antimicrob. Chemother. 58, 960–965. + + + REF + ref + 24753 + Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307–326. + + + REF + ref + 24821 + Pan, X.-S. & Fisher, L. M. (1996). J. Bacteriol. 178, 4060–4069. + + + REF + ref + 24888 + Pommier, Y. (2013). ACS Chem. Biol. 8, 82–95. + + + REF + ref + 24936 + Schoeffler, A. J. & Berger, J. M. (2008). Q. Rev. Biophys. 41, 41–101. + + + REF + ref + 25009 + Sissi, C. & Palumbo, M. (2009). Nucleic Acids Res. 37, 702–711. + + + REF + ref + 25075 + Sohi, M. K., Veselkov, D. A., Laponogov, I., Pan, X.-S., Fisher, L. M. & Sanderson, M. R. (2008). PLoS One, 3, e3201. + + + REF + ref + 25193 + Srikannathasan, V., Wohlkonig, A., Shillings, A., Singh, O., Chan, P. F., Huang, J., Gwynn, M. N., Fosberry, A. P., Homes, P., Hibbs, M., Theobald, A. J., Spitzfaden, C. & Bax, B. D. (2015). Acta Cryst. F71, 1242–1246. + + + REF + ref + 25414 + Tomašić, T. & Mašič, L. P. (2014). Curr. Top. Med. Chem. 14, 130–151. + + + REF + ref + 25490 + Vos, S. M., Tretter, E. M., Schmidt, B. H. & Berger, J. M. (2011). Nature Rev. Mol. Cell Biol. 12, 827–841. + + + REF + ref + 25600 + Wendorff, T. J., Schmidt, B. H., Heslop, P., Austin, C. A. & Berger, J. M. (2012). J. Mol. Biol. 424, 109–124. + + + REF + ref + 25713 + Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242. + + + REF + ref + 25768 + Wu, C.-C., Li, T.-K., Farh, L., Lin, L.-Y., Lin, T.-S., Yu, Y.-J., Yen, T.-J., Chiang, C.-W. & Chan, N.-L. (2011). Science, 333, 459–462. + + + REF + ref + 25908 + Wu, C.-C., Li, Y.-C., Wang, Y.-R., Li, T.-K. & Chan, N.-L. (2013). Nucleic Acids Res. 41, 10630–10640. + + + d-72-00488-fig1.jpg + fig1 + FIG + fig_caption + 26013 + Protein and DNA used in the co-crystallization experiment. (a) Coloured diagram of the protein constructs used in crystallization. (b) DNA sequences used in crystallization. The 4 bp overhang is shown in red. Cleavage points are indicated by arrows. + + 0.8724873 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.99885994 + experimental_method + cleaner0 + 2023-07-25T14:53:13Z + MESH: + + co-crystallization + + + 0.7814805 + experimental_method + cleaner0 + 2023-07-25T14:53:16Z + MESH: + + crystallization + + + 0.895672 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.9635726 + experimental_method + cleaner0 + 2023-07-25T14:53:18Z + MESH: + + crystallization + + + + d-72-00488-fig2.jpg + fig2 + FIG + fig_caption + 26265 + Chemical structure of levofloxacin (a) and its conformations observed within the active sites of S. pneumoniae topoisomerase IV (b) and K. pneumoniae topoisomerase IV (c, d). Electron-density maps (2F +obs − F +calc) are shown as meshes for the drug molecules contoured at 1.5σ and are limited to a distance of 2.3 Å from the drug atoms. + + 0.99906665 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.9990804 + site + cleaner0 + 2023-07-25T14:54:00Z + SO: + + active sites + + + 0.9983861 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.85898304 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.9984185 + species + cleaner0 + 2023-07-25T14:41:46Z + MESH: + + K. pneumoniae + + + 0.9489127 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.99863946 + evidence + cleaner0 + 2023-07-25T14:49:40Z + DUMMY: + + Electron-density maps + + + evidence + DUMMY: + cleaner0 + 2023-07-25T14:49:54Z + + 2F +obs − F +calc + + + + d-72-00488-fig3.jpg + fig3 + FIG + fig_caption + 26608 + Overall orthogonal views of the cleavage complex of topoisomerase IV from K. pneumoniae in surface (left) and cartoon (right) representations. The ParC subunit is in blue, ParE is in yellow and DNA is in cyan. The bound quinolone molecules (levofloxacin) are in red and are shown using van der Waals representation. + + 0.9031069 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.998235 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.99896073 + protein + cleaner0 + 2023-07-25T14:03:41Z + PR: + + ParC + + + 0.9989273 + protein + cleaner0 + 2023-07-25T14:03:46Z + PR: + + ParE + + + 0.9880157 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.9796252 + protein_state + cleaner0 + 2023-07-25T14:34:17Z + DUMMY: + + bound + + + 0.99837327 + chemical + cleaner0 + 2023-07-25T14:47:22Z + CHEBI: + + quinolone + + + 0.999288 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + + d-72-00488-fig4.jpg + fig4 + FIG + fig_caption + 26924 + Schematic representation of the catalytic cycle of type II topoisomerases. The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. Bound ATP is indicated by pink circles in the ATPase domains (reproduced with permission from Fig. 1 of Lapanogov et al., 2013). + + 0.9978693 + protein_type + cleaner0 + 2023-07-25T14:44:02Z + MESH: + + type II topoisomerases + + + 0.99913293 + protein + cleaner0 + 2023-07-25T14:03:41Z + PR: + + ParC + + + 0.99659085 + protein + cleaner0 + 2023-07-25T14:34:03Z + PR: + + ParC55 + + + 0.999258 + protein + cleaner0 + 2023-07-25T14:03:41Z + PR: + + ParC + + + 0.9991524 + structure_element + cleaner0 + 2023-07-25T14:52:37Z + SO: + + β-­pinwheel domain + + + 0.9992643 + protein + cleaner0 + 2023-07-25T14:03:46Z + PR: + + ParE + + + 0.99911124 + structure_element + cleaner0 + 2023-07-25T14:52:40Z + SO: + + ATPase domain + + + 0.9991266 + protein + cleaner0 + 2023-07-25T14:03:46Z + PR: + + ParE + + + 0.85989636 + structure_element + cleaner0 + 2023-07-25T14:52:43Z + SO: + + C-terminal domain + + + 0.99709535 + protein + cleaner0 + 2023-07-25T14:34:07Z + PR: + + ParE30 + + + 0.77977353 + structure_element + cleaner0 + 2023-07-25T14:21:46Z + SO: + + G-gate + + + 0.9974456 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.96818286 + structure_element + cleaner0 + 2023-07-25T14:52:45Z + SO: + + T-segment + + + 0.9974764 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.99823666 + protein_state + cleaner0 + 2023-07-25T14:34:17Z + DUMMY: + + Bound + + + 0.99909747 + chemical + cleaner0 + 2023-07-25T14:30:36Z + CHEBI: + + ATP + + + 0.9989592 + structure_element + cleaner0 + 2023-07-25T14:52:47Z + SO: + + ATPase domains + + + + d-72-00488-fig5.jpg + fig5 + FIG + fig_caption + 27389 + Detailed views of the active sites of topoisomerase IV from S. pneumoniae and K. pneumoniae with quinolone molecules bound. The magnesium ions and their coordinating amino acids are shown in purple. The drug molecules and residues known to lead to drug resistance upon mutation are in red. The active-site tyrosine and arginine are in orange. The DNA is shown in silver/cyan. The ParC and ParE backbones are shown in blue and yellow, respectively. + + 0.9991249 + site + cleaner0 + 2023-07-25T14:54:08Z + SO: + + active sites + + + complex_assembly + GO: + cleaner0 + 2023-07-25T14:25:38Z + + topoisomerase IV + + + 0.9985092 + species + cleaner0 + 2023-07-25T13:57:01Z + MESH: + + S. pneumoniae + + + 0.9983072 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.99641657 + chemical + cleaner0 + 2023-07-25T14:47:27Z + CHEBI: + + quinolone + + + 0.995401 + protein_state + cleaner0 + 2023-07-25T14:34:17Z + DUMMY: + + bound + + + 0.99900204 + chemical + cleaner0 + 2023-07-25T14:47:32Z + CHEBI: + + magnesium + + + 0.99899036 + site + cleaner0 + 2023-07-25T14:54:10Z + SO: + + active-site + + + 0.99725467 + residue_name + cleaner0 + 2023-07-25T14:31:46Z + SO: + + tyrosine + + + 0.9973659 + residue_name + cleaner0 + 2023-07-25T14:31:48Z + SO: + + arginine + + + 0.9882323 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + 0.9971631 + protein + cleaner0 + 2023-07-25T14:03:41Z + PR: + + ParC + + + 0.9974571 + protein + cleaner0 + 2023-07-25T14:03:46Z + PR: + + ParE + + + + d-72-00488-fig6.jpg + fig6 + FIG + fig_caption + 27837 + Comparison of DNA cleavage by topoisomerase IV core ParE-ParC fusion proteins from K. pneumoniae (KP) and S. pneumoniae (SP) promoted by levofloxacin. Supercoiled plasmid pBR322 (400 ng) was incubated with topoisomerase IV proteins (400 ng) in the absence or presence of levofloxacin at the indicated concentrations. After 60 min incubation, samples were treated with SDS and proteinase K to remove proteins covalent bound to DNA, and the DNA products were examined by gel electrophoresis in 1% agarose. Lane A, supercoiled pBR322 DNA; N, L and S, nicked, linear and supercoiled pBR322, respectively. + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + 0.9077884 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + 0.98855954 + complex_assembly + cleaner0 + 2023-07-25T14:44:56Z + GO: + + ParE-ParC + + + 0.9982917 + species + cleaner0 + 2023-07-25T13:56:28Z + MESH: + + K. pneumoniae + + + 0.9960282 + species + cleaner0 + 2023-07-25T14:41:54Z + MESH: + + KP + + + 0.99837327 + species + cleaner0 + 2023-07-25T14:41:57Z + MESH: + + S. pneumoniae + + + 0.99792224 + species + cleaner0 + 2023-07-25T14:42:00Z + MESH: + + SP + + + 0.99899095 + chemical + cleaner0 + 2023-07-25T14:01:07Z + CHEBI: + + levofloxacin + + + 0.9897747 + complex_assembly + cleaner0 + 2023-07-25T14:25:38Z + GO: + + topoisomerase IV + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T14:23:38Z + + presence of + + + 0.99899036 + chemical + cleaner0 + 2023-07-25T14:01:08Z + CHEBI: + + levofloxacin + + + 0.7885971 + chemical + cleaner0 + 2023-07-25T14:00:32Z + CHEBI: + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-25T14:00:32Z + + DNA + + + + + table1 + TABLE + table_title_caption + 28445 + Macromolecule-production information + + + d36e1283.xml + table1 + TABLE + table_caption + 28482 + +K. pneumoniae topoisomerase IV. + + + d36e1283.xml + table1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><tbody valign="top"><tr><td rowspan="1" colspan="1" align="left" valign="top">Source organism</td><td rowspan="1" colspan="1" align="left" valign="top"> +<italic>K. pneumoniae</italic> (strain ATCC 35657)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Expression vector</td><td rowspan="1" colspan="1" align="left" valign="top">pET-29a</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Expression host</td><td rowspan="1" colspan="1" align="left" valign="top"> +<italic>E. coli</italic> BL21(λDE3) pLysS</td></tr><tr><td rowspan="1" colspan="2" align="left" valign="top">Complete amino-acid sequence of the construct produced</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Topoisomerase IV (ParE CTD 390–631 and ParC NTD 1–490 fused) </td><td rowspan="1" colspan="1" align="left" valign="top"> +<underline>M</underline>KKLTSGPALPGKLADCTAQDLNRTELFLVEGDSAGGSAKQARDREYQAIMPLKGKILNTWEVSSDEVLASQEVHDISVAIGIDPDSDDLSQLRYGKICILADADSDGLHIATLLCALFVRHFRTLVKEGHVYVALPPLYRIDLGKEVYYALTEEEKTGVLEQLKRKKGKPNVQRFKGLGEMNPMQLRETTLDPNTRRLVQLVISDEDEQQTTAIMDMLLAKKRSEDRRNWLQEKGDMADLEV<underline>EF</underline>MSDMAERLALHEFTENAYLNYSMYVIMDRALPFIGDGLKPVQRRIVYAMSELGLNASAKFKKSARTVGDVLGKYHPHGDSACYEAMVLMAQPFSYRYPLGDGQGNWGAPDDPKSFAAMRYTESRLSKYAELLLSELGQGTVDWVPNFDGTLQEPKMLPARLPNILLNGTTGIAVGMATDIPPHNLREVAKAAITLIEQPKTTLDELLDIVQGPDFPTEAEIITSRAEIRKIYQNGRGSVRMRAVWSKEDGAVVITALPHQVSGAKVLEQIAAQMRNKKLPMVDDLRDESDHENPTRLVIVPRSNRVDMEQVMNHLFATTDLEKSYRINLNMIGLDGRPAVKNLLEILSEWLVFRRDTVRRRLNHRLEKVLKRLHILEGLLVAFLNIDEVIEIIRTEDEPKPALMSRFGISETQAEAILELKLRHLAKLEEMKIRGEQSELEKERDQLQAILASERKMNNLLKKELQADADAFGDDRRSPLHEREEAKAMS<underline>HHHHHH</underline> +</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Symmetrized E-site (pre-cut) DNA1</td><td rowspan="1" colspan="1" align="left" valign="top">5′-CGTATTACGTTGTAT-3′</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Symmetrized E-site (pre-cut) DNA2</td><td rowspan="1" colspan="1" align="left" valign="top">5′-GATCATACAACGTAATACG-3′</td></tr></tbody></table> + + 28515 + Source organism K. pneumoniae (strain ATCC 35657) Expression vector pET-29a Expression host E. coli BL21(λDE3) pLysS Complete amino-acid sequence of the construct produced  Topoisomerase IV (ParE CTD 390–631 and ParC NTD 1–490 fused) MKKLTSGPALPGKLADCTAQDLNRTELFLVEGDSAGGSAKQARDREYQAIMPLKGKILNTWEVSSDEVLASQEVHDISVAIGIDPDSDDLSQLRYGKICILADADSDGLHIATLLCALFVRHFRTLVKEGHVYVALPPLYRIDLGKEVYYALTEEEKTGVLEQLKRKKGKPNVQRFKGLGEMNPMQLRETTLDPNTRRLVQLVISDEDEQQTTAIMDMLLAKKRSEDRRNWLQEKGDMADLEVEFMSDMAERLALHEFTENAYLNYSMYVIMDRALPFIGDGLKPVQRRIVYAMSELGLNASAKFKKSARTVGDVLGKYHPHGDSACYEAMVLMAQPFSYRYPLGDGQGNWGAPDDPKSFAAMRYTESRLSKYAELLLSELGQGTVDWVPNFDGTLQEPKMLPARLPNILLNGTTGIAVGMATDIPPHNLREVAKAAITLIEQPKTTLDELLDIVQGPDFPTEAEIITSRAEIRKIYQNGRGSVRMRAVWSKEDGAVVITALPHQVSGAKVLEQIAAQMRNKKLPMVDDLRDESDHENPTRLVIVPRSNRVDMEQVMNHLFATTDLEKSYRINLNMIGLDGRPAVKNLLEILSEWLVFRRDTVRRRLNHRLEKVLKRLHILEGLLVAFLNIDEVIEIIRTEDEPKPALMSRFGISETQAEAILELKLRHLAKLEEMKIRGEQSELEKERDQLQAILASERKMNNLLKKELQADADAFGDDRRSPLHEREEAKAMSHHHHHH  Symmetrized E-site (pre-cut) DNA1 5′-CGTATTACGTTGTAT-3′  Symmetrized E-site (pre-cut) DNA2 5′-GATCATACAACGTAATACG-3′ + + + d36e1342.xml + table1 + TABLE + table_caption + 29644 + +S. pneumoniae topoisomerase IV. + + + d36e1342.xml + table1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><tbody valign="top"><tr><td rowspan="1" colspan="1" align="left" valign="top">Source organism</td><td rowspan="1" colspan="1" align="left" valign="top"> +<italic>S. pneumoniae</italic> (isolate 7785 St George’s Hospital; Pan &amp; Fisher, 1996<xref ref-type="bibr" rid="bb50"> ▸</xref>)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Expression vector</td><td rowspan="1" colspan="1" align="left" valign="top">pET-19b (N-terminal His<sub>10</sub>), pET-29a (C-terminal His<sub>6</sub>)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Expression host</td><td rowspan="1" colspan="1" align="left" valign="top"> +<italic>E. coli</italic> BL21(λDE3) pLysS</td></tr><tr><td rowspan="1" colspan="2" align="left" valign="top">Complete amino-acid sequence of the construct produced</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> ParC55</td><td rowspan="1" colspan="1" align="left" valign="top">MSNIQNMSLEDIMGERFGRYSKYIIQDRALPDIRDGLKPVQRRILYSMNKDSNTFDKSYRKSAKSVGNIMGNFHPHGDSSIYDAMVRMSQNWKNREILVEMHGNNGSMDGDPPAAMRYTEARLSEIAGYLLQDIEKKTVPFAWNFDDTEKEPTVLPAAFPNLLVNGSTGISAGYATDIPPHNLAEVIDAAVYMIDHPTAKIDKLMEFLPGPDFPTGAIIQGRDEIKKAYETGKGRVVVRSKTEIEKLKGGKEQIVITEIPYEINKANLVKKIDDVRVNNKVAGIAEVRDESDRDGLRIAIELKKDANTELVLNYLFKYTDLQINYNFNMVAIDNFTPRQVGIVPILSSYIAHRREVILARSRFDKEKAEKRLHIVEGLIRVISILDEVIALIRASENKADAKENLKVSYDFTEEQAEAIVTLQLYRLTNTDVVVLQEEEAELREKIAMLAAIIGDERTMYNLMKKELREVKKKFATPRLSSLEDTAKA<underline>L</underline>E<underline>HHHHHH</underline> +</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> ParE30</td><td rowspan="1" colspan="1" align="left" valign="top"> +<underline>MGHHHHHHHHHHSSGHIDDDDKHM</underline>KNKKDKGLLSGKLTPAQSKNPAKNELYLVEGDSAGGSAKQGRDRKFQAILPLRGKVINTAKAKMADILKNEEINTMIYTIGAGVGADFSIEDANYDKIIIMTDADTDGAHIQTLLLTFFYRYMRPLVEAGHVYIALPPLYKMSKGKGKKEEVAYAWTDGELEELRKQFGKGATLQRYKGLGEMNADQLWETTMNPETRTLIRVTIEDLARAERRVNVLMGDKVEPRRKWIEDNVKFTLEEATVF </td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> E-site DNA1 </td><td rowspan="1" colspan="1" align="left" valign="top">5′-CATGAATGACTATGCACG-3′</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> E-site DNA2 </td><td rowspan="1" colspan="1" align="left" valign="top">5′-CGTGCATAGTCATTCATG-3′</td></tr></tbody></table> + + 29677 + Source organism S. pneumoniae (isolate 7785 St George’s Hospital; Pan & Fisher, 1996) Expression vector pET-19b (N-terminal His10), pET-29a (C-terminal His6) Expression host E. coli BL21(λDE3) pLysS Complete amino-acid sequence of the construct produced  ParC55 MSNIQNMSLEDIMGERFGRYSKYIIQDRALPDIRDGLKPVQRRILYSMNKDSNTFDKSYRKSAKSVGNIMGNFHPHGDSSIYDAMVRMSQNWKNREILVEMHGNNGSMDGDPPAAMRYTEARLSEIAGYLLQDIEKKTVPFAWNFDDTEKEPTVLPAAFPNLLVNGSTGISAGYATDIPPHNLAEVIDAAVYMIDHPTAKIDKLMEFLPGPDFPTGAIIQGRDEIKKAYETGKGRVVVRSKTEIEKLKGGKEQIVITEIPYEINKANLVKKIDDVRVNNKVAGIAEVRDESDRDGLRIAIELKKDANTELVLNYLFKYTDLQINYNFNMVAIDNFTPRQVGIVPILSSYIAHRREVILARSRFDKEKAEKRLHIVEGLIRVISILDEVIALIRASENKADAKENLKVSYDFTEEQAEAIVTLQLYRLTNTDVVVLQEEEAELREKIAMLAAIIGDERTMYNLMKKELREVKKKFATPRLSSLEDTAKALEHHHHHH  ParE30 MGHHHHHHHHHHSSGHIDDDDKHMKNKKDKGLLSGKLTPAQSKNPAKNELYLVEGDSAGGSAKQGRDRKFQAILPLRGKVINTAKAKMADILKNEEINTMIYTIGAGVGADFSIEDANYDKIIIMTDADTDGAHIQTLLLTFFYRYMRPLVEAGHVYIALPPLYKMSKGKGKKEEVAYAWTDGELEELRKQFGKGATLQRYKGLGEMNADQLWETTMNPETRTLIRVTIEDLARAERRVNVLMGDKVEPRRKWIEDNVKFTLEEATVF  E-site DNA1 5′-CATGAATGACTATGCACG-3′  E-site DNA2 5′-CGTGCATAGTCATTCATG-3′ + + + table2.xml + table2 + TABLE + table_title_caption + 30828 + Crystallization + + + table2.xml + table2 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><thead valign="top"><tr><th style="border-bottom:1px solid black;" rowspan="1" colspan="1" align="left" valign="bottom"> </th><th style="border-bottom:1px solid black;" rowspan="1" colspan="1" align="left" valign="bottom"> +<italic>K. pneumoniae</italic> topoisomerase IV</th><th style="border-bottom:1px solid black;" rowspan="1" colspan="1" align="left" valign="bottom"> +<italic>S. pneumoniae</italic> topoisomerase IV</th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1" align="left" valign="top">Method</td><td rowspan="1" colspan="1" align="left" valign="top">Vapour diffusion</td><td rowspan="1" colspan="1" align="left" valign="top">Capillary counter-diffusion</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Plate type</td><td rowspan="1" colspan="1" align="left" valign="top">24-well Limbro</td><td rowspan="1" colspan="1" align="left" valign="top">N/A</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Temperature (K)</td><td rowspan="1" colspan="1" align="left" valign="top">298</td><td rowspan="1" colspan="1" align="left" valign="top">304</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Protein concentration (mg ml<sup>−1</sup>)</td><td rowspan="1" colspan="1" align="left" valign="top">4.5 </td><td rowspan="1" colspan="1" align="left" valign="top">4</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Buffer composition of protein solution</td><td rowspan="1" colspan="2" align="left" valign="top">20 m<italic>M</italic> Tris pH 7.5, 100 m<italic>M</italic> NaCl, 1 m<italic>M</italic> β-mercaptoethanol, 0.05% NaN<sub>3</sub> +</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Composition of reservoir solution</td><td rowspan="1" colspan="1" align="left" valign="top">0.1 <italic>M</italic> Tris pH 7.5–8.0, 0–50 m<italic>M</italic> NaCl, 4–8% PEG 4000, 12–15% glycerol</td><td rowspan="1" colspan="1" align="left" valign="top">50 m<italic>M</italic> sodium cacodylate pH 6.5, 2.5% Tacsimate, 7% 2-propanol, 62.5 m<italic>M</italic> KCl, 7.5 m<italic>M</italic> MgCl<sub>2</sub> +</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Volume and ratio of drop</td><td rowspan="1" colspan="1" align="left" valign="top">4 + 2 µl</td><td rowspan="1" colspan="1" align="left" valign="top">N/A</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Volume of reservoir (ml)</td><td rowspan="1" colspan="1" align="left" valign="top">0.5</td><td rowspan="1" colspan="1" align="left" valign="top">N/A</td></tr></tbody></table> + + 30844 +   K. pneumoniae topoisomerase IV S. pneumoniae topoisomerase IV Method Vapour diffusion Capillary counter-diffusion Plate type 24-well Limbro N/A Temperature (K) 298 304 Protein concentration (mg ml−1) 4.5 4 Buffer composition of protein solution 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM β-mercaptoethanol, 0.05% NaN3 Composition of reservoir solution 0.1 M Tris pH 7.5–8.0, 0–50 mM NaCl, 4–8% PEG 4000, 12–15% glycerol 50 mM sodium cacodylate pH 6.5, 2.5% Tacsimate, 7% 2-propanol, 62.5 mM KCl, 7.5 mM MgCl2 Volume and ratio of drop 4 + 2 µl N/A Volume of reservoir (ml) 0.5 N/A + + + table3.xml + table3 + TABLE + table_title_caption + 31473 + Data collection and processing + + + table3.xml + table3 + TABLE + table_caption + 31504 + Values in parentheses are for the outer shell. + + + table3.xml + table3 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><thead valign="bottom"><tr><th style="border-bottom:1px solid black;" rowspan="1" colspan="1" align="left" valign="bottom"> </th><th style="border-bottom:1px solid black;" rowspan="1" colspan="1" align="left" valign="bottom"> +<italic>K. pneumoniae</italic> topoisomerase IV</th><th style="border-bottom:1px solid black;" rowspan="1" colspan="1" align="left" valign="bottom"> +<italic>S. pneumoniae</italic> topoisomerase IV</th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1" align="left" valign="top">Diffraction source</td><td rowspan="1" colspan="2" align="left" valign="top">Beamline I03, Diamond Light Source</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Wavelength (Å)</td><td rowspan="1" colspan="1" align="left" valign="top">0.97620</td><td rowspan="1" colspan="1" align="left" valign="top">0.97630</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Temperature (K)</td><td rowspan="1" colspan="1" align="left" valign="top">100.0</td><td rowspan="1" colspan="1" align="left" valign="top">100.0</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Detector</td><td rowspan="1" colspan="1" align="left" valign="top">Pilatus 6M-F</td><td rowspan="1" colspan="1" align="left" valign="top">ADSC Quantum 315</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Crystal-to-detector distance (mm)</td><td rowspan="1" colspan="1" align="left" valign="top">502.22</td><td rowspan="1" colspan="1" align="left" valign="top">377.629</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Rotation range per image (°)</td><td rowspan="1" colspan="1" align="left" valign="top">0.2</td><td rowspan="1" colspan="1" align="left" valign="top">0.25</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Total rotation range (°)</td><td rowspan="1" colspan="1" align="left" valign="top">180</td><td rowspan="1" colspan="1" align="left" valign="top">75</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Exposure time per image (s)</td><td rowspan="1" colspan="1" align="left" valign="top">0.2</td><td rowspan="1" colspan="1" align="left" valign="top">1.0</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Space group</td><td rowspan="1" colspan="1" align="left" valign="top"> +<italic>P</italic>2<sub>1</sub> +</td><td rowspan="1" colspan="1" align="left" valign="top"> +<italic>P</italic>3<sub>1</sub>21</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> +<italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td rowspan="1" colspan="1" align="left" valign="top">102.07, 161.53, 138.60</td><td rowspan="1" colspan="1" align="left" valign="top">157.83, 157.83, 211.15</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">α, β, γ (°)</td><td rowspan="1" colspan="1" align="left" valign="top">90, 94.22, 90</td><td rowspan="1" colspan="1" align="left" valign="top"> </td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Mosaicity (°)</td><td rowspan="1" colspan="1" align="left" valign="top">0.237</td><td rowspan="1" colspan="1" align="left" valign="top">0.466</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Resolution range (Å)</td><td rowspan="1" colspan="1" align="left" valign="top">86.12–3.35 (3.53–3.35)</td><td rowspan="1" colspan="1" align="left" valign="top">50–2.90 (3.00–2.90)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Total No. of reflections</td><td rowspan="1" colspan="1" align="left" valign="top">160764</td><td rowspan="1" colspan="1" align="left" valign="top">311576</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">No. of unique reflections</td><td rowspan="1" colspan="1" align="left" valign="top">63406</td><td rowspan="1" colspan="1" align="left" valign="top">67471</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Completeness (%)</td><td rowspan="1" colspan="1" align="left" valign="top">98.5 (98.4)</td><td rowspan="1" colspan="1" align="left" valign="top">99.4 (99.9)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Multiplicity</td><td rowspan="1" colspan="1" align="left" valign="top">2.53 (2.59)</td><td rowspan="1" colspan="1" align="left" valign="top">4.6 (4.7)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">〈<italic>I</italic>/σ(<italic>I</italic>)〉</td><td rowspan="1" colspan="1" align="left" valign="top">3.48 (1.95)</td><td rowspan="1" colspan="1" align="left" valign="top">16.14 (3.48)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> +<italic>R</italic> +<sub>r.i.m.</sub> +<xref ref-type="table-fn" rid="tfn1">†</xref> +</td><td rowspan="1" colspan="1" align="left" valign="top">0.116 (0.434)</td><td rowspan="1" colspan="1" align="left" valign="top">0.08 (0.515)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Overall <italic>B</italic> factor from Wilson plot (Å<sup>2</sup>)</td><td rowspan="1" colspan="1" align="left" valign="top">53.29</td><td rowspan="1" colspan="1" align="left" valign="top">73.37</td></tr></tbody></table> + + 31551 +   K. pneumoniae topoisomerase IV S. pneumoniae topoisomerase IV Diffraction source Beamline I03, Diamond Light Source Wavelength (Å) 0.97620 0.97630 Temperature (K) 100.0 100.0 Detector Pilatus 6M-F ADSC Quantum 315 Crystal-to-detector distance (mm) 502.22 377.629 Rotation range per image (°) 0.2 0.25 Total rotation range (°) 180 75 Exposure time per image (s) 0.2 1.0 Space group P21 P3121 a, b, c (Å) 102.07, 161.53, 138.60 157.83, 157.83, 211.15 α, β, γ (°) 90, 94.22, 90   Mosaicity (°) 0.237 0.466 Resolution range (Å) 86.12–3.35 (3.53–3.35) 50–2.90 (3.00–2.90) Total No. of reflections 160764 311576 No. of unique reflections 63406 67471 Completeness (%) 98.5 (98.4) 99.4 (99.9) Multiplicity 2.53 (2.59) 4.6 (4.7) 〈I/σ(I)〉 3.48 (1.95) 16.14 (3.48) Rr.i.m.† 0.116 (0.434) 0.08 (0.515) Overall B factor from Wilson plot (Å2) 53.29 73.37 + + + table3.xml + table3 + TABLE + table_footnote + 32465 + Estimated R +r.i.m. = R +merge[N/(N − 1)]1/2, where N is the data multiplicity. + + + table4.xml + table4 + TABLE + table_title_caption + 32545 + Structure solution and refinement + + + table4.xml + table4 + TABLE + table_caption + 32579 + Values in parentheses are for the outer shell. + + + table4.xml + table4 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><thead valign="bottom"><tr><th style="border-bottom:1px solid black;" rowspan="1" colspan="1" align="left" valign="bottom"> </th><th style="border-bottom:1px solid black;" rowspan="1" colspan="1" align="left" valign="bottom"> +<italic>K. pneumoniae</italic> topoisomerase IV</th><th style="border-bottom:1px solid black;" rowspan="1" colspan="1" align="left" valign="bottom"> +<italic>S. pneumoniae</italic> topoisomerase IV</th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1" align="left" valign="top">Resolution range (Å)</td><td rowspan="1" colspan="1" align="left" valign="top">85.01–3.35 (3.40–3.35)</td><td rowspan="1" colspan="1" align="left" valign="top">41.83–2.90 (2.93–2.90)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Completeness (%)</td><td rowspan="1" colspan="1" align="left" valign="top">98.3</td><td rowspan="1" colspan="1" align="left" valign="top">99.5</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">σ Cutoff</td><td rowspan="1" colspan="1" align="left" valign="top"> +<italic>F</italic> &gt; 1.350σ(<italic>F</italic>)</td><td rowspan="1" colspan="1" align="left" valign="top"> +<italic>F</italic> &gt; 1.34σ(<italic>F</italic>)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">No. of reflections, working set</td><td rowspan="1" colspan="1" align="left" valign="top">60158 (2615)</td><td rowspan="1" colspan="1" align="left" valign="top">67471 (1992)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">No. of reflections, test set</td><td rowspan="1" colspan="1" align="left" valign="top">3208 (142)</td><td rowspan="1" colspan="1" align="left" valign="top">6838 (218)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Final <italic>R</italic> +<sub>cryst</sub> +</td><td rowspan="1" colspan="1" align="left" valign="top">0.224 (0.2990)</td><td rowspan="1" colspan="1" align="left" valign="top">0.186 (0.2806)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top">Final <italic>R</italic> +<sub>free</sub> +</td><td rowspan="1" colspan="1" align="left" valign="top">0.259 (0.3537)</td><td rowspan="1" colspan="1" align="left" valign="top">0.226 (0.3562)</td></tr><tr><td rowspan="1" colspan="3" align="left" valign="top">No. of non-H atoms</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Protein</td><td rowspan="1" colspan="1" align="left" valign="top">18741</td><td rowspan="1" colspan="1" align="left" valign="top">10338</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Nucleic acid</td><td rowspan="1" colspan="1" align="left" valign="top">1608</td><td rowspan="1" colspan="1" align="left" valign="top">730</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Ligand</td><td rowspan="1" colspan="1" align="left" valign="top">104</td><td rowspan="1" colspan="1" align="left" valign="top">52</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Ion</td><td rowspan="1" colspan="1" align="left" valign="top">8</td><td rowspan="1" colspan="1" align="left" valign="top">6</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Water</td><td rowspan="1" colspan="1" align="left" valign="top">—</td><td rowspan="1" colspan="1" align="left" valign="top">54</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Total</td><td rowspan="1" colspan="1" align="left" valign="top">20461</td><td rowspan="1" colspan="1" align="left" valign="top">11180</td></tr><tr><td rowspan="1" colspan="3" align="left" valign="top">R.m.s. deviations</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Bonds (Å)</td><td rowspan="1" colspan="1" align="left" valign="top">0.002</td><td rowspan="1" colspan="1" align="left" valign="top">0.008</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Angles (°)</td><td rowspan="1" colspan="1" align="left" valign="top">0.611</td><td rowspan="1" colspan="1" align="left" valign="top">1.221</td></tr><tr><td rowspan="1" colspan="3" align="left" valign="top">Average <italic>B</italic> factors (Å<sup>2</sup>)</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Protein</td><td rowspan="1" colspan="1" align="left" valign="top">58.05</td><td rowspan="1" colspan="1" align="left" valign="top">76.7</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Nucleic acid</td><td rowspan="1" colspan="1" align="left" valign="top">64.85</td><td rowspan="1" colspan="1" align="left" valign="top">90.7</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Ligand</td><td rowspan="1" colspan="1" align="left" valign="top">60.14</td><td rowspan="1" colspan="1" align="left" valign="top">95.7</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Ion</td><td rowspan="1" colspan="1" align="left" valign="top">42.62</td><td rowspan="1" colspan="1" align="left" valign="top">84.5</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Water</td><td rowspan="1" colspan="1" align="left" valign="top">—</td><td rowspan="1" colspan="1" align="left" valign="top">64.2</td></tr><tr><td rowspan="1" colspan="3" align="left" valign="top">Ramachandran plot</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Most favoured (%)</td><td rowspan="1" colspan="1" align="left" valign="top">93</td><td rowspan="1" colspan="1" align="left" valign="top">94</td></tr><tr><td rowspan="1" colspan="1" align="left" valign="top"> Allowed (%)</td><td rowspan="1" colspan="1" align="left" valign="top">6</td><td rowspan="1" colspan="1" align="left" valign="top">6</td></tr></tbody></table> + + 32626 +   K. pneumoniae topoisomerase IV S. pneumoniae topoisomerase IV Resolution range (Å) 85.01–3.35 (3.40–3.35) 41.83–2.90 (2.93–2.90) Completeness (%) 98.3 99.5 σ Cutoff F > 1.350σ(F) F > 1.34σ(F) No. of reflections, working set 60158 (2615) 67471 (1992) No. of reflections, test set 3208 (142) 6838 (218) Final Rcryst 0.224 (0.2990) 0.186 (0.2806) Final Rfree 0.259 (0.3537) 0.226 (0.3562) No. of non-H atoms  Protein 18741 10338  Nucleic acid 1608 730  Ligand 104 52  Ion 8 6  Water — 54  Total 20461 11180 R.m.s. deviations  Bonds (Å) 0.002 0.008  Angles (°) 0.611 1.221 Average B factors (Å2)  Protein 58.05 76.7  Nucleic acid 64.85 90.7  Ligand 60.14 95.7  Ion 42.62 84.5  Water — 64.2 Ramachandran plot  Most favoured (%) 93 94  Allowed (%) 6 6 + + + diff --git a/BioC_XML/4831588_v0.xml b/BioC_XML/4831588_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..b8e1a866049f5f330515097695464ac1196ad515 --- /dev/null +++ b/BioC_XML/4831588_v0.xml @@ -0,0 +1,11149 @@ + + + + PMC + 20201216 + pmc.key + + 4831588 + NO-CC CODE + no + 0 + 0 + + 10.1021/jacs.6b01332 + 4831588 + 26967810 + 4634 + 13 + This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. + 4642 + surname:Kreutzer;given-names:Adam G. + surname:Hamza;given-names:Imane L. + surname:Spencer;given-names:Ryan K. + surname:Nowick;given-names:James S. + TITLE + front + 138 + 2017 + 0 + X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an Aβ17–36 β-Hairpin + + 0.9994534 + evidence + cleaner0 + 2023-07-10T14:56:28Z + DUMMY: + + X-ray Crystallographic Structures + + + 0.9992366 + oligomeric_state + cleaner0 + 2023-07-10T09:57:04Z + DUMMY: + + Trimer + + + 0.9990484 + oligomeric_state + cleaner0 + 2023-07-10T10:07:15Z + DUMMY: + + Dodecamer + + + 0.92726004 + site + cleaner0 + 2023-07-10T14:08:21Z + SO: + + Annular Pore + + + protein + PR: + cleaner0 + 2023-07-10T10:08:53Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:09:12Z + + 17–36 + + + 0.998604 + structure_element + cleaner0 + 2023-07-10T10:09:18Z + SO: + + β-Hairpin + + + + ABSTRACT + abstract + 112 + High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are needed to understand the molecular basis of Alzheimer’s disease and develop therapies. This paper presents the X-ray crystallographic structures of oligomers formed by a 20-residue peptide segment derived from Aβ. The development of a peptide in which Aβ17–36 is stabilized as a β-hairpin is described, and the X-ray crystallographic structures of oligomers it forms are reported. Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. An N-methyl group at position 33 blocks uncontrolled aggregation. The peptide readily crystallizes as a folded β-hairpin, which assembles hierarchically in the crystal lattice. Three β-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore. This hierarchical assembly provides a model, in which full-length Aβ transitions from an unfolded monomer to a folded β-hairpin, which assembles to form oligomers that further pack to form an annular pore. This model may provide a better understanding of the molecular basis of Alzheimer’s disease at atomic resolution. + + 0.9979809 + evidence + cleaner0 + 2023-07-10T14:56:36Z + DUMMY: + + structures + + + 0.97725874 + oligomeric_state + cleaner0 + 2023-07-10T09:57:13Z + DUMMY: + + oligomers + + + 0.86354554 + protein + cleaner0 + 2023-07-10T14:36:01Z + PR: + + β-amyloid peptide + + + 0.7519912 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.9995192 + evidence + cleaner0 + 2023-07-10T14:56:29Z + DUMMY: + + X-ray crystallographic structures + + + 0.9574868 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + 0.8060632 + residue_range + cleaner0 + 2023-07-10T14:42:56Z + DUMMY: + + 20-residue peptide segment + + + 0.5019251 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + protein + PR: + cleaner0 + 2023-07-10T14:38:49Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T14:39:01Z + + 17–36 + + + 0.99956745 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.9995325 + evidence + cleaner0 + 2023-07-10T14:56:29Z + DUMMY: + + X-ray crystallographic structures + + + 0.70311177 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + protein + PR: + cleaner0 + 2023-07-10T14:39:36Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T14:39:47Z + + 17–36 + + + structure_element + SO: + cleaner0 + 2023-07-10T14:40:06Z + + hairpin + + + protein_state + DUMMY: + cleaner0 + 2023-07-10T10:26:42Z + + δ-linked + + + residue_name + SO: + cleaner0 + 2023-07-10T10:26:21Z + + ornithine + + + structure_element + SO: + cleaner0 + 2023-07-10T14:02:39Z + + turn + + + 0.99510807 + residue_number + cleaner0 + 2023-07-10T14:43:03Z + DUMMY: + + 17 + + + 0.9962352 + residue_number + cleaner0 + 2023-07-10T14:43:06Z + DUMMY: + + 36 + + + 0.9945172 + ptm + cleaner0 + 2023-07-10T10:10:01Z + MESH: + + disulfide linkage + + + 0.9947225 + residue_number + cleaner0 + 2023-07-10T14:43:09Z + DUMMY: + + 24 + + + 0.9957502 + residue_number + cleaner0 + 2023-07-10T14:43:14Z + DUMMY: + + 29 + + + 0.9970932 + residue_number + cleaner0 + 2023-07-10T14:43:17Z + DUMMY: + + 33 + + + evidence + DUMMY: + cleaner0 + 2023-07-10T15:36:42Z + + readily crystallizes + + + 0.9996369 + protein_state + cleaner0 + 2023-07-10T14:40:19Z + DUMMY: + + folded + + + 0.99950814 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.8951478 + evidence + cleaner0 + 2023-07-10T14:56:41Z + DUMMY: + + crystal lattice + + + 0.9995174 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.9990044 + oligomeric_state + cleaner0 + 2023-07-10T09:57:24Z + DUMMY: + + monomers + + + 0.94550765 + protein_state + cleaner0 + 2023-07-10T09:57:45Z + DUMMY: + + triangular + + + 0.9986405 + oligomeric_state + cleaner0 + 2023-07-10T09:57:06Z + DUMMY: + + trimer + + + 0.9976846 + oligomeric_state + cleaner0 + 2023-07-10T09:58:03Z + DUMMY: + + trimers + + + 0.80919456 + oligomeric_state + cleaner0 + 2023-07-10T10:07:47Z + DUMMY: + + dodecamer + + + 0.66039157 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + 0.9301689 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.99956006 + protein_state + cleaner0 + 2023-07-10T14:29:13Z + DUMMY: + + full-length + + + 0.8096459 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.9996525 + protein_state + cleaner0 + 2023-07-10T14:40:26Z + DUMMY: + + unfolded + + + 0.99926764 + oligomeric_state + cleaner0 + 2023-07-10T09:58:16Z + DUMMY: + + monomer + + + 0.99964523 + protein_state + cleaner0 + 2023-07-10T14:40:20Z + DUMMY: + + folded + + + 0.99954224 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.998198 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + 0.9254092 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + + INTRO + title_1 + 1545 + Introduction + + + INTRO + paragraph + 1558 + High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are desperately needed to understand the molecular basis of Alzheimer’s disease and ultimately develop preventions or treatments. In Alzheimer’s disease, monomeric Aβ aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils. Over the last two decades the role of Aβ oligomers in the pathophysiology of Alzheimer’s disease has begun to unfold. + + 0.99909747 + evidence + cleaner0 + 2023-07-10T14:56:46Z + DUMMY: + + structures + + + 0.99652416 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + 0.94002265 + protein + cleaner0 + 2023-07-10T14:40:39Z + PR: + + β-amyloid peptide + + + 0.93936694 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.9981597 + oligomeric_state + cleaner0 + 2023-07-10T09:58:24Z + DUMMY: + + monomeric + + + 0.89636207 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.9967926 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + 0.99898165 + oligomeric_state + cleaner0 + 2023-07-10T09:58:30Z + DUMMY: + + dimers + + + 0.9987111 + oligomeric_state + cleaner0 + 2023-07-10T09:58:05Z + DUMMY: + + trimers + + + 0.9987313 + oligomeric_state + cleaner0 + 2023-07-10T09:58:36Z + DUMMY: + + tetramers + + + 0.9982185 + oligomeric_state + cleaner0 + 2023-07-10T09:58:41Z + DUMMY: + + hexamers + + + 0.9954139 + oligomeric_state + cleaner0 + 2023-07-10T09:58:47Z + DUMMY: + + nonamers + + + 0.73250586 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + complex_assembly + GO: + cleaner0 + 2023-07-10T10:16:52Z + + annular protofibrils + + + 0.871762 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.9886619 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + + INTRO + paragraph + 2136 + Mouse models for Alzheimer’s disease have helped shape our current understanding about the Aβ oligomerization that precedes neurodegeneration. Aβ isolated from the brains of young plaque-free Tg2576 mice forms a mixture of low molecular weight oligomers. A 56 kDa soluble oligomer identified by SDS-PAGE was found to be especially important within this mixture. This oligomer was termed Aβ*56 and appears to be a dodecamer of Aβ. Purified Aβ*56 injected intercranially into healthy rats was found to impair memory, providing evidence that this Aβ oligomer may cause memory loss in Alzheimer’s disease. Smaller oligomers with molecular weights consistent with trimers, hexamers, and nonamers were also identified within the mixture of low molecular weight oligomers. Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. Recently, Aβ trimers and Aβ*56 were identified in the brains of cognitively normal humans and were found to increase with age. + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-10T10:17:54Z + + Mouse + + + 0.95153725 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.76898766 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-10T10:18:10Z + + mice + + + 0.99578744 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + 0.9961333 + oligomeric_state + cleaner0 + 2023-07-10T09:59:25Z + DUMMY: + + oligomer + + + 0.99951744 + experimental_method + cleaner0 + 2023-07-10T10:22:56Z + MESH: + + SDS-PAGE + + + 0.99662304 + oligomeric_state + cleaner0 + 2023-07-10T09:59:26Z + DUMMY: + + oligomer + + + 0.99874526 + complex_assembly + cleaner0 + 2023-07-10T10:16:25Z + GO: + + Aβ*56 + + + 0.9969062 + oligomeric_state + cleaner0 + 2023-07-10T10:14:51Z + DUMMY: + + dodecamer + + + 0.524894 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.999077 + complex_assembly + cleaner0 + 2023-07-10T10:16:25Z + GO: + + Aβ*56 + + + 0.930322 + experimental_method + cleaner0 + 2023-07-10T15:36:51Z + MESH: + + injected intercranially + + + 0.71981853 + taxonomy_domain + cleaner0 + 2023-07-10T14:41:27Z + DUMMY: + + rats + + + 0.7839674 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.99745375 + oligomeric_state + cleaner0 + 2023-07-10T09:59:26Z + DUMMY: + + oligomer + + + 0.9809374 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + 0.99799526 + oligomeric_state + cleaner0 + 2023-07-10T09:58:05Z + DUMMY: + + trimers + + + 0.9979175 + oligomeric_state + cleaner0 + 2023-07-10T09:58:42Z + DUMMY: + + hexamers + + + 0.99625224 + oligomeric_state + cleaner0 + 2023-07-10T09:58:48Z + DUMMY: + + nonamers + + + 0.9794788 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + 0.9043188 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + 0.99964046 + chemical + cleaner0 + 2023-07-10T14:46:54Z + CHEBI: + + hexafluoroisopropanol + + + 0.7555674 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + 0.9777452 + oligomeric_state + cleaner0 + 2023-07-10T09:58:48Z + DUMMY: + + nonamers + + + 0.9961927 + oligomeric_state + cleaner0 + 2023-07-10T09:58:42Z + DUMMY: + + hexamers + + + 0.9981608 + oligomeric_state + cleaner0 + 2023-07-10T09:58:05Z + DUMMY: + + trimers + + + 0.9986059 + oligomeric_state + cleaner0 + 2023-07-10T09:57:24Z + DUMMY: + + monomers + + + 0.9974662 + oligomeric_state + cleaner0 + 2023-07-10T09:58:05Z + DUMMY: + + trimers + + + 0.9601586 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + 0.9466172 + oligomeric_state + cleaner0 + 2023-07-10T09:58:48Z + DUMMY: + + nonamers + + + 0.9969951 + oligomeric_state + cleaner0 + 2023-07-10T09:58:42Z + DUMMY: + + hexamers + + + 0.4964463 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.99813914 + oligomeric_state + cleaner0 + 2023-07-10T09:58:05Z + DUMMY: + + trimers + + + 0.9990856 + complex_assembly + cleaner0 + 2023-07-10T10:16:25Z + GO: + + Aβ*56 + + + 0.9989365 + species + cleaner0 + 2023-07-10T14:41:36Z + MESH: + + humans + + + + INTRO + paragraph + 3339 + A type of large oligomers called annular protofibrils (APFs) have also been observed in the brains of transgenic mice and isolated from the brains of Alzheimer’s patients. APFs were first discovered in vitro using chemically synthesized Aβ that aggregated into porelike structures that could be observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The sizes of APFs prepared in vitro vary among different studies. Lashuel et al. observed APFs with an outer diameter that ranged from 7–10 nm and an inner diameter that ranged from 1.5–2 nm, consistent with molecular weights of 150–250 kDa. Quist et al. observed APFs with an outer diameter of 16 nm embedded in a lipid bilayer. Kayed et al. observed APFs with an outer diameter that ranged from 8–25 nm, which were composed of small spherical Aβ oligomers, 3–5 nm in diameter. Although the APFs in these studies differ in size, they share a similar annular morphology and appear to be composed of smaller oligomers. + + 0.9700486 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + 0.8665932 + complex_assembly + cleaner0 + 2023-07-10T10:16:51Z + GO: + + annular protofibrils + + + 0.7713709 + complex_assembly + cleaner0 + 2023-07-10T10:13:51Z + GO: + + APFs + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-10T10:18:10Z + + mice + + + 0.5791445 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + 0.9984447 + protein_state + cleaner0 + 2023-07-10T15:45:05Z + DUMMY: + + chemically synthesized + + + 0.63466835 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.8028501 + structure_element + cleaner0 + 2023-07-11T09:33:21Z + SO: + + porelike structures + + + 0.9995325 + experimental_method + cleaner0 + 2023-07-10T10:17:10Z + MESH: + + atomic force microscopy + + + 0.99963975 + experimental_method + cleaner0 + 2023-07-10T10:17:15Z + MESH: + + AFM + + + 0.99952656 + experimental_method + cleaner0 + 2023-07-10T10:17:20Z + MESH: + + transmission electron microscopy + + + 0.9996375 + experimental_method + cleaner0 + 2023-07-10T10:17:25Z + MESH: + + TEM + + + 0.95248115 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + 0.91114235 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + 0.9306856 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + 0.8881248 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + 0.97571397 + protein_state + cleaner0 + 2023-07-10T15:45:13Z + DUMMY: + + small spherical + + + 0.77268577 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.94128937 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + 0.9496165 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + 0.99063766 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + + INTRO + paragraph + 4352 + APFs have also been observed in the brains of APP23 transgenic mice by immunofluorescence with an anti-APF antibody and were found to accumulate in neuronal processes and synapses. In a subsequent study, APFs were isolated from the brains of Alzheimer’s patients by immunoprecipitation with an anti-APF antibody. These APFs had an outer diameter that ranged from 11–14 nm and an inner diameter that ranged from 2.5–4 nm. + + 0.98581684 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-10T10:18:09Z + + mice + + + 0.9995883 + experimental_method + cleaner0 + 2023-07-10T15:37:02Z + MESH: + + immunofluorescence + + + complex_assembly + GO: + cleaner0 + 2023-07-10T14:29:04Z + + APF + + + 0.95454985 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + 0.99961036 + experimental_method + cleaner0 + 2023-07-10T15:37:06Z + MESH: + + immunoprecipitation + + + complex_assembly + GO: + cleaner0 + 2023-07-10T14:29:04Z + + APF + + + 0.91438216 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + + INTRO + paragraph + 4779 + Dimers of Aβ have also been isolated from the brains of Alzheimer’s patients.− Aβ dimers inhibit long-term potentiation in mice and promote hyperphosphorylation of the microtubule-associated protein tau, leading to neuritic damage. Aβ dimers have only been isolated from human or transgenic mouse brains that contain the pathognomonic fibrillar Aβ plaques associated with Alzheimer’s disease. Furthermore, the endogenous rise of Aβ dimers in the brains of Tg2576 and J20 transgenic mice coincides with the deposition of Aβ plaques. These observations suggest that the Aβ trimers, hexamers, dodecamers, and related assemblies may be associated with presymptomatic neurodegeneration, while Aβ dimers are more closely associated with fibril formation and plaque deposition during symptomatic Alzheimer’s disease.− + + 0.99945194 + oligomeric_state + cleaner0 + 2023-07-10T09:58:31Z + DUMMY: + + Dimers + + + 0.8021377 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.5438175 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.99941444 + oligomeric_state + cleaner0 + 2023-07-10T09:58:31Z + DUMMY: + + dimers + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-10T10:18:10Z + + mice + + + 0.9493683 + ptm + cleaner0 + 2023-07-10T15:27:09Z + MESH: + + hyperphosphorylation + + + protein + PR: + cleaner0 + 2023-07-10T15:26:50Z + + microtubule-associated protein tau + + + 0.6077137 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.99936277 + oligomeric_state + cleaner0 + 2023-07-10T09:58:31Z + DUMMY: + + dimers + + + 0.999215 + species + cleaner0 + 2023-07-10T14:41:54Z + MESH: + + human + + + 0.47958887 + taxonomy_domain + cleaner0 + 2023-07-10T10:17:54Z + DUMMY: + + mouse + + + protein_state + DUMMY: + cleaner0 + 2023-07-10T15:49:03Z + + fibrillar + + + 0.5131039 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.7966062 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.99929655 + oligomeric_state + cleaner0 + 2023-07-10T09:58:31Z + DUMMY: + + dimers + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-10T10:18:10Z + + mice + + + protein + PR: + cleaner0 + 2023-07-10T10:08:54Z + + Aβ + + + 0.72620815 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.9988053 + oligomeric_state + cleaner0 + 2023-07-10T09:58:05Z + DUMMY: + + trimers + + + 0.9966085 + oligomeric_state + cleaner0 + 2023-07-10T09:58:42Z + DUMMY: + + hexamers + + + 0.8557124 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + 0.646234 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.9993901 + oligomeric_state + cleaner0 + 2023-07-10T09:58:31Z + DUMMY: + + dimers + + + + INTRO + paragraph + 5614 + The approach of isolating and characterizing Aβ oligomers has not provided any high-resolution structures of Aβ oligomers. Techniques such as SDS-PAGE, TEM, and AFM have only provided information about the molecular weights, sizes, morphologies, and stoichiometry of Aβ oligomers. High-resolution structural studies of Aβ have primarily focused on Aβ fibrils and Aβ monomers. Solid-state NMR spectroscopy studies of Aβ fibrils revealed that Aβ fibrils are generally composed of extended networks of in-register parallel β-sheets.− X-ray crystallographic studies using fragments of Aβ have provided additional information about how Aβ fibrils pack. Solution-phase NMR and solid-state NMR have been used to study the structures of the Aβ monomers within oligomeric assemblies.− A major finding from these studies is that oligomeric assemblies of Aβ are primarily composed of antiparallel β-sheets. Many of these studies have reported the monomer subunit as adopting a β-hairpin conformation, in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. + + 0.5039002 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.99614125 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + 0.99699545 + evidence + cleaner0 + 2023-07-10T14:42:04Z + DUMMY: + + structures + + + 0.7964468 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.9962723 + oligomeric_state + cleaner0 + 2023-07-10T09:57:14Z + DUMMY: + + oligomers + + + 0.99959564 + experimental_method + cleaner0 + 2023-07-10T10:22:55Z + MESH: + + SDS-PAGE + + + 0.9996464 + experimental_method + cleaner0 + 2023-07-10T10:17:25Z + MESH: + + TEM + + + 0.9996631 + experimental_method + cleaner0 + 2023-07-10T10:17:16Z + MESH: + + AFM + + + 0.60583735 + protein + cleaner0 + 2023-07-10T10:08:54Z + PR: + + Aβ + + + 0.99693775 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.7642248 + experimental_method + cleaner0 + 2023-07-10T15:37:11Z + MESH: + + structural studies + + + 0.9772439 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.693741 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.7077935 + oligomeric_state + cleaner0 + 2023-07-10T10:00:00Z + DUMMY: + + fibrils + + + 0.9717112 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.99897325 + oligomeric_state + cleaner0 + 2023-07-10T09:57:24Z + DUMMY: + + monomers + + + 0.99954873 + experimental_method + cleaner0 + 2023-07-10T10:23:01Z + MESH: + + Solid-state NMR spectroscopy + + + 0.82302004 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.47209287 + oligomeric_state + cleaner0 + 2023-07-10T10:00:00Z + DUMMY: + + fibrils + + + 0.65992016 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.39904255 + oligomeric_state + cleaner0 + 2023-07-10T10:00:01Z + DUMMY: + + fibrils + + + 0.9986865 + structure_element + cleaner0 + 2023-07-11T09:33:37Z + SO: + + in-register parallel β-sheets + + + 0.9992774 + experimental_method + cleaner0 + 2023-07-10T15:37:15Z + MESH: + + X-ray crystallographic studies + + + 0.8482603 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.5764966 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-10T10:00:01Z + + fibrils + + + 0.99956304 + experimental_method + cleaner0 + 2023-07-10T10:23:09Z + MESH: + + Solution-phase NMR + + + 0.9995558 + experimental_method + cleaner0 + 2023-07-10T10:23:13Z + MESH: + + solid-state NMR + + + 0.9957182 + evidence + cleaner0 + 2023-07-10T14:42:06Z + DUMMY: + + structures + + + 0.85935736 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.9992181 + oligomeric_state + cleaner0 + 2023-07-10T09:57:24Z + DUMMY: + + monomers + + + 0.99018073 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.9996321 + structure_element + cleaner0 + 2023-07-11T09:33:41Z + SO: + + antiparallel β-sheets + + + 0.9992317 + oligomeric_state + cleaner0 + 2023-07-10T09:58:17Z + DUMMY: + + monomer + + + 0.93509406 + structure_element + cleaner0 + 2023-07-10T10:00:19Z + SO: + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-10T10:09:19Z + + β-hairpin + + + 0.98106134 + structure_element + cleaner0 + 2023-07-11T09:33:44Z + SO: + + central + + + 0.95612884 + structure_element + cleaner0 + 2023-07-11T09:33:47Z + SO: + + C-terminal regions + + + 0.99963534 + structure_element + cleaner0 + 2023-07-11T09:33:50Z + SO: + + antiparallel β-sheet + + + + INTRO + paragraph + 6738 + In 2008, Hoyer et al. reported the NMR structure of an Aβ monomer bound to an artificial binding protein called an affibody (PDB 2OTK). The structure revealed that monomeric Aβ forms a β-hairpin when bound to the affibody. This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. Sequestering Aβ within the affibody prevents its fibrilization and reduces its neurotoxicity, providing evidence that the β-hairpin structure may contribute to the ability of Aβ to form neurotoxic oligomers. In a related study, Sandberg et al. constrained Aβ in a β-hairpin conformation by mutating residues A21 and A30 to cysteine and forming an intramolecular disulfide bond. Locking Aβ into a β-hairpin structure resulted in the formation Aβ oligomers, which were observed by size exclusion chromatography (SEC) and SDS-PAGE. The oligomers with a molecular weight of ∼100 kDa that were isolated by SEC were toxic toward neuronally derived SH-SY5Y cells. This study provides evidence for the role of β-hairpin structure in Aβ oligomerization and neurotoxicity. + + 0.99964845 + experimental_method + cleaner0 + 2023-07-10T15:37:25Z + MESH: + + NMR + + + 0.99663997 + evidence + cleaner0 + 2023-07-10T14:42:10Z + DUMMY: + + structure + + + 0.89516926 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.99936324 + oligomeric_state + cleaner0 + 2023-07-10T09:58:17Z + DUMMY: + + monomer + + + 0.9994837 + protein_state + cleaner0 + 2023-07-10T15:45:45Z + DUMMY: + + bound to + + + 0.98704666 + chemical + cleaner0 + 2023-07-10T15:29:47Z + CHEBI: + + artificial binding protein + + + 0.8710008 + chemical + cleaner0 + 2023-07-10T15:29:59Z + CHEBI: + + affibody + + + 0.99931574 + evidence + cleaner0 + 2023-07-10T14:42:13Z + DUMMY: + + structure + + + 0.9989262 + oligomeric_state + cleaner0 + 2023-07-10T09:58:25Z + DUMMY: + + monomeric + + + 0.57115346 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.9996558 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.9994358 + protein_state + cleaner0 + 2023-07-10T15:45:48Z + DUMMY: + + bound to + + + 0.4964339 + chemical + cleaner0 + 2023-07-10T15:29:59Z + CHEBI: + + affibody + + + 0.9803711 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.9994075 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.9986763 + residue_range + cleaner0 + 2023-07-10T14:42:52Z + DUMMY: + + 17–37 + + + 0.9996045 + structure_element + cleaner0 + 2023-07-11T09:33:54Z + SO: + + β-strands + + + protein + PR: + cleaner0 + 2023-07-10T10:20:14Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:20:26Z + + 17–24 + + + protein + PR: + cleaner0 + 2023-07-10T10:20:42Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:20:58Z + + 30–37 + + + protein + PR: + cleaner0 + 2023-07-10T10:21:19Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:21:31Z + + 25–29 + + + structure_element + SO: + cleaner0 + 2023-07-10T10:21:42Z + + loop + + + 0.6987664 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.2828719 + chemical + cleaner0 + 2023-07-10T15:29:59Z + CHEBI: + + affibody + + + 0.99965763 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.92513806 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.9674943 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.63113844 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + structure_element + SO: + cleaner0 + 2023-07-10T10:09:19Z + + β-hairpin + + + 0.9923884 + experimental_method + cleaner0 + 2023-07-10T15:37:29Z + MESH: + + mutating + + + 0.9999056 + residue_name_number + cleaner0 + 2023-07-10T13:57:12Z + DUMMY: + + A21 + + + 0.99990606 + residue_name_number + cleaner0 + 2023-07-10T10:49:30Z + DUMMY: + + A30 + + + 0.9987962 + residue_name + cleaner0 + 2023-07-10T10:36:04Z + SO: + + cysteine + + + 0.8780202 + ptm + cleaner0 + 2023-07-10T10:22:21Z + MESH: + + disulfide bond + + + 0.47184205 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.99963254 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.4833778 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.99662304 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.99956506 + experimental_method + cleaner0 + 2023-07-10T15:37:32Z + MESH: + + size exclusion chromatography + + + 0.9996662 + experimental_method + cleaner0 + 2023-07-10T15:37:36Z + MESH: + + SEC + + + 0.9995844 + experimental_method + cleaner0 + 2023-07-10T10:22:56Z + MESH: + + SDS-PAGE + + + 0.99566936 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.99965584 + experimental_method + cleaner0 + 2023-07-10T15:37:36Z + MESH: + + SEC + + + 0.9996759 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.85829324 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + + INTRO + paragraph + 7914 + Inspired by these β-hairpin structures, our laboratory developed a macrocyclic β-sheet peptide derived from Aβ17–36 designed to mimic an Aβ β-hairpin and reported its X-ray crystallographic structure. This peptide (peptide 1) consists of two β-strands comprising Aβ17–23 and Aβ30–36 covalently linked by two δ-linked ornithine (δOrn) β-turn mimics. The δOrn that connects residues D23 and A30 replaces the Aβ24–29 loop. The δOrn that connects residues L17 and V36 enforces β-hairpin structure. We incorporated an N-methyl group at position G33 to prevent uncontrolled aggregation and precipitation of the peptide. To improve the solubility of the peptide we replaced M35 with the hydrophilic isostere of methionine, ornithine (α-linked) (Figure 1B). The X-ray crystallographic structure of peptide 1 reveals that it folds to form a β-hairpin that assembles to form trimers and that the trimers further assemble to form hexamers and dodecamers. + + 0.99959207 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.83836514 + evidence + cleaner0 + 2023-07-10T14:42:23Z + DUMMY: + + structures + + + 0.8475345 + structure_element + cleaner0 + 2023-07-11T09:34:01Z + SO: + + β-sheet + + + protein + PR: + cleaner0 + 2023-07-10T10:24:08Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:24:19Z + + 17–36 + + + 0.7955069 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.9995086 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.9995098 + evidence + cleaner0 + 2023-07-10T10:23:28Z + DUMMY: + + X-ray crystallographic structure + + + 0.9646842 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + 0.99670887 + structure_element + cleaner0 + 2023-07-11T09:34:09Z + SO: + + β-strands + + + protein + PR: + cleaner0 + 2023-07-10T10:24:34Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:24:46Z + + 17–23 + + + protein + PR: + cleaner0 + 2023-07-10T10:25:01Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:25:11Z + + 30–36 + + + protein_state + DUMMY: + cleaner0 + 2023-07-10T10:26:41Z + + δ-linked + + + residue_name + SO: + cleaner0 + 2023-07-10T10:26:21Z + + ornithine + + + 0.8010554 + structure_element + cleaner0 + 2023-07-10T10:25:39Z + SO: + + δOrn + + + structure_element + SO: + cleaner0 + 2023-07-10T10:27:37Z + + β-turn + + + 0.99973744 + structure_element + cleaner0 + 2023-07-10T14:03:39Z + SO: + + δOrn + + + 0.99988973 + residue_name_number + cleaner0 + 2023-07-10T10:49:38Z + DUMMY: + + D23 + + + 0.99989367 + residue_name_number + cleaner0 + 2023-07-10T10:49:30Z + DUMMY: + + A30 + + + protein + PR: + cleaner0 + 2023-07-10T10:27:55Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:28:07Z + + 24–29 + + + structure_element + SO: + cleaner0 + 2023-07-10T10:28:15Z + + loop + + + 0.9997259 + structure_element + cleaner0 + 2023-07-10T14:03:39Z + SO: + + δOrn + + + 0.9998894 + residue_name_number + cleaner0 + 2023-07-10T13:57:03Z + DUMMY: + + L17 + + + 0.99989426 + residue_name_number + cleaner0 + 2023-07-10T13:57:33Z + DUMMY: + + V36 + + + 0.9995308 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.99989927 + residue_name_number + cleaner0 + 2023-07-10T13:58:11Z + DUMMY: + + G33 + + + 0.9414554 + experimental_method + cleaner0 + 2023-07-10T15:37:42Z + MESH: + + replaced + + + 0.9999063 + residue_name_number + cleaner0 + 2023-07-10T13:58:16Z + DUMMY: + + M35 + + + 0.8500073 + residue_name + cleaner0 + 2023-07-10T10:35:57Z + SO: + + methionine + + + 0.8644892 + residue_name + cleaner0 + 2023-07-10T10:26:21Z + SO: + + ornithine + + + protein_state + DUMMY: + cleaner0 + 2023-07-10T10:27:18Z + + α-linked + + + 0.9995065 + evidence + cleaner0 + 2023-07-10T10:23:29Z + DUMMY: + + X-ray crystallographic structure + + + 0.85178244 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + 0.9996161 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.99884737 + oligomeric_state + cleaner0 + 2023-07-10T09:58:05Z + DUMMY: + + trimers + + + 0.9988152 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.9988193 + oligomeric_state + cleaner0 + 2023-07-10T09:58:42Z + DUMMY: + + hexamers + + + 0.9941906 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + + ja-2016-013325_0002.jpg + fig1 + FIG + fig_caption + 8914 + (A) Cartoon +illustrating the design of peptides 1 and 2 and their relationship to an Aβ17–36 β-hairpin. +(B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked +ornithine turns. (C) Chemical structure of peptide 2 illustrating +Aβ17–36, the N-methyl group, +the disulfide bond across positions 24 and 29, and the δ-linked +ornithine turn. + + chemical + CHEBI: + cleaner0 + 2023-07-10T10:33:20Z + + peptides 1 and 2 + + + protein + PR: + cleaner0 + 2023-07-10T10:33:04Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:37:44Z + + 17–36 + + + 0.999314 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + mutant + MESH: + cleaner0 + 2023-07-10T10:38:32Z + + peptide 1 + + + protein + PR: + cleaner0 + 2023-07-10T10:31:49Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:32:00Z + + 17–23 + + + protein + PR: + cleaner0 + 2023-07-10T10:32:15Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:32:30Z + + 30–36 + + + protein_state + DUMMY: + cleaner0 + 2023-07-10T10:26:42Z + + δ-linked + + + 0.9810042 + residue_name + cleaner0 + 2023-07-10T10:26:21Z + SO: + + ornithine + + + structure_element + SO: + cleaner0 + 2023-07-10T10:34:22Z + + turns + + + mutant + MESH: + cleaner0 + 2023-07-10T10:39:07Z + + peptide 2 + + + protein + PR: + cleaner0 + 2023-07-10T10:33:54Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:34:07Z + + 17–36 + + + 0.9905417 + ptm + cleaner0 + 2023-07-10T10:22:22Z + MESH: + + disulfide bond + + + 0.99705064 + residue_number + cleaner0 + 2023-07-10T14:43:23Z + DUMMY: + + 24 + + + 0.9969585 + residue_number + cleaner0 + 2023-07-10T14:43:26Z + DUMMY: + + 29 + + + protein_state + DUMMY: + cleaner0 + 2023-07-10T10:26:42Z + + δ-linked + + + 0.9834495 + residue_name + cleaner0 + 2023-07-10T10:26:21Z + SO: + + ornithine + + + structure_element + SO: + cleaner0 + 2023-07-10T10:34:30Z + + turn + + + + INTRO + paragraph + 9331 + Our design of peptide 1 omitted the Aβ24–29 loop. To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. These studies provided a working model for a trimer of Aβ17–36 β-hairpins and demonstrated that the trimer should be capable of accommodating the Aβ24–29 loop. + + mutant + MESH: + cleaner0 + 2023-07-10T10:38:33Z + + peptide 1 + + + protein + PR: + cleaner0 + 2023-07-10T10:41:20Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:41:42Z + + 24–29 + + + structure_element + SO: + cleaner0 + 2023-07-10T10:41:52Z + + loop + + + protein + PR: + cleaner0 + 2023-07-10T10:43:00Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:43:10Z + + 24–29 + + + structure_element + SO: + cleaner0 + 2023-07-10T10:44:39Z + + loop + + + 0.999436 + experimental_method + cleaner0 + 2023-07-10T15:37:48Z + MESH: + + replica-exchange molecular dynamics + + + 0.999634 + experimental_method + cleaner0 + 2023-07-10T15:37:50Z + MESH: + + REMD + + + 0.9984914 + experimental_method + cleaner0 + 2023-07-10T15:37:52Z + MESH: + + simulations + + + protein + PR: + cleaner0 + 2023-07-10T10:42:10Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:42:23Z + + 17–36 + + + 0.9992975 + evidence + cleaner0 + 2023-07-10T14:56:54Z + DUMMY: + + X-ray crystallographic coordinates + + + protein + PR: + cleaner0 + 2023-07-10T10:42:37Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:42:47Z + + 17–23 + + + protein + PR: + cleaner0 + 2023-07-10T10:43:37Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:43:49Z + + 30–36 + + + mutant + MESH: + cleaner0 + 2023-07-10T10:38:33Z + + peptide 1 + + + 0.9994091 + oligomeric_state + cleaner0 + 2023-07-10T09:57:06Z + DUMMY: + + trimer + + + protein + PR: + cleaner0 + 2023-07-10T10:44:15Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:44:26Z + + 17–36 + + + 0.9996304 + structure_element + cleaner0 + 2023-07-10T10:51:04Z + SO: + + β-hairpins + + + 0.9994087 + oligomeric_state + cleaner0 + 2023-07-10T09:57:06Z + DUMMY: + + trimer + + + protein + PR: + cleaner0 + 2023-07-10T10:45:53Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:46:04Z + + 24–29 + + + structure_element + SO: + cleaner0 + 2023-07-10T10:44:40Z + + loop + + + + INTRO + paragraph + 9759 + In the current study we set out to restore the Aβ24–29 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form. We designed peptide 2 as a homologue of peptide 1 that embodies these ideas. Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). Here, we describe the development of peptide 2 and report the X-ray crystallographic structures of the trimer, dodecamer, and annular pore observed within the crystal structure. + + 0.9742589 + experimental_method + cleaner0 + 2023-07-10T15:37:57Z + MESH: + + restore + + + protein + PR: + cleaner0 + 2023-07-10T10:46:20Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:46:36Z + + 24–29 + + + structure_element + SO: + cleaner0 + 2023-07-10T10:44:40Z + + loop + + + 0.9987637 + experimental_method + cleaner0 + 2023-07-10T15:37:59Z + MESH: + + reintroduce + + + 0.99891746 + residue_name + cleaner0 + 2023-07-10T10:35:56Z + SO: + + methionine + + + 0.9964091 + residue_number + cleaner0 + 2023-07-10T14:43:31Z + DUMMY: + + 35 + + + 0.9995488 + evidence + cleaner0 + 2023-07-10T14:56:29Z + DUMMY: + + X-ray crystallographic structures + + + 0.96816325 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.9973719 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + 0.98622614 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + 0.9961827 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + Peptide 2 + + + 0.99892515 + residue_name + cleaner0 + 2023-07-10T10:35:57Z + SO: + + methionine + + + 0.9962081 + residue_number + cleaner0 + 2023-07-10T14:43:33Z + DUMMY: + + 35 + + + protein + PR: + cleaner0 + 2023-07-10T10:47:13Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:47:25Z + + 24–29 + + + structure_element + SO: + cleaner0 + 2023-07-10T10:44:40Z + + loop + + + 0.99221075 + residue_number + cleaner0 + 2023-07-10T14:43:36Z + DUMMY: + + 24 + + + 0.9959067 + residue_number + cleaner0 + 2023-07-10T14:43:39Z + DUMMY: + + 29 + + + 0.99940336 + residue_name + cleaner0 + 2023-07-10T14:44:57Z + SO: + + Val + + + 0.99938273 + residue_name + cleaner0 + 2023-07-10T14:45:00Z + SO: + + Gly + + + 0.9951917 + experimental_method + cleaner0 + 2023-07-10T15:38:06Z + MESH: + + mutated + + + 0.9989937 + residue_name + cleaner0 + 2023-07-10T10:36:03Z + SO: + + cysteine + + + 0.99533427 + ptm + cleaner0 + 2023-07-10T10:22:22Z + MESH: + + disulfide bond + + + 0.9937948 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + 0.99953794 + evidence + cleaner0 + 2023-07-10T14:56:29Z + DUMMY: + + X-ray crystallographic structures + + + 0.99918133 + oligomeric_state + cleaner0 + 2023-07-10T09:57:06Z + DUMMY: + + trimer + + + 0.99606377 + oligomeric_state + cleaner0 + 2023-07-10T14:43:47Z + DUMMY: + + dodecamer + + + 0.98689055 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.99962056 + evidence + cleaner0 + 2023-07-10T14:42:40Z + DUMMY: + + crystal structure + + + + RESULTS + title_1 + 10385 + Results + + + RESULTS + title_2 + 10393 + Development of Peptide 2 + + 0.99923146 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + Peptide 2 + + + + RESULTS + paragraph + 10418 + We developed peptide 2 from peptide 1 by an iterative process, in which we first attempted to restore the Aβ24–29 loop without a disulfide linkage. We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. We routinely use p-iodophenylalanine to determine the X-ray crystallographic phases. After determining the X-ray crystallographic structure of the p-iodophenylalanine variant we attempt to determine the structure of the native phenylalanine compound by isomorphous replacement. Upon synthesizing peptide 3, we found that it formed an amorphous precipitate in most crystallization conditions screened and failed to afford crystals in any condition. + + 0.9892849 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + 0.9937219 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + protein + PR: + cleaner0 + 2023-07-10T10:48:36Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:48:46Z + + 24–29 + + + structure_element + SO: + cleaner0 + 2023-07-10T10:44:40Z + + loop + + + 0.9951011 + ptm + cleaner0 + 2023-07-10T10:10:02Z + MESH: + + disulfide linkage + + + 0.9922428 + mutant + cleaner0 + 2023-07-10T10:48:10Z + MESH: + + peptide 3 + + + 0.97108465 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + protein + PR: + cleaner0 + 2023-07-10T10:49:01Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:49:11Z + + 24–29 + + + structure_element + SO: + cleaner0 + 2023-07-10T10:44:40Z + + loop + + + 0.9992563 + structure_element + cleaner0 + 2023-07-10T14:03:39Z + SO: + + δOrn + + + 0.9998549 + residue_name_number + cleaner0 + 2023-07-10T10:49:38Z + DUMMY: + + D23 + + + 0.99987125 + residue_name_number + cleaner0 + 2023-07-10T10:49:29Z + DUMMY: + + A30 + + + 0.9996927 + chemical + cleaner0 + 2023-07-10T10:49:17Z + CHEBI: + + p-iodophenylalanine + + + 0.99924856 + chemical + cleaner0 + 2023-07-10T10:49:22Z + CHEBI: + + FI + + + 0.9998852 + residue_name_number + cleaner0 + 2023-07-10T10:49:33Z + DUMMY: + + F19 + + + 0.99968237 + chemical + cleaner0 + 2023-07-10T10:49:18Z + CHEBI: + + p-iodophenylalanine + + + 0.999561 + evidence + cleaner0 + 2023-07-10T14:57:01Z + DUMMY: + + X-ray crystallographic phases + + + 0.99957705 + evidence + cleaner0 + 2023-07-10T10:23:29Z + DUMMY: + + X-ray crystallographic structure + + + 0.99928826 + chemical + cleaner0 + 2023-07-10T10:49:18Z + CHEBI: + + p-iodophenylalanine + + + 0.9989945 + evidence + cleaner0 + 2023-07-10T14:57:05Z + DUMMY: + + structure + + + 0.98784876 + residue_name + cleaner0 + 2023-07-10T10:49:44Z + SO: + + phenylalanine + + + 0.9995549 + experimental_method + cleaner0 + 2023-07-10T15:38:10Z + MESH: + + isomorphous replacement + + + 0.99354255 + mutant + cleaner0 + 2023-07-10T10:48:17Z + MESH: + + peptide 3 + + + 0.9983418 + evidence + cleaner0 + 2023-07-10T14:57:08Z + DUMMY: + + crystals + + + + RESULTS + paragraph + 11191 + We postulate that the loss of the δOrn constraint leads to conformational heterogeneity that prevents peptide 3 from crystallizing. To address this issue, we next incorporated a disulfide bond between residues 24 and 29 as a conformational constraint that serves as a surrogate for δOrn. We designed peptide 4 to embody this idea, mutating Val24 and Gly29 to cysteine and forming an interstrand disulfide linkage. We mutated these residues because they occupy the same position as the δOrn that connects D23 and A30 in peptide 1. Residues V24 and G29 form a non-hydrogen-bonded pair, which can readily accommodate disulfide linkages in antiparallel β-sheets. Disulfide bonds across non-hydrogen-bonded pairs stabilize β-hairpins, while disulfide bonds across hydrogen-bonded pairs do not. Although the disulfide bond between positions 24 and 29 helps stabilize the β-hairpin, it does not alter the charge or substantially change the hydrophobicity of the Aβ17–36 β-hairpin. We were gratified to find that peptide 4 afforded crystals suitable for X-ray crystallography. As the next step in the iterative process, we determined the X-ray crystallographic structure of this peptide (PDB 5HOW). + + 0.999203 + structure_element + cleaner0 + 2023-07-10T14:03:39Z + SO: + + δOrn + + + 0.9848524 + mutant + cleaner0 + 2023-07-10T10:48:17Z + MESH: + + peptide 3 + + + 0.9961566 + ptm + cleaner0 + 2023-07-10T10:22:22Z + MESH: + + disulfide bond + + + 0.9908619 + residue_number + cleaner0 + 2023-07-10T14:43:54Z + DUMMY: + + 24 + + + 0.99191636 + residue_number + cleaner0 + 2023-07-10T14:43:56Z + DUMMY: + + 29 + + + 0.99907047 + structure_element + cleaner0 + 2023-07-10T14:03:39Z + SO: + + δOrn + + + 0.9774096 + mutant + cleaner0 + 2023-07-10T10:50:20Z + MESH: + + peptide 4 + + + 0.96276623 + experimental_method + cleaner0 + 2023-07-10T15:38:19Z + MESH: + + mutating + + + 0.99990726 + residue_name_number + cleaner0 + 2023-07-10T10:51:45Z + DUMMY: + + Val24 + + + 0.9999074 + residue_name_number + cleaner0 + 2023-07-10T10:51:49Z + DUMMY: + + Gly29 + + + 0.99879634 + residue_name + cleaner0 + 2023-07-10T10:36:04Z + SO: + + cysteine + + + 0.84402835 + ptm + cleaner0 + 2023-07-10T10:10:02Z + MESH: + + disulfide linkage + + + 0.96369773 + experimental_method + cleaner0 + 2023-07-10T15:38:33Z + MESH: + + mutated + + + 0.9994572 + structure_element + cleaner0 + 2023-07-10T14:03:39Z + SO: + + δOrn + + + 0.99991214 + residue_name_number + cleaner0 + 2023-07-10T10:49:38Z + DUMMY: + + D23 + + + 0.999912 + residue_name_number + cleaner0 + 2023-07-10T10:49:30Z + DUMMY: + + A30 + + + 0.9778849 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + 0.99990845 + residue_name_number + cleaner0 + 2023-07-10T10:52:02Z + DUMMY: + + V24 + + + 0.9999087 + residue_name_number + cleaner0 + 2023-07-10T10:52:07Z + DUMMY: + + G29 + + + 0.9766631 + bond_interaction + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + MESH: + + non-hydrogen-bonded pair + + + 0.99633384 + ptm + cleaner0 + 2023-07-10T13:58:29Z + MESH: + + disulfide linkages + + + 0.999683 + structure_element + cleaner0 + 2023-07-11T09:34:23Z + SO: + + antiparallel β-sheets + + + 0.99575865 + ptm + cleaner0 + 2023-07-10T15:28:01Z + MESH: + + Disulfide bonds + + + 0.96280503 + bond_interaction + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + MESH: + + non-hydrogen-bonded pairs + + + 0.99969673 + structure_element + cleaner0 + 2023-07-10T10:51:04Z + SO: + + β-hairpins + + + 0.995991 + ptm + cleaner0 + 2023-07-10T15:28:01Z + MESH: + + disulfide bonds + + + 0.9655484 + bond_interaction + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + MESH: + + hydrogen-bonded pairs + + + 0.9962245 + ptm + cleaner0 + 2023-07-10T10:22:22Z + MESH: + + disulfide bond + + + 0.9932238 + residue_number + cleaner0 + 2023-07-10T14:43:59Z + DUMMY: + + 24 + + + 0.99372065 + residue_number + cleaner0 + 2023-07-10T14:44:01Z + DUMMY: + + 29 + + + 0.99970675 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + protein + PR: + cleaner0 + 2023-07-10T10:52:24Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T10:52:34Z + + 17–36 + + + 0.9996972 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.9822699 + mutant + cleaner0 + 2023-07-10T10:50:26Z + MESH: + + peptide 4 + + + 0.99896896 + evidence + cleaner0 + 2023-07-10T14:57:11Z + DUMMY: + + crystals + + + 0.9995647 + experimental_method + cleaner0 + 2023-07-10T15:38:26Z + MESH: + + X-ray crystallography + + + 0.9332236 + experimental_method + cleaner0 + 2023-07-10T15:38:28Z + MESH: + + determined + + + 0.9994993 + evidence + cleaner0 + 2023-07-10T10:23:29Z + DUMMY: + + X-ray crystallographic structure + + + + RESULTS + paragraph + 12409 + After determining the X-ray crystallographic structure of peptide 4 we reintroduced the native phenylalanine at position 19 and the methionine at position 35 to afford peptide 2. We completed the iterative process—from 1 to 3 to 4 to 2—by successfully determining the X-ray crystallographic structure of peptide 2 (PDB 5HOX and 5HOY). The following sections describe the synthesis of peptides 2–4 and the X-ray crystallographic structure of peptide 2. + + 0.999524 + evidence + cleaner0 + 2023-07-10T10:23:29Z + DUMMY: + + X-ray crystallographic structure + + + mutant + MESH: + cleaner0 + 2023-07-10T10:50:26Z + + peptide 4 + + + 0.99385285 + experimental_method + cleaner0 + 2023-07-10T15:38:37Z + MESH: + + reintroduced + + + 0.99925715 + residue_name + cleaner0 + 2023-07-10T10:49:45Z + SO: + + phenylalanine + + + 0.99681 + residue_number + cleaner0 + 2023-07-10T14:44:06Z + DUMMY: + + 19 + + + 0.99917006 + residue_name + cleaner0 + 2023-07-10T10:35:57Z + SO: + + methionine + + + 0.996888 + residue_number + cleaner0 + 2023-07-10T14:44:09Z + DUMMY: + + 35 + + + 0.90846217 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + 0.9995273 + evidence + cleaner0 + 2023-07-10T10:23:29Z + DUMMY: + + X-ray crystallographic structure + + + 0.90822935 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + mutant + MESH: + cleaner0 + 2023-07-10T10:54:05Z + + peptides 2–4 + + + 0.99954253 + evidence + cleaner0 + 2023-07-10T10:23:29Z + DUMMY: + + X-ray crystallographic structure + + + 0.9377295 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + + RESULTS + title_2 + 12867 + Synthesis of Peptides 2–4 + + mutant + MESH: + cleaner0 + 2023-07-10T10:54:06Z + + Peptides 2–4 + + + + RESULTS + paragraph + 12895 + We synthesized peptides 2–4 by similar procedures to those we have developed for other macrocyclic peptides. Our laboratory routinely prepares macrocyclic peptides by solid-phase synthesis of the corresponding linear peptide on 2-chlorotrityl resin, followed by cleavage of the protected linear peptide from the resin, solution-phase macrolactamization, and deprotection of the resulting macrocyclic peptide. In synthesizing peptides 2 and 4 we formed the disulfide linkage after macrolactamization and deprotection of the acid-labile side chain protecting groups. We used acid-stable Acm-protected cysteine residues at positions 24 and 29 and removed the Acm groups by oxidation with I2 in aqueous acetic acid to afford the disulfide linkage. Peptides 2–4 were purified by RP-HPLC. + + 0.76412606 + mutant + cleaner0 + 2023-07-10T10:54:06Z + MESH: + + peptides 2–4 + + + mutant + MESH: + cleaner0 + 2023-07-10T13:56:23Z + + peptides 2 and 4 + + + 0.93680143 + ptm + cleaner0 + 2023-07-10T10:10:02Z + MESH: + + disulfide linkage + + + 0.7893932 + protein_state + cleaner0 + 2023-07-10T15:46:41Z + DUMMY: + + acid-stable + + + 0.9989392 + protein_state + cleaner0 + 2023-07-10T15:46:44Z + DUMMY: + + Acm-protected + + + 0.99904937 + residue_name + cleaner0 + 2023-07-10T10:36:04Z + SO: + + cysteine + + + 0.9967083 + residue_number + cleaner0 + 2023-07-10T14:44:14Z + DUMMY: + + 24 + + + 0.99653554 + residue_number + cleaner0 + 2023-07-10T14:44:16Z + DUMMY: + + 29 + + + 0.80693245 + chemical + cleaner0 + 2023-07-10T15:33:50Z + CHEBI: + + acetic acid + + + 0.9724722 + ptm + cleaner0 + 2023-07-10T10:10:02Z + MESH: + + disulfide linkage + + + mutant + MESH: + cleaner0 + 2023-07-10T10:54:06Z + + Peptides 2–4 + + + 0.99951 + experimental_method + cleaner0 + 2023-07-10T15:38:42Z + MESH: + + RP-HPLC + + + + RESULTS + title_2 + 13682 + Crystallization, X-ray Crystallographic Data Collection, Data Processing, and Structure Determination of Peptides 2 and 4 + + 0.99958974 + experimental_method + cleaner0 + 2023-07-10T15:38:45Z + MESH: + + Crystallization + + + 0.99932367 + experimental_method + cleaner0 + 2023-07-10T15:38:51Z + MESH: + + X-ray Crystallographic Data Collection + + + 0.9984472 + experimental_method + cleaner0 + 2023-07-10T15:38:54Z + MESH: + + Structure Determination + + + mutant + MESH: + cleaner0 + 2023-07-10T13:56:23Z + + Peptides 2 and 4 + + + + RESULTS + paragraph + 13804 + We screened crystallization conditions for peptide 4 in a 96-well-plate format using three different Hampton Research crystallization kits (Crystal Screen, Index, and PEG/Ion) with three ratios of peptide and mother liquor per condition (864 experiments). Peptide 4 afforded crystals in a single set of conditions containing HEPES buffer and Jeffamine M-600—the same crystallization conditions that afforded crystals of peptide 1. Peptide 2 also afforded crystals in these conditions. We further optimized these conditions to rapidly (∼72 h) yield crystals suitable for X-ray crystallography. The optimized conditions consist of 0.1 M HEPES at pH 6.4 with 31% Jeffamine M-600 for peptide 4 and 0.1 M HEPES pH 7.1 with 29% Jeffamine M-600 for peptide 2. + + experimental_method + MESH: + cleaner0 + 2023-07-10T15:39:20Z + + screened crystallization conditions + + + 0.85154676 + mutant + cleaner0 + 2023-07-10T10:50:26Z + MESH: + + peptide 4 + + + 0.76433134 + mutant + cleaner0 + 2023-07-10T10:50:26Z + MESH: + + Peptide 4 + + + 0.99602854 + evidence + cleaner0 + 2023-07-10T14:44:20Z + DUMMY: + + crystals + + + 0.9996301 + chemical + cleaner0 + 2023-07-10T14:44:29Z + CHEBI: + + Jeffamine M-600 + + + 0.9981212 + evidence + cleaner0 + 2023-07-10T14:44:22Z + DUMMY: + + crystals + + + 0.9948926 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + 0.9947971 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + Peptide 2 + + + 0.99759406 + evidence + cleaner0 + 2023-07-10T14:44:26Z + DUMMY: + + crystals + + + 0.99853027 + evidence + cleaner0 + 2023-07-10T14:44:24Z + DUMMY: + + crystals + + + 0.99957484 + experimental_method + cleaner0 + 2023-07-10T15:39:35Z + MESH: + + X-ray crystallography + + + 0.92050093 + chemical + cleaner0 + 2023-07-10T14:44:31Z + CHEBI: + + HEPES + + + 0.9996245 + chemical + cleaner0 + 2023-07-10T14:44:33Z + CHEBI: + + Jeffamine M-600 + + + 0.77729464 + mutant + cleaner0 + 2023-07-10T10:50:26Z + MESH: + + peptide 4 + + + 0.9420801 + chemical + cleaner0 + 2023-07-10T15:33:54Z + CHEBI: + + HEPES + + + 0.9996414 + chemical + cleaner0 + 2023-07-10T14:44:36Z + CHEBI: + + Jeffamine M-600 + + + 0.9898722 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + + RESULTS + paragraph + 14561 + Crystal diffraction data for peptides 4 and 2 were collected in-house with a Rigaku MicroMax 007HF X-ray diffractometer at 1.54 Å wavelength. Crystal diffraction data for peptide 2 were also collected at the Advanced Light Source at Lawrence Berkeley National Laboratory with a synchrotron source at 1.00 Å wavelength to achieve higher resolution. Data from peptides 4 and 2 suitable for refinement at 2.30 Å were obtained from the diffractometer; data from peptide 2 suitable for refinement at 1.90 Å were obtained from the synchrotron. + + 0.999041 + evidence + cleaner0 + 2023-07-10T14:44:38Z + DUMMY: + + Crystal diffraction data + + + mutant + MESH: + cleaner0 + 2023-07-10T10:57:10Z + + peptides 4 and 2 + + + 0.9987157 + evidence + cleaner0 + 2023-07-10T14:44:40Z + DUMMY: + + Crystal diffraction data + + + 0.6537781 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + mutant + MESH: + cleaner0 + 2023-07-10T10:57:11Z + + peptides 4 and 2 + + + mutant + MESH: + cleaner0 + 2023-07-10T10:39:08Z + + peptide 2 + + + + RESULTS + paragraph + 15103 + Data for peptides 4 and 2 were scaled and merged using XDS. Phases for peptide 4 were determined by single-wavelength anomalous diffraction (SAD) phasing by using the coordinates of the iodine anomalous signal from p-iodophenylalanine. Phases for peptide 2 were determined by isomorphous replacement of peptide 4. The structures of peptides 2 and 4 were solved and refined in the P6122 space group. Coordinates for hydrogens were generated by phenix.refine during refinement. The asymmetric unit of each peptide consists of six monomers, arranged as two trimers. Peptides 2 and 4 form morphologically identical structures and assemblies in the crystal lattice. + + mutant + MESH: + cleaner0 + 2023-07-10T10:57:11Z + + peptides 4 and 2 + + + 0.9662267 + evidence + cleaner0 + 2023-07-10T14:57:16Z + DUMMY: + + Phases + + + mutant + MESH: + cleaner0 + 2023-07-10T10:50:26Z + + peptide 4 + + + 0.9994998 + experimental_method + cleaner0 + 2023-07-10T15:40:11Z + MESH: + + single-wavelength anomalous diffraction + + + 0.9996172 + experimental_method + cleaner0 + 2023-07-10T15:40:13Z + MESH: + + SAD + + + 0.87384784 + experimental_method + cleaner0 + 2023-07-10T15:40:16Z + MESH: + + phasing + + + 0.95816034 + evidence + cleaner0 + 2023-07-10T14:57:23Z + DUMMY: + + iodine anomalous signal + + + 0.99971867 + chemical + cleaner0 + 2023-07-10T10:49:18Z + CHEBI: + + p-iodophenylalanine + + + 0.96701795 + evidence + cleaner0 + 2023-07-10T14:57:27Z + DUMMY: + + Phases + + + mutant + MESH: + cleaner0 + 2023-07-10T10:39:08Z + + peptide 2 + + + 0.99955964 + experimental_method + cleaner0 + 2023-07-10T15:40:18Z + MESH: + + isomorphous replacement + + + mutant + MESH: + cleaner0 + 2023-07-10T10:50:26Z + + peptide 4 + + + 0.99867237 + evidence + cleaner0 + 2023-07-10T14:57:30Z + DUMMY: + + structures + + + mutant + MESH: + cleaner0 + 2023-07-10T13:56:23Z + + peptides 2 and 4 + + + 0.607354 + experimental_method + cleaner0 + 2023-07-10T15:40:20Z + MESH: + + solved + + + chemical + CHEBI: + cleaner0 + 2023-07-10T10:31:20Z + + peptide + + + 0.99909973 + oligomeric_state + cleaner0 + 2023-07-10T09:57:24Z + DUMMY: + + monomers + + + 0.99905604 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + mutant + MESH: + cleaner0 + 2023-07-10T13:56:22Z + + Peptides 2 and 4 + + + 0.9846275 + evidence + cleaner0 + 2023-07-10T14:57:33Z + DUMMY: + + crystal lattice + + + + RESULTS + title_2 + 15764 + X-ray Crystallographic Structure of Peptide 2 and the Oligomers It Forms + + 0.9994813 + evidence + cleaner0 + 2023-07-10T10:23:29Z + DUMMY: + + X-ray Crystallographic Structure + + + 0.9979197 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + Peptide 2 + + + 0.9978307 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + Oligomers + + + + RESULTS + paragraph + 15837 + The X-ray crystallographic structure of peptide 2 reveals that it folds to form a twisted β-hairpin comprising two β-strands connected by a loop (Figure 2A). Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. The β-strands of the monomers in the asymmetric unit are virtually identical, differing primarily in rotamers of F20, E22, C24, C29, I31, and M35 (Figure S1). The disulfide linkages suffered radiation damage under synchrotron radiation. We refined three of the β-hairpins with intact disulfide linkages and three with thiols to represent cleaved disulfide linkages in the synchrotron data set (PDB 5HOX). No evidence for cleavage of the disulfides was observed in the refinement of the data set collected on the X-ray diffractometer, and we refined all disulfide linkages as intact (PDB 5HOY). + + 0.99944 + evidence + cleaner0 + 2023-07-10T10:23:29Z + DUMMY: + + X-ray crystallographic structure + + + 0.99803936 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + 0.995728 + structure_element + cleaner0 + 2023-07-11T09:34:28Z + SO: + + twisted β-hairpin + + + 0.9995902 + structure_element + cleaner0 + 2023-07-11T09:34:31Z + SO: + + β-strands + + + 0.99972075 + structure_element + cleaner0 + 2023-07-10T10:44:40Z + SO: + + loop + + + 0.99963075 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.99987936 + residue_name_number + cleaner0 + 2023-07-10T13:57:03Z + DUMMY: + + L17 + + + 0.99988186 + residue_name_number + cleaner0 + 2023-07-10T10:49:34Z + DUMMY: + + F19 + + + 0.9998845 + residue_name_number + cleaner0 + 2023-07-10T13:57:11Z + DUMMY: + + A21 + + + 0.9998822 + residue_name_number + cleaner0 + 2023-07-10T10:49:38Z + DUMMY: + + D23 + + + 0.9998852 + residue_name_number + cleaner0 + 2023-07-10T10:49:30Z + DUMMY: + + A30 + + + 0.9998814 + residue_name_number + cleaner0 + 2023-07-10T13:57:22Z + DUMMY: + + I32 + + + 0.9998826 + residue_name_number + cleaner0 + 2023-07-10T13:57:27Z + DUMMY: + + L34 + + + 0.9998826 + residue_name_number + cleaner0 + 2023-07-10T13:57:32Z + DUMMY: + + V36 + + + 0.99988556 + residue_name_number + cleaner0 + 2023-07-10T13:57:38Z + DUMMY: + + V18 + + + 0.9998834 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.9998845 + residue_name_number + cleaner0 + 2023-07-10T13:57:48Z + DUMMY: + + E22 + + + 0.9998807 + residue_name_number + cleaner0 + 2023-07-10T13:57:53Z + DUMMY: + + C24 + + + 0.9998834 + residue_name_number + cleaner0 + 2023-07-10T13:57:59Z + DUMMY: + + C29 + + + 0.9998833 + residue_name_number + cleaner0 + 2023-07-10T13:58:04Z + DUMMY: + + I31 + + + 0.9998894 + residue_name_number + cleaner0 + 2023-07-10T13:58:10Z + DUMMY: + + G33 + + + 0.9998834 + residue_name_number + cleaner0 + 2023-07-10T13:58:15Z + DUMMY: + + M35 + + + 0.9996355 + structure_element + cleaner0 + 2023-07-11T09:34:35Z + SO: + + β-strands + + + 0.9992638 + oligomeric_state + cleaner0 + 2023-07-10T09:57:24Z + DUMMY: + + monomers + + + 0.99987507 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.9998801 + residue_name_number + cleaner0 + 2023-07-10T13:57:49Z + DUMMY: + + E22 + + + 0.99987435 + residue_name_number + cleaner0 + 2023-07-10T13:57:54Z + DUMMY: + + C24 + + + 0.9998753 + residue_name_number + cleaner0 + 2023-07-10T13:57:59Z + DUMMY: + + C29 + + + 0.99987805 + residue_name_number + cleaner0 + 2023-07-10T13:58:04Z + DUMMY: + + I31 + + + 0.99987507 + residue_name_number + cleaner0 + 2023-07-10T13:58:16Z + DUMMY: + + M35 + + + 0.9944804 + ptm + cleaner0 + 2023-07-10T13:58:29Z + MESH: + + disulfide linkages + + + 0.98165166 + experimental_method + cleaner0 + 2023-07-10T15:40:28Z + MESH: + + refined + + + 0.9996164 + structure_element + cleaner0 + 2023-07-10T10:51:04Z + SO: + + β-hairpins + + + 0.99951124 + protein_state + cleaner0 + 2023-07-10T14:46:11Z + DUMMY: + + intact + + + 0.98381037 + ptm + cleaner0 + 2023-07-10T13:58:29Z + MESH: + + disulfide linkages + + + 0.9994874 + protein_state + cleaner0 + 2023-07-10T14:46:15Z + DUMMY: + + cleaved + + + 0.9916744 + ptm + cleaner0 + 2023-07-10T13:58:29Z + MESH: + + disulfide linkages + + + 0.9787425 + ptm + cleaner0 + 2023-07-10T15:28:10Z + MESH: + + disulfides + + + 0.9752783 + experimental_method + cleaner0 + 2023-07-10T15:40:30Z + MESH: + + refinement + + + 0.90610075 + experimental_method + cleaner0 + 2023-07-10T15:40:39Z + MESH: + + refined + + + 0.9930679 + ptm + cleaner0 + 2023-07-10T13:58:29Z + MESH: + + disulfide linkages + + + 0.9995315 + protein_state + cleaner0 + 2023-07-10T14:46:11Z + DUMMY: + + intact + + + + ja-2016-013325_0003.jpg + fig2 + FIG + fig_caption + 16792 + X-ray crystallographic +structure of peptide 2 (PDB 5HOX, synchrotron data +set). (A) X-ray crystallographic structure of a representative β-hairpin +monomer formed by peptide 2. (B) Overlay of the six β-hairpin +monomers in the asymmetric unit. The β-hairpins are shown as +cartoons to illustrate the differences in the Aβ25–28 loops. + + 0.9995028 + evidence + cleaner0 + 2023-07-10T14:57:44Z + DUMMY: + + X-ray crystallographic +structure + + + 0.97852206 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + 0.9995206 + evidence + cleaner0 + 2023-07-10T10:23:29Z + DUMMY: + + X-ray crystallographic structure + + + 0.9994331 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.999218 + oligomeric_state + cleaner0 + 2023-07-10T09:58:17Z + DUMMY: + + monomer + + + 0.9582621 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + 0.99906534 + experimental_method + cleaner0 + 2023-07-10T15:40:45Z + MESH: + + Overlay + + + 0.9994893 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.9991929 + oligomeric_state + cleaner0 + 2023-07-10T09:57:24Z + DUMMY: + + monomers + + + 0.999655 + structure_element + cleaner0 + 2023-07-10T10:51:04Z + SO: + + β-hairpins + + + protein + PR: + cleaner0 + 2023-07-10T14:21:43Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T14:21:55Z + + 25–28 + + + structure_element + SO: + cleaner0 + 2023-07-10T14:22:05Z + + loops + + + + RESULTS + paragraph + 17135 + The Aβ25–28 loops of the six monomers within the asymmetric unit vary substantially in backbone geometry and side chain rotamers (Figures 2B and S1). The electron density for the loops is weak and diffuse compared to the electron density for the β-strands. The B values for the loops are large, indicating that the loops are dynamic and not well ordered. Thus, the differences in backbone geometry and side chain rotamers among the loops are likely of little significance and should be interpreted with caution. + + protein + PR: + cleaner0 + 2023-07-10T14:00:00Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T14:00:11Z + + 25–28 + + + 0.9992186 + structure_element + cleaner0 + 2023-07-11T09:34:40Z + SO: + + loops + + + 0.99915147 + oligomeric_state + cleaner0 + 2023-07-10T09:57:24Z + DUMMY: + + monomers + + + 0.999589 + evidence + cleaner0 + 2023-07-10T14:57:50Z + DUMMY: + + electron density + + + 0.9990503 + structure_element + cleaner0 + 2023-07-11T09:34:45Z + SO: + + loops + + + 0.9995669 + evidence + cleaner0 + 2023-07-10T14:57:51Z + DUMMY: + + electron density + + + 0.99967366 + structure_element + cleaner0 + 2023-07-11T09:34:48Z + SO: + + β-strands + + + 0.9993787 + evidence + cleaner0 + 2023-07-10T14:57:55Z + DUMMY: + + B values + + + 0.99822277 + structure_element + cleaner0 + 2023-07-11T09:34:51Z + SO: + + loops + + + 0.9990182 + structure_element + cleaner0 + 2023-07-11T09:34:54Z + SO: + + loops + + + 0.99886763 + structure_element + cleaner0 + 2023-07-11T09:34:56Z + SO: + + loops + + + + RESULTS + paragraph + 17653 + Peptide 2 assembles into oligomers similar in morphology to those formed by peptide 1. Like peptide 1, peptide 2 forms a triangular trimer, and four trimers assemble to form a dodecamer. In the higher-order assembly of the dodecamers formed by peptide 2 a new structure emerges, not seen in peptide 1, an annular pore consisting of five dodecamers. + + 0.99781215 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + Peptide 2 + + + 0.99908066 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.99880075 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + 0.9989558 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + 0.99889326 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + 0.98909026 + protein_state + cleaner0 + 2023-07-10T09:57:46Z + DUMMY: + + triangular + + + 0.99917245 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9990213 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.99811757 + oligomeric_state + cleaner0 + 2023-07-10T15:22:02Z + DUMMY: + + dodecamer + + + 0.9985569 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + 0.9981346 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + 0.6090316 + evidence + cleaner0 + 2023-07-10T15:21:46Z + DUMMY: + + structure + + + 0.9986092 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + 0.83510053 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.9990969 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + + RESULTS + title_3 + 18002 + Trimer + + 0.99737906 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + Trimer + + + + RESULTS + paragraph + 18009 + Peptide 2 forms a trimer, much like that which we observed previously for peptide 1, in which three β-hairpins assemble to form an equilateral triangle (Figure 3A). The trimer maintains all of the same stabilizing contacts as those of peptide 1. Hydrogen bonding and hydrophobic interactions between residues on the β-strands comprising Aβ17–23 and Aβ30–36 stabilize the core of the trimer. The disulfide bonds between residues 24 and 29 are adjacent to the structural core of the trimer and do not make any substantial intermolecular contacts. Two crystallographically distinct trimers comprise the peptide portion of the asymmetric unit. The two trimers are almost identical in structure, differing slightly among side chain rotamers and loop conformations. + + 0.9708526 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + Peptide 2 + + + 0.999243 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.98338246 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + 0.9996609 + structure_element + cleaner0 + 2023-07-10T10:51:04Z + SO: + + β-hairpins + + + 0.6800581 + structure_element + cleaner0 + 2023-07-11T09:35:01Z + SO: + + equilateral triangle + + + 0.9992054 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.8864662 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + Hydrogen bonding + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + hydrophobic interactions + + + 0.99971074 + structure_element + cleaner0 + 2023-07-11T09:35:04Z + SO: + + β-strands + + + protein + PR: + cleaner0 + 2023-07-10T14:00:50Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T14:01:02Z + + 17–23 + + + protein + PR: + cleaner0 + 2023-07-10T14:01:16Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T14:01:33Z + + 30–36 + + + 0.80977035 + structure_element + cleaner0 + 2023-07-11T09:35:16Z + SO: + + core + + + 0.9992182 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9961841 + ptm + cleaner0 + 2023-07-10T15:28:01Z + MESH: + + disulfide bonds + + + 0.9793444 + residue_number + cleaner0 + 2023-07-10T14:44:47Z + DUMMY: + + 24 + + + 0.9923781 + residue_number + cleaner0 + 2023-07-10T14:44:51Z + DUMMY: + + 29 + + + 0.7002436 + structure_element + cleaner0 + 2023-07-11T09:35:18Z + SO: + + structural core + + + 0.9992055 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9988949 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + chemical + CHEBI: + cleaner0 + 2023-07-10T10:31:21Z + + peptide + + + 0.9986878 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.99829334 + structure_element + cleaner0 + 2023-07-10T10:44:40Z + SO: + + loop + + + + ja-2016-013325_0004.jpg + fig3 + FIG + fig_caption + 18781 + X-ray crystallographic structure of the trimer formed +by peptide 2. (A) Triangular trimer. The three water +molecules in the +center hole of the trimer are shown as spheres. (B) Detailed view +of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The +F20 face of the trimer, with key side chains as spheres. + + 0.9992159 + evidence + cleaner0 + 2023-07-10T10:23:29Z + DUMMY: + + X-ray crystallographic structure + + + 0.99929285 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + mutant + MESH: + cleaner0 + 2023-07-10T10:39:08Z + + peptide 2 + + + 0.9195634 + protein_state + cleaner0 + 2023-07-10T09:57:46Z + DUMMY: + + Triangular + + + 0.9992619 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9997776 + chemical + cleaner0 + 2023-07-10T15:34:00Z + CHEBI: + + water + + + 0.9992895 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + hydrogen bonds + + + 0.9998975 + residue_name_number + cleaner0 + 2023-07-10T13:57:38Z + DUMMY: + + V18 + + + 0.99990034 + residue_name_number + cleaner0 + 2023-07-10T13:57:49Z + DUMMY: + + E22 + + + 0.9998609 + structure_element + cleaner0 + 2023-07-10T14:03:39Z + SO: + + δOrn + + + 0.9998971 + residue_name_number + cleaner0 + 2023-07-10T13:57:54Z + DUMMY: + + C24 + + + 0.8647282 + protein_state + cleaner0 + 2023-07-10T09:57:46Z + DUMMY: + + triangular + + + 0.9991831 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.99988556 + residue_name_number + cleaner0 + 2023-07-10T10:49:34Z + DUMMY: + + F19 + + + 0.999286 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9998834 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.99926513 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + + RESULTS + paragraph + 19252 + A network of 18 intermolecular hydrogen bonds helps stabilize the trimer. At the corners of the trimer, the pairs of β-hairpin monomers form four hydrogen bonds: two between the main chains of V18 and E22 and two between δOrn and the main chain of C24 (Figure 3B). Three ordered water molecules fill the hole in the center of the trimer, hydrogen bonding to each other and to the main chain of F20 (Figure 3A). + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + hydrogen bonds + + + 0.999288 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9992549 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9994802 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.99830544 + oligomeric_state + cleaner0 + 2023-07-10T09:57:24Z + DUMMY: + + monomers + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + hydrogen bonds + + + 0.99987984 + residue_name_number + cleaner0 + 2023-07-10T13:57:38Z + DUMMY: + + V18 + + + 0.9998851 + residue_name_number + cleaner0 + 2023-07-10T13:57:49Z + DUMMY: + + E22 + + + 0.9997837 + structure_element + cleaner0 + 2023-07-10T14:03:39Z + SO: + + δOrn + + + 0.9998801 + residue_name_number + cleaner0 + 2023-07-10T13:57:54Z + DUMMY: + + C24 + + + 0.9997751 + chemical + cleaner0 + 2023-07-10T15:34:03Z + CHEBI: + + water + + + 0.9992343 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + hydrogen bonding + + + 0.9998777 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + + RESULTS + paragraph + 19670 + Hydrophobic contacts between residues at the three corners of the trimer, where the β-hairpins meet, further stabilize the trimer. At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. The other face of the trimer displays a smaller hydrophobic surface, which includes the side chains of residues V18, F20, and I31 of the three β-hairpins (Figure 3D). In subsequent discussion, we designate the former surface the “F19 face” and the latter surface the “F20 face”. + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + Hydrophobic contacts + + + 0.99907696 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9996171 + structure_element + cleaner0 + 2023-07-10T10:51:04Z + SO: + + β-hairpins + + + 0.9992318 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9999 + residue_name_number + cleaner0 + 2023-07-10T13:57:03Z + DUMMY: + + L17 + + + 0.99989927 + residue_name_number + cleaner0 + 2023-07-10T10:49:34Z + DUMMY: + + F19 + + + 0.9999001 + residue_name_number + cleaner0 + 2023-07-10T13:57:33Z + DUMMY: + + V36 + + + 0.99961406 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.9999045 + residue_name_number + cleaner0 + 2023-07-10T13:57:12Z + DUMMY: + + A21 + + + 0.9999031 + residue_name_number + cleaner0 + 2023-07-10T13:57:22Z + DUMMY: + + I32 + + + 0.99990225 + residue_name_number + cleaner0 + 2023-07-10T13:57:27Z + DUMMY: + + L34 + + + 0.999902 + residue_name_number + cleaner0 + 2023-07-10T10:49:38Z + DUMMY: + + D23 + + + 0.99961996 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.99905705 + site + cleaner0 + 2023-07-10T14:54:17Z + SO: + + hydrophobic cluster + + + 0.99815303 + site + cleaner0 + 2023-07-10T14:04:47Z + SO: + + hydrophobic clusters + + + 0.99938655 + site + cleaner0 + 2023-07-10T14:04:53Z + SO: + + hydrophobic surface + + + 0.99910957 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9990772 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.99921834 + site + cleaner0 + 2023-07-10T14:04:53Z + SO: + + hydrophobic surface + + + 0.9999033 + residue_name_number + cleaner0 + 2023-07-10T13:57:38Z + DUMMY: + + V18 + + + 0.99990296 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.99990034 + residue_name_number + cleaner0 + 2023-07-10T13:58:04Z + DUMMY: + + I31 + + + 0.9995834 + structure_element + cleaner0 + 2023-07-10T10:51:04Z + SO: + + β-hairpins + + + 0.99988294 + residue_name_number + cleaner0 + 2023-07-10T10:49:34Z + DUMMY: + + F19 + + + 0.99988115 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + + RESULTS + title_3 + 20404 + Dodecamer + + 0.9991642 + oligomeric_state + cleaner0 + 2023-07-11T09:35:39Z + DUMMY: + + Dodecamer + + + + RESULTS + paragraph + 20414 + Four trimers assemble to form a dodecamer. The four trimers arrange in a tetrahedral fashion, creating a central cavity inside the dodecamer. Because each trimer is triangular, the resulting arrangement resembles an octahedron. Each of the 12 β-hairpins constitutes an edge of the octahedron, and the triangular trimers occupy four of the eight faces of the octahedron. Figure 4A illustrates the octahedral shape of the dodecamer. Figure 4B illustrates the tetrahedral arrangement of the four trimers. + + 0.92943406 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.9883305 + oligomeric_state + cleaner0 + 2023-07-11T09:35:47Z + DUMMY: + + dodecamer + + + 0.9677702 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.68210614 + protein_state + cleaner0 + 2023-07-10T15:46:55Z + DUMMY: + + tetrahedral + + + 0.94942534 + site + cleaner0 + 2023-07-10T14:06:25Z + SO: + + central cavity + + + 0.987146 + oligomeric_state + cleaner0 + 2023-07-11T09:35:56Z + DUMMY: + + dodecamer + + + 0.99367625 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.89434457 + protein_state + cleaner0 + 2023-07-10T09:57:46Z + DUMMY: + + triangular + + + 0.9869421 + protein_state + cleaner0 + 2023-07-10T10:01:19Z + DUMMY: + + octahedron + + + 0.9996304 + structure_element + cleaner0 + 2023-07-10T10:51:04Z + SO: + + β-hairpins + + + 0.6038043 + protein_state + cleaner0 + 2023-07-10T10:01:20Z + DUMMY: + + octahedron + + + 0.9887107 + protein_state + cleaner0 + 2023-07-10T09:57:46Z + DUMMY: + + triangular + + + 0.97976136 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.6768757 + protein_state + cleaner0 + 2023-07-10T10:01:20Z + DUMMY: + + octahedron + + + protein_state + DUMMY: + cleaner0 + 2023-07-11T09:36:15Z + + octahedral + + + 0.97393256 + oligomeric_state + cleaner0 + 2023-07-11T09:36:05Z + DUMMY: + + dodecamer + + + 0.99546045 + protein_state + cleaner0 + 2023-07-10T15:47:25Z + DUMMY: + + tetrahedral + + + 0.9308589 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + + ja-2016-013325_0005.jpg + fig4 + FIG + fig_caption + 20919 + X-ray +crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral +shape. (B) View of the dodecamer that illustrates the tetrahedral +arrangement of the four trimers that comprise the dodecamer. (C) View +of two trimer subunits from inside the cavity of the dodecamer. Residues +L17, L34, and V36 are shown as spheres, +illustrating the hydrophobic packing that occurs at the six vertices +of the dodecamer. (D) Detailed view of one of the six vertices of +the dodecamer. + + 0.99948007 + evidence + cleaner0 + 2023-07-10T15:22:14Z + DUMMY: + + X-ray +crystallographic structure + + + 0.9991375 + oligomeric_state + cleaner0 + 2023-07-10T14:22:51Z + DUMMY: + + dodecamer + + + 0.9928001 + mutant + cleaner0 + 2023-07-10T10:39:08Z + MESH: + + peptide 2 + + + 0.99905556 + oligomeric_state + cleaner0 + 2023-07-10T14:23:00Z + DUMMY: + + dodecamer + + + protein_state + DUMMY: + cleaner0 + 2023-07-10T15:47:46Z + + octahedral + + + 0.99870574 + oligomeric_state + cleaner0 + 2023-07-10T14:23:06Z + DUMMY: + + dodecamer + + + protein_state + DUMMY: + cleaner0 + 2023-07-10T15:48:02Z + + tetrahedral + + + 0.9834343 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.9980312 + oligomeric_state + cleaner0 + 2023-07-10T14:23:14Z + DUMMY: + + dodecamer + + + 0.9939126 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.6427129 + structure_element + cleaner0 + 2023-07-10T14:28:53Z + SO: + + subunits + + + 0.96798074 + site + cleaner0 + 2023-07-10T14:54:30Z + SO: + + cavity + + + 0.99848217 + oligomeric_state + cleaner0 + 2023-07-10T14:23:20Z + DUMMY: + + dodecamer + + + 0.999864 + residue_name_number + cleaner0 + 2023-07-10T13:57:03Z + DUMMY: + + L17 + + + 0.9998683 + residue_name_number + cleaner0 + 2023-07-10T13:57:27Z + DUMMY: + + L34 + + + 0.99987245 + residue_name_number + cleaner0 + 2023-07-10T13:57:33Z + DUMMY: + + V36 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + hydrophobic packing + + + 0.9971776 + oligomeric_state + cleaner0 + 2023-07-10T14:23:25Z + DUMMY: + + dodecamer + + + 0.9980862 + oligomeric_state + cleaner0 + 2023-07-10T14:23:32Z + DUMMY: + + dodecamer + + + + RESULTS + paragraph + 21447 + The F19 faces of the trimers line the interior of the dodecamer. At the six vertices, hydrophobic packing between the side chains of L17, L34, and V36 helps stabilize the dodecamer (Figures 4C and D). Salt bridges between the side chains of D23 and δOrn at the vertices further stabilize the dodecamer. Each of the six vertices includes two Aβ25–28 loops that extend past the core of the dodecamer without making any substantial intermolecular contacts. The exterior of the dodecamer displays four F20 faces (Figure S3). In the crystal lattice, each F20 face of one dodecamer packs against an F20 face of another dodecamer. Although the asymmetric unit comprises half a dodecamer, the crystal lattice may be thought of as being built of dodecamers. + + 0.99988055 + residue_name_number + cleaner0 + 2023-07-10T10:49:34Z + DUMMY: + + F19 + + + 0.9881462 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.9855783 + oligomeric_state + cleaner0 + 2023-07-10T14:23:53Z + DUMMY: + + dodecamer + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + hydrophobic packing + + + 0.99987864 + residue_name_number + cleaner0 + 2023-07-10T13:57:03Z + DUMMY: + + L17 + + + 0.99987817 + residue_name_number + cleaner0 + 2023-07-10T13:57:27Z + DUMMY: + + L34 + + + 0.9998815 + residue_name_number + cleaner0 + 2023-07-10T13:57:33Z + DUMMY: + + V36 + + + 0.9894287 + oligomeric_state + cleaner0 + 2023-07-10T14:24:01Z + DUMMY: + + dodecamer + + + 0.99987495 + residue_name_number + cleaner0 + 2023-07-10T10:49:38Z + DUMMY: + + D23 + + + 0.9998466 + structure_element + cleaner0 + 2023-07-10T14:03:39Z + SO: + + δOrn + + + 0.98633343 + oligomeric_state + cleaner0 + 2023-07-10T14:24:12Z + DUMMY: + + dodecamer + + + protein + PR: + cleaner0 + 2023-07-10T14:05:44Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T14:05:55Z + + 25–28 + + + structure_element + SO: + cleaner0 + 2023-07-10T14:06:05Z + + loops + + + 0.99597794 + structure_element + cleaner0 + 2023-07-11T09:36:22Z + SO: + + core + + + 0.99218833 + oligomeric_state + cleaner0 + 2023-07-10T14:24:22Z + DUMMY: + + dodecamer + + + 0.9943877 + oligomeric_state + cleaner0 + 2023-07-10T14:24:30Z + DUMMY: + + dodecamer + + + 0.70828927 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.9666192 + evidence + cleaner0 + 2023-07-10T15:22:19Z + DUMMY: + + crystal lattice + + + 0.9916802 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.9694082 + oligomeric_state + cleaner0 + 2023-07-10T14:24:40Z + DUMMY: + + dodecamer + + + 0.98894006 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.9841942 + oligomeric_state + cleaner0 + 2023-07-10T14:24:51Z + DUMMY: + + dodecamer + + + 0.9857307 + oligomeric_state + cleaner0 + 2023-07-10T14:25:02Z + DUMMY: + + dodecamer + + + 0.856898 + evidence + cleaner0 + 2023-07-10T15:22:21Z + DUMMY: + + crystal lattice + + + 0.9417257 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + + RESULTS + paragraph + 22203 + The electron density map for the X-ray crystallographic structure of peptide 2 has long tubes of electron density inside the central cavity of the dodecamer. The shape and length of the electron density is consistent with the structure of Jeffamine M-600, which is an essential component of the crystallization conditions. Jeffamine M-600 is a polypropylene glycol derivative with a 2-methoxyethoxy unit at one end and a 2-aminopropyl unit at the other end. Its average molecular weight is about 600 Da, which corresponds to nine propylene glycol units. Although Jeffamine M-600 is a heterogeneous mixture with varying chain lengths and stereochemistry, we modeled a single stereoisomer with nine propylene glycol units (n = 9) to fit the electron density. The Jeffamine M-600 appears to stabilize the dodecamer by occupying the central cavity and making hydrophobic contacts with residues lining the cavity (Figure S3). In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer. + + 0.99958426 + evidence + cleaner0 + 2023-07-10T15:22:29Z + DUMMY: + + electron density map + + + 0.9995545 + evidence + cleaner0 + 2023-07-10T10:23:29Z + DUMMY: + + X-ray crystallographic structure + + + 0.97934306 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.9995809 + evidence + cleaner0 + 2023-07-10T14:57:51Z + DUMMY: + + electron density + + + 0.99335635 + site + cleaner0 + 2023-07-10T14:06:25Z + SO: + + central cavity + + + 0.9986966 + oligomeric_state + cleaner0 + 2023-07-10T14:16:52Z + DUMMY: + + dodecamer + + + 0.9995684 + evidence + cleaner0 + 2023-07-10T14:57:51Z + DUMMY: + + electron density + + + 0.99932253 + evidence + cleaner0 + 2023-07-10T15:22:42Z + DUMMY: + + structure + + + 0.9993839 + chemical + cleaner0 + 2023-07-10T15:34:09Z + CHEBI: + + Jeffamine M-600 + + + 0.999519 + chemical + cleaner0 + 2023-07-10T15:34:12Z + CHEBI: + + Jeffamine M-600 + + + 0.99952114 + chemical + cleaner0 + 2023-07-10T15:34:50Z + CHEBI: + + Jeffamine M-600 + + + 0.99959373 + evidence + cleaner0 + 2023-07-10T14:57:51Z + DUMMY: + + electron density + + + 0.9994647 + chemical + cleaner0 + 2023-07-10T15:34:52Z + CHEBI: + + Jeffamine M-600 + + + 0.99720436 + oligomeric_state + cleaner0 + 2023-07-10T14:17:02Z + DUMMY: + + dodecamer + + + 0.9966955 + site + cleaner0 + 2023-07-10T14:06:24Z + SO: + + central cavity + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + hydrophobic contacts + + + 0.975463 + site + cleaner0 + 2023-07-10T14:54:47Z + SO: + + cavity + + + 0.9955487 + oligomeric_state + cleaner0 + 2023-07-10T14:17:12Z + DUMMY: + + dodecamer + + + 0.9995497 + protein_state + cleaner0 + 2023-07-10T14:29:13Z + DUMMY: + + full-length + + + 0.4306851 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + protein + PR: + cleaner0 + 2023-07-10T14:06:48Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T14:07:02Z + + 37–40 + + + protein + PR: + cleaner0 + 2023-07-10T14:07:18Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T14:07:38Z + + 37–42 + + + 0.99829847 + oligomeric_state + cleaner0 + 2023-07-10T14:17:22Z + DUMMY: + + dodecamer + + + 0.9977617 + site + cleaner0 + 2023-07-10T14:54:50Z + SO: + + hydrophobic core + + + 0.9987234 + site + cleaner0 + 2023-07-10T14:06:25Z + SO: + + central cavity + + + 0.9980034 + oligomeric_state + cleaner0 + 2023-07-10T14:17:30Z + DUMMY: + + dodecamer + + + + RESULTS + title_3 + 23368 + Annular Pore + + 0.55672383 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + Annular Pore + + + + RESULTS + paragraph + 23381 + Five dodecamers assemble to form an annular porelike structure (Figure 5A). Hydrophobic packing between the F20 faces of trimers displayed on the outer surface of each dodecamer stabilizes the porelike assembly. Two morphologically distinct interactions between trimers occur at the interfaces of the five dodecamers: one in which the trimers are eclipsed (Figure 5B), and one in which the trimers are staggered (Figure 5C). Hydrophobic packing between the side chains of F20, I31, and E22 stabilizes these interfaces (Figure 5D and E). The annular pore contains three eclipsed interfaces and two staggered interfaces. The eclipsed interfaces occur between dodecamers 1 and 2, 1 and 5, and 3 and 4, as shown in Figure 5A. The staggered interfaces occur between dodecamers 2 and 3 and 4 and 5. The annular pore is not completely flat, instead, adopting a slightly puckered shape, which accommodates the eclipsed and staggered interfaces. Ten Aβ25–28 loops from the vertices of the five dodecamers line the hole in the center of the pore. The hydrophilic side chains of S26, N27, and K28 decorate the hole. + + 0.9947389 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + 0.46617872 + structure_element + cleaner0 + 2023-07-11T09:36:26Z + SO: + + porelike + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + Hydrophobic packing + + + 0.9994198 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.9937476 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.99768627 + oligomeric_state + cleaner0 + 2023-07-10T14:16:35Z + DUMMY: + + dodecamer + + + 0.98369724 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.99890244 + site + cleaner0 + 2023-07-10T14:09:34Z + SO: + + interfaces + + + 0.9975661 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + 0.9835196 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + protein_state + DUMMY: + cleaner0 + 2023-07-10T15:48:24Z + + eclipsed + + + 0.98756206 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.9984919 + protein_state + cleaner0 + 2023-07-10T15:48:30Z + DUMMY: + + staggered + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + Hydrophobic packing + + + 0.999902 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.9998994 + residue_name_number + cleaner0 + 2023-07-10T13:58:05Z + DUMMY: + + I31 + + + 0.9999013 + residue_name_number + cleaner0 + 2023-07-10T13:57:49Z + DUMMY: + + E22 + + + 0.99853134 + site + cleaner0 + 2023-07-10T14:09:35Z + SO: + + interfaces + + + site + SO: + cleaner0 + 2023-07-10T14:08:21Z + + annular pore + + + 0.98955053 + protein_state + cleaner0 + 2023-07-10T15:48:24Z + DUMMY: + + eclipsed + + + 0.9773628 + site + cleaner0 + 2023-07-10T14:09:33Z + SO: + + interfaces + + + 0.9603877 + protein_state + cleaner0 + 2023-07-10T15:48:30Z + DUMMY: + + staggered + + + 0.9564764 + site + cleaner0 + 2023-07-10T14:09:35Z + SO: + + interfaces + + + 0.99811506 + protein_state + cleaner0 + 2023-07-10T15:48:24Z + DUMMY: + + eclipsed + + + 0.98816967 + site + cleaner0 + 2023-07-10T14:09:35Z + SO: + + interfaces + + + 0.9447598 + structure_element + cleaner0 + 2023-07-10T10:06:34Z + SO: + + dodecamers 1 and 2 + + + 0.8105362 + structure_element + cleaner0 + 2023-07-11T09:36:31Z + SO: + + 1 and 5 + + + 0.7737394 + structure_element + cleaner0 + 2023-07-11T09:36:35Z + SO: + + 3 and 4 + + + 0.99496907 + protein_state + cleaner0 + 2023-07-10T15:48:30Z + DUMMY: + + staggered + + + 0.9717439 + site + cleaner0 + 2023-07-10T14:09:35Z + SO: + + interfaces + + + structure_element + SO: + cleaner0 + 2023-07-10T10:06:51Z + + dodecamers 2 and 3 + + + 0.9016544 + structure_element + cleaner0 + 2023-07-11T09:36:38Z + SO: + + 4 and 5 + + + site + SO: + cleaner0 + 2023-07-10T14:08:22Z + + annular pore + + + 0.99846435 + protein_state + cleaner0 + 2023-07-10T15:48:24Z + DUMMY: + + eclipsed + + + 0.9233372 + protein_state + cleaner0 + 2023-07-10T15:48:30Z + DUMMY: + + staggered + + + 0.9233138 + site + cleaner0 + 2023-07-10T14:09:35Z + SO: + + interfaces + + + protein + PR: + cleaner0 + 2023-07-10T14:09:53Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T14:10:10Z + + 25–28 + + + 0.9773431 + structure_element + cleaner0 + 2023-07-11T09:36:41Z + SO: + + loops + + + 0.9940684 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + 0.9651338 + site + cleaner0 + 2023-07-10T14:54:57Z + SO: + + pore + + + 0.9999002 + residue_name_number + cleaner0 + 2023-07-10T14:10:48Z + DUMMY: + + S26 + + + 0.99989796 + residue_name_number + cleaner0 + 2023-07-10T14:10:43Z + DUMMY: + + N27 + + + 0.9998975 + residue_name_number + cleaner0 + 2023-07-10T14:10:38Z + DUMMY: + + K28 + + + + ja-2016-013325_0006.jpg + fig5 + FIG + fig_caption + 24489 + X-ray crystallographic structure of the annular pore formed by +peptide 2. (A) Annular porelike structure illustrating +the relationship of the five dodecamers that form the pore (top view). +(B) Eclipsed interface between dodecamers 1 and 2 (side view). The +same eclipsed interface also occurs between dodecamers 1 and 5 and +3 and 4. (C) Staggered interface between dodecamers 2 and 3 (side +view). The same staggered interface also occurs between dodecamers +4 and 5. (D) Eclipsed interface between dodecamers 1 and 5 (top view). +Residues F20, I31, and E22 are shown +as spheres to detail the hydrophobic packing. (E) Staggered interface +between dodecamers 2 and 3 (top view). Residues F20, I31, and E22 are shown as spheres to detail the hydrophobic +packing. + + 0.99919903 + evidence + cleaner0 + 2023-07-10T10:23:29Z + DUMMY: + + X-ray crystallographic structure + + + 0.7619935 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.9839363 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.9952337 + structure_element + cleaner0 + 2023-07-11T09:36:50Z + SO: + + Annular porelike + + + 0.76622033 + evidence + cleaner0 + 2023-07-10T15:22:49Z + DUMMY: + + structure + + + 0.99862015 + oligomeric_state + cleaner0 + 2023-07-10T09:58:53Z + DUMMY: + + dodecamers + + + 0.99661726 + site + cleaner0 + 2023-07-10T14:55:02Z + SO: + + pore + + + 0.99857295 + site + cleaner0 + 2023-07-10T14:55:12Z + SO: + + Eclipsed interface + + + structure_element + SO: + cleaner0 + 2023-07-10T10:04:45Z + + dodecamers 1 and 2 + + + 0.99860144 + site + cleaner0 + 2023-07-10T14:55:13Z + SO: + + eclipsed interface + + + structure_element + SO: + cleaner0 + 2023-07-10T10:05:01Z + + dodecamers 1 and 5 + + + structure_element + SO: + cleaner0 + 2023-07-10T10:03:23Z + + 3 and 4 + + + 0.9986403 + site + cleaner0 + 2023-07-10T14:55:19Z + SO: + + Staggered interface + + + structure_element + SO: + cleaner0 + 2023-07-10T10:05:16Z + + dodecamers 2 and 3 + + + 0.9983357 + site + cleaner0 + 2023-07-10T14:55:19Z + SO: + + staggered interface + + + structure_element + SO: + cleaner0 + 2023-07-10T10:05:31Z + + dodecamers +4 and 5 + + + 0.9980626 + site + cleaner0 + 2023-07-10T14:55:13Z + SO: + + Eclipsed interface + + + structure_element + SO: + cleaner0 + 2023-07-10T10:05:46Z + + dodecamers 1 and 5 + + + 0.9998859 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.9998832 + residue_name_number + cleaner0 + 2023-07-10T13:58:05Z + DUMMY: + + I31 + + + 0.99988735 + residue_name_number + cleaner0 + 2023-07-10T13:57:49Z + DUMMY: + + E22 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + hydrophobic packing + + + 0.9983897 + site + cleaner0 + 2023-07-10T14:55:19Z + SO: + + Staggered interface + + + structure_element + SO: + cleaner0 + 2023-07-10T10:06:01Z + + dodecamers 2 and 3 + + + 0.9998871 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.9998814 + residue_name_number + cleaner0 + 2023-07-10T13:58:05Z + DUMMY: + + I31 + + + 0.99988616 + residue_name_number + cleaner0 + 2023-07-10T13:57:49Z + DUMMY: + + E22 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + hydrophobic +packing + + + + RESULTS + paragraph + 25245 + The annular pore is comparable in size to other large protein assemblies. The outer diameter is ∼11–12 nm. The diameter of the hole in the center of the pore is ∼2 nm. The thickness of the pore is ∼5 nm, which is comparable to that of a lipid bilayer membrane. It is important to note that the annular pore formed by peptide 2 is not a discrete unit in the crystal lattice. Rather, the crystal lattice is composed of conjoined annular pores in which all four F20 faces on the surface of each dodecamer contact F20 faces on other dodecamers (Figure S4). The crystal lattice shows how the dodecamers can further assemble to form larger structures. Each dodecamer may be thought of as a tetravalent building block with the potential to assemble on all four faces to form higher-order supramolecular assemblies. + + 0.6856659 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.9704287 + site + cleaner0 + 2023-07-10T14:55:26Z + SO: + + pore + + + 0.9694308 + site + cleaner0 + 2023-07-10T14:55:29Z + SO: + + pore + + + 0.67850506 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.994282 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.99795693 + evidence + cleaner0 + 2023-07-10T15:22:54Z + DUMMY: + + crystal lattice + + + 0.9919589 + evidence + cleaner0 + 2023-07-10T15:22:56Z + DUMMY: + + crystal lattice + + + site + SO: + cleaner0 + 2023-07-10T14:25:54Z + + annular pores + + + 0.88399476 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.99921536 + oligomeric_state + cleaner0 + 2023-07-10T14:16:05Z + DUMMY: + + dodecamer + + + 0.92644376 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.99911577 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + 0.9943179 + evidence + cleaner0 + 2023-07-10T15:22:58Z + DUMMY: + + crystal lattice + + + 0.99896383 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + 0.99923635 + oligomeric_state + cleaner0 + 2023-07-10T14:16:15Z + DUMMY: + + dodecamer + + + + DISCUSS + title_1 + 26061 + Discussion + + + DISCUSS + paragraph + 26072 + The X-ray crystallographic study of peptide 2 described here provides high-resolution structures of oligomers formed by an Aβ17–36 β-hairpin. The crystallographic assembly of peptide 2 into a trimer, dodecamer, and annular pore provides a model for the assembly of the full-length Aβ peptide to form oligomers. In this model Aβ folds to form a β-hairpin comprising the hydrophobic central and C-terminal regions. Three β-hairpins assemble to form a trimer, and four trimers assemble to form a dodecamer. The dodecamers further assemble to form an annular pore (Figure 6). + + 0.99939936 + experimental_method + cleaner0 + 2023-07-10T15:40:51Z + MESH: + + X-ray crystallographic study + + + 0.99834687 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.9981528 + evidence + cleaner0 + 2023-07-10T15:23:06Z + DUMMY: + + structures + + + 0.99295276 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + protein + PR: + cleaner0 + 2023-07-10T14:14:44Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T14:14:58Z + + 17–36 + + + 0.99963754 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.8816129 + evidence + cleaner0 + 2023-07-10T15:41:09Z + DUMMY: + + crystallographic assembly + + + 0.9982991 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.9985261 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9975731 + oligomeric_state + cleaner0 + 2023-07-10T14:15:26Z + DUMMY: + + dodecamer + + + site + SO: + cleaner0 + 2023-07-10T14:08:22Z + + annular pore + + + 0.9995305 + protein_state + cleaner0 + 2023-07-10T14:29:13Z + DUMMY: + + full-length + + + protein + PR: + cleaner0 + 2023-07-10T10:08:55Z + + Aβ + + + 0.99453884 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.9411563 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.9996684 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.9953944 + structure_element + cleaner0 + 2023-07-11T09:36:57Z + SO: + + central and C-terminal regions + + + 0.99965125 + structure_element + cleaner0 + 2023-07-10T10:51:04Z + SO: + + β-hairpins + + + 0.9991762 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9989819 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.9929763 + oligomeric_state + cleaner0 + 2023-07-10T14:15:35Z + DUMMY: + + dodecamer + + + 0.9912322 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + site + SO: + cleaner0 + 2023-07-10T14:08:22Z + + annular pore + + + + ja-2016-013325_0007.jpg + fig6 + FIG + fig_caption + 26662 + Model for the hierarchical assembly of an Aβ +β-hairpin +into a trimer, dodecamer, and annular pore based on the crystallographic +assembly of peptide 2. Monomeric Aβ folds to form +a β-hairpin in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. Three β-hairpin +monomers assemble to form a triangular trimer. Four triangular trimers +assemble to form a dodecamer. Five dodecamers assemble to form an +annular pore. The molecular weights shown correspond to an Aβ42 monomer (∼4.5 kDa), an Aβ42 trimer +(∼13.5 kDa), an Aβ42 dodecamer (∼54 +kDa), and an Aβ42 annular pore composed of five dodecamers +(∼270 kDa). + + 0.46425954 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.99969107 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.9990308 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.879043 + oligomeric_state + cleaner0 + 2023-07-10T15:23:21Z + DUMMY: + + dodecamer + + + 0.8754952 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.8207643 + evidence + cleaner0 + 2023-07-10T15:41:22Z + DUMMY: + + crystallographic +assembly + + + 0.99860716 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.9989329 + oligomeric_state + cleaner0 + 2023-07-10T09:58:25Z + DUMMY: + + Monomeric + + + 0.4228502 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.99969894 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.8335692 + structure_element + cleaner0 + 2023-07-11T09:37:00Z + SO: + + central + + + 0.96587986 + structure_element + cleaner0 + 2023-07-11T09:37:04Z + SO: + + C-terminal regions + + + 0.99966925 + structure_element + cleaner0 + 2023-07-11T09:37:10Z + SO: + + antiparallel β-sheet + + + 0.999692 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.9991974 + oligomeric_state + cleaner0 + 2023-07-10T09:57:24Z + DUMMY: + + monomers + + + 0.9414926 + protein_state + cleaner0 + 2023-07-10T09:57:46Z + DUMMY: + + triangular + + + 0.99896777 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.79635763 + protein_state + cleaner0 + 2023-07-10T09:57:46Z + DUMMY: + + triangular + + + 0.9984816 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.8916394 + oligomeric_state + cleaner0 + 2023-07-10T14:26:46Z + DUMMY: + + dodecamer + + + 0.89428616 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + 0.8921617 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.99917895 + protein + cleaner0 + 2023-07-10T15:30:12Z + PR: + + Aβ42 + + + 0.99933344 + oligomeric_state + cleaner0 + 2023-07-10T09:58:17Z + DUMMY: + + monomer + + + 0.9991387 + protein + cleaner0 + 2023-07-10T15:30:15Z + PR: + + Aβ42 + + + 0.999338 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.99744 + protein + cleaner0 + 2023-07-10T15:30:18Z + PR: + + Aβ42 + + + 0.99199814 + oligomeric_state + cleaner0 + 2023-07-10T14:26:53Z + DUMMY: + + dodecamer + + + 0.9983273 + protein + cleaner0 + 2023-07-10T15:30:24Z + PR: + + Aβ42 + + + 0.9344156 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.99022186 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + + DISCUSS + paragraph + 27327 + The model put forth in Figure 6 is consistent with the current understanding of endogenous Aβ oligomerization and explains at atomic resolution many key observations about Aβ oligomers. Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. Nonfibrillar oligomers accumulate early in Alzheimer’s disease before plaque deposition. + + 0.98362213 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.69500244 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.9968413 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.69728047 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.9842993 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.4827744 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.97100955 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.9607778 + oligomeric_state + cleaner0 + 2023-07-10T10:00:01Z + DUMMY: + + fibrils + + + 0.99556965 + protein_state + cleaner0 + 2023-07-10T15:49:02Z + DUMMY: + + fibrillar + + + 0.90404606 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.59141874 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.90367794 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + protein_state + DUMMY: + cleaner0 + 2023-07-10T15:49:03Z + + fibrillar + + + 0.99723345 + protein_state + cleaner0 + 2023-07-10T15:49:08Z + DUMMY: + + nonfibrillar + + + 0.93664545 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.99050224 + protein_state + cleaner0 + 2023-07-10T15:49:03Z + DUMMY: + + Fibrillar + + + 0.97474265 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.99849355 + protein_state + cleaner0 + 2023-07-10T15:49:08Z + DUMMY: + + nonfibrillar + + + 0.98835844 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.99834716 + protein_state + cleaner0 + 2023-07-10T15:49:08Z + DUMMY: + + Nonfibrillar + + + 0.9744353 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + + DISCUSS + paragraph + 27970 + Fibrillar and nonfibrillar oligomers have structurally distinct characteristics, which are reflected in their reactivity with the fibril-specific OC antibody and the oligomer-specific A11 antibody. Fibrillar oligomers are recognized by the OC antibody but not the A11 antibody, whereas nonfibrillar oligomers are recognized by the A11 antibody but not the OC antibody. These criteria have been used to classify the Aβ oligomers that accumulate in vivo. Aβ dimers have been classified as fibrillar oligomers, whereas Aβ trimers, Aβ*56, and APFs have been classified as nonfibrillar oligomers. + + 0.99884295 + protein_state + cleaner0 + 2023-07-10T15:49:03Z + DUMMY: + + Fibrillar + + + 0.99918383 + protein_state + cleaner0 + 2023-07-10T15:49:08Z + DUMMY: + + nonfibrillar + + + 0.9958556 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-10T09:59:26Z + + oligomer + + + 0.9987214 + protein_state + cleaner0 + 2023-07-10T15:49:03Z + DUMMY: + + Fibrillar + + + 0.9927113 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.99916637 + protein_state + cleaner0 + 2023-07-10T15:49:08Z + DUMMY: + + nonfibrillar + + + 0.9938916 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.7839367 + protein + cleaner0 + 2023-07-10T10:08:55Z + PR: + + Aβ + + + 0.99323857 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.7455043 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.9992397 + oligomeric_state + cleaner0 + 2023-07-10T09:58:31Z + DUMMY: + + dimers + + + 0.99877805 + protein_state + cleaner0 + 2023-07-10T15:49:03Z + DUMMY: + + fibrillar + + + 0.9936293 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.8332771 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.99881977 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.999163 + complex_assembly + cleaner0 + 2023-07-10T10:16:25Z + GO: + + Aβ*56 + + + 0.7249561 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + 0.99906164 + protein_state + cleaner0 + 2023-07-10T15:49:08Z + DUMMY: + + nonfibrillar + + + 0.9969014 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + + DISCUSS + paragraph + 28574 + Larson and Lesné proposed a model for the endogenous production of nonfibrillar oligomers that explains these observations. In this model, folded Aβ monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils. The hierarchical assembly of peptide 2 is consistent with this model; and the trimer, dodecamer, and annular pore formed by peptide 2 may share similarities to the trimers, Aβ*56, and APFs observed in vivo. At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to Aβ trimers and Aβ*56, as little is known about the structure of Aβ trimers and Aβ*56. + + 0.82338506 + protein_state + cleaner0 + 2023-07-10T15:49:08Z + DUMMY: + + nonfibrillar + + + 0.46907452 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.99957055 + protein_state + cleaner0 + 2023-07-10T14:40:20Z + DUMMY: + + folded + + + 0.8741774 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.99933285 + oligomeric_state + cleaner0 + 2023-07-10T09:58:17Z + DUMMY: + + monomer + + + 0.9993048 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.99879164 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9681349 + oligomeric_state + cleaner0 + 2023-07-10T09:58:42Z + DUMMY: + + hexamers + + + 0.99741024 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + 0.9988606 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + complex_assembly + GO: + cleaner0 + 2023-07-10T10:16:52Z + + annular protofibrils + + + 0.9990848 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.9990043 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.99851674 + oligomeric_state + cleaner0 + 2023-07-10T14:27:45Z + DUMMY: + + dodecamer + + + 0.93985254 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.9988367 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.9990896 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.9994509 + complex_assembly + cleaner0 + 2023-07-10T10:16:25Z + GO: + + Aβ*56 + + + 0.67791164 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + 0.99890137 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.99848807 + oligomeric_state + cleaner0 + 2023-07-10T14:27:52Z + DUMMY: + + dodecamer + + + 0.99874556 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.61046284 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.9991455 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.9994221 + complex_assembly + cleaner0 + 2023-07-10T10:16:25Z + GO: + + Aβ*56 + + + 0.9986656 + evidence + cleaner0 + 2023-07-10T15:23:29Z + DUMMY: + + structure + + + 0.5242032 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.99914396 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.9994405 + complex_assembly + cleaner0 + 2023-07-10T10:16:25Z + GO: + + Aβ*56 + + + + DISCUSS + paragraph + 29315 + The crystallographically observed annular pore formed by peptide 2 is morphologically similar to the APFs formed by full-length Aβ. The annular pore formed by peptide 2 is comparable in size to the APFs prepared in vitro or isolated from Alzheimer’s brains (Figure 7 and Table 1). The varying sizes of APFs formed by full-length Aβ might result from differences in the number of oligomer subunits comprising each APF. Although the annular pore formed by peptide 2 contains five dodecamer subunits, pores containing fewer or more subunits can easily be envisioned. The dodecamers that comprise the annular pore exhibit two modes of assembly—eclipsed interactions and staggered interactions between the F20 faces of trimers within dodecamers. These two modes of assembly might reflect a dynamic interaction between dodecamers, which could permit assemblies of more dodecamers into larger annular pores. + + evidence + DUMMY: + cleaner0 + 2023-07-10T15:43:20Z + + crystallographically observed + + + 0.74659073 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.99914885 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.9904997 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + 0.9995325 + protein_state + cleaner0 + 2023-07-10T14:29:12Z + DUMMY: + + full-length + + + 0.8696307 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.8513384 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.9992081 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.9876654 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + 0.99124134 + complex_assembly + cleaner0 + 2023-07-10T10:13:52Z + GO: + + APFs + + + 0.9995335 + protein_state + cleaner0 + 2023-07-10T14:29:13Z + DUMMY: + + full-length + + + 0.7001555 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.99407035 + oligomeric_state + cleaner0 + 2023-07-10T09:59:26Z + DUMMY: + + oligomer + + + 0.6554335 + structure_element + cleaner0 + 2023-07-10T14:28:52Z + SO: + + subunits + + + 0.9955497 + complex_assembly + cleaner0 + 2023-07-10T14:29:03Z + GO: + + APF + + + 0.818646 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.9992809 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.99894255 + oligomeric_state + cleaner0 + 2023-07-10T14:55:46Z + DUMMY: + + dodecamer + + + 0.9971551 + structure_element + cleaner0 + 2023-07-10T14:28:53Z + SO: + + subunits + + + 0.83031535 + site + cleaner0 + 2023-07-10T14:55:36Z + SO: + + pores + + + 0.9976739 + structure_element + cleaner0 + 2023-07-10T14:28:53Z + SO: + + subunits + + + 0.99886453 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + 0.7511022 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + protein_state + DUMMY: + cleaner0 + 2023-07-10T15:48:24Z + + eclipsed + + + protein_state + DUMMY: + cleaner0 + 2023-07-10T15:48:30Z + + staggered + + + 0.9966365 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + 0.9572813 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.9883255 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + 0.99812466 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + 0.9975942 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + 0.7614624 + site + cleaner0 + 2023-07-10T14:55:50Z + SO: + + annular pores + + + + ja-2016-013325_0008.jpg + fig7 + FIG + fig_caption + 30222 + Surface views of the annular pore formed +by peptide 2. (A) Top view. (B) Side view. + + 0.9367285 + site + cleaner0 + 2023-07-10T14:08:22Z + SO: + + annular pore + + + 0.9604572 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + + tbl1.xml + tbl1 + TABLE + table_title_caption + 30306 + Annular Pores Formed by Aβ and +Peptide 2 + + site + SO: + cleaner0 + 2023-07-10T14:29:49Z + + Annular Pores + + + 0.49635306 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.9986495 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + Peptide 2 + + + + tbl1.xml + tbl1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups" border="0"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="left"/></colgroup><thead><tr><th style="border:none;" align="center">annular pore +source</th><th style="border:none;" align="center">outer diameter</th><th style="border:none;" align="center">inner diameter</th><th style="border:none;" align="center">observation +method</th></tr></thead><tbody><tr><td style="border:none;" align="left">peptide <bold>2</bold></td><td style="border:none;" align="center"> ∼11–12 nm</td><td style="border:none;" align="center">∼2 nm</td><td style="border:none;" align="left">X-ray crystallography</td></tr><tr><td style="border:none;" align="left">synthetic Aβ<sup><xref ref-type="bibr" rid="ref6">6</xref></sup></td><td style="border:none;" align="center">7–10 nm</td><td style="border:none;" align="center">1.5–2 nm</td><td style="border:none;" align="left">TEM</td></tr><tr><td style="border:none;" align="left">synthetic Aβ<sup><xref ref-type="bibr" rid="ref7">7</xref></sup></td><td style="border:none;" align="center">16 nm</td><td style="border:none;" align="center">not reported</td><td style="border:none;" align="left">AFM</td></tr><tr><td style="border:none;" align="left">synthetic Aβ<sup><xref ref-type="bibr" rid="ref8">8</xref></sup></td><td style="border:none;" align="center">8–25 nm</td><td style="border:none;" align="center">not reported</td><td style="border:none;" align="left">TEM</td></tr><tr><td style="border:none;" align="left">Alzheimer’s brain<sup><xref ref-type="bibr" rid="ref10">10</xref></sup></td><td style="border:none;" align="center">11–14 nm</td><td style="border:none;" align="center">2.5–4 nm</td><td style="border:none;" align="left">TEM</td></tr></tbody></table> + + 30350 + annular pore source outer diameter inner diameter observation method peptide 2 ∼11–12 nm ∼2 nm X-ray crystallography synthetic Aβ 7–10 nm 1.5–2 nm TEM synthetic Aβ 16 nm not reported AFM synthetic Aβ 8–25 nm not reported TEM Alzheimer’s brain 11–14 nm 2.5–4 nm TEM + + site + SO: + cleaner0 + 2023-07-10T14:08:22Z + + annular pore + + + chemical + CHEBI: + cleaner0 + 2023-07-10T10:31:21Z + + peptide + + + 0.9993832 + experimental_method + cleaner0 + 2023-07-10T15:43:27Z + MESH: + + X-ray crystallography + + + 0.50929236 + protein_state + cleaner0 + 2023-07-10T15:49:22Z + DUMMY: + + synthetic + + + 0.72633183 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + experimental_method + MESH: + cleaner0 + 2023-07-10T10:17:25Z + + TEM + + + 0.68735087 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + experimental_method + MESH: + cleaner0 + 2023-07-10T10:17:16Z + + AFM + + + 0.725103 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + experimental_method + MESH: + cleaner0 + 2023-07-10T10:17:25Z + + TEM + + + experimental_method + MESH: + cleaner0 + 2023-07-10T10:17:25Z + + TEM + + + + DISCUSS + paragraph + 30663 + Dot blot analysis shows that peptide 2 is reactive toward the A11 antibody (Figure S5). This reactivity suggests that peptide 2 forms oligomers in solution that share structural similarities to the nonfibrillar oligomers formed by full-length Aβ. Further studies are needed to elucidate the species that peptide 2 forms in solution and to study their biological properties. This is an active area of research in our laboratory. Preliminary attempts to study these species by SEC and SDS-PAGE have not provided a clear measure of the structures formed in solution. The difficulty in studying the oligomers formed in solution may reflect the propensity of the dodecamer to assemble on all four F20 faces. + + 0.9996084 + experimental_method + cleaner0 + 2023-07-10T15:43:34Z + MESH: + + Dot blot + + + 0.9719767 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.9831927 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.9981207 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.98931164 + protein_state + cleaner0 + 2023-07-10T15:49:08Z + DUMMY: + + nonfibrillar + + + 0.99711084 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.9995144 + protein_state + cleaner0 + 2023-07-10T14:29:13Z + DUMMY: + + full-length + + + 0.8415795 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.9940018 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.9996823 + experimental_method + cleaner0 + 2023-07-10T15:37:37Z + MESH: + + SEC + + + 0.9995587 + experimental_method + cleaner0 + 2023-07-10T10:22:56Z + MESH: + + SDS-PAGE + + + 0.7605614 + evidence + cleaner0 + 2023-07-10T15:23:34Z + DUMMY: + + structures + + + 0.99833256 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.9996724 + oligomeric_state + cleaner0 + 2023-07-10T15:23:43Z + DUMMY: + + dodecamer + + + 0.99944156 + residue_name_number + cleaner0 + 2023-07-10T13:57:43Z + DUMMY: + + F20 + + + + DISCUSS + paragraph + 31369 + The X-ray crystallographic structure and A11 reactivity of peptide 2 support the model proposed by Larsen and Lesné and suggest that β-hairpins constitute a fundamental building block for nonfibrillar oligomers. What makes β-hairpins special is that three β-hairpins can nestle together to form trimers, stabilized by a network of hydrogen bonds and hydrophobic interactions. This mode of assembly is not unique to Aβ. The foldon domain of bacteriophage T4 fibritin is composed of three β-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2. Additionally, our research group has observed a similar assembly of a β-hairpin peptide derived from β2-microglobulin. + + 0.99950874 + evidence + cleaner0 + 2023-07-10T10:23:30Z + DUMMY: + + X-ray crystallographic structure + + + 0.99778897 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.99964684 + structure_element + cleaner0 + 2023-07-10T10:51:04Z + SO: + + β-hairpins + + + 0.9959486 + protein_state + cleaner0 + 2023-07-10T15:49:08Z + DUMMY: + + nonfibrillar + + + 0.9490833 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + 0.99963474 + structure_element + cleaner0 + 2023-07-10T10:51:04Z + SO: + + β-hairpins + + + 0.99953574 + structure_element + cleaner0 + 2023-07-10T10:51:05Z + SO: + + β-hairpins + + + 0.99923205 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + hydrogen bonds + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:41Z + + hydrophobic interactions + + + 0.9831617 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.9996187 + structure_element + cleaner0 + 2023-07-11T09:37:39Z + SO: + + foldon domain + + + species + MESH: + cleaner0 + 2023-07-10T14:32:13Z + + bacteriophage T4 + + + 0.8102835 + protein + cleaner0 + 2023-07-10T14:31:00Z + PR: + + fibritin + + + 0.99958324 + structure_element + cleaner0 + 2023-07-10T10:51:05Z + SO: + + β-hairpins + + + 0.80067027 + protein_state + cleaner0 + 2023-07-10T09:57:46Z + DUMMY: + + triangular + + + 0.9992448 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.8109537 + protein_state + cleaner0 + 2023-07-10T09:57:46Z + DUMMY: + + triangular + + + 0.99926907 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.9844508 + mutant + cleaner0 + 2023-07-10T10:39:09Z + MESH: + + peptide 2 + + + 0.99943477 + structure_element + cleaner0 + 2023-07-10T10:09:19Z + SO: + + β-hairpin + + + 0.97846574 + protein + cleaner0 + 2023-07-10T15:32:24Z + PR: + + β2-microglobulin + + + + CONCL + title_1 + 32100 + Conclusion + + + CONCL + paragraph + 32111 + Although we began these studies with a relatively simple hypothesis—that the trimers and dodecamers formed by peptide 1 could accommodate the Aβ24–29 loop—an even more exciting finding has emerged—that the dodecamers can assemble to form annular pores. This finding could not have been anticipated from the X-ray crystallographic structure of peptide 1 and reveals a new level of hierarchical assembly that recapitulates micrographic observations of annular protofibrils. The crystallographically observed dodecamer, in turn, recapitulates the observation of Aβ*56, which appears to be a dodecamer of Aβ. The crystallographically observed trimer recapitulates the Aβ trimers that are observed even before the onset of symptoms in Alzheimer’s disease. + + 0.9979778 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + 0.96598125 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + 0.99939597 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + protein + PR: + cleaner0 + 2023-07-10T14:32:48Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-10T14:32:59Z + + 24–29 + + + structure_element + SO: + cleaner0 + 2023-07-10T10:44:40Z + + loop + + + 0.9943329 + oligomeric_state + cleaner0 + 2023-07-10T09:58:54Z + DUMMY: + + dodecamers + + + site + SO: + cleaner0 + 2023-07-10T14:56:18Z + + annular pores + + + 0.99947774 + evidence + cleaner0 + 2023-07-10T10:23:30Z + DUMMY: + + X-ray crystallographic structure + + + 0.99930394 + mutant + cleaner0 + 2023-07-10T10:38:33Z + MESH: + + peptide 1 + + + complex_assembly + GO: + cleaner0 + 2023-07-10T10:16:52Z + + annular protofibrils + + + evidence + DUMMY: + cleaner0 + 2023-07-10T15:24:14Z + + crystallographically observed + + + 0.996816 + oligomeric_state + cleaner0 + 2023-07-10T14:33:42Z + DUMMY: + + dodecamer + + + 0.99949056 + complex_assembly + cleaner0 + 2023-07-10T10:16:25Z + GO: + + Aβ*56 + + + 0.9902602 + oligomeric_state + cleaner0 + 2023-07-10T14:33:51Z + DUMMY: + + dodecamer + + + 0.9434316 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + evidence + DUMMY: + cleaner0 + 2023-07-10T15:43:49Z + + crystallographically observed + + + 0.9990804 + oligomeric_state + cleaner0 + 2023-07-10T09:57:07Z + DUMMY: + + trimer + + + 0.74936247 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.9990004 + oligomeric_state + cleaner0 + 2023-07-10T09:58:06Z + DUMMY: + + trimers + + + + CONCL + paragraph + 32876 + Our approach of constraining Aβ17–36 into a β-hairpin conformation and blocking aggregation with an N-methyl group has allowed us to crystallize a large fragment of what is generally considered to be an uncrystallizable peptide. We believe this iterative, “bottom up” approach of identifying the minimal modification required to crystallize Aβ peptides will ultimately allow larger fragments of Aβ to be crystallized, thus providing greater insights into the structures of Aβ oligomers. + + protein + PR: + cleaner0 + 2023-07-11T09:38:52Z + + Aβ + + + residue_range + DUMMY: + cleaner0 + 2023-07-11T09:39:09Z + + 17–36 + + + structure_element + SO: + cleaner0 + 2023-07-10T10:09:19Z + + β-hairpin + + + 0.9996012 + experimental_method + cleaner0 + 2023-07-10T15:44:11Z + MESH: + + crystallize + + + 0.9995086 + experimental_method + cleaner0 + 2023-07-10T15:44:15Z + MESH: + + crystallize + + + 0.9806091 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.98060685 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.9995915 + experimental_method + cleaner0 + 2023-07-10T15:44:18Z + MESH: + + crystallized + + + 0.735821 + evidence + cleaner0 + 2023-07-10T15:24:26Z + DUMMY: + + structures + + + 0.9897104 + protein + cleaner0 + 2023-07-10T10:08:56Z + PR: + + Aβ + + + 0.6001843 + oligomeric_state + cleaner0 + 2023-07-10T09:57:15Z + DUMMY: + + oligomers + + + + SUPPL + title + 33378 + Supporting Information Available + + + SUPPL + paragraph + 33411 + Procedures for the synthesis and crystallization of peptides 2–4; details of X-ray crystallographic data collection, processing, and refinement; procedure and data for dot blot analysis (PDF) + + + SUPPL + paragraph + 33605 + Crystallographic coordinates of peptide 2 deposited into the Protein Data Bank (PDB) with code 5HOX (data collected on a synchrotron at 1.00 Å wavelength) (PDB) + + + SUPPL + paragraph + 33767 + Crystallographic coordinates of peptide 2 deposited into the Protein Data Bank (PDB) with code 5HOY (data collected on an X-ray diffractometer at 1.54 Å wavelength) (PDB) + + + SUPPL + paragraph + 33939 + Crystallographic coordinates of peptide 4 deposited into the Protein Data Bank (PDB) with code 5HOW (PDB) + + + SUPPL + paragraph + 34045 + Crystallographic coordinates of the dodecamer formed by peptide 2 (PDB) + + + SUPPL + paragraph + 34117 + Crystallographic coordinates of the annular pore formed by peptide 2 (PDB) + + + SUPPL + paragraph + 34192 + Crystallographic data for 5HOW (CIF) + + + SUPPL + paragraph + 34229 + Crystallographic data for 5HOX (CIF) + + + SUPPL + paragraph + 34266 + Crystallographic data for 5HOY (CIF) + + + SUPPL + paragraph + 34303 + The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.6b01332. + + + SUPPL + title_1 + 34420 + Supplementary Material + + + SUPPL + paragraph + 34443 + The authors declare no competing financial interest. + + + REF + title + 34496 + References + + + 349 + 357 + surname:Benilova;given-names:I. + surname:Karran;given-names:E. + surname:De Strooper;given-names:B. + 10.1038/nn.3028 + 22286176 + REF + Nat. Neurosci. + ref + 15 + 2012 + 34507 + + + 352 + 357 + surname:Lesné;given-names:S. + surname:Koh;given-names:M. T. + surname:Kotilinek;given-names:L. + surname:Kayed;given-names:R. + surname:Glabe;given-names:C. G. + surname:Yang;given-names:A. + surname:Gallagher;given-names:M. + surname:Ashe;given-names:K. H. + 10.1038/nature04533 + 16541076 + REF + Nature + ref + 440 + 2006 + 34508 + + + 1383 + 1398 + surname:Lesné;given-names:S. E. + surname:Sherman;given-names:M. A. + surname:Grant;given-names:M. + surname:Kuskowski;given-names:M. + surname:Schneider;given-names:J. A. + surname:Bennett;given-names:D. A. + surname:Ashe;given-names:K. H. + 10.1093/brain/awt062 + 23576130 + REF + Brain + ref + 136 + 2013 + 34509 + + + 73 + 110 + surname:Hafner;given-names:J. H. + surname:Cheung;given-names:C. L. + surname:Woolley;given-names:A. T. + surname:Lieber;given-names:C. M. + 10.1016/B978-008044031-6/50037-9 + 11473787 + REF + Prog. Biophys. Mol. Biol. + ref + 77 + 2001 + 34510 + + + 2433 + 2444 + surname:Lin;given-names:H. + surname:Bhatia;given-names:R. + surname:Lal;given-names:R. + 10.1096/fj.01-0377com + 11689468 + REF + FASEB J. + ref + 15 + 2001 + 34511 + + + 291 + 292 + surname:Lashuel;given-names:H. A. + surname:Hartley;given-names:D. + surname:Petre;given-names:B. M. + surname:Walz;given-names:T. + surname:Lansbury;given-names:P. T.;suffix:Jr. + 10.1038/418291a + REF + Nature + ref + 418 + 2002 + 34512 + + + 10427 + 10432 + surname:Quist;given-names:A. + surname:Doudevski;given-names:I. + surname:Lin;given-names:H. + surname:Azimova;given-names:R. + surname:Ng;given-names:D. + surname:Frangione;given-names:B. + surname:Kagan;given-names:B. + surname:Ghiso;given-names:J. + surname:Lal;given-names:R. + 10.1073/pnas.0502066102 + 16020533 + REF + Proc. Natl. Acad. +Sci. U. S. A. + ref + 102 + 2005 + 34513 + + + 4230 + 4237 + surname:Kayed;given-names:R. + surname:Pensalfini;given-names:A. + surname:Margol;given-names:L. + surname:Sokolov;given-names:Y. + surname:Sarsoza;given-names:F. + surname:Head;given-names:E. + surname:Hall;given-names:J. + surname:Glabe;given-names:C. + 10.1074/jbc.M808591200 + 19098006 + REF + J. Biol. Chem. + ref + 284 + 2009 + 34514 + + + 689285 + surname:Kokubo;given-names:H. + surname:Kayed;given-names:R. + surname:Glabe;given-names:C. G. + surname:Staufenbiel;given-names:M. + surname:Saido;given-names:T. C. + surname:Iwata;given-names:N. + surname:Yamaguchi;given-names:H. + 10.4061/2009/689285 + 20798763 + REF + Int. J. Alzheimer's +Dis. + ref + 2009 + 2009 + 34515 + + + 22122 + 22130 + surname:Lasagna-Reeves;given-names:C. A. + surname:Glabe;given-names:C. G. + surname:Kayed;given-names:R. + 10.1074/jbc.M111.236257 + 21507938 + REF + J. Biol. Chem. + ref + 286 + 2011 + 34516 + + + 20631 + 20635 + surname:Roher;given-names:A. E. + surname:Chaney;given-names:M. O. + surname:Bonnell;given-names:B. S. + surname:Emmerling;given-names:M. R. + surname:Kuo;given-names:Y. M. + surname:Webster;given-names:S. D. + surname:Stine;given-names:W. B. + surname:Haverkamp;given-names:L. J. + surname:Woods;given-names:A. S. + surname:Cotter;given-names:R. J. + surname:Tuohy;given-names:J. M. + surname:Krafft;given-names:G. A. + 10.1074/jbc.271.34.20631 + 8702810 + REF + J. Biol. Chem. + ref + 271 + 1996 + 34517 + + + 860 + 866 + surname:McLean;given-names:C. A. + surname:Cherny;given-names:R. A. + surname:Fraser;given-names:F. W. + surname:Fuller;given-names:S. J. + surname:Smith;given-names:M. J. + surname:Beyreuther;given-names:K. + surname:Bush;given-names:A. I. + surname:Masters;given-names:C. L. + 10.1002/1531-8249(199912)46:6<860::AID-ANA8>3.0.CO;2-M + 10589538 + REF + Ann. Neurol. + ref + 46 + 1999 + 34518 + + + 1328 + 1341 + surname:McDonald;given-names:J. M. + surname:Savva;given-names:G. M. + surname:Brayne;given-names:C. + surname:Welzel;given-names:A. T. + surname:Forster;given-names:G. + surname:Shankar;given-names:G. M. + surname:Selkoe;given-names:D. J. + surname:Ince;given-names:P. G. + surname:Walsh;given-names:D. M. + 10.1093/brain/awq065 + 20403962 + REF + Brain + ref + 133 + 2010 + 34519 + + + 837 + 842 + surname:Shankar;given-names:G. M. + surname:Li;given-names:S. + surname:Regan;given-names:C. M. + surname:Walsh;given-names:D. M. + surname:Sabatini;given-names:B. L. + surname:Selkoe;given-names:D. J. + surname:Mehta;given-names:T. H. + surname:Garcia-Munoz;given-names:A. + surname:Shepardson;given-names:N. E. + surname:Smith;given-names:I. + surname:Brett;given-names:F. M. + surname:Farrell;given-names:M. A. + surname:Rowan;given-names:M. J. + surname:Lemere;given-names:C. A. + 10.1038/nm1782 + 18568035 + REF + Nat. Med. + ref + 14 + 2008 + 34520 + + + 5819 + 5824 + surname:Jin;given-names:M. + surname:Shepardson;given-names:N. + surname:Yang;given-names:T. + surname:Chen;given-names:G. + surname:Walsh;given-names:D. + surname:Selkoe;given-names:D. J. + 10.1073/pnas.1017033108 + 21421841 + REF + Proc. Natl. Acad. +Sci. U. S. A. + ref + 108 + 2011 + 34521 + + + 4050 + 4058 + surname:Mucke;given-names:L. + surname:Masliah;given-names:E. + surname:Yu;given-names:G. Q. + surname:Mallory;given-names:M. + surname:Rockenstein;given-names:E. M. + surname:Tatsuno;given-names:G. + surname:Hu;given-names:K. + surname:Kholodenko;given-names:D. + surname:Johnson-Wood;given-names:K. + surname:McConlogue;given-names:L. + 10818140 + REF + J. Neurosci. + ref + 20 + 2000 + 34522 + + + 372 + 381 + surname:Kawarabayashi;given-names:T. + surname:Younkin;given-names:L. H. + surname:Saido;given-names:T. C. + surname:Shoji;given-names:M. + surname:Ashe;given-names:K. H. + surname:Younkin;given-names:S. G. + 11160418 + REF + J. Neurosci. + ref + 21 + 2001 + 34523 + + + 3801 + 3809 + surname:Kawarabayashi;given-names:T. + surname:Shoji;given-names:M. + surname:Younkin;given-names:L. H. + surname:Wen-Lang;given-names:L. + surname:Dickson;given-names:D. W. + surname:Murakami;given-names:T. + surname:Matsubara;given-names:E. + surname:Abe;given-names:K. + surname:Ashe;given-names:K. H. + surname:Younkin;given-names:S. G. + 10.1523/JNEUROSCI.5543-03.2004 + 15084661 + REF + J. Neurosci. + ref + 24 + 2004 + 34524 + + + 1977 + 1986 + surname:Meilandt;given-names:W. J. + surname:Cisse;given-names:M. + surname:Ho;given-names:K. + surname:Wu;given-names:T. + surname:Esposito;given-names:L. A. + surname:Scearce-Levie;given-names:K. + surname:Cheng;given-names:I. H. + surname:Yu;given-names:G. Q. + surname:Mucke;given-names:L. + 10.1523/JNEUROSCI.2984-08.2009 + 19228952 + REF + J. +Neurosci. + ref + 29 + 2009 + 34525 + + + 293 + 302 + surname:Shankar;given-names:G. M. + surname:Leissring;given-names:M. A. + surname:Adame;given-names:A. + surname:Sun;given-names:X. + surname:Spooner;given-names:E. + surname:Masliah;given-names:E. + surname:Selkoe;given-names:D. J. + surname:Lemere;given-names:C. A. + surname:Walsh;given-names:D. M. + 10.1016/j.nbd.2009.07.021 + 19660551 + REF + Neurobiol. Dis. + ref + 36 + 2009 + 34526 + + + 13407 + 13412 + surname:Benzinger;given-names:T. L. + surname:Gregory;given-names:D. M. + surname:Burkoth;given-names:T. S. + surname:Miller-Auer;given-names:H. + surname:Lynn;given-names:D. G. + surname:Botto;given-names:R. E. + surname:Meredith;given-names:S. C. + 10.1073/pnas.95.23.13407 + 9811813 + REF + Proc. Natl. Acad. +Sci. +U. S. A. + ref + 95 + 1998 + 34527 + + + 262 + 265 + surname:Petkova;given-names:A. T. + surname:Leapman;given-names:R. D. + surname:Guo;given-names:Z. + surname:Yau;given-names:W. M. + surname:Mattson;given-names:M. P. + surname:Tycko;given-names:R. + 10.1126/science.1105850 + 15653506 + REF + Science + ref + 307 + 2005 + 34528 + + + 17342 + 17347 + surname:Lührs;given-names:T. + surname:Ritter;given-names:C. + surname:Adrian;given-names:M. + surname:Riek-Loher;given-names:D. + surname:Bohrmann;given-names:B. + surname:Döbeli;given-names:H. + surname:Schubert;given-names:D. + surname:Riek;given-names:R. + 10.1073/pnas.0506723102 + 16293696 + REF + Proc. Natl. Acad. +Sci. U. S. A. + ref + 102 + 2005 + 34529 + + + 498 + 512 + surname:Petkova;given-names:A. T. + surname:Yau;given-names:W. M. + surname:Tycko;given-names:R. + 10.1021/bi051952q + 16401079 + REF + Biochemistry + ref + 45 + 2006 + 34530 + + + 4618 + 4629 + surname:Paravastu;given-names:A. K. + surname:Petkova;given-names:A. T. + surname:Tycko;given-names:R. + 10.1529/biophysj.105.076927 + 16565054 + REF + Biophys. J. + ref + 90 + 2006 + 34531 + + + 1257 + 1268 + surname:Lu;given-names:J. X. + surname:Qiang;given-names:W. + surname:Yau;given-names:W. M. + surname:Schwieters;given-names:C. D. + surname:Meredith;given-names:S. C. + surname:Tycko;given-names:R. + 10.1016/j.cell.2013.08.035 + 24034249 + REF + Cell + ref + 154 + 2013 + 34532 + + + 499 + 505 + surname:Xiao;given-names:Y. + surname:Ma;given-names:B. + surname:McElheny;given-names:D. + surname:Parthasarathy;given-names:S. + surname:Long;given-names:F. + surname:Hoshi;given-names:M. + surname:Nussinov;given-names:R. + surname:Ishii;given-names:Y. + 10.1038/nsmb.2991 + 25938662 + REF + Nat. +Struct. Mol. Biol. + ref + 22 + 2015 + 34533 + + + 453 + 457 + surname:Sawaya;given-names:M. R. + surname:Sambashivan;given-names:S. + surname:Madsen;given-names:A. Ø. + surname:Riekel;given-names:C. + surname:Eisenberg;given-names:D. + surname:Nelson;given-names:R. + surname:Ivanova;given-names:M. I. + surname:Sievers;given-names:S. A. + surname:Apostol;given-names:M. I. + surname:Thompson;given-names:M. J. + surname:Balbirnie;given-names:M. + surname:Wiltzius;given-names:J. J. + surname:McFarlane;given-names:H. T. + 10.1038/nature05695 + 17468747 + REF + Nature + ref + 447 + 2007 + 34534 + + + 16938 + 16943 + surname:Colletier;given-names:J. P. + surname:Laganowsky;given-names:A. + surname:Landau;given-names:M. + surname:Zhao;given-names:M. + surname:Soriaga;given-names:A. B. + surname:Goldschmidt;given-names:L. + surname:Flot;given-names:D. + surname:Cascio;given-names:D. + surname:Sawaya;given-names:M. R. + surname:Eisenberg;given-names:D. + 10.1073/pnas.1112600108 + 21949245 + REF + Proc. Natl. Acad. +Sci. U. S. A. + ref + 108 + 2011 + 34535 + + + 1870 + 1877 + surname:Yu;given-names:L. + surname:Edalji;given-names:R. + surname:Miesbauer;given-names:L. + surname:Solomon;given-names:L. + surname:Bartley;given-names:D. + surname:Walter;given-names:K. + surname:Johnson;given-names:R. W. + surname:Hajduk;given-names:P. J. + surname:Olejniczak;given-names:E. T. + surname:Harlan;given-names:J. E. + surname:Holzman;given-names:T. F. + surname:Lopez;given-names:A. P. + surname:Labkovsky;given-names:B. + surname:Hillen;given-names:H. + surname:Barghorn;given-names:S. + surname:Ebert;given-names:U. + surname:Richardson;given-names:P. L. + 10.1021/bi802046n + 19216516 + REF + Biochemistry + ref + 48 + 2009 + 34536 + + + 22822 + 22826 + surname:Scheidt;given-names:H. A. + surname:Morgado;given-names:I. + surname:Huster;given-names:D. + 10.1074/jbc.M112.367474 + 22589542 + REF + J. Biol. Chem. + ref + 287 + 2012 + 34537 + + + 458 + 462 + surname:Doi;given-names:T. + surname:Masuda;given-names:Y. + surname:Irie;given-names:K. + surname:Akagi;given-names:K. + surname:Monobe;given-names:Y. + surname:Imazawa;given-names:T. + surname:Takegoshi;given-names:K. + 10.1016/j.bbrc.2012.10.096 + 23131555 + REF + Biochem. Biophys. +Res. Commun. + ref + 428 + 2012 + 34538 + + + 18673 + 18683 + surname:Gu;given-names:L. + surname:Liu;given-names:C. + surname:Guo;given-names:Z. + 10.1074/jbc.M113.457739 + 23687299 + REF + J. +Biol. Chem. + ref + 288 + 2013 + 34539 + + + 2494 + 2508 + surname:Tay;given-names:W. M. + surname:Huang;given-names:D. + surname:Rosenberry;given-names:T. L. + surname:Paravastu;given-names:A. K. + 10.1016/j.jmb.2013.04.003 + 23583777 + REF + J. Mol. Biol. + ref + 425 + 2013 + 34540 + + + 8294 + 8307 + surname:Potapov;given-names:A. + surname:Yau;given-names:W.-M. + surname:Ghirlando;given-names:R. + surname:Thurber;given-names:K. R. + surname:Tycko;given-names:R. + 10.1021/jacs.5b04843 + 26068174 + REF + J. Am. Chem. Soc. + ref + 137 + 2015 + 34541 + + + 5099 + 6104 + surname:Hoyer;given-names:W. + surname:Grönwall;given-names:C. + surname:Jonsson;given-names:A. + surname:Ståhl;given-names:S. + surname:Härd;given-names:T. + 10.1073/pnas.0711731105 + 18375754 + REF + Proc. Natl. Acad. +Sci. U. S. A. + ref + 105 + 2008 + 34542 + + + 15595 + 15600 + surname:Sandberg;given-names:A. + surname:Luheshi;given-names:L. M. + surname:Lannfelt;given-names:L. + surname:Dobson;given-names:C. M. + surname:Härd;given-names:T. + surname:Söllvander;given-names:S. + surname:Pereira de Barros;given-names:T. + surname:Macao;given-names:B. + surname:Knowles;given-names:T. P. + surname:Biverstål;given-names:H. + surname:Lendel;given-names:C. + surname:Ekholm-Petterson;given-names:F. + surname:Dubnovitsky;given-names:A. + 10.1073/pnas.1001740107 + 20713699 + REF + Proc. Natl. Acad. +Sci. U. S. A. + ref + 107 + 2010 + 34543 + + + 12756 + 12760 + surname:Lendel + 10.1002/anie.201406357 + REF + Angew. Chem., Int. Ed. + ref + 53 + 2014 + 34544 + recently reported the NMR structure of a hexameric peptide barrel formed by this disulfide constrained Aβ: Lendel, C.; Bjerring, M.; Dubnovitsky, A.; Kelly, R. T.; Filippov, A.; Antzutkin, O. N.; Nielsen, N. C.; Härd, T + + + 5595 + 5598 + surname:Spencer;given-names:R. K. + surname:Li;given-names:H. + surname:Nowick;given-names:J. S. + 10.1021/ja5017409 + 24669800 + REF + J. Am. Chem. Soc. + ref + 136 + 2014 + 34767 + + + 4972 + 4973 + surname:Nowick;given-names:J. S. + surname:Lam;given-names:K. S. + surname:Khasanova;given-names:T. V. + surname:Kemnitzer;given-names:W. E. + surname:Maitra;given-names:S. + surname:Mee;given-names:H. T. + surname:Liu;given-names:R. + 10.1021/ja025699i + 11982357 + REF + J. Am. Chem. Soc. + ref + 124 + 2002 + 34768 + + + We also created a peptide +with an N-methyl group at position F20. This peptide +forms oligomers with structures similar to those formed by peptide 1. + REF + ref + 34769 + + + 698 + 710 + surname:Spencer;given-names:R. K. + surname:Nowick;given-names:J. S. + 10.1002/ijch.201400179 + 26213415 + REF + Isr. J. Chem. + ref + 55 + 2015 + 34770 + + + 488 + 499 + surname:Santiveri;given-names:C. M. + surname:León;given-names:E. + surname:Rico;given-names:M. + surname:Jiménez;given-names:M. A. + 10.1002/chem.200700845 + 17943702 + REF + Chem. - Eur. J. + ref + 14 + 2008 + 34771 + + + 6304 + 6311 + surname:Spencer;given-names:R. K. + surname:Kreutzer;given-names:A. G. + surname:Salveson;given-names:P. J. + surname:Li;given-names:H. + surname:Nowick;given-names:J. S. + 10.1021/jacs.5b01673 + 25915729 + REF + J. Am. Chem. Soc. + ref + 137 + 2015 + 34772 + + + 3523 + 3528 + surname:Spencer;given-names:R. + surname:Chen;given-names:K. H. + surname:Manuel;given-names:G. + surname:Nowick;given-names:J. S. + 10.1002/ejoc.201300221 + REF + Eur. J. Org. Chem. + ref + 2013 + 2013 + 34773 + + + 125 + 132 + surname:Kabsch;given-names:W. + 10.1107/S0907444909047337 + 20124692 + REF + Acta Crystallogr., +Sect. D: Biol. Crystallogr. + ref + 66 + 2010 + 34774 + + + 623 + 628 + surname:Weik;given-names:M. + surname:Ravelli;given-names:R. B. + surname:Kryger;given-names:G. + surname:McSweeney;given-names:S. + surname:Raves;given-names:M. L. + surname:Harel;given-names:M. + surname:Gros;given-names:P. + surname:Silman;given-names:I. + surname:Kroon;given-names:J. + surname:Sussman;given-names:J. L. + 10.1073/pnas.97.2.623 + 10639129 + REF + Proc. Natl. Acad. Sci. U. S. A. + ref + 97 + 2000 + 34775 + + + 488 + 497 + surname:Leiros;given-names:H. K. + surname:McSweeney;given-names:S. M. + surname:Smalås;given-names:A. O. + 10.1107/S0907444901000646 + 11264577 + REF + Acta Crystallogr., Sect. D: Biol. Crystallogr. + ref + 57 + 2001 + 34776 + + + The δOrn in peptide 2 replaces K16 in Aβ. +In a dodecamer of full-length +Aβ, the side chain of K16 could form a salt bridge +with the side chain of D23. + REF + ref + 34777 + + + 24 + 39 + surname:Pieters;given-names:B. J. + surname:van Eldijk;given-names:M. B. + surname:Nolte;given-names:R. J. + surname:Mecinović;given-names:J. + 10.1039/C5CS00157A + 26497225 + REF + Chem. Soc. Rev. + ref + 45 + 2016 + 34778 + + + 5628 + 5654 + surname:Butterfield;given-names:S. M. + surname:Lashuel;given-names:H. A. + 10.1002/anie.200906670 + REF + Angew. Chem., Int. Ed. + ref + 49 + 2010 + 34779 + + + 29639 + 29643 + surname:Glabe;given-names:C. G. + 10.1074/jbc.R800016200 + 18723507 + REF + J. Biol. Chem. + ref + 283 + 2008 + 34780 + + + 125 + 139 + surname:Larson;given-names:M. E. + surname:Lesné;given-names:S. E. + 10.1111/j.1471-4159.2011.07478.x + REF + J. Neurochem. + ref + 120 + 2012 + 34781 + + + 1760 + 1771 + surname:Liu;given-names:P. + surname:Reed;given-names:M. N. + surname:Wilmot;given-names:C. M. + surname:Cleary;given-names:J. P. + surname:Zahs;given-names:K. R. + surname:Ashe;given-names:K. H. + surname:Kotilinek;given-names:L. A. + surname:Grant;given-names:M. K. + surname:Forster;given-names:C. L. + surname:Qiang;given-names:W. + surname:Shapiro;given-names:S. L. + surname:Reichl;given-names:J. H. + surname:Chiang;given-names:A. C. + surname:Jankowsky;given-names:J. L. + 10.1016/j.celrep.2015.05.021 + 26051935 + REF + Cell Rep. + ref + 11 + 2015 + 34782 + + + 486 + 489 + surname:Kayed;given-names:R. + surname:Head;given-names:E. + surname:Thompson;given-names:J. L. + surname:McIntire;given-names:T. M. + surname:Milton;given-names:S. C. + surname:Cotman;given-names:C. W. + surname:Glabe;given-names:C. G. + 10.1126/science.1079469 + 12702875 + REF + Science + ref + 300 + 2003 + 34783 + + + 789 + 798 + surname:Tao;given-names:Y. + surname:Strelkov;given-names:S. V. + surname:Mesyanzhinov;given-names:V. V. + surname:Rossmann;given-names:M. G. + 10.1016/S0969-2126(97)00233-5 + 9261070 + REF + Structure + ref + 5 + 1997 + 34784 + + + diff --git a/BioC_XML/4832331_v0.xml b/BioC_XML/4832331_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..56842da91a06852d4c294413dfb891681ef04bd3 --- /dev/null +++ b/BioC_XML/4832331_v0.xml @@ -0,0 +1,7109 @@ + + + + PMC + 20140719 + pmc.key + + 4832331 + CC BY + no + 0 + 0 + + 10.1038/srep24601 + srep24601 + 4832331 + 27080013 + 24601 + This work is licensed under a Creative Commons Attribution 4.0 +International License. The images or other third party material in this article are +included in the article’s Creative Commons license, unless indicated +otherwise in the credit line; if the material is not included under the Creative +Commons license, users will need to obtain permission from the license holder to +reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ + surname:Kandiah;given-names:Eaazhisai + surname:Carriel;given-names:Diego + surname:Gutsche;given-names:Irina + surname:Perard;given-names:Julien + surname:Malet;given-names:Hélène + surname:Bacia;given-names:Maria + surname:Liu;given-names:Kaiyin + surname:Chan;given-names:Sze W. S. + surname:Houry;given-names:Walid A. + surname:Ollagnier de Choudens;given-names:Sandrine + surname:Elsen;given-names:Sylvie + TITLE + front + 6 + 2016 + 0 + Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:43Z + + Escherichia coli + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:01:09Z + + AAA+ ATPase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:17Z + + RavA + + + + ABSTRACT + abstract + 136 + The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:15Z + + inducible + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:28:58Z + + lysine decarboxylase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:44Z + + enterobacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:52:58Z + + acid stress response enzyme + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:53:28Z + + decamers + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:43Z + + Escherichia coli + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:53:41Z + + hexamers + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:01:09Z + + AAA+ ATPase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:17Z + + RavA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:53:55Z + + pseudoatomic model + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-06-15T10:33:58Z + + LdcI-RavA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:02:51Z + + cryo-electron microscopy + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:03:07Z + + map + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:54:52Z + + crystal structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:03Z + + inactive + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:19Z + + decamer + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:17Z + + RavA + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:12Z + + monomer + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:03:42Z + + cryo-electron microscopy + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:03:55Z + + 3D reconstructions + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:55:31Z + + E. coli + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:40Z + + improved map + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:48Z + + bound to + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T08:55:55Z + + LARA domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:17Z + + RavA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:56:31Z + + pH optimal + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:56:35Z + + Comparison + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:56:39Z + + structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:52:58Z + + acid stress response enzyme + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:17Z + + RavA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:57:12Z + + pH-dependent + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:17Z + + RavA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:32:28Z + + Multiple sequence alignment + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:57:19Z + + phylogenetic analysis + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:57:25Z + + enterobacteria + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:29:01Z + + lysine decarboxylase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:17Z + + RavA + + + + INTRO + paragraph + 1452 + Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. They counteract acid stress experienced by the bacterium in the host digestive and urinary tract, and in particular in the extremely acidic stomach. Each decarboxylase is induced by an excess of the target amino acid and a specific range of extracellular pH, and works in conjunction with a cognate inner membrane antiporter. Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. Consequently, these enzymes buffer both the bacterial cytoplasm and the local extracellular environment. These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:44Z + + Enterobacterial + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:15Z + + inducible + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:01:14Z + + decarboxylases + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:01:02Z + + basic + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:01:09Z + + amino acids + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:01:19Z + + lysine + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:01:22Z + + arginine + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:01:24Z + + ornithine + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:01:33Z + + α-family + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:01:53Z + + pyridoxal-5′-phosphate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:03Z + + PLP + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:02:10Z + + bacterium + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:02:21Z + + decarboxylase + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:56Z + + amino acid + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:02:26Z + + inner membrane antiporter + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:57Z + + amino acid + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:33Z + + polyamine + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:16Z + + PLP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:45Z + + proton + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:48Z + + CO2 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:33Z + + polyamine + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:29:05Z + + antiporter + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:57Z + + amino acid + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:03:05Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:03:24Z + + amino acid decarboxylases + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:15Z + + inducible + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:03:46Z + + biodegradative + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:03:54Z + + biosynthetic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:03:58Z + + lysine and ornithine decarboxylase + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:33Z + + polyamine + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:04:09Z + + neutral pH + + + + INTRO + paragraph + 2662 + Inducible enterobacterial amino acid decarboxylases have been intensively studied since the early 1940 because the ability of bacteria to withstand acid stress can be linked to their pathogenicity in humans. In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI). This effect is attributed to cadaverine, the diamine produced by decarboxylation of lysine by LdcI and LdcC, that was shown to enhance UPEC colonisation of the bladder. In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. Both acid pH and cadaverine induce closure of outer membrane porins thereby contributing to bacterial protection from acid stress, but also from certain antibiotics, by reduction in membrane permeability. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:15Z + + Inducible + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:44Z + + enterobacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:03:24Z + + amino acid decarboxylases + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:09:20Z + + bacteria + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:09:26Z + + humans + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:15Z + + inducible + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:29:12Z + + lysine decarboxylase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:09:34Z + + CadA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:10:09Z + + broad pH range + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:57:26Z + + enterobacteria + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:10:22Z + + Salmonella enterica serovar Typhimurium + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:10:27Z + + Vibrio cholerae + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:10:30Z + + Vibrio vulnificus + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:03:54Z + + biosynthetic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:29:19Z + + lysine decarboxylase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:11:04Z + + uropathogenic Escherichia coli + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:11:15Z + + UPEC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:11:19Z + + nitric oxide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:11:26Z + + cadaverine + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:12:00Z + + lysine + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:11:15Z + + UPEC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:03:54Z + + biosynthetic + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:55:31Z + + E. coli + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:29:16Z + + lysine decarboxylase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:12:07Z + + fluoroquinolones + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:12:13Z + + RpoS + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:11:26Z + + cadaverine + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:55:31Z + + E. coli + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:12:39Z + + acid pH + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:11:26Z + + cadaverine + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:29:22Z + + porins + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:03:05Z + + bacterial + + + + INTRO + paragraph + 4295 + The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:14:46Z + + crystal structure + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:55:31Z + + E. coli + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:14:53Z + + electron microscopy + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:14:59Z + + EM + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:19Z + + decamer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:15:13Z + + pentameric + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:15:25Z + + rings + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:12Z + + monomer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:15:32Z + + wing domain + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:15:36Z + + 1–129 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:15:42Z + + PLP-binding core domain + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:15:45Z + + 130–563 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:15:48Z + + C-terminal domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:15:54Z + + CTD + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:15:58Z + + 564–715 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:16:06Z + + Monomers + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:17Z + + core domains + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:16:20Z + + 2-fold symmetrical + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:16:27Z + + dimers + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:34Z + + active sites + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:39Z + + toroidal D5-symmetrical structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T10:25:52Z + + wing + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T10:26:02Z + + core domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:51Z + + central pore + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:57Z + + CTDs + + + + INTRO + paragraph + 4931 + Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins. This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation. The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal β-sheet of LdcI. + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:55:31Z + + E. coli + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:01:09Z + + AAA+ ATPase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:17Z + + RavA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:15:13Z + + pentameric + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:15:25Z + + rings + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:20:38Z + + hexameric + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:15:25Z + + rings + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:17Z + + RavA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:02:10Z + + bacterium + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:20:48Z + + solved the structure + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:55:31Z + + E. coli + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:20:56Z + + LdcI-RavA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:21:06Z + + cryo-electron microscopy + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:21:13Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:54:52Z + + crystal structures + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:21:24Z + + pseudoatomic model + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:04:28Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:04:37Z + + map + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:45Z + + LdcI-LARA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:56Z + + LARA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:59Z + + LdcI associating domain of RavA + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:20:56Z + + LdcI-RavA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:33Z + + loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T08:55:55Z + + LARA domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:17Z + + RavA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:52Z + + β-sheet + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + + INTRO + paragraph + 6005 + In spite of this wealth of structural information, the fact that LdcC does not interact with RavA, although the two lysine decarboxylases are 69% identical and 84% similar, and the physiological significance of the absence of this interaction remained unexplored. To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures. Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. Finally, we performed multiple sequence alignment of 22 lysine decarboxylases from Enterobacteriaceae containing the ravA-viaA operon in their genome. Remarkably, this analysis revealed that several specific residues in the above-mentioned β-sheet, independently of the rest of the protein sequence, are sufficient to define if a particular lysine decarboxylase should be classified as an “LdcC-like” or an “LdcI-like”. Moreover, this classification perfectly agrees with the genetic environment of the lysine decarboxylase genes. This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:28:19Z + + structural information + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:17Z + + RavA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:05:16Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:05:26Z + + reconstruction + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:20:56Z + + LdcI-RavA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:28:55Z + + structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:54:52Z + + crystal structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:29:23Z + + high pH + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:03Z + + inactive + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:29:50Z + + pH 8.5 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:05:42Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:05:54Z + + reconstructions + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:20:56Z + + LdcI-RavA + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:45Z + + LdcI-LARA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:31:54Z + + acidic pH optimal + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:30:36Z + + 3D reconstruction + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:30:51Z + + active pH + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:31:06Z + + pH 6.2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:08:49Z + + biodegradative + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:03:54Z + + biosynthetic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:31:37Z + + pH-dependent + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:17Z + + RavA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:31:50Z + + RavA binding site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:52Z + + β-sheet + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:31Z + + LdcI + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:32:38Z + + LdcI-LdcC chimeras + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:32:32Z + + interchanged + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:32:21Z + + β-sheets + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:32:28Z + + multiple sequence alignment + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:32:46Z + + Enterobacteriaceae + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T11:37:18Z + + ravA-viaA operon + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:32:56Z + + specific residues + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:52Z + + β-sheet + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:29:29Z + + lysine decarboxylase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:04Z + + LdcC-like + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:10Z + + LdcI-like + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:18Z + + RavA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:44Z + + enterobacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:29:31Z + + lysine decarboxylase + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:33:22Z + + high degree of conservation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:33:25Z + + small structural motif + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:08:49Z + + biodegradative + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:44Z + + enterobacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:01:09Z + + AAA+ ATPase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:18Z + + RavA + + + + RESULTS + title_1 + 8130 + Results and Discussion + + + RESULTS + title_2 + 8153 + CryoEM 3D reconstructions of LdcC, LdcIa and LdcI-LARA + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:06:20Z + + CryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:06:31Z + + 3D reconstructions + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:45Z + + LdcI-LARA + + + + RESULTS + paragraph + 8208 + In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). In addition, we improved our earlier cryoEM map of the LdcI-LARA complex from 7.5 Å to 6.2 Å resolution (Figs 1E,F and S3). Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1). Significant differences between these pseudoatomic models can be interpreted as movements between specific biological states of the proteins as described below. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:06:47Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:06:59Z + + reconstructions + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:55:31Z + + E. coli + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:36:34Z + + pH optimal + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:07:12Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:07:25Z + + map + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:37:01Z + + pH 7.5 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:07:40Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:07:49Z + + map + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:31:06Z + + pH 6.2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:08:04Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:08:13Z + + map + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:45Z + + LdcI-LARA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:08:25Z + + reconstructions + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:37:13Z + + pseudoatomic models + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:37:18Z + + flexible fitting of + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:14:46Z + + crystal structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:37:21Z + + structural homology model + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:37:13Z + + pseudoatomic models + + + + RESULTS + title_2 + 9121 + The wing domains as a stable anchor at the center of the double-ring + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:25:25Z + + wing domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:38:09Z + + double-ring + + + + RESULTS + paragraph + 9190 + As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). This superposition reveals that the densities lining the central hole of the toroid are roughly at the same location, while the rest of the structure exhibits noticeable changes. Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. This preservation of the central part of the double-ring assembly may help the enzymes to maintain their decameric state upon activation and incorporation into the LdcI-RavA cage. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:24:41Z + + superimposed + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:08:40Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:08:48Z + + reconstructions + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:45Z + + LdcI-LARA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:05Z + + LdcC + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:14:46Z + + crystal structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:19Z + + decamer + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:24:59Z + + superposition + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:27:25Z + + densities + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:35:26Z + + central hole + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:35:30Z + + toroid + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:27:22Z + + structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:25:09Z + + double-ring + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:25:25Z + + wing domains + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:25:47Z + + conserved + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:26:09Z + + lowest root mean square deviation + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:26:21Z + + RMSD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:57Z + + CTDs + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:26:50Z + + RavA binding interface + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:26:32Z + + RMSD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:25:25Z + + wing domains + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:27:17Z + + structures + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:26:32Z + + RMSD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:38:43Z + + RMSDmin + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:09:02Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:09:15Z + + maps + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:25:25Z + + wing domains + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:27:20Z + + structures + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:26:32Z + + RMSD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:27:45Z + + central part + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:33:29Z + + decameric + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:20:56Z + + LdcI-RavA + + + + RESULTS + title_2 + 10525 + The core domain and the active site rearrangements upon pH-dependent enzyme activation and LARA binding + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:28:35Z + + core domain + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:28:38Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:28:31Z + + pH-dependent + + + + RESULTS + paragraph + 10629 + Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). The decameric enzyme is built of five dimers associating into a 5-fold symmetrical double-ring (two monomers making a dimer are delineated in Fig. 1). As common for the α family of the PLP-dependent decarboxylases, dimerization is required for the enzymatic activity because the active site is buried in the dimer interface (Fig. 3A,B). This interface is formed essentially by the core domains with some contribution of the CTDs. The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). Zooming in the variations in the PLP-SD shows that most of the structural changes concern displacements in the active site (Fig. 3C–F). The most conspicuous differences between the PLP-SDs can be linked to the pH-dependent activation of the enzymes. The resolution of the cryoEM maps does not allow modeling the position of the PLP moiety and calls for caution in detailed mechanistic interpretations in terms of individual amino acids. Therefore we restrict our analysis to secondary structure elements. In particular, transition from LdcIi to LdcI-LARA involves ~3.5 Å and ~4.5 Å shifts away from the 5-fold axis in the active site α-helices spanning residues 218–232 and 246–254 respectively (Fig. 3C–E). Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin = 0.9 Å, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity. In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA. Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:34:11Z + + visual inspection + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:34:14Z + + RMSD calculations + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:34:17Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:30:51Z + + active pH + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:45Z + + LdcI-LARA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:06Z + + LdcC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:34:35Z + + high pH + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:33:29Z + + decameric + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:16:27Z + + dimers + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:34:50Z + + 5-fold symmetrical double-ring + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:16:06Z + + monomers + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:34:58Z + + dimer + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:35:02Z + + α family + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:35:06Z + + PLP-dependent decarboxylases + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:36:57Z + + active site + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:35:49Z + + dimer interface + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:37:00Z + + interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:17Z + + core domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:57Z + + CTDs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:36:05Z + + core domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:36:08Z + + PLP-binding subdomain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:36:13Z + + PLP-SD + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:36:17Z + + 184–417 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:36:35Z + + subdomains + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:36:59Z + + partly disordered + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:37:13Z + + loops + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:37:16Z + + linker region + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:37:19Z + + 130–183 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:37:22Z + + subdomain 4 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:37:25Z + + 418–563 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:36:13Z + + PLP-SD + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:37:04Z + + active site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:37:36Z + + PLP-SDs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:37:54Z + + pH-dependent + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:09:40Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:09:48Z + + maps + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:16Z + + PLP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:01:09Z + + amino acids + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:45Z + + LdcI-LARA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:37:07Z + + active site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:38:09Z + + α-helices + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:38:11Z + + 218–232 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:38:14Z + + 246–254 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:37:36Z + + PLP-SDs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:06Z + + LdcC + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:19Z + + decamer + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:38:38Z + + RMSDmin + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:39:00Z + + optimal pH + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T18:23:01Z + + biochemical observation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:18Z + + RavA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:37:11Z + + active site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:45Z + + LdcI-LARA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:14:46Z + + crystal structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:15Z + + inducible + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:29:39Z + + arginine decarboxylase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T10:46:24Z + + AdiA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:39:47Z + + high conservation + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:39:51Z + + PLP-coordinating residues + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:39:54Z + + patch of negatively charged residues + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:39:56Z + + active site channel + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:39:59Z + + binding site + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:57Z + + amino acid + + + + RESULTS + title_2 + 13132 + Rearrangements of the ppGpp binding pocket upon pH-dependent enzyme activation and LARA binding + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:40:28Z + + ppGpp binding pocket + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:40:42Z + + pH-dependent + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:48:37Z + + LARA + + + + RESULTS + paragraph + 13228 + An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4). The ppGpp binding pocket is made up by residues from all domains and is located approximately 30 Å away from the PLP moiety. Whereas the crystal structure of the ppGpp-LdcIi was solved to 2 Å resolution, only a 4.1 Å resolution structure of the ppGpp-free LdcIi could be obtained. At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ). Thus, we speculated that inhibition of LdcI by ppGpp would be accompanied by a transduction of subtle structural changes at the level of individual amino acid side chains between the ppGpp binding pocket and the active site of the enzyme. All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study. While differences in the ppGpp binding site could indeed be visualized (Fig. S4), the level of resolution warns against speculations about their significance. The fact that interaction with RavA reduces the ppGpp affinity for LdcI despite the long distance of ~30 Å between the LARA domain binding site and the closest ppGpp binding pocket (Fig. S5) seems to favor an allosteric regulation mechanism. Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:06Z + + LdcC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T10:44:21Z + + stringent response alarmone + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T10:44:24Z + + ppGpp + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:44:26Z + + interface + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:16:06Z + + monomers + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:44:30Z + + ring + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:40:28Z + + ppGpp binding pocket + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:16Z + + PLP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:14:46Z + + crystal structure + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T10:44:41Z + + ppGpp-LdcIi + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:44:44Z + + solved + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:44:47Z + + structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:44:52Z + + ppGpp-free + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:44:58Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T10:44:41Z + + ppGpp-LdcIi + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:45:01Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:29:51Z + + pH 8.5 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T10:45:12Z + + ppGpp + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T10:45:16Z + + ppGpp + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:57Z + + amino acid + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:40:28Z + + ppGpp binding pocket + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:45:25Z + + active site + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:10:14Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:10:24Z + + reconstructions + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:45:43Z + + absence of + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T10:45:46Z + + ppGpp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:45:48Z + + active + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:45:56Z + + ppGpp binding site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:18Z + + RavA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-06-15T10:26:42Z + + ppGpp + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:46:06Z + + LARA domain binding site + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:40:28Z + + ppGpp binding pocket + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:46:13Z + + ppGpp binding residues + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:46:19Z + + strictly conserved + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T10:46:24Z + + AdiA + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:53:28Z + + decamers + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:47:39Z + + low pH optimal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:47:12Z + + arginine decarboxylase + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T10:46:50Z + + ppGpp + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T10:46:24Z + + AdiA + + + + RESULTS + title_2 + 14916 + Swinging and stretching of the CTDs upon pH-dependent LdcI activation and LARA binding + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:57Z + + CTDs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:40:43Z + + pH-dependent + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:48:17Z + + LARA + + + + RESULTS + paragraph + 15003 + Inspection of the superimposed decameric structures (Figs 2 and S6) suggests a depiction of the wing domains as an anchor around which the peripheral CTDs swing. This swinging movement seems to be mediated by the core domains and is accompanied by a stretching of the whole LdcI subunits attracted by the RavA magnets. Indeed, all CTDs have very similar structures (RMSDmin <1 Å). Yet the superposition of the decamers lays bare a progressive movement of the CTD as a whole upon enzyme activation by pH and the binding of LARA. The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). These small but noticeable swinging and stretching (up to ~4 Å) may be related to the incorporation of the LdcI decamer into the LdcI-RavA cage. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:24:41Z + + superimposed + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:33:29Z + + decameric + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:33:41Z + + structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:25:25Z + + wing domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:57Z + + CTDs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:17Z + + core domains + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:35:34Z + + subunits + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:18Z + + RavA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:57Z + + CTDs + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:38:43Z + + RMSDmin + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:24:59Z + + superposition + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:53:28Z + + decamers + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:15:54Z + + CTD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:35:38Z + + LARA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:12Z + + monomer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:51:21Z + + most compact + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:45Z + + LdcI-LARA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:51:23Z + + gradually extend + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:16:57Z + + CTDs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T08:55:55Z + + LARA domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:18Z + + RavA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:19Z + + decamer + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:20:56Z + + LdcI-RavA + + + + RESULTS + title_2 + 15836 + The C-terminal β-sheet of a lysine decarboxylase as a major determinant of the interaction with RavA + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:52Z + + β-sheet + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:51:51Z + + lysine decarboxylase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:18Z + + RavA + + + + RESULTS + paragraph + 15940 + In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. However, at the level of this structural element the two proteins are actually surpisingly similar. Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). Both constructs could be purified and could form decamers visually indistinguishable from the wild-type proteins. As expected, binding of LdcI to RavA was completely abolished by this procedure and no LdcIC-RavA complex could be detected. On the contrary, introduction of the C-terminal β-sheet of LdcI into LdcC led to an assembly of the LdcCI-RavA complex. On the negative stain EM grid, the chimeric cages appeared less rigid than the native LdcI-RavA, which probably means that the environment of the β-sheet contributes to the efficiency of the interaction and the stability of the entire architecture (Fig. 5D–F). + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:35:42Z + + LARA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:54:52Z + + crystal structures + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:45Z + + LdcI-LARA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:10:38Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:10:50Z + + density + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-06-15T10:27:39Z + + LdcI-RavA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T10:56:05Z + + two-stranded β-sheet + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:11:12Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:11:01Z + + maps + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:37:13Z + + pseudoatomic models + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:15Z + + inducible + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:56:16Z + + constitutive + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:18Z + + RavA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T18:23:01Z + + swapped + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:32:21Z + + β-sheets + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:56:46Z + + chimeras + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:56:53Z + + LdcIC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:52Z + + β-sheet + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:06Z + + LdcC + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:57:02Z + + LdcCI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:06Z + + LdcC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:52Z + + β-sheet + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:57:09Z + + Both constructs + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:53:28Z + + decamers + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:57:13Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:19Z + + RavA + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T10:57:27Z + + LdcIC-RavA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:57:30Z + + introduction + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:52Z + + β-sheet + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:06Z + + LdcC + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T10:57:39Z + + LdcCI-RavA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:57:41Z + + negative stain EM grid + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:57:44Z + + chimeric + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:57:50Z + + native + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:20:57Z + + LdcI-RavA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:53Z + + β-sheet + + + + RESULTS + title_2 + 17457 + The C-terminal β-sheet of a lysine decarboxylase is a highly conserved signature allowing to distinguish between LdcI and LdcC + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:53Z + + β-sheet + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:58:36Z + + lysine decarboxylase + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:58:41Z + + highly conserved + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:06Z + + LdcC + + + + RESULTS + paragraph + 17587 + Alignment of the primary sequences of the E. coli LdcI and LdcC shows that some amino acid residues of the C-terminal β-sheet are the same in the two proteins, whereas others are notably different in chemical nature. Importantly, most of the amino acid differences between the two enzymes are located in this very region. Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. We inspected the genetic environment of lysine decarboxylases from 22 enterobacterial species referenced in the NCBI database, corrected the gene annotation where necessary (Tables S3 and S4), and performed multiple sequence alignment coupled to a phylogenetic analysis (see Methods). This procedure yielded several unexpected and exciting results. First of all, consensus sequence for the entire lysine decarboxylase family was derived. Second, the phylogenetic analysis clearly split the lysine decarboxylases into two groups (Fig. 6A). All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. Thus, consensus sequences could also be determined for each of the two groups (Figs 6B,C and S7). Inspection of these consensus sequences revealed important differences between the groups regarding charge, size and hydrophobicity of several residues precisely at the level of the C-terminal β-sheet that is responsible for the interaction with RavA (Fig. 6B–D). For example, in our previous study, site-directed mutations identified Y697 as critically required for the RavA binding. Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. The third and most remarkable finding was that exactly the same separation into “LdcI-like” and “LdcC”-like groups can be obtained based on a comparison of the C-terminal β-sheets only, without taking the rest of the primary sequence into account. Therefore the C-terminal β-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the “LdcI-like” and “LdcC-like” enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7). Our structures show that this motif is not involved in the enzymatic activity or the oligomeric state of the proteins. Thus, enterobacteria identified here (Fig. 6, Table S4) appear to exert evolutionary pressure on the biodegradative lysine decarboxylase towards the RavA binding. One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp. Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5 MDa assembly. The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. Finally, cadaverine being an important platform chemical for the production of industrial polymers such as nylon, structural information is valuable for optimisation of bacterial lysine decarboxylases used for its production in biotechnology. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:01:58Z + + Alignment of the primary sequences + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:55:31Z + + E. coli + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:32Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:06Z + + LdcC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:53Z + + β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:02:22Z + + very region + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:53Z + + β-sheet + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:02:26Z + + lysine decarboxylase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:19Z + + RavA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:02:32Z + + certain residues + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:53Z + + β-sheet + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:02:34Z + + conserved + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:57:26Z + + enterobacteria + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-20T11:37:18Z + + ravA-viaA operon + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:02:39Z + + inspected the genetic environment + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:44Z + + enterobacterial + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:32:28Z + + multiple sequence alignment + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:02:47Z + + phylogenetic analysis + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:02:54Z + + consensus sequence + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:02:58Z + + lysine decarboxylase + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:03:02Z + + phylogenetic analysis + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:10Z + + LdcI-like + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:03:17Z + + biodegradable + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T11:03:36Z + + CadB + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:03:39Z + + antiporter + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:03:24Z + + enzymes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:04Z + + LdcC-like + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:03:54Z + + biosynthetic + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:33:46Z + + consensus sequences + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:33:49Z + + consensus sequences + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:53Z + + β-sheet + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:19Z + + RavA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:36:28Z + + site-directed mutations + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:37:25Z + + Y697 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:19Z + + RavA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:37:25Z + + Y697 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:46:19Z + + strictly conserved + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:10Z + + LdcI-like + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:04Z + + LdcC-like + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:31:24Z + + always have + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:36:39Z + + lysine + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:19Z + + RavA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:36:25Z + + site-directed mutagenesis + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:10Z + + LdcI-like + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:30:05Z + + LdcC”-like + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:32:21Z + + β-sheets + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:53Z + + β-sheet + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:58:41Z + + highly conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:08:28Z + + signature sequence + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:10Z + + LdcI-like + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:04Z + + LdcC-like + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:44Z + + enterobacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:08:32Z + + structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:08:35Z + + this motif + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:57:26Z + + enterobacteria + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:08:49Z + + biodegradative + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:08:57Z + + lysine decarboxylase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:20Z + + RavA + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:20:57Z + + LdcI-RavA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:33Z + + LdcI + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:44Z + + enterobacterial + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:33Z + + LdcI + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T11:09:09Z + + stringent response alarmone + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T11:36:35Z + + ppGpp + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:33Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:20Z + + RavA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:09:13Z + + subunits + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:09:15Z + + respiratory complex I + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:20Z + + RavA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:09:17Z + + iron-sulfur proteins + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:33Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T23:01:20Z + + RavA + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-06-15T10:28:21Z + + LdcI-RavA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:44Z + + enterobacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:09:24Z + + lysine decarboxylase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:10Z + + LdcI-like + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:04Z + + LdcC-like + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:20:57Z + + LdcI-RavA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:11:03Z + + structures + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:37:13Z + + pseudoatomic models + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:11:08Z + + active + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:44:52Z + + ppGpp-free + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:08:49Z + + biodegradative + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:03:54Z + + biosynthetic + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:55:31Z + + E. coli + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:11:15Z + + UPEC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:44:58Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:33Z + + LdcI + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T10:44:41Z + + ppGpp-LdcIi + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:54:53Z + + crystal structures + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:11:33Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:11:44Z + + reconstructions + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:57:26Z + + enterobacteria + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:32:46Z + + Enterobacteriaceae + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:11:31Z + + lysine decarboxylase + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:11:33Z + + Eikenella corrodens + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:11:26Z + + cadaverine + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:03:05Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:51Z + + lysine decarboxylases + + + + METHODS + title_1 + 22628 + Methods + + + METHODS + title_2 + 22636 + Protein expression and purification + + + METHODS + paragraph + 22672 + LdcI and LdcC were expressed and purified as described from an E. coli strain that cannot produce ppGpp (MG1655 ΔrelA ΔspoT strain). LdcI was stored in 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM PLP, pH 6.8 (buffer A) and LdcC in 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM PLP, pH 7.5 (buffer B). + + + METHODS + paragraph + 22993 + Chimeric LdcIC and LdcCI were constructed, expressed and purified as follows. The chimeras were designed by exchange, between LdcI and LdcC, of residues from 631 to 640 and from 697 to the C-terminus, corresponding to the regions around the two strands of the C-terminal β-sheet (Fig. 5B,C), while leaving the rest of the sequence unaltered. The synthetic ldcIC and ldcCI genes (2148 bp and 2154 bp respectively), provided within a pUC57 vector (GenScript) were subcloned into pET-TEV vector based on pET-28a (Invitrogen) containing an N-terminal TEV-cleavable 6x-His-Tag. Proteins were expressed in Rosetta 2 (DE3) cells (Novagen) in LB medium supplemented with kanamycin and chloramphenicol at 37 °C, upon induction with 0.5 mM IPTG at 18 °C. Cells were harvested by centrifugation, the pellet resuspended in a 50 mM Tris-HCl, 150 mM NaCl, pH 8 buffer supplemented with Complete EDTA free (Roche) and 0.1 mM PMSF (Sigma), and disrupted by sonication at 4 °C. After centrifugation at 75000 g at 4 °C for 20 min, the supernatant was loaded on a Ni-NTA column. The eluted protein-containing fractions were pooled and the His-Tag removed by incubation with the TEV protease at 1/100 ratio and an extensive dialysis in a 50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5 mM EDTA, pH 8 buffer. After a second dialysis in a 50 mM Tris-HCl, 150 mM NaCl, pH 8 buffer supplemented with 10 mM imidazole, the sample was loaded on a Ni-NTA column in the same buffer, which allowed to separate the TEV protease and LdcCI/LdcIC. Finally, the pure proteins were obtained by size exclusion chromatography on a Superdex-S200 column in buffer A. + + + METHODS + title_2 + 24652 + LdcIa -cryoEM data collection and 3D reconstruction + + + METHODS + paragraph + 24704 + LdcI was prepared at 2 mg/ml in a buffer containing 25 mM MES, 100 mM NaCl, 0.2 mM PLP, 1 mM DTT, pH 6.2. 3 μl of sample were applied to glow-discharged quantifoil grids 300 mesh 2/1 (Quantifoil Micro Tools GmbH, Germany), excess solution was blotted during 2.5 s with a Vitrobot (FEI) and the grid frozen in liquid ethane. Data collection was performed on a FEI Polara microscope operated at 300 kV under low dose conditions. Micrographs were recorded on Kodak SO-163 film at 59,000 magnification, with defocus ranging from 0.6 to 4.9 μm. Films were digitized on a Zeiss scanner (Photoscan) at a step size of 7 μm giving a pixel size of 1.186 Å. The contrast transfer function (CTF) for each micrograph was determined with CTFFIND3. + + + METHODS + paragraph + 25462 + Initially ~2500 particles of 256 × 256 pixels were extracted manually, binned 4 times and subjected to one round of multivariate statistical analysis and classification using IMAGIC. Representative class averages corresponding to projections in different orientations were used as input for an ab-initio 3D reconstruction by RICOserver (rico.ibs.fr/). The resulting 3D reconstruction was refined using RELION, which yielded an 18 Å resolution map. Using projections of this model as a template, particles of size 256 × 256 pixels were semi-automatically selected from all the micrographs using the Fast Projection Matching (FPM) algorithm. The resulting dataset of ~46000 particles was processed in RELION with the previous map as an initial model and with a full CTF correction after the first peak. The final map comprised 44207 particles with a resolution of 7.4 Å as per the gold-standard FSC = 0.143 criterion. It was sharpened with EMBfactor using calculated B-factor of −350 Å2 and masked with a soft mask to obtain a final map with a resolution of 6.1 Å (Fig. S3, Table S1). + + + METHODS + title_2 + 26571 + LdcC - cryoEM data collection and 3D reconstruction + + + METHODS + paragraph + 26623 + LdcC was prepared at 2 mg/ml in a buffer containing 25 mM HEPES, 100 mM NaCl, 0.2 mM PLP, 1 mM DTT, pH 7.2. Grids were prepared and sample imaged as LdcIa. Data were processed essentially as LdcIa described above. Briefly, an initial ~20 Å resolution model was generated by angular reconstitution after manual picking of circa 3000 particles from the first micrographs, filtered to 60 Å resolution, refined with RELION and used for a semi-automatic selection with FPM. The dataset was processed in RELION with a full CTF correction to yield a final reconstruction comprising 61000 particles. The map was sharpened with Relion postprocessing, using a soft mask and automated B-factor estimation (−690 Å2), yielding a map at 5.5 Å resolution (Fig. S1, Table S1). + + + METHODS + title_2 + 27400 + LdcI-LARA - 3D reconstruction + + + METHODS + paragraph + 27430 + In our first study, the dataset was processed in SPIDER and the CTF correction involved a simple phase-flipping. Therefore, for consistency with the present work, we revisited the dataset and processed it in RELION with a full CTF correction after the first peak. It was sharpened with EMBfactor using calculated B-factor of −350 Å2 and masked with a soft mask to obtain a final map with a resolution of 6.2 Å (Fig. S2). This reconstruction is of a slightly better quality in terms of the continuity of the internal density. Therefore we used this improved map for fitting of the atomic model and further analysis (Fig. S2, Table S1). + + + METHODS + title_2 + 28073 + Additional image processing + + + METHODS + paragraph + 28101 + As a crosscheck, each data set was also refined either from other initial models: a “featureless donut” with approximate dimensions of the decamer, and low pass-filtered reconstructions from the two other data sets (i.e. the LdcC reconstruction was used as a model for the LdcIa and LdcI-LARA data sets, etc). All refinements converged to the same solutions independently of the starting model. Local resolution of all maps was determined with ResMap. + + + METHODS + title_2 + 28557 + LdcCI and LdcIC chimeras —negative stain EM and 2D image analysis + + + METHODS + paragraph + 28625 + 0.4 mg/ml of RavA (in a 20 mM Tris-HCl, 500 mM NaCl, 10 mM MgCl2, 1 mM DTT, 5% glycerol, pH 6.8 buffer) was mixed with 0.3 mg/ml of either LdcI, LdcC, LdcCI or LdcIC in the presence of 2 mM ADP and 10 mM MgCl2 in a buffer containing 20 mM Hepes and 150 mM NaCl at pH 7.4. After 10 minutes incubation at room temperature, 3 μl of mixture were applied to the clear side of the carbon on a carbon-mica interface and negatively stained with 2% uranyl acetate. Images were recorded with a JEOL 1200 EX II microscope at 100 kV at a nominal magnification of 15000 on a CCD camera yielding a pixel size of 4.667 Å. No complexes between RavA and LdcC or LdcIC could be observed, whereas the LdcCI-RavA preparation manifested cage-like particles similar to the previously published LdcI-RavA, but also unbound RavA and LdcCI, which implies that the affinity of RavA to the LdcCI chimera is lower than its affinity to the native LdcI. 1260 particles of 96 × 96 pixels were extracted interactively from several micrographs. 2D centering, multivariate statistical analysis and classification were performed using IMAGIC. Class-averages similar to the cage-like LdcI-RavA complex were used as references for multi-reference alignment followed by multivariate statistical analysis and classification. + + + METHODS + title_2 + 29946 + Fitting of atomic models into cryoEM maps + + + METHODS + paragraph + 29988 + A homology model of LdcC was obtained using the atomic coordinates of the LdcI monomer (3N75) as the template in SWISS-MODEL server. The RMSD between the template and the resulting model was 0.26 Å. The LdcC model was then fitted as a rigid body into the LdcC cryoEM map using the fit-in-map module of UCSF Chimera. This rigid fit indicated movements of several parts of the protein. Therefore, the density corresponding to one LdcC monomer was extracted and flexible fitting was performed using IMODFIT at 8 Å resolution. This monomeric model was then docked into the decameric cryoEM map with URO and its graphical version VEDA that use symmetry information for fitting in Fourier space. The Cα RMSDmin between the initial model of the LdcC monomer and the final IMODFIT LdcC model is 1.2 Å. In the case of LdcIa, the density corresponding to an individual monomer was extracted and the fit performed similarly to the one described above, with the final model of the decameric particle obtained with URO and VEDA. The Cα RMSDmin between the LdcIi monomer and the final IMODFIT model is 1.4 Å. For LdcI-LARA, the density accounting for one LdcI monomer bound to a LARA domain was extracted and further separated into individual densities corresponding to LdcI and to LARA. The fit of LdcI was performed as for LdcC and LdcIa, while the crystal structure of LARA was docked into the monomeric LdcI-LARA map as a rigid body using SITUS. The resulting pseudoatomic models were used to create the final model of the LdcI-LARA decamer with URO and VEDA. The Cα RMSDmin between the LdcIi monomer and the final IMODFIT model is 1.4 Å. A brief summary of relevant parameters is provided in Table S1. + + + METHODS + title_2 + 31699 + Sequence analysis + + + METHODS + paragraph + 31717 + Out of a non-exhaustive list of 50 species of Enterobacteriaceae (Table S3), 22 were found to contain genes annotated as ldcI or ldcC and containing the ravA-viaA operon (Table S4). An analysis using MUSCLE with default parameters showed that these genes share more than 65% identity. To verify annotation of these genes, we compared their genetic environment with that of E. coli ldcI and ldcC. Indeed, in E. coli the ldcI gene is in operon with the cadB gene encoding a lysine-cadaverine antiporter, whereas the ldcC gene is present between the accA gene (encoding an acetyl-CoA carboxylase alpha subunit carboxyltransferase) and the yaeR gene (coding for an unknown protein belonging to the family of Glyoxalase/Dioxygenase/Bleomycin resistance proteins). Compared with this genetic environment, the annotation of several ldcI and ldcC genes in enterobacteria was found to be inconsistent (Table S4). For example, several strains contain genes annotated as ldcC in the genetic background of ldcI and vice versa, as in the case of Salmonella enterica and Trabulsiella guamensi. Furthermore, the gene with an “ldcC-like” environment was found to be annotated as cadA in particular strains of Citrobacter freundii, Cronobacter sakazakii, Enterobacter cloacae subsp. Cloaca, Erwinia amylovora, Pantoea agglomerans, Rahnella aquatilis, Shigella dysenteriae, and Yersinia enterocolitica subsp. enterocolitica, whereas in Hafnia alvei, Kluyvera ascorbata, and Serratia marcescens subsp. marcescens, the gene with an “ldcI-like” environment was found to be annotated as ldcC. In addition, as far as the genetic environment is concerned, Plesiomonas appears to have two ldc genes with the organization of the E. coli ldcI (operon cadA-cadB). Consequently, we corrected for gene annotation consistent with the genetic environment and made multiple sequence alignments using version 8.0.1 of the CLC Genomics Workbench software. A phylogenetic tree was generated based on Maximum Likelihood and following the Neighbor-Joining method with the WAG protein substitution model. The reliability of the generated phylogenetic tree was assessed by bootstrap analysis. The presented unrooted phylogenetic tree shows the nodes that are reliable over 95% (Fig. 6A). Remarkably, the multiple sequence alignment and the resulting phylogenetic tree clearly grouped together all sequences annotated as ldcI on the one side, and all sequences annotated as ldcC on the other side. Thus, we conclude that all modifications in gene annotations that we introduced for the sake of consistency with the genetic environment are perfectly corroborated by the multiple sequence alignment and the phylogenetic analysis. Since the regulation of the lysine decarboxylase gene (i.e. inducible or constitutive) cannot be assessed by this analysis, we call the resulting groups “ldcI-like” and “ldcC-like” as referred to the E. coli enzymes, and make a parallel between the membership in a given group and the ability of the protein to form a cage complex with RavA. + + + METHODS + title_1 + 34762 + Additional Information + + + METHODS + paragraph + 34785 + Accession codes: CryoEM maps and corresponding fitted atomic structures (main chain atoms) have been deposited in the Electron Microscopy Data Bank and Protein Data Bank, respectively, with accession codes EMD-3205 and 5FKZ for LdcC, EMD-3204 and 5FKX for LdcIa and EMD-3206 and 5FL2 for LdcI-LARA. + + + METHODS + paragraph + 35084 + How to cite this article: Kandiah, E. et al. Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA. Sci. Rep. 6, 24601; doi: 10.1038/srep24601 (2016). + + + SUPPL + title_1 + 35317 + Supplementary Material + + + 436 + 447 + surname:Christen;given-names:P. + surname:Mehta;given-names:P. K. + REF + Chem. Rec. +N. Y. N + ref + 1 + 2001 + 35340 + From cofactor to enzymes. The molecular evolution of pyridoxal-5′-phosphate-dependent enzymes + + + 383 + 415 + surname:Eliot;given-names:A. C. + surname:Kirsch;given-names:J. F. + 15189147 + REF + Annu. Rev. Biochem. + ref + 73 + 2004 + 35436 + Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations + + + 301 + 314 + surname:Zhao;given-names:B. + surname:Houry;given-names:W. A. + REF + Biochem. Cell Biol. Biochim. Biol. Cell. + ref + 88 + 2010 + 35522 + Acid stress response in enteropathogenic gammaproteobacteria: an aptitude for survival + + + 65 + 81 + surname:Kanjee;given-names:U. + surname:Houry;given-names:W. A. + 23701194 + REF + Annu. Rev. +Microbiol. + ref + 67 + 2013 + 35609 + Mechanisms of acid resistance in Escherichia coli + + + e22397 + surname:Viala;given-names:J. P. M. + 21799843 + REF + PloS One + ref + 6 + 2011 + 35659 + Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella + + + 600 + 618 + surname:Gale;given-names:E. F. + surname:Epps;given-names:H. M. + 16747564 + REF + Biochem. +J. + ref + 36 + 1942 + 35754 + The effect of the pH of the medium during growth on the enzymic activities of bacteria (Escherichia coli and Micrococcus lysodeikticus) and the biological significance of the changes produced + + + 232 + 242 + surname:Gale;given-names:E. F. + surname:Epps;given-names:H. M. + 16747785 + REF + Biochem. J. + ref + 38 + 1944 + 35946 + Studies on bacterial amino-acid decarboxylases: 1. l(+)-lysine decarboxylase + + + 836 + 849 + surname:Merrell;given-names:D. S. + surname:Camilli;given-names:A. + 10564522 + REF + Mol. Microbiol. + ref + 34 + 1999 + 36023 + The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance + + + 8586 + 8592 + surname:Kim;given-names:J.-S. + surname:Choi;given-names:S. H. + surname:Lee;given-names:J. +K. + 17012399 + REF + J. Bacteriol. + ref + 188 + 2006 + 36119 + Lysine decarboxylase expression by Vibrio vulnificus is induced by SoxR in response to superoxide stress + + + 928 + 933 + surname:Bower;given-names:J. M. + surname:Mulvey;given-names:M. A. + 16428396 + REF + J. Bacteriol. + ref + 188 + 2006 + 36224 + Polyamine-mediated resistance of uropathogenic Escherichia coli to nitrosative stress + + + 2104 + 2112 + surname:Bower;given-names:J. M. + surname:Gordon-Raagas;given-names:H. B. + surname:Mulvey;given-names:M. A. + 19255192 + REF + Infect. Immun. + ref + 77 + 2009 + 36310 + Conditioning of uropathogenic Escherichia coli for enhanced colonization of host + + + 575 + 579 + surname:Akhova;given-names:A. V. + surname:Tkachenko;given-names:A. G. + REF + Microbiology + ref + 78 + 2009 + 36391 + Lysine Decarboxylase Activity as a Factor of Fluoroquinolone Resistance in Escherichia coli + + + 1267 + 1270 + surname:Kikuchi;given-names:Y. + surname:Kurahashi;given-names:O. + surname:Nagano;given-names:T. + surname:Kamio;given-names:Y. + 9692215 + REF + Biosci. Biotechnol. Biochem. + ref + 62 + 1998 + 36483 + RpoS-dependent expression of the second lysine decarboxylase gene in Escherichia coli + + + 13 + 19 + surname:Samartzidou;given-names:H. + surname:Mehrazin;given-names:M. + surname:Xu;given-names:Z. + surname:Benedik;given-names:M. +J. + surname:Delcour;given-names:A. +H. + 12486035 + REF + J. Bacteriol. + ref + 185 + 2003 + 36569 + Cadaverine inhibition of porin plays a role in cell survival at acidic pH + + + 330 + 334 + surname:Bekhit;given-names:A. + surname:Fukamachi;given-names:T. + surname:Saito;given-names:H. + surname:Kobayashi;given-names:H. + 21372380 + REF + Biol. Pharm. Bull. + ref + 34 + 2011 + 36643 + The role of OmpC and OmpF in acidic resistance in Escherichia coli + + + 1042 + 1049 + surname:Tkachenko;given-names:A. G. + surname:Pozhidaeva;given-names:O. N. + surname:Shumkov;given-names:M. S. + REF + Biochem. +Biokhimiia + ref + 71 + 2006 + 36710 + Role of polyamines in formation of multiple antibiotic resistance of Escherichia coli under stress conditions + + + 931 + 944 + surname:Kanjee;given-names:U. + 21278708 + REF + EMBO J. + ref + 30 + 2011 + 36820 + Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase + + + 662 + 670 + surname:Sabo;given-names:D. L. + surname:Boeker;given-names:E. A. + surname:Byers;given-names:B. + surname:Waron;given-names:H. + surname:Fischer;given-names:E. H. + REF + Biochemistry (Mosc.) + ref + 13 + 1974 + 36939 + Purification and physical properties of inducible Escherichia coli lysine decarboxylase + + + 1532 + 1546 + surname:Snider;given-names:J. + 16301313 + REF + J. Biol. Chem. + ref + 281 + 2006 + 37027 + Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase + + + 211 + 221 + surname:Wong;given-names:K. S. + surname:Houry;given-names:W. A. + 22491058 + REF + J. Struct. Biol. + ref + 179 + 2012 + 37139 + Novel structural and functional insights into the MoxR family of AAA+ ATPases + + + 22499 + 22504 + surname:El Bakkouri;given-names:M. + 21148420 + REF + Proc. Natl. Acad. Sci. USA + ref + 107 + 2010 + 37217 + Structure of RavA MoxR AAA+ protein reveals the design principles of a molecular cage modulating the inducible lysine decarboxylase activity + + + e85529 + surname:Wong;given-names:K. S. + 24454883 + REF + PloS +One + ref + 9 + 2014 + 37358 + The MoxR ATPase RavA and its cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I in Escherichia coli + + + e03653 + surname:Malet;given-names:H. + 25097238 + REF + eLife + ref + 3 + 2014 + 37472 + Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins + + + 167 + 172 + surname:Yamamoto;given-names:Y. + surname:Miwa;given-names:Y. + surname:Miyoshi;given-names:K. + surname:Furuyama;given-names:J. + surname:Ohmori;given-names:H. + 9339543 + REF + Genes Genet. Syst. + ref + 72 + 1997 + 37560 + The Escherichia coli ldcC gene encodes another lysine decarboxylase, probably a constitutive enzyme + + + 857 + 876 + surname:Käck;given-names:H. + surname:Sandmark;given-names:J. + surname:Gibson;given-names:K. + surname:Schneider;given-names:G. + surname:Lindqvist;given-names:Y. + 10452893 + REF + J. Mol. Biol. + ref + 291 + 1999 + 37660 + Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal-5′-phosphate-dependent enzymes + + + 9388 + 9398 + surname:Kanjee;given-names:U. + surname:Gutsche;given-names:I. + surname:Ramachandran;given-names:S. + surname:Houry;given-names:W. +A. + REF + Biochemistry +(Mosc.) + ref + 50 + 2011 + 37792 + The enzymatic activities of the Escherichia coli basic aliphatic amino acid decarboxylases exhibit a pH zone of inhibition + + + 863 + 871 + surname:Erhardt;given-names:H. + 22063474 + REF + Biochim. Biophys. Acta + ref + 1817 + 2012 + 37915 + Disruption of individual nuo-genes leads to the formation of partially assembled NADH:ubiquinone oxidoreductase (complex I) in Escherichia coli + + + 1176 + 1184 + surname:Lohinai;given-names:Z. + 26110450 + REF + J. Periodontol. + ref + 86 + 2015 + 38059 + Biofilm Lysine Decarboxylase, a New Therapeutic Target for Periodontal Inflammation + + + 6706 + 6712 + surname:Peters;given-names:J. L. + 22975025 + REF + Vaccine + ref + 30 + 2012 + 38143 + Effects of immunization with natural and recombinant lysine decarboxylase on canine gingivitis development + + + 1048 + 1056 + surname:Lohinai;given-names:Z. + 22141361 + REF + J. Periodontol. + ref + 83 + 2012 + 38250 + Bacterial lysine decarboxylase influences human dental biofilm lysine content, biofilm accumulation, and subclinical gingival inflammation + + + 1108 + 1113 + surname:Kim;given-names:H. J. + 25674800 + REF + J. Microbiol. Biotechnol + ref + 25 + 2015 + 38389 + Optimization of Direct Lysine Decarboxylase Biotransformation for Cadaverine Production with Whole-Cell Biocatalysts at High Lysine Concentration + + + 799 + 806 + surname:Ma;given-names:W. + 25515797 + REF + Biotechnol. Lett. + ref + 37 + 2015 + 38535 + Enhanced cadaverine production from L-lysine using recombinant Escherichia coli co-overexpressing CadA and CadB + + + 965 + 972 + surname:Li;given-names:N. + surname:Chou;given-names:H. + surname:Yu;given-names:L. + surname:Xu;given-names:Y. + REF + Biotechnol. Bioprocess Eng. + ref + 19 + 2014 + 38647 + Cadaverine production by heterologous expression of Klebsiella oxytoca lysine decarboxylase + + + 129 + 228 + surname:Dubochet;given-names:J. + 3043536 + REF + Q. Rev. +Biophys. + ref + 21 + 1988 + 38739 + Cryo-electron microscopy of vitrified specimens + + + 334 + 347 + surname:Mindell;given-names:J. A. + surname:Grigorieff;given-names:N. + 12781660 + REF + J. Struct. Biol. + ref + 142 + 2003 + 38787 + Accurate determination of local defocus and specimen tilt in electron microscopy + + + 17 + 24 + surname:van Heel;given-names:M. + surname:Harauz;given-names:G. + surname:Orlova;given-names:E. +V. + surname:Schmidt;given-names:R. + surname:Schatz;given-names:M. + 8742718 + REF + J. +Struct. Biol. + ref + 116 + 1996 + 38868 + A new generation of the IMAGIC image processing system + + + 13 + 23 + surname:Navaza;given-names:J. + 14643206 + REF + J. Struct. Biol. + ref + 144 + 2003 + 38923 + On the three-dimensional reconstruction of icosahedral particles + + + 519 + 530 + surname:Scheres;given-names:S. H. W. + 23000701 + REF + J. Struct. Biol. + ref + 180 + 2012 + 38988 + RELION: implementation of a Bayesian approach to cryo-EM structure determination + + + 253 + 260 + surname:Estrozi;given-names:L. F. + surname:Navaza;given-names:J. + 20599509 + REF + J. Struct. Biol. + ref + 172 + 2010 + 39069 + Ab initio high-resolution single-particle 3D reconstructions: the symmetry adapted functions way + + + 853 + 854 + surname:Scheres;given-names:S. H. W. + surname:Chen;given-names:S. + 22842542 + REF + Nat. Methods + ref + 9 + 2012 + 39166 + Prevention of overfitting in cryo-EM structure determination + + + 170 + 175 + surname:Fernández;given-names:J. J. + surname:Luque;given-names:D. + surname:Castón;given-names:J. R. + surname:Carrascosa;given-names:J. L. + 18614378 + REF + J. Struct. Biol. + ref + 164 + 2008 + 39227 + Sharpening high resolution information in single particle electron cryomicroscopy + + + 63 + 65 + surname:Kucukelbir;given-names:A. + surname:Sigworth;given-names:F. J. + surname:Tagare;given-names:H. D. + 24213166 + REF + Nat. +Methods + ref + 11 + 2014 + 39309 + Quantifying the local resolution of cryo-EM density maps + + + 3381 + 3385 + surname:Schwede;given-names:T. + surname:Kopp;given-names:J. + surname:Guex;given-names:N. + surname:Peitsch;given-names:M. C. + 12824332 + REF + Nucleic Acids Res. + ref + 31 + 2003 + 39366 + SWISS-MODEL: An automated protein homology-modeling server + + + 1605 + 1612 + surname:Pettersen;given-names:E. F. + 15264254 + REF + J. Comput. Chem. + ref + 25 + 2004 + 39425 + UCSF Chimera–a visualization system for exploratory research and analysis + + + 261 + 270 + surname:Lopéz-Blanco;given-names:J. R. + surname:Chacón;given-names:P. + 23999189 + REF + J. Struct. Biol. + ref + 184 + 2013 + 39501 + iMODFIT: efficient and robust flexible fitting based on vibrational analysis in internal coordinates + + + 1820 + 1825 + surname:Navaza;given-names:J. + surname:Lepault;given-names:J. + surname:Rey;given-names:F. A. + surname:Alvarez-Rúa;given-names:C. + surname:Borge;given-names:J. + 12351826 + REF + Acta Crystallogr. D Biol. +Crystallogr + ref + 58 + 2002 + 39602 + On the fitting of model electron densities into EM reconstructions: a reciprocal-space formulation + + + 651 + 658 + surname:Siebert;given-names:X. + surname:Navaza;given-names:J. + 19564685 + REF + Acta Crystallogr. D Biol. +Crystallogr + ref + 65 + 2009 + 39701 + UROX 2.0: an interactive tool for fitting atomic models into electron-microscopy reconstructions + + + 1792 + 1797 + surname:Edgar;given-names:R. C. + 15034147 + REF + Nucleic Acids Res. + ref + 32 + 2004 + 39798 + MUSCLE: multiple sequence alignment with high accuracy and high throughput + + + 691 + 699 + surname:Whelan;given-names:S. + surname:Goldman;given-names:N. + 11319253 + REF + Mol. Biol. +Evol. + ref + 18 + 2001 + 39873 + A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach + + + SUPPL + footnote + 39995 + Author Contributions E.K., H.M. and I.G. carried out EM data collection with assistance of M.B. and analyzed the data. D.C. performed cloning, multiple sequence alignment and phylogenetic analysis under the direction of S.E. and I.G., J.P. cloned and purified chimeric proteins under the direction of S.O.C., K.L. and S.W.S.C. purified LdcI, LdcC and LARA under the direction of W.A.H., I.G. conceived and directed the studies and wrote the manuscript with input from E.K. + + + srep24601-f1.jpg + f1 + FIG + fig_title_caption + 40468 + 3D cryoEM reconstructions of LdcC, LdcI-LARA and LdcIa. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:12:13Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:12:22Z + + reconstructions + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:06Z + + LdcC + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:46Z + + LdcI-LARA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + + srep24601-f1.jpg + f1 + FIG + fig_caption + 40524 + (A,C,E) cryoEM map of the LdcC (A), LdcIa +(C) and LdcI-LARA (E) decamers with one protomer in light +grey. In the rest of the protomers, the wing, core and C-terminal domains +are colored from light to dark in shades of green for LdcC (A), pink +for LdcIa (C) and blue for LdcI in LdcI-LARA (E). +In (E), the LARA domain density is shown in dark grey. Two monomers +making a dimer are delineated. Scale bar 50 Å. +(B,D,F) One protomer from the cryoEM map of the LdcC (B), +LdcIa (D) and LdcI-LARA (F) in light grey with +the pseudoatomic model represented as cartoons and colored as the densities +in (A,C,E). Each domain is indicated for clarity. Scale bar +50 Å. See also Figs S1 and S3. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:12:39Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:12:48Z + + map + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:07Z + + LdcC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:46Z + + LdcI-LARA + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:53:28Z + + decamers + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:18:25Z + + protomer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:18:23Z + + protomers + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:35:46Z + + wing + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:35:49Z + + core + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:36:19Z + + C-terminal domains + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:07Z + + LdcC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:35Z + + LdcI + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:46Z + + LdcI-LARA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T08:55:55Z + + LARA domain + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:16:06Z + + monomers + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:33:36Z + + dimer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:18:28Z + + protomer + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:13:01Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:13:10Z + + map + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:07Z + + LdcC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:46Z + + LdcI-LARA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:33:55Z + + pseudoatomic model + + + + srep24601-f2.jpg + f2 + FIG + fig_title_caption + 41210 + Analysis of conformational rearrangements. + + + srep24601-f2.jpg + f2 + FIG + fig_caption + 41253 + Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and +LdcIa colored as in Fig. 1, and the +crystal structure of LdcIi in shades of yellow. Only one of the +two rings of the double toroid is shown for clarity. The dashed circle +indicates the central region that remains virtually unchanged between all +the structures, while the periphery undergoes visible movements. Scale bar +50 Å. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:24:59Z + + Superposition + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:37:14Z + + pseudoatomic models + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:07Z + + LdcC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:35Z + + LdcI + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:46Z + + LdcI-LARA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:14:46Z + + crystal structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:15:25Z + + rings + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:19:32Z + + double toroid + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:35:54Z + + region + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:19:38Z + + structures + + + + srep24601-f3.jpg + f3 + FIG + fig_title_caption + 41656 + Conformational rearrangements in the enzyme active site. + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:19:54Z + + active site + + + + srep24601-f3.jpg + f3 + FIG + fig_caption + 41713 + (A) LdcIi crystal structure, with one ring represented as a +grey surface and the second as a cartoon. A monomer with its PLP cofactor is +delineated. The PLP moieties of the cartoon ring are shown in red. +(B) The LdcIi dimer extracted from the crystal structure +of the decamer. One monomer is colored in shades of yellow as in Figs 1 and 2, while the monomer +related by C2 symmetry is grey. The PLP is red. The active site is boxed. +(C–F) Close-up views of the active site. The PLP +moiety in red is from the LdcIi crystal structure. We did not +attempt to model it in the cryoEM maps. The dimer interface is shown as a +dashed line and the active site α-helices mentioned in the text +are highlighted. (C) Compares LdcIi (yellow) and +LdcIa (pink), (D) compares LdcIa (pink) and +LdcI-LARA (blue), and (E) compares LdcIi (yellow), +LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to +show the progressive shift described in the text. (F) Shows the +similarity between LdcIa and LdcC at the level of the secondary +structure elements composing the active site. Colors are as in the other +figures. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:14:46Z + + crystal structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:22:32Z + + ring + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:13Z + + monomer + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:16Z + + PLP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:16Z + + PLP + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:22:38Z + + ring + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:22:42Z + + dimer + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:14:46Z + + crystal structure + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:20Z + + decamer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:13Z + + monomer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:13Z + + monomer + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:16Z + + PLP + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:22:52Z + + active site + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:22:55Z + + active site + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-20T09:02:16Z + + PLP + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:14:46Z + + crystal structure + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:13:28Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:13:45Z + + maps + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:22:57Z + + dimer interface + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:23:03Z + + active site α-helices + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:34Z + + LdcIi + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:46Z + + LdcI-LARA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:35Z + + LdcIi + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:46Z + + LdcI-LARA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:07Z + + LdcIa + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:07Z + + LdcC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:23:09Z + + secondary +structure elements + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:23:12Z + + active site + + + + srep24601-f4.jpg + f4 + FIG + fig_title_caption + 42813 + Stretching of the LdcI monomer upon pH-dependent enzyme activation and LARA +binding. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:35Z + + LdcI + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:55:13Z + + monomer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T10:40:43Z + + pH-dependent + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:23:46Z + + LARA + + + + srep24601-f4.jpg + f4 + FIG + fig_caption + 42898 + (A–C) A slice through the pseudoatomic models of the LdcI +monomers extracted from the superimposed decamers (Fig. +2) The rectangle indicates the regions enlarged in +(D–F). (A) compares LdcIi (yellow) +and LdcIa (pink), (B) compares LdcIa (pink) and +LdcI-LARA (blue), and (C) compares LdcIi (yellow), +LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to +show the progressive stretching described in the text. The cryoEM density of +the LARA domain is represented as a grey surface to show the position of the +binding site and the direction of the movement. (D–F) +Inserts zooming at the CTD part in proximity of the LARA binding site. Loop +regions are removed for a clearer visual comparison. An arrow indicates a +swinging movement. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:37:14Z + + pseudoatomic models + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:35Z + + LdcI + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:16:06Z + + monomers + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:24:41Z + + superimposed + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:53:28Z + + decamers + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:35Z + + LdcIi + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:08Z + + LdcIa + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:08Z + + LdcIa + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:46Z + + LdcI-LARA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:29:35Z + + LdcIi + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:08Z + + LdcIa + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:21:46Z + + LdcI-LARA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:14:01Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:14:10Z + + density + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T08:55:55Z + + LARA domain + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:25:09Z + + binding site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:15:55Z + + CTD + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:25:11Z + + LARA binding site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:25:13Z + + Loop +regions + + + + srep24601-f5.jpg + f5 + FIG + fig_title_caption + 43641 + Analysis of the LdcIC and LdcCI chimeras. + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:56:53Z + + LdcIC + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:57:02Z + + LdcCI + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:25:32Z + + chimeras + + + + srep24601-f5.jpg + f5 + FIG + fig_caption + 43683 + (A) A slice through the pseudoatomic models of the LdcIa +(purple) and LdcC (green) monomers extracted from the superimposed decamers +(Fig. 2). (B) The C-terminal +β-sheet in LdcIa and LdcC enlarged from +(A,C) Exchanged primary sequences (capital letters) and +their immediate vicinity (lower case letters) colored as in +(A,B), with the corresponding secondary structure elements +and the amino acid numbering shown. (D,E) A gallery of negative stain +EM images of (D) the wild type LdcI-RavA cage and (E) the +LdcCI-RavA cage-like particles. (F) Some representative class +averages of the LdcCI-RavA cage-like particles. Scale bar +20 nm. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:37:14Z + + pseudoatomic models + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:08Z + + LdcIa + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:07Z + + LdcC + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T09:16:06Z + + monomers + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-20T10:24:41Z + + superimposed + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:53:28Z + + decamers + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:53Z + + β-sheet + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T09:34:08Z + + LdcIa + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:07Z + + LdcC + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:26:54Z + + negative stain +EM images + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:31:29Z + + wild type + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-20T09:20:57Z + + LdcI-RavA + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:26:48Z + + LdcCI-RavA cage-like particles + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-20T11:26:51Z + + LdcCI-RavA cage-like particles + + + + srep24601-f6.jpg + f6 + FIG + fig_title_caption + 44318 + Sequence analysis of enterobacterial lysine decarboxylases. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-21T18:23:01Z + + Sequence analysis + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T08:52:44Z + + enterobacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T23:00:52Z + + lysine decarboxylases + + + + srep24601-f6.jpg + f6 + FIG + fig_caption + 44378 + (A) Maximum likelihood tree with the +“LdcC-like” and the +“LdcI-like” groups highlighted in green and pink, +respectively. Only nodes with higher values than 95% are shown (500 +replicates of the original dataset, see Methods for details). Scale bar +indicates the average number of substitutions per site. (B) Analysis +of consensus “LdcI-like” and +“LdcC-like” sequences around the first and second +C-terminal β-strands. The height of the bars and the letters +representing the amino acids reflects the degree of conservation of each +particular position is in the alignment. Amino acids are colored according +to a polarity color scheme with hydrophobic residues in black, hydrophilic +in green, acidic in red and basic in blue. Numbering as in E. coli. +(C) Signature sequences of LdcI and LdcC in the C-terminal +β-sheet. Polarity differences are highlighted. (D) +Position and nature of these differences at the surface of the respective +cryoEM maps with the color code as in B. See also Fig. S7 and Tables S3 and S4. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-20T11:28:40Z + + Maximum likelihood tree + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:04Z + + LdcC-like + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:10Z + + LdcI-like + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:10Z + + LdcI-like + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-20T09:33:05Z + + LdcC-like + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T11:35:59Z + + β-strands + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-20T08:55:31Z + + E. coli + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:52:35Z + + LdcI + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-20T08:53:07Z + + LdcC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-20T09:22:53Z + + β-sheet + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-06-15T14:14:36Z + + cryoEM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:14:44Z + + maps + + + + diff --git a/BioC_XML/4833862_v0.xml b/BioC_XML/4833862_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..5da500ea503021d2101691bb968b0de863f88f8c --- /dev/null +++ b/BioC_XML/4833862_v0.xml @@ -0,0 +1,9854 @@ + + + + PMC + 20140719 + pmc.key + + 4833862 + CC BY + no + 0 + 0 + + 10.1038/ncomms11196 + ncomms11196 + 4833862 + 27073141 + 11196 + This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ + surname:Hunkeler;given-names:Moritz + surname:Stuttfeld;given-names:Edward + surname:Hagmann;given-names:Anna + surname:Imseng;given-names:Stefan + surname:Maier;given-names:Timm + TITLE + front + 7 + 2016 + 0 + The dynamic organization of fungal acetyl-CoA carboxylase + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:55:28Z + + dynamic + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:48:20Z + + acetyl-CoA carboxylase + + + + ABSTRACT + abstract + 58 + Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:58:07Z + + Acetyl-CoA carboxylases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:22:38Z + + ATP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:51:14Z + + acetyl-CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:51:34Z + + malonyl-CoA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:05:54Z + + Eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:51Z + + single-chain multienzymes + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:51:54Z + + non-catalytic + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:51:56Z + + central domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:03Z + + CD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:44Z + + ACC + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:14Z + + crystal structure + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:22Z + + yeast + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:03Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:48Z + + regulatory loop + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:53:27Z + + phosphorylation site + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:03Z + + CD + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:22Z + + yeast + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:03Z + + CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:55:04Z + + structure + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:10Z + + larger fragments + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:55:17Z + + intact + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:55:28Z + + dynamic + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:56:12Z + + carboxylases + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:03Z + + CD + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + + ABSTRACT + abstract + 1127 + Acetyl-CoA carboxylases are central regulatory hubs of fatty acid metabolism and are important targets for drug development in obesity and cancer. Here, the authors demonstrate that the regulation of these highly dynamic enzymes in fungi is governed by a mechanism based on phosphorylation-dependent conformational variability. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:58:07Z + + Acetyl-CoA carboxylases + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:58:13Z + + highly dynamic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:58:16Z + + enzymes + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:58:19Z + + fungi + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + + INTRO + paragraph + 1456 + Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase. By catalysing this rate-limiting step in fatty-acid biosynthesis, ACC plays a key role in anabolic metabolism. ACC inhibition and knock-out studies show the potential of targeting ACC for treatment of the metabolic syndrome. Furthermore, elevated ACC activity is observed in malignant tumours. A direct link between ACC and cancer is provided by cancer-associated mutations in the breast cancer susceptibility gene 1 (BRCA1), which relieve inhibitory interactions of BRCA1 with ACC. Thus, ACC is a relevant drug target for type 2 diabetes and cancer. Microbial ACCs are also the principal target of antifungal and antibiotic compounds, such as Soraphen A. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:52:09Z + + Biotin-dependent acetyl-CoA carboxylases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:22:53Z + + ATP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:51:14Z + + acetyl-CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:51:34Z + + malonyl-CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:23:23Z + + fatty acids + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:00:40Z + + fatty-acid synthase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:00:49Z + + ACC inhibition and knock-out studies + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T10:23:04Z + + ACC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:01:06Z + + mutations + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:01:02Z + + breast cancer susceptibility gene 1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:01:12Z + + BRCA1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:01:12Z + + BRCA1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:01:23Z + + Microbial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T17:01:17Z + + Soraphen A + + + + INTRO + paragraph + 2376 + The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. Eukaryotic ACCs, instead, are multienzymes, which integrate all functional components into a single polypeptide chain of ∼2,300 amino acids. Human ACC occurs in two closely related isoforms, ACC1 and 2, located in the cytosol and at the outer mitochondrial membrane, respectively. In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). The CD comprises one-third of the protein and is a unique feature of eukaryotic ACCs without homologues in other proteins. The function of this domain remains poorly characterized, although phosphorylation of several serine residues in the CD regulates ACC activity. The BT domain has been visualized in bacterial carboxylases, where it mediates contacts between α- and β-subunits. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:04:36Z + + Escherichia coli + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:04:43Z + + E. coli + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:04:49Z + + Biotin carboxylase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:04:52Z + + BC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:23:40Z + + ATP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T17:04:55Z + + biotin + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:05:00Z + + biotin carboxyl carrier protein + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:05:03Z + + BCCP + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:05:06Z + + Carboxyltransferase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:05:09Z + + CT + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T17:05:16Z + + carboxyl + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T17:05:19Z + + carboxybiotin + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:51:14Z + + acetyl-CoA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:51:34Z + + malonyl-CoA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:05:32Z + + Prokaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:13:06Z + + transient + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:05:43Z + + BC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:05:46Z + + CT + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:05:49Z + + BCCP + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:05:54Z + + Eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:05:59Z + + multienzymes + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + Human + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:06:46Z + + isoforms + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:18:52Z + + ACC1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:06:52Z + + 2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:07:03Z + + ACC components + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:05:54Z + + eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:07:12Z + + non-catalytic + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:07:16Z + + regions + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:07:20Z + + central domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:03Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:07:30Z + + BC–CT interaction domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:07:37Z + + BT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:03Z + + CD + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:07:43Z + + unique feature of + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:05:54Z + + eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:08:22Z + + serine + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:03Z + + CD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T10:23:56Z + + ACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:07:37Z + + BT + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:08:41Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:56:12Z + + carboxylases + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:08:31Z + + α- + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:08:44Z + + β-subunits + + + + INTRO + paragraph + 3706 + Structural studies on the functional architecture of intact ACCs have been hindered by their huge size and pronounced dynamics, as well as the transient assembly mode of bacterial ACCs. However, crystal structures of individual components or domains from prokaryotic and eukaryotic ACCs, respectively, have been solved. The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. In these structures, the BC and CT active sites are at distances between 40 and 80 Å, such that substrate transfer could be mediated solely by the mobility of the flexibly tethered BCCP. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:12:57Z + + Structural studies + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:55:17Z + + intact + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:13:06Z + + transient + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:08:41Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:54:18Z + + crystal structures + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:05:32Z + + prokaryotic + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:05:54Z + + eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:13:21Z + + structure determination + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:13:24Z + + holoenzymes + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:08:41Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:13:28Z + + biotin-dependent carboxylases + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:13:58Z + + lack + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:03Z + + CD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:13:47Z + + pyruvate carboxylase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:14:02Z + + PC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:14:05Z + + propionyl-CoA carboxylase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:14:08Z + + 3-methyl-crotonyl-CoA carboxylase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:14:14Z + + long-chain acyl-CoA carboxylase + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T14:33:06Z + + structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:14:21Z + + BC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:14:24Z + + CT + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:14:31Z + + active sites + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:14:37Z + + flexibly tethered + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:14:42Z + + BCCP + + + + INTRO + paragraph + 4584 + Human ACC1 is regulated allosterically, via specific protein–protein interactions, and by reversible phosphorylation. Dynamic polymerization of human ACC1 is linked to increased activity and is regulated allosterically by the activator citrate and the inhibitor palmitate, or by binding of the small protein MIG-12 (ref.). Human ACC1 is further regulated by specific phosphorylation-dependent binding of BRCA1 to Ser1263 in the CD. BRCA1 binds only to the phosphorylated form of ACC1 and prevents ACC activation by phosphatase-mediated dephosphorylation. Furthermore, phosphorylation by AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA) leads to a decrease in ACC1 activity. AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201. However, regulatory effects on ACC1 activity are mainly mediated by phosphorylation of Ser80 and Ser1201 (refs). Phosphorylated Ser80, which is highly conserved only in higher eukaryotes, presumably binds into the Soraphen A-binding pocket. The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes. However, no effect of Ser1216 phosphorylation on ACC activity has been reported in higher eukaryotes. + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + Human + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:18:52Z + + ACC1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:18:39Z + + regulated allosterically + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + human + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:18:52Z + + ACC1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:18:58Z + + regulated allosterically + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T17:19:11Z + + citrate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T17:19:17Z + + palmitate + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:19:24Z + + MIG-12 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + Human + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:18:52Z + + ACC1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:01:12Z + + BRCA1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:19:35Z + + Ser1263 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:03Z + + CD + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:01:12Z + + BRCA1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:18:52Z + + ACC1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T10:24:12Z + + ACC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T10:24:39Z + + phosphatase + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:19:47Z + + AMP-activated protein kinase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:19:53Z + + AMPK + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:19:56Z + + cAMP-dependent protein kinase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:20:03Z + + PKA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:18:52Z + + ACC1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:19:53Z + + AMPK + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:18:52Z + + ACC1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:13Z + + Ser80 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:20Z + + Ser1201 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:28Z + + Ser1216 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:20:03Z + + PKA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:35Z + + Ser78 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:20Z + + Ser1201 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:18:52Z + + ACC1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:13Z + + Ser80 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:20Z + + Ser1201 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + Phosphorylated + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:13Z + + Ser80 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:22:23Z + + highly conserved + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:22:30Z + + higher eukaryotes + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:22:35Z + + Soraphen A-binding pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:20Z + + Ser1201 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:24:21Z + + moderate conservation + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:22:30Z + + higher eukaryotes + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:28Z + + Ser1216 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:22:23Z + + highly conserved + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:24:48Z + + eukaryotes + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:28Z + + Ser1216 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T10:24:22Z + + ACC + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:22:30Z + + higher eukaryotes + + + + INTRO + paragraph + 5882 + For fungal ACC, neither spontaneous nor inducible polymerization has been detected despite considerable sequence conservation to human ACC1. The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein–protein interactions of fungal ACC have been described. In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.). Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1. Its phosphorylation by the AMPK homologue SNF1 results in strongly reduced ACC activity. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + human + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:18:52Z + + ACC1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-06-15T09:02:41Z + + BRCA1 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:44:14Z + + phosphoserine + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:14Z + + not conserved + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:22Z + + yeast + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:28:30Z + + phosphorylation sites + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:39Z + + Ser2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:45Z + + Ser735 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:52Z + + Ser1148 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:58Z + + Ser1157 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:29:07Z + + Ser1162 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:58Z + + Ser1157 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:22:23Z + + highly conserved + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:30:29Z + + aligns to + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:28Z + + Ser1216 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + human + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:18:52Z + + ACC1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:25:15Z + + AMPK + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T22:55:30Z + + SNF1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T10:25:26Z + + ACC + + + + INTRO + paragraph + 6491 + Despite the outstanding relevance of ACC in primary metabolism and disease, the dynamic organization and regulation of the giant eukaryotic, and in particular fungal ACC, remain poorly characterized. Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). Integrating these data with small-angle X-ray scattering (SAXS) and electron microscopy (EM) observations yield a comprehensive representation of the dynamic structure and regulation of fungal ACC. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:05:54Z + + eukaryotic + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:33:50Z + + structure + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:05Z + + Saccharomyces cerevisiae + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:33:58Z + + Sce + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:03Z + + CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:34:09Z + + structures + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + human + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:15Z + + Hsa + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-07-25T09:33:01Z + + larger fragments + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:25Z + + Chaetomium thermophilum + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:32Z + + Cth + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:39Z + + small-angle X-ray scattering + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:45Z + + SAXS + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:52Z + + electron microscopy + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:58Z + + EM + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + + RESULTS + title_1 + 7107 + Results + + + RESULTS + title_2 + 7115 + The organization of the yeast ACC CD + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:22Z + + yeast + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + + RESULTS + paragraph + 7152 + First, we focused on structure determination of the 82-kDa CD. The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). The overall extent of the SceCD is 70 by 75 Å (Fig. 1b and Supplementary Fig. 1a,b), and the attachment points of the N-terminal 26-residue linker to the BCCP domain and the C-terminal CT domain are separated by 46 Å (the N- and C termini are indicated with spheres in Fig. 1b). SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). CDL is composed of a small, irregular four-helix bundle (Lα1–4) and tightly interacts with the open face of CDC1 via an interface of 1,300 Å2 involving helices Lα3 and Lα4. CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between Lα2/α3 and the N-terminal end of Lα1, with an interface area of 400 Å2. CDC1/CDC2 share a common fold; they are composed of six-stranded β-sheets flanked on one side by two long, bent helices inserted between strands β3/β4 and β4/β5. CDC2 is extended at its C terminus by an additional β-strand and an irregular β-hairpin. On the basis of a root mean square deviation of main chain atom positions of 2.2 Å, CDC1/CDC2 are structurally more closely related to each other than to any other protein (Fig. 1c); they may thus have evolved by duplication. Close structural homologues could not be found for the CDN or the CDC domains. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:39:40Z + + structure determination + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:14Z + + crystal structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:39:48Z + + SceACC + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:16:24Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:16:33Z + + CD + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:40:02Z + + experimental phasing + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:40:05Z + + refined + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:40:23Z + + Rwork + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:40:34Z + + Rfree + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:15:20Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:15:46Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:40:45Z + + 26-residue linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:41:57Z + + BCCP + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:52:19Z + + CT + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:14:36Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:14:51Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:20Z + + α-helical domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:46Z + + four-helix bundle linker domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:01Z + + α–β-fold C-terminal domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:17Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:43:29Z + + C shape + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:35Z + + regular four-helix bundle + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:39Z + + Nα3-6 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:42Z + + helical hairpin + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:45Z + + Nα8,9 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:44:05Z + + bridging region + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:44:30Z + + helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:44:34Z + + Nα1,2,7,10–12 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:44:39Z + + small, irregular four-helix bundle + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:44:42Z + + Lα1–4 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:45:12Z + + interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:45:16Z + + helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:45:19Z + + Lα3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:45:22Z + + Lα4 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:17Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:45:28Z + + loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:45:30Z + + Lα2/α3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:45:40Z + + Lα1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:45:43Z + + interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:17Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:45:49Z + + six-stranded β-sheets + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:46:28Z + + long, bent helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:46:32Z + + strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:46:36Z + + β3/β4 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:46:39Z + + β4/β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:17Z + + CDC2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:46:42Z + + extended + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:46:59Z + + β-strand + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:47:02Z + + irregular β-hairpin + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:47:04Z + + root mean square deviation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:17Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:52:23Z + + CDC + + + + RESULTS + title_2 + 9025 + A regulatory loop mediates interdomain interactions + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:48Z + + regulatory loop + + + + RESULTS + paragraph + 9077 + To define the functional state of insect-cell-expressed ACC variants, we employed mass spectrometry (MS) for phosphorylation site detection. In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae. Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast. MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals. The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:06:42Z + + insect-cell-expressed + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:06:45Z + + mass spectrometry + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:06:50Z + + MS + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:06:56Z + + phosphorylation site detection + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:07:01Z + + insect-cell-expressed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:07:07Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:39:48Z + + SceACC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:22:23Z + + highly conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:58Z + + Ser1157 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:07:40Z + + fully occupied + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T19:07:22Z + + phosphorylation site + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:07:49Z + + S. cerevisiae + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:08:02Z + + Ser2101 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:08:08Z + + Tyr2179 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:42:02Z + + neither conserved + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:51:50Z + + nor natively phosphorylated + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:22Z + + yeast + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:06:50Z + + MS + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:09:56Z + + dissolved crystals + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:58Z + + Ser1157 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:17:12Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:17:25Z + + CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:54:25Z + + crystals + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:17:46Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:17:58Z + + CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:54:27Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:39:48Z + + SceACC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T22:55:36Z + + enzyme + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:10:50Z + + inhibited + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:10:35Z + + SNF1-dependent phosphorylation + + + + RESULTS + paragraph + 9788 + In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. This regulatory loop wedges between the CDC1 and CDC2 domains and provides the largest contribution to the interdomain interface. The N-terminal region of the regulatory loop also directly contacts the C-terminal region of CDC2 leading into CT. Phosphoserine 1157 is tightly bound by two highly conserved arginines (Arg1173 and Arg1260) of CDC1 (Fig. 1d). Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. Phosphorylation of the regulatory loop thus determines interdomain interactions of CDC1 and CDC2, suggesting that it may exert its regulatory function by modifying the overall structure and dynamics of the CD. + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:19:21Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:19:33Z + + CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:14Z + + crystal structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:58Z + + Ser1157 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T19:13:59Z + + regulatory 36-amino-acid loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T19:14:02Z + + strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T19:14:05Z + + β2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T19:14:07Z + + β3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:14:13Z + + less-conserved + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:28:30Z + + phosphorylation sites + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:52Z + + Ser1148 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:29:07Z + + Ser1162 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:22Z + + yeast + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:48Z + + regulatory loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:17Z + + CDC2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T19:14:31Z + + interdomain interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:48Z + + regulatory loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:17Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:52:29Z + + CT + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:56:31Z + + Phosphoserine 1157 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:22:23Z + + highly conserved + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T19:15:15Z + + arginines + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:15:21Z + + Arg1173 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:15:27Z + + Arg1260 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:58Z + + Ser1157 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:48Z + + regulatory loop + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:28:30Z + + phosphorylation sites + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:52Z + + Ser1148 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:29:07Z + + Ser1162 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T19:16:03Z + + same loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:48Z + + regulatory loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:17Z + + CDC2 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + Phosphorylation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:48Z + + regulatory loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:17Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + + RESULTS + paragraph + 10924 + The functional role of Ser1157 was confirmed by an activity assay based on the incorporation of radioactive carbonate into acid non-volatile material. Phosphorylated SceACC shows only residual activity (kcat=0.4±0.2 s−1, s.d. based on five replicate measurements), which increases 16-fold (kcat=6.5±0.3 s−1) after dephosphorylation with λ protein phosphatase. The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli. + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:58Z + + Ser1157 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:17:19Z + + activity assay + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + Phosphorylated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:39:48Z + + SceACC + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:17:37Z + + kcat + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:17:39Z + + kcat + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:18:38Z + + λ protein phosphatase + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:18:44Z + + dephosphorylated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:39:48Z + + SceACC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:18:51Z + + non-phosphorylated + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:22Z + + yeast + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:18:55Z + + expressed in + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:04:43Z + + E. coli + + + + RESULTS + title_2 + 11436 + The variable CD is conserved between yeast and human + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:30:43Z + + conserved + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:22Z + + yeast + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + human + + + + RESULTS + paragraph + 11489 + To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). An experimentally phased map was obtained at 3.7 Å resolution for a cadmium-derivatized crystal and was interpreted by a poly-alanine model (Fig. 1e and Table 1). Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures. In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). As a result, the N terminus of CDL at helix Lα1, which connects to CDN, is shifted by 12 Å. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD. With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160° around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d). This rotation displaces the N terminus of CDN in HsaBT-CD by 51 Å compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67 Å (the attachment points are indicated with spheres in Fig. 1e). The BT domain of HsaBT-CD consists of a helix that is surrounded at its N terminus by an antiparallel eight-stranded β-barrel. It resembles the BT of propionyl-CoA carboxylase; only the four C-terminal strands of the β-barrel are slightly tilted. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + human + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:09Z + + determined the structure + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + human + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:12Z + + ACC1 fragment + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:07:37Z + + BT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:20Z + + HsaBT-CD + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:36:43Z + + lacks + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:37:59Z + + BCCP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:36:48Z + + experimentally phased map + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:26:06Z + + cadmium + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:20Z + + HsaBT-CD + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:12:38Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:12:47Z + + CD + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:22Z + + yeast + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:52:34Z + + CDs + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:37:08Z + + structures + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:23:04Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:23:14Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:20Z + + HsaBT-CD + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + human + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:37:11Z + + superposition + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:22Z + + yeast + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:52:41Z + + helix + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:52:45Z + + Lα1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:21Z + + HsaBT-CD + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:13:08Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:13:21Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:37:14Z + + superposed + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:21Z + + HsaBT-CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:52:49Z + + hinge + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:13:31Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:13:40Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:21Z + + HsaBT-CD + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:11:43Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:11:56Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:52:52Z + + linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:41:11Z + + BCCP domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:38:21Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:07:37Z + + BT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:21Z + + HsaBT-CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:52:57Z + + helix + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:37:44Z + + antiparallel eight-stranded β-barrel + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:07:37Z + + BT + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:52:14Z + + propionyl-CoA carboxylase + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:37:52Z + + strands of the β-barrel + + + + RESULTS + paragraph + 13128 + On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%). Ser29 and Ser1263, implicated in insulin-dependent phosphorylation and BRCA1 binding, respectively, are phosphorylated at intermediate levels (40%). The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated. However, residual phosphorylation levels were detected for Ser1204 (7%) and Ser1218 (7%) in the same loop. MS analysis of the HsaBT-CD crystallization sample reveals partial proteolytic digestion of the regulatory loop. Accordingly, most of this loop is not represented in the HsaBT-CD crystal structure. The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains. Besides the regulatory loop, also the phosphopeptide target region for BRCA1 interaction is not resolved presumably because of pronounced flexibility. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:06:50Z + + MS + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:41:25Z + + insect-cell-expressed + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:06Z + + human + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:07:07Z + + full-length + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:13Z + + Ser80 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:56:37Z + + Ser29 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:19:35Z + + Ser1263 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:44:38Z + + insulin-dependent phosphorylation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:26:23Z + + BRCA1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:22:23Z + + highly conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:28Z + + Ser1216 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:07:49Z + + S. cerevisiae + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:58Z + + Ser1157 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:20Z + + Ser1201 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:44:58Z + + not phosphorylated + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:45:13Z + + Ser1204 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:45:23Z + + Ser1218 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:44:52Z + + same loop + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:06:50Z + + MS + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:21Z + + HsaBT-CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:54:32Z + + crystallization sample + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:45:34Z + + this loop + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:21Z + + HsaBT-CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:14Z + + crystal structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:56:48Z + + absence of + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:45:49Z + + less-restrained + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:45:54Z + + interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:10:23Z + + domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:46:18Z + + phosphopeptide target region + + + protein + PR: + melaniev@ebi.ac.uk + 2023-06-15T09:03:56Z + + BRCA1 + + + + RESULTS + paragraph + 14183 + At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:47:47Z + + isolated + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:22Z + + yeast + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:07Z + + human + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:47:50Z + + structural analysis + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:53:11Z + + hinges + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:01:03Z + + CDN/CDL connection + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + + RESULTS + title_2 + 14487 + The integration of CD into the fungal ACC multienzyme + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:49:03Z + + ACC multienzyme + + + + RESULTS + paragraph + 14541 + To further obtain insights into the functional architecture of fungal ACC, we characterized larger multidomain fragments up to the intact enzymes. Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). No crystals diffracting to sufficient resolution were obtained for larger BC-containing fragments, or for full-length Cth or SceACC. To improve crystallizability, we generated ΔBCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a–c). For CthΔBCCP, crystals diffracting to 8.4 Å resolution were obtained. However, molecular replacement did not reveal a unique positioning of the BC domain. Owing to the limited resolution the discussion of structures of CthCD-CT and CthΔBCCP is restricted to the analysis of domain localization. Still, these structures contribute considerably to the visualization of an intrinsically dynamic fungal ACC. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:52:24Z + + larger multidomain fragments + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:55:17Z + + intact + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T22:52:31Z + + enzymes + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:52:34Z + + molecular replacement + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:45Z + + ACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:52:38Z + + CT + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:52:42Z + + structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:52:45Z + + variant + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:52:51Z + + Cth + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:52:55Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:52:59Z + + two crystal forms + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:32Z + + CthCD-CTCter1/2 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:53:06Z + + Cth + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:53:09Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:24Z + + CthCD-CT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:53:33Z + + larger BC-containing fragments + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:07:07Z + + full-length + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:32Z + + Cth + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:39:48Z + + SceACC + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:53:49Z + + improve crystallizability + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:53:52Z + + generated + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:53:54Z + + ΔBCCP variants + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:07:07Z + + full-length + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:00Z + + SAXS analysis + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:55:17Z + + intact + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:09Z + + CthΔBCCP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:54:14Z + + crystals + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:17Z + + molecular replacement + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:53:18Z + + BC + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:54:39Z + + structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:24Z + + CthCD-CT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:09Z + + CthΔBCCP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:54:46Z + + these structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:55:28Z + + dynamic + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + + RESULTS + paragraph + 15756 + In all these crystal structures, the CT domains build a canonical head-to-tail dimer, with active sites formed by contributions from both protomers (Fig. 2 and Supplementary Fig. 3a). The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular β-hairpin/β-strand extension of CDC2 (Supplementary Fig. 3b). The connecting region is remarkably similar in isolated CD and CthCD-CTCter structures, indicating inherent conformational stability. CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. The neighbouring loop on the CT side (between CT β1/β2) is displaced by 2.5 Å compared to isolated CT structures (Supplementary Fig. 3c). On the basis of an interface area of ∼600 Å2 and its edge-to-edge connection characteristics, the interface between CT and CD might be classified as conformationally variable. Indeed, the comparison of the positioning of eight instances of the C-terminal part of CD relative to CT in crystal structures determined here, reveals flexible interdomain linking (Fig. 3a). The CDC2/CT interface acts as a true hinge with observed rotation up to 16°, which results in a translocation of the distal end of CDC2 by 8 Å. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:57:28Z + + crystal structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:57:31Z + + CT + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:57:34Z + + head-to-tail + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:57:36Z + + dimer + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:14:31Z + + active sites + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:57:46Z + + protomers + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:57:50Z + + connection + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:57:53Z + + CT + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:57:57Z + + 10-residue peptide stretch + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:58:01Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:58:05Z + + irregular β-hairpin/β-strand extension + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:58:09Z + + connecting region + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:58:13Z + + isolated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:58:20Z + + CthCD-CTCter + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:58:27Z + + structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:04:33Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:04:42Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:58:41Z + + β-hairpin extension + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:58:45Z + + loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:58:48Z + + strands β2/β3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:58:55Z + + CT N-lobe + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:30:43Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:59:03Z + + RxxGxN motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:53:26Z + + loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-22T10:26:44Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:59:54Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:53:31Z + + β1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:53:34Z + + β2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:59:45Z + + isolated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:59:51Z + + CT + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:59:48Z + + structures + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:00:01Z + + interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T22:59:58Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:00:14Z + + CT + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:00:17Z + + crystal structures + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T23:00:19Z + + determined + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:00:22Z + + CDC2/CT interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:00:24Z + + true hinge + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + + RESULTS + paragraph + 17149 + The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution. However, MS analysis of CthCD-CT and CthΔBCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157). + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:01:42Z + + interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:10Z + + CDC1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:20:32Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:20:45Z + + CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:01:49Z + + structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:01:52Z + + CD–CT junction + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:01:56Z + + interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T23:02:04Z + + CthACC variant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:02:07Z + + structures + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:06:51Z + + MS + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:24Z + + CthCD-CT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:09Z + + CthΔBCCP + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:02:17Z + + Ser1170 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:39:48Z + + SceACC + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:59Z + + Ser1157 + + + + RESULTS + paragraph + 17691 + The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:04:03Z + + structures + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:21:27Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:21:41Z + + CD + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T23:04:06Z + + larger CthACC fragments + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:04:19Z + + protomers + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:24Z + + CthCD-CT + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:04:36Z + + protomer + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:09Z + + CthΔBCCP + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T23:04:40Z + + CthCD-CT1/2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:55:14Z + + CthΔBCCP1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:04:44Z + + hinges + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:04:36Z + + protomer + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:09Z + + CthΔBCCP + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T23:05:20Z + + CthΔBCCP2 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:22:20Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:22:30Z + + CD + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:05:31Z + + anchor site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:53:40Z + + BCCP linker + + + + RESULTS + paragraph + 18366 + Conformational variability in the CD thus contributes considerably to variations in the spacing between the BC and CT domains, and may extend to distance variations beyond the mobility range of the flexibly tethered BCCP. On the basis of the occurrence of related conformational changes between fungal and human ACC fragments, the observed set of conformations may well represent general states present in all eukaryotic ACCs. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:53:44Z + + BC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:06:29Z + + CT + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:14:37Z + + flexibly tethered + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:06:34Z + + BCCP + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:07Z + + human + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T23:06:37Z + + ACC fragments + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:05:54Z + + eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + + RESULTS + title_2 + 18793 + Large-scale conformational variability of fungal ACC + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + + RESULTS + paragraph + 18846 + To obtain a comprehensive view of fungal ACC dynamics in solution, we employed SAXS and EM. SAXS analysis of CthACC agrees with a dimeric state and an elongated shape with a maximum extent of 350 Å (Supplementary Table 1). The smooth appearance of scattering curves and derived distance distributions might indicate substantial interdomain flexibility (Supplementary Fig. 2a–c). Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. They identify the connections between CDN/CDL and between CDC2/CT as major contributors to conformational heterogeneity (Supplementary Fig. 4a,b). The flexibility in the CDC2/CT hinge appears substantially larger than the variations observed in the set of crystal structures. The BC domain is not completely disordered, but laterally attached to BT/CDN in a generally conserved position, albeit with increased flexibility. Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110 Å. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase. Furthermore, based on an average length of the BCCP–CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT. Consequently, increased flexibility or additional modes of conformational changes may be required for productive catalysis. The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T23:11:14Z + + ACC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:11:20Z + + in solution + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:45Z + + SAXS + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:59Z + + EM + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:45Z + + SAXS + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T22:55:55Z + + CthACC + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:11:25Z + + dimeric + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:11:28Z + + elongated shape + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:11:30Z + + scattering curves + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:11:33Z + + derived distance distributions + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:07:07Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T22:55:55Z + + CthACC + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:11:37Z + + particles + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T19:06:51Z + + MS + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:11:39Z + + low-activity state + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T23:11:42Z + + negative stain EM + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:11:45Z + + rod-like extended + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:11:48Z + + U-shaped + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:11:50Z + + particles + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:11:53Z + + Class averages + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T23:11:55Z + + maximum-likelihood-based two-dimensional (2D) classification + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:11:58Z + + dimeric + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:12:01Z + + CT + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:12:03Z + + full + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T23:12:06Z + + BC–BCCP–CD + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:04:36Z + + protomer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:12:12Z + + BC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:12:20Z + + CT + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:12:24Z + + dimer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:12:36Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:12:39Z + + CDC2/CT hinge + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:12:42Z + + crystal structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:12:45Z + + BC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:07:38Z + + BT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:13:12Z + + generally conserved position + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:13:14Z + + linear and U-shaped conformations + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:13:17Z + + BC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:13:20Z + + CT + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:14:31Z + + active sites + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:51:56Z + + static + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:54:38Z + + structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T23:13:25Z + + biotin-dependent carboxylase + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:13:28Z + + BCCP–CD linker + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:13:31Z + + 26 amino acids + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:13:34Z + + BCCP + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:14:31Z + + active sites + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:13:39Z + + BC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:13:42Z + + CT + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T23:13:53Z + + CDC1/CDC2 interface + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:59Z + + Ser1157 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:24:08Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:24:18Z + + CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:14Z + + crystal structure + + + + DISCUSS + title_1 + 21030 + Discussion + + + DISCUSS + paragraph + 21041 + Altogether, the architecture of fungal ACC is based on the central dimeric CT domain (Fig. 4d). The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain. The CD has no direct role in substrate recognition or catalysis but contributes to the regulation of all eukaryotic ACCs. In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263. In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity. In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface. However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer. The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT. A comparison between fungal and human ACC will help to further discriminate mechanistic differences that contribute to the extended control and polymerization of human ACC. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:19:20Z + + dimeric + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:19:23Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:04Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:53:51Z + + subdomains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:19:38Z + + CT + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:19:33Z + + mobile + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:19:30Z + + BCCP + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:19:36Z + + oriented + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:19:27Z + + BC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:05Z + + CD + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:05:54Z + + eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:52:04Z + + higher eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:13Z + + Ser80 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:20:20Z + + Ser1201 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:19:35Z + + Ser1263 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:11Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:59Z + + Ser1157 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:05Z + + CD + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:19:47Z + + phosphorylation site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T10:27:20Z + + ACC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:59Z + + Ser1157 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:20:02Z + + conformational freedom + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:20:06Z + + interdomain interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:20:10Z + + hinge + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:20:42Z + + full ACC activity + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:20:45Z + + BCCP anchor points + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:14:31Z + + active sites + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:20:51Z + + BC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:20:53Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:20:56Z + + BCCP + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:12Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:21:01Z + + unique + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:05Z + + CD + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:14:31Z + + active sites + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:21:06Z + + BC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:21:08Z + + CT + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:12Z + + fungal + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:07Z + + human + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:06:07Z + + human + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + + DISCUSS + paragraph + 22541 + Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong. In flACC, the ACC dimer obeys twofold symmetry and assembles in a triangular architecture with dimeric BC domains (Supplementary Fig. 5a). In their study, mutational data indicate a requirement for BC dimerization for catalytic activity. The transition from the elongated open shape, observed in our experiments, towards a compact triangular shape is based on an intricate interplay of several hinge-bending motions in the CD (Fig. 4d). Comparison of flACC with our CthΔBCCP structure reveals the CDC2/CT hinge as a major contributor to conformational flexibility (Supplementary Fig. 5b,c). In flACC, CDC2 rotates ∼120° with respect to the CT domain. A second hinge can be identified between CDC1/CDC2. On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:14Z + + crystal structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:24:18Z + + near full-length + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:18:51Z + + non-phosphorylated + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:56:21Z + + S. cerevisae + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:24:24Z + + lacking only + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:56:58Z + + 21 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-07-21T08:50:55Z + + flACC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-07-21T08:50:55Z + + flACC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:24:50Z + + dimer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:24:53Z + + triangular architecture + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:24:55Z + + dimeric + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:24:58Z + + BC + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:25:00Z + + mutational data + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:25:03Z + + elongated open shape + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:25:06Z + + compact triangular shape + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:05Z + + CD + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:25:09Z + + Comparison + + + protein + PR: + melaniev@ebi.ac.uk + 2023-07-21T08:50:55Z + + flACC + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:09Z + + CthΔBCCP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:25:18Z + + structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:25:21Z + + CDC2/CT hinge + + + protein + PR: + melaniev@ebi.ac.uk + 2023-07-21T08:50:55Z + + flACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:25:27Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:25:30Z + + second hinge + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:25:32Z + + superposition + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:25:04Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:25:23Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:18:51Z + + non-phosphorylated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-07-21T08:50:55Z + + flACC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:18:51Z + + non-phosphorylated + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:21Z + + HsaBT-CD + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:26:16Z + + inspecting + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:26:34Z + + protomer + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:26:57Z + + fragment + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:27:08Z + + structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:27:12Z + + CDN/CDC1 connection + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:27:14Z + + highly flexible + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:27:17Z + + hinge + + + + DISCUSS + paragraph + 23895 + The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation. In flACC, the regulatory loop is mostly disordered, illustrating the increased flexibility due to the absence of the phosphoryl group. Only in three out of eight observed protomers a short peptide stretch (including Ser1157) was modelled. In those instances the Ser1157 residue is located at a distance of 14–20 Å away from the location of the phosphorylated serine observed here, based on superposition of either CDC1 or CDC2. Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). Thus, in accordance with the results presented here, phosphorylation of Ser1157 in SceACC most likely limits flexibility in the CDC1/CDC2 hinge such that activation through BC dimerization is not possible (Fig. 4d), which however does not exclude intermolecular dimerization. In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7). This implicates that the triangular shape with dimeric BC domains has a low population also in the active form, even though a biasing influence of grid preparation cannot be excluded completely. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:29:53Z + + regulatory + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:29:56Z + + phophorylation site + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:12Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:05:34Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:05:42Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:30:03Z + + hinge conformation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-07-21T08:50:55Z + + flACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:30:08Z + + mostly disordered + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-18T22:30:13Z + + phosphoryl + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:30:17Z + + protomers + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:30:20Z + + short peptide + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:59Z + + Ser1157 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:30:23Z + + modelled + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:59Z + + Ser1157 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:08:22Z + + serine + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:30:36Z + + superposition + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:30:39Z + + Applying + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:30:51Z + + CDC1/CDC2 hinge + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:25:49Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:25:58Z + + CD + + + protein + PR: + melaniev@ebi.ac.uk + 2023-07-21T08:50:55Z + + flACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:07:38Z + + BT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:26Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:30:58Z + + CT + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:59Z + + Ser1157 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:39:48Z + + SceACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:31:03Z + + CDC1/CDC2 hinge + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-22T10:27:50Z + + BC + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:34:59Z + + EM + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:31:06Z + + micrographs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:18:44Z + + dephosphorylated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:39:48Z + + SceACC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:31:10Z + + elongated and U-shaped conformations + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:31:12Z + + particle shape distributions + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:31:14Z + + triangular shape + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:31:17Z + + dimeric + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:31:20Z + + BC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:31:23Z + + active form + + + + DISCUSS + paragraph + 25397 + Large-scale conformational variability has also been observed in most other carrier protein-based multienzymes, including polyketide and fatty-acid synthases (with the exception of fungal-type fatty-acid synthases), non-ribosomal peptide synthetases and the pyruvate dehydrogenase complexes, although based on completely different architectures. Together, this structural information suggests that variable carrier protein tethering is not sufficient for efficient substrate transfer and catalysis in any of these systems. The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d). The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC. It disfavours the adoption of a rare, compact conformation, in which intramolecular dimerization of the BC domains results in catalytic turnover. The regulation of activity thus results from restrained large-scale conformational dynamics rather than a direct or indirect influence on active site structure. To our best knowledge, ACC is the first multienzyme for which such a phosphorylation-dependent mechanical control mechanism has been visualized. However, the example of ACC now demonstrates the possibility of regulating activity by controlled dynamics of non-enzymatic linker regions also in other families of carrier-dependent multienzymes. Understanding such structural and dynamic constraints imposed by scaffolding and linking in carrier protein-based multienzyme systems is a critical prerequisite for engineering of efficient biosynthetic assembly lines. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:33:37Z + + carrier protein-based multienzymes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:33:40Z + + polyketide and fatty-acid synthases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:33:43Z + + fungal-type fatty-acid synthases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:33:46Z + + non-ribosomal peptide synthetases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:33:48Z + + pyruvate dehydrogenase complexes + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:33:52Z + + structural information + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:33:54Z + + determination of a set of + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:54:42Z + + crystal structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T17:39:48Z + + SceACC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:33:58Z + + unphosphorylated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:34:01Z + + major regulatory site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:59Z + + Ser1157 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:34:07Z + + allosteric site + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:34:25Z + + interface + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:34:56Z + + non-catalytic + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:35:18Z + + hinges + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:55:28Z + + dynamic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:35:47Z + + rare, compact conformation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:35:50Z + + BC + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:35:54Z + + active site structure + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:36:05Z + + multienzyme + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:52Z + + phosphorylation + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:46Z + + ACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:36:10Z + + non-enzymatic linker regions + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:36:08Z + + carrier-dependent multienzymes + + + + METHODS + title_1 + 27220 + Methods + + + METHODS + title_2 + 27228 + Protein expression and purification + + + METHODS + paragraph + 27264 + All proteins were expressed in the Baculovirus Expression Vector System. The MultiBac insect cell expression plasmid pACEBACI (Geneva Biotech) was modified to host a GATEWAY (LifeTechnologies) cassette with an N-terminal 10xHis-tag, named pAB1GW-NH10 hereafter. Full-length HsaACC (Genebank accession #Q13085), SceACC (#Q00955) and CthACC (#G0S3L5) were cloned into pAB1GW-NH10 using GATEWAY according to the manufacturer's manual. Truncated variants were constructed by PCR amplification, digestion of the template DNA with DpnI, phosphorylation of the PCR product and religation of the linear fragment to a circular plasmid. The following constructs were used for this study: SceACC (1–2,233), CthACC (1–2,297), CthΔBCCP (1–2,297, Δ700–765), CthCD-CT (788–2,297), CthCD-CTCter (1,114–2,297), SceCD (768–1,494) and HsaBT-CD (622–1,584, Δ753–818). Bacmid and virus production was carried out according to MultiBac instructions. Baculovirus generation and amplification as well as protein expression were performed in Sf21 cells (Expression Systems) in Insect-Xpress medium (Lonza). The cells were harvested 68–96 h post infection by centrifugation and stored at −80 °C until being processed. + + + METHODS + paragraph + 28486 + Cells were lysed by sonication and the lysate was cleared by ultracentrifugation. Soluble protein was purified using Ni-NTA (Genscript) and size exclusion chromatography (Superose 6, GE Healthcare). The affinity tag was removed by tobacco etch virus (TEV) protease cleavage overnight at 4 °C. TEV protease and uncleaved protein were removed by orthogonal Ni-NTA purification before size exclusion chromatography. SceACC, CthACC and CthΔBCCP were further purified by high-resolution anion exchange chromatography before size exclusion chromatography. Purified SceCD, CthCD-CTCter, CthCD-CT, CthΔBCCP, CthACC and SceACC were concentrated to 10 mg ml−1 in 30 mM 3-(N-morpholino) propanesulfonic acid (MOPS) pH 7, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Purified HsaBT-CD was concentrated to 20 mg ml−1 in 20 mM bicine pH 8.0, 200 mM NaCl, 5% glycerol and 5 mM tris(2-carboxyethyl) phosphine (TCEP). Proteins were used directly or were stored at −80 °C after flash-freezing in liquid nitrogen. + + + METHODS + title_2 + 29533 + Protein crystallization + + + METHODS + paragraph + 29557 + All crystallization experiments were conducted using sitting drop vapour diffusion. SceCD crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M BisTrisPropane pH 6.5, 0.05–0.2 M di-sodium malonate, 20–30% polyethylene glycol (PEG) 3350, 10 mM trimethylamine or 2% benzamidine) in a 1:1 or 2:1 ratio. Crystals appeared after several days and continued to grow for 20–200 days. Crystals were cryoprotected by short incubation in mother liquor supplemented with 22% ethylene glycol and flash-cooled in liquid nitrogen. For heavy metal derivatization the crystals were incubated in stabilization solution supplemented with 1 mM Thimerosal or 10 mM EuCl2, and then backsoaked for 15 s in stabilization solution without heavy metal. + + + METHODS + paragraph + 30328 + Initial crystals of HsaBT-CD grew in 0.1 M Tris pH 8.5, 0.35 M tri-potassium citrate and 2–3.5% PEG10000 at 19 °C. After several rounds of optimization, good-quality diffraction crystals were obtained at 19 °C in 0.1 M MES pH 6, 0.25–0.35 M tri-potassium citrate, 2–5% PEG10000 and 0.01–0.04 M cadmium chloride. The protein drop contained a 1:1 ratio of protein and reservoir solution. Crystals grew immediately and stopped growing after 3 days. They were dehydrated and cryoprotected in several steps in artificial mother liquor containing incrementally increasing concentrations of tri-potassium citrate, PEG10000 and ethylene glycol and then flash-cooled in liquid nitrogen. The final solution was composed of 0.1 M MES pH 6, 0.5 M tri-potassium citrate, 6.75% PEG10000, 0.01 M cadmium chloride and 22% ethylene glycol. + + + METHODS + paragraph + 31179 + CthCD-CTCter crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M HEPES pH 7.5, 2–7% Tacsimate pH 7, 7.5–15% PEG monomethyl ether 5000) in a 1:1 ratio. Crystals appeared after several days and continued to grow for up to 2 weeks. Crystals were cryoprotected by short incubation in mother liquor supplemented with 22% ethylene glycol. + + + METHODS + paragraph + 31546 + CthCD-CT ACC crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M Bicine pH 8.5–9.5, 4–8% PEG8000) in a 1:1 or 1:2 ratio. Crystals grew 8– 10 days and were cryoprotected by short incubation in mother liquor supplemented with 22% ethylene glycol before flash-cooling in liquid nitrogen. + + + METHODS + paragraph + 31867 + CthΔBCCP ACC crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M Morpheus buffer 3 (Molecular Dimensions, MD2-100-102), 7–12% Morpheus ethylene glycols mix (MD2-100-74), 8–12% PEG4000, 17–23% glycerol) in a 1:1 or 1:2 ratio. Crystals grew up to 3 weeks and were cryoprotected in reservoir solution before flash-cooling in liquid nitrogen. + + + METHODS + title_2 + 32243 + Structure determination and analysis of phosphorylation + + + METHODS + paragraph + 32299 + All X-ray diffraction data were collected at beamlines X06SA (PXI) or X06DA (PXIII) at the Swiss Light Source (SLS, Paul Scherrer Institute, Villigen, Switzerland) equipped with PILATUS detectors. The wavelength of data collection was 1.000 Å for native crystals, and 1.527 and 1.907 Å for crystals derivatized with europium and cadmium, respectively. Raw data were processed using XDS. Molecular replacement was carried out using Phaser 2.5.7 and 2.6.0, density modification was performed using Parrot and resolve, multicrystal averaging was carried out using phenix. All model building procedures were conducted using Coot and figures were prepared using PyMOL (Schrödinger LLC). + + + METHODS + paragraph + 32988 + Diffraction of initial SceCD crystals in space group P43212 with unit cell dimensions of a=b=110.3 Å and c=131.7 Å was limited to 3.5 Å. The resolution was improved to 3 Å by addition of trimethylamine or benzamidine to the reservoir solution without significant changes in unit cell dimensions. Crystals derivatized with thimerosal and europium were used for initial SAD phase determination using the SHELXC/D package. Two mercury and four europium sites were located, and an initial model was placed in the resulting maps. Since crystals derivatized with europium were slightly non-isomorphous with a c axis length of 127 Å, multicrystal averaging was used for density modification and provided directly interpretable maps. Iterative cycles of model building and refinement in Buster (version 2.10.2; Global Phasing Ltd) converged at Rwork/Rfree of 0.20/0.24. The final model lacks the disordered N terminus (amino acids 768–789), an extended loop in the CDC1 domain (1,203–1,215), a short stretch (1,147–1,149) preceding the regulatory loop and the two very C-terminal residues (1,493–1,494). On the basis of temperature factor analysis, the start and end of the regulatory loop show higher disorder than the region around the interacting phosphoserine 1157. MS analysis of dissolved crystals detected quantitative phosphorylation of the regulatory Ser1157, as also found for full-length SceACC, and additionally albeit with much lower occurrence, phosphorylation of Ser790, Ser1137, Ser1148 and Ser1159. A modelled phosphoryl position for Ser1159 could overlap with the one of Ser1157, and might be represented in the crystal. For all other phosphorylation sites no difference density could be observed, probably because of very low occupancy. PDBeFold was used to search for structural homologues. The thresholds for lowest acceptable percentage of matched secondary structure elements were 70% for the search query and 20% for the result. + + + METHODS + paragraph + 34956 + Initial HsaBT-CD crystals were obtained in space group I4122 with a=b=240.1 Å and c=768.9 Å and diffracted to 7.5 Å. Optimized and dehydrated crystals also belonged to space group I4122 but with unit cell parameters a=b=267.3 Å and c=210.6 Å and diffracted to a resolution of 3.7 Å. Phase information was obtained from SAD based on bound cadmium ions from the crystallization condition. Six cadmium positions were located in a 4.0-Å resolution data set at 1.9 Å wavelength using SHELXC/D via the HKL2MAP interface. Density modification and phasing based on this anomalous data set, a 3.7-Å resolution data set at 1.0 Å wavelength and additional non-isomorphous lower-resolution data sets led to a high-quality electron density map. At the intermediate resolution obtained, the map was interpreted by a poly-alanine model, which was guided by predicted secondary structure as well as sequence and structural alignment with SceCD. The final model contains five cadmium ions and refines using phenix against experimental data with Rwork/Rfree of 0.35/0.38, as expected for a poly-alanine model. Two HsaBT-CD monomers are packed in the asymmetric unit via the CDN and BT domains. Density on top of the β-barrel of one BT most likely representing parts of the BT–CD linker guided the assignment of this BT to its linked CD partner domain. This BT-to-CD assignment was further supported by the analysis of an additional lower-resolution crystal form. Cadmium ions were found to participate in crystal packing. + + + METHODS + paragraph + 36489 + In HsaACC, phosphorylation at regulatory sites was detected as provided in the main text. No phosphorylation was detected for other phosphosites previously identified in large-scale phosphoproteomics studies, namely serines 5, 23, 25, 48, 53, 78, 488, 786, 1273 (refs). + + + METHODS + paragraph + 36759 + Two different crystal forms were obtained for CthCD-CTCter (denoted as CthCD-CTCter1 and CthCD-CTCter2), diffracting to 3.6 and 4.5 Å. Both forms packed in space group P212121 with unit cell constants of a=97.7 Å, b=165.3 Å and c=219.2 Å or a=100.2 Å, b=153.5 Å and c=249.2 Å, respectively. Phases were determined by molecular replacement using a homology model based on SceCT (pdb 1od2) as search model in Phaser; multicrystal averaging was applied in density modification. The CT domain was rebuilt and an initial homology model based on the SceCD structure was fitted into difference density for CthCD-CTCter1. Iterative cycles of rebuilding and refinement in Buster converged at Rwork/Rfree of 0.20/0.24. The refined CD fragment served as a starting model for rebuilding CthCD-CTCter2 at lower resolution. Coordinate refinement in Buster was additionally guided by reference model restraints and converged at Rwork/Rfree of 0.24/0.24. Residues 1,114–1,185, 1,213–1,252, 1,380–1,385, 1,465–1,468 and 2,188–2,195 were disordered in both crystal forms and are not included in the models. Helical regions C terminal to Glu2264 of both protomers of CthCD-CTCter1 and C terminal to Leu2259 and Arg2261 of the two protomers of CthCD-CTCter2, respectively, could not be built unambiguously and were therefore interpreted by placing poly-alanine stretches. Conservation was mapped on the CthCD-CTCter1 crystal structure using al2co based on a sequence alignment of 367 fungal ACC sequences calculated by Clustal Omega. MS analysis of purified protein detected 7% phosphorylation at Ser1170 (corresponding to Ser1157 in SceCD). + + + METHODS + paragraph + 38412 + CthCD-CT crystallized in space group P31212 with unit cell constants of a=b=195.0 Å and c=189.5 Å and crystals diffracted to a resolution of 7.2 Å. The structure was solved by molecular replacement using a model composed of CthCT and CDC2 as search model in Phaser. CDC1 and CDN were placed manually into the resulting maps, and the model was refined using rigid-body, domain-wise TLS and B-factor refinement and NCS- and reference model-restrained coordinate refinement in Buster to Rwork/Rfree of 0.23/0.25. Owing to the low resolution, the maximum allowed B-factor in Buster refinement was increased from the default value of 300–500 Å2, minimizing B-factor clipping to 5% of all atoms. Residues 1,033–1,035, 1,134–1,152, 1,213–1,252, 1,380–1,385, 1,465–1,468 and 2,188–2,195 were not included in the models. Helical regions C terminal to Leu2259 and Arg2261 on the two protomers, respectively, were interpreted as described for CthCD-CTCter. Loop conformations, including the regulatory loop, were modelled as observed in SceCD. MS analysis of purified protein detected 60% phosphorylation at Ser1170 (corresponding to Ser1157 in SceCD). Conservation was mapped on the CthCD-CT crystal structure as for CthCD-CTCter. + + + METHODS + paragraph + 39659 + CthΔBCCP ACC crystallized in space group P6422 with unit cell constants of a=b=462.2 Å and c=204.6 Å, resolution was limited to 8.4 Å. Structure determination and refinement was performed as for CthCD-CT, with a maximum allowed B-factor of 500 Å2, minimizing B-factor clipping to 3% of all atoms. Although substantial difference density is observed, no defined positions of the BT and BC domains could be derived because of disorder or partial in situ proteolysis or combinations thereof. In addition, residues 1,032–1,039, 1,134–1,152, 1,213–1,252, 1,380–1,385, 1,465–1,468 and 2,188–2,195 were not included in the model. The MissingAtom macro implemented in Buster was employed to account for missing atoms, the final Rwork/Rfree were 0.30/0.32. A region C terminal to Leu2259 on one protomer was interpreted as poly-alanine. Loop conformations, including the regulatory loop, were modelled as observed in SceCD. MS analysis of purified protein detected 70% phosphorylation at Ser1170 (corresponding to Ser1157 in SceCD). + + + METHODS + title_2 + 40708 + Small-angle X-ray scattering + + + METHODS + paragraph + 40737 + Proteins were thawed on ice and dialysed overnight against 30 mM MOPS pH 7, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Raw scattering data were measured at SAXS beamline B21 at Diamond Light Source. The samples were measured at concentrations of 2.5, 5 and 10 mg ml−1. Data were processed using the ATSAS package according to standard procedures. A slight increase in scattering in the very low-resolution range was observed with increasing protein concentrations, which may be because of interparticle attraction or minor aggregation. Scattering intensities were thus extrapolated to zero concentration using point-wise extrapolation implemented in Primus. Direct comparison of raw scattering curves demonstrates the similarity of CthACC and CthΔBCCP, and the derived values such as Rg and Porod Volume match within expected error margins. Molecular mass estimations based on the SAXS–MOW method derive values of 534.7 and 534.0 kDa for CthACC and CthΔBCCP, respectively. The relative discrepancies to the theoretical weights of 516.8 kDa (CthACC) and 503.0 kDa (CthΔBCCP) are 3.5% and 6.2%, respectively, which is in a typical range for this method. + + + METHODS + title_2 + 41932 + Electron microscopy + + + METHODS + paragraph + 41952 + Full-length CthACC was diluted to 0.01 mg ml−1 in 30 mM MOPS pH 7.0, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Protein sample was adsorbed to a 200-μm copper grid and stained with 2% uranyl acetate. Grids of CthACC were imaged on a CM-200 microscope (Philips) equipped with a TVIPS F416 4k CMOS camera (Tietz Video and Image Processing Systems). The voltage used was 200 kV, and a magnification of × 50,000 results in a pixel size of 2.14 Å. Initial image processing and particle picking was carried out using Xmipp. Overall, 22,309 particles were picked semi-automatically from 236 micrographs with a box size of 300 × 300 pixels. After extraction, particles with a z-score of more than three were discarded and 22,257 particles were aligned and classified into 48 2D class averages using the maximum-likelihood target function in Fourier space (MLF2D). After 72 iterations, 4,226 additional particles were discarded and the remaining 18,031 particles were re-aligned and classified into 36 classes using MLF2D with a high-resolution cutoff of 30 Å. After 44 iterations the alignment converged and class averages were extracted. + + + METHODS + title_2 + 43128 + In vitro biotinylation and activity assay + + + METHODS + paragraph + 43170 + To ensure full functionality, SceACC was biotinylated in vitro using the E. coli biotin ligase BirA. The reaction mixture contained 10 μM ACC, 3.7 μM BirA, 50 mM Tris-HCl, pH 8, 5.5 mM MgCl2, 0.5 mM biotin, 60 mM NaCl, 3 mM ATP and 10% glycerol, and the reaction was allowed to proceed for 7 h at 30 °C. + + + METHODS + paragraph + 43495 + The catalytic activity of phosphorylated and dephosphorylated SceACC was measured by following the incorporation of radioactive 14C into acid-stable non-volatile material. Dephosphorylated ACC was prepared by overnight treatment with λ protein phosphatase (New England Biolabs) of partially purified ACC before the final gel filtration step. The removal of the phosphoryl group from Ser1157 was confirmed by MS. The reaction mixture contained 0.5 μg recombinant ACC in 100 mM potassium phosphate, pH 8, 3 mM ATP, 5 mM MgCl2, 50 mM NaH14CO3 (specific activity 7.4 MBq mmol−1) and 1 mM acetyl-CoA in a total reaction volume of 100 μl. The reaction mixture was incubated for 15 min at 30 °C, stopped by addition of 200 μl 6 M HCl and subsequently evaporated to dryness at 85 °C. The non-volatile residue was redissolved in 100 μl of water, 1 ml Ultima Gold XR scintillation medium (Perkin Elmer) was added and the 14C radioactivity was measured in a Packard Tricarb 2000CA liquid scintillation analyser. Measurements were carried out in five replicates and catalytic activities were calculated using a standard curve derived from measurements of varying concentrations of NaH14CO3 in reaction buffer. + + + METHODS + title_1 + 44734 + Additional information + + + METHODS + paragraph + 44757 + Accession codes: Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5I6E (SceCD), 5I87 (HsaBT-CD), 5I6F/5I6G (CthCD-CTCter1/2), 5I6H (CthCD-CT) and 5I6I (CthΔBCCP). + + + METHODS + paragraph + 44982 + How to cite this article: Hunkeler, M. et al. The dynamic organization of fungal acetyl-CoA carboxylase. Nat. Commun. 7:11196 doi: 10.1038/ncomms11196 (2016). + + + SUPPL + title_1 + 45141 + Supplementary Material + + + 225 + 226 + surname:Wakil;given-names:S. J. + surname:Titchener;given-names:E. B. + surname:Gibson;given-names:D. M. + 13560478 + REF + Biochim. Biophys. Acta + ref + 29 + 1958 + 45164 + Evidence for the participation of biotin in the enzymic synthesis of fatty acids + + + 537 + 579 + surname:Wakil;given-names:S. J. + surname:Stoops;given-names:J. K. + surname:Joshi;given-names:V. C. + 6137188 + REF + Annu. Rev. Biochem. + ref + 52 + 1983 + 45245 + Fatty acid synthesis and its regulation + + + 2613 + 2616 + surname:Abu-Elheiga;given-names:L. + surname:Matzuk;given-names:M. M. + surname:Abo-Hashema;given-names:K. A. + surname:Wakil;given-names:S. J. + 11283375 + REF + Science + ref + 291 + 2001 + 45285 + Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2 + + + 10207 + 10212 + surname:Abu-Elheiga;given-names:L. + surname:Oh;given-names:W. + surname:Kordari;given-names:P. + surname:Wakil;given-names:S. J. + 12920182 + REF + Proc. Natl Acad. Sci. USA + ref + 100 + 2003 + 45382 + Acetyl-CoA carboxylase 2 mutant mice are protected against obesity and diabetes induced by high-fat/high-carbohydrate diets + + + 283 + 289 + surname:Harwood;given-names:H. J. J. + REF + Curr. Opin. Invest. Drugs + ref + 5 + 2004 + 45506 + Acetyl-CoA carboxylase inhibition for the treatment of metabolic syndrome + + + 2115 + 2120 + surname:Milgraum;given-names:L. Z. + surname:Witters;given-names:L. A. + surname:Pasternack;given-names:G. R. + surname:Kuhajda;given-names:F. P. + 9815604 + REF + Clin. Cancer Res. + ref + 3 + 1997 + 45580 + Enzymes of the fatty acid synthesis pathway are highly expressed in in situ breast carcinoma + + + 358 + 365 + surname:Swinnen;given-names:J. V. + surname:Brusselmans;given-names:K. + surname:Verhoeven;given-names:G. + 16778563 + REF + Curr. Opin. Clin. Nutr. Metab. Care + ref + 9 + 2006 + 45673 + Increased lipogenesis in cancer cells: new players, novel targets + + + 519 + 525 + surname:Williams;given-names:R. S. + surname:Lee;given-names:M. S. + surname:Hau;given-names:D. D. + surname:Glover;given-names:J. N. + 15133503 + REF + Nat. Struct. Mol. Biol. + ref + 11 + 2004 + 45739 + Structural basis of phosphopeptide recognition by the BRCT domain of BRCA1 + + + 5767 + 5773 + surname:Shen;given-names:Y. + surname:Tong;given-names:L. + 18452305 + REF + Biochemistry + ref + 47 + 2008 + 45814 + Structural evidence for direct interactions between the BRCT domains of human BRCA1 and a phospho-peptide from human ACC1 + + + 1784 + 1803 + surname:Tong;given-names:L. + 15968460 + REF + Cell Mol. Life Sci. + ref + 62 + 2005 + 45936 + Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and attractive target for drug discovery + + + S138 + S143 + surname:Wakil;given-names:S. J. + surname:Abu-Elheiga;given-names:L. A. + 19047759 + REF + J. Lipid Res. + ref + 50 + 2009 + 46033 + Fatty acid metabolism: target for metabolic syndrome + + + 881 + 891 + surname:Shen;given-names:Y. + surname:Volrath;given-names:S. L. + surname:Weatherly;given-names:S. C. + surname:Elich;given-names:T. D. + surname:Tong;given-names:L. + 15610732 + REF + Mol. Cell + ref + 16 + 2004 + 46086 + A mechanism for the potent inhibition of eukaryotic acetyl-coenzyme A carboxylase by soraphen A, a macrocyclic polyketide natural product + + + 305 + 332 + surname:Campbell;given-names:J. W. + surname:Cronan;given-names:J. E.;suffix:Jr. + 11544358 + REF + Annu. Rev. Microbiol. + ref + 55 + 2001 + 46224 + Bacterial fatty acid biosynthesis: targets for antibacterial drug discovery + + + 105 + 110 + surname:Weatherly;given-names:S. C. + surname:Volrath;given-names:S. L. + surname:Elich;given-names:T. D. + 14766011 + REF + Biochem. J. + ref + 380 + 2004 + 46300 + Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain + + + 561 + 568 + surname:Alberts;given-names:A. W. + surname:Vagelos;given-names:P. R. + 4868901 + REF + Proc. Natl Acad. Sci. USA + ref + 59 + 1968 + 46420 + Acetyl CoA carboxylase. I. Requirement for two protein fractions + + + 1319 + 1326 + surname:Alberts;given-names:A. W. + surname:Nervi;given-names:A. M. + surname:Vagelos;given-names:P. R. + 4901473 + REF + Proc. Natl Acad. Sci. USA + ref + 63 + 1969 + 46485 + Acetyl CoA carboxylase, II. Deomonstration of biotin-protein and biotin carboxylase subunits + + + 407 + 435 + surname:Cronan;given-names:J. E.;suffix:Jr. + surname:Waldrop;given-names:G. L. + 12121720 + REF + Prog. Lipid Res. + ref + 41 + 2002 + 46578 + Multi-subunit acetyl-CoA carboxylases + + + 1502 + 1509 + surname:Bianchi;given-names:A. + 1967254 + REF + J. Biol. Chem. + ref + 265 + 1990 + 46616 + Identification of an isozymic form of acetyl-CoA carboxylase + + + 1444 + 1449 + surname:Abu-Elheiga;given-names:L. + 10677481 + REF + Proc. Natl Acad. Sci. USA + ref + 97 + 2000 + 46677 + The subcellular localization of acetyl-CoA carboxylase 2 + + + 223 + 227 + surname:Brownsey;given-names:R. W. + surname:Boone;given-names:A. N. + surname:Elliott;given-names:J. E. + surname:Kulpa;given-names:J. E. + surname:Lee;given-names:W. M. + 16545081 + REF + Biochem. Soc. Trans. + ref + 34 + 2006 + 46734 + Regulation of acetyl-CoA carboxylase + + + 1059 + 1064 + surname:Munday;given-names:M. R. + 12440972 + REF + Biochem. Soc. Trans. + ref + 30 + 2002 + 46771 + Regulation of mammalian acetyl-CoA carboxylase + + + e01130 + 01114 + surname:Shi;given-names:S. + surname:Chen;given-names:Y. + surname:Siewers;given-names:V. + surname:Nielsen;given-names:J. + 24803522 + REF + MBio + ref + 5 + 2014 + 46818 + Improving production of malonyl coenzyme A-derived metabolites by abolishing Snf1-dependent regulation of Acc1 + + + 1001 + 1005 + surname:Huang;given-names:C. S. + 20725044 + REF + Nature + ref + 466 + 2010 + 46929 + Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase + + + 219 + 223 + surname:Huang;given-names:C. S. + surname:Ge;given-names:P. + surname:Zhou;given-names:Z. H. + surname:Tong;given-names:L. + 22158123 + REF + Nature + ref + 481 + 2012 + 47017 + An unanticipated architecture of the 750-kDa alpha6beta6 holoenzyme of 3-methylcrotonyl-CoA carboxylase + + + 10249 + 10256 + surname:Waldrop;given-names:G. L. + surname:Rayment;given-names:I. + surname:Holden;given-names:H. M. + 7915138 + REF + Biochemistry + ref + 33 + 1994 + 47121 + Three-dimensional structure of the biotin carboxylase subunit of acetyl-CoA carboxylase + + + 1407 + 1419 + surname:Athappilly;given-names:F. K. + surname:Hendrickson;given-names:W. A. + 8747466 + REF + Structure + ref + 3 + 1995 + 47209 + Structure of the biotinyl domain of acetyl-coenzyme A carboxylase determined by MAD phasing + + + 2064 + 2067 + surname:Zhang;given-names:H. + surname:Yang;given-names:Z. + surname:Shen;given-names:Y. + surname:Tong;given-names:L. + 12663926 + REF + Science + ref + 299 + 2003 + 47301 + Crystal structure of the carboxyltransferase domain of acetyl-coenzyme A carboxylase + + + 1712 + 1722 + surname:Bilder;given-names:P. + 16460018 + REF + Biochemistry + ref + 45 + 2006 + 47386 + The structure of the carboxyltransferase component of acetyl-coA carboxylase reveals a zinc-binding motif unique to the bacterial enzyme + + + 863 + 891 + surname:Tong;given-names:L. + 22869039 + REF + Cell Mol. Life Sci. + ref + 70 + 2013 + 47523 + Structure and function of biotin-dependent carboxylases + + + 1076 + 1079 + surname:St Maurice;given-names:M. + 17717183 + REF + Science + ref + 317 + 2007 + 47579 + Domain architecture of pyruvate carboxylase, a biotin-dependent multifunctional enzyme + + + 120 + 124 + surname:Tran;given-names:T. H. + 25383525 + REF + Nature + ref + 518 + 2015 + 47666 + Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase + + + 9626 + 9631 + surname:Kim;given-names:C. W. + 20457939 + REF + Proc. Natl Acad. Sci. USA + ref + 107 + 2010 + 47753 + Induced polymerization of mammalian acetyl-CoA carboxylase by MIG12 provides a tertiary level of regulation of fatty acid synthesis + + + 973 + 982 + surname:Ray;given-names:H. + surname:Moreau;given-names:K. + surname:Dizin;given-names:E. + surname:Callebaut;given-names:I. + surname:Venezia;given-names:N. D. + 16698035 + REF + J. Mol. Biol. + ref + 359 + 2006 + 47885 + ACCA phosphopeptide recognition by the BRCT repeats of BRCA1 + + + 183 + 190 + surname:Davies;given-names:S. P. + surname:Sim;given-names:A. T. + surname:Hardie;given-names:D. G. + 1967580 + REF + Eur. J. Biochem. + ref + 187 + 1990 + 47946 + Location and function of three sites phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase + + + 22162 + 22168 + surname:Ha;given-names:J. + surname:Daniel;given-names:S. + surname:Broyles;given-names:S. S. + surname:Kim;given-names:K. H. + 7915280 + REF + J. Biol. Chem. + ref + 269 + 1994 + 48064 + Critical phosphorylation sites for acetyl-CoA carboxylase activity + + + 187 + 192 + surname:Cho;given-names:Y. S. + 19900410 + REF + Biochem. Biophys. Res. Commun. + ref + 391 + 2010 + 48131 + Molecular mechanism for the regulation of human ACC2 through phosphorylation by AMPK + + + 301 + 305 + surname:Ficarro;given-names:S. B. + 11875433 + REF + Nat. Biotechnol. + ref + 20 + 2002 + 48216 + Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae + + + 19509 + 19515 + surname:Woods;given-names:A. + 7913470 + REF + J. Biol. Chem. + ref + 269 + 1994 + 48310 + Yeast SNF1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo + + + 1682 + 1686 + surname:Holt;given-names:L. J. + 19779198 + REF + Science + ref + 325 + 2009 + 48432 + Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution + + + 31228 + 31236 + surname:Diacovich;given-names:L. + 12048195 + REF + J. Biol. Chem. + ref + 277 + 2002 + 48521 + Kinetic and structural analysis of a new group of Acyl-CoA carboxylases found in Streptomyces coelicolor A3(2) + + + 723 + 727 + surname:Wei;given-names:J. + surname:Tong;given-names:L. + 26458104 + REF + Nature + ref + 526 + 2015 + 48632 + Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase holoenzyme dimer + + + 769 + 780 + surname:Bernado;given-names:P. + 19844700 + REF + Eur. Biophys. J. + ref + 39 + 2010 + 48711 + Effect of interdomain dynamics on the structure determination of modular proteins by small-angle scattering + + + 9 + 19 + surname:Smith;given-names:J. L. + surname:Skiniotis;given-names:G. + surname:Sherman;given-names:D. H. + 25791608 + REF + Curr. Opin. Struct. Biol. + ref + 31 + 2015 + 48819 + Architecture of the polyketide synthase module: surprises from electron cryo-microscopy + + + 1315 + 1322 + surname:Maier;given-names:T. + surname:Leibundgut;given-names:M. + surname:Ban;given-names:N. + 18772430 + REF + Science + ref + 321 + 2008 + 48907 + The crystal structure of a mammalian fatty acid synthase + + + 190 + 197 + surname:Brignole;given-names:E. J. + surname:Smith;given-names:S. + 19151726 + REF + Nat. Struct. Mol. Biol. + ref + 16 + 2009 + 48964 + Asturias FJ. Conformational flexibility of metazoan fatty acid synthase enables catalysis + + + 234 + 240 + surname:Strieker;given-names:M. + surname:Tanovic;given-names:A. + surname:Marahiel;given-names:M. A. + 20153164 + REF + Curr. Opin. Struct. Biol. + ref + 20 + 2010 + 49054 + Nonribosomal peptide synthetases: structures and dynamics + + + 16615 + 16623 + surname:Patel;given-names:M. S. + surname:Nemeria;given-names:N. S. + surname:Furey;given-names:W. + surname:Jordan;given-names:F. + 24798336 + REF + J. Biol. Chem. + ref + 289 + 2014 + 49112 + The pyruvate dehydrogenase complexes: structure-based function and regulation + + + 1021 + 1032 + surname:Fitzgerald;given-names:D. J. + 17117155 + REF + Nat. Methods + ref + 3 + 2006 + 49190 + Protein complex expression by using multigene baculoviral vectors + + + 125 + 132 + surname:Kabsch;given-names:W. + 20124692 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 49256 + XDS + + + 53 + 64 + surname:Zhang;given-names:K. Y. + surname:Cowtan;given-names:K. + surname:Main;given-names:P. + 18488305 + REF + Methods Enzymol. + ref + 277 + 1997 + 49260 + Combining constraints for electron-density modification + + + 235 + 242 + surname:Winn;given-names:M. D. + 21460441 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 67 + 2011 + 49316 + Overview of the CCP4 suite and current developments + + + 213 + 221 + surname:Adams;given-names:P. D. + 20124702 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 49368 + PHENIX: a comprehensive Python-based system for macromolecular structure solution + + + 486 + 501 + surname:Emsley;given-names:P. + surname:Lohkamp;given-names:B. + surname:Scott;given-names:W. G. + surname:Cowtan;given-names:K. + 20383002 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 49450 + Features and development of Coot + + + 112 + 122 + surname:Sheldrick;given-names:G. M. + 18156677 + REF + Acta Crystallogr. A + ref + 64 + 2008 + 49483 + A short history of SHELX + + + 2256 + 2268 + surname:Krissinel;given-names:E. + surname:Henrick;given-names:K. + 15572779 + REF + Acta Crystallogr. D + ref + 60 + 2004 + 49508 + Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions + + + 843 + 844 + surname:Pape;given-names:T. + surname:Schneider;given-names:T. R. + REF + J. Appl. Crystallogr. + ref + 37 + 2004 + 49612 + HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs + + + ra3 + surname:Olsen;given-names:J. V. + 20068231 + REF + Sci. Signal. + ref + 3 + 2010 + 49695 + Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis + + + 253 + 262 + surname:Bian;given-names:Y. + 24275569 + REF + J. Proteomics + ref + 96 + 2014 + 49796 + An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome + + + 1346 + 1351 + surname:Cantin;given-names:G. T. + 18220336 + REF + J. Proteome Res. + ref + 7 + 2008 + 49885 + Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis + + + 658 + 674 + surname:McCoy;given-names:A. J. + 19461840 + REF + J. Appl. Crystallogr. + ref + 40 + 2007 + 49986 + Phaser crystallographic software + + + W252 + W258 + surname:Biasini;given-names:M. + 24782522 + REF + Nucleic Acids Res. + ref + 42 + 2014 + 50019 + SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information + + + 700 + 712 + surname:Pei;given-names:J. + surname:Grishin;given-names:N. V. + 11524371 + REF + Bioinformatics + ref + 17 + 2001 + 50115 + AL2CO: calculation of positional conservation in a protein sequence alignment + + + 539 + surname:Sievers;given-names:F. + 21988835 + REF + Mol. Syst. Biol. + ref + 7 + 2011 + 50193 + Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega + + + 342 + 350 + surname:Petoukhov;given-names:M. V. + 25484842 + REF + J. Appl. Crystallogr. + ref + 45 + 2012 + 50292 + New developments in the program package for small-angle scattering data analysis + + + 1727 + 1739 + surname:Skou;given-names:S. + surname:Gillilan;given-names:R. E. + surname:Ando;given-names:N. + 24967622 + REF + Nat. Protoc. + ref + 9 + 2014 + 50373 + Synchrotron-based small-angle X-ray scattering of proteins in solution + + + 642 + 657 + surname:Jacques;given-names:D. A. + surname:Trewhella;given-names:J. + 20120026 + REF + Protein Sci. + ref + 19 + 2010 + 50444 + Small-angle scattering for structural biology-Expanding the frontier while avoiding the pitfalls + + + 1277 + 1282 + surname:Konarev;given-names:P. V. + surname:Volkov;given-names:V. V. + surname:Sokolova;given-names:A. V. + surname:Koch;given-names:M. H. J. + surname:Svergun;given-names:D. I. + REF + J. Appl. Crystallogr. + ref + 36 + 2003 + 50541 + PRIMUS: a Windows PC-based system for small-angle scattering data analysis + + + 101 + 109 + surname:Fischer;given-names:H. + surname:Neto;given-names:M. D. + surname:Napolitano;given-names:H. B. + surname:Polikarpov;given-names:I. + surname:Craievich;given-names:A. F. + REF + J. Appl. Crystallogr. + ref + 43 + 2010 + 50616 + Determination of the molecular weight of proteins in solution from a single small-angle X-ray scattering measurement on a relative scale + + + 406 + 418 + surname:Scheres;given-names:S. H. + 22100448 + REF + J. Mol. Biol. + ref + 415 + 2012 + 50753 + A Bayesian view on cryo-EM structure determination + + + 237 + 240 + surname:Marabini;given-names:R. + 8812978 + REF + J. Struct. Biol. + ref + 116 + 1996 + 50804 + Xmipp: an image processing package for electron microscopy + + + SUPPL + footnote + 50863 + Author contributions M.H. cloned, expressed, purified and crystallized fungal ACC constructs, determined their structure and carried out SAXS analysis. E.S. cloned, expressed and crystallized human ACC CD and determined its structure. EM analysis was carried out by E.S., M.H. and A.H. S.I. contributed to structural analysis and figure preparation. T.M. designed and supervised work and analysed crystallographic data; all authors contributed to manuscript preparation. + + + ncomms11196-f1.jpg + f1 + FIG + fig_title_caption + 51334 + The phosphorylated central domain of yeast ACC. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:43:06Z + + central domain + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:22Z + + yeast + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:47Z + + ACC + + + + ncomms11196-f1.jpg + f1 + FIG + fig_caption + 51382 + (a) Schematic overview of the domain organization of eukaryotic ACCs. Crystallized constructs are indicated. (b) Cartoon representation of the SceCD crystal structure. CDN is linked by a four-helix bundle (CDL) to two α–β-fold domains (CDC1 and CDC2). The regulatory loop is shown as bold cartoon, and the phosphorylated Ser1157 is marked by a red triangle. The N- and C termini are indicated by spheres. (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. (e) Structural overview of HsaBT-CD. The attachment points to the N-terminal BCCP domain and the C-terminal CT domain are indicated with spheres. All colourings are according to scheme a. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:05:54Z + + eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:51:46Z + + ACCs + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:45:28Z + + Crystallized constructs + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:26:45Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:26:57Z + + CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:14Z + + crystal structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:27Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:54:05Z + + four-helix bundle + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:53Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:54:09Z + + two α–β-fold domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:59Z + + Ser1157 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:45:17Z + + Superposition + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:22:23Z + + highly conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:54:13Z + + folds + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:59Z + + Ser1157 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:30:43Z + + conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:15:21Z + + Arg1173 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:15:27Z + + Arg1260 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-18T22:45:05Z + + phosphoryl + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:36:21Z + + HsaBT-CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:45:08Z + + BCCP + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:45:03Z + + CT + + + + ncomms11196-f2.jpg + f2 + FIG + fig_title_caption + 52228 + Architecture of the CD–CT core of fungal ACC. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:05Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:45:52Z + + CT + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:12Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:47Z + + ACC + + + + ncomms11196-f2.jpg + f2 + FIG + fig_caption + 52276 + Cartoon representation of crystal structures of multidomain constructs of CthACC. One protomer is shown in colour and one in grey. Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:54:47Z + + crystal structures + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:46:20Z + + multidomain constructs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-18T22:55:55Z + + CthACC + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:04:36Z + + protomer + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:46:26Z + + active site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:46:28Z + + CT + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T22:30:43Z + + conserved + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:52:00Z + + regulatory + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:46:31Z + + phosphoserine site + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:27:38Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:27:49Z + + CD + + + + ncomms11196-f3.jpg + f3 + FIG + fig_title_caption + 52600 + Variability of the connections of CDC2 to CT and CDC1 in fungal ACC. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:47:01Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:12Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:47Z + + ACC + + + + ncomms11196-f3.jpg + f3 + FIG + fig_caption + 52669 + (a) Hinge properties of the CDC2–CT connection analysed by a CT-based superposition of eight instances of the CDC2-CT segment. For clarity, only one protomer of CthCD-CTCter1 is shown in full colour as reference. For other instances, CDC2 domains are shown in transparent tube representation with only one helix each highlighted. The range of hinge bending is indicated and the connection points between CDC2 and CT (blue) as well as between CDC1 and CDC2 (green and grey) are marked as spheres. (b) The interdomain interface of CDC1 and CDC2 exhibits only limited plasticity. Representation as in a, but the CDC1 and CDC2 are superposed based on CDC2. One protomer of CthΔBCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle. The connection points from CDC1 to CDC2 and to CDL are represented by green spheres. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:48:32Z + + Hinge + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:48:30Z + + CDC2–CT connection + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:48:55Z + + CT-based superposition + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:48:45Z + + CDC2-CT segment + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:04:36Z + + protomer + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:55:19Z + + CthCD-CTCter1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:49:08Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:18Z + + CDC2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:48:49Z + + interdomain interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:19Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:19Z + + CDC2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:48:52Z + + superposed + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:19Z + + CDC2 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T23:04:36Z + + protomer + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T22:54:10Z + + CthΔBCCP + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:54Z + + CDL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:08:22Z + + serine + + + species + MESH: + melaniev@ebi.ac.uk + 2023-06-15T09:28:56Z + + Sce + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-15T09:29:06Z + + CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:19Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:54Z + + CDL + + + + ncomms11196-f4.jpg + f4 + FIG + fig_title_caption + 53591 + The conformational dynamics of fungal ACC. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:12Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:47Z + + ACC + + + + ncomms11196-f4.jpg + f4 + FIG + fig_caption + 53634 + (a–c) Large-scale conformational variability of the CDN domain relative to the CDL/CDC1 domain. CthCD-CT1 (in colour) serves as reference, the compared structures (as indicated, numbers after construct name differentiate between individual protomers) are shown in grey. Domains other than CDN and CDL/CDC1 are omitted for clarity. The domains are labelled and the distances between the N termini of CDN (spheres) in the compared structures are indicated. (d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state. Flexibility of the CDC2/CT and CDN/CDL hinges is illustrated by arrows. The Ser1157 phosphorylation site and the regulatory loop are schematically indicated in magenta. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:27Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:54Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:55:24Z + + CthCD-CT1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-18T22:50:49Z + + compared structures + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:50:52Z + + protomers + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:27Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:54Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:11Z + + CDC1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:27Z + + CDN + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:48:12Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:55:47Z + + ACC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:05Z + + CD + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:52:59Z + + phosphorylated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:50:57Z + + inhibited + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T19:18:51Z + + non-phosphorylated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-18T22:51:00Z + + activated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:43:19Z + + CDC2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:51:03Z + + CT + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:27Z + + CDN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T17:42:54Z + + CDL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-18T22:51:06Z + + hinges + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T17:28:59Z + + Ser1157 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T17:07:53Z + + phosphorylation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:52:49Z + + regulatory loop + + + + t1.xml + t1 + TABLE + table_title_caption + 54420 + Crystallographic data collection and refinement statistics. + + + t1.xml + t1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th align="center" valign="top" charoff="50"><italic><bold>Sce</bold></italic><bold>CD</bold></th><th align="center" valign="top" charoff="50"><italic><bold>Sce</bold></italic><bold>CD Thimerosal</bold></th><th align="center" valign="top" charoff="50"><italic><bold>Sce</bold></italic><bold>CD Eu</bold></th><th align="center" valign="top" charoff="50"><italic><bold>Hsa</bold></italic><bold>BT-CD</bold></th><th align="center" valign="top" charoff="50"><italic><bold>Hsa</bold></italic><bold>BT-CD Cd</bold><sup><bold>2+</bold></sup></th><th align="center" valign="top" charoff="50"><italic><bold>Cth</bold></italic><bold>CD-CT</bold><sub><bold>Cter1</bold></sub></th><th align="center" valign="top" charoff="50"><italic><bold>Cth</bold></italic><bold>CD-CT</bold><sub><bold>Cter2</bold></sub></th><th align="center" valign="top" charoff="50"><italic><bold>Cth</bold></italic><bold>CD-CT</bold></th><th align="center" valign="top" charoff="50"><italic><bold>Cth</bold></italic>Δ<bold>BCCP</bold></th></tr></thead><tbody valign="top"><tr><td colspan="10" align="left" valign="top" charoff="50"><italic>Data collection</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Space group</td><td align="center" valign="top" charoff="50">P4<sub>3</sub>2<sub>1</sub>2</td><td align="center" valign="top" charoff="50">P4<sub>3</sub>2<sub>1</sub>2</td><td align="center" valign="top" charoff="50">P4<sub>3</sub>2<sub>1</sub>2</td><td align="center" valign="top" charoff="50">I4<sub>1</sub>22</td><td align="center" valign="top" charoff="50">I4<sub>1</sub>22</td><td align="center" valign="top" charoff="50">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="center" valign="top" charoff="50">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="center" valign="top" charoff="50">P3<sub>1</sub>2<sub>1</sub>2</td><td align="center" valign="top" charoff="50">P6<sub>4</sub>22</td></tr><tr><td align="left" valign="top" charoff="50"> Cell dimensions</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  <italic>a, b, c</italic> (Å)</td><td align="center" valign="top" charoff="50">110.86, 110.86, 131.12</td><td align="center" valign="top" charoff="50">111.22, 111.22, 131.49</td><td align="center" valign="top" charoff="50">108.65, 108.65, 127.36</td><td align="center" valign="top" charoff="50">267.27, 267.27, 210.61</td><td align="center" valign="top" charoff="50">267.67, 267.67, 210.46</td><td align="center" valign="top" charoff="50">97.66, 165.34, 219.23</td><td align="center" valign="top" charoff="50">100.17, 153.45, 249,24</td><td align="center" valign="top" charoff="50">295.02, 295.02, 189.52</td><td align="center" valign="top" charoff="50">462.20, 462.20, 204.64</td></tr><tr><td align="left" valign="top" charoff="50">  α, β, γ (°)</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 120</td><td align="center" valign="top" charoff="50">90, 90, 120</td></tr><tr><td align="left" valign="top" charoff="50"> Resolution<xref ref-type="fn" rid="t1-fn1">*</xref> (Å)</td><td align="center" valign="top" charoff="50">3.0</td><td align="center" valign="top" charoff="50">3.4</td><td align="center" valign="top" charoff="50">4.0</td><td align="center" valign="top" charoff="50">3.7</td><td align="center" valign="top" charoff="50">4.1</td><td align="center" valign="top" charoff="50">3.6</td><td align="center" valign="top" charoff="50">4.5</td><td align="center" valign="top" charoff="50">7.2</td><td align="center" valign="top" charoff="50">8.4</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>Merge</sub><xref ref-type="fn" rid="t1-fn2">†</xref></td><td align="center" valign="top" charoff="50">18.2 (389.6)</td><td align="center" valign="top" charoff="50">20.5 (306.1)</td><td align="center" valign="top" charoff="50">40.6 (327.0)</td><td align="center" valign="top" charoff="50">7.5 (400.9)</td><td align="center" valign="top" charoff="50">15 (730.5)</td><td align="center" valign="top" charoff="50">14.5 (384.5)</td><td align="center" valign="top" charoff="50">27.4 (225.6)</td><td align="center" valign="top" charoff="50">5.6 (302.6)</td><td align="center" valign="top" charoff="50">29.4 (381.7)</td></tr><tr><td align="left" valign="top" charoff="50"> CC ½<xref ref-type="fn" rid="t1-fn1">*</xref><xref ref-type="fn" rid="t1-fn2">†</xref></td><td align="center" valign="top" charoff="50">100 (58.3)</td><td align="center" valign="top" charoff="50">99.9 (42.6)</td><td align="center" valign="top" charoff="50">99.9 (48.5)</td><td align="center" valign="top" charoff="50">100 (59.4)</td><td align="center" valign="top" charoff="50">99.8 (73.2)</td><td align="center" valign="top" charoff="50">99.9 (50.9)</td><td align="center" valign="top" charoff="50">99.5 (46.7)</td><td align="center" valign="top" charoff="50">100 (33.3)</td><td align="center" valign="top" charoff="50">99.7 (35)</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>I</italic>/<italic>σI</italic><xref ref-type="fn" rid="t1-fn2">†</xref></td><td align="center" valign="top" charoff="50">24.68 (1.46)</td><td align="center" valign="top" charoff="50">7.99 (0.89)</td><td align="center" valign="top" charoff="50">17.92 (1.85)</td><td align="center" valign="top" charoff="50">21.24 (1.07)</td><td align="center" valign="top" charoff="50">16.53 (1.41)</td><td align="center" valign="top" charoff="50">10.61 (0.97)</td><td align="center" valign="top" charoff="50">6.35 (1.00)</td><td align="center" valign="top" charoff="50">18.95 (0.92)</td><td align="center" valign="top" charoff="50">9.05 (0.9)</td></tr><tr><td align="left" valign="top" charoff="50"> Completeness<xref ref-type="fn" rid="t1-fn2">†</xref></td><td align="center" valign="top" charoff="50">99.9 (99.9)</td><td align="center" valign="top" charoff="50">99.6 (100)</td><td align="center" valign="top" charoff="50">99.7 (96.8)</td><td align="center" valign="top" charoff="50">99.8 (99.1)</td><td align="center" valign="top" charoff="50">99.8 (99.7)</td><td align="center" valign="top" charoff="50">99.7 (99.9)</td><td align="center" valign="top" charoff="50">99.4 (98.6)</td><td align="center" valign="top" charoff="50">99.6 (100)</td><td align="center" valign="top" charoff="50">99.1 (99.9)</td></tr><tr><td align="left" valign="top" charoff="50"> Redundancy<xref ref-type="fn" rid="t1-fn2">†</xref></td><td align="center" valign="top" charoff="50">39.1 (39.8)</td><td align="center" valign="top" charoff="50">12.1 (14.3)</td><td align="center" valign="top" charoff="50">81.6 (65.2)</td><td align="center" valign="top" charoff="50">13.7 (13.7)</td><td align="center" valign="top" charoff="50">20.9 (19.1)</td><td align="center" valign="top" charoff="50">12.7 (13.5)</td><td align="center" valign="top" charoff="50">6.1 (6.5)</td><td align="center" valign="top" charoff="50">9.9 (10.4)</td><td align="center" valign="top" charoff="50">18.5 (18.2)</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td></tr><tr><td colspan="10" align="left" valign="top" charoff="50"><italic>Refinement</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Resolution<xref ref-type="fn" rid="t1-fn1">*</xref> (Å)</td><td align="center" valign="top" charoff="50">46.4–3.0</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">84.5–3.7</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">49.2–3.6</td><td align="center" valign="top" charoff="50">49.1–4.5</td><td align="center" valign="top" charoff="50">49.9–7.2</td><td align="center" valign="top" charoff="50">50.0–8.4</td></tr><tr><td align="left" valign="top" charoff="50"> Reflections</td><td align="center" valign="top" charoff="50">16,928</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">40,647</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">41,799</td><td align="center" valign="top" charoff="50">23,340</td><td align="center" valign="top" charoff="50">14,046</td><td align="center" valign="top" charoff="50">12,111</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub></td><td align="center" valign="top" charoff="50">0.20/0.24</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">0.35/0.38<xref ref-type="fn" rid="t1-fn3">‡</xref></td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">0.20/0.24</td><td align="center" valign="top" charoff="50">0.24/0.24</td><td align="center" valign="top" charoff="50">0.23/0.25</td><td align="center" valign="top" charoff="50">0.30/0.32</td></tr><tr><td align="left" valign="top" charoff="50"> Number of atoms</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  Protein</td><td align="center" valign="top" charoff="50">5,465</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">6,925</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">16,592</td><td align="center" valign="top" charoff="50">16,405</td><td align="center" valign="top" charoff="50">22,543</td><td align="center" valign="top" charoff="50">22,445</td></tr><tr><td align="left" valign="top" charoff="50">  Waters</td><td align="center" valign="top" charoff="50">43</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td></tr><tr><td align="left" valign="top" charoff="50">  Ligand/ion</td><td align="center" valign="top" charoff="50">7</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">5</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>B</italic>-factors</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  Protein</td><td align="center" valign="top" charoff="50">130</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">158</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">226</td><td align="center" valign="top" charoff="50">275</td><td align="center" valign="top" charoff="50">272</td><td align="center" valign="top" charoff="50">250</td></tr><tr><td align="left" valign="top" charoff="50">  Waters</td><td align="center" valign="top" charoff="50">84</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td></tr><tr><td align="left" valign="top" charoff="50">  Ligand/ion</td><td align="center" valign="top" charoff="50">90</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">189</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td></tr><tr><td align="left" valign="top" charoff="50"> R.m.s.d.</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  RMS (angles, °)</td><td align="center" valign="top" charoff="50">0.97</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">0.83</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">1.07</td><td align="center" valign="top" charoff="50">1.11</td><td align="center" valign="top" charoff="50">1.15</td><td align="center" valign="top" charoff="50">1.01</td></tr><tr><td align="left" valign="top" charoff="50">  RMS (bonds, Å)</td><td align="center" valign="top" charoff="50">0.01</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">0.01</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">0.01</td><td align="center" valign="top" charoff="50">0.01</td><td align="center" valign="top" charoff="50">0.01</td><td align="center" valign="top" charoff="50">0.01</td></tr></tbody></table> + + 54480 +   SceCD SceCD Thimerosal SceCD Eu HsaBT-CD HsaBT-CD Cd2+ CthCD-CTCter1 CthCD-CTCter2 CthCD-CT CthΔBCCP Data collection  Space group P43212 P43212 P43212 I4122 I4122 P212121 P212121 P31212 P6422  Cell dimensions                     a, b, c (Å) 110.86, 110.86, 131.12 111.22, 111.22, 131.49 108.65, 108.65, 127.36 267.27, 267.27, 210.61 267.67, 267.67, 210.46 97.66, 165.34, 219.23 100.17, 153.45, 249,24 295.02, 295.02, 189.52 462.20, 462.20, 204.64   α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 120 90, 90, 120  Resolution* (Å) 3.0 3.4 4.0 3.7 4.1 3.6 4.5 7.2 8.4  RMerge† 18.2 (389.6) 20.5 (306.1) 40.6 (327.0) 7.5 (400.9) 15 (730.5) 14.5 (384.5) 27.4 (225.6) 5.6 (302.6) 29.4 (381.7)  CC ½*† 100 (58.3) 99.9 (42.6) 99.9 (48.5) 100 (59.4) 99.8 (73.2) 99.9 (50.9) 99.5 (46.7) 100 (33.3) 99.7 (35)  I/σI† 24.68 (1.46) 7.99 (0.89) 17.92 (1.85) 21.24 (1.07) 16.53 (1.41) 10.61 (0.97) 6.35 (1.00) 18.95 (0.92) 9.05 (0.9)  Completeness† 99.9 (99.9) 99.6 (100) 99.7 (96.8) 99.8 (99.1) 99.8 (99.7) 99.7 (99.9) 99.4 (98.6) 99.6 (100) 99.1 (99.9)  Redundancy† 39.1 (39.8) 12.1 (14.3) 81.6 (65.2) 13.7 (13.7) 20.9 (19.1) 12.7 (13.5) 6.1 (6.5) 9.9 (10.4) 18.5 (18.2)                     Refinement  Resolution* (Å) 46.4–3.0     84.5–3.7   49.2–3.6 49.1–4.5 49.9–7.2 50.0–8.4  Reflections 16,928 — — 40,647 — 41,799 23,340 14,046 12,111  Rwork/Rfree 0.20/0.24 — — 0.35/0.38‡ — 0.20/0.24 0.24/0.24 0.23/0.25 0.30/0.32  Number of atoms                     Protein 5,465     6,925   16,592 16,405 22,543 22,445   Waters 43 — — — — — — — —   Ligand/ion 7 — — 5 — — — — —  B-factors                     Protein 130     158   226 275 272 250   Waters 84 — — — — — — — —   Ligand/ion 90 — — 189 — — — — —  R.m.s.d.                     RMS (angles, °) 0.97 — — 0.83 — 1.07 1.11 1.15 1.01   RMS (bonds, Å) 0.01 — — 0.01 — 0.01 0.01 0.01 0.01 + + + t1.xml + t1 + TABLE + table_footnote + 56655 + *Resolution cutoffs determined based on internal correlation significant at the 0.1% level as calculated by XDS. + + + t1.xml + t1 + TABLE + table_footnote + 56768 + †Highest-resolution shell is shown in parentheses. + + + t1.xml + t1 + TABLE + table_footnote + 56821 + ‡Modelled only as poly-alanine. + + + diff --git a/BioC_XML/4841544_v0.xml b/BioC_XML/4841544_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..f8bf6267fbdfb1e4651578026bfcdd78a21740b8 --- /dev/null +++ b/BioC_XML/4841544_v0.xml @@ -0,0 +1,14587 @@ + + + + PMC + 20201223 + pmc.key + + 4841544 + CC BY + no + 0 + 0 + + Molecular Basis of NadR Regulation + 10.1371/journal.ppat.1005557 + 4841544 + 27105075 + PPATHOGENS-D-15-00389 + e1005557 + 4 + This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. + surname:Liguori;given-names:Alessia + surname:Malito;given-names:Enrico + surname:Nassif;given-names:Xavier + surname:Lo Surdo;given-names:Paola + surname:Fagnocchi;given-names:Luca + surname:Cantini;given-names:Francesca + surname:Haag;given-names:Andreas F. + surname:Brier;given-names:Sébastien + surname:Pizza;given-names:Mariagrazia + surname:Delany;given-names:Isabel + surname:Bottomley;given-names:Matthew J. + The atomic coordinates of the NadR structures (with or without 4-HPA) have been deposited in the Protein Data Bank (PDB) and have been released with codes 5aip and 5aiq. + TITLE + Data Availability + front + 12 + 2016 + 0 + Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA + + 0.67065054 + protein + cleaner0 + 2023-07-05T16:01:44Z + PR: + + NadR + + + 0.99641824 + protein_type + cleaner0 + 2023-07-06T08:13:04Z + MESH: + + Transcriptional Repressor + + + 0.66509914 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:15Z + DUMMY: + + Meningococcal + + + 0.9822619 + protein + cleaner0 + 2023-07-05T16:01:59Z + PR: + + NadA + + + + ABSTRACT + abstract + 125 + Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis. + + protein + PR: + cleaner0 + 2023-07-05T16:03:40Z + + Neisseria adhesin A + + + 0.9674451 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.8832868 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + 0.8363549 + species + cleaner0 + 2023-07-05T16:04:50Z + MESH: + + human + + + 0.841002 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-05T16:07:12Z + + serogroup B meningococcus + + + 0.9365269 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.8454706 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:57Z + DUMMY: + + bacterial + + + 0.5582895 + species + cleaner0 + 2023-07-05T16:04:51Z + MESH: + + human + + + 0.55552936 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:56Z + DUMMY: + + bacterial + + + 0.9443213 + gene + cleaner0 + 2023-07-05T16:04:00Z + GENE: + + nadA + + + 0.9836204 + protein_type + cleaner0 + 2023-07-06T08:13:08Z + MESH: + + transcriptional regulator + + + 0.9970018 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + protein + PR: + cleaner0 + 2023-07-05T16:03:46Z + + Neisseria adhesin A Regulator + + + 0.9965952 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.93591195 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.7677482 + protein_state + cleaner0 + 2023-07-06T08:39:25Z + DUMMY: + + presence of + + + 0.9972517 + chemical + cleaner0 + 2023-07-05T16:05:05Z + CHEBI: + + 4-hydroxyphenylacetate + + + 0.9971226 + chemical + cleaner0 + 2023-07-05T16:05:11Z + CHEBI: + + 4-HPA + + + 0.98793995 + species + cleaner0 + 2023-07-05T16:04:51Z + MESH: + + human + + + 0.9820755 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:57Z + DUMMY: + + bacterial + + + 0.99725217 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.9253285 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.7519901 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.98988193 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.8327364 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + 0.9927263 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.9970572 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.9963371 + experimental_method + cleaner0 + 2023-07-06T11:58:41Z + MESH: + + structural, biochemical, and mutagenesis studies + + + 0.9963485 + evidence + cleaner0 + 2023-07-06T08:26:47Z + DUMMY: + + crystal structures + + + 0.9965448 + protein_state + cleaner0 + 2023-07-05T16:12:38Z + DUMMY: + + ligand-free + + + 0.9960804 + protein_state + cleaner0 + 2023-07-06T08:39:31Z + DUMMY: + + ligand-bound + + + 0.9977785 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.99697524 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.9944887 + oligomeric_state + cleaner0 + 2023-07-05T16:23:05Z + DUMMY: + + dimeric + + + 0.9976907 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.99785906 + chemical + cleaner0 + 2023-07-06T08:23:28Z + CHEBI: + + hydroxyphenylacetate + + + chemical + CHEBI: + cleaner0 + 2023-07-05T16:12:21Z + + 3Cl,4-HPA + + + 0.98984224 + residue_name + cleaner0 + 2023-07-06T08:22:53Z + SO: + + leucine + + + 0.9918474 + protein_state + cleaner0 + 2023-07-06T08:39:37Z + DUMMY: + + conserved + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.9988482 + residue_name_number + cleaner0 + 2023-07-06T08:02:27Z + DUMMY: + + His7 + + + 0.99881124 + residue_name_number + cleaner0 + 2023-07-05T16:30:04Z + DUMMY: + + Ser9 + + + 0.99891496 + residue_name_number + cleaner0 + 2023-07-06T08:02:37Z + DUMMY: + + Asn11 + + + 0.9988734 + residue_name_number + cleaner0 + 2023-07-05T16:37:34Z + DUMMY: + + Phe25 + + + 0.99702144 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.99452037 + taxonomy_domain + cleaner0 + 2023-07-05T16:09:53Z + DUMMY: + + bacteria + + + 0.9725539 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.99542 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.48914933 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + + ABSTRACT + abstract_title_1 + 2618 + Author Summary + + + ABSTRACT + abstract + 2633 + Serogroup B meningococcus (MenB) causes fatal sepsis and invasive meningococcal disease, particularly in young children and adolescents, as highlighted by recent MenB outbreaks in universities of the United States and Canada. The Bexsero vaccine protects against MenB and has recently been approved in > 35 countries worldwide. Neisseria adhesin A (NadA) present on the meningococcal surface can mediate binding to human cells and is one of the three MenB vaccine protein antigens. The amount of NadA exposed on the meningococcal surface also influences the antibody-mediated serum bactericidal response measured in vitro. A deep understanding of nadA expression is therefore important, otherwise the contribution of NadA to vaccine-induced protection against meningococcal meningitis may be underestimated. The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. These findings shed light on the regulation of NadR, a key MarR-family virulence factor of this important human pathogen. + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-05T16:07:13Z + + Serogroup B meningococcus + + + 0.9293316 + species + cleaner0 + 2023-07-06T08:18:39Z + MESH: + + MenB + + + 0.46876094 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + species + MESH: + cleaner0 + 2023-07-06T08:18:39Z + + MenB + + + 0.5040738 + species + cleaner0 + 2023-07-06T08:18:39Z + MESH: + + MenB + + + protein + PR: + cleaner0 + 2023-07-05T16:05:50Z + + Neisseria adhesin A + + + 0.9725767 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.80491525 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + 0.82990414 + species + cleaner0 + 2023-07-05T16:04:51Z + MESH: + + human + + + 0.9468687 + species + cleaner0 + 2023-07-06T08:18:39Z + MESH: + + MenB + + + 0.97354656 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.5592224 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + 0.9241102 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.9674066 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.51698756 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + 0.99396306 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.99102986 + protein_type + cleaner0 + 2023-07-06T08:14:00Z + MESH: + + ligand-responsive transcriptional repressor + + + 0.9980363 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + evidence + DUMMY: + cleaner0 + 2023-07-06T08:27:21Z + + functional, biochemical and high-resolution structural data + + + 0.998453 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.9978891 + chemical + cleaner0 + 2023-07-06T08:23:34Z + CHEBI: + + hydroxyphenylacetate + + + 0.9983973 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.9967884 + protein_state + cleaner0 + 2023-07-06T08:39:49Z + DUMMY: + + inactive + + + 0.9979068 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.645245 + species + cleaner0 + 2023-07-05T16:04:51Z + MESH: + + human + + + + INTRO + title_1 + 3999 + Introduction + + + INTRO + paragraph + 4012 + The ‘Reverse Vaccinology’ approach was pioneered to identify antigens for a protein-based vaccine against serogroup B Neisseria meningitidis (MenB), a human pathogen causing potentially-fatal sepsis and invasive meningococcal disease. Indeed, Reverse Vaccinology identified Neisseria adhesin A (NadA), a surface-exposed protein involved in epithelial cell invasion and found in ~30% of clinical isolates. Recently, we reported the crystal structure of NadA, providing insights into its biological and immunological functions. Recombinant NadA elicits a strong bactericidal immune response and is therefore included in the Bexsero vaccine that protects against MenB and which was recently approved in over 35 countries worldwide. + + 0.9534057 + experimental_method + cleaner0 + 2023-07-06T11:58:52Z + MESH: + + Reverse Vaccinology + + + species + MESH: + cleaner0 + 2023-07-06T08:18:01Z + + serogroup B Neisseria meningitidis + + + 0.97182584 + species + cleaner0 + 2023-07-06T08:18:38Z + MESH: + + MenB + + + 0.7824973 + species + cleaner0 + 2023-07-05T16:04:51Z + MESH: + + human + + + 0.76409507 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + 0.98660016 + experimental_method + cleaner0 + 2023-07-06T11:58:58Z + MESH: + + Reverse Vaccinology + + + 0.7993973 + protein + cleaner0 + 2023-07-05T16:05:50Z + PR: + + Neisseria adhesin A + + + 0.99444807 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.9971548 + evidence + cleaner0 + 2023-07-06T08:27:27Z + DUMMY: + + crystal structure + + + 0.99714524 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.9969074 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.69539905 + species + cleaner0 + 2023-07-06T08:18:39Z + MESH: + + MenB + + + + INTRO + paragraph + 4745 + Previous studies revealed that nadA expression levels are mainly regulated by the Neisseria adhesin A Regulator (NadR). Although additional factors influence nadA expression, we focused on its regulation by NadR, the major mediator of NadA phase variable expression. Studies of NadR also have broader implications, since a genome-wide analysis of MenB wild-type and nadR knock-out strains revealed that NadR influences the regulation of > 30 genes, including maf genes, from the multiple adhesin family. These genes encode a wide variety of proteins connected to many biological processes contributing to bacterial survival, adaptation in the host niche, colonization and invasion. + + 0.99617 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + protein + PR: + cleaner0 + 2023-07-05T16:08:44Z + + Neisseria adhesin A Regulator + + + 0.9830526 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.9966336 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.99631846 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.9956279 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.97683835 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.997136 + species + cleaner0 + 2023-07-06T08:18:39Z + MESH: + + MenB + + + 0.99319696 + protein_state + cleaner0 + 2023-07-05T16:24:28Z + DUMMY: + + wild-type + + + 0.31171978 + gene + cleaner0 + 2023-07-05T16:09:00Z + GENE: + + nadR + + + 0.6257549 + protein_state + cleaner0 + 2023-07-06T08:41:05Z + DUMMY: + + knock-out + + + 0.9666791 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:10:30Z + + adhesin + + + 0.9946519 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:57Z + DUMMY: + + bacterial + + + + INTRO + paragraph + 5427 + NadR belongs to the MarR (Multiple Antibiotic Resistance Regulator) family, a group of ligand-responsive transcriptional regulators ubiquitous in bacteria and archaea. MarR family proteins can promote bacterial survival in the presence of antibiotics, toxic chemicals, organic solvents or reactive oxygen species and can regulate virulence factor expression. MarR homologues can act either as transcriptional repressors or as activators. Although > 50 MarR family structures are known, a molecular understanding of their ligand-dependent regulatory mechanisms is still limited, often hampered by lack of identification of their ligands and/or DNA targets. A potentially interesting exception comes from the ligand-free and salicylate-bound forms of the Methanobacterium thermoautotrophicum protein MTH313 which revealed that two salicylate molecules bind to one MTH313 dimer and induce large conformational changes, apparently sufficient to prevent DNA binding. However, the homologous archeal Sulfolobus tokodaii protein ST1710 presented essentially the same structure in ligand-free and salicylate-bound forms, apparently contrasting the mechanism proposed for MTH313. Despite these apparent differences, MTH313 and ST1710 bind salicylate in approximately the same site, between their dimerization and DNA-binding domains. However, it is unknown whether salicylate is a relevant in vivo ligand of either of these two proteins, which share ~20% sequence identity with NadR, rendering unclear the interpretation of these findings in relation to the regulatory mechanisms of NadR or other MarR family proteins. + + 0.9835437 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.9859337 + protein_type + cleaner0 + 2023-07-05T16:09:31Z + MESH: + + MarR + + + 0.98408157 + protein_type + cleaner0 + 2023-07-06T08:14:12Z + MESH: + + Multiple Antibiotic Resistance Regulator + + + 0.9902781 + protein_type + cleaner0 + 2023-07-06T08:14:21Z + MESH: + + ligand-responsive transcriptional regulators + + + 0.9964108 + taxonomy_domain + cleaner0 + 2023-07-05T16:09:52Z + DUMMY: + + bacteria + + + 0.99622107 + taxonomy_domain + cleaner0 + 2023-07-05T16:09:46Z + DUMMY: + + archaea + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.99675155 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:57Z + DUMMY: + + bacterial + + + 0.99439716 + protein_type + cleaner0 + 2023-07-05T16:09:39Z + MESH: + + MarR + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.9917734 + evidence + cleaner0 + 2023-07-06T08:27:31Z + DUMMY: + + structures + + + 0.995853 + protein_state + cleaner0 + 2023-07-05T16:12:38Z + DUMMY: + + ligand-free + + + 0.9960255 + protein_state + cleaner0 + 2023-07-05T16:12:43Z + DUMMY: + + salicylate-bound + + + 0.995173 + species + cleaner0 + 2023-07-05T16:10:49Z + MESH: + + Methanobacterium thermoautotrophicum + + + 0.99857247 + protein + cleaner0 + 2023-07-05T16:10:56Z + PR: + + MTH313 + + + 0.99865437 + chemical + cleaner0 + 2023-07-05T16:11:41Z + CHEBI: + + salicylate + + + 0.9986519 + protein + cleaner0 + 2023-07-05T16:10:57Z + PR: + + MTH313 + + + 0.9956377 + oligomeric_state + cleaner0 + 2023-07-06T08:36:40Z + DUMMY: + + dimer + + + 0.98779243 + taxonomy_domain + cleaner0 + 2023-07-05T16:11:16Z + DUMMY: + + archeal + + + 0.9956137 + species + cleaner0 + 2023-07-05T16:11:10Z + MESH: + + Sulfolobus tokodaii + + + 0.99848586 + protein + cleaner0 + 2023-07-05T16:11:05Z + PR: + + ST1710 + + + 0.99651754 + evidence + cleaner0 + 2023-07-06T08:27:36Z + DUMMY: + + structure + + + 0.9961021 + protein_state + cleaner0 + 2023-07-05T16:12:38Z + DUMMY: + + ligand-free + + + 0.996548 + protein_state + cleaner0 + 2023-07-05T16:12:43Z + DUMMY: + + salicylate-bound + + + 0.9985191 + protein + cleaner0 + 2023-07-05T16:10:57Z + PR: + + MTH313 + + + 0.9986349 + protein + cleaner0 + 2023-07-05T16:10:57Z + PR: + + MTH313 + + + 0.9985905 + protein + cleaner0 + 2023-07-05T16:11:06Z + PR: + + ST1710 + + + 0.99850345 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.9700515 + structure_element + cleaner0 + 2023-07-06T12:15:50Z + SO: + + dimerization and DNA-binding domains + + + 0.9986278 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.9977221 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.9915398 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + + INTRO + paragraph + 7037 + NadR binds the nadA promoter and represses gene transcription. NadR binds nadA on three different operators (OpI, OpII and OpIII). The DNA-binding activity of NadR is attenuated in vitro upon addition of various hydroxyphenylacetate (HPA) derivatives, including 4-HPA. 4-HPA is a small molecule derived from mammalian aromatic amino acid catabolism and is released in human saliva, where it has been detected at micromolar concentration. In the presence of 4-HPA, NadR is unable to bind the nadA promoter and nadA gene expression is induced. In vivo, the presence of 4-HPA in the host niche of N. meningitidis serves as an inducer of NadA production, thereby promoting bacterial adhesion to host cells. Further, we recently reported that 3Cl,4-HPA, produced during inflammation, is another inducer of nadA expression. + + 0.9984029 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.85982156 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.99811816 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.99614644 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.99832064 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.99852234 + chemical + cleaner0 + 2023-07-06T08:23:40Z + CHEBI: + + hydroxyphenylacetate + + + 0.9960388 + chemical + cleaner0 + 2023-07-06T08:23:45Z + CHEBI: + + HPA + + + 0.9979666 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.9978723 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.9187701 + taxonomy_domain + cleaner0 + 2023-07-06T08:16:45Z + DUMMY: + + mammalian + + + 0.99355847 + species + cleaner0 + 2023-07-05T16:04:51Z + MESH: + + human + + + 0.9978695 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.99806863 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.91204 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.8036804 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.9978263 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.99539036 + species + cleaner0 + 2023-07-05T16:12:02Z + MESH: + + N. meningitidis + + + 0.9113778 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-05T16:04:57Z + + bacterial + + + 0.99766195 + chemical + cleaner0 + 2023-07-05T16:12:20Z + CHEBI: + + 3Cl,4-HPA + + + 0.4188783 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + + INTRO + paragraph + 7855 + Extending our previous studies based on hydrogen-deuterium exchange mass spectrometry (HDX-MS), here we sought to reveal the molecular mechanisms and effects of NadR/HPA interactions via X-ray crystallography, NMR spectroscopy and complementary biochemical and in vivo mutagenesis studies. We obtained detailed new insights into ligand specificity, how the ligand allosterically influences the DNA-binding ability of NadR, and the regulation of nadA expression, thus also providing a deeper structural understanding of the ligand-responsive MarR super-family. Moreover, these findings are important because the activity of NadR impacts the potential coverage provided by anti-NadA antibodies elicited by the Bexsero vaccine and influences host-bacteria interactions that contribute to meningococcal pathogenesis. + + 0.99628466 + experimental_method + cleaner0 + 2023-07-06T08:14:38Z + MESH: + + hydrogen-deuterium exchange mass spectrometry + + + 0.9962923 + experimental_method + cleaner0 + 2023-07-06T08:14:41Z + MESH: + + HDX-MS + + + 0.45488164 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.7351569 + chemical + cleaner0 + 2023-07-06T08:15:04Z + CHEBI: + + HPA + + + 0.99648964 + experimental_method + cleaner0 + 2023-07-06T08:14:44Z + MESH: + + X-ray crystallography + + + 0.99516284 + experimental_method + cleaner0 + 2023-07-06T08:14:34Z + MESH: + + NMR spectroscopy + + + 0.98606044 + experimental_method + cleaner0 + 2023-07-06T08:14:48Z + MESH: + + biochemical and in vivo mutagenesis studies + + + 0.99073 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.49103263 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.9970283 + protein_type + cleaner0 + 2023-07-05T16:09:39Z + MESH: + + MarR + + + 0.76515985 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.858279 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-05T16:09:53Z + + bacteria + + + 0.8999263 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + + RESULTS + title_1 + 8668 + Results + + + RESULTS + title_2 + 8676 + NadR is dimeric and is stabilized by specific hydroxyphenylacetate ligands + + 0.7011899 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.98950446 + oligomeric_state + cleaner0 + 2023-07-05T16:23:05Z + DUMMY: + + dimeric + + + 0.9979918 + chemical + cleaner0 + 2023-07-06T08:23:58Z + CHEBI: + + hydroxyphenylacetate + + + + RESULTS + paragraph + 8751 + Recombinant NadR was produced in E. coli using an expression construct prepared from N. meningitidis serogroup B strain MC58. Standard chromatographic techniques were used to obtain a highly purified sample of NadR (see Materials and Methods). In analytical size-exclusion high-performance liquid chromatography (SE-HPLC) experiments coupled with multi-angle laser light scattering (MALLS), NadR presented a single species with an absolute molecular mass of 35 kDa (S1 Fig). These data showed that NadR was dimeric in solution, since the theoretical molecular mass of the NadR dimer is 33.73 kDa; and, there was no change in oligomeric state on addition of 4-HPA. + + 0.9955266 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + species + MESH: + cleaner0 + 2023-07-05T16:14:02Z + + E. coli + + + 0.9589244 + experimental_method + cleaner0 + 2023-07-06T11:59:04Z + MESH: + + expression construct + + + species + MESH: + cleaner0 + 2023-07-05T16:13:44Z + + N. meningitidis serogroup B strain MC58 + + + 0.9976324 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.9955736 + experimental_method + cleaner0 + 2023-07-06T11:59:08Z + MESH: + + analytical size-exclusion high-performance liquid chromatography + + + 0.9805386 + experimental_method + cleaner0 + 2023-07-06T11:59:12Z + MESH: + + SE-HPLC + + + 0.9925471 + experimental_method + cleaner0 + 2023-07-06T11:59:17Z + MESH: + + multi-angle laser light scattering + + + 0.7785794 + experimental_method + cleaner0 + 2023-07-06T11:59:22Z + MESH: + + MALLS + + + 0.9957093 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.9982318 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.98969907 + oligomeric_state + cleaner0 + 2023-07-05T16:23:05Z + DUMMY: + + dimeric + + + 0.9982332 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.99625266 + oligomeric_state + cleaner0 + 2023-07-06T08:36:45Z + DUMMY: + + dimer + + + 0.9959236 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + + RESULTS + paragraph + 9415 + The thermal stability of NadR was examined using differential scanning calorimetry (DSC). Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313. The Tm of NadR was 67.4 ± 0.1°C in the absence of ligand, and was unaffected by salicylate. However, an increased thermal stability was induced by 4-HPA and, to a lesser extent, by 3-HPA. Interestingly, NadR displayed the greatest Tm increase upon addition of 3Cl,4-HPA (Table 1 and Fig 1B). + + 0.9988651 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.99587494 + experimental_method + cleaner0 + 2023-07-06T11:59:27Z + MESH: + + differential scanning calorimetry + + + 0.9905911 + experimental_method + cleaner0 + 2023-07-06T11:59:30Z + MESH: + + DSC + + + 0.80012095 + chemical + cleaner0 + 2023-07-05T16:18:02Z + CHEBI: + + HPAs + + + 0.99425703 + evidence + cleaner0 + 2023-07-06T08:27:41Z + DUMMY: + + melting temperature + + + 0.9845307 + evidence + cleaner0 + 2023-07-05T16:14:20Z + DUMMY: + + Tm + + + 0.9988589 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.99864787 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.98750615 + evidence + cleaner0 + 2023-07-05T16:14:21Z + DUMMY: + + Tm + + + 0.99866354 + protein + cleaner0 + 2023-07-05T16:11:06Z + PR: + + ST1710 + + + 0.998628 + protein + cleaner0 + 2023-07-05T16:10:57Z + PR: + + MTH313 + + + 0.9938285 + evidence + cleaner0 + 2023-07-05T16:14:21Z + DUMMY: + + Tm + + + 0.99881953 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + 0.9731813 + protein_state + cleaner0 + 2023-07-06T08:41:34Z + DUMMY: + + absence of ligand + + + 0.9985411 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.9962826 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.9964277 + chemical + cleaner0 + 2023-07-05T16:14:40Z + CHEBI: + + 3-HPA + + + 0.99867177 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + evidence + DUMMY: + cleaner0 + 2023-07-05T16:14:21Z + + Tm + + + chemical + CHEBI: + cleaner0 + 2023-07-05T16:12:21Z + + 3Cl,4-HPA + + + + ppat.1005557.g001.jpg + ppat.1005557.g001 + FIG + fig_title_caption + 10107 + Stability of NadR is increased by small molecule ligands. + + 0.9982602 + protein + cleaner0 + 2023-07-05T16:01:45Z + PR: + + NadR + + + + ppat.1005557.g001.jpg + ppat.1005557.g001 + FIG + fig_caption + 10165 + +(A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). All DSC profiles are representative of triplicate experiments. + + 0.99785185 + chemical + cleaner0 + 2023-07-05T16:14:41Z + CHEBI: + + 3-HPA + + + 0.9977923 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + chemical + CHEBI: + cleaner0 + 2023-07-05T16:12:21Z + + 3Cl,4-HPA + + + 0.9974121 + chemical + cleaner0 + 2023-07-06T08:24:03Z + CHEBI: + + salicylic acid + + + 0.650066 + experimental_method + cleaner0 + 2023-07-06T11:59:38Z + MESH: + + DSC + + + 0.61258185 + evidence + cleaner0 + 2023-07-06T08:27:49Z + DUMMY: + + profiles + + + 0.9971584 + protein_state + cleaner0 + 2023-07-05T16:18:27Z + DUMMY: + + apo + + + 0.9981931 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + complex_assembly + GO: + cleaner0 + 2023-07-05T16:16:02Z + + NadR+salicylate + + + complex_assembly + GO: + cleaner0 + 2023-07-05T16:16:28Z + + NadR+3-HPA + + + complex_assembly + GO: + cleaner0 + 2023-07-05T16:16:50Z + + NadR+4-HPA + + + complex_assembly + GO: + cleaner0 + 2023-07-05T16:17:12Z + + NadR+3Cl,4-HPA + + + experimental_method + MESH: + cleaner0 + 2023-07-06T08:28:04Z + + DSC + + + evidence + DUMMY: + cleaner0 + 2023-07-06T08:28:13Z + + profiles + + + + ppat.1005557.t001.xml + ppat.1005557.t001 + TABLE + table_title_caption + 10487 + Melting-point (Tm) and its ligand-induced increase (ΔTm) derived from DSC thermostability experiments. + + 0.99452204 + evidence + cleaner0 + 2023-07-06T08:28:22Z + DUMMY: + + Melting-point + + + 0.98550236 + evidence + cleaner0 + 2023-07-05T16:14:21Z + DUMMY: + + Tm + + + evidence + DUMMY: + cleaner0 + 2023-07-06T11:59:55Z + + ΔTm + + + 0.9887888 + experimental_method + cleaner0 + 2023-07-06T11:59:43Z + MESH: + + DSC + + + 0.64275366 + experimental_method + cleaner0 + 2023-07-06T11:59:45Z + MESH: + + thermostability experiments + + + + ppat.1005557.t001.xml + ppat.1005557.t001 + TABLE + table_caption + 10594 + Dissociation constants (KD) of the NadR/ligand interactions from SPR steady-state binding experiments. + + 0.96362484 + evidence + cleaner0 + 2023-07-06T08:28:25Z + DUMMY: + + Dissociation constants + + + 0.97385705 + evidence + cleaner0 + 2023-07-06T08:28:28Z + DUMMY: + + KD + + + 0.8992532 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + 0.9748532 + experimental_method + cleaner0 + 2023-07-06T12:00:06Z + MESH: + + SPR steady-state binding experiments + + + + ppat.1005557.t001.xml + ppat.1005557.t001 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><thead><tr><th align="center" rowspan="1" colspan="1">Ligand</th><th align="center" rowspan="1" colspan="1">T<sub>m</sub> (°C)</th><th align="center" rowspan="1" colspan="1">ΔT<sub>m</sub> (°C)</th><th align="center" rowspan="1" colspan="1">K<sub>D</sub> (mM)</th></tr></thead><tbody><tr><td align="center" rowspan="1" colspan="1">No ligand</td><td align="center" rowspan="1" colspan="1">67.4 ± 0.1</td><td align="center" rowspan="1" colspan="1">n.a.</td><td align="center" rowspan="1" colspan="1">n.a.</td></tr><tr><td align="center" rowspan="1" colspan="1">Salicylate</td><td align="center" rowspan="1" colspan="1">67.5 ± 0.1</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">n.d.</td></tr><tr><td align="center" rowspan="1" colspan="1">3-HPA</td><td align="center" rowspan="1" colspan="1">70.0 ± 0.1</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">2.7 ± 0.1</td></tr><tr><td align="center" rowspan="1" colspan="1">4-HPA</td><td align="center" rowspan="1" colspan="1">70.7 ± 0.1</td><td align="center" rowspan="1" colspan="1">3.3</td><td align="center" rowspan="1" colspan="1">1.5 ± 0.1</td></tr><tr><td align="center" rowspan="1" colspan="1">3Cl,4-HPA</td><td align="center" rowspan="1" colspan="1">71.3 ± 0.2</td><td align="center" rowspan="1" colspan="1">3.9</td><td align="center" rowspan="1" colspan="1">1.1 ± 0.1</td></tr></tbody></table> + + 10697 + Ligand Tm (°C) ΔTm (°C) KD (mM) No ligand 67.4 ± 0.1 n.a. n.a. Salicylate 67.5 ± 0.1 0 n.d. 3-HPA 70.0 ± 0.1 2.7 2.7 ± 0.1 4-HPA 70.7 ± 0.1 3.3 1.5 ± 0.1 3Cl,4-HPA 71.3 ± 0.2 3.9 1.1 ± 0.1 + + 0.98275363 + evidence + cleaner0 + 2023-07-05T16:14:21Z + DUMMY: + + Tm + + + 0.98464245 + evidence + cleaner0 + 2023-07-06T08:28:33Z + DUMMY: + + ΔTm + + + 0.9940189 + evidence + cleaner0 + 2023-07-06T08:28:36Z + DUMMY: + + KD + + + 0.9986681 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + Salicylate + + + 0.997032 + chemical + cleaner0 + 2023-07-05T16:14:41Z + CHEBI: + + 3-HPA + + + 0.99667215 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + chemical + CHEBI: + cleaner0 + 2023-07-05T16:12:21Z + + 3Cl,4-HPA + + + + ppat.1005557.t001.xml + ppat.1005557.t001 + TABLE + table_footnote + 10911 + n.a.: not applicable; n.d.: not determinable + + + RESULTS + title_2 + 10956 + NadR displays distinct binding affinities for hydroxyphenylacetate ligands + + 0.6722496 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + 0.9190013 + evidence + cleaner0 + 2023-07-06T08:28:39Z + DUMMY: + + binding affinities + + + 0.9986161 + chemical + cleaner0 + 2023-07-06T08:24:09Z + CHEBI: + + hydroxyphenylacetate + + + + RESULTS + paragraph + 11031 + To further investigate the binding of HPAs to NadR, we used surface plasmon resonance (SPR). The SPR sensorgrams revealed very fast association and dissociation events, typical of small molecule ligands, thus prohibiting a detailed study of binding kinetics. However, steady-state SPR analyses of the NadR-HPA interactions allowed determination of the equilibrium dissociation constants (KD) (Table 1 and S2 Fig). The interactions of 4-HPA and 3Cl,4-HPA with NadR exhibited KD values of 1.5 mM and 1.1 mM, respectively. 3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments. Although these KD values indicate rather weak interactions, they are similar to the values reported previously for the MarR/salicylate interaction (KD ~1 mM) and the MTH313/salicylate interaction (KD 2–3 mM), and approximately 20-fold tighter than the ST1710/salicylate interaction (KD ~20 mM). + + 0.99240065 + chemical + cleaner0 + 2023-07-05T16:18:03Z + CHEBI: + + HPAs + + + 0.9953414 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + 0.99554175 + experimental_method + cleaner0 + 2023-07-06T12:00:11Z + MESH: + + surface plasmon resonance + + + 0.99126047 + experimental_method + cleaner0 + 2023-07-06T12:00:15Z + MESH: + + SPR + + + 0.8462134 + experimental_method + cleaner0 + 2023-07-06T12:00:18Z + MESH: + + SPR + + + 0.8488718 + evidence + cleaner0 + 2023-07-06T08:28:43Z + DUMMY: + + sensorgrams + + + 0.994028 + experimental_method + cleaner0 + 2023-07-06T12:00:23Z + MESH: + + steady-state SPR + + + 0.977904 + complex_assembly + cleaner0 + 2023-07-06T08:22:36Z + GO: + + NadR-HPA + + + 0.99436253 + evidence + cleaner0 + 2023-07-06T08:28:46Z + DUMMY: + + equilibrium dissociation constants + + + 0.9904281 + evidence + cleaner0 + 2023-07-06T08:28:49Z + DUMMY: + + KD + + + 0.99725914 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.9930995 + chemical + cleaner0 + 2023-07-05T16:12:21Z + CHEBI: + + 3Cl,4-HPA + + + 0.99311405 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + 0.9946077 + evidence + cleaner0 + 2023-07-06T08:28:52Z + DUMMY: + + KD + + + 0.9976005 + chemical + cleaner0 + 2023-07-05T16:14:41Z + CHEBI: + + 3-HPA + + + 0.9771876 + evidence + cleaner0 + 2023-07-06T08:28:55Z + DUMMY: + + KD + + + 0.9987431 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.9925982 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + 0.9888996 + evidence + cleaner0 + 2023-07-06T08:28:57Z + DUMMY: + + KD + + + 0.99542093 + chemical + cleaner0 + 2023-07-05T16:12:21Z + CHEBI: + + 3Cl,4-HPA + + + evidence + DUMMY: + cleaner0 + 2023-07-05T16:14:21Z + + Tm + + + 0.98787653 + experimental_method + cleaner0 + 2023-07-06T12:00:26Z + MESH: + + DSC + + + 0.99358 + evidence + cleaner0 + 2023-07-06T08:28:59Z + DUMMY: + + KD + + + 0.722346 + protein_type + cleaner0 + 2023-07-05T16:09:39Z + MESH: + + MarR + + + 0.9977836 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.9962682 + protein + cleaner0 + 2023-07-05T16:10:57Z + PR: + + MTH313 + + + 0.9972759 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.9920671 + protein + cleaner0 + 2023-07-05T16:11:06Z + PR: + + ST1710 + + + 0.997875 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + + RESULTS + title_2 + 12203 + Crystal structures of holo-NadR and apo-NadR + + 0.9974237 + evidence + cleaner0 + 2023-07-06T08:29:07Z + DUMMY: + + Crystal structures + + + 0.99786276 + protein_state + cleaner0 + 2023-07-05T16:18:20Z + DUMMY: + + holo + + + 0.9929733 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + 0.9979085 + protein_state + cleaner0 + 2023-07-05T16:18:26Z + DUMMY: + + apo + + + 0.9956548 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + + RESULTS + paragraph + 12248 + To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms. First, we crystallized NadR (a selenomethionine-labelled derivative) in the presence of a 200-fold molar excess of 4-HPA. The structure of the NadR/4-HPA complex was determined at 2.3 Å resolution using a combination of the single-wavelength anomalous dispersion (SAD) and molecular replacement (MR) methods, and was refined to R work/R free values of 20.9/26.0% (Table 2). Despite numerous attempts, we were unable to obtain high-quality crystals of NadR complexed with 3Cl,4-HPA, 3,4-HPA, 3-HPA or DNA targets. However, it was eventually possible to crystallize apo-NadR, and the structure was determined at 2.7 Å resolution by MR methods using the NadR/4-HPA complex as the search model. The apo-NadR structure was refined to R work/R free values of 19.1/26.8% (Table 2). + + 0.9539873 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + 0.9201813 + chemical + cleaner0 + 2023-07-06T08:24:19Z + CHEBI: + + HPA + + + 0.9969667 + evidence + cleaner0 + 2023-07-06T08:29:11Z + DUMMY: + + crystal structures + + + 0.9985066 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + 0.9969062 + protein_state + cleaner0 + 2023-07-06T08:41:39Z + DUMMY: + + ligand-bound + + + 0.99781823 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.99661463 + protein_state + cleaner0 + 2023-07-05T16:12:38Z + DUMMY: + + ligand-free + + + 0.997442 + protein_state + cleaner0 + 2023-07-05T16:18:27Z + DUMMY: + + apo + + + 0.9914547 + experimental_method + cleaner0 + 2023-07-06T12:00:30Z + MESH: + + crystallized + + + 0.99856746 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + 0.8083253 + experimental_method + cleaner0 + 2023-07-06T12:00:34Z + MESH: + + selenomethionine-labelled derivative + + + 0.99723 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.9960259 + evidence + cleaner0 + 2023-07-06T08:29:15Z + DUMMY: + + structure + + + 0.9751709 + complex_assembly + cleaner0 + 2023-07-05T16:22:40Z + GO: + + NadR/4-HPA + + + 0.99386454 + experimental_method + cleaner0 + 2023-07-06T12:00:38Z + MESH: + + single-wavelength anomalous dispersion + + + 0.83583677 + experimental_method + cleaner0 + 2023-07-06T12:00:41Z + MESH: + + SAD + + + 0.9825908 + experimental_method + cleaner0 + 2023-07-06T12:00:44Z + MESH: + + molecular replacement + + + 0.48077685 + experimental_method + cleaner0 + 2023-07-06T12:00:48Z + MESH: + + MR + + + 0.99208534 + evidence + cleaner0 + 2023-07-06T08:29:20Z + DUMMY: + + R work/R free + + + 0.99488586 + evidence + cleaner0 + 2023-07-06T08:29:24Z + DUMMY: + + crystals + + + 0.9974232 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + 0.99201286 + protein_state + cleaner0 + 2023-07-06T08:41:46Z + DUMMY: + + complexed with + + + chemical + CHEBI: + cleaner0 + 2023-07-05T16:12:21Z + + 3Cl,4-HPA + + + 0.99713355 + chemical + cleaner0 + 2023-07-06T08:24:34Z + CHEBI: + + 3,4-HPA + + + 0.99740076 + chemical + cleaner0 + 2023-07-05T16:14:41Z + CHEBI: + + 3-HPA + + + 0.8414009 + experimental_method + cleaner0 + 2023-07-06T12:00:59Z + MESH: + + crystallize + + + 0.99761933 + protein_state + cleaner0 + 2023-07-05T16:18:27Z + DUMMY: + + apo + + + 0.9981098 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + 0.990086 + evidence + cleaner0 + 2023-07-06T08:29:27Z + DUMMY: + + structure + + + 0.699113 + experimental_method + cleaner0 + 2023-07-06T12:00:55Z + MESH: + + MR + + + 0.953306 + complex_assembly + cleaner0 + 2023-07-05T16:22:40Z + GO: + + NadR/4-HPA + + + 0.99776614 + protein_state + cleaner0 + 2023-07-05T16:18:27Z + DUMMY: + + apo + + + 0.99626714 + protein + cleaner0 + 2023-07-05T16:01:46Z + PR: + + NadR + + + 0.99615306 + evidence + cleaner0 + 2023-07-06T08:29:29Z + DUMMY: + + structure + + + evidence + DUMMY: + cleaner0 + 2023-07-06T08:29:46Z + + R work/R free + + + + ppat.1005557.t002.xml + ppat.1005557.t002 + TABLE + table_title_caption + 13176 + Data collection and refinement statistics for NadR structures. + + 0.99552464 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.99634415 + evidence + cleaner0 + 2023-07-06T08:29:50Z + DUMMY: + + structures + + + + ppat.1005557.t002.xml + ppat.1005557.t002 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="left" rowspan="1" colspan="1">NadR SeMet + 4-HPA (SAD peak) (PDB code 5aip)</th><th align="left" rowspan="1" colspan="1">NadR apo-form (PDB code 5aiq)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"> +<bold><italic>Data collection</italic></bold> +</td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">Wavelength (Å)</td><td align="left" rowspan="1" colspan="1">0.9792</td><td align="left" rowspan="1" colspan="1">1.0</td></tr><tr><td align="left" rowspan="1" colspan="1">Beamline</td><td align="left" rowspan="1" colspan="1">SLS (PXII-X10SA)</td><td align="left" rowspan="1" colspan="1">SLS (PXII-X10SA)</td></tr><tr><td align="left" rowspan="1" colspan="1">Resolution range (Å)</td><td align="left" rowspan="1" colspan="1">39.2–2.3</td><td align="left" rowspan="1" colspan="1">48.2–2.7</td></tr><tr><td align="left" rowspan="1" colspan="1">Space group</td><td align="left" rowspan="1" colspan="1">P 43 21 2</td><td align="left" rowspan="1" colspan="1">P 43 21 2</td></tr><tr><td align="left" rowspan="1" colspan="1">Unit cell dimensions (Å)</td><td align="left" rowspan="1" colspan="1">75.3, 75.3, 91.8</td><td align="left" rowspan="1" colspan="1">69.4, 69.4, 253.8</td></tr><tr><td align="left" rowspan="1" colspan="1">Total reflections</td><td align="left" rowspan="1" colspan="1">291132 (41090)</td><td align="left" rowspan="1" colspan="1">225521 (35809)</td></tr><tr><td align="left" rowspan="1" colspan="1">Unique reflections</td><td align="left" rowspan="1" colspan="1">12320 (1773)</td><td align="left" rowspan="1" colspan="1">17700 (2780)</td></tr><tr><td align="left" rowspan="1" colspan="1">Multiplicity</td><td align="left" rowspan="1" colspan="1">23.6 (23.2)</td><td align="left" rowspan="1" colspan="1">12.7 (12.8)</td></tr><tr><td align="left" rowspan="1" colspan="1">Completeness (%)</td><td align="left" rowspan="1" colspan="1">100.0 (100.00)</td><td align="left" rowspan="1" colspan="1">99.9 (99.7)</td></tr><tr><td align="left" rowspan="1" colspan="1">Mean I/sigma(I)</td><td align="left" rowspan="1" colspan="1">25.5 (9.0)</td><td align="left" rowspan="1" colspan="1">22.6 (3.8)</td></tr><tr><td align="left" rowspan="1" colspan="1">Wilson B-factor</td><td align="left" rowspan="1" colspan="1">23.9</td><td align="left" rowspan="1" colspan="1">49.1</td></tr><tr><td align="left" rowspan="1" colspan="1"> +<italic>R</italic> +<sub>sym</sub> +<xref ref-type="table-fn" rid="t002fn002">*</xref> +</td><td align="left" rowspan="1" colspan="1">10.9 (39.4)</td><td align="left" rowspan="1" colspan="1">11.4 (77.6)</td></tr><tr><td align="left" rowspan="1" colspan="1"> +<italic>R</italic> +<sub>meas</sub> +<xref ref-type="table-fn" rid="t002fn003">**</xref> +</td><td align="left" rowspan="1" colspan="1">11.3</td><td align="left" rowspan="1" colspan="1">11.8</td></tr><tr><td align="left" rowspan="1" colspan="1"> +<bold><italic>Refinement</italic></bold> +</td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> +<italic>R</italic> +<sub>work</sub> +<xref ref-type="table-fn" rid="t002fn004"> +<sup>♯</sup> +</xref> +</td><td align="left" rowspan="1" colspan="1">20.9</td><td align="left" rowspan="1" colspan="1">21.7</td></tr><tr><td align="left" rowspan="1" colspan="1"> +<italic>R</italic> +<sub>free</sub> +<xref ref-type="table-fn" rid="t002fn005"> +<sup>♯♯</sup> +</xref> +</td><td align="left" rowspan="1" colspan="1">26.0</td><td align="left" rowspan="1" colspan="1">27.2</td></tr><tr><td align="left" rowspan="1" colspan="1"> +<italic>Number of atoms</italic> +</td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">  Non-hydrogen atoms</td><td align="left" rowspan="1" colspan="1">2263</td><td align="left" rowspan="1" colspan="1">4163</td></tr><tr><td align="left" rowspan="1" colspan="1">  Macromolecules</td><td align="left" rowspan="1" colspan="1">2207</td><td align="left" rowspan="1" colspan="1">4144</td></tr><tr><td align="left" rowspan="1" colspan="1">  Ligands</td><td align="left" rowspan="1" colspan="1">11</td><td align="left" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">  Water</td><td align="left" rowspan="1" colspan="1">45</td><td align="left" rowspan="1" colspan="1">19</td></tr><tr><td align="left" rowspan="1" colspan="1">Protein residues</td><td align="left" rowspan="1" colspan="1">275</td><td align="left" rowspan="1" colspan="1">521</td></tr><tr><td align="left" rowspan="1" colspan="1">RMS(bonds)</td><td align="left" rowspan="1" colspan="1">0.008</td><td align="left" rowspan="1" colspan="1">0.003</td></tr><tr><td align="left" rowspan="1" colspan="1">RMS(angles)</td><td align="left" rowspan="1" colspan="1">1.09</td><td align="left" rowspan="1" colspan="1">0.823</td></tr><tr><td align="left" rowspan="1" colspan="1"> +<italic>Ramachandran (%)</italic> +<xref ref-type="table-fn" rid="t002fn006"> +<sup>§</sup> +</xref> +</td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">  Favored</td><td align="left" rowspan="1" colspan="1">100</td><td align="left" rowspan="1" colspan="1">98.4</td></tr><tr><td align="left" rowspan="1" colspan="1">  Outliers</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">Clash score</td><td align="left" rowspan="1" colspan="1">5.0</td><td align="left" rowspan="1" colspan="1">3.9</td></tr><tr><td align="left" rowspan="1" colspan="1"> +<italic>Average B-factor</italic> +</td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">  Macromolecules</td><td align="left" rowspan="1" colspan="1">34.8</td><td align="left" rowspan="1" colspan="1">53.3</td></tr><tr><td align="left" rowspan="1" colspan="1">  Ligands</td><td align="left" rowspan="1" colspan="1">32.9</td><td align="left" rowspan="1" colspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1">  Solvent</td><td align="left" rowspan="1" colspan="1">37.3 (H<sub>2</sub>O)</td><td align="left" rowspan="1" colspan="1">29.0 (H<sub>2</sub>O)</td></tr></tbody></table> + + 13239 + NadR SeMet + 4-HPA (SAD peak) (PDB code 5aip) NadR apo-form (PDB code 5aiq) Data collection Wavelength (Å) 0.9792 1.0 Beamline SLS (PXII-X10SA) SLS (PXII-X10SA) Resolution range (Å) 39.2–2.3 48.2–2.7 Space group P 43 21 2 P 43 21 2 Unit cell dimensions (Å) 75.3, 75.3, 91.8 69.4, 69.4, 253.8 Total reflections 291132 (41090) 225521 (35809) Unique reflections 12320 (1773) 17700 (2780) Multiplicity 23.6 (23.2) 12.7 (12.8) Completeness (%) 100.0 (100.00) 99.9 (99.7) Mean I/sigma(I) 25.5 (9.0) 22.6 (3.8) Wilson B-factor 23.9 49.1 Rsym* 10.9 (39.4) 11.4 (77.6) Rmeas** 11.3 11.8 Refinement Rwork♯ 20.9 21.7 Rfree♯♯ 26.0 27.2 Number of atoms   Non-hydrogen atoms 2263 4163   Macromolecules 2207 4144   Ligands 11 0   Water 45 19 Protein residues 275 521 RMS(bonds) 0.008 0.003 RMS(angles) 1.09 0.823 Ramachandran (%)§   Favored 100 98.4   Outliers 0 0 Clash score 5.0 3.9 Average B-factor   Macromolecules 34.8 53.3   Ligands 32.9 -   Solvent 37.3 (H2O) 29.0 (H2O) + + + ppat.1005557.t002.xml + ppat.1005557.t002 + TABLE + table_footnote + 14314 + Statistics for the highest-resolution shell are shown in parentheses. + + + ppat.1005557.t002.xml + ppat.1005557.t002 + TABLE + table_footnote + 14384 + *R +sym = Σhkl Σi |Ii(hkl)—<I(hkl)>| / Σhkl Σi Ii(hkl) + + + ppat.1005557.t002.xml + ppat.1005557.t002 + TABLE + table_footnote + 14454 + ** R +meas = redundancy-independent (multiplicity-weighted) R +merge as reported from AIMLESS. + + + ppat.1005557.t002.xml + ppat.1005557.t002 + TABLE + table_footnote + 14547 + +♯ +R +work = Σ||F(obs)|- |F(calc)||/Σ|F(obs)| + + + ppat.1005557.t002.xml + ppat.1005557.t002 + TABLE + table_footnote + 14600 + +♯♯ +R +free = as for R +work, calculated for 5.0% of the total reflections, chosen at random, and omitted from refinement. + + + ppat.1005557.t002.xml + ppat.1005557.t002 + TABLE + table_footnote + 14725 + +§ Values obtained using Molprobity. + + + RESULTS + paragraph + 14763 + The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. In the apo-NadR crystals, the two homodimers were related by a rotation of ~90°; the observed association of the two dimers was presumably merely an effect of crystal packing, since the interface between the two homodimers is small (< 550 Å2 of buried surface area), and is not predicted to be physiologically relevant by the PISA software. Moreover, our SE-HPLC/MALLS analyses (see above) revealed that in solution NadR is dimeric, and previous studies using native mass spectrometry (MS) revealed dimers, not tetramers. + + complex_assembly + GO: + cleaner0 + 2023-07-05T16:22:35Z + + NadR/4-HPA + + + 0.86533326 + evidence + cleaner0 + 2023-07-06T08:31:13Z + DUMMY: + + crystals + + + 0.9976197 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.98081243 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.99743205 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.9954763 + oligomeric_state + cleaner0 + 2023-07-05T16:22:48Z + DUMMY: + + homodimer + + + 0.9976458 + protein_state + cleaner0 + 2023-07-05T16:18:27Z + DUMMY: + + apo + + + 0.97368956 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.95074636 + evidence + cleaner0 + 2023-07-06T08:31:15Z + DUMMY: + + crystals + + + 0.99451524 + oligomeric_state + cleaner0 + 2023-07-05T16:51:20Z + DUMMY: + + homodimers + + + 0.99760973 + protein_state + cleaner0 + 2023-07-05T16:18:27Z + DUMMY: + + apo + + + 0.9667072 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.9422732 + evidence + cleaner0 + 2023-07-06T08:31:18Z + DUMMY: + + crystals + + + 0.99336135 + oligomeric_state + cleaner0 + 2023-07-05T16:51:20Z + DUMMY: + + homodimers + + + 0.9952251 + oligomeric_state + cleaner0 + 2023-07-05T16:22:58Z + DUMMY: + + dimers + + + 0.9958224 + site + cleaner0 + 2023-07-06T12:08:25Z + SO: + + interface + + + 0.9900866 + oligomeric_state + cleaner0 + 2023-07-05T16:51:20Z + DUMMY: + + homodimers + + + 0.98433065 + experimental_method + cleaner0 + 2023-07-06T12:01:05Z + MESH: + + SE-HPLC/MALLS + + + 0.997324 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.9947507 + oligomeric_state + cleaner0 + 2023-07-05T16:23:04Z + DUMMY: + + dimeric + + + 0.99470735 + experimental_method + cleaner0 + 2023-07-06T12:01:09Z + MESH: + + native mass spectrometry + + + 0.9927449 + experimental_method + cleaner0 + 2023-07-06T12:01:12Z + MESH: + + MS + + + 0.9962018 + oligomeric_state + cleaner0 + 2023-07-05T16:22:59Z + DUMMY: + + dimers + + + 0.9957474 + oligomeric_state + cleaner0 + 2023-07-05T16:23:10Z + DUMMY: + + tetramers + + + + RESULTS + paragraph + 15430 + The NadR homodimer bound to 4-HPA has a dimerization interface mostly involving the top of its ‘triangular’ form, while the two DNA-binding domains are located at the base (Fig 2A). High-quality electron density maps allowed clear identification of the bound ligand, 4-HPA (Fig 2B). The overall structure of NadR shows dimensions of ~50 × 65 × 50 Å and a large homodimer interface that buries a total surface area of ~ 4800 Å2. Each NadR monomer consists of six α-helices and two short β-strands, with helices α1, α5, and α6 forming the dimer interface. Helices α3 and α4 form a helix-turn-helix motif, followed by the “wing motif” comprised of two short antiparallel β-strands (β1-β2) linked by a relatively long and flexible loop. Interestingly, in the α4-β2 region, the stretch of residues from R64-R91 presents seven positively-charged side chains, all available for potential interactions with DNA. Together, these structural elements constitute the winged helix-turn-helix (wHTH) DNA-binding domain and, together with the dimeric organization, are the hallmarks of MarR family structures. + + 0.99825007 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.9960361 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.9944947 + protein_state + cleaner0 + 2023-07-06T08:41:54Z + DUMMY: + + bound to + + + 0.9968419 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.99718595 + site + cleaner0 + 2023-07-06T12:08:28Z + SO: + + dimerization interface + + + 0.48700365 + protein_state + cleaner0 + 2023-07-06T08:42:03Z + DUMMY: + + triangular + + + 0.9804043 + structure_element + cleaner0 + 2023-07-06T12:16:29Z + SO: + + DNA-binding domains + + + 0.9945665 + evidence + cleaner0 + 2023-07-06T08:31:20Z + DUMMY: + + electron density maps + + + 0.9670418 + protein_state + cleaner0 + 2023-07-06T08:42:17Z + DUMMY: + + bound + + + 0.9967594 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.9968761 + evidence + cleaner0 + 2023-07-06T08:31:26Z + DUMMY: + + structure + + + 0.99832994 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.99712276 + site + cleaner0 + 2023-07-06T12:08:31Z + SO: + + homodimer interface + + + 0.9982451 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.99608815 + oligomeric_state + cleaner0 + 2023-07-05T16:23:33Z + DUMMY: + + monomer + + + 0.96665907 + structure_element + cleaner0 + 2023-07-06T12:16:37Z + SO: + + α-helices + + + 0.9856715 + structure_element + cleaner0 + 2023-07-06T12:16:41Z + SO: + + short β-strands + + + 0.96922195 + structure_element + cleaner0 + 2023-07-06T12:16:44Z + SO: + + helices + + + 0.99701667 + structure_element + cleaner0 + 2023-07-06T12:16:47Z + SO: + + α1 + + + 0.99716765 + structure_element + cleaner0 + 2023-07-06T12:16:50Z + SO: + + α5 + + + 0.99717206 + structure_element + cleaner0 + 2023-07-06T12:16:53Z + SO: + + α6 + + + 0.9954803 + site + cleaner0 + 2023-07-06T12:08:37Z + SO: + + dimer interface + + + 0.9688121 + structure_element + cleaner0 + 2023-07-06T12:16:56Z + SO: + + Helices + + + 0.99735874 + structure_element + cleaner0 + 2023-07-06T12:16:59Z + SO: + + α3 + + + 0.9970772 + structure_element + cleaner0 + 2023-07-06T12:17:02Z + SO: + + α4 + + + 0.99675316 + structure_element + cleaner0 + 2023-07-06T12:17:05Z + SO: + + helix-turn-helix motif + + + 0.9934369 + structure_element + cleaner0 + 2023-07-06T12:17:08Z + SO: + + wing motif + + + 0.9623122 + structure_element + cleaner0 + 2023-07-06T12:17:12Z + SO: + + short antiparallel β-strands + + + 0.9967703 + structure_element + cleaner0 + 2023-07-06T12:17:15Z + SO: + + β1-β2 + + + 0.99074954 + structure_element + cleaner0 + 2023-07-06T12:17:18Z + SO: + + loop + + + 0.9239757 + structure_element + cleaner0 + 2023-07-06T12:17:21Z + SO: + + α4-β2 region + + + 0.49355826 + residue_range + cleaner0 + 2023-07-06T08:38:03Z + DUMMY: + + R64-R91 + + + 0.9922071 + chemical + cleaner0 + 2023-07-06T08:24:47Z + CHEBI: + + DNA + + + 0.99746746 + structure_element + cleaner0 + 2023-07-06T12:17:25Z + SO: + + winged helix-turn-helix + + + 0.9777044 + structure_element + cleaner0 + 2023-07-06T12:17:28Z + SO: + + wHTH + + + 0.9843573 + structure_element + cleaner0 + 2023-07-06T12:17:30Z + SO: + + DNA-binding domain + + + 0.9876031 + oligomeric_state + cleaner0 + 2023-07-05T16:23:05Z + DUMMY: + + dimeric + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.98246956 + evidence + cleaner0 + 2023-07-06T08:31:29Z + DUMMY: + + structures + + + + ppat.1005557.g002.jpg + ppat.1005557.g002 + FIG + fig_title_caption + 16568 + The crystal structure of NadR in complex with 4-HPA. + + 0.99741197 + evidence + cleaner0 + 2023-07-06T08:31:33Z + DUMMY: + + crystal structure + + + 0.99870706 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.9840052 + protein_state + cleaner0 + 2023-07-06T08:42:22Z + DUMMY: + + in complex with + + + 0.99743825 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + + ppat.1005557.g002.jpg + ppat.1005557.g002 + FIG + fig_caption + 16621 + +(A) The holo-NadR homodimer is depicted in green and blue for chains A and B respectively, while yellow sticks depict the 4-HPA ligand (labelled). For simplicity, secondary structure elements are labelled for chain B only. Red dashes show hypothetical positions of chain B residues 88–90 that were not modeled due to lack of electron density. (B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix. The map is contoured at 1σ and the figure was prepared with a density mesh carve factor of 1.7, using Pymol (www.pymol.org). + + protein_state + DUMMY: + cleaner0 + 2023-07-05T16:18:21Z + + holo + + + 0.99378645 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.9962703 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:43Z + + chains A and B + + + 0.9972606 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:13Z + + chain B + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:13Z + + chain B + + + 0.97810286 + residue_range + cleaner0 + 2023-07-06T08:38:08Z + DUMMY: + + 88–90 + + + 0.98408073 + evidence + cleaner0 + 2023-07-06T08:31:38Z + DUMMY: + + electron density + + + 0.9705171 + site + cleaner0 + 2023-07-06T12:08:43Z + SO: + + pocket + + + 0.99676806 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:43Z + + chains A and B + + + 0.9881984 + evidence + cleaner0 + 2023-07-06T08:31:42Z + DUMMY: + + electron density + + + 0.996697 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.9852703 + evidence + cleaner0 + 2023-07-06T08:31:46Z + DUMMY: + + composite omit map + + + 0.9935761 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.96732014 + experimental_method + cleaner0 + 2023-07-06T12:01:20Z + MESH: + + phenix + + + 0.99295795 + evidence + cleaner0 + 2023-07-06T08:31:49Z + DUMMY: + + map + + + 0.6867331 + evidence + cleaner0 + 2023-07-06T08:31:54Z + DUMMY: + + density mesh + + + + RESULTS + title_2 + 17306 + A single conserved leucine residue (L130) is crucial for dimerization + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:42:44Z + + conserved + + + 0.9880569 + residue_name + cleaner0 + 2023-07-06T08:22:59Z + SO: + + leucine + + + 0.9985177 + residue_name_number + cleaner0 + 2023-07-06T08:20:21Z + DUMMY: + + L130 + + + + RESULTS + paragraph + 17376 + The NadR dimer interface is formed by at least 32 residues, which establish numerous inter-chain salt bridges or hydrogen bonds, and many hydrophobic packing interactions (Fig 3A and 3B). To determine which residues were most important for dimerization, we studied the interface in silico and identified several residues as potential mediators of key stabilizing interactions. Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. Each mutant NadR protein was purified, and then its oligomeric state was examined by analytical SE-HPLC. Almost all the mutants showed the same elution profile as the wild-type (WT) NadR protein. Only the L130K mutation induced a notable change in the oligomeric state of NadR (Fig 3C). Further, in SE-MALLS analyses, the L130K mutant displayed two distinct species in solution, approximately 80% being monomeric (a 19 kDa species), and only 20% retaining the typical native dimeric state (a 35 kDa species) (Fig 3D), demonstrating that Leu130 is crucial for stable dimerization. It is notable that L130 is usually present as Leu, or an alternative bulky hydrophobic amino acid (e.g. Phe, Val), in many MarR family proteins, suggesting a conserved role in stabilizing the dimer interface. In contrast, most of the other residues identified in the NadR dimer interface were poorly conserved in the MarR family. + + 0.5062376 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.99676085 + site + cleaner0 + 2023-07-06T12:08:47Z + SO: + + dimer interface + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + salt bridges + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + hydrogen bonds + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + hydrophobic packing interactions + + + 0.97324896 + site + cleaner0 + 2023-07-06T12:08:50Z + SO: + + interface + + + 0.994365 + experimental_method + cleaner0 + 2023-07-06T12:01:24Z + MESH: + + site-directed mutagenesis + + + 0.5038502 + protein_state + cleaner0 + 2023-07-05T16:26:10Z + DUMMY: + + mutant + + + 0.7035019 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.9982158 + mutant + cleaner0 + 2023-07-05T16:25:18Z + MESH: + + H7A + + + 0.99785584 + mutant + cleaner0 + 2023-07-05T16:25:23Z + MESH: + + S9A + + + 0.99816763 + mutant + cleaner0 + 2023-07-05T16:25:28Z + MESH: + + N11A + + + 0.9981578 + mutant + cleaner0 + 2023-07-05T16:25:33Z + MESH: + + D112A + + + 0.99808407 + mutant + cleaner0 + 2023-07-05T16:25:38Z + MESH: + + R114A + + + 0.99788886 + mutant + cleaner0 + 2023-07-05T16:25:43Z + MESH: + + Y115A + + + 0.99800426 + mutant + cleaner0 + 2023-07-05T16:25:48Z + MESH: + + K126A + + + 0.9981881 + mutant + cleaner0 + 2023-07-05T16:25:54Z + MESH: + + L130K + + + 0.99821556 + mutant + cleaner0 + 2023-07-05T16:25:59Z + MESH: + + L133K + + + 0.9961478 + site + cleaner0 + 2023-07-06T12:08:54Z + SO: + + dimer interface + + + 0.8433007 + protein_state + cleaner0 + 2023-07-05T16:26:10Z + DUMMY: + + mutant + + + 0.6565402 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.9888089 + experimental_method + cleaner0 + 2023-07-06T12:01:28Z + MESH: + + analytical SE-HPLC + + + 0.9974766 + protein_state + cleaner0 + 2023-07-05T16:24:27Z + DUMMY: + + wild-type + + + 0.99762636 + protein_state + cleaner0 + 2023-07-05T16:24:32Z + DUMMY: + + WT + + + 0.99548244 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.99832827 + mutant + cleaner0 + 2023-07-05T16:25:55Z + MESH: + + L130K + + + 0.9947925 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.99607325 + experimental_method + cleaner0 + 2023-07-06T12:01:31Z + MESH: + + SE-MALLS + + + 0.99842083 + mutant + cleaner0 + 2023-07-05T16:25:55Z + MESH: + + L130K + + + 0.98589516 + protein_state + cleaner0 + 2023-07-05T16:26:09Z + DUMMY: + + mutant + + + 0.6365736 + oligomeric_state + cleaner0 + 2023-07-05T16:24:59Z + DUMMY: + + monomeric + + + 0.87424535 + oligomeric_state + cleaner0 + 2023-07-05T16:23:05Z + DUMMY: + + dimeric + + + 0.99873704 + residue_name_number + cleaner0 + 2023-07-06T08:20:25Z + DUMMY: + + Leu130 + + + 0.9986596 + residue_name_number + cleaner0 + 2023-07-06T08:20:28Z + DUMMY: + + L130 + + + 0.99059427 + residue_name + cleaner0 + 2023-07-06T08:23:04Z + SO: + + Leu + + + 0.9906516 + residue_name + cleaner0 + 2023-07-06T08:23:07Z + SO: + + Phe + + + 0.9905082 + residue_name + cleaner0 + 2023-07-06T08:23:09Z + SO: + + Val + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.8940407 + protein_state + cleaner0 + 2023-07-06T08:42:52Z + DUMMY: + + conserved + + + 0.99682724 + site + cleaner0 + 2023-07-06T12:08:57Z + SO: + + dimer interface + + + 0.39978522 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.9964477 + site + cleaner0 + 2023-07-06T12:09:01Z + SO: + + dimer interface + + + 0.9971421 + protein_state + cleaner0 + 2023-07-06T08:42:56Z + DUMMY: + + poorly conserved + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + + ppat.1005557.g003.jpg + ppat.1005557.g003 + FIG + fig_title_caption + 18880 + Analysis of the NadR dimer interface. + + 0.8886324 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.99709916 + site + cleaner0 + 2023-07-06T12:09:07Z + SO: + + dimer interface + + + + ppat.1005557.g003.jpg + ppat.1005557.g003 + FIG + fig_caption + 18918 + +(A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). (C) SE-HPLC analyses of all mutant forms of NadR are compared with the wild-type (WT) protein. The WT and most of the mutants show a single elution peak with an absorbance maximum at 17.5 min. Only the mutation L130K has a noteworthy effect on the oligomeric state, inducing a second peak with a longer retention time and a second peak maximum at 18.6 min. To a much lesser extent, the L133K mutation also appears to induce a ‘shoulder’ to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer. The curves plotted correspond to Absorbance Units (mAU) at 280nm wavelength (green), light scattering (red), and refractive index (blue). + + structure_element + SO: + cleaner0 + 2023-07-05T16:34:01Z + + chain A + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + salt bridges + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + hydrogen bonds + + + 0.99885666 + residue_name_number + cleaner0 + 2023-07-05T16:26:25Z + DUMMY: + + Q4 + + + 0.99869007 + residue_name_number + cleaner0 + 2023-07-05T16:26:30Z + DUMMY: + + S5 + + + 0.99884427 + residue_name_number + cleaner0 + 2023-07-05T16:26:35Z + DUMMY: + + K6 + + + 0.9984061 + residue_name_number + cleaner0 + 2023-07-05T16:26:41Z + DUMMY: + + H7 + + + 0.9984659 + residue_name_number + cleaner0 + 2023-07-05T16:26:48Z + DUMMY: + + S9 + + + 0.99881685 + residue_name_number + cleaner0 + 2023-07-05T16:26:53Z + DUMMY: + + I10 + + + 0.9988582 + residue_name_number + cleaner0 + 2023-07-05T16:26:59Z + DUMMY: + + N11 + + + 0.99883896 + residue_name_number + cleaner0 + 2023-07-05T16:27:05Z + DUMMY: + + I15 + + + 0.9989189 + residue_name_number + cleaner0 + 2023-07-05T16:27:11Z + DUMMY: + + Q16 + + + 0.99889046 + residue_name_number + cleaner0 + 2023-07-05T16:27:17Z + DUMMY: + + R18 + + + 0.9989405 + residue_name_number + cleaner0 + 2023-07-05T16:27:24Z + DUMMY: + + D36 + + + 0.99892056 + residue_name_number + cleaner0 + 2023-07-05T16:27:33Z + DUMMY: + + R43 + + + 0.99886984 + residue_name_number + cleaner0 + 2023-07-05T16:27:38Z + DUMMY: + + A46 + + + 0.99889106 + residue_name_number + cleaner0 + 2023-07-05T16:27:44Z + DUMMY: + + Q59 + + + 0.9988123 + residue_name_number + cleaner0 + 2023-07-05T16:27:50Z + DUMMY: + + C61 + + + 0.9988758 + residue_name_number + cleaner0 + 2023-07-05T16:27:55Z + DUMMY: + + Y104 + + + 0.9988651 + residue_name_number + cleaner0 + 2023-07-05T16:28:01Z + DUMMY: + + D112 + + + 0.99886703 + residue_name_number + cleaner0 + 2023-07-05T16:28:08Z + DUMMY: + + R114 + + + 0.99889946 + residue_name_number + cleaner0 + 2023-07-05T16:28:14Z + DUMMY: + + Y115 + + + 0.9988416 + residue_name_number + cleaner0 + 2023-07-05T16:28:20Z + DUMMY: + + D116 + + + 0.99883765 + residue_name_number + cleaner0 + 2023-07-05T16:28:25Z + DUMMY: + + E119 + + + 0.99891067 + residue_name_number + cleaner0 + 2023-07-05T16:28:30Z + DUMMY: + + K126 + + + 0.9989189 + residue_name_number + cleaner0 + 2023-07-05T16:28:35Z + DUMMY: + + E136 + + + 0.9989262 + residue_name_number + cleaner0 + 2023-07-05T16:28:41Z + DUMMY: + + E141 + + + 0.99893135 + residue_name_number + cleaner0 + 2023-07-05T16:28:46Z + DUMMY: + + N145 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + hydrophobic packing interactions + + + 0.9988618 + residue_name_number + cleaner0 + 2023-07-05T16:26:54Z + DUMMY: + + I10 + + + 0.99886155 + residue_name_number + cleaner0 + 2023-07-06T08:20:34Z + DUMMY: + + I12 + + + 0.9988972 + residue_name_number + cleaner0 + 2023-07-06T08:20:37Z + DUMMY: + + L14 + + + 0.9988487 + residue_name_number + cleaner0 + 2023-07-05T16:27:06Z + DUMMY: + + I15 + + + 0.9988752 + residue_name_number + cleaner0 + 2023-07-05T16:27:18Z + DUMMY: + + R18 + + + 0.9988952 + residue_name_number + cleaner0 + 2023-07-05T16:28:15Z + DUMMY: + + Y115 + + + 0.99885106 + residue_name_number + cleaner0 + 2023-07-06T08:20:43Z + DUMMY: + + I118 + + + 0.9989354 + residue_name_number + cleaner0 + 2023-07-06T08:20:47Z + DUMMY: + + L130 + + + 0.9989448 + residue_name_number + cleaner0 + 2023-07-06T08:20:51Z + DUMMY: + + L133 + + + 0.99895287 + residue_name_number + cleaner0 + 2023-07-06T08:20:54Z + DUMMY: + + L134 + + + 0.9989623 + residue_name_number + cleaner0 + 2023-07-06T08:20:58Z + DUMMY: + + L137 + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:13Z + + Chain B + + + 0.98969185 + experimental_method + cleaner0 + 2023-07-06T12:01:37Z + MESH: + + site-directed mutagenesis + + + 0.9988722 + residue_name_number + cleaner0 + 2023-07-05T16:28:36Z + DUMMY: + + E136 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + salt bridge + + + 0.9989114 + residue_name_number + cleaner0 + 2023-07-05T16:28:31Z + DUMMY: + + K126 + + + 0.99832875 + mutant + cleaner0 + 2023-07-05T16:25:49Z + MESH: + + K126A + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + ionic interaction + + + 0.96227586 + residue_name_number + cleaner0 + 2023-07-05T16:26:42Z + DUMMY: + + H7 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-05T16:23:34Z + + monomer + + + 0.17348053 + structure_element + cleaner0 + 2023-07-05T16:35:54Z + SO: + + A + + + 0.9874846 + evidence + cleaner0 + 2023-07-06T08:31:59Z + DUMMY: + + electron density + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-05T16:23:34Z + + monomer + + + 0.19094652 + structure_element + cleaner0 + 2023-07-05T16:36:15Z + SO: + + B + + + 0.98470205 + structure_element + cleaner0 + 2023-07-06T12:17:36Z + SO: + + helix + + + 0.94984627 + structure_element + cleaner0 + 2023-07-06T12:17:39Z + SO: + + α6 + + + 0.9989864 + residue_name_number + cleaner0 + 2023-07-06T08:21:04Z + DUMMY: + + L130 + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:13Z + + chain B + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + hydrophobic packing interactions + + + 0.9989598 + residue_name_number + cleaner0 + 2023-07-06T08:21:08Z + DUMMY: + + L130 + + + 0.998961 + residue_name_number + cleaner0 + 2023-07-06T08:21:12Z + DUMMY: + + L133 + + + 0.998955 + residue_name_number + cleaner0 + 2023-07-06T08:21:15Z + DUMMY: + + L134 + + + 0.99896586 + residue_name_number + cleaner0 + 2023-07-06T08:21:18Z + DUMMY: + + L137 + + + structure_element + SO: + cleaner0 + 2023-07-05T16:34:02Z + + chain A + + + 0.99414223 + experimental_method + cleaner0 + 2023-07-06T12:01:42Z + MESH: + + SE-HPLC + + + 0.71256983 + protein_state + cleaner0 + 2023-07-05T16:26:10Z + DUMMY: + + mutant + + + 0.99403435 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.9974262 + protein_state + cleaner0 + 2023-07-05T16:24:28Z + DUMMY: + + wild-type + + + 0.9963147 + protein_state + cleaner0 + 2023-07-05T16:24:33Z + DUMMY: + + WT + + + 0.99613416 + protein_state + cleaner0 + 2023-07-05T16:24:33Z + DUMMY: + + WT + + + 0.998431 + mutant + cleaner0 + 2023-07-05T16:25:55Z + MESH: + + L130K + + + 0.9983797 + mutant + cleaner0 + 2023-07-05T16:26:00Z + MESH: + + L133K + + + 0.9958579 + oligomeric_state + cleaner0 + 2023-07-06T08:36:49Z + DUMMY: + + dimer + + + 0.9769496 + experimental_method + cleaner0 + 2023-07-06T12:01:45Z + MESH: + + SE-HPLC/MALLS + + + 0.99842215 + mutant + cleaner0 + 2023-07-05T16:25:55Z + MESH: + + L130K + + + 0.9901176 + protein_state + cleaner0 + 2023-07-05T16:26:10Z + DUMMY: + + mutant + + + 0.99585176 + oligomeric_state + cleaner0 + 2023-07-06T08:36:53Z + DUMMY: + + dimer + + + 0.9947885 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + + RESULTS + title_2 + 20621 + The holo-NadR structure presents only one occupied ligand-binding pocket + + 0.9975821 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.77789515 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.9968785 + evidence + cleaner0 + 2023-07-06T08:32:23Z + DUMMY: + + structure + + + 0.9981352 + site + cleaner0 + 2023-07-06T12:09:12Z + SO: + + ligand-binding pocket + + + + RESULTS + paragraph + 20694 + The NadR/4-HPA structure revealed the ligand-binding site nestled between the dimerization and DNA-binding domains (Fig 2). The ligand showed a different position and orientation compared to salicylate complexed with MTH313 and ST1710 (see Discussion). The binding pocket was almost entirely filled by 4-HPA and one water molecule, although there also remained a small tunnel 2-4Å in diameter and 5-6Å long leading from the pocket (proximal to the 4-hydroxyl position) to the protein surface. The tunnel was lined with rather hydrophobic amino acids, and did not contain water molecules. Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions. + + complex_assembly + GO: + cleaner0 + 2023-07-05T16:22:40Z + + NadR/4-HPA + + + 0.9955249 + evidence + cleaner0 + 2023-07-06T08:32:28Z + DUMMY: + + structure + + + 0.99813354 + site + cleaner0 + 2023-07-06T12:09:16Z + SO: + + ligand-binding site + + + structure_element + SO: + cleaner0 + 2023-07-06T12:17:57Z + + dimerization and DNA-binding domains + + + 0.9985886 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.7387806 + protein_state + cleaner0 + 2023-07-06T08:43:01Z + DUMMY: + + complexed with + + + 0.99255496 + protein + cleaner0 + 2023-07-05T16:10:57Z + PR: + + MTH313 + + + 0.97436315 + protein + cleaner0 + 2023-07-05T16:11:06Z + PR: + + ST1710 + + + 0.9978281 + site + cleaner0 + 2023-07-06T12:09:19Z + SO: + + binding pocket + + + 0.9974978 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.9983492 + chemical + cleaner0 + 2023-07-06T08:24:52Z + CHEBI: + + water + + + 0.9884349 + site + cleaner0 + 2023-07-06T12:09:36Z + SO: + + tunnel + + + 0.98912305 + site + cleaner0 + 2023-07-06T12:09:38Z + SO: + + pocket + + + 0.9682272 + site + cleaner0 + 2023-07-06T12:09:41Z + SO: + + tunnel + + + 0.99737287 + chemical + cleaner0 + 2023-07-06T08:24:55Z + CHEBI: + + water + + + 0.9947184 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T16:18:21Z + + holo + + + 0.996798 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.9954543 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.9967265 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.9977387 + site + cleaner0 + 2023-07-06T12:09:45Z + SO: + + binding pocket + + + 0.60767347 + site + cleaner0 + 2023-07-06T12:09:55Z + SO: + + pocket + + + 0.9937448 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + 0.99742675 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + + RESULTS + paragraph + 21535 + Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). At the other ‘end’ of the ligand, the 4-hydroxyl group was proximal to AspB36, with which it may establish an H-bond (see bond distances in Table 3). The water molecule observed in the pocket was bound by the carboxylate group and the side chains of SerA9 and AsnA11. + + 0.68997246 + site + cleaner0 + 2023-07-06T12:10:00Z + SO: + + protein-ligand interaction network + + + 0.97603875 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + hydrogen (H)-bonds + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + ionic interactions + + + 0.996655 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.999091 + residue_name_number + cleaner0 + 2023-07-05T16:30:03Z + DUMMY: + + Ser9 + + + structure_element + SO: + cleaner0 + 2023-07-05T16:34:01Z + + chain A + + + 0.7951284 + residue_name_number + cleaner0 + 2023-07-05T16:31:00Z + DUMMY: + + SerA9 + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:13Z + + chain B + + + 0.9991043 + residue_name_number + cleaner0 + 2023-07-05T16:30:09Z + DUMMY: + + TrpB39 + + + 0.999084 + residue_name_number + cleaner0 + 2023-07-05T16:30:14Z + DUMMY: + + ArgB43 + + + 0.99910814 + residue_name_number + cleaner0 + 2023-07-05T16:30:19Z + DUMMY: + + TyrB115 + + + 0.99911577 + residue_name_number + cleaner0 + 2023-07-05T16:30:27Z + DUMMY: + + AspB36 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + H-bond + + + 0.99828917 + chemical + cleaner0 + 2023-07-06T08:24:58Z + CHEBI: + + water + + + 0.9987155 + residue_name_number + cleaner0 + 2023-07-05T16:31:00Z + DUMMY: + + SerA9 + + + 0.99903935 + residue_name_number + cleaner0 + 2023-07-05T16:30:36Z + DUMMY: + + AsnA11 + + + + ppat.1005557.g004.jpg + ppat.1005557.g004 + FIG + fig_title_caption + 22137 + Atomic details of NadR/HPA interactions. + + 0.9616745 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.5170488 + chemical + cleaner0 + 2023-07-06T08:15:18Z + CHEBI: + + HPA + + + + ppat.1005557.g004.jpg + ppat.1005557.g004 + FIG + fig_caption + 22178 + +A) A stereo-view zoom into the binding pocket showing side chain sticks for all interactions between NadR and 4-HPA. Green and blue ribbons depict NadR chains A and B, respectively. 4-HPA is shown in yellow sticks, with oxygen atoms in red. A water molecule is shown by the red sphere. H-bonds up to 3.6Å are shown as dashed lines. The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). Residues AsnA11 and ArgB18 likely make indirect yet local contributions to ligand binding, mainly by stabilizing the position of AspB36. Bond distances for interacting polar atoms are provided in Table 3. Side chains mediating hydrophobic interactions are shown in orange. (B) A model was prepared to visualize putative interactions of 3Cl,4-HPA (pink) with NadR, revealing the potential for additional contacts (dashed lines) of the chloro moiety (green stick) with LeuB29 and AspB36. + + 0.9978131 + site + cleaner0 + 2023-07-06T12:10:06Z + SO: + + binding pocket + + + 0.99591124 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.99723047 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.99690944 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:43Z + + chains A and B + + + 0.9973424 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.99820757 + chemical + cleaner0 + 2023-07-06T08:25:03Z + CHEBI: + + water + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + H-bonds + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + H-bonds + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + non-bonded contacts + + + 0.9970533 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.9989214 + residue_name_number + cleaner0 + 2023-07-05T16:30:59Z + DUMMY: + + SerA9 + + + 0.9991154 + residue_name_number + cleaner0 + 2023-07-05T16:30:37Z + DUMMY: + + AsnA11 + + + 0.9990701 + residue_name_number + cleaner0 + 2023-07-05T16:31:08Z + DUMMY: + + LeuB21 + + + 0.999102 + residue_name_number + cleaner0 + 2023-07-05T16:31:14Z + DUMMY: + + MetB22 + + + 0.9990681 + residue_name_number + cleaner0 + 2023-07-05T16:31:20Z + DUMMY: + + PheB25 + + + 0.99906904 + residue_name_number + cleaner0 + 2023-07-05T16:31:26Z + DUMMY: + + LeuB29 + + + 0.9991032 + residue_name_number + cleaner0 + 2023-07-05T16:30:30Z + DUMMY: + + AspB36 + + + 0.999113 + residue_name_number + cleaner0 + 2023-07-05T16:30:10Z + DUMMY: + + TrpB39 + + + 0.9991092 + residue_name_number + cleaner0 + 2023-07-05T16:30:15Z + DUMMY: + + ArgB43 + + + 0.9991091 + residue_name_number + cleaner0 + 2023-07-05T16:31:48Z + DUMMY: + + ValB111 + + + 0.9991191 + residue_name_number + cleaner0 + 2023-07-05T16:30:20Z + DUMMY: + + TyrB115 + + + 0.59851336 + experimental_method + cleaner0 + 2023-07-06T12:01:54Z + MESH: + + PDBsum + + + 0.9991345 + residue_name_number + cleaner0 + 2023-07-05T16:30:37Z + DUMMY: + + AsnA11 + + + 0.9991111 + residue_name_number + cleaner0 + 2023-07-05T16:32:02Z + DUMMY: + + ArgB18 + + + 0.9991405 + residue_name_number + cleaner0 + 2023-07-05T16:30:30Z + DUMMY: + + AspB36 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + hydrophobic interactions + + + chemical + CHEBI: + cleaner0 + 2023-07-05T16:12:21Z + + 3Cl,4-HPA + + + 0.9978667 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.99910825 + residue_name_number + cleaner0 + 2023-07-05T16:31:26Z + DUMMY: + + LeuB29 + + + 0.99913305 + residue_name_number + cleaner0 + 2023-07-05T16:30:30Z + DUMMY: + + AspB36 + + + + ppat.1005557.t003.xml + ppat.1005557.t003 + TABLE + table_title_caption + 23247 + List of 4-HPA atoms bound to NadR via ionic interactions and/or H-bonds. + + 0.9970317 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + 0.996888 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + ionic interactions + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + H-bonds + + + + ppat.1005557.t003.xml + ppat.1005557.t003 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><thead><tr><th align="center" rowspan="1" colspan="1">4-HPA atom</th><th align="center" rowspan="1" colspan="1">NadR residue/atom</th><th align="center" rowspan="1" colspan="1">Distance (Å)</th></tr></thead><tbody><tr><td align="center" rowspan="1" colspan="1">O2</td><td align="center" rowspan="1" colspan="1">TrpB39/NE1</td><td align="center" rowspan="1" colspan="1">2.83</td></tr><tr><td align="center" rowspan="1" colspan="1">O2</td><td align="center" rowspan="1" colspan="1">ArgB43/NH1</td><td align="center" rowspan="1" colspan="1">2.76</td></tr><tr><td align="center" rowspan="1" colspan="1">O1</td><td align="center" rowspan="1" colspan="1">ArgB43/NH1</td><td align="center" rowspan="1" colspan="1">3.84</td></tr><tr><td align="center" rowspan="1" colspan="1">O1</td><td align="center" rowspan="1" colspan="1">SerA9/OG</td><td align="center" rowspan="1" colspan="1">2.75</td></tr><tr><td align="center" rowspan="1" colspan="1">O1</td><td align="center" rowspan="1" colspan="1">TyrB115/OH</td><td align="center" rowspan="1" colspan="1">2.50</td></tr><tr><td align="center" rowspan="1" colspan="1">O2</td><td align="center" rowspan="1" colspan="1">Water (<xref ref-type="table-fn" rid="t003fn001">*</xref>Ser9/Asn11)</td><td align="center" rowspan="1" colspan="1">2.88</td></tr><tr><td align="center" rowspan="1" colspan="1">OH</td><td align="center" rowspan="1" colspan="1">AspB36/OD1/OD2</td><td align="center" rowspan="1" colspan="1">3.6/3.7</td></tr></tbody></table> + + 23320 + 4-HPA atom NadR residue/atom Distance (Å) O2 TrpB39/NE1 2.83 O2 ArgB43/NH1 2.76 O1 ArgB43/NH1 3.84 O1 SerA9/OG 2.75 O1 TyrB115/OH 2.50 O2 Water (*Ser9/Asn11) 2.88 OH AspB36/OD1/OD2 3.6/3.7 + + 0.99659586 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + protein + PR: + cleaner0 + 2023-07-05T16:01:47Z + + NadR + + + 0.99603003 + residue_name_number + cleaner0 + 2023-07-05T16:30:10Z + DUMMY: + + TrpB39 + + + 0.99567515 + residue_name_number + cleaner0 + 2023-07-05T16:30:15Z + DUMMY: + + ArgB43 + + + 0.99616563 + residue_name_number + cleaner0 + 2023-07-05T16:30:15Z + DUMMY: + + ArgB43 + + + 0.9866859 + residue_name_number + cleaner0 + 2023-07-05T16:31:00Z + DUMMY: + + SerA9 + + + 0.996403 + residue_name_number + cleaner0 + 2023-07-05T16:30:20Z + DUMMY: + + TyrB115 + + + 0.9975382 + chemical + cleaner0 + 2023-07-06T08:25:17Z + CHEBI: + + Water + + + 0.9978388 + residue_name_number + cleaner0 + 2023-07-05T16:30:05Z + DUMMY: + + Ser9 + + + 0.99788034 + residue_name_number + cleaner0 + 2023-07-06T08:02:37Z + DUMMY: + + Asn11 + + + 0.99583423 + residue_name_number + cleaner0 + 2023-07-05T16:30:30Z + DUMMY: + + AspB36 + + + + ppat.1005557.t003.xml + ppat.1005557.t003 + TABLE + table_footnote + 23527 + * Bond distance between the ligand carboxylate group and the water molecule, which in turn makes H-bond to the SerA9 and AsnA11 side chains. + + 0.9985153 + chemical + cleaner0 + 2023-07-06T08:25:25Z + CHEBI: + + water + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + H-bond + + + 0.9948244 + residue_name_number + cleaner0 + 2023-07-05T16:31:00Z + DUMMY: + + SerA9 + + + 0.99785656 + residue_name_number + cleaner0 + 2023-07-05T16:30:37Z + DUMMY: + + AsnA11 + + + + RESULTS + paragraph + 23668 + In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals’ contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A). Notably, the phenyl ring of PheB25 was positioned parallel to the phenyl ring of 4-HPA, potentially forming π-π parallel-displaced stacking interactions. Consequently, residues in the 4-HPA binding pocket are mostly contributed by NadR chain B, and effectively created a polar ‘floor’ and a hydrophobic ‘ceiling’, which house the ligand. Collectively, this mixed network of polar and hydrophobic interactions endows NadR with a strong recognition pattern for HPAs, with additional medium-range interactions potentially established with the hydroxyl group at the 4-position. + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + H-bonds + + + 0.99728394 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + van der Waals’ contacts + + + 0.9991007 + residue_name_number + cleaner0 + 2023-07-05T16:31:09Z + DUMMY: + + LeuB21 + + + 0.99911135 + residue_name_number + cleaner0 + 2023-07-05T16:31:15Z + DUMMY: + + MetB22 + + + 0.9991079 + residue_name_number + cleaner0 + 2023-07-05T16:31:21Z + DUMMY: + + PheB25 + + + 0.99910295 + residue_name_number + cleaner0 + 2023-07-05T16:31:26Z + DUMMY: + + LeuB29 + + + 0.9991049 + residue_name_number + cleaner0 + 2023-07-05T16:31:48Z + DUMMY: + + ValB111 + + + 0.9991109 + residue_name_number + cleaner0 + 2023-07-05T16:31:21Z + DUMMY: + + PheB25 + + + 0.99720365 + chemical + cleaner0 + 2023-07-05T16:05:12Z + CHEBI: + + 4-HPA + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + π-π parallel-displaced stacking interactions + + + 0.9981514 + site + cleaner0 + 2023-07-06T12:10:13Z + SO: + + 4-HPA binding pocket + + + 0.9982968 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:13Z + + chain B + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + polar and hydrophobic interactions + + + 0.9981133 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.98768824 + chemical + cleaner0 + 2023-07-05T16:18:03Z + CHEBI: + + HPAs + + + + RESULTS + title_2 + 24546 + Structure-activity relationships: molecular basis of enhanced stabilization by 3Cl,4-HPA + + chemical + CHEBI: + cleaner0 + 2023-07-05T16:12:21Z + + 3Cl,4-HPA + + + + RESULTS + paragraph + 24635 + We modelled the binding of other HPAs by in silico superposition onto 4-HPA in the holo-NadR structure, and thereby obtained molecular explanations for the binding specificities of diverse ligands. For example, similar to 4-HPA, the binding of 3Cl,4-HPA could involve multiple bonds towards the carboxylate group of the ligand and some to the 4-hydroxyl group. Additionally, the side chains of LeuB29 and AspB36 would be only 2.6–3.5 Å from the chlorine atom, thus providing van der Waals’ interactions or H-bonds to generate the additional binding affinity observed for 3Cl,4-HPA (Fig 4B). The presence of a single hydroxyl group at position 2, as in 2-HPA, rather than at position 4, would eliminate the possibility of favorable interactions with AspB36, resulting in the lack of NadR regulation by 2-HPA described previously. Finally, salicylate is presumably unable to specifically bind NadR due to the 2-hydroxyl substitution and the shorter aliphatic chain connecting its carboxylate group (Fig 1A): the compound simply seems too small to simultaneously establish the network of beneficial bonds observed in the NadR/HPA interactions. + + 0.9514532 + experimental_method + cleaner0 + 2023-07-06T12:02:02Z + MESH: + + modelled + + + 0.9974298 + chemical + cleaner0 + 2023-07-05T16:18:03Z + CHEBI: + + HPAs + + + 0.83580416 + experimental_method + cleaner0 + 2023-07-06T12:02:05Z + MESH: + + in silico superposition + + + 0.9979047 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.99764794 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.99511176 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.99702317 + evidence + cleaner0 + 2023-07-06T08:32:33Z + DUMMY: + + structure + + + 0.997828 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9963824 + chemical + cleaner0 + 2023-07-05T16:12:21Z + CHEBI: + + 3Cl,4-HPA + + + 0.9990632 + residue_name_number + cleaner0 + 2023-07-05T16:31:26Z + DUMMY: + + LeuB29 + + + 0.9991033 + residue_name_number + cleaner0 + 2023-07-05T16:30:30Z + DUMMY: + + AspB36 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + van der Waals’ interactions + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:15Z + + H-bonds + + + 0.84820175 + evidence + cleaner0 + 2023-07-06T08:32:38Z + DUMMY: + + binding affinity + + + 0.9964987 + chemical + cleaner0 + 2023-07-05T16:12:21Z + CHEBI: + + 3Cl,4-HPA + + + 0.9977615 + chemical + cleaner0 + 2023-07-06T08:25:31Z + CHEBI: + + 2-HPA + + + 0.99911207 + residue_name_number + cleaner0 + 2023-07-05T16:30:30Z + DUMMY: + + AspB36 + + + protein + PR: + cleaner0 + 2023-07-05T16:01:47Z + + NadR + + + 0.9978326 + chemical + cleaner0 + 2023-07-06T08:25:47Z + CHEBI: + + 2-HPA + + + 0.99866676 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.99530065 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + protein + PR: + cleaner0 + 2023-07-05T16:34:50Z + + NadR + + + chemical + CHEBI: + cleaner0 + 2023-07-05T16:34:59Z + + HPA + + + + RESULTS + title_2 + 25781 + Analysis of the pockets reveals the molecular basis for asymmetric binding and stoichiometry + + 0.9929965 + site + cleaner0 + 2023-07-06T12:10:20Z + SO: + + pockets + + + + RESULTS + paragraph + 25874 + We attempted to investigate further the binding stoichiometry using solution-based techniques. However, studies based on tryptophan fluorescence were confounded by the fluorescence of the HPA ligands, and isothermal titration calorimetry (ITC) was unfeasible due to the need for very high concentrations of NadR in the ITC chamber (due to the relatively low affinity), which exceeded the solubility limits of the protein. However, it was possible to calculate the binding stoichiometry of the NadR-HPA interactions using an SPR-based approach. In SPR, the signal measured is proportional to the total molecular mass proximal to the sensor surface; consequently, if the molecular weights of the interactors are known, then the stoichiometry of the resulting complex can be determined. This approach relies on the assumption that the captured protein (‘the ligand’, according to SPR conventions) is 100% active and freely-accessible to potential interactors (‘the analytes’). This assumption is likely valid for this pair of interactors, for two main reasons. Firstly, NadR is expected to be covalently immobilized on the sensor chip as a dimer in random orientations, since it is a stable dimer in solution and has sixteen lysines well-distributed around its surface, all able to act as potential sites for amine coupling to the chip, and none of which are close to the ligand-binding pocket. Secondly, the HPA analytes are all very small (MW 150–170, Fig 1A) and therefore are expected to be able to diffuse readily into all potential binding sites, irrespective of the random orientations of the immobilized NadR dimers on the chip.The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule. + + 0.51442033 + experimental_method + cleaner0 + 2023-07-06T12:02:17Z + MESH: + + tryptophan fluorescence + + + 0.95978224 + chemical + cleaner0 + 2023-07-06T08:25:51Z + CHEBI: + + HPA + + + 0.9953435 + experimental_method + cleaner0 + 2023-07-06T12:02:20Z + MESH: + + isothermal titration calorimetry + + + 0.98154825 + experimental_method + cleaner0 + 2023-07-06T12:02:23Z + MESH: + + ITC + + + 0.9986652 + protein + cleaner0 + 2023-07-05T16:01:47Z + PR: + + NadR + + + 0.48151696 + experimental_method + cleaner0 + 2023-07-06T12:02:27Z + MESH: + + ITC + + + 0.6601409 + evidence + cleaner0 + 2023-07-06T08:32:43Z + DUMMY: + + binding stoichiometry + + + 0.9863785 + complex_assembly + cleaner0 + 2023-07-06T08:22:40Z + GO: + + NadR-HPA + + + 0.9919219 + experimental_method + cleaner0 + 2023-07-06T12:02:30Z + MESH: + + SPR + + + 0.9925154 + experimental_method + cleaner0 + 2023-07-06T12:02:34Z + MESH: + + SPR + + + 0.9252812 + experimental_method + cleaner0 + 2023-07-06T12:02:39Z + MESH: + + SPR + + + 0.997682 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.9963148 + oligomeric_state + cleaner0 + 2023-07-06T08:36:58Z + DUMMY: + + dimer + + + 0.9510204 + protein_state + cleaner0 + 2023-07-06T08:43:25Z + DUMMY: + + stable + + + 0.99628484 + oligomeric_state + cleaner0 + 2023-07-06T08:37:02Z + DUMMY: + + dimer + + + 0.9714164 + residue_name + cleaner0 + 2023-07-06T08:23:16Z + SO: + + lysines + + + 0.9982489 + site + cleaner0 + 2023-07-06T12:10:27Z + SO: + + ligand-binding pocket + + + 0.93585426 + chemical + cleaner0 + 2023-07-06T08:25:57Z + CHEBI: + + HPA + + + 0.992237 + site + cleaner0 + 2023-07-06T12:10:29Z + SO: + + binding sites + + + 0.9982815 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99626595 + oligomeric_state + cleaner0 + 2023-07-05T16:22:59Z + DUMMY: + + dimers + + + 0.98445946 + complex_assembly + cleaner0 + 2023-07-06T08:22:44Z + GO: + + NadR-HPA + + + 0.578732 + evidence + cleaner0 + 2023-07-06T08:32:51Z + DUMMY: + + stoichiometries + + + 0.99664 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9967003 + chemical + cleaner0 + 2023-07-05T16:14:41Z + CHEBI: + + 3-HPA + + + 0.8855335 + chemical + cleaner0 + 2023-07-05T16:12:21Z + CHEBI: + + 3Cl,4-HPA + + + 0.998278 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99647456 + oligomeric_state + cleaner0 + 2023-07-06T08:37:05Z + DUMMY: + + dimer + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:43:42Z + + bound to + + + 0.99778146 + chemical + cleaner0 + 2023-07-06T08:26:00Z + CHEBI: + + HPA + + + + RESULTS + paragraph + 27780 + The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins. To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B). Overall, the superposition revealed a high degree of structural similarity (Cα root mean square deviation (rmsd) of 1.5Å), though on closer inspection a rotational difference of ~9 degrees along the long axis of helix α6 was observed, suggesting that 4-HPA induced a slight conformational change (Fig 5A). However, since residues of helix α6 were not directly involved in ligand binding, an explanation for the lack of 4-HPA in monomer A did not emerge by analyzing only these backbone atom positions, suggesting that a more complex series of allosteric events may occur. Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly. Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 Å3, while the pocket in chain A was occupied by these three side chains that adopted ‘inward’ positions and thereby divided the space into a few much smaller pockets, each with volume < 50 Å3, evidently rendering chain A unfavorable for ligand binding. Most notably, atomic clashes between the ligand and the side chains of MetA22, PheA25 and ArgA43 would occur if 4-HPA were present in the monomer A pocket (Fig 5B). Subsequently, analyses of the pockets in apo-NadR revealed that in the absence of ligand the long Arg43 side chain was always in the open ‘outward’ position compatible with binding to the 4-HPA carboxylate group. In contrast, the apo-form Met22 and Phe25 residues were still encroaching the spaces of the 4-hydroxyl group and the phenyl ring of the ligand, respectively (Fig 5C). The ‘outward’ position of Arg43 generated an open apo-form pocket with volume approximately 380Å3. Taken together, these observations suggest that Arg43 is a major determinant of ligand binding, and that its ‘inward’ position inhibits the binding of 4-HPA to the empty pocket of holo-NadR. + + 0.9309345 + evidence + cleaner0 + 2023-07-06T08:32:54Z + DUMMY: + + crystallographic data + + + 0.9882294 + experimental_method + cleaner0 + 2023-07-06T12:03:00Z + MESH: + + SPR + + + 0.961169 + evidence + cleaner0 + 2023-07-06T08:32:57Z + DUMMY: + + binding stoichiometry + + + 0.99608153 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9959656 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.80875015 + protein + cleaner0 + 2023-07-05T16:10:58Z + PR: + + MTH313 + + + 0.99761474 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.9970886 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9930714 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99597245 + experimental_method + cleaner0 + 2023-07-06T12:03:04Z + MESH: + + superposed + + + 0.99558645 + protein_state + cleaner0 + 2023-07-05T16:12:38Z + DUMMY: + + ligand-free + + + 0.99573404 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + structure_element + SO: + cleaner0 + 2023-07-05T16:34:02Z + + chain A + + + 0.99643254 + protein_state + cleaner0 + 2023-07-06T08:43:48Z + DUMMY: + + ligand-occupied + + + 0.9957568 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:13Z + + chain B + + + 0.9947714 + experimental_method + cleaner0 + 2023-07-06T12:03:08Z + MESH: + + superposition + + + 0.8987919 + evidence + cleaner0 + 2023-07-06T08:33:09Z + DUMMY: + + root mean square deviation + + + 0.9872866 + evidence + cleaner0 + 2023-07-05T16:51:11Z + DUMMY: + + rmsd + + + 0.98954785 + structure_element + cleaner0 + 2023-07-06T12:18:04Z + SO: + + helix + + + 0.870803 + structure_element + cleaner0 + 2023-07-06T12:18:06Z + SO: + + α6 + + + 0.9959488 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9914029 + structure_element + cleaner0 + 2023-07-06T12:18:08Z + SO: + + helix + + + 0.8272526 + structure_element + cleaner0 + 2023-07-06T12:18:11Z + SO: + + α6 + + + 0.99483943 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.56652486 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + 0.3225745 + structure_element + cleaner0 + 2023-07-05T16:35:52Z + SO: + + A + + + 0.9989826 + residue_name_number + cleaner0 + 2023-07-05T16:37:27Z + DUMMY: + + Met22 + + + 0.9989274 + residue_name_number + cleaner0 + 2023-07-05T16:37:33Z + DUMMY: + + Phe25 + + + 0.99899143 + residue_name_number + cleaner0 + 2023-07-05T16:37:39Z + DUMMY: + + Arg43 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-05T16:23:34Z + + monomer + + + 0.27231634 + structure_element + cleaner0 + 2023-07-05T16:36:14Z + SO: + + B + + + 0.43374527 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + 0.27271384 + structure_element + cleaner0 + 2023-07-05T16:35:54Z + SO: + + A + + + 0.7804215 + site + cleaner0 + 2023-07-06T12:10:35Z + SO: + + pocket + + + 0.9258107 + experimental_method + cleaner0 + 2023-07-06T12:03:13Z + MESH: + + CASTp + + + 0.9522308 + site + cleaner0 + 2023-07-06T12:10:38Z + SO: + + pocket + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:13Z + + chain B + + + 0.995066 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.89990324 + site + cleaner0 + 2023-07-06T12:10:41Z + SO: + + pocket + + + structure_element + SO: + cleaner0 + 2023-07-05T16:34:02Z + + chain A + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:44:58Z + + inward + + + structure_element + SO: + cleaner0 + 2023-07-05T16:34:02Z + + chain A + + + 0.9989052 + residue_name_number + cleaner0 + 2023-07-05T16:37:48Z + DUMMY: + + MetA22 + + + 0.99899954 + residue_name_number + cleaner0 + 2023-07-05T16:37:52Z + DUMMY: + + PheA25 + + + 0.99904007 + residue_name_number + cleaner0 + 2023-07-05T16:37:58Z + DUMMY: + + ArgA43 + + + 0.9953595 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.55017084 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + 0.2799846 + structure_element + cleaner0 + 2023-07-05T16:35:55Z + SO: + + A + + + 0.89701104 + site + cleaner0 + 2023-07-06T12:10:45Z + SO: + + pocket + + + 0.90599453 + site + cleaner0 + 2023-07-06T12:10:48Z + SO: + + pockets + + + 0.997891 + protein_state + cleaner0 + 2023-07-05T16:18:27Z + DUMMY: + + apo + + + 0.99532133 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.94971794 + protein_state + cleaner0 + 2023-07-06T08:43:55Z + DUMMY: + + absence of ligand + + + 0.9989594 + residue_name_number + cleaner0 + 2023-07-05T16:37:40Z + DUMMY: + + Arg43 + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:45:05Z + + outward + + + 0.9965679 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9979519 + protein_state + cleaner0 + 2023-07-05T16:18:27Z + DUMMY: + + apo + + + 0.99890244 + residue_name_number + cleaner0 + 2023-07-05T16:37:28Z + DUMMY: + + Met22 + + + 0.99886096 + residue_name_number + cleaner0 + 2023-07-05T16:37:34Z + DUMMY: + + Phe25 + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:45:05Z + + outward + + + 0.99894184 + residue_name_number + cleaner0 + 2023-07-05T16:37:40Z + DUMMY: + + Arg43 + + + 0.9927199 + protein_state + cleaner0 + 2023-07-06T08:44:20Z + DUMMY: + + open + + + 0.99767154 + protein_state + cleaner0 + 2023-07-05T16:18:27Z + DUMMY: + + apo + + + 0.9245357 + site + cleaner0 + 2023-07-06T12:10:51Z + SO: + + pocket + + + 0.9989795 + residue_name_number + cleaner0 + 2023-07-05T16:37:40Z + DUMMY: + + Arg43 + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:44:58Z + + inward + + + 0.99592376 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9391805 + site + cleaner0 + 2023-07-06T12:10:53Z + SO: + + pocket + + + 0.9977241 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + protein + PR: + cleaner0 + 2023-07-05T16:01:48Z + + NadR + + + + ppat.1005557.g005.jpg + ppat.1005557.g005 + FIG + fig_title_caption + 30286 + Structural differences of NadR in ligand-bound or free forms. + + 0.99626154 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.9965341 + protein_state + cleaner0 + 2023-07-06T08:44:36Z + DUMMY: + + ligand-bound + + + 0.99479055 + protein_state + cleaner0 + 2023-07-06T08:44:40Z + DUMMY: + + free + + + + ppat.1005557.g005.jpg + ppat.1005557.g005 + FIG + fig_caption + 30348 + +(A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. In these crystals, the ArgA43 side chain showed two alternate conformations, modelled with 50% occupancy in each state, as indicated by the two ‘mirrored’ arrows. The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. + + 0.8240444 + experimental_method + cleaner0 + 2023-07-06T12:03:19Z + MESH: + + Aligned + + + 0.9941704 + oligomeric_state + cleaner0 + 2023-07-06T08:37:15Z + DUMMY: + + monomers + + + 0.9975924 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9966384 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + structure_element + SO: + cleaner0 + 2023-07-05T16:34:02Z + + chain A + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:13Z + + chain B + + + 0.9901239 + structure_element + cleaner0 + 2023-07-06T12:18:17Z + SO: + + helix + + + 0.991663 + structure_element + cleaner0 + 2023-07-06T12:18:20Z + SO: + + α6 + + + experimental_method + MESH: + cleaner0 + 2023-07-06T12:11:16Z + + Comparison + + + 0.9876643 + site + cleaner0 + 2023-07-06T12:11:05Z + SO: + + binding pockets + + + 0.99701047 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9959848 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99530977 + protein_state + cleaner0 + 2023-07-05T16:12:38Z + DUMMY: + + ligand-free + + + 0.95212543 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + 0.34092307 + structure_element + cleaner0 + 2023-07-05T16:35:55Z + SO: + + A + + + 0.9991235 + residue_name_number + cleaner0 + 2023-07-05T16:37:28Z + DUMMY: + + Met22 + + + 0.9990852 + residue_name_number + cleaner0 + 2023-07-05T16:37:34Z + DUMMY: + + Phe25 + + + 0.9991242 + residue_name_number + cleaner0 + 2023-07-05T16:37:40Z + DUMMY: + + Arg43 + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:44:58Z + + inward + + + 0.7328055 + protein_state + cleaner0 + 2023-07-06T08:44:44Z + DUMMY: + + ligand-occupied + + + 0.92590094 + site + cleaner0 + 2023-07-06T12:11:21Z + SO: + + pocket + + + 0.50661314 + protein_state + cleaner0 + 2023-07-06T08:44:58Z + DUMMY: + + inward + + + 0.9969907 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.98822 + evidence + cleaner0 + 2023-07-06T08:33:23Z + DUMMY: + + crystals + + + 0.9991375 + residue_name_number + cleaner0 + 2023-07-05T16:37:59Z + DUMMY: + + ArgA43 + + + 0.99679786 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + site + SO: + cleaner0 + 2023-07-06T08:45:44Z + + pocket + + + 0.997463 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9966234 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.923355 + site + cleaner0 + 2023-07-06T12:11:25Z + SO: + + pockets + + + 0.9979037 + protein_state + cleaner0 + 2023-07-05T16:18:27Z + DUMMY: + + apo + + + 0.99683505 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.9961276 + protein_state + cleaner0 + 2023-07-06T08:45:55Z + DUMMY: + + absence of + + + 0.9955799 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.99911577 + residue_name_number + cleaner0 + 2023-07-05T16:37:40Z + DUMMY: + + Arg43 + + + 0.50120544 + protein_state + cleaner0 + 2023-07-06T08:45:04Z + DUMMY: + + outward + + + + RESULTS + paragraph + 31380 + Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). The 1H-15N TROSY-HSQC spectrum of apo-NadR, acquired at 25°C, displayed approximately 140 distinct peaks (Fig 6A), most of which correspond to backbone amide N-H groups. The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25°C, two or more distinct conformations of apo-NadR monomers were not readily apparent. Upon the addition of 4-HPA, over 45 peaks showed chemical shift perturbations, i.e. changed position in the spectrum or disappeared, while the remaining peaks remained unchanged. This observation showed that 4-HPA was able to bind NadR and induce notable changes in specific regions of the protein. + + 0.9907934 + experimental_method + cleaner0 + 2023-07-06T12:03:23Z + MESH: + + 15N heteronuclear solution NMR spectroscopy + + + 0.99501413 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.99779665 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9982626 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + experimental_method + MESH: + cleaner0 + 2023-07-05T16:48:33Z + + NMR + + + evidence + DUMMY: + cleaner0 + 2023-07-05T16:48:22Z + + spectra + + + 0.998331 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:46:17Z + + in the presence + + + 0.9639535 + protein_state + cleaner0 + 2023-07-06T08:46:22Z + DUMMY: + + absence of + + + 0.9935445 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9916582 + experimental_method + cleaner0 + 2023-07-06T12:03:30Z + MESH: + + 1H-15N TROSY-HSQC + + + 0.94737655 + evidence + cleaner0 + 2023-07-06T08:33:30Z + DUMMY: + + spectrum + + + 0.9978265 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9972324 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.9978796 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9976908 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:46:44Z + + well-folded + + + experimental_method + MESH: + cleaner0 + 2023-07-05T16:48:34Z + + NMR + + + 0.9822833 + experimental_method + cleaner0 + 2023-07-05T16:48:34Z + MESH: + + NMR + + + 0.99777395 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.99778455 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.9940479 + oligomeric_state + cleaner0 + 2023-07-06T08:37:18Z + DUMMY: + + monomers + + + 0.9915989 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9925506 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.998434 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + + ppat.1005557.g006.jpg + ppat.1005557.g006 + FIG + fig_title_caption + 32464 + NMR spectra of NadR in the presence and absence of 4-HPA. + + experimental_method + MESH: + cleaner0 + 2023-07-05T16:48:34Z + + NMR + + + evidence + DUMMY: + cleaner0 + 2023-07-05T16:49:00Z + + spectra + + + 0.9984425 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:47:17Z + + in the presence + + + 0.9574306 + protein_state + cleaner0 + 2023-07-06T08:47:20Z + DUMMY: + + absence of + + + 0.99724704 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + + ppat.1005557.g006.jpg + ppat.1005557.g006 + FIG + fig_caption + 32522 + +(A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25°C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red). (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). The spectra acquired at 10°C are excluded from panel A for simplicity. + + 0.9234609 + experimental_method + cleaner0 + 2023-07-06T12:03:48Z + MESH: + + Superposition + + + 0.98206204 + experimental_method + cleaner0 + 2023-07-06T12:03:51Z + MESH: + + 1H-15N TROSY-HSQC + + + 0.4906918 + evidence + cleaner0 + 2023-07-05T16:49:01Z + DUMMY: + + spectra + + + 0.9975116 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9983646 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99781954 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.6589855 + protein_state + cleaner0 + 2023-07-06T08:47:24Z + DUMMY: + + presence of + + + 0.996272 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.6536667 + experimental_method + cleaner0 + 2023-07-06T12:03:55Z + MESH: + + Overlay + + + 0.9817009 + experimental_method + cleaner0 + 2023-07-06T12:03:59Z + MESH: + + 1H-15N TROSY-HSQC + + + evidence + DUMMY: + cleaner0 + 2023-07-05T16:49:01Z + + spectra + + + 0.9968502 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9982101 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + complex_assembly + GO: + cleaner0 + 2023-07-05T16:22:40Z + + NadR/4-HPA + + + 0.5712608 + experimental_method + cleaner0 + 2023-07-06T12:04:03Z + MESH: + + superimposed + + + evidence + DUMMY: + cleaner0 + 2023-07-05T16:49:01Z + + spectra + + + 0.99734455 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.99837685 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + complex_assembly + GO: + cleaner0 + 2023-07-05T16:22:40Z + + NadR/4-HPA + + + evidence + DUMMY: + cleaner0 + 2023-07-05T16:49:02Z + + spectra + + + + RESULTS + paragraph + 32943 + However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable. Considering the small size, fast diffusion and relatively low binding affinity of 4-HPA, it would not be surprising if the ligand associates and dissociates rapidly on the NMR time scale, resulting in only one set of peaks whose chemical shifts represent the average environment of the bound and unbound states. Interestingly, by cooling the samples to 10°C, we observed that a number of those peaks strongly affected by 4-HPA (and therefore likely to be in the ligand-binding site) demonstrated evidence of peak splitting, i.e. a tendency to become two distinct peaks rather than one single peak (Fig 6B and 6C). These doubled peaks may therefore reveal that the cooler temperature partially trapped the existence in solution of two distinct states, in presence or absence of 4-HPA, with minor conformational differences occurring at least in proximity to the binding pocket. Although more comprehensive NMR experiments and full chemical shift assignment of the spectra would be required to precisely define this multi-state behavior, the NMR data clearly demonstrate that NadR exhibits conformational flexibility which is modulated by 4-HPA in solution. + + 0.98322904 + protein_state + cleaner0 + 2023-07-06T08:47:27Z + DUMMY: + + presence of + + + 0.9958422 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9887062 + experimental_method + cleaner0 + 2023-07-06T12:04:07Z + MESH: + + 1H-15N TROSY-HSQC + + + 0.96818626 + evidence + cleaner0 + 2023-07-06T08:33:35Z + DUMMY: + + spectrum + + + 0.99864155 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99783957 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.99860746 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.90495 + experimental_method + cleaner0 + 2023-07-06T12:04:10Z + MESH: + + crystallographically + + + evidence + DUMMY: + cleaner0 + 2023-07-06T08:33:54Z + + binding affinity + + + 0.99629325 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.97597426 + experimental_method + cleaner0 + 2023-07-05T16:48:34Z + MESH: + + NMR + + + 0.99651694 + protein_state + cleaner0 + 2023-07-06T08:47:32Z + DUMMY: + + bound + + + 0.99608624 + protein_state + cleaner0 + 2023-07-06T08:47:35Z + DUMMY: + + unbound + + + 0.9951579 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9980556 + site + cleaner0 + 2023-07-06T12:11:30Z + SO: + + ligand-binding site + + + 0.94725573 + protein_state + cleaner0 + 2023-07-06T08:47:42Z + DUMMY: + + presence + + + 0.9762615 + protein_state + cleaner0 + 2023-07-06T08:47:44Z + DUMMY: + + absence of + + + 0.9945387 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9978014 + site + cleaner0 + 2023-07-06T12:11:33Z + SO: + + binding pocket + + + 0.9899743 + experimental_method + cleaner0 + 2023-07-05T16:48:34Z + MESH: + + NMR + + + evidence + DUMMY: + cleaner0 + 2023-07-05T16:49:02Z + + spectra + + + 0.9846875 + experimental_method + cleaner0 + 2023-07-05T16:48:34Z + MESH: + + NMR + + + 0.99864143 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99530214 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + + RESULTS + title_2 + 34376 + Apo-NadR structures reveal intrinsic conformational flexibility + + 0.9978946 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + Apo + + + 0.8863035 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99732757 + evidence + cleaner0 + 2023-07-06T08:34:02Z + DUMMY: + + structures + + + + RESULTS + paragraph + 34440 + The apo-NadR crystal structure contained two homodimers in the asymmetric unit (chains A+B and chains C+D). Upon overall structural superposition, these dimers revealed a few minor differences in the α6 helix (a major component of the dimer interface) and the helices α4-α5 (the DNA binding region), and an rmsd of 1.55Å (Fig 7A). Similarly, the entire holo-homodimer could be closely superposed onto each of the apo-homodimers, showing rmsd values of 1.29Å and 1.31Å, and with more notable differences in the α6 helix positions (Fig 7B). The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility. + + 0.99693346 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9919003 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.9970949 + evidence + cleaner0 + 2023-07-06T08:34:05Z + DUMMY: + + crystal structure + + + 0.9959527 + oligomeric_state + cleaner0 + 2023-07-05T16:51:19Z + DUMMY: + + homodimers + + + structure_element + SO: + cleaner0 + 2023-07-05T16:50:42Z + + chains A+B + + + structure_element + SO: + cleaner0 + 2023-07-05T16:50:52Z + + chains C+D + + + 0.995749 + experimental_method + cleaner0 + 2023-07-06T12:04:22Z + MESH: + + structural superposition + + + 0.9959527 + oligomeric_state + cleaner0 + 2023-07-05T16:22:59Z + DUMMY: + + dimers + + + 0.9938742 + structure_element + cleaner0 + 2023-07-06T12:18:28Z + SO: + + α6 helix + + + 0.988893 + site + cleaner0 + 2023-07-06T12:11:36Z + SO: + + dimer interface + + + 0.93791986 + structure_element + cleaner0 + 2023-07-06T12:18:36Z + SO: + + helices + + + 0.9968322 + structure_element + cleaner0 + 2023-07-06T12:18:39Z + SO: + + α4-α5 + + + 0.9906445 + site + cleaner0 + 2023-07-06T12:11:39Z + SO: + + DNA binding region + + + 0.9954869 + evidence + cleaner0 + 2023-07-05T16:51:09Z + DUMMY: + + rmsd + + + 0.99730045 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.99183285 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.9215127 + experimental_method + cleaner0 + 2023-07-06T12:04:29Z + MESH: + + closely superposed + + + 0.9976131 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.98759776 + oligomeric_state + cleaner0 + 2023-07-05T16:51:20Z + DUMMY: + + homodimers + + + 0.9925884 + evidence + cleaner0 + 2023-07-05T16:51:11Z + DUMMY: + + rmsd + + + 0.9951743 + structure_element + cleaner0 + 2023-07-06T12:18:42Z + SO: + + α6 helix + + + 0.9944622 + evidence + cleaner0 + 2023-07-05T16:51:11Z + DUMMY: + + rmsd + + + 0.99771786 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.989388 + oligomeric_state + cleaner0 + 2023-07-05T16:51:20Z + DUMMY: + + homodimers + + + 0.9976179 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9975793 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.98964864 + oligomeric_state + cleaner0 + 2023-07-05T16:51:20Z + DUMMY: + + homodimers + + + 0.9975782 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9933122 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + + ppat.1005557.g007.jpg + ppat.1005557.g007 + FIG + fig_title_caption + 35197 + Overall apo- and holo-NadR structures are similar. + + 0.9978346 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9977877 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9330376 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.9972395 + evidence + cleaner0 + 2023-07-06T08:34:10Z + DUMMY: + + structures + + + + ppat.1005557.g007.jpg + ppat.1005557.g007 + FIG + fig_caption + 35248 + +(A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. Here, larger differences are observed in the α6 helices (top). + + 0.9300884 + experimental_method + cleaner0 + 2023-07-06T12:04:33Z + MESH: + + Pairwise alignment + + + 0.99730194 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.98105603 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99527574 + oligomeric_state + cleaner0 + 2023-07-05T16:51:20Z + DUMMY: + + homodimers + + + 0.96431 + structure_element + cleaner0 + 2023-07-05T16:53:24Z + SO: + + AB + + + 0.98471916 + structure_element + cleaner0 + 2023-07-05T16:54:33Z + SO: + + CD + + + 0.9975815 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9542532 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.988332 + evidence + cleaner0 + 2023-07-06T08:34:14Z + DUMMY: + + crystals + + + 0.979324 + experimental_method + cleaner0 + 2023-07-06T12:04:35Z + MESH: + + Alignment + + + 0.997494 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.99012506 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.9950531 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.9975956 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9803587 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99435633 + oligomeric_state + cleaner0 + 2023-07-05T16:51:20Z + DUMMY: + + homodimers + + + 0.995286 + structure_element + cleaner0 + 2023-07-06T12:18:47Z + SO: + + α6 helices + + + + RESULTS + title_2 + 35520 + 4-HPA stabilizes concerted conformational changes in NadR that prevent DNA-binding + + 0.9963954 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9955106 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + + RESULTS + paragraph + 35603 + To further investigate the conformational rearrangements of NadR, we performed local structural alignments using only a subset of residues in the DNA-binding helix (α4). By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. While one monomer from each structure was closely superimposable (Fig 8A, left side), the second monomer displayed quite large differences (Fig 8A, right side). Most notably, the position of the DNA-binding helix α4 shifted by as much as 6 Å (Fig 8B). Accordingly, helix α4 was also found to be one of the most dynamic regions in previous HDX-MS analyses of apo-NadR in solution. + + 0.9982886 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99280906 + experimental_method + cleaner0 + 2023-07-06T12:04:41Z + MESH: + + local structural alignments + + + 0.91794753 + structure_element + cleaner0 + 2023-07-06T12:18:52Z + SO: + + DNA-binding helix + + + 0.99741924 + structure_element + cleaner0 + 2023-07-06T12:18:57Z + SO: + + α4 + + + 0.52089626 + experimental_method + cleaner0 + 2023-07-06T12:04:51Z + MESH: + + selecting + + + 0.6833694 + experimental_method + cleaner0 + 2023-07-06T12:04:54Z + MESH: + + aligning + + + 0.9459782 + residue_range + cleaner0 + 2023-07-06T08:38:14Z + DUMMY: + + Arg64-Ala77 + + + 0.99575436 + structure_element + cleaner0 + 2023-07-06T12:19:05Z + SO: + + α4 helix + + + 0.9961479 + oligomeric_state + cleaner0 + 2023-07-06T08:37:24Z + DUMMY: + + dimer + + + 0.9952786 + experimental_method + cleaner0 + 2023-07-06T12:04:57Z + MESH: + + superposition + + + 0.99776876 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9934657 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.9978448 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9930607 + oligomeric_state + cleaner0 + 2023-07-05T16:51:20Z + DUMMY: + + homodimers + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-05T16:23:34Z + + monomer + + + 0.99148434 + evidence + cleaner0 + 2023-07-06T08:34:18Z + DUMMY: + + structure + + + 0.9931491 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + 0.9811638 + evidence + cleaner0 + 2023-07-06T08:34:20Z + DUMMY: + + structure + + + 0.9917691 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + chemical + CHEBI: + cleaner0 + 2023-07-06T12:11:59Z + + DNA + + + 0.9013841 + structure_element + cleaner0 + 2023-07-06T12:19:00Z + SO: + + helix + + + 0.99405414 + structure_element + cleaner0 + 2023-07-06T12:19:02Z + SO: + + α4 + + + 0.9898205 + structure_element + cleaner0 + 2023-07-06T12:19:07Z + SO: + + helix + + + 0.99336183 + structure_element + cleaner0 + 2023-07-06T12:19:10Z + SO: + + α4 + + + 0.9958964 + experimental_method + cleaner0 + 2023-07-06T08:14:42Z + MESH: + + HDX-MS + + + 0.9979127 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9984351 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + + ppat.1005557.g008.jpg + ppat.1005557.g008 + FIG + fig_title_caption + 36376 + Structural comparisons of NadR and modelling of interactions with DNA. + + 0.98863584 + experimental_method + cleaner0 + 2023-07-06T12:05:13Z + MESH: + + Structural comparisons + + + 0.9943613 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.9958268 + chemical + cleaner0 + 2023-07-06T08:26:05Z + CHEBI: + + DNA + + + + ppat.1005557.g008.jpg + ppat.1005557.g008 + FIG + fig_caption + 36447 + +(A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). The α4 helices aligned closely, Cα rmsd 0.2Å for 14 residues. (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. Dashes indicate the Ala77 Cα atoms, in the most highly shifted region of the ‘non-fixed’ α4 helix. (C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR α4 helices in the DNA major grooves. The proteins share ~30% amino acid sequence identity. For clarity, only the α4 helices are shown in panels (B) and (C). (D) Upon comparison with the experimentally-determined OhrR:ohrA structure (grey), the α4 helix of holo-NadR (blue) is shifted ~8Å out of the major groove. + + 0.99718 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9837816 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.99679875 + evidence + cleaner0 + 2023-07-06T08:34:25Z + DUMMY: + + structure + + + structure_element + SO: + cleaner0 + 2023-07-05T16:52:30Z + + chain A and B + + + 0.99501824 + oligomeric_state + cleaner0 + 2023-07-05T16:51:20Z + DUMMY: + + homodimers + + + 0.99778533 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9952924 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + structure_element + SO: + cleaner0 + 2023-07-05T16:52:48Z + + A/C + + + structure_element + SO: + cleaner0 + 2023-07-05T16:53:05Z + + B/D + + + 0.9940654 + oligomeric_state + cleaner0 + 2023-07-05T16:51:20Z + DUMMY: + + homodimers + + + 0.9471725 + structure_element + cleaner0 + 2023-07-05T16:53:23Z + SO: + + AB + + + 0.99380106 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9343996 + structure_element + cleaner0 + 2023-07-05T16:53:24Z + SO: + + AB + + + 0.99271977 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.90673345 + structure_element + cleaner0 + 2023-07-05T16:54:33Z + SO: + + CD + + + 0.99611545 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.52363145 + experimental_method + cleaner0 + 2023-07-06T12:05:17Z + MESH: + + overlaid + + + experimental_method + MESH: + cleaner0 + 2023-07-06T12:05:44Z + + structural alignment + + + 0.97950435 + residue_range + cleaner0 + 2023-07-06T08:38:20Z + DUMMY: + + R64-A77 + + + structure_element + SO: + cleaner0 + 2023-07-05T16:35:55Z + + A + + + 0.9969573 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + structure_element + SO: + cleaner0 + 2023-07-05T16:35:55Z + + A + + + 0.99714524 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + structure_element + SO: + cleaner0 + 2023-07-05T16:53:39Z + + C + + + 0.9969393 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.8825526 + structure_element + cleaner0 + 2023-07-06T12:19:15Z + SO: + + helix + + + 0.9969965 + structure_element + cleaner0 + 2023-07-06T12:19:18Z + SO: + + α4 + + + 0.9915956 + structure_element + cleaner0 + 2023-07-06T12:19:21Z + SO: + + α4 helices + + + 0.95211196 + evidence + cleaner0 + 2023-07-05T16:51:11Z + DUMMY: + + rmsd + + + 0.9872302 + structure_element + cleaner0 + 2023-07-06T12:19:24Z + SO: + + α4 helices + + + 0.9448308 + protein_state + cleaner0 + 2023-07-06T08:47:49Z + DUMMY: + + 4-HPA-bound + + + 0.99755234 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9775006 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:13Z + + chain B + + + 0.9976858 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.96839976 + oligomeric_state + cleaner0 + 2023-07-05T16:51:20Z + DUMMY: + + homodimers + + + 0.86218876 + structure_element + cleaner0 + 2023-07-05T16:53:24Z + SO: + + AB + + + 0.87432945 + structure_element + cleaner0 + 2023-07-05T16:54:32Z + SO: + + CD + + + structure_element + SO: + cleaner0 + 2023-07-05T16:54:16Z + + chains B and D + + + 0.99893063 + residue_name_number + cleaner0 + 2023-07-06T08:21:23Z + DUMMY: + + Ala77 + + + 0.97499454 + structure_element + cleaner0 + 2023-07-06T12:19:27Z + SO: + + α4 helix + + + 0.99629563 + chemical + cleaner0 + 2023-07-06T08:26:09Z + CHEBI: + + DNA + + + 0.9949884 + complex_assembly + cleaner0 + 2023-07-06T08:12:42Z + GO: + + OhrR-ohrA + + + 0.9925903 + experimental_method + cleaner0 + 2023-07-06T12:05:51Z + MESH: + + superposition + + + 0.99717915 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.9952188 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.98921365 + structure_element + cleaner0 + 2023-07-06T12:19:32Z + SO: + + α4 helices + + + 0.9845547 + chemical + cleaner0 + 2023-07-06T08:26:14Z + CHEBI: + + DNA + + + 0.98696077 + structure_element + cleaner0 + 2023-07-06T12:19:35Z + SO: + + α4 helices + + + 0.99406904 + complex_assembly + cleaner0 + 2023-07-06T08:12:51Z + GO: + + OhrR:ohrA + + + 0.99007696 + evidence + cleaner0 + 2023-07-06T08:34:28Z + DUMMY: + + structure + + + 0.988043 + structure_element + cleaner0 + 2023-07-06T12:19:38Z + SO: + + α4 helix + + + 0.99767274 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9952963 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + + RESULTS + paragraph + 37749 + However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). In summary, compared to ligand-stabilized holo-NadR, apo-NadR displayed an intrinsic flexibility focused in the DNA-binding region. This was also evident in the greater disorder (i.e. less well-defined electron density) in the β1-β2 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing). + + 0.9929446 + experimental_method + cleaner0 + 2023-07-06T12:05:54Z + MESH: + + structural comparisons + + + 0.9967393 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.99476093 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.952134 + structure_element + cleaner0 + 2023-07-06T12:19:42Z + SO: + + helix + + + 0.99507433 + structure_element + cleaner0 + 2023-07-06T12:19:45Z + SO: + + α4 + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:48:23Z + + presence of + + + 0.99623346 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9973326 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9907912 + site + cleaner0 + 2023-07-06T12:12:06Z + SO: + + dimer interface + + + 0.9975399 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9863324 + site + cleaner0 + 2023-07-06T12:12:10Z + SO: + + dimer interfaces + + + 0.9945024 + structure_element + cleaner0 + 2023-07-06T12:19:48Z + SO: + + α6 helices + + + 0.9942922 + protein_state + cleaner0 + 2023-07-06T08:48:28Z + DUMMY: + + ligand-stabilized + + + 0.99697816 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9928624 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99738103 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.99429804 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.9873474 + site + cleaner0 + 2023-07-06T12:12:14Z + SO: + + DNA-binding region + + + 0.9962344 + evidence + cleaner0 + 2023-07-06T08:34:32Z + DUMMY: + + electron density + + + 0.9968533 + structure_element + cleaner0 + 2023-07-06T12:19:52Z + SO: + + β1-β2 loops + + + 0.99763954 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.98444253 + oligomeric_state + cleaner0 + 2023-07-05T16:22:59Z + DUMMY: + + dimers + + + 0.9690363 + evidence + cleaner0 + 2023-07-06T08:34:35Z + DUMMY: + + density + + + 0.9940493 + oligomeric_state + cleaner0 + 2023-07-06T08:37:28Z + DUMMY: + + dimer + + + 0.9976184 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.9817718 + oligomeric_state + cleaner0 + 2023-07-06T08:37:32Z + DUMMY: + + dimer + + + 0.9886591 + evidence + cleaner0 + 2023-07-06T08:34:37Z + DUMMY: + + density + + + + RESULTS + paragraph + 38470 + In holo-NadR, the distance separating the two DNA-binding α4 helices was 32 Å, while in apo-NadR it was 29 Å for homodimer AB, and 34 Å for homodimer CD (Fig 8C). Thus, the apo-homodimer AB presented the DNA-binding helices in a conformation similar to that observed in the protein:DNA complex of OhrR:ohrA from Bacillus subtilis (Fig 8C). Interestingly, OhrR contacts ohrA across 22 base pairs (bp), and similarly the main NadR target sites identified in the nadA promoter (the operators Op I and Op II) both span 22 bp. Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. Specifically, in addition to the different inter-helical translational distances, the α4 helices in the holo-NadR homodimer were also reoriented, resulting in movement of α4 out of the major groove, by up to 8Å, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D). When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9Å), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above. + + 0.99745196 + protein_state + cleaner0 + 2023-07-05T16:18:21Z + DUMMY: + + holo + + + 0.99483335 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.97909606 + structure_element + cleaner0 + 2023-07-06T12:19:56Z + SO: + + α4 helices + + + 0.997521 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9924177 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.98887575 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.9837299 + structure_element + cleaner0 + 2023-07-05T16:53:24Z + SO: + + AB + + + 0.9894264 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.9882554 + structure_element + cleaner0 + 2023-07-05T16:54:33Z + SO: + + CD + + + 0.9966793 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.6534773 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.9956454 + structure_element + cleaner0 + 2023-07-05T16:53:24Z + SO: + + AB + + + 0.9174837 + structure_element + cleaner0 + 2023-07-06T12:20:01Z + SO: + + DNA-binding helices + + + complex_assembly + GO: + cleaner0 + 2023-07-05T16:56:09Z + + OhrR:ohrA + + + 0.9951938 + species + cleaner0 + 2023-07-06T08:18:51Z + MESH: + + Bacillus subtilis + + + 0.99838316 + protein + cleaner0 + 2023-07-05T16:56:42Z + PR: + + OhrR + + + 0.99838257 + gene + cleaner0 + 2023-07-06T08:11:09Z + GENE: + + ohrA + + + 0.39123315 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.7473986 + site + cleaner0 + 2023-07-06T12:12:25Z + SO: + + target sites + + + gene + GENE: + cleaner0 + 2023-07-05T16:04:01Z + + nadA + + + 0.99486613 + experimental_method + cleaner0 + 2023-07-06T12:06:00Z + MESH: + + Pairwise superpositions + + + 0.9952264 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T16:18:28Z + + apo + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-05T16:22:49Z + + homodimer + + + 0.99704534 + structure_element + cleaner0 + 2023-07-05T16:53:24Z + SO: + + AB + + + 0.99840873 + protein + cleaner0 + 2023-07-05T16:56:43Z + PR: + + OhrR + + + 0.9321035 + evidence + cleaner0 + 2023-07-05T16:51:11Z + DUMMY: + + rmsd + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T16:18:21Z + + holo + + + 0.581259 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.79532903 + evidence + cleaner0 + 2023-07-05T16:51:11Z + DUMMY: + + rmsd + + + chemical + CHEBI: + cleaner0 + 2023-07-06T08:49:17Z + + DNA + + + 0.9984114 + protein + cleaner0 + 2023-07-05T16:56:43Z + PR: + + OhrR + + + 0.9982993 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.99724054 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.79037267 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.9975526 + structure_element + cleaner0 + 2023-07-05T16:53:24Z + SO: + + AB + + + chemical + CHEBI: + cleaner0 + 2023-07-06T08:48:50Z + + DNA + + + 0.9900882 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9970386 + protein_state + cleaner0 + 2023-07-05T16:18:22Z + DUMMY: + + holo + + + 0.9963247 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + chemical + CHEBI: + cleaner0 + 2023-07-06T08:49:07Z + + DNA + + + 0.9482872 + evidence + cleaner0 + 2023-07-06T08:34:46Z + DUMMY: + + inter-helical translational distances + + + 0.99293673 + structure_element + cleaner0 + 2023-07-06T12:20:12Z + SO: + + α4 helices + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T16:18:22Z + + holo + + + 0.9932461 + protein + cleaner0 + 2023-07-05T16:01:48Z + PR: + + NadR + + + 0.992219 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.99768054 + structure_element + cleaner0 + 2023-07-06T12:20:18Z + SO: + + α4 + + + chemical + CHEBI: + cleaner0 + 2023-07-06T08:48:58Z + + DNA + + + 0.9934158 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9555606 + experimental_method + cleaner0 + 2023-07-06T12:06:04Z + MESH: + + aligned + + + 0.9983878 + protein + cleaner0 + 2023-07-05T16:56:43Z + PR: + + OhrR + + + 0.99690175 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.78264475 + oligomeric_state + cleaner0 + 2023-07-05T16:22:49Z + DUMMY: + + homodimer + + + 0.993802 + structure_element + cleaner0 + 2023-07-05T16:54:33Z + SO: + + CD + + + 0.93124425 + evidence + cleaner0 + 2023-07-05T16:51:11Z + DUMMY: + + rmsd + + + chemical + CHEBI: + cleaner0 + 2023-07-06T08:49:31Z + + DNA + + + 0.6082675 + structure_element + cleaner0 + 2023-07-05T16:53:24Z + SO: + + AB + + + + RESULTS + title_2 + 39993 + NadR residues His7, Ser9, Asn11 and Phe25 are essential for regulation of NadA expression in vivo + + 0.944478 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.99892396 + residue_name_number + cleaner0 + 2023-07-06T08:02:27Z + DUMMY: + + His7 + + + 0.99898106 + residue_name_number + cleaner0 + 2023-07-05T16:30:05Z + DUMMY: + + Ser9 + + + 0.9990349 + residue_name_number + cleaner0 + 2023-07-06T08:02:37Z + DUMMY: + + Asn11 + + + 0.9990089 + residue_name_number + cleaner0 + 2023-07-05T16:37:35Z + DUMMY: + + Phe25 + + + 0.9928429 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + + RESULTS + paragraph + 40092 + While previous studies had correctly suggested the involvement of several NadR residues in ligand binding, the crystal structures presented here revealed additional residues with previously unknown roles in dimerization and/or binding to 4-HPA. To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. NadA protein abundance levels were assessed by Western blotting to evaluate the ability of the NadR mutants to repress the nadA promoter, in the presence or absence of 4-HPA. The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). Interestingly, and on the contrary, the nadR N11A complemented strain showed hypo-repression (i.e. exhibited high expression of nadA both in absence and presence of 4-HPA). This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo. + + 0.92497444 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.99734956 + evidence + cleaner0 + 2023-07-06T08:34:50Z + DUMMY: + + crystal structures + + + 0.9965666 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.45507443 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9967115 + mutant + cleaner0 + 2023-07-05T16:25:19Z + MESH: + + H7A + + + 0.9961534 + mutant + cleaner0 + 2023-07-05T16:25:24Z + MESH: + + S9A + + + 0.9967585 + mutant + cleaner0 + 2023-07-05T16:25:29Z + MESH: + + N11A + + + 0.99725634 + mutant + cleaner0 + 2023-07-06T08:21:39Z + MESH: + + F25A + + + 0.9463827 + mutant + cleaner0 + 2023-07-06T08:21:44Z + MESH: + + MC58-Δ1843 + + + gene + GENE: + cleaner0 + 2023-07-05T16:09:01Z + + nadR + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T16:26:10Z + + mutant + + + 0.9969253 + protein_state + cleaner0 + 2023-07-05T16:24:28Z + DUMMY: + + wild-type + + + 0.9982344 + gene + cleaner0 + 2023-07-05T16:09:01Z + GENE: + + nadR + + + 0.41754237 + gene + cleaner0 + 2023-07-05T16:09:01Z + GENE: + + nadR + + + 0.6736878 + protein_state + cleaner0 + 2023-07-06T08:21:59Z + DUMMY: + + mutants + + + 0.93231297 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.94588625 + experimental_method + cleaner0 + 2023-07-06T12:06:07Z + MESH: + + Western blotting + + + 0.33933762 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.5745186 + protein_state + cleaner0 + 2023-07-06T08:22:12Z + DUMMY: + + mutants + + + gene + GENE: + cleaner0 + 2023-07-05T16:04:01Z + + nadA + + + 0.99555683 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9613765 + gene + cleaner0 + 2023-07-05T16:09:01Z + GENE: + + nadR + + + 0.99275607 + mutant + cleaner0 + 2023-07-05T16:25:19Z + MESH: + + H7A + + + 0.994399 + mutant + cleaner0 + 2023-07-05T16:25:24Z + MESH: + + S9A + + + 0.99687046 + mutant + cleaner0 + 2023-07-06T08:22:16Z + MESH: + + F25A + + + gene + GENE: + cleaner0 + 2023-07-05T16:04:01Z + + nadA + + + gene + GENE: + cleaner0 + 2023-07-05T16:04:01Z + + nadA + + + 0.9222919 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.98351914 + protein_state + cleaner0 + 2023-07-05T16:24:33Z + DUMMY: + + WT + + + 0.9954092 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9972098 + protein_state + cleaner0 + 2023-07-05T16:24:28Z + DUMMY: + + wild-type + + + 0.99799526 + gene + cleaner0 + 2023-07-05T16:09:01Z + GENE: + + nadR + + + 0.96869344 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.9950135 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.99465454 + gene + cleaner0 + 2023-07-05T16:09:01Z + GENE: + + nadR + + + 0.99013466 + mutant + cleaner0 + 2023-07-05T16:25:29Z + MESH: + + N11A + + + 0.81234396 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.9941128 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9766634 + experimental_method + cleaner0 + 2023-07-06T12:06:12Z + MESH: + + mutagenesis + + + 0.77991426 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9988035 + residue_name_number + cleaner0 + 2023-07-06T08:02:27Z + DUMMY: + + His7 + + + 0.99889284 + residue_name_number + cleaner0 + 2023-07-05T16:30:05Z + DUMMY: + + Ser9 + + + 0.99894816 + residue_name_number + cleaner0 + 2023-07-06T08:02:37Z + DUMMY: + + Asn11 + + + 0.9988921 + residue_name_number + cleaner0 + 2023-07-05T16:37:35Z + DUMMY: + + Phe25 + + + 0.9938478 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.7965298 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.9934442 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.99594617 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + + ppat.1005557.g009.jpg + ppat.1005557.g009 + FIG + fig_title_caption + 41588 + Structure-based point mutations shed light on ligand-induced regulation of NadR. + + 0.99432194 + experimental_method + cleaner0 + 2023-07-06T12:06:17Z + MESH: + + Structure-based point mutations + + + 0.9983051 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + + ppat.1005557.g009.jpg + ppat.1005557.g009 + FIG + fig_caption + 41669 + Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. Complementation of ΔNadR with WT NadR enables induction of nadA expression by 4-HPA. The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). + + 0.97901464 + experimental_method + cleaner0 + 2023-07-06T12:06:20Z + MESH: + + Western blot + + + 0.995406 + protein_state + cleaner0 + 2023-07-05T16:24:28Z + DUMMY: + + wild-type + + + 0.9735041 + protein_state + cleaner0 + 2023-07-05T16:24:33Z + DUMMY: + + WT + + + 0.6550133 + gene + cleaner0 + 2023-07-05T16:09:01Z + GENE: + + nadR + + + 0.9981198 + mutant + cleaner0 + 2023-07-06T08:22:21Z + MESH: + + ΔNadR + + + 0.99103373 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.99277824 + protein_state + cleaner0 + 2023-07-05T16:24:33Z + DUMMY: + + WT + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T16:26:10Z + + mutant + + + 0.99599695 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.4534085 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.47945735 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9983777 + mutant + cleaner0 + 2023-07-06T08:22:25Z + MESH: + + ΔNadR + + + 0.9974239 + protein_state + cleaner0 + 2023-07-05T16:24:33Z + DUMMY: + + WT + + + 0.9969547 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.74495965 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.9963741 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.99826473 + mutant + cleaner0 + 2023-07-05T16:25:19Z + MESH: + + H7A + + + 0.99798965 + mutant + cleaner0 + 2023-07-05T16:25:24Z + MESH: + + S9A + + + 0.9983475 + mutant + cleaner0 + 2023-07-06T08:22:29Z + MESH: + + F25A + + + 0.7451092 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.99741817 + protein_state + cleaner0 + 2023-07-05T16:24:33Z + DUMMY: + + WT + + + 0.9977149 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9984787 + mutant + cleaner0 + 2023-07-05T16:25:29Z + MESH: + + N11A + + + 0.9429268 + protein_state + cleaner0 + 2023-07-05T16:26:10Z + DUMMY: + + mutant + + + 0.47659972 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.9960682 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.84731996 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + protein + PR: + cleaner0 + 2023-07-06T08:12:12Z + + factor H binding protein + + + 0.9949124 + protein + cleaner0 + 2023-07-06T08:12:15Z + PR: + + fHbp + + + + DISCUSS + title_1 + 42414 + Discussion + + + DISCUSS + paragraph + 42425 + NadA is a surface-exposed meningococcal protein contributing to pathogenesis, and is one of three main antigens present in the vaccine Bexsero. A detailed understanding of the in vitro repression of nadA expression by the transcriptional regulator NadR is important, both because it is a relevant disease-related model of how small-molecule ligands can regulate MarR family proteins and thereby impact bacterial virulence, and because nadA expression levels are linked to the prediction of vaccine coverage. The repressive activity of NadR can be relieved by hydroxyphenylacetate (HPA) ligands, and HDX-MS studies previously indicated that 4-HPA stabilizes dimeric NadR in a configuration incompatible with DNA binding. Despite these and other studies, the molecular mechanisms by which ligands regulate MarR family proteins are relatively poorly understood and likely differ depending on the specific ligand. Given the importance of NadR-mediated regulation of NadA levels in the contexts of meningococcal pathogenesis, we sought to characterize NadR, and its interaction with ligands, at atomic resolution. + + 0.97509515 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.55074143 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + 0.9294955 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.9943236 + protein_type + cleaner0 + 2023-07-06T08:15:27Z + MESH: + + transcriptional regulator + + + 0.99856794 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.99289215 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:57Z + DUMMY: + + bacterial + + + 0.473951 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.9984724 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9982064 + chemical + cleaner0 + 2023-07-06T08:26:21Z + CHEBI: + + hydroxyphenylacetate + + + 0.99808073 + chemical + cleaner0 + 2023-07-06T08:26:25Z + CHEBI: + + HPA + + + 0.9962147 + experimental_method + cleaner0 + 2023-07-06T08:14:42Z + MESH: + + HDX-MS + + + 0.997154 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9923085 + oligomeric_state + cleaner0 + 2023-07-05T16:23:05Z + DUMMY: + + dimeric + + + 0.9986915 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.9868035 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.6850392 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.72020483 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + 0.99818844 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + + DISCUSS + paragraph + 43534 + Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. We also used structure-guided site-directed mutagenesis to identify an important conserved residue, Leu130, which stabilizes the NadR dimer interface, knowledge of which may also inform future studies to explore the regulatory mechanisms of other MarR family proteins. Secondly, we assessed the thermal stability and unfolding of NadR in the presence or absence of ligands. All DSC profiles showed a single peak, suggesting that a single unfolding event simultaneously disrupted the dimer and the monomer. HPA ligands specifically increased the stability of NadR. The largest effects were induced by the naturally-occurring compounds 4-HPA and 3Cl,4-HPA, which, in SPR assays, were found to bind NadR with KD values of 1.5 mM and 1.1 mM, respectively. Although these NadR/HPA interactions appeared rather weak, their distinct affinities and specificities matched their in vitro effects and their biological relevance appears similar to previous proposals that certain small molecules, including some antibiotics, in the millimolar concentration range may be broad inhibitors of MarR family proteins. Indeed, 4-HPA is found in human saliva and 3Cl,4-HPA is produced during inflammatory processes, suggesting that these natural ligands are encountered by N. meningitidis in the mucosa of the oropharynx during infections. It is also possible that NadR responds to currently unidentified HPA analogues. Indeed, in the NadR/4-HPA complex there was a water molecule close to the carboxylate group and also a small unfilled tunnel ~5Å long, both factors suggesting that alternative larger ligands could occupy the pocket. It is conceivable that such putative ligands may establish different bonding networks, potentially binding in a 2:2 ratio, rather than the 1:2 ratio observed herein. The ability to respond to various ligands might enable NadR in vivo to orchestrate multiple response mechanisms and modulate expression of genes other than nadA. Ultimately, confirmation of the relevance of each ligand will require a deeper understanding of the available concentration in vivo in the host niche during bacterial colonization and inflammation. + + 0.99859005 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.973828 + oligomeric_state + cleaner0 + 2023-07-05T16:23:05Z + DUMMY: + + dimeric + + + 0.96735674 + oligomeric_state + cleaner0 + 2023-07-05T16:23:05Z + DUMMY: + + dimeric + + + 0.9712814 + protein_state + cleaner0 + 2023-07-06T08:49:46Z + DUMMY: + + presence of + + + 0.9975283 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.73995596 + oligomeric_state + cleaner0 + 2023-07-05T16:25:00Z + DUMMY: + + monomeric + + + 0.99705654 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9986633 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.99819344 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9925506 + experimental_method + cleaner0 + 2023-07-06T12:06:26Z + MESH: + + SEC + + + 0.93368363 + experimental_method + cleaner0 + 2023-07-06T12:06:29Z + MESH: + + mass spectrometry + + + 0.98113465 + experimental_method + cleaner0 + 2023-07-06T12:06:32Z + MESH: + + crystallographic studies + + + 0.9954275 + protein_type + cleaner0 + 2023-07-05T16:09:39Z + MESH: + + MarR + + + 0.943355 + oligomeric_state + cleaner0 + 2023-07-05T16:23:05Z + DUMMY: + + dimeric + + + 0.9960764 + experimental_method + cleaner0 + 2023-07-06T12:06:35Z + MESH: + + structure-guided site-directed mutagenesis + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:50:06Z + + conserved + + + 0.9986517 + residue_name_number + cleaner0 + 2023-07-06T08:21:28Z + DUMMY: + + Leu130 + + + 0.9982626 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.99668217 + site + cleaner0 + 2023-07-06T12:13:01Z + SO: + + dimer interface + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + experimental_method + MESH: + cleaner0 + 2023-07-06T08:35:15Z + + assessed the thermal stability + + + 0.9985331 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:50:27Z + + in the presence + + + 0.910316 + protein_state + cleaner0 + 2023-07-06T08:50:30Z + DUMMY: + + absence of + + + 0.7912865 + experimental_method + cleaner0 + 2023-07-06T12:06:42Z + MESH: + + DSC + + + 0.6052191 + evidence + cleaner0 + 2023-07-06T08:35:20Z + DUMMY: + + profiles + + + 0.9964078 + oligomeric_state + cleaner0 + 2023-07-06T08:37:37Z + DUMMY: + + dimer + + + 0.9960084 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + 0.9986971 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.99759865 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.99718535 + chemical + cleaner0 + 2023-07-05T16:12:21Z + CHEBI: + + 3Cl,4-HPA + + + 0.9872149 + experimental_method + cleaner0 + 2023-07-06T12:06:46Z + MESH: + + SPR assays + + + 0.9986644 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9551488 + evidence + cleaner0 + 2023-07-06T08:35:48Z + DUMMY: + + KD + + + 0.8284178 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-07-21T12:29:34Z + + HPA + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.99768394 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.99437547 + species + cleaner0 + 2023-07-05T16:04:51Z + MESH: + + human + + + 0.9973361 + chemical + cleaner0 + 2023-07-05T16:12:21Z + CHEBI: + + 3Cl,4-HPA + + + 0.9945516 + species + cleaner0 + 2023-07-05T16:12:03Z + MESH: + + N. meningitidis + + + 0.9974638 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9980355 + chemical + cleaner0 + 2023-07-06T08:26:30Z + CHEBI: + + HPA + + + complex_assembly + GO: + cleaner0 + 2023-07-05T16:22:40Z + + NadR/4-HPA + + + 0.9984682 + chemical + cleaner0 + 2023-07-06T08:26:34Z + CHEBI: + + water + + + 0.7275508 + site + cleaner0 + 2023-07-06T12:13:06Z + SO: + + tunnel + + + 0.99801755 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.99730766 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.9845227 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:57Z + DUMMY: + + bacterial + + + + DISCUSS + paragraph + 46104 + Here, we determined the first crystal structures of apo-NadR and holo-NadR. These experimentally-determined structures enabled a new detailed characterization of the ligand-binding pocket. In holo-NadR, 4-HPA interacted directly with at least 11 polar and hydrophobic residues. Several, but not all, of these interactions were predicted previously by homology modelling combined with ligand docking in silico. Subsequently, we established the functional importance of His7, Ser9, Asn11 and Phe25 in the in vitro response of meningococcus to 4-HPA, via site-directed mutagenesis. More unexpectedly, the crystal structure revealed that only one molecule of 4-HPA was bound per NadR dimer. We confirmed this stoichiometry in solution using SPR methods. We also used heteronuclear NMR spectroscopy to detect substantial conformational changes of NadR occurring in solution upon addition of 4-HPA. Moreover, NMR spectra at 10°C suggested the existence of two distinct conformations of NadR in the vicinity of the ligand-binding pocket. More powerfully, our unique crystallographic observation of this ‘occupied vs unoccupied site’ asymmetry in the NadR/4-HPA interaction is, to our knowledge, the first example reported for a MarR family protein. Structural analyses suggested that ‘inward’ side chain positions of Met22, Phe25 and especially Arg43 precluded binding of a second ligand molecule. Such a mechanism indicates negative cooperativity, which may enhance the ligand-responsiveness of NadR. + + 0.9971545 + evidence + cleaner0 + 2023-07-06T08:35:52Z + DUMMY: + + crystal structures + + + 0.9977964 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9986632 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9977132 + protein_state + cleaner0 + 2023-07-05T16:18:22Z + DUMMY: + + holo + + + 0.9986835 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9959116 + evidence + cleaner0 + 2023-07-06T08:35:58Z + DUMMY: + + structures + + + 0.99816763 + site + cleaner0 + 2023-07-06T12:13:11Z + SO: + + ligand-binding pocket + + + 0.9975103 + protein_state + cleaner0 + 2023-07-05T16:18:22Z + DUMMY: + + holo + + + 0.9985104 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9960315 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9938643 + experimental_method + cleaner0 + 2023-07-06T12:07:01Z + MESH: + + homology modelling + + + 0.99227476 + experimental_method + cleaner0 + 2023-07-06T12:07:04Z + MESH: + + ligand docking + + + 0.9991386 + residue_name_number + cleaner0 + 2023-07-06T08:02:27Z + DUMMY: + + His7 + + + 0.9991304 + residue_name_number + cleaner0 + 2023-07-05T16:30:05Z + DUMMY: + + Ser9 + + + 0.9991472 + residue_name_number + cleaner0 + 2023-07-06T08:02:37Z + DUMMY: + + Asn11 + + + 0.9991252 + residue_name_number + cleaner0 + 2023-07-05T16:37:35Z + DUMMY: + + Phe25 + + + 0.986343 + taxonomy_domain + cleaner0 + 2023-07-06T08:19:08Z + DUMMY: + + meningococcus + + + 0.99670297 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.99458283 + experimental_method + cleaner0 + 2023-07-06T12:07:07Z + MESH: + + site-directed mutagenesis + + + 0.9972843 + evidence + cleaner0 + 2023-07-06T08:36:02Z + DUMMY: + + crystal structure + + + 0.996538 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.76325077 + protein_state + cleaner0 + 2023-07-06T08:50:36Z + DUMMY: + + bound + + + 0.9986249 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9958813 + oligomeric_state + cleaner0 + 2023-07-06T08:37:41Z + DUMMY: + + dimer + + + 0.9931855 + experimental_method + cleaner0 + 2023-07-06T12:07:12Z + MESH: + + SPR + + + 0.9955998 + experimental_method + cleaner0 + 2023-07-06T12:07:15Z + MESH: + + heteronuclear NMR spectroscopy + + + 0.99871445 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9958806 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9211322 + experimental_method + cleaner0 + 2023-07-05T16:48:34Z + MESH: + + NMR + + + 0.7642749 + evidence + cleaner0 + 2023-07-05T16:49:02Z + DUMMY: + + spectra + + + 0.99845326 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.99803483 + site + cleaner0 + 2023-07-06T12:13:15Z + SO: + + ligand-binding pocket + + + evidence + DUMMY: + cleaner0 + 2023-07-06T12:13:41Z + + crystallographic observation + + + 0.6236123 + protein_state + cleaner0 + 2023-07-06T08:50:42Z + DUMMY: + + occupied + + + 0.66957355 + protein_state + cleaner0 + 2023-07-06T08:50:45Z + DUMMY: + + unoccupied + + + complex_assembly + GO: + cleaner0 + 2023-07-05T16:22:40Z + + NadR/4-HPA + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.9818895 + experimental_method + cleaner0 + 2023-07-06T12:07:21Z + MESH: + + Structural analyses + + + protein_state + DUMMY: + cleaner0 + 2023-07-06T08:44:58Z + + inward + + + 0.9991411 + residue_name_number + cleaner0 + 2023-07-05T16:37:28Z + DUMMY: + + Met22 + + + 0.99911517 + residue_name_number + cleaner0 + 2023-07-05T16:37:35Z + DUMMY: + + Phe25 + + + 0.9991381 + residue_name_number + cleaner0 + 2023-07-05T16:37:40Z + DUMMY: + + Arg43 + + + 0.9987212 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + + DISCUSS + paragraph + 47608 + Comparisons of the NadR/4-HPA complex with available MarR family/salicylate complexes revealed that 4-HPA has a previously unobserved binding mode. Briefly, in the M. thermoautotrophicum MTH313 dimer, one molecule of salicylate binds in the pocket of each monomer, though with two rather different positions and orientations, only one of which (site-1) is thought to be biologically relevant (Fig 10A). In the S. tokodaii protein ST1710, salicylate binds to the same position in each monomer of the dimer, in a site equivalent to the putative biologically relevant site of MTH313 (Fig 10B). Unlike other MarR family proteins which revealed multiple ligand binding interactions, we observed only 1 molecule of 4-HPA bound to NadR, suggesting a more specific and less promiscuous interaction. In NadR, the single molecule of 4-HPA binds in a position distinctly different from the salicylate binding site: translated by > 10 Å and with a 180° inverted orientation (Fig 10C). + + complex_assembly + GO: + cleaner0 + 2023-07-05T16:22:40Z + + NadR/4-HPA + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.4382097 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.9972873 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.99461573 + species + cleaner0 + 2023-07-06T08:19:13Z + MESH: + + M. thermoautotrophicum + + + 0.9984688 + protein + cleaner0 + 2023-07-05T16:10:58Z + PR: + + MTH313 + + + 0.99616396 + oligomeric_state + cleaner0 + 2023-07-06T08:37:45Z + DUMMY: + + dimer + + + 0.9982845 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.9935482 + site + cleaner0 + 2023-07-06T12:13:55Z + SO: + + pocket + + + 0.9945979 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + 0.9863995 + site + cleaner0 + 2023-07-06T12:14:01Z + SO: + + site-1 + + + 0.99450547 + species + cleaner0 + 2023-07-06T08:19:18Z + MESH: + + S. tokodaii + + + 0.99853384 + protein + cleaner0 + 2023-07-05T16:11:06Z + PR: + + ST1710 + + + 0.989588 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.9952158 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + 0.99607986 + oligomeric_state + cleaner0 + 2023-07-06T08:37:48Z + DUMMY: + + dimer + + + 0.99854845 + protein + cleaner0 + 2023-07-05T16:10:58Z + PR: + + MTH313 + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.997176 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.99584013 + protein_state + cleaner0 + 2023-07-06T08:50:48Z + DUMMY: + + bound to + + + 0.9988686 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.99867034 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9970718 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9979003 + site + cleaner0 + 2023-07-06T12:14:23Z + SO: + + salicylate binding site + + + + ppat.1005557.g010.jpg + ppat.1005557.g010 + FIG + fig_title_caption + 48583 + NadR shows a ligand binding site distinct from other MarR homologues. + + 0.74721384 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9980111 + site + cleaner0 + 2023-07-06T12:14:27Z + SO: + + ligand binding site + + + 0.99762696 + protein_type + cleaner0 + 2023-07-05T16:09:39Z + MESH: + + MarR + + + + ppat.1005557.g010.jpg + ppat.1005557.g010 + FIG + fig_caption + 48653 + +(A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7Å (indicated by black dotted line) and also by ligand orientation. (B) A structural alignment of MTH313 chain A and ST1710 (pink) (Cα rmsd 2.3Å), shows that they bind salicylate in equivalent sites (differing by only ~3Å) and with the same orientation. (C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by > 10 Å compared to salicylate, and adopts a novel orientation. + + 0.9951623 + experimental_method + cleaner0 + 2023-07-06T12:07:26Z + MESH: + + structural alignment + + + 0.99830675 + protein + cleaner0 + 2023-07-05T16:10:58Z + PR: + + MTH313 + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:44Z + + chains A and B + + + 0.99855167 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.5704616 + protein_state + cleaner0 + 2023-07-06T08:50:54Z + DUMMY: + + bound + + + 0.98944247 + oligomeric_state + cleaner0 + 2023-07-05T16:23:34Z + DUMMY: + + monomer + + + 0.9937976 + site + cleaner0 + 2023-07-06T12:14:34Z + SO: + + site-1 + + + 0.9936908 + site + cleaner0 + 2023-07-06T12:14:37Z + SO: + + site-2 + + + 0.9943106 + experimental_method + cleaner0 + 2023-07-06T12:07:29Z + MESH: + + structural alignment + + + 0.998104 + protein + cleaner0 + 2023-07-05T16:10:58Z + PR: + + MTH313 + + + structure_element + SO: + cleaner0 + 2023-07-05T16:34:02Z + + chain A + + + 0.755813 + protein + cleaner0 + 2023-07-05T16:11:06Z + PR: + + ST1710 + + + 0.9025636 + evidence + cleaner0 + 2023-07-05T16:51:11Z + DUMMY: + + rmsd + + + 0.99835485 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.9980702 + protein_state + cleaner0 + 2023-07-05T16:18:22Z + DUMMY: + + holo + + + 0.9984768 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + structure_element + SO: + cleaner0 + 2023-07-05T16:33:13Z + + chain B + + + 0.82494926 + experimental_method + cleaner0 + 2023-07-06T12:07:42Z + MESH: + + alignment + + + 0.9933189 + protein_state + cleaner0 + 2023-07-06T08:50:58Z + DUMMY: + + bound + + + 0.997561 + chemical + cleaner0 + 2023-07-05T16:05:13Z + CHEBI: + + 4-HPA + + + 0.9984068 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + + DISCUSS + paragraph + 49281 + Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. It can be speculated that MarR family members have evolved separately to engage distinct signaling molecules, thus enabling bacteria to use the overall conserved MarR scaffold to adapt and respond to diverse changing environmental stimuli experienced in their natural niches. Alternatively, it is possible that other MarR homologues (e.g. NMB1585) may have no extant functional binding pocket and thus may have lost the ability to respond to a ligand, acting instead as constitutive DNA-binding regulatory proteins. + + 0.9972241 + evidence + cleaner0 + 2023-07-06T08:36:07Z + DUMMY: + + crystal structure + + + 0.65245795 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + 0.99733835 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.9971601 + protein + cleaner0 + 2023-07-06T08:12:27Z + PR: + + NMB1585 + + + 0.99822205 + protein + cleaner0 + 2023-07-05T16:01:49Z + PR: + + NadR + + + 0.99524575 + evidence + cleaner0 + 2023-07-06T08:36:10Z + DUMMY: + + structures + + + 0.9727957 + evidence + cleaner0 + 2023-07-05T16:51:11Z + DUMMY: + + rmsd + + + 0.9963366 + protein + cleaner0 + 2023-07-06T08:12:28Z + PR: + + NMB1585 + + + 0.98902774 + chemical + cleaner0 + 2023-07-05T16:18:03Z + CHEBI: + + HPAs + + + 0.95218843 + site + cleaner0 + 2023-07-06T12:15:04Z + SO: + + ‘pocket + + + protein_type + MESH: + cleaner0 + 2023-07-05T16:09:39Z + + MarR + + + 0.9961553 + taxonomy_domain + cleaner0 + 2023-07-05T16:09:53Z + DUMMY: + + bacteria + + + 0.9724381 + protein_type + cleaner0 + 2023-07-05T16:09:39Z + MESH: + + MarR + + + 0.99568754 + protein_type + cleaner0 + 2023-07-05T16:09:39Z + MESH: + + MarR + + + 0.99731016 + protein + cleaner0 + 2023-07-06T08:12:28Z + PR: + + NMB1585 + + + 0.9018167 + site + cleaner0 + 2023-07-06T12:15:13Z + SO: + + binding pocket + + + chemical + CHEBI: + cleaner0 + 2023-07-06T08:16:12Z + + DNA + + + + DISCUSS + paragraph + 50188 + The apo-NadR crystal structures revealed two dimers with slightly different conformations, most divergent in the DNA-binding domain. It is not unusual for a crystal structure to reveal multiple copies of the same protein in very slightly different conformations, which are likely representative of the lowest-energy conformations sampled by the dynamic ensemble of molecular states occurring in solution, and which likely have only small energetic differences, as described previously for MexR (a MarR protein) or more recently for the solute-binding protein FhuD2. Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. + + 0.99787676 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9971002 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.99730563 + evidence + cleaner0 + 2023-07-06T08:36:13Z + DUMMY: + + crystal structures + + + 0.99617815 + oligomeric_state + cleaner0 + 2023-07-05T16:22:59Z + DUMMY: + + dimers + + + 0.9942808 + structure_element + cleaner0 + 2023-07-06T12:20:25Z + SO: + + DNA-binding domain + + + 0.9972534 + evidence + cleaner0 + 2023-07-06T08:36:16Z + DUMMY: + + crystal structure + + + 0.99853015 + protein + cleaner0 + 2023-07-06T07:59:32Z + PR: + + MexR + + + 0.9948894 + protein_type + cleaner0 + 2023-07-05T16:09:39Z + MESH: + + MarR + + + 0.9842086 + protein_type + cleaner0 + 2023-07-06T08:16:23Z + MESH: + + solute-binding protein + + + 0.99863726 + protein + cleaner0 + 2023-07-06T07:59:36Z + PR: + + FhuD2 + + + 0.9976864 + protein_state + cleaner0 + 2023-07-05T16:18:22Z + DUMMY: + + holo + + + 0.9967859 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.99675167 + evidence + cleaner0 + 2023-07-06T08:36:19Z + DUMMY: + + structure + + + 0.9978709 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9969388 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.99600583 + evidence + cleaner0 + 2023-07-06T08:36:22Z + DUMMY: + + structures + + + 0.99534804 + evidence + cleaner0 + 2023-07-05T16:51:11Z + DUMMY: + + rmsd + + + 0.9987024 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.99678 + chemical + cleaner0 + 2023-07-05T16:05:14Z + CHEBI: + + 4-HPA + + + 0.9960879 + protein_state + cleaner0 + 2023-07-06T08:51:26Z + DUMMY: + + active + + + 0.9954644 + protein_state + cleaner0 + 2023-07-06T08:51:29Z + DUMMY: + + inactive + + + 0.9986125 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.9232329 + chemical + cleaner0 + 2023-07-06T08:26:39Z + CHEBI: + + DNA + + + + DISCUSS + paragraph + 51217 + Comparisons of the apo- and holo-NadR structures revealed that the largest differences occurred in the DNA-binding helix α4. The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. While some flexibility of helix α4 was also observed in the two apo-structures, concomitant changes in the dimer interfaces were not observed, possibly due to the absence of ligand. One of the two conformations of apo-NadR appeared ideally suited for DNA-binding. Overall, these analyses suggest that the apo-NadR dimer has a pre-existing equilibrium that samples a variety of conformations, some of which are compatible with DNA binding. This intrinsically dynamic nature underlies the possibility for different conformations to inter-convert or to be preferentially selected by a regulatory ligand, as generally described in the ‘conformational selection’ model for protein-ligand interactions (the Monod-Wyman-Changeux model), rather than an ‘induced fit’ model (Koshland-Nemethy-Filmer). The noted flexibility may also explain how NadR can adapt to bind various DNA target sequences with slightly different structural features. Subsequently, upon ligand binding, holo-NadR adopts a structure less suited for DNA-binding and this conformation is selected and stabilized by a network of protein-ligand interactions and concomitant rearrangements at the NadR holo dimer interface. In an alternative and less extensive manner, the binding of two salicylate molecules to the M. thermoautotrophicum protein MTH313 appeared to induce large changes in the wHTH domain, which was associated with reduced DNA-binding activity. + + 0.9973699 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9971554 + protein_state + cleaner0 + 2023-07-05T16:18:22Z + DUMMY: + + holo + + + 0.9959662 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.9974324 + evidence + cleaner0 + 2023-07-06T08:36:25Z + DUMMY: + + structures + + + chemical + CHEBI: + cleaner0 + 2023-07-06T12:15:27Z + + DNA + + + 0.5043439 + structure_element + cleaner0 + 2023-07-06T12:20:28Z + SO: + + helix + + + 0.9908263 + structure_element + cleaner0 + 2023-07-06T12:20:31Z + SO: + + α4 + + + 0.98037887 + structure_element + cleaner0 + 2023-07-06T12:20:36Z + SO: + + helix + + + 0.99362767 + structure_element + cleaner0 + 2023-07-06T12:20:39Z + SO: + + α4 + + + 0.9971974 + protein_state + cleaner0 + 2023-07-05T16:18:22Z + DUMMY: + + holo + + + 0.9961991 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.99508715 + site + cleaner0 + 2023-07-06T12:15:31Z + SO: + + dimer interface + + + 0.95091134 + structure_element + cleaner0 + 2023-07-06T12:20:42Z + SO: + + helices + + + 0.9958883 + structure_element + cleaner0 + 2023-07-06T12:20:44Z + SO: + + α1 + + + 0.9955355 + structure_element + cleaner0 + 2023-07-06T12:20:47Z + SO: + + α5 + + + 0.9956929 + structure_element + cleaner0 + 2023-07-06T12:20:50Z + SO: + + α6 + + + 0.9974261 + protein_state + cleaner0 + 2023-07-05T16:18:22Z + DUMMY: + + holo + + + chemical + CHEBI: + cleaner0 + 2023-07-06T08:52:26Z + + DNA + + + 0.9916764 + complex_assembly + cleaner0 + 2023-07-06T08:16:35Z + GO: + + OhrR:ohrA + + + 0.9877594 + structure_element + cleaner0 + 2023-07-06T12:20:54Z + SO: + + helix + + + 0.9935834 + structure_element + cleaner0 + 2023-07-06T12:20:56Z + SO: + + α4 + + + 0.9978315 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.979077 + evidence + cleaner0 + 2023-07-06T08:36:28Z + DUMMY: + + structures + + + 0.9937589 + site + cleaner0 + 2023-07-06T12:15:34Z + SO: + + dimer interfaces + + + 0.7910685 + protein_state + cleaner0 + 2023-07-06T08:51:39Z + DUMMY: + + absence of ligand + + + 0.9975109 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9974704 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + chemical + CHEBI: + cleaner0 + 2023-07-06T08:51:48Z + + DNA + + + 0.99754024 + protein_state + cleaner0 + 2023-07-05T16:18:28Z + DUMMY: + + apo + + + 0.9964143 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.99657625 + oligomeric_state + cleaner0 + 2023-07-06T08:37:53Z + DUMMY: + + dimer + + + chemical + CHEBI: + cleaner0 + 2023-07-06T08:52:00Z + + DNA + + + 0.99644935 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + chemical + CHEBI: + cleaner0 + 2023-07-06T08:52:09Z + + DNA + + + 0.9972614 + protein_state + cleaner0 + 2023-07-05T16:18:22Z + DUMMY: + + holo + + + 0.9973586 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + chemical + CHEBI: + cleaner0 + 2023-07-06T08:52:17Z + + DNA + + + 0.9977654 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.99550545 + protein_state + cleaner0 + 2023-07-05T16:18:22Z + DUMMY: + + holo + + + 0.9939954 + site + cleaner0 + 2023-07-06T12:15:38Z + SO: + + dimer interface + + + 0.9988034 + chemical + cleaner0 + 2023-07-05T16:11:42Z + CHEBI: + + salicylate + + + 0.9949896 + species + cleaner0 + 2023-07-06T08:19:22Z + MESH: + + M. thermoautotrophicum + + + 0.9984168 + protein + cleaner0 + 2023-07-05T16:10:58Z + PR: + + MTH313 + + + 0.9724839 + structure_element + cleaner0 + 2023-07-06T12:21:04Z + SO: + + wHTH domain + + + + DISCUSS + paragraph + 53025 + Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. The latter may influence the surface abundance or secretion of maf proteins, an emerging class of highly conserved meningococcal putative adhesins and toxins with many important roles. Further work is required to investigate how the two different promoter types influence the ligand-responsiveness of NadR during bacterial infection and may provide insights into the regulatory mechanisms occurring during these host-pathogen interactions. Ultimately, knowledge of the ligand-dependent activity of NadR will continue to deepen our understanding of nadA expression levels, which influence meningococcal pathogenesis. + + 0.996686 + evidence + cleaner0 + 2023-07-06T08:36:33Z + DUMMY: + + crystal structures + + + 0.9821497 + protein_type + cleaner0 + 2023-07-06T08:16:39Z + MESH: + + transcription factor + + + 0.9978492 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.7136107 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + 0.9967894 + protein + cleaner0 + 2023-07-05T16:02:00Z + PR: + + NadA + + + 0.97987914 + experimental_method + cleaner0 + 2023-07-06T12:07:47Z + MESH: + + structural analyses + + + 0.9983432 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.9701741 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + 0.99673766 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.9928474 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.99358 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.9957644 + gene + cleaner0 + 2023-07-06T08:00:45Z + GENE: + + mafA + + + 0.99762875 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.9962601 + chemical + cleaner0 + 2023-07-05T16:05:14Z + CHEBI: + + 4-HPA + + + 0.99696594 + protein_state + cleaner0 + 2023-07-06T08:52:33Z + DUMMY: + + highly conserved + + + 0.97295785 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + 0.9980994 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.99093115 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:57Z + DUMMY: + + bacterial + + + 0.9976197 + protein + cleaner0 + 2023-07-05T16:01:50Z + PR: + + NadR + + + 0.9941636 + gene + cleaner0 + 2023-07-05T16:04:01Z + GENE: + + nadA + + + 0.8491299 + taxonomy_domain + cleaner0 + 2023-07-05T16:04:16Z + DUMMY: + + meningococcal + + + + METHODS + title_1 + 54246 + Materials and Methods + + + METHODS + title_2 + 54268 + Bacterial strains, culture conditions and mutant generation + + + METHODS + paragraph + 54328 + In this study we used N. meningitidis MC58 wild type strain and related mutant derivatives. The MC58 isolate was kindly provided to us by Professor E. Richard Moxon, University of Oxford, UK, and was previously submitted to the Meningococcal Reference Laboratory, Manchester, UK. Strains were routinely cultured, stocked, and transformed as described previously. To generate N. meningitidis MC58 mutant strains expressing only the amino acid substituted forms of NadR, plasmids containing the sequence of nadR mutated to insert alanine codons to replace His7, Ser9, Asn11 or Phe25 were constructed using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). The nadR gene (also termed NMB1843) was mutated in the pComEry-1843 plasmid using couples of mutagenic primers (forward and reverse). The resulting plasmids were named pComEry-1843H7A, -1843S9A, -1843N11A or -1843F25A, and contained a site-directed mutant allele of the nadR gene, in which the selected codons were respectively substituted by a GCG alanine codon, and were used for transformation of the MC-Δ1843 strain. Total lysates from single colonies of all transformants were used as a template for PCR analysis to confirm the correct insertion by double homologous recombinant event. When indicated, bacterial strains were grown in presence of 5 mM 4-HPA (MW 152, Sigma-Aldrich). + + + METHODS + title_2 + 55688 + Molecular cloning + + + METHODS + paragraph + 55706 + The preparation of the expression construct enabling production of soluble NadR with an N-terminal His-tag followed by a thrombin cleavage site (MGSSHHHHHHSSGLVPR↓GSH-) (where the arrow indicates the cleavage site) and then NadR residues M1-S146 (Uniprot code Q7DD70), and methods to generate site-directed mutants, were described previously. Briefly, site-directed mutagenesis was performed using two overlapping primers containing the desired mutation to amplify pET15b containing several NadR variants. (Full oligonucleotide sequences of primers are available upon request). 1–10 ng of plasmid DNA template were amplified using Kapa HiFi DNA polymerase (Kapa Biosystems) and with the following cycling conditions: 98°C for 5 min, 15 cycles of (98°C for 30 s, 60°C for 30 s, 72°C for 6 min) followed by a final extension of 10 min at 72°C. Residual template DNA was digested by 30 min incubation with FastDigest DpnI (Thermo Scientific) at 37°C and 1 μl of this reaction was used to transform E. coli DH5α competent cells. The full recombinant tagged NadR protein generated contained 166 residues, with a theoretical MW of 18746, while after thrombin-cleavage the untagged protein contained 149 residues, with a theoretical MW of 16864. + + + METHODS + title_2 + 56957 + Protein production and purification + + + METHODS + paragraph + 56993 + The NadR expression constructs (wild-type or mutant clones) were transformed into E. coli BL21 (DE3) cells and were grown at 37°C in Luria-Bertani (LB) medium supplemented with 100 μg/mL ampicillin, until an OD600 of 0.5 was reached. Target protein production was induced by the addition of 1 mM IPTG followed by incubation with shaking overnight at 21°C. For production of the selenomethionine (SeMet) derivative form of NadR for crystallization studies, essentially the same procedure was followed, but using the E. coli B834 strain grown in a modified M9 minimal medium supplemented with 40 mg/L L-SeMet. For production of 15N-labeled NadR for NMR analyses, the EnPresso B Defined Nitrogen-free medium (Sigma-Aldrich) was used; in brief, BL21 (DE3) cells were grown in BioSilta medium at 30°C for 30 h, and production of the 15N-labeled NadR was enabled by the addition of 2.5 g/L 15NH4Cl and further incubation for 2 days. + + + METHODS + paragraph + 57924 + Cells were harvested by centrifugation (6400 g, 30 min, 4°C), resuspended in 20 mM HEPES pH 8.0, 300 mM NaCl, 20 mM imidazole, and were lysed by sonication (Qsonica Q700). Cell lysates were clarified by centrifugation at 2800 g for 30 min, and the supernatant was filtered using a 0.22 μm membrane (Corning filter system) prior to protein purification. NadR was purified by affinity chromatography using an AKTA purifier (GE Healthcare). All steps were performed at room temperature (18–26°C), unless stated otherwise. The filtered supernatant was loaded onto an Ni-NTA resin (5 mL column, GE Healthcare), and NadR was eluted using 4 steps of imidazole at 20, 30, 50 and 250 mM concentration, at a flow rate of 5 mL/min. Eluted fractions were examined by reducing and denaturing SDS-PAGE analysis. Fractions containing NadR were identified by a band migrating at ~17 kDa, and were pooled. The N-terminal 6-His tag was removed enzymatically using the Thrombin CleanCleave Kit (Sigma-Aldrich). Subsequently, the sample was reloaded on the Ni-NTA resin to capture the free His tag (or unprocessed tagged protein), thus allowing elution in the column flow-through of tagless NadR protein, which was used in all subsequent studies. The NadR sample was concentrated and loaded onto a HiLoad Superdex 75 (16/60) preparative size-exclusion chromatography (SEC) column equilibrated in buffer containing 20 mM HEPES pH 8.0, 150 mM NaCl, at a flow-rate of 1 mL/min. NadR protein was collected and the final yield of purified protein obtained from 0.5 L LB growth medium was approximately 8 mg (~2 mg protein per g wet biomass). Samples were used immediately for crystallization or analytical experiments, or were frozen for storage at -20°C. + + + METHODS + title_2 + 59661 + SE-HPLC/MALLS analyses + + + METHODS + paragraph + 59684 + MALLS analyses were performed online with SE-HPLC using a Dawn TREOS MALLS detector (Wyatt Corp., Santa Barbara, CA, USA) and an incident laser wavelength of 658 nm. The intensity of the scattered light was measured at 3 angles simultaneously. Data analysis was performed using the Astra V software (Wyatt) to determine the weighted-average absolute molecular mass (MW), the polydispersity index (MW/Mn) and homogeneity (Mz/Mn) for each oligomer present in solution. Normalization of the MALLS detectors was performed in each analytical session by use of bovine serum albumin. + + + METHODS + title_2 + 60261 + Differential scanning calorimetry + + + METHODS + paragraph + 60295 + The thermal stability of NadR proteins was assessed by differential scanning calorimetry (DSC) using a MicroCal VP-Capillary DSC instrument (GE Healthcare). NadR samples were prepared at a protein concentration of 0.5 mg/mL (~30 μM) in buffer containing 20 mM HEPES, 300 mM NaCl, pH 7.4, with or without 6 mM HPA or salicylate. The DSC temperature scan ranged from 10°C to 110°C, with a thermal ramping rate of 200°C per hour and a 4 second filter period. Data were analyzed by subtraction of the reference data for a sample containing buffer only, using the Origin 7 software. All experiments were performed in triplicate, and mean values of the melting temperature (Tm) were determined. + + + METHODS + title_2 + 60988 + Surface plasmon resonance (SPR) + + + METHODS + title_3 + 61020 + Determination of equilibrium dissociation constant, KD + + + METHODS + paragraph + 61076 + Surface plasmon resonance binding analyses were performed using a Biacore T200 instrument (GE Healthcare) equilibrated at 25°C. The ligand (NadR) was covalently immobilized by amine-coupling on a CM-5 sensor chip (GE Healthcare), using 20 μg/mL purified protein in 10 mM sodium acetate buffer pH 5, injected at 10 μl/min for 120 s until ~9000 response units (RU) were captured. A high level of ligand immobilization was required due to the small size of the analytes. An unmodified surface was used as the reference channel. Titrations with analytes (HPAs or salicylate) were performed with a flow-rate of 30 μl/min, injecting the compounds in a concentration range of 10 μM to 20 mM, using filtered running buffer containing Phosphate Buffered Saline (PBS) with 0.05% Tween-20, pH 7.4. Following each injection, sensor chip surfaces were regenerated with a 30-second injection of 10 mM Glycine pH 2.5. Each titration series contained 20 analyte injections and was performed in triplicate. Titration experiments with long injection phases (> 15 mins) were used to enable steady-state analyses. Data were analyzed using the BIAcore T200 evaluation software and the steady-state affinity model. A buffer injection was subtracted from each curve, and reference sensorgrams were subtracted from experimental sensorgrams to yield curves representing specific binding. The equilibrium dissociation constant, KD, was determined from the plot of RUeq against analyte concentration (S2 Fig), as described previously. Determination of binding stoichiometry: From each plot of RUeq against analyte concentration, obtained from triplicate experiments, the Rmax value (maximum analyte binding capacity of the surface) was extrapolated from the experimental data (S2 Fig). Stoichiometry was calculated using the molecular weight of dimeric NadR as ligand molecule (MWligand) and the molecular weights of the HPA analyte molecules (MWanalyte), and the following equation: where Rligand is recorded directly from the sensorgram during ligand immobilization prior to the titration series, as described previously. The stoichiometry derived therefore represented the number of HPA molecules bound to one dimeric NadR protein. + + + METHODS + title_2 + 63290 + Crystallization of NadR in the presence or absence of 4-HPA + + + METHODS + paragraph + 63350 + Purified NadR was concentrated to 2.7 mg/mL (~160 μM) using a centrifugal concentration device (Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane with cut-off size 10 kDa; Millipore) running at 600 g in a bench top centrifuge (Thermo Scientific IEC CL40R) refrigerated at 2–8°C. To prepare holo-NadR samples, HPA ligands were added at a 200-fold molar excess prior to the centrifugal concentration step. The concentrated holo- or apo-NadR was subjected to crystallization trials performed in 96-well low-profile Intelli-Plates (Art Robbins) or 96-well low-profile Greiner crystallization plates, using a nanodroplet sitting-drop vapour-diffusion format and mixing equal volumes (200 nL) of protein samples and crystallization buffers using a Gryphon robot (Art Robbins). Crystallization trays were incubated at 20°C. Crystals of apo-NadR were obtained in 50% PEG 3350 and 0.13 M di-Ammonium hydrogen citrate, whereas crystals of SeMet–NadR in complex with 4-HPA grew in condition H4 of the Morpheus screen (Molecular Dimensions), which contains 37.5% of the pre-mixed precipitant stock MPD_P1K_PEG 3350, buffer system 1 and 0.1 M amino acids, at pH 6.5. All crystals were mounted in cryo-loops using 10% ethylene glycol or 10% glycerol as cryo-protectant before cooling to 100 K for data collection. + + + METHODS + title_2 + 64673 + X-ray diffraction data collection and structure determination + + + METHODS + paragraph + 64735 + X-ray diffraction data from crystals of apo-NadR and SeMet–NadR/4-HPA were collected on beamline PXII-X10SA of the Swiss Light Source (SLS) at the Paul Scherrer Institut (PSI), Villigen, Switzerland. All diffraction data were processed with XDS and programs from the CCP4 suite. Crystals of apo-NadR and 4-HPA-bound SeMet-NadR belonged to space group P43 21 2 (see Table 2). Apo-NadR crystals contained four molecules (two dimers) in the asymmetric unit (Matthews coefficient 2.25 Å3 Da−1, for a solvent content of 45%), while crystals of SeMet–NadR/4-HPA contained two molecules (one dimer) in the asymmetric unit (Matthews coefficient 1.98 Å3 Da−1, for a solvent content of 38%). In solving the holo-NadR structure, an initial and marginal molecular replacement (MR) solution was obtained using as template search model the crystal structure of the transcriptional regulator PA4135 (PBD entry 2FBI), with which NadR shares ~54% sequence identity. This solution was combined with SAD data to aid identification of two selenium sites in NadR, using autosol in phenix and this allowed generation of high-quality electron density maps that were used to build and refine the structure of the complex. Electron densities were clearly observed for almost the entire dimeric holo-NadR protein, except for a new N-terminal residues and residues 88–90 of chain B. + + + METHODS + paragraph + 66103 + The crystal structure of apo-NadR was subsequently solved by MR in Phaser at 2.7 Å, using the final refined model of SeMet-NadR/4-HPA as the search model. For apo-NadR, electron densities were clearly observed for almost the entire protein, although residues 84–91 of chains A, C, and D, and residues 84–90 of chain B lacked densities suggesting local disorder. + + + METHODS + paragraph + 66470 + Both structures were refined and rebuilt using phenix and Coot, and structural validation was performed using Molprobity. Data collection and refinement statistics are reported in Table 2. Atomic coordinates of the two NadR structures have been deposited in the Protein Data Bank, with entry codes 5aip (NadR bound to 4-HPA) and 5aiq (apo-NadR). All crystallographic software was compiled, installed and maintained by SBGrid. + + + METHODS + title_2 + 66896 + NMR spectroscopy + + + METHODS + paragraph + 66913 + For heteronuclear NMR experiments, the NadR protein concentration used was 85 μM (~ 1.4 mg/mL) in a solution containing 100 mM sodium phosphate buffer (90% H2O and 10% D2O) and 200 mM NaCl, prepared in the apo-form or in the presence of a 200-fold molar excess of 4-HPA, at pH 6.5. The stability, integrity and dimeric state of the protein in the NMR buffer was confirmed by analytical SEC (Superdex 75, 10/300 column) prior to NMR studies. 1H-15N transverse relaxation-optimized spectroscopy (TROSY)-heteronuclear single quantum coherence (HSQC) experiments on apo-NadR and NadR in the presence of 4-HPA were acquired using an Avance 950 Bruker spectrometer, operating at a proton frequency of 949.2 MHz and equipped with triple resonance cryogenically-cooled probe at two different temperatures (298 K and 283 K). 1H-15N TROSY-HSQC experiments were recorded for 12 h, with a data size of 1024 x 232 points. Spectra were processed using the Bruker TopSpin 2.1 and 3.1 software packages. + + + METHODS + title_2 + 67902 + Western blot + + + METHODS + paragraph + 67915 + Western blot analysis was performed as described previously. + + + SUPPL + title_1 + 67976 + Supporting Information + + + REF + title + 67999 + References + + + 1816 + 5459 + 20 + surname:Pizza;given-names:M + surname:Scarlato;given-names:V + surname:Masignani;given-names:V + surname:Giuliani;given-names:MM + surname:Arico;given-names:B + surname:Comanducci;given-names:M + 10710308 + REF + Science + ref + 287 + 2000 + 68010 + Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing + + + 966 + 7 + 71 + surname:Bambini;given-names:S + surname:De Chiara;given-names:M + surname:Muzzi;given-names:A + surname:Mora;given-names:M + surname:Lucidarme;given-names:J + surname:Brehony;given-names:C + 10.1128/CVI.00825-13 + 24807056 + REF + Clin Vaccine Immunol + ref + 21 + 2014 + 68108 + Neisseria adhesin A variation and revised nomenclature scheme + + + 687 + 3 + 98 + surname:Capecchi;given-names:B + surname:Adu-Bobie;given-names:J + surname:Di Marcello;given-names:F + surname:Ciucchi;given-names:L + surname:Masignani;given-names:V + surname:Taddei;given-names:A + 10.1111/j.1365-2958.2004.04423.x + 15660996 + REF + Molecular microbiology + ref + 55 + 2005 + 68171 + Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells + + + 1445 + 11 + 54 + surname:Comanducci;given-names:M + surname:Bambini;given-names:S + surname:Brunelli;given-names:B + surname:Adu-Bobie;given-names:J + surname:Arico;given-names:B + surname:Capecchi;given-names:B + 12045242 + REF + J Exp Med + ref + 195 + 2002 + 68298 + NadA, a novel vaccine candidate of Neisseria meningitidis + + + 17128 + 48 + 33 + surname:Malito;given-names:E + surname:Biancucci;given-names:M + surname:Faleri;given-names:A + surname:Ferlenghi;given-names:I + surname:Scarselli;given-names:M + surname:Maruggi;given-names:G + 10.1073/pnas.1419686111 + 25404323 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 111 + 2014 + 68357 + Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody + + + 15 + 1 + 30 + surname:O'Ryan;given-names:M + surname:Stoddard;given-names:J + surname:Toneatto;given-names:D + surname:Wassil;given-names:J + surname:Dull;given-names:PM + 10.1007/s40265-013-0155-7 + 24338083 + REF + Drugs + ref + 74 + 2014 + 68456 + A multi-component meningococcal serogroup B vaccine (4CMenB): the clinical development program + + + 1054 + 4 + 67 + surname:Schielke;given-names:S + surname:Huebner;given-names:C + surname:Spatz;given-names:C + surname:Nagele;given-names:V + surname:Ackermann;given-names:N + surname:Frosch;given-names:M + 10.1111/j.1365-2958.2009.06710.x + 19400792 + REF + Molecular microbiology + ref + 72 + 2009 + 68551 + Expression of the meningococcal adhesin NadA is controlled by a transcriptional regulator of the MarR family + + + e56097 + 2 + surname:Cloward;given-names:JM + surname:Shafer;given-names:WM + 10.1371/journal.pone.0056097 + 23409129 + REF + PloS one + ref + 8 + 2013 + 68660 + . MtrR control of a transcriptional regulatory pathway in Neisseria meningitidis that influences expression of a gene (nadA) encoding a vaccine candidate + + + e1000710 + 12 + surname:Metruccio;given-names:MM + surname:Pigozzi;given-names:E + surname:Roncarati;given-names:D + surname:Berlanda Scorza;given-names:F + surname:Norais;given-names:N + surname:Hill;given-names:SA + 10.1371/journal.ppat.1000710 + 20041170 + REF + PLoS Pathog + ref + 5 + 2009 + 68814 + A novel phase variation mechanism in the meningococcus driven by a ligand-responsive repressor and differential spacing of distal promoter elements + + + 460 + 2 + 74 + surname:Fagnocchi;given-names:L + surname:Pigozzi;given-names:E + surname:Scarlato;given-names:V + surname:Delany;given-names:I + 10.1128/JB.06161-11 + 22081399 + REF + J Bacteriol + ref + 194 + 2012 + 68962 + In the NadR Regulon, Adhesins and Diverse Meningococcal Functions Are Regulated in Response to Signals in Human Saliva + + + e1004592 + 1 + surname:Jamet;given-names:A + surname:Jousset;given-names:AB + surname:Euphrasie;given-names:D + surname:Mukorako;given-names:P + surname:Boucharlat;given-names:A + surname:Ducousso;given-names:A + 10.1371/journal.ppat.1004592 + 25569427 + REF + PLoS Pathog + ref + 11 + 2015 + 69081 + A new family of secreted toxins in pathogenic Neisseria species + + + e01974 + 5 + 14 + surname:Lamelas;given-names:A + surname:Harris;given-names:SR + surname:Roltgen;given-names:K + surname:Dangy;given-names:JP + surname:Hauser;given-names:J + surname:Kingsley;given-names:RA + 10.1128/mBio.01974-14 + 25336458 + REF + mBio + ref + 5 + 2014 + 69145 + Emergence of a new epidemic Neisseria meningitidis serogroup A Clone in the African meningitis belt: high-resolution picture of genomic changes that mediate immune evasion + + + 410 + 10 + 3 + surname:Alekshun;given-names:MN + surname:Levy;given-names:SB + 10498949 + REF + Trends Microbiol + ref + 7 + 1999 + 69317 + The mar regulon: multiple resistance to antibiotics and other toxic chemicals + + + R142 + 4 + 3 + surname:Grove;given-names:A + 10.1016/j.cub.2013.01.013 + 23428319 + REF + Curr Biol + ref + 23 + 2013 + 69395 + MarR family transcription factors + + + 153 + 2 + 9 + surname:Ellison;given-names:DW + surname:Miller;given-names:VL + 10.1016/j.mib.2006.02.003 + 16529980 + REF + Curr Opin Microbiol + ref + 9 + 2006 + 69429 + Regulation of virulence by members of the MarR/SlyA family + + + 243 + 5 + 54 + surname:Perera;given-names:IC + surname:Grove;given-names:A + 10.1093/jmcb/mjq021 + 20716550 + REF + J Mol Cell Biol + ref + 2 + 2010 + 69488 + Molecular mechanisms of ligand-mediated attenuation of DNA binding by MarR family transcriptional regulators + + + 655 + 3 + 67 + surname:Saridakis;given-names:V + surname:Shahinas;given-names:D + surname:Xu;given-names:X + surname:Christendat;given-names:D + 10.1016/j.jmb.2008.01.001 + 18272181 + REF + Journal of molecular biology + ref + 377 + 2008 + 69597 + Structural insight on the mechanism of regulation of the MarR family of proteins: high-resolution crystal structure of a transcriptional repressor from Methanobacterium thermoautotrophicum + + + 4723 + 14 + 35 + surname:Kumarevel;given-names:T + surname:Tanaka;given-names:T + surname:Umehara;given-names:T + surname:Yokoyama;given-names:S + 10.1093/nar/gkp496 + 19509310 + REF + Nucleic acids research + ref + 37 + 2009 + 69787 + ST1710-DNA complex crystal structure reveals the DNA binding mechanism of the MarR family of regulators + + + 116 + 1–3 + 8 + surname:Takahama;given-names:U + surname:Oniki;given-names:T + surname:Murata;given-names:H + 11997029 + REF + FEBS Lett + ref + 518 + 2002 + 69891 + The presence of 4-hydroxyphenylacetic acid in human saliva and the possibility of its nitration by salivary nitrite in the stomach + + + 560 + 2 + 9 + surname:Fagnocchi;given-names:L + surname:Biolchi;given-names:A + surname:Ferlicca;given-names:F + surname:Boccadifuoco;given-names:G + surname:Brunelli;given-names:B + surname:Brier;given-names:S + 10.1128/IAI.01085-12 + 23230289 + REF + Infect Immun + ref + 81 + 2013 + 70022 + Transcriptional regulation of the nadA gene in Neisseria meningitidis impacts the prediction of coverage of a multicomponent meningococcal serogroup B vaccine + + + 6738 + 34 + 52 + surname:Brier;given-names:S + surname:Fagnocchi;given-names:L + surname:Donnarumma;given-names:D + surname:Scarselli;given-names:M + surname:Rappuoli;given-names:R + surname:Nissum;given-names:M + 10.1021/bi300656w + 22834735 + REF + Biochemistry + ref + 51 + 2012 + 70181 + Structural Insight into the Mechanism of DNA-Binding Attenuation of the Neisserial Adhesin Repressor NadR by the Small Natural Ligand 4-Hydroxyphenylacetic Acid + + + 5456 + 12 + 60 + surname:Martin;given-names:RG + surname:Rosner;given-names:JL + 7777530 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 92 + 1995 + 70342 + Binding of purified multiple antibiotic-resistance repressor protein (MarR) to mar operator sequences + + + 774 + 3 + 97 + surname:Krissinel;given-names:E + surname:Henrick;given-names:K + 10.1016/j.jmb.2007.05.022 + 17681537 + REF + Journal of molecular biology + ref + 372 + 2007 + 70444 + Inference of macromolecular assemblies from crystalline state + + + 271 + 2 + 7 + surname:Mistrik;given-names:P + surname:Moreau;given-names:F + surname:Allen;given-names:JM + 10.1016/j.ab.2004.01.022 + 15051545 + REF + Anal Biochem + ref + 327 + 2004 + 70506 + BiaCore analysis of leptin-leptin receptor interaction: evidence for 1:1 stoichiometry + + + 1 + 1 + 17 + surname:Liang;given-names:J + surname:Edelsbrunner;given-names:H + surname:Fu;given-names:P + surname:Sudhakar;given-names:PV + surname:Subramaniam;given-names:S + 9741840 + REF + Proteins + ref + 33 + 1998 + 70593 + Analytical shape computation of macromolecules: I. Molecular area and volume through alpha shape + + + 131 + 1 + 41 + surname:Hong;given-names:M + surname:Fuangthong;given-names:M + surname:Helmann;given-names:JD + surname:Brennan;given-names:RG + 10.1016/j.molcel.2005.09.013 + 16209951 + REF + Mol Cell + ref + 20 + 2005 + 70690 + Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of the MarR family + + gene + GENE: + cleaner0 + 2023-07-06T08:11:09Z + + ohrA + + + + 19490 + 45 + 5 + surname:Donnelly;given-names:J + surname:Medini;given-names:D + surname:Boccadifuoco;given-names:G + surname:Biolchi;given-names:A + surname:Ward;given-names:J + surname:Frasch;given-names:C + 10.1073/pnas.1013758107 + 20962280 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 107 + 2010 + 70786 + Qualitative and quantitative assessment of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines + + + 29114 + 40 + 21 + surname:Mani;given-names:AR + surname:Ippolito;given-names:S + surname:Moreno;given-names:JC + surname:Visser;given-names:TJ + surname:Moore;given-names:KP + 10.1074/jbc.M704270200 + 17686770 + REF + J Biol Chem + ref + 282 + 2007 + 70920 + The metabolism and dechlorination of chlorotyrosine in vivo + + + 204 + Pt 3 + 9 + surname:Nichols;given-names:CE + surname:Sainsbury;given-names:S + surname:Ren;given-names:J + surname:Walter;given-names:TS + surname:Verma;given-names:A + surname:Stammers;given-names:DK + 10.1107/S174430910900414X + 19255465 + REF + Acta Crystallogr Sect F Struct Biol Cryst Commun + ref + 65 + 2009 + 70980 + The structure of NMB1585, a MarR-family regulator from Neisseria meningitidis + + protein + PR: + cleaner0 + 2023-07-06T08:12:28Z + + NMB1585 + + + + 29253 + 32 + 9 + surname:Lim;given-names:D + surname:Poole;given-names:K + surname:Strynadka;given-names:NC + 10.1074/jbc.M111381200 + 12034710 + REF + J Biol Chem + ref + 277 + 2002 + 71059 + Crystal structure of the MexR repressor of the mexRAB-oprM multidrug efflux operon of Pseudomonas aeruginosa + + + 683 + 3 + 93 + surname:Mariotti;given-names:P + surname:Malito;given-names:E + surname:Biancucci;given-names:M + surname:Lo Surdo;given-names:P + surname:Mishra;given-names:RP + surname:Nardi-Dei;given-names:V + 10.1042/BJ20121426 + 23113737 + REF + The Biochemical journal + ref + 449 + 2013 + 71169 + Structural and functional characterization of the Staphylococcus aureus virulence factor and vaccine candidate FhuD2 + + + 2017 + 12 + 31 + surname:Podkowa;given-names:KJ + surname:Briere;given-names:LA + surname:Heinrichs;given-names:DE + surname:Shilton;given-names:BH + 10.1021/bi401349d + 24606332 + REF + Biochemistry + ref + 53 + 2014 + 71286 + Crystal and solution structure analysis of FhuD2 from Staphylococcus aureus in multiple unliganded conformations and bound to ferrioxamine-B + + + 19 + surname:Changeux;given-names:JP + surname:Edelstein;given-names:S + 10.3410/B3-19 + 21941598 + REF + F1000 biology reports + ref + 3 + 2011 + 71427 + Conformational selection or induced fit? 50 years of debate resolved + + + 514 + 8740 + 7 + surname:McGuinness;given-names:BT + surname:Clarke;given-names:IN + surname:Lambden;given-names:PR + surname:Barlow;given-names:AK + surname:Poolman;given-names:JT + surname:Jones;given-names:DM + 1705642 + REF + Lancet + ref + 337 + 1991 + 71496 + Point mutation in meningococcal por A gene associated with increased endemic disease + + + 1300 + 12 + 5 + surname:Lo Surdo;given-names:P + surname:Bottomley;given-names:MJ + surname:Calzetta;given-names:A + surname:Settembre;given-names:EC + surname:Cirillo;given-names:A + surname:Pandit;given-names:S + 10.1038/embor.2011.205 + 22081141 + REF + EMBO Rep + ref + 12 + 2011 + 71581 + Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH + + + 125 + Pt 2 + 32 + surname:Kabsch;given-names:W + 10.1107/S0907444909047337 + 20124692 + REF + Acta crystallographica Section D, Biological crystallography + ref + 66 + 2010 + 71679 + Xds + + + 760 + Pt 5 + 3 + 15299374 + REF + Acta crystallographica Section D, Biological crystallography + ref + 50 + 1994 + 71683 + The CCP4 suite: programs for protein crystallography + + + 213 + Pt 2 + 21 + surname:Adams;given-names:PD + surname:Afonine;given-names:PV + surname:Bunkoczi;given-names:G + surname:Chen;given-names:VB + surname:Davis;given-names:IW + surname:Echols;given-names:N + 10.1107/S0907444909052925 + 20124702 + REF + Acta crystallographica Section D, Biological crystallography + ref + 66 + 2010 + 71736 + PHENIX: a comprehensive Python-based system for macromolecular structure solution + + + 658 + Pt 4 + 74 + surname:McCoy;given-names:AJ + surname:Grosse-Kunstleve;given-names:RW + surname:Adams;given-names:PD + surname:Winn;given-names:MD + surname:Storoni;given-names:LC + surname:Read;given-names:RJ + 10.1107/S0021889807021206 + 19461840 + REF + J Appl Crystallogr + ref + 40 + 2007 + 71818 + Phaser crystallographic software + + + 486 + Pt 4 + 501 + surname:Emsley;given-names:P + surname:Lohkamp;given-names:B + surname:Scott;given-names:WG + surname:Cowtan;given-names:K + 10.1107/S0907444910007493 + 20383002 + REF + Acta crystallographica Section D, Biological crystallography + ref + 66 + 2010 + 71851 + Features and development of Coot + + + 12 + Pt 1 + 21 + surname:Chen;given-names:VB + surname:Arendall;given-names:WB;suffix:3rd + surname:Headd;given-names:JJ + surname:Keedy;given-names:DA + surname:Immormino;given-names:RM + surname:Kapral;given-names:GJ + 10.1107/S0907444909042073 + 20057044 + REF + Acta crystallographica Section D, Biological crystallography + ref + 66 + 2010 + 71884 + MolProbity: all-atom structure validation for macromolecular crystallography + + + e01456 + surname:Morin;given-names:A + surname:Eisenbraun;given-names:B + surname:Key;given-names:J + surname:Sanschagrin;given-names:PC + surname:Timony;given-names:MA + surname:Ottaviano;given-names:M + 10.7554/eLife.01456 + 24040512 + REF + Elife + ref + 2 + 2013 + 71961 + Collaboration gets the most out of software + + + 72 + Pt 1 + 82 + surname:Evans;given-names:P + 10.1107/S0907444905036693 + 16369096 + REF + Acta crystallographica Section D, Biological crystallography + ref + 62 + 2006 + 72005 + Scaling and assessment of data quality + + + D292 + 6 + surname:de Beer;given-names:TA + surname:Berka;given-names:K + surname:Thornton;given-names:JM + surname:Laskowski;given-names:RA + 10.1093/nar/gkt940 + 24153109 + REF + Nucleic acids research + ref + 42 + 2014 + 72044 + PDBsum additions + + + diff --git a/BioC_XML/4848090_v0.xml b/BioC_XML/4848090_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..1e0823427cb5294235714dea01bfc882d67fe8df --- /dev/null +++ b/BioC_XML/4848090_v0.xml @@ -0,0 +1,10265 @@ + + + + PMC + 20140719 + pmc.key + + 4848090 + CC BY + no + 0 + 0 + + 10.7554/eLife.15075 + 4848090 + 27058169 + 15075 + e15075 + membrane signaling receptor kinase peptide hormone floral abscission plant development protein complexes <i>A. thaliana</i> + This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. + surname:Santiago;given-names:Julia + surname:Brandt;given-names:Benjamin + surname:Hothorn;given-names:Michael + surname:Butenko;given-names:Melinka A + surname:Brandt;given-names:Benjamin + surname:Santiago;given-names:Julia + surname:Wildhagen;given-names:Mari + surname:Hohmann;given-names:Ulrich + surname:Hothorn;given-names:Ludwig A + surname:Butenko;given-names:Melinka A + surname:Hothorn;given-names:Michael + surname:Zhang;given-names:Mingjie + surname:Hothorn;given-names:Michael + surname:Hothorn;given-names:Michael + TITLE + Author Keywords Research Organism + front + 5 + 2016 + 0 + Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:29:24Z + + peptide hormone + + + + ABSTRACT + abstract + 95 + Plants constantly renew during their life cycle and thus require to shed senescent and damaged organs. Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. It is unknown how expression of IDA in the abscission zone leads to HAESA activation. Here we show that IDA is sensed directly by the HAESA ectodomain. Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. A central hydroxyproline residue anchors IDA to the receptor. The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + Plants + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:34:57Z + + leucine-rich repeat receptor kinase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:35:06Z + + LRR-RK + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:29:24Z + + peptide hormone + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:33Z + + ectodomain + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:25Z + + Crystal structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:35:29Z + + in complex with + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:30:04Z + + hormone binding pocket + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:12Z + + active + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:45Z + + dodecamer + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:28:58Z + + peptide + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:36:09Z + + hydroxyproline + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:36:27Z + + co-receptor + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:29:24Z + + peptide hormone + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:01:51Z + + Arg-His-Asn motif + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:36:42Z + + conserved + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:01Z + + plant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:29:02Z + + peptides + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:01Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:36:36Z + + peptide hormone receptors + + + + ABSTRACT + abstract + 1019 + DOI: http://dx.doi.org/10.7554/eLife.15075.001 + + + ABSTRACT + abstract_title_1 + 1066 + eLife digest + + + ABSTRACT + abstract + 1079 + Plants can shed their leaves, flowers or other organs when they no longer need them. But how does a leaf or a flower know when to let go? A receptor protein called HAESA is found on the surface of the cells that surround a future break point on the plant. When its time to shed an organ, a hormone called IDA instructs HAESA to trigger the shedding process. However, the molecular details of how IDA triggers organ shedding are not clear. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + Plants + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:38:22Z + + receptor protein + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T08:38:42Z + + hormone + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + + ABSTRACT + abstract + 1518 + The shedding of floral organs (or leaves) can be easily studied in a model plant called Arabidopsis. Santiago et al. used protein biochemistry, structural biology and genetics to uncover how the IDA hormone activates HAESA. The experiments show that IDA binds directly to a canyon shaped pocket in HAESA that extends out from the surface of the cell. IDA binding to HAESA allows another receptor protein called SERK1 to bind to HAESA, which results in the release of signals inside the cell that trigger the shedding of organs. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:01Z + + plant + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:40:57Z + + Arabidopsis + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:41:04Z + + protein biochemistry + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:41:10Z + + structural biology + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:41:13Z + + genetics + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T08:41:17Z + + hormone + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:42:02Z + + binds directly to + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:41:22Z + + canyon shaped + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:41:25Z + + pocket + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:16:55Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:41:40Z + + receptor protein + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:42:20Z + + to bind to + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + + ABSTRACT + abstract + 2046 + The next step following on from this work is to understand what signals are produced when IDA activates HAESA. Another challenge will be to find out where IDA is produced in the plant and what causes it to accumulate in specific places in preparation for organ shedding. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:01Z + + plant + + + + ABSTRACT + abstract + 2317 + DOI: http://dx.doi.org/10.7554/eLife.15075.002 + + + INTRO + title_1 + 2364 + Introduction + + + elife-15075-fig1-figsupp1.jpg + fig1s1 + FIG + fig_title_caption + 2377 + The HAESA ectodomain folds into a superhelical assembly of 21 leucine-rich repeats. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:05Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:33Z + + ectodomain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:43:20Z + + superhelical assembly + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:44:11Z + + leucine-rich repeats + + + + elife-15075-fig1-figsupp1.jpg + fig1s1 + FIG + fig_caption + 2461 + (A) SDS PAGE analysis of the purified Arabidopsis thaliana HAESA ectodomain (residues 20–620) obtained by secreted expression in insect cells. The calculated molecular mass is 65.7 kDa, the actual molecular mass obtained by mass spectrometry is 74,896 Da, accounting for the N-glycans. (B) Ribbon diagrams showing front (left panel) and side views (right panel) of the isolated HAESA LRR domain. The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. The HAESA ectodomain is shown in blue (in surface representation), the glycan structures are shown in yellow. Molecular surfaces were calculated with the program MSMS, with a probe radius of 1.5 Å. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:45:35Z + + SDS PAGE + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:47:13Z + + Arabidopsis thaliana + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:06Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:33Z + + ectodomain + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:47:22Z + + 20–620 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:47:18Z + + secreted expression in insect cells + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:47:25Z + + mass spectrometry + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T08:47:39Z + + N-glycans + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:06Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:47:45Z + + LRR domain + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:47:57Z + + 20–88 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:48:04Z + + 593–615 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:48:11Z + + capping domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:48:52Z + + LRR motifs + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:49:02Z + + disulphide bonds + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:49:09Z + + Structure based sequence alignment + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:44:13Z + + leucine-rich repeats + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:06Z + + HAESA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:01Z + + plant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:49:42Z + + LRR + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:49:46Z + + Conserved + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:49:56Z + + hydrophobic + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:50:01Z + + residues + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:50:06Z + + N-glycosylation sites + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:50:12Z + + structures + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:50:25Z + + cysteine + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:50:33Z + + disulphide bridge + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:50:57Z + + Asn-linked glycans + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:06Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:33Z + + ectodomain + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T08:51:14Z + + Oligomannose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T08:51:20Z + + N-actylglucosamines + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T08:51:29Z + + mannose + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:51:37Z + + Trichoplusia ni + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:52:19Z + + glycosylation sites + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:06Z + + HAESA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:51:47Z + + structures + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T08:51:55Z + + carbohydrate + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:06Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:33Z + + ectodomain + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T08:52:03Z + + glycan + + + + elife-15075-fig1-figsupp1.jpg + fig1s1 + FIG + fig_caption + 3975 + DOI: +http://dx.doi.org/10.7554/eLife.15075.004 + + + elife-15075-fig1-figsupp2.jpg + fig1s2 + FIG + fig_title_caption + 4022 + Hydrophobic contacts and a hydrogen-bond network mediate the interaction between HAESA and the peptide hormone IDA. + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:28Z + + Hydrophobic contacts + + + site + SO: + melaniev@ebi.ac.uk + 2023-06-15T08:44:48Z + + hydrogen-bond network + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:06Z + + HAESA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:29:24Z + + peptide hormone + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + + elife-15075-fig1-figsupp2.jpg + fig1s2 + FIG + fig_caption + 4138 + (A) Details of the IDA binding pocket. HAESA is shown in blue (ribbon diagram), the C-terminal Arg-His-Asn motif (left panel), the central Hyp anchor (center) and the N-terminal Pro-rich motif in IDA (right panel) are shown in yellow (in bonds representation). HAESA interface residues are shown as sticks, selected hydrogen bond interactions are denoted as dotted lines (in magenta). (B) View of the complete IDA (in bonds representation, in yellow) binding pocket in HAESA (surface view, in blue). Orientation as in (A). (C) Structure based sequence alignment of leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. Residues mediating hydrophobic interactions with the IDA peptide are highlighted in blue, residues contributing to hydrogen bond interactions and/or salt bridges are shown in red. The IDA binding pocket covers LRRs 2–14 and all residues originate from the inner surface of the HAESA superhelix. + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:01:38Z + + IDA binding pocket + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:06Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:01:51Z + + Arg-His-Asn motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:02:42Z + + Hyp anchor + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:02:59Z + + Pro-rich motif + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:03:09Z + + HAESA interface residues + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:28Z + + hydrogen bond interactions + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:42Z + + IDA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:03:27Z + + binding pocket + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:06Z + + HAESA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:03:30Z + + Structure based sequence alignment + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:44:13Z + + leucine-rich repeats + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:06Z + + HAESA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:01Z + + plant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:03:37Z + + LRR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:03:40Z + + consensus sequence + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:28Z + + hydrophobic interactions + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:08:50Z + + IDA peptide + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:28Z + + hydrogen bond interactions + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:28Z + + salt bridges + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:01:38Z + + IDA binding pocket + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:04:34Z + + LRRs 2–14 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:06Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:04:41Z + + superhelix + + + + elife-15075-fig1-figsupp2.jpg + fig1s2 + FIG + fig_caption + 5090 + DOI: +http://dx.doi.org/10.7554/eLife.15075.005 + + + elife-15075-fig1-figsupp3.jpg + fig1s3 + FIG + fig_title_caption + 5137 + The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like proteins from different plant species. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-17T09:05:48Z + + IDA-HAESA + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-17T09:05:55Z + + SERK1-HAESA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:06:00Z + + interfaces + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:06:03Z + + conserved + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:06Z + + HAESA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:06:10Z + + HAESA-like proteins + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:01Z + + plant + + + + elife-15075-fig1-figsupp3.jpg + fig1s3 + FIG + fig_caption + 5267 + Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). The alignment includes a secondary structure assignment calculated with the program DSSP and colored according to Figure 1, with the N- and C-terminal caps and the 21 LRR motifs indicated in orange and blue, respectively. Cysteine residues engaged in disulphide bonds are depicted in green. HAESA residues interacting with the IDA peptide and/or the SERK1 co-receptor kinase ectodomain are highlighted in blue and orange, respectively. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:07:34Z + + Structure-based sequence alignment + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:07:37Z + + HAESA family members + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:47:13Z + + Arabidopsis thaliana + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:47:13Z + + Arabidopsis thaliana + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:07:45Z + + HSL2 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:07:54Z + + Capsella rubella + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:08:02Z + + Citrus clementina + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:07:45Z + + HSL2 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:08:11Z + + Vitis vinifera + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:08:27Z + + caps + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:09:03Z + + LRR motifs + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:50:25Z + + Cysteine + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:49:02Z + + disulphide bonds + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:08:59Z + + IDA peptide + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:09:39Z + + co-receptor kinase + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:33Z + + ectodomain + + + + elife-15075-fig1-figsupp3.jpg + fig1s3 + FIG + fig_caption + 6017 + DOI: +http://dx.doi.org/10.7554/eLife.15075.006 + + + elife-15075-fig1.jpg + fig1 + FIG + fig_title_caption + 6064 + The peptide hormone IDA binds to the HAESA LRR ectodomain. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:29:24Z + + peptide hormone + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:10:10Z + + LRR ectodomain + + + + elife-15075-fig1.jpg + fig1 + FIG + fig_caption + 6123 + (A) Multiple sequence alignment of selected IDA family members. The conserved PIP motif is highlighted in yellow, the central Hyp in blue. The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. (C) Structure of the HAESA – IDA complex with HAESA shown in blue (ribbon diagram). IDA (in bonds representation, surface view included) is depicted in yellow. The peptide binding pocket covers HAESA LRRs 2–14. (D) Close-up view of the entire IDA (in yellow) peptide binding site in HAESA (in blue). Details of the interactions between the central Hyp anchor in IDA and the C-terminal Arg-His-Asn motif with HAESA are highlighted in (E) and (F), respectively. Hydrogren bonds are depicted as dotted lines (in magenta), a water molecule is shown as a red sphere. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:12:20Z + + Multiple sequence alignment + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:19:03Z + + IDA family members + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:12:51Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:12:59Z + + PIP motif + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:13:21Z + + Hyp + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:13:30Z + + PKGV motif + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:28:24Z + + N-terminally extended + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:09:00Z + + IDA peptide + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:14:02Z + + Isothermal titration calorimetry + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:33Z + + ectodomain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:14:06Z + + synthetic + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T09:14:10Z + + peptide + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-17T16:29:32Z + + HAESA – IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:14:20Z + + peptide binding pocket + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:04:34Z + + LRRs 2–14 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:14:28Z + + peptide binding site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:02:42Z + + Hyp anchor + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:01:51Z + + Arg-His-Asn motif + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T09:15:02Z + + water + + + + elife-15075-fig1.jpg + fig1 + FIG + fig_caption + 7031 + DOI: +http://dx.doi.org/10.7554/eLife.15075.003 + + + INTRO + paragraph + 7078 + During their growth, development and reproduction plants use cell separation processes to detach no-longer required, damaged or senescent organs. Abscission of floral organs in Arabidopsis is a model system to study these cell separation processes in molecular detail. The LRR-RKs HAESA (greek: to adhere to) and HAESA-LIKE 2 (HSL2) redundantly control floral abscission. Loss-of-function of the secreted small protein INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) causes floral organs to remain attached while its over-expression leads to premature shedding. Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A). It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and HSL2 in transient assays in tobacco cells. This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). The available genetic and biochemical evidence suggests that IDA and HAESA together control floral abscission, but it is poorly understood if IDA is directly sensed by the receptor kinase HAESA and how IDA binding at the cell surface would activate the receptor. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:19:27Z + + Arabidopsis + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:19:47Z + + LRR-RKs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:19:35Z + + HAESA-LIKE 2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:07:45Z + + HSL2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:20:13Z + + INFLORESCENCE DEFICIENT IN ABSCISSION + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:19:56Z + + Full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:20:35Z + + proteolytically processed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:20:38Z + + conserved + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:20:41Z + + stretch of 20 amino-acids + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:21:35Z + + EPIP + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:45Z + + dodecamer + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T09:21:18Z + + peptide + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:21:35Z + + EPIP + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:07:45Z + + HSL2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:21:42Z + + transient assays + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:21:57Z + + tobacco + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:22:02Z + + This sequence motif + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:22:05Z + + highly conserved + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:19:03Z + + IDA family members + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:22:49Z + + IDA-LIKE PROTEINS + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:23:05Z + + IDLs + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:23:11Z + + Pro + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:23:35Z + + post-translationally modified + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:23:58Z + + hydroxyproline + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:24:15Z + + Hyp + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:24:20Z + + receptor kinase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:17:44Z + + IDA + + + + RESULTS + title_1 + 8403 + Results + + + RESULTS + title_2 + 8411 + IDA directly binds to the LRR domain of HAESA + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:24:55Z + + LRR domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + + elife-15075-fig2.jpg + fig2 + FIG + fig_title_caption + 8457 + Active IDA-family peptide hormones are hydroxyprolinated dodecamers. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:12Z + + Active + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:25:46Z + + IDA-family peptide hormones + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:26:07Z + + hydroxyprolinated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:26:18Z + + dodecamers + + + + elife-15075-fig2.jpg + fig2 + FIG + fig_caption + 8526 + Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain. Root mean square deviation (r.m.s.d.) is 1.0 Å comparing 100 corresponding atoms. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:28:29Z + + N-terminally extended + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:30:14Z + + PKGV-IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:29:55Z + + IDL1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:30:41Z + + bound to + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:30:04Z + + hormone binding pocket + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:30:18Z + + simulated annealing + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:30:26Z + + 2Fo–Fc omit electron density maps + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:31:16Z + + Pro58 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:30:56Z + + IDA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:31:22Z + + Leu67 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:31:07Z + + IDA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:30:31Z + + electron density + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:30:41Z + + bound to + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:07Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:33Z + + ectodomain + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:31:30Z + + equilibrium dissociation constants + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:31:37Z + + Kd + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:31:42Z + + binding enthalpies + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:31:48Z + + ΔH + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:31:52Z + + binding entropies + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:31:59Z + + ΔS + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:10:22Z + + IDA peptides + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:08Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:33Z + + ectodomain + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:32:08Z + + Structural superposition + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:12Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:43Z + + IDA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:10:54Z + + IDL1 peptide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:30:41Z + + bound to + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:08Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:33Z + + ectodomain + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:33:22Z + + Root mean square deviation + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:33:38Z + + r.m.s.d. + + + + elife-15075-fig2.jpg + fig2 + FIG + fig_caption + 9382 + DOI: +http://dx.doi.org/10.7554/eLife.15075.007 + + + elife-15075-fig3.jpg + fig3 + FIG + fig_title_caption + 9429 + The receptor kinase SERK1 acts as a HAESA co-receptor and promotes high-affinity IDA sensing. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:35:33Z + + receptor kinase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:36:35Z + + HAESA co-receptor + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:18:04Z + + IDA + + + + elife-15075-fig3.jpg + fig3 + FIG + fig_caption + 9523 + (A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. Petal break-strength is measured from positions 1 to 8 along the primary inflorescence where positions 1 is defined as the flower at anthesis (n=15, bars=SD). This treatment-by-position balanced two-way layout was analyzed separately per position, because of the serious interaction, by means of a Dunnett-type comparison against the Col-0 control, allowing for heterogeneous variances. Petal break-strength was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. Calculations were performed in R (version 3.2.3). (B) Analytical size-exclusion chromatography. The HAESA LRR domain elutes as a monomer (black dotted line), as does the isolated SERK1 ectodomain (blue dotted line). A HAESA – IDA – SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line). Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). A SDS PAGE of the peak fractions is shown alongside. Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. The titration of IDA wild-type versus the isolated HAESA ectodomain from Figure 1B is shown for comparison (red line; n.d. no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. A SDS PAGE of the peak fractions is shown alongside. (E) In vitro kinase assays of the HAESA and SERK1 kinase domains. Wild-type HAESA and SERK1 kinase domains (KDs) exhibit auto-phosphorylation activities (lanes 1 + 3). Mutant (m) versions, which carry point mutations in their active sites (Asp837HAESA→Asn, Asp447SERK1→Asn) possess no autophosphorylation activity (lanes 2+4). Transphosphorylation activity from the active kinase to the mutated form can be observed in both directions (lanes 5+6). A coomassie-stained gel loading control is shown below. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:39:22Z + + Petal break-strength assays + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T16:29:45Z + + serk + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:55Z + + mutant + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T09:43:03Z + + haesa + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T09:43:21Z + + hsl2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:44Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:50Z + + wild-type + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T09:43:05Z + + haesa + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T09:43:23Z + + hsl2 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T09:44:19Z + + serk1-1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:58Z + + mutant + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:39:31Z + + Analytical size-exclusion chromatography + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:08Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:44:40Z + + LRR domain + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:37Z + + monomer + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:33Z + + ectodomain + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-17T09:45:13Z + + HAESA – IDA – SERK1 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:45:27Z + + heterodimer + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:08Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:45:22Z + + monomeric + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:08Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:47:16Z + + Thyroglobulin + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:47:30Z + + Ferritin + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:47:46Z + + Aldolase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:48:00Z + + Conalbumin + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:48:15Z + + Ovalbumin + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:48:30Z + + Carbonic anhydrase + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:45:35Z + + SDS PAGE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:08Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:29:07Z + + Isothermal titration calorimetry + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:50Z + + wild-type + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T12:37:35Z + + Hyp64→Pro + + + protein + PR: + melaniev@ebi.ac.uk + 2023-07-21T12:36:07Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:08Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:27:48Z + + ectodomains + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:46:53Z + + titration + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:44Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:50Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:08Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:33Z + + ectodomain + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:29:11Z + + Analytical size-exclusion chromatography + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:35:15Z + + presence of + + + protein + PR: + melaniev@ebi.ac.uk + 2023-07-21T12:36:27Z + + IDA + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T12:37:35Z + + Hyp64→Pro + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:46:39Z + + mutant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T09:46:42Z + + peptide + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:08Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:27:52Z + + ectodomains + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:45:35Z + + SDS PAGE + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:45:40Z + + In vitro kinase assays + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:08Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:18:32Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:45:46Z + + kinase domains + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:50Z + + Wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:08Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:27:56Z + + kinase domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:27:59Z + + KDs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:28:35Z + + Mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:45:42Z + + point mutations + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:46:35Z + + active sites + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:46:22Z + + Asp837HAESA→Asn + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:46:29Z + + Asp447SERK1→Asn + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:12Z + + active + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:45:58Z + + mutated + + + + elife-15075-fig3.jpg + fig3 + FIG + fig_caption + 12096 + DOI: +http://dx.doi.org/10.7554/eLife.15075.008 + + + tbl1.xml + tbl1 + TABLE + table_caption + 12143 + Crystallographic data collection, phasing and refinement statistics for the isolated A. thaliana HAESA ectodomain. + + + tbl1.xml + tbl1 + TABLE + table_caption + 12258 + DOI: +http://dx.doi.org/10.7554/eLife.15075.009 + + + tbl1.xml + tbl1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><thead><tr><th valign="top" rowspan="1" colspan="1"/><th valign="top" rowspan="1" colspan="1">HAESA NaI shortsoak</th><th valign="top" rowspan="1" colspan="1">HAESA apo</th></tr></thead><tbody><tr><td valign="top" rowspan="1" colspan="1"><bold>PDB-ID</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">5IXO</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>Data collection</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Space group</td><td valign="top" rowspan="1" colspan="1"><italic>P</italic>3<sub>1</sub> 21</td><td valign="top" rowspan="1" colspan="1"><italic>P</italic>3<sub>1</sub> 21</td></tr><tr><td valign="top" rowspan="1" colspan="1">Cell dimensions</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>a</italic>, <italic>b, c</italic> (Å)</td><td valign="top" rowspan="1" colspan="1">148.55, 148.55, 58.30</td><td valign="top" rowspan="1" colspan="1">149.87, 149.87, 58.48</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>α</italic>, β, γ (°)</td><td valign="top" rowspan="1" colspan="1">90, 90, 120</td><td valign="top" rowspan="1" colspan="1">90, 90, 120</td></tr><tr><td valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td valign="top" rowspan="1" colspan="1">48.63–2.39 (2.45–2.39)</td><td valign="top" rowspan="1" colspan="1">45.75–1.74 (1.85–1.74)</td></tr><tr><td valign="top" rowspan="1" colspan="1">R<sub>meas</sub><sup>#</sup></td><td valign="top" rowspan="1" colspan="1">0.096 (0.866)</td><td valign="top" rowspan="1" colspan="1">0.038 (1.02)</td></tr><tr><td valign="top" rowspan="1" colspan="1">CC(1/2)<italic><sup>#</sup></italic></td><td valign="top" rowspan="1" colspan="1">100/86.6</td><td valign="top" rowspan="1" colspan="1">100/75.6</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>I/σ I<sup>#</sup></italic></td><td valign="top" rowspan="1" colspan="1">27.9 (4.9)</td><td valign="top" rowspan="1" colspan="1">18.7 (1.8)</td></tr><tr><td valign="top" rowspan="1" colspan="1">Completeness (%)<italic><sup>#</sup></italic></td><td valign="top" rowspan="1" colspan="1">99.9 (98.6)</td><td valign="top" rowspan="1" colspan="1">99.6 (97.4)</td></tr><tr><td valign="top" rowspan="1" colspan="1">Redundancy<italic><sup>#</sup></italic></td><td valign="top" rowspan="1" colspan="1">53.1 (29.9)</td><td valign="top" rowspan="1" colspan="1">14.4 (14.0)</td></tr><tr><td valign="top" rowspan="1" colspan="1">Wilson B-factor (Å<sup>2</sup>)<italic><sup>#</sup></italic></td><td valign="top" rowspan="1" colspan="1">84.45</td><td valign="top" rowspan="1" colspan="1">81.10</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>Refinement</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">45.75 – 1.74</td></tr><tr><td valign="top" rowspan="1" colspan="1">No. reflections</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">71,213</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>R</italic><sub>work/</sub><italic>R</italic><sub>free</sub><sup>$</sup></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">0.188/0.218</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>No. atoms</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Protein/glycan</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">4,533/126</td></tr><tr><td valign="top" rowspan="1" colspan="1">Water</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">71</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>Res. B-factors (Å<sup>2</sup>)</bold><sup>$</sup></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Protein</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">77.54</td></tr><tr><td valign="top" rowspan="1" colspan="1">Glycan</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">95.98</td></tr><tr><td valign="top" rowspan="1" colspan="1">Water</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">73.20</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>R.m.s deviations</bold><sup>$</sup></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Bond lengths (Å)</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">0.0095</td></tr><tr><td valign="top" rowspan="1" colspan="1">Bond angles (°)</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1">1.51</td></tr></tbody></table> + + 12305 + HAESA NaI shortsoak HAESA apo PDB-ID 5IXO Data collection Space group P31 21 P31 21 Cell dimensions a, b, c (Å) 148.55, 148.55, 58.30 149.87, 149.87, 58.48 α, β, γ (°) 90, 90, 120 90, 90, 120 Resolution (Å) 48.63–2.39 (2.45–2.39) 45.75–1.74 (1.85–1.74) Rmeas# 0.096 (0.866) 0.038 (1.02) CC(1/2)# 100/86.6 100/75.6 I/σ I# 27.9 (4.9) 18.7 (1.8) Completeness (%)# 99.9 (98.6) 99.6 (97.4) Redundancy# 53.1 (29.9) 14.4 (14.0) Wilson B-factor (Å2)# 84.45 81.10 Refinement Resolution (Å) 45.75 – 1.74 No. reflections 71,213 Rwork/Rfree$ 0.188/0.218 No. atoms Protein/glycan 4,533/126 Water 71 Res. B-factors (Å2)$ Protein 77.54 Glycan 95.98 Water 73.20 R.m.s deviations$ Bond lengths (Å) 0.0095 Bond angles (°) 1.51 + + + tbl1.xml + tbl1 + TABLE + table_footnote + 13117 + Highest resolution shell is shown in parenthesis. + + + tbl1.xml + tbl1 + TABLE + table_footnote + 13167 + #As defined in XDS. + + + tbl1.xml + tbl1 + TABLE + table_footnote + 13187 + $As defined in Refmac5. + + + tbl2.xml + tbl2 + TABLE + table_caption + 13211 + Crystallographic data collection and refinement statistics for the HAESA – IDA, – PKGV-IDA, – IDL1 and – IDA – SERK1 complexes. + + + tbl2.xml + tbl2 + TABLE + table_caption + 13349 + DOI: +http://dx.doi.org/10.7554/eLife.15075.010 + + + tbl2.xml + tbl2 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><thead><tr><th valign="top" rowspan="1" colspan="1"/><th valign="top" rowspan="1" colspan="1">HAESA – IDA</th><th valign="top" rowspan="1" colspan="1">HAESA – PKGV-IDA</th><th valign="top" rowspan="1" colspan="1">HAESA – IDL1</th><th valign="top" rowspan="1" colspan="1">HAESA – IDA – SERK1</th></tr></thead><tbody><tr><td valign="top" rowspan="1" colspan="1"><bold>PDB-ID</bold></td><td valign="top" rowspan="1" colspan="1">5IXQ</td><td valign="top" rowspan="1" colspan="1">5IXT</td><td valign="top" rowspan="1" colspan="1">5IYN</td><td valign="top" rowspan="1" colspan="1">5IYX</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>Data collection</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Space group</td><td valign="top" rowspan="1" colspan="1"><italic>P</italic>3<sub>1</sub> 21</td><td valign="top" rowspan="1" colspan="1"><italic>P</italic>3<sub>1</sub> 21</td><td valign="top" rowspan="1" colspan="1"><italic>P</italic>3<sub>1</sub> 21</td><td valign="top" rowspan="1" colspan="1"><italic>P</italic>2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td></tr><tr><td valign="top" rowspan="1" colspan="1">Cell dimensions</td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1"> +<italic>a</italic>, <italic>b, c</italic> (Å)</td><td valign="top" rowspan="1" colspan="1">148.55, 148.55, 58.30</td><td valign="top" rowspan="1" colspan="1">148.92, 148.92, 58.02</td><td valign="top" rowspan="1" colspan="1">150.18, 150.18, 60.07</td><td valign="top" rowspan="1" colspan="1">74.51, 100.46, 142.76</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>α</italic>, β, γ (°)</td><td valign="top" rowspan="1" colspan="1">90, 90, 120</td><td valign="top" rowspan="1" colspan="1">90, 90, 120</td><td valign="top" rowspan="1" colspan="1">90, 90, 120</td><td valign="top" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td valign="top" rowspan="1" colspan="1">48.54–1.86 <break/>(1.97–1.86)</td><td valign="top" rowspan="1" colspan="1">48.75–1.94 (2,06–1.94)</td><td valign="top" rowspan="1" colspan="1">49.16–2.56 (2.72–2.56)</td><td valign="top" rowspan="1" colspan="1">47.59–2.43 (2.57–2.43)</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>R<sub>meas</sub><sup>#</sup></italic></td><td valign="top" rowspan="1" colspan="1">0.057 (1.35)</td><td valign="top" rowspan="1" colspan="1">0.037 (0.97)</td><td valign="top" rowspan="1" colspan="1">0.056 (1.27)</td><td valign="top" rowspan="1" colspan="1">0.113 (1.37)</td></tr><tr><td valign="top" rowspan="1" colspan="1">CC(1/2)<italic><sup>#</sup></italic></td><td valign="top" rowspan="1" colspan="1">100/77.9</td><td valign="top" rowspan="1" colspan="1">100/80.3</td><td valign="top" rowspan="1" colspan="1">100/89.5</td><td valign="top" rowspan="1" colspan="1">100/77.6</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>I</italic>/σ<italic>I<sup>#</sup></italic></td><td valign="top" rowspan="1" colspan="1">16.7 (2.0)</td><td valign="top" rowspan="1" colspan="1">20.9 (2.4)</td><td valign="top" rowspan="1" colspan="1">26.0 (1.9)</td><td valign="top" rowspan="1" colspan="1">16.12 (2.0)</td></tr><tr><td valign="top" rowspan="1" colspan="1">Completeness<italic><sup>#</sup></italic> (%)</td><td valign="top" rowspan="1" colspan="1">99.8 (98.6)</td><td valign="top" rowspan="1" colspan="1">99.4 (97.9)</td><td valign="top" rowspan="1" colspan="1">99.5 (98.8)</td><td valign="top" rowspan="1" colspan="1">99.4 (96.4s</td></tr><tr><td valign="top" rowspan="1" colspan="1">Redundancy<italic><sup>#</sup></italic></td><td valign="top" rowspan="1" colspan="1">20.3 (19.1)</td><td valign="top" rowspan="1" colspan="1">11.2 (11.1)</td><td valign="top" rowspan="1" colspan="1">14.7 (14.7)</td><td valign="top" rowspan="1" colspan="1">9.7 (9.3)</td></tr><tr><td valign="top" rowspan="1" colspan="1">Wilson B-factor (Å<sup>2</sup>)<italic><sup>#</sup></italic></td><td valign="top" rowspan="1" colspan="1">80.0</td><td valign="top" rowspan="1" colspan="1">81.7</td><td valign="top" rowspan="1" colspan="1">89.5</td><td valign="top" rowspan="1" colspan="1">59.3</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>Refinement</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td valign="top" rowspan="1" colspan="1">48.54–1.86</td><td valign="top" rowspan="1" colspan="1">48.75–1.94</td><td valign="top" rowspan="1" colspan="1">49.16–2.56</td><td valign="top" rowspan="1" colspan="1">47.59–2.43</td></tr><tr><td valign="top" rowspan="1" colspan="1">No. reflections</td><td valign="top" rowspan="1" colspan="1">58,551</td><td valign="top" rowspan="1" colspan="1">51,557</td><td valign="top" rowspan="1" colspan="1">23,835</td><td valign="top" rowspan="1" colspan="1">38,969</td></tr><tr><td valign="top" rowspan="1" colspan="1"><italic>R</italic><sub>work/</sub><italic>R</italic><sub>free</sub><sup>$</sup></td><td valign="top" rowspan="1" colspan="1">0.190/0.209</td><td valign="top" rowspan="1" colspan="1">0.183/0.208</td><td valign="top" rowspan="1" colspan="1">0.199/0.236</td><td valign="top" rowspan="1" colspan="1">0.199/0.235</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>No. atoms</bold></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Protein/Glycan</td><td valign="top" rowspan="1" colspan="1">4,541/176</td><td valign="top" rowspan="1" colspan="1">4,545/176</td><td valign="top" rowspan="1" colspan="1">4,499/176</td><td valign="top" rowspan="1" colspan="1">5,965/168</td></tr><tr><td valign="top" rowspan="1" colspan="1">Peptide</td><td valign="top" rowspan="1" colspan="1">93</td><td valign="top" rowspan="1" colspan="1">93</td><td valign="top" rowspan="1" colspan="1">90</td><td valign="top" rowspan="1" colspan="1">112</td></tr><tr><td valign="top" rowspan="1" colspan="1">Water</td><td valign="top" rowspan="1" colspan="1">39</td><td valign="top" rowspan="1" colspan="1">40</td><td valign="top" rowspan="1" colspan="1">9</td><td valign="top" rowspan="1" colspan="1">136</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>Res. B-factors (Å<sup>2</sup>)</bold><sup>$</sup></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Protein/Glycan</td><td valign="top" rowspan="1" colspan="1">79.48/109.02</td><td valign="top" rowspan="1" colspan="1">79.63/113.24</td><td valign="top" rowspan="1" colspan="1">102.12/132.49</td><td valign="top" rowspan="1" colspan="1">60.05/73.48</td></tr><tr><td valign="top" rowspan="1" colspan="1">Peptide</td><td valign="top" rowspan="1" colspan="1">87.19</td><td valign="top" rowspan="1" colspan="1">89.50</td><td valign="top" rowspan="1" colspan="1">125.74</td><td valign="top" rowspan="1" colspan="1">51.06</td></tr><tr><td valign="top" rowspan="1" colspan="1">Water</td><td valign="top" rowspan="1" colspan="1">75.32</td><td valign="top" rowspan="1" colspan="1">71.92</td><td valign="top" rowspan="1" colspan="1">74.65</td><td valign="top" rowspan="1" colspan="1">51.47</td></tr><tr><td valign="top" rowspan="1" colspan="1"><bold>R.m.s deviations</bold><sup>$</sup></td><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/><td valign="top" rowspan="1" colspan="1"/></tr><tr><td valign="top" rowspan="1" colspan="1">Bond lengths (Å)</td><td valign="top" rowspan="1" colspan="1">0.0087</td><td valign="top" rowspan="1" colspan="1">0.0091</td><td valign="top" rowspan="1" colspan="1">0.0081</td><td valign="top" rowspan="1" colspan="1">0.0074</td></tr><tr><td valign="top" rowspan="1" colspan="1">Bond angles (°)</td><td valign="top" rowspan="1" colspan="1">1.48</td><td valign="top" rowspan="1" colspan="1">1.47</td><td valign="top" rowspan="1" colspan="1">1.36</td><td valign="top" rowspan="1" colspan="1">1.34</td></tr></tbody></table> + + 13396 + HAESA – IDA HAESA – PKGV-IDA HAESA – IDL1 HAESA – IDA – SERK1 PDB-ID 5IXQ 5IXT 5IYN 5IYX Data collection Space group P31 21 P31 21 P31 21 P212121 Cell dimensions a, b, c (Å) 148.55, 148.55, 58.30 148.92, 148.92, 58.02 150.18, 150.18, 60.07 74.51, 100.46, 142.76 α, β, γ (°) 90, 90, 120 90, 90, 120 90, 90, 120 90, 90, 90 Resolution (Å) 48.54–1.86 (1.97–1.86) 48.75–1.94 (2,06–1.94) 49.16–2.56 (2.72–2.56) 47.59–2.43 (2.57–2.43) Rmeas# 0.057 (1.35) 0.037 (0.97) 0.056 (1.27) 0.113 (1.37) CC(1/2)# 100/77.9 100/80.3 100/89.5 100/77.6 I/σI# 16.7 (2.0) 20.9 (2.4) 26.0 (1.9) 16.12 (2.0) Completeness# (%) 99.8 (98.6) 99.4 (97.9) 99.5 (98.8) 99.4 (96.4s Redundancy# 20.3 (19.1) 11.2 (11.1) 14.7 (14.7) 9.7 (9.3) Wilson B-factor (Å2)# 80.0 81.7 89.5 59.3 Refinement Resolution (Å) 48.54–1.86 48.75–1.94 49.16–2.56 47.59–2.43 No. reflections 58,551 51,557 23,835 38,969 Rwork/Rfree$ 0.190/0.209 0.183/0.208 0.199/0.236 0.199/0.235 No. atoms Protein/Glycan 4,541/176 4,545/176 4,499/176 5,965/168 Peptide 93 93 90 112 Water 39 40 9 136 Res. B-factors (Å2)$ Protein/Glycan 79.48/109.02 79.63/113.24 102.12/132.49 60.05/73.48 Peptide 87.19 89.50 125.74 51.06 Water 75.32 71.92 74.65 51.47 R.m.s deviations$ Bond lengths (Å) 0.0087 0.0091 0.0081 0.0074 Bond angles (°) 1.48 1.47 1.36 1.34 + + + tbl2.xml + tbl2 + TABLE + table_footnote + 14804 + Highest resolution shell is shown in parenthesis. + + + tbl2.xml + tbl2 + TABLE + table_footnote + 14854 + #As defined in XDS. + + + tbl2.xml + tbl2 + TABLE + table_footnote + 14874 + $As defined in Refmac5. + + + RESULTS + paragraph + 14898 + We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20 μM (Figure 1B). We next determined crystal structures of the apo HAESA ectodomain and of a HAESA-IDA complex, at 1.74 and 1.86 Å resolution, respectively (Figure 1C; Figure 1—figure supplement 1B–D; Tables 1,2). IDA binds in a completely extended conformation along the inner surface of the HAESA ectodomain, covering LRRs 2–14 (Figure 1C,D, Figure 1—figure supplement 2). The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones. The C-terminal Arg-His-Asn motif in IDA maps to a cavity formed by HAESA LRRs 11–14 (Figure 1D,F). The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein. Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features. Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and in other HAESA-type receptors in different plant species (Figure 1—figure supplement 3). A N-terminal Pro-rich motif in IDA makes contacts with LRRs 2–6 of the receptor (Figure 1D, Figure 1—figure supplement 2A–C). Other hydrophobic and polar interactions are mediated by Ser62IDA, Ser65IDA and by backbone atoms along the IDA peptide (Figure 1D, Figure 1—figure supplement 2A–C). + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:58:05Z + + purified + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:09Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:34Z + + ectodomain + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:58:08Z + + 20–620 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:58:11Z + + baculovirus-infected insect cells + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:58:14Z + + glycoprotein + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:58:36Z + + synthetic + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:11:53Z + + IDA peptides + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:58:41Z + + isothermal titration calorimetry + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:58:49Z + + ITC + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:59:09Z + + Hyp-modified + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:45Z + + dodecamer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:59:16Z + + highly conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:12:59Z + + PIP motif + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:44Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:09Z + + HAESA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:59:53Z + + dissociation constant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:31:37Z + + Kd + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:25Z + + crystal structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:00:06Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:09Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:34Z + + ectodomain + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-17T10:00:16Z + + HAESA-IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:44Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:00:36Z + + completely extended conformation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:09Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:34Z + + ectodomain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:04:34Z + + LRRs 2–14 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T12:39:35Z + + Hyp64 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:01:33Z + + IDA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:01:41Z + + pocket + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:09Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:01:50Z + + LRRs 8–10 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:28Z + + hydrogen bonds + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:02:03Z + + strictly conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:02:08Z + + Glu266 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:02:15Z + + HAESA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T09:15:02Z + + water + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:02:29Z + + Phe289 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:02:37Z + + HAESA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:02:42Z + + Ser311 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:02:56Z + + HAESA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:03:01Z + + Hyp pocket + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:44Z + + IDA + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:03:05Z + + arabinosylation + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T12:39:50Z + + Hyp64 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:03:28Z + + IDA + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:03:54Z + + Hyp + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:01Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:04:09Z + + CLE peptide hormones + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:01:51Z + + Arg-His-Asn motif + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:44Z + + IDA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:06:21Z + + cavity + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:09Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:06:28Z + + LRRs 11–14 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:06:35Z + + Asn69 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:06:46Z + + IDA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:06:54Z + + Arg407 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:06:56Z + + HAESA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:06:59Z + + Arg409 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:07:02Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:09Z + + HAESA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:07:10Z + + C-terminally extended + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:07:16Z + + IDA-SFVN + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:07:20Z + + conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:07:23Z + + Asn69 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:07:32Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:07:37Z + + mature + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:09:00Z + + IDA peptide + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:08:00Z + + planta + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:12Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:44Z + + IDA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:08:09Z + + Mutation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:08:11Z + + Arg417 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:08:14Z + + HSL2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:08:17Z + + Arg409 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:08:19Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:07:45Z + + HSL2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:08:23Z + + peptide binding pockets + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:08:25Z + + HAESA receptors + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:08:29Z + + IDA binding surface + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:09Z + + HAESA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:08:32Z + + conserved + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:07:45Z + + HSL2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:08:36Z + + HAESA-type receptors + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:01Z + + plant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:02:59Z + + Pro-rich motif + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:44Z + + IDA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:08:48Z + + LRRs 2–6 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:28Z + + hydrophobic and polar interactions + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:09:02Z + + Ser62 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:09:13Z + + IDA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:09:16Z + + Ser65 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:09:25Z + + IDA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:09:00Z + + IDA peptide + + + + RESULTS + title_2 + 17511 + HAESA specifically senses IDA-family dodecamer peptides + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:09Z + + HAESA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:12:54Z + + IDA-family + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:06:17Z + + dodecamer + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:13:06Z + + peptides + + + + RESULTS + paragraph + 17567 + We next investigated whether HAESA binds N-terminally extended versions of IDA. We obtained a structure of HAESA in complex with a PKGV-IDA peptide at 1.94 Å resolution (Table 2). In this structure, no additional electron density accounts for the PKGV motif at the IDA N-terminus (Figure 2A,B). Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D). + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:09Z + + HAESA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:12:37Z + + N-terminally extended + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:44Z + + IDA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:13:01Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:09Z + + HAESA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:35:29Z + + in complex with + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:30:14Z + + PKGV-IDA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T10:13:12Z + + peptide + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:13:16Z + + structure + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:13:19Z + + electron density + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:13:30Z + + PKGV motif + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:44Z + + IDA + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:30:14Z + + PKGV-IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:44Z + + IDA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:13:35Z + + binding affinities + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:13:38Z + + ITC assays + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:45Z + + dodecamer + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T10:14:02Z + + peptide + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:14:06Z + + 58-69 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:14:20Z + + IDA + + + + RESULTS + paragraph + 18046 + We next tested if HAESA binds other IDA peptide family members. IDL1, which can rescue IDA loss-of-function mutants when introduced in abscission zone cells, can also be sensed by HAESA, albeit with lower affinity (Figure 2D). A 2.56 Å co-crystal structure with IDL1 reveals that different IDA family members use a common binding mode to interact with HAESA-type receptors (Figure 2A–C,E, Table 2). We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA → Ala double-mutant discussed below (Figure 1A, 2D). Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7). + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:14:03Z + + IDA peptide family members + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:29:55Z + + IDL1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:44Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:18:36Z + + affinity + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:18:39Z + + co-crystal structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:29:55Z + + IDL1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:19:03Z + + IDA family members + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:19:14Z + + HAESA-type receptors + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:19:21Z + + synthetic + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T10:19:25Z + + peptide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:19:28Z + + missing the C-terminal + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:19:34Z + + Asn69 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:19:46Z + + IDA + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:19:53Z + + ΔN69 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:28Z + + polar interactions + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:45Z + + IDA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:20:03Z + + Arg407 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:20:06Z + + HAESA + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:20:09Z + + Arg409 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:20:12Z + + HAESA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:20:15Z + + Replacing + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T12:37:23Z + + Hyp64 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:20:32Z + + IDA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:23:05Z + + IDLs + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:20:44Z + + proline + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:20:55Z + + Lys66IDA/Arg67IDA → Ala + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:20:58Z + + double-mutant + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:23:05Z + + IDLs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:21:07Z + + functionally unrelated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:45Z + + dodecamer + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T10:21:28Z + + peptides + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:21:33Z + + Hyp modifications + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:21:54Z + + CLV3 + + + + RESULTS + title_2 + 19026 + The co-receptor kinase SERK1 allows for high-affinity IDA sensing + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:22:26Z + + co-receptor kinase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + + RESULTS + paragraph + 19092 + Our binding assays reveal that IDA family peptides are sensed by the isolated HAESA ectodomain with relatively weak binding affinities (Figures 1B, 2A–D). It has been recently reported that SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) are positive regulators of floral abscission and can interact with HAESA and HSL2 in an IDA-dependent manner. As all five SERK family members appear to be expressed in the Arabidopsis abscission zone, we quantified their relative contribution to floral abscission in Arabidopsis using a petal break-strength assay. Our experiments suggest that among the SERK family members, SERK1 is a positive regulator of floral abscission. We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A). The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants. Possibly because SERKs have additional roles in plant development such as in pollen formation and brassinosteroid signaling, we found that higher-order SERK mutants exhibit pleiotropic phenotypes in the flower, rendering their analysis and comparison by quantitative petal break-strength assays difficult. We thus focused on analyzing the contribution of SERK1 to HAESA ligand sensing and receptor activation. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:27:24Z + + binding assays + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T10:27:28Z + + IDA family peptides + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:27:31Z + + isolated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:34Z + + ectodomain + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:27:34Z + + binding affinities + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:27:38Z + + SOMATIC EMBRYOGENESIS RECEPTOR KINASES + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:27:47Z + + SERKs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:07:45Z + + HSL2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:28:50Z + + SERK family members + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:28:16Z + + Arabidopsis + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:28:38Z + + Arabidopsis + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:28:43Z + + petal break-strength assay + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:28:50Z + + SERK family members + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T09:44:19Z + + serk1-1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:29:05Z + + mutants + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:51Z + + wild-type + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T09:43:05Z + + haesa + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T09:43:23Z + + hsl2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:29:10Z + + mutants + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T09:44:19Z + + serk1-1 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T10:29:18Z + + serk2-2 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T10:29:27Z + + serk3-1 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T10:29:36Z + + serk4-1 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T10:29:45Z + + serk5-1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:29:49Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:51Z + + wild-type + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:27:47Z + + SERKs + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:30:01Z + + quantitative petal break-strength assays + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + + RESULTS + paragraph + 20558 + In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). We next quantified the contribution of SERK1 to IDA recognition by HAESA. We found that HAESA senses IDA with a ~60 fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C). We next titrated SERK1 into a solution containing only the HAESA ectodomain. In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range (Figure 3C). This suggests that IDA itself promotes receptor – co-receptor association, as previously described for the steroid hormone brassinolide and for other LRR-RK complexes. Importantly, hydroxyprolination of IDA is critical for HAESA-IDA-SERK1 complex formation (Figure 3C,D). Our calorimetry experiments now reveal that SERKs may render HAESA, and potentially other receptor kinases, competent for high-affinity sensing of their cognate ligands. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:10:10Z + + LRR ectodomain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:34:12Z + + 24–213 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:34:19Z + + stable + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:34:23Z + + IDA-dependent + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:34:39Z + + heterodimeric + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:34:43Z + + complexes with + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:34:47Z + + size exclusion chromatography + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:29Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:19:27Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:45Z + + IDA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:34:51Z + + binding affinity + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:35:13Z + + presence of + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:29:24Z + + peptide hormone + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:35:24Z + + titrated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:34Z + + ectodomain + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:35:15Z + + presence of + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:45Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:35:53Z + + dissociation constant + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:45Z + + IDA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T10:35:59Z + + steroid hormone + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T10:36:07Z + + brassinolide + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T18:20:42Z + + LRR-RK + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:36:41Z + + hydroxyprolination + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:45Z + + IDA + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T18:20:42Z + + HAESA-IDA-SERK1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:37:07Z + + calorimetry + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:27:47Z + + SERKs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:37:12Z + + receptor kinases + + + + RESULTS + paragraph + 21738 + Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases, may interact in the cytoplasm to activate each other. Consistently, the HAESA kinase domain can transphosphorylate SERK1 and vice versa in in vitro transphosphorylation assays (Figure 3E). Together, our genetic and biochemical experiments implicate SERK1 as a HAESA co-receptor in the Arabidopsis abscission zone. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:19:41Z + + IDA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:39:05Z + + kinase domains + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:12Z + + active + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:39:17Z + + protein kinases + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:39:23Z + + kinase domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:39:26Z + + transphosphorylation assays + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:39:45Z + + genetic and biochemical experiments + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:39:59Z + + HAESA co-receptor + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:40:29Z + + Arabidopsis + + + + RESULTS + title_2 + 22181 + SERK1 senses a conserved motif in IDA family peptides + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:41:04Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:41:07Z + + motif + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T10:41:10Z + + IDA family peptides + + + + elife-15075-fig4.jpg + fig4 + FIG + fig_title_caption + 22235 + Crystal structure of a HAESA – IDA – SERK1 signaling complex. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:41:33Z + + Crystal structure + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-17T09:45:14Z + + HAESA – IDA – SERK1 + + + + elife-15075-fig4.jpg + fig4 + FIG + fig_caption + 22301 + (A) Overview of the ternary complex with HAESA in blue (surface representation), IDA in yellow (bonds representation) and SERK1 in orange (surface view). (B) The HAESA ectodomain undergoes a conformational change upon SERK1 co-receptor binding. Shown are Cα traces of a structural superposition of the unbound (yellow) and SERK1-bound (blue) HAESA ectodomains (r.m.s.d. is 1.5 Å between 572 corresponding Cα atoms). SERK1 (in orange) and IDA (in red) are shown alongside. The conformational change in the C-terminal LRRs and capping domain is indicated by an arrow. (C) SERK1 forms an integral part of the receptor's peptide binding pocket. The N-terminal capping domain of SERK1 (in orange) directly contacts the C-terminal part of IDA (in yellow, in bonds representation) and the receptor HAESA (in blue). Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface. Ribbon diagrams of HAESA (in blue) and SERK1 (in orange) are shown with selected interface residues (in bonds representation). Polar interactions are highlighted as dotted lines (in magenta). + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:45Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:10Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:34Z + + ectodomain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:20:00Z + + SERK1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:32:08Z + + structural superposition + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:44:20Z + + unbound + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:44:23Z + + SERK1-bound + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:11Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:28:03Z + + ectodomains + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:44:48Z + + r.m.s.d. + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:45Z + + IDA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:45:02Z + + LRRs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:51:15Z + + capping domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:14:20Z + + peptide binding pocket + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:51:15Z + + capping domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:45Z + + IDA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:45:34Z + + receptor + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:11Z + + HAESA + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:28Z + + Polar contacts + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:45Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:11Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:45:41Z + + LRR domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:46:04Z + + zipper-like + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:46:09Z + + SERK1-HAESA interface + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:11Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:46:15Z + + interface residues + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:28Z + + Polar interactions + + + + elife-15075-fig4.jpg + fig4 + FIG + fig_caption + 23453 + DOI: +http://dx.doi.org/10.7554/eLife.15075.011 + + + RESULTS + paragraph + 23500 + To understand in molecular terms how SERK1 contributes to high-affinity IDA recognition, we solved a 2.43 Å crystal structure of the ternary HAESA – IDA – SERK1 complex (Figure 4A, Table 2). HAESA LRRs 16–21 and its C-terminal capping domain undergo a conformational change upon SERK1 binding (Figure 4B). The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C). SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C). SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1—figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:20:17Z + + IDA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:50:47Z + + crystal structure + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-17T09:45:14Z + + HAESA – IDA – SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:11Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:51:05Z + + LRRs 16–21 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:51:15Z + + capping domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:20:28Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:34Z + + ectodomain + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:51:25Z + + IDA peptide binding site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:51:29Z + + loop region + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:51:32Z + + 51-59 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:51:35Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:51:43Z + + cap + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:51:46Z + + loop + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:28Z + + hydrophobic and polar contacts + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:51:55Z + + Lys66 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T10:52:10Z + + IDA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:01:51Z + + Arg-His-Asn motif + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:45Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:52:15Z + + LRRs 1–5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:51:15Z + + capping domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:52:29Z + + zipper-like + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:52:33Z + + interface + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:11Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:52:36Z + + LRRs 15–21 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:11Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:52:39Z + + cap + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:11Z + + HAESA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:52:44Z + + interaction surfaces + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:52:47Z + + N-cap + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:52:51Z + + LRR domain + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:52:56Z + + IDA peptide binding cleft + + + + elife-15075-fig5.jpg + fig5 + FIG + fig_title_caption + 24478 + The IDA C-terminal motif is required for HAESA-SERK1 complex formation and for IDA bioactivity. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:45Z + + IDA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:56:03Z + + C-terminal motif + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T18:20:42Z + + HAESA-SERK1 + + + + elife-15075-fig5.jpg + fig5 + FIG + fig_caption + 24574 + (A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes. Deletion of the C-terminal Asn69IDA completely inhibits complex formation. Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. Left panel: IDA K66A/R67A; center: IDA ΔN69, right panel: SDS-PAGE of peak fractions. Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. Table summaries for calorimetric binding constants and stoichoimetries for different IDA peptides binding to the HAESA – SERK1 ectodomain mixture ( ± fitting errors; n.d. no detectable binding) are shown alongside. (C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. Petal break is measured from positions 1 to 8 along the primary inflorescence where positions 1 is defined as the flower at anthesis (n=15, bars=SD). The three treatment groups in this unbalanced one-way layout were compared by Tukey’s all-pairs comparison procedure using the package multcomp in R (version 3.2.3). 35S::IDA plants showed significantly increased abscission compared to Col-0 controls in inflorescence positions 2 and 3 (a). Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. 15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed). + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:45:52Z + + Size exclusion chromatography + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:45Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T14:45:44Z + + mutant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T14:45:47Z + + peptides + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T14:45:50Z + + C-terminal motif + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T14:45:56Z + + biochemically stable + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-17T14:45:41Z + + HAESA-IDA-SERK1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:45:59Z + + Deletion + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T14:46:02Z + + Asn69 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T14:44:34Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T14:45:11Z + + inhibits + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:47:16Z + + Thyroglobulin + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:47:30Z + + Ferritin + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:47:46Z + + Aldolase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:48:00Z + + Conalbumin + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:48:15Z + + Ovalbumin + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:48:30Z + + Carbonic anhydrase + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:29:17Z + + Purified + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:11Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:46:11Z + + IDA K66A/R67A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:46:14Z + + IDA ΔN69 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:46:23Z + + SDS-PAGE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:11Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T14:46:31Z + + Isothermal titration thermographs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:51Z + + wild-type + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T14:46:34Z + + mutant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:11:55Z + + IDA peptides + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:47:02Z + + titrated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:11Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T14:47:09Z + + calorimetric binding constants + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:11:55Z + + IDA peptides + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:11Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:34Z + + ectodomain + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:47:42Z + + petal break-strength assay + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:51Z + + wild-type + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T14:52:18Z + + 35S + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:51Z + + wild-type + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T14:52:19Z + + 35S + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:51:06Z + + IDA K66A/R67A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T14:51:10Z + + mutant + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T14:52:20Z + + 35S + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T14:52:20Z + + 35S + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:54:27Z + + IDA K66A/R67A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T14:54:31Z + + mutant + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T14:52:20Z + + 35S + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T14:52:20Z + + 35S + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T14:52:20Z + + 35S + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:55:03Z + + IDA K66A/R67A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:51Z + + wild-type + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T14:55:07Z + + mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:55:28Z + + 35S promoter over-expression lines + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T14:52:20Z + + 35S + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:51Z + + wild-type + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T14:52:20Z + + 35S + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:56:01Z + + IDA K66A/R67A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T14:56:05Z + + double-mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:56:07Z + + T3 transgenic lines + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T14:52:20Z + + 35S + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T14:52:20Z + + 35S + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T14:56:22Z + + IDA K66A/R67A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T14:56:25Z + + double-mutant + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + + elife-15075-fig5.jpg + fig5 + FIG + fig_caption + 27046 + DOI: +http://dx.doi.org/10.7554/eLife.15075.012 + + + RESULTS + paragraph + 27093 + The four C-terminal residues in IDA (Lys66IDA-Asn69IDA) are conserved among IDA family members and are in direct contact with SERK1 (Figures 1A, 4C). We thus assessed their contribution to HAESA – SERK1 complex formation. Deletion of the buried Asn69IDA completely inhibits receptor – co-receptor complex formation and HSL2 activation (Figure 5A,B). A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). We found that over-expression of wild-type IDA leads to early floral abscission and an enlargement of the abscission zone (Figure 5C–E). In contrast, over-expression of the IDA Lys66IDA/Arg67IDA → Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C–E). Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA → Ala may cause a weak dominant negative effect (Figure 5C–E). In agreement with our structures and biochemical assays, this experiment suggests a role of the conserved IDA C-terminus in the control of floral abscission. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:01:23Z + + Lys66IDA-Asn69IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:01:26Z + + conserved + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:19:03Z + + IDA family members + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:30Z + + SERK1 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-21T18:20:42Z + + HAESA – SERK1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:02:08Z + + Deletion + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:02:11Z + + Asn69 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:02:22Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:02:41Z + + completely inhibits + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:03:03Z + + synthetic + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:03:05Z + + Lys66IDA/Arg67IDA → Ala + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:03:08Z + + mutant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T15:03:10Z + + peptide + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:03:20Z + + IDA K66A/R66A + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:03:24Z + + binding affinity + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:03:27Z + + titrated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:03:29Z + + over-expressed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:19:56Z + + full-length + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:51Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:03:32Z + + Lys66IDA/Arg67IDA → Ala + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:03:35Z + + double-mutant + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:03:48Z + + Arabidopsis + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:03:57Z + + over-expression + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:51Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:04:00Z + + over-expression + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:04:04Z + + IDA Lys66IDA/Arg67IDA → Ala + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:04:06Z + + double mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:51Z + + wild-type + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:49Z + + plants + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:04:14Z + + mutant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:09:00Z + + IDA peptide + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:08:00Z + + planta + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T14:52:20Z + + 35S + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:44:51Z + + wild-type + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:04:49Z + + mutant + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:50Z + + plants + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:04:52Z + + mutation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:04:54Z + + Lys66IDA/Arg67IDA → Ala + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:04:56Z + + structures + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:05:00Z + + biochemical assays + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:05:02Z + + conserved + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + + DISCUSS + title_1 + 28494 + Discussion + + + DISCUSS + paragraph + 28505 + In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation. For a rapidly growing number of plant signaling pathways, SERK proteins act as these essential co-receptors (; ). SERK1 has been previously reported as a positive regulator in plant embryogenesis, male sporogenesis, brassinosteroid signaling and in phytosulfokine perception. Recent findings by and our mechanistic studies now also support a positive role for SERK1 in floral abscission. As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A). It has been previously suggested that SERK1 can inhibit cell separation. However our results show that SERK1 also can activate this process upon IDA sensing, indicating that SERKs may fulfill several different functions in the course of the abscission process. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:08:22Z + + animal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:08:27Z + + LRR receptors + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:01Z + + plant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:19:47Z + + LRR-RKs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:08:34Z + + spiral-shaped + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:28:09Z + + ectodomains + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:08:38Z + + shape-complementary + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:08:42Z + + co-receptor proteins + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:01Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:08:47Z + + SERK proteins + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:08:50Z + + co-receptors + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:02Z + + plant + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T09:44:19Z + + serk1-1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:28:40Z + + mutant + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:33:50Z + + plants + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T09:43:05Z + + haesa + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-17T09:43:23Z + + hsl2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:28:44Z + + mutants + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:27:47Z + + SERKs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:21:16Z + + IDA + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T10:27:47Z + + SERKs + + + + DISCUSS + paragraph + 29513 + While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. It will be thus interesting to see if proteolytic processing of full-length IDA in vivo is regulated in a cell-type or tissue-specific manner. The central Hyp residue in IDA is found buried in the HAESA peptide binding surface and thus this post-translational modification may regulate IDA bioactivity. Our comparative structural and biochemical analysis further suggests that IDLs share a common receptor binding mode, but may preferably bind to HAESA, HSL1 or HSL2 in different plant tissues and organs. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:11:07Z + + mature + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:09:00Z + + IDA peptide + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:08:00Z + + planta + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-17T10:00:16Z + + HAESA-IDA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:11:39Z + + structures + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:11:42Z + + calorimetry assays + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:12Z + + active + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:23:05Z + + IDLs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:11:57Z + + hydroxyprolinated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:16:45Z + + dodecamers + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:19:56Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:12:16Z + + Hyp + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:12:28Z + + peptide binding surface + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:21:31Z + + IDA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:12:43Z + + comparative structural and biochemical analysis + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:23:05Z + + IDLs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T16:27:35Z + + HSL1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T09:07:46Z + + HSL2 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:02Z + + plant + + + + DISCUSS + paragraph + 30229 + In our quantitative biochemical assays, the presence of SERK1 dramatically increases the HAESA binding specificity and affinity for IDA. This observation is consistent with our complex structure in which receptor and co-receptor together form the IDA binding pocket. The fact that SERK1 specifically interacts with the very C-terminus of IDLs may allow for the rational design of peptide hormone antagonists, as previously demonstrated for the brassinosteroid pathway. Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C). This ligand-induced formation of a receptor – co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm. It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:29:22Z + + quantitative biochemical assays + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:35:15Z + + presence of + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:31:21Z + + structure + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:01:38Z + + IDA binding pocket + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:23:05Z + + IDLs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T15:31:45Z + + peptide hormone antagonists + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:31:48Z + + calorimetry assays + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T08:34:34Z + + ectodomain + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:32:04Z + + binds + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T10:35:15Z + + presence of + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:32:32Z + + kinase domains + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:28:18Z + + binding affinities + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:32:35Z + + synthetic + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T15:32:38Z + + peptides + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:32:41Z + + isolated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:32:49Z + + ectodomains + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:19:56Z + + full-length + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:32:45Z + + membrane-embedded + + + + elife-15075-fig6.jpg + fig6 + FIG + fig_title_caption + 31293 + SERK1 uses partially overlapping surface areas to activate different plant signaling receptors. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:02Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:33:24Z + + signaling receptors + + + + elife-15075-fig6.jpg + fig6 + FIG + fig_caption + 31389 + (A) Structural comparison of plant steroid and peptide hormone membrane signaling complexes. Left panel: Ribbon diagram of HAESA (in blue), SERK1 (in orange) and IDA (in bonds and surface represention). Right panel: Ribbon diagram of the plant steroid receptor BRI1 (in blue) bound to brassinolide (in gray, in bonds representation) and to SERK1, shown in the same orientation (PDB-ID. 4lsx). (B) View of the inner surface of the SERK1 LRR domain (PDB-ID 4lsc, surface representation, in gray). A ribbon diagram of SERK1 in the same orientation is shown alongside. Residues interacting with the HAESA or BRI1 LRR domains are shown in orange or magenta, respectively. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:29:27Z + + Structural comparison + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:02Z + + plant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T15:44:58Z + + steroid + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:51:22Z + + peptide hormone + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:45:38Z + + membrane signaling complexes + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:46Z + + IDA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:02Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:45:44Z + + steroid receptor + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:45:52Z + + BRI1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T09:30:41Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T15:46:03Z + + brassinolide + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:46:33Z + + LRR domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:45:52Z + + BRI1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:46:36Z + + LRR domains + + + + elife-15075-fig6.jpg + fig6 + FIG + fig_caption + 32056 + DOI: +http://dx.doi.org/10.7554/eLife.15075.013 + + + DISCUSS + paragraph + 32103 + Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). In addition, residues 53-55SERK1 from the SERK1 N-terminal cap mediate specific interactions with the IDA peptide (Figures 4C, 6B). These residues are not involved in the sensing of the steroid hormone brassinolide. In both cases however, the co-receptor completes the hormone binding pocket. This fact together with the largely overlapping SERK1 binding surfaces in HAESA and BRI1 allows us to speculate that SERK1 may promote high-affinity peptide hormone and brassinosteroid sensing by simply slowing down dissociation of the ligand from its cognate receptor. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:52:04Z + + Comparison + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-17T09:45:14Z + + HAESA – IDA – SERK1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:52:17Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:52:55Z + + co-receptor + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:52:32Z + + conserved + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:52:39Z + + ligand binding pockets + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:53:01Z + + LRRs 2 – 14 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:53:05Z + + LRRs 21 – 25 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:45:52Z + + BRI1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:45:52Z + + BRI1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T10:51:15Z + + capping domain + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:53:40Z + + Thr59 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:53:42Z + + SERK1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:53:46Z + + Phe61 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:53:49Z + + SERK1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:53:58Z + + LRR inner surface + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:54:01Z + + Asp75 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:54:03Z + + SERK1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:54:06Z + + Tyr101 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:54:09Z + + SERK1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:54:12Z + + SER121 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:54:15Z + + SERK1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:54:18Z + + Phe145 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:54:21Z + + SERK1 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T15:54:27Z + + 53-55 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:54:31Z + + SERK1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:54:42Z + + cap + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:09:00Z + + IDA peptide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T15:55:13Z + + steroid hormone + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T15:55:15Z + + brassinolide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:30:04Z + + hormone binding pocket + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:55:24Z + + SERK1 binding surfaces + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T15:45:52Z + + BRI1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:29:25Z + + peptide hormone + + + + elife-15075-fig7.jpg + fig7 + FIG + fig_title_caption + 33296 + Different plant peptide hormone families contain a C-terminal (Arg)-His-Asn motif, which in IDA represents the co-receptor recognition site. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:02Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:56:16Z + + peptide hormone families + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:56:20Z + + (Arg)-His-Asn motif + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:47Z + + IDA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T15:56:23Z + + co-receptor recognition site + + + + elife-15075-fig7.jpg + fig7 + FIG + fig_caption + 33437 + Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. The conserved (Arg)-His-Asn motif is highlighted in red, the central Hyp residue in IDLs and CLEs is marked in blue. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:57:40Z + + Structure-guided multiple sequence alignment + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:47Z + + IDA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T15:58:54Z + + IDA-like peptides + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:02Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:59:07Z + + peptide hormone families + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:59:18Z + + CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:59:28Z + + CLV3/CLE + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:59:39Z + + ROOT GROWTH FACTOR – GOLVEN + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:59:49Z + + RGF/GLV + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T15:59:59Z + + PRECURSOR GENE PROPEP1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:00:10Z + + PEP1 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-17T08:47:13Z + + Arabidopsis thaliana + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:00:15Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:00:17Z + + (Arg)-His-Asn motif + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:00:30Z + + Hyp + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T09:23:05Z + + IDLs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:00:44Z + + CLEs + + + + elife-15075-fig7.jpg + fig7 + FIG + fig_caption + 33838 + DOI: +http://dx.doi.org/10.7554/eLife.15075.014 + + + DISCUSS + paragraph + 33885 + Our experiments reveal that SERK1 recognizes a C-terminal Arg-His-Asn motif in IDA. Importantly, this motif can also be found in other peptide hormone families (Figure 7). Among these are the CLE peptides regulating stem cell maintenance in the shoot and the root. It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA. Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:31Z + + SERK1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T09:01:51Z + + Arg-His-Asn motif + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:32:47Z + + IDA + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:03:02Z + + this motif + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:03:13Z + + peptide hormone families + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-17T16:03:28Z + + CLE peptides + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:03:38Z + + CLEs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:03:41Z + + mature form + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:04:05Z + + hydroxyprolinated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:16:25Z + + dodecamers + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:04:41Z + + surface area + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:04:44Z + + BARELY ANY MERISTEM 1 receptor + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:04:48Z + + IDA binding cleft + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-17T08:33:12Z + + HAESA + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T08:34:02Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:05:02Z + + peptide hormones + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:05:07Z + + LRR-RK receptors + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:05:10Z + + extended conformation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-17T16:28:14Z + + LRR domain + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:28:49Z + + small + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-17T16:28:53Z + + shape-complementary + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-17T16:05:29Z + + co-receptors + + + + METHODS + title_1 + 34637 + Materials and methods + + + METHODS + title_2 + 34659 + Protein expression and purification + + + METHODS + paragraph + 34695 + Synthetic genes coding for the Arabidopsis thaliana HAESA (residues 20–620) and SERK1 ectodomains (residues 24–213, carrying Asn115→Asp and Asn163→Gln mutations), codon optimized for expression in Trichoplusia ni (Geneart, Germany), were cloned into a modified pBAC-6 transfer vector (Novagen, Billerica, MA), providing an azurocidin signal peptide and a C-terminal TEV (tobacco etch virus protease) cleavable Strep-9xHis tandem affinity tag. Recombinant baculoviruses were generated by co-transfecting transfer vectors with linearised baculovirus DNA (ProFold-ER1, AB vector, San Diego, CA) followed by viral amplification in Spodoptera frugiperda Sf9 cells. The HAESA and SERK1 ectodomains were individually expressed in Trichoplusia ni Tnao38 cells using a multiplicity of infection of 3, and harvested from the medium 2 days post infection by tangential flow filtration using 30 kDa MWCO and 10 kDa MWCO (molecular weight cut-off) filter membranes (GE Healthcare Life Sciences, Pittsburgh, PA), respectively. Proteins were purified separately by sequential Ni2+ (HisTrap HP, GE Healthcare) and Strep (Strep-Tactin Superflow high-capacity, IBA, Germany) affinity chromatography. Next, affinity tags were removed by incubating the purified proteins with recombinant Strep-tagged TEV protease in 1:100 molar ratio. The cleaved tag and the protease were separated from HAESA and SERK1 by a second Strep affinity step. The purified HAESA ectodomain was incubated with a synthetic IDA peptide (YVPIPPSA-Hyp-SKRHN, the N-terminal Tyr residue was added to allow for peptide quantification by UV absorbance) and the SERK1 ectodomain in 1:1:1.5 molar ratio. The HAESA-IDA-SERK1 complex was purified by size exclusion chromatography on a Superdex 200 HR10/30 column (GE Healthcare) equilibrated in 20 mM citric acid pH 5.0, 100 mM NaCl). Peak fractions containing the complex were concentrated to ~10 mg/mL and immediately used for crystallization. About 0.2 mg of purified HAESA and 0.1 mg of purified SERK1 protein were obtained from 1 L of insect cell culture, respectively. + + + METHODS + title_2 + 36785 + Crystallization and data collection + + + METHODS + paragraph + 36821 + Hexagonal crystals of the isolated HAESA ectodomain developed at room-temperature in hanging drops composed of 1.0 μL of protein solution (5.5 mg/mL) and 1.0 μL of crystallization buffer (21% PEG 3,350, 0.2 M MgCl2 · 6 H2O, 0.1 M citric acid pH 4.0), suspended above 1.0 mL of crystallization buffer. For structure solution crystals were derivatized and cryo-protected by serial transfer into crystallization buffer supplemented with 0.5 M NaI and 15% ethylene glycol and cryo-cooled in liquid nitrogen. Redundant single-wavelength anomalous diffraction (SAD) data to 2.39 Å resolution were collected at beam-line PXII at the Swiss Light Source (SLS), Villigen, CH with λ=1.7 Å. A native data set to 1.74 Å resolution was collected on a crystal from the same drop cryo-protected by serial transfer into crystallization buffer supplemented with 15% (v/v) ethylene glycol only (λ=1.0 Å; Table 1). + + + METHODS + paragraph + 37745 + HAESA complexes with IDA (PIPPSA-Hyp-SKRHN), PKGV-IDA (YPKGVPIPPSA-Hyp-SKRHN) and IDL1 (LVPPSG-Hyp-SMRHN) peptide hormones were obtained by soaking apo crystals in crystallization buffer containing the respective synthetic peptide at a final concentration of 15 mM. Soaked crystals diffracted to 1.86 Å (HAESA – IDA), 1.94 Å (HAESA-PKGV-IDA) and 2.56 Å resolution (HAESA – IDL1), respectively (Table 2). Orthorhombic crystals of the HAESA-IDA-SERK1 complex developed in 18% PEG 8000, MgCl2 · 6 H2O, 0.1 M citric acid and diffracted to 2.43 Å resolution (Table 2). Data processing and scaling was done in XDS (version: Nov 2014). + + + METHODS + title_2 + 38395 + Structure solution and refinement + + + METHODS + paragraph + 38429 + The SAD method was used to determine the structure of the isolated HAESA ectodomain. SHELXD located 32 iodine sites (CC All/Weak 37.7/14.9). 20 consistent sites were input into the program SHARP for phasing and identification of 8 additional sites at 2.39 Å resolution. Refined heavy atom sites and phases were provided to PHENIX.AUTOBUILD for density modification and automated model building. The structure was completed in alternating cycles of model building in COOT and restrained TLS refinement in REFMAC5 (version 5.8.0107) against an isomorphous high resolution native data set. Crystals contain one HAESA monomer per asymmetric unit with a solvent content of ~55%, the final model comprises residues 20 – 615. The refined structure has excellent stereochemistry, with 93.8% of all residues in the favored region of the Ramachandran plot, no outliers and a PHENIX.MOLPROBITY score of 1.34 (Table 1). + + + METHODS + paragraph + 39342 + The HAESA – IDA – SERK1 complex structure was determined by molecular replacement with the program PHASER, using the isolated HAESA and SERK1 (PDB-ID: 4LSC) LRR domain structures as search models. The solution comprises one HASEA-IDA-SERK1 complex in the asymmetric unit. The structure was completed in iterative cycles of manual model-building in COOT and restrained TLS refinement in REFMAC5. Amino acids whose side-chain position could not be modeled with confidence were truncated to alanine (0.6 – 1% of total residues), the stereochemistry of N-linked glycan structures was assessed with the CCP4 program PRIVATEER-VALIDATE. The refined model has 94.44% of all residues in the favored region of the Ramachandran plot, no outliers and a PHENIX.MOLPROBITY score of 1.17 (Table 2). Structural visualization was done with POVScript+ and POV-Ray (http://www.povray.org). + + + METHODS + title_2 + 40220 + Size-exclusion chromatography + + + METHODS + paragraph + 40250 + Gel filtration experiments were performed using a Superdex 200 HR 10/30 column (GE Healthcare) pre-equilibrated in 20 mM citric acid (pH 5) and 100 mM NaCl. 100 μL of the isolated HAESA ectodomain (5.5 mg/mL), of the purified SERK1 LRR domain (3 mg/mL) or of mixtures of HAESA and SERK1 (either in the presence or absence of synthetic wild-type IDA, wild-type IDL1 or mutant IDA peptides at a concentration of 25 μM; 10 mg/mL; samples contained HAESA and SERK1 in 1:1 molar ratio) were loaded sequentially onto the column and elution at 0.5 mL/min was monitored by ultraviolet absorbance at 280 nm. + + + METHODS + title_2 + 40861 + Isothermal titration calorimetry + + + METHODS + paragraph + 40894 + ITC experiments were performed using a Nano ITC (TA Instruments, New Castle, DE) with a 1.0 mL standard cell and a 250 μL titration syringe. Proteins were dialyzed extensively against ITC buffer (20 mM citric acid pH 5.0, 100 mM NaCl) and synthetic wild-type or point-mutant peptides (with wild-type IDA sequence YVPIPPSA-Hyp-SKRHN, PKGV-IDA YPKGVPIPPSA-Hyp-SKRHN, IDA-SFVN YPIPPSA-Hyp-SKRHNSFVN, IDL1 YLVPPSG-Hyp-SMRHN and CLV3 sequence YRTV-Hyp-SG-Hyp-DPLHH) were dissolved in ITC buffer prior to all titrations. Molar protein concentrations for SERK1 and HAESA were calculated using their molar extinction coefficient and a molecular weight of 27,551 and 74,896 Da, respectively (determined by MALDI-TOF mass spectrometry). Experiments were performed at 25°C. A typical experiment consisted of injecting 10 μL aliquots of peptide solution (250 μM) into 20 μM HAESA. The concentrations for the complex titrations were 150 μM of ligand (either wild-type or point-mutant IDA peptides) in the syringe and 10 μM of a 1:1 HAESA – SERK1 protein mixture in the cell at time intervals of 150 s to ensure that the titration peak returned to the baseline. Binding of SERK1 to HAESA was assessed by titrating SERK1 (100 μM) into a solution containing HAESA (10 μM) in the pre- or absence of 150 μM wild-type IDA peptide. ITC data were corrected for the heat of dilution by subtracting the mixing enthalpies for titrant solution injections into protein free ITC buffer. Data were analyzed using the NanoAnalyze program (version 2.3.6) as provided by the manufacturer. + + + METHODS + title_2 + 42490 + In vitro kinase trans-phosphorylation assay + + + METHODS + paragraph + 42534 + Coding sequences of SERK1 kinase domain (SERK1-KD) (residues 264–625) and HAESA-KD (residues 671–969) were cloned into a modified pET (Novagen) vector providing an TEV-cleavable N-terminal 8xHis-StrepII-Thioredoxin tag. Point mutations were introduced into the SERK1 (Asp447→Asn; mSERK1) and HAESA (Asp837→Asn; mHAESA) coding sequences by site directed mutagenesis, thereby rendering the kinases inactive. The plasmids were transformed into E.coli Rosetta 2 (DE3) (Novagen). Protein expression was induced by adding IPTG to final concentration of 0.5 mM to cell cultures grown to an OD600 = 0.6. Cells were then incubated at 16°C for 18 hr, pelleted by centrifugation at 5000 x g and 4°C for 15 min, and resuspended in buffer A (20 mM Tris-HCl pH 8, 500 mM NaCl, 4 mM MgCl2 and 2 mM β-Mercaptoethanol) supplemented with 15 mM Imidazole and 0.1% (v/v) Igepal. After cell lysis by sonication, cell debris was removed by centrifugation at 35,000 x g and 4°C for 30 min. The recombinant proteins were isolated by Co2+ metal affinity purification using a combination of batch and gravity flow approaches (HIS-Select Cobalt Affinity Gel, Sigma, St. Louis, MO). After washing the resin with the wash buffer (buffer A + 15 mM Imidazole) proteins were eluted in buffer A supplemented with 250 mM Imidazole. All elutions were then dialyzed against 20 mM Tris-HCl pH 8, 250 mM NaCl, 4 mM MgCl2 and 0.5 mM TCEP. For SERK1-KD and mSERK1-KD the 8xHis-StrepII-Thioredoxin tag was removed with 6xHis tagged TEV protease. TEV and the cleaved tag were removed by a second metal affinity purification step. Subsequently, all proteins were purified by gel filtration on a Superdex 200 10/300 GL column equilibrated in 20 mM Tris pH 8, 250 mM NaCl, 4 mM MgCl2 and 0.5 mM TCEP. Peak fractions were collected and concentrated using Amicon Ultra centrifugation devices (10,000 MWCO). For in vitro kinase assays, 1 μg of HAESA-KD, 0.25 μg of SERK1-KD and 2 μg of mSERK1 and mHAESA were used in a final reaction volume of 20 μl. The reaction buffer consisted of 20 mM Tris pH 8, 250 mM NaCl, 4 mM MgCl2 and 0.5 mM TCEP. The reactions were started by the addition of 4 μCi [γ-32P]-ATP (Perkin-Elmer, Waltham, MA), incubated at room temperature for 45 min and stopped by the addition of 6x SDS-loading dye immediately followed by incubating the samples at 95°C. Proteins of the whole reaction were subsequently separated via SDS-PAGE in 4–15% gradient gels (TGX, Biorad, Hercules, CA) and stained with Instant Blue (Expedeon, San Diego, CA). After pictures were taken of the stained gel, 32P-derived signals were visualized by exposing the gel to an X-ray film (Fuji, SuperRX, Valhalla, NY). + + + METHODS + title_2 + 45274 + Plant material and generation of transgenic lines + + + METHODS + paragraph + 45324 + 35S::IDA wild-type and 35S::IDA (R66 → Ala/K67 → Ala) over-expressing transgenic lines in Col-0 background were generated as follows: The constructs were introduced in the destination vector pB7m34GW2 and transferred to A. tumefaciens strain pGV2260. Plants were transformed using the floral dip method. Transformants were selected in medium supplemented with BASTA up to the T3 generation. For phenotyping, plants were grown at 21°C with 50% humidity and a 16h light: 8 hr dark cycle. + + + METHODS + title_2 + 45816 + RNA analyses + + + METHODS + paragraph + 45829 + Plants were grown on ½ Murashige and Skoog (MS) plates supplemented with 1% sucrose. After 7 d, ∼30 to 40 seedlings were collected and frozen in liquid nitrogen. Total RNA was extracted using a RNeasy plant mini kit (Qiagen, Valencia, CA), and 1 μg of the RNA solution obtained was reverse-transcribed using the SuperScritpVILO cDNA synthesis kit (Invitrogen, Grand Island, NY). RT-qPCR amplifications and measurements were performed using a 7900HT Fast Real Time PCR-System by Applied Biosystems (Carlsbad, CA). RT-qPCR amplifications were monitored using SYBR-Green fluorescent stain (Applied Biosystems). Relative quantification of gene expression data was performed using the 2−ΔΔCT (or comparative CT) method. Expression levels were normalized using the CT values obtained for the actin2 gene (forward: TGCCAATCTACGAGGGTTTC; reverse: TTCTCGATGGAAGAGCTGGT). For detection and amplification of IDA sequence we used specific primers (forward: TCGTACGATGATGGTTCTGC; reverse: GAATGGGAACGCCTTTAGGT). The presence of a single PCR product was further verified by dissociation analysis in all amplifications. All quantifications were made in quadruplicates on RNA samples obtained from three independent experiments. + + + METHODS + title_2 + 47053 + Petal break measurements + + + METHODS + paragraph + 47078 + serk1-1, serk2-2, serk3-1, serk4-1 and serk5-1 and Col-0 wild-type plants were grown in growth chambers at 22°C under long days (16 hr day/8 hr dark) at a light intensity of 100 µE·m-2·sec-1. Petal break-strength was quantified as the force in gram equivalents required for removal of a petal from a flower when the plants had a minimum of twenty flowers and siliques. Measurements were performed using a load transducer as described in. Break-strength was measured for 15 plants and a minimum of 15 measurements at each position. + + + ACK_FUND + title_1 + 47617 + Funding Information + + + ACK_FUND + paragraph + 47637 + This paper was supported by the following grants: + + + ACK_FUND + paragraph + 47687 + to Michael Hothorn. + + + ACK_FUND + paragraph + 47709 + to Michael Hothorn. + + + ACK_FUND + paragraph + 47731 + to Michael Hothorn. + + + ACK_FUND + paragraph + 47753 + to Melinka A Butenko. + + + ACK_FUND + paragraph + 47777 + to Benjamin Brandt. + + + ACK_FUND + paragraph + 47799 + to Julia Santiago. + + + ACK_FUND + title_1 + 47820 + Additional information + + + COMP_INT + title_1 + 47843 + Competing interests + + + COMP_INT + footnote + 47863 + The authors declare that no competing interests exist. + + + AUTH_CONT + title_1 + 47918 + Author contributions + + + AUTH_CONT + footnote + 47939 + JS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. + + + AUTH_CONT + footnote + 48058 + BB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. + + + AUTH_CONT + footnote + 48154 + MW, Acquisition of data, Analysis and interpretation of data. + + + AUTH_CONT + footnote + 48216 + UH, Acquisition of data, Analysis and interpretation of data. + + + AUTH_CONT + footnote + 48278 + LAH, Analysis and interpretation of data, Drafting or revising the article. + + + AUTH_CONT + footnote + 48354 + MAB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. + + + AUTH_CONT + footnote + 48474 + MH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. + + + REF + title + 48593 + References + + + 5253 + 5261 + surname:Aalen;given-names:RB + surname:Wildhagen;given-names:M + surname:Stø;given-names:IM + surname:Butenko;given-names:MA + 10.1093/jxb/ert338 + 24151306 + REF + Journal of Experimental Botany + ref + 64 + 2013 + 48604 + Ida: A peptide ligand regulating cell separation processes in Arabidopsis + + + 3337 + 3349 + surname:Albrecht;given-names:C + surname:Russinova;given-names:E + surname:Hecht;given-names:V + surname:Baaijens;given-names:E + surname:de Vries;given-names:S + 10.1105/tpc.105.036814 + 16284305 + REF + The Plant Cell + ref + 17 + 2005 + 48678 + The Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES1 and 2 control male sporogenesis + + + 611 + 619 + surname:Albrecht;given-names:C + surname:Russinova;given-names:E + surname:Kemmerling;given-names:B + surname:Kwaaitaal;given-names:M + surname:de Vries;given-names:SC + 10.1104/pp.108.123216 + 18667726 + REF + Plant Physiology + ref + 148 + 2008 + 48780 + Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE proteins serve brassinosteroid-dependent and -independent signaling pathways + + + 31 + 43 + surname:Bojar;given-names:D + surname:Martinez;given-names:J + surname:Santiago;given-names:J + surname:Rybin;given-names:V + surname:Bayliss;given-names:R + surname:Hothorn;given-names:M + 10.1111/tpj.12445 + 24461462 + REF + The Plant Journal + ref + 78 + 2014 + 48907 + Crystal structures of the phosphorylated BRI1 kinase domain and implications for brassinosteroid signal initiation + + + R225 + R226 + surname:Brandt;given-names:B + surname:Hothorn;given-names:M + 10.1016/j.cub.2015.12.014 + 27003880 + REF + Current Biology + ref + 26 + 2016 + 49022 + SERK co-receptor kinases + + + 2023 + 2030 + surname:Bricogne;given-names:G + surname:Vonrhein;given-names:C + surname:Flensburg;given-names:C + surname:Schiltz;given-names:M + surname:Paciorek;given-names:W + 10.1107/S0907444903017694 + 14573958 + REF + Acta Crystallographica. Section D, Biological Crystallography + ref + 59 + 2003 + 49047 + Generation, representation and flow of phase information in structure determination: Recent developments in and around SHARP 2.0 + + + 2296 + 2307 + surname:Butenko;given-names:MA + surname:Patterson;given-names:SE + surname:Grini;given-names:PE + surname:Stenvik;given-names:GE + surname:Amundsen;given-names:SS + surname:Mandal;given-names:A + surname:Aalen;given-names:RB + 10.1105/tpc.014365 + 12972671 + REF + The Plant Cell + ref + 15 + 2003 + 49176 + Inflorescence deficient in abscission controls floral organ abscission in Arabidopsis and identifies a novel family of putative ligands in plants + + + 255 + 263 + surname:Butenko;given-names:MA + surname:Vie;given-names:AK + surname:Brembu;given-names:T + surname:Aalen;given-names:RB + surname:Bones;given-names:AM + 10.1016/j.tplants.2009.02.002 + 19362511 + REF + Trends in Plant Science + ref + 14 + 2009 + 49322 + Plant peptides in signalling: Looking for new partners + + + 1838 + 1847 + surname:Butenko;given-names:MA + surname:Wildhagen;given-names:M + surname:Albert;given-names:M + surname:Jehle;given-names:A + surname:Kalbacher;given-names:H + surname:Aalen;given-names:RB + surname:Felix;given-names:G + 10.1105/tpc.113.120071 + 24808051 + REF + The Plant Cell + ref + 26 + 2014 + 49377 + Tools and strategies to match peptide-ligand receptor pairs + + + 15629 + 15634 + surname:Cho;given-names:SK + surname:Larue;given-names:CT + surname:Chevalier;given-names:D + surname:Wang;given-names:H + surname:Jinn;given-names:TL + surname:Zhang;given-names:S + surname:Walker;given-names:JC + 10.1073/pnas.0805539105 + 18809915 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 105 + 2008 + 49437 + Regulation of floral organ abscission in Arabidopsis thaliana + + + 2057 + 2067 + surname:Clark;given-names:SE + surname:Running;given-names:MP + surname:Meyerowitz;given-names:EM + REF + Development + ref + 121 + 1995 + 49499 + CLAVATA3 is a specific regulator of shoot and floral meristem development affecting the same processes as CLAVATA1 + + + 735 + 743 + surname:Clough;given-names:SJ + surname:Bent;given-names:AF + 10.1046/j.1365-313x.1998.00343.x + 10069079 + REF + The Plant Journal + ref + 16 + 1998 + 49614 + Floral dip: A simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana + + + 3350 + 3361 + surname:Colcombet;given-names:J + surname:Boisson-Dernier;given-names:A + surname:Ros-Palau;given-names:R + surname:Vera;given-names:CE + surname:Schroeder;given-names:JI + 10.1105/tpc.105.036731 + 16284306 + REF + The Plant Cell + ref + 17 + 2005 + 49712 + Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 are essential for tapetum development and microspore maturation + + + W375 + W383 + surname:Davis;given-names:IW + surname:Leaver-Fay;given-names:A + surname:Richardson;given-names:DC + surname:Chen;given-names:VB + surname:Block;given-names:JN + surname:Kapral;given-names:GJ + surname:Wang;given-names:X + surname:Murray;given-names:LW + surname:Arendall;given-names:WB + surname:Snoeyink;given-names:J + surname:Richardson;given-names:JS + 10.1093/nar/gkm216 + 17452350 + REF + Nucleic Acids Research + ref + 35 + 2007 + 49834 + Molprobity: All-atom contacts and structure validation for proteins and nucleic acids + + + 2126 + 2132 + surname:Emsley;given-names:P + surname:Cowtan;given-names:K + 10.1107/S0907444904019158 + 15572765 + REF + Acta Crystallographica Section D Biological Crystallography + ref + 60 + 2004 + 49920 + Coot : Model-building tools for molecular graphics + + + 944 + 947 + surname:Fenn;given-names:TD + surname:Ringe;given-names:D + surname:Petsko;given-names:GA + 10.1107/S0021889803006721 + REF + Journal of Applied Crystallography + ref + 36 + 2003 + 49971 + Povscript+ : A program for model and data visualization using persistence of vision ray-tracing + + + e15075 + surname:Gou;given-names:X + surname:Yin;given-names:H + surname:He;given-names:K + surname:Du;given-names:J + surname:Yi;given-names:J + surname:Xu;given-names:S + surname:Lin;given-names:H + surname:Clouse;given-names:SD + surname:Li;given-names:J + 10.1371/journal.pgen.1002452 + REF + PLoS Genetics + ref + 8 + 2012 + 50067 + Genetic evidence for an indispensable role of somatic embryogenesis receptor kinases in brassinosteroid signaling + + + e15075 + surname:Hashimoto;given-names:Y + surname:Zhang;given-names:S + surname:Blissard;given-names:GW + 10.1186/1472-6750-10-50 + REF + BMC Biotechnology + ref + 10 + 2010 + 50181 + Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins + + + 793 + 800 + surname:Hasler;given-names:M + surname:Hothorn;given-names:LA + 10.1002/bimj.200710466 + 18932141 + REF + Biometrical Journal + ref + 50 + 2008 + 50366 + Multiple contrast tests in the presence of heteroscedasticity + + + 803 + 816 + surname:Hecht;given-names:V + surname:Vielle-Calzada;given-names:JP + surname:Hartog;given-names:MV + surname:Schmidt;given-names:ED + surname:Boutilier;given-names:K + surname:Grossniklaus;given-names:U + surname:de Vries;given-names:SC + 10.1104/pp.010324 + 11706164 + REF + Plant Physiology + ref + 127 + 2001 + 50428 + The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture + + + 346 + 363 + surname:Hothorn;given-names:T + surname:Bretz;given-names:F + surname:Westfall;given-names:P + 10.1002/bimj.200810425 + 18481363 + REF + Biometrical Journal + ref + 50 + 2008 + 50582 + Simultaneous inference in general parametric models + + + 467 + 471 + surname:Hothorn;given-names:M + surname:Belkhadir;given-names:Y + surname:Dreux;given-names:M + surname:Dabi;given-names:T + surname:Noel;given-names:JP + surname:Wilson;given-names:IA + surname:Chory;given-names:J + 10.1038/nature10153 + 21666665 + REF + Nature + ref + 474 + 2011 + 50634 + Structural basis of steroid hormone perception by the receptor kinase BRI1 + + + 108 + 117 + surname:Jinn;given-names:TL + surname:Stone;given-names:JM + surname:Walker;given-names:JC + 10.1101/gad.14.1.108 + 10640280 + REF + Genes & Development + ref + 14 + 2000 + 50709 + HAESA, an Arabidopsis leucine-rich repeat receptor kinase, controls floral organ abscission + + + 2577 + 2637 + surname:Kabsch;given-names:W + surname:Sander;given-names:C + 10.1002/bip.360221211 + 6667333 + REF + Biopolymers + ref + 22 + 1983 + 50801 + Dictionary of protein secondary structure: Pattern recognition of hydrogen-bonded and geometrical features + + + 795 + 800 + surname:Kabsch;given-names:W + 10.1107/S0021889893005588 + REF + Journal of Applied Crystallography + ref + 26 + 1993 + 50908 + Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants + + + 845 + 848 + surname:Kondo;given-names:T + surname:Sawa;given-names:S + surname:Kinoshita;given-names:A + surname:Mizuno;given-names:S + surname:Kakimoto;given-names:T + surname:Fukuda;given-names:H + surname:Sakagami;given-names:Y + 10.1126/science.1128439 + 16902141 + REF + Science + ref + 313 + 2006 + 51021 + A plant peptide encoded by CLV3 identified by in situ MALDI-TOF MS analysis + + + 2 + surname:Lease;given-names:KA + surname:Cho;given-names:SK + surname:Walker;given-names:JC + 10.1186/1746-4811-2-2 + 16483376 + REF + Plant Methods + ref + 2 + 2006 + 51097 + A petal breakstrength meter for Arabidopsis abscission studies + + + 817 + 828 + surname:Lewis;given-names:MW + surname:Leslie;given-names:ME + surname:Fulcher;given-names:EH + surname:Darnielle;given-names:L + surname:Healy;given-names:PN + surname:Youn;given-names:JY + surname:Liljegren;given-names:SJ + 10.1111/j.1365-313X.2010.04194.x + 20230490 + REF + The Plant Journal + ref + 62 + 2010 + 51160 + The SERK1 receptor-like kinase regulates organ separation in Arabidopsis flowers + + + 402 + 408 + surname:Livak;given-names:KJ + surname:Schmittgen;given-names:TD + 10.1006/meth.2001.1262 + 11846609 + REF + Methods + ref + 25 + 2001 + 51241 + Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method + + + 1065 + 1067 + surname:Matsuzaki;given-names:Y + surname:Ogawa-Ohnishi;given-names:M + surname:Mori;given-names:A + surname:Matsubayashi;given-names:Y + 10.1126/science.1191132 + 20798316 + REF + Science + ref + 329 + 2010 + 51352 + Secreted peptide signals required for maintenance of root stem cell niche in Arabidopsis + + + 658 + 674 + surname:McCoy;given-names:AJ + surname:Grosse-Kunstleve;given-names:RW + surname:Adams;given-names:PD + surname:Winn;given-names:MD + surname:Storoni;given-names:LC + surname:Read;given-names:RJ + 10.1107/S0021889807021206 + 19461840 + REF + Journal of Applied Crystallography + ref + 40 + 2007 + 51441 + Phaser crystallographic software + + + 2361 + 2372 + surname:Meng;given-names:X + surname:Chen;given-names:X + surname:Mang;given-names:H + surname:Liu;given-names:C + surname:Yu;given-names:X + surname:Gao;given-names:X + surname:Torii;given-names:KU + surname:He;given-names:P + surname:Shan;given-names:L + 10.1016/j.cub.2015.07.068 + 26320950 + REF + Current Biology + ref + 25 + 2015 + 51474 + Differential function of Arabidopsis SERK family receptor-like kinases in stomatal patterning + + + 1330 + 1338 + surname:Meng;given-names:X + surname:Zhou;given-names:J + surname:Tang;given-names:J + surname:Li;given-names:B + surname:de Oliveira;given-names:MVV + surname:Chai;given-names:J + surname:He;given-names:P + surname:Shan;given-names:L + 10.1016/j.celrep.2016.01.023 + 26854226 + REF + Cell Reports + ref + 14 + 2016 + 51568 + Ligand-induced receptor-like kinase complex regulates floral organ abscission in Arabidopsis + + + 240 + 255 + surname:Murshudov;given-names:GN + surname:Vagin;given-names:AA + surname:Dodson;given-names:EJ + 10.1107/S0907444996012255 + 15299926 + REF + Acta Crystallographica Section D Biological Crystallography + ref + 53 + 1997 + 51661 + Refinement of macromolecular structures by the maximum-likelihood method + + + 4413 + 4419 + surname:Muto;given-names:T + surname:Todoroki;given-names:Y + 10.1016/j.bmc.2013.04.048 + 23673217 + REF + Bioorganic & Medicinal Chemistry + ref + 21 + 2013 + 51734 + Brassinolide-2,3-acetonide: A brassinolide-induced rice lamina joint inclination antagonist + + + 1251 + 1263 + surname:Niederhuth;given-names:CE + surname:Cho;given-names:SK + surname:Seitz;given-names:K + surname:Walker;given-names:JC + 10.1111/jipb.12116 + 24138310 + REF + Journal of Integrative Plant Biology + ref + 55 + 2013 + 51826 + Letting go is never easy: Abscission and receptor-like protein kinases + + + e15075 + surname:Niederhuth;given-names:CE + surname:Patharkar;given-names:OR + surname:Walker;given-names:JC + 10.1186/1471-2164-14-37 + REF + BMC Genomics + ref + 14 + 2013 + 51897 + Transcriptional profiling of the Arabidopsis abscission mutant hae hsl2 by RNA-Seq + + + 294 + surname:Ogawa;given-names:M + surname:Shinohara;given-names:H + surname:Sakagami;given-names:Y + surname:Matsubayashi;given-names:Y + 10.1126/science.1150083 + 18202283 + REF + Science + ref + 319 + 2008 + 51980 + Arabidopsis CLV3 peptide directly binds CLV1 ectodomain + + + 578 + 580 + surname:Ohyama;given-names:K + surname:Shinohara;given-names:H + surname:Ogawa-Ohnishi;given-names:M + surname:Matsubayashi;given-names:Y + 10.1038/nchembio.182 + 19525968 + REF + Nature Chemical Biology + ref + 5 + 2009 + 52036 + A glycopeptide regulating stem cell fate in Arabidopsis thaliana + + + REF + R Foundation for Statistical Computing + ref + 2014 + 52101 + + + 709 + 714 + surname:Salaj;given-names:J + surname:von Recklinghausen;given-names:IR + surname:Hecht;given-names:V + surname:de Vries;given-names:SC + surname:Schel;given-names:JH + surname:van Lammeren;given-names:AA + 10.1016/j.plaphy.2008.04.011 + 18515128 + REF + Plant Physiology and Biochemistry + ref + 46 + 2008 + 52102 + AtSERK1 expression precedes and coincides with early somatic embryogenesis in Arabidopsis thaliana + + + 305 + 320 + surname:Sanner;given-names:MF + surname:Olson;given-names:AJ + surname:Spehner;given-names:JC + 10.1002/(SICI)1097-0282(199603)38:3&amp;lt;305::AID-BIP4&amp;gt;3.0.CO;2-Y + 8906967 + REF + Biopolymers + ref + 38 + 1996 + 52201 + Reduced surface: An efficient way to compute molecular surfaces + + + 889 + 892 + surname:Santiago;given-names:J + surname:Henzler;given-names:C + surname:Hothorn;given-names:M + 10.1126/science.1242468 + 23929946 + REF + Science + ref + 341 + 2013 + 52265 + Molecular mechanism for plant steroid receptor activation by somatic embryogenesis co-receptor kinases + + + 41263 + 41269 + surname:Shah;given-names:K + surname:Vervoort;given-names:J + surname:de Vries;given-names:SC + 10.1074/jbc.M102381200 + 11509554 + REF + The Journal of Biological Chemistry + ref + 276 + 2001 + 52368 + Role of threonines in the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 activation loop in phosphorylation + + + 112 + 122 + surname:Sheldrick;given-names:GM + 10.1107/S0108767307043930 + 18156677 + REF + Acta Crystallographica Section A Foundations of Crystallography + ref + 64 + 2008 + 52490 + A short history of SHELX + + + 845 + 854 + surname:Shinohara;given-names:H + surname:Moriyama;given-names:Y + surname:Ohyama;given-names:K + surname:Matsubayashi;given-names:Y + 10.1111/j.1365-313X.2012.04934.x + 22321211 + REF + The Plant Journal + ref + 70 + 2012 + 52515 + Biochemical mapping of a ligand-binding domain within Arabidopsis BAM1 reveals diversified ligand recognition mechanisms of plant LRR-RKs + + + 1467 + 1476 + surname:Stenvik;given-names:GE + surname:Butenko;given-names:MA + surname:Urbanowicz;given-names:BR + surname:Rose;given-names:JK + surname:Aalen;given-names:RB + 10.1105/tpc.106.042036 + 16679455 + REF + The Plant Cell + ref + 18 + 2006 + 52653 + Overexpression of INFLORESCENCE DEFICIENT IN ABSCISSION activates cell separation in vestigial abscission zones in Arabidopsis + + + 1805 + 1817 + surname:Stenvik;given-names:GE + surname:Tandstad;given-names:NM + surname:Guo;given-names:Y + surname:Shi;given-names:CL + surname:Kristiansen;given-names:W + surname:Holmgren;given-names:A + surname:Clark;given-names:SE + surname:Aalen;given-names:RB + surname:Butenko;given-names:MA + 10.1105/tpc.108.059139 + 18660431 + REF + The Plant Cell + ref + 20 + 2008 + 52780 + The EPIP peptide of INFLORESCENCE DEFICIENT IN ABSCISSION is sufficient to induce abscission in Arabidopsis through the receptor-like kinases HAESA and HAESA-LIKE2 + + + 1326 + 1329 + surname:Sun;given-names:Y + surname:Han;given-names:Z + surname:Tang;given-names:J + surname:Hu;given-names:Z + surname:Chai;given-names:C + surname:Zhou;given-names:B + surname:Chai;given-names:J + 10.1038/cr.2013.131 + 24126715 + REF + Cell Research + ref + 23 + 2013 + 52944 + Structure reveals that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide + + + 110 + 120 + surname:Tang;given-names:J + surname:Han;given-names:Z + surname:Sun;given-names:Y + surname:Zhang;given-names:H + surname:Gong;given-names:X + surname:Chai;given-names:J + 10.1038/cr.2014.161 + 25475059 + REF + Cell Research + ref + 25 + 2015 + 53028 + Structural basis for recognition of an endogenous peptide by the plant receptor kinase PEPR1 + + + e15075 + surname:Taylor;given-names:I + surname:Wang;given-names:Y + surname:Seitz;given-names:K + surname:Baer;given-names:J + surname:Bennewitz;given-names:S + surname:Mooney;given-names:BP + surname:Walker;given-names:JC + 10.1371/journal.pone.0147203 + REF + PLOS ONE + ref + 11 + 2016 + 53121 + Analysis of phosphorylation of the receptor-like protein kinase HAESA during arabidopsis floral abscission + + + 61 + 69 + surname:Terwilliger;given-names:TC + surname:Grosse-Kunstleve;given-names:RW + surname:Afonine;given-names:PV + surname:Moriarty;given-names:NW + surname:Zwart;given-names:PH + surname:Hung;given-names:L-W + surname:Read;given-names:RJ + surname:Adams;given-names:PD + 10.1107/S090744490705024X + 18094468 + REF + Acta Crystallographica Section D Biological Crystallography + ref + 64 + 2008 + 53228 + Iterative model building, structure refinement and density modification with the PHENIX autobuild wizard + + + 265 + 268 + surname:Wang;given-names:J + surname:Li;given-names:H + surname:Han;given-names:Z + surname:Zhang;given-names:H + surname:Wang;given-names:T + surname:Lin;given-names:G + surname:Chang;given-names:J + surname:Yang;given-names:W + surname:Chai;given-names:J + 10.1038/nature14858 + 26308901 + REF + Nature + ref + 525 + 2015 + 53333 + Allosteric receptor activation by the plant peptide hormone phytosulfokine + + + REF + paragraph + 53408 + 10.7554/eLife.15075.017 + + + REVIEW_INFO + title + 53432 + Decision letter + + + REVIEW_INFO + paragraph + 53448 + Zhang + + + REVIEW_INFO + paragraph + 53454 + Mingjie + + + REVIEW_INFO + paragraph + 53462 + In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. + + + REVIEW_INFO + paragraph + 53746 + Thank you for submitting your article "Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission" to eLife for consideration by eLife. Your article has been favorably evaluated by John Kuyiyan (Senior editor) and three reviewers, one of whom, (Mingjie Zhang) is a member of our Board of Reviewing Editors. + + + REVIEW_INFO + paragraph + 54089 + The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you will see that the required revisions are essentially clarifications and some additional analysis of existing data in nature. + + + REVIEW_INFO + paragraph + 54372 + Summary: + + + REVIEW_INFO + paragraph + 54381 + In this work, Hothorn and colleagues investigated the structural basis governing the recognition of peptide hormone IDA during plant floral abscission process. Through an array of complex structures, supplemented with biochemical and genetic experiments, the authors uncovered the IDA recognition mechanism by a co-receptor (HAESA and SERK1) detection mechanism. The structures also reveal the specific recognition mechanism of the 12-residue IDA core peptide sequence by the co-receptors, and suggest that this 12-residue IDA sequence is likely to be the mature peptide hormone functioning in plants. The comparison of the structures of the HAESA/SERK1/IDA complex and the previously determined BRA1/SERK1/brassinolide complex by the same group also suggests a co-receptor pairing mechanism for various plant hormones. The story gives detailed and novel mechanistic insights in the perception of IDA during floral abscission, and is convincing and worthy to be considered for publication in eLife with the following revisions. + + + REVIEW_INFO + paragraph + 55409 + Key issues which need to be addressed: + + + REVIEW_INFO + paragraph + 55448 + 1) As the results reported here rest largely on the interpretation of the structural data, the following points need to be addressed by the authors. i) Temperature factors (Wilson B and residual) are unusually high for the reported resolution. Are there portions of the structure that exhibit more disorder or are these high temperature factors throughout the structure? Are there potential problems with radiation damage due to the high multiplicity? ii) A simulated annealing omit map figure should be provided as the peptides all exhibit very high temperature factors. iii) How many side chains were trimmed due to poor electron density? If this is a significant percentage, it should be noted. + + + REVIEW_INFO + paragraph + 56146 + 2) A further discussion comparing the mechanisms of BRI1-BL-SERK1 and HAESA-IDA-SERK1 would be helpful as a structural comparison is presented. As BR1 binds its ligand with high affinity, the low affinity of HAESA for IDA could be further discussed. + + + REVIEW_INFO + paragraph + 56396 + 3) Are the results here applicable to the related receptor HSL2? Are the residues that interact with SERK1 and IDA conserved in HSL2? + + + REVIEW_INFO + paragraph + 56530 + 4) The authors show that N-terminal extension of the peptide does not impact on binding efficiency, but what would happen if the peptide was extended at the C-terminal end, at the suggested cleavage site? Would cleavage be required for recognition? A brief discussion on this point may help. + + + REVIEW_INFO + paragraph + 56822 + 5) Figure 3C: Could the authors comment on the difference between the blue (SERK1 vs. HAESA-IDA) and the black (IDA vs. HAESA-SERK1) line? SERK1 vs. HAESA-IDA gives a Kd of 75 nM, yet IDA vs. SERK1-HAESA gives a Kd of 350 nM. In the text the authors keep referring to the 75 nM Kd, but not the 350 nM Kd. Is it really fair to say the Kd is 75 nM? + + + REVIEW_INFO + paragraph + 57169 + 6) In the Discussion, can the authors comment further on the discrepancy between their study and the study of Lewis et al. (Plant J, 2010) concerning the role of SERK1 in floral abscission? Similarly, could the authors comment on the fact that the LRR-RLP EVD/SOBIR seems to be a negative regulator of the HAESA/HSL2 pathway (Leslie et al., Development, 2010), which seems puzzling given that EVD/SOBIR function is normally restricted to LRR-RLPs (Gust & Felix, Curr Op Plant Biol, 2014). + + + REVIEW_INFO + paragraph + 57658 + 7) Given that the central hydroxyproline in IDA is of such crucial importance for binding, isn't it surprising that IDAΔ69N does not bind to HAESA at all? Wouldn't it be expected that the remaining part of the peptide still binds to HAESA? + + + REVIEW_INFO + paragraph + 57902 + 8) Figure 1—figure supplement 2: How is it possible that charged amino acids are involved in hydrophobic interactions? + + + REVIEW_INFO + paragraph + 58023 + 9) Are the distances shown in the graphical representation of the structures proportional? It would seem that some of the aromatic rings could cause steric hindrance. + + + REVIEW_INFO + paragraph + 58190 + 10) Why did the authors decide to express HAESA and SERK1 without signal peptide? Would it make a difference for binding of IDA, if they leave the SP on? + + + REVIEW_INFO + paragraph + 58344 + 11) Figure 7: Are the homologous regions also the active parts of these peptides? And could the authors display amino acids numbers on either side of the fragments? + + + REVIEW_INFO + paragraph + 58509 + 12) Have the authors ever measured dissociation of the peptide from the complex? And in this regard, to what does "highly stable receptor – co-receptor complex" refer/compare to? + + + REVIEW_INFO + paragraph + 58690 + 13) Figures 3A and 5C require statistical analyses. + + + REVIEW_INFO + paragraph + 58742 + 10.7554/eLife.15075.018 + + + REVIEW_INFO + title + 58766 + Author response + + + REVIEW_INFO + paragraph + 58782 + 1) As the results reported here rest largely on the interpretation of the structural data, the following points need to be addressed by the authors. i) Temperature factors (Wilson B and residual) are unusually high for the reported resolution. + + + REVIEW_INFO + paragraph + 59028 + We thank the reviewer(s) for pointing out this issue: Indeed our mean B-values deviate substantially from the expected mean B-values (resolution 1.75 – 2.00 A, B(Wilson) ~ 27.0 over 5,510 structures in the PDB; Pavel Afonine, personal communication). We would like to note that due to the many N-glycosylation sites dispersed over the HAESA LRR domain (shown in Figure 1—figure supplement 1D), we find relatively few crystal contacts in our P3121 crystal form, which may rationalize our high B-values. We have reanalyzed our space group assignment (using the CCP4 program ZANUDA) and checked for any signs of problems during data collection (ice rings, multiple crystal lattices, splitting, using the programs XDS and XDSSTAT), as well as for twinning and pseudosymmetry (using phenix.xtriage). No such problems appear to exist, our structures refine very well and our refined B-values are in good agreement with our Wilson B-factors (see Table 2). Thus, the high B-values appear to represent an intrinsic property of our crystals and are not the result of a poor data collection strategy or inappropriate crystallographic analysis. + + + REVIEW_INFO + paragraph + 60165 + Are there portions of the structure that exhibit more disorder or are these high temperature factors throughout the structure? + + + elife-15075-resp-fig1.jpg + fig8 + FIG + fig_title_caption + 60293 + Cα trace of the HAESA LRR domain and IDA peptide colored according to B-factor from low (60.9, in blue) to high (134.7, in red). + + + elife-15075-resp-fig1.jpg + fig8 + FIG + fig_caption + 60426 + Mean B-value is 79.5. + + + elife-15075-resp-fig1.jpg + fig8 + FIG + fig_caption + 60448 + DOI: +http://dx.doi.org/10.7554/eLife.15075.015 + + + REVIEW_INFO + paragraph + 60495 + Yes. As shown in revised Figure 4B, the Cterminal LRRs of HAESA in contact with SERK1 in our complex structure appear to be somewhat flexible. Author response image 1. illustrates that the B-values are significantly higher in the C-terminal part of the HAESA LRR domain (with the Cterminal capping domain being the most flexible), while both the N-terminal LRRs of HAESA (with exception of the LRR N-terminal capping domain) and the IDA peptide appear better ordered in our P3121 crystals form. + + + REVIEW_INFO + paragraph + 60990 + Are there potential problems with radiation damage due to the high multiplicity? + + + REVIEW_INFO + paragraph + 61072 + No. Data were collected at SLS beamline PXIII equipped with a Dectris Pilatus 2M-F detector. We perform all our data collections at very low dose and high multiplicity of measurement, which at this beam-line produces similar results compared to exposing the crystal at higher dose for a smaller angular range. We collected 360 deg with 0.1 deg slices and obtained a Wilson B-value of 80, with no sign of radiation damage in our data processing (subroutine COLSPOT in XDS over all frames). To test the reviewer's hypothesis we cut the data after 90 deg (when completeness approaches 100%) and we obtained a Wilson B-value of 78 and a refined mean B-value of around 75. These value do not significantly differ from our presented 360 deg data set and thus it is unlikely that radiation damage produces these high B-values. Again, they rather appear to be an intrinsic property of our crystals. + + + REVIEW_INFO + paragraph + 61963 + ii) A simulated annealing omit map figure should be provided as the peptides all exhibit very high temperature factors. + + + REVIEW_INFO + paragraph + 62083 + Thank you for this suggestion. In our first submission, we presented 2Fo-Fc omit electron density maps for our HAESA-IDA/IDL complex structures. As suggested, we now present simulated annealing omit maps in revised Figure 2A, B, C. The maps were generated like this: phenix.composite_omit_map *.pdb *.mtz *.cif nproc=8 anneal=True We would like to note that our peptide are well ordered in our structures, and their B-values match the B-values of their interacting LRR surface (compare Author response image 1). + + + REVIEW_INFO + paragraph + 62595 + iii) How many side chains were trimmed due to poor electron density? If this is a significant percentage, it should be noted. + + + REVIEW_INFO + paragraph + 62722 + Here are the requested numbers (trimmed residues out of total residues in asymmetric unit, percentage): + + + REVIEW_INFO + paragraph + 62826 + HAESA apo: 7 out of 595 (1%) + + + REVIEW_INFO + paragraph + 62855 + HAESA IDA: 6 out of 597 + 12 (1%) + + + REVIEW_INFO + paragraph + 62889 + HAESA IDL1: 6 out of 597 + 12 (1%) + + + REVIEW_INFO + paragraph + 62924 + HAESA – IDA – SERK1: 5 out of 594 + 12 + 185 (0.6%) + + + REVIEW_INFO + paragraph + 62980 + We have included a statement in the Methods section that reads: “Amino acids whose side-chain position could not be modeled with confidence were truncated to alanine (0.6 – 1% of total residues)[…]” + + + REVIEW_INFO + paragraph + 63187 + 2) A further discussion comparing the mechanisms of BRI1-BL-SERK1 and HAESA-IDA-SERK1 would be helpful as a structural comparison is presented. + + + REVIEW_INFO + paragraph + 63333 + We have expanded our discussion of the HAESA – SERK1 and BRI1 – SERK1 interfaces. We now specify the SERK1 residues in contact with both receptors and the SERK1 residues unique to HAESA/IDA sensing. We also comment on the very different ligand binding modes in HAESA and BRI1 and specify that different LRR segments contribute to the formation of the respective steroid and peptide hormone binding pockets. We feel however that an in-depth comparison of the interacting surfaces is beyond the scope of this report and partially redundant with our earlier work (Santiago et al., Science, 2013). In our opinion, such an analysis seems more appropriate for a review on the subject, which we are currently preparing. Our revised Discussion now reads: “Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor (Santiago, Henzler, and Hothorn 2013), reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A).[…] These residues are not involved in the sensing of the steroid hormone brassinolide (Santiago, Henzler, and Hothorn 2013). In both cases however, the co-receptor completes the hormone binding pocket.” + + + REVIEW_INFO + paragraph + 64732 + As BR1 binds its ligand with high affinity, the low affinity of HAESA for IDA could be further discussed. + + + REVIEW_INFO + paragraph + 64839 + High affinity brassinosteroid binding to BRI1 was previously shown using BRI1-enriched plant extracts and radiolabeled brassinolide (Wang et al., Nature410:380-383, 2001). We now know that co-immunoprecipitations of BRI1 from Arabidopsis contain SERK proteins (compare for example Jaillais et al., PNAS, 2011) and thus the reported binding constants likely correspond to steroid binding to BRI1-SERK complexes, not to BRI1 alone. We would thus prefer not to compare the binding affinities for brassinosteroid and peptide hormone ligands at this point. + + + REVIEW_INFO + paragraph + 65391 + 3) Are the results here applicable to the related receptor HSL2? Are the residues that interact with SERK1 and IDA conserved in HSL2? + + + REVIEW_INFO + paragraph + 65526 + Yes. We present a structure-based sequence alignment of AtHAESA and AtHSL2, as well as other HAESA-type receptors from different plant species in Figure 1—figure supplement 3. In the peptide binding surface, 17 out of 26 contributing amino-acids are conserved among AtHAESA and AtHSL2. 13 out of 19 interacting residues in the HAESA – SERK1 complex are also present in AtHSL2. We feel that this is strong conservation given that the AtHAESA and AtHSL2 ectodomains share 45% overall sequence identity. We have included a statement in our manuscript that reads: “Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and other HAESA-type receptors in different plant species (Figure 1—figure supplement 3).” + + + REVIEW_INFO + paragraph + 66315 + 4) The authors show that N-terminal extension of the peptide does not impact on binding efficiency, but what would happen if the peptide was extended at the C-terminal end, at the suggested cleavage site? + + + elife-15075-resp-fig2.jpg + fig9 + FIG + fig_title_caption + 66521 + Isothermal titration calorimetry thermograph of the C-terminally extended IDA-SFVN peptide (200 μM) titrated into a solution containing 20 μM of the purified HAESA ectodomain. + + + elife-15075-resp-fig2.jpg + fig9 + FIG + fig_caption + 66699 + No detectable binding is observed. + + + elife-15075-resp-fig2.jpg + fig9 + FIG + fig_caption + 66734 + DOI: +http://dx.doi.org/10.7554/eLife.15075.016 + + + REVIEW_INFO + paragraph + 66781 + Thank you for suggesting this experiment. We synthesized a C-terminally extended version of the IDA peptide (IDA-SFVN with sequence YPIPPSA-Hyp- SKRHN SFVN) and performed quantitative binding assays by ITC. As shown in Author response image 2, we cannot observe any detectable binding of this C-terminally extended peptide to the HAESA ectodomain, consistent with our crystallographic models that suggest that HAESA specifically senses an active IDA 12mer. We have incorporated this new result in Figure 2D. We have included a new statement in the manuscript that reads: “The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D, F, 2D).” + + + REVIEW_INFO + paragraph + 67523 + Would cleavage be required for recognition? A brief discussion on this point may help. + + + REVIEW_INFO + paragraph + 67611 + Yes. We have modified our manuscript accordingly: “This suggests that the conserved Asn69 may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein (Stenvik et al. 2008).“ + + + REVIEW_INFO + paragraph + 67885 + 5) Figure 3C: Could the authors comment on the difference between the blue (SERK1 vs. HAESA-IDA) and the black (IDA vs. HAESA-SERK1) line? SERK1 vs. HAESA-IDA gives a Kd of 75 nM, yet IDA vs. SERK1-HAESA gives a Kd of 350 nM. In the text the authors keep referring to the 75 nM Kd, but not the 350 nM Kd. Is it really fair to say the Kd is 75 nM? + + + REVIEW_INFO + paragraph + 68232 + Yes and No. These are two different experiments. We first measured (by ITC) the binding affinity of IDA (in the syringe) binding to a protein solution containing equimolar ratios of HAESA and SERK1 (Kdis 350 nM in this case). Next, we titrated a concentrated SERK1 solution (in the syringe) into a solution of HAESA containing IDA in 10fold molar excess (Kdin this case is 75 nM). Given that the experimental conditions (protein and peptide concentrations and molar ratios between the components) are very different, we feel that the Kd 's obtained by these experiments are in good agreement (4.5 fold difference vs. a 60-260 fold difference when compared to the isolated HAESA ectodomain). Nevertheless, we addressed the reviewer's concern by modifying our manuscript which now states: “In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range. (Figure 3C).” + + + REVIEW_INFO + paragraph + 69236 + 6) In the Discussion, can the authors comment further on the discrepancy between their study and the study of Lewis et al. (Plant J, 2010) concerning the role of SERK1 in floral abscission? + + + REVIEW_INFO + paragraph + 69427 + The process of floral organ abscission in Arabidopsis is divided into distinct steps where a gradual loosening of the cell wall between abscising cells can be measured as a reduction in petal breakstrength (Bleecker and Patterson, 1997). During floral abscission in wild-type plants a significant drop in breakstrength occurs shortly before the petals drop (shown in Figure 3A in our manuscript). Previously reported negative regulators of abscission, such as the transcription factor KNAT1, have an earlier reduction in breakstrength, indicative of early cell wall remodeling (Shi et al. 2011). Our results show that serk1 mutant plants, contrary to knat1 mutants and wild type, have a delay in cell wall loosening and organ separation (Figure 3A) thus positively regulating organ separation during abscission. The weaker phenotype when compared to haesa/hsl2 mutants is likely due to the redundant nature of other SERKs inthe abscission zone (recent work of Meng et al. 2016, cited in the Results and Discussion sections of our manuscript). + + + REVIEW_INFO + paragraph + 70470 + It has previously been reported that mutations in SERK1 can rescue the block in abscission in plants without the functional ADP-ribosylation factor GTPase-activating protein NEVERSHED (NEV) (Lewis et al. 2010). However, as a mutation in SERK1 is not capable of rescuing the ida mutant phenotype (Lewis et al. 2010) and revertant mutants capable of rescuing the abscission defect of ida do not complement nev, it has been suggested that NEV and IDA function in parallel pathways to promote cell separation (Liu 2013). Our work does not rule out a function for SERK1 in such a parallel pathway, we merely report SERK1 can ALSO act as a positive regulator of abscission by interacting with HAESA in an IDA-dependent manner. We do not observe negative regulation of floral abscission using our SERK1 mutant alleles. Based on the available evidence there is thus little to discuss and speculate about the different functions of SERK1 in abscission, as no molecular mechanism for the negative role of SERK1 in this pathway has been reported thus far. We feel that it is beyond the scope of our manuscript to clarify the different roles of SERK1 in the Arabidopsis abscission zone. + + + REVIEW_INFO + paragraph + 71645 + Similarly, could the authors comment on the fact that the LRR-RLP EVD/SOBIR seems to be a negative regulator of the HAESA/HSL2 pathway (Leslie et al., Development, 2010), which seems puzzling given that EVD/SOBIR function is normally restricted to LRR-RLPs (Gust & Felix, Curr Op Plant Biol, 2014). + + + REVIEW_INFO + paragraph + 71944 + We did attempt to express and purify the EVR/SOBIR extracellular domain, but in our hands the protein is not properly secreted and hence unfolded. We thus could not further investigate the potential mechanism of EVR/SOBIR in the HAESA pathway. + + + REVIEW_INFO + paragraph + 72188 + 7) Given that the central hydroxyproline in IDA is of such crucial importance for binding, isn't it surprising that IDAΔ69N does not bind to HAESA at all? Wouldn't it be expected that the remaining part of the peptide still binds to HAESA? + + + REVIEW_INFO + paragraph + 72433 + We thank the reviewers for pointing this out to us. Indeed, we find several structural and sequence features in IDA peptide to be important determinants for HAESA binding, namely the correct size of the peptide, the presence of a central Hyp residue and an intact C-terminal Arg-His-Asn motif that is buried in the structure. In the revised manuscript we now provide new experiments (binding of a C-terminal extended IDA peptide to HAESA) that clarifies this point (summarized in revised Figure 2D). We have revised our statement in the Discussion accordingly: “The central Hyp residue in IDA is found buried in the HAESA peptide binding surfaceand thus this post-translational modification may regulateIDA bioactivity.” + + + REVIEW_INFO + paragraph + 73158 + 8) Figure 1—figure supplement 2: How is it possible that charged amino acids are involved in hydrophobic interactions? + + + REVIEW_INFO + paragraph + 73280 + We apologize for this confusing statement. It now reads: “A N-terminal Pro-rich motif in IDA makes contacts LRRs 2-6 of the receptor(Figure 1D, Figure 1—figure supplement 2A-C).” + + + REVIEW_INFO + paragraph + 73465 + 9) Are the distances shown in the graphical representation of the structures proportional? It would seem that some of the aromatic rings could cause steric hindrance. + + + REVIEW_INFO + paragraph + 73633 + No. The graphical representation are proportional and e.g. Trp218 in the back of the binding pocket is not producing steric clashes with the peptide with the closest distance being 4.5 A. + + + REVIEW_INFO + paragraph + 73821 + 10) Why did the authors decide to express HAESA and SERK1 without signal peptide? Would it make a difference for binding of IDA, if they leave the SP on? + + + REVIEW_INFO + paragraph + 73976 + No. We did express both the HAESA and SERK1 ectodomains fused to the signal peptide of human azurocidin, which provides very efficient secretion of LRR proteins in insect cells (Olczak & Olczak, Anal. Biochem., 2006) (see Methods, subsection “Protein Expression and Purification“). Both the native signal peptides for SERK1 and HAESA as well as the azurocidin signal peptide are being recognized and cleaved by the Trichoplusia ni signal peptidase. This results, just like in planta, in a mature receptor/coreceptor ectodomain starting with the first α-helix of the N-terminal capping domain (residues 20 and 24, respectively). Thus, there is no reason to believe that the signal peptide would play a role in IDA sensing. Using our system, we cannot produce HAESA and/or SERK1 ectodomains with an intact signal peptide, as this would impair folding and proper secretion of the recombinant proteins. + + + REVIEW_INFO + paragraph + 74880 + 11) Figure 7: Are the homologous regions also the active parts of these peptides? + + + REVIEW_INFO + paragraph + 74963 + Yes. We have included three additional references in the Discussion section of our manuscript, which report the bioactive regions of CLV3/CLE, RGF and PEP peptides shown in Figure 7. The revised section now reads: “Importantly, this motif can also be found in other peptide hormone families (Kondo et al. 2006; Matsuzaki et al. 2010; Tang et al. 2015)(Figure 7). Among these are the CLE peptides regulating stem cell maintenance in the shoot and the root (Clark, Running, and Meyerowitz 1995). It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA (Kondo et al. 2006;Ogawa et al. 2008; Shinohara et al. 2012).” + + + REVIEW_INFO + paragraph + 75752 + And could the authors display amino acids numbers on either side of the fragments? + + + REVIEW_INFO + paragraph + 75836 + Yes. We have now included the residues number of each peptide in Figure 7. + + + REVIEW_INFO + paragraph + 75911 + 12) Have the authors ever measured dissociation of the peptide from the complex? + + + REVIEW_INFO + paragraph + 75993 + No. We have not performed any biochemical experiment that would allow us to quantify the dissociation of the peptide from the ternary complex. In qualitative terms it is however of note that HAESA-IDA-SERK1 complexes do not dissociate in size exclusion chromatography experiments, even when the peptide is not provided in excess or supplied in the running buffer. + + + REVIEW_INFO + paragraph + 76357 + And in this regard, to what does "highly stable receptor – co-receptor complex" refer/compare to? + + + REVIEW_INFO + paragraph + 76458 + The reviewers are correct, we should not claim that the complex is 'highly stable' if we have not quantified the dissociation rate. The revised sentence reads: “This ligand-induced formation of a receptor – co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm.” + + + REVIEW_INFO + paragraph + 76814 + 13) Figures 3A and 5C require statistical analyses. + + + REVIEW_INFO + paragraph + 76866 + Thank you for pointing this out to us. The statistical analysis of the petal break-strength assays shown in Figures 3A and 5C has been carried out by Prof. Ludwig A. Hothorn, Institute for Biostatistics, University of Hannover, Germany, whom we have added as an author on our manuscript: + + + REVIEW_INFO + paragraph + 77154 + Statistical analysis for Figure 3A: The statistical analysis is described in the figure legend of Figure 3A; statistical significant changes are indicated by a * in the Figure itself. The revised figure legend reads: “Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. […] Petal break was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. Calculations were performed in R (R Core Team 2014) (version 3.2.3).” The two new references have been added to the Reference section of the manuscript. + + + REVIEW_INFO + paragraph + 77907 + We have changed our Results section accordingly: “Our experiments suggest that among the SERK family members, SERK1 is a positive regulator offloral abscission. We found that the force required to remove the petals of serk1-1 mutants is significantlyhigher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants (Stenvik et al. 2008), and that floral abscission is delayed in serk1-1 (Figure 3A). The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines (Albrecht et al. 2008) showed a petal break-strength profile not significantly differentfrom wild-type plants.” + + + REVIEW_INFO + paragraph + 78505 + Statistical analysis for Figure 5C: The statistical analysis is described in the figure legend of Figure 5C; statistical significant changes are indicated by * and # symbols in the Figure itself. The revised figure legend reads:”Quantitative petal break assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. […] Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c).” + + + REVIEW_INFO + paragraph + 79041 + We have changed our Results section accordingly: “We overexpressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). […] Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA→ Ala may cause a weak dominant negative effect (Figure 5C-E).” + + + REVIEW_INFO + paragraph + 79420 + Following the suggestions from for example Nuzzo (Nature506:150-152, 2014) and Trafirmow and Marks (Basic and Applied Social Psychology37:1-2, 2015), we decided not to report p-values. + + + diff --git a/BioC_XML/4848761_v0.xml b/BioC_XML/4848761_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..7e6e1c6aab8bb1a17e861baca540a72bcefa198e --- /dev/null +++ b/BioC_XML/4848761_v0.xml @@ -0,0 +1,9309 @@ + + + + PMC + 20201216 + pmc.key + + 4848761 + CC BY + no + 0 + 0 + + Jerome C Nwachukwu et al + 10.15252/msb.20156701 + 4848761 + 27107013 + MSB156701 + 864 + 4 + Breast cancer Chemical biology Crystal structure Nuclear receptor Signal transduction Chemical Biology Structural Biology Transcription + This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. + surname:Nwachukwu;given-names:Jerome C + surname:Srinivasan;given-names:Sathish + surname:Carlson;given-names:Kathryn E + surname:Josan;given-names:Jatinder S + surname:Elemento;given-names:Olivier + surname:Katzenellenbogen;given-names:John A + surname:Zhou;given-names:Hai‐Bing + surname:Nettles;given-names:Kendall W + surname:Zheng;given-names:Yangfan + surname:Wang;given-names:Song + surname:Min;given-names:Jian + surname:Dong;given-names:Chune + surname:Liao;given-names:Zongquan + surname:Nowak;given-names:Jason + surname:Wright;given-names:Nicholas J + surname:Houtman;given-names:René + + +. () : 864 + + TITLE + Mol Syst Biol + Subject Categories + front + 12 + 2016 + 0 + Predictive features of ligand‐specific signaling through the estrogen receptor + + 0.99101317 + protein_type + cleaner0 + 2023-07-05T12:38:48Z + MESH: + + estrogen receptor + + + + ABSTRACT + abstract_title_1 + 81 + Abstract + + + ABSTRACT + abstract + 90 + Some estrogen receptor‐α (ERα)‐targeted breast cancer therapies such as tamoxifen have tissue‐selective or cell‐specific activities, while others have similar activities in different cell types. To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. For some ligand series, a single inter‐atomic distance in the ligand‐binding domain predicted their proliferative effects. In contrast, the N‐terminal coactivator‐binding site, activation function‐1 (AF‐1), determined cell‐specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα. + + 0.98243415 + protein + cleaner0 + 2023-07-05T12:38:53Z + PR: + + estrogen receptor‐α + + + 0.9781553 + protein + cleaner0 + 2023-07-05T12:38:58Z + PR: + + ERα + + + 0.9961785 + chemical + cleaner0 + 2023-07-05T12:38:43Z + CHEBI: + + tamoxifen + + + 0.9045924 + experimental_method + cleaner0 + 2023-07-05T15:11:00Z + MESH: + + synthesized + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + 0.98368347 + experimental_method + cleaner0 + 2023-07-05T15:11:05Z + MESH: + + quantitative bioassays + + + 0.5754821 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.9963126 + experimental_method + cleaner0 + 2023-07-05T15:11:07Z + MESH: + + X‐ray crystallography + + + 0.99831116 + site + cleaner0 + 2023-07-05T12:39:07Z + SO: + + coactivator‐binding site + + + 0.99466616 + structure_element + cleaner0 + 2023-07-05T12:39:13Z + SO: + + ligand‐binding domain + + + 0.99521536 + structure_element + cleaner0 + 2023-07-05T12:39:17Z + SO: + + activation function‐2 + + + 0.9947168 + structure_element + cleaner0 + 2023-07-05T12:39:23Z + SO: + + AF‐2 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + 0.90314347 + protein + cleaner0 + 2023-07-05T12:39:51Z + PR: + + GREB1 + + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:40:15Z + + inter‐atomic distance + + + 0.9391775 + structure_element + cleaner0 + 2023-07-05T12:39:13Z + SO: + + ligand‐binding domain + + + 0.99817234 + site + cleaner0 + 2023-07-05T12:39:08Z + SO: + + coactivator‐binding site + + + 0.9880258 + structure_element + cleaner0 + 2023-07-05T12:40:21Z + SO: + + activation function‐1 + + + 0.9855774 + structure_element + cleaner0 + 2023-07-05T12:40:25Z + SO: + + AF‐1 + + + 0.9663252 + experimental_method + cleaner0 + 2023-07-05T15:11:12Z + MESH: + + systems structural analyses + + + 0.9850201 + experimental_method + cleaner0 + 2023-07-05T15:11:15Z + MESH: + + quantitative chemical biology + + + 0.98969567 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + + INTRO + title_1 + 1503 + Introduction + + + INTRO + paragraph + 1516 + Many drugs are small‐molecule ligands of allosteric signaling proteins, including G protein‐coupled receptors (GPCRs) and nuclear receptors such as ERα. These receptors regulate distinct phenotypic outcomes (i.e., observable characteristics of cells and tissues, such as cell proliferation or the inflammatory response) in a ligand‐dependent manner. Small‐molecule ligands control receptor activity by modulating recruitment of effector enzymes to distal regions of the receptor, relative to the ligand‐binding site. Some of these ligands achieve selectivity for a subset of tissue‐ or pathway‐specific signaling outcomes, which is called selective modulation, functional selectivity, or biased signaling, through structural mechanisms that are poorly understood (Frolik et al, 1996; Nettles & Greene, 2005; Overington et al, 2006; Katritch et al, 2012; Wisler et al, 2014). For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003). + + 0.99419004 + protein_type + cleaner0 + 2023-07-05T13:48:39Z + MESH: + + G protein‐coupled receptors + + + 0.9895253 + protein_type + cleaner0 + 2023-07-05T12:41:17Z + MESH: + + GPCRs + + + 0.9876436 + protein_type + cleaner0 + 2023-07-05T12:41:43Z + MESH: + + nuclear receptors + + + 0.9964353 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.99821043 + site + cleaner0 + 2023-07-05T15:08:47Z + SO: + + ligand‐binding site + + + 0.6081143 + protein_type + cleaner0 + 2023-07-05T13:48:43Z + MESH: + + estrogen receptor modulators + + + protein_type + MESH: + cleaner0 + 2023-07-05T12:41:05Z + + SERMs + + + 0.9969007 + chemical + cleaner0 + 2023-07-05T12:38:44Z + CHEBI: + + tamoxifen + + + chemical + CHEBI: + cleaner0 + 2023-07-05T12:40:51Z + + Nolvadex® + + + 0.9951567 + chemical + cleaner0 + 2023-07-05T12:43:17Z + CHEBI: + + raloxifene + + + 0.80817395 + chemical + cleaner0 + 2023-07-05T13:51:27Z + CHEBI: + + Evista® + + + 0.9869108 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.9966714 + chemical + cleaner0 + 2023-07-05T13:51:32Z + CHEBI: + + estrogen + + + 0.9966848 + chemical + cleaner0 + 2023-07-05T12:49:49Z + CHEBI: + + 17β‐estradiol + + + 0.9977841 + chemical + cleaner0 + 2023-07-05T12:49:41Z + CHEBI: + + E2 + + + + MSB-12-864-g002.jpg + msb156701-fig-0001 + FIG + fig_title_caption + 2912 + Allosteric control of ERα activity + + 0.5108732 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + + MSB-12-864-g002.jpg + msb156701-fig-0001 + FIG + fig_caption + 2951 + Chemical structures of some common ERα ligands. BSC, basic side chain. E2‐rings are numbered A‐D. The E‐ring is the common site of attachment for BSC found in many SERMS. + + 0.722546 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.98871267 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + 0.994365 + protein_type + cleaner0 + 2023-07-05T12:41:05Z + MESH: + + SERMS + + + + MSB-12-864-g002.jpg + msb156701-fig-0001 + FIG + fig_caption + 3129 + ERα domain organization lettered, A‐F. DBD, DNA‐binding domain; LBD, ligand‐binding domain; AF, activation function + + 0.9844996 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.99813074 + structure_element + cleaner0 + 2023-07-05T12:42:14Z + SO: + + DBD + + + 0.97538567 + structure_element + cleaner0 + 2023-07-05T12:42:18Z + SO: + + DNA‐binding domain + + + 0.9980124 + structure_element + cleaner0 + 2023-07-05T12:42:23Z + SO: + + LBD + + + 0.97787577 + structure_element + cleaner0 + 2023-07-05T12:39:13Z + SO: + + ligand‐binding domain + + + 0.99472946 + structure_element + cleaner0 + 2023-07-05T12:42:37Z + SO: + + AF + + + 0.6370711 + structure_element + cleaner0 + 2023-07-05T12:42:51Z + SO: + + activation function + + + + MSB-12-864-g002.jpg + msb156701-fig-0001 + FIG + fig_caption + 3252 + Schematic illustration of the canonical ERα signaling pathway. + + 0.48197246 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + + MSB-12-864-g002.jpg + msb156701-fig-0001 + FIG + fig_caption + 3319 + Linear causality model for ERα‐mediated cell proliferation. + + 0.9659611 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + + MSB-12-864-g002.jpg + msb156701-fig-0001 + FIG + fig_caption + 3383 + Branched causality model for ERα‐mediated cell proliferation. + + 0.96815825 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + + INTRO + paragraph + 3449 + ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). The LBD contains a ligand‐dependent coactivator‐binding site called activation function‐2 (AF‐2). However, the agonist activity of SERMs derives from activation function‐1 (AF‐1)—a coactivator recruitment site located in the AB domain (Berry et al, 1990; Shang & Brown, 2002; Abot et al, 2013). + + 0.9963446 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.996717 + protein_state + cleaner0 + 2023-07-05T15:17:59Z + DUMMY: + + structurally conserved + + + 0.99633765 + structure_element + cleaner0 + 2023-07-05T15:06:29Z + SO: + + globular domains + + + 0.96176726 + protein_type + cleaner0 + 2023-07-05T13:48:49Z + MESH: + + nuclear receptor superfamily + + + 0.9852898 + structure_element + cleaner0 + 2023-07-05T12:42:19Z + SO: + + DNA‐binding domain + + + 0.99855286 + structure_element + cleaner0 + 2023-07-05T12:42:15Z + SO: + + DBD + + + 0.49088258 + protein_state + cleaner0 + 2023-07-05T15:18:10Z + DUMMY: + + flexible + + + 0.9973324 + structure_element + cleaner0 + 2023-07-05T15:06:34Z + SO: + + hinge region + + + 0.95946825 + structure_element + cleaner0 + 2023-07-05T12:39:13Z + SO: + + ligand‐binding domain + + + 0.9976718 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.9132317 + protein_state + cleaner0 + 2023-07-05T15:18:12Z + DUMMY: + + unstructured + + + 0.99860966 + structure_element + cleaner0 + 2023-07-05T12:51:03Z + SO: + + AB + + + structure_element + SO: + cleaner0 + 2023-07-05T12:51:12Z + + F + + + 0.99767905 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + site + SO: + cleaner0 + 2023-07-05T12:39:08Z + + coactivator‐binding site + + + 0.7260005 + structure_element + cleaner0 + 2023-07-05T12:39:18Z + SO: + + activation function‐2 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:39:23Z + + AF‐2 + + + 0.9952769 + protein_type + cleaner0 + 2023-07-05T12:41:06Z + MESH: + + SERMs + + + structure_element + SO: + cleaner0 + 2023-07-05T12:40:21Z + + activation function‐1 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:40:26Z + + AF‐1 + + + 0.9973503 + site + cleaner0 + 2023-07-05T15:08:54Z + SO: + + coactivator recruitment site + + + structure_element + SO: + cleaner0 + 2023-07-05T12:51:03Z + + AB + + + + INTRO + paragraph + 4073 + AF‐1 and AF‐2 bind distinct but overlapping sets of coregulators (Webb et al, 1998; Endoh et al, 1999; Delage‐Mourroux et al, 2000; Yi et al, 2015). AF‐2 binds the signature LxxLL motif peptides of coactivators such as NCOA1/2/3 (also known as SRC‐1/2/3). AF‐1 binds a separate surface on these coactivators (Webb et al, 1998; Yi et al, 2015). Yet, it is unknown how different ERα ligands control AF‐1 through the LBD, and whether this inter‐domain communication is required for cell‐specific signaling or anti‐proliferative responses. + + 0.98835874 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.98771614 + structure_element + cleaner0 + 2023-07-05T12:39:23Z + SO: + + AF‐2 + + + 0.988358 + structure_element + cleaner0 + 2023-07-05T12:39:23Z + SO: + + AF‐2 + + + 0.9694072 + structure_element + cleaner0 + 2023-07-05T15:06:42Z + SO: + + LxxLL motif + + + 0.9784671 + protein + cleaner0 + 2023-07-05T12:39:46Z + PR: + + NCOA1/2/3 + + + 0.94810975 + protein + cleaner0 + 2023-07-05T13:49:47Z + PR: + + SRC‐1/2/3 + + + 0.9889884 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.9961467 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.9896169 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.99829656 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + + INTRO + paragraph + 4636 + In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson & O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). However, ERα‐mediated proliferative responses vary in a ligand‐dependent manner (Srinivasan et al, 2013); thus, it is not known whether this canonical model is widely applicable across diverse ERα ligands. + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + 0.9966757 + protein_state + cleaner0 + 2023-07-05T15:18:15Z + DUMMY: + + E2‐bound + + + 0.99311686 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.996216 + oligomeric_state + cleaner0 + 2023-07-05T14:07:01Z + DUMMY: + + homodimer + + + 0.9631549 + site + cleaner0 + 2023-07-05T15:09:21Z + SO: + + estrogen‐response elements + + + 0.4373797 + site + cleaner0 + 2023-07-05T15:09:24Z + SO: + + EREs + + + 0.6682438 + protein + cleaner0 + 2023-07-05T12:39:46Z + PR: + + NCOA1/2/3 + + + 0.80179566 + protein + cleaner0 + 2023-07-05T12:39:51Z + PR: + + GREB1 + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + 0.552018 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.51123303 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + + INTRO + paragraph + 5284 + Our long‐term goal is to be able to predict proliferative or anti‐proliferative activity of a ligand in different tissues from its crystal structure by identifying different structural perturbations that lead to specific signaling outcomes. The simplest response model for ligand‐specific proliferative effects is a linear causality model, where the degree of NCOA1/2/3 recruitment determines GREB1 expression, which in turn drives ligand‐specific cell proliferation (Fig 1D). Alternatively, a more complicated branched causality model could explain ligand‐specific proliferative responses (Fig 1E). In this signaling model, multiple coregulator binding events and target genes (Won Jeong et al, 2012; Nwachukwu et al, 2014), LBD conformation, nucleocytoplasmic shuttling, the occupancy and dynamics of DNA binding, and other biophysical features could contribute independently to cell proliferation (Lickwar et al, 2012). + + 0.99724627 + evidence + cleaner0 + 2023-07-05T14:07:07Z + DUMMY: + + crystal structure + + + 0.85250056 + protein + cleaner0 + 2023-07-05T12:39:46Z + PR: + + NCOA1/2/3 + + + 0.99367493 + protein + cleaner0 + 2023-07-05T12:39:51Z + PR: + + GREB1 + + + 0.9958538 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + + INTRO + paragraph + 6222 + To test these signaling models, we profiled a diverse library of ERα ligands using systems biology approaches to X‐ray crystallography and chemical biology (Srinivasan et al, 2013), including a series of quantitative bioassays for ERα function that were statistically robust and reproducible, based on the Z’‐statistic (Fig EV1A and B; see Materials and Methods). We also determined the structures of 76 distinct ERα LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ERα LBD. Our findings here indicate that specific structural perturbations can be tied to ligand‐selective domain usage and signaling patterns, thus providing a framework for structure‐based design of improved breast cancer therapeutics, and understanding the different phenotypic effects of environmental estrogens. + + 0.54615736 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.9938122 + experimental_method + cleaner0 + 2023-07-05T15:11:21Z + MESH: + + X‐ray crystallography + + + 0.7150245 + experimental_method + cleaner0 + 2023-07-05T15:11:24Z + MESH: + + chemical biology + + + 0.65988773 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.6892823 + evidence + cleaner0 + 2023-07-05T14:07:11Z + DUMMY: + + Z’‐statistic + + + 0.9331319 + experimental_method + cleaner0 + 2023-07-05T15:11:27Z + MESH: + + determined + + + 0.99378294 + evidence + cleaner0 + 2023-07-05T14:07:15Z + DUMMY: + + structures + + + 0.9661832 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.47582763 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.97217715 + protein_state + cleaner0 + 2023-07-05T15:18:20Z + DUMMY: + + bound to + + + 0.99364096 + protein_state + cleaner0 + 2023-07-05T15:18:26Z + DUMMY: + + active + + + 0.99790215 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.99694806 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.997186 + chemical + cleaner0 + 2023-07-05T12:45:41Z + CHEBI: + + estrogens + + + + MSB-12-864-g003.jpg + msb156701-fig-0001ev + FIG + fig_title_caption + 7118 + High‐throughput screens for ERα ligand profiling + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + + MSB-12-864-g003.jpg + msb156701-fig-0001ev + FIG + fig_caption + 7171 + Summary of ligand screening assays used to measure ER‐mediated activities. ERE, estrogen‐response element; Luc, luciferase reporter gene; M2H, mammalian 2‐hybrid; UAS, upstream‐activating sequence. + + 0.9710028 + experimental_method + cleaner0 + 2023-07-05T15:11:35Z + MESH: + + ligand screening assays + + + 0.898545 + structure_element + cleaner0 + 2023-07-05T15:06:51Z + SO: + + ERE + + + structure_element + SO: + cleaner0 + 2023-07-05T12:46:07Z + + estrogen‐response element + + + experimental_method + MESH: + cleaner0 + 2023-07-05T13:01:23Z + + Luc + + + 0.44347644 + experimental_method + cleaner0 + 2023-07-05T15:11:39Z + MESH: + + luciferase reporter gene + + + 0.95992655 + experimental_method + cleaner0 + 2023-07-05T15:11:42Z + MESH: + + M2H + + + 0.9920692 + experimental_method + cleaner0 + 2023-07-05T15:11:45Z + MESH: + + mammalian 2‐hybrid + + + 0.9925506 + structure_element + cleaner0 + 2023-07-05T15:06:55Z + SO: + + UAS + + + 0.9208929 + structure_element + cleaner0 + 2023-07-05T15:06:59Z + SO: + + upstream‐activating sequence + + + + MSB-12-864-g003.jpg + msb156701-fig-0001ev + FIG + fig_caption + 7377 + Controls for screening assays described in panel (A), above. Error bars indicate mean ± SEM, n = 3. + + + RESULTS + title_1 + 7478 + Results + + + RESULTS + title_2 + 7486 + Strength of AF‐1 signaling does not determine cell‐specific signaling + + structure_element + SO: + cleaner0 + 2023-07-05T12:40:26Z + + AF‐1 + + + + RESULTS + paragraph + 7560 + To compare ERα signaling induced by diverse ligand types, we synthesized and assayed a library of 241 ERα ligands containing 19 distinct molecular scaffolds. These include 15 indirect modulator series, which lack a SERM‐like side chain and modulate coactivator binding indirectly from the ligand‐binding pocket (Fig 2A–E; Dataset EV1) (Zheng et al, 2012) (Zhu et al, 2012) (Muthyala et al, 2003; Seo et al, 2006) (Srinivasan et al, 2013) (Wang et al, 2012) (Liao et al, 2014) (Min et al, 2013). We also generated four direct modulator series with side chains designed to directly dislocate h12 and thereby completely occlude the AF‐2 surface (Fig 2C and E; Dataset EV1) (Kieser et al, 2010). Ligand profiling using our quantitative bioassays revealed a wide range of ligand‐induced GREB1 expression, reporter gene activities, ERα‐coactivator interactions, and proliferative effects on MCF‐7 breast cancer cells (Figs EV1 and EV2A–J). This wide variance enabled us to probe specific features of ERα signaling using ligand class analyses, and identify signaling patterns shared by specific ligand series or scaffolds. + + 0.50055975 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.97331995 + experimental_method + cleaner0 + 2023-07-05T15:11:50Z + MESH: + + synthesized and assayed + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + 0.8351258 + protein_state + cleaner0 + 2023-07-05T15:18:32Z + DUMMY: + + lack + + + protein_type + MESH: + cleaner0 + 2023-07-05T12:47:26Z + + SERM‐like + + + 0.9979671 + site + cleaner0 + 2023-07-05T15:09:30Z + SO: + + ligand‐binding pocket + + + 0.97495544 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.9794798 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + 0.92169464 + experimental_method + cleaner0 + 2023-07-05T15:11:53Z + MESH: + + Ligand profiling + + + 0.9695189 + experimental_method + cleaner0 + 2023-07-05T15:11:55Z + MESH: + + quantitative bioassays + + + protein + PR: + cleaner0 + 2023-07-05T12:39:51Z + + GREB1 + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + 0.77025014 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.97555035 + experimental_method + cleaner0 + 2023-07-05T15:11:59Z + MESH: + + ligand class analyses + + + + MSB-12-864-g004.jpg + msb156701-fig-0002 + FIG + fig_title_caption + 8708 + Classes of compounds in the ERα ligand library + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + + MSB-12-864-g004.jpg + msb156701-fig-0002 + FIG + fig_caption + 8759 + Structure of the E2‐bound ERα LBD in complex with an NCOA2 peptide of (PDB 1GWR). + + 0.994959 + evidence + cleaner0 + 2023-07-05T14:07:28Z + DUMMY: + + Structure + + + 0.9947484 + protein_state + cleaner0 + 2023-07-05T15:18:39Z + DUMMY: + + E2‐bound + + + 0.99766695 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.9940242 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.9758456 + protein_state + cleaner0 + 2023-07-05T15:18:42Z + DUMMY: + + in complex with + + + 0.88555133 + protein + cleaner0 + 2023-07-05T12:49:10Z + PR: + + NCOA2 + + + + MSB-12-864-g004.jpg + msb156701-fig-0002 + FIG + fig_caption + 8845 + Structural details of the ERα LBD bound to the indicated ligands. Unlike E2 (PDB 1GWR), TAM is a direct modulator with a BSC that dislocates h12 to block the NCOA2‐binding site (PDB 3ERT). OBHS is an indirect modulator that dislocates the h11 C‐terminus to destabilize the h11–h12 interface (PDB 4ZN9). + + 0.9972567 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.99309206 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.98950016 + protein_state + cleaner0 + 2023-07-05T15:18:44Z + DUMMY: + + bound to + + + 0.9974898 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + 0.9979062 + chemical + cleaner0 + 2023-07-05T13:01:45Z + CHEBI: + + TAM + + + 0.50051475 + structure_element + cleaner0 + 2023-07-05T12:48:50Z + SO: + + h12 + + + 0.99800706 + site + cleaner0 + 2023-07-05T15:09:35Z + SO: + + NCOA2‐binding site + + + 0.9172119 + chemical + cleaner0 + 2023-07-05T13:03:37Z + CHEBI: + + OBHS + + + 0.99688464 + structure_element + cleaner0 + 2023-07-05T12:48:35Z + SO: + + h11 + + + 0.9972157 + site + cleaner0 + 2023-07-05T15:09:38Z + SO: + + h11–h12 interface + + + + MSB-12-864-g004.jpg + msb156701-fig-0002 + FIG + fig_caption + 9155 + The ERα ligand library contains 241 ligands representing 15 indirect modulator scaffolds, plus 4 direct modulator scaffolds. The number of compounds per scaffold is shown in parentheses (see Dataset EV1 for individual compound information and Appendix Supplementary Methods for synthetic protocols). + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + + MSB-12-864-g005.jpg + msb156701-fig-0002ev + FIG + fig_title_caption + 9459 + ERα ligands induced a range of agonist activity profiles + + 0.45072588 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + + MSB-12-864-g005.jpg + msb156701-fig-0002ev + FIG + fig_caption + 9520 + Screening data from individual ligands are shown, grouped by scaffold. Each data point represents the activity of a distinct compound. Error bars indicate the class average (mean) ± range. *Direct modulator. + + + MSB-12-864-g005.jpg + msb156701-fig-0002ev + FIG + fig_caption + 9731 + +Source data are available online for this figure. + + + + RESULTS + paragraph + 9783 + We first asked whether direct modulation of the receptor with an extended side chain is required for cell‐specific signaling. To this end, we compared the average ligand‐induced GREB1 mRNA levels in MCF‐7 cells and 3×ERE‐Luc reporter gene activity in Ishikawa endometrial cancer cells (E‐Luc) or in HepG2 cells transfected with wild‐type ERα (L‐Luc ERα‐WT) (Figs 3A and EV2A–C). Direct modulators showed significant differences in average activity between cell types except OBHS‐ASC analogs, which had similar low agonist activities in the three cell types. The other direct modulators had low agonist activity in Ishikawa cells, no or inverse agonist activity in MCF‐7 cells, and more variable activity in HepG2 liver cells. While it was known that direct modulators such as tamoxifen drive cell‐specific signaling, these experiments reveal that indirect modulators also drive cell‐specific signaling, since eight of fourteen classes showed significant differences in average activity (Figs 3A and EV2A–C). + + 0.69088733 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + 0.81260395 + experimental_method + cleaner0 + 2023-07-05T15:12:07Z + MESH: + + 3×ERE‐Luc + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:55Z + + E‐Luc + + + 0.9967254 + protein_state + cleaner0 + 2023-07-05T12:52:35Z + DUMMY: + + wild‐type + + + 0.99650925 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:31Z + + L‐Luc + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T12:54:01Z + + WT + + + 0.94454867 + chemical + cleaner0 + 2023-07-05T13:02:08Z + CHEBI: + + OBHS‐ASC + + + 0.99727494 + chemical + cleaner0 + 2023-07-05T12:38:44Z + CHEBI: + + tamoxifen + + + + MSB-12-864-g006.jpg + msb156701-fig-0003 + FIG + fig_title_caption + 10824 + Ligand‐specific signaling underlies ERα‐mediated cell proliferation + + 0.5306793 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + + MSB-12-864-g006.jpg + msb156701-fig-0003 + FIG + fig_caption + 10897 + (A) Ligand‐specific ERα activities in HepG2, Ishikawa and MCF‐7 cells. The ligand‐induced L‐Luc ERα‐WT and E‐Luc activities and GREB1 mRNA levels are shown by scaffold (mean + SD). (B) Ligand class analysis of the L‐Luc ERα‐WT and ERα‐ΔAB activities in HepG2 cells. Significant sensitivity to AB domain deletion was determined by Student's t‐test (n = number of ligands per scaffold in Fig 2). The average activities of ligands classes are shown (mean + SEM). + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + 0.51193875 + experimental_method + cleaner0 + 2023-07-05T12:54:30Z + MESH: + + L‐Luc + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T12:54:01Z + + WT + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:55Z + + E‐Luc + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + 0.84152174 + experimental_method + cleaner0 + 2023-07-05T15:12:16Z + MESH: + + class analysis + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:31Z + + L‐Luc + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T12:54:01Z + + WT + + + 0.9970581 + mutant + cleaner0 + 2023-07-05T12:52:46Z + MESH: + + ERα‐ΔAB + + + 0.8804764 + structure_element + cleaner0 + 2023-07-05T12:51:02Z + SO: + + AB + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:55:58Z + + Student's t‐test + + + + MSB-12-864-g006.jpg + msb156701-fig-0003 + FIG + fig_caption + 11389 + Correlation and regression analyses in a large test set. The r +2 values are plotted as a heat map. In cluster 1, the first three comparisons (rows) showed significant positive correlations (F‐test for nonzero slope, P ≤ 0.05). In cluster 2, only one of these comparisons revealed a significant positive correlation, while none was significant in cluster 3. +, statistically significant correlations gained by deletion of the AB or F domains. −, significant correlations lost upon deletion of AB or F domains. + + 0.98742986 + experimental_method + cleaner0 + 2023-07-05T15:12:19Z + MESH: + + Correlation and regression analyses + + + 0.8578758 + evidence + cleaner0 + 2023-07-05T14:07:34Z + DUMMY: + + r +2 values + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:51:37Z + + F‐test + + + evidence + DUMMY: + cleaner0 + 2023-07-05T13:26:18Z + + P + + + 0.51995146 + experimental_method + cleaner0 + 2023-07-05T15:12:23Z + MESH: + + deletion + + + 0.9976948 + structure_element + cleaner0 + 2023-07-05T12:51:03Z + SO: + + AB + + + 0.9931163 + structure_element + cleaner0 + 2023-07-05T12:51:12Z + SO: + + F + + + 0.9974043 + structure_element + cleaner0 + 2023-07-05T12:51:03Z + SO: + + AB + + + 0.97985506 + structure_element + cleaner0 + 2023-07-05T12:51:11Z + SO: + + F + + + + MSB-12-864-g006.jpg + msb156701-fig-0003 + FIG + fig_caption + 11906 + +Source data are available online for this figure. + + + + RESULTS + paragraph + 11958 + Tamoxifen depends on AF‐1 for its cell‐specific activity (Sakamoto et al, 2002); therefore, we asked whether cell‐specific signaling observed here is due to a similar dependence on AF‐1 for activity (Fig EV1). To test this idea, we compared the average L‐Luc activities of each scaffold in HepG2 cells co‐transfected with wild‐type ERα or with ERα lacking the AB domain (Figs 1B and EV1). While E2 showed similar L‐Luc ERα‐WT and ERα‐ΔAB activities, tamoxifen showed complete loss of activity without the AB domain (Fig EV1B). Deletion of the AB domain significantly reduced the average L‐Luc activities of 14 scaffolds (Student's t‐test, P ≤ 0.05) (Fig 3B). These “AF‐1‐sensitive” activities were exhibited by both direct and indirect modulators, and were not limited to scaffolds that showed cell‐specific signaling (Fig 3A and B). Thus, the strength of AF‐1 signaling does not determine cell‐specific signaling. + + 0.99671257 + chemical + cleaner0 + 2023-07-05T12:38:44Z + CHEBI: + + Tamoxifen + + + 0.95681185 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.9622051 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:56:28Z + + average L‐Luc activities + + + 0.97614646 + experimental_method + cleaner0 + 2023-07-05T15:12:27Z + MESH: + + co‐transfected + + + 0.99681187 + protein_state + cleaner0 + 2023-07-05T12:52:35Z + DUMMY: + + wild‐type + + + 0.9976277 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.99342316 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.9647014 + protein_state + cleaner0 + 2023-07-05T15:18:49Z + DUMMY: + + lacking the + + + structure_element + SO: + cleaner0 + 2023-07-05T12:51:03Z + + AB + + + 0.99783427 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:31Z + + L‐Luc + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T12:54:00Z + + WT + + + 0.9962079 + mutant + cleaner0 + 2023-07-05T12:52:46Z + MESH: + + ERα‐ΔAB + + + 0.996908 + chemical + cleaner0 + 2023-07-05T12:38:44Z + CHEBI: + + tamoxifen + + + 0.87945706 + protein_state + cleaner0 + 2023-07-05T15:18:53Z + DUMMY: + + without + + + structure_element + SO: + cleaner0 + 2023-07-05T12:51:03Z + + AB + + + 0.7637515 + experimental_method + cleaner0 + 2023-07-05T15:12:31Z + MESH: + + Deletion of + + + structure_element + SO: + cleaner0 + 2023-07-05T12:51:03Z + + AB + + + evidence + DUMMY: + cleaner0 + 2023-07-05T14:08:00Z + + average L‐Luc activities + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:53:06Z + + Student's t‐test + + + evidence + DUMMY: + cleaner0 + 2023-07-05T13:26:18Z + + P + + + structure_element + SO: + cleaner0 + 2023-07-05T12:40:26Z + + AF‐1 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:40:26Z + + AF‐1 + + + + RESULTS + title_2 + 12926 + Identifying cell‐specific signaling clusters in ERα ligand classes + + 0.9333595 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + + RESULTS + paragraph + 12997 + As another approach to identifying cell‐specific signaling, we determined the degree of correlation between ligand‐induced activities in the different cell types. Here, we compared ligands within each class (Fig 3C), instead of comparing average activities (Fig 3A and B). For each ligand class or scaffold, we calculated the Pearson's correlation coefficient, r, for pairwise comparison of activity profiles in breast (GREB1), liver (L‐Luc), and endometrial cells (E‐Luc). The value of r ranges from −1 to 1, and it defines the extent to which the data fit a straight line when compounds show similar agonist/antagonist activity profiles between cell types (Fig EV3A). We also calculated the coefficient of determination, r 2, which describes the percentage of variance in a dependent variable such as proliferation that can be predicted by an independent variable such as GREB1 expression. We present both calculations as r 2 to readily compare signaling specificities using a heat map on which the red–yellow palette indicates significant positive correlations (P ≤ 0.05, F‐test for nonzero slope), while the blue palette denotes negative correlations (Fig 3C–F). + + 0.8269178 + evidence + cleaner0 + 2023-07-05T14:08:09Z + DUMMY: + + Pearson's correlation coefficient + + + 0.8409841 + evidence + cleaner0 + 2023-07-05T14:08:12Z + DUMMY: + + r + + + 0.9782397 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:31Z + + L‐Luc + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:55Z + + E‐Luc + + + 0.8962522 + evidence + cleaner0 + 2023-07-05T14:08:17Z + DUMMY: + + r + + + evidence + DUMMY: + cleaner0 + 2023-07-05T14:08:50Z + + coefficient of determination + + + 0.8511789 + evidence + cleaner0 + 2023-07-05T14:08:53Z + DUMMY: + + r 2 + + + 0.98343074 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + 0.9520688 + evidence + cleaner0 + 2023-07-05T14:08:57Z + DUMMY: + + r 2 + + + evidence + DUMMY: + cleaner0 + 2023-07-05T13:26:18Z + + P + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:57:27Z + + F‐test + + + + MSB-12-864-g007.jpg + msb156701-fig-0003ev + FIG + fig_title_caption + 14188 + The side chain of OBHS‐BSC analogs induces cell‐specific signaling + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:02:52Z + + OBHS‐BSC + + + + MSB-12-864-g007.jpg + msb156701-fig-0003ev + FIG + fig_caption + 14259 + Correlation analysis of OBHS versus OBHS‐BSC activity across cell types. + + 0.99562293 + chemical + cleaner0 + 2023-07-05T13:03:37Z + CHEBI: + + OBHS + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:02:52Z + + OBHS‐BSC + + + + MSB-12-864-g007.jpg + msb156701-fig-0003ev + FIG + fig_caption + 14334 + Correlation analysis of L‐Luc ERα‐ΔAB activity versus endogenous ERα activity of OBHS analogs. In panel (D), L‐Luc ERα‐WT activity from panel (B) is shown for comparison. + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:31Z + + L‐Luc + + + 0.99583405 + mutant + cleaner0 + 2023-07-05T12:52:47Z + MESH: + + ERα‐ΔAB + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + 0.97782236 + chemical + cleaner0 + 2023-07-05T13:03:37Z + CHEBI: + + OBHS + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:31Z + + L‐Luc + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T12:54:01Z + + WT + + + + MSB-12-864-g007.jpg + msb156701-fig-0003ev + FIG + fig_caption + 14521 + Correlation analysis of L‐Luc ERα‐ΔF activity versus endogenous ERα activities of OBHS analogs. + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:31Z + + L‐Luc + + + 0.9871301 + mutant + cleaner0 + 2023-07-05T14:06:47Z + MESH: + + ERα‐ΔF + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + 0.9828555 + chemical + cleaner0 + 2023-07-05T13:03:37Z + CHEBI: + + OBHS + + + + MSB-12-864-g007.jpg + msb156701-fig-0003ev + FIG + fig_caption + 14629 + Correlation analysis of MCF‐7 cell proliferation versus NCOA2/3 recruitment or GREB1 levels observed in response to (G) OBHS‐N and (H) OBHS‐BSC analogs. + + 0.6739586 + protein + cleaner0 + 2023-07-05T13:49:52Z + PR: + + NCOA2/3 + + + 0.95227623 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + 0.9852438 + chemical + cleaner0 + 2023-07-05T13:02:40Z + CHEBI: + + OBHS‐N + + + 0.9810481 + chemical + cleaner0 + 2023-07-05T13:02:51Z + CHEBI: + + OBHS‐BSC + + + + MSB-12-864-g007.jpg + msb156701-fig-0003ev + FIG + fig_caption + 14788 + +Data information: In each panel, a data point indicates the activity of a distinct compound.Source data are available online for this figure. + + + + RESULTS + paragraph + 14932 + This analysis revealed diverse signaling specificities that we grouped into three clusters. Scaffolds in cluster 1 exhibited strongly correlated GREB1 levels, E‐Luc and L‐Luc activity profiles across the three cell types (Fig 3C lanes 1–4), suggesting these ligands use similar ERα signaling pathways in the breast, endometrial, and liver cell types. This cluster includes WAY‐C, OBHS, OBHS‐N, and triaryl‐ethylene analogs, all of which are indirect modulators. Cluster 2 contains scaffolds with activities that were positively correlated in only two of the three cell types, indicating cell‐specific signaling (Fig 3C lanes 5–12). This cluster includes two classes of direct modulators (cyclofenil‐ASC and WAY dimer), and six classes of indirect modulators (2,5‐DTP, 3,4‐DTP, S‐OBHS‐2 and S‐OBHS‐3, furan, and WAY‐D). In this cluster, the correlated activities varied by scaffold. For example, 3,4‐DTP, furan, and S‐OBHS‐2 drove positively correlated GREB1 levels and E‐Luc but not L‐Luc ERα‐WT activity (Fig 3C lanes 5–7). In contrast, WAY dimer and WAY‐D analogs drove positively correlated GREB1 levels and L‐Luc ERα‐WT but not E‐Luc activity (Fig 3C lanes 8 and 9). The last set of scaffolds, cluster 3, displayed cell‐specific activities that were not correlated in any of the three cell types (Fig 3C lanes 13–19). This cluster includes two direct modulator scaffolds (OBHS‐ASC and OBHS‐BSC), and five indirect modulator scaffolds (A‐CD, cyclofenil, 3,4‐DTPD, imine, and imidazopyridine). + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:55Z + + E‐Luc + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:31Z + + L‐Luc + + + 0.96300846 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.9935394 + chemical + cleaner0 + 2023-07-05T13:04:18Z + CHEBI: + + WAY‐C + + + 0.5436915 + chemical + cleaner0 + 2023-07-05T13:03:37Z + CHEBI: + + OBHS + + + 0.6676753 + chemical + cleaner0 + 2023-07-05T13:02:41Z + CHEBI: + + OBHS‐N + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:08:31Z + + triaryl‐ethylene + + + 0.98814267 + chemical + cleaner0 + 2023-07-05T13:06:19Z + CHEBI: + + cyclofenil‐ASC + + + 0.9942509 + chemical + cleaner0 + 2023-07-05T14:01:20Z + CHEBI: + + WAY dimer + + + 0.99742794 + chemical + cleaner0 + 2023-07-05T14:01:24Z + CHEBI: + + 2,5‐DTP + + + 0.99740154 + chemical + cleaner0 + 2023-07-05T14:01:27Z + CHEBI: + + 3,4‐DTP + + + 0.6645348 + chemical + cleaner0 + 2023-07-05T13:05:14Z + CHEBI: + + S‐OBHS‐2 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:04:56Z + + S‐OBHS‐3 + + + 0.9984285 + chemical + cleaner0 + 2023-07-05T14:01:30Z + CHEBI: + + furan + + + 0.99470615 + chemical + cleaner0 + 2023-07-05T13:05:03Z + CHEBI: + + WAY‐D + + + 0.9973963 + chemical + cleaner0 + 2023-07-05T14:01:33Z + CHEBI: + + 3,4‐DTP + + + 0.99861944 + chemical + cleaner0 + 2023-07-05T14:01:36Z + CHEBI: + + furan + + + 0.6933242 + chemical + cleaner0 + 2023-07-05T13:05:14Z + CHEBI: + + S‐OBHS‐2 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:55Z + + E‐Luc + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:31Z + + L‐Luc + + + 0.9050587 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T12:54:01Z + + WT + + + 0.99606335 + chemical + cleaner0 + 2023-07-05T14:01:40Z + CHEBI: + + WAY dimer + + + 0.9956608 + chemical + cleaner0 + 2023-07-05T13:05:03Z + CHEBI: + + WAY‐D + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:31Z + + L‐Luc + + + 0.83370477 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T12:54:01Z + + WT + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:55Z + + E‐Luc + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:02:09Z + + OBHS‐ASC + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:02:52Z + + OBHS‐BSC + + + 0.9929027 + chemical + cleaner0 + 2023-07-05T13:05:54Z + CHEBI: + + A‐CD + + + 0.99842876 + chemical + cleaner0 + 2023-07-05T13:06:00Z + CHEBI: + + cyclofenil + + + 0.9973632 + chemical + cleaner0 + 2023-07-05T13:05:49Z + CHEBI: + + 3,4‐DTPD + + + 0.99747247 + chemical + cleaner0 + 2023-07-05T14:01:45Z + CHEBI: + + imine + + + 0.9938916 + chemical + cleaner0 + 2023-07-05T13:06:06Z + CHEBI: + + imidazopyridine + + + + RESULTS + paragraph + 16503 + These results suggest that addition of an extended side chain to an ERα ligand scaffold is sufficient to induce cell‐specific signaling, where the relative activity profiles of the individual ligands change between cell types. This is demonstrated by directly comparing the signaling specificities of matched OBHS (indirect modulator, cluster 1) and OBHS‐BSC analogs (direct modulator, cluster 3), which differ only in the basic side chain (Fig 2E). The activities of OBHS analogs were positively correlated across the three cell types, but the side chain of OBHS‐BSC analogs was sufficient to abolish these correlations (Figs 3C lanes 1 and 19, and EV3A–C). + + 0.9594942 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.99750704 + chemical + cleaner0 + 2023-07-05T13:03:37Z + CHEBI: + + OBHS + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:02:52Z + + OBHS‐BSC + + + 0.7002744 + chemical + cleaner0 + 2023-07-05T13:03:37Z + CHEBI: + + OBHS + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:02:52Z + + OBHS‐BSC + + + + RESULTS + paragraph + 17173 + The indirect modulator scaffolds in clusters 2 and 3 showed cell‐specific signaling patterns without the extended side chain typically viewed as the primary chemical and structural mechanism driving cell‐specific activity. Many of these scaffolds drove similar average activities of the ligand class in the different cell types (Fig 3A), but the individual ligands in each class had different cell‐specific activities (Fig EV2A–C). Thus, examining the correlated patterns of ERα activity within each scaffold demonstrates that an extended side chain is not required for cell‐specific signaling. + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + + RESULTS + title_2 + 17781 + Modulation of signaling specificity by AF‐1 + + 0.80650944 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + + RESULTS + paragraph + 17827 + To evaluate the role of AF‐1 and the F domain in ERα signaling specificity, we compared activity of truncated ERα constructs in HepG2 liver cells with endogenous ERα activity in the other cell types. The positive correlation between the L‐Luc and E‐Luc activities or GREB1 levels induced by scaffolds in cluster 1 was generally retained without the AB domain, or the F domain (Fig 3D lanes 1–4). This demonstrates that the signaling specificities underlying these positive correlations are not modified by AF‐1. OBHS analogs showed an average L‐Luc ERα‐ΔAB activity of 3.2% ± 3 (mean + SEM) relative to E2. Despite this nearly complete lack of activity, the pattern of L‐Luc ERα‐ΔAB activity was still highly correlated with the E‐Luc activity and GREB1 expression (Fig EV3D and E), demonstrating that very small AF‐2 activities can be amplified by AF‐1 to produce robust signals. Similarly, deletion of the F domain did not abolish correlations between the L‐Luc and E‐Luc or GREB1 levels induced by OBHS analogs (Fig EV3F). These similar patterns of ligand activity in the wild‐type and deletion mutants suggest that AF‐1 and the F domain purely amplify the AF‐2 activities of ligands in cluster 1. + + 0.9814356 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:51:12Z + + F + + + 0.91343945 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.9525181 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.9613005 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.92285633 + experimental_method + cleaner0 + 2023-07-05T12:54:31Z + MESH: + + L‐Luc + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:55Z + + E‐Luc + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:51:03Z + + AB + + + structure_element + SO: + cleaner0 + 2023-07-05T12:51:12Z + + F + + + 0.9698805 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:03:37Z + + OBHS + + + 0.9410656 + experimental_method + cleaner0 + 2023-07-05T12:54:31Z + MESH: + + L‐Luc + + + 0.9903858 + mutant + cleaner0 + 2023-07-05T12:52:47Z + MESH: + + ERα‐ΔAB + + + 0.9981902 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + 0.9430773 + experimental_method + cleaner0 + 2023-07-05T12:54:31Z + MESH: + + L‐Luc + + + 0.992173 + mutant + cleaner0 + 2023-07-05T12:52:47Z + MESH: + + ERα‐ΔAB + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:55Z + + E‐Luc + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:39:24Z + + AF‐2 + + + 0.96515197 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.7311077 + experimental_method + cleaner0 + 2023-07-05T15:12:37Z + MESH: + + deletion of + + + 0.9980215 + structure_element + cleaner0 + 2023-07-05T12:51:12Z + SO: + + F + + + 0.916756 + experimental_method + cleaner0 + 2023-07-05T12:54:31Z + MESH: + + L‐Luc + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:55Z + + E‐Luc + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:03:37Z + + OBHS + + + 0.99662095 + protein_state + cleaner0 + 2023-07-05T12:52:36Z + DUMMY: + + wild‐type + + + 0.4187774 + protein_state + cleaner0 + 2023-07-05T15:18:59Z + DUMMY: + + mutants + + + 0.9841226 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:51:12Z + + F + + + structure_element + SO: + cleaner0 + 2023-07-05T12:39:24Z + + AF‐2 + + + + RESULTS + paragraph + 19077 + In contrast, AF‐1 was a determinant of signaling specificity for scaffolds in cluster 2. Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). Comparing Fig 3C and D, the + and − signs indicate where the deletion mutant assays led to a gain or loss of statically significant correlation, respectively. Thus, in cluster 2, AF‐1 substantially modulated the specificity of ligands with cell‐specific activity (Fig 3D lanes 5–12). For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). Thus, ligands in cluster 2 rely on AF‐1 for both activity (Fig 3B) and signaling specificity (Fig 3D). As discussed below, this cell specificity derives from alternate coactivator preferences. + + 0.9907372 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.9822912 + experimental_method + cleaner0 + 2023-07-05T15:12:41Z + MESH: + + Deletion of + + + 0.99755126 + structure_element + cleaner0 + 2023-07-05T12:51:03Z + SO: + + AB + + + 0.9729634 + structure_element + cleaner0 + 2023-07-05T12:51:12Z + SO: + + F + + + 0.9972523 + chemical + cleaner0 + 2023-07-05T14:01:50Z + CHEBI: + + 2,5‐DTP + + + 0.9971563 + chemical + cleaner0 + 2023-07-05T14:01:52Z + CHEBI: + + 3,4‐DTP + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:13:51Z + + S‐OBHS‐3 + + + 0.92636657 + chemical + cleaner0 + 2023-07-05T13:05:03Z + CHEBI: + + WAY‐D + + + 0.9874922 + chemical + cleaner0 + 2023-07-05T14:01:56Z + CHEBI: + + WAY dimer + + + 0.9927645 + chemical + cleaner0 + 2023-07-05T13:06:20Z + CHEBI: + + cyclofenil‐ASC + + + 0.97313994 + experimental_method + cleaner0 + 2023-07-05T15:12:45Z + MESH: + + deletion mutant assays + + + 0.9914057 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.9862086 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.978631 + experimental_method + cleaner0 + 2023-07-05T15:12:49Z + MESH: + + deletion of + + + 0.997577 + structure_element + cleaner0 + 2023-07-05T12:51:03Z + SO: + + AB + + + 0.9891452 + structure_element + cleaner0 + 2023-07-05T12:51:12Z + SO: + + F + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:05:55Z + + A‐CD + + + 0.97583896 + chemical + cleaner0 + 2023-07-05T13:02:09Z + CHEBI: + + OBHS‐ASC + + + 0.96934676 + experimental_method + cleaner0 + 2023-07-05T15:12:51Z + MESH: + + deletion of + + + 0.99685115 + structure_element + cleaner0 + 2023-07-05T12:51:03Z + SO: + + AB + + + 0.9968815 + structure_element + cleaner0 + 2023-07-05T12:51:12Z + SO: + + F + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:55Z + + E‐Luc + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + 0.98908633 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + + RESULTS + title_2 + 20306 + Ligand‐specific control of GREB1 expression + + 0.99805826 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + + RESULTS + paragraph + 20352 + To determine whether ligand classes control expression of native ERα target genes through the canonical linear signaling pathway, we performed pairwise linear regression analyses using ERα–NCOA1/2/3 interactions in M2H assay as independent predictors of GREB1 expression (the dependent variable) (Figs EV1 and EV2A, F–H). In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). The average induction of GREB1 by cluster 1 ligands showed greater variance, with a range between ~25 and ~75% for OBHS and a range from full agonist to inverse agonist for the others in cluster 1 (Fig EV2A). GREB1 levels induced by OBHS analogs were determined by recruitment of NCOA1 but not NCOA2/3 (Fig 3E lane 1), suggesting that there may be alternate or preferential use of these coactivators by different classes. However, in cluster 1, NCOA1/2/3 recruitment generally predicted GREB1 levels (Fig 3E lanes 1–4), consistent with the canonical signaling model (Fig 1D). + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + 0.98062885 + experimental_method + cleaner0 + 2023-07-05T15:12:56Z + MESH: + + pairwise linear regression analyses + + + 0.96621025 + complex_assembly + cleaner0 + 2023-07-05T13:34:55Z + GO: + + ERα–NCOA1/2/3 + + + 0.9850155 + experimental_method + cleaner0 + 2023-07-05T15:12:59Z + MESH: + + M2H assay + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + 0.9985461 + protein + cleaner0 + 2023-07-05T13:49:56Z + PR: + + NCOA1 + + + 0.9983197 + protein + cleaner0 + 2023-07-05T12:49:11Z + PR: + + NCOA2 + + + 0.99309796 + chemical + cleaner0 + 2023-07-05T13:04:18Z + CHEBI: + + WAY‐C + + + 0.9910898 + chemical + cleaner0 + 2023-07-05T13:08:31Z + CHEBI: + + triaryl‐ethylene + + + 0.9588286 + chemical + cleaner0 + 2023-07-05T13:02:41Z + CHEBI: + + OBHS‐N + + + 0.6048767 + chemical + cleaner0 + 2023-07-05T13:03:37Z + CHEBI: + + OBHS + + + 0.9985275 + protein + cleaner0 + 2023-07-05T13:49:59Z + PR: + + NCOA3 + + + 0.93739086 + chemical + cleaner0 + 2023-07-05T13:02:41Z + CHEBI: + + OBHS‐N + + + 0.8894773 + chemical + cleaner0 + 2023-07-05T13:03:37Z + CHEBI: + + OBHS + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + 0.97384655 + chemical + cleaner0 + 2023-07-05T13:03:37Z + CHEBI: + + OBHS + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + 0.97890204 + chemical + cleaner0 + 2023-07-05T13:03:37Z + CHEBI: + + OBHS + + + 0.9983368 + protein + cleaner0 + 2023-07-05T13:50:03Z + PR: + + NCOA1 + + + 0.8513415 + protein + cleaner0 + 2023-07-05T13:50:07Z + PR: + + NCOA2/3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + + RESULTS + paragraph + 21517 + For clusters 2 and 3, GREB1 activity was generally not predicted by NCOA1/2/3 recruitment. Direct modulators showed low NCOA1/2/3 recruitment (Fig EV2F–H), but only OBHS‐ASC analogs had NCOA2 recruitment profiles that predicted a full range of effects on GREB1 levels (Figs 3E lanes 9, 11, 18–19, and EV2A). The indirect modulators in clusters 2 and 3 stimulated NCOA1/2/3 recruitment and GREB1 expression with substantial variance (Figs 3A and EV2F–H). However, ligand‐induced GREB1 levels were generally not determined by NCOA1/2/3 recruitment (Fig 3E lanes 5–19), consistent with an alternate causality model (Fig 1E). Out of 11 indirect modulator series in cluster 2 or 3, only the S‐OBHS‐3 class had NCOA1/2/3 recruitment profiles that predicted GREB1 levels (Fig 3E lane 12). These results suggest that compounds that show cell‐specific signaling do not activate GREB1, or use coactivators other than NCOA1/2/3 to control GREB1 expression (Fig 1E). + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + 0.93991566 + chemical + cleaner0 + 2023-07-05T13:02:09Z + CHEBI: + + OBHS‐ASC + + + protein + PR: + cleaner0 + 2023-07-05T12:49:11Z + + NCOA2 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:13:51Z + + S‐OBHS‐3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + 0.92096794 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + 0.66645485 + protein + cleaner0 + 2023-07-05T12:39:46Z + PR: + + NCOA1/2/3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + + RESULTS + title_2 + 22497 + Ligand‐specific control of cell proliferation + + + RESULTS + paragraph + 22545 + To determine mechanisms for ligand‐dependent control of breast cancer cell proliferation, we performed linear regression analyses across the 19 scaffolds using MCF‐7 cell proliferation as the dependent variable, and the other activities as independent variables (Fig 3F). In cluster 1, E‐Luc and L‐Luc activities, NCOA1/2/3 recruitment, and GREB1 levels generally predicted the proliferative response (Fig 3F lanes 2–4). With the OBHS‐N compounds, NCOA3 and GREB1 showed near perfect prediction of proliferation (Fig EV3G), with unexplained variance similar to the noise in the assays. The lack of significant predictors for OBHS analogs (Fig 3F lane 1) reflects their small range of proliferative effects on MCF‐7 cells (Fig EV2I). The significant correlations with GREB1 expression and NCOA1/2/3 recruitment observed in this cluster are consistent with the canonical signaling model (Fig 1D), where NCOA1/2/3 recruitment determines GREB1 expression, which then drives proliferation. + + 0.98775655 + experimental_method + cleaner0 + 2023-07-05T15:13:05Z + MESH: + + linear regression analyses + + + 0.8465802 + experimental_method + cleaner0 + 2023-07-05T12:54:55Z + MESH: + + E‐Luc + + + 0.81952065 + experimental_method + cleaner0 + 2023-07-05T12:54:31Z + MESH: + + L‐Luc + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + 0.94380665 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:02:41Z + + OBHS‐N + + + 0.99678516 + protein + cleaner0 + 2023-07-05T13:50:11Z + PR: + + NCOA3 + + + 0.99611676 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:03:38Z + + OBHS + + + 0.9898878 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + 0.9906052 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + + RESULTS + paragraph + 23550 + Ligands in cluster 2 and cluster 3 showed a wide range of proliferative effects on MCF‐7 cells (Fig EV2I). Despite this phenotypic variance, proliferation was not generally predicted by correlated NCOA1/2/3 recruitment and GREB1 induction (Figs 3F lanes 5–19, and EV3H). Out of 15 ligand series in these clusters, only 2,5‐DTP analogs induced a proliferative response that was predicted by GREB1 levels, which were not determined by NCOA1/2/3 recruitment (Fig 3E and F lane 10). 3,4‐DTP, cyclofenil, 3,4‐DTPD, and imidazopyridine analogs had NCOA1/3 recruitment profiles that predicted their proliferative effects, without determining GREB1 levels (Fig 3E and F, lanes 5 and 14–16). Similarly, S‐OBHS‐3, cyclofenil‐ASC, and OBHS‐ASC had positively correlated NCOA1/2/3 recruitment and GREB1 levels, but none of these activities determined their proliferative effects (Fig 3E and F lanes 11–12 and 18). For ligands that show cell‐specific signaling, ERα‐mediated recruitment of other coregulators and activation of other target genes likely determine their proliferative effects on MCF‐7 cells. + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + 0.99721795 + chemical + cleaner0 + 2023-07-05T14:02:03Z + CHEBI: + + 2,5‐DTP + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + 0.9969743 + chemical + cleaner0 + 2023-07-05T14:02:06Z + CHEBI: + + 3,4‐DTP + + + 0.99792635 + chemical + cleaner0 + 2023-07-05T13:06:01Z + CHEBI: + + cyclofenil + + + 0.99604523 + chemical + cleaner0 + 2023-07-05T13:05:49Z + CHEBI: + + 3,4‐DTPD + + + 0.97465694 + chemical + cleaner0 + 2023-07-05T13:06:07Z + CHEBI: + + imidazopyridine + + + protein + PR: + cleaner0 + 2023-07-05T15:13:20Z + + NCOA1/3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + 0.8801133 + chemical + cleaner0 + 2023-07-05T13:13:51Z + CHEBI: + + S‐OBHS‐3 + + + 0.9819379 + chemical + cleaner0 + 2023-07-05T13:06:20Z + CHEBI: + + cyclofenil‐ASC + + + 0.92578393 + chemical + cleaner0 + 2023-07-05T13:02:09Z + CHEBI: + + OBHS‐ASC + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + 0.9874334 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + + RESULTS + title_2 + 24678 + NCOA3 occupancy at GREB1 did not predict the proliferative response + + 0.99655735 + protein + cleaner0 + 2023-07-05T13:50:15Z + PR: + + NCOA3 + + + 0.8558061 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + + RESULTS + paragraph + 24746 + We also questioned whether promoter occupancy by coactivators is statistically robust and reproducible for ligand class analysis using a chromatin immunoprecipitation (ChIP)‐based quantitative assay, and whether it has a better predictive power than the M2H assay. ERα and NCOA3 cycle on and off the GREB1 promoter (Nwachukwu et al, 2014). Therefore, we first performed a time‐course study, and found that E2 and the WAY‐C analog, AAPII‐151‐4, induced recruitment of NCOA3 to the GREB1 promoter in a temporal cycle that peaked after 45 min in MCF‐7 cells (Fig 4A). At this time point, other WAY‐C analogs also induced recruitment of NCOA3 at this site to varying degrees (Fig 4B). The Z’ for this assay was 0.6, showing statistical robustness (see Materials and Methods). We prepared biological replicates with different cell passage numbers and separately prepared samples, which showed r 2 of 0.81, demonstrating high reproducibility (Fig 4C). + + experimental_method + MESH: + cleaner0 + 2023-07-05T15:13:54Z + + chromatin immunoprecipitation (ChIP)‐based quantitative assay, + + + 0.8732414 + experimental_method + cleaner0 + 2023-07-05T15:13:58Z + MESH: + + M2H assay + + + 0.99798834 + protein + cleaner0 + 2023-07-05T12:38:59Z + PR: + + ERα + + + 0.9980611 + protein + cleaner0 + 2023-07-05T13:50:21Z + PR: + + NCOA3 + + + 0.9242139 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + 0.91339153 + experimental_method + cleaner0 + 2023-07-05T15:14:01Z + MESH: + + time‐course study + + + 0.9979705 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:04:18Z + + WAY‐C + + + 0.97044486 + chemical + cleaner0 + 2023-07-05T14:02:11Z + CHEBI: + + AAPII‐151‐4 + + + 0.9977604 + protein + cleaner0 + 2023-07-05T13:50:24Z + PR: + + NCOA3 + + + 0.8418753 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:04:18Z + + WAY‐C + + + 0.9977362 + protein + cleaner0 + 2023-07-05T13:50:26Z + PR: + + NCOA3 + + + 0.9782229 + evidence + cleaner0 + 2023-07-05T14:09:08Z + DUMMY: + + Z’ + + + 0.9696659 + evidence + cleaner0 + 2023-07-05T14:09:11Z + DUMMY: + + r 2 + + + + MSB-12-864-g008.jpg + msb156701-fig-0004 + FIG + fig_title_caption + 25714 + +NCOA3 occupancy at GREB1 is statistically robust but does not predict transcriptional activity + + 0.9965205 + protein + cleaner0 + 2023-07-05T13:50:31Z + PR: + + NCOA3 + + + 0.7577269 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + + MSB-12-864-g008.jpg + msb156701-fig-0004 + FIG + fig_caption + 25810 + Kinetic ChIP assay examining recruitment of NCOA3 to the GREB1 gene in MCF‐7 cells stimulated with E2 or the indicated WAY‐C analog. The average of duplicate experiments (mean ± SEM) is shown. + + 0.9934087 + experimental_method + cleaner0 + 2023-07-05T15:14:10Z + MESH: + + Kinetic ChIP assay + + + 0.9978503 + protein + cleaner0 + 2023-07-05T13:50:33Z + PR: + + NCOA3 + + + 0.75698066 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + 0.99739885 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:04:18Z + + WAY‐C + + + + MSB-12-864-g008.jpg + msb156701-fig-0004 + FIG + fig_caption + 26010 + NCOA3 occupancy at GREB1 was compared by ChIP assay 45 min after stimulation with vehicle, E2, or the WAY‐C analogs. In panel (B), the average recruitment of two biological replicates are shown as mean + SEM, and the Z‐score is indicated. In panel (C), correlation analysis was performed for two biological replicates. + + 0.9935454 + protein + cleaner0 + 2023-07-05T13:50:36Z + PR: + + NCOA3 + + + 0.5045772 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + 0.99326444 + experimental_method + cleaner0 + 2023-07-05T15:14:13Z + MESH: + + ChIP assay + + + 0.99746776 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:04:18Z + + WAY‐C + + + evidence + DUMMY: + cleaner0 + 2023-07-05T13:25:46Z + + Z‐score + + + 0.85250723 + experimental_method + cleaner0 + 2023-07-05T15:14:17Z + MESH: + + correlation analysis + + + + MSB-12-864-g008.jpg + msb156701-fig-0004 + FIG + fig_caption + 26336 + Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY‐C analogs. *Significant positive correlation (F‐test for nonzero slope, P‐value). + + 0.8993063 + experimental_method + cleaner0 + 2023-07-05T15:14:21Z + MESH: + + Linear regression analyses + + + 0.9973508 + protein + cleaner0 + 2023-07-05T13:50:42Z + PR: + + NCOA3 + + + 0.9898379 + experimental_method + cleaner0 + 2023-07-05T15:14:23Z + MESH: + + ChIP + + + 0.9818325 + experimental_method + cleaner0 + 2023-07-05T15:14:26Z + MESH: + + M2H + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:04:18Z + + WAY‐C + + + experimental_method + MESH: + cleaner0 + 2023-07-05T13:20:04Z + + F‐test + + + evidence + DUMMY: + cleaner0 + 2023-07-05T13:26:05Z + + P‐value + + + + MSB-12-864-g008.jpg + msb156701-fig-0004 + FIG + fig_caption + 26563 + +Source data are available online for this figure. + + + + RESULTS + paragraph + 26615 + The M2H assay for NCOA3 recruitment broadly correlated with the other assays, and was predictive for GREB1 expression and cell proliferation (Fig 3E). However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). Thus, the simplified coactivator‐binding assay showed much greater predictive power than the ChIP assay for ligand‐specific effects on GREB1 expression and cell proliferation. + + 0.98578024 + experimental_method + cleaner0 + 2023-07-05T15:14:29Z + MESH: + + M2H assay + + + 0.99369276 + protein + cleaner0 + 2023-07-05T13:50:45Z + PR: + + NCOA3 + + + 0.96231395 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + 0.9859141 + experimental_method + cleaner0 + 2023-07-05T15:14:32Z + MESH: + + ChIP assays + + + 0.974938 + chemical + cleaner0 + 2023-07-05T13:04:18Z + CHEBI: + + WAY‐C + + + 0.997054 + protein + cleaner0 + 2023-07-05T13:50:48Z + PR: + + NCOA3 + + + 0.90827537 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + 0.92724705 + chemical + cleaner0 + 2023-07-05T13:04:19Z + CHEBI: + + WAY‐C + + + 0.9725241 + experimental_method + cleaner0 + 2023-07-05T15:14:35Z + MESH: + + ChIP assays + + + 0.99389195 + protein + cleaner0 + 2023-07-05T13:50:50Z + PR: + + NCOA3 + + + 0.98268086 + experimental_method + cleaner0 + 2023-07-05T15:14:38Z + MESH: + + M2H assay + + + evidence + DUMMY: + cleaner0 + 2023-07-05T13:25:29Z + + r 2 + + + evidence + DUMMY: + cleaner0 + 2023-07-05T13:26:18Z + + P + + + experimental_method + MESH: + cleaner0 + 2023-07-05T13:19:37Z + + F‐test + + + 0.97863764 + experimental_method + cleaner0 + 2023-07-05T15:14:43Z + MESH: + + coactivator‐binding assay + + + 0.96783495 + experimental_method + cleaner0 + 2023-07-05T15:14:45Z + MESH: + + ChIP assay + + + 0.95587784 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + + RESULTS + title_2 + 27302 + ERβ activity is not an independent predictor of cell‐specific activity + + 0.933958 + protein + cleaner0 + 2023-07-05T13:49:12Z + PR: + + ERβ + + + + RESULTS + paragraph + 27376 + One difference between MCF‐7 breast cancer cells and Ishikawa endometrial cancer cells is the contribution of ERβ to estrogenic response, as Ishikawa cells may express ERβ (Bhat & Pezzuto, 2001). When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). + + 0.8390067 + protein + cleaner0 + 2023-07-05T13:50:58Z + PR: + + ERβ + + + 0.9147357 + protein + cleaner0 + 2023-07-05T13:51:03Z + PR: + + ERβ + + + 0.98094374 + experimental_method + cleaner0 + 2023-07-05T15:14:50Z + MESH: + + overexpressed + + + 0.9563717 + protein + cleaner0 + 2023-07-05T13:51:05Z + PR: + + ERβ + + + 0.9714489 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + 0.44195107 + protein + cleaner0 + 2023-07-05T13:49:21Z + PR: + + ERβ + + + 0.88420296 + experimental_method + cleaner0 + 2023-07-05T12:54:55Z + MESH: + + E‐Luc + + + 0.95827705 + experimental_method + cleaner0 + 2023-07-05T12:54:55Z + MESH: + + E‐Luc + + + 0.9432032 + experimental_method + cleaner0 + 2023-07-05T12:54:31Z + MESH: + + L‐Luc + + + protein + PR: + cleaner0 + 2023-07-05T12:38:59Z + + ERα + + + 0.7737816 + protein_state + cleaner0 + 2023-07-05T12:54:01Z + DUMMY: + + WT + + + 0.96682733 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + + RESULTS + paragraph + 27993 + To test this idea, we determined the L‐Luc ERβ activity profiles of the ligands (Fig EV1). All direct modulator and two indirect modulator scaffolds (OBHS and S‐OBHS‐3) lacked ERβ agonist activity. However, the other ligands showed a range of ERβ activities (Fig EV2J). For most scaffolds, L‐Luc ERβ and E‐Luc activities were not correlated, except for 2,5‐DTP and cyclofenil analogs, which showed moderate but significant correlations (Fig EV4A). Nevertheless, the E‐Luc activities of both 2,5‐DTP and cyclofenil analogs were better predicted by their L‐Luc ERα‐WT than L‐Luc ERβ activities (Fig EV4A and B). Thus, ERβ activity was not an independent determinant of the observed activity profiles. + + 0.85501605 + experimental_method + cleaner0 + 2023-07-05T12:54:31Z + MESH: + + L‐Luc + + + 0.9511798 + chemical + cleaner0 + 2023-07-05T13:03:38Z + CHEBI: + + OBHS + + + 0.80254567 + chemical + cleaner0 + 2023-07-05T13:13:51Z + CHEBI: + + S‐OBHS‐3 + + + 0.7573789 + experimental_method + cleaner0 + 2023-07-05T12:54:31Z + MESH: + + L‐Luc + + + 0.39837897 + experimental_method + cleaner0 + 2023-07-05T12:54:56Z + MESH: + + E‐Luc + + + 0.997215 + chemical + cleaner0 + 2023-07-05T14:02:18Z + CHEBI: + + 2,5‐DTP + + + 0.9970463 + chemical + cleaner0 + 2023-07-05T13:06:01Z + CHEBI: + + cyclofenil + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:56Z + + E‐Luc + + + 0.9972227 + chemical + cleaner0 + 2023-07-05T14:02:21Z + CHEBI: + + 2,5‐DTP + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:06:01Z + + cyclofenil + + + 0.74976367 + experimental_method + cleaner0 + 2023-07-05T12:54:31Z + MESH: + + L‐Luc + + + 0.60933095 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T12:54:01Z + + WT + + + 0.7702003 + experimental_method + cleaner0 + 2023-07-05T12:54:31Z + MESH: + + L‐Luc + + + 0.57141453 + protein + cleaner0 + 2023-07-05T13:51:13Z + PR: + + ERβ + + + + MSB-12-864-g009.jpg + msb156701-fig-0004ev + FIG + fig_title_caption + 28725 + ERβ activity is not an independent predictor of E‐Luc activity + + 0.5572566 + protein + cleaner0 + 2023-07-05T13:49:32Z + PR: + + ERβ + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:56Z + + E‐Luc + + + + MSB-12-864-g009.jpg + msb156701-fig-0004ev + FIG + fig_caption + 28791 + ERβ activity in HepG2 cells rarely correlates with E‐Luc activity. + + 0.85604817 + protein + cleaner0 + 2023-07-05T13:49:40Z + PR: + + ERβ + + + 0.7764904 + experimental_method + cleaner0 + 2023-07-05T12:54:56Z + MESH: + + E‐Luc + + + + MSB-12-864-g009.jpg + msb156701-fig-0004ev + FIG + fig_caption + 28861 + ERα activity of 2,5‐DTP and cyclofenil analogs correlates with E‐Luc activity. + + protein + PR: + cleaner0 + 2023-07-05T12:39:00Z + + ERα + + + 0.99681073 + chemical + cleaner0 + 2023-07-05T14:02:26Z + CHEBI: + + 2,5‐DTP + + + 0.9913715 + chemical + cleaner0 + 2023-07-05T13:06:01Z + CHEBI: + + cyclofenil + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:54:56Z + + E‐Luc + + + + MSB-12-864-g009.jpg + msb156701-fig-0004ev + FIG + fig_caption + 28945 + +Data information: The r +2 and P values for the indicated correlations are shown in both panels. *Significant positive correlation (F‐test for nonzero slope, P‐value) + + 0.9492532 + evidence + cleaner0 + 2023-07-05T14:09:16Z + DUMMY: + + r +2 + + + evidence + DUMMY: + cleaner0 + 2023-07-05T14:09:36Z + + P values + + + experimental_method + MESH: + cleaner0 + 2023-07-05T13:20:04Z + + F‐test + + + evidence + DUMMY: + cleaner0 + 2023-07-05T13:26:06Z + + P‐value + + + + RESULTS + title_2 + 29116 + Structural features of consistent signaling across cell types + + + RESULTS + paragraph + 29178 + To overcome barriers to crystallization of ERα LBD complexes, we developed a conformation‐trapping X‐ray crystallography approach using the ERα‐Y537S mutation (Nettles et al, 2008; Bruning et al, 2010; Srinivasan et al, 2013). To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). Using this approach, we solved 76 ERα LBD structures in the active conformation and bound to ligands studied here (Appendix Fig S1C). Eleven of these structures have been published, while 65 are new, including the DES‐bound ERα‐Y537S LBD. We present 57 of these new structures here (Dataset EV2), while the remaining eight new structures bound to OBHS‐N analogs will be published elsewhere (S. Srinivasan et al, in preparation). Examining many closely related structures allows us to visualize subtle structural differences, in effect using X‐ray crystallography as a systems biology tool. + + 0.9824072 + experimental_method + cleaner0 + 2023-07-05T15:14:56Z + MESH: + + crystallization + + + 0.99178946 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.6792933 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.99458843 + experimental_method + cleaner0 + 2023-07-05T15:15:00Z + MESH: + + conformation‐trapping X‐ray crystallography + + + 0.9950919 + mutant + cleaner0 + 2023-07-05T13:07:41Z + MESH: + + ERα‐Y537S + + + 0.9639338 + experimental_method + cleaner0 + 2023-07-05T15:15:03Z + MESH: + + solved + + + 0.9943229 + evidence + cleaner0 + 2023-07-05T14:09:54Z + DUMMY: + + structure + + + 0.9968536 + mutant + cleaner0 + 2023-07-05T13:07:42Z + MESH: + + ERα‐Y537S + + + 0.98712665 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.9900389 + protein_state + cleaner0 + 2023-07-05T15:19:05Z + DUMMY: + + in complex with + + + 0.9972697 + chemical + cleaner0 + 2023-07-05T14:02:32Z + CHEBI: + + diethylstilbestrol + + + 0.9976993 + chemical + cleaner0 + 2023-07-05T14:02:35Z + CHEBI: + + DES + + + 0.9972461 + protein_state + cleaner0 + 2023-07-05T12:52:36Z + DUMMY: + + wild‐type + + + 0.9963813 + mutant + cleaner0 + 2023-07-05T13:07:42Z + MESH: + + ERα‐Y537S + + + 0.97803456 + structure_element + cleaner0 + 2023-07-05T15:07:21Z + SO: + + LBDs + + + 0.7712057 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.92050976 + experimental_method + cleaner0 + 2023-07-05T15:15:07Z + MESH: + + solved + + + 0.99171996 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.9457268 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.99098295 + evidence + cleaner0 + 2023-07-05T14:10:00Z + DUMMY: + + structures + + + 0.9952619 + protein_state + cleaner0 + 2023-07-05T15:19:17Z + DUMMY: + + active conformation + + + 0.8746801 + protein_state + cleaner0 + 2023-07-05T15:19:20Z + DUMMY: + + bound to ligands + + + 0.98621434 + evidence + cleaner0 + 2023-07-05T14:10:02Z + DUMMY: + + structures + + + 0.9961564 + protein_state + cleaner0 + 2023-07-05T15:19:22Z + DUMMY: + + DES‐bound + + + 0.996548 + mutant + cleaner0 + 2023-07-05T13:07:42Z + MESH: + + ERα‐Y537S + + + 0.9871116 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.991763 + evidence + cleaner0 + 2023-07-05T14:10:08Z + DUMMY: + + structures + + + 0.9558419 + evidence + cleaner0 + 2023-07-05T14:10:11Z + DUMMY: + + structures + + + 0.98350334 + protein_state + cleaner0 + 2023-07-05T15:19:25Z + DUMMY: + + bound to + + + 0.9924657 + chemical + cleaner0 + 2023-07-05T13:02:41Z + CHEBI: + + OBHS‐N + + + 0.99455065 + evidence + cleaner0 + 2023-07-05T14:10:18Z + DUMMY: + + structures + + + 0.9958229 + experimental_method + cleaner0 + 2023-07-05T15:15:24Z + MESH: + + X‐ray crystallography + + + + RESULTS + paragraph + 30436 + The indirect modulator scaffolds in cluster 1 did not show cell‐specific signaling (Fig 3C), but shared common structural perturbations that we designed to modulate h12 dynamics. Based on our original OBHS structure, the OBHS, OBHS‐N, and triaryl‐ethylene compounds were modified with h11‐directed pendant groups (Zheng et al, 2012; Zhu et al, 2012; Liao et al, 2014). Superposing the LBDs based on the class of bound ligands provides an ensemble view of the structural variance and clarifies what part of the ligand‐binding pocket is differentially perturbed or targeted. + + 0.7064927 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.76004595 + chemical + cleaner0 + 2023-07-05T13:03:38Z + CHEBI: + + OBHS + + + 0.9969669 + evidence + cleaner0 + 2023-07-05T14:10:40Z + DUMMY: + + structure + + + 0.9129474 + chemical + cleaner0 + 2023-07-05T13:03:38Z + CHEBI: + + OBHS + + + 0.9696711 + chemical + cleaner0 + 2023-07-05T13:02:41Z + CHEBI: + + OBHS‐N + + + 0.9701834 + chemical + cleaner0 + 2023-07-05T13:08:30Z + CHEBI: + + triaryl‐ethylene + + + structure_element + SO: + cleaner0 + 2023-07-05T12:48:36Z + + h11 + + + 0.99451905 + experimental_method + cleaner0 + 2023-07-05T15:15:29Z + MESH: + + Superposing + + + 0.9976539 + structure_element + cleaner0 + 2023-07-05T15:07:25Z + SO: + + LBDs + + + 0.997629 + site + cleaner0 + 2023-07-05T15:09:45Z + SO: + + ligand‐binding pocket + + + + RESULTS + paragraph + 31023 + The 24 structures containing OBHS, OBHS‐N, or triaryl‐ethylene analogs showed structural diversity in the same part of the scaffolds (Figs 5A and EV5A), and the same region of the LBD—the C‐terminal end of h11 (Figs 5B and C, and EV5B), which in turn nudges h12 (Fig 5C and D). We observed that the OBHS‐N analogs displaced h11 along a vector away from Leu354 in a region of h3 that is unaffected by the ligands, and toward the dimer interface. For the triaryl‐ethylene analogs, the displacement of h11 was in a perpendicular direction, away from Ile424 in h8 and toward h12. Remarkably, these individual inter‐atomic distances showed a ligand class‐specific ability to significantly predict proliferative effects (Fig 5E and F), demonstrating the feasibility of developing a minimal set of activity predictors from crystal structures. + + 0.9963756 + evidence + cleaner0 + 2023-07-05T15:02:47Z + DUMMY: + + structures + + + 0.96527314 + chemical + cleaner0 + 2023-07-05T13:03:38Z + CHEBI: + + OBHS + + + 0.8041764 + chemical + cleaner0 + 2023-07-05T13:02:41Z + CHEBI: + + OBHS‐N + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:08:31Z + + triaryl‐ethylene + + + 0.99776983 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.949963 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + 0.8083586 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:02:41Z + + OBHS‐N + + + 0.9745204 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + 0.99872535 + residue_name_number + cleaner0 + 2023-07-05T14:05:46Z + DUMMY: + + Leu354 + + + 0.8633965 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + 0.9823445 + site + cleaner0 + 2023-07-05T15:09:51Z + SO: + + dimer interface + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:08:31Z + + triaryl‐ethylene + + + 0.9856156 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + 0.9987231 + residue_name_number + cleaner0 + 2023-07-05T14:05:49Z + DUMMY: + + Ile424 + + + 0.8637221 + structure_element + cleaner0 + 2023-07-05T13:09:13Z + SO: + + h8 + + + 0.4989166 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.9894757 + evidence + cleaner0 + 2023-07-05T15:03:07Z + DUMMY: + + inter‐atomic distances + + + 0.9974425 + evidence + cleaner0 + 2023-07-05T15:03:11Z + DUMMY: + + crystal structures + + + + MSB-12-864-g010.jpg + msb156701-fig-0005 + FIG + fig_title_caption + 31879 + Structural determinants of consistent signaling + + + MSB-12-864-g010.jpg + msb156701-fig-0005 + FIG + fig_caption + 31927 + Structure‐class analysis of triaryl‐ethylene analogs. Triaryl‐ethylene analogs bound to the superposed crystal structures of the ERα LBD are shown. Arrows indicate chemical variance in the orientation of the different h11‐directed ligand side groups (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF and 5DP0). + + 0.99104416 + experimental_method + cleaner0 + 2023-07-05T15:15:36Z + MESH: + + Structure‐class analysis + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:08:31Z + + triaryl‐ethylene + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:08:31Z + + Triaryl‐ethylene + + + 0.9835514 + protein_state + cleaner0 + 2023-07-05T15:19:30Z + DUMMY: + + bound to + + + 0.99520504 + experimental_method + cleaner0 + 2023-07-05T15:15:39Z + MESH: + + superposed + + + 0.9967205 + evidence + cleaner0 + 2023-07-05T15:03:19Z + DUMMY: + + crystal structures + + + 0.9971552 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.9955042 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.3932451 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + + MSB-12-864-g010.jpg + msb156701-fig-0005 + FIG + fig_caption + 32255 + Triaryl‐ethylene analogs induce variance of ERα conformations at the C‐terminal region of h11. Panel (B) shows the crystal structure of a triaryl‐ethylene analog‐bound ERα LBD (PDB 5DLR). The h11–h12 interface (circled) includes the C‐terminal part of h11. This region was expanded in panel (C), where the 10 triaryl‐ethylene analog‐bound ERα LBD structures (see Datasets EV1 and EV2) were superposed to show variations in the h11 C‐terminus (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, and 5DP0). + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:08:31Z + + Triaryl‐ethylene + + + 0.99039906 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.9382732 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + 0.9974959 + evidence + cleaner0 + 2023-07-05T15:03:25Z + DUMMY: + + crystal structure + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:08:31Z + + triaryl‐ethylene + + + 0.99623007 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.9846935 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.99638605 + site + cleaner0 + 2023-07-05T15:09:56Z + SO: + + h11–h12 interface + + + 0.96403486 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:08:31Z + + triaryl‐ethylene + + + 0.99468887 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.9621993 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.99664575 + evidence + cleaner0 + 2023-07-05T15:03:29Z + DUMMY: + + structures + + + 0.9946602 + experimental_method + cleaner0 + 2023-07-05T15:15:43Z + MESH: + + superposed + + + 0.9513391 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + + MSB-12-864-g010.jpg + msb156701-fig-0005 + FIG + fig_caption + 32789 + ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC). + + 0.9905766 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.98728615 + structure_element + cleaner0 + 2023-07-05T15:07:31Z + SO: + + LBDs + + + 0.95576185 + protein_state + cleaner0 + 2023-07-05T15:19:33Z + DUMMY: + + in complex with + + + 0.9977882 + chemical + cleaner0 + 2023-07-05T14:02:40Z + CHEBI: + + diethylstilbestrol + + + 0.9981653 + chemical + cleaner0 + 2023-07-05T14:02:42Z + CHEBI: + + DES + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:08:31Z + + triaryl‐ethylene + + + 0.9955011 + experimental_method + cleaner0 + 2023-07-05T15:15:47Z + MESH: + + superposed + + + 0.9965508 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + 0.9915627 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + + MSB-12-864-g010.jpg + msb156701-fig-0005 + FIG + fig_caption + 33012 + Inter‐atomic distances predict the proliferative effects of specific ligand series. Ile424–His524 distance measured in the crystal structures correlates with the proliferative effect of triaryl‐ethylene analogs in MCF‐7 cells. In contrast, the Leu354–Leu525 distance correlates with the proliferative effects of OBHS‐N analogs in MCF‐7 cells. + + 0.9898077 + evidence + cleaner0 + 2023-07-05T15:03:38Z + DUMMY: + + Inter‐atomic distances + + + 0.98629063 + residue_name_number + cleaner0 + 2023-07-05T14:05:53Z + DUMMY: + + Ile424 + + + 0.73622644 + residue_name_number + cleaner0 + 2023-07-05T13:11:24Z + DUMMY: + + His524 + + + evidence + DUMMY: + cleaner0 + 2023-07-05T13:11:37Z + + distance + + + 0.9973312 + evidence + cleaner0 + 2023-07-05T15:03:49Z + DUMMY: + + crystal structures + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:08:31Z + + triaryl‐ethylene + + + 0.9721175 + residue_name_number + cleaner0 + 2023-07-05T14:06:04Z + DUMMY: + + Leu354 + + + 0.64800006 + residue_name_number + cleaner0 + 2023-07-05T13:11:08Z + DUMMY: + + Leu525 + + + evidence + DUMMY: + cleaner0 + 2023-07-05T13:11:46Z + + distance + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:02:41Z + + OBHS‐N + + + + MSB-12-864-g010.jpg + msb156701-fig-0005 + FIG + fig_caption + 33369 + Structure‐class analysis of WAY‐C analogs. WAY‐C side groups subtly nudge h12 Leu540. ERα LBD structures bound to 4 distinct WAY‐C analogs were superposed (PDB 4 IU7, 4IV4, 4IVW, 4IW6) (see Datasets EV1 and EV2). + + 0.984905 + experimental_method + cleaner0 + 2023-07-05T15:15:52Z + MESH: + + Structure‐class analysis + + + 0.99775034 + chemical + cleaner0 + 2023-07-05T13:04:19Z + CHEBI: + + WAY‐C + + + 0.9974684 + chemical + cleaner0 + 2023-07-05T13:04:19Z + CHEBI: + + WAY‐C + + + 0.6735084 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.99860686 + residue_name_number + cleaner0 + 2023-07-05T14:06:11Z + DUMMY: + + Leu540 + + + 0.99446446 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.97007364 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.99538004 + evidence + cleaner0 + 2023-07-05T15:04:00Z + DUMMY: + + structures + + + 0.9724702 + protein_state + cleaner0 + 2023-07-05T15:19:37Z + DUMMY: + + bound to + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:04:19Z + + WAY‐C + + + 0.9926271 + experimental_method + cleaner0 + 2023-07-05T15:15:54Z + MESH: + + superposed + + + + MSB-12-864-g010.jpg + msb156701-fig-0005 + FIG + fig_caption + 33592 + +Source data are available online for this figure. + + + + MSB-12-864-g011.jpg + msb156701-fig-0005ev + FIG + fig_title_caption + 33644 + Structure‐class analysis of indirect modulators + + 0.96463877 + experimental_method + cleaner0 + 2023-07-05T15:15:58Z + MESH: + + Structure‐class analysis + + + + MSB-12-864-g011.jpg + msb156701-fig-0005ev + FIG + fig_caption + 33694 + Structure‐class analysis of indirect modulators in cluster 1. Crystal structures of the ERα LBD bound to OBHS and OBHS‐N analogs were superposed. The bound ligands are shown in panel (A). Arrows indicate chemical variance in the orientation of the different h11‐directed ligand side groups. Panel (B) shows the ligand‐induced conformational variation at the C‐terminal region of h11 (OBHS: PDB 4ZN9, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, and 4ZNW; OBHS‐N: PDB 4ZUB, 4ZUC, 4ZWH, 4ZWK, 5BNU, 5BP6, 5BPR, and 5BQ4). + + 0.9879625 + experimental_method + cleaner0 + 2023-07-05T15:16:01Z + MESH: + + Structure‐class analysis + + + 0.99701476 + evidence + cleaner0 + 2023-07-05T15:04:05Z + DUMMY: + + Crystal structures + + + 0.99095917 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.98924434 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.9945032 + protein_state + cleaner0 + 2023-07-05T15:19:42Z + DUMMY: + + bound to + + + 0.80187863 + chemical + cleaner0 + 2023-07-05T13:03:38Z + CHEBI: + + OBHS + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:02:41Z + + OBHS‐N + + + 0.9949473 + experimental_method + cleaner0 + 2023-07-05T15:16:04Z + MESH: + + superposed + + + 0.30305144 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + 0.88866675 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + 0.98454535 + chemical + cleaner0 + 2023-07-05T13:03:38Z + CHEBI: + + OBHS + + + 0.88782674 + chemical + cleaner0 + 2023-07-05T13:02:41Z + CHEBI: + + OBHS‐N + + + + MSB-12-864-g011.jpg + msb156701-fig-0005ev + FIG + fig_caption + 34213 + Structure‐class analysis of indirect modulators in clusters 2 and 3. Crystal structures of the ERα LBD bound to ligands with cell‐specific activities were superposed. The bound ligands are shown, and arrows indicate considerable variation in the orientation of the different h3‐, h8‐, h11‐, or h12‐directed ligand side groups. + + 0.9906344 + experimental_method + cleaner0 + 2023-07-05T15:16:07Z + MESH: + + Structure‐class analysis + + + 0.99684536 + evidence + cleaner0 + 2023-07-05T15:04:12Z + DUMMY: + + Crystal structures + + + 0.9966181 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.99585813 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.99386704 + protein_state + cleaner0 + 2023-07-05T15:19:50Z + DUMMY: + + bound to + + + 0.99511755 + experimental_method + cleaner0 + 2023-07-05T15:16:09Z + MESH: + + superposed + + + 0.51302165 + structure_element + cleaner0 + 2023-07-05T13:12:38Z + SO: + + h3 + + + 0.8291484 + structure_element + cleaner0 + 2023-07-05T13:09:14Z + SO: + + h8 + + + 0.81261206 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + 0.56028163 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + + RESULTS + paragraph + 34553 + As visualized in four LBD structures (Srinivasan et al, 2013), WAY‐C analogs were designed with small substitutions that slightly nudge h12 Leu540, without exiting the ligand‐binding pocket (Fig 5G and H). Therefore, changing h12 dynamics maintains the canonical signaling pathway defined by E2 (Fig 1D) to support AF‐2‐driven signaling and recruit NCOA1/2/3 for GREB1‐stimulated proliferation. + + 0.9919625 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.99573463 + evidence + cleaner0 + 2023-07-05T15:04:16Z + DUMMY: + + structures + + + 0.9974811 + chemical + cleaner0 + 2023-07-05T13:04:19Z + CHEBI: + + WAY‐C + + + 0.62945586 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.99864286 + residue_name_number + cleaner0 + 2023-07-05T14:06:16Z + DUMMY: + + Leu540 + + + 0.9961742 + site + cleaner0 + 2023-07-05T15:10:02Z + SO: + + ligand‐binding pocket + + + 0.58697456 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.9964024 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + 0.8013544 + structure_element + cleaner0 + 2023-07-05T12:39:24Z + SO: + + AF‐2 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + 0.9558981 + protein + cleaner0 + 2023-07-05T12:39:52Z + PR: + + GREB1 + + + + RESULTS + title_2 + 34961 + Ligands with cell‐specific activity alter the shape of the AF‐2 surface + + site + SO: + cleaner0 + 2023-07-05T13:15:13Z + + AF‐2 surface + + + + RESULTS + paragraph + 35037 + Direct modulators like tamoxifen drive AF‐1‐dependent cell‐specific activity by completely occluding AF‐2, but it is not known how indirect modulators produce cell‐specific ERα activity. Therefore, we examined another 50 LBD structures containing ligands in clusters 2 and 3. These structures demonstrated that cell‐specific activity derived from altering the shape of the AF‐2 surface without an extended side chain. + + 0.99780315 + chemical + cleaner0 + 2023-07-05T12:38:44Z + CHEBI: + + tamoxifen + + + structure_element + SO: + cleaner0 + 2023-07-05T12:40:26Z + + AF‐1 + + + 0.92056084 + structure_element + cleaner0 + 2023-07-05T12:39:24Z + SO: + + AF‐2 + + + 0.93319297 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.9463632 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.99304503 + evidence + cleaner0 + 2023-07-05T15:04:20Z + DUMMY: + + structures + + + 0.996619 + evidence + cleaner0 + 2023-07-05T15:04:26Z + DUMMY: + + structures + + + site + SO: + cleaner0 + 2023-07-05T13:15:13Z + + AF‐2 surface + + + + RESULTS + paragraph + 35470 + Ligands in cluster 2 and cluster 3 showed conformational heterogeneity in parts of the scaffold that were directed toward multiple regions of the receptor including h3, h8, h11, h12, and/or the β‐sheets (Fig EV5C–G). For instance, S‐OBHS‐2 and S‐OBHS‐3 analogs (Fig 2) had similar ERα activity profiles in the different cell types (Fig EV2A–C), but the 2‐ versus 3‐methyl substituted phenol rings altered the correlated signaling patterns in different cell types (Fig 3B lanes 7 and 12). Structurally, the 2‐ versus 3‐methyl substitutions changed the binding position of the A‐ and E‐ring phenols by 1.0 Å and 2.2 Å, respectively (Fig EV5C). This difference in ligand positioning altered the AF‐2 surface via a shift in the N‐terminus of h12, which directly contacts the coactivator. This effect is evident in a single structure due to its 1 Å magnitude (Fig 6A and B). The shifts in h12 residues Asp538 and Leu539 led to rotation of the coactivator peptide (Fig 6C). Thus, cell‐specific activity can stem from perturbation of the AF‐2 surface without an extended side chain, which presumably alters the receptor–coregulator interaction profile. + + 0.6167863 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + 0.99027514 + structure_element + cleaner0 + 2023-07-05T13:09:14Z + SO: + + h8 + + + 0.9901378 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + 0.5148027 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.99690086 + structure_element + cleaner0 + 2023-07-05T15:07:35Z + SO: + + β‐sheets + + + 0.8626019 + chemical + cleaner0 + 2023-07-05T13:05:14Z + CHEBI: + + S‐OBHS‐2 + + + 0.8850729 + chemical + cleaner0 + 2023-07-05T13:13:50Z + CHEBI: + + S‐OBHS‐3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:00Z + + ERα + + + 0.9642925 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + 0.9710217 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.99724615 + evidence + cleaner0 + 2023-07-05T15:04:34Z + DUMMY: + + structure + + + 0.89150023 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.9988188 + residue_name_number + cleaner0 + 2023-07-05T14:06:19Z + DUMMY: + + Asp538 + + + 0.9988593 + residue_name_number + cleaner0 + 2023-07-05T14:06:22Z + DUMMY: + + Leu539 + + + 0.95563185 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + + MSB-12-864-g012.jpg + msb156701-fig-0006 + FIG + fig_title_caption + 36665 + Structural correlates of cell‐specific signaling + + + MSB-12-864-g012.jpg + msb156701-fig-0006 + FIG + fig_caption + 36716 + S‐OBHS‐2/3 analogs subtly distort the AF‐2 surface. Panel (A) shows the crystal structure of an S‐OBHS‐3‐bound ERα LBD (PDB 5DUH). The h3–h12 interface (circled) at AF‐2 (pink) was expanded in panels (B, C). The S‐OBHS‐2/3‐bound ERα LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF‐2 surface (panel C). + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:14:59Z + + S‐OBHS‐2/3 + + + 0.77102524 + site + cleaner0 + 2023-07-05T13:15:12Z + SO: + + AF‐2 surface + + + 0.9972326 + evidence + cleaner0 + 2023-07-05T15:04:42Z + DUMMY: + + crystal structure + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T13:15:54Z + + S‐OBHS‐3‐bound + + + 0.9974546 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.98308796 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.99694616 + site + cleaner0 + 2023-07-05T13:15:30Z + SO: + + h3–h12 interface + + + structure_element + SO: + cleaner0 + 2023-07-05T12:39:24Z + + AF‐2 + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T13:16:18Z + + S‐OBHS‐2/3‐bound + + + 0.99649495 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.9761948 + structure_element + cleaner0 + 2023-07-05T15:07:44Z + SO: + + LBDs + + + 0.9918316 + experimental_method + cleaner0 + 2023-07-05T15:16:26Z + MESH: + + superposed + + + 0.6813256 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + 0.77806485 + protein + cleaner0 + 2023-07-05T12:49:11Z + PR: + + NCOA2 + + + site + SO: + cleaner0 + 2023-07-05T13:15:13Z + + AF‐2 surface + + + + MSB-12-864-g012.jpg + msb156701-fig-0006 + FIG + fig_caption + 37088 + Crystal structures show that 2,5‐DTP analogs shift h3 and h11 further apart compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DRM, 5DRJ). The 2F +o‐F +c electron density map and F +o‐F +c difference map of a 2,5‐DTP‐bound structure (PDB 5DRJ) were contoured at 1.0 sigma and ± 3.0 sigma, respectively. + + 0.9974384 + evidence + cleaner0 + 2023-07-05T15:04:46Z + DUMMY: + + Crystal structures + + + 0.98484725 + chemical + cleaner0 + 2023-07-05T14:02:48Z + CHEBI: + + 2,5‐DTP + + + 0.9452825 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + 0.99413896 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:16:59Z + + estrogen + + + 0.978199 + evidence + cleaner0 + 2023-07-05T13:17:26Z + DUMMY: + + 2F +o‐F +c electron density map + + + 0.9819667 + evidence + cleaner0 + 2023-07-05T13:17:11Z + DUMMY: + + F +o‐F +c difference map + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T13:17:52Z + + 2,5‐DTP‐bound + + + 0.9718598 + evidence + cleaner0 + 2023-07-05T15:04:52Z + DUMMY: + + structure + + + + MSB-12-864-g012.jpg + msb156701-fig-0006 + FIG + fig_caption + 37398 + Average (mean + SEM) α‐carbon distance measured from h3 Thr347 to h11 Leu525 of A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound ERα LBDs. *Two‐tailed Student's t‐test, P = 0.002 (PDB A‐CD: 5DI7, 5DID, 5DIE, 5DIG, and 4PPS; 2,5‐DTP: 4IWC, 5DRM, and 5DRJ; 3,4‐DTPD: 5DTV and 5DU5). + + 0.9582201 + evidence + cleaner0 + 2023-07-05T15:04:57Z + DUMMY: + + α‐carbon distance + + + 0.9764398 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + 0.9974693 + residue_name_number + cleaner0 + 2023-07-05T14:06:26Z + DUMMY: + + Thr347 + + + 0.9939917 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + 0.9983132 + residue_name_number + cleaner0 + 2023-07-05T14:06:29Z + DUMMY: + + Leu525 + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T13:28:54Z + + A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound + + + 0.996784 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.99171036 + structure_element + cleaner0 + 2023-07-05T15:07:48Z + SO: + + LBDs + + + experimental_method + MESH: + cleaner0 + 2023-07-05T13:28:17Z + + Student's t‐test + + + evidence + DUMMY: + cleaner0 + 2023-07-05T13:26:19Z + + P + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:28:04Z + + A‐CD + + + 0.9906206 + chemical + cleaner0 + 2023-07-05T14:02:52Z + CHEBI: + + 2,5‐DTP + + + 0.98730147 + chemical + cleaner0 + 2023-07-05T13:05:49Z + CHEBI: + + 3,4‐DTPD + + + + MSB-12-864-g012.jpg + msb156701-fig-0006 + FIG + fig_caption + 37696 + Crystal structures show that a 3,4‐DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DTV). + + 0.9977703 + evidence + cleaner0 + 2023-07-05T15:05:02Z + DUMMY: + + Crystal structures + + + 0.99749833 + chemical + cleaner0 + 2023-07-05T13:05:49Z + CHEBI: + + 3,4‐DTPD + + + 0.7596846 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:51:12Z + + F + + + 0.5140214 + protein + cleaner0 + 2023-07-05T12:49:11Z + PR: + + NCOA2 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:05:55Z + + A‐CD + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:29:12Z + + estrogen + + + + MSB-12-864-g012.jpg + msb156701-fig-0006 + FIG + fig_caption + 37841 + Hierarchical clustering of ligand‐specific binding of 154 interacting peptides to the ERα LBD was performed in triplicate by MARCoNI analysis. + + 0.7017504 + experimental_method + cleaner0 + 2023-07-05T15:16:31Z + MESH: + + Hierarchical clustering + + + 0.99713767 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.99558806 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.97988975 + experimental_method + cleaner0 + 2023-07-05T15:16:34Z + MESH: + + MARCoNI analysis + + + + MSB-12-864-g012.jpg + msb156701-fig-0006 + FIG + fig_caption + 37988 + +Source data are available online for this figure. + + + + RESULTS + paragraph + 38040 + The 2,5‐DTP analogs showed perturbation of h11, as well as h3, which forms part of the AF‐2 surface. These compounds bind the LBD in an unusual fashion because they have a phenol‐to‐phenol length of ~12 Å, which is longer than steroids and other prototypical ERα agonists that are ~10 Å in length. One phenol pushed further toward h3 (Fig 6D), while the other phenol pushed toward the C‐terminus of h11 to a greater extent than A‐CD‐ring estrogens (Nwachukwu et al, 2014), which are close structural analogs of E2 that lack a B‐ring (Fig 2). To quantify this difference, we compared the distance between α‐carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5‐DTP analogs (n = 3) or A‐CD‐ring analogs (n = 5) (Fig 6E). We observed a difference of 0.4 Å that was significant (two‐tailed Student's t‐test, P = 0.002) due to the very tight clustering of the 2,5‐DTP‐induced LBD conformation. The shifts in h3 suggest these compounds are positioned to alter coregulator preferences. + + chemical + CHEBI: + cleaner0 + 2023-07-05T14:03:10Z + + 2,5‐DTP + + + 0.99617827 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + 0.9889631 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + 0.9715357 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + 0.97476834 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.43669656 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.98127764 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + 0.9963342 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:05:55Z + + A‐CD + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:29:51Z + + estrogens + + + 0.99769753 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + 0.9318791 + evidence + cleaner0 + 2023-07-05T15:05:15Z + DUMMY: + + distance + + + 0.9763156 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + 0.9987458 + residue_name_number + cleaner0 + 2023-07-05T14:06:33Z + DUMMY: + + Thr347 + + + 0.99291295 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + 0.99873775 + residue_name_number + cleaner0 + 2023-07-05T14:06:35Z + DUMMY: + + Leu525 + + + 0.99461865 + evidence + cleaner0 + 2023-07-05T15:05:18Z + DUMMY: + + structures + + + 0.9943036 + chemical + cleaner0 + 2023-07-05T14:03:35Z + CHEBI: + + 2,5‐DTP + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:05:55Z + + A‐CD + + + experimental_method + MESH: + cleaner0 + 2023-07-05T13:28:18Z + + Student's t‐test + + + evidence + DUMMY: + cleaner0 + 2023-07-05T13:26:19Z + + P + + + 0.9942724 + chemical + cleaner0 + 2023-07-05T14:03:38Z + CHEBI: + + 2,5‐DTP + + + 0.8768127 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.85706687 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + + RESULTS + paragraph + 39088 + The 2,5‐DTP and 3,4‐DTP scaffolds are isomeric, but with aryl groups at obtuse and acute angles, respectively (Fig 2). The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). The 3,4‐DTPD analogs also induced a shift in h3 positioning, which translated again into a shift in the bound coactivator peptide (Fig 6F). Therefore, these indirect modulators, including S‐OBHS‐2, S‐OBHS‐3, 2,5‐DTP, and 3,4‐DTPD analogs—all of which show cell‐specific activity profiles—induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF‐2 surface. + + 0.99704283 + chemical + cleaner0 + 2023-07-05T14:03:43Z + CHEBI: + + 2,5‐DTP + + + 0.9970254 + chemical + cleaner0 + 2023-07-05T14:03:46Z + CHEBI: + + 3,4‐DTP + + + 0.99742705 + evidence + cleaner0 + 2023-07-05T15:05:27Z + DUMMY: + + crystal structure + + + 0.9969002 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.9694765 + protein_state + cleaner0 + 2023-07-05T15:19:59Z + DUMMY: + + in complex with + + + 0.9962179 + chemical + cleaner0 + 2023-07-05T14:03:51Z + CHEBI: + + 3,4‐DTP + + + 0.79860765 + experimental_method + cleaner0 + 2023-07-05T15:16:39Z + MESH: + + solved + + + 0.9974173 + evidence + cleaner0 + 2023-07-05T15:05:31Z + DUMMY: + + crystal structures + + + 0.99612707 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.9932176 + protein_state + cleaner0 + 2023-07-05T15:20:02Z + DUMMY: + + bound to + + + 0.9937948 + chemical + cleaner0 + 2023-07-05T13:05:50Z + CHEBI: + + 3,4‐DTPD + + + 0.7612082 + evidence + cleaner0 + 2023-07-05T15:05:34Z + DUMMY: + + structure + + + 0.9876172 + chemical + cleaner0 + 2023-07-05T14:03:55Z + CHEBI: + + furan + + + 0.9957991 + evidence + cleaner0 + 2023-07-05T15:05:38Z + DUMMY: + + structures + + + 0.99678385 + chemical + cleaner0 + 2023-07-05T13:05:50Z + CHEBI: + + 3,4‐DTPD + + + 0.47391936 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + 0.99885285 + residue_name_number + cleaner0 + 2023-07-05T14:06:39Z + DUMMY: + + Glu353 + + + 0.5352343 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:04Z + + hydrogen bond + + + 0.99898213 + residue_name_number + cleaner0 + 2023-07-05T14:06:42Z + DUMMY: + + Thr347 + + + 0.98964405 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.99798995 + site + cleaner0 + 2023-07-05T15:10:19Z + SO: + + ligand‐binding pocket + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:05:50Z + + 3,4‐DTPD + + + 0.5454983 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:05:14Z + + S‐OBHS‐2 + + + 0.7148489 + chemical + cleaner0 + 2023-07-05T13:13:51Z + CHEBI: + + S‐OBHS‐3 + + + 0.99419975 + chemical + cleaner0 + 2023-07-05T14:05:01Z + CHEBI: + + 2,5‐DTP + + + 0.99482787 + chemical + cleaner0 + 2023-07-05T13:05:50Z + CHEBI: + + 3,4‐DTPD + + + 0.85888684 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + 0.7486747 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.9619484 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + + RESULTS + paragraph + 40164 + To test whether the AF‐2 surface shows changes in shape in solution, we used the microarray assay for real‐time coregulator–nuclear receptor interaction (MARCoNI) analysis (Aarts et al, 2013). Here, the ligand‐dependent interactions of the ERα LBD with over 150 distinct LxxLL motif peptides were assayed to define structural fingerprints for the AF‐2 surface, in a manner similar to the use of phage display peptides as structural probes (Connor et al, 2001). Despite the similar average activities of these ligand classes (Fig 3A and B), 2,5‐DTP and 3,4‐DTP analogs displayed remarkably different peptide recruitment patterns (Fig 6H), consistent with the structural analyses. + + 0.9325462 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + 0.8547747 + experimental_method + cleaner0 + 2023-07-05T15:16:47Z + MESH: + + microarray assay for real‐time coregulator–nuclear receptor interaction + + + experimental_method + MESH: + cleaner0 + 2023-07-05T15:17:11Z + + MARCoNI + + + 0.997502 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.99503595 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + structure_element + SO: + cleaner0 + 2023-07-05T13:31:29Z + + LxxLL motif + + + 0.9527356 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + 0.8179392 + experimental_method + cleaner0 + 2023-07-05T15:17:15Z + MESH: + + phage display peptides + + + 0.9957962 + chemical + cleaner0 + 2023-07-05T14:05:07Z + CHEBI: + + 2,5‐DTP + + + 0.9952011 + chemical + cleaner0 + 2023-07-05T14:05:10Z + CHEBI: + + 3,4‐DTP + + + 0.95486695 + experimental_method + cleaner0 + 2023-07-05T15:17:17Z + MESH: + + structural analyses + + + + RESULTS + paragraph + 40862 + Hierarchical clustering revealed that many of the 2,5‐DTP analogs recapitulated most of the peptide recruitment and dismissal patterns observed with E2 (Fig 6H). However, there was a unique cluster of peptides that were recruited by E2 but not the 2,5‐DTP analogs. In contrast, 3,4‐DTP analogs dismissed most of the peptides from the AF‐2 surface (Fig 6H). Thus, the isomeric attachment of diaryl groups to the thiophene core changed the AF‐2 surface from inside the ligand‐binding pocket, as predicted by the crystal structures. Together, these findings suggest that without an extended side chain, cell‐specific activity stems from different coregulator recruitment profiles, due to unique ligand‐induced conformations of the AF‐2 surface, in addition to differential usage of AF‐1. Indirect modulators in cluster 1 avoid this by perturbing the h11–h12 interface, and modulating the dynamics of h12 without changing the shape of AF‐2 when stabilized. + + 0.9910126 + experimental_method + cleaner0 + 2023-07-05T15:17:21Z + MESH: + + Hierarchical clustering + + + 0.9818026 + chemical + cleaner0 + 2023-07-05T14:05:13Z + CHEBI: + + 2,5‐DTP + + + 0.9958525 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + 0.9956453 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:32:03Z + + 2,5‐DTP + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:32:20Z + + 3,4‐DTP + + + 0.8935431 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + 0.28013456 + chemical + cleaner0 + 2023-07-05T14:05:17Z + CHEBI: + + thiophene + + + 0.9795056 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + 0.9978263 + site + cleaner0 + 2023-07-05T15:10:24Z + SO: + + ligand‐binding pocket + + + 0.99719036 + evidence + cleaner0 + 2023-07-05T15:05:44Z + DUMMY: + + crystal structures + + + 0.98265505 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + 0.8201614 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.99679905 + site + cleaner0 + 2023-07-05T15:10:28Z + SO: + + h11–h12 interface + + + 0.98348224 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:39:24Z + + AF‐2 + + + + DISCUSS + title_1 + 41841 + Discussion + + + DISCUSS + paragraph + 41852 + Our goal was to identify a minimal set of predictors that would link specific structural perturbations to ERα signaling pathways that control cell‐specific signaling and proliferation. We found a very strong set of predictors, where ligands in cluster 1, defined by similar signaling across cell types, showed indirect modulation of h12 dynamics via the h11–12 interface or slight contact with h12. This perturbation determined proliferation that correlated strongly with AF‐2 activity, recruitment of NCOA1/2/3 family members, and induction of the GREB1 gene, consistent with the canonical ERα signaling pathway (Fig 1D). For ligands in cluster 1, deletion of AF‐1 reduced activity to varying degrees, but did not change the underlying signaling patterns established through AF‐2. In contrast, an extended side chain designed to directly reposition h12 and completely disrupt the AF‐2 surface results in cell‐specific signaling. This was demonstrated with direct modulators in clusters 2 and 3. Cluster 2 was defined by ligand classes that showed correlated activities in two of the three cell types tested, while ligand classes in cluster 3 did not show correlated activities among any of the three cell types. Compared to cluster 1, the structural rules are less clear in clusters 2 and 3, but a number of indirect modulator classes perturbed the LBD conformation at the intersection of h3, the h12 N‐terminus, and the AF‐2 surface. Ligands in these classes altered the shape of AF‐2 to affect coregulator preferences. For direct and indirect modulators in cluster 2 or 3, the canonical ERα signaling pathway involving recruitment of NCOA1/2/3 and induction of GREB1 did not generally predict their proliferative effects, indicating an alternate causal model (Fig 1E). + + 0.8802955 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.5235491 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.99708647 + site + cleaner0 + 2023-07-05T13:33:15Z + SO: + + h11–12 interface + + + 0.7182491 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.6642222 + structure_element + cleaner0 + 2023-07-05T12:39:24Z + SO: + + AF‐2 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + 0.92598903 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.9340975 + experimental_method + cleaner0 + 2023-07-05T15:17:26Z + MESH: + + deletion + + + structure_element + SO: + cleaner0 + 2023-07-05T12:40:26Z + + AF‐1 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:39:24Z + + AF‐2 + + + 0.7415674 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.9791953 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + 0.9899205 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.94423753 + structure_element + cleaner0 + 2023-07-05T13:12:39Z + SO: + + h3 + + + 0.9293732 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.96612656 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + structure_element + SO: + cleaner0 + 2023-07-05T12:39:24Z + + AF‐2 + + + 0.9450942 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + protein + PR: + cleaner0 + 2023-07-05T12:39:46Z + + NCOA1/2/3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:52Z + + GREB1 + + + + DISCUSS + paragraph + 43650 + These principles outlined above provide a structural basis for how the ligand–receptor interface leads to different signaling specificities through AF‐1 and AF‐2. It is noteworthy that regulation of h12 dynamics indirectly through h11 can virtually abolish AF‐2 activity, and yet still drive robust transcriptional activity through AF‐1, as demonstrated with the OBHS series. This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF‐1 and AF‐2 (McInerney et al, 1996; Webb et al, 1998), which allows ligands to nucleate ERα–NCOA1/2/3 interaction through AF‐2, and reinforce this interaction with additional binding to AF‐1. Completely blocking AF‐2 with an extended side chain or altering the shape of AF‐2 changes the preference away from NCOA1/2/3 for determining GREB1 levels and proliferation of breast cancer cells. AF‐2 blockade also allows AF‐1 to function independently, which is important since AF‐1 drives tissue‐selective effects in vivo. This was demonstrated with AF‐1 knockout mice that show E2‐dependent vascular protection, but not uterine proliferation, thus highlighting the role of AF‐1 in tissue‐selective or cell‐specific signaling (Billon‐Gales et al, 2009; Abot et al, 2013). + + 0.9938232 + site + cleaner0 + 2023-07-05T15:10:33Z + SO: + + ligand–receptor interface + + + 0.9457522 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.96084327 + structure_element + cleaner0 + 2023-07-05T12:39:24Z + SO: + + AF‐2 + + + 0.97882164 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.9980751 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:39:24Z + + AF‐2 + + + 0.9495473 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.43619075 + chemical + cleaner0 + 2023-07-05T13:03:38Z + CHEBI: + + OBHS + + + 0.6342545 + protein + cleaner0 + 2023-07-05T12:39:46Z + PR: + + NCOA1/2/3 + + + 0.9928863 + site + cleaner0 + 2023-07-05T15:10:41Z + SO: + + binding sites + + + 0.943999 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.94957405 + structure_element + cleaner0 + 2023-07-05T12:39:24Z + SO: + + AF‐2 + + + 0.9724985 + complex_assembly + cleaner0 + 2023-07-05T13:34:54Z + GO: + + ERα–NCOA1/2/3 + + + 0.95173496 + structure_element + cleaner0 + 2023-07-05T12:39:24Z + SO: + + AF‐2 + + + 0.92101926 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.7640235 + structure_element + cleaner0 + 2023-07-05T12:39:24Z + SO: + + AF‐2 + + + 0.92421794 + structure_element + cleaner0 + 2023-07-05T12:39:24Z + SO: + + AF‐2 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:47Z + + NCOA1/2/3 + + + 0.9842743 + protein + cleaner0 + 2023-07-05T12:39:53Z + PR: + + GREB1 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:39:24Z + + AF‐2 + + + 0.9037087 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.89438206 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.43650404 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.73675174 + chemical + cleaner0 + 2023-07-05T12:49:42Z + CHEBI: + + E2 + + + 0.88954836 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + + DISCUSS + paragraph + 44954 + One current limitation to our approach is the identification of statistical variables that predict ligand‐specific activity. Here, we examined many LBD structures and tested several variables that were not predictive, including ERβ activity, the strength of AF‐1 signaling, and NCOA3 occupancy at the GREB1 gene. Similarly, we visualized structures to identify patterns. There are many systems biology approaches that could contribute to the unbiased identification of predictive variables for statistical modeling. For example, phage display was used to identify the androgen receptor interactome, which was cloned into an M2H library and used to identify clusters of ligand‐selective interactions (Norris et al, 2009). Also, we have used siRNA screening to identify a number of coregulators required for ERα‐mediated repression of the IL‐6 gene (Nwachukwu et al, 2014). However, the use of larger datasets to identify such predictor variables has its own limitations, one of the major ones being the probability of false positives from multiple hypothesis testing. If we calculated inter‐atomic distance matrices containing 4,000 atoms per structure × 76 ligand–receptor complexes, we would have 3 × 105 predictions. One way to address this issue is to use the cross‐validation concept, where hypotheses are generated on training sets of ligands and tested with another set of ligands. + + 0.970414 + structure_element + cleaner0 + 2023-07-05T12:42:24Z + SO: + + LBD + + + 0.99617624 + evidence + cleaner0 + 2023-07-05T15:05:50Z + DUMMY: + + structures + + + 0.99014485 + protein + cleaner0 + 2023-07-05T13:51:17Z + PR: + + ERβ + + + structure_element + SO: + cleaner0 + 2023-07-05T12:40:26Z + + AF‐1 + + + 0.9916755 + protein + cleaner0 + 2023-07-05T13:51:22Z + PR: + + NCOA3 + + + protein + PR: + cleaner0 + 2023-07-05T12:39:53Z + + GREB1 + + + 0.9942068 + evidence + cleaner0 + 2023-07-05T15:05:53Z + DUMMY: + + structures + + + 0.9900647 + experimental_method + cleaner0 + 2023-07-05T15:17:30Z + MESH: + + phage display + + + 0.4478848 + experimental_method + cleaner0 + 2023-07-05T15:17:33Z + MESH: + + M2H + + + 0.9732868 + experimental_method + cleaner0 + 2023-07-05T15:17:35Z + MESH: + + siRNA screening + + + 0.9902525 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.9951785 + evidence + cleaner0 + 2023-07-05T15:06:00Z + DUMMY: + + inter‐atomic distance matrices + + + + DISCUSS + paragraph + 46367 + Based on this work, we propose several testable hypotheses for drug discovery. We have identified atomic vectors for the OBHS‐N and triaryl‐ethylene classes that predict ligand response (Fig 5E and F). These ligands in cluster 1 drive consistent, canonical signaling across cell types, which is desirable for generating full antagonists. Indeed, the most anti‐proliferative compound in the OBHS‐N series had a fulvestrant‐like profile across a battery of assays (S. Srinivasan et al, in preparation). Secondly, our finding that WAY‐C compounds do not rely of AF‐1 for signaling efficacy may derive from the slight contacts with h12 observed in crystal structures (Figs 3B and 5H), unlike other compounds in cluster 1 that dislocate h11 and rely on AF‐1 for signaling efficacy (Figs 3B and 5C, and EV5B). Thirdly, we found ligands that achieved cell‐specific activity without a prototypical extended side chain. Some of these ligands altered the shape of the AF‐2 surface by perturbing the h3–h12 interface, thus providing a route to new SERM‐like activity profiles by combining indirect and direct modulation of receptor structure. Incorporation of statistical approaches to understand relationships between structure and signaling variables moves us toward predictive models for complex ERα‐mediated responses such as in vivo uterine proliferation or tumor growth, and more generally toward structure‐based design for other allosteric drug targets including GPCRs and other nuclear receptors. + + 0.9962663 + evidence + cleaner0 + 2023-07-05T15:06:12Z + DUMMY: + + atomic vectors + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:02:41Z + + OBHS‐N + + + 0.8877854 + chemical + cleaner0 + 2023-07-05T13:08:31Z + CHEBI: + + triaryl‐ethylene + + + chemical + CHEBI: + cleaner0 + 2023-07-05T13:02:41Z + + OBHS‐N + + + 0.96572393 + chemical + cleaner0 + 2023-07-05T13:04:19Z + CHEBI: + + WAY‐C + + + 0.89169496 + structure_element + cleaner0 + 2023-07-05T12:40:26Z + SO: + + AF‐1 + + + 0.98411536 + structure_element + cleaner0 + 2023-07-05T12:48:51Z + SO: + + h12 + + + 0.9975318 + evidence + cleaner0 + 2023-07-05T15:06:21Z + DUMMY: + + crystal structures + + + 0.9966354 + structure_element + cleaner0 + 2023-07-05T12:48:36Z + SO: + + h11 + + + structure_element + SO: + cleaner0 + 2023-07-05T12:40:26Z + + AF‐1 + + + 0.992412 + site + cleaner0 + 2023-07-05T13:15:13Z + SO: + + AF‐2 surface + + + 0.99735576 + site + cleaner0 + 2023-07-05T13:15:30Z + SO: + + h3–h12 interface + + + 0.9914342 + protein + cleaner0 + 2023-07-05T12:39:00Z + PR: + + ERα + + + 0.95777607 + protein_type + cleaner0 + 2023-07-05T12:41:18Z + MESH: + + GPCRs + + + 0.83924866 + protein_type + cleaner0 + 2023-07-05T12:41:44Z + MESH: + + nuclear receptors + + + + METHODS + title_1 + 47895 + Materials and Methods + + + METHODS + title_2 + 47917 + Statistical analysis + + + METHODS + paragraph + 47938 + Correlation and linear regression analyses were performed using GraphPad Prism software. For correlation analysis, the degree to which two datasets vary together was calculated with the Pearson correlation coefficient (r). However, we reported r 2 rather than r, to facilitate comparison with the linear regression results for which we calculated and reported r 2 (Fig 3C–F). Significance for r 2 was determined using the F‐test for nonzero slope. High‐throughput assays were considered statistically robust if they show Z’ > 0.5, where Z’ = 1 − (3(σp+σn)/|μp−μn|), for the mean (σ) and standard deviations (μ) of the positive and negative controls (Fig EV1A and B). + + + METHODS + title_2 + 48631 + ERα ligand library + + + METHODS + paragraph + 48654 + The library of compounds examined includes both previously reported (Srinivasan et al, 2013) and newly synthesized compound series (see Dataset EV1 for individual compound information, and Appendix Supplementary Methods for synthetic protocols). + + + METHODS + title_2 + 48902 + Luciferase reporter assays + + + METHODS + paragraph + 48929 + Cells were transfected with FugeneHD reagent (Roche Applied Sciences, Indianapolis, IN) in 384‐well plates. After 24 h, cells were stimulated with 10 μM compounds dispensed using a 100‐nl pintool Biomeck NXP workstation (Beckman Coulter Inc.). Luciferase activity was measured 24 h later (see Appendix Supplementary Methods for more details). + + + METHODS + title_2 + 49281 + Mammalian 2‐hybrid (M2H) assays + + + METHODS + paragraph + 49315 + HEK293T cells were transfected with 5× UAS‐luciferase reporter, and wild‐type ERα‐VP16 activation domain plus full‐length NCOA1/2/3‐GAL4 DBD fusion protein expression plasmids, using the TransIT‐LT1 transfection reagent (Mirus Bio LLC, Madison, WI). The next day, cells were stimulated with 10 μM compounds using a 100‐nl pintool Biomeck NXP workstation (Beckman Coulter Inc.). Luciferase activity was measured after 24 h (see Appendix Supplementary Methods for more details). + + + METHODS + title_2 + 49813 + Cell proliferation assay + + + METHODS + paragraph + 49838 + MCF‐7 cells were plated on 384‐well plates in phenol red‐free media plus 10% FBS and stimulated with 10 μM compounds using 100‐nl pintool Biomeck NXP workstation (Beckman Coulter Inc.). Cell numbers determined 1 week later (see Appendix Supplementary Methods for more details). + + + METHODS + title_2 + 50128 + Quantitative RT–PCR + + + METHODS + paragraph + 50150 + MCF‐7 cells were steroid‐deprived and stimulated with compounds for 24 h. Total RNA was extracted and reverse‐transcribed. The cDNA was analyzed using TaqMan Gene Expression Master Mix (Life Technologies, Grand Island, NY), GREB1 and GAPDH (control) primers, and hybridization probes (see Appendix Supplementary Methods for more details). + + + METHODS + title_2 + 50497 + MARCoNI coregulator‐interaction profiling + + + METHODS + paragraph + 50541 + This assay was performed as previously described with the ERα LBD, 10 μM compounds, and a PamChiP peptide microarray (PamGene International) containing 154 unique coregulator peptides (Aarts et al, 2013) (see Appendix Supplementary Methods for more details). + + + METHODS + title_2 + 50805 + Protein production and X‐ray crystallography + + + METHODS + paragraph + 50852 + ERα protein was produced as previously described (Bruning et al, 2010). New ERα LBD structures (see Dataset EV2 for data collection and refinement statistics) were solved by molecular replacement using PHENIX (Adams et al, 2010), refined using ExCoR as previously described (Nwachukwu et al, 2013), and COOT (Emsley & Cowtan, 2004) for ligand‐docking and rebuilding. + + + METHODS + title_2 + 51227 + Data availability + + + METHODS + paragraph + 51245 + Crystal structures analyzed in this study include the following: 1GWR (Warnmark et al, 2002), 3ERD and 3ERT (Shiau et al, 1998), 4ZN9 (Zheng et al, 2012), 4IWC, 4 IU7, 4IV4, 4IVW, 4IW6, 4IUI, 4IV2, 4IVY and 4IW8 (Srinivasan et al, 2013), and 4PPS (Nwachukwu et al, 2014). New crystal structures analyzed in this study were deposited in the RCSB protein data bank (http://www.pdb.org): 4ZN7, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, 4ZNW, 5DI7, 5DID, 5DIE, 5DIG, 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, 5DP0, 5DRM, 5DRJ, 5DTV, 5DU5, 5DUE, 5DUG, 5DUH, 5DXK, 5DXM, 5DXP, 5DXQ, 5DXR, 5EHJ, 5DY8, 5DYB, 5DYD, 5DZ0, 5DZ1, 5DZ3, 5DZH, 5DZI, 5E0W, 5E0X, 5E14, 5E15, 5E19, 5E1C, 5DVS, 5DVV, 5DWE, 5DWG, 5DWI, 5DWJ, 5EGV, 5EI1, 5EIT. + + + AUTH_CONT + title_1 + 51978 + Author contributions + + + AUTH_CONT + paragraph + 51999 + JCN and SS contributed equally to this work. JCN and SS designed and performed experiments and wrote the manuscript; YZ, KEC, SW, JM, CD, ZL, VC, JN, NJW, JSJ, and RH performed experiments; HBZ designed experiments; and JAK and KWN designed experiments and wrote the manuscript. + + + COMP_INT + title_1 + 52278 + Conflict of Interest + + + COMP_INT + paragraph + 52299 + The authors declare that they have no conflict of interest. + + + SUPPL + title_1 + 52359 + Supporting information + + + REF + title + 52382 + References + + + 336 + 346 + 23383871 + REF + Chem Res Toxicol + ref + 26 + 2013 + 52393 + Robust array‐based coregulator binding assay predicting ERalpha‐agonist potency and generating binding profiles reflecting ligand structure + + + 2222 + 2233 + 23580568 + REF + Endocrinology + ref + 154 + 2013 + 52537 + The AF‐1 activation function of estrogen receptor alpha is necessary and sufficient for uterine epithelial cell proliferation in vivo + + + 213 + 221 + 20124702 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 52674 + PHENIX: a comprehensive Python‐based system for macromolecular structure solution + + + 2811 + 2818 + 2118104 + REF + EMBO J + ref + 9 + 1990 + 52758 + Role of the two activating domains of the oestrogen receptor in the cell‐type and promoter‐context dependent agonistic activity of the anti‐oestrogen 4‐hydroxytamoxifen + + + 6137 + 6144 + 11507064 + REF + Cancer Res + ref + 61 + 2001 + 52935 + Resveratrol exhibits cytostatic and antiestrogenic properties with human endometrial adenocarcinoma (Ishikawa) cells + + + 2053 + 2058 + 19188600 + REF + Proc Natl Acad Sci USA + ref + 106 + 2009 + 53052 + The transactivating function 1 of estrogen receptor alpha is dispensable for the vasculoprotective actions of 17beta‐estradiol + + + 837 + 843 + 20924370 + REF + Nat Chem Biol + ref + 6 + 2010 + 53181 + Coupling of receptor conformation and ligand orientation determine graded activity + + + 2917 + 2922 + 11306468 + REF + Cancer Res + ref + 61 + 2001 + 53264 + Circumventing tamoxifen resistance in breast cancers using antiestrogens that induce unique conformational changes in the estrogen receptor + + + 35848 + 35856 + 10960470 + REF + J Biol Chem + ref + 275 + 2000 + 53404 + Analysis of estrogen receptor interaction with a repressor of estrogen receptor activity (REA) and the regulation of estrogen receptor transcriptional activity by REA + + + 14930 + 14935 + 22927406 + REF + Proc Natl Acad Sci USA + ref + 109 + 2012 + 53571 + Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes + + + 17335 + 17339 + 17463000 + REF + J Biol Chem + ref + 282 + 2007 + 53710 + Regulation of GREB1 transcription by estrogen receptor alpha through a multipartite enhancer spread over 20 kb of upstream flanking sequences + + + 2126 + 2132 + 15572765 + REF + Acta Crystallogr D Biol Crystallogr + ref + 60 + 2004 + 53853 + Coot: model‐building tools for molecular graphics + + + 5363 + 5372 + 10409727 + REF + Mol Cell Biol + ref + 19 + 1999 + 53905 + Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor alpha + + + 1371 + 1388 + 9747868 + REF + J Natl Cancer Inst + ref + 90 + 1998 + 54069 + Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P‐1 Study + + + 621 + 627 + 8806005 + REF + Bone + ref + 18 + 1996 + 54190 + Time‐dependent changes in biochemical bone markers and serum cholesterol in ovariectomized rats: effects of raloxifene HCl, tamoxifen, estrogen, and alendronate + + + 6367 + 6375 + 11103799 + REF + Cancer Res + ref + 60 + 2000 + 54353 + PDZK1 and GREB1 are estrogen‐regulated genes expressed in hormone‐responsive breast cancer + + + 430 + 439 + 21664237 + REF + Mol Cell Endocrinol + ref + 348 + 2012 + 54448 + Steroid receptor coactivators 1, 2, and 3: critical regulators of nuclear receptor activity and steroid receptor modulator (SRM)‐based cancer therapy + + + 883 + 908 + 12620065 + REF + J Med Chem + ref + 46 + 2003 + 54600 + Antiestrogens and selective estrogen receptor modulators as multifunctional medicines. 1. Receptor interactions + + + 17 + 27 + 22032986 + REF + Trends Pharmacol Sci + ref + 33 + 2012 + 54712 + Diversity and modularity of G protein‐coupled receptor structures + + + 3320 + 3329 + 20334372 + REF + J Med Chem + ref + 53 + 2010 + 54780 + Characterization of the pharmacophore properties of novel selective estrogen receptor downregulators (SERDs) + + + 3532 + 3545 + 24708493 + REF + J Med Chem + ref + 57 + 2014 + 54889 + Triaryl‐substituted Schiff bases are high‐affinity subtype‐selective ligands for the estrogen receptor + + + 251 + 255 + 22498630 + REF + Nature + ref + 484 + 2012 + 54998 + Genome‐wide protein‐DNA binding dynamics suggest a molecular clutch for transcription factor function + + + e46410 + 23056300 + REF + PLoS ONE + ref + 7 + 2012 + 55104 + GREB1 functions as a growth promoter and is modulated by IL6/STAT3 in breast cancer + + + 295 + 316 + 12017549 + REF + Recent Prog Horm Res + ref + 57 + 2002 + 55188 + Definition of the molecular and cellular mechanisms underlying the tissue‐selective agonist/antagonist activities of selective estrogen receptor modulators + + + 10069 + 10073 + 8816752 + REF + Proc Natl Acad Sci USA + ref + 93 + 1996 + 55346 + Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator + + + 751 + 763 + 14675539 + REF + Cell + ref + 115 + 2003 + 55446 + Estrogen receptor‐alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter + + + 3346 + 3366 + 23586645 + REF + J Med Chem + ref + 56 + 2013 + 55571 + Thiophene‐core estrogen receptor ligands having superagonist activity + + + 1589 + 1602 + 12699377 + REF + J Med Chem + ref + 46 + 2003 + 55643 + Bridged bicyclic cores containing a 1,1‐diarylethylene motif are high‐affinity subtype‐selective ligands for the estrogen receptor + + + 309 + 333 + 15709961 + REF + Annu Rev Physiol + ref + 67 + 2005 + 55780 + Ligand control of coregulator recruitment to nuclear receptors + + + 241 + 247 + 18344977 + REF + Nat Chem Biol + ref + 4 + 2008 + 55843 + NFκB selectivity of estrogen receptor ligands revealed by comparative crystallographic analyses + + + 452 + 460 + 19389631 + REF + Chem Biol + ref + 16 + 2009 + 55943 + Differential presentation of protein interaction surfaces on the androgen receptor defines the pharmacological actions of bound ligands + + + 1923 + 1930 + 24076406 + REF + Structure + ref + 21 + 2013 + 56079 + Improved crystallographic structures using extensive combinatorial refinement + + + e02057 + 24771768 + REF + eLife + ref + 3 + 2014 + 56157 + Resveratrol modulates the inflammatory response via an estrogen receptor‐signal integration network + + + 993 + 996 + 17139284 + REF + Nat Rev Drug Discov + ref + 5 + 2006 + 56259 + How many drug targets are there? + + + 141 + 149 + 15986123 + REF + Breast Cancer Res Treat + ref + 92 + 2005 + 56292 + GREB 1 is a critical regulator of hormone dependent breast cancer growth + + + 93 + 104 + 12088871 + REF + Mol Cell Endocrinol + ref + 192 + 2002 + 56365 + Estrogen receptor‐mediated effects of tamoxifen on human endometrial cancer cells + + + 2496 + 2511 + 16610793 + REF + J Med Chem + ref + 49 + 2006 + 56449 + Fluorine‐substituted cyclofenil derivatives as estrogen receptor ligands: synthesis and structure‐affinity relationship study of potential positron emission tomography agents for imaging estrogen receptors in breast cancer + + + 2465 + 2468 + 11923541 + REF + Science + ref + 295 + 2002 + 56676 + Molecular determinants for the tissue specificity of SERMs + + + 927 + 937 + 9875847 + REF + Cell + ref + 95 + 1998 + 56735 + The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen + + + 326 + 332 + 23524984 + REF + Nat Chem Biol + ref + 9 + 2013 + 56853 + Ligand‐binding dynamics rewire cellular signaling via estrogen receptor‐alpha + + + 2324 + 2341 + 22283328 + REF + J Med Chem + ref + 55 + 2012 + 56935 + Identification and structure‐activity relationships of a novel series of estrogen receptor ligands based on 7‐thiabicyclo[2.2.1]hept‐2‐ene‐7‐oxide + + + 21862 + 21868 + 11937504 + REF + J Biol Chem + ref + 277 + 2002 + 57094 + Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha + + + 1605 + 1618 + 9773983 + REF + Mol Endocrinol + ref + 12 + 1998 + 57238 + Estrogen receptor activation function 1 works by binding p160 coactivator proteins + + + 18 + 24 + 24680426 + REF + Curr Opin Cell Biol + ref + 27 + 2014 + 57321 + Recent developments in biased agonism + + + 955 + 966 + 22543272 + REF + Mol Endocrinol + ref + 26 + 2012 + 57359 + Gene‐specific patterns of coregulator requirements by estrogen receptor‐alpha in breast cancer cells + + + R27 + 21392396 + REF + Breast Cancer Res + ref + 13 + 2011 + 57464 + Estrogen receptor‐beta sensitizes breast cancer cells to the anti‐estrogenic actions of endoxifen + + + 1047 + 1058 + 25728767 + REF + Mol Cell + ref + 57 + 2015 + 57566 + Structure of a biologically active estrogen receptor‐coactivator complex on DNA + + + 1094 + 1100 + 22517684 + REF + ChemMedChem + ref + 7 + 2012 + 57648 + Development of selective estrogen receptor modulator (SERM)‐like activity through an indirect mechanism of estrogen receptor antagonism: defining the binding mode of 7‐oxabicyclo[2.2.1]hept‐5‐ene scaffold core ligands + + + 8692 + 8700 + 23033157 + REF + Org Biomol Chem + ref + 10 + 2012 + 57874 + Bicyclic core estrogens as full antagonists: synthesis, biological evaluation and structure‐activity relationships of estrogen receptor ligands based on bridged oxabicyclic core arylsulfonamides + + + diff --git a/BioC_XML/4850273_v0.xml b/BioC_XML/4850273_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..633a38dbbc88c49dc445da0362e3e9b8a77b7e6e --- /dev/null +++ b/BioC_XML/4850273_v0.xml @@ -0,0 +1,10760 @@ + + + + PMC + 20140719 + pmc.key + + 4850273 + CC BY-NC-SA + no + 0 + 0 + + Xyloglucan Recognition by Gut Bacteria Tauzin et al. + 10.1128/mBio.02134-15 + 4850273 + 27118585 + mBio02134-15 + e02134-15 + 2 + This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. + surname:Tauzin;given-names:Alexandra S. + surname:Kwiatkowski;given-names:Kurt J. + surname:Orlovsky;given-names:Nicole I. + surname:Smith;given-names:Christopher J. + surname:Creagh;given-names:A. Louise + surname:Haynes;given-names:Charles A. + surname:Wawrzak;given-names:Zdzislaw + surname:Brumer;given-names:Harry + surname:Koropatkin;given-names:Nicole M. + TITLE + front + 7 + 2016 + 0 + Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:58:24Z + + Xyloglucan + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + Human + + + + ABSTRACT + abstract_title_1 + 81 + ABSTRACT + + + ABSTRACT + abstract + 90 + Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:22Z + + Polysaccharide utilization loci + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:41:37Z + + carbohydrates + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:30Z + + bacterial + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:56:33Z + + complex polysaccharide + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:20Z + + glycosidases + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:08Z + + biochemical, structural, and reverse genetic characterization + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:44:49Z + + cell surface glycan-binding proteins + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:02Z + + SGBPs + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:06Z + + xyloglucan utilization locus + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:48:01Z + + Bacteroides ovatus + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:50:28Z + + vegetable + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:50:41Z + + polysaccharide + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:46:06Z + + Biochemical analysis + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:44:54Z + + outer membrane-anchored proteins + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:34Z + + xyloglucan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:43Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:50:41Z + + polysaccharide + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:50:03Z + + crystal structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:51:30Z + + bound + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:43Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:51:35Z + + tetradecasaccharide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:48:38Z + + carbohydrate-binding platform + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:41:40Z + + β-glucan + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T21:48:14Z + + tetra-modular + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:46:31Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:48Z + + SGBP-B + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:49:26Z + + tandem Ig-like folds + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:58:35Z + + XyG + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:49:16Z + + C-terminal domain + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:42:56Z + + affinities + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:43Z + + XyG + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:50:16Z + + reverse-genetic analysis + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:48Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:51:46Z + + oligosaccharides + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:59:07Z + + carbohydrate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:43Z + + XyG + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:48Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:58:56Z + + carbohydrate + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:51Z + + B. ovatus + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:50:28Z + + vegetable + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:52:04Z + + xyloglucans + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + + ABSTRACT + abstract_title_1 + 1947 + IMPORTANCE + + + ABSTRACT + abstract + 1958 + The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:54:54Z + + bacteria + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:55:37Z + + complex polysaccharides + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:55:04Z + + plant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T17:14:06Z + + polysaccharides + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:55:51Z + + biochemical, crystallographic, and genetic insight + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:00Z + + surface glycan-binding proteins + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:48:01Z + + Bacteroides ovatus + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:55:59Z + + xyloglucan utilization locus + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:50:28Z + + vegetable + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:50:41Z + + polysaccharide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:56:25Z + + complex polysaccharide + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:54:54Z + + bacteria + + + + INTRO + title_1 + 2852 + INTRODUCTION + + + INTRO + paragraph + 2865 + The human gut microbiota influences the course of human development and health, playing key roles in immune stimulation, intestinal cell proliferation, and metabolic balance. This microbial community is largely bacterial, with the Bacteroidetes, Firmicutes, and Actinobacteria comprising the dominant phyla. The ability to acquire energy from carbohydrates of dietary or host origin is central to the adaptation of human gut bacterial species to their niche. More importantly, this makes diet a tractable way to manipulate the abundance and metabolic output of the microbiota toward improved human health. However, there is a paucity of data regarding how the vast array of complex carbohydrate structures are selectively recognized and imported by members of the microbiota, a critical process that enables these organisms to thrive in the competitive gut environment. The human gut bacteria Bacteroidetes share a profound capacity for dietary glycan degradation, with many species containing >250 predicted carbohydrate-active enzymes (CAZymes), compared to 50 to 100 within many Firmicutes and only 17 in the human genome devoted toward carbohydrate utilization. A remarkable feature of the Bacteroidetes is the packaging of genes for carbohydrate catabolism into discrete polysaccharide utilization loci (PUL), which are transcriptionally regulated by specific substrate signatures. The archetypal PUL-encoded system is the starch utilization system (Sus) (Fig. 1B) of Bacteroides thetaiotaomicron. The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. The importance of PUL as a successful evolutionary strategy is underscored by the observation that Bacteroidetes such as B. thetaiotaomicron and Bacteroides ovatus devote ~18% of their genomes to these systems. Moving beyond seminal genomic and transcriptomic analyses, the current state-of-the-art PUL characterization involves combined reverse-genetic, biochemical, and structural studies to illuminate the molecular details of PUL function. + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:03:40Z + + microbiota + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:03:30Z + + microbial + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:30Z + + bacterial + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:03:51Z + + Firmicutes + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:04:02Z + + Actinobacteria + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:41:45Z + + carbohydrates + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:30Z + + bacterial + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:03:40Z + + microbiota + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:04:30Z + + complex carbohydrate + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:03:40Z + + microbiota + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:54:54Z + + bacteria + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:27Z + + glycan + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:03:51Z + + Firmicutes + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T21:41:11Z + + polysaccharide utilization loci + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-16T16:06:19Z + + starch utilization system + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-16T16:06:28Z + + Sus + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:06:08Z + + Bacteroides thetaiotaomicron + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-16T16:06:28Z + + Sus + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:05:57Z + + lipid-anchored + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:05:48Z + + endo-amylase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:06:49Z + + SusG + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:06:34Z + + TonB-dependent transporter + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:06:38Z + + TBDT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:06:58Z + + SusC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:07:03Z + + oligosaccharides + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:05:44Z + + starch-binding protein + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:07:24Z + + carbohydrate-binding lipoproteins + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:33Z + + SusE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:44Z + + SusF + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:05:37Z + + exo-glucosidases + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:58Z + + SusA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:08:08Z + + SusB + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:08:13Z + + glucose + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:08:28Z + + B. thetaiotaomicron + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:48:01Z + + Bacteroides ovatus + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:08:40Z + + reverse-genetic, biochemical, and structural studies + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + + mbo0021627940001.jpg + fig1 + FIG + fig_caption + 5194 + Xyloglucan and the Bacteroides ovatus xyloglucan utilization locus (XyGUL). (A) Representative structures of common xyloglucans using the Consortium for Functional Glycomics Symbol Nomenclature (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml). Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. The location of SGBP-A/B is presented in this work; the location of GH5 has been empirically determined, and the enzymes have been placed based upon their predicted cellular location. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:11:10Z + + Xyloglucan + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:48:01Z + + Bacteroides ovatus + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:11:16Z + + xyloglucan utilization locus + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:11:20Z + + structures + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:52:04Z + + xyloglucans + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:12:52Z + + BoXyGUL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:20Z + + glycosidases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:12:08Z + + GHs + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:12:17Z + + solanaceous + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:12:23Z + + xyloglucan + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:12:37Z + + BtSus + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:12:52Z + + BoXyGUL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:12:52Z + + BoXyGUL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:52:04Z + + xyloglucans + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:13:18Z + + B + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:13:26Z + + GH5 + + + + INTRO + paragraph + 5909 + We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B. ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG). XyG variants (Fig. 1A) constitute up to 25% of the dry weight of common vegetables. Analogous to the Sus locus, the xyloglucan utilization locus (XyGUL) encodes a cohort of carbohydrate-binding, -hydrolyzing, and -importing proteins (Fig. 1B and C). The number of glycoside hydrolases (GHs) encoded by the XyGUL is, however, more expansive than that by the Sus locus (Fig. 1B), which reflects the greater complexity of glycosidic linkages found in XyG vis-à-vis starch. Whereas our previous study focused on the characterization of the linkage specificity of these GHs, a key outstanding question regarding this locus is how XyG recognition is mediated at the cell surface. + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:25:03Z + + B. ovatus ATCC 8483 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:55:04Z + + plant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:41:52Z + + glycans + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:52:04Z + + xyloglucans + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:43Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:43Z + + XyG + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:25:25Z + + vegetables + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:25:36Z + + Sus locus + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:25:42Z + + xyloglucan utilization locus + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:26:24Z + + carbohydrate-binding, -hydrolyzing, and -importing proteins + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:25:57Z + + glycoside hydrolases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:12:08Z + + GHs + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:25:37Z + + Sus locus + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:26:10Z + + starch + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:12:08Z + + GHs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:59:58Z + + XyG + + + + INTRO + paragraph + 6811 + In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as “putative lipoproteins”. The genes coding for these proteins, sometimes referred to as “susE/F positioned,” display products with a wide variation in amino acid sequence and which have little or no homology to other PUL-encoded proteins or known carbohydrate-binding proteins. As the Sus SGBPs remain the only structurally characterized cohort to date, we therefore wondered whether such glycan binding and function are extended to other PUL that target more complex and heterogeneous polysaccharides, such as XyG. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-16T16:06:19Z + + starch utilization system + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:08:28Z + + B. thetaiotaomicron + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T16:30:12Z + + starch-binding sites + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:30:21Z + + surface glycan-binding proteins + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:02Z + + SGBPs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:30:34Z + + amylase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:06:49Z + + SusG + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:33Z + + SusE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:44Z + + SusF + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:31:00Z + + starch + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:33Z + + SusE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:44Z + + SusF + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:06:49Z + + SusG + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T16:31:05Z + + binding sites + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T17:14:34Z + + polysaccharide + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:06:58Z + + SusC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:31:21Z + + susD + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:31:30Z + + susE/F + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:31:36Z + + putative + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:31:39Z + + lipoproteins + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:31:30Z + + susE/F + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:31:44Z + + carbohydrate-binding proteins + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-16T16:06:29Z + + Sus + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:02Z + + SGBPs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:27Z + + glycan + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T17:14:06Z + + polysaccharides + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + + INTRO + paragraph + 8045 + We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo. Combined biochemical, structural, and reverse-genetic approaches clearly illuminate the distinct, yet complementary, functions that these two proteins play in XyG recognition as it impacts the physiology of B. ovatus. These data extend our current understanding of the Sus-like glycan uptake paradigm within the Bacteroidetes and reveals how the complex dietary polysaccharide xyloglucan is recognized at the cell surface. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:42:46Z + + functional and structural characterization + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:42:51Z + + noncatalytic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:02Z + + SGBPs + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:42:55Z + + Bacova_02651 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:43:00Z + + Bacova_02650 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:48Z + + SGBP-B + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:43:05Z + + biochemical, structural, and reverse-genetic approaches + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:00:18Z + + XyG + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:52Z + + B. ovatus + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:27Z + + glycan + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T17:14:34Z + + polysaccharide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:43:22Z + + xyloglucan + + + + RESULTS + title_1 + 8734 + RESULTS AND DISCUSSION + + + RESULTS + title_2 + 8757 + SGBP-A and SGBP-B are cell-surface-localized, xyloglucan-specific binding proteins. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:48Z + + SGBP-B + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:43:47Z + + cell-surface-localized, xyloglucan-specific binding proteins + + + + RESULTS + paragraph + 8841 + SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig. 1B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B. thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work. In contrast, SGBP-B, encoded by locus tag Bacova_02650, displays little sequence similarity to the products of similarly positioned genes in syntenic XyGUL nor to any other gene product among the diversity of Bacteroidetes PUL. Whereas sequence similarity among SusC/SusD homolog pairs often serves as a hallmark for PUL identification, the sequence similarities of downstream genes encoding SGBPs are generally too low to allow reliable bioinformatic classification of their products into protein families, let alone prediction of function. Hence, there is a critical need for the elucidation of detailed structure-function relationships among PUL SGBPs, in light of the manifold glycan structures in nature. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:45:44Z + + Bacova_02651 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:08:28Z + + B. thetaiotaomicron + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:45:54Z + + SusD-like proteins + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:48Z + + SGBP-B + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T21:41:16Z + + Bacova_02650 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:06:58Z + + SusC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:02Z + + SGBPs + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:02Z + + SGBPs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + + RESULTS + paragraph + 9828 + Immunofluorescence of formaldehyde-fixed, nonpermeabilized cells grown in minimal medium with XyG as the sole carbon source to induce XyGUL expression, reveals that both SGBP-A and SGBP-B are presented on the cell surface by N-terminal lipidation, as predicted by signal peptide analysis with SignalP (Fig. 2). Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B. ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig. 1B and C). + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:47:57Z + + Immunofluorescence + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:48Z + + SGBP-B + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:48:08Z + + lipidation + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:02Z + + SGBPs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:48:15Z + + cell-surface-localized endo-xyloglucanase + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:52Z + + B. ovatus + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:13:26Z + + GH5 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:48:42Z + + BoGH5 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:48:31Z + + SusC-like TBDT + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + + mbo0021627940002.jpg + fig2 + FIG + fig_caption + 10372 + SGBP-A and SGBP-B visualized by immunofluorescence. Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. (D) FITC images of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. Cells lacking SGBP-A (ΔSGBP-A) do not grow on XyG and therefore could not be tested in parallel. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:48Z + + SGBP-B + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:03Z + + immunofluorescence + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:52Z + + B. ovatus + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:48Z + + SGBP-B + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:07Z + + Overlay + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:51:11Z + + bright-field and FITC images + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:52Z + + B. ovatus + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:46:13Z + + Overlay + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:51:15Z + + bright-field and FITC images + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:52Z + + B. ovatus + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:51:19Z + + Bright-field image + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:26Z + + ΔSGBP-B + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:51:35Z + + FITC images + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:26Z + + ΔSGBP-B + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:51:31Z + + lacking + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:43Z + + ΔSGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + + RESULTS + paragraph + 11064 + In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. In contrast, SGBP-B bound to HEC more weakly than SGBP-A and did not bind to MLG or GM. Neither SGBP recognized galactomannan (GGM), starch, carboxymethylcellulose, or mucin (see Fig. S1 in the supplemental material). Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). Notably, the absence of carbohydrate-binding modules in the GHs encoded by the XyGUL implies that noncatalytic recognition of xyloglucan is mediated entirely by SGBP-A and -B. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:25:57Z + + glycoside hydrolases + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:57:12Z + + affinity PAGE + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:56:57Z + + isothermal titration calorimetry + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:57:05Z + + ITC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:48Z + + SGBP-B + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:05Z + + xyloglucan-binding proteins + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:34:50Z + + affinity constant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:05Z + + Ka + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:57:12Z + + affinity PAGE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:18Z + + hydroxyethyl cellulose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:24Z + + HEC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:29Z + + β(1 → 4)-glucan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:32Z + + mixed-linkage β(1→3)/β(1→4)-glucan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:39Z + + MLG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:44Z + + glucomannan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:51Z + + GM + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:55Z + + glucosyl + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:58Z + + mannosyl + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T17:14:34Z + + polysaccharide + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:48Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:24Z + + HEC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:39Z + + MLG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:51Z + + GM + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:08Z + + SGBP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:58:03Z + + galactomannan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:58:09Z + + GGM + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:58:13Z + + starch + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:58:16Z + + carboxymethylcellulose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:58:20Z + + mucin + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:01Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:58:25Z + + XyG-specific GHs + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:52Z + + B. ovatus + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:30Z + + PUL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:39Z + + MLG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:57:51Z + + GM + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:58:09Z + + GGM + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T16:58:32Z + + carbohydrate-binding modules + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:12:09Z + + GHs + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T16:58:35Z + + xyloglucan + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:58:39Z + + -B + + + + mbo0021627940003.jpg + fig3 + FIG + fig_caption + 12557 + SGBP-A and SGBP-B preferentially bind xyloglucan. Affinity electrophoresis (10% acrylamide) of SGBP-A and SGBP-B with BSA as a control protein. All samples were loaded on the same gel next to the BSA controls; thin black lines indicate where intervening lanes were removed from the final image for both space and clarity. The percentage of polysaccharide incorporated into each native gel is displayed. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T17:00:22Z + + xyloglucan + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T17:00:26Z + + Affinity electrophoresis + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T17:00:34Z + + BSA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T17:00:34Z + + BSA + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:50:42Z + + polysaccharide + + + + RESULTS + paragraph + 12960 + The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. Together, these data clearly suggest that polysaccharide binding of both SGBPs is fulfilled by a dimer of the minimal repeat, corresponding to XyGO2 (cf. Fig. 1A). The observation by affinity PAGE that these proteins specifically recognize XyG is further substantiated by their lack of binding for the undecorated oligosaccharide cellotetraose (Table 1; see Fig. S3). Furthermore, SGBP-A binds cellohexaose with ~770-fold weaker affinity than XyG, while SGBP-B displays no detectable binding to this linear hexasaccharide. To provide molecular-level insight into how the XyGUL SGBPs equip B. ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:13Z + + endo-xyloglucanase + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:48:42Z + + BoGH5 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:50:42Z + + polysaccharide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:41:57Z + + glucosyl + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:42:01Z + + xylogluco-oligosaccharides + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:42:05Z + + XyGOs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T21:48:31Z + + Glc4 backbone + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T21:48:36Z + + variable side-chain galactosylation + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:36:33Z + + XyGO1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:46:18Z + + controlled digestion and fractionation + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:46:22Z + + size exclusion chromatography + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:42:08Z + + oligosaccharides + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:21Z + + XyGO2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:57:05Z + + ITC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:50:42Z + + polysaccharide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:21Z + + XyGO2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T21:49:23Z + + Glc8 backbone + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:42:55Z + + affinities + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:36:33Z + + XyGO1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T21:49:29Z + + Glc4 backbone + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:08:44Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:21Z + + XyGO2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:42:57Z + + affinities + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:07Z + + Ka + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:54Z + + bound to + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:36:33Z + + XyGO1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:36Z + + affinity + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:36:41Z + + minimal repeating unit + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:07Z + + Ka + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:21Z + + XyGO2 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T17:14:34Z + + polysaccharide + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:02Z + + SGBPs + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:50:04Z + + dimer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T21:49:33Z + + minimal repeat + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:21Z + + XyGO2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:57:12Z + + affinity PAGE + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:42:13Z + + oligosaccharide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:42:18Z + + cellotetraose + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:42:25Z + + cellohexaose + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:09:08Z + + affinity + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:43:12Z + + hexasaccharide + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:02Z + + SGBPs + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:52Z + + B. ovatus + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:46:26Z + + X-ray crystallography + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T21:46:39Z + + SGPB-B + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-16T21:49:37Z + + oligosaccharide-complex forms + + + + tab1.xml + tab1 + TABLE + table_caption + 14796 + Summary of thermodynamic parameters for wild-type SGBP-A and SGBP-B obtained by isothermal titration calorimetry at 25°Ca + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:34:04Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T18:33:57Z + + isothermal titration calorimetry + + + + tab1.xml + tab1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup span="1"><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/></colgroup><thead><tr><th rowspan="2" colspan="1">Carbohydrate</th><th colspan="2" rowspan="1"><italic>K<sub>a</sub></italic> (M<sup>−1</sup>)<hr/></th><th colspan="2" rowspan="1">Δ<italic>G</italic> (kcal ⋅ mol<sup>−1</sup>)<hr/></th><th colspan="2" rowspan="1">Δ<italic>H</italic> (kcal ⋅ mol<sup>−1</sup>)<hr/></th><th colspan="2" rowspan="1"><italic>T</italic>Δ<italic>S</italic> (kcal ⋅ mol<sup>−1</sup>)<hr/></th></tr><tr><th rowspan="1" colspan="1">SGBP-A</th><th rowspan="1" colspan="1">SGBP-B</th><th rowspan="1" colspan="1">SGBP-A</th><th rowspan="1" colspan="1">SGBP-B</th><th rowspan="1" colspan="1">SGBP-A</th><th rowspan="1" colspan="1">SGBP-B</th><th rowspan="1" colspan="1">SGBP-A</th><th rowspan="1" colspan="1">SGBP-B</th></tr></thead><tbody><tr><td rowspan="1" colspan="1">XyG<xref ref-type="table-fn" rid="ngtab1.2"><sup>b</sup></xref></td><td rowspan="1" colspan="1">(4.4 ± 0.1) × 10<sup>5</sup></td><td rowspan="1" colspan="1">(5.7 ± 0.2) × 10<sup>4</sup></td><td rowspan="1" colspan="1">−7.7</td><td rowspan="1" colspan="1">−6.5</td><td rowspan="1" colspan="1">−14 ± 3</td><td rowspan="1" colspan="1">−14 ± 2</td><td rowspan="1" colspan="1">−6.5</td><td rowspan="1" colspan="1">−7.6</td></tr><tr><td rowspan="1" colspan="1">XyGO<sub>2</sub><xref ref-type="table-fn" rid="ngtab1.3"><sup>c</sup></xref></td><td rowspan="1" colspan="1">3.0 × 10<sup>5</sup></td><td rowspan="1" colspan="1">2.0 × 10<sup>4</sup></td><td rowspan="1" colspan="1">−7.5</td><td rowspan="1" colspan="1">−5.9</td><td rowspan="1" colspan="1">−17.2</td><td rowspan="1" colspan="1">−17.6</td><td rowspan="1" colspan="1">−9.7</td><td rowspan="1" colspan="1">−11.7</td></tr><tr><td rowspan="1" colspan="1">XyGO<sub>1</sub></td><td rowspan="1" colspan="1">NB<xref ref-type="table-fn" rid="ngtab1.4"><sup>d</sup></xref></td><td rowspan="1" colspan="1">(2.4 ± 0.1) × 10<sup>3</sup></td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">−4.6</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">−4.4 ± 0.2</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">0.2</td></tr><tr><td rowspan="1" colspan="1">Cellohexaose</td><td rowspan="1" colspan="1">568.0 ± 291.0</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">−3.8</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">−16 ± 8</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">−12.7</td><td rowspan="1" colspan="1">NB</td></tr><tr><td rowspan="1" colspan="1">Cellotetraose</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td></tr></tbody></table> + + 14919 + Carbohydrate Ka (M−1) ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) SGBP-A SGBP-B SGBP-A SGBP-B SGBP-A SGBP-B SGBP-A SGBP-B XyGb (4.4 ± 0.1) × 105 (5.7 ± 0.2) × 104 −7.7 −6.5 −14 ± 3 −14 ± 2 −6.5 −7.6 XyGO2c 3.0 × 105 2.0 × 104 −7.5 −5.9 −17.2 −17.6 −9.7 −11.7 XyGO1 NBd (2.4 ± 0.1) × 103 NB −4.6 NB −4.4 ± 0.2 NB 0.2 Cellohexaose 568.0 ± 291.0 NB −3.8 NB −16 ± 8 NB −12.7 NB Cellotetraose NB NB NB NB NB NB NB NB + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-07-21T08:36:57Z + + ΔG + + + + tab1.xml + tab1 + TABLE + table_footnote + 15427 + Shown are average values ± standard errors from two independent titrations, unless otherwise indicated. + + + tab1.xml + tab1 + TABLE + table_footnote + 15532 + Binding thermodynamics for XyG based on the concentration of the binding unit, XyGO2. + + + tab1.xml + tab1 + TABLE + table_footnote + 15618 + Values from a single titration. + + + tab1.xml + tab1 + TABLE + table_footnote + 15650 + NB, no binding observed. + + + RESULTS + title_2 + 15675 + SGBP-A is a SusD homolog with an extensive glycan-binding platform. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:37:53Z + + glycan-binding platform + + + + RESULTS + paragraph + 15743 + As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). Specifically, SGBP-A overlays B. thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2 Å for 363 Cα pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig. 4C). Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. The SGBP-A:XyGO2 complex superimposes closely with the apo structure (RMSD of 0.6 Å) and demonstrates that no major conformational change occurs upon substrate binding; small deviations in the orientation of several surface loops are likely the result of differential crystal packing. It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:42:28Z + + structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:42:35Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:43:15Z + + Rwork + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:43:40Z + + Rfree + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:43:51Z + + 28 to 546 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:43:55Z + + “SusD-like” protein fold + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:44:02Z + + tetratrico-peptide repeat + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:44:05Z + + TPR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:44:09Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T18:44:12Z + + overlays + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:08:28Z + + B. thetaiotaomicron + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T18:44:26Z + + BtSusD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:44:47Z + + root mean square deviation + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:45:02Z + + RMSD + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T18:45:13Z + + Cocrystallization + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:21Z + + XyGO2 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-16T18:45:27Z + + substrate complex + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:45:31Z + + structure + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:43:17Z + + Rwork + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:43:43Z + + Rfree + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:46:04Z + + 36 to 546 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T21:48:27Z + + binding-site + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:18Z + + XyG binding protein + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-16T18:42:50Z + + SGBP-A:XyGO2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T18:46:14Z + + superimposes + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:42:35Z + + apo + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-15T08:35:30Z + + structure + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:45:04Z + + RMSD + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:46:24Z + + ligand-binding site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:46:27Z + + conserved + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:46:33Z + + aromatic platform + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:47:02Z + + site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:46:53Z + + amylose + + + + mbo0021627940004.jpg + fig4 + FIG + fig_caption + 17245 + Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures. The apo structure is color ramped from blue to red. An omit map (2σ) for XyGO2 (orange and red sticks) is displayed. (B) Close-up view of the omit map as in panel A, rotated 90° clockwise. (C) Overlay of the Cα backbones of SGBP-A (black) with XyGO2 (orange and red spheres) and BtSusD (blue) with maltoheptaose (pink and red spheres), highlighting the conservation of the glycan-binding site location. (D) Close-up of the SGBP-A (black and orange) and SusD (blue and pink) glycan-binding sites. The approximate length of each glycan-binding site is displayed, colored to match the protein structures. (E) Stereo view of the xyloglucan-binding site of SGBP-A, displaying all residues within 4 Å of the ligand. The backbone glucose residues are numbered from the nonreducing end; xylose residues are labeled X1 and X2. Potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:56:14Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T18:56:21Z + + Bacova_02651 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T18:56:18Z + + Overlay + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:42:35Z + + apo + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:21Z + + XyGO2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:56:26Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:42:35Z + + apo + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:47:03Z + + structure + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:24:59Z + + omit map + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:21Z + + XyGO2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:24:59Z + + omit map + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T18:56:34Z + + Overlay + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:21Z + + XyGO2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T18:44:26Z + + BtSusD + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:56:41Z + + maltoheptaose + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:56:50Z + + glycan-binding site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:56:58Z + + glycan-binding sites + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:56:50Z + + glycan-binding site + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:57:12Z + + protein structures + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:57:08Z + + xyloglucan-binding site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:57:18Z + + glucose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:57:16Z + + xylose + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:57:21Z + + X1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:57:24Z + + X2 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:16Z + + hydrogen-bonding interactions + + + + tab2.xml + tab2 + TABLE + table_caption + 18298 + X-ray data collection and refinement statistics + + + tab2.xml + tab2 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup span="1"><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/></colgroup><thead><tr><th rowspan="2" colspan="1">Parameter</th><th colspan="4" rowspan="1">Value(s) for<xref ref-type="table-fn" rid="ngtab2.1"><sup>a</sup></xref>:<hr/></th></tr><tr><th rowspan="1" colspan="1">SGBP-A <italic>apo</italic></th><th rowspan="1" colspan="1">SGBP-A/XyGO<sub>2</sub></th><th rowspan="1" colspan="1">SGBP-B/XyGO<sub>2</sub></th><th rowspan="1" colspan="1">SGBP-B (CD)/XyGO<sub>2</sub></th></tr></thead><tbody><tr><td rowspan="1" colspan="1">PDB ID no.</td><td rowspan="1" colspan="1">5E75</td><td rowspan="1" colspan="1">5E76</td><td rowspan="1" colspan="1">5E7G</td><td rowspan="1" colspan="1">5E7H</td></tr><tr><td rowspan="1" colspan="1">Resolution (Å)</td><td rowspan="1" colspan="1">21.48–1.36 (1.409–1.36)</td><td rowspan="1" colspan="1">56.13–2.3 (2.382–2.3)</td><td rowspan="1" colspan="1">39.19–2.37 (2.455–2.37)</td><td rowspan="1" colspan="1">30.69–1.57 (1.626–1.570)</td></tr><tr><td rowspan="1" colspan="1">Space group</td><td rowspan="1" colspan="1">P2<sub>1</sub></td><td rowspan="1" colspan="1">I422</td><td rowspan="1" colspan="1">R32</td><td rowspan="1" colspan="1">P6<sub>1</sub>22</td></tr><tr><td rowspan="1" colspan="1">Unit cell dimensions, <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td rowspan="1" colspan="1">52.8, 81.4, 57.7; β = 107.85°</td><td rowspan="1" colspan="1">131.5, 131.5, 188</td><td rowspan="1" colspan="1">207.4, 207.4, 117.9</td><td rowspan="1" colspan="1">87.1, 87.1, 201.6</td></tr><tr><td rowspan="1" colspan="1">No. of reflections</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">    Total</td><td rowspan="1" colspan="1">355,272 (26,772)</td><td rowspan="1" colspan="1">1,068,014 (102,923)</td><td rowspan="1" colspan="1">324,544 (32,355)</td><td rowspan="1" colspan="1">1,366,812 (129,645)</td></tr><tr><td rowspan="1" colspan="1">    Unique</td><td rowspan="1" colspan="1">99,136 (9,762)</td><td rowspan="1" colspan="1">36,775 (3,625)</td><td rowspan="1" colspan="1">39,362 (3,898)</td><td rowspan="1" colspan="1">62,808 (6,068)</td></tr><tr><td rowspan="1" colspan="1">Multiplicity</td><td rowspan="1" colspan="1">3.6 (2.7)</td><td rowspan="1" colspan="1">29.0 (28.4)</td><td rowspan="1" colspan="1">8.2 (8.3)</td><td rowspan="1" colspan="1">21.8 (21.4)</td></tr><tr><td rowspan="1" colspan="1">Completeness (%)</td><td rowspan="1" colspan="1">99.71 (98.82)</td><td rowspan="1" colspan="1">99.63 (99.42)</td><td rowspan="1" colspan="1">99.96 (100.00)</td><td rowspan="1" colspan="1">98.4 (96.98)</td></tr><tr><td rowspan="1" colspan="1">Mean <italic>I</italic>/σ〈<italic>I</italic>〉</td><td rowspan="1" colspan="1">15.57 (2.29)</td><td rowspan="1" colspan="1">24.93 (6.71)</td><td rowspan="1" colspan="1">20.98 (2.36)</td><td rowspan="1" colspan="1">38.52 (5.03)</td></tr><tr><td rowspan="1" colspan="1">Wilson B-factor</td><td rowspan="1" colspan="1">11.91</td><td rowspan="1" colspan="1">31.14</td><td rowspan="1" colspan="1">43.91</td><td rowspan="1" colspan="1">17.86</td></tr><tr><td rowspan="1" colspan="1"><italic>R</italic><sub>merge</sub></td><td rowspan="1" colspan="1">0.04759 (0.4513)</td><td rowspan="1" colspan="1">0.1428 (0.7178)</td><td rowspan="1" colspan="1">0.09159 (1.197)</td><td rowspan="1" colspan="1">0.05559 (0.7748)</td></tr><tr><td rowspan="1" colspan="1">CC<sub>1/2</sub><xref ref-type="table-fn" rid="ngtab2.2"><sup>b</sup></xref></td><td rowspan="1" colspan="1">0.999 (0.759)</td><td rowspan="1" colspan="1">0.999 (0.982)</td><td rowspan="1" colspan="1">0.999 (0.794)</td><td rowspan="1" colspan="1">1.000 (0.933)</td></tr><tr><td rowspan="1" colspan="1">CC*<xref ref-type="table-fn" rid="ngtab2.3"><sup>c</sup></xref></td><td rowspan="1" colspan="1">1.000 (0.929)</td><td rowspan="1" colspan="1">1.000 (0.995)</td><td rowspan="1" colspan="1">1.000 (0.941)</td><td rowspan="1" colspan="1">1.000 (0.982)</td></tr><tr><td rowspan="1" colspan="1"><italic>R</italic><sub>work</sub></td><td rowspan="1" colspan="1">0.1468 (0.2597)</td><td rowspan="1" colspan="1">0.2178 (0.2788)</td><td rowspan="1" colspan="1">0.1975 (0.3018)</td><td rowspan="1" colspan="1">0.1560 (0.2008)</td></tr><tr><td rowspan="1" colspan="1"><italic>R</italic><sub>free</sub></td><td rowspan="1" colspan="1">0.1738 (0.2632)</td><td rowspan="1" colspan="1">0.2482 (0.2978)</td><td rowspan="1" colspan="1">0.2260 (0.3219)</td><td rowspan="1" colspan="1">0.1712 (0.2019)</td></tr><tr><td rowspan="1" colspan="1">No. of non-hydrogen atoms</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">    All</td><td rowspan="1" colspan="1">4,562</td><td rowspan="1" colspan="1">4,319</td><td rowspan="1" colspan="1">3,678</td><td rowspan="1" colspan="1">2,328</td></tr><tr><td rowspan="1" colspan="1">    Macromolecules</td><td rowspan="1" colspan="1">4,079</td><td rowspan="1" colspan="1">3,974</td><td rowspan="1" colspan="1">3,425</td><td rowspan="1" colspan="1">1,985</td></tr><tr><td rowspan="1" colspan="1">    Ligands</td><td rowspan="1" colspan="1">39</td><td rowspan="1" colspan="1">116</td><td rowspan="1" colspan="1">127</td><td rowspan="1" colspan="1">25</td></tr><tr><td rowspan="1" colspan="1">    Water</td><td rowspan="1" colspan="1">444</td><td rowspan="1" colspan="1">229</td><td rowspan="1" colspan="1">126</td><td rowspan="1" colspan="1">318</td></tr><tr><td rowspan="1" colspan="1">No. of protein residues</td><td rowspan="1" colspan="1">506</td><td rowspan="1" colspan="1">492</td><td rowspan="1" colspan="1">446</td><td rowspan="1" colspan="1">260</td></tr><tr><td rowspan="1" colspan="1">RMSD</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">    Bond length (Å)</td><td rowspan="1" colspan="1">0.008</td><td rowspan="1" colspan="1">0.007</td><td rowspan="1" colspan="1">0.005</td><td rowspan="1" colspan="1">0.009</td></tr><tr><td rowspan="1" colspan="1">    Bond angle (°)</td><td rowspan="1" colspan="1">1.15</td><td rowspan="1" colspan="1">0.96</td><td rowspan="1" colspan="1">0.87</td><td rowspan="1" colspan="1">1.18</td></tr><tr><td rowspan="1" colspan="1">Ramachandran statistics</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">    Favored (%)</td><td rowspan="1" colspan="1">98</td><td rowspan="1" colspan="1">95</td><td rowspan="1" colspan="1">97</td><td rowspan="1" colspan="1">98</td></tr><tr><td rowspan="1" colspan="1">    Outliers (%)</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">0.41</td><td rowspan="1" colspan="1">0.23</td><td rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">    Clash score</td><td rowspan="1" colspan="1">0.5</td><td rowspan="1" colspan="1">2.13</td><td rowspan="1" colspan="1">0.86</td><td rowspan="1" colspan="1">1.27</td></tr><tr><td rowspan="1" colspan="1">Avg B-factors</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">    All</td><td rowspan="1" colspan="1">16.1</td><td rowspan="1" colspan="1">53.2</td><td rowspan="1" colspan="1">53</td><td rowspan="1" colspan="1">25.4</td></tr><tr><td rowspan="1" colspan="1">    Macromolecules</td><td rowspan="1" colspan="1">15.2</td><td rowspan="1" colspan="1">53.5</td><td rowspan="1" colspan="1">52.5</td><td rowspan="1" colspan="1">22.9</td></tr><tr><td rowspan="1" colspan="1">    Ligands</td><td rowspan="1" colspan="1">24.7</td><td rowspan="1" colspan="1">61</td><td rowspan="1" colspan="1">71.1</td><td rowspan="1" colspan="1">47</td></tr><tr><td rowspan="1" colspan="1">    Solvent</td><td rowspan="1" colspan="1">24.4</td><td rowspan="1" colspan="1">42.9</td><td rowspan="1" colspan="1">47.6</td><td rowspan="1" colspan="1">39</td></tr></tbody></table> + + 18346 + Parameter Value(s) fora: SGBP-A apo SGBP-A/XyGO2 SGBP-B/XyGO2 SGBP-B (CD)/XyGO2 PDB ID no. 5E75 5E76 5E7G 5E7H Resolution (Å) 21.48–1.36 (1.409–1.36) 56.13–2.3 (2.382–2.3) 39.19–2.37 (2.455–2.37) 30.69–1.57 (1.626–1.570) Space group P21 I422 R32 P6122 Unit cell dimensions, a, b, c (Å) 52.8, 81.4, 57.7; β = 107.85° 131.5, 131.5, 188 207.4, 207.4, 117.9 87.1, 87.1, 201.6 No. of reflections     Total 355,272 (26,772) 1,068,014 (102,923) 324,544 (32,355) 1,366,812 (129,645)     Unique 99,136 (9,762) 36,775 (3,625) 39,362 (3,898) 62,808 (6,068) Multiplicity 3.6 (2.7) 29.0 (28.4) 8.2 (8.3) 21.8 (21.4) Completeness (%) 99.71 (98.82) 99.63 (99.42) 99.96 (100.00) 98.4 (96.98) Mean I/σ〈I〉 15.57 (2.29) 24.93 (6.71) 20.98 (2.36) 38.52 (5.03) Wilson B-factor 11.91 31.14 43.91 17.86 Rmerge 0.04759 (0.4513) 0.1428 (0.7178) 0.09159 (1.197) 0.05559 (0.7748) CC1/2b 0.999 (0.759) 0.999 (0.982) 0.999 (0.794) 1.000 (0.933) CC*c 1.000 (0.929) 1.000 (0.995) 1.000 (0.941) 1.000 (0.982) Rwork 0.1468 (0.2597) 0.2178 (0.2788) 0.1975 (0.3018) 0.1560 (0.2008) Rfree 0.1738 (0.2632) 0.2482 (0.2978) 0.2260 (0.3219) 0.1712 (0.2019) No. of non-hydrogen atoms     All 4,562 4,319 3,678 2,328     Macromolecules 4,079 3,974 3,425 1,985     Ligands 39 116 127 25     Water 444 229 126 318 No. of protein residues 506 492 446 260 RMSD     Bond length (Å) 0.008 0.007 0.005 0.009     Bond angle (°) 1.15 0.96 0.87 1.18 Ramachandran statistics     Favored (%) 98 95 97 98     Outliers (%) 0 0.41 0.23 0     Clash score 0.5 2.13 0.86 1.27 Avg B-factors     All 16.1 53.2 53 25.4     Macromolecules 15.2 53.5 52.5 22.9     Ligands 24.7 61 71.1 47     Solvent 24.4 42.9 47.6 39 + + + tab2.xml + tab2 + TABLE + table_footnote + 20239 + Numbers in parentheses are for the highest-resolution shell. + + + tab2.xml + tab2 + TABLE + table_footnote + 20300 + CC1/2, Pearson correlation coefficient between the average intensities of each subset. + + + tab2.xml + tab2 + TABLE + table_footnote + 20387 + CC*, Pearson correlation coefficient for correlation between the observed data set and true signal. + + + RESULTS + paragraph + 20487 + Seven of the eight backbone glucosyl residues of XyGO2 could be convincingly modeled in the ligand electron density, and only two α(1→6)-linked xylosyl residues were observed (Fig. 4B; cf. Fig. 1). Indeed, the electron density for the ligand suggests some disorder, which may arise from multiple oligosaccharide orientations along the binding site. Three aromatic residues—W82, W283, W306—comprise the flat platform that stacks along the naturally twisted β-glucan backbone (Fig. 4E). The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table 3; see Fig. S4 in the supplemental material). Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding. Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3. Most surprising in light of the saccharide-binding data, however, was a lack of extensive recognition of the XyG side chains; only Y84 appeared to provide a hydrophobic interface for a xylosyl residue (Xyl1). + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:43:17Z + + glucosyl + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:02:43Z + + ligand electron density + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:43:25Z + + α(1→6)-linked xylosyl + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:02:47Z + + electron density + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:02:17Z + + oligosaccharide + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:02:32Z + + binding site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:03:44Z + + W82 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:03:51Z + + W283 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:03:58Z + + W306 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:03:27Z + + flat platform + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:16Z + + stacks + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:03:37Z + + β-glucan + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:03:30Z + + platform + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:12Z + + W82A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:19Z + + W283A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:27Z + + W306A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:03:19Z + + mutant + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:49:42Z + + SGBP-A* + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:45:29Z + + completely devoid of XyG affinity + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:12Z + + W82A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:04:32Z + + mutant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:07Z + + Ka + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:27Z + + W306A + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:04Z + + substitution + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:45:00Z + + abolishes XyG binding + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:16Z + + hydrophobic interactions + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:16Z + + hydrogen-bonding interactions + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:43:30Z + + ligand + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:04:50Z + + R65 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:04:56Z + + N83 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:05:03Z + + S308 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:05:10Z + + Glc5 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:05:16Z + + Glc3 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:04:40Z + + saccharide-binding data + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:05:13Z + + XyG + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:05:24Z + + Y84 + + + site + SO: + melaniev@ebi.ac.uk + 2023-06-15T08:28:18Z + + hydrophobic interface + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:49:40Z + + xylosyl + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:05:32Z + + Xyl1 + + + + tab3.xml + tab3 + TABLE + table_caption + 21867 + Summary of thermodynamic parameters for site-directed mutants of SGBP-A and SGBP-B obtained by ITC with XyG at 25°Ca + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:57:05Z + + ITC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + + tab3.xml + tab3 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup span="1"><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/><col span="1"/></colgroup><thead><tr><th rowspan="2" colspan="1">Protein name</th><th colspan="2" rowspan="1"><italic>K<sub>a</sub></italic><hr/></th><th rowspan="2" colspan="1">Δ<italic>G</italic> (kcal ⋅ mol<sup>−1</sup>)</th><th rowspan="2" colspan="1">Δ<italic>H</italic> (kcal ⋅ mol<sup>−1</sup>)</th><th rowspan="2" colspan="1"><italic>T</italic>Δ<italic>S</italic> (kcal ⋅ mol<sup>−1</sup>)</th></tr><tr><th rowspan="1" colspan="1">Fold change<xref ref-type="table-fn" rid="ngtab3.2"><sup>b</sup></xref></th><th rowspan="1" colspan="1">M<sup>−1</sup></th></tr></thead><tbody><tr><td rowspan="1" colspan="1">SGBP-A(W82A W283A W306A)</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td></tr><tr><td rowspan="1" colspan="1">SGBP-A(W82A)<xref ref-type="table-fn" rid="ngtab3.3"><sup>c</sup></xref></td><td rowspan="1" colspan="1">4.9</td><td rowspan="1" colspan="1">9.1 × 10<sup>4</sup></td><td rowspan="1" colspan="1">−6.8</td><td rowspan="1" colspan="1">−6.3</td><td rowspan="1" colspan="1">0.5</td></tr><tr><td rowspan="1" colspan="1">SGBP-A(W306)</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td><td rowspan="1" colspan="1">NB</td></tr><tr><td rowspan="1" colspan="1">SGBP-B(230–489)</td><td rowspan="1" colspan="1">0.7</td><td rowspan="1" colspan="1">(8.6 ± 0.20) × 10<sup>4</sup></td><td rowspan="1" colspan="1">−6.7</td><td rowspan="1" colspan="1">−14.9 ± 0.1</td><td rowspan="1" colspan="1">−8.2</td></tr><tr><td rowspan="1" colspan="1">SGBP-B(Y363A)</td><td rowspan="1" colspan="1">19.7</td><td rowspan="1" colspan="1">(2.9 ± 0.10) × 10<sup>3</sup></td><td rowspan="1" colspan="1">−4.7</td><td rowspan="1" colspan="1">−18.1 ± 0.1</td><td rowspan="1" colspan="1">−13.3</td></tr><tr><td rowspan="1" colspan="1">SGBP-B(W364A)</td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">Weak</td><td rowspan="1" colspan="1">Weak</td><td rowspan="1" colspan="1">Weak</td><td rowspan="1" colspan="1">Weak</td></tr><tr><td rowspan="1" colspan="1">SGBP-B(F414A)</td><td rowspan="1" colspan="1">3.2</td><td rowspan="1" colspan="1">(1.80 ± 0.03) × 10<sup>4</sup></td><td rowspan="1" colspan="1">−5.8</td><td rowspan="1" colspan="1">−11.4 ± 0.1</td><td rowspan="1" colspan="1">−5.6</td></tr></tbody></table> + + 21985 + Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:07Z + + Ka + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-07-21T08:36:57Z + + ΔG + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-22T10:05:33Z + + ΔH + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-22T10:05:42Z + + TΔS + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:12Z + + W82A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:19Z + + W283A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:27Z + + W306A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:12Z + + W82A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:02Z + + SGBP-A + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:03:58Z + + W306 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:07:33Z + + 230–489 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:47:43Z + + Y363A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:47:55Z + + W364A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:49:54Z + + F414A + + + + tab3.xml + tab3 + TABLE + table_footnote + 22478 + Shown are average values ± standard deviations from two independent titrations, unless otherwise indicated. Binding thermodynamics are based on the concentration of the binding unit, XyGO2. Weak binding represents a Ka of <500 M−1. ND, not determined; NB, no binding. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:07Z + + Ka + + + + tab3.xml + tab3 + TABLE + table_footnote + 22749 + Ka fold change = Ka of wild-type protein/Ka of mutant protein for xyloglucan binding. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:07Z + + Ka + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:07Z + + Ka + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:34:04Z + + wild-type + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:07Z + + Ka + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:06:02Z + + xyloglucan + + + + tab3.xml + tab3 + TABLE + table_footnote + 22835 + Values from a single titration. + + + RESULTS + title_2 + 22867 + SGBP-B has a multimodular structure with a single, C-terminal glycan-binding domain. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:08:13Z + + glycan-binding domain + + + + RESULTS + paragraph + 22952 + The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). These domains also display similarity to the C-terminal β-sandwich domains of many GH13 enzymes, including the cyclodextrin glucanotransferase of Geobacillus stearothermophilus (Fig. 5B). Such domains are not typically involved in carbohydrate binding. Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig. S5 in the supplemental material), indicates that these domains do not contribute to XyG capture. On the other hand, production of the fused domains C and D in tandem (SGBP-B residues 230 to 489) retains complete binding of xyloglucan in vitro, with the observed slight increase in affinity likely arising from a reduced potential for steric hindrance of the smaller protein construct during polysaccharide interactions (Table 3). While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). The structure-based alignment of these proteins reveals 17% sequence identity, with a core RMSD of 3.6 Å for 253 aligned residues. While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:50:03Z + + crystal structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:15:21Z + + full-length + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:15:29Z + + in complex with + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:43:17Z + + Rwork + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:43:43Z + + Rfree + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:15:34Z + + 34 to 489 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:47:27Z + + structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:15:50Z + + tandem immunoglobulin (Ig)-like domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:16:02Z + + A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:16:05Z + + B + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:16:08Z + + C + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:16:21Z + + xyloglucan-binding domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:17:18Z + + D + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:16:27Z + + A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:16:31Z + + B + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:16:34Z + + C + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:16:43Z + + β-sandwich folds + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:16:46Z + + B + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:16:49Z + + 134 to 230 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:16:53Z + + C + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:16:55Z + + 231 to 313 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:17:22Z + + superimposed + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:17:37Z + + A + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:17:40Z + + 34 to 133 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:18:01Z + + RMSDs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:18:07Z + + These domains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:18:15Z + + β-sandwich domains + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:18:18Z + + GH13 enzymes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:18:21Z + + cyclodextrin glucanotransferase + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:18:24Z + + Geobacillus stearothermophilus + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:18:28Z + + Such domains + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:06:17Z + + carbohydrate + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:18:31Z + + visual inspection + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:18:34Z + + structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:18:39Z + + A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:18:42Z + + B + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:57:12Z + + affinity PAGE + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:06:29Z + + XyG + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:18:49Z + + production + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:18:56Z + + fused domains C and D + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:19:04Z + + 230 to 489 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:19:23Z + + xyloglucan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:50:42Z + + polysaccharide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:15:21Z + + full-length + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:19:50Z + + D + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:19:54Z + + XyG-binding proteins + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:19:58Z + + xylan-binding protein + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T19:20:01Z + + Bacova_04391 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:07:21Z + + xylan + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:31Z + + PUL + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:52Z + + B. ovatus + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:20:08Z + + structure-based alignment + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:45:04Z + + RMSD + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T19:20:21Z + + Bacova_04391 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:20:25Z + + binding site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:20:14Z + + W241 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:20:17Z + + Y404 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:20:28Z + + XyGO binding site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:20:34Z + + opposing, clamp-like arrangement + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:20:36Z + + these residues + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T19:20:39Z + + Bacova_04391 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:20:42Z + + planar surface arrangement + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:20:45Z + + residues + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + + mbo0021627940005.jpg + fig5 + FIG + fig_caption + 25179 + Multimodular structure of SGBP-B (Bacova_02650). (A) Full-length structure of SGBP-B, color coded by domain as indicated. Prolines between domains are indicated as spheres. An omit map (2σ) for XyGO2 is displayed to highlight the location of the glycan-binding site. (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). (D) Close-up omit map for the XyGO2 ligand, contoured at 2σ. (E) Stereo view of the xyloglucan-binding site of SGBP-B, displaying all residues within 4 Å of the ligand. The backbone glucose residues are numbered from the nonreducing end, xylose residues are shown as X1, X2, and X3, potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T19:24:43Z + + Bacova_02650 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:15:21Z + + Full-length + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:24:48Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:24:52Z + + Prolines + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:24:59Z + + omit map + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:56:50Z + + glycan-binding site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:25:09Z + + A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:25:13Z + + B + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:25:16Z + + C + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:25:25Z + + Ig-like domain + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:25:29Z + + G. stearothermophilus + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:25:34Z + + cyclodextrin glucanotransferase + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:25:38Z + + 375 to 493 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:25:44Z + + overlay + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T19:25:48Z + + Bacova_04391 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:24:59Z + + omit map + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:57:08Z + + xyloglucan-binding site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:25:52Z + + glucose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:25:55Z + + xylose + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:25:59Z + + X1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:26:02Z + + X2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:26:07Z + + X3 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:16Z + + hydrogen-bonding interactions + + + + RESULTS + paragraph + 26120 + Inspection of the tertiary structure indicates that domains C and D are effectively inseparable, with a contact interface of 396 Å2. Domains A, B, and C do not pack against each other. Moreover, the five-residue linkers between these first three domains all feature a proline as the middle residue, suggesting significant conformational rigidity (Fig. 5A). Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface. Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-β-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:31:26Z + + structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:31:30Z + + C + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:31:34Z + + D + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:31:37Z + + A + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:31:41Z + + B + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:31:44Z + + C + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:31:48Z + + five-residue linkers + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:31:52Z + + proline + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:31:55Z + + middle residue + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:31:58Z + + proline + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:32:02Z + + Ig-like domains + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T19:32:05Z + + Bacova_04391 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:32:09Z + + starch-binding proteins + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:33Z + + SusE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:44Z + + SusF + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:32:14Z + + glycan binding site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:32:22Z + + eight-residue linker + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:32:49Z + + lipidated + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:32:55Z + + Cys + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:32:58Z + + C28 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:33:01Z + + first β-strand + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:33:21Z + + A + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:33:26Z + + outer membrane proteins + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-16T19:33:29Z + + Sus-like systems + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:33:33Z + + 10- to 20-amino-acid flexible linker + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:32:49Z + + lipidated + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:33:38Z + + Cys + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:33:41Z + + outer membrane-anchored + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:33:44Z + + endo-xyloglucanase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:48:42Z + + BoGH5 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:33:51Z + + 100-amino-acid, all-β-strand + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:33:54Z + + N-terminal module + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:33:57Z + + flexible linker + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:34:00Z + + catalytic module + + + + RESULTS + paragraph + 27551 + XyG binds to domain D of SGBP-B at the concave interface of the top β-sheet, with binding mediated by loops connecting the β-strands. Six glucosyl residues, comprising the main chain, and three branching xylosyl residues of XyGO2 can be modeled in the density (Fig. 5D; cf. Fig. 1A). The backbone is flat, with less of the “twisted-ribbon” geometry observed in some cello- and xylogluco-oligosaccharides. The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table 3; see Fig. S6 in the supplemental material). There are no additional contacts between the protein and the β-glucan backbone and surprisingly few interactions with the side-chain xylosyl residues, despite that fact that ITC data demonstrate that SGBP-B does not measurably bind the cellohexaose (Table 1). F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1. Surprisingly, an F414A mutant of SGBP-B displays only a mild 3-fold decrease in the Ka value for XyG, again suggesting that glycan recognition is primarily mediated via contact with the β-glucan backbone (Table 3; see Fig. S6). Additional residues surrounding the binding site, including Y369 and E412, may contribute to the recognition of more highly decorated XyG, but precisely how this is mediated is presently unclear. Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). The CD domains of the truncated and full-length proteins superimpose with a 0.4-Å RMSD of the Cα backbone, with no differences in the position of any of the glycan-binding residues (see Fig. S7A in the supplemental material). While density is observed for XyGO2, the ligand could not be unambiguously modeled into this density to achieve a reasonable fit between the X-ray data and the known stereochemistry of the sugar (see Fig. S7B and C). While this may occur for a number of reasons in crystal structures, it is likely that the poor ligand density even at higher resolution is due to movement or multiple orientations of the sugar averaged throughout the lattice. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:46:03Z + + binds to + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:46:07Z + + D + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:46:10Z + + concave interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:46:14Z + + β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:46:17Z + + loops + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:46:21Z + + β-strands + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:46:25Z + + glucosyl + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:49:40Z + + xylosyl + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:46:42Z + + density + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:46:47Z + + cello- and xylogluco-oligosaccharides + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T18:46:34Z + + aromatic platform + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:46:59Z + + W330 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:47:07Z + + W364 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:47:14Z + + Y363 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:47:21Z + + glucosyl + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:47:24Z + + longer + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:47:27Z + + platform + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:03Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:47:36Z + + glucosyl + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:47:43Z + + Y363A + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:47:46Z + + site-directed mutant + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:49Z + + SGBP-B + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:07Z + + Ka + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:44Z + + XyG + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:47:55Z + + W364A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:47:59Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:48:08Z + + lacks XyG binding + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:48:15Z + + β-glucan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:49:40Z + + xylosyl + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:57:05Z + + ITC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:08:04Z + + cellohexaose + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:49:03Z + + F414 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:16Z + + stacks + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:49:40Z + + xylosyl + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:05:16Z + + Glc3 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:49:21Z + + Q407 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:16Z + + hydrogen bonding + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:49:40Z + + xylosyl + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:05:33Z + + Xyl1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:49:54Z + + F414A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:49:59Z + + mutant + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:35:07Z + + Ka + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:45Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:08:17Z + + glycan + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:50:04Z + + residues + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:50:07Z + + binding site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:50:14Z + + Y369 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:50:22Z + + E412 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:45Z + + XyG + + + protein + PR: + melaniev@ebi.ac.uk + 2023-06-15T08:38:53Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-06-15T08:39:01Z + + xyloglucan + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:50:33Z + + solved + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:50:03Z + + crystal structure + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:50:38Z + + fused CD domains + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:15:29Z + + in complex with + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:43:17Z + + Rwork + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:43:43Z + + Rfree + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:50:46Z + + 230 to 489 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:50:49Z + + CD domains + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:50:52Z + + truncated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:15:21Z + + full-length + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:50:57Z + + superimpose + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:45:04Z + + RMSD + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T19:51:01Z + + glycan-binding residues + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:51:05Z + + density + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:51:08Z + + density + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-22T10:08:41Z + + X-ray data + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:51:14Z + + crystal structures + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:51:11Z + + sugar + + + + RESULTS + title_2 + 30050 + SGBP-A and SGBP-B have distinct, coordinated functions in vivo. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:03Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + + RESULTS + paragraph + 30114 + The similarity of the glycan specificity of SGBP-A and SGBP-B presents an intriguing conundrum regarding their individual roles in XyG utilization by B. ovatus. To disentangle the functions of SGBP-A and SGBP-B in XyG recognition and uptake, we created individual in-frame deletion and complementation mutant strains of B. ovatus. In these growth experiments, overnight cultures of strains grown on minimal medium plus glucose were back-diluted 1:100-fold into minimal medium containing 5 mg/ml of the reported carbohydrate. Growth on glucose displayed the shortest lag time for each strain, and so lag times were normalized for each carbohydrate by subtracting the lag time of that strain in glucose (Fig. 6; see Fig. S8 in the supplemental material). A strain in which the entire XyGUL is deleted displays a lag of 24.5 h during growth on glucose compared to the isogenic parental wild-type (WT) Δtdk strain, for which exponential growth lags for 19.8 h (see Fig. S8D). It is unknown whether this is because cultures were not normalized by the starting optical density (OD) or viable cells or reflects a minor defect for glucose utilization. The former seems more likely as the growth rates are nearly identical for these strains on glucose and xylose. The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). The reason for this observation on XyGO2 is unclear, as the ΔSGBP-B mutant does not have a significantly different growth rate from the WT on XyGO2. Therefore, we limit our discussion to those mutants that displayed the most obvious defects in growth on particular substrates. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:03Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:08:58Z + + XyG + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:52Z + + B. ovatus + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:03Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:09:09Z + + XyG + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:57:08Z + + in-frame deletion and complementation mutant + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:52Z + + B. ovatus + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:57:11Z + + growth experiments + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:57:19Z + + glucose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:57:16Z + + carbohydrate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:57:24Z + + glucose + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:24:50Z + + lag time + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:25:13Z + + lag times + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:57:21Z + + carbohydrate + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:24:52Z + + lag time + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:57:27Z + + glucose + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:57:30Z + + deleted + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:58:46Z + + lag + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:57:32Z + + glucose + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:34:04Z + + wild-type + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:57:38Z + + WT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:57:45Z + + Δtdk + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:59:09Z + + lags + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:09:23Z + + glucose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:57:58Z + + glucose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:58:01Z + + xylose + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:57:53Z + + ΔXyGUL + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:57:38Z + + WT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:57:45Z + + Δtdk + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:25:15Z + + lag times + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:58:03Z + + xylose + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:24:52Z + + lag time + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:58:05Z + + xylose + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:57:38Z + + WT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:57:45Z + + Δtdk + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:58:28Z + + Complementation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:43Z + + ΔSGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:43Z + + ΔSGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:03Z + + SGBP-A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:34:04Z + + wild-type + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T19:58:24Z + + xyloglucan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:36:33Z + + XyGO1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:57:38Z + + WT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:57:45Z + + Δtdk + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:26Z + + ΔSGBP-B + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:58:14Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:57:38Z + + WT + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + + mbo0021627940006.jpg + fig6 + FIG + fig_caption + 32273 + Growth of select XyGUL mutants on xyloglucan and oligosaccharides. B. ovatus mutants were created in a thymidine kinase deletion (Δtdk) mutant as described previously. SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649. Growth was measured over time in minimal medium containing (A) XyG, (B) XyGO2, (C) XyGO1, (D) glucose, and (E) xylose. In panel F, the growth rate of each strain on the five carbon sources is displayed, and in panel G, the normalized lag time of each culture, relative to its growth on glucose, is displayed. Solid bars indicate conditions that are not statistically significant from the WT Δtdk cultures grown on the indicated carbohydrate, while open bars indicate a P value of <0.005 compared to the WT Δtdk strain. Conditions denoted by the same letter (b, c, or d) are not statistically significant from each other but are significantly different from the condition labeled “a.” Complementation of ΔSGBP-A and ΔSBGP-B was performed by allelic exchange of the wild-type genes back into the genome for expression via the native promoter: these growth curves, quantified rates and lag times are displayed in Fig. S8 in the supplemental material. + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:01:10Z + + xyloglucan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:01:14Z + + oligosaccharides + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:40:58Z + + B. ovatus + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:01:17Z + + thymidine kinase deletion + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:57:45Z + + Δtdk + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:01:24Z + + SGBP-A* + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T20:01:28Z + + Bacova_02651 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:12Z + + W82A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:19Z + + W283A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:27Z + + W306A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T20:01:33Z + + GH9 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T20:01:36Z + + Bacova_02649 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:45Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:36:33Z + + XyGO1 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:01:44Z + + glucose + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:01:50Z + + xylose + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:24:52Z + + lag time + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:01:53Z + + glucose + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:57:38Z + + WT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:57:45Z + + Δtdk + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:01:56Z + + carbohydrate + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:57:38Z + + WT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:57:45Z + + Δtdk + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:43Z + + ΔSGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:49:49Z + + ΔSBGP-B + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T18:34:04Z + + wild-type + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:25:15Z + + lag times + + + + RESULTS + paragraph + 33503 + The ΔSGBP-A (ΔBacova_02651) strain (cf. Fig. 1B) was completely incapable of growth on XyG, XyGO1, and XyGO2, indicating that SGBP-A is essential for XyG utilization (Fig. 6). This result mirrors our previous data for the canonical Sus of B. thetaiotaomicron, which revealed that a homologous ΔsusD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins. More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. Similarly, the function of SGBP-A extends beyond glycan binding. Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5 g/liter maltose was required to restore growth of the ΔsusD::SusD* strain on starch, which nonetheless occurred following an extended lag phase. In contrast, the ΔSGBP-A::SGBP-A* strain does not display an extended lag time on any of the xyloglucan substrates compared to the WT (Fig. 6). The specific glycan signal that upregulates BoXyGUL is currently unknown. From our present data, we cannot eliminate the possibility that the glycan binding by SGBP-A enhances transcriptional activation of the XyGUL. However, the modest rate defect displayed by the SGBP-A::SGBP-A* strain suggests that recognition of XyG and product import is somewhat less efficient in these cells. + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:43Z + + ΔSGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:07:26Z + + ΔBacova_02651 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:45Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:36:33Z + + XyGO1 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:03Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:09:56Z + + XyG + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-16T16:06:29Z + + Sus + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:08:28Z + + B. thetaiotaomicron + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:07:49Z + + ΔsusD + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:07:42Z + + mutant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:07:54Z + + starch + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:07:56Z + + malto-oligosaccharides + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:31Z + + PUL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:07:59Z + + complementation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:07:50Z + + ΔsusD + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:08:03Z + + SusD* + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:08:06Z + + triple site-directed mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:49:53Z + + W96A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:49:56Z + + W320A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:49:58Z + + Y296A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:08:15Z + + ablates glycan binding + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:08:28Z + + B. thetaiotaomicron + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:08:21Z + + malto-oligosaccharides + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:08:25Z + + starch + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T21:41:21Z + + sus + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:08:27Z + + maltose + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:03Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:08:31Z + + Complementation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:43Z + + ΔSGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:08:34Z + + SGBP-A* + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:12Z + + W82A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:20Z + + W283A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:04:27Z + + W306A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:09:06Z + + not bind + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:45Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:45Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:43:36Z + + XyGOs + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:43Z + + ΔSGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:08:37Z + + SGBP-A* + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:57:38Z + + WT + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:10:09Z + + carbohydrate + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:09:22Z + + maltose + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:07:50Z + + ΔsusD + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:09:24Z + + SusD* + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:09:26Z + + starch + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:26:19Z + + lag phase + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:43Z + + ΔSGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:09:29Z + + SGBP-A* + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:24:52Z + + lag time + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T20:09:32Z + + xyloglucan + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:57:38Z + + WT + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:12:53Z + + BoXyGUL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:03Z + + SGBP-A + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:03Z + + SGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:09:39Z + + SGBP-A* + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:45Z + + XyG + + + + RESULTS + paragraph + 35481 + Intriguingly, the ΔSGBP-B strain (ΔBacova_02650) (cf. Fig. 1B) exhibited a minor growth defect on both XyG and XyGO2, with rates 84.6% and 93.9% that of the WT Δtdk strain. However, growth of the ΔSGBP-B strain on XyGO1 was 54.2% the rate of the parental strain, despite the fact that SGBP-B binds this substrate ca. 10-fold more weakly than XyGO2 and XyG (Fig. 6; Table 1). As such, the data suggest that SGBP-A can compensate for the loss of function of SGBP-B on longer oligo- and polysaccharides, while SGBP-B may adapt the cell to recognize smaller oligosaccharides efficiently. Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B. ovatus to scavenge smaller XyGOs liberated by other gut commensals. This additional role of SGBP-B is especially notable in the context of studies on BtSusE and BtSusF (positioned similarly in the archetypal Sus locus) (Fig. 1B), for which growth defects on starch or malto-oligosaccharides have never been observed. Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface. However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig. 6; ΔSGBP-B/ΔGH9). + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:26Z + + ΔSGBP-B + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:23:05Z + + ΔBacova_02650 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:45Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T19:57:38Z + + WT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T19:57:45Z + + Δtdk + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:26Z + + ΔSGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:36:33Z + + XyGO1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:45Z + + XyG + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:03Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:43:40Z + + oligo- and polysaccharides + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:43:44Z + + oligosaccharides + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:23:11Z + + double mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:23:14Z + + crippled + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:03Z + + SGBP-A + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:23:17Z + + deletion of + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:43Z + + ΔSGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:23:20Z + + SGBP-A* + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:26Z + + ΔSGBP-B + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:24:52Z + + lag time + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:45Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:45:22Z + + XyGO2 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:36:33Z + + XyGO1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:04Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:45Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:50:42Z + + polysaccharide + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:52Z + + B. ovatus + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:43:50Z + + XyGOs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T20:23:32Z + + BtSusE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T20:23:34Z + + BtSusF + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:25:37Z + + Sus locus + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:43:53Z + + starch + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:43:56Z + + malto-oligosaccharides + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:04Z + + SGBP-A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:23:37Z + + catalytically feeble + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:23:41Z + + endo-xyloglucanase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T20:23:44Z + + GH9 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:13:27Z + + GH5 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:24:01Z + + combined deletion of the genes encoding + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T21:47:32Z + + GH9 + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T20:23:56Z + + Bacova_02649 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T18:36:33Z + + XyGO1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:26Z + + ΔSGBP-B + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:24:05Z + + ΔGH9 + + + + RESULTS + paragraph + 37123 + The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B. thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs. Our previous work demonstrates that B. ovatus cells grown in minimal medium plus glucose express low levels of the XyGUL transcript. Thus, in our experiments, we presume that each strain, initially grown in glucose, expresses low levels of the XyGUL transcript and thus low levels of the XyGUL-encoded surface proteins, including the vanguard GH5. Presumably without glycan binding by the SGBPs, the GH5 protein cannot efficiently process xyloglucan, and/or the lack of SGBP function prevents efficient capture and import of the processed oligosaccharides. It may then be that only after a sufficient amount of glycan is processed and imported by the cell is XyGUL upregulated and exponential growth on the glycan can begin. We hypothesize that during exponential growth the essential role of SGBP-A extends beyond glycan recognition, perhaps due to a critical interaction with the TBDT. In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. Likewise, such cognate interactions between homologous protein pairs such as SGBP-A and its TBDT may underlie our observation that a ΔSGBP-A mutant cannot grow on xyloglucan. However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:18:27Z + + SGBP-A* + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:43Z + + ΔSGBP-A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:18:30Z + + SGBP-A* + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:26Z + + ΔSGBP-B + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:18:33Z + + mutant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:24:17Z + + lag + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:49:45Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:18:37Z + + xylogluco-oligosaccharides + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:24:17Z + + lag + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:18:54Z + + malto-oligosaccharide + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:08:28Z + + B. thetaiotaomicron + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T20:24:17Z + + lag + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:44:01Z + + xyloglucan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:02Z + + SGBPs + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:52Z + + B. ovatus + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:19:03Z + + glucose + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:19:05Z + + glucose + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:13:27Z + + GH5 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:03Z + + SGBPs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:13:27Z + + GH5 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:19:12Z + + xyloglucan + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T10:10:53Z + + SGBP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:19:16Z + + oligosaccharides + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:15Z + + XyGUL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:04Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:19:25Z + + TBDT + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T21:19:38Z + + BtSus + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:18Z + + SusD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:19:27Z + + TBDT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:06:59Z + + SusC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:25Z + + glycan + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:07:50Z + + ΔsusD + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:48:22Z + + mutant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:19:45Z + + starch + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T20:07:50Z + + ΔsusD + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:19:54Z + + SusD* + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:20:06Z + + transcriptional activator + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T21:19:42Z + + sus operon + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:04Z + + SGBP-A + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:23Z + + TBDT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:51:43Z + + ΔSGBP-A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:20:20Z + + mutant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:20:23Z + + xyloglucan + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-16T16:06:29Z + + Sus + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:20:16Z + + elimination of + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:33Z + + SusE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:45Z + + SusF + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:20:26Z + + starch + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:20:28Z + + xylogluco-oligosaccharides + + + + RESULTS + title_2 + 39216 + Conclusions. + + + RESULTS + paragraph + 39229 + The ability of gut-adapted microorganisms to thrive in the gastrointestinal tract is critically dependent upon their ability to efficiently recognize, cleave, and import glycans. The human gut, in particular, is a densely packed ecosystem with hundreds of species, in which there is potential for both competition and synergy in the utilization of different substrates. Recent work has elucidated that Bacteroidetes cross-feed during growth on many glycans; the glycoside hydrolases expressed by one species liberate oligosaccharides for consumption by other members of the community. Thus, understanding glycan capture at the cell surface is fundamental to explaining, and eventually predicting, how the carbohydrate content of the diet shapes the gut community structure as well as its causative health effects. Here, we demonstrate that the surface glycan binding proteins encoded within the BoXyGUL play unique and essential roles in the acquisition of the ubiquitous and abundant vegetable polysaccharide xyloglucan. Yet, a number of questions remain regarding the molecular interplay of SGBPs with their cotranscribed cohort of glycoside hydrolases and TonB-dependent transporters. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:41:31Z + + microorganisms + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:23:32Z + + glycans + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:23:38Z + + glycans + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:25:57Z + + glycoside hydrolases + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:24:00Z + + oligosaccharides + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:23:42Z + + surface glycan binding proteins + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:12:53Z + + BoXyGUL + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:50:28Z + + vegetable + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:50:42Z + + polysaccharide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:23:54Z + + xyloglucan + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:03Z + + SGBPs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:25:57Z + + glycoside hydrolases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:23:47Z + + TonB-dependent transporters + + + + RESULTS + paragraph + 40417 + A particularly understudied aspect of glycan utilization is the mechanism of import via TBDTs (SusC homologs) (Fig. 1), which are ubiquitous and defining components of all PUL. PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP. A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus. Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig. 1C). It is yet presently unclear whether this interaction is static or dynamic and to what extent the association of cognate TBDT/SGBPs is dependent upon the structure of the carbohydrate to be imported. On the other hand, there is clear evidence for independent TBDTs in Bacteroidetes that do not require SGBP association for activity. For example, it was recently demonstrated that expression of nanO, which encodes a SusC-like TBDT as part of a sialic-acid-targeting PUL from B. fragilis, restored growth on this monosaccharide in a mutant strain of E. coli. In this instance, coexpression of the susD-like gene nanU was not required, nor did the expression of the nanU gene enhance growth kinetics. Similarly, the deletion of BT1762 encoding a fructan-targeting SusD-like protein in B. thetaiotaomicron did not result in a dramatic loss of growth on fructans. Thus, the strict dependence on a SusD-like SGBP for glycan uptake in the Bacteroidetes may be variable and substrate dependent. Furthermore, considering the broader distribution of TBDTs in PUL lacking SGBPs (sometimes known as carbohydrate utilization containing TBDT [CUT] loci; see reference and reviewed in reference) across bacterial phyla, it appears that the intimate biophysical association of these substrate-transport and -binding proteins is the result of specific evolution within the Bacteroidetes. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:28Z + + glycan + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:29:12Z + + TBDTs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:06:59Z + + SusC + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:31Z + + PUL + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:31Z + + PUL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:29:12Z + + TBDTs + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:30Z + + iron-targeting TBDTs + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:29:42Z + + Proteobacteria + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:33Z + + glycan-importing TBDTs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:37Z + + SGBP + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T21:29:33Z + + BtSusC + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:40Z + + TBDT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:19Z + + SusD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:43Z + + SGBP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:29Z + + glycan + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:31Z + + PUL + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:41:02Z + + Capnocytophaga canimorsus + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T16:07:19Z + + SusD + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:04Z + + SGBP-A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:12:13Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:12:05Z + + XyG + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:29Z + + glycan + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:47Z + + SusC-like TBDTs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:51Z + + SusD-like SGBPs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:29Z + + glycan + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:53Z + + TBDT + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:03Z + + SGBPs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:29:51Z + + carbohydrate + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:29:12Z + + TBDTs + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:56Z + + SGBP + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T21:29:59Z + + nanO + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:45:59Z + + SusC-like TBDT + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:31Z + + PUL + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:30:06Z + + B. fragilis + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:29:53Z + + monosaccharide + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:41:05Z + + E. coli + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T16:31:21Z + + susD + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T21:41:26Z + + nanU + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T21:30:01Z + + nanU + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T21:30:03Z + + BT1762 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:30:12Z + + fructan-targeting SusD-like protein + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:08:28Z + + B. thetaiotaomicron + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:29:55Z + + fructans + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:30:14Z + + SusD-like SGBP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:29Z + + glycan + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:29:12Z + + TBDTs + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:31Z + + PUL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:03Z + + SGBPs + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T21:30:23Z + + carbohydrate utilization containing TBDT [CUT] loci + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:31Z + + bacterial + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + + RESULTS + paragraph + 42733 + Equally intriguing is the observation that while SusD-like proteins such as SGBP-A share moderate primary and high tertiary structural conservation, the genes for the SGBPs encoded immediately downstream (Fig. 1B [sometimes referred to as “susE positioned”]) encode glycan-binding lipoproteins with little or no sequence or structural conservation, even among syntenic PUL that target the same polysaccharide. Such is the case for XyGUL from related Bacteroides species, which may encode either one or two of these predicted SGBPs, and these proteins vary considerably in length. The extremely low similarity of these SGBPs is striking in light of the moderate sequence conservation observed among homologous GHs in syntenic PUL. This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface. Because the intestinal ecosystem is a dense consortium of bacteria that must compete for their nutrients, these multimodular SGBPs may reflect ongoing evolutionary experiments to enhance glycan uptake efficiency. Whether organisms that express longer SGBPs, extending further above the cell surface toward the extracellular environment, are better equipped to compete for available carbohydrates is presently unknown. However, the natural diversity of these proteins represents a rich source for the discovery of unique carbohydrate-binding motifs to both inform gut microbiology and generate new, specific carbohydrate analytical reagents. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:32:20Z + + SusD-like proteins + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:49:04Z + + SGBP-A + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:03Z + + SGBPs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:32:23Z + + glycan-binding lipoproteins + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:31Z + + PUL + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:50:42Z + + polysaccharide + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:16Z + + XyGUL + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T21:32:51Z + + Bacteroides + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:03Z + + SGBPs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:03Z + + SGBPs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T16:12:09Z + + GHs + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:31Z + + PUL + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:03Z + + SGBPs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T21:32:57Z + + BtSusE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T21:33:00Z + + BtSusF + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T15:48:16Z + + XyGUL + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:48:50Z + + SGBP-B + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:50:42Z + + polysaccharide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:29Z + + glycan + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:54:55Z + + bacteria + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:03Z + + SGBPs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:29Z + + glycan + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:47:03Z + + SGBPs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:33:08Z + + carbohydrates + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T21:33:10Z + + carbohydrate-binding motifs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:33:12Z + + carbohydrate + + + + RESULTS + paragraph + 44381 + In conclusion, the present study further illuminates the essential role that surface-glycan binding proteins play in facilitating the catabolism of complex dietary carbohydrates by Bacteroidetes. The ability of our resident gut bacteria to recognize polysaccharides is the first committed step of glycan consumption by these organisms, a critical process that influences the community structure and thus the metabolic output (i.e., short-chain fatty acid and metabolite profile) of these organisms. A molecular understanding of glycan uptake by human gut bacteria is therefore central to the development of strategies to improve human health through manipulation of the microbiota. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T21:34:13Z + + surface-glycan binding proteins + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T21:34:16Z + + carbohydrates + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:47:40Z + + Bacteroidetes + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:54:55Z + + bacteria + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T17:14:06Z + + polysaccharides + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:29Z + + glycan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:11:30Z + + glycan + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:54:55Z + + bacteria + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:41:08Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T16:03:40Z + + microbiota + + + + METHODS + title_1 + 45063 + MATERIALS AND METHODS + + + METHODS + title_2 + 45085 + Protein production and purification. + + + METHODS + paragraph + 45122 + The gene fragments corresponding to Bacova_02650 (encoding SGBP-B residues 34 to 489) and Bacova_02651 (encoding SGBP-A residues 28 to 546) were amplified from Bacteroidetes ovatus ATCC 8483 genomic DNA by PCR using forward primers, including NdeI restriction sites, and reverse primers, including XhoI. The gene products were ligated into a modified version of pET-28a (EMD Biosciences) containing a recombinant tobacco etch virus (rTEV) protease recognition site (pET-28aTEV) preceding an N-terminal 6-His tag for affinity purification. The expression vector (pET-28TEV) containing SGBP-B was used for subsequent cloning of the domains A (residues 34 to 133), B (residues 134 to 229), and CD (residues 230 to 489). The pET-28TEV vector expressing residues 28 to 546 of SGBP-A was utilized for carbohydrate-binding experiments and crystallization of the apo structure. To obtain crystals of SGBP-A with XyGO2, the DNA sequence coding for residues 36 to 546 was PCR amplified from genomic DNA for ligation-independent cloning into the pETite N-His vector (Lucigen, Madison, WI) according to the manufacturer’s instructions. The N-terminal primer for pETite N-His insertion contained a TEV cleavage site immediately downstream of the complementary 18-bp overlap (encoding the His tag) to create a TEV-cleavable His-tagged protein. The site-directed mutants of SGBP-A and SGBP-B in pET-28TEV were created using the QuikChange II site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. The sequences of all primers to generate these constructs are displayed in Table S1 in the supplemental material. + + + METHODS + paragraph + 46758 + The plasmids containing the SGBP-A and SGBP-B genes were transformed into Escherichia coli BL21(DE3) or Rosetta(DE3) cells. For native protein expression, cells were cultured in Terrific Broth containing kanamycin (50 µg/ml) and chloramphenicol (20 µg/ml) at 37°C to the mid-exponential phase (A600 of ≈0.6), induced by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and then incubated for 2 days at 16°C or 1 day at 20°C. Cells were harvested by centrifugation and frozen at −80°C prior to protein purification. For selenomethionine-substituted SGBP-B, the pET-28TEV-SGBP-B plasmid was transformed into E. coli Rosetta(DE3)/pLysS and plated onto LB supplemented with kanamycin (50 µg/ml) and chloramphenicol (20 µg/ml). After 16 h of growth at 37°C, colonies were harvested from the plates, used to inoculate 100 ml of M9 minimal medium supplemented with kanamycin (30 µg/ml) and chloramphenicol (20 µg/ml), and then grown at 37°C for 16 h. This overnight culture was used to inoculate a 2-liter baffled flask containing 1 liter of Molecular Dimensions SelenoMet premade medium supplemented with 50 ml of the recommended sterile nutrient mix, chloramphenicol, and kanamycin. Cultures were grown at 37°C to an A600 of ≈0.45 before adjusting the temperature to 20°C and supplementing each flask with 100 mg each of l-lysine, l-threonine, and l-phenylalanine and 50 mg each of l-leucine, l-isoleucine, l-valine, and l-selenomethionine. After 20 additional minutes of growth, the cells were induced with 0.5 mM IPTG, and cultures were grown for an additional 48 h. + + + METHODS + paragraph + 48382 + For the purification of native and selenomethionine-substituted protein, cells were thawed and lysed via sonication in His buffer (25 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, pH 7.5) and purified via immobilized nickel affinity chromatography (His-Trap; GE Healthcare) using a gradient of 20 to 300 mM imidazole, according to the manufacturer’s instructions. The His tag was removed by incubation with TEV protease (1:100 molar ratio relative to protein) at room temperature for 2 h and then overnight at 4°C while being dialyzed against His buffer. The cleaved protein was then repurified via nickel affinity chromatography to remove undigested target protein, the cleaved His tag, and His-tagged TEV protease. Purified proteins were dialyzed against 20 mM HEPES–100 mM NaCl (pH 7.0) prior to crystallization and concentrated using Vivaspin 15 (10,000-molecular-weight-cutoff [MWCO]) centrifugal concentrators (Vivaproducts, Inc.). + + + METHODS + title_2 + 49330 + Glycans. + + + METHODS + paragraph + 49339 + Xyloglucan from tamarind seed, β-glucan from barley, and konjac glucomannan were purchased from Megazyme. Starch, guar, and mucin were purchased from Sigma. Hydroxyethyl cellulose was purchased from AMRESCO. Carboxymethyl cellulose was purchased from Acros Organics. Xylogluco-oligosaccharides XyGO1 and XyGO2 for biophysical analyses were prepared from tamarind seed XyG according to the method of Martinez-Fleites et al. with minor modifications. XyGO2 for cocrystallization was purchased from Megazyme (O-XGHDP). + + + METHODS + title_2 + 49858 + Affinity gel electrophoresis. + + + METHODS + paragraph + 49888 + Affinity PAGE was performed as described previously, with minor modification. Various polysaccharides were used at a concentration of 0.05 to 0.1% (wt/vol), and electrophoresis was carried out for 90 min at room temperature in native 10% (wt/vol) polyacrylamide gels. BSA was used as noninteracting negative-control protein. + + + METHODS + title_2 + 50214 + ITC. + + + METHODS + paragraph + 50219 + Isothermal titration calorimetry (ITC) of glycan binding by the SGPB-A mutants was performed using the TA Nano isothermal titration calorimeter calibrated to 25°C. Proteins were dialyzed against 20 mM HEPES–100 mM NaCl (pH 7.0), and sugars were prepared using the dialysis buffer. The protein (45 to 50 µM) was placed in the sample cell, and the syringe was loaded with 2.5 to 4 mg/ml XyG polysaccharide. Following an initial injection of 0.5 µl, 26 subsequent injections of 2 µl were performed with stirring at 350 rpm, and the resulting heat of reaction was recorded. Data were analyzed using the Nano Analyze software. All other ITC experiments were performed using a MicroCal VP-ITC titration calorimeter calibrated to 25°C. Proteins were dialyzed into 20 mM HEPES–100 mM NaCl (pH 7.0), and polysaccharides were prepared using the dialysis buffer. Proteins (micromolar concentrations) were placed in the sample cell, and a first injection of 2 µl was performed followed by 24 subsequent injections of 10 µl of 2 to 20 mM oligosaccharide (cellotetraose, cellohexaose, XyGO1, or XyGO2) or 1 to 2.5 mg/ml XyG polysaccharide. The solution was stirred at 280 rpm, and the resulting heat of reaction was recorded. Data were analyzed using the Origin software program. + + + METHODS + title_2 + 51512 + DSC. + + + METHODS + paragraph + 51517 + Structural integrity of the SGBP-B mutants was verified by differential scanning calorimetry (DSC). DSC studies were performed on a MicroCal VP-DSC (Malvern Instruments). Experiments were carried out in 50 mM HEPES (pH 7.0) at a scan rate of 60°C/h. All samples (40 µM protein) were degassed for 7 min with gentle stirring under vacuum prior to being loaded into the calorimeter. Background excess thermal power scans were obtained with buffer in both the sample and reference cells and subtracted from the scans for each sample solution to generate excess heat capacity versus temperature thermograms. + + + METHODS + paragraph + 52126 + The melting temperature decreased from 57.8 ± 0.9°C for the wild-type SGBP-B protein to 54.6 ± 0.1°C for the Y363A mutant, 54.2 ± 0.1°C for the W364A mutant, and 52 ± 1°C for the F414A mutant. All proteins were therefore in their stable folded state for the ITC measurements (see Fig. S9 in the supplemental material). + + + METHODS + title_2 + 52454 + Bacteroides ovatus mutagenesis. + + + METHODS + paragraph + 52486 + Gene deletions and complementations were performed via allelic exchange in a Bacteroides ovatus thymidine kinase gene (Bacova_03071) deletion (Δtdk) derivative strain of ATCC 8483 using the vector pExchange-tdk, as previously described. Primers for the construction of B. ovatus mutants are listed in Table S1. The B. ovatus Δtdk strain and the B. ovatus ΔXyGUL mutant were a generous gift from Eric Martens, University of Michigan Medical School. + + + METHODS + title_2 + 52946 + Bacteroides growth experiments. + + + METHODS + paragraph + 52978 + All Bacteroides ovatus culturing was performed in a Coy anaerobic chamber (85% N2, 10% H2, 5% CO2) at 37°C. Prior to growth on minimal medium plus the carbohydrates indicated (Fig. 6; see Fig. S8 in the supplemental material), each strain was grown for 16 h from a freezer stock in tryptone-yeast extract-glucose (TYG) medium and then back diluted 1:100 into Bacteroides minimal medium supplemented with 5 mg/ml glucose, as previously described. After growth for 24 h, cultures were back-diluted 1:100 into Bacteroides minimal medium supplemented with 5 mg/ml of glucose, xylose, XyG, XyGO1, or XyGO2. Growth experiments were performed in replicates of 12 (glucose, xylose, and xyloglucan) or 5 (XyGO1 and XyGO2) as 200-µl cultures in 96-well plates. Plates were loaded into a Biostack automated plate handling device coupled to a Powerwave HT absorbance reader (both devices from Biotek Instruments, Winooski, VT). Absorbance at 600 nm (A600; i.e., optical density at 600 nm [OD600]) was measured for each well at 20-min intervals. Data were processed using Gen5 software (BioTek) and Microsoft Excel. Growth was quantified in each assay by first identifying a minimum time point (Amin) at which A600 had increased by 15% over a baseline reading taken during the first 500 min of incubation. Next, we identified the time point at which A600 reached its maximum (Amax) immediately after exponential growth. The growth rate for each well was defined by (Amax − Amin)/(Tmax − Tmin), where Tmax and Tmin are the corresponding time values for each absorbance. To account for variations in inoculum density, for each strain, the lag time (Tmin) on glucose was subtracted from the lag time for the substrate of interest; in all cases, cultures had shorter lag times on glucose than other glycans. + + + METHODS + title_2 + 54783 + Immunofluorescence. + + + METHODS + paragraph + 54803 + Custom rabbit antibodies to recombinant SGBP-A and SGBP-B were generated by Cocalico Biologicals, Inc. (Reamstown, PA). The B. ovatus ATCC 8483 Δtdk and ΔSGBP-B strains were grown in 1 ml minimal Bacteroides medium supplemented with 5 mg/ml tamarind xyloglucan to an A600 of ≈0.6 and then harvested via centrifugation (7,000 × g for 3 min) and washed twice with phosphate-buffered saline (PBS). Cells were resuspended in 0.25 ml PBS, and 0.75 ml of 6% formalin in PBS was added. Cells were incubated with rocking at 20°C for 1.5 h and then washed twice with PBS. Cells were resuspended in 0.5 to 1 ml blocking solution (2% goat serum, 0.02% NaN3 in PBS) and incubated for 16 h at 4°C. Cells were centrifuged and resuspended in 0.5 ml of a 1/100 dilution of custom rabbit antibody sera in blocking solution and incubated by rocking for 2 h at 20°C. Cells were washed with PBS and then resuspended in 0.4 ml of a 1/500 dilution of Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) in blocking solution and incubated with rocking for 1 h at 20°C. Cells were washed three times with an excess of PBS and then resuspended in 20 µl of PBS plus 1 µl of ProLong Gold antifade (Life Technologies). Cells were spotted on 0.8% agarose pads and imaged at the Center for Live Cell Imaging at the University of Michigan Medical School, using an Olympus IX70 inverted confocal microscope. Images were processed with Metamorph Software. + + + METHODS + title_2 + 56256 + Crystallization and data collection. + + + METHODS + paragraph + 56293 + All X-ray diffraction data for both native and selenomethionine-substituted protein crystals were collected at the Life Science Consortium (LS-CAT) at the Advance Photon Source at Argonne National Laboratory in Argonne, IL. The native protein SGBP-B (residues 34 to 489) was concentrated to an A280 of 12.25 prior to crystallization and mixed with 10 mM XyGO2 (Megazyme, O-XGHDP). Hanging drop vapor diffusion was performed against mother liquor consisting of 1.1 to 1.5 M ammonium sulfate and 30 to 70 mM sodium cacodylate (pH 6.5). To decrease crystal nucleation, 0.3 ml of paraffin oil was overlaid on top of 0.5 ml of mother liquor yielding diffraction-quality crystals within 2 weeks. Selenomethionine-substituted crystals of SGBP-B were generated using the same conditions as the native crystals. Crystals of the truncated SGBP-B (domains CD, residues 230 to 489) were obtained by mixing concentrated protein (A600 of 20.6) with 10 mM XyGO2 for hanging drop vapor diffusion against a solution containing 2 M sodium formate and 0.1 M sodium acetate (pH 4.6). All SGBP-B crystals were flash-frozen prior to data collection by briefly soaking in a solution of 80% mother liquor–20% glycerol plus 10 mM xylogluco-oligosaccharide. Data were processed and scaled using HKL2000 and Scalepack. SAD phasing from a selenomethionine-substituted protein crystals was used to determine the structure of SGBP-B. The AutoSol and Autobuild algorithms within the Phenix suite of programs were used to locate and refine the selenium positions and automatically build an initial model of the protein structure, respectively. Successive rounds of manual model building and refinement in Coot and Phenix, respectively, were utilized to build a 2.7-Å model of the selenomethionine-substituted protein, which then was placed in the unit cell of the native data set. Additional rounds of manual model building and refinement were performed to complete the 2.37-Å structure of SGBP-B with XyGO2. The structure of the truncated protein (CD domains, residues 230 to 489) was solved via molecular replacement with Phaser using the CD domains of the full-length protein as a model. + + + METHODS + paragraph + 58465 + The native protein SGBP-A (residues 28 to 546) was concentrated to an A280 of 28.6 and crystallized via hanging drop vapor diffusion from the Morpheus crystal screen (Molecular Dimensions). Crystals formed in well A1 (30 mM MgCl2, 30 mM CaCl2, 20% polyethylene glycol [PEG 500], 10% PEG 20K, 0.1 M imidazole-MES [morpholinethanesulfonic acid], pH 6.5), and were flash-frozen in liquid nitrogen without additional cryoprotectant. The truncated SGBP-A (residues 36 to 546) concentrated to an A280 of 38.2 yielded crystals with 10 mM XyGO2 via hanging drop vapor diffusion against 1.2 to 1.8 M sodium citrate (pH 6.15 to 6.25), and were flash-frozen in a cryoprotectant solution of 80% mother liquor–20% ethylene glycol with the glycan. Data were processed and scaled using HKL2000 and Scalepack. The structure of the apo protein was solved via molecular replacement with BALBES using the homologous structure PDB 3JYS, followed by successive rounds of automatic and manual model building with Autobuild and Coot. The structure of SGBP-A with XyGO2 was solved via molecular replacement with Phaser and refined with Phenix. X-data collection and refinement statistics are presented in Table 2. + + + SUPPL + title_1 + 59664 + SUPPLEMENTAL MATERIAL + + + SUPPL + footnote + 59686 + Citation Tauzin AS, Kwiatkowski KJ, Orlovsky NI, Smith CJ, Creagh AL, Haynes CA, Wawrzak Z, Brumer H, Koropatkin NM. 2016. Molecular dissection of xyloglucan recognition in a prominent human gut symbiont. mBio 7(2):e02134-15. doi:10.1128/mBio.02134-15. + + + REF + title + 59939 + REFERENCES + + + 107 + 118 + surname:Mazmanian;given-names:SK + surname:Liu;given-names:CH + surname:Tzianabos;given-names:AO + surname:Kasper;given-names:DL + 10.1016/j.cell.2005.05.007 + 16009137 + REF + Cell + ref + 122 + 2005 + 59950 + An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system + + + 62 + surname:Sommer;given-names:F + surname:Nookaew;given-names:I + surname:Sommer;given-names:N + surname:Fogelstrand;given-names:P + surname:Bäckhed;given-names:F + 10.1186/s13059-015-0614-4 + 25887251 + REF + Genome Biol + ref + 16 + 2015 + 60046 + Site-specific programming of the host epithelial transcriptome by the gut microbiota + + + 1241214 + surname:Ridaura;given-names:VK + surname:Faith;given-names:JJ + surname:Muehlbauer;given-names:MJ + surname:Ilkayeva;given-names:O + surname:Semenkovich;given-names:CF + surname:Funai;given-names:K + surname:Hayashi;given-names:DK + surname:Lyle;given-names:BJ + surname:Martini;given-names:MC + surname:Ursell;given-names:LK + surname:Clemente;given-names:JC + surname:Van Treuren;given-names:W + surname:Rey;given-names:FE + surname:Cheng;given-names:J + surname:Duncan;given-names:AE + surname:Kau;given-names:AL + surname:Griffin;given-names:NW + surname:Lombard;given-names:V + surname:Henrissat;given-names:B + surname:Bain;given-names:JR + 10.1126/science.1241214 + REF + Science + ref + 341 + 2013 + 60131 + Gut microbiota from twins discordant for obesity modulate metabolism in mice + + + 979 + 984 + surname:Bäckhed;given-names:F + surname:Manchester;given-names:JK + surname:Semenkovich;given-names:CF + surname:Gordon;given-names:JI + 10.1073/pnas.0605374104 + 17210919 + REF + Proc Natl Acad Sci U S A + ref + 104 + 2007 + 60208 + Mechanisms underlying the resistance to diet-induced obesity in germ-free mice + + + 1635 + 1638 + surname:Eckburg;given-names:PB + surname:Bik;given-names:EM + surname:Bernstein;given-names:CN + surname:Purdom;given-names:E + surname:Dethlefsen;given-names:L + surname:Sargent;given-names:M + surname:Gill;given-names:SR + surname:Nelson;given-names:KE + surname:Relman;given-names:DA + 10.1126/science.1110591 + 15831718 + REF + Science + ref + 308 + 2005 + 60287 + Diversity of the human intestinal microbial flora + + + 357 + 360 + surname:Ding;given-names:T + surname:Schloss;given-names:PD + 10.1038/nature13178 + 24739969 + REF + Nature + ref + 509 + 2014 + 60337 + Dynamics and associations of microbial community types across the human body + + + 497 + 504 + surname:El Kaoutari;given-names:A + surname:Armougom;given-names:F + surname:Gordon;given-names:JI + surname:Raoult;given-names:D + surname:Henrissat;given-names:B + 10.1038/nrmicro3050 + 23748339 + REF + Nat Rev Microbiol + ref + 11 + 2013 + 60414 + The abundance and variety of carbohydrate-active enzymes in the human gut microbiota + + + e156 + surname:Xu;given-names:J + surname:Mahowald;given-names:MA + surname:Cordum;given-names:H + surname:Van Brunt;given-names:A + surname:Kim;given-names:K + surname:Fulton;given-names:RS + surname:Fulton;given-names:LA + surname:Clifton;given-names:SW + surname:Wilson;given-names:RK + surname:Knight;given-names:RD + surname:Gordon;given-names:JI + surname:Ley;given-names:RE + surname:Lozupone;given-names:CA + surname:Hamady;given-names:M + surname:Martens;given-names:EC + surname:Henrissat;given-names:B + surname:Coutinho;given-names:PM + surname:Minx;given-names:P + surname:Latreille;given-names:P + 10.1371/journal.pbio.0050156 + 17579514 + REF + PLoS Biol + ref + 5 + 2007 + 60499 + Evolution of symbiotic bacteria in the distal human intestine + + + 2074 + 2076 + surname:Xu;given-names:J + surname:Bjursell;given-names:MK + surname:Himrod;given-names:J + surname:Deng;given-names:S + surname:Carmichael;given-names:LK + surname:Chiang;given-names:HC + surname:Hooper;given-names:LV + surname:Gordon;given-names:JI + 10.1126/science.1080029 + 12663928 + REF + Science + ref + 299 + 2003 + 60561 + A genomic view of the human-Bacteroides thetaiotaomicron symbiosis + + + 3851 + 3865 + surname:Martens;given-names:EC + surname:Kelly;given-names:AG + surname:Tauzin;given-names:AS + surname:Brumer;given-names:H + 10.1016/j.jmb.2014.06.022 + 25026064 + REF + J Mol Biol + ref + 426 + 2014 + 60628 + The devil lies in the details: how variations in polysaccharide fine-structure impact the physiology and evolution of gut microbes + + + e1001221 + surname:Martens;given-names:EC + surname:Lowe;given-names:EC + surname:Gordon;given-names:JI + surname:Chiang;given-names:H + surname:Pudlo;given-names:NA + surname:Wu;given-names:M + surname:McNulty;given-names:NP + surname:Abbott;given-names:DW + surname:Henrissat;given-names:B + surname:Gilbert;given-names:HJ + surname:Bolam;given-names:DN + 10.1371/journal.pbio.1001221 + 22205877 + REF + PLoS Biol + ref + 9 + 2011 + 60759 + Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts + + + 5609 + 5616 + surname:Tancula;given-names:E + surname:Feldhaus;given-names:MJ + surname:Bedzyk;given-names:LA + surname:Salyers;given-names:AA + 1512196 + REF + J Bacteriol + ref + 174 + 1992 + 60849 + Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron + + + 5365 + 5372 + surname:Shipman;given-names:JA + surname:Berleman;given-names:JE + surname:Salyers;given-names:AA + 10.1128/JB.182.19.5365-5372.2000 + 10986238 + REF + J Bacteriol + ref + 182 + 2000 + 60965 + Characterization of four outer membrane proteins involved in binding starch to the cell surface of Bacteroides thetaiotaomicron + + + 7206 + 7211 + surname:Shipman;given-names:JA + surname:Cho;given-names:KH + surname:Siegel;given-names:HA + surname:Salyers;given-names:AA + 10572122 + REF + J Bacteriol + ref + 181 + 1999 + 61093 + Physiological characterization of SusG, an outer membrane protein essential for starch utilization by Bacteroides thetaiotaomicron + + + 200 + 215 + surname:Koropatkin;given-names:NM + surname:Smith;given-names:TJ + 10.1016/j.str.2009.12.010 + 20159465 + REF + Structure + ref + 18 + 2010 + 61224 + SusG: A unique cell-membrane-associated alpha-amylase from a prominent human gut symbiont targets complex starch molecules + + + 1105 + 1115 + surname:Koropatkin;given-names:NM + surname:Martens;given-names:EC + surname:Gordon;given-names:JI + surname:Smith;given-names:TJ + 10.1016/j.str.2008.03.017 + 18611383 + REF + Structure + ref + 16 + 2008 + 61347 + Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices + + + 823 + 830 + surname:Reeves;given-names:AR + surname:D’Elia;given-names:JN + surname:Frias;given-names:J + surname:Salyers;given-names:AA + 8550519 + REF + J Bacteriol + ref + 178 + 1996 + 61449 + A Bacteroides thetaiotaomicron outer membrane protein that is essential for utilization of maltooligosaccharides and starch + + + 34614 + 34625 + surname:Cameron;given-names:EA + surname:Maynard;given-names:MA + surname:Smith;given-names:CJ + surname:Smith;given-names:TJ + surname:Koropatkin;given-names:NM + surname:Martens;given-names:EC + 10.1074/jbc.M112.397380 + 22910908 + REF + J Biol Chem + ref + 287 + 2012 + 61573 + Multidomain carbohydrate-binding proteins involved in Bacteroides thetaiotaomicron starch metabolism + + + 7173 + 7179 + surname:D’Elia;given-names:JN + surname:Salyers;given-names:AA + 8955399 + REF + J Bacteriol + ref + 178 + 1996 + 61674 + Contribution of a neopullulanase, a pullulanase, and an alpha-glucosidase to growth of Bacteroides thetaiotaomicron on starch + + + 19786 + 19791 + surname:Hehemann;given-names:JH + surname:Kelly;given-names:AG + surname:Pudlo;given-names:NA + surname:Martens;given-names:EC + surname:Boraston;given-names:AB + 10.1073/pnas.1211002109 + 23150581 + REF + Proc Natl Acad Sci U S A + ref + 109 + 2012 + 61800 + Bacteria of the human gut microbiome catabolize red seaweed glycans with carbohydrate-active enzyme updates from extrinsic microbes + + + 165 + 169 + surname:Cuskin;given-names:F + surname:Lowe;given-names:EC + surname:Rogowski;given-names:A + surname:Hamilton;given-names:BS + surname:Chen;given-names:R + surname:Tolbert;given-names:TJ + surname:Piens;given-names:K + surname:Bracke;given-names:D + surname:Vervecken;given-names:W + surname:Hakki;given-names:Z + surname:Speciale;given-names:G + surname:Munōz-Munōz;given-names:JL + surname:Temple;given-names:MJ + surname:Day;given-names:A + surname:Pena;given-names:MJ + surname:McLean;given-names:R + surname:Suits;given-names:MD + surname:Boraston;given-names:AB + surname:Atherly;given-names:T + surname:Ziemer;given-names:CJ + surname:Williams;given-names:SJ + surname:Davies;given-names:GJ + surname:Abbott;given-names:DW + surname:Zhu;given-names:Y + surname:Martens;given-names:EC + surname:Gilbert;given-names:HJ + surname:Cameron;given-names:EA + surname:Pudlo;given-names:NA + surname:Porter;given-names:NT + surname:Urs;given-names:K + surname:Thompson;given-names:AJ + surname:Cartmell;given-names:A + 10.1038/nature13995 + 25567280 + REF + Nature + ref + 517 + 2015 + 61932 + Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism + + + 7481 + surname:Rogowski;given-names:A + surname:Briggs;given-names:JA + surname:Rogers;given-names:TE + surname:Thompson;given-names:P + surname:Hawkins;given-names:AR + surname:Yadav;given-names:MP + surname:Henrissat;given-names:B + surname:Martens;given-names:EC + surname:Dupree;given-names:P + surname:Gilbert;given-names:HJ + surname:Bolam;given-names:DN + surname:Mortimer;given-names:JC + surname:Tryfona;given-names:T + surname:Terrapon;given-names:N + surname:Lowe;given-names:EC + surname:Basle;given-names:A + surname:Morland;given-names:C + surname:Day;given-names:AM + surname:Zheng;given-names:H + 10.1038/ncomms8481 + 26112186 + REF + Nat Commun + ref + 6 + 2015 + 62009 + Glycan complexity dictates microbial resource allocation in the large intestine + + + 498 + 502 + surname:Larsbrink;given-names:J + surname:Rogers;given-names:TE + surname:Creagh;given-names:AL + surname:Haynes;given-names:CA + surname:Kelly;given-names:AG + surname:Cederholm;given-names:SN + surname:Davies;given-names:GJ + surname:Martens;given-names:EC + surname:Brumer;given-names:H + surname:Hemsworth;given-names:GR + surname:McKee;given-names:LS + surname:Tauzin;given-names:AS + surname:Spadiut;given-names:O + surname:Klinter;given-names:S + surname:Pudlo;given-names:NA + surname:Urs;given-names:K + surname:Koropatkin;given-names:NM + 10.1038/nature12907 + 24463512 + REF + Nature + ref + 506 + 2014 + 62089 + A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes + + + 133 + 150 + surname:McDougall;given-names:GJ + surname:Morrison;given-names:IM + surname:Stewart;given-names:D + surname:Hillman;given-names:JR + REF + J Sci Food Agric + ref + 70 + 1996 + 62178 + Plant cell walls as dietary fibre: range, structure, processing and function + + + 526 + 542 + surname:Schultink;given-names:A + surname:Liu;given-names:L + surname:Zhu;given-names:L + surname:Pauly;given-names:M + 10.3390/plants3040526 + REF + Plants + ref + 3 + 2014 + 62255 + Structural diversity and function of xyloglucan sidechain substituents + + + e01441-01414 + surname:Cameron;given-names:EA + surname:Kwiatkowski;given-names:KJ + surname:Lee;given-names:BH + surname:Hamaker;given-names:BR + surname:Koropatkin;given-names:NM + surname:Martens;given-names:EC + 10.1128/mBio.01441-14 + 25205092 + REF + mBio + ref + 5 + 2014 + 62326 + Multifunctional nutrient-binding proteins adapt human symbiotic bacteria for glycan competition in the gut by separately promoting enhanced sensing and catalysis + + + 647 + 655 + surname:Terrapon;given-names:N + surname:Lombard;given-names:V + surname:Gilbert;given-names:HJ + surname:Henrissat;given-names:B + 10.1093/bioinformatics/btu716 + 25355788 + REF + Bioinformatics + ref + 31 + 2015 + 62488 + Automatic prediction of polysaccharide utilization loci in Bacteroidetes species + + + 24673 + 24677 + surname:Martens;given-names:EC + surname:Koropatkin;given-names:NM + surname:Smith;given-names:TJ + surname:Gordon;given-names:JI + 10.1074/jbc.R109.022848 + 19553672 + REF + J Biol Chem + ref + 284 + 2009 + 62569 + Complex glycan catabolism by the human gut microbiota: the Bacteroidetes Sus-like paradigm + + + 86 + 90 + surname:Bolam;given-names:DN + surname:Sonnenburg;given-names:JL + 10.4161/gmic.2.2.15232 + 21637023 + REF + Gut Microbes + ref + 2 + 2011 + 62660 + Mechanistic insight into polysaccharide use within the intestinal microbiota + + + e1000798 + surname:Ellrott;given-names:K + surname:Jaroszewski;given-names:L + surname:Li;given-names:W + surname:Wooley;given-names:JC + surname:Godzik;given-names:A + 10.1371/journal.pcbi.1000798 + 20532204 + REF + PLoS Comput Biol + ref + 6 + 2010 + 62737 + Expansion of the protein repertoire in newly explored environments: human gut microbiome specific protein families + + + 563 + 569 + surname:Bolam;given-names:DN + surname:Koropatkin;given-names:NM + 10.1016/j.sbi.2012.06.006 + 22819666 + REF + Curr Opin Struct Biol + ref + 22 + 2012 + 62852 + Glycan recognition by the Bacteroidetes Sus-like systems + + + 625 + 641 + surname:Zhou;given-names:Q + surname:Rutland;given-names:MW + surname:Teeri;given-names:TT + surname:Brumer;given-names:H + 10.1007/s10570-007-9109-0 + REF + Cellulose + ref + 14 + 2007 + 62909 + Xyloglucan in cellulose modification + + + 1532 + 1542 + surname:Koropatkin;given-names:N + surname:Martens;given-names:EC + surname:Gordon;given-names:JI + surname:Smith;given-names:TJ + 10.1021/bi801942a + 19191477 + REF + Biochemistry + ref + 48 + 2009 + 62946 + Structure of a SusD homologue, BT1043, involved in mucin O-glycan utilization in a prominent human gut symbiont + + + 3466 + 3475 + surname:von Schantz;given-names:L + surname:Håkansson;given-names:M + surname:Logan;given-names:DT + surname:Nordberg-Karlsson;given-names:E + surname:Ohlin;given-names:M + 10.1002/prot.24700 + 25302425 + REF + Proteins + ref + 82 + 2014 + 63058 + Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH-Pi interactions + + + 4799 + 4809 + surname:Luís;given-names:AS + surname:Venditto;given-names:I + surname:Najmudin;given-names:S + surname:Gilbert;given-names:HJ + surname:Temple;given-names:MJ + surname:Rogowski;given-names:A + surname:Baslé;given-names:A + surname:Xue;given-names:J + surname:Knox;given-names:JP + surname:Prates;given-names:JA + surname:Ferreira;given-names:LM + surname:Fontes;given-names:CM + 10.1074/jbc.M112.432781 + 23229556 + REF + J Biol Chem + ref + 288 + 2013 + 63184 + Understanding how noncatalytic carbohydrate binding modules can display specificity for xyloglucan + + + 1205 + 1211 + surname:Tsukimoto;given-names:K + surname:Takada;given-names:R + surname:Araki;given-names:Y + surname:Suzuki;given-names:K + surname:Karita;given-names:S + surname:Wakagi;given-names:T + surname:Shoun;given-names:H + surname:Watanabe;given-names:T + surname:Fushinobu;given-names:S + 10.1016/j.febslet.2010.02.027 + 20159017 + REF + FEBS Lett + ref + 584 + 2010 + 63283 + Recognition of cellooligosaccharides by a family 28 carbohydrate-binding module + + + 7224 + 7230 + surname:Cho;given-names:KH + surname:Salyers;given-names:AA + 10.1128/JB.183.24.7224-7230.2001 + 11717282 + REF + J Bacteriol + ref + 183 + 2001 + 63363 + Biochemical analysis of interactions between outer membrane proteins that contribute to starch utilization by Bacteroides thetaiotaomicron + + + 40 + 49 + surname:Rakoff-Nahoum;given-names:S + surname:Coyne;given-names:MJ + surname:Comstock;given-names:LE + 10.1016/j.cub.2013.10.077 + 24332541 + REF + Curr Biol + ref + 24 + 2014 + 63502 + An ecological network of polysaccharide utilization among human intestinal symbionts + + + e00909-14 + surname:Elhenawy;given-names:W + surname:Debelyy;given-names:MO + surname:Feldman;given-names:MF + 10.1128/mBio.00909-14 + 24618254 + REF + mBio + ref + 5 + 2014 + 63587 + Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles + + + 94 + 108 + surname:Hemsworth;given-names:GR + surname:Déjean;given-names:G + surname:Davies;given-names:GJ + surname:Brumer;given-names:H + 10.1042/BST20150180 + 26862194 + REF + Biochem Soc Trans + ref + 44 + 2016 + 63686 + Learning from microbial strategies for polysaccharide degradation + + + 43 + 60 + surname:Noinaj;given-names:N + surname:Guillier;given-names:M + surname:Barnard;given-names:TJ + surname:Buchanan;given-names:SK + 10.1146/annurev.micro.112408.134247 + 20420522 + REF + Annu Rev Microbiol + ref + 64 + 2010 + 63752 + TonB-dependent transporters: regulation, structure, and function + + + e1002118 + surname:Renzi;given-names:F + surname:Manfredi;given-names:P + surname:Mally;given-names:M + surname:Moes;given-names:S + surname:enö;given-names:P + surname:Cornelis;given-names:GR + 10.1371/journal.ppat.1002118 + 21738475 + REF + PLoS Pathog + ref + 7 + 2011 + 63817 + The N-glycan glycoprotein deglycosylation complex (Gpd) from Capnocytophaga canimorsus deglycosylates human IgG + + + 499 + 511 + surname:Phansopa;given-names:C + surname:Roy;given-names:S + surname:Rafferty;given-names:JB + surname:Douglas;given-names:CW + surname:Pandhal;given-names:J + surname:Wright;given-names:PC + surname:Kelly;given-names:DJ + surname:Stafford;given-names:GP + 10.1042/BJ20131415 + 24351045 + REF + Biochem J + ref + 458 + 2014 + 63929 + Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species + + + 1241 + 1252 + surname:Sonnenburg;given-names:ED + surname:Zheng;given-names:H + surname:Joglekar;given-names:P + surname:Higginbottom;given-names:SK + surname:Firbank;given-names:SJ + surname:Bolam;given-names:DN + surname:Sonnenburg;given-names:JL + 10.1016/j.cell.2010.05.005 + 20603004 + REF + Cell + ref + 141 + 2010 + 64088 + Specificity of polysaccharide use in intestinal Bacteroides species determines diet-induced microbiota alterations + + + e224 + surname:Blanvillain;given-names:S + surname:Meyer;given-names:D + surname:Boulanger;given-names:A + surname:Lautier;given-names:M + surname:Guynet;given-names:C + surname:Denancé;given-names:N + surname:Vasse;given-names:J + surname:Lauber;given-names:E + surname:Arlat;given-names:M + 10.1371/journal.pone.0000224 + 17311090 + REF + PLoS One + ref + 2 + 2007 + 64203 + Plant carbohydrate scavenging through tonB-dependent receptors: a feature shared by phytopathogenic and aquatic bacteria + + + 669 + 677 + surname:Gilbert;given-names:HJ + surname:Knox;given-names:JP + surname:Boraston;given-names:AB + 10.1016/j.sbi.2013.05.005 + 23769966 + REF + Curr Opin Struct Biol + ref + 23 + 2013 + 64324 + Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules + + + 1251 + 1253 + surname:Gordon;given-names:JI + 10.1126/science.1224686 + 22674326 + REF + Science + ref + 336 + 2012 + 64448 + Honor thy gut symbionts redux + + + 1 + 7 + surname:Hutkins;given-names:RW + surname:Krumbeck;given-names:JA + surname:Vaughan;given-names:E + surname:Sanders;given-names:ME + surname:Bindels;given-names:LB + surname:Cani;given-names:PD + surname:Fahey;given-names:G;suffix:Jr + surname:Goh;given-names:YJ + surname:Hamaker;given-names:B + surname:Martens;given-names:EC + surname:Mills;given-names:DA + surname:Rastal;given-names:RA + 10.1016/j.copbio.2015.09.001 + 26431716 + REF + Curr Opin Biotechnol + ref + 37 + 2016 + 64478 + Prebiotics: why definitions matter + + + 105 + 124 + surname:Van Duyne;given-names:GD + surname:Standaert;given-names:RF + surname:Karplus;given-names:PA + surname:Schreiber;given-names:SL + surname:Clardy;given-names:J + 10.1006/jmbi.1993.1012 + 7678431 + REF + J Mol Biol + ref + 229 + 1993 + 64513 + Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin + + + 43010 + 43017 + surname:Freelove;given-names:AC + surname:Bolam;given-names:DN + surname:White;given-names:P + surname:Hazlewood;given-names:GP + surname:Gilbert;given-names:HJ + 10.1074/jbc.M107143200 + 11560933 + REF + J Biol Chem + ref + 276 + 2001 + 64600 + A novel carbohydrate-binding protein is a component of the plant cell wall-degrading complex of Piromyces equi + + + surname:Holdeman;given-names:LV + surname:Cato;given-names:ED + surname:Moore;given-names:WEC + REF + Anaerobe laboratory manual + ref + 1977 + 64711 + + + 447 + 457 + surname:Martens;given-names:EC + surname:Chiang;given-names:HC + surname:Gordon;given-names:JI + 10.1016/j.chom.2008.09.007 + 18996345 + REF + Cell Host Microbe + ref + 4 + 2008 + 64712 + Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont + + + 307 + 326 + surname:Otwinowski;given-names:Z + surname:Minor;given-names:W + REF + Methods Enzymol + ref + 276 + 1997 + 64818 + Processing of X-ray diffraction data collected in oscillation mode + + + 582 + 601 + surname:Terwilliger;given-names:TC + surname:Adams;given-names:PD + surname:Read;given-names:RJ + surname:McCoy;given-names:AJ + surname:Moriarty;given-names:NW + surname:Grosse-Kunstleve;given-names:RW + surname:Afonine;given-names:PV + surname:Zwart;given-names:PH + surname:Hung;given-names:LW + 10.1107/S0907444909012098 + 19465773 + REF + Acta Crystallogr D Biol Crystallogr + ref + 65 + 2009 + 64885 + Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard + + + 61 + 69 + surname:Terwilliger;given-names:TC + surname:Grosse-Kunstleve;given-names:RW + surname:Afonine;given-names:PV + surname:Moriarty;given-names:NW + surname:Zwart;given-names:PH + surname:Hung;given-names:LW + surname:Read;given-names:RJ + surname:Adams;given-names:PD + 10.1107/S090744490705024X + 18094468 + REF + Acta Crystallogr D Biol Crystallogr + ref + 64 + 2008 + 64990 + Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard + + + 1948 + 1954 + surname:Adams;given-names:PD + surname:Grosse-Kunstleve;given-names:RW + surname:Hung;given-names:LW + surname:Ioerger;given-names:TR + surname:McCoy;given-names:AJ + surname:Moriarty;given-names:NW + surname:Read;given-names:RJ + surname:Sacchettini;given-names:JC + surname:Sauter;given-names:NK + surname:Terwilliger;given-names:TC + 10.1107/S0907444902016657 + 12393927 + REF + Acta Crystallogr D Biol Crystallogr + ref + 58 + 2002 + 65095 + PHENIX: building new software for automated crystallographic structure determination + + + 2126 + 2132 + surname:Emsley;given-names:P + surname:Cowtan;given-names:K + 10.1107/S0907444904019158 + 15572765 + REF + Acta Crystallogr D Biol Crystallogr + ref + 60 + 2004 + 65180 + Coot: model-building tools for molecular graphics + + + 658 + 674 + surname:McCoy;given-names:AJ + surname:Grosse-Kunstleve;given-names:RW + surname:Adams;given-names:PD + surname:Winn;given-names:MD + surname:Storoni;given-names:LC + surname:Read;given-names:RJ + 10.1107/S0021889807021206 + 19461840 + REF + J Appl Crystallogr + ref + 40 + 2007 + 65230 + Phaser crystallographic software + + + 125 + 132 + surname:Long;given-names:F + surname:Vagin;given-names:AA + surname:Young;given-names:P + surname:Murshudov;given-names:GN + 10.1107/S0907444907050172 + 18094476 + REF + Acta Crystallogr D Biol Crystallogr + ref + 64 + 2008 + 65263 + BALBES: a molecular-replacement pipeline + + + 56 + 66 + surname:Tuomivaara;given-names:ST + surname:Yaoi;given-names:K + surname:O’Neill;given-names:MA + surname:York;given-names:WS + 10.1016/j.carres.2014.06.031 + 25497333 + REF + Carbohydr Res + ref + 402 + 2015 + 65304 + Generation and structural validation of a library of diverse xyloglucan-derived oligosaccharides, including an update on xyloglucan nomenclature + + + 24922 + 24933 + surname:Martinez-Fleites;given-names:C + surname:Guerreiro;given-names:CI + surname:Baumann;given-names:MJ + surname:Taylor;given-names:EJ + surname:Prates;given-names:JA + surname:Ferreira;given-names:LM + surname:Fontes;given-names:CM + surname:Brumer;given-names:H + surname:Davies;given-names:GJ + 10.1074/jbc.M603583200 + 16772298 + REF + J Biol Chem + ref + 281 + 2006 + 65449 + Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation + + + diff --git a/BioC_XML/4850288_v0.xml b/BioC_XML/4850288_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..6640527bfee6d41b4c8b32721e2482083f89586c --- /dev/null +++ b/BioC_XML/4850288_v0.xml @@ -0,0 +1,7298 @@ + + + + PMC + 20140719 + pmc.key + + 4850288 + CC BY + no + 0 + 0 + + P. merdae C11 Cysteine Peptidase + 10.1074/jbc.M115.706143 + 4850288 + 26940874 + M115.706143 + 9482 + 18 + C-terminal domain (carboxyl tail domain, CTD) crystal structure cysteine protease enzyme proteolysis active site domain kinteoplast + Author's Choice—Final version free via Creative Commons CC-BY license. + 9491 + surname:McLuskey;given-names:Karen + surname:Grewal;given-names:Jaspreet S. + surname:Das;given-names:Debanu + surname:Godzik;given-names:Adam + surname:Lesley;given-names:Scott A. + surname:Deacon;given-names:Ashley M. + surname:Coombs;given-names:Graham H. + surname:Elsliger;given-names:Marc-André + surname:Wilson;given-names:Ian A. + surname:Mottram;given-names:Jeremy C. + TITLE + front + 291 + 2016 + 0 + Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome* + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:28:56Z + + Crystal Structure + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:29:22Z + + Activity Studies + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:18Z + + C11 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:29:34Z + + Cysteine Peptidase + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:29:41Z + + Parabacteroides merdae + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:29:46Z + + Human + + + + ABSTRACT + abstract + 126 + Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys147, resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:31:49Z + + Clan CD cysteine peptidases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:31:52Z + + peptidases + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:31:59Z + + mammalian + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:04Z + + caspases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:11Z + + clostripain family + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:18Z + + C11 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:28:56Z + + crystal structure + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:18Z + + C11 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:29:46Z + + human + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:32:46Z + + bacterium + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:29:41Z + + Parabacteroides merdae + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:21Z + + monomeric + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:29:34Z + + cysteine peptidase + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:33:11Z + + extended caspase-like α/β/α sandwich + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:33:06Z + + C-terminal domain + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:41:33Z + + clan CD cysteine peptidases + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:27Z + + Lys147 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:34:26Z + + active site + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:34:21Z + + Biochemical and kinetic analysis + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:27Z + + Lys147 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:33:50Z + + intramolecular processing site + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:34:11Z + + cleavage + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:34:16Z + + full activation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:34:18Z + + enzyme + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:34:33Z + + acidic binding pocket + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:54:55Z + + Arg + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:55:14Z + + Lys + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:48:22Z + + P1 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T13:34:44Z + + Ca2+ + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:37:55Z + + C11 peptidases + + + + INTRO + title_1 + 1565 + Introduction + + + INTRO + paragraph + 1578 + Cysteine peptidases play crucial roles in the virulence of bacterial and other eukaryotic pathogens. In the MEROPS peptidase database, clan CD contains groups (or families) of cysteine peptidases that share some highly conserved structural elements. Clan CD families are typically described using the name of their archetypal, or founding, member and also given an identification number preceded by a “C,” to denote cysteine peptidase. Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). No structural information is available for clostripain (C11), separase (C50), or PrtH-peptidase (C85). + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:31Z + + Cysteine peptidases + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:39:44Z + + bacterial + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:39:50Z + + eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:59Z + + clan CD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:31Z + + cysteine peptidases + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:48:11Z + + highly conserved + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:40:10Z + + Clan CD families + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:29:34Z + + cysteine peptidase + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:40:27Z + + crystal structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:44:14Z + + legumain + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:40:35Z + + C13 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:44:28Z + + caspase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:49:48Z + + C14a + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:46:27Z + + paracaspase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:40:42Z + + C14b(P + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:46:38Z + + metacaspase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:40:47Z + + C14b(M + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:46:53Z + + gingipain + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:40:52Z + + C25 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:40:54Z + + cysteine peptidase domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:40:57Z + + CPD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:48:35Z + + C80 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:12Z + + clostripain + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:18Z + + C11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:47:03Z + + separase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:48:40Z + + C50 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:47:15Z + + PrtH-peptidase + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:48:43Z + + C85 + + + + INTRO + paragraph + 2445 + Clan CD enzymes have a highly conserved His/Cys catalytic dyad and exhibit strict specificity for the P1 residue of their substrates. However, despite these similarities, clan CD forms a functionally diverse group of enzymes: the overall structural diversity between (and at times within) the various families provides these peptidases with a wide variety of substrate specificities and activation mechanisms. Several members are initially expressed as proenzymes, demonstrating self-inhibition prior to full activation. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:48:05Z + + Clan CD enzymes + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:48:11Z + + highly conserved + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:48:17Z + + His/Cys catalytic dyad + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:48:22Z + + P1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:59Z + + clan CD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:48:29Z + + peptidases + + + + INTRO + paragraph + 2966 + The archetypal and arguably most notable family in the clan is that of the mammalian caspases (C14a), although clan CD members are distributed throughout the entire phylogenetic kingdom and are often required in fundamental biological processes. Interestingly, little is known about the structure or function of the C11 proteins, despite their widespread distribution and its archetypal member, clostripain from Clostridium histolyticum, first reported in the literature in 1938. Clostripain has been described as an arginine-specific peptidase with a requirement for Ca2+ and loss of an internal nonapeptide for full activation; lack of structural information on the family appears to have prohibited further investigation. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:31:59Z + + mammalian + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:04Z + + caspases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:49:48Z + + C14a + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:59Z + + clan CD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:18Z + + C11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:12Z + + clostripain + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:50:06Z + + Clostridium histolyticum + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:12Z + + Clostripain + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:50:19Z + + arginine-specific peptidase + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T13:50:22Z + + Ca2+ + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:50:26Z + + internal nonapeptide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:50:54Z + + full activation + + + + INTRO + paragraph + 3691 + As part of an ongoing project to characterize commensal bacteria in the microbiome that inhabit the human gut, the structure of C11 peptidase, PmC11, from Parabacteroides merdae was determined using the Joint Center for Structural Genomics (JCSG)4 HTP structural biology pipeline. The structure was analyzed, and the enzyme was biochemically characterized to provide the first structure/function correlation for a C11 peptidase. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:51:49Z + + bacteria + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:29:46Z + + human + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:51:44Z + + structure + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:51:40Z + + C11 peptidase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:29:41Z + + Parabacteroides merdae + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:51:56Z + + structure was analyzed + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:51:58Z + + biochemically characterized + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:51:40Z + + C11 peptidase + + + + METHODS + title_1 + 4120 + Experimental Procedures + + + METHODS + paragraph + 4144 + Cloning, expression, purification, crystallization, and structure determination of PmC11 were carried out using standard JCSG protocols as follows. + + + METHODS + title_4 + 4292 + Cloning + + + METHODS + paragraph + 4300 + Clones were generated using the polymerase incomplete primer extension (PIPE) cloning method. The gene encoding PmC11 (SP5111E) was amplified by polymerase chain reaction (PCR) from P. merdae genomic DNA using PfuTurbo DNA polymerase (Stratagene), using I-PIPE primers that included sequences for the predicted 5′ and 3′ ends (shown below). The expression vector, pSpeedET, which encodes an amino-terminal tobacco etch virus protease-cleavable expression and purification tag (MGSDKIHHHHHHENLYFQ/G), was PCR amplified with V-PIPE (Vector) primers. V-PIPE and I-PIPE PCR products were mixed to anneal the amplified DNA fragments together. Escherichia coli GeneHogs (Invitrogen) competent cells were transformed with the I-PIPE/V-PIPE mixture and dispensed on selective LB-agar plates. The cloning junctions were confirmed by DNA sequencing. The plasmid encoding the full-length protein was deposited in the PSI:Biology Materials Repository at the DNASU plasmid repository (PmCD00547516). For structure determination, to obtain soluble protein using the PIPE, method the gene segment encoding residues Met1-Asn22 was deleted because these residues were predicted to correspond to a signal peptide using SignalP. + + + METHODS + title_4 + 5514 + Protein Expression and Selenomethionine Incorporation + + + METHODS + paragraph + 5568 + The expression plasmid for the truncated PmC11 construct was transformed into E. coli GeneHogs competent cells and grown in minimal media supplemented with selenomethionine and 30 μg ml−1 of kanamycin at 37 °C using a GNF fermentor. A methionine auxotrophic strain was not required as selenomethionine is incorporated via the inhibition of methionine biosynthesis. Protein expression was induced using 0.1% (w/v) l-arabinose and the cells were left to grow for a further 3 h at 37 °C. At the end of the cell culture, lysozyme was added to all samples to a final concentration of 250 μg ml−1 and the cells were harvested and stored at −20 °C, until required. Primers used in this section are as follows: I-PIPE (forward): CTGTACTTCCAGGGCGAGACTCCGGAACCCCGGACAACCCGC; I-PIPE (reverse): AATTAAGTCGCGTTATTCATAAACTGCCTTATACCAGCCGAC; V-PIPE (forward): TAACGCGACTTAATTAACTCGTTTAAACGGTCTCCAGC; and V-PIPE (reverse): GCCCTGGAAGTACAGGTTTTCGTGATGATGATGATGAT. + + + METHODS + title_4 + 6526 + Protein Purification for Crystallization + + + METHODS + paragraph + 6567 + Cells were resuspended, homogenized, and lysed by sonication in 40 mm Tris (pH 8.0), 300 mm NaCl, 10 mm imidazole, and 1 mm Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Lysis Buffer 1) containing 0.4 mm MgSO4 and 1 μl of 250 unit/μl−1 of benzonase (Sigma). The cell lysate was then clarified by centrifugation (32,500 × g for 25 min at 4 °C) before being passed over Ni2+-chelating resin equilibrated in Lysis Buffer 1 and washed in the same buffer supplemented with 40 mm imidazole and 10% (v/v) glycerol. The protein was subsequently eluted in 20 mm Tris (pH 8.0), 150 mm NaCl, 10% (v/v) glycerol, 1 mm TCEP, and 300 mm imidazole, and the fractions containing the protein were pooled. + + + METHODS + paragraph + 7267 + To remove the His tag, PmC11 was exchanged into 20 mm Tris (pH 8.0), 150 mm NaCl, 30 mm imidazole, and 1 mm TCEP using a PD-10 column (GE Healthcare), followed by incubation with 1 mg of His-tagged tobacco etch virus protease per 15 mg of protein for 2 h at room temperature and subsequent overnight incubation at 4 °C. The sample was centrifuged to remove any precipitated material (13,000 × g for 10 min at 4 °C) and the supernatant loaded onto Ni2+-chelating resin equilibrated with 20 mm Tris (pH 8.0), 150 mm NaCl, 30 mm imidazole, and 1 mm TCEP and washed with the same buffer. The flow-through and wash fractions were collected and concentrated to 13.3 mg ml−1 using Amicon Ultra-15 5K centrifugal concentrators (Millipore). + + + METHODS + title_4 + 8004 + Crystallization and Data Collection + + + METHODS + paragraph + 8040 + PmC11 was crystallized using the nanodroplet vapor diffusion method using standard JCSG crystallization protocols. Drops were comprised of 200 nl of protein solution mixed with 200 nl of crystallization solution in 96-well sitting-drop plates, equilibrated against a 50-μl reservoir. Crystals of PmC11 were grown at 4 °C in mother liquor consisting of 0.2 m NH4H2PO4, 20% PEG-3350 (JCSG Core Suite I). Crystals were flash cooled in liquid nitrogen using 10% ethylene glycol as a cryoprotectant prior to data collection and initial screening for diffraction was carried out using the Stanford Automated Mounting system at the Stanford Synchrotron Radiation Lightsource (SSRL, Menlo Park, CA). Single wavelength anomalous dispersion data were collected using a wavelength of 0.9793 Å, at the Advanced Light Source (ALS, beamline 8.2.2, Berkeley, CA) on an ADSC Quantum 315 CCD detector. The data were indexed and integrated with XDS and scaled using XSCALE. The diffraction data were indexed in space group P21 with a = 39.11, b = 108.68, c = 77.97 Å, and β = 94.32°. The unit cell contained two molecules in the asymmetric unit resulting in a solvent content of 39% (Matthews' coefficient (Vm) of 2.4 Å3 Da−1). + + + METHODS + title_4 + 9262 + Structure Determination + + + METHODS + paragraph + 9286 + The PmC11 structure was determined by the single wavelength anomalous dispersion method using an x-ray wavelength corresponding to the peak of the selenium K edge. Initial phases were derived using the autoSHARP interface, which included density modification with SOLOMON. Good quality electron density was obtained at 1.7-Å resolution, allowing an initial model to be obtained by automated model building with ARP/wARP. Model completion and refinement were iteratively performed with COOT and REFMAC to produce a final model with an Rcryst and Rfree of 14.3 and 17.5%, respectively. The refinement included experimental phase restraints in the form of Hendrickson-Lattman coefficients, TLS refinement with one TLS group per molecule in the asymmetric unit, and NCS restraints. The refined structure contains residues 24–375 and 28–375 for the two molecules in the crystallographic asymmetric unit. Structural validation was carried using the JCSG Quality Control Server that analyzes both the coordinates and data using a variety of structural validation tools to confirm the stereochemical quality of the model (ADIT, MOLPROBITY, and WHATIF 5.0) and agreement between model and data (SGCHECK and RESOLVE). All of the main-chain torsion angles were in the allowed regions of the Ramachandran plot and the MolProbity overall clash score for the structure was 2.09 (within the 99th percentile for its resolution). The atomic coordinates and structure factors for PmC11 have been deposited in the Protein Data Bank (PDB) with the accession code 3UWS. Data collection, model, and refinement statistics are reported in Table 1. + + + T1.xml + T1 + TABLE + table_caption + 10916 + Crystallographic statistics for PDB code 3UWS + + + T1.xml + T1 + TABLE + table_caption + 10962 + Values in parentheses are for the highest resolution shell. + + + T1.xml + T1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><tbody valign="top"><tr><td align="left" rowspan="1" colspan="1"><bold>Data collection</bold></td><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">    Wavelength (Å)</td><td align="left" rowspan="1" colspan="1">0.9793</td></tr><tr><td align="left" rowspan="1" colspan="1">    Space group</td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub></td></tr><tr><td align="left" rowspan="1" colspan="1">    Unit cell dimensions <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å); β<sup>°</sup></td><td align="left" rowspan="1" colspan="1">39.11, 108.68, 77.97; β = 94.32°</td></tr><tr><td align="left" rowspan="1" colspan="1">    Resolution range (Å)</td><td align="left" rowspan="1" colspan="1">28.73–1.70 (1.79–1.70)</td></tr><tr><td align="left" rowspan="1" colspan="1">    Unique reflections</td><td align="left" rowspan="1" colspan="1">70,913</td></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>R</italic><sub>merge</sub><xref ref-type="table-fn" rid="TF1-1"><italic><sup>a</sup></italic></xref> on <italic>I</italic> (%)</td><td align="left" rowspan="1" colspan="1">10.2 (49.0)</td></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>R</italic><sub>meas</sub><xref ref-type="table-fn" rid="TF1-2"><italic><sup>b</sup></italic></xref> on <italic>I</italic> (%)</td><td align="left" rowspan="1" colspan="1">11.0 (52.7)</td></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>R</italic><sub>pim</sub><xref ref-type="table-fn" rid="TF1-3"><italic><sup>c</sup></italic></xref> on <italic>I</italic> (%)</td><td align="left" rowspan="1" colspan="1">4.1 (19.2)</td></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>I</italic>/σ<italic><sub>I</sub></italic></td><td align="left" rowspan="1" colspan="1">15.6 (4.6)</td></tr><tr><td align="left" rowspan="1" colspan="1">    Wilson B (Å<sup>2</sup>)</td><td align="left" rowspan="1" colspan="1">15.9</td></tr><tr><td align="left" rowspan="1" colspan="1">    Completeness (%)</td><td align="left" rowspan="1" colspan="1">99.6 (99.8)</td></tr><tr><td align="left" rowspan="1" colspan="1">    Multiplicity</td><td align="left" rowspan="1" colspan="1">7.3 (7.5)</td></tr><tr><td colspan="2" rowspan="1"><hr/></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Model and refinement</bold></td><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">    Reflections (total/test)</td><td align="left" rowspan="1" colspan="1">70,883/3,577</td></tr><tr><td align="left" rowspan="1" colspan="1">    <italic>R</italic><sub>cryst</sub>/<italic>R</italic><sub>free</sub><xref ref-type="table-fn" rid="TF1-4"><italic><sup>d</sup></italic></xref> (%)</td><td align="left" rowspan="1" colspan="1">14.3/17.5</td></tr><tr><td align="left" rowspan="1" colspan="1">    No. protein residues/atoms</td><td align="left" rowspan="1" colspan="1">700/5612</td></tr><tr><td align="left" rowspan="1" colspan="1">    No. of water/EDO molecules</td><td align="left" rowspan="1" colspan="1">690/7</td></tr><tr><td align="left" rowspan="1" colspan="1">    ESU<xref ref-type="table-fn" rid="TF1-5"><italic><sup>e</sup></italic></xref> based on <italic>R</italic><sub>free</sub> (Å)</td><td align="left" rowspan="1" colspan="1">0.095</td></tr><tr><td align="left" rowspan="1" colspan="1">    B-values (Å<sup>2</sup>)</td><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">        Average isotropic B (overall)</td><td align="left" rowspan="1" colspan="1">20.0</td></tr><tr><td align="left" rowspan="1" colspan="1">        Protein overall</td><td align="left" rowspan="1" colspan="1">18.8</td></tr><tr><td align="left" rowspan="1" colspan="1">        All main/side chains</td><td align="left" rowspan="1" colspan="1">16.7/20.8</td></tr><tr><td align="left" rowspan="1" colspan="1">        Solvent/EDO</td><td align="left" rowspan="1" colspan="1">29.4/35.6</td></tr><tr><td align="left" rowspan="1" colspan="1">    RMSD<xref ref-type="table-fn" rid="TF1-7"><italic><sup>g</sup></italic></xref></td><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">        Bond lengths (Å)</td><td align="left" rowspan="1" colspan="1">0.01</td></tr><tr><td align="left" rowspan="1" colspan="1">        Bond angles (°)</td><td align="left" rowspan="1" colspan="1">1.6</td></tr><tr><td align="left" rowspan="1" colspan="1">    Ramachandran analysis (%)</td><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">        Favored regions</td><td align="left" rowspan="1" colspan="1">97.0</td></tr><tr><td align="left" rowspan="1" colspan="1">        Allowed regions</td><td align="left" rowspan="1" colspan="1">3.0</td></tr><tr><td align="left" rowspan="1" colspan="1">        Outliers</td><td align="left" rowspan="1" colspan="1">0.0</td></tr></tbody></table> + + 11022 + Data collection     Wavelength (Å) 0.9793     Space group P21     Unit cell dimensions a, b, c (Å); β° 39.11, 108.68, 77.97; β = 94.32°     Resolution range (Å) 28.73–1.70 (1.79–1.70)     Unique reflections 70,913     Rmergea on I (%) 10.2 (49.0)     Rmeasb on I (%) 11.0 (52.7)     Rpimc on I (%) 4.1 (19.2)     I/σI 15.6 (4.6)     Wilson B (Å2) 15.9     Completeness (%) 99.6 (99.8)     Multiplicity 7.3 (7.5) Model and refinement     Reflections (total/test) 70,883/3,577     Rcryst/Rfreed (%) 14.3/17.5     No. protein residues/atoms 700/5612     No. of water/EDO molecules 690/7     ESUe based on Rfree (Å) 0.095     B-values (Å2)         Average isotropic B (overall) 20.0         Protein overall 18.8         All main/side chains 16.7/20.8         Solvent/EDO 29.4/35.6     RMSDg         Bond lengths (Å) 0.01         Bond angles (°) 1.6     Ramachandran analysis (%)         Favored regions 97.0         Allowed regions 3.0         Outliers 0.0 + + + T1.xml + T1 + TABLE + table_footnote + 12367 + a Rmerge = ΣhklΣi|Ii(hkl) − 〈I(hkl)〉|/Σhkl Σi(hkl). + + + T1.xml + T1 + TABLE + table_footnote + 12435 + b Rmeas = Σhkl[N/(N-1)]1/2Σi|Ii(hkl) − 〈I(hkl)〉|/ΣhklΣiIi(hkl). + + + T1.xml + T1 + TABLE + table_footnote + 12515 + c Rpim (precision-indicating Rmerge) = Σhkl[(1/(N-1)]1/2 Σi|Ii (hkl) − 〈I(hkl)〉|/ΣhklΣi Ii(hkl), where n is the multiplicity of reflection hkl, and Ii(hkl) and 〈I(hkl)〉 are the intensity of the ith measurement and the average intensity of reflection hkl, respectively. + + + T1.xml + T1 + TABLE + table_footnote + 12800 + d Rcryst and Rfree = Σ‖Fobs| − |Fcalc‖/Σ|Fobs| for reflections in the working and test sets, respectively, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively. Rfree is the same as Rcryst but for 5% of the total reflections chosen at random and omitted from structural refinement. + + + T1.xml + T1 + TABLE + table_footnote + 13134 + e ESU is the estimated standard uncertainties of atoms. + + + T1.xml + T1 + TABLE + table_footnote + 13190 + f The average isotropic B includes TLS and residual B components. + + + T1.xml + T1 + TABLE + table_footnote + 13256 + g RMSD, root-mean-square deviation. + + + METHODS + title_4 + 13292 + Structural Analysis + + + METHODS + paragraph + 13312 + The primary sequence alignment with assigned secondary structure was prepared using CLUSTAL OMEGA and ALINE. The topology diagram was produced with TOPDRAW and all three-dimensional structural figures were prepared with PyMol with the electrostatic surface potential calculated with APBS and contoured at ±5 kT/e. Architectural comparisons with known structures revealed that PmC11 was most structurally similar to caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively). The statistical significance of the structural alignment between PmC11 and both caspase-7 and gingipain-K is equivalent (Z-score of 9.2) with legumain giving a very similar result (Z-score of 9.1). Of note, the β-strand topology of the CDP domains of Clostridium difficile toxin B (family C80; TcdB; PDB code 3pee) is identical to that observed in the PmC11 β-sheet, but the Z-score from DaliLite was notably less at 7.6. It is possible that the PmC11 structure is more closely related to the C80 family than other families in clan CD, and appear to reside on the same branch of the phylogenetic tree based on structure. + + + METHODS + title_4 + 14440 + Protein Production for Biochemical Assays + + + METHODS + paragraph + 14482 + The PmCD00547516 plasmid described above was obtained from the PSI:Biology Materials Repository and used to generate a cleavage site mutant PmC11K147A and an active-site mutant PmC11C179A using the QuikChange Site-directed Mutagenesis kit (Stratagene) as per the manufacturer's instructions using the following primers: K147A mutant (forward): CAGAATAAGCTGGCAGCGTTCGGACAGGACG, and K147A mutant (reverse): CGTCCTGTCCGAACGCTGCCAGCTTATTCTG; C179A mutant (forward): CCTGTTCGATGCCGCCTACATGGCAAGC, and C179A mutant (reverse): GCTTGCCATGTAGGCGGCATCGAACAGG. The expression plasmids containing PmC11 were transformed into E. coli BL21 Star (DE3) and grown in Luria-Bertani media containing 30 μg ml−1 of kanamycin at 37 °C until an optical density (600 nm) of ∼0.6 was reached. l-Arabinose was added to a final concentration of 0.2% (w/v) and the cells incubated overnight at 25 °C. + + + METHODS + paragraph + 15363 + Compared with the protein production for crystallography, a slightly modified purification protocol was employed for biochemical assays. Initially, the cells were resuspended in 20 mm sodium phosphate (pH 7.5), 150 mm NaCl (Lysis Buffer 2) containing an EDTA-free protease inhibitor mixture (cOmplete, Roche Applied Science). Cells were disrupted by three passages (15 KPSI) through a One-Shot cell disruptor (Constant Systems) followed by centrifugation at 20,000 × g for 20 min at 4 °C. The supernatant was collected and sterile-filtered (0.2 μm) before being applied to a 5-ml HisTrap HP column (GE Healthcare) equilibrated in Lysis Buffer 2 containing 25 mm imidazole, and the protein was eluted in the same buffer containing 250 mm imidazole. The peak fractions were pooled and buffer exchanged into the assay buffer (20 mm Tris, 150 mm NaCl, pH 8.0) using a PD-10 column. When required, purified PmC11 was concentrated using Vivaspin 2 30-K centrifugal concentrators (Sartorius). Protein concentration was routinely measured using Bradford's reagent (Bio-Rad) with a BSA standard. + + + METHODS + title_4 + 16453 + Fluorogenic Substrate Activity Assays + + + METHODS + paragraph + 16491 + The release of the fluorescent group AMC (7-amino-4-methylcoumarin) from potential peptide substrates was used to assess the activity of PmC11. Peptidase activity was tested using 20 μg of PmC11 and 100 μm substrate (unless otherwise stated) in assay buffer to a final reaction volume of 200 μl and all samples were incubated (without substrate) at 37 °C for 16 h prior to carrying out the assay. The substrate and plate reader were brought to 37 °C for 20 min prior to the addition of the PmC11 and samples prepared without PmC11 were used as blanks (negative controls). The curves were plotted using the blank-corrected fluorescence units against the time of acquisition (in min). The assays were carried out in black 96-well flat-bottomed plates (Greiner). AMC fluorescence was measured using a PHERAstar FS plate reader (BMG Labtech) with excitation and emission wavelengths of 355 and 460 nm, respectively. + + + METHODS + paragraph + 17408 + To investigate the substrate specificity of PmC11, substrates Z-GGR-AMC, Bz-R-AMC, Z-GP-AMC, Z-HGP-AMC, Ac-DEVD-AMC (all Bachem), BOC-VLK-AMC, and BOC-K-AMC (both PeptaNova) were prepared at 100 mm in 100% dimethyl sulfoxide. The amount of AMC (micromoles) released was calculated by generating an AMC standard curve (as described in Ref.) and the specific activity of PmC11 was calculated as picomoles of AMC released per min per mg of the protein preparation. + + + METHODS + paragraph + 17870 + The reaction rates (Vmax) and Km values were determined for mutants PmC11K147A and PmC11C179A by carrying out the activity assay at varying concentrations of Bz-R-AMC between 0 and 600 μm. The blank-corrected relative fluorescence units were plotted against time (min) with ΔFU/T giving the reaction rate. The Km and Vmax of PmC11 and PmC11K147A against an R-AMC substrate were determined from the Lineweaver-Burk plot as described, calculated using GraphPad Prism6 software. All experiments were carried out in triplicate. + + + METHODS + title_4 + 18399 + Effect of VRPR-FMK on PmC11 + + + METHODS + paragraph + 18427 + To test the effect of the inhibitor on the activity of PmC11, 25 μm Z-VRPR-FMK (100 mm stock in 100% dimethyl sulfoxide, Enzo Life Sciences), 20 μg of PmC11, 100 μm R-AMC substrate, 1 mm EGTA were prepared in the assay buffer and the activity assay carried out as described above. A gel-shift assay, to observe Z-VRPR-FMK binding to PmC11, was also set up using 20 μg of PmC11, 25 μm inhibitor, 1 mm EGTA in assay buffer. The reactions were incubated at 37 °C for 20 min before being stopped by the addition SDS-PAGE sample buffer. Samples were analyzed by loading the reaction mixture on a 10% NuPAGE BisTris gel using MES buffer. + + + METHODS + title_4 + 19065 + Effect of Cations on PmC11 + + + METHODS + paragraph + 19092 + The enzyme activity of PmC11 was tested in the presence of various divalent cations: Mg2+, Ca2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+. The final concentration of the salts (MgSO4, CaCl2, MnCl2, CoCl2, FeSO4, ZnCl2, and CuSO4) was 1 mm and the control was set up without divalent ions but with addition of 1 mm EGTA. The assay was set up using 20 mg of PmC11, 1 mm salts, 100 μm R-AMC substrate, and the assay buffer, and incubated at 37 °C for 16 h. The activity assay was carried out as described above. + + + METHODS + title_4 + 19595 + Size Exclusion Chromatography + + + METHODS + paragraph + 19625 + Affinity-purified PmC11 was loaded onto a HiLoad 16/60 Superdex 200 gel filtration column (GE Healthcare) equilibrated in the assay buffer. The apparent molecular weight of PmC11 was determined from calibration curves based on protein standards of known molecular weights. + + + METHODS + title_4 + 19898 + Autoprocessing Profile of PmC11 + + + METHODS + paragraph + 19930 + Autoprocessing of PmC11 was evaluated by incubating the enzyme at 37 °C and removing samples at 1-h intervals from 0 to 16 h and placing into SDS-PAGE loading buffer to stop the processing. Samples were then analyzed on a 4–12% NuPAGE (Thermo Fisher) Novex BisTris gel run in MES buffer. + + + METHODS + title_4 + 20221 + Autoprocessing Cleavage Site Analysis + + + METHODS + paragraph + 20259 + To investigate whether processing is a result of intra- or inter-molecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of activated PmC11 (0, 0.1, 0.2, 0.5, 1, 2, and 5 μg). The final assay volume was 40 μl and the proteins were incubated at 37 °C for 16 h in the PmC11 assay buffer. To stop the reaction, NuPAGE sample buffer was added to the protein samples and 20 μl was analyzed on 10% NuPAGE Novex BisTris gel using MES buffer. These studies revealed no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11: PmC11C179A) was increased to ∼1:10 and 1:4. + + + RESULTS + title_1 + 20969 + Results + + + RESULTS + title_4 + 20977 + Structure of PmC11 + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:54:14Z + + Structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + + RESULTS + paragraph + 20996 + The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like α/β/α sandwich architecture comprised of a central nine-stranded β-sheet, with an unusual C-terminal domain (CTD), starting at Lys250. A single cleavage was observed in the polypeptide chain at Lys147 (Fig. 1, A and B), where both ends of the cleavage site are fully visible and well ordered in the electron density. The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. Helices α1, α7, and α6 are located on one side of the β-sheet with α2, α4, and α5 on the opposite side (Fig. 1A). Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:28:56Z + + crystal structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:37:40Z + + catalytically active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:37:30Z + + extended caspase-like α/β/α sandwich + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:37:44Z + + nine-stranded β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:33:06Z + + C-terminal domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:58:04Z + + CTD + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:57:54Z + + Lys250 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:38:37Z + + single cleavage + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:27Z + + Lys147 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:57:19Z + + cleavage site + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:57:49Z + + electron density + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:37:50Z + + nine-stranded β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:58:11Z + + β1–β9 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:00:05Z + + parallel + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:00:22Z + + anti-parallel β-strands + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:00:29Z + + structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:01:00Z + + α-helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:01:26Z + + α1–α2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:01:28Z + + α4–α7 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:01:40Z + + β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:01:42Z + + Helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:01:45Z + + α1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:01:47Z + + α7 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:01:49Z + + α6 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:01:56Z + + β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:01:58Z + + α2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:02:00Z + + α4 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:13:55Z + + α5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:02:30Z + + Helix + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:02:40Z + + α3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:02:48Z + + loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:02:50Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:03:12Z + + L5 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:27Z + + Lys147 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:57:19Z + + cleavage site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:03:12Z + + L5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:03:18Z + + α3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:03:26Z + + β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:58:04Z + + CTD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:14:05Z + + α8 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:03:32Z + + structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:04:11Z + + β-hairpins + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:04:13Z + + βA–βB + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:21:20Z + + βD–βE + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:04:18Z + + small β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:04:20Z + + βC–βF + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:04:28Z + + βC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:14:10Z + + α11 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:05:30Z + + α12 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:21:41Z + + β9 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:05:37Z + + βF + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:05:43Z + + βD-βE + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:05:46Z + + hairpin + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:58:04Z + + CTD + + + + zbc0191642560001.jpg + F1 + FIG + fig_caption + 22424 + Crystal structure of a C11 peptidase from P. merdae. +A, primary sequence alignment of PmC11 (Uniprot ID A7A9N3) and clostripain (Uniprot ID P09870) from C. histolyticum with identical residues highlighted in gray shading. The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. Connecting loops are colored gray, the main β-sheet is in orange, with other strands in olive, α-helices are in blue, and the nonapeptide linker of clostripain that is excised upon autocleavage is underlined in red. Sequences around the catalytic site of clostripain and PmC11 align well. B, topology diagram of PmC11 colored as in A except that additional (non-core) β-strands are in yellow. Helices found on either side of the central β-sheet are shown above and below the sheet, respectively. The position of the catalytic dyad (H, C) and the processing site (Lys147) are highlighted. Helices (1–14) and β-strands (1–9 and A-F) are numbered from the N terminus. The core caspase-fold is highlighted in a box. C, tertiary structure of PmC11. The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. Loops are colored gray, the main β-sheet is in orange, with other β-strands in yellow, and α-helices are in blue. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:28:56Z + + Crystal structure + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:51:40Z + + C11 peptidase + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:10:05Z + + P. merdae + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:10:10Z + + primary sequence alignment + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:12Z + + clostripain + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:10:15Z + + C. histolyticum + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:28:56Z + + crystal structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:10:24Z + + catalytic dyad + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:10:52Z + + autocatalytic cleavage site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:27Z + + Lys147 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:10:59Z + + S1 binding pocket + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:11:03Z + + Asp + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:11:08Z + + Asp177 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:11:15Z + + loops + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:11:17Z + + β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:11:20Z + + α-helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:11:23Z + + nonapeptide linker + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:12Z + + clostripain + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:11:44Z + + autocleavage + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:11:52Z + + catalytic site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:12Z + + clostripain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T14:12:20Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:12:32Z + + β-strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:12:34Z + + Helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:12:46Z + + β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:12:48Z + + sheet + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:10:24Z + + catalytic dyad + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:12:51Z + + H + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:12:53Z + + C + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:13:13Z + + processing site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:27Z + + Lys147 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:13:24Z + + Helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:13:27Z + + β-strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:12:57Z + + core caspase-fold + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:57Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:13:47Z + + β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:13:50Z + + helix + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:13:55Z + + α5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:13:59Z + + helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:14:05Z + + α8 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:14:10Z + + α11 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:14:15Z + + α13 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:33:06Z + + C-terminal domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:14:19Z + + Loops + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:14:21Z + + β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:14:24Z + + β-strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:14:26Z + + α-helices + + + + RESULTS + paragraph + 24015 + The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. Of the interacting secondary structure elements, α5 is perhaps the most interesting. This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C). + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:58:04Z + + CTD + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:20:20Z + + tight helical bundle + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:20:51Z + + helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:21:03Z + + α8–α14 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:21:06Z + + strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:21:09Z + + βC + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:21:12Z + + βF + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:21:15Z + + β-hairpin + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:21:20Z + + βD–βE + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:58:04Z + + CTD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:03:18Z + + α3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:13:55Z + + α5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:21:41Z + + β9 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:22:08Z + + loops + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:22:18Z + + β8 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:13:55Z + + α5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:22:34Z + + This helix + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:03Z + + hydrogen bonds + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:58:04Z + + CTD + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:22:03Z + + salt bridge + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:22:52Z + + Arg191 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:22:58Z + + Asp255 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:58:04Z + + CTD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:23:07Z + + main core + + + + RESULTS + title_4 + 24647 + Structural Comparisons + + + RESULTS + paragraph + 24670 + PmC11 is, as expected, most structurally similar to other members of clan CD with the top hits in a search of known structures being caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively) (Table 2). The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold. As the archetypal and arguably most well studied member of clan CD, the caspases were used as the basis to investigate the structure/function relationships in PmC11, with caspase-7 as the representative member. Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). A multiple sequence alignment of C11 proteins revealed that these residues are highly conserved (data not shown). + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:59Z + + clan CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:25:20Z + + structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T14:25:28Z + + caspase-7 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T14:25:31Z + + gingipain-K + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:44:14Z + + legumain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:33:06Z + + C-terminal domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:59Z + + clan CD + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:25:41Z + + structure comparisons + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:25:45Z + + this domain alone + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:26:12Z + + DaliLite + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:26:25Z + + PDBeFold + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:59Z + + clan CD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:04Z + + caspases + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T14:26:53Z + + caspase-7 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:27:24Z + + β-strands + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:27:20Z + + β1–β2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:27:31Z + + β5–β8 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:27:38Z + + six-stranded β-sheet + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:04Z + + caspases + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:27:43Z + + strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:27:46Z + + β3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:27:49Z + + β4 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:21:41Z + + β9 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:28:03Z + + core structure + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:28:08Z + + His133 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:28:13Z + + Cys179 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:36:41Z + + caspase + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:10:24Z + + catalytic dyad + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:59Z + + clan CD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:28:26Z + + structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:28:37Z + + strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:28:40Z + + β5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:28:43Z + + β6 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:28:46Z + + multiple sequence alignment + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:18Z + + C11 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:48:11Z + + highly conserved + + + + T2.xml + T2 + TABLE + table_caption + 25879 + Summary of PDBeFOLD superposition of structures found to be most similar to PmC11 in the PBD based on DaliLite + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:29:05Z + + PDBeFOLD superposition + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:29:14Z + + DaliLite + + + + T2.xml + T2 + TABLE + table_caption + 25991 + The results are ordered in terms of structural homology (QH), where %SSEPC-X is the percentage of the SSEs in the PmC11 that can be identified in the target X (where X = caspase-7, legumain, gingipain, and TcdB-CPD; % SSEX-PC is the percentage of SSEs in X (as above) that can be identified in PmC11 (as above); % sequence ID is the percentage sequence identity after structural alignment; Nalign is the number of matched residues; and r.m.s. deviation the root mean squared deviation on the Cα positions of the matched residues. + + + T2.xml + T2 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table xmlns:xlink="http://www.w3.org/1999/xlink" frame="hsides" rules="groups"><thead valign="bottom"><tr><th align="center" rowspan="1" colspan="1">Enzyme</th><th align="center" rowspan="1" colspan="1">Family</th><th align="center" rowspan="1" colspan="1">PDB code</th><th align="center" rowspan="1" colspan="1">Q<sup>H</sup></th><th align="center" rowspan="1" colspan="1">Z-score</th><th align="center" rowspan="1" colspan="1">%SSE<sup>PC-X</sup></th><th align="center" rowspan="1" colspan="1">%SSE<sup>X-PC</sup></th><th align="center" rowspan="1" colspan="1">% Seq. ID</th><th align="center" rowspan="1" colspan="1"><italic>N</italic><sub>align</sub></th><th align="center" rowspan="1" colspan="1">RMSD (Å)</th><th align="center" rowspan="1" colspan="1"><italic>N</italic><sub>Strands</sub></th></tr></thead><tbody valign="top"><tr><td align="left" rowspan="1" colspan="1">PmC11</td><td align="left" rowspan="1" colspan="1">C11</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="3UWS">3UWS</ext-link></td><td align="left" rowspan="1" colspan="1">1.00</td><td align="left" rowspan="1" colspan="1">33.4</td><td align="left" rowspan="1" colspan="1">100</td><td align="left" rowspan="1" colspan="1">100</td><td align="left" rowspan="1" colspan="1">100</td><td align="left" rowspan="1" colspan="1">352</td><td align="left" rowspan="1" colspan="1">0.00</td><td align="left" rowspan="1" colspan="1">9</td></tr><tr><td align="left" rowspan="1" colspan="1">Caspase-7</td><td align="left" rowspan="1" colspan="1">C14A</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="4HQ0">4HQ0</ext-link></td><td align="left" rowspan="1" colspan="1">0.16</td><td align="left" rowspan="1" colspan="1">4.3</td><td align="left" rowspan="1" colspan="1">38</td><td align="left" rowspan="1" colspan="1">79</td><td align="left" rowspan="1" colspan="1">14</td><td align="left" rowspan="1" colspan="1">162</td><td align="left" rowspan="1" colspan="1">3.27</td><td align="left" rowspan="1" colspan="1">6</td></tr><tr><td align="left" rowspan="1" colspan="1">Legumain</td><td align="left" rowspan="1" colspan="1">C13</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="4AW9">4AW9</ext-link></td><td align="left" rowspan="1" colspan="1">0.13</td><td align="left" rowspan="1" colspan="1">5.5</td><td align="left" rowspan="1" colspan="1">31</td><td align="left" rowspan="1" colspan="1">53</td><td align="left" rowspan="1" colspan="1">13</td><td align="left" rowspan="1" colspan="1">161</td><td align="left" rowspan="1" colspan="1">2.05</td><td align="left" rowspan="1" colspan="1">6</td></tr><tr><td align="left" rowspan="1" colspan="1">TcdB-CPD</td><td align="left" rowspan="1" colspan="1">C80</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="3PEE">3PEE</ext-link></td><td align="left" rowspan="1" colspan="1">0.10</td><td align="left" rowspan="1" colspan="1">4.9</td><td align="left" rowspan="1" colspan="1">28</td><td align="left" rowspan="1" colspan="1">50</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">138</td><td align="left" rowspan="1" colspan="1">3.18</td><td align="left" rowspan="1" colspan="1">9</td></tr><tr><td align="left" rowspan="1" colspan="1">Gingipain</td><td align="left" rowspan="1" colspan="1">C25</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="4TKX">4TKX</ext-link></td><td align="left" rowspan="1" colspan="1">0.07</td><td align="left" rowspan="1" colspan="1">5.4</td><td align="left" rowspan="1" colspan="1">28</td><td align="left" rowspan="1" colspan="1">27</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">153</td><td align="left" rowspan="1" colspan="1">2.97</td><td align="left" rowspan="1" colspan="1">10</td></tr></tbody></table> + + 26525 + Enzyme Family PDB code QH Z-score %SSEPC-X %SSEX-PC % Seq. ID Nalign RMSD (Å) NStrands PmC11 C11 3UWS 1.00 33.4 100 100 100 352 0.00 9 Caspase-7 C14A 4HQ0 0.16 4.3 38 79 14 162 3.27 6 Legumain C13 4AW9 0.13 5.5 31 53 13 161 2.05 6 TcdB-CPD C80 3PEE 0.10 4.9 28 50 12 138 3.18 9 Gingipain C25 4TKX 0.07 5.4 28 27 12 153 2.97 10 + + + zbc0191642560002.jpg + F2 + FIG + fig_caption + 26866 + Biochemical and structural characterization of PmC11. +A, ribbon representation of the overall structure of PmC11 illustrating the catalytic site, cleavage site displacement, and potential S1 binding site. The overall structure of PmC11 is shown in gray, looking down into the catalytic site with the catalytic dyad in red. The two ends of the autolytic cleavage site (Lys147 and Ala148, green) are displaced by 19.5 Å (thin black line) from one another and residues in the potential substrate binding pocket are highlighted in blue. B, size exclusion chromatography of PmC11. PmC11 migrates as a monomer with a molecular mass around 41 kDa calculated from protein standards of known molecular weights. Elution fractions across the major peak (1–6) were analyzed by SDS-PAGE on a 4–12% gel in MES buffer. C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. D, cysteine peptidase activity of PmC11. Km and Vmax of PmC11 and K147A mutant were determined by monitoring change in the fluorescence corresponding to AMC release from Bz-R-AMC. Reactions were performed in triplicate and representative values ± S.D. are shown. E, intermolecular processing of PmC11C179A by PmC11. PmC11C179A (20 μg) was incubated overnight at 37 °C with increasing amounts of processed PmC11 and analyzed on a 10% SDS-PAGE gel. Inactive PmC11C179A was not processed to a major extent by active PmC11 until around a ratio of 1:4 (5 μg of active PmC11). A single lane of 20 μg of active PmC11 (labeled 20) is shown for comparison. F, activity of PmC11 against basic substrates. Specific activity is shown ± S.D. from three independent experiments. G, electrostatic surface potential of PmC11 shown in a similar orientation, where blue and red denote positively and negatively charged surface potential, respectively, contoured at ±5 kT/e. The position of the catalytic dyad, one potential key substrate binding residue Asp177, and the ends of the cleavage site Lys147 and Ala148 are indicated. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:32:47Z + + Biochemical and structural characterization + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:11:52Z + + catalytic site + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:32:59Z + + S1 binding site + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:32:52Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:11:52Z + + catalytic site + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:10:24Z + + catalytic dyad + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:33:21Z + + autolytic cleavage site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:27Z + + Lys147 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:30Z + + Ala148 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:33:35Z + + substrate binding pocket + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:33:38Z + + size exclusion chromatography + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:43Z + + monomer + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:33:49Z + + SDS-PAGE + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:56Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:03Z + + PmC11C179A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:09Z + + PmC11K147A + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:33:49Z + + SDS-PAGE + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:17Z + + Western blot + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:33Z + + autoprocesses + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:03Z + + PmC11C179A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:09Z + + PmC11K147A + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:45Z + + autoprocessing + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:34:54Z + + Km + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:34:59Z + + Vmax + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:35:04Z + + K147A + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T14:54:37Z + + Bz-R-AMC + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:35:16Z + + intermolecular processing + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:03Z + + PmC11C179A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:03Z + + PmC11C179A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:33:49Z + + SDS-PAGE + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:35:32Z + + Inactive + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:03Z + + PmC11C179A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:56Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:57Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:57Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:35:38Z + + activity + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:10:24Z + + catalytic dyad + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:35:48Z + + key substrate binding residue + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:11:08Z + + Asp177 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:57:19Z + + cleavage site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:27Z + + Lys147 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:30Z + + Ala148 + + + + RESULTS + paragraph + 29083 + Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. Other than its more extended β-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven α-helices and four β-strands after β8. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:38:43Z + + α-helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:38:52Z + + β-sheet + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:39:00Z + + α1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:39:03Z + + α2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:39:07Z + + α4 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:39:09Z + + α6 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:39:13Z + + α7 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:38:39Z + + structurally conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:39:17Z + + helices + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:04Z + + caspases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:59Z + + clan CD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:39:22Z + + C80 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:39:25Z + + extended β-sheet + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:59Z + + clan CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:58:04Z + + CTD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:40:08Z + + α-helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:40:43Z + + β-strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:22:18Z + + β8 + + + + RESULTS + title_4 + 29537 + Autoprocessing of PmC11 + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:41:11Z + + Autoprocessing + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + + RESULTS + paragraph + 29561 + Purification of recombinant PmC11 (molecular mass = 42.6 kDa) revealed partial processing into two cleavage products of 26.4 and 16.2 kDa, related to the observed cleavage at Lys147 in the crystal structure (Fig. 2A). Incubation of PmC11 at 37 °C for 16 h, resulted in a fully processed enzyme that remained as an intact monomer when applied to a size-exclusion column (Fig. 2B). The single cleavage site of PmC11 at Lys147 is found immediately after α3, in loop L5 within the central β-sheet (Figs. 1, A and B, and 2A). The two ends of the cleavage site are remarkably well ordered in the crystal structure and displaced from one another by 19.5 Å (Fig. 2A). Moreover, the C-terminal side of the cleavage site resides near the catalytic dyad with Ala148 being 4.5 and 5.7 Å from His133 and Cys179, respectively. Consequently, it appears feasible that the helix attached to Lys147 (α3) could be responsible for steric autoinhibition of PmC11 when Lys147 is covalently bonded to Ala148. Thus, the cleavage would be required for full activation of PmC11. To investigate this possibility, two mutant forms of the enzyme were created: PmC11C179A (a catalytically inactive mutant) and PmC11K147A (a cleavage-site mutant). Initial SDS-PAGE and Western blot analysis of both mutants revealed no discernible processing occurred as compared with active PmC11 (Fig. 2C). The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D). Taken together, these data reveal that PmC11 requires processing at Lys147 for optimum activity. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:44:17Z + + Purification + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:34:11Z + + cleavage + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:28Z + + Lys147 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:28:56Z + + crystal structure + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:44:25Z + + Incubation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:44:29Z + + fully processed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:44:38Z + + intact + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:43Z + + monomer + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:57:19Z + + cleavage site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:28Z + + Lys147 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:03:18Z + + α3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:37:56Z + + loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:03:12Z + + L5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:38:00Z + + β-sheet + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:57:19Z + + cleavage site + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:28:56Z + + crystal structure + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:57:19Z + + cleavage site + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:10:24Z + + catalytic dyad + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:30Z + + Ala148 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:28:08Z + + His133 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:28:13Z + + Cys179 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:45:34Z + + helix + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:28Z + + Lys147 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:03:18Z + + α3 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:28Z + + Lys147 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:30Z + + Ala148 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:34:11Z + + cleavage + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:45:43Z + + full activation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:03Z + + PmC11C179A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:46:11Z + + catalytically inactive mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:09Z + + PmC11K147A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:46:17Z + + cleavage-site mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:33:49Z + + SDS-PAGE + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:17Z + + Western blot + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:57Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:09Z + + PmC11K147A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:46:21Z + + mutant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:46:33Z + + reaction rate + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:34:59Z + + Vmax + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:46:27Z + + WT + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:46:31Z + + reaction velocity + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:09Z + + PmC11K147A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:28Z + + Lys147 + + + + RESULTS + paragraph + 31216 + To investigate whether processing is a result of intra- or intermolecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of processed and activated PmC11. These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). This suggests that cleavage of PmC11C179A was most likely an effect of the increasing concentration of PmC11 and intermolecular cleavage. Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147. This cleavage subsequently allows movement of the region containing Lys147 and the active site to open up for substrate access. + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:03Z + + PmC11C179A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:49:25Z + + mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:49:28Z + + incubated with increasing concentrations + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:49:31Z + + processed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:49:35Z + + activated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:03Z + + PmC11C179A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:57Z + + active + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:49:46Z + + at low concentrations + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:57Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:03Z + + PmC11C179A + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:49:53Z + + increased to ∼1:10 and 1:4 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:49:56Z + + ratio of 1:1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:34:11Z + + cleavage + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:34:03Z + + PmC11C179A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:50:12Z + + pro-form + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:50:16Z + + autoinhibited + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:03:12Z + + L5 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:34:26Z + + active site + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:50:36Z + + intramolecular cleavage + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:28Z + + Lys147 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-06-15T08:23:33Z + + cleavage + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:28Z + + Lys147 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:34:26Z + + active site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:51:01Z + + open + + + + RESULTS + title_4 + 32158 + Substrate Specificity of PmC11 + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + + RESULTS + paragraph + 32189 + The autocatalytic cleavage of PmC11 at Lys147 (sequence KLK∧A) demonstrates that the enzyme accepts substrates with Lys in the P1 position. The substrate specificity of the enzyme was further tested using a variety of fluorogenic substrates. As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. The rate of cleavage was ∼3-fold greater toward the single Arg substrate Bz-R-AMC than for the other two (Fig. 2F) and, unexpectedly, PmC11 showed no activity toward BOC-K-AMC. These results confirm that PmC11 accepts substrates containing Arg or Lys in P1 with a possible preference for Arg. + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:54:06Z + + autocatalytic cleavage + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:28Z + + Lys147 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:55:14Z + + Lys + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:48:22Z + + P1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:54:23Z + + Pro + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:54:26Z + + Asp + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:48:22Z + + P1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:57Z + + active + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:48:22Z + + P1 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T14:54:37Z + + Bz-R-AMC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T14:54:42Z + + Z-GGR-AMC + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T14:54:48Z + + BOC-VLK-AMC + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:54:55Z + + Arg + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T14:54:37Z + + Bz-R-AMC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T14:55:07Z + + BOC-K-AMC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:54:55Z + + Arg + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:55:14Z + + Lys + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:48:22Z + + P1 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:54:55Z + + Arg + + + + RESULTS + paragraph + 32913 + The catalytic dyad of PmC11 sits near the bottom of an open pocket on the surface of the enzyme at a conserved location in the clan CD family. The PmC11 structure reveals that the catalytic dyad forms part of a large acidic pocket (Fig. 2G), consistent with a binding site for a basic substrate. This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A). Interestingly, these residues are in regions that are structurally similar to those involved in the S1 binding pockets of other clan CD members (shown in Ref.). + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:10:24Z + + catalytic dyad + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:56:36Z + + open + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:56:39Z + + pocket + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:57:02Z + + conserved location + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:57:14Z + + CD family + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:57:19Z + + structure + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:10:24Z + + catalytic dyad + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:57:24Z + + acidic pocket + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:57:27Z + + binding site + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:57:30Z + + pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:57:35Z + + Asn50 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:11:08Z + + Asp177 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:57:45Z + + Thr204 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:57:50Z + + Gly134 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:57:55Z + + Asp207 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:58:02Z + + Met205 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:58:05Z + + pocket + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:58:11Z + + structurally similar + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:58:14Z + + S1 binding pockets + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:58:18Z + + clan CD members + + + + RESULTS + paragraph + 33537 + Because PmC11 recognizes basic substrates, the tetrapeptide inhibitor Z-VRPR-FMK was tested as an enzyme inhibitor and was found to inhibit both the autoprocessing and activity of PmC11 (Fig. 3A). Z-VRPR-FMK was also shown to bind to the enzyme: a size-shift was observed, by SDS-PAGE analysis, in the larger processed product of PmC11 suggesting that the inhibitor bound to the active site (Fig. 3B). A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). Asp177 is located near the catalytic cysteine and is conserved throughout the C11 family, suggesting it is the primary S1 binding site residue. In the structure of PmC11, Asp207 resides on a flexible loop pointing away from the S1 binding pocket (Fig. 3C). However, this loop has been shown to be important for substrate binding in clan CD and this residue could easily rotate and be involved in substrate binding in PmC11. Thus, Asn50, Asp177, and Asp207 are most likely responsible for the substrate specificity of PmC11. Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C). + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:02:52Z + + Z-VRPR-FMK + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:02:33Z + + inhibit + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:02:44Z + + autoprocessing + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:02:52Z + + Z-VRPR-FMK + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:03:03Z + + size-shift + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:33:49Z + + SDS-PAGE + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:03:26Z + + inhibitor bound + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:34:26Z + + active site + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:03:33Z + + structure overlay + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:03:45Z + + MALT1-paracacaspase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:04:29Z + + MALT1-P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:04:01Z + + complex + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:02:52Z + + Z-VRPR-FMK + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:38:12Z + + dyad + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:33:57Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:04:29Z + + MALT1-P + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:57:35Z + + Asn50 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:11:08Z + + Asp177 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:57:55Z + + Asp207 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:04:29Z + + MALT1-P + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:05:32Z + + inhibitor binding residues + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:05:39Z + + Asp365 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:05:44Z + + Asp462 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:05:49Z + + Glu500 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:05:57Z + + VRPR-FMK + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:04:29Z + + MALT1-P + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:06:02Z + + structural overlay + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:11:08Z + + Asp177 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:06:05Z + + catalytic + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:06:08Z + + cysteine + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:06:11Z + + conserved throughout + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:06:13Z + + C11 family + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:06:20Z + + S1 binding site residue + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:06:23Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:57:55Z + + Asp207 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:38:04Z + + loop + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:10:59Z + + S1 binding pocket + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:38:07Z + + loop + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:59Z + + clan CD + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:57:35Z + + Asn50 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:11:08Z + + Asp177 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:57:55Z + + Asp207 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:11:08Z + + Asp177 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:48:11Z + + highly conserved + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:06:36Z + + clan CD C11 peptidases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:06:45Z + + clan CD enzymes + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:02:52Z + + Z-VRPR-FMK + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:06:50Z + + overlaid + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:04:29Z + + MALT1-P + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:06:52Z + + structure + + + + zbc0191642560003.jpg + F3 + FIG + fig_caption + 35240 + PmC11 binds and is inhibited by Z-VRPR-FMK and does not require Ca2+ for activity. +A, PmC11 activity is inhibited by Z-VRPR-FMK. Cleavage of Bz-R-AMC by PmC11 was measured in a fluorometric activity assay with (+, purple) and without (−, red) Z-VRPR-FMK. The relative fluorescence units of AMC released are plotted against time (min) (n = 3; ±S.D.). B, gel-shift assay reveals that Z-VRPR-FMK binds to PmC11. PmC11 was incubated with (+) or without (−) Z-VRPR-FMK and the samples analyzed on a 10% SDS-PAGE gel. A size shift can be observed in the larger processed product of PmC11 (26.1 kDa). C, PmC11 with the Z-VRPR-FMK from the MALT1-paracacaspase (MALT1-P) superimposed. A three-dimensional structural overlay of Z-VRPR-FMK from the MALT1-P complex onto PmC11. The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11. Residues surrounding the inhibitor are labeled and represent potentially important binding site residues, labeled in black and shown in an atomic representation. Carbon atoms are shown in gray, nitrogen in blue, and oxygen in red. C, divalent cations do not increase the activity of PmC11. The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). The addition of cations produced no improvement in activity of PmC11 when compared in the presence of EGTA, suggesting that PmC11 does not require metal ions for proteolytic activity. Furthermore, Cu2+, Fe2+, and Zn2+ appear to inhibit PmC11. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:02:52Z + + Z-VRPR-FMK + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:10:14Z + + Ca2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:02:52Z + + Z-VRPR-FMK + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T14:54:37Z + + Bz-R-AMC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:10:42Z + + fluorometric activity assay + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:02:52Z + + Z-VRPR-FMK + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:10:46Z + + gel-shift assay + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:02:52Z + + Z-VRPR-FMK + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:10:49Z + + incubated + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:02:52Z + + Z-VRPR-FMK + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:33:49Z + + SDS-PAGE + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:10:54Z + + size shift + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:02:52Z + + Z-VRPR-FMK + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:10:59Z + + MALT1-paracacaspase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:04:29Z + + MALT1-P + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:11:02Z + + superimposed + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:11:05Z + + three-dimensional structural overlay + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:02:52Z + + Z-VRPR-FMK + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:04:29Z + + MALT1-P + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:02:52Z + + Z-VRPR-FMK + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:11:08Z + + superposition + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:37:07Z + + MALTI_P + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:11:11Z + + structures + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:34:26Z + + active site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:11:18Z + + binding site residues + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:58Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T14:54:37Z + + Bz-R-AMC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:11:41Z + + Ca2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:11:46Z + + Mn2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:11:49Z + + Zn2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:11:52Z + + Co2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:11:56Z + + Cu2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:11:59Z + + Mg2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:12:01Z + + Fe3+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:12:04Z + + EGTA + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:12:11Z + + relative fluorescence measured against time + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:12:14Z + + addition of cations + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:12:17Z + + EGTA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:12:23Z + + Cu2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:12:25Z + + Fe2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:12:28Z + + Zn2+ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:13:11Z + + inhibit + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + + RESULTS + title_4 + 36912 + Comparison with Clostripain + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:12Z + + Clostripain + + + + RESULTS + paragraph + 36940 + Clostripain from C. histolyticum is the founding member of the C11 family of peptidases and contains an additional 149 residues compared with PmC11. A multiple sequence alignment revealed that most of the secondary structural elements are conserved between the two enzymes, although they are only ∼23% identical (Fig. 1A). Nevertheless, PmC11 may be a good model for the core structure of clostripain. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:12Z + + Clostripain + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T14:10:15Z + + C. histolyticum + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:14:10Z + + C11 family + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:14:14Z + + peptidases + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:14:20Z + + 149 residues + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:14:23Z + + multiple sequence alignment + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:14:35Z + + secondary structural elements + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:14:38Z + + conserved + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:12Z + + clostripain + + + + RESULTS + paragraph + 37344 + The primary structural alignment also shows that the catalytic dyad in PmC11 is structurally conserved in clostripain (Fig. 1A). Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A). Interestingly, Arg190 was found to align with Lys147 in PmC11. In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A). + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:16:18Z + + primary structural alignment + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:10:24Z + + catalytic dyad + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:16:21Z + + structurally conserved + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:13Z + + clostripain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:13Z + + clostripain + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:16:37Z + + cleavage sites + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:17:03Z + + Arg181 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:17:09Z + + Arg190 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:17:16Z + + nonapeptide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:17:19Z + + full activation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:17:09Z + + Arg190 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:33:28Z + + Lys147 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:17:37Z + + S1-binding residue + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T14:11:08Z + + Asp177 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:17:41Z + + overlays + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:17:47Z + + P1 specificity determining residue + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:13Z + + clostripain + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:17:51Z + + Asp229 + + + + RESULTS + paragraph + 37919 + As studies on clostripain revealed addition of Ca2+ ions are required for full activation, the Ca2+ dependence of PmC11 was examined. Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). In support of these findings, EGTA did not inhibit PmC11 suggesting that, unlike clostripain, PmC11 does not require Ca2+ or other divalent cations, for activity. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:13Z + + clostripain + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:19:08Z + + Ca2+ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:19:12Z + + full activation + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:19:15Z + + Ca2+ + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:19:18Z + + Ca2+ + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T09:56:52Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:19:22Z + + Mg2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:19:25Z + + Mn2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:19:28Z + + Co2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:19:31Z + + Fe2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:19:34Z + + Zn2+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:19:38Z + + Cu2+ + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T09:57:02Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:19:42Z + + EGTA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:13Z + + clostripain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T15:19:44Z + + Ca2+ + + + + DISCUSS + title_1 + 38397 + Discussion + + + DISCUSS + paragraph + 38408 + The crystal structure of PmC11 now provides three-dimensional information for a member of the clostripain C11 family of cysteine peptidases. The enzyme exhibits all of the key structural elements of clan CD members, but is unusual in that it has a nine-stranded central β-sheet with a novel C-terminal domain. The structural similarity of PmC11 with its nearest structural neighbors in the PDB is decidedly low, overlaying better with six-stranded caspase-7 than any of the other larger members of the clan (Table 2). The substrate specificity of PmC11 is Arg/Lys and the crystal structure revealed an acidic pocket for specific binding of such basic substrates. In addition, the structure suggested a mechanism of self-inhibition in both PmC11 and clostripain and an activation mechanism that requires autoprocessing. PmC11 differs from clostripain in that is does not appear to require divalent cations for activation. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:28:56Z + + crystal structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:13Z + + clostripain + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:26:34Z + + C11 family + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:31Z + + cysteine peptidases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:26:43Z + + clan CD members + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:26:50Z + + β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:33:06Z + + C-terminal domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:26:56Z + + caspase-7 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:54:55Z + + Arg + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T14:55:14Z + + Lys + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T13:28:57Z + + crystal structure + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:27:08Z + + acidic pocket + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:27:11Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:13Z + + clostripain + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:27:18Z + + autoprocessing + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:50:13Z + + clostripain + + + + DISCUSS + paragraph + 39332 + Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7. To date, the effector caspases are the only group of enzymes that require cleavage of a loop within the central β-sheet. This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:39:59Z + + clan CD + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:38:44Z + + processing + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:28:29Z + + full activation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:44:14Z + + legumain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:37:13Z + + gingipain-R + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:28:33Z + + MARTX-CPD + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:28:36Z + + effector caspases + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:37:18Z + + caspase-7 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:28:41Z + + effector caspases + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:34:11Z + + cleavage + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:28:45Z + + loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:28:49Z + + β-sheet + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:29:29Z + + cleavage + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:29:39Z + + loop + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:04Z + + caspases + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:29:57Z + + catalytic + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:29:51Z + + His + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:29:47Z + + Cys + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:04Z + + caspases + + + + DISCUSS + paragraph + 39810 + All other clan CD members requiring cleavage for full activation do so at sites external to their central sheets. The caspases and gingipain-R both undergo intermolecular (trans) cleavage and legumain and MARTX-CPD are reported to perform intramolecular (cis) cleavage. In addition, several members of clan CD exhibit self-inhibition, whereby regions of the enzyme block access to the active site. Like PmC11, these structures show preformed catalytic machinery and, for a substrate to gain access, movement and/or cleavage of the blocking region is required. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:31:05Z + + clan CD members + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:34:11Z + + cleavage + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:31:08Z + + full activation + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:38:18Z + + sites + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:31:27Z + + sheets + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:32:04Z + + caspases + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:37:21Z + + gingipain-R + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:32:28Z + + intermolecular (trans) cleavage + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:44:14Z + + legumain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:37:25Z + + MARTX-CPD + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:32:39Z + + intramolecular (cis) cleavage + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:40:00Z + + clan CD + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:32:54Z + + regions + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T13:34:26Z + + active site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-16T13:34:11Z + + cleavage + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T15:32:57Z + + blocking region + + + + DISCUSS + paragraph + 40370 + The structure of PmC11 gives the first insight into this class of relatively unexplored family of proteins and should allow important catalytic and substrate binding residues to be identified in a variety of orthologues. Indeed, insights gained from an analysis of the PmC11 structure revealed the identity of the Trypanosoma brucei PNT1 protein as a C11 cysteine peptidase with an essential role in organelle replication. The PmC11 structure should provide a good basis for structural modeling and, given the importance of other clan CD enzymes, this work should also advance the exploration of these peptidases and potentially identify new biologically important substrates. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:33:43Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:33:45Z + + structure + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:33:36Z + + Trypanosoma brucei + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T15:33:40Z + + PNT1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:33:58Z + + C11 cysteine peptidase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T13:32:59Z + + PmC11 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T15:33:47Z + + structure + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:34:20Z + + structural modeling + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:34:03Z + + clan CD enzymes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T15:33:55Z + + peptidases + + + + AUTH_CONT + title_1 + 41047 + Author Contributions + + + AUTH_CONT + paragraph + 41068 + K. M., J. S. G., D. D., I. A. W., and J. C. M. designed the research; K. M., J. S. G., and D. D. performed the research; K. M., J. S. G., D. D., G. H. C., A. S., M. A. E., and J. C. M. analyzed the data; A. G., S. A. L., A. M. D., M. A. E., and I. A. W. supervised various components of the JCSG structural genomics pipeline; M. K. G., A. G., S. A. L., A. M. D., and M. A. E. contributed reagents, materials, and analysis tools; and K. M., J. S. G., G. H. C., M. A. E., I. A. W., and J. C. M. wrote the paper. + + + AUTH_CONT + footnote + 41578 + This work was supported by the Medical Research Council Grant MR/K019384, Wellcome Trust Grants 091790 and 104111, and National Institutes of Health Grant U54 GM094586 (JCSG). The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and National Institutes of Health (NIH), National Center for Research Resources, Biomedical Technology Program Grant P41RR001209, and the NIMGS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIGMS or NIH. The authors declare that they have no conflicts of interest with the contents of this article. + + + AUTH_CONT + footnote + 42243 + The atomic coordinates and structure factors (code 3UWS) have been deposited in the Protein Data Bank (http://wwpdb.org/). + + + AUTH_CONT + footnote + 42366 + JCSG + + + AUTH_CONT + footnote + 42371 + Joint Center for Structural Genomics + + + AUTH_CONT + footnote + 42408 + PIPE + + + AUTH_CONT + footnote + 42413 + polymerase incomplete primer extension + + + AUTH_CONT + footnote + 42452 + TCEP + + + AUTH_CONT + footnote + 42457 + Tris(2-carboxyethyl)phosphine + + + AUTH_CONT + footnote + 42487 + AMC + + + AUTH_CONT + footnote + 42491 + 7-amino-4-methylcoumarin + + + AUTH_CONT + footnote + 42516 + PDB + + + AUTH_CONT + footnote + 42520 + Protein Data Bank + + + AUTH_CONT + footnote + 42538 + BisTris + + + AUTH_CONT + footnote + 42546 + 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol + + + AUTH_CONT + footnote + 42609 + Z + + + AUTH_CONT + footnote + 42611 + benzyloxycarbonyl + + + AUTH_CONT + footnote + 42629 + FMK + + + AUTH_CONT + footnote + 42633 + fluoromethyl ketone + + + AUTH_CONT + footnote + 42653 + CTD + + + AUTH_CONT + footnote + 42657 + C-terminal domain + + + AUTH_CONT + footnote + 42675 + Bz-R-AMC + + + AUTH_CONT + footnote + 42684 + benzoyl-l-Arg-4-methylcoumaryl-7-amide + + + AUTH_CONT + footnote + 42723 + Z-GGR-AMC + + + AUTH_CONT + footnote + 42733 + benzyloxycarbonyl-Gly-Gly-Arg-AMC + + + AUTH_CONT + footnote + 42767 + BOC-VLK-AMC + + + AUTH_CONT + footnote + 42779 + t-butyloxycarbonyl-Val-Leu-Lys. + + + AUTH_CONT + footnote + 42811 + The abbreviations used are: + + + REF + title + 42840 + References + + + D343 + 350 + surname:Rawlings;given-names:N. D. + surname:Barrett;given-names:A. J. + surname:Bateman;given-names:A. + 22086950 + REF + Nucleic Acids Res. + ref + 40 + 2012 + 42851 + MEROPS: the database of proteolytic enzymes, their substrates and inhibitors + + + 219 + 232 + surname:McLuskey;given-names:K. + surname:Mottram;given-names:J. C. + 25697094 + REF + Biochem. J. + ref + 466 + 2015 + 42928 + Comparative structural analysis of the caspase family with other clan CD cysteine peptidases + + + 10940 + 10945 + surname:Dall;given-names:E. + surname:Brandstetter;given-names:H. + 23776206 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 110 + 2013 + 43021 + Mechanistic and structural studies on legumain explain its zymogenicity, distinct activation pathways, and regulation + + + 343 + 352 + surname:Walker;given-names:N. P. + surname:Talanian;given-names:R. V. + surname:Brady;given-names:K. D. + surname:Dang;given-names:L. C. + surname:Bump;given-names:N. J. + surname:Ferenz;given-names:C. R. + surname:Franklin;given-names:S. + surname:Ghayur;given-names:T. + surname:Hackett;given-names:M. C. + surname:Hammill;given-names:L. D. + 8044845 + REF + Cell + ref + 78 + 1994 + 43139 + Crystal structure of the cysteine protease interleukin-1β-converting enzyme: a (p20/p10)2 homodimer + + + 21004 + 21009 + surname:Yu;given-names:J. W. + surname:Jeffrey;given-names:P. D. + surname:Ha;given-names:J. Y. + surname:Yang;given-names:X. + surname:Shi;given-names:Y. + 22158899 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 108 + 2011 + 43242 + Crystal structure of the mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase region + + + 7469 + 7474 + surname:McLuskey;given-names:K. + surname:Rudolf;given-names:J. + surname:Proto;given-names:W. R. + surname:Isaacs;given-names:N. W. + surname:Coombs;given-names:G. H. + surname:Moss;given-names:C. X. + surname:Mottram;given-names:J. C. + 22529389 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 109 + 2012 + 43353 + Crystal structure of a Trypanosoma brucei metacaspase + + + 5453 + 5462 + surname:Eichinger;given-names:A. + surname:Beisel;given-names:H. G. + surname:Jacob;given-names:U. + surname:Huber;given-names:R. + surname:Medrano;given-names:F. J. + surname:Banbula;given-names:A. + surname:Potempa;given-names:J. + surname:Travis;given-names:J. + surname:Bode;given-names:W. + 10523290 + REF + EMBO J. + ref + 18 + 1999 + 43407 + Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinase with a caspase-like fold + + + 265 + 268 + surname:Lupardus;given-names:P. J. + surname:Shen;given-names:A. + surname:Bogyo;given-names:M. + surname:Garcia;given-names:K. C. + 18845756 + REF + Science + ref + 322 + 2008 + 43512 + Small molecule-induced allosteric activation of the Vibrio cholerae RTX cysteine protease domain + + + 1685 + 1690 + surname:Kocholaty;given-names:W. + surname:Weil;given-names:L. + surname:Smith;given-names:L. + 16746798 + REF + Biochem. J. + ref + 32 + 1938 + 43609 + Proteinase secretion and growth of Clostridium histolyticum + + + 277 + 280 + surname:Kembhavi;given-names:A. A. + surname:Buttle;given-names:D. J. + surname:Rauber;given-names:P. + surname:Barrett;given-names:A. J. + 2044766 + REF + FEBS Lett. + ref + 283 + 1991 + 43669 + Clostripain: characterization of the active site + + + 1137 + 1142 + surname:Elsliger;given-names:M. A. + surname:Deacon;given-names:A. M. + surname:Godzik;given-names:A. + surname:Lesley;given-names:S. A. + surname:Wooley;given-names:J. + surname:Wüthrich;given-names:K. + surname:Wilson;given-names:I. A. + REF + Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. + ref + 66 + 2010 + 43718 + The JCSG high-throughput structural biology pipeline + + + 91 + 103 + surname:Klock;given-names:H. E. + surname:Lesley;given-names:S. A. + 18988020 + REF + Methods Mol. Biol. + ref + 498 + 2009 + 43771 + The Polymerase Incomplete Primer Extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis + + + 785 + 786 + surname:Petersen;given-names:T. N. + surname:Brunak;given-names:S. + surname:von Heijne;given-names:G. + surname:Nielsen;given-names:H. + 21959131 + REF + Nat. Methods + ref + 8 + 2011 + 43893 + SignalP 4.0: discriminating signal peptides from transmembrane regions + + + 11664 + 11669 + surname:Lesley;given-names:S. A. + surname:Kuhn;given-names:P. + surname:Vincent;given-names:J. + surname:Robb;given-names:A. + surname:Brinen;given-names:L. S. + surname:Miller;given-names:M. D. + surname:McPhillips;given-names:T. M. + surname:Miller;given-names:M. A. + surname:Scheibe;given-names:D. + surname:Canaves;given-names:J. M. + surname:Guda;given-names:C. + surname:Jaroszewski;given-names:L. + surname:Godzik;given-names:A. + surname:Selby;given-names:T. L. + surname:Elsliger;given-names:M. A. + surname:Wooley;given-names:J. + surname:Taylor;given-names:S. S. + surname:Hodgson;given-names:K. O. + surname:Wilson;given-names:I. A. + surname:Schultz;given-names:P. G. + surname:Stevens;given-names:R. C. + surname:Deacon;given-names:A. M. + surname:Mathews;given-names:I. + surname:Kreusch;given-names:A. + surname:Spraggon;given-names:G. + surname:Klock;given-names:H. E. + surname:McMullan;given-names:D. + surname:Shin;given-names:T. + 12193646 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 99 + 2002 + 43964 + Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline + + + 91 + 108 + surname:Doublié;given-names:S. + 17272838 + REF + Methods Mol. Biol. + ref + 363 + 2007 + 44086 + Production of selenomethionyl proteins in prokaryotic and eukaryotic expression systems + + + 105 + 124 + surname:Van Duyne;given-names:G. D. + surname:Standaert;given-names:R. F. + surname:Karplus;given-names:P. A. + surname:Schreiber;given-names:S. L. + surname:Clardy;given-names:J. + 7678431 + REF + J. Mol. Biol. + ref + 229 + 1993 + 44174 + Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin + + + 720 + 726 + surname:Cohen;given-names:A. E. + surname:Ellis;given-names:P. J. + surname:Miller;given-names:M. D. + surname:Deacon;given-names:A. M. + surname:Phizackerley;given-names:R. P. + 24899734 + REF + J. Appl. Crystallogr. + ref + 35 + 2002 + 44261 + An automated system to mount cryo-cooled protein crystals on a synchrotron beam line, using compact sample cassettes and a small-scale robot + + + 125 + 132 + surname:Kabsch;given-names:W. + 20124692 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 44402 + XDS + + + 215 + 230 + surname:Vonrhein;given-names:C. + surname:Blanc;given-names:E. + surname:Roversi;given-names:P. + surname:Bricogne;given-names:G. + 17172768 + REF + Methods Mol. Biol. + ref + 364 + 2007 + 44406 + Automated structure solution with autoSHARP + + + 30 + 42 + surname:Abrahams;given-names:J. P. + surname:Leslie;given-names:A. G. + 15299723 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 52 + 1996 + 44450 + Methods used in the structure determination of bovine mitochondrial F1 ATPase + + + 1171 + 1179 + surname:Langer;given-names:G. + surname:Cohen;given-names:S. X. + surname:Lamzin;given-names:V. S. + surname:Perrakis;given-names:A. + 18600222 + REF + Nat. Protoc. + ref + 3 + 2008 + 44528 + Automated macromolecular model building for x-ray crystallography using ARP/wARP version 7 + + + 486 + 501 + surname:Emsley;given-names:P. + surname:Lohkamp;given-names:B. + surname:Scott;given-names:W. G. + surname:Cowtan;given-names:K. + 20383002 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 44619 + Features and development of Coot + + + 235 + 242 + surname:Winn;given-names:M. D. + surname:Ballard;given-names:C. C. + surname:McNicholas;given-names:S. J. + surname:Murshudov;given-names:G. N. + surname:Pannu;given-names:N. S. + surname:Potterton;given-names:E. A. + surname:Powell;given-names:H. R. + surname:Cowtan;given-names:K. D. + surname:Dodson;given-names:E. J. + surname:Emsley;given-names:P. + surname:Evans;given-names:P. R. + surname:Keegan;given-names:R. M. + surname:Krissinel;given-names:E. B. + surname:Leslie;given-names:A. G. + surname:McCoy;given-names:A. + 21460441 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 67 + 2011 + 44652 + Overview of the CCP4 suite and current developments + + + 355 + 367 + surname:Murshudov;given-names:G. N. + surname:Skubák;given-names:P. + surname:Lebedev;given-names:A. A. + surname:Pannu;given-names:N. S. + surname:Steiner;given-names:R. A. + surname:Nicholls;given-names:R. A. + surname:Winn;given-names:M. D. + surname:Long;given-names:F. + surname:Vagin;given-names:A. A. + 21460454 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 67 + 2011 + 44704 + REFMAC5 for the refinement of macromolecular crystal structures + + + 1833 + 1839 + surname:Yang;given-names:H. + surname:Guranovic;given-names:V. + surname:Dutta;given-names:S. + surname:Feng;given-names:Z. + surname:Berman;given-names:H. M. + surname:Westbrook;given-names:J. D. + 15388930 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 60 + 2004 + 44768 + Automated and accurate deposition of structures solved by X-ray diffraction to the Protein Data Bank + + + 12 + 21 + surname:Chen;given-names:V. B. + surname:Arendall;given-names:W. B.;suffix:3rd + surname:Headd;given-names:J. J. + surname:Keedy;given-names:D. A. + surname:Immormino;given-names:R. M. + surname:Kapral;given-names:G. J. + surname:Murray;given-names:L. W. + surname:Richardson;given-names:J. S. + surname:Richardson;given-names:D. C. + 20057044 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 44869 + MolProbity: all-atom structure validation for macromolecular crystallography + + + 52 + 56 + surname:Vriend;given-names:G. + 2268628 + REF + J. Mol. Graph. + ref + 8 + 1990 + 44946 + WHAT IF: a molecular modeling and drug design program + + + 191 + 205 + surname:Vaguine;given-names:A. A. + surname:Richelle;given-names:J. + surname:Wodak;given-names:S. J. + 10089410 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 55 + 1999 + 45000 + SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model + + + 49 + 52 + surname:Terwilliger;given-names:T. + 14646132 + REF + J. Synchrotron Radiat. + ref + 11 + 2004 + 45146 + SOLVE and RESOLVE: automated structure solution, density modification and model building + + + 539 + surname:Sievers;given-names:F. + surname:Wilm;given-names:A. + surname:Thompson;given-names:J. D. + surname:Higgins;given-names:D. G. + surname:Dineen;given-names:D. + surname:Gibson;given-names:T. J. + surname:Karplus;given-names:K. + surname:Li;given-names:W. + surname:Lopez;given-names:R. + surname:McWilliam;given-names:H. + surname:Remmert;given-names:M. + surname:Söding;given-names:J. + 21988835 + REF + Mol. Syst. Biol. + ref + 7 + 2011 + 45235 + Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega + + + 510 + 512 + surname:Bond;given-names:C. S. + surname:Schüttelkopf;given-names:A. W. + 19390156 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 65 + 2009 + 45334 + ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments + + + 311 + 312 + surname:Bond;given-names:C. S. + 12538265 + REF + Bioinformatics + ref + 19 + 2003 + 45420 + TopDraw: a sketchpad for protein structure topology cartoons + + + version 1.2r3pre + surname:DeLano;given-names:W. + REF + The PyMOL Molecular Graphic System + ref + 2002 + 45481 + + + 203 + 221 + surname:McLuskey;given-names:K. + surname:Moss;given-names:C. X. + surname:Mottram;given-names:J. C. + 24567104 + REF + Methods Mol. Biol. + ref + 1133 + 2014 + 45482 + Purification, characterization, and crystallization of Trypanosoma metacaspases + + + 4 + 21 + surname:Wiesmann;given-names:C. + surname:Leder;given-names:L. + surname:Freuler;given-names:F. + surname:Nikolay;given-names:R. + surname:Alves;given-names:J. + surname:Bornancin;given-names:F. + surname:Renatus;given-names:M. + surname:Blank;given-names:J. + surname:Bernardi;given-names:A. + surname:Melkko;given-names:S. + surname:Decock;given-names:A. + surname:D'Arcy;given-names:A. + surname:Villard;given-names:F. + surname:Erbel;given-names:P. + surname:Hughes;given-names:N. + 22366302 + REF + J. Mol. Biol. + ref + 419 + 2012 + 45562 + Structural determinants of MALT1 protease activity + + + 865 + 872 + surname:Ullmann;given-names:D. + surname:Jakubke;given-names:H. D. + 8055964 + REF + Eur. J. Biochem. + ref + 223 + 1994 + 45613 + The specificity of clostripain from Clostridium histolyticum: mapping the S′ subsites via acyl transfer to amino acid amides and peptides + + + 281 + 286 + surname:Witte;given-names:V. + surname:Wolf;given-names:N. + surname:Dargatz;given-names:H. + 8875906 + REF + Curr. Microbiol. + ref + 33 + 1996 + 45753 + Clostripain linker deletion variants yield active enzyme in Escherichia coli: a possible function of the linker peptide as intramolecular inhibitor of clostripain automaturation + + + 983 + 992 + surname:Labrou;given-names:N. E. + surname:Rigden;given-names:D. J. + 15009210 + REF + Eur. J. Biochem. + ref + 271 + 2004 + 45931 + The structure-function relationship in the clostripain family of peptidases + + + 38980 + 38990 + surname:Li;given-names:D. N. + surname:Matthews;given-names:S. P. + surname:Antoniou;given-names:A. N. + surname:Mazzeo;given-names:D. + surname:Watts;given-names:C. + 12860980 + REF + J. Biol. Chem. + ref + 278 + 2003 + 46007 + Multistep autoactivation of asparaginyl endopeptidase in vitro and in vivo + + + 10458 + 10464 + surname:Mikolajczyk;given-names:J. + surname:Boatright;given-names:K. M. + surname:Stennicke;given-names:H. R. + surname:Nazif;given-names:T. + surname:Potempa;given-names:J. + surname:Bogyo;given-names:M. + surname:Salvesen;given-names:G. S. + 12533545 + REF + J. Biol. Chem. + ref + 278 + 2003 + 46082 + Sequential autolytic processing activates the zymogen of Arg-gingipain + + + 34042 + 34050 + surname:Denault;given-names:J. B. + surname:Salvesen;given-names:G. S. + 12824163 + REF + J. Biol. Chem. + ref + 278 + 2003 + 46153 + Human caspase-7 activity and regulation by its N-terminal peptide + + + 9492 + 9500 + surname:Grewal;given-names:J. S. + surname:McLuskey;given-names:K. + surname:Schnaufer;given-names:A. + surname:Mottram;given-names:J. C. + surname:Das;given-names:D. + surname:Myburgh;given-names:E. + surname:Wilkes;given-names:J. + surname:Brown;given-names:E. + surname:Lemgruber;given-names:L. + surname:Gould;given-names:M. K. + surname:Burchmore;given-names:R. J. + surname:Coombs;given-names:G. H. + REF + J. Biol. Chem. + ref + 291 + 2016 + 46219 + PNT1 is a C11 cysteine peptidase essential for replication of the Trypanosome kinetoplast + + + 291 + 296 + surname:Weiss;given-names:M. S. + surname:Metzner;given-names:H. J. + surname:Hilgenfeld;given-names:R. + 9515726 + REF + FEBS Lett. + ref + 423 + 1998 + 46309 + Two non-proline cis peptide bonds may be important for factor XIII function + + + 269 + 275 + surname:Diederichs;given-names:K. + surname:Karplus;given-names:P. A. + 9095194 + REF + Nat. Struct. Biol. + ref + 4 + 1997 + 46385 + Improved R-factors for diffraction data analysis in macromolecular crystallography + + + 2256 + 2268 + surname:Krissinel;given-names:E. + surname:Henrick;given-names:K. + 15572779 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 60 + 2004 + 46468 + Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions + + + 566 + 567 + surname:Holm;given-names:L. + surname:Park;given-names:J. + 10980157 + REF + Bioinformatics + ref + 16 + 2000 + 46572 + DaliLite workbench for protein structure comparison + + + 1514 + 1521 + surname:Kang;given-names:H. J. + surname:Lee;given-names:Y. M. + surname:Bae;given-names:K. H. + surname:Kim;given-names:S. J. + surname:Chung;given-names:S. J. + 23897474 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 69 + 2013 + 46624 + Structural asymmetry of procaspase-7 bound to a specific inhibitor + + + 162 + 166 + surname:Gorman;given-names:M. A. + surname:Seers;given-names:C. A. + surname:Michell;given-names:B. J. + surname:Feil;given-names:S. C. + surname:Huq;given-names:N. L. + surname:Cross;given-names:K. J. + surname:Reynolds;given-names:E. C. + surname:Parker;given-names:M. W. + 25327141 + REF + Protein Sci. + ref + 24 + 2015 + 46691 + Structure of the lysine specific protease Kgp from Porphyromonas gingivalis, a target for improved oral health + + + 364 + 371 + surname:Shen;given-names:A. + surname:Lupardus;given-names:P. J. + surname:Gersch;given-names:M. M. + surname:Puri;given-names:A. W. + surname:Albrow;given-names:V. E. + surname:Garcia;given-names:K. C. + surname:Bogyo;given-names:M. + 21317893 + REF + Nat. Struct. Mol. Biol. + ref + 18 + 2011 + 46802 + Defining an allosteric circuit in the cysteine protease domain of Clostridium difficile toxins + + + diff --git a/BioC_XML/4852598_v0.xml b/BioC_XML/4852598_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..a9015335673c0a2de1fa1e71eb1c2c60c2aeb99c --- /dev/null +++ b/BioC_XML/4852598_v0.xml @@ -0,0 +1,11397 @@ + + + + PMC + 20140719 + pmc.key + + 4852598 + CC BY + no + 0 + 0 + + 10.1038/ncomms11337 + ncomms11337 + 4852598 + 27088325 + 11337 + This work is licensed under a Creative Commons Attribution 4.0 +International License. The images or other third party material in this article are +included in the article's Creative Commons license, unless indicated otherwise +in the credit line; if the material is not included under the Creative Commons +license, users will need to obtain permission from the license holder to reproduce +the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ + surname:van den Berg;given-names:Bert + surname:Chembath;given-names:Anupama + surname:Jefferies;given-names:Damien + surname:Basle;given-names:Arnaud + surname:Khalid;given-names:Syma + surname:Rutherford;given-names:Julian C. + TITLE + front + 7 + 2016 + 0 + Structural basis for Mep2 ammonium transceptor activation by phosphorylation + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:34:03Z + + Mep2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:33:50Z + + ammonium transceptor + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:13:59Z + + phosphorylation + + + + ABSTRACT + abstract + 77 + Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:02Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:44:35Z + + transceptors + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:36:41Z + + ammonium + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:02Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:34:16Z + + Mep2 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:13:59Z + + phosphorylation + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:21Z + + X-ray crystal structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:28:54Z + + Mep2 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:36:35Z + + Saccharomyces cerevisiae + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:36:48Z + + Candida albicans + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:57Z + + transporters + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:39:34Z + + not phosphorylated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:01Z + + closed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:11Z + + inactive + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:39:42Z + + non-phosphorylated + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:28:58Z + + Mep2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:18:42Z + + cytoplasmic loops + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:37:56Z + + C-terminal region + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:05Z + + CTR + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:36:27Z + + exit + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:36:32Z + + channel + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:47Z + + His2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:37:38Z + + twin-His motif + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:22Z + + phosphorylation site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:05Z + + CTR + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:32:08Z + + solvent accessible + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:36:37Z + + negatively charged pocket + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:37:46Z + + channel exit + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:44Z + + crystal structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:38:37Z + + phosphorylation-mimicking + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:40:36Z + + Mep2 variants + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:40:10Z + + C. albicans + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:32:12Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:05Z + + CTR + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:05Z + + eukaryotic + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:36:54Z + + ammonium + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:13:59Z + + phosphorylation + + + + ABSTRACT + abstract + 1224 + Mep2 proteins are tightly regulated fungal ammonium transporters. Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:02Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:57:59Z + + crystal structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:01Z + + closed + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:46:37Z + + by comparing them with + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:38Z + + bacteria + + + + INTRO + paragraph + 1481 + Transceptors are membrane proteins that function not only as transporters but also as receptors/sensors during nutrient sensing to activate downstream signalling pathways. A common feature of transceptors is that they are induced when cells are starved for their substrate. While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). One of the most important unresolved questions in the field is how the transceptors couple to downstream signalling pathways. One hypothesis is that downstream signalling is dependent on a specific conformation of the transporter. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:44:35Z + + Transceptors + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:46:25Z + + membrane proteins + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:44:35Z + + transceptors + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:36:35Z + + Saccharomyces cerevisiae + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:44:35Z + + transceptors + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:45:39Z + + phosphate + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:45:43Z + + Pho84 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:45:47Z + + amino acids + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:45:50Z + + Gap1 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:45:56Z + + ammonium + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:45:59Z + + Mep2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:44:35Z + + transceptors + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:45:06Z + + higher eukaryotes + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:44:57Z + + mammalian + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:42:27Z + + SNAT2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:46:20Z + + amino-acid transporter + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:42:30Z + + GLUT2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:46:16Z + + glucose transporter + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:44:35Z + + transceptors + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:46:12Z + + transporter + + + + INTRO + paragraph + 2271 + Mep2 (methylammonium (MA) permease) proteins are ammonium transceptors that are ubiquitous in fungi. They belong to the Amt/Mep/Rh family of transporters that are present in all kingdoms of life and they take up ammonium from the extracellular environment. Fungi typically have more than one Mep paralogue, for example, Mep1-3 in S. cerevisiae. Of these, only Mep2 proteins function as ammonium receptors/sensors in fungal development. Under conditions of nitrogen limitation, Mep2 initiates a signalling cascade that results in a switch from the yeast form to filamentous (pseudohyphal) growth that may be required for fungal pathogenicity. As is the case for other transceptors, it is not clear how Mep2 interacts with downstream signalling partners, but the protein kinase A and mitogen-activated protein kinase pathways have been proposed as downstream effectors of Mep2 (refs). Compared with Mep1 and Mep3, Mep2 is highly expressed and functions as a low-capacity, high-affinity transporter in the uptake of MA. In addition, Mep2 is also important for uptake of ammonium produced by growth on other nitrogen sources. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:29:04Z + + Mep2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:52:34Z + + (methylammonium (MA) permease) proteins + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:33Z + + ammonium transceptors + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:50:14Z + + fungi + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:52:31Z + + Amt/Mep/Rh family of transporters + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:52:27Z + + all kingdoms of life + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:50:22Z + + ammonium + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:50:14Z + + Fungi + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:29:08Z + + Mep + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:52:17Z + + Mep1-3 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:01Z + + S. cerevisiae + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:36:23Z + + ammonium + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:02Z + + fungal + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:42:34Z + + Mep2 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:02Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:44:35Z + + transceptors + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:51:43Z + + Mep2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:51:49Z + + Mep2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:52:13Z + + Mep1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:52:05Z + + Mep3 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:51:56Z + + Mep2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:51:52Z + + highly expressed + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:51:28Z + + MA + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:51:40Z + + Mep2 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:37:37Z + + ammonium + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:51:17Z + + nitrogen + + + + INTRO + paragraph + 3393 + With the exception of the human RhCG structure, no structural information is available for eukaryotic ammonium transporters. By contrast, several bacterial Amt orthologues have been characterized in detail via high-resolution crystal structures and a number of molecular dynamics (MD) studies. All the solved structures including that of RhCG are very similar, establishing the basic architecture of ammonium transporters. The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. All structures show the transporters in open conformations. Intriguingly, fundamental questions such as the nature of the transported substrate and the transport mechanism are still controversial. Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport). A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport. + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:58:24Z + + human + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:58:35Z + + RhCG + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:00:00Z + + structure + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:05Z + + eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:59:54Z + + Amt + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:57:59Z + + crystal structures + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:58:04Z + + molecular dynamics + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:58:11Z + + MD + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:00:06Z + + structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:58:35Z + + RhCG + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:59:28Z + + stable + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:57:46Z + + trimers + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:27Z + + monomer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:57:08Z + + transmembrane + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:57:20Z + + TM + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:57:35Z + + helices + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T17:00:25Z + + central channel + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T10:48:09Z + + ammonium + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:57:52Z + + structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:29:12Z + + transporters + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T11:41:53Z + + ammonia + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:29:14Z + + Amt/Mep proteins + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:59:44Z + + active + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:00:14Z + + electrogenic transporters + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:58:43Z + + NH4+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:58:55Z + + NH3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:59:01Z + + H+ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:32:17Z + + highly conserved + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:32:52Z + + channel + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-15T17:08:15Z + + histidine + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:37:38Z + + twin-His motif + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:58:55Z + + NH3 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:36:48Z + + channel + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:58:55Z + + NH3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:59:10Z + + H+ + + + + INTRO + paragraph + 4562 + Ammonium transport is tightly regulated. In animals, this is due to toxicity of elevated intracellular ammonium levels, whereas for microorganisms ammonium is a preferred nitrogen source. In bacteria, amt genes are present in an operon with glnK, encoding a PII-like signal transduction class protein. By binding tightly to Amt proteins without inducing a conformational change in the transporter, GlnK sterically blocks ammonium conductance when nitrogen levels are sufficient. Under conditions of nitrogen limitation, GlnK becomes uridylated, blocking its ability to bind and inhibit Amt proteins. Importantly, eukaryotes do not have GlnK orthologues and have a different mechanism for regulation of ammonium transport activity. In plants, transporter phosphorylation and dephosphorylation are known to regulate activity. In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:35:58Z + + Ammonium + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:16Z + + animals + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:35:30Z + + ammonium + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:08:43Z + + microorganisms + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T11:41:58Z + + ammonium + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:38Z + + bacteria + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T11:44:27Z + + amt + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-16T11:44:30Z + + glnK + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:08:38Z + + PII-like signal transduction class protein + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:29:21Z + + Amt proteins + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:29:25Z + + transporter + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:29:31Z + + GlnK + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:35:42Z + + ammonium + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:38:07Z + + nitrogen + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:29:34Z + + GlnK + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:13:36Z + + uridylated + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:29:37Z + + Amt proteins + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:24Z + + eukaryotes + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:29:41Z + + GlnK + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:38:17Z + + ammonium + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:06:27Z + + plants + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:29:44Z + + transporter + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:13:59Z + + phosphorylation + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:11Z + + dephosphorylation + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:01Z + + S. cerevisiae + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:13:59Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:06:11Z + + Ser457 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:37:56Z + + C-terminal region + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:05Z + + CTR + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:35:14Z + + Mep2 + + + + INTRO + paragraph + 5574 + To elucidate the mechanism of Mep2 transport regulation, we present here X-ray crystal structures of the Mep2 transceptors from S. cerevisiae and C. albicans. The structures are similar to each other but show considerable differences to all other ammonium transporter structures. The most striking difference is the fact that the Mep2 proteins have closed conformations. The putative phosphorylation site is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR. Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:38:32Z + + Mep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:21Z + + X-ray crystal structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:40:14Z + + Mep2 transceptors + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:01Z + + S. cerevisiae + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:40:10Z + + C. albicans + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:40:28Z + + structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:40:11Z + + ammonium transporter + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:40:31Z + + structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:01Z + + closed + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:22Z + + phosphorylation site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:32:59Z + + solvent accessible + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T18:40:07Z + + negatively charged pocket + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:37:46Z + + channel exit + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T18:41:07Z + + channels + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:40:53Z + + phosphorylation-mimicking mutants + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:40:10Z + + C. albicans + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:42:39Z + + Mep2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:01Z + + closed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:40:43Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:05Z + + CTR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:50Z + + structure + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:39:45Z + + Mep2 variant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:38:55Z + + lacking + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T18:38:29Z + + segment + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:22Z + + phosphorylation site + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:05Z + + eukaryotic + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:38:50Z + + ammonium + + + + RESULTS + title_1 + 6478 + Results + + + RESULTS + title_2 + 6486 + General architecture of Mep2 ammonium transceptors + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:41:31Z + + Mep2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:33Z + + ammonium transceptors + + + + RESULTS + paragraph + 6537 + The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure–function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals. Of these, Mep2 from C. albicans (CaMep2) showed superior stability in relatively harsh detergents such as nonyl-glucoside, allowing structure determination in two different crystal forms to high resolution (up to 1.5 Å). Despite different crystal packing (Supplementary Table 1), the two CaMep2 structures are identical to each other and very similar to ScMep2 (Cα r.m.s.d. (root mean square deviation)=0.7 Å for 434 residues), with the main differences confined to the N terminus and the CTR (Fig. 1). Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered. Both Mep2 proteins show the archetypal trimeric assemblies in which each monomer consists of 11 TM helices surrounding a central pore. Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG. In the remainder of the manuscript, we will specifically discuss CaMep2 due to the superior resolution of the structure. Unless specifically stated, the drawn conclusions also apply to ScMep2. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:53:13Z + + Mep2 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:01Z + + S. cerevisiae + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:51:54Z + + overexpressed + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:01Z + + S. cerevisiae + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:51:57Z + + structure determination + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:52:00Z + + X-ray crystallography + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:52:03Z + + molecular replacement + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:52:07Z + + MR + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:52:14Z + + archaebacterial + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:52:23Z + + Amt-1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:52:30Z + + structure + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:52:35Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:52:39Z + + structure–function studies + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:02Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:53:21Z + + Mep2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:53:53Z + + overexpressed and screened for + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:53:57Z + + crystals + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:53:44Z + + Mep2 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:40:10Z + + C. albicans + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:52Z + + CaMep2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:54:00Z + + structure determination + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-14T09:45:50Z + + crystal forms + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:52Z + + CaMep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:54:07Z + + structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:48:17Z + + r.m.s.d. + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:48:45Z + + root mean square deviation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:05Z + + CTR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:26:42Z + + Electron density + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:44:36Z + + 43 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:44:39Z + + 25 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:52Z + + CaMep2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:39Z + + poorly conserved + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:51:49Z + + disordered + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:12Z + + trimeric + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:27Z + + monomer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T18:49:54Z + + TM helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T18:55:30Z + + central pore + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T18:55:52Z + + ammonium binding site + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T18:56:01Z + + Phe gate + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:37:38Z + + twin-His motif + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T18:51:24Z + + hydrophobic channel + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T18:56:19Z + + transporters + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:58:35Z + + RhCG + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:52Z + + CaMep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:55Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + + RESULTS + paragraph + 8338 + While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. The N termini of the Mep2 proteins are ∼20–25 residues longer compared with their bacterial counterparts (Figs 1 and 2), substantially increasing the size of the extracellular domain. Moreover, the N terminus of one monomer interacts with the extended extracellular loop ECL5 of a neighbouring monomer. Together with additional, smaller differences in other extracellular loops, these changes generate a distinct vestibule leading to the ammonium binding site that is much more pronounced than in the bacterial proteins. The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins. However, given that an N-terminal deletion mutant (2-27Δ) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T19:01:22Z + + Mep2 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:00:53Z + + prokaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:29:54Z + + transporters + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:59Z + + r.m.s.d. + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:52:23Z + + Amt-1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:37Z + + intracellular loops + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:44Z + + ICLs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:13Z + + ICL3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:05Z + + CTR + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:01:29Z + + 20–25 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:04:29Z + + extracellular domain + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:27Z + + monomer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:55Z + + extracellular loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:02:03Z + + ECL5 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:27Z + + monomer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:04:38Z + + extracellular loops + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:03:25Z + + vestibule + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T18:55:52Z + + ammonium binding site + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:03:25Z + + vestibule + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-07-21T08:26:16Z + + monomer + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T19:02:59Z + + Mep2 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:08:50Z + + trimer + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:03:15Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T19:03:08Z + + AMT proteins + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:42:36Z + + deletion mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T19:04:22Z + + 2-27Δ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:43Z + + wild-type + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:51Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T19:03:02Z + + Mep2 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:39:36Z + + ammonium + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T09:39:51Z + + Mep2 + + + + RESULTS + title_2 + 9498 + Mep2 channels are closed by a two-tier channel block + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T19:05:28Z + + Mep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:05:39Z + + channels + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:01Z + + closed + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:35:06Z + + channel block + + + + RESULTS + paragraph + 9551 + The largest differences between the Mep2 structures and the other known ammonium transporter structures are located on the intracellular side of the membrane. In the vicinity of the Mep2 channel exit, the cytoplasmic end of TM2 has unwound, generating a longer ICL1 even though there are no insertions in this region compared to the bacterial proteins (Figs 2 and 4). ICL1 has also moved inwards relative to its position in the bacterial Amts. The largest backbone movements of equivalent residues within ICL1 are ∼10 Å, markedly affecting the conserved basic RxK motif (Fig. 4). The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). In addition to changing the RxK motif, the movement of ICL1 has another, crucial functional consequence. At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). In bacterial Amt proteins, this Tyr side chain is rotated ∼4 Å away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2). + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:43:54Z + + Mep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:32:03Z + + structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:30:57Z + + ammonium transporter + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:05:14Z + + structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:42:45Z + + Mep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:37:46Z + + channel exit + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:35:31Z + + TM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:49:46Z + + Amts + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:06:39Z + + conserved + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:06:42Z + + basic + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:06:50Z + + RxK motif + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:03:56Z + + Arg54 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:52:23Z + + Amt-1 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:03:47Z + + Lys55 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:04Z + + Lys56 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:07:07Z + + basic + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:07:10Z + + motif + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:42:49Z + + Mep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:07:01Z + + structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:52:23Z + + Amt-1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:06:51Z + + RxK motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:34:46Z + + TM1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:07:38Z + + relatively conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:15Z + + Tyr49 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:24Z + + Tyr53 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:51Z + + hydrogen bond + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:53Z + + absolutely conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:42Z + + His342 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:37:38Z + + twin-His motif + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:33Z + + His348 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:07:58Z + + channel + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:08:23Z + + Amt proteins + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:08:06Z + + Tyr + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:34:46Z + + TM1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:14:12Z + + channel + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-15T17:08:15Z + + histidine + + + + RESULTS + paragraph + 11074 + Compared with ICL1, the backbone conformational changes observed for the neighbouring ICL2 are smaller, but large shifts are nevertheless observed for the conserved residues Glu140 and Arg141 (Fig. 4). Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to ∼10 Å and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5). This two-tier channel block likely ensures that very little ammonium transport will take place under nitrogen-sufficient conditions. The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. Significantly, this is also true for ScMep2, which was crystallized in the presence of 0.2 M ammonium ions (see Methods section). + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:13:11Z + + ICL2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:13:23Z + + conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:13:31Z + + Glu140 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:13:39Z + + Arg141 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:13Z + + ICL3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:13:45Z + + pseudo-symmetrical halves + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:13:48Z + + TM1-5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:13:52Z + + TM6-10 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:13:57Z + + transporter + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:14:05Z + + channel + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:35:06Z + + channel block + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:40:28Z + + ammonium + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:40:40Z + + nitrogen + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:01Z + + closed + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:14:16Z + + channel + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:14:52Z + + no density + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T21:14:30Z + + ammonium + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T21:14:39Z + + water + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T21:14:25Z + + Mep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:14:21Z + + channel + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:37:38Z + + twin-His motif + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:14:44Z + + crystallized + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T21:12:00Z + + ammonium + + + + RESULTS + paragraph + 11967 + The final region in Mep2 that shows large differences compared with the bacterial transporters is the CTR. In Mep2, the CTR has moved away and makes relatively few contacts with the main body of the transporter, generating a more elongated protein (Figs 1 and 4). By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). These residues include those of the ‘ExxGxD' motif, which when mutated generate inactive transporters. In Amt-1 and other bacterial ammonium transporters, these CTR residues interact with residues within the N-terminal half of the protein. On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3. At the other end of ICL3, the backbone carbonyl groups of Gly172 and Lys173 are hydrogen bonded to the side chain of Arg370. Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). The result of these interactions is that the CTR ‘hugs' the N-terminal half of the transporters (Fig. 4). Also noteworthy is Asp381, the side chain of which interacts strongly with the positive dipole on the N-terminal end of TM2. This interaction in the centre of the protein may be particularly important to stabilize the open conformations of ammonium transporters. In the Mep2 structures, none of the interactions mentioned above are present. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T21:22:19Z + + Mep2 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:22:16Z + + transporters + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:05Z + + CTR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T21:22:23Z + + Mep2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:05Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:22:36Z + + main body + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:22:40Z + + transporter + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:22:44Z + + elongated + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:22:51Z + + structures + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:05Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:25:08Z + + N-terminal half + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:23:02Z + + transporters + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:23:06Z + + TM1-5 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:10Z + + compact + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:13Z + + structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:17Z + + universally conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:05Z + + CTR + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:25Z + + Arg415 + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:29Z + + 370 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:37Z + + Glu421 + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:41Z + + 376 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:47Z + + Gly424 + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:51Z + + 379 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:59Z + + Asp426 + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:24:04Z + + 381 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:24:11Z + + Tyr 435 + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:24:16Z + + 390 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:52Z + + CaMep2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:52:23Z + + Amt-1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:24:27Z + + ‘ExxGxD' motif + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:24:43Z + + mutated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:11Z + + inactive + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:24:51Z + + transporters + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:52:23Z + + Amt-1 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:05Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:25:08Z + + N-terminal half + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:25:33Z + + Tyr390 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:52:23Z + + Amt-1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:51Z + + hydrogen bonded + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:25:41Z + + conserved + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:25:49Z + + His185 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:26:04Z + + loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:26:18Z + + Gly172 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:26:26Z + + Lys173 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:51Z + + hydrogen bonded + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:26:44Z + + Arg370 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:41:16Z + + modelled + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:59:44Z + + active + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:39:42Z + + non-phosphorylated + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:03:15Z + + plant + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T21:27:13Z + + AtAmt-1;1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:27:17Z + + structure + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:27:26Z + + Y467 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:27:35Z + + H239 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:27:42Z + + D458 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:27:50Z + + K71 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:25:08Z + + N-terminal half + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:27:00Z + + transporters + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:28:10Z + + Asp381 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:35:31Z + + TM2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T21:29:23Z + + Mep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:29:26Z + + structures + + + + RESULTS + title_2 + 13717 + Phosphorylation target site is at the periphery of Mep2 + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:31:00Z + + Phosphorylation target site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T21:31:10Z + + Mep2 + + + + RESULTS + paragraph + 13773 + Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). Conversely, the phosphorylation-mimicking S457D variant is active both in the triple mepΔ background and in a triple mepΔ npr1Δ strain (Fig. 3). Mutation of other potential phosphorylation sites in the CTR did not support growth in the npr1Δ background. Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake. Ser457 is located in a part of the CTR that is conserved in a subgroup of Mep2 proteins, but which is not present in bacterial proteins (Fig. 2). This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:06:11Z + + Ser457 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:35:25Z + + Ser453 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:52Z + + CaMep2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:35:48Z + + phosphorylated + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:35:57Z + + TORC1 effector kinase + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T21:36:03Z + + Npr1 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:41:18Z + + nitrogen + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:36:25Z + + absence of + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:42:54Z + + Npr1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:36:33Z + + plasmid-encoded + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:51Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T21:36:40Z + + Mep2 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:01Z + + S. cerevisiae + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:36:44Z + + mep1-3Δ + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:37:26Z + + triple mepΔ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T21:36:52Z + + ammonium + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:36:57Z + + transporter + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:11Z + + inactive + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:38:37Z + + phosphorylation-mimicking + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:37:14Z + + S457D + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:59:44Z + + active + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:37:26Z + + triple mepΔ + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:37:41Z + + triple mepΔ npr1Δ + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:37:47Z + + Mutation + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:38:02Z + + phosphorylation sites + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:38:12Z + + npr1Δ + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:00Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:06:11Z + + Ser457 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:06:11Z + + Ser457 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:42:58Z + + Mep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:38:52Z + + channel + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:41:31Z + + ammonium + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:06:11Z + + Ser457 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:39:03Z + + conserved + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-06-14T09:48:13Z + + segment + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:39:11Z + + 450–457 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:39:14Z + + 446–453 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:52Z + + CaMep2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:39:24Z + + autoinhibitory (AI) region + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:39:27Z + + removal + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:59:44Z + + active + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:39:30Z + + transporter + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:39:49Z + + absence of + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:43:02Z + + Npr1 + + + + RESULTS + paragraph + 14939 + Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). The AI regions have very similar conformations in CaMep2 and ScMep2, despite considerable differences in the rest of the CTR (Fig. 6). Strikingly, the Npr1 target serine residue is located at the periphery of the trimer, far away (∼30 Å) from any channel exit (Fig. 6). Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6). This makes sense since the proteins were expressed in rich medium and confirms the recent suggestion by Boeckstaens et al. that the non-phosphorylated form of Mep2 corresponds to the inactive state. For ScMep2, Ser457 is the most C-terminal residue for which electron density is visible, indicating that the region beyond Ser457 is disordered. In CaMep2, the visible part of the sequence extends for two residues beyond Ser453 (Fig. 6). The peripheral location and disorder of the CTR beyond the kinase target site should facilitate the phosphorylation by Npr1. The disordered part of the CTR is not conserved in ammonium transporters (Fig. 2), suggesting that it is not important for transport. Interestingly, a ScMep2 457Δ truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1. The same mutant lacking the His-tag has WT properties (Supplementary Fig. 1b), confirming that the region following the phosphorylation site is dispensable for function. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:39Z + + AI region + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T08:27:01Z + + Npr1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:22Z + + phosphorylation site + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:26:48Z + + structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:39Z + + AI region + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:08:50Z + + trimer + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:26:51Z + + structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:39Z + + AI region + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:35:31Z + + TM2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:35:50Z + + TM4 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:22:36Z + + main body + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:27:09Z + + transporter + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:28:07Z + + Val447 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:28:14Z + + Asp449 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:28:23Z + + Pro450 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:28:37Z + + Arg452 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:56Z + + AI regions + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:53Z + + CaMep2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:29:21Z + + Npr1 target serine + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:08:50Z + + trimer + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:37:46Z + + channel exit + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:08:50Z + + trimer + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:26:42Z + + electron density + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:44:10Z + + serine + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:43:07Z + + Mep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:26:54Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:39:42Z + + non-phosphorylated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:39:42Z + + non-phosphorylated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:43:10Z + + Mep2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:11Z + + inactive + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:06:11Z + + Ser457 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:26:42Z + + electron density + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:06:11Z + + Ser457 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:27:21Z + + disordered + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:53Z + + CaMep2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:35:25Z + + Ser453 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:33:39Z + + disorder + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:29:08Z + + kinase target site + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:00Z + + phosphorylation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:43:15Z + + Npr1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:29:14Z + + disordered + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:33:44Z + + not conserved + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:28:58Z + + 457Δ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:27:31Z + + truncation mutant + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:06:11Z + + Ser457 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:33:48Z + + low activity + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:00Z + + phosphorylation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:43:18Z + + Npr1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-06-14T09:50:49Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:27:55Z + + lacking the His-tag + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-22T09:42:02Z + + WT + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:22Z + + phosphorylation site + + + + RESULTS + title_2 + 16987 + Mep2 lacking the AI region is conformationally heterogeneous + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T08:34:05Z + + Mep2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:34:08Z + + lacking + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:39Z + + AI region + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:33:59Z + + conformationally heterogeneous + + + + RESULTS + paragraph + 17048 + Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. Boeckstaens et al. proposed that phosphorylation does not affect channel activity directly, but instead relieves inhibition by the AI region. The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. The structure shows that removal of the AI region markedly increases the dynamics of the cytoplasmic parts of the transporter. This is not unexpected given the fact that the AI region bridges the CTR and the main body of Mep2 (Fig. 6). Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). Interestingly, however, the Tyr49-His342 hydrogen bond that closes the channel in the WT protein is still present (Fig. 7 and Supplementary Fig. 2). Why then does this mutant appear to be constitutively active? We propose two possibilities. The first one is that the open state is disfavoured by crystallization because of lower stability or due to crystal packing constraints. The second possibility is that the Tyr–His hydrogen bond has to be disrupted by the incoming substrate to open the channel. The latter model would fit well with the NH3/H+ symport model in which the proton is relayed by the twin-His motif. The importance of the Tyr–His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3). + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:06:11Z + + Ser457 + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:41:29Z + + 453 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:37:46Z + + channel exit + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:00Z + + phosphorylation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T08:41:36Z + + Mep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:41:39Z + + channel + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:59:44Z + + active + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:41:46Z + + transporter + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:00Z + + phosphorylation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:39Z + + AI region + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:42:09Z + + 449-485Δ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:42:36Z + + deletion mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:42:13Z + + lacking + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:39Z + + AI region + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:41:52Z + + highly active + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:42:20Z + + MA + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:37:27Z + + triple mepΔ + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:37:41Z + + triple mepΔ npr1Δ + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:42:21Z + + Mep2 variant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:42:03Z + + constitutively open + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:37:36Z + + channel + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:42:28Z + + 446Δ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:33:56Z + + mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:43:04Z + + constructed and purified + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:53Z + + CaMep2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:40:50Z + + 442Δ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:27:31Z + + truncation mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:42:58Z + + determined + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:43:19Z + + crystal structure + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:43:22Z + + structure + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:43:00Z + + removal of + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:39Z + + AI region + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:30:32Z + + cytoplasmic parts + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:30:23Z + + transporter + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:39Z + + AI region + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:22:36Z + + main body + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:43:23Z + + Mep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:43:31Z + + Density + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:25Z + + Arg415 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:43:11Z + + 442Δ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:43:14Z + + mutant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:43:34Z + + density + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:44Z + + ICLs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:43:28Z + + structure + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:15Z + + Tyr49 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:42Z + + His342 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:51Z + + hydrogen bond + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:51Z + + WT + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:59:44Z + + active + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:42:55Z + + crystallization + + + site + SO: + melaniev@ebi.ac.uk + 2023-06-14T09:37:58Z + + Tyr–His hydrogen bond + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:58:55Z + + NH3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T11:42:21Z + + H+ + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:37:38Z + + twin-His motif + + + site + SO: + melaniev@ebi.ac.uk + 2023-06-14T09:38:06Z + + Tyr–His hydrogen bond + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:41:21Z + + removal + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:06Z + + ScMep2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:43:46Z + + Y53A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:43:49Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:43:55Z + + constitutively active + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T08:44:01Z + + transporter + + + + RESULTS + title_2 + 19155 + Phosphorylation causes a conformational change in the CTR + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:00Z + + Phosphorylation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + + RESULTS + paragraph + 19213 + Do the Mep2 structures provide any clues regarding the potential effect of phosphorylation? The side-chain hydroxyl of Ser457/453 is located in a well-defined electronegative pocket that is solvent accessible (Fig. 6). The closest atoms to the serine hydroxyl group are the backbone carbonyl atoms of Asp419, Glu420 and Glu421, which are 3–4 Å away. We therefore predict that phosphorylation of Ser453 will result in steric clashes as well as electrostatic repulsion, which in turn might cause substantial conformational changes within the CTR. To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). Unexpectedly, the AI segment containing the mutated residues has only undergone a slight shift compared with the WT protein (Fig. 8 and Supplementary Fig. 3). By contrast, the conserved part of the CTR has undergone a large conformational change involving formation of a 12-residue-long α-helix from Leu427 to Asp438. In addition, residues Glu420-Leu423 including Glu421 of the ExxGxD motif are now disordered (Fig. 8 and Supplementary Fig. 3). Overall, ∼20 residues are affected by the introduced mutations. This is the first time a large conformational change has been observed in an ammonium transporter as a result of a mutation, and confirms previous hypotheses that phosphorylation causes structural changes in the CTR. To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the ‘single D' S453D mutant. As shown in Supplementary Fig. 4, the consequence of the single D mutation is very similar to that of the DD substitution, with conformational changes and increased dynamics confined to the conserved part of the CTR (Supplementary Fig. 4). To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453. After 200 ns of MD simulation, the interactions between these residues and Ser453 remain intact. The protein backbone has an average r.m.s.d. of only ∼3 Å during the 200-ns simulation, indicating that the protein is stable. There is flexibility in the side chains of the acidic residues so that they are able to form stable hydrogen bonds with Ser453. In particular, persistent hydrogen bonds are observed between the Ser453 hydroxyl group and the acidic group of Glu420, and also between the amine group of Ser453 and the backbone carbonyl of Glu420 (Supplementary Fig. 5). The DD mutant is also stable during the simulations, but the average backbone r.m.s.d of ∼3.6 Å suggests slightly more conformational flexibility than WT. As the simulation proceeds, the side chains of the acidic residues move away from Asp452 and Asp453, presumably to avoid electrostatic repulsion. For example, the distance between the Asp453 acidic oxygens and the Glu420 acidic oxygens increases from ∼7 to >22 Å after 200 ns simulations, and thus these residues are not interacting. The protein is structurally stable throughout the simulation with little deviation in the other parts of the protein. Finally, the S453J mutant is also stable throughout the 200-ns simulation and has an average backbone deviation of ∼3.8 Å, which is similar to the DD mutant. The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant. The distance between the phosphate of Sep453 and the acidic oxygen atoms of Glu420 is initially ∼11 Å, but increases to >30 Å after 200 ns. The short helix formed by residues Leu427 to Asp438 unravels during the simulations to a disordered state. The remainder of the protein is not affected (Supplementary Fig. 5). Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR. However, the conformational changes for the phosphomimetic mutants in the crystals are confined to the CTR (Fig. 8), and the channels are still closed (Supplementary Fig. 2). One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). The fact that the S453D structure was obtained in the presence of 10 mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:08:40Z + + Mep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:08:43Z + + structures + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:00Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:06:11Z + + Ser457 + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:08:47Z + + 453 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:08:57Z + + electronegative pocket + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:09:01Z + + solvent accessible + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:44:16Z + + serine + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:02:02Z + + Asp419 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:02:11Z + + Glu420 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:37Z + + Glu421 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:35:25Z + + Ser453 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:01:54Z + + determined + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:07:35Z + + structure + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:38:37Z + + phosphorylation-mimicking + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:08:33Z + + R452D/S453D + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:01:49Z + + DD mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:02:25Z + + additional mutation of + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:02:30Z + + arginine + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:22Z + + phosphorylation site + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T09:09:46Z + + phosphate + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:39Z + + AI region + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:22:36Z + + main body + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:09:59Z + + transporter + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:42:48Z + + ammonium + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:01Z + + S. cerevisiae + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:01:49Z + + DD mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:51Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:10:08Z + + Mep2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:08:22Z + + S453D + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:10:11Z + + mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:37:27Z + + triple mepΔ + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:10:25Z + + AI segment + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:51Z + + WT + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:10:43Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:07:53Z + + 12-residue-long α-helix + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:07:58Z + + Leu427 to Asp438 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:07:49Z + + Glu420-Leu423 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:23:37Z + + Glu421 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:08:08Z + + ExxGxD motif + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:08:12Z + + disordered + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:30:39Z + + ammonium transporter + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:11:08Z + + mutation + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:11:01Z + + R452D + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:11:13Z + + determined + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:11:16Z + + structure + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:11:22Z + + single D + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:08:22Z + + S453D + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:11:29Z + + mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:11:22Z + + single D + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:11:32Z + + mutation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:11:37Z + + DD substitution + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:11:44Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:58:00Z + + crystal structures + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:11:52Z + + modelling + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:58:11Z + + MD + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:51Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:53Z + + CaMep2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:01:49Z + + DD mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:28:47Z + + phosphorylated + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:12:09Z + + S453J + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:51Z + + WT + + + evidence + 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melaniev@ebi.ac.uk + 2023-03-15T16:59:28Z + + stable + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:59:28Z + + stable + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:51Z + + hydrogen bonds + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:35:25Z + + Ser453 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:51Z + + hydrogen bonds + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:35:25Z + + Ser453 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:02:11Z + + Glu420 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:35:25Z + + Ser453 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:02:11Z + + Glu420 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:01:49Z + + DD mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:59:28Z + + stable + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:14:52Z + + simulations + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:14:28Z + + r.m.s.d + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:51Z + + WT + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:14:41Z + + simulation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:38:34Z + + Asp452 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:38:54Z + + Asp453 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:15:34Z + + distance + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:38:54Z + + Asp453 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:02:11Z + + Glu420 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:14:52Z + + simulations + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:15:01Z + + structurally stable + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:14:41Z + + simulation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:12:09Z + + S453J + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:15:08Z + + mutant + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:59:28Z + + stable + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:14:41Z + + simulation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:01:49Z + + DD mutant + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:28:37Z + + Arg452 + + + residue_name_number + DUMMY: + means Ser453 + melaniev@ebi.ac.uk + 2023-03-16T11:40:07Z + + Sep453 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:14:41Z + + simulation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:40:14Z + + Asp452 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:38:54Z + + Asp453 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:01:49Z + + DD mutant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:15:37Z + + distance + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T09:15:43Z + + phosphate + + + residue_name_number + DUMMY: + means Ser453 + melaniev@ebi.ac.uk + 2023-03-16T11:40:25Z + + Sep453 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:02:11Z + + Glu420 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:15:48Z + + short helix + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:44:49Z + + Leu427 to Asp438 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:14:52Z + + simulations + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:34:10Z + + disordered + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:58:11Z + + MD + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:16:32Z + + simulations + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:58:00Z + + crystal structures + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:16:38Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:16:43Z + + phosphomimetic mutants + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:16:47Z + + crystals + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:16:51Z + + channels + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:01Z + + closed + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:16:58Z + + mutants + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:17:30Z + + phosphoserine + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:08:22Z + + S453D + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:41:10Z + + DD mutants + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:17:36Z + + fully active + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:17:49Z + + absence of + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:43:33Z + + Npr1 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:17:55Z + + mutations + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:08:22Z + + S453D + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:17:59Z + + structure + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T09:18:29Z + + ammonium + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:18:06Z + + crystallization + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:01Z + + closed + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:18:02Z + + Mep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:18:08Z + + channels + + + + DISCUSS + title_1 + 24519 + Discussion + + + DISCUSS + paragraph + 24530 + Knowledge about ammonium transporter structure has been obtained from experimental and theoretical studies on bacterial family members. In addition, a number of biochemical and genetic studies are available for bacterial, fungal and plant proteins. These efforts have advanced our knowledge considerably but have not yet yielded atomic-level answers to several important mechanistic questions, including how ammonium transport is regulated in eukaryotes and the mechanism of ammonium signalling. In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation. Owing to the lack of structural information for plant AMTs, the details of channel closure and inter-monomer crosstalk are not yet clear. Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:44:16Z + + ammonium transporter + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-22T09:44:20Z + + structure + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:24:22Z + + biochemical and genetic studies + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:02Z + + fungal + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:03:15Z + + plant + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:43:45Z + + ammonium + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:24Z + + eukaryotes + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:44:55Z + + ammonium + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:24:37Z + + Arabidopsis thaliana + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:24:49Z + + Amt-1;1 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:24:57Z + + T460 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:44:48Z + + ammonium + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:39:42Z + + non-phosphorylated + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:03:15Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:25:05Z + + transporter + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:25:01Z + + phosphomimetic mutations + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:28Z + + monomer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:08:50Z + + trimer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T10:51:45Z + + ammonium + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:03:15Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:49:46Z + + AMTs + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:25:15Z + + channel + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:03:15Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:25:19Z + + transporters + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:11Z + + inactive + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:45:13Z + + ammonium + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:39:42Z + + non-phosphorylated + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:25:37Z + + channels + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:02Z + + closed + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:25:51Z + + transporters + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:25:32Z + + A. thaliana + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:24:49Z + + Amt-1;1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:43:39Z + + Mep2 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:34:16Z + + inactivation + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T17:06:27Z + + plants + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:34:20Z + + activation + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:50:14Z + + fungi + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:50:14Z + + fungi + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:45:24Z + + ammonium + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:45:35Z + + ammonium + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:45:45Z + + ammonium + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:24:10Z + + Ato + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:25:46Z + + transporters + + + + DISCUSS + paragraph + 26215 + By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a). In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group. Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9). Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel. How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3. Owing to the crosstalk between monomers, a single phosphorylation event might lead to opening of the entire trimer, although this has not yet been tested (Fig. 9b). Whether or not Mep2 channel opening requires, in addition to phosphorylation, disruption of the Tyr–His2 interaction by the ammonium substrate is not yet clear. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:31:18Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:02Z + + closed + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:31:21Z + + comparing + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:31:24Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:31:28Z + + permanently open + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:46:14Z + + Mep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-22T09:46:31Z + + channel + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:31:47Z + + transporters + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:31:51Z + + channel + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-22T09:46:57Z + + channel + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:47Z + + His2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:37:38Z + + twin-His motif + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:51Z + + hydrogen bonding + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:32:07Z + + highly conserved + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:32:10Z + + tyrosine + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:43:44Z + + Npr1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:32:16Z + + kinase + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:46:44Z + + nitrogen + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:32:20Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:08:08Z + + ExxGxD motif + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:32:25Z + + channel + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:32:28Z + + structural similarities + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:32:32Z + + TM parts + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:32:35Z + + Mep2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:42:52Z + + AfAmt-1 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-22T09:47:11Z + + channel + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:32:39Z + + channel + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-22T09:47:52Z + + channel + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:28Z + + monomer + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:32:57Z + + channel + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:43:58Z + + monomers + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:08:50Z + + trimer + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:47:27Z + + Mep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-22T09:47:36Z + + channel + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + site + SO: + melaniev@ebi.ac.uk + 2023-06-14T09:39:23Z + + Tyr–His2 interaction + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T09:33:15Z + + ammonium + + + + DISCUSS + paragraph + 27848 + Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. However, even the otherwise highly similar Mep2 proteins of S. cerevisiae and C. albicans have different structures for their CTRs (Fig. 1 and Supplementary Fig. 6). In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary. Nevertheless, given the central role of absolutely conserved residues within the ICL1-ICL3-CTR interaction network (Fig. 4), we propose that the structural basics of fungal ammonium transporter activation are conserved. The fact that Mep2 orthologues of distantly related fungi are fully functional in ammonium transport and signalling in S. cerevisiae supports this notion. It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:36:43Z + + Mep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:36:47Z + + channels + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:05Z + + eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:36:54Z + + structural data + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:18:42Z + + cytoplasmic loops + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:02Z + + closed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:01Z + + S. cerevisiae + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:40:10Z + + C. albicans + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:37:06Z + + structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:37:12Z + + CTRs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:39Z + + AI region + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:37:19Z + + Npr1 kinase site + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:37:22Z + + conserved + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:02Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:37:27Z + + transporters + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:53Z + + absolutely conserved + + + site + SO: + melaniev@ebi.ac.uk + 2023-06-14T09:39:36Z + + ICL1-ICL3-CTR interaction network + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:02Z + + fungal + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:48:32Z + + ammonium + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:37:39Z + + conserved + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:37:42Z + + Mep2 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:50:14Z + + fungi + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:48:41Z + + ammonium + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:02Z + + S. cerevisiae + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:37:49Z + + tyrosine + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:47Z + + His2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:37:54Z + + highly conserved + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:02Z + + fungal + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:37:59Z + + Mep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-06-14T09:39:47Z + + Tyr–His2 hydrogen bond + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:34:30Z + + close + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + + DISCUSS + paragraph + 29070 + With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus. This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. Our Mep2 structures show that this assumption may not be correct (Fig. 4 and Supplementary Fig. 6). In addition, the considerable differences between structurally resolved CTR domains means that the exact environment of T460 in Amt-1;1 is also not known (Supplementary Fig. 6). Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters. + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:03:15Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:49:46Z + + AMTs + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:24:57Z + + T460 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:42:14Z + + pore + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:42:36Z + + C terminus + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:42:40Z + + homology model + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:24:49Z + + Amt-1;1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:52:15Z + + archaebacterial + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:42:52Z + + AfAmt-1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:42:55Z + + structure + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:43:00Z + + C terminus + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:24:49Z + + Amt-1;1 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:28Z + + monomer + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:43:06Z + + Mep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:43:09Z + + structures + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:24:57Z + + T460 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:24:49Z + + Amt-1;1 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:43:14Z + + structural information + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:24:49Z + + Amt-1;1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:06Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:27:26Z + + Y467 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:27:35Z + + H239 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:27:42Z + + D458 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:27:50Z + + K71 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:53Z + + CaMep2 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:15Z + + Tyr49 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:03:15Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:49:46Z + + AMTs + + + site + SO: + melaniev@ebi.ac.uk + 2023-06-14T09:40:12Z + + Tyr–His2 hydrogen bond + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:43:43Z + + Mep2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:02Z + + closed + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:03:15Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:43:45Z + + transporters + + + + DISCUSS + paragraph + 30177 + We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. The need to regulate in opposite ways may be the reason why the phosphorylation sites are in different parts of the CTR, that is, centrally located close to the ExxGxD motif in AMTs and peripherally in Mep2. In this way, phosphorylation can either lead to channel closing (in the case of AMTs) or channel opening in the case of Mep2. Our model also provides an explanation for the observation that certain mutations within the CTR completely abolish transport activity. An example of an inactivating residue is the glycine of the ExxGxD motif of the CTR. Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2. + + site + SO: + melaniev@ebi.ac.uk + 2023-06-14T09:40:25Z + + intra-monomeric CTR-ICL1/ICL3 interactions + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:02Z + + fungal + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:03:15Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:48:17Z + + channels + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:48:13Z + + lack of + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:11Z + + inactive + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:38:02Z + + phosphorylation sites + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:08:08Z + + ExxGxD motif + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:49:46Z + + AMTs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:49:31Z + + Mep2 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-22T09:49:17Z + + channel + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:49:46Z + + AMTs + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-22T09:49:28Z + + channel + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:49:51Z + + Mep2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:49:39Z + + certain mutations + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:44:21Z + + glycine + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:08:08Z + + ExxGxD motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:49:56Z + + Mutation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:50:01Z + + G393 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:50:05Z + + EcAmtB + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T09:50:08Z + + G456 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T21:27:13Z + + AtAmt-1;1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:50:13Z + + transporters + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:50:17Z + + Escherichia coli + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:50:21Z + + AmtB + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:25:32Z + + A. thaliana + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:24:49Z + + Amt-1;1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:02Z + + closed + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:50:31Z + + Mep2 + + + + DISCUSS + paragraph + 31335 + Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters. Recently, phosphorylation was also shown to modulate substrate affinity in nitrate transporters. With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs). However, the absence of GlnK proteins in eukaryotes suggests that phosphorylation-based regulation of ammonium transport may be widespread. Nevertheless, as discussed above, considerable differences may exist between different species. + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T10:57:58Z + + aquaporins + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T10:58:00Z + + urea transporters + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:05Z + + eukaryotic + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T10:58:06Z + + channels + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T10:58:09Z + + transporters + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T10:58:14Z + + nitrate transporters + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:49:43Z + + ammonium + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:25:32Z + + A. thaliana + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:49:46Z + + AMTs + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:02Z + + S. cerevisiae + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:01:16Z + + Mep2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:01:05Z + + absence of + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:30:49Z + + GlnK proteins + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:24Z + + eukaryotes + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:50:00Z + + phosphorylation + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:50:14Z + + ammonium + + + + DISCUSS + paragraph + 32018 + With respect to Mep2-mediated signalling to induce pseudohyphal growth, two models have been put forward as to how this occurs and why it is specific to Mep2 proteins. In one model, signalling is proposed to depend on the nature of the transported substrate, which might be different in certain subfamilies of ammonium transporters (for example, Mep1/Mep3 versus Mep2). For example, NH3 uniport or symport of NH3/H+ might result in changes in local pH, but NH4+ uniport might not, and this difference might determine signalling. In the other model, signalling is thought to require a distinct conformation of the Mep2 transporter occurring during the transport cycle. While the current study does not specifically address the mechanism of signalling underlying pseudohyphal growth, our structures do show that Mep2 proteins can assume different conformations. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:50:30Z + + Mep2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:02:50Z + + Mep1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:52:05Z + + Mep3 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:02:56Z + + Mep2 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:58:55Z + + NH3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T16:58:55Z + + NH3 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T11:03:06Z + + H+ + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T11:03:03Z + + NH4+ + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:03:10Z + + Mep2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:03:12Z + + transporter + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:03:14Z + + structures + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:42:15Z + + Mep2 proteins + + + + DISCUSS + paragraph + 32878 + It is clear that ammonium transport across biomembranes remains a fascinating and challenging field in large part due to the unique properties of the substrate. Our Mep2 structural work now provides a foundation for future studies to uncover the details of the structural changes that occur during eukaryotic ammonium transport and signaling, and to assess the possibility to utilize small molecules to shut down ammonium sensing and downstream signalling pathways in pathogenic fungi. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:50:51Z + + ammonium + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:03:44Z + + Mep2 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:31:05Z + + eukaryotic + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:51:02Z + + ammonium + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:51:15Z + + ammonium + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:50:14Z + + fungi + + + + METHODS + title_1 + 33364 + Methods + + + METHODS + title_2 + 33372 + Mep2 overexpression and purification + + + METHODS + paragraph + 33409 + Ammonium transporter genes were amplified from genomic DNA or cDNA by PCR (Phusion, New England Biolabs). In both ScMEP2 and CaMEP2, Asn4 was replaced by a glutamine to prevent glycosylation. In order to allow transformation of yeast by recombination, the following primer extensions were used: forward 5′-GAAAAAACCCCGGATTCTAGAACTAGTGGATCCTCC-3′ and reverse 5′-TGACTCGAGTTATGCACCGTGGTGGTGATGGTGATG-3′. These primers result in a construct that lacks the cleavable N- and C-terminal tags present in the original vector, and replaces these with a C-terminal hexa-histidine tag. Recombination in yeast strain W303 pep4Δ was carried out using ∼50–100 ng of SmaI-digested vector 83νΔ (ref.) and at least a fourfold molar excess of PCR product via the lithium acetate method. Transformants were selected on SCD -His plates incubated at 30 °C. Construction of mutant CaMEP2 genes was done using the Q5 site-directed mutagenesis kit (NEB) per manufacturer's instructions. Three CaMep2 mutants were made for crystallization: the first mutant is a C-terminal truncation mutant 442Δ, lacking residues 443–480 including the AI domain. The second mutant, R452D/S453D, mimics the protein phosphorylated at Ser453. Given that phosphate is predominantly charged −2 at physiological pH, we introduced the second aspartate residue for Arg452. However, we also constructed the ‘single D', S453D CaMep2 variant. + + + METHODS + paragraph + 34827 + For expression, cells were grown in shaker flasks at 30 °C for ∼24 h in synthetic minimal medium lacking histidine and with 1% (w/v) glucose to a typical OD600 of 6–8. Cells were subsequently spun down for 15 min at 4,000g and resuspended in YP medium containing 1.5% (w/v) galactose, followed by another 16–20 h growth at 30 °C/160 r.p.m. and harvesting by centrifugation. Final OD600 values typically reached 18–20. Cells were lysed by bead beating (Biospec) for 5 × 1 min with 1 min intervals on ice, or by 1–2 passes through a cell disrupter operated at 35,000 p.s.i. (TS-Series 0.75 kW; Constant Systems). Membranes were collected from the suspension by centrifugation at 200,000g for 90 min (45Ti rotor; Beckmann Coulter). Membrane protein extraction was performed by homogenization in a 1:1 (w/w) mixture of dodecyl-β-D-maltoside and decyl-β-D-maltoside (DDM/DM) followed by stirring at 4 °C overnight. Typically, 1 g (1% w/v) of total detergent was used for membranes from 2 l of cells. The membrane extract was centrifuged for 35 min at 200,000g and the supernatant was loaded onto a 10-ml Nickel column (Chelating Sepharose; GE Healthcare) equilibrated in 20 mM Tris/300 mM NaCl/0.2% DDM, pH 8. The column was washed with 15 column volumes buffer containing 30 mM imidazole and eluted in 3 column volumes with 250 mM imidazole. Proteins were purified to homogeneity by gel filtration chromatography in 10 mM HEPES/100 mM NaCl/0.05% DDM, pH 7–7.5. For polishing and detergent exchange, a second gel filtration column was performed using various detergents. In the case of ScMep2, diffracting crystals were obtained only with 0.05% decyl-maltose neopentyl glycol. For the more stable CaMep2 protein, we obtained crystals in, for example, nonyl-glucoside, decyl-maltoside and octyl-glucose neopentyl glycol. Proteins were concentrated to 7–15 mg ml−1 using 100 kDa cutoff centrifugal devices (Millipore), flash-frozen and stored at −80 °C before use. + + + METHODS + title_2 + 36860 + Crystallization and structure determination + + + METHODS + paragraph + 36904 + Crystallization screening trials by sitting drop vapour diffusion were set up at 4 and 20 °C using in-house screens and the MemGold 1 and 2 screens (Molecular Dimensions) with a Mosquito crystallization robot. Crystals were harvested directly from the initial trials or optimized by sitting or hanging drop vapour diffusion using larger drops (typically 2–3 μl total volume). Bar-shaped crystals for ScMep2 diffracting to 3.2 Å resolution were obtained from 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)/0.2 M di-ammonium hydrogen phosphate/30% PEG 400, pH 6. They belong to space group P212121 and have nine molecules (three trimers) in the asymmetric unit (AU). Well-diffracting crystals for CaMep2 were obtained in space group P3 from 0.1 M MES/0.2 M lithium sulphate/20% PEG400, pH 6 (two molecules per AU). An additional crystal form in space group R3 was grown in 0.04 M Tris/0.04 M NaCl/27% PEG350 MME, pH 8 (one molecule per AU). Diffracting crystals for the phosporylation-mimicking CaMep2 DD mutant were obtained in space group P6322 from 0.1 M sodium acetate/15–20% PEG400, pH 5 (using decyl-maltoside as detergent; one molecule per AU), while S453D mutant crystals grew in 24% PEG400/0.05 M sodium acetate, pH 5.4/0.05 M magnesium acetate tetrahydrate/10 mM NH4Cl (space group R32; one molecule per AU). Finally, the 442Δ truncation mutant gave crystals under many different conditions, but most of these diffracted poorly or not at all. A reasonable low-resolution data set (3.4 Å resolution) was eventually obtained from a crystal grown in 24% PEG400/0.05 M sodium acetate/0.05 M magnesium acetate, pH 6.1 (space group R32). Diffraction data were collected at the Diamond Light Source and processed with XDS or HKL2000 (ref. ). + + + METHODS + paragraph + 38689 + For MR, a search model was constructed with Sculptor within Phenix, using a sequence alignment of ScMep2 with Archaeoglobus fulgidus Amt-1 (PDB ID 2B2H; ∼40% sequence identity to ScMep2). A clear solution with nine molecules (three trimers) in the AU was obtained using Phaser. The model was subsequently completed by iterative rounds of manual building within Coot followed by refinement within Phenix. The structures for WT CaMep2 were solved using the best-defined monomer of ScMep2 (60% sequence identity with CaMep2) in MR with Phaser, followed by automated model building within Phenix. Finally, the structures of the three mutant CaMep2 proteins were solved using WT CaMep2 as the search model. The data collection and refinement statistics for all six solved structures have been summarized in Supplementary Tables 1 and 2. + + + METHODS + title_2 + 39523 + Growth assays + + + METHODS + paragraph + 39537 + The S. cerevisiae haploid triple mepΔ strain (Σ1278b MATα mep1::LEU2 mep2::LEU2 mep3::G418 ura3-52) and triple mepΔ npr1Δ strain (Σ1278b MATα mep1::LEU2 mep2::LEU2 mep3::G418 npr1::NAT1 ura3-52) were generated by integrating the NAT1 resistance gene at one NPR1 locus in the diploid strain MLY131 (ref.), followed by isolation of individual haploid strains. Cells were grown in synthetic minimal medium with glucose (2%) as the carbon source and ammonium sulphate (1 mM) or glutamate (0.1%) as the nitrogen source. Yeast cells were transformed as described. All DNA sequences encoding epitope-tagged ScMep2 and its mutant derivatives were generated by PCR and homologous recombination using the vector pRS316 (ref. ). In each case, the ScMEP2 sequences included the ScMEP2 promoter (1 kb), the ScMEP2 terminator and sequences coding for a His-6 epitope at the C-terminal end of the protein. All Mep2-His fusions contain the N4Q mutation to prevent glycosylation of Mep2 (ref.). All newly generated plasmid inserts were verified by DNA sequencing. For growth assays, S. cerevisiae cells containing plasmids expressing ScMep2 or mutant derivatives were grown overnight in synthetic minimal glutamate medium, washed, spotted by robot onto solid agar plates and culture growth followed by time course photography. Images were then processed to quantify the growth of each strain over 3 days as described. + + + METHODS + title_2 + 40966 + Protein modelling + + + METHODS + paragraph + 40984 + The MODELLER (version 9.15) software package was used to build protein structures for MD simulations. This method was required to construct two complete protein models, the double mutant R452D/S453D (with the four missing residues from the X-ray structure added) and also the construct in which the mutation at position 452 is reverted to R, and D453 is replaced with a phosphoserine. The quality of these models was assessed using normalized Discrete Optimized Protein Energy (DOPE) values and the molpdf assessment function within the MODELLER package. The model R452D/S453D mutant has a molpdf assessment score of 1854.05, and a DOPE assessment score of -60920.55. The model of the S453J mutant has a molpdf assessment score of 1857.01 and a DOPE assessment score of −61032.15. + + + METHODS + title_2 + 41767 + MD simulations + + + METHODS + paragraph + 41782 + WT and model structures were embedded into a pre-equilibrated lipid bilayer composed of 512 dipalmitoylphosphatidylcholine lipids using the InflateGRO2 computer programme. The bilayers were then solvated with the SPC water model and counterions were added to achieve a charge neutral state. All simulations were performed with the GROMACS package (version 4.5.5), and the GROMOS96 43a1p force field. During simulation time, the temperature was maintained at 310 K using the Nosé-Hoover thermostat with a coupling constant of 0.5 ps. Pressure was maintained at 1 bar using semi-isotropic coupling with the Parrinello-Rahman barostat and a time constant of 5 ps. Electrostatic interactions were treated using the smooth particle mesh Ewald algorithm with a short-range cutoff of 0.9 nm. Van der Waals interactions were truncated at 1.4 nm with a long-range dispersion correction applied to energy and pressure. The neighbour list was updated every five steps. All bonds were constrained with the LINCS algorithm, so that a 2-fs time step could be applied throughout. The phospholipid parameters for the dipalmitoylphosphatidylcholine lipids were based on the work of Berger. The embedded proteins were simulated for 200 ns each; a repeat simulation was performed for each system with different initial velocities to ensure reproducibility. To keep the c.p.u. times within reasonable limits, all simulations were performed on Mep2 monomers. This is also consistent with previous simulations for E. coli AmtB. + + + METHODS + title_1 + 43303 + Additional information + + + METHODS + paragraph + 43326 + Accession codes: The atomic coordinates and the associated structure factors have been deposited in the Protein Data Bank (http:// www.pdbe.org) with accession codes 5AEX (ScMep2), 5AEZ(CaMep2; R3), 5AF1(CaMep2; P3), 5AID(CaMep2; 442D), 5AH3 (CaMep2; R452D/S453D) and 5FUF (CaMep2; S453D). + + + METHODS + paragraph + 43616 + How to cite this article: van den Berg, B. et al. Structural basis for Mep2 ammonium transceptor activation by phosphorylation. Nat. Commun. 7:11337 doi: 10.1038/ncomms11337 (2016). + + + SUPPL + title_1 + 43798 + Supplementary Material + + + 556 + 564 + surname:Holsbeeks;given-names:I. + surname:Lagatie;given-names:O. + surname:Van +Nuland;given-names:A. + surname:Van de Velde;given-names:S. + surname:Thevelein;given-names:J. +M. + 15450611 + REF + Trends Biochem. Sci. + ref + 29 + 2004 + 43821 + The eukaryotic plasma membrane as a nutrient-sensing device + + + 254 + 299 + surname:Conrad;given-names:M. + 24483210 + REF + FEMS Microbiol. Rev. + ref + 38 + 2014 + 43881 + Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae + + + D251 + D258 + surname:Saier;given-names:M. H. + surname:Reddy;given-names:V. S. + surname:Tamang;given-names:D. +G. + surname:Vastermark;given-names:A. + 24225317 + REF + Nucleic Acids +Res. + ref + 42 + 2014 + 43950 + The transporter classification database + + + 4282 + 4293 + surname:Marini;given-names:A. M. + surname:Soussi-Boudekou;given-names:S. + surname:Vissers;given-names:S. + surname:Andre;given-names:B. + 9234685 + REF + Mol. Cell Biol. + ref + 17 + 1997 + 43990 + A family of ammonium transporters in Saccharomyces cerevisiae + + + e24275 + surname:Van Zeebroeck;given-names:G. + surname:Kimpe;given-names:M. + surname:Vandormael;given-names:P. + surname:Thevelein;given-names:J. M. + 21912684 + REF + PLoS ONE + ref + 6 + 2011 + 44052 + A split-ubiquitin two-hybrid screen for proteins physically interacting with the yeast amino acid transceptor Gap1 and ammonium transceptor Mep2 + + + 1236 + 1247 + surname:Lorenz;given-names:M. C. + surname:Heitman;given-names:J. + 9482721 + REF + EMBO J. + ref + 17 + 1998 + 44197 + The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae + + + 345 + 355 + surname:Shnaiderman;given-names:C. + surname:Miyara;given-names:I. + surname:Kobiler;given-names:I. + surname:Sherman;given-names:A. + surname:Prusky;given-names:D. + 23387470 + REF + Mol. Plant Microbe Interact. + ref + 26 + 2013 + 44291 + Differential activation of ammonium transporters during the accumulation of ammonia by Colletotrichum gloeosporioides and its effect on appressoria formation andpathogenicity + + + 3028 + 3039 + surname:Rutherford;given-names:J. C. + surname:Chua;given-names:G. + surname:Hughes;given-names:T. + surname:Cardenas;given-names:M. +E. + surname:Heitman;given-names:J. + 18434596 + REF + Mol. Biol. Cell + ref + 19 + 44466 + A Mep2-dependent transcriptional profile links permease function to gene expression during pseudohyphal growth in Saccharomyces cerevisiae + + + 649 + 669 + surname:Biswas;given-names:K. + surname:Morschhäuser;given-names:J. + 15819622 + REF + Mol. Microbiol. + ref + 56 + 2005 + 44605 + The Mep2p ammonium permease controls nitrogen starvation-induced filamentous growth in Candida albicans + + + 21362 + 21370 + surname:Boeckstaens;given-names:M. + surname:André;given-names:B. + surname:Marini;given-names:A. M. + 18508774 + REF + J. Biol. +Chem. + ref + 283 + 2008 + 44709 + Distinct transport mechanisms in yeast ammonium transport/sensor proteins of the Mep/Amt/Rh family and impact on filamentation + + + 534 + 546 + surname:Boeckstaens;given-names:M. + surname:André;given-names:B. + surname:Marini;given-names:A. M. + 17493133 + REF + Mol. Microbiol. + ref + 64 + 2007 + 44836 + The yeast ammonium transport protein Mep2 and its positive regulator, the Npr1 kinase, play an important role in normal and pseudohyphal growth on various nitrogen media through retrieval of excreted ammonium + + + 9638 + 9643 + surname:Gruswitz;given-names:F. + 20457942 + REF + Proc. +Natl Acad. Sci. USA + ref + 107 + 2010 + 45045 + Function of human Rh based on structure of RhCG at 2.1A + + + 1587 + 1594 + surname:Khademi;given-names:S. + 15361618 + REF + Science + ref + 305 + 2004 + 45101 + Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35A + + + 14994 + 14999 + surname:Andrade;given-names:S. L. + surname:Dickmanns;given-names:A. + surname:Ficner;given-names:R. + surname:Einsle;given-names:O. + 16214888 + REF + Proc. Natl Acad. Sci. USA + ref + 102 + 2005 + 45174 + Crystal structure of the archaeal ammonium transporter Amt-1 from Archaeoglobus fulgidus + + + 39492 + 39498 + surname:Javelle;given-names:A. + 17040913 + REF + J. Biol. Chem. + ref + 281 + 2006 + 45263 + An unusual twin-his arrangement in the pore of ammonia channels is essential for substrate conductance + + + 10419 + 10427 + surname:Wang;given-names:S. + surname:Orabi;given-names:E. A. + surname:Baday;given-names:S. + surname:Bernèche;given-names:S. + surname:Lamoureux;given-names:G. + 22631217 + REF + J. Am. Chem. Soc. + ref + 134 + 2012 + 45366 + Ammonium transporters achieve charge transfer by fragmenting their substrate + + + e62745 + surname:Wang;given-names:J. + 23667517 + REF + PLoS ONE + ref + 8 + 2013 + 45443 + Ammonium transport proteins with changes in one of the conserved pore histidines have different performance in ammonia and methylamine conduction + + + 10876 + 10884 + surname:Lin;given-names:Y. + surname:Cao;given-names:Z. + surname:Mo;given-names:Y. + 16910683 + REF + J. Am. +Chem. Soc. + ref + 128 + 2006 + 45589 + Molecular dynamics simulations on the Escherichia coli ammonia channel protein AmtB: mechanism of ammonia/ammonium transport + + + 3970 + 3975 + surname:Akgun;given-names:U. + surname:Khademi;given-names:S. + 21368153 + REF + Proc. Natl +Acad. Sci. USA + ref + 108 + 2011 + 45714 + Periplasmic vestibule plays an important role for solute recruitment, selectivity, and gating in the Rh/Amt/MEP superfamily + + + 17090 + 17095 + surname:Zheng;given-names:L. + surname:Kostrewa;given-names:D. + surname:Bernèche;given-names:S. + surname:Winkler;given-names:F. K. + surname:Li;given-names:X. D. + 15563598 + REF + Proc. Natl Acad. Sci. USA + ref + 101 + 2004 + 45838 + The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli + + + 7091 + 7098 + surname:Baday;given-names:S. + surname:Wang;given-names:S. + surname:Lamoureux;given-names:G. + surname:Bernèche;given-names:S. + 24021113 + REF + Biochemistry + ref + 52 + 2013 + 45932 + Different hydration patterns in the pores of AmtB and RhCG could determine their transport mechanisms + + + 628 + 695 + surname:van Heeswijk;given-names:W. C. + surname:Westerhoff;given-names:H. V. + surname:Boogerd;given-names:F. C. + 24296575 + REF + Microbiol. Mol. Biol. Rev. + ref + 77 + 2013 + 46034 + Nitrogen assimilation in Escherichia coli: putting molecular data into a systems perspective + + + 9995 + 10000 + surname:Wacker;given-names:T. + surname:Garcia-Celma;given-names:J. J. + surname:Lewe;given-names:P. + surname:Andrade;given-names:S. L. + 24958855 + REF + Proc. Natl Acad. Sci. USA + ref + 111 + 2014 + 46127 + Direct observation of electrogenic NH4(+) transport in ammonium transport (Amt) proteins + + + 11650 + 11655 + surname:Neuhäuser;given-names:B. + surname:Ludewig;given-names:U. + 24634212 + REF + J. Biol. Chem. + ref + 289 + 2014 + 46216 + Uncoupling of ionic currents from substrate transport in the plant ammonium transporter AtAMT1;2 + + + 95 + 102 + surname:Monfort;given-names:P. + surname:Kosenko;given-names:E. + surname:Erceg;given-names:S. + surname:Canales;given-names:J. J. + surname:Felipo;given-names:V. + 12020609 + REF + Neurochem. Int. + ref + 41 + 2002 + 46313 + Molecular mechanism of acute ammonia toxicity: role of NMDA receptors + + + 11 + 14 + surname:Thomas;given-names:G. + surname:Coutts;given-names:G. + surname:Merrick;given-names:M. + 10637624 + REF + Trends Genet. + ref + 16 + 2000 + 46383 + The glnKamtB operon. A conserved gene pair in prokaryotes + + + 42 + 47 + surname:Gruswitz;given-names:F. + surname:O'Connell;given-names:J.;suffix:III + surname:Stroud;given-names:R. +M. + 17190799 + REF + Proc. Natl Acad. +Sci. USA + ref + 104 + 2006 + 46441 + Inhibitory complex of the transmembrane ammonia channel, AmtB, and the cytosolic regulatory protein, GlnK, at 1.96A + + + 29558 + 29567 + surname:Durand;given-names:A. + surname:Merrick;given-names:M. + 16864585 + REF + J. Biol. Chem. + ref + 281 + 2006 + 46557 + In vitro analysis of the Escherichia coli AmtB-GlnK complex reveals a stoichiometric interaction and sensitivity to ATP and 2-oxoglutarate + + + 736 + 738 + surname:Lanquar;given-names:V. + surname:Frommer;given-names:W. B. + 20418663 + REF + Plant Signal. +Behav. + ref + 5 + 2010 + 46696 + Adjusting ammonium uptake via phosphorylation + + + 3101 + 3110 + surname:Boeckstaens;given-names:M. + surname:Llinares;given-names:E. + surname:Van +Vooren;given-names:P. + surname:Marini;given-names:A. M. + 24476960 + REF + Nat. Commun. + ref + 5 + 2014 + 46742 + The TORC1 effector kinase Npr1 fine tunes the inherent activity of the Mep2 ammonium transport protein + + + 1361 + 1373 + surname:Graff;given-names:L. + 21127027 + REF + J. Exp. Bot. + ref + 62 + 2011 + 46845 + N- terminal cysteines affect oligomer stability of the allosterically regulated ammonium transporter LeAMT1;1 + + + 161 + 171 + surname:Severi;given-names:E. + surname:Javelle;given-names:A. + surname:Merrick;given-names:M. + 17453422 + REF + Mol. Membr. Biol. + ref + 24 + 2007 + 46955 + The conserved carboxy-terminal region of the ammonia channel AmtB plays a critical role in channel function + + + 195 + 198 + surname:Loqué;given-names:D. + surname:Lalonde;given-names:S. + surname:Looger;given-names:L. L. + surname:von +Wirén;given-names:N. + surname:Frommer;given-names:W. B. + 17293878 + REF + Nature + ref + 446 + 2007 + 47063 + A cytosolic trans-activation domain essential for ammonium uptake + + + 974 + 984 + surname:Yuan;given-names:L. + 23463773 + REF + Plant +Cell + ref + 25 + 2013 + 47129 + Allosteric regulation of transport activity by heterotrimerization of Arabidopsis ammonium transporter complexes in vivo + + + e351 + surname:Hess;given-names:D. C. + surname:Lu;given-names:W. + surname:Rabinowitz;given-names:J. +D. + surname:Botstein;given-names:D. + 17048990 + REF + PLoS +Biol. + ref + 4 + 2006 + 47250 + Ammonium toxicity and potassium limitation in yeast + + + 259 + 275 + surname:Smith;given-names:D. G. + surname:Garcia-Pedrajas;given-names:M. D. + surname:Gold;given-names:S. E. + surname:Perlin;given-names:M. H. + 14507379 + REF + Mol. +Microbiol. + ref + 50 + 2003 + 47302 + Isolation and characterization from pathogenic fungi of genes encoding ammonium permeases and their roles in dimorphism + + + 187 + 201 + surname:Teichert;given-names:S. + surname:Rutherford;given-names:J. C. + surname:Wottawa;given-names:M. + surname:Heitman;given-names:J. + surname:Tudzynski;given-names:B. + 18083831 + REF + Eukaryot. Cell + ref + 7 + 2008 + 47422 + Impact of ammonium permeases mepA, mepB, and mepC on nitrogen-regulated secondary metabolism in Fusarium fujikuroi + + + 411 + 430 + surname:Javelle;given-names:A. + 12519192 + REF + Mol. +Microbiol. + ref + 47 + 2003 + 47537 + Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS, NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum + + + 2580 + 2588 + surname:Törnroth-Horsefield;given-names:S. + surname:Hedfalk;given-names:K. + surname:Fischer;given-names:G. + surname:Lindkvist-Petersson;given-names:K. + surname:Neutze;given-names:R. + REF + FEBS +Lett. + ref + 584 + 2006 + 47723 + Structural insights into eukaryotic aquaporin regulation + + + 79 + 107 + surname:Klein;given-names:J. D. + 25298340 + REF + Subcell. +Biochem. + ref + 73 + 2014 + 47780 + Expression of urea transporters and their regulation + + + 68 + 72 + surname:Parker;given-names:J. L. + surname:Newstead;given-names:S. + 24572366 + REF + Nature + ref + 507 + 2014 + 47833 + Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1 + + + 695 + 707 + surname:Hays;given-names:F. A. + surname:Roe-Zurz;given-names:Z. + surname:Stroud;given-names:R. M. + 20946832 + REF + Methods Enzymol. + ref + 470 + 2010 + 47907 + Overexpression and purification of integral membrane proteins in yeast + + + 125 + 132 + surname:Kabsch;given-names:W. + REF + Acta Crystallogr. + ref + D66 + 2010 + 47978 + XDS + + + 307 + 326 + surname:Otwinowski;given-names:Z. + surname:Minor;given-names:W. + REF + Methods Enzymol. + ref + 276 + 2003 + 47982 + Processing of X-ray diffraction data collected in oscillation mode + + + 352 + 367 + surname:Afonine;given-names:P. V. + REF + Acta Crystallogr. + ref + D68 + 2012 + 48049 + Towards automated crystallographic structure refinement with phenix.refine + + + 458 + 464 + surname:McCoy;given-names:A. J. + surname:Grosse-Kunstleve;given-names:R. W. + surname:Storoni;given-names:L. C. + surname:Read;given-names:R. J. + REF + Acta +Crystallogr. + ref + D61 + 2005 + 48124 + Likelihood-enhanced fast translation functions + + + 2126 + 2132 + surname:Emsley;given-names:P. + surname:Cowtan;given-names:K. + REF + Acta +Crystallogr. + ref + D60 + 2004 + 48171 + Coot: model-building tools for molecular graphics + + + 339 + 346 + surname:Schiestl;given-names:R. H. + surname:Gietz;given-names:R. D. + 2692852 + REF + Curr. Genet. + ref + 16 + 1989 + 48221 + High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier + + + 201 + 216 + surname:Ma;given-names:H. + surname:Kunes;given-names:S. + surname:Schatz;given-names:P. +J. + surname:Botstein;given-names:D. + 2828185 + REF + Gene + ref + 58 + 1987 + 48323 + Plasmid construction by homologous recombination in yeast + + + 552 + 564 + surname:Marini;given-names:A. M. + surname:André;given-names:B. + 11069679 + REF + Mol. Microbiol. + ref + 38 + 2000 + 48381 + In vivo N-glycosylation of the Mep2 high-affinity ammonium transporter of Saccharomyces cerevisiae reveals an extracytosolic N-terminus + + + e1001362 + surname:Addinall;given-names:S. G. + 21490951 + REF + PLoS Genet. + ref + 7 + 2011 + 48517 + Quantitative fitness analysis shows that NMD proteins and many other protein complexes suppress or enhance distinct telomere cap defects + + + 291 + 325 + surname:Marti-Renom;given-names:M. A. + 10940251 + REF + Annu. Rev. Biophys. Biomol. Struct. + ref + 29 + 2000 + 48654 + Comparative protein structure modeling of genes and genomes + + + 2657 + 2669 + surname:Schmidt;given-names:T. H. + surname:Kandt;given-names:C. + 22989154 + REF + J. Chem. +Inf. Model. + ref + 52 + 2012 + 48714 + LAMBADA and InflateGRO2: efficient membrane alignment and insertion of membrane proteins for molecular dynamics simulations + + + 331 + 342 + surname:Berendsen;given-names:H. J. C. + surname:Postma;given-names:J. P. + surname:van +Gunsteren;given-names:W. F. + surname:Hermans;given-names:J. + REF + Interaction models for water in relation to protein hydration Intermolecular Forces + ref + 1981 + 48838 + + + 1701 + 1718 + surname:Van Der Spoel;given-names:D. + 16211538 + REF + J. Comput. Chem. + ref + 26 + 2005 + 48839 + GROMACS: fast, flexible, and free + + + 511 + 519 + surname:Nosé;given-names:S. + REF + J. Chem. Phys. + ref + 81 + 1984 + 48873 + A unified formulation of the constant temperature molecular dynamics methods + + + 1695 + surname:Hoover;given-names:W. G. + REF + Phys. +Rev. A + ref + 31 + 1985 + 48950 + Canonical dynamics: equilibrium phase-space distributions + + + 7182 + 7190 + surname:Parrinello;given-names:M. + surname:Rahman;given-names:A. + REF + J. Appl. Phys. + ref + 52 + 1981 + 49008 + Polymorphic transitions in single crystals: a new molecular dynamics method + + + 8577 + 8593 + surname:Essmann;given-names:U. + REF + J. Chem. Phys. + ref + 103 + 1995 + 49084 + A smooth particle mesh Ewald method + + + 1463 + 1472 + surname:Hess;given-names:B. + surname:Bekker;given-names:H. + surname:Berendsen;given-names:H. +J. + surname:Fraaije;given-names:J. G. + REF + J. +Comput. Chem. + ref + 18 + 1997 + 49120 + LINCS: a linear constraint solver for molecular simulations + + + 2002 + 2013 + surname:Berger;given-names:O. + surname:Edholm;given-names:O. + surname:Jähnig;given-names:F. + 9129804 + REF + Biophys. J. + ref + 72 + 1997 + 49180 + Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature + + + REF + ref + 49327 + The PyMOL Molecular Graphics System. version 1.7.4 (Schrödinger, LLC). + + + SUPPL + footnote + 49400 + Author contributions B.v.d.B. performed the experiments related to Mep2 structure determination, designed research and wrote the paper. A.C. performed ammonium growth experiments of Mep variants. D.J. and S.K. performed modelling studies and MD simulations. A.B. collected the X-ray synchrotron data and maintained the Newcastle Structural Biology Laboratory. J.C.R. designed research related to the S. cerevisiae growth assays. + + + ncomms11337-f1.jpg + f1 + FIG + fig_title_caption + 49829 + X-ray crystal structures of Mep2 transceptors. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:22Z + + X-ray crystal structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:07:14Z + + Mep2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:44:36Z + + transceptors + + + + ncomms11337-f1.jpg + f1 + FIG + fig_caption + 49876 + (a) Monomer cartoon models viewed from the side for (left) A. +fulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and +C. albicans Mep2 (right). The cartoons are in rainbow +representation. The region showing ICL1 (blue), ICL3 (green) and the CTR +(red) is boxed for comparison. (b) CaMep2 trimer viewed from the +intracellular side (right). One monomer is coloured as in a and one +monomer is coloured by B-factor (blue, low; red; high). The CTR is boxed. +(c) Overlay of ScMep2 (grey) and CaMep2 (rainbow), illustrating +the differences in the CTRs. All structure figures were generated with +Pymol. + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:28Z + + Monomer + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:08:55Z + + A. +fulgidus + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:52:24Z + + Amt-1 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:50:02Z + + S. cerevisiae + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:43:49Z + + Mep2 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:40:10Z + + C. albicans + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:09:00Z + + Mep2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:08:50Z + + trimer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:28Z + + monomer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:28Z + + monomer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:41:29Z + + Overlay + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:07Z + + ScMep2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:37:12Z + + CTRs + + + + ncomms11337-f2.jpg + f2 + FIG + fig_title_caption + 50476 + Sequence conservation in ammonium transporters. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:09:27Z + + Sequence conservation + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T16:41:48Z + + ammonium transporters + + + + ncomms11337-f2.jpg + f2 + FIG + fig_caption + 50524 + ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E. +coli AmtB and A. thaliana Amt-1;1. The secondary structure +elements observed for CaMep2 are indicated, with the numbers corresponding +to the centre of the TM segment. Important regions are labelled. The +conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, +the conserved ExxGxD motif of the CTR in red and the AI region in yellow. +Coloured residues are functionally important and correspond to those of the +Phe gate (blue), the binding site Trp residue (magenta) and the twin-His +motif (red). The Npr1 kinase site in the AI region is highlighted pink. The +grey sequences at the C termini of CaMep2 and ScMep2 are not visible in the +structures and are likely disordered. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:41:36Z + + ClustalW alignment + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:07Z + + ScMep2 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:11:32Z + + A. fulgidus + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:52:24Z + + Amt-1 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:11:38Z + + E. +coli + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:11:41Z + + AmtB + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:25:32Z + + A. thaliana + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:24:49Z + + Amt-1;1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:12:14Z + + TM segment + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:11:44Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:06:51Z + + RxK motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:11:48Z + + ER motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:13:11Z + + ICL2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:11:51Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:08:08Z + + ExxGxD motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:40Z + + AI region + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T18:56:01Z + + Phe gate + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:11:57Z + + binding site + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:11:54Z + + Trp + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:35:57Z + + twin-His +motif + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:12:01Z + + Npr1 kinase site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:40Z + + AI region + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:07Z + + ScMep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:12:04Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:12:08Z + + likely disordered + + + + ncomms11337-f3.jpg + f3 + FIG + fig_title_caption + 51276 + Growth of ScMep2 variants on low ammonium medium. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:14:22Z + + Growth + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:13:11Z + + ScMep2 variants + + + + ncomms11337-f3.jpg + f3 + FIG + fig_caption + 51326 + (a) The triple mepΔ strain (black) and triple +mepΔ npr1Δ strain (grey) containing plasmids +expressing WT and variant ScMep2 were grown on minimal medium containing +1 mM ammonium sulphate. The quantified cell density reflects +logarithmic growth after 24 h. Error bars are the s.d. for three +replicates of each strain (b) The strains used in a were also +serially diluted and spotted onto minimal agar plates containing glutamate +(0.1%) or ammonium sulphate (1 mM), and grown for 3 days at +30 °C. + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T21:37:27Z + + triple mepΔ + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:14:11Z + + triple +mepΔ npr1Δ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:52Z + + WT + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:14:15Z + + variant ScMep2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:14:19Z + + grown on minimal medium + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-16T11:14:25Z + + ammonium sulphate + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:14:28Z + + cell density + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-06-15T15:03:58Z + + glutamate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:52:07Z + + ammonium sulphate + + + + ncomms11337-f4.jpg + f4 + FIG + fig_title_caption + 51832 + Structural differences between Mep2 and bacterial ammonium +transporters. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:14:51Z + + Mep2 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:41:28Z + + bacterial + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:14:55Z + + ammonium +transporters + + + + ncomms11337-f4.jpg + f4 + FIG + fig_caption + 51905 + (a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing +unwinding and inward movement in the fungal protein. (b) Stereo +diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in +AfAmt-1 (light colours) and CaMep2 (dark colours). The side chains of +residues in the RxK motif as well as those of Tyr49 and His342 are labelled. +The numbering is for CaMep2. (c) Conserved residues in ICL1-3 and the +CTR. Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the +large differences in conformation of the conserved residues in ICL1 (RxK +motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). The +labelled residues are analogous within both structures. In b and +c, the centre of the trimer is at top. + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:42:52Z + + AfAmt-1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:36:02Z + + fungal + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:42:52Z + + AfAmt-1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:06:51Z + + RxK motif + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:15Z + + Tyr49 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:42Z + + His342 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:16:55Z + + Conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:36:09Z + + ICL1-3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:42:52Z + + AfAmt-1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:17:09Z + + conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:04Z + + ICL1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:36:16Z + + RxK +motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T21:13:11Z + + ICL2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:17:06Z + + ER motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:17:12Z + + structures + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:08:50Z + + trimer + + + + ncomms11337-f5.jpg + f5 + FIG + fig_title_caption + 52652 + Channel closures in Mep2. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:17:25Z + + Mep2 + + + + ncomms11337-f5.jpg + f5 + FIG + fig_caption + 52678 + (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of +the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 +that forms a hydrogen bond with His2 in CaMep2. (b) Surface views +from the side in rainbow colouring, showing the two-tier channel block +(indicated by the arrows) in CaMep2. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:18:26Z + + superposition + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T09:42:52Z + + AfAmt-1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T18:56:01Z + + Phe gate + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:47Z + + His2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:37:38Z + + twin-His motif + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:18:29Z + + tyrosine + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:40:31Z + + Y49 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:34:46Z + + TM1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:51Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:47Z + + His2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:35:06Z + + channel block + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + + ncomms11337-f6.jpg + f6 + FIG + fig_title_caption + 53000 + The Npr1 kinase target Ser453 is dephosphorylated and located in an +electronegative pocket. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:19:02Z + + Npr1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:19:05Z + + kinase + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:35:26Z + + Ser453 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:19:07Z + + dephosphorylated + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:08:57Z + + electronegative pocket + + + + ncomms11337-f6.jpg + f6 + FIG + fig_caption + 53092 + (a) Stereoviews of CaMep2 showing 2Fo–Fc +electron density (contoured at 1.0 σ) for CTR residues +Asp419-Met422 and for Tyr446-Thr455 of the AI region. For clarity, the +residues shown are coloured white, with oxygen atoms in red and nitrogen +atoms in blue. The phosphorylation target residue Ser453 is labelled in +bold. (b) Overlay of the CTRs of ScMep2 (grey) and CaMep2 (green), +showing the similar electronegative environment surrounding the +phosphorylation site (P). The AI regions are coloured magenta. (c) +Cytoplasmic view of the Mep2 trimer indicating the large distance between +Ser453 and the channel exits (circles; Ile52 lining the channel exit is +shown). + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:20:42Z + + 2Fo–Fc +electron density + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:20:49Z + + Asp419-Met422 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:20:52Z + + Tyr446-Thr455 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:40Z + + AI region + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + phosphorylation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:35:26Z + + Ser453 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:21:09Z + + Overlay + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:37:12Z + + CTRs + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:51:08Z + + ScMep2 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:22Z + + phosphorylation site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:56Z + + AI regions + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:21:18Z + + Mep2 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:08:50Z + + trimer + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:35:26Z + + Ser453 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:21:23Z + + channel exits + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:21:25Z + + Ile52 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:37:46Z + + channel exit + + + + ncomms11337-f7.jpg + f7 + FIG + fig_title_caption + 53761 + Effect of removal of the AI region on Mep2 structure. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:21:55Z + + removal + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:40Z + + AI region + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:21:58Z + + Mep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:22:00Z + + structure + + + + ncomms11337-f7.jpg + f7 + FIG + fig_caption + 53815 + (a) Side views for WT CaMep2 (left) and the truncation mutant +442Δ (right). The latter is shown as a putty model according to +B-factors to illustrate the disorder in the protein on the cytoplasmic side. +Missing regions are labelled. (b) Stereo superpositions of WT CaMep2 +and the truncation mutant. 2Fo–Fc electron +density (contoured at 1.0 σ) for residues Tyr49 and His342 is +shown for the truncation mutant. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:52Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:27:31Z + + truncation mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:22:48Z + + 442Δ + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:22:51Z + + disorder + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:22:54Z + + superpositions + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:52Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:27:31Z + + truncation mutant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:22:58Z + + 2Fo–Fc electron +density + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:15Z + + Tyr49 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T21:04:42Z + + His342 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T08:27:31Z + + truncation mutant + + + + ncomms11337-f8.jpg + f8 + FIG + fig_title_caption + 54233 + Phosphorylation causes conformational changes in the CTR. + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:01Z + + Phosphorylation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + + ncomms11337-f8.jpg + f8 + FIG + fig_caption + 54291 + (a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2 +superposed in grey for one of the monomers. The arrow indicates the +phosphorylation site. The AI region is coloured magenta. (b) Monomer +side-view superposition of WT CaMep2 and the DD mutant, showing the +conformational change and disorder around the ExxGxD motif. Side chains for +residues 452 and 453 are shown as stick models. + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:01:49Z + + DD mutant + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:08:50Z + + trimer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:52Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:24:27Z + + superposed + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:44:04Z + + monomers + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:22Z + + phosphorylation site + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T08:25:40Z + + AI region + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:28Z + + Monomer + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:24:36Z + + superposition + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:02:52Z + + WT + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:01:49Z + + DD mutant + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:08:08Z + + ExxGxD motif + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:24:40Z + + 452 + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:24:42Z + + 453 + + + + ncomms11337-f9.jpg + f9 + FIG + fig_title_caption + 54681 + Schematic model for phosphorylation-based regulation of Mep2 ammonium +transporters. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:25:03Z + + Mep2 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:25:06Z + + ammonium +transporters + + + + ncomms11337-f9.jpg + f9 + FIG + fig_caption + 54765 + (a) In the closed, non-phosphorylated state (i), the CTR (magenta) and +ICL3 (green) are far apart with the latter blocking the intracellular +channel exit (indicated with a hatched circle). Upon phosphorylation and +mimicked by the CaMep2 S453D and DD mutants (ii), the region around the +ExxGxD motif undergoes a conformational change that results in the CTR +interacting with the inward-moving ICL3, opening the channel (full circle) +(iii). The arrows depict the movements of important structural elements. The +open-channel Mep2 structure is represented by archaebacterial Amt-1 and +shown in lighter colours consistent with Fig. 4. As +discussed in the text, similar structural arrangements may occur in plant +AMTs. In this case however, the open channel corresponds to the +non-phosphorylated state; phosphorylation breaks the CTR–ICL3 +interactions leading to channel closure. (b) Model based on AMT +transporter analogy showing how phosphorylation of a +Mep2 monomer might allosterically open channels in the entire trimer via +disruption of the interactions between the CTR and ICL3 of a neighbouring +monomer (arrow). + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:02Z + + closed + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:39:42Z + + non-phosphorylated + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:37:46Z + + channel exit + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:02Z + + phosphorylation + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:27:54Z + + mimicked + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:50:54Z + + CaMep2 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:08:22Z + + S453D + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:41:10Z + + DD mutants + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-16T09:08:08Z + + ExxGxD motif + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:37:51Z + + channel + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:28:01Z + + channel + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:28:04Z + + Mep2 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:28:07Z + + structure + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:52:15Z + + archaebacterial + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T18:52:24Z + + Amt-1 + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T19:03:15Z + + plant + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T09:49:46Z + + AMTs + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-16T11:28:11Z + + channel + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:39:42Z + + non-phosphorylated + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:02Z + + phosphorylation + + + site + SO: + melaniev@ebi.ac.uk + 2023-06-14T09:41:01Z + + CTR–ICL3 +interactions + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-22T09:53:12Z + + channel + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-16T11:28:14Z + + AMT +transporter + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T17:14:02Z + + phosphorylation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-16T11:28:22Z + + Mep2 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:28Z + + monomer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:37:21Z + + open + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-22T09:53:22Z + + channels + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-16T11:08:50Z + + trimer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:38:07Z + + CTR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T19:01:14Z + + ICL3 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T18:50:28Z + + monomer + + + + diff --git a/BioC_XML/4854314_v0.xml b/BioC_XML/4854314_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..3fead0a81677d8704d0d51b45010799a91de7ef2 --- /dev/null +++ b/BioC_XML/4854314_v0.xml @@ -0,0 +1,5113 @@ + + + + PMC + 20201222 + pmc.key + + 4854314 + CC BY + no + 0 + 0 + + RNA protects a nucleoprotein complex against radiation damage + ACSDAD + 10.1107/S2059798316003351 + S2059798316003351 + 4854314 + 27139628 + rr5121 + 648 + Pt 5 + radiation damage protein–RNA complex electron difference density specific damage decarboxylation + This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits +unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. + 657 + surname:Bury;given-names:Charles S. + surname:McGeehan;given-names:John E. + surname:Antson;given-names:Alfred A. + surname:Carmichael;given-names:Ian + surname:Gerstel;given-names:Markus + surname:Shevtsov;given-names:Mikhail B. + surname:Garman;given-names:Elspeth F. + TITLE + front + 72 + 2016 + 0 + RNA protects a nucleoprotein complex against radiation damage + + 0.9980228 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + complex_assembly + GO: + cleaner0 + 2023-07-05T10:34:01Z + + nucleoprotein + + + + ABSTRACT + abstract + 62 + Systematic analysis of radiation damage within a protein–RNA complex over a large dose range (1.3–25 MGy) reveals significant differential susceptibility of RNA and protein. A new method of difference electron-density quantification is presented. + + complex_assembly + GO: + cleaner0 + 2023-07-05T12:04:56Z + + protein–RNA + + + 0.9902272 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.9882428 + experimental_method + cleaner0 + 2023-07-05T12:16:13Z + MESH: + + difference electron-density quantification + + + + ABSTRACT + abstract + 315 + Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. Even when an eventual model results from the crystallographic pipeline, the manifestations of radiation-induced structural and conformation changes, the so-called specific damage, within crystalline macromolecules can lead to false interpretations of biological mechanisms. Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes. Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3–25.0 MGy) range and at higher resolution (1.98 Å) than the previous systematic specific damage study on a protein–DNA complex. Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein. The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density. + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:16:36Z + + macromolecular X-ray crystallographic data collection + + + experimental_method + MESH: + cleaner0 + 2023-07-05T10:26:50Z + + macromolecular structure determinations + + + 0.93006164 + evidence + cleaner0 + 2023-07-05T10:27:59Z + DUMMY: + + crystals + + + 0.9697803 + evidence + cleaner0 + 2023-07-05T10:28:01Z + DUMMY: + + per-atom density changes + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:36:34Z + + DNA + + + 0.9563537 + protein_type + cleaner0 + 2023-07-05T12:26:59Z + MESH: + + trp RNA-binding attenuation protein + + + 0.53643787 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.99148065 + protein_state + cleaner0 + 2023-07-05T12:33:37Z + DUMMY: + + bound to + + + 0.9971852 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.99032986 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.937143 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.9731102 + protein_state + cleaner0 + 2023-07-05T10:43:39Z + DUMMY: + + bound + + + 0.99720335 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:26:21Z + + RNA + + + 0.8899272 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + structure_element + SO: + cleaner0 + 2023-07-05T11:58:12Z + + ring + + + 0.9860465 + residue_name + cleaner0 + 2023-07-05T10:28:06Z + SO: + + Glu + + + 0.9869811 + residue_name + cleaner0 + 2023-07-05T10:28:09Z + SO: + + Asp + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:26:21Z + + RNA + + + 0.9977442 + site + cleaner0 + 2023-07-05T12:01:06Z + SO: + + RNA-binding pockets + + + 0.9823989 + residue_name + cleaner0 + 2023-07-05T10:28:11Z + SO: + + Lys + + + 0.9763282 + residue_name + cleaner0 + 2023-07-05T10:28:13Z + SO: + + Phe + + + 0.652342 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.9961912 + evidence + cleaner0 + 2023-07-05T10:28:04Z + DUMMY: + + electron density + + + + INTRO + title_1 + 2173 + Introduction   + + + INTRO + paragraph + 2190 + With the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100 K). Significant progress has been made in recent years in understanding the inevitable manifestations of X-ray-induced RD within protein crystals, and there is now a body of literature on possible strategies to mitigate the effects of RD (e.g. Zeldin, Brockhauser et al., 2013; Bourenkov & Popov, 2010). However, there is still no general consensus within the field on how to minimize RD during MX data collection, and debates on the dependence of RD progression on incident X-ray energy (Shimizu et al., 2007; Liebschner et al., 2015) and the efficacy of radical scavengers (Allan et al., 2013) have yet to be resolved. + + 0.8348757 + experimental_method + cleaner0 + 2023-07-05T10:28:23Z + MESH: + + structure determination + + + 0.98041725 + experimental_method + cleaner0 + 2023-07-05T10:28:25Z + MESH: + + macromolecular crystallography + + + 0.56438595 + experimental_method + cleaner0 + 2023-07-05T10:28:29Z + MESH: + + MX + + + 0.9865131 + evidence + cleaner0 + 2023-07-05T10:28:34Z + DUMMY: + + crystals + + + 0.9509965 + experimental_method + cleaner0 + 2023-07-05T10:28:30Z + MESH: + + MX + + + + INTRO + paragraph + 3116 + RD manifests in two forms. Global radiation damage is observed within reciprocal space as the overall decay of the summed intensity of reflections detected within the diffraction pattern as dose increases (Garman, 2010; Murray & Garman, 2002). Dose is defined as the absorbed energy per unit mass of crystal in grays (Gy; 1 Gy = 1 J kg−1), and is the metric against which damage progression should be monitored during MX data collection, as opposed to time. At 100 K, an experimental dose limit of 30 MGy has been recommended as an upper limit beyond which the biological information derived from any macromolecular crystal may be compromised (Owen et al., 2006). + + 0.9363032 + evidence + cleaner0 + 2023-07-05T12:19:18Z + DUMMY: + + diffraction pattern + + + 0.96844155 + experimental_method + cleaner0 + 2023-07-05T10:28:30Z + MESH: + + MX + + + 0.91692525 + evidence + cleaner0 + 2023-07-05T10:31:01Z + DUMMY: + + crystal + + + + INTRO + paragraph + 3792 + Specific radiation damage (SRD) is observed in the real-space electron density, and has been detected at much lower doses than any observable decay in the intensity of reflections. Indeed, the C—Se bond in selenomethionine, the stability of which is key for the success of experimental phasing methods, can be cleaved at a dose as low as 2 MGy for a crystal maintained at 100 K (Holton, 2007). SRD has been well characterized in a large range of proteins, and is seen to follow a reproducible order: metallo-centre reduction, disulfide-bond cleavage, acidic residue decarboxylation and methionine methylthio cleavage (Ravelli & McSweeney, 2000; Burmeister, 2000; Weik et al., 2000; Yano et al., 2005). Furthermore, damage susceptibility within each residue type follows a preferential ordering influenced by a combination of local environment factors (solvent accessibility, conformational strain, proximity to active sites/high X-ray cross-section atoms; Holton, 2009). Deconvoluting the individual roles of these parameters has been surprisingly challenging, with factors such as solvent accessibility currently under active investigation (Weik et al., 2000; Fioravanti et al., 2007; Gerstel et al., 2015). + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:19:00Z + + Specific radiation damage + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:19:10Z + + SRD + + + 0.9674265 + evidence + cleaner0 + 2023-07-05T10:31:05Z + DUMMY: + + real-space electron density + + + 0.28211054 + chemical + cleaner0 + 2023-07-05T10:29:47Z + CHEBI: + + Se + + + 0.9843067 + chemical + cleaner0 + 2023-07-05T10:29:51Z + CHEBI: + + selenomethionine + + + 0.9706023 + evidence + cleaner0 + 2023-07-05T10:31:07Z + DUMMY: + + crystal + + + ptm + MESH: + cleaner0 + 2023-07-05T10:30:47Z + + disulfide-bond + + + + INTRO + paragraph + 5008 + There are a number of cases where SRD manifestations have compromised the biological information extracted from MX-determined structures at much lower doses than the recommended 30 MGy limit, leading to false structural interpretations of protein mechanisms. Active-site residues appear to be particularly susceptible, particularly for photosensitive proteins and in instances where chemical strain is an intrinsic feature of the reaction mechanism. For instance, structure determination of the purple membrane protein bacterio­rhodopsin required careful corrections for radiation-induced structural changes before the correct photosensitive intermediate states could be isolated (Matsui et al., 2002). The significant chemical strain required for catalysis within the active site of phosphoserine aminotransferase has been observed to diminish during X-ray exposure (Dubnovitsky et al., 2005). + + 0.9724948 + experimental_method + cleaner0 + 2023-07-05T10:31:22Z + MESH: + + MX-determined + + + 0.9942268 + evidence + cleaner0 + 2023-07-05T12:19:53Z + DUMMY: + + structures + + + 0.9978055 + site + cleaner0 + 2023-07-05T12:01:17Z + SO: + + Active-site residues + + + 0.99299383 + experimental_method + cleaner0 + 2023-07-05T10:31:25Z + MESH: + + structure determination + + + 0.84919345 + protein_type + cleaner0 + 2023-07-05T10:31:44Z + MESH: + + bacterio­rhodopsin + + + 0.99806416 + site + cleaner0 + 2023-07-05T12:01:21Z + SO: + + active site + + + 0.9870368 + protein_type + cleaner0 + 2023-07-05T10:31:34Z + MESH: + + phosphoserine aminotransferase + + + + INTRO + paragraph + 5906 + Since the majority of SRD studies to date have focused on proteins, much less is known about the effects of X-ray irradiation on the wider class of crystalline nucleoprotein complexes or how to correct for such radiation-induced structural changes. Understanding RD to such complexes is crucial, since DNA is rarely naked within a cell, instead dynamically interacting with proteins, facilitating replication, transcription, modification and DNA repair. As of early 2016, >5400 nucleoprotein complex structures have been deposited within the PDB, with 91% solved by MX. It is essential to understand how these increasingly complex macromolecular structures are affected by the radiation used to solve them. Nucleoproteins also represent one of the main targets of radiotherapy, and an insight into the damage mechanisms induced by X-ray irradiation could inform innovative treatments. + + experimental_method + MESH: + cleaner0 + 2023-07-05T10:32:31Z + + SRD studies + + + 0.5266972 + complex_assembly + cleaner0 + 2023-07-05T11:49:23Z + GO: + + nucleoprotein + + + 0.6261449 + chemical + cleaner0 + 2023-07-05T10:36:34Z + CHEBI: + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:36:34Z + + DNA + + + complex_assembly + GO: + cleaner0 + 2023-07-05T10:33:26Z + + nucleoprotein + + + 0.9936459 + evidence + cleaner0 + 2023-07-05T12:19:56Z + DUMMY: + + structures + + + 0.9480319 + experimental_method + cleaner0 + 2023-07-05T10:28:30Z + MESH: + + MX + + + 0.9201258 + evidence + cleaner0 + 2023-07-05T12:19:59Z + DUMMY: + + structures + + + 0.78504515 + complex_assembly + cleaner0 + 2023-07-05T10:33:41Z + GO: + + Nucleoproteins + + + + INTRO + paragraph + 6791 + When a typical macromolecular crystal is irradiated with ionizing X-rays, each photoelectron produced via interactions with both the macromolecule (direct damage) and solvent (indirect damage) can induce cascades of up to 500 secondary low-energy electrons (LEEs) that are capable of inducing further ionizations. Investigations on sub-ionization-level LEEs (0–15 eV) interacting with both dried and aqueous oligonucleotides (Alizadeh & Sanche, 2014; Simons, 2006) concluded that resonant electron attachment to DNA bases and the sugar-phosphate backbone could lead to the preferential cleavage of strong (∼4 eV, 385 kJ mol−1) sugar-phosphate C—O covalent bonds within the DNA backbone and then base-sugar N1—C bonds, eventually leading to single-strand breakages (SSBs; Ptasińska & Sanche, 2007). Electrons have been shown to be mobile at 77 K by electron spin resonance spectroscopy studies (Symons, 1997; Jones et al., 1987), with rapid electron quantum tunnelling and positive hole migration along the protein backbone and through stacked DNA bases indicated as a dominant mechanism by which oxidative and reductive damage localizes at distances from initial ionization sites at 100 K (O’Neill et al., 2002). + + 0.9642742 + chemical + cleaner0 + 2023-07-05T10:36:34Z + CHEBI: + + DNA + + + 0.94849676 + chemical + cleaner0 + 2023-07-05T10:36:34Z + CHEBI: + + DNA + + + 0.99424225 + experimental_method + cleaner0 + 2023-07-05T10:34:36Z + MESH: + + electron spin resonance spectroscopy + + + 0.8569334 + chemical + cleaner0 + 2023-07-05T10:36:34Z + CHEBI: + + DNA + + + 0.97740066 + site + cleaner0 + 2023-07-05T12:01:27Z + SO: + + ionization sites + + + + INTRO + paragraph + 8029 + The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein–DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8 Å; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1–44.6 MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015). Only at doses above 20 MGy were precursors of phosphodiester-bond cleavage observed within AT-rich regions of the 35-mer DNA. + + 0.92757994 + experimental_method + cleaner0 + 2023-07-05T10:35:56Z + MESH: + + crystallized + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-05T10:35:49Z + + bacterial + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:36:34Z + + DNA + + + 0.99454117 + complex_assembly + cleaner0 + 2023-07-05T12:31:03Z + GO: + + C.Esp1396I + + + 0.99493873 + chemical + cleaner0 + 2023-07-05T10:36:33Z + CHEBI: + + DNA + + + 0.9964142 + evidence + cleaner0 + 2023-07-05T12:20:04Z + DUMMY: + + crystal + + + structure_element + SO: + cleaner0 + 2023-07-05T10:36:28Z + + AT-rich regions + + + 0.9971282 + chemical + cleaner0 + 2023-07-05T10:36:34Z + CHEBI: + + DNA + + + + INTRO + paragraph + 8670 + For crystalline complexes such as C.Esp1396I, whether the protein is intrinsically more susceptible to X-ray-induced damage or whether the protein scavenges electrons to protect the DNA remains unclear in the absence of a non-nucleic acid-bound protein control obtained under exactly the same crystallization and data-collection conditions. To monitor the effects of nucleic acid binding on protein damage susceptibility, a crystal containing two protein molecules per asymmetric unit, only one of which was bound to RNA, is reported here (Fig. 1 ▸). Using newly developed methodology, we present a controlled SRD investigation at 1.98 Å resolution using a large (∼91 kDa) crystalline protein–RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53 bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002). TRAP consists of 11 identical subunits assembled into a ring with 11-fold rotational symmetry. It binds with high affinity (K d ≃ 1.0 nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999). In this structure, the bases of the G1-A2-G3 nucleotides form direct hydrogen bonds to the protein, unlike the U4-U5 nucleotides, which appear to be more flexible. + + 0.99455875 + complex_assembly + cleaner0 + 2023-07-05T12:31:03Z + GO: + + C.Esp1396I + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:36:34Z + + DNA + + + 0.9853733 + evidence + cleaner0 + 2023-07-05T12:20:08Z + DUMMY: + + crystal + + + 0.98737174 + protein_state + cleaner0 + 2023-07-05T12:33:43Z + DUMMY: + + bound to + + + 0.9977902 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.9718507 + experimental_method + cleaner0 + 2023-07-05T12:16:48Z + MESH: + + controlled SRD + + + complex_assembly + GO: + cleaner0 + 2023-07-05T12:04:56Z + + protein–RNA + + + 0.9377823 + protein_type + cleaner0 + 2023-07-05T12:27:03Z + MESH: + + trp RNA-binding attenuation protein + + + 0.8530336 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.9948477 + protein_state + cleaner0 + 2023-07-05T11:51:21Z + DUMMY: + + bound to + + + 0.99753857 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:38:29Z + + (GAGUU)10GAG + + + 0.9524646 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + structure_element + SO: + cleaner0 + 2023-07-05T12:15:54Z + + subunits + + + 0.5846764 + structure_element + cleaner0 + 2023-07-05T11:58:12Z + SO: + + ring + + + 0.9860402 + evidence + cleaner0 + 2023-07-05T12:20:10Z + DUMMY: + + K d + + + 0.9952761 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.9207858 + structure_element + cleaner0 + 2023-07-05T10:47:16Z + SO: + + GAG/UAG triplets + + + structure_element + SO: + cleaner0 + 2023-07-05T11:54:57Z + + spacer nucleotides + + + 0.90775585 + chemical + cleaner0 + 2023-07-05T12:14:09Z + CHEBI: + + tryptophan + + + 0.99550605 + species + cleaner0 + 2023-07-05T10:37:26Z + MESH: + + Bacillus subtilis + + + 0.9971539 + evidence + cleaner0 + 2023-07-05T12:20:16Z + DUMMY: + + structure + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:39:38Z + + G1-A2-G3 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + hydrogen bonds + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:39:55Z + + U4-U5 + + + + INTRO + paragraph + 10044 + Ten successive 1.98 Å resolution MX data sets were collected from the same TRAP–RNA crystal to analyse X-ray-induced structural changes over a large dose range (d 1 = 1.3 MGy to d 10 = 25.0 MGy). To avoid the previous necessity for visual inspection of electron-density maps to detect SRD sites, a computational approach was designed to quantify the electron-density change for each refined atom with increasing dose, thus providing a rapid systematic method for SRD study on such large multimeric complexes. By employing the high 11-fold structural symmetry within each TRAP macromolecule, this approach permitted a thorough statistical quantification of the RD effects of RNA binding to TRAP. + + 0.947729 + experimental_method + cleaner0 + 2023-07-05T10:28:30Z + MESH: + + MX + + + 0.98999834 + complex_assembly + cleaner0 + 2023-07-05T10:38:43Z + GO: + + TRAP–RNA + + + 0.9876061 + evidence + cleaner0 + 2023-07-05T10:40:23Z + DUMMY: + + crystal + + + 0.99650353 + evidence + cleaner0 + 2023-07-05T10:40:25Z + DUMMY: + + electron-density maps + + + 0.99333715 + site + cleaner0 + 2023-07-05T12:01:33Z + SO: + + SRD sites + + + 0.9945757 + evidence + cleaner0 + 2023-07-05T10:40:28Z + DUMMY: + + electron-density change + + + 0.9496573 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.99639255 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.9609993 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + + METHODS + title_1 + 10748 + Materials and methods   + + + METHODS + title_2 + 10774 + RNA synthesis and protein preparation   + + + METHODS + paragraph + 10816 + As previously described (Hopcroft et al., 2002), the 53-base RNA (GAGUU)10GAG was synthesized by in vitro transcription with T7 RNA polymerase and gel-purified. TRAP from B. stearothermophilus was overexpressed in Escherichia coli and purified. + + + METHODS + title_2 + 11061 + Crystallization   + + + METHODS + paragraph + 11081 + TRAP–RNA crystals were prepared using a previously established hanging-drop crystallization protocol (Antson et al., 1999). By using a 2:1 molar ratio of TRAP to RNA, crystals successfully formed from the protein–RNA complex (∼15 mg ml−1) in a solution containing 70 mM potassium phosphate pH 7.8 and 10 mM l-tryptophan. The reservoir consisted of 0.2 M potassium glutamate, 50 mM triethanol­amine pH 8.0, 10 mM MgCl2, 8–11% monomethyl ether PEG 2000. In order to accelerate crystallization, a further gradient was induced by adding 0.4 M KCl to the reservoir after 1.5 µl protein solution had been mixed with an equal volume of the reservoir solution. Wedge-shaped crystals of approximate length 70 µm (longest dimension) grew within 3 d and were vitrified and stored in liquid nitrogen immediately after growth. The cryosolution consisted of 12% monomethyl ether PEG 2000, 30 mM triethanolamine pH 8.0, 6 mM l-tryptophan, 0.1 M potassium glutamate, 35 mM potassium phosphate pH 7.8, 5 mM MgCl2 with 25% 2-methyl-2,4-pentanediol (MPD) included as a cryoprotectant. + + + METHODS + title_2 + 12192 + X-ray data collection   + + + METHODS + paragraph + 12218 + Data were collected at 100 K from a wedge-shaped TRAP–RNA crystal of approximate dimensions 70 × 20 × 40 µm (see Supplementary Fig. S2) on beamline ID14-4 at the ESRF using an incident wavelength of 0.940 Å (13.2 keV) and an ADSC Q315R mosaic CCD detector at 304.5 mm from the crystal throughout the data collection. The beam size was slitted to 0.100 mm (vertical) × 0.160 mm (horizontal), with a uniformly distributed profile, such that the crystal was completely bathed within the beam throughout data collection. Ten successive (1.98 Å resolution) 180° data sets (with Δφ = 1°) were collected over the same angular range from a TRAP–RNA crystal at 28.9% beam transmission. The TRAP–RNA macromolecule crystallized in space group C2, with unit-cell parameters a = 140.9, b = 110.9, c = 137.8 Å, α = γ = 90, β = 137.8° (the values quoted are for the first data set; see Supplementary Table S1 for subsequent values). For the first nine data sets the attenuated flux was recorded to be ∼5 × 1011 photons s−1. A beam refill took place immediately before data set 10, requiring a flux-scale factor increase of 1.42 to be applied, based on the ratio of observed relative intensity I D/I 1 at data set 10 to that extrapolated from data set 9. + + + METHODS + title_2 + 13507 + Dose calculation   + + + METHODS + paragraph + 13528 + RADDOSE-3D (Zeldin, Gerstel et al., 2013) was used to calculate the absorbed dose distribution during each data set (see input file; Supplementary Figs. S1 and S2). The crystal composition was calculated from the deposited TRAP–RNA structure (PDB entry 1gtf; Hopcroft et al., 2002). Crystal absorption coefficients were calculated in RADDOSE-3D using the concentration (mmol l−1) of solvent heavy elements from the crystallization conditions. The beam-intensity profile was modelled as a uniform (‘top-hat’) distribution. The diffraction-weighted dose (DWD) values (Zeldin, Brock­hauser et al., 2013) are given in Supplementary Table S1. + + + METHODS + title_2 + 14178 + Data processing and model refinement   + + + METHODS + paragraph + 14219 + Each data set was integrated using iMosflm (Leslie & Powell, 2007) and was scaled using AIMLESS (Evans & Murshudov, 2013; Winn et al., 2011) using the same 5% R free set of test reflections for each data set. To phase the structure obtained from the first data set, molecular replacement was carried out with Phaser (McCoy et al., 2007), using an identical TRAP–RNA structure (PDB entry 1gtf; resolution 1.75 Å; Hopcroft et al., 2002) as a search model. The resulting TRAP–RNA structure (TR1) was refined using REFMAC5 (Murshudov et al., 2011), initially using rigid-body refinement, followed by repeated cycles of restrained, TLS and isotropic B-factor refinement, coupled with visual inspection in Coot (Emsley et al., 2010). TR1 was refined to 1.98 Å resolution, with a dimeric assembly of non-RNA-bound and RNA-bound TRAP rings within the asymmetric unit. Consistent with previous structures of the TRAP–RNA complex, the RNA sequence termini were not observed within the 2F o − F c map; the first spacer (U4) was then modelled at all 11 repeats around the TRAP ring and the second spacer (U5) was omitted from the final refined structure. For the later data sets, the observed structure-factor amplitudes from each separately scaled data set (output from AIMLESS) were combined with the phases of TR1 and the resulting higher-dose model was refined with phenix.refine (Adams et al., 2010) using only rigid-body and isotropic B-factor refinement. During this refinement, the TRAP–RNA complex and nonbound TRAP ring were treated as two separate rigid bodies within the asymmetric unit. Supplementary Table S1 shows the relevant summary statistics. + + + METHODS + title_2 + 15885 + D loss metric calculation   + + + METHODS + paragraph + 15916 + The CCP4 program CAD was used to create a series of nine merged .mtz files combining observed structure-factor amplitudes for the first data set F obs(d 1) with each later data set F obs(d n) (for n = 2, …, 10). All later data sets were scaled against the initial low-dose data set in SCALEIT. For each data set an atom-tagged .map file was generated using the ATMMAP mode in SFALL (Winn et al., 2011). A full set of nine Fourier difference maps F obs(d n) − F obs(d 1) were calculated using FFT (Ten Eyck, 1973) over the full TRAP–RNA unit-cell dimensions, with the same grid-sampling dimensions as the atom-tagged .map file. All maps were cropped to the TRAP asymmetric unit in MAPMASK. Comparing the atom-tagged .map file and F obs(d n) − F obs(d 1) difference map at each dose, each refined atom was assigned a set of density-change values X. The maximum density-loss metric, D loss (units of e Å−3), was calculated to quantify the per-atom electron-density decay at each dose, assigned as the absolute magnitude of the most negative Fourier difference map voxel value in a local volume around each atom as defined by the set X. + + + METHODS + title_2 + 17062 + Model system calculation   + + + METHODS + paragraph + 17091 + Model calculations were run for the simple amino acids glutamate and aspartate. In order to avoid decarboxylation at the C-terminus instead of the side chain on the Cα atom, the C-terminus of each amino acid was methylated. While the structures of the closed shell acids are well known, the same is not true of those in the oxidized state. The quantum-chemical calculations employed were chosen to provide a satisfactory description of the structure of such radical species and also provide a reliable estimation of the relative C—C(O2) bond strengths, which are otherwise not available. + + + METHODS + paragraph + 17683 + Structures of methyl-terminated (at the N- and C-termini) carboxylates were determined using analytic energy gradients with density functional theory (B3LYP functional; Becke, 1993) and a flexible basis set of polarized valence triple-zeta size with diffuse functions on the non-H atoms [6-311+G(d,p)] in the Gaussian 09 computational chemistry package (Frisch et al., 2009). The stationary points obtained were characterized as at least local minima by examination of the associated analytic Hessian. Effects of the medium were modelled using a dielectric cavity approach (Tomasi et al., 1999) parameterized for water. + + + RESULTS + title_1 + 18303 + Results   + + + RESULTS + title_2 + 18315 + Per-atom quantification of electron density   + + experimental_method + MESH: + cleaner0 + 2023-07-05T11:50:30Z + + Per-atom quantification of electron density + + + + RESULTS + paragraph + 18363 + To quantify the exact effects of nucleic acid binding to a protein on SRD susceptibility, a high-throughput and automated pipeline was created to systematically calculate the electron-density change for every refined atom within the TRAP–RNA structure as a function of dose. This provides an atom-specific quantification of density–dose dynamics, which was previously lacking within the field. Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) − F obs(d 1) Fourier difference map peaks in terms of the sigma (σ) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible. However, these σ levels depend on the standard deviation values of the map, which can deviate between data sets, and are thus unsuitable for quantitative comparison of density between different dose data sets. Instead, we use here a maximum density-loss metric (D loss), which is the per-atom equivalent of the magnitude of these negative Fourier difference map peaks in units of e Å−3. Large positive D loss values indicate radiation-induced atomic disordering reproducibly throughout the unit cells with respect to the initial low-dose data set. + + 0.993571 + evidence + cleaner0 + 2023-07-05T12:20:24Z + DUMMY: + + electron-density change + + + 0.9888622 + complex_assembly + cleaner0 + 2023-07-05T10:38:43Z + GO: + + TRAP–RNA + + + 0.9903577 + evidence + cleaner0 + 2023-07-05T12:20:28Z + DUMMY: + + structure + + + 0.82108414 + evidence + cleaner0 + 2023-07-05T12:20:32Z + DUMMY: + + density–dose dynamics + + + 0.9954945 + site + cleaner0 + 2023-07-05T12:01:39Z + SO: + + SRD sites + + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:01:56Z + + F obs(d n) − F obs(d 1) Fourier difference map peaks + + + 0.88435054 + evidence + cleaner0 + 2023-07-05T12:20:37Z + DUMMY: + + sigma + + + 0.58001226 + evidence + cleaner0 + 2023-07-05T12:20:40Z + DUMMY: + + σ + + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:03:31Z + + standard deviations + + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:03:02Z + + mean map electron-density value + + + 0.7078795 + evidence + cleaner0 + 2023-07-05T12:20:44Z + DUMMY: + + σ + + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:03:19Z + + standard deviation + + + 0.9917287 + evidence + cleaner0 + 2023-07-05T12:20:48Z + DUMMY: + + map + + + 0.6958162 + evidence + cleaner0 + 2023-07-05T12:20:51Z + DUMMY: + + density + + + 0.9909112 + evidence + cleaner0 + 2023-07-05T12:20:55Z + DUMMY: + + maximum density-loss metric + + + 0.97732365 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + 0.9871848 + evidence + cleaner0 + 2023-07-05T12:21:00Z + DUMMY: + + negative Fourier difference map peaks + + + 0.98676395 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + + RESULTS + paragraph + 19592 + For each TRAP–RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 ▸ a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9 MGy; Fig. 3 ▸ a). The main sequence of TRAP does not contain any Trp and Cys residues (and thus contains no disulfide bonds). The substrate Trp amino-acid ligands also exhibited disordering of the free terminal carboxyl groups at higher doses (Fig. 2 ▸ a); however, no clear Fourier difference peaks could be observed visually. Even for radiation-insensitive residues (e.g. Gly) the average D loss increases with dose: this is the effect of global radiation damage, since as dose increases the electron density associated with each refined atom becomes weaker as the atomic occupancy decreases (Fig. 2 ▸ b). Only Glu and Asp residues exhibit a rate of D loss increase that consistently exceeds the average decay (Fig. 2 ▸ b, dashed line) at each dose. Additionally, the density surrounding ordered solvent molecules was determined to significantly diminish with increasing dose (Fig. 2 ▸ b). The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts. + + 0.96854377 + complex_assembly + cleaner0 + 2023-07-05T10:38:43Z + GO: + + TRAP–RNA + + + 0.9839508 + evidence + cleaner0 + 2023-07-05T12:21:04Z + DUMMY: + + D loss metric + + + 0.98396283 + experimental_method + cleaner0 + 2023-07-05T12:27:21Z + MESH: + + SRD + + + 0.9861578 + residue_name + cleaner0 + 2023-07-05T12:07:07Z + SO: + + Glu + + + 0.98857224 + residue_name + cleaner0 + 2023-07-05T10:42:18Z + SO: + + Asp + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-07-06T15:26:33Z + + difference map + + + 0.91976935 + complex_assembly + cleaner0 + 2023-07-05T12:07:53Z + GO: + + TRAP + + + 0.9860378 + residue_name + cleaner0 + 2023-07-05T10:42:35Z + SO: + + Trp + + + 0.99085087 + residue_name + cleaner0 + 2023-07-05T10:42:39Z + SO: + + Cys + + + 0.9699916 + chemical + cleaner0 + 2023-07-05T12:14:14Z + CHEBI: + + Trp + + + 0.9950683 + evidence + cleaner0 + 2023-07-05T12:21:30Z + DUMMY: + + Fourier difference peaks + + + 0.97884846 + residue_name + cleaner0 + 2023-07-05T10:42:43Z + SO: + + Gly + + + 0.9714825 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + 0.99046993 + evidence + cleaner0 + 2023-07-05T12:21:33Z + DUMMY: + + electron density + + + 0.98477256 + residue_name + cleaner0 + 2023-07-05T12:07:11Z + SO: + + Glu + + + 0.98784196 + residue_name + cleaner0 + 2023-07-05T10:42:19Z + SO: + + Asp + + + 0.7084436 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + evidence + DUMMY: + cleaner0 + 2023-07-05T10:43:57Z + + D loss + + + 0.9841786 + residue_name + cleaner0 + 2023-07-05T12:07:17Z + SO: + + Glu + + + 0.9849412 + residue_name + cleaner0 + 2023-07-05T10:42:19Z + SO: + + Asp + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + hydrogen-bonding + + + 0.993453 + chemical + cleaner0 + 2023-07-05T12:14:18Z + CHEBI: + + CO2 + + + 0.9969453 + protein_state + cleaner0 + 2023-07-05T12:31:56Z + DUMMY: + + oxidized + + + 0.98660594 + residue_name + cleaner0 + 2023-07-05T12:07:19Z + SO: + + Glu + + + 0.98063844 + residue_name + cleaner0 + 2023-07-05T10:42:19Z + SO: + + Asp + + + + RESULTS + title_2 + 21189 + RNA is less susceptible to electron-density loss than protein within the TRAP–RNA complex   + + 0.99770075 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.83079296 + evidence + cleaner0 + 2023-07-05T10:43:12Z + DUMMY: + + electron-density + + + 0.9937124 + complex_assembly + cleaner0 + 2023-07-05T10:38:43Z + GO: + + TRAP–RNA + + + + RESULTS + paragraph + 21285 + Visual inspection of Fourier difference maps illustrated the clear lack of RNA electron-density degradation with increasing dose compared with the obvious protein damage manifestations (Figs. 3 ▸ b and 3 ▸ c). Only at the highest doses investigated (>20 MGy) was density loss observed at the RNA phosphate and C—O bonds of the phosphodiester backbone. However, the median D loss was lower by a factor of >2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0 MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly Cα atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3). + + 0.9262293 + experimental_method + cleaner0 + 2023-07-05T12:16:54Z + MESH: + + Visual inspection of + + + 0.9843981 + evidence + cleaner0 + 2023-07-05T10:43:15Z + DUMMY: + + Fourier difference maps + + + 0.99798906 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.8665679 + evidence + cleaner0 + 2023-07-05T10:43:17Z + DUMMY: + + electron-density degradation + + + 0.9983839 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.88909703 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + 0.9981768 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.9861231 + residue_name + cleaner0 + 2023-07-05T12:07:24Z + SO: + + Glu + + + 0.9858482 + residue_name + cleaner0 + 2023-07-05T10:42:19Z + SO: + + Asp + + + 0.9861166 + residue_name + cleaner0 + 2023-07-05T10:42:44Z + SO: + + Gly + + + 0.97520036 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + + RESULTS + title_2 + 21965 + RNA binding protects radiation-sensitive residues   + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:26:21Z + + RNA + + + + RESULTS + paragraph + 22019 + For the large number of acidic residues per TRAP ring (four Asp and six Glu residues per protein monomer), a strong dependence of decarboxylation susceptibility on local environment was observed (Fig. 4 ▸). For each Glu Cδ or Asp Cγ atom, D loss provided a direct measure of the rate of side-chain carboxyl-group disordering and subsequent decarboxylation. For acidic residues with no differing interactions between nonbound and bound TRAP (Fig. 4 ▸ a), similar damage was apparent between the two rings within the asymmetric unit, as expected. However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 ▸ b), and for residues at the ring–ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 ▸ c). + + 0.9912158 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + structure_element + SO: + cleaner0 + 2023-07-05T11:58:12Z + + ring + + + 0.989652 + residue_name + cleaner0 + 2023-07-05T10:42:19Z + SO: + + Asp + + + 0.98959446 + residue_name + cleaner0 + 2023-07-05T12:34:05Z + SO: + + Glu + + + 0.9787446 + oligomeric_state + cleaner0 + 2023-07-05T10:43:50Z + DUMMY: + + monomer + + + 0.9698287 + residue_name + cleaner0 + 2023-07-05T12:34:09Z + SO: + + Glu + + + 0.95125747 + residue_name + cleaner0 + 2023-07-05T10:42:19Z + SO: + + Asp + + + evidence + DUMMY: + cleaner0 + 2023-07-05T10:43:57Z + + D loss + + + 0.9959565 + protein_state + cleaner0 + 2023-07-05T10:44:09Z + DUMMY: + + nonbound + + + 0.9911316 + protein_state + cleaner0 + 2023-07-05T10:43:39Z + DUMMY: + + bound + + + 0.99624735 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.8192733 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.99758184 + site + cleaner0 + 2023-07-05T10:43:27Z + SO: + + RNA-binding interfaces + + + 0.9959115 + protein_state + cleaner0 + 2023-07-05T10:44:10Z + DUMMY: + + nonbound + + + 0.99391973 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.9969743 + site + cleaner0 + 2023-07-05T10:43:29Z + SO: + + ring–ring interfaces + + + 0.9965004 + protein_state + cleaner0 + 2023-07-05T10:43:39Z + DUMMY: + + bound + + + 0.9917121 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + + RESULTS + paragraph + 22856 + Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. Hotelling’s T-squared test (the multivariate counterpart of Student’s t-test) was used to reject the null hypothesis that the means of the D loss metric were equal for the bound and nonbound groups in Fig. 5 ▸. + + 0.99910825 + residue_name_number + cleaner0 + 2023-07-05T10:44:41Z + DUMMY: + + Glu36 + + + 0.99910176 + residue_name_number + cleaner0 + 2023-07-05T10:44:46Z + DUMMY: + + Asp39 + + + 0.99909663 + residue_name_number + cleaner0 + 2023-07-05T10:44:50Z + DUMMY: + + Glu42 + + + 0.9915868 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.7789828 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.4135374 + structure_element + cleaner0 + 2023-07-05T11:58:12Z + SO: + + ring + + + structure_element + SO: + cleaner0 + 2023-07-05T12:15:54Z + + subunits + + + 0.9754962 + evidence + cleaner0 + 2023-07-05T12:21:41Z + DUMMY: + + density changes + + + 0.9385281 + experimental_method + cleaner0 + 2023-07-05T10:44:16Z + MESH: + + Hotelling’s T-squared test + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:17:44Z + + Student’s t-test + + + 0.9535044 + evidence + cleaner0 + 2023-07-05T12:21:49Z + DUMMY: + + D loss metric + + + 0.99416935 + protein_state + cleaner0 + 2023-07-05T10:43:39Z + DUMMY: + + bound + + + 0.98500234 + protein_state + cleaner0 + 2023-07-05T10:44:10Z + DUMMY: + + nonbound + + + + RESULTS + paragraph + 23260 + A significant reduction in D loss is seen for Glu36 in RNA-bound compared with nonbound TRAP, indicative of a lower rate of side-chain decarboxylation (Fig. 5 ▸ a; p = 6.06 × 10−5). For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base. In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). + + 0.95402676 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + 0.9990717 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + 0.99607676 + protein_state + cleaner0 + 2023-07-05T12:32:01Z + DUMMY: + + RNA-bound + + + 0.9939761 + protein_state + cleaner0 + 2023-07-05T10:44:10Z + DUMMY: + + nonbound + + + 0.94174945 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + complex_assembly + GO: + cleaner0 + 2023-07-05T12:07:58Z + + TRAP + + + structure_element + SO: + cleaner0 + 2023-07-05T11:58:12Z + + ring + + + structure_element + SO: + cleaner0 + 2023-07-05T12:15:42Z + + subunit + + + 0.9990544 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + hydrogen bonds + + + 0.98224247 + residue_name_number + cleaner0 + 2023-07-05T10:52:11Z + DUMMY: + + G3 + + + 0.996082 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.99904436 + residue_name_number + cleaner0 + 2023-07-05T10:44:46Z + DUMMY: + + Asp39 + + + complex_assembly + GO: + cleaner0 + 2023-07-05T12:00:19Z + + TRAP–(GAGUU)10GAG + + + 0.9971259 + evidence + cleaner0 + 2023-07-05T12:21:52Z + DUMMY: + + structure + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + hydrogen bonds + + + 0.43124557 + residue_name_number + cleaner0 + 2023-07-05T10:52:24Z + DUMMY: + + G1 + + + site + SO: + cleaner0 + 2023-07-05T10:45:21Z + + TRAP–RNA interfaces + + + 0.99898416 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + 0.5508266 + residue_name_number + cleaner0 + 2023-07-05T10:52:11Z + DUMMY: + + G3 + + + 0.8253004 + evidence + cleaner0 + 2023-07-05T12:21:57Z + DUMMY: + + density + + + 0.59934 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.9990036 + residue_name_number + cleaner0 + 2023-07-05T10:44:46Z + DUMMY: + + Asp39 + + + 0.99900705 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + + RESULTS + title_2 + 23913 + RNA binding reduces radiation-induced disorder on the atomic scale   + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:26:21Z + + RNA + + + + RESULTS + paragraph + 23984 + One oxygen (O∊1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O∊2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999). Salt-bridge interactions have previously been suggested to reduce the glutamate decarboxylation rate within the large (∼62.4 kDa) myrosinase protein structure (Burmeister, 2000). A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate. + + 0.9987974 + residue_name_number + cleaner0 + 2023-07-05T10:44:50Z + DUMMY: + + Glu42 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + hydrogen bond + + + 0.9982838 + chemical + cleaner0 + 2023-07-05T12:14:22Z + CHEBI: + + water + + + site + SO: + cleaner0 + 2023-07-05T10:45:45Z + + TRAP RNA-binding pocket + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + salt-bridge + + + 0.99884206 + residue_name_number + cleaner0 + 2023-07-05T10:45:53Z + DUMMY: + + Arg58 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + Salt-bridge + + + 0.99792117 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:26:50Z + SO: + + glutamate + + + 0.99548495 + protein_type + cleaner0 + 2023-07-05T12:27:28Z + MESH: + + myrosinase + + + 0.996011 + evidence + cleaner0 + 2023-07-05T12:22:00Z + DUMMY: + + structure + + + 0.98592824 + evidence + cleaner0 + 2023-07-05T12:22:03Z + DUMMY: + + D loss dynamics + + + 0.995103 + protein_state + cleaner0 + 2023-07-05T10:44:10Z + DUMMY: + + nonbound + + + 0.98567003 + protein_state + cleaner0 + 2023-07-05T10:43:39Z + DUMMY: + + bound + + + 0.9971819 + residue_name_number + cleaner0 + 2023-07-05T10:44:50Z + DUMMY: + + Glu42 + + + 0.99736005 + residue_name_number + cleaner0 + 2023-07-05T10:44:50Z + DUMMY: + + Glu42 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + salt-bridge + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:26:21Z + + RNA + + + 0.6723021 + chemical + cleaner0 + 2023-07-05T12:14:46Z + CHEBI: + + water + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + hydrogen bond + + + 0.99513197 + protein_state + cleaner0 + 2023-07-05T12:32:17Z + DUMMY: + + absence of + + + 0.97005063 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.99278337 + evidence + cleaner0 + 2023-07-05T12:22:06Z + DUMMY: + + density-change dynamics + + + 0.99656266 + protein_state + cleaner0 + 2023-07-05T10:43:39Z + DUMMY: + + bound + + + 0.9923832 + protein_state + cleaner0 + 2023-07-05T10:44:10Z + DUMMY: + + nonbound + + + 0.23798376 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.99764353 + residue_name_number + cleaner0 + 2023-07-05T10:44:50Z + DUMMY: + + Glu42 + + + 0.4976674 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + salt-bridge + + + 0.9930019 + residue_name_number + cleaner0 + 2023-07-05T10:44:50Z + DUMMY: + + Glu42 + + + + RESULTS + paragraph + 25115 + The RNA-stabilizing effect was not restricted to radiation-sensitive acidic residues. The side chain of Phe32 stacks against the G3 base within the 11 TRAP RNA-binding interfaces (Antson et al., 1999). With increasing dose, the D loss associated with the Phe32 side chain was significantly reduced upon RNA binding (Fig. 5 ▸ e; Phe32 Cζ; p = 0.0014), an indication that radiation-induced conformation disordering of Phe32 had been reduced. The extended aliphatic Lys37 side chain stacks against the nearby G1 base, making a series of nonpolar contacts within each RNA-binding interface. The D loss for Lys37 side-chain atoms was also reduced when stacked against the G1 base (Fig. 5 ▸ f; p = 0.0243 for Lys37 C∊ atoms). Representative Phe32 and Lys37 atoms were selected to illustrate these trends. + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:26:21Z + + RNA + + + 0.99905163 + residue_name_number + cleaner0 + 2023-07-05T10:51:36Z + DUMMY: + + Phe32 + + + 0.9022129 + residue_name_number + cleaner0 + 2023-07-05T10:52:10Z + DUMMY: + + G3 + + + site + SO: + cleaner0 + 2023-07-05T10:46:58Z + + TRAP RNA-binding interfaces + + + 0.9752722 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + 0.999049 + residue_name_number + cleaner0 + 2023-07-05T10:51:36Z + DUMMY: + + Phe32 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:26:21Z + + RNA + + + 0.99901557 + residue_name_number + cleaner0 + 2023-07-05T10:51:36Z + DUMMY: + + Phe32 + + + 0.99905235 + residue_name_number + cleaner0 + 2023-07-05T10:51:36Z + DUMMY: + + Phe32 + + + 0.99905616 + residue_name_number + cleaner0 + 2023-07-05T10:51:41Z + DUMMY: + + Lys37 + + + 0.54564714 + residue_name_number + cleaner0 + 2023-07-05T10:52:23Z + DUMMY: + + G1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + nonpolar contacts + + + 0.99768096 + site + cleaner0 + 2023-07-05T12:04:01Z + SO: + + RNA-binding interface + + + 0.9751469 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + 0.99891317 + residue_name_number + cleaner0 + 2023-07-05T10:51:42Z + DUMMY: + + Lys37 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + stacked + + + 0.7176499 + residue_name_number + cleaner0 + 2023-07-05T10:52:24Z + DUMMY: + + G1 + + + 0.9988103 + residue_name_number + cleaner0 + 2023-07-05T10:51:42Z + DUMMY: + + Lys37 + + + 0.99893457 + residue_name_number + cleaner0 + 2023-07-05T10:51:36Z + DUMMY: + + Phe32 + + + 0.9989254 + residue_name_number + cleaner0 + 2023-07-05T10:51:42Z + DUMMY: + + Lys37 + + + + DISCUSS + title_1 + 25925 + Discussion   + + + DISCUSS + paragraph + 25940 + Here, MX radiation-induced specific structural changes within the large TRAP–RNA assembly over a large dose range (1.3–25.0 MGy) have been analysed using a high-throughput quantitative approach, providing a measure of the electron-density distribution for each refined atom with increasing dose, D loss. Compared with previous studies, the results provide a further step in the detailed characterization of SRD effects in MX. Our method­ology, which eliminated tedious and error-prone visual inspection, permitted the determination on a per-atom basis of the most damaged sites, as characterized by F obs(d n) − F obs(d 1) Fourier difference map peaks between successive data sets collected from the same crystal. Here, it provided the precision required to quantify the role of RNA in the damage susceptibilities of equivalent atoms between RNA-bound and nonbound TRAP, but it is applicable to any MX SRD study. + + experimental_method + MESH: + cleaner0 + 2023-07-05T10:28:30Z + + MX + + + 0.9915398 + complex_assembly + cleaner0 + 2023-07-05T10:38:43Z + GO: + + TRAP–RNA + + + 0.9947875 + evidence + cleaner0 + 2023-07-05T12:22:11Z + DUMMY: + + electron-density distribution + + + 0.78213966 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + 0.64419585 + experimental_method + cleaner0 + 2023-07-05T10:28:30Z + MESH: + + MX + + + evidence + DUMMY: + cleaner0 + 2023-07-05T10:54:22Z + + F obs(d n) − F obs(d 1) Fourier difference map peaks + + + 0.98756266 + evidence + cleaner0 + 2023-07-05T12:22:22Z + DUMMY: + + crystal + + + 0.9878491 + chemical + cleaner0 + 2023-07-05T10:26:21Z + CHEBI: + + RNA + + + 0.9950579 + protein_state + cleaner0 + 2023-07-05T12:32:29Z + DUMMY: + + RNA-bound + + + 0.9650826 + protein_state + cleaner0 + 2023-07-05T10:44:10Z + DUMMY: + + nonbound + + + 0.45595852 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + experimental_method + MESH: + cleaner0 + 2023-07-05T10:28:30Z + + MX + + + + DISCUSS + paragraph + 26862 + The RNA was found to be substantially more radiation-resistant than the protein, even at the highest doses investigated (∼25.0 MGy), which is in strong concurrence with our previous SRD investigation of the C.Esp1396I protein–DNA complex (Bury et al., 2015). Consistent with that study, at high doses of above ∼20 MGy, F obs(d n) − F obs(d 1) map density was detected around P, O3′ and O5′ atoms of the RNA backbone, with no significant difference density localized to RNA ribose and basic subunits. RNA backbone disordering thus appears to be the main radiation-induced effect in RNA, with the protein–base interactions maintained even at high doses (>20 MGy). The U4 phosphate exhibited marginally larger D loss values above 20 MGy than G1, A2 and G3 (Supplementary Fig. S4). Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. At 25.0 MGy, the magnitude of the RNA backbone D loss was of the same order as for the radiation-insensitive Gly Cα atoms and on average less than half that of the acidic residues of the protein (Supplementary Fig. S3). Consequently, no clear single-strand breaks could be located, and since RNA-binding within the current TRAP–(GAGUU)10GAG complex is mediated predominantly through base–protein interactions, the biological integrity of the RNA complex was dictated by the rate at which protein decarboxylation occurred. + + 0.97414523 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + 0.7477479 + protein_state + cleaner0 + 2023-07-05T12:32:46Z + DUMMY: + + radiation-resistant + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:17:38Z + + SRD investigation + + + 0.99410534 + complex_assembly + cleaner0 + 2023-07-05T12:31:03Z + GO: + + C.Esp1396I + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:36:34Z + + DNA + + + evidence + DUMMY: + cleaner0 + 2023-07-05T10:54:03Z + + F obs(d n) − F obs(d 1) map density + + + 0.9961176 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + 0.9903002 + evidence + cleaner0 + 2023-07-05T12:22:26Z + DUMMY: + + difference density + + + 0.99770594 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + structure_element + SO: + cleaner0 + 2023-07-05T12:15:54Z + + subunits + + + 0.9490286 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + 0.98482555 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + 0.6900701 + residue_name_number + cleaner0 + 2023-07-05T10:53:16Z + DUMMY: + + U4 + + + 0.5953651 + chemical + cleaner0 + 2023-07-05T12:14:51Z + CHEBI: + + phosphate + + + 0.9657221 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + 0.48627108 + residue_name_number + cleaner0 + 2023-07-05T10:52:24Z + DUMMY: + + G1 + + + 0.43101338 + residue_name_number + cleaner0 + 2023-07-05T10:54:30Z + DUMMY: + + A2 + + + 0.69601625 + residue_name_number + cleaner0 + 2023-07-05T10:52:11Z + DUMMY: + + G3 + + + 0.97514343 + residue_name_number + cleaner0 + 2023-07-05T10:53:17Z + DUMMY: + + U4 + + + 0.8990134 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.9884177 + chemical + cleaner0 + 2023-07-05T12:15:01Z + CHEBI: + + water + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + hydrogen bond + + + structure_element + SO: + cleaner0 + 2023-07-05T12:15:54Z + + subunits + + + 0.99823785 + residue_name_number + cleaner0 + 2023-07-05T10:45:53Z + DUMMY: + + Arg58 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + hydrogen bond + + + structure_element + SO: + cleaner0 + 2023-07-05T12:15:54Z + + subunits + + + 0.9919527 + residue_name_number + cleaner0 + 2023-07-05T10:53:17Z + DUMMY: + + U4 + + + 0.7579533 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:26:22Z + + RNA + + + 0.66986865 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + 0.9113056 + residue_name + cleaner0 + 2023-07-05T10:42:44Z + SO: + + Gly + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:26:22Z + + RNA + + + complex_assembly + GO: + cleaner0 + 2023-07-05T10:53:41Z + + TRAP–(GAGUU)10GAG + + + 0.86929446 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + + DISCUSS + paragraph + 28527 + RNA interacting with TRAP was shown to offer significant protection against radiation-induced structural changes. Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively). However, compared with Asp39, Glu36 is strikingly less decarboxylated when bound to RNA (Fig. 4 ▸). This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). For Glu36 and Asp39, no direct quantitative correlation could be established between hydrogen-bond length and D loss (linear R 2 of <0.23 for all doses; Supplementary Fig. S5). Thus, another factor must be responsible for this clear reduction in Glu36 CO2 decarboxyl­ation in RNA-bound TRAP. The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. Radiation-induced decarboxylation has been proposed to be mediated by preferential positive-hole migration to the side-chain carboxyl group, with rapid proton transfer trapping the hole at the carboxyl group (Burmeister, 2000; Symons, 1997):where the forward rate is K 1 and the backward rate is K −1, where the forward rate is K 2. + + 0.99296665 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + 0.98447126 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.9987546 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + 0.99878544 + residue_name_number + cleaner0 + 2023-07-05T10:44:46Z + DUMMY: + + Asp39 + + + 0.99426657 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + hydrogen bonds + + + 0.97147286 + chemical + cleaner0 + 2023-07-05T12:15:09Z + CHEBI: + + guanine + + + 0.92182314 + residue_name_number + cleaner0 + 2023-07-05T10:52:11Z + DUMMY: + + G3 + + + 0.95111024 + residue_name_number + cleaner0 + 2023-07-05T10:52:24Z + DUMMY: + + G1 + + + 0.998728 + residue_name_number + cleaner0 + 2023-07-05T10:44:46Z + DUMMY: + + Asp39 + + + 0.99873954 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + 0.9912282 + protein_state + cleaner0 + 2023-07-05T12:32:51Z + DUMMY: + + bound to + + + 0.9868809 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:18:19Z + + mutagenesis and nucleoside analogue studies + + + 0.7989518 + residue_name_number + cleaner0 + 2023-07-05T10:52:24Z + DUMMY: + + G1 + + + 0.98919463 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.78089446 + residue_name_number + cleaner0 + 2023-07-05T10:54:30Z + DUMMY: + + A2 + + + 0.8507618 + residue_name_number + cleaner0 + 2023-07-05T10:52:11Z + DUMMY: + + G3 + + + 0.98744667 + evidence + cleaner0 + 2023-07-05T12:22:33Z + DUMMY: + + RNA-binding affinity + + + 0.99020547 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.99115556 + evidence + cleaner0 + 2023-07-05T12:22:36Z + DUMMY: + + K d + + + 0.99872905 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + 0.998755 + residue_name_number + cleaner0 + 2023-07-05T10:44:46Z + DUMMY: + + Asp39 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + hydrogen-bond + + + evidence + DUMMY: + cleaner0 + 2023-07-05T10:43:57Z + + D loss + + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:23:06Z + + linear R 2 + + + 0.9987036 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + 0.9961844 + protein_state + cleaner0 + 2023-07-05T12:32:54Z + DUMMY: + + RNA-bound + + + 0.9805304 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.9988978 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:54Z + + hydrogen bonds + + + 0.9990214 + residue_name_number + cleaner0 + 2023-07-05T10:55:21Z + DUMMY: + + His34 + + + 0.99905556 + residue_name_number + cleaner0 + 2023-07-05T10:55:26Z + DUMMY: + + Lys56 + + + 0.95854217 + protein_state + cleaner0 + 2023-07-05T12:32:59Z + DUMMY: + + conserved + + + 0.996298 + residue_name_number + cleaner0 + 2023-07-05T10:52:11Z + DUMMY: + + G3 + + + 0.9989222 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + 0.9960868 + protein_state + cleaner0 + 2023-07-05T12:33:02Z + DUMMY: + + RNA-bound + + + 0.9864499 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + + DISCUSS + paragraph + 30034 + When bound to RNA, the average solvent-accessible area of the Glu36 side-chain O atoms is reduced from ∼15 to 0 Å2. We propose that with no solvent accessibility Glu36 decarboxylation is inhibited, since the CO2-formation rate K 2 is greatly reduced, and suggest that steric hindrance prevents each radicalized Glu36 CO2 group from achieving the planar conformation required for complete dissociation from TRAP. The electron-recombination rate K −1 remains high, however, owing to rapid electron migration through the protein–RNA complex to refill the Glu36 positive hole (the precursor for Glu decarboxylation). Upon RNA binding, the Asp39 side-chain carboxyl group solvent-accessible area changes from ∼75 to 35 Å2, still allowing a high CO2-formation rate K 2. + + 0.9948141 + protein_state + cleaner0 + 2023-07-05T12:33:06Z + DUMMY: + + bound to + + + 0.99623907 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + 0.99894124 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + 0.9985372 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + 0.76363486 + evidence + cleaner0 + 2023-07-05T12:23:16Z + DUMMY: + + CO2-formation rate K 2 + + + 0.9988926 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + complex_assembly + GO: + cleaner0 + 2023-07-05T12:07:58Z + + TRAP + + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:23:39Z + + electron-recombination rate K −1 + + + complex_assembly + GO: + cleaner0 + 2023-07-05T12:04:50Z + + protein–RNA + + + 0.99887615 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + 0.97079873 + site + cleaner0 + 2023-07-05T12:34:26Z + SO: + + positive hole + + + 0.8287284 + residue_name + cleaner0 + 2023-07-05T12:34:16Z + SO: + + Glu + + + 0.9915752 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + 0.998931 + residue_name_number + cleaner0 + 2023-07-05T10:44:46Z + DUMMY: + + Asp39 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T12:23:52Z + + CO2 + + + 0.59207654 + evidence + cleaner0 + 2023-07-05T12:23:58Z + DUMMY: + + rate K 2 + + + + DISCUSS + paragraph + 30812 + Previous studies have reported inconsistent results concerning the dependence of the acidic residue decarboxylation rate on solvent accessibility (Weik et al., 2000; Fioravanti et al., 2007; Gerstel et al., 2015). The prevalence of radical attack from solvent channels surrounding the protein in the crystal is a questionable cause, considering previous observations indicating that the strongly oxidizing hydroxyl radical is immobile at 100 K (Allan et al., 2013; Owen et al., 2012). Furthermore, the suggested electron hole-trapping mechanism which induces decarboxylation within proteins at 100 K has no clear mechanistic dependence on the solvent-accessible area of each carboxyl group. By comparing equivalent acidic residues with and without RNA, we have now deconvoluted the role of solvent accessibility from other local protein environment factors, and thus propose a suitable mechanism by which exceptionally low solvent accessibility can reduce the rate of decarboxylation. Overall, no direct correlation between solvent accessibility and decarboxylation susceptibility was observed, but it is very clear that inaccessible residues are protected. + + 0.99064636 + evidence + cleaner0 + 2023-07-05T12:24:02Z + DUMMY: + + crystal + + + 0.86443084 + protein_state + cleaner0 + 2023-07-05T12:33:09Z + DUMMY: + + with + + + 0.7418846 + protein_state + cleaner0 + 2023-07-05T12:33:11Z + DUMMY: + + without + + + 0.9973688 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + + DISCUSS + paragraph + 31974 + Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 ▸ c). However, for each of these residues the exact crystal contacts are not preserved between bound and nonbound TRAP or even between monomers within one TRAP ring. For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. Thus, the dependence of decarboxylation rates on these interactions could not be established. + + 0.99785984 + site + cleaner0 + 2023-07-05T12:34:37Z + SO: + + RNA-binding interfaces + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:26:22Z + + RNA + + + 0.999079 + residue_name_number + cleaner0 + 2023-07-05T10:56:05Z + DUMMY: + + Glu50 + + + 0.99905926 + residue_name_number + cleaner0 + 2023-07-05T10:56:10Z + DUMMY: + + Glu71 + + + 0.9990657 + residue_name_number + cleaner0 + 2023-07-05T10:56:14Z + DUMMY: + + Glu73 + + + complex_assembly + GO: + cleaner0 + 2023-07-05T12:07:58Z + + TRAP + + + structure_element + SO: + cleaner0 + 2023-07-05T12:05:25Z + + rings + + + 0.9930227 + protein_state + cleaner0 + 2023-07-05T10:43:39Z + DUMMY: + + bound + + + 0.9660058 + protein_state + cleaner0 + 2023-07-05T10:44:10Z + DUMMY: + + nonbound + + + 0.9864768 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + complex_assembly + GO: + cleaner0 + 2023-07-05T12:07:58Z + + TRAP + + + structure_element + SO: + cleaner0 + 2023-07-05T11:58:12Z + + ring + + + 0.9962069 + protein_state + cleaner0 + 2023-07-05T10:43:39Z + DUMMY: + + bound + + + 0.9905319 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.9990728 + residue_name_number + cleaner0 + 2023-07-05T10:56:14Z + DUMMY: + + Glu73 + + + 0.99010086 + residue_name + cleaner0 + 2023-07-05T10:56:37Z + SO: + + lysine + + + structure_element + SO: + cleaner0 + 2023-07-05T12:15:54Z + + subunits + + + 0.9937517 + protein_state + cleaner0 + 2023-07-05T10:44:10Z + DUMMY: + + nonbound + + + 0.9874014 + complex_assembly + cleaner0 + 2023-07-05T12:07:58Z + GO: + + TRAP + + + 0.999064 + residue_name_number + cleaner0 + 2023-07-05T10:56:14Z + DUMMY: + + Glu73 + + + 0.9762691 + chemical + cleaner0 + 2023-07-05T12:15:18Z + CHEBI: + + waters + + + structure_element + SO: + cleaner0 + 2023-07-05T12:15:42Z + + subunit + + + + DISCUSS + paragraph + 32661 + Radiation-induced side-chain conformational changes have been poorly characterized in previous SRD investigations owing to their strong dependence on packing density and geometric strain. Such structural changes are known to have significant roles within enzymatic pathways, and experimenters must be aware of these possible confounding factors when assigning true functional mechanisms using MX. Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively. It has been suggested (Burmeister, 2000) that Tyr residues can lose their aromatic –OH group owing to radiation-induced effects; however, no energetically favourable pathway for –OH cleavage exists and this has not been detected in aqueous radiation-chemistry studies. In TRAP, D loss increased at a similar rate for both the Tyr O atoms and aromatic ring atoms, suggesting that full ring conformational disordering is more likely. Indeed, no convincing reproducible Fourier difference peaks above the background map noise were observed around any Tyr terminal –OH groups. + + experimental_method + MESH: + cleaner0 + 2023-07-05T12:18:42Z + + SRD investigations + + + 0.74215937 + experimental_method + cleaner0 + 2023-07-05T10:28:30Z + MESH: + + MX + + + 0.95398015 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + 0.9726016 + complex_assembly + cleaner0 + 2023-07-05T12:07:59Z + GO: + + TRAP + + + 0.8587137 + complex_assembly + cleaner0 + 2023-07-05T12:07:59Z + GO: + + TRAP + + + 0.9990075 + residue_name_number + cleaner0 + 2023-07-05T10:51:42Z + DUMMY: + + Lys37 + + + 0.99903595 + residue_name_number + cleaner0 + 2023-07-05T10:51:36Z + DUMMY: + + Phe32 + + + 0.65299237 + residue_name_number + cleaner0 + 2023-07-05T10:52:24Z + DUMMY: + + G1 + + + 0.5014381 + residue_name_number + cleaner0 + 2023-07-05T10:52:11Z + DUMMY: + + G3 + + + 0.98193204 + residue_name + cleaner0 + 2023-07-05T10:56:31Z + SO: + + Tyr + + + 0.9829249 + complex_assembly + cleaner0 + 2023-07-05T12:07:59Z + GO: + + TRAP + + + 0.6355414 + evidence + cleaner0 + 2023-07-05T10:43:57Z + DUMMY: + + D loss + + + 0.97448653 + residue_name + cleaner0 + 2023-07-05T10:56:34Z + SO: + + Tyr + + + structure_element + SO: + cleaner0 + 2023-07-05T11:58:12Z + + ring + + + structure_element + SO: + cleaner0 + 2023-07-05T11:58:12Z + + ring + + + 0.9927671 + evidence + cleaner0 + 2023-07-05T12:24:08Z + DUMMY: + + Fourier difference peaks + + + 0.56909597 + evidence + cleaner0 + 2023-07-05T12:24:17Z + DUMMY: + + map + + + 0.9574091 + residue_name + cleaner0 + 2023-07-05T10:56:40Z + SO: + + Tyr + + + + DISCUSS + paragraph + 33837 + The RNA-stabilization effects on protein are observed at short ranges and are restricted to within the RNA-binding interfaces around the TRAP ring. For example, Asp17 is located ∼6.8 Å from the G1 base, outside the RNA-binding interfaces, and has indistinguishable Cγ atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p > 0.9). An increase in the dose at which functionally important residues remain intact has biological ramifications for understanding the mechanisms at which ionizing radiation damage is mitigated within naturally forming DNA–protein and RNA–protein complexes. Observations of lower protein radiation-sensitivity in DNA-bound forms have been recorded in solution at RT at much lower doses (∼1 kGy) than those used for typical MX experiments [e.g. an oestrogen response element–receptor complex (Stísová et al., 2006) and a DNA glycosylase and its abasic DNA target site (Gillard et al., 2004)]. In these studies, the main damaging species is predicted to be the oxidizing hydroxyl radical produced through solvent irradiation, which is known to add to double covalent bonds within both DNA and RNA bases to induce strand breaks and base modification (Spotheim-Maurizot & Davídková, 2011; Chance et al., 1997). It was suggested that physical screening of DNA by protein shielded the DNA–protein interaction sites from radical damage, yielding an extended life-dose for the nucleoprotein complex compared with separate protein and DNA constituents at RT. + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:26:22Z + + RNA + + + 0.99812096 + site + cleaner0 + 2023-07-05T12:34:42Z + SO: + + RNA-binding interfaces + + + complex_assembly + GO: + cleaner0 + 2023-07-05T12:07:59Z + + TRAP + + + structure_element + SO: + cleaner0 + 2023-07-05T11:58:12Z + + ring + + + 0.9991387 + residue_name_number + cleaner0 + 2023-07-05T12:00:25Z + DUMMY: + + Asp17 + + + 0.48927557 + residue_name_number + cleaner0 + 2023-07-05T10:52:24Z + DUMMY: + + G1 + + + 0.9979715 + site + cleaner0 + 2023-07-05T12:34:45Z + SO: + + RNA-binding interfaces + + + 0.9856348 + evidence + cleaner0 + 2023-07-05T12:24:21Z + DUMMY: + + loss dose-dynamics + + + 0.99598575 + protein_state + cleaner0 + 2023-07-05T12:33:16Z + DUMMY: + + RNA-bound + + + 0.9913564 + protein_state + cleaner0 + 2023-07-05T10:44:10Z + DUMMY: + + nonbound + + + 0.9880157 + complex_assembly + cleaner0 + 2023-07-05T12:07:59Z + GO: + + TRAP + + + complex_assembly + GO: + cleaner0 + 2023-07-05T12:06:23Z + + DNA–protein + + + complex_assembly + GO: + cleaner0 + 2023-07-05T12:06:42Z + + RNA–protein + + + 0.99518895 + protein_state + cleaner0 + 2023-07-05T12:33:22Z + DUMMY: + + DNA-bound + + + experimental_method + MESH: + cleaner0 + 2023-07-05T10:28:30Z + + MX + + + 0.9768747 + protein_type + cleaner0 + 2023-07-05T12:27:33Z + MESH: + + DNA glycosylase + + + 0.992797 + site + cleaner0 + 2023-07-05T12:35:08Z + SO: + + abasic DNA target site + + + 0.98401004 + chemical + cleaner0 + 2023-07-05T10:36:34Z + CHEBI: + + DNA + + + 0.98465955 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:36:34Z + + DNA + + + 0.99563825 + site + cleaner0 + 2023-07-05T12:35:12Z + SO: + + DNA–protein interaction sites + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:36:34Z + + DNA + + + + DISCUSS + paragraph + 35345 + However, in the current MX study at 100 K, the main damaging species are believed to be migrating LEEs and holes produced directly within the protein–RNA components or in closely associated solvent. The results presented here suggest that biologically relevant nucleoprotein complexes also exhibit prolonged life-doses under the effect of LEE-induced structural changes, involving direct physical protection of key RNA-binding residues. Such reduced radiation-sensitivity in this case ensures that the interacting protein remains bound long enough to the RNA to complete its function, even whilst exposed to ionizing radiation. Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 ▸). When exposed to X-rays, these residues will be preferentially damaged by X-rays and subsequently reduce the affinity with which TRAP binds to RNA. Within the cellular environment, this mechanism could reduce the risk that radiation-damaged proteins might bind to RNA, thus avoiding the detrimental introduction of incorrect DNA-repair, transcriptional and base-modification pathways. + + 0.62370133 + experimental_method + cleaner0 + 2023-07-05T10:28:30Z + MESH: + + MX + + + complex_assembly + GO: + cleaner0 + 2023-07-05T12:04:56Z + + protein–RNA + + + 0.53609496 + complex_assembly + cleaner0 + 2023-07-05T12:13:58Z + GO: + + nucleoprotein + + + 0.9972315 + site + cleaner0 + 2023-07-05T12:34:49Z + SO: + + RNA-binding residues + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T10:43:39Z + + bound + + + 0.9973586 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + 0.98719054 + protein_state + cleaner0 + 2023-07-05T10:44:10Z + DUMMY: + + nonbound + + + 0.6919731 + complex_assembly + cleaner0 + 2023-07-05T12:07:59Z + GO: + + TRAP + + + 0.9982071 + site + cleaner0 + 2023-07-05T12:34:52Z + SO: + + RNA-binding interfaces + + + 0.9990889 + residue_name_number + cleaner0 + 2023-07-05T10:44:46Z + DUMMY: + + Asp39 + + + 0.99907863 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + 0.9990723 + residue_name_number + cleaner0 + 2023-07-05T10:44:50Z + DUMMY: + + Glu42 + + + 0.87554866 + complex_assembly + cleaner0 + 2023-07-05T12:07:59Z + GO: + + TRAP + + + 0.99667054 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + 0.9962542 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + chemical + CHEBI: + cleaner0 + 2023-07-05T10:36:34Z + + DNA + + + + DISCUSS + paragraph + 36579 + The Python scripts written to calculate the per atom D loss metric are available from the authors on request. + + + INTRO + title_1 + 36689 + Related literature   + + + INTRO + paragraph + 36712 + The following references are cited in the Supporting Information for this article: Chen et al. (2010). + + + SUPPL + title_1 + 36815 + Supplementary Material + + + REF + title + 36838 + References + + + REF + ref + 36849 + Adams, P. D. et al. (2010). Acta Cryst. D66, 213–221. + + + REF + ref + 36905 + Alizadeh, E. & Sanche, L. (2014). Eur. Phys. J. D, 68, 97. + + + REF + ref + 36964 + Allan, E. G., Kander, M. C., Carmichael, I. & Garman, E. F. (2013). J. Synchrotron Rad. 20, 23–36. + + + REF + ref + 37065 + Antson, A. A., Dodson, E. J., Dodson, G., Greaves, R. B., Chen, X. & Gollnick, P. (1999). Nature (London), 401, 235–242. + + + REF + ref + 37188 + Becke, A. D. (1993). J. Chem. Phys. 98, 5648–5652. + + + REF + ref + 37241 + Bourenkov, G. P. & Popov, A. N. (2010). Acta Cryst. D66, 409–419. + + + REF + ref + 37309 + Burmeister, W. P. (2000). Acta Cryst. D56, 328–341. + + + REF + ref + 37363 + Bury, C., Garman, E. F., Ginn, H. M., Ravelli, R. B. G., Carmichael, I., Kneale, G. & McGeehan, J. E. (2015). J. Synchrotron Rad. 22, 213–224. + + + REF + ref + 37508 + Chance, M. R., Sclavi, B., Woodson, S. A. & Brenowitz, M. (1997). Structure, 5, 865–869. + + + REF + ref + 37599 + Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. & Richardson, D. C. (2010). Acta Cryst. D66, 12–21. + + + REF + ref + 37777 + Dubnovitsky, A. P., Ravelli, R. B. G., Popov, A. N. & Papageorgiou, A. C. (2005). Protein Sci. 14, 1498–1507. + + + REF + ref + 37889 + Elliott, M. B., Gottlieb, P. A. & Gollnick, P. (2001). RNA, 7, 85–93. + + + REF + ref + 37961 + Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486–501. + + + REF + ref + 38048 + Evans, P. R. & Murshudov, G. N. (2013). Acta Cryst. D69, 1204–1214. + + + REF + ref + 38118 + Fioravanti, E., Vellieux, F. M. D., Amara, P., Madern, D. & Weik, M. (2007). J. Synchrotron Rad. 14, 84–91. + + + REF + ref + 38228 + Frisch, M. J. et al. (2009). Gaussian 09. Gaussian Inc., Wallingford, Connecticut, USA. + + + REF + ref + 38316 + Garman, E. F. (2010). Acta Cryst. D66, 339–351. + + + REF + ref + 38366 + Gerstel, M., Deane, C. M. & Garman, E. F. (2015). J. Synchrotron Rad. 22, 201–212. + + + REF + ref + 38451 + Gillard, N., Begusova, M., Castaing, B. & Spotheim-Maurizot, M. (2004). Radiat. Res. 162, 566–571. + + + REF + ref + 38552 + Holton, J. M. (2007). J. Synchrotron Rad. 14, 51–72. + + + REF + ref + 38607 + Holton, J. M. (2009). J. Synchrotron Rad. 16, 133–142. + + + REF + ref + 38664 + Hopcroft, N. H., Wendt, A. L., Gollnick, P. & Antson, A. A. (2002). Acta Cryst. D58, 615–621. + + + REF + ref + 38760 + Jones, G. D., Lea, J. S., Symons, M. C. & Taiwo, F. A. (1987). Nature (London), 330, 772–773. + + + REF + ref + 38856 + Leslie, A. G. W. & Powell, H. R. (2007). Evolving Methods for Macromolecular Crystallography, edited by R. J. Read & J. L. Sussman, pp. 41–51. Dordrecht: Springer. + + + REF + ref + 39022 + Liebschner, D., Rosenbaum, G., Dauter, M. & Dauter, Z. (2015). Acta Cryst. D71, 772–778. + + + REF + ref + 39113 + Matsui, Y., Sakai, K., Murakami, M., Shiro, Y., Adachi, S., Okumura, H. & Kouyama, T. (2002). J. Mol. Biol. 324, 469–481. + + + REF + ref + 39237 + McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658–674. + + + REF + ref + 39371 + McGeehan, J. E., Streeter, S. D., Thresh, S. J., Ball, N., Ravelli, R. B. G. & Kneale, G. G. (2008). Nucleic Acids Res. 36, 4778–4787. + + + REF + ref + 39508 + Murray, J. & Garman, E. (2002). J. Synchrotron Rad. 9, 347–354. + + + REF + ref + 39574 + Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355–367. + + + REF + ref + 39741 + O’Neill, P., Stevens, D. L. & Garman, E. (2002). J. Synchrotron Rad. 9, 329–332. + + + REF + ref + 39826 + Owen, R. L., Axford, D., Nettleship, J. E., Owens, R. J., Robinson, J. I., Morgan, A. W., Doré, A. S., Lebon, G., Tate, C. G., Fry, E. E., Ren, J., Stuart, D. I. & Evans, G. (2012). Acta Cryst. D68, 810–818. + + + REF + ref + 40037 + Owen, R. L., Rudiño-Piñera, E. & Garman, E. F. (2006). Proc. Natl Acad. Sci. USA, 103, 4912–4917. + + + REF + ref + 40139 + Ptasińska, S. & Sanche, L. (2007). Phys. Rev. E, 75, 031915. + + + REF + ref + 40201 + Ravelli, R. B. G. & McSweeney, S. M. (2000). Structure, 8, 315–328. + + + REF + ref + 40271 + Shimizu, N., Hirata, K., Hasegawa, K., Ueno, G. & Yamamoto, M. (2007). J. Synchrotron Rad. 14, 4–10. + + + REF + ref + 40374 + Simons, J. (2006). Acc. Chem. Res. 39, 772–779. + + + REF + ref + 40424 + Spotheim-Maurizot, M. & Davídková, M. (2011). Mutat. Res. 711, 41–48. + + + REF + ref + 40498 + Stísová, V., Goffinont, S., Spotheim-Maurizot, M. & Davídková, M. (2006). Radiat. Prot. Dosimetry, 122, 106–109. + + + REF + ref + 40617 + Symons, M. C. R. (1997). Free Radical Biol. Med. 22, 1271–1276. + + + REF + ref + 40683 + Ten Eyck, L. F. (1973). Acta Cryst. A29, 183–191. + + + REF + ref + 40735 + Tomasi, J., Mennucci, B. & Cancès, E. (1999). J. Mol. Struct. 464, 211–226. + + + REF + ref + 40814 + Weik, M., Ravelli, R. B. G., Kryger, G., McSweeney, S., Raves, M. L., Harel, M., Gros, P., Silman, I., Kroon, J. & Sussman, J. L. (2000). Proc. Natl Acad. Sci. USA, 97, 623–628. + + + REF + ref + 40994 + Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242. + + + REF + ref + 41049 + Yano, J., Kern, J., Irrgang, K. D., Latimer, M. J., Bergmann, U., Glatzel, P., Pushkar, Y., Biesiadka, J., Loll, B., Sauer, K., Messinger, J., Zouni, A. & Yachandra, V. K. (2005). Proc. Natl Acad. Sci. USA, 102, 12047–12052. + + + REF + ref + 41276 + Zeldin, O. B., Brockhauser, S., Bremridge, J., Holton, J. M. & Garman, E. F. (2013). Proc. Natl Acad. Sci. USA, 110, 20551–20556. + + + REF + ref + 41408 + Zeldin, O. B., Gerstel, M. & Garman, E. F. (2013). J. Appl. Cryst. 46, 1225–1230. + + + d-72-00648-fig1.jpg + fig1 + FIG + fig_caption + 41492 + The TRAP–(GAGUU)10GAG complex asymmetric unit (PDB entry 1gtf; Hopcroft et al., 2002). Bound tryptophan ligands are represented as coloured spheres. RNA is shown is yellow. + + complex_assembly + GO: + cleaner0 + 2023-07-05T10:58:32Z + + TRAP–(GAGUU)10GAG + + + 0.99105424 + protein_state + cleaner0 + 2023-07-05T10:43:39Z + DUMMY: + + Bound + + + 0.9840899 + chemical + cleaner0 + 2023-07-05T12:16:01Z + CHEBI: + + tryptophan + + + 0.9981456 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + + d-72-00648-fig2.jpg + fig2 + FIG + fig_caption + 41667 + (a) Electron-density loss sites as indicated by D +loss in the TRAP–RNA complex crystal by residue/nucleotide type for five doses [sites determined above the 4× average D +loss threshold, calculated over the TRAP–RNA structure for the first difference map: F +obs(d +2) − F +obs(d +1)]. Cumulative frequencies are normalized to both the total number of non-H atoms per residue/nucleotide and the total number of that residue/nucleotide type present. (b) Average D +loss for each residue/nucleotide type with respect to the DWD (diffraction-weighted dose; Zeldin, Brock­hauser et al., 2013). 95% confidence intervals (CI) are shown. Only a subset of key TRAP residue types are included. The average D +loss (calculated over the whole TRAP asymmetric unit) is shown at each dose (dashed line). + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:12:10Z + + D +loss + + + 0.9932771 + complex_assembly + cleaner0 + 2023-07-05T10:38:44Z + GO: + + TRAP–RNA + + + 0.9954058 + evidence + cleaner0 + 2023-07-05T12:24:38Z + DUMMY: + + crystal + + + 0.7991534 + evidence + cleaner0 + 2023-07-05T12:24:42Z + DUMMY: + + D +loss threshold + + + 0.99266356 + complex_assembly + cleaner0 + 2023-07-05T10:38:44Z + GO: + + TRAP–RNA + + + 0.98201466 + evidence + cleaner0 + 2023-07-05T12:24:45Z + DUMMY: + + structure + + + 0.9719779 + evidence + cleaner0 + 2023-07-05T12:24:49Z + DUMMY: + + difference map + + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:25:15Z + + F +obs(d +2) − F +obs(d +1) + + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:13:13Z + + Average D +loss + + + 0.96845776 + evidence + cleaner0 + 2023-07-05T12:25:20Z + DUMMY: + + DWD + + + 0.98903877 + evidence + cleaner0 + 2023-07-05T12:25:23Z + DUMMY: + + diffraction-weighted dose + + + 0.5221266 + complex_assembly + cleaner0 + 2023-07-05T12:07:59Z + GO: + + TRAP + + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:25:43Z + + average D +loss + + + 0.7290474 + complex_assembly + cleaner0 + 2023-07-05T12:07:59Z + GO: + + TRAP + + + + d-72-00648-fig3.jpg + fig3 + FIG + fig_caption + 42459 + +F +obs(d +n) − F +obs(d +1) Fourier difference maps for (a) n = 2 (3.9 MGy), (b) n = 3 (6.5 MGy) and (c) n = 7 (16.7 MGy) contoured at ±4σ (a) and ±3.5σ (b, c). In (a) clear difference density is observed around the Glu42 carboxyl side chain in chain H, within the lowest dose difference map at d +2 = 3.9 MGy. Radiation-induced protein disordering is evident across the large dose range (b, c); in comparison, no clear deterioration of the RNA density was observed. + + evidence + DUMMY: + cleaner0 + 2023-07-05T12:26:14Z + + F +obs(d +n) − F +obs(d +1) Fourier difference maps + + + 0.9945644 + evidence + cleaner0 + 2023-07-05T12:26:18Z + DUMMY: + + difference density + + + 0.9991478 + residue_name_number + cleaner0 + 2023-07-05T10:44:51Z + DUMMY: + + Glu42 + + + 0.8718893 + evidence + cleaner0 + 2023-07-05T12:26:29Z + DUMMY: + + lowest dose difference map + + + 0.997905 + chemical + cleaner0 + 2023-07-05T10:26:22Z + CHEBI: + + RNA + + + 0.9303295 + evidence + cleaner0 + 2023-07-05T12:26:36Z + DUMMY: + + density + + + + d-72-00648-fig4.jpg + fig4 + FIG + fig_caption + 42938 + +D +loss calculated for all side-chain carboxyl group Glu Cδ and Asp Cγ atoms within the TRAP–RNA complex for a dose of 19.3 MGy (d +8). Residues have been grouped by amino-acid number, and split into bound and nonbound groupings, with each bar representing the mean calculated over 11 equivalent atoms around a TRAP ring. Whiskers indicate 95% CI. The D +loss behaviour shown here was consistently exhibited across the entire investigated dose range. + + 0.89408696 + evidence + cleaner0 + 2023-07-05T12:26:41Z + DUMMY: + + D +loss + + + 0.9847978 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:26:50Z + SO: + + Glu + + + 0.97973424 + residue_name + cleaner0 + 2023-07-05T10:42:19Z + SO: + + Asp + + + 0.9919364 + complex_assembly + cleaner0 + 2023-07-05T10:38:44Z + GO: + + TRAP–RNA + + + 0.98781407 + protein_state + cleaner0 + 2023-07-05T10:43:40Z + DUMMY: + + bound + + + 0.94484127 + protein_state + cleaner0 + 2023-07-05T10:44:10Z + DUMMY: + + nonbound + + + 0.72071564 + complex_assembly + cleaner0 + 2023-07-05T12:07:59Z + GO: + + TRAP + + + structure_element + SO: + cleaner0 + 2023-07-05T11:58:12Z + + ring + + + 0.93198663 + evidence + cleaner0 + 2023-07-05T12:26:44Z + DUMMY: + + D +loss + + + + d-72-00648-fig5.jpg + fig5 + FIG + fig_caption + 43397 + +D +loss against dose for (a) Glu36 Cδ, (b) Asp39 Cγ, (c) Glu42 O∊1, (d) Glu42 O∊2, (e) Phe32 Cζ and (f) Lys37 C∊ atoms. 95% CI are included for each set of 11 equivalent atoms grouped as bound/nonbound. RNA-binding interface interactions are shown for TRAP chain N, with the F +obs(d +7) − F +obs(d +1) Fourier difference map (dose 16.7 MGy) overlaid and contoured at a ±4σ level. + + 0.7863771 + evidence + cleaner0 + 2023-07-05T12:26:53Z + DUMMY: + + D +loss + + + 0.99909854 + residue_name_number + cleaner0 + 2023-07-05T10:44:42Z + DUMMY: + + Glu36 + + + 0.9990896 + residue_name_number + cleaner0 + 2023-07-05T10:44:46Z + DUMMY: + + Asp39 + + + 0.9990759 + residue_name_number + cleaner0 + 2023-07-05T10:44:51Z + DUMMY: + + Glu42 + + + 0.9990729 + residue_name_number + cleaner0 + 2023-07-05T10:44:51Z + DUMMY: + + Glu42 + + + 0.9990627 + residue_name_number + cleaner0 + 2023-07-05T10:51:36Z + DUMMY: + + Phe32 + + + 0.9990792 + residue_name_number + cleaner0 + 2023-07-05T10:51:42Z + DUMMY: + + Lys37 + + + 0.9931265 + protein_state + cleaner0 + 2023-07-05T10:43:40Z + DUMMY: + + bound + + + 0.9739872 + protein_state + cleaner0 + 2023-07-05T10:44:10Z + DUMMY: + + nonbound + + + 0.9972383 + site + cleaner0 + 2023-07-05T12:34:57Z + SO: + + RNA-binding interface + + + 0.95457613 + complex_assembly + cleaner0 + 2023-07-05T12:07:59Z + GO: + + TRAP + + + evidence + DUMMY: + cleaner0 + 2023-07-05T10:59:56Z + + F +obs(d +7) − F +obs(d +1) Fourier difference map + + + + diff --git a/BioC_XML/4855620_v0.xml b/BioC_XML/4855620_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..55dd1deb52f459aaa28a3091b453409b86584453 --- /dev/null +++ b/BioC_XML/4855620_v0.xml @@ -0,0 +1,5797 @@ + + + + PMC + 20201222 + pmc.key + + 4855620 + CC BY + no + 0 + 0 + + D. E. Scott et al. + 10.1002/1873-3468.12139 + 4855620 + 26992456 + FEB212139 + Edited by Alfonso Valencia + 1094 + 8 + alanine scanning biophysics/ITC peptides protein–protein interaction RAD51 X‐ray crystallography + This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. + 1102 + surname:Scott;given-names:Duncan E. + surname:Marsh;given-names:May + surname:Blundell;given-names:Tom L. + surname:Abell;given-names:Chris + surname:Hyvönen;given-names:Marko + TITLE + front + 590 + 2016 + 0 + Structure‐activity relationship of the peptide binding‐motif mediating the BRCA2:RAD51 protein–protein interaction + + 0.99913776 + structure_element + cleaner0 + 2023-07-26T17:03:49Z + SO: + + peptide binding‐motif + + + 0.8988559 + complex_assembly + cleaner0 + 2023-07-26T16:03:56Z + GO: + + BRCA2:RAD51 + + + + ABSTRACT + abstract + 121 + RAD51 is a recombinase involved in the homologous recombination of double‐strand breaks in DNA. RAD51 forms oligomers by binding to another molecule of RAD51 via an ‘FxxA’ motif, and the same recognition sequence is similarly utilised to bind BRCA2. We have tabulated the effects of mutation of this sequence, across a variety of experimental methods and from relevant mutations observed in the clinic. We use mutants of a tetrapeptide sequence to probe the binding interaction, using both isothermal titration calorimetry and X‐ray crystallography. Where possible, comparison between our tetrapeptide mutational study and the previously reported mutations is made, discrepancies are discussed and the importance of secondary structure in interpreting alanine scanning and mutational data of this nature is considered. + + 0.99919516 + protein + cleaner0 + 2023-07-26T16:04:03Z + PR: + + RAD51 + + + 0.9950399 + protein_type + cleaner0 + 2023-07-26T16:04:14Z + MESH: + + recombinase + + + 0.9991793 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9961217 + oligomeric_state + cleaner0 + 2023-07-26T16:04:09Z + DUMMY: + + oligomers + + + 0.99924916 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:04:46Z + + FxxA + + + 0.9992681 + protein + cleaner0 + 2023-07-26T16:05:12Z + PR: + + BRCA2 + + + 0.9946819 + experimental_method + cleaner0 + 2023-07-26T17:11:30Z + MESH: + + mutation + + + 0.7667018 + protein_state + cleaner0 + 2023-07-26T17:06:01Z + DUMMY: + + mutants + + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:39:12Z + + tetrapeptide + + + 0.99898463 + experimental_method + cleaner0 + 2023-07-26T17:11:34Z + MESH: + + isothermal titration calorimetry + + + 0.9989825 + experimental_method + cleaner0 + 2023-07-26T16:29:24Z + MESH: + + X‐ray crystallography + + + 0.9988322 + experimental_method + cleaner0 + 2023-07-26T16:56:06Z + MESH: + + tetrapeptide mutational study + + + 0.99887955 + experimental_method + cleaner0 + 2023-07-26T16:56:14Z + MESH: + + alanine scanning + + + + ABBR + title_1 + 949 + Abbreviations + + + ABBR + paragraph + 963 + BRCA2, breast cancer type‐2 susceptibility protein + + + ABBR + paragraph + 1017 + HR, homologous recombination + + + ABBR + paragraph + 1047 + ITC, isothermal titration calorimetry + + + ABBR + paragraph + 1086 + PPI, protein–protein interaction + + + ABBR + paragraph + 1122 + SAR, structure activity relationship + + + ABBR + paragraph + 1160 + Eukaryotic RAD51, archeal RadA and prokaryotic RecA are a family of ATP‐dependent recombinases involved in homologous recombination (HR) of double‐strand breaks in DNA 1. RAD51 interacts with BRCA2, and is thought to localise RAD51 to sites of DNA damage 2, 3. Both BRCA2 and RAD51 together are vital for helping to repair and maintain a high fidelity in DNA replication. BRCA2 especially has garnered much attention in a clinical context, as many mutations have been identified that drive an increased risk of cancer in individuals 4, 5. Although the inactivation of the BRCA2:RAD51 DNA repair pathway can cause genomic instability and eventual tumour development, an inability to repair breaks in DNA may also engender a sensitivity to ionising radiation 6, 7. For this reason it is hypothesised that in tumour cells with an intact BRCA2:RAD51 repair pathway, small molecules which prevent the interaction between RAD51 and BRCA2 may confer radiosensitivity by disabling the HR pathway 8. The interaction between the two proteins is mediated by eight BRC repeats, which are characterised by a conserved ‘FxxA’ motif 9. RAD51 and RadA proteins also contain an ‘FxxA’ sequence (FTTA for human RAD51) through which it can bind to other RAD51 and RadA molecules, and oligomerise to form higher order filament structures on DNA. The common FxxA motifs of both the BRC repeats and RAD51 oligomerisation sequence are recognised by the same FxxA‐binding site of RAD51. + + 0.99842775 + taxonomy_domain + cleaner0 + 2023-07-26T16:15:10Z + DUMMY: + + Eukaryotic + + + 0.9990484 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.99834526 + taxonomy_domain + cleaner0 + 2023-07-26T16:15:39Z + DUMMY: + + archeal + + + 0.9979638 + protein + cleaner0 + 2023-07-26T16:15:47Z + PR: + + RadA + + + 0.998509 + taxonomy_domain + cleaner0 + 2023-07-26T16:15:43Z + DUMMY: + + prokaryotic + + + 0.87125826 + protein + cleaner0 + 2023-07-26T16:15:51Z + PR: + + RecA + + + 0.9985747 + protein_type + cleaner0 + 2023-07-26T17:03:33Z + MESH: + + ATP‐dependent recombinases + + + 0.99920446 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9992754 + protein + cleaner0 + 2023-07-26T16:05:12Z + PR: + + BRCA2 + + + 0.9992434 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.99925226 + protein + cleaner0 + 2023-07-26T16:05:12Z + PR: + + BRCA2 + + + 0.99922514 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9992255 + protein + cleaner0 + 2023-07-26T16:05:12Z + PR: + + BRCA2 + + + 0.99644136 + complex_assembly + cleaner0 + 2023-07-26T16:03:57Z + GO: + + BRCA2:RAD51 + + + 0.996708 + complex_assembly + cleaner0 + 2023-07-26T16:03:57Z + GO: + + BRCA2:RAD51 + + + 0.99927634 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9992718 + protein + cleaner0 + 2023-07-26T16:05:12Z + PR: + + BRCA2 + + + 0.99885535 + structure_element + cleaner0 + 2023-07-26T16:16:47Z + SO: + + BRC repeats + + + 0.9988293 + protein_state + cleaner0 + 2023-07-26T16:18:15Z + DUMMY: + + conserved + + + structure_element + SO: + cleaner0 + 2023-07-26T16:17:23Z + + ‘FxxA’ motif 9 + + + 0.99902153 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + protein + PR: + cleaner0 + 2023-07-26T16:15:47Z + + RadA + + + structure_element + SO: + cleaner0 + 2023-07-26T16:04:46Z + + FxxA + + + 0.97832024 + structure_element + cleaner0 + 2023-07-26T16:20:15Z + SO: + + FTTA + + + 0.99859184 + species + cleaner0 + 2023-07-26T16:16:41Z + MESH: + + human + + + 0.99923325 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.99917173 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.99886394 + protein + cleaner0 + 2023-07-26T16:15:47Z + PR: + + RadA + + + structure_element + SO: + cleaner0 + 2023-07-26T16:04:46Z + + FxxA + + + 0.9991766 + structure_element + cleaner0 + 2023-07-26T16:16:47Z + SO: + + BRC repeats + + + 0.99913067 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9915185 + structure_element + cleaner0 + 2023-07-26T16:19:35Z + SO: + + oligomerisation sequence + + + 0.9979475 + site + cleaner0 + 2023-07-26T16:16:59Z + SO: + + FxxA‐binding site + + + 0.99926895 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + + ABBR + paragraph + 2637 + In general, the dominant contribution of certain residues to the overall binding energy of a protein–protein interaction are known as ‘hot‐spot’ residues. Interestingly, small molecule inhibitors of PPIs are often found to occupy the same pockets which are otherwise occupied by hot‐spot residues in the native complex. It is therefore of great interest to identify hot‐spots in an effort to guide drug discovery efforts against a PPI. Further, a correlation between residues that are strongly conserved and hot‐spot residues has been reported 10. Purely based on the amino acid consensus sequence reported by Pellegrini et al., 11 phenylalanine and alanine would both be expected to be hot‐spots and to a lesser extent, threonine. However, whilst the identification of highly conserved residues may be a good starting point for identifying hot‐spots, experimental validation by mutation of these sequences is vital. + + 0.9907151 + evidence + cleaner0 + 2023-07-26T17:09:13Z + DUMMY: + + binding energy + + + 0.9954261 + site + cleaner0 + 2023-07-26T16:17:43Z + SO: + + hot‐spot + + + 0.9654628 + site + cleaner0 + 2023-07-26T17:08:21Z + SO: + + pockets + + + site + SO: + cleaner0 + 2023-07-26T16:17:44Z + + hot‐spot + + + 0.73989046 + protein_state + cleaner0 + 2023-07-26T17:06:09Z + DUMMY: + + native + + + 0.9973863 + site + cleaner0 + 2023-07-26T17:08:30Z + SO: + + hot‐spots + + + 0.9987445 + protein_state + cleaner0 + 2023-07-26T16:18:08Z + DUMMY: + + strongly conserved + + + site + SO: + cleaner0 + 2023-07-26T16:17:44Z + + hot‐spot + + + 0.99785006 + residue_name + cleaner0 + 2023-07-26T16:18:03Z + SO: + + phenylalanine + + + 0.9975369 + residue_name + cleaner0 + 2023-07-26T16:18:06Z + SO: + + alanine + + + 0.9974454 + site + cleaner0 + 2023-07-26T17:08:30Z + SO: + + hot‐spots + + + 0.9978544 + residue_name + cleaner0 + 2023-07-26T16:18:01Z + SO: + + threonine + + + 0.998924 + protein_state + cleaner0 + 2023-07-26T16:18:10Z + DUMMY: + + highly conserved + + + 0.99733776 + site + cleaner0 + 2023-07-26T17:08:30Z + SO: + + hot‐spots + + + 0.9932742 + experimental_method + cleaner0 + 2023-07-26T17:11:41Z + MESH: + + mutation + + + + ABBR + paragraph + 3572 + The importance of residues in the FxxA motif has been probed by a variety of techniques, collated in Table 1. Briefly, mutating phenylalanine to glutamic acid inactivated the BRC4 peptide and prevented RAD51 oligomerisation 11, 12. A phenylalanine‐truncated BRC4 is also found to be inactive 13, however, introducing a tryptophan for phenylalanine was found to have no significant effect on BRC4 affinity 12. A glutamine replacing the histidine in BRC4 maintains BRC4 activity 13. The ability of BRC3 to interact with RAD51 nucleoprotein filaments is disrupted when threonine is mutated to an alanine 3. Similarly, mutating alanine to glutamic acid in the RAD51 oligomerisation sequence 11 or to serine in BRC4 13 leads to loss of interaction in both cases. The BRC5 repeat in humans has serine in the place of alanine, and is thought to be a nonbinding repeat 12. Mutations identified in the clinic, in the FxxA region of the BRC repeats of BRCA2 are collated in Table 1 14. It is difficult to state the clinical relevance of these mutations as they are annotated as ‘unvalidated’, that is, it is not known whether they contribute to the disease phenotype or are neutral variants. For completeness, we present them here with this caveat, and to make the comment that these clinical mutations are consistent with abrogating the interaction with RAD51. + + structure_element + SO: + cleaner0 + 2023-07-26T16:04:46Z + + FxxA + + + 0.97577786 + experimental_method + cleaner0 + 2023-07-26T17:11:45Z + MESH: + + mutating + + + 0.99699545 + residue_name + cleaner0 + 2023-07-26T17:00:46Z + SO: + + phenylalanine + + + residue_name + SO: + cleaner0 + 2023-07-26T16:18:42Z + + glutamic acid + + + 0.982657 + protein_state + cleaner0 + 2023-07-26T17:06:14Z + DUMMY: + + inactivated + + + 0.99903286 + chemical + cleaner0 + 2023-07-26T16:34:55Z + CHEBI: + + BRC4 + + + 0.99923897 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.99665284 + protein_state + cleaner0 + 2023-07-26T16:18:26Z + DUMMY: + + phenylalanine‐truncated + + + 0.99904925 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9963792 + protein_state + cleaner0 + 2023-07-26T17:06:19Z + DUMMY: + + inactive + + + 0.8490267 + experimental_method + cleaner0 + 2023-07-26T17:11:48Z + MESH: + + introducing + + + 0.99701047 + residue_name + cleaner0 + 2023-07-26T16:18:55Z + SO: + + tryptophan + + + 0.9974559 + residue_name + cleaner0 + 2023-07-26T17:00:49Z + SO: + + phenylalanine + + + 0.9984425 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9841949 + evidence + cleaner0 + 2023-07-26T17:09:26Z + DUMMY: + + affinity + + + 0.99741167 + residue_name + cleaner0 + 2023-07-26T16:18:47Z + SO: + + glutamine + + + 0.47801086 + experimental_method + cleaner0 + 2023-07-26T17:11:51Z + MESH: + + replacing + + + 0.9974899 + residue_name + cleaner0 + 2023-07-26T16:18:49Z + SO: + + histidine + + + 0.99859816 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9987394 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9989969 + chemical + cleaner0 + 2023-07-26T16:35:06Z + CHEBI: + + BRC3 + + + 0.9992268 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9974733 + residue_name + cleaner0 + 2023-07-26T16:39:50Z + SO: + + threonine + + + 0.9941875 + experimental_method + cleaner0 + 2023-07-26T17:11:56Z + MESH: + + mutated + + + 0.99733514 + residue_name + cleaner0 + 2023-07-26T16:19:01Z + SO: + + alanine + + + 0.99345124 + experimental_method + cleaner0 + 2023-07-26T17:11:59Z + MESH: + + mutating + + + 0.99758816 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + residue_name + SO: + cleaner0 + 2023-07-26T16:19:10Z + + glutamic acid + + + 0.9992557 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9967948 + structure_element + cleaner0 + 2023-07-26T16:19:34Z + SO: + + oligomerisation sequence + + + 0.9974023 + residue_name + cleaner0 + 2023-07-26T17:00:52Z + SO: + + serine + + + 0.9991073 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.86205214 + chemical + cleaner0 + 2023-07-26T16:35:20Z + CHEBI: + + BRC5 + + + 0.99791235 + species + cleaner0 + 2023-07-26T17:02:08Z + MESH: + + humans + + + 0.99766123 + residue_name + cleaner0 + 2023-07-26T17:00:55Z + SO: + + serine + + + 0.9975331 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.9990456 + structure_element + cleaner0 + 2023-07-26T17:04:06Z + SO: + + nonbinding repeat + + + structure_element + SO: + cleaner0 + 2023-07-26T16:04:46Z + + FxxA + + + 0.9990586 + structure_element + cleaner0 + 2023-07-26T16:16:47Z + SO: + + BRC repeats + + + 0.99931264 + protein + cleaner0 + 2023-07-26T16:05:12Z + PR: + + BRCA2 + + + 0.9992446 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + + feb212139-tbl-0001.xml + feb212139-tbl-0001 + TABLE + table_caption + 4930 + Summary of FxxA‐relevant mutations previously reported and degree of characterisation. The mutation, relevant peptide context, resulting FxxA motif sequence and experimental technique for each entry is given. For clarity, mutated residues are shown in bold + + 0.99378055 + structure_element + cleaner0 + 2023-07-26T16:04:46Z + SO: + + FxxA + + + structure_element + SO: + cleaner0 + 2023-07-26T16:04:46Z + + FxxA + + + + feb212139-tbl-0001.xml + feb212139-tbl-0001 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><col style="border-right:solid 1px #000000" span="1"/><col style="border-right:solid 1px #000000" span="1"/><col style="border-right:solid 1px #000000" span="1"/><col style="border-right:solid 1px #000000" span="1"/><col style="border-right:solid 1px #000000" span="1"/><thead valign="top"><tr style="border-bottom:solid 1px #000000"><th align="left" valign="top" rowspan="1" colspan="1">Mutation context<xref ref-type="fn" rid="feb212139-note-0002">a</xref> +</th><th align="left" valign="top" rowspan="1" colspan="1">Mutation</th><th align="left" valign="top" rowspan="1" colspan="1">FxxA motif</th><th align="left" valign="top" rowspan="1" colspan="1">Technique used</th><th align="left" valign="top" rowspan="1" colspan="1">Effect</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">RAD51 (FTTA)</td><td align="left" rowspan="1" colspan="1">F86E</td><td align="left" rowspan="1" colspan="1"> +<monospace><bold>E</bold>TTA</monospace> +</td><td align="left" rowspan="1" colspan="1">Immunoprecipitation <xref rid="feb212139-bib-0011" ref-type="ref">11</xref> +</td><td align="left" rowspan="1" colspan="1">No binding</td></tr><tr><td align="left" rowspan="1" colspan="1">BRC4 (FHTA)</td><td align="left" rowspan="1" colspan="1">F1524E</td><td align="left" rowspan="1" colspan="1"> +<monospace><bold>E</bold>HTA</monospace> +</td><td align="left" rowspan="1" colspan="1">Competitive ELISA <xref rid="feb212139-bib-0012" ref-type="ref">12</xref> +</td><td align="left" rowspan="1" colspan="1">Peptide inactive</td></tr><tr><td align="left" rowspan="1" colspan="1">BRC4 (FHTA)</td><td align="left" rowspan="1" colspan="1">F1524W</td><td align="left" rowspan="1" colspan="1"> +<monospace><bold>W</bold>HTA</monospace> +</td><td align="left" rowspan="1" colspan="1">Competitive ELISA <xref rid="feb212139-bib-0012" ref-type="ref">12</xref> +</td><td align="left" rowspan="1" colspan="1">Comparable activity to WT</td></tr><tr><td align="left" rowspan="1" colspan="1">BRC4 (FHTA)</td><td align="left" rowspan="1" colspan="1">F1524V</td><td align="left" rowspan="1" colspan="1"> +<monospace><bold>V</bold>HTA</monospace> +</td><td align="left" rowspan="1" colspan="1">BRCA2 mutations database <xref rid="feb212139-bib-0014" ref-type="ref">14</xref> +</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" rowspan="1" colspan="1">BRC4 (FHTA)</td><td align="left" rowspan="1" colspan="1">ΔF1524</td><td align="left" rowspan="1" colspan="1"> +<monospace><bold>‐</bold>HTA</monospace> +</td><td align="left" rowspan="1" colspan="1">Dissociation of RAD51‐DNA complex <xref rid="feb212139-bib-0013" ref-type="ref">13</xref> +</td><td align="left" rowspan="1" colspan="1">Peptide inactive</td></tr><tr><td align="left" rowspan="1" colspan="1">BRC4 (FHTA)</td><td align="left" rowspan="1" colspan="1">H1525Q</td><td align="left" rowspan="1" colspan="1"> +<monospace>F<bold>Q</bold>TA</monospace> +</td><td align="left" rowspan="1" colspan="1">Dissociation of RAD51‐DNA complex <xref rid="feb212139-bib-0013" ref-type="ref">13</xref> +</td><td align="left" rowspan="1" colspan="1">Comparable activity</td></tr><tr><td align="left" rowspan="1" colspan="1">BRC7 (FSTA)</td><td align="left" rowspan="1" colspan="1">S1979R</td><td align="left" rowspan="1" colspan="1"> +<monospace>F<bold>R</bold>TA</monospace> +</td><td align="left" rowspan="1" colspan="1">BRCA2 mutations database <xref rid="feb212139-bib-0014" ref-type="ref">14</xref> +</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" rowspan="1" colspan="1">BRC3 (FQTA)</td><td align="left" rowspan="1" colspan="1">T1430A</td><td align="left" rowspan="1" colspan="1"> +<monospace>FQ<bold>A</bold>A</monospace> +</td><td align="left" rowspan="1" colspan="1">RAD51:DNA bandshift assay <xref rid="feb212139-bib-0003" ref-type="ref">3</xref> +</td><td align="left" rowspan="1" colspan="1">Peptide inactive</td></tr><tr><td align="left" rowspan="1" colspan="1">BRC3 (FQTA)</td><td align="left" rowspan="1" colspan="1">T1430A</td><td align="left" rowspan="1" colspan="1"> +<monospace>FQ<bold>A</bold>A</monospace> +</td><td align="left" rowspan="1" colspan="1">Electron microscopic visualisation of nucleoprotein filaments <xref rid="feb212139-bib-0003" ref-type="ref">3</xref> +</td><td align="left" rowspan="1" colspan="1">Peptide inactive</td></tr><tr><td align="left" rowspan="1" colspan="1">BRC1 (FRTA)</td><td align="left" rowspan="1" colspan="1">T1011R</td><td align="left" rowspan="1" colspan="1"> +<monospace>FR<bold>R</bold>A</monospace> +</td><td align="left" rowspan="1" colspan="1">BRCA2 mutations database <xref rid="feb212139-bib-0014" ref-type="ref">14</xref> +</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" rowspan="1" colspan="1">BRC2 (FYSA)</td><td align="left" rowspan="1" colspan="1">S1221P</td><td align="left" rowspan="1" colspan="1"> +<monospace>FY<bold>P</bold>A</monospace> +</td><td align="left" rowspan="1" colspan="1">BRCA2 mutations database <xref rid="feb212139-bib-0014" ref-type="ref">14</xref> +</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" rowspan="1" colspan="1">BRC2 (FYSA)</td><td align="left" rowspan="1" colspan="1">S1221Y</td><td align="left" rowspan="1" colspan="1"> +<monospace>FY<bold>Y</bold>A</monospace> +</td><td align="left" rowspan="1" colspan="1">BRCA2 mutations database <xref rid="feb212139-bib-0014" ref-type="ref">14</xref> +</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" rowspan="1" colspan="1">RAD51 (FTTA)</td><td align="left" rowspan="1" colspan="1">A89E</td><td align="left" rowspan="1" colspan="1"> +<monospace>FTT<bold>E</bold></monospace> +</td><td align="left" rowspan="1" colspan="1">Immunoprecipitation <xref rid="feb212139-bib-0011" ref-type="ref">11</xref> +</td><td align="left" rowspan="1" colspan="1">No binding</td></tr><tr><td align="left" rowspan="1" colspan="1">BRC4 (FHTA)</td><td align="left" rowspan="1" colspan="1">A1527S</td><td align="left" rowspan="1" colspan="1"> +<monospace>FHT<bold>S</bold></monospace> +</td><td align="left" rowspan="1" colspan="1">Dissociation of RAD51‐DNA complex <xref rid="feb212139-bib-0013" ref-type="ref">13</xref> +</td><td align="left" rowspan="1" colspan="1">Peptide inactive</td></tr></tbody></table> + + 5189 + Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive + + structure_element + SO: + cleaner0 + 2023-07-26T16:04:46Z + + FxxA + + + 0.534558 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:20:16Z + + FTTA + + + 0.84453404 + mutant + cleaner0 + 2023-07-26T16:20:55Z + MESH: + + F86E + + + 0.7938878 + structure_element + cleaner0 + 2023-07-26T16:20:50Z + SO: + + ETTA + + + 0.800821 + experimental_method + cleaner0 + 2023-07-26T17:12:04Z + MESH: + + Immunoprecipitation + + + 0.8440317 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.5206655 + structure_element + cleaner0 + 2023-07-26T16:21:33Z + SO: + + FHTA + + + 0.66016483 + mutant + cleaner0 + 2023-07-26T16:22:07Z + MESH: + + F1524E + + + structure_element + SO: + cleaner0 + 2023-07-26T16:22:03Z + + EHTA + + + experimental_method + MESH: + cleaner0 + 2023-07-26T16:22:27Z + + Competitive ELISA + + + 0.9012287 + protein_state + cleaner0 + 2023-07-26T17:06:24Z + DUMMY: + + inactive + + + 0.7178567 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:21:34Z + + FHTA + + + 0.78329676 + mutant + cleaner0 + 2023-07-26T16:22:36Z + MESH: + + F1524W + + + structure_element + SO: + cleaner0 + 2023-07-26T16:22:51Z + + WHTA + + + experimental_method + MESH: + cleaner0 + 2023-07-26T16:22:27Z + + Competitive ELISA + + + 0.9991561 + protein_state + cleaner0 + 2023-07-26T17:06:33Z + DUMMY: + + WT + + + 0.55990845 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:21:34Z + + FHTA + + + 0.8656532 + mutant + cleaner0 + 2023-07-26T16:59:04Z + MESH: + + F1524V + + + structure_element + SO: + cleaner0 + 2023-07-26T16:23:14Z + + VHTA + + + protein + PR: + cleaner0 + 2023-07-26T16:05:12Z + + BRCA2 + + + 0.98529506 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9320015 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + mutant + MESH: + cleaner0 + 2023-07-26T16:59:33Z + + ΔF1524 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:59:43Z + + HTA + + + 0.9833138 + complex_assembly + cleaner0 + 2023-07-26T17:02:17Z + GO: + + RAD51‐DNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T16:26:26Z + + inactive + + + 0.8127769 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:21:34Z + + FHTA + + + 0.7130153 + mutant + cleaner0 + 2023-07-26T16:59:48Z + MESH: + + H1525Q + + + structure_element + SO: + cleaner0 + 2023-07-26T16:23:33Z + + FQTA + + + 0.9634507 + complex_assembly + cleaner0 + 2023-07-26T17:02:18Z + GO: + + RAD51‐DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:35:45Z + + BRC7 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:28:39Z + + FSTA + + + 0.8763732 + mutant + cleaner0 + 2023-07-26T16:59:51Z + MESH: + + S1979R + + + structure_element + SO: + cleaner0 + 2023-07-26T16:27:06Z + + FRTA + + + protein + PR: + cleaner0 + 2023-07-26T16:05:12Z + + BRCA2 + + + 0.9441306 + chemical + cleaner0 + 2023-07-26T16:35:07Z + CHEBI: + + BRC3 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:23:33Z + + FQTA + + + 0.82190853 + mutant + cleaner0 + 2023-07-26T16:59:57Z + MESH: + + T1430A + + + 0.9656751 + structure_element + cleaner0 + 2023-07-26T16:23:46Z + SO: + + FQAA + + + 0.99068993 + complex_assembly + cleaner0 + 2023-07-26T17:02:27Z + GO: + + RAD51:DNA + + + 0.972504 + experimental_method + cleaner0 + 2023-07-26T17:12:07Z + MESH: + + bandshift assay + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T17:06:53Z + + inactive + + + 0.97438896 + chemical + cleaner0 + 2023-07-26T16:35:07Z + CHEBI: + + BRC3 + + + 0.8860515 + structure_element + cleaner0 + 2023-07-26T16:23:33Z + SO: + + FQTA + + + 0.5953698 + mutant + cleaner0 + 2023-07-26T17:00:25Z + MESH: + + T1430A + + + 0.97964513 + structure_element + cleaner0 + 2023-07-26T16:23:45Z + SO: + + FQAA + + + 0.9761479 + experimental_method + cleaner0 + 2023-07-26T17:12:13Z + MESH: + + Electron microscopic + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T16:26:52Z + + inactive + + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:35:56Z + + BRC1 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:27:05Z + + FRTA + + + 0.81862295 + mutant + cleaner0 + 2023-07-26T16:59:59Z + MESH: + + T1011R + + + structure_element + SO: + cleaner0 + 2023-07-26T16:27:40Z + + FRRA + + + protein + PR: + cleaner0 + 2023-07-26T16:05:12Z + + BRCA2 + + + 0.78808254 + chemical + cleaner0 + 2023-07-26T17:00:18Z + CHEBI: + + BRC2 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:27:50Z + + FYSA + + + 0.81237847 + mutant + cleaner0 + 2023-07-26T17:00:32Z + MESH: + + S1221P + + + structure_element + SO: + cleaner0 + 2023-07-26T16:28:10Z + + FYPA + + + protein + PR: + cleaner0 + 2023-07-26T16:05:12Z + + BRCA2 + + + 0.8775508 + chemical + cleaner0 + 2023-07-26T17:00:18Z + CHEBI: + + BRC2 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:27:50Z + + FYSA + + + 0.85255635 + mutant + cleaner0 + 2023-07-26T17:00:34Z + MESH: + + S1221Y + + + structure_element + SO: + cleaner0 + 2023-07-26T16:27:58Z + + FYYA + + + protein + PR: + cleaner0 + 2023-07-26T16:05:12Z + + BRCA2 + + + 0.5648615 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:20:16Z + + FTTA + + + 0.9408125 + mutant + cleaner0 + 2023-07-26T17:00:37Z + MESH: + + A89E + + + structure_element + SO: + cleaner0 + 2023-07-26T16:57:10Z + + FTTE + + + experimental_method + MESH: + cleaner0 + 2023-07-26T16:57:20Z + + Immunoprecipitation + + + 0.67835486 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:21:34Z + + FHTA + + + 0.6297939 + mutant + cleaner0 + 2023-07-26T17:00:40Z + MESH: + + A1527S + + + structure_element + SO: + cleaner0 + 2023-07-26T16:28:32Z + + FHTS + + + 0.8125436 + complex_assembly + cleaner0 + 2023-07-26T17:02:18Z + GO: + + RAD51‐DNA + + + 0.6590094 + protein_state + cleaner0 + 2023-07-26T17:06:56Z + DUMMY: + + inactive + + + + feb212139-tbl-0001.xml + feb212139-tbl-0001 + TABLE + table_footnote + 6227 + The wild‐type FxxA sequence is indicated in parenthesis. + + 0.99905777 + protein_state + cleaner0 + 2023-07-26T16:29:13Z + DUMMY: + + wild‐type + + + 0.99849474 + structure_element + cleaner0 + 2023-07-26T16:04:47Z + SO: + + FxxA + + + + ABBR + paragraph + 6286 + In this work, we report the most detailed study of systematic mutations of peptides to probe the FxxA‐binding motif to date. We have chosen to focus on tetrapeptides, which allows us to examine the effect of mutation on the fundamental unit of binding, FxxA, rather than in the context of either the BRC repeat or self‐oligomerisation sequence. Affinities of peptides were measured directly using Isothermal Titration Calorimetry (ITC) and the structures of many of the peptides bound to humanised RadA were determined by X‐ray crystallography. The use of ITC is generally perceived as a gold‐standard in protein–ligand characterisation, rather than a competitive assay which may be prone to aggregation artefacts. Wild‐type human RAD51, however, is a heterogeneous mixture of oligomers and when monomerised by mutation, is highly unstable. In this context, we have previously reported the use of stable monomeric forms of RAD51, humanised from Pyrococcus furiosus homologue RadA, for ITC experiments and X‐ray crystallography 8, 15. + + 0.9980581 + experimental_method + cleaner0 + 2023-07-26T17:12:21Z + MESH: + + systematic mutations + + + 0.9990543 + structure_element + cleaner0 + 2023-07-26T16:29:06Z + SO: + + FxxA‐binding motif + + + 0.85504335 + chemical + cleaner0 + 2023-07-26T16:45:14Z + CHEBI: + + tetrapeptides + + + 0.9969074 + experimental_method + cleaner0 + 2023-07-26T17:12:24Z + MESH: + + mutation + + + 0.9983365 + structure_element + cleaner0 + 2023-07-26T16:04:47Z + SO: + + FxxA + + + 0.99906564 + structure_element + cleaner0 + 2023-07-26T17:04:12Z + SO: + + BRC repeat + + + 0.9682815 + structure_element + cleaner0 + 2023-07-26T16:29:04Z + SO: + + self‐oligomerisation sequence + + + 0.9963232 + evidence + cleaner0 + 2023-07-26T17:09:32Z + DUMMY: + + Affinities + + + 0.9990049 + experimental_method + cleaner0 + 2023-07-26T17:12:27Z + MESH: + + Isothermal Titration Calorimetry + + + 0.99859995 + experimental_method + cleaner0 + 2023-07-26T16:29:17Z + MESH: + + ITC + + + 0.99843127 + evidence + cleaner0 + 2023-07-26T17:09:35Z + DUMMY: + + structures + + + 0.9990597 + protein_state + cleaner0 + 2023-07-26T16:43:25Z + DUMMY: + + bound to + + + 0.9876819 + protein_state + cleaner0 + 2023-07-26T16:37:09Z + DUMMY: + + humanised + + + 0.99935275 + protein + cleaner0 + 2023-07-26T16:15:47Z + PR: + + RadA + + + 0.99897313 + experimental_method + cleaner0 + 2023-07-26T16:29:25Z + MESH: + + X‐ray crystallography + + + 0.998669 + experimental_method + cleaner0 + 2023-07-26T16:29:17Z + MESH: + + ITC + + + 0.88887405 + experimental_method + cleaner0 + 2023-07-26T17:12:31Z + MESH: + + competitive assay + + + 0.99905324 + protein_state + cleaner0 + 2023-07-26T16:29:12Z + DUMMY: + + Wild‐type + + + 0.99876416 + species + cleaner0 + 2023-07-26T16:16:41Z + MESH: + + human + + + 0.9993426 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9963168 + oligomeric_state + cleaner0 + 2023-07-26T16:04:09Z + DUMMY: + + oligomers + + + 0.99779105 + oligomeric_state + cleaner0 + 2023-07-26T16:29:41Z + DUMMY: + + monomerised + + + 0.98276305 + experimental_method + cleaner0 + 2023-07-26T17:12:35Z + MESH: + + mutation + + + 0.8997078 + protein_state + cleaner0 + 2023-07-26T17:07:02Z + DUMMY: + + highly unstable + + + 0.9989635 + protein_state + cleaner0 + 2023-07-26T17:07:05Z + DUMMY: + + stable + + + 0.99876297 + oligomeric_state + cleaner0 + 2023-07-26T16:29:36Z + DUMMY: + + monomeric + + + 0.9993001 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9828095 + protein_state + cleaner0 + 2023-07-26T16:37:09Z + DUMMY: + + humanised + + + 0.9986927 + species + cleaner0 + 2023-07-26T16:29:30Z + MESH: + + Pyrococcus furiosus + + + 0.99930453 + protein + cleaner0 + 2023-07-26T16:15:47Z + PR: + + RadA + + + 0.998747 + experimental_method + cleaner0 + 2023-07-26T16:29:17Z + MESH: + + ITC + + + 0.9989815 + experimental_method + cleaner0 + 2023-07-26T16:29:24Z + MESH: + + X‐ray crystallography + + + + METHODS + title_1 + 7334 + Materials and methods + + + METHODS + title_2 + 7356 + Peptide synthesis + + + METHODS + paragraph + 7374 + Peptides were synthesised using solid‐phase FMOC chemistry by Alta Biosciences (Birmingham, UK) or the Protein and Nucleic Acid Service at the Department of Biochemistry (University of Cambridge). All peptides prepared and used in the study were N‐acetylated and C‐amide terminated. + + + METHODS + title_2 + 7663 + Protein preparation + + + METHODS + paragraph + 7683 + Protein expression and purification was performed as described previously 15. In brief, monomeric HumRadA2 was expressed in E. coli using T7‐based expression vector at 37 °C for 3 h. Soluble cell lysate was heat treated to precipitate most of the cellular proteins and the soluble fraction containing HumRadA2 was purified using a combination of cation exchange chromatography at pH 6.0 and size‐exclusion chromatography in 10 mm MES, 100 mm NaCl pH 6.0 buffer. Protein concentration was determined using the calculated extinction coefficient at 280 nm, and stored at −80 °C in small aliquots after flash freezing. + + + METHODS + title_2 + 8306 + Isothermal titration calorimetry + + + METHODS + paragraph + 8339 + Isothermal titration calorimetry experiments were performed at 25 °C on a MicroCal iTC200. HumRadA2 (600 μm in 20 mm MES pH 6.0 with 100 mm NaCl and 0.5 mm EDTA) was diluted with Tris buffer (200 mm, pH 7.5 with 100 mm NaCl) to 64–83 μm. Peptides were dissolved in MilliQ water (50 mm) and an aliquot taken and diluted with 200 mm Tris, pH 7.5, 100 mm NaCl to give a ligand solution of 2.5–5 mm. The peptide solution was titrated into the protein solution; 16 injections (2.4 μL) of 4.8 s duration were made at 80‐s intervals. The initial injection of ligand (0.4 μL) was discarded during data analysis. Control experiments of peptides to buffer showed insignificant heats. The data were processed and thermodynamic parameters obtained by fitting the data to a single‐site‐binding model using Origin software and fixing the stoichiometry as 1.0 for weak‐binding ligands 16. All data from ITC measurements are shown in the Figs S1 and S2. + + + METHODS + title_2 + 9294 + X‐ray crystallography + + + METHODS + paragraph + 9318 + Monomerised RadA proteins were crystallised in the same conditions as described previously 15. Peptides were soaked into the crystals at 2–5 mm concentration overnight in the presence of 10% glycerol as a cryoprotectant. Crystals were cryo‐cooled in liquid nitrogen and data collected at synchrotron light sources and processed using XDS: details of this are found in crystallographic table (Table S1 in Supporting Information). Structures were solved by molecular replacement using unliganded, monomeric RadA coordinates (PDB: 4b3b, after removal of FHTA peptide) as a search model and refined with an automated procedure using Refmac5 17. After inspection of the resulting electron density, the bound peptides were modelled into the density and structures were further refined using Refmac5 18 and phenix.refine 19, and manually rebuilt using Coot 20. Coordinates and structure factors have been deposited in the PDB under accession codes as listed in Table 2 and in the crystallographic data table in the Supporting Information. With the exception of FATA peptide complex, which was crystallised with wild‐type RadA, the structures are determined using HumRadA1 mutant. + + + feb212139-tbl-0002.xml + feb212139-tbl-0002 + TABLE + table_caption + 10497 + Summary of peptide‐binding data determined by ITC against HumRadA2. Mutated residues are highlighted in bold. All peptides were N‐acetylated and C‐amide terminated + + + feb212139-tbl-0002.xml + feb212139-tbl-0002 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table xmlns:xlink="http://www.w3.org/1999/xlink" frame="hsides" rules="groups"><col style="border-right:solid 1px #000000" span="1"/><col style="border-right:solid 1px #000000" span="1"/><col style="border-right:solid 1px #000000" span="1"/><col style="border-right:solid 1px #000000" span="1"/><col style="border-right:solid 1px #000000" span="1"/><col style="border-right:solid 1px #000000" span="1"/><thead valign="top"><tr style="border-bottom:solid 1px #000000"><th align="left" valign="top" rowspan="1" colspan="1">Table entry</th><th align="left" valign="top" rowspan="1" colspan="1">Peptide</th><th align="char" valign="top" rowspan="1" colspan="1"> +<italic>K</italic> +<sub>D</sub>/μ<sc>m</sc> +</th><th align="char" char=" " valign="top" rowspan="1" colspan="1">Δ<italic>H</italic>/cal·mol<sup>−1</sup> +</th><th align="char" char=" " valign="top" rowspan="1" colspan="1"> +<italic>T</italic>Δ<italic>S</italic>/cal·mol<sup>−1</sup> +</th><th align="left" valign="top" rowspan="1" colspan="1">PDB code</th></tr></thead><tbody><tr><td align="left" colspan="6" rowspan="1">First position variation</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"> +<monospace>FHTA</monospace> +</td><td align="char" char="±" rowspan="1" colspan="1">280 ± 20</td><td align="char" char="±" rowspan="1" colspan="1">−2388 ± 94</td><td align="char" char="." rowspan="1" colspan="1">2453</td><td align="left" rowspan="1" colspan="1"> +<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4b3b">4b3b</ext-link> +<xref rid="feb212139-bib-0015" ref-type="ref">15</xref> +</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1"> +<monospace><bold>W</bold>HTA</monospace> +</td><td align="char" char="±" rowspan="1" colspan="1">93 ± 3</td><td align="char" char="±" rowspan="1" colspan="1">−2768 ± 34</td><td align="char" char="." rowspan="1" colspan="1">2727</td><td align="left" rowspan="1" colspan="1"> +<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5fow">5fow</ext-link> +</td></tr><tr><td align="left" colspan="6" rowspan="1">Second position variation</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1"> +<monospace>F<bold>A</bold>TA</monospace> +</td><td align="char" char="±" rowspan="1" colspan="1">280 ± 29</td><td align="char" char="±" rowspan="1" colspan="1">−1820 ± 109</td><td align="char" char="." rowspan="1" colspan="1">3010</td><td align="left" rowspan="1" colspan="1"> +<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5fpk">5fpk</ext-link> +<xref ref-type="fn" rid="feb212139-note-0003">a</xref> +</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1"> +<monospace>F<bold>N</bold>TA</monospace> +</td><td align="char" char="±" rowspan="1" colspan="1">613 ± 44</td><td align="char" char="±" rowspan="1" colspan="1">−4036 ± 177</td><td align="char" char="." rowspan="1" colspan="1">346</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1"> +<monospace>F<bold>P</bold>TA</monospace> +</td><td align="char" rowspan="1" colspan="1">No detectable binding</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" colspan="6" rowspan="1">Third position variation</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">6</td><td align="left" rowspan="1" colspan="1"> +<monospace>FH<bold>P</bold>A</monospace> +</td><td align="char" char="±" rowspan="1" colspan="1">113 ± 3</td><td align="char" char="±" rowspan="1" colspan="1">−2155 ± 26</td><td align="char" char="." rowspan="1" colspan="1">3218</td><td align="left" rowspan="1" colspan="1"> +<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5fou">5fou</ext-link> +</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1"> +<monospace>FH<bold>A</bold>A</monospace> +</td><td align="char" char="±" rowspan="1" colspan="1">675 ± 60</td><td align="char" char="±" rowspan="1" colspan="1">−7948 ± 466</td><td align="char" char="." rowspan="1" colspan="1">−3636</td><td align="left" rowspan="1" colspan="1"> +<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5fox">5fox</ext-link> +</td></tr><tr><td align="left" colspan="6" rowspan="1">Fourth position variation</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">8</td><td align="left" rowspan="1" colspan="1"> +<monospace>FHT<bold>G</bold></monospace> +</td><td align="char" char="±" rowspan="1" colspan="1">1590 ± 300</td><td align="char" char="±" rowspan="1" colspan="1">−5518 ± 924</td><td align="char" char="." rowspan="1" colspan="1">−1702</td><td align="left" rowspan="1" colspan="1"> +<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5fov">5fov</ext-link> +</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">9</td><td align="left" rowspan="1" colspan="1"> +<monospace>FHT<bold>U</bold></monospace> +</td><td align="char" char="±" rowspan="1" colspan="1">680 ± 51</td><td align="char" char="±" rowspan="1" colspan="1">−14 600 ± 771</td><td align="char" char="." rowspan="1" colspan="1">−10 281</td><td align="left" rowspan="1" colspan="1"> +<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5fot">5fot</ext-link> +</td></tr><tr><td align="left" colspan="6" rowspan="1">Combination</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1"> +<monospace><bold>W</bold>H<bold>P</bold>A</monospace> +</td><td align="char" char="±" rowspan="1" colspan="1">330 ± 25</td><td align="char" char="±" rowspan="1" colspan="1">−6801 ± 318</td><td align="char" char="." rowspan="1" colspan="1">−2044</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" colspan="6" rowspan="1">Peptide truncations</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">11</td><td align="left" rowspan="1" colspan="1"> +<monospace>FH</monospace> +</td><td align="char" rowspan="3" colspan="1"> +No binding detected<break/> +No binding detected<break/> +No binding detected +</td><td align="char" rowspan="3" colspan="1"/><td align="char" rowspan="3" colspan="1"/><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1"> +<monospace>FHT</monospace> +</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">13</td><td align="left" rowspan="1" colspan="1"> +<monospace>HTA</monospace> +</td><td align="left" rowspan="1" colspan="1">–</td></tr></tbody></table> + + 10667 + Table entry Peptide KD/μm ΔH/cal·mol−1 TΔS/cal·mol−1 PDB code First position variation 1 FHTA 280 ± 20 −2388 ± 94 2453 4b3b15 2 WHTA 93 ± 3 −2768 ± 34 2727 5fow Second position variation 3 FATA 280 ± 29 −1820 ± 109 3010 5fpka 4 FNTA 613 ± 44 −4036 ± 177 346 – 5 FPTA No detectable binding – Third position variation 6 FHPA 113 ± 3 −2155 ± 26 3218 5fou 7 FHAA 675 ± 60 −7948 ± 466 −3636 5fox Fourth position variation 8 FHTG 1590 ± 300 −5518 ± 924 −1702 5fov 9 FHTU 680 ± 51 −14 600 ± 771 −10 281 5fot Combination 10 WHPA 330 ± 25 −6801 ± 318 −2044 – Peptide truncations 11 FH No binding detected No binding detected No binding detected – 12 FHT – 13 HTA – + + + feb212139-tbl-0002.xml + feb212139-tbl-0002 + TABLE + table_footnote + 11443 + Structure solved with wild‐type RadA. + + + METHODS + title_2 + 11483 + Sequence analysis + + + METHODS + paragraph + 11501 + Sequences of mammalian RAD51 proteins and archeal RadA orthologues were obtained from Ensembl (www.ensembl.org) and Uniprot (www.uniprot.org) databases. Sequences were aligned using ClustalX2, and aligned sequences for the FxxA motifs were used in WebLogo (weblogo.berkely.edu/logo.cgi) server 21 to derive the consensus diagrams shown in Figs 1 and 4. All the sequences used in these analyses are shown in Figs S4, S5 and S6. + + + FEB2-590-1094-g001.jpg + feb212139-fig-0001 + FIG + fig_caption + 11928 + Conservation of FxxA motif (A) BRC4 peptide (green cartoon) bound to truncated human RAD51 (grey surface) (PDB: 1n0w, 11). The blue dashed box highlights the FxxA interaction pocket. (B) Two interacting protein molecules of RAD51 from Saccharomyces cerevisiae are shown. One RAD51 (green cartoon) interacts with another molecule of RAD51 (grey and pink surface) via the FxxA pocket indicated by the dashed blue box. The N‐terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation. Sequence figures generated using Weblogo 3.0 21, sequence details are found in the Supporting Information. + + structure_element + SO: + cleaner0 + 2023-07-26T16:04:47Z + + FxxA + + + 0.9506754 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.999126 + protein_state + cleaner0 + 2023-07-26T16:43:25Z + DUMMY: + + bound to + + + 0.9981902 + protein_state + cleaner0 + 2023-07-26T17:07:10Z + DUMMY: + + truncated + + + 0.9987091 + species + cleaner0 + 2023-07-26T16:16:41Z + MESH: + + human + + + 0.9991491 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.99895304 + site + cleaner0 + 2023-07-26T16:42:49Z + SO: + + FxxA interaction pocket + + + 0.9990839 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9974071 + species + cleaner0 + 2023-07-26T16:43:29Z + MESH: + + Saccharomyces cerevisiae + + + 0.99901354 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.99908805 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.99897516 + site + cleaner0 + 2023-07-26T17:08:37Z + SO: + + FxxA pocket + + + 0.99899083 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9984875 + oligomeric_state + cleaner0 + 2023-07-26T17:05:20Z + DUMMY: + + protomer + + + 0.99657625 + oligomeric_state + cleaner0 + 2023-07-26T17:05:20Z + DUMMY: + + protomer + + + 0.8748951 + structure_element + cleaner0 + 2023-07-26T16:43:10Z + SO: + + FxxA oligomerisation sequence + + + structure_element + SO: + cleaner0 + 2023-07-26T16:04:47Z + + FxxA + + + 0.9984786 + species + cleaner0 + 2023-07-26T16:16:41Z + MESH: + + human + + + 0.99905276 + structure_element + cleaner0 + 2023-07-26T16:16:47Z + SO: + + BRC repeats + + + 0.99825853 + taxonomy_domain + cleaner0 + 2023-07-26T16:15:11Z + DUMMY: + + eukaryotic + + + 0.73481506 + protein_type + cleaner0 + 2023-07-26T17:02:57Z + MESH: + + RAD51s + + + 0.9801996 + protein_type + cleaner0 + 2023-07-26T17:03:42Z + MESH: + + RadAs + + + + RESULTS + title_1 + 12824 + Results + + + RESULTS + paragraph + 12832 + We have mutated and truncated the tetrapeptide epitope FHTA, and examined the effects both structurally and on the binding affinity with humanised RadA. As a comparative reference, we are using the FHTA sequence derived from the most tightly binding BRC repeat, BRC4 22. The peptides used are N‐acetylated and C‐amide terminated in order to provide the most relevant peptide in the context of a longer peptide chain. A summary of the peptide sequence, PDB codes and K D data measured by ITC with the corresponding ΔH and TΔS values are collated in Table 2. + + 0.99530095 + experimental_method + cleaner0 + 2023-07-26T17:12:47Z + MESH: + + mutated and truncated + + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:39:12Z + + tetrapeptide + + + 0.9974577 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.9988854 + evidence + cleaner0 + 2023-07-26T16:30:32Z + DUMMY: + + binding affinity + + + 0.99665076 + protein_state + cleaner0 + 2023-07-26T16:37:09Z + DUMMY: + + humanised + + + 0.99918216 + protein + cleaner0 + 2023-07-26T16:15:48Z + PR: + + RadA + + + 0.5367598 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.9974935 + structure_element + cleaner0 + 2023-07-26T17:04:17Z + SO: + + BRC repeat + + + 0.99721956 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T16:30:21Z + + N‐acetylated + + + 0.98856413 + evidence + cleaner0 + 2023-07-26T16:30:27Z + DUMMY: + + K D + + + 0.99875176 + experimental_method + cleaner0 + 2023-07-26T16:29:17Z + MESH: + + ITC + + + 0.9984059 + evidence + cleaner0 + 2023-07-26T16:30:36Z + DUMMY: + + ΔH + + + 0.9976464 + evidence + cleaner0 + 2023-07-26T16:30:40Z + DUMMY: + + TΔS + + + + RESULTS + paragraph + 13397 + Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210. The residue is highly conserved across BRC repeats and oligomerisation sequences. Consistent with this, the truncated HTA tripeptide could not be detected to bind to humanised, monomeric RadA, HumRadA2 (Table 2, entry 13). As previously discussed, there is some evidence that substituting a tryptophan for the phenylalanine at this position was tolerated in the context of BRC4 12. Therefore, the WHTA peptide was tested and found to not only be tolerated, but to increase the binding affinity of the peptide approximately threefold. + + 0.99960774 + residue_name_number + cleaner0 + 2023-07-26T16:38:19Z + DUMMY: + + Phe1524 + + + 0.99871147 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9695183 + site + cleaner0 + 2023-07-26T17:08:42Z + SO: + + surface pocket + + + 0.998021 + species + cleaner0 + 2023-07-26T16:16:41Z + MESH: + + human + + + 0.99930155 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9995981 + residue_name_number + cleaner0 + 2023-07-26T16:38:23Z + DUMMY: + + Met158 + + + 0.99960023 + residue_name_number + cleaner0 + 2023-07-26T16:38:27Z + DUMMY: + + Ile160 + + + 0.99960023 + residue_name_number + cleaner0 + 2023-07-26T16:38:31Z + DUMMY: + + Ala192 + + + 0.99959713 + residue_name_number + cleaner0 + 2023-07-26T16:38:35Z + DUMMY: + + Leu203 + + + 0.9996026 + residue_name_number + cleaner0 + 2023-07-26T16:38:40Z + DUMMY: + + Met210 + + + 0.9988133 + protein_state + cleaner0 + 2023-07-26T16:38:43Z + DUMMY: + + highly conserved + + + 0.99884677 + structure_element + cleaner0 + 2023-07-26T16:16:47Z + SO: + + BRC repeats + + + 0.9984584 + structure_element + cleaner0 + 2023-07-26T17:04:21Z + SO: + + oligomerisation sequences + + + 0.9989083 + protein_state + cleaner0 + 2023-07-26T16:38:46Z + DUMMY: + + truncated + + + 0.6846549 + structure_element + cleaner0 + 2023-07-26T16:37:37Z + SO: + + HTA + + + 0.8202713 + chemical + cleaner0 + 2023-07-26T16:37:48Z + CHEBI: + + tripeptide + + + 0.98587054 + protein_state + cleaner0 + 2023-07-26T16:37:09Z + DUMMY: + + humanised + + + 0.99878544 + oligomeric_state + cleaner0 + 2023-07-26T16:29:36Z + DUMMY: + + monomeric + + + 0.9992279 + protein + cleaner0 + 2023-07-26T16:15:48Z + PR: + + RadA + + + 0.99922657 + mutant + cleaner0 + 2023-07-26T17:03:14Z + MESH: + + HumRadA2 + + + 0.9976732 + experimental_method + cleaner0 + 2023-07-26T17:12:51Z + MESH: + + substituting + + + 0.9969066 + residue_name + cleaner0 + 2023-07-26T16:18:55Z + SO: + + tryptophan + + + 0.99715364 + residue_name + cleaner0 + 2023-07-26T16:38:13Z + SO: + + phenylalanine + + + 0.9989423 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.99292654 + structure_element + cleaner0 + 2023-07-26T16:22:51Z + SO: + + WHTA + + + 0.9988109 + evidence + cleaner0 + 2023-07-26T16:30:33Z + DUMMY: + + binding affinity + + + + RESULTS + paragraph + 14089 + The second position of the tetrapeptide was found to be largely invariant to changes in the side chains that were investigated. The residue makes no interactions with the RAD51 protein, but may make an internal hydrogen bond with Thr1520 in the context of BRC4, Fig. 3A. Replacing the histidine with an asparagine, chosen to potentially mimic the hydrogen bond donor–acceptor nature of histidine, resulted in a moderate, twofold decrease in potency (Table 2, entry 4). Mutating to an alanine, recapitulated the potency of FHTA, implying that the interactions made by histidine do not contribute overall to binding affinity (Table 2, entry 3). FPTA was also tested, but was found to have no affinity for the protein (Table 2, entry 5). Modelling suggests that a proline in the second position would be expected to clash sterically with the surface of the protein, and provides a rationale for the lack of binding observed. + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:39:11Z + + tetrapeptide + + + 0.99936825 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9963764 + bond_interaction + cleaner0 + 2023-07-26T16:38:51Z + MESH: + + hydrogen bond + + + 0.99956983 + residue_name_number + cleaner0 + 2023-07-26T16:39:17Z + DUMMY: + + Thr1520 + + + 0.9991948 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9979494 + experimental_method + cleaner0 + 2023-07-26T17:12:55Z + MESH: + + Replacing + + + 0.99749786 + residue_name + cleaner0 + 2023-07-26T16:39:23Z + SO: + + histidine + + + 0.9974106 + residue_name + cleaner0 + 2023-07-26T16:39:26Z + SO: + + asparagine + + + 0.9945632 + bond_interaction + cleaner0 + 2023-07-26T16:38:52Z + MESH: + + hydrogen bond + + + 0.99764866 + residue_name + cleaner0 + 2023-07-26T16:39:23Z + SO: + + histidine + + + 0.9978922 + experimental_method + cleaner0 + 2023-07-26T17:12:57Z + MESH: + + Mutating + + + 0.99710673 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.9993806 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.9977756 + residue_name + cleaner0 + 2023-07-26T16:39:23Z + SO: + + histidine + + + 0.998498 + evidence + cleaner0 + 2023-07-26T16:30:33Z + DUMMY: + + binding affinity + + + 0.9980749 + structure_element + cleaner0 + 2023-07-26T16:38:04Z + SO: + + FPTA + + + evidence + DUMMY: + cleaner0 + 2023-07-26T16:40:02Z + + affinity + + + 0.9975682 + residue_name + cleaner0 + 2023-07-26T16:39:31Z + SO: + + proline + + + + RESULTS + paragraph + 15013 + Threonine was mutated to an alanine, resulting in only a moderately weaker K D (twofold, Table 2, entry 7). In the context of a tetrapeptide at least, this result implies a lack of importance of a threonine at this position. Interestingly, it was found that a proline at this position improved the affinity almost threefold, to 113 μm (Table 2, entry 6). This beneficial mutation was incorporated with another previously identified variant to produce the peptide WHPA. Disappointingly, the combined effect of the mutations was not additive and the potency was weakened to 690 μm. + + 0.9973579 + residue_name + cleaner0 + 2023-07-26T16:39:50Z + SO: + + Threonine + + + 0.9983125 + experimental_method + cleaner0 + 2023-07-26T17:13:02Z + MESH: + + mutated + + + 0.9973705 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.99819696 + evidence + cleaner0 + 2023-07-26T16:30:28Z + DUMMY: + + K D + + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:39:12Z + + tetrapeptide + + + 0.99734503 + residue_name + cleaner0 + 2023-07-26T16:39:50Z + SO: + + threonine + + + 0.9977114 + residue_name + cleaner0 + 2023-07-26T16:39:32Z + SO: + + proline + + + 0.99879825 + evidence + cleaner0 + 2023-07-26T17:09:41Z + DUMMY: + + affinity + + + experimental_method + MESH: + cleaner0 + 2023-07-26T16:40:18Z + + mutation + + + 0.9978607 + structure_element + cleaner0 + 2023-07-26T16:45:53Z + SO: + + WHPA + + + + RESULTS + paragraph + 15595 + While the importance of the phenylalanine may be possible to predict from examination of the crystal structure, the alanine appears to be of much less importance in this regard. It is, however, a highly conserved residue and clearly of interest for systematic mutation. Removing the alanine residue entirely produced the truncated tripeptide FHT, which did not bind (Table 2, entry 12). The unnatural amino acid, α‐amino butyric acid (U), was introduced at the fourth position, positioning an ethyl group into the alanine pocket (Table 2, entry 9). Perhaps surprisingly, it was accommodated and the affinity dropped only by twofold as compared to FHTA. The effect of simply removing the β‐carbon of alanine, by mutation to glycine (FHTG), produced an approximately sixfold drop in binding affinity (Table 2, entry 8). This is in line with the observation that alanine is not 100% conserved and some archeal RadA proteins contain a glycine in the place of alanine 23. + + 0.9974511 + residue_name + cleaner0 + 2023-07-26T17:01:00Z + SO: + + phenylalanine + + + 0.9988898 + evidence + cleaner0 + 2023-07-26T17:09:44Z + DUMMY: + + crystal structure + + + 0.99755496 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.99900615 + protein_state + cleaner0 + 2023-07-26T16:41:38Z + DUMMY: + + highly conserved + + + 0.98846674 + experimental_method + cleaner0 + 2023-07-26T17:13:05Z + MESH: + + Removing + + + 0.99754447 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.9974942 + protein_state + cleaner0 + 2023-07-26T16:41:40Z + DUMMY: + + truncated + + + 0.4026408 + chemical + cleaner0 + 2023-07-26T16:37:49Z + CHEBI: + + tripeptide + + + 0.9564891 + structure_element + cleaner0 + 2023-07-26T16:40:56Z + SO: + + FHT + + + 0.9989956 + chemical + cleaner0 + 2023-07-26T16:41:05Z + CHEBI: + + α‐amino butyric acid + + + 0.99806684 + chemical + cleaner0 + 2023-07-26T16:41:08Z + CHEBI: + + U + + + 0.99861443 + site + cleaner0 + 2023-07-26T16:40:46Z + SO: + + alanine pocket + + + 0.99823797 + evidence + cleaner0 + 2023-07-26T17:09:48Z + DUMMY: + + affinity + + + 0.9994129 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.94364685 + experimental_method + cleaner0 + 2023-07-26T17:13:13Z + MESH: + + removing + + + 0.9975478 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.99640715 + experimental_method + cleaner0 + 2023-07-26T17:13:16Z + MESH: + + mutation to + + + 0.99719185 + residue_name + cleaner0 + 2023-07-26T16:41:46Z + SO: + + glycine + + + 0.5550107 + structure_element + cleaner0 + 2023-07-26T16:41:21Z + SO: + + FHTG + + + 0.9985664 + evidence + cleaner0 + 2023-07-26T16:30:33Z + DUMMY: + + binding affinity + + + 0.9978498 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.891015 + protein_state + cleaner0 + 2023-07-26T16:41:35Z + DUMMY: + + not 100% conserved + + + 0.9965121 + taxonomy_domain + cleaner0 + 2023-07-26T16:15:39Z + DUMMY: + + archeal + + + 0.99841964 + protein_type + cleaner0 + 2023-07-26T16:41:30Z + MESH: + + RadA proteins + + + 0.9978029 + residue_name + cleaner0 + 2023-07-26T16:41:47Z + SO: + + glycine + + + 0.997543 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + + RESULTS + title_2 + 16569 + Structural characterisation of peptide complexes + + 0.9981247 + experimental_method + cleaner0 + 2023-07-26T17:13:20Z + MESH: + + Structural characterisation + + + + RESULTS + paragraph + 16618 + Structures of the key tetrapeptides were solved by soaking into crystals of a humanised form of RAD51, HumRadA1, which we have previously reported as a convenient surrogate system for RAD51 crystallography 15. The corresponding PDB codes are indicated in Table 2 and crystallographic data are found in the Supporting Information. All structures are of high resolution (1.2–1.7 Å) and the electron density for the peptide was clearly visible after the first refinement using unliganded RadA coordinates (Fig. S1). + + 0.9936168 + evidence + cleaner0 + 2023-07-26T17:09:53Z + DUMMY: + + Structures + + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:45:09Z + + tetrapeptides + + + 0.9863721 + experimental_method + cleaner0 + 2023-07-26T17:13:24Z + MESH: + + soaking into + + + 0.8061884 + evidence + cleaner0 + 2023-07-26T17:09:57Z + DUMMY: + + crystals + + + 0.9140547 + protein_state + cleaner0 + 2023-07-26T16:37:09Z + DUMMY: + + humanised + + + 0.9993393 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9992865 + mutant + cleaner0 + 2023-07-26T16:44:10Z + MESH: + + HumRadA1 + + + 0.9993542 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + experimental_method + MESH: + cleaner0 + 2023-07-26T16:44:42Z + + crystallography + + + evidence + DUMMY: + cleaner0 + 2023-07-26T16:44:53Z + + crystallographic data + + + 0.9978719 + evidence + cleaner0 + 2023-07-26T17:09:59Z + DUMMY: + + structures + + + 0.99863166 + evidence + cleaner0 + 2023-07-26T17:10:02Z + DUMMY: + + electron density + + + 0.99918324 + protein_state + cleaner0 + 2023-07-26T17:07:15Z + DUMMY: + + unliganded + + + 0.99934083 + protein + cleaner0 + 2023-07-26T16:15:48Z + PR: + + RadA + + + + RESULTS + paragraph + 17134 + Some of the SAR observed in the binding analysis can be interpreted in terms of these X‐ray crystal structures. For example, an overlay of the bound poses of the ligands FHTA and FHPA (Fig. 2B) reveals a high similarity in the binding modes, indicating that the conformational rigidity conferred by the proline is compatible with the FHTA‐binding mode, and a reduction in an entropic penalty of binding may be the source of the improvement in affinity. WHTA peptide shows a relative dislocation when compared to FHTA (Fig 2A), with the entire ligand backbone of WHTA shifted to accommodate the change in the position of the main chain carbon of the first residue, as the larger indole side chain fills the Phe pocket. This shift is translated all the way to the alanine side chain. It is possible that this mutation is beneficial in the tetrapeptide context and neutral in the full‐length BRC4 context because the smaller peptide is less constrained and allowed to explore more conformations. An attempt to combine both the tryptophan and proline mutations, however, led to no improvement for WHPA peptide compared to FHTA. One possible explanation is that the ‘shifted’ binding mode observed in WHTA was not compatible with the conformational restriction that the proline of WHPA introduced. + + 0.99807745 + experimental_method + cleaner0 + 2023-07-26T17:13:29Z + MESH: + + binding analysis + + + experimental_method + MESH: + cleaner0 + 2023-07-26T17:10:23Z + + X‐ray + + + evidence + DUMMY: + cleaner0 + 2023-07-26T17:10:31Z + + crystal structures + + + 0.9986412 + experimental_method + cleaner0 + 2023-07-26T17:13:36Z + MESH: + + overlay + + + 0.99830616 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.9984145 + structure_element + cleaner0 + 2023-07-26T16:31:48Z + SO: + + FHPA + + + 0.99802244 + residue_name + cleaner0 + 2023-07-26T16:39:32Z + SO: + + proline + + + 0.99196225 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + evidence + DUMMY: + cleaner0 + 2023-07-26T16:45:35Z + + entropic penalty + + + 0.7549844 + evidence + cleaner0 + 2023-07-26T17:10:36Z + DUMMY: + + affinity + + + 0.82944214 + structure_element + cleaner0 + 2023-07-26T16:22:51Z + SO: + + WHTA + + + 0.99929047 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.99935335 + structure_element + cleaner0 + 2023-07-26T16:22:51Z + SO: + + WHTA + + + 0.99852115 + site + cleaner0 + 2023-07-26T17:08:47Z + SO: + + Phe pocket + + + 0.9962748 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.8962439 + experimental_method + cleaner0 + 2023-07-26T17:13:40Z + MESH: + + mutation + + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:39:12Z + + tetrapeptide + + + 0.9989994 + protein_state + cleaner0 + 2023-07-26T16:32:06Z + DUMMY: + + full‐length + + + 0.99927825 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9950858 + residue_name + cleaner0 + 2023-07-26T16:18:55Z + SO: + + tryptophan + + + 0.9965922 + residue_name + cleaner0 + 2023-07-26T16:39:32Z + SO: + + proline + + + 0.91066885 + experimental_method + cleaner0 + 2023-07-26T17:13:43Z + MESH: + + mutations + + + 0.5357877 + structure_element + cleaner0 + 2023-07-26T16:45:53Z + SO: + + WHPA + + + 0.99910384 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.9993487 + structure_element + cleaner0 + 2023-07-26T16:22:51Z + SO: + + WHTA + + + 0.99779344 + residue_name + cleaner0 + 2023-07-26T16:39:32Z + SO: + + proline + + + 0.99865997 + structure_element + cleaner0 + 2023-07-26T16:45:53Z + SO: + + WHPA + + + + FEB2-590-1094-g002.jpg + feb212139-fig-0002 + FIG + fig_caption + 18437 + Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan). + + 0.9981914 + experimental_method + cleaner0 + 2023-07-26T17:13:49Z + MESH: + + Overlay + + + 0.99882334 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.9988022 + structure_element + cleaner0 + 2023-07-26T16:22:51Z + SO: + + WHTA + + + 0.99801457 + experimental_method + cleaner0 + 2023-07-26T17:13:52Z + MESH: + + Superposition + + + 0.9988606 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.99897075 + structure_element + cleaner0 + 2023-07-26T16:31:49Z + SO: + + FHPA + + + 0.9978636 + experimental_method + cleaner0 + 2023-07-26T17:13:55Z + MESH: + + Overlay + + + 0.99792933 + structure_element + cleaner0 + 2023-07-26T16:46:22Z + SO: + + FHTU + + + 0.99839157 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.9958371 + structure_element + cleaner0 + 2023-07-26T16:41:22Z + SO: + + FHTG + + + + RESULTS + paragraph + 18744 + The thermodynamic data of peptide binding are also shown in Table 2. Although we have both thermodynamic data and high‐quality X‐ray structural information for some of the mutant peptides, we do not attempt to interpret differences in thermodynamic profiles between ligands, that is, to analyse ΔΔH and ΔΔS. Although ΔH and ΔS are tabulated, the K Ds measured are relatively weak and necessarily performed under low c‐value conditions. In this experimental regime, nonsigmoidal curves are generated and therefore errors in ΔH are expected to be much higher than the errors from model fitting given in Table 2 16. As ΔS is derived from ΔG by subtracting ΔH, errors in ΔH will be correlated with errors in ΔS, giving rise to a ‘phantom’ enthalpy–entropy compensation. Such effects have been discussed by Klebe 24 and Chodera and Mobley 25 and will frustrate attempts to interpret the measured ΔΔH and ΔΔS. + + evidence + DUMMY: + cleaner0 + 2023-07-26T16:47:31Z + + thermodynamic data + + + evidence + DUMMY: + cleaner0 + 2023-07-26T16:47:41Z + + thermodynamic data + + + experimental_method + MESH: + cleaner0 + 2023-07-26T16:47:17Z + + X‐ray + + + evidence + DUMMY: + cleaner0 + 2023-07-26T16:47:25Z + + structural information + + + 0.9914076 + protein_state + cleaner0 + 2023-07-26T17:07:32Z + DUMMY: + + mutant + + + 0.9803295 + chemical + cleaner0 + 2023-07-26T17:05:52Z + CHEBI: + + peptides + + + evidence + DUMMY: + cleaner0 + 2023-07-26T16:47:53Z + + thermodynamic profiles + + + 0.99760973 + evidence + cleaner0 + 2023-07-26T16:46:29Z + DUMMY: + + ΔΔH + + + 0.9968677 + evidence + cleaner0 + 2023-07-26T17:10:43Z + DUMMY: + + ΔΔS + + + 0.99740845 + evidence + cleaner0 + 2023-07-26T16:30:37Z + DUMMY: + + ΔH + + + 0.9965479 + evidence + cleaner0 + 2023-07-26T16:46:47Z + DUMMY: + + ΔS + + + 0.99775016 + evidence + cleaner0 + 2023-07-26T16:46:34Z + DUMMY: + + K Ds + + + 0.99671614 + evidence + cleaner0 + 2023-07-26T16:30:37Z + DUMMY: + + ΔH + + + 0.9966466 + evidence + cleaner0 + 2023-07-26T16:46:47Z + DUMMY: + + ΔS + + + 0.9972613 + evidence + cleaner0 + 2023-07-26T16:46:39Z + DUMMY: + + ΔG + + + 0.9966577 + evidence + cleaner0 + 2023-07-26T16:30:37Z + DUMMY: + + ΔH + + + 0.996783 + evidence + cleaner0 + 2023-07-26T16:30:37Z + DUMMY: + + ΔH + + + 0.9965578 + evidence + cleaner0 + 2023-07-26T16:46:47Z + DUMMY: + + ΔS + + + 0.99783725 + evidence + cleaner0 + 2023-07-26T16:46:29Z + DUMMY: + + ΔΔH + + + 0.9971649 + evidence + cleaner0 + 2023-07-26T17:10:47Z + DUMMY: + + ΔΔS + + + + RESULTS + title_2 + 19712 + Understanding mutations, residue conservation and epitope secondary structure + + + RESULTS + paragraph + 19790 + The conserved phenylalanine and alanine residues of the FHTA sequence were both found to be essential for binding by ITC. Conversely the second position histidine residue, corresponding to the unconserved His1525 in the BRC4 sequence, could be mutated without significant effect on the peptide affinity. The more general correlation between hot‐spot residues in protein–protein interactions and the high conservation of such residues has been previously reported 10, 26. Interestingly, however, the highly conserved threonine residue could be mutated without affecting the peptide affinity. This unexpected result, in the light of its very high conservation in the BRC and oligomerisation sequences, begs the question of what the role of Thr1526 is and highlights a potential pitfall and need for caution in the experimental design of alanine mutation studies. + + 0.9987602 + protein_state + cleaner0 + 2023-07-26T16:48:07Z + DUMMY: + + conserved + + + 0.9973533 + residue_name + cleaner0 + 2023-07-26T17:01:07Z + SO: + + phenylalanine + + + 0.9974299 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.970415 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.9984049 + experimental_method + cleaner0 + 2023-07-26T16:29:17Z + MESH: + + ITC + + + 0.99729925 + residue_name + cleaner0 + 2023-07-26T16:39:23Z + SO: + + histidine + + + 0.9990958 + protein_state + cleaner0 + 2023-07-26T16:48:09Z + DUMMY: + + unconserved + + + 0.99955827 + residue_name_number + cleaner0 + 2023-07-26T16:48:14Z + DUMMY: + + His1525 + + + 0.9971667 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.99749196 + experimental_method + cleaner0 + 2023-07-26T17:14:03Z + MESH: + + mutated + + + 0.99572706 + evidence + cleaner0 + 2023-07-26T17:10:50Z + DUMMY: + + peptide affinity + + + site + SO: + cleaner0 + 2023-07-26T16:17:44Z + + hot‐spot + + + 0.99891305 + protein_state + cleaner0 + 2023-07-26T16:48:34Z + DUMMY: + + high conservation + + + 0.9990065 + protein_state + cleaner0 + 2023-07-26T16:48:29Z + DUMMY: + + highly conserved + + + 0.9973839 + residue_name + cleaner0 + 2023-07-26T16:39:50Z + SO: + + threonine + + + 0.99731064 + experimental_method + cleaner0 + 2023-07-26T17:14:06Z + MESH: + + mutated + + + 0.9958695 + evidence + cleaner0 + 2023-07-26T17:10:54Z + DUMMY: + + peptide affinity + + + 0.9374611 + protein_state + cleaner0 + 2023-07-26T16:48:31Z + DUMMY: + + high conservation + + + 0.94251853 + structure_element + cleaner0 + 2023-07-26T17:04:26Z + SO: + + BRC + + + 0.91794825 + structure_element + cleaner0 + 2023-07-26T17:04:29Z + SO: + + oligomerisation sequences + + + 0.9996178 + residue_name_number + cleaner0 + 2023-07-26T16:48:39Z + DUMMY: + + Thr1526 + + + 0.9945902 + experimental_method + cleaner0 + 2023-07-26T16:48:48Z + MESH: + + alanine mutation studies + + + + RESULTS + paragraph + 20655 + As the FHTA peptide is potentially a surrogate peptide for both the BRC repeat peptides and the RAD51 self‐oligomerisation peptide, it is useful to examine the role of Thr1526 (BRC4) and the analogous Thr87 (RAD51) in both binding contexts in more detail. Structural information for these two interactions is limited. Only one structure of BRC4 is published in complex with human RAD51 (PDB: 1n0w). Figure 3A shows the binding pose of BRC4 when bound to RAD51 and the intrapeptide hydrogen bonds that are made by BRC4. While Phe1524 and Ala1527 are buried in hydrophobic pockets on the surface, His1525 is close enough to form a hydrogen bond with the carbonyl of Thr1520, but the rotamer of His1525, supported by clearly positioned water molecules, is not compatible with hydrogen bonding. Also, Thr1520 is constrained by crystal contacts in this structure. Lack of conservation of this residue supports the idea that this interaction is not crucial for RAD51:BRC repeat binding. + + structure_element + SO: + cleaner0 + 2023-07-26T16:21:34Z + + FHTA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:49:07Z + + peptide + + + 0.9626839 + structure_element + cleaner0 + 2023-07-26T17:04:33Z + SO: + + BRC repeat + + + 0.9991685 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + structure_element + SO: + cleaner0 + 2023-07-26T17:05:12Z + + self‐oligomerisation peptide + + + 0.9995678 + residue_name_number + cleaner0 + 2023-07-26T16:48:40Z + DUMMY: + + Thr1526 + + + 0.99928313 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9995701 + residue_name_number + cleaner0 + 2023-07-26T16:49:27Z + DUMMY: + + Thr87 + + + 0.99928457 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9969598 + evidence + cleaner0 + 2023-07-26T17:10:59Z + DUMMY: + + structure + + + 0.99872345 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9985256 + protein_state + cleaner0 + 2023-07-26T17:07:40Z + DUMMY: + + in complex with + + + 0.9986804 + species + cleaner0 + 2023-07-26T16:16:41Z + MESH: + + human + + + 0.9991341 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.99864966 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.99823594 + protein_state + cleaner0 + 2023-07-26T16:43:25Z + DUMMY: + + bound to + + + 0.9991392 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9966239 + bond_interaction + cleaner0 + 2023-07-26T16:50:15Z + MESH: + + hydrogen bonds + + + 0.9987847 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9995983 + residue_name_number + cleaner0 + 2023-07-26T16:38:20Z + DUMMY: + + Phe1524 + + + 0.9996031 + residue_name_number + cleaner0 + 2023-07-26T16:49:35Z + DUMMY: + + Ala1527 + + + 0.99880517 + site + cleaner0 + 2023-07-26T16:49:18Z + SO: + + hydrophobic pockets + + + 0.999587 + residue_name_number + cleaner0 + 2023-07-26T16:48:15Z + DUMMY: + + His1525 + + + 0.9967305 + bond_interaction + cleaner0 + 2023-07-26T16:38:52Z + MESH: + + hydrogen bond + + + 0.9995628 + residue_name_number + cleaner0 + 2023-07-26T16:39:18Z + DUMMY: + + Thr1520 + + + 0.99955875 + residue_name_number + cleaner0 + 2023-07-26T16:48:15Z + DUMMY: + + His1525 + + + 0.9987618 + chemical + cleaner0 + 2023-07-26T17:08:14Z + CHEBI: + + water + + + 0.9963473 + bond_interaction + cleaner0 + 2023-07-26T16:50:23Z + MESH: + + hydrogen bonding + + + 0.99956197 + residue_name_number + cleaner0 + 2023-07-26T16:39:18Z + DUMMY: + + Thr1520 + + + 0.9982121 + evidence + cleaner0 + 2023-07-26T17:11:02Z + DUMMY: + + structure + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T16:50:10Z + + Lack of conservation + + + 0.99651295 + complex_assembly + cleaner0 + 2023-07-26T16:49:51Z + GO: + + RAD51:BRC repeat + + + + FEB2-590-1094-g003.jpg + feb212139-fig-0003 + FIG + fig_caption + 21638 + (A) Highlight of intra‐BRC4 interactions when bound to RAD51 (omitted for clarity) (PDB: 1n0w), with key residues shown in colour. (B) Intrapeptide interactions from oligomerisation epitope of S. cerevisiae +RAD51 when bound to next RAD51 in the filament (PDB: 1szp). Colouring as in (A). Residue numbering relates to the S. cerevisiae +RAD51 protein, the corresponding human residues are in parentheses. + + 0.6920761 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9990468 + protein_state + cleaner0 + 2023-07-26T16:43:25Z + DUMMY: + + bound to + + + 0.99925846 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.8725573 + structure_element + cleaner0 + 2023-07-26T17:09:02Z + SO: + + oligomerisation epitope + + + 0.99856406 + species + cleaner0 + 2023-07-26T16:50:34Z + MESH: + + S. cerevisiae + + + 0.99927527 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9988258 + protein_state + cleaner0 + 2023-07-26T16:43:25Z + DUMMY: + + bound to + + + 0.99918395 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.99847794 + species + cleaner0 + 2023-07-26T16:50:35Z + MESH: + + S. cerevisiae + + + 0.9993026 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.998708 + species + cleaner0 + 2023-07-26T16:16:41Z + MESH: + + human + + + + RESULTS + paragraph + 22045 + Either a threonine or serine is most commonly found in the third position of the FxxA motif. Thr1526 makes no direct interactions with the RAD51 protein, but instead forms a hydrogen bond network with the highly conserved S1528 and K1530 (Fig. 1C). The high degree of conservation of these three residues suggests an important possible role in facilitating a turn and stabilising the conformation of the peptide as it continues its way to a second interaction site on the side of RAD51. With respect to understanding the RAD51:RAD51 interaction, no human crystal structure has been published, however, several oligomeric structures of archaeal RadA as well that of Saccharomyces cerevisiae RAD51 have been reported 27, 28, 29. Figure 3B shows a highlight of the FxxA portion of oligomerisation peptide from the S. cerevisiae RAD51 structure, with residues in parentheses corresponding to the human RAD51 protein. The conserved threonine residue at the third position forms a hydrogen bond with the peptide backbone amide, which forms the base of an α‐helix. + + 0.99722326 + residue_name + cleaner0 + 2023-07-26T16:39:50Z + SO: + + threonine + + + 0.9970481 + residue_name + cleaner0 + 2023-07-26T17:01:23Z + SO: + + serine + + + structure_element + SO: + cleaner0 + 2023-07-26T16:04:47Z + + FxxA + + + 0.999584 + residue_name_number + cleaner0 + 2023-07-26T16:48:40Z + DUMMY: + + Thr1526 + + + 0.9992059 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.99766254 + bond_interaction + cleaner0 + 2023-07-26T16:50:56Z + MESH: + + hydrogen bond network + + + 0.99887633 + protein_state + cleaner0 + 2023-07-26T17:07:45Z + DUMMY: + + highly conserved + + + 0.999589 + residue_name_number + cleaner0 + 2023-07-26T17:01:55Z + DUMMY: + + S1528 + + + 0.9995921 + residue_name_number + cleaner0 + 2023-07-26T17:01:58Z + DUMMY: + + K1530 + + + 0.997444 + protein_state + cleaner0 + 2023-07-26T17:07:48Z + DUMMY: + + high degree of conservation + + + 0.95321923 + site + cleaner0 + 2023-07-26T17:09:07Z + SO: + + interaction site + + + 0.9991904 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9950401 + complex_assembly + cleaner0 + 2023-07-26T16:51:17Z + GO: + + RAD51:RAD51 + + + 0.99872005 + species + cleaner0 + 2023-07-26T16:16:42Z + MESH: + + human + + + 0.9986067 + evidence + cleaner0 + 2023-07-26T17:11:07Z + DUMMY: + + crystal structure + + + 0.98462796 + evidence + cleaner0 + 2023-07-26T17:11:10Z + DUMMY: + + structures + + + 0.99880326 + taxonomy_domain + cleaner0 + 2023-07-26T17:05:28Z + DUMMY: + + archaeal + + + 0.99924856 + protein + cleaner0 + 2023-07-26T16:15:48Z + PR: + + RadA + + + 0.9982946 + species + cleaner0 + 2023-07-26T16:43:29Z + MESH: + + Saccharomyces cerevisiae + + + 0.9992536 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9992311 + structure_element + cleaner0 + 2023-07-26T16:04:47Z + SO: + + FxxA + + + structure_element + SO: + cleaner0 + 2023-07-26T16:52:07Z + + oligomerisation peptide + + + 0.99861413 + species + cleaner0 + 2023-07-26T16:50:35Z + MESH: + + S. cerevisiae + + + 0.9992016 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.99821633 + evidence + cleaner0 + 2023-07-26T17:11:12Z + DUMMY: + + structure + + + 0.9987942 + species + cleaner0 + 2023-07-26T16:16:42Z + MESH: + + human + + + 0.9992667 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + 0.9983101 + protein_state + cleaner0 + 2023-07-26T16:51:23Z + DUMMY: + + conserved + + + 0.99705935 + residue_name + cleaner0 + 2023-07-26T16:39:50Z + SO: + + threonine + + + 0.99639153 + bond_interaction + cleaner0 + 2023-07-26T16:38:52Z + MESH: + + hydrogen bond + + + 0.99917835 + structure_element + cleaner0 + 2023-07-26T17:04:38Z + SO: + + α‐helix + + + + RESULTS + paragraph + 23107 + In both structural contexts, the role of the third position threonine in FxxA seems to be in stabilising secondary structure; a β‐turn in the case of BRC binding and an α‐helix in the case of RAD51 oligomerisation. In the tetrapeptide context these secondary interactions are not present and mutation of threonine to alanine would be expected to have little effect on affinity. In line with this, although we observe a slight twofold weakening of peptide affinity, the effect is far from being as drastic or inactivating as reported in longer peptide backgrounds 3. It would be interesting to investigate the importance of this residue in the context of the BRC4 peptide, and the oligomerisation peptide. Rather than indifference to alanine mutation, a significant effect, via lack of secondary structure stabilisation, would be predicted, as indeed has been reported for BRC3 3. + + 0.99690884 + residue_name + cleaner0 + 2023-07-26T16:39:50Z + SO: + + threonine + + + 0.99244726 + structure_element + cleaner0 + 2023-07-26T16:04:47Z + SO: + + FxxA + + + 0.9991738 + structure_element + cleaner0 + 2023-07-26T17:04:42Z + SO: + + β‐turn + + + 0.998428 + structure_element + cleaner0 + 2023-07-26T17:03:26Z + SO: + + BRC + + + 0.9992311 + structure_element + cleaner0 + 2023-07-26T17:04:45Z + SO: + + α‐helix + + + 0.99922276 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + RAD51 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:39:12Z + + tetrapeptide + + + 0.9984964 + experimental_method + cleaner0 + 2023-07-26T17:14:14Z + MESH: + + mutation + + + 0.9964101 + residue_name + cleaner0 + 2023-07-26T16:39:50Z + SO: + + threonine + + + 0.9965353 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.99826944 + evidence + cleaner0 + 2023-07-26T17:11:15Z + DUMMY: + + affinity + + + 0.9126651 + evidence + cleaner0 + 2023-07-26T17:11:18Z + DUMMY: + + peptide affinity + + + 0.9941453 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + structure_element + SO: + cleaner0 + 2023-07-26T16:52:06Z + + oligomerisation peptide + + + 0.9957846 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.9758036 + experimental_method + cleaner0 + 2023-07-26T17:14:17Z + MESH: + + mutation + + + 0.9989441 + chemical + cleaner0 + 2023-07-26T16:35:07Z + CHEBI: + + BRC3 + + + + CONCL + title_1 + 23994 + Conclusions + + + CONCL + paragraph + 24006 + The key observations from this work are shown in Fig 4. Two residues in the FxxA motif, phenylalanine and alanine, are highly conserved (Fig 4a). Phenylalanine mutated to tryptophan, in the context of the tetrapeptide improved potency, contrary to the reported result of comparable activity in the context of BRC4 12. Proline at the third position similarly improved potency. Activity was lost by mutating the terminal alanine to glycine, but recovered somewhat with the novel α‐amino butyric acid (U). Threonine was found to be relatively unimportant in the tetrapeptides but has been previously reported to be crucial in the context of BRC3. The reason for this disconnection is suggested to be that threonine plays a role in stabilising the β‐turn in the BRC repeats, which is absent in the tetrapeptides studied. This may lead to a more general caution, that hot‐spot data should be interpreted by considering the bound interaction with the protein, as well as the potential role in stabilising the bound peptide secondary structure. In either case, the requirement for structural data in correctly interpreting alanine‐scanning experiments is reinforced. + + structure_element + SO: + cleaner0 + 2023-07-26T16:04:47Z + + FxxA + + + 0.9977926 + residue_name + cleaner0 + 2023-07-26T17:01:37Z + SO: + + phenylalanine + + + 0.99750537 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.99875915 + protein_state + cleaner0 + 2023-07-26T17:07:53Z + DUMMY: + + highly conserved + + + 0.99754864 + residue_name + cleaner0 + 2023-07-26T17:01:41Z + SO: + + Phenylalanine + + + 0.99062407 + experimental_method + cleaner0 + 2023-07-26T17:14:21Z + MESH: + + mutated to + + + 0.99714524 + residue_name + cleaner0 + 2023-07-26T16:18:55Z + SO: + + tryptophan + + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:39:12Z + + tetrapeptide + + + 0.99805427 + chemical + cleaner0 + 2023-07-26T16:34:56Z + CHEBI: + + BRC4 + + + 0.9978619 + residue_name + cleaner0 + 2023-07-26T16:39:32Z + SO: + + Proline + + + 0.9956429 + experimental_method + cleaner0 + 2023-07-26T17:14:41Z + MESH: + + mutating + + + 0.99792 + residue_name + cleaner0 + 2023-07-26T16:19:02Z + SO: + + alanine + + + 0.99754554 + residue_name + cleaner0 + 2023-07-26T16:41:47Z + SO: + + glycine + + + 0.9961217 + chemical + cleaner0 + 2023-07-26T17:01:45Z + CHEBI: + + α‐amino butyric acid + + + 0.7480348 + chemical + cleaner0 + 2023-07-26T17:01:48Z + CHEBI: + + U + + + 0.9980652 + residue_name + cleaner0 + 2023-07-26T16:39:50Z + SO: + + Threonine + + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:45:14Z + + tetrapeptides + + + 0.99815035 + chemical + cleaner0 + 2023-07-26T16:35:07Z + CHEBI: + + BRC3 + + + 0.9980634 + residue_name + cleaner0 + 2023-07-26T16:39:50Z + SO: + + threonine + + + 0.99932057 + structure_element + cleaner0 + 2023-07-26T17:04:50Z + SO: + + β‐turn + + + 0.99917924 + structure_element + cleaner0 + 2023-07-26T16:16:47Z + SO: + + BRC repeats + + + chemical + CHEBI: + cleaner0 + 2023-07-26T16:45:14Z + + tetrapeptides + + + 0.7696015 + site + cleaner0 + 2023-07-26T16:17:44Z + SO: + + hot‐spot + + + experimental_method + MESH: + cleaner0 + 2023-07-26T17:14:38Z + + alanine‐scanning experiments + + + + FEB2-590-1094-g004.jpg + feb212139-fig-0004 + FIG + fig_caption + 25176 + Summary of key observations (A) FxxA motif sequence conservation of Rad51 oligomerisation sequences and BRC repeats. (B) Highlight of SAR identified for the tetrapeptide. The differences in ΔG for different peptide variants relative to FHTA are shown in the bar chart with colouring matching with the structural overlay below. (C) Overlay of tetrapeptide structures, with wild‐type FHTA peptide across the figure for reference and truncated segments of mutated residues shown in each panel. Purple carbon is WHTA, light blue is FATA, yellow is FHPA, cyan is FHTG and grey carbon is FHTA. Note the C‐terminal amide changes position in FHTG without the anchoring methyl group. + + structure_element + SO: + cleaner0 + 2023-07-26T16:04:47Z + + FxxA + + + 0.99931145 + protein + cleaner0 + 2023-07-26T16:04:04Z + PR: + + Rad51 + + + 0.99892724 + structure_element + cleaner0 + 2023-07-26T16:16:47Z + SO: + + BRC repeats + + + 0.90110344 + chemical + cleaner0 + 2023-07-26T16:39:12Z + CHEBI: + + tetrapeptide + + + 0.997654 + evidence + cleaner0 + 2023-07-26T16:46:39Z + DUMMY: + + ΔG + + + 0.999315 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.99883795 + experimental_method + cleaner0 + 2023-07-26T17:14:46Z + MESH: + + structural overlay + + + 0.9988518 + experimental_method + cleaner0 + 2023-07-26T17:14:48Z + MESH: + + Overlay + + + 0.9542874 + chemical + cleaner0 + 2023-07-26T16:39:12Z + CHEBI: + + tetrapeptide + + + 0.9985379 + evidence + cleaner0 + 2023-07-26T17:11:23Z + DUMMY: + + structures + + + 0.9989162 + protein_state + cleaner0 + 2023-07-26T16:29:13Z + DUMMY: + + wild‐type + + + 0.99930334 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.9992341 + structure_element + cleaner0 + 2023-07-26T16:22:51Z + SO: + + WHTA + + + 0.99928206 + structure_element + cleaner0 + 2023-07-26T16:53:17Z + SO: + + FATA + + + 0.9991596 + structure_element + cleaner0 + 2023-07-26T16:31:49Z + SO: + + FHPA + + + 0.99848 + structure_element + cleaner0 + 2023-07-26T16:41:22Z + SO: + + FHTG + + + 0.9990269 + structure_element + cleaner0 + 2023-07-26T16:21:34Z + SO: + + FHTA + + + 0.99308217 + structure_element + cleaner0 + 2023-07-26T16:41:22Z + SO: + + FHTG + + + + SUPPL + title_1 + 25856 + Supporting information + + + REF + title + 25879 + References + + + 457 + 470 + 1581961 + REF + Cell + ref + 69 + 1992 + 25890 + RAD51 protein involved in repair and recombination in S. cerevisiae is a RecA‐like protein + + + 171 + 182 + 11832208 + REF + Cell + ref + 108 + 2002 + 25983 + Cancer susceptibility and the functions of BRCA1 and BRCA2 + + + 273 + 282 + 11239456 + REF + Mol Cell + ref + 7 + 2001 + 26042 + Role of BRCA2 in control of the RAD51 recombination and DNA repair protein + + + 17 + 22 + 18163131 + REF + Nat Genet + ref + 40 + 2008 + 26117 + The emerging landscape of breast cancer susceptibility + + + 194 + 201 + 18182601 + REF + JAMA + ref + 299 + 2008 + 26172 + Variation of breast cancer risk among BRCA1/2 carriers + + + 100 + 106 + 25557581 + REF + Breast + ref + 24 + 2015 + 26227 + Clinical relevance of normal and tumour cell radiosensitivity in BRCA1/BRCA2 mutation carriers: a review + + + 804 + 810 + 9126738 + REF + Nature + ref + 386 + 1997 + 26332 + Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2 + + + 296 + 303 + 25470112 + REF + ChemMedChem + ref + 10 + 2015 + 26423 + Small‐molecule inhibitors that target protein‐protein interactions in the RAD51 family of recombinases + + + 1015 + 1028 + 12967658 + REF + DNA Repair + ref + 2 + 2003 + 26530 + Sequence fingerprints in BRCA2 and RAD51: implications for DNA repair and cancer + + + 1281 + 1294 + 15644221 + REF + J Mol Biol + ref + 345 + 2005 + 26611 + Hot regions in protein–protein interactions: the organization and contribution of structurally conserved hot spot residues + + + 287 + 293 + 12442171 + REF + Nature + ref + 420 + 2002 + 26736 + Insights into DNA recombination from the structure of a RAD51‐BRCA2 complex + + + 82 + 96 + 19875419 + REF + Nucleic Acids Res + ref + 38 + 2010 + 26814 + Two modules in the BRC repeats of BRCA2 mediate structural and functional interactions with the RAD51 recombinase + + + 5782 + 5791 + 20684611 + REF + J Med Chem + ref + 53 + 2010 + 26928 + Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide + + + D992 + D1002 + 22144684 + REF + Nucleic Acids Res + ref + 40 + 2012 + 27079 + Description and analysis of genetic variants in French hereditary breast and ovarian cancer families recorded in the UMD‐BRCA1/BRCA2 databases + + + 332 + 342 + 23344974 + REF + ChemBioChem + ref + 14 + 2013 + 27224 + Using a fragment‐based approach to target protein‐protein interactions + + + 14859 + 14866 + 14640663 + REF + J Am Chem Soc + ref + 125 + 2003 + 27299 + On the value of c: can low affinity systems be studied by isothermal titration calorimetry? + + + 355 + 367 + 21460454 + REF + Acta Crystallogr D Biol Crystallogr + ref + 67 + 2011 + 27391 + REFMAC5 for the refinement of macromolecular crystal structures + + + 240 + 255 + 15299926 + REF + Acta Crystallogr D Biol Crystallogr + ref + 53 + 1997 + 27455 + Refinement of macromolecular structures by the maximum‐likelihood method + + + 213 + 221 + 20124702 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 27530 + PHENIX: a comprehensive Python‐based system for macromolecular structure solution + + + 2126 + 2132 + 15572765 + REF + Acta Crystallogr D Biol Crystallogr + ref + 60 + 2004 + 27614 + Coot: model‐building tools for molecular graphics + + + 1188 + 1190 + 15173120 + REF + Genome Res + ref + 14 + 2004 + 27666 + WebLogo: a sequence logo generator + + + 10448 + 10453 + 21670257 + REF + Proc Natl Acad Sci USA + ref + 108 + 2011 + 27701 + Two classes of BRC repeats in BRCA2 promote RAD51 nucleoprotein filament function by distinct mechanisms + + + 423 + 435 + 15304222 + REF + Mol Cell + ref + 15 + 2004 + 27806 + Crystal structure of archaeal recombinase RADA: a snapshot of its extended conformation + + + 95 + 110 + 25614222 + REF + Nat Rev Drug Discov + ref + 14 + 2015 + 27894 + Applying thermodynamic profiling in lead finding and optimization + + + 121 + 142 + 23654303 + REF + Annu Rev Biophys + ref + 42 + 2013 + 27960 + Entropy‐enthalpy compensation: role and ramifications in biomolecular ligand recognition and design + + + 21 + 27 + 11839485 + REF + Curr Opin Struct Biol + ref + 12 + 2002 + 28062 + Evolutionary predictions of binding surfaces and interactions + + + 791 + 796 + 15235592 + REF + Nat Struct Mol Biol + ref + 11 + 2004 + 28124 + Crystal structure of a Rad51 filament + + + 13753 + 13761 + 16229465 + REF + Biochemistry + ref + 44 + 2005 + 28162 + Crystal structure of Methanococcus voltae RadA in complex with ADP: hydrolysis‐induced conformational change + + + 4566 + 4576 + 12941707 + REF + EMBO J + ref + 22 + 2003 + 28273 + Full‐length archaeal RAD51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2 + + + diff --git a/BioC_XML/4857006_v0.xml b/BioC_XML/4857006_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..5c3c76a8f11e5fea568fbc628598ee9d731a795c --- /dev/null +++ b/BioC_XML/4857006_v0.xml @@ -0,0 +1,13334 @@ + + + + PMC + 20201223 + pmc.key + + 4857006 + NO-CC CODE + no + 0 + 0 + + 10.1093/nar/gkw228 + 4857006 + 27060144 + 3829 + 8 + 3844 + surname:Schellenberg;given-names:Matthew J. + surname:Perera;given-names:Lalith + surname:Strom;given-names:Christina N. + surname:Waters;given-names:Crystal A. + surname:Monian;given-names:Brinda + surname:Appel;given-names:C. Denise + surname:Vilas;given-names:Caroline K. + surname:Williams;given-names:Jason G. + surname:Ramsden;given-names:Dale A. + surname:Williams;given-names:R. Scott + TITLE + front + 44 + 2016 + 0 + Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2 + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:35Z + + DNA + + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:01Z + + Topoisomerase 2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.99900573 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + + ABSTRACT + abstract + 80 + Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. + + 0.99843246 + taxonomy_domain + cleaner0 + 2023-07-26T12:30:55Z + DUMMY: + + Mammalian + + + 0.9967226 + protein + cleaner0 + 2023-07-26T12:31:00Z + PR: + + Tyrosyl-DNA phosphodiesterase 2 + + + 0.9992386 + protein + cleaner0 + 2023-07-26T12:31:05Z + PR: + + Tdp2 + + + 0.99735916 + protein_type + cleaner0 + 2023-07-26T12:34:00Z + MESH: + + Topoisomerase 2 + + + 0.9955147 + protein_type + cleaner0 + 2023-07-26T12:34:11Z + MESH: + + Top2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.9907197 + protein_type + cleaner0 + 2023-07-26T12:34:12Z + MESH: + + Top2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.99176073 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:12Z + + Top2 + + + 0.9986221 + evidence + cleaner0 + 2023-07-26T15:36:55Z + DUMMY: + + X-ray crystal structures + + + 0.99878114 + protein_state + cleaner0 + 2023-07-26T12:42:39Z + DUMMY: + + ligand-free + + + 0.9990239 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9990631 + complex_assembly + cleaner0 + 2023-07-26T12:32:18Z + GO: + + Tdp2-DNA + + + 0.9968876 + chemical + cleaner0 + 2023-07-26T12:31:41Z + CHEBI: + + DNA + + + 0.98043025 + protein_state + cleaner0 + 2023-07-26T15:03:53Z + DUMMY: + + dynamic + + + 0.99904484 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9921932 + structure_element + cleaner0 + 2023-07-26T14:16:26Z + SO: + + active site lid + + + 0.9969414 + site + cleaner0 + 2023-07-26T12:57:33Z + SO: + + substrate binding trench + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.90231067 + protein_type + cleaner0 + 2023-07-26T12:34:12Z + MESH: + + Top2 + + + 0.99860317 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9981152 + experimental_method + cleaner0 + 2023-07-26T12:33:22Z + MESH: + + mutagenesis + + + 0.844049 + experimental_method + cleaner0 + 2023-07-26T12:33:24Z + MESH: + + biochemical studies + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:32:48Z + + Mg2+ + + + 0.99893266 + site + cleaner0 + 2023-07-26T14:32:43Z + SO: + + phosphotyrosyl-arginine cation-π interface + + + 0.99872833 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9907745 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.9897452 + protein_state + cleaner0 + 2023-07-26T15:04:00Z + DUMMY: + + ablates + + + 0.9990169 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9969199 + chemical + cleaner0 + 2023-07-26T12:32:56Z + CHEBI: + + Mg2+ + + + 0.99809676 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9949307 + residue_name + cleaner0 + 2023-07-26T14:30:02Z + SO: + + tyrosine + + + 0.998818 + chemical + cleaner0 + 2023-07-26T12:35:43Z + CHEBI: + + etoposide + + + 0.9985922 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:12Z + + Top2 + + + 0.9976163 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:12Z + + Top2 + + + + INTRO + title_1 + 1348 + INTRODUCTION + + + INTRO + paragraph + 1361 + Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division. The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions. Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.88209844 + chemical + cleaner0 + 2023-07-26T12:31:41Z + CHEBI: + + DNA + + + 0.81101096 + protein_type + cleaner0 + 2023-07-26T12:34:27Z + MESH: + + RNA polymerases + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.9987746 + protein_type + cleaner0 + 2023-07-26T12:34:29Z + MESH: + + Topoisomerases + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.9978592 + taxonomy_domain + cleaner0 + 2023-07-26T12:30:55Z + DUMMY: + + mammalian + + + 0.99836165 + protein_type + cleaner0 + 2023-07-26T12:34:33Z + MESH: + + type II topoisomerases + + + 0.9987889 + protein + cleaner0 + 2023-07-26T12:34:37Z + PR: + + Top2α + + + 0.9984995 + protein + cleaner0 + 2023-07-26T12:34:42Z + PR: + + Top2β + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.6967313 + protein_type + cleaner0 + 2023-07-26T12:34:12Z + MESH: + + Top2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + ptm + MESH: + cleaner0 + 2023-07-26T12:52:01Z + + phosphotyrosyl linkages + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:51:52Z + + 5′-phosphate + + + 0.9983624 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.9972882 + protein_type + cleaner0 + 2023-07-26T12:34:12Z + MESH: + + Top2 + + + 0.9917122 + residue_name + cleaner0 + 2023-07-26T12:35:12Z + SO: + + tyrosine + + + 0.66438603 + protein_type + cleaner0 + 2023-07-26T12:34:12Z + MESH: + + Top2 + + + 0.9765489 + complex_assembly + cleaner0 + 2023-07-26T12:35:26Z + GO: + + Top2cc + + + + INTRO + paragraph + 2166 + The Top2cc protein–DNA adduct is a unique threat to genomic integrity which must be resolved to prevent catastrophic Top2cc collisions with the cellular replication and transcription machineries. To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin. Importantly, Top2 is also poisoned when it engages abundant endogenous DNA damage not limited to but including ribonucleotides, abasic sites and alkylation damage such as exocyclic DNA adducts arising from bioactivation of the vinyl chloride carcinogen (Figure 1A). In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc. Precisely how the cellular DNA repair machinery navigates these complex lesions is an important aspect of Top2cc repair that has not yet been explored. + + 0.83297205 + complex_assembly + cleaner0 + 2023-07-26T12:35:27Z + GO: + + Top2cc + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.73943245 + complex_assembly + cleaner0 + 2023-07-26T12:35:27Z + GO: + + Top2cc + + + 0.88264203 + protein_type + cleaner0 + 2023-07-26T12:34:12Z + MESH: + + Top2 + + + 0.9992494 + chemical + cleaner0 + 2023-07-26T12:35:42Z + CHEBI: + + etoposide + + + 0.9993249 + chemical + cleaner0 + 2023-07-26T12:35:47Z + CHEBI: + + teniposide + + + 0.999252 + chemical + cleaner0 + 2023-07-26T12:35:53Z + CHEBI: + + doxorubicin + + + 0.9692414 + protein_type + cleaner0 + 2023-07-26T12:34:12Z + MESH: + + Top2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.66714185 + complex_assembly + cleaner0 + 2023-07-26T12:35:27Z + GO: + + Top2cc + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.94664997 + protein_type + cleaner0 + 2023-07-26T12:34:12Z + MESH: + + Top2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.79155755 + complex_assembly + cleaner0 + 2023-07-26T12:35:27Z + GO: + + Top2cc + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + 0.47465068 + complex_assembly + cleaner0 + 2023-07-26T12:35:27Z + GO: + + Top2cc + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:41Z + + DNA + + + complex_assembly + GO: + cleaner0 + 2023-07-26T12:35:27Z + + Top2cc + + + + gkw228fig1.jpg + F1 + FIG + fig_caption + 3357 + Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts. (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow). DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. 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Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair. Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression. The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. It is possible that TDP2 single nucleotide polymorphisms (SNPs) encode mutations that impact Tdp2 function, but the molecular underpinnings for such Tdp2 deficiencies are not understood. + + 0.98642004 + protein + cleaner0 + 2023-07-26T12:40:33Z + PR: + + Tyrosyl DNA phosphodiesterase 2 + + + 0.99919206 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + ptm + MESH: + cleaner0 + 2023-07-26T14:34:43Z + + 5′-phosphotyrosyl + + + 0.9862006 + ptm + cleaner0 + 2023-07-26T14:35:05Z + MESH: + + 5′-Y + + + ptm + MESH: + cleaner0 + 2023-07-26T14:34:53Z + + linkages + + + 0.56705964 + protein_type + cleaner0 + 2023-07-26T12:34:12Z + MESH: + + Top2 + + + 0.87840337 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.8811085 + experimental_method + cleaner0 + 2023-07-26T15:46:25Z + MESH: + + knockdown + + + 0.99879384 + chemical + cleaner0 + 2023-07-26T12:35:43Z + CHEBI: + + etoposide + + + 0.99888426 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + complex_assembly + GO: + cleaner0 + 2023-07-26T12:35:27Z + + Top2cc + + + 0.9987218 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.99906737 + protein_state + cleaner0 + 2023-07-26T12:41:31Z + DUMMY: + + mutant + + + 0.9947148 + protein + cleaner0 + 2023-07-26T12:41:36Z + PR: + + p53 + + + 0.9987332 + protein_state + cleaner0 + 2023-07-26T12:41:32Z + DUMMY: + + mutant + + + 0.9982451 + protein + cleaner0 + 2023-07-26T12:41:37Z + PR: + + p53 + + + 0.9989644 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.6971097 + protein_type + cleaner0 + 2023-07-26T14:14:39Z + MESH: + + topoisomerase + + + 0.9985305 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + 0.98522377 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + TDP2 + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T12:41:23Z + + loss of + + + 0.9987111 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9975921 + protein + cleaner0 + 2023-07-26T12:34:43Z + PR: + + Top2β + + + 0.884141 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + TDP2 + + + 0.99856347 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.94919825 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + + INTRO + paragraph + 6320 + Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. However, important questions regarding the mechanism of Tdp2 engagement and processing of DNA damage remain. First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization. Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. We further establish that DNA damage binding in the Tdp2 active site is linked to conformational change and binding of metal cofactor. Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. + + experimental_method + MESH: + cleaner0 + 2023-07-26T15:37:56Z + + X-ray + + + evidence + DUMMY: + cleaner0 + 2023-07-26T15:38:06Z + + crystal structures + + + 0.9860983 + protein_state + cleaner0 + 2023-07-26T12:41:57Z + DUMMY: + + minimal catalytically active + + + 0.97164345 + structure_element + cleaner0 + 2023-07-26T12:42:12Z + SO: + + endonuclease/exonuclease/phosphatase + + + 0.9820685 + structure_element + cleaner0 + 2023-07-26T12:42:16Z + SO: + + EEP + + + 0.9538989 + taxonomy_domain + cleaner0 + 2023-07-26T12:42:25Z + DUMMY: + + mouse + + + 0.9993767 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9992173 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.999075 + protein_state + cleaner0 + 2023-07-26T12:40:14Z + DUMMY: + + bound to + + + 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2023-07-26T15:38:32Z + DUMMY: + + structures + + + 0.99878 + chemical + cleaner0 + 2023-07-26T14:52:18Z + CHEBI: + + Mg2+ + + + 0.94777775 + experimental_method + cleaner0 + 2023-07-26T15:46:32Z + MESH: + + structure-function study + + + 0.9991704 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + experimental_method + MESH: + cleaner0 + 2023-07-26T15:38:24Z + + X-ray + + + evidence + DUMMY: + cleaner0 + 2023-07-26T15:38:29Z + + structures + + + 0.99901646 + protein_state + cleaner0 + 2023-07-26T12:42:38Z + DUMMY: + + ligand-free + + + 0.9992428 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9989625 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9990712 + protein_state + cleaner0 + 2023-07-26T12:40:14Z + DUMMY: + + bound to + + + 0.9987847 + chemical + cleaner0 + 2023-07-26T14:53:37Z + CHEBI: + + 1-N6-etheno-adenine + + + 0.99304974 + chemical + cleaner0 + 2023-07-26T12:31:41Z + CHEBI: + + DNA + + + 0.99384344 + experimental_method 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2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.99843264 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.9606471 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.81276083 + protein_state + cleaner0 + 2023-07-26T15:04:12Z + DUMMY: + + ablates + + + 0.9989931 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9838504 + site + cleaner0 + 2023-07-26T15:19:55Z + SO: + + single metal binding site + + + 0.99788207 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9951066 + protein_type + cleaner0 + 2023-07-26T12:34:12Z + MESH: + + Top2 + + + 0.99776757 + taxonomy_domain + cleaner0 + 2023-07-26T12:30:55Z + DUMMY: + + mammalian + + + + METHODS + title_1 + 7916 + MATERIALS AND METHODS + + + METHODS + title_2 + 7938 + Generation of PNP modified oligonucleotide substrates + + + METHODS + paragraph + 7992 + Oligonucleotides with a 5′-phosphate modification were obtained from IDT and diluted to a concentration of 2 mmol l−1 in water. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC; Pierce) was used to couple p-nitrophenol to the oligonucleotide with a modified version of the manufacturer's instructions. Briefly, 25 mg EDC were dissolved in 150 μl oligonucleotide solution. After the EDC dissolved, 400 μl DDW, 60 μl 100 mmol l−1 Imidazole pH 6 and 100 μl of PNP from a saturated water/PNP mixture preheated to 55°C were added. Reactions were heated at 55°C for 30 min to ensure the PNP dissolved, then incubated at 37°C overnight. Reactions were quenched with 500 μl 2 mol l−1 acetic acid and heated to 55°C for 1 h, followed by neutralization with 500 μl 2 mol l−1 Tris base. Reactions were twice diluted to 15 ml in DDW then concentrated to 300 μl with a 3K MWT cutoff spin concentrator (Amicon), then run on 20% (w/v) 19:1 8M urea-TBE PAGE to resolve reaction products. Bands were visualized by UV shadow, excised, soaked in 15 ml DDW for 16 h at 15°C, and purified on a C18 reverse-phase Sep-Pac (Waters). + + + METHODS + title_2 + 9126 + Protein expression, purification and crystallization + + + METHODS + paragraph + 9179 + Mouse Tdp2 catalytic domain (mTdp2cat, residues 118 to 370), human Tdp2 catalytic domain (hTdp2cat, residues 108–362) or full-length human Tdp2 (hTdp2FL, residues 1–362) were expressed and purified as previously described. Plasmids containing mutant Tdp2 sequences were generated using the Quickchange kit (Stratagene). Crystals containing mTdp2cat and 5′-phosphorylated DNA (product) with modified 5′ nucleotides (ϵA, dA, THF) were grown and cryoprotected as described. Sequences, modifications and synthesis sources for oligonucleotides used for co-crystallization are indicated in Supplementary Table S1. For alternate divalent metal complexes, a 5′-phosphorylated DNA substrate (substrate dC) was co-crystallized in the presence of Mg2+, and divalent metals were swapped by soaking the crystals in crystallization buffer containing 5 mmol l−1 MnCl2 or 10 mmol l−1 Ca(OAc)2 for 1 h prior to cryoprotection. Cryoprotectant solutions contained mother liquor plus 25% PEG-3350, 8% glycerol and 5% glucose, and either 5 mmol l−1 MnCl2 or 10 mmol l−1 Ca(OAc)2. + + + METHODS + paragraph + 10257 + Crystals of hTdp2cat bound to 5′-phosphate DNA (substrate dC) were prepared by mixing a 1.2-fold molar excess of DNA, and grown in 90 mmol l−1 TRIS pH 7.0, 27% (w/v) PEG600, 9% (v/v) glycerol and 450 mmol l−1 (NH4)2SO4. Crystals of apo-mTdp2cat were grown in 14–18% (w/v) PEG3350, 100 mmol l−1 HEPES pH 7.5, 200 mmol l−1 Li2SO4 and 10 mmol l−1 Mg(OAc)2, and soaked into the same buffer containing 25% PEG3350 and 12% (v/v) glycerol prior to flash-freezing in liquid nitrogen for data collection. + + + METHODS + title_2 + 10767 + X-ray diffraction data collection, phasing and refinement + + + METHODS + paragraph + 10825 + X-ray data (Table 1) for all structures except the manganese soak (PDB entry 5INP) were collected at 100 K on beamline 22-ID of the Advanced Photon Source at a wavelength of 1.000 Å. X-ray data for the manganese soak (PDB entry 5INP) were collected on a Rigaku HF007 Cu rotating anode X-ray source at a wavelength of 1.5418 Å. X-ray diffraction data were processed and scaled using the HKL2000 suite. The hTdp2cat-DNA and mTdp2cat-apo crystals were phased by molecular replacement in PHASER using chain A of PDB entry 4GZ1. Initial solutions were improved by iterative rounds of manual fitting in COOT and refinement in PHENIX. + + + tbl1.xml + tbl1 + TABLE + table_title_caption + 11455 + Data collection and refinement statistics + + + tbl1.xml + tbl1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="left" rowspan="1" colspan="1">mTdp2 118–370 5′-P +ϵA</th><th align="left" rowspan="1" colspan="1">mTdp2 118–370 5′-P + THF</th><th align="left" rowspan="1" colspan="1">mTdp2 118–370 5′-P + dA</th><th align="left" rowspan="1" colspan="1">mTdp2 118–370 wt, apo</th><th align="left" rowspan="1" colspan="1">mTdp2 118–370 D358N, apo</th><th align="left" rowspan="1" colspan="1">hTdp2 108–362 5′-P DNA</th><th align="left" rowspan="1" colspan="1">mTdp2 118–370 5′-P DNA Mn<sup>2+</sup> soak</th><th align="left" rowspan="1" colspan="1">mTdp2 118–370 5′-P DNA Ca<sup>2+</sup> soak</th></tr><tr><th align="left" rowspan="1" colspan="1">PDB entry ID</th><th align="left" rowspan="1" colspan="1">5HT2</th><th align="left" rowspan="1" colspan="1">5INK</th><th align="left" rowspan="1" colspan="1">5INL</th><th align="left" rowspan="1" colspan="1">5INM</th><th align="left" rowspan="1" colspan="1">5INN</th><th align="left" rowspan="1" colspan="1">5INO</th><th align="left" rowspan="1" colspan="1">5INP</th><th align="left" rowspan="1" colspan="1">5INQ</th></tr></thead><tbody><tr><td colspan="9" align="left" rowspan="1"><bold>Data collection</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">Space group</td><td align="left" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P 3<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P 2<sub>1</sub> 2<sub>1</sub> 2<sub>1</sub></td></tr><tr><td colspan="9" align="left" rowspan="1">Cell dimensions</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>a, b, c</italic> (Å)</td><td align="left" rowspan="1" colspan="1">54.88, 68.60, 167.25</td><td align="left" rowspan="1" colspan="1">54.62, 67.66, 166.74</td><td align="left" rowspan="1" colspan="1">54.90, 69.05, 167.80</td><td align="left" rowspan="1" colspan="1">95.85, 113.44, 114.96</td><td align="left" rowspan="1" colspan="1">95.44, 114.88, 115.45</td><td align="left" rowspan="1" colspan="1">69.66, 69.66, 120.68</td><td align="left" rowspan="1" colspan="1">54.92, 68.52, 166.90</td><td align="left" rowspan="1" colspan="1">54.70, 67.50, 167.52</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>α, β, γ</italic> (°)</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 120</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="left" rowspan="1" colspan="1">Resolution (Å)</td><td align="left" rowspan="1" colspan="1">50–1.43 (1.48–1.43)</td><td align="left" rowspan="1" colspan="1">50–2.15 (2.23–2.15)</td><td align="left" rowspan="1" colspan="1">50–1.55 (1.61–1.55)</td><td align="left" rowspan="1" colspan="1">50–2.40 (2.49–2.40)</td><td align="left" rowspan="1" colspan="1">50–2.80 (2.90–2.80)</td><td align="left" rowspan="1" colspan="1">50–3.20 (3.31–3.20)</td><td align="left" rowspan="1" colspan="1">50–1.95 (2.02–1.95)</td><td align="left" rowspan="1" colspan="1">50–1.85 (1.92–1.85)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>R</italic><sub>sym</sub> or <italic>R</italic><sub>merge</sub></td><td align="left" rowspan="1" colspan="1">0.051 (0.599)</td><td align="left" rowspan="1" colspan="1">0.125 (0.650)</td><td align="left" rowspan="1" colspan="1">0.067 (0.588)</td><td align="left" rowspan="1" colspan="1">0.124 (0.518)</td><td align="left" rowspan="1" colspan="1">0.113 (0.459)</td><td align="left" rowspan="1" colspan="1">0.082 (0.574)</td><td align="left" rowspan="1" colspan="1">0.054 (0.181)</td><td align="left" rowspan="1" colspan="1">0.068 (0.564)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>I</italic> / σ<italic>I</italic></td><td align="left" rowspan="1" colspan="1">21.0 (2.1)</td><td align="left" rowspan="1" colspan="1">17.5 (2.7)</td><td align="left" rowspan="1" colspan="1">19.4 (2.4)</td><td align="left" rowspan="1" colspan="1">9.8 (2.1)</td><td align="left" rowspan="1" colspan="1">10.6 (2.0)</td><td align="left" rowspan="1" colspan="1">15.5 (2.2)</td><td align="left" rowspan="1" colspan="1">25.5 (4.8)</td><td align="left" rowspan="1" colspan="1">21.1 (3.1)</td></tr><tr><td align="left" rowspan="1" colspan="1">Completeness (%)</td><td align="left" rowspan="1" colspan="1">97.4 (86.6)</td><td align="left" rowspan="1" colspan="1">98.4 (95.4)</td><td align="left" rowspan="1" colspan="1">99.0 (91.5)</td><td align="left" rowspan="1" colspan="1">98.0 (94.2)</td><td align="left" rowspan="1" colspan="1">95.7 (93.0)</td><td align="left" rowspan="1" colspan="1">100 (100)</td><td align="left" rowspan="1" colspan="1">97.1 (80.7)</td><td align="left" rowspan="1" colspan="1">99.9 (100)</td></tr><tr><td align="left" rowspan="1" colspan="1">Redundancy</td><td align="left" rowspan="1" colspan="1">3.9 (3.0)</td><td align="left" rowspan="1" colspan="1">6.7 (6.5)</td><td align="left" rowspan="1" colspan="1">5.4 (3.7)</td><td align="left" rowspan="1" colspan="1">4.2 (3.6)</td><td align="left" rowspan="1" colspan="1">4.2 (3.4)</td><td align="left" rowspan="1" colspan="1">3.9 (3.9)</td><td align="left" rowspan="1" colspan="1">5.5 (2.0)</td><td align="left" rowspan="1" colspan="1">6.0 (6.0)</td></tr><tr><td colspan="9" align="left" rowspan="1"><bold>Refinement</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">Resolution (Å)</td><td align="left" rowspan="1" colspan="1">29.9–1.43</td><td align="left" rowspan="1" colspan="1">45.7–2.15</td><td align="left" rowspan="1" colspan="1">34.1–1.55</td><td align="left" rowspan="1" colspan="1">48.8–2.4</td><td align="left" rowspan="1" colspan="1">49.3–2.80</td><td align="left" rowspan="1" colspan="1">34.8–3.205</td><td align="left" rowspan="1" colspan="1">29.8–1.95</td><td align="left" rowspan="1" colspan="1">39.1–1.85</td></tr><tr><td align="left" rowspan="1" colspan="1">No. reflections</td><td align="left" rowspan="1" colspan="1">114 115</td><td align="left" rowspan="1" colspan="1">34 369</td><td align="left" rowspan="1" colspan="1">91 871</td><td align="left" rowspan="1" colspan="1">49 905</td><td align="left" rowspan="1" colspan="1">29 807</td><td align="left" rowspan="1" colspan="1">10 757</td><td align="left" rowspan="1" colspan="1">45 238</td><td align="left" rowspan="1" colspan="1">53 889</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>R</italic><sub>work</sub> / <italic>R</italic><sub>free</sub></td><td align="left" rowspan="1" colspan="1">0.116/0.150</td><td align="left" rowspan="1" colspan="1">0.167/0.201</td><td align="left" rowspan="1" colspan="1">0.129/0.168</td><td align="left" rowspan="1" colspan="1">0.183/0.233</td><td align="left" rowspan="1" colspan="1">0.214/0.255</td><td align="left" rowspan="1" colspan="1">0.214/0.264</td><td align="left" rowspan="1" colspan="1">0.152/0.179</td><td align="left" rowspan="1" colspan="1">0.164/0.200</td></tr><tr><td colspan="9" align="left" rowspan="1">Non-H atoms</td></tr><tr><td align="left" rowspan="1" colspan="1">Protein/DNA</td><td align="left" rowspan="1" colspan="1">4552</td><td align="left" rowspan="1" colspan="1">4372</td><td align="left" rowspan="1" colspan="1">4600</td><td align="left" rowspan="1" colspan="1">9621</td><td align="left" rowspan="1" colspan="1">9411</td><td align="left" rowspan="1" colspan="1">4363</td><td align="left" rowspan="1" colspan="1">4512</td><td align="left" rowspan="1" colspan="1">4452</td></tr><tr><td align="left" rowspan="1" colspan="1">Ligand/ion</td><td align="left" rowspan="1" colspan="1">25</td><td align="left" rowspan="1" colspan="1">22</td><td align="left" rowspan="1" colspan="1">25</td><td align="left" rowspan="1" colspan="1">25</td><td align="left" rowspan="1" colspan="1">28</td><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">15</td><td align="left" rowspan="1" colspan="1">21</td></tr><tr><td align="left" rowspan="1" colspan="1">Water</td><td align="left" rowspan="1" colspan="1">726</td><td align="left" rowspan="1" colspan="1">449</td><td align="left" rowspan="1" colspan="1">721</td><td align="left" rowspan="1" colspan="1">365</td><td align="left" rowspan="1" colspan="1">148</td><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">444</td><td align="left" rowspan="1" colspan="1">374</td></tr><tr><td colspan="9" align="left" rowspan="1"><italic>B</italic>-factors (Å<sup>2</sup>)</td></tr><tr><td align="left" rowspan="1" colspan="1">Protein/DNA</td><td align="left" rowspan="1" colspan="1">26.5</td><td align="left" rowspan="1" colspan="1">35.8</td><td align="left" rowspan="1" colspan="1">26.7</td><td align="left" rowspan="1" colspan="1">47.7</td><td align="left" rowspan="1" colspan="1">53.3</td><td align="left" rowspan="1" colspan="1">147.9</td><td align="left" rowspan="1" colspan="1">35.6</td><td align="left" rowspan="1" colspan="1">50.4</td></tr><tr><td align="left" rowspan="1" colspan="1">Ligand/ion</td><td align="left" rowspan="1" colspan="1">52.5</td><td align="left" rowspan="1" colspan="1">50.2</td><td align="left" rowspan="1" colspan="1">53.0</td><td align="left" rowspan="1" colspan="1">73.5</td><td align="left" rowspan="1" colspan="1">82.2</td><td align="left" rowspan="1" colspan="1">225.8</td><td align="left" rowspan="1" colspan="1">53.7</td><td align="left" rowspan="1" colspan="1">54.7</td></tr><tr><td align="left" rowspan="1" colspan="1">Water</td><td align="left" rowspan="1" colspan="1">44.2</td><td align="left" rowspan="1" colspan="1">43.0</td><td align="left" rowspan="1" colspan="1">46.0</td><td align="left" rowspan="1" colspan="1">38.2</td><td align="left" rowspan="1" colspan="1">28.3</td><td align="left" rowspan="1" colspan="1">83.3</td><td align="left" rowspan="1" colspan="1">44.7</td><td align="left" rowspan="1" colspan="1">52.4</td></tr><tr><td colspan="9" align="left" rowspan="1">R.m.s. deviations</td></tr><tr><td align="left" rowspan="1" colspan="1">Bond lengths (Å)</td><td align="left" rowspan="1" colspan="1">0.007</td><td align="left" rowspan="1" colspan="1">0.007</td><td align="left" rowspan="1" colspan="1">0.005</td><td align="left" rowspan="1" colspan="1">0.004</td><td align="left" rowspan="1" colspan="1">0.002</td><td align="left" rowspan="1" colspan="1">0.003</td><td align="left" rowspan="1" colspan="1">0.007</td><td align="left" rowspan="1" colspan="1">0.008</td></tr><tr><td align="left" rowspan="1" colspan="1">Bond angles (°)</td><td align="left" rowspan="1" colspan="1">1.18</td><td align="left" rowspan="1" colspan="1">1.01</td><td align="left" rowspan="1" colspan="1">0.85</td><td align="left" rowspan="1" colspan="1">0.81</td><td align="left" rowspan="1" colspan="1">0.58</td><td align="left" rowspan="1" colspan="1">0.78</td><td align="left" rowspan="1" colspan="1">1.03</td><td align="left" rowspan="1" colspan="1">1.08</td></tr></tbody></table> + + 11497 + mTdp2 118–370 5′-P +ϵA mTdp2 118–370 5′-P + THF mTdp2 118–370 5′-P + dA mTdp2 118–370 wt, apo mTdp2 118–370 D358N, apo hTdp2 108–362 5′-P DNA mTdp2 118–370 5′-P DNA Mn2+ soak mTdp2 118–370 5′-P DNA Ca2+ soak PDB entry ID 5HT2 5INK 5INL 5INM 5INN 5INO 5INP 5INQ Data collection Space group P 21 21 21 P 21 21 21 P 21 21 21 P 21 21 21 P 21 21 21 P 31 P 21 21 21 P 21 21 21 Cell dimensions a, b, c (Å) 54.88, 68.60, 167.25 54.62, 67.66, 166.74 54.90, 69.05, 167.80 95.85, 113.44, 114.96 95.44, 114.88, 115.45 69.66, 69.66, 120.68 54.92, 68.52, 166.90 54.70, 67.50, 167.52 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 120 90, 90, 90 90, 90, 90 Resolution (Å) 50–1.43 (1.48–1.43) 50–2.15 (2.23–2.15) 50–1.55 (1.61–1.55) 50–2.40 (2.49–2.40) 50–2.80 (2.90–2.80) 50–3.20 (3.31–3.20) 50–1.95 (2.02–1.95) 50–1.85 (1.92–1.85) Rsym or Rmerge 0.051 (0.599) 0.125 (0.650) 0.067 (0.588) 0.124 (0.518) 0.113 (0.459) 0.082 (0.574) 0.054 (0.181) 0.068 (0.564) I / σI 21.0 (2.1) 17.5 (2.7) 19.4 (2.4) 9.8 (2.1) 10.6 (2.0) 15.5 (2.2) 25.5 (4.8) 21.1 (3.1) Completeness (%) 97.4 (86.6) 98.4 (95.4) 99.0 (91.5) 98.0 (94.2) 95.7 (93.0) 100 (100) 97.1 (80.7) 99.9 (100) Redundancy 3.9 (3.0) 6.7 (6.5) 5.4 (3.7) 4.2 (3.6) 4.2 (3.4) 3.9 (3.9) 5.5 (2.0) 6.0 (6.0) Refinement Resolution (Å) 29.9–1.43 45.7–2.15 34.1–1.55 48.8–2.4 49.3–2.80 34.8–3.205 29.8–1.95 39.1–1.85 No. reflections 114 115 34 369 91 871 49 905 29 807 10 757 45 238 53 889 Rwork / Rfree 0.116/0.150 0.167/0.201 0.129/0.168 0.183/0.233 0.214/0.255 0.214/0.264 0.152/0.179 0.164/0.200 Non-H atoms Protein/DNA 4552 4372 4600 9621 9411 4363 4512 4452 Ligand/ion 25 22 25 25 28 5 15 21 Water 726 449 721 365 148 5 444 374 B-factors (Å2) Protein/DNA 26.5 35.8 26.7 47.7 53.3 147.9 35.6 50.4 Ligand/ion 52.5 50.2 53.0 73.5 82.2 225.8 53.7 54.7 Water 44.2 43.0 46.0 38.2 28.3 83.3 44.7 52.4 R.m.s. deviations Bond lengths (Å) 0.007 0.007 0.005 0.004 0.002 0.003 0.007 0.008 Bond angles (°) 1.18 1.01 0.85 0.81 0.58 0.78 1.03 1.08 + + + tbl1.xml + tbl1 + TABLE + table_footnote + 13636 + Each data set was collected from a single crystal. Values in parentheses are for highest-resolution shell (10% of relections). + + + METHODS + title_2 + 13763 + Limited proteolysis assays + + + METHODS + paragraph + 13790 + For proteolysis experiments, 4 μl reactions containing 40 μmol l−1 mTdp2cat (aa 118 to 370) in reaction buffer (10 mmol l−1 HEPES pH 7.5, 200 mmol l−1 NaCl, 0.5 mmol L−1 TCEP) with 4 mmol l−1 Mg(OAc)2 (indicated +Mg) or 8 mmol l−1 NaOAc (indicated -Mg), with 0 or 60 μmol l−1 12 nt DNA were incubated with 0, 5, 1.7 or 0.6 μg l−1 Trypsin for 1 h at 22°C. Reactions were quenched by addition of Laemmli SDS-PAGE dye, heated at 70°C for 10 min, and analyzed by SDS-PAGE. For mass-spectrometry analysis of peptide masses, reactions were quenched with 1% (v/v) trifluoroacetic acid, purified on a C18 ZipTip (Millipore) and an ESI-MS mass measurement made on a Q-ToF Ultima/Global (Micromass/Waters) using flow injection from a pressurized bomb. The instrument was operated in the positive ion, V-mode and calibrated using the multiply-charged ion envelope of horse heart cytochrome C. The molecular ion mass was determined using the Max Ent 1 routine from the MassLynx software. + + + METHODS + title_2 + 14788 + Intrinsic tryptophan fluorescence + + + METHODS + paragraph + 14822 + Reactions contained 50 μl with 1 μmol l−1 mTdp2cat (residues 118 to 370) in buffer (6 mmol l−1 HEPES pH 7.5, 300 mmol l−1 NaCl, 0.3 mmol L−1 TCEP and 0.01% (v/v) TWEEN-20) with 0–20 mmol l−1 Mg(OAc)2 or ultrapure Ca(OAc)2 (99.9965%, Alfa Aesar) titrated against 0–40 mmol l−1 NaOAc to maintain a constant concentration of acetate, with 0 or 1.5 μmol l−1 12 nt DNA. Reactions were incubated at room temperature for 20 minutes in a 96-well black plastic plate (Corning), then tryptophan fluorescence was measured in a Polarstar Omega platereader (BMG Labtech) with 4 readings of 150 pulses per well using 280/10 excitation and 350/10 emission filters. The increase in fluorescent intensity was calculated by subtracting the fluorescent intensity from samples with no divalent metal ions and plotted as a function of divalent metal ion concentration. Kd values and Hill coefficients were calculated using PRISM6. + + + METHODS + title_2 + 15753 + Tdp2 enzyme assays + + + METHODS + paragraph + 15772 + Assays on MBP-fusion proteins of the human catalytic domain (MBP-hTdp2cat) mutant proteins with 5′ -tyrosylated DNA substrates (Figures 5E and 6C and Supplementary Figure S5C) were performed as described. For experiments measuring the effect of divalent metal ions on reaction rates, 50 μl reactions contained 1 μmol l−1 mTdp2cat (residues 118 to 370, Figure 4B), hTdp2cat (residues 108–362) or hTdp2FL (residues 108–362) in buffer (10 mmol l−1 HEPES pH 7.5, 100 mmol l−1 NaCl, 0.5 mmol l−1 TCEP, 0.4 mg ml−1 BSA, 0.02% (v/v) TWEEN-20, 1 mmol l−1 Thymidine 5′–p-nitrophenyl phosphate) with 0–100 mmol l−1 Mg(OAc)2 titrated against 20–220 mmol l−1 NaOAc to maintain a constant concentration of acetate. Reactions contained 0, 1 or 10 mmol l−1 ultrapure Ca(OAc)2 (99.9965%, Alfa Aesar), with 0, 2 or 20 mmol l−1 less NaOAc to maintain a constant acetate concentration. PNP formation was monitored by the absorbance at 415 nm with a background correction at 515 nm. The change in absorbance at 415 nm at 10 min as a function of Mg2+ concentration was plotted. + + + METHODS + title_2 + 16866 + Preparation of NHEJ substrates + + + METHODS + paragraph + 16897 + Oligonucleotide 300 bp substrates with 5′ phosphorylated GATC overhangs were generated as previously described. Comparable substrate with 5′-phophotyrosine adducted GATC overhangs were generated by annealing the modified strand to complementary strands that generate caps for substrate head and tail ends. Head and tail caps have a 5′-phosphotyrosine-GATC overhang terminus on one end; the 5′-phosphorylated, non-adducted overhangs on the other end are made complementary to the head and tail ends of a 270 bp double stranded core fragment generated by polymerase chain reaction. Ligation of an excess of these caps to the 270 bp fragment generates a 300 bp substrate with 5′-phosphotyrosine end structures as described in Figure 7. Unligated caps are removed using a Qiaquick PCR cleanup kit. + + + METHODS + title_2 + 17701 + In vitro NHEJ reactions + + + METHODS + paragraph + 17725 + Purified NHEJ proteins (Ku, XRCC4-ligase IV, XLF) were prepared as previously described. End joining reactions were performed using 5 nM DNA substrate, 25 nM Ku, 25 nM XRCC4/LigaseIV complex, 50 nM XLF and hTdp2FL proteins as indicated. Reactions contained 25 mM Tris-HCl pH 7.5, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 2 mM dithiothreitol (DTT), 125 mM KCl, 5 mM MgCl2, 100 μM ATP, 8% (w/v) polyethyleneglycol, 0.05% (w/v) Triton X-100, 50 μg ml−1 bovine serum albumin (BSA) and 50 ng supercoiled plasmid DNA. Reactions were carried out in a final volume of 10 μl and incubated at 37°C for 5 min. Reactions were stopped by the addition of 0.1% (w/v) SDS and 5 mM EDTA and analyzed by 5% native PAGE. + + + METHODS + title_2 + 18439 + Cellular NHEJ assays + + + METHODS + paragraph + 18460 + Mouse embryo fibroblast (MEF) cells from matched Tdp2+/+ and Tdp2−/− mice were a gift from F. Cortes-Ledesma, and were immortalized by transformation with a construct that expresses SV40 T-antigen (Addgene #1779). HCT-116 cells and a ligase IV deficient variant were the gift of E. A. Hendrickson. The cDNAs with wild-type human Tdp2 and polymorphic variants generated by mutagenesis were introduced into pLX302 (Addgene#25896) to prepare lentivirus. Tdp2−/− cells were infected with lentivirus and bulk cell cultures expressing lentiviral delivered construct purified by treatment with puromycin. Expression of human Tdp2 (hTdp2FL) was validated by Western analysis (12203-1-AP; Proteintech). Fifty nanograms of the 300 bp substrates used in in vitro experiments and 2 μg of carrier supercoiled plasmid DNA were introduced into 2 × 105 MEF or HCT 116 cells by electroporation (Neon, Invitrogen) using a 10 μl chamber and a single 1350 V, 30 ms pulse. Cells were recovered 1 h later, washed with phosphate buffered saline and DNA purified using a Qiamp DNA mini kit on a Qiacube. NHEJ products were quantified by qPCR and characterized by amplicon sequencing as previously described. + + + METHODS + title_2 + 19655 + Etoposide Clonogenic survival assays + + + METHODS + paragraph + 19692 + Clonogenic survival assays were carried out by treating log phase cells with Etoposide as described in the legend to Figure 7 before seeding treated and mock treated cells in 10 cm dishes. Colonies formed after 10 days post-treatment were strained using a crystal violet (0.5% w/v) solution. Plates containing a minimum of 50 colonies were counted by hand, and at least three plates were counted for each dose. + + + METHODS + title_2 + 20103 + QM/MM + + + METHODS + paragraph + 20109 + In the QM/MM calculation, in addition to the water nucleophile and the putative catalytic Lewis base (Asp272), the side chains of proximal residues (Asn130, Asn274, His236, His326, His359, Ser239 and Glu162), the bound phosphate moiety, the Mg2+ and three waters in its coordinate shell are all included in the QM sub-system. In addition, we modeled the position of the Top2 peptide tyrosine based on conformations of the substrate analog. + + + METHODS + paragraph + 20549 + The QM sub-system of this study consisted of 110 atoms with a zero net charge on the sub-system. QM/MM calculations are performed using Gaussian09.D01. Since the QM sub-system contains a large number of buried atoms and commonly used electrostatic potential fitting schemes to obtain the charge distribution at atomic positions become unreliable for such systems with buried atoms, we have selected the CM5 charge model in the current study to calculate the charges on atomic positions at each step of the QM/MM calculation. This CM5 scheme is an extension to the Hirshfeld population analysis and is adapted to handle buried charges properly. This charge distribution of the QM sub-system was used to evaluate contributions of residues in the MM region to the net stability of the transition and product states as compared to that of the initial reactive state. As reported previously, this residue analysis is solely based on the electrostatic energy contributions to the initial, transition and product states. According to this estimation, the residues Arg142, Lys213, Ser235, Asp277, Glu279, Asp292, Glu295, Asp308, Asp343, Arg354 and Trp360 contribute toward the stability of both the transition and product states as compared with the reactant. However, the electrostatic contributions from residues Asp132, Asp135, Glu186, Glu242, Arg247, Thr273, Arg276, Lys299, Lys322, Arg324, Arg327 and Asp358 have the opposite effect toward the stabilities of the transition and product states. + + + RESULTS + title_1 + 22040 + RESULTS + + + RESULTS + title_2 + 22048 + Tdp2 processing of compound DNA damage + + 0.9985752 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + 0.9772528 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + + RESULTS + paragraph + 22087 + Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A). Whether Tdp2 processes phosphotyrosyl linkages within these diverse structural contexts is not known. To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods). We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage. + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:12Z + + Top2 + + + 0.8562551 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + 0.7553812 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + 0.9991347 + chemical + cleaner0 + 2023-07-26T12:46:54Z + CHEBI: + + 1-N6-ethenoadenine + + + 0.9992362 + chemical + cleaner0 + 2023-07-26T12:38:42Z + CHEBI: + + ϵA + + + 0.9992888 + protein + cleaner0 + 2023-07-26T12:31:06Z + PR: + + Tdp2 + + + ptm + MESH: + cleaner0 + 2023-07-26T12:52:01Z + + phosphotyrosyl linkages + + + 0.9945329 + experimental_method + cleaner0 + 2023-07-26T15:46:39Z + MESH: + + EDC coupling method + + + 0.998858 + chemical + cleaner0 + 2023-07-26T12:47:01Z + CHEBI: + + p-nitrophenol + + + 0.9992225 + chemical + cleaner0 + 2023-07-26T12:39:08Z + CHEBI: + + PNP + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + 0.6169123 + taxonomy_domain + cleaner0 + 2023-07-26T12:42:26Z + DUMMY: + + mouse + + + 0.99939454 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99920964 + structure_element + cleaner0 + 2023-07-26T15:13:35Z + SO: + + catalytic domain + + + 0.99887484 + structure_element + cleaner0 + 2023-07-26T15:13:56Z + SO: + + mTdp2cat + + + 0.99916923 + chemical + cleaner0 + 2023-07-26T12:39:08Z + CHEBI: + + PNP + + + 0.9973947 + protein_type + cleaner0 + 2023-07-26T14:35:55Z + MESH: + + topoisomerase + + + 0.99488384 + residue_name + cleaner0 + 2023-07-26T14:30:22Z + SO: + + tyrosine + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + 0.998505 + experimental_method + cleaner0 + 2023-07-26T15:46:44Z + MESH: + + colorimetric assay + + + 0.9991352 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9992424 + chemical + cleaner0 + 2023-07-26T12:39:08Z + CHEBI: + + PNP + + + 0.99925834 + chemical + cleaner0 + 2023-07-26T12:55:49Z + CHEBI: + + dA-PNP + + + 0.9992699 + chemical + cleaner0 + 2023-07-26T12:56:05Z + CHEBI: + + ϵA-PNP + + + 0.9985287 + chemical + cleaner0 + 2023-07-26T12:54:54Z + CHEBI: + + tetrahydrofuran spacer + + + 0.99913245 + chemical + cleaner0 + 2023-07-26T12:38:53Z + CHEBI: + + THF + + + 0.9992937 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9356308 + ptm + cleaner0 + 2023-07-26T12:52:01Z + MESH: + + phosphotyrosyl linkages + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + + RESULTS + paragraph + 23152 + To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. One half of the molecule contributes to formation of the walls of the DNA-binding cleft that embraces the terminal position of the damaged DNA substrate. In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. + + 0.99859506 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99526465 + complex_assembly + cleaner0 + 2023-07-26T12:35:27Z + GO: + + Top2cc + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + 0.94103336 + experimental_method + cleaner0 + 2023-07-26T15:46:47Z + MESH: + + crystallized and determined + + + experimental_method + MESH: + cleaner0 + 2023-07-26T15:39:23Z + + X-ray + + + evidence + DUMMY: + cleaner0 + 2023-07-26T15:39:32Z + + crystal structures + + + 0.9992999 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9990647 + protein_state + cleaner0 + 2023-07-26T12:40:14Z + DUMMY: + + bound to + + + 0.9614989 + chemical + cleaner0 + 2023-07-26T12:39:37Z + CHEBI: + + 5′-phosphate DNA + + + 0.99694085 + chemical + cleaner0 + 2023-07-26T12:55:13Z + CHEBI: + + 5′-ϵA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + 0.9990707 + chemical + cleaner0 + 2023-07-26T12:55:26Z + CHEBI: + + 5′-THF + + + 0.9986968 + complex_assembly + cleaner0 + 2023-07-26T12:32:18Z + GO: + + Tdp2-DNA + + + 0.9970637 + evidence + cleaner0 + 2023-07-26T15:39:37Z + DUMMY: + + structures + + + 0.99927646 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9987038 + structure_element + cleaner0 + 2023-07-26T15:16:53Z + SO: + + mixed α-β fold + + + 0.99913585 + structure_element + cleaner0 + 2023-07-26T15:16:58Z + SO: + + 12-stranded anti-parallel β-sandwich + + + 0.99926144 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9988234 + site + cleaner0 + 2023-07-26T12:57:41Z + SO: + + active site + + + 0.99865127 + site + cleaner0 + 2023-07-26T12:56:35Z + SO: + + DNA-binding cleft + + + 0.9775187 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + 0.99890137 + protein_state + cleaner0 + 2023-07-26T12:56:49Z + DUMMY: + + DNA lesion-bound + + + 0.5233519 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + 0.9992761 + structure_element + cleaner0 + 2023-07-26T12:57:11Z + SO: + + β-2-helix-β + + + 0.9992574 + structure_element + cleaner0 + 2023-07-26T12:57:16Z + SO: + + β2Hβ + + + 0.98709863 + structure_element + cleaner0 + 2023-07-26T15:17:19Z + SO: + + grasp + + + 0.9978013 + structure_element + cleaner0 + 2023-07-26T15:17:22Z + SO: + + helical cap + + + 0.9332749 + site + cleaner0 + 2023-07-26T12:57:32Z + SO: + + substrate binding trench + + + 0.9977112 + chemical + cleaner0 + 2023-07-26T14:53:45Z + CHEBI: + + ssDNA + + + 0.9984056 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.99799395 + evidence + cleaner0 + 2023-07-26T15:39:40Z + DUMMY: + + structure + + + 0.99881727 + protein_state + cleaner0 + 2023-07-26T12:52:35Z + DUMMY: + + DNA-free + + + 0.99919254 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9993724 + protein_state + cleaner0 + 2023-07-26T12:40:07Z + DUMMY: + + apo + + + 0.9987128 + structure_element + cleaner0 + 2023-07-26T15:17:26Z + SO: + + loop + + + 0.99721 + protein_state + cleaner0 + 2023-07-26T15:04:28Z + DUMMY: + + conformationally mobile + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + + RESULTS + paragraph + 24285 + The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage. The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). + + 0.9916313 + chemical + cleaner0 + 2023-07-26T12:38:42Z + CHEBI: + + ϵA + + + 0.9993426 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9951747 + protein_state + cleaner0 + 2023-07-26T12:57:01Z + DUMMY: + + 5′-tyrosylated + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + 0.79590106 + chemical + cleaner0 + 2023-07-26T12:55:14Z + CHEBI: + + 5′-ϵA + + + 0.99912304 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99814034 + site + cleaner0 + 2023-07-26T12:57:46Z + SO: + + van Der Waals interaction surface + + + 0.99452126 + site + cleaner0 + 2023-07-26T15:20:19Z + SO: + + hydrophobic wall + + + 0.9995802 + residue_name_number + cleaner0 + 2023-07-26T12:57:51Z + DUMMY: + + Leu315 + + + 0.99957186 + residue_name_number + cleaner0 + 2023-07-26T12:57:55Z + DUMMY: + + Ile317 + + + + gkw228fig2.jpg + F2 + FIG + fig_caption + 24692 + Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning. (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). mTdp2cat is colored by electrostatic surface potential (red = negative, blue = positive, gray = neutral/hydrophobic). PDB entry 5HT2. (B) σ-A weighted 2Fo-Fc electron density map (at 1.43 Å resolution, contoured at 2.0 σ) for the ϵA DNA complex. The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). mTdp2cat is colored with red (electronegative), blue (electropositive) and gray (hydrophobic) electrostatic surface potential displayed. PDB entry 5INK is displayed. (D) σ-A weighted 2Fo-Fc electron density map (at 2.15 Å resolution, contoured at 2.0 σ) for THF-DNA complex. The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes. + + 0.7720775 + evidence + cleaner0 + 2023-07-26T15:39:46Z + DUMMY: + + Structures + + + 0.99914205 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9991491 + protein_state + cleaner0 + 2023-07-26T12:40:14Z + DUMMY: + + bound to + + + 0.64154464 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + 0.8514806 + protein_type + cleaner0 + 2023-07-26T12:34:12Z + MESH: + + Top2 + + + 0.9866431 + evidence + cleaner0 + 2023-07-26T15:39:49Z + DUMMY: + + Structure + + + 0.99914145 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9990815 + protein_state + cleaner0 + 2023-07-26T12:40:14Z + DUMMY: + + bound to + + + 0.9113455 + chemical + cleaner0 + 2023-07-26T12:39:37Z + CHEBI: + + 5′-phosphate DNA + + + 0.9940754 + chemical + cleaner0 + 2023-07-26T12:38:42Z + CHEBI: + + ϵA + + + 0.9987432 + chemical + cleaner0 + 2023-07-26T12:58:17Z + CHEBI: + + Mg2+ + + + 0.9987256 + chemical + cleaner0 + 2023-07-26T12:58:19Z + CHEBI: + + waters + + + 0.99873 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9868148 + evidence + cleaner0 + 2023-07-26T12:59:40Z + DUMMY: + + σ-A weighted 2Fo-Fc electron density map + + + 0.9443636 + chemical + cleaner0 + 2023-07-26T14:55:21Z + CHEBI: + + ϵA DNA + + + 0.7369393 + chemical + cleaner0 + 2023-07-26T12:38:42Z + CHEBI: + + ϵA + + + 0.9969584 + bond_interaction + cleaner0 + 2023-07-26T12:59:14Z + MESH: + + hydrogen bond + + + 0.90411705 + chemical + cleaner0 + 2023-07-26T12:38:42Z + CHEBI: + + ϵA + + + 0.9988483 + chemical + cleaner0 + 2023-07-26T12:58:13Z + CHEBI: + + water + + + 0.9956351 + evidence + cleaner0 + 2023-07-26T15:39:52Z + DUMMY: + + Structure + + + 0.99918884 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9990744 + protein_state + cleaner0 + 2023-07-26T12:40:14Z + DUMMY: + + bound to + + + 0.87784576 + chemical + cleaner0 + 2023-07-26T12:39:37Z + CHEBI: + + 5′-phosphate DNA + + + 0.99923897 + chemical + cleaner0 + 2023-07-26T12:38:53Z + CHEBI: + + THF + + + 0.9988057 + chemical + cleaner0 + 2023-07-26T12:59:48Z + CHEBI: + + Mg2+ + + + 0.9986425 + chemical + cleaner0 + 2023-07-26T14:55:24Z + CHEBI: + + waters + + + 0.9988708 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9829815 + evidence + cleaner0 + 2023-07-26T12:59:43Z + DUMMY: + + σ-A weighted 2Fo-Fc electron density map + + + 0.9986307 + complex_assembly + cleaner0 + 2023-07-26T12:59:32Z + GO: + + THF-DNA + + + 0.99896157 + chemical + cleaner0 + 2023-07-26T12:38:53Z + CHEBI: + + THF + + + 0.99660724 + bond_interaction + cleaner0 + 2023-07-26T12:59:14Z + MESH: + + hydrogen bond + + + 0.99889404 + chemical + cleaner0 + 2023-07-26T12:38:53Z + CHEBI: + + THF + + + 0.9986707 + chemical + cleaner0 + 2023-07-26T12:59:53Z + CHEBI: + + water + + + + RESULTS + paragraph + 25848 + For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. Likewise, the abasic deoxyribose analog THF substrate binds similar to the alkylated and non-alkylated substrates, but with a slight alteration in the approach of the 5′-terminus (Figure 2C). Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D). This shift is coincident with a small adjustment in the position of the +2 and +3 nucleotides (Supplementary Figure S1E). These collective differences may explain the slight, but statistically significant elevated activity on the THF substrate (Figure 1C). + + 0.9249608 + experimental_method + cleaner0 + 2023-07-26T15:46:52Z + MESH: + + determined + + + 0.97238356 + evidence + cleaner0 + 2023-07-26T15:39:57Z + DUMMY: + + structure + + + 0.99888366 + chemical + cleaner0 + 2023-07-26T13:00:07Z + CHEBI: + + 5′-adenine + + + 0.999053 + chemical + cleaner0 + 2023-07-26T13:00:02Z + CHEBI: + + 5′-dA + + + 0.9991252 + protein_state + cleaner0 + 2023-07-26T12:40:14Z + DUMMY: + + bound to + + + 0.9993679 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99883926 + experimental_method + cleaner0 + 2023-07-26T15:46:57Z + MESH: + + structural overlay + + + 0.99381423 + protein_state + cleaner0 + 2023-07-26T15:04:40Z + DUMMY: + + bound + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + 0.99256223 + chemical + cleaner0 + 2023-07-26T12:38:42Z + CHEBI: + + ϵA + + + 0.9938169 + chemical + cleaner0 + 2023-07-26T12:38:32Z + CHEBI: + + dA + + + 0.9904856 + chemical + cleaner0 + 2023-07-26T14:55:54Z + CHEBI: + + dC + + + 0.9927987 + chemical + cleaner0 + 2023-07-26T14:55:59Z + CHEBI: + + ϵG + + + 0.9920523 + chemical + cleaner0 + 2023-07-26T14:56:03Z + CHEBI: + + ϵT + + + 0.99941397 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99863046 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.88645285 + bond_interaction + cleaner0 + 2023-07-26T14:28:42Z + MESH: + + planar base stacking + + + 0.9984208 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.65205204 + site + cleaner0 + 2023-07-26T15:20:24Z + SO: + + hydrophobic wall + + + structure_element + SO: + cleaner0 + 2023-07-26T12:57:17Z + + β2Hβ + + + 0.8869473 + chemical + cleaner0 + 2023-07-26T14:57:24Z + CHEBI: + + abasic deoxyribose + + + 0.9991879 + chemical + cleaner0 + 2023-07-26T12:38:53Z + CHEBI: + + THF + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T13:07:47Z + + absence of + + + 0.9991715 + chemical + cleaner0 + 2023-07-26T12:38:53Z + CHEBI: + + THF + + + 0.9990872 + chemical + cleaner0 + 2023-07-26T13:31:32Z + CHEBI: + + water + + + 0.99596417 + chemical + cleaner0 + 2023-07-26T13:00:40Z + CHEBI: + + Mg2+ + + + 0.88189507 + bond_interaction + cleaner0 + 2023-07-26T15:20:42Z + MESH: + + ion coordination shell + + + 0.99915457 + chemical + cleaner0 + 2023-07-26T12:38:53Z + CHEBI: + + THF + + + + RESULTS + title_2 + 27087 + Structural plasticity in the Tdp2 DNA binding trench + + 0.9991358 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + site + SO: + cleaner0 + 2023-07-26T15:21:09Z + + DNA binding trench + + + + RESULTS + paragraph + 27140 + An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358. The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement. To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). + + 0.99895585 + protein_state + cleaner0 + 2023-07-26T13:00:53Z + DUMMY: + + DNA-damage bound + + + 0.99934393 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9989356 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + chemical + CHEBI: + cleaner0 + 2023-07-26T13:31:32Z + + water + + + 0.86871845 + site + cleaner0 + 2023-07-26T15:21:31Z + SO: + + catalytic core + + + site + SO: + cleaner0 + 2023-07-26T15:21:47Z + + DNA binding β2Hβ-grasp + + + 0.9989495 + site + cleaner0 + 2023-07-26T15:27:32Z + SO: + + β2Hβ-grasp + + + 0.9975083 + bond_interaction + cleaner0 + 2023-07-26T13:01:20Z + MESH: + + hydrogen bonds + + + 0.99809754 + chemical + cleaner0 + 2023-07-26T13:01:53Z + CHEBI: + + Mg2+ + + + 0.9995534 + residue_name_number + cleaner0 + 2023-07-26T13:01:49Z + DUMMY: + + Asp358 + + + 0.9974086 + bond_interaction + cleaner0 + 2023-07-26T13:02:06Z + MESH: + + hydrophobic interactions + + + 0.9751883 + structure_element + cleaner0 + 2023-07-26T12:57:17Z + SO: + + β2Hβ + + + 0.9990463 + protein_state + cleaner0 + 2023-07-26T13:09:24Z + DUMMY: + + DNA-bound + + + 0.99850863 + structure_element + cleaner0 + 2023-07-26T12:57:17Z + SO: + + β2Hβ + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + 0.9985183 + experimental_method + cleaner0 + 2023-07-26T15:47:23Z + MESH: + + crystallized + + + 0.99932575 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T13:07:47Z + + absence of + + + 0.9939926 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + 0.9989264 + protein_state + cleaner0 + 2023-07-26T13:01:33Z + DUMMY: + + DNA free + + + 0.9993586 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9971194 + evidence + cleaner0 + 2023-07-26T15:40:03Z + DUMMY: + + structure + + + + gkw228fig3.jpg + F3 + FIG + fig_caption + 27931 + Conformational plasticity in the Tdp2 active site. (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop. Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. + + 0.99939024 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99889517 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.99921846 + protein_state + cleaner0 + 2023-07-26T13:10:20Z + DUMMY: + + open + + + 0.86148614 + structure_element + cleaner0 + 2023-07-26T15:17:42Z + SO: + + 3-helix + + + 0.9991924 + protein_state + cleaner0 + 2023-07-26T15:05:17Z + DUMMY: + + flexible + + + 0.9731954 + structure_element + cleaner0 + 2023-07-26T13:05:43Z + SO: + + active-site loop + + + 0.993626 + oligomeric_state + cleaner0 + 2023-07-26T13:06:50Z + DUMMY: + + monomer + + + 0.9974331 + structure_element + cleaner0 + 2023-07-26T15:17:46Z + SO: + + E + + + 0.99895936 + protein_state + cleaner0 + 2023-07-26T12:52:35Z + DUMMY: + + DNA-free + + + 0.99920815 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.997035 + evidence + cleaner0 + 2023-07-26T15:40:08Z + DUMMY: + + structure + + + 0.9995196 + residue_name_number + cleaner0 + 2023-07-26T13:06:01Z + DUMMY: + + T309 + + + 0.9539218 + structure_element + cleaner0 + 2023-07-26T12:42:17Z + SO: + + EEP + + + site + SO: + cleaner0 + 2023-07-26T15:22:32Z + + β2Hβ docking pocket + + + 0.99943167 + residue_name_number + cleaner0 + 2023-07-26T13:06:06Z + DUMMY: + + N312 + + + 0.99943453 + residue_name_number + cleaner0 + 2023-07-26T13:06:10Z + DUMMY: + + N314 + + + 0.9994703 + residue_name_number + cleaner0 + 2023-07-26T13:06:15Z + DUMMY: + + L315 + + + 0.9954908 + protein_state + cleaner0 + 2023-07-26T15:05:22Z + DUMMY: + + solvent-exposed + + + 0.9993517 + protein_state + cleaner0 + 2023-07-26T13:09:49Z + DUMMY: + + closed + + + 0.9978934 + structure_element + cleaner0 + 2023-07-26T12:57:17Z + SO: + + β2Hβ + + + complex_assembly + GO: + cleaner0 + 2023-07-26T13:04:49Z + + mTdp2cat–DNA + + + 0.99420965 + evidence + cleaner0 + 2023-07-26T15:40:11Z + DUMMY: + + structure + + + 0.99893767 + chemical + cleaner0 + 2023-07-26T12:55:14Z + CHEBI: + + 5′-ϵA + + + 0.99954814 + residue_name_number + cleaner0 + 2023-07-26T13:06:02Z + DUMMY: + + T309 + + + site + SO: + cleaner0 + 2023-07-26T15:25:53Z + + β2Hβ DNA-binding grasp + + + 0.99370754 + bond_interaction + cleaner0 + 2023-07-26T13:01:20Z + MESH: + + hydrogen bonds + + + 0.9994844 + residue_name_number + cleaner0 + 2023-07-26T13:06:20Z + DUMMY: + + Y321 + + + 0.99949145 + residue_name_number + cleaner0 + 2023-07-26T13:06:11Z + DUMMY: + + N314 + + + site + SO: + cleaner0 + 2023-07-26T15:22:32Z + + β2Hβ docking pocket + + + structure_element + SO: + cleaner0 + 2023-07-26T13:03:05Z + + active site loop + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-26T13:06:45Z + + promoters + + + 0.9989384 + protein_state + cleaner0 + 2023-07-26T12:52:35Z + DUMMY: + + DNA-free + + + 0.99923813 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.997685 + experimental_method + cleaner0 + 2023-07-26T15:47:30Z + MESH: + + sequence alignment + + + 0.9949455 + evidence + cleaner0 + 2023-07-26T15:40:15Z + DUMMY: + + electron density + + + 0.99373347 + experimental_method + cleaner0 + 2023-07-26T15:47:32Z + MESH: + + Limited trypsin proteolysis + + + 0.99897814 + protein_state + cleaner0 + 2023-07-26T15:05:31Z + DUMMY: + + flexible + + + 0.9811407 + structure_element + cleaner0 + 2023-07-26T13:05:42Z + SO: + + active-site loop + + + 0.9982493 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9991867 + protein_state + cleaner0 + 2023-07-26T13:04:57Z + DUMMY: + + WT + + + 0.9967235 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9966536 + mutant + cleaner0 + 2023-07-26T13:05:26Z + MESH: + + D358N + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T13:07:28Z + + presence + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T13:07:46Z + + absence of + + + 0.9987406 + chemical + cleaner0 + 2023-07-26T13:07:55Z + CHEBI: + + Mg2+ + + + 0.9972529 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + 0.9982733 + experimental_method + cleaner0 + 2023-07-26T15:47:36Z + MESH: + + SDS-PAGE + + + 0.9888363 + experimental_method + cleaner0 + 2023-07-26T15:47:40Z + MESH: + + Limited chymotrypsin proteolysis + + + 0.9988778 + protein_state + cleaner0 + 2023-07-26T15:05:34Z + DUMMY: + + flexible + + + 0.930664 + structure_element + cleaner0 + 2023-07-26T13:05:43Z + SO: + + active-site loop + + + 0.9989303 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.999265 + protein_state + cleaner0 + 2023-07-26T13:04:58Z + DUMMY: + + WT + + + 0.99579656 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9958763 + mutant + cleaner0 + 2023-07-26T13:05:27Z + MESH: + + D358N + + + experimental_method + MESH: + cleaner0 + 2023-07-26T15:48:04Z + + chymotrypsin + + + experimental_method + MESH: + cleaner0 + 2023-07-26T15:48:16Z + + trypsin + + + + RESULTS + paragraph + 29648 + This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers. The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B). In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density. The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding. + + 0.99904555 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9984268 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.99710524 + chemical + cleaner0 + 2023-07-26T13:08:17Z + CHEBI: + + Mg2+ + + + structure_element + SO: + cleaner0 + 2023-07-26T13:08:34Z + + substrate binding loops + + + 0.99879104 + oligomeric_state + cleaner0 + 2023-07-26T14:29:36Z + DUMMY: + + protomers + + + 0.99862456 + protein_state + cleaner0 + 2023-07-26T13:08:45Z + DUMMY: + + DNA ligand-free + + + site + SO: + cleaner0 + 2023-07-26T15:28:09Z + + active site β2Hβ-grasp + + + 0.9988449 + protein_state + cleaner0 + 2023-07-26T13:09:23Z + DUMMY: + + DNA-bound + + + 0.99933976 + protein_state + cleaner0 + 2023-07-26T13:09:49Z + DUMMY: + + closed + + + site + SO: + cleaner0 + 2023-07-26T15:28:16Z + + β2Hβ DNA binding grasp + + + 0.99883777 + oligomeric_state + cleaner0 + 2023-07-26T13:06:51Z + DUMMY: + + monomer + + + 0.97328824 + structure_element + cleaner0 + 2023-07-26T15:17:51Z + SO: + + chain ‘E’ + + + 0.9993617 + structure_element + cleaner0 + 2023-07-26T15:17:55Z + SO: + + grasp + + + 0.9992951 + protein_state + cleaner0 + 2023-07-26T13:10:20Z + DUMMY: + + open + + + 0.99883795 + structure_element + cleaner0 + 2023-07-26T15:17:59Z + SO: + + 3-helix loop + + + 0.55258197 + structure_element + cleaner0 + 2023-07-26T12:42:17Z + SO: + + EEP + + + 0.97456133 + site + cleaner0 + 2023-07-26T15:22:46Z + SO: + + catalytic core + + + 0.99876237 + oligomeric_state + cleaner0 + 2023-07-26T13:10:10Z + DUMMY: + + monomers + + + 0.77713597 + protein_state + cleaner0 + 2023-07-26T13:09:53Z + DUMMY: + + disordered + + + 0.9942424 + structure_element + cleaner0 + 2023-07-26T15:18:05Z + SO: + + DNA binding loop + + + 0.9979557 + evidence + cleaner0 + 2023-07-26T15:40:20Z + DUMMY: + + electron density + + + 0.9986331 + protein_state + cleaner0 + 2023-07-26T12:52:35Z + DUMMY: + + DNA-free + + + 0.6881894 + evidence + cleaner0 + 2023-07-26T15:40:24Z + DUMMY: + + crystal form + + + 0.9993507 + protein_state + cleaner0 + 2023-07-26T13:09:48Z + DUMMY: + + closed + + + 0.999046 + structure_element + cleaner0 + 2023-07-26T12:57:17Z + SO: + + β2Hβ + + + 0.9989207 + protein_state + cleaner0 + 2023-07-26T13:09:41Z + DUMMY: + + DNA bound + + + 0.9982318 + evidence + cleaner0 + 2023-07-26T15:40:27Z + DUMMY: + + structures + + + 0.99921954 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + 0.9992812 + protein_state + cleaner0 + 2023-07-26T13:09:49Z + DUMMY: + + closed + + + site + SO: + cleaner0 + 2023-07-26T15:28:55Z + + β2Hβ grasp + + + + RESULTS + paragraph + 30597 + A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes. In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site. + + 0.99728405 + protein_state + cleaner0 + 2023-07-26T15:05:40Z + DUMMY: + + extended + + + 0.96280384 + structure_element + cleaner0 + 2023-07-26T15:18:28Z + SO: + + 3-helix + + + 0.9978262 + structure_element + cleaner0 + 2023-07-26T15:18:32Z + SO: + + substrate-binding loop + + + 0.9992514 + protein_state + cleaner0 + 2023-07-26T13:10:19Z + DUMMY: + + open + + + residue_range + DUMMY: + cleaner0 + 2023-07-26T13:10:56Z + + Asn312-Leu315 + + + 0.9987749 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.99515945 + protein_state + cleaner0 + 2023-07-26T15:05:45Z + DUMMY: + + solvent-exposed + + + 0.9994771 + residue_name_number + cleaner0 + 2023-07-26T13:11:38Z + DUMMY: + + Thr309 + + + 0.99763954 + site + cleaner0 + 2023-07-26T15:22:53Z + SO: + + pocket + + + 0.98731893 + structure_element + cleaner0 + 2023-07-26T12:42:17Z + SO: + + EEP + + + 0.9990565 + structure_element + cleaner0 + 2023-07-26T15:18:37Z + SO: + + loop + + + 0.99948716 + residue_name_number + cleaner0 + 2023-07-26T13:11:38Z + DUMMY: + + Thr309 + + + bond_interaction + MESH: + cleaner0 + 2023-07-26T13:11:27Z + + cis–peptide bond + + + 0.9994842 + residue_name_number + cleaner0 + 2023-07-26T13:11:32Z + DUMMY: + + Asp308 + + + 0.9994766 + residue_name_number + cleaner0 + 2023-07-26T13:11:37Z + DUMMY: + + Thr309 + + + 0.9987033 + structure_element + cleaner0 + 2023-07-26T15:18:41Z + SO: + + short antiparallel beta-strand + + + structure_element + SO: + cleaner0 + 2023-07-26T12:57:17Z + + β2Hβ + + + 0.9992895 + protein_state + cleaner0 + 2023-07-26T13:09:49Z + DUMMY: + + closed + + + site + SO: + cleaner0 + 2023-07-26T15:29:21Z + + β2Hβ grasp + + + 0.99957484 + residue_name_number + cleaner0 + 2023-07-26T14:27:40Z + DUMMY: + + Asn312 + + + 0.9995746 + residue_name_number + cleaner0 + 2023-07-26T14:27:42Z + DUMMY: + + Asn314 + + + site + SO: + cleaner0 + 2023-07-26T13:13:29Z + + β2Hβ docking pockets + + + 0.9995832 + residue_name_number + cleaner0 + 2023-07-26T12:57:52Z + DUMMY: + + Leu315 + + + 0.9992415 + protein_state + cleaner0 + 2023-07-26T13:09:49Z + DUMMY: + + closed + + + 0.9987795 + structure_element + cleaner0 + 2023-07-26T12:57:17Z + SO: + + β2Hβ + + + 0.99950314 + residue_name_number + cleaner0 + 2023-07-26T13:11:38Z + DUMMY: + + Thr309 + + + 0.92933995 + structure_element + cleaner0 + 2023-07-26T12:42:17Z + SO: + + EEP + + + 0.99783933 + site + cleaner0 + 2023-07-26T15:22:56Z + SO: + + pocket + + + 0.99929917 + structure_element + cleaner0 + 2023-07-26T12:57:17Z + SO: + + β2Hβ + + + 0.9585682 + structure_element + cleaner0 + 2023-07-26T15:18:49Z + SO: + + antiparallel β-sheet + + + 0.9992459 + protein_state + cleaner0 + 2023-07-26T13:09:49Z + DUMMY: + + closed + + + site + SO: + cleaner0 + 2023-07-26T15:29:25Z + + β2Hβ-grasp + + + 0.99887127 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.9965538 + bond_interaction + cleaner0 + 2023-07-26T12:59:14Z + MESH: + + hydrogen bond + + + 0.9995154 + residue_name_number + cleaner0 + 2023-07-26T13:12:56Z + DUMMY: + + Trp307 + + + 0.922613 + site + cleaner0 + 2023-07-26T13:12:40Z + SO: + + Mg2+ coordinating residue + + + 0.99948764 + residue_name_number + cleaner0 + 2023-07-26T13:01:50Z + DUMMY: + + Asp358 + + + 0.99866587 + protein_state + cleaner0 + 2023-07-26T13:01:34Z + DUMMY: + + DNA free + + + 0.99789596 + evidence + cleaner0 + 2023-07-26T15:40:32Z + DUMMY: + + structure + + + 0.9992829 + protein_state + cleaner0 + 2023-07-26T13:09:49Z + DUMMY: + + closed + + + 0.99863595 + oligomeric_state + cleaner0 + 2023-07-26T13:10:11Z + DUMMY: + + monomers + + + 0.9988458 + chemical + cleaner0 + 2023-07-26T13:12:42Z + CHEBI: + + Mg2+ + + + 0.99890983 + site + cleaner0 + 2023-07-26T15:23:03Z + SO: + + active sites + + + 0.99861944 + oligomeric_state + cleaner0 + 2023-07-26T13:10:11Z + DUMMY: + + monomers + + + 0.9992842 + protein_state + cleaner0 + 2023-07-26T13:10:20Z + DUMMY: + + open + + + 0.99887437 + site + cleaner0 + 2023-07-26T15:23:07Z + SO: + + metal binding site + + + 0.99310863 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + site + SO: + cleaner0 + 2023-07-26T15:28:29Z + + β2Hβ DNA binding grasp + + + 0.99875396 + chemical + cleaner0 + 2023-07-26T13:12:45Z + CHEBI: + + Mg2+ + + + 0.99943167 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99897933 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + + RESULTS + paragraph + 32206 + To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E). In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage. Interestingly, addition of Mg2+ alone protects against proteolysis as well. This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). + + 0.97640836 + chemical + cleaner0 + 2023-07-26T13:14:12Z + CHEBI: + + Mg2+ + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + 0.9990816 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99923956 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9882291 + experimental_method + cleaner0 + 2023-07-26T15:47:51Z + MESH: + + limited trypsin and chymotrypsin proteolysis + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T13:00:59Z + + In the absence of + + + 0.99738055 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + 0.9947781 + chemical + cleaner0 + 2023-07-26T13:14:14Z + CHEBI: + + Mg2+, + + + 0.99924123 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9199223 + site + cleaner0 + 2023-07-26T15:29:44Z + SO: + + DNA binding grasp + + + 0.41016766 + experimental_method + cleaner0 + 2023-07-26T13:14:52Z + MESH: + + trypsin + + + 0.9995902 + residue_name_number + cleaner0 + 2023-07-26T13:15:03Z + DUMMY: + + Arg316 + + + 0.32405397 + experimental_method + cleaner0 + 2023-07-26T13:14:58Z + MESH: + + chymotrypsin + + + 0.9995851 + residue_name_number + cleaner0 + 2023-07-26T13:12:57Z + DUMMY: + + Trp307 + + + 0.9995977 + residue_name_number + cleaner0 + 2023-07-26T12:57:52Z + DUMMY: + + Leu315 + + + 0.99511623 + chemical + cleaner0 + 2023-07-26T13:15:23Z + CHEBI: + + Mg2+, + + + 0.9951626 + chemical + cleaner0 + 2023-07-26T13:15:26Z + CHEBI: + + Mg2+ + + + 0.951914 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + 0.99928135 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9988735 + chemical + cleaner0 + 2023-07-26T13:15:29Z + CHEBI: + + Mg2+ + + + 0.9989096 + chemical + cleaner0 + 2023-07-26T13:15:32Z + CHEBI: + + Mg2+ + + + 0.99929166 + protein_state + cleaner0 + 2023-07-26T13:09:49Z + DUMMY: + + closed + + + 0.998343 + site + cleaner0 + 2023-07-26T15:29:51Z + SO: + + β2Hβ-grasp + + + 0.9973899 + bond_interaction + cleaner0 + 2023-07-26T15:33:06Z + MESH: + + hydrogen-bonding network + + + 0.999526 + residue_name_number + cleaner0 + 2023-07-26T13:01:50Z + DUMMY: + + Asp358 + + + 0.9970352 + site + cleaner0 + 2023-07-26T15:29:56Z + SO: + + β2Hβ-grasp + + + 0.9995309 + residue_name_number + cleaner0 + 2023-07-26T13:12:57Z + DUMMY: + + Trp307 + + + 0.9991842 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99477494 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + + RESULTS + paragraph + 33042 + To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B). Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. + + 0.9991805 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9983687 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + 0.9924873 + taxonomy_domain + cleaner0 + 2023-07-26T12:42:26Z + DUMMY: + + mouse + + + 0.9992368 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.97244793 + experimental_method + cleaner0 + 2023-07-26T15:48:21Z + MESH: + + determined + + + 0.99115354 + evidence + cleaner0 + 2023-07-26T15:40:36Z + DUMMY: + + structure + + + 0.9986696 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + 0.99517787 + structure_element + cleaner0 + 2023-07-26T15:16:04Z + SO: + + Tdp2cat + + + 0.9991405 + protein_state + cleaner0 + 2023-07-26T12:40:14Z + DUMMY: + + bound to + + + 0.9366652 + chemical + cleaner0 + 2023-07-26T13:17:21Z + CHEBI: + + DNA 5′-PO4 + + + 0.9987079 + species + cleaner0 + 2023-07-26T13:16:29Z + MESH: + + human + + + 0.9990139 + complex_assembly + cleaner0 + 2023-07-26T13:16:24Z + GO: + + hTdp2cat-DNA + + + 0.97620994 + evidence + cleaner0 + 2023-07-26T15:40:48Z + DUMMY: + + structure + + + 0.99730194 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9990261 + protein_state + cleaner0 + 2023-07-26T13:09:42Z + DUMMY: + + DNA bound + + + 0.9990485 + protein_state + cleaner0 + 2023-07-26T13:09:24Z + DUMMY: + + DNA-bound + + + 0.99933916 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.99800044 + structure_element + cleaner0 + 2023-07-26T15:15:37Z + SO: + + hTdp2cat + + + 0.93132704 + structure_element + cleaner0 + 2023-07-26T13:17:54Z + SO: + + substrate binding loop + + + 0.99901927 + chemical + cleaner0 + 2023-07-26T13:17:38Z + CHEBI: + + Mg2+ + + + 0.9979335 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + 0.8249772 + experimental_method + cleaner0 + 2023-07-26T15:48:24Z + MESH: + + X-ray + + + 0.89792025 + evidence + cleaner0 + 2023-07-26T15:40:40Z + DUMMY: + + structures + + + 0.9983575 + experimental_method + cleaner0 + 2023-07-26T15:48:27Z + MESH: + + limited proteolysis analysis + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:42Z + + DNA + + + 0.98338115 + protein_state + cleaner0 + 2023-07-26T15:05:54Z + DUMMY: + + conserved + + + 0.9981893 + taxonomy_domain + cleaner0 + 2023-07-26T14:42:32Z + DUMMY: + + vertebrate + + + 0.99938107 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + + RESULTS + title_2 + 33806 + Tdp2 metal ion dependence + + 0.99817944 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + + RESULTS + paragraph + 33832 + Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site. This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction. In these experiments, at limiting Mg2+ concentrations, Ca2+ addition to Tdp2 reactions stimulated activity. While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site. In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies. To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). + + 0.9950906 + experimental_method + cleaner0 + 2023-07-26T15:48:31Z + MESH: + + X-ray structural analyses + + + 0.9991188 + chemical + cleaner0 + 2023-07-26T12:53:34Z + CHEBI: + + Mg2+ + + + 0.98035765 + protein_state + cleaner0 + 2023-07-26T12:52:51Z + DUMMY: + + bound in + + + 0.9992648 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99903685 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.99869794 + protein_state + cleaner0 + 2023-07-26T12:52:35Z + DUMMY: + + DNA-free + + + 0.9985156 + protein_state + cleaner0 + 2023-07-26T12:52:45Z + DUMMY: + + DNA damage bound + + + 0.99734735 + protein_state + cleaner0 + 2023-07-26T15:06:00Z + DUMMY: + + reaction product-bound + + + 0.9974191 + evidence + cleaner0 + 2023-07-26T15:40:51Z + DUMMY: + + crystal forms + + + 0.96647906 + taxonomy_domain + cleaner0 + 2023-07-26T12:42:26Z + DUMMY: + + mouse + + + 0.99816585 + species + cleaner0 + 2023-07-26T13:18:14Z + MESH: + + D. rerio + + + 0.9983072 + species + cleaner0 + 2023-07-26T13:18:19Z + MESH: + + C. elegans + + + 0.9991033 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + experimental_method + MESH: + cleaner0 + 2023-07-26T15:48:49Z + + biochemical analysis + + + 0.99910516 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99198085 + protein_type + cleaner0 + 2023-07-26T12:54:02Z + MESH: + + phosphotyrosyl phosphodiesterase + + + 0.9991456 + chemical + cleaner0 + 2023-07-26T12:53:36Z + CHEBI: + + Mg2+ + + + 0.9991478 + chemical + cleaner0 + 2023-07-26T12:53:39Z + CHEBI: + + Ca2+ + + + 0.99804735 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9162536 + ptm + cleaner0 + 2023-07-26T14:31:48Z + MESH: + + phosphotyrosyl + + + 0.9992785 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9989072 + site + cleaner0 + 2023-07-26T15:30:57Z + SO: + + metal ion binding sites + + + 0.9990357 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:53:19Z + + divalent metals + + + 0.9987674 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.995423 + chemical + cleaner0 + 2023-07-26T12:31:42Z + CHEBI: + + DNA + + + 0.99894774 + site + cleaner0 + 2023-07-26T15:31:02Z + SO: + + active center + + + 0.99914515 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9626226 + protein_type + cleaner0 + 2023-07-26T12:54:09Z + MESH: + + phosphodiesterase + + + 0.99879843 + experimental_method + cleaner0 + 2023-07-26T15:48:54Z + MESH: + + metal ion binding assays + + + 0.9953145 + evidence + cleaner0 + 2023-07-26T15:40:54Z + DUMMY: + + crystal structures + + + 0.92679715 + protein_state + cleaner0 + 2023-07-26T12:52:55Z + DUMMY: + + presence of + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:53:19Z + + divalent metals + + + 0.9992048 + chemical + cleaner0 + 2023-07-26T12:53:45Z + CHEBI: + + Mn2+ + + + 0.9982317 + chemical + cleaner0 + 2023-07-26T12:53:48Z + CHEBI: + + Ca2+) + + + 0.9991887 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9865973 + protein_type + cleaner0 + 2023-07-26T12:54:01Z + MESH: + + phosphotyrosyl phosphodiesterase + + + + gkw228fig4.jpg + F4 + FIG + fig_caption + 35149 + Metal cofactor interactions with Tdp2. (A) Intrinsic tryptophan fluorescence of mTdp2cat was used to monitor a conformational response to divalent metal ion binding. Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. A 53σ peak in the anomalous difference Fourier map (data collected at λ = 1.5418 Å) supports Mn2+ as the identity of this atom. (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. + + 0.9991905 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + evidence + DUMMY: + cleaner0 + 2023-07-26T15:41:49Z + + Intrinsic tryptophan fluorescence + + + 0.99917656 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9990857 + chemical + cleaner0 + 2023-07-26T13:20:10Z + CHEBI: + + Mg2+ + + + 0.9991176 + chemical + cleaner0 + 2023-07-26T13:20:12Z + CHEBI: + + Ca2+ + + + 0.99648654 + experimental_method + cleaner0 + 2023-07-26T15:49:27Z + MESH: + + titrated + + + 0.8527634 + protein_state + cleaner0 + 2023-07-26T15:06:06Z + DUMMY: + + presence + + + 0.9986613 + protein_state + cleaner0 + 2023-07-26T13:07:47Z + DUMMY: + + absence of + + + 0.9011693 + chemical + cleaner0 + 2023-07-26T13:19:28Z + CHEBI: + + 5′-P DNA + + + 0.9180519 + evidence + cleaner0 + 2023-07-26T15:41:53Z + DUMMY: + + tryptophan fluorescence + + + 0.99909085 + chemical + cleaner0 + 2023-07-26T13:20:15Z + CHEBI: + + Mg2+ + + + 0.99906313 + chemical + cleaner0 + 2023-07-26T13:20:17Z + CHEBI: + + Ca2+ + + + 0.96642387 + evidence + cleaner0 + 2023-07-26T15:41:56Z + DUMMY: + + tryptophan fluorescence + + + 0.99924517 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9982895 + protein_state + cleaner0 + 2023-07-26T15:06:16Z + DUMMY: + + presence + + + 0.99903226 + protein_state + cleaner0 + 2023-07-26T13:07:47Z + DUMMY: + + absence of + + + 0.997184 + chemical + cleaner0 + 2023-07-26T12:31:43Z + CHEBI: + + DNA + + + 0.9990357 + mutant + cleaner0 + 2023-07-26T13:05:27Z + MESH: + + D358N + + + site + SO: + cleaner0 + 2023-07-26T12:57:42Z + + active site + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T12:41:32Z + + mutant + + + 0.999131 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T15:49:45Z + + unresponsive + + + 0.9990889 + chemical + cleaner0 + 2023-07-26T13:20:25Z + CHEBI: + + Mg2+ + + + 0.99853384 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.99932754 + chemical + cleaner0 + 2023-07-26T13:21:16Z + CHEBI: + + T5PNP + + + 0.9990814 + chemical + cleaner0 + 2023-07-26T13:20:20Z + CHEBI: + + Mg2+ + + + 0.99908483 + chemical + cleaner0 + 2023-07-26T13:20:23Z + CHEBI: + + Ca2+ + + + 0.99935204 + chemical + cleaner0 + 2023-07-26T12:39:08Z + CHEBI: + + PNP + + + 0.9990599 + chemical + cleaner0 + 2023-07-26T13:20:30Z + CHEBI: + + Mg2+ + + + 0.9761267 + protein_state + cleaner0 + 2023-07-26T15:06:21Z + DUMMY: + + absence + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T12:52:56Z + + presence of + + + 0.99911594 + chemical + cleaner0 + 2023-07-26T13:20:28Z + CHEBI: + + Ca2+ + + + evidence + DUMMY: + cleaner0 + 2023-07-26T13:19:47Z + + σ-A weighted 2Fo-Fc electron density map + + + evidence + DUMMY: + cleaner0 + 2023-07-26T15:50:08Z + + model-phased anomalous difference Fourier + + + 0.6339561 + evidence + cleaner0 + 2023-07-26T15:42:05Z + DUMMY: + + maps + + + 0.9986401 + complex_assembly + cleaner0 + 2023-07-26T13:20:03Z + GO: + + mTdp2cat–DNA–Mn2+ + + + 0.9990269 + chemical + cleaner0 + 2023-07-26T13:20:33Z + CHEBI: + + Mn2+ + + + 0.9979833 + evidence + cleaner0 + 2023-07-26T15:42:09Z + DUMMY: + + anomalous difference Fourier map + + + 0.9990164 + chemical + cleaner0 + 2023-07-26T13:20:36Z + CHEBI: + + Mn2+ + + + 0.999102 + chemical + cleaner0 + 2023-07-26T13:20:43Z + CHEBI: + + Ca2+ + + + 0.9989511 + chemical + cleaner0 + 2023-07-26T13:20:45Z + CHEBI: + + Ca2+ + + + 0.99540246 + chemical + cleaner0 + 2023-07-26T12:31:43Z + CHEBI: + + DNA + + + 0.99907815 + chemical + cleaner0 + 2023-07-26T13:20:38Z + CHEBI: + + Mg2+ + + + 0.999029 + chemical + cleaner0 + 2023-07-26T13:20:40Z + CHEBI: + + Mg2+ + + + 0.9924315 + chemical + cleaner0 + 2023-07-26T12:31:43Z + CHEBI: + + DNA + + + 0.9978165 + complex_assembly + cleaner0 + 2023-07-26T13:04:50Z + GO: + + mTdp2cat–DNA + + + 0.99594927 + evidence + cleaner0 + 2023-07-26T15:42:12Z + DUMMY: + + structures + + + 0.9989953 + chemical + cleaner0 + 2023-07-26T13:20:47Z + CHEBI: + + Ca2+ + + + 0.80895495 + site + cleaner0 + 2023-07-26T13:21:52Z + SO: + + 5′-phosphate binding mode + + + + RESULTS + paragraph + 36620 + Our proteolysis results indicate a Mg2+-dependent Tdp2 conformational response to metal binding. The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. These data were an excellent fit to a single-site binding model both in the presence and absence of DNA (Figure 4A). This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5′-phosphate by mTdp2cat. This small molecule substrate is not expected to be influenced by metal–DNA coordination outside of the active site. Inclusion of ultrapure Ca2+ (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting Mg2+ (Figure 4B). We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. Overall, these metal binding analyses are consistent with a single metal ion mediated reaction. + + 0.9979517 + experimental_method + cleaner0 + 2023-07-26T15:50:15Z + MESH: + + proteolysis + + + chemical + CHEBI: + cleaner0 + 2023-07-26T13:22:10Z + + Mg2+ + + + 0.99918836 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9992749 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99902856 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.99684274 + residue_name + cleaner0 + 2023-07-26T14:30:26Z + SO: + + tryptophan + + + 0.9989566 + site + cleaner0 + 2023-07-26T15:31:07Z + SO: + + metal binding center + + + 0.98998046 + evidence + cleaner0 + 2023-07-26T15:50:32Z + DUMMY: + + intrinsic tryptophan fluorescence + + + 0.9993517 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9909933 + protein_state + cleaner0 + 2023-07-26T15:06:26Z + DUMMY: + + presence + + + 0.99886477 + protein_state + cleaner0 + 2023-07-26T13:07:47Z + DUMMY: + + absence of + + + 0.99456245 + chemical + cleaner0 + 2023-07-26T12:31:43Z + CHEBI: + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T13:22:45Z + + Mg2+ + + + 0.9723421 + evidence + cleaner0 + 2023-07-26T15:42:16Z + DUMMY: + + Kd + + + 0.99801743 + evidence + cleaner0 + 2023-07-26T15:42:18Z + DUMMY: + + Hill coefficients + + + 0.99856025 + site + cleaner0 + 2023-07-26T15:31:12Z + SO: + + metal binding site + + + 0.99141264 + protein_state + cleaner0 + 2023-07-26T15:06:29Z + DUMMY: + + presence + + + 0.9989222 + protein_state + cleaner0 + 2023-07-26T13:07:47Z + DUMMY: + + absence of + + + 0.99371064 + chemical + cleaner0 + 2023-07-26T12:31:43Z + CHEBI: + + DNA + + + 0.99917245 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9991501 + chemical + cleaner0 + 2023-07-26T13:22:21Z + CHEBI: + + p-nitrophenyl-thymidine-5′-phosphate + + + 0.99933296 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.81853044 + chemical + cleaner0 + 2023-07-26T12:31:43Z + CHEBI: + + DNA + + + 0.9990066 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.99913436 + chemical + cleaner0 + 2023-07-26T13:23:14Z + CHEBI: + + Ca2+ + + + 0.9989423 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9990857 + chemical + cleaner0 + 2023-07-26T13:23:18Z + CHEBI: + + Mg2+ + + + 0.9532363 + experimental_method + cleaner0 + 2023-07-26T15:50:40Z + MESH: + + titrations + + + 0.9985998 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + 0.9865117 + protein + cleaner0 + 2023-07-26T13:22:57Z + PR: + + hTdp2FL + + + 0.9694039 + structure_element + cleaner0 + 2023-07-26T15:15:36Z + SO: + + hTdp2cat + + + 0.9991063 + chemical + cleaner0 + 2023-07-26T13:23:16Z + CHEBI: + + Mg2+ + + + 0.9991188 + chemical + cleaner0 + 2023-07-26T13:23:22Z + CHEBI: + + Ca2+ + + + 0.99810153 + experimental_method + cleaner0 + 2023-07-26T15:50:43Z + MESH: + + metal binding analyses + + + + RESULTS + paragraph + 37916 + To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site. Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site. Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. + + 0.99901974 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9988178 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.998683 + evidence + cleaner0 + 2023-07-26T15:42:25Z + DUMMY: + + crystal structures + + + 0.99810183 + experimental_method + cleaner0 + 2023-07-26T15:51:06Z + MESH: + + soaking crystals + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T14:58:47Z + + support + + + 0.9990768 + chemical + cleaner0 + 2023-07-26T13:23:38Z + CHEBI: + + Mn2+ + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T14:58:57Z + + inhibit + + + chemical + CHEBI: + cleaner0 + 2023-07-26T13:24:02Z + + Ca2+ + + + 0.99799085 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9985504 + evidence + cleaner0 + 2023-07-26T15:42:28Z + DUMMY: + + Anomalous difference Fourier maps + + + 0.9992215 + complex_assembly + cleaner0 + 2023-07-26T13:23:34Z + GO: + + Tdp2–DNA–Mn2+ + + + 0.9972949 + site + cleaner0 + 2023-07-26T15:31:20Z + SO: + + binding site + + + 0.9989053 + chemical + cleaner0 + 2023-07-26T13:24:06Z + CHEBI: + + Mn2+ + + + 0.99915457 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9988585 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + bond_interaction + MESH: + cleaner0 + 2023-07-26T15:06:55Z + + octahedral coordination + + + 0.9989402 + chemical + cleaner0 + 2023-07-26T13:24:10Z + CHEBI: + + Mn2+ + + + 0.9989435 + chemical + cleaner0 + 2023-07-26T13:24:12Z + CHEBI: + + Mn2+ + + + 0.99896514 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99874413 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.9985651 + protein_state + cleaner0 + 2023-07-26T15:07:01Z + DUMMY: + + Mg2+-bound + + + 0.99906534 + chemical + cleaner0 + 2023-07-26T13:24:15Z + CHEBI: + + Mn2+ + + + 0.9989944 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + evidence + DUMMY: + cleaner0 + 2023-07-26T15:51:28Z + + co-complex structures + + + 0.99900204 + chemical + cleaner0 + 2023-07-26T13:24:20Z + CHEBI: + + Ca2+ + + + 0.99896026 + chemical + cleaner0 + 2023-07-26T13:24:17Z + CHEBI: + + Ca2+ + + + 0.99670094 + site + cleaner0 + 2023-07-26T13:24:26Z + SO: + + Mg2+ site + + + 0.9989062 + chemical + cleaner0 + 2023-07-26T13:24:29Z + CHEBI: + + Ca2+ + + + 0.64549816 + bond_interaction + cleaner0 + 2023-07-26T15:07:15Z + MESH: + + octahedrally coordinated + + + 0.99886703 + chemical + cleaner0 + 2023-07-26T13:24:56Z + CHEBI: + + Ca2+ + + + 0.99878913 + chemical + cleaner0 + 2023-07-26T13:24:53Z + CHEBI: + + Ca2+ + + + site + SO: + cleaner0 + 2023-07-26T13:24:50Z + + Mg2+ ion site + + + bond_interaction + MESH: + cleaner0 + 2023-07-26T14:29:10Z + + bi-dentate inner sphere metal contacts + + + 0.99859476 + chemical + cleaner0 + 2023-07-26T13:24:59Z + CHEBI: + + Ca2+ + + + 0.9992447 + residue_name_number + cleaner0 + 2023-07-26T13:25:11Z + DUMMY: + + Glu162 + + + site + SO: + cleaner0 + 2023-07-26T13:25:35Z + + active site phosphate-binding mode + + + 0.9966893 + chemical + cleaner0 + 2023-07-26T13:25:57Z + CHEBI: + + 5′-PO4 + + + 0.99908304 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99874544 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.99904895 + chemical + cleaner0 + 2023-07-26T13:25:03Z + CHEBI: + + Ca2+ + + + 0.99864763 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99898195 + protein_state + cleaner0 + 2023-07-26T15:07:24Z + DUMMY: + + divalent metal bound + + + 0.9988103 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99823064 + evidence + cleaner0 + 2023-07-26T15:42:34Z + DUMMY: + + structures + + + 0.9987406 + chemical + cleaner0 + 2023-07-26T13:25:05Z + CHEBI: + + Ca2+ + + + 0.99761885 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + + RESULTS + title_2 + 39369 + Modeling the Tdp2 reaction coordinate + + 0.9985037 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + + RESULTS + paragraph + 39407 + Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct. + + 0.9952079 + chemical + cleaner0 + 2023-07-26T14:59:41Z + CHEBI: + + Mg2+ + + + 0.9923113 + experimental_method + cleaner0 + 2023-07-26T15:51:37Z + MESH: + + simulated + + + 0.9987791 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9978865 + experimental_method + cleaner0 + 2023-07-26T13:25:48Z + MESH: + + hybrid QM/MM modeling + + + 0.9987839 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.90296316 + protein_state + cleaner0 + 2023-07-26T15:07:32Z + DUMMY: + + substrate analog- + + + 0.99698424 + protein_state + cleaner0 + 2023-07-26T15:07:35Z + DUMMY: + + product-bound + + + 0.99819833 + evidence + cleaner0 + 2023-07-26T15:42:38Z + DUMMY: + + structures + + + 0.99502784 + experimental_method + cleaner0 + 2023-07-26T15:51:42Z + MESH: + + structural analyses + + + 0.9985355 + experimental_method + cleaner0 + 2023-07-26T15:51:46Z + MESH: + + superposition + + + 0.9951969 + chemical + cleaner0 + 2023-07-26T12:31:43Z + CHEBI: + + DNA + + + 0.9991888 + chemical + cleaner0 + 2023-07-26T13:26:13Z + CHEBI: + + 5′-aminohexanol + + + 0.99886715 + chemical + cleaner0 + 2023-07-26T13:25:56Z + CHEBI: + + 5′-PO4 + + + 0.99889874 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9970548 + chemical + cleaner0 + 2023-07-26T13:26:40Z + CHEBI: + + water + + + 0.9926974 + bond_interaction + cleaner0 + 2023-07-26T13:26:45Z + MESH: + + hydrogen bonded + + + 0.9994535 + residue_name_number + cleaner0 + 2023-07-26T13:26:30Z + DUMMY: + + Asp272 + + + 0.99943703 + residue_name_number + cleaner0 + 2023-07-26T13:26:34Z + DUMMY: + + Asn274 + + + + gkw228fig5.jpg + F5 + FIG + fig_caption + 40112 + Structure-function analysis of the Tdp2 reaction mechanism. (A) Proposed mechanism for hydrolysis of phosphotyrosine bond by Tdp2. Residues in green form the binding-site for the 5′-tyrosine (red) and phosphate, yellow bind the 5′ nucleotide and blue bind nucleotides 2–3. Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom. Reaction proceeds from right to left. (C) Models for the mTdp2cat-DNA complex during the QM/MM reaction path simulation showing the substrate (left, tan), transition state intermediate (center, cyan) and product (right, pink) states. Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm. Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d. n = 3. Mutants of hTdp2 (black) and the equivalent residue in mTdp2 (tan) are indicated. + + 0.9991591 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9853335 + residue_name + cleaner0 + 2023-07-26T14:30:32Z + SO: + + phosphotyrosine + + + 0.9992422 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99896973 + site + cleaner0 + 2023-07-26T15:31:38Z + SO: + + binding-site + + + 0.6851773 + residue_name + cleaner0 + 2023-07-26T15:31:58Z + SO: + + 5′-tyrosine + + + 0.9872578 + chemical + cleaner0 + 2023-07-26T14:59:58Z + CHEBI: + + phosphate + + + 0.9989473 + protein + cleaner0 + 2023-07-26T14:38:25Z + PR: + + mTdp2 + + + 0.99686766 + evidence + cleaner0 + 2023-07-26T15:42:42Z + DUMMY: + + Free energy + + + 0.99843395 + experimental_method + cleaner0 + 2023-07-26T13:27:33Z + MESH: + + QM/MM simulation + + + 0.99903286 + chemical + cleaner0 + 2023-07-26T13:31:32Z + CHEBI: + + water + + + 0.99904007 + complex_assembly + cleaner0 + 2023-07-26T13:27:45Z + GO: + + mTdp2cat-DNA + + + 0.99856716 + experimental_method + cleaner0 + 2023-07-26T13:29:11Z + MESH: + + QM/MM reaction path simulation + + + 0.99888355 + protein + cleaner0 + 2023-07-26T14:38:33Z + PR: + + mTdp2 + + + 0.9944894 + evidence + cleaner0 + 2023-07-26T15:42:47Z + DUMMY: + + Electrostatic surface potential + + + residue_name + SO: + cleaner0 + 2023-07-26T13:29:56Z + + 5′-phosphotyrosine + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T12:52:56Z + + presence of + + + 0.993049 + bond_interaction + cleaner0 + 2023-07-26T13:27:57Z + MESH: + + cation–π interaction + + + 0.9994918 + residue_name_number + cleaner0 + 2023-07-26T14:04:47Z + DUMMY: + + Arg216 + + + 0.7602875 + evidence + cleaner0 + 2023-07-26T15:42:54Z + DUMMY: + + Electrostatic potential + + + 0.9991514 + protein_state + cleaner0 + 2023-07-26T13:30:07Z + DUMMY: + + wild-type + + + 0.9988102 + protein_state + cleaner0 + 2023-07-26T12:41:32Z + DUMMY: + + mutant + + + 0.9986695 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + experimental_method + MESH: + cleaner0 + 2023-07-26T13:28:20Z + + MBP + + + structure_element + SO: + cleaner0 + 2023-07-26T15:15:37Z + + hTdp2cat + + + experimental_method + MESH: + cleaner0 + 2023-07-26T13:28:35Z + + fusion proteins + + + 0.99930274 + chemical + cleaner0 + 2023-07-26T12:39:08Z + CHEBI: + + PNP + + + 0.9993006 + chemical + cleaner0 + 2023-07-26T12:39:08Z + CHEBI: + + PNP + + + 0.9947207 + chemical + cleaner0 + 2023-07-26T15:00:39Z + CHEBI: + + phosphate + + + 0.99907947 + chemical + cleaner0 + 2023-07-26T13:21:17Z + CHEBI: + + T5PNP + + + 0.9491028 + evidence + cleaner0 + 2023-07-26T15:42:58Z + DUMMY: + + Reaction rates + + + 0.99921954 + protein_state + cleaner0 + 2023-07-26T13:30:03Z + DUMMY: + + wildtype + + + experimental_method + MESH: + cleaner0 + 2023-07-26T13:28:57Z + + MBP + + + structure_element + SO: + cleaner0 + 2023-07-26T15:15:37Z + + hTdp2cat + + + 0.9990615 + protein + cleaner0 + 2023-07-26T14:38:40Z + PR: + + hTdp2 + + + 0.9991819 + protein + cleaner0 + 2023-07-26T14:38:34Z + PR: + + mTdp2 + + + + RESULTS + paragraph + 41640 + We examined the energy profile of the nucleophilic attack of the water molecule by using the distance between the water oxygen and the P atom on the phosphate moiety as the sole reaction coordinate in the present calculation (Figure 5B and C). A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å). No appreciable energy penalty is observed during the first 0.5 Å of the reaction coordinate. When the reaction reaches an O–P distance of 2.18 Å, formation of a transition state with an energy maximum of +7.4 kcal mol−1 is observed. Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length. In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction. Concomitant with this, the phosphotyrosyl O–P bond weakens (d = 1.89 Å), and the formation of the penta-covalent transition state (Figure 5C ‘ii-transition state’) is observed. The final steps show inversion of stereochemistry at the phosphate, along with lengthening and breaking of the phosphotyrosyl O–P bond. Product formation is coupled to a transfer of a proton from the nucleophillic water to Asp272, consistent with the proposed function for this residue as the catalytic base. + + 0.9987967 + chemical + cleaner0 + 2023-07-26T13:31:32Z + CHEBI: + + water + + + 0.9988035 + chemical + cleaner0 + 2023-07-26T13:31:32Z + CHEBI: + + water + + + 0.98136073 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.99678147 + chemical + cleaner0 + 2023-07-26T13:31:13Z + CHEBI: + + 5′–aminohexanol + + + 0.85370886 + evidence + cleaner0 + 2023-07-26T15:43:01Z + DUMMY: + + structure + + + 0.9973348 + residue_name + cleaner0 + 2023-07-26T13:30:36Z + SO: + + tyrosine + + + 0.99907035 + chemical + cleaner0 + 2023-07-26T15:00:45Z + CHEBI: + + 5′-aminohexanol + + + 0.9988524 + chemical + cleaner0 + 2023-07-26T13:31:18Z + CHEBI: + + Mg2+ + + + 0.99862885 + chemical + cleaner0 + 2023-07-26T15:00:49Z + CHEBI: + + waters + + + 0.99895006 + complex_assembly + cleaner0 + 2023-07-26T13:30:24Z + GO: + + mTdp2-DNA + + + 0.81706643 + evidence + cleaner0 + 2023-07-26T15:43:05Z + DUMMY: + + structure + + + 0.9959628 + experimental_method + cleaner0 + 2023-07-26T13:30:41Z + MESH: + + molecular dynamics simulation + + + 0.9975905 + experimental_method + cleaner0 + 2023-07-26T13:30:33Z + MESH: + + QM/MM optimization + + + 0.97578055 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9957191 + chemical + cleaner0 + 2023-07-26T15:00:52Z + CHEBI: + + 5′-aminohexanol + + + 0.9717063 + evidence + cleaner0 + 2023-07-26T15:43:16Z + DUMMY: + + structure + + + 0.9987042 + chemical + cleaner0 + 2023-07-26T13:31:31Z + CHEBI: + + water + + + 0.9993772 + residue_name_number + cleaner0 + 2023-07-26T13:26:30Z + DUMMY: + + Asp272 + + + 0.9969578 + bond_interaction + cleaner0 + 2023-07-26T12:59:14Z + MESH: + + hydrogen bond + + + 0.847118 + ptm + cleaner0 + 2023-07-26T14:31:47Z + MESH: + + phosphotyrosyl + + + 0.99797827 + experimental_method + cleaner0 + 2023-07-26T15:51:52Z + MESH: + + simulation + + + 0.9975592 + chemical + cleaner0 + 2023-07-26T13:31:32Z + CHEBI: + + water + + + 0.99617916 + bond_interaction + cleaner0 + 2023-07-26T12:59:14Z + MESH: + + hydrogen bond + + + 0.9986946 + chemical + cleaner0 + 2023-07-26T13:31:22Z + CHEBI: + + water + + + 0.99932206 + residue_name_number + cleaner0 + 2023-07-26T13:26:30Z + DUMMY: + + Asp272 + + + 0.998473 + chemical + cleaner0 + 2023-07-26T13:31:32Z + CHEBI: + + water + + + 0.99807954 + chemical + cleaner0 + 2023-07-26T13:31:25Z + CHEBI: + + water + + + 0.9966124 + bond_interaction + cleaner0 + 2023-07-26T12:59:14Z + MESH: + + hydrogen bond + + + 0.9993255 + residue_name_number + cleaner0 + 2023-07-26T13:26:35Z + DUMMY: + + Asn274 + + + 0.99835783 + chemical + cleaner0 + 2023-07-26T13:31:27Z + CHEBI: + + water + + + 0.83306664 + ptm + cleaner0 + 2023-07-26T14:31:48Z + MESH: + + phosphotyrosyl + + + 0.9027897 + chemical + cleaner0 + 2023-07-26T15:00:58Z + CHEBI: + + phosphate + + + 0.7681125 + ptm + cleaner0 + 2023-07-26T14:31:48Z + MESH: + + phosphotyrosyl + + + 0.9983329 + chemical + cleaner0 + 2023-07-26T13:31:32Z + CHEBI: + + water + + + 0.9993311 + residue_name_number + cleaner0 + 2023-07-26T13:26:30Z + DUMMY: + + Asp272 + + + + RESULTS + paragraph + 43916 + Of note, both nitrogens of the imidazole side chain of His 359 require protonation for stability of the simulation. Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. In the final optimized structure, the observed product state (Figure 5C, ‘iii-product’) is found in a conformation that is 7.4 kcal mol−1 more stable than the initial reactive state (Figure 5B). + + 0.9964541 + residue_name_number + cleaner0 + 2023-07-26T13:33:06Z + DUMMY: + + His 359 + + + 0.9942985 + experimental_method + cleaner0 + 2023-07-26T15:51:59Z + MESH: + + simulation + + + 0.997066 + residue_name_number + cleaner0 + 2023-07-26T13:33:12Z + DUMMY: + + Asp 326 + + + 0.99698794 + bond_interaction + cleaner0 + 2023-07-26T12:59:14Z + MESH: + + hydrogen bond + + + 0.9993562 + residue_name_number + cleaner0 + 2023-07-26T14:27:49Z + DUMMY: + + His359 + + + 0.99433935 + bond_interaction + cleaner0 + 2023-07-26T13:32:57Z + MESH: + + salt bridge + + + 0.9985972 + protein_state + cleaner0 + 2023-07-26T15:07:42Z + DUMMY: + + protonated + + + 0.9993844 + residue_name_number + cleaner0 + 2023-07-26T14:28:22Z + DUMMY: + + His359 + + + 0.589665 + residue_name + cleaner0 + 2023-07-26T14:32:02Z + SO: + + Asp + + + residue_name + SO: + cleaner0 + 2023-07-26T14:28:14Z + + His + + + structure_element + SO: + cleaner0 + 2023-07-26T12:42:17Z + + EEP + + + 0.99917275 + protein + cleaner0 + 2023-07-26T13:32:50Z + PR: + + APE1 + + + evidence + DUMMY: + cleaner0 + 2023-07-26T13:33:32Z + + pKa + + + 0.9912129 + residue_name + cleaner0 + 2023-07-26T13:33:23Z + SO: + + His + + + 0.9970705 + bond_interaction + cleaner0 + 2023-07-26T12:59:14Z + MESH: + + hydrogen bond + + + 0.9964067 + protein_state + cleaner0 + 2023-07-26T15:07:46Z + DUMMY: + + doubly protonated + + + 0.9994153 + residue_name_number + cleaner0 + 2023-07-26T14:28:26Z + DUMMY: + + His359 + + + 0.989944 + chemical + cleaner0 + 2023-07-26T15:01:04Z + CHEBI: + + phosphate + + + 0.9986948 + chemical + cleaner0 + 2023-07-26T15:01:09Z + CHEBI: + + Mg2+ + + + 0.9993716 + residue_name_number + cleaner0 + 2023-07-26T14:28:29Z + DUMMY: + + His359 + + + 0.99631864 + bond_interaction + cleaner0 + 2023-07-26T13:33:19Z + MESH: + + H-bond + + + 0.999406 + residue_name_number + cleaner0 + 2023-07-26T14:28:32Z + DUMMY: + + Asp326 + + + 0.99722195 + evidence + cleaner0 + 2023-07-26T15:43:38Z + DUMMY: + + structure + + + + RESULTS + paragraph + 44776 + The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group. A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. We propose this cation–π interaction further contributes to tuned stabilization of the negatively charged phenolate reaction product. Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A). Removing Arg216 from the quantum subsystem incurs an ∼2 kcal mol−1 penalty in the transition state and product complex. Removing the +1 charge on the Arg216 has a minimal impact on the transition state, but incurs an additional ∼2 kcal mol−1 penalty in the product complex. Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis. + + 0.8954392 + residue_name + cleaner0 + 2023-07-26T13:33:44Z + SO: + + tyrosine + + + 0.9534459 + bond_interaction + cleaner0 + 2023-07-26T14:29:17Z + MESH: + + coordinated to + + + 0.9983754 + chemical + cleaner0 + 2023-07-26T13:33:55Z + CHEBI: + + Mg2+ + + + 0.99876976 + chemical + cleaner0 + 2023-07-26T13:33:58Z + CHEBI: + + Mg2+ + + + 0.9985714 + chemical + cleaner0 + 2023-07-26T13:31:32Z + CHEBI: + + water + + + 0.8796977 + chemical + cleaner0 + 2023-07-26T15:01:18Z + CHEBI: + + phosphate + + + 0.9994289 + residue_name_number + cleaner0 + 2023-07-26T13:25:11Z + DUMMY: + + Glu162 + + + 0.9985143 + chemical + cleaner0 + 2023-07-26T13:34:00Z + CHEBI: + + Mg2+ + + + 0.9978571 + experimental_method + cleaner0 + 2023-07-26T13:33:52Z + MESH: + + QM/MM modeling + + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:12Z + + Top2 + + + 0.958332 + residue_name + cleaner0 + 2023-07-26T13:34:10Z + SO: + + tyrosine + + + 0.98768425 + protein_state + cleaner0 + 2023-07-26T15:07:52Z + DUMMY: + + conserved + + + 0.9956136 + residue_name_number + cleaner0 + 2023-07-26T13:34:21Z + DUMMY: + + Tyr 188 + + + 0.9971628 + residue_name_number + cleaner0 + 2023-07-26T13:34:25Z + DUMMY: + + Arg 216 + + + 0.9965563 + residue_name_number + cleaner0 + 2023-07-26T13:34:30Z + DUMMY: + + Ser 239 + + + 0.99910384 + protein_state + cleaner0 + 2023-07-26T13:34:05Z + DUMMY: + + conserved + + + 0.70152825 + protein_type + cleaner0 + 2023-07-26T12:34:12Z + MESH: + + Top2 + + + 0.9989686 + site + cleaner0 + 2023-07-26T13:34:08Z + SO: + + tyrosine binding pocket + + + 0.9964851 + bond_interaction + cleaner0 + 2023-07-26T13:27:57Z + MESH: + + cation–π interaction + + + 0.98839015 + evidence + cleaner0 + 2023-07-26T15:43:43Z + DUMMY: + + electrostatic potential + + + 0.45142025 + ptm + cleaner0 + 2023-07-26T14:31:48Z + MESH: + + phosphotyrosyl + + + 0.9982492 + protein_state + cleaner0 + 2023-07-26T15:07:55Z + DUMMY: + + presence + + + 0.9986373 + protein_state + cleaner0 + 2023-07-26T13:07:47Z + DUMMY: + + absence of + + + 0.9994874 + residue_name_number + cleaner0 + 2023-07-26T14:04:47Z + DUMMY: + + Arg216 + + + 0.99950695 + residue_name_number + cleaner0 + 2023-07-26T14:04:47Z + DUMMY: + + Arg216 + + + 0.99622107 + bond_interaction + cleaner0 + 2023-07-26T13:27:57Z + MESH: + + cation–π interaction + + + 0.99941087 + residue_name_number + cleaner0 + 2023-07-26T14:04:47Z + DUMMY: + + Arg216 + + + 0.9803389 + experimental_method + cleaner0 + 2023-07-26T15:52:05Z + MESH: + + QM + + + 0.9599018 + experimental_method + cleaner0 + 2023-07-26T15:52:10Z + MESH: + + MM + + + 0.99449956 + evidence + cleaner0 + 2023-07-26T15:43:51Z + DUMMY: + + energy penalties + + + 0.9993787 + residue_name_number + cleaner0 + 2023-07-26T14:04:47Z + DUMMY: + + Arg216 + + + 0.99938774 + residue_name_number + cleaner0 + 2023-07-26T14:04:47Z + DUMMY: + + Arg216 + + + 0.9925355 + experimental_method + cleaner0 + 2023-07-26T13:34:45Z + MESH: + + QM/MM modeling + + + 0.99879587 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99813336 + chemical + cleaner0 + 2023-07-26T15:01:24Z + CHEBI: + + Mg2+ + + + 0.99851614 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.8226487 + residue_name + cleaner0 + 2023-07-26T13:34:59Z + SO: + + 5′-phosphotyrosine + + + + RESULTS + title_2 + 46512 + Tdp2 mutational analysis + + 0.99812895 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9983307 + experimental_method + cleaner0 + 2023-07-26T15:52:14Z + MESH: + + mutational analysis + + + + RESULTS + paragraph + 46537 + To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). By analyzing activities on this nested set of chemically related substrates we aimed to dissect structure-activity relationships of Tdp2 catalysis. For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. + + 0.9989396 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99524313 + taxonomy_domain + cleaner0 + 2023-07-26T12:42:26Z + DUMMY: + + mouse + + + 0.9990645 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9988365 + evidence + cleaner0 + 2023-07-26T15:44:08Z + DUMMY: + + crystal structures + + + 0.9973043 + taxonomy_domain + cleaner0 + 2023-07-26T12:42:26Z + DUMMY: + + mouse + + + 0.9328153 + experimental_method + cleaner0 + 2023-07-26T15:52:17Z + MESH: + + engineered and purified + + + 0.99854726 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + experimental_method + MESH: + cleaner0 + 2023-07-26T13:35:19Z + + MBP + + + structure_element + SO: + cleaner0 + 2023-07-26T15:15:37Z + + hTdp2cat + + + 0.99700445 + protein_state + cleaner0 + 2023-07-26T12:41:32Z + DUMMY: + + mutant + + + 0.9987233 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + 0.8504147 + experimental_method + cleaner0 + 2023-07-26T15:52:22Z + MESH: + + mutations + + + 0.99911267 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.9968714 + protein_state + cleaner0 + 2023-07-26T13:35:32Z + DUMMY: + + tyrosylated + + + 0.9931571 + chemical + cleaner0 + 2023-07-26T12:31:43Z + CHEBI: + + DNA + + + 0.9988423 + ptm + cleaner0 + 2023-07-26T14:35:06Z + MESH: + + 5′-Y + + + 0.99913514 + chemical + cleaner0 + 2023-07-26T13:35:45Z + CHEBI: + + p-nitrophenyl phosphate + + + 0.9992067 + chemical + cleaner0 + 2023-07-26T13:35:50Z + CHEBI: + + PNPP + + + 0.998497 + chemical + cleaner0 + 2023-07-26T13:35:55Z + CHEBI: + + thymidine 5′-monophosphate p-nitrophenyl ester + + + 0.9993 + chemical + cleaner0 + 2023-07-26T13:21:17Z + CHEBI: + + T5PNP + + + 0.9992094 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.54402196 + experimental_method + cleaner0 + 2023-07-26T15:52:29Z + MESH: + + mutations + + + 0.99925834 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99123883 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.95041716 + ptm + cleaner0 + 2023-07-26T14:31:48Z + MESH: + + phosphotyrosyl + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.99901456 + ptm + cleaner0 + 2023-07-26T14:35:06Z + MESH: + + 5′-Y + + + 0.9992015 + chemical + cleaner0 + 2023-07-26T13:21:17Z + CHEBI: + + T5PNP + + + 0.99919766 + chemical + cleaner0 + 2023-07-26T13:35:51Z + CHEBI: + + PNPP + + + + RESULTS + paragraph + 47605 + Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage. To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis. Next, we mutated key elements of the mobile loop (β2Hβ hydrophobic wall, Figure 2A and C). Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity). The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). Third, we altered properties of the proposed enzyme substrate cation–π interface. No activity was detected for a mutant that removes the positive charge at this position (hR206A). The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair. We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. + + 0.79008055 + evidence + cleaner0 + 2023-07-26T15:44:21Z + DUMMY: + + Structural results + + + 0.9975596 + experimental_method + cleaner0 + 2023-07-26T13:36:16Z + MESH: + + QM/MM modeling + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-26T13:37:30Z + + mAsp272 + + + 0.999032 + chemical + cleaner0 + 2023-07-26T13:31:32Z + CHEBI: + + water + + + ptm + MESH: + cleaner0 + 2023-07-26T13:38:42Z + + phosphotyrosyl linkage + + + 0.9958906 + experimental_method + cleaner0 + 2023-07-26T15:52:33Z + MESH: + + mutated + + + 0.7221521 + experimental_method + cleaner0 + 2023-07-26T15:52:36Z + MESH: + + to + + + 0.99744344 + residue_name + cleaner0 + 2023-07-26T13:38:01Z + SO: + + His + + + 0.9977508 + residue_name + cleaner0 + 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protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + 0.99860847 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.9985455 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + Tdp2 + + + + RESULTS + paragraph + 50028 + Recently, it was found that inactivation of TDP2 by a splice-site mutation is associated with neurological disease and confers hypersensitivity to Top2 poisons. We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini. We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). We show the hD350N substitution severely impairs activity on all substrates tested in vitro, whereas hI307V only has a mild impact on catalysis (Figure 6B–D). To better understand the basis for the D350N catalytic defect, we analyzed the structural environment of this substitution based on the high-resolution structures of mTdp2cat (Figure 6A). Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations. + + 0.9992699 + protein + cleaner0 + 2023-07-26T12:31:07Z + PR: + + TDP2 + + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:12Z + + Top2 + + + 0.9981804 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + 0.9992962 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.99913436 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.99846315 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + 0.99936324 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + TDP2 + + + 0.99834377 + site + cleaner0 + 2023-07-26T13:43:32Z + SO: + + DNA processing active site + + + 0.42868415 + gene + cleaner0 + 2023-07-26T13:43:43Z + GENE: + + rs199602263 + + + 0.9707081 + residue_name_number + cleaner0 + 2023-07-26T13:42:31Z + DUMMY: + + hAsp350 + + + 0.992804 + residue_name + cleaner0 + 2023-07-26T13:44:42Z + SO: + + Asn + + + 0.7287399 + gene + cleaner0 + 2023-07-26T13:44:00Z + GENE: + + rs77273535 + + + 0.950774 + residue_name_number + cleaner0 + 2023-07-26T13:42:49Z + DUMMY: + + hIle307 + + + 0.99489695 + residue_name + cleaner0 + 2023-07-26T13:44:39Z + SO: + + Val + + + 0.9990108 + mutant + cleaner0 + 2023-07-26T13:44:12Z + MESH: + + hD350N + + + 0.760592 + experimental_method + cleaner0 + 2023-07-26T15:53:13Z + MESH: + + substitution + + + 0.9989466 + mutant + cleaner0 + 2023-07-26T13:44:07Z + MESH: + + hI307V + + + 0.99899906 + mutant + cleaner0 + 2023-07-26T14:27:22Z + MESH: + + D350N + + + 0.99863416 + evidence + cleaner0 + 2023-07-26T15:44:34Z + DUMMY: + + structures + + + 0.9992316 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9994 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.99658155 + chemical + cleaner0 + 2023-07-26T13:44:46Z + CHEBI: + + Mg2+ + + + bond_interaction + MESH: + cleaner0 + 2023-07-26T15:32:30Z + + octahedral coordination shell + + + 0.9951826 + bond_interaction + cleaner0 + 2023-07-26T15:33:06Z + MESH: + + hydrogen-bonding network + + + 0.9983719 + residue_name_number + cleaner0 + 2023-07-26T13:42:37Z + DUMMY: + + hAsp350 + + + 0.9982931 + residue_name_number + cleaner0 + 2023-07-26T13:43:10Z + DUMMY: + + mAsp358 + + + 0.99897057 + protein_state + cleaner0 + 2023-07-26T13:09:24Z + DUMMY: + + DNA-bound + + + structure_element + SO: + cleaner0 + 2023-07-26T13:44:33Z + + β2Hβ substrate-binding loop + + + 0.9971111 + bond_interaction + cleaner0 + 2023-07-26T14:29:27Z + MESH: + + hydrogen bonding + + + 0.99771726 + residue_name_number + cleaner0 + 2023-07-26T13:43:21Z + DUMMY: + + mTrp307 + + + 0.99843055 + residue_name_number + cleaner0 + 2023-07-26T13:42:37Z + DUMMY: + + hAsp350 + + + 0.9984785 + residue_name_number + cleaner0 + 2023-07-26T13:43:09Z + DUMMY: + + mAsp358 + + + 0.9943887 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.8859374 + structure_element + cleaner0 + 2023-07-26T15:19:25Z + SO: + + substrate binding loop + + + + gkw228fig6.jpg + F6 + FIG + fig_caption + 51446 + Tdp2 SNPs impair function. (A) Active site residues mutated by TDP2 SNPs. D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. Samples were withdrawn from reactions, neutralized with TBE-urea loading dye at the indicated timepoints, and electrophoresed on a 20% TBE-urea PAGE. (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C. Error bars, s.d. n = 3. + + 0.90539426 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9987093 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + Active site + + + 0.6554838 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + TDP2 + + + 0.99902546 + mutant + cleaner0 + 2023-07-26T14:27:23Z + MESH: + + D350N + + + 0.9868723 + protein + cleaner0 + 2023-07-26T14:38:34Z + PR: + + mTdp2 + + + 0.99899143 + mutant + cleaner0 + 2023-07-26T13:05:27Z + MESH: + + D358N + + + 0.9990601 + mutant + cleaner0 + 2023-07-26T14:00:51Z + MESH: + + I307V + + + 0.9781138 + protein + cleaner0 + 2023-07-26T14:38:34Z + PR: + + mTdp2 + + + 0.99900824 + mutant + cleaner0 + 2023-07-26T14:27:30Z + MESH: + + I317V + + + 0.64577967 + experimental_method + cleaner0 + 2023-07-26T15:54:20Z + MESH: + + substitutions + + + 0.999169 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9989639 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.9964228 + structure_element + cleaner0 + 2023-07-26T15:13:57Z + SO: + + mTdp2cat + + + 0.9972203 + evidence + cleaner0 + 2023-07-26T15:44:39Z + DUMMY: + + structure + + + 0.9977994 + experimental_method + cleaner0 + 2023-07-26T15:54:49Z + MESH: + + SDS-PAGE + + + 0.99927455 + protein_state + cleaner0 + 2023-07-26T13:04:58Z + DUMMY: + + WT + + + 0.99888164 + protein_state + cleaner0 + 2023-07-26T12:41:32Z + DUMMY: + + mutant + + + experimental_method + MESH: + cleaner0 + 2023-07-26T13:45:29Z + + MBP + + + structure_element + SO: + cleaner0 + 2023-07-26T15:15:37Z + + hTdp2cat + + + 0.99929047 + protein_state + cleaner0 + 2023-07-26T13:04:58Z + DUMMY: + + WT + + + 0.99909174 + protein_state + cleaner0 + 2023-07-26T12:41:32Z + DUMMY: + + mutant + + + experimental_method + MESH: + cleaner0 + 2023-07-26T13:45:40Z + + MBP + + + structure_element + SO: + cleaner0 + 2023-07-26T15:15:37Z + + hTdp2cat + + + 0.9898276 + chemical + cleaner0 + 2023-07-26T13:46:09Z + CHEBI: + + 5′–phosphotyrosyl–DNA oligonucleotides + + + 0.9969799 + chemical + cleaner0 + 2023-07-26T13:46:11Z + CHEBI: + + fluorescein + + + 0.99651134 + experimental_method + cleaner0 + 2023-07-26T15:54:52Z + MESH: + + TBE-urea PAGE + + + 0.99931324 + protein_state + cleaner0 + 2023-07-26T13:04:58Z + DUMMY: + + WT + + + 0.9989936 + protein_state + cleaner0 + 2023-07-26T12:41:32Z + DUMMY: + + mutant + + + 0.9986303 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + experimental_method + MESH: + cleaner0 + 2023-07-26T13:45:40Z + + MBP + + + structure_element + SO: + cleaner0 + 2023-07-26T15:15:37Z + + hTdp2cat + + + 0.48587248 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + experimental_method + MESH: + cleaner0 + 2023-07-26T13:45:40Z + + MBP + + + structure_element + SO: + cleaner0 + 2023-07-26T15:15:37Z + + hTdp2cat + + + 0.9992694 + protein_state + cleaner0 + 2023-07-26T13:04:58Z + DUMMY: + + WT + + + 0.98641795 + chemical + cleaner0 + 2023-07-26T13:46:42Z + CHEBI: + + 5′-Y DNA oligonucleotide + + + 0.99929273 + chemical + cleaner0 + 2023-07-26T13:21:17Z + CHEBI: + + T5PNP + + + 0.9992957 + chemical + cleaner0 + 2023-07-26T13:35:51Z + CHEBI: + + PNPP + + + 0.99924445 + chemical + cleaner0 + 2023-07-26T12:39:08Z + CHEBI: + + PNP + + + 0.9970171 + chemical + cleaner0 + 2023-07-26T13:46:50Z + CHEBI: + + PNP phosphate + + + 0.9993144 + chemical + cleaner0 + 2023-07-26T13:35:51Z + CHEBI: + + PNPP + + + 0.98813766 + ptm + cleaner0 + 2023-07-26T14:35:06Z + MESH: + + 5′-Y + + + 0.9984953 + experimental_method + cleaner0 + 2023-07-26T15:54:58Z + MESH: + + gel based assay + + + + RESULTS + paragraph + 52589 + To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. Consequently, poor electron density is visible for the β2Hβ loop which is mostly disordered (Supplementary Figure S6). Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. Therefore, metal-regulated opening/closure of the active site may modulate Tdp2 activity, and D350N is sufficient to block both metal binding and conformational change. In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). + + 0.99912316 + mutant + cleaner0 + 2023-07-26T13:44:13Z + MESH: + + hD350N + + + 0.9990528 + mutant + cleaner0 + 2023-07-26T13:47:03Z + MESH: + + mD358N + + + 0.9052841 + experimental_method + cleaner0 + 2023-07-26T15:55:03Z + MESH: + + crystallized and determined + + + 0.99290943 + evidence + cleaner0 + 2023-07-26T15:44:44Z + DUMMY: + + structure + + + 0.99883103 + protein_state + cleaner0 + 2023-07-26T12:52:35Z + DUMMY: + + DNA-free + + + 0.9990723 + mutant + cleaner0 + 2023-07-26T13:47:04Z + MESH: + + mD358N + + + 0.99838674 + evidence + cleaner0 + 2023-07-26T15:44:48Z + DUMMY: + + structure + + + 0.9991359 + mutant + cleaner0 + 2023-07-26T13:05:27Z + MESH: + + D358N + + + experimental_method + MESH: + cleaner0 + 2023-07-26T15:45:03Z + + mutation + + + 0.9956249 + bond_interaction + cleaner0 + 2023-07-26T12:59:14Z + MESH: + + hydrogen bond + + + 0.99949586 + residue_name_number + cleaner0 + 2023-07-26T13:01:50Z + DUMMY: + + Asp358 + + + 0.99950325 + residue_name_number + cleaner0 + 2023-07-26T13:12:57Z + DUMMY: + + Trp307 + + + 0.99952376 + residue_name_number + cleaner0 + 2023-07-26T14:28:37Z + DUMMY: + + Asn358 + + + 0.99950814 + residue_name_number + cleaner0 + 2023-07-26T13:12:57Z + DUMMY: + + Trp307 + + + 0.99758136 + evidence + cleaner0 + 2023-07-26T15:45:07Z + DUMMY: + + electron density + + + 0.9990127 + structure_element + cleaner0 + 2023-07-26T13:47:17Z + SO: + + β2Hβ loop + + + 0.8247247 + protein_state + cleaner0 + 2023-07-26T15:08:25Z + DUMMY: + + disordered + + + 0.9990962 + chemical + cleaner0 + 2023-07-26T13:47:35Z + CHEBI: + + Mg2+ + + + 0.9985459 + protein_state + cleaner0 + 2023-07-26T13:04:58Z + DUMMY: + + WT + + + 0.99796176 + protein + cleaner0 + 2023-07-26T14:38:57Z + PR: + + mTdpcat + + + 0.9982697 + evidence + cleaner0 + 2023-07-26T15:45:11Z + DUMMY: + + crystals + + + 0.9980352 + site + cleaner0 + 2023-07-26T15:34:19Z + SO: + + metal site + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T15:45:27Z + + unoccupied + + + 0.99911433 + mutant + cleaner0 + 2023-07-26T13:47:04Z + MESH: + + mD358N + + + 0.99801105 + evidence + cleaner0 + 2023-07-26T15:45:15Z + DUMMY: + + crystals + + + 0.9988834 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.9993944 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9991404 + mutant + cleaner0 + 2023-07-26T14:27:23Z + MESH: + + D350N + + + 0.9990422 + mutant + cleaner0 + 2023-07-26T13:44:13Z + MESH: + + hD350N + + + 0.99904007 + mutant + cleaner0 + 2023-07-26T13:47:04Z + MESH: + + mD358N + + + 0.99887985 + chemical + cleaner0 + 2023-07-26T13:47:37Z + CHEBI: + + Mg2+ + + + 0.9873295 + evidence + cleaner0 + 2023-07-26T15:55:20Z + DUMMY: + + intrinsic tryptophan fluorescence + + + 0.98958737 + chemical + cleaner0 + 2023-07-26T13:47:40Z + CHEBI: + + Mg2+ + + + 0.9983114 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.999418 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + structure_element + SO: + cleaner0 + 2023-07-26T15:19:47Z + + loop + + + + RESULTS + title_2 + 53660 + Tdp2 facilitates NHEJ repair of 5′-phosphotyrosine adducted DSBs + + 0.9983936 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9844601 + residue_name + cleaner0 + 2023-07-26T15:02:33Z + SO: + + 5′-phosphotyrosine + + + + RESULTS + paragraph + 53727 + Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. Accordingly, we demonstrate here that 5′-tyrosylated ends are sufficient to severely impair an in vitro reconstituted mammalian NHEJ reaction (Figure 7A, lanes 3 and 6), unless supplemented with catalytic quantities of hTdp2FL (Figure 7A, lane 8). Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). We confirmed that efficient joining of the same tyrosine-adducted substrate in cells (Figure 7B) was dependent on both NHEJ (reduced over 10-fold in ligase IV deficient HCT 116 cells; Supplementary Figure S7B), and Tdp2 (reduced 5-fold in Tdp2 deficient MEFs; Figure 7C). Moreover, products with error (i.e. junctions have missing sequence flanking the adducted terminus) are twice as frequent in cells deficient in Tdp2 (Figure 7D). Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex). + + 0.9991026 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9957433 + experimental_method + cleaner0 + 2023-07-26T15:55:24Z + MESH: + + structure/activity studies + + + 0.58879304 + ptm + cleaner0 + 2023-07-26T14:32:26Z + MESH: + + 5′-detyrosylation + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.9990109 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.96590036 + protein_type + cleaner0 + 2023-07-26T12:34:13Z + MESH: + + Top2 + + + 0.99530303 + protein_state + cleaner0 + 2023-07-26T12:57:02Z + DUMMY: + + 5′-tyrosylated + + + 0.9981232 + taxonomy_domain + cleaner0 + 2023-07-26T12:30:55Z + DUMMY: + + mammalian + + + 0.39539015 + protein + cleaner0 + 2023-07-26T13:22:57Z + PR: + + hTdp2FL + + + 0.88032275 + structure_element + cleaner0 + 2023-07-26T15:15:37Z + SO: + + hTdp2cat + + + 0.6381468 + protein + cleaner0 + 2023-07-26T13:22:57Z + PR: + + hTdp2FL + + + 0.99213654 + protein_state + cleaner0 + 2023-07-26T15:08:29Z + DUMMY: + + catalytically deficient + + + 0.99908483 + mutant + cleaner0 + 2023-07-26T14:27:34Z + MESH: + + E152Q + + + 0.99922144 + protein_state + cleaner0 + 2023-07-26T12:41:32Z + DUMMY: + + mutant + + + 0.5497484 + protein_state + cleaner0 + 2023-07-26T15:08:32Z + DUMMY: + + inactive + + + 0.9991979 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.4748861 + ptm + cleaner0 + 2023-07-26T14:31:48Z + MESH: + + phosphotyrosyl + + + 0.9506391 + residue_name + cleaner0 + 2023-07-26T14:32:08Z + SO: + + tyrosine + + + 0.99922705 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + protein + PR: + cleaner0 + 2023-07-26T12:31:08Z + + Tdp2 + + + 0.9990403 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.90651315 + residue_name + cleaner0 + 2023-07-26T14:32:10Z + SO: + + tyrosine + + + 0.9990497 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.99426055 + ptm + cleaner0 + 2023-07-26T14:32:31Z + MESH: + + detyrosylation + + + 0.9984225 + protein + cleaner0 + 2023-07-26T14:41:07Z + PR: + + Artemis + + + 0.99692726 + complex_assembly + cleaner0 + 2023-07-26T13:48:47Z + GO: + + Mre11/Rad50/Nbs1 + + + + gkw228fig7.jpg + F7 + FIG + fig_caption + 55210 + Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. Concatemer ligation products were detected by 5% native PAGE. (B) Workflow diagram of cellular end joining assays. DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells. After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. Error bars, s.d, n = 3. (D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate. Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3. + + 0.9991573 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9970032 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.9988662 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.89477986 + chemical + cleaner0 + 2023-07-26T15:02:40Z + CHEBI: + + Cy5 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:51:53Z + + 5′-phosphate + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T12:57:02Z + + 5′-tyrosylated + + + 0.99820864 + protein + cleaner0 + 2023-07-26T14:37:32Z + PR: + + Ku + + + 0.9856509 + protein_type + cleaner0 + 2023-07-26T14:36:09Z + MESH: + + NHEJ ligase + + + 0.9962369 + protein + cleaner0 + 2023-07-26T14:37:37Z + PR: + + XRCC4 + + + protein + PR: + cleaner0 + 2023-07-26T14:37:29Z + + ligase IV + + + 0.9835473 + protein + cleaner0 + 2023-07-26T14:37:35Z + PR: + + XLF + + + 0.91570735 + protein + cleaner0 + 2023-07-26T13:22:57Z + PR: + + hTdp2FL + + + 0.9926214 + experimental_method + cleaner0 + 2023-07-26T15:55:35Z + MESH: + + native PAGE + + + experimental_method + MESH: + cleaner0 + 2023-07-26T15:57:05Z + + cellular end joining assays + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + residue_name + SO: + cleaner0 + 2023-07-26T15:56:17Z + + 5′-phosphotyrosine + + + 0.99737763 + taxonomy_domain + cleaner0 + 2023-07-26T12:30:55Z + DUMMY: + + mammalian + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.99741226 + experimental_method + cleaner0 + 2023-07-26T15:56:32Z + MESH: + + qPCR + + + 0.9362941 + experimental_method + cleaner0 + 2023-07-26T15:56:34Z + MESH: + + sequencing + + + 0.9971861 + experimental_method + cleaner0 + 2023-07-26T15:56:37Z + MESH: + + qPCR + + + 0.9987417 + protein_state + cleaner0 + 2023-07-26T15:08:58Z + DUMMY: + + tyrosylated + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T13:30:04Z + + wildtype + + + protein + PR: + cleaner0 + 2023-07-26T12:31:08Z + + Tdp2 + + + protein + PR: + cleaner0 + 2023-07-26T12:31:08Z + + Tdp2 + + + 0.99691737 + protein_state + cleaner0 + 2023-07-26T13:30:04Z + DUMMY: + + wildtype + + + 0.97012746 + protein + cleaner0 + 2023-07-26T13:22:57Z + PR: + + hTDP2FL + + + 0.9991529 + protein_state + cleaner0 + 2023-07-26T13:04:58Z + DUMMY: + + WT + + + experimental_method + MESH: + cleaner0 + 2023-07-26T15:57:28Z + + cellular end-joining assays + + + 0.9900619 + experimental_method + cleaner0 + 2023-07-26T15:57:33Z + MESH: + + sequencing + + + evidence + DUMMY: + cleaner0 + 2023-07-26T15:57:46Z + + end-joining error rate + + + 0.9922305 + experimental_method + cleaner0 + 2023-07-26T15:57:55Z + MESH: + + Clonogenic survival assay + + + 0.9984871 + protein_state + cleaner0 + 2023-07-26T13:04:58Z + DUMMY: + + WT + + + protein + PR: + cleaner0 + 2023-07-26T12:31:08Z + + Tdp2 + + + 0.99866784 + chemical + cleaner0 + 2023-07-26T12:35:43Z + CHEBI: + + etoposide + + + + RESULTS + paragraph + 56550 + We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C). Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N. Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). The rare D350N variant is thus inactive by all metrics analyzed. By comparison the more frequent I307V has only mild effects on in vitro activity, and no detectable impact on cellular assays. + + 0.9991307 + protein_state + cleaner0 + 2023-07-26T13:30:08Z + DUMMY: + + wild-type + + + 0.9985868 + protein_state + cleaner0 + 2023-07-26T12:41:32Z + DUMMY: + + mutant + + + 0.9400883 + protein + cleaner0 + 2023-07-26T13:22:57Z + PR: + + hTdp2FL + + + 0.52100265 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.99739563 + taxonomy_domain + cleaner0 + 2023-07-26T12:42:26Z + DUMMY: + + mouse + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.7648256 + residue_name + cleaner0 + 2023-07-26T15:02:58Z + SO: + + phosphotyrosine + + + 0.9986171 + protein_state + cleaner0 + 2023-07-26T13:30:08Z + DUMMY: + + wild-type + + + 0.99561113 + taxonomy_domain + cleaner0 + 2023-07-26T12:42:26Z + DUMMY: + + mouse + + + protein + PR: + cleaner0 + 2023-07-26T12:31:08Z + + Tdp2 + + + 0.94059974 + taxonomy_domain + cleaner0 + 2023-07-26T12:42:26Z + DUMMY: + + mouse + + + 0.99913245 + protein_state + cleaner0 + 2023-07-26T13:30:08Z + DUMMY: + + wild-type + + + 0.9979085 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + 0.99898106 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + protein + PR: + cleaner0 + 2023-07-26T12:31:08Z + + Tdp2 + + + 0.99879104 + mutant + cleaner0 + 2023-07-26T14:00:50Z + MESH: + + I307V + + + 0.9328563 + protein_state + cleaner0 + 2023-07-26T13:49:18Z + DUMMY: + + variant + + + 0.9982362 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + 0.99898344 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + protein + PR: + cleaner0 + 2023-07-26T12:31:08Z + + Tdp2 + + + protein + PR: + cleaner0 + 2023-07-26T12:31:08Z + + Tdp2 + + + 0.8572738 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9989687 + mutant + cleaner0 + 2023-07-26T14:27:23Z + MESH: + + D350N + + + protein + PR: + cleaner0 + 2023-07-26T12:31:08Z + + Tdp2 + + + protein + PR: + cleaner0 + 2023-07-26T12:31:08Z + + Tdp2 + + + 0.99891436 + mutant + cleaner0 + 2023-07-26T14:27:23Z + MESH: + + D350N + + + 0.99910986 + protein_state + cleaner0 + 2023-07-26T13:49:29Z + DUMMY: + + wild type + + + 0.99873346 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.41842 + protein + cleaner0 + 2023-07-26T14:38:40Z + PR: + + hTdp2 + + + 0.9986695 + mutant + cleaner0 + 2023-07-26T14:00:51Z + MESH: + + I307V + + + 0.99910665 + protein_state + cleaner0 + 2023-07-26T13:49:28Z + DUMMY: + + wild type + + + 0.99881375 + mutant + cleaner0 + 2023-07-26T14:00:51Z + MESH: + + I307V + + + 0.9984975 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + 0.99901104 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + protein + PR: + cleaner0 + 2023-07-26T12:31:08Z + + Tdp2 + + + 0.99726605 + chemical + cleaner0 + 2023-07-26T12:35:43Z + CHEBI: + + etoposide + + + 0.9990652 + protein_state + cleaner0 + 2023-07-26T13:30:08Z + DUMMY: + + wild-type + + + 0.5926537 + experimental_method + cleaner0 + 2023-07-26T15:58:00Z + MESH: + + overexpression + + + 0.9975042 + species + cleaner0 + 2023-07-26T13:16:30Z + MESH: + + human + + + 0.99878615 + mutant + cleaner0 + 2023-07-26T14:27:23Z + MESH: + + D350N + + + 0.99857616 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.99891496 + mutant + cleaner0 + 2023-07-26T14:27:23Z + MESH: + + D350N + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T13:49:19Z + + variant + + + 0.99815565 + protein_state + cleaner0 + 2023-07-26T15:09:15Z + DUMMY: + + inactive + + + 0.9983854 + mutant + cleaner0 + 2023-07-26T14:00:51Z + MESH: + + I307V + + + + DISCUSS + title_1 + 57868 + DISCUSSION + + + DISCUSS + paragraph + 57879 + Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable. Understanding how cells cope with complex DNA breaks bearing topoisomerase–DNA protein crosslinks is key to deciphering individual responses to chemotherapeutic outcomes and genotoxic agents that poison Top2. Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing. + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:13Z + + Top2 + + + 0.5547223 + chemical + cleaner0 + 2023-07-26T12:31:43Z + CHEBI: + + DNA + + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:13Z + + Top2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.982362 + protein_type + cleaner0 + 2023-07-26T12:34:13Z + MESH: + + Top2 + + + 0.99664575 + experimental_method + cleaner0 + 2023-07-26T15:58:03Z + MESH: + + mutagenesis + + + 0.9245626 + experimental_method + cleaner0 + 2023-07-26T15:58:06Z + MESH: + + functional assays + + + 0.9989999 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9976665 + evidence + cleaner0 + 2023-07-26T15:46:06Z + DUMMY: + + structures + + + protein_state + DUMMY: + cleaner0 + 2023-07-26T13:00:59Z + + in the absence of + + + chemical + CHEBI: + cleaner0 + 2023-07-26T14:01:23Z + + ligands + + + 0.998565 + protein_state + cleaner0 + 2023-07-26T15:09:19Z + DUMMY: + + in complex with + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.99887747 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.9989502 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.99840856 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.7127077 + chemical + cleaner0 + 2023-07-26T12:31:43Z + CHEBI: + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.7769798 + protein_type + cleaner0 + 2023-07-26T12:34:13Z + MESH: + + Top2 + + + 0.9978385 + experimental_method + cleaner0 + 2023-07-26T15:58:10Z + MESH: + + structural analysis + + + 0.99858177 + experimental_method + cleaner0 + 2023-07-26T15:58:16Z + MESH: + + mutational studies + + + 0.9989797 + experimental_method + cleaner0 + 2023-07-26T14:01:32Z + MESH: + + QM/MM molecular modeling + + + 0.9990037 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + structure_element + SO: + cleaner0 + 2023-07-26T12:42:17Z + + EEP + + + 0.996482 + protein_type + cleaner0 + 2023-07-26T14:38:07Z + MESH: + + phosphoryl hydrolase + + + 0.9989484 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9985404 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.6557976 + protein_state + cleaner0 + 2023-07-26T15:09:29Z + DUMMY: + + conformationally plastic + + + 0.9476443 + chemical + cleaner0 + 2023-07-26T12:31:43Z + CHEBI: + + DNA + + + 0.9987576 + chemical + cleaner0 + 2023-07-26T15:03:04Z + CHEBI: + + Mg2+ + + + 0.9637267 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.99900526 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.7581892 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.9989201 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.818567 + chemical + cleaner0 + 2023-07-26T12:31:43Z + CHEBI: + + DNA + + + 0.9981352 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + + DISCUSS + paragraph + 59305 + Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways. First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal. The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group. To our knowledge, this is the first proposed example of a substrate cation–π interface exploited to promote a phosphoryl-transfer reaction. This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. + + 0.9983854 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.77449465 + protein_type + cleaner0 + 2023-07-26T14:41:30Z + MESH: + + Top1 + + + 0.70503974 + protein_type + cleaner0 + 2023-07-26T12:34:13Z + MESH: + + Top2 + + + 0.9987135 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.9564418 + protein_type + cleaner0 + 2023-07-26T12:34:13Z + MESH: + + Top2 + + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:13Z + + Top2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.7941354 + protein_type + cleaner0 + 2023-07-26T12:34:13Z + MESH: + + Top2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.9990264 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.80130893 + protein_type + cleaner0 + 2023-07-26T14:38:10Z + MESH: + + topoisomerase + + + 0.9990449 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.998955 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9987481 + site + cleaner0 + 2023-07-26T15:34:32Z + SO: + + substrate interaction groove + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.9982162 + site + cleaner0 + 2023-07-26T15:34:37Z + SO: + + nucleic acid binding trench + + + 0.98721373 + protein_state + cleaner0 + 2023-07-26T15:09:34Z + DUMMY: + + dynamic + + + structure_element + SO: + cleaner0 + 2023-07-26T14:02:16Z + + β2Hβ DNA damage-binding loop + + + ptm + MESH: + cleaner0 + 2023-07-26T12:52:02Z + + phosphotyrosyl linkages + + + 0.9980977 + chemical + cleaner0 + 2023-07-26T12:38:42Z + CHEBI: + + ϵA + + + bond_interaction + MESH: + cleaner0 + 2023-07-26T15:36:18Z + + base-stacking + + + site + SO: + cleaner0 + 2023-07-26T15:36:36Z + + hydrophobic wall + + + 0.99921054 + structure_element + cleaner0 + 2023-07-26T14:04:20Z + SO: + + β2Hβ-loop + + + 0.9951184 + experimental_method + cleaner0 + 2023-07-26T12:43:25Z + MESH: + + QM/MM + + + 0.85165536 + bond_interaction + cleaner0 + 2023-07-26T13:27:57Z + MESH: + + cation–π interaction + + + 0.9990927 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.9989152 + protein_state + cleaner0 + 2023-07-26T14:04:50Z + DUMMY: + + strictly conserved + + + 0.998353 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.9994487 + residue_name_number + cleaner0 + 2023-07-26T14:04:46Z + DUMMY: + + Arg216 + + + 0.97798014 + ptm + cleaner0 + 2023-07-26T14:31:48Z + MESH: + + phosphotyrosyl + + + 0.9949945 + site + cleaner0 + 2023-07-26T15:36:42Z + SO: + + substrate cation–π interface + + + 0.99916697 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + complex_assembly + GO: + cleaner0 + 2023-07-26T12:35:27Z + + Top2cc + + + 0.9965321 + taxonomy_domain + cleaner0 + 2023-07-26T14:42:42Z + DUMMY: + + picornaviral + + + chemical + CHEBI: + cleaner0 + 2023-07-26T15:11:59Z + + RNA + + + 0.9415552 + taxonomy_domain + cleaner0 + 2023-07-26T15:12:16Z + DUMMY: + + Hepatitis B Virus + + + 0.9940905 + taxonomy_domain + cleaner0 + 2023-07-26T15:12:25Z + DUMMY: + + HBV + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + structure_element + SO: + cleaner0 + 2023-07-26T12:42:17Z + + EEP + + + protein_type + MESH: + cleaner0 + 2023-07-26T14:04:40Z + + nucleases + + + 0.9991364 + protein + cleaner0 + 2023-07-26T13:32:51Z + PR: + + Ape1 + + + 0.99911577 + protein + cleaner0 + 2023-07-26T14:41:35Z + PR: + + Ape2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.99906904 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + + DISCUSS + paragraph + 61294 + The dynamic nature of the Tdp2 active site presents opportunities for enzyme regulation. However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 β2Hβ-loop is unknown. We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. + + 0.9991721 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.99902624 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.9991653 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9992085 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9993501 + structure_element + cleaner0 + 2023-07-26T14:05:08Z + SO: + + β2Hβ-loop + + + 0.9952834 + protein_type + cleaner0 + 2023-07-26T12:34:13Z + MESH: + + Top2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.9991738 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.9990084 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.99902844 + protein + cleaner0 + 2023-07-26T12:31:08Z + PR: + + Tdp2 + + + 0.99012405 + protein_type + cleaner0 + 2023-07-26T12:34:13Z + MESH: + + Top2 + + + 0.9971257 + protein_type + cleaner0 + 2023-07-26T12:34:13Z + MESH: + + Top2 + + + 0.9985684 + evidence + cleaner0 + 2023-07-26T15:46:14Z + DUMMY: + + structures + + + 0.97867566 + taxonomy_domain + cleaner0 + 2023-07-26T12:42:26Z + DUMMY: + + mouse + + + 0.99823874 + species + cleaner0 + 2023-07-26T13:18:20Z + MESH: + + C. elegans + + + 0.99915874 + protein + cleaner0 + 2023-07-26T12:31:09Z + PR: + + Tdp2 + + + 0.99923694 + protein + cleaner0 + 2023-07-26T12:31:09Z + PR: + + Tdp2 + + + 0.99902004 + site + cleaner0 + 2023-07-26T12:57:42Z + SO: + + active site + + + 0.99806255 + taxonomy_domain + cleaner0 + 2023-07-26T14:05:14Z + DUMMY: + + worms + + + 0.6359281 + taxonomy_domain + cleaner0 + 2023-07-26T14:05:21Z + DUMMY: + + man + + + 0.9975939 + experimental_method + cleaner0 + 2023-07-26T14:06:00Z + MESH: + + intrinsic tryptophan fluorescence + + + 0.99865973 + experimental_method + cleaner0 + 2023-07-26T14:06:02Z + MESH: + + crystallographic analysis + + + 0.99818724 + experimental_method + cleaner0 + 2023-07-26T14:06:06Z + MESH: + + mutagenesis + + + experimental_method + MESH: + cleaner0 + 2023-07-26T14:05:57Z + + Ca2+ inhibition studies + + + 0.99872404 + experimental_method + cleaner0 + 2023-07-26T14:05:32Z + MESH: + + QM/MM analysis + + + 0.99904025 + chemical + cleaner0 + 2023-07-26T14:06:10Z + CHEBI: + + Mg2+ + + + 0.9991862 + protein + cleaner0 + 2023-07-26T12:31:09Z + PR: + + Tdp2 + + + + DISCUSS + paragraph + 62328 + The advent of personalized medical screening opens doors for assessment of individual vulnerabilities to commonly used chemotherapeutic drugs. It would be beneficial to employ this knowledge during the early decision making processes regarding treatment. Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection. Lastly, Tdp2 inhibitors may synergize or potentiate cytotoxic effects of current anticancer treatments that target Tdp2. Thus, we anticipate this atomic-level and mechanistic definition of the molecular determinants of Tdp2 catalysis and conformational changes driven by DNA–protein and protein–protein interactions will foster unique strategies for the development of Tdp2 targeted small molecule interventions. + + 0.9990723 + chemical + cleaner0 + 2023-07-26T12:35:43Z + CHEBI: + + Etoposide + + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:13Z + + Top2 + + + 0.8624938 + protein + cleaner0 + 2023-07-26T12:31:09Z + PR: + + Tdp2 + + + 0.99850404 + species + cleaner0 + 2023-07-26T13:16:31Z + MESH: + + human + + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:13Z + + Top2 + + + 0.99926907 + chemical + cleaner0 + 2023-07-26T12:35:43Z + CHEBI: + + etoposide + + + 0.9992687 + chemical + cleaner0 + 2023-07-26T15:03:26Z + CHEBI: + + doxyrubicin + + + 0.9938152 + protein + cleaner0 + 2023-07-26T12:31:09Z + PR: + + Tdp2 + + + 0.9984585 + species + cleaner0 + 2023-07-26T13:16:31Z + MESH: + + human + + + 0.93653077 + protein + cleaner0 + 2023-07-26T12:31:09Z + PR: + + TDP2 + + + protein_type + MESH: + cleaner0 + 2023-07-26T12:34:13Z + + Top2 + + + 0.79797673 + taxonomy_domain + cleaner0 + 2023-07-26T14:06:20Z + DUMMY: + + viral + + + 0.54016036 + protein + cleaner0 + 2023-07-26T12:31:09Z + PR: + + Tdp2 + + + 0.99850464 + protein + cleaner0 + 2023-07-26T12:31:09Z + PR: + + Tdp2 + + + 0.9987066 + protein + cleaner0 + 2023-07-26T12:31:09Z + PR: + + Tdp2 + + + chemical + CHEBI: + cleaner0 + 2023-07-26T12:31:43Z + + DNA + + + 0.99685895 + protein + cleaner0 + 2023-07-26T12:31:09Z + PR: + + Tdp2 + + + + KEYWORD + title_1 + 63495 + ACCESSION NUMBERS + + + KEYWORD + paragraph + 63513 + Coordinates and structure factors have been deposited in the RCSB Protein Data Bank under accession code 5HT2 (mTdp2-Mg-ϵA-DNA complex), 5INK (mTdp2-Mg-THF complex), 5INL (mTdp2-Mg-dA-DNA-product complex), 5INM (mTdp2-apo structure), 5INN (mTdp2-D350N structure), 5INO (hTdp2-Mg-DNA product complex), 5INP (mTdp2-Mn-DNA product complex) and 5INQ (mTdp2-Ca-DNA product complex). + + + SUPPL + title_1 + 63897 + Supplementary Material + + + SUPPL + title_1 + 63920 + SUPPLEMENTARY DATA + + + SUPPL + paragraph + 63939 + Supplementary Data are available at NAR Online. + + + ACK_FUND + title_1 + 63987 + FUNDING + + + ACK_FUND + paragraph + 63995 + US National Institute of Health Intramural Program; US National Institute of Environmental Health Sciences (NIEHS) [1Z01ES102765 to R.S.W.]; National Cancer Institute (NCI) [R01 CA084442 to D.A.R]. Use of the APS was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. Funding for open access publication: NIEHS [1Z01ES102765 to R.S.W]. + + + ACK_FUND + paragraph + 64413 + Conflict of interest statement. None declared. + + + REF + title + 64460 + REFERENCES + + + 338 + 350 + surname:Nitiss;given-names:J.L. + 19377506 + REF + Nat. Rev. Cancer + ref + 9 + 2009 + 64471 + Targeting DNA topoisomerase II in cancer chemotherapy + + + 327 + 337 + surname:Nitiss;given-names:J.L. + 19377505 + REF + Nat. Rev. Cancer + ref + 9 + 2009 + 64525 + DNA topoisomerase II and its growing repertoire of biological functions + + + 114 + 129 + surname:Pommier;given-names:Y. + surname:Huang;given-names:S.Y. + surname:Gao;given-names:R. + surname:Das;given-names:B.B. + surname:Murai;given-names:J. + surname:Marchand;given-names:C. + 24856239 + REF + DNA Repair (Amst) + ref + 19 + 2014 + 64597 + Tyrosyl-DNA-phosphodiesterases (TDP1 and TDP2) + + + 82 + 95 + surname:Pommier;given-names:Y. + 23259582 + REF + ACS Chem. Biol. + ref + 8 + 2013 + 64644 + Drugging topoisomerases: lessons and challenges + + + 1 + 21 + surname:Andres;given-names:S.N. + surname:Schellenberg;given-names:M.J. + surname:Wallace;given-names:B.D. + surname:Tumbale;given-names:P. + surname:Williams;given-names:R.S. + 25111769 + REF + Environ. Mol. Mutagen. + ref + 56 + 2015 + 64692 + Recognition and repair of chemically heterogeneous structures at DNA ends + + + 1340 + 1346 + surname:Wallace;given-names:B.D. + surname:Williams;given-names:R.S. + 25692233 + REF + RNA Biol. + ref + 11 + 2014 + 64766 + Ribonucleotide triggered DNA damage and RNA-DNA damage responses + + + 137 + 151 + surname:Ashour;given-names:M.E. + surname:Atteya;given-names:R. + surname:El-Khamisy;given-names:S.F. + 25693836 + REF + Nat. Rev. Cancer + ref + 15 + 2015 + 64831 + Topoisomerase-mediated chromosomal break repair: an emerging player in many games + + + 17960 + 17969 + surname:Gao;given-names:R. + surname:Schellenberg;given-names:M.J. + surname:Huang;given-names:S.Y. + surname:Abdelmalak;given-names:M. + surname:Marchand;given-names:C. + surname:Nitiss;given-names:K.C. + surname:Nitiss;given-names:J.L. + surname:Williams;given-names:R.S. + surname:Pommier;given-names:Y. + 24808172 + REF + J. Biol. Chem. + ref + 289 + 2014 + 64913 + Proteolytic degradation of topoisomerase II (Top2) enables the processing of Top2.DNA and Top2.RNA covalent complexes by Tyrosyl-DNA-Phosphodiesterase 2 (TDP2) + + + 22839 + 22846 + surname:Wang;given-names:Y. + surname:Knudsen;given-names:B.R. + surname:Bjergbaek;given-names:L. + surname:Westergaard;given-names:O. + surname:Andersen;given-names:A.H. + 10428869 + REF + J. Biol. Chem. + ref + 274 + 1999 + 65073 + Stimulated activity of human topoisomerases IIalpha and IIbeta on RNA-containing substrates + + + 46290 + 46296 + surname:Wilstermann;given-names:A.M. + surname:Osheroff;given-names:N. + 11591703 + REF + J. Biol. Chem. + ref + 276 + 2001 + 65165 + Base excision repair intermediates as topoisomerase II poisons + + + 21441 + 21444 + surname:Kingma;given-names:P.S. + surname:Corbett;given-names:A.H. + surname:Burcham;given-names:P.C. + surname:Marnett;given-names:L.J. + surname:Osheroff;given-names:N. + 7665552 + REF + J. Biol. Chem. + ref + 270 + 1995 + 65228 + Abasic sites stimulate double-stranded DNA cleavage mediated by topoisomerase II. DNA lesions as endogenous topoisomerase II poisons + + + 7488 + 7493 + surname:Kingma;given-names:P.S. + surname:Osheroff;given-names:N. + 9054451 + REF + J. Biol. Chem. + ref + 272 + 1997 + 65361 + Spontaneous DNA damage stimulates topoisomerase II-mediated DNA cleavage + + + 1148 + 1155 + surname:Kingma;given-names:P.S. + surname:Osheroff;given-names:N. + 8995415 + REF + J. Biol. Chem. + ref + 272 + 1997 + 65434 + Apurinic sites are position-specific topoisomerase II poisons + + + 223 + 232 + surname:Kingma;given-names:P.S. + surname:Osheroff;given-names:N. + 9748592 + REF + Biochim. Biophys. Acta + ref + 1400 + 1998 + 65496 + The response of eukaryotic topoisomerases to DNA damage + + + 307 + 323 + surname:Bolt;given-names:H.M. + 15989139 + REF + Crit. Rev. Toxicol. + ref + 35 + 2005 + 65552 + Vinyl chloride-a classical industrial toxicant of new interest + + + 1947 + 1954 + surname:Sabourin;given-names:M. + surname:Osheroff;given-names:N. + 10756196 + REF + Nucleic Acids Res. + ref + 28 + 2000 + 65615 + Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme-mediated DNA cleavage by abasic, oxidized and alkylated lesions + + + 674 + 678 + surname:Cortes Ledesma;given-names:F. + surname:El Khamisy;given-names:S.F. + surname:Zuma;given-names:M.C. + surname:Osborn;given-names:K. + surname:Caldecott;given-names:K.W. + 19794497 + REF + Nature + ref + 461 + 2009 + 65760 + A human 5'-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damage + + + 516 + 521 + surname:Gomez-Herreros;given-names:F. + surname:Schuurs-Hoeijmakers;given-names:J.H. + surname:McCormack;given-names:M. + surname:Greally;given-names:M.T. + surname:Rulten;given-names:S. + surname:Romero-Granados;given-names:R. + surname:Counihan;given-names:T.J. + surname:Chaila;given-names:E. + surname:Conroy;given-names:J. + surname:Ennis;given-names:S. + 24658003 + REF + Nat. Genet. + ref + 46 + 2014 + 65848 + TDP2 protects transcription from abortive topoisomerase activity and is required for normal neural function + + + e1003226 + surname:Gomez-Herreros;given-names:F. + surname:Romero-Granados;given-names:R. + surname:Zeng;given-names:Z. + surname:Alvarez-Quilon;given-names:A. + surname:Quintero;given-names:C. + surname:Ju;given-names:L. + surname:Umans;given-names:L. + surname:Vermeire;given-names:L. + surname:Huylebroeck;given-names:D. + surname:Caldecott;given-names:K.W. + 23505375 + REF + PLoS Genet. + ref + 9 + 2013 + 65956 + TDP2-dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks and genome instability in cells and in vivo + + + 1363 + 1371 + surname:Schellenberg;given-names:M.J. + surname:Appel;given-names:C.D. + surname:Adhikari;given-names:S. + surname:Robertson;given-names:P.D. + surname:Ramsden;given-names:D.A. + surname:Williams;given-names:R.S. + 23104055 + REF + Nat. Struct. Mol. Biol. + ref + 19 + 2012 + 66095 + Mechanism of repair of 5'-topoisomerase II-DNA adducts by mammalian tyrosyl-DNA phosphodiesterase 2 + + + 830 + 845 + surname:Do;given-names:P.M. + surname:Varanasi;given-names:L. + surname:Fan;given-names:S. + surname:Li;given-names:C. + surname:Kubacka;given-names:I. + surname:Newman;given-names:V. + surname:Chauhan;given-names:K. + surname:Daniels;given-names:S.R. + surname:Boccetta;given-names:M. + surname:Garrett;given-names:M.R. + 22508727 + REF + Genes Dev. + ref + 26 + 66195 + Mutant p53 cooperates with ETS2 to promote etoposide resistance + + + 307 + 326 + surname:Otwinowski;given-names:Z. + surname:Minor;given-names:W. + surname:Carter;given-names:C.W.;suffix:Jr + surname:Sweets;given-names:R.M. + REF + Methods in Enzymology + ref + 276 + 1997 + 66259 + + + 658 + 674 + surname:McCoy;given-names:A.J. + surname:Grosse-Kunstleve;given-names:R.W. + surname:Adams;given-names:P.D. + surname:Winn;given-names:M.D. + surname:Storoni;given-names:L.C. + surname:Read;given-names:R.J. + 19461840 + REF + J. Appl. Crystallogr. + ref + 40 + 2007 + 66260 + Phaser crystallographic software + + + 486 + 501 + surname:Emsley;given-names:P. + surname:Lohkamp;given-names:B. + surname:Scott;given-names:W.G. + surname:Cowtan;given-names:K. + 20383002 + REF + Acta Crystallogr. D Biol. Crystallogr. + ref + 66 + 2010 + 66293 + Features and development of Coot + + + 94 + 106 + surname:Adams;given-names:P.D. + surname:Afonine;given-names:P.V. + surname:Bunkoczi;given-names:G. + surname:Chen;given-names:V.B. + surname:Echols;given-names:N. + surname:Headd;given-names:J.J. + surname:Hung;given-names:L.W. + surname:Jain;given-names:S. + surname:Kapral;given-names:G.J. + surname:Grosse Kunstleve;given-names:R.W. + 21821126 + REF + Methods + ref + 55 + 2011 + 66326 + The Phenix software for automated determination of macromolecular structures + + + 3085 + 3094 + surname:Davis;given-names:B.J. + surname:Havener;given-names:J.M. + surname:Ramsden;given-names:D.A. + 18397950 + REF + Nucleic Acids Res. + ref + 36 + 2008 + 66403 + End-bridging is required for pol mu to efficiently promote repair of noncomplementary ends by nonhomologous end joining + + + 4286 + surname:Waters;given-names:C.A. + surname:Strande;given-names:N.T. + surname:Pryor;given-names:J.M. + surname:Strom;given-names:C.N. + surname:Mieczkowski;given-names:P. + surname:Burkhalter;given-names:M.D. + surname:Oh;given-names:S. + surname:Qaqish;given-names:B.F. + surname:Moore;given-names:D.T. + surname:Hendrickson;given-names:E.A. + 24989324 + REF + Nat. Commun. + ref + 5 + 2014 + 66523 + The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining + + + e1000855 + surname:Fattah;given-names:F. + surname:Lee;given-names:E.H. + surname:Weisensel;given-names:N. + surname:Wang;given-names:Y. + surname:Lichter;given-names:N. + surname:Hendrickson;given-names:E.A. + 20195511 + REF + PLoS Genet. + ref + 6 + 2010 + 66623 + Ku regulates the non-homologous end joining pathway choice of DNA double-strand break repair in human somatic cells + + + 659 + 661 + surname:Yang;given-names:X. + surname:Boehm;given-names:J.S. + surname:Yang;given-names:X. + surname:Salehi-Ashtiani;given-names:K. + surname:Hao;given-names:T. + surname:Shen;given-names:Y. + surname:Lubonja;given-names:R. + surname:Thomas;given-names:S.R. + surname:Alkan;given-names:O. + surname:Bhimdi;given-names:T. + 21706014 + REF + Nat. Methods + ref + 8 + 2011 + 66739 + A public genome-scale lentiviral expression library of human ORFs + + + surname:Frisch;given-names:M.J. + surname:Trucks;given-names:G.W. + surname:Schlegel;given-names:H.B. + surname:Scuseria;given-names:G.E. + surname:Robb;given-names:M.A. + surname:Cheeseman;given-names:J.R. + surname:Scalmani;given-names:G. + surname:Barone;given-names:V. + surname:Mennucci;given-names:B. + surname:Petersson;given-names:G.A. + REF + ref + 2009 + 66805 + + + 8078 + 8088 + surname:Batra;given-names:V.K. + surname:Perera;given-names:L. + surname:Lin;given-names:P. + surname:Shock;given-names:D.D. + surname:Beard;given-names:W.A. + surname:Pedersen;given-names:L.C. + surname:Pedersen;given-names:L.G. + surname:Wilson;given-names:S.H. + 23647366 + REF + J. Am. Chem. Soc. + ref + 135 + 2013 + 66806 + Amino acid substitution in the active site of DNA polymerase beta explains the energy barrier of the nucleotidyl transfer reaction + + + 31201 + 31206 + surname:Bromberg;given-names:K.D. + surname:Hendricks;given-names:C. + surname:Burgin;given-names:A.B. + surname:Osheroff;given-names:N. + 12050172 + REF + J. Biol. Chem. + ref + 277 + 2002 + 66937 + Human topoisomerase IIalpha possesses an intrinsic nucleic acid specificity for DNA ligation. Use of 5' covalently activated oligonucleotide substrates to study enzyme mechanism + + + 3323 + 3331 + surname:Woodfield;given-names:G. + surname:Cheng;given-names:C. + surname:Shuman;given-names:S. + surname:Burgin;given-names:A.B. + 10954601 + REF + Nucleic Acids Res. + ref + 28 + 2000 + 67115 + Vaccinia topoisomerase and Cre recombinase catalyze direct ligation of activated DNA substrates containing a 3'-para-nitrophenyl phosphate ester + + + 3972 + 3981 + surname:Velez-Cruz;given-names:R. + surname:Riggins;given-names:J.N. + surname:Daniels;given-names:J.S. + surname:Cai;given-names:H. + surname:Guengerich;given-names:F.P. + surname:Marnett;given-names:L.J. + surname:Osheroff;given-names:N. + 15751973 + REF + Biochemistry + ref + 44 + 2005 + 67260 + Exocyclic DNA lesions stimulate DNA cleavage mediated by human topoisomerase II alpha in vitro and in cultured cells + + + 738 + 748 + surname:Deweese;given-names:J.E. + surname:Osheroff;given-names:N. + 19042970 + REF + Nucleic Acids Res. + ref + 37 + 2009 + 67377 + The DNA cleavage reaction of topoisomerase II: wolf in sheep's clothing + + + 1372 + 1377 + surname:Shi;given-names:K. + surname:Kurahashi;given-names:K. + surname:Gao;given-names:R. + surname:Tsutakawa;given-names:S.E. + surname:Tainer;given-names:J.A. + surname:Pommier;given-names:Y. + surname:Aihara;given-names:H. + 23104058 + REF + Nat. Struct. Mol. Biol. + ref + 19 + 2012 + 67449 + Structural basis for recognition of 5'-phosphotyrosine adducts by Tdp2 + + + 30842 + 30852 + surname:Gao;given-names:R. + surname:Huang;given-names:S.Y. + surname:Marchand;given-names:C. + surname:Pommier;given-names:Y. + 22822062 + REF + J. Biol. Chem. + ref + 287 + 2012 + 67520 + Biochemical characterization of human tyrosyl-DNA phosphodiesterase 2 (TDP2/TTRAP): a Mg(2+)/Mn(2+)-dependent phosphodiesterase specific for the repair of topoisomerase cleavage complexes + + + 250 + 255 + surname:Dupureur;given-names:C.M. + 18261473 + REF + Curr. Opin. Chem. Biol. + ref + 12 + 2008 + 67708 + Roles of metal ions in nucleases + + + 112 + 116 + surname:Adhikari;given-names:S. + surname:Karmahapatra;given-names:S.K. + surname:Elias;given-names:H. + surname:Dhopeshwarkar;given-names:P. + surname:Williams;given-names:R.S. + surname:Byers;given-names:S. + surname:Uren;given-names:A. + surname:Roy;given-names:R. + 21620793 + REF + Anal. Biochem. + ref + 416 + 2011 + 67741 + Development of a novel assay for human tyrosyl DNA phosphodiesterase 2 + + + 311 + 322 + surname:Lowry;given-names:D.F. + surname:Hoyt;given-names:D.W. + surname:Khazi;given-names:F.A. + surname:Bagu;given-names:J. + surname:Lindsey;given-names:A.G. + surname:Wilson;given-names:D.M. 3rd + 12758078 + REF + J. Mol. Biol. + ref + 329 + 2003 + 67812 + Investigation of the role of the histidine-aspartate pair in the human exonuclease III-like abasic endonuclease, Ape1 + + + 308 + 311 + surname:Sherry;given-names:S.T. + surname:Ward;given-names:M.H. + surname:Kholodov;given-names:M. + surname:Baker;given-names:J. + surname:Phan;given-names:L. + surname:Smigielski;given-names:E.M. + surname:Sirotkin;given-names:K. + 11125122 + REF + Nucleic Acids Res. + ref + 29 + 2001 + 67930 + dbSNP: the NCBI database of genetic variation + + + 14634 + 14639 + surname:Virgen-Slane;given-names:R. + surname:Rozovics;given-names:J.M. + surname:Fitzgerald;given-names:K.D. + surname:Ngo;given-names:T. + surname:Chou;given-names:W. + surname:van der Heden van Noort;given-names:G.J. + surname:Filippov;given-names:D.V. + surname:Gershon;given-names:P.D. + surname:Semler;given-names:B.L. + 22908287 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 109 + 2012 + 67976 + An RNA virus hijacks an incognito function of a DNA repair enzyme + + + E4244 + E4253 + surname:Koniger;given-names:C. + surname:Wingert;given-names:I. + surname:Marsmann;given-names:M. + surname:Rosler;given-names:C. + surname:Beck;given-names:J. + surname:Nassal;given-names:M. + 25201958 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 111 + 2014 + 68042 + Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses + + + 924 + 931 + surname:Freudenthal;given-names:B.D. + surname:Beard;given-names:W.A. + surname:Cuneo;given-names:M.J. + surname:Dyrkheeva;given-names:N.S. + surname:Wilson;given-names:S.H. + 26458045 + REF + Nat. Struct. Mol. Biol. + ref + 22 + 2015 + 68185 + Capturing snapshots of APE1 processing DNA damage + + + 853 + 866 + surname:Hadi;given-names:M.Z. + surname:Ginalski;given-names:K. + surname:Nguyen;given-names:L.H. + surname:Wilson;given-names:D.M. 3rd + 11866537 + REF + J. Mol. Biol. + ref + 316 + 2002 + 68235 + Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III + + + diff --git a/BioC_XML/4869123_v0.xml b/BioC_XML/4869123_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..228f171ec757a84b35f1f9a3f8425435aaa1f86f --- /dev/null +++ b/BioC_XML/4869123_v0.xml @@ -0,0 +1,9401 @@ + + + + PMC + 20201220 + pmc.key + + 4869123 + CC BY + no + 0 + 0 + + 10.1038/srep26071 + srep26071 + 4869123 + 27184415 + 26071 + This work is licensed under a Creative Commons Attribution 4.0 International License. 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To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ + surname:Liu;given-names:Shenping + surname:Desharnais;given-names:Joel + surname:Feng;given-names:Xidong + surname:Griffor;given-names:Matt + surname:Jimenez;given-names:Judith + surname:Chen;given-names:Gang + surname:Tumelty;given-names:David + surname:Bhat;given-names:Abhijit + surname:Bradshaw;given-names:Curt W. + surname:Woodnutt;given-names:Gary + surname:Lappe;given-names:Rodney W. + surname:Thorarensen;given-names:Atli + surname:Sahasrabudhe;given-names:Parag V. + surname:Qiu;given-names:Xiayang + surname:Withka;given-names:Jane M. + surname:Wood;given-names:Lauren D. + surname:Jin;given-names:Ping + surname:Li;given-names:Wei + surname:Oates;given-names:Bryan D. + surname:Shanker;given-names:Suman + surname:Banker;given-names:Mary Ellen + surname:Chrunyk;given-names:Boris A. + surname:Song;given-names:Xi + TITLE + front + 6 + 2016 + 0 + Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide + + 0.95569223 + protein + cleaner0 + 2023-07-20T08:16:49Z + PR: + + IL-17A + + + 0.9845403 + protein + cleaner0 + 2023-07-20T08:16:59Z + PR: + + IL-17RA + + + 0.99911207 + chemical + cleaner0 + 2023-07-20T09:57:34Z + CHEBI: + + peptide + + + + ABSTRACT + abstract + 73 + IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target. + + 0.9583933 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:50:51Z + + cytokine + + + 0.8298379 + protein_type + cleaner0 + 2023-07-20T09:25:45Z + MESH: + + antibodies + + + protein + PR: + cleaner0 + 2023-07-20T08:16:50Z + + IL-17A + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:18:25Z + + high affinity IL-17A peptide antagonist + + + 0.4951907 + chemical + cleaner0 + 2023-07-20T08:18:35Z + CHEBI: + + HAP + + + 0.9906646 + residue_range + cleaner0 + 2023-07-20T09:26:24Z + DUMMY: + + 15 residues + + + 0.99955106 + experimental_method + cleaner0 + 2023-07-20T09:18:38Z + MESH: + + phage-display screening + + + 0.99955803 + experimental_method + cleaner0 + 2023-07-20T09:29:41Z + MESH: + + saturation mutagenesis optimization + + + 0.9994239 + experimental_method + cleaner0 + 2023-07-20T09:29:45Z + MESH: + + amino acid substitutions + + + 0.98556024 + chemical + cleaner0 + 2023-07-20T08:18:35Z + CHEBI: + + HAP + + + 0.99852103 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:50:51Z + + cytokine + + + protein_type + MESH: + cleaner0 + 2023-07-20T09:25:36Z + + receptor + + + 0.996836 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.9989361 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.97730803 + chemical + cleaner0 + 2023-07-20T08:18:35Z + CHEBI: + + HAP + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:45:34Z + + cytokines + + + experimental_method + MESH: + cleaner0 + 2023-07-20T09:29:32Z + + Crystal structure studies + + + 0.9854585 + chemical + cleaner0 + 2023-07-20T08:18:35Z + CHEBI: + + HAP + + + 0.9989205 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + 0.99925786 + oligomeric_state + cleaner0 + 2023-07-20T09:18:26Z + DUMMY: + + dimer + + + 0.97047263 + chemical + cleaner0 + 2023-07-20T08:18:35Z + CHEBI: + + HAP + + + 0.99970937 + structure_element + cleaner0 + 2023-07-20T09:45:39Z + SO: + + β-strand + + + 0.99877185 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + 0.99928147 + oligomeric_state + cleaner0 + 2023-07-20T08:23:13Z + DUMMY: + + monomers + + + 0.99972767 + structure_element + cleaner0 + 2023-07-20T09:45:43Z + SO: + + α helix + + + 0.9988974 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.9989691 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + + INTRO + paragraph + 1118 + The family of IL-17 cytokines and receptors consists of six polypeptides, IL-17A-F, and five receptors, IL-17RA-E. IL-17A is secreted from activated Th17 cells, and several innate immune T cell types including macrophages, neutrophils, natural killer cells, and dendritic cells. IL-17A signals through a specific cell surface receptor complex which consists of IL-17RA and IL-17RC. IL-17A’s downstream signaling leads to increased production of inflammatory cytokines such as IL-6, IL-8, CCL-20 and CXCL1 by various mechanisms including stimulation of transcription and stabilization of mRNA. Although various cell types have been reported to express IL-17RA, the highest responses to IL-17A come from epithelial cells, endothelial cells, keratinocytes and fibroblasts. + + 0.9996173 + protein_type + cleaner0 + 2023-07-20T08:20:45Z + MESH: + + IL-17 cytokines + + + protein + PR: + cleaner0 + 2023-07-20T08:19:52Z + + IL-17A-F + + + 0.98434764 + protein + cleaner0 + 2023-07-20T09:24:28Z + PR: + + IL-17RA-E + + + protein + PR: + cleaner0 + 2023-07-20T08:19:34Z + + IL-17A + + + 0.9625089 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + protein_type + MESH: + cleaner0 + 2023-07-20T09:25:36Z + + receptor + + + 0.99856997 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99870443 + protein + cleaner0 + 2023-07-20T08:20:06Z + PR: + + IL-17RC + + + 0.9706386 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:45:34Z + + cytokines + + + 0.99858046 + protein_type + cleaner0 + 2023-07-20T08:20:58Z + MESH: + + IL-6 + + + 0.99673206 + protein_type + cleaner0 + 2023-07-20T08:21:00Z + MESH: + + IL-8 + + + 0.9613981 + protein_type + cleaner0 + 2023-07-20T08:21:02Z + MESH: + + CCL-20 + + + 0.64120615 + protein_type + cleaner0 + 2023-07-20T08:21:05Z + MESH: + + CXCL1 + + + 0.98661 + chemical + cleaner0 + 2023-07-20T08:21:09Z + CHEBI: + + mRNA + + + 0.9991738 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99757344 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + + INTRO + paragraph + 1890 + IL-17A and its signaling is important in host defense against certain fungal and bacterial infections as demonstrated by patients with autoantibodies against IL-17A and IL-17F, or with inborn errors of IL-17 immunity. In addition to its physiological role, IL-17A is a key pathogenic factor in inflammatory and autoimmune diseases. In phase II and III clinical trials, neutralizing monoclonal antibodies against IL-17A (secukinumab and ixekizumab) or its receptor IL-17RA (brodalumab) are highly efficacious in treating moderate to severe plaque psoriasis and psoriatic arthritis. Secukinumab has been approved recently as a new psoriasis drug by the US Food and Drug Administration (Cosentyx™). In addition to psoriasis and psoriatic arthritis, IL-17A blockade has also shown preclinical and clinical efficacies in ankylosing spondylitis and rheumatoid arthritis. + + protein + PR: + cleaner0 + 2023-07-20T08:16:50Z + + IL-17A + + + 0.94309646 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + 0.9504142 + protein + cleaner0 + 2023-07-20T09:24:32Z + PR: + + IL-17F + + + 0.9994386 + protein_type + cleaner0 + 2023-07-20T08:20:35Z + MESH: + + IL-17 + + + protein + PR: + cleaner0 + 2023-07-20T08:16:50Z + + IL-17A + + + protein_type + MESH: + cleaner0 + 2023-07-20T09:26:14Z + + antibodies + + + 0.86277634 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + 0.99755496 + chemical + cleaner0 + 2023-07-20T08:21:18Z + CHEBI: + + secukinumab + + + 0.99573684 + chemical + cleaner0 + 2023-07-20T08:21:23Z + CHEBI: + + ixekizumab + + + 0.95846945 + protein_type + cleaner0 + 2023-07-20T09:25:29Z + MESH: + + receptor + + + 0.92973715 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99208236 + chemical + cleaner0 + 2023-07-20T08:21:27Z + CHEBI: + + brodalumab + + + 0.9984517 + chemical + cleaner0 + 2023-07-20T08:21:19Z + CHEBI: + + Secukinumab + + + 0.9591042 + chemical + cleaner0 + 2023-07-20T08:21:33Z + CHEBI: + + Cosentyx™ + + + 0.99504143 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + + INTRO + paragraph + 2757 + Among IL-17 cytokines, IL-17A and IL-17F share the highest homology. These polypeptides form covalent homodimers, and IL-17A and IL-17F also form an IL-17A/IL-17F hetereodimer. Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. In these structures, both IL-17A and IL-17F adopt a cysteine-knot fold with two intramolecular disulfides and two interchain disulfide bonds that covalently link two monomers. + + 0.99963903 + protein_type + cleaner0 + 2023-07-20T08:20:46Z + MESH: + + IL-17 cytokines + + + 0.9972264 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + 0.997863 + protein + cleaner0 + 2023-07-20T09:24:38Z + PR: + + IL-17F + + + 0.57897425 + protein_state + cleaner0 + 2023-07-20T08:22:03Z + DUMMY: + + covalent + + + 0.99931896 + oligomeric_state + cleaner0 + 2023-07-20T08:23:07Z + DUMMY: + + homodimers + + + 0.99898124 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + 0.9990733 + protein + cleaner0 + 2023-07-20T09:24:38Z + PR: + + IL-17F + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:23:37Z + + IL-17A/IL-17F + + + 0.99701786 + oligomeric_state + cleaner0 + 2023-07-20T08:23:02Z + DUMMY: + + hetereodimer + + + 0.9988896 + evidence + cleaner0 + 2023-07-20T09:40:29Z + DUMMY: + + Structures + + + 0.9996731 + protein_state + cleaner0 + 2023-07-20T08:21:58Z + DUMMY: + + apo + + + 0.9991986 + protein + cleaner0 + 2023-07-20T09:24:38Z + PR: + + IL-17F + + + 0.99873376 + protein_state + cleaner0 + 2023-07-20T08:21:53Z + DUMMY: + + complex with + + + 0.998748 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.9996706 + protein_state + cleaner0 + 2023-07-20T08:21:59Z + DUMMY: + + apo + + + 0.99925786 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + 0.99880755 + protein_state + cleaner0 + 2023-07-20T08:21:53Z + DUMMY: + + complex with + + + 0.9991448 + protein_type + cleaner0 + 2023-07-20T08:47:54Z + MESH: + + antibody + + + 0.99974674 + structure_element + cleaner0 + 2023-07-20T08:47:40Z + SO: + + Fab + + + 0.9989835 + protein_state + cleaner0 + 2023-07-20T08:21:52Z + DUMMY: + + complex with + + + 0.9982937 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99912375 + evidence + cleaner0 + 2023-07-20T09:40:33Z + DUMMY: + + structures + + + 0.99910766 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + 0.99922085 + protein + cleaner0 + 2023-07-20T09:24:38Z + PR: + + IL-17F + + + structure_element + SO: + cleaner0 + 2023-07-20T08:22:32Z + + cysteine-knot + + + 0.99184275 + ptm + cleaner0 + 2023-07-20T08:22:49Z + MESH: + + disulfides + + + 0.87225294 + ptm + cleaner0 + 2023-07-20T08:22:56Z + MESH: + + disulfide bonds + + + 0.99904376 + oligomeric_state + cleaner0 + 2023-07-20T08:23:12Z + DUMMY: + + monomers + + + + INTRO + paragraph + 3256 + There has been active research in identifying orally available chemical entities that would functionally antagonize IL-17A-mediated signaling. Developing small molecules targeting protein-protein interactions is difficult with particular challenges associated with the large, shallow IL-17A/IL-17RA interfaces. Since IL-17RA is a shared receptor for at least IL-17A, IL-17F, IL-17A/IL-17F and IL-17E, we chose to seek IL-17A-specific inhibitors that may have more defined pharmacological responses than IL-17RA inhibitors. Our initial approach was to identify peptide inhibitors which could serve as leads for the development of anti-inflammatory therapeutics that could be used alone or in combination with other agents. Our efforts resulted in discovery of a high affinity IL-17A peptide antagonist (HAP), which we attempted to increase the functional production and pharmacokinetics after fusing HAP to antibodies for evaluation as a bispecific therapeutic in animal studies. Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR). + + 0.9965642 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + 0.99520123 + site + cleaner0 + 2023-07-20T08:39:44Z + SO: + + IL-17A/IL-17RA interfaces + + + protein + PR: + cleaner0 + 2023-07-20T08:17:00Z + + IL-17RA + + + protein_type + MESH: + cleaner0 + 2023-07-20T09:25:36Z + + receptor + + + 0.994631 + protein + cleaner0 + 2023-07-20T08:16:50Z + PR: + + IL-17A + + + 0.9799486 + protein + cleaner0 + 2023-07-20T09:24:38Z + PR: + + IL-17F + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:23:38Z + + IL-17A/IL-17F + + + 0.9883621 + protein + cleaner0 + 2023-07-20T08:24:20Z + PR: + + IL-17E + + + protein + PR: + cleaner0 + 2023-07-20T08:16:51Z + + IL-17A + + + protein + PR: + cleaner0 + 2023-07-20T08:17:00Z + + IL-17RA + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:24:35Z + + high affinity IL-17A peptide antagonist + + + 0.9493229 + chemical + cleaner0 + 2023-07-20T08:18:35Z + CHEBI: + + HAP + + + 0.87955695 + experimental_method + cleaner0 + 2023-07-20T09:29:52Z + MESH: + + fusing + + + 0.8208474 + chemical + cleaner0 + 2023-07-20T08:18:35Z + CHEBI: + + HAP + + + 0.54800403 + protein_type + cleaner0 + 2023-07-20T09:25:44Z + MESH: + + antibodies + + + protein_state + DUMMY: + cleaner0 + 2023-07-20T08:41:30Z + + uncapped + + + 0.84635955 + chemical + cleaner0 + 2023-07-20T08:18:35Z + CHEBI: + + HAP + + + 0.70992947 + chemical + cleaner0 + 2023-07-20T08:18:35Z + CHEBI: + + HAP + + + evidence + DUMMY: + cleaner0 + 2023-07-20T09:42:22Z + + complex structure + + + 0.9984722 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9962735 + chemical + cleaner0 + 2023-07-20T08:18:35Z + CHEBI: + + HAP + + + 0.8284904 + experimental_method + cleaner0 + 2023-07-20T09:29:55Z + MESH: + + peptide optimization + + + 0.76640385 + experimental_method + cleaner0 + 2023-07-20T09:30:00Z + MESH: + + structure activity relationship + + + experimental_method + MESH: + cleaner0 + 2023-07-20T09:04:27Z + + SAR + + + + RESULTS + title_1 + 4573 + Results + + + RESULTS + title_2 + 4581 + Identification of IL-17A peptide inhibitors + + 0.97984284 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + + RESULTS + paragraph + 4625 + Peptides specifically binding to human IL-17A were identified from phage panning using cyclic and linear peptide libraries (Supplementary Figure S1). Positive phage pools were then sub-cloned into a maltose-binding protein (MBP) fusion system. Single clones were isolated and sub-cultured in growth medium, and culture supernatants were used in an enzyme-linked immunosorbent assay (ELISA) to identify specific IL-17A-binding clones. The positive binding supernatants were tested for the ability to block biotinylated IL-17A signaling through IL-17RA in an IL-17A/IL-17RA competition ELISA assay where unlabeled IL-17A was used as positive control to inhibit biotinylated IL-17A binding. Approximately 10% of the clones that specifically bound to IL-17A also prevented the cytokine from binding to IL-17RA. Sequences identified from phage clones were chemically synthesized (Supplementary Table 1) and tested for inhibition of IL-17A binding to IL-17RA (Table 1). A 15-mer linear peptide 1 was shown to block IL-17A/IL-17RA binding with an IC50 of 80 nM in the competition ELISA assay (Table 1). This peptide was then tested in a cell-based functional assay wherein production of GRO-α in BJ human fibroblast cells was measured as a function of IL-17A stimulation using 1 ng/ml IL-17A. Peptide 1 was found to be active in this functional assay with an IC50 of 370 nM. + + 0.9993475 + species + cleaner0 + 2023-07-20T08:25:03Z + MESH: + + human + + + 0.9993885 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.999529 + experimental_method + cleaner0 + 2023-07-20T09:19:10Z + MESH: + + phage panning + + + experimental_method + MESH: + cleaner0 + 2023-07-20T08:26:37Z + + cyclic and linear peptide libraries + + + experimental_method + MESH: + cleaner0 + 2023-07-20T08:26:18Z + + Positive phage pools + + + 0.9991283 + experimental_method + cleaner0 + 2023-07-20T09:30:04Z + MESH: + + sub-cloned + + + experimental_method + MESH: + cleaner0 + 2023-07-20T08:26:02Z + + maltose-binding protein (MBP) fusion system + + + 0.9995176 + experimental_method + cleaner0 + 2023-07-20T08:26:46Z + MESH: + + enzyme-linked immunosorbent assay + + + 0.9980411 + experimental_method + cleaner0 + 2023-07-20T08:26:51Z + MESH: + + ELISA + + + 0.98475736 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99689853 + protein_state + cleaner0 + 2023-07-20T09:15:20Z + DUMMY: + + biotinylated + + + 0.99892646 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99871516 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:28:04Z + + IL-17A/IL-17RA + + + 0.9978862 + experimental_method + cleaner0 + 2023-07-20T08:27:13Z + MESH: + + competition ELISA assay + + + 0.9931461 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99169886 + protein_state + cleaner0 + 2023-07-20T09:15:20Z + DUMMY: + + biotinylated + + + 0.99915606 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99945474 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:50:51Z + + cytokine + + + 0.9991376 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.9994713 + experimental_method + cleaner0 + 2023-07-20T09:19:24Z + MESH: + + phage clones + + + 0.99924475 + experimental_method + cleaner0 + 2023-07-20T09:19:26Z + MESH: + + chemically synthesized + + + 0.9984878 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.999304 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:38:10Z + + peptide 1 + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:28:05Z + + IL-17A/IL-17RA + + + 0.9994924 + evidence + cleaner0 + 2023-07-20T08:32:48Z + DUMMY: + + IC50 + + + 0.99933887 + experimental_method + cleaner0 + 2023-07-20T08:27:13Z + MESH: + + competition ELISA assay + + + 0.9960262 + experimental_method + cleaner0 + 2023-07-20T08:39:25Z + MESH: + + cell-based functional assay + + + 0.99944496 + protein + cleaner0 + 2023-07-20T09:24:45Z + PR: + + GRO-α + + + 0.99842834 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.99569863 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99933106 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:38:10Z + + Peptide 1 + + + experimental_method + MESH: + cleaner0 + 2023-07-20T09:30:35Z + + functional assay + + + 0.9993832 + evidence + cleaner0 + 2023-07-20T08:32:48Z + DUMMY: + + IC50 + + + + RESULTS + title_2 + 6000 + Optimization of IL-17A peptide inhibitors + + 0.9963889 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + + RESULTS + paragraph + 6042 + A SAR campaign was undertaken to improve the potency of peptide 1. An alanine scan of peptide 2, an analogue of 1 with a lysine to arginine substitution at position 14, was initiated. When alanine was already present (positions 7 and 15), substitution was made with lysine (Table 1, peptides 3–17). Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA. In particular, at position 5 (13), substitution of methionine with alanine resulted in a seven fold improvement in potency (80 nM versus 11 nM respectively). In order to rapidly evaluate the effects of substitution of natural amino acids at tolerant positions identified by the alanine scan, the lead sequence was subjected to site-specific saturation mutagenesis using MBP. Each of the seven positions identified by the alanine scan was individually modified while keeping the rest of the sequence constant. Modifications at positions 2 and 14 were shown to display improvement in binding affinity (data not shown). + + experimental_method + MESH: + cleaner0 + 2023-07-20T09:04:27Z + + SAR + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:38:09Z + + peptide 1 + + + 0.99952424 + experimental_method + cleaner0 + 2023-07-20T09:31:46Z + MESH: + + alanine scan + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:38:27Z + + peptide 2 + + + 0.60970217 + chemical + cleaner0 + 2023-07-20T08:38:30Z + CHEBI: + + 1 + + + 0.99725467 + residue_name + cleaner0 + 2023-07-20T09:21:46Z + SO: + + lysine + + + 0.9975005 + residue_name + cleaner0 + 2023-07-20T09:21:48Z + SO: + + arginine + + + 0.99908924 + experimental_method + cleaner0 + 2023-07-20T09:31:49Z + MESH: + + substitution + + + 0.99520063 + residue_number + cleaner0 + 2023-07-20T09:27:33Z + DUMMY: + + 14 + + + 0.9989141 + residue_name + cleaner0 + 2023-07-20T09:21:52Z + SO: + + alanine + + + 0.9927239 + residue_number + cleaner0 + 2023-07-20T09:27:36Z + DUMMY: + + 7 + + + 0.99312204 + residue_number + cleaner0 + 2023-07-20T09:27:39Z + DUMMY: + + 15 + + + 0.9978138 + experimental_method + cleaner0 + 2023-07-20T09:31:52Z + MESH: + + substitution + + + 0.99909055 + residue_name + cleaner0 + 2023-07-20T09:22:33Z + SO: + + lysine + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:37:43Z + + peptides 3–17 + + + 0.9782874 + residue_number + cleaner0 + 2023-07-20T09:27:42Z + DUMMY: + + 1 + + + 0.94353265 + residue_number + cleaner0 + 2023-07-20T09:27:45Z + DUMMY: + + 2 + + + 0.9745618 + residue_number + cleaner0 + 2023-07-20T09:27:48Z + DUMMY: + + 4 + + + 0.9761025 + residue_number + cleaner0 + 2023-07-20T09:27:51Z + DUMMY: + + 5 + + + 0.98406637 + residue_number + cleaner0 + 2023-07-20T09:27:54Z + DUMMY: + + 7 + + + 0.98588353 + residue_number + cleaner0 + 2023-07-20T09:27:56Z + DUMMY: + + 14 + + + 0.989995 + residue_number + cleaner0 + 2023-07-20T09:27:59Z + DUMMY: + + 15 + + + 0.9992334 + evidence + cleaner0 + 2023-07-20T09:40:48Z + DUMMY: + + binding affinity + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:28:05Z + + IL-17A/IL-17RA + + + 0.9994817 + experimental_method + cleaner0 + 2023-07-20T08:28:33Z + MESH: + + competition ELISA + + + 0.9915571 + residue_number + cleaner0 + 2023-07-20T09:28:02Z + DUMMY: + + 5 + + + 0.99155676 + chemical + cleaner0 + 2023-07-20T08:38:44Z + CHEBI: + + 13 + + + 0.99961156 + experimental_method + cleaner0 + 2023-07-20T09:32:01Z + MESH: + + substitution + + + 0.99922335 + residue_name + cleaner0 + 2023-07-20T09:21:56Z + SO: + + methionine + + + 0.9991792 + residue_name + cleaner0 + 2023-07-20T09:21:59Z + SO: + + alanine + + + experimental_method + MESH: + cleaner0 + 2023-07-20T09:32:25Z + + substitution + + + 0.9994993 + experimental_method + cleaner0 + 2023-07-20T09:32:30Z + MESH: + + alanine scan + + + 0.99943316 + experimental_method + cleaner0 + 2023-07-20T09:32:37Z + MESH: + + site-specific saturation mutagenesis + + + 0.5736163 + experimental_method + cleaner0 + 2023-07-20T08:38:58Z + MESH: + + MBP + + + 0.99947476 + experimental_method + cleaner0 + 2023-07-20T09:32:42Z + MESH: + + alanine scan + + + 0.9931477 + residue_number + cleaner0 + 2023-07-20T09:28:04Z + DUMMY: + + 2 + + + 0.992432 + residue_number + cleaner0 + 2023-07-20T09:28:07Z + DUMMY: + + 14 + + + 0.9993174 + evidence + cleaner0 + 2023-07-20T09:40:52Z + DUMMY: + + binding affinity + + + + RESULTS + paragraph + 7158 + Peptides with beneficial point mutations at positions 2, 5, and 14 were synthesized and evaluated in the competition ELISA (Table 1). Two of the changes, V2H (18) or V2T (21) displayed improved binding in the competition ELISA. Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. Introduction of a methionine (27) or a carboxamide (28 and 29) at position 14 was shown to improve the binding affinity of the lead peptide. In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion. + + experimental_method + MESH: + cleaner0 + 2023-07-20T09:33:09Z + + point mutations + + + 0.9938885 + residue_number + cleaner0 + 2023-07-20T09:28:10Z + DUMMY: + + 2 + + + 0.99447745 + residue_number + cleaner0 + 2023-07-20T09:28:13Z + DUMMY: + + 5 + + + 0.9924971 + residue_number + cleaner0 + 2023-07-20T09:28:16Z + DUMMY: + + 14 + + + 0.99920183 + experimental_method + cleaner0 + 2023-07-20T09:33:13Z + MESH: + + synthesized + + + 0.9996035 + experimental_method + cleaner0 + 2023-07-20T08:28:52Z + MESH: + + competition ELISA + + + mutant + MESH: + cleaner0 + 2023-07-20T08:32:11Z + + V2H + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:36:11Z + + 18 + + + mutant + MESH: + cleaner0 + 2023-07-20T08:32:27Z + + V2T + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:36:20Z + + 21 + + + 0.9995967 + experimental_method + cleaner0 + 2023-07-20T08:29:00Z + MESH: + + competition ELISA + + + 0.9990429 + experimental_method + cleaner0 + 2023-07-20T09:33:18Z + MESH: + + replacement + + + 0.9991487 + residue_name + cleaner0 + 2023-07-20T09:22:03Z + SO: + + methionine + + + 0.9952172 + residue_number + cleaner0 + 2023-07-20T09:28:18Z + DUMMY: + + 5 + + + 0.998944 + residue_name + cleaner0 + 2023-07-20T09:22:05Z + SO: + + alanine + + + 0.9989441 + residue_name + cleaner0 + 2023-07-20T09:22:08Z + SO: + + isoleucine + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:36:29Z + + 24 + + + 0.99902976 + residue_name + cleaner0 + 2023-07-20T09:22:10Z + SO: + + leucine + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:36:39Z + + 25 + + + 0.99911135 + residue_name + cleaner0 + 2023-07-20T09:22:12Z + SO: + + valine + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:36:47Z + + 26 + + + 0.99879634 + residue_name + cleaner0 + 2023-07-20T09:22:16Z + SO: + + valine + + + 0.99913895 + residue_name + cleaner0 + 2023-07-20T09:22:14Z + SO: + + isoleucine + + + 0.5152386 + experimental_method + cleaner0 + 2023-07-20T09:33:22Z + MESH: + + replacements + + + 0.99869436 + residue_name + cleaner0 + 2023-07-20T09:22:18Z + SO: + + alanine + + + 0.9977946 + experimental_method + cleaner0 + 2023-07-20T09:33:25Z + MESH: + + Introduction + + + 0.9992287 + residue_name + cleaner0 + 2023-07-20T09:22:20Z + SO: + + methionine + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:36:59Z + + 27 + + + 0.9798034 + chemical + cleaner0 + 2023-07-20T08:41:41Z + CHEBI: + + carboxamide + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:37:09Z + + 28 + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:37:17Z + + 29 + + + 0.9943374 + residue_number + cleaner0 + 2023-07-20T09:28:21Z + DUMMY: + + 14 + + + 0.9994836 + evidence + cleaner0 + 2023-07-20T09:40:55Z + DUMMY: + + binding affinity + + + 0.99957854 + evidence + cleaner0 + 2023-07-20T09:40:59Z + DUMMY: + + binding affinities + + + 0.88407314 + experimental_method + cleaner0 + 2023-07-20T08:29:16Z + MESH: + + MBP fusion + + + 0.99947506 + experimental_method + cleaner0 + 2023-07-20T09:33:47Z + MESH: + + substitution + + + 0.9991304 + residue_name + cleaner0 + 2023-07-20T09:22:23Z + SO: + + valine + + + 0.9938672 + residue_number + cleaner0 + 2023-07-20T09:28:24Z + DUMMY: + + 2 + + + 0.9991211 + residue_name + cleaner0 + 2023-07-20T09:22:27Z + SO: + + tryptophan + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:37:27Z + + 22 + + + 0.86052316 + evidence + cleaner0 + 2023-07-20T09:41:03Z + DUMMY: + + affinity + + + 0.97281766 + experimental_method + cleaner0 + 2023-07-20T08:29:17Z + MESH: + + MBP fusion + + + + RESULTS + paragraph + 8161 + Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17 nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2). We also examined the effect of removing the acetyl group at the N-terminus of HAP (which is present in all the peptides made, see Supplementary Material). The un-capped peptide (31) had an IC50 of 420 nM in the cell-based assay. The loss of cellular activity of 31 was most likely due to the degradation of the N-terminus of 31, since peptide 31 was shown to be able to bind to IL-17A with similar affinity as HAP itself. Furthermore, our previous work had reported that in antibody fusions the uncapped peptide was degraded under cell assay conditions with removal of the first 1-3 residues to inactive products with the same N-terminal sequences as peptides 32–34. In this work, 32–34 are capped by protective acetyl group and reflect the same inactivity as reported. C-terminal truncations showed a more gradual reduction in activity (35–37; Table 2). After deletion of three amino acids from the C-terminal end (37), the peptide is no longer active. + + 0.99909127 + experimental_method + cleaner0 + 2023-07-20T09:04:27Z + MESH: + + SAR + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:35:01Z + + peptide 30 + + + 0.94325596 + chemical + cleaner0 + 2023-07-20T09:57:42Z + CHEBI: + + high affinity peptide + + + 0.98649454 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + protein + PR: + cleaner0 + 2023-07-20T08:16:51Z + + IL-17A + + + species + MESH: + cleaner0 + 2023-07-20T08:25:04Z + + human + + + 0.995522 + evidence + cleaner0 + 2023-07-20T08:32:48Z + DUMMY: + + IC50 + + + experimental_method + MESH: + cleaner0 + 2023-07-20T09:01:15Z + + phage + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:35:59Z + + peptide 1 + + + 0.99545926 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9994185 + protein_state + cleaner0 + 2023-07-20T08:41:22Z + DUMMY: + + un-capped + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:35:37Z + + peptide (31) + + + 0.9992348 + evidence + cleaner0 + 2023-07-20T08:32:48Z + DUMMY: + + IC50 + + + 0.97057736 + experimental_method + cleaner0 + 2023-07-20T09:34:06Z + MESH: + + cell-based assay + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:35:16Z + + 31 + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:35:17Z + + 31 + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:35:17Z + + 31 + + + protein + PR: + cleaner0 + 2023-07-20T08:16:51Z + + IL-17A + + + 0.9978257 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99392676 + experimental_method + cleaner0 + 2023-07-20T09:19:48Z + MESH: + + antibody fusions + + + 0.999673 + protein_state + cleaner0 + 2023-07-20T08:41:29Z + DUMMY: + + uncapped + + + 0.6190488 + chemical + cleaner0 + 2023-07-20T09:57:58Z + CHEBI: + + peptide + + + 0.9911684 + experimental_method + cleaner0 + 2023-07-20T09:34:11Z + MESH: + + removal of + + + 0.90224326 + residue_range + cleaner0 + 2023-07-20T09:26:32Z + DUMMY: + + first 1-3 residues + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:40:30Z + + peptides 32–34 + + + 0.9917441 + chemical + cleaner0 + 2023-07-20T08:40:45Z + CHEBI: + + 32–34 + + + 0.95190495 + protein_state + cleaner0 + 2023-07-20T08:41:07Z + DUMMY: + + capped + + + 0.883352 + experimental_method + cleaner0 + 2023-07-20T09:34:19Z + MESH: + + truncations + + + 0.9986224 + chemical + cleaner0 + 2023-07-20T08:40:53Z + CHEBI: + + 35–37 + + + 0.9165045 + experimental_method + cleaner0 + 2023-07-20T09:34:22Z + MESH: + + deletion of + + + 0.991111 + residue_range + cleaner0 + 2023-07-20T09:26:39Z + DUMMY: + + three amino acids + + + 0.94737333 + chemical + cleaner0 + 2023-07-20T08:40:08Z + CHEBI: + + 37 + + + + RESULTS + title_2 + 9469 + Dimerization of HAP can further increase its potency + + 0.9830784 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + + RESULTS + paragraph + 9522 + We reasoned that since the IL-17A protein is almost exclusively present in a dimeric form, dimerizing the IL-17A binding peptides could result in an improvement in binding affinity and inhibitory activity. Homodimers of HAP were made through attachment of polyethylene glycol (PEG) spacers of different lengths at amino acids 4, 7 and 14, as these positions were identified in the alanine scan analysis as not contributing significantly to the activity, and at each N-terminus (Supplementary Table S2). Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. This exercise revealed that several dimeric peptides with the longer PEG21 spacer were significantly more potent than the monomer peptide in the cell-based assay (Supplementary Table S2). Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1 nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21 nM (Supplementary Scheme S2). + + 0.9925688 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9993999 + oligomeric_state + cleaner0 + 2023-07-20T08:33:39Z + DUMMY: + + dimeric + + + 0.99754333 + oligomeric_state + cleaner0 + 2023-07-20T08:33:46Z + DUMMY: + + dimerizing + + + 0.97856927 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.85114086 + evidence + cleaner0 + 2023-07-20T09:41:09Z + DUMMY: + + binding affinity + + + 0.99938464 + oligomeric_state + cleaner0 + 2023-07-20T08:23:08Z + DUMMY: + + Homodimers + + + 0.995388 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9979036 + chemical + cleaner0 + 2023-07-20T08:33:14Z + CHEBI: + + polyethylene glycol + + + 0.99892706 + chemical + cleaner0 + 2023-07-20T08:33:18Z + CHEBI: + + PEG + + + 0.99253124 + residue_number + cleaner0 + 2023-07-20T09:28:30Z + DUMMY: + + 4 + + + 0.99313086 + residue_number + cleaner0 + 2023-07-20T09:28:33Z + DUMMY: + + 7 + + + 0.992668 + residue_number + cleaner0 + 2023-07-20T09:28:39Z + DUMMY: + + 14 + + + 0.9994256 + experimental_method + cleaner0 + 2023-07-20T09:34:44Z + MESH: + + alanine scan analysis + + + 0.99972945 + chemical + cleaner0 + 2023-07-20T08:33:24Z + CHEBI: + + pentafluoroester + + + 0.99973756 + chemical + cleaner0 + 2023-07-20T08:33:28Z + CHEBI: + + PFP + + + 0.99917716 + chemical + cleaner0 + 2023-07-20T08:33:19Z + CHEBI: + + PEG + + + 0.99935335 + residue_name + cleaner0 + 2023-07-20T09:22:40Z + SO: + + histidine + + + 0.99485767 + residue_number + cleaner0 + 2023-07-20T09:28:42Z + DUMMY: + + 2 + + + 0.9993436 + residue_name + cleaner0 + 2023-07-20T09:22:42Z + SO: + + lysine + + + 0.995314 + residue_number + cleaner0 + 2023-07-20T09:28:45Z + DUMMY: + + 15 + + + 0.99937785 + residue_name + cleaner0 + 2023-07-20T09:22:48Z + SO: + + threonine + + + 0.99938715 + residue_name + cleaner0 + 2023-07-20T09:22:56Z + SO: + + dimethyllysine + + + chemical + CHEBI: + cleaner0 + 2023-07-20T09:23:12Z + + peptide 38 + + + 0.9922083 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9993723 + oligomeric_state + cleaner0 + 2023-07-20T08:33:38Z + DUMMY: + + dimeric + + + 0.9179296 + chemical + cleaner0 + 2023-07-20T09:58:17Z + CHEBI: + + peptides + + + 0.9063272 + chemical + cleaner0 + 2023-07-20T09:58:21Z + CHEBI: + + PEG21 + + + 0.9993399 + oligomeric_state + cleaner0 + 2023-07-20T09:23:52Z + DUMMY: + + monomer + + + experimental_method + MESH: + cleaner0 + 2023-07-20T09:35:09Z + + cell-based assay + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:34:30Z + + Peptide 45 + + + 0.99911827 + oligomeric_state + cleaner0 + 2023-07-20T08:33:53Z + DUMMY: + + dimerized + + + 0.9993223 + chemical + cleaner0 + 2023-07-20T09:58:25Z + CHEBI: + + PEG21 + + + 0.99526054 + residue_number + cleaner0 + 2023-07-20T09:28:48Z + DUMMY: + + 14 + + + 0.99950886 + evidence + cleaner0 + 2023-07-20T08:32:48Z + DUMMY: + + IC50 + + + 0.999368 + oligomeric_state + cleaner0 + 2023-07-20T09:23:53Z + DUMMY: + + monomer + + + 0.9993444 + chemical + cleaner0 + 2023-07-20T09:58:29Z + CHEBI: + + PEG21 + + + 0.99590635 + residue_number + cleaner0 + 2023-07-20T09:28:51Z + DUMMY: + + 14 + + + chemical + CHEBI: + cleaner0 + 2023-07-20T09:23:29Z + + peptide 48 + + + 0.9994103 + evidence + cleaner0 + 2023-07-20T08:32:48Z + DUMMY: + + IC50 + + + + RESULTS + paragraph + 10901 + The species cross-reactivity of the dimeric peptide 45 and HAP were assessed in a murine functional cell assay using 15 ng/ml murine IL-17A. Peptide 45 blocked the receptor binding of murine IL-17A although with potency two orders of magnitude weaker than that observed against human IL-17A (IC50 = 41 nM vs IC50 = 0.1 nM, respectively). The monomer HAP was much weaker (IC50 >1 μM) in inhibiting murine IL-17A signaling (Supplementary Figure S4). + + 0.99936944 + oligomeric_state + cleaner0 + 2023-07-20T08:33:39Z + DUMMY: + + dimeric + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:34:30Z + + peptide 45 + + + 0.9996276 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9974878 + experimental_method + cleaner0 + 2023-07-20T08:42:45Z + MESH: + + murine functional cell assay + + + 0.9954389 + taxonomy_domain + cleaner0 + 2023-07-20T08:42:12Z + DUMMY: + + murine + + + 0.99672765 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:34:30Z + + Peptide 45 + + + protein_type + MESH: + cleaner0 + 2023-07-20T09:25:36Z + + receptor + + + 0.995782 + taxonomy_domain + cleaner0 + 2023-07-20T08:42:12Z + DUMMY: + + murine + + + 0.9957366 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9992908 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.9978351 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.93804055 + evidence + cleaner0 + 2023-07-20T08:32:48Z + DUMMY: + + IC50 + + + 0.8690941 + evidence + cleaner0 + 2023-07-20T08:32:48Z + DUMMY: + + IC50 + + + 0.9993467 + oligomeric_state + cleaner0 + 2023-07-20T09:23:53Z + DUMMY: + + monomer + + + 0.99963975 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + evidence + DUMMY: + cleaner0 + 2023-07-20T08:32:48Z + + IC50 + + + 0.9985342 + taxonomy_domain + cleaner0 + 2023-07-20T08:42:11Z + DUMMY: + + murine + + + 0.9880951 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + + RESULTS + paragraph + 11366 + Although the dimeric peptide 45 is much more potent than HAP in the cell-based assay, in subsequent studies we decided to focus our efforts solely on characterizations of the monomeric peptide HAP in hopes to identify smaller peptide inhibitors containing the best minimal functional group. + + 0.9993104 + oligomeric_state + cleaner0 + 2023-07-20T08:33:39Z + DUMMY: + + dimeric + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:34:31Z + + peptide 45 + + + 0.9993166 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + experimental_method + MESH: + cleaner0 + 2023-07-20T08:42:36Z + + cell-based assay + + + 0.9989555 + oligomeric_state + cleaner0 + 2023-07-20T09:24:03Z + DUMMY: + + monomeric + + + 0.99938715 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + + RESULTS + title_2 + 11657 + Orthogonal assays to confirm HAP antagonism + + 0.9997316 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + + RESULTS + paragraph + 11701 + To further characterize the interaction of HAP with IL-17A, we set out to determine its in vitro binding affinity, specificity and kinetic profile using Surface Plasmon Resonance (SPR) methods (Fig. 1A). HAP binds to immobilized human IL-17A homodimer tightly (Table 3). It has slightly weaker affinity for human IL-17A/F heterodimer and >10 fold weaker affinity for mouse IL-17A (Table 3). HAP does not show significant binding to immobilized human IL-17F homodimer or IL-17RA at concentrations up to 100 nM. + + 0.99962664 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9991302 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9748456 + evidence + cleaner0 + 2023-07-20T09:41:17Z + DUMMY: + + binding affinity + + + 0.9721006 + evidence + cleaner0 + 2023-07-20T09:41:22Z + DUMMY: + + kinetic profile + + + 0.9995649 + experimental_method + cleaner0 + 2023-07-20T08:43:31Z + MESH: + + Surface Plasmon Resonance + + + 0.9996598 + experimental_method + cleaner0 + 2023-07-20T08:43:35Z + MESH: + + SPR + + + 0.9996233 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99923086 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.99936104 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99927896 + oligomeric_state + cleaner0 + 2023-07-20T09:24:10Z + DUMMY: + + homodimer + + + 0.907098 + evidence + cleaner0 + 2023-07-20T09:41:26Z + DUMMY: + + affinity + + + 0.999366 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.9975115 + complex_assembly + cleaner0 + 2023-07-20T08:43:17Z + GO: + + IL-17A/F + + + 0.9991142 + oligomeric_state + cleaner0 + 2023-07-20T09:24:17Z + DUMMY: + + heterodimer + + + 0.99288344 + evidence + cleaner0 + 2023-07-20T09:41:30Z + DUMMY: + + affinity + + + 0.9979754 + taxonomy_domain + cleaner0 + 2023-07-20T08:42:57Z + DUMMY: + + mouse + + + 0.99932736 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9996487 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9993154 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.99919397 + protein + cleaner0 + 2023-07-20T09:24:38Z + PR: + + IL-17F + + + 0.9992888 + oligomeric_state + cleaner0 + 2023-07-20T09:24:11Z + DUMMY: + + homodimer + + + 0.9994171 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + + RESULTS + paragraph + 12213 + Additionally, we investigated the antagonism of the human IL-17A/IL-17RA interaction by HAP using orthogonal methods including SPR and Förster resonance energy transfer (FRET) competition assays (Fig. 1B,C). In both assays, incubation of IL-17A with HAP effectively blocks the binding of IL-17A to immobilized IL-17RA with similar sub-nM IC50 (Table 3). + + 0.99922895 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:28:05Z + + IL-17A/IL-17RA + + + 0.99973136 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9996264 + experimental_method + cleaner0 + 2023-07-20T08:43:35Z + MESH: + + SPR + + + experimental_method + MESH: + cleaner0 + 2023-07-20T08:44:07Z + + Förster resonance energy transfer (FRET) competition assays + + + 0.9995489 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9997228 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99951553 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9434454 + protein_state + cleaner0 + 2023-07-20T09:55:35Z + DUMMY: + + immobilized + + + 0.9993718 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.9993969 + evidence + cleaner0 + 2023-07-20T08:32:49Z + DUMMY: + + IC50 + + + + RESULTS + title_2 + 12568 + HAP blocks IL-17A signaling in a human primary cell assay + + 0.9997081 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.93761903 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99930716 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + + RESULTS + paragraph + 12626 + While either IL-17A or TNF-α alone can stimulate the release of multiple inflammatory cytokines, when acting together they can synergistically enhance each other’s effects (Supplementary Figure S5). These integrative responses to IL-17A and TNF-α in human keratinocytes have been reported to account for key inflammatory pathogenic circuits in psoriasis. Thus, we chose to study HAP’s efficacy in blocking the production of IL-8, IL-6 and CCL-20 by primary human keratinocytes stimulated by IL-17A in the presence of TNF-α, an assay which may be more disease-relevant. HAP inhibits the production of all three cytokines in a dose-dependent fashion (Fig. 1D). Significantly, the baseline levels of IL-8, IL-6 and CCL-20 stimulated by TNF-α alone are not inhibited by HAP, further indicating the selectivity of HAP (Fig. 1D). Such pharmacological selectivity may be important to suppress inflammatory pathogenic circuits in psoriasis, while sparing the anti-infectious immune responses produced by TNF-α. The relatively high IC50 values in this assay (Table 3) are probably due to the high IL-17A concentration (100 ng/ml) needed for detection of IL-6. As a reference, a commercial anti-IL-17A antibody (R&D Systems) inhibits the production of IL-8 with an IC50 of 13(±6) nM (N = 3). Indeed, the IC50 was 14(±9) nM (N = 12) for HAP inhibition of IL-8 production when only 5 ng/ml IL-17A was used in this assay. In patients, the concentration of IL-17A in psoriatic lesions is reported to be 0.01 ng/ml, well below the EC50 (5–10ng/ml) of IL-17A induced IL-8 production in vitro. + + 0.9989521 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9993132 + protein + cleaner0 + 2023-07-20T08:44:22Z + PR: + + TNF-α + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:45:33Z + + cytokines + + + 0.99909925 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99937916 + protein + cleaner0 + 2023-07-20T08:44:23Z + PR: + + TNF-α + + + 0.9993874 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.99958223 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.999372 + protein_type + cleaner0 + 2023-07-20T08:44:31Z + MESH: + + IL-8 + + + 0.9994655 + protein_type + cleaner0 + 2023-07-20T08:44:36Z + MESH: + + IL-6 + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:44:54Z + + CCL-20 + + + 0.99926895 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.9993224 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9896974 + protein + cleaner0 + 2023-07-20T08:44:23Z + PR: + + TNF-α + + + 0.99960405 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:45:34Z + + cytokines + + + 0.9993946 + protein_type + cleaner0 + 2023-07-20T08:44:32Z + MESH: + + IL-8 + + + 0.9994555 + protein_type + cleaner0 + 2023-07-20T08:44:36Z + MESH: + + IL-6 + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:44:55Z + + CCL-20 + + + 0.9222906 + protein + cleaner0 + 2023-07-20T08:44:23Z + PR: + + TNF-α + + + 0.99961793 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9995413 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99793345 + protein + cleaner0 + 2023-07-20T08:44:23Z + PR: + + TNF-α + + + 0.99960846 + evidence + cleaner0 + 2023-07-20T08:32:49Z + DUMMY: + + IC50 + + + 0.99871665 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99944514 + protein_type + cleaner0 + 2023-07-20T08:44:36Z + MESH: + + IL-6 + + + protein + PR: + cleaner0 + 2023-07-20T08:16:51Z + + IL-17A + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:47:54Z + + antibody + + + 0.99719936 + protein_type + cleaner0 + 2023-07-20T08:44:32Z + MESH: + + IL-8 + + + 0.999605 + evidence + cleaner0 + 2023-07-20T08:32:49Z + DUMMY: + + IC50 + + + 0.99960977 + evidence + cleaner0 + 2023-07-20T08:32:49Z + DUMMY: + + IC50 + + + 0.9996481 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9992235 + protein_type + cleaner0 + 2023-07-20T08:44:32Z + MESH: + + IL-8 + + + 0.9984741 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9984421 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9857058 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9992984 + protein_type + cleaner0 + 2023-07-20T08:44:32Z + MESH: + + IL-8 + + + + RESULTS + paragraph + 14230 + Similar to keratinocytes assay results, while HAP inhibits IL-17A stimulated IL-6 production by BJ human fibroblast potently (IC50 of 17 nM), it does not inhibit TNF-α stimulated IL-6 production at concentrations up to 10 μM (Supplementary Figure S2). + + 0.9993535 + experimental_method + cleaner0 + 2023-07-20T08:45:53Z + MESH: + + keratinocytes assay + + + 0.99973947 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + protein + PR: + cleaner0 + 2023-07-20T08:16:51Z + + IL-17A + + + 0.99953794 + protein_type + cleaner0 + 2023-07-20T08:44:37Z + MESH: + + IL-6 + + + 0.99904794 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.9992768 + evidence + cleaner0 + 2023-07-20T08:32:49Z + DUMMY: + + IC50 + + + 0.9035897 + protein + cleaner0 + 2023-07-20T08:44:23Z + PR: + + TNF-α + + + 0.99948907 + protein_type + cleaner0 + 2023-07-20T08:44:37Z + MESH: + + IL-6 + + + + RESULTS + title_2 + 14488 + Crystallization and structure determination + + 0.9995088 + experimental_method + cleaner0 + 2023-07-20T08:48:17Z + MESH: + + Crystallization and structure determination + + + + RESULTS + paragraph + 14532 + Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals. We theorized that HAP binding induced large conformational changes in IL-17A that led to the difficulty of getting an IL-17A/HAP binary complex crystal. It is known that an antibody antigen-binding fragment (Fab) can be used as crystallization chaperones in crystallizing difficult targets. We hypothesized that HAP may target the N-terminal of IL-17A which is known to be more flexible than its C-terminal and conformational changes needed for HAP binding may be more likely there. We designed an antibody Fab known to target the C-terminal half of IL-17A based on a published IL-17A/Fab complex crystal structure, and produced it in HEK293 cells. In an SPR assay HAP and this Fab were able to co-bind IL-17A without large changes in their binding affinities and kinetics, confirming our hypothesis (Supplementary Figure S6). Furthermore, since it binds to an area far away from that of HAP (see below), this Fab should have minimum effects on HAP binding conformation. Crystals of Fab/IL-17A/HAP ternary complex were obtained readily in crystallization screens. + + 0.99862516 + experimental_method + cleaner0 + 2023-07-20T08:48:14Z + MESH: + + crystallization trials + + + 0.9995484 + experimental_method + cleaner0 + 2023-07-20T08:48:27Z + MESH: + + co-crystallization + + + 0.99957675 + experimental_method + cleaner0 + 2023-07-20T08:48:30Z + MESH: + + soaking + + + 0.9997243 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9996561 + protein_state + cleaner0 + 2023-07-20T08:21:59Z + DUMMY: + + apo + + + 0.9994383 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99938405 + evidence + cleaner0 + 2023-07-20T08:48:04Z + DUMMY: + + crystals + + + 0.99961644 + complex_assembly + cleaner0 + 2023-07-20T08:46:25Z + GO: + + IL-17A/HAP + + + 0.9993255 + evidence + cleaner0 + 2023-07-20T08:48:06Z + DUMMY: + + crystals + + + 0.999076 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.999286 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99953806 + complex_assembly + cleaner0 + 2023-07-20T08:46:26Z + GO: + + IL-17A/HAP + + + 0.9993813 + evidence + cleaner0 + 2023-07-20T08:48:09Z + DUMMY: + + crystal + + + 0.99914825 + protein_type + cleaner0 + 2023-07-20T08:47:53Z + MESH: + + antibody + + + 0.99950033 + structure_element + cleaner0 + 2023-07-20T08:47:33Z + SO: + + antigen-binding fragment + + + 0.9997451 + structure_element + cleaner0 + 2023-07-20T08:47:39Z + SO: + + Fab + + + 0.99963105 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9990602 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99944824 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9992513 + protein_type + cleaner0 + 2023-07-20T08:47:54Z + MESH: + + antibody + + + 0.9997279 + structure_element + cleaner0 + 2023-07-20T08:47:40Z + SO: + + Fab + + + 0.9996767 + structure_element + cleaner0 + 2023-07-20T09:45:49Z + SO: + + C-terminal half + + + 0.9992643 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99960023 + complex_assembly + cleaner0 + 2023-07-20T08:46:48Z + GO: + + IL-17A/Fab + + + 0.99963623 + evidence + cleaner0 + 2023-07-20T08:48:11Z + DUMMY: + + crystal structure + + + 0.9992395 + experimental_method + cleaner0 + 2023-07-20T08:46:37Z + MESH: + + SPR assay + + + 0.9995968 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9997446 + structure_element + cleaner0 + 2023-07-20T08:47:40Z + SO: + + Fab + + + 0.9992985 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9995063 + evidence + cleaner0 + 2023-07-20T08:48:37Z + DUMMY: + + binding affinities + + + evidence + DUMMY: + cleaner0 + 2023-07-20T08:47:19Z + + kinetics + + + 0.9996674 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9997683 + structure_element + cleaner0 + 2023-07-20T08:47:40Z + SO: + + Fab + + + 0.9984326 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99895763 + evidence + cleaner0 + 2023-07-20T08:48:34Z + DUMMY: + + Crystals + + + 0.99862987 + complex_assembly + cleaner0 + 2023-07-20T08:47:06Z + GO: + + Fab/IL-17A/HAP + + + 0.9885818 + experimental_method + cleaner0 + 2023-07-20T09:35:48Z + MESH: + + crystallization screens + + + + RESULTS + paragraph + 15763 + Crystallization of IL-17A and its binding partners was accomplished using two forms of IL-17A. These were, respectively, a presumably more homogeneous form of IL-17A that lacked the disordered N-terminal peptide and a full-length form of the cytokine with a full complement of disulfide bonds. (see Method). Crystals of the Fab/truncated IL-17A/HAP complex diffracted to 2.2 Å, and the Fab/full length IL-17A/HAP complex diffracted to 3.0 Å (Supplementary Table S3). Both structures were solved by molecular replacement. Both complexes crystallized in the space group of P321, with half the complex (1 Fab/1 IL-17A monomer/1 HAP) in the asymmetric unit. The intact complex can be generated by applying crystallographic 2-fold symmetry. Electron densities for HAP residues Ile1-Asn14 were readily interpretable with the exception of Lys15, which is disordered. When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered. Cys106 is covalently linked to Cys10 that resides in the disordered N-terminal peptide in the full length IL-17A. + + 0.9996735 + experimental_method + cleaner0 + 2023-07-20T09:35:54Z + MESH: + + Crystallization + + + 0.99947786 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9995449 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9994836 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99967206 + protein_state + cleaner0 + 2023-07-20T09:55:41Z + DUMMY: + + lacked + + + 0.99911493 + protein_state + cleaner0 + 2023-07-20T08:50:12Z + DUMMY: + + disordered + + + 0.9991087 + structure_element + cleaner0 + 2023-07-20T09:45:53Z + SO: + + N-terminal peptide + + + 0.99956125 + protein_state + cleaner0 + 2023-07-20T09:55:47Z + DUMMY: + + full-length + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:50:50Z + + cytokine + + + 0.96609986 + ptm + cleaner0 + 2023-07-20T08:22:57Z + MESH: + + disulfide bonds + + + 0.99891126 + evidence + cleaner0 + 2023-07-20T09:41:38Z + DUMMY: + + Crystals + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:49:08Z + + Fab/truncated IL-17A/HAP + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:49:32Z + + Fab/full length IL-17A/HAP + + + 0.9788112 + evidence + cleaner0 + 2023-07-20T09:41:47Z + DUMMY: + + structures + + + 0.99959016 + experimental_method + cleaner0 + 2023-07-20T09:38:09Z + MESH: + + molecular replacement + + + 0.8246216 + experimental_method + cleaner0 + 2023-07-20T09:38:28Z + MESH: + + crystallized + + + 0.9996136 + structure_element + cleaner0 + 2023-07-20T08:47:40Z + SO: + + Fab + + + 0.99951524 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99906343 + oligomeric_state + cleaner0 + 2023-07-20T09:23:53Z + DUMMY: + + monomer + + + 0.8256031 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9996556 + protein_state + cleaner0 + 2023-07-20T09:56:06Z + DUMMY: + + intact + + + 0.99957204 + evidence + cleaner0 + 2023-07-20T09:41:51Z + DUMMY: + + Electron densities + + + 0.85543257 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9728047 + residue_range + cleaner0 + 2023-07-20T09:20:04Z + DUMMY: + + Ile1-Asn14 + + + 0.99989474 + residue_name_number + cleaner0 + 2023-07-20T08:52:46Z + DUMMY: + + Lys15 + + + 0.9995851 + protein_state + cleaner0 + 2023-07-20T08:50:12Z + DUMMY: + + disordered + + + evidence + DUMMY: + cleaner0 + 2023-07-20T09:42:18Z + + complex structure + + + 0.9996083 + protein_state + cleaner0 + 2023-07-20T08:49:57Z + DUMMY: + + full length + + + 0.99952394 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9996094 + protein_state + cleaner0 + 2023-07-20T08:50:17Z + DUMMY: + + truncated + + + 0.9994805 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9998896 + residue_name_number + cleaner0 + 2023-07-20T08:52:50Z + DUMMY: + + Cys106 + + + 0.9998876 + residue_name_number + cleaner0 + 2023-07-20T08:52:55Z + DUMMY: + + Ser106 + + + 0.9996117 + protein_state + cleaner0 + 2023-07-20T08:50:16Z + DUMMY: + + truncated + + + 0.9994609 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.999613 + protein_state + cleaner0 + 2023-07-20T08:50:12Z + DUMMY: + + disordered + + + 0.9998901 + residue_name_number + cleaner0 + 2023-07-20T08:52:51Z + DUMMY: + + Cys106 + + + 0.9998889 + residue_name_number + cleaner0 + 2023-07-20T08:53:02Z + DUMMY: + + Cys10 + + + 0.9996817 + protein_state + cleaner0 + 2023-07-20T08:50:12Z + DUMMY: + + disordered + + + 0.9992804 + structure_element + cleaner0 + 2023-07-20T09:45:56Z + SO: + + N-terminal peptide + + + 0.9996056 + protein_state + cleaner0 + 2023-07-20T08:49:57Z + DUMMY: + + full length + + + 0.9995246 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + + RESULTS + title_2 + 16959 + Overall structure of Fab/IL-17A/HAP complex + + 0.9995376 + evidence + cleaner0 + 2023-07-20T09:42:36Z + DUMMY: + + structure + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:47:06Z + + Fab/IL-17A/HAP + + + + RESULTS + paragraph + 17003 + In a similar manner to the published structure of Fab/IL-17A complex, two Fab molecules bind symmetrically to the C-terminal of the cytokine dimer, interacting with epitopes from both monomers (Fig. 2A). Two copies of HAP bind to the N-terminal of the cytokine dimer, also symmetrically, and each HAP molecule also interacts with both IL-17A monomers (Fig. 2). Based on disclosed epitopes of Secukinumab and Ixekizumab, HAP binds to IL-17A at an area that is also different from those of those two antibodies. The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. This β-strand is parallel to both strands 0 and 4 (Fig. 3B). Strands 0 of two IL-17A monomer are antiparallel, as appeared in other IL-17A structures. The C-terminal 8 residues of the HAP that are ordered in the structure, 7ADLWDWIN, form an amphipathic α-helix interacting with the second IL-17A monomer. Pro6 of HAP makes a transition between the N-terminal β-strand and the C-terminal α-helix of HAP. As a comparison, an IL-17A/IL-17RA complex structure (PDB code 4HSA) is also shown with IL-17A in the same orientation (Fig. 2C). + + 0.99806315 + evidence + cleaner0 + 2023-07-20T09:42:39Z + DUMMY: + + structure + + + 0.9996273 + complex_assembly + cleaner0 + 2023-07-20T08:51:15Z + GO: + + Fab/IL-17A + + + 0.9994886 + structure_element + cleaner0 + 2023-07-20T08:47:40Z + SO: + + Fab + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:50:51Z + + cytokine + + + 0.9993357 + oligomeric_state + cleaner0 + 2023-07-20T09:18:27Z + DUMMY: + + dimer + + + 0.9993024 + oligomeric_state + cleaner0 + 2023-07-20T08:23:13Z + DUMMY: + + monomers + + + 0.61343205 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:50:51Z + + cytokine + + + 0.99925727 + oligomeric_state + cleaner0 + 2023-07-20T09:18:27Z + DUMMY: + + dimer + + + 0.5626085 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99929076 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9993086 + oligomeric_state + cleaner0 + 2023-07-20T08:23:13Z + DUMMY: + + monomers + + + 0.99596775 + chemical + cleaner0 + 2023-07-20T08:21:19Z + CHEBI: + + Secukinumab + + + 0.9903983 + chemical + cleaner0 + 2023-07-20T08:21:23Z + CHEBI: + + Ixekizumab + + + 0.88919455 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99936366 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9986572 + protein_type + cleaner0 + 2023-07-20T09:25:45Z + MESH: + + antibodies + + + 0.99149716 + residue_range + cleaner0 + 2023-07-20T09:26:46Z + DUMMY: + + 5 residues + + + 0.55224633 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:52:18Z + + 1IHVTI + + + 0.7852521 + protein_state + cleaner0 + 2023-07-20T08:52:30Z + DUMMY: + + amphipathic + + + 0.99962467 + structure_element + cleaner0 + 2023-07-20T09:46:01Z + SO: + + β-strand + + + 0.99964887 + structure_element + cleaner0 + 2023-07-20T09:46:05Z + SO: + + β-strand 4 + + + 0.9992595 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9992736 + oligomeric_state + cleaner0 + 2023-07-20T09:23:53Z + DUMMY: + + monomer + + + 0.99960977 + structure_element + cleaner0 + 2023-07-20T08:55:11Z + SO: + + β-strand 0 + + + 0.9992409 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99922407 + oligomeric_state + cleaner0 + 2023-07-20T09:23:53Z + DUMMY: + + monomer + + + 0.999587 + structure_element + cleaner0 + 2023-07-20T09:46:09Z + SO: + + β-strand + + + 0.99940616 + structure_element + cleaner0 + 2023-07-20T09:46:12Z + SO: + + strands 0 and 4 + + + 0.99930954 + structure_element + cleaner0 + 2023-07-20T09:46:16Z + SO: + + Strands 0 + + + 0.99928087 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9992731 + oligomeric_state + cleaner0 + 2023-07-20T09:23:53Z + DUMMY: + + monomer + + + 0.9980819 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99938834 + evidence + cleaner0 + 2023-07-20T09:42:42Z + DUMMY: + + structures + + + 0.98815715 + residue_range + cleaner0 + 2023-07-20T09:26:50Z + DUMMY: + + 8 residues + + + 0.7171196 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9983701 + evidence + cleaner0 + 2023-07-20T09:42:46Z + DUMMY: + + structure + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:52:00Z + + 7ADLWDWIN + + + 0.91809016 + protein_state + cleaner0 + 2023-07-20T08:52:31Z + DUMMY: + + amphipathic + + + 0.9996877 + structure_element + cleaner0 + 2023-07-20T09:46:19Z + SO: + + α-helix + + + 0.9993891 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99920493 + oligomeric_state + cleaner0 + 2023-07-20T09:23:53Z + DUMMY: + + monomer + + + 0.9998714 + residue_name_number + cleaner0 + 2023-07-20T08:53:09Z + DUMMY: + + Pro6 + + + 0.58119506 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9996755 + structure_element + cleaner0 + 2023-07-20T09:46:22Z + SO: + + β-strand + + + 0.9996958 + structure_element + cleaner0 + 2023-07-20T09:46:25Z + SO: + + α-helix + + + 0.54212296 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99954754 + complex_assembly + cleaner0 + 2023-07-20T08:28:05Z + GO: + + IL-17A/IL-17RA + + + evidence + DUMMY: + cleaner0 + 2023-07-20T09:42:22Z + + complex structure + + + 0.99945027 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + + RESULTS + title_2 + 18271 + Inhibition mechanism of IL-17A signaling by HAP + + 0.97919995 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9996896 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + + RESULTS + paragraph + 18319 + IL-17RA binds IL-17A at three regions on the IL-17A homodimer. HAP binds IL-17A at region I. Region I is formed by residues at the ends of β strands 0 and 4, and from loops 1–2 and 3–4 of IL-17A (Fig. 2). Conformational changes in region I induced by HAP binding alone may allosterically affect IL-17RA binding, but more importantly, the α-helix of HAP directly competes with IL-17RA for binding to IL-17A (Fig. 3). The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). The Trp12 side chain of HAP donates a hydrogen bond to the main chain oxygen of Pro69 of IL-17A. The positively charged Arg101 side chain of the IL-17A engages in a charge-helix dipole interaction with the main chain oxygen of Trp12. Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. + + 0.99949485 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99935526 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99940854 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99936646 + oligomeric_state + cleaner0 + 2023-07-20T09:24:11Z + DUMMY: + + homodimer + + + 0.99929655 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9995146 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9996282 + structure_element + cleaner0 + 2023-07-20T08:54:15Z + SO: + + region I + + + 0.99964935 + structure_element + cleaner0 + 2023-07-20T08:54:15Z + SO: + + Region I + + + 0.999601 + structure_element + cleaner0 + 2023-07-20T09:46:28Z + SO: + + β strands 0 and 4 + + + 0.99961 + structure_element + cleaner0 + 2023-07-20T09:46:33Z + SO: + + loops 1–2 + + + 0.999551 + structure_element + cleaner0 + 2023-07-20T09:46:35Z + SO: + + 3–4 + + + 0.99930185 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9996196 + structure_element + cleaner0 + 2023-07-20T08:54:15Z + SO: + + region I + + + 0.99903524 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99943143 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99970704 + structure_element + cleaner0 + 2023-07-20T09:46:39Z + SO: + + α-helix + + + 0.9990969 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9995093 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99942046 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9997335 + structure_element + cleaner0 + 2023-07-20T09:46:42Z + SO: + + α helix + + + 0.9985568 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9992655 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9999037 + residue_name_number + cleaner0 + 2023-07-20T08:53:16Z + DUMMY: + + Trp12 + + + 0.9994375 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99956673 + site + cleaner0 + 2023-07-20T09:39:43Z + SO: + + hydrophobic pocket + + + 0.9994507 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99990094 + residue_name_number + cleaner0 + 2023-07-20T08:53:21Z + DUMMY: + + Phe110 + + + 0.9999037 + residue_name_number + cleaner0 + 2023-07-20T08:53:25Z + DUMMY: + + Tyr62 + + + 0.9999033 + residue_name_number + cleaner0 + 2023-07-20T08:53:30Z + DUMMY: + + Pro59 + + + 0.99990165 + residue_name_number + cleaner0 + 2023-07-20T08:53:35Z + DUMMY: + + Arg101 + + + 0.9998971 + residue_name_number + cleaner0 + 2023-07-20T08:53:17Z + DUMMY: + + Trp12 + + + 0.9993279 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:31Z + + hydrogen bond + + + 0.9999037 + residue_name_number + cleaner0 + 2023-07-20T08:53:42Z + DUMMY: + + Pro69 + + + 0.99942374 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99989986 + residue_name_number + cleaner0 + 2023-07-20T08:53:36Z + DUMMY: + + Arg101 + + + 0.9994616 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:31Z + + charge-helix dipole interaction + + + 0.99990106 + residue_name_number + cleaner0 + 2023-07-20T08:53:17Z + DUMMY: + + Trp12 + + + 0.99990165 + residue_name_number + cleaner0 + 2023-07-20T08:53:50Z + DUMMY: + + Leu9 + + + 0.9999014 + residue_name_number + cleaner0 + 2023-07-20T08:53:55Z + DUMMY: + + Ile13 + + + 0.9982498 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:31Z + + hydrophobic interactions + + + 0.99939984 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9999045 + residue_name_number + cleaner0 + 2023-07-20T08:54:00Z + DUMMY: + + Asp8 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:31Z + + hydrogen bond + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:31Z + + ion pair interactions + + + 0.99990094 + residue_name_number + cleaner0 + 2023-07-20T08:53:26Z + DUMMY: + + Tyr62 + + + 0.99989843 + residue_name_number + cleaner0 + 2023-07-20T08:54:06Z + DUMMY: + + Lys114 + + + 0.99948734 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + + RESULTS + paragraph + 19432 + In region I, an IL-17RA peptide interacts with IL-17A in a very similar fashion to the α-helix of HAP. The IL-17RA peptide has sequences of 27LDDSWI, and part of the peptide is also α-helical (Fig. 3B). Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B). In this sense, the α-helix of HAP with a sequence of 9LWDWI is a good mimetic of the 27LDDSWI peptide of IL-17RA. + + 0.9996127 + structure_element + cleaner0 + 2023-07-20T08:54:15Z + SO: + + region I + + + 0.99875784 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.9995494 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99970263 + structure_element + cleaner0 + 2023-07-20T09:46:49Z + SO: + + α-helix + + + 0.9991284 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9990926 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.7019358 + chemical + cleaner0 + 2023-07-20T08:54:33Z + CHEBI: + + 27LDDSWI + + + 0.95826167 + structure_element + cleaner0 + 2023-07-20T09:46:54Z + SO: + + α-helical + + + 0.99990034 + residue_name_number + cleaner0 + 2023-07-20T08:55:19Z + DUMMY: + + Leu7 + + + 0.9999012 + residue_name_number + cleaner0 + 2023-07-20T08:55:24Z + DUMMY: + + Trp31 + + + 0.9999027 + residue_name_number + cleaner0 + 2023-07-20T08:55:28Z + DUMMY: + + Ile32 + + + 0.99933296 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.999542 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.99989986 + residue_name_number + cleaner0 + 2023-07-20T08:53:51Z + DUMMY: + + Leu9 + + + 0.9999027 + residue_name_number + cleaner0 + 2023-07-20T08:53:17Z + DUMMY: + + Trp12 + + + 0.9999013 + residue_name_number + cleaner0 + 2023-07-20T08:53:56Z + DUMMY: + + Ile13 + + + 0.99930537 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9996958 + structure_element + cleaner0 + 2023-07-20T09:46:58Z + SO: + + α-helix + + + 0.99933165 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9877137 + chemical + cleaner0 + 2023-07-20T08:54:46Z + CHEBI: + + 9LWDWI + + + 0.28522155 + chemical + cleaner0 + 2023-07-20T08:54:33Z + CHEBI: + + 27LDDSWI + + + 0.9992437 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + + RESULTS + paragraph + 19894 + The β-strand of HAP has no equivalent in IL-17RA. However, it mimics the β-strand 0 of IL-17A. The amphipathic β-strand of HAP orients the hydrophilic side chains of His2 and Thr4 outwards, and the hydrophobic side chains of Ile1, Val3 and Ile5 inward (Fig. 3A). β-strand 0 in IL-17A is also amphipathic with the sequence of 21TVMVNLNI. In all IL-17A structures obtained to date, β-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward. + + 0.999598 + structure_element + cleaner0 + 2023-07-20T09:47:04Z + SO: + + β-strand + + + 0.99895287 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99928015 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99962753 + structure_element + cleaner0 + 2023-07-20T08:55:10Z + SO: + + β-strand 0 + + + 0.99929506 + protein + cleaner0 + 2023-07-20T08:16:51Z + PR: + + IL-17A + + + 0.9992919 + protein_state + cleaner0 + 2023-07-20T08:52:31Z + DUMMY: + + amphipathic + + + 0.99960834 + structure_element + cleaner0 + 2023-07-20T09:47:02Z + SO: + + β-strand + + + 0.9991375 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9998934 + residue_name_number + cleaner0 + 2023-07-20T08:55:43Z + DUMMY: + + His2 + + + 0.999887 + residue_name_number + cleaner0 + 2023-07-20T08:55:47Z + DUMMY: + + Thr4 + + + 0.99989235 + residue_name_number + cleaner0 + 2023-07-20T08:55:52Z + DUMMY: + + Ile1 + + + 0.999892 + residue_name_number + cleaner0 + 2023-07-20T08:55:56Z + DUMMY: + + Val3 + + + 0.9998919 + residue_name_number + cleaner0 + 2023-07-20T08:56:01Z + DUMMY: + + Ile5 + + + 0.9996271 + structure_element + cleaner0 + 2023-07-20T08:55:11Z + SO: + + β-strand 0 + + + 0.9993189 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.99940896 + protein_state + cleaner0 + 2023-07-20T08:52:31Z + DUMMY: + + amphipathic + + + 0.9022796 + chemical + cleaner0 + 2023-07-20T08:54:59Z + CHEBI: + + 21TVMVNLNI + + + 0.99885064 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9995154 + evidence + cleaner0 + 2023-07-20T09:42:53Z + DUMMY: + + structures + + + 0.9996283 + structure_element + cleaner0 + 2023-07-20T08:55:11Z + SO: + + β-strand 0 + + + 0.9998964 + residue_name_number + cleaner0 + 2023-07-20T08:56:08Z + DUMMY: + + Thr21 + + + 0.99989736 + residue_name_number + cleaner0 + 2023-07-20T08:56:12Z + DUMMY: + + Asn25 + + + 0.99989593 + residue_name_number + cleaner0 + 2023-07-20T08:56:16Z + DUMMY: + + Asn27 + + + 0.99989736 + residue_name_number + cleaner0 + 2023-07-20T08:56:21Z + DUMMY: + + Val22 + + + 0.99989736 + residue_name_number + cleaner0 + 2023-07-20T08:56:25Z + DUMMY: + + Val24 + + + 0.99989676 + residue_name_number + cleaner0 + 2023-07-20T08:56:29Z + DUMMY: + + Leu26 + + + 0.9998976 + residue_name_number + cleaner0 + 2023-07-20T08:56:34Z + DUMMY: + + Ile28 + + + + RESULTS + paragraph + 20444 + The binding pocket occupied by either Trp12 of HAP or Trp31 of IL-17RA is not formed in the apo IL-17A structure (Fig. 3C). Conformational changes of IL-17A are needed for both HAP and IL-17RA to bind to that region. Particularly for HAP, β-strands 0 have to shift out of the hydrophobic cleft formed by the main body of the IL-17A by as much as 10 Å between Cα atoms (Fig. 3C). Disruptions of the apo IL-17A structure by HAP binding are apparently compensated for by formation of the new interactions that involve almost the entire HAP molecule (Fig. 3B). + + 0.99961424 + site + cleaner0 + 2023-07-20T09:39:47Z + SO: + + binding pocket + + + 0.99989605 + residue_name_number + cleaner0 + 2023-07-20T08:53:17Z + DUMMY: + + Trp12 + + + 0.9997621 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99989736 + residue_name_number + cleaner0 + 2023-07-20T08:55:25Z + DUMMY: + + Trp31 + + + 0.99956274 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99963915 + protein_state + cleaner0 + 2023-07-20T08:21:59Z + DUMMY: + + apo + + + 0.99955493 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9995809 + evidence + cleaner0 + 2023-07-20T09:42:58Z + DUMMY: + + structure + + + 0.9994552 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9997068 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99957436 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99975723 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.9995387 + structure_element + cleaner0 + 2023-07-20T09:47:12Z + SO: + + β-strands 0 + + + 0.99959135 + site + cleaner0 + 2023-07-20T09:39:50Z + SO: + + hydrophobic cleft + + + 0.99966615 + structure_element + cleaner0 + 2023-07-20T09:47:17Z + SO: + + main body + + + 0.99951905 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9996426 + protein_state + cleaner0 + 2023-07-20T08:21:59Z + DUMMY: + + apo + + + 0.99947864 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.99891293 + evidence + cleaner0 + 2023-07-20T09:43:00Z + DUMMY: + + structure + + + 0.99960774 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99975985 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + + RESULTS + title_2 + 21008 + Structure basis for the observed SAR of peptides + + experimental_method + MESH: + cleaner0 + 2023-07-20T09:04:27Z + + SAR + + + + RESULTS + paragraph + 21057 + The IL-17A/HAP complex structure obtained is very consistent with the observed SAR of our identified peptide inhibitors, explaining well how the evolution of the initial phage peptide 1 to HAP and 45 improved its potency (Supplementary Figure S7). The important interactions involving Trp12 of HAP explain the >90 times drop in potency of the W12A variant (6 vs 1, Table 1). The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). The C-terminal Asn14 and Lys15 of HAP are not directly involved in interactions with IL-17A, and this is reflected in the gradual reduction in activity caused by C-terminal truncations (35 and 36, Table 2). Each peptide monomer in 45 may not necessarily be more potent than HAP, but two monomer peptides within the same molecule that can simultaneously bind to IL-17A can greatly improve its potency due to avidity effects. + + 0.9996457 + complex_assembly + cleaner0 + 2023-07-20T08:46:26Z + GO: + + IL-17A/HAP + + + evidence + DUMMY: + cleaner0 + 2023-07-20T09:42:22Z + + complex structure + + + experimental_method + MESH: + cleaner0 + 2023-07-20T09:04:27Z + + SAR + + + experimental_method + MESH: + cleaner0 + 2023-07-20T09:01:14Z + + phage + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:38:10Z + + peptide 1 + + + 0.98668355 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.98723143 + chemical + cleaner0 + 2023-07-20T09:58:35Z + CHEBI: + + 45 + + + 0.99989724 + residue_name_number + cleaner0 + 2023-07-20T08:53:17Z + DUMMY: + + Trp12 + + + 0.67716074 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99962044 + mutant + cleaner0 + 2023-07-20T09:02:54Z + MESH: + + W12A + + + 0.9936667 + protein_state + cleaner0 + 2023-07-20T08:52:31Z + DUMMY: + + amphipathic + + + 0.68529487 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99964666 + structure_element + cleaner0 + 2023-07-20T09:47:22Z + SO: + + β-strand + + + 0.9873102 + residue_number + cleaner0 + 2023-07-20T09:28:56Z + DUMMY: + + 2 + + + 0.9828635 + residue_number + cleaner0 + 2023-07-20T09:28:59Z + DUMMY: + + 4 + + + 0.41763604 + chemical + cleaner0 + 2023-07-20T09:58:41Z + CHEBI: + + 14 + + + 0.40545538 + chemical + cleaner0 + 2023-07-20T09:58:44Z + CHEBI: + + 18 + + + 0.3360236 + chemical + cleaner0 + 2023-07-20T09:58:47Z + CHEBI: + + 19 + + + 0.3754059 + chemical + cleaner0 + 2023-07-20T09:58:49Z + CHEBI: + + 21 + + + 0.3401372 + chemical + cleaner0 + 2023-07-20T09:58:52Z + CHEBI: + + 23 + + + chemical + CHEBI: + cleaner0 + 2023-07-20T09:01:41Z + + 1 + + + chemical + CHEBI: + cleaner0 + 2023-07-20T09:01:51Z + + 22 + + + 0.47717986 + chemical + cleaner0 + 2023-07-20T08:18:36Z + CHEBI: + + HAP + + + 0.99965686 + structure_element + cleaner0 + 2023-07-20T09:47:25Z + SO: + + β-sheet + + + 0.99937516 + structure_element + cleaner0 + 2023-07-20T09:47:28Z + SO: + + β-stands 0 and 4 + + + 0.99845576 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.95655215 + experimental_method + cleaner0 + 2023-07-20T09:38:42Z + MESH: + + removal of + + + residue_range + DUMMY: + cleaner0 + 2023-07-20T09:27:17Z + + first 1–3 residues + + + 0.5255684 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + protein + PR: + cleaner0 + 2023-07-20T08:16:52Z + + IL-17A + + + chemical + CHEBI: + cleaner0 + 2023-07-20T08:35:17Z + + 31 + + + chemical + CHEBI: + cleaner0 + 2023-07-20T09:02:03Z + + 32 + + + chemical + CHEBI: + cleaner0 + 2023-07-20T09:02:12Z + + 33 + + + 0.99989414 + residue_name_number + cleaner0 + 2023-07-20T09:21:02Z + DUMMY: + + Asn14 + + + 0.9998921 + residue_name_number + cleaner0 + 2023-07-20T08:52:46Z + DUMMY: + + Lys15 + + + 0.7684114 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.99879616 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.5781626 + experimental_method + cleaner0 + 2023-07-20T09:38:48Z + MESH: + + truncations + + + chemical + CHEBI: + cleaner0 + 2023-07-20T09:02:24Z + + 35 + + + chemical + CHEBI: + cleaner0 + 2023-07-20T09:02:35Z + + 36 + + + 0.9993181 + oligomeric_state + cleaner0 + 2023-07-20T09:23:53Z + DUMMY: + + monomer + + + 0.96388507 + chemical + cleaner0 + 2023-07-20T09:58:56Z + CHEBI: + + 45 + + + 0.5796342 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.99935955 + oligomeric_state + cleaner0 + 2023-07-20T09:23:53Z + DUMMY: + + monomer + + + 0.9987038 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + + RESULTS + paragraph + 22280 + HAP targets region I of IL-17A, an area that has the least sequence conservation in IL-17 cytokines. This lack of sequence conservation in the HAP binding site explains the observed specificity of HAP binding to human IL-17A. For example, inspection of the published IL-17F crystal structure (PDB code 1JPY) revealed a pocket of IL-17F similar to that of IL-17A for W12 of HAP binding, but it is occupied by a Phe-Phe motif at the N-terminal peptide of IL-17F. This Phe-Phe motif is missing in IL-17A. Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal β-strand of HAP. In human IL-17A the sequences are 21TVMVNLNI, and in mouse they are 21NVKVNLKV. + + 0.9994549 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.99918175 + structure_element + cleaner0 + 2023-07-20T08:54:15Z + SO: + + region I + + + 0.99885875 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9995997 + protein_type + cleaner0 + 2023-07-20T08:20:46Z + MESH: + + IL-17 cytokines + + + 0.9996042 + site + cleaner0 + 2023-07-20T09:03:35Z + SO: + + HAP binding site + + + 0.9994111 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9990902 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.9993593 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9993753 + protein + cleaner0 + 2023-07-20T09:24:38Z + PR: + + IL-17F + + + 0.99959993 + evidence + cleaner0 + 2023-07-20T09:43:11Z + DUMMY: + + crystal structure + + + 0.99950004 + site + cleaner0 + 2023-07-20T09:39:56Z + SO: + + pocket + + + 0.99945974 + protein + cleaner0 + 2023-07-20T09:24:38Z + PR: + + IL-17F + + + 0.99938995 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9996131 + residue_name_number + cleaner0 + 2023-07-20T09:21:05Z + DUMMY: + + W12 + + + 0.9995919 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9995097 + structure_element + cleaner0 + 2023-07-20T09:03:47Z + SO: + + Phe-Phe motif + + + 0.9994707 + protein + cleaner0 + 2023-07-20T09:24:38Z + PR: + + IL-17F + + + 0.99954486 + structure_element + cleaner0 + 2023-07-20T09:03:48Z + SO: + + Phe-Phe motif + + + 0.99655885 + protein_state + cleaner0 + 2023-07-20T09:56:43Z + DUMMY: + + missing + + + 0.99916726 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.99954736 + experimental_method + cleaner0 + 2023-07-20T09:38:53Z + MESH: + + Sequence alignments + + + 0.9992693 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.9207432 + taxonomy_domain + cleaner0 + 2023-07-20T08:42:57Z + DUMMY: + + mouse + + + 0.9992469 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9975853 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9996598 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9996507 + structure_element + cleaner0 + 2023-07-20T09:47:34Z + SO: + + strand 0 + + + 0.9992854 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.99962634 + structure_element + cleaner0 + 2023-07-20T09:47:37Z + SO: + + β-strand + + + 0.9995598 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.99916685 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.99937075 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.6848422 + chemical + cleaner0 + 2023-07-20T08:55:01Z + CHEBI: + + 21TVMVNLNI + + + 0.99622476 + taxonomy_domain + cleaner0 + 2023-07-20T08:42:58Z + DUMMY: + + mouse + + + 0.67692673 + chemical + cleaner0 + 2023-07-20T09:03:58Z + CHEBI: + + 21NVKVNLKV + + + + DISCUSS + title_1 + 23082 + Discussion + + + DISCUSS + paragraph + 23093 + Using a combination of phage display and SAR we have discovered novel peptides that are IL-17A antagonists. One of those peptides, HAP, also shows activity in inhibiting the production of multiple inflammatory cytokines by primary human keratinocytes stimulated by IL-17A and TNF-α, a disease relevant-model. We have also determined the complex structure of IL-17A/HAP, which provides the structural basis for HAP’s antagonism to IL-17A signaling. + + 0.99951345 + experimental_method + cleaner0 + 2023-07-20T09:04:21Z + MESH: + + phage display + + + 0.99884343 + experimental_method + cleaner0 + 2023-07-20T09:04:27Z + MESH: + + SAR + + + 0.94560504 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9995239 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:45:34Z + + cytokines + + + 0.99935263 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.9938674 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.99192315 + protein + cleaner0 + 2023-07-20T08:44:23Z + PR: + + TNF-α + + + 0.9757987 + experimental_method + cleaner0 + 2023-07-20T09:39:01Z + MESH: + + determined + + + evidence + DUMMY: + cleaner0 + 2023-07-20T09:42:22Z + + complex structure + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:46:26Z + + IL-17A/HAP + + + 0.999521 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9747037 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + + DISCUSS + paragraph + 23545 + During IL-17A signaling, IL-17A binds to one copy of IL-17RA and one copy of IL-17RC. Since apo IL-17A is a homodimer with 2 fold symmetry, IL-17RA potentially can bind to either face of the IL-17A dimer. With two HAP molecules covering both faces of the IL-17A dimer, HAP can block IL-17RA approaching from either face. To form the 1:2 complex observed in crystal structure, it is important that there is no strong negative cooperativity in the binding of two HAP molecules. In fact, in native electrospray ionization mass spectrometry analysis only 1:2 IL-17A/HAP complex was observed even when IL-17A was in excess (Supplementary Figure S8), indicating a positive binding cooperativity that favors inhibition of IL-17RA binding by HAP. + + 0.99425274 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.99880075 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.99937516 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99941367 + protein + cleaner0 + 2023-07-20T08:20:07Z + PR: + + IL-17RC + + + 0.9996289 + protein_state + cleaner0 + 2023-07-20T08:21:59Z + DUMMY: + + apo + + + 0.99945265 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.99932516 + oligomeric_state + cleaner0 + 2023-07-20T09:24:11Z + DUMMY: + + homodimer + + + 0.9992736 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99930763 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.999328 + oligomeric_state + cleaner0 + 2023-07-20T09:18:27Z + DUMMY: + + dimer + + + 0.99969614 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.999307 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9993388 + oligomeric_state + cleaner0 + 2023-07-20T09:18:27Z + DUMMY: + + dimer + + + 0.9996923 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9994337 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.9996326 + evidence + cleaner0 + 2023-07-20T09:43:22Z + DUMMY: + + crystal structure + + + 0.99968565 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9995424 + experimental_method + cleaner0 + 2023-07-20T09:39:09Z + MESH: + + native electrospray ionization mass spectrometry + + + 0.9995264 + complex_assembly + cleaner0 + 2023-07-20T08:46:26Z + GO: + + IL-17A/HAP + + + 0.99953824 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9984725 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.9996768 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + + DISCUSS + paragraph + 24284 + HAP, with only 15 residues, can achieve almost the same binding affinity as the much larger IL-17RA molecule, indicating a more efficient way of binding to IL-17A. The interaction of IL-17A with IL-17RA has an extensive interface, covering ~2,200 Å2 surface area of IL-17A. Due to the discontinuous nature of the IL-17A/IL-17RA binding interface, it is classified as having tertiary structural epitopes on both binding partners, and is therefore hard to target using small molecules. Our studies of HAP demonstrated an uncommon mode of action for a peptide in inhibiting such a difficult protein-protein interaction target, and suggest further possible improvements in its binding potency. + + 0.99971944 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.93260455 + residue_range + cleaner0 + 2023-07-20T09:27:23Z + DUMMY: + + 15 residues + + + 0.9994731 + evidence + cleaner0 + 2023-07-20T09:43:25Z + DUMMY: + + binding affinity + + + 0.9992774 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.9994075 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9994039 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.99942493 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.9994136 + site + cleaner0 + 2023-07-20T09:40:05Z + SO: + + interface + + + 0.9994745 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9989624 + site + cleaner0 + 2023-07-20T09:05:20Z + SO: + + IL-17A/IL-17RA binding interface + + + 0.99971825 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + + DISCUSS + paragraph + 24977 + One way of further improving HAP’s potency is by dimerization. Homo-dimerization of HAP (45) achieved sub-nanomolar potency against human IL-17A in cell assay. In the crystal structure, the distance between the carbonyl of Asn14 of one HAP molecule and the N-terminus of the second is only 15.7 Å, suggesting the potential for more potent dimeric peptides to be designed by using linkers of different lengths at different positions. + + 0.9989919 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9997008 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + chemical + CHEBI: + cleaner0 + 2023-07-20T09:05:36Z + + 45 + + + 0.9993666 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.8267777 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9995927 + evidence + cleaner0 + 2023-07-20T09:43:30Z + DUMMY: + + crystal structure + + + 0.9998982 + residue_name_number + cleaner0 + 2023-07-20T09:21:10Z + DUMMY: + + Asn14 + + + 0.99961996 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9993316 + oligomeric_state + cleaner0 + 2023-07-20T08:33:39Z + DUMMY: + + dimeric + + + 0.99874264 + chemical + cleaner0 + 2023-07-20T09:59:17Z + CHEBI: + + peptides + + + + DISCUSS + paragraph + 25415 + Another direction of improving HAP is by reducing its size. As demonstrated by the crystal structure, binding of the α-helix of HAP should be sufficient for preventing IL-17RA binding to IL-17A. Theoretically, it is possible to design chemicals such as stapled α-helical peptides to block α-helix-mediated IL-17A/IL-17RA interactions. Such peptides may have smaller sizes with more favorable physical properties (proteolytic resistance, serum half-life, permeability, etc). + + 0.9902175 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9996195 + evidence + cleaner0 + 2023-07-20T09:43:34Z + DUMMY: + + crystal structure + + + 0.99971706 + structure_element + cleaner0 + 2023-07-20T09:47:54Z + SO: + + α-helix + + + 0.9993011 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9988205 + protein + cleaner0 + 2023-07-20T08:17:00Z + PR: + + IL-17RA + + + 0.99886304 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.9997104 + structure_element + cleaner0 + 2023-07-20T09:47:57Z + SO: + + α-helix + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:28:05Z + + IL-17A/IL-17RA + + + + DISCUSS + paragraph + 25901 + In summary, these peptide-based anti-IL-17A modalities could be further developed as alternative therapeutic options to the reported monoclonal antibodies. We are also very interested in finding non-peptidic small molecule IL-17A antagonists, and HAP can be used as an excellent tool peptide. The strategy utilized in generating the complex structures of HAP may also be useful for enabling structure based design of some known small molecule IL-17A antagonists. + + 0.8363242 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.92506737 + protein_type + cleaner0 + 2023-07-20T09:25:45Z + MESH: + + antibodies + + + 0.9988014 + protein + cleaner0 + 2023-07-20T08:16:52Z + PR: + + IL-17A + + + 0.99968266 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9987901 + evidence + cleaner0 + 2023-07-20T09:43:37Z + DUMMY: + + structures + + + 0.9997285 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + + METHODS + title_1 + 26364 + Methods + + + METHODS + title_2 + 26372 + Peptide synthesis + + + METHODS + paragraph + 26390 + Peptides were synthesized either on a PTI Symphony or Biotage Syro II synthesizer employing standard Fmoc chemistry on Rink resin with N-terminal amine capped with acetyl group unless noted otherwise. Resin, amino acids and solvents were purchased from EMD Chemicals. Peptides were then removed from polymer support using the cleavage cocktail (88:5:5:2, TFA:PhOH:H2O:TIPS) and precipitated with cold diethyl ether. For the dimers, the native peptides incorporating a primary amine (N-terminal amine or lysine at specific positions in the peptide sequence) were mixed with the bis-pentafluoroesters of the desired PEG (purchased from Quanta BioDesign) in the presence of DIEA in DMF. Peptides were purified by reversed phase HPLC on Phenomenex preparative Luna C18 columns using water:acetonitrile gradients and lyophilized. + + + METHODS + title_2 + 27215 + HPLC-MS conditions for peptide purity analysis + + + METHODS + paragraph + 27262 + All peptides were assessed for purity by analytical C18 RP-HP-LCMS prior to use in biological assays. Buffers used were 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B). The standard method (1) consisted of a linear gradient of 5% to 95% B over 10 minutes on Agilent 1100 series HPLC-MSD and method (2) consisted of a linear gradient of 5% to 95% B over 8 minutes on Waters 2767 series HPLC-MSD. The C18 column (Phenomenex, Luna C18, 4.6 × 150mm) effluent was immediately mass analyzed in electrospray positive mode. Accurate mass measurements of final peptides were performed using C18 reversed-phase chromatography mass spectrometry (RPHPLCMS) and mass detected on a Waters Synapt G2 Q-Tof mass spectrometer tuned to a resolution (FWHM) of 25,000. Exact intact masses were calculated based on the monoisotopic m/z value of the base peak charge state. All peptides were analyzed using these methods. Supporting data of peptide 45 is shown (Supplementary Figure S2) as representative data sets of the all the molecules investigated (characterization of all peptides presented on Table 1S). + + + METHODS + title_2 + 28401 + ELISA assay + + + METHODS + paragraph + 28413 + rhIL-17 (#317-ILB), IL-17R (#2269-IL) were from R&D Systems. ELISA wash buffer (50-63-01) and TMB SureBlue Microwell Peroxidase substrate (#50-63-01 or #52-00-00) were from KPL. Half-well high-binding ELISA plates (#3690) and full-well plates (#9018) were from Costar. Superblock (#2011 or AAA500) was from ScyTek. No-Weigh NSH-PEG4-Biotin (21329) and Zeba Desalt Spin Columns (89882) were from Pierce. + + + METHODS + paragraph + 28816 + ELISAs were as follows in duplicate unless otherwise specified. Plates were coated in PBS overnight at 4 °C and blocked using Superblock for one hour at RT. Subsequent incubations were for 1 hr at room temperature with dilutions in Superblock. Plates were washed 3X between steps using a Biotek ELx405 plate washer and developed using TMB substrate. Reactions were stopped in 2 M H2SO4 and OD450 values were read on a Molecular Devices SpectraMax Plus plate reader. + + + METHODS + paragraph + 29289 + IL-17 RA competition: half-well plates were coated with 0.5 μg/mL IL-17R-Fc and blocked. Peptides were titrated in Superblock containing 0.4 μg/mL Biotinylated IL-17A. Biotinylated IL-17A was detected by adding tetramethylbenzidine (TMB). Reactions were stopped in 2 M H2SO4 and OD450 values were read on a Molecular Devices SpectraMax Plus plate reader. + + + METHODS + title_2 + 29652 + Human BJ fibroblast and murine MLE-12 cell assays for inhibitor screen + + + METHODS + paragraph + 29723 + For inhibitor screen, human BJ fibroblast cells (ATCC CRL2522) (American Type Culture Collection, VA) were used. For mouse cell based assay MLE-12 mouse epithelial cells (ATCC CRL2110) were used. Both cell lines were maintained in ATCC recommended media. Cells were seeded at 5 × 103 cells/well into 96-well flat-bottom microtiter plates in which peptides that had been pre-diluted with cytokines (1 ng/mL for human IL-17A or 15 ng/mL for mouse IL-17A) in culture medium. Cells were incubated at 37 °C for 16–24 hrs, and supernatants were collected and analyzed by ELISA for either human CXCL1/GRO-α (R&D Systems DY275) or mouse CXCL1/KC (R&D Systems DY453). + + + METHODS + title_2 + 30399 + Primary human keratinocytes cell assay + + + METHODS + paragraph + 30438 + Primary human keratinocytes were cultured in Epilife medium with EDGS (Life Technology, Cascade Biologics) following product instructions. 5 days after establishing the culture from frozen vials, cells were plated at 10,000/well (80 μl) in culture media in 384 well plate. 4 hours after plating the cells, 10 μl of 10X peptide stocks were added. Final DMSO concentration was 1%. Immediately after peptide addition, 10 μl of a mixture of recombinant human IL-17A (endotoxin Level <0.10 EU per 1 μg of the protein, E.coli expression, >97% pure judged by SDS/page, R&D System, Minneapolis, MN) 100 ng /ml (final) and TNF-α (Sigma-Aldrich, St Louis, MO) 10 ng/ ml (final), or TNF-α 10 ng/ml only were added to the cells. Cell assay plates were incubated for 48 hours at 37 °C in a tissue culture incubator before harvesting culture supernatants for analysis of IL-6, IL-8 and CCL-20 production using kits K211AKB-2, K211ANB-2, and K211BEB-2, respectively (Meso Scale Discovery, Rockville, MD). + + + METHODS + title_2 + 31455 + Protein production + + + METHODS + paragraph + 31474 + Details of non-commercial protein constructs used in this study and their purifications are in the Supplementary Material. + + + METHODS + title_2 + 31597 + SPR binding assays + + + METHODS + paragraph + 31616 + The IL-17 SPR binding assay was run on a Biacore 3000 SPR instrument (GE Healthcare). Biotinylated human IL-17A, or IL-17A/F heteromer, or IL-17F, or mouse IL-17A (Cell Signaling) was captured on a Biacore Streptavidin chip to achieve protein density of about 2500 to 3500 RUs on the surface. The SPR running buffer was 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.01% P20 with 3% DMSO. Peptide samples were injected at a flow rate of 50 μl/min for 180 seconds association and at least 600 seconds dissociation using a 2–3 fold dilution series. Multiple blank injections were run before and after each peptide series for references. The data were processed and analyzed with Scrubber 2.0 (BioLogical Software) and Biaeval software (GE Healthcare) to calculate binding constants and on and off rates. + + + METHODS + paragraph + 32416 + The IL-17A SPR competition assay was run on a BioRAD ProteON instrument (BioRAD Laboratories). IL-17RA-Fc fusion protein (R&D Systems) was captured on a GLM chip using the standard amine coupling reaction. The chip surface activated using a mixture of sulfo-NHS and EDC, was exposed to the receptor dissolved in acetate buffer pH 5.0 at 0.01 mg/ml concentration. The surface was deactivated using 1 M ethanolamine HCl. An adjacent flow cell on the chip was treated identically without the protein and was used as a reference surface for subsequent data analysis. Three-fold dilution series of peptides mixed with 5 nM IL-17A were injected at a flow rate of 50 μl/min for 120 seconds, followed by 300 seconds of dissociation. The receptor on the surface was regenerated using a 30 second injection of 3 M MgCl2. Observed signal between 180 and 200 seconds was averaged for each sample. Average response observed for multiple 5 nM IL-17A samples without any peptide was used as 100% signal for calculating % inhibition for each concentration of compound. The data were fit with Microcal Origin software (OriginLab, MA) to calculate IC50 for peptides. + + + METHODS + title_2 + 33578 + FRET Assay + + + METHODS + paragraph + 33589 + The FRET signal of a Eu3+-labeled IL-17A donor and an Alexa Fluor 647 labeled IL-17RA acceptor was measured to monitor the interaction of IL-17A and IL-17RA. Maximal FRET was observed when IL-17A was bound to IL-17RA and diminished FRET was observed when IL-17A was separated from IL-17RA. The excitation of the donor at a wavelength of 320 nm triggers fluorescence at 615 nM and this in turn serves to excite the acceptor, which then fluoresces at a wavelength of 665 nm. The fluorescence at both 615 nm and 665 nm were measured and the ratio of 665 nm/615 nm was used to monitor the IL-17A/IL-17RA binding. Final assay concentrations were 1 nM biotinylated IL-17A labeled with 0.67 nM Europium-Streptavidin (Invitrogen), 6 nM IL-17RA-Fc fusion protein (R&D Systems) labeled with 1 nM Alexa Fluor 647 antibody (BioLegand) in a buffer containing 10 mM HEPES pH 7.4, 150 mM NaCl, 0.02% BSA, and 0.01% Tween 20. Peptides were tested using a half log dilution series of 11 concentrations. The IL-17A was incubated with the Europium-Streptavidin to 1.5X final assay concentration for one hour at room temperature. Peptides were prepared at 50X final concentration in 100% DMSO and 300 nl were added to a 384-well white assay plate (Greiner). 10 μl of the Eu3+-labeled IL-17A was added to the peptides and incubated at room temperature for one hour. During this pre-incubation of peptide and IL-17A, IL-17RA was incubated with Alexa Fluor 647 antibody to 3X final assay concentration at room temperature for one hour and then 5 μl of the 3X Alexa Fluor 647 labeled IL-17RA was added to the assay for a total volume of 15.3 μl and a final DMSO concentration of 2%. The plates were covered and incubated at room temperature for 3 hours. The FRET signal of the IL-17A/IL-17RA interaction was measured using an EnVision Multilabel plate reader (PerkinElmer). The peptide data was converted into % inhibition, using 0% (no HAP) and 100% inhibition (100 nM HAP) as controls. A four parameter logistic nonlinear regression model using the percent inhibition at each concentration was used to calculate an IC50 for each peptide. + + + METHODS + title_2 + 35746 + Protein crystallization and data collection + + + METHODS + paragraph + 35790 + To crystallize Fab/IL-17A/HAP complex, 30 mM HAP in DMSO stock was added to the Fab/IL-17A complex to a final concentration of 1 mM. The complex was then screened for crystals using commercial screen kits using a sitting drop vapor diffusion format. Crystals of Fab/truncated IL-17A/HAP complex were obtained under condition of 0.02 mM CdCl2, 0.02 M MgCl2, 0.02 M NiCl2, 0.1 M NaOAc pH = 4.2–4.9, and 24–28% PEG MME 2000. Crystals of Fab/full length IL-17A covalent dimer/HAP complex were obtained under conditions of 0.1 M NaOAc, pH = 4.5 and 30% PEG MME 5000. Crystals were soaked briefly in cryo solutions of the mother liquor supplemented with 25% glycerol before flash cooled in liquid nitrogen. Crystal data sets were collected at APS IMCA 17ID beamline (Chicago, IL), processed with autoPROC. Data collection statistics are listed in Supplementary Table 2. + + + METHODS + title_2 + 36680 + Structure determination and refinement + + + METHODS + paragraph + 36719 + Fab/IL-17A/HAP complex structures were solved with molecular replacement method using the published FAN/IL-17A crystal structure (pdb code 2VXS), using program Phaser. Structure refinements was carried out using program Buster and manual model building using program COOT. Final refinement statistics are listed in Supplementary Table 2. + + + METHODS + title_1 + 37057 + Additional Information + + + METHODS + paragraph + 37080 + How to cite this article: Liu, S. et al. Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide. Sci. Rep. 6, 26071; doi: 10.1038/srep26071 (2016). + + + SUPPL + title_1 + 37246 + Supplementary Material + + + 556 + 567 + surname:Gaffen;given-names:S. L. + 10.1038/nri2586 + 19575028 + REF + Nat Rev Immunol + ref + 9 + 2009 + 37269 + Structure and signalling in the IL-17 receptor family + + + 479 + 489 + surname:Cua;given-names:D. J. + surname:Tato;given-names:C. M. + 20559326 + REF + Nat Rev Immunol + ref + 10 + 2010 + 37323 + Innate IL-17-producing cells: the sentinels of the immune system + + + 36 + 39 + surname:Toy;given-names:D. + 10.4049/jimmunol.177.1.36 + 16785495 + REF + J Immunol. + ref + 177 + 2006 + 37388 + Cutting Edge: Interleukin 17 Signals through a Heteromeric Receptor Complex + + + 311 + 321 + surname:Onishi;given-names:R. M. + surname:Gaffen;given-names:S. L. + 10.1111/j.1365-2567.2009.03240.x + 20409152 + REF + Immunology + ref + 129 + 2010 + 37464 + Interleukin-17 and its target genes: mechanisms of interleukin-17 function in disease + + + R120 + 128 + surname:Hwang;given-names:S. Y. + 10.1186/ar1038 + 15059275 + REF + Arthritis Res Ther + ref + 6 + 2004 + 37550 + IL-17 induces production of IL-6 and IL-8 in rheumatoid arthritis synovial fibroblasts via NF-kappaB- and PI3-kinase/Akt-dependent pathways + + + 2636 + 2649 + surname:Mills;given-names:K. H. + 10.1002/eji.200838535 + 18958872 + REF + Eur J Immunol + ref + 38 + 2008 + 37690 + Induction, function and regulation of IL-17-producing T cells + + + 549 + 553 + surname:van den Berg;given-names:W. B. + surname:Miossec;given-names:P. + 10.1038/nrrheum.2009.179 + 19798029 + REF + Nat Rev Rheumatol + ref + 5 + 2009 + 37752 + IL-17 as a future therapeutic target for rheumatoid arthritis + + + 291 + 297 + surname:Puel;given-names:A. + 10.1084/jem.20091983 + 20123958 + REF + J Exp Med. + ref + 207 + 2010 + 37814 + Autoantibodies against IL-17A, IL-17F, and IL-22 in patients with chronic mucocutaneous candidiasis and autoimmune polyendocrine syndrome type I + + + 65 + 68 + surname:Puel;given-names:A. + 10.1126/science.1200439 + 21350122 + REF + Science + ref + 332 + 2011 + 37959 + Chronic Mucocutaneous Candidiasis in Humans with Inborn Errors of Interleukin-17 Immunity + + + surname:Griffiths;given-names:C. E. M. + 10.1016/S0140-6736(15)60125-8 + REF + Lancet + ref + 2015 + 38049 + Comparison of ixekizumab with etanercept or placebo in moderate-to-severe psoriasis (UNCOVER-2 and UNCOVER-3): results from two phase 3 randomised trials + + + 326 + 338 + surname:Langley;given-names:R. G. + 10.1056/NEJMoa1314258 + 25007392 + REF + N Engl J Med. + ref + 371 + 2014 + 38203 + Secukinumab in Plaque Psoriasis — Results of Two Phase 3 Trials + + + 1137 + 1146 + surname:McInnes;given-names:I. B. + 10.1016/S0140-6736(15)61134-5 + 26135703 + REF + Lancet + ref + 386 + 2015 + 38269 + Secukinumab, a human anti-interleukin-17A monoclonal antibody, in patients with psoriatic arthritis (FUTURE 2): a randomised, double-blind, placebo-controlled, phase 3 trial + + + surname:Papp;given-names:K. + 10.1016/j.jaad.2014.08.039 + REF + J Am Acad Dermatol. + ref + 71 + 2014 + 38443 + Safety and efficacy of brodalumab for psoriasis after 120 weeks of treatment + + + REF + ref + 38520 + U.S. Food and Drug Administration. FDA approves new psoriasis drug Cosentyx. U.S. FDA press release (2015). + + + 1345 + 1352 + surname:Kotake;given-names:S. + 10.1172/JCI5703 + 10225978 + REF + J Clin Invest + ref + 103 + 1999 + 38628 + IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis + + + 963 + 970 + surname:Chabaud;given-names:M. + 10.1002/1529-0131(199905)42:5 + 10323452 + REF + Arthritis Rheum + ref + 42 + 1999 + 38738 + Human interleukin-17: A T cell-derived proinflammatory cytokine produced by the rheumatoid synovium + + + 2832 + 2838 + surname:Ziolkowska;given-names:M. + 10679127 + REF + J Immunol + ref + 164 + 2000 + 38838 + High levels of IL-17 in rheumatoid arthritis patients: IL-15 triggers in vitro IL-17 production via cyclosporin A-sensitive mechanism + + + R93 + surname:Shahrara;given-names:S. + surname:Huang;given-names:Q. + surname:Mandelin;given-names:A. M.;suffix:2nd + surname:Pope;given-names:R. M. + 10.1186/ar2477 + 18710567 + REF + Arthritis Res Ther + ref + 10 + 2008 + 38972 + TH-17 cells in rheumatoid arthritis + + + 1004 + 1013 + surname:Lubberts;given-names:E. + 11441109 + REF + J Immunol + ref + 167 + 2001 + 39008 + IL-1-independent role of IL-17 in synovial inflammation and joint destruction during collagen-induced arthritis + + + 650 + 659 + surname:Lubberts;given-names:E. + 10.1002/art.20001 + 14872510 + REF + Arthritis Rheum + ref + 50 + 2004 + 39120 + Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion + + + 2799 + 2805 + surname:Wright;given-names:J. F. + 18684971 + REF + J Immunol + ref + 181 + 2008 + 39300 + The human IL-17F/IL-17A heterodimeric cytokine signals through the IL-17RA/IL-17RC receptor complex + + + 5332 + 5341 + surname:Hymowitz;given-names:S. G. + 10.1093/emboj/20.19.5332 + 11574464 + REF + EMBO J. + ref + 20 + 2001 + 39400 + IL‐17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL‐17F, and implications for receptor binding + + + 1245 + 1251 + surname:Ely;given-names:L. K. + surname:Fischer;given-names:S. + surname:Garcia;given-names:K. C. + 10.1038/ni.1813 + 19838198 + REF + Nat. Immunol. + ref + 10 + 2009 + 39528 + Structural basis of receptor sharing by interleukin 17 cytokines + + + 1888 + surname:Liu;given-names:S. + 10.1038/ncomms2880 + 23695682 + REF + Nat Commun + ref + 4 + 2013 + 39593 + Crystal structures of interleukin 17A and its complex with IL-17 receptor A + + + 905 + 921 + surname:Gerhardt;given-names:S. + 10.1016/j.jmb.2009.10.008 + 19835883 + REF + J Mol Biol. + ref + 394 + 2009 + 39669 + Structure of IL-17A in Complex with a Potent, Fully Human Neutralizing Antibody + + + 4299 + 4310 + surname:Rickel;given-names:E. A. + 10.4049/jimmunol.181.6.4299 + 18768888 + REF + J Immunol. + ref + 181 + 2008 + 39749 + Identification of Functional Roles for Both IL-17RB and IL-17RA in Mediating IL-25-Induced Activities + + + 1409 + 1419 + surname:Zhong;given-names:X. + 10.1074/jbc.M112.417717 + 23184956 + REF + J Biol Chem. + ref + 288 + 2013 + 39851 + Pyroglutamate and O-Linked Glycan Determine Functional Production of Anti-IL17A and Anti-IL22 Peptide-Antibody Bispecific Genetic Fusions + + + 225 + 227 + surname:Vugmeyster;given-names:Y. + surname:Zhang;given-names:Y. + surname:Zhong;given-names:X. + surname:Wright;given-names:J. + surname:Leung;given-names:S. S. + 10.1016/j.intimp.2013.11.013 + 24295652 + REF + Int Immunopharmacol. + ref + 18 + 2014 + 39989 + Pharmacokinetics of anti-IL17A and anti-IL22 peptide–antibody bispecific genetic fusions in mice + + + 87 + 97 + surname:Zwick;given-names:M. B. + 9784192 + REF + Anal Biochem + ref + 264 + 1998 + 40088 + The maltose-binding protein as a scaffold for monovalent display of peptides derived from phage libraries + + + 435 + 440 + surname:Chang;given-names:S. H. + surname:Dong;given-names:C. + 10.1038/cr.2007.35 + 17452998 + REF + Cell Res + ref + 17 + 2007 + 40194 + A novel heterodimeric cytokine consisting of IL-17 and IL-17F regulates inflammatory responses + + + 87 + 102 + surname:Fouser;given-names:L. A. + surname:Wright;given-names:J. F. + surname:Dunussi-Joannopoulos;given-names:K. + surname:Collins;given-names:M. + 19161418 + REF + Immunol Rev. + ref + 226 + 2008 + 40289 + Th17 cytokines and their emerging roles in inflammation and autoimmunity + + + 677 + 687 + surname:Chiricozzi;given-names:A. + 10.1038/jid.2010.340 + 21085185 + REF + J Invest Dermatol. + ref + 131 + 2011 + 40362 + Integrative responses to IL-17 and TNF-alpha in human keratinocytes account for key inflammatory pathogenic circuits in psoriasis + + + AB177 + surname:Loesche;given-names:C. + surname:Polus;given-names:F. + surname:Sinner;given-names:F. + surname:Gerard;given-names:B. + surname:Valentin;given-names:M. A. + 10.1016/j.jaad.2014.01.735 + REF + J Am Acad Dermatol. + ref + 70 + 2015 + 40492 + Novel microperfusion method confirms that psoriasis lesional skin contains higher protein levels of IL-17A and β-defensin-2 compared to nonlesional skin + + + 285 + 291 + surname:Griffin;given-names:L. + surname:Lawson;given-names:A. + 10.1111/j.1365-2249.2011.04427.x + 21649648 + REF + Clin. Exp. Immunol + ref + 165 + 2011 + 40648 + Antibody fragments as tools in crystallography + + + surname:Guettner;given-names:A. + surname:Machacek;given-names:M. + surname:Papavassilis;given-names:C. + surname:Sander;given-names:O. + REF + ref + 40695 + + + surname:Allan;given-names:B. + surname:Chow;given-names:C. K. + surname:Liu;given-names:L. + surname:Lu;given-names:J. + surname:Tetreault;given-names:J. W. + REF + ref + 40696 + + + 1102 + 1114 + surname:Arkin;given-names:Michelle R. + surname:Tang;given-names:Y. + surname:Wells;given-names:James A. + 10.1016/j.chembiol.2014.09.001 + 25237857 + REF + Chem. Biol. + ref + 21 + 2014 + 40697 + Small-Molecule Inhibitors of Protein-Protein Interactions: Progressing toward the Reality + + + 3 + 33 + surname:Verdine;given-names:G. L. + surname:Hilinski;given-names:G. J. + surname:Wittrup;given-names:K. Dane + surname:Verdine Gregory;given-names:L. + REF + Methods in Enzymology Vol. Volume 503 + ref + 2012 + 40787 + + + 161 + 173 + surname:Azzarito;given-names:V. + surname:Long;given-names:K. + surname:Murphy;given-names:N. S. + surname:Wilson;given-names:A. J. + 23422557 + REF + Nat Chem + ref + 5 + 2013 + 40788 + Inhibition of [alpha]-helix-mediated protein-protein interactions using designed molecules + + + 315 + 323 + surname:Boersma;given-names:M. D. + 10.1021/ja207148m + 22040025 + REF + J. Am. Chem. Soc. + ref + 134 + 2012 + 40879 + Evaluation of Diverse α/β-Backbone Patterns for Functional α-Helix Mimicry: Analogues of the Bim BH3 Domain + + + 1810 + surname:Livingston;given-names:D. + 10.1002/art.39542 + REF + Arthritis Rheum + ref + 64 + 2012 + 40998 + Identification and Characterization of Synthetic Small Molecule Macrocycle Antagonists of Human IL17A + + + 293 + 302 + surname:Vonrhein;given-names:C. + 10.1107/S0907444911007773 + 21460447 + REF + Acta Crystallogr D Biol Crystallogr + ref + 67 + 2011 + 41100 + Data processing and analysis with the autoPROC toolbox + + + 658 + 674 + surname:McCoy;given-names:A. J. + 19461840 + REF + J Appl Crystallogr + ref + 40 + 2007 + 41155 + Phaser crystallographic software + + + 2210 + 2221 + surname:Blanc;given-names:E. + 15572774 + REF + Acta Crystallogr D Biol Crystallogr + ref + 60 + 2004 + 41188 + Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT + + + 486 + 501 + surname:Emsley;given-names:P. + surname:Lohkamp;given-names:B. + surname:Scott;given-names:W. G. + surname:Cowtan;given-names:K. + 20383002 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 41271 + Features and development of Coot + + + 386 + 394 + surname:McNicholas;given-names:S. + surname:Potterton;given-names:E. + surname:Wilson;given-names:K. S. + surname:Noble;given-names:M. E. + 10.1107/S0907444911007281 + 21460457 + REF + Acta Crystallogr D Biol Crystallogr + ref + 67 + 2011 + 41304 + Presenting your structures: the CCP4mg molecular-graphics software + + + SUPPL + footnote + 41371 + Author Contributions P.J., L.D.W., J.J. and G.C. designed and performed phage display and mutagenesis optimization experiments. J.D. and B.O. designed and synthesized peptides. S.S., X.S. and M.G. created protein constructs and purified in house protein reagents. S.L. designed and performed crystallographic studies and analyzed the structures, made Figures 2 and 3, Supplementary Table S3 and Supplementary Figure S7. M.E.B., P.V.S. and W.L. performed FRET, SPR and cell assay, respectively, and made Table 3 and Figure 1. W.L. made Supplementary Figures S2 and S5. J.D., B.D.O., D.T., A.B. and C.W.B. contributed to peptide SAR. J.D., L.D.W. and P.J. prepared Tables 1 and 2, Supplementary Schemes S1 and S2, Supplementary Figures S1, S3 and S4 and Supplementary Tables S1 and S2. P.V.S. and B.A.C. performed Fab/HAP co-binding SPR and made Supplementary Figure S6. X.F. performed mass spectral analysis of the stoichiometry of HAP binding to IL-17A and made Supplementary Figure S8. L.D.W., G.C., D.T., A.B., C.W.B., G.W., R.W.L., A.T., J.M.W. and X.Q. supervised the study. S.L., J.D., J.M.W. and L.D.W. wrote the main text. All authors reviewed the manuscript. + + + srep26071-f1.jpg + f1 + FIG + fig_title_caption + 42538 + Binding of HAP to IL-17A and inhibition of IL-17A/IL-17RA are measured by SPR, FRET and cell-based assays. + + 0.9997749 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9971237 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:28:05Z + + IL-17A/IL-17RA + + + 0.99964595 + experimental_method + cleaner0 + 2023-07-20T08:43:35Z + MESH: + + SPR + + + 0.99958235 + experimental_method + cleaner0 + 2023-07-20T09:39:17Z + MESH: + + FRET + + + 0.9995019 + experimental_method + cleaner0 + 2023-07-20T09:39:20Z + MESH: + + cell-based assays + + + + srep26071-f1.jpg + f1 + FIG + fig_caption + 42645 + (A) Typical SPR sensorgrams (black) of HAP at indicated concentrations binding to biotinylated human IL-17A immobilized on a streptavidin chip surface, fitted with single site binding model curves (red). Kinetic parameters (ka, kd) were obtained by a global fit using three concentrations in triplicate. KD determined by the standard equation, KD = kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA. Data are mean and error bars of +/− standard deviation of three measurements. (C) Inhibition of IL-17A and IL-17RA binding by HAP measured by FRET assay. Data are mean and error bars of +/− standard deviation from 299 experiments, each performed in duplicate. (D) Example of HAP selective inhibition of the production of IL-8 (triangles), IL-6 (squares) and CCL-20 (circles) by primary human keratinocyte cells synergistically stimulated by 100 ng/ml IL-17A and 10 ng/ml TNF-α. HAP does not inhibit the baseline production of IL-6, IL-8 and CCL-20 stimulated by 10 ng/ml TNF-α alone (gray lines and symbols). Data are mean and error bars of +/− standard deviation of duplicated experiments. + + 0.9996934 + experimental_method + cleaner0 + 2023-07-20T08:43:35Z + MESH: + + SPR + + + 0.9996536 + evidence + cleaner0 + 2023-07-20T09:14:36Z + DUMMY: + + sensorgrams + + + 0.9997179 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9992107 + protein_state + cleaner0 + 2023-07-20T09:15:19Z + DUMMY: + + biotinylated + + + 0.9993474 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.9994132 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + evidence + DUMMY: + cleaner0 + 2023-07-20T09:14:28Z + + single site binding model curves + + + 0.99936134 + evidence + cleaner0 + 2023-07-20T09:15:12Z + DUMMY: + + ka + + + 0.998982 + evidence + cleaner0 + 2023-07-20T09:15:04Z + DUMMY: + + kd + + + 0.9994641 + evidence + cleaner0 + 2023-07-20T09:15:03Z + DUMMY: + + KD + + + 0.9994006 + evidence + cleaner0 + 2023-07-20T09:15:04Z + DUMMY: + + KD + + + 0.9980882 + evidence + cleaner0 + 2023-07-20T09:15:04Z + DUMMY: + + kd + + + 0.9948657 + evidence + cleaner0 + 2023-07-20T09:15:12Z + DUMMY: + + ka + + + 0.9997172 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9996642 + experimental_method + cleaner0 + 2023-07-20T08:43:35Z + MESH: + + SPR + + + 0.9989503 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.8585394 + protein_state + cleaner0 + 2023-07-20T09:56:49Z + DUMMY: + + immobilized + + + 0.9993916 + protein + cleaner0 + 2023-07-20T08:17:01Z + PR: + + IL-17RA + + + 0.99624085 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.9984536 + protein + cleaner0 + 2023-07-20T08:17:01Z + PR: + + IL-17RA + + + 0.99971646 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.99960434 + experimental_method + cleaner0 + 2023-07-20T09:39:26Z + MESH: + + FRET assay + + + 0.9997013 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9994752 + protein_type + cleaner0 + 2023-07-20T08:44:32Z + MESH: + + IL-8 + + + 0.99951357 + protein_type + cleaner0 + 2023-07-20T08:44:37Z + MESH: + + IL-6 + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:44:55Z + + CCL-20 + + + 0.9990013 + species + cleaner0 + 2023-07-20T08:25:04Z + MESH: + + human + + + 0.9985098 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.93264896 + protein + cleaner0 + 2023-07-20T08:44:23Z + PR: + + TNF-α + + + 0.9997168 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9995456 + protein_type + cleaner0 + 2023-07-20T08:44:37Z + MESH: + + IL-6 + + + 0.9994187 + protein_type + cleaner0 + 2023-07-20T08:44:32Z + MESH: + + IL-8 + + + protein_type + MESH: + cleaner0 + 2023-07-20T08:44:55Z + + CCL-20 + + + 0.99070376 + protein + cleaner0 + 2023-07-20T08:44:23Z + PR: + + TNF-α + + + + srep26071-f2.jpg + f2 + FIG + fig_title_caption + 43783 + Overall structure of the Fab/IL-17A/HAP complex in ribbon presentation. + + 0.99678874 + evidence + cleaner0 + 2023-07-20T09:43:41Z + DUMMY: + + structure + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:47:07Z + + Fab/IL-17A/HAP + + + + srep26071-f2.jpg + f2 + FIG + fig_caption + 43855 + For clarity, different molecules are colored differently. Two HAP molecules are colored blue and red, and IL-17A monomers are colored ice blue and pink, respectively. Picture prepared using program CCP4MG. (A) Overview of the distinct binding sites of Fab and HAP to IL-17A. (B) Close-in view of the IL-17A/HAP structure. IL-17A β-strands are labelled. Each of the two bound HAP interacts with both monomers of the IL-17A dimer. (C) As a comparison, the IL-17A/IL-17RA complex was shown with IL-17A in the same orientation. Three distinct areas IL-17A/IL-17RA interface are labeled. + + 0.999759 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.99955463 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.9991665 + oligomeric_state + cleaner0 + 2023-07-20T08:23:13Z + DUMMY: + + monomers + + + 0.99946296 + site + cleaner0 + 2023-07-20T09:40:12Z + SO: + + binding sites + + + 0.9997955 + structure_element + cleaner0 + 2023-07-20T08:47:40Z + SO: + + Fab + + + 0.9996724 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9994636 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.9994233 + complex_assembly + cleaner0 + 2023-07-20T08:46:26Z + GO: + + IL-17A/HAP + + + 0.9993218 + evidence + cleaner0 + 2023-07-20T09:43:45Z + DUMMY: + + structure + + + 0.9994388 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.9996136 + structure_element + cleaner0 + 2023-07-20T09:48:08Z + SO: + + β-strands + + + 0.99904794 + protein_state + cleaner0 + 2023-07-20T09:56:54Z + DUMMY: + + bound + + + 0.99970585 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9992748 + oligomeric_state + cleaner0 + 2023-07-20T08:23:13Z + DUMMY: + + monomers + + + 0.9994292 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.99929965 + oligomeric_state + cleaner0 + 2023-07-20T09:18:27Z + DUMMY: + + dimer + + + 0.9996103 + complex_assembly + cleaner0 + 2023-07-20T08:28:05Z + GO: + + IL-17A/IL-17RA + + + 0.9995683 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.9995848 + site + cleaner0 + 2023-07-20T09:15:57Z + SO: + + IL-17A/IL-17RA interface + + + + srep26071-f3.jpg + f3 + FIG + fig_title_caption + 44441 + Mechanism of the inhibition of the IL-17A/IL-17RA interaction by HAP. + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:28:06Z + + IL-17A/IL-17RA + + + 0.9997354 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + + srep26071-f3.jpg + f3 + FIG + fig_caption + 44511 + (A) HAP binds at region I of IL-17A. IL-17A dimer is in surface presentation (β-strands 0 shown as ribbons for clarity). Polar interactions are shown in dashes. HAP residues as well as key IL-17A residues are labeled. For clarity, a few HAP residues are also shown in stick model with carbon atoms colored green, oxygen in red and nitrogen in blue. (B) I-17RA (ribbon in gold) peptide Leu27-Ile32 binds to the same area as the HAP α-helix. Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. Notice that the Trp binding pocket for W12 of HAP or W31 of IL-17RA is missing in the apo structure. + + 0.99956113 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.99846333 + structure_element + cleaner0 + 2023-07-20T08:54:15Z + SO: + + region I + + + 0.99849606 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.99864507 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.9993493 + oligomeric_state + cleaner0 + 2023-07-20T09:18:27Z + DUMMY: + + dimer + + + 0.99935997 + structure_element + cleaner0 + 2023-07-20T09:48:13Z + SO: + + β-strands 0 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:31Z + + Polar interactions + + + 0.9993606 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.99775904 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.9994431 + chemical + cleaner0 + 2023-07-20T08:18:37Z + CHEBI: + + HAP + + + 0.9994113 + protein + cleaner0 + 2023-07-20T09:25:04Z + 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2023-07-20T09:48:25Z + SO: + + β-strands 0 + + + 0.996832 + complex_assembly + cleaner0 + 2023-07-20T08:46:26Z + GO: + + IL-17A/HAP + + + 0.9996488 + protein_state + cleaner0 + 2023-07-20T08:21:59Z + DUMMY: + + apo + + + 0.9991872 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.99958926 + evidence + cleaner0 + 2023-07-20T09:43:55Z + DUMMY: + + structure + + + 0.999281 + structure_element + cleaner0 + 2023-07-20T08:54:15Z + SO: + + region I + + + 0.99816847 + protein + cleaner0 + 2023-07-20T08:16:53Z + PR: + + IL-17A + + + 0.99966127 + structure_element + cleaner0 + 2023-07-20T09:48:29Z + SO: + + β-stand + + + 0.99964935 + structure_element + cleaner0 + 2023-07-20T09:48:32Z + SO: + + α-helix + + + 0.9995005 + chemical + cleaner0 + 2023-07-20T08:18:38Z + CHEBI: + + HAP + + + 0.9996323 + site + cleaner0 + 2023-07-20T09:16:41Z + SO: + + Trp binding pocket + + + 0.99988747 + residue_name_number + cleaner0 + 2023-07-20T09:21:15Z + DUMMY: + + W12 + + + 0.9996706 + chemical + cleaner0 + 2023-07-20T08:18:38Z + CHEBI: + + HAP + + + 0.9998939 + residue_name_number + cleaner0 + 2023-07-20T09:21:18Z + DUMMY: + + W31 + + + 0.99928457 + protein + cleaner0 + 2023-07-20T08:17:01Z + PR: + + IL-17RA + + + 0.9996501 + protein_state + cleaner0 + 2023-07-20T08:21:59Z + DUMMY: + + apo + + + 0.99953055 + evidence + cleaner0 + 2023-07-20T09:43:59Z + DUMMY: + + structure + + + + t1.xml + t1 + TABLE + table_title_caption + 45380 + ELISA competition activity of peptide analogues of 1. + + 0.9044505 + experimental_method + cleaner0 + 2023-07-20T09:16:56Z + MESH: + + ELISA competition activity + + + + t1.xml + t1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50">Compound #</th><th align="center" valign="top" charoff="50">Competition IC<sub>50</sub>(nM)</th><th align="center" valign="top" charoff="50">1</th><th align="center" valign="top" charoff="50">2</th><th align="center" valign="top" charoff="50">3</th><th align="center" valign="top" charoff="50">4</th><th align="center" valign="top" charoff="50">5</th><th align="center" valign="top" charoff="50">6</th><th align="center" valign="top" charoff="50">7</th><th align="center" valign="top" charoff="50">8</th><th align="center" valign="top" charoff="50">9</th><th align="center" valign="top" charoff="50">10</th><th align="center" valign="top" charoff="50">11</th><th align="center" valign="top" charoff="50">12</th><th align="center" valign="top" charoff="50">13</th><th align="center" valign="top" charoff="50">14</th><th align="center" valign="top" charoff="50">15</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50"><bold>1</bold></td><td align="center" valign="top" charoff="50">80</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">K</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>2</bold></td><td align="center" valign="top" charoff="50">80</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>3</bold></td><td align="center" valign="top" charoff="50">66</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50"><bold>K</bold></td></tr><tr><td align="left" valign="top" charoff="50"><bold>4</bold></td><td align="center" valign="top" charoff="50">172</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>5</bold></td><td align="center" valign="top" charoff="50">402</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>6</bold></td><td align="center" valign="top" charoff="50">7451</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>7</bold></td><td align="center" valign="top" charoff="50">474</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>8</bold></td><td align="center" valign="top" charoff="50">524</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>9</bold></td><td align="center" valign="top" charoff="50">640</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>10</bold></td><td align="center" valign="top" charoff="50">4016</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>11</bold></td><td align="center" valign="top" charoff="50">84</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>12</bold></td><td align="center" valign="top" charoff="50">2651</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>13</bold></td><td align="center" valign="top" charoff="50">11</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>14</bold></td><td align="center" valign="top" charoff="50">195</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>15</bold></td><td align="center" valign="top" charoff="50">5717</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>16</bold></td><td align="center" valign="top" charoff="50">149</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>17</bold></td><td align="center" valign="top" charoff="50">167</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>18</bold></td><td align="center" valign="top" charoff="50">45</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>H</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>19</bold></td><td align="center" valign="top" charoff="50">71</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>Q</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>20</bold></td><td align="center" valign="top" charoff="50">117</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>R</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>21</bold></td><td align="center" valign="top" charoff="50">31</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>T</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>22</bold></td><td align="center" valign="top" charoff="50">522</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>W</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>23</bold></td><td align="center" valign="top" charoff="50">64</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>Y</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>24</bold></td><td align="center" valign="top" charoff="50">3</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50"><bold>I</bold></td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>25</bold></td><td align="center" valign="top" charoff="50">33</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50"><bold>L</bold></td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>26</bold></td><td align="center" valign="top" charoff="50">6</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50"><bold>V</bold></td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>27</bold></td><td align="center" valign="top" charoff="50">31</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>M</bold></td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>28</bold></td><td align="center" valign="top" charoff="50">26</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>N</bold></td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>29</bold></td><td align="center" valign="top" charoff="50">33</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>Q</bold></td><td align="center" valign="top" charoff="50">A</td></tr></tbody></table> + + 45434 + Compound # Competition IC50(nM) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 80 I V V T M P A D L W D W I K A 2 80 I V V T M P A D L W D W I R A 3 66 I V V T M P A D L W D W I R K 4 172 I V V T M P A D L W D W I A A 5 402 I V V T M P A D L W D W A R A 6 7451 I V V T M P A D L W D A I R A 7 474 I V V T M P A D L W A W I R A 8 524 I V V T M P A D L A D W I R A 9 640 I V V T M P A D A W D W I R A 10 4016 I V V T M P A A L W D W I R A 11 84 I V V T M P A D L W D W I R A 12 2651 I V V T M A A D L W D W I R A 13 11 I V V T A P A D L W D W I R A 14 195 I V V A M P A D L W D W I R A 15 5717 I V A T M P A D L W D W I R A 16 149 I A V T M P A D L W D W I R A 17 167 A V V T M P A D L W D W I R A 18 45 I H V T M P A D L W D W I R A 19 71 I Q V T M P A D L W D W I R A 20 117 I R V T M P A D L W D W I R A 21 31 I T V T M P A D L W D W I R A 22 522 I W V T M P A D L W D W I R A 23 64 I Y V T M P A D L W D W I R A 24 3 I V V T I P A D L W D W I R A 25 33 I V V T L P A D L W D W I R A 26 6 I V V T V P A D L W D W I R A 27 31 I V V T M P A D L W D W I M A 28 26 I V V T M P A D L W D W I N A 29 33 I V V T M P A D L W D W I Q A + + + t1.xml + t1 + TABLE + table_footnote + 46614 + All peptides are acetylated at their N-termini. + + + t2.xml + t2 + TABLE + table_title_caption + 46662 + Cell-based activity of amino acid deletions of 30. + + + t2.xml + t2 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50">Compound #</th><th align="center" valign="top" charoff="50">Cell IC<sub>50</sub>(nM)</th><th align="center" valign="top" charoff="50">1</th><th align="center" valign="top" charoff="50">2</th><th align="center" valign="top" charoff="50">3</th><th align="center" valign="top" charoff="50">4</th><th align="center" valign="top" charoff="50">5</th><th align="center" valign="top" charoff="50">6</th><th align="center" valign="top" charoff="50">7</th><th align="center" valign="top" charoff="50">8</th><th align="center" valign="top" charoff="50">9</th><th align="center" valign="top" charoff="50">10</th><th align="center" valign="top" charoff="50">11</th><th align="center" valign="top" charoff="50">12</th><th align="center" valign="top" charoff="50">13</th><th align="center" valign="top" charoff="50">14</th><th align="center" valign="top" charoff="50">15</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50"><bold>1</bold></td><td align="center" valign="top" charoff="50">370</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">K</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>24</bold></td><td align="center" valign="top" charoff="50">18</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>26</bold></td><td align="center" valign="top" charoff="50">137</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>30</bold></td><td align="center" valign="top" charoff="50">17</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">H</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">N</td><td align="center" valign="top" charoff="50">K</td></tr><tr><td align="left" valign="top" charoff="50"><bold>32</bold></td><td align="center" valign="top" charoff="50">&gt;1000</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">H</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">N</td><td align="center" valign="top" charoff="50">K</td></tr><tr><td align="left" valign="top" charoff="50"><bold>33</bold></td><td align="center" valign="top" charoff="50">&gt;1000</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">N</td><td align="center" valign="top" charoff="50">K</td></tr><tr><td align="left" valign="top" charoff="50"><bold>34</bold></td><td align="center" valign="top" charoff="50">&gt;1000</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">N</td><td align="center" valign="top" charoff="50">K</td></tr><tr><td align="left" valign="top" charoff="50"><bold>35</bold></td><td align="center" valign="top" charoff="50">12</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">H</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">N</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"><bold>36</bold></td><td align="center" valign="top" charoff="50">183</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">H</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"><bold>37</bold></td><td align="center" valign="top" charoff="50">&gt;1000</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">H</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr></tbody></table> + + 46713 + Compound # Cell IC50(nM) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 370 I V V T M P A D L W D W I K A 24 18 I V V T I P A D L W D W I R A 26 137 I V V T L P A D L W D W I R A 30 17 I H V T I P A D L W D W I N K 32 >1000   H V T I P A D L W D W I N K 33 >1000     V T I P A D L W D W I N K 34 >1000       T I P A D L W D W I N K 35 12 I H V T I P A D L W D W I N   36 183 I H V T I P A D L W D W I     37 >1000 I H V T I P A D L W D W       + + + t3.xml + t3 + TABLE + table_title_caption + 47183 + Binding affinity/IC50 of HAP in orthogonal in vitro assays/primary keratinocyte cell assay. + + + t3.xml + t3 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50">Assay</th><th align="center" valign="top" charoff="50">K<sub>D</sub>/IC<sub>50</sub> (nM)</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">SPR binding (N = 3)<xref ref-type="fn" rid="t3-fn1">*</xref>: hIL-17A, hIL-17A/F, mIL-17A</td><td align="center" valign="top" charoff="50">2.6(±0.5), 4.6(±0.7), 30.1(±0.1)</td></tr><tr><td align="left" valign="top" charoff="50">SPR competition</td><td align="center" valign="top" charoff="50">0.9(±0.3)</td></tr><tr><td align="left" valign="top" charoff="50">FRET IL-17RA competition (N = 598)</td><td align="center" valign="top" charoff="50">0.8 ± 0.2</td></tr><tr><td align="left" valign="top" charoff="50">Human primary keratinocytes IC-8 (N = 9), IC-6 (N = 4), CCL-20 (N = 4)</td><td align="center" valign="top" charoff="50">151(±49), 136(±37), 253(±64)</td></tr></tbody></table> + + 47275 + Assay KD/IC50 (nM) SPR binding (N = 3)*: hIL-17A, hIL-17A/F, mIL-17A 2.6(±0.5), 4.6(±0.7), 30.1(±0.1) SPR competition 0.9(±0.3) FRET IL-17RA competition (N = 598) 0.8 ± 0.2 Human primary keratinocytes IC-8 (N = 9), IC-6 (N = 4), CCL-20 (N = 4) 151(±49), 136(±37), 253(±64) + + + t3.xml + t3 + TABLE + table_footnote + 47591 + *KD = kd/ka. ka (M−1s−1)and kd (s−1): 2(±1) × 105, 6(±5) × 10−4 (hIL-17A); 4.6(±0.7) × 105, 2.1(±0.2) × 10−3 (hIL-17A/F); 2.7(±0.2) × 105, 8.2(±0.8) × 10−3 (mIL-17A). + + + diff --git a/BioC_XML/4871749_v0.xml b/BioC_XML/4871749_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..407de21bc2a7508e8acefb385377a12bbe46fac8 --- /dev/null +++ b/BioC_XML/4871749_v0.xml @@ -0,0 +1,4175 @@ + + + + PMC + 20201221 + pmc.key + + 4871749 + NO-CC CODE + no + 0 + 0 + + 10.1038/nchembio.2065 + NIHMS769551 + 4871749 + 27089029 + 396 + 6 + YEATS domain crotonylated lysine chromatin Taf14 histone PTM + Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: + + 398 + surname:Andrews;given-names:Forest H. + surname:Shinsky;given-names:Stephen A. + surname:Shanle;given-names:Erin K. + surname:Bridgers;given-names:Joseph B. + surname:Gest;given-names:Anneliese + surname:Tsun;given-names:Ian K. + surname:Krajewski;given-names:Krzysztof + surname:Shi;given-names:Xiaobing + surname:Strahl;given-names:Brian D. + surname:Kutateladze;given-names:Tatiana G. + TITLE + front + 12 + 2016 + 0 + The Taf14 YEATS domain is a reader of histone crotonylation + + 0.99879897 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + 0.9920379 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + 0.9768092 + protein_type + cleaner0 + 2023-07-04T16:21:00Z + MESH: + + histone + + + 0.60347056 + ptm + cleaner0 + 2023-07-04T16:18:43Z + MESH: + + crotonylation + + + + ABSTRACT + abstract + 60 + The discovery of new histone modifications is unfolding at startling rates, however, the identification of effectors capable of interpreting these modifications has lagged behind. Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity. + + protein_type + MESH: + cleaner0 + 2023-07-04T16:21:00Z + + histone + + + 0.98157585 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + 0.6092492 + protein_type + cleaner0 + 2023-07-04T16:21:00Z + MESH: + + histone + + + 0.64678025 + residue_name + cleaner0 + 2023-07-05T08:52:59Z + SO: + + lysine + + + 0.7005873 + ptm + cleaner0 + 2023-07-04T16:18:43Z + MESH: + + crotonylation + + + 0.9987513 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + 0.98611736 + structure_element + cleaner0 + 2023-07-04T16:20:27Z + SO: + + YEATS domain + + + 0.99818015 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + π-π-π-stacking + + + 0.8457508 + structure_element + cleaner0 + 2023-07-05T08:49:39Z + SO: + + YEATS domains + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + + crotonyllysine + + + + INTRO + paragraph + 560 + Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin. This epigenetic PTM is widespread and enriched at active gene promoters and potentially enhancers. The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. Owing to some differences in their genomic distribution, the crotonyllysine and acetyllysine (Kac) modifications have been linked to distinct functional outcomes. p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation. The discovery of individual biological roles for the crotonyllysine and acetyllysine marks suggests that these PTMs can be read by distinct readers. While a number of acetyllysine readers have been identified and characterized, a specific reader of the crotonyllysine mark remains unknown (reviewed in). A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity. + + 0.97012514 + ptm + cleaner0 + 2023-07-04T16:18:43Z + MESH: + + Crotonylation + + + 0.8647513 + residue_name + cleaner0 + 2023-07-05T08:53:04Z + SO: + + lysine + + + 0.99726224 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + 0.9968893 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + Kcr + + + protein_type + MESH: + cleaner0 + 2023-07-04T16:21:00Z + + histone + + + 0.9958947 + taxonomy_domain + cleaner0 + 2023-07-04T16:21:35Z + DUMMY: + + mammalian + + + 0.7653515 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + protein_type + MESH: + cleaner0 + 2023-07-04T16:21:00Z + + histone + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:36:55Z + + H3 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-05T08:37:14Z + + K18 + + + 0.98865753 + protein + cleaner0 + 2023-07-04T16:20:32Z + PR: + + p300 + + + 0.98868376 + protein_type + cleaner0 + 2023-07-04T16:22:54Z + MESH: + + histone acetyltransferase + + + 0.7823207 + ptm + cleaner0 + 2023-07-04T16:18:49Z + MESH: + + acetylation + + + 0.9841765 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + 0.9894874 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + acetyllysine + + + 0.98921156 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + Kac + + + 0.9711801 + protein + cleaner0 + 2023-07-04T16:20:33Z + PR: + + p300 + + + protein_type + MESH: + cleaner0 + 2023-07-04T16:21:00Z + + histone + + + 0.9621592 + ptm + cleaner0 + 2023-07-04T16:18:43Z + MESH: + + crotonylation + + + 0.9124805 + protein + cleaner0 + 2023-07-04T16:20:33Z + PR: + + p300 + + + 0.87436926 + ptm + cleaner0 + 2023-07-04T16:18:49Z + MESH: + + acetylation + + + 0.91712606 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + 0.9561159 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + acetyllysine + + + 0.8240044 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + acetyllysine + + + 0.6335516 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + 0.94788533 + structure_element + cleaner0 + 2023-07-04T16:21:48Z + SO: + + bromodomains + + + 0.8961469 + structure_element + cleaner0 + 2023-07-04T16:21:53Z + SO: + + BDs + + + 0.92061985 + structure_element + cleaner0 + 2023-07-05T08:51:22Z + SO: + + BD + + + 0.9774638 + protein_state + cleaner0 + 2023-07-04T16:22:06Z + DUMMY: + + crotonylated + + + 0.98200285 + protein_state + cleaner0 + 2023-07-04T16:22:11Z + DUMMY: + + acetylated + + + 0.7515075 + structure_element + cleaner0 + 2023-07-04T16:21:48Z + SO: + + bromodomains + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + + crotonyllysine + + + + INTRO + paragraph + 1865 + The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. Similarly, activation of a subset of genes and DNA damage repair in yeast require the acetyllysine binding activity of the Taf14 YEATS domain. Consistent with its role in gene regulation, Taf14 was identified as a core component of the transcription factor complexes TFIID and TFIIF. However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. We found that H3K9cr is present in yeast and is dynamically regulated. + + 0.9015483 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + acetyllysine + + + 0.9973253 + structure_element + cleaner0 + 2023-07-05T08:51:28Z + SO: + + YEATS + + + 0.99829453 + protein + cleaner0 + 2023-07-05T07:54:03Z + PR: + + Yaf9 + + + 0.99791545 + protein + cleaner0 + 2023-07-05T07:54:08Z + PR: + + ENL + + + 0.9983627 + protein + cleaner0 + 2023-07-05T07:54:14Z + PR: + + AF9 + + + 0.9985991 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + 0.9987198 + protein + cleaner0 + 2023-07-05T07:54:25Z + PR: + + Sas5 + + + 0.96409446 + species + cleaner0 + 2023-07-04T16:22:25Z + MESH: + + human + + + 0.99878556 + protein + cleaner0 + 2023-07-05T07:54:15Z + PR: + + AF9 + + + 0.9893267 + taxonomy_domain + cleaner0 + 2023-07-04T16:22:20Z + DUMMY: + + yeast + + + 0.9988275 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + protein_type + MESH: + cleaner0 + 2023-07-04T16:21:00Z + + histone + + + protein_type + MESH: + cleaner0 + 2023-07-05T07:55:54Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:53:13Z + + K9ac + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + + acetyllysine + + + 0.99856865 + protein + cleaner0 + 2023-07-05T07:54:15Z + PR: + + AF9 + + + 0.9379061 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + 0.9904277 + protein_type + cleaner0 + 2023-07-04T16:22:40Z + MESH: + + histone methyltransferase + + + 0.99846435 + protein + cleaner0 + 2023-07-05T07:57:04Z + PR: + + DOT1L + + + protein_type + MESH: + cleaner0 + 2023-07-05T07:56:42Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T12:00:58Z + + K9ac + + + 0.9973254 + protein + cleaner0 + 2023-07-05T07:57:04Z + PR: + + DOT1L + + + protein_type + MESH: + cleaner0 + 2023-07-05T07:57:26Z + + H3 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-05T07:57:40Z + + K79 + + + ptm + MESH: + cleaner0 + 2023-07-05T07:57:58Z + + methylation + + + 0.9963744 + taxonomy_domain + cleaner0 + 2023-07-04T16:22:20Z + DUMMY: + + yeast + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + + acetyllysine + + + 0.9987602 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + 0.94719505 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + 0.9980909 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + 0.84213173 + complex_assembly + cleaner0 + 2023-07-04T16:23:51Z + GO: + + TFIID + + + 0.6936775 + complex_assembly + cleaner0 + 2023-07-04T16:24:02Z + GO: + + TFIIF + + + 0.9974746 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + 0.7698535 + complex_assembly + cleaner0 + 2023-07-05T07:58:48Z + GO: + + INO80 + + + 0.67576855 + complex_assembly + cleaner0 + 2023-07-05T07:58:53Z + GO: + + SWI/SNF + + + 0.5300177 + complex_assembly + cleaner0 + 2023-07-05T07:59:00Z + GO: + + RSC + + + 0.8560897 + protein_type + cleaner0 + 2023-07-04T16:22:53Z + MESH: + + histone acetyltransferase + + + 0.77899855 + complex_assembly + cleaner0 + 2023-07-05T08:50:53Z + GO: + + NuA3 + + + 0.99761933 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + 0.9986816 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + 0.93180734 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + 0.9986914 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + protein_type + MESH: + cleaner0 + 2023-07-04T16:21:00Z + + histone + + + protein_type + MESH: + cleaner0 + 2023-07-05T07:55:54Z + + H3 + + + 0.6754686 + protein_state + cleaner0 + 2023-07-04T16:22:07Z + DUMMY: + + crotonylated + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-05T07:58:27Z + + lysine 9 + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:00:12Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:52:22Z + + K9cr + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:00:12Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:52:32Z + + K9cr + + + 0.9966305 + taxonomy_domain + cleaner0 + 2023-07-04T16:22:20Z + DUMMY: + + yeast + + + + INTRO + paragraph + 3072 + To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c). + + protein_type + MESH: + cleaner0 + 2023-07-05T08:00:50Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:52:40Z + + K9cr + + + 0.9973307 + evidence + cleaner0 + 2023-07-05T08:56:20Z + DUMMY: + + crystal structure + + + 0.9986278 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + 0.9957862 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + 0.9311703 + protein_state + cleaner0 + 2023-07-05T08:53:17Z + DUMMY: + + in complex with + + + 0.97627616 + chemical + cleaner0 + 2023-07-05T08:36:15Z + CHEBI: + + H3K9cr5-13 + + + 0.96639997 + residue_range + cleaner0 + 2023-07-05T08:49:48Z + DUMMY: + + 5–13 + + + 0.9895627 + protein_type + cleaner0 + 2023-07-05T07:55:54Z + MESH: + + H3 + + + 0.998461 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + 0.9965588 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + 0.99343634 + structure_element + cleaner0 + 2023-07-05T08:51:32Z + SO: + + immunoglobin-like β sandwich fold + + + 0.96191806 + structure_element + cleaner0 + 2023-07-05T08:51:36Z + SO: + + anti-parallel β strands + + + 0.6941337 + structure_element + cleaner0 + 2023-07-05T08:51:44Z + SO: + + loops + + + 0.9979013 + site + cleaner0 + 2023-07-05T08:51:58Z + SO: + + binding site + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:01:22Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:52:54Z + + K9cr + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:01:47Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:52:47Z + + K9cr + + + 0.9527997 + protein_state + cleaner0 + 2023-07-05T08:53:22Z + DUMMY: + + extended conformation + + + 0.99544466 + structure_element + cleaner0 + 2023-07-05T08:51:48Z + SO: + + β strands + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-07-21T12:02:44Z + + water + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + hydrogen bonds + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + salt bridge + + + + INTRO + paragraph + 3741 + The most striking feature of the crotonyllysine recognition mechanism is the unique coordination of crotonylated lysine residue. The fully extended side chain of K9cr transverses the narrow tunnel, crossing the β sandwich at right angle in a corkscrew-like manner (Fig. 1b and Supplementary Figure 1b). The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic π-π-π-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c). The side chain of Trp81 appears to adopt two conformations, one of which provides maximum π-stacking with the alkene functional group while the other rotamer affords maximum π-stacking with the amide π electrons (Supplementary Fig. 1c). The dual conformation of Trp81 is likely due to the conjugated nature of the C=C and C=O π-orbitals within the crotonyl functional group. + + 0.9968951 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + 0.884992 + protein_state + cleaner0 + 2023-07-04T16:22:07Z + DUMMY: + + crotonylated + + + 0.98747534 + residue_name + cleaner0 + 2023-07-05T08:02:11Z + SO: + + lysine + + + 0.99885833 + ptm + melaniev@ebi.ac.uk + 2023-07-21T11:52:08Z + MESH: + + K9cr + + + 0.9932809 + structure_element + cleaner0 + 2023-07-05T08:52:02Z + SO: + + β sandwich + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-07-21T11:51:04Z + + crotonyl + + + 0.99896455 + residue_name_number + cleaner0 + 2023-07-05T08:02:27Z + DUMMY: + + Trp81 + + + 0.99897707 + residue_name_number + cleaner0 + 2023-07-05T08:02:31Z + DUMMY: + + Phe62 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-07-21T11:51:04Z + + crotonyl + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + π-π-π-stacking + + + 0.99895763 + residue_name_number + cleaner0 + 2023-07-05T08:02:27Z + DUMMY: + + Trp81 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + π-stacking + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + π-stacking + + + 0.9987826 + residue_name_number + cleaner0 + 2023-07-05T08:02:27Z + DUMMY: + + Trp81 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-07-21T11:51:04Z + + crotonyl + + + + INTRO + paragraph + 4832 + In addition to π-π-π stacking, the crotonyl group is stabilized by a set of hydrogen bonds and electrostatic interactions. The π bond conjugation of the crotonyl group gives rise to a dipole moment of the alkene moiety, resulting in a partial positive charge on the β-carbon (Cβ) and a partial negative charge on the α-carbon (Cα). This provides the capability for the alkene moiety to form electrostatic contacts, as Cα and Cβ lay within electrostatic interaction distances of the carbonyl oxygen of Gln79 and of the hydroxyl group of Thr61, respectively. The hydroxyl group of Thr61 also participates in a hydrogen bond with the amide nitrogen of the K9cr side chain (Fig. 1d). The fixed position of the Thr61 hydroxyl group, which facilitates interactions with both the amide and Cα of K9cr, is achieved through a hydrogen bond with imidazole ring of His59. Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d). This distinctive mechanism was corroborated through mapping the Taf14 YEATS-H3K9cr binding interface in solution using NMR chemical shift perturbation analysis (Supplementary Fig. 2a, b). + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + π-π-π stacking + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-07-21T11:51:04Z + + crotonyl + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + hydrogen bonds + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + electrostatic interactions + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + π bond + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-07-21T11:51:04Z + + crotonyl + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + electrostatic contacts + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + electrostatic interaction + + + 0.9991684 + residue_name_number + cleaner0 + 2023-07-05T08:02:41Z + DUMMY: + + Gln79 + + + 0.9991742 + residue_name_number + cleaner0 + 2023-07-05T08:02:45Z + DUMMY: + + Thr61 + + + 0.9991697 + residue_name_number + cleaner0 + 2023-07-05T08:02:44Z + DUMMY: + + Thr61 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + hydrogen bond + + + 0.9990522 + ptm + melaniev@ebi.ac.uk + 2023-07-21T11:52:08Z + MESH: + + K9cr + + + 0.99915934 + residue_name_number + cleaner0 + 2023-07-05T08:02:45Z + DUMMY: + + Thr61 + + + 0.9990495 + ptm + melaniev@ebi.ac.uk + 2023-07-21T11:52:08Z + MESH: + + K9cr + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + hydrogen bond + + + 0.99916136 + residue_name_number + cleaner0 + 2023-07-05T08:02:52Z + DUMMY: + + His59 + + + 0.9988856 + ptm + melaniev@ebi.ac.uk + 2023-07-21T11:52:08Z + MESH: + + K9cr + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + hydrogen bond + + + 0.9991516 + residue_name_number + cleaner0 + 2023-07-05T08:02:27Z + DUMMY: + + Trp81 + + + 0.9917144 + chemical + cleaner0 + 2023-07-05T08:55:01Z + CHEBI: + + water + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + hydrogen bond + + + 0.99916196 + residue_name_number + cleaner0 + 2023-07-05T08:02:57Z + DUMMY: + + Gly82 + + + 0.9983511 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + 0.9934926 + site + cleaner0 + 2023-07-05T08:03:03Z + SO: + + YEATS-H3K9cr binding interface + + + 0.9922565 + experimental_method + cleaner0 + 2023-07-05T08:03:06Z + MESH: + + NMR chemical shift perturbation analysis + + + + INTRO + paragraph + 6134 + Binding of the Taf14 YEATS domain to H3K9cr is robust. The dissociation constant (Kd) for the Taf14 YEATS-H3K9cr5-13 complex was found to be 9.5 μM, as measured by fluorescence spectroscopy (Supplementary Fig. 2c). This value is in the range of binding affinities exhibited by the majority of histone readers, thus attesting to the physiological relevance of the H3K9cr recognition by Taf14. + + 0.99884135 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + 0.94870484 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:03:24Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:54:19Z + + K9cr + + + 0.995806 + evidence + cleaner0 + 2023-07-05T08:03:41Z + DUMMY: + + dissociation constant + + + 0.9937781 + evidence + cleaner0 + 2023-07-05T08:03:45Z + DUMMY: + + Kd + + + 0.9887676 + complex_assembly + cleaner0 + 2023-07-05T08:35:55Z + GO: + + Taf14 YEATS-H3K9cr5-13 + + + 0.9937454 + experimental_method + cleaner0 + 2023-07-05T08:04:07Z + MESH: + + fluorescence spectroscopy + + + 0.99432206 + evidence + cleaner0 + 2023-07-05T08:04:01Z + DUMMY: + + binding affinities + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:04:40Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:54:27Z + + K9cr + + + 0.9988524 + protein + cleaner0 + 2023-07-04T16:20:21Z + PR: + + Taf14 + + + + INTRO + paragraph + 6527 + To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains. Furthermore, fluctuations in the H3K9cr levels were more substantial than fluctuations in the corresponding H3K9ac levels. Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs. + + protein_type + MESH: + cleaner0 + 2023-07-05T08:08:27Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:54:36Z + + K9cr + + + 0.99658173 + taxonomy_domain + cleaner0 + 2023-07-04T16:22:20Z + DUMMY: + + yeast + + + 0.8414014 + experimental_method + cleaner0 + 2023-07-05T08:57:36Z + MESH: + + whole cell extracts + + + 0.9963198 + taxonomy_domain + cleaner0 + 2023-07-04T16:22:20Z + DUMMY: + + yeast + + + 0.9293416 + experimental_method + cleaner0 + 2023-07-05T08:57:39Z + MESH: + + Western blot analysis + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:09:10Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:54:53Z + + K9cr + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:09:38Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:55:01Z + + K9ac + + + 0.7814144 + protein_type + cleaner0 + 2023-07-05T07:55:54Z + MESH: + + H3 + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:10:08Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:54:45Z + + K9cr + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:10:36Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:55:10Z + + K9ac + + + 0.99671257 + taxonomy_domain + cleaner0 + 2023-07-04T16:22:20Z + DUMMY: + + yeast + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:57:52Z + + histones + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:38:01Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:55:18Z + + K9cr + + + 0.9966935 + taxonomy_domain + cleaner0 + 2023-07-04T16:22:20Z + DUMMY: + + yeast + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:38:30Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:55:26Z + + K9cr + + + 0.99616396 + protein_type + cleaner0 + 2023-07-05T08:06:46Z + MESH: + + histone acetyltransferases + + + 0.9961572 + protein_type + cleaner0 + 2023-07-05T08:06:50Z + MESH: + + HATs + + + 0.99562824 + protein_type + cleaner0 + 2023-07-05T08:07:02Z + MESH: + + histone deacetylases + + + 0.9862266 + protein_type + cleaner0 + 2023-07-05T08:07:06Z + MESH: + + HDACs + + + 0.9961592 + taxonomy_domain + cleaner0 + 2023-07-04T16:22:20Z + DUMMY: + + yeast + + + 0.99671113 + taxonomy_domain + cleaner0 + 2023-07-04T16:22:20Z + DUMMY: + + yeast + + + 0.9969458 + protein_type + cleaner0 + 2023-07-05T08:06:51Z + MESH: + + HATs + + + 0.99758315 + protein + cleaner0 + 2023-07-05T08:07:12Z + PR: + + HAT1 + + + 0.9978067 + protein + cleaner0 + 2023-07-05T08:07:16Z + PR: + + Gcn5 + + + 0.9977192 + protein + cleaner0 + 2023-07-05T08:07:20Z + PR: + + Rtt109 + + + 0.9930031 + protein_type + cleaner0 + 2023-07-05T08:07:06Z + MESH: + + HDACs + + + 0.99792385 + protein + cleaner0 + 2023-07-05T08:07:24Z + PR: + + Rpd3 + + + 0.99804014 + protein + cleaner0 + 2023-07-05T08:07:27Z + PR: + + Hos1 + + + 0.9978777 + protein + cleaner0 + 2023-07-05T08:07:31Z + PR: + + Hos2 + + + 0.53316224 + experimental_method + cleaner0 + 2023-07-05T09:00:08Z + MESH: + + deleted + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:11:11Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:55:34Z + + K9cr + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:58:28Z + + HAT + + + 0.47926757 + experimental_method + cleaner0 + 2023-07-05T09:00:12Z + MESH: + + deletion + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:58:22Z + + HDAC + + + experimental_method + MESH: + cleaner0 + 2023-07-05T09:04:29Z + + deletion + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:11:41Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:55:48Z + + K9cr + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:12:21Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:55:41Z + + K9ac + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:12:47Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:55:56Z + + K9cr + + + 0.9963509 + taxonomy_domain + cleaner0 + 2023-07-04T16:22:20Z + DUMMY: + + yeast + + + 0.9969043 + protein_type + cleaner0 + 2023-07-05T08:06:51Z + MESH: + + HATs + + + 0.9925954 + protein_type + cleaner0 + 2023-07-05T08:07:06Z + MESH: + + HDACs + + + + INTRO + paragraph + 7741 + We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). Binding of H3K9cr induced resonance changes in slow exchange regime on the NMR time scale, indicative of strong interaction. In contrast, binding of H3K9ac resulted in an intermediate exchange, which is characteristic of a weaker association. Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c). One of the conformations, characterized by the π stacking involving two aromatic residues and the alkene group, is observed only in the YEATS-H3K9cr complex. + + protein_state + DUMMY: + cleaner0 + 2023-07-04T16:22:11Z + + acetylated + + + protein_type + MESH: + cleaner0 + 2023-07-04T16:21:00Z + + histone + + + 0.9988348 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.9854789 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + 0.9957682 + protein_state + cleaner0 + 2023-07-04T16:22:11Z + DUMMY: + + acetylated + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:13:59Z + + H3 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-05T08:14:10Z + + K9 + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:14:26Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:56:09Z + + K9cr + + + 0.99873716 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.9983891 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + 0.9863785 + experimental_method + cleaner0 + 2023-07-05T08:18:57Z + MESH: + + 1H,15N HSQC + + + 0.990856 + chemical + cleaner0 + 2023-07-05T08:36:15Z + CHEBI: + + H3K9cr5-13 + + + 0.9934884 + chemical + cleaner0 + 2023-07-05T08:38:55Z + CHEBI: + + H3K9ac5-13 + + + 0.96406466 + experimental_method + cleaner0 + 2023-07-05T09:00:19Z + MESH: + + titrated + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T08:53:55Z + + 15N-labeled + + + 0.9987318 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.98387945 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:15:14Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:56:22Z + + K9cr + + + evidence + DUMMY: + cleaner0 + 2023-07-05T08:59:29Z + + resonance changes + + + experimental_method + MESH: + cleaner0 + 2023-07-05T09:04:29Z + + NMR + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:15:40Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:56:29Z + + K9ac + + + 0.49922895 + evidence + cleaner0 + 2023-07-05T08:56:27Z + DUMMY: + + crosspeaks + + + 0.99908006 + residue_name_number + cleaner0 + 2023-07-05T08:15:58Z + DUMMY: + + Gly80 + + + 0.9990513 + residue_name_number + cleaner0 + 2023-07-05T08:02:27Z + DUMMY: + + Trp81 + + + 0.98532 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:16:15Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:56:52Z + + K9cr + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:16:48Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:56:37Z + + K9ac + + + site + SO: + cleaner0 + 2023-07-05T08:13:40Z + + crotonyllysine and acetyllysine binding pockets + + + 0.998936 + residue_name_number + cleaner0 + 2023-07-05T08:02:27Z + DUMMY: + + Trp81 + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:17:15Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:57:01Z + + K9cr + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:17:45Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:56:45Z + + K9ac + + + 0.99369735 + complex_assembly + cleaner0 + 2023-07-05T08:18:01Z + GO: + + YEATS-H3K9cr + + + + INTRO + paragraph + 8976 + To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). As shown in Figure 2d and Supplementary Fig. 5a, the Taf14 YEATS domain binds more strongly to H3K9cr1-20, as compared to other acylated histone peptides. The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). We concluded that H3K9cr is the preferred target of this domain. From comparative structural analysis of the YEATS complexes, Gly80 emerged as candidate residue potentially responsible for the preference for crotonyllysine. In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. In contrast, mutation of Val24, a residue located on another side of Trp81, had no effect on binding (Fig. 2d and Supplementary Fig. 5a, c). + + 0.99862266 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.9849653 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + + acyllysine + + + 0.99509495 + experimental_method + cleaner0 + 2023-07-05T09:00:34Z + MESH: + + solution pull-down assays + + + 0.99270695 + protein_type + cleaner0 + 2023-07-05T07:55:54Z + MESH: + + H3 + + + 0.9932382 + protein_state + cleaner0 + 2023-07-04T16:22:11Z + DUMMY: + + acetylated + + + 0.956428 + protein_state + cleaner0 + 2023-07-05T08:40:11Z + DUMMY: + + propionylated + + + 0.9795297 + protein_state + cleaner0 + 2023-07-05T08:40:15Z + DUMMY: + + butyrylated + + + 0.97335297 + protein_state + cleaner0 + 2023-07-04T16:22:07Z + DUMMY: + + crotonylated + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-05T08:18:20Z + + lysine 9 + + + 0.9283707 + residue_range + cleaner0 + 2023-07-05T08:50:16Z + DUMMY: + + 1–20 + + + 0.951781 + protein_type + cleaner0 + 2023-07-05T07:55:54Z + MESH: + + H3 + + + 0.99854577 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.98509496 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + chemical + CHEBI: + cleaner0 + 2023-07-05T08:33:09Z + + H3K9cr1-20 + + + 0.78338593 + protein_state + cleaner0 + 2023-07-05T08:54:04Z + DUMMY: + + acylated + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:20:37Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:57:16Z + + K9cr + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:21:11Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:57:35Z + + K9ac + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:21:37Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:57:44Z + + K9pr + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:22:10Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:57:54Z + + K9bu + + + 0.98849183 + experimental_method + cleaner0 + 2023-07-05T08:19:00Z + MESH: + + 1H,15N HSQC titration experiments + + + chemical + CHEBI: + cleaner0 + 2023-07-05T08:33:21Z + + H3K9ac1-20 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T08:33:34Z + + H3K9pr1-20 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T08:33:45Z + + H3K9bu1-20 + + + evidence + DUMMY: + cleaner0 + 2023-07-05T09:01:01Z + + chemical shift perturbations + + + 0.9984143 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.9821952 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + chemical + CHEBI: + cleaner0 + 2023-07-05T08:33:09Z + + H3K9cr1-20 + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:22:49Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:58:04Z + + K9cr + + + 0.99294585 + experimental_method + cleaner0 + 2023-07-05T09:01:12Z + MESH: + + comparative structural analysis + + + 0.9990103 + residue_name_number + cleaner0 + 2023-07-05T08:15:59Z + DUMMY: + + Gly80 + + + 0.9981839 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-07-21T11:51:04Z + + crotonyl + + + 0.99031365 + protein_state + cleaner0 + 2023-07-05T08:49:33Z + DUMMY: + + mutated + + + 0.9989976 + residue_name_number + cleaner0 + 2023-07-05T08:15:59Z + DUMMY: + + Gly80 + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T09:01:51Z + + mutants of + + + 0.9989625 + residue_name_number + cleaner0 + 2023-07-05T08:15:59Z + DUMMY: + + Gly80 + + + 0.885494 + protein_state + cleaner0 + 2023-07-05T08:54:04Z + DUMMY: + + acylated + + + 0.9990196 + residue_name_number + cleaner0 + 2023-07-05T08:15:59Z + DUMMY: + + Gly80 + + + 0.9797934 + experimental_method + cleaner0 + 2023-07-05T09:01:59Z + MESH: + + mutation + + + 0.99903935 + residue_name_number + cleaner0 + 2023-07-05T08:24:25Z + DUMMY: + + Val24 + + + 0.9990657 + residue_name_number + cleaner0 + 2023-07-05T08:02:27Z + DUMMY: + + Trp81 + + + + INTRO + paragraph + 10484 + To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). We found that all YEATS domains tested are capable of binding to crotonyllysine peptides, though they display variable preferences for the acyl moieties. While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well. + + 0.99843556 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + 0.984743 + protein_state + cleaner0 + 2023-07-05T08:54:12Z + DUMMY: + + conserved + + + 0.99253696 + species + cleaner0 + 2023-07-04T16:22:25Z + MESH: + + human + + + structure_element + SO: + cleaner0 + 2023-07-05T08:49:39Z + + YEATS domains + + + 0.9765474 + experimental_method + cleaner0 + 2023-07-05T09:02:03Z + MESH: + + pull-down experiments + + + 0.9461614 + protein_state + cleaner0 + 2023-07-04T16:22:11Z + DUMMY: + + acetylated + + + 0.9879154 + protein_state + cleaner0 + 2023-07-05T08:40:11Z + DUMMY: + + propionylated + + + 0.9869178 + protein_state + cleaner0 + 2023-07-05T08:40:15Z + DUMMY: + + butyrylated + + + 0.9762997 + protein_state + cleaner0 + 2023-07-04T16:22:07Z + DUMMY: + + crotonylated + + + protein_type + MESH: + cleaner0 + 2023-07-05T09:02:17Z + + histone + + + structure_element + SO: + cleaner0 + 2023-07-05T08:49:39Z + + YEATS domains + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + + crotonyllysine + + + 0.9987778 + protein + cleaner0 + 2023-07-05T08:39:48Z + PR: + + YEATS2 + + + 0.99860966 + protein + cleaner0 + 2023-07-05T07:54:09Z + PR: + + ENL + + + 0.9837325 + protein_state + cleaner0 + 2023-07-04T16:22:07Z + DUMMY: + + crotonylated + + + 0.99857676 + protein + cleaner0 + 2023-07-05T08:39:56Z + PR: + + GAS41 + + + 0.9983223 + protein + cleaner0 + 2023-07-05T07:54:15Z + PR: + + AF9 + + + 0.99105185 + protein_state + cleaner0 + 2023-07-05T08:54:04Z + DUMMY: + + acylated + + + + INTRO + paragraph + 11007 + Unlike the YEATS domain, a known acetyllysine reader, bromodomain, does not recognize crotonyllysine. We assayed a large set of BDs in pull-down experiments and found that this module is highly specific for acetyllysine and propionyllysine containing peptides (Supplementary Fig. 7). However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports. These results demonstrate that the YEATS domain is currently the sole reader of crotonyllysine. + + 0.9855747 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + 0.80789137 + protein_type + cleaner0 + 2023-07-05T08:52:52Z + MESH: + + acetyllysine reader + + + 0.9922646 + structure_element + cleaner0 + 2023-07-05T08:52:07Z + SO: + + bromodomain + + + 0.99855334 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + 0.7588632 + structure_element + cleaner0 + 2023-07-04T16:21:53Z + SO: + + BDs + + + 0.9541701 + experimental_method + cleaner0 + 2023-07-05T09:02:34Z + MESH: + + pull-down experiments + + + 0.99803704 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + acetyllysine + + + 0.9968952 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + propionyllysine + + + 0.9787652 + structure_element + cleaner0 + 2023-07-04T16:21:48Z + SO: + + bromodomains + + + 0.99844176 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + 0.8739639 + structure_element + cleaner0 + 2023-07-04T16:21:53Z + SO: + + BDs + + + 0.99858606 + protein + cleaner0 + 2023-07-05T08:51:13Z + PR: + + TAF1 + + + 0.9986286 + protein + cleaner0 + 2023-07-05T08:51:16Z + PR: + + BRD2 + + + 0.98622835 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + 0.9985879 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + + INTRO + paragraph + 11579 + In conclusion, we have identified the YEATS domain of Taf14 as the first reader of histone crotonylation. The unique and previously unobserved aromatic-amide/aliphatic-aromatic π-π-π-stacking mechanism facilitates the specific recognition of the crotonyl moiety. We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs. As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. Furthermore, the functional significance of crotonyllysine recognition by other YEATS proteins will be of great importance to elucidate and compare. + + 0.99071395 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + 0.9987459 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.5421235 + protein_type + cleaner0 + 2023-07-04T16:21:00Z + MESH: + + histone + + + 0.672082 + ptm + cleaner0 + 2023-07-04T16:18:43Z + MESH: + + crotonylation + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + π-π-π-stacking + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-07-21T11:51:04Z + + crotonyl + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:41:07Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T12:01:20Z + + K9cr + + + 0.99646616 + taxonomy_domain + cleaner0 + 2023-07-04T16:22:20Z + DUMMY: + + yeast + + + 0.9973616 + protein_type + cleaner0 + 2023-07-05T08:06:51Z + MESH: + + HATs + + + 0.9948607 + protein_type + cleaner0 + 2023-07-05T08:07:06Z + MESH: + + HDACs + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + + acyllysine + + + 0.998776 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.9936651 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + + crotonyllysine + + + 0.9987233 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.99826485 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + 0.9959139 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + acetyllysine + + + residue_name + SO: + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + + crotonyllysine + + + 0.9981281 + protein_type + cleaner0 + 2023-07-05T08:52:37Z + MESH: + + YEATS + + + + METHODS + title_1 + 12449 + ONLINE METHODS + + + METHODS + title_2 + 12464 + Protein expression and purification + + + METHODS + paragraph + 12500 + The Taf14 YEATS constructs (residues 1–132 or 1–137) were expressed in E. coli BL21 (DE3) RIL in either Luria Broth or M19 minimal media supplemented with 15NH4Cl and purified as N-terminal GST fusion proteins. Cells were harvested by centrifugation and resuspended in 50 mM HEPES (pH 7.5) supplemented with 150 mM NaCl and 1 mM TCEP. Cells are lysed by freeze-thaw followed by sonication. Proteins were purified on glutathione Sepharose 4B beads and the GST tag was cleaved with PreScission protease. + + + METHODS + title_2 + 13006 + X-ray data collection and structure determination + + + METHODS + paragraph + 13056 + Taf14 YEATS (residues 1–137) was concentrated to 9 mg/mL in 25 mM MES (pH 6.5) and incubated with 2 molar equivalence of the H3K9cr5-13 at RT for 30 mins prior to crystallization. Crystals were obtain via sitting drop diffusion method at 18°C by mixing 800 nL of protein/peptide solution with 800 nL of well solution composed of 44% PEG600 (v/v) and 0.2 M citric acid (pH 6.0). X-ray diffraction data was collected at a wavelength of 1.54 Å at 100 K from a single crystal on the UC Denver Biophysical Core home source composed of a Rigaku Micromax 007 high frequency microfocus X-ray generator with a Pilatus 200K 2D area detector. HKL3000 was used for indexing, scaling, and data reduction. Solution was solved via molecular replacement with Phaser using the Taf14 YEATS domain (PDB 5D7E) as search model with waters, ligands, and peptide removed. Phenix was used for refinement of structure and waters were manually placed by inception of difference maps in Coot. Ramachandran plot indicates good stereochemistry of the three-dimensional structure with 100% of all residues falling within the favored (98%) and allowed (2%) regions. The crystallographic statistics are shown in Supplementary Table 1. + + + METHODS + title_2 + 14263 + NMR spectroscopy + + + METHODS + paragraph + 14280 + NMR spectroscopy was carried out on a Varian INOVA 600 MHz spectrometer outfitted with a cryogenic probe. Chemical shift perturbation (CSP) analysis was performed using uniformly 15N-labeled Taf14 (1–132). 1H,15N heteronuclear single quantum coherence (HSQC) spectra of the Taf14 YEATS domain were collected in the presence of increasing concentrations of either H3K9cr5-13, H3K9ac5-13, H3K9cr1-20, H3K9ac1-20 H3K9pr1-20, H3K9bu1-20 or free Kcr in PBS buffer pH 6.8, 8% D2O. + + + METHODS + title_2 + 14757 + Fluorescence binding assays + + + METHODS + paragraph + 14785 + Tryptophan fluorescence measurements were performed on a Fluorolog spectrofluorometer at room temperature as described. The samples containing 2 μM of Taf14 YEATS in PBS (pH 7.4) and increasing concentrations of H3K9cr5-13 were excited at 295 nm. Emission spectra were recorded from 310 to 340 nm with a 1 nm step size and a 0.5 sec integration time. The Kd value was determined using a nonlinear least-squares analysis and the equation: where [L] is the concentration of the peptide, [P] is the concentration of the protein, ΔI is the observed change of signal intensity, and ΔImax is the difference in signal intensity of the free and bound states. The Kd values were averaged over 3 separate experiments, with error calculated as the standard deviation (SD). + + + METHODS + title_2 + 15558 + Peptide pull-downs + + + METHODS + paragraph + 15577 + YEATS domains in pGEX vectors were expressed in SoluBL21 cells (Amsbio) by induction with 1 mM IPTG at 16–18°C overnight with shaking. Cells were lysed by freeze-thaw and sonication then purified over glutathione agarose (Pierce) in a buffer containing 50 mM Tris pH 8.0, 500 mM NaCl, 20% glycerol (v/v) and 1 mM dithiothreitol (DTT). Peptide pull-downs were performed essentially as described except that the assay buffer contained 50 mM Tris pH 8.0, 500 mM NaCl, and 0.1% NP-40, and 500 pmols of biotinylated histone peptides were loaded onto streptavidin coated magnetic beads before incubation with 40 pmols of protein. Bound proteins were detected with rabbit GST antibody (Sigma, G7781). Point mutants were generated by site-directed mutagenesis and purified/assayed as described above. The YEATS domains of Taf14, AF9, ENL, and GAS41 were previously described. + + + METHODS + title_2 + 16448 + Western blotting + + + METHODS + paragraph + 16465 + Yeast cultures were grown in YPD media at 30°C to mid-log phase and extracts were prepared as previously described. Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. Anti-H3K9ac (Millipore, 07-352) and anti-H3K9cr (PTM Biolabs, PTM-516) were diluted to 1:2000 and 1:1000, respectively, in 1x Superblock (ThermoScientific). An HRP-conjugated anti-rabbit (GE Healthcare) was used for detection. Bands were quantified using the ImageJ program. + + + METHODS + title_2 + 16947 + Dot blotting + + + METHODS + paragraph + 16960 + Increasing concentrations of biotinylated histone peptides (0.06–1.5 μg) were spotted onto a PVDF membrane then probed with the anti-H3K9ac (Millipore, 07-352) or H3K9cr (PTM Biolabs, PTM-516) at 1:2000 in a 5% non-fat milk solution and detected with an HRP-conjugated anti-rabbit by enhanced chemiluminesence (ECL). + + + METHODS + title_2 + 17280 + Bromodomains pull-downs + + + METHODS + paragraph + 17304 + cDNAs of GST-fused bromodomains were obtained either from EpiCypher Inc. or as a kind gift from Katrin Chua (Stanford University). GST fusions were expressed as described above except that the preparation buffer contained 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol (v/v), and 1 mM DTT. Pull-down assays were preformed as described above except that the assay buffer contained 50 mM Tris (pH 8.0), 300 mM NaCl, and 0.1% NP-40. + + + SUPPL + title_1 + 17734 + Supplementary Material + + + SUPPL + footnote + 17757 + Accession codes. Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5IOK. + + + SUPPL + footnote + 17881 + Author contributions + + + SUPPL + footnote + 17902 + F.H.A., S.A.S., E.K.S., J.B.B., A.G., I.K.T and K.K. performed experiments and together with X.S., B.D.S and T.G.K. analyzed the data. F.H.A., S.A.S., B.D.S. and T.G.K. wrote the manuscript with input from all authors. + + + SUPPL + footnote + 18121 + Competing Financial Interest + + + SUPPL + footnote + 18150 + The authors declare no competing financial interests. + + + SUPPL + footnote + 18204 + Additional information + + + SUPPL + footnote + 18227 + Any supplementary information is available in the online version of this paper. + + + 1016 + 28 + surname:Tan;given-names:M + 21925322 + REF + Cell + ref + 146 + 2011 + 18307 + Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification + + + 203 + 15 + surname:Sabari;given-names:BR + 25818647 + REF + Mol Cell + ref + 58 + 2015 + 18413 + Intracellular Crotonyl-CoA Stimulates Transcription through p300-Catalyzed Histone Crotonylation + + + 947 + 60 + surname:Lin;given-names:H + surname:Su;given-names:X + surname:He;given-names:B + 22571489 + REF + ACS Chem Biol + ref + 7 + 2012 + 18510 + Protein lysine acylation and cysteine succination by intermediates of energy metabolism + + + surname:Bao;given-names:X + REF + Elife + ref + 3 + 2014 + 18598 + Identification of ‘erasers’ for lysine crotonylated histone marks using a chemical proteomics approach + + + 1218 + 27 + surname:Musselman;given-names:CA + surname:Lalonde;given-names:ME + surname:Cote;given-names:J + surname:Kutateladze;given-names:TG + 23211769 + REF + Nat Struct Mol Biol + ref + 19 + 2012 + 18705 + Perceiving the epigenetic landscape through histone readers + + + 627 + 43 + surname:Rothbart;given-names:SB + surname:Strahl;given-names:BD + 24631868 + REF + Biochim Biophys Acta + ref + 1839 + 2014 + 18765 + Interpreting the language of histone and DNA modifications + + + 1801 + 14 + surname:Flynn;given-names:EM + 26365797 + REF + Structure + ref + 23 + 2015 + 18824 + A Subset of Human Bromodomains Recognizes Butyryllysine and Crotonyllysine Histone Peptide Modifications + + + 214 + 31 + surname:Filippakopoulos;given-names:P + 22464331 + REF + Cell + ref + 149 + 2012 + 18929 + Histone recognition and large-scale structural analysis of the human bromodomain family + + + 65 + 75 + surname:Schulze;given-names:JM + surname:Wang;given-names:AY + surname:Kobor;given-names:MS + 19234524 + REF + Biochem Cell Biol + ref + 87 + 2009 + 19017 + YEATS domain proteins: a diverse family with many links to chromatin modification and transcription + + + 558 + 71 + surname:Li;given-names:Y + 25417107 + REF + Cell + ref + 159 + 2014 + 19117 + AF9 YEATS domain links histone acetylation to DOT1L-mediated H3K79 methylation + + + 1795 + 800 + surname:Shanle;given-names:EK + 26341557 + REF + Genes Dev + ref + 29 + 2015 + 19196 + Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response + + + 398 + 403 + surname:Kabani;given-names:M + surname:Michot;given-names:K + surname:Boschiero;given-names:C + surname:Werner;given-names:M + 15896708 + REF + Biochem Biophys Res Commun + ref + 332 + 2005 + 19299 + Anc1 interacts with the catalytic subunits of the general transcription factors TFIID and TFIIF, the chromatin remodeling complexes RSC and INO80, and the histone acetyltransferase complex NuA3 + + + 401 + 12 + surname:Shen;given-names:X + 14979041 + REF + Methods Enzymol + ref + 377 + 2004 + 19493 + Preparation and analysis of the INO80 complex + + + 3308 + 16 + surname:Cairns;given-names:BR + surname:Henry;given-names:NL + surname:Kornberg;given-names:RD + 8668146 + REF + Mol Cell Biol + ref + 16 + 1996 + 19539 + TFG/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukemogenic proteins ENL and AF-9 + + + 1196 + 208 + surname:John;given-names:S + 10817755 + REF + Genes Dev + ref + 14 + 2000 + 19654 + The something about silencing protein, Sas3, is the catalytic subunit of NuA3, a yTAF(II)30-containing HAT complex that interacts with the Spt16 subunit of the yeast CP (Cdc68/Pob3)-FACT complex + + + 14 + 20 + surname:Andrews;given-names:FH + surname:Shanle;given-names:EK + surname:Strahl;given-names:BD + surname:Kutateladze;given-names:TG + 26934307 + REF + Transcription + ref + 7 + 2016 + 19849 + The essential role of acetyllysine binding by the YEATS domain in transcriptional regulation + + + 658 + 674 + surname:McCoy;given-names:AJ + 19461840 + REF + J Appl Crystallogr + ref + 40 + 2007 + 19942 + Phaser crystallographic software + + + 213 + 21 + surname:Adams;given-names:PD + 20124702 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 19975 + PHENIX: a comprehensive Python-based system for macromolecular structure solution + + + 486 + 501 + surname:Emsley;given-names:P + surname:Lohkamp;given-names:B + surname:Scott;given-names:WG + surname:Cowtan;given-names:K + 20383002 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 20057 + Features and development of Coot + + + 11296 + 301 + surname:Ali;given-names:M + 23798402 + REF + Proc Natl Acad Sci U S A + ref + 110 + 2013 + 20090 + Molecular basis for chromatin binding and regulation of MLL5 + + + 1155 + 60 + surname:Rothbart;given-names:SB + 23022729 + REF + Nat Struct Mol Biol + ref + 19 + 2012 + 20151 + Association of UHRF1 with methylated H3K9 directs the maintenance of DNA methylation + + + 1795 + 800 + surname:Shanle;given-names:EK + 26341557 + REF + Genes Dev + ref + 29 + 2015 + 20236 + Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response + + + 660 + 5 + surname:Keogh;given-names:MC + 16543219 + REF + Genes Dev + ref + 20 + 2006 + 20339 + The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4 + + + 497 + 501 + surname:Keogh;given-names:MC + 16299494 + REF + Nature + ref + 439 + 2006 + 20415 + A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery + + + nihms769551f1.jpg + F1 + FIG + fig_title_caption + 20510 + The structural mechanism for the recognition of H3K9cr + + protein_type + MESH: + cleaner0 + 2023-07-05T08:46:18Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:59:06Z + + K9cr + + + + nihms769551f1.jpg + F1 + FIG + fig_caption + 20565 + (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. (d) The π-π-π stacking mechanism involving the alkene moiety of crotonyllysine. + + 0.99838877 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + 0.99736893 + evidence + cleaner0 + 2023-07-05T08:56:31Z + DUMMY: + + crystal structure + + + 0.99835616 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.97807664 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + protein_state + DUMMY: + cleaner0 + 2023-07-05T08:54:40Z + + in complex with + + + 0.80304146 + chemical + cleaner0 + 2023-07-05T08:36:15Z + CHEBI: + + H3K9cr5-13 + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:46:58Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:58:57Z + + K9cr + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + electrostatic and polar interactions + + + 0.99865216 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.9790957 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:44Z + + π-π-π stacking + + + 0.9983652 + residue_name + melaniev@ebi.ac.uk + 2023-07-06T15:25:20Z + SO: + + crotonyllysine + + + + nihms769551f2.jpg + F2 + FIG + fig_title_caption + 20932 + H3K9cr is a selective target of the Taf14 YEATS domain + + protein_type + MESH: + cleaner0 + 2023-07-05T08:47:40Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:59:33Z + + K9cr + + + 0.9988475 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.9892417 + structure_element + cleaner0 + 2023-07-04T16:20:28Z + SO: + + YEATS domain + + + + nihms769551f2.jpg + F2 + FIG + fig_caption + 20987 + (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. Total H3 was used as a loading control. (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. Spectra are color coded according to the protein:peptide molar ratio. (d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides. + + 0.98307806 + experimental_method + cleaner0 + 2023-07-05T09:03:32Z + MESH: + + Western blot + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:48:14Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T11:59:41Z + + K9cr + + + protein_type + MESH: + cleaner0 + 2023-07-05T08:48:42Z + + H3 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-21T12:00:02Z + + K9ac + + + 0.9924296 + protein_state + cleaner0 + 2023-07-05T08:49:13Z + DUMMY: + + wild type + + + 0.98962665 + protein_state + cleaner0 + 2023-07-05T08:49:18Z + DUMMY: + + WT + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-07-21T12:00:29Z + + HAT + + + protein_type + MESH: + cleaner0 + 2023-07-05T09:03:46Z + + HDAC + + + experimental_method + MESH: + cleaner0 + 2023-07-05T09:04:00Z + + deletion + + + 0.99134904 + taxonomy_domain + cleaner0 + 2023-07-04T16:22:20Z + DUMMY: + + yeast + + + 0.9896226 + protein_type + cleaner0 + 2023-07-05T07:55:54Z + MESH: + + H3 + + + 0.90694416 + experimental_method + cleaner0 + 2023-07-05T09:03:35Z + MESH: + + 1H,15N HSQC + + + 0.65750146 + evidence + cleaner0 + 2023-07-05T08:57:05Z + DUMMY: + + spectra + + + 0.9981918 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.99271107 + structure_element + cleaner0 + 2023-07-05T08:52:13Z + SO: + + YEATS + + + 0.7894704 + chemical + cleaner0 + 2023-07-05T08:36:15Z + CHEBI: + + H3K9cr5-13 + + + chemical + CHEBI: + cleaner0 + 2023-07-05T08:38:55Z + + H3K9ac5-13 + + + experimental_method + MESH: + cleaner0 + 2023-07-05T09:04:10Z + + titrated + + + 0.6348235 + evidence + cleaner0 + 2023-07-05T08:57:07Z + DUMMY: + + Spectra + + + 0.98869205 + experimental_method + cleaner0 + 2023-07-05T08:49:22Z + MESH: + + Western blot + + + 0.99291915 + experimental_method + cleaner0 + 2023-07-05T08:49:24Z + MESH: + + peptide pull-down assays + + + 0.99699277 + protein_state + cleaner0 + 2023-07-05T08:49:30Z + DUMMY: + + wild-type + + + 0.959159 + protein_state + cleaner0 + 2023-07-05T08:49:33Z + DUMMY: + + mutated + + + 0.9981369 + protein + cleaner0 + 2023-07-04T16:20:22Z + PR: + + Taf14 + + + 0.981699 + structure_element + cleaner0 + 2023-07-05T08:49:39Z + SO: + + YEATS domains + + + + diff --git a/BioC_XML/4872110_v0.xml b/BioC_XML/4872110_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..383c406f7db661cf5223ca57093203bfafe52546 --- /dev/null +++ b/BioC_XML/4872110_v0.xml @@ -0,0 +1,12951 @@ + + + + PMC + 20201219 + pmc.key + + 4872110 + CC BY-NC + no + 0 + 0 + + 10.1093/nar/gkw244 + 4872110 + 27084949 + 4304 + 9 + This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com + 4316 + surname:Meyer;given-names:Britta + surname:Wurm;given-names:Jan Philip + surname:Sharma;given-names:Sunny + surname:Immer;given-names:Carina + surname:Pogoryelov;given-names:Denys + surname:Kötter;given-names:Peter + surname:Lafontaine;given-names:Denis L. J. + surname:Wöhnert;given-names:Jens + surname:Entian;given-names:Karl-Dieter + TITLE + front + 44 + 2016 + 0 + Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans + + 0.9939031 + protein_type + cleaner0 + 2023-07-04T14:27:44Z + MESH: + + Ribosome biogenesis factor + + + 0.99808204 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.9947394 + protein_type + cleaner0 + 2023-07-04T12:19:28Z + MESH: + + aminocarboxypropyl transferase + + + 0.9733748 + chemical + cleaner0 + 2023-07-04T12:17:53Z + CHEBI: + + 18S rRNA + + + 0.9940844 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:25Z + DUMMY: + + yeast + + + 0.9955108 + species + cleaner0 + 2023-07-04T12:18:31Z + MESH: + + humans + + + + ABSTRACT + abstract + 133 + The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes. + + 0.996774 + taxonomy_domain + cleaner0 + 2023-07-04T12:20:32Z + DUMMY: + + eukaryotic + + + 0.3907103 + chemical + cleaner0 + 2023-07-04T12:17:20Z + CHEBI: + + rRNA + + + 0.9956499 + protein_state + cleaner0 + 2023-07-04T16:03:17Z + DUMMY: + + conserved + + + 0.9075137 + protein_state + cleaner0 + 2023-07-04T14:38:36Z + DUMMY: + + hypermodified + + + 0.9943597 + chemical + cleaner0 + 2023-07-04T14:29:32Z + CHEBI: + + nucleotide + + + 0.9960326 + chemical + cleaner0 + 2023-07-04T12:18:06Z + CHEBI: + + N1-methyl-N3-aminocarboxypropyl-pseudouridine + + + 0.97784287 + chemical + cleaner0 + 2023-07-04T12:18:11Z + CHEBI: + + m1acp3Ψ + + + site + SO: + cleaner0 + 2023-07-04T14:28:59Z + + P-site + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:29:09Z + + tRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:17:54Z + + 18S rRNA + + + 0.99749947 + chemical + cleaner0 + 2023-07-04T12:30:02Z + CHEBI: + + S-adenosylmethionine + + + 0.99658644 + chemical + cleaner0 + 2023-07-04T14:29:37Z + CHEBI: + + aminocarboxypropyl + + + 0.9577856 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.99857605 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.9746249 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:25Z + DUMMY: + + yeast + + + 0.9947389 + species + cleaner0 + 2023-07-04T12:27:15Z + MESH: + + human + + + protein + PR: + cleaner0 + 2023-07-04T14:08:01Z + + Tsr3 + + + 0.59193677 + protein_state + cleaner0 + 2023-07-04T16:03:23Z + DUMMY: + + mutants + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:18:51Z + + 20S pre-rRNA + + + 0.9973525 + evidence + cleaner0 + 2023-07-04T15:08:12Z + DUMMY: + + crystal structure + + + 0.9966714 + taxonomy_domain + cleaner0 + 2023-07-04T12:34:33Z + DUMMY: + + archaeal + + + 0.985493 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.9943765 + protein_type + cleaner0 + 2023-07-04T12:19:34Z + MESH: + + SPOUT-class RNA-methyltransferases + + + 0.78219205 + site + cleaner0 + 2023-07-04T12:19:43Z + SO: + + SAM binding mode + + + 0.71633095 + site + cleaner0 + 2023-07-04T12:19:44Z + SO: + + SAM binding mode + + + 0.99791557 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.9964366 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + 0.9982572 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + 0.9923056 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.7530078 + protein_type + cleaner0 + 2023-07-04T12:36:04Z + MESH: + + acp transferase + + + + INTRO + title_1 + 1127 + INTRODUCTION + + + INTRO + paragraph + 1140 + Eukaryotic ribosome biogenesis is highly complex and requires a large number of non-ribosomal proteins and small non-coding RNAs in addition to ribosomal RNAs (rRNAs) and proteins. An increasing number of diseases—so called ribosomopathies—are associated with disturbed ribosome biogenesis. + + 0.996716 + taxonomy_domain + cleaner0 + 2023-07-04T12:20:32Z + DUMMY: + + Eukaryotic + + + 0.99148196 + chemical + cleaner0 + 2023-07-04T14:29:53Z + CHEBI: + + small non-coding RNAs + + + 0.99014926 + chemical + cleaner0 + 2023-07-04T14:29:57Z + CHEBI: + + ribosomal RNAs + + + 0.9890884 + chemical + cleaner0 + 2023-07-04T14:30:00Z + CHEBI: + + rRNAs + + + + INTRO + paragraph + 1435 + During eukaryotic ribosome biogenesis several dozens of rRNA nucleotides become chemically modified. The most abundant rRNA modifications are methylations at the 2′-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs). In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes. In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA. While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. + + 0.99573874 + taxonomy_domain + cleaner0 + 2023-07-04T12:20:32Z + DUMMY: + + eukaryotic + + + 0.8191487 + chemical + cleaner0 + 2023-07-04T12:20:15Z + CHEBI: + + rRNA + + + 0.9955368 + chemical + cleaner0 + 2023-07-04T14:30:03Z + CHEBI: + + nucleotides + + + 0.3742851 + chemical + cleaner0 + 2023-07-04T14:12:44Z + CHEBI: + + rRNA + + + 0.92576516 + ptm + cleaner0 + 2023-07-04T15:56:23Z + MESH: + + methylations + + + 0.75269485 + chemical + cleaner0 + 2023-07-04T14:30:08Z + CHEBI: + + ribose + + + 0.55292654 + chemical + cleaner0 + 2023-07-04T14:30:15Z + CHEBI: + + uridine + + + 0.94387376 + chemical + cleaner0 + 2023-07-04T12:27:44Z + CHEBI: + + pseudouridine + + + 0.85888165 + complex_assembly + cleaner0 + 2023-07-04T12:20:49Z + GO: + + small nucleolar ribonucleoprotein particles + + + 0.8105936 + complex_assembly + cleaner0 + 2023-07-04T12:20:53Z + GO: + + snoRNPs + + + 0.6118344 + chemical + cleaner0 + 2023-07-04T12:21:09Z + CHEBI: + + 18S + + + 0.39413956 + chemical + cleaner0 + 2023-07-04T12:21:18Z + CHEBI: + + 25S + + + 0.9883475 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:25Z + DUMMY: + + yeast + + + 0.52910745 + chemical + cleaner0 + 2023-07-04T12:21:28Z + CHEBI: + + 28S + + + 0.9891657 + species + cleaner0 + 2023-07-04T12:18:31Z + MESH: + + humans + + + 0.29764622 + chemical + cleaner0 + 2023-07-04T14:30:19Z + CHEBI: + + rRNAs + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:21:56Z + + snoRNA + + + 0.9952312 + species + cleaner0 + 2023-07-04T12:22:09Z + MESH: + + Saccharomyces cerevisiae + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:17:54Z + + 18S rRNA + + + 0.81744164 + ptm + cleaner0 + 2023-07-04T15:56:27Z + MESH: + + methylations + + + 0.9603707 + ptm + cleaner0 + 2023-07-04T15:56:32Z + MESH: + + acetylations + + + chemical + CHEBI: + cleaner0 + 2023-07-04T13:52:12Z + + 3-amino-3-carboxypropyl + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.8290558 + ptm + cleaner0 + 2023-07-04T15:56:36Z + MESH: + + methylations + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:23:02Z + + 25S rRNA + + + 0.9947161 + species + cleaner0 + 2023-07-04T12:18:31Z + MESH: + + humans + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:17:55Z + + 18S rRNA + + + 0.99721646 + protein_state + cleaner0 + 2023-07-04T16:03:48Z + DUMMY: + + highly conserved + + + 0.99383414 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:25Z + DUMMY: + + yeast + + + 0.9987739 + protein + cleaner0 + 2023-07-04T12:26:28Z + PR: + + ScRrp8 + + + 0.9982759 + protein + cleaner0 + 2023-07-04T12:26:36Z + PR: + + HsNML + + + 0.9987858 + protein + cleaner0 + 2023-07-04T12:26:42Z + PR: + + ScRcm1 + + + 0.99836296 + protein + cleaner0 + 2023-07-04T12:26:47Z + PR: + + HsNSUN5 + + + 0.9988186 + protein + cleaner0 + 2023-07-04T12:26:52Z + PR: + + ScNop2 + + + 0.9983693 + protein + cleaner0 + 2023-07-04T12:26:57Z + PR: + + HsNSUN1 + + + 0.9948597 + species + cleaner0 + 2023-07-04T12:27:14Z + MESH: + + human + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:22:27Z + + 28S rRNA + + + + INTRO + paragraph + 2321 + Ribosomal RNA modifications have been suggested to optimize ribosome function, although in most cases this remains to be clearly established. They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations). Most modified rRNA nucleotides cluster in the vicinity of the decoding or the peptidyl transferase center, suggesting an influence on ribosome functionality and stability. Defects of rRNA modification enzymes often lead to disturbed ribosome biogenesis or functionally impaired ribosomes, although the lack of individual rRNA modifications often has no or only a slight influence on the cell. Importantly, malfunctions of several base modifying enzymes are linked to developmental diseases, aging or tumorigenesis. + + 0.6964196 + chemical + cleaner0 + 2023-07-04T14:30:23Z + CHEBI: + + Ribosomal RNA + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:24:16Z + + RNA + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:28Z + + hydrogen bonds + + + 0.9017562 + chemical + cleaner0 + 2023-07-04T12:28:01Z + CHEBI: + + pseudouridines + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:28Z + + base stacking + + + 0.8979499 + chemical + cleaner0 + 2023-07-04T12:28:02Z + CHEBI: + + pseudouridines + + + 0.9240885 + ptm + cleaner0 + 2023-07-04T15:56:41Z + MESH: + + base methylations + + + ptm + MESH: + cleaner0 + 2023-07-04T15:57:20Z + + ribose methylations + + + 0.5139551 + chemical + cleaner0 + 2023-07-04T12:23:53Z + CHEBI: + + rRNA + + + 0.94696105 + chemical + cleaner0 + 2023-07-04T14:30:27Z + CHEBI: + + nucleotides + + + 0.5629403 + site + cleaner0 + 2023-07-04T15:16:28Z + SO: + + decoding + + + 0.9937131 + site + cleaner0 + 2023-07-04T15:16:31Z + SO: + + peptidyl transferase center + + + 0.6016872 + chemical + cleaner0 + 2023-07-04T12:24:06Z + CHEBI: + + rRNA + + + 0.6801251 + chemical + cleaner0 + 2023-07-04T14:30:34Z + CHEBI: + + rRNA + + + + INTRO + paragraph + 3215 + The chemically most complex modification is located in the loop capping helix 31 of 18S rRNA (Supplementary Figure S1B). There a uridine (U1191 in yeast) is modified to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ, Figure 1A). This base modification was first described in 1968 for hamster cells and is conserved in eukaryotes. This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. In a second step, the essential SPOUT-class methyltransferase Nep1/Emg1 modifies the pseudouridine to N1-methylpseudouridine. Methylation can only occur once pseudouridylation has taken place, as the latter reaction generates the substrate for the former. The final acp modification leading to N1-methyl-N3-aminocarboxypropyl-pseudouridine occurs late during 40S biogenesis in the cytoplasm, while the two former reactions are taking place in the nucleolus and nucleus, and is independent from pseudouridylation or methylation. Both the methyl and the acp group are derived from S-adenosylmethionine (SAM), but the enzyme responsible for acp modification remained elusive for more than 40 years. + + 0.90391713 + structure_element + cleaner0 + 2023-07-04T15:58:02Z + SO: + + loop capping helix 31 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:17:55Z + + 18S rRNA + + + 0.5255018 + residue_name + cleaner0 + 2023-07-04T15:26:56Z + SO: + + uridine + + + 0.9984127 + residue_name_number + cleaner0 + 2023-07-04T15:01:12Z + DUMMY: + + U1191 + + + 0.9965745 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:25Z + DUMMY: + + yeast + + + 0.9967144 + chemical + cleaner0 + 2023-07-04T12:24:41Z + CHEBI: + + 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine + + + 0.9299609 + chemical + cleaner0 + 2023-07-04T12:18:12Z + CHEBI: + + m1acp3Ψ + + + 0.8263748 + taxonomy_domain + cleaner0 + 2023-07-04T12:24:54Z + DUMMY: + + hamster + + + 0.90488756 + protein_state + cleaner0 + 2023-07-04T16:03:57Z + DUMMY: + + conserved in + + + 0.9952035 + taxonomy_domain + cleaner0 + 2023-07-04T14:58:46Z + DUMMY: + + eukaryotes + + + 0.7234122 + protein_state + cleaner0 + 2023-07-04T14:38:36Z + DUMMY: + + hypermodified + + + 0.99725276 + chemical + cleaner0 + 2023-07-04T14:30:39Z + CHEBI: + + nucleotide + + + 0.9953161 + site + cleaner0 + 2023-07-04T15:16:37Z + SO: + + P-site + + + 0.38184616 + chemical + cleaner0 + 2023-07-04T14:30:43Z + CHEBI: + + tRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:08:48Z + + snR35 + + + structure_element + SO: + cleaner0 + 2023-07-04T14:04:38Z + + H/ACA + + + 0.9066868 + complex_assembly + cleaner0 + 2023-07-04T12:29:16Z + GO: + + snoRNP + + + 0.8301994 + chemical + cleaner0 + 2023-07-04T14:30:46Z + CHEBI: + + uridine + + + 0.96616 + chemical + cleaner0 + 2023-07-04T12:27:45Z + CHEBI: + + pseudouridine + + + 0.9949054 + protein_type + cleaner0 + 2023-07-04T13:56:16Z + MESH: + + SPOUT-class methyltransferase + + + 0.99870765 + protein + cleaner0 + 2023-07-04T14:39:37Z + PR: + + Nep1 + + + 0.99626595 + protein + cleaner0 + 2023-07-04T14:39:44Z + PR: + + Emg1 + + + 0.9889643 + chemical + cleaner0 + 2023-07-04T12:27:45Z + CHEBI: + + pseudouridine + + + 0.9949991 + chemical + cleaner0 + 2023-07-04T14:30:51Z + CHEBI: + + N1-methylpseudouridine + + + ptm + MESH: + cleaner0 + 2023-07-04T14:40:35Z + + Methylation + + + 0.9898722 + ptm + cleaner0 + 2023-07-04T15:57:29Z + MESH: + + pseudouridylation + + + 0.6104194 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + 0.99630696 + chemical + cleaner0 + 2023-07-04T14:30:59Z + CHEBI: + + N1-methyl-N3-aminocarboxypropyl-pseudouridine + + + 0.9727791 + complex_assembly + cleaner0 + 2023-07-04T12:48:58Z + GO: + + 40S + + + 0.98769104 + ptm + cleaner0 + 2023-07-04T15:57:32Z + MESH: + + pseudouridylation + + + 0.99729675 + chemical + cleaner0 + 2023-07-04T12:30:01Z + CHEBI: + + S-adenosylmethionine + + + 0.9979736 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + 0.90244967 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + + gkw244fig1.jpg + F1 + FIG + fig_caption + 4441 + Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. The asterisk indicates the C1-atom labeled in the 14C-incorporation assay. (B) RP-HPLC elution profile of yeast 18S rRNA nucleosides. Hypermodified m1acp3Ψ elutes at 7.4 min (wild type, left profile) and is missing in Δtsr3 (middle profile) and Δnep1 Δnop6 mutants (right profile). (C) 14C-acp labeling of 18S rRNAs. Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). Upper lanes show the ethidium bromide staining of the 18S rRNAs for quantification. All samples were loaded on the gel with two different amounts of 5 and 10 μl. (D) Primer extension analysis of acp modification in yeast 18S rRNA (right gel) including a sequencing ladder (left gel). The primer extension stop at nucleotide 1191 is missing exclusively in Δtsr3 mutants and Δtsr3 Δsnr35 recombinants. (E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel). The primer extension arrest is reduced in HTC116 cells transfected with siRNAs 544 and 545. The efficiency of siRNA mediated HsTSR3 repression correlates with the primer extension signals (see Supplementary Figure S2A). As a loading control, a structural stop is shown (asterisks). + + 0.9977913 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.66196173 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.98787004 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:25Z + DUMMY: + + yeast + + + 0.99423885 + species + cleaner0 + 2023-07-04T12:27:15Z + MESH: + + human + + + 0.48301774 + protein_state + cleaner0 + 2023-07-04T14:38:36Z + DUMMY: + + Hypermodified + + + 0.9966366 + chemical + cleaner0 + 2023-07-04T14:31:26Z + CHEBI: + + nucleotide + + + 0.9877445 + chemical + cleaner0 + 2023-07-04T12:18:12Z + CHEBI: + + m1acp3Ψ + + + 0.98037624 + ptm + cleaner0 + 2023-07-04T15:57:36Z + MESH: + + pseudouridylation + + + 0.99872464 + complex_assembly + cleaner0 + 2023-07-04T12:33:33Z + GO: + + snoRNP35 + + + 0.810374 + ptm + cleaner0 + 2023-07-04T15:57:38Z + MESH: + + N1-methylation + + + 0.96947104 + protein_type + cleaner0 + 2023-07-04T13:56:57Z + MESH: + + methyltransferase + + + 0.99881953 + protein + cleaner0 + 2023-07-04T14:13:21Z + PR: + + Nep1 + + + 0.4573778 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + 0.99871767 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.9611237 + experimental_method + cleaner0 + 2023-07-04T15:30:01Z + MESH: + + 14C-incorporation assay + + + 0.9936349 + experimental_method + cleaner0 + 2023-07-04T15:30:06Z + MESH: + + RP-HPLC + + + 0.862252 + evidence + cleaner0 + 2023-07-04T15:08:23Z + DUMMY: + + elution profile + + + 0.99435884 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:25Z + DUMMY: + + yeast + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:17:55Z + + 18S rRNA + + + 0.9959521 + chemical + cleaner0 + 2023-07-04T14:36:41Z + CHEBI: + + nucleosides + + + 0.88696 + protein_state + cleaner0 + 2023-07-04T14:38:36Z + DUMMY: + + Hypermodified + + + 0.9843954 + chemical + cleaner0 + 2023-07-04T12:18:12Z + CHEBI: + + m1acp3Ψ + + + 0.9969202 + protein_state + cleaner0 + 2023-07-04T12:31:11Z + DUMMY: + + wild type + + + 0.99864334 + mutant + cleaner0 + 2023-07-04T12:32:05Z + MESH: + + Δtsr3 + + + mutant + MESH: + cleaner0 + 2023-07-04T13:24:04Z + + Δnep1 Δnop6 + + + 0.9283411 + chemical + cleaner0 + 2023-07-04T14:36:53Z + CHEBI: + + 14C-acp + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:33:54Z + + 18S rRNAs + + + 0.9971446 + protein_state + cleaner0 + 2023-07-04T12:31:11Z + DUMMY: + + Wild type + + + 0.991848 + protein_state + cleaner0 + 2023-07-04T12:31:16Z + DUMMY: + + WT + + + 0.59906983 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.9985305 + mutant + cleaner0 + 2023-07-04T12:31:47Z + MESH: + + U1191U + + + 0.98280305 + chemical + cleaner0 + 2023-07-04T14:36:57Z + CHEBI: + + 14C-acp + + + 0.9748735 + chemical + cleaner0 + 2023-07-04T14:37:00Z + CHEBI: + + 14C-acp + + + 0.99853575 + mutant + cleaner0 + 2023-07-04T13:24:17Z + MESH: + + U1191A + + + 0.9680972 + protein_state + cleaner0 + 2023-07-04T16:04:06Z + DUMMY: + + mutant + + + 0.64080244 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.9984263 + mutant + cleaner0 + 2023-07-04T13:24:17Z + MESH: + + U1191A + + + mutant + MESH: + cleaner0 + 2023-07-04T12:32:05Z + + Δtsr3 + + + 0.9984206 + mutant + cleaner0 + 2023-07-04T12:32:05Z + MESH: + + Δtsr3 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:38:51Z + + ethidium bromide + + + 0.6604001 + chemical + cleaner0 + 2023-07-04T12:33:54Z + CHEBI: + + 18S rRNAs + + + 0.82524186 + experimental_method + cleaner0 + 2023-07-04T15:30:12Z + MESH: + + Primer extension analysis + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.9893718 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.6189569 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.86530745 + residue_number + cleaner0 + 2023-07-04T15:32:51Z + DUMMY: + + 1191 + + + mutant + MESH: + cleaner0 + 2023-07-04T12:32:05Z + + Δtsr3 + + + mutant + MESH: + cleaner0 + 2023-07-04T12:46:37Z + + Δtsr3 Δsnr35 + + + 0.9750293 + experimental_method + cleaner0 + 2023-07-04T15:30:16Z + MESH: + + Primer extension analysis + + + 0.9907601 + species + cleaner0 + 2023-07-04T12:27:15Z + MESH: + + human + + + 0.62276906 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.9582116 + experimental_method + cleaner0 + 2023-07-04T15:30:21Z + MESH: + + siRNA knockdown + + + 0.99881566 + protein + cleaner0 + 2023-07-04T12:30:51Z + PR: + + HsNEP1 + + + 0.99794334 + protein + cleaner0 + 2023-07-04T13:57:13Z + PR: + + EMG1 + + + 0.99872357 + protein + cleaner0 + 2023-07-04T12:30:27Z + PR: + + HsTSR3 + + + 0.6693486 + chemical + cleaner0 + 2023-07-04T15:30:34Z + CHEBI: + + siRNAs + + + 0.8873454 + chemical + cleaner0 + 2023-07-04T12:32:52Z + CHEBI: + + siRNA + + + 0.9972561 + protein + cleaner0 + 2023-07-04T12:30:26Z + PR: + + HsTSR3 + + + 0.67660683 + evidence + cleaner0 + 2023-07-04T15:08:32Z + DUMMY: + + primer extension signals + + + + INTRO + paragraph + 6133 + Only a few acp transferring enzymes have been characterized until now. During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. Archaeal Tyw2 has a structure very similar to Rossmann-fold (class I) RNA-methyltransferases, but its distinctive SAM-binding mode enables the transfer of the acp group instead of the methyl group of the cofactor. Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism. + + 0.689649 + chemical + cleaner0 + 2023-07-04T12:34:58Z + CHEBI: + + acp + + + 0.9988757 + chemical + cleaner0 + 2023-07-04T14:37:17Z + CHEBI: + + wybutosine + + + 0.60589993 + chemical + cleaner0 + 2023-07-04T14:37:22Z + CHEBI: + + nucleoside + + + 0.99640274 + taxonomy_domain + cleaner0 + 2023-07-04T12:20:33Z + DUMMY: + + eukaryotic + + + 0.9962257 + taxonomy_domain + cleaner0 + 2023-07-04T12:34:33Z + DUMMY: + + archaeal + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:37:48Z + + phenylalanine + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:38:00Z + + tRNA + + + 0.9990005 + protein + cleaner0 + 2023-07-04T14:13:38Z + PR: + + Tyw2 + + + 0.99896145 + protein + cleaner0 + 2023-07-04T12:35:12Z + PR: + + Trm12 + + + 0.9952987 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.99641234 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + 0.9981183 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + 0.99501246 + taxonomy_domain + cleaner0 + 2023-07-04T12:34:33Z + DUMMY: + + Archaeal + + + 0.9989735 + protein + cleaner0 + 2023-07-04T14:13:49Z + PR: + + Tyw2 + + + 0.9959848 + evidence + cleaner0 + 2023-07-04T15:08:39Z + DUMMY: + + structure + + + protein_type + MESH: + cleaner0 + 2023-07-04T13:58:12Z + + Rossmann-fold (class I) RNA-methyltransferases + + + 0.84350294 + site + cleaner0 + 2023-07-04T15:17:12Z + SO: + + SAM-binding mode + + + 0.9315737 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.8116939 + chemical + cleaner0 + 2023-07-04T14:38:06Z + CHEBI: + + diphtamide + + + 0.9980925 + chemical + cleaner0 + 2023-07-04T12:37:15Z + CHEBI: + + acp + + + 0.9985505 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + 0.9903178 + residue_name + cleaner0 + 2023-07-04T15:27:03Z + SO: + + histidine + + + 0.99253607 + taxonomy_domain + cleaner0 + 2023-07-04T12:20:33Z + DUMMY: + + eukaryotic + + + 0.9430889 + protein_type + cleaner0 + 2023-07-04T13:52:57Z + MESH: + + translation elongation factor 2 + + + + INTRO + paragraph + 6861 + In a recent bioinformatic study, the uncharacterized yeast gene YOR006c was predicted to be involved in ribosome biogenesis. It is highly conserved among eukaryotes and archaea (Supplementary Figure S1A) and its deletion leads to an accumulation of the 20S pre-rRNA precursor of 18S rRNA, suggesting an influence on D-site cleavage during the maturation of the small ribosomal subunit. On this basis, YOR006C was renamed ‘Twenty S rRNA accumulation 3′ (TSR3). However, its function remained unclear although recently a putative nuclease function during 18S rRNA maturation was predicted. + + 0.9967096 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.9333779 + gene + cleaner0 + 2023-07-04T12:35:31Z + GENE: + + YOR006c + + + 0.99700546 + protein_state + cleaner0 + 2023-07-04T16:04:14Z + DUMMY: + + highly conserved + + + 0.99592763 + taxonomy_domain + cleaner0 + 2023-07-04T14:58:51Z + DUMMY: + + eukaryotes + + + 0.9961767 + taxonomy_domain + cleaner0 + 2023-07-04T12:35:37Z + DUMMY: + + archaea + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:35:58Z + + 20S pre-rRNA + + + 0.99367166 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + site + SO: + cleaner0 + 2023-07-04T12:37:54Z + + D-site + + + 0.95902884 + gene + cleaner0 + 2023-07-04T12:39:20Z + GENE: + + YOR006C + + + protein + PR: + cleaner0 + 2023-07-04T14:14:03Z + + Twenty S rRNA accumulation 3 + + + 0.9505563 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + TSR3 + + + 0.9926686 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + + INTRO + paragraph + 7453 + Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3Ψ in yeast and human cells. Furthermore using catalytically defective mutants of yeast Tsr3 we demonstrated that the acp modification is required for 18S rRNA maturation. Surprisingly, the crystal structures of archaeal homologs revealed that Tsr3 is structurally similar to the SPOUT-class RNA methyltransferases. In contrast, the only other structurally characterized acp transferase enzyme Tyw2 belongs to the Rossmann-fold class of methyltransferase proteins. Interestingly, the two structurally very different enzymes use similar strategies in binding the SAM-cofactor in order to ensure that in contrast to methyltransferases the acp and not the methyl group of SAM is transferred to the substrate. + + 0.99876356 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.99231577 + protein_type + cleaner0 + 2023-07-04T12:36:03Z + MESH: + + acp transferase + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T14:38:36Z + + hypermodified + + + 0.99090844 + chemical + cleaner0 + 2023-07-04T14:38:16Z + CHEBI: + + nucleotide + + + 0.9904343 + chemical + cleaner0 + 2023-07-04T12:18:12Z + CHEBI: + + m1acp3Ψ + + + 0.97093236 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.99499077 + species + cleaner0 + 2023-07-04T12:27:15Z + MESH: + + human + + + 0.9358644 + protein_state + cleaner0 + 2023-07-04T16:04:21Z + DUMMY: + + catalytically defective + + + 0.97263247 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.9987662 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.9625655 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + 0.92116916 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.9973514 + evidence + cleaner0 + 2023-07-04T15:08:47Z + DUMMY: + + crystal structures + + + 0.9961379 + taxonomy_domain + cleaner0 + 2023-07-04T12:34:33Z + DUMMY: + + archaeal + + + 0.9986999 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.98849475 + protein_type + cleaner0 + 2023-07-04T12:36:16Z + MESH: + + SPOUT-class RNA methyltransferases + + + 0.99062526 + protein_type + cleaner0 + 2023-07-04T12:36:04Z + MESH: + + acp transferase + + + 0.9989262 + protein + cleaner0 + 2023-07-04T14:14:17Z + PR: + + Tyw2 + + + protein_type + MESH: + cleaner0 + 2023-07-04T14:14:42Z + + Rossmann-fold class of methyltransferase proteins + + + 0.99854136 + chemical + cleaner0 + 2023-07-04T12:36:25Z + CHEBI: + + SAM + + + 0.9936279 + protein_type + cleaner0 + 2023-07-04T12:36:21Z + MESH: + + methyltransferases + + + 0.9980083 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + 0.9983473 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + + METHODS + title_1 + 8305 + MATERIALS AND METHODS + + + METHODS + title_2 + 8327 + Genetic constructions, growth conditions and yeast media + + + METHODS + paragraph + 8384 + Detailed descriptions are available in Supplementary Data. + + + METHODS + title_2 + 8443 + Cell culture + + + METHODS + paragraph + 8456 + HCT116(+/+) cells (CCL-247; ATCC) were grown at 37°C in a humidified incubator under 5% CO2 in the McCoy's 5a modified (Sigma-Aldrich)/10% FBS media. All media were supplemented with 50 U/ml penicillin and 50 μg/ml streptomycin (Life Technologies). + + + METHODS + title_2 + 8707 + DsiRNA inactivation and RT-qPCR + + + METHODS + paragraph + 8739 + Reverse transfection of HCT116 cells, DsiRNA inactivation and RT-qPCR using total human RNA are described in Supplementary Data. + + + METHODS + title_2 + 8868 + Sucrose gradient analysis + + + METHODS + paragraph + 8894 + Detailed descriptions for analytical or preparative separations of ribosomal subunits or polysome gradients are provided in Supplementary Data. + + + METHODS + title_2 + 9038 + HPLC analysis of 18S rRNA nucleosides + + + METHODS + paragraph + 9076 + 40S subunits from 200 ml yeast culture were isolated by sucrose gradient centrifugation in a SW28 rotor as described above, and precipitated with 2.5 vol of 100% ethanol (−20°C over night). Precipitated 40S subunits were dissolved in water and the 18S rRNA was purified via spin columns (Ambion PureLink RNA Mini Kit). RNA fragments were hydrolysed and dephosphorylated as described by Gehrke and Kuo. HPLC analysis of rRNA nucleoside composition was performed using a Supelcosil LC-18S column (Sigma; 250 × 4.6 mm, 5 μm) with a pre-column (4.6 × 20 mm) as previously described. + + + METHODS + title_2 + 9661 + 14C labeling of 18S rRNA nucleotide Ψ(U)1191 + + + METHODS + paragraph + 9708 + To enhance 14C-labeling, mutants of interest were recombined with a Δmet13 deletion. Resulting strains were cultivated with l-[1-14C]-methionine (Hartmann Analytic, 0.1 mCi/ml, 54 mCi/mmol) as described before. From isotope labeled cells total RNA was isolated with the PureLink RNA Mini Kit (Ambion) after enzymatic cell lysis with zymolyase. Ribosomal RNAs were separated on a 4% denaturing polyacrylamide gel. After ethidium bromide straining gels were dried and analyzed by autoradiography for 3–5 days using a storage phosphor screen. Signals were visualized with the Typhoon 9100 (GE Healthcare). + + + METHODS + title_2 + 10315 + Northern blot analysis + + + METHODS + paragraph + 10338 + 5 μg of total yeast RNAs extracted with phenol/chloroform were separated on 1.2% agarose gels in BPTE buffer for 16 h at 60V and afterwards transferred to a Biodyne B membrane by vacuum blotting. Oligonucleotides D/A2 or +1-A0 were radiolabeled using γ-[32P]-ATP and T4-polynucleotide kinase and hybridized to the membrane at 37°C. Signals were visualized by phosphoimaging with the Typhoon 9100 (GE Healthcare). RNA extraction from human cells, gel-electrophoresis and northern blotting were performed as described before. + + + METHODS + title_2 + 10867 + Primer extension + + + METHODS + paragraph + 10884 + 20 pmol of oligonucleotide PE-1191 complementary to yeast 18S rRNA nucleotides 1247–1228 were labeled with 50 μCi γ-[32P]-ATP using T4-polynucleotide kinase, purified via Sephadex G-25 and annealed to 500 ng of 18S rRNA. Primer annealing and reverse transcription were carried out as described by Sharma et al.. After precipitation with ethanol and 3 M NaAc pH 5.2 pellets were washed with 70% ethanol, dried and dissolved in 12 μl formamide loading dye. 2 μl of primer extension samples were separated on sequencing or mini gels which were dried after running and exposed on a storage phosphor screen. Signals were visualized with the Typhoon 9100 (GE Healthcare). + + + METHODS + paragraph + 11557 + Primer extension on human RNA was performed using 5 μg of total RNA with AMV Reverse Transcriptase (Promega) and oligonucleotide PE_1248. Following alkaline hydrolysis, cDNAs were precipitated with ethanol, resuspended in acrylamide loading buffer and separated on a 6% (v/v) denaturing acrylamide gel in 0.5× TBE at 80 W for 1.5 h. After migration, the gels were dried and exposed to Fuji Imaging plates (Fujifilm). The signal was acquired with a Phosphor imager (FLA-7000, Fujifilm). + + + METHODS + title_2 + 12045 + Protein detection and localization + + + METHODS + paragraph + 12080 + A description of the western blot detection of HA-fused Tsr3 in yeast crude extracts or sucrose gradients fractions is provided in Supplementary Data. For cellular localization Tsr3 was expressed as N-terminal fusion with yEGFP in a yeast strain encoding for ScNop56-mRFP. Protein localization in exponentially growing cells was visualized using a Leica TCS SP5. + + + METHODS + title_2 + 12443 + In vitro SAM binding + + + METHODS + paragraph + 12464 + Purified SsTsr3 protein in 25 mM Tris–HCl pH 7.8 250 mM NaCl was mixed with S-[methyl-14C]-adenosyl-l-methionine (PerkinElmer; 20 μCi/ml, 58 mCi/mmol) and 0–10 mM non-labeled SAM in a binding buffer (50 mM Tris–HCl pH 7.8, 250 mM NaCl) in a total volume of 50 μl and incubated at 30°C for 10 min. Samples were passed over HAWP02500 membrane filters (Millipore) and unbound 14C-SAM was removed by washing three times with 5 ml buffer using a vacuum filtering equipment. Filter bound 14C-SAM was measured by liquid scintillation spectrometry in a Wallac 1401 scintillation counter. + + + METHODS + title_2 + 13053 + Protein expression and purification + + + METHODS + paragraph + 13089 + Genes coding for archaeal Tsr3 homologs without any tags were obtained commercially (Genscript) in pET11a vectors and overexpressed in Escherichia coli BL21(DE3). Proteins were purified by a combination of heat shock and appropriate column chromatography steps as described in detail in the Supplementary Data. + + + METHODS + title_2 + 13400 + Crystallization, X-ray data collection, structure calculation and refinement + + + METHODS + paragraph + 13477 + Initial hits for VdTsr3 and SsTsr3 were obtained using the Morpheus Screen (Molecular dimensions) and further refined as described in the Supplementary Data. Diffraction data were collected at the Swiss Light Source (Paul Scherer Institut). The structure of VdTsr3 was determined at 1.6 Å by SAD using a selenomethionine derivative. The structure of SsTsr3 was determined at 2.25 Å by molecular replacement using VdTsr3 as the search model. A detailed description of the data collection, processing, structure calculation and refinement procedures can be found in the Supplementary Data and in Supplementary Table S1. Structures were deposited in the Protein Data Bank as entries 5APG (VdTsr3) and 5AP8 (SsTsr3). + + + METHODS + title_2 + 14192 + Analytical gel filtration + + + METHODS + paragraph + 14218 + For analytical gel filtration experiments a Sephadex S75 10/300 GL column (GE Healthcare) was used. 100 μl protein samples (25 mM Tris–HCl pH 7.8, 250 mM NaCl, 2 mM β-mercaptoethanol) with a protein concentration of 150 μM were used. The flow rate was 0.5 ml/min. The column was calibrated using the marker proteins of the LMW gel filtration calibration kit (GE Healthcare). Protein elution was followed by recording the adsorption at a wavelength of λ = 280 nm. + + + METHODS + title_2 + 14691 + Fluorescence quenching and fluorescence anisotropy measurements + + + METHODS + paragraph + 14755 + Fluorescence quenching and fluorescence anisotropy measurements were carried out in triplicates at 25°C on a Fluorolog 3 spectrometer (Horiba Jobin Yvon) equipped with polarizers. For fluorescence quenching with SAM, SAH and 5′-methylthioadenosine experiments the tryptophan fluorescence of SsTsr3 (200 nM in 25 mM Tris–HCl pH 7.8, 250 mM NaCl, 2 mM β-mercaptoethanol) was excited at 295 nm and emission spectra were recorded from 250 to 450 nm for each titration step. The fluorescence intensity at 351 nm for each titration step was normalized with regard to the fluorescence of the free protein and was used for deriving binding curves. KD's were derived by nonlinear regression with Origin 8.0 (Origin Labs) using Equation (1): (F is the normalized fluorescence intensity, a is the change in fluorescence intensity, c is the ligand concentration and KD is the dissociation constant). + + + METHODS + paragraph + 15650 + 5′-Fluoresceine labeled RNAs for fluorescence anisotropy measurements were obtained commercially (Dharmacon), deprotected according to the manufacturer's protocol and the RNA concentration adjusted to 50 nM in 25 mM Tris–HCl pH 7.8, 250 mM NaCl. Fluoresceine fluorescence was excited at 492 nm and emission was recorded at 516 nm. The data were fitted to Equation (1) (F is the normalized fluorescence anisotropy, a is the change in fluorescence anisotropy). + + + RESULTS + title_1 + 16113 + RESULTS + + + RESULTS + title_2 + 16121 + Tsr3 is the enzyme responsible for 18S rRNA acp modification in yeast and humans + + 0.97725815 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.9920869 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.76246583 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + 0.996209 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.99572146 + species + cleaner0 + 2023-07-04T12:18:31Z + MESH: + + humans + + + + RESULTS + paragraph + 16202 + The S. cerevisiae 18S rRNA acp transferase was identified in a systematic genetic screen where numerous deletion mutants from the EUROSCARF strain collection (www.euroscarf.de) were analyzed by HPLC for alterations in 18S rRNA base modifications. + + 0.995652 + species + cleaner0 + 2023-07-04T14:59:06Z + MESH: + + S. cerevisiae + + + protein_type + MESH: + cleaner0 + 2023-07-04T14:59:27Z + + 18S rRNA acp transferase + + + 0.9912554 + experimental_method + cleaner0 + 2023-07-04T15:30:46Z + MESH: + + HPLC + + + 0.7911035 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + + RESULTS + paragraph + 16449 + For the Δtsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant Δnep1, where a shoulder at ∼ 7.4 min elution time was missing in the elution profile. As previously reported this shoulder was identified by ESI-MS as corresponding to m1acp3Ψ. In order to directly analyze the presence of the acp modification of nucleotide 1191 we used an in vivo14C incorporation assay with 1-14C-methionine. Whereas the acp labeling of 18S rRNA was clearly present in the wild type strain no radioactive labeling could be observed in a Δtsr3 strain (Figure 1C). No radioactive labeling was detected in the 18S U1191A mutant which served as a control for the specificity of the 14C-aminocarboxypropyl incorporation. + + 0.9983985 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + evidence + DUMMY: + cleaner0 + 2023-07-04T15:09:17Z + + HPLC elution profile + + + 0.6619743 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.96492976 + chemical + cleaner0 + 2023-07-04T14:40:57Z + CHEBI: + + nucleosides + + + 0.9782849 + protein_type + cleaner0 + 2023-07-04T13:58:27Z + MESH: + + pseudouridine-N1 methyltransferase + + + 0.58084774 + protein_state + cleaner0 + 2023-07-04T16:04:34Z + DUMMY: + + mutant + + + 0.99856335 + mutant + cleaner0 + 2023-07-04T13:24:30Z + MESH: + + Δnep1 + + + 0.9661906 + experimental_method + cleaner0 + 2023-07-04T15:30:50Z + MESH: + + ESI-MS + + + 0.44394964 + chemical + cleaner0 + 2023-07-04T12:18:12Z + CHEBI: + + m1acp3Ψ + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.6568178 + chemical + cleaner0 + 2023-07-04T15:33:08Z + CHEBI: + + nucleotide + + + 0.8943901 + residue_number + cleaner0 + 2023-07-04T15:33:13Z + DUMMY: + + 1191 + + + 0.9051086 + experimental_method + cleaner0 + 2023-07-04T15:30:55Z + MESH: + + in vivo14C incorporation assay + + + 0.99287385 + chemical + cleaner0 + 2023-07-04T14:41:08Z + CHEBI: + + 1-14C-methionine + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.7277 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.99619067 + protein_state + cleaner0 + 2023-07-04T12:31:11Z + DUMMY: + + wild type + + + 0.9984763 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + 0.9542309 + mutant + cleaner0 + 2023-07-04T12:42:59Z + MESH: + + 18S U1191A + + + 0.9941788 + protein_state + cleaner0 + 2023-07-04T16:04:37Z + DUMMY: + + mutant + + + 0.9883127 + chemical + cleaner0 + 2023-07-04T12:43:07Z + CHEBI: + + 14C-aminocarboxypropyl + + + + RESULTS + paragraph + 17262 + As previously shown, only the acp but none of the other modifications at U1191 of yeast 18S rRNA blocks reverse transcriptase activity. Therefore the presence of the acp modification can be directly assessed by primer extension. Indeed, in wild-type yeast a strong primer extension stop signal occurred at position 1192. In contrast, in a Δtsr3 mutant no primer extension stop signal was present at this position. As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. In a Δtsr3 Δsnr35 double deletion strain the 18S rRNA contains an unmodified U and the primer extension stop signal was missing (Figure 1D). + + 0.95631903 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + 0.9972254 + residue_name_number + cleaner0 + 2023-07-04T15:01:18Z + DUMMY: + + U1191 + + + 0.9948807 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.6937653 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.96929646 + experimental_method + cleaner0 + 2023-07-04T15:31:01Z + MESH: + + primer extension + + + 0.9969904 + protein_state + cleaner0 + 2023-07-04T16:04:42Z + DUMMY: + + wild-type + + + 0.99475217 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + evidence + DUMMY: + cleaner0 + 2023-07-04T15:10:10Z + + primer extension stop signal + + + 0.50405174 + residue_number + cleaner0 + 2023-07-04T14:41:25Z + DUMMY: + + 1192 + + + 0.9984048 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + 0.49695376 + protein_state + cleaner0 + 2023-07-04T16:04:47Z + DUMMY: + + mutant + + + 0.9985353 + mutant + cleaner0 + 2023-07-04T12:32:25Z + MESH: + + Δsnr35 + + + 0.69914734 + experimental_method + cleaner0 + 2023-07-04T15:31:06Z + MESH: + + deletion + + + 0.98630756 + ptm + cleaner0 + 2023-07-04T15:57:43Z + MESH: + + pseudouridylation + + + 0.8456991 + ptm + cleaner0 + 2023-07-04T15:57:46Z + MESH: + + N1-methylation + + + 0.91452855 + chemical + cleaner0 + 2023-07-04T13:24:53Z + CHEBI: + + acp3U + + + 0.9984842 + mutant + cleaner0 + 2023-07-04T13:24:57Z + MESH: + + Δnep1 + + + 0.9438199 + chemical + cleaner0 + 2023-07-04T12:27:45Z + CHEBI: + + pseudouridine + + + 0.8533305 + protein_state + cleaner0 + 2023-07-04T16:04:50Z + DUMMY: + + not methylated + + + 0.82459676 + chemical + cleaner0 + 2023-07-04T13:25:20Z + CHEBI: + + acp3Ψ + + + evidence + DUMMY: + cleaner0 + 2023-07-04T15:10:27Z + + primer extension stop signal + + + 0.99689096 + protein_state + cleaner0 + 2023-07-04T12:31:11Z + DUMMY: + + wild type + + + 0.95136213 + mutant + cleaner0 + 2023-07-04T12:46:37Z + MESH: + + Δtsr3 Δsnr35 + + + 0.63465166 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.9961945 + protein_state + cleaner0 + 2023-07-04T16:04:54Z + DUMMY: + + unmodified + + + 0.5297126 + chemical + cleaner0 + 2023-07-04T14:41:17Z + CHEBI: + + U + + + + RESULTS + paragraph + 18125 + The Tsr3 protein is highly conserved in yeast and humans (50% identity). Human 18S rRNA has also been shown to contain m1acp3Ψ in the 18S rRNA at position 1248. After siRNA-mediated depletion of Tsr3 in human colon carcinoma HCT116(+/+) cells the acp primer extension arrest was reduced in comparison to cells transfected with a non-targeting scramble siRNA control (Figure 1E, compare lanes 544 and scramble). The efficiency of siRNA-mediated depletion was established by RT-qPCR and found to be very high with siRNA 544 (Supplementary Figure S2A, remaining TSR3 mRNA level of 2%). By comparison, treating cells with siRNA 545, which only reduced the TSR3 mRNA to 20%, did not markedly reduced the acp signal. This suggests that low residual levels of HsTsr3 are sufficient to modify the RNA. As a control for loading, a structural stop is shown (asterisk, Figure 1E). Thus, HsTsr3 is also responsible for the acp modification of 18S rRNA nucleotide Ψ1248 in helix 31. Similar to yeast, siRNA-mediated depletion of the Ψ1248 N1-methyltransferase Nep1/Emg1 had no influence on the primer extension arrest (Figure 1E). + + 0.99776614 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.9970249 + protein_state + cleaner0 + 2023-07-04T16:04:59Z + DUMMY: + + highly conserved + + + 0.9957001 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.99552494 + species + cleaner0 + 2023-07-04T12:18:31Z + MESH: + + humans + + + 0.9954537 + species + cleaner0 + 2023-07-04T12:27:15Z + MESH: + + Human + + + 0.97678864 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.994372 + ptm + cleaner0 + 2023-07-04T14:43:21Z + MESH: + + m1acp3Ψ + + + 0.9758189 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.76458 + residue_number + cleaner0 + 2023-07-04T15:33:17Z + DUMMY: + + 1248 + + + 0.9573526 + experimental_method + cleaner0 + 2023-07-04T15:31:16Z + MESH: + + siRNA-mediated depletion + + + 0.9982256 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + species + MESH: + cleaner0 + 2023-07-04T12:27:15Z + + human + + + 0.92925614 + evidence + cleaner0 + 2023-07-04T15:10:37Z + DUMMY: + + acp primer extension arrest + + + 0.37355578 + chemical + cleaner0 + 2023-07-04T12:32:53Z + CHEBI: + + siRNA + + + 0.8478326 + chemical + cleaner0 + 2023-07-04T12:32:53Z + CHEBI: + + siRNA + + + 0.9939262 + experimental_method + cleaner0 + 2023-07-04T15:31:19Z + MESH: + + RT-qPCR + + + 0.48623383 + chemical + cleaner0 + 2023-07-04T12:32:53Z + CHEBI: + + siRNA + + + 0.99075913 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + TSR3 + + + 0.39353594 + chemical + cleaner0 + 2023-07-04T12:32:53Z + CHEBI: + + siRNA + + + 0.99643326 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + TSR3 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.9987041 + protein + cleaner0 + 2023-07-04T12:30:27Z + PR: + + HsTsr3 + + + 0.9926917 + chemical + cleaner0 + 2023-07-04T14:41:46Z + CHEBI: + + RNA + + + 0.9985796 + protein + cleaner0 + 2023-07-04T12:30:27Z + PR: + + HsTsr3 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.89912796 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.91223305 + chemical + cleaner0 + 2023-07-04T14:43:53Z + CHEBI: + + nucleotide + + + 0.9950204 + ptm + cleaner0 + 2023-07-04T14:43:37Z + MESH: + + Ψ1248 + + + 0.94343853 + structure_element + cleaner0 + 2023-07-04T15:58:08Z + SO: + + helix 31 + + + 0.9956008 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.85411745 + experimental_method + cleaner0 + 2023-07-04T15:31:23Z + MESH: + + siRNA-mediated depletion + + + 0.7850956 + protein_type + cleaner0 + 2023-07-04T12:44:26Z + MESH: + + Ψ1248 N1-methyltransferase + + + 0.9987066 + protein + cleaner0 + 2023-07-04T14:19:15Z + PR: + + Nep1 + + + 0.99816966 + protein + cleaner0 + 2023-07-04T14:19:24Z + PR: + + Emg1 + + + 0.9749694 + evidence + cleaner0 + 2023-07-04T15:10:40Z + DUMMY: + + primer extension arrest + + + + RESULTS + title_2 + 19249 + Phenotypic characterization of Δtsr3 mutants + + 0.99842864 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + + RESULTS + paragraph + 19298 + Although the acp modification of 18S rRNA is highly conserved in eukaryotes, yeast Δtsr3 mutants showed only a minor growth defect. However, the Δtsr3 deletion was synthetic sick with a Δsnr35 deletion preventing pseudouridylation and Nep1-catalyzed methylation of nucleotide 1191 (Figure 2A). Interestingly, no increased growth defect could be observed for Δtsr3 Δnep1 recombinants containing the nep1 suppressor mutation Δnop6 as well as for Δtsr3 Δsnr35 Δnep1 recombinants with unmodified U1191 (Supplementary Figure S2D and E). + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.83371437 + chemical + cleaner0 + 2023-07-04T12:17:55Z + CHEBI: + + 18S rRNA + + + 0.9968771 + protein_state + cleaner0 + 2023-07-04T16:05:11Z + DUMMY: + + highly conserved + + + 0.9964318 + taxonomy_domain + cleaner0 + 2023-07-04T14:58:55Z + DUMMY: + + eukaryotes + + + 0.99618405 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.9985261 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + 0.9985886 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + 0.9986823 + mutant + cleaner0 + 2023-07-04T12:32:25Z + MESH: + + Δsnr35 + + + 0.9297403 + ptm + cleaner0 + 2023-07-04T15:57:51Z + MESH: + + pseudouridylation + + + 0.9752127 + protein + cleaner0 + 2023-07-04T14:19:42Z + PR: + + Nep1 + + + 0.94331276 + residue_number + cleaner0 + 2023-07-04T15:01:31Z + DUMMY: + + 1191 + + + 0.99091315 + mutant + cleaner0 + 2023-07-04T13:27:48Z + MESH: + + Δtsr3 Δnep1 + + + gene + GENE: + cleaner0 + 2023-07-04T14:19:51Z + + nep1 + + + 0.9982834 + mutant + cleaner0 + 2023-07-04T12:32:33Z + MESH: + + Δnop6 + + + 0.95741963 + mutant + cleaner0 + 2023-07-04T13:38:41Z + MESH: + + Δtsr3 Δsnr35 Δnep1 + + + 0.9960886 + protein_state + cleaner0 + 2023-07-04T16:05:13Z + DUMMY: + + unmodified + + + 0.99495137 + residue_name_number + cleaner0 + 2023-07-04T15:01:36Z + DUMMY: + + U1191 + + + + gkw244fig2.jpg + F2 + FIG + fig_caption + 19866 + Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. The Δtsr3 deletion is synthetic sick with a Δsnr35 deletion preventing U1191 pseudouridylation. (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. The accumulation of 18SE and 47S and/or 45S pre-RNAs is enforced upon HsTSR3 depletion. Right gel: Ethidium bromide staining showing 18S and 28S rRNAs. (D) Cytoplasmic localization of yeast Tsr3 shown by fluorescence microscopy of GFP-fused Tsr3. From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). The TSR3 gene was genetically modified at its native locus, resulting in a C-terminal fusion of Tsr3 with a 3xHA epitope expressed by the native promotor in yeast strain CEN.BM258-5B. + + 0.9915274 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.5132569 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + TSR3 + + + 0.9977635 + mutant + cleaner0 + 2023-07-04T13:27:59Z + MESH: + + Δtrs3 + + + 0.98250157 + species + cleaner0 + 2023-07-04T12:27:15Z + MESH: + + human + + + 0.99517316 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + TSR3 + + + 0.60299724 + chemical + cleaner0 + 2023-07-04T14:43:58Z + CHEBI: + + siRNAs + + + 0.993353 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.99692875 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.99348444 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.9944301 + protein_state + cleaner0 + 2023-07-04T12:31:11Z + DUMMY: + + wild type + + 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mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + 0.9782717 + protein_state + cleaner0 + 2023-07-04T16:05:28Z + DUMMY: + + deletion mutant + + + 0.5368966 + chemical + cleaner0 + 2023-07-04T14:44:02Z + CHEBI: + + paromomycin + + + 0.7071477 + chemical + cleaner0 + 2023-07-04T14:44:04Z + CHEBI: + + hygromycin B + + + 0.99770373 + mutant + cleaner0 + 2023-07-04T12:32:25Z + MESH: + + Δsnr35 + + + 0.8474186 + experimental_method + cleaner0 + 2023-07-04T15:31:49Z + MESH: + + Northern blot analysis + + + 0.96813726 + experimental_method + cleaner0 + 2023-07-04T15:31:53Z + MESH: + + siRNA depletion + + + 0.9944193 + protein + cleaner0 + 2023-07-04T12:30:27Z + PR: + + HsTSR3 + + + 0.5565004 + chemical + cleaner0 + 2023-07-04T14:44:12Z + CHEBI: + + siRNAs + + + 0.9163474 + chemical + cleaner0 + 2023-07-04T12:32:53Z + CHEBI: + + siRNA + + + 0.9965947 + chemical + cleaner0 + 2023-07-04T14:44:23Z + CHEBI: + + 18SE + + + 0.994317 + chemical + cleaner0 + 2023-07-04T14:44:25Z + CHEBI: + + 47S + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:44:45Z + + 45S pre-RNAs + + + 0.9552973 + protein + cleaner0 + 2023-07-04T12:30:27Z + PR: + + HsTSR3 + + + 0.9932202 + chemical + cleaner0 + 2023-07-04T14:45:09Z + CHEBI: + + 18S + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:45:01Z + + 28S rRNAs + + + 0.9947326 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.9974341 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.94156015 + experimental_method + cleaner0 + 2023-07-04T15:31:57Z + MESH: + + fluorescence microscopy + + + mutant + MESH: + cleaner0 + 2023-07-04T13:31:12Z + + GFP-fused Tsr3 + + + experimental_method + MESH: + cleaner0 + 2023-07-04T13:30:33Z + + differential interference contrast + + + experimental_method + MESH: + cleaner0 + 2023-07-04T13:30:45Z + + DIC + + + mutant + MESH: + cleaner0 + 2023-07-04T13:29:17Z + + GFP-Tsr3 + + + mutant + MESH: + cleaner0 + 2023-07-04T13:29:35Z + + Nop56-mRFP + + + mutant + MESH: + cleaner0 + 2023-07-04T13:29:53Z + + GFP-Tsr3 + + + mutant + MESH: + cleaner0 + 2023-07-04T13:30:14Z + + Nop56-mRFP + + + experimental_method + MESH: + cleaner0 + 2023-07-04T13:30:46Z + + DIC + + + 0.9706644 + evidence + cleaner0 + 2023-07-04T15:10:47Z + DUMMY: + + Elution profile + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-07-06T15:24:22Z + + sucrose gradient separation + + + 0.9942629 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.5915116 + complex_assembly + cleaner0 + 2023-07-04T15:51:06Z + GO: + + ribosomal subunits + + + 0.53156686 + complex_assembly + cleaner0 + 2023-07-04T15:51:10Z + GO: + + polysomes + + + 0.9763651 + experimental_method + cleaner0 + 2023-07-04T15:32:02Z + MESH: + + western blot + + + 0.38005278 + chemical + cleaner0 + 2023-07-04T13:32:16Z + CHEBI: + + 3xHA + + + 0.99773884 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.99546224 + mutant + cleaner0 + 2023-07-04T12:49:50Z + MESH: + + Tsr3-3xHA + + + 0.9745136 + experimental_method + cleaner0 + 2023-07-04T15:32:06Z + MESH: + + SDS-PAGE + + + 0.7641838 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + TSR3 + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T15:48:29Z + + fusion + + + 0.99737096 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.7335174 + chemical + cleaner0 + 2023-07-04T14:45:13Z + CHEBI: + + 3xHA + + + 0.9918937 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + + RESULTS + paragraph + 21519 + The influence of the acp modification of nucleotide 1191 on ribosome function was analyzed by treating Δtsr3 mutants with protein synthesis inhibitors. Similar to a temperature-sensitive nep1 mutant, the Δtsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown). In accordance with the synthetic sick growth phenotype the paromomycin and hygromycin B hypersensitivity further increased in a Δtsr3 Δsnr35 recombination strain (Figure 2B). + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.6377991 + chemical + cleaner0 + 2023-07-04T15:33:35Z + CHEBI: + + nucleotide + + + 0.9465645 + residue_number + cleaner0 + 2023-07-04T15:33:41Z + DUMMY: + + 1191 + + + mutant + MESH: + cleaner0 + 2023-07-04T12:32:06Z + + Δtsr3 + + + 0.98296434 + gene + cleaner0 + 2023-07-04T14:20:33Z + GENE: + + nep1 + + + 0.9572939 + protein_state + cleaner0 + 2023-07-04T16:05:41Z + DUMMY: + + mutant + + + 0.99866676 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + 0.99684036 + chemical + cleaner0 + 2023-07-04T14:45:21Z + CHEBI: + + paromomycin + + + 0.9956605 + chemical + cleaner0 + 2023-07-04T14:45:26Z + CHEBI: + + hygromycin B + + + 0.99802125 + chemical + cleaner0 + 2023-07-04T14:46:17Z + CHEBI: + + G418 + + + 0.9970824 + chemical + cleaner0 + 2023-07-04T14:46:19Z + CHEBI: + + cycloheximide + + + 0.5927976 + chemical + cleaner0 + 2023-07-04T14:46:23Z + CHEBI: + + paromomycin + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:46:40Z + + hygromycin B + + + mutant + MESH: + cleaner0 + 2023-07-04T12:46:31Z + + Δtsr3 Δsnr35 + + + + RESULTS + paragraph + 22073 + In a yeast Δtsr3 strain as well as in the Δtsr3 Δsnr35 recombinant 20S pre-rRNA accumulated significantly and the level of mature 18S rRNA was reduced (Supplementary Figures S2C and S3D), as reported previously. A minor effect on 20S rRNA accumulation was also observed for Δsnr35, but - probably due to different strain backgrounds – to a weaker extent than described earlier. In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B). Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast Δtsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B). + + 0.9893476 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.99783283 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + mutant + MESH: + cleaner0 + 2023-07-04T12:46:37Z + + Δtsr3 Δsnr35 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:48:25Z + + 20S pre-rRNA + + + 0.994905 + chemical + cleaner0 + 2023-07-04T12:17:56Z + CHEBI: + + 18S rRNA + + + 0.8788376 + chemical + cleaner0 + 2023-07-04T14:46:48Z + CHEBI: + + 20S rRNA + + + 0.9984927 + mutant + cleaner0 + 2023-07-04T12:32:25Z + MESH: + + Δsnr35 + + + 0.98830533 + species + cleaner0 + 2023-07-04T12:27:15Z + MESH: + + human + + + 0.78692925 + experimental_method + cleaner0 + 2023-07-04T15:34:08Z + MESH: + + depletion of + + + 0.9969137 + protein + cleaner0 + 2023-07-04T12:30:27Z + PR: + + HsTsr3 + + + 0.98755056 + species + cleaner0 + 2023-07-04T12:27:15Z + MESH: + + human + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:48:26Z + + 20S pre-rRNA + + + 0.9749642 + chemical + cleaner0 + 2023-07-04T14:46:53Z + CHEBI: + + 18S-E + + + 0.9874466 + protein + cleaner0 + 2023-07-04T14:08:01Z + PR: + + Tsr3 + + + 0.9903704 + chemical + cleaner0 + 2023-07-04T12:17:56Z + CHEBI: + + 18S rRNA + + + 0.9830163 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.99749655 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + 0.98898333 + complex_assembly + cleaner0 + 2023-07-04T14:47:21Z + GO: + + 35S + + + protein + PR: + cleaner0 + 2023-07-04T16:06:14Z + + Tsr3 + + + 0.9809749 + species + cleaner0 + 2023-07-04T12:27:15Z + MESH: + + human + + + 0.92224807 + complex_assembly + cleaner0 + 2023-07-04T14:47:31Z + GO: + + 47S + + + 0.9139037 + complex_assembly + cleaner0 + 2023-07-04T14:47:39Z + GO: + + 45S + + + 0.9626368 + experimental_method + cleaner0 + 2023-07-04T15:34:12Z + MESH: + + Northern blot + + + + RESULTS + paragraph + 23037 + Consistent with its role in late 18S rRNA processing, TSR3 deletion leads to a ribosomal subunit imbalance with a reduced 40S to 60S ratio of 0.81 (σ = 0.024) which was further increased in a Δtsr3 Δsnr35 recombinant to 0.73 (σ = 0.023) (Supplementary Figure S2F). In polysome profiles, a reduced level of 80S ribosomes and a strong signal for free 60S subunits was observed in line with the 40S subunit deficiency (Supplementary Figure S2G). + + 0.8237121 + chemical + cleaner0 + 2023-07-04T12:17:56Z + CHEBI: + + 18S rRNA + + + 0.9034129 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + TSR3 + + + 0.94708794 + complex_assembly + cleaner0 + 2023-07-04T12:48:57Z + GO: + + 40S + + + complex_assembly + GO: + cleaner0 + 2023-07-04T12:49:13Z + + 60S + + + 0.8403921 + mutant + cleaner0 + 2023-07-04T12:46:37Z + MESH: + + Δtsr3 Δsnr35 + + + 0.63479966 + evidence + cleaner0 + 2023-07-04T15:19:48Z + DUMMY: + + polysome profiles + + + complex_assembly + GO: + cleaner0 + 2023-07-04T15:12:16Z + + 80S ribosomes + + + 0.8691986 + complex_assembly + cleaner0 + 2023-07-04T12:49:13Z + GO: + + 60S + + + 0.6724853 + complex_assembly + cleaner0 + 2023-07-04T12:48:58Z + GO: + + 40S + + + + RESULTS + title_2 + 23496 + Cellular localization of Tsr3 in S. cerevisiae + + 0.9898936 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9955408 + species + cleaner0 + 2023-07-04T14:59:36Z + MESH: + + S. cerevisiae + + + + RESULTS + paragraph + 23543 + Fluorescence microscopy of GFP-tagged Tsr3 localized the fusion protein in the cytoplasm of yeast cells and no co-localization with the nucleolar marker protein Nop56 could be observed (Figure 2D). This agrees with previous biochemical data suggesting that the acp modification of 18S rRNA occurs late during 40S subunit biogenesis in the cytoplasm, and makes an additional nuclear localization as reported in a previous large-scale analysis unlikely. After polysome gradient separation C-terminally epitope-labeled Tsr3-3xHA was exclusively detectable in the low-density fraction (Figure 2E). Such distribution on a density gradient suggests that Tsr3 only interacts transiently with pre-40S subunits, which presumably explains why it was not characterized in pre-ribosome affinity purifications. + + 0.98735833 + experimental_method + cleaner0 + 2023-07-04T15:34:16Z + MESH: + + Fluorescence microscopy + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T14:10:17Z + + GFP-tagged + + + 0.99816704 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9956487 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.9889811 + protein + cleaner0 + 2023-07-04T14:25:18Z + PR: + + Nop56 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.9948225 + chemical + cleaner0 + 2023-07-04T12:17:56Z + CHEBI: + + 18S rRNA + + + 0.94876456 + complex_assembly + cleaner0 + 2023-07-04T12:48:58Z + GO: + + 40S + + + 0.8942659 + experimental_method + cleaner0 + 2023-07-04T15:34:20Z + MESH: + + polysome gradient separation + + + 0.99817866 + mutant + cleaner0 + 2023-07-04T12:49:50Z + MESH: + + Tsr3-3xHA + + + evidence + DUMMY: + cleaner0 + 2023-07-04T15:34:54Z + + distribution on a density gradient + + + 0.9985233 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.8188459 + complex_assembly + cleaner0 + 2023-07-04T15:52:58Z + GO: + + pre-40S subunits + + + 0.988572 + experimental_method + cleaner0 + 2023-07-04T15:35:02Z + MESH: + + pre-ribosome affinity purifications + + + + RESULTS + title_2 + 24341 + Structure of Tsr3 + + 0.9653325 + evidence + cleaner0 + 2023-07-04T15:19:53Z + DUMMY: + + Structure + + + 0.99821293 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + + RESULTS + paragraph + 24359 + Searches for sequence homologs of S. cerevisiae Tsr3 (ScTsr3) by us and others revealed that the genomes of many archaea contain genes encoding Tsr3-like proteins. However, these archaeal homologs are significantly smaller than ScTsr3 (∼190 aa in archaea vs. 313 aa in yeast) due to shortened N- and C-termini (Supplementary Figure S1A). + + 0.99540585 + species + cleaner0 + 2023-07-04T14:59:40Z + MESH: + + S. cerevisiae + + + 0.8784227 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.99813855 + protein + cleaner0 + 2023-07-04T12:52:43Z + PR: + + ScTsr3 + + + 0.99666363 + taxonomy_domain + cleaner0 + 2023-07-04T12:35:38Z + DUMMY: + + archaea + + + 0.91221446 + protein_type + cleaner0 + 2023-07-04T14:27:49Z + MESH: + + Tsr3-like proteins + + + 0.99677074 + taxonomy_domain + cleaner0 + 2023-07-04T12:34:33Z + DUMMY: + + archaeal + + + 0.9985812 + protein + cleaner0 + 2023-07-04T12:52:43Z + PR: + + ScTsr3 + + + 0.99671614 + taxonomy_domain + cleaner0 + 2023-07-04T12:35:38Z + DUMMY: + + archaea + + + 0.9966486 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + + RESULTS + paragraph + 24699 + To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a Δtsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C). N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected. Even a Tsr3 fragment with a 90 aa C-terminal truncation showed a residual primer extension stop, whereas N-terminal truncations exceeding 46 aa almost completely abolished the primer extension arrest (Figure 3B). + + protein + PR: + cleaner0 + 2023-07-04T14:08:02Z + + Tsr3 + + + 0.9229415 + protein + cleaner0 + 2023-07-04T12:52:43Z + PR: + + ScTsr3 + + + 0.9971113 + protein_state + cleaner0 + 2023-07-04T16:06:21Z + DUMMY: + + highly conserved + + + 0.9294225 + experimental_method + cleaner0 + 2023-07-04T15:35:08Z + MESH: + + expressed + + + 0.99860305 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + 0.9922924 + protein_state + cleaner0 + 2023-07-04T16:06:24Z + DUMMY: + + mutant + + + 0.9922793 + experimental_method + cleaner0 + 2023-07-04T15:35:13Z + MESH: + + primer extension + + + 0.9610331 + experimental_method + cleaner0 + 2023-07-04T15:35:16Z + MESH: + + Northern blotting + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-07-06T15:24:22Z + + truncations + + + 0.6994033 + residue_range + cleaner0 + 2023-07-04T15:42:34Z + DUMMY: + + 45 aa + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-07-06T15:24:22Z + + truncations + + + 0.7585292 + residue_range + cleaner0 + 2023-07-04T15:42:53Z + DUMMY: + + 76 aa + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.9966659 + protein_state + cleaner0 + 2023-07-04T16:06:28Z + DUMMY: + + full-length + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:50:34Z + + 20S pre-RNA + + + protein + PR: + cleaner0 + 2023-07-04T14:08:02Z + + Tsr3 + + + 0.73044395 + residue_range + cleaner0 + 2023-07-04T15:42:56Z + DUMMY: + + 90 aa + + + 0.8059091 + residue_range + cleaner0 + 2023-07-04T15:42:59Z + DUMMY: + + 46 aa + + + + gkw244fig3.jpg + F3 + FIG + fig_caption + 25394 + Domain characterization of yeast Tsr3 and correlation of acp modification with late 18S rRNA processing steps. (A) Scheme of the TSR3 gene with truncation positions in the open reading frame. TSR3 fragments of different length were expressed under the native promotor from multicopy plasmids in a Δtsr3 deletion strain. (B) Primer extension analysis of 18S rRNA acp modification in yeast cells expressing the indicated TSR3 fragments. N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type. Tsr3 fragments 37–223 or 46–223 cause a nearly complete loss of the arrest signal. The box highlights the shortest Tsr3 fragment (aa 46–270) with wild type activity (strong primer extension block). (C) Northern blot analysis of 20S pre-rRNA accumulation. A weak 20S rRNA signal, indicating normal processing, is observed for Tsr3 fragment 46–270 (highlighted in a box) showing its functionality. Strong 20S rRNA accumulation similar to that of the Δtsr3 deletion is observed for Tsr3 fragments 37–223 or 46–223. + + 0.9951761 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.9630516 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:17:56Z + + 18S rRNA + + + 0.94555557 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + TSR3 + + + protein + PR: + cleaner0 + 2023-07-04T14:08:02Z + + TSR3 + + + 0.9973393 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + 0.990005 + experimental_method + cleaner0 + 2023-07-04T15:35:22Z + MESH: + + Primer extension analysis + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:17:56Z + + 18S rRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.99398273 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + protein + PR: + cleaner0 + 2023-07-04T14:08:02Z + + TSR3 + + + 0.33643785 + experimental_method + cleaner0 + 2023-07-04T15:35:29Z + MESH: + + deletions + + + 0.9236323 + residue_range + cleaner0 + 2023-07-04T15:43:08Z + DUMMY: + + 36 + + + 0.8441494 + residue_range + cleaner0 + 2023-07-04T15:43:11Z + DUMMY: + + 45 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-07-06T15:24:22Z + + deletions + + + 0.9539571 + residue_range + cleaner0 + 2023-07-04T15:43:24Z + DUMMY: + + 43 + + + 0.8959835 + residue_range + cleaner0 + 2023-07-04T15:43:27Z + DUMMY: + + 76 + + + evidence + DUMMY: + cleaner0 + 2023-07-04T15:20:24Z + + primer extension stop + + + 0.99560976 + protein_state + cleaner0 + 2023-07-04T12:31:11Z + DUMMY: + + wild type + + + 0.99218893 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.87861127 + residue_range + cleaner0 + 2023-07-04T15:43:30Z + DUMMY: + + 37–223 + + + 0.8790083 + residue_range + cleaner0 + 2023-07-04T15:43:32Z + DUMMY: + + 46–223 + + + 0.9892195 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9662222 + residue_range + cleaner0 + 2023-07-04T15:43:36Z + DUMMY: + + 46–270 + + + 0.9844143 + protein_state + cleaner0 + 2023-07-04T12:31:11Z + DUMMY: + + wild type + + + evidence + DUMMY: + cleaner0 + 2023-07-04T15:21:09Z + + primer extension block + + + 0.9899976 + experimental_method + cleaner0 + 2023-07-04T15:35:32Z + MESH: + + Northern blot + + + 0.88851964 + chemical + cleaner0 + 2023-07-04T12:48:26Z + CHEBI: + + 20S pre-rRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-04T15:43:53Z + + 20S rRNA + + + 0.99354035 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9015322 + residue_range + cleaner0 + 2023-07-04T15:43:40Z + DUMMY: + + 46–270 + + + 0.99736816 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-07-06T15:24:22Z + + deletion + + + protein + PR: + cleaner0 + 2023-07-04T14:08:02Z + + Tsr3 + + + 0.8548698 + residue_range + cleaner0 + 2023-07-04T15:43:56Z + DUMMY: + + 37–223 + + + 0.69136864 + residue_range + cleaner0 + 2023-07-04T15:43:59Z + DUMMY: + + 46–223 + + + + RESULTS + paragraph + 26504 + Thus, the archaeal homologs correspond to the functional core of Tsr3. In order to define the structural basis for Tsr3 function, homologs from thermophilic archaea were screened for crystallization. We focused on archaeal species containing a putative Nep1 homolog suggesting that these species are in principle capable of synthesizing N1-methyl-N3-acp-pseudouridine. Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). While for S. solfataricus the existence of a modified nucleotide of unknown chemical composition in the loop capping helix 31 of its 16S rRNA has been demonstrated, no information regarding rRNA modifications is yet available for V. distributa. + + 0.99613106 + taxonomy_domain + cleaner0 + 2023-07-04T12:34:34Z + DUMMY: + + archaeal + + + 0.9906142 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9757283 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-04T12:53:34Z + + thermophilic archaea + + + 0.9104248 + experimental_method + cleaner0 + 2023-07-04T15:35:36Z + MESH: + + crystallization + + + 0.9963098 + taxonomy_domain + cleaner0 + 2023-07-04T12:34:34Z + DUMMY: + + archaeal + + + 0.9979527 + protein + cleaner0 + 2023-07-04T14:21:07Z + PR: + + Nep1 + + + 0.9948228 + chemical + cleaner0 + 2023-07-04T14:47:49Z + CHEBI: + + N1-methyl-N3-acp-pseudouridine + + + 0.99405485 + evidence + cleaner0 + 2023-07-04T15:21:14Z + DUMMY: + + crystals + + + 0.9711174 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9944575 + taxonomy_domain + cleaner0 + 2023-07-04T12:53:09Z + DUMMY: + + crenarchaeal + + + 0.994915 + species + cleaner0 + 2023-07-04T12:52:13Z + MESH: + + Vulcanisaeta distributa + + + 0.99615246 + protein + cleaner0 + 2023-07-04T12:52:36Z + PR: + + VdTsr3 + + + 0.99521303 + species + cleaner0 + 2023-07-04T12:52:19Z + MESH: + + Sulfolobus solfataricus + + + 0.99852824 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.9984504 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.9986474 + protein + cleaner0 + 2023-07-04T12:52:48Z + PR: + + SsTsr3 + + + 0.99852055 + protein + cleaner0 + 2023-07-04T12:52:42Z + PR: + + ScTsr3 + + + 0.98933065 + structure_element + cleaner0 + 2023-07-04T15:58:31Z + SO: + + core region + + + 0.99867487 + protein + cleaner0 + 2023-07-04T12:52:43Z + PR: + + ScTsr3 + + + 0.9788138 + residue_range + cleaner0 + 2023-07-04T15:44:20Z + DUMMY: + + 46–223 + + + 0.9926 + species + cleaner0 + 2023-07-04T12:52:24Z + MESH: + + S. solfataricus + + + 0.9942549 + chemical + cleaner0 + 2023-07-04T14:47:54Z + CHEBI: + + nucleotide + + + structure_element + SO: + cleaner0 + 2023-07-04T15:58:50Z + + loop capping helix 31 + + + 0.7716433 + chemical + cleaner0 + 2023-07-04T12:52:59Z + CHEBI: + + 16S rRNA + + + 0.9940617 + species + cleaner0 + 2023-07-04T12:52:30Z + MESH: + + V. distributa + + + + RESULTS + paragraph + 27381 + Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM). The structure of SsTsr3 was solved by molecular replacement using VdTsr3 as a search model (see Supplementary Table S1 for data collection and refinement statistics). The structure of VdTsr3 can be divided into two domains (Figure 4A). The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. The C-terminal domain (aa 93–184) has a globular all α-helical structure comprising α-helices α4 to α9. Both domains are tightly packed against each other. Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects β-strand β3 and α-helix α2 of the N-terminal domain. Thus, the VdTsr3 structure contains a deep trefoil knot. The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. + + 0.894146 + evidence + cleaner0 + 2023-07-04T15:21:18Z + DUMMY: + + Crystals + + + 0.99865985 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.9334132 + evidence + cleaner0 + 2023-07-04T15:21:21Z + DUMMY: + + crystals + + + 0.99864453 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.9986304 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.98529214 + experimental_method + cleaner0 + 2023-07-04T15:35:47Z + MESH: + + crystallized + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T16:06:55Z + + in complex with + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T16:07:13Z + + endogenous + + + species + MESH: + cleaner0 + 2023-07-04T12:53:57Z + + E. coli + + + 0.9981237 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + 0.9985421 + protein + cleaner0 + 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structure_element + SO: + cleaner0 + 2023-07-04T12:57:43Z + + α8 + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T16:07:46Z + + absence of + + + structure_element + SO: + cleaner0 + 2023-07-04T12:58:00Z + + α-helix + + + structure_element + SO: + cleaner0 + 2023-07-04T12:58:10Z + + α9 + + + 0.99849546 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + + gkw244fig4.jpg + F4 + FIG + fig_caption + 29350 + Tsr3 has a fold similar to SPOUT-class RNA methyltransferases. (A) Cartoon representation of the X-ray structure of VdTsr3 in two orientations. β-strands are colored in crimson whereas α-helices in the N-terminal domain are colored light blue and α-helices in the C-terminal domain are colored dark blue. The bound S-adenosylmethionine is shown in a stick representation and colored by atom type. A red arrow marks the location of the topological knot in the structure. (B) Secondary structure representation of the VdTsr3 structure. The color coding is the same as in (A). (C) Structural superposition of the X-ray structures of VdTsr3 in the SAM-bound state (red) and SsTsr3 (blue) in the apo state. The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. Vertical lines indicate the elution volumes of molecular weight markers. Vd, Vulcanisaeta distributa; Ss, Sulfolobus solfataricus. + + 0.99868983 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9898712 + protein_type + cleaner0 + 2023-07-04T13:58:35Z + MESH: + + SPOUT-class RNA methyltransferases + + + 0.98756117 + evidence + cleaner0 + 2023-07-04T15:21:51Z + DUMMY: + + X-ray structure + + + 0.9984108 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.9932092 + structure_element + cleaner0 + 2023-07-04T15:58:55Z + SO: + + β-strands + + + 0.9937828 + structure_element + cleaner0 + 2023-07-04T15:58:59Z + SO: + + α-helices + + + 0.9317618 + structure_element + cleaner0 + 2023-07-04T12:54:50Z + SO: + + N-terminal domain + + + 0.99329805 + structure_element + cleaner0 + 2023-07-04T15:59:02Z + SO: + + α-helices + + + 0.97738254 + structure_element + cleaner0 + 2023-07-04T12:56:54Z + SO: + + C-terminal domain + + + 0.9962704 + chemical + cleaner0 + 2023-07-04T12:30:02Z + CHEBI: + + S-adenosylmethionine + + + 0.8493673 + structure_element + cleaner0 + 2023-07-04T15:59:06Z + SO: + + topological knot + + + 0.9957776 + evidence + cleaner0 + 2023-07-04T15:21:55Z + DUMMY: + + structure + + + 0.9981481 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.9955183 + evidence + cleaner0 + 2023-07-04T15:21:57Z + DUMMY: + + structure + + + 0.9939426 + experimental_method + cleaner0 + 2023-07-04T15:36:03Z + MESH: + + Structural superposition + + + 0.97521293 + evidence + cleaner0 + 2023-07-04T12:59:20Z + DUMMY: + + X-ray structures + + + 0.99852186 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.99706334 + protein_state + cleaner0 + 2023-07-04T16:07:59Z + DUMMY: + + SAM-bound + + + 0.99863017 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.99736446 + protein_state + cleaner0 + 2023-07-04T16:08:02Z + DUMMY: + + apo + + + 0.99600226 + structure_element + cleaner0 + 2023-07-04T15:59:09Z + SO: + + α-helix + + + 0.52897996 + structure_element + cleaner0 + 2023-07-04T12:59:08Z + SO: + + α8 + + + 0.99844724 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.99594086 + structure_element + cleaner0 + 2023-07-04T15:59:13Z + SO: + + α-helix + + + 0.66921145 + structure_element + cleaner0 + 2023-07-04T12:59:01Z + SO: + + α9 + + + 0.9980621 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.9956155 + species + cleaner0 + 2023-07-04T12:58:29Z + MESH: + + S. pombe + + + 0.99886984 + protein + cleaner0 + 2023-07-04T12:58:54Z + PR: + + Trm10 + + + 0.98729503 + protein_type + cleaner0 + 2023-07-04T13:58:56Z + MESH: + + SPOUT-class RNA methyltransferase + + + 0.9988362 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9872602 + experimental_method + cleaner0 + 2023-07-04T15:36:06Z + MESH: + + superposition + + + 0.9986639 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.99880326 + protein + cleaner0 + 2023-07-04T12:58:54Z + PR: + + Trm10 + + + 0.9941336 + evidence + cleaner0 + 2023-07-04T12:59:20Z + DUMMY: + + X-ray structures + + + 0.9899495 + experimental_method + cleaner0 + 2023-07-04T15:36:10Z + MESH: + + Analytical gel filtration + + + 0.7014155 + evidence + cleaner0 + 2023-07-04T15:22:56Z + DUMMY: + + profiles + + + 0.99863523 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.99863416 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.6680443 + oligomeric_state + cleaner0 + 2023-07-04T15:59:23Z + DUMMY: + + monomeric + + + 0.92951286 + species + cleaner0 + 2023-07-04T12:58:35Z + MESH: + + Vd + + + 0.9925858 + species + cleaner0 + 2023-07-04T12:52:14Z + MESH: + + Vulcanisaeta distributa + + + 0.97483873 + species + cleaner0 + 2023-07-04T12:58:44Z + MESH: + + Ss + + + 0.9946715 + species + cleaner0 + 2023-07-04T12:52:20Z + MESH: + + Sulfolobus solfataricus + + + + RESULTS + paragraph + 30665 + Structure predictions suggested that Tsr3 might contain a so-called RLI domain which contains a ‘bacterial like’ ferredoxin fold and binds two iron-sulfur clusters through eight conserved cysteine residues. However, no structural similarity to an RLI-domain was detectable. This is in accordance with the functional analysis of alanine replacement mutations of cysteine residues in ScTsr3 (Supplementary Figure S3). + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-07-06T15:24:22Z + + Structure predictions + + + 0.9984465 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9907023 + structure_element + cleaner0 + 2023-07-04T15:59:34Z + SO: + + RLI domain + + + 0.8769801 + structure_element + cleaner0 + 2023-07-04T15:59:45Z + SO: + + bacterial like’ ferredoxin fold + + + 0.7951602 + protein_state + cleaner0 + 2023-07-04T16:08:09Z + DUMMY: + + conserved + + + 0.99081624 + residue_name + cleaner0 + 2023-07-04T12:59:30Z + SO: + + cysteine + + + 0.9892566 + structure_element + cleaner0 + 2023-07-04T15:59:50Z + SO: + + RLI-domain + + + 0.9950836 + experimental_method + cleaner0 + 2023-07-04T15:37:01Z + MESH: + + alanine replacement mutations + + + 0.9880019 + residue_name + cleaner0 + 2023-07-04T12:59:31Z + SO: + + cysteine + + + 0.9986821 + protein + cleaner0 + 2023-07-04T12:52:43Z + PR: + + ScTsr3 + + + + RESULTS + paragraph + 31085 + The β-strand topology and the deep C-terminal trefoil knot of archaeal Tsr3 are the structural hallmarks of the SPOUT-class RNA-methyltransferase fold. The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. In comparison to Tsr3 the central β-sheet element of Trm10 is extended by one additional β-strand pairing to β2. Furthermore, the trefoil knot of Trm10 is not as deep as that of Tsr3 (Figure 4D). Interestingly, Nep1—the enzyme preceding Tsr3 in the biosynthetic pathway for the synthesis of m1acp3Ψ—also belongs to the SPOUT-class of RNA methyltransferases. However, the structural similarities between Nep1 and Tsr3 (DALI Z-score 4.4) are less pronounced than between Tsr3 and Trm10. Most SPOUT-class RNA-methyltransferases are homodimers. A notable exception is Trm10. Gel filtration experiments with both VdTsr3 and SsTsr3 (Figure 4E) showed that both proteins are monomeric in solution thereby extending the structural similarities to Trm10. + + 0.9482632 + structure_element + cleaner0 + 2023-07-04T13:00:51Z + SO: + + β-strand topology + + + 0.9970552 + structure_element + cleaner0 + 2023-07-04T13:00:42Z + SO: + + trefoil knot + + + 0.9952211 + taxonomy_domain + cleaner0 + 2023-07-04T12:34:34Z + DUMMY: + + archaeal + + + 0.99835443 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.991478 + protein_type + cleaner0 + 2023-07-04T13:00:23Z + MESH: + + SPOUT-class RNA-methyltransferase + + + 0.9869234 + experimental_method + cleaner0 + 2023-07-04T15:37:04Z + MESH: + + DALI search + + + 0.9954552 + protein_type + cleaner0 + 2023-07-04T13:00:15Z + MESH: + + tRNA methyltransferase + + + 0.9988763 + protein + cleaner0 + 2023-07-04T12:58:54Z + PR: + + Trm10 + + + evidence + DUMMY: + cleaner0 + 2023-07-04T15:02:13Z + + DALI Z-score + + + 0.6525595 + residue_name_number + cleaner0 + 2023-07-04T15:02:48Z + DUMMY: + + G9 + + + 0.58903307 + residue_name_number + cleaner0 + 2023-07-04T15:02:54Z + DUMMY: + + A9 + + + 0.99626356 + taxonomy_domain + cleaner0 + 2023-07-04T12:34:34Z + DUMMY: + + archaeal + + + 0.99567163 + taxonomy_domain + cleaner0 + 2023-07-04T12:20:33Z + DUMMY: + + eukaryotic + + + 0.9963192 + chemical + cleaner0 + 2023-07-04T14:48:45Z + CHEBI: + + tRNAs + + + 0.99841475 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + 0.99876726 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9794103 + structure_element + cleaner0 + 2023-07-04T13:00:48Z + SO: + + β-sheet element + + + 0.99884915 + protein + cleaner0 + 2023-07-04T12:58:54Z + PR: + + Trm10 + + + 0.97613883 + structure_element + cleaner0 + 2023-07-04T13:00:46Z + SO: + + β-strand + + + 0.98917216 + structure_element + cleaner0 + 2023-07-04T12:55:22Z + SO: + + β2 + + + 0.99724126 + structure_element + cleaner0 + 2023-07-04T13:00:39Z + SO: + + trefoil knot + + + 0.9988411 + protein + cleaner0 + 2023-07-04T12:58:54Z + PR: + + Trm10 + + + 0.99886334 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9988117 + protein + cleaner0 + 2023-07-04T14:21:38Z + PR: + + Nep1 + + + 0.9986298 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9943409 + chemical + cleaner0 + 2023-07-04T12:18:12Z + CHEBI: + + m1acp3Ψ + + + 0.98864883 + protein_type + cleaner0 + 2023-07-04T13:00:26Z + MESH: + + SPOUT-class of RNA methyltransferases + + + 0.9988576 + protein + cleaner0 + 2023-07-04T14:21:45Z + PR: + + Nep1 + + + 0.9988325 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + evidence + DUMMY: + cleaner0 + 2023-07-04T15:02:13Z + + DALI Z-score + + + 0.9988111 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.99880946 + protein + cleaner0 + 2023-07-04T12:58:54Z + PR: + + Trm10 + + + 0.9966035 + protein_type + cleaner0 + 2023-07-04T12:19:35Z + MESH: + + SPOUT-class RNA-methyltransferases + + + 0.9852149 + oligomeric_state + cleaner0 + 2023-07-04T15:00:04Z + DUMMY: + + homodimers + + + 0.99888307 + protein + cleaner0 + 2023-07-04T12:58:54Z + PR: + + Trm10 + + + 0.991344 + experimental_method + cleaner0 + 2023-07-04T15:37:08Z + MESH: + + Gel filtration + + + 0.9986413 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.9987723 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.9127686 + oligomeric_state + cleaner0 + 2023-07-04T15:00:07Z + DUMMY: + + monomeric + + + 0.9987997 + protein + cleaner0 + 2023-07-04T12:58:55Z + PR: + + Trm10 + + + + RESULTS + paragraph + 32231 + So far, structural information is only available for one other enzyme that transfers the acp group from SAM to an RNA nucleotide. This enzyme, Tyw2, is part of the biosynthesis pathway of wybutosine nucleotides in tRNAs. However, there are no structural similarities between Tsr3 and Tyw2, which contains an all-parallel β-sheet of a different topology and no knot structure. Instead, Tyw2 has a fold typical for the class-I-or Rossmann-fold class of methyltransferases (Supplementary Figure S5B). + + 0.99843997 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + 0.9985026 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + 0.9970258 + chemical + cleaner0 + 2023-07-04T14:48:49Z + CHEBI: + + RNA + + + 0.98358846 + chemical + cleaner0 + 2023-07-04T14:48:52Z + CHEBI: + + nucleotide + + + 0.99894077 + protein + cleaner0 + 2023-07-04T14:22:01Z + PR: + + Tyw2 + + + 0.98726386 + chemical + cleaner0 + 2023-07-04T14:48:55Z + CHEBI: + + wybutosine nucleotides + + + 0.9973935 + chemical + cleaner0 + 2023-07-04T14:48:59Z + CHEBI: + + tRNAs + + + 0.99888283 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9989672 + protein + cleaner0 + 2023-07-04T14:22:10Z + PR: + + Tyw2 + + + 0.9862101 + structure_element + cleaner0 + 2023-07-04T13:01:04Z + SO: + + all-parallel β-sheet + + + 0.60714936 + structure_element + cleaner0 + 2023-07-04T13:01:10Z + SO: + + knot structure + + + 0.99880314 + protein + cleaner0 + 2023-07-04T14:22:17Z + PR: + + Tyw2 + + + 0.9827729 + protein_type + cleaner0 + 2023-07-04T13:01:07Z + MESH: + + class-I-or Rossmann-fold class of methyltransferases + + + + RESULTS + title_2 + 32732 + Cofactor binding of Tsr3 + + 0.9985978 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + + RESULTS + paragraph + 32757 + The SAM-binding site of Tsr3 is located in a deep crevice between the N- and C-terminal domains in the vicinity of the trefoil knot as typical for SPOUT-class RNA-methyltransferases (Figure 4A). The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. The ribose 2′ and 3′ hydroxyl groups of SAM are hydrogen bonded to the backbone carbonyl group of I69. The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in α1 as well as the backbone amide group of T112 in α4 (C-terminal domain). Most importantly, the methyl group of SAM is buried in a hydrophobic pocket formed by the sidechains of W73 and A76 both located in α3 (Figure 5A and B). W73 is highly conserved in all known Tsr3 proteins, whereas A76 can be replaced by other hydrophobic amino acids. Consequently, the accessibility of this methyl group for a nucleophilic attack is strongly reduced in comparison with RNA-methyltransferases such as Trm10 (Figure 5B, C). In contrast, the acp side chain of SAM is accessible for reactions in the Tsr3-bound state (Figure 5B). + + 0.99810785 + site + cleaner0 + 2023-07-04T15:17:20Z + SO: + + SAM-binding site + + + 0.9987777 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.924005 + structure_element + cleaner0 + 2023-07-04T16:00:03Z + SO: + + N- and C-terminal domains + + + 0.9967799 + structure_element + cleaner0 + 2023-07-04T16:00:07Z + SO: + + trefoil knot + + + 0.9960068 + protein_type + cleaner0 + 2023-07-04T12:19:35Z + MESH: + + SPOUT-class RNA-methyltransferases + + + 0.84465826 + chemical + cleaner0 + 2023-07-04T14:49:03Z + CHEBI: + + adenine + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:28Z + + hydrogen bonds + + + 0.9990513 + residue_name_number + cleaner0 + 2023-07-04T15:03:01Z + DUMMY: + + L93 + + + 0.9896722 + structure_element + cleaner0 + 2023-07-04T12:55:42Z + SO: + + β5 + + + 0.99908054 + residue_name_number + cleaner0 + 2023-07-04T15:03:04Z + DUMMY: + + Y108 + + + 0.9925528 + structure_element + cleaner0 + 2023-07-04T16:00:13Z + SO: + + loop + + + 0.99131435 + structure_element + cleaner0 + 2023-07-04T12:55:42Z + SO: + + β5 + + + 0.6410846 + structure_element + cleaner0 + 2023-07-04T16:00:24Z + SO: + + N-terminal + + + 0.9918751 + structure_element + cleaner0 + 2023-07-04T16:00:31Z + SO: + + α4 + + + 0.8204628 + structure_element + cleaner0 + 2023-07-04T12:56:55Z + SO: + + C-terminal domain + + + 0.6286845 + chemical + cleaner0 + 2023-07-04T14:49:06Z + CHEBI: + + adenine + + + 0.99832124 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:28Z + + hydrophobic packing interactions + + + 0.9990734 + residue_name_number + cleaner0 + 2023-07-04T15:03:07Z + DUMMY: + + L45 + + + 0.99424934 + structure_element + cleaner0 + 2023-07-04T12:55:34Z + SO: + + β3 + + + 0.99873954 + residue_name_number + cleaner0 + 2023-07-04T15:03:10Z + DUMMY: + + P47 + + + 0.99907047 + residue_name_number + cleaner0 + 2023-07-04T15:03:12Z + DUMMY: + + W73 + + + 0.9933528 + structure_element + cleaner0 + 2023-07-04T12:56:18Z + SO: + + α3 + + + 0.73925114 + structure_element + cleaner0 + 2023-07-04T12:54:50Z + SO: + + N-terminal domain + + + 0.9990564 + residue_name_number + cleaner0 + 2023-07-04T15:03:15Z + DUMMY: + + L93 + + + 0.99907124 + residue_name_number + cleaner0 + 2023-07-04T15:03:19Z + DUMMY: + + L110 + + + 0.9804981 + structure_element + cleaner0 + 2023-07-04T16:00:38Z + SO: + + loop + + + 0.99220556 + structure_element + cleaner0 + 2023-07-04T12:55:42Z + SO: + + β5 + + + 0.9943011 + structure_element + cleaner0 + 2023-07-04T16:00:41Z + SO: + + α4 + + + 0.99905497 + residue_name_number + cleaner0 + 2023-07-04T15:03:24Z + DUMMY: + + A115 + + + 0.9954548 + structure_element + cleaner0 + 2023-07-04T16:00:44Z + SO: + + α5 + + + 0.8100722 + structure_element + cleaner0 + 2023-07-04T12:56:55Z + SO: + + C-terminal domain + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:49:27Z + + ribose + + + 0.99836034 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:28Z + + hydrogen bonded + + + 0.9990386 + residue_name_number + cleaner0 + 2023-07-04T15:03:28Z + DUMMY: + + I69 + + + 0.99780697 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + 0.99834704 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:28Z + + hydrogen bonding + + + 0.99905115 + residue_name_number + cleaner0 + 2023-07-04T15:03:32Z + DUMMY: + + T19 + + + 0.9845276 + structure_element + cleaner0 + 2023-07-04T12:56:07Z + SO: + + α1 + + + 0.9990582 + residue_name_number + cleaner0 + 2023-07-04T15:03:34Z + DUMMY: + + T112 + + + 0.98914295 + structure_element + cleaner0 + 2023-07-04T16:00:49Z + SO: + + α4 + + + structure_element + SO: + cleaner0 + 2023-07-04T12:56:55Z + + C-terminal domain + + + 0.99828535 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + 0.9978372 + site + cleaner0 + 2023-07-04T15:17:30Z + SO: + + hydrophobic pocket + + + 0.9990553 + residue_name_number + cleaner0 + 2023-07-04T15:03:39Z + DUMMY: + + W73 + + + 0.9990553 + residue_name_number + cleaner0 + 2023-07-04T15:03:41Z + DUMMY: + + A76 + + + 0.9903403 + structure_element + cleaner0 + 2023-07-04T12:56:18Z + SO: + + α3 + + + 0.999012 + residue_name_number + cleaner0 + 2023-07-04T15:03:44Z + DUMMY: + + W73 + + + 0.9969107 + protein_state + cleaner0 + 2023-07-04T16:08:14Z + DUMMY: + + highly conserved + + + 0.7578435 + protein_type + cleaner0 + 2023-07-04T14:27:55Z + MESH: + + Tsr3 proteins + + + 0.99897087 + residue_name_number + cleaner0 + 2023-07-04T15:03:48Z + DUMMY: + + A76 + + + 0.728626 + chemical + cleaner0 + 2023-07-04T14:49:33Z + CHEBI: + + amino acids + + + 0.9963153 + protein_type + cleaner0 + 2023-07-04T13:59:17Z + MESH: + + RNA-methyltransferases + + + 0.99891496 + protein + cleaner0 + 2023-07-04T12:58:55Z + PR: + + Trm10 + + + 0.99788874 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + 0.9983612 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T16:12:29Z + + Tsr3-bound + + + + gkw244fig5.jpg + F5 + FIG + fig_caption + 34453 + SAM-binding by Tsr3. (A) Close-up view of the SAM-binding pocket of VdTsr3. Nitrogen atoms are dark blue, oxygen atoms red, sulfur atoms orange, carbon atoms of the protein light blue and carbon atoms of SAM yellow. Hydrogen bonds are indicated by dashed lines. (B) Solvent accessibility of the acp group of SAM bound to VdTsr3. The solvent accessible surface of the protein is shown in semitransparent gray whereas SAM is show in a stick representation. Atoms are colored as in (A). A red arrow indicates the reactive CH2-moiety of the acp group. (C) Solvent accessibility of the SAM methyl group for SAM bound to the RNA methyltransferase Trm10. Bound SAM was modelled based on the X-ray structure of the Trm10/SAH-complex (pdb4jwf). A red arrow indicates the SAM methyl group. (D) Binding of SAM analogs to SsTsr3. Tryptophan fluorescence quenching curves upon addition of SAM (blue), 5′-methyl-thioadenosine (red) and SAH (black). (E) Binding of 14C-labeled SAM to SsTsr3. Radioactively labeled SAM is retained on a filter in the presence of SsTsr3. Addition of unlabeled SAM competes with the binding of labeled SAM. A W66A-mutant of SsTsr3 (W73 in VdTsr3) does not bind SAM. (F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in Δtsr3 cells expressing Tsr3-S62D, -E111A or –W114A. This correlates with a 20S pre-rRNA accumulation comparable to the Δtsr3 deletion (right: northern blot). 3xHA tagged Tsr3 mutants are expressed comparable to the wild type as shown by western blot (lower left). + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:36:26Z + + SAM + + + 0.9984106 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9982338 + site + cleaner0 + 2023-07-04T15:17:35Z + SO: + + SAM-binding pocket + + + 0.9986333 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.93889225 + chemical + cleaner0 + 2023-07-04T14:49:39Z + CHEBI: + + sulfur + + + 0.9985293 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:28Z + + Hydrogen bonds + + + 0.84584075 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + 0.9984308 + chemical + cleaner0 + 2023-07-04T12:36:26Z + CHEBI: + + SAM + + + 0.8229357 + protein_state + cleaner0 + 2023-07-04T16:08:21Z + DUMMY: 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+ + + 0.9987343 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:50:00Z + + SAM + + + 0.99836856 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.98396105 + evidence + cleaner0 + 2023-07-04T15:23:18Z + DUMMY: + + Tryptophan fluorescence quenching curves + + + 0.99866307 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.9976242 + chemical + cleaner0 + 2023-07-04T14:50:05Z + CHEBI: + + 5′-methyl-thioadenosine + + + 0.9985287 + chemical + cleaner0 + 2023-07-04T13:02:55Z + CHEBI: + + SAH + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:50:38Z + + 14C-labeled SAM + + + 0.99834895 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.99848664 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T16:08:57Z + + presence of + + + 0.99842 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.9985732 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.9985936 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.9953957 + mutant + cleaner0 + 2023-07-04T13:34:09Z + MESH: + + W66A + + + 0.7282947 + protein_state + cleaner0 + 2023-07-04T16:09:01Z + DUMMY: + + mutant + + + 0.9984365 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.99610424 + residue_name_number + cleaner0 + 2023-07-04T15:03:58Z + DUMMY: + + W73 + + + 0.9983192 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.9986083 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.97968125 + experimental_method + cleaner0 + 2023-07-04T15:37:32Z + MESH: + + Primer extension + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.99094594 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.8286558 + chemical + cleaner0 + 2023-07-04T12:17:56Z + CHEBI: + + 18S rRNA + + + 0.9967998 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + 0.99414736 + mutant + cleaner0 + 2023-07-04T13:34:14Z + MESH: + + Tsr3-S62D + + + 0.96076906 + mutant + cleaner0 + 2023-07-04T13:34:17Z + MESH: + + -E111A + + + 0.98010665 + mutant + cleaner0 + 2023-07-04T13:34:19Z + MESH: + + –W114A + + + 0.8035865 + chemical + cleaner0 + 2023-07-04T12:48:26Z + CHEBI: + + 20S pre-rRNA + + + 0.99793786 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + 0.8783016 + experimental_method + cleaner0 + 2023-07-04T15:37:43Z + MESH: + + northern blot + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T13:34:46Z + + 3xHA tagged + + + 0.3629817 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.71937597 + protein_state + cleaner0 + 2023-07-04T13:35:01Z + DUMMY: + + mutants + + + 0.9967206 + protein_state + cleaner0 + 2023-07-04T12:31:11Z + DUMMY: + + wild type + + + 0.92499804 + experimental_method + cleaner0 + 2023-07-04T15:37:46Z + MESH: + + western blot + + + + RESULTS + paragraph + 36003 + Binding affinities for SAM and its analogs 5′-methylthioadenosin and SAH to SsTsr3 were measured using tryptophan fluorescence quenching. VdTsr3 could not be used in these experiments since we could not purify it in a stable SAM-free form. SsTsr3 bound SAM with a KD of 6.5 μM, which is similar to SAM-KD's reported for several SPOUT-class methyltransferases. 5′-methylthioadenosin—the reaction product after the acp-transfer—binds only ∼2.5-fold weaker (KD = 16.7 μM) compared to SAM. S-adenosylhomocysteine which lacks the methyl group of SAM binds with significantly lower affinity (KD = 55.5 μM) (Figure 5D). This suggests that the hydrophobic interaction between SAM's methyl group and the hydrophobic pocket of Tsr3 is thermodynamically important for the interaction. On the other hand, the loss of hydrogen bonds between the acp sidechain carboxylate group and the protein appears to be thermodynamically less important but these hydrogen bonds might play a crucial role for the proper orientation of the cofactor side chain in the substrate binding pocket. + + 0.9926367 + evidence + cleaner0 + 2023-07-04T15:23:25Z + DUMMY: + + Binding affinities + + + 0.99860746 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.9977343 + chemical + cleaner0 + 2023-07-04T14:50:45Z + CHEBI: + + 5′-methylthioadenosin + + + 0.9984421 + chemical + cleaner0 + 2023-07-04T13:02:56Z + CHEBI: + + SAH + + + 0.99856395 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.99246246 + experimental_method + cleaner0 + 2023-07-04T15:37:49Z + MESH: + + tryptophan fluorescence quenching + + + 0.99861467 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.8481856 + protein_state + cleaner0 + 2023-07-04T16:09:07Z + DUMMY: + + stable + + + 0.9949457 + protein_state + cleaner0 + 2023-07-04T16:09:12Z + DUMMY: + + SAM-free + + + 0.99857163 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.9594057 + protein_state + cleaner0 + 2023-07-04T16:09:15Z + DUMMY: + + bound + + + 0.9980363 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.9939552 + evidence + cleaner0 + 2023-07-04T15:23:29Z + DUMMY: + + KD + + + 0.9500855 + evidence + cleaner0 + 2023-07-04T15:23:32Z + DUMMY: + + SAM-KD's + + + 0.99625754 + protein_type + cleaner0 + 2023-07-04T13:59:48Z + MESH: + + SPOUT-class methyltransferases + + + 0.99783933 + chemical + cleaner0 + 2023-07-04T14:50:48Z + CHEBI: + + 5′-methylthioadenosin + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.9985222 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.9977331 + chemical + cleaner0 + 2023-07-04T14:50:53Z + CHEBI: + + S-adenosylhomocysteine + + + 0.998492 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.6109229 + evidence + cleaner0 + 2023-07-04T15:23:39Z + DUMMY: + + affinity + + + 0.5327044 + evidence + cleaner0 + 2023-07-04T15:23:43Z + DUMMY: + + KD + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:28Z + + hydrophobic interaction + + + 0.99848396 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.9978147 + site + cleaner0 + 2023-07-04T15:17:52Z + SO: + + hydrophobic pocket + + + 0.99859685 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:28Z + + hydrogen bonds + + + 0.9986084 + chemical + cleaner0 + 2023-07-04T12:37:16Z + CHEBI: + + acp + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:28Z + + hydrogen bonds + + + 0.99764055 + site + cleaner0 + 2023-07-04T15:18:02Z + SO: + + substrate binding pocket + + + + RESULTS + paragraph + 37081 + Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E). Furthermore, a W to A mutation at the equivalent position W114 in ScTsr3 strongly reduced the in vivo acp transferase activity (Figure 5F). The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F). + + 0.9705291 + mutant + cleaner0 + 2023-07-04T13:35:40Z + MESH: + + W66A + + + experimental_method + MESH: + cleaner0 + 2023-07-04T13:35:36Z + + mutation + + + 0.99866235 + residue_name_number + cleaner0 + 2023-07-04T15:04:06Z + DUMMY: + + W73 + + + 0.9982755 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.9987612 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.6666495 + evidence + cleaner0 + 2023-07-04T15:23:48Z + DUMMY: + + SAM-binding + + + 0.98855877 + experimental_method + cleaner0 + 2023-07-04T15:37:56Z + MESH: + + filter binding assay + + + 0.99703217 + protein_state + cleaner0 + 2023-07-04T12:31:11Z + DUMMY: + + wild type + + + 0.8402462 + experimental_method + cleaner0 + 2023-07-04T15:38:13Z + MESH: + + W to A mutation + + + 0.9988821 + residue_name_number + cleaner0 + 2023-07-04T15:04:11Z + DUMMY: + + W114 + + + 0.9987276 + protein + cleaner0 + 2023-07-04T12:52:43Z + PR: + + ScTsr3 + + + protein_type + MESH: + cleaner0 + 2023-07-04T12:36:04Z + + acp transferase + + + 0.99874383 + residue_name_number + cleaner0 + 2023-07-04T15:04:14Z + DUMMY: + + T19 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:36:27Z + + SAM + + + 0.9686928 + experimental_method + cleaner0 + 2023-07-04T15:38:21Z + MESH: + + mutations + + + 0.9987029 + residue_name_number + cleaner0 + 2023-07-04T15:04:22Z + DUMMY: + + T17 + + + 0.9986645 + residue_name_number + cleaner0 + 2023-07-04T15:04:24Z + DUMMY: + + T19 + + + 0.99822766 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.9717845 + residue_name + cleaner0 + 2023-07-04T15:27:23Z + SO: + + A + + + 0.97430634 + residue_name + cleaner0 + 2023-07-04T15:27:26Z + SO: + + D + + + 0.99270034 + evidence + cleaner0 + 2023-07-04T15:23:52Z + DUMMY: + + SAM-binding affinity + + + 0.9986903 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.87185836 + evidence + cleaner0 + 2023-07-04T15:23:55Z + DUMMY: + + KD + + + 0.98011154 + experimental_method + cleaner0 + 2023-07-04T15:38:24Z + MESH: + + mutation + + + 0.9986607 + residue_name_number + cleaner0 + 2023-07-04T15:04:16Z + DUMMY: + + S62 + + + 0.99876213 + protein + cleaner0 + 2023-07-04T12:52:43Z + PR: + + ScTsr3 + + + 0.9630524 + residue_name + cleaner0 + 2023-07-04T15:27:31Z + SO: + + D + + + 0.96706134 + residue_name + cleaner0 + 2023-07-04T15:27:34Z + SO: + + A + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.96824175 + experimental_method + cleaner0 + 2023-07-04T15:38:27Z + MESH: + + primer extension analysis + + + + RESULTS + paragraph + 37802 + The acp-transfer reaction catalyzed by Tsr3 most likely requires the presence of a catalytic base in order to abstract a proton from the N3 imino group of the modified pseudouridine. The side chain of D70 (VdTsr3) located in β4 is ∼5 Å away from the SAM sulfur atom. This residue is conserved as D or E both in archaeal and eukaryotic Tsr3 homologs. Mutations of the corresponding residue in SsTsr3 to A (D63) does not significantly alter the SAM-binding affinity of the protein (KD = 11.0 μM). However, the mutation of the corresponding residue of ScTsr3 (E111A) leads to a significant decrease of the acp transferase activity in vivo (Figure 5F). + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:16Z + + acp + + + 0.99884063 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9921693 + chemical + cleaner0 + 2023-07-04T12:27:45Z + CHEBI: + + pseudouridine + + + 0.9988368 + residue_name_number + cleaner0 + 2023-07-04T15:04:28Z + DUMMY: + + D70 + + + 0.99746466 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.99062866 + structure_element + cleaner0 + 2023-07-04T12:55:27Z + SO: + + β4 + + + 0.9985306 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.9653026 + protein_state + cleaner0 + 2023-07-04T16:09:22Z + DUMMY: + + conserved as + + + 0.9061018 + residue_name + cleaner0 + 2023-07-04T15:27:46Z + SO: + + D + + + 0.91264516 + residue_name + cleaner0 + 2023-07-04T15:27:49Z + SO: + + E + + + 0.99593264 + taxonomy_domain + cleaner0 + 2023-07-04T12:34:34Z + DUMMY: + + archaeal + + + 0.9953687 + taxonomy_domain + cleaner0 + 2023-07-04T12:20:33Z + DUMMY: + + eukaryotic + + + 0.9853893 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9630044 + experimental_method + cleaner0 + 2023-07-04T15:38:32Z + MESH: + + Mutations + + + 0.9986683 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.93534106 + residue_name + cleaner0 + 2023-07-04T15:27:59Z + SO: + + A + + + 0.99817395 + residue_name_number + cleaner0 + 2023-07-04T15:04:34Z + DUMMY: + + D63 + + + 0.99293053 + evidence + cleaner0 + 2023-07-04T15:24:07Z + DUMMY: + + SAM-binding affinity + + + evidence + DUMMY: + cleaner0 + 2023-07-04T15:24:16Z + + KD + + + 0.96987814 + experimental_method + cleaner0 + 2023-07-04T15:38:37Z + MESH: + + mutation + + + 0.9986234 + protein + cleaner0 + 2023-07-04T12:52:43Z + PR: + + ScTsr3 + + + 0.99509156 + mutant + cleaner0 + 2023-07-04T13:03:48Z + MESH: + + E111A + + + protein_type + MESH: + cleaner0 + 2023-07-04T12:36:04Z + + acp transferase + + + + RESULTS + title_2 + 38456 + RNA-binding of Tsr3 + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-07-21T10:07:58Z + + RNA + + + 0.9985703 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + + RESULTS + paragraph + 38476 + Analysis of the electrostatic surface properties of VdTsr3 clearly identified positively charged surface patches in the vicinity of the SAM-binding site suggesting a putative RNA-binding site (Figure 6A). Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix α1. Its negatively charged sulfate group might mimic an RNA backbone phosphate. Helix α1 contains two more positively charged amino acids K17 and R25 as does the loop preceding it (R9). A second cluster of positively charged residues is found in or near helix α3 (K74, R75, K82, R85 and K87). Some of these amino acids are conserved between archaeal and eukaryotic Tsr3 (Supplementary Figure S1A). In the C-terminal domain, the surface exposed α-helices α5 and α7 carry a significant amount of positively charged amino acids. A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-07-06T15:24:22Z + + Analysis of the electrostatic surface properties + + + 0.99870753 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.9920173 + site + cleaner0 + 2023-07-04T15:18:08Z + SO: + + positively charged surface patches + + + 0.99826443 + site + cleaner0 + 2023-07-04T15:18:12Z + SO: + + SAM-binding site + + + 0.99828744 + site + cleaner0 + 2023-07-04T15:18:16Z + SO: + + RNA-binding site + + + 0.6921352 + chemical + cleaner0 + 2023-07-04T14:51:02Z + CHEBI: + + MES + + + 0.99710965 + evidence + cleaner0 + 2023-07-04T15:24:55Z + DUMMY: + + crystal structure + + + 0.99869245 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.9235827 + protein_state + cleaner0 + 2023-07-04T16:09:32Z + DUMMY: + + complexed to + + + 0.99885 + residue_name_number + cleaner0 + 2023-07-04T15:04:48Z + DUMMY: + + K22 + + + 0.9862877 + structure_element + cleaner0 + 2023-07-04T16:00:56Z + SO: + + helix + + + 0.91419095 + structure_element + cleaner0 + 2023-07-04T12:56:07Z + SO: + + α1 + + + 0.8520645 + chemical + cleaner0 + 2023-07-04T14:51:05Z + CHEBI: + + sulfate + + + 0.9982864 + chemical + cleaner0 + 2023-07-04T14:51:10Z + CHEBI: + + RNA + + + 0.99472684 + structure_element + cleaner0 + 2023-07-04T16:01:02Z + SO: + + Helix + + + 0.7284624 + structure_element + cleaner0 + 2023-07-04T12:56:07Z + SO: + + α1 + + + 0.9987287 + residue_name_number + cleaner0 + 2023-07-04T15:04:52Z + DUMMY: + + K17 + + + 0.9987393 + residue_name_number + cleaner0 + 2023-07-04T15:04:54Z + DUMMY: + + R25 + + + 0.98964345 + structure_element + cleaner0 + 2023-07-04T16:01:08Z + SO: + + loop + + + 0.99876547 + residue_name_number + cleaner0 + 2023-07-04T15:04:57Z + DUMMY: + + R9 + + + 0.9933456 + structure_element + cleaner0 + 2023-07-04T16:01:11Z + SO: + + helix + + + 0.7019198 + structure_element + cleaner0 + 2023-07-04T12:56:18Z + SO: + + α3 + + + 0.99877614 + residue_name_number + cleaner0 + 2023-07-04T15:05:00Z + DUMMY: + + K74 + + + 0.9987398 + residue_name_number + cleaner0 + 2023-07-04T15:05:03Z + DUMMY: + + R75 + + + 0.99875665 + residue_name_number + cleaner0 + 2023-07-04T15:05:06Z + DUMMY: + + K82 + + + 0.9987343 + residue_name_number + cleaner0 + 2023-07-04T15:05:08Z + DUMMY: + + R85 + + + 0.9987583 + residue_name_number + cleaner0 + 2023-07-04T15:05:11Z + DUMMY: + + K87 + + + 0.9972134 + protein_state + cleaner0 + 2023-07-04T16:09:36Z + DUMMY: + + conserved + + + 0.9950637 + taxonomy_domain + cleaner0 + 2023-07-04T12:34:34Z + DUMMY: + + archaeal + + + 0.99446905 + taxonomy_domain + cleaner0 + 2023-07-04T12:20:33Z + DUMMY: + + eukaryotic + + + 0.9987722 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.91891503 + structure_element + cleaner0 + 2023-07-04T12:56:55Z + SO: + + C-terminal domain + + + 0.993428 + structure_element + cleaner0 + 2023-07-04T16:01:14Z + SO: + + α-helices + + + 0.9057671 + structure_element + cleaner0 + 2023-07-04T16:01:18Z + SO: + + α5 + + + 0.9876522 + structure_element + cleaner0 + 2023-07-04T16:01:21Z + SO: + + α7 + + + 0.98904145 + experimental_method + cleaner0 + 2023-07-04T15:38:43Z + MESH: + + triple mutation + + + 0.99426585 + protein_state + cleaner0 + 2023-07-04T16:09:50Z + DUMMY: + + conserved + + + 0.9986034 + residue_name_number + cleaner0 + 2023-07-04T15:05:14Z + DUMMY: + + R60 + + + 0.9987668 + residue_name_number + cleaner0 + 2023-07-04T15:05:17Z + DUMMY: + + K65 + + + 0.9987972 + residue_name_number + cleaner0 + 2023-07-04T15:05:19Z + DUMMY: + + R131 + + + 0.9349181 + residue_name + cleaner0 + 2023-07-04T15:28:11Z + SO: + + A + + + 0.99876946 + protein + cleaner0 + 2023-07-04T12:52:43Z + PR: + + ScTsr3 + + + protein_type + MESH: + cleaner0 + 2023-07-04T12:36:04Z + + acp transferase + + + + gkw244fig6.jpg + F6 + FIG + fig_caption + 39635 + RNA-binding of Tsr3. (A) Electrostatic charge distribution on the surface of VdTsr3. Surface areas colored in blue are positively charged whereas red areas are negatively charged. SAM is shown in a stick representation. Also shown in stick representation is a negatively charged MES ion. Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red. For S. solfataricus the chemical identity of the hypermodified nucleotide is not known but the existence of NEP1 and TSR3 homologs suggest that it is indeed N1-methyl-N3-acp-pseudouridine. (C) Binding of SsTsr3 to RNA. 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). Oligo-U9-RNA was used for comparison (black). The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. Combination of the three point mutations (R60A/K65A/R131A) leads to a strongly reduced acp modification of 18S rRNA. + + 0.99881566 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.99865013 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.9986539 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.86521053 + chemical + cleaner0 + 2023-07-04T14:51:17Z + CHEBI: + + MES + + + 0.6367843 + protein_state + cleaner0 + 2023-07-04T16:10:01Z + DUMMY: + + Conserved + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:51:36Z + + amino acids + + + 0.9185542 + structure_element + cleaner0 + 2023-07-04T16:01:27Z + SO: + + helix 31 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:00:32Z + + rRNAs + + + 0.99534893 + species + cleaner0 + 2023-07-04T14:59:47Z + MESH: + + S. cerevisiae + + + 0.9950297 + species + cleaner0 + 2023-07-04T12:52:25Z + MESH: + + S. solfataricus + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T14:38:37Z + + hypermodified + + + 0.99140173 + chemical + cleaner0 + 2023-07-04T14:51:42Z + CHEBI: + + nucleotide + + + 0.994428 + species + cleaner0 + 2023-07-04T12:52:25Z + MESH: + + S. solfataricus + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T14:38:37Z + + hypermodified + + + 0.9902706 + chemical + cleaner0 + 2023-07-04T14:51:46Z + CHEBI: + + nucleotide + + + 0.99886656 + protein + cleaner0 + 2023-07-04T14:23:09Z + PR: + + NEP1 + + + 0.99883705 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + TSR3 + + + 0.9968796 + chemical + cleaner0 + 2023-07-04T14:51:49Z + CHEBI: + + N1-methyl-N3-acp-pseudouridine + + + 0.99873513 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.9947417 + chemical + cleaner0 + 2023-07-04T14:51:53Z + CHEBI: + + RNA + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:53:25Z + + fluoresceine + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:52:18Z + + RNA + + + 0.97259057 + protein_state + cleaner0 + 2023-07-04T16:10:05Z + DUMMY: + + native + + + 0.79190457 + oligomeric_state + cleaner0 + 2023-07-04T13:36:35Z + DUMMY: + + 20mer + + + 0.7217474 + protein_state + cleaner0 + 2023-07-04T16:10:09Z + DUMMY: + + stabilized + + + 0.8586677 + oligomeric_state + cleaner0 + 2023-07-04T13:36:47Z + DUMMY: + + 20mer_GC + + + structure_element + SO: + cleaner0 + 2023-07-04T16:01:44Z + + helix 31 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:00:55Z + + rRNA + + + 0.99525017 + species + cleaner0 + 2023-07-04T12:52:25Z + MESH: + + S. solfataricus + + + 0.7044766 + experimental_method + cleaner0 + 2023-07-04T15:38:51Z + MESH: + + titrated with increasing amounts + + + 0.9987336 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.74837214 + chemical + cleaner0 + 2023-07-04T14:52:35Z + CHEBI: + + fluoresceine + + + 0.9626857 + evidence + cleaner0 + 2023-07-04T15:25:14Z + DUMMY: + + fluorescence anisotropy + + + 0.8108233 + evidence + cleaner0 + 2023-07-04T15:25:18Z + DUMMY: + + binding curve + + + 0.5670902 + oligomeric_state + cleaner0 + 2023-07-04T13:37:00Z + DUMMY: + + 20mer + + + 0.8563897 + oligomeric_state + cleaner0 + 2023-07-04T13:37:11Z + DUMMY: + + 20mer_GC + + + 0.99646825 + chemical + cleaner0 + 2023-07-04T14:52:39Z + CHEBI: + + Oligo-U9-RNA + + + 0.8098977 + oligomeric_state + cleaner0 + 2023-07-04T13:37:23Z + DUMMY: + + 20mer_GC + + + 0.98672026 + chemical + cleaner0 + 2023-07-04T14:52:43Z + CHEBI: + + RNA + + + 0.8701082 + experimental_method + cleaner0 + 2023-07-04T15:39:51Z + MESH: + + titrated + + + 0.9987534 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.99848866 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.4945502 + protein_state + cleaner0 + 2023-07-04T15:40:04Z + DUMMY: + + Mutants + + + 0.99845564 + protein + cleaner0 + 2023-07-04T12:52:43Z + PR: + + ScTsr3 + + + 0.99465525 + residue_name_number + cleaner0 + 2023-07-04T15:05:31Z + DUMMY: + + R60 + + + 0.99793184 + residue_name_number + cleaner0 + 2023-07-04T15:05:32Z + DUMMY: + + K65 + + + 0.99827754 + residue_name_number + cleaner0 + 2023-07-04T15:05:35Z + DUMMY: + + R131 + + + 0.9979954 + residue_name_number + cleaner0 + 2023-07-04T15:05:37Z + DUMMY: + + K17 + + + 0.9981592 + residue_name_number + cleaner0 + 2023-07-04T15:05:40Z + DUMMY: + + K22 + + + 0.998417 + residue_name_number + cleaner0 + 2023-07-04T15:05:42Z + DUMMY: + + R91 + + + 0.9984724 + protein + cleaner0 + 2023-07-04T12:52:37Z + PR: + + VdTsr3 + + + 0.7232749 + experimental_method + cleaner0 + 2023-07-04T15:40:11Z + MESH: + + expressed + + + 0.99725455 + mutant + cleaner0 + 2023-07-04T12:32:06Z + MESH: + + Δtsr3 + + + 0.98106325 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.64899063 + evidence + cleaner0 + 2023-07-04T15:25:23Z + DUMMY: + + primer extension stop + + + 0.99662733 + protein_state + cleaner0 + 2023-07-04T12:31:12Z + DUMMY: + + wild type + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-07-06T15:24:22Z + + Combination of the three point mutations + + + 0.9958204 + mutant + cleaner0 + 2023-07-04T13:37:27Z + MESH: + + R60A + + + 0.9970393 + mutant + cleaner0 + 2023-07-04T13:37:30Z + MESH: + + K65A + + + 0.997195 + mutant + cleaner0 + 2023-07-04T13:37:32Z + MESH: + + R131A + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + 0.5971787 + chemical + cleaner0 + 2023-07-04T12:17:56Z + CHEBI: + + 18S rRNA + + + + RESULTS + paragraph + 41195 + In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5′-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements. SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). A single stranded oligoU-RNA bound with a 10-fold-reduced affinity (6.0 μM). The presence of saturating amounts of SAM (2 mM) did not have a significant influence on the RNA-affinity of SsTsr3 (KD of 1.7 μM for the 20mer-GC-RNA) suggesting no cooperativity in substrate binding. + + 0.99782616 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9878314 + experimental_method + cleaner0 + 2023-07-04T15:40:24Z + MESH: + + titrated + + + 0.9984755 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + 0.9882069 + protein_state + cleaner0 + 2023-07-04T16:10:28Z + DUMMY: + + RNase-free + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:53:44Z + + fluoresceine + + + 0.99855906 + chemical + cleaner0 + 2023-07-04T14:53:51Z + CHEBI: + + RNA + + + 0.9866698 + evidence + cleaner0 + 2023-07-04T15:25:31Z + DUMMY: + + affinity + + + 0.9863573 + experimental_method + cleaner0 + 2023-07-04T15:40:28Z + MESH: + + fluorescence anisotropy measurements + + + 0.99838126 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T16:10:48Z + + apo + + + 0.9739899 + protein_state + cleaner0 + 2023-07-04T16:10:51Z + DUMMY: + + bound + + + 0.7132142 + oligomeric_state + cleaner0 + 2023-07-04T14:54:04Z + DUMMY: + + 20mer + + + 0.99687374 + chemical + cleaner0 + 2023-07-04T14:54:08Z + CHEBI: + + RNA + + + 0.964221 + structure_element + cleaner0 + 2023-07-04T16:01:48Z + SO: + + helix 31 + + + 0.9939218 + species + cleaner0 + 2023-07-04T12:52:25Z + MESH: + + S. solfataricus + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:52:59Z + + 16S rRNA + + + 0.8649587 + evidence + cleaner0 + 2023-07-04T15:26:21Z + DUMMY: + + KD + + + 0.9732021 + structure_element + cleaner0 + 2023-07-04T16:01:51Z + SO: + + hairpin + + + 0.9424103 + oligomeric_state + cleaner0 + 2023-07-04T13:37:48Z + DUMMY: + + 20mer-GC + + + 0.55589414 + evidence + cleaner0 + 2023-07-04T15:26:24Z + DUMMY: + + KD + + + 0.99726105 + chemical + cleaner0 + 2023-07-04T14:54:13Z + CHEBI: + + oligoU-RNA + + + 0.98908454 + protein_state + cleaner0 + 2023-07-04T16:10:59Z + DUMMY: + + bound + + + 0.9814804 + evidence + cleaner0 + 2023-07-04T15:25:36Z + DUMMY: + + affinity + + + 0.99879575 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.9905457 + evidence + cleaner0 + 2023-07-04T15:25:48Z + DUMMY: + + RNA-affinity + + + 0.99852633 + protein + cleaner0 + 2023-07-04T12:52:49Z + PR: + + SsTsr3 + + + evidence + DUMMY: + cleaner0 + 2023-07-04T15:25:45Z + + KD + + + 0.8882387 + oligomeric_state + cleaner0 + 2023-07-04T13:38:02Z + DUMMY: + + 20mer-GC + + + chemical + CHEBI: + cleaner0 + 2023-07-04T13:38:18Z + + RNA + + + + DISCUSS + title_1 + 41928 + DISCUSSION + + + DISCUSS + paragraph + 41939 + U1191 is the only hypermodified base in the yeast 18S rRNA and is strongly conserved in eukaryotes. The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3). This makes it unique in eukaryotic rRNA modification. The m1acp3Ψ base is located at the tip of helix 31 on the 18S rRNA (Supplementary Figure S1B) which, together with helices 18, 24, 34 and 44, contribute to building the decoding center of the small ribosomal subunit. A similar modification (acp3U) was identified in Haloferax volcanii and corresponding modified nucleotides were also shown to occur in other archaea. + + 0.9954489 + residue_name_number + cleaner0 + 2023-07-04T15:05:48Z + DUMMY: + + U1191 + + + 0.5176854 + protein_state + cleaner0 + 2023-07-04T14:38:37Z + DUMMY: + + hypermodified + + + 0.9957968 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.4961639 + chemical + cleaner0 + 2023-07-04T12:17:56Z + CHEBI: + + 18S rRNA + + + 0.99721104 + protein_state + cleaner0 + 2023-07-04T16:11:05Z + DUMMY: + + strongly conserved + + + 0.996487 + taxonomy_domain + cleaner0 + 2023-07-04T14:59:00Z + DUMMY: + + eukaryotes + + + 0.99650055 + chemical + cleaner0 + 2023-07-04T12:24:42Z + CHEBI: + + 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine + + + 0.9825304 + chemical + cleaner0 + 2023-07-04T12:18:12Z + CHEBI: + + m1acp3Ψ + + + structure_element + SO: + cleaner0 + 2023-07-04T14:04:37Z + + H/ACA + + + complex_assembly + GO: + cleaner0 + 2023-07-04T12:29:22Z + + snoRNP + + + 0.9979875 + protein + cleaner0 + 2023-07-04T14:25:34Z + PR: + + snR35 + + + 0.9965995 + protein + cleaner0 + 2023-07-04T14:23:24Z + PR: + + Nep1 + + + 0.9951173 + protein + cleaner0 + 2023-07-04T14:23:31Z + PR: + + Emg1 + + + 0.99738914 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.99688345 + taxonomy_domain + cleaner0 + 2023-07-04T12:20:33Z + DUMMY: + + eukaryotic + + + 0.399971 + chemical + cleaner0 + 2023-07-04T14:54:17Z + CHEBI: + + rRNA + + + 0.9253921 + chemical + cleaner0 + 2023-07-04T12:18:12Z + CHEBI: + + m1acp3Ψ + + + 0.84618723 + structure_element + cleaner0 + 2023-07-04T16:01:56Z + SO: + + helix 31 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:17:56Z + + 18S rRNA + + + structure_element + SO: + cleaner0 + 2023-07-04T16:02:39Z + + helices 18, 24, 34 and 44 + + + 0.9734116 + chemical + cleaner0 + 2023-07-04T14:54:23Z + CHEBI: + + acp3U + + + 0.99549747 + species + cleaner0 + 2023-07-04T13:05:16Z + MESH: + + Haloferax volcanii + + + 0.9920008 + chemical + cleaner0 + 2023-07-04T14:54:41Z + CHEBI: + + nucleotides + + + 0.9965738 + taxonomy_domain + cleaner0 + 2023-07-04T12:35:38Z + DUMMY: + + archaea + + + + DISCUSS + paragraph + 42689 + As shown here TSR3 encodes the transferase catalyzing the acp modification as the last step in the biosynthesis of m1acp3Ψ in yeast and human cells. Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. Similar to the structurally unrelated Rossmann-fold Tyw2 acp transferase, the SAM methyl group of Tsr3 is bound in an inaccessible hydrophobic pocket whereas the acp side chain becomes accessible for a nucleophilic attack by the N3 of pseudouridine. In contrast, in the structurally closely related RNA methyltransferase Trm10 the methyl group of the cofactor SAM is accessible whereas its acp side chain is buried inside the protein. This suggests that enzymes with a SAM-dependent acp transferase activity might have evolved from SAM-dependent methyltransferases by slight modifications of the SAM-binding pocket. Thus, additional examples for acp transferase enzymes might be found with similarities to other structural classes of methyltransferases. In contrast to Nep1, the enzyme preceding Tsr3 in the m1acp3Ψ biosynthesis pathway, Tsr3 binds rather weakly and with little specificity to its isolated substrate RNA. This suggests that Tsr3 is not stably incorporated into pre-ribosomal particles and that its binding to the nascent ribosomal subunit possibly requires additional interactions with other pre-ribosomal components. Consistently, in sucrose gradient analysis, Tsr3 was found in low-molecular weight fractions rather than with pre-ribosome containing high-molecular weight fractions. + + 0.9975333 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + TSR3 + + + 0.8691466 + chemical + cleaner0 + 2023-07-04T12:37:17Z + CHEBI: + + acp + + + 0.99790925 + chemical + cleaner0 + 2023-07-04T12:18:12Z + CHEBI: + + m1acp3Ψ + + + 0.89228064 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.9943726 + species + cleaner0 + 2023-07-04T12:27:15Z + MESH: + + human + + + 0.9943803 + taxonomy_domain + cleaner0 + 2023-07-04T12:34:34Z + DUMMY: + + archaeal + + + 0.9955882 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9956197 + evidence + cleaner0 + 2023-07-04T15:26:31Z + DUMMY: + + structure + + + 0.99729 + protein_type + cleaner0 + 2023-07-04T14:01:04Z + MESH: + + SPOUT-class RNA methyltransferases + + + 0.98861283 + chemical + cleaner0 + 2023-07-04T12:37:17Z + CHEBI: + + acp + + + 0.99829775 + site + cleaner0 + 2023-07-04T15:18:21Z + SO: + + SAM-binding pocket + + + 0.99807334 + chemical + cleaner0 + 2023-07-04T12:37:17Z + CHEBI: + + acp + + + 0.9986235 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.99667263 + chemical + cleaner0 + 2023-07-04T14:54:47Z + CHEBI: + + RNA + + + protein_type + MESH: + cleaner0 + 2023-07-04T13:06:51Z + + Rossmann-fold Tyw2 acp transferase + + + 0.99843293 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.9988778 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.99789596 + site + cleaner0 + 2023-07-04T15:18:26Z + SO: + + hydrophobic pocket + + + 0.9977308 + chemical + cleaner0 + 2023-07-04T12:37:17Z + CHEBI: + + acp + + + 0.99758387 + chemical + cleaner0 + 2023-07-04T12:27:45Z + CHEBI: + + pseudouridine + + + 0.9967475 + protein_type + cleaner0 + 2023-07-04T14:01:13Z + MESH: + + RNA methyltransferase + + + 0.9989367 + protein + cleaner0 + 2023-07-04T12:58:55Z + PR: + + Trm10 + + + 0.9985428 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.9958383 + chemical + cleaner0 + 2023-07-04T12:37:17Z + CHEBI: + + acp + + + 0.96537554 + protein_type + cleaner0 + 2023-07-04T14:01:18Z + MESH: + + SAM-dependent acp transferase + + + 0.995602 + protein_type + cleaner0 + 2023-07-04T14:01:21Z + MESH: + + SAM-dependent methyltransferases + + + 0.9983296 + site + cleaner0 + 2023-07-04T15:18:30Z + SO: + + SAM-binding pocket + + + 0.86682975 + protein_type + cleaner0 + 2023-07-04T12:36:04Z + MESH: + + acp transferase + + + 0.9942889 + protein_type + cleaner0 + 2023-07-04T14:01:25Z + MESH: + + methyltransferases + + + 0.9989317 + protein + cleaner0 + 2023-07-04T14:23:41Z + PR: + + Nep1 + + + 0.99891746 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9979054 + chemical + cleaner0 + 2023-07-04T12:18:12Z + CHEBI: + + m1acp3Ψ + + + 0.9988238 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.99848574 + chemical + cleaner0 + 2023-07-04T14:54:55Z + CHEBI: + + RNA + + + 0.9985832 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.9387523 + complex_assembly + cleaner0 + 2023-07-04T15:53:03Z + GO: + + pre-ribosomal particles + + + 0.6695276 + complex_assembly + cleaner0 + 2023-07-04T15:53:08Z + GO: + + ribosomal subunit + + + 0.96781635 + experimental_method + cleaner0 + 2023-07-04T15:40:50Z + MESH: + + sucrose gradient analysis + + + 0.99829954 + protein + cleaner0 + 2023-07-04T14:08:02Z + PR: + + Tsr3 + + + 0.8778073 + complex_assembly + cleaner0 + 2023-07-04T15:53:12Z + GO: + + pre-ribosome + + + + DISCUSS + paragraph + 44433 + In contrast to several enzymes that catalyze base specific modifications in rRNAs Tsr3 is not an essential protein. Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7). In contrast, for several Tsr3 mutants (SAM-binding and cysteine mutations) we found a systematic correlation between the loss of acp modification and the efficiency of 18S rRNA maturation. This demonstrates that, unlike the other small subunit rRNA base modifications, the acp modification is required for efficient pre-rRNA processing. + + 0.50096446 + chemical + cleaner0 + 2023-07-04T14:56:08Z + CHEBI: + + rRNAs + + + 0.9946498 + protein + cleaner0 + 2023-07-04T14:08:03Z + PR: + + Tsr3 + + + protein_type + MESH: + cleaner0 + 2023-07-04T14:01:55Z + + small subunit rRNA methyltransferases + + + 0.9988682 + protein + cleaner0 + 2023-07-04T14:25:50Z + PR: + + Dim1 + + + 0.9988501 + protein + cleaner0 + 2023-07-04T14:25:53Z + PR: + + Bud23 + + + 0.9988293 + protein + cleaner0 + 2023-07-04T14:23:56Z + PR: + + Nep1 + + + 0.99859124 + protein + cleaner0 + 2023-07-04T14:24:03Z + PR: + + Emg1 + + + 0.66774464 + chemical + cleaner0 + 2023-07-04T14:56:12Z + CHEBI: + + rRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:02:13Z + + pre-RNA + + + 0.90541875 + protein + cleaner0 + 2023-07-04T14:08:03Z + PR: + + Tsr3 + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T15:29:19Z + + SAM-binding + + + 0.6111105 + protein_state + cleaner0 + 2023-07-04T15:29:42Z + DUMMY: + + cysteine mutations + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + 0.8981632 + chemical + cleaner0 + 2023-07-04T12:17:56Z + CHEBI: + + 18S rRNA + + + 0.81034946 + chemical + cleaner0 + 2023-07-04T14:24:14Z + CHEBI: + + rRNA + + + 0.7638087 + chemical + cleaner0 + 2023-07-04T12:37:17Z + CHEBI: + + acp + + + 0.84768337 + chemical + cleaner0 + 2023-07-04T14:56:16Z + CHEBI: + + pre-rRNA + + + + DISCUSS + paragraph + 45169 + Recently, structural, functional, and CRAC (cross-linking and cDNA analysis) experiments of late assembly factors involved in cytoplasmic processing of 40S subunits, along with cryo-EM studies of the late pre-40S subunits have provided important insights into late pre-40S processing. Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-07-06T15:24:22Z + + structural, functional, and CRAC (cross-linking and cDNA analysis) experiments + + + complex_assembly + GO: + cleaner0 + 2023-07-04T15:41:59Z + + 40S subunits + + + 0.99610144 + experimental_method + cleaner0 + 2023-07-04T15:41:34Z + MESH: + + cryo-EM + + + 0.9612105 + protein_state + cleaner0 + 2023-07-04T16:11:17Z + DUMMY: + + late + + + 0.94691247 + complex_assembly + cleaner0 + 2023-07-04T15:53:18Z + GO: + + pre-40S subunits + + + 0.9573801 + complex_assembly + cleaner0 + 2023-07-04T15:53:26Z + GO: + + pre-40S + + + 0.98362994 + complex_assembly + cleaner0 + 2023-07-04T15:53:29Z + GO: + + pre-40S particles + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:56:36Z + + 20S rRNA + + + 0.8642103 + protein_type + cleaner0 + 2023-07-04T14:26:48Z + MESH: + + non-ribosomal proteins + + + 0.993677 + protein_type + cleaner0 + 2023-07-04T14:26:51Z + MESH: + + D-site endonuclease + + + 0.9986266 + protein + cleaner0 + 2023-07-04T14:25:57Z + PR: + + Nob1 + + + 0.99859494 + protein + cleaner0 + 2023-07-04T14:26:00Z + PR: + + Tsr1 + + + 0.9910274 + protein_type + cleaner0 + 2023-07-04T14:26:55Z + MESH: + + GTPase + + + 0.9984989 + protein + cleaner0 + 2023-07-04T14:26:02Z + PR: + + Rio2 + + + 0.9762112 + site + cleaner0 + 2023-07-04T15:18:42Z + SO: + + mRNA channel + + + 0.9976265 + site + cleaner0 + 2023-07-04T15:18:44Z + SO: + + initiator tRNA binding site + + + + DISCUSS + paragraph + 45789 + After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. The cleavage step most likely acts as a quality control check that ensures the proper 40S subunit assembly with only completely processed precursors. Finally, termination factor Rli1, an ATPase, promotes the dissociation of assembly factors and the 80S-like complex dissociates and releases the mature 40S subunit. + + 0.9948684 + chemical + cleaner0 + 2023-07-04T14:56:41Z + CHEBI: + + GTP + + + 0.9979454 + site + cleaner0 + 2023-07-04T15:18:52Z + SO: + + decoding site + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:48:26Z + + 20S pre-rRNA + + + 0.82476866 + protein + cleaner0 + 2023-07-04T13:09:12Z + PR: + + Nob1 + + + 0.8359637 + site + cleaner0 + 2023-07-04T15:18:58Z + SO: + + site D + + + 0.8337764 + complex_assembly + cleaner0 + 2023-07-04T15:53:33Z + GO: + + pre-40S + + + 0.6900945 + complex_assembly + cleaner0 + 2023-07-04T15:53:36Z + GO: + + 60S subunits + + + 0.98824817 + complex_assembly + cleaner0 + 2023-07-04T15:53:39Z + GO: + + 80S-like particles + + + 0.9983012 + protein + cleaner0 + 2023-07-04T13:09:10Z + PR: + + eIF5B + + + complex_assembly + GO: + cleaner0 + 2023-07-04T15:54:02Z + + 40S subunit + + + 0.73086596 + protein_type + cleaner0 + 2023-07-04T14:27:00Z + MESH: + + termination factor + + + 0.9985043 + protein + cleaner0 + 2023-07-04T13:09:15Z + PR: + + Rli1 + + + 0.965042 + protein_type + cleaner0 + 2023-07-04T13:09:19Z + MESH: + + ATPase + + + 0.98697394 + complex_assembly + cleaner0 + 2023-07-04T15:54:07Z + GO: + + 80S-like complex + + + 0.98814327 + protein_state + cleaner0 + 2023-07-04T16:11:28Z + DUMMY: + + mature + + + complex_assembly + GO: + cleaner0 + 2023-07-04T15:54:25Z + + 40S subunit + + + + DISCUSS + paragraph + 46413 + Interestingly, differences in the level of acp modification were demonstrated for different steps of the cytoplasmic pre-40S subunit maturation after analyzing purified 20S pre-rRNAs using different purification bait proteins. Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified. In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits. Thus, the acp transfer to m1Ψ1191 occurs during the step at which Rio2 leaves the pre-40S particle. + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + 0.9643731 + complex_assembly + cleaner0 + 2023-07-04T15:54:32Z + GO: + + pre-40S subunit + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:57:00Z + + 20S pre-rRNAs + + + 0.96552056 + complex_assembly + cleaner0 + 2023-07-04T15:54:35Z + GO: + + pre-40S subunits + + + protein_type + MESH: + cleaner0 + 2023-07-04T14:27:20Z + + ribosome assembly factors + + + 0.9985605 + protein + cleaner0 + 2023-07-04T13:08:13Z + PR: + + Tsr1 + + + 0.9984236 + protein + cleaner0 + 2023-07-04T13:08:15Z + PR: + + Ltv1 + + + 0.9982217 + protein + cleaner0 + 2023-07-04T13:08:18Z + PR: + + Enp1 + + + 0.99821365 + protein + cleaner0 + 2023-07-04T13:08:21Z + PR: + + Rio2 + + + protein_state + DUMMY: + cleaner0 + 2023-07-04T16:11:55Z + + acp modified + + + 0.96248454 + complex_assembly + cleaner0 + 2023-07-04T15:54:38Z + GO: + + pre-40S subunits + + + 0.99809605 + protein + cleaner0 + 2023-07-04T13:09:05Z + PR: + + Nob1 + + + 0.99777716 + protein + cleaner0 + 2023-07-04T13:09:02Z + PR: + + Rio1 + + + 0.8839221 + complex_assembly + cleaner0 + 2023-07-04T15:54:42Z + GO: + + 60S subunits + + + 0.78486603 + complex_assembly + cleaner0 + 2023-07-04T15:54:45Z + GO: + + 80S-like particles + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + 0.99625635 + protein_state + cleaner0 + 2023-07-04T16:12:04Z + DUMMY: + + mature + + + 0.7907378 + complex_assembly + cleaner0 + 2023-07-04T15:54:48Z + GO: + + 40S subunits + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + 0.99692446 + residue_name_number + cleaner0 + 2023-07-04T15:06:37Z + DUMMY: + + m1Ψ1191 + + + 0.9954157 + protein + cleaner0 + 2023-07-04T13:09:07Z + PR: + + Rio2 + + + 0.9807246 + complex_assembly + cleaner0 + 2023-07-04T15:54:51Z + GO: + + pre-40S particle + + + + DISCUSS + paragraph + 47079 + These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1. The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the Ψ1191 residue that receives the acp modification. Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + 0.94139224 + chemical + cleaner0 + 2023-07-04T14:57:42Z + CHEBI: + + pre-20S rRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + 0.9972498 + protein + cleaner0 + 2023-07-04T14:26:08Z + PR: + + Rio2 + + + 0.9969145 + site + cleaner0 + 2023-07-04T15:19:18Z + SO: + + decoding site + + + site + SO: + cleaner0 + 2023-07-04T14:58:25Z + + D-site + + + 0.99823534 + protein + cleaner0 + 2023-07-04T14:26:11Z + PR: + + Nob1 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + 0.99523795 + protein + cleaner0 + 2023-07-04T14:26:15Z + PR: + + Rio2 + + + 0.842754 + experimental_method + cleaner0 + 2023-07-04T15:42:06Z + MESH: + + CRAC analysis + + + 0.995242 + protein + cleaner0 + 2023-07-04T14:26:18Z + PR: + + Rio2 + + + 0.957628 + structure_element + cleaner0 + 2023-07-04T16:02:48Z + SO: + + helix 31 + + + 0.99894506 + residue_name_number + cleaner0 + 2023-07-04T15:06:43Z + DUMMY: + + Ψ1191 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + 0.9965959 + protein + cleaner0 + 2023-07-04T14:26:22Z + PR: + + Rio2 + + + 0.52873844 + protein + cleaner0 + 2023-07-04T14:08:03Z + PR: + + Tsr3 + + + 0.89228296 + structure_element + cleaner0 + 2023-07-04T16:02:50Z + SO: + + helix 31 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + 0.9940521 + protein + cleaner0 + 2023-07-04T14:26:25Z + PR: + + Rio2 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + 0.9988901 + residue_name_number + cleaner0 + 2023-07-04T15:06:48Z + DUMMY: + + m1Ψ1191 + + + chemical + CHEBI: + cleaner0 + 2023-07-04T14:57:58Z + + 20S rRNA + + + 0.99593145 + protein + cleaner0 + 2023-07-04T14:26:28Z + PR: + + Rio2 + + + 0.9363694 + structure_element + cleaner0 + 2023-07-04T16:02:53Z + SO: + + helix 31 + + + 0.92609435 + chemical + cleaner0 + 2023-07-04T14:58:14Z + CHEBI: + + pre-rRNA + + + + DISCUSS + paragraph + 47812 + In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. The current data together with the finding that acp modification takes place at the very last step in pre-40S subunit maturation indicate that the acp modification probably supports the formation of the decoding site and efficient 20S pre-rRNA D-site cleavage. Furthermore, our structural data unravelled how the regioselectivity of SAM-dependent group transfer reactions can be tuned by distinct small evolutionary adaptions of the ligand binding pocket of SAM-binding enzymes. + + 0.9977992 + protein + cleaner0 + 2023-07-04T14:08:03Z + PR: + + Tsr3 + + + 0.8789575 + chemical + cleaner0 + 2023-07-04T12:37:17Z + CHEBI: + + acp + + + 0.9624326 + protein_state + cleaner0 + 2023-07-04T14:38:37Z + DUMMY: + + hypermodified + + + 0.8184547 + chemical + cleaner0 + 2023-07-04T12:18:12Z + CHEBI: + + m1acp3Ψ + + + 0.8651114 + chemical + cleaner0 + 2023-07-04T14:58:30Z + CHEBI: + + nucleotide + + + 0.65205365 + residue_number + cleaner0 + 2023-07-04T15:33:46Z + DUMMY: + + 1191 + + + 0.9899032 + taxonomy_domain + cleaner0 + 2023-07-04T12:18:26Z + DUMMY: + + yeast + + + 0.49469864 + residue_number + cleaner0 + 2023-07-04T15:06:57Z + DUMMY: + + 1248 + + + 0.987317 + species + cleaner0 + 2023-07-04T12:18:32Z + MESH: + + humans + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:17:56Z + + 18S rRNA + + + 0.9969258 + taxonomy_domain + cleaner0 + 2023-07-04T12:20:33Z + DUMMY: + + eukaryotic + + + chemical + CHEBI: + cleaner0 + 2023-07-04T15:55:21Z + + small ribosomal subunit rRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + complex_assembly + GO: + cleaner0 + 2023-07-04T15:55:41Z + + pre-40S subunit + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:37:17Z + + acp + + + 0.9980171 + site + cleaner0 + 2023-07-04T15:19:27Z + SO: + + decoding site + + + chemical + CHEBI: + cleaner0 + 2023-07-04T12:48:26Z + + 20S pre-rRNA + + + 0.84141666 + site + cleaner0 + 2023-07-04T15:19:33Z + SO: + + D-site + + + 0.9965247 + evidence + cleaner0 + 2023-07-04T15:26:40Z + DUMMY: + + structural data + + + 0.61613 + chemical + cleaner0 + 2023-07-04T12:36:27Z + CHEBI: + + SAM + + + 0.9981494 + site + cleaner0 + 2023-07-04T15:19:39Z + SO: + + ligand binding pocket + + + 0.958201 + protein_type + cleaner0 + 2023-07-04T14:27:25Z + MESH: + + SAM-binding enzymes + + + + KEYWORD + title_1 + 48587 + ACCESSION NUMBERS + + + KEYWORD + paragraph + 48605 + Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes PDB 5APG (VdTsr3/SAM-complex) and PDB 5AP8 (SsTsr3). + + + SUPPL + title_1 + 48759 + Supplementary Material + + + SUPPL + title_1 + 48782 + SUPPLEMENTARY DATA + + + SUPPL + paragraph + 48801 + Supplementary Data are available at NAR Online. + + + ACK_FUND + title_1 + 48849 + FUNDING + + + ACK_FUND + paragraph + 48857 + DFG grant [En134/9-1]; SFB 902 (Molecular Principles of RNA-based Regulation); DFG SPP1784 (Chemical Biology of Native Nucleic Acid Modifications, DFG grants) [En134/13-1, Wo 901/5-1]; European Community's Seventh Framework Programme [FP7/2007-2013] under BioStruct-X [283570]; Goethe University and the State of Hesse; EMBO long-term fellowship [ALTF 644-2014 to S.S.]; Research in the Lab of DLJL is supported by the Université Libre de Bruxelles (ULB); Fonds National de la Recherche (F.R.S./FNRS); Walloon Region [DGO6]; Fédération Wallonie-Bruxelles; European Research Development Fund (ERDF). Funding for open access charge: DFG SPP1784 (Chemical Biology of Native Nucleic Acid Modifications, DFG grants) [En134/13-1, Wo 901/5-1]. + + + ACK_FUND + paragraph + 49597 + Conflict of interest statement. None declared. + + + REF + title + 49644 + REFERENCES + + + 643 + 681 + surname:Woolford;given-names:J.L.;suffix:Jr + surname:Baserga;given-names:S.J. + 24190922 + REF + Genetics + ref + 195 + 2013 + 49655 + Ribosome biogenesis in the yeast Saccharomyces cerevisiae + + + 1491 + 1500 + surname:Armistead;given-names:J. + surname:Triggs-Raine;given-names:B. + 24657617 + REF + FEBS Lett. + ref + 588 + 2014 + 49713 + Diverse diseases from a ubiquitous process: the ribosomopathy paradox + + + 849 + 850 + surname:McCann;given-names:K.L. + surname:Baserga;given-names:S.J. + REF + Science (New York, N.Y.) + ref + 341 + 2013 + 49783 + Genetics. Mysterious ribosomopathies + + + 758 + 764 + surname:Sondalle;given-names:S.B. + surname:Baserga;given-names:S.J. + 24240090 + REF + Biochim. Biophys. Acta + ref + 1842 + 2014 + 49820 + Human diseases of the SSU processome + + + 1077 + 1088 + surname:Kiss-Laszlo;given-names:Z. + surname:Henry;given-names:Y. + surname:Bachellerie;given-names:J.P. + surname:Caizergues-Ferrer;given-names:M. + surname:Kiss;given-names:T. + 8674114 + REF + Cell + ref + 85 + 1996 + 49857 + Site-specific ribose methylation of preribosomal RNA. a novel function for small nucleolar RNAs + + + 799 + 809 + surname:Ganot;given-names:P. + surname:Bortolin;given-names:M.L. + surname:Kiss;given-names:T. + 9182768 + REF + Cell + ref + 89 + 1997 + 49953 + Site-specific pseudouridine formation in preribosomal RNA is guided by small nucleolar RNAs + + + 560 + 575 + surname:Sharma;given-names:S. + surname:Lafontaine;given-names:D.L.J. + 26410597 + REF + Trends Biochem. Sci. + ref + 40 + 2015 + 50045 + 'View from a bridge': a new perspective on eukaryotic rRNA base modification + + + 1151 + 1163 + surname:Peifer;given-names:C. + surname:Sharma;given-names:S. + surname:Watzinger;given-names:P. + surname:Lamberth;given-names:S. + surname:Kötter;given-names:P. + surname:Entian;given-names:K.-D. + 23180764 + REF + Nucleic Acids Res. + ref + 41 + 2013 + 50122 + Yeast Rrp8p, a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA + + + 9062 + 9076 + surname:Sharma;given-names:S. + surname:Yang;given-names:J. + surname:Watzinger;given-names:P. + surname:Kotter;given-names:P. + surname:Entian;given-names:K.-D. + 23913415 + REF + Nucleic Acids Res. + ref + 41 + 2013 + 50215 + Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively + + + 6158 + surname:Schosserer;given-names:M. + surname:Minois;given-names:N. + surname:Angerer;given-names:T.B. + surname:Amring;given-names:M. + surname:Dellago;given-names:H. + surname:Harreither;given-names:E. + surname:Calle-Perez;given-names:A. + surname:Pircher;given-names:A. + surname:Gerstl;given-names:M.P. + surname:Pfeifenberger;given-names:S. + 25635753 + REF + Nat. Commun. + ref + 6 + 2015 + 50291 + Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan + + + 721 + 733 + surname:Helm;given-names:M. + 16452298 + REF + Nucleic Acids Res. + ref + 34 + 2006 + 50385 + Post-transcriptional nucleotide modification and alternative folding of RNA + + + 610 + 619 + surname:Chow;given-names:C.S. + surname:Lamichhane;given-names:T.N. + surname:Mahto;given-names:S.K. + 17894445 + REF + ACS Chem. Biol. + ref + 2 + 2007 + 50461 + Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications + + + 330 + 339 + surname:Ishitani;given-names:R. + surname:Yokoyama;given-names:S. + surname:Nureki;given-names:O. + 18539024 + REF + Curr. Opin. Struct. Biol. + ref + 18 + 2008 + 50553 + Structure, dynamics, and function of RNA modification enzymes + + + 344 + 351 + surname:Decatur;given-names:W.A. + surname:Fournier;given-names:M.J. + 12114023 + REF + Trends Biochem. Sci. + ref + 27 + 2002 + 50615 + rRNA modifications and ribosome function + + + e174 + surname:Baxter-Roshek;given-names:J.L. + surname:Petrov;given-names:A.N. + surname:Dinman;given-names:J.D. + 17245450 + REF + PLoS One + ref + 2 + 2007 + 50656 + Optimization of ribosome structure and function by rRNA base modification + + + 1716 + 1728 + surname:Liang;given-names:X.H. + surname:Liu;given-names:Q. + surname:Fournier;given-names:M.J. + 19628622 + REF + RNA + ref + 15 + 2009 + 50730 + Loss of rRNA modifications in the decoding center of the ribosome impairs translation and strongly delays pre-rRNA processing + + + 425 + 435 + surname:King;given-names:T.H. + surname:Liu;given-names:B. + surname:McCully;given-names:R.R. + surname:Fournier;given-names:M.J. + 12620230 + REF + Mol. Cell + ref + 11 + 2003 + 50856 + Ribosome structure and activity are altered in cells lacking snoRNPs that form pseudouridines in the peptidyl transferase center + + + 3196 + 3205 + surname:Narla;given-names:A. + surname:Ebert;given-names:B.L. + 20194897 + REF + Blood + ref + 115 + 2010 + 50985 + Ribosomopathies: human disorders of ribosome dysfunction + + + 11 + 19 + surname:Lafontaine;given-names:D.L.J. + 25565028 + REF + Nat. Struct. Mol. Biol. + ref + 22 + 2015 + 51042 + Noncoding RNAs in eukaryotic ribosome biogenesis and function + + + 2721 + 2727 + surname:Sato;given-names:G. + surname:Saijo;given-names:Y. + surname:Uchiyama;given-names:B. + surname:Kumano;given-names:N. + surname:Sugawara;given-names:S. + surname:Fujimura;given-names:S. + surname:Sato;given-names:M. + surname:Sagawa;given-names:M. + surname:Ohkuda;given-names:K. + surname:Koike;given-names:K. + 10561346 + REF + J. Clin. Oncol. + ref + 17 + 1999 + 51104 + Prognostic value of nucleolar protein p120 in patients with resected lung adenocarcinoma + + + 319 + 322 + surname:Enger;given-names:M.D. + surname:Saponara;given-names:A.G. + 5646651 + REF + J. Mol. Biol. + ref + 33 + 1968 + 51193 + Incorporation of 14C from [2-14C]methionine into 18 s but not 28 s RNA of Chinese hamster cells + + + 2548 + 2554 + surname:Samarsky;given-names:D.A. + surname:Balakin;given-names:A.G. + surname:Fournier;given-names:M.J. + 7630735 + REF + Nucleic Acids Res. + ref + 23 + 1995 + 51289 + Characterization of three new snRNAs from Saccharomyces cerevisiae. snR34, snR35 and snR36 + + + 1542 + 1554 + surname:Taylor;given-names:A.B. + surname:Meyer;given-names:B. + surname:Leal;given-names:B.Z. + surname:Kötter;given-names:P. + surname:Schirf;given-names:V. + surname:Demeler;given-names:B. + surname:Hart;given-names:P.J. + surname:Entian;given-names:K.-D. + surname:Wöhnert;given-names:J. + 18208838 + REF + Nucleic Acids Res. + ref + 36 + 2008 + 51380 + The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site + + + 2387 + 2398 + surname:Wurm;given-names:J.P. + surname:Meyer;given-names:B. + surname:Bahr;given-names:U. + surname:Held;given-names:M. + surname:Frolow;given-names:O. + surname:Kotter;given-names:P. + surname:Engels;given-names:J.W. + surname:Heckel;given-names:A. + surname:Karas;given-names:M. + surname:Entian;given-names:K.D. + 20047967 + REF + Nucleic Acids Res. + ref + 38 + 2010 + 51502 + The ribosome assembly factor Nep1 responsible for Bowen-Conradi syndrome is a pseudouridine-N1-specific methyltransferase + + + 1526 + 1537 + surname:Meyer;given-names:B. + surname:Wurm;given-names:J.P. + surname:Kötter;given-names:P. + surname:Leisegang;given-names:M.S. + surname:Schilling;given-names:V. + surname:Buchhaupt;given-names:M. + surname:Held;given-names:M. + surname:Bahr;given-names:U. + surname:Karas;given-names:M. + surname:Heckel;given-names:A. + 20972225 + REF + Nucleic Acids Res. + ref + 39 + 2011 + 51624 + The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA + + + 71 + 77 + surname:Brand;given-names:R.C. + surname:Klootwijk;given-names:J. + surname:Planta;given-names:R.J. + surname:Maden;given-names:B.E. + 629754 + REF + Biochem J. + ref + 169 + 1978 + 51806 + Biosynthesis of a hypermodified nucleotide in Saccharomyces carlsbergensis 17S and HeLa-cell 18S ribosomal ribonucleic acid + + + 12138 + 12154 + surname:Hector;given-names:R.D. + surname:Burlacu;given-names:E. + surname:Aitken;given-names:S. + surname:Le Bihan;given-names:T. + surname:Tuijtel;given-names:M. + surname:Zaplatina;given-names:A. + surname:Cook;given-names:A.G. + surname:Granneman;given-names:S. + 25200078 + REF + Nucleic Acids Res. + ref + 42 + 2014 + 51930 + Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution + + + 161 + 170 + surname:Lin;given-names:H. + 21762947 + REF + Bioorg. Chem. + ref + 39 + 2011 + 52040 + S-Adenosylmethionine-dependent alkylation reactions: when are radical reactions used? + + + 2142 + 2154 + surname:Noma;given-names:A. + surname:Kirino;given-names:Y. + surname:Ikeuchi;given-names:Y. + surname:Suzuki;given-names:T. + 16642040 + REF + EMBO J. + ref + 25 + 2006 + 52126 + Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA + + + 15616 + 15621 + surname:Umitsu;given-names:M. + surname:Nishimasu;given-names:H. + surname:Noma;given-names:A. + surname:Suzuki;given-names:T. + surname:Ishitani;given-names:R. + surname:Nureki;given-names:O. + 19717466 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 106 + 2009 + 52215 + Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2 + + + 1213 + 1226 + surname:Schaffrath;given-names:R. + surname:Abdel-Fattah Mohamed;given-names:W. + surname:Klassen;given-names:R. + surname:Stark;given-names:M.J. + 25352115 + REF + Mol. Microbiol. + ref + 94 + 2014 + 52344 + The diphthamide modification pathway from Saccharomyces cerevisiae - revisited + + + 149 + 154 + surname:Mattheakis;given-names:L.C. + surname:Sor;given-names:F. + surname:Collier;given-names:R.J. + 8406038 + REF + Gene + ref + 132 + 1993 + 52423 + Diphthamide synthesis in Saccharomyces cerevisiae: structure of the DPH2 gene + + + 9487 + 9497 + surname:Liu;given-names:S. + surname:Milne;given-names:G.T. + surname:Kuremsky;given-names:J.G. + surname:Fink;given-names:G.R. + surname:Leppla;given-names:S.H. + 15485916 + REF + Mol. Cell. Biol. + ref + 24 + 2004 + 52501 + Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2 + + + e1000213 + surname:Li;given-names:Z. + surname:Lee;given-names:I. + surname:Moradi;given-names:E. + surname:Hung;given-names:N.-J. + surname:Johnson;given-names:A.W. + surname:Marcotte;given-names:E.M. + 19806183 + REF + PLoS Biol. + ref + 7 + 2009 + 52657 + Rational extension of the ribosome biogenesis pathway using network-guided genetics + + + 424 + surname:Burroughs;given-names:A.M. + surname:Aravind;given-names:L. + 25566315 + REF + Front. Genet. + ref + 5 + 2014 + 52741 + Analysis of two domains with novel RNA-processing activities throws light on the complex evolution of ribosomal RNA biogenesis + + + 3 + 36 + surname:Gehrke;given-names:C.W. + surname:Kuo;given-names:K.C. + 2670985 + REF + J. Chromatogr. + ref + 471 + 1989 + 52868 + Ribonucleoside analysis by reversed-phase high-performance liquid chromatography + + + surname:Sambrook;given-names:J. + surname:Russell;given-names:D. + REF + Molecular Cloning. A Laboratory Manual + ref + 2001 + 52949 + + + 2242 + 2258 + surname:Sharma;given-names:S. + surname:Langhendries;given-names:J.-L. + surname:Watzinger;given-names:P. + surname:Kotter;given-names:P. + surname:Entian;given-names:K.-D. + surname:Lafontaine;given-names:D.L.J. + 25653167 + REF + Nucleic Acids Res. + ref + 43 + 2015 + 52950 + Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1 + + + 3246 + 3260 + surname:Sharma;given-names:S. + surname:Yang;given-names:J. + surname:Düttmann;given-names:S. + surname:Watzinger;given-names:P. + surname:Kötter;given-names:P. + surname:Entian;given-names:K.-D. + 24335083 + REF + Nucleic Acids Res + ref + 42 + 2014 + 53084 + Identification of novel methyltransferases, Bmt5 and Bmt6, responsible for the m3U methylations of 25S rRNA in Saccharomyces cerevisiae + + + 686 + 691 + surname:Huh;given-names:W.K. + surname:Falvo;given-names:J.V. + surname:Gerke;given-names:L.C. + surname:Carroll;given-names:A.S. + surname:Howson;given-names:R.W. + surname:Weissman;given-names:J.S. + surname:O'Shea;given-names:E.K. + 14562095 + REF + Nature + ref + 425 + 2003 + 53220 + Global analysis of protein localization in budding yeast + + + 60 + 63 + surname:Maden;given-names:B.E. + surname:Forbes;given-names:J. + surname:de Jonge;given-names:P. + surname:Klootwijk;given-names:J. + 1225623 + REF + FEBS Lett. + ref + 59 + 1975 + 53277 + Presence of a hypermodified nucleotide in HeLa cell 18 S and Saccharomyces carlsbergensis 17 S ribosomal RNAs + + + 771 + 781 + surname:Buchhaupt;given-names:M. + surname:Kötter;given-names:P. + surname:Entian;given-names:K.D. + 17425675 + REF + FEMS Yeast Res. + ref + 7 + 2007 + 53387 + Mutations in the nucleolar proteins Tma23 and Nop6 suppress the malfunction of the Nep1 protein + + + 326 + 338 + surname:Eschrich;given-names:D. + surname:Buchhaupt;given-names:M. + surname:Kötter;given-names:P. + surname:Entian;given-names:K.D. + 11935223 + REF + Curr. Genet. + ref + 40 + 2002 + 53483 + Nep1p (Emg1p), a novel protein conserved in eukaryotes and archaea, is involved in ribosome biogenesis + + + 3 + surname:Armengaud;given-names:J. + surname:Dedieu;given-names:A. + surname:Solques;given-names:O. + surname:Pellequer;given-names:J.-L. + surname:Quemeneur;given-names:E. + 15701177 + REF + BMC Struct. Biol. + ref + 5 + 2005 + 53586 + Deciphering structure and topology of conserved COG2042 orphan proteins + + + 97 + 105 + surname:Woese;given-names:C.R. + surname:Gupta;given-names:R. + surname:Hahn;given-names:C.M. + surname:Zillig;given-names:W. + surname:Tu;given-names:J. + 11541975 + REF + Syst. Appl. Microbiol. + ref + 5 + 1984 + 53658 + The phylogenetic relationships of three sulfur dependent archaebacteria + + + 7962 + 7971 + surname:Karcher;given-names:A. + surname:Schele;given-names:A. + surname:Hopfner;given-names:K.-P. + 18160405 + REF + J. Biol. Chem. + ref + 283 + 2008 + 53730 + X-ray structure of the complete ABC enzyme ABCE1 from Pyrococcus abyssi + + + 574 + 585 + surname:Jackman;given-names:J.E. + surname:Montange;given-names:R.K. + surname:Malik;given-names:H.S. + surname:Phizicky;given-names:E.M. + 12702816 + REF + RNA + ref + 9 + 2003 + 53802 + Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9 + + + 509 + 525 + surname:Shao;given-names:Z. + surname:Yan;given-names:W. + surname:Peng;given-names:J. + surname:Zuo;given-names:X. + surname:Zou;given-names:Y. + surname:Li;given-names:F. + surname:Gong;given-names:D. + surname:Ma;given-names:R. + surname:Wu;given-names:J. + surname:Shi;given-names:Y. + 24081582 + REF + Nucleic Acids Res. + ref + 42 + 2014 + 53918 + Crystal structure of tRNA m1G9 methyltransferase Trm10: insight into the catalytic mechanism and recognition of tRNA substrate + + + 1203 + 1209 + surname:Ben-Shem;given-names:A. + surname:Jenner;given-names:L. + surname:Yusupova;given-names:G. + surname:Yusupov;given-names:M. + REF + Science (New York, N.Y.) + ref + 330 + 2010 + 54045 + Crystal structure of the eukaryotic ribosome + + + 24484 + 24489 + surname:Kowalak;given-names:J.A. + surname:Bruenger;given-names:E. + surname:Crain;given-names:P.F. + surname:McCloskey;given-names:J.A. + 10818097 + REF + J. Biol. Chem. + ref + 275 + 2000 + 54090 + Identities and phylogenetic comparisons of posttranscriptional modifications in 16 S ribosomal RNA from Haloferax volcanii + + + 254 + 261 + surname:Jones;given-names:W. + surname:Leigh;given-names:J. + surname:Mayer;given-names:F. + surname:Woese;given-names:C.R. + surname:Wolfe;given-names:R. + REF + Arch. Microbiol. + ref + 136 + 1983 + 54213 + Methanococcus jannaschii sp. nov., an extremely thermophilic methanogen from a submarine hydrothermal vent + + + 492 + 497 + surname:Lafontaine;given-names:D. + surname:Delcour;given-names:J. + surname:Glasser;given-names:A.L. + surname:Desgres;given-names:J. + surname:Vandenhaute;given-names:J. + 8064863 + REF + J. Mol. Biol. + ref + 241 + 1994 + 54320 + The DIM1 gene responsible for the conserved m6(2)Am6(2)A dimethylation in the 3'-terminal loop of 18 S rRNA is essential in yeast + + + 3151 + 3161 + surname:White;given-names:J. + surname:Li;given-names:Z. + surname:Sardana;given-names:R. + surname:Bujnicki;given-names:J.M. + surname:Marcotte;given-names:E.M. + surname:Johnson;given-names:A.W. + 18332120 + REF + Mol. Cell. Biol. + ref + 28 + 2008 + 54450 + Bud23 methylates G1575 of 18S rRNA and is required for efficient nuclear export of pre-40S subunits + + + 2080 + 2095 + surname:Zorbas;given-names:C. + surname:Nicolas;given-names:E. + surname:Wacheul;given-names:L. + surname:Huvelle;given-names:E. + surname:Heurgué-Hamard;given-names:V. + surname:Lafontaine;given-names:D.L.J. + 25851604 + REF + Mol. Biol. Cell + ref + 26 + 2015 + 54550 + The human 18S rRNA base methyltransferases DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis + + + 629 + 639 + surname:Leulliot;given-names:N. + surname:Bohnsack;given-names:M.T. + surname:Graille;given-names:M. + surname:Tollervey;given-names:D. + surname:van Tilbeurgh;given-names:H. + 18063569 + REF + Nucleic Acids Res. + ref + 36 + 2008 + 54683 + The yeast ribosome synthesis factor Emg1 is a novel member of the superfamily of alpha/beta knot fold methyltransferases + + + 2026 + 2036 + surname:Granneman;given-names:S. + surname:Petfalski;given-names:E. + surname:Swiatkowska;given-names:A. + surname:Tollervey;given-names:D. + 20453830 + REF + EMBO J. + ref + 29 + 2010 + 54804 + Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA-protein cross-linking + + + 1449 + 1453 + surname:Strunk;given-names:B.S. + surname:Loucks;given-names:C.R. + surname:Su;given-names:M. + surname:Vashisth;given-names:H. + surname:Cheng;given-names:S. + surname:Schilling;given-names:J. + surname:Brooks;given-names:C.L. + surname:Karbstein;given-names:K. + surname:Skiniotis;given-names:G. + REF + Science (New York, N.Y.) + ref + 333 + 2011 + 54901 + Ribosome assembly factors prevent premature translation initiation by 40S assembly intermediates + + + 15253 + 15258 + surname:Hellmich;given-names:U.A. + surname:Weis;given-names:B.L. + surname:Lioutikov;given-names:A. + surname:Wurm;given-names:J.P. + surname:Kaiser;given-names:M. + surname:Christ;given-names:N.A. + surname:Hantke;given-names:K. + surname:Kötter;given-names:P. + surname:Entian;given-names:K.-D. + surname:Schleiff;given-names:E. + 24003121 + REF + Proc. Natl. Acad. Sci. U.S.A. + ref + 110 + 2013 + 54998 + Essential ribosome assembly factor Fap7 regulates a hierarchy of RNA-protein interactions during small ribosomal subunit biogenesis + + + 744 + 753 + surname:Lebaron;given-names:S. + surname:Schneider;given-names:C. + surname:van Nues;given-names:R.W. + surname:Swiatkowska;given-names:A. + surname:Walsh;given-names:D. + surname:Böttcher;given-names:B. + surname:Granneman;given-names:S. + surname:Watkins;given-names:N.J. + surname:Tollervey;given-names:D. + 22751017 + REF + Nat. Struct. Mol. Biol. + ref + 19 + 2012 + 55130 + Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits + + + 111 + 121 + surname:Strunk;given-names:B.S. + surname:Novak;given-names:M.N. + surname:Young;given-names:C.L. + surname:Karbstein;given-names:K. + 22770215 + REF + Cell + ref + 150 + 2012 + 55226 + A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits + + + diff --git a/BioC_XML/4880283_v0.xml b/BioC_XML/4880283_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..e4c9227e08345e33d4c5af4e625cbc1b91eb9ca2 --- /dev/null +++ b/BioC_XML/4880283_v0.xml @@ -0,0 +1,7767 @@ + + + + PMC + 20201215 + pmc.key + + 4880283 + CC BY + no + 0 + 0 + + Crystal Structures of Putative Sugar Kinases + 10.1371/journal.pone.0156067 + 4880283 + 27223615 + PONE-D-16-05184 + e0156067 + 5 + This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + surname:Xie;given-names:Yuan + surname:Li;given-names:Mei + surname:Chang;given-names:Wenrui + surname:Zeth;given-names:Kornelius + surname:Chang;given-names:Wenrui + surname:Li;given-names:Mei + surname:Chang;given-names:Wenrui + surname:Li;given-names:Mei + All structural files are available from the Protein Data Bank (accession numbers 5HTN, 5HTP, 5HUX, 5HV7, 5HTJ, 5HU2, 5HTY, 5HTR, 5HTV and 5HTX). + TITLE + Data Availability + front + 11 + 2016 + 0 + Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana + + 0.99754834 + evidence + cleaner0 + 2023-06-29T13:10:44Z + DUMMY: + + Crystal Structures + + + 0.9963206 + protein_type + cleaner0 + 2023-06-29T13:00:43Z + MESH: + + Sugar Kinases + + + 0.99516356 + species + cleaner0 + 2023-06-29T13:05:32Z + MESH: + + Synechococcus Elongatus PCC 7942 + + + 0.9951167 + species + cleaner0 + 2023-06-29T12:30:25Z + MESH: + + Arabidopsis Thaliana + + + + ABSTRACT + abstract + 108 + The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. However, their exact physiological function and real substrates remain unknown. Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates. In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose. In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. Using mutation and activity analysis, we further verified the key residues important for its catalytic activity. Moreover, our structural comparison with other family members suggests that there are major conformational changes in SePSK upon substrate binding, facilitating the catalytic process. Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1. + + 0.98088884 + species + cleaner0 + 2023-06-29T12:30:19Z + MESH: + + Synechococcus elongatus strain PCC 7942 + + + 0.9968517 + protein_type + cleaner0 + 2023-06-29T13:00:47Z + MESH: + + sugar kinase + + + 0.9925585 + protein + cleaner0 + 2023-06-29T12:30:02Z + PR: + + SePSK + + + 0.9407749 + protein + cleaner0 + 2023-06-29T13:03:44Z + PR: + + xylulose kinase-1 + + + 0.9969945 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.99455905 + species + cleaner0 + 2023-06-29T12:30:24Z + MESH: + + Arabidopsis thaliana + + + 0.9910189 + experimental_method + cleaner0 + 2023-06-29T13:06:49Z + MESH: + + Sequence alignment + + + 0.9706698 + protein_type + cleaner0 + 2023-06-29T12:31:13Z + MESH: + + kinases + + + 0.99740136 + protein_type + cleaner0 + 2023-06-29T12:31:04Z + MESH: + + ribulokinase-like carbohydrate kinases + + + 0.9950826 + protein_type + cleaner0 + 2023-06-29T12:31:09Z + MESH: + + FGGY family carbohydrate kinases + + + 0.8419162 + experimental_method + cleaner0 + 2023-06-29T13:06:57Z + MESH: + + solved + + + 0.9908178 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.9986952 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9972751 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.99609536 + protein_state + cleaner0 + 2023-06-29T12:30:32Z + DUMMY: + + apo + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T13:59:21Z + + in complex with + + + 0.8922534 + chemical + cleaner0 + 2023-06-29T13:44:06Z + CHEBI: + + nucleotide + + + protein_type + MESH: + cleaner0 + 2023-06-29T14:04:45Z + + kinases + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.8761011 + protein_state + cleaner0 + 2023-06-29T13:59:31Z + DUMMY: + + absence of substrates + + + 0.99225545 + experimental_method + cleaner0 + 2023-06-29T13:07:02Z + MESH: + + enzymatic assays + + + 0.9987288 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9976764 + chemical + cleaner0 + 2023-06-29T12:31:20Z + CHEBI: + + D-ribulose + + + 0.99876344 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9303222 + experimental_method + cleaner0 + 2023-06-29T13:07:05Z + MESH: + + solved + + + 0.9951799 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.99875295 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.83708745 + protein_state + cleaner0 + 2023-06-29T13:59:21Z + DUMMY: + + in complex with + + + 0.99768275 + chemical + cleaner0 + 2023-06-29T12:31:20Z + CHEBI: + + D-ribulose + + + 0.99790317 + site + cleaner0 + 2023-06-29T12:30:58Z + SO: + + substrate binding pockets + + + 0.9986987 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9952727 + experimental_method + cleaner0 + 2023-06-29T13:07:09Z + MESH: + + mutation and activity analysis + + + 0.9891796 + experimental_method + cleaner0 + 2023-06-29T13:07:12Z + MESH: + + structural comparison + + + 0.99874437 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9987619 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.995307 + taxonomy_domain + cleaner0 + 2023-06-29T12:30:50Z + DUMMY: + + plant + + + 0.997238 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + + INTRO + title_1 + 1612 + Introduction + + + INTRO + paragraph + 1625 + Carbohydrates are essential cellular compounds involved in the metabolic processes present in all organisms. Phosphorylation is one of the various pivotal modifications of carbohydrates, and is catalyzed by specific sugar kinases. These kinases exhibit considerable differences in their folding pattern and substrate specificity. Based on sequence analysis, they can be divided into four families, namely HSP 70_NBD family, FGGY family, Mer_B like family and Parm_like family. The FGGY family carbohydrate kinases contain different types of sugar kinases, all of which possess different catalytic substrates with preferences for short-chained sugar substrates, ranging from triose to heptose. These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. Structures reported in the Protein Data Bank of the FGGY family carbohydrate kinases exhibit a similar overall architecture containing two protein domains, one of which is responsible for the binding of substrate, while the second is used for binding cofactor ATP. While the binding pockets for substrates are at the same position, each FGGY family carbohydrate kinases uses different substrate-binding residues, resulting in high substrate specificity. + + 0.9974124 + chemical + cleaner0 + 2023-06-29T12:31:27Z + CHEBI: + + Carbohydrates + + + 0.99177116 + ptm + cleaner0 + 2023-06-29T12:31:34Z + MESH: + + Phosphorylation + + + 0.99604684 + chemical + cleaner0 + 2023-06-29T12:31:28Z + CHEBI: + + carbohydrates + + + 0.9974047 + protein_type + cleaner0 + 2023-06-29T13:00:53Z + MESH: + + sugar kinases + + + 0.9926248 + protein_type + cleaner0 + 2023-06-29T13:00:57Z + MESH: + + kinases + + + 0.95107186 + experimental_method + cleaner0 + 2023-06-29T13:07:18Z + MESH: + + sequence analysis + + + 0.99077827 + protein_type + cleaner0 + 2023-06-29T13:01:01Z + MESH: + + HSP 70_NBD family + + + 0.9724302 + protein_type + cleaner0 + 2023-06-29T13:01:03Z + MESH: + + FGGY family + + + 0.96071815 + protein_type + cleaner0 + 2023-06-29T13:01:05Z + MESH: + + Mer_B like family + + + 0.9917743 + protein_type + cleaner0 + 2023-06-29T13:01:08Z + MESH: + + Parm_like family + + + 0.9966028 + protein_type + cleaner0 + 2023-06-29T12:31:09Z + MESH: + + FGGY family carbohydrate kinases + + + 0.9971162 + protein_type + cleaner0 + 2023-06-29T13:00:54Z + MESH: + + sugar kinases + + + 0.6016427 + chemical + cleaner0 + 2023-06-29T13:44:10Z + CHEBI: + + sugar + + + 0.9989779 + chemical + cleaner0 + 2023-06-29T13:44:14Z + CHEBI: + + triose + + + 0.9988084 + chemical + cleaner0 + 2023-06-29T13:44:16Z + CHEBI: + + heptose + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:05:17Z + + sugar + + + 0.9977336 + chemical + cleaner0 + 2023-06-29T12:33:14Z + CHEBI: + + L-ribulose + + + 0.998863 + chemical + cleaner0 + 2023-06-29T12:33:19Z + CHEBI: + + erythritol + + + 0.997777 + chemical + cleaner0 + 2023-06-29T12:33:23Z + CHEBI: + + L-fuculose + + + 0.99766636 + chemical + cleaner0 + 2023-06-29T12:33:28Z + CHEBI: + + D-glycerol + + + 0.99771184 + chemical + cleaner0 + 2023-06-29T12:33:32Z + CHEBI: + + D-gluconate + + + 0.99763614 + chemical + cleaner0 + 2023-06-29T12:33:36Z + CHEBI: + + L-xylulose + + + 0.99765563 + chemical + cleaner0 + 2023-06-29T12:31:20Z + CHEBI: + + D-ribulose + + + 0.99760133 + chemical + cleaner0 + 2023-06-29T12:33:45Z + CHEBI: + + L-rhamnulose + + + 0.9977079 + chemical + cleaner0 + 2023-06-29T12:33:50Z + CHEBI: + + D-xylulose + + + 0.99341464 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + Structures + + + 0.99726695 + protein_type + cleaner0 + 2023-06-29T12:31:09Z + MESH: + + FGGY family carbohydrate kinases + + + 0.99870217 + chemical + cleaner0 + 2023-06-29T12:33:55Z + CHEBI: + + ATP + + + 0.9979714 + site + cleaner0 + 2023-06-29T12:34:00Z + SO: + + binding pockets + + + 0.9970257 + protein_type + cleaner0 + 2023-06-29T12:31:09Z + MESH: + + FGGY family carbohydrate kinases + + + 0.99784184 + site + cleaner0 + 2023-06-29T12:34:04Z + SO: + + substrate-binding residues + + + + INTRO + paragraph + 2917 + Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids. The At2g21370 gene product from Arabidopsis thaliana, xylulose kinase-1 (AtXK-1), whose mature form contains 436 amino acids, is located in the chloroplast (ChloroP 1.1 Server). SePSK and AtXK-1 display a sequence identity of 44.9%, and belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. Members of this sub-family are responsible for the phosphorylation of sugars similar to L-ribulose and D-ribulose. The sequence and the substrate specificity of ribulokinase-like carbohydrate kinases are different, but they share the common folding feature with two domains. Domain I exhibits a ribonuclease H-like folding pattern, and is responsible for the substrate binding, while domain II possesses an actin-like ATPase domain that binds cofactor ATP. + + gene + GENE: + cleaner0 + 2023-06-29T13:43:36Z + + Synpcc7942_2462 + + + 0.9932019 + taxonomy_domain + cleaner0 + 2023-06-29T12:31:44Z + DUMMY: + + cyanobacteria + + + 0.9948773 + species + cleaner0 + 2023-06-29T13:05:38Z + MESH: + + Synechococcus elongatus PCC 7942 + + + 0.9971451 + protein_type + cleaner0 + 2023-06-29T13:01:17Z + MESH: + + sugar kinase + + + 0.63160545 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9100555 + protein_type + cleaner0 + 2023-06-29T12:34:54Z + MESH: + + kinase + + + 0.85704523 + residue_range + cleaner0 + 2023-06-29T13:42:07Z + DUMMY: + + 426 + + + 0.9747736 + gene + cleaner0 + 2023-06-29T12:32:25Z + GENE: + + At2g21370 + + + 0.9906274 + species + cleaner0 + 2023-06-29T12:30:25Z + MESH: + + Arabidopsis thaliana + + + protein + PR: + cleaner0 + 2023-06-29T13:03:44Z + + xylulose kinase-1 + + + 0.993728 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.9908381 + protein_state + cleaner0 + 2023-06-29T13:59:36Z + DUMMY: + + mature form + + + residue_range + DUMMY: + cleaner0 + 2023-06-29T13:42:24Z + + 436 + + + 0.9935946 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9964879 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.9976362 + protein_type + cleaner0 + 2023-06-29T12:31:04Z + MESH: + + ribulokinase-like carbohydrate kinases + + + 0.9972199 + protein_type + cleaner0 + 2023-06-29T12:31:09Z + MESH: + + FGGY family carbohydrate kinases + + + 0.69236356 + ptm + cleaner0 + 2023-06-29T12:31:34Z + MESH: + + phosphorylation + + + 0.9973399 + chemical + cleaner0 + 2023-06-29T13:44:21Z + CHEBI: + + sugars + + + 0.9979513 + chemical + cleaner0 + 2023-06-29T12:33:15Z + CHEBI: + + L-ribulose + + + 0.9979672 + chemical + cleaner0 + 2023-06-29T12:31:20Z + CHEBI: + + D-ribulose + + + 0.99770737 + protein_type + cleaner0 + 2023-06-29T12:31:04Z + MESH: + + ribulokinase-like carbohydrate kinases + + + structure_element + SO: + cleaner0 + 2023-06-29T12:45:50Z + + Domain I + + + structure_element + SO: + cleaner0 + 2023-06-29T13:51:28Z + + ribonuclease H-like folding pattern + + + structure_element + SO: + cleaner0 + 2023-06-29T12:39:03Z + + domain II + + + 0.98265535 + structure_element + cleaner0 + 2023-06-29T13:51:37Z + SO: + + actin-like ATPase domain + + + 0.9985184 + chemical + cleaner0 + 2023-06-29T12:33:56Z + CHEBI: + + ATP + + + + INTRO + paragraph + 3870 + Two possible xylulose kinases (xylulose kinase-1: XK-1 and xylulose kinase-2: XK-2) from Arabidopsis thaliana were previously proposed. It was shown that XK-2 (At5g49650) located in the cytosol is indeed xylulose kinase. However, the function of XK-1 (At2g21370) inside the chloroplast stroma has remained unknown. SePSK from Synechococcus elongatus strain PCC 7942 is the homolog of AtXK-1, though its physiological function and substrates remain unclear. In order to obtain functional and structural information about these two proteins, here we reported the crystal structures of SePSK and AtXK-1. Our findings provide new details of the catalytic mechanism of SePSK and lay the foundation for future studies into its homologs in eukaryotes. + + 0.99621874 + protein_type + cleaner0 + 2023-06-29T13:01:23Z + MESH: + + xylulose kinases + + + 0.9020749 + protein + cleaner0 + 2023-06-29T13:03:44Z + PR: + + xylulose kinase-1 + + + 0.9957288 + protein + cleaner0 + 2023-06-29T13:06:07Z + PR: + + XK-1 + + + protein + PR: + cleaner0 + 2023-06-29T13:03:56Z + + xylulose kinase-2 + + + 0.99588984 + protein + cleaner0 + 2023-06-29T13:06:17Z + PR: + + XK-2 + + + 0.99466956 + species + cleaner0 + 2023-06-29T12:30:25Z + MESH: + + Arabidopsis thaliana + + + 0.9965222 + protein + cleaner0 + 2023-06-29T13:06:17Z + PR: + + XK-2 + + + gene + GENE: + cleaner0 + 2023-06-29T12:32:39Z + + At5g49650 + + + 0.99674535 + protein_type + cleaner0 + 2023-06-29T13:01:29Z + MESH: + + xylulose kinase + + + 0.99666864 + protein + cleaner0 + 2023-06-29T13:06:07Z + PR: + + XK-1 + + + gene + GENE: + cleaner0 + 2023-06-29T12:32:26Z + + At2g21370 + + + 0.997888 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.98438454 + species + cleaner0 + 2023-06-29T12:30:19Z + MESH: + + Synechococcus elongatus strain PCC 7942 + + + 0.9970591 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.99739194 + evidence + cleaner0 + 2023-06-29T13:10:49Z + DUMMY: + + crystal structures + + + 0.9986594 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99723387 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.99841964 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99531025 + taxonomy_domain + cleaner0 + 2023-06-29T12:31:52Z + DUMMY: + + eukaryotes + + + + RESULTS + title_1 + 4615 + Results and Discussion + + + RESULTS + title_2 + 4638 + Overall structures of apo-SePSK and apo-AtXK-1 + + 0.9962684 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.9976174 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.998877 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9975981 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.9971158 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + + RESULTS + paragraph + 4685 + The attempt to solve the SePSK structure by molecular replacement method failed with ribulokinase from Bacillus halodurans (PDB code: 3QDK, 15.7% sequence identity) as an initial model. We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study. + + 0.99876773 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9933109 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.97644377 + experimental_method + cleaner0 + 2023-06-29T13:07:25Z + MESH: + + molecular replacement method + + + 0.99807465 + protein + cleaner0 + 2023-06-29T13:06:31Z + PR: + + ribulokinase + + + 0.99563575 + species + cleaner0 + 2023-06-29T13:05:46Z + MESH: + + Bacillus halodurans + + + 0.994757 + experimental_method + cleaner0 + 2023-06-29T13:07:31Z + MESH: + + single isomorphous replacement anomalous scattering method + + + 0.8514184 + experimental_method + cleaner0 + 2023-06-29T13:07:36Z + MESH: + + SIRAS + + + 0.9976591 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.99857235 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9953922 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.9976841 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.9983925 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.996648 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.9473977 + experimental_method + cleaner0 + 2023-06-29T13:07:41Z + MESH: + + molecular replacement model + + + 0.9923522 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + + RESULTS + paragraph + 5168 + Our structural analysis showed that apo-SePSK consists of one SePSK protein molecule in an asymmetric unit. The amino-acid residues were traced from Val2 to His419, except for the Met1 residue and the seven residues at the C-termini. Apo-SePSK contains two domains referred to further on as domain I and domain II (Fig 1A). Domain I consists of non-contiguous portions of the polypeptide chains (aa. 2–228 and aa. 402–419), exhibiting 11 α-helices and 11 β-sheets. Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. Domain II is comprised of aa. 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). The overall folding of SePSK resembles a clip, with A2 of domain I acting as a hinge region. As a consequence, a deep cleft is formed between the two domains. + + 0.9923353 + experimental_method + cleaner0 + 2023-06-29T13:07:44Z + MESH: + + structural analysis + + + 0.9976369 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.9985624 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99793905 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.8815784 + residue_name_number + cleaner0 + 2023-06-29T13:42:37Z + DUMMY: + + Val2 + + + 0.5677447 + residue_name_number + cleaner0 + 2023-06-29T13:42:42Z + DUMMY: + + His419 + + + 0.99703836 + residue_name_number + cleaner0 + 2023-06-29T13:39:17Z + DUMMY: + + Met1 + + + 0.99758613 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + Apo + + + 0.99850345 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + structure_element + SO: + cleaner0 + 2023-06-29T12:45:50Z + + domain I + + + structure_element + SO: + cleaner0 + 2023-06-29T12:39:03Z + + domain II + + + 0.7847574 + structure_element + cleaner0 + 2023-06-29T12:45:50Z + SO: + + Domain I + + + 0.97053427 + residue_range + cleaner0 + 2023-06-29T13:42:48Z + DUMMY: + + 2–228 + + + 0.9677195 + residue_range + cleaner0 + 2023-06-29T13:42:51Z + DUMMY: + + 402–419 + + + 0.98251957 + structure_element + cleaner0 + 2023-06-29T13:51:43Z + SO: + + α-helices + + + 0.95828867 + structure_element + cleaner0 + 2023-06-29T13:51:46Z + SO: + + β-sheets + + + 0.9965893 + structure_element + cleaner0 + 2023-06-29T13:51:48Z + SO: + + α4 + + + 0.5221494 + structure_element + cleaner0 + 2023-06-29T13:51:51Z + SO: + + α5 + + + 0.98980695 + structure_element + cleaner0 + 2023-06-29T13:51:54Z + SO: + + α11 + + + 0.97880346 + structure_element + cleaner0 + 2023-06-29T13:51:56Z + SO: + + α18 + + + 0.991999 + structure_element + cleaner0 + 2023-06-29T13:51:59Z + SO: + + β3 + + + 0.97956353 + structure_element + cleaner0 + 2023-06-29T13:52:01Z + SO: + + β2 + + + 0.97985715 + structure_element + cleaner0 + 2023-06-29T13:52:03Z + SO: + + β1 + + + 0.97420245 + structure_element + cleaner0 + 2023-06-29T13:52:06Z + SO: + + β6 + + + 0.9660877 + structure_element + cleaner0 + 2023-06-29T13:52:08Z + SO: + + β19 + + + 0.9405666 + structure_element + cleaner0 + 2023-06-29T13:52:11Z + SO: + + β20 + + + 0.9089153 + structure_element + cleaner0 + 2023-06-29T13:52:13Z + SO: + + β17 + + + 0.9609138 + structure_element + cleaner0 + 2023-06-29T13:52:16Z + SO: + + α21 + + + 0.85766864 + structure_element + cleaner0 + 2023-06-29T13:52:20Z + SO: + + α32 + + + 0.9635641 + structure_element + cleaner0 + 2023-06-29T13:52:24Z + SO: + + A1 + + + 0.94301206 + structure_element + cleaner0 + 2023-06-29T13:52:26Z + SO: + + B1 + + + 0.9472735 + structure_element + cleaner0 + 2023-06-29T13:52:28Z + SO: + + A2 + + + 0.9584068 + structure_element + cleaner0 + 2023-06-29T13:52:31Z + SO: + + core region + + + 0.8511612 + structure_element + cleaner0 + 2023-06-29T13:52:33Z + SO: + + β-sheets + + + 0.9793401 + structure_element + cleaner0 + 2023-06-29T13:52:36Z + SO: + + β7 + + + 0.9741762 + structure_element + cleaner0 + 2023-06-29T13:52:39Z + SO: + + β10 + + + 0.9605815 + structure_element + cleaner0 + 2023-06-29T13:52:42Z + SO: + + β12 + + + 0.955182 + structure_element + cleaner0 + 2023-06-29T13:52:44Z + SO: + + β16 + + + 0.9639299 + structure_element + cleaner0 + 2023-06-29T13:52:46Z + SO: + + α-helices + + + 0.96275705 + structure_element + cleaner0 + 2023-06-29T13:52:48Z + SO: + + α8 + + + 0.95128447 + structure_element + cleaner0 + 2023-06-29T13:52:51Z + SO: + + α9 + + + 0.9159867 + structure_element + cleaner0 + 2023-06-29T13:52:53Z + SO: + + α13 + + + 0.9050932 + structure_element + cleaner0 + 2023-06-29T13:52:56Z + SO: + + α14 + + + 0.9102981 + structure_element + cleaner0 + 2023-06-29T13:52:58Z + SO: + + α15 + + + 0.92403173 + structure_element + cleaner0 + 2023-06-29T13:53:00Z + SO: + + core region + + + 0.7898005 + structure_element + cleaner0 + 2023-06-29T12:39:04Z + SO: + + Domain II + + + 0.97022057 + residue_range + cleaner0 + 2023-06-29T13:42:54Z + DUMMY: + + 229–401 + + + 0.9894503 + structure_element + cleaner0 + 2023-06-29T13:53:05Z + SO: + + B2 + + + 0.8862108 + structure_element + cleaner0 + 2023-06-29T13:53:07Z + SO: + + β31 + + + 0.8667047 + structure_element + cleaner0 + 2023-06-29T13:53:09Z + SO: + + β29 + + + 0.84616303 + structure_element + cleaner0 + 2023-06-29T13:53:13Z + SO: + + β22 + + + 0.8279116 + structure_element + cleaner0 + 2023-06-29T13:53:15Z + SO: + + β23 + + + 0.8425631 + structure_element + cleaner0 + 2023-06-29T13:53:17Z + SO: + + β25 + + + 0.8620087 + structure_element + cleaner0 + 2023-06-29T13:53:19Z + SO: + + β24 + + + 0.98577106 + structure_element + cleaner0 + 2023-06-29T13:53:22Z + SO: + + A3 + + + 0.92820394 + structure_element + cleaner0 + 2023-06-29T13:53:24Z + SO: + + α26 + + + 0.8721497 + structure_element + cleaner0 + 2023-06-29T13:53:26Z + SO: + + α27 + + + 0.875759 + structure_element + cleaner0 + 2023-06-29T13:53:28Z + SO: + + α28 + + + 0.8336133 + structure_element + cleaner0 + 2023-06-29T13:53:31Z + SO: + + α30 + + + 0.99854624 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99730116 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.9805611 + structure_element + cleaner0 + 2023-06-29T13:53:35Z + SO: + + B1 + + + 0.9538362 + structure_element + cleaner0 + 2023-06-29T13:53:38Z + SO: + + B2 + + + 0.94712037 + structure_element + cleaner0 + 2023-06-29T13:53:40Z + SO: + + A1 + + + 0.9093556 + structure_element + cleaner0 + 2023-06-29T13:53:43Z + SO: + + A2 + + + 0.9046779 + structure_element + cleaner0 + 2023-06-29T13:53:45Z + SO: + + A3 + + + 0.9966517 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.99050295 + structure_element + cleaner0 + 2023-06-29T13:53:48Z + SO: + + A1 + + + 0.9724399 + structure_element + cleaner0 + 2023-06-29T13:53:51Z + SO: + + B1 + + + 0.9742293 + structure_element + cleaner0 + 2023-06-29T13:53:53Z + SO: + + A2 + + + 0.97901684 + structure_element + cleaner0 + 2023-06-29T13:53:56Z + SO: + + B2 + + + 0.97291994 + structure_element + cleaner0 + 2023-06-29T13:53:59Z + SO: + + A3 + + + structure_element + SO: + cleaner0 + 2023-06-29T13:54:10Z + + α + + + 0.3309372 + structure_element + cleaner0 + 2023-06-29T13:54:13Z + SO: + + β + + + structure_element + SO: + cleaner0 + 2023-06-29T13:54:28Z + + α + + + structure_element + SO: + cleaner0 + 2023-06-29T13:54:38Z + + β + + + structure_element + SO: + cleaner0 + 2023-06-29T13:54:48Z + + α + + + 0.9194723 + protein_type + cleaner0 + 2023-06-29T12:31:09Z + MESH: + + FGGY family carbohydrate kinases + + + 0.99868494 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.97997653 + structure_element + cleaner0 + 2023-06-29T13:54:53Z + SO: + + A2 + + + structure_element + SO: + cleaner0 + 2023-06-29T12:45:50Z + + domain I + + + 0.85653365 + structure_element + cleaner0 + 2023-06-29T13:54:56Z + SO: + + hinge region + + + + pone.0156067.g001.jpg + pone.0156067.g001 + FIG + fig_title_caption + 6599 + Overall structures of SePSK and AtXK-1. + + 0.99674416 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.9987685 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9974437 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + + pone.0156067.g001.jpg + pone.0156067.g001 + FIG + fig_caption + 6639 + (A) Three-dimensional structure of apo-SePSK. The secondary structural elements are indicated (α-helix: cyan, β-sheet: yellow). (B) Three-dimensional structure of apo-AtXK-1. The secondary structural elements are indicated (α-helix: green, β-sheet: wheat). + + evidence + DUMMY: + cleaner0 + 2023-06-29T12:36:04Z + + structure + + + 0.99773824 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.99876773 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99562484 + structure_element + cleaner0 + 2023-06-29T13:55:01Z + SO: + + α-helix + + + 0.98599243 + structure_element + cleaner0 + 2023-06-29T13:55:03Z + SO: + + β-sheet + + + 0.5304055 + evidence + cleaner0 + 2023-06-29T12:36:03Z + DUMMY: + + structure + + + 0.99765897 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.9961786 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.9946821 + structure_element + cleaner0 + 2023-06-29T13:55:05Z + SO: + + α-helix + + + 0.9832224 + structure_element + cleaner0 + 2023-06-29T13:55:07Z + SO: + + β-sheet + + + + RESULTS + paragraph + 6910 + Apo-AtXK-1 exhibits a folding pattern similar to that of SePSK in line with their high sequence identity (Fig 1B and S1 Fig). However, superposition of structures of AtXK-1 and SePSK shows some differences, especially at the loop regions. A considerable difference is found in the loop3 linking β3 and α4, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part. The corresponding residues between these two structures (SePSK-Lys35 and AtXK-1-Lys48) have a distance of 15.4 Å (S3 Fig). + + 0.99779034 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + Apo + + + 0.99601835 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.9987596 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99529535 + experimental_method + cleaner0 + 2023-06-29T13:07:51Z + MESH: + + superposition + + + 0.995905 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.9961788 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.9985306 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.8974517 + structure_element + cleaner0 + 2023-06-29T13:55:10Z + SO: + + loop regions + + + 0.9978461 + structure_element + cleaner0 + 2023-06-29T13:55:13Z + SO: + + loop3 + + + 0.9979189 + structure_element + cleaner0 + 2023-06-29T13:55:15Z + SO: + + β3 + + + 0.9976839 + structure_element + cleaner0 + 2023-06-29T13:55:17Z + SO: + + α4 + + + 0.9932323 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.99584335 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.99782026 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9960836 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.9948949 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.99823296 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9940054 + residue_name_number + cleaner0 + 2023-06-29T13:39:44Z + DUMMY: + + Lys35 + + + 0.99420935 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.992396 + residue_name_number + cleaner0 + 2023-06-29T13:39:50Z + DUMMY: + + Lys48 + + + + RESULTS + title_2 + 7464 + Activity assays of SePSK and AtXK-1 + + 0.994784 + experimental_method + cleaner0 + 2023-06-29T13:07:58Z + MESH: + + Activity assays + + + 0.9980216 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.997217 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + + RESULTS + paragraph + 7500 + In order to understand the function of these two kinases, we performed structural comparison using Dali server. The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases. We first tested whether both enzymes possessed ATP hydrolysis activity in the absence of substrates. As shown in Fig 2A, both SePSK and AtXK-1 exhibited ATP hydrolysis activity. This finding is in agreement with a previous result showing that xylulose kinase (PDB code: 2ITM) possessed ATP hydrolysis activity without adding substrate. To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays. As shown in Fig 2B, the ATP hydrolysis activity of SePSK greatly increased upon adding D-ribulose than adding other potential substrates, suggesting that it has D-ribulose kinase activity. In contrary, limited increasing of ATP hydrolysis activity was detected for AtXK-1 upon addition of D-ribulose (Fig 2C), despite its structural similarity with SePSK. + + 0.9927877 + experimental_method + cleaner0 + 2023-06-29T13:08:02Z + MESH: + + structural comparison + + + 0.98594457 + experimental_method + cleaner0 + 2023-06-29T13:08:06Z + MESH: + + Dali server + + + 0.9860701 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.9985549 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9904278 + protein_type + cleaner0 + 2023-06-29T13:01:30Z + MESH: + + xylulose kinase + + + 0.9862346 + protein_type + cleaner0 + 2023-06-29T13:01:37Z + MESH: + + glycerol kinase + + + 0.9149159 + protein_type + cleaner0 + 2023-06-29T13:01:42Z + MESH: + + ribulose kinase + + + 0.99882716 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.996994 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + protein_type + MESH: + cleaner0 + 2023-06-29T14:06:05Z + + kinases + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.97429013 + protein_state + cleaner0 + 2023-06-29T13:59:44Z + DUMMY: + + absence of + + + 0.99876404 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99695987 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.98557127 + protein_type + cleaner0 + 2023-06-29T13:01:30Z + MESH: + + xylulose kinase + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.9988098 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99698764 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.9979536 + chemical + cleaner0 + 2023-06-29T12:31:20Z + CHEBI: + + D-ribulose + + + 0.99782056 + chemical + cleaner0 + 2023-06-29T12:33:15Z + CHEBI: + + L-ribulose + + + 0.99789363 + chemical + cleaner0 + 2023-06-29T12:33:50Z + CHEBI: + + D-xylulose + + + 0.9978849 + chemical + cleaner0 + 2023-06-29T12:33:37Z + CHEBI: + + L-xylulose + + + 0.9986665 + chemical + cleaner0 + 2023-06-29T13:44:27Z + CHEBI: + + Glycerol + + + 0.9742984 + experimental_method + cleaner0 + 2023-06-29T13:08:11Z + MESH: + + enzymatic activity assays + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.99883 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99776167 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.64733255 + protein_type + cleaner0 + 2023-06-29T13:04:22Z + MESH: + + D-ribulose kinase + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.99709326 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.99760944 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.9988199 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + + pone.0156067.g002.jpg + pone.0156067.g002 + FIG + fig_title_caption + 8686 + The enzymatic activity assays of SePSK and AtXK-1. + + 0.99519634 + experimental_method + cleaner0 + 2023-06-29T13:08:11Z + MESH: + + enzymatic activity assays + + + 0.9988117 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9974227 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + + pone.0156067.g002.jpg + pone.0156067.g002 + FIG + fig_caption + 8737 + (A) The ATP hydrolysis activity of SePSK and AtXK-1. Both SePSK and AtXK-1 showed ATP hydrolysis activity in the absence of substrate. While the ATP hydrolysis activity of SePSK greatly increases upon addition of D-ribulose (DR). (B) The ATP hydrolysis activity of SePSK with addition of five different substrates. The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK. The ATP hydrolysis activity measured via luminescent ADP-Glo assay (Promega). + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.99884677 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99608546 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.9987903 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99594265 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.98278284 + protein_state + cleaner0 + 2023-06-29T13:59:45Z + DUMMY: + + absence of + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.9987889 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99793214 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.9982743 + chemical + cleaner0 + 2023-06-29T13:44:34Z + CHEBI: + + DR + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.9987832 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99842155 + chemical + cleaner0 + 2023-06-29T13:44:38Z + CHEBI: + + DR + + + 0.9979138 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.9984219 + chemical + cleaner0 + 2023-06-29T13:44:43Z + CHEBI: + + LR + + + 0.99779844 + chemical + cleaner0 + 2023-06-29T12:33:15Z + CHEBI: + + L-ribulose + + + 0.99827313 + chemical + cleaner0 + 2023-06-29T13:44:46Z + CHEBI: + + DX + + + 0.99786544 + chemical + cleaner0 + 2023-06-29T12:33:50Z + CHEBI: + + D-xylulose + + + 0.9981193 + chemical + cleaner0 + 2023-06-29T13:44:51Z + CHEBI: + + LX + + + 0.99784297 + chemical + cleaner0 + 2023-06-29T12:33:37Z + CHEBI: + + L-xylulose + + + 0.9980958 + chemical + cleaner0 + 2023-06-29T13:44:54Z + CHEBI: + + GLY + + + 0.99852884 + chemical + cleaner0 + 2023-06-29T13:44:58Z + CHEBI: + + Glycerol + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.998868 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99602515 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.99775594 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.99538165 + protein_state + cleaner0 + 2023-06-29T13:59:52Z + DUMMY: + + wild-type + + + 0.99654883 + protein_state + cleaner0 + 2023-06-29T12:45:29Z + DUMMY: + + WT + + + 0.9987507 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99850804 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.99760216 + mutant + cleaner0 + 2023-06-29T13:38:51Z + MESH: + + D8A + + + 0.91232306 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.997962 + mutant + cleaner0 + 2023-06-29T13:38:53Z + MESH: + + T11A + + + 0.8812514 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9981262 + mutant + cleaner0 + 2023-06-29T13:38:56Z + MESH: + + D221A + + + 0.91704124 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.9585148 + experimental_method + cleaner0 + 2023-06-29T13:08:17Z + MESH: + + luminescent ADP-Glo assay + + + + RESULTS + paragraph + 9478 + To understand the catalytic mechanism of SePSK, we performed structural comparisons among xylulose kinase, glycerol kinase, ribulose kinase and SePSK. Our results suggested that three conserved residues (D8, T11 and D221 of SePSK) play an important role in SePSK function. Mutations of the corresponding residue in xylulose kinase and glycerol kinase from Escherichia coli greatly reduced their activity. To identify the function of these three residues of SePSK, we constructed D8A, T11A and D221A mutants. Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D). + + 0.99626297 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9939593 + experimental_method + cleaner0 + 2023-06-29T13:08:23Z + MESH: + + structural comparisons + + + 0.98220813 + protein_type + cleaner0 + 2023-06-29T13:01:30Z + MESH: + + xylulose kinase + + + 0.99061966 + protein_type + cleaner0 + 2023-06-29T13:01:37Z + MESH: + + glycerol kinase + + + protein_type + MESH: + cleaner0 + 2023-06-29T13:01:42Z + + ribulose kinase + + + 0.99799514 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9935463 + residue_name_number + cleaner0 + 2023-06-29T13:39:54Z + DUMMY: + + D8 + + + 0.99717474 + residue_name_number + cleaner0 + 2023-06-29T13:39:57Z + DUMMY: + + T11 + + + 0.9975109 + residue_name_number + cleaner0 + 2023-06-29T13:40:00Z + DUMMY: + + D221 + + + 0.99862444 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9833528 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9122575 + experimental_method + cleaner0 + 2023-06-29T13:08:31Z + MESH: + + Mutations + + + 0.96090376 + protein_type + cleaner0 + 2023-06-29T13:01:30Z + MESH: + + xylulose kinase + + + 0.98994493 + protein_type + cleaner0 + 2023-06-29T13:01:37Z + MESH: + + glycerol kinase + + + 0.9943024 + species + cleaner0 + 2023-06-29T13:05:53Z + MESH: + + Escherichia coli + + + 0.99813 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9982863 + mutant + cleaner0 + 2023-06-29T13:38:59Z + MESH: + + D8A + + + 0.9981589 + mutant + cleaner0 + 2023-06-29T13:39:01Z + MESH: + + T11A + + + 0.9984445 + mutant + cleaner0 + 2023-06-29T13:39:03Z + MESH: + + D221A + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T14:06:44Z + + mutants + + + 0.9937988 + experimental_method + cleaner0 + 2023-06-29T13:08:12Z + MESH: + + enzymatic activity assays + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.99685556 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.9964043 + protein_state + cleaner0 + 2023-06-29T12:45:24Z + DUMMY: + + wild type + + + ptm + MESH: + cleaner0 + 2023-06-29T12:31:34Z + + phosphorylation + + + 0.9972331 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.99854565 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + + RESULTS + title_2 + 10319 + SePSK and AtXK-1 possess a similar ATP binding site + + 0.9988085 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9974907 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.9981734 + site + cleaner0 + 2023-06-29T14:02:02Z + SO: + + ATP binding site + + + + RESULTS + paragraph + 10371 + To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. In both structures, a strong electron density was found in the conserved ATP binding pocket, but can only be fitted with an ADP molecule (S4 Fig). Thus the two structures were named ADP-SePSK and ADP-AtXK-1, respectively. The extremely weak electron densities of ATP γ-phosphate in both structures suggest that the γ-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1. This result was consistent with our enzymatic activity assays where SePSK and AtXK-1 showed ATP hydrolysis activity without adding any substrates (Fig 2A and 2C). + + 0.9988488 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9968018 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.9100135 + protein_state + cleaner0 + 2023-06-29T13:59:21Z + DUMMY: + + in complex with + + + 0.9981864 + chemical + cleaner0 + 2023-06-29T12:33:56Z + CHEBI: + + ATP + + + 0.98961544 + experimental_method + cleaner0 + 2023-06-29T13:08:38Z + MESH: + + soaked + + + 0.99751925 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.9718859 + evidence + cleaner0 + 2023-06-29T12:58:48Z + DUMMY: + + crystals + + + 0.99853814 + chemical + cleaner0 + 2023-06-29T12:33:56Z + CHEBI: + + ATP + + + 0.996439 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.99883145 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9963374 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.96040344 + protein_state + cleaner0 + 2023-06-29T14:00:00Z + DUMMY: + + bound with + + + 0.99664634 + chemical + cleaner0 + 2023-06-29T12:33:56Z + CHEBI: + + ATP + + + 0.9949189 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.9952722 + evidence + cleaner0 + 2023-06-29T12:58:56Z + DUMMY: + + electron density + + + 0.990589 + protein_state + cleaner0 + 2023-06-29T14:00:07Z + DUMMY: + + conserved + + + 0.99830914 + site + cleaner0 + 2023-06-29T14:02:06Z + SO: + + ATP binding pocket + + + 0.99864465 + chemical + cleaner0 + 2023-06-29T13:45:01Z + CHEBI: + + ADP + + + 0.99441355 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-06-29T12:39:32Z + + ADP-SePSK + + + complex_assembly + GO: + cleaner0 + 2023-06-29T12:39:49Z + + ADP-AtXK-1 + + + 0.9916402 + evidence + cleaner0 + 2023-06-29T12:40:10Z + DUMMY: + + electron densities + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:07:52Z + + phosphate + + + 0.9949497 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:07:52Z + + phosphate + + + 0.9984862 + chemical + cleaner0 + 2023-06-29T12:33:56Z + CHEBI: + + ATP + + + 0.99891424 + protein + cleaner0 + 2023-06-29T12:30:03Z + PR: + + SePSK + + + 0.9967306 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.9894783 + experimental_method + cleaner0 + 2023-06-29T13:08:12Z + MESH: + + enzymatic activity assays + + + 0.9988708 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9964905 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + + RESULTS + paragraph + 11193 + To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP. However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. The γ-phosphate group of ATP is transferred to the sugar substrate during the reaction process, so this flexibility might be important for the ability of these kinases. The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. As shown in Fig 3A, one SePSK protein molecule is in an asymmetric unit with one AMP-PNP molecule. The AMP-PNP is bound at the domain II, where it fits well inside a positively charged groove. The AMP-PNP binding pocket consists of four α-helices (α26, α28, α27 and α30) and forms a shape resembling a half-fist (Fig 3A and 3B). The head group of the AMP-PNP is embedded in a pocket surrounded by Trp383, Asn380, Gly376 and Gly377. The purine ring of AMP-PNP is positioned in parallel to the indole ring of Trp383. In addition, it is hydrogen-bonded with the side chain amide of Asn380 (Fig 3B). The tail of AMP-PNP points to the hinge region of SePSK, and its α-phosphate and β-phosphate groups are stabilized by Gly376 and Ser243, respectively. Together, this structure clearly shows that the AMP-PNP-β-phosphate is sticking out of the ATP binding pocket, thus the γ-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein. + + 0.99848014 + chemical + cleaner0 + 2023-06-29T12:33:56Z + CHEBI: + + ATP + + + experimental_method + MESH: + cleaner0 + 2023-06-29T13:08:39Z + + soaked + + + 0.9708657 + evidence + cleaner0 + 2023-06-29T12:59:03Z + DUMMY: + + crystals + + + 0.9975642 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.9987382 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.99758816 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.934169 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.997727 + chemical + cleaner0 + 2023-06-29T12:40:24Z + CHEBI: + + AMP-PNP + + + 0.9949826 + evidence + cleaner0 + 2023-06-29T12:40:10Z + DUMMY: + + electron densities + + + chemical + CHEBI: 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0.99482995 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.99865365 + chemical + cleaner0 + 2023-06-29T13:45:06Z + CHEBI: + + ADP + + + 0.99787015 + chemical + cleaner0 + 2023-06-29T12:40:24Z + CHEBI: + + AMP-PNP + + + 0.92048323 + complex_assembly + cleaner0 + 2023-06-29T13:37:58Z + GO: + + AMP-PNP-AtXK-1 + + + 0.80632174 + complex_assembly + cleaner0 + 2023-06-29T12:40:00Z + GO: + + ADP-AtXK-1 + + + complex_assembly + GO: + cleaner0 + 2023-06-29T12:38:21Z + + ADP-SePSK + + + 0.858922 + complex_assembly + cleaner0 + 2023-06-29T12:47:00Z + GO: + + AMP-PNP-SePSK + + + 0.99605405 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.993999 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.9145252 + complex_assembly + cleaner0 + 2023-06-29T12:47:00Z + GO: + + AMP-PNP-SePSK + + + 0.95755285 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.99582964 + chemical + cleaner0 + 2023-06-29T12:40:24Z + CHEBI: + + AMP-PNP + + + 0.99785537 + chemical + cleaner0 + 2023-06-29T12:40:24Z + CHEBI: + + AMP-PNP + + + 0.93380296 + structure_element + cleaner0 + 2023-06-29T12:39:03Z + SO: + + domain II + + + 0.99633616 + site + cleaner0 + 2023-06-29T14:01:22Z + SO: + + positively charged groove + + + 0.99805367 + site + cleaner0 + 2023-06-29T14:02:11Z + SO: + + AMP-PNP binding pocket + + + 0.9860923 + structure_element + cleaner0 + 2023-06-29T13:55:23Z + SO: + + four α-helices + + + 0.9966859 + structure_element + cleaner0 + 2023-06-29T13:55:25Z + SO: + + α26 + + + 0.99637395 + structure_element + cleaner0 + 2023-06-29T13:55:27Z + SO: + + α28 + + + 0.9963959 + structure_element + cleaner0 + 2023-06-29T13:55:30Z + SO: + + α27 + + + 0.99632263 + structure_element + cleaner0 + 2023-06-29T13:55:32Z + SO: + + α30 + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T13:56:12Z + + shape resembling a half-fist + + + 0.9978635 + chemical + cleaner0 + 2023-06-29T12:40:24Z + CHEBI: + + AMP-PNP + + + 0.91757286 + site + cleaner0 + 2023-06-29T14:01:27Z + SO: + + pocket + + + 0.99911684 + residue_name_number + cleaner0 + 2023-06-29T13:40:05Z + DUMMY: + + Trp383 + + + 0.99911386 + residue_name_number + cleaner0 + 2023-06-29T13:40:07Z + DUMMY: + + Asn380 + + + 0.9991211 + residue_name_number + cleaner0 + 2023-06-29T13:40:10Z + DUMMY: + + Gly376 + + + 0.99910307 + residue_name_number + cleaner0 + 2023-06-29T13:40:12Z + DUMMY: + + Gly377 + + + 0.997886 + chemical + cleaner0 + 2023-06-29T12:40:24Z + CHEBI: + + AMP-PNP + + + 0.9991265 + residue_name_number + cleaner0 + 2023-06-29T13:40:15Z + DUMMY: + + Trp383 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:17Z + + hydrogen-bonded + + + 0.9991429 + residue_name_number + cleaner0 + 2023-06-29T13:40:17Z + DUMMY: + + Asn380 + + + 0.9977692 + chemical + cleaner0 + 2023-06-29T12:40:24Z + CHEBI: + + AMP-PNP + + + 0.99663115 + structure_element + cleaner0 + 2023-06-29T13:56:17Z + SO: + + hinge region + + + 0.99869126 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:07:52Z + + phosphate + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:07:52Z + + phosphate + + + 0.99915516 + residue_name_number + cleaner0 + 2023-06-29T13:40:20Z + DUMMY: + + Gly376 + + + 0.9991394 + residue_name_number + cleaner0 + 2023-06-29T13:40:22Z + DUMMY: + + Ser243 + + + 0.99610484 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + chemical + CHEBI: + cleaner0 + 2023-06-29T13:47:47Z + + AMP-PNP + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:07:52Z + + phosphate + + + 0.99785566 + site + cleaner0 + 2023-06-29T14:02:14Z + SO: + + ATP binding pocket + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:07:52Z + + phosphate + + + 0.9083584 + structure_element + cleaner0 + 2023-06-29T12:45:50Z + SO: + + domain I + + + 0.8696945 + structure_element + cleaner0 + 2023-06-29T12:39:04Z + SO: + + domain II + + + + pone.0156067.g003.jpg + pone.0156067.g003 + FIG + fig_title_caption + 13053 + Structure of SePSK in complex with AMP-PNP. + + 0.99607456 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + Structure + + + 0.99878126 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9349039 + protein_state + cleaner0 + 2023-06-29T13:59:21Z + DUMMY: + + in complex with + + + 0.99783736 + chemical + cleaner0 + 2023-06-29T12:40:25Z + CHEBI: + + AMP-PNP + + + + pone.0156067.g003.jpg + pone.0156067.g003 + FIG + fig_caption + 13097 + (A) The electron density of AMP-PNP. The SePSK structure is shown in the electrostatic potential surface mode. The AMP-PNP is depicted as sticks with its ǀFoǀ-ǀFcǀ map contoured at 3 σ shown as cyan mesh. (B) The AMP-PNP binding pocket. The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383). The protein skeleton is shown as cartoon (cyan). The four α-helices (α26, α28, α27 and α30) are labeled in red. The AMP-PNP and coordinated residues are shown as sticks. The interactions between them are represented as black dashed lines. The numerical note near the black dashed line indicates the distance (Å). + + 0.99501157 + evidence + cleaner0 + 2023-06-29T12:58:57Z + DUMMY: + + electron density + + + 0.9978182 + chemical + cleaner0 + 2023-06-29T12:40:25Z + CHEBI: + + AMP-PNP + + + 0.9988563 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.99646336 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.99791497 + chemical + cleaner0 + 2023-06-29T12:40:25Z + CHEBI: + + AMP-PNP + + + 0.99490136 + evidence + cleaner0 + 2023-06-29T12:59:33Z + DUMMY: + + ǀFoǀ-ǀFcǀ map + + + 0.99810237 + site + cleaner0 + 2023-06-29T14:02:19Z + SO: + + AMP-PNP binding pocket + + + 0.99770594 + chemical + cleaner0 + 2023-06-29T12:40:25Z + CHEBI: + + AMP-PNP + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:17Z + + sandwiched by + + + 0.9989385 + residue_name_number + cleaner0 + 2023-06-29T13:40:26Z + DUMMY: + + Leu293 + + + 0.9988998 + residue_name_number + cleaner0 + 2023-06-29T13:40:29Z + DUMMY: + + Gly376 + + + 0.99890697 + residue_name_number + cleaner0 + 2023-06-29T13:40:31Z + DUMMY: + + Gly377 + + + 0.9988945 + residue_name_number + cleaner0 + 2023-06-29T13:40:34Z + DUMMY: + + Trp383 + + + 0.9038727 + structure_element + cleaner0 + 2023-06-29T13:56:21Z + SO: + + α-helices + + + 0.9949256 + structure_element + cleaner0 + 2023-06-29T13:56:23Z + SO: + + α26 + + + 0.9944654 + structure_element + cleaner0 + 2023-06-29T13:56:26Z + SO: + + α28 + + + 0.994235 + structure_element + cleaner0 + 2023-06-29T13:56:28Z + SO: + + α27 + + + 0.99400276 + structure_element + cleaner0 + 2023-06-29T13:56:30Z + SO: + + α30 + + + 0.99779725 + chemical + cleaner0 + 2023-06-29T12:40:25Z + CHEBI: + + AMP-PNP + + + + RESULTS + title_2 + 13759 + The potential substrate binding site in SePSK + + 0.99813384 + site + cleaner0 + 2023-06-29T14:01:32Z + SO: + + substrate binding site + + + 0.998784 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + + RESULTS + paragraph + 13805 + The results from our activity assays suggested that SePSK has D-ribulose kinase activity. To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. As shown in S6 Fig, two residual electron densities are visible in domain I, which can be interpreted as two D-ribulose molecules with reasonable fit. + + 0.9912284 + experimental_method + cleaner0 + 2023-06-29T13:08:46Z + MESH: + + activity assays + + + 0.99871147 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + protein_type + MESH: + cleaner0 + 2023-06-29T13:05:04Z + + D-ribulose kinase + + + 0.99872583 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9973994 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.9976351 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.9985197 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.81197965 + experimental_method + cleaner0 + 2023-06-29T13:10:28Z + MESH: + + crystals were soaked into + + + 0.5801219 + experimental_method + cleaner0 + 2023-06-29T13:09:05Z + MESH: + + reservoir + + + 0.9971234 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.76671934 + chemical + cleaner0 + 2023-06-29T12:44:15Z + CHEBI: + + RBL + + + 0.96759635 + complex_assembly + cleaner0 + 2023-06-29T13:38:03Z + GO: + + RBL-SePSK + + + 0.98671824 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.7476223 + experimental_method + cleaner0 + 2023-06-29T13:09:09Z + MESH: + + solved + + + 0.79123193 + evidence + cleaner0 + 2023-06-29T12:40:10Z + DUMMY: + + electron densities + + + structure_element + SO: + cleaner0 + 2023-06-29T12:45:50Z + + domain I + + + 0.99729663 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + + RESULTS + paragraph + 14243 + As shown in Fig 4A, the nearest distance between the carbon skeleton of two D-ribulose molecules are approx. 7.1 Å (RBL1-C4 and RBL2-C1). RBL1 is located in the pocket consisting of α21 and the loop between β6 and β7. The O4 and O5 of RBL1 are coordinated with the side chain carboxyl group of Asp221. Furthermore, the O2 of RBL1 interacts with the main chain amide nitrogen of Ser72 (Fig 4B). This pocket is at a similar position of substrate binding site of other sugar kinase, such as L-ribulokinase (PDB code: 3QDK) (S7 Fig). However, structural comparison shows that the substrate ligating residues between the two structures are not strictly conserved. Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). Glu329 in 3QDK has no counterpart in RBL-SePSK structure. In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. These differences might account for their different substrate specificity. + + 0.99797374 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.9956892 + residue_name_number + cleaner0 + 2023-06-29T12:51:27Z + DUMMY: + + RBL1 + + + 0.9937836 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.9795904 + residue_name_number + cleaner0 + 2023-06-29T12:51:28Z + DUMMY: + + RBL1 + + + 0.9959525 + site + cleaner0 + 2023-06-29T14:01:36Z + SO: + + pocket + + + 0.9964199 + structure_element + cleaner0 + 2023-06-29T13:56:39Z + SO: + + α21 + + + 0.99509996 + structure_element + cleaner0 + 2023-06-29T13:56:41Z + SO: + + loop + + + 0.96590996 + structure_element + cleaner0 + 2023-06-29T13:56:44Z + SO: + + β6 and β7 + + + 0.9849681 + residue_name_number + cleaner0 + 2023-06-29T12:51:28Z + DUMMY: + + RBL1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:17Z + + coordinated with + + + 0.9991665 + residue_name_number + cleaner0 + 2023-06-29T13:40:38Z + DUMMY: + + Asp221 + + + 0.9860649 + residue_name_number + cleaner0 + 2023-06-29T12:51:28Z + DUMMY: + + RBL1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:17Z + + interacts with + + + 0.99916434 + residue_name_number + cleaner0 + 2023-06-29T13:40:42Z + DUMMY: + + Ser72 + + + 0.9924704 + site + cleaner0 + 2023-06-29T14:01:39Z + SO: + + pocket + + + 0.99791 + site + cleaner0 + 2023-06-29T14:01:43Z + SO: + + substrate binding site + + + 0.99478817 + protein_type + cleaner0 + 2023-06-29T13:01:17Z + MESH: + + sugar kinase + + + 0.58787787 + protein + cleaner0 + 2023-06-29T13:06:42Z + PR: + + L-ribulokinase + + + 0.9893616 + experimental_method + cleaner0 + 2023-06-29T13:08:03Z + MESH: + + structural comparison + + + 0.984846 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.93839407 + protein_state + cleaner0 + 2023-06-29T14:00:12Z + DUMMY: + + not strictly conserved + + + 0.96085685 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.99409026 + residue_name_number + cleaner0 + 2023-06-29T12:51:28Z + DUMMY: + + RBL1 + + + 0.98868424 + complex_assembly + cleaner0 + 2023-06-29T13:38:07Z + GO: + + RBL-SePSK + + + 0.9924574 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.9991571 + residue_name_number + cleaner0 + 2023-06-29T13:40:46Z + DUMMY: + + Ser72 + + + 0.99915314 + residue_name_number + cleaner0 + 2023-06-29T13:40:49Z + DUMMY: + + Asp221 + + + 0.99915004 + residue_name_number + cleaner0 + 2023-06-29T13:40:51Z + DUMMY: + + Ser222 + + + 0.9974332 + chemical + cleaner0 + 2023-06-29T12:33:15Z + CHEBI: + + L-ribulose + + + 0.7296011 + protein + cleaner0 + 2023-06-29T13:06:42Z + PR: + + L-ribulokinase + + + 0.99916625 + residue_name_number + cleaner0 + 2023-06-29T13:40:55Z + DUMMY: + + Ala96 + + + 0.99914515 + residue_name_number + cleaner0 + 2023-06-29T13:40:58Z + DUMMY: + + Lys208 + + + 0.99915195 + residue_name_number + cleaner0 + 2023-06-29T13:41:00Z + DUMMY: + + Asp274 + + + 0.9991542 + residue_name_number + cleaner0 + 2023-06-29T13:41:03Z + DUMMY: + + Glu329 + + + 0.9991345 + residue_name_number + cleaner0 + 2023-06-29T13:41:06Z + DUMMY: + + Glu329 + + + 0.9835308 + complex_assembly + cleaner0 + 2023-06-29T13:38:10Z + GO: + + RBL-SePSK + + + 0.99096465 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.9991315 + residue_name_number + cleaner0 + 2023-06-29T13:41:12Z + DUMMY: + + Lys208 + + + 0.6947624 + protein + cleaner0 + 2023-06-29T13:06:42Z + PR: + + L-ribulokinase + + + 0.9991339 + residue_name_number + cleaner0 + 2023-06-29T13:41:15Z + DUMMY: + + Lys163 + + + 0.97491217 + complex_assembly + cleaner0 + 2023-06-29T13:38:12Z + GO: + + RBL-SePSK + + + 0.99150014 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:17Z + + hydrogen bond + + + 0.99914443 + residue_name_number + cleaner0 + 2023-06-29T13:41:18Z + DUMMY: + + Lys163 + + + 0.97993106 + structure_element + cleaner0 + 2023-06-29T13:56:47Z + SO: + + α-helices + + + 0.9958072 + structure_element + cleaner0 + 2023-06-29T13:56:50Z + SO: + + α9 + + + 0.99544024 + structure_element + cleaner0 + 2023-06-29T13:56:52Z + SO: + + α13 + + + 0.99875915 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + + pone.0156067.g004.jpg + pone.0156067.g004 + FIG + fig_title_caption + 15500 + The binding of D-ribulose (RBL) with SePSK. + + 0.9978377 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.9984383 + chemical + cleaner0 + 2023-06-29T12:44:14Z + CHEBI: + + RBL + + + 0.99876654 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + + pone.0156067.g004.jpg + pone.0156067.g004 + FIG + fig_caption + 15544 + (A) The electrostatic potential surface map of RBL-SePSK and a zoom-in view of RBL binding site. The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK. The RBL molecules (carbon atoms colored yellow) and amino acid residues of SePSK (carbon atoms colored green) involved in RBL interaction are shown as sticks. The hydrogen bonds are indicated by the black dashed lines and the numbers near the dashed lines are the distances (Å). (C) The binding affinity assays of SePSK with D-ribulose. Single-cycle kinetic data are reflecting the interaction of SePSK and D8A-SePSK with D-ribulose. It shows two experimental sensorgrams after minus the empty sensorgrams. The original data is shown as black curve, and the fitted data is shown as different color (wild type SePSK: red curve, D8A-SePSK: green curve). Dissociation rate constant of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. + + 0.9894691 + evidence + cleaner0 + 2023-06-29T12:59:39Z + DUMMY: + + electrostatic potential surface map + + + 0.9804246 + complex_assembly + cleaner0 + 2023-06-29T13:38:17Z + GO: + + RBL-SePSK + + + 0.9982365 + site + cleaner0 + 2023-06-29T14:02:25Z + SO: + + RBL binding site + + + 0.94320077 + residue_name_number + cleaner0 + 2023-06-29T12:51:28Z + DUMMY: + + RBL1 + + + 0.89772403 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.99795705 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.6757883 + residue_name_number + cleaner0 + 2023-06-29T12:51:28Z + DUMMY: + + RBL1 + + + 0.7007424 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.9981646 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:44:15Z + + RBL + + + 0.9984713 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:44:15Z + + RBL + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:17Z + + hydrogen bonds + + + 0.9919452 + experimental_method + cleaner0 + 2023-06-29T13:10:32Z + MESH: + + binding affinity assays + + + 0.99844223 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9978544 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.87909096 + experimental_method + cleaner0 + 2023-06-29T13:09:16Z + MESH: + + Single-cycle kinetic data + + + 0.9979601 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + mutant + MESH: + cleaner0 + 2023-06-29T12:43:33Z + + D8A + + + 0.7842108 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9978338 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.98476356 + evidence + cleaner0 + 2023-06-29T12:59:45Z + DUMMY: + + sensorgrams + + + 0.9796078 + evidence + cleaner0 + 2023-06-29T12:59:45Z + DUMMY: + + sensorgrams + + + 0.9971192 + protein_state + cleaner0 + 2023-06-29T12:45:24Z + DUMMY: + + wild type + + + 0.9978951 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + mutant + MESH: + cleaner0 + 2023-06-29T12:43:10Z + + D8A + + + protein + PR: + cleaner0 + 2023-06-29T12:44:03Z + + SePSK + + + 0.9945125 + evidence + cleaner0 + 2023-06-29T12:59:51Z + DUMMY: + + Dissociation rate constant + + + 0.9971053 + protein_state + cleaner0 + 2023-06-29T12:45:24Z + DUMMY: + + wild type + + + mutant + MESH: + cleaner0 + 2023-06-29T12:43:51Z + + D8A + + + 0.7984277 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + + RESULTS + paragraph + 16499 + The binding pocket of RBL2 with relatively weak electron density is near the N-terminal region of SePSK and is negatively charged. The side chain of Asp8 interacts strongly with O3 and O4 of RBL2. The hydroxyl group of Ser12 coordinates with O2 of RBL2. The backbone amide nitrogens of Gly13 and Arg15 also keep hydrogen bonds with RBL2 (Fig 4B). Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. This break is probably induced by the conformational change of the two β-sheets (β1 and β2), with the result that the linking loop (loop 1) is located further away from the RBL2 binding site. This change might be the reason that AtXK-1 only shows limited increasing in its ATP hydrolysis ability upon adding D-ribulose as a substrate after comparing with SePSK (Fig 2C). + + 0.99783593 + site + cleaner0 + 2023-06-29T14:01:48Z + SO: + + binding pocket + + + 0.9875448 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.9649341 + evidence + cleaner0 + 2023-06-29T12:58:57Z + DUMMY: + + electron density + + + 0.99888295 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.99913883 + residue_name_number + cleaner0 + 2023-06-29T13:41:22Z + DUMMY: + + Asp8 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:17Z + + interacts strongly with + + + 0.71752113 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.9991345 + residue_name_number + cleaner0 + 2023-06-29T13:41:26Z + DUMMY: + + Ser12 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:17Z + + coordinates with + + + 0.7989556 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.9991352 + residue_name_number + cleaner0 + 2023-06-29T13:41:29Z + DUMMY: + + Gly13 + + + 0.9991284 + residue_name_number + cleaner0 + 2023-06-29T13:41:31Z + DUMMY: + + Arg15 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:17Z + + hydrogen bonds + + + 0.5216133 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.9917146 + experimental_method + cleaner0 + 2023-06-29T13:08:03Z + MESH: + + Structural comparison + + + 0.99887604 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.99594635 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.998116 + site + cleaner0 + 2023-06-29T14:02:31Z + SO: + + RBL1 binding pocket + + + 0.99576545 + protein_state + cleaner0 + 2023-06-29T14:00:18Z + DUMMY: + + conserved + + + 0.99765503 + site + cleaner0 + 2023-06-29T14:02:34Z + SO: + + RBL2 pocket + + + 0.9941177 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.99615026 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.9577052 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.99713546 + protein_state + cleaner0 + 2023-06-29T14:00:21Z + DUMMY: + + highly conserved + + + 0.99430466 + complex_assembly + cleaner0 + 2023-06-29T13:38:21Z + GO: + + RBL-SePSK + + + 0.9921163 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:17Z + + hydrogen bond + + + 0.64399064 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.99910873 + residue_name_number + cleaner0 + 2023-06-29T13:41:39Z + DUMMY: + + Ser12 + + + 0.9906059 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.7845717 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:17Z + + hydrogen bond + + + 0.9990852 + residue_name_number + cleaner0 + 2023-06-29T13:41:42Z + DUMMY: + + Ser22 + + + 0.9911086 + structure_element + cleaner0 + 2023-06-29T13:56:58Z + SO: + + β-sheets + + + 0.99780756 + structure_element + cleaner0 + 2023-06-29T13:57:01Z + SO: + + β1 + + + 0.9975147 + structure_element + cleaner0 + 2023-06-29T13:57:03Z + SO: + + β2 + + + 0.99682295 + structure_element + cleaner0 + 2023-06-29T13:57:05Z + SO: + + linking loop + + + 0.99523675 + structure_element + cleaner0 + 2023-06-29T13:57:08Z + SO: + + loop 1 + + + 0.9978866 + site + cleaner0 + 2023-06-29T14:02:38Z + SO: + + RBL2 binding site + + + 0.99623936 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:33:56Z + + ATP + + + 0.9972736 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.9988605 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + + RESULTS + paragraph + 17671 + Our SePSK structure shows that the Asp8 residue forms strong hydrogen bond with RBL2 (Fig 4B). In addition, our enzymatic assays indicated that Asp8 is important for the activity of SePSK (Fig 2D). To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method. The results showed that the affinity of D8A-SePSK with D-ribulose is weaker than that of WT with a reduction of approx. two third (Fig 4C). Dissociation rate constant (Kd) of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. The results implied that the second RBL binding site plays a role in the D-ribulose kinase function of SePSK. However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact. + + 0.99849105 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9971219 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.9986168 + residue_name_number + cleaner0 + 2023-06-29T13:41:46Z + DUMMY: + + Asp8 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:17Z + + hydrogen bond + + + 0.85944766 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.9923231 + experimental_method + cleaner0 + 2023-06-29T13:09:20Z + MESH: + + enzymatic assays + + + 0.99869055 + residue_name_number + cleaner0 + 2023-06-29T13:41:51Z + DUMMY: + + Asp8 + + + 0.9987179 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.99342453 + evidence + cleaner0 + 2023-06-29T13:00:02Z + DUMMY: + + binding affinity + + + 0.9973588 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.99713755 + protein_state + cleaner0 + 2023-06-29T12:45:23Z + DUMMY: + + wild type + + + 0.99644285 + protein_state + cleaner0 + 2023-06-29T12:45:29Z + DUMMY: + + WT + + + 0.99761707 + mutant + cleaner0 + 2023-06-29T13:39:07Z + MESH: + + D8A + + + 0.6049578 + protein_state + cleaner0 + 2023-06-29T14:00:26Z + DUMMY: + + mutant + + + 0.9978137 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9624258 + experimental_method + cleaner0 + 2023-06-29T13:09:25Z + MESH: + + surface plasmon resonance method + + + 0.9864738 + evidence + cleaner0 + 2023-06-29T13:00:20Z + DUMMY: + + affinity + + + mutant + MESH: + cleaner0 + 2023-06-29T12:47:51Z + + D8A + + + 0.37831542 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9971581 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.9970625 + protein_state + cleaner0 + 2023-06-29T12:45:29Z + DUMMY: + + WT + + + 0.99627906 + evidence + cleaner0 + 2023-06-29T12:59:52Z + DUMMY: + + Dissociation rate constant + + + 0.99015814 + evidence + cleaner0 + 2023-06-29T13:00:28Z + DUMMY: + + Kd + + + 0.99728435 + protein_state + cleaner0 + 2023-06-29T12:45:24Z + DUMMY: + + wild type + + + mutant + MESH: + cleaner0 + 2023-06-29T12:47:32Z + + D8A + + + 0.852059 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9975878 + site + cleaner0 + 2023-06-29T14:02:41Z + SO: + + second RBL binding site + + + protein_type + MESH: + cleaner0 + 2023-06-29T12:45:10Z + + D-ribulose kinase + + + 0.9986615 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.99736065 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.99453807 + experimental_method + cleaner0 + 2023-06-29T13:09:35Z + MESH: + + crystal soaking + + + 0.99286854 + evidence + cleaner0 + 2023-06-29T12:58:57Z + DUMMY: + + electron density + + + 0.45815828 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.99792475 + site + cleaner0 + 2023-06-29T14:01:52Z + SO: + + second binding site + + + 0.99537796 + chemical + cleaner0 + 2023-06-29T12:31:21Z + CHEBI: + + D-ribulose + + + 0.9986771 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + + RESULTS + title_2 + 18614 + Simulated conformational change of SePSK during the catalytic process + + 0.9988053 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + + RESULTS + paragraph + 18684 + It was reported earlier that the crossing angle between the domain I and domain II in FGGY family carbohydrate kinases is different. In addition, this difference may be caused by the binding of substrates and/or ATP. As reported previously, members of the sugar kinase family undergo a conformational change to narrow the crossing angle between two domains and reduce the distance between substrate and ATP in order to facilitate the catalytic reaction of phosphorylation of sugar substrates. After comparing structures of apo-SePSK, RBL-SePSK and AMP-PNP-SePSK, we noticed that these structures presented here are similar. Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). This distance is too long to transfer the γ-phosphate group from ATP to the substrate. Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates. + + structure_element + SO: + cleaner0 + 2023-06-29T12:45:50Z + + domain I + + + structure_element + SO: + cleaner0 + 2023-06-29T12:39:04Z + + domain II + + + 0.99632156 + protein_type + cleaner0 + 2023-06-29T12:31:09Z + MESH: + + FGGY family carbohydrate kinases + + + 0.99829966 + chemical + cleaner0 + 2023-06-29T12:33:56Z + CHEBI: + + ATP + + + 0.99598795 + protein_type + cleaner0 + 2023-06-29T13:01:17Z + MESH: + + sugar kinase + + + 0.9983065 + chemical + cleaner0 + 2023-06-29T12:33:56Z + CHEBI: + + ATP + + + ptm + MESH: + cleaner0 + 2023-06-29T12:31:34Z + + phosphorylation + + + 0.9965299 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.99774003 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.9985146 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.8958693 + complex_assembly + cleaner0 + 2023-06-29T13:38:25Z + GO: + + RBL-SePSK + + + complex_assembly + GO: + cleaner0 + 2023-06-29T12:46:36Z + + AMP-PNP-SePSK + + + 0.9932585 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.99514425 + experimental_method + cleaner0 + 2023-06-29T13:09:38Z + MESH: + + Superposing + + + 0.9604764 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.93207264 + complex_assembly + cleaner0 + 2023-06-29T13:38:30Z + GO: + + RBL-SePSK + + + complex_assembly + GO: + cleaner0 + 2023-06-29T12:47:00Z + + AMP-PNP-SePSK + + + chemical + CHEBI: + cleaner0 + 2023-06-29T13:47:47Z + + AMP-PNP + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:07:52Z + + phosphate + + + 0.96116656 + residue_name_number + cleaner0 + 2023-06-29T12:51:28Z + DUMMY: + + RBL1 + + + 0.9353864 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.8891231 + residue_name_number + cleaner0 + 2023-06-29T12:51:28Z + DUMMY: + + RBL1 + + + 0.8204545 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:07:52Z + + phosphate + + + 0.9982406 + chemical + cleaner0 + 2023-06-29T12:33:56Z + CHEBI: + + ATP + + + 0.99869305 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9967721 + evidence + cleaner0 + 2023-06-29T12:36:04Z + DUMMY: + + structure + + + 0.9964838 + evidence + cleaner0 + 2023-06-29T12:36:09Z + DUMMY: + + structures + + + 0.99859387 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9968291 + protein_state + cleaner0 + 2023-06-29T14:00:30Z + DUMMY: + + open + + + 0.9961675 + protein_state + cleaner0 + 2023-06-29T14:00:33Z + DUMMY: + + closed + + + ptm + MESH: + cleaner0 + 2023-06-29T12:31:34Z + + phosphorylation + + + + RESULTS + paragraph + 19898 + For studying such potential conformational change, a simulation on the Hingeprot Server was performed to predict the movement of different SePSK domains. The results showed that domain I and domain II are closer to each other with Ala228 and Thr401 in A2 as Hinge-residues. Based on the above results, SePSK is divided into two rigid parts. The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). The results of superposition displayed different crossing angle between these two domains. After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). This distance between RBL2 and AMP-PNP-γ-phosphate is close enough to facilitate phosphate transferring. Together, our superposition results provided snapshots of the conformational changes at different catalytic stages of SePSK and potentially revealed the closed form of SePSK. + + 0.9933035 + experimental_method + cleaner0 + 2023-06-29T13:09:44Z + MESH: + + simulation + + + 0.9314792 + experimental_method + cleaner0 + 2023-06-29T13:09:47Z + MESH: + + Hingeprot Server + + + 0.9705146 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.8122375 + structure_element + cleaner0 + 2023-06-29T12:45:50Z + SO: + + domain I + + + 0.81924534 + structure_element + cleaner0 + 2023-06-29T12:39:04Z + SO: + + domain II + + + 0.99919015 + residue_name_number + cleaner0 + 2023-06-29T13:41:56Z + DUMMY: + + Ala228 + + + 0.9991621 + residue_name_number + cleaner0 + 2023-06-29T13:41:58Z + DUMMY: + + Thr401 + + + 0.9935452 + structure_element + cleaner0 + 2023-06-29T13:57:16Z + SO: + + A2 + + + structure_element + SO: + cleaner0 + 2023-06-29T12:49:01Z + + Hinge-residues + + + 0.9967945 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.97336656 + structure_element + cleaner0 + 2023-06-29T12:45:50Z + SO: + + domain I + + + 0.8564523 + complex_assembly + cleaner0 + 2023-06-29T13:38:38Z + GO: + + RBL-SePSK + + + 0.9739439 + residue_range + cleaner0 + 2023-06-29T13:43:01Z + DUMMY: + + 1–228 + + + 0.9688861 + residue_range + cleaner0 + 2023-06-29T13:43:03Z + DUMMY: + + 402–421 + + + 0.9628848 + structure_element + cleaner0 + 2023-06-29T12:39:04Z + SO: + + domain II + + + complex_assembly + GO: + cleaner0 + 2023-06-29T12:47:00Z + + AMP-PNP-SePSK + + + 0.9754912 + residue_range + cleaner0 + 2023-06-29T13:43:06Z + DUMMY: + + 229–401 + + + 0.9953492 + experimental_method + cleaner0 + 2023-06-29T13:09:52Z + MESH: + + superposed + + + evidence + DUMMY: + cleaner0 + 2023-06-29T12:36:10Z + + structures + + + 0.9979248 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.98395985 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.9979678 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.9985403 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.6744378 + protein_type + cleaner0 + 2023-06-29T13:01:30Z + MESH: + + xylulose kinase + + + 0.99515074 + species + cleaner0 + 2023-06-29T13:06:00Z + MESH: + + Lactobacillus acidophilus + + + 0.99639267 + mutant + cleaner0 + 2023-06-29T13:39:12Z + MESH: + + S58W + + + 0.98757553 + protein_state + cleaner0 + 2023-06-29T14:00:36Z + DUMMY: + + mutant + + + 0.82658494 + protein_type + cleaner0 + 2023-06-29T13:01:37Z + MESH: + + glycerol kinase + + + 0.995497 + species + cleaner0 + 2023-06-29T13:05:53Z + MESH: + + Escherichia coli + + + 0.99414694 + experimental_method + cleaner0 + 2023-06-29T13:07:51Z + MESH: + + superposition + + + 0.99029946 + experimental_method + cleaner0 + 2023-06-29T13:07:51Z + MESH: + + superposition + + + chemical + CHEBI: + cleaner0 + 2023-06-29T13:47:47Z + + AMP-PNP + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:07:52Z + + phosphate + + + 0.47843257 + residue_name_number + cleaner0 + 2023-06-29T12:51:28Z + DUMMY: + + RBL1 + + + 0.9856103 + experimental_method + cleaner0 + 2023-06-29T13:09:52Z + MESH: + + superposed + + + 0.973536 + protein + cleaner0 + 2023-06-29T12:30:10Z + PR: + + AtXK-1 + + + 0.9896381 + experimental_method + cleaner0 + 2023-06-29T13:09:52Z + MESH: + + superposed + + + 0.9963516 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9665063 + experimental_method + cleaner0 + 2023-06-29T13:09:52Z + MESH: + + superposed + + + 0.96540505 + experimental_method + cleaner0 + 2023-06-29T13:09:52Z + MESH: + + superposed + + + chemical + CHEBI: + cleaner0 + 2023-06-29T13:47:47Z + + AMP-PNP + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:07:52Z + + phosphate + + + 0.65726316 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.9834472 + experimental_method + cleaner0 + 2023-06-29T13:09:52Z + MESH: + + superposed + + + 0.97864777 + protein + cleaner0 + 2023-06-29T12:30:11Z + PR: + + AtXK-1 + + + 0.9775199 + experimental_method + cleaner0 + 2023-06-29T13:09:52Z + MESH: + + superposed + + + 0.99551105 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9601499 + experimental_method + cleaner0 + 2023-06-29T13:09:53Z + MESH: + + superposed + + + chemical + CHEBI: + cleaner0 + 2023-06-29T13:47:47Z + + AMP-PNP + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:07:52Z + + phosphate + + + 0.47177985 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + 0.9726828 + experimental_method + cleaner0 + 2023-06-29T13:07:51Z + MESH: + + superposition + + + 0.5351513 + residue_name_number + cleaner0 + 2023-06-29T12:51:47Z + DUMMY: + + RBL2 + + + chemical + CHEBI: + cleaner0 + 2023-06-29T13:47:47Z + + AMP-PNP + + + chemical + CHEBI: + cleaner0 + 2023-06-29T14:07:52Z + + phosphate + + + chemical + CHEBI: + cleaner0 + 2023-06-29T13:58:27Z + + phosphate + + + 0.9943678 + experimental_method + cleaner0 + 2023-06-29T13:07:51Z + MESH: + + superposition + + + 0.99855286 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.99732554 + protein_state + cleaner0 + 2023-06-29T14:00:39Z + DUMMY: + + closed + + + 0.99819595 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + + pone.0156067.g005.jpg + pone.0156067.g005 + FIG + fig_title_caption + 21433 + Simulated conformational change of SePSK during the catalytic process. + + 0.99879944 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + + pone.0156067.g005.jpg + pone.0156067.g005 + FIG + fig_caption + 21504 + The structures are shown as cartoon and the ligands are shown as sticks. Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. The numbers near the black dashed lines show the distances (Å) between two nearest atoms of RBL and AMP-PNP. + + 0.9796005 + evidence + cleaner0 + 2023-06-29T12:36:10Z + DUMMY: + + structures + + + 0.71441394 + structure_element + cleaner0 + 2023-06-29T12:45:50Z + SO: + + Domain I + + + complex_assembly + GO: + cleaner0 + 2023-06-29T12:53:01Z + + D-ribulose-SePSK + + + 0.7680092 + structure_element + cleaner0 + 2023-06-29T12:39:04Z + SO: + + Domain II + + + complex_assembly + GO: + cleaner0 + 2023-06-29T12:47:00Z + + AMP-PNP-SePSK + + + 0.99386805 + experimental_method + cleaner0 + 2023-06-29T13:09:53Z + MESH: + + superposed + + + 0.99759597 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.9718533 + protein + cleaner0 + 2023-06-29T12:30:11Z + PR: + + AtXK-1 + + + 0.9976769 + protein_state + cleaner0 + 2023-06-29T12:30:33Z + DUMMY: + + apo + + + 0.9985405 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9970982 + chemical + cleaner0 + 2023-06-29T12:44:15Z + CHEBI: + + RBL + + + 0.9981644 + chemical + cleaner0 + 2023-06-29T12:40:25Z + CHEBI: + + AMP-PNP + + + + RESULTS + paragraph + 21862 + In summary, our structural and enzymatic analyses provide evidence that SePSK shows D-ribulose kinase activity, and exhibits the conserved features of FGGY family carbohydrate kinases. Three conserved residues in SePSK were identified to be essential for this function. Our results provide the detailed information about the interaction of SePSK with ATP and substrates. Moreover, structural superposition results enable us to visualize the conformational change of SePSK during the catalytic process. In conclusion, our results provide important information for a more detailed understanding of the mechanisms of SePSK and other members of FGGY family carbohydrate kinases. + + 0.9951409 + experimental_method + cleaner0 + 2023-06-29T13:10:02Z + MESH: + + structural and enzymatic analyses + + + 0.99875736 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + protein_type + MESH: + cleaner0 + 2023-06-29T12:53:50Z + + D-ribulose kinase + + + 0.9950805 + protein_type + cleaner0 + 2023-06-29T12:31:09Z + MESH: + + FGGY family carbohydrate kinases + + + 0.29086307 + site + cleaner0 + 2023-06-29T14:01:57Z + SO: + + conserved + + + 0.99886703 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.998818 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9985494 + chemical + cleaner0 + 2023-06-29T12:33:56Z + CHEBI: + + ATP + + + 0.9941108 + experimental_method + cleaner0 + 2023-06-29T13:10:37Z + MESH: + + structural superposition + + + 0.99877065 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.9988788 + protein + cleaner0 + 2023-06-29T12:30:04Z + PR: + + SePSK + + + 0.99508905 + protein_type + cleaner0 + 2023-06-29T12:31:09Z + MESH: + + FGGY family carbohydrate kinases + + + + METHODS + title_1 + 22537 + Materials and Methods + + + METHODS + title_2 + 22559 + Cloning, expression and purification of SePSK + + + METHODS + paragraph + 22605 + The gene encoding SePSK was amplified by polymerase chain reaction (PCR) with forward primer 5' CATGCCATGGGCATGGTCGTTGCACTTGGCCTCG 3' containing an internal Nco I restriction site (underlined) and reverse primer 5' CCGCTCGAGGGTTCTCTTTAACCCCGCCG 3' including an internal Xho I restriction site (underlined) from Synechococcus elongatus PCC 7942 genomic DNA. The amplified PCR product was digested with Nco I and Xho I (Takara) and ligated into linearized pET28-a vector (Novagen) between Nco I and Xho I restriction sites with a C-terminal his6 tag. The recombinant plasmids were transformed into competent Escherichia coli Trans10 cells for DNA production and purification, and the final constructs were verified by sequencing. The recombinant vectors were transformed into Escherichia coli BL21 (DE3) to express the protein. After induction with the 1 mM IPTG at 289 K in Luria-Bertani medium until the cell density reached an OD 600 nm of 0.6–0.8, the cells were harvested by centrifugation at 6,000 g at 4°C for 15 min, re-suspended in buffer A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole) and disrupted by sonication. After centrifuge 40,000 g for 30 min, the protein was purified by passage through a Ni2+ affinity column in buffer A, and then washed the unbound protein with buffer B (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 60 mM imidazole), and eluted the fraction with the buffer C (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole). After that, the protein was further purified by size exclusion chromatography with Superdex 200 10/300 GL (GE Healthcare) equilibrated with the buffer D (20 mM Tris-HCl, pH 8.0, 300 mM NaCl). The eluted major peak fraction was concentrated to 20 mg/mL protein using 10,000 MCWO centrifugal filter units (Millipore) and stored at -80°C for crystallization trials. The purified product was analyzed by SDS-PAGE with a single band visible only. + + + METHODS + title_2 + 24505 + Cloning, expression and purification of AtXK-1 + + + METHODS + paragraph + 24552 + The gene encoding AtXK-1 was amplified by PCR using a forward primer 5' TACTTCCAATCCAATGCTGTTATGAGTGGCAATAAAGGAACGA 3' and reverse primer 5' TTATCCACTTCCAATGTTACAAACCACTGTTCTGTTTTGCGCCC 3' from cDNA library of Arabidopsis thaliana. The underlined nucleotides were used for the ligation-independent cloning. The PCR product was treated by T4 DNA polymerase (LIC-qualified, Novagen) and then cloned into linearized pMCSG7 vector treated by T4 DNA polymerase (LIC-qualified, Novagen) with an N-terminal his6 tag though ligation-independent cloning method. The final construct was confirmed by DNA sequencing after amplified in competent Escherichia coli Trans10 cells. The recombinant vectors were transformed into Escherichia coli BL21 (DE3) for protein expression. After induction with 1 mM IPTG at 289 K in Luria-Bertani medium, cells were grown until the cell density reached an OD 600 nm of 0.6–0.8. Subsequent purification was identical to that used for SePSK, except that there was one additional step, during which tobacco etch virus protease was used to digest the crude AtXK-1 protein for removal of the N-terminal his6 tag following Ni2+ affinity purification. Ni2+ affinity column buffer contained extra 20% glycerol. The protein was further purified by size exclusion chromatography with Superdex 200 10/300 GL (GE Healthcare) in elution buffer consisting of 20 mM HEPES, pH 7.5, 100 mM NaCl. Finally, AtXK-1 protein was concentrated to 40 mg/mL protein using 10,000 MCWO centrifugal filter units (Millipore) and stored at -80°C prior to crystallization trials. Purity was verified by SDS-PAGE with a single band visible only. + + + METHODS + title_2 + 26192 + Site-directed mutagenesis of SePSK + + + METHODS + paragraph + 26227 + The gene of D8A and T11A mutations were amplified by PCR with the forward primer 5' CATGCCATGGGCATGGTCGTTGCACTTGGCCTCGCCTTCGGCAC 3' and forward primer 5' CATGCCATGGGCATGGTCGTTGCACTTGGCCTCGACTTCGGCGCCTCTGGAGCCC 3' (mismatched base pairs are underlined). The reverse primers of D8A and T11A mutants, the further constructions and purification procedures were identical with those used for wild type SePSK. + + + METHODS + paragraph + 26631 + The N-terminal sequence of D221A was amplified with forward primer (T7 promoter primer) 5' TAATACGACTCACTATA 3' and reverse primer 5' AGCAGCAATGCTAGCCGTTGTACCG 3’, and the C-terminal sequence of D221A was amplified with forward primer 5' TGCCGGTACAACGGCTAGCATTGCT 3' and reverse primer (T7 terminator primer) CGATCAATAACGAGTCGCC (mismatched base pairs are underlined). The second cycle PCR used the above PCR products as templates, and the construction and purification procedures were identical to those used for wild type SePSK. + + + METHODS + title_2 + 27164 + Crystallization and data collection + + + METHODS + paragraph + 27200 + Crystallization trials of SePSK and AtXK-1 were carried out at 281 K by mixing equal volume of 20 mg/ml protein and reservoir solution with the sitting-drop vapor diffusion method. The reservoir solution was PEG Rx I-35 (0.1 M BIS-TRIS pH 6.5, 20% w/v Polyethylene glycol monomethyl ether 5,000) (Hampton research). After 2 or 3 days, the rod-like crystals could be observed. For phasing, the high-quality apo-SePSK crystals were soaked in mother liquor containing 1 mM ethylmercuricthiosalicylic acid, sodium salt (Hampton research, heavy atom kit) overnight at 281 K. In order to get the complexes with ATP and AMP-PNP, the crystals of apo-SePSK and apo-AtXK-1 were incubated with the reservoir including 10 mM ATP and 20 mM MgCl2 as well as 10 mM AMP-PNP and 20 mM MgCl2, respectively. The apo-SePSK crystals were incubated with the reservoir including 10 mM D-ribulose in order to obtain the complex D-ribulose-bound SePSK (RBL-SePSK). The crystals of three mutants (D8A, T11A and D221A) grew in the same condition as that of the wild type SePSK. The crystals were dipped into reservoir solution supplemented with 15% glycerol and then flash frozen in a nitrogen gas stream at 100 K. All data sets were collected at Shanghai Synchrotron Radiation Facility, Photo Factory in Japan and Institute of Biophysics, Chinese Academy of Sciences. Diffraction data were processed using the HKL2000 package. + + + METHODS + title_2 + 28601 + Structure determination and refinement + + + METHODS + paragraph + 28640 + The initial phases of SePSK were obtained from the Hg-derivative crystals by single isomorphous replacement anomalous scattering (SIRAS) using AutoSol from the PHENIX suite. AutoBuild from the PHENIX suite was used to build 75% of the main chain of apo-SePSK, and the remaining residues were built manually by Coot. All other structures were solved by molecular replacement method using apo-SePSK as an initial model. The model was refined using phenix.refine and REFMAC5. The final model was checked with PROCHECK. All structural figures were prepared by PyMOL. The summary of the data-collection and structure-refinement statistics is shown in Table 1 and S1 Table. Atomic coordinates and structure factors in this article have been deposited in the Protein Data Bank. The deposited codes of all structures listed in the Table 1 and S1 Table. + + + pone.0156067.t001.xml + pone.0156067.t001 + TABLE + table_title_caption + 29485 + Data collection and refinement statistics. + + + pone.0156067.t001.xml + pone.0156067.t001 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><thead><tr><th align="justify" rowspan="1" colspan="1">Data set</th><th align="justify" rowspan="1" colspan="1">Hg-SePSK</th><th align="justify" rowspan="1" colspan="1">apo-SePSK</th><th align="justify" rowspan="1" colspan="1">AMP-PNP-SePSK</th><th align="justify" rowspan="1" colspan="1">RBL-SePSK</th><th align="justify" rowspan="1" colspan="1">apo-AtXK-1</th></tr></thead><tbody><tr><td align="justify" rowspan="1" colspan="1">Data collection</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">Space group</td><td align="justify" rowspan="1" colspan="1">C 1 2 1</td><td align="justify" rowspan="1" colspan="1">C 1 2 1</td><td align="justify" rowspan="1" colspan="1">C 1 2 1</td><td align="justify" rowspan="1" colspan="1">C 1 2 1</td><td align="justify" rowspan="1" colspan="1">P21</td></tr><tr><td align="justify" rowspan="1" colspan="1">Wavelength (Å)</td><td align="justify" rowspan="1" colspan="1">1.54178</td><td align="justify" rowspan="1" colspan="1">1.54178</td><td align="justify" rowspan="1" colspan="1">1.54178</td><td align="justify" rowspan="1" colspan="1">1.54178</td><td align="justify" rowspan="1" colspan="1">1.54178</td></tr><tr><td align="justify" rowspan="1" colspan="1">Cell parameters</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">a/b/c(Å)</td><td align="justify" rowspan="1" colspan="1">103.1, 46.6, 88.3</td><td align="justify" rowspan="1" colspan="1">110.2, 49.0, 86.9</td><td align="justify" rowspan="1" colspan="1">103.5, 46.6, 88.0</td><td align="justify" rowspan="1" colspan="1">102.6, 47.0, 88.7</td><td align="justify" rowspan="1" colspan="1">49.7, 87.9, 53.6</td></tr><tr><td align="justify" rowspan="1" colspan="1">α/β/γ(°)</td><td align="justify" rowspan="1" colspan="1">90.0, 91.9, 90.0</td><td align="justify" rowspan="1" colspan="1">90.0, 110.3, 90.0</td><td align="justify" rowspan="1" colspan="1">90.0, 91.0, 90.0</td><td align="justify" rowspan="1" colspan="1">90.0, 91.4, 90.0</td><td align="justify" rowspan="1" colspan="1">90.0, 97.0, 90.0</td></tr><tr><td align="justify" rowspan="1" colspan="1">Resolution (Å)<xref ref-type="table-fn" rid="t001fn001"><sup>a</sup></xref></td><td align="justify" rowspan="1" colspan="1">50.00–2.20(2.28–2.20)</td><td align="justify" rowspan="1" colspan="1">50.00–2.30(2.38–2.30)</td><td align="justify" rowspan="1" colspan="1">50.00–2.30(2.38–2.30)</td><td align="justify" rowspan="1" colspan="1">50.00–2.35(2.43–2.35)</td><td align="justify" rowspan="1" colspan="1">50.00–2.00(2.07–2.00)</td></tr><tr><td align="justify" rowspan="1" colspan="1">R merge<xref ref-type="table-fn" rid="t001fn002"><sup>b</sup></xref></td><td align="justify" rowspan="1" colspan="1">0.105(0.514)</td><td align="justify" rowspan="1" colspan="1">0.149(0.501)</td><td align="justify" rowspan="1" colspan="1">0.082(0.503)</td><td align="justify" rowspan="1" colspan="1">0.095(0.507)</td><td align="justify" rowspan="1" colspan="1">0.106(0.454)</td></tr><tr><td align="justify" rowspan="1" colspan="1">〈 I/σ(I)〉</td><td align="justify" rowspan="1" colspan="1">28.89(4.07)</td><td align="justify" rowspan="1" colspan="1">13.85(4.10)</td><td align="justify" rowspan="1" colspan="1">10.18(1.79)</td><td align="justify" rowspan="1" colspan="1">19.4(4.6)</td><td align="justify" rowspan="1" colspan="1">12.91(4.08)</td></tr><tr><td align="justify" rowspan="1" colspan="1">Completeness (%)</td><td align="justify" rowspan="1" colspan="1">92.3(99.2)</td><td align="justify" rowspan="1" colspan="1">96.1(94.2)</td><td align="justify" rowspan="1" colspan="1">98.9(99.8)</td><td align="justify" rowspan="1" colspan="1">99.8(100.0)</td><td align="justify" rowspan="1" colspan="1">97.1(94.5)</td></tr><tr><td align="justify" rowspan="1" colspan="1">Redundancy</td><td align="justify" rowspan="1" colspan="1">6.7(5.1)</td><td align="justify" rowspan="1" colspan="1">7.4(7.5)</td><td align="justify" rowspan="1" colspan="1">2.4(2.4)</td><td align="justify" rowspan="1" colspan="1">6.9(6.7)</td><td align="justify" rowspan="1" colspan="1">7.2(6.9)</td></tr><tr><td align="justify" rowspan="1" colspan="1">Refinement statistics</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">Resolution (Å)</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">32.501–2.301</td><td align="justify" rowspan="1" colspan="1">24.707–2.300</td><td align="justify" rowspan="1" colspan="1">24.475–2.344</td><td align="justify" rowspan="1" colspan="1">23.771–1.998</td></tr><tr><td align="justify" rowspan="1" colspan="1">R<sub>work</sub>/ R<sub>free</sub><xref ref-type="table-fn" rid="t001fn003"><sup>c</sup></xref></td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">0.1834/0.2276</td><td align="justify" rowspan="1" colspan="1">0.1975/0.2327</td><td align="justify" rowspan="1" colspan="1">0.2336/0.2687</td><td align="justify" rowspan="1" colspan="1">0.1893/0.2161</td></tr><tr><td align="justify" rowspan="1" colspan="1">No. atoms</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">Protein</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">3503</td><td align="justify" rowspan="1" colspan="1">3196</td><td align="justify" rowspan="1" colspan="1">3209</td><td align="justify" rowspan="1" colspan="1">3256</td></tr><tr><td align="justify" rowspan="1" colspan="1">ligand/ion</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">-</td><td align="justify" rowspan="1" colspan="1">31</td><td align="justify" rowspan="1" colspan="1">20</td><td align="justify" rowspan="1" colspan="1">-</td></tr><tr><td align="justify" rowspan="1" colspan="1">Water</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">313</td><td align="justify" rowspan="1" colspan="1">146</td><td align="justify" rowspan="1" colspan="1">143</td><td align="justify" rowspan="1" colspan="1">486</td></tr><tr><td align="justify" rowspan="1" colspan="1">RMSD Bond lengths (Å)<xref ref-type="table-fn" rid="t001fn004"><sup>d</sup></xref></td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">0.003</td><td align="justify" rowspan="1" colspan="1">0.005</td><td align="justify" rowspan="1" colspan="1">0.003</td><td align="justify" rowspan="1" colspan="1">0.003</td></tr><tr><td align="justify" rowspan="1" colspan="1">RMSD Bond angles (°)<xref ref-type="table-fn" rid="t001fn004"><sup>d</sup></xref></td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">0.674</td><td align="justify" rowspan="1" colspan="1">0.886</td><td align="justify" rowspan="1" colspan="1">0.649</td><td align="justify" rowspan="1" colspan="1">0.838</td></tr><tr><td align="justify" rowspan="1" colspan="1">Ramachandran plot (%)</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1"/></tr><tr><td align="justify" rowspan="1" colspan="1">favoured</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">98.1</td><td align="justify" rowspan="1" colspan="1">97.8</td><td align="justify" rowspan="1" colspan="1">96.7</td><td align="justify" rowspan="1" colspan="1">99.1</td></tr><tr><td align="justify" rowspan="1" colspan="1">allowed</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">1.9</td><td align="justify" rowspan="1" colspan="1">2.2</td><td align="justify" rowspan="1" colspan="1">3.3</td><td align="justify" rowspan="1" colspan="1">0.9</td></tr><tr><td align="justify" rowspan="1" colspan="1">disallowed</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">0.0</td><td align="justify" rowspan="1" colspan="1">0.0</td><td align="justify" rowspan="1" colspan="1">0.0</td><td align="justify" rowspan="1" colspan="1">0.0</td></tr><tr><td align="justify" rowspan="1" colspan="1">PDB code</td><td align="justify" rowspan="1" colspan="1"/><td align="justify" rowspan="1" colspan="1">5HTN</td><td align="justify" rowspan="1" colspan="1">5HTP</td><td align="justify" rowspan="1" colspan="1">5HV7</td><td align="justify" rowspan="1" colspan="1">5HTR</td></tr></tbody></table> + + 29528 + Data set Hg-SePSK apo-SePSK AMP-PNP-SePSK RBL-SePSK apo-AtXK-1 Data collection Space group C 1 2 1 C 1 2 1 C 1 2 1 C 1 2 1 P21 Wavelength (Å) 1.54178 1.54178 1.54178 1.54178 1.54178 Cell parameters a/b/c(Å) 103.1, 46.6, 88.3 110.2, 49.0, 86.9 103.5, 46.6, 88.0 102.6, 47.0, 88.7 49.7, 87.9, 53.6 α/β/γ(°) 90.0, 91.9, 90.0 90.0, 110.3, 90.0 90.0, 91.0, 90.0 90.0, 91.4, 90.0 90.0, 97.0, 90.0 Resolution (Å)a 50.00–2.20(2.28–2.20) 50.00–2.30(2.38–2.30) 50.00–2.30(2.38–2.30) 50.00–2.35(2.43–2.35) 50.00–2.00(2.07–2.00) R mergeb 0.105(0.514) 0.149(0.501) 0.082(0.503) 0.095(0.507) 0.106(0.454) 〈 I/σ(I)〉 28.89(4.07) 13.85(4.10) 10.18(1.79) 19.4(4.6) 12.91(4.08) Completeness (%) 92.3(99.2) 96.1(94.2) 98.9(99.8) 99.8(100.0) 97.1(94.5) Redundancy 6.7(5.1) 7.4(7.5) 2.4(2.4) 6.9(6.7) 7.2(6.9) Refinement statistics Resolution (Å) 32.501–2.301 24.707–2.300 24.475–2.344 23.771–1.998 Rwork/ Rfreec 0.1834/0.2276 0.1975/0.2327 0.2336/0.2687 0.1893/0.2161 No. atoms Protein 3503 3196 3209 3256 ligand/ion - 31 20 - Water 313 146 143 486 RMSD Bond lengths (Å)d 0.003 0.005 0.003 0.003 RMSD Bond angles (°)d 0.674 0.886 0.649 0.838 Ramachandran plot (%) favoured 98.1 97.8 96.7 99.1 allowed 1.9 2.2 3.3 0.9 disallowed 0.0 0.0 0.0 0.0 PDB code 5HTN 5HTP 5HV7 5HTR + + + pone.0156067.t001.xml + pone.0156067.t001 + TABLE + table_footnote + 30913 + a The values in parentheses correspond to the highest resolution shell. + + + pone.0156067.t001.xml + pone.0156067.t001 + TABLE + table_footnote + 30985 + b Rmerge = ∑j∑h|Ij,h-<Ih>|/∑j∑h<Ih> where h are unique reflection indices and Ij,h are intensities of symmetry-related reflections and <Ih> is the mean intensity. + + + pone.0156067.t001.xml + pone.0156067.t001 + TABLE + table_footnote + 31156 + c R-work and R-free were calculated as follows: R = Σ (|Fobs-Fcalc|)/Σ |Fobs| ×100, where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. + + + pone.0156067.t001.xml + pone.0156067.t001 + TABLE + table_footnote + 31344 + d Root mean square deviations (r.m.s.d.) from standard values. + + + METHODS + title_2 + 31407 + ADP-Glo kinase assay + + + METHODS + paragraph + 31428 + ADP-Glo kinase assay was used according to the manufacturer’s instructions (Promega). Each reaction mixture system consisted of 8 uM enzyme, 100 uM ATP, 1 mM MgCl2, 20 mM HEPES (pH 7.4), 5 mM substrate. The reaction was initiated by adding the purified enzyme into the reaction system. After incubation at 298 K for different time, equal volume ADP-Glo™ reagent was added to terminate the kinase reaction and to deplete any remaining ATP. Subsequently, kinase detection reagent with double volume of reaction system was added to convert ADP to ATP and allowed the newly synthesized ATP to be measured using a luciferase/luciferin reaction which produced luminescence signal and could be recorded. After incubation at room temperature for another 60 min, luminescence was detected by Varioskan Flash Multimode Reader (Thermo). The reference experiment was carried out in the same reaction system without the enzyme. For each assay, at least three repeats were performed for the calculation of mean values and standard deviations (SDs). The purity of five substrates in the activity assays was ≥98% (D-ribulose, Santa cruz), 99.7% (L-ribulose, Carbosynth), 99.3% (D-xylulose, Carbosynth), 99.5% (L-xylulose, Carbosynth) and 99.0% (Glycerol, AMRESCO). + + + METHODS + title_2 + 32683 + Surface plasmon resonance + + + METHODS + paragraph + 32709 + Surface plasmon resonance (SPR) was used to analyze the interaction of SePSK and D-ribulose. The SPR experiments were performed on a Biacore T100 system using series S CM5 sensor chips (GE Healthcare). All sensorgrams were recorded at 298 K. The proteins in buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, was diluted to 40 ug/ml by 10 mM sodium acetate buffer at pH 4.5. All flow cells on a CM5 sensor chip were activated with a freshly prepared solution of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS) in a ratio of 1:1 at a constant flow rate of 10 ul/min for 420 s. Deactivation of the surface was performed with an injection of a 1 M solution of ethanolamine-HCl (pH 8.5) using the same flow rate and duration. Kinetic parameters were derived from data sets acquired in single-cycle mode. Each run consisted of five consecutive analytic injections at 125, 250, 500, 1000 and 2000 uM. Analytic injections lasted for 60 s, separated by 30 s dissociation periods. Each cycle was completed with an extended dissociation period of 300 s. The specific binding to a blank flow cell was subtracted to obtain corrected sensorgrams. Biacore data were analyzed using BiaEvaluation software (GE Healthcare) by fitting to a 1:1 Langmuir binding fitting model. + + + METHODS + title_2 + 34017 + Accession Codes + + + METHODS + paragraph + 34033 + Coordinates and structure factors for all the structures in this article have been deposited in the Protein Data Bank. These accession codes are 5HTN, 5HTP, 5HUX, 5HV7, 5HTJ, 5HU2, 5HTY, 5HTR, 5HTV and 5HTX. The corresponding-structures are apo-SePSK, AMP-PNP-SePSK, ADP-SePSK, RBL-SePSK, D8A-SePSK, T11A-SePSK, D221A-SePSK, apo-AtXK1, AMP-PNP-AtXK1 and ADP-AtXK1, respectively. + + + SUPPL + title_1 + 34412 + Supporting Information + + + REF + title + 34435 + References + + + e1002318 + 12 + surname:Zhang;given-names:Y + surname:Zagnitko;given-names:O + surname:Rodionova;given-names:I + surname:Osterman;given-names:A + surname:Godzik;given-names:A + 10.1371/journal.pcbi.1002318 + 22215998 + REF + PLoS computational biology + ref + 7 + 2011 + 34446 + The FGGY carbohydrate kinase family: insights into the evolution of functional specificities + + + 1643 + 3 + 52 + surname:Bunker;given-names:RD + surname:Bulloch;given-names:EM + surname:Dickson;given-names:JM + surname:Loomes;given-names:KM + surname:Baker;given-names:EN + 10.1074/jbc.M112.427997 + 23179721 + REF + Journal of Biological Chemistry + ref + 288 + 2013 + 34539 + Structure and function of human xylulokinase, an enzyme with important roles in carbohydrate metabolism + + + 261 + 1 + 8 + surname:Agarwal;given-names:R + surname:Burley;given-names:SK + surname:Swaminathan;given-names:S + 10.1002/prot.23202 + 22072612 + REF + Proteins + ref + 80 + 2012 + 34643 + Structural insight into mechanism and diverse substrate selection strategy of L-ribulokinase + + + 783 + 3 + 98 + surname:Di Luccio;given-names:E + surname:Petschacher;given-names:B + surname:Voegtli;given-names:J + surname:Chou;given-names:H-T + surname:Stahlberg;given-names:H + surname:Nidetzky;given-names:B + 17123542 + REF + Journal of molecular biology + ref + 365 + 2007 + 34736 + Structural and Kinetic Studies of Induced Fit in Xylulose Kinase from Escherichia coli + + + 978 + 5 + 84 + surname:Emanuelsson;given-names:O + surname:Nielsen;given-names:H + surname:Von Heijne;given-names:G + 10338008 + REF + Protein Sci + ref + 8 + 1999 + 34823 + ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites + + + 441 + 2 + 57 + surname:Hemmerlin;given-names:A + surname:Tritsch;given-names:D + surname:Hartmann;given-names:M + surname:Pacaud;given-names:K + surname:Hoeffler;given-names:J-F + surname:van Dorsselaer;given-names:A + 16920870 + REF + Plant physiology + ref + 142 + 2006 + 34931 + A cytosolic Arabidopsis D-xylulose kinase catalyzes the phosphorylation of 1-deoxy-D-xylulose into a precursor of the plastidial isoprenoid pathway + + + 3508 + 12 + 18 + surname:Bystrom;given-names:CE + surname:Pettigrew;given-names:DW + surname:Branchaud;given-names:BP + surname:O'Brien;given-names:P + surname:Remington;given-names:SJ + 10090737 + REF + Biochemistry + ref + 38 + 1999 + 35079 + Crystal structures of Escherichia coli glycerol kinase variant S58→ W in complex with nonhydrolyzable ATP analogues reveal a putative active conformation of the enzyme as a result of domain motion + + + 1407 + 11 + 18 + surname:Feese;given-names:MD + surname:Faber;given-names:HR + surname:Bystrom;given-names:CE + surname:Pettigrew;given-names:DW + surname:Remington;given-names:SJ + 9817843 + REF + Structure + ref + 6 + 1998 + 35278 + Glycerol kinase from Escherichia coli and an Ala65→ Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation + + + 787 + 3 + 97 + surname:Grueninger;given-names:D + surname:Schulz;given-names:GE + 16674975 + REF + Journal of molecular biology + ref + 359 + 2006 + 35441 + Structure and Reaction Mechanism of l-Rhamnulose Kinase from Escherichia coli + + + 1469 + 7 + 82 + surname:Higgins;given-names:MA + surname:Suits;given-names:MD + surname:Marsters;given-names:C + surname:Boraston;given-names:AB + 10.1016/j.jmb.2013.12.006 + 24333485 + REF + Journal of molecular biology + ref + 426 + 2014 + 35519 + Structural and Functional Analysis of Fucose-Processing Enzymes from Streptococcus pneumoniae + + + 236 + 2 + 45 + surname:Pettigrew;given-names:DW + surname:Smith;given-names:GB + surname:Thomas;given-names:KP + surname:D'Nette;given-names:CD + 9448710 + REF + Archives of biochemistry and biophysics + ref + 349 + 1998 + 35613 + Conserved active site aspartates and domain–domain interactions in regulatory properties of the sugar kinase superfamily + + + 136 + 1 + 47 + surname:Karlsson;given-names:R + surname:Katsamba;given-names:PS + surname:Nordin;given-names:H + surname:Pol;given-names:E + surname:Myszka;given-names:DG + 16337141 + REF + Analytical biochemistry + ref + 349 + 2006 + 35736 + Analyzing a kinetic titration series using affinity biosensors + + + 362 + 2 + 73 + surname:Yeh;given-names:JI + surname:Charrier;given-names:V + surname:Paulo;given-names:J + surname:Hou;given-names:L + surname:Darbon;given-names:E + surname:Claiborne;given-names:A + 14717590 + REF + Biochemistry + ref + 43 + 2004 + 35799 + Structures of enterococcal glycerol kinase in the absence and presence of glycerol: correlation of conformation to substrate binding and a mechanism of activation by phosphorylation + + + 137 + 62 + surname:Hurley;given-names:JH + 8800467 + REF + Annual review of biophysics and biomolecular structure + ref + 25 + 1996 + 35981 + The sugar kinase/heat shock protein 70/actin superfamily: implications of conserved structure for mechanism + + + 1219 + 4 + 27 + surname:Emekli;given-names:U + surname:Schneidman-Duhovny;given-names:D + surname:Wolfson;given-names:HJ + surname:Nussinov;given-names:R + surname:Haliloglu;given-names:T + 17847101 + REF + Proteins + ref + 70 + 2008 + 36089 + HingeProt: automated prediction of hinges in protein structures + + + 8 + 1 + 15 + surname:Stols;given-names:L + surname:Gu;given-names:M + surname:Dieckman;given-names:L + surname:Raffen;given-names:R + surname:Collart;given-names:FR + surname:Donnelly;given-names:MI + 12071693 + REF + Protein Expr Purif + ref + 25 + 2002 + 36153 + A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site + + + 307 + 26 + surname:Otwinowski;given-names:Z + surname:Minor;given-names:W + REF + Methods In Enzymology + ref + 276 + 1997 + 36269 + Processing of X-ray diffraction data collected in oscillation mode + + + 213 + 2 + 21 + surname:Adams;given-names:PD + surname:Afonine;given-names:PV + surname:Bunkóczi;given-names:G + surname:Chen;given-names:VB + surname:Davis;given-names:IW + surname:Echols;given-names:N + 20124702 + REF + Acta Crystallographica Section D: Biological Crystallography + ref + 66 + 2010 + 36336 + PHENIX: a comprehensive Python-based system for macromolecular structure solution + + + 2126 + 12 + 32 + surname:Emsley;given-names:P + surname:Cowtan;given-names:K + 15572765 + REF + Acta Crystallographica Section D: Biological Crystallography + ref + 60 + 2004 + 36418 + Coot: model-building tools for molecular graphics + + + 1194 + 11 + 7 + surname:Afonine;given-names:PV + surname:Grosse-Kunstleve;given-names:RW + surname:Adams;given-names:PD + surname:Lunin;given-names:VY + surname:Urzhumtsev;given-names:A + 18007035 + REF + Acta Crystallographica Section D: Biological Crystallography + ref + 63 + 2007 + 36468 + On macromolecular refinement at subatomic resolution with interatomic scatterers + + + 283 + 2 + 91 + surname:Laskowski;given-names:RA + surname:MacArthur;given-names:MW + surname:Moss;given-names:DS + surname:Thornton;given-names:JM + REF + Journal of applied crystallography + ref + 26 + 1993 + 36549 + PROCHECK: a program to check the stereochemical quality of protein structures + + + REF + ref + 36627 + DeLano. W. The PyMOL Molecular Graphics System. Available: http://www.pymol.org. + + + 615 + 6 + 22 + surname:Sanghera;given-names:J + surname:Li;given-names:R + surname:Yan;given-names:J + 10.1089/adt.2009.0237 + 20105027 + REF + Assay and drug development technologies + ref + 7 + 2009 + 36708 + Comparison of the luminescent ADP-Glo assay to a standard radiometric assay for measurement of protein kinase activity + + + diff --git a/BioC_XML/4885502_v0.xml b/BioC_XML/4885502_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..9c6b0428318b7ea34d94564f49618bd5b0d76509 --- /dev/null +++ b/BioC_XML/4885502_v0.xml @@ -0,0 +1,5893 @@ + + + + PMC + 20201220 + pmc.key + + 4885502 + NO-CC CODE + no + 0 + 0 + + Cryo-EM Studies with Glutamate Dehydrogenase + 10.1124/mol.116.103382 + 4885502 + 27036132 + MOL_103382 + 645 + 6 + 651 + surname:Borgnia;given-names:Mario J. + surname:Banerjee;given-names:Soojay + surname:Milne;given-names:Jacqueline L. S. + surname:Merk;given-names:Alan + surname:Matthies;given-names:Doreen + surname:Bartesaghi;given-names:Alberto + surname:Rao;given-names:Prashant + surname:Pierson;given-names:Jason + surname:Earl;given-names:Lesley A. + surname:Falconieri;given-names:Veronica + surname:Subramaniam;given-names:Sriram + TITLE + front + 89 + 2016 + 0 + Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase + + 0.9987944 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + Cryo-EM + + + 0.9987963 + protein_type + cleaner0 + 2023-07-27T15:32:06Z + MESH: + + Glutamate Dehydrogenase + + + + ABSTRACT + abstract + 102 + Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an “open” or “closed” state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes. + + 0.9989001 + experimental_method + cleaner0 + 2023-07-27T15:28:19Z + MESH: + + Cryo-electron microscopy + + + 0.99874353 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + cryo-EM + + + 0.99726605 + evidence + cleaner0 + 2023-07-27T16:13:01Z + DUMMY: + + structures + + + 0.9116105 + protein_type + cleaner0 + 2023-07-27T15:32:06Z + MESH: + + glutamate dehydrogenase + + + 0.8540359 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.9758364 + chemical + cleaner0 + 2023-07-27T15:28:57Z + CHEBI: + + glutamate + + + 0.8643728 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.9988768 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + cryo-EM + + + 0.99851555 + evidence + cleaner0 + 2023-07-27T16:13:04Z + DUMMY: + + structures + + + 0.9978859 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.9963707 + evidence + cleaner0 + 2023-07-27T16:13:08Z + DUMMY: + + crystal structures + + + 0.999298 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.9992781 + chemical + cleaner0 + 2023-07-27T15:29:01Z + CHEBI: + + GTP + + + 0.9991974 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + open + + + 0.9992416 + protein_state + cleaner0 + 2023-07-27T15:28:46Z + DUMMY: + + closed + + + 0.99849856 + evidence + cleaner0 + 2023-07-27T16:13:11Z + DUMMY: + + structure + + + 0.9992193 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.9989946 + site + cleaner0 + 2023-07-27T15:36:23Z + SO: + + active site + + + 0.9992312 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + open + + + 0.99926895 + protein_state + cleaner0 + 2023-07-27T15:28:46Z + DUMMY: + + closed + + + 0.9990865 + site + cleaner0 + 2023-07-27T15:38:43Z + SO: + + regulatory site + + + 0.99889517 + experimental_method + cleaner0 + 2023-07-27T15:28:24Z + MESH: + + X-ray crystallography + + + 0.9988362 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + cryo-EM + + + + INTRO + title_1 + 1392 + Introduction + + + INTRO + paragraph + 1405 + Recent advances in cryo-electron microscopy (cryo-EM) allow determination of structures of small protein complexes and membrane proteins at near-atomic resolution, marking a critical shift in the structural biology field. One specific area of broad general interest in drug discovery is the localization of bound ligands and cofactors under conditions in which efforts at crystallization have not been successful because of structural heterogeneity. Recent cryo-EM analyses have already demonstrated that it is now possible to use single-particle cryo-EM methods to localize small bound ligands or inhibitors on target proteins. Whether ligand binding can be visualized at high resolution is an important question, even in the more general case when multiple conformations are present simultaneously. Here, we address this question using mammalian glutamate dehydrogenase as an example. + + 0.9989047 + experimental_method + cleaner0 + 2023-07-27T15:28:19Z + MESH: + + cryo-electron microscopy + + + 0.9988249 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + cryo-EM + + + 0.99750286 + evidence + cleaner0 + 2023-07-27T16:13:13Z + DUMMY: + + structures + + + 0.9772683 + experimental_method + cleaner0 + 2023-07-27T16:11:30Z + MESH: + + crystallization + + + 0.99887514 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + cryo-EM + + + 0.99884266 + experimental_method + cleaner0 + 2023-07-27T15:29:24Z + MESH: + + single-particle cryo-EM + + + 0.9985678 + taxonomy_domain + cleaner0 + 2023-07-27T16:07:56Z + DUMMY: + + mammalian + + + 0.99871063 + protein_type + cleaner0 + 2023-07-27T15:32:05Z + MESH: + + glutamate dehydrogenase + + + + INTRO + paragraph + 2292 + Glutamate dehydrogenase (GDH) is a highly conserved enzyme expressed in most organisms. GDH plays a central role in glutamate metabolism by catalyzing the reversible oxidative deamination of glutamate to generate α-ketoglutarate and ammonia, with the concomitant transfer of a pair of electrons to either NAD+ or NADP+. Regulation of GDH is tightly controlled through multiple allosteric mechanisms. Extensive biochemical and crystallographic studies have characterized the enzymatic activity of GDH and its modulation by a chemically diverse group of compounds such as nucleotides, amino acids, steroid hormones, antipsychotic drugs, and natural products. X-ray crystallographic studies have shown that the functional unit of GDH is a homohexamer composed of a trimer of dimers, with a 3-fold axis and an equatorial plane that define its D3 symmetry (Fig. 1A). Each 56-kDa protomer consists of three domains. The first is located near the dimer interface and forms the core of the hexamer. The second, a nucleotide-binding domain (NBD) with a Rossmann fold, defines one face of the catalytic cleft bounded by the core domain. During the catalytic cycle, the NBD executes a large movement, hinged around a “pivot” helix, that closes the catalytic cleft, and drives a large conformational change in the hexamer from open to closed states (Fig. 1B). The third domain, dubbed the “antenna,” is an evolutionary acquisition in protista and animals. Antennae of adjacent protomers in each trimer intercalate to form a bundle, perpendicular to the pivot helices, that protrudes along the distal extremes of the 3-fold axis. When a protomer undergoes a conformational change, the rotation of its pivot helix is transferred through the antenna to the adjacent subunit. The influence of the antenna, present only in protozoan and metazoan enzymes, has been proposed to explain its cooperative behavior, which is absent in bacterial homologs. Deletion of this domain leads to loss of cooperativity. + + 0.99880797 + protein_type + cleaner0 + 2023-07-27T15:32:06Z + MESH: + + Glutamate dehydrogenase + + + 0.99915147 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.9990763 + protein_state + cleaner0 + 2023-07-27T15:36:33Z + DUMMY: + + highly conserved + + + 0.9990645 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.9654128 + chemical + cleaner0 + 2023-07-27T15:28:57Z + CHEBI: + + glutamate + + + 0.995365 + chemical + cleaner0 + 2023-07-27T15:28:57Z + CHEBI: + + glutamate + + + 0.99907213 + chemical + cleaner0 + 2023-07-27T15:32:16Z + CHEBI: + + α-ketoglutarate + + + 0.99903846 + chemical + cleaner0 + 2023-07-27T15:32:21Z + CHEBI: + + ammonia + + + 0.99906206 + chemical + cleaner0 + 2023-07-27T15:32:26Z + CHEBI: + + NAD+ + + + 0.9990859 + chemical + cleaner0 + 2023-07-27T15:32:31Z + CHEBI: + + NADP+ + + + 0.99913496 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.9988078 + experimental_method + cleaner0 + 2023-07-27T16:11:33Z + MESH: + + biochemical and crystallographic studies + + + 0.99890316 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.99894965 + experimental_method + cleaner0 + 2023-07-27T16:11:36Z + MESH: + + X-ray crystallographic studies + + + 0.985707 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.9984865 + oligomeric_state + cleaner0 + 2023-07-27T15:35:47Z + DUMMY: + + homohexamer + + + 0.9985979 + oligomeric_state + cleaner0 + 2023-07-27T15:35:50Z + DUMMY: + + trimer + + + 0.99865305 + oligomeric_state + cleaner0 + 2023-07-27T15:35:59Z + DUMMY: + + dimers + + + 0.99866486 + oligomeric_state + cleaner0 + 2023-07-27T15:35:55Z + DUMMY: + + protomer + + + 0.99872315 + site + cleaner0 + 2023-07-27T15:35:07Z + SO: + + dimer interface + + + 0.99862754 + oligomeric_state + cleaner0 + 2023-07-27T15:35:43Z + DUMMY: + + hexamer + + + 0.9992577 + structure_element + cleaner0 + 2023-07-27T15:34:50Z + SO: + + nucleotide-binding domain + + + 0.9992562 + structure_element + cleaner0 + 2023-07-27T15:34:54Z + SO: + + NBD + + + 0.9680246 + structure_element + cleaner0 + 2023-07-27T15:35:00Z + SO: + + Rossmann fold + + + 0.99891114 + site + cleaner0 + 2023-07-27T15:35:10Z + SO: + + catalytic cleft + + + 0.9994492 + structure_element + cleaner0 + 2023-07-27T15:34:55Z + SO: + + NBD + + + structure_element + SO: + cleaner0 + 2023-07-27T15:36:59Z + + “pivot” helix + + + 0.9986644 + site + cleaner0 + 2023-07-27T15:35:11Z + SO: + + catalytic cleft + + + 0.9985611 + oligomeric_state + cleaner0 + 2023-07-27T15:35:44Z + DUMMY: + + hexamer + + + 0.9992925 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + open + + + 0.99928766 + protein_state + cleaner0 + 2023-07-27T15:28:46Z + DUMMY: + + closed + + + 0.9992238 + structure_element + cleaner0 + 2023-07-27T15:38:09Z + SO: + + antenna + + + 0.99862075 + taxonomy_domain + cleaner0 + 2023-07-27T15:35:16Z + DUMMY: + + protista + + + 0.9982576 + taxonomy_domain + cleaner0 + 2023-07-27T15:35:22Z + DUMMY: + + animals + + + 0.9992933 + structure_element + cleaner0 + 2023-07-27T15:38:13Z + SO: + + Antennae + + + 0.99870396 + oligomeric_state + cleaner0 + 2023-07-27T16:06:37Z + DUMMY: + + protomers + + + 0.9986652 + oligomeric_state + cleaner0 + 2023-07-27T15:35:51Z + DUMMY: + + trimer + + + 0.998791 + structure_element + cleaner0 + 2023-07-27T15:37:50Z + SO: + + pivot helices + + + 0.9987753 + oligomeric_state + cleaner0 + 2023-07-27T15:35:55Z + DUMMY: + + protomer + + + 0.99898434 + structure_element + cleaner0 + 2023-07-27T15:37:54Z + SO: + + pivot helix + + + 0.9989784 + structure_element + cleaner0 + 2023-07-27T15:38:09Z + SO: + + antenna + + + 0.80029213 + structure_element + cleaner0 + 2023-07-27T16:06:57Z + SO: + + subunit + + + 0.99908864 + structure_element + cleaner0 + 2023-07-27T15:38:09Z + SO: + + antenna + + + 0.99850154 + taxonomy_domain + cleaner0 + 2023-07-27T15:35:27Z + DUMMY: + + protozoan + + + 0.99777126 + taxonomy_domain + cleaner0 + 2023-07-27T15:35:30Z + DUMMY: + + metazoan + + + 0.9988238 + taxonomy_domain + cleaner0 + 2023-07-27T15:35:34Z + DUMMY: + + bacterial + + + + mol.116.103382f1.jpg + F1 + FIG + fig_caption + 4289 + Structure and quaternary conformational changes in GDH. (A) Views of open (PDB ID 1NR7) and closed (PDB 3MW9) states of the GDH hexamer, shown in ribbon representation perpendicular to the 2-fold symmetry axis (side view, top) and 3-fold symmetry axis (top view, bottom). Only three protomers are shown in the top view for purposes of visual clarity. The dashed lines and arrows, respectively, highlight the slight extension in length, and twist in shape that occurs with transition from open to the closed state. The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. + + 0.718332 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.99922514 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + open + + + 0.99928147 + protein_state + cleaner0 + 2023-07-27T15:28:46Z + DUMMY: + + closed + + + 0.89957345 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.99871457 + oligomeric_state + cleaner0 + 2023-07-27T15:35:44Z + DUMMY: + + hexamer + + + 0.9986778 + oligomeric_state + cleaner0 + 2023-07-27T16:07:04Z + DUMMY: + + protomers + + + 0.99914074 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + open + + + 0.99912757 + protein_state + cleaner0 + 2023-07-27T15:28:46Z + DUMMY: + + closed + + + 0.9990472 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + open + + + 0.9991609 + protein_state + cleaner0 + 2023-07-27T15:45:16Z + DUMMY: + + unliganded + + + 0.9177118 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.99912745 + protein_state + cleaner0 + 2023-07-27T15:28:46Z + DUMMY: + + closed + + + 0.99924785 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.9992212 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9989837 + chemical + cleaner0 + 2023-07-27T15:28:57Z + CHEBI: + + glutamate + + + 0.9989303 + protein_state + cleaner0 + 2023-07-27T16:10:36Z + DUMMY: + + bound + + + 0.9985043 + experimental_method + cleaner0 + 2023-07-27T16:11:58Z + MESH: + + Superposition + + + 0.99673766 + evidence + cleaner0 + 2023-07-27T16:13:19Z + DUMMY: + + structures + + + 0.99926716 + protein_state + cleaner0 + 2023-07-27T15:28:46Z + DUMMY: + + closed + + + 0.9992894 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + open + + + + INTRO + paragraph + 5155 + The transition between “closed” and “open” states of GDH is modulated by two allosteric sites in each protomer (Fig. 1A), which are differentially bound by GTP (an inhibitor) and ADP (an activator). These allosteric modulators tightly control GDH function in vivo. In the first site, which sits next to the pivot helix at the base of the antenna (the “GTP binding site”), GTP binding is known to act as an inhibitor, preventing release of the reaction product from the catalytic site by stabilizing the closed conformation of the catalytic cleft. In the second “regulatory site”, which is situated near the pivot helix between adjacent protomers, ADP acts as an activator of enzymatic activity, presumably by hastening the opening of the catalytic cleft that leads to the release of the reaction product. Interestingly, it has also been shown that the coenzyme NADH can bind to the regulatory site (also bound by the activator ADP), exerting a converse, inhibitory effect on GDH product release, although the role this may play in vivo is not entirely clear. + + 0.99919957 + protein_state + cleaner0 + 2023-07-27T15:28:46Z + DUMMY: + + closed + + + 0.99915993 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + open + + + 0.99916553 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.99809766 + site + cleaner0 + 2023-07-27T15:38:47Z + SO: + + allosteric sites + + + 0.99857914 + oligomeric_state + cleaner0 + 2023-07-27T15:35:55Z + DUMMY: + + protomer + + + 0.91330147 + protein_state + cleaner0 + 2023-07-27T15:39:07Z + DUMMY: + + bound by + + + 0.9992711 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99927336 + chemical + cleaner0 + 2023-07-27T15:38:53Z + CHEBI: + + ADP + + + 0.9991462 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.9991275 + structure_element + cleaner0 + 2023-07-27T15:37:54Z + SO: + + pivot helix + + + 0.9787057 + structure_element + cleaner0 + 2023-07-27T15:38:09Z + SO: + + antenna + + + 0.9988017 + site + cleaner0 + 2023-07-27T15:38:36Z + SO: + + GTP binding site + + + 0.99916077 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99892986 + site + cleaner0 + 2023-07-27T16:09:05Z + SO: + + catalytic site + + + 0.99922884 + protein_state + cleaner0 + 2023-07-27T15:28:46Z + DUMMY: + + closed + + + 0.9989816 + site + cleaner0 + 2023-07-27T15:35:11Z + SO: + + catalytic cleft + + + 0.9832761 + site + cleaner0 + 2023-07-27T15:38:43Z + SO: + + regulatory site + + + 0.9990891 + structure_element + cleaner0 + 2023-07-27T15:37:54Z + SO: + + pivot helix + + + 0.998329 + oligomeric_state + cleaner0 + 2023-07-27T16:07:29Z + DUMMY: + + protomers + + + 0.9992016 + chemical + cleaner0 + 2023-07-27T15:38:54Z + CHEBI: + + ADP + + + 0.99899286 + site + cleaner0 + 2023-07-27T15:35:11Z + SO: + + catalytic cleft + + + 0.99929297 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.9987782 + site + cleaner0 + 2023-07-27T15:38:43Z + SO: + + regulatory site + + + 0.9827585 + protein_state + cleaner0 + 2023-07-27T15:39:07Z + DUMMY: + + bound by + + + 0.99925977 + chemical + cleaner0 + 2023-07-27T15:38:54Z + CHEBI: + + ADP + + + 0.99798226 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + + INTRO + paragraph + 6230 + Although there are numerous crystal structures available for GDH in complex with cofactors and nucleotides, they are limited to the combinations that have been amenable to crystallization. Nearly all X-ray structures of mammalian GDH are in the closed conformation, and the few structures that are in the open conformation are at lower resolution (Table 1). Of those structures in the closed conformation, most include NAD[P]H, GTP, and glutamate (or, alternately, NAD+, GTP, and α-ketoglutarate). However, the effects of coenzyme and GTP, bound alone or in concert in the absence of glutamate, have not been analyzed by crystallographic methods. Here, we report single-particle cryo-electron microscopy (cryo-EM) studies that show that under these conditions enzyme complexes coexist in both closed and open conformations. We show that the structures in both states can be resolved at near-atomic resolution, suggesting a molecular mechanism for synergistic inhibition of GDH by NADH and GTP (see Table 2 for detailed information on all cryo-EM-derived structures that we report in this work). + + 0.99880433 + evidence + cleaner0 + 2023-07-27T16:13:08Z + DUMMY: + + crystal structures + + + 0.9965873 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.99895054 + protein_state + cleaner0 + 2023-07-27T15:39:14Z + DUMMY: + + in complex with + + + 0.99693906 + experimental_method + cleaner0 + 2023-07-27T16:12:03Z + MESH: + + crystallization + + + experimental_method + MESH: + cleaner0 + 2023-07-27T16:13:37Z + + X-ray + + + evidence + DUMMY: + cleaner0 + 2023-07-27T16:13:46Z + + structures + + + 0.9984755 + taxonomy_domain + cleaner0 + 2023-07-27T16:08:02Z + DUMMY: + + mammalian + + + 0.983615 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.9992649 + protein_state + cleaner0 + 2023-07-27T15:28:46Z + DUMMY: + + closed + + + 0.9981117 + evidence + cleaner0 + 2023-07-27T16:13:50Z + DUMMY: + + structures + + + 0.9992205 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + open + + + 0.9985306 + evidence + cleaner0 + 2023-07-27T16:13:53Z + DUMMY: + + structures + + + 0.99927074 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9990926 + chemical + cleaner0 + 2023-07-27T15:39:35Z + CHEBI: + + NAD[P]H + + + 0.9991685 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99859256 + chemical + cleaner0 + 2023-07-27T15:28:57Z + CHEBI: + + glutamate + + + 0.9992113 + chemical + cleaner0 + 2023-07-27T15:39:31Z + CHEBI: + + NAD+ + + + 0.9992071 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99916536 + chemical + cleaner0 + 2023-07-27T15:32:17Z + CHEBI: + + α-ketoglutarate + + + 0.99878365 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99912345 + protein_state + cleaner0 + 2023-07-27T16:10:45Z + DUMMY: + + bound alone + + + 0.99913263 + protein_state + cleaner0 + 2023-07-27T16:10:54Z + DUMMY: + + absence of + + + 0.9949268 + chemical + cleaner0 + 2023-07-27T15:28:57Z + CHEBI: + + glutamate + + + 0.99887645 + experimental_method + cleaner0 + 2023-07-27T15:39:25Z + MESH: + + single-particle cryo-electron microscopy + + + 0.99877906 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + cryo-EM + + + 0.9992718 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9992467 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + open + + + 0.99840206 + evidence + cleaner0 + 2023-07-27T16:13:56Z + DUMMY: + + structures + + + 0.99060386 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.99922943 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.99920636 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99880105 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + cryo-EM + + + 0.9982753 + evidence + cleaner0 + 2023-07-27T16:13:58Z + DUMMY: + + structures + + + + T1.xml + T1 + TABLE + table_caption + 7329 + X-ray structures of mammalian GDH reported in both the open and closed conformations + + experimental_method + MESH: + cleaner0 + 2023-07-27T16:14:11Z + + X-ray + + + evidence + DUMMY: + cleaner0 + 2023-07-27T16:14:24Z + + structures + + + 0.99856323 + taxonomy_domain + cleaner0 + 2023-07-27T16:08:06Z + DUMMY: + + mammalian + + + 0.9988951 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + 0.9992053 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + open + + + 0.9992336 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + + T1.xml + T1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><col width="17.06%" span="1"/><col width="33.34%" span="1"/><col width="13.13%" span="1"/><col width="20.31%" span="1"/><col width="16.16%" span="1"/><thead><tr><th valign="top" align="center" scope="col" rowspan="1" colspan="1">GDH</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Ligands</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">PDB ID</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Conformation</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Resolution</th></tr></thead><tbody><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADH + GLU + GTP</td><td valign="top" align="left" rowspan="1" colspan="1">3MW9</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">2.4</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">Glu, GTP, NADPH, and Bithionol</td><td valign="top" align="left" rowspan="1" colspan="1">3ETD</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">2.5</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">Glu, NADPH, GTP + GW5074</td><td valign="top" align="left" rowspan="1" colspan="1">3ETG</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">2.5</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">apo</td><td valign="top" align="left" rowspan="1" colspan="1">1L1F</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">2.7</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADPH, glutamate, and GTP</td><td valign="top" align="left" rowspan="1" colspan="1">1HWZ</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">2.8</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADPH + GLU + GTP + Zinc</td><td valign="top" align="left" rowspan="1" colspan="1">3MVQ</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">2.94</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADPH, Glu, GTP, Hexachlorophene</td><td valign="top" align="left" rowspan="1" colspan="1">3ETE</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">3</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NAD, PO4, and 2-oxoglutarate</td><td valign="top" align="left" rowspan="1" colspan="1">1HWY</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">3.2</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADPH + GLU + Eu</td><td valign="top" align="left" rowspan="1" colspan="1">3MVO</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">3.23</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">R463A mutant</td><td valign="top" align="left" rowspan="1" colspan="1">apo</td><td valign="top" align="left" rowspan="1" colspan="1">1NR1</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.3</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">apo</td><td valign="top" align="left" rowspan="1" colspan="1">1NR7</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.3</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">ADP</td><td valign="top" align="left" rowspan="1" colspan="1">1NQT</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.5</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADPH and Epicatechin-3-gallate (Ecg)</td><td valign="top" align="left" rowspan="1" colspan="1">3QMU</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.62</td></tr></tbody></table> + + 7414 + GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 + + protein + PR: + cleaner0 + 2023-07-27T16:14:40Z + + GDH + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:36Z + + NADH + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:44:08Z + + GLU + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:42Z + + GTP + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:30Z + + Closed + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:44:08Z + + Glu + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:42Z + + GTP + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:57Z + + NADPH + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:30Z + + Closed + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:44:07Z + + Glu + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:57Z + + NADPH + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:42Z + + GTP + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:30Z + + Closed + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:41:05Z + + apo + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:26Z + + Open + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:57Z + + NADPH + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:43:30Z + + glutamate + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:42Z + + GTP + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:30Z + + Closed + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:57Z + + NADPH + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:44:08Z + + GLU + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:42Z + + GTP + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:43:06Z + + Zinc + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:30Z + + Closed + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:57Z + + NADPH + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:44:08Z + + Glu + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:42Z + + GTP + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:30Z + + Closed + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:41:33Z + + NAD + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:41:44Z + + PO4 + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:42:32Z + + 2-oxoglutarate + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:30Z + + Closed + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:57Z + + NADPH + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:44:08Z + + GLU + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:30Z + + Closed + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:41:21Z + + mutant + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:41:05Z + + apo + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:26Z + + Open + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:41:05Z + + apo + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:26Z + + Open + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:42:21Z + + ADP + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:26Z + + Open + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + chemical + CHEBI: + cleaner0 + 2023-07-27T15:40:57Z + + NADPH + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:26Z + + Open + + + + T2.xml + T2 + TABLE + table_caption + 7991 + Cryo-EM structures of mammalian GDH determined for this study + + 0.9988289 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + Cryo-EM + + + 0.99778193 + evidence + cleaner0 + 2023-07-27T16:14:46Z + DUMMY: + + structures + + + 0.9982822 + taxonomy_domain + cleaner0 + 2023-07-27T16:08:08Z + DUMMY: + + mammalian + + + 0.9989058 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + + T2.xml + T2 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><col width="7.86%" span="1"/><col width="15.31%" span="1"/><col width="18.73%" span="1"/><col width="14.12%" span="1"/><col width="16.09%" span="1"/><col width="12.62%" span="1"/><col width="15.27%" span="1"/><thead><tr><th valign="top" align="center" scope="col" rowspan="1" colspan="1">GDH</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Ligands</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">EMDB ID</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">PDB ID</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Conformation</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Resolution</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Particles</th></tr></thead><tbody><tr><td valign="top" align="center" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">apo</td><td valign="top" align="left" rowspan="1" colspan="1">EMD-6630</td><td valign="top" align="center" rowspan="1" colspan="1">3JCZ</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.26</td><td valign="top" align="center" rowspan="1" colspan="1">22462</td></tr><tr><td valign="top" align="center" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">GTP</td><td valign="top" align="left" rowspan="1" colspan="1">EMD-6631</td><td valign="top" align="center" rowspan="1" colspan="1">3JD0</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.47</td><td valign="top" align="center" rowspan="1" colspan="1">39439</td></tr><tr><td valign="top" align="center" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADH</td><td valign="top" align="left" rowspan="1" colspan="1">EMD-6635</td><td valign="top" align="center" rowspan="1" colspan="1">3JD2</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.27</td><td valign="top" align="center" rowspan="1" colspan="1">34716</td></tr><tr><td valign="top" align="center" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADH</td><td valign="top" align="left" rowspan="1" colspan="1">EMD-6634</td><td valign="top" align="center" rowspan="1" colspan="1">3JD1</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">3.27</td><td valign="top" align="center" rowspan="1" colspan="1">34926</td></tr><tr><td valign="top" align="center" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADH + GTP</td><td valign="top" align="left" rowspan="1" colspan="1">EMD-6632</td><td valign="top" align="center" rowspan="1" colspan="1">3JD3</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.55</td><td valign="top" align="center" rowspan="1" colspan="1">14793</td></tr><tr><td valign="top" align="center" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADH + GTP</td><td valign="top" align="left" rowspan="1" colspan="1">EMD-6633</td><td valign="top" align="center" rowspan="1" colspan="1">3JD4</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">3.40</td><td valign="top" align="center" rowspan="1" colspan="1">20429</td></tr></tbody></table> + + 8053 + GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 + + 0.98749566 + protein_type + cleaner0 + 2023-07-27T15:32:11Z + MESH: + + GDH + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:40:17Z + + WT + + + 0.9988914 + protein_state + cleaner0 + 2023-07-27T15:41:05Z + DUMMY: + + apo + + + 0.9940724 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + Open + + + 0.99706405 + protein_state + cleaner0 + 2023-07-27T15:40:17Z + DUMMY: + + WT + + + 0.99901605 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9962424 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + Open + + + 0.99640036 + protein_state + cleaner0 + 2023-07-27T15:40:17Z + DUMMY: + + WT + + + 0.9986166 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.99558604 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + Open + + + 0.9958423 + protein_state + cleaner0 + 2023-07-27T15:40:17Z + DUMMY: + + WT + + + 0.9985378 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.9955289 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + Closed + + + 0.99510795 + protein_state + cleaner0 + 2023-07-27T15:40:17Z + DUMMY: + + WT + + + 0.9025693 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.6816098 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99488443 + protein_state + cleaner0 + 2023-07-27T15:28:41Z + DUMMY: + + Open + + + 0.9968978 + protein_state + cleaner0 + 2023-07-27T15:40:17Z + DUMMY: + + WT + + + 0.965978 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.70165646 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9965994 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + Closed + + + + METHODS + title_1 + 8371 + Materials and Methods + + + METHODS + title_3 + 8393 + Specimen Preparation. + + + METHODS + paragraph + 8415 + Bovine glutamate dehydrogenase (Enzyme Commission 1.4.1.3; Sigma-Aldrich/MilliporeSigma, St. Louis, MO) was dialyzed overnight against fractionation buffer (100 mM potassium phosphate, pH 6.8) prior to fractionation by size-exclusion chromatography using a Superdex 200 10/30 column connected to an ÄKTA FPLC apparatus (GE Healthcare Bio-Sciences, Piscataway, NJ ). The concentration of GDH was adjusted to ∼2 mg/ml by rapid mixing with potassium phosphate buffer containing the concentration of ligand as necessary and with n-octyl glucopyranoside at a final concentration of 0.1%. The final concentration of each ligand was 20 mM. Small volumes of sample, typically 3 µl, were deposited on 200 mesh Quantifoil R2/2 grids (Quantifoil Micro Tools, Großlöbichaum, Germany), blotted, and plunge-frozen in liquid ethane using an FEI Vitrobot Mark IV (FEI Company, Hillsboro, OR). Frozen grids were mounted into autoloader cartridges and transferred to the microscope. + + + METHODS + title_3 + 9386 + Cryo-Electron Microscopy. + + + METHODS + paragraph + 9412 + Specimens were imaged on an FEI Titan Krios microscope (FEI Company) aligned for parallel illumination and operated at 300 kV. The instrument was furnished with a Gatan K2 Summit camera placed at the end of a GIF Quantum energy filter (Gatan Inc., Pleasanton, CA), operated in zero-energy-loss mode with a slit width of 20 eV. Images were collected manually at a dose rate of ∼5 e– pixel−1 s−1, i.e., in the linear range of the detector. The physical pixel size at the plane of the specimen was 1.275 Å, corresponding to a super-resolution pixel size of 0.6375 Å. The total exposure time was 15.2 s, and intermediate frames were recorded every 0.4 s, giving an accumulated dose of ∼45 e–/Å2 and a total of 38 frames per image. The majority of images were collected at under-focus values between 1 μm and 3 μm. + + + METHODS + title_3 + 10239 + Data Processing. + + + METHODS + paragraph + 10256 + Drift and beam-induced motion were compensated by whole-frame alignment of movies, and CTF was estimated as described in. Integrated frames were manually examined and selected on the basis of the quality of the CTF estimation, astigmatism, drift, and particle distribution. Molecular images were automatically identified from selected integrated micrographs by detecting the local maxima of correlation of each image with a Gaussian disk of 150 Å in radius. Individual particle projections were extracted from integrated super-resolution images using a binning factor of 4 and a box size of 96 × 96 pixels and assigned into 20–100 groups by iterative reference free two-dimensional classification as implemented in EMAN2. Following 8 iterations of classification, a subset of classes depicting intact particles were used to build symmetric (D3) density maps using the program e2initialmodel.py from the EMAN2 suite. One or more maps were selected as reference for further processing on the basis of consistency between projections and the original classes. These “initial models” were refined to ∼15–20 Å using e2refine_easy.py. Unbinned particles were then re-extracted from the original super-resolution images using a binning of 2 and a box size of 384 × 384, and subject to classification in three dimensions using the maximum likelihood method implemented in RELION (; MRC Laboratory, Cambridge, UK) (regularization parameter of T=4). Unless noted otherwise, D3 symmetry was imposed for three-dimensional classification runs, the number of classes was initially determined on the basis of the number of particles included in the analysis and later adjusted on the basis of the number of conformations detected in the sample (see Supplemental Table 1 for details). Iteration over classification in three dimensions was continued until convergence as judged by resolution and distribution of particles among the classes. Particles belonging to “good” three-dimensional classes were pooled into one or more classes, depending on the conformational landscape of the complex, and refined using the “gold standard” method in RELION. The refined maps were corrected for the MTF of the camera and for B-factor in the framework of RELION. Figures were generated using UCSF Chimera and Maxon Cinema4D (Maxon Computer Inc., Newbury Park, CA), and two-dimensionally composited in Adobe Photoshop and Illustrator. + + + METHODS + title_3 + 12684 + Building of Atomic Models. + + + METHODS + paragraph + 12711 + The deposited models for unliganded GDH (1NR7), the binary complex with ADP (1NQT), and the quaternary complex with NADH, GTP, and Glu (3MW9) were used to derive models from the six structures reported here. Conflicts in sequence between the deposited models were solved by conforming to the primary sequence as reported in 3MW9. The models were placed in the corresponding map (open or closed) by rigid body fitting as implemented in Chimera. In the closed state, in which all relative orientations of the ligands are known, only the ligands known to be present in each structure were retained, all other nonstandard residues were deleted. For the open state, ligands were initially placed on the basis of their orientation relative to the corresponding binding site. The models were refined against a map derived from one-half of the dataset using Rosetta as described (https://faculty.washington.edu/dimaio/files/density_tutorial.pdf), followed by real space refinement in PHENIX (; PHENIX, Berkeley, CA) and evaluated by calculating the Fourier shell correlation between the model and the map derived from the second half of the dataset. For each complex, ten best scoring instances on the basis of the Fourier shell correlation were selected among one hundred runs, visually examined, and the one deemed best interactively corrected in Coot. Each corrected model was subject to a final instance of real space refinement using PHENIX. + + + RESULTS + title_1 + 14150 + Results and Discussion + + + RESULTS + paragraph + 14173 + To explore the conformational landscape of apo-GDH, we first determined its structure in the absence of any added ligands (Supplemental Fig. 1, Fig. 2, A–C). The density map, refined to an average resolution of ∼3.0 Å (Supplemental Fig. 2), is in the open conformation and closely matches the model of unliganded GDH derived by X-ray crystallography at 3.3 Å resolution (PDB ID 1NR7). The variation in local resolution from the core to the periphery, as reported by ResMap (Supplemental Fig. 3D), is consistent with the B-factor gradient observed in the crystal structure (Supplemental Fig. 3A). Extensive classification without imposing symmetry yielded only open structures and failed to detect any closed catalytic cleft in the unliganded enzyme, suggesting that all six protomers are in the open conformation. Consistent with this conclusion, the loops connecting the β-strands of the Rossmann fold are well-defined (Fig. 2B), implying that there is little movement at the NBD, as the transition between closed and open states is associated with NBD movement (Fig. 1B). + + 0.99936336 + protein_state + cleaner0 + 2023-07-27T15:41:05Z + DUMMY: + + apo + + + 0.99453545 + protein + cleaner0 + 2023-07-27T15:44:42Z + PR: + + GDH + + + 0.981202 + evidence + cleaner0 + 2023-07-27T16:14:51Z + DUMMY: + + structure + + + 0.99872416 + protein_state + cleaner0 + 2023-07-27T16:10:54Z + DUMMY: + + absence of + + + 0.9985163 + evidence + cleaner0 + 2023-07-27T16:14:54Z + DUMMY: + + density map + + + 0.9992907 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99930954 + protein_state + cleaner0 + 2023-07-27T15:45:16Z + DUMMY: + + unliganded + + + 0.8116277 + protein + cleaner0 + 2023-07-27T15:44:50Z + PR: + + GDH + + + 0.9986926 + experimental_method + cleaner0 + 2023-07-27T15:28:24Z + MESH: + + X-ray crystallography + + + 0.9988036 + experimental_method + cleaner0 + 2023-07-27T16:12:09Z + MESH: + + ResMap + + + 0.845573 + evidence + cleaner0 + 2023-07-27T16:15:00Z + DUMMY: + + B-factor gradient + + + 0.9979139 + evidence + cleaner0 + 2023-07-27T16:15:07Z + DUMMY: + + crystal structure + + + 0.9992725 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.9983443 + evidence + cleaner0 + 2023-07-27T16:15:03Z + DUMMY: + + structures + + + 0.9992125 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99904025 + site + cleaner0 + 2023-07-27T15:35:11Z + SO: + + catalytic cleft + + + 0.999288 + protein_state + cleaner0 + 2023-07-27T15:45:16Z + DUMMY: + + unliganded + + + 0.997468 + oligomeric_state + cleaner0 + 2023-07-27T16:07:35Z + DUMMY: + + protomers + + + 0.99926406 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.9990702 + structure_element + cleaner0 + 2023-07-27T15:44:55Z + SO: + + loops + + + 0.9990347 + structure_element + cleaner0 + 2023-07-27T15:44:58Z + SO: + + β-strands + + + 0.99909985 + structure_element + cleaner0 + 2023-07-27T15:35:00Z + SO: + + Rossmann fold + + + 0.99953437 + structure_element + cleaner0 + 2023-07-27T15:34:55Z + SO: + + NBD + + + 0.99922514 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9991897 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99954224 + structure_element + cleaner0 + 2023-07-27T15:34:55Z + SO: + + NBD + + + + mol.116.103382f2.jpg + F2 + FIG + fig_caption + 15254 + Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at ∼3 Å resolution. (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. Each protomer is shown in a different color and densities for NADH bound in both regulatory (red) and catalytic (purple) sites on one protomer are indicated. The overall quaternary structures of the two conformations are essentially the same as that of the open and closed states observed by X-ray crystallography. + + 0.9984913 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + Cryo-EM + + + 0.9980799 + evidence + cleaner0 + 2023-07-27T16:15:13Z + DUMMY: + + structures + + + 0.94350004 + protein + cleaner0 + 2023-07-27T16:15:45Z + PR: + + GDH + + + 0.99929535 + protein_state + cleaner0 + 2023-07-27T15:45:16Z + DUMMY: + + unliganded + + + 0.9991093 + protein_state + cleaner0 + 2023-07-27T15:45:12Z + DUMMY: + + NADH-bound + + + 0.9986468 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + cryo-EM + + + 0.998569 + evidence + cleaner0 + 2023-07-27T16:15:15Z + DUMMY: + + map + + + 0.9992817 + protein_state + cleaner0 + 2023-07-27T15:45:15Z + DUMMY: + + unliganded + + + 0.9956453 + protein + cleaner0 + 2023-07-27T16:15:53Z + PR: + + GDH + + + 0.9983629 + evidence + cleaner0 + 2023-07-27T16:15:17Z + DUMMY: + + density map + + + 0.9992018 + structure_element + cleaner0 + 2023-07-27T15:45:29Z + SO: + + Rossmann nucleotide binding fold + + + 0.9991868 + structure_element + cleaner0 + 2023-07-27T15:45:30Z + SO: + + pivot and antenna helices + + + 0.99929273 + protein_state + cleaner0 + 2023-07-27T15:45:16Z + DUMMY: + + unliganded + + + 0.9963735 + protein + cleaner0 + 2023-07-27T16:16:08Z + PR: + + GDH + + + 0.9970074 + evidence + cleaner0 + 2023-07-27T16:15:29Z + DUMMY: + + map + + + 0.9986481 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + Cryo-EM + + + 0.9985701 + evidence + cleaner0 + 2023-07-27T16:15:19Z + DUMMY: + + density maps + + + 0.99651164 + protein + cleaner0 + 2023-07-27T16:16:01Z + PR: + + GDH + + + 0.99913245 + protein_state + cleaner0 + 2023-07-27T15:45:34Z + DUMMY: + + bound to + + + 0.9992711 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.998767 + oligomeric_state + cleaner0 + 2023-07-27T15:35:55Z + DUMMY: + + protomer + + + 0.9957604 + evidence + cleaner0 + 2023-07-27T16:15:21Z + DUMMY: + + densities + + + 0.99877113 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.88400507 + protein_state + cleaner0 + 2023-07-27T15:45:39Z + DUMMY: + + bound in + + + 0.9983589 + site + cleaner0 + 2023-07-27T16:09:12Z + SO: + + regulatory + + + 0.9984016 + site + cleaner0 + 2023-07-27T16:09:16Z + SO: + + catalytic + + + 0.9940574 + site + cleaner0 + 2023-07-27T16:09:18Z + SO: + + sites + + + 0.99874145 + oligomeric_state + cleaner0 + 2023-07-27T15:35:55Z + DUMMY: + + protomer + + + 0.9992987 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99929917 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99866635 + experimental_method + cleaner0 + 2023-07-27T15:28:24Z + MESH: + + X-ray crystallography + + + + RESULTS + paragraph + 15982 + When GDH is bound to NADH, GTP, and glutamate, the enzyme adopts a closed conformation; this “abortive complex” has been determined to 2.4-Å resolution by X-ray crystallography (PDB 3MW9). However, crystal structures of GDH bound only to NADH or to GTP have not yet been reported. To test the effect of NADH binding on GDH conformation in solution, we determined the structure of this binary complex using cryo-EM methods combined with three-dimensional classification. Two dominant conformational states, in an all open or all closed conformation were detected, segregated (Fig. 2D), and further refined to near-atomic resolution (∼3.3 Å; Supplemental Fig. 2). Densities for 12 molecules of bound NADH were identified in maps of both open and closed states (Supplemental Fig. 4). The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map. + + 0.96874183 + protein + cleaner0 + 2023-07-27T16:16:20Z + PR: + + GDH + + + 0.9990741 + protein_state + cleaner0 + 2023-07-27T15:45:35Z + DUMMY: + + bound to + + + 0.9990263 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.998454 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9938263 + chemical + cleaner0 + 2023-07-27T15:28:57Z + CHEBI: + + glutamate + + + 0.9992925 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99878615 + experimental_method + cleaner0 + 2023-07-27T15:28:24Z + MESH: + + X-ray crystallography + + + 0.9983753 + evidence + cleaner0 + 2023-07-27T16:13:08Z + DUMMY: + + crystal structures + + + 0.9649907 + protein + cleaner0 + 2023-07-27T16:16:27Z + PR: + + GDH + + + 0.9990327 + protein_state + cleaner0 + 2023-07-27T15:45:45Z + DUMMY: + + bound only to + + + 0.9990496 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.71146953 + protein_state + cleaner0 + 2023-07-27T15:45:51Z + DUMMY: + + to + + + 0.9990275 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99858487 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.8397284 + protein + cleaner0 + 2023-07-27T16:16:34Z + PR: + + GDH + + + 0.996267 + evidence + cleaner0 + 2023-07-27T16:16:39Z + DUMMY: + + structure + + + 0.9987752 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + cryo-EM + + + 0.99817914 + experimental_method + cleaner0 + 2023-07-27T16:12:18Z + MESH: + + three-dimensional classification + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:28:42Z + + open + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:28:47Z + + closed + + + 0.9983753 + evidence + cleaner0 + 2023-07-27T16:16:42Z + DUMMY: + + Densities + + + 0.99764234 + protein_state + cleaner0 + 2023-07-27T15:46:04Z + DUMMY: + + bound + + + 0.99921286 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.998577 + evidence + cleaner0 + 2023-07-27T16:16:44Z + DUMMY: + + maps + + + 0.999295 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.9992986 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99908704 + protein_state + cleaner0 + 2023-07-27T15:45:12Z + DUMMY: + + NADH-bound + + + 0.9989404 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9974841 + evidence + cleaner0 + 2023-07-27T16:16:46Z + DUMMY: + + structure + + + 0.9987444 + experimental_method + cleaner0 + 2023-07-27T15:28:24Z + MESH: + + X-ray crystallography + + + 0.9972446 + evidence + cleaner0 + 2023-07-27T16:16:48Z + DUMMY: + + density + + + 0.9980788 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99235004 + chemical + cleaner0 + 2023-07-27T15:28:57Z + CHEBI: + + glutamate + + + 0.99871415 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + cryo-EM + + + 0.99860364 + evidence + cleaner0 + 2023-07-27T16:16:51Z + DUMMY: + + map + + + + RESULTS + paragraph + 16995 + Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by ∼7°, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D). A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. Thus, closure of the catalytic cleft is accompanied by a quaternary structural change that can be described as a global bending of the structure about an axis that runs parallel to the pivot helix, accompanied by an expansion of the core (Figs. 1A and 2D). + + 0.9990828 + protein_state + cleaner0 + 2023-07-27T15:45:12Z + DUMMY: + + NADH-bound + + + 0.9991443 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9990894 + protein_state + cleaner0 + 2023-07-27T15:45:12Z + DUMMY: + + NADH-bound + + + 0.9990368 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99876136 + site + cleaner0 + 2023-07-27T15:35:11Z + SO: + + catalytic cleft + + + 0.9992411 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99957377 + structure_element + cleaner0 + 2023-07-27T15:46:45Z + SO: + + NBDs + + + 0.99929595 + structure_element + cleaner0 + 2023-07-27T15:37:54Z + SO: + + pivot helix + + + structure_element + SO: + cleaner0 + 2023-07-27T15:38:14Z + + antennae + + + 0.999078 + protein_state + cleaner0 + 2023-07-27T15:45:12Z + DUMMY: + + NADH-bound + + + 0.9991357 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.9993374 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9993145 + structure_element + cleaner0 + 2023-07-27T15:46:42Z + SO: + + helix 5 + + + 0.9978593 + residue_range + cleaner0 + 2023-07-27T16:06:18Z + DUMMY: + + 171–186 + + + 0.9982042 + structure_element + cleaner0 + 2023-07-27T15:46:35Z + SO: + + β-sheets + + + 0.99780947 + residue_range + cleaner0 + 2023-07-27T16:06:22Z + DUMMY: + + 57–97 + + + 0.997643 + residue_range + cleaner0 + 2023-07-27T16:06:25Z + DUMMY: + + 122–130 + + + 0.99933445 + structure_element + cleaner0 + 2023-07-27T15:46:40Z + SO: + + α-helix 2 + + + 0.9977677 + residue_range + cleaner0 + 2023-07-27T16:06:28Z + DUMMY: + + 36–54 + + + 0.9845567 + structure_element + cleaner0 + 2023-07-27T15:46:38Z + SO: + + helix + + + 0.99878156 + site + cleaner0 + 2023-07-27T15:35:11Z + SO: + + catalytic cleft + + + 0.9939615 + evidence + cleaner0 + 2023-07-27T16:16:57Z + DUMMY: + + structure + + + 0.9993472 + structure_element + cleaner0 + 2023-07-27T15:37:54Z + SO: + + pivot helix + + + + RESULTS + paragraph + 17908 + Detailed analysis of the GDH/NADH structures shows that both the adenosine and nicotinamide moieties of NADH bind to the catalytic site within the NBD in nearly the same orientation in both the open and the closed states, and display closely comparable interactions with the Rossmann fold (Fig. 3, A and B). At the regulatory site, where either ADP can bind as an activator or NADH can bind as an inhibitor, the binding of the adenine moiety of NADH is nearly identical between the two conformers. However, there is a significant difference in the orientation of the nicotinamide and phosphate moieties in the two conformational states (Fig. 3, C and D). In the closed state, the nicotinamide group is oriented toward the center of the hexamer, inserted into a narrow cavity between two adjacent subunits in the trimer. There are extensive interactions between NADH and the residues lining this cavity, which may explain the well-defined density of this portion of NADH in the closed state. In contrast, in the open conformation, the cavity present in the closed state becomes too narrow for the nicotinamide group; instead, the group is oriented in the opposite direction, parallel to the pivot helix with the amido group extending toward the C-terminal end of the helix. + + 0.9989229 + complex_assembly + cleaner0 + 2023-07-27T15:47:00Z + GO: + + GDH/NADH + + + 0.9978829 + evidence + cleaner0 + 2023-07-27T16:17:02Z + DUMMY: + + structures + + + 0.99905795 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.99909294 + site + cleaner0 + 2023-07-27T16:09:23Z + SO: + + catalytic site + + + 0.99957985 + structure_element + cleaner0 + 2023-07-27T15:34:55Z + SO: + + NBD + + + 0.99925643 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99927026 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9992144 + structure_element + cleaner0 + 2023-07-27T15:35:00Z + SO: + + Rossmann fold + + + 0.99911773 + site + cleaner0 + 2023-07-27T15:38:43Z + SO: + + regulatory site + + + 0.9991086 + chemical + cleaner0 + 2023-07-27T15:38:54Z + CHEBI: + + ADP + + + 0.99898523 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.9990736 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.99927324 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9986626 + oligomeric_state + cleaner0 + 2023-07-27T15:35:44Z + DUMMY: + + hexamer + + + 0.996176 + site + cleaner0 + 2023-07-27T16:09:28Z + SO: + + cavity + + + 0.99576217 + structure_element + cleaner0 + 2023-07-27T16:07:46Z + SO: + + subunits + + + 0.998681 + oligomeric_state + cleaner0 + 2023-07-27T15:35:51Z + DUMMY: + + trimer + + + 0.9987847 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.9952844 + site + cleaner0 + 2023-07-27T16:09:30Z + SO: + + cavity + + + 0.99668294 + evidence + cleaner0 + 2023-07-27T16:17:11Z + DUMMY: + + density + + + 0.99878377 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.99926203 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99923563 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.998295 + site + cleaner0 + 2023-07-27T16:09:32Z + SO: + + cavity + + + 0.9992661 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9983104 + structure_element + cleaner0 + 2023-07-27T15:37:54Z + SO: + + pivot helix + + + 0.9987325 + structure_element + cleaner0 + 2023-07-27T16:08:59Z + SO: + + helix + + + + mol.116.103382f3.jpg + F3 + FIG + fig_caption + 19181 + Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. + + 0.9990859 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.99858665 + site + cleaner0 + 2023-07-27T16:09:37Z + SO: + + catalytic and regulatory sites + + + 0.9988092 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.9816166 + evidence + cleaner0 + 2023-07-27T16:17:15Z + DUMMY: + + density + + + 0.9990692 + site + cleaner0 + 2023-07-27T16:09:41Z + SO: + + catalytic sites + + + 0.9993414 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9993337 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99882585 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.98555994 + evidence + cleaner0 + 2023-07-27T16:17:17Z + DUMMY: + + density + + + 0.9990089 + site + cleaner0 + 2023-07-27T16:09:43Z + SO: + + regulatory sites + + + 0.99933225 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9993401 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + + RESULTS + paragraph + 19457 + Although there is a difference in orientation of the nicotinamide moiety between the closed and open states in the regulatory site, in both structures the adenine portion of NADH has a similar binding pocket and is located in almost exactly the same position as ADP, a potent activator of GDH function (Supplemental Fig. 5). In the open state, the binding of ADP or NADH is further stabilized by His209, a residue that undergoes a large movement during the transition from open to closed conformation (Fig. 3, C and D). In the open conformation, the distance between His209 and the α-phosphate of NADH is ∼4.4 Å, which is comparable with the corresponding distance in the ADP-bound conformation. In the closed conformation, however, this key histidine residue is >10.5 Å away from the nearest phosphate group on NADH, altering a critical stabilization point within the regulatory site. This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity. + + 0.99932384 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.999311 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99911374 + site + cleaner0 + 2023-07-27T15:38:43Z + SO: + + regulatory site + + + 0.9983247 + evidence + cleaner0 + 2023-07-27T16:17:21Z + DUMMY: + + structures + + + 0.99916303 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.99900854 + site + cleaner0 + 2023-07-27T16:09:47Z + SO: + + binding pocket + + + 0.9990884 + chemical + cleaner0 + 2023-07-27T15:38:54Z + CHEBI: + + ADP + + + 0.9989911 + protein + cleaner0 + 2023-07-27T15:58:10Z + PR: + + GDH + + + 0.9992756 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99897265 + chemical + cleaner0 + 2023-07-27T15:38:54Z + CHEBI: + + ADP + + + 0.9990429 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.9995297 + residue_name_number + cleaner0 + 2023-07-27T16:00:21Z + DUMMY: + + His209 + + + 0.99926263 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.999252 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99930274 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.9995384 + residue_name_number + cleaner0 + 2023-07-27T16:00:21Z + DUMMY: + + His209 + + + 0.99889994 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.9991041 + protein_state + cleaner0 + 2023-07-27T15:57:49Z + DUMMY: + + ADP-bound + + + 0.99930465 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9826623 + residue_name + cleaner0 + 2023-07-27T16:06:11Z + SO: + + histidine + + + 0.9988972 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.99906135 + site + cleaner0 + 2023-07-27T15:38:43Z + SO: + + regulatory site + + + 0.9986971 + chemical + cleaner0 + 2023-07-27T15:28:52Z + CHEBI: + + NADH + + + 0.9992798 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.999044 + site + cleaner0 + 2023-07-27T15:38:43Z + SO: + + regulatory site + + + 0.9988551 + chemical + cleaner0 + 2023-07-27T15:38:54Z + CHEBI: + + ADP + + + 0.9987978 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.999305 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99903715 + site + cleaner0 + 2023-07-27T15:38:43Z + SO: + + regulatory site + + + 0.998934 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.99881065 + chemical + cleaner0 + 2023-07-27T15:38:54Z + CHEBI: + + ADP + + + 0.99901974 + protein + cleaner0 + 2023-07-27T15:58:21Z + PR: + + GDH + + + + RESULTS + paragraph + 20664 + In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. The density for GTP is not very well defined, suggesting considerable wobble in the binding site. Subtraction of the GTP-bound map with that of the apo state shows that GTP binding can nevertheless be visualized specifically in the GTP binding site (Supplemental Fig. 6). Importantly, the binding of GTP alone does not appear to drive the transition from the open to the closed state of GDH. + + 0.9991131 + protein_state + cleaner0 + 2023-07-27T16:10:54Z + DUMMY: + + absence of + + + 0.99894017 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.99902225 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99219173 + protein + cleaner0 + 2023-07-27T15:58:55Z + PR: + + GDH + + + 0.9977647 + evidence + cleaner0 + 2023-07-27T16:17:25Z + DUMMY: + + dissociation constant + + + 0.9987583 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + Cryo-EM + + + 0.9932561 + protein + cleaner0 + 2023-07-27T15:59:03Z + PR: + + GDH + + + 0.8448001 + protein_state + cleaner0 + 2023-07-27T16:11:08Z + DUMMY: + + incubated with + + + 0.9988732 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99858713 + evidence + cleaner0 + 2023-07-27T16:17:28Z + DUMMY: + + structure + + + 0.99923015 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99954456 + structure_element + cleaner0 + 2023-07-27T15:46:45Z + SO: + + NBDs + + + 0.99920124 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99864095 + evidence + cleaner0 + 2023-07-27T16:17:30Z + DUMMY: + + density + + + 0.9989548 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9989122 + site + cleaner0 + 2023-07-27T16:09:54Z + SO: + + binding site + + + 0.9963775 + experimental_method + cleaner0 + 2023-07-27T16:12:27Z + MESH: + + Subtraction + + + 0.9990127 + protein_state + cleaner0 + 2023-07-27T15:58:35Z + DUMMY: + + GTP-bound + + + 0.99843186 + evidence + cleaner0 + 2023-07-27T16:17:32Z + DUMMY: + + map + + + 0.9993505 + protein_state + cleaner0 + 2023-07-27T15:41:05Z + DUMMY: + + apo + + + 0.9970625 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99904937 + site + cleaner0 + 2023-07-27T15:38:36Z + SO: + + GTP binding site + + + 0.9990978 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9992861 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99928904 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9957417 + protein + cleaner0 + 2023-07-27T15:59:21Z + PR: + + GDH + + + + RESULTS + paragraph + 21349 + To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. When NADH and GTP are both present, classification reveals the presence of both closed and open GDH conformations, similar to the condition when only NADH is present (Fig. 4, A and B). Reconstruction without classification, however, yields a structure clearly in the closed conformation, suggesting that, in coordination with NADH, GTP may further stabilize the closed conformation. The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate. Likewise, the position of NADH in the open and closed states closely resembles the position of NADH in the GDH/NADH open and closed structures. One key difference between the open and closed states of these structures is the position of the His209 residue: As mentioned above, His209 swings away from the adenine moiety of NADH in the closed state. When GTP is present in the GTP binding site, His209 instead interacts with GTP, probably stabilizing the closed conformation (Fig. 4, C and D). Thus, GTP binding to GDH appears synergistic with NADH and displaces the conformational landscape toward the closed state. + + 0.9990538 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.99909663 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9992446 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99927586 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.8281136 + experimental_method + cleaner0 + 2023-07-27T16:12:31Z + MESH: + + determined + + + 0.99192166 + evidence + cleaner0 + 2023-07-27T16:17:36Z + DUMMY: + + structures + + + 0.98979175 + protein + cleaner0 + 2023-07-27T15:59:30Z + PR: + + GDH + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T15:39:15Z + + in complex with + + + 0.99887913 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.9989507 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9970987 + protein_state + cleaner0 + 2023-07-27T15:59:32Z + DUMMY: + + without + + + 0.6332602 + chemical + cleaner0 + 2023-07-27T15:28:57Z + CHEBI: + + glutamate + + + 0.99893636 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.9988495 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.995776 + experimental_method + cleaner0 + 2023-07-27T16:12:37Z + MESH: + + classification + + + protein_state + DUMMY: + cleaner0 + 2023-07-27T16:11:14Z + + presence of + + + 0.9992207 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99923444 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.8307806 + protein + cleaner0 + 2023-07-27T16:00:04Z + PR: + + GDH + + + 0.99833554 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.9285469 + experimental_method + cleaner0 + 2023-07-27T16:12:40Z + MESH: + + Reconstruction without classification + + + 0.9980426 + evidence + cleaner0 + 2023-07-27T16:17:38Z + DUMMY: + + structure + + + 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GTP + + + 0.95512336 + chemical + cleaner0 + 2023-07-27T15:28:57Z + CHEBI: + + glutamate + + + 0.9990609 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.9992643 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99928856 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9990212 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.99880105 + complex_assembly + cleaner0 + 2023-07-27T15:47:00Z + GO: + + GDH/NADH + + + 0.9992926 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.9993414 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9984408 + evidence + cleaner0 + 2023-07-27T16:17:46Z + DUMMY: + + structures + + + 0.9992549 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.9992506 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99817526 + evidence + cleaner0 + 2023-07-27T16:17:49Z + DUMMY: + + structures + + + 0.999451 + residue_name_number + cleaner0 + 2023-07-27T16:00:21Z + DUMMY: + + His209 + + + 0.99944216 + residue_name_number + cleaner0 + 2023-07-27T16:00:21Z + DUMMY: + + His209 + + + 0.99909997 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.99925214 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9987779 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99891776 + site + cleaner0 + 2023-07-27T15:38:36Z + SO: + + GTP binding site + + + 0.999456 + residue_name_number + cleaner0 + 2023-07-27T16:00:21Z + DUMMY: + + His209 + + + 0.99750525 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9992095 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99874985 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9704605 + protein + cleaner0 + 2023-07-27T16:00:33Z + PR: + + GDH + + + 0.9989549 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.99925166 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + + mol.116.103382f4.jpg + F4 + FIG + fig_caption + 22721 + Cryo-EM structure of GDH bound to both NADH and GTP. (A, B) Observation of co-existing open (A) and closed (B) conformations in the GDH-NADH-GTP ternary complex. Densities for GTP (yellow) as well as NADH bound to both catalytic (purple) and regulatory (red) sites in each protomer are shown. (C, D) Detailed inspection of the interactions near the regulatory site show that the orientation of His209 switches between the two states, which may allow interactions with bound GTP in the closed (D), but not open (C) conformation. + + 0.99880934 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + Cryo-EM + + + 0.9970041 + evidence + cleaner0 + 2023-07-27T16:17:54Z + DUMMY: + + structure + + + 0.66216755 + protein + cleaner0 + 2023-07-27T16:01:08Z + PR: + + GDH + + + 0.9991266 + protein_state + cleaner0 + 2023-07-27T15:45:35Z + DUMMY: + + bound to + + + 0.9992132 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.999258 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9993253 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99934715 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9992854 + complex_assembly + cleaner0 + 2023-07-27T16:00:47Z + GO: + + GDH-NADH-GTP + + + 0.99802315 + evidence + cleaner0 + 2023-07-27T16:17:56Z + DUMMY: + + Densities + + + 0.99926347 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9991916 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.9990061 + protein_state + cleaner0 + 2023-07-27T15:45:35Z + DUMMY: + + bound to + + + 0.99908483 + site + cleaner0 + 2023-07-27T16:10:01Z + SO: + + catalytic + + + 0.99892175 + site + cleaner0 + 2023-07-27T16:10:03Z + SO: + + regulatory + + + 0.99871826 + site + cleaner0 + 2023-07-27T16:10:05Z + SO: + + sites + + + 0.99875784 + oligomeric_state + cleaner0 + 2023-07-27T15:35:55Z + DUMMY: + + protomer + + + 0.9990251 + site + cleaner0 + 2023-07-27T15:38:43Z + SO: + + regulatory site + + + 0.9995421 + residue_name_number + cleaner0 + 2023-07-27T16:00:21Z + DUMMY: + + His209 + + + 0.9986702 + protein_state + cleaner0 + 2023-07-27T16:11:19Z + DUMMY: + + bound + + + 0.9990889 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.99931157 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99916506 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + + RESULTS + paragraph + 23249 + Our structural studies thus establish that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states. Whereas the orientation in which NADH binds at the catalytic site is similar for both conformations, the orientation of the nicotinamide portion of NADH in the regulatory site is different between the open and closed conformations (Figs. 3 and 4). In the closed state, the nicotinamide moiety is inserted into a well-defined cavity at the interface between two adjacent protomers in the trimer. As mentioned above, this cavity is much narrower in the open state, suggesting that this cavity may be unavailable to the NADH nicotinamide moiety when the enzyme is in the open conformation. These structural features provide a potential explanation of the weaker density for the nicotinamide moiety of NADH in the open state, and may account for the higher reported affinity of NADH for the closed state. The role of the nicotinamide moiety in acting as a wedge that prevents the transition to the open conformation also suggests a structural explanation of the mechanism by which NADH binding inhibits the activity of the enzyme by stabilizing the closed conformation state. + + 0.9984971 + experimental_method + cleaner0 + 2023-07-27T16:12:47Z + MESH: + + structural studies + + + 0.9985751 + chemical + cleaner0 + 2023-07-27T15:29:02Z + CHEBI: + + GTP + + + 0.9976909 + protein_state + cleaner0 + 2023-07-27T16:11:23Z + DUMMY: + + bound + + + 0.99857664 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.99882007 + site + cleaner0 + 2023-07-27T16:10:09Z + SO: + + catalytic and regulatory sites + + + 0.9992549 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.9992772 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.99857247 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.99903965 + site + cleaner0 + 2023-07-27T16:10:13Z + SO: + + catalytic site + + + 0.99883884 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.9990834 + site + cleaner0 + 2023-07-27T15:38:43Z + SO: + + regulatory site + + + 0.9992575 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.9992855 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9992781 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.96565443 + site + cleaner0 + 2023-07-27T16:10:17Z + SO: + + cavity + + + 0.9969772 + site + cleaner0 + 2023-07-27T16:10:20Z + SO: + + interface + + + 0.9985948 + oligomeric_state + cleaner0 + 2023-07-27T16:07:50Z + DUMMY: + + protomers + + + 0.9988117 + oligomeric_state + cleaner0 + 2023-07-27T15:35:51Z + DUMMY: + + trimer + + + 0.99401975 + site + cleaner0 + 2023-07-27T16:10:23Z + SO: + + cavity + + + 0.9992507 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.975312 + site + cleaner0 + 2023-07-27T16:10:25Z + SO: + + cavity + + + 0.9984908 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.99924904 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.9779223 + evidence + cleaner0 + 2023-07-27T16:18:00Z + DUMMY: + + density + + + 0.9989441 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.99927527 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.998892 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.9992582 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + 0.9988876 + protein_state + cleaner0 + 2023-07-27T15:28:42Z + DUMMY: + + open + + + 0.99809366 + chemical + cleaner0 + 2023-07-27T15:28:53Z + CHEBI: + + NADH + + + 0.9991322 + protein_state + cleaner0 + 2023-07-27T15:28:47Z + DUMMY: + + closed + + + + RESULTS + paragraph + 24506 + The rapid emergence of cryo-EM as a tool for near-atomic resolution structure determination provides new opportunities for complementing atomic resolution information from X-ray crystallography, as illustrated here with GDH. Perhaps the most important contribution of these methods is the prospect that when there are discrete subpopulations present, the structure of each state can be determined at near-atomic resolution. What we demonstrate here with GDH is that by employing three-dimensional image classification approaches, we not only can isolate distinct, coexisting conformations, but we can also localize small molecule ligands in each of these conformations. These kinds of approaches will probably become increasingly important in molecular pharmacology, especially in the context of better understanding drug-target interactions in dynamic protein complexes. + + 0.99890405 + experimental_method + cleaner0 + 2023-07-27T15:28:14Z + MESH: + + cryo-EM + + + 0.83094627 + experimental_method + cleaner0 + 2023-07-27T16:12:51Z + MESH: + + structure determination + + + 0.998904 + experimental_method + cleaner0 + 2023-07-27T15:28:25Z + MESH: + + X-ray crystallography + + + 0.79175967 + protein + cleaner0 + 2023-07-27T16:01:19Z + PR: + + GDH + + + 0.9978859 + evidence + cleaner0 + 2023-07-27T16:18:02Z + DUMMY: + + structure + + + 0.9639614 + protein + cleaner0 + 2023-07-27T16:01:27Z + PR: + + GDH + + + 0.98971945 + experimental_method + cleaner0 + 2023-07-27T16:12:55Z + MESH: + + three-dimensional image classification approaches + + + + SUPPL + title_1 + 25378 + Supplementary Material + + + SUPPL + footnote + 25401 + This research was supported by funds from the National Cancer Institute Center for Cancer Research, the IATAP program at NIH, and the NIH-FEI Living Laboratory for Structural Biology (S.S., J.L.S.M.). This work was supported by the Intramural Research Program of the National Institutes of Health National Cancer Institute. + + + SUPPL + footnote + 25726 + dx.doi.org/10.1124/mol.116.103382. + + + SUPPL + footnote + 25761 + This article has supplemental material available at molpharm.aspetjournals.org. + + + ABBR + title + 25841 + Abbreviations + + + ABBR + paragraph + 25855 + cryo-EM + + + ABBR + paragraph + 25863 + cryo-electron microscopy + + + ABBR + paragraph + 25888 + GDH + + + ABBR + paragraph + 25892 + glutamate dehydrogenase + + + ABBR + paragraph + 25916 + NBD + + + ABBR + paragraph + 25920 + nucleotide binding domain + + + AUTH_CONT + title + 25946 + Authorship Contributions + + + AUTH_CONT + paragraph + 25971 + Participated in research design: Borgnia, Banerjee, Merk, Subramaniam, Milne. + + + AUTH_CONT + paragraph + 26049 + Conducted experiments: Borgnia, Banerjee, Merk, Rao, Pierson. + + + AUTH_CONT + paragraph + 26111 + Performed data analysis: Borgnia, Banerjee, Merk, Matthies, Bartesaghi, Earl, Falconieri, Subramaniam, Milne. + + + AUTH_CONT + paragraph + 26221 + Wrote or contributed to the writing of the manuscript: Borgnia, Banerjee, Earl, Falconieri, Subramaniam, Milne. + + + REF + title + 26333 + References + + + 213 + 221 + surname:Adams;given-names:PD + surname:Afonine;given-names:PV + surname:Bunkóczi;given-names:G + surname:Chen;given-names:VB + surname:Davis;given-names:IW + surname:Echols;given-names:N + surname:Headd;given-names:JJ + surname:Hung;given-names:LW + surname:Kapral;given-names:GJ + surname:Grosse-Kunstleve;given-names:RW + 20124702 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 26344 + PHENIX: a comprehensive Python-based system for macromolecular structure solution + + + 14431 + 14443 + surname:Allen;given-names:A + surname:Kwagh;given-names:J + surname:Fang;given-names:J + surname:Stanley;given-names:CA + surname:Smith;given-names:TJ + 15533048 + REF + Biochemistry + ref + 43 + 2004 + 26426 + Evolution of glutamate dehydrogenase regulation of insulin homeostasis is an example of molecular exaptation + + + 5579 + 5583 + surname:Bailey;given-names:J + surname:Bell;given-names:ET + surname:Bell;given-names:JE + 7068608 + REF + J Biol Chem + ref + 257 + 1982 + 26535 + Regulation of bovine glutamate dehydrogenase. The effects of pH and ADP + + + 3446 + 3456 + surname:Banerjee;given-names:S + surname:Schmidt;given-names:T + surname:Fang;given-names:J + surname:Stanley;given-names:CA + surname:Smith;given-names:TJ + 12653548 + REF + Biochemistry + ref + 42 + 2003 + 26607 + Structural studies on ADP activation of mammalian glutamate dehydrogenase and the evolution of regulation + + + 11709 + 11714 + surname:Bartesaghi;given-names:A + surname:Matthies;given-names:D + surname:Banerjee;given-names:S + surname:Merk;given-names:A + surname:Subramaniam;given-names:S + 25071206 + REF + Proc Natl Acad Sci USA + ref + 111 + 2014 + 26713 + Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy + + + 1167 + 1173 + surname:Couée;given-names:I + surname:Tipton;given-names:KF + 2322301 + REF + Biochem Pharmacol + ref + 39 + 1990 + 26802 + Inhibition of ox brain glutamate dehydrogenase by perphenazine + + + 217 + 226 + surname:Dieter;given-names:H + surname:Koberstein;given-names:R + surname:Sund;given-names:H + 7227367 + REF + Eur J Biochem + ref + 115 + 1981 + 26865 + Studies of glutamate dehydrogenase. The interaction of ADP, GTP, and NADPH in complexes with glutamate dehydrogenase + + + 486 + 501 + surname:Emsley;given-names:P + surname:Lohkamp;given-names:B + surname:Scott;given-names:WG + surname:Cowtan;given-names:K + 20383002 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 26982 + Features and development of Coot + + + 3286 + 3299 + surname:Frieden;given-names:C + 14085375 + REF + J Biol Chem + ref + 238 + 1963 + 27015 + Glutamate Dehydrogenase. V. The relation of enzyme structure to the catalytic function + + + 2028 + 2035 + surname:Frieden;given-names:C + 14299621 + REF + J Biol Chem + ref + 240 + 1965 + 27102 + Glutamate Dehydrogenase. VI. Survey of purine nucleotide and other effects on the enzyme from various sources + + + 6057 + 6061 + surname:George;given-names:A + surname:Bell;given-names:JE + 7470450 + REF + Biochemistry + ref + 19 + 1980 + 27212 + Effects of adenosine 5′-diphosphate on bovine glutamate dehydrogenase: diethyl pyrocarbonate modification + + + 767 + 792 + surname:Hudson;given-names:RC + surname:Daniel;given-names:RM + 8299344 + REF + Comp Biochem Physiol B + ref + 106 + 1993 + 27320 + L-glutamate dehydrogenases: distribution, properties and mechanism + + + 545 + 552 + surname:Koberstein;given-names:R + surname:Sund;given-names:H + 4147202 + REF + Eur J Biochem + ref + 36 + 1973 + 27387 + Studies of glutamate dehydrogenase. The influence of ADP, GTP, and L-glutamate on the binding of the reduced coenzyme to beef-liver glutamate dehydrogenase + + + 63 + 65 + surname:Kucukelbir;given-names:A + surname:Sigworth;given-names:FJ + surname:Tagare;given-names:HD + 24213166 + REF + Nat Methods + ref + 11 + 2014 + 27543 + Quantifying the local resolution of cryo-EM density maps + + + 34164 + 34174 + surname:Li;given-names:C + surname:Li;given-names:M + surname:Chen;given-names:P + surname:Narayan;given-names:S + surname:Matschinsky;given-names:FM + surname:Bennett;given-names:MJ + surname:Stanley;given-names:CA + surname:Smith;given-names:TJ + 21813650 + REF + J Biol Chem + ref + 286 + 2011 + 27600 + Green tea polyphenols control dysregulated glutamate dehydrogenase in transgenic mice by hijacking the ADP activation site + + + 69 + 80 + surname:Li;given-names:M + surname:Li;given-names:C + surname:Allen;given-names:A + surname:Stanley;given-names:CA + surname:Smith;given-names:TJ + 22079166 + REF + Arch Biochem Biophys + ref + 519 + 2012 + 27723 + The structure and allosteric regulation of mammalian glutamate dehydrogenase + + + 1605 + 1612 + surname:Pettersen;given-names:EF + surname:Goddard;given-names:TD + surname:Huang;given-names:CC + surname:Couch;given-names:GS + surname:Greenblatt;given-names:DM + surname:Meng;given-names:EC + surname:Ferrin;given-names:TE + 15264254 + REF + J Comput Chem + ref + 25 + 2004 + 27800 + UCSF Chimera--a visualization system for exploratory research and analysis + + + 2321 + 2328 + surname:Rife;given-names:JE + surname:Cleland;given-names:WW + 7190024 + REF + Biochemistry + ref + 19 + 1980 + 27875 + Kinetic mechanism of glutamate dehydrogenase + + + 519 + 530 + surname:Scheres;given-names:SH + 23000701 + REF + J Struct Biol + ref + 180 + 2012 + 27920 + RELION: implementation of a Bayesian approach to cryo-EM structure determination + + + 166 + 171 + surname:Shafer;given-names:JA + surname:Chiancone;given-names:E + surname:Vittorelli;given-names:LM + surname:Spagnuolo;given-names:C + surname:Mackler;given-names:B + surname:Antonini;given-names:E + 4344911 + REF + Eur J Biochem + ref + 31 + 1972 + 28001 + Binding of reduced cofactor to glutamate dehydrogenase + + + 707 + 720 + surname:Smith;given-names:TJ + surname:Peterson;given-names:PE + surname:Schmidt;given-names:T + surname:Fang;given-names:J + surname:Stanley;given-names:CA + 11254391 + REF + J Mol Biol + ref + 307 + 2001 + 28056 + Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation + + + 1352 + 1357 + surname:Stanley;given-names:CA + surname:Lieu;given-names:YK + surname:Hsu;given-names:BY + surname:Burlina;given-names:AB + surname:Greenberg;given-names:CR + surname:Hopwood;given-names:NJ + surname:Perlman;given-names:K + surname:Rich;given-names:BH + surname:Zammarchi;given-names:E + surname:Poncz;given-names:M + 9571255 + REF + N Engl J Med + ref + 338 + 1998 + 28156 + Hyperinsulinism and hyperammonemia in infants with regulatory mutations of the glutamate dehydrogenase gene + + + 38 + 46 + surname:Tang;given-names:G + surname:Peng;given-names:L + surname:Baldwin;given-names:PR + surname:Mann;given-names:DS + surname:Jiang;given-names:W + surname:Rees;given-names:I + surname:Ludtke;given-names:SJ + 16859925 + REF + J Struct Biol + ref + 157 + 2007 + 28264 + EMAN2: an extensible image processing suite for electron microscopy + + + 1704 + 1708 + surname:Tomkins;given-names:GM + surname:Yielding;given-names:KL + surname:Curran;given-names:JF + 13921784 + REF + J Biol Chem + ref + 237 + 1962 + 28332 + The influence of diethylstilbestrol and adenosine diphosphate on pyridine nucleotide coenzyme binding by glutamic dehydrogenase + + + 983 + 989 + surname:Yielding;given-names:KL + surname:Tomkins;given-names:GM + 13787322 + REF + Proc Natl Acad Sci USA + ref + 47 + 1961 + 28460 + An effect of L-leucine and other essential amino acids on the structure and activity of glutamic dehydrogenase + + + diff --git a/BioC_XML/4887163_v0.xml b/BioC_XML/4887163_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..621cc502d84862c3541edc55e3de9914c399fa0a --- /dev/null +++ b/BioC_XML/4887163_v0.xml @@ -0,0 +1,9285 @@ + + + + PMC + 20201223 + pmc.key + + 4887163 + CC BY + no + 0 + 0 + + 10.1172/JCI85679 + 4887163 + 27183389 + 85679 + 2191 + 6 + This paper is licensed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + + 2204 + surname:Cole;given-names:David K. + surname:Bulek;given-names:Anna M. + surname:Rossjohn;given-names:Jamie + surname:Zhu;given-names:Cheng + surname:Miles;given-names:John J. + surname:Peakman;given-names:Mark + surname:Wooldridge;given-names:Linda + surname:Rizkallah;given-names:Pierre J. + surname:Sewell;given-names:Andrew K. + surname:Dolton;given-names:Garry + surname:Schauenberg;given-names:Andrea J. + surname:Szomolay;given-names:Barbara + surname:Rittase;given-names:William + surname:Trimby;given-names:Andrew + surname:Jothikumar;given-names:Prithiviraj + surname:Fuller;given-names:Anna + surname:Skowera;given-names:Ania + TITLE + front + 126 + 2016 + 0 + Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity + + 0.9898953 + protein_type + cleaner0 + 2023-07-19T20:52:37Z + MESH: + + T cell receptor + + + 0.9988882 + chemical + cleaner0 + 2023-07-20T07:57:25Z + CHEBI: + + insulin + + + + ABSTRACT + abstract + 99 + The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease. + + 0.9838819 + complex_assembly + cleaner0 + 2023-07-19T20:54:37Z + GO: + + T cell antigen receptor + + + 0.9382497 + complex_assembly + cleaner0 + 2023-07-19T20:53:35Z + GO: + + TCR + + + 0.9977353 + complex_assembly + cleaner0 + 2023-07-20T07:56:15Z + GO: + + peptide–major histocompatibility complexes + + + 0.99710053 + complex_assembly + cleaner0 + 2023-07-19T16:24:26Z + GO: + + pMHC + + + 0.9991755 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + protein + PR: + cleaner0 + 2023-07-19T20:09:34Z + + preproinsulin + + + complex_assembly + GO: + cleaner0 + 2023-07-20T07:49:27Z + + MHC + + + 0.99922645 + evidence + cleaner0 + 2023-07-19T20:44:44Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:50Z + + 1E6 TCR + + + 0.999249 + protein_state + cleaner0 + 2023-07-19T16:13:16Z + DUMMY: + + bound to + + + chemical + CHEBI: + cleaner0 + 2023-07-19T20:10:47Z + + altered peptide ligands + + + 0.9994267 + evidence + cleaner0 + 2023-07-19T20:44:48Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.80075455 + evidence + cleaner0 + 2023-07-19T20:44:51Z + DUMMY: + + antipathogen-like binding affinity + + + + INTRO + title_1 + 1445 + Introduction + + + INTRO + paragraph + 1458 + T cells perform an essential role in adaptive immunity by interrogating the host proteome for anomalies, classically by recognizing peptides bound in major histocompatibility (MHC) molecules at the cell surface. Recent data supports the notion that, to perform this role, the highly variable αβ T cell antigen receptor (TCR) must be able to recognize thousands, if not millions, of different peptide ligands. This ability is required to enable the estimated 25 million distinct TCRs expressed in humans to provide effective immune coverage against all possible foreign peptide antigens. Although essential to avoid blind spots during pathogen recognition, T cell cross-reactivity has also been implicated as a pathway to autoimmunity, possibly mediated by highly reactive pathogen-specific T cells weakly recognizing self-ligands. + + 0.9515432 + complex_assembly + cleaner0 + 2023-07-20T07:49:14Z + GO: + + major histocompatibility + + + 0.9665209 + complex_assembly + cleaner0 + 2023-07-20T07:49:26Z + GO: + + MHC + + + 0.99938995 + protein_state + cleaner0 + 2023-07-20T07:59:48Z + DUMMY: + + highly variable + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:55:10Z + + αβ T cell antigen receptor + + + 0.9842329 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.5031044 + complex_assembly + cleaner0 + 2023-07-19T20:55:23Z + GO: + + TCRs + + + 0.99924266 + species + cleaner0 + 2023-07-19T20:43:39Z + MESH: + + humans + + + + INTRO + paragraph + 2296 + Several mechanisms, by which TCRs could bind to a large number of different peptide-MHC (pMHC), have been proposed. Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations. Both MHC and peptide have also been shown to undergo structural changes upon TCR binding, mediating an induced fit between the TCR and pMHC. Other studies, mainly in the murine system, have demonstrated that the same TCR can interact with different pMHCs using a common or divergent modality. Recent studies in model murine systems demonstrate that TCR cross-reactivity can be governed by recognition of a conserved region in the peptide that allows tolerance of peptide sequence variation outside of this hotspot. + + 0.9945287 + complex_assembly + cleaner0 + 2023-07-19T20:55:24Z + GO: + + TCRs + + + complex_assembly + GO: + cleaner0 + 2023-07-19T16:24:59Z + + peptide-MHC + + + 0.59893274 + complex_assembly + cleaner0 + 2023-07-19T16:24:26Z + GO: + + pMHC + + + 0.9992618 + evidence + cleaner0 + 2023-07-19T20:44:54Z + DUMMY: + + Structures + + + 0.99964464 + protein_state + cleaner0 + 2023-07-19T16:17:41Z + DUMMY: + + unligated + + + 0.9996339 + protein_state + cleaner0 + 2023-07-19T20:05:05Z + DUMMY: + + ligated + + + 0.966741 + complex_assembly + cleaner0 + 2023-07-19T20:55:24Z + GO: + + TCRs + + + 0.99959594 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.9739079 + structure_element + cleaner0 + 2023-07-19T16:28:16Z + SO: + + complementarity determining region + + + 0.999788 + structure_element + cleaner0 + 2023-07-19T16:28:27Z + SO: + + CDR + + + 0.99899834 + structure_element + cleaner0 + 2023-07-19T16:28:21Z + SO: + + loops + + + 0.9993062 + structure_element + cleaner0 + 2023-07-19T16:28:24Z + SO: + + loop + + + 0.40489408 + complex_assembly + cleaner0 + 2023-07-20T07:49:27Z + GO: + + MHC + + + 0.5937887 + chemical + cleaner0 + 2023-07-20T07:57:32Z + CHEBI: + + peptide + + + 0.80366987 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.9463298 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T16:24:26Z + + pMHC + + + 0.99782526 + taxonomy_domain + cleaner0 + 2023-07-19T20:08:40Z + DUMMY: + + murine + + + 0.9772652 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.45551145 + complex_assembly + cleaner0 + 2023-07-19T16:28:10Z + GO: + + pMHCs + + + 0.99696213 + taxonomy_domain + cleaner0 + 2023-07-19T20:08:40Z + DUMMY: + + murine + + + 0.9929351 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + + INTRO + paragraph + 3125 + We recently reported that the 1E6 human CD8+ T cell clone — which mediates the destruction of β cells through the recognition of a major, HLA-A*0201–restricted, preproinsulin signal peptide (ALWGPDPAAA15–24) — can recognize upwards of 1 million different peptides. CD8+ T cells that recognize HLA-A*0201–ALWGPDPAAA have been shown to populate insulitic lesions in patients with type 1 diabetes (T1D). We demonstrated that the TCR from the 1E6 T cell clone bound to HLA-A*0201–ALWGPDPAAA using a limited footprint and very weak binding affinity. This first experimental evidence of a high level of CD8+ T cell cross-reactivity in a human autoimmune disease system hinted toward molecular mimicry by a more potent pathogenic peptide as a potential mechanism leading to β cell destruction. Here, we solved the structure of the 1E6 TCR with 7 altered peptide ligands (APLs) determined by our previously published combinatorial peptide library (CPL) screening, 2 of which mapped within human pathogens. These APLs differed from the natural preproinsulin peptide by up to 7 of 10 residues. We also solved the structure of each unligated APL to investigate whether structural changes occurred before or after binding — which, combined with an in-depth cellular and biophysical analysis of the 1E6 interaction with each APL, demonstrated the molecular mechanism mediating the high level of cross-reactivity exhibited by this preproinsulin-reactive human CD8+ T cell clone. + + 0.9989231 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + protein + PR: + cleaner0 + 2023-07-19T16:27:26Z + + HLA-A*0201 + + + 0.75353974 + protein + cleaner0 + 2023-07-19T20:09:34Z + PR: + + preproinsulin + + + 0.8835323 + structure_element + cleaner0 + 2023-07-20T08:09:34Z + SO: + + signal peptide + + + 0.5604127 + chemical + cleaner0 + 2023-07-19T20:09:47Z + CHEBI: + + ALWGPDPAAA15–24 + + + 0.98721063 + complex_assembly + cleaner0 + 2023-07-19T16:27:47Z + GO: + + HLA-A*0201–ALWGPDPAAA + + + 0.662854 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.99923784 + protein_state + cleaner0 + 2023-07-19T16:13:16Z + DUMMY: + + bound to + + + 0.946986 + complex_assembly + cleaner0 + 2023-07-19T16:27:48Z + GO: + + HLA-A*0201–ALWGPDPAAA + + + 0.9991387 + evidence + cleaner0 + 2023-07-19T16:20:13Z + DUMMY: + + binding affinity + + + 0.99896324 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + 0.99941254 + experimental_method + cleaner0 + 2023-07-20T08:05:04Z + MESH: + + solved + + + 0.9995402 + evidence + cleaner0 + 2023-07-19T20:44:58Z + DUMMY: + + structure + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:29:05Z + + altered peptide ligands + + + 0.9808608 + chemical + cleaner0 + 2023-07-19T16:29:09Z + CHEBI: + + APLs + + + experimental_method + MESH: + cleaner0 + 2023-07-20T08:05:31Z + + combinatorial peptide library (CPL) screening + + + 0.9985892 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + 0.8167679 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.9198488 + protein + cleaner0 + 2023-07-19T20:09:34Z + PR: + + preproinsulin + + + 0.9994512 + experimental_method + cleaner0 + 2023-07-20T08:05:40Z + MESH: + + solved + + + 0.999616 + evidence + cleaner0 + 2023-07-19T20:45:01Z + DUMMY: + + structure + + + 0.99964905 + protein_state + cleaner0 + 2023-07-19T16:17:41Z + DUMMY: + + unligated + + + 0.9893568 + chemical + cleaner0 + 2023-07-19T16:29:16Z + CHEBI: + + APL + + + 0.9985127 + experimental_method + cleaner0 + 2023-07-20T08:05:43Z + MESH: + + cellular and biophysical analysis + + + 0.97832096 + chemical + cleaner0 + 2023-07-19T16:29:15Z + CHEBI: + + APL + + + protein + PR: + cleaner0 + 2023-07-19T20:09:34Z + + preproinsulin + + + 0.9987363 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + + RESULTS + title_1 + 4606 + Results + + + RESULTS + title_2 + 4614 + The 1E6 T cell clone recognizes APLs across a large dynamic range. + + 0.9728898 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + + RESULTS + paragraph + 4681 + We have previously demonstrated that the 1E6 T cell clone can recognize over 1 million different peptides with a potency comparable with, or better than, the cognate preproinsulin peptide ALWGPDPAAA. From this large functional scan, we selected 7 different APLs that activated the 1E6 T cell clone across a wide (4-log) functional range (Table 1). Two of these peptides, MVWGPDPLYV and RQFGPDWIVA (bold text signifies amino acids that are different from the index preproinsulin–derived sequence), are contained within the proteomes of the human pathogens Bacteroides fragilis/thetaiotaomicron and Clostridium asparagiforme, respectively. Competitive functional testing revealed that the preproinsulin-derived sequence ALWGPDPAAA was one of the least potent targets for 1E6, with only the MVWGPDPLYV and YLGGPDFPTI demonstrating a similar low-activity profile in MIP-1β secretion and target killing assays (Figure 1, A and B). The RQFGPDWIVA sequence (present in C. asparagiforme) activated the 1E6 T cell with around 1 log–greater potency compared with ALWGPDPAAA. At the other end of the spectrum, the RQFGPDFPTI peptide stimulated MIP-1β release and killing by 1E6 at an exogenous peptide concentration 2–3 logs lower compared with ALWGPDPAAA. The pattern of peptide potency was closely mirrored by pMHC tetramer staining experiments (Figure 1C and plots shown in Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI85679DS1). Here, the A2-RQFGPDFPTI tetramer stained 1E6 with the greatest MFI, gradually decreasing to the weakest tetramers: A2-MVWGPDPLYV and -YLGGPDFPTI. To parallel the functional analysis, we also performed thermal melt (Tm) experiments using synchrotron radiation circular dichroism (SRCD) to investigate the stability of each APL (Figure 1D). The range of Tm was between 49.4°C (RQFGPDWIVA) and 60.3°C (YQFGPDFPIA), with an average approximately 55°C, similar to our previous findings. This pattern of stability did not correlate with the T cell activation or tetramer staining experiments, indicating that peptide binding to the MHC do not explain ligand potency. + + 0.9016905 + protein + cleaner0 + 2023-07-19T20:09:35Z + PR: + + preproinsulin + + + 0.7047767 + chemical + cleaner0 + 2023-07-19T16:08:19Z + CHEBI: + + ALWGPDPAAA + + + 0.9961451 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.98260164 + chemical + cleaner0 + 2023-07-19T16:08:35Z + CHEBI: + + MVWGPDPLYV + + + 0.96969604 + chemical + cleaner0 + 2023-07-19T16:08:55Z + CHEBI: + + RQFGPDWIVA + + + 0.8399371 + protein + cleaner0 + 2023-07-19T20:09:35Z + PR: + + preproinsulin + + + 0.9994413 + species + cleaner0 + 2023-07-19T16:09:39Z + MESH: + + human + + + 0.9981843 + species + cleaner0 + 2023-07-19T16:09:27Z + MESH: + + Bacteroides fragilis/thetaiotaomicron + + + 0.9994335 + species + cleaner0 + 2023-07-19T16:09:22Z + MESH: + + Clostridium asparagiforme + + + 0.999571 + experimental_method + cleaner0 + 2023-07-20T08:06:01Z + MESH: + + Competitive functional testing + + + 0.55834925 + protein + cleaner0 + 2023-07-19T20:09:35Z + PR: + + preproinsulin + + + 0.8603277 + chemical + cleaner0 + 2023-07-19T16:08:19Z + CHEBI: + + ALWGPDPAAA + + + 0.9908454 + chemical + cleaner0 + 2023-07-19T16:08:36Z + CHEBI: + + MVWGPDPLYV + + + 0.89793074 + chemical + cleaner0 + 2023-07-19T16:09:09Z + CHEBI: + + YLGGPDFPTI + + + protein + PR: + cleaner0 + 2023-07-19T20:12:00Z + + MIP-1β + + + 0.9913579 + chemical + cleaner0 + 2023-07-19T16:08:56Z + CHEBI: + + RQFGPDWIVA + + + 0.99943805 + species + cleaner0 + 2023-07-19T16:09:33Z + MESH: + + C. asparagiforme + + + 0.89824975 + chemical + cleaner0 + 2023-07-19T16:08:19Z + CHEBI: + + ALWGPDPAAA + + + 0.9736738 + chemical + cleaner0 + 2023-07-19T16:30:12Z + CHEBI: + + RQFGPDFPTI + + + protein + PR: + cleaner0 + 2023-07-19T20:12:01Z + + MIP-1β + + + 0.811304 + chemical + cleaner0 + 2023-07-19T16:08:19Z + CHEBI: + + ALWGPDPAAA + + + 0.7686303 + complex_assembly + cleaner0 + 2023-07-19T16:24:26Z + GO: + + pMHC + + + 0.97690034 + experimental_method + cleaner0 + 2023-07-20T08:06:08Z + MESH: + + tetramer staining + + + 0.834167 + chemical + cleaner0 + 2023-07-19T16:12:27Z + CHEBI: + + A2-RQFGPDFPTI + + + 0.7076429 + oligomeric_state + cleaner0 + 2023-07-20T07:58:17Z + DUMMY: + + tetramer + + + 0.9878656 + oligomeric_state + cleaner0 + 2023-07-20T07:58:19Z + DUMMY: + + tetramers + + + 0.9034901 + chemical + cleaner0 + 2023-07-19T16:12:10Z + CHEBI: + + A2-MVWGPDPLYV + + + 0.5810209 + chemical + cleaner0 + 2023-07-19T16:09:10Z + CHEBI: + + YLGGPDFPTI + + + 0.999585 + experimental_method + cleaner0 + 2023-07-20T08:06:17Z + MESH: + + thermal melt + + + 0.9986412 + evidence + cleaner0 + 2023-07-19T16:29:57Z + DUMMY: + + Tm + + + 0.99956584 + experimental_method + cleaner0 + 2023-07-20T08:06:25Z + MESH: + + synchrotron radiation circular dichroism + + + 0.9995995 + experimental_method + cleaner0 + 2023-07-20T08:06:28Z + MESH: + + SRCD + + + 0.99415684 + chemical + cleaner0 + 2023-07-19T16:29:16Z + CHEBI: + + APL + + + 0.99952316 + evidence + cleaner0 + 2023-07-19T16:29:58Z + DUMMY: + + Tm + + + 0.7603012 + chemical + cleaner0 + 2023-07-19T16:08:56Z + CHEBI: + + RQFGPDWIVA + + + 0.9138942 + chemical + cleaner0 + 2023-07-19T20:12:21Z + CHEBI: + + YQFGPDFPIA + + + 0.9968275 + experimental_method + cleaner0 + 2023-07-20T08:06:31Z + MESH: + + tetramer staining + + + 0.9852401 + complex_assembly + cleaner0 + 2023-07-20T07:49:27Z + GO: + + MHC + + + + RESULTS + title_2 + 6826 + The 1E6 TCR can bind peptides with strong antipathogen-like affinities. + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + + RESULTS + paragraph + 6898 + We, and others, have previously demonstrated that antipathogenic TCRs tend to bind with stronger affinity compared with self-reactive TCRs, likely a consequence of the deletion of T cells with high-affinity self-reactive TCR during thymic selection. In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell–derived TCR with a biologically relevant ligand (KD > 200 μM; KD, equilibrium binding constant). Surface plasmon resonance (SPR) analysis of the 1E6 TCR–pMHC interaction for all 7 APLs (Figure 2, A–H) demonstrated that stronger binding affinity (represented as ΔG°, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson’s correlation analysis value of 0.8 (P = 0.01) (Figure 2I). It should be noted that this correlation, although consistent with the T cell killing experiments, uses only approximate affinities calculated for the 2 weakest ligands. These experiments revealed 4 important findings. First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 μM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 μM, equivalent to the binding affinity of the very strongest antipathogen TCRs. Third, the 1E6 TCR bound to A2-RQFGPDWIVA peptide, within the C. asparagiforme proteome, with approximately 4-fold stronger affinity than A2-ALWGPDPAAA, demonstrating the potential for a pathogen-derived antigen to initiate a response to the self-derived sequence. Finally, these data demonstrate the largest range of binding affinities reported for a natural, endogenous human TCR of more than 3 logs of magnitude (A2-MVWGPDPLYV vs. A2-RQFGPDFPTI). To confirm the affinity spread detected by SPR, and to evaluate whether experiments performed using soluble molecules were biologically relevant to events at the T cell surface, we determined the effective 2D affinity of each APL using an adhesion frequency assay in which a human rbc coated in pMHC acted as an adhesion sensor. In agreement with SPR experiments, the range of 2D affinities we detected differed by around 3 logs, with the A2-MVWGPDPLYV generating the weakest 2D affinity (2.6 × 10–5 AcKa μm4) and A2-RQFGPDFPTI the strongest (4.5 × 10–2 AcKa μm4) (Figure 2J). As with the 3D affinity measurements, the 2D affinity measurements correlated well with the EC50 values for each ligand (Figure 2K) demonstrating a strong correlation (Pearson’s correlation = 0.8, P = 0.01) between T cell antigen sensitivity and TCR binding affinity. Of note, these data demonstrate a close agreement between the 3D affinity values generated using SPR and 2D affinity values generated using adhesion frequency assays. + + 0.9443769 + complex_assembly + cleaner0 + 2023-07-19T20:55:24Z + GO: + + TCRs + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + 0.9800795 + complex_assembly + cleaner0 + 2023-07-19T20:55:24Z + GO: + + TCRs + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + 0.9109045 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.9852579 + protein_state + cleaner0 + 2023-07-19T20:56:07Z + DUMMY: + + bound + + + 0.54183936 + protein + cleaner0 + 2023-07-19T20:09:35Z + PR: + + preproinsulin + + + 0.9975931 + chemical + cleaner0 + 2023-07-19T16:08:19Z + CHEBI: + + ALWGPDPAAA + + + 0.87047696 + evidence + cleaner0 + 2023-07-19T16:19:49Z + DUMMY: + + affinity + + + 0.99915814 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + 0.99290437 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.9996025 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.99964154 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.9994817 + evidence + cleaner0 + 2023-07-19T18:02:09Z + DUMMY: + + equilibrium binding constant + + + 0.99956703 + experimental_method + cleaner0 + 2023-07-19T18:02:05Z + MESH: + + Surface plasmon resonance + + + 0.9996811 + experimental_method + cleaner0 + 2023-07-19T16:32:30Z + MESH: + + SPR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T16:24:26Z + + pMHC + + + 0.9805093 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.999523 + evidence + cleaner0 + 2023-07-19T16:20:13Z + DUMMY: + + binding affinity + + + 0.99963284 + evidence + cleaner0 + 2023-07-19T20:15:38Z + DUMMY: + + ΔG° + + + 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2023-07-19T16:20:13Z + DUMMY: + + binding affinity + + + 0.98072755 + complex_assembly + cleaner0 + 2023-07-19T20:55:24Z + GO: + + TCRs + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.9973413 + protein_state + cleaner0 + 2023-07-19T16:13:16Z + DUMMY: + + bound to + + + 0.9959906 + chemical + cleaner0 + 2023-07-19T16:12:47Z + CHEBI: + + A2-RQFGPDWIVA + + + 0.99946743 + species + cleaner0 + 2023-07-19T16:09:34Z + MESH: + + C. asparagiforme + + + 0.9960381 + evidence + cleaner0 + 2023-07-19T16:19:49Z + DUMMY: + + affinity + + + 0.9956104 + chemical + cleaner0 + 2023-07-19T16:16:49Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.9995508 + evidence + cleaner0 + 2023-07-19T20:45:17Z + DUMMY: + + binding affinities + + + 0.70571 + protein_state + cleaner0 + 2023-07-20T08:00:03Z + DUMMY: + + endogenous + + + 0.9993166 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + 0.9945815 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 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A2-MVWGPDPLYV + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:32:22Z + + 2D affinity + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:51:27Z + + AcKa + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:12:27Z + + A2-RQFGPDFPTI + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:51:36Z + + AcKa + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:33:07Z + + 3D affinity + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:32:22Z + + 2D affinity + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:51:31Z + + EC50 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:14:28Z + + Pearson’s correlation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:51:21Z + + P + + + evidence + DUMMY: + cleaner0 + 2023-07-19T18:06:46Z + + TCR binding affinity + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:33:03Z + + 3D affinity + + + experimental_method + MESH: + cleaner0 + 2023-07-19T16:32:30Z + + SPR + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:32:22Z + + 2D affinity + + + + RESULTS + title_2 + 9844 + The 1E6 TCR uses a consensus binding mode to engage multiple APLs. + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.51438355 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + + RESULTS + paragraph + 9911 + Our previous structure of the 1E6-A2-ALWGPDPAAA complex demonstrated a limited binding footprint between the TCR and pMHC. The low number of contacts between the 2 molecules most likely contributed to the weak binding affinity of the interaction. In order to examine the mechanism by which the 1E6 TCR engaged a wide range of peptides with divergent binding affinities, we solved the structure of the 1E6 TCR in complex with all 7 APLs used in Figure 2. All structures were solved in space group P1 to 2–3 Å resolution with crystallographic Rwork/Rfree ratios within accepted limits as shown in the theoretically expected distribution (ref. and Supplemental Table 1). The 1E6 TCR used a very similar overall binding modality to engage all of the APLs, with root mean square deviation ranging between 0.81 and 1.12 Å2 (compared with 1E6-A2-ALWGPDPAAA). The relatively broad range of buried surface areas (1,670–1,920 Å2) did not correlate well with TCR binding affinity (Pearson’s correlation = 0.45, P = 0.2). The surface complementarity values (0.52–0.7) correlated slightly with affinity (Pearson’s correlation = 0.7, P = 0.05) but could not explain all differences in binding (Figure 3A and Table 2). The TCR CDR loops were in a very similar position in all complexes, apart from some slight deviations in the TCR β-chain (Figure 3B); the peptides were all presented in a similar conformation (Figure 3C); and there was minimal variation in crossing angles of the TCR (42.3°–45.6°) (Figure 3D). Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. However, subtle differences in the respective interfaces were apparent (discussed below) and resulted in altered binding affinities of the respective complexes. + + 0.9994141 + evidence + cleaner0 + 2023-07-19T20:13:22Z + DUMMY: + + structure + + + 0.9996247 + complex_assembly + cleaner0 + 2023-07-19T16:14:42Z + GO: + + 1E6-A2-ALWGPDPAAA + + + 0.8440795 + site + cleaner0 + 2023-07-20T08:12:19Z + SO: + + binding footprint + + + 0.66571075 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.86722314 + complex_assembly + cleaner0 + 2023-07-19T16:24:26Z + GO: + + pMHC + + + 0.9874364 + evidence + cleaner0 + 2023-07-19T16:20:13Z + DUMMY: + + binding affinity + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.5872233 + evidence + cleaner0 + 2023-07-19T20:45:24Z + DUMMY: + + binding affinities + + + 0.99880445 + experimental_method + cleaner0 + 2023-07-20T08:06:37Z + MESH: + + solved + + + 0.99947244 + evidence + cleaner0 + 2023-07-19T20:13:19Z + DUMMY: + + structure + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.99926734 + protein_state + cleaner0 + 2023-07-19T18:02:49Z + DUMMY: + + in complex with + + + 0.74618053 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.9814917 + evidence + cleaner0 + 2023-07-19T20:13:14Z + DUMMY: + + structures + + + 0.7286678 + experimental_method + cleaner0 + 2023-07-20T08:06:42Z + MESH: + + solved + + + 0.99763364 + evidence + cleaner0 + 2023-07-19T20:13:11Z + DUMMY: + + Rwork/Rfree ratios + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.5221809 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.9993834 + evidence + cleaner0 + 2023-07-19T20:13:17Z + DUMMY: + + root mean square deviation + + + 0.99953204 + complex_assembly + cleaner0 + 2023-07-19T16:14:43Z + GO: + + 1E6-A2-ALWGPDPAAA + + + 0.87155503 + evidence + cleaner0 + 2023-07-19T17:58:44Z + DUMMY: + + TCR binding affinity + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:14:23Z + + Pearson’s correlation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:51:59Z + + P + + + 0.99492294 + evidence + cleaner0 + 2023-07-19T20:13:26Z + DUMMY: + + surface complementarity values + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:14:28Z + + Pearson’s correlation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:52:03Z + + P + + + 0.5172308 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.9997113 + structure_element + cleaner0 + 2023-07-19T17:58:59Z + SO: + + CDR loops + + + 0.5146105 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.99949646 + structure_element + cleaner0 + 2023-07-19T17:59:47Z + SO: + + β-chain + + + 0.92718446 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.5723033 + chemical + cleaner0 + 2023-07-19T16:29:16Z + CHEBI: + + APL + + + 0.72113127 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.99952 + structure_element + cleaner0 + 2023-07-19T17:59:52Z + SO: + + α-chain + + + complex_assembly + GO: + cleaner0 + 2023-07-20T07:49:55Z + + MHC class I + + + 0.9365372 + complex_assembly + cleaner0 + 2023-07-20T07:50:06Z + GO: + + MHCI + + + 0.99968386 + structure_element + cleaner0 + 2023-07-20T08:09:40Z + SO: + + α2-helix + + + 0.5055066 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.99942774 + structure_element + cleaner0 + 2023-07-19T17:59:48Z + SO: + + β-chain + + + 0.5436469 + complex_assembly + cleaner0 + 2023-07-20T07:50:07Z + GO: + + MHCI + + + 0.9996726 + structure_element + cleaner0 + 2023-07-20T08:09:44Z + SO: + + α-1 helix + + + 0.99937385 + site + cleaner0 + 2023-07-20T08:12:23Z + SO: + + interfaces + + + 0.9975358 + evidence + cleaner0 + 2023-07-19T20:45:30Z + DUMMY: + + binding affinities + + + + RESULTS + title_2 + 11806 + Interactions between the 1E6 TCR and different APLs are focused around a conserved GPD peptide motif. + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.9785179 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.99952507 + protein_state + cleaner0 + 2023-07-20T08:00:12Z + DUMMY: + + conserved + + + structure_element + SO: + cleaner0 + 2023-07-19T18:04:37Z + + GPD peptide motif + + + + RESULTS + paragraph + 11908 + We next performed an in-depth atomic analysis of the contacts between the 1E6 TCR and each APL to determine the structural basis for the altered T cell peptide sensitivities and TCR binding affinities (Table 2). Concomitant with our global analysis of 1E6 TCR binding to the APLs, we observed a common interaction element, consistent with our previous findings, that utilized TCR residues Tyr97α and Trp97β, forming an aromatic cap over a central GPD motif that was present in all of the APLs (Figure 4). Interactions between these 2 TCR and 3 peptide residues accounted for 41%–50% of the total contacts across all complexes (Table 2), demonstrating the conserved peptide centric binding mode utilized by the 1E6 TCR. This fixed anchoring between the 2 molecules was important for stabilization of the TCR-pMHC complex, as — although other peptides without the ‘GDP’ motif were tested and shown to activate the 1E6 T cell clone — we were unable to measure robust affinities using SPR (data not shown). These data support the requirement for a conserved interaction between the 1E6 TCR and the GPD motif, as we observed in our previously published 1E6-A2-ALWGPDPAAA structure. + + 0.94664764 + experimental_method + cleaner0 + 2023-07-20T08:06:47Z + MESH: + + atomic analysis + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.7138178 + chemical + cleaner0 + 2023-07-19T16:29:16Z + CHEBI: + + APL + + + 0.9993618 + evidence + cleaner0 + 2023-07-19T18:03:25Z + DUMMY: + + TCR binding affinities + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.7065273 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.9956772 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.99990237 + residue_name_number + cleaner0 + 2023-07-19T18:00:25Z + DUMMY: + + Tyr97α + + + 0.9999002 + residue_name_number + cleaner0 + 2023-07-19T18:00:43Z + DUMMY: + + Trp97β + + + 0.8847289 + structure_element + cleaner0 + 2023-07-20T08:09:51Z + SO: + + aromatic cap + + + 0.9996864 + structure_element + cleaner0 + 2023-07-19T18:05:06Z + SO: + + GPD motif + + + 0.49906126 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.99199945 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.9910685 + protein_state + cleaner0 + 2023-07-19T20:52:22Z + DUMMY: + + conserved + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.99948066 + complex_assembly + cleaner0 + 2023-07-19T18:04:07Z + GO: + + TCR-pMHC + + + structure_element + SO: + cleaner0 + 2023-07-19T18:05:00Z + + ‘GDP’ motif + + + 0.99936527 + evidence + cleaner0 + 2023-07-19T20:13:53Z + DUMMY: + + affinities + + + 0.99967253 + experimental_method + cleaner0 + 2023-07-19T16:32:30Z + MESH: + + SPR + + + 0.9992118 + protein_state + cleaner0 + 2023-07-19T20:52:25Z + DUMMY: + + conserved + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.99967885 + structure_element + cleaner0 + 2023-07-19T18:05:06Z + SO: + + GPD motif + + + 0.9997021 + complex_assembly + cleaner0 + 2023-07-19T16:14:43Z + GO: + + 1E6-A2-ALWGPDPAAA + + + 0.9996151 + evidence + cleaner0 + 2023-07-19T20:13:48Z + DUMMY: + + structure + + + + RESULTS + title_2 + 13097 + Focused hotspot binding around a conserved GPD motif enables the 1E6 TCR to tolerate peptide degeneracy. + + 0.99951005 + protein_state + cleaner0 + 2023-07-20T08:00:19Z + DUMMY: + + conserved + + + 0.9997299 + structure_element + cleaner0 + 2023-07-19T18:05:05Z + SO: + + GPD motif + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + + RESULTS + paragraph + 13202 + Although the 1E6 TCR formed a similar overall interaction with each APL, the stabilization between the TCR and the GPD motif enabled fine differences in the contact network with both the peptide and MHC surface that allowed discrimination between each ligand (Figure 5). For example, the 1E6 TCR made only 47 peptide contacts with A2-MVWGPDPLYV (KD = ~600 μM) compared with 63 and 57 contacts with A2-YQFGPDFPIA (KD = 7.4 μM) and A2-RQFGPDFPTI (KD = 0.5 μM), respectively. Although the number of peptide contacts was a good predictor of TCR binding affinity for some of the APLs, for others, the correlation was poor (Pearson’s correlation = 0.045, P = 0.92), possibly because of different resolutions for each complex structure. For example, the 1E6 TCR made 64 peptide contacts with A2-YLGGPDFPTI (KD = ~400 μM) compared with 43 contacts with A2-RQWGPDPAAV (KD = 7.8 μM). + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.9926323 + chemical + cleaner0 + 2023-07-19T16:29:16Z + CHEBI: + + APL + + + 0.9984389 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.99968 + structure_element + cleaner0 + 2023-07-19T18:05:06Z + SO: + + GPD motif + + + 0.99865997 + site + cleaner0 + 2023-07-20T08:12:28Z + SO: + + contact network + + + 0.9031253 + chemical + cleaner0 + 2023-07-19T20:14:20Z + CHEBI: + + peptide + + + 0.9292942 + site + cleaner0 + 2023-07-20T07:50:45Z + SO: + + MHC surface + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.9980581 + chemical + cleaner0 + 2023-07-19T16:12:10Z + CHEBI: + + A2-MVWGPDPLYV + + + 0.9408846 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.998652 + chemical + cleaner0 + 2023-07-19T16:15:51Z + CHEBI: + + A2-YQFGPDFPIA + + + 0.97073865 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.9988263 + chemical + cleaner0 + 2023-07-19T16:12:27Z + CHEBI: + + A2-RQFGPDFPTI + + + 0.83079475 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.99772674 + evidence + cleaner0 + 2023-07-19T18:06:16Z + DUMMY: + + TCR binding affinity + + + 0.99602747 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.9775787 + evidence + cleaner0 + 2023-07-19T16:14:28Z + DUMMY: + + Pearson’s correlation + + + 0.9996146 + evidence + cleaner0 + 2023-07-19T20:14:33Z + DUMMY: + + structure + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.9981677 + chemical + cleaner0 + 2023-07-19T16:16:10Z + CHEBI: + + A2-YLGGPDFPTI + + + 0.9152873 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.9987187 + chemical + cleaner0 + 2023-07-19T16:16:23Z + CHEBI: + + A2-RQWGPDPAAV + + + 0.9516478 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + + RESULTS + paragraph + 14083 + The most important peptide modification in terms of generating new contacts was peptide position 1. The stronger ligands all encoded larger side chains (Arg or Tyr) at peptide position 1 (Figure 5, E–H), enabling interactions with 1E6 that were not present in the weaker APLs that lacked large side chains in this position (Figure 5, A, C, and D). We have previously shown that the 1E6 TCR uses a rigid lock-and-key mechanism during binding to A2-ALWGPDPAAA. These data demonstrated that the unligated structure of the 1E6 TCR was virtually identical to its ligated counterparts. In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (ΔG) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2). The unligated A2-MVWGPDPLYV (KD = ~600 μM) structure revealed that the side chain Tyr9 swung around 8 Å in the complex structure, subsequently making contacts with TCR residues Asp30β and Asn51β (Figure 6A and Figure 5A, respectively). This movement could result in an entropic penalty contributing to the weak TCR binding affinity we observed for this ligand. Additional small movements in the Cα backbone of the peptide around peptide residue Asp6 were apparent in the A2-YLGGPDFPTI (KD = ~400 μM), A2-ALWGPDPAAA (KD = ~208 μM), and A2-RQFGPDWIVA (KD = 44.4 μM) structures (Figure 6, B, C, and E). The unligated structures of A2-AQWGPDAAA, A2-RQWGPDPAAV, A2-YQFGPDFPIA, and A2-RQFGPDFPTI were virtually identical when in complex with 1E6 (Figure 6, D and F–H). Apart from the case of A2-AQWGPDAAA (KD = 61.9 μM), these observations support the conclusion that the higher-affinity ligands required less conformational melding during binding, which could be energetically beneficial (lower entopic cost) during ligation with the 1E6 TCR. + + 0.98920166 + residue_number + cleaner0 + 2023-07-20T08:04:04Z + DUMMY: + + 1 + + + 0.99930096 + residue_name + cleaner0 + 2023-07-19T20:03:56Z + SO: + + Arg + + + 0.99929297 + residue_name + cleaner0 + 2023-07-19T20:03:59Z + SO: + + Tyr + + + 0.98596257 + residue_number + cleaner0 + 2023-07-20T08:04:08Z + DUMMY: + + 1 + + + 0.529526 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.9851528 + chemical + cleaner0 + 2023-07-19T16:16:49Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.9996426 + protein_state + cleaner0 + 2023-07-19T16:17:41Z + DUMMY: + + unligated + + + 0.99905044 + evidence + cleaner0 + 2023-07-19T20:14:36Z + DUMMY: + + structure + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.99959403 + protein_state + cleaner0 + 2023-07-19T20:05:05Z + DUMMY: + + ligated + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:29:10Z + + APLs + + + 0.99935204 + evidence + cleaner0 + 2023-07-19T20:14:40Z + DUMMY: + + free binding energy + + + 0.9987571 + evidence + cleaner0 + 2023-07-19T20:14:44Z + DUMMY: + + ΔG + + + 0.99803597 + experimental_method + cleaner0 + 2023-07-20T07:50:57Z + MESH: + + solved + + + 0.99954385 + protein_state + cleaner0 + 2023-07-19T16:17:41Z + DUMMY: + + unligated + + + 0.99930704 + evidence + cleaner0 + 2023-07-19T20:14:38Z + DUMMY: + + structures + + + 0.6730447 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.9470319 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.99931955 + evidence + cleaner0 + 2023-07-19T20:45:35Z + DUMMY: + + structure + + + 0.99965835 + protein_state + cleaner0 + 2023-07-19T16:17:40Z + DUMMY: + + unligated + + + 0.9856176 + chemical + cleaner0 + 2023-07-19T16:12:10Z + CHEBI: + + A2-MVWGPDPLYV + + + 0.99568236 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.99956495 + evidence + cleaner0 + 2023-07-19T20:14:51Z + DUMMY: + + structure + + + 0.9999052 + residue_name_number + cleaner0 + 2023-07-19T20:04:10Z + DUMMY: + + Tyr9 + + + 0.9995851 + evidence + cleaner0 + 2023-07-19T20:14:54Z + DUMMY: + + structure + + + 0.87050486 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.9999012 + residue_name_number + cleaner0 + 2023-07-19T20:04:14Z + DUMMY: + + Asp30β + + + 0.9999014 + residue_name_number + cleaner0 + 2023-07-19T20:04:16Z + DUMMY: + + Asn51β + + + 0.99930316 + evidence + cleaner0 + 2023-07-19T18:06:45Z + DUMMY: + + TCR binding affinity + + + 0.99990344 + residue_name_number + cleaner0 + 2023-07-19T20:04:19Z + DUMMY: + + Asp6 + + + 0.98224705 + chemical + cleaner0 + 2023-07-19T16:16:10Z + CHEBI: + + A2-YLGGPDFPTI + + + 0.98664665 + evidence + cleaner0 + 2023-07-19T16:17:16Z + DUMMY: + + KD + + + 0.97232896 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.9939036 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.9855407 + chemical + cleaner0 + 2023-07-19T16:12:47Z + CHEBI: + + A2-RQFGPDWIVA + + + 0.9931004 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.99954164 + evidence + cleaner0 + 2023-07-19T20:14:57Z + DUMMY: + + structures + + + 0.999648 + protein_state + cleaner0 + 2023-07-19T16:17:41Z + DUMMY: + + unligated + + + 0.99961734 + evidence + cleaner0 + 2023-07-19T20:15:00Z + DUMMY: + + structures + + + 0.98120284 + chemical + cleaner0 + 2023-07-19T16:17:31Z + CHEBI: + + A2-AQWGPDAAA + + + 0.96967775 + chemical + cleaner0 + 2023-07-19T16:16:24Z + CHEBI: + + A2-RQWGPDPAAV + + + 0.9569805 + chemical + cleaner0 + 2023-07-19T16:15:52Z + CHEBI: + + A2-YQFGPDFPIA + + + 0.9750207 + chemical + cleaner0 + 2023-07-19T16:12:27Z + CHEBI: + + A2-RQFGPDFPTI + + + 0.9942916 + protein_state + cleaner0 + 2023-07-19T18:02:49Z + DUMMY: + + in complex with + + + 0.9857505 + chemical + cleaner0 + 2023-07-19T16:17:32Z + CHEBI: + + A2-AQWGPDAAA + + + 0.9792589 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + + RESULTS + title_2 + 16093 + Peptide modifications alter the interaction between the 1E6 TCR and the MHC surface. + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.98852634 + site + cleaner0 + 2023-07-20T07:51:05Z + SO: + + MHC surface + + + + RESULTS + paragraph + 16178 + In addition to changes between the TCR and peptide component, we also observed that different APLs had different knock-on effects between the TCR and MHC. MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). Generally, the weaker-affinity APLs made fewer contacts with the MHC surface (27–29 interactions) compared with the stronger-affinity APLs (29–35 contacts), consistent with a better Pearson’s correlation value (0.55) compared with TCR-peptide interactions versus affinity (0.045). For instance, contacts were made between TCR residue Val53β and MHC residue Gln72 in all APLs except for in the weakest affinity ligand pair, 1E6-A2-MVWGPDPLYV, in which a subtle change in TCR conformation — probably mediated by different peptide contacts — abrogated this interaction (Figure 7A). + + 0.9057284 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:29:10Z + + APLs + + + 0.98210955 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.9670497 + complex_assembly + cleaner0 + 2023-07-20T07:49:27Z + GO: + + MHC + + + 0.9803191 + complex_assembly + cleaner0 + 2023-07-20T07:49:27Z + GO: + + MHC + + + 0.99990535 + residue_name_number + cleaner0 + 2023-07-19T18:07:15Z + DUMMY: + + Arg65 + + + 0.9910882 + site + cleaner0 + 2023-07-20T07:51:19Z + SO: + + MHC restriction triad + + + 0.9999044 + residue_name_number + cleaner0 + 2023-07-19T18:07:15Z + DUMMY: + + Arg65 + + + 0.9999025 + residue_name_number + cleaner0 + 2023-07-19T18:07:21Z + DUMMY: + + Ala69 + + + 0.9999012 + residue_name_number + cleaner0 + 2023-07-19T18:07:25Z + DUMMY: + + Gln155 + + + 0.6897632 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-20T07:49:27Z + + MHC + + + 0.9999025 + residue_name_number + cleaner0 + 2023-07-19T18:07:26Z + DUMMY: + + Gln155 + + + 0.9999045 + residue_name_number + cleaner0 + 2023-07-19T18:07:21Z + DUMMY: + + Ala69 + + + 0.99446267 + site + cleaner0 + 2023-07-20T08:12:34Z + SO: + + interface + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + 0.9721638 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.98300236 + complex_assembly + cleaner0 + 2023-07-20T07:49:27Z + GO: + + MHC + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + 0.9680678 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.9995654 + evidence + cleaner0 + 2023-07-19T20:45:41Z + DUMMY: + + Pearson’s correlation value + + + 0.40218425 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.9993118 + evidence + cleaner0 + 2023-07-19T16:19:49Z + DUMMY: + + affinity + + + 0.9918806 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + 0.9999056 + residue_name_number + cleaner0 + 2023-07-19T18:10:06Z + DUMMY: + + Val53β + + + 0.9934963 + complex_assembly + cleaner0 + 2023-07-20T07:49:27Z + GO: + + MHC + + + 0.99990606 + residue_name_number + cleaner0 + 2023-07-19T18:10:12Z + DUMMY: + + Gln72 + + + 0.9320513 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + 0.9955964 + complex_assembly + cleaner0 + 2023-07-19T16:18:16Z + GO: + + 1E6-A2-MVWGPDPLYV + + + 0.9805279 + complex_assembly + cleaner0 + 2023-07-19T20:53:36Z + GO: + + TCR + + + + RESULTS + title_2 + 17164 + An energetic switch from unfavorable to favorable entropy (order-to-disorder) correlates with antigen potency. + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:43Z + + entropy + + + + RESULTS + paragraph + 17275 + Our analysis of the contact network provided some clues that could explain the different antigen potencies and binding affinities between the 1E6 TCR and the different APLs. However, there were clear outliers in which the number of contacts did not match with the strength/potency of the interaction. For example, the 1E6 TCR bound to A2-RQWGPDPAAV with the third strongest affinity (KD = 7.8 μM) but made fewer contacts than with A2-ALWGPDPAAA (KD = ~208 μM) (Table 2). However, it is not necessarily the quantity of contacts that determines the strength of an interaction, but the quality of the contacts. Thus, we performed an in-depth thermodynamic analysis of 6 of the ligands under investigation (Figure 8 and Supplemental Table 3). The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (ΔH°) and entropic (TΔS°) changes that contributed to the different binding affinities/potencies for each APL. The overall free binding energies (ΔG°) were between –4.4 and –8.6 kcal/mol, reflecting the wide range of TCR binding affinities we observed for the different APLs. The enthalpic contribution in each complex did not follow a clear trend with affinity, with all but the 1E6-A2-RQFGPDFPTI interaction (ΔH° = 6.3 kcal/mol) generating an energetically favorable enthalpy value (ΔH° = –3.7 to –11.4 kcal/mol); this indicated a net gain in electrostatic interactions during complex formation. However, there was a clear switch in entropy between the weaker-affinity and stronger-affinity ligands, indicated by a strong Pearson’s correlation value between entropy and affinity (Pearson’s correlation value 0.93, P =0.007). For instance, the A2-ALWGPDPAAA, A2-AQWGPDAAA, and A2-RQFGPDWIVA (KD = ~208 μM, KD = 61.9 μM, and KD = 44.4 μM, respectively) were all entropically unfavorable (TΔS° = –2.9 to –5.6 kcal/mol), indicating a net change from disorder to order. Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 μM), A2-YQFGPDFPIA (KD = 7.4 μM), and A2-RQFGPDFPTI (KD = 0.5 μM) exhibited favorable entropy (TΔS° = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules. Furthermore, the structures of the unligated pMHCs demonstrated that, for these stronger-affinity ligands, there was less conformational difference between the TCR ligated pMHCs compared with the weaker-affinity ligands (Figure 6). The potential requirement for a larger degree of induced fit during binding to these weaker-affinity ligands is consistent with the larger entropic penalties observed for these interactions. + + 0.99799347 + site + cleaner0 + 2023-07-20T07:51:25Z + SO: + + contact network + + + 0.9993336 + evidence + cleaner0 + 2023-07-19T20:45:58Z + DUMMY: + + binding affinities + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:29:10Z + + APLs + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.9994259 + protein_state + cleaner0 + 2023-07-19T16:13:16Z + DUMMY: + + bound to + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:16:24Z + + A2-RQWGPDPAAV + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + 0.9945314 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.67255753 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.99652267 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evidence + cleaner0 + 2023-07-19T16:19:58Z + DUMMY: + + ΔG° + + + 0.9992965 + evidence + cleaner0 + 2023-07-19T18:10:40Z + DUMMY: + + TCR binding affinities + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:29:10Z + + APLs + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + 0.99789983 + complex_assembly + cleaner0 + 2023-07-19T16:19:12Z + GO: + + 1E6-A2-RQFGPDFPTI + + + 0.99929667 + evidence + cleaner0 + 2023-07-19T20:46:15Z + DUMMY: + + ΔH° + + + 0.9959241 + evidence + cleaner0 + 2023-07-19T16:19:38Z + DUMMY: + + enthalpy + + + 0.9993475 + evidence + cleaner0 + 2023-07-19T16:19:34Z + DUMMY: + + ΔH° + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:43Z + + entropy + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + 0.9310463 + evidence + cleaner0 + 2023-07-19T20:46:20Z + DUMMY: + + Pearson’s correlation value + + + 0.99341667 + evidence + cleaner0 + 2023-07-19T16:19:43Z + DUMMY: + + entropy + + + 0.9612975 + evidence + cleaner0 + 2023-07-19T16:19:48Z + DUMMY: + + affinity + + + 0.94222593 + evidence + cleaner0 + 2023-07-19T20:46:24Z + DUMMY: + + Pearson’s correlation value + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:46:32Z + + P + + + 0.96640253 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.8315219 + chemical + cleaner0 + 2023-07-19T16:17:32Z + CHEBI: + + A2-AQWGPDAAA + + + 0.9568971 + chemical + cleaner0 + 2023-07-19T16:12:47Z + CHEBI: + + A2-RQFGPDWIVA + + + 0.9870682 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.968549 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.94423616 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.9992269 + evidence + cleaner0 + 2023-07-19T20:46:40Z + DUMMY: + + TΔS° + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:16:24Z + + A2-RQWGPDPAAV + + + 0.85928726 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:15:52Z + + A2-YQFGPDFPIA + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:17:17Z + + KD + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:12:27Z + + A2-RQFGPDFPTI + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:17:17Z + + KD + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:43Z + + entropy + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:16:15Z + + TΔS° + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:17:41Z + + unligated + + + complex_assembly + GO: + cleaner0 + 2023-07-19T16:28:11Z + + pMHCs + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:53:37Z + + TCR + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T20:05:05Z + + ligated + + + complex_assembly + GO: + cleaner0 + 2023-07-19T16:28:11Z + + pMHCs + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + + RESULTS + title_2 + 20013 + Potential epitopes for 1E6 TCR occur commonly in the viral proteome. + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.99776137 + taxonomy_domain + cleaner0 + 2023-07-19T20:08:47Z + DUMMY: + + viral + + + + RESULTS + paragraph + 20082 + We searched a database of over 1,924,572 unique decamer peptides from the proteome of viral pathogens that are known, or strongly suspected, to infect humans. Three hundred forty-two of these decamers conformed to the motif xxxGPDxxxx. Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4). Thus, there are many pathogen-encoded peptides that could act as agonists for the 1E6 T cell beyond the MVWGPDPLYV and RQFGPDWIVA sequences studied here. Extension of these analyses to include the larger genomes of bacterial pathogens would be expected to considerably increase these numbers. The binding affinity of the 1E6 TCR interaction with A2-RQFGPDWIVA is considerably higher than with the disease-implicated A2-ALWGPDPAAA sequence (KD = 44.4 μM and KD > 200 μM, respectively), highlighting how a pathogen-derived sequence might be capable of priming a 1E6-like T cell. + + 0.73867536 + chemical + cleaner0 + 2023-07-20T07:57:42Z + CHEBI: + + peptides + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-19T20:08:49Z + + viral + + + 0.9992586 + species + cleaner0 + 2023-07-19T20:43:40Z + MESH: + + humans + + + 0.9991959 + structure_element + cleaner0 + 2023-07-19T18:11:28Z + SO: + + xxxGPDxxxx + + + 0.99927825 + structure_element + cleaner0 + 2023-07-19T18:11:34Z + SO: + + xOxGPDxxxO + + + 0.99843377 + residue_name + cleaner0 + 2023-07-19T18:11:44Z + SO: + + A + + + 0.9987311 + residue_name + cleaner0 + 2023-07-19T18:11:47Z + SO: + + V + + + 0.99887043 + residue_name + cleaner0 + 2023-07-19T18:11:50Z + SO: + + I + + + 0.9988965 + residue_name + cleaner0 + 2023-07-19T18:11:53Z + SO: + + L + + + 0.998949 + residue_name + cleaner0 + 2023-07-19T18:11:56Z + SO: + + M + + + 0.999042 + residue_name + cleaner0 + 2023-07-19T18:11:58Z + SO: + + Y + + + 0.9989599 + residue_name + cleaner0 + 2023-07-19T18:12:01Z + SO: + + F + + + 0.99902046 + residue_name + cleaner0 + 2023-07-19T18:12:04Z + SO: + + W + + + 0.9695014 + protein + cleaner0 + 2023-07-19T20:43:55Z + PR: + + HLA-A*0201 + + + 0.6446219 + chemical + cleaner0 + 2023-07-19T16:08:36Z + CHEBI: + + MVWGPDPLYV + + + 0.58860224 + chemical + cleaner0 + 2023-07-19T16:08:56Z + CHEBI: + + RQFGPDWIVA + + + 0.99951077 + taxonomy_domain + cleaner0 + 2023-07-19T20:08:55Z + DUMMY: + + bacterial + + + 0.99958223 + evidence + cleaner0 + 2023-07-19T16:20:13Z + DUMMY: + + binding affinity + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.981877 + chemical + cleaner0 + 2023-07-19T16:12:47Z + CHEBI: + + A2-RQFGPDWIVA + + + 0.98185354 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.99964726 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.9996582 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + + DISCUSS + title_1 + 21094 + Discussion + + + DISCUSS + paragraph + 21105 + T cell antigen discrimination is governed by an interaction between the clonally expressed TCR and pMHC, mediated by the chemical characteristics of the interacting molecules. It has recently become clear that TCR cross-reactivity with large numbers of different pMHC ligands is essential to plug holes in T cell immune coverage that pathogens could exploit. Flexibility at the interface between the TCR and pMHC, demonstrated in various studies, has been suggested as a mechanism mediating T cell cross-reactivity with multiple distinct epitopes. This notion is attractive because the CDR loops, which form the TCR antigen-binding site, are usually the most flexible part of the TCR and have the ability to mold around differently shaped ligands. Focused binding around a minimal peptide motif has also been implicated as an alternative mechanism enabling TCR cross-reactivity. Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues. + + 0.9691974 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + 0.93291223 + complex_assembly + cleaner0 + 2023-07-19T16:24:26Z + GO: + + pMHC + + + 0.9701488 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + 0.6247094 + complex_assembly + cleaner0 + 2023-07-19T16:24:26Z + GO: + + pMHC + + + 0.99802315 + site + cleaner0 + 2023-07-20T08:13:53Z + SO: + + interface + + + 0.45847034 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + 0.6944931 + complex_assembly + cleaner0 + 2023-07-19T16:24:26Z + GO: + + pMHC + + + 0.9996326 + structure_element + cleaner0 + 2023-07-19T17:58:59Z + SO: + + CDR loops + + + 0.9995467 + site + cleaner0 + 2023-07-19T18:12:20Z + SO: + + TCR antigen-binding site + + + 0.98521197 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + 0.9578315 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + 0.8868667 + protein_state + cleaner0 + 2023-07-20T08:00:59Z + DUMMY: + + alloreactive + + + 0.9976369 + taxonomy_domain + cleaner0 + 2023-07-19T20:08:39Z + DUMMY: + + murine + + + 0.8430462 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-20T07:49:27Z + + MHC + + + 0.71081364 + protein + cleaner0 + 2023-07-19T20:21:31Z + PR: + + 42F3 + + + 0.7886316 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + protein + PR: + cleaner0 + 2023-07-19T20:18:16Z + + H2-Ld + + + 0.9117572 + protein_state + cleaner0 + 2023-07-20T08:01:10Z + DUMMY: + + conserved + + + 0.996566 + site + cleaner0 + 2023-07-20T08:13:57Z + SO: + + hotspot + + + + DISCUSS + paragraph + 22253 + Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85–99 bound to HLA-DQ1). This motif was conserved in at least 2 potential foreign peptides, originating from Herpes simplex virus and Pseudomonas aeruginosa, enabling TCR recognition of foreign epitopes. Although these data provided some clues into the molecular mechanism of T cell recognition, there still remain several gaps in our understanding. First, we currently know nothing about how human MHCI–restricted TCRs mediate cross-reactivity in the context of a clinically relevant model of autoimmunity, thought to be a major pathway of disease initiation in several autoimmune diseases. Second, molecular studies have not yet revealed a broad set of rules that determine TCR cross-reactivity because, with the exception of the allo–TCR-MHC pair of the 42F3 TCR and H2-Ld that did not encounter each other during T cell development, studies have been limited to structures of a TCR with only 2 or 3 different ligands. Finally, no studies have included characterization of pathogen-derived ligands recognized by self-reactive T cells with greater potency than the autoantigen, a potentially important facet to break self-tolerance. + + 0.35402265 + protein_type + cleaner0 + 2023-07-19T20:18:21Z + MESH: + + MHCII + + + protein + PR: + cleaner0 + 2023-07-19T20:20:30Z + + Hy.1B11 + + + 0.8917636 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + 0.99890864 + site + cleaner0 + 2023-07-20T08:14:02Z + SO: + + deep pocket + + + 0.991907 + residue_number + cleaner0 + 2023-07-20T08:04:15Z + DUMMY: + + 2 + + + 0.97971445 + residue_number + cleaner0 + 2023-07-20T08:04:18Z + DUMMY: + + 3 + + + 0.9611459 + residue_number + cleaner0 + 2023-07-20T08:04:22Z + DUMMY: + + 5 + + + 0.98578435 + protein + cleaner0 + 2023-07-19T20:20:51Z + PR: + + MBP85–99 + + + 0.9992782 + protein_state + cleaner0 + 2023-07-19T16:13:16Z + DUMMY: + + bound to + + + 0.9985524 + protein + cleaner0 + 2023-07-19T20:43:59Z + PR: + + HLA-DQ1 + + + 0.99849296 + protein_state + cleaner0 + 2023-07-19T20:21:23Z + DUMMY: + + conserved + + + 0.7983127 + species + cleaner0 + 2023-07-19T20:08:26Z + MESH: + + Herpes simplex virus + + + 0.9946457 + species + cleaner0 + 2023-07-19T20:43:47Z + MESH: + + Pseudomonas aeruginosa + + + 0.91344637 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + 0.99882895 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + 0.5277726 + complex_assembly + cleaner0 + 2023-07-20T07:50:07Z + GO: + + MHCI + + + 0.93057656 + complex_assembly + cleaner0 + 2023-07-19T20:55:24Z + GO: + + TCRs + + + 0.9568025 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + protein_state + DUMMY: + cleaner0 + 2023-07-20T08:03:53Z + + allo + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:03:43Z + + TCR-MHC + + + 0.9717623 + protein + cleaner0 + 2023-07-19T20:21:30Z + PR: + + 42F3 + + + 0.6603948 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + protein + PR: + cleaner0 + 2023-07-19T20:18:27Z + + H2-Ld + + + 0.99895006 + evidence + cleaner0 + 2023-07-19T20:46:46Z + DUMMY: + + structures + + + 0.98545176 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + + DISCUSS + paragraph + 23617 + Here, we investigated a highly cross-reactive MHCI-restricted TCR isolated from a patient with T1D that recognizes an HLA-A*0201–restricted preproinsulin signal peptide (ALWGPDPAAA15–24). Human CD8+ T cell clones expressing TCRs with this specificity mediate the destruction of β cells, have been found in islets early in infection, and are proposed to be a major driver of disease. We solved the structure of the 1E6 TCR with 7 APLs to enable a comprehensive analysis of the molecular basis of TCR degeneracy. The epitopes we selected exhibited a broad range of potencies and could activate the 1E6 T cell clone at exogenously supplied concentrations more than 4 logs apart. Overall, the difference in antigen potency correlated well with the binding energy (ΔG° kcal/mol) of the 1E6 TCR for the different epitopes, which ranged from values of ΔG° = ~–4.4 to –8.6 kcal/mol (calculated from 3D affinity data) or 2D affinity values of AcKa = 2.5 × 10–5 to 4.4 × 10–2 μm4. The weaker end of this spectrum extends our understanding of the limits in which T cells can functionally operate in terms of TCR 3D binding affinity and is in line with the types of very low affinity, yet fully functional self-reactive CD8+ T cells we have observed in tumor-infiltrating lymphocytes. Previous studies of autoreactive TCRs have shown that their binding mode is generally atypical, either due to an unusual binding manner, weak TCR binding affinity, an unstable pMHC, or a combination of these factors. Our data demonstrate the potential for an autoreactive TCR to bind with a conventional binding mode to a stable pMHC with antipathogen-like affinity (KD = 0.5 μM) depending on the peptide sequence. Our structural analysis revealed that the 1E6 TCR bound with a conserved conformation across all APLs investigated. This binding orientation was mediated through a focused interaction with TCR residues Tyr97α and Trp97β that formed an aromatic cap over a central ‘GDP’ motif that was common to all APLs. We have previously demonstrated the importance of the GPD motif using a peptide library scan, as well as a CPL scan approach. Although the 1E6 T cell was able to activate weakly with peptides that lacked this motif, we were unable to robustly measure binding affinities or generate complex structures with these ligands, highlighting the central role of this interaction during 1E6 T cell antigen recognition. This hotspot binding, defined as a localized cluster of interactions that dominate binding energy during protein-protein interactions, has been previously shown to contribute to TCR recognition of MHC as a mechanism that tunes T cell cross-reactivity by providing fixed anchor points that enable TCRs to tolerate a variable peptide cargo. Alternatively, interactions between the TCR and peptide have been shown to dominate the energetic landscape during ligand engagement, ensuring that T cells retain peptide specificity. The binding mechanism utilized by the 1E6 TCR during pMHC recognition is consistent with both of these models. Ligand engagement is dominated by peptide interactions, but hotspot-like interactions with the central GPD motif enable the 1E6 TCR to tolerate peptide residues that vary outside of this region, explaining how T cells expressing this TCR may cross-react with a large number of different peptides. These findings are also analogous to the observed binding mode of the Hy.1B11 TCR, in which one aromatic residue of the TCR CDR3α loop anchored into a pocket created by a conserved peptide motif. In both of these examples, self-recognition is mediated by TCR residues with aromatic side chains. These large, generally hydrophobic amino acids can form strong interactions with other residues through π-π stacking. Combined with evidence demonstrating that aromatic side chains are conserved in the CDR2 loops of TCRs from many species, we speculate that these aromatic residues could impart a level of “stickiness” to TCRs, which might be enriched in an autoimmune setting when the TCR often binds in a nonoptimal fashion. + + 0.81052303 + complex_assembly + cleaner0 + 2023-07-20T07:50:07Z + GO: + + MHCI + + + 0.979186 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + protein + PR: + cleaner0 + 2023-07-19T20:21:49Z + + HLA-A*0201 + + + 0.8616006 + protein + cleaner0 + 2023-07-19T20:09:35Z + PR: + + preproinsulin + + + structure_element + SO: + cleaner0 + 2023-07-20T07:58:57Z + + signal peptide + + + chemical + CHEBI: + cleaner0 + 2023-07-19T18:13:27Z + + ALWGPDPAAA15–24 + + + 0.99920386 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + Human + + + 0.9869667 + complex_assembly + cleaner0 + 2023-07-19T20:55:24Z + GO: + + TCRs + + + 0.9993399 + experimental_method + cleaner0 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experimental_method + cleaner0 + 2023-07-20T07:51:55Z + MESH: + + CPL scan + + + 0.9993992 + protein_state + cleaner0 + 2023-07-20T08:01:55Z + DUMMY: + + lacked + + + 0.9995267 + evidence + cleaner0 + 2023-07-19T20:47:04Z + DUMMY: + + binding affinities + + + 0.99743986 + evidence + cleaner0 + 2023-07-19T20:47:07Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:53:37Z + + TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-20T07:49:27Z + + MHC + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:55:24Z + + TCRs + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:53:37Z + + TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T16:24:26Z + + pMHC + + + structure_element + SO: + cleaner0 + 2023-07-19T18:05:06Z + + GPD motif + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:53:37Z + + TCR + + + protein + PR: + cleaner0 + 2023-07-19T20:20:31Z + + Hy.1B11 + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:53:37Z + + TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:53:37Z + + TCR + + + structure_element + SO: + cleaner0 + 2023-07-19T20:07:36Z + + CDR3α loop + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:53:37Z + + TCR + + + structure_element + SO: + cleaner0 + 2023-07-19T20:07:49Z + + CDR2 loops + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:55:24Z + + TCRs + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:55:24Z + + TCRs + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:53:37Z + + TCR + + + + DISCUSS + paragraph + 27706 + Despite some weak statistical correlation between the surface complementarity (SC) and affinity, closer inspection of the interface revealed no obvious structural signature that could definitively explain the differences in antigen potency and TCR binding strength between the different ligands. However, similar to our findings in other systems, modifications to residues outside of the canonical central peptide bulge were important for generating new interactions. For example, all of the stronger ligands encoded larger side chains (Arg or Tyr) at peptide position 1 that enabled new interactions with 1E6 not present with the Ala at this position in the natural preproinsulin peptide. These data also explain our previous findings that alteration of the anchor residue at peptide position 2 (Leu-Gln) has a direct effect on 1E6 TCR binding affinity because our structural analysis demonstrated that 1E6 made 3 additional bonds with A2-AQWGPDPAAA compared with A2-ALWGPDPAAA, consistent with the >3-fold stronger binding affinity. We have recently demonstrated how a suboptimal position 2 anchor in a melanoma-derived antigen can improve TCR binding through a similar mechanism. These results challenge the notion that the most potent peptide antigens exhibit the greatest pMHC stability and have implications for the design of anchor residue–modified heteroclitic peptides for vaccination. + + 0.9992144 + evidence + cleaner0 + 2023-07-19T20:19:30Z + DUMMY: + + surface complementarity + + + 0.9990908 + evidence + cleaner0 + 2023-07-19T20:19:34Z + DUMMY: + + SC + + + 0.95532745 + evidence + cleaner0 + 2023-07-19T16:19:49Z + DUMMY: + + affinity + + + 0.99941313 + site + cleaner0 + 2023-07-20T08:14:22Z + SO: + + interface + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:07:00Z + + antigen potency + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:07:13Z + + TCR binding strength + + + 0.9919825 + structure_element + cleaner0 + 2023-07-20T08:10:25Z + SO: + + central peptide bulge + + + 0.9993142 + residue_name + cleaner0 + 2023-07-19T18:16:23Z + SO: + + Arg + + + 0.9993111 + residue_name + cleaner0 + 2023-07-19T18:16:26Z + SO: + + Tyr + + + 0.8965547 + residue_number + cleaner0 + 2023-07-20T08:04:28Z + DUMMY: + + 1 + + + 0.9992756 + residue_name + cleaner0 + 2023-07-19T18:16:28Z + SO: + + Ala + + + 0.80057234 + protein + cleaner0 + 2023-07-19T20:09:35Z + PR: + + preproinsulin + + + 0.7066019 + chemical + cleaner0 + 2023-07-20T07:59:05Z + CHEBI: + + peptide + + + 0.9753542 + structure_element + cleaner0 + 2023-07-20T08:10:30Z + SO: + + anchor residue + + + 0.79387724 + residue_number + cleaner0 + 2023-07-20T08:04:31Z + DUMMY: + + 2 + + + mutant + MESH: + cleaner0 + 2023-07-19T20:05:49Z + + Leu-Gln + + + evidence + DUMMY: + cleaner0 + 2023-07-19T18:15:42Z + + 1E6 TCR binding affinity + + + 0.99653417 + experimental_method + cleaner0 + 2023-07-20T08:07:18Z + MESH: + + structural analysis + + + 0.97112995 + chemical + cleaner0 + 2023-07-19T18:15:59Z + CHEBI: + + A2-AQWGPDPAAA + + + 0.97348905 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.99940276 + evidence + cleaner0 + 2023-07-19T16:20:13Z + DUMMY: + + binding affinity + + + residue_number + DUMMY: + cleaner0 + 2023-07-20T08:04:50Z + + 2 + + + 0.5003262 + structure_element + cleaner0 + 2023-07-20T08:10:33Z + SO: + + anchor + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:53:37Z + + TCR + + + 0.62192553 + complex_assembly + cleaner0 + 2023-07-19T16:24:26Z + GO: + + pMHC + + + + DISCUSS + paragraph + 29103 + Early thermodynamic analysis of TCR-pMHC interactions suggested a common energetic signature, driven by favorable enthalpy (generally mediated through an increase in electrostatic interactions) and unfavorable entropy (changes from disorder to order). These parameters aligned well with structural data, demonstrating that TCRs engaged pMHC using an induced fit binding mode. However, more recent data have shown that TCRs can utilize a range of energetic strategies during pMHC binding, currently with no obvious pattern in terms of TCR affinity, binding mechanism, or specificity (pathogen, cancer, or self-ligands). Although no energetic signature appears to exist for different TCRs, we used thermodynamic analysis here to explore whether changes in energetics could help explain ligand discrimination by a single TCR. This analysis demonstrated a strong relationship (according to the Pearson’s correlation analysis) between the energetic signature used by the 1E6 TCR and the sensitivity of the 1E6 T cell clone to different APLs. The weaker APL ligands were characterized by favorable enthalpy and unfavorable entropy, whereas the stronger ligands progressively shifted to favorable entropy. These differences were consistent with a greater degree of movement between the unligated and ligated pMHCs for the weaker ligands, suggesting a greater requirement for disorder-to-order changes during TCR binding. Thus, the enhanced antigen potency was probably mediated through a shift from an induced fit to a lock-and-key interaction between the stronger ligands (less requirement for energetically unfavorable disorder-to-order changes), resulting in a more energetically favorable ΔG value. + + 0.99953127 + experimental_method + cleaner0 + 2023-07-19T21:01:01Z + MESH: + + thermodynamic analysis + + + 0.9553528 + complex_assembly + cleaner0 + 2023-07-19T18:04:07Z + GO: + + TCR-pMHC + + + 0.9950689 + evidence + cleaner0 + 2023-07-19T16:19:39Z + DUMMY: + + enthalpy + + + 0.9896561 + evidence + cleaner0 + 2023-07-19T16:19:43Z + DUMMY: + + entropy + + + 0.9974977 + evidence + cleaner0 + 2023-07-19T20:47:13Z + DUMMY: + + structural data + + + 0.9933831 + complex_assembly + cleaner0 + 2023-07-19T20:55:24Z + GO: + + TCRs + + + 0.7360548 + complex_assembly + cleaner0 + 2023-07-19T16:24:26Z + GO: + + pMHC + + + 0.99634874 + complex_assembly + cleaner0 + 2023-07-19T20:55:24Z + GO: + + TCRs + + + 0.5931357 + complex_assembly + cleaner0 + 2023-07-19T16:24:26Z + GO: + + pMHC + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:06:15Z + + TCR affinity + + + 0.9940952 + complex_assembly + cleaner0 + 2023-07-19T20:55:24Z + GO: + + TCRs + + + 0.9995968 + experimental_method + cleaner0 + 2023-07-19T21:00:58Z + MESH: + + thermodynamic analysis + + + 0.99539065 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + 0.9399544 + experimental_method + cleaner0 + 2023-07-19T16:31:19Z + MESH: + + Pearson’s correlation analysis + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:51Z + + 1E6 TCR + + + 0.9980301 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.99799025 + chemical + cleaner0 + 2023-07-19T16:29:16Z + CHEBI: + + APL + + + 0.9958858 + evidence + cleaner0 + 2023-07-19T16:19:39Z + DUMMY: + + enthalpy + + + 0.9963213 + evidence + cleaner0 + 2023-07-19T16:19:43Z + DUMMY: + + entropy + + + 0.9973941 + evidence + cleaner0 + 2023-07-19T16:19:43Z + DUMMY: + + entropy + + + 0.9996511 + protein_state + cleaner0 + 2023-07-19T16:17:41Z + DUMMY: + + unligated + + + 0.99963677 + protein_state + cleaner0 + 2023-07-19T20:05:05Z + DUMMY: + + ligated + + + 0.6019646 + complex_assembly + cleaner0 + 2023-07-19T16:28:11Z + GO: + + pMHCs + + + 0.85927975 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + 0.9993516 + evidence + cleaner0 + 2023-07-19T20:23:20Z + DUMMY: + + ΔG value + + + + DISCUSS + paragraph + 30803 + Importantly, the preproinsulin-derived epitope was one of the least potent peptides, demonstrating that the 1E6 T cell clone had the ability to respond to different peptide sequences with far greater potency. The RQFGPDWIVA peptide, which was substantially more potent than the preproinsulin peptide, is within the proteome of a common human pathogen (C. asparagiforme), demonstrating the potential for an encounter between a naive 1E6-like T cell and a foreign peptide with a more potent ligand that might then break self-tolerance. Indeed, we found over 50 decamer peptides from the proteome of likely, or known, human viral pathogens alone that contained both the conserved central GPD motif and anchor residues at positions 2 and 10 that would enable binding to HLA-A*02:01. Further experiments will be required to determine whether any naturally presented, human pathogen–derived peptides act as active ligands for 1E6, but our work presented here demonstrates that it is at least feasible for an autoimmune TCR to bind to a different peptide sequence that could be present in a pathogen proteome with substantially higher affinity and potency than the interaction it might use to attack self-tissue. + + 0.28811467 + protein + cleaner0 + 2023-07-19T20:09:35Z + PR: + + preproinsulin + + + 0.3508806 + chemical + cleaner0 + 2023-07-19T16:08:56Z + CHEBI: + + RQFGPDWIVA + + + 0.3288183 + protein + cleaner0 + 2023-07-19T20:09:35Z + PR: + + preproinsulin + + + 0.99934274 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + 0.9992831 + species + cleaner0 + 2023-07-19T16:09:34Z + MESH: + + C. asparagiforme + + + 0.99928576 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-19T20:08:49Z + + viral + + + 0.99951875 + protein_state + cleaner0 + 2023-07-20T08:02:00Z + DUMMY: + + conserved + + + 0.99971485 + structure_element + cleaner0 + 2023-07-19T18:05:06Z + SO: + + GPD motif + + + 0.9981116 + structure_element + cleaner0 + 2023-07-20T08:10:39Z + SO: + + anchor residues + + + 0.99485046 + residue_number + cleaner0 + 2023-07-20T08:04:56Z + DUMMY: + + 2 + + + 0.9937171 + residue_number + cleaner0 + 2023-07-20T08:04:58Z + DUMMY: + + 10 + + + protein + PR: + cleaner0 + 2023-07-19T20:23:42Z + + HLA-A*02:01 + + + 0.9993616 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + 0.9938782 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + evidence + DUMMY: + cleaner0 + 2023-07-19T16:19:49Z + + affinity + + + + DISCUSS + paragraph + 32011 + In summary, this investigation into the molecular basis of T cell cross-reactivity using a clinically relevant cytotoxic CD8+ T cell clone that kills human pancreatic β cells provides answers to a number of previously outstanding questions. First, our data shows that a single TCR has the potential to functionally (assessed through T cell activation) bind to different ligands with affinities ranging across 3 orders of magnitude. Second, this is the first example in which ligands have been identified and characterized for a human autoreactive TCR that are substantially more potent than the natural self-ligand, demonstrating the potential for a pathogenic ligand to break self-tolerance and prime self-reactive T cells. Third, this first structural analysis of a cross-reactive human MHCI–restricted autoimmune TCR showed that degeneracy was mediated through TCR-pMHC anchoring by a conserved minimal binding peptide motif. Finally, TCR ligand discrimination was characterized by an energetic shift from an enthalpically to entropically driven interaction. Our demonstration of the molecular mechanism governing cross-reactivity by this preproinsulin reactive human CD8+ T cell clone supports the notion first put forward by Wucherpfennig and Strominger that molecular mimicry could mediate autoimmunity and has far-reaching implications for the complex nature of T cell antigen discrimination. + + 0.99927574 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + 0.99455017 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + 0.9996008 + evidence + cleaner0 + 2023-07-19T20:47:18Z + DUMMY: + + affinities + + + 0.9992525 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + 0.99530643 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + 0.9987658 + experimental_method + cleaner0 + 2023-07-19T20:24:12Z + MESH: + + structural analysis + + + 0.9992393 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + complex_assembly + GO: + cleaner0 + 2023-07-20T07:50:07Z + + MHCI + + + 0.9947507 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T18:04:07Z + + TCR-pMHC + + + 0.99901617 + protein_state + cleaner0 + 2023-07-20T08:02:04Z + DUMMY: + + conserved + + + 0.9989063 + structure_element + cleaner0 + 2023-07-20T08:10:43Z + SO: + + minimal binding peptide motif + + + 0.9823903 + complex_assembly + cleaner0 + 2023-07-19T20:53:37Z + GO: + + TCR + + + 0.99480104 + protein + cleaner0 + 2023-07-19T20:09:35Z + PR: + + preproinsulin + + + 0.9992594 + species + cleaner0 + 2023-07-19T16:09:40Z + MESH: + + human + + + + METHODS + title_1 + 33414 + Methods + + + METHODS + title_2 + 33422 + T cell maintenance and culture. + + + METHODS + paragraph + 33454 + The 1E6 T cell clone was generated as previously described and stored in vapor phase liquid nitrogen in freezing buffer (90% FCS and 10% DMSO). Cells were defrosted rapidly in a 37°C water bath until a small amount of frozen cells were left and then immediately washed in 15–20 ml of R10 media (RPMI 1640 with 10% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine) by centrifuging at 300 g for 5 minutes. Defrosted cells were cultured in T cell media: R10 with 1× nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES buffer (all from Invitrogen), 20 IU/ml of IL-2 (aldesleukin, brand name Proleukin, Prometheus) and 25 ng/ml IL-15 (PeproTech), for 24 hours; then, 0.75 × 106 to 1.5 × 106 cells expanded by coculture with 15 × 106 irradiated (3,100 cGy) PBMCs from 3 donors in a 25 cm2 tissue culture flask with 1 μg/ml of phytoheamagglutinin (Alere Inc.) and T cell media as above. The clone was transferred to 24-well tissue culture plates (3 × 106 to 4 × 106 per well in 2 ml) at day 7, and the IL-2 increased to 200 IU/ml. For the purpose of this study, the clone was passaged 3 times and used between weeks 2 and 4 after expansion. + + + METHODS + title_2 + 34626 + T cell activation assays and tetramer staining. + + + METHODS + paragraph + 34674 + The [51Cr] release cytotoxicity assay was performed as previously described. Target A2 CIR cells were labeled for 1 hour at 37°C with 30 μCi chromium (sodium chromate in normal saline, PerkinElmer) per 1 × 106 cells, washed with R10, and allowed to leach for a further hour at 37°C in R10 to remove any excess chromium from the cells. After chromium labeling, target cells were washed and plated at 1,000 cells/well in 96-well tissue culture plates and pulsed with peptide at the indicated concentrations for 2 hours at 37°C. T cells were added to give the desired T cell/target cell (5:1) ratio and a final volume of 150 μl R10. Target cells were also incubated alone or with 1% Triton X-100 detergent (Sigma-Aldrich) to give the spontaneous and total chromium released from the target cells, respectively. After overnight incubation, at 37°C and 5% CO2, the supernatants were both (i) assayed for MIP1β by ELISA (R&D Systems) and (ii) harvested (10% of total volume), mixed with 150 μl Optipahse supermix scintillation mixture (PerkinElmer) in 96-well polyethylene terephthalate plates (PerkinElmer), and sealed; the amount of released chromium was measured indirectly on a 1450 Microbeta counter (PerkinElmer). The percentage of specific target cell lysis by T cells was calculated according to the following formula: (experimental release [with T cells and target cells] − spontaneous release from target cells)/(total release from target cells − spontaneous release from target cells) × 100. Experiments were independently completed in triplicate. Tetrameric pMHCI reagents (tetramers) were constructed by the addition of PE-conjugated streptavidin (Invitrogen) at a pMHCI/streptavidin molar ratio of 4:1. A total of 50,000 T cells were stained with PE-conjugated tetramer (25 μg/ml) folded around the indicated peptides for 30 minutes on ice and washed with PBS before staining with 2 μl (1:40 dilution of the DMSO stock in PBS) of the violet LIVE/DEAD fixable dead cell stain Vivid (Invitrogen) for 5 minutes at room temperature before direct addition of 2 μl of anti–CD8-APC antibody (clone BW135/80, Miltenyi Biotec) and incubated for a further 20 minutes on ice before being washed in FACS buffer (2% FCS in PBS). Data were acquired using a FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc.). + + + METHODS + title_2 + 37041 + Protein expression, refolding, and purification. + + + METHODS + paragraph + 37090 + The 1E6 TCR, HLA-A*0201, and human β2m chain were generated as described previously. The 1E6 TCR and HLA-A*0201 peptide variants were refolded and purified as described previously. Biotinylated pMHCI and pMHC tetramer were prepared as previously described. + + + METHODS + title_2 + 37350 + pMHC stability assays. + + + METHODS + paragraph + 37373 + Thermal stability of the HLA-A*0201–peptide complexes was assessed by circular dichroism (CD) spectroscopy monitoring the change in ellipticities at 218 nm. Data were collected, in duplicate, using a nitrogen-flushed Module B end-station spectrophotometer at the B23 Synchrotron Radiation CD Beamline at the Diamond Light Source (DLS). Samples were prepared in phosphate buffered saline, pH 7.4, and concentrated to ~10 mM. Spectra were measured every 5°C over a temperature range between 5°C and 90°C with 5 minutes of equilibration time for each temperature. Four scans were acquired using an integration time of 1 second, a path length of 0.02 cm, and a slit width of 0.5 mm equivalent to a 1.2-nm bandwidth. Reversibility was monitored by measuring the spectrum at 20°C after cooling from 90°C with 30 minutes of incubation. Melting curves were analyzed assuming a 2-state trimer-to-monomer transition from the native (N) to unfolded (U) conformation N3 ↔ 3U with an equilibrium constant K = (U)3/(N3) = F/(3c2[1-F]3), where F and c are the degree of folding and protein concentration, respectively. Data were fitted as described. Fitted parameters were the melting temperature Tm, van’t Hoff’s enthalpy ΔHvH, and the slope and intercept of the native baseline. As all protein complexes aggregated to various degrees upon unfolding, the ellipticity of the unfolded state was set as a constant of –4,500 deg cm2/dmol. + + + METHODS + title_2 + 38810 + SPR analysis. + + + METHODS + paragraph + 38824 + Binding analysis was performed using a BIAcore T200 equipped with a CM5 sensor chip as previously described. Binding analysis was performed 3× in independent experiments using pMHC monomers generated in-house. Approximately 200–500 RU of each HLA-A*0201–peptide complex was attached to the CM5 sensor chip at a slow flow rate of 10 μl/min to ensure uniform distribution on the chip surface. The 1E6 TCR was purified and concentrated to approximately 40–350 μM on the same day of SPR analysis to reduce the likelihood of TCR aggregation. For equilibrium analysis, 10 serial dilutions were prepared in triplicate for each sample and injected over the relevant sensor chips at 25°C. TCR was injected over the chip surface using kinetic injections at a flow rate of 45 μl/min using HLA-A*0201–ELAGIGILTV as a negative control surface on flow cell 1. For the thermodynamics experiments, this method was repeated at the following temperatures: 5°C, 13°C, 18°C, 25°C, 30°C, and 37°C. Results were analyzed using BIAevaluation 3.1, Excel, and Origin 6.0 software. The KD values were calculated assuming a 1:1 interaction by plotting specific equilibrium-binding responses against protein concentrations, followed by nonlinear least squares fitting of the Langmuir binding equation. The thermodynamic parameters were calculated using the nonlinear van’t Hoff equation (RT ln KD = ΔH° –TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0]) with T0=298 K. + + + METHODS + title_2 + 40285 + Adhesion frequency assay. + + + METHODS + paragraph + 40311 + We used an adhesion frequency assay to measure the 2D affinity of TCR-pMHC interactions at the cell membrane as previously described. Briefly, human 1E6 T cells were mounted onto 1 micropipette, and, on the other pipette, human rbcs coated with pMHC by biotin-streptavidin coupling served as both a surrogate APC and an adhesion sensor for detecting the TCR-pMHC interaction. Site densities of TCR and pMHC were measured by flow cytometry as previously described. All assays were performed using at least 5 cell pairs and calculated as an average of 100 cell-cell contacts. + + + METHODS + title_2 + 40885 + Crystal structure determination. + + + METHODS + paragraph + 40918 + All protein crystals were grown at 18°C by vapor diffusion via the sitting drop technique. Each pMHCI (200 nl, 10 mg/ml) in crystallization buffer (10 mM TRIS [pH 8.1] and 10 mM NaCl) was added to 200 nl of reservoir solution. HLA-A*0201–MVWGPDPLYV (A2-MVW) crystals were grown in 0.2 M ammonium chloride, 0.1 M TRIS (pH 8), 20% PEG 6000; HLA-A*0201–YLGGPDFPTI (A2-YLG) crystals were grown in 0.2 M sodium nitrate, 0.1 M BIS TRIS propane (pH 6.5), 20% PEG 3350; HLA-A*0201–AQWGPDPAAA (A2-AQW) crystals were grown in 0.2 M sodium malonate, 0.1 M BIS TRIS propane (pH 6.5), 20% PEG3350; HLA-A*0201–RQFGPDWIVA (A2-RQF[A]) crystals were grown in 0.2 M sodium sulphate, 0.1 M BIS TRIS propane (pH 6.5), 20% PEG 3350; HLA-A*0201–RQWGPDPAAV (A2-RQW) crystals were grown in 0.1 M TRIS (pH 8), 20% PEG 8000, 15% glycerol; HLA-A*0201–YQFGPDFPTA (A2-YQF) crystals were grown in 0.1 M TRIS (pH 8), 25% PEG 4000, 15% glycerol; HLA-A*0201–RQFGPDFPTI (A2-RQF[I]) crystals were grown in 0.2 M potassium/sodium tartrate, 0.1 M BIS TRIS propane (pH 8.5), 20% PEG 3350; 1E6-A2-MVW crystals were grown in 0.1 M HEPES (pH 7.5), 15% PEG 4000, 0.2 M sodium acetate; 1E6-A2-YLG crystals were grown in 0.1 M sodium cacodylate (pH 6.5), 15% PEG 4000, 0.2 M sodium acetate; 1E6-A2-AQW crystals were grown in 0.2 M sodium citrate, 0.1 M BIS TRIS propane (pH 6.5), 20% PEG 3350; 1E6-A2-RQF(A) crystals were grown in 0.1 M HEPES (pH 7), 15% PEG 4000, 0.2 M sodium acetate; 1E6-A2-RQW crystals were grown in 0.2 M sodium cholride, 0.1 M MES (pH 6), 20% PEG 6000; 1E6-A2-YQF crystals were grown in 0.2 M sodium cholride, 0.1 M HEPES (pH 7), 20% PEG 3350; and 1E6-A2-RQF(I) crystals were grown in 0.1 M HEPES (pH 7.5), 15% PEG 4000, 0.2 M sodium acetate. Crystallization screens were conducted using an Art-Robbins Phoenix dispensing robot (Alpha Biotech Ltd.), and data were collected at 100 K at the DLS at a wavelength of 0.98 Å using an ADSC Q315 CCD detector. Reflection intensities were estimated using XIA2, and the data were analyzed with Scala and the CCP4 package. Structures were solved with molecular replacement using Phaser. Sequences were adjusted with Coot, and the models were refined with REFMAC5. Graphical representations were prepared with PyMOL. The reflection data and final model coordinates were deposited with the PDB database (A2-MVW PDB: 5C0H; A2-YLG PDB: 5C0G; A2-AQW PDB: 5C0D; A2-RQF[A] PDB: 5C0J; A2-RQW PDB: 5C0F; A2-YQF PDB: 5C0E; A2-RQF[I] PDB: 5C0I; 1E6-A2-MVW PDB: 5C0A; 1E6-A2-YLG PDB: 5C09; 1E6-A2-AQW PDB: 5HYJ; 1E6-A2-RQF[A] PDB: 5C0C; 1E6-A2-RQW PDB: 5C08; 1E6-A2-YQF PDB: 5C07; and 1E6-A2-RQF[I] PDB: 5C0B). + + + METHODS + title_2 + 43552 + Peptide motif predictions. + + + METHODS + paragraph + 43579 + Peptide motif predictions were performed by searching a viral database compiled using publicly available protein sequences of over 1,924,572 unique decamer peptides from the proteome of viral pathogens. The motif xOxGPDxxxO — where O is anyone of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 — was used as the search parameter. + + + METHODS + title_2 + 43967 + Statistics. + + + METHODS + paragraph + 43979 + Pearson’s correlation analysis was performed to determine the relationship between TCR binding affinity and antigen potency, structural correlates, or thermodynamics using Origin Lab 9.0 pro. + + + AUTH_CONT + title_1 + 44173 + Author contributions + + + AUTH_CONT + paragraph + 44194 + AMB, GD, AJS, BS, WR, AT, PJ, AF, AS, JJM, LW, PJR, and DKC performed experiments and analyzed the data. AKS, JR, CZ, JJM, MP, and DKC wrote the manuscript. AKS and DKC conceived and directed the study. AKS and DKC funded the study. All authors contributed to discussions. + + + SUPPL + title_1 + 44467 + Supplementary Material + + + SUPPL + footnote + 44490 + Conflict of interest: The authors have declared that no conflict of interest exists. + + + SUPPL + footnote + 44575 + License: This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. + + + SUPPL + footnote + 44755 + Reference information:J Clin Invest. 2016;126(6):2191–2204. doi:10.1172/JCI85679. + + + SUPPL + footnote + 44839 + See the related Commentary beginning on page . + + + 1073 + 5 + 1087 + surname:Birnbaum;given-names:ME + 10.1016/j.cell.2014.03.047 + 24855945 + REF + Cell + ref + 157 + 2014 + 44886 + Deconstructing the peptide-MHC specificity of T cell recognition + + + 1047 + 14–15 + 1055 + surname:Wilson;given-names:DB + 15036909 + REF + Mol Immunol + ref + 40 + 2004 + 44951 + Specificity and degeneracy of T cells + + + 1168 + 2 + 1177 + surname:Wooldridge;given-names:L + 10.1074/jbc.M111.289488 + 22102287 + REF + J Biol Chem + ref + 287 + 2012 + 44989 + A single autoimmune T cell receptor recognizes more than a million different peptides + + + 395 + 9 + 404 + surname:Mason;given-names:D + 10.1016/S0167-5699(98)01299-7 + 9745202 + REF + Immunol Today + ref + 19 + 1998 + 45075 + A very high level of crossreactivity is an essential feature of the T-cell receptor + + + 669 + 9 + 677 + surname:Sewell;given-names:AK + 10.1038/nri3279 + 22918468 + REF + Nat Rev Immunol + ref + 12 + 2012 + 45159 + Why must T cells be cross-reactive? + + + 958 + 5441 + 961 + surname:Arstila;given-names:TP + surname:Casrouge;given-names:A + surname:Baron;given-names:V + surname:Even;given-names:J + surname:Kanellopoulos;given-names:J + surname:Kourilsky;given-names:P + 10.1126/science.286.5441.958 + 10542151 + REF + Science + ref + 286 + 1999 + 45195 + A direct estimate of the human alphabeta T cell receptor diversity + + + 417 + 4 + 423 + surname:Christen;given-names:U + surname:Bender;given-names:C + surname:von Herrath;given-names:MG + 10.1097/BOR.0b013e3283533719 + 22504578 + REF + Curr Opin Rheumatol + ref + 24 + 2012 + 45262 + Infection as a cause of type 1 diabetes? + + + 205 + 3 + 206 + surname:Coppieters;given-names:KT + surname:von Herrath;given-names:MG + 10.1038/ni.2226 + 22344274 + REF + Nat Immunol + ref + 13 + 2012 + 45303 + Motifs for a deadly encounter + + + 695 + 5 + 705 + surname:Wucherpfennig;given-names:KW + surname:Strominger;given-names:JL + 10.1016/0092-8674(95)90348-8 + 7534214 + REF + Cell + ref + 80 + 1995 + 45333 + Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein + + + 348 + 3 + 357 + surname:Harkiolaki;given-names:M + 10.1016/j.immuni.2009.01.009 + 19303388 + REF + Immunity + ref + 30 + 2009 + 45462 + T cell-mediated autoimmune disease due to low-affinity crossreactivity to common microbial peptides + + + 241 + 3 + 247 + surname:Reiser;given-names:JB + 10.1038/ni891 + 12563259 + REF + Nat Immunol + ref + 4 + 2003 + 45562 + CDR3 loop flexibility contributes to the degeneracy of TCR recognition + + + 183 + 2 + 196 + surname:Armstrong;given-names:KM + surname:Piepenbrink;given-names:KH + surname:Baker;given-names:BM + 10.1042/BJ20080850 + 18800968 + REF + Biochem J + ref + 415 + 2008 + 45633 + Conformational changes and flexibility in T-cell receptor recognition of peptide-MHC complexes + + + 885 + 6 + 896 + surname:Borbulevych;given-names:OY + 10.1016/j.immuni.2009.11.003 + 20064447 + REF + Immunity + ref + 31 + 2009 + 45728 + T cell receptor cross-reactivity directed by antigen-dependent tuning of peptide-MHC molecular flexibility + + + 897 + 6 + 908 + surname:Macdonald;given-names:WA + 10.1016/j.immuni.2009.09.025 + 20064448 + REF + Immunity + ref + 31 + 2009 + 45835 + T cell allorecognition via molecular mimicry + + + 1114 + 11 + 1122 + surname:Tynan;given-names:FE + 10.1038/ni1257 + 16186824 + REF + Nat Immunol + ref + 6 + 2005 + 45880 + T cell receptor recognition of a ‘super-bulged’ major histocompatibility complex class I-bound peptide + + + 268 + 3 + 276 + surname:Tynan;given-names:FE + 10.1038/ni1432 + 17259989 + REF + Nat Immunol + ref + 8 + 2007 + 45987 + A T cell receptor flattens a bulged antigenic peptide presented by a major histocompatibility complex class I molecule + + + 681 + 5 + 693 + surname:Adams;given-names:JJ + 10.1016/j.immuni.2011.09.013 + 22101157 + REF + Immunity + ref + 35 + 2011 + 46106 + T cell receptor signaling is limited by docking geometry to peptide-major histocompatibility complex + + + 291 + 4 + 297 + surname:Reiser;given-names:JB + 10.1038/79728 + 11017099 + REF + Nat Immunol + ref + 1 + 2000 + 46207 + Crystal structure of a T cell receptor bound to an allogeneic MHC molecule + + + surname:Sethi;given-names:DK + surname:Gordo;given-names:S + surname:Schubert;given-names:DA + surname:Wucherpfennig;given-names:KW + 24136005 + REF + Nat Commun + ref + 4 + 2013 + 46282 + Crossreactivity of a human autoimmune TCR is dominated by a single TCR loop + + + surname:Holland;given-names:CJ + 22953050 + REF + Sci Rep + ref + 2 + 2012 + 46358 + Minimal conformational plasticity enables TCR cross-reactivity to different MHC class II heterodimers + + + 135 + 1 + 146 + surname:Colf;given-names:LA + 10.1016/j.cell.2007.01.048 + 17418792 + REF + Cell + ref + 129 + 2007 + 46460 + How a single T cell receptor recognizes both self and foreign MHC + + + 87 + 1 + 94 + surname:Adams;given-names:JJ + 26523866 + REF + Nat Immunol + ref + 17 + 2016 + 46526 + Structural interplay between germline interactions and adaptive recognition determines the bandwidth of TCR-peptide-MHC cross-reactivity + + + 283 + 3 + 289 + surname:Bulek;given-names:AM + 10.1038/ni.2206 + 22245737 + REF + Nat Immunol + ref + 13 + 2012 + 46663 + Structural basis for the killing of human β cells by CD8(+) T cells in type 1 diabetes + + + 205 + 1 + 213 + surname:Knight;given-names:RR + 10.2337/db12-0315 + 22936177 + REF + Diabetes + ref + 62 + 2013 + 46753 + Human beta-cell killing by autoreactive preproinsulin-specific CD8 T cells is predominantly granule-mediated with the potency dependent upon T-cell receptor avidity + + + 3390 + 10 + 3402 + surname:Skowera;given-names:A + 10.1172/JCI35449 + 18802479 + REF + J Clin Invest + ref + 118 + 2008 + 46918 + CTLs are targeted to kill β cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope + + + 51 + 1 + 60 + surname:Coppieters;given-names:KT + 10.1084/jem.20111187 + 22213807 + REF + J Exp Med + ref + 209 + 2012 + 47053 + Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients + + + surname:Kloverpris;given-names:HN + 25808313 + REF + Retrovirology + ref + 12 + 2015 + 47179 + A molecular switch in immunodominant HIV-1-specific CD8 T-cell epitopes shapes differential HLA-restricted escape + + + 9 + 1 + 18 + surname:Bridgeman;given-names:JS + surname:Sewell;given-names:AK + surname:Miles;given-names:JJ + surname:Price;given-names:DA + surname:Cole;given-names:DK + 10.1111/j.1365-2567.2011.03515.x + 22044041 + REF + Immunology + ref + 135 + 2012 + 47293 + Structural and biophysical determinants of αβ T-cell antigen recognition + + + 169 + 200 + surname:Rossjohn;given-names:J + surname:Gras;given-names:S + surname:Miles;given-names:JJ + surname:Turner;given-names:SJ + surname:Godfrey;given-names:DI + surname:McCluskey;given-names:J + 10.1146/annurev-immunol-032414-112334 + 25493333 + REF + Annu Rev Immunol + ref + 33 + 2015 + 47373 + T cell antigen receptor recognition of antigen-presenting molecules + + + 1553 + 3 + 1572 + surname:Chesla;given-names:SE + surname:Selvaraj;given-names:P + surname:Zhu;given-names:C + 10.1016/S0006-3495(98)74074-3 + 9726957 + REF + Biophys J + ref + 75 + 1998 + 47441 + Measuring two-dimensional receptor-ligand binding kinetics by micropipette + + + 932 + 7290 + 936 + surname:Huang;given-names:J + 10.1038/nature08944 + 20357766 + REF + Nature + ref + 464 + 2010 + 47516 + The kinetics of two-dimensional TCR and pMHC interactions determine T-cell responsiveness + + + 442 + pt 4 + 450 + surname:Tickle;given-names:IJ + surname:Laskowski;given-names:RA + surname:Moss;given-names:DS + 10739917 + REF + Acta Crystallogr D Biol Crystallogr + ref + 56 + 2000 + 47606 + Rfree and the rfree ratio + + + 10608 + 23 + 10613 + surname:Burrows;given-names:SR + 10.1073/pnas.1004926107 + 20483993 + REF + Proc Natl Acad Sci U S A + ref + 107 + 2010 + 47632 + Hard wiring of T cell receptor specificity for the major histocompatibility complex is underpinned by TCR adaptability + + + 34324 + 45 + 34332 + surname:Archbold;given-names:JK + 10.1074/jbc.M606755200 + 16963442 + REF + J Biol Chem + ref + 281 + 2006 + 47751 + Alloreactivity between disparate cognate and allogeneic pMHC-I complexes is the result of highly focused, peptide-dependent structural mimicry + + + 151 + 2 + 157 + surname:von Herrath;given-names:MG + surname:Fujinami;given-names:RS + surname:Whitton;given-names:JL + 10.1038/nrmicro754 + 15035044 + REF + Nat Rev Microbiol + ref + 1 + 2003 + 47894 + Microorganisms and autoimmunity: making the barren field fertile? + + + 2453 + 5 + 2463 + surname:Borbulevych;given-names:OY + surname:Santhanagopolan;given-names:SM + surname:Hossain;given-names:M + surname:Baker;given-names:BM + 10.4049/jimmunol.1101268 + 21795600 + REF + J Immunol + ref + 187 + 2011 + 47960 + TCRs used in cancer gene therapy cross-react with MART-1/Melan-A tumor antigens via distinct mechanisms + + + 45 + 1 + 56 + surname:Ding;given-names:YH + surname:Baker;given-names:BM + surname:Garboczi;given-names:DN + surname:Biddison;given-names:WE + surname:Wiley;given-names:DC + 10.1016/S1074-7613(00)80080-1 + 10435578 + REF + Immunity + ref + 11 + 1999 + 48064 + Four A6-TCR/peptide/HLA-A2 structures that generate very different T cell signals are nearly identical + + + 324 + 3 + 334 + surname:Dai;given-names:S + 10.1016/j.immuni.2008.01.008 + 18308592 + REF + Immunity + ref + 28 + 2008 + 48167 + Crossreactive T Cells spotlight the germline rules for alphabeta T cell-receptor interactions with MHC molecules + + + 14960 + 37 + 14965 + surname:Deng;given-names:L + surname:Langley;given-names:RJ + surname:Wang;given-names:Q + surname:Topalian;given-names:SL + surname:Mariuzza;given-names:RA + 10.1073/pnas.1207186109 + 22930819 + REF + Proc Natl Acad Sci U S A + ref + 109 + 2012 + 48280 + Structural insights into the editing of germ-line-encoded interactions between T-cell receptor and MHC class II by Valpha CDR3 + + + 311 + 1 + 321 + surname:Gras;given-names:S + 10.4049/jimmunol.1102686 + 22140258 + REF + J Immunol + ref + 188 + 2012 + 48407 + A structural basis for varied αβ TCR usage against an immunodominant EBV antigen restricted to a HLA-B8 molecule + + + 47 + 1 + 63 + surname:Dolton;given-names:G + 10.1111/cei.12339 + 24673376 + REF + Clin Exp Immunol + ref + 177 + 2014 + 48527 + Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells + + + 11 + 1 + 22 + surname:Dolton;given-names:G + 10.1111/imm.12499 + 26076649 + REF + Immunology + ref + 146 + 2015 + 48658 + More tricks with tetramers: a practical guide to staining T cells with peptide-MHC multimers + + + 463 + 1 + 474 + surname:Tungatt;given-names:K + 10.4049/jimmunol.1401785 + 25452566 + REF + J Immunol + ref + 194 + 2015 + 48751 + Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs + + + 490 + 5 + 496 + surname:Hahn;given-names:M + surname:Nicholson;given-names:MJ + surname:Pyrdol;given-names:J + surname:Wucherpfennig;given-names:KW + 10.1038/ni1187 + 15821740 + REF + Nat Immunol + ref + 6 + 2005 + 48862 + Unconventional topology of self peptide-major histocompatibility complex binding by a human autoimmune T cell receptor + + + 2968 + 17 + 2979 + surname:Li;given-names:Y + surname:Huang;given-names:Y + surname:Lue;given-names:J + surname:Quandt;given-names:JA + surname:Martin;given-names:R + surname:Mariuzza;given-names:RA + 10.1038/sj.emboj.7600771 + 16079912 + REF + EMBO J + ref + 24 + 2005 + 48981 + Structure of a human autoimmune TCR bound to a myelin basic protein self-peptide and a multiple sclerosis-associated MHC class II molecule + + + 91 + 1 + 102 + surname:Sethi;given-names:DK + 10.1084/jem.20100725 + 21199956 + REF + J Exp Med + ref + 208 + 2011 + 49120 + A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC + + + 1153 + 11 + 1161 + surname:Beringer;given-names:DX + 10.1038/ni.3271 + 26437244 + REF + Nat Immunol + ref + 16 + 2015 + 49233 + T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex + + + 1137 + 6 + 1148 + surname:Yin;given-names:Y + surname:Li;given-names:Y + surname:Kerzic;given-names:MC + surname:Martin;given-names:R + surname:Mariuzza;given-names:RA + 10.1038/emboj.2011.21 + 21297580 + REF + EMBO J + ref + 30 + 2011 + 49330 + Structure of a TCR with high affinity for self-antigen reveals basis for escape from negative selection + + + 18924 + 31 + 18933 + surname:Motozono;given-names:C + 10.1074/jbc.M114.622522 + 26085090 + REF + J Biol Chem + ref + 290 + 2015 + 49434 + Distortion of the MHC class I binding groove to accommodate an insulin-derived 10-mer peptide + + + 383 + 5196 + 386 + surname:Clackson;given-names:T + surname:Wells;given-names:JA + 10.1126/science.7529940 + 7529940 + REF + Science + ref + 267 + 1995 + 49528 + A hot spot of binding energy in a hormone-receptor interface + + + 551 + 5 + 562 + surname:Baker;given-names:BM + surname:Turner;given-names:RV + surname:Gagnon;given-names:SJ + surname:Wiley;given-names:DC + surname:Biddison;given-names:WE + 10.1084/jem.193.5.551 + 11238586 + REF + J Exp Med + ref + 193 + 2001 + 49589 + Identification of a crucial energetic footprint on the alpha1 helix of human histocompatibility leukocyte antigen (HLA)-A2 that provides functional interactions for recognition by tax peptide/HLA-A2-specific T cell receptors + + + 1191 + 11 + 1199 + surname:Huseby;given-names:ES + surname:Crawford;given-names:F + surname:White;given-names:J + surname:Marrack;given-names:P + surname:Kappler;given-names:JW + 10.1038/ni1401 + 17041605 + REF + Nat Immunol + ref + 7 + 2006 + 49814 + Interface-disrupting amino acids establish specificity between T cell receptors and complexes of major histocompatibility complex and peptide + + + 552 + 6897 + 556 + surname:Wu;given-names:LC + surname:Tuot;given-names:DS + surname:Lyons;given-names:DS + surname:Garcia;given-names:KC + surname:Davis;given-names:MM + 10.1038/nature00920 + 12152083 + REF + Nature + ref + 418 + 2002 + 49956 + Two-step binding mechanism for T-cell receptor recognition of peptide MHC + + + 171 + 2 + 180 + surname:Borg;given-names:NA + 10.1038/ni1155 + 15640805 + REF + Nat Immunol + ref + 6 + 2005 + 50030 + The CDR3 regions of an immunodominant T cell receptor dictate the ‘energetic landscape’ of peptide-MHC recognition + + + 628 + 2 + 638 + surname:Cole;given-names:DK + 10.1074/jbc.M113.522110 + 24196962 + REF + J Biol Chem + ref + 289 + 2014 + 50149 + TCR-peptide specificity overrides affinity enhancing TCR-MHC interactions + + + 526 + 4 + 535 + surname:Scott-Browne;given-names:JP + surname:Crawford;given-names:F + surname:Young;given-names:MH + surname:Kappler;given-names:JW + surname:Marrack;given-names:P + surname:Gapin;given-names:L + 10.1016/j.immuni.2011.09.005 + 21962492 + REF + Immunity + ref + 35 + 2011 + 50223 + Evolutionarily conserved features contribute to alphabeta T cell receptor specificity + + + 27281 + 40 + 27289 + surname:Cole;given-names:DK + 10.1074/jbc.M109.022509 + 19605354 + REF + J Biol Chem + ref + 284 + 2009 + 50309 + Germ line-governed recognition of a cancer epitope by an immunodominant human T-cell receptor + + + 584 + 2 + 591 + surname:Madura;given-names:F + 10.1002/eji.201445114 + 25471691 + REF + Eur J Immunol + ref + 45 + 2015 + 50403 + Structural basis for ineffective T-cell responses to MHC anchor residue-improved “heteroclitic” peptides + + + 37269 + 44 + 37281 + surname:Ekeruche-Makinde;given-names:J + 10.1074/jbc.M112.386409 + 22952231 + REF + J Biol Chem + ref + 287 + 2012 + 50512 + T-cell receptor-optimized peptide skewing of the T-cell repertoire can enhance antigen targeting + + + 2600 + 4 + 2610 + surname:Cole;given-names:DK + 10.4049/jimmunol.1000629 + 20639478 + REF + J Immunol + ref + 185 + 2010 + 50609 + Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition + + + 11446 + 20 + 11451 + surname:Boniface;given-names:JJ + surname:Reich;given-names:Z + surname:Lyons;given-names:DS + surname:Davis;given-names:MM + 10.1073/pnas.96.20.11446 + 10500196 + REF + Proc Natl Acad Sci U S A + ref + 96 + 1999 + 50723 + Thermodynamics of T cell receptor binding to peptide-MHC: evidence for a general mechanism of molecular scanning + + + 357 + 3 + 365 + surname:Willcox;given-names:BE + 10.1016/S1074-7613(00)80035-7 + 10204491 + REF + Immunity + ref + 10 + 1999 + 50836 + TCR binding to peptide-MHC stabilizes a flexible recognition interface + + + 369 + 397 + surname:Garcia;given-names:KC + surname:Teyton;given-names:L + surname:Wilson;given-names:IA + 10.1146/annurev.immunol.17.1.369 + 10358763 + REF + Annu Rev Immunol + ref + 17 + 1999 + 50907 + Structural basis of T cell recognition + + + 533 + 2 + 550 + surname:Davis-Harrison;given-names:RL + surname:Armstrong;given-names:KM + surname:Baker;given-names:BM + 10.1016/j.jmb.2004.11.063 + 15670602 + REF + J Mol Biol + ref + 346 + 2005 + 50946 + Two different T cell receptors use different thermodynamic strategies to recognize the same peptide/MHC ligand + + + 275 + 4 + 287 + surname:Armstrong;given-names:KM + surname:Insaidoo;given-names:FK + surname:Baker;given-names:BM + 10.1002/jmr.896 + 18496839 + REF + J Mol Recognit + ref + 21 + 2008 + 51057 + Thermodynamics of T-cell receptor-peptide/MHC interactions: progress and opportunities + + + 171 + 2 + 182 + surname:Ishizuka;given-names:J + surname:Stewart-Jones;given-names:GB + surname:van der Merwe;given-names:A + surname:Bell;given-names:JI + surname:McMichael;given-names:AJ + surname:Jones;given-names:EY + 10.1016/j.immuni.2007.12.018 + 18275829 + REF + Immunity + ref + 28 + 2008 + 51144 + The structural dynamics and energetics of an immunodominant T cell receptor are programmed by its Vbeta domain + + + 12267 + 15 + 12276 + surname:Liu;given-names:YC + 10.1074/jbc.M112.344689 + 22343629 + REF + J Biol Chem + ref + 287 + 2012 + 51255 + The energetic basis underpinning T-cell receptor recognition of a super-bulged peptide bound to a major histocompatibility complex class I molecule + + + 27491 + 30 + 27501 + surname:Wooldridge;given-names:L + 10.1074/jbc.M500555200 + 15837791 + REF + J Biol Chem + ref + 280 + 2005 + 51403 + Interaction between the CD8 coreceptor and major histocompatibility complex class I stabilizes T cell receptor-antigen complexes at the cell surface + + + E149 + suppl 1 + E153 + surname:Javorfi;given-names:T + surname:Hussain;given-names:R + surname:Myatt;given-names:D + surname:Siligardi;given-names:G + 21038386 + REF + Chirality + ref + 22 + 2010 + 51552 + Measuring circular dichroism in a capillary cell using the b23 synchrotron radiation CD beamline at diamond light source + + + 282 + 317 + surname:Greenfield;given-names:NJ + 15063655 + REF + Methods Enzymol + ref + 383 + 2004 + 51673 + Analysis of circular dichroism data + + + 2411 + 11 + 2423 + surname:Pace;given-names:CN + surname:Vajdos;given-names:F + surname:Fee;given-names:L + surname:Grimsley;given-names:G + surname:Gray;given-names:T + 10.1002/pro.5560041120 + 8563639 + REF + Protein Sci + ref + 4 + 1995 + 51709 + How to measure and predict the molar absorption coefficient of a protein + + + 17 + 1 + 24 + surname:Venyaminov;given-names:S + surname:Baikalov;given-names:IA + surname:Shen;given-names:ZM + surname:Wu;given-names:CS + surname:Yang;given-names:JT + 10.1006/abio.1993.1450 + 8250221 + REF + Anal Biochem + ref + 214 + 1993 + 51782 + Circular dichroic analysis of denatured proteins: inclusion of denatured proteins in the reference set + + + 2700 + 9 + 2709 + surname:Cole;given-names:DK + surname:Dunn;given-names:SM + surname:Sami;given-names:M + surname:Boulter;given-names:JM + surname:Jakobsen;given-names:BK + surname:Sewell;given-names:AK + 10.1016/j.molimm.2007.12.009 + 18243322 + REF + Mol Immunol + ref + 45 + 2008 + 51885 + T cell receptor engagement of peptide-major histocompatibility complex class I does not modify CD8 binding + + + 7653 + 11 + 7662 + surname:Huang;given-names:J + surname:Edwards;given-names:LJ + surname:Evavold;given-names:BD + surname:Zhu;given-names:C + 10.4049/jimmunol.179.11.7653 + 18025211 + REF + J Immunol + ref + 179 + 2007 + 51992 + Kinetics of MHC-CD8 interaction at the T cell membrane + + + 1260 + pt 7 + 1273 + surname:Winter;given-names:G + surname:Lobley;given-names:CM + surname:Prince;given-names:SM + 23793152 + REF + Acta Crystallogr D Biol Crystallogr + ref + 69 + 2013 + 52047 + Decision making in xia2 + + + 620 + 633 + surname:Dodson;given-names:EJ + surname:Winn;given-names:M + surname:Ralph;given-names:A + 18488327 + REF + Methods Enzymol + ref + 277 + 1997 + 52071 + Collaborative Computational Project, number 4: providing programs for protein crystallography + + + 658 + pt 4 + 674 + surname:McCoy;given-names:AJ + surname:Grosse-Kunstleve;given-names:RW + surname:Adams;given-names:PD + surname:Winn;given-names:MD + surname:Storoni;given-names:LC + surname:Read;given-names:RJ + 19461840 + REF + J Appl Crystallogr + ref + 40 + 2007 + 52165 + Phaser crystallographic software + + + 2126 + pt 12 pt 1 + 2132 + surname:Emsley;given-names:P + surname:Cowtan;given-names:K + 15572765 + REF + Acta Crystallogr D Biol Crystallogr + ref + 60 + 2004 + 52198 + Coot: model-building tools for molecular graphics + + + The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC. + REF + ref + 52248 + + + [published online ahead of print February 5, 2016]. doi:10.1038/icb.2016.12 + surname:Szomolay;given-names:B + 10.1038/icb.2016.12 + 26846725 + REF + Immunol Cell Biol + ref + 52249 + Identification of human viral protein-derived ligands recognized by individual major histocompatibility complex class I (MHCI)-restricted T-cell receptors + + + JCI85679.f1.jpg + F1 + FIG + fig_title_caption + 52404 + The 1E6 T cell clone reacts with a broad sensitivity range to APLs. + + 0.9845797 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + + JCI85679.f1.jpg + F1 + FIG + fig_caption + 52472 + (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). (C) The 1E6 T cell clone was stained, in duplicate, with tetramers composed of each APL (colored as above) presented by HLA-A*0201. (D) The stability of each APL (colored as above) was tested, in duplicate, using CD by recording the peak at 218 nm absorbance from 5°C–90°C. Tm values were calculated using a Boltzmann fit to each set of data. + + 0.999573 + experimental_method + cleaner0 + 2023-07-19T20:25:01Z + MESH: + + peptide dilution assay + + + 0.9895058 + chemical + cleaner0 + 2023-07-19T16:08:36Z + CHEBI: + + MVWGPDPLYV + + + 0.9752729 + chemical + cleaner0 + 2023-07-19T16:09:11Z + CHEBI: + + YLGGPDFPTI + + + 0.9628796 + chemical + cleaner0 + 2023-07-19T16:08:20Z + CHEBI: + + ALWGPDPAAA + + + 0.96822727 + chemical + cleaner0 + 2023-07-19T18:27:26Z + CHEBI: + + AQWGPDPAAA + + + 0.99096864 + chemical + cleaner0 + 2023-07-19T16:08:56Z + CHEBI: + + RQFGPDWIVA + + + 0.99126 + chemical + cleaner0 + 2023-07-19T18:27:35Z + CHEBI: + + RQWGPDPAAV + + + 0.986967 + chemical + cleaner0 + 2023-07-19T18:27:44Z + CHEBI: + + YQFGPDFPTA + + + 0.98250383 + chemical + cleaner0 + 2023-07-19T18:27:52Z + CHEBI: + + RQFGPDFPTI + + + 0.89255476 + protein + cleaner0 + 2023-07-19T20:44:04Z + PR: + + HLA-A*0201 + + + 0.8122008 + protein + cleaner0 + 2023-07-19T20:12:01Z + PR: + + MIP-1β + + + 0.9930211 + oligomeric_state + cleaner0 + 2023-07-20T07:58:34Z + DUMMY: + + tetramers + + + 0.9965977 + chemical + cleaner0 + 2023-07-19T16:29:17Z + CHEBI: + + APL + + + 0.99024296 + protein + cleaner0 + 2023-07-19T20:44:08Z + PR: + + HLA-A*0201 + + + 0.997544 + chemical + cleaner0 + 2023-07-19T16:29:17Z + CHEBI: + + APL + + + 0.99962974 + experimental_method + cleaner0 + 2023-07-19T20:24:56Z + MESH: + + CD + + + 0.99954647 + evidence + cleaner0 + 2023-07-19T16:29:58Z + DUMMY: + + Tm + + + experimental_method + MESH: + cleaner0 + 2023-07-19T18:28:20Z + + Boltzmann fit to each set of data + + + + JCI85679.f2.jpg + F2 + FIG + fig_title_caption + 53174 + 3D and 2D binding analysis of the 1E6 TCR with A2-ALW and the APLs. + + experimental_method + MESH: + cleaner0 + 2023-07-20T08:07:41Z + + 3D and 2D binding analysis + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.8336919 + chemical + cleaner0 + 2023-07-19T20:41:38Z + CHEBI: + + A2-ALW + + + 0.5477331 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + + JCI85679.f2.jpg + F2 + FIG + fig_caption + 53242 + (A–H) Binding affinity of the 1E6 TCR interaction at 25°C using SPR. Eight serial dilutions of the 1E6 TCR were measured (shown in the inset); representative data from 3 independent experiments are plotted. The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y= [P1x]/[P2 + X]). In order to calculate each response, the 1E6 TCR was also injected over a control sample (HLA-A*0201–ILAKFLHWL) that was deducted from the experimental data. (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). (J) Effective 2D affinity (AcKa) calculated using adhesion frequency assays, using at least 5 cell pairs, and calculated as an average of 100 cell cell contacts. (K) Effective 2D affinity plotted against 1/EC50 showing Pearson’s coefficient analysis (r) and P value. + + 0.9995432 + evidence + cleaner0 + 2023-07-19T16:20:13Z + DUMMY: + + Binding affinity + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.99968934 + experimental_method + cleaner0 + 2023-07-19T16:32:30Z + MESH: + + SPR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.99949384 + evidence + cleaner0 + 2023-07-19T18:28:45Z + DUMMY: + + equilibrium binding constant + + + 0.9995623 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.9307487 + experimental_method + cleaner0 + 2023-07-19T21:06:13Z + MESH: + + nonlinear curve fit + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:42:26Z + + HLA-A*0201–ILAKFLHWL + + + 0.9859878 + complex_assembly + cleaner0 + 2023-07-19T16:18:17Z + GO: + + 1E6-A2-MVWGPDPLYV + + + complex_assembly + GO: + cleaner0 + 2023-07-19T18:29:39Z + + 1E6-A2-YLGGPDFPTI + + + 0.9805851 + complex_assembly + cleaner0 + 2023-07-19T16:14:43Z + GO: + + 1E6-A2-ALWGPDPAAA + + + 0.9903617 + complex_assembly + cleaner0 + 2023-07-19T18:30:04Z + GO: + + 1E6-A2-AQWGPDPAAA + + + 0.9882841 + complex_assembly + cleaner0 + 2023-07-19T18:30:24Z + GO: + + 1E6-A2-RQFGPDWIVA + + + 0.9882075 + complex_assembly + cleaner0 + 2023-07-19T18:30:45Z + GO: + + 1E6-A2-RQWGPDPAAV + + + 0.9757681 + complex_assembly + cleaner0 + 2023-07-19T18:29:50Z + GO: + + 1E6-A2-YQFGPDFPTA + + + 0.98563766 + complex_assembly + cleaner0 + 2023-07-19T16:19:13Z + GO: + + 1E6-A2-RQFGPDFPTI + + + 0.9887948 + evidence + cleaner0 + 2023-07-19T20:25:35Z + DUMMY: + + ΔG values + + + 0.9996978 + experimental_method + cleaner0 + 2023-07-19T16:32:30Z + MESH: + + SPR + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:42:36Z + + EC50 + + + 0.9450487 + experimental_method + cleaner0 + 2023-07-20T08:07:47Z + MESH: + + Pearson’s coefficient analysis + + + 0.84214836 + evidence + cleaner0 + 2023-07-19T20:25:14Z + DUMMY: + + r + + + 0.7447264 + evidence + cleaner0 + 2023-07-19T20:25:17Z + DUMMY: + + P + + + 0.99866694 + evidence + cleaner0 + 2023-07-19T20:47:26Z + DUMMY: + + Effective 2D affinity + + + 0.99921787 + evidence + cleaner0 + 2023-07-19T20:47:31Z + DUMMY: + + AcKa + + + 0.99958515 + experimental_method + cleaner0 + 2023-07-20T08:07:51Z + MESH: + + adhesion frequency assays + + + 0.9983494 + evidence + cleaner0 + 2023-07-19T20:47:35Z + DUMMY: + + Effective 2D affinity + + + 0.5637522 + evidence + cleaner0 + 2023-07-19T20:47:38Z + DUMMY: + + EC50 + + + 0.97026414 + experimental_method + cleaner0 + 2023-07-20T08:07:54Z + MESH: + + Pearson’s coefficient analysis + + + 0.8508054 + evidence + cleaner0 + 2023-07-19T20:25:20Z + DUMMY: + + r + + + 0.8390739 + evidence + cleaner0 + 2023-07-19T20:25:26Z + DUMMY: + + P value + + + + JCI85679.f3.jpg + F3 + FIG + fig_title_caption + 54482 + The 1E6 TCR uses a conserved binding mode to engage A2-ALWGPDPAAA and the APLs. + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9342026 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.99728084 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + + JCI85679.f3.jpg + F3 + FIG + fig_caption + 54562 + (A) Superposition of the 1E6 TCR (multicolored illustration) in complex with all 7 APLs (multicolored sticks) and the A2-ALWGPDPAAA ligand using the HLA-A*0201 (gray illustration) molecule to align all of the structures. The 1E6 TCR and each peptide are colored according to the APL used in the complex as in Figure 1. (B) Position of the 1E6 TCR CDR loops (multicolored lines) in each complex. The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The Cα backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 α1 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface). + + 0.999629 + experimental_method + cleaner0 + 2023-07-20T08:07:59Z + MESH: + + Superposition + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T18:02:50Z + + in complex with + + + 0.8016965 + chemical + cleaner0 + 2023-07-19T16:29:10Z + CHEBI: + + APLs + + + 0.6889396 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.9861517 + protein + cleaner0 + 2023-07-19T20:44:14Z + PR: + + HLA-A*0201 + + + 0.66666234 + experimental_method + cleaner0 + 2023-07-19T21:06:42Z + MESH: + + align + + + 0.9962159 + evidence + cleaner0 + 2023-07-19T20:47:43Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:29:17Z + + APL + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9996439 + structure_element + cleaner0 + 2023-07-19T17:58:59Z + SO: + + CDR loops + + + 0.99761826 + chemical + cleaner0 + 2023-07-19T16:08:20Z + CHEBI: + + ALWGPDPAAA + + + site + SO: + cleaner0 + 2023-07-20T08:08:20Z + + HLA-A*0201 binding groove + + + 0.9049958 + chemical + cleaner0 + 2023-07-19T16:29:17Z + CHEBI: + + APL + + + 0.9774972 + protein + cleaner0 + 2023-07-19T20:44:17Z + PR: + + HLA-A*0201 + + + 0.9996949 + structure_element + cleaner0 + 2023-07-20T08:10:49Z + SO: + + α1 helices + + + 0.9993441 + evidence + cleaner0 + 2023-07-19T20:47:49Z + DUMMY: + + Crossing angle + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9979407 + chemical + cleaner0 + 2023-07-19T16:08:20Z + CHEBI: + + ALWGPDPAAA + + + 0.99416566 + protein_state + cleaner0 + 2023-07-20T08:02:23Z + DUMMY: + + bound in + + + site + SO: + cleaner0 + 2023-07-19T20:43:29Z + + HLA-A*0201 binding groove + + + + JCI85679.f4.jpg + F4 + FIG + fig_title_caption + 55414 + A conserved interaction with a GPD motif underpins the 1E6 TCR interaction with the APLs. + + 0.9997474 + structure_element + cleaner0 + 2023-07-19T18:05:06Z + SO: + + GPD motif + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.94330746 + chemical + cleaner0 + 2023-07-19T16:29:11Z + CHEBI: + + APLs + + + + JCI85679.f4.jpg + F4 + FIG + fig_caption + 55504 + Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). The rest of the peptide, and the MHCα1 helix, are shown as a gray illustration. + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.99990344 + residue_name_number + cleaner0 + 2023-07-19T18:00:26Z + DUMMY: + + Tyr97α + + + 0.9999007 + residue_name_number + cleaner0 + 2023-07-19T20:04:48Z + DUMMY: + + Tyr97β + + + 0.63116497 + complex_assembly + cleaner0 + 2023-07-19T20:53:38Z + GO: + + TCR + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T18:02:50Z + + in complex with + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:29:11Z + + APLs + + + 0.99554044 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + structure_element + SO: + cleaner0 + 2023-07-19T21:07:22Z + + GPD peptide motif + + + chemical + CHEBI: + cleaner0 + 2023-07-19T16:29:11Z + + APLs + + + 0.99627393 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.98936254 + protein_state + cleaner0 + 2023-07-19T18:02:50Z + DUMMY: + + in complex with + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:11:09Z + + MHC + + + structure_element + SO: + cleaner0 + 2023-07-20T08:11:19Z + + α1 helix + + + + JCI85679.f5.jpg + F5 + FIG + fig_title_caption + 55989 + The 1E6 TCR makes distinct peptide contacts with peripheral APL residues. + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9978986 + chemical + cleaner0 + 2023-07-19T16:29:17Z + CHEBI: + + APL + + + + JCI85679.f5.jpg + F5 + FIG + fig_caption + 56063 + Interactions between the 1E6 TCR and peptide residues outside of the conserved GPD motif. The MHCα1 helix is shown in gray illustrations. Hydrogen bonds are shown as red dotted lines; van der Waals (vdW) contacts are shown as black dotted lines. Boxes show total contacts between the 1E6 TCR and each peptide ligand. (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.99947935 + protein_state + cleaner0 + 2023-07-20T08:02:45Z + DUMMY: + + conserved + + + 0.9997277 + structure_element + cleaner0 + 2023-07-19T18:05:06Z + SO: + + GPD motif + + + complex_assembly + GO: + cleaner0 + 2023-07-20T08:03:02Z + + MHC + + + structure_element + SO: + cleaner0 + 2023-07-20T08:03:13Z + + α1 helix + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:18Z + + Hydrogen bonds + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:18Z + + van der Waals (vdW) contacts + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9976714 + chemical + cleaner0 + 2023-07-19T16:12:10Z + CHEBI: + + A2-MVWGPDPLYV + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.99183035 + chemical + cleaner0 + 2023-07-19T16:16:11Z + CHEBI: + + A2-YLGGPDFPTI + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9978401 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.99737835 + chemical + cleaner0 + 2023-07-19T18:34:05Z + CHEBI: + + A2-AQWGPDPAAA + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.99795914 + chemical + cleaner0 + 2023-07-19T16:12:48Z + CHEBI: + + A2-RQFGPDWIVA + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9971487 + chemical + cleaner0 + 2023-07-19T16:16:24Z + CHEBI: + + A2-RQWGPDPAAV + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.99718714 + chemical + cleaner0 + 2023-07-19T18:33:55Z + CHEBI: + + A2-YQFGPDFPTA + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9949441 + chemical + cleaner0 + 2023-07-19T16:12:27Z + CHEBI: + + A2-RQFGPDFPTI + + + + JCI85679.f6.jpg + F6 + FIG + fig_title_caption + 57380 + Comparison of ligated and unligated APLs. + + 0.9996076 + protein_state + cleaner0 + 2023-07-19T20:05:06Z + DUMMY: + + ligated + + + 0.99960905 + protein_state + cleaner0 + 2023-07-19T16:17:41Z + DUMMY: + + unligated + + + 0.64326084 + chemical + cleaner0 + 2023-07-19T16:29:11Z + CHEBI: + + APLs + + + + JCI85679.f6.jpg + F6 + FIG + fig_caption + 57422 + Superposition of each APL in unligated form and ligated to the 1E6 TCR. All unligated pMHCs are shown as light green illustrations. Peptide sequences are shown underneath each structure aligned with the peptide structure. Black arrows denote the direction of the side chain. Upward arrows indicates solvent exposed, downward arrows indicates anchor position, and no arrow indicates an intermediate position. (A) A2-MVWGPDPLYV (black sticks). A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks). + + 0.99966896 + experimental_method + cleaner0 + 2023-07-20T08:08:32Z + MESH: + + Superposition + + + 0.9908853 + chemical + cleaner0 + 2023-07-19T16:29:17Z + CHEBI: + + APL + + + 0.99965394 + protein_state + cleaner0 + 2023-07-19T16:17:41Z + DUMMY: + + unligated + + + 0.99964714 + protein_state + cleaner0 + 2023-07-19T20:05:06Z + DUMMY: + + ligated + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9996667 + protein_state + cleaner0 + 2023-07-19T16:17:41Z + DUMMY: + + unligated + + + 0.9887504 + complex_assembly + cleaner0 + 2023-07-19T16:28:11Z + GO: + + pMHCs + + + 0.9992514 + evidence + cleaner0 + 2023-07-19T20:47:54Z + DUMMY: + + structure + + + 0.66176826 + evidence + cleaner0 + 2023-07-19T20:48:20Z + DUMMY: + + structure + + + 0.9982674 + chemical + cleaner0 + 2023-07-19T16:12:10Z + CHEBI: + + A2-MVWGPDPLYV + + + 0.99989426 + residue_name_number + cleaner0 + 2023-07-19T20:04:53Z + DUMMY: + + Tyr8 + + + 0.9996716 + protein_state + cleaner0 + 2023-07-19T20:05:05Z + DUMMY: + + ligated + + + 0.99966383 + protein_state + cleaner0 + 2023-07-19T16:17:41Z + DUMMY: + + unligated + + + 0.99798316 + chemical + cleaner0 + 2023-07-19T16:16:11Z + CHEBI: + + A2-YLGGPDFPTI + + + 0.9979634 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.9983786 + chemical + cleaner0 + 2023-07-19T18:34:47Z + CHEBI: + + A2-AQWGPDPAAA + + + 0.99870086 + chemical + cleaner0 + 2023-07-19T16:12:48Z + CHEBI: + + A2-RQFGPDWIVA + + + 0.9987801 + chemical + cleaner0 + 2023-07-19T16:16:24Z + CHEBI: + + A2-RQWGPDPAAV + + + 0.9979759 + chemical + cleaner0 + 2023-07-19T18:34:39Z + CHEBI: + + A2-YQFGPDFPTA + + + 0.99866563 + chemical + cleaner0 + 2023-07-19T16:12:28Z + CHEBI: + + A2-RQFGPDFPTI + + + + JCI85679.f7.jpg + F7 + FIG + fig_title_caption + 58250 + The 1E6 TCR makes distinct peptide contacts with the MHC surface depending on the peptide cargo. + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9990809 + site + cleaner0 + 2023-07-20T07:55:06Z + SO: + + MHC surface + + + + JCI85679.f7.jpg + F7 + FIG + fig_caption + 58347 + Interactions between the 1E6 TCR and the MHC α1 helix residues Arg65, Lys66, and Gln72. Hydrogen bonds are shown as red dotted lines; vdW contacts are shown as black dotted lines. MHCα1 helix are shown in gray illustrations. Boxes show total contacts between the 1E6 TCR and these key residues (green boxes are MHC residues; white boxes are TCR residues). (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.835831 + complex_assembly + cleaner0 + 2023-07-20T07:49:28Z + GO: + + MHC + + + 0.9996875 + structure_element + cleaner0 + 2023-07-20T08:11:24Z + SO: + + α1 helix + + + 0.99990416 + residue_name_number + cleaner0 + 2023-07-19T18:07:16Z + DUMMY: + + Arg65 + + + 0.9999031 + residue_name_number + cleaner0 + 2023-07-19T20:05:14Z + DUMMY: + + Lys66 + + + 0.999902 + residue_name_number + cleaner0 + 2023-07-19T18:10:13Z + DUMMY: + + Gln72 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:18Z + + Hydrogen bonds + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:18Z + + vdW + + + complex_assembly + GO: + cleaner0 + 2023-07-20T07:55:49Z + + MHC + + + structure_element + SO: + cleaner0 + 2023-07-20T07:56:02Z + + α1 helix + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-20T07:49:28Z + + MHC + + + 0.99735093 + complex_assembly + cleaner0 + 2023-07-19T20:53:38Z + GO: + + TCR + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9922688 + chemical + cleaner0 + 2023-07-19T16:12:10Z + CHEBI: + + A2-MVWGPDPLYV + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9202607 + chemical + cleaner0 + 2023-07-19T16:16:11Z + CHEBI: + + A2-YLGGPDFPTI + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.99592036 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9899547 + chemical + cleaner0 + 2023-07-19T19:59:25Z + CHEBI: + + A2-AQWGPDPAAA + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9949196 + chemical + cleaner0 + 2023-07-19T16:12:48Z + CHEBI: + + A2-RQFGPDWIVA + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.992281 + chemical + cleaner0 + 2023-07-19T16:16:24Z + CHEBI: + + A2-RQWGPDPAAV + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.9855128 + chemical + cleaner0 + 2023-07-19T19:59:37Z + CHEBI: + + A2-YQFGPDFPTA + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.98272467 + chemical + cleaner0 + 2023-07-19T16:12:28Z + CHEBI: + + A2-RQFGPDFPTI + + + + JCI85679.f8.jpg + F8 + FIG + fig_title_caption + 59707 + Thermodynamic analysis of the 1E6 TCR with A2-ALWGPDPAAA and the APLs. + + 0.9992712 + experimental_method + cleaner0 + 2023-07-20T08:08:39Z + MESH: + + Thermodynamic analysis + + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.902253 + chemical + cleaner0 + 2023-07-19T16:16:50Z + CHEBI: + + A2-ALWGPDPAAA + + + 0.99184835 + chemical + cleaner0 + 2023-07-19T16:29:11Z + CHEBI: + + APLs + + + + JCI85679.f8.jpg + F8 + FIG + fig_caption + 59778 + Eight serial dilutions of the 1E6 TCR were injected, in duplicate, over each immobilized APL and A2-ALW at 5°C, 13°C, 18°C, 25°C, 30°C, and 37°C. The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = [P1x]/[P2 + X]), and thermodynamic parameters were calculated according to the Gibbs-Helmholtz equation (ΔG° = ΔH − TΔS°). The binding free energies, ΔG° (ΔG° = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van’t Hoff equation (RT ln KD = ΔH° – TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0] with T0 = 298 K). (A) 1E6-A2-ALWGPDPAAA; (B) 1E6-A2-AQWGPDPAAA; (C) 1E6-A2-RQFGPDWIVA; (D) 1E6-A2-RQWGPDPAAV, (E) 1E6-A2-YQFGPDFPTA; and (F) 1E6-A2-RQFGPDFPTI. + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:54:52Z + + 1E6 TCR + + + 0.84157336 + chemical + cleaner0 + 2023-07-19T16:29:17Z + CHEBI: + + APL + + + 0.9946752 + chemical + cleaner0 + 2023-07-19T20:42:55Z + CHEBI: + + A2-ALW + + + 0.99944896 + evidence + cleaner0 + 2023-07-19T20:48:28Z + DUMMY: + + equilibrium binding constant + + + 0.9995074 + evidence + cleaner0 + 2023-07-19T16:17:17Z + DUMMY: + + KD + + + 0.97272396 + experimental_method + cleaner0 + 2023-07-20T08:08:43Z + MESH: + + nonlinear curve fit + + + experimental_method + MESH: + cleaner0 + 2023-07-19T20:00:21Z + + Gibbs-Helmholtz equation + + + 0.9981754 + evidence + cleaner0 + 2023-07-19T20:48:41Z + DUMMY: + + ΔG° + + + 0.99414825 + evidence + cleaner0 + 2023-07-19T20:49:03Z + DUMMY: + + ΔH + + + 0.6460132 + evidence + cleaner0 + 2023-07-19T20:49:06Z + DUMMY: + + TΔS° + + + 0.9993293 + evidence + cleaner0 + 2023-07-19T20:49:08Z + DUMMY: + + binding free energies + + + 0.9992066 + evidence + cleaner0 + 2023-07-19T20:48:52Z + DUMMY: + + ΔG° + + + 0.998804 + evidence + cleaner0 + 2023-07-19T20:49:00Z + DUMMY: + + ΔG° + + + 0.9961696 + experimental_method + cleaner0 + 2023-07-20T08:08:48Z + MESH: + + nonlinear regression + + + experimental_method + MESH: + cleaner0 + 2023-07-19T20:02:29Z + + van’t Hoff equation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:50:04Z + + RT ln KD + + + 0.9869153 + evidence + cleaner0 + 2023-07-19T20:49:19Z + DUMMY: + + ΔH° + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:02:12Z + + TΔS° + + + 0.9905539 + evidence + cleaner0 + 2023-07-19T20:49:24Z + DUMMY: + + ΔCp° + + + evidence + DUMMY: + cleaner0 + 2023-07-19T20:50:20Z + + TΔCp° + + + 0.99468195 + complex_assembly + cleaner0 + 2023-07-19T16:14:43Z + GO: + + 1E6-A2-ALWGPDPAAA + + + 0.9910844 + complex_assembly + cleaner0 + 2023-07-19T20:01:23Z + GO: + + 1E6-A2-AQWGPDPAAA + + + 0.99196416 + complex_assembly + cleaner0 + 2023-07-19T20:01:31Z + GO: + + 1E6-A2-RQFGPDWIVA + + + 0.99309886 + complex_assembly + cleaner0 + 2023-07-19T20:01:39Z + GO: + + 1E6-A2-RQWGPDPAAV + + + 0.992071 + complex_assembly + cleaner0 + 2023-07-19T20:01:49Z + GO: + + 1E6-A2-YQFGPDFPTA + + + 0.9890793 + complex_assembly + cleaner0 + 2023-07-19T16:19:13Z + GO: + + 1E6-A2-RQFGPDFPTI + + + + + T2 + TABLE + table_title_caption + 60546 + 1E6 TCR-pMHC contacts, affinity measurements and thermodynamics + + complex_assembly + GO: + cleaner0 + 2023-07-19T20:03:08Z + + 1E6 TCR-pMHC + + + 0.9994793 + experimental_method + cleaner0 + 2023-07-19T20:03:19Z + MESH: + + affinity measurements + + + experimental_method + MESH: + cleaner0 + 2023-07-19T20:03:30Z + + thermodynamics + + + + + T1 + TABLE + table_title_caption + 60610 + Peptides used in this study + + + diff --git a/BioC_XML/4887326_v0.xml b/BioC_XML/4887326_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..af851878418834fc531dfa519621726d96365899 --- /dev/null +++ b/BioC_XML/4887326_v0.xml @@ -0,0 +1,9011 @@ + + + + PMC + 20140719 + pmc.key + + 4887326 + CC BY + no + 0 + 0 + + 10.1007/s13238-016-0264-7 + 4887326 + 27113583 + 264 + 403 + 6 + the YfiBNR system c-di-GMP Vitamin B6 L-Trp peptidoglycan layer bioflim formation + +Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. + 416 + surname:Xu;given-names:Min + surname:Yang;given-names:Xuan + surname:Yang;given-names:Xiu-An + surname:Zhou;given-names:Lei + surname:Liu;given-names:Tie-Zheng + surname:Fan;given-names:Zusen + surname:Jiang;given-names:Tao + TITLE + KEYWORDS + front + 7 + 2016 + 0 + Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + Pseudomonas aeruginosa + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T11:33:15Z + + YfiBNR + + + + ABSTRACT + abstract + 94 + YfiBNR is a recently identified bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T11:33:15Z + + YfiBNR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:33:30Z + + bis-(3’-5’)-cyclic dimeric GMP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:33:38Z + + c-di-GMP + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:11Z + + YfiN + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:11Z + + YfiN + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T12:55:59Z + + c-di-GMP + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:06Z + + crystal structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:14Z + + alone + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:21Z + + active + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:27Z + + mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:43Z + + complexed with + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:52Z + + Structural analyses + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:03Z + + compact conformation + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:11Z + + dimeric + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:14Z + + alone + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:20Z + + stretched conformation + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:28Z + + activated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:36:36Z + + peptidoglycan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:01Z + + PG + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T11:36:51Z + + PG-binding pocket + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:57Z + + PG binding affinity + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:37:06Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:37:15Z + + crystallographic analyses + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:37:22Z + + Vitamin B6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:05Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:08Z + + L-Trp + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T11:38:14Z + + YfiB-binding site + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:11Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:14Z + + L-Trp + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:09Z + + structural and biochemical data + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T11:33:15Z + + YfiBNR + + + + INTRO + title_1 + 1498 + INTRODUCTION + + + INTRO + paragraph + 1511 + Bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) is a ubiquitous second messenger that bacteria use to facilitate behavioral adaptations to their ever-changing environment. An increase in c-di-GMP promotes biofilm formation, and a decrease results in biofilm degradation (Boehm et al.,; Duerig et al.,; Hickman et al.,; Jenal,; Romling et al.,). The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). Intriguingly, studies in diverse species have revealed that a single bacterium can have dozens of DGCs and PDEs (Hickman et al.,; Kirillina et al.,; Kulasakara et al.,; Tamayo et al.,). In Pseudomonas aeruginosa in particular, 42 genes containing putative DGCs and/or PDEs were identified (Kulasakara et al.,). The functional role of a number of downstream effectors of c-di-GMP has been characterized as affecting exopolysaccharide (EPS) production, transcription, motility, and surface attachment (Caly et al.,; Camilli and Bassler,; Ha and O’Toole,; Pesavento and Hengge,). However, due to the intricacy of c-di-GMP signaling networks and the diversity of experimental cues, the detailed mechanisms by which these signaling pathways specifically sense and integrate different inputs remain largely elusive. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:33:30Z + + Bis-(3’-5’)-cyclic dimeric GMP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:33:38Z + + c-di-GMP + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T12:56:28Z + + bacteria + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T12:55:59Z + + c-di-GMP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T12:55:59Z + + c-di-GMP + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T12:56:36Z + + diguanylate cyclases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T12:56:42Z + + DGCs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:33:38Z + + c-di-GMP + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T12:56:50Z + + phosphodiesterases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T12:56:57Z + + PDEs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T12:55:59Z + + c-di-GMP + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T12:57:53Z + + N-terminal domain + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T12:57:05Z + + relatively conserved + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T12:57:11Z + + GGDEF motif + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T12:56:42Z + + DGCs + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T12:57:44Z + + EAL/HD-GYP domains + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T12:56:57Z + + PDEs + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T12:57:19Z + + bacterium + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T12:56:42Z + + DGCs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T12:56:57Z + + PDEs + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + Pseudomonas aeruginosa + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T12:56:42Z + + DGCs + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T12:56:57Z + + PDEs + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:33:38Z + + c-di-GMP + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T12:57:28Z + + exopolysaccharide + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T12:57:35Z + + EPS + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:33:38Z + + c-di-GMP + + + + INTRO + paragraph + 3186 + Biofilm formation protects pathogenic bacteria from antibiotic treatment, and c-di-GMP-regulated biofilm formation has been extensively studied in P. aeruginosa (Evans,; Kirisits et al.,; Malone,; Reinhardt et al.,). In the lungs of cystic fibrosis (CF) patients, adherent biofilm formation and the appearance of small colony variant (SCV) morphologies of P. aeruginosa correlate with prolonged persistence of infection and poor lung function (Govan and Deretic,; Haussler et al.,; Haussler et al.,; Parsek and Singh,; Smith et al.,). Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,). The YfiBNR system contains three protein members and modulates intracellular c-di-GMP levels in response to signals received in the periplasm (Malone et al.,). More recently, this system was also reported in other Gram-negative bacteria, such as Escherichia coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), Klebsiella pneumonia (Huertas et al.,) and Yersinia pestis (Ren et al.,). YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). YfiN is repressed by specific interaction between its periplasmic PAS domain and the periplasmic protein YfiR (Malone et al.,). YfiB is an OmpA/Pal-like outer-membrane lipoprotein (Parsons et al.,) that can activate YfiN by sequestering YfiR (Malone et al.,) in an unknown manner. Whether YfiB directly recruits YfiR or recruits YfiR via a third partner is an open question. After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,). + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T12:56:28Z + + bacteria + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + P. aeruginosa + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + P. aeruginosa + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:03:59Z + + tripartite + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:24:01Z + + c-di-GMP + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T11:33:15Z + + YfiBNR + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T13:04:19Z + + AwsXRO + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T13:04:22Z + + Tbp + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:04:31Z + + genetic screening + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + P. aeruginosa PAO1 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T11:33:15Z + + YfiBNR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:33:38Z + + c-di-GMP + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:35:04Z + + Gram-negative bacteria + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + Escherichia coli + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + Klebsiella pneumonia + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + Yersinia pestis + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:11Z + + YfiN + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:06:10Z + + transmembrane helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:42:41Z + + Per-Arnt-Sim + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:06:27Z + + PAS + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:06:48Z + + HAMP domain + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:06:55Z + + histidine kinases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:06:58Z + + adenylyl cyclases + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:07:00Z + + methyl-accepting chemotaxis proteins + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:07:03Z + + phosphatases + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:42:46Z + + GGDEF domain + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:08:50Z + + DGC + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:11Z + + YfiN + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:10:58Z + + repressed by + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:10:22Z + + PAS domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:10:50Z + + OmpA/Pal-like + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:10:56Z + + lipoprotein + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:11Z + + YfiN + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:33:38Z + + c-di-GMP + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:28Z + + activated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:11Z + + YfiN + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T13:11:09Z + + Pel + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T13:11:16Z + + Psl + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T13:11:22Z + + EPSs + + + + INTRO + paragraph + 5437 + It has been reported that the activation of YfiN may be induced by redox-driven misfolding of YfiR (Giardina et al.,; Malone et al.,; Malone et al.,). It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). In addition, quorum sensing-related dephosphorylation of the PAS domain of YfiN may also be involved in the regulation (Ueda and Wood,; Xu et al.,). Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:11Z + + YfiN + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T09:24:38Z + + YfiB + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:13:48Z + + mutagenesis studies + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:42:57Z + + activation residues + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:13:42Z + + predicted + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:43:21Z + + first helix + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:43:32Z + + periplasmic domain + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:43:41Z + + PAS domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:11Z + + YfiN + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:19Z + + crystal structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:13:56Z + + non-oxidized + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:11:03Z + + oxidized + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:14:32Z + + disulfide bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:24Z + + Cys71 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:31Z + + Cys110 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:12Z + + Cys145 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:18Z + + Cys152 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:12Z + + Cys145 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:18Z + + Cys152 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + + INTRO + paragraph + 6386 + In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. Most recently, Li and coworkers reported the crystal structures of YfiB (27–168) alone and YfiRC71S in complex with YfiB (59–168) (Li et al.,). Compared with the reported complex structure, YfiBL43P in our YfiB-YfiR complex structure has additional visible N-terminal residues 44–58 that are shown to play essential roles in YfiB activation and biofilm formation. Therefore, we are able to visualize the detailed allosteric arrangement of the N-terminal structure of YfiB and its important role in YfiB-YfiR interaction. In addition, we found that the YfiBL43P shows a much higher PG-binding affinity than wild-type YfiB, most likely due to its more compact PG-binding pocket. Moreover, we found that Vitamin B6 (VB6) or L-Trp can bind YfiR with an affinity in the ten millimolar range. Together with functional data, these results provide new mechanistic insights into how activated YfiB sequesters YfiR and releases the suppression of YfiN. These findings may facilitate the development and optimization of anti-biofilm drugs for the treatment of chronic infections. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:28Z + + crystal structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:35:23Z + + truncated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:38:46Z + + 34–168 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:35:33Z + + in complex with + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:21Z + + active + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:27Z + + mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:28Z + + crystal structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:38:52Z + + 27–168 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:14Z + + alone + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:39:23Z + + YfiRC71S + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:35:33Z + + in complex with + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:38:55Z + + 59–168 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T13:36:20Z + + YfiB-YfiR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:37:31Z + + structure + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:38:57Z + + 44–58 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T10:51:22Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-06-14T09:13:35Z + + YfiB-YfiR + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:46:46Z + + PG-binding affinity + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:37:06Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:49Z + + YfiB + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T11:36:51Z + + PG-binding pocket + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:37:22Z + + Vitamin B6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:20Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:23Z + + L-Trp + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:39Z + + affinity + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:28Z + + activated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:00Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:11Z + + YfiN + + + + RESULTS + title_1 + 7619 + RESULTS + + + RESULTS + title_2 + 7627 + Overall structure of YfiB + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:37:28Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + + RESULTS + paragraph + 7653 + We obtained two crystal forms of YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33), crystal forms I and II, belonging to space groups P21 and P41, respectively. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:38:39Z + + crystal forms + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:38:47Z + + 34–168 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:11:28Z + + lacking + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:39:01Z + + signal peptide + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:39:03Z + + 1–26 + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:39:06Z + + 27–33 + + + + 13238_2016_264_Fig1_HTML.jpg + Fig1 + FIG + fig_caption + 7871 + Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:40:01Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:39:59Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:39:54Z + + monomer + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:39:54Z + + monomer + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:40:04Z + + analytical ultracentrifugation + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:37:06Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + + 13238_2016_264_Fig2_HTML.jpg + Fig2 + FIG + fig_caption + 8090 + Two dimeric types of YfiB dimer. (A–C) The “head to head” dimer. (D–F) The “back to back” dimer. (A) and (E) indicate the front views of the two dimers, (B) and (F) indicate the top views of the two dimers, and (C) and (D) indicate the details of the two dimeric interfaces + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:11Z + + dimeric + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:12Z + + dimer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:29Z + + head to head + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:12Z + + dimer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:35Z + + back to back + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:21Z + + dimers + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:21Z + + dimers + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:41:54Z + + dimeric interfaces + + + + RESULTS + paragraph + 8376 + The crystal structure of YfiB monomer consists of a five-stranded β-sheet (β1-2-5-3-4) flanked by five α-helices (α1–5) on one side. In addition, there is a short helix turn connecting the β4 strand and α4 helix (Fig. 1A and 1B). Each crystal form contains three different dimeric types of YfiB, two of which are present in both, suggesting that the rest of the dimeric types may result from crystal packing. Here, we refer to the two dimeric types as “head to head” and “back to back” according to the interacting mode (Fig. 2A and 2E), with the total buried surface areas being 316.8 Å2 and 554.3 Å2, respectively. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:19Z + + crystal structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:39:55Z + + monomer + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:43:04Z + + five-stranded β-sheet + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:43:09Z + + β1-2-5-3-4 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:43:14Z + + five α-helices + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:43:17Z + + α1–5 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:43:20Z + + helix turn + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:43:28Z + + β4 strand + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:43:31Z + + α4 helix + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:11Z + + dimeric + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:11Z + + dimeric + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:11Z + + dimeric + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:29Z + + head to head + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:36Z + + back to back + + + + RESULTS + paragraph + 9014 + The “head to head” dimer exhibits a clamp shape. The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C). + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:29Z + + head to head + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:11:35Z + + clamp shape + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:40Z + + hydrophobic interactions + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:44:27Z + + A37 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:44:33Z + + I40 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:44:53Z + + α1 helices + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:44:39Z + + L50 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:44:55Z + + β1 strands + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:44:46Z + + W55 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:44:58Z + + β2 strands + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:41:13Z + + hydrophobic interacting core + + + + RESULTS + paragraph + 9293 + The “back to back” dimer presents a Y shape. The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:36Z + + back to back + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:46:00Z + + Y shape + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-07-21T08:13:24Z + + dimeric + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:40Z + + interaction is mainly hydrophilic + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:46:21Z + + N68 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:46:27Z + + L69 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:46:33Z + + D70 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:46:38Z + + R71 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:46:42Z + + α2-α3 loops + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:46:47Z + + R116 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:46:52Z + + S120 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T13:46:55Z + + α4 helices + + + site + SO: + melaniev@ebi.ac.uk + 2023-06-14T09:12:22Z + + hydrogen bond network + + + + RESULTS + title_2 + 9606 + The YfiB-YfiR interaction + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-06-14T09:14:18Z + + YfiB-YfiR + + + + 13238_2016_264_Fig3_HTML.jpg + Fig3 + FIG + fig_caption + 9632 + Overall structure of the YfiB-YfiR complex and the conserved surface in YfiR. (A) The overall structure of the YfiB-YfiR complex. The YfiBL43P molecules are shown in cyan and yellow. The YfiR molecules are shown in green and magenta. Two interacting regions are highlighted by red rectangles. (B) Structural superposition of apo YfiB and YfiR-bound YfiBL43P. To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. The residues proposed to contribute to YfiB activation are illustrated in sticks. The key residues in apo YfiB are shown in red and those in YfiBL43P are shown in blue. (D) Close-up views showing interactions in regions I and II. YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. The red sticks, which represent the YfiB-interacting residues, are also responsible for the proposed interactions with YfiN + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:44Z + + structure + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T13:36:21Z + + YfiB-YfiR + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:48:07Z + + conserved surface + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:47Z + + structure + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T13:36:21Z + + YfiB-YfiR + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:30:57Z + + Structural superposition + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:38Z + + YfiR-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:38Z + + YfiR-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:22:46Z + + 34–70 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:38Z + + YfiR-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:22:50Z + + 44–70 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:38Z + + YfiR-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T09:25:27Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:25:17Z + + regions I and II + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:48:07Z + + conserved surface + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:25:22Z + + residues + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:41:34Z + + YfiN-interacting residues + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:41:39Z + + YfiB-interacting residues + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:12Z + + YfiN + + + + RESULTS + paragraph + 10966 + To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,). It is likely that these residues may be involved in the conformational changes of YfiB that are related to YfiR sequestration (Fig. 3C). Therefore, we constructed two such single mutants of YfiB (YfiBL43P and YfiBF48S). As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-06-14T09:15:02Z + + YfiB-YfiR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:39:41Z + + co-expressed + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:38:47Z + + 34–168 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:22:55Z + + 35–190 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:11:42Z + + lacking + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:25:25Z + + signal peptide + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:11:57Z + + no stable + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T13:36:21Z + + YfiB-YfiR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:39:45Z + + single mutants of + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:23:15Z + + Q39 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:27:28Z + + L43 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:20:18Z + + F48 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:44:46Z + + W55 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T09:25:43Z + + YfiB + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + P. aeruginosa PAO1 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T09:25:55Z + + YfiR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:13:09Z + + constructed two such single mutants + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:39:30Z + + YfiBF48S + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:12:40Z + + stable + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:12:45Z + + complex with + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:13:16Z + + crystalized + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:35:33Z + + in complex with + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:27Z + + mutant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:51Z + + structure + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:13:06Z + + molecular replacement + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + + RESULTS + paragraph + 11897 + The YfiB-YfiR complex is a 2:2 heterotetramer (Fig. 3A) in which the YfiR dimer is clamped by two separated YfiBL43P molecules with a total buried surface area of 3161.2 Å2. The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. The observed changes in conformation of YfiB and the results of mutagenesis suggest a mechanism by which YfiB sequesters YfiR. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T13:36:21Z + + YfiB-YfiR + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:41:01Z + + heterotetramer + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:13:56Z + + non-oxidized + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:14Z + + alone + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:12Z + + Cys145 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:18Z + + Cys152 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:12:38Z + + disulfide bonds + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:24Z + + Cys71 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:31Z + + Cys110 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:14:32Z + + disulfide bond + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T13:36:21Z + + YfiB-YfiR + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:46:33Z + + D70 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:14:03Z + + α1 helix + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:14:23Z + + 34–43 + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:58Z + + electron density map + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:25:30Z + + α2 helix + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:25:33Z + + β1 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:25:36Z + + β2 strands + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:19:31Z + + loop + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:25:41Z + + α-helix turns + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:39:51Z + + mutagenesis + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + + RESULTS + paragraph + 12877 + The YfiB-YfiR interface can be divided into two regions (Fig. 3A and 3D). Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). These two regions contribute a robust hydrogen-bonding network to the YfiB-YfiR interface, resulting in a tightly bound complex. + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:23:18Z + + YfiB-YfiR interface + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:20:25Z + + Region I + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:40Z + + hydrophilic interactions + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:19:07Z + + E45 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:19:15Z + + G47 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:19:22Z + + E53 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:19:31Z + + loop + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:19:52Z + + S57 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:19:58Z + + R60 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:20:04Z + + A89 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:20:10Z + + H177 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:41:44Z + + hydrophobic anchoring sites + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:20:25Z + + region I + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:20:18Z + + F48 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:44:46Z + + W55 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:41:49Z + + hydrophobic cores + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:19:52Z + + S57 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:20:43Z + + Q88 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:20:04Z + + A89 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:20:53Z + + N90 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:19:58Z + + R60 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:05Z + + R175 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:20:10Z + + H177 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:14Z + + F57 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:23:36Z + + hydrophobic pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:23Z + + L166 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:29Z + + I169 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:36Z + + V176 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:43Z + + P178 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:49Z + + L181 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:21:57Z + + region II + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:22:08Z + + R96 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:22:15Z + + E98 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:22:23Z + + E157 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:22:33Z + + E163 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:22:40Z + + S146 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:22:48Z + + R171 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:22:55Z + + I163 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:23:02Z + + V165 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:40Z + + hydrogen bonds + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:23Z + + L166 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:23:22Z + + A164 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:23:26Z + + P166 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:23:09Z + + R185 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:24:13Z + + hydrogen-bonding network + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:23:18Z + + YfiB-YfiR interface + + + + RESULTS + paragraph + 14067 + Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P. The results showed that wild-type YfiB exists in both monomeric and dimeric states in solution, while YfiBL43P primarily adopts the monomer state in solution (Fig. 1C–D). This suggests that the N-terminus of YfiB plays an important role in forming the dimeric YfiB in solution and that the conformational change of residue L43 is associated with the stretch of the N-terminus and opening of the dimer. Therefore, it is possible that both dimeric types might exist in solution. For simplicity, we only discuss the “head to head” dimer in the following text. + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:27:48Z + + structure + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:01Z + + YfiR + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:27:12Z + + analytical ultracentrifugation + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:37:06Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:37:06Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:41:06Z + + monomeric + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:11Z + + dimeric + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:39:55Z + + monomer + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:11Z + + dimeric + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:27:28Z + + L43 + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:11Z + + dimeric + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:29Z + + head to head + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + + RESULTS + title_2 + 14862 + The PG-binding site of YfiB + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:28:20Z + + PG-binding site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + + 13238_2016_264_Fig4_HTML.jpg + Fig4 + FIG + fig_caption + 14890 + The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. (B) Close-up view showing the key residues of Pal interacting with the m-Dap5 ε-carboxylate group of PG-P. Pal is shown in wheat and PG-P is in magenta. (C) Close-up view showing the key residues of YfiR-bound YfiBL43P interacting with a sulfate ion. YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:28:20Z + + PG-binding site + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:30:57Z + + Structural superposition + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:31:43Z + + PG-binding sites + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + H. influenzae + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T14:31:27Z + + Pal/PG-P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:38Z + + YfiR-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:43Z + + complexed with + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:32:02Z + + sulfate + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:31:16Z + + Pal + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:28Z + + m-Dap5 ε-carboxylate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:32:13Z + + PG-P + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:31:16Z + + Pal + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:32:13Z + + PG-P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:38Z + + YfiR-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:32:02Z + + sulfate + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:38Z + + YfiR-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:32:02Z + + sulfate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:31:56Z + + water + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:30:57Z + + Structural superposition + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:31:43Z + + PG-binding sites + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:38Z + + YfiR-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + Apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:39Z + + YfiR-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:46:26Z + + MST + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:57Z + + binding affinities + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:37:06Z + + wild-type + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:32:25Z + + PG + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:39:57Z + + sequence alignment + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + P. aeruginosa + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + E. coli + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:31:16Z + + Pal + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:43:32Z + + periplasmic domain + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:32:45Z + + OmpA + + + + RESULTS + paragraph + 15823 + PG-associated lipoprotein (Pal) is highly conserved in Gram-negative bacteria and anchors to the outer membrane through an N-terminal lipid attachment and to PG layer through its periplasmic domain, which is implicated in maintaining outer membrane integrity. Previous homology modeling studies suggested that YfiB contains a Pal-like PG-binding site (Parsons et al.,), and the mutation of two residues at this site, D102 and G105, reduces the ability for biofilm formation and surface attachment (Malone et al.,). In the YfiB-YfiR complex, one sulfate ion is found at the bottom of each YfiBL43P molecule (Fig. 3A) and forms a strong hydrogen bond with D102 of YfiBL43P (Fig. 4A and 4C). Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). In the Pal/PG-P complex structure, the m-Dap5 ϵ-carboxylate group interacts with the side-chain atoms of D71 and the main-chain amide of D37 (Fig. 4B). Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). Moreover, a water molecule was found to bridge the sulfate ion and the side chains of N67 and D102, strengthening the hydrogen bond network (Fig. 4C). In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:40:05Z + + PG-associated lipoprotein + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:31:16Z + + Pal + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:40:31Z + + highly conserved + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:35:11Z + + Gram-negative bacteria + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-07-21T08:16:44Z + + PG + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:43:32Z + + periplasmic domain + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:40:51Z + + homology modeling + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:50Z + + YfiB + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:40:00Z + + Pal-like PG-binding site + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:40:40Z + + mutation of two residues + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:29Z + + D102 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:35Z + + G105 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T13:36:21Z + + YfiB-YfiR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:32:02Z + + sulfate + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:40Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:29Z + + D102 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:30:57Z + + Structural superposition + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + Haemophilus influenzae + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:31:16Z + + Pal + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:43Z + + complexed with + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:36Z + + peptidoglycan precursor + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:32:13Z + + PG-P + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:39Z + + UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:42Z + + m-Dap + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:46Z + + meso-diaminopimelate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:32:02Z + + sulfate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:50Z + + m-Dap5 ϵ-carboxylate + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T15:36:46Z + + Pal/PG-P + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T15:36:50Z + + Pal/PG-P + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:39:46Z + + structure + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:37:53Z + + m-Dap5 ϵ-carboxylate + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:43Z + + D71 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:50Z + + D37 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:39Z + + YfiR-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:39:49Z + + structure + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:32:02Z + + sulfate + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:29Z + + D102 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:43Z + + D71 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:31:16Z + + Pal + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:59Z + + R117 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:39:05Z + + R86 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:31:16Z + + Pal + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:46:21Z + + N68 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:50Z + + D37 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:31:16Z + + Pal + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:31:56Z + + water + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:32:02Z + + sulfate + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:39:26Z + + N67 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:29Z + + D102 + + + site + SO: + melaniev@ebi.ac.uk + 2023-06-14T09:16:41Z + + hydrogen bond network + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:40:44Z + + sequence alignment + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:31:16Z + + Pal + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:43:32Z + + periplasmic domain + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:39:31Z + + OmpA + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:28:20Z + + PG-binding site + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:46:21Z + + N68 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:29Z + + D102 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:40:32Z + + highly conserved + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + + RESULTS + paragraph + 17719 + Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. The results indicated that the PG-binding affinity of YfiBL43P is 65.5 μmol/L, which is about 16-fold stronger than that of wild-type YfiB (Kd = 1.1 mmol/L) (Fig. 4E–F). As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:47:10Z + + superposition + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:39Z + + YfiR-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:41:54Z + + PG-binding region + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:47:00Z + + different conformation + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:46:21Z + + N68 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:19:31Z + + loop + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:46:21Z + + N68 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:19:31Z + + loop + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:29Z + + D102 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:38:59Z + + R117 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T11:36:51Z + + PG-binding pocket + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:32:02Z + + sulfate + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:31:56Z + + water + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:27:19Z + + PG + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:46:54Z + + inactive + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:21Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:46:20Z + + microscale thermophoresis + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:46:26Z + + MST + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:46:39Z + + binding affinities + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:38:00Z + + PG + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:37:06Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:46:46Z + + PG-binding affinity + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:37:06Z + + wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-07-21T08:17:12Z + + Kd + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:17:35Z + + in the absence of + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:46:46Z + + PG-binding affinity + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-06-14T09:17:01Z + + YfiB-YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T16:17:54Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:18:26Z + + stretched N-terminal conformation + + + + RESULTS + title_2 + 18822 + The conserved surface in YfiR is functional for binding YfiB and YfiN + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:48:07Z + + conserved surface + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:12Z + + YfiN + + + + RESULTS + paragraph + 18892 + Calculation using the ConSurf Server (http://consurf.tau.ac.il/), which estimates the evolutionary conservation of amino acid positions and visualizes information on the structure surface, revealed a conserved surface on YfiR that contributes to the interaction with YfiB (Fig. 3E and 3F). Interestingly, the majority of this conserved surface contributes to the interaction with YfiB (Fig. 3E and 3F). Malone JG et al. have reported that F151, E163, I169 and Q187, located near the C-terminus of YfiR, comprise a putative YfiN binding site (Malone et al.,). Interestingly, these residues are part of the conserved surface of YfiR (Fig. 3G). F151, E163 and I169 form a hydrophobic core while, Q187 is located at the end of the α6 helix. E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). Collectively, a part of the YfiB-YfiR interface overlaps with the proposed YfiR-YfiN interface, suggesting alteration in the association-disassociation equilibrium of YfiR-YfiN and hence the ability of YfiB to sequester YfiR. + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:40:03Z + + ConSurf Server + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:39:06Z + + evolutionary conservation + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:42:00Z + + structure surface + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:48:07Z + + conserved surface + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:48:07Z + + conserved surface + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:23:32Z + + F151 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:22:33Z + + E163 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:29Z + + I169 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:23:36Z + + Q187 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:42:08Z + + YfiN binding site + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:48:07Z + + conserved surface + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:23:45Z + + F151 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:22:33Z + + E163 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:29Z + + I169 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:51:31Z + + hydrophobic core + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:23:49Z + + Q187 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:25:52Z + + α6 helix + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:22:33Z + + E163 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:29Z + + I169 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:42:11Z + + YfiB-interacting residues + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:22:33Z + + E163 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:21:40Z + + hydrogen bond + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:22:08Z + + R96 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:29Z + + I169 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:23Z + + L166 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:29Z + + I169 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:36Z + + V176 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:43Z + + P178 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:49Z + + L181 + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:51:31Z + + hydrophobic core + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:14Z + + F57 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:23:18Z + + YfiB-YfiR interface + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:42:18Z + + YfiR-YfiN interface + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:12Z + + YfiN + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + + RESULTS + title_2 + 20101 + YfiR binds small molecules + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + + RESULTS + paragraph + 20128 + Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). However, whether YfiR is involved in other regulatory mechanisms is still an open question. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T09:27:42Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:12Z + + YfiN + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:53:31Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:13:56Z + + non-oxidized + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:53:28Z + + oxidized + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:12Z + + Cys145 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:12:18Z + + Cys152 + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T13:14:32Z + + disulfide bond + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + + 13238_2016_264_Fig5_HTML.jpg + Fig5 + FIG + fig_caption + 20602 + Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:55:21Z + + Structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:58:52Z + + VB6-bound + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:59:00Z + + Trp-bound + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:55:52Z + + Superposition + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:55:16Z + + structures + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:58:52Z + + VB6-bound + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:59:00Z + + Trp-bound + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:55:49Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:55:46Z + + L-Trp + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:55:13Z + + electron densities + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:55:38Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:55:35Z + + Trp + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:55:19Z + + |Fo|-|Fc| maps + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:55:24Z + + Superposition + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:23:36Z + + hydrophobic pocket + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:55:44Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:55:41Z + + L-Trp + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:15Z + + F57 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + + RESULTS + paragraph + 21003 + Intriguingly, a Dali search (Holm and Rosenstrom,) indicated that the closest homologs of YfiR shared the characteristic of being able to bind several structurally similar small molecules, such as L-Trp, L-Phe, B-group vitamins and their analogs, encouraging us to test whether YfiR can recognize these molecules. For this purpose, we co-crystallized YfiR or soaked YfiR crystals with different small molecules, including L-Trp and B-group vitamins. Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:59:05Z + + Dali search + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:59:16Z + + L-Trp + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:59:19Z + + L-Phe + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:59:10Z + + co-crystallized + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:59:13Z + + soaked + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:59:33Z + + crystals + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T14:59:22Z + + L-Trp + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:59:31Z + + small-molecule density + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:58:52Z + + VB6-bound + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:59:00Z + + Trp-bound + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:02Z + + YfiR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:28Z + + crystal structures + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-06-14T09:23:30Z + + structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:21Z + + dimers + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:59:45Z + + oxidized + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:59:29Z + + structure + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-03-15T14:12:38Z + + disulfide bonds + + + + 13238_2016_264_Fig6_HTML.jpg + Fig6 + FIG + fig_caption + 21723 + Functional analysis of VB6 and L-Trp. (A and B) The effect of increasing concentrations of VB6 or L-Trp on YfiBL43P-induced attachment (bars). The relative optical density is represented as curves. Wild-type YfiB is used as negative control. (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:01:47Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:01:50Z + + L-Trp + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:01:56Z + + effect of increasing concentrations + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:01:59Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:02:02Z + + L-Trp + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:28:17Z + + YfiBL43P + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:02:05Z + + relative optical density + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:37:06Z + + Wild-type + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:02:11Z + + BIAcore + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:02:15Z + + binding affinities + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:02:18Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:02:21Z + + L-Trp + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:02:26Z + + ITC + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:02:30Z + + titration + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:37:06Z + + wild-type + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:06:07Z + + F57A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + + RESULTS + paragraph + 22173 + Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface. Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:52Z + + Structural analyses + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:06:21Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:06:23Z + + L-Trp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:05:24Z + + bound at + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:05:55Z + + dimer interface + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:06:26Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:06:28Z + + L-Trp + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:23:36Z + + hydrophobic pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:23Z + + L166 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:29Z + + I169 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:36Z + + V176 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:43Z + + P178 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:49Z + + L181 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:05:48Z + + binding pocket + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:15Z + + F57 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T13:36:21Z + + YfiB-YfiR + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:21:15Z + + F57 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-06-14T09:18:10Z + + YfiBL43P-YfiR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:06:11Z + + binding affinities + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:28:32Z + + YfiBL43P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:06:07Z + + F57A + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:05:08Z + + isothermal titration calorimetry + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:02:26Z + + ITC + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-07-21T08:17:12Z + + Kd + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:28:47Z + + YfiBL43P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:06:07Z + + F57A + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:06:07Z + + F57A + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:27Z + + mutant + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:06:13Z + + binding affinity + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:35Z + + YfiBL43P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:27Z + + mutant + + + + RESULTS + paragraph + 22983 + In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. Growth and surface attachment assays were carried out for the yfiB-L43P strain in the presence of increasing concentrations of VB6 or L-Trp. As shown in Fig. 6A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4–6 mmol/L for VB6 and 6–10 mmol/L for L-Trp) could reduce the surface attachment. Interestingly, at a concentration higher than 8 mmol/L, VB6 lost its ability to inhibit biofilm formation, implying that the VB6-involving regulatory mechanism is highly complicated and remains to be further investigated. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:11:44Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:11:48Z + + L-Trp + + + gene + GENE: + melaniev@ebi.ac.uk + 2023-03-15T15:11:04Z + + yfiB + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:12:47Z + + deleted + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + PAO1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:37:06Z + + wild-type + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:12:38Z + + construct expressing + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:36Z + + YfiBL43P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:27Z + + mutant + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:12:44Z + + transformed into + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + PAO1 + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:12:11Z + + ΔyfiB + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-22T09:29:21Z + + YfiBL43P + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:12:35Z + + Growth and surface attachment assays + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:11:30Z + + yfiB-L43P + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:12:41Z + + increasing concentrations + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:11:57Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:12:00Z + + L-Trp + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:12:33Z + + over-expression + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:36Z + + YfiBL43P + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:11:40Z + + yfiB-L43P + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:12:15Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:12:18Z + + L-Trp + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:12:20Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:12:23Z + + L-Trp + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:12:26Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:29:34Z + + VB6 + + + + RESULTS + paragraph + 23891 + Of note, both VB6 and L-Trp have been reported to correlate with biofilm formation in certain Gram-negative bacteria (Grubman et al.,; Shimazaki et al.,). In Helicobacter pylori in particular, VB6 biosynthetic enzymes act as novel virulence factors, and VB6 is required for full motility and virulence (Grubman et al.,). In E. coli, mutants with decreased tryptophan synthesis show greater biofilm formation, and matured biofilm is degraded by L-tryptophan addition (Shimazaki et al.,). However, the detailed mechanism remains elusive. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:14:14Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:14:12Z + + L-Trp + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:35:11Z + + Gram-negative bacteria + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + Helicobacter pylori + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:14:07Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:14:04Z + + VB6 + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + E. coli + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-06-15T15:01:48Z + + tryptophan + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:14:01Z + + L-tryptophan + + + + RESULTS + paragraph + 24430 + To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. The results showed relatively weak Kd values of 35.2 mmol/L and 76.9 mmol/L for VB6 and L-Trp, respectively (Fig. 6C and 6D). Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation. + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:17:51Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:17:54Z + + L-Trp + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + site + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:17:58Z + + F57-binding pocket + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:17:40Z + + binding affinities + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:17:43Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:17:46Z + + L-Trp + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:17:30Z + + BIAcore + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-07-21T08:17:12Z + + Kd + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:17:37Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:17:34Z + + L-Trp + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:17:22Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:17:19Z + + L-Trp + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:17:16Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:17:14Z + + L-Trp + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:14Z + + alone + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-06-14T09:18:52Z + + YfiB-YfiR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:16:51Z + + VB6 + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T15:17:03Z + + L-Trp + + + + DISCUSS + title_1 + 25080 + DISCUSSION + + + DISCUSS + paragraph + 25091 + Previous studies suggested that in response to cell stress, YfiB in the outer membrane sequesters the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus inducing the diguanylate cyclase activity of YfiN to allow c-di-GMP production (Giardina et al.,; Malone et al.,; Malone et al.,). However, the pattern of interaction between these proteins and the detailed regulatory mechanism remain unknown due to a lack of structural information. + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:12Z + + YfiN + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:12Z + + YfiN + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:30:47Z + + c-di-GMP + + + + DISCUSS + paragraph + 25567 + Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. In addition to the preceding 8 aa loop (from the lipid acceptor Cys26 to Gly34), the full length of the periplasmic portion of apo YfiB can reach approximately 60 Å. It was reported that the distance between the outer membrane and the cell wall is approximately 50 Å and that the thickness of the PG layer is approximately 70 Å (Matias et al.,). Thus, YfiB alone represents an inactive form that may only partially insert into the PG matrix. By contrast, YfiR-bound YfiBL43P (residues 44–168) has a stretched conformation of approximately 55 Å in length. In addition to the 17 preceding intracellular residues (from the lipid acceptor Cys26 to Leu43), the length of the intracellular portion of active YfiB may extend over 100 Å, assuming a fully stretched conformation. Provided that the diameter of the widest part of the YfiB dimer is approximately 64 Å, which is slightly smaller than the smallest diameter of the PG pore (70 Å) (Meroueh et al.,), the YfiB dimer should be able to penetrate the PG layer. + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:28Z + + crystal structures + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:14Z + + alone + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:21Z + + active + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:27Z + + mutant + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:36Z + + YfiBL43P + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:35:34Z + + in complex with + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:24:18Z + + complex with + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:24:00Z + + structural data analysis + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:28Z + + activated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:26:07Z + + N-terminal portion + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:21Z + + stretched conformation + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:03Z + + compact conformation + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:24:06Z + + structure + + + residue_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:22:41Z + + 34 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:03Z + + compact conformation + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:23:03Z + + preceding 8 aa + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:19:31Z + + loop + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:24:34Z + + Cys26 to Gly34 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:18:54Z + + full length + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:30Z + + apo + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:14Z + + alone + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:46:54Z + + inactive + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:51:39Z + + YfiR-bound + + + mutant + MESH: + melaniev@ebi.ac.uk + 2023-03-15T11:35:36Z + + YfiBL43P + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:24:40Z + + 44–168 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:21Z + + stretched conformation + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:23:07Z + + 17 preceding intracellular residues + + + residue_range + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:24:37Z + + Cys26 to Leu43 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:35:21Z + + active + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:19:01Z + + fully stretched conformation + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + + 13238_2016_264_Fig7_HTML.jpg + Fig7 + FIG + fig_caption + 27106 + Regulatory model of the YfiBNR tripartite system. The periplasmic domain of YfiB and the YfiB-YfiR complex are depicted according to the crystal structures. The lipid acceptor Cys26 is indicated as blue ball. The loop connecting Cys26 and Gly34 of YfiB is modeled. The PAS domain of YfiN is shown as pink oval. Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T11:33:15Z + + YfiBNR + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:03:59Z + + tripartite + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:43:32Z + + periplasmic domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T13:36:21Z + + YfiB-YfiR + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:38:28Z + + crystal structures + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:27:57Z + + Cys26 + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:19:31Z + + loop + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:27:49Z + + Cys26 + + + residue_name_number + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:27:52Z + + Gly34 + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:43:41Z + + PAS domain + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:12Z + + YfiN + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:28Z + + activated + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:11Z + + dimeric + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:03Z + + compact conformation + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:21Z + + stretched conformation + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T15:43:32Z + + periplasmic domain + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:19:09Z + + membrane-anchored + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:12Z + + YfiN + + + + DISCUSS + paragraph + 27698 + These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig. 7). In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity. This allows the C-terminal portion of the membrane-anchored YfiB to reach, bind and penetrate the cell wall and sequester the YfiR dimer. The YfiBNR system provides a good example of a delicate homeostatic system that integrates multiple signals to regulate the c-di-GMP level. Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation. The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance. + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:28Z + + activated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:51Z + + YfiB + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T15:39:17Z + + cell wall binding affinity + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:31:40Z + + PG + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-22T09:32:09Z + + YfiB + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T14:19:31Z + + loop + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:31:59Z + + PG + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T11:33:15Z + + YfiBNR + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:11Z + + dimeric + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:52Z + + YfiB + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:28Z + + activated + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:20:26Z + + compact + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T14:46:54Z + + inactive + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:20:32Z + + conformation + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:21Z + + stretched conformation + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:31:50Z + + PG + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-03-15T16:26:12Z + + C-terminal portion + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:20:37Z + + membrane-anchored + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:52Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:03Z + + YfiR + + + oligomeric_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T13:41:13Z + + dimer + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T11:33:15Z + + YfiBNR + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-15T11:33:38Z + + c-di-GMP + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T11:33:15Z + + YfiBNR + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T16:20:48Z + + functionally conserved + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + P. aeruginosa + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + E. coli + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + K. pneumonia + + + species + MESH: + melaniev@ebi.ac.uk + 2023-03-15T15:14:23Z + + Y. pestis + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:32:20Z + + c-di-GMP + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T11:36:28Z + + activated + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:33:52Z + + YfiB + + + protein + PR: + melaniev@ebi.ac.uk + 2023-03-15T11:34:12Z + + YfiN + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-03-15T11:33:15Z + + YfiBNR + + + taxonomy_domain + DUMMY: + melaniev@ebi.ac.uk + 2023-03-15T12:56:28Z + + bacteria + + + chemical + CHEBI: + melaniev@ebi.ac.uk + 2023-03-22T09:32:34Z + + c-di-GMP + + + + METHODS + title_1 + 29236 + MATERIALS AND METHODS + + + METHODS + title_2 + 29258 + Protein expression and purification + + + METHODS + paragraph + 29294 + P. aeruginosa YfiR (residues 35–190, lacking the predicted N-terminal periplasmic localization signaling peptide) and YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33) were cloned into ORF1 of the pETDuet-1 (Merck Millipore, Darmstadt, Germany) vector via the BamHI and HindIII restriction sites, with a constructed N-terminal His6 and a TEV cleavage site, respectively. In addition, YfiB (residues 34–168) was ligated into the NdeI and XhoI restriction sites of ORF2 in the previously constructed YfiR expression vector. Site-directed mutagenesis was carried out using a QuikChange kit (Agilent Technologies, Santa Clara, CA), following the manufacturer’s instructions. + + + METHODS + paragraph + 30028 + The proteins were over-expressed in the E. coli BL21-CodonPlus(DE3)-RIPL strain. Protein expression was induced by adding 0.5–1 mmol/L IPTG at an OD600 of approximately 0.8. The cell cultures were then incubated for an additional 4.5 h at 37°C. The cells were subsequently harvested by centrifugation and stored at −80°C. + + + METHODS + paragraph + 30356 + Cell suspensions were thawed and homogenized using a high-pressure homogenizer (JNBIO, Beijing, China). YfiR was first purified by Ni affinity chromatography and then incubated with His6-tagged TEV protease overnight. The His6-TEV cleavage site was subsequently removed by incubation with Ni-NTA resin. Finally, YfiR was purified with a HiTrap STM column (GE Healthcare), followed by a Superdex 200 (GE Healthcare) column. YfiB was purified with Ni affinity chromatography, followed by a Superdex 200 (GE Healthcare) column. The YfiB-YfiR complex was first purified by Ni affinity chromatography, then by a Superdex 200 (GE Healthcare) column, and finally by a HiTrap STM column (GE Healthcare). All of the purified fractions were collected and concentrated to ~40 mg/mL in 20 mmol/L Tris-HCl (pH 8.0) and 200 mmol/L NaCl, frozen in liquid nitrogen and stored at −80°C. + + + METHODS + title_2 + 31229 + Crystallization and data collection + + + METHODS + paragraph + 31265 + Crystal screening was performed with commercial screening kits (Hampton Research, CA, USA) using the sitting-drop vapor diffusion method, and positive hits were optimized using the hanging-drop vapor diffusion method at 293 K. Crystals of the YfiB protein were obtained and optimized in buffer containing 0.2 mol/L lithium sulfate monohydrate, 0.1 mol/L Tris-HCl (pH 8.0) and 30% w/v polyethylene glycol 4000. After being soaked for a few seconds in cryoprotection solution (well solution complemented with 25% xylitol), the crystals were cooled by plunging them into liquid nitrogen. Diffraction-quality crystals of the YfiB-YfiR complex were grown in buffer containing 0.2 mol/L ammonium sulfate, 0.1 mol/L Tris-HCl (pH 8.0) and 12% w/v polyethylene glycol 8000. The crystals were cryoprotected with 8% (w/v) polyethylene glycol 8000 and 0.1 mol/L Tris-HCl (pH 7.5) supplemented with saturated sucrose prior to being flash frozen. Crystals of the native YfiR were obtained and optimized in 0.1 mol/L HEPES (pH 7.5) and 1.8 mol/L ammonium sulfate. VB6-bound YfiR crystals were obtained by soaking the native YfiR crystals in 2 mmol/L VB6 molecules. Trp-bound YfiR crystals were obtained by co-crystalizing the YfiR protein and 4 mmol/L L-Trp molecules in 0.2 mol/L NaCl, 0.1 mol/L BIS-TRIS (pH 5.5), and 25% w/v polyethylene glycol 3350. For cryoprotection, both the VB6-bound and the L-Trp-bound YfiR crystals were soaked in 2.5 mol/L lithium sulfate monohydrate for a few seconds before data collection. Diffraction data for the YfiB crystal belonging to space group P21 was collected in house, the data for the YfiB crystal belonging to space group P41 and for the Trp-bound YfiR crystal were collected on beamline BL17U at the Shanghai Synchrotron Radiation Facility (SSRF), and the data for the VB6-bound YfiR crystal were collected on beamline BL18U at SSRF. Finally, the data for the YfiB-YfiR complex crystal were collected on beamline BL-1A at the Photon Factory in Japan. The diffraction data were processed with the HKL2000 software program (Otwinowski and Minor,). + + + METHODS + title_2 + 33343 + Structure determination and refinement + + + Tab1.xml + Tab1 + TABLE + table_caption + 33382 + Data collection, phasing and refinement statistics + + + Tab1.xml + Tab1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><thead><tr><th align="left"> +<bold>Data collection</bold> +</th><th align="left">YfiB (crystal form I)</th><th align="left">YfiB (crystal form II)</th><th align="left">VB6-bound YfiR</th><th align="left">Trp-bound YfiR</th><th align="left">YfiBL43P-YfiR</th></tr></thead><tbody><tr><td align="left">Space group</td><td align="left"> +<italic>P</italic>21</td><td align="left"> +<italic>P</italic>41</td><td align="left"> +<italic>P</italic>43212</td><td align="left"> +<italic>P</italic>43212</td><td align="left"> +<italic>P</italic>1</td></tr><tr><td align="left">Wavelength (Å)</td><td align="left">1.54187</td><td align="left">0.9791</td><td align="left">0.97861</td><td align="left">0.9791</td><td align="left">1.10000</td></tr><tr><td align="left">Resolution (Å)<sup>a</sup> +</td><td align="left">50.0–2.15 (2.19–2.15)</td><td align="left">50.0–2.80 (2.85–2.8)</td><td align="left">50.0–2.4 (2.44–2.4)</td><td align="left">50.0–2.5 (2.54–2.5)</td><td align="left">50–1.78 (1.86–1.78)</td></tr><tr><td align="left" colspan="6">Cell dimensions</td></tr><tr><td align="left"> a, b, c (Å)</td><td align="left">65.85, 90.45, 66.30</td><td align="left">46.95, 46.95, 154.24</td><td align="left">120.24, 120.24, 84.99</td><td align="left">120.88, 120.88, 88.46</td><td align="left">49.50, 58.57, 69.86</td></tr><tr><td align="left"> α, β, γ (°)</td><td align="left">90, 113.87, 90</td><td align="left">90, 90, 90</td><td align="left">90, 90, 90</td><td align="left">90, 90, 90</td><td align="left">72.93, 96.98, 90.19</td></tr><tr><td align="left"> Unique reflections</td><td align="left">37,625 (1866)</td><td align="left">8,105 (412)</td><td align="left">24,776 (1202)</td><td align="left">23170 (1132)</td><td align="left">67,774 (6615)</td></tr><tr><td align="left"> +<italic> I/</italic>σ<italic>I</italic> +</td><td align="left">19.59 (2.62)</td><td align="left">12.36 (4.15)</td><td align="left">20.17 (2.4)</td><td align="left">39.5 (4.68)</td><td align="left">17.75 (1.89)</td></tr><tr><td align="left"> Completeness (%)</td><td align="left">97.1 (95.4)</td><td align="left">97.8 (100)</td><td align="left">99.1 (98.8)</td><td align="left">99.9 (100)</td><td align="left">96.5 (94.6)</td></tr><tr><td align="left"> <italic>R</italic> +<sub>merge</sub> (%)</td><td align="left">6.5 (44.5)</td><td align="left">14.6 (49.7)</td><td align="left">8.9 (56.8)</td><td align="left">9.4 (89.2)</td><td align="left">5.6 (46.3)</td></tr><tr><td align="left"> +<italic> R</italic> +<sub>meas</sub> (%)</td><td align="left">7.4 (51.6)</td><td align="left">15.4 (52.0)</td><td align="left">9.6 (61.7)</td><td align="left">9.6 (90.8)</td><td align="left">6.6 (55.1)</td></tr><tr><td align="left"> CC1/2<sup>b</sup> +</td><td align="left">0.747</td><td align="left">0.952</td><td align="left">0.899</td><td align="left">0.974</td><td align="left">0.849</td></tr><tr><td align="left" colspan="6"> +<bold>Refinement</bold> +</td></tr><tr><td align="left"> +<italic> R</italic> +<sub>work</sub> (%)</td><td align="left">20.14</td><td align="left">19.17</td><td align="left">17.82</td><td align="left">18.66</td><td align="left">17.90</td></tr><tr><td align="left"> +<italic> R</italic> +<sub>free</sub>(%)</td><td align="left">26.29</td><td align="left">26.49</td><td align="left">19.81</td><td align="left">23.05</td><td align="left">20.61</td></tr><tr><td align="left" colspan="6">Average B factors (Å<sup>2</sup>)</td></tr><tr><td align="left"> Protein</td><td align="left">25.54</td><td align="left">42.70</td><td align="left">38.68</td><td align="left">35.03</td><td align="left">32.54</td></tr><tr><td align="left"> VB6</td><td align="left">-</td><td align="left">-</td><td align="left">44.08</td><td align="left">-</td><td align="left">-</td></tr><tr><td align="left"> Trp</td><td align="left">-</td><td align="left">-</td><td align="left">-</td><td align="left">87.51</td><td align="left">-</td></tr><tr><td align="left"> SO<sub>4</sub> +<sup>2−</sup> +</td><td align="left">37.16</td><td align="left">66.52</td><td align="left">51.55</td><td align="left">41.93</td><td align="left">45.51</td></tr><tr><td align="left"> H<sub>2</sub>O</td><td align="left">32.91</td><td align="left">36.09</td><td align="left">40.58</td><td align="left">34.75</td><td align="left">43.52</td></tr><tr><td align="left" colspan="6">Root mean square deviations</td></tr><tr><td align="left"> Bond lengths (Å)</td><td align="left">0.009</td><td align="left">0.009</td><td align="left">0.007</td><td align="left">0.007</td><td align="left">0.007</td></tr><tr><td align="left"> Bond angles (°)</td><td align="left">1.085</td><td align="left">1.132</td><td align="left">1.021</td><td align="left">0.977</td><td align="left">1.110</td></tr><tr><td align="left" colspan="6">Ramachandran plot</td></tr><tr><td align="left"> Most favored (%)</td><td align="left">92.6</td><td align="left">87.7</td><td align="left">96.5</td><td align="left">98.1</td><td align="left">94.2</td></tr><tr><td align="left"> Additionally allowed (%)</td><td align="left">7.4</td><td align="left">12.3</td><td align="left">3.5</td><td align="left">1.9</td><td align="left">5.8</td></tr><tr><td align="left"> Generously allowed (%)</td><td align="left">0</td><td align="left">0</td><td align="left">0</td><td align="left">0</td><td align="left">0</td></tr><tr><td align="left"> Disallowed</td><td align="left">0</td><td align="left">0</td><td align="left">0</td><td align="left">0</td><td align="left">0</td></tr></tbody></table> + + 33433 + Data collection YfiB (crystal form I) YfiB (crystal form II) VB6-bound YfiR Trp-bound YfiR YfiBL43P-YfiR Space group P21 P41 P43212 P43212 P1 Wavelength (Å) 1.54187 0.9791 0.97861 0.9791 1.10000 Resolution (Å)a 50.0–2.15 (2.19–2.15) 50.0–2.80 (2.85–2.8) 50.0–2.4 (2.44–2.4) 50.0–2.5 (2.54–2.5) 50–1.78 (1.86–1.78) Cell dimensions  a, b, c (Å) 65.85, 90.45, 66.30 46.95, 46.95, 154.24 120.24, 120.24, 84.99 120.88, 120.88, 88.46 49.50, 58.57, 69.86  α, β, γ (°) 90, 113.87, 90 90, 90, 90 90, 90, 90 90, 90, 90 72.93, 96.98, 90.19  Unique reflections 37,625 (1866) 8,105 (412) 24,776 (1202) 23170 (1132) 67,774 (6615)  I/σI 19.59 (2.62) 12.36 (4.15) 20.17 (2.4) 39.5 (4.68) 17.75 (1.89)  Completeness (%) 97.1 (95.4) 97.8 (100) 99.1 (98.8) 99.9 (100) 96.5 (94.6)  Rmerge (%) 6.5 (44.5) 14.6 (49.7) 8.9 (56.8) 9.4 (89.2) 5.6 (46.3)  Rmeas (%) 7.4 (51.6) 15.4 (52.0) 9.6 (61.7) 9.6 (90.8) 6.6 (55.1)  CC1/2b 0.747 0.952 0.899 0.974 0.849 Refinement  Rwork (%) 20.14 19.17 17.82 18.66 17.90  Rfree(%) 26.29 26.49 19.81 23.05 20.61 Average B factors (Å2)  Protein 25.54 42.70 38.68 35.03 32.54  VB6 - - 44.08 - -  Trp - - - 87.51 -  SO42− 37.16 66.52 51.55 41.93 45.51  H2O 32.91 36.09 40.58 34.75 43.52 Root mean square deviations  Bond lengths (Å) 0.009 0.009 0.007 0.007 0.007  Bond angles (°) 1.085 1.132 1.021 0.977 1.110 Ramachandran plot  Most favored (%) 92.6 87.7 96.5 98.1 94.2  Additionally allowed (%) 7.4 12.3 3.5 1.9 5.8  Generously allowed (%) 0 0 0 0 0  Disallowed 0 0 0 0 0 + + + Tab1.xml + Tab1 + TABLE + table_foot + 35033 + +a Numbers in parentheses are for the highest resolution shell + + + Tab1.xml + Tab1 + TABLE + table_foot + 35096 + +b The values of CC1/2 are for the highest resolution shell + + + METHODS + paragraph + 35156 + The two YfiB crystal structures respectively belonging to space groups P21 and P41 were both solved by molecular replacement (Lebedev et al.,) using the putative MotB-like protein DVU_2228 from D. vulgaris as a model (PDB code: 3khn) at 2.15 Å and 2.8 Å resolution, respectively. Both the VB6-bound and the Trp-bound YfiR crystals belonging to space group P43212, with a dimer in the asymmetric unit, were solved by molecular replacement (Lebedev et al.,) using native YfiR as a model (PDB code: 4YN7) at 2.4 Å and 2.5 Å resolution, respectively. The YfiB-YfiR crystal belonging to space group P1, with a 2:2 heterotetramer in the asymmetric unit, was solved by molecular replacement using YfiR and YfiB as models. Electron density maps were calculated using PHENIX (Adams et al.,). Model building was performed using COOT (Emsley et al.,) and refined with PHENIX (Adams et al.,; Afonine et al.,). The final structures were analyzed with PROCHECK (Laskowski et al.,). Data collection and refinement statistics are presented in Table 1. The figures depicting structures were prepared using PyMOL (http://www.pymol.org). Atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank (http://www.pdb.org) under accession codes 5EAZ, 5EB0, 5EB1, 5EB2 and 5EB3. + + + METHODS + title_2 + 36447 + Analytical ultracentrifugation + + + METHODS + paragraph + 36478 + Sedimentation velocity measurements were performed on a Beckman ProteomeLab XL-I at 25°C. All protein samples were diluted to an OD280 of 0.7 in 20 mmol/L Tris (pH 8.0) and 200 mmol/L NaCl. Data were collected at 60,000 rpm. (262,000 ×g) every 3 min at a wavelength of 280 nm. Interference sedimentation coefficient distributions, or c(M), were calculated from the sedimentation velocity data using SEDFIT (Schuck,). + + + METHODS + title_2 + 36897 + PG preparation + + + METHODS + paragraph + 36912 + PG was extracted from the E. coli DH5α strain by following a method described previously (Desmarais et al.,). Briefly, cells were cultured until they reached an OD600 of 0.7–0.8 and then collected at 5,000 ×g, 4°C. The collected bacteria were dripped into the boiling 6% (w/v) SDS and stirred at 500 rpm in a boiling water bath for 3 h before incubating overnight at room temperature. The large PG polymers were collected by ultracentrifugation at 130,000 ×g for 1 h at room temperature and washed repeatedly to remove SDS. The pellet was treated with Pronase E (200 μg/mL final concentration) for 3 h at 60°C followed by SDS to remove contaminating proteins and washed three times to remove the SDS by ultracentrifugation. Next, the samples were treated with lysozyme (200 μg/mL final concentration) for 16 h at 37°C. Finally, the purified PG is obtained by treating the samples in a boiling water bath for 10 min and centrifuging it at 13,000 ×g to remove the contaminating lysozyme. + + + METHODS + title_2 + 37909 + Microscale thermophoresis (MST) + + + METHODS + paragraph + 37941 + Purified YfiB wild-type and it mutant YfiBL43P were fluorescently labeled using the NanoTemper blue protein-labeling kit according to the manufacturer’s protocol. This resulted in coupling of the fluorescent dye NT-495. PG was titrated in 1:1 dilutions starting at 1 mmol/L. To determine of the Kd values, 10 μL labeled protein was mixed with 10 μL PG at various concentrations in Hepes buffer (20 mmol/L Hepes, 200 mmol/L NaCl, 0.005% Tween-20, pH 7.5). After 10 min of incubation, all binding reaction mixtures were loaded into the MST-grade glass capillaries (NanoTemper Technologies), and thermophoresis was measured with a NanoTemper Monolith-NT115 system (20% light-emitting diode, 20% IR laser power). + + + METHODS + title_2 + 38654 + Deletion of the yfiB genes + + + METHODS + paragraph + 38681 + The yfiB deletion construct was produced by SOE-PCR (Hmelo et al.,) and contained homologous flanking regions to the target gene. This construct was ligated into the pEX18Gm vector between the HindIII and the KpnI sites. The resulting vector was then used to delete yfiB by two-step allelic exchange (Hmelo et al.,). After being introduced into PAO1 via biparental mating with E. coli SM10 (λpir), single crossovers were selected on Vogel-Bonner Minimal Medium (VBMM), which was used for counter-selection against E. coli (P. aeruginosa can utilize citrate as a sole carbon source and energy source, whereas E. coli cannot), containing 50 μg/mL gentamycin. Restreaking was then performed on no-salt Luria-Bertani (NSLB) agar that contained 15% sucrose to force the resolution of double crossovers. Deletion of yfiB in the strains was confirmed by colony PCR. + + + METHODS + paragraph + 39546 + For complementation experiments, yfiB wild-type and L43P mutant genes were cloned into the pJN105 vector via the EcoRI and XbaI restriction sites, respectively. The plasmids were then individually transformed into the PAO1 ΔyfiB strain using the rapid electroporation method described in Choi KH et al. (Choi et al.,). Transformants were selected on LB plates containing 50 μg/mL gentamycin. For induction, arabinose was added to a final concentration of 0.2%. + + + METHODS + title_2 + 40012 + Attachment assays + + + METHODS + paragraph + 40030 + The attachment assays were carried out using the MBECTM (Minimum Biofilm Eradication Concentration, Innovotech, Inc.) biofilm inoculator, which consists of a plastic lid with 96 pegs and 96 individual wells. The MBEC plates containing 150 μL LB medium/well were inoculated with 1% overnight cultures of the yfiB-L43P strain and incubated overnight at 37°C without shaking. VB6, L-Trp and arabinose were added as appropriate. The peg lids were washed with distilled water, and the attached cell material was then stained with 0.1% crystal violet solution (5% methanol, 5% isopropanol) before further washing to remove excess dye. The crystal violet was re-dissolved in 20% acetic acid solution, and the absorbance was measured at 600 nm. Assays were performed with 12 wells/strain and repeated independently for each experiment. + + + METHODS + title_2 + 40860 + BIAcore analysis + + + METHODS + paragraph + 40877 + The interaction kinetics of YfiR with VB6 and L-Trp were examined on a SPR machine Biacore 3000 (GE Healthcare) at 25°C. The running buffer (20 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 0.005% (v/v) Tween-20) was vacuum filtered, and degassed immediately prior to use. YfiR at 10 μg/mL in 10 mmol/L sodium acetate (pH 5.5) was immobilized to 3000 response units on the carboxymethylated dextran surface-modified chip (CM5 chip). The binding affinities were evaluated over a range of 2.5–40 mmol/L concentrations. Meanwhile, for both binding assays, the concentration of 10 mmol/L was repeated as an internal control. All of the data collected were analyzed using BIAevaluation software version 4.1. + + + METHODS + title_2 + 41577 + ITC assays + + + METHODS + paragraph + 41588 + ITC experiments were performed in a buffer composed of 20 mmol/L Tris (pH 8.0) and 150 mmol/L NaCl at 25°C using an iTC200 calorimeter (GE Healthcare). YfiB wild-type or its mutants (YfiBL43P, YfiBL43P/F57A) (0.4 mmol/L, in the syringe) was titrated into YfiR (0.04 mmol/L, in the cell), respectively. The titration sequence included a single 0.5 µL injection, followed by 19 injections of 2 µL each, with a 2-min interval between injections and a stirring rate of 1000 rpm. The calorimetric data were then analyzed with OriginLab software (GE Healthcare). + + + METHODS + footnote + 42153 + Min Xu, Xuan Yang and Xiu-An Yang have contributed equally to this work. + + + REF + title + 42226 + References + + + 213 + 221 + surname:Adams;given-names:PD + surname:Afonine;given-names:PV + surname:Bunkoczi;given-names:G + surname:Chen;given-names:VB + surname:Davis;given-names:IW + surname:Echols;given-names:N + surname:Headd;given-names:JJ + surname:Hung;given-names:LW + surname:Kapral;given-names:GJ + surname:Grosse-Kunstleve;given-names:RW + 10.1107/S0907444909052925 + 20124702 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 42237 + PHENIX: a comprehensive Python-based system for macromolecular structure solution + + + 352 + 367 + surname:Afonine;given-names:PV + surname:Grosse-Kunstleve;given-names:RW + surname:Echols;given-names:N + surname:Headd;given-names:JJ + surname:Moriarty;given-names:NW + surname:Mustyakimov;given-names:M + surname:Terwilliger;given-names:TC + surname:Urzhumtsev;given-names:A + surname:Zwart;given-names:PH + surname:Adams;given-names:PD + 10.1107/S0907444912001308 + 22505256 + REF + Acta Crystallogr D Biol Crystallogr + ref + 68 + 2012 + 42319 + Towards automated crystallographic structure refinement with phenix.refine + + + 90 + 93 + surname:Beaumont;given-names:HJ + surname:Gallie;given-names:J + surname:Kost;given-names:C + surname:Ferguson;given-names:GC + surname:Rainey;given-names:PB + 10.1038/nature08504 + 19890329 + REF + Nature + ref + 462 + 2009 + 42394 + Experimental evolution of bet hedging + + + 107 + 116 + surname:Boehm;given-names:A + surname:Kaiser;given-names:M + surname:Li;given-names:H + surname:Spangler;given-names:C + surname:Kasper;given-names:CA + surname:Ackermann;given-names:M + surname:Kaever;given-names:V + surname:Sourjik;given-names:V + surname:Roth;given-names:V + surname:Jenal;given-names:U + 10.1016/j.cell.2010.01.018 + 20303158 + REF + Cell + ref + 141 + 2010 + 42432 + Second messenger-mediated adjustment of bacterial swimming velocity + + + 12 + 24 + surname:Caly;given-names:DL + surname:Bellini;given-names:D + surname:Walsh;given-names:MA + surname:Dow;given-names:JM + surname:Ryan;given-names:RP + 10.2174/1381612820666140905124701 + 25189859 + REF + Curr Pharm Des + ref + 21 + 2015 + 42500 + Targeting cyclic di-GMP signalling: a strategy to control biofilm formation? + + + 1113 + 1116 + surname:Camilli;given-names:A + surname:Bassler;given-names:BL + 10.1126/science.1121357 + 16497924 + REF + Science + ref + 311 + 2006 + 42577 + Bacterial small-molecule signaling pathways + + + 391 + 397 + surname:Choi;given-names:KH + surname:Kumar;given-names:A + surname:Schweizer;given-names:HP + 10.1016/j.mimet.2005.06.001 + 15987659 + REF + J Microbiol Methods + ref + 64 + 2006 + 42621 + A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation + + + REF + ref + 42795 + Desmarais SM, Cava F, de Pedro MA, Huang KC (2014) Isolation and preparation of bacterial cell walls for compositional analysis by ultra performance liquid chromatography. J Vis Exp 83:e51183 + + + 93 + 104 + surname:Duerig;given-names:A + surname:Abel;given-names:S + surname:Folcher;given-names:M + surname:Nicollier;given-names:M + surname:Schwede;given-names:T + surname:Amiot;given-names:N + surname:Giese;given-names:B + surname:Jenal;given-names:U + 10.1101/gad.502409 + 19136627 + REF + Genes Dev + ref + 23 + 2009 + 42987 + Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression + + + 486 + 501 + surname:Emsley;given-names:P + surname:Lohkamp;given-names:B + surname:Scott;given-names:WG + surname:Cowtan;given-names:K + 10.1107/S0907444910007493 + 20383002 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 43102 + Features and development of Coot + + + 231 + 239 + surname:Evans;given-names:TJ + 10.2217/fmb.14.107 + 25689535 + REF + Future Microbiol + ref + 10 + 2015 + 43135 + Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis + + + e81324 + surname:Giardina;given-names:G + surname:Paiardini;given-names:A + surname:Fernicola;given-names:S + surname:Franceschini;given-names:S + surname:Rinaldo;given-names:S + surname:Stelitano;given-names:V + surname:Cutruzzola;given-names:F + 10.1371/journal.pone.0081324 + 24278422 + REF + PLoS One + ref + 8 + 2013 + 43245 + Investigating the allosteric regulation of YfiN from Pseudomonas aeruginosa: clues from the structure of the catalytic domain + + + 18247 + 18252 + surname:Giddens;given-names:SR + surname:Jackson;given-names:RW + surname:Moon;given-names:CD + surname:Jacobs;given-names:MA + surname:Zhang;given-names:XX + surname:Gehrig;given-names:SM + surname:Rainey;given-names:PB + 10.1073/pnas.0706739104 + 17989226 + REF + Proc Natl Acad Sci USA + ref + 104 + 2007 + 43371 + Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment + + + 539 + 574 + surname:Govan;given-names:JR + surname:Deretic;given-names:V + 8840786 + REF + Microbiol Rev + ref + 60 + 1996 + 43507 + Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia + + + REF + ref + 43605 + Grubman A, Phillips A, Thibonnier M, Kaparakis-Liaskos M, Johnson C, Thiberge JM, Radcliff FJ, Ecobichon C, Labigne A, de Reuse H. et al (2010) Vitamin B6 is required for full motility and virulence in Helicobacter pylori. MBio 1 + + + REF + ref + 43835 + Ha DG, O’Toole GA (2015) c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review. Microbiol Spectr 3, MB-0003-2014 + + + 621 + 625 + surname:Haussler;given-names:S + surname:Tummler;given-names:B + surname:Weissbrodt;given-names:H + surname:Rohde;given-names:M + surname:Steinmetz;given-names:I + 10.1086/598644 + 10530458 + REF + Clin Infect Dis + ref + 29 + 1999 + 43990 + Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis + + + 295 + 301 + surname:Haussler;given-names:S + surname:Ziegler;given-names:I + surname:Lottel;given-names:A + surname:von Gotz;given-names:F + surname:Rohde;given-names:M + surname:Wehmhohner;given-names:D + surname:Saravanamuthu;given-names:S + surname:Tummler;given-names:B + surname:Steinmetz;given-names:I + 10.1099/jmm.0.05069-0 + 12676867 + REF + J Med Microbiol + ref + 52 + 2003 + 44057 + Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection + + + 263 + 273 + surname:Hengge;given-names:R + 10.1038/nrmicro2109 + 19287449 + REF + Nat Rev Microbiol + ref + 7 + 2009 + 44155 + Principles of c-di-GMP signalling in bacteria + + + 14422 + 14427 + surname:Hickman;given-names:JW + surname:Tifrea;given-names:DF + surname:Harwood;given-names:CS + 10.1073/pnas.0507170102 + 16186483 + REF + Proc Natl Acad Sci USA + ref + 102 + 2005 + 44201 + A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels + + + 1820 + 1841 + surname:Hmelo;given-names:LR + surname:Borlee;given-names:BR + surname:Almblad;given-names:H + surname:Love;given-names:ME + surname:Randall;given-names:TE + surname:Tseng;given-names:BS + surname:Lin;given-names:C + surname:Irie;given-names:Y + surname:Storek;given-names:KM + surname:Yang;given-names:JJ + 10.1038/nprot.2015.115 + 26492139 + REF + Nat Protoc + ref + 10 + 2015 + 44304 + Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange + + + W545 + W549 + surname:Holm;given-names:L + surname:Rosenstrom;given-names:P + 10.1093/nar/gkq366 + 20457744 + REF + Nucleic Acids Res + ref + 38 + 2010 + 44391 + Dali server: conservation mapping in 3D + + + 2595 + 2606 + surname:Huertas;given-names:MG + surname:Zarate;given-names:L + surname:Acosta;given-names:IC + surname:Posada;given-names:L + surname:Cruz;given-names:DP + surname:Lozano;given-names:M + surname:Zambrano;given-names:MM + 10.1099/mic.0.081992-0 + 25261190 + REF + Microbiology + ref + 160 + 2014 + 44431 + Klebsiella pneumoniae yfiRNB operon affects biofilm formation, polysaccharide production and drug susceptibility + + + 3690 + 3699 + surname:Hufnagel;given-names:DA + surname:DePas;given-names:WH + surname:Chapman;given-names:MR + 10.1128/JB.02019-14 + 25112475 + REF + J Bacteriol + ref + 196 + 2014 + 44544 + The disulfide bonding system suppresses CsgD-independent cellulose production in Escherichia coli + + + 185 + 191 + surname:Jenal;given-names:U + 10.1016/j.mib.2004.02.007 + 15063857 + REF + Curr Opin Microbiol + ref + 7 + 2004 + 44642 + Cyclic di-guanosine-monophosphate comes of age: a novel secondary messenger involved in modulating cell surface structures in bacteria? + + + 75 + 88 + surname:Kirillina;given-names:O + surname:Fetherston;given-names:JD + surname:Bobrov;given-names:AG + surname:Abney;given-names:J + surname:Perry;given-names:RD + 10.1111/j.1365-2958.2004.04253.x + 15458406 + REF + Mol Microbiol + ref + 54 + 2004 + 44778 + HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis + + + 4809 + 4821 + surname:Kirisits;given-names:MJ + surname:Prost;given-names:L + surname:Starkey;given-names:M + surname:Parsek;given-names:MR + 10.1128/AEM.71.8.4809-4821.2005 + 16085879 + REF + Appl Environ Microbiol + ref + 71 + 2005 + 44915 + Characterization of colony morphology variants isolated from Pseudomonas aeruginosa biofilms + + + 2839 + 2844 + surname:Kulasakara;given-names:H + surname:Lee;given-names:V + surname:Brencic;given-names:A + surname:Liberati;given-names:N + surname:Urbach;given-names:J + surname:Miyata;given-names:S + surname:Lee;given-names:DG + surname:Neely;given-names:AN + surname:Hyodo;given-names:M + surname:Hayakawa;given-names:Y + 10.1073/pnas.0511090103 + 16477007 + REF + Proc Natl Acad Sci USA + ref + 103 + 2006 + 45008 + Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence + + + 283 + 291 + surname:Laskowski;given-names:RA + surname:MacArthur;given-names:MW + surname:Moss;given-names:DS + surname:Thornton;given-names:JM + 10.1107/S0021889892009944 + REF + J Appl Crystallogr + ref + 26 + 1993 + 45146 + PROCHECK: a program to check the stereochemical quality of protein structures + + + 33 + 39 + surname:Lebedev;given-names:AA + surname:Vagin;given-names:AA + surname:Murshudov;given-names:GN + 10.1107/S0907444907049839 + 18094465 + REF + Acta Crystallogr D Biol Crystallogr + ref + 64 + 2008 + 45224 + Model preparation in MOLREP and examples of model improvement using X-ray data + + + 16915 + surname:Li;given-names:S + surname:Li;given-names:T + surname:Xu;given-names:Y + surname:Zhang;given-names:Q + surname:Zhang;given-names:W + surname:Che;given-names:S + surname:Liu;given-names:R + surname:Wang;given-names:Y + surname:Bartlam;given-names:M + 10.1038/srep16915 + 26593397 + REF + Sci Rep + ref + 5 + 2015 + 45303 + Structural insights into YfiR sequestering by YfiB in Pseudomonas aeruginosa PAO1 + + + 237 + 247 + surname:Malone;given-names:JG + 10.2147/IDR.S68214 + 26251621 + REF + Infect Drug Resist + ref + 8 + 2015 + 45385 + Role of small colony variants in persistence of Pseudomonas aeruginosa infections in cystic fibrosis lungs + + + e1000804 + surname:Malone;given-names:JG + surname:Jaeger;given-names:T + surname:Spangler;given-names:C + surname:Ritz;given-names:D + surname:Spang;given-names:A + surname:Arrieumerlou;given-names:C + surname:Kaever;given-names:V + surname:Landmann;given-names:R + surname:Jenal;given-names:U + 10.1371/journal.ppat.1000804 + 20300602 + REF + PLoS Pathog + ref + 6 + 2010 + 45492 + YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa + + + e1002760 + surname:Malone;given-names:JG + surname:Jaeger;given-names:T + surname:Manfredi;given-names:P + surname:Dotsch;given-names:A + surname:Blanka;given-names:A + surname:Bos;given-names:R + surname:Cornelis;given-names:GR + surname:Haussler;given-names:S + surname:Jenal;given-names:U + 10.1371/journal.ppat.1002760 + 22719254 + REF + PLoS Pathog + ref + 8 + 2012 + 45605 + The YfiBNR signal transduction mechanism reveals novel targets for the evolution of persistent Pseudomonas aeruginosa in cystic fibrosis airways + + + 6112 + 6118 + surname:Matias;given-names:VR + surname:Al-Amoudi;given-names:A + surname:Dubochet;given-names:J + surname:Beveridge;given-names:TJ + 10.1128/JB.185.20.6112-6118.2003 + 14526023 + REF + J Bacteriol + ref + 185 + 2003 + 45750 + Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa + + + 4404 + 4409 + surname:Meroueh;given-names:SO + surname:Bencze;given-names:KZ + surname:Hesek;given-names:D + surname:Lee;given-names:M + surname:Fisher;given-names:JF + surname:Stemmler;given-names:TL + surname:Mobashery;given-names:S + 10.1073/pnas.0510182103 + 16537437 + REF + Proc Natl Acad Sci USA + ref + 103 + 2006 + 45863 + Three-dimensional structure of the bacterial cell wall peptidoglycan + + + e1000588 + surname:Navarro;given-names:MV + surname:Newell;given-names:PD + surname:Krasteva;given-names:PV + surname:Chatterjee;given-names:D + surname:Madden;given-names:DR + surname:O’Toole;given-names:GA + surname:Sondermann;given-names:H + 10.1371/journal.pbio.1000588 + 21304926 + REF + PLoS Biol + ref + 9 + 2011 + 45932 + Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis + + + 307 + 326 + surname:Otwinowski;given-names:Z + surname:Minor;given-names:W + 10.1016/S0076-6879(97)76066-X + REF + Methods Enzymol + ref + 276 + 1997 + 46028 + Processing of X-ray diffraction data collected in oscillation mode + + + 677 + 701 + surname:Parsek;given-names:MR + surname:Singh;given-names:PK + 10.1146/annurev.micro.57.030502.090720 + 14527295 + REF + Annu Rev Microbiol + ref + 57 + 2003 + 46095 + Bacterial biofilms: an emerging link to disease pathogenesis + + + 2122 + 2128 + surname:Parsons;given-names:LM + surname:Lin;given-names:F + surname:Orban;given-names:J + 10.1021/bi052227i + 16475801 + REF + Biochemistry + ref + 45 + 2006 + 46156 + Peptidoglycan recognition by Pal, an outer membrane lipoprotein + + + 170 + 176 + surname:Pesavento;given-names:C + surname:Hengge;given-names:R + 10.1016/j.mib.2009.01.007 + 19318291 + REF + Curr Opin Microbiol + ref + 12 + 2009 + 46220 + Bacterial nucleotide-based second messengers + + + 3089 + 3098 + surname:Raterman;given-names:EL + surname:Shapiro;given-names:DD + surname:Stevens;given-names:DJ + surname:Schwartz;given-names:KJ + surname:Welch;given-names:RA + 10.1128/IAI.01396-12 + 23774594 + REF + Infect Immun + ref + 81 + 2013 + 46265 + Genetic analysis of the role of yfiR in the ability of Escherichia coli CFT073 to control cellular cyclic dimeric GMP levels and to persist in the urinary tract + + + 1341 + 1350 + surname:Reinhardt;given-names:A + surname:Kohler;given-names:T + surname:Wood;given-names:P + surname:Rohner;given-names:P + surname:Dumas;given-names:JL + surname:Ricou;given-names:B + surname:van Delden;given-names:C + 10.1128/AAC.01278-06 + 17261619 + REF + Antimicrob Agents Chemother + ref + 51 + 2007 + 46426 + Development and persistence of antimicrobial resistance in Pseudomonas aeruginosa: a longitudinal observation in mechanically ventilated patients + + + 1202 + 1216 + surname:Ren;given-names:GX + surname:Yan;given-names:HQ + surname:Zhu;given-names:H + surname:Guo;given-names:XP + surname:Sun;given-names:YC + 10.1111/1462-2920.12323 + 24192006 + REF + Environ Microbiol + ref + 16 + 2014 + 46572 + HmsC, a periplasmic protein, controls biofilm formation via repression of HmsD, a diguanylate cyclase in Yersinia pestis + + + 1 + 52 + surname:Romling;given-names:U + surname:Galperin;given-names:MY + surname:Gomelsky;given-names:M + 10.1128/MMBR.00043-12 + 23471616 + REF + Microbiol Mol Biol Rev + ref + 77 + 2013 + 46693 + Cyclic di-GMP: the first 25 years of a universal bacterial second messenger + + + 279 + 281 + surname:Ross;given-names:P + surname:Weinhouse;given-names:H + surname:Aloni;given-names:Y + surname:Michaeli;given-names:D + surname:Weinberger-Ohana;given-names:P + surname:Mayer;given-names:R + surname:Braun;given-names:S + surname:de Vroom;given-names:E + surname:van der Marel;given-names:GA + surname:van Boom;given-names:JH + 10.1038/325279a0 + 18990795 + REF + Nature + ref + 325 + 1987 + 46769 + Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid + + + 35 + 58 + surname:Ross;given-names:P + surname:Mayer;given-names:R + surname:Benziman;given-names:M + 2030672 + REF + Microbiol Rev + ref + 55 + 1991 + 46852 + Cellulose biosynthesis and function in bacteria + + + 651 + 658 + surname:Sanchez-Torres;given-names:V + surname:Hu;given-names:H + surname:Wood;given-names:TK + 10.1007/s00253-010-3074-5 + 21181144 + REF + Appl Microbiol Biotechnol + ref + 90 + 2011 + 46900 + GGDEF proteins YeaI, YedQ, and YfiN reduce early biofilm formation and swimming motility in Escherichia coli + + + 724 + 735 + surname:Schirmer;given-names:T + surname:Jenal;given-names:U + 10.1038/nrmicro2203 + 19756011 + REF + Nat Rev Microbiol + ref + 7 + 2009 + 47009 + Structural and mechanistic determinants of c-di-GMP signalling + + + 1606 + 1619 + surname:Schuck;given-names:P + 10.1016/S0006-3495(00)76713-0 + 10692345 + REF + Biophys J + ref + 78 + 2000 + 47072 + Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling + + + 715 + 718 + surname:Shimazaki;given-names:J + surname:Furukawa;given-names:S + surname:Ogihara;given-names:H + surname:Morinaga;given-names:Y + 10.1016/j.bbrc.2012.02.085 + 22386992 + REF + Biochem Biophys Res Commun + ref + 419 + 2012 + 47190 + L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation + + + 8487 + 8492 + surname:Smith;given-names:EE + surname:Buckley;given-names:DG + surname:Wu;given-names:Z + surname:Saenphimmachak;given-names:C + surname:Hoffman;given-names:LR + surname:D’Argenio;given-names:DA + surname:Miller;given-names:SI + surname:Ramsey;given-names:BW + surname:Speert;given-names:DP + surname:Moskowitz;given-names:SM + 10.1073/pnas.0602138103 + 16687478 + REF + Proc Natl Acad Sci USA + ref + 103 + 2006 + 47280 + Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients + + + 33324 + 33330 + surname:Tamayo;given-names:R + surname:Tischler;given-names:AD + surname:Camilli;given-names:A + 10.1074/jbc.M506500200 + 16081414 + REF + J Biol Chem + ref + 280 + 2005 + 47368 + The EAL domain protein VieA is a cyclic diguanylate phosphodiesterase + + + e1000483 + surname:Ueda;given-names:A + surname:Wood;given-names:TK + 10.1371/journal.ppat.1000483 + 19543378 + REF + PLoS Pathog + ref + 5 + 2009 + 47438 + Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885) + + + e0124330 + surname:Xu;given-names:K + surname:Li;given-names:S + surname:Yang;given-names:W + surname:Li;given-names:K + surname:Bai;given-names:Y + surname:Xu;given-names:Y + surname:Jin;given-names:J + surname:Wang;given-names:Y + surname:Bartlam;given-names:M + 10.1371/journal.pone.0124330 + 25909591 + REF + PLoS One + ref + 10 + 2015 + 47586 + Structural and biochemical analysis of tyrosine phosphatase related to biofilm formation A (TpbA) from the opportunistic pathogen Pseudomonas aeruginosa PAO1 + + + 14 + 20 + surname:Yang;given-names:X + surname:Yang;given-names:XA + surname:Xu;given-names:M + surname:Zhou;given-names:L + surname:Fan;given-names:Z + surname:Jiang;given-names:T + 10.1016/j.bbrc.2015.03.160 + 25849887 + REF + Biochem Biophys Res Commun + ref + 461 + 2015 + 47744 + Crystal structures of YfiR from Pseudomonas aeruginosa in two redox states + + + diff --git a/BioC_XML/4888278_v0.xml b/BioC_XML/4888278_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..d0c69c81d2a73f4598e7fac090c0bb6d01fce2b3 --- /dev/null +++ b/BioC_XML/4888278_v0.xml @@ -0,0 +1,6219 @@ + + + + PMC + 20201221 + pmc.key + + 4888278 + CC BY + no + 0 + 0 + + 10.1186/s12900-016-0059-3 + 4888278 + 27246200 + 59 + 7 + RORγ Agonist Inverse Agonist Activation Function 2 Helix (AF2) TH17cells IL-17 Autoimmune Disease + +Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. + surname:Marcotte;given-names:Douglas J. + surname:Liu;given-names:YuTing + surname:Little;given-names:Kevin + surname:Jones;given-names:John H. + surname:Powell;given-names:Noel A. + surname:Wildes;given-names:Craig P. + surname:Silvian;given-names:Laura F. + surname:Chodaparambil;given-names:Jayanth V. + TITLE + Keywords + front + 16 + 2016 + 0 + Structural determinant for inducing RORgamma specific inverse agonism triggered by a synthetic benzoxazinone ligand + + 0.9996841 + protein + cleaner0 + 2023-07-19T15:12:51Z + PR: + + RORgamma + + + 0.9997347 + chemical + cleaner0 + 2023-07-19T15:13:56Z + CHEBI: + + benzoxazinone + + + + ABSTRACT + abstract_title_1 + 116 + Background + + + ABSTRACT + abstract + 127 + The nuclear hormone receptor RORγ regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. Our goal was to develop a RORγ specific inverse agonist that would help down regulate pro-inflammatory gene transcription by disrupting the protein protein interaction with coactivator proteins as a therapeutic agent. + + 0.9994586 + protein_type + cleaner0 + 2023-07-19T15:12:34Z + MESH: + + nuclear hormone receptor + + + 0.9995332 + protein + cleaner0 + 2023-07-19T15:12:44Z + PR: + + RORγ + + + protein_type + MESH: + cleaner0 + 2023-07-19T15:55:50Z + + interleukin + + + protein_type + MESH: + cleaner0 + 2023-07-19T15:55:24Z + + IL-17 + + + 0.99969506 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + structure_element + SO: + cleaner0 + 2023-07-19T15:13:36Z + + activation function 2 (AF2) helix + + + 0.9996891 + structure_element + cleaner0 + 2023-07-19T15:13:07Z + SO: + + ligand binding domain + + + 0.9997353 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9995486 + protein_state + cleaner0 + 2023-07-19T15:59:12Z + DUMMY: + + conserved + + + 0.99966925 + structure_element + cleaner0 + 2023-07-19T15:54:48Z + SO: + + LXXLL helix motif + + + 0.99968076 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + + ABSTRACT + abstract_title_1 + 855 + Results + + + ABSTRACT + abstract + 863 + We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. We show that the AF2 helix of RORγ is proteolytically sensitive when inverse agonist BIO399 binds. Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of RORγ. Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and β. + + 0.99951196 + chemical + cleaner0 + 2023-07-19T15:13:55Z + CHEBI: + + benzoxazinone + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9995297 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.9994516 + chemical + cleaner0 + 2023-07-19T15:14:08Z + CHEBI: + + BIO399 + + + 0.9995902 + experimental_method + cleaner0 + 2023-07-19T15:14:47Z + MESH: + + FRET based assay + + + 0.9997276 + structure_element + cleaner0 + 2023-07-19T15:14:25Z + SO: + + AF2 helix + + + 0.99978596 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.7023409 + protein_state + cleaner0 + 2023-07-19T15:59:28Z + DUMMY: + + proteolytically sensitive + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.9991371 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9995466 + experimental_method + cleaner0 + 2023-07-19T15:14:51Z + MESH: + + x-ray crystallography + + + 0.9994616 + chemical + cleaner0 + 2023-07-19T15:13:56Z + CHEBI: + + benzoxazinone + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9995976 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.99979097 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9995693 + experimental_method + cleaner0 + 2023-07-19T15:14:56Z + MESH: + + in vivo reporter assay + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.99939096 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9997814 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99713814 + protein_type + cleaner0 + 2023-07-19T15:14:21Z + MESH: + + ROR + + + 0.99299026 + protein + cleaner0 + 2023-07-19T15:16:12Z + PR: + + α + + + 0.9972766 + protein + cleaner0 + 2023-07-19T15:16:15Z + PR: + + β + + + + ABSTRACT + abstract_title_1 + 1409 + Conclusion + + + ABSTRACT + abstract + 1420 + The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. The proteolytic sensitivity of the AF2 helix of RORγ demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site. Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism. + + 0.9997489 + chemical + cleaner0 + 2023-07-19T15:13:56Z + CHEBI: + + benzoxazinone + + + 0.99962234 + experimental_method + cleaner0 + 2023-07-19T15:15:18Z + MESH: + + FRET assay + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.999524 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.9993519 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9997969 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9997283 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.99979514 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9988256 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.99966204 + site + cleaner0 + 2023-07-19T15:15:29Z + SO: + + coactivator protein binding site + + + 0.9996029 + experimental_method + cleaner0 + 2023-07-19T15:15:07Z + MESH: + + structural investigation + + + 0.99946314 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9992617 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.9992576 + evidence + cleaner0 + 2023-07-19T15:50:19Z + DUMMY: + + structures + + + 0.9998889 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.9997987 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99973875 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + + ABSTRACT + abstract_title_1 + 1972 + Electronic supplementary material + + + ABSTRACT + abstract + 2006 + The online version of this article (doi:10.1186/s12900-016-0059-3) contains supplementary material, which is available to authorized users. + + + INTRO + title_1 + 2146 + Background + + + INTRO + paragraph + 2157 + Retinoid-related orphan receptor gamma (RORγ) is a transcription factor belonging to a sub-family of nuclear receptors that includes two closely related members RORα and RORβ. Even though a high degree of sequence similarity exists between the RORs, their functional roles in regulation for physiological processes involved in development and immunity are distinct. During development, RORγ regulates the transcriptional genes involved in the functioning of multiple pro-inflammatory lymphocyte lineages including T helper cells (TH17cells) which are necessary for IL-17 production. IL-17 is a pro-inflammatory interleukin linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease; making its transcriptional regulation through RORγ an attractive therapeutic target. + + protein + PR: + cleaner0 + 2023-07-19T15:15:55Z + + Retinoid-related orphan receptor gamma + + + 0.99976176 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.96197236 + protein_type + cleaner0 + 2023-07-19T15:16:22Z + MESH: + + transcription factor + + + 0.9992901 + protein_type + cleaner0 + 2023-07-19T15:16:28Z + MESH: + + nuclear receptors + + + 0.9998398 + protein + cleaner0 + 2023-07-19T15:16:01Z + PR: + + RORα + + + 0.99984014 + protein + cleaner0 + 2023-07-19T15:16:06Z + PR: + + RORβ + + + 0.99961334 + protein_type + cleaner0 + 2023-07-19T15:16:33Z + MESH: + + RORs + + + 0.99953103 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9956913 + protein_type + cleaner0 + 2023-07-19T15:55:24Z + MESH: + + IL-17 + + + 0.99601126 + protein_type + cleaner0 + 2023-07-19T15:55:24Z + MESH: + + IL-17 + + + protein_type + MESH: + cleaner0 + 2023-07-19T15:55:50Z + + interleukin + + + 0.9997352 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + + INTRO + paragraph + 2995 + RORγ consists of an N-terminal DNA binding domain (DBD) connected to a C-terminal ligand binding domain (LBD) via a flexible hinge region. The DBD is composed of two zinc fingers that allow it to interact with specifically encoded regions on the DNA called the nuclear receptor response elements. The LBD consists of a coactivator protein binding pocket and a hydrophobic ligand binding site (LBS) which are responsible for regulating transcription. The coactivator binding pocket of RORγ recognizes a conserved helix motif LXXLL (where X can be any amino acid) on transcriptional coactivator complexes and recruits it to activate transcription. Like other nuclear hormone receptors, RORγ’s helix12 which makes up the C-termini of the LBD is an essential part of the coactivator binding pocket and is commonly referred to as the activation function helix 2 (AF2). In RORγ, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to π- π interactions between Tyr502 and Phe506. The conformation of the AF2 helix can be modulated through targeted ligands which bind the LBS and increase the binding of the coactivator protein (agonists) or disrupt binding (inverse agonists) thereby enhancing or inhibiting transcription. Since RORγ has been demonstrated to play an important role in pro-inflammatory gene expression patterns implicated in several major autoimmune diseases, our aim was to develop RORγ inverse agonists that would help down regulate pro-inflammatory gene transcription. + + 0.9995522 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99970484 + structure_element + cleaner0 + 2023-07-19T15:16:52Z + SO: + + DNA binding domain + + + 0.9998134 + structure_element + cleaner0 + 2023-07-19T15:16:57Z + SO: + + DBD + + + 0.999716 + structure_element + cleaner0 + 2023-07-19T15:13:08Z + SO: + + ligand binding domain + + + 0.9998136 + structure_element + cleaner0 + 2023-07-19T15:17:06Z + SO: + + LBD + + + 0.9997424 + structure_element + cleaner0 + 2023-07-19T15:17:15Z + SO: + + hinge region + + + 0.9998217 + structure_element + cleaner0 + 2023-07-19T15:16:57Z + SO: + + DBD + + + 0.99940574 + structure_element + cleaner0 + 2023-07-19T15:17:20Z + SO: + + zinc fingers + + + structure_element + SO: + cleaner0 + 2023-07-19T15:17:55Z + + nuclear receptor response elements + + + 0.99982005 + structure_element + cleaner0 + 2023-07-19T15:17:06Z + SO: + + LBD + + + 0.9995779 + site + cleaner0 + 2023-07-19T15:17:29Z + SO: + + coactivator protein binding pocket + + + 0.99728817 + site + cleaner0 + 2023-07-19T15:18:04Z + SO: + + hydrophobic ligand binding site + + + 0.97560805 + site + cleaner0 + 2023-07-19T15:18:14Z + SO: + + LBS + + + 0.9996037 + site + cleaner0 + 2023-07-19T15:18:21Z + SO: + + coactivator binding pocket + + + 0.99960774 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9993538 + protein_state + cleaner0 + 2023-07-19T16:00:23Z + DUMMY: + + conserved + + + structure_element + SO: + cleaner0 + 2023-07-19T15:58:57Z + + helix motif LXXLL + + + 0.99955034 + protein_type + cleaner0 + 2023-07-19T15:18:38Z + MESH: + + nuclear hormone receptors + + + 0.99942905 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9998221 + structure_element + cleaner0 + 2023-07-19T15:18:45Z + SO: + + helix12 + + + 0.99983406 + structure_element + cleaner0 + 2023-07-19T15:17:06Z + SO: + + LBD + + + 0.9996125 + site + cleaner0 + 2023-07-19T15:18:21Z + SO: + + coactivator binding pocket + + + 0.9996948 + structure_element + cleaner0 + 2023-07-19T15:18:49Z + SO: + + activation function helix 2 + + + 0.99982625 + structure_element + cleaner0 + 2023-07-19T15:18:51Z + SO: + + AF2 + + + 0.9995863 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99973696 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.99961215 + site + cleaner0 + 2023-07-19T15:18:21Z + SO: + + coactivator binding pocket + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + salt bridge + + + 0.9999027 + residue_name_number + cleaner0 + 2023-07-19T15:25:24Z + DUMMY: + + His479 + + + 0.99989915 + residue_name_number + cleaner0 + 2023-07-19T15:18:56Z + DUMMY: + + Tyr502 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + π- π interactions + + + 0.99990165 + residue_name_number + cleaner0 + 2023-07-19T15:18:56Z + DUMMY: + + Tyr502 + + + 0.99990034 + residue_name_number + cleaner0 + 2023-07-19T15:19:01Z + DUMMY: + + Phe506 + + + 0.9997456 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.99979895 + site + cleaner0 + 2023-07-19T15:18:14Z + SO: + + LBS + + + 0.9995858 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9994648 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + + 12900_2016_59_Fig1_HTML.jpg + Fig1 + FIG + fig_caption + 4599 + FRET results for agonist BIO592 (a) and Inverse Agonist BIO399 (b) + + evidence + DUMMY: + cleaner0 + 2023-07-19T15:20:05Z + + FRET results + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9992667 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + Inverse Agonist + + + 0.99943894 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + + INTRO + paragraph + 4666 + Here we present the identification of two synthetic benzoxazinone RORγ ligands, a weak agonist BIO592 (Fig. 1a) and an inverse agonist BIO399 (Fig. 1b) which were identified using a Fluorescence Resonance Energy transfer (FRET) based assay that monitored coactivator peptide recruitment. Using partial proteolysis in combination with mass spectrometry analysis we demonstrate that the AF2 helix of RORγ destabilizes upon BIO399 (inverse agonist) binding. Finally, comparing binding modes of our benzoxazinone RORγ crystal structures to other ROR structures, we hypothesize a new mode of action for achieving inverse agonism and selectivity. + + 0.9993067 + chemical + cleaner0 + 2023-07-19T15:13:56Z + CHEBI: + + benzoxazinone + + + 0.99667335 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9994783 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.99941254 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + experimental_method + MESH: + cleaner0 + 2023-07-19T15:19:33Z + + Fluorescence Resonance Energy transfer (FRET) based assay + + + 0.99953055 + experimental_method + cleaner0 + 2023-07-19T15:19:44Z + MESH: + + partial proteolysis + + + 0.99952245 + experimental_method + cleaner0 + 2023-07-19T15:19:46Z + MESH: + + mass spectrometry + + + 0.9997479 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.999608 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9987185 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.9855926 + evidence + cleaner0 + 2023-07-19T15:50:30Z + DUMMY: + + binding modes + + + 0.99849415 + chemical + cleaner0 + 2023-07-19T15:13:56Z + CHEBI: + + benzoxazinone + + + 0.99864763 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99943864 + evidence + cleaner0 + 2023-07-19T15:50:34Z + DUMMY: + + crystal structures + + + 0.99912757 + protein_type + cleaner0 + 2023-07-19T15:14:21Z + MESH: + + ROR + + + 0.9982961 + evidence + cleaner0 + 2023-07-19T15:20:17Z + DUMMY: + + structures + + + + METHODS + title_1 + 5319 + Methods + + + METHODS + title_2 + 5327 + Cloning, protein expression and purification of RORγ518 + + + METHODS + paragraph + 5387 + GST-RORγ518 was constructed by sub-cloning residues 259 to 518 of a human RORγ cDNA into a pGEX-6P vector with a cleavable N-terminal GST fusion tag. BL21 (DE3) Escherichia coli cells were transformed with the plasmid encoding the GST-PreScission-hRORgamma 259–518 protein (GST-RORγ518) and were grown at 37 °C in LB media supplemented with ampicillin to an OD of 1. The temperature was reduced to 18 °C and protein expression was induced by adding 1 mM IPTG and was shaking for an additional 16 h. The cells were harvested and resuspended in lysis buffer (25 mM TRIS pH 8.0, 250 mM NaCl, 10 % Glycerol, 5 mM DTT and Roche EDTA-free protease inhibitor cocktail) and were lysed using a microfluidizer. The lysate was clarified by centrifugation at 20,000 × g for 1 h at 4 °C and GST-RORγ518 was captured by batch binding to Glutathione Sepharose resin overnight at 4 °C. The resin was washed with buffer A (25 mM TRIS pH 8.0, 250 mM NaCl, 10 % glycerol, 5 mM DTT) and loaded onto a XK column and washed until no non-specific unbound protein was detected. GST- RORγ518 was eluted from the column using buffer A supplemented with 10 mM Glutathione pH 8.0 and analyzed by SDS-PAGE. The eluate was then treated with PreScission Protease (10units/mg of protein) and further purified on a Superdex 75 column equilibrated in buffer B (25 mM TRIS pH 8.0, 250 mM NaCl, 5 % glycerol and 2 mM DTT). RORγ518 eluted as a monomer and was approximately 95 % pure as observed by SDS-PAGE. + + + METHODS + paragraph + 6901 + Additional constructs including c-terminal truncations, surface entropy reduction and cysteine scrubbed mutations were also expressed and purified in the same manner as RORγ518 if an expression level of >1 mg/L was achieved. + + + METHODS + title_2 + 7130 + RORγ FRET based assay and GAL4 reporter assay + + + METHODS + paragraph + 7180 + FRET-based (Fluorescence Resonance Energy Transfer) assay and the GAL4 Reporter assay were performed as described previously. BIO592 and BIO399 were synthesized (Additional file 1) and belonged to a proprietary library where they were identified as RORγ activity modulators using the FRET-based assay. + + + METHODS + title_2 + 7486 + Partial proteolysis of RORγ518 + + + METHODS + paragraph + 7521 + RORγ518 at 8 mg/ml or in complex with 1 mM BIO399 or 1 mM BIO592 and 0.5 mM coactivator peptide EBI96 EFPYLLSLLGEVSPQ (New England Peptide) were treated with Actinase E (Hampton Research) added at a ratio of 1.25ugs of protease/1 mg of RORγ518 for 6 h at 4 °C. The reactions were quenched using 1X Protease inhibitor cocktail (Roche) + 1 mM EDTA and subjected to mass spectrometry analysis. + + + METHODS + title_2 + 7927 + Mass spectrometry of partially proteolyzed RORγ518 + + + METHODS + paragraph + 7982 + Proteolyzed RORγ518 samples were reduced with 50 mM dithiothreitol in 50 mM Tris pH 8.0, 150 mM NaCl containing 4 M urea and 5 mM EDTA. The sample was then analyzed on a LC-MS system comprised of a UPLC (ACQUITY, Waters Corp.), a TUV dual-wavelength UV detector (Waters Corp.), and a ZQ mass spectrometer (Waters Corp.). A Vydac C4 cartridge was used for desalting. Molecular masses for the Actinase E treated RORγ518 samples were obtained by deconvoluting the raw mass spectra using MaxLynx 4.1 software (Waters Corp.). + + + METHODS + title_2 + 8511 + Crystallization of RORγ518 with agonist BIO592 and inverse agonist BIO399 + + + METHODS + paragraph + 8589 + RORγ518 was concentrated to 8 mg/ml and EBI96 was added to a final concentration of 0.5 mM and agonist BIO592 to 1 mM and incubated on ice for 1 h. The coactivator peptide EBI96 which was identified by phage display was chosen for crystallization because of its strong interaction with RORγ in a mammalian two-hybrid analysis system that assessed the transactivation of RORγ. Diffraction quality crystals were grown through vapor diffusion in a buffer containing 0.1 M HEPES pH 8.0, 25 % PEG3350 and 0.2 M NaCl at 18 °C. Crystals were cryoprotected in the mother liquor containing 20 % glycerol as cryoprotectant prior to being frozen in liquid nitrogen for data collection. + + + METHODS + paragraph + 9278 + Actinase E proteolyzed RORγ518 BIO399 concentrated to 8 mg/ml was crystallized using vapor diffusion in a buffer containing 0.1 M BisTRIS pH 5.5, 0.2 M ammonium acetate and 15 % PEG3350 at 18 °C. Crystals were cryoprotected for data collection by transferring them to a mother liquor containing 15 % PEG400 prior to being frozen in liquid nitrogen. + + + METHODS + title_2 + 9636 + Data collection and structure determination for RORγ518 BIO592 and BIO399 complexes + + + METHODS + paragraph + 9724 + X-ray diffraction data for all the crystals were measured at beam line ID31 at the Argonne Photon Source. The data were processed with Mosflm in case of the RORγ518-BIO592-EBI96 ternary complex and with HKL2000 in the case of the Actinase E treated aeRORγ518/BIO399 complex. For both datasets, PDB ID: 3LOL was used as the search model, and the molecular replacement solutions were determined using MOLREP. The refinement was carried out using Refmac5 and model building was carried out in Coot. The data processing and refinement statistics are provided in Additional file 2. + + + METHODS + paragraph + 10309 + RORγ518-BIO592-EBI96 ternary complex: + + + METHODS + paragraph + 10351 + The data for the ternary complex were measured to 2.63 Å. It crystallized in a P21 space group with four molecules of the ternary complex in the asymmetric unit. The final model was refined to a Rcryst of 19.9 % and Rfree of 25.5 %. + + + METHODS + paragraph + 10588 + aeRORγ518/BIO399 complex: + + + METHODS + paragraph + 10618 + Diffraction data for the aeRORγ518-BIO399 complex were measured to 2.35 Å. It crystallized in C2 space group with two molecules in the asymmetric unit. The final model was refined to a Rcryst of 21.1 % and Rfree of 26.3 %. + + + RESULTS + title_1 + 10846 + Results and discussion + + + RESULTS + title_2 + 10869 + Identification of BIO592 and BIO399 as ligands that modulate RORγ coactivator peptide recruitment + + + RESULTS + paragraph + 10971 + Using a FRET based assay we discovered agonist BIO592 (Fig. 1a) which increased the coactivator peptide TRAP220 recruitment to RORγ (EC50 0f 58nM and Emax of 130 %) and a potent inverse agonist BIO399 (Fig. 1b) which inhibited coactivator recruitment (IC50: 4.7nM). Interestingly, the structural difference between the agonist BIO592 and inverse agonist BIO399 was minor; with the 2,3-dihydrobenzo[1,4]oxazepin-4-one ring system of BIO399 being 3 atoms larger than the benzo[1,4]oxazine-3-one ring system of BIO592. In order to understand how small changes in the core ring system leads to inverse agonism, we wanted to structurally determine the binding mode of both BIO592 and BIO399 in the LBS of RORγ using x-ray crystallography. + + 0.99959534 + experimental_method + cleaner0 + 2023-07-19T15:14:47Z + MESH: + + FRET based assay + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9988141 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.48274022 + chemical + cleaner0 + 2023-07-19T16:06:11Z + CHEBI: + + TRAP220 + + + 0.99982065 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99535024 + evidence + cleaner0 + 2023-07-19T15:20:56Z + DUMMY: + + EC50 + + + 0.9963708 + evidence + cleaner0 + 2023-07-19T15:21:01Z + DUMMY: + + Emax + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.9961767 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9992803 + evidence + cleaner0 + 2023-07-19T15:50:45Z + DUMMY: + + IC50 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9986708 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.9949522 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9993613 + chemical + cleaner0 + 2023-07-19T15:21:29Z + CHEBI: + + 2,3-dihydrobenzo[1,4]oxazepin-4-one + + + 0.9886368 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99949 + chemical + cleaner0 + 2023-07-19T15:21:40Z + CHEBI: + + benzo[1,4]oxazine-3-one + + + 0.9971795 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.9968554 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.9926582 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9996377 + site + cleaner0 + 2023-07-19T15:18:14Z + SO: + + LBS + + + 0.99981266 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99956167 + experimental_method + cleaner0 + 2023-07-19T15:14:52Z + MESH: + + x-ray crystallography + + + + RESULTS + title_2 + 11713 + Structure of the RORγ518-BIO592-EBI96 ternary complex is in a transcriptionally active conformation + + 0.71792406 + evidence + cleaner0 + 2023-07-19T15:50:51Z + DUMMY: + + Structure + + + 0.9997385 + complex_assembly + cleaner0 + 2023-07-19T15:21:15Z + GO: + + RORγ518-BIO592-EBI96 + + + 0.6499308 + protein_state + cleaner0 + 2023-07-19T15:21:52Z + DUMMY: + + active + + + + 12900_2016_59_Fig2_HTML.jpg + Fig2 + FIG + fig_caption + 11817 + +a The ternary structure of RORγ518 BIO592 and EBI96. b RORγ AF2 helix in the agonist conformation. c EBI96 coactivator peptide bound in the coactivator pocket of RORγ + + 0.88747376 + evidence + cleaner0 + 2023-07-19T15:50:58Z + DUMMY: + + ternary structure + + + 0.99852496 + protein + cleaner0 + 2023-07-19T15:23:34Z + PR: + + RORγ518 + + + 0.97716755 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.99978215 + chemical + cleaner0 + 2023-07-19T15:23:28Z + CHEBI: + + EBI96 + + + 0.9997538 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99961114 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9998048 + chemical + cleaner0 + 2023-07-19T15:23:28Z + CHEBI: + + EBI96 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T15:24:07Z + + bound in + + + 0.9995923 + site + cleaner0 + 2023-07-19T15:23:47Z + SO: + + coactivator pocket + + + 0.9994972 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + + RESULTS + paragraph + 11997 + RORγ518 bound to agonist BIO592 was crystallized with a truncated form of the coactivator peptide EBI96 to a resolution of 2.6 Å (Fig. 2a). The structure of the ternary complex had features similar to other ROR agonist coactivator structures in a transcriptionally active canonical three layer helix fold with the AF2 helix in the agonist conformation. The agonist conformation is stabilized by a hydrogen bond between His479 and Tyr502, in addition to π-π interactions between His479, Tyr502 and Phe506 (Fig. 2b). The hydrogen bond between His479 and Tyr502 has been reported to be critical for RORγ agonist activity. Disrupting this interaction through mutagenesis reduced transcriptional activity of RORγ. This reduced transcriptional activity has been attributed to the inability of the AF2 helix to complete the formation of the coactivator binding pocket necessary for coactivator proteins to bind. + + 0.9997739 + protein + cleaner0 + 2023-07-19T15:23:34Z + PR: + + RORγ518 + + + 0.9995284 + protein_state + cleaner0 + 2023-07-19T15:24:12Z + DUMMY: + + bound to + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.99953663 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.9996308 + experimental_method + cleaner0 + 2023-07-19T15:52:21Z + MESH: + + crystallized + + + 0.9987739 + protein_state + cleaner0 + 2023-07-19T16:00:29Z + DUMMY: + + truncated + + + 0.96395886 + chemical + cleaner0 + 2023-07-19T15:23:27Z + CHEBI: + + EBI96 + + + 0.9991172 + evidence + cleaner0 + 2023-07-19T15:51:05Z + DUMMY: + + structure + + + 0.99873656 + protein_type + cleaner0 + 2023-07-19T15:14:21Z + MESH: + + ROR + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9898212 + evidence + cleaner0 + 2023-07-19T15:51:13Z + DUMMY: + + structures + + + 0.99713844 + protein_state + cleaner0 + 2023-07-19T15:25:01Z + DUMMY: + + transcriptionally active + + + 0.99960726 + protein_state + cleaner0 + 2023-07-19T15:25:14Z + DUMMY: + + canonical three layer helix fold + + + 0.9997512 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.9995945 + protein_state + cleaner0 + 2023-07-19T16:00:34Z + DUMMY: + + agonist + + + 0.9995764 + protein_state + cleaner0 + 2023-07-19T16:00:38Z + DUMMY: + + agonist + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + hydrogen bond + + + 0.99990356 + residue_name_number + cleaner0 + 2023-07-19T15:25:24Z + DUMMY: + + His479 + + + 0.9998977 + residue_name_number + cleaner0 + 2023-07-19T15:18:57Z + DUMMY: + + Tyr502 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + π-π interactions + + + 0.99989974 + residue_name_number + cleaner0 + 2023-07-19T15:25:24Z + DUMMY: + + His479 + + + 0.9998951 + residue_name_number + cleaner0 + 2023-07-19T15:18:57Z + DUMMY: + + Tyr502 + + + 0.9998956 + residue_name_number + cleaner0 + 2023-07-19T15:19:02Z + DUMMY: + + Phe506 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + hydrogen bond + + + 0.99989986 + residue_name_number + cleaner0 + 2023-07-19T15:25:24Z + DUMMY: + + His479 + + + 0.99989367 + residue_name_number + cleaner0 + 2023-07-19T15:18:57Z + DUMMY: + + Tyr502 + + + 0.83831507 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9996239 + experimental_method + cleaner0 + 2023-07-19T15:25:38Z + MESH: + + mutagenesis + + + 0.99961555 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99976134 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.99962956 + site + cleaner0 + 2023-07-19T15:18:21Z + SO: + + coactivator binding pocket + + + + RESULTS + paragraph + 12916 + Electron density for the coactivator peptide EBI96 was observed for residues EFPYLLSLLG which formed a α-helix stabilized through hydrophobic interactions with the coactivator binding pocket on RORγ (Fig. 2c). This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. Formation of this charged clamp is essential for RORγ’s function for playing a role in transcriptional activation and this has been corroborated through mutagenic studies in this region. + + 0.99952424 + evidence + cleaner0 + 2023-07-19T15:25:47Z + DUMMY: + + Electron density + + + 0.9981281 + chemical + cleaner0 + 2023-07-19T15:23:28Z + CHEBI: + + EBI96 + + + 0.7580885 + structure_element + cleaner0 + 2023-07-19T15:25:52Z + SO: + + EFPYLLSLLG + + + 0.99970657 + structure_element + cleaner0 + 2023-07-19T15:25:58Z + SO: + + α-helix + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + hydrophobic interactions + + + 0.999589 + site + cleaner0 + 2023-07-19T15:18:21Z + SO: + + coactivator binding pocket + + + 0.9997904 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99953485 + protein_state + cleaner0 + 2023-07-19T15:26:40Z + DUMMY: + + conserved + + + 0.9152874 + structure_element + cleaner0 + 2023-07-19T15:26:31Z + SO: + + charged clamp + + + 0.9999007 + residue_name_number + cleaner0 + 2023-07-19T15:26:12Z + DUMMY: + + Tyr7 + + + 0.99990094 + residue_name_number + cleaner0 + 2023-07-19T15:26:17Z + DUMMY: + + Leu11 + + + 0.99767834 + chemical + cleaner0 + 2023-07-19T15:23:28Z + CHEBI: + + EBI96 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + hydrogen bonds + + + 0.9999043 + residue_name_number + cleaner0 + 2023-07-19T15:26:23Z + DUMMY: + + Glu504 + + + 0.9997838 + structure_element + cleaner0 + 2023-07-19T15:18:45Z + SO: + + helix12 + + + 0.9999033 + residue_name_number + cleaner0 + 2023-07-19T15:26:27Z + DUMMY: + + Lys336 + + + 0.9997806 + structure_element + cleaner0 + 2023-07-19T15:26:49Z + SO: + + helix3 + + + 0.9997863 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.85428715 + structure_element + cleaner0 + 2023-07-19T15:26:32Z + SO: + + charged clamp + + + 0.999788 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9995812 + experimental_method + cleaner0 + 2023-07-19T15:26:37Z + MESH: + + mutagenic studies + + + + RESULTS + title_2 + 13539 + BIO592 binds in a collapsed conformation stabilizing the agonist conformation of RORγ + + 0.9965342 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.999559 + protein_state + cleaner0 + 2023-07-19T15:27:02Z + DUMMY: + + collapsed + + + 0.6351255 + protein_state + cleaner0 + 2023-07-19T15:27:05Z + DUMMY: + + agonist + + + 0.99970454 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + + 12900_2016_59_Fig3_HTML.jpg + Fig3 + FIG + fig_caption + 13629 + +a Collapsed binding mode of agonist BIO592 in the hydrophobic LBS of RORγ. b Benzoxazinone ring system of agonist BIO592 packing against His479 of RORγ stabilizing agonist conformation of the AF2 helix + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9995141 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.6673667 + site + cleaner0 + 2023-07-19T15:18:14Z + SO: + + LBS + + + 0.999788 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + chemical + CHEBI: + cleaner0 + 2023-07-19T15:13:56Z + + Benzoxazinone + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.99953103 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.9998983 + residue_name_number + cleaner0 + 2023-07-19T15:25:24Z + DUMMY: + + His479 + + + 0.9997874 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.99967206 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + + RESULTS + paragraph + 13840 + BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). The sulfonyl group faces the entrance of the pocket, while the CF3 makes a hydrophobic contact with Ala327. Hydrophobic interaction between the ethyl group of the benzoxazinone and His479 reinforce the His479 sidechain position for making the hydrogen bond with Tyr502 thereby stabilizing the agonist conformation (Fig. 3b). + + 0.9990957 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.9857471 + protein_state + cleaner0 + 2023-07-19T15:24:08Z + DUMMY: + + bound in + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T15:27:48Z + + collapsed + + + 0.99963737 + site + cleaner0 + 2023-07-19T15:18:14Z + SO: + + LBS + + + 0.99978584 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99809307 + chemical + cleaner0 + 2023-07-19T15:29:29Z + CHEBI: + + xylene + + + 0.99882764 + site + cleaner0 + 2023-07-19T15:54:20Z + SO: + + pocket + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + hydrophobic interactions + + + 0.9999033 + residue_name_number + cleaner0 + 2023-07-19T15:28:06Z + DUMMY: + + Val376 + + + 0.9998995 + residue_name_number + cleaner0 + 2023-07-19T15:28:10Z + DUMMY: + + Phe378 + + + 0.99989617 + residue_name_number + cleaner0 + 2023-07-19T15:28:14Z + DUMMY: + + Phe388 + + + 0.9998977 + residue_name_number + cleaner0 + 2023-07-19T15:28:19Z + DUMMY: + + Phe401 + + + 0.9988957 + chemical + cleaner0 + 2023-07-19T15:29:27Z + CHEBI: + + ethyl-benzoxazinone + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + hydrophobic interactions + + + 0.9999012 + residue_name_number + cleaner0 + 2023-07-19T15:28:25Z + DUMMY: + + Trp317 + + + 0.999899 + residue_name_number + cleaner0 + 2023-07-19T15:28:29Z + DUMMY: + + Leu324 + + + 0.9999006 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.9999014 + residue_name_number + cleaner0 + 2023-07-19T15:28:37Z + DUMMY: + + Leu391 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T15:28:56Z + + Ile 400 + + + 0.9999039 + residue_name_number + cleaner0 + 2023-07-19T15:25:24Z + DUMMY: + + His479 + + + 0.97242635 + chemical + cleaner0 + 2023-07-19T15:29:34Z + CHEBI: + + sulfonyl + + + 0.9925991 + site + cleaner0 + 2023-07-19T15:54:25Z + SO: + + pocket + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + hydrophobic contact + + + 0.99990606 + residue_name_number + cleaner0 + 2023-07-19T15:29:06Z + DUMMY: + + Ala327 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + Hydrophobic interaction + + + 0.9993363 + chemical + cleaner0 + 2023-07-19T15:13:56Z + CHEBI: + + benzoxazinone + + + 0.9999043 + residue_name_number + cleaner0 + 2023-07-19T15:25:24Z + DUMMY: + + His479 + + + 0.999907 + residue_name_number + cleaner0 + 2023-07-19T15:25:24Z + DUMMY: + + His479 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + hydrogen bond + + + 0.99990344 + residue_name_number + cleaner0 + 2023-07-19T15:18:57Z + DUMMY: + + Tyr502 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + + RESULTS + title_2 + 14529 + RORγ AF2 helix is sensitive to proteolysis in the presence of Inverse Agonist BIO399 + + 0.999711 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99974036 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.9675162 + protein_state + cleaner0 + 2023-07-19T15:29:49Z + DUMMY: + + presence of + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + Inverse Agonist + + + 0.9995938 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + + RESULTS + paragraph + 14618 + Next, we attempted co-crystallization with the inverse agonist BIO399. However, extensive crystallization efforts with BIO399 and RORγ518 or other AF2 intact constructs did not produce crystals. We hypothesized that the RORγ518 coactivator peptide interaction in the FRET assay was disrupted upon BIO399 binding and that a conformational rearrangement of the AF2 helix could have occurred, hindering crystallization. + + 0.999572 + experimental_method + cleaner0 + 2023-07-19T15:30:01Z + MESH: + + co-crystallization + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.9988544 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99946946 + experimental_method + cleaner0 + 2023-07-19T15:29:54Z + MESH: + + crystallization + + + 0.9979844 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9996563 + protein + cleaner0 + 2023-07-19T15:23:34Z + PR: + + RORγ518 + + + 0.9412052 + structure_element + cleaner0 + 2023-07-19T15:59:05Z + SO: + + AF2 + + + 0.9938573 + protein_state + cleaner0 + 2023-07-19T15:30:12Z + DUMMY: + + intact + + + 0.9034121 + evidence + cleaner0 + 2023-07-19T15:30:17Z + DUMMY: + + crystals + + + 0.9997154 + protein + cleaner0 + 2023-07-19T15:23:34Z + PR: + + RORγ518 + + + 0.9995203 + experimental_method + cleaner0 + 2023-07-19T15:15:19Z + MESH: + + FRET assay + + + 0.99598217 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9997167 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.9927527 + experimental_method + cleaner0 + 2023-07-19T15:30:05Z + MESH: + + crystallization + + + + 12900_2016_59_Fig4_HTML.jpg + Fig4 + FIG + fig_caption + 15043 + Specific proteolytic positions on RORγ518 when treated with Actinase E alone (Green) or in the presence of BIO399 (Red) and shared proteolytic sites (Yellow) + + 0.99923706 + protein + cleaner0 + 2023-07-19T15:23:34Z + PR: + + RORγ518 + + + 0.9101149 + experimental_method + cleaner0 + 2023-07-19T15:52:27Z + MESH: + + treated with + + + 0.9971443 + protein + cleaner0 + 2023-07-19T15:53:23Z + PR: + + Actinase E + + + 0.99917436 + protein_state + cleaner0 + 2023-07-19T15:29:49Z + DUMMY: + + presence of + + + 0.999374 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99941564 + site + cleaner0 + 2023-07-19T15:54:37Z + SO: + + proteolytic sites + + + + RESULTS + paragraph + 15205 + The unfolding of the AF2 helix has been observed for other nuclear hormone receptors when bound to an inverse agonist or antagonist. We used partial proteolysis in combination with mass spectrometry to determine if BIO399 was causing the AF2 helix to unfold. Results of the Actinase E proteolysis experiments on RORγ518, the ternary complex of RORγ518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis. Analysis of the fragmentation pattern showed minimal proteolytic removal of the AF2 helix by Actinase E on RORγ518 alone (ending at 504 to 506) and the ternary complex remained primarily intact (ending at 515/518) (Additional file 4). However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig. 4, Additional file 5). + + 0.9997374 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.9996216 + protein_type + cleaner0 + 2023-07-19T15:18:39Z + MESH: + + nuclear hormone receptors + + + 0.99951684 + protein_state + cleaner0 + 2023-07-19T15:24:12Z + DUMMY: + + bound to + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.99952674 + experimental_method + cleaner0 + 2023-07-19T15:31:34Z + MESH: + + partial proteolysis + + + 0.9995297 + experimental_method + cleaner0 + 2023-07-19T15:31:30Z + MESH: + + mass spectrometry + + + 0.99214536 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9997202 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.7968726 + experimental_method + cleaner0 + 2023-07-19T15:31:25Z + MESH: + + Actinase E proteolysis + + + 0.9997969 + protein + cleaner0 + 2023-07-19T15:23:34Z + PR: + + RORγ518 + + + 0.99978536 + protein + cleaner0 + 2023-07-19T15:23:34Z + PR: + + RORγ518 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9989819 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.9997379 + chemical + cleaner0 + 2023-07-19T15:23:28Z + CHEBI: + + EBI96 + + + 0.99913347 + protein_state + cleaner0 + 2023-07-19T15:29:49Z + DUMMY: + + presence of + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.99921584 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99508756 + evidence + cleaner0 + 2023-07-19T15:31:16Z + DUMMY: + + fragmentation pattern + + + 0.9997283 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.9967369 + protein + cleaner0 + 2023-07-19T15:53:23Z + PR: + + Actinase E + + + 0.99980134 + protein + cleaner0 + 2023-07-19T15:23:34Z + PR: + + RORγ518 + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T15:48:58Z + + 504 to 506 + + + 0.80447483 + protein_state + cleaner0 + 2023-07-19T16:00:48Z + DUMMY: + + ternary complex + + + 0.43031177 + residue_number + cleaner0 + 2023-07-19T15:49:44Z + DUMMY: + + 515 + + + residue_number + DUMMY: + cleaner0 + 2023-07-19T15:49:25Z + + 518 + + + 0.999481 + protein_state + cleaner0 + 2023-07-19T15:29:49Z + DUMMY: + + presence of + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.99928194 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9842393 + evidence + cleaner0 + 2023-07-19T15:31:19Z + DUMMY: + + proteolytic pattern + + + 0.49895975 + residue_number + cleaner0 + 2023-07-19T15:49:48Z + DUMMY: + + 494 + + + residue_number + DUMMY: + cleaner0 + 2023-07-19T15:49:40Z + + 495 + + + 0.9997306 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.99967384 + protein_state + cleaner0 + 2023-07-19T16:00:53Z + DUMMY: + + APO + + + 0.7635996 + protein_state + cleaner0 + 2023-07-19T16:01:01Z + DUMMY: + + ternary agonist complex + + + + RESULTS + paragraph + 16237 + Several rounds of cocrystallization attempts with RORγ518 or other RORγ AF2 helix containing constructs complexed with BIO399 had not produced crystals. We attributed the inability to form crystals to the unfolding of the AF2 helix induced by BIO399. We reasoned that if we could remove the unfolded AF2 helix using proteolysis we could produce a binary complex more amenable to crystallization. + + 0.999603 + experimental_method + cleaner0 + 2023-07-19T15:32:05Z + MESH: + + cocrystallization + + + 0.9997676 + protein + cleaner0 + 2023-07-19T15:23:34Z + PR: + + RORγ518 + + + 0.9812258 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99960935 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.99924374 + protein_state + cleaner0 + 2023-07-19T15:31:42Z + DUMMY: + + complexed with + + + 0.88344735 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.97513825 + evidence + cleaner0 + 2023-07-19T15:32:18Z + DUMMY: + + crystals + + + 0.8911621 + evidence + cleaner0 + 2023-07-19T15:32:16Z + DUMMY: + + crystals + + + 0.9997282 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.96908194 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99956316 + protein_state + cleaner0 + 2023-07-19T15:32:14Z + DUMMY: + + unfolded + + + 0.99973404 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.99730253 + experimental_method + cleaner0 + 2023-07-19T15:32:09Z + MESH: + + proteolysis + + + 0.99929774 + experimental_method + cleaner0 + 2023-07-19T15:32:11Z + MESH: + + crystallization + + + + RESULTS + title_2 + 16641 + AF2 truncated RORγ BIO399 complex is more amenable to crystallization + + 0.99842143 + protein_state + cleaner0 + 2023-07-19T15:32:36Z + DUMMY: + + AF2 truncated + + + 0.99530745 + complex_assembly + cleaner0 + 2023-07-19T15:32:42Z + GO: + + RORγ BIO399 + + + 0.9996778 + experimental_method + cleaner0 + 2023-07-19T15:32:39Z + MESH: + + crystallization + + + + 12900_2016_59_Fig5_HTML.jpg + Fig5 + FIG + fig_caption + 16715 + +a The binary structure of AF2-truncated RORγ and BIO399. b The superposition of inverse agonist BIO399 (Cyan) and agonist BIO592 (Green). c Movement of Met358 and His479 in the BIO399 (Cyan) and BIO592 (Green) structures + + 0.9753916 + evidence + cleaner0 + 2023-07-19T15:32:53Z + DUMMY: + + structure + + + 0.99942464 + protein_state + cleaner0 + 2023-07-19T16:01:07Z + DUMMY: + + AF2-truncated + + + 0.9997619 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9949635 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9996861 + experimental_method + cleaner0 + 2023-07-19T15:32:49Z + MESH: + + superposition + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.9971846 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.99816895 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.9998957 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.99989843 + residue_name_number + cleaner0 + 2023-07-19T15:25:24Z + DUMMY: + + His479 + + + 0.9886769 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9916482 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.99948066 + evidence + cleaner0 + 2023-07-19T15:32:55Z + DUMMY: + + structures + + + + RESULTS + paragraph + 16941 + The Actinase E treated RORγ518 BIO399 ternary complex (aeRORγ493/4) co-crystallized readily in several PEG based conditions. The structure of aeRORγ493/4 BIO399 complex was solved to 2.3 Å and adopted a similar core fold to the BIO592 agonist crystal structure (Fig. 5a, Additional file 3). The aeRORγ493/4 BIO399 structure diverged at the c-terminal end of Helix 11 from the RORγ518 BIO592 EBI96 structure, where helix 11 unwinds into a random coil after residue L475. + + protein + PR: + cleaner0 + 2023-07-19T15:53:22Z + + Actinase E + + + 0.99942446 + complex_assembly + cleaner0 + 2023-07-19T15:33:06Z + GO: + + RORγ518 BIO399 + + + 0.99912286 + complex_assembly + cleaner0 + 2023-07-19T15:33:12Z + GO: + + aeRORγ493/4 + + + 0.99949425 + experimental_method + cleaner0 + 2023-07-19T15:34:49Z + MESH: + + co-crystallized + + + 0.99952996 + evidence + cleaner0 + 2023-07-19T15:34:46Z + DUMMY: + + structure + + + 0.9994172 + complex_assembly + cleaner0 + 2023-07-19T15:33:28Z + GO: + + aeRORγ493/4 BIO399 + + + 0.84812504 + experimental_method + cleaner0 + 2023-07-19T15:34:51Z + MESH: + + solved + + + 0.98904616 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.999605 + evidence + cleaner0 + 2023-07-19T15:34:41Z + DUMMY: + + crystal structure + + + 0.9969038 + complex_assembly + cleaner0 + 2023-07-19T15:33:29Z + GO: + + aeRORγ493/4 BIO399 + + + 0.99960536 + evidence + cleaner0 + 2023-07-19T15:34:38Z + DUMMY: + + structure + + + 0.99971807 + structure_element + cleaner0 + 2023-07-19T15:34:34Z + SO: + + Helix 11 + + + complex_assembly + GO: + cleaner0 + 2023-07-19T15:34:27Z + + RORγ518 BIO592 EBI96 + + + 0.9993129 + evidence + cleaner0 + 2023-07-19T15:34:43Z + DUMMY: + + structure + + + 0.99970734 + structure_element + cleaner0 + 2023-07-19T15:34:33Z + SO: + + helix 11 + + + 0.99849415 + residue_name_number + cleaner0 + 2023-07-19T15:33:49Z + DUMMY: + + L475 + + + + RESULTS + title_2 + 17431 + Inverse agonist BIO399 uses Met358 as a trigger for inverse agonism + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + Inverse agonist + + + 0.9986848 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9999069 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + + RESULTS + paragraph + 17499 + BIO399 binds to the ligand binding site of RORγ adopting a collapsed conformation as seen with BIO592 where the two compounds superimpose with an RMSD of 0.72 Å (Fig. 5b). The majority of the side chains within 4 Å of BIO399 and BIO592 adopt similar rotomer conformations with the exceptions of Met358 and His479 (Fig. 5c). The difference density map showed clear positive density for Met358 in an alternate rotomer conformation compared to the one observed in the molecular replacement model or the other agonist containing models (Additional file 6). We tried to refine Met358 in the same conformation as the molecular replacement model or the other agonist containing models, but the results clearly indicated that this was not possible, thus confirming the new rotamer conformation for the Met358 sidechain in the inverse agonist bound structure. The change in rotomer conformation of Met358 between the agonist and inverse agonist structures is attributed to the gem-dimethyl group on the larger 7 membered benzoxazinone ring system of BIO399. The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c). + + 0.9960114 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99961 + site + cleaner0 + 2023-07-19T15:34:59Z + SO: + + ligand binding site + + + 0.99980706 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T15:35:15Z + + collapsed + + + 0.9943234 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.998801 + experimental_method + cleaner0 + 2023-07-19T15:53:32Z + MESH: + + superimpose + + + 0.99946564 + evidence + cleaner0 + 2023-07-19T15:35:28Z + DUMMY: + + RMSD + + + 0.9960063 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99231964 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.9998167 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.9998622 + residue_name_number + cleaner0 + 2023-07-19T15:25:24Z + DUMMY: + + His479 + + + 0.9993809 + evidence + cleaner0 + 2023-07-19T15:35:33Z + DUMMY: + + difference density map + + + 0.9976566 + evidence + cleaner0 + 2023-07-19T15:35:38Z + DUMMY: + + positive density + + + 0.99972934 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.99948543 + experimental_method + cleaner0 + 2023-07-19T15:35:43Z + MESH: + + molecular replacement model + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.99954575 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.9995101 + experimental_method + cleaner0 + 2023-07-19T15:35:43Z + MESH: + + molecular replacement model + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.99965596 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.99805737 + protein_state + cleaner0 + 2023-07-19T16:01:14Z + DUMMY: + + inverse agonist bound + + + 0.9991708 + evidence + cleaner0 + 2023-07-19T15:35:59Z + DUMMY: + + structure + + + 0.999673 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.9903914 + protein_state + cleaner0 + 2023-07-19T16:01:37Z + DUMMY: + + agonist + + + 0.9016677 + protein_state + cleaner0 + 2023-07-19T16:01:19Z + DUMMY: + + inverse agonist + + + 0.9906707 + evidence + cleaner0 + 2023-07-19T15:36:01Z + DUMMY: + + structures + + + 0.99437535 + chemical + cleaner0 + 2023-07-19T15:13:56Z + CHEBI: + + benzoxazinone + + + 0.99684626 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.8270343 + experimental_method + cleaner0 + 2023-07-19T15:53:36Z + MESH: + + comparison + + + 0.74309015 + evidence + cleaner0 + 2023-07-19T15:36:04Z + DUMMY: + + structures + + + 0.88217115 + protein_state + cleaner0 + 2023-07-19T16:01:38Z + DUMMY: + + agonist + + + 0.53165466 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.9989053 + evidence + cleaner0 + 2023-07-19T15:36:06Z + DUMMY: + + structure + + + 0.80424184 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9997873 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.9998599 + residue_name_number + cleaner0 + 2023-07-19T15:19:02Z + DUMMY: + + Phe506 + + + 0.99969506 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.9998171 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.9997954 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + + RESULTS + title_2 + 18820 + BIO399 and Inverse agonist T0901317 bind in a collapsed conformation distinct from other RORγ Inverse Agonists Cocrystal structures + + 0.9986603 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + Inverse agonist + + + 0.9993025 + chemical + cleaner0 + 2023-07-19T15:36:21Z + CHEBI: + + T0901317 + + + 0.9996755 + protein_state + cleaner0 + 2023-07-19T15:36:15Z + DUMMY: + + collapsed + + + 0.6189321 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99943864 + evidence + cleaner0 + 2023-07-19T15:36:12Z + DUMMY: + + Cocrystal structures + + + + 12900_2016_59_Fig6_HTML.jpg + Fig6 + FIG + fig_caption + 18956 + +a Overlay of RORγ structures bound to BIO596 (Green), BIO399 (Cyan) and T0901317 (Pink). b Overlay of M358 in RORγ structure BIO596 (Green), BIO399 (Cyan), Digoxin (Yellow), Compound 2 (Grey), Compound 48 (Salmon) and Compound 4j (Orange) + + 0.99830246 + experimental_method + cleaner0 + 2023-07-19T15:36:34Z + MESH: + + Overlay + + + 0.99475783 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9991398 + evidence + cleaner0 + 2023-07-19T15:36:36Z + DUMMY: + + structures + + + 0.9994433 + protein_state + cleaner0 + 2023-07-19T15:24:12Z + DUMMY: + + bound to + + + 0.9967662 + chemical + cleaner0 + 2023-07-19T16:06:49Z + CHEBI: + + BIO596 + + + 0.9978752 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.96408117 + chemical + cleaner0 + 2023-07-19T15:36:22Z + CHEBI: + + T0901317 + + + 0.99945694 + experimental_method + cleaner0 + 2023-07-19T15:36:42Z + MESH: + + Overlay + + + 0.99989235 + residue_name_number + cleaner0 + 2023-07-19T15:50:13Z + DUMMY: + + M358 + + + 0.99910957 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9986027 + evidence + cleaner0 + 2023-07-19T15:36:44Z + DUMMY: + + structure + + + 0.997511 + chemical + cleaner0 + 2023-07-19T16:07:00Z + CHEBI: + + BIO596 + + + 0.99866104 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9995783 + chemical + cleaner0 + 2023-07-19T15:36:55Z + CHEBI: + + Digoxin + + + + RESULTS + paragraph + 19204 + The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. The two compounds superimpose with an RMSD of 0.81 Å (Fig. 6a). The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the RORγ BIO592 agonist structure. We hypothesize that since the Met358 sidechain conformation in the T0901317 RORγ structure is not in the BIO399 conformation, this difference could account for the 10-fold reduction in the inverse agonism for T0901317 compared to BIO399 in the FRET assay. + + 0.9994807 + evidence + cleaner0 + 2023-07-19T15:37:12Z + DUMMY: + + co-crystal structure + + + 0.99975437 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9983985 + chemical + cleaner0 + 2023-07-19T15:36:22Z + CHEBI: + + T0901317 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.9997485 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9804152 + evidence + cleaner0 + 2023-07-19T15:51:33Z + DUMMY: + + IC50 + + + 0.99956346 + experimental_method + cleaner0 + 2023-07-19T15:37:06Z + MESH: + + SRC1 displacement FRET assay + + + 0.9948265 + evidence + cleaner0 + 2023-07-19T15:51:37Z + DUMMY: + + IC50 + + + 0.99950325 + experimental_method + cleaner0 + 2023-07-19T15:15:19Z + MESH: + + FRET assay + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T15:37:44Z + + collapsed + + + 0.63860226 + evidence + cleaner0 + 2023-07-19T15:51:41Z + DUMMY: + + structure + + + 0.9829714 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9992175 + experimental_method + cleaner0 + 2023-07-19T15:37:15Z + MESH: + + superimpose + + + 0.99965835 + evidence + cleaner0 + 2023-07-19T15:35:28Z + DUMMY: + + RMSD + + + 0.99770904 + chemical + cleaner0 + 2023-07-19T15:37:28Z + CHEBI: + + hexafluoropropanol + + + 0.9959305 + chemical + cleaner0 + 2023-07-19T15:36:22Z + CHEBI: + + T0901317 + + + 0.9994836 + evidence + cleaner0 + 2023-07-19T15:25:47Z + DUMMY: + + electron density + + + 0.99987984 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.9998841 + residue_name_number + cleaner0 + 2023-07-19T15:19:02Z + DUMMY: + + Phe506 + + + 0.9995697 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.8003477 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.9993617 + evidence + cleaner0 + 2023-07-19T15:37:20Z + DUMMY: + + structure + + + 0.99987173 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.9912252 + chemical + cleaner0 + 2023-07-19T15:36:22Z + CHEBI: + + T0901317 + + + 0.99891305 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99936765 + evidence + cleaner0 + 2023-07-19T15:37:23Z + DUMMY: + + structure + + + 0.86961484 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99782795 + chemical + cleaner0 + 2023-07-19T15:36:22Z + CHEBI: + + T0901317 + + + 0.9925011 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99927664 + experimental_method + cleaner0 + 2023-07-19T15:15:19Z + MESH: + + FRET assay + + + + RESULTS + paragraph + 20050 + Co-crystal structures of RORγ have been generated with several potent inverse agonists adopting a linear conformation distinct from the collapsed conformations seen for BIO399 and T090131718. The inverse agonist activity for these compounds has been attributed to orientating Trp317 to clash with Tyr502 or a direct inverse agonist hydrogen bonding event with His479, both of which would perturb the agonist conformation of RORγ. BIO399 neither orients the sidechain of Trp317 toward Tyr502 nor forms a hydrogen bond with His479 suggesting its mode of action is distinct from linear inverse agonists (Additional file 8). In the linear inverse agonist crystal structures the side chain of Met358 resides in a similar position as the rotomer observed in RORγ agonist structures with BIO592 described here or as observed in the hydroxycholesterol derivatives and therefore would not trigger inverse agonism with these ligands (Fig. 6b). + + 0.9995247 + evidence + cleaner0 + 2023-07-19T15:37:57Z + DUMMY: + + Co-crystal structures + + + 0.9997024 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9989466 + protein_state + cleaner0 + 2023-07-19T15:37:52Z + DUMMY: + + linear + + + 0.9995504 + protein_state + cleaner0 + 2023-07-19T15:37:54Z + DUMMY: + + collapsed + + + 0.9995369 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9996209 + chemical + cleaner0 + 2023-07-19T16:07:05Z + CHEBI: + + T090131718 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.99989784 + residue_name_number + cleaner0 + 2023-07-19T15:28:25Z + DUMMY: + + Trp317 + + + 0.99989426 + residue_name_number + cleaner0 + 2023-07-19T15:18:57Z + DUMMY: + + Tyr502 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + hydrogen bonding + + + 0.9999031 + residue_name_number + cleaner0 + 2023-07-19T15:25:24Z + DUMMY: + + His479 + + + 0.99489343 + protein_state + cleaner0 + 2023-07-19T16:01:38Z + DUMMY: + + agonist + + + 0.9996356 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9995697 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9999018 + residue_name_number + cleaner0 + 2023-07-19T15:28:25Z + DUMMY: + + Trp317 + + + 0.9998988 + residue_name_number + cleaner0 + 2023-07-19T15:18:57Z + DUMMY: + + Tyr502 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:18:02Z + + hydrogen bond + + + 0.9999014 + residue_name_number + cleaner0 + 2023-07-19T15:25:24Z + DUMMY: + + His479 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.9996216 + evidence + cleaner0 + 2023-07-19T15:38:08Z + DUMMY: + + crystal structures + + + 0.9999008 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.9991548 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.7199689 + evidence + cleaner0 + 2023-07-19T15:38:11Z + DUMMY: + + structures + + + 0.99945503 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.99954826 + chemical + cleaner0 + 2023-07-19T15:38:05Z + CHEBI: + + hydroxycholesterol + + + + RESULTS + title_2 + 20996 + BIO399 shows selectivity for RORγ over RORα and RORβ in a GAL4 Cellular Reporter Assay + + 0.9948939 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9997609 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99983096 + protein + cleaner0 + 2023-07-19T15:16:02Z + PR: + + RORα + + + 0.99983597 + protein + cleaner0 + 2023-07-19T15:16:06Z + PR: + + RORβ + + + 0.9995607 + experimental_method + cleaner0 + 2023-07-19T15:38:17Z + MESH: + + GAL4 Cellular Reporter Assay + + + + Tab1.xml + Tab1 + TABLE + table_caption + 21094 + GAL4 cell assay selectivity profile for BIO399 toward RORα and RORβ in GAL4 + + 0.9994817 + experimental_method + cleaner0 + 2023-07-19T15:38:20Z + MESH: + + GAL4 cell assay + + + 0.9978909 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.9998468 + protein + cleaner0 + 2023-07-19T15:16:02Z + PR: + + RORα + + + 0.9998485 + protein + cleaner0 + 2023-07-19T15:16:06Z + PR: + + RORβ + + + 0.98739153 + protein + cleaner0 + 2023-07-19T15:57:30Z + PR: + + GAL4 + + + + Tab1.xml + Tab1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><thead><tr><th>ROR</th><th>γ</th><th>α</th><th>β</th></tr></thead><tbody><tr><td>IC50 (uM)</td><td>0.043 (+/− 0.01uM; N = 6)</td><td>&gt;10 (N = 2)</td><td>&gt;1.2 (N = 2)</td></tr><tr><td>Selectivity (X)</td><td>-</td><td>&gt;235</td><td>&gt;28.2</td></tr></tbody></table> + + 21177 + ROR γ α β IC50 (uM) 0.043 (+/− 0.01uM; N = 6) >10 (N = 2) >1.2 (N = 2) Selectivity (X) - >235 >28.2 + + + 12900_2016_59_Fig7_HTML.jpg + Fig7 + FIG + fig_caption + 21301 + +a Overlay of RORα (yellow), β (pink) and γ (cyan) showing side chain differences at Met358 inverse agonism trigger position and (b) around the benzoxazinone ring system of BIO399 + + 0.9992525 + experimental_method + cleaner0 + 2023-07-19T15:38:33Z + MESH: + + Overlay + + + 0.99982053 + protein + cleaner0 + 2023-07-19T15:16:02Z + PR: + + RORα + + + 0.9997744 + protein + cleaner0 + 2023-07-19T15:38:36Z + PR: + + β + + + 0.999747 + protein + cleaner0 + 2023-07-19T15:38:39Z + PR: + + γ + + + 0.9998907 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T15:13:56Z + + benzoxazinone + + + 0.99398804 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + + RESULTS + paragraph + 21492 + In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. The ROR-GAL4 fusion proteins were expressed in cells with the luciferase reporter gene under the control of a GAL4 promoter. BIO399 inhibited the luciferase activity when added to the cells expressing the RORγ-GAL4 fusion with an in vivo IC50 of 42.5nM while showing >235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR α or β, respectively (Table 1). The LBS of RORs share a high degree of similarity. However, the inverse agonism trigger of BIO399, residue Met358, is a leucine in both RORα and β. This selectivity profile for BIO399 is attributed to the shorter leucine side chain in RORα and β which would not reach the phenylalanine on the AF2 helix further underscoring the role of Met358 as a trigger for RORγ specific inverse agonism (Fig. 7a). Furthermore, RORα contains two phenylalanine residues in its LBS whereas RORβ and γ have a leucine in the same position (Fig. 6b). We hypothesize that the two phenylalanine residues in the LBS of RORα occlude the dihydrobenzoxazepinone ring system of BIO399 from binding it and responsible for the increase in selectivity for RORα over β. + + 0.99780947 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99894136 + experimental_method + cleaner0 + 2023-07-19T15:39:38Z + MESH: + + cellular reporter assay + + + 0.9995373 + structure_element + cleaner0 + 2023-07-19T15:39:40Z + SO: + + ligand binding domains + + + 0.9841285 + protein_type + cleaner0 + 2023-07-19T15:14:21Z + MESH: + + ROR + + + 0.9995415 + protein + cleaner0 + 2023-07-19T15:39:27Z + PR: + + α + + + 0.9996561 + protein + cleaner0 + 2023-07-19T15:39:30Z + PR: + + β + + + 0.9997303 + protein + cleaner0 + 2023-07-19T15:39:33Z + PR: + + γ + + + 0.774799 + experimental_method + cleaner0 + 2023-07-19T15:39:35Z + MESH: + + fused to + + + 0.99931574 + structure_element + cleaner0 + 2023-07-19T15:16:53Z + SO: + + DNA binding domain + + + 0.96916354 + protein_type + cleaner0 + 2023-07-19T15:39:49Z + MESH: + + transcriptional factor + + + 0.8299078 + protein + cleaner0 + 2023-07-19T15:39:52Z + PR: + + GAL4 + + + protein_type + MESH: + cleaner0 + 2023-07-19T15:14:21Z + + ROR + + + protein + PR: + cleaner0 + 2023-07-19T15:57:30Z + + GAL4 + + + protein + PR: + cleaner0 + 2023-07-19T15:57:30Z + + GAL4 + + + 0.99855095 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + protein + PR: + cleaner0 + 2023-07-19T15:12:45Z + + RORγ + + + protein + PR: + cleaner0 + 2023-07-19T15:57:30Z + + GAL4 + + + 0.99954826 + evidence + cleaner0 + 2023-07-19T15:52:08Z + DUMMY: + + IC50 + + + 0.34280136 + protein + cleaner0 + 2023-07-19T15:57:30Z + PR: + + GAL4 + + + 0.9998186 + structure_element + cleaner0 + 2023-07-19T15:17:06Z + SO: + + LBD + + + 0.6574821 + protein_type + cleaner0 + 2023-07-19T15:14:21Z + MESH: + + ROR + + + 0.9995084 + protein + cleaner0 + 2023-07-19T15:40:06Z + PR: + + α + + + 0.9996867 + protein + cleaner0 + 2023-07-19T15:40:09Z + PR: + + β + + + 0.99981755 + site + cleaner0 + 2023-07-19T15:18:14Z + SO: + + LBS + + + 0.9994504 + protein_type + cleaner0 + 2023-07-19T15:16:33Z + MESH: + + RORs + + + 0.99703026 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99989223 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.9991722 + residue_name + cleaner0 + 2023-07-19T15:40:17Z + SO: + + leucine + + + 0.9998022 + protein + cleaner0 + 2023-07-19T15:16:02Z + PR: + + RORα + + + 0.9997577 + protein + cleaner0 + 2023-07-19T15:56:20Z + PR: + + β + + + 0.99747974 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99865365 + residue_name + cleaner0 + 2023-07-19T15:40:17Z + SO: + + leucine + + + 0.9997924 + protein + cleaner0 + 2023-07-19T15:16:02Z + PR: + + RORα + + + 0.99976355 + protein + cleaner0 + 2023-07-19T15:40:25Z + PR: + + β + + + 0.9991916 + residue_name + cleaner0 + 2023-07-19T15:40:21Z + SO: + + phenylalanine + + + 0.9997171 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.99989223 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + 0.9997229 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9998173 + protein + cleaner0 + 2023-07-19T15:16:02Z + PR: + + RORα + + + 0.9992132 + residue_name + cleaner0 + 2023-07-19T15:40:21Z + SO: + + phenylalanine + + + 0.9998124 + site + cleaner0 + 2023-07-19T15:18:14Z + SO: + + LBS + + + 0.9997999 + protein + cleaner0 + 2023-07-19T15:16:06Z + PR: + + RORβ + + + 0.9997799 + protein + cleaner0 + 2023-07-19T15:40:28Z + PR: + + γ + + + 0.99925333 + residue_name + cleaner0 + 2023-07-19T15:40:17Z + SO: + + leucine + + + 0.99922276 + residue_name + cleaner0 + 2023-07-19T15:40:21Z + SO: + + phenylalanine + + + 0.99981564 + site + cleaner0 + 2023-07-19T15:18:14Z + SO: + + LBS + + + 0.999821 + protein + cleaner0 + 2023-07-19T15:16:02Z + PR: + + RORα + + + 0.99927276 + chemical + cleaner0 + 2023-07-19T15:40:31Z + CHEBI: + + dihydrobenzoxazepinone + + + 0.99807584 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.999803 + protein + cleaner0 + 2023-07-19T15:16:02Z + PR: + + RORα + + + 0.999731 + protein + cleaner0 + 2023-07-19T15:56:26Z + PR: + + β + + + + CONCL + title_1 + 22900 + Conclusions + + + CONCL + paragraph + 22912 + We have identified a novel series of synthetic benzoxazinone ligands which modulate the transcriptional activity of RORγ in a FRET based assay. Using partial proteolysis we show a conformational change which destabilizes the AF2 helix of RORγ when the inverse agonist BIO399 binds. The two RORγ co-crystal structures reported here show how a small change to the core ring system can modulate the mode of action from agonist (BIO592) to inverse agonism (BIO399). Finally, we are reporting a newly identified trigger for achieving RORγ specific inverse agonism in an in vivo setting through Met358 which perturbs the agonist conformation of the AF2 helix and prevents coactivator protein binding. + + 0.9997615 + chemical + cleaner0 + 2023-07-19T15:13:56Z + CHEBI: + + benzoxazinone + + + 0.9997459 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99949485 + experimental_method + cleaner0 + 2023-07-19T15:14:47Z + MESH: + + FRET based assay + + + 0.9995538 + experimental_method + cleaner0 + 2023-07-19T15:40:50Z + MESH: + + partial proteolysis + + + 0.9997312 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + 0.99975103 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:20Z + + inverse agonist + + + 0.9993443 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99973184 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.99941427 + evidence + cleaner0 + 2023-07-19T15:40:53Z + DUMMY: + + co-crystal structures + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.99953425 + chemical + cleaner0 + 2023-07-19T15:14:02Z + CHEBI: + + BIO592 + + + 0.9995059 + chemical + cleaner0 + 2023-07-19T15:14:09Z + CHEBI: + + BIO399 + + + 0.99942756 + protein + cleaner0 + 2023-07-19T15:12:45Z + PR: + + RORγ + + + 0.9998945 + residue_name_number + cleaner0 + 2023-07-19T15:15:13Z + DUMMY: + + Met358 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T16:01:38Z + + agonist + + + 0.99972713 + structure_element + cleaner0 + 2023-07-19T15:14:26Z + SO: + + AF2 helix + + + + ABBR + title_1 + 23623 + Abbreviations + + + ABBR + paragraph + 23637 + AF2, activation function 2; BisTRIS, 2-[Bis(2-hydroxyethyl)amino]-a-(hydroxymethyl)propane-1,3-diol; DND, DNA binding domain; DTT, 1,4-Dithiothreitol; EDTA, 2-({2-[Bis(carboxymethyl)amino]ethyl}(carboxymethyl)amino)acetic acid; FRET, fluorescence resonance energy transfer; GST, Glutathione-S-Transferase; HEPES, 2-[4(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IC50, half maximal inhibitory concentration; IL-17, Interleukin-17; IPTG, isopropyl β-D-1-thiogalactopyranoside; LBD, Ligand Binding Domain; LBS, ligand binding site; LC-MS, liquid chromatography/mass spectrometry; PDB, Protein Data Bank; ROR, retinoid orphan receptor; SRC-1, steroid receptor coactivator-1; TH17 Cells, T helper cells; TRIS, 2-amino-2-hydroxymethyl-propane-1,3,diol. + + + ABBR + title_1 + 24392 + Additional files + + + COMP_INT + title_1 + 24409 + Competing interests + + + COMP_INT + paragraph + 24429 + The authors declare that they have no competing interests. + + + COMP_INT + title_1 + 24488 + Consent to publish + + + COMP_INT + paragraph + 24507 + Not applicable. + + + COMP_INT + title_1 + 24523 + Ethics + + + COMP_INT + paragraph + 24530 + Not applicable. + + + REF + title + 24546 + References + + + REF + ref + 24557 + Jetten AM. Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism. Nuclear Receptor Signaingl. 2007;4:e003. + + + 1121 + 33 + surname:Ivanov;given-names:II + surname:McKenzie;given-names:BS + surname:Zhou;given-names:L + surname:Tadokoro;given-names:CE + surname:Lepelley;given-names:A + surname:Lafaille;given-names:JJ + surname:Cua;given-names:DJ + surname:Littman;given-names:DR + 10.1016/j.cell.2006.07.035 + 16990136 + REF + Cell + ref + 126 + 2006 + 24735 + The Orphan Nuclear Receptor RORγt Directs the Differentiation Program of Proinflammatory IL-17+ T Helper Cells + + + 963 + 70 + surname:Chabaud;given-names:M + surname:Durand;given-names:JM + surname:Buchs;given-names:N + surname:Fossiez;given-names:F + surname:Page;given-names:G + surname:Frappart;given-names:L + surname:Miossec;given-names:P + 10.1002/1529-0131(199905)42:5<963::AID-ANR15>3.0.CO;2-E + 10323452 + REF + Arthritis Rheum + ref + 42 + 1999 + 24850 + Human interleukin-17: A T cell-derived proinflammatory cytokine produced by the rheumatoid synovium + + + 500 + 8 + surname:Lock;given-names:C + surname:Hermans;given-names:G + surname:Klonowski;given-names:P + surname:Austin;given-names:A + surname:Lad;given-names:N + surname:Kaminski;given-names:N + surname:Galli;given-names:SJ + surname:Oksenberg;given-names:JR + surname:Raine;given-names:CS + surname:Heller;given-names:R + surname:Steinman;given-names:L + surname:Pedotti;given-names:R + surname:Brendolan;given-names:A + surname:Schadt;given-names:E + surname:Garren;given-names:H + surname:Langer-Gould;given-names:A + surname:Strober;given-names:S + surname:Cannella;given-names:B + surname:Allard;given-names:J + 10.1038/nm0502-500 + 11984595 + REF + Nat Med + ref + 8 + 2002 + 24950 + Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis + + + 65 + 70 + surname:Fujino;given-names:S + surname:Andoh;given-names:A + surname:Bamba;given-names:S + surname:Ogawa;given-names:A + surname:Hata;given-names:K + surname:Araki;given-names:Y + surname:Bamba;given-names:T + surname:Fujiyama;given-names:Y + 10.1136/gut.52.1.65 + 12477762 + REF + Gut + ref + 52 + 2003 + 25066 + Increased expression of interleukin 17 in inflammatory bowel disease + + + 3800 + 6 + surname:Xie;given-names:H + surname:Sadim;given-names:MS + surname:Sun;given-names:Z + 10.4049/jimmunol.175.6.3800 + 16148126 + REF + J Immunol + ref + 175 + 2005 + 25135 + RORgammat recruits steroid receptor coactivators to ensure thymocyte survival + + + 1025 + 33 + surname:Danielian;given-names:PS + surname:White;given-names:R + surname:Lees;given-names:JA + surname:Parker;given-names:MG + 1372244 + REF + EMBO J + ref + 11 + 1992 + 25213 + Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors + + + 1697 + 707 + surname:Kallen;given-names:JA + surname:Schlaeppi;given-names:JM + surname:Bitsch;given-names:F + surname:Geisse;given-names:S + surname:Geiser;given-names:M + surname:Delhon;given-names:I + surname:Fournier;given-names:B + 10.1016/S0969-2126(02)00912-7 + 12467577 + REF + Structure + ref + 10 + 2002 + 25337 + X-ray structure of the hRORalpha LBD at 1.63 A: structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of RORalpha + + + 5293 + 302 + surname:Wang;given-names:Y + surname:Tang;given-names:T + surname:Zhang;given-names:W + surname:Wu;given-names:Q + surname:Zhang;given-names:K + surname:Li;given-names:Y + surname:Xiang;given-names:JN + surname:Elliot;given-names:JD + surname:Leung;given-names:S + surname:Ren;given-names:F + surname:Lin;given-names:X + surname:Liu;given-names:Q + surname:Ma;given-names:Y + surname:Yang;given-names:L + surname:Zhou;given-names:L + surname:Xiang;given-names:Z + surname:Cheng;given-names:Z + surname:Lu;given-names:S + surname:Orband-Miller;given-names:LA + 10.1016/j.bmc.2015.07.068 + 26277758 + REF + Bioorg Med Chem + ref + 23 + 2015 + 25495 + Discovery of N-(4-aryl-5-aryloxy-thiazol-2-yl)-amides as potent RORγt inverse agonists + + + REF + ref + 25586 + Wang Y, Cai W, Cheng Y, Yang T, Liu Q, Zhang G, Meng Q, Han F, Huang Y, Zhou L2 Xiang Z, Zhao YG, Xu Y, Cheng Z, Lu S, Wu Q, Xiang JN, Elliott JD, Leung S, Ren F, Lin X. Discovery of Biaryl Amides as Potent, Orally Bioavailable, and CNS Penetrant RORγt Inhibitors. ACS Med Chem Lett. 2015;26:787–92. + + + REF + ref + 25890 + Fauber BP, René O, Deng Y, DeVoss J, Eidenschenk C, Everett C, Ganguli A1, Gobbi A, Hawkins J1, Johnson AR, La H, Lesch J, Lockey P1, Norman M1, Ouyang W, Summerhill S1, Wong H. Discovery of 1-{4-[3-fluoro-4-((3s,6r)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone (GNE-3500): a potent, selective, and orally bioavailable retinoic acid receptor-related orphan receptor C (RORc or RORγ) inverse agonist. J Med Chem. 2015;58:5308–22. + + + 431 + 41 + surname:Zhang;given-names:Y + surname:Xue;given-names:X + surname:Jin;given-names:X + surname:Song;given-names:Y + surname:Li;given-names:J + surname:Luo;given-names:X + surname:Song;given-names:M + surname:Yan;given-names:W + surname:Song;given-names:H + surname:Xu;given-names:Y + 10.1016/j.ejmech.2014.03.065 + 24704616 + REF + Eur J Med Chem + ref + 78 + 2014 + 26366 + Discovery of 2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide derivatives as new RORγ inhibitors using virtual screening, synthesis and biological evaluation + + + REF + ref + 26526 + Chao J, Enyedy IJ, Hutchings RH, Jones HJ, Powell NA, Vanvloten KD. Biaryl-containing compounds as inverse agonists of ror-gamma receptors. PCT Int Appl. 2014, WO2014008214 A1. + + + 919 + 27 + surname:Kurebayashi;given-names:S + surname:Nakajima;given-names:T + surname:Kim;given-names:SC + surname:Chang;given-names:CY + surname:McDonnell;given-names:DP + surname:Renaud;given-names:JP + surname:Jetten;given-names:AM + 10.1016/j.bbrc.2004.01.131 + 14985100 + REF + Biochem Biophys Res Commun + ref + 315 + 2004 + 26703 + Selective LXXLL peptides antagonize transcriptional activation by the retinoid-related orphan receptor RORγ + + + 271 + 81 + surname:Battye;given-names:TGG + surname:Kontogiannis;given-names:L + surname:Johnson;given-names:O + surname:Powell;given-names:HR + surname:Leslie;given-names:AGW + 10.1107/S0907444910048675 + 21460445 + REF + Acta Crystallogr Sect D: Biol Crystallogr + ref + 67 + 2011 + 26815 + iMOSFLM : a new graphical interface for diffraction-image processing with MOSFLM + + + REF + ref + 26896 + Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997;276:307–26 + + + 923 + 9 + surname:Jin;given-names:L + surname:Martynowski;given-names:D + surname:Zheng;given-names:S + surname:Wada;given-names:T + surname:Xie;given-names:W + surname:Li;given-names:Y + 10.1210/me.2009-0507 + 20203100 + REF + Mol Endocrinol + ref + 24 + 2010 + 27022 + Structural basis for hydroxycholesterols as natural ligands of orphan nuclear receptor RORgamma + + + 22 + 5 + surname:Vagin;given-names:A + surname:Teplyakov;given-names:A + 10.1107/S0907444909042589 + 20057045 + REF + Acta Crystallogr Sect D: Biol Crystallogr + ref + 66 + 2010 + 27118 + Molecular replacement with MOLREP + + + 247 + 55 + surname:Murshudov;given-names:GN + surname:Vagin;given-names:A + surname:Lebedev;given-names:A + surname:Wilson;given-names:KS + surname:Dodson;given-names:EJ + 10.1107/S090744499801405X + 10089417 + REF + Acta Crystallogr D Biol Crystallogr + ref + 55 + 1999 + 27152 + Efficient anisotropic refinement of macromolecular structures using FFT + + + 2126 + 32 + surname:Emsley;given-names:P + surname:Cowtan;given-names:K + 10.1107/S0907444904019158 + 15572765 + REF + Acta Crystallogr Sect D: Biol Crystallogr + ref + 60 + 2004 + 27224 + Coot: Model-building tools for molecular graphics + + + 71 + 87 + surname:Zhang;given-names:Y + surname:Luo;given-names:X + surname:Wu;given-names:D + surname:Xu;given-names:Y + 10.1038/aps.2014.120 + 25500868 + REF + Acta Pharmacol Sin + ref + 36 + 2015 + 27274 + ROR nuclear receptors: structures, related diseases, and drug discovery + + + 927 + 37 + surname:Shiau;given-names:AK + surname:Barstad;given-names:D + surname:Loria;given-names:PM + surname:Cheng;given-names:L + surname:Kushner;given-names:PJ + surname:Agard;given-names:DA + surname:Greene;given-names:GL + 10.1016/S0092-8674(00)81717-1 + 9875847 + REF + Cell + ref + 95 + 1998 + 27346 + The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen + + + 614 + 6 + surname:Nahoum;given-names:V + surname:Lipski;given-names:A + surname:Quillard;given-names:F + surname:Guichou;given-names:JF + surname:Boublik;given-names:Y + surname:Pérez;given-names:E + surname:Germain;given-names:P + surname:de Lera;given-names:AR + surname:Bourguet;given-names:W + 10.1107/S1744309108015492 + REF + Acta Crystallogr Sect F: Struct Biol Cryst Commun + ref + 64 + 2008 + 27464 + Nuclear receptor ligand-binding domains: reduction of helix H12 dynamics to favor crystallization + + + 11025 + 33 + surname:Singarapu;given-names:KK + surname:Zhu;given-names:J + surname:Tonelli;given-names:M + surname:Rao;given-names:H + surname:Assadi-Porter;given-names:FM + surname:Westler;given-names:WM + surname:DeLuca;given-names:HF + surname:Markley;given-names:JL + 10.1021/bi201637p + 22112050 + REF + Biochemistry + ref + 50 + 2011 + 27562 + Ligand-specific structural changes in the vitamin D receptor in solution + + + 77 + 84 + surname:Koth;given-names:CM + surname:Orlicky;given-names:SM + surname:Larson;given-names:SM + surname:Edwards;given-names:AM + 10.1016/S0076-6879(03)68005-5 + REF + Methods Enzym + ref + 368 + 2003 + 27635 + Use of limited proteolysis to identify protein domains suitable for structural analysis + + + 6604 + 9 + surname:Fauber;given-names:BP + surname:de Leon;given-names:BG + surname:Norman;given-names:M + surname:Ouyang;given-names:W + surname:René;given-names:O + surname:Wong;given-names:H + surname:Burton;given-names:B + surname:Eidenschenk;given-names:C + surname:Everett;given-names:C + surname:Gobbi;given-names:A + surname:Hymowitz;given-names:SG + surname:Johnson;given-names:AR + surname:Liimatta;given-names:M + surname:Lockey;given-names:P + 10.1016/j.bmcl.2013.10.054 + 24239186 + REF + Bioorg Med Chem Lett + ref + 23 + 2013 + 27723 + Structure-based design of substituted hexafluoroisopropanol-arylsulfonamides as modulators of RORc + + + 31409 + 17 + surname:Fujita-Sato;given-names:S + 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surname:Lin;given-names:EY + surname:Marcotte;given-names:D + 10.1016/j.bmcl.2015.05.028 + 26048789 + REF + Bioorg Med Chem Lett + ref + 25 + 2015 + 28160 + Discovery of novel pyrazole-containing benzamides as potent RORγ inverse agonists + + + 2991 + 7 + surname:Chao;given-names:J + surname:Enyedy;given-names:I + surname:Silvian;given-names:L + surname:Hong;given-names:VS + surname:Little;given-names:K + surname:Banerjee;given-names:D + surname:Peng;given-names:L + surname:Taveras;given-names:A + surname:Viney;given-names:JL + surname:Fontenot;given-names:J + surname:Van Vloten;given-names:K + surname:Marcotte;given-names:D + surname:Guertin;given-names:K + surname:Hutchings;given-names:R + surname:Powell;given-names:N + surname:Jones;given-names:H + surname:Bohnert;given-names:T + surname:Peng;given-names:C-C + 10.1016/j.bmcl.2015.05.026 + 26048806 + REF + Bioorg Med Chem Lett + ref + 25 + 2015 + 28246 + Discovery of biaryl carboxylamides as potent RORγ inverse agonists + + + diff --git a/BioC_XML/4896748_v0.xml b/BioC_XML/4896748_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..eabdb4293eebe6fbd01742e17143f8612a176947 --- /dev/null +++ b/BioC_XML/4896748_v0.xml @@ -0,0 +1,26649 @@ + + + + PMC + 20201215 + pmc.key + + 4896748 + CC BY + no + 0 + 0 + + 10.7554/eLife.14874 + 4896748 + 27159452 + 14874 + e14874 + Taura syndrome virus ribosome internal ribosome entry site IRES translocation elongation factor eEF2 S. cerevisiae + This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. + surname:Abeyrathne;given-names:Priyanka D + surname:Koh;given-names:Cha San + surname:Grant;given-names:Timothy + surname:Grigorieff;given-names:Nikolaus + surname:Korostelev;given-names:Andrei A + surname:Subramaniam;given-names:Sriram + surname:Grigorieff;given-names:Nikolaus + surname:Grigorieff;given-names:Nikolaus + surname:Korostelev;given-names:Andrei A + surname:Korostelev;given-names:Andrei A + TITLE + Author Keywords Research Organism + front + 5 + 2016 + 0 + Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome + + 0.99953175 + experimental_method + cleaner0 + 2023-07-17T08:27:30Z + MESH: + + cryo-EM + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T10:13:02Z + + inchworm + + + 0.99878675 + taxonomy_domain + cleaner0 + 2023-07-14T09:20:19Z + DUMMY: + + viral + + + 0.5541505 + site + cleaner0 + 2023-07-14T09:20:57Z + SO: + + IRES + + + 0.9956189 + complex_assembly + cleaner0 + 2023-07-14T09:32:55Z + GO: + + ribosome + + + + ABSTRACT + abstract + 91 + Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation. + + 0.97262037 + site + cleaner0 + 2023-07-19T09:54:24Z + SO: + + Internal ribosome entry sites + + + 0.4109444 + site + cleaner0 + 2023-07-14T09:20:07Z + SO: + + IRESs + + + 0.85950506 + taxonomy_domain + cleaner0 + 2023-07-14T09:20:21Z + DUMMY: + + viral + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:30Z + + mRNAs + + + 0.9995241 + experimental_method + cleaner0 + 2023-07-17T08:27:44Z + MESH: + + electron cryo-microscopy + + + 0.9442826 + complex_assembly + cleaner0 + 2023-07-14T09:32:55Z + GO: + + ribosome + + + 0.9963812 + evidence + cleaner0 + 2023-07-14T16:19:22Z + DUMMY: + + structures + + + 0.7445826 + species + cleaner0 + 2023-07-14T09:24:11Z + MESH: + + Taura syndrome virus + + + 0.84979075 + site + cleaner0 + 2023-07-14T09:20:59Z + SO: + + IRES + + + 0.9981079 + protein_type + cleaner0 + 2023-07-17T08:38:45Z + MESH: + + translocase + + + 0.9993623 + complex_assembly + cleaner0 + 2023-07-14T09:31:04Z + GO: + + eEF2•GTP + + + 0.99943036 + protein_state + cleaner0 + 2023-07-17T08:30:36Z + DUMMY: + + bound with + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:54Z + + sordarin + + + 0.99468297 + evidence + cleaner0 + 2023-07-14T16:19:22Z + DUMMY: + + structures + + + 0.36434925 + site + cleaner0 + 2023-07-14T09:20:59Z + SO: + + IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:10Z + + initiation + + + 0.9997938 + protein + cleaner0 + 2023-07-14T09:30:41Z + PR: + + eEF2 + + + 0.9629238 + site + cleaner0 + 2023-07-14T09:20:59Z + SO: + + IRES + + + 0.9996594 + protein_state + cleaner0 + 2023-07-17T08:34:20Z + DUMMY: + + extended + + + 0.99965954 + protein_state + cleaner0 + 2023-07-19T12:26:28Z + DUMMY: + + bent + + + 0.99962234 + protein_state + cleaner0 + 2023-07-17T08:34:20Z + DUMMY: + + extended + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T10:13:02Z + + inchworm + + + 0.791828 + complex_assembly + cleaner0 + 2023-07-17T08:54:51Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + 0.9996791 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + complex_assembly + GO: + cleaner0 + 2023-07-18T13:49:56Z + + 60S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:11Z + + subunit + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-18T13:46:36Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:12Z + + subunit + + + 0.99909836 + site + cleaner0 + 2023-07-14T09:28:51Z + SO: + + A site + + + 0.9991868 + ptm + cleaner0 + 2023-07-19T09:17:14Z + MESH: + + diphthamide + + + structure_element + SO: + cleaner0 + 2023-07-19T10:38:17Z + + IV + + + 0.9991062 + structure_element + cleaner0 + 2023-07-19T13:22:30Z + SO: + + tRNA-mRNA-like pseudoknot I + + + 0.9989575 + structure_element + cleaner0 + 2023-07-14T09:27:39Z + SO: + + PKI + + + 0.63113517 + site + cleaner0 + 2023-07-14T09:20:59Z + SO: + + IRES + + + site + SO: + cleaner0 + 2023-07-18T14:50:01Z + + decoding center + + + 0.9523076 + complex_assembly + cleaner0 + 2023-07-17T09:02:37Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + 0.560403 + site + cleaner0 + 2023-07-14T09:20:59Z + SO: + + IRES + + + 0.99070907 + structure_element + cleaner0 + 2023-07-14T09:27:39Z + SO: + + PKI + + + 0.99939346 + site + cleaner0 + 2023-07-19T09:54:34Z + SO: + + A and P sites + + + 0.9984297 + evidence + cleaner0 + 2023-07-14T16:19:22Z + DUMMY: + + structures + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + + ABSTRACT + abstract + 1139 + DOI: http://dx.doi.org/10.7554/eLife.14874.001 + + + INTRO + title_1 + 1186 + Introduction + + + INTRO + paragraph + 1199 + Virus propagation relies on the host translational apparatus. To efficiently compete with host mRNAs and engage in translation under stress, some viral mRNAs undergo cap-independent translation. To this end, internal ribosome entry site (IRES) RNAs are employed (reviewed in. An IRES is located at the 5’ untranslated region of the viral mRNA, preceding an open reading frame (ORF). To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit. The canonical scenario of cap-dependent and IRES-dependent initiation involves positioning of the AUG start codon and the initiator tRNAMet in the ribosomal peptidyl-tRNA (P) site, facilitated by interaction with initiation factors. Subsequent binding of an elongator aminoacyl-tRNA to the ribosomal A site transitions the initiation complex into the elongation cycle of translation. Upon peptide bond formation, the two tRNAs and their respective mRNA codons translocate from the A and P to P and E (exit) sites, freeing the A site for the next elongator tRNA. + + 0.99361897 + taxonomy_domain + cleaner0 + 2023-07-17T08:49:06Z + DUMMY: + + Virus + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:29Z + + mRNAs + + + 0.9985154 + taxonomy_domain + cleaner0 + 2023-07-14T09:20:21Z + DUMMY: + + viral + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:30Z + + mRNAs + + + 0.98197454 + site + cleaner0 + 2023-07-14T09:23:04Z + SO: + + internal ribosome entry site + + + 0.36906794 + site + cleaner0 + 2023-07-14T09:21:00Z + SO: + + IRES + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:19Z + + RNAs + + + 0.7004662 + site + cleaner0 + 2023-07-14T09:21:00Z + SO: + + IRES + + + 0.9973719 + structure_element + cleaner0 + 2023-07-19T14:13:02Z + SO: + + 5’ untranslated region + + + 0.99826705 + taxonomy_domain + cleaner0 + 2023-07-14T09:20:22Z + DUMMY: + + viral + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:01Z + + mRNA + + + 0.9646409 + structure_element + cleaner0 + 2023-07-19T09:59:13Z + SO: + + open reading frame + + + 0.98755205 + structure_element + cleaner0 + 2023-07-19T09:44:51Z + SO: + + ORF + + + 0.9995622 + protein_state + cleaner0 + 2023-07-19T12:27:02Z + DUMMY: + + structured + + + 0.8683453 + site + cleaner0 + 2023-07-14T09:21:00Z + SO: + + IRES + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:16Z + + RNA + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:12Z + + subunit + + + complex_assembly + GO: + cleaner0 + 2023-07-18T13:51:20Z + + 80S ribosome + + + 0.9970957 + protein_state + cleaner0 + 2023-07-19T12:28:13Z + DUMMY: + + small + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:12Z + + subunit + + + 0.61591065 + site + cleaner0 + 2023-07-14T09:21:00Z + SO: + + IRES + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:47Z + + tRNAMet + + + 0.99957794 + site + cleaner0 + 2023-07-14T09:31:22Z + SO: + + peptidyl-tRNA (P) site + + + protein_type + MESH: + cleaner0 + 2023-07-19T12:27:45Z + + initiation factors + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:40Z + + aminoacyl-tRNA + + + 0.9993316 + site + cleaner0 + 2023-07-14T09:28:48Z + SO: + + A site + + + complex_assembly + GO: + cleaner0 + 2023-07-19T12:27:22Z + + initiation complex + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:04Z + + tRNAs + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:03Z + + mRNA + + + site + SO: + cleaner0 + 2023-07-14T09:29:28Z + + A and P + + + 0.9991889 + site + cleaner0 + 2023-07-19T09:54:59Z + SO: + + P and E (exit) sites + + + 0.9995337 + site + cleaner0 + 2023-07-14T09:28:51Z + SO: + + A site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:19Z + + tRNA + + + + INTRO + paragraph + 2334 + An unusual strategy of initiation is used by intergenic-region (IGR) IRESs found in Dicistroviridae arthropod-infecting viruses. These include shrimp-infecting Taura syndrome virus (TSV), and insect viruses Plautia stali intestine virus (PSIV) and Cricket paralysis virus (CrPV). The IGR IRES mRNAs do not contain an AUG start codon. The IGR-IRES-driven initiation does not involve initiator tRNAMet and initiation factors. As such, this group of IRESs represents the most streamlined mechanism of eukaryotic translation initiation. A recent demonstration of bacterial translation initiation by an IGR IRES indicates that the IRESs take advantage of conserved structural and dynamic properties of the ribosome. Early electron cryo-microscopy (cryo-EM) studies have found that the CrPV IRES packs in the ribosome intersubunit space. Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. The ~200-nt IRES RNAs span from the A site beyond the E site. A conserved tRNA-mRNA–like structural element of pseudoknot I (PKI) interacts with the decoding center in the A site of the 40S subunit. The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). The downstream initiation codon—coding for alanine—is placed in the mRNA tunnel, preceding the decoding center. PKI of IGR IRESs therefore mimics an A-site elongator tRNA interacting with an mRNA sense codon, but not a P-site initiator tRNAMet and the AUG start codon. + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:10Z + + initiation + + + structure_element + SO: + cleaner0 + 2023-07-14T09:25:52Z + + intergenic-region + + + 0.78682446 + structure_element + cleaner0 + 2023-07-14T09:26:09Z + SO: + + IGR + + + 0.6738639 + site + cleaner0 + 2023-07-14T09:20:11Z + SO: + + IRESs + + + species + MESH: + cleaner0 + 2023-07-14T09:23:36Z + + Dicistroviridae arthropod + + + taxonomy_domain + DUMMY: + cleaner0 + 2023-07-14T09:24:01Z + + viruses + + + 0.6145182 + taxonomy_domain + cleaner0 + 2023-07-17T08:49:12Z + DUMMY: + + shrimp + + + 0.87469715 + species + cleaner0 + 2023-07-14T09:24:09Z + MESH: + + Taura syndrome virus + + + 0.9599049 + species + cleaner0 + 2023-07-14T09:24:16Z + MESH: + + TSV + + + 0.94927967 + taxonomy_domain + cleaner0 + 2023-07-17T08:49:16Z + DUMMY: + + insect + + + 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2023-07-19T13:15:21Z + + tRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:03Z + + mRNA + + + 0.99148136 + site + cleaner0 + 2023-07-14T09:32:34Z + SO: + + P-site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:48Z + + tRNAMet + + + + INTRO + paragraph + 4024 + How this non-canonical initiation complex transitions to the elongation step is not fully understood. For a cognate aminoacyl-tRNA to bind the first viral mRNA codon, PKI has to be translocated from the A site, so that the first codon can be presented in the A site. A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state. In this structure, PKI is positioned in the P site and the first mRNA codon is located in the A site. How the large IRES RNA translocates within the ribosome, allowing PKI translocation from the A to P site is not known. + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:10Z + + initiation + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:46Z + + aminoacyl-tRNA + + + 0.99630934 + taxonomy_domain + cleaner0 + 2023-07-14T09:20:22Z + DUMMY: + + viral + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:03Z + + mRNA + + + 0.537322 + structure_element + cleaner0 + 2023-07-14T09:27:39Z + SO: + + PKI + + + 0.9994906 + site + cleaner0 + 2023-07-14T09:28:51Z + SO: + + A site + + + 0.99944466 + site + cleaner0 + 2023-07-14T09:28:51Z + SO: + + A site + + + 0.9993964 + experimental_method + cleaner0 + 2023-07-17T08:27:34Z + MESH: + + cryo-EM + + + 0.9979473 + evidence + cleaner0 + 2023-07-14T16:19:11Z + DUMMY: + + structure + + + 0.99900335 + complex_assembly + cleaner0 + 2023-07-14T09:32:55Z + GO: + + ribosome + + + 0.9994935 + protein_state + cleaner0 + 2023-07-17T08:30:36Z + DUMMY: + + bound with + + + 0.98907185 + species + cleaner0 + 2023-07-14T09:25:05Z + MESH: + + CrPV + + + 0.9706527 + site + cleaner0 + 2023-07-14T09:21:00Z + SO: + + IRES + + + 0.99888396 + protein_type + cleaner0 + 2023-07-19T09:17:26Z + MESH: + + release factor + + + 0.99953234 + protein + cleaner0 + 2023-07-19T09:25:03Z + PR: + + eRF1 + + + 0.99944097 + site + cleaner0 + 2023-07-14T09:28:51Z + SO: + + A site + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:27:19Z + + post-translocation + + + 0.9993575 + evidence + cleaner0 + 2023-07-14T16:19:11Z + DUMMY: + + structure + + + 0.94575197 + structure_element + cleaner0 + 2023-07-14T09:27:39Z + SO: + + PKI + + + 0.9994904 + site + cleaner0 + 2023-07-19T09:56:12Z + SO: + + P site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:03Z + + mRNA + + + 0.9994333 + site + cleaner0 + 2023-07-14T09:28:51Z + SO: + + A site + + + 0.8747202 + protein_state + cleaner0 + 2023-07-19T12:28:37Z + DUMMY: + + large + + + 0.7590424 + site + cleaner0 + 2023-07-14T09:21:00Z + SO: + + IRES + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:17Z + + RNA + + + 0.9992211 + complex_assembly + cleaner0 + 2023-07-14T09:32:55Z + GO: + + ribosome + + + 0.9958423 + structure_element + cleaner0 + 2023-07-14T09:27:39Z + SO: + + PKI + + + site + SO: + cleaner0 + 2023-07-17T08:57:28Z + + A to P site + + + + INTRO + paragraph + 4668 + The structural similarity of PKI and the tRNA anticodon stem loop (ASL) bound to a codon suggests that their mechanisms of translocation are similar to some extent. Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). Translocation of 2tRNA•mRNA involves two major large-scale ribosome rearrangements (Figure 1—figure supplement 1) (reviewed in). First, studies of bacterial ribosomes showed that a ~10° rotation of the small subunit relative to the large subunit, known as intersubunit rotation, or ratcheting, is required for translocation. Intersubunit rotation occurs spontaneously upon peptidyl transfer, and is coupled with formation of hybrid tRNA states. In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). Concurrently, the deacyl-tRNA interacts with the P site of the small subunit and the E site of the large subunit (P/E hybrid state). The ribosome can undergo spontaneous, thermally-driven forward-reverse rotation that shifts the two tRNAs between the hybrid and 'classical' states while the anticodon stem loops remain non-translocated. Binding of EF-G next to the A site and reverse rotation of the small subunit results in translocation of both ASLs on the small subunit. EF-G is thought to 'unlock' the pre-translocation ribosome, allowing movement of the 2tRNA•mRNA complex, however the structural details of this unlocking are not known. + + 0.6377333 + structure_element + cleaner0 + 2023-07-14T09:27:39Z + SO: + + PKI + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + 0.9995071 + structure_element + cleaner0 + 2023-07-14T09:34:46Z + SO: + + anticodon stem loop + + + 0.99966896 + structure_element + cleaner0 + 2023-07-14T09:34:54Z + SO: + + ASL + + + 0.9995195 + protein_state + cleaner0 + 2023-07-19T12:28:43Z + DUMMY: + + bound to + + + 0.63755476 + site + cleaner0 + 2023-07-14T09:21:01Z + SO: + + IRES + + + 0.9917429 + complex_assembly + cleaner0 + 2023-07-14T09:36:30Z + GO: + + tRNA-mRNA + + + 0.999411 + taxonomy_domain + cleaner0 + 2023-07-14T09:35:54Z + DUMMY: + + eukaryotic + + + 0.9992318 + protein + cleaner0 + 2023-07-14T09:35:43Z + PR: + + elongation factor 2 + + + 0.99962425 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: 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2023-07-14T09:36:39Z + GO: + + 2tRNA•mRNA + + + + INTRO + paragraph + 6437 + The second large-scale rearrangement involves rotation, or swiveling, of the head of the small subunit relative to the body. The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. The structural role of head swivel is not fully understood. The head swivel was proposed to facilitate transition of the tRNA from the P to E site by widening a constriction between these sites on the 30S subunit. This widening allows the ASL to sample positions between the P and E sites. Whether and how the head swivel mediates tRNA transition from the A to P site remains unknown. + + 0.9985524 + structure_element + cleaner0 + 2023-07-17T08:56:45Z + SO: + + head + + + 0.7539265 + structure_element + cleaner0 + 2023-07-14T09:39:03Z + SO: + + small subunit + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:33Z + + body + + + 0.9990159 + structure_element + cleaner0 + 2023-07-17T08:56:47Z + SO: + + head + + + 0.99955404 + protein_state + cleaner0 + 2023-07-14T09:55:35Z + DUMMY: + + absence of + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + 0.9994737 + protein_state + cleaner0 + 2023-07-14T09:55:43Z + DUMMY: + + presence of + + + 0.79971176 + site + cleaner0 + 2023-07-17T08:58:06Z + SO: + + P + + + 0.75976425 + site + cleaner0 + 2023-07-17T08:58:14Z + SO: + + E + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + 0.99949706 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + 0.9994564 + protein + cleaner0 + 2023-07-14T09:36:12Z + PR: + + EF-G + + + 0.98532176 + experimental_method + cleaner0 + 2023-07-14T09:40:59Z + MESH: + + Förster resonance energy transfer + + + 0.7090511 + experimental_method + cleaner0 + 2023-07-14T09:41:06Z + MESH: + + FRET + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + 0.9996474 + protein_state + cleaner0 + 2023-07-17T08:58:24Z + DUMMY: + + rotated + + + 0.6217345 + structure_element + cleaner0 + 2023-07-14T09:39:03Z + SO: + + small subunit + + + 0.99879694 + protein + cleaner0 + 2023-07-14T09:36:12Z + PR: + + EF-G + + + 0.9994505 + complex_assembly + cleaner0 + 2023-07-14T09:36:39Z + GO: + + 2tRNA•mRNA + + + 0.9994387 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + Structures + + + 0.9997114 + complex_assembly + cleaner0 + 2023-07-14T09:39:49Z + GO: + + 70S•EF-G + + + 0.9995264 + protein_state + cleaner0 + 2023-07-17T08:30:36Z + DUMMY: + + bound with + + + 0.74690247 + protein_state + cleaner0 + 2023-07-17T08:58:29Z + DUMMY: + + nearly translocated + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:06Z + + tRNAs + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + 0.9994354 + protein_state + cleaner0 + 2023-07-17T08:58:42Z + DUMMY: + + mid-rotated + + + 0.98889226 + structure_element + cleaner0 + 2023-07-18T13:50:12Z + SO: + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + 0.9995543 + protein_state + cleaner0 + 2023-07-17T08:58:33Z + DUMMY: + + fully rotated + + + 0.9844136 + protein_state + cleaner0 + 2023-07-14T15:24:43Z + DUMMY: + + pre-translocation + + + 0.9993871 + protein_state + cleaner0 + 2023-07-17T08:58:36Z + DUMMY: + + non-rotated + + + 0.92643744 + protein_state + cleaner0 + 2023-07-14T15:27:19Z + DUMMY: + + post-translocation + + + 0.9997191 + complex_assembly + cleaner0 + 2023-07-14T09:39:55Z + GO: + + 70S•2tRNA•EF-G + + + 0.99933076 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + structures + + + 0.95221514 + structure_element + cleaner0 + 2023-07-17T08:56:47Z + SO: + + head + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + 0.9970267 + site + cleaner0 + 2023-07-17T08:57:36Z + SO: + + P to E site + + + site + SO: + cleaner0 + 2023-07-19T10:25:25Z + + constriction + + + complex_assembly + GO: + cleaner0 + 2023-07-18T13:52:44Z + + 30S + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-18T13:46:36Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:12Z + + subunit + + + 0.9996208 + structure_element + cleaner0 + 2023-07-14T09:34:55Z + SO: + + ASL + + + 0.9991023 + site + cleaner0 + 2023-07-17T08:57:43Z + SO: + + P and E sites + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + 0.9073696 + site + cleaner0 + 2023-07-17T08:57:27Z + SO: + + A to P site + + + + elife-14874-fig1-figsupp1.jpg + fig1s1 + FIG + fig_title_caption + 7577 + Comparison of 70S•2tRNA•mRNA and 80S•IRES translocation complexes. + + 0.9997015 + complex_assembly + cleaner0 + 2023-07-14T09:40:38Z + GO: + + 70S•2tRNA•mRNA + + + 0.9997058 + complex_assembly + cleaner0 + 2023-07-14T09:40:43Z + GO: + + 80S•IRES + + + + elife-14874-fig1-figsupp1.jpg + fig1s1 + FIG + fig_caption + 7650 + (a) Structures of bacterial 70S•2tRNA•mRNA translocation complexes, ordered according to the position of the translocating A->P tRNA (orange). The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body), elongation factor G (EF-G) is shown in green. Nucleotides C1054, G966 and G693 of 16S rRNA are shown in black to denote the A, P and E sites, respectively. The extents of the 30S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows. References and PDB codes of the structures are shown. (b) Structures of the 80S•IRES complexes in the absence and presence of eEF2 (this work). The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. Unresolved regions of the IRES in densities for Structures III and V are shown in gray. The extents of the 40S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows. + + 0.6640606 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + Structures + + + 0.9995146 + taxonomy_domain + cleaner0 + 2023-07-14T09:36:04Z + DUMMY: + + bacterial + + + 0.99969876 + complex_assembly + cleaner0 + 2023-07-14T09:40:39Z + GO: + + 70S•2tRNA•mRNA + + + site + SO: + cleaner0 + 2023-07-17T08:59:28Z + + A->P + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:12Z + + subunit + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-20T15:10:40Z + + small subunit + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:33Z + + body + + + 0.9799428 + protein + cleaner0 + 2023-07-19T09:22:15Z + PR: + + elongation factor G + + + 0.9816089 + protein + cleaner0 + 2023-07-14T09:36:12Z + PR: + + EF-G + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:32Z + + C1054 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:29:10Z + + G966 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T08:07:58Z + + G693 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:26:06Z + + 16S rRNA + + + 0.99627763 + site + cleaner0 + 2023-07-17T08:59:05Z + SO: + + A, P and E sites + + + complex_assembly + GO: + cleaner0 + 2023-07-18T13:52:45Z + + 30S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:12Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:27:19Z + + post-translocation + + + 0.9771657 + evidence + cleaner0 + 2023-07-14T16:19:11Z + DUMMY: + + structure + + + 0.9966439 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + structures + + + 0.992148 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + Structures + + + 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+ DUMMY: + cleaner0 + 2023-07-19T13:26:32Z + + Structures III and V + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:12Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:27:19Z + + post-translocation + + + 0.9831435 + evidence + cleaner0 + 2023-07-14T16:19:11Z + DUMMY: + + structure + + + + elife-14874-fig1-figsupp1.jpg + fig1s1 + FIG + fig_caption + 8924 + DOI: +http://dx.doi.org/10.7554/eLife.14874.004 + + + elife-14874-fig1-figsupp2.jpg + fig1s2 + FIG + fig_title_caption + 8971 + Schematic of cryo-EM refinement and classification procedures. + + 0.9995087 + experimental_method + cleaner0 + 2023-07-17T08:27:35Z + MESH: + + cryo-EM + + + + elife-14874-fig1-figsupp2.jpg + fig1s2 + FIG + fig_caption + 9034 + All particles were initially aligned to a single model. 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). + + 0.9984871 + experimental_method + cleaner0 + 2023-07-19T14:09:56Z + MESH: + + particles + + + 0.9994972 + experimental_method + cleaner0 + 2023-07-17T08:28:01Z + MESH: + + 3D classification + + + evidence + DUMMY: + cleaner0 + 2023-07-17T08:28:32Z + + 3D mask + + + 0.9884299 + complex_assembly + cleaner0 + 2023-07-17T08:59:59Z + GO: + + 40S + + + 0.95528805 + structure_element + cleaner0 + 2023-07-17T08:56:47Z + SO: + + head + + + 0.96366817 + species + cleaner0 + 2023-07-14T09:24:18Z + MESH: + + TSV + + + 0.9968683 + site + cleaner0 + 2023-07-14T09:21:01Z + SO: + + IRES + + + 0.99964654 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:52Z + + stack + + + 0.9960376 + experimental_method + cleaner0 + 2023-07-19T14:09:58Z + MESH: + + particles + + + 0.9980515 + site + cleaner0 + 2023-07-14T09:21:02Z + SO: + + IRES + + + 0.99976474 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + 0.999455 + experimental_method + cleaner0 + 2023-07-17T08:28:03Z + MESH: + + 3D classification + + + evidence + DUMMY: + cleaner0 + 2023-07-17T08:28:50Z + + 2D mask + + + 0.9998017 + structure_element + cleaner0 + 2023-07-14T09:27:39Z + SO: + + PKI + + + structure_element + SO: + cleaner0 + 2023-07-19T10:39:43Z + + IV + + + 0.9997911 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:39:27Z + + Structures I through V + + + 0.9956601 + experimental_method + cleaner0 + 2023-07-17T08:29:03Z + MESH: + + Sub-classification + + + 0.99904877 + evidence + cleaner0 + 2023-07-19T14:10:06Z + DUMMY: + + density + + + 0.999783 + structure_element + cleaner0 + 2023-07-14T09:27:39Z + SO: + + PKI + + + 0.9988875 + experimental_method + cleaner0 + 2023-07-19T14:09:58Z + MESH: + + particles + + + 0.8339024 + experimental_method + cleaner0 + 2023-07-17T08:29:18Z + MESH: + + sub-classified + + + 0.99856925 + evidence + cleaner0 + 2023-07-19T14:10:10Z + DUMMY: + + reconstruction + + + + elife-14874-fig1-figsupp2.jpg + fig1s2 + FIG + fig_caption + 9640 + DOI: +http://dx.doi.org/10.7554/eLife.14874.005 + + + elife-14874-fig1-figsupp3.jpg + fig1s3 + FIG + fig_title_caption + 9687 + Cryo-EM density of Structures I-V. + + 0.9995305 + experimental_method + cleaner0 + 2023-07-17T08:27:35Z + MESH: + + Cryo-EM + + + 0.9992926 + evidence + cleaner0 + 2023-07-19T14:10:16Z + DUMMY: + + density + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:40:22Z + + Structures I-V + + + + elife-14874-fig1-figsupp3.jpg + fig1s3 + FIG + fig_caption + 9722 + In panels (a-e), the maps are segmented and colored as in Figure 1. The maps in all panels were B-softened by applying a B-factor of 30 Å2. (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). + + 0.99940383 + evidence + cleaner0 + 2023-07-19T14:10:20Z + DUMMY: + + maps + + + 0.9994673 + evidence + cleaner0 + 2023-07-19T14:10:23Z + DUMMY: + + maps + + + 0.9990313 + experimental_method + cleaner0 + 2023-07-17T08:27:35Z + MESH: + + Cryo-EM + + + 0.95958966 + evidence + cleaner0 + 2023-07-19T14:10:25Z + DUMMY: + + map + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:40:35Z + + Structures I, II, III, IV and V + + + 0.999253 + experimental_method + cleaner0 + 2023-07-17T08:27:35Z + MESH: + + cryo-EM + + + 0.9922787 + evidence + cleaner0 + 2023-07-19T14:10:29Z + DUMMY: + + reconstructions + + + 0.99884653 + experimental_method + cleaner0 + 2023-07-17T08:30:07Z + MESH: + + Blocres + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:40:52Z + + Structures I, II, III, IV and V + + + 0.998946 + experimental_method + cleaner0 + 2023-07-17T08:27:35Z + MESH: + + Cryo-EM + + + 0.9651188 + evidence + cleaner0 + 2023-07-19T14:10:33Z + DUMMY: + + density + + + 0.97608155 + species + cleaner0 + 2023-07-14T09:24:18Z + MESH: + + TSV + + + 0.9965508 + site + cleaner0 + 2023-07-14T09:21:02Z + SO: + + IRES + + + 0.999648 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:41:05Z + + Structures I, II, III, IV and V + + + 0.9994685 + evidence + cleaner0 + 2023-07-14T09:43:28Z + DUMMY: + + Fourier shell correlation + + + 0.9995912 + evidence + cleaner0 + 2023-07-14T09:43:33Z + DUMMY: + + FSC + + + 0.7466353 + evidence + cleaner0 + 2023-07-19T14:10:39Z + DUMMY: + + curves + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:41:18Z + + Structures I-V + + + 0.99947685 + evidence + cleaner0 + 2023-07-14T09:43:35Z + DUMMY: + + FSC + + + 0.99933165 + experimental_method + cleaner0 + 2023-07-17T08:30:50Z + MESH: + + FREALIGN + + + 0.99954766 + evidence + cleaner0 + 2023-07-14T09:43:35Z + DUMMY: + + FSC + + + + elife-14874-fig1-figsupp3.jpg + fig1s3 + FIG + fig_caption + 10470 + DOI: +http://dx.doi.org/10.7554/eLife.14874.006 + + + elife-14874-fig1.jpg + fig1 + FIG + fig_title_caption + 10517 + Cryo-EM structures of the 80S•TSV IRES bound with eEF2•GDP•sordarin. + + 0.99955845 + experimental_method + cleaner0 + 2023-07-17T08:27:35Z + MESH: + + Cryo-EM + + + 0.9989022 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:45:12Z + + 80S•TSV IRES + + + 0.9995089 + protein_state + cleaner0 + 2023-07-17T08:30:34Z + DUMMY: + + bound with + + + 0.9996313 + complex_assembly + cleaner0 + 2023-07-17T09:01:03Z + GO: + + eEF2•GDP•sordarin + + + + elife-14874-fig1.jpg + fig1 + FIG + fig_caption + 10592 + (a) Structures I through V. In all panels, the large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. Unresolved regions of the IRES in densities for Structures III and V are shown in gray. (b) Schematic representation of the structures shown in panel a, denoting the conformations of the small subunit relative to the large subunit. A, P and E sites are shown as rectangles. All measurements are relative to the non-rotated 80S•2tRNA•mRNA structure. The colors are as in panel a. + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:41:39Z + + Structures I through V + + + structure_element + SO: + cleaner0 + 2023-07-17T09:00:41Z + + large ribosomal subunit + + + structure_element + SO: + cleaner0 + 2023-07-14T09:39:03Z + + small subunit + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:33Z + + body + + + 0.827816 + species + cleaner0 + 2023-07-14T09:24:18Z + MESH: + + TSV + + + 0.9558331 + site + cleaner0 + 2023-07-14T09:21:02Z + SO: + + IRES + + + 0.9962767 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:19Z + + C1274 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:58Z + + U1191 + + + 0.9931932 + complex_assembly + cleaner0 + 2023-07-17T09:01:24Z + GO: + + 40S + + + 0.6128699 + structure_element + cleaner0 + 2023-07-17T08:56:47Z + SO: + + head + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T08:07:42Z + + G904 + + + 0.9848712 + site + cleaner0 + 2023-07-19T09:56:48Z + SO: + + platform + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:32Z + + C1054 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:29:10Z + + G966 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T08:07:57Z + + G693 + + + 0.999288 + species + cleaner0 + 2023-07-14T09:31:43Z + MESH: + + E. coli + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:26:08Z + + 16S rRNA + + + 0.99937105 + site + cleaner0 + 2023-07-17T08:59:41Z + SO: + + A, P and E sites + + + 0.981008 + site + cleaner0 + 2023-07-14T09:21:02Z + SO: + + IRES + + + 0.9910972 + evidence + cleaner0 + 2023-07-19T14:11:05Z + DUMMY: + + densities + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:42:00Z + + Structures III and V + + + 0.9976392 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-07-14T09:39:03Z + + small subunit + + + structure_element + SO: + cleaner0 + 2023-07-14T09:49:05Z + + large subunit + + + 0.99750715 + site + cleaner0 + 2023-07-17T08:59:41Z + SO: + + A, P and E sites + + + 0.9950251 + protein_state + cleaner0 + 2023-07-19T12:29:27Z + DUMMY: + + non-rotated + + + 0.999683 + complex_assembly + cleaner0 + 2023-07-14T09:44:23Z + GO: + + 80S•2tRNA•mRNA + + + 0.9991591 + evidence + cleaner0 + 2023-07-14T16:19:11Z + DUMMY: + + structure + + + + elife-14874-fig1.jpg + fig1 + FIG + fig_caption + 11341 + DOI: +http://dx.doi.org/10.7554/eLife.14874.002 + + + INTRO + paragraph + 11388 + We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? We used cryo-EM to visualize 80S•TSV IRES complexes formed in the presence of eEF2•GTP and the translation inhibitor sordarin, which stabilizes eEF2 on the ribosome. Although the mechanism of sordarin action is not fully understood, the inhibitor does not affect the conformation of eEF2•GDPNP on the ribosome, rendering it an excellent tool in translocation studies. Maximum-likelihood classification using FREALIGN identified five IRES-eEF2-bound ribosome structures within a single sample (Figures 1 and 2). The structures differ in the positions and conformations of ribosomal subunits (Figures 1b and 2), IRES RNA (Figures 3 and 4) and eEF2 (Figures 5 and 6). This ensemble of structures allowed us to reconstruct a sequence of steps in IRES translocation induced by eEF2. + + 0.9995842 + experimental_method + cleaner0 + 2023-07-17T08:31:00Z + MESH: + + structural visualization + + + 0.9997401 + complex_assembly + cleaner0 + 2023-07-14T09:44:47Z + GO: + + 80S•IRES•eEF2 + + + site + SO: + cleaner0 + 2023-07-14T09:21:02Z + + IRES + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:17Z + + RNA + + + 0.816472 + structure_element + cleaner0 + 2023-07-14T09:27:39Z + SO: + + PKI + + + 0.99950457 + site + cleaner0 + 2023-07-17T08:57:28Z + SO: + + A to P site + + + structure_element + SO: + cleaner0 + 2023-07-14T09:39:03Z + + small subunit + + + 0.99959093 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + site + SO: + cleaner0 + 2023-07-14T09:21:02Z + + IRES + + + site + SO: + cleaner0 + 2023-07-14T09:21:02Z + + IRES + + + 0.9975649 + complex_assembly + cleaner0 + 2023-07-14T09:32:55Z + GO: + + ribosome + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + 0.9349918 + complex_assembly + cleaner0 + 2023-07-17T09:01:28Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + site + SO: + cleaner0 + 2023-07-14T09:21:02Z + + IRES + + + 0.99958605 + experimental_method + cleaner0 + 2023-07-17T08:27:35Z + MESH: + + cryo-EM + + + 0.99875283 + complex_assembly + cleaner0 + 2023-07-14T09:45:10Z + GO: + + 80S•TSV IRES + + + 0.99009824 + protein_state + cleaner0 + 2023-07-14T09:55:43Z + DUMMY: + + presence of + + + 0.9996914 + complex_assembly + cleaner0 + 2023-07-14T09:31:05Z + GO: + + eEF2•GTP + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:54Z + + sordarin + + + 0.9997973 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:32:56Z + + ribosome + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:54Z + + sordarin + + + 0.9997215 + complex_assembly + cleaner0 + 2023-07-14T09:45:47Z + GO: + + eEF2•GDPNP + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:32:56Z + + ribosome + + + 0.99953794 + experimental_method + cleaner0 + 2023-07-17T08:31:36Z + MESH: + + Maximum-likelihood classification + + + 0.99951017 + experimental_method + cleaner0 + 2023-07-17T08:31:41Z + MESH: + + FREALIGN + + + 0.99900323 + protein_state + cleaner0 + 2023-07-17T08:31:22Z + DUMMY: + + IRES-eEF2-bound + + + 0.99925774 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + 0.9992119 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + structures + + + 0.9978788 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + structures + + + site + SO: + cleaner0 + 2023-07-14T09:21:03Z + + IRES + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:17Z + + RNA + + + 0.999419 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + 0.9989291 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + structures + + + site + SO: + cleaner0 + 2023-07-14T09:21:03Z + + IRES + + + 0.9996069 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + + RESULTS + title_1 + 12662 + Results + + + RESULTS + paragraph + 12670 + We used single-particle cryo-EM and maximum-likelihood image classification in FREALIGN to obtain three-dimensional density maps from a single specimen. The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. Unsupervised cryo-EM data classification was combined with the use of three-dimensional and two-dimensional masking around the ribosomal A site (Figure 1—figure supplement 2). This approach revealed five 80S•IRES•eEF2•GDP structures at average resolutions of 3.5 to 4.2 Å, sufficient to locate IRES domains and to resolve individual residues in the core regions of the ribosome and eEF2 (Figures 3c,d, and 5f,h; see also Figure 1—figure supplement 2 and Figure 5—figure supplement 2), including the post-translational modification diphthamide 699 (Figure 3c). + + 0.99952173 + experimental_method + cleaner0 + 2023-07-17T08:31:49Z + MESH: + + single-particle cryo-EM + + + 0.99954903 + experimental_method + cleaner0 + 2023-07-17T08:31:54Z + MESH: + + maximum-likelihood image classification + + + 0.9982401 + experimental_method + cleaner0 + 2023-07-17T08:31:57Z + MESH: + + FREALIGN + + + 0.9972732 + evidence + cleaner0 + 2023-07-19T13:53:36Z + DUMMY: + + density maps + + + 0.99904794 + species + cleaner0 + 2023-07-14T10:07:58Z + MESH: + + S. cerevisiae + + + 0.99906874 + complex_assembly + cleaner0 + 2023-07-17T09:01:37Z + GO: + + 80S ribosomes + + + 0.9007337 + species + cleaner0 + 2023-07-14T09:24:11Z + MESH: + + Taura syndrome virus + + + 0.9833481 + site + cleaner0 + 2023-07-14T09:21:03Z + SO: + + IRES + + + 0.99910754 + species + cleaner0 + 2023-07-14T10:07:58Z + MESH: + + S. cerevisiae + + + 0.9996774 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T09:55:43Z + + presence of + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:12:27Z + + GTP + + + 0.9299479 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:54Z + + sordarin + + + experimental_method + MESH: + cleaner0 + 2023-07-17T08:32:27Z + + Unsupervised cryo-EM data classification + + + 0.9989114 + experimental_method + cleaner0 + 2023-07-17T08:32:52Z + MESH: + + three-dimensional and two-dimensional masking + + + site + SO: + cleaner0 + 2023-07-14T09:28:51Z + + A site + + + 0.99971926 + complex_assembly + cleaner0 + 2023-07-14T09:46:09Z + GO: + + 80S•IRES•eEF2•GDP + + + 0.999582 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + structures + + + 0.99777067 + site + cleaner0 + 2023-07-14T09:21:03Z + SO: + + IRES + + + 0.99455357 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + 0.99969053 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + ptm + MESH: + cleaner0 + 2023-07-18T14:01:42Z + + diphthamide 699 + + + + elife-14874-fig2-figsupp1.jpg + fig2s1 + FIG + fig_title_caption + 13597 + Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site. + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:42:40Z + + Structures I through V + + + 0.99945265 + structure_element + cleaner0 + 2023-07-14T09:27:39Z + SO: + + PKI + + + 0.99961084 + site + cleaner0 + 2023-07-17T08:57:28Z + SO: + + A to P site + + + 0.9994778 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + 0.99959016 + site + cleaner0 + 2023-07-14T09:28:51Z + SO: + + A site + + + + elife-14874-fig2-figsupp1.jpg + fig2s1 + FIG + fig_caption + 13737 + (a) Rotational states of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). For each structure, the triangle outlines the contours of the 40S body; the lower angle illustrates the extent of intersubunit (body) rotation. The sizes of the arrows correspond to the extent of the head swivel (yellow) and subunit rotation (black). The views were obtained by structural alignment of the 25S rRNAs; the sarcin-ricin loop (SRL) of 25S rRNA is shown in gray for reference. (b) Solvent view (opposite from that shown in (a)) of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). The structures are colored as in Figure 1. + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:12Z + + subunit + + + 0.9997216 + complex_assembly + cleaner0 + 2023-07-14T09:40:45Z + GO: + + 80S•IRES + + + 0.99910873 + evidence + cleaner0 + 2023-07-14T16:19:11Z + DUMMY: + + structure + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:57:16Z + + INIT + + + 0.9997345 + complex_assembly + cleaner0 + 2023-07-14T09:44:49Z + GO: + + 80S•IRES•eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:42:50Z + + Structures I, II, III, IV and V + + + 0.99464685 + evidence + cleaner0 + 2023-07-14T16:19:11Z + DUMMY: + + structure + + + 0.8802642 + complex_assembly + cleaner0 + 2023-07-17T09:01:55Z + GO: + + 40S + + + 0.9393941 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + 0.8593879 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + 0.92288214 + structure_element + cleaner0 + 2023-07-17T08:56:47Z + SO: + + head + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:12Z + + subunit + + + 0.9995775 + experimental_method + cleaner0 + 2023-07-17T08:33:04Z + MESH: + + structural alignment + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:28:28Z + + 25S rRNAs + + + 0.99961627 + structure_element + cleaner0 + 2023-07-14T09:47:32Z + SO: + + sarcin-ricin loop + + + 0.9996407 + structure_element + cleaner0 + 2023-07-14T09:47:39Z + SO: + + SRL + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:28:12Z + + 25S rRNA + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.9997192 + complex_assembly + cleaner0 + 2023-07-14T09:40:45Z + GO: + + 80S•IRES + + + 0.99903995 + evidence + cleaner0 + 2023-07-14T16:19:11Z + DUMMY: + + structure + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:57:16Z + + INIT + + + 0.999736 + complex_assembly + cleaner0 + 2023-07-14T09:44:49Z + GO: + + 80S•IRES•eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:42:29Z + + Structures I, II, III, IV and V + + + 0.9907845 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + structures + + + + elife-14874-fig2-figsupp1.jpg + fig2s1 + FIG + fig_caption + 14510 + DOI: +http://dx.doi.org/10.7554/eLife.14874.009 + + + elife-14874-fig2.jpg + fig2 + FIG + fig_title_caption + 14557 + Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site. + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:43:09Z + + Structures I through V + + + 0.99945265 + structure_element + cleaner0 + 2023-07-14T09:27:39Z + SO: + + PKI + + + 0.99961084 + site + cleaner0 + 2023-07-17T08:57:28Z + SO: + + A to P site + + + 0.9994778 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + 0.99959016 + site + cleaner0 + 2023-07-14T09:28:51Z + SO: + + A site + + + + elife-14874-fig2.jpg + fig2 + FIG + fig_caption + 14697 + (a) Comparison of the 40S-subunit rotational states in Structures I through V, sampling a ~10° range between Structure I (fully rotated) and Structure V (non-rotated). 18S ribosomal RNA is shown and ribosomal proteins are omitted for clarity. The superpositions of Structures I-V were performed by structural alignments of the 25S ribosomal RNAs. (b) Bar graph of the angles characterizing the 40S rotational and 40S head swiveling states in Structures I through V. Measurements for the two 80S•IRES (INIT) structures are included for comparison. All measurements are relative to the non-rotated 80S•2tRNA•mRNA structure. (c) Comparison of the 40S conformations in Structures I through V shows distinct positions of the head relative to the body of the 40S subunit (head swivel). Conformation of the non-swiveled 40S subunit in the S. cerevisiae 80S ribosome bound with two tRNAs is shown for reference (blue). (d) Comparison of conformations of the L1 and P stalks of the large subunit in Structures I through V with those in the 80S•IRES and tRNA-bound 80S structures. Superpositions were performed by structural alignments of 25S ribosomal RNAs. The central protuberance (CP) is labeled. (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. The superpositions of structures were performed by structural alignments of the 18S ribosomal RNAs excluding the head region (nt 1150–1620). + + 0.9992841 + complex_assembly + cleaner0 + 2023-07-17T09:02:34Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:43:20Z + + Structures I through V + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:43:33Z + + Structure I + + + 0.999404 + protein_state + cleaner0 + 2023-07-19T12:29:35Z + DUMMY: + + fully rotated + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.99928814 + protein_state + cleaner0 + 2023-07-19T12:29:40Z + DUMMY: + + non-rotated + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:29:01Z + + 18S ribosomal RNA + + + 0.99929976 + experimental_method + cleaner0 + 2023-07-17T08:33:33Z + MESH: + + superpositions + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:43:48Z + + Structures I-V + + + 0.9995618 + experimental_method + cleaner0 + 2023-07-17T08:33:41Z + MESH: + + structural alignments + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:29:27Z + + 25S ribosomal RNAs + + + 0.9989073 + complex_assembly + cleaner0 + 2023-07-17T09:02:37Z + GO: + + 40S + + + 0.9897857 + complex_assembly + cleaner0 + 2023-07-17T09:02:37Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:43:57Z + + Structures I through V + + + 0.9996868 + complex_assembly + cleaner0 + 2023-07-14T09:40:46Z + GO: + + 80S•IRES + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:57:16Z + + INIT + + + evidence + DUMMY: + cleaner0 + 2023-07-14T16:19:23Z + + structures + + + 0.9992688 + protein_state + cleaner0 + 2023-07-19T12:29:42Z + DUMMY: + + non-rotated + + + 0.9997202 + complex_assembly + cleaner0 + 2023-07-14T09:44:25Z + GO: + + 80S•2tRNA•mRNA + + + 0.8293142 + evidence + cleaner0 + 2023-07-14T16:19:11Z + DUMMY: + + structure + + + 0.99936455 + complex_assembly + cleaner0 + 2023-07-17T09:02:37Z + GO: + + 40S + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:44:10Z + + Structures I through V + + + 0.996555 + structure_element + cleaner0 + 2023-07-17T08:56:47Z + SO: + + head + + + 0.7652675 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.8441466 + structure_element + cleaner0 + 2023-07-17T08:56:47Z + SO: + + head + + + 0.99903923 + protein_state + cleaner0 + 2023-07-19T12:29:57Z + DUMMY: + + non-swiveled + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.9994119 + species + cleaner0 + 2023-07-14T10:07:58Z + MESH: + + S. cerevisiae + + + 0.9996078 + complex_assembly + cleaner0 + 2023-07-14T09:26:32Z + GO: + + 80S ribosome + + + 0.9992867 + protein_state + cleaner0 + 2023-07-17T08:30:36Z + DUMMY: + + bound with + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:06Z + + tRNAs + + + 0.9998221 + structure_element + cleaner0 + 2023-07-19T14:14:44Z + SO: + + L1 + + + 0.9994683 + structure_element + cleaner0 + 2023-07-19T14:14:54Z + SO: + + P stalks + + + 0.90232724 + structure_element + cleaner0 + 2023-07-14T09:48:56Z + SO: + + large subunit + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:44:21Z + + Structures I through V + + + 0.9996479 + complex_assembly + cleaner0 + 2023-07-14T09:40:46Z + GO: + + 80S•IRES + + + 0.9994848 + protein_state + cleaner0 + 2023-07-14T09:48:14Z + DUMMY: + + tRNA-bound + + + 0.999627 + complex_assembly + cleaner0 + 2023-07-18T13:50:41Z + GO: + + 80S + + + 0.98382646 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + structures + + + 0.9994832 + experimental_method + cleaner0 + 2023-07-17T08:33:34Z + MESH: + + Superpositions + + + 0.99953955 + experimental_method + cleaner0 + 2023-07-17T08:33:43Z + MESH: + + structural alignments + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:29:28Z + + 25S ribosomal RNAs + + + structure_element + SO: + cleaner0 + 2023-07-19T13:30:23Z + + central protuberance + + + 0.960011 + structure_element + cleaner0 + 2023-07-19T13:30:29Z + SO: + + CP + + + 0.99775046 + structure_element + cleaner0 + 2023-07-14T09:27:39Z + SO: + + PKI + + + structure_element + SO: + cleaner0 + 2023-07-19T10:44:40Z + + IV + + + 0.99980396 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + 0.99868816 + site + cleaner0 + 2023-07-19T09:57:16Z + SO: + + P site + + + 0.99832207 + structure_element + cleaner0 + 2023-07-17T08:56:47Z + SO: + + head + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:58Z + + U1191 + + + 0.998409 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:29:21Z + + C1637 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T13:29:59Z + + Structures I through V + + + 0.9990062 + site + cleaner0 + 2023-07-19T09:57:20Z + SO: + + A and P sites + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:10Z + + initiation + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:57:16Z + + INIT + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:27:19Z + + post-translocation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.98836297 + complex_assembly + cleaner0 + 2023-07-17T09:02:37Z + GO: + + 40S + + + 0.7802401 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + 0.98842585 + complex_assembly + cleaner0 + 2023-07-17T09:02:37Z + GO: + + 40S + + + 0.9775685 + structure_element + cleaner0 + 2023-07-17T08:56:47Z + SO: + + head + + + 0.9995332 + experimental_method + cleaner0 + 2023-07-17T08:33:34Z + MESH: + + superpositions + + + 0.93230164 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + structures + + + 0.9995551 + experimental_method + cleaner0 + 2023-07-17T08:33:43Z + MESH: + + structural alignments + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:30:55Z + + 18S ribosomal RNAs + + + 0.99933124 + structure_element + cleaner0 + 2023-07-17T08:56:47Z + SO: + + head + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:06:57Z + + 1150–1620 + + + + elife-14874-fig2.jpg + fig2 + FIG + fig_caption + 16490 + DOI: +http://dx.doi.org/10.7554/eLife.14874.007 + + + RESULTS + paragraph + 16537 + Our structures represent hitherto uncharacterized translocation complexes of the TSV IRES captured within globally distinct 80S conformations (Figures 1b and 2). We numbered the structures from I to V, according to the position of the tRNA-mRNA-like PKI on the 40S subunit (Figure 2—source data 1). Specifically, PKI is partially withdrawn from the A site in Structure I, and fully translocated to the P site in Structure V (Figure 4; see also Figure 3—figure supplement 1). Thus Structures I to IV represent different positions of PKI between the A and P sites (Figure 2—source data 1), suggesting that these structures describe intermediate states of translocation. Structure V corresponds to the post-translocation state. + + 0.9955178 + evidence + cleaner0 + 2023-07-14T16:19:23Z + DUMMY: + + structures + + + 0.7319587 + species + cleaner0 + 2023-07-14T09:24:19Z + MESH: + + TSV + + + 0.844797 + site + cleaner0 + 2023-07-14T09:21:03Z + SO: + + IRES + + + 0.99952984 + complex_assembly + cleaner0 + 2023-07-18T13:50:41Z + GO: + + 80S + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:45:21Z + + structures from I to V + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:36:32Z + + tRNA-mRNA + + + structure_element + SO: + cleaner0 + 2023-07-14T09:27:39Z + + PKI + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.9885704 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.99954414 + site + cleaner0 + 2023-07-14T09:28:51Z + SO: + + A site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:45:36Z + + Structure I + + + 0.9993441 + protein_state + cleaner0 + 2023-07-17T08:37:56Z + DUMMY: + + fully translocated + + + 0.9995669 + site + cleaner0 + 2023-07-19T09:57:27Z + SO: + + P site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:45Z + + Structures I to IV + + + 0.9872119 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.9779728 + site + cleaner0 + 2023-07-19T09:57:32Z + SO: + + A and P sites + + + 0.8598577 + evidence + cleaner0 + 2023-07-14T16:19:21Z + DUMMY: + + structures + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.9568593 + protein_state + cleaner0 + 2023-07-14T15:27:19Z + DUMMY: + + post-translocation + + + + RESULTS + title_2 + 17268 + Changes in ribosome conformation and eEF2 positions are coupled with IRES movement through the ribosome + + 0.6416077 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + 0.66172516 + protein + cleaner0 + 2023-07-14T09:30:44Z + PR: + + eEF2 + + + 0.89029235 + site + cleaner0 + 2023-07-14T09:21:04Z + SO: + + IRES + + + 0.9118381 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + + RESULTS + title_3 + 17372 + Intersubunit rotation + + + RESULTS + paragraph + 17394 + Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). Structure I comprises the most rotated ribosome conformation (~10°), characteristic of pre-translocation hybrid-tRNA states. From Structure I to V, the body of the small subunit undergoes backward (reverse) rotation (Figure 2b; see also Figure 1—figure supplement 2 and Figure 2—figure supplement 1). Structures II and III are in mid-rotation conformations (~5°). Structure IV adopts a slightly rotated conformation (~1°). Structure V is in a nearly non-rotated conformation (0.5°), very similar to that of post-translocation ribosome-tRNA complexes. Thus, intersubunit rotation of ~9° from Structure I to V covers a nearly complete range of relative subunit positions, similar to what was reported for tRNA-bound yeast, bacterial and mammalian ribosomes. + + 0.99220747 + protein_state + cleaner0 + 2023-07-14T15:27:20Z + DUMMY: + + post-translocation + + + 0.99947834 + species + cleaner0 + 2023-07-14T10:07:58Z + MESH: + + S. cerevisiae + + + 0.998595 + complex_assembly + cleaner0 + 2023-07-14T09:26:32Z + GO: + + 80S ribosome + + + 0.99935925 + protein_state + cleaner0 + 2023-07-17T08:30:36Z + DUMMY: + + bound with + + + 0.99726045 + site + cleaner0 + 2023-07-19T09:57:37Z + SO: + + P and E site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:06Z + + tRNAs + + + 0.99971163 + complex_assembly + cleaner0 + 2023-07-14T09:44:25Z + GO: + + 80S•2tRNA•mRNA + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:34Z + + body + + + 0.9979474 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:46:13Z + + Structures I through V + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:46:23Z + + Structure I + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T10:47:51Z + + most rotated + + + 0.95640725 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + 0.9972074 + protein_state + cleaner0 + 2023-07-14T15:24:43Z + DUMMY: + + pre-translocation + + + 0.7733402 + protein_state + cleaner0 + 2023-07-19T12:30:08Z + DUMMY: + + hybrid-tRNA + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:46:37Z + + Structure I to V + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:34Z + + body + + + 0.95990837 + structure_element + cleaner0 + 2023-07-14T09:39:03Z + SO: + + small subunit + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:17:32Z + + Structures II and III + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T10:47:22Z + + mid-rotation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:46:58Z + + Structure IV + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T10:47:37Z + + slightly rotated + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.7309394 + protein_state + cleaner0 + 2023-07-19T12:29:42Z + DUMMY: + + non-rotated + + + 0.99596024 + protein_state + cleaner0 + 2023-07-14T15:27:20Z + DUMMY: + + post-translocation + + + 0.99951434 + complex_assembly + cleaner0 + 2023-07-14T09:51:53Z + GO: + + ribosome-tRNA + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:47:08Z + + Structure I to V + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.9994242 + protein_state + cleaner0 + 2023-07-14T09:48:16Z + DUMMY: + + tRNA-bound + + + 0.99951315 + taxonomy_domain + cleaner0 + 2023-07-17T08:49:33Z + DUMMY: + + yeast + + + 0.9995684 + taxonomy_domain + cleaner0 + 2023-07-14T09:36:04Z + DUMMY: + + bacterial + + + 0.9995504 + taxonomy_domain + cleaner0 + 2023-07-17T08:49:36Z + DUMMY: + + mammalian + + + 0.9663577 + complex_assembly + cleaner0 + 2023-07-19T09:22:31Z + GO: + + ribosomes + + + + RESULTS + title_3 + 18535 + 40S head swivel + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + + RESULTS + paragraph + 18551 + The pattern of 40S head swivel between the structures is different from that of intersubunit rotation (Figures 2c and d; see also Figure 2—source data 1). As with the intersubunit rotation, the small head swivel (~1°) in the non-rotated Structure V is closest to that in the 80S•2tRNA•mRNA post-translocation ribosome. However in the pre-translocation intermediates (from Structure I to IV), the beak of the head domain first turns toward the large subunit and then backs off (Figure 2—figure supplement 1). This movement reflects the forward and reverse swivel. The head samples a mid-swiveled position in Structure I (12°), then a highly-swiveled position in Structures II and III (17°) and a less swiveled position in Structure IV (14°). The maximum head swivel is observed in the mid-rotated complexes II and III, in which PKI transitions from the A to P site, while eEF2 occupies the A site partially. By comparison, the similarly mid-rotated (4°) 80S•TSV IRES initiation complex, in the absence of eEF2, adopts a mid-swiveled position (~10°) (Figure 2c). These observations suggest that eEF2 is necessary for inducing or stabilizing the large head swivel of the 40S subunit characteristic for IRES translocation intermediates. + + 0.9986701 + complex_assembly + cleaner0 + 2023-07-17T09:02:37Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + 0.9972216 + evidence + cleaner0 + 2023-07-14T16:19:24Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + 0.99944735 + protein_state + cleaner0 + 2023-07-19T12:29:42Z + DUMMY: + + non-rotated + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.99970686 + complex_assembly + cleaner0 + 2023-07-14T09:44:25Z + GO: + + 80S•2tRNA•mRNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:27:20Z + + post-translocation + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:32:56Z + + ribosome + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:24:43Z + + pre-translocation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:48:11Z + + Structure I to IV + + + 0.99885476 + structure_element + cleaner0 + 2023-07-17T08:56:47Z + SO: + + head + + + 0.6931741 + structure_element + cleaner0 + 2023-07-14T09:49:05Z + SO: + + large subunit + + + 0.97973186 + structure_element + cleaner0 + 2023-07-17T08:56:47Z + SO: + + head + + + 0.9279712 + protein_state + cleaner0 + 2023-07-18T13:58:05Z + DUMMY: + + mid-swiveled + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:48:58Z + + Structure I + + + 0.99621826 + protein_state + cleaner0 + 2023-07-18T13:58:10Z + DUMMY: + + highly-swiveled + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:17:32Z + + Structures II and III + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T12:45:58Z + + less swiveled + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:48:40Z + + Structure IV + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + 0.9993841 + protein_state + cleaner0 + 2023-07-18T13:57:54Z + DUMMY: + + mid-rotated + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:48:29Z + + II and III + + + 0.9864746 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.99730706 + site + cleaner0 + 2023-07-17T08:57:29Z + SO: + + A to P site + + + 0.9989791 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + 0.9994684 + site + cleaner0 + 2023-07-14T09:28:51Z + SO: + + A site + + + 0.94117004 + protein_state + cleaner0 + 2023-07-18T13:57:52Z + DUMMY: + + mid-rotated + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:45:12Z + + 80S•TSV IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:10Z + + initiation + + + 0.99954855 + protein_state + cleaner0 + 2023-07-14T09:55:35Z + DUMMY: + + absence of + + + 0.9993574 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + 0.9826918 + protein_state + cleaner0 + 2023-07-18T13:58:03Z + DUMMY: + + mid-swiveled + + + 0.9996069 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:47Z + + head + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + site + SO: + cleaner0 + 2023-07-14T09:21:04Z + + IRES + + + + RESULTS + title_3 + 19800 + IRES rearrangements + + 0.9982077 + site + cleaner0 + 2023-07-14T09:21:04Z + SO: + + IRES + + + + elife-14874-fig3-figsupp1.jpg + fig3s1 + FIG + fig_title_caption + 19820 + Comparison of the TSV IRES and eEF2 positions in Structures I through V. + + 0.9165737 + species + cleaner0 + 2023-07-14T09:24:19Z + MESH: + + TSV + + + 0.9817011 + site + cleaner0 + 2023-07-14T09:21:04Z + SO: + + IRES + + + 0.99873954 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:49:18Z + + Structures I through V + + + + elife-14874-fig3-figsupp1.jpg + fig3s1 + FIG + fig_caption + 19893 + (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). Superpositions were obtained by structural alignments of the 18S rRNAs excluding the head domains (nt 1150–1620). + + 0.9924055 + site + cleaner0 + 2023-07-14T09:21:04Z + SO: + + IRES + + + 0.9995933 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:10Z + + initiation + + + 0.9773825 + protein_state + cleaner0 + 2023-07-14T15:24:43Z + DUMMY: + + pre-translocation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:49:42Z + + I + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T09:53:31Z + + post-translocation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:49:36Z + + V + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:34Z + + body + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-18T13:46:36Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.99283445 + site + cleaner0 + 2023-07-14T09:21:04Z + SO: + + IRES + + + 0.999634 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:10Z + + initiation + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:57:16Z + + INIT + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:49:30Z + + II, III and IV + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:34Z + + body + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-18T13:46:37Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.9995757 + experimental_method + cleaner0 + 2023-07-17T08:33:34Z + MESH: + + Superpositions + + + 0.99957794 + experimental_method + cleaner0 + 2023-07-17T08:33:43Z + MESH: + + structural alignments + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:32:50Z + + 18S rRNAs + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:48Z + + head + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:06:12Z + + 1150–1620 + + + + elife-14874-fig3-figsupp1.jpg + fig3s1 + FIG + fig_caption + 20350 + DOI: +http://dx.doi.org/10.7554/eLife.14874.011 + + + elife-14874-fig3-figsupp2.jpg + fig3s2 + FIG + fig_title_caption + 20397 + Positions of the IRES relative to proteins uS7, uS11 and eS25. + + 0.9545575 + site + cleaner0 + 2023-07-14T09:21:04Z + SO: + + IRES + + + 0.99954516 + protein + cleaner0 + 2023-07-18T14:35:30Z + PR: + + uS7 + + + 0.9995259 + protein + cleaner0 + 2023-07-18T14:35:37Z + PR: + + uS11 + + + 0.9995547 + protein + cleaner0 + 2023-07-18T14:35:43Z + PR: + + eS25 + + + + elife-14874-fig3-figsupp2.jpg + fig3s2 + FIG + fig_caption + 20460 + (a) Intra-IRES rearrangements from the 80S*IRES initiation structure (INIT; PDB 3J6Y,) to Structures I through V. For each structure (shown in red), the conformation from a preceding structure is shown in light red for comparison. Superpositions were obtained by structural alignments of 18S rRNA. (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). In all panels, superpositions were obtained by structural alignments of the 18S rRNAs. Ribosomal proteins of the initiation state are shown in gray for comparison. + + site + SO: + cleaner0 + 2023-07-14T09:21:04Z + + IRES + + + 0.9997053 + complex_assembly + cleaner0 + 2023-07-14T09:54:07Z + GO: + + 80S*IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:10Z + + initiation + + + evidence + DUMMY: + cleaner0 + 2023-07-14T16:19:12Z + + structure + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:57:16Z + + INIT + + + evidence + DUMMY: + cleaner0 + 2023-07-19T13:33:34Z + + Structures I through V + + + 0.84493774 + evidence + cleaner0 + 2023-07-14T16:19:12Z + DUMMY: + + structure + + + evidence + DUMMY: + cleaner0 + 2023-07-14T16:19:12Z + + structure + + + 0.9873174 + experimental_method + cleaner0 + 2023-07-17T08:33:34Z + MESH: + + Superpositions + + + 0.9995508 + experimental_method + cleaner0 + 2023-07-17T08:33:43Z + MESH: + + structural alignments + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:33:19Z + + 18S rRNA + + + 0.9971047 + site + cleaner0 + 2023-07-14T09:21:04Z + SO: + + IRES + + + 0.9997212 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + 0.9983177 + site + cleaner0 + 2023-07-19T09:57:45Z + SO: + + P- and E-site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:06Z + + tRNAs + + + 0.99970275 + complex_assembly + cleaner0 + 2023-07-14T09:54:20Z + GO: + + 80S•tRNA + + + 0.9968957 + site + cleaner0 + 2023-07-14T09:21:04Z + SO: + + IRES + + + 0.9988563 + protein + cleaner0 + 2023-07-18T14:35:37Z + PR: + + uS11 + + + 0.6837357 + site + cleaner0 + 2023-07-19T09:58:10Z + SO: + + 40S platform + + + 0.99955755 + protein + cleaner0 + 2023-07-18T14:35:30Z + PR: + + uS7 + + + 0.9996861 + protein + cleaner0 + 2023-07-18T14:35:43Z + PR: + + eS25 + + + 0.54895943 + complex_assembly + cleaner0 + 2023-07-17T09:02:37Z + GO: + + 40S + + + 0.8590382 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.9993345 + structure_element + cleaner0 + 2023-07-19T14:15:17Z + SO: + + 5′ domain + + + 0.99839693 + site + cleaner0 + 2023-07-14T09:21:04Z + SO: + + IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:10Z + + initiation + + + 0.9983734 + experimental_method + cleaner0 + 2023-07-17T08:33:34Z + MESH: + + superpositions + + + 0.9995617 + experimental_method + cleaner0 + 2023-07-17T08:33:43Z + MESH: + + structural alignments + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:32:52Z + + 18S rRNAs + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + + elife-14874-fig3-figsupp2.jpg + fig3s2 + FIG + fig_caption + 21217 + DOI: +http://dx.doi.org/10.7554/eLife.14874.012 + + + elife-14874-fig3-figsupp3.jpg + fig3s3 + FIG + fig_title_caption + 21264 + Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S•tRNA structures and 80S•IRES structures I and V (this work). + + 0.6030796 + structure_element + cleaner0 + 2023-07-19T14:15:26Z + SO: + + L1stalk + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + 0.49342766 + species + cleaner0 + 2023-07-14T09:24:19Z + MESH: + + TSV + + + 0.63299435 + site + cleaner0 + 2023-07-14T09:21:04Z + SO: + + IRES + + + 0.9997242 + protein + cleaner0 + 2023-07-18T14:35:30Z + PR: + + uS7 + + + 0.99974793 + protein + cleaner0 + 2023-07-18T14:35:44Z + PR: + + eS25 + + + 0.9996447 + complex_assembly + cleaner0 + 2023-07-14T09:54:22Z + GO: + + 80S•tRNA + + + 0.9993544 + evidence + cleaner0 + 2023-07-14T16:19:24Z + DUMMY: + + structures + + + 0.99965173 + complex_assembly + cleaner0 + 2023-07-14T09:40:46Z + GO: + + 80S•IRES + + + evidence + DUMMY: + cleaner0 + 2023-07-19T11:57:42Z + + structures I and V + + + + elife-14874-fig3-figsupp3.jpg + fig3s3 + FIG + fig_caption + 21415 + The view shows the vicinity of the ribosomal E site. Loop 1.1 and stem loops 4 and 5 of the IRES are labeled. + + 0.9886788 + site + cleaner0 + 2023-07-14T09:35:32Z + SO: + + E site + + + 0.99967754 + structure_element + cleaner0 + 2023-07-19T14:15:35Z + SO: + + Loop 1.1 + + + 0.99966127 + structure_element + cleaner0 + 2023-07-19T14:15:42Z + SO: + + stem loops 4 and 5 + + + 0.9996101 + site + cleaner0 + 2023-07-14T09:21:04Z + SO: + + IRES + + + + elife-14874-fig3-figsupp3.jpg + fig3s3 + FIG + fig_caption + 21525 + DOI: +http://dx.doi.org/10.7554/eLife.14874.013 + + + elife-14874-fig3-figsupp4.jpg + fig3s4 + FIG + fig_title_caption + 21572 + Interactions of the stem loops 4 and 5 of the TSV with proteins uS7 and eS25. + + 0.9995259 + structure_element + cleaner0 + 2023-07-19T14:15:44Z + SO: + + stem loops 4 and 5 + + + 0.9827891 + species + cleaner0 + 2023-07-14T09:24:19Z + MESH: + + TSV + + + 0.9995628 + protein + cleaner0 + 2023-07-18T14:35:30Z + PR: + + uS7 + + + 0.999608 + protein + cleaner0 + 2023-07-18T14:35:44Z + PR: + + eS25 + + + + elife-14874-fig3-figsupp4.jpg + fig3s4 + FIG + fig_caption + 21650 + DOI: +http://dx.doi.org/10.7554/eLife.14874.014 + + + elife-14874-fig3-figsupp5.jpg + fig3s5 + FIG + fig_title_caption + 21697 + Position and interactions of loop 3 (variable loop region) of the PKI domain in Structure V (this work) resembles those of the anticodon stem loop of the E-site tRNA (blue) in the 80S•2tRNA•mRNA complex. + + 0.99972403 + structure_element + cleaner0 + 2023-07-19T14:16:03Z + SO: + + loop 3 + + + 0.9996975 + structure_element + cleaner0 + 2023-07-19T14:16:10Z + SO: + + variable loop region + + + structure_element + SO: + cleaner0 + 2023-07-14T09:27:40Z + + PKI + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.95514816 + structure_element + cleaner0 + 2023-07-14T09:34:47Z + SO: + + anticodon stem loop + + + 0.99940234 + site + cleaner0 + 2023-07-19T09:58:33Z + SO: + + E-site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + 0.99971503 + complex_assembly + cleaner0 + 2023-07-14T09:44:25Z + GO: + + 80S•2tRNA•mRNA + + + + elife-14874-fig3-figsupp5.jpg + fig3s5 + FIG + fig_caption + 21905 + DOI: +http://dx.doi.org/10.7554/eLife.14874.015 + + + elife-14874-fig3-figsupp6.jpg + fig3s6 + FIG + fig_title_caption + 21952 + Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008–1043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt. 2668–2687) and protein uL5 (collectively labeled as central protuberance, CP, in the upper-row first figure, and individually labeled in the lower-row first figure). + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:06Z + + tRNAs + + + 0.9799182 + species + cleaner0 + 2023-07-14T09:24:19Z + MESH: + + TSV + + + 0.52040637 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + 0.9895371 + structure_element + cleaner0 + 2023-07-19T14:19:09Z + SO: + + A-site finger + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:05:31Z + + 1008–1043 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:28:14Z + + 25S rRNA + + + 0.9992714 + site + cleaner0 + 2023-07-19T09:58:45Z + SO: + + P site + + + 0.69923544 + structure_element + cleaner0 + 2023-07-14T09:49:05Z + SO: + + large subunit + + + 0.9996331 + structure_element + cleaner0 + 2023-07-19T14:16:21Z + SO: + + helix 84 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:28:14Z + + 25S rRNA + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:05:43Z + + 2668–2687 + + + 0.9996697 + protein + cleaner0 + 2023-07-19T09:25:13Z + PR: + + uL5 + + + structure_element + SO: + cleaner0 + 2023-07-19T13:30:24Z + + central protuberance + + + structure_element + SO: + cleaner0 + 2023-07-19T13:30:30Z + + CP + + + + elife-14874-fig3-figsupp6.jpg + fig3s6 + FIG + fig_caption + 22295 + Structures of translocation complexes of the bacterial 70S ribosome bound with two tRNAs and yeast 80S complexes with tRNAs are shown in the upper row and labeled. Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the lower row and labeled. + + 0.787759 + evidence + cleaner0 + 2023-07-14T16:19:24Z + DUMMY: + + Structures + + + 0.9994966 + taxonomy_domain + cleaner0 + 2023-07-14T09:36:04Z + DUMMY: + + bacterial + + + 0.99806744 + complex_assembly + cleaner0 + 2023-07-14T09:56:37Z + GO: + + 70S ribosome + + + 0.99931896 + protein_state + cleaner0 + 2023-07-17T08:30:36Z + DUMMY: + + bound with + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:06Z + + tRNAs + + + 0.9994647 + taxonomy_domain + cleaner0 + 2023-07-17T08:49:42Z + DUMMY: + + yeast + + + 0.9995678 + complex_assembly + cleaner0 + 2023-07-18T13:50:41Z + GO: + + 80S + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T09:56:02Z + + complexes with + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:06Z + + tRNAs + + + 0.9689409 + evidence + cleaner0 + 2023-07-14T16:19:24Z + DUMMY: + + Structures + + + 0.99964684 + complex_assembly + cleaner0 + 2023-07-14T09:40:46Z + GO: + + 80S•IRES + + + 0.9990853 + protein_state + cleaner0 + 2023-07-14T09:55:35Z + DUMMY: + + absence of + + + 0.73675007 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:57:16Z + + INIT + + + 0.9957364 + protein_state + cleaner0 + 2023-07-14T09:55:43Z + DUMMY: + + presence of + + + 0.8072261 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + + elife-14874-fig3-figsupp6.jpg + fig3s6 + FIG + fig_caption + 22615 + DOI: +http://dx.doi.org/10.7554/eLife.14874.016 + + + elife-14874-fig3-figsupp7.jpg + fig3s7 + FIG + fig_title_caption + 22662 + Interactions of the TSV IRES with uL5 and eL42. + + 0.9965576 + species + cleaner0 + 2023-07-14T09:24:19Z + MESH: + + TSV + + + 0.901585 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + 0.9838537 + protein + cleaner0 + 2023-07-19T09:25:14Z + PR: + + uL5 + + + 0.9647332 + protein + cleaner0 + 2023-07-19T09:25:23Z + PR: + + eL42 + + + + elife-14874-fig3-figsupp7.jpg + fig3s7 + FIG + fig_caption + 22710 + Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the upper row and labeled. Structures of the 80S complexes with tRNAs are shown in the lower row in a view similar to that for the 80S•IRES complex. + + 0.97064203 + evidence + cleaner0 + 2023-07-14T16:19:24Z + DUMMY: + + Structures + + + 0.99967486 + complex_assembly + cleaner0 + 2023-07-14T09:40:46Z + GO: + + 80S•IRES + + + 0.9992517 + protein_state + cleaner0 + 2023-07-14T09:55:33Z + DUMMY: + + absence of + + + 0.6100859 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:57:16Z + + INIT + + + 0.999338 + protein_state + cleaner0 + 2023-07-14T09:55:40Z + DUMMY: + + presence of + + + 0.58906734 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + 0.98536354 + evidence + cleaner0 + 2023-07-14T16:19:24Z + DUMMY: + + Structures + + + 0.99963903 + complex_assembly + cleaner0 + 2023-07-18T13:50:41Z + GO: + + 80S + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T09:56:01Z + + complexes with + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:06Z + + tRNAs + + + 0.99968195 + complex_assembly + cleaner0 + 2023-07-14T09:40:46Z + GO: + + 80S•IRES + + + + elife-14874-fig3-figsupp7.jpg + fig3s7 + FIG + fig_caption + 22990 + DOI: +http://dx.doi.org/10.7554/eLife.14874.017 + + + elife-14874-fig3.jpg + fig3 + FIG + fig_title_caption + 23037 + Positions of the IRES relative to eEF2 and elements of the ribosome in Structures I through V. + + 0.994894 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + 0.9987348 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + 0.9971704 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + evidence + DUMMY: + cleaner0 + 2023-07-19T11:58:20Z + + Structures I through V + + + + elife-14874-fig3.jpg + fig3 + FIG + fig_caption + 23132 + (a) Secondary structure of the TSV IRES. The TSV IRES comprises two domains: the 5' domain (blue) and the PKI domain (red). The open reading frame (gray) is immediately following pseudoknot I (PKI). (b) Three-dimensional structure of the TSV IRES (Structure II). Pseudoknots and stem loops are labeled and colored as in (a). (c) Positions of the IRES and eEF2 on the small subunit in Structures I to V. The initiation-state IRES is shown in gray. The insert shows density for interaction of diphthamide 699 (eEF2; green) with the codon-anticodon-like helix (PKI; red) in Structure V. (d and e) Density of the P site in Structure V shows that interactions of PKI with the 18S rRNA nucleotides (c) are nearly identical to those in the P site of the 2tRNA•mRNA-bound 70S ribosome (d). + + evidence + DUMMY: + cleaner0 + 2023-07-14T16:19:12Z + + structure + + + 0.9950825 + species + cleaner0 + 2023-07-14T09:24:19Z + MESH: + + TSV + + + 0.90993303 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + 0.99355936 + species + cleaner0 + 2023-07-14T09:24:19Z + MESH: + + TSV + + + 0.9564812 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + 0.9997061 + structure_element + cleaner0 + 2023-07-19T14:16:32Z + SO: + + 5' domain + + + 0.99972063 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + structure_element + SO: + cleaner0 + 2023-07-19T09:59:12Z + + open reading frame + + + 0.9996612 + structure_element + cleaner0 + 2023-07-14T09:27:32Z + SO: + + pseudoknot I + + + 0.9996896 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.75437105 + evidence + cleaner0 + 2023-07-14T16:19:12Z + DUMMY: + + structure + + + 0.99480766 + species + cleaner0 + 2023-07-14T09:24:19Z + MESH: + + TSV + + + 0.96464014 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + evidence + DUMMY: + cleaner0 + 2023-07-19T11:58:35Z + + Structure II + + + 0.99979013 + structure_element + cleaner0 + 2023-07-19T14:16:37Z + SO: + + Pseudoknots + + + 0.99973154 + structure_element + cleaner0 + 2023-07-19T14:16:41Z + SO: + + stem loops + + + 0.95425636 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + 0.9992661 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + 0.986463 + structure_element + cleaner0 + 2023-07-14T09:39:03Z + SO: + + small subunit + + + evidence + DUMMY: + cleaner0 + 2023-07-19T13:34:51Z + + Structures I to V + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + 0.9783975 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + 0.9983353 + evidence + cleaner0 + 2023-07-19T14:11:11Z + DUMMY: + + density + + + 0.9997157 + ptm + cleaner0 + 2023-07-18T14:01:39Z + MESH: + + diphthamide 699 + + + 0.99809605 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + 0.9996616 + structure_element + cleaner0 + 2023-07-19T14:13:13Z + SO: + + codon-anticodon-like helix + + + 0.99958616 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.99908054 + evidence + cleaner0 + 2023-07-19T14:11:16Z + DUMMY: + + Density + + + 0.9988978 + site + cleaner0 + 2023-07-19T09:59:27Z + SO: + + P site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.99917597 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:33:20Z + + 18S rRNA + + + 0.99879754 + site + cleaner0 + 2023-07-19T09:59:32Z + SO: + + P site + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:36:39Z + + 2tRNA•mRNA + + + 0.9991086 + complex_assembly + cleaner0 + 2023-07-14T09:56:35Z + GO: + + 70S ribosome + + + + elife-14874-fig3.jpg + fig3 + FIG + fig_caption + 23918 + DOI: +http://dx.doi.org/10.7554/eLife.14874.010 + + + RESULTS + paragraph + 23965 + In each structure, the TSV IRES adopts a distinct conformation in the intersubunit space of the ribosome (Figures 3 and 4). The IRES (nt 6758–6952) consists of two globular parts (Figure 3a): the 5’-region (domains I and II, nt 6758–6888) and the PKI domain (domain III, nt 6889–6952). We collectively term domains I and II the 5’ domain. The PKI domain comprises PKI and stem loop 3 (SL3), which stacks on top of the stem of PKI. The 6953GCU triplet immediately following the PKI domain is the first codon of the open reading frame. In the eEF2-free 80S•IRES initiation complex (INIT), the bulk of the 5’-domain (nt. 6758–6888) binds near the E site, contacting the ribosome mostly by means of three protruding structural elements: the L1.1 region and stem loops 4 and 5 (SL4 and SL5). In Structures I to IV, these contacts remain as in the initiation complex (Figure 1a). Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to). In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). The tip of SL4 binds in the vicinity of R157 in the β-hairpin of uS7 and of Y58 in uS11. The minor groove of SL5 (at nt 6862–6868) contacts the positively charged region of eS25 (R49, R58 and R68) (Figure 3—figure supplement 4). In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20 Å away from its initial position (Figure 3—figure supplement 2c). The L1.1 region remains in contact with the L1 stalk (Figure 3—figure supplement 3). + + 0.9970613 + evidence + cleaner0 + 2023-07-14T16:19:12Z + DUMMY: + + structure + + + 0.99605346 + species + cleaner0 + 2023-07-14T09:24:19Z + MESH: + + TSV + + + 0.8422998 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + 0.95629233 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + 0.9447189 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:04:47Z + + 6758–6952 + + + 0.99967647 + structure_element + cleaner0 + 2023-07-19T14:17:52Z + SO: + + 5’-region + + + structure_element + SO: + cleaner0 + 2023-07-19T11:59:16Z + + I + + + structure_element + SO: + cleaner0 + 2023-07-19T11:59:25Z + + II + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:04:36Z + + 6758–6888 + + + structure_element + SO: + cleaner0 + 2023-07-14T09:27:40Z + + PKI + + + structure_element + SO: + cleaner0 + 2023-07-19T11:59:40Z + + III + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:04:58Z + + 6889–6952 + + + structure_element + SO: + cleaner0 + 2023-07-19T14:18:10Z + + I + + + structure_element + SO: + cleaner0 + 2023-07-19T14:18:21Z + + II + + + 0.99967736 + structure_element + cleaner0 + 2023-07-19T14:18:26Z + SO: + + 5’ domain + + + 0.99978715 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.99979395 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.9997096 + structure_element + cleaner0 + 2023-07-19T14:16:50Z + SO: + + stem loop 3 + + + 0.99975425 + structure_element + cleaner0 + 2023-07-19T14:16:55Z + SO: + + SL3 + + + 0.999703 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.9997857 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + structure_element + SO: + cleaner0 + 2023-07-19T09:59:13Z + + open reading frame + + + 0.9993868 + protein_state + cleaner0 + 2023-07-14T09:57:26Z + DUMMY: + + eEF2-free + + + complex_assembly + GO: + cleaner0 + 2023-07-18T14:02:32Z + + 80S•IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + 0.9994357 + complex_assembly + cleaner0 + 2023-07-14T09:57:14Z + GO: + + INIT + + + 0.9996576 + structure_element + cleaner0 + 2023-07-19T14:18:43Z + SO: + + 5’-domain + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:04:23Z + + 6758–6888 + + + 0.9979558 + site + cleaner0 + 2023-07-14T09:35:33Z + SO: + + E site + + + 0.9804565 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + 0.9945699 + structure_element + cleaner0 + 2023-07-19T12:21:22Z + SO: + + L1.1 region + + + 0.9996824 + structure_element + cleaner0 + 2023-07-19T14:15:44Z + SO: + + stem loops 4 and 5 + + + 0.9997663 + structure_element + cleaner0 + 2023-07-19T14:17:19Z + SO: + + SL4 + + + 0.99975306 + structure_element + cleaner0 + 2023-07-19T14:17:26Z + SO: + + SL5 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:45Z + + Structures I to IV + + + 0.9967144 + complex_assembly + cleaner0 + 2023-07-19T09:22:51Z + GO: + + initiation complex + + + 0.9954308 + structure_element + cleaner0 + 2023-07-19T12:21:22Z + SO: + + L1.1 region + + + 0.99948716 + structure_element + cleaner0 + 2023-07-19T12:21:14Z + SO: + + L1 stalk + + + structure_element + SO: + cleaner0 + 2023-07-14T09:49:05Z + + large subunit + + + 0.9997856 + structure_element + cleaner0 + 2023-07-19T14:17:21Z + SO: + + SL4 + + + 0.99977416 + structure_element + cleaner0 + 2023-07-19T14:17:27Z + SO: + + SL5 + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:48Z + + head + + + 0.99570835 + protein + cleaner0 + 2023-07-18T14:35:29Z + PR: + + uS7 + + + 0.9891466 + protein + cleaner0 + 2023-07-18T14:35:35Z + PR: + + uS11 + + + 0.9916831 + protein + cleaner0 + 2023-07-18T14:35:42Z + PR: + + eS25 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:00:18Z + + Structures I-IV + + + 0.9255752 + site + cleaner0 + 2023-07-19T10:02:12Z + SO: + + minor groove + + + 0.9997675 + structure_element + cleaner0 + 2023-07-19T14:17:21Z + SO: + + SL4 + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:04:11Z + + 6840–6846 + + + 0.99971604 + structure_element + cleaner0 + 2023-07-19T14:18:47Z + SO: + + α-helix + + + 0.8886787 + protein + cleaner0 + 2023-07-18T14:35:30Z + PR: + + uS7 + + + 0.9998964 + residue_name_number + cleaner0 + 2023-07-18T14:34:14Z + DUMMY: + + K212 + + + 0.99989355 + residue_name_number + cleaner0 + 2023-07-18T14:34:20Z + DUMMY: + + K213 + + + 0.99989116 + residue_name_number + cleaner0 + 2023-07-18T14:34:26Z + DUMMY: + + R219 + + + 0.99989223 + residue_name_number + cleaner0 + 2023-07-18T14:34:33Z + DUMMY: + + K222 + + + 0.9997558 + structure_element + cleaner0 + 2023-07-19T14:17:21Z + SO: + + SL4 + + + 0.99989307 + residue_name_number + cleaner0 + 2023-07-18T14:34:41Z + DUMMY: + + R157 + + + 0.99970436 + structure_element + cleaner0 + 2023-07-19T14:18:53Z + SO: + + β-hairpin + + + 0.9571371 + protein + cleaner0 + 2023-07-18T14:35:30Z + PR: + + uS7 + + + 0.99989974 + residue_name_number + cleaner0 + 2023-07-18T14:35:02Z + DUMMY: + + Y58 + + + 0.98084277 + protein + cleaner0 + 2023-07-18T14:35:38Z + PR: + + uS11 + + + 0.9051391 + site + cleaner0 + 2023-07-19T10:02:15Z + SO: + + minor groove + + + 0.9997534 + structure_element + cleaner0 + 2023-07-19T14:17:27Z + SO: + + SL5 + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:04:01Z + + 6862–6868 + + + 0.9662515 + protein + cleaner0 + 2023-07-18T14:35:44Z + PR: + + eS25 + + + 0.99989974 + residue_name_number + cleaner0 + 2023-07-18T14:35:10Z + DUMMY: + + R49 + + + 0.9998976 + residue_name_number + cleaner0 + 2023-07-18T14:35:15Z + DUMMY: + + R58 + + + 0.99989545 + residue_name_number + cleaner0 + 2023-07-18T14:35:21Z + DUMMY: + + R68 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.9994122 + evidence + cleaner0 + 2023-07-19T14:11:21Z + DUMMY: + + density + + + 0.99968743 + structure_element + cleaner0 + 2023-07-19T14:17:27Z + SO: + + SL5 + + + 0.9996635 + structure_element + cleaner0 + 2023-07-19T14:17:27Z + SO: + + SL5 + + + 0.97760105 + protein_state + cleaner0 + 2023-07-19T12:46:11Z + DUMMY: + + mobile + + + 0.9997112 + structure_element + cleaner0 + 2023-07-19T14:17:21Z + SO: + + SL4 + + + 0.9993094 + evidence + cleaner0 + 2023-07-19T14:11:23Z + DUMMY: + + density + + + 0.9996848 + structure_element + cleaner0 + 2023-07-19T14:17:21Z + SO: + + SL4 + + + 0.6528504 + protein + cleaner0 + 2023-07-18T14:35:30Z + PR: + + uS7 + + + 0.98740685 + structure_element + cleaner0 + 2023-07-19T12:21:22Z + SO: + + L1.1 region + + + 0.9992199 + structure_element + cleaner0 + 2023-07-19T12:21:14Z + SO: + + L1 stalk + + + + elife-14874-fig4.jpg + fig4 + FIG + fig_title_caption + 25878 + Inchworm-like translocation of the TSV IRES. + + protein_state + DUMMY: + cleaner0 + 2023-07-19T10:13:02Z + + Inchworm + + + 0.91477746 + species + cleaner0 + 2023-07-14T09:24:19Z + MESH: + + TSV + + + 0.98869705 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + + elife-14874-fig4.jpg + fig4 + FIG + fig_caption + 25923 + Conformations and positions of the IRES in the initiation state and in Structures I-V are shown relative to those of the A-, P- and E-site tRNAs. The view was obtained by structural alignment of the body domains of 18S rRNAs of the corresponding 80S structures. Distances between nucleotides 6848 and 6913 in SL4 and PKI, respectively, are shown (see also Figure 2—source data 1). + + 0.9905737 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + 0.8976364 + protein_state + cleaner0 + 2023-07-17T08:39:11Z + DUMMY: + + initiation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:02:31Z + + Structures I-V + + + 0.99613404 + site + cleaner0 + 2023-07-19T10:03:07Z + SO: + + A-, P- and E-site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:06Z + + tRNAs + + + 0.9995541 + experimental_method + cleaner0 + 2023-07-17T08:33:57Z + MESH: + + structural alignment + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:34Z + + body + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:32:52Z + + 18S rRNAs + + + 0.999451 + complex_assembly + cleaner0 + 2023-07-18T13:50:41Z + GO: + + 80S + + + 0.93994606 + evidence + cleaner0 + 2023-07-14T16:19:24Z + DUMMY: + + structures + + + 0.930004 + residue_number + cleaner0 + 2023-07-19T14:35:02Z + DUMMY: + + 6848 + + + 0.943204 + residue_number + cleaner0 + 2023-07-19T14:35:05Z + DUMMY: + + 6913 + + + 0.9998031 + structure_element + cleaner0 + 2023-07-19T14:17:21Z + SO: + + SL4 + + + 0.99979204 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + + elife-14874-fig4.jpg + fig4 + FIG + fig_caption + 26306 + DOI: +http://dx.doi.org/10.7554/eLife.14874.018 + + + RESULTS + paragraph + 26353 + The shape of the IRES changes considerably from the initiation state to Structures I through V, from an extended to compact to extended conformation (Figure 4; see also Figure 3—figure supplement 2a). Because in Structures I to IV the PKI domain shifts toward the P site, while the 5’ remains unchanged near the E site, the distance between the domains shortens (Figure 4). In the 80S•IRES initiation state, the A-site-bound PKI is separated from SL4 by almost 50 Å (Figure 4). In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35 Å. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25 Å of SL4. Because the 5’-domain in the following structure (V) moves by ~20 Å along the 40S head, the IRES returns to an extended conformation (~45 Å) that is similar to that in the 80S•IRES initiation complex. + + 0.99249226 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:02:48Z + + Structures I through V + + + 0.9996642 + protein_state + cleaner0 + 2023-07-17T08:34:19Z + DUMMY: + + extended + + + 0.99965656 + protein_state + cleaner0 + 2023-07-17T08:34:26Z + DUMMY: + + compact + + + 0.9996586 + protein_state + cleaner0 + 2023-07-17T08:34:20Z + DUMMY: + + extended + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:45Z + + Structures I to IV + + + 0.9997297 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.99946684 + site + cleaner0 + 2023-07-19T10:03:15Z + SO: + + P site + + + 0.99946356 + site + cleaner0 + 2023-07-14T09:35:33Z + SO: + + E site + + + 0.9997106 + complex_assembly + cleaner0 + 2023-07-14T09:40:46Z + GO: + + 80S•IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + 0.99922115 + protein_state + cleaner0 + 2023-07-19T12:46:19Z + DUMMY: + + A-site-bound + + + 0.99880826 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.99958056 + structure_element + cleaner0 + 2023-07-19T14:17:21Z + SO: + + SL4 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:03:10Z + + Structures I and II + + + 0.99573106 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.99949205 + site + cleaner0 + 2023-07-14T09:28:51Z + SO: + + A site + + + 0.9995528 + structure_element + cleaner0 + 2023-07-19T14:17:21Z + SO: + + SL4 + + + 0.8595263 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.9995056 + site + cleaner0 + 2023-07-19T10:03:20Z + SO: + + P site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:03:22Z + + Structures III and IV + + + 0.9995981 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.99947983 + structure_element + cleaner0 + 2023-07-19T14:17:21Z + SO: + + SL4 + + + 0.9996444 + structure_element + cleaner0 + 2023-07-19T14:19:01Z + SO: + + 5’-domain + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:03:39Z + + structure (V) + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:48Z + + head + + + 0.9931164 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + 0.99966455 + protein_state + cleaner0 + 2023-07-17T08:34:20Z + DUMMY: + + extended + + + complex_assembly + GO: + cleaner0 + 2023-07-18T14:04:04Z + + 80S•IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + + RESULTS + paragraph + 27280 + Rearrangements of the IRES involve restructuring of several interactions with the ribosome. In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. 2668–2687) and protein uL5 (Figure 3—figure supplement 6). This position of SL3 is ~25 Å away from that in the 80S•IRES initiation state, in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively. As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA. The second set of major structural changes involves interaction of the P site region of the large subunit with the hinge point of the IRES bending between the 5´ domain and the PKI domain (nt. 6886–6890). In the highly bent Structures III and IV, the hinge region interacts with the universally conserved uL5 and the C-terminal tail of eL42 (Figure 3—figure supplement 7). However, in the extended conformations, these parts of the IRES and the 60S subunit are separated by more than 10 Å, suggesting that an interaction between them stabilizes the bent conformations but not the extended ones. Another local rearrangement concerns loop 3, also known as the variable loop region , which connects the ASL- and mRNA-like parts of PKI. This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI and biochemical studies of unbound IRESs. In Structure V, loop 3 is bound in the 40S E site and the backbone of loop 3 near the codon-like part of PKI (at nt. 6945–6946) interacts with R148 and R157 in β-hairpin of uS7. The interaction of loop 3 backbone with uS7 resembles that of the anticodon-stem loop of E-site tRNA in the post-translocation 80S•2tRNA•mRNA structure (Figure 3—figure supplement 5). Ordering of loop 3 suggests that this flexible region contributes to the stabilization of the PKI domain in the post-translocation state. This interpretation is consistent with the recent observation that alterations in loop 3 of the CrPV IRES result in decreased efficiency of translocation. + + 0.7381755 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + 0.9867508 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:03:59Z + + Structure I + + + 0.9997814 + structure_element + cleaner0 + 2023-07-19T14:16:56Z + SO: + + SL3 + + + 0.99781585 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.9058745 + structure_element + cleaner0 + 2023-07-19T14:19:08Z + SO: + + A-site finger + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:03:18Z + + 1008–1043 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:28:14Z + + 25S rRNA + + + 0.99828005 + site + cleaner0 + 2023-07-19T10:03:24Z + SO: + + P site + + + complex_assembly + GO: + cleaner0 + 2023-07-18T13:49:58Z + + 60S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.9996959 + structure_element + cleaner0 + 2023-07-19T14:16:23Z + SO: + + helix 84 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:28:14Z + + 25S rRNA + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:03:05Z + + 2668–2687 + + + 0.9980566 + protein + cleaner0 + 2023-07-19T09:25:14Z + PR: + + uL5 + + + 0.999749 + structure_element + cleaner0 + 2023-07-19T14:16:56Z + SO: + + SL3 + + + 0.9996088 + complex_assembly + cleaner0 + 2023-07-14T09:40:46Z + GO: + + 80S•IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + 0.99866784 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.99974304 + structure_element + cleaner0 + 2023-07-19T14:16:56Z + SO: + + SL3 + + + 0.9813192 + structure_element + cleaner0 + 2023-07-14T09:34:56Z + SO: + + ASL + + + 0.5455373 + structure_element + cleaner0 + 2023-07-19T14:19:17Z + SO: + + elbow + + + 0.9991398 + site + cleaner0 + 2023-07-19T10:03:28Z + SO: + + A-site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:04:10Z + + Structure I + + + 0.99976534 + structure_element + cleaner0 + 2023-07-19T14:16:56Z + SO: + + SL3 + + + 0.88417906 + structure_element + cleaner0 + 2023-07-19T14:19:09Z + SO: + + A-site finger + + + 0.9985389 + protein_state + cleaner0 + 2023-07-14T15:24:43Z + DUMMY: + + pre-translocation + + + 0.9702811 + site + cleaner0 + 2023-07-19T09:24:30Z + SO: + + A/P + + + site + SO: + cleaner0 + 2023-07-19T09:24:47Z + + A/P* + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + 0.8752671 + site + cleaner0 + 2023-07-19T10:03:34Z + SO: + + P site region + + + 0.8531384 + structure_element + cleaner0 + 2023-07-14T09:49:05Z + SO: + + large subunit + + + 0.9981436 + structure_element + cleaner0 + 2023-07-19T14:19:24Z + SO: + + hinge point + + + 0.99713683 + site + cleaner0 + 2023-07-14T09:21:05Z + SO: + + IRES + + + 0.9996864 + structure_element + cleaner0 + 2023-07-19T14:19:28Z + SO: + + 5´ domain + + + 0.9993082 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:02:35Z + + 6886–6890 + + + 0.9995686 + protein_state + cleaner0 + 2023-07-19T12:46:24Z + DUMMY: + + highly bent + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:04:23Z + + Structures III and IV + + + 0.9996611 + structure_element + cleaner0 + 2023-07-19T14:19:32Z + SO: + + hinge region + + + 0.9994885 + protein_state + cleaner0 + 2023-07-19T12:46:29Z + DUMMY: + + universally conserved + + + 0.4882759 + protein + cleaner0 + 2023-07-19T09:25:14Z + PR: + + uL5 + + + 0.9570417 + structure_element + cleaner0 + 2023-07-19T14:19:38Z + SO: + + C-terminal tail + + + 0.9966792 + protein + cleaner0 + 2023-07-19T09:25:24Z + PR: + + eL42 + + + 0.99959904 + protein_state + cleaner0 + 2023-07-17T08:34:20Z + DUMMY: + + extended + + + 0.5358189 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + complex_assembly + GO: + cleaner0 + 2023-07-18T13:49:58Z + + 60S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.9994918 + protein_state + cleaner0 + 2023-07-19T12:46:35Z + DUMMY: + + bent + + + 0.9995733 + protein_state + cleaner0 + 2023-07-17T08:34:20Z + DUMMY: + + extended + + + 0.9997194 + structure_element + cleaner0 + 2023-07-19T14:16:05Z + SO: + + loop 3 + + + 0.99959445 + structure_element + cleaner0 + 2023-07-19T14:16:12Z + SO: + + variable loop region + + + 0.9992625 + structure_element + cleaner0 + 2023-07-19T14:19:47Z + SO: + + ASL- and mRNA-like parts + + + 0.9064679 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.52141404 + structure_element + cleaner0 + 2023-07-19T14:19:52Z + SO: + + loop + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:04:35Z + + Structures I through IV + + + 0.999429 + experimental_method + cleaner0 + 2023-07-17T08:34:44Z + MESH: + + structural studies + + + 0.985685 + protein_state + cleaner0 + 2023-07-19T12:46:41Z + DUMMY: + + isolated + + + 0.8800568 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.99564946 + experimental_method + cleaner0 + 2023-07-17T08:34:51Z + MESH: + + biochemical studies + + + 0.9996556 + protein_state + cleaner0 + 2023-07-19T12:46:44Z + DUMMY: + + unbound + + + 0.48086995 + site + cleaner0 + 2023-07-14T09:20:11Z + SO: + + IRESs + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.9997057 + structure_element + cleaner0 + 2023-07-19T14:16:05Z + SO: + + loop 3 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T12:48:08Z + + bound in + + + 0.87549275 + complex_assembly + cleaner0 + 2023-07-17T09:02:37Z + GO: + + 40S + + + 0.99951816 + site + cleaner0 + 2023-07-14T09:35:33Z + SO: + + E site + + + 0.99970174 + structure_element + cleaner0 + 2023-07-19T14:16:05Z + SO: + + loop 3 + + + 0.99952507 + structure_element + cleaner0 + 2023-07-19T14:19:57Z + SO: + + codon-like part + + + 0.92538077 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T08:02:48Z + + 6945–6946 + + + 0.9998919 + residue_name_number + cleaner0 + 2023-07-18T14:35:54Z + DUMMY: + + R148 + + + 0.99988735 + residue_name_number + cleaner0 + 2023-07-18T14:34:43Z + DUMMY: + + R157 + + + 0.9996516 + structure_element + cleaner0 + 2023-07-19T14:18:55Z + SO: + + β-hairpin + + + 0.5180033 + protein + cleaner0 + 2023-07-18T14:35:30Z + PR: + + uS7 + + + 0.999694 + structure_element + cleaner0 + 2023-07-19T14:16:05Z + SO: + + loop 3 + + + 0.65433675 + protein + cleaner0 + 2023-07-18T14:35:31Z + PR: + + uS7 + + + 0.9988688 + structure_element + cleaner0 + 2023-07-19T14:21:03Z + SO: + + anticodon-stem loop + + + 0.99718076 + site + cleaner0 + 2023-07-19T10:03:41Z + SO: + + E-site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + 0.9982068 + protein_state + cleaner0 + 2023-07-14T15:27:20Z + DUMMY: + + post-translocation + + + 0.9996589 + complex_assembly + cleaner0 + 2023-07-14T09:44:25Z + GO: + + 80S•2tRNA•mRNA + + + 0.9967224 + evidence + cleaner0 + 2023-07-14T16:19:12Z + DUMMY: + + structure + + + 0.9997008 + structure_element + cleaner0 + 2023-07-19T14:16:05Z + SO: + + loop 3 + + + 0.9987172 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.9985666 + protein_state + cleaner0 + 2023-07-14T15:27:20Z + DUMMY: + + post-translocation + + + 0.999707 + structure_element + cleaner0 + 2023-07-19T14:16:05Z + SO: + + loop 3 + + + 0.8578052 + species + cleaner0 + 2023-07-14T09:25:05Z + MESH: + + CrPV + + + 0.52434605 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + + RESULTS + title_3 + 29581 + eEF2 structures + + 0.9997577 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + 0.9993443 + evidence + cleaner0 + 2023-07-14T16:19:24Z + DUMMY: + + structures + + + + elife-14874-fig5-figsupp1.jpg + fig5s1 + FIG + fig_title_caption + 29597 + Elements of the 80S ribosome that contact eEF2 in Structures I through V. + + 0.9989406 + complex_assembly + cleaner0 + 2023-07-14T09:26:32Z + GO: + + 80S ribosome + + + 0.99974626 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:05:07Z + + Structures I through V + + + + elife-14874-fig5-figsupp1.jpg + fig5s1 + FIG + fig_caption + 29671 + The view and colors are as in Figure 5b: eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. + + 0.9992322 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + site + SO: + cleaner0 + 2023-07-19T13:00:44Z + + IRES + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:17Z + + RNA + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-18T13:46:37Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.9994413 + complex_assembly + cleaner0 + 2023-07-18T13:49:58Z + GO: + + 60S + + + + elife-14874-fig5-figsupp1.jpg + fig5s1 + FIG + fig_caption + 29803 + DOI: +http://dx.doi.org/10.7554/eLife.14874.020 + + + elife-14874-fig5-figsupp2.jpg + fig5s2 + FIG + fig_title_caption + 29850 + Cryo-EM density of the GTPase region in Structures I and II. + + 0.99951786 + experimental_method + cleaner0 + 2023-07-17T08:27:35Z + MESH: + + Cryo-EM + + + 0.9994723 + evidence + cleaner0 + 2023-07-19T14:11:30Z + DUMMY: + + density + + + 0.99432194 + structure_element + cleaner0 + 2023-07-19T14:21:10Z + SO: + + GTPase region + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:05:19Z + + Structures I and II + + + + elife-14874-fig5-figsupp2.jpg + fig5s2 + FIG + fig_caption + 29911 + The switch loop I in Structure I is shown in blue. The putative position of the switch loop I, unresolved in the density of Structure II, is shown with a dashed line. Colors for the ribosome and eEF2 are as in Figure 1. + + 0.99477166 + structure_element + cleaner0 + 2023-07-19T12:12:07Z + SO: + + switch loop I + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:05:46Z + + Structure I + + + 0.99388623 + structure_element + cleaner0 + 2023-07-19T12:12:07Z + SO: + + switch loop I + + + 0.99963343 + evidence + cleaner0 + 2023-07-19T14:11:33Z + DUMMY: + + density + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:05:35Z + + Structure II + + + 0.80866975 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + 0.9986211 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + + elife-14874-fig5-figsupp2.jpg + fig5s2 + FIG + fig_caption + 30131 + DOI: +http://dx.doi.org/10.7554/eLife.14874.021 + + + elife-14874-fig5.jpg + fig5 + FIG + fig_title_caption + 30178 + Conformations and interactions of eEF2. + + 0.9998418 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + + elife-14874-fig5.jpg + fig5 + FIG + fig_caption + 30218 + (a) Conformations of eEF2 in Structures I-V and domain organization of eEF2 are shown. Roman numerals denote eEF2 domains. Superposition was obtained by structural alignment of domains I and II. (b) Elements of the 80S ribosome in Structures I and V that contact eEF2. eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. (c) Comparison of conformations of eEF2•sordarin in Structure I (light green) with those of free apo-eEF2 (magenta) and eEF2•sordarin (teal). (d) Interactions of the GTPase domains with the 40S and 60S subunits in Structure I (colored in green/blue, eEF2; orange, 40S; cyan/teal, 60S) and in Structure II (gray). Switch loop I (SWI) in Structure I is in blue; dashed line shows the putative location of unresolved switch loop I in Structure II. Superposition was obtained by structural alignment of the 25S rRNAs. (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). (h) Cryo-EM density showing the sordarin-binding pocket of eEF2 (Structure II). Sordarin is shown in pink with oxygen atoms in red. + + 0.99985135 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:06:05Z + + Structures I-V + + + 0.99985003 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + 0.9998006 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + 0.873961 + experimental_method + cleaner0 + 2023-07-17T08:35:00Z + MESH: + + Superposition + + + 0.9995562 + experimental_method + cleaner0 + 2023-07-17T08:35:09Z + MESH: + + structural alignment + + + structure_element + SO: + cleaner0 + 2023-07-19T12:06:33Z + + I + + + structure_element + SO: + cleaner0 + 2023-07-19T12:06:43Z + + II + + + 0.9990153 + complex_assembly + cleaner0 + 2023-07-14T09:26:32Z + GO: + + 80S ribosome + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:06:18Z + + Structures I and V + + + 0.9998424 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + 0.9998312 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + site + SO: + cleaner0 + 2023-07-14T09:21:06Z + + IRES + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:17Z + + RNA + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.99963224 + complex_assembly + cleaner0 + 2023-07-18T13:49:58Z + GO: + + 60S + + + 0.9996764 + complex_assembly + cleaner0 + 2023-07-14T10:01:27Z + GO: + + eEF2•sordarin + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:08:13Z + + Structure I + + + 0.99968135 + protein_state + cleaner0 + 2023-07-19T12:47:11Z + DUMMY: + + free + + + 0.99966586 + protein_state + cleaner0 + 2023-07-19T12:47:16Z + DUMMY: + + apo + + + 0.9998547 + protein + cleaner0 + 2023-07-14T09:30:45Z + PR: + + eEF2 + + + 0.99971074 + complex_assembly + cleaner0 + 2023-07-14T10:01:24Z + GO: + + eEF2•sordarin + + + 0.8062828 + structure_element + cleaner0 + 2023-07-19T14:21:16Z + SO: + + GTPase domains + + + 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2023-07-17T08:27:35Z + MESH: + + Cryo-EM + + + 0.89887357 + evidence + cleaner0 + 2023-07-19T14:11:39Z + DUMMY: + + density + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:24Z + + guanosine diphosphate + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T12:48:06Z + + bound in + + + 0.9806194 + site + cleaner0 + 2023-07-19T10:05:19Z + SO: + + GTPase center + + + 0.9995697 + structure_element + cleaner0 + 2023-07-14T09:47:34Z + SO: + + sarcin-ricin loop + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:28:14Z + + 25S rRNA + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:08:28Z + + Structure II + + + 0.9995969 + site + cleaner0 + 2023-07-19T10:05:23Z + SO: + + sordarin-binding sites + + + 0.9994927 + protein_state + cleaner0 + 2023-07-14T09:33:14Z + DUMMY: + + ribosome-bound + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:08:41Z + + Structure II + + + 0.99986005 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.9989149 + experimental_method + cleaner0 + 2023-07-17T08:27:35Z + MESH: + + Cryo-EM + + + 0.78451294 + evidence + cleaner0 + 2023-07-19T14:11:42Z + DUMMY: + + density + + + 0.99963415 + site + cleaner0 + 2023-07-19T10:05:27Z + SO: + + sordarin-binding pocket + + + 0.99985826 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:08:55Z + + Structure II + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:55Z + + Sordarin + + + + elife-14874-fig5.jpg + fig5 + FIG + fig_caption + 31716 + DOI: +http://dx.doi.org/10.7554/eLife.14874.019 + + + RESULTS + paragraph + 31763 + Elongation factor eEF2 in all five structures is bound with GDP and sordarin (Figure 5). The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation. In post-translocation-like 80S•tRNA•eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site. GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex. + + 0.999284 + protein_type + cleaner0 + 2023-07-19T09:17:40Z + MESH: + + Elongation factor + + + 0.99977976 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.99828666 + evidence + cleaner0 + 2023-07-14T16:19:24Z + DUMMY: + + structures + + + 0.999545 + protein_state + cleaner0 + 2023-07-17T08:30:36Z + DUMMY: + + bound with + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:39Z + + GDP + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:53Z + + sordarin + + + 0.99952257 + protein_type + cleaner0 + 2023-07-19T09:17:41Z + MESH: + + elongation factor + + + 0.99952376 + structure_element + cleaner0 + 2023-07-19T14:21:20Z + SO: + + superdomains + + + structure_element + SO: + cleaner0 + 2023-07-19T12:23:38Z + + superdomain + + + structure_element + SO: + cleaner0 + 2023-07-19T14:21:49Z + + G (GTPase) domain + + + structure_element + SO: + cleaner0 + 2023-07-19T12:09:24Z + + I + + + structure_element + SO: + cleaner0 + 2023-07-19T12:09:39Z + + II + + + structure_element + SO: + cleaner0 + 2023-07-19T08:01:13Z + + linker domain III + + + 0.99970895 + structure_element + cleaner0 + 2023-07-19T12:23:38Z + SO: + + superdomain + + + structure_element + SO: + cleaner0 + 2023-07-19T12:09:59Z + + IV + + + structure_element + SO: + cleaner0 + 2023-07-19T12:10:07Z + + V + + + structure_element + SO: + cleaner0 + 2023-07-19T13:38:54Z + + IV + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:34Z + + body + + + 0.9998116 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.99928755 + protein + cleaner0 + 2023-07-14T09:36:12Z + PR: + + EF-G + + + 0.98917866 + ptm + cleaner0 + 2023-07-19T14:34:33Z + MESH: + + ADP-ribosylation + + + 0.9998412 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.98412645 + structure_element + cleaner0 + 2023-07-19T14:21:54Z + SO: + + IV + + + 0.99782866 + experimental_method + cleaner0 + 2023-07-17T08:36:38Z + MESH: + + deletion + + + 0.9721094 + structure_element + cleaner0 + 2023-07-19T14:22:01Z + SO: + + IV + + + 0.9992158 + protein + cleaner0 + 2023-07-14T09:36:12Z + PR: + + EF-G + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:27:20Z + + post-translocation + + + 0.99970853 + complex_assembly + cleaner0 + 2023-07-14T10:02:51Z + GO: + + 80S•tRNA•eEF2 + + + 0.98174274 + structure_element + cleaner0 + 2023-07-19T14:22:05Z + SO: + + IV + + + complex_assembly + GO: + cleaner0 + 2023-07-14T10:03:24Z + + 40S + + + site + SO: + cleaner0 + 2023-07-14T09:28:51Z + + A site + + + structure_element + SO: + cleaner0 + 2023-07-19T12:10:51Z + + IV + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:21Z + + tRNA + + + 0.9992967 + site + cleaner0 + 2023-07-17T08:57:29Z + SO: + + A to P site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:40Z + + GDP + + + 0.99895847 + evidence + cleaner0 + 2023-07-14T16:19:24Z + DUMMY: + + structures + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T12:48:08Z + + bound in + + + 0.99005324 + site + cleaner0 + 2023-07-19T10:05:32Z + SO: + + GTPase center + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:55Z + + sordarin + + + 0.9994995 + structure_element + cleaner0 + 2023-07-19T14:22:11Z + SO: + + β-platforms + + + structure_element + SO: + cleaner0 + 2023-07-19T12:10:25Z + + III + + + structure_element + SO: + cleaner0 + 2023-07-19T12:10:34Z + + V + + + 0.9964886 + evidence + cleaner0 + 2023-07-14T16:19:12Z + DUMMY: + + structure + + + 0.99968815 + protein_state + cleaner0 + 2023-07-19T12:48:32Z + DUMMY: + + free + + + 0.99971986 + complex_assembly + cleaner0 + 2023-07-14T10:01:27Z + GO: + + eEF2•sordarin + + + + RESULTS + paragraph + 32707 + The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD ≤ 2 Å), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2—source data 1). From Structure I to V, eEF2 is rigidly attached to the GTPase-associated center of the 60S subunit. The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). The sarcin-ricin loop interacts with the GTP-binding site of eEF2 (Figures 5d and f). While the overall mode of this interaction is similar to that seen in 70S•EF-G crystal structures, there is an important local difference between Structure I and Structures II-V in switch loop I, as discussed below. + + 0.99981755 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.997101 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.8492427 + evidence + cleaner0 + 2023-07-19T14:11:48Z + DUMMY: + + RMSD + + + 0.9998332 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:11:08Z + + Structure I to V + + + 0.9997104 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.9939383 + site + cleaner0 + 2023-07-19T10:05:40Z + SO: + + GTPase-associated center + + + complex_assembly + GO: + cleaner0 + 2023-07-18T13:49:58Z + + 60S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.99371576 + site + cleaner0 + 2023-07-19T10:05:43Z + SO: + + GTPase-associated center + + + 0.9997053 + structure_element + cleaner0 + 2023-07-19T12:11:57Z + SO: + + P stalk + + + 0.9997775 + structure_element + cleaner0 + 2023-07-19T14:22:17Z + SO: + + L11 + + + 0.9998105 + structure_element + cleaner0 + 2023-07-19T14:22:20Z + SO: + + L7 + + + 0.99622995 + structure_element + cleaner0 + 2023-07-19T14:22:24Z + SO: + + L12 + + + 0.90342546 + structure_element + cleaner0 + 2023-07-19T14:22:42Z + SO: + + stalk + + + 0.9993674 + taxonomy_domain + cleaner0 + 2023-07-17T08:49:56Z + DUMMY: + + bacteria + + + 0.9996973 + structure_element + cleaner0 + 2023-07-14T09:47:34Z + SO: + + sarcin-ricin loop + + + 0.9998029 + structure_element + cleaner0 + 2023-07-14T09:47:41Z + SO: + + SRL + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T07:59:59Z + + 3012–3042 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:28:14Z + + 25S rRNA + + + structure_element + SO: + cleaner0 + 2023-07-19T14:23:08Z + + helices 43 and 44 + + + 0.9997042 + structure_element + cleaner0 + 2023-07-19T12:11:59Z + SO: + + P stalk + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T08:00:15Z + + G1242 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T08:00:29Z + + A1270 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:52Z + + stack + + + 0.99989116 + residue_name_number + cleaner0 + 2023-07-18T14:36:23Z + DUMMY: + + V754 + + + 0.9998895 + residue_name_number + cleaner0 + 2023-07-18T14:36:29Z + DUMMY: + + Y744 + + + 0.99813145 + structure_element + cleaner0 + 2023-07-19T14:23:13Z + SO: + + V + + + 0.9997227 + structure_element + cleaner0 + 2023-07-19T14:23:17Z + SO: + + αββ motif + + + 0.94464266 + taxonomy_domain + cleaner0 + 2023-07-17T08:50:28Z + DUMMY: + + eukaryote + + + 0.9955135 + protein + cleaner0 + 2023-07-19T09:25:36Z + PR: + + P0 + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T07:59:23Z + + 126–154 + + + structure_element + SO: + cleaner0 + 2023-07-19T14:23:39Z + + long α-helix D + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T07:59:37Z + + 172–188 + + + 0.99969864 + structure_element + cleaner0 + 2023-07-19T14:23:44Z + SO: + + GTPase domain + + + 0.99960715 + structure_element + cleaner0 + 2023-07-19T14:23:48Z + SO: + + β-sheet region + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T07:31:21Z + + 246–263 + + + 0.9996789 + structure_element + cleaner0 + 2023-07-19T14:23:54Z + SO: + + GTPase domain insert + + + 0.9996882 + structure_element + cleaner0 + 2023-07-19T14:23:57Z + SO: + + G’ insert + + + 0.9998374 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.99983907 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.99964243 + protein + cleaner0 + 2023-07-14T09:36:12Z + PR: + + EF-G + + + 0.9937736 + structure_element + cleaner0 + 2023-07-19T14:24:01Z + SO: + + P/L11 stalk + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:11:20Z + + Structure I to V + + + 0.99692535 + evidence + cleaner0 + 2023-07-19T14:11:52Z + DUMMY: + + root-mean-square differences + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:28:14Z + + 25S rRNA + + + 0.9997188 + structure_element + cleaner0 + 2023-07-19T12:11:59Z + SO: + + P stalk + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T07:31:08Z + + 1223–1286 + + + 0.99971396 + complex_assembly + cleaner0 + 2023-07-14T09:40:46Z + GO: + + 80S•IRES + + + 0.99948514 + protein_state + cleaner0 + 2023-07-14T09:55:35Z + DUMMY: + + absence of + + + 0.9996878 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.9997269 + complex_assembly + cleaner0 + 2023-07-14T09:44:25Z + GO: + + 80S•2tRNA•mRNA + + + 0.9996971 + structure_element + cleaner0 + 2023-07-19T12:12:00Z + SO: + + P stalk + + + 0.99947333 + site + cleaner0 + 2023-07-14T09:28:51Z + SO: + + A site + + + 0.9996922 + structure_element + cleaner0 + 2023-07-14T09:47:34Z + SO: + + sarcin-ricin loop + + + 0.99954873 + site + cleaner0 + 2023-07-19T10:05:49Z + SO: + + GTP-binding site + + + 0.99984443 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.9997264 + complex_assembly + cleaner0 + 2023-07-14T09:39:50Z + GO: + + 70S•EF-G + + + 0.99957466 + evidence + cleaner0 + 2023-07-17T08:45:17Z + DUMMY: + + crystal structures + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:11:31Z + + Structure I + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:11:42Z + + Structures II-V + + + 0.9861694 + structure_element + cleaner0 + 2023-07-19T12:12:06Z + SO: + + switch loop I + + + + elife-14874-fig6-figsupp1.jpg + fig6s1 + FIG + fig_title_caption + 34340 + Repositioning (sliding) of the positively-charged cluster of domain IV of eEF2 over the phosphate backbone (red) of the 18S helices 33 and 34. + + site + SO: + cleaner0 + 2023-07-19T12:12:44Z + + positively-charged cluster + + + structure_element + SO: + cleaner0 + 2023-07-19T12:12:31Z + + IV + + + 0.99980754 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.99446136 + structure_element + cleaner0 + 2023-07-19T14:24:11Z + SO: + + 18S helices 33 and 34 + + + + elife-14874-fig6-figsupp1.jpg + fig6s1 + FIG + fig_caption + 34483 + Structures I through V are shown. Electrostatic surface of eEF2 is shown; negatively and positively charged regions are shown in red and blue, respectively. The view was obtained by structural alignment of the 18S rRNAs. + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:12:56Z + + Structures I through V + + + 0.99984944 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.9995648 + experimental_method + cleaner0 + 2023-07-17T08:35:10Z + MESH: + + structural alignment + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:32:52Z + + 18S rRNAs + + + + elife-14874-fig6-figsupp1.jpg + fig6s1 + FIG + fig_caption + 34704 + DOI: +http://dx.doi.org/10.7554/eLife.14874.023 + + + elife-14874-fig6.jpg + fig6 + FIG + fig_title_caption + 34751 + Interactions of eEF2 with the 40S subunit. + + 0.99976 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:37Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + + elife-14874-fig6.jpg + fig6 + FIG + fig_caption + 34794 + (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. The view was obtained by superpositions of the body domains of 18S rRNAs. (c) Rearrangements, from Structure I through V, of a positively charged cluster of eEF2 (K613, R617 and R631) positioned over the phosphate backbone of 18S helices 33 and 34, suggesting a role of electrostatic interactions in eEF2 diffusion over the 40S surface. (d) Shift of the tip of domain III of eEF2, interacting with uS12 upon reverse subunit rotation from Structure I to Structure V. Structure I colored as in Figure 1, except uS12, which is in purple; Structure V is in gray. + + 0.9998191 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.99936455 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:13:13Z + + Structure I + + + 0.9998184 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.99891543 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.9991998 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:13:23Z + + Structures II through V + + + 0.99656504 + complex_assembly + cleaner0 + 2023-07-17T09:02:37Z + GO: + + 40S + + + 0.9998185 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.9998473 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.9523571 + complex_assembly + cleaner0 + 2023-07-17T09:02:37Z + GO: + + 40S + + + 0.9768433 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:14:08Z + + Structure I through V + + + 0.99959284 + site + cleaner0 + 2023-07-19T10:05:56Z + SO: + + A-site + + + 0.9998487 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.9996791 + experimental_method + cleaner0 + 2023-07-17T08:33:35Z + MESH: + + superpositions + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:34Z + + body + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:32:52Z + + 18S rRNAs + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:14:18Z + + Structure I through V + + + 0.9998417 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.99987113 + residue_name_number + cleaner0 + 2023-07-18T14:36:39Z + DUMMY: + + K613 + + + 0.99987674 + residue_name_number + cleaner0 + 2023-07-18T14:36:52Z + DUMMY: + + R617 + + + 0.99987566 + residue_name_number + cleaner0 + 2023-07-18T14:37:00Z + DUMMY: + + R631 + + + structure_element + SO: + cleaner0 + 2023-07-19T14:24:31Z + + 18S helices 33 and 34 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:52Z + + electrostatic interactions + + + 0.9998312 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.96340984 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-19T12:14:53Z + + III + + + 0.9998385 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.99918574 + protein + cleaner0 + 2023-07-18T14:37:08Z + PR: + + uS12 + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:13:56Z + + Structure I to Structure V + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:14:39Z + + Structure I + + + 0.9986628 + protein + cleaner0 + 2023-07-18T14:37:07Z + PR: + + uS12 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + + elife-14874-fig6.jpg + fig6 + FIG + fig_caption + 35792 + DOI: +http://dx.doi.org/10.7554/eLife.14874.022 + + + RESULTS + paragraph + 35839 + There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50 Å of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c), eEF2 undergoes moderate repositioning of domain IV (~3 Å; Figure 5a) and domain III (~5 Å; Figure 6d). This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit. Domain IV of eEF2 binds at the 40S A site in Structures I to V but the mode of interaction differs in each complex (Figure 6). Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. eEF2 settles into the A site from Structure I to V, as the tip of domain IV shifts by ~10 Å relative to the body and by ~20 Å relative to the swiveling head. Modest intra-eEF2 shifts of domain IV between Structures I to V outline a stochastic trajectory (Figure 5a), consistent with local adjustments of the domain in the A site. At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). From Structure I to V, these central domains migrate by ~10 Å along the 40S surface (Figure 6c). Comparison of eEF2 conformations reveals that in Structure V, domain III is displaced as a result of interaction with uS12, as discussed below. + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:15:18Z + + Structures I and V + + + 0.99964416 + protein_state + cleaner0 + 2023-07-19T12:48:46Z + DUMMY: + + free + + + 0.9998368 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.9996706 + structure_element + cleaner0 + 2023-07-19T12:23:38Z + SO: + + superdomain + + + 0.9996793 + structure_element + cleaner0 + 2023-07-19T12:23:38Z + SO: + + superdomain + + + 0.99982363 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.51207805 + structure_element + cleaner0 + 2023-07-19T14:24:43Z + SO: + + IV + + + 0.987593 + structure_element + cleaner0 + 2023-07-19T14:24:46Z + SO: + + III + + + 0.99947435 + protein_state + cleaner0 + 2023-07-14T09:33:14Z + DUMMY: + + ribosome-bound + + + 0.99983263 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.999838 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + 0.9995493 + structure_element + cleaner0 + 2023-07-19T14:24:50Z + SO: + + superdomains + + + 0.8187614 + structure_element + cleaner0 + 2023-07-19T14:24:54Z + SO: + + I + + + 0.91360235 + structure_element + cleaner0 + 2023-07-19T14:24:56Z + SO: + + V + + + site + SO: + cleaner0 + 2023-07-19T10:06:19Z + + GTPase-associated center + + + 0.8994949 + structure_element + cleaner0 + 2023-07-14T09:49:05Z + SO: + + large subunit + + + structure_element + SO: + cleaner0 + 2023-07-19T12:15:38Z + + IV + + + 0.99981886 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + complex_assembly + GO: + cleaner0 + 2023-07-14T10:05:52Z + + 40S + + + site + SO: + cleaner0 + 2023-07-14T09:28:52Z + + A site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:15:58Z + + Structures I to V + + + 0.99981755 + protein + cleaner0 + 2023-07-14T09:30:46Z + PR: + + eEF2 + + + complex_assembly + GO: + cleaner0 + 2023-07-18T13:49:58Z + + 60S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-19T14:25:14Z + + IV + + + 0.999347 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:15:50Z + + Structures I and V + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:48Z + + head + + + 0.99980146 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.9995426 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:16:10Z + + Structure I to V + + + 0.7244747 + structure_element + cleaner0 + 2023-07-19T14:25:21Z + SO: + + IV + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:34Z + + body + + + 0.9873011 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + protein + PR: + cleaner0 + 2023-07-14T09:30:47Z + + eEF2 + + + 0.54072547 + structure_element + cleaner0 + 2023-07-19T14:25:25Z + SO: + + IV + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:16:22Z + + Structures I to V + + + 0.99956334 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.99982905 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + structure_element + SO: + cleaner0 + 2023-07-19T12:16:54Z + + II + + + structure_element + SO: + cleaner0 + 2023-07-19T12:17:02Z + + III + + + 0.9813007 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + 0.86465216 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T07:30:15Z + + 48–52 + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T07:30:28Z + + 429–432 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:33:20Z + + 18S rRNA + + + 0.99123967 + structure_element + cleaner0 + 2023-07-19T14:25:31Z + SO: + + helix 5 + + + 0.8519885 + protein + cleaner0 + 2023-07-18T14:37:08Z + PR: + + uS12 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:16:36Z + + Structure I to V + + + 0.77772486 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + 0.9997981 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.6926696 + structure_element + cleaner0 + 2023-07-19T14:25:37Z + SO: + + III + + + 0.9610733 + protein + cleaner0 + 2023-07-18T14:37:08Z + PR: + + uS12 + + + + RESULTS + paragraph + 37466 + In summary, between Structures I and V, a step-wise translocation of PKI by ~15 Å from the A to P site - within the 40S subunit – occurs simultaneously with the ~11 Å side-way entry of domain IV into the A site coupled with ~3 to 5 Å inter-domain rearrangements in eEF2. These shifts occur during the reverse rotation of the 40S body coupled with the forward-then-reverse head swivel. To elucidate the detailed structural mechanism of IRES translocation and the roles of eEF2 and ribosome rearrangements, we describe in the following sections the interactions of PKI and eEF2 with the ribosomal A and P sites in Structures I through V (Figure 2g; see also Figure 1—figure supplement 1). + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:17:28Z + + Structures I and V + + + 0.99981135 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.9982888 + site + cleaner0 + 2023-07-17T08:57:29Z + SO: + + A to P site + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.95864165 + structure_element + cleaner0 + 2023-07-19T12:17:47Z + SO: + + IV + + + 0.99957335 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.9997912 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.9582871 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + 0.4921384 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:48Z + + head + + + 0.73164666 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + 0.99975234 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:32:56Z + + ribosome + + + 0.9998122 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.99979895 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.9992882 + site + cleaner0 + 2023-07-19T10:06:35Z + SO: + + A and P sites + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:17:40Z + + Structures I through V + + + + RESULTS + title_2 + 38166 + Structure I represents a pre-translocation IRES and initial entry of eEF2 in a GTP-like state + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:18:01Z + + Structure I + + + 0.97620755 + protein_state + cleaner0 + 2023-07-14T15:24:43Z + DUMMY: + + pre-translocation + + + 0.72210205 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + 0.9996119 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:12:27Z + + GTP + + + + RESULTS + paragraph + 38260 + In the fully rotated Structure I, PKI is shifted toward the P site by ~3 Å relative to its position in the initiation complex but maintains interactions with the partially swiveled head. At the head, C1274 of the 18S rRNA (C1054 in E. coli) base pairs with the first nucleotide of the ORF immediately downstream of PKI. The C1274:G6953 base pair provides a stacking platform for the codon-anticodon–like helix of PKI. We therefore define C1274 as the foundation of the 'head A site'. Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. + + 0.99953103 + protein_state + cleaner0 + 2023-07-19T12:49:02Z + DUMMY: + + fully rotated + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:18:12Z + + Structure I + + + 0.99581677 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + 0.99951875 + site + cleaner0 + 2023-07-19T10:06:39Z + SO: + + P site + + + 0.96036226 + complex_assembly + cleaner0 + 2023-07-18T14:08:05Z + GO: + + initiation complex + + + 0.9994416 + protein_state + cleaner0 + 2023-07-19T12:49:09Z + DUMMY: + + partially swiveled + + + 0.7870612 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.979624 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:18Z + + C1274 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:33:20Z + + 18S rRNA + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:31Z + + C1054 + + + 0.9993178 + species + cleaner0 + 2023-07-14T09:31:43Z + MESH: + + E. coli + + + 0.6925956 + structure_element + cleaner0 + 2023-07-19T09:44:51Z + SO: + + ORF + + + 0.8231169 + structure_element + cleaner0 + 2023-07-14T09:27:40Z + SO: + + PKI + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:19Z + + C1274 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:44Z + + G6953 + + + site + SO: + melaniev@ebi.ac.uk + 2023-07-21T13:08:33Z + + stacking platform + + + 0.9618395 + structure_element + cleaner0 + 2023-07-19T14:25:43Z + SO: + + codon-anticodon–like helix + + + 0.9990872 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:19Z + + C1274 + + + 0.9973279 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.9724035 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:57Z + + U1191 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:29:09Z + + G966 + + + 0.999213 + species + cleaner0 + 2023-07-14T09:31:43Z + MESH: + + E. coli + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:29:20Z + + C1637 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:29:35Z + + C1400 + + + 0.999264 + species + cleaner0 + 2023-07-14T09:31:43Z + MESH: + + E. coli + + + 0.9984804 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.9994645 + site + cleaner0 + 2023-07-19T10:06:43Z + SO: + + P site + + + 0.99849963 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + 0.9975149 + site + cleaner0 + 2023-07-19T10:06:46Z + SO: + + P site + + + 0.99953794 + protein_state + cleaner0 + 2023-07-17T08:37:56Z + DUMMY: + + fully translocated + + + structure_element + SO: + cleaner0 + 2023-07-19T13:05:39Z + + mRNA-tRNA helix + + + 0.99953336 + protein_state + cleaner0 + 2023-07-14T09:48:16Z + DUMMY: + + tRNA-bound + + + 0.99887365 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.9613529 + protein_state + cleaner0 + 2023-07-14T15:27:20Z + DUMMY: + + post-translocation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + + elife-14874-fig7.jpg + fig7 + FIG + fig_title_caption + 39092 + Interactions of the residues at the eEF2 tip with the decoding center of the IRES-bound ribosome. + + 0.99824035 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.9988792 + site + cleaner0 + 2023-07-18T14:50:01Z + SO: + + decoding center + + + 0.99951357 + protein_state + cleaner0 + 2023-07-14T10:07:05Z + DUMMY: + + IRES-bound + + + 0.9953264 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + + elife-14874-fig7.jpg + fig7 + FIG + fig_caption + 39190 + Key elements of the decoding center of the 'locked' initiation structure, 'unlocked' Structure I, and post-translocation Structure V (this work) are shown. The histidine-diphthamide tip of eEF2 is shown in green. The codon-anticodon-like helix of PKI is shown in red, the downstream first codon of the ORF in magenta. Nucleotides of the 18S rRNA body are in orange and head in yellow; 25S rRNA nucleotide A2256 is blue. A and P sites are schematically demarcated by dotted lines. + + 0.9964236 + site + cleaner0 + 2023-07-18T14:50:01Z + SO: + + decoding center + + + 0.9996582 + protein_state + cleaner0 + 2023-07-19T12:49:16Z + DUMMY: + + locked + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + evidence + DUMMY: + cleaner0 + 2023-07-14T16:19:13Z + + structure + + + 0.99964154 + protein_state + cleaner0 + 2023-07-19T12:49:20Z + DUMMY: + + unlocked + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:18:36Z + + Structure I + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:27:20Z + + post-translocation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + site + SO: + cleaner0 + 2023-07-19T10:10:33Z + + histidine-diphthamide tip + + + 0.999668 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.9995654 + structure_element + cleaner0 + 2023-07-19T14:13:13Z + SO: + + codon-anticodon-like helix + + + 0.9991404 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.48675555 + structure_element + cleaner0 + 2023-07-19T09:44:51Z + SO: + + ORF + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:33:20Z + + 18S rRNA + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:34Z + + body + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:48Z + + head + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:28:14Z + + 25S rRNA + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:26:31Z + + A2256 + + + 0.9995314 + site + cleaner0 + 2023-07-19T10:07:00Z + SO: + + A and P sites + + + + elife-14874-fig7.jpg + fig7 + FIG + fig_caption + 39670 + DOI: +http://dx.doi.org/10.7554/eLife.14874.024 + + + RESULTS + paragraph + 39717 + The interaction of PKI with the 40S body is substantially rearranged relative to that in the initiation state. In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes. In Structure I, PKI does not contact these nucleotides (Figures 2g and 7). + + 0.9996917 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:32Z + + body + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + 0.99935657 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.99939984 + protein_state + cleaner0 + 2023-07-19T12:49:27Z + DUMMY: + + universally conserved + + + 0.99936384 + site + cleaner0 + 2023-07-19T10:07:08Z + SO: + + decoding-center + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:26:45Z + + G577 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:26:58Z + + A1755 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:12Z + + A1756 + + + structure_element + SO: + cleaner0 + 2023-07-18T14:13:59Z + + body + + + site + SO: + cleaner0 + 2023-07-18T14:15:40Z + + A site + + + site + SO: + cleaner0 + 2023-07-17T08:51:14Z + + A-site + + + 0.99787134 + protein_state + cleaner0 + 2023-07-14T10:07:26Z + DUMMY: + + tRNA bound + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:27:45Z + + Structure I + + + 0.9993703 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + + RESULTS + paragraph + 40089 + The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). Domain IV is partially engaged with the body A site. The tip of domain IV is wedged between PKI and decoding-center nucleotides A1755 and A1756, which are bulged out of h44. This tip contains the histidine-diphthamide triad (H583, H694 and Diph699), which interacts with the codon-anticodon-like helix of PKI and A1756 (Figure 7). Histidines 583 and 694 interact with the phosphate backbone of the anticodon-like strand (at G6907 and C6908). Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-Å long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1. The trimethylamino end of Diph699 packs over A1756 (Figure 7). The opposite surface of the tail is oriented toward the minor-groove side of the second base pair of the codon-anticodon helix (G6906:C6951). Thus, in comparison with the initiation state, the histidine-diphthamide tip of eEF2 replaces the codon-anticodon–like helix of PKI. The splitting of the interaction of A1755-A1756 and PKI is achieved by providing the histidine-diphthamine tip as a binding partner for both A1756 and the minor groove of the codon-anticodon helix (Figure 7). + + 0.9997521 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-18T13:46:37Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:19:45Z + + Structure I + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:27:59Z + + Structures II to V + + + 0.98683816 + protein_type + cleaner0 + 2023-07-17T08:38:45Z + MESH: + + translocase + + + 0.989749 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + 0.73024505 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + 0.8404604 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.9073289 + structure_element + cleaner0 + 2023-07-19T14:25:51Z + SO: + + IV + + + structure_element + SO: + cleaner0 + 2023-07-18T14:13:59Z + + body + + + site + SO: + cleaner0 + 2023-07-18T14:15:22Z + + A site + + + structure_element + SO: + cleaner0 + 2023-07-19T12:19:04Z + + IV + + + 0.9950157 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + site + SO: + cleaner0 + 2023-07-19T13:42:01Z + + decoding-center + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:00Z + + A1755 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:13Z + + A1756 + + + 0.9980547 + site + cleaner0 + 2023-07-19T10:07:21Z + SO: + + histidine-diphthamide triad + + + 0.99989474 + residue_name_number + cleaner0 + 2023-07-18T14:40:23Z + DUMMY: + + H583 + + + 0.99989736 + residue_name_number + cleaner0 + 2023-07-18T14:40:28Z + DUMMY: + + H694 + + + 0.9998921 + ptm + cleaner0 + 2023-07-18T14:38:53Z + MESH: + + Diph699 + + + 0.99952817 + structure_element + cleaner0 + 2023-07-19T14:13:13Z + SO: + + codon-anticodon-like helix + + + 0.99750704 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:13Z + + A1756 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-18T14:40:59Z + + Histidines 583 and 694 + + + 0.99910843 + structure_element + cleaner0 + 2023-07-19T14:26:01Z + SO: + + anticodon-like strand + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:49Z + + G6907 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:35Z + + C6908 + + + 0.8746058 + ptm + cleaner0 + 2023-07-18T14:09:05Z + MESH: + + Diphthamide + + + 0.9992817 + protein_state + cleaner0 + 2023-07-19T12:49:31Z + DUMMY: + + conserved + + + 0.9993942 + taxonomy_domain + cleaner0 + 2023-07-17T08:51:41Z + DUMMY: + + archaeal + + + 0.9991135 + taxonomy_domain + cleaner0 + 2023-07-14T09:35:56Z + DUMMY: + + eukaryotic + + + 0.99899644 + protein + cleaner0 + 2023-07-19T09:25:45Z + PR: + + EF2 + + + 0.99668854 + residue_number + cleaner0 + 2023-07-19T14:35:12Z + DUMMY: + + 699 + + + 0.99941987 + species + cleaner0 + 2023-07-14T10:07:55Z + MESH: + + S. cerevisiae + + + 0.99890375 + residue_name + cleaner0 + 2023-07-19T09:16:47Z + SO: + + histidine + + + 0.99988616 + ptm + cleaner0 + 2023-07-18T14:40:10Z + MESH: + + Diph699 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:13Z + + A1756 + + + 0.92978686 + site + cleaner0 + 2023-07-19T10:07:45Z + SO: + + minor-groove + + + 0.9991538 + structure_element + cleaner0 + 2023-07-19T14:26:24Z + SO: + + codon-anticodon helix + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T10:28:28Z + + G6906 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T10:28:40Z + + C6951 + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + site + SO: + cleaner0 + 2023-07-19T10:10:33Z + + histidine-diphthamide tip + + + 0.99968684 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.99952126 + structure_element + cleaner0 + 2023-07-19T14:26:31Z + SO: + + codon-anticodon–like helix + + + 0.9989303 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:01Z + + A1755 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:13Z + + A1756 + + + 0.99835086 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + site + SO: + cleaner0 + 2023-07-19T10:30:01Z + + histidine-diphthamine tip + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:13Z + + A1756 + + + 0.9950962 + site + cleaner0 + 2023-07-19T10:02:15Z + SO: + + minor groove + + + 0.9993161 + structure_element + cleaner0 + 2023-07-19T14:26:26Z + SO: + + codon-anticodon helix + + + + RESULTS + paragraph + 41573 + Unlike in Structures II to V, the conformation of the eEF2 GTPase center in Structure I resembles that of a GTP-bound translocase (Figure 5e). In translational GTPases, switch loops I and II are involved in the GTPase activity (reviewed in). Switch loop II (aa 105–110), which carries the catalytic H108 (H92 in E. coli EF-G; is well resolved in all five structures. The histidine resides next to the backbone of G3028 of the sarcin-ricin loop and near the diphosphate of GDP (Figure 5e). By contrast, switch loop I (aa 50–70 in S. cerevisiae eEF2) is resolved only in Structure I (Figure 5—figure supplement 2). The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). Bulged A416 interacts with the switch loop in the vicinity of D53. Next to GDP, the C-terminal part of the switch loop (aa 61–67) adopts a helical fold. As such, the conformations of SWI and the GTPase center in general are similar to those observed in ribosome-bound EF-Tu and EF-G in the presence of GTP analogs. + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:29:09Z + + Structures II to V + + + 0.9998616 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.99855995 + site + cleaner0 + 2023-07-19T10:07:49Z + SO: + + GTPase center + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:29:20Z + + Structure I + + + 0.9995056 + protein_state + cleaner0 + 2023-07-17T08:40:56Z + DUMMY: + + GTP-bound + + + 0.9988091 + protein_type + cleaner0 + 2023-07-17T08:38:45Z + MESH: + + translocase + + + 0.98535705 + protein_type + cleaner0 + 2023-07-17T08:43:31Z + MESH: + + translational GTPases + + + 0.9974408 + structure_element + cleaner0 + 2023-07-19T14:26:36Z + SO: + + switch loops I and II + + + protein_type + MESH: + cleaner0 + 2023-07-19T09:20:23Z + + GTPase + + + 0.99858505 + structure_element + cleaner0 + 2023-07-19T14:26:40Z + SO: + + Switch loop II + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T09:18:58Z + + 105–110 + + + 0.99701595 + protein_state + cleaner0 + 2023-07-19T12:49:39Z + DUMMY: + + catalytic + + + 0.99989295 + residue_name_number + cleaner0 + 2023-07-18T14:42:00Z + DUMMY: + + H108 + + + 0.99989355 + residue_name_number + cleaner0 + 2023-07-18T14:42:07Z + DUMMY: + + H92 + + + 0.99941176 + species + cleaner0 + 2023-07-14T09:31:43Z + MESH: + + E. coli + + + 0.9996832 + protein + cleaner0 + 2023-07-14T09:36:12Z + PR: + + EF-G + + + 0.99816775 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.9992586 + residue_name + cleaner0 + 2023-07-19T09:16:52Z + SO: + + histidine + + + 0.9999068 + residue_name_number + cleaner0 + 2023-07-18T14:42:18Z + DUMMY: + + G3028 + + + 0.99961376 + structure_element + cleaner0 + 2023-07-14T09:47:34Z + SO: + + sarcin-ricin loop + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:40Z + + GDP + + + 0.99871475 + structure_element + cleaner0 + 2023-07-19T12:12:07Z + SO: + + switch loop I + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T09:19:09Z + + 50–70 + + + 0.9994602 + species + cleaner0 + 2023-07-14T10:07:58Z + MESH: + + S. cerevisiae + + + 0.9998534 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:29:34Z + + Structure I + + + 0.99975973 + structure_element + cleaner0 + 2023-07-19T14:26:46Z + SO: + + loop + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T09:19:20Z + + 50–60 + + + 0.99965966 + structure_element + cleaner0 + 2023-07-19T14:26:53Z + SO: + + helix 14 + + + structure_element + SO: + cleaner0 + 2023-07-19T14:32:32Z + + 415CAAA418 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:33:20Z + + 18S rRNA + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-18T13:46:37Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.9996563 + structure_element + cleaner0 + 2023-07-19T14:27:09Z + SO: + + helix A + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T09:19:32Z + + 32–42 + + + 0.9998455 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.9982673 + protein_state + cleaner0 + 2023-07-19T12:49:44Z + DUMMY: + + Bulged + + + 0.9999093 + residue_name_number + cleaner0 + 2023-07-18T14:42:27Z + DUMMY: + + A416 + + + 0.99888873 + structure_element + cleaner0 + 2023-07-19T14:27:16Z + SO: + + switch loop + + + 0.99990916 + residue_name_number + cleaner0 + 2023-07-18T14:42:33Z + DUMMY: + + D53 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:40Z + + GDP + + + 0.9993894 + structure_element + cleaner0 + 2023-07-19T14:27:20Z + SO: + + switch loop + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T09:19:42Z + + 61–67 + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T09:20:59Z + + helical fold + + + 0.9956304 + structure_element + cleaner0 + 2023-07-19T09:20:10Z + SO: + + SWI + + + 0.99862015 + site + cleaner0 + 2023-07-19T10:07:54Z + SO: + + GTPase center + + + 0.9995088 + protein_state + cleaner0 + 2023-07-14T09:33:14Z + DUMMY: + + ribosome-bound + + + 0.9996894 + protein + cleaner0 + 2023-07-19T09:25:59Z + PR: + + EF-Tu + + + 0.9996955 + protein + cleaner0 + 2023-07-14T09:36:12Z + PR: + + EF-G + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T09:55:43Z + + presence of + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:12:27Z + + GTP + + + + RESULTS + title_2 + 42690 + Structure II reveals PKI between the body A and P sites and eEF2 partially advanced into the A site + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:30:38Z + + Structure II + + + 0.7873689 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.99036425 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + 0.8719973 + site + cleaner0 + 2023-07-19T10:07:58Z + SO: + + A and P sites + + + 0.99751306 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.99937135 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + + RESULTS + paragraph + 42790 + In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4 Å toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site. Thus, the intermediate position of PKI is possible due to a large swivel of the head relative to the body, which brings the head A site close to the body P site. + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:30:50Z + + Structure II + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:31:02Z + + Structure I + + + 0.9993813 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:34Z + + body + + + 0.9995806 + site + cleaner0 + 2023-07-19T10:08:03Z + SO: + + P site + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:52Z + + stacking + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:19Z + + C1274 + + + 0.98957705 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.840852 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.99969447 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.9964796 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.94224715 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + 0.99084234 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.989069 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.9223027 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + 0.99935853 + site + cleaner0 + 2023-07-19T10:08:07Z + SO: + + P site + + + + RESULTS + paragraph + 43142 + Domain IV of eEF2 is further entrenched in the A site by ~3 Å relative to the body and ~8 Å relative to the head, preserving its interactions with PKI. The decoding center residues A1755 and A1756 are rearranged to pack inside helix 44, making room for eEF2. This conformation of decoding center residues is also observed in the absence of A-site ligands. The head interface of domain IV interacts with the 40S head (Figure 6a). Here, a positively charged surface of eEF2, formed by K613, R617 and R631 contacts the phosphate backbone of helix 33 (Figures 6c; see also Figure 6—figure supplement 1). + + structure_element + SO: + cleaner0 + 2023-07-19T10:31:40Z + + IV + + + 0.9998313 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.9995843 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.8641417 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + 0.99907506 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.9995809 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.99852085 + site + cleaner0 + 2023-07-18T14:50:02Z + SO: + + decoding center + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:01Z + + A1755 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:14Z + + A1756 + + + 0.9996426 + structure_element + cleaner0 + 2023-07-19T14:27:24Z + SO: + + helix 44 + + + 0.9998375 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.9980835 + site + cleaner0 + 2023-07-18T14:50:02Z + SO: + + decoding center + + + 0.9994034 + protein_state + cleaner0 + 2023-07-14T09:55:35Z + DUMMY: + + absence of + + + 0.99924415 + site + cleaner0 + 2023-07-19T10:08:18Z + SO: + + A-site + + + 0.999521 + site + cleaner0 + 2023-07-19T10:08:35Z + SO: + + head interface + + + structure_element + SO: + cleaner0 + 2023-07-19T10:31:24Z + + IV + + + 0.94184136 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + 0.9168235 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.9993641 + site + cleaner0 + 2023-07-19T10:08:44Z + SO: + + positively charged surface + + + 0.9998305 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.999892 + residue_name_number + cleaner0 + 2023-07-18T14:36:41Z + DUMMY: + + K613 + + + 0.9998951 + residue_name_number + cleaner0 + 2023-07-18T14:36:54Z + DUMMY: + + R617 + + + 0.9998938 + residue_name_number + cleaner0 + 2023-07-18T14:37:01Z + DUMMY: + + R631 + + + 0.99963105 + structure_element + cleaner0 + 2023-07-19T14:27:29Z + SO: + + helix 33 + + + + RESULTS + title_2 + 43751 + Structure III represents a highly bent IRES with PKI captured between the head A and P sites + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:31:59Z + + Structure III + + + 0.9995066 + protein_state + cleaner0 + 2023-07-19T12:49:51Z + DUMMY: + + highly bent + + + 0.9398213 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + 0.99536395 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.8582808 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.84949577 + site + cleaner0 + 2023-07-19T10:08:48Z + SO: + + A and P sites + + + + RESULTS + paragraph + 43844 + Consistent with the similar head swivels in Structure III and Structure II, relative positions of the 40S head A site and body P site remain as in Structure II. Among the five structures, the PKI domain is least ordered in Structure III and lacks density for SL3. The map allows placement of PKI at the body P site (Figure 1—figure supplement 3). Thus, in Structure III, PKI has translocated along the 40S body, but the head remains fully swiveled so that PKI is between the head A and P sites. Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191). The position of eEF2 is similar to that in Structure II. + + 0.9960608 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:32:10Z + + Structure III + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:32:22Z + + Structure II + + + 0.9972505 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T14:13:01Z + + head + + + site + SO: + cleaner0 + 2023-07-18T14:13:11Z + + A site + + + structure_element + SO: + cleaner0 + 2023-07-18T14:13:27Z + + body + + + site + SO: + cleaner0 + 2023-07-18T14:13:39Z + + P site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:32:34Z + + Structure II + + + 0.99680847 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.99970204 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:32:46Z + + Structure III + + + 0.9980464 + evidence + cleaner0 + 2023-07-19T14:11:58Z + DUMMY: + + density + + + 0.99959975 + structure_element + cleaner0 + 2023-07-19T14:16:56Z + SO: + + SL3 + + + 0.9996086 + evidence + cleaner0 + 2023-07-19T14:12:03Z + DUMMY: + + map + + + 0.9989035 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + structure_element + SO: + cleaner0 + 2023-07-18T14:13:59Z + + body + + + site + SO: + cleaner0 + 2023-07-18T14:14:31Z + + P site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:32:59Z + + Structure III + + + 0.9979552 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.9871639 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + 0.83239126 + structure_element + cleaner0 + 2023-07-18T14:09:34Z + SO: + + body + + + 0.98784155 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.9990567 + protein_state + cleaner0 + 2023-07-19T12:49:55Z + DUMMY: + + fully swiveled + + + 0.99851257 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.96826124 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.99928623 + site + cleaner0 + 2023-07-19T10:08:58Z + SO: + + A and P sites + + + 0.99964607 + evidence + cleaner0 + 2023-07-19T14:12:08Z + DUMMY: + + map + + + 0.99689925 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + structure_element + SO: + cleaner0 + 2023-07-18T14:13:58Z + + body + + + site + SO: + cleaner0 + 2023-07-18T14:14:13Z + + P site + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T09:55:35Z + + absence of + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:52Z + + stacking + + + structure_element + SO: + cleaner0 + 2023-07-18T14:14:38Z + + head + + + site + SO: + cleaner0 + 2023-07-18T14:14:57Z + + A site + + + 0.9998996 + residue_name_number + cleaner0 + 2023-07-18T14:33:17Z + DUMMY: + + C1274 + + + 0.9994837 + site + cleaner0 + 2023-07-19T10:09:03Z + SO: + + P site + + + 0.9998987 + residue_name_number + cleaner0 + 2023-07-18T14:33:25Z + DUMMY: + + U1191 + + + 0.9988851 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:33:12Z + + Structure II + + + + RESULTS + title_2 + 44614 + Structure IV represents a highly bent IRES with PKI partially accommodated in the P site + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:33:24Z + + Structure IV + + + 0.9995568 + protein_state + cleaner0 + 2023-07-19T12:49:58Z + DUMMY: + + highly bent + + + 0.721137 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + 0.95081747 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.99952155 + site + cleaner0 + 2023-07-19T10:09:09Z + SO: + + P site + + + + RESULTS + paragraph + 44703 + In Structure IV, the 40S subunit is almost non-rotated relative to the 60S subunit, and the 40S head is mid-swiveled. Unwinding of the head moves the head P-site residue U1191 and body P-site residue C1637 closer together, resulting in a partially restored 40S P site. Whereas C1637 forms a stacking platform for the last base pair of PKI, U1191 does not yet stack on PKI because the head remains partially swiveled. This renders PKI partially accommodated in the P site (Figure 2g). + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:33:35Z + + Structure IV + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-18T13:46:37Z + + subunit + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T12:29:42Z + + non-rotated + + + 0.9996226 + complex_assembly + cleaner0 + 2023-07-18T13:49:58Z + GO: + + 60S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.99892324 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + 0.99740845 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + protein_state + DUMMY: + cleaner0 + 2023-07-18T13:58:05Z + + mid-swiveled + + + 0.99835205 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.9910733 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.9992142 + site + cleaner0 + 2023-07-14T09:32:36Z + SO: + + P-site + + + 0.99990165 + residue_name_number + cleaner0 + 2023-07-18T14:33:25Z + DUMMY: + + U1191 + + + 0.9396259 + structure_element + cleaner0 + 2023-07-18T14:09:35Z + SO: + + body + + + 0.9993933 + site + cleaner0 + 2023-07-14T09:32:36Z + SO: + + P-site + + + 0.99990094 + residue_name_number + cleaner0 + 2023-07-18T14:34:03Z + DUMMY: + + C1637 + + + 0.9982626 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + 0.9994377 + site + cleaner0 + 2023-07-19T10:09:14Z + SO: + + P site + + + 0.9998981 + residue_name_number + cleaner0 + 2023-07-18T14:34:03Z + DUMMY: + + C1637 + + + 0.99662864 + site + cleaner0 + 2023-07-19T10:09:33Z + SO: + + stacking platform + + + 0.7164754 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.9998995 + residue_name_number + cleaner0 + 2023-07-18T14:33:25Z + DUMMY: + + U1191 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:52Z + + stack + + + 0.7873554 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.9956104 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.99063313 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.9995845 + site + cleaner0 + 2023-07-19T10:09:45Z + SO: + + P site + + + + RESULTS + paragraph + 45187 + Unwinding of the 40S head also positions the head A site closer to the body A site. This results in rearrangements of eEF2 interactions with the head, allowing eEF2 to advance further into the A site. To this end, the head-interacting interface of domain IV slides along the surface of the head by 5 Å. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6—figure supplement 1). + + 0.99870026 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + 0.9922685 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.93732303 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.99877226 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.918403 + structure_element + cleaner0 + 2023-07-18T14:09:35Z + SO: + + body + + + 0.9943743 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.9997671 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.9647755 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.99979585 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.9995562 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.9996025 + site + cleaner0 + 2023-07-18T14:16:53Z + SO: + + head-interacting interface + + + structure_element + SO: + cleaner0 + 2023-07-19T10:34:10Z + + IV + + + 0.9610157 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.99934053 + structure_element + cleaner0 + 2023-07-19T14:27:35Z + SO: + + Helix A + + + structure_element + SO: + cleaner0 + 2023-07-19T10:33:57Z + + IV + + + 0.98424226 + structure_element + cleaner0 + 2023-07-19T14:27:40Z + SO: + + h34 + + + 0.99989235 + residue_name_number + cleaner0 + 2023-07-18T14:36:41Z + DUMMY: + + K613 + + + 0.9998957 + residue_name_number + cleaner0 + 2023-07-18T14:36:54Z + DUMMY: + + R617 + + + 0.99989617 + residue_name_number + cleaner0 + 2023-07-18T14:37:01Z + DUMMY: + + R631 + + + 0.5743242 + structure_element + cleaner0 + 2023-07-19T14:27:44Z + SO: + + h33 + + + + RESULTS + title_2 + 45700 + Structure V represents an extended IRES with PKI fully accommodated in the P site and domain IV of eEF2 in the A site + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.99963784 + protein_state + cleaner0 + 2023-07-17T08:34:20Z + DUMMY: + + extended + + + 0.95457494 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + 0.79774344 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.9995582 + site + cleaner0 + 2023-07-19T10:09:49Z + SO: + + P site + + + structure_element + SO: + cleaner0 + 2023-07-19T12:20:23Z + + IV + + + 0.9996544 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.9995189 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + + RESULTS + paragraph + 45818 + In the nearly non-rotated and non-swiveled ribosome conformation in Structure V closely resembling that of the post-translocation 80S•2tRNA•mRNA complex, PKI is fully accommodated in the P site. The codon-anticodon–like helix is stacked on P-site residues U1191 and C1637 (Figure 3d), analogous to stacking of the tRNA-mRNA helix (Figure 3e). + + 0.99578774 + protein_state + cleaner0 + 2023-07-19T12:56:07Z + DUMMY: + + nearly non-rotated + + + 0.99908614 + protein_state + cleaner0 + 2023-07-19T12:29:59Z + DUMMY: + + non-swiveled + + + 0.9979869 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:27:20Z + + post-translocation + + + 0.9996662 + complex_assembly + cleaner0 + 2023-07-14T09:44:25Z + GO: + + 80S•2tRNA•mRNA + + + 0.9804216 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.9995103 + site + cleaner0 + 2023-07-19T10:09:54Z + SO: + + P site + + + 0.9994028 + structure_element + cleaner0 + 2023-07-19T13:43:35Z + SO: + + codon-anticodon–like helix + + + 0.99938446 + site + cleaner0 + 2023-07-14T09:32:36Z + SO: + + P-site + + + 0.9999014 + residue_name_number + cleaner0 + 2023-07-18T14:33:25Z + DUMMY: + + U1191 + + + 0.999902 + residue_name_number + cleaner0 + 2023-07-18T14:34:03Z + DUMMY: + + C1637 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:52Z + + stacking + + + 0.8938687 + complex_assembly + cleaner0 + 2023-07-14T09:36:32Z + GO: + + tRNA-mRNA + + + 0.99245244 + structure_element + cleaner0 + 2023-07-19T14:27:55Z + SO: + + helix + + + + RESULTS + paragraph + 46167 + A notable conformational change in eEF2 from that in the preceding Structures is visible in the position of domain III, which contacts uS12 (Figure 6d). In Structure V, protein uS12 is shifted along with the 40S body as a result of intersubunit rotation. In this position, uS12 forms extensive interactions with eEF2 domains II and III. Specifically, the C-terminal tail of uS12 packs against the β-barrel of domain II, while the β-barrel of uS12 packs against helix A of domain III. This shifts the tip of helix A of domain III (at aa 500) by ~5 Å (relative to that in Structure I) toward domain I. Although domain III remains in contact with domain V, the shift occurs in the direction that could eventually disconnect the β-platforms of these domains. + + 0.9998037 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + 0.98563147 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + Structures + + + structure_element + SO: + cleaner0 + 2023-07-19T10:34:55Z + + III + + + 0.9951474 + protein + cleaner0 + 2023-07-18T14:37:08Z + PR: + + uS12 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.99933136 + protein + cleaner0 + 2023-07-18T14:37:08Z + PR: + + uS12 + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:35Z + + body + + + 0.99940455 + protein + cleaner0 + 2023-07-18T14:37:08Z + PR: + + uS12 + + + 0.9998267 + protein + cleaner0 + 2023-07-14T09:30:47Z + PR: + + eEF2 + + + structure_element + SO: + cleaner0 + 2023-07-19T10:35:12Z + + II + + + structure_element + SO: + cleaner0 + 2023-07-19T10:35:25Z + + III + + + 0.99852663 + structure_element + cleaner0 + 2023-07-19T14:19:39Z + SO: + + C-terminal tail + + + 0.99970776 + protein + cleaner0 + 2023-07-18T14:37:08Z + PR: + + uS12 + + + 0.9995521 + structure_element + cleaner0 + 2023-07-19T14:28:02Z + SO: + + β-barrel + + + structure_element + SO: + cleaner0 + 2023-07-19T10:35:55Z + + II + + + 0.9995248 + structure_element + cleaner0 + 2023-07-19T14:28:06Z + SO: + + β-barrel + + + 0.9996666 + protein + cleaner0 + 2023-07-18T14:37:08Z + PR: + + uS12 + + + 0.99963236 + structure_element + cleaner0 + 2023-07-19T14:28:10Z + SO: + + helix A + + + structure_element + SO: + cleaner0 + 2023-07-19T10:35:40Z + + III + + + 0.9996503 + structure_element + cleaner0 + 2023-07-19T14:28:15Z + SO: + + helix A + + + 0.99836904 + structure_element + cleaner0 + 2023-07-19T14:28:22Z + SO: + + III + + + residue_number + DUMMY: + cleaner0 + 2023-07-19T14:28:49Z + + 500 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:36:06Z + + Structure I + + + structure_element + SO: + cleaner0 + 2023-07-19T10:36:21Z + + I + + + structure_element + SO: + cleaner0 + 2023-07-19T10:36:35Z + + III + + + 0.99888307 + structure_element + cleaner0 + 2023-07-19T14:28:30Z + SO: + + V + + + 0.99947006 + structure_element + cleaner0 + 2023-07-19T14:28:57Z + SO: + + β-platforms + + + + RESULTS + paragraph + 46931 + Domain IV of eEF2 is fully accommodated in the A site. The first codon of the open reading frame is also positioned in the A site, with bases exposed toward eEF2 (Figure 7), resembling the conformations of the A-site codons in EF-G-bound 70S complexes. As in the preceding Structures, the histidine-diphthamide tip is bound in the minor groove of the P-site codon-anticodon helix. Diph699 slightly rearranges, relative to that in Structure I (Figure 7), and interacts with four out of six codon-anticodon nucleotides. The imidazole moiety stacks on G6907 (corresponding to nt 36 in the tRNA anticodon) and hydrogen bonds with O2’ of G6906 (nt 35 of tRNA). The amide at the diphthamide end interacts with N2 of G6906 and O2 and O2’ of C6951 (corresponding to nt 2 of the codon). The trimethylamino-group is positioned over the ribose of C6952 (codon nt 3). + + structure_element + SO: + cleaner0 + 2023-07-19T14:29:18Z + + IV + + + 0.9998129 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.99953854 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.69675964 + structure_element + cleaner0 + 2023-07-19T09:59:13Z + SO: + + open reading frame + + + 0.9995544 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.99983454 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.99938416 + site + cleaner0 + 2023-07-19T10:10:00Z + SO: + + A-site + + + 0.99941826 + protein_state + cleaner0 + 2023-07-14T10:09:41Z + DUMMY: + + EF-G-bound + + + complex_assembly + GO: + cleaner0 + 2023-07-19T09:26:43Z + + 70S + + + 0.7774972 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + Structures + + + site + SO: + cleaner0 + 2023-07-19T10:10:31Z + + histidine-diphthamide tip + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T12:48:08Z + + bound in + + + 0.9989767 + site + cleaner0 + 2023-07-19T10:02:15Z + SO: + + minor groove + + + 0.99924606 + site + cleaner0 + 2023-07-14T09:32:36Z + SO: + + P-site + + + 0.91299975 + structure_element + cleaner0 + 2023-07-19T14:26:26Z + SO: + + codon-anticodon helix + + + 0.9998822 + ptm + cleaner0 + 2023-07-18T14:40:10Z + MESH: + + Diph699 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:36:52Z + + Structure I + + + 0.9998859 + residue_name_number + cleaner0 + 2023-07-18T14:41:09Z + DUMMY: + + G6907 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:22Z + + tRNA + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:52Z + + hydrogen bonds + + + 0.999887 + residue_name_number + cleaner0 + 2023-07-18T14:41:35Z + DUMMY: + + G6906 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:22Z + + tRNA + + + 0.99800164 + ptm + cleaner0 + 2023-07-19T10:11:05Z + MESH: + + diphthamide + + + 0.9998913 + residue_name_number + cleaner0 + 2023-07-18T14:41:35Z + DUMMY: + + G6906 + + + 0.99988925 + residue_name_number + cleaner0 + 2023-07-18T14:41:48Z + DUMMY: + + C6951 + + + 0.9998864 + residue_name_number + cleaner0 + 2023-07-18T14:42:45Z + DUMMY: + + C6952 + + + + DISCUSS + title_1 + 47791 + Discussion + + + DISCUSS + title_2 + 47802 + IRES translocation mechanism + + 0.9654568 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + + .jpg + media1 + FIG + fig_title_caption + 47831 + Animation showing the transition from the initiation 80S•TSV IRES structures (Koh et al., 2014) to eEF2-bound Structures I through V (this work). + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:45:12Z + + 80S•TSV IRES + + + 0.99882346 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.9995427 + protein_state + cleaner0 + 2023-07-14T10:10:09Z + DUMMY: + + eEF2-bound + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:20:52Z + + Structures I through V + + + + .jpg + media1 + FIG + fig_caption + 47979 + Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. Colors are as in Figure 1. In scenes 1, 2 and 3, nucleotides C1274, U1191 of the 40S head and G904 of the 40S platform are shown in black to denote the A, P and E sites, respectively. In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange. + + 0.54897505 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + complex_assembly + GO: + cleaner0 + 2023-07-14T10:10:44Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-14T10:10:52Z + + head + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:13Z + + subunit + + + 0.9994383 + site + cleaner0 + 2023-07-18T14:50:02Z + SO: + + decoding center + + + 0.9994782 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.9995713 + site + cleaner0 + 2023-07-19T10:11:58Z + SO: + + P site + + + 0.9998884 + residue_name_number + cleaner0 + 2023-07-18T14:33:17Z + DUMMY: + + C1274 + + + 0.9998902 + residue_name_number + cleaner0 + 2023-07-18T14:33:25Z + DUMMY: + + U1191 + + + 0.9773158 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + 0.52976966 + structure_element + cleaner0 + 2023-07-17T08:56:48Z + SO: + + head + + + 0.9998908 + residue_name_number + cleaner0 + 2023-07-18T14:33:31Z + DUMMY: + + G904 + + + 0.90101135 + site + cleaner0 + 2023-07-18T14:18:01Z + SO: + + 40S platform + + + 0.9991433 + site + cleaner0 + 2023-07-17T08:59:41Z + SO: + + A, P and E sites + + + 0.9998864 + residue_name_number + cleaner0 + 2023-07-18T14:33:17Z + DUMMY: + + C1274 + + + 0.99988925 + residue_name_number + cleaner0 + 2023-07-18T14:33:25Z + DUMMY: + + U1191 + + + 0.9998865 + residue_name_number + cleaner0 + 2023-07-18T14:32:04Z + DUMMY: + + G577 + + + 0.9998846 + residue_name_number + cleaner0 + 2023-07-18T14:32:13Z + DUMMY: + + A1755 + + + 0.9998816 + residue_name_number + cleaner0 + 2023-07-18T14:32:20Z + DUMMY: + + A1756 + + + 0.8844175 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + 0.9850679 + structure_element + cleaner0 + 2023-07-18T14:09:35Z + SO: + + body + + + 0.9992986 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.9998901 + residue_name_number + cleaner0 + 2023-07-18T14:34:03Z + DUMMY: + + C1637 + + + 0.8927989 + structure_element + cleaner0 + 2023-07-18T14:09:35Z + SO: + + body + + + 0.997367 + site + cleaner0 + 2023-07-19T10:12:04Z + SO: + + P site + + + + .jpg + media1 + FIG + fig_caption + 48776 + DOI: +http://dx.doi.org/10.7554/eLife.14874.025 + + + DISCUSS + paragraph + 48823 + In this work we have captured the structures of the TSV IRES, whose PKI samples positions between the A and P sites (Structures I–IV), as well as in the P site (Structure V). We propose that together with the previously reported initiation state, these structures represent the trajectory of eEF2-induced IRES translocation (shown as an animation in http://labs.umassmed.edu/korostelevlab/msc/iresmovie.gif and Video 1). Our structures reveal previously unseen intermediate states of eEF2 or EF-G engagement with the A site, providing the structural basis for the mechanism of translocase action. Furthermore, they provide insight into the mechanism of eEF2•GTP association with the pre-translocation ribosome and eEF2•GDP dissociation from the post-translocation ribosome, also delineating the mechanism of translation inhibition by the antifungal drug sordarin. In summary, the reported ensemble of structures substantially enhances our understanding of the translocation mechanism, including that of tRNAs as discussed below. + + 0.99793327 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.9551537 + species + cleaner0 + 2023-07-14T09:24:19Z + MESH: + + TSV + + + 0.80141485 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + 0.80973506 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.9995227 + site + cleaner0 + 2023-07-19T10:12:08Z + SO: + + A and P sites + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:15:05Z + + Structures I–IV + + + 0.99955225 + site + cleaner0 + 2023-07-19T10:13:51Z + SO: + + P site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:20Z + + Structure V + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + 0.9376766 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.96496916 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + site + SO: + cleaner0 + 2023-07-14T09:21:06Z + + IRES + + + 0.99713254 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.99933076 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.95569724 + protein + cleaner0 + 2023-07-14T09:36:12Z + PR: + + EF-G + + + 0.99951375 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + 0.9086211 + protein_type + cleaner0 + 2023-07-17T08:38:45Z + MESH: + + translocase + + + 0.99914217 + complex_assembly + cleaner0 + 2023-07-14T09:31:05Z + GO: + + eEF2•GTP + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:24:43Z + + pre-translocation + + + 0.95648843 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + 0.9993141 + complex_assembly + cleaner0 + 2023-07-14T15:19:52Z + GO: + + eEF2•GDP + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:27:20Z + + post-translocation + + + 0.9844319 + complex_assembly + cleaner0 + 2023-07-14T09:32:56Z + GO: + + ribosome + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:55Z + + sordarin + + + 0.98899066 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:06Z + + tRNAs + + + + DISCUSS + paragraph + 49858 + Translocation of the TSV IRES on the 40S subunit globally resembles a step of an inchworm (Figure 4; see also Figure 3—figure supplement 2). At the start (initiation state), the IRES adopts an extended conformation (extended inchworm). The front 'legs' (SL4 and SL5) of the 5’-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2). PKI, representing the hind end, is bound in the A site. In the first sub-step (Structures I to IV), the hind end advances from the A to the P site and approaches the front end, which remains attached to the 40S surface. This shortens the distance between PKI and SL4 by up to 20 Å relative to the initiating IRES structure, resulting in a bent IRES conformation (bent inchworm). Finally (Structures IV to V), as the hind end is accommodated in the P site, the front 'legs' advance by departing from their initial binding sites. This converts the IRES into an extended conformation, rendering the inchworm prepared for the next translocation step. Notably, at all steps, the head of the IRES inchworm (L1.1 region) is supported by the mobile L1 stalk. In the post-translocation CrPV IRES structure, the 5’-domain similarly protrudes between the subunits and interacts with the L1 stalk, as in the initiation state for this IRES. This underlines structural similarity for the TSV and CrPV IRES translocation mechanisms. + + 0.9863063 + species + cleaner0 + 2023-07-14T09:24:20Z + MESH: + + TSV + + + 0.5959895 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:14Z + + subunit + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T10:13:00Z + + inchworm + + + 0.5983167 + protein_state + cleaner0 + 2023-07-17T08:39:11Z + DUMMY: + + initiation + + + 0.7090151 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:34:20Z + + extended + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T10:13:19Z + + extended inchworm + + + 0.9987333 + structure_element + cleaner0 + 2023-07-19T14:29:28Z + SO: + + front 'legs + + + 0.99978346 + structure_element + cleaner0 + 2023-07-19T14:17:21Z + SO: + + SL4 + + + 0.99977344 + structure_element + cleaner0 + 2023-07-19T14:17:27Z + SO: + + SL5 + + + 0.9996127 + structure_element + cleaner0 + 2023-07-19T14:29:33Z + SO: + + 5’-domain + + + 0.9726136 + structure_element + cleaner0 + 2023-07-19T14:29:41Z + SO: + + front end + + + 0.996906 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + 0.9940593 + protein + cleaner0 + 2023-07-18T14:35:31Z + PR: + + uS7 + + + 0.99744236 + protein + cleaner0 + 2023-07-18T14:35:38Z + PR: + + uS11 + + + 0.9980939 + protein + cleaner0 + 2023-07-18T14:35:44Z + PR: + + eS25 + + + 0.65140986 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.9880641 + structure_element + cleaner0 + 2023-07-19T14:29:50Z + SO: + + hind end + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T12:48:08Z + + bound in + + + 0.9993156 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:44Z + + Structures I to IV + + + 0.98847455 + structure_element + cleaner0 + 2023-07-19T14:29:52Z + SO: + + hind end + + + site + SO: + cleaner0 + 2023-07-19T10:15:58Z + + A to the P site + + + 0.9903552 + structure_element + cleaner0 + 2023-07-19T14:29:43Z + SO: + + front end + + + 0.997926 + complex_assembly + cleaner0 + 2023-07-17T09:02:38Z + GO: + + 40S + + + 0.756115 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.9997739 + structure_element + cleaner0 + 2023-07-19T14:17:21Z + SO: + + SL4 + + + 0.4570263 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + 0.9970169 + evidence + cleaner0 + 2023-07-14T16:19:14Z + DUMMY: + + structure + + + 0.99966156 + protein_state + cleaner0 + 2023-07-19T12:50:20Z + DUMMY: + + bent + + + 0.50760794 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T10:13:36Z + + bent inchworm + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:15:27Z + + Structures IV to V + + + 0.9929627 + structure_element + cleaner0 + 2023-07-19T14:29:52Z + SO: + + hind end + + + 0.9993958 + site + cleaner0 + 2023-07-19T10:16:03Z + SO: + + P site + + + 0.9455021 + structure_element + cleaner0 + 2023-07-19T14:30:19Z + SO: + + front 'legs' + + + 0.9993908 + site + cleaner0 + 2023-07-19T10:16:32Z + SO: + + initial binding sites + + + 0.75255346 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:34:20Z + + extended + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T10:13:02Z + + inchworm + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + 0.7248495 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T10:13:02Z + + inchworm + + + 0.9987351 + structure_element + cleaner0 + 2023-07-19T12:21:20Z + SO: + + L1.1 region + + + 0.9996345 + protein_state + cleaner0 + 2023-07-19T12:50:29Z + DUMMY: + + mobile + + + 0.999406 + structure_element + cleaner0 + 2023-07-19T12:21:13Z + SO: + + L1 stalk + + + 0.982758 + protein_state + cleaner0 + 2023-07-14T15:27:20Z + DUMMY: + + post-translocation + + + 0.97469056 + species + cleaner0 + 2023-07-14T09:25:05Z + MESH: + + CrPV + + + 0.37083906 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + 0.99950397 + evidence + cleaner0 + 2023-07-14T16:19:14Z + DUMMY: + + structure + + + 0.99961686 + structure_element + cleaner0 + 2023-07-19T14:30:27Z + SO: + + 5’-domain + + + 0.9983759 + structure_element + cleaner0 + 2023-07-19T12:21:14Z + SO: + + L1 stalk + + + 0.9272192 + protein_state + cleaner0 + 2023-07-17T08:39:11Z + DUMMY: + + initiation + + + 0.44835773 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + 0.99326384 + species + cleaner0 + 2023-07-14T09:24:20Z + MESH: + + TSV + + + 0.9852445 + species + cleaner0 + 2023-07-14T09:25:05Z + MESH: + + CrPV + + + 0.41263622 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + + DISCUSS + paragraph + 51272 + Upon translocation, the GCU start codon is positioned in the A site (Structure V), ready for interaction with Ala-tRNAAla upon eEF2 departure. Recent studies have shown that in some cases a fraction of IGR IRES-driven translation results from an alternative reading frame, which is shifted by one nucleotide relative to the normal ORF. One of the mechanistic scenarios (discussed in) involves binding of the first aminoacyl-tRNA to the post-translocated IRES mRNA frame shifted by one nucleotide (predominantly a +1 frame shift). In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs. It is likely that alternative frame setting occurs following eEF2 release and that this depends on transient displacement of the start codon in the decoding center, allowing binding of the corresponding amino acyl-tRNA to an off-frame codon. Further structural studies involving 80S•IRES•tRNA complexes are necessary to understand the mechanisms underlying alternative reading frame selection. + + 0.99959457 + site + cleaner0 + 2023-07-14T09:28:52Z + SO: + + A site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:44:46Z + + Ala-tRNAAla + + + 0.9994394 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.752359 + structure_element + cleaner0 + 2023-07-14T09:26:12Z + SO: + + IGR + + + 0.59038293 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + structure_element + SO: + cleaner0 + 2023-07-19T09:44:51Z + + ORF + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:46Z + + aminoacyl-tRNA + + + 0.9929593 + protein_state + cleaner0 + 2023-07-17T08:37:34Z + DUMMY: + + post-translocated + + + 0.51287735 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:04Z + + mRNA + + + 0.9989116 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.8084409 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + site + SO: + cleaner0 + 2023-07-18T14:50:02Z + + decoding center + + + 0.9994006 + protein_state + cleaner0 + 2023-07-17T08:37:48Z + DUMMY: + + pre-translocated + + + 0.99937904 + protein_state + cleaner0 + 2023-07-17T08:37:55Z + DUMMY: + + fully translocated + + + 0.49451753 + structure_element + cleaner0 + 2023-07-19T09:44:49Z + SO: + + ORF + + + structure_element + SO: + cleaner0 + 2023-07-19T09:44:51Z + + ORF + + + 0.9986228 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.6783173 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:31Z + + mRNAs + + + 0.9995797 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.99145734 + site + cleaner0 + 2023-07-18T14:50:02Z + SO: + + decoding center + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:45:24Z + + amino acyl-tRNA + + + 0.9933733 + experimental_method + cleaner0 + 2023-07-17T08:36:43Z + MESH: + + structural studies + + + 0.99899083 + complex_assembly + cleaner0 + 2023-07-19T09:26:49Z + GO: + + 80S•IRES•tRNA + + + + DISCUSS + paragraph + 52439 + The presence of several translocation complexes in a single sample suggests that the structures represent equilibrium states of forward and reverse translocation of the IRES, which interconvert among each other. This is consistent with the observations that the intergenic IRESs are prone to reverse translocation. Specifically, biochemical toe-printing studies in the presence of eEF2•GTP identified IRES in a non-translocated position unless eEF1a•aa-tRNA is also present. These findings indicate that IRES translocation by eEF2 is futile: the IRES returns to the A site upon releasing eEF2•GDP unless an amino-acyl tRNA enters the A site and blocks IRES back-translocation. This contrasts with the post-translocated 2tRNA•mRNA complex, in which the classical P and E-site tRNAs are stabilized in the non-rotated ribosome after translocase release. Thus, the meta-stability of the post-translocation IRES is likely due to the absence of stabilizing structural features present in the 2tRNA•mRNA complex. In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. Because the anticodon-stem loop of the A-tRNA is sufficient for translocation completion, we ascribe the meta-stability of the post-translocation IRES to the absence of the P/E-tRNA elements, either the ASL or the acceptor arm, or both. Furthermore, interactions of SL4 and SL5 with the 40S subunit likely contribute to stabilization of pre-translocation structures. + + protein_state + DUMMY: + cleaner0 + 2023-07-14T09:55:43Z + + presence of + + + 0.99862504 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.9799646 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + 0.60521096 + site + cleaner0 + 2023-07-14T09:20:11Z + SO: + + IRESs + + + 0.99808633 + experimental_method + cleaner0 + 2023-07-17T08:36:56Z + MESH: + + biochemical toe-printing studies + + + 0.9920868 + protein_state + cleaner0 + 2023-07-14T09:55:43Z + DUMMY: + + presence of + + + 0.9993641 + complex_assembly + cleaner0 + 2023-07-14T09:31:05Z + GO: + + eEF2•GTP + + + 0.93958384 + site + cleaner0 + 2023-07-14T09:21:06Z + SO: + + IRES + + + 0.999259 + protein_state + cleaner0 + 2023-07-17T08:38:06Z + DUMMY: + + non-translocated + + + 0.9734882 + complex_assembly + cleaner0 + 2023-07-14T15:19:32Z + GO: + + eEF1a•aa-tRNA + + 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2023-07-19T12:29:42Z + DUMMY: + + non-rotated + + + 0.99886465 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + protein_type + MESH: + cleaner0 + 2023-07-17T08:38:45Z + + translocase + + + 0.9989495 + protein_state + cleaner0 + 2023-07-14T15:27:20Z + DUMMY: + + post-translocation + + + 0.9693661 + site + cleaner0 + 2023-07-14T09:21:07Z + SO: + + IRES + + + 0.9974084 + protein_state + cleaner0 + 2023-07-14T09:55:35Z + DUMMY: + + absence of + + + 0.99960643 + complex_assembly + cleaner0 + 2023-07-14T09:36:40Z + GO: + + 2tRNA•mRNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + 0.98280346 + site + cleaner0 + 2023-07-14T09:21:07Z + SO: + + IRES + + + 0.998801 + protein_state + cleaner0 + 2023-07-14T15:24:43Z + DUMMY: + + pre-translocation + + + 0.9994915 + complex_assembly + cleaner0 + 2023-07-14T09:36:40Z + GO: + + 2tRNA•mRNA + + + site + SO: + cleaner0 + 2023-07-19T13:09:38Z + + A/P + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:22Z + + tRNA + + + 0.9990821 + structure_element + cleaner0 + 2023-07-19T14:30:33Z + SO: + + anticodon-stem loop + + + 0.9661361 + structure_element + cleaner0 + 2023-07-19T14:19:19Z + SO: + + elbow + + + 0.9994403 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + site + SO: + cleaner0 + 2023-07-19T13:10:12Z + + P/E + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:22Z + + tRNA + + + 0.7541472 + structure_element + cleaner0 + 2023-07-19T14:19:19Z + SO: + + elbow + + + 0.99874556 + structure_element + cleaner0 + 2023-07-19T12:21:14Z + SO: + + L1 stalk + + + 0.9993862 + structure_element + cleaner0 + 2023-07-19T14:30:44Z + SO: + + anticodon-stem loop + + + site + SO: + cleaner0 + 2023-07-19T13:10:42Z + + A + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:22Z + + tRNA + + + 0.99891806 + protein_state + cleaner0 + 2023-07-14T15:27:20Z + DUMMY: + + post-translocation + + + 0.9638772 + site + cleaner0 + 2023-07-14T09:21:07Z + SO: + + IRES + + + 0.99940073 + protein_state + cleaner0 + 2023-07-14T09:55:35Z + DUMMY: + + absence of + + + site + SO: + cleaner0 + 2023-07-19T13:11:10Z + + P/E + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:22Z + + tRNA + + + 0.99958044 + structure_element + cleaner0 + 2023-07-14T09:34:56Z + SO: + + ASL + + + 0.99979967 + structure_element + cleaner0 + 2023-07-19T14:17:21Z + SO: + + SL4 + + + 0.9997967 + structure_element + cleaner0 + 2023-07-19T14:17:27Z + SO: + + SL5 + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:14Z + + subunit + + + 0.9979062 + protein_state + cleaner0 + 2023-07-14T15:24:43Z + DUMMY: + + pre-translocation + + + 0.5761848 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + + DISCUSS + title_2 + 54023 + Partitioned roles of 40S subunit rearrangements + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:38Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:14Z + + subunit + + + + DISCUSS + paragraph + 54071 + Our structures delineate the mechanistic functions for intersubunit rotation and head swivel in translocation. These functions are partitioned. Specifically, intersubunit rotation allows eEF2 entry into the A site, while the head swivel mediates PKI translocation. Various degrees of intersubunit rotation have been observed in cryo-EM studies of the 80S•IRES initiation complexes. This suggests that the subunits are capable of spontaneous rotation, as is the case for tRNA-bound pre-translocation complexes. The pre-translocation Structure I with eEF2 least advanced into the A site adopts a fully rotated conformation. Reverse intersubunit rotation from Structure I to V shifts the translocation tunnel (the tunnel between the A, P and E sites) toward eEF2, which is rigidly attached to the 60S subunit. This allows eEF2 to move into the A site. As such, reverse intersubunit rotation facilitates full docking of eEF2 in the A site. + + 0.9964527 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + 0.9995925 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.9994972 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + 0.99975497 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.99249893 + experimental_method + cleaner0 + 2023-07-17T08:46:04Z + MESH: + + cryo-EM studies + + + complex_assembly + GO: + cleaner0 + 2023-07-17T08:46:29Z + + 80S•IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + 0.972903 + structure_element + cleaner0 + 2023-07-19T14:31:00Z + SO: + + subunits + + + 0.9995444 + protein_state + cleaner0 + 2023-07-14T09:48:16Z + DUMMY: + + tRNA-bound + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:24:43Z + + pre-translocation + + + 0.9458094 + protein_state + cleaner0 + 2023-07-14T15:24:43Z + DUMMY: + + pre-translocation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:21:44Z + + Structure I + + + 0.9992386 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.9994122 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + 0.93447584 + protein_state + cleaner0 + 2023-07-19T12:50:51Z + DUMMY: + + fully rotated conformation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:21:57Z + + Structure I to V + + + 0.9994348 + site + cleaner0 + 2023-07-19T10:16:56Z + SO: + + translocation tunnel + + + 0.9945496 + site + cleaner0 + 2023-07-19T10:17:01Z + SO: + + tunnel + + + 0.9994321 + site + cleaner0 + 2023-07-17T08:59:41Z + SO: + + A, P and E sites + + + 0.99843365 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + complex_assembly + GO: + cleaner0 + 2023-07-18T13:49:59Z + + 60S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:14Z + + subunit + + + 0.999355 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.9995361 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + 0.9995158 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.99955 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + + DISCUSS + paragraph + 55009 + Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site. The head swivel allows gradual translocation of PKI to the P site, first with respect to the body and then to the head. The fully swiveled conformations of Structures II and III represent the mid-point of translocation, in which PKI relocates between the head A site and body P site. We note that such mid-states have not been observed for 2tRNA•mRNA, but their formation can explain the formation of subsequent pe/E hybrid and ap/P chimeric structures (Figure 1—figure supplement 1). Reverse swivel from Structure III to V brings the head to the non-swiveled position, restoring the A and P sites on the small subunit. + + site + SO: + cleaner0 + 2023-07-19T10:10:33Z + + histidine-diphthamide tip + + + 0.9997118 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.99985754 + residue_name_number + cleaner0 + 2023-07-18T14:40:24Z + DUMMY: + + H583 + + + 0.9998585 + residue_name_number + cleaner0 + 2023-07-18T14:40:30Z + DUMMY: + + H694 + + + 0.99985206 + ptm + cleaner0 + 2023-07-18T14:19:47Z + MESH: + + Diph699 + + + 0.99960977 + structure_element + cleaner0 + 2023-07-19T14:13:14Z + SO: + + codon-anticodon-like helix + + + 0.9998061 + structure_element + cleaner0 + 2023-07-14T09:27:41Z + SO: + + PKI + + + 0.99902654 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.99981576 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.99955726 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + 0.99943393 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + 0.99979264 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.99957776 + site + cleaner0 + 2023-07-19T10:17:16Z + SO: + + P site + + + 0.95581007 + structure_element + cleaner0 + 2023-07-18T14:09:35Z + SO: + + body + + + 0.8795007 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + 0.9994571 + protein_state + cleaner0 + 2023-07-19T12:51:27Z + DUMMY: + + fully swiveled + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:17:30Z + + Structures II and III + + + 0.99976593 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.57131225 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + 0.99952805 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + 0.7615037 + structure_element + cleaner0 + 2023-07-18T14:09:35Z + SO: + + body + + + 0.9995496 + site + cleaner0 + 2023-07-19T10:18:07Z + SO: + + P site + + + 0.9987424 + complex_assembly + cleaner0 + 2023-07-14T09:36:40Z + GO: + + 2tRNA•mRNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T09:51:08Z + + pe/E hybrid + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T09:51:25Z + + ap/P chimeric + + + 0.9976616 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:17:54Z + + Structure III to V + + + 0.9472916 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + 0.9992423 + protein_state + cleaner0 + 2023-07-19T12:29:59Z + DUMMY: + + non-swiveled + + + 0.999267 + site + cleaner0 + 2023-07-19T10:18:11Z + SO: + + A and P sites + + + 0.90010196 + structure_element + cleaner0 + 2023-07-14T09:39:03Z + SO: + + small subunit + + + + DISCUSS + title_2 + 55809 + The functions of eEF2 in translocation + + 0.99973947 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + + DISCUSS + paragraph + 55848 + To our knowledge, our work provides the first high-resolution view of the dynamics of a ribosomal translocase that is inferred from an ensemble of structures sampled under uniform conditions. The structures, therefore, offer a unique opportunity to address the role of the elongation factors during translocation. Translocases are efficient enzymes. While the ribosome itself has the capacity to translocate in the absence of the translocase, spontaneous translocation is slow. EF-G enhances the translocation rate by several orders of magnitude, aided by an additional 2- to 50-fold boost from GTP hydrolysis. Due to the lack of structures of translocation intermediates, the mechanistic role of eEF2/EF-G is not fully understood. + + protein_type + MESH: + cleaner0 + 2023-07-19T09:21:34Z + + ribosomal translocase + + + 0.996012 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.9986753 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.996802 + protein_type + cleaner0 + 2023-07-19T09:21:42Z + MESH: + + elongation factors + + + 0.99910754 + protein_type + cleaner0 + 2023-07-19T09:21:47Z + MESH: + + Translocases + + + 0.9989417 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.9994695 + protein_state + cleaner0 + 2023-07-14T09:55:35Z + DUMMY: + + absence of + + + 0.9979169 + protein_type + cleaner0 + 2023-07-17T08:38:45Z + MESH: + + translocase + + + 0.99960035 + protein + cleaner0 + 2023-07-14T09:36:12Z + PR: + + EF-G + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:12:25Z + + GTP + + + 0.843711 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.99973124 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.99966127 + protein + cleaner0 + 2023-07-14T09:36:12Z + PR: + + EF-G + + + + DISCUSS + paragraph + 56580 + The 80S•IRES•eEF2 structures reported here suggest two main roles for eEF2 in translocation. As discussed above, the first role is to directly shift PKI out of the A site upon spontaneous reverse intersubunit rotation. In our structures, the tip of domain IV docks next to PKI, with diphthamide 699 fit into the minor groove of the codon-anticodon-like helix of PKI (Figure 7). This arrangement rationalizes inactivation of eEF2 by diphtheria toxin, which catalyzes ADP-ribosylation of the diphthamide (reviewed in). The enzyme ADP-ribosylates the NE2 atom of the imidazole ring, which in our structures interacts with the first two residues of the anticodon-like strand of PKI. The bulky ADP-ribosyl moiety at this position would disrupt the interaction, rendering eEF2 unable to bind to the A site and/or stalled on ribosomes in a non-productive conformation. + + 0.99970853 + complex_assembly + cleaner0 + 2023-07-14T09:44:49Z + GO: + + 80S•IRES•eEF2 + + + 0.99891007 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.99980456 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.9995877 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.9995156 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + 0.999129 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-07-19T12:22:22Z + + IV + + + 0.581891 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + ptm + MESH: + cleaner0 + 2023-07-18T14:01:42Z + + diphthamide 699 + + + 0.99847627 + site + cleaner0 + 2023-07-19T10:02:15Z + SO: + + minor groove + + + 0.99963886 + structure_element + cleaner0 + 2023-07-19T14:13:14Z + SO: + + codon-anticodon-like helix + + + 0.9945262 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.9998211 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + protein_type + MESH: + cleaner0 + 2023-07-14T15:23:19Z + + diphtheria toxin + + + 0.99228305 + ptm + cleaner0 + 2023-07-19T14:34:49Z + MESH: + + ADP-ribosylation + + + 0.97661567 + ptm + cleaner0 + 2023-07-19T09:46:56Z + MESH: + + diphthamide + + + 0.9664541 + ptm + cleaner0 + 2023-07-19T14:34:55Z + MESH: + + ADP-ribosylates + + + 0.9991998 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + structures + + + 0.9996568 + structure_element + cleaner0 + 2023-07-19T14:31:20Z + SO: + + anticodon-like strand + + + 0.9931681 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:12:44Z + + ADP + + + 0.99978954 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.9994521 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + 0.9782526 + complex_assembly + cleaner0 + 2023-07-19T09:51:36Z + GO: + + ribosomes + + + + DISCUSS + paragraph + 57446 + As eEF2 shifts PKI toward the P site in the course of reverse intersubunit rotation, the 60S-attached translocase migrates along the surface of the 40S subunit, guided by electrostatic interactions. Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. The Structures reveal hopping of the positive clusters over rRNA helices. For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19 Å (for Cα of R617) from the phosphate backbone of h33 (at nt 1261–1264) to that of the neighboring h34 (at nt 1442–1445). Thus, sliding of eEF2 involves reorganization of electrostatic, perhaps isoenergetic interactions, echoing those implied in extraordinarily fast ribosome inactivation rates by the small-protein ribotoxins and in fast protein association and diffusion along DNA. + + 0.99974936 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + 0.93826824 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.99952894 + site + cleaner0 + 2023-07-19T10:18:17Z + SO: + + P site + + + 0.9881857 + protein_state + cleaner0 + 2023-07-18T14:43:13Z + DUMMY: + + 60S-attached + + + 0.93992156 + protein_type + cleaner0 + 2023-07-17T08:38:45Z + MESH: + + translocase + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:39Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:14Z + + subunit + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:52Z + + electrostatic interactions + + + 0.99779767 + site + cleaner0 + 2023-07-19T10:18:21Z + SO: + + Positively-charged patches + + + structure_element + SO: + cleaner0 + 2023-07-19T12:22:50Z + + II + + + structure_element + SO: + cleaner0 + 2023-07-19T12:22:57Z + + III + + + 0.9998037 + residue_name_number + cleaner0 + 2023-07-18T14:43:17Z + DUMMY: + + R391 + + + 0.9997944 + residue_name_number + cleaner0 + 2023-07-18T14:43:26Z + DUMMY: + + K394 + + + 0.99980396 + residue_name_number + cleaner0 + 2023-07-18T14:43:33Z + DUMMY: + + R433 + + + 0.9998085 + residue_name_number + cleaner0 + 2023-07-18T14:43:40Z + DUMMY: + + R510 + + + 0.99766445 + structure_element + cleaner0 + 2023-07-19T14:31:27Z + SO: + + IV + + + 0.9998011 + residue_name_number + cleaner0 + 2023-07-18T14:36:41Z + DUMMY: + + K613 + + + 0.99982196 + residue_name_number + cleaner0 + 2023-07-18T14:36:54Z + DUMMY: + + R617 + + + 0.99981934 + residue_name_number + cleaner0 + 2023-07-18T14:43:54Z + DUMMY: + + R609 + + + 0.9998204 + residue_name_number + cleaner0 + 2023-07-18T14:37:02Z + DUMMY: + + R631 + + + 0.9997899 + residue_name_number + cleaner0 + 2023-07-18T14:44:04Z + DUMMY: + + K651 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:12:08Z + + rRNA + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:39Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:35Z + + body + + + 0.99733657 + structure_element + cleaner0 + 2023-07-19T14:31:32Z + SO: + + h5 + + + 0.45621443 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + 0.92518467 + structure_element + cleaner0 + 2023-07-19T14:31:36Z + SO: + + h18 + + + 0.9868046 + structure_element + cleaner0 + 2023-07-19T14:31:39Z + SO: + + h33 + + + 0.9810987 + structure_element + cleaner0 + 2023-07-19T14:31:42Z + SO: + + h34 + + + 0.9985941 + evidence + cleaner0 + 2023-07-14T16:19:25Z + DUMMY: + + Structures + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:12:10Z + + rRNA + + + 0.49309957 + structure_element + cleaner0 + 2023-07-19T14:31:48Z + SO: + + helices + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:19:35Z + + Structures II and V + + + 0.999716 + residue_name_number + cleaner0 + 2023-07-18T14:36:41Z + DUMMY: + + K613 + + + 0.99978894 + residue_name_number + cleaner0 + 2023-07-18T14:36:54Z + DUMMY: + + R617 + + + 0.99978656 + residue_name_number + cleaner0 + 2023-07-18T14:37:02Z + DUMMY: + + R631 + + + 0.9058253 + structure_element + cleaner0 + 2023-07-19T14:31:53Z + SO: + + IV + + + 0.9997923 + residue_name_number + cleaner0 + 2023-07-18T14:36:54Z + DUMMY: + + R617 + + + structure_element + SO: + cleaner0 + 2023-07-19T10:19:11Z + + h33 + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T10:18:50Z + + 1261–1264 + + + structure_element + SO: + cleaner0 + 2023-07-19T10:19:20Z + + h34 + + + residue_range + DUMMY: + cleaner0 + 2023-07-19T10:19:03Z + + 1442–1445 + + + 0.99978274 + protein + cleaner0 + 2023-07-14T09:30:48Z + PR: + + eEF2 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:52Z + + electrostatic, perhaps isoenergetic interactions + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:32:57Z + + ribosome + + + + DISCUSS + paragraph + 58396 + Comparison of our structures with the 80S•IRES initiation structure reveals the structural basis for the second key function of the translocase: 'unlocking' of intrasubunit rearrangements that are required for step-wise translocation of PKI on the small subunit. The unlocking model of the ribosome•2tRNA•mRNA pre-translocation complex has been proposed decades ago and functional requirement of the translocase in this process has been implicated. However, the structural and mechanistic definitions of the locked and unlocked states have remained unclear, ranging from the globally distinct ribosome conformations to unknown local rearrangements, e.g. those in the decoding center. FRET data indicate that translocation of 2tRNA•mRNA on the 70S ribosome requires a forward-and-reverse head swivel, which may be related to the unlocking phenomenon. Whereas intersubunit rotation of the pre-translocation complex occurs spontaneously, the head swivel is induced by the eEF2/EF-G translocase, consistent with requirement of eEF2 for unlocking. Structural studies revealed large head swivels in various 70S•tRNA•EF-G and 80S•tRNA•eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase. + + 0.89813346 + experimental_method + cleaner0 + 2023-07-17T08:38:23Z + MESH: + + Comparison + + + 0.9969137 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + 0.9997051 + complex_assembly + cleaner0 + 2023-07-14T09:40:46Z + GO: + + 80S•IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:38:34Z + + initiation + + + 0.9052483 + evidence + cleaner0 + 2023-07-14T16:19:14Z + DUMMY: + + structure + + + 0.9980654 + protein_type + cleaner0 + 2023-07-17T08:38:45Z + MESH: + + translocase + + + 0.99966836 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.98800325 + structure_element + cleaner0 + 2023-07-14T09:39:03Z + SO: + + small subunit + + + 0.99971324 + complex_assembly + cleaner0 + 2023-07-19T09:51:43Z + GO: + + ribosome•2tRNA•mRNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:24:41Z + + pre-translocation + + + 0.9982248 + protein_type + cleaner0 + 2023-07-17T08:38:45Z + MESH: + + translocase + + + 0.9996582 + protein_state + cleaner0 + 2023-07-19T12:51:33Z + DUMMY: + + locked + + + 0.9996425 + protein_state + cleaner0 + 2023-07-19T12:51:36Z + DUMMY: + + unlocked + + + 0.9709593 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + site + SO: + cleaner0 + 2023-07-18T14:50:02Z + + decoding center + + + 0.9691931 + evidence + cleaner0 + 2023-07-19T14:12:17Z + DUMMY: + + FRET data + + + 0.9987307 + complex_assembly + cleaner0 + 2023-07-14T09:36:40Z + GO: + + 2tRNA•mRNA + + + 0.9992502 + complex_assembly + cleaner0 + 2023-07-14T09:56:37Z + GO: + + 70S ribosome + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:24:43Z + + pre-translocation + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + 0.9152942 + protein + cleaner0 + 2023-07-14T09:30:49Z + PR: + + eEF2 + + + 0.8970521 + protein + cleaner0 + 2023-07-14T09:36:13Z + PR: + + EF-G + + + 0.99846315 + protein_type + cleaner0 + 2023-07-17T08:38:45Z + MESH: + + translocase + + + 0.9997421 + protein + cleaner0 + 2023-07-14T09:30:49Z + PR: + + eEF2 + + + 0.9994819 + experimental_method + cleaner0 + 2023-07-17T08:39:29Z + MESH: + + Structural studies + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + 0.99972045 + complex_assembly + cleaner0 + 2023-07-14T15:26:12Z + GO: + + 70S•tRNA•EF-G + + + 0.99971277 + complex_assembly + cleaner0 + 2023-07-14T10:02:55Z + GO: + + 80S•tRNA•eEF2 + + + 0.9996203 + protein_state + cleaner0 + 2023-07-19T12:51:40Z + DUMMY: + + locked + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T09:56:03Z + + complexes with + + + 0.9992039 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:23Z + + tRNA + + + 0.99949956 + protein_state + cleaner0 + 2023-07-14T09:55:35Z + DUMMY: + + absence of + + + 0.9987398 + protein_type + cleaner0 + 2023-07-17T08:38:43Z + MESH: + + translocase + + + + DISCUSS + paragraph + 59658 + Our structures suggest that eEF2 induces head swivel by 'unlocking' the head-body interactions (Figure 7). Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains. The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). This 'locked' state is identical to that observed for PKI in the 80S•IRES initiation structures in the absence of eEF2. Structure I demonstrates that at an early pre-translocation step, the histidine-diphthamide tip of eEF2 is wedged between A1755 and A1756 and PKI. This destabilization allows PKI to detach from the body A site upon spontaneous reverse 40S body rotation, while maintaining interactions with the head A site. Destabilization of the head-bound PKI at the body A site thus allows mobility of the head relative to the body. The histidine-diphthamide-induced disengagement of PKI from A1755 and A1756 therefore provides the structural definition for the 'unlocking' mode of eEF2 action. + + 0.99587005 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + 0.99974424 + protein + cleaner0 + 2023-07-14T09:30:49Z + PR: + + eEF2 + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:35Z + + body + + + 0.99927324 + structure_element + cleaner0 + 2023-07-14T09:34:56Z + SO: + + ASL + + + 0.99937326 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + 0.9993887 + experimental_method + cleaner0 + 2023-07-17T08:39:34Z + MESH: + + structural studies + + + 0.9994765 + taxonomy_domain + cleaner0 + 2023-07-14T09:36:04Z + DUMMY: + + bacterial + + + 0.9977017 + complex_assembly + cleaner0 + 2023-07-19T09:51:46Z + GO: + + ribosomes + + + 0.7095232 + protein_state + cleaner0 + 2023-07-19T12:51:44Z + DUMMY: + + domain closure + + + 0.9678366 + structure_element + cleaner0 + 2023-07-14T09:39:03Z + SO: + + small subunit + + + 0.9994672 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + 0.999673 + structure_element + cleaner0 + 2023-07-19T14:32:21Z + SO: + + shoulder + + + 0.99939096 + structure_element + cleaner0 + 2023-07-18T14:09:35Z + SO: + + body + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:23Z + + tRNA + + + 0.9994721 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:52Z + + stacking + + + 0.97418755 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + 0.9974241 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:19Z + + C1274 + + + 0.9993815 + species + cleaner0 + 2023-07-14T10:07:58Z + MESH: + + S. cerevisiae + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:28:32Z + + C1054 + + + 0.99935037 + species + cleaner0 + 2023-07-14T09:31:43Z + MESH: + + E. coli + + + 0.9744485 + structure_element + cleaner0 + 2023-07-18T14:09:35Z + SO: + + body + + + 0.9972685 + site + cleaner0 + 2023-07-18T14:47:03Z + SO: + + A-site + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:01Z + + A1755 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:14Z + + A1756 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T08:09:17Z + + A1492 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T08:09:31Z + + A1493 + + + 0.99930114 + species + cleaner0 + 2023-07-14T09:31:43Z + MESH: + + E. coli + + + 0.9996037 + protein_state + cleaner0 + 2023-07-19T12:51:51Z + DUMMY: + + locked + + + 0.9996692 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.99954915 + complex_assembly + cleaner0 + 2023-07-14T09:40:46Z + GO: + + 80S•IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:39:11Z + + initiation + + + evidence + DUMMY: + cleaner0 + 2023-07-14T16:19:26Z + + structures + + + 0.99952143 + protein_state + cleaner0 + 2023-07-14T09:55:35Z + DUMMY: + + absence of + + + 0.9996743 + protein + cleaner0 + 2023-07-14T09:30:49Z + PR: + + eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:20:18Z + + Structure I + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:24:43Z + + pre-translocation + + + site + SO: + cleaner0 + 2023-07-19T10:10:33Z + + histidine-diphthamide tip + + + 0.99966717 + protein + cleaner0 + 2023-07-14T09:30:49Z + PR: + + eEF2 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:01Z + + A1755 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:14Z + + A1756 + + + 0.9992706 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.9996315 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.9429087 + structure_element + cleaner0 + 2023-07-18T14:09:35Z + SO: + + body + + + 0.99893606 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + 0.98754764 + complex_assembly + cleaner0 + 2023-07-17T09:02:39Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:35Z + + body + + + 0.9702828 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + 0.9870522 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + 0.9995422 + protein_state + cleaner0 + 2023-07-18T14:22:30Z + DUMMY: + + head-bound + + + 0.99971634 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:35Z + + body + + + site + SO: + cleaner0 + 2023-07-14T09:28:53Z + + A site + + + 0.9808553 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + 0.93796915 + structure_element + cleaner0 + 2023-07-18T14:09:35Z + SO: + + body + + + 0.9992714 + ptm + cleaner0 + 2023-07-18T14:20:20Z + MESH: + + histidine-diphthamide + + + 0.9996124 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:01Z + + A1755 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-19T07:27:14Z + + A1756 + + + 0.9997342 + protein + cleaner0 + 2023-07-14T09:30:49Z + PR: + + eEF2 + + + + DISCUSS + paragraph + 60900 + In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2. Observation of different PKI conformations sampling a range of positions between the A and P sites in the presence of eEF2•GDP implies that thermal fluctuations of the 40S head domain are sufficient for translocation along the energetically flat trajectory. + + 0.9975625 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + protein + PR: + cleaner0 + 2023-07-14T09:30:49Z + + eEF2 + + + 0.7392025 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.99839884 + protein + cleaner0 + 2023-07-14T09:30:49Z + PR: + + eEF2 + + + site + SO: + cleaner0 + 2023-07-19T10:20:42Z + + P and A site + + + 0.9945557 + complex_assembly + cleaner0 + 2023-07-17T09:02:39Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:35Z + + body + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + 0.9995337 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + 0.9990865 + protein + cleaner0 + 2023-07-14T09:30:49Z + PR: + + eEF2 + + + 0.54781204 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.87531567 + site + cleaner0 + 2023-07-19T10:20:45Z + SO: + + A and P sites + + + 0.9993807 + protein_state + cleaner0 + 2023-07-14T09:55:43Z + DUMMY: + + presence of + + + 0.9994604 + complex_assembly + cleaner0 + 2023-07-14T15:19:52Z + GO: + + eEF2•GDP + + + 0.9986683 + complex_assembly + cleaner0 + 2023-07-17T09:02:39Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + + DISCUSS + title_2 + 61444 + Insights into eEF2 association with and dissociation from the ribosome + + 0.9994549 + protein + cleaner0 + 2023-07-14T09:30:49Z + PR: + + eEF2 + + + 0.9963135 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + + DISCUSS + paragraph + 61515 + The conformational rearrangements in eEF2 from Structure I through Structure V provide insights into the mechanisms of eEF2 association with the pre-translocation ribosome and dissociation from the post-translocation ribosome. In all five structures, the GTPase domain is attached to the P stalk and the sarcin-ricin loop. In the fully-rotated pre-translocation-like Structure I, an additional interaction exists. Here, switch loop I interacts with helix 14 (415CAAA418) of the 18S rRNA. This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). Mutations of residues flanking A344 in E. coli 16S rRNA modestly inhibit translation but do not specifically affect EF-G-mediated translocation. However, the effect of A344 mutation on translation was not addressed in that study, leaving the question open whether this residue is critical for eEF2/EF-G function. The interaction between h14 and switch loop I is not resolved in Structures II to V, in all of which the small subunit is partially rotated or non-rotated, so that helix 14 is placed at least 6 Å farther from eEF2 (Figure 5d). We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase. This structural basis rationalizes the observation of transient stabilization of the rotated 70S ribosome upon EF-G•GTP binding and prior to translocation. + + 0.9998493 + protein + cleaner0 + 2023-07-14T09:30:49Z + PR: + + eEF2 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:21:08Z + + Structure I + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:22Z + + Structure V + + + 0.9998442 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + 0.97487086 + protein_state + cleaner0 + 2023-07-14T15:24:43Z + DUMMY: + + pre-translocation + + + 0.8318909 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.82947904 + protein_state + cleaner0 + 2023-07-14T15:27:18Z + DUMMY: + + post-translocation + + + 0.7084811 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.9796959 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + 0.9992837 + structure_element + cleaner0 + 2023-07-19T14:32:26Z + SO: + + GTPase domain + + + 0.99973273 + structure_element + cleaner0 + 2023-07-19T12:12:00Z + SO: + + P stalk + + + 0.99968755 + structure_element + cleaner0 + 2023-07-14T09:47:34Z + SO: + + sarcin-ricin loop + + + 0.99955994 + protein_state + cleaner0 + 2023-07-19T12:51:56Z + DUMMY: + + fully-rotated + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:24:43Z + + pre-translocation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:21:20Z + + Structure I + + + 0.9984552 + structure_element + cleaner0 + 2023-07-19T12:12:07Z + SO: + + switch loop I + + + 0.9996377 + structure_element + cleaner0 + 2023-07-19T14:32:37Z + SO: + + helix 14 + + + 0.5314314 + structure_element + cleaner0 + 2023-07-19T14:32:31Z + SO: + + 415CAAA418 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:33:20Z + + 18S rRNA + + + 0.9990754 + site + cleaner0 + 2023-07-19T10:22:44Z + SO: + + GTPase center + + + 0.99953556 + protein_state + cleaner0 + 2023-07-17T08:40:56Z + DUMMY: + + GTP-bound + + + 0.95360684 + protein_type + cleaner0 + 2023-07-17T08:43:29Z + MESH: + + translational GTPases + + + 0.7912144 + protein_state + cleaner0 + 2023-07-14T09:55:43Z + DUMMY: + + presence of + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:12:27Z + + GTP + + + 0.99969083 + complex_assembly + cleaner0 + 2023-07-14T15:27:34Z + GO: + + 80S•eEF2 + + + 0.99943185 + protein_state + cleaner0 + 2023-07-17T08:30:36Z + DUMMY: + + bound with + + + 0.92701703 + complex_assembly + cleaner0 + 2023-07-17T08:40:02Z + GO: + + GDP•AlF4– + + + 0.99840194 + structure_element + cleaner0 + 2023-07-19T14:32:42Z + SO: + + switch loop + + + 0.9999075 + residue_name_number + cleaner0 + 2023-07-18T14:42:29Z + DUMMY: + + A416 + + + 0.99923444 + protein_state + cleaner0 + 2023-07-19T12:52:14Z + DUMMY: + + invariable + + + 0.99990714 + residue_name_number + cleaner0 + 2023-07-14T15:28:27Z + DUMMY: + + A344 + + + 0.9993867 + species + cleaner0 + 2023-07-14T09:31:43Z + MESH: + + E. coli + + + 0.9999064 + residue_name_number + cleaner0 + 2023-07-14T15:28:34Z + DUMMY: + + A463 + + + 0.9994647 + species + cleaner0 + 2023-07-14T15:28:13Z + MESH: + + H. sapiens + + + 0.9980787 + experimental_method + cleaner0 + 2023-07-17T08:39:43Z + MESH: + + Mutations + + + 0.9999037 + residue_name_number + cleaner0 + 2023-07-14T15:28:26Z + DUMMY: + + A344 + + + 0.9993077 + species + cleaner0 + 2023-07-14T09:31:43Z + MESH: + + E. coli + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:26:08Z + + 16S rRNA + + + 0.8618088 + protein + cleaner0 + 2023-07-14T09:36:13Z + PR: + + EF-G + + + 0.9999007 + residue_name_number + cleaner0 + 2023-07-14T15:28:27Z + DUMMY: + + A344 + + + 0.9947273 + experimental_method + cleaner0 + 2023-07-17T08:43:03Z + MESH: + + mutation + + + 0.9997851 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + 0.99825007 + protein + cleaner0 + 2023-07-14T09:36:13Z + PR: + + EF-G + + + structure_element + SO: + cleaner0 + 2023-07-19T13:52:41Z + + h14 + + + 0.99813884 + structure_element + cleaner0 + 2023-07-19T12:12:07Z + SO: + + switch loop I + + + evidence + DUMMY: + melaniev@ebi.ac.uk + 2023-07-20T18:23:17Z + + Structures II to V + + + structure_element + SO: + cleaner0 + 2023-07-14T09:39:03Z + + small subunit + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T12:52:34Z + + partially rotated + + + 0.9993487 + protein_state + cleaner0 + 2023-07-19T12:29:42Z + DUMMY: + + non-rotated + + + 0.99959505 + structure_element + cleaner0 + 2023-07-19T14:32:48Z + SO: + + helix 14 + + + 0.99983156 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + 0.9993599 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.9996027 + protein_state + cleaner0 + 2023-07-19T12:52:38Z + DUMMY: + + fully rotated + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:39Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:14Z + + subunit + + + 0.9950142 + protein_state + cleaner0 + 2023-07-14T15:24:43Z + DUMMY: + + pre-translocation + + + 0.7444801 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.99955964 + site + cleaner0 + 2023-07-19T10:22:57Z + SO: + + interaction surface + + + 0.999715 + structure_element + cleaner0 + 2023-07-19T12:12:00Z + SO: + + P stalk + + + 0.9997533 + structure_element + cleaner0 + 2023-07-14T09:47:41Z + SO: + + SRL + + + 0.99957496 + protein_state + cleaner0 + 2023-07-17T08:40:55Z + DUMMY: + + GTP-bound + + + 0.9986099 + protein_type + cleaner0 + 2023-07-17T08:38:45Z + MESH: + + translocase + + + 0.9995628 + protein_state + cleaner0 + 2023-07-19T12:52:43Z + DUMMY: + + rotated + + + 0.99885076 + complex_assembly + cleaner0 + 2023-07-14T09:56:37Z + GO: + + 70S ribosome + + + 0.9991972 + complex_assembly + cleaner0 + 2023-07-14T15:28:04Z + GO: + + EF-G•GTP + + + + DISCUSS + paragraph + 63283 + The least rotated conformation of the post-translocation Structure V suggests conformational changes that may trigger eEF2 release from the ribosome at the end of translocation. The most pronounced inter-domain rearrangement in eEF2 involves movement of domain III. In the rotated or mid-rotated Structures I through III, this domain remains rigidly associated with domain V and the N-terminal superdomain and does not undergo noticeable rearrangements. In Structure V, however, the tip of helix A of domain III is displaced toward domain I by ~5 Å relative to that in mid-rotated or fully rotated structures. This displacement is caused by the 8 Å movement of the 40S body protein uS12 upon reverse intersubunit rotation from Structure I to V (Figure 6d). We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the β-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the β-platforms are nearly perpendicular. As we discuss below, Structure V captures a 'pre-unstacking' state due to stabilization of the interface between domains III and V by sordarin. + + 0.9994625 + protein_state + cleaner0 + 2023-07-19T12:52:46Z + DUMMY: + + least rotated + + + 0.98654073 + protein_state + cleaner0 + 2023-07-14T15:27:20Z + DUMMY: + + post-translocation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:23Z + + Structure V + + + 0.9998485 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + 0.8951123 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.99985516 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + 0.8255967 + structure_element + cleaner0 + 2023-07-19T14:32:54Z + SO: + + III + + + 0.99959224 + protein_state + cleaner0 + 2023-07-19T12:52:52Z + DUMMY: + + rotated + + + 0.9994974 + protein_state + cleaner0 + 2023-07-18T13:57:54Z + DUMMY: + + mid-rotated + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:22:06Z + + Structures I through III + + + 0.97735584 + structure_element + cleaner0 + 2023-07-19T14:32:58Z + SO: + + V + + + 0.9997249 + structure_element + cleaner0 + 2023-07-19T12:23:37Z + SO: + + superdomain + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:23Z + + Structure V + + + 0.9994612 + structure_element + cleaner0 + 2023-07-19T14:33:02Z + SO: + + helix A + + + 0.9780176 + structure_element + cleaner0 + 2023-07-19T14:33:06Z + SO: + + III + + + 0.99887127 + structure_element + cleaner0 + 2023-07-19T14:33:11Z + SO: + + I + + + 0.9994686 + protein_state + cleaner0 + 2023-07-18T13:57:54Z + DUMMY: + + mid-rotated + + + 0.9995203 + protein_state + cleaner0 + 2023-07-19T12:52:55Z + DUMMY: + + fully rotated + + + 0.9939659 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-07-17T09:02:39Z + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:35Z + + body + + + 0.9996605 + protein + cleaner0 + 2023-07-18T14:37:08Z + PR: + + uS12 + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:22:22Z + + Structure I to V + + + 0.9943276 + structure_element + cleaner0 + 2023-07-19T14:33:17Z + SO: + + III + + + 0.9997677 + protein + cleaner0 + 2023-07-18T14:37:08Z + PR: + + uS12 + + + 0.999859 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + 0.9613572 + structure_element + cleaner0 + 2023-07-19T12:23:43Z + SO: + + β-platform + + + 0.6897788 + structure_element + cleaner0 + 2023-07-19T14:33:21Z + SO: + + III + + + 0.9956507 + structure_element + cleaner0 + 2023-07-19T14:33:26Z + SO: + + V + + + 0.99965215 + protein_state + cleaner0 + 2023-07-19T12:53:00Z + DUMMY: + + free + + + 0.9998503 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + 0.99952966 + protein + cleaner0 + 2023-07-14T09:36:13Z + PR: + + EF-G + + + 0.9500897 + structure_element + cleaner0 + 2023-07-19T14:33:30Z + SO: + + β-platforms + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:23Z + + Structure V + + + 0.99900514 + protein_state + cleaner0 + 2023-07-19T12:53:04Z + DUMMY: + + pre-unstacking + + + 0.9990054 + site + cleaner0 + 2023-07-19T10:23:02Z + SO: + + interface + + + 0.9948738 + structure_element + cleaner0 + 2023-07-19T14:33:35Z + SO: + + III + + + 0.99739707 + structure_element + cleaner0 + 2023-07-19T14:33:39Z + SO: + + V + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:55Z + + sordarin + + + + DISCUSS + title_2 + 64477 + Sordarin stabilizes GDP-bound eEF2 on the ribosome + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:55Z + + Sordarin + + + 0.9995802 + protein_state + cleaner0 + 2023-07-17T08:40:25Z + DUMMY: + + GDP-bound + + + 0.9996613 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + 0.9434002 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + + DISCUSS + paragraph + 64528 + Sordarin is a potent antifungal antibiotic that inhibits translation. Based on biochemical experiments, two alternative mechanisms of action were proposed: sordarin either prevents eEF2 departure by inhibiting GTP hydrolysis or acts after GTP hydrolysis. Although our complex was assembled using eEF2•GTP, density maps clearly show GDP and Mg2+ in each structure (Figure 5g). Our structures therefore indicate that sordarin stalls eEF2 on the ribosome in the GDP-bound form, i.e. following GTP hydrolysis and phosphate release. + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:55Z + + Sordarin + + + 0.99928516 + experimental_method + cleaner0 + 2023-07-17T08:43:39Z + MESH: + + biochemical experiments + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:55Z + + sordarin + + + 0.99967504 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:12:27Z + + GTP + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:12:27Z + + GTP + + + 0.9996948 + complex_assembly + cleaner0 + 2023-07-14T09:31:05Z + GO: + + eEF2•GTP + + + 0.99957275 + evidence + cleaner0 + 2023-07-19T13:53:35Z + DUMMY: + + density maps + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:41Z + + GDP + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:53:19Z + + Mg2+ + + + 0.9992229 + evidence + cleaner0 + 2023-07-14T16:19:14Z + DUMMY: + + structure + + + 0.9995141 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:55Z + + sordarin + + + 0.99974555 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + 0.88018614 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.99953574 + protein_state + cleaner0 + 2023-07-17T08:40:26Z + DUMMY: + + GDP-bound + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:12:27Z + + GTP + + + + DISCUSS + paragraph + 65058 + The mechanism of stalling is suggested by comparison of pre-translocation and post-translocation structures in our ensemble. In all five structures, sordarin is bound between domains III and V of eEF2, stabilized by hydrophobic interactions identical to those in the isolated eEF2•sordarin complex (Figures 5g and h). In the nearly non-rotated post-translocation Structure V, the tip of domain III is shifted, however the interface between domains III and V remains unchanged, suggesting strong stabilization of this interface by sordarin. We note that Structure V is slightly more rotated than the 80S•2tRNA•mRNA complex in the absence of eEF2•sordarin, implying that sordarin interferes with the final stages of reverse rotation of the post-translocation ribosome. We propose that sordarin acts to prevent full reverse rotation and release of eEF2•GDP by stabilizing the interdomain interface and thus blocking uS12-induced disengagement of domain III from domain V. + + 0.9816342 + protein_state + cleaner0 + 2023-07-14T15:24:43Z + DUMMY: + + pre-translocation + + + 0.9316675 + protein_state + cleaner0 + 2023-07-14T15:27:20Z + DUMMY: + + post-translocation + + + 0.9989109 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + 0.99879503 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:55Z + + sordarin + + + 0.99920243 + protein_state + cleaner0 + 2023-07-19T12:53:17Z + DUMMY: + + bound + + + structure_element + SO: + cleaner0 + 2023-07-19T10:23:24Z + + III + + + structure_element + SO: + cleaner0 + 2023-07-19T10:23:32Z + + V + + + 0.9998517 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:52Z + + hydrophobic interactions + + + 0.91515946 + protein_state + cleaner0 + 2023-07-19T12:53:24Z + DUMMY: + + isolated + + + 0.9997354 + complex_assembly + cleaner0 + 2023-07-14T10:01:27Z + GO: + + eEF2•sordarin + + + 0.9235951 + protein_state + cleaner0 + 2023-07-19T12:53:33Z + DUMMY: + + nearly non-rotated + + + 0.96038 + protein_state + cleaner0 + 2023-07-14T15:27:20Z + DUMMY: + + post-translocation + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:23Z + + Structure V + + + 0.8961695 + structure_element + cleaner0 + 2023-07-19T14:33:43Z + SO: + + III + + + 0.99937916 + site + cleaner0 + 2023-07-19T10:23:07Z + SO: + + interface + + + 0.79765296 + structure_element + cleaner0 + 2023-07-19T14:33:47Z + SO: + + III + + + 0.99194676 + structure_element + cleaner0 + 2023-07-19T14:33:51Z + SO: + + V + + + 0.99939907 + site + cleaner0 + 2023-07-19T10:24:15Z + SO: + + interface + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:55Z + + sordarin + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:14:23Z + + Structure V + + + 0.99972975 + complex_assembly + cleaner0 + 2023-07-14T09:44:25Z + GO: + + 80S•2tRNA•mRNA + + + 0.99957705 + protein_state + cleaner0 + 2023-07-14T09:55:35Z + DUMMY: + + absence of + + + 0.9997294 + complex_assembly + cleaner0 + 2023-07-14T10:01:27Z + GO: + + eEF2•sordarin + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:55Z + + sordarin + + + 0.83613044 + protein_state + cleaner0 + 2023-07-14T15:27:20Z + DUMMY: + + post-translocation + + + 0.9993555 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:37:55Z + + sordarin + + + 0.9991319 + complex_assembly + cleaner0 + 2023-07-14T15:19:52Z + GO: + + eEF2•GDP + + + 0.99960303 + site + cleaner0 + 2023-07-19T10:24:19Z + SO: + + interdomain interface + + + 0.7847563 + protein + cleaner0 + 2023-07-18T14:37:08Z + PR: + + uS12 + + + structure_element + SO: + cleaner0 + 2023-07-19T10:24:03Z + + III + + + 0.99102354 + structure_element + cleaner0 + 2023-07-19T14:33:57Z + SO: + + V + + + + DISCUSS + title_2 + 66037 + Implications for tRNA and mRNA translocation during translation + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:23Z + + tRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:04Z + + mRNA + + + + DISCUSS + paragraph + 66101 + Because translocation of tRNA must involve large-scale dynamics, this step has long been regarded as the most puzzling step of translation. Intersubunit rearrangements and tRNA hybrid states have been proposed to play key roles half a century ago. Despite an impressive body of biochemical, fluorescence and structural data accumulated since then, translocation remains the least understood step of elongation. The structural understanding of ribosome and tRNA dynamics has been greatly aided by a wealth of X-ray and cryo-EM structures (reviewed in). However, visualization of the eEF2/EF-G-induced translocation is confined to very early pre-EF-G-entry states and late (almost translocated or fully translocated) states, leaving most of the path from the A to the P site uncharacterized (Figure 1—figure supplement 1). + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:23Z + + tRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:23Z + + tRNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T13:54:31Z + + hybrid + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:35Z + + body + + + evidence + DUMMY: + cleaner0 + 2023-07-19T13:54:55Z + + biochemical, fluorescence and structural data + + + 0.99352056 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:23Z + + tRNA + + + experimental_method + MESH: + cleaner0 + 2023-07-17T08:42:27Z + + X-ray + + + experimental_method + MESH: + cleaner0 + 2023-07-17T08:27:36Z + + cryo-EM + + + 0.9832516 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + 0.9422131 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + protein + PR: + cleaner0 + 2023-07-14T09:36:13Z + + EF-G + + + protein_state + DUMMY: + cleaner0 + 2023-07-17T08:42:06Z + + pre-EF-G-entry + + + 0.99901545 + protein_state + cleaner0 + 2023-07-17T08:42:43Z + DUMMY: + + almost translocated + + + 0.9991677 + protein_state + cleaner0 + 2023-07-17T08:37:56Z + DUMMY: + + fully translocated + + + site + SO: + cleaner0 + 2023-07-19T10:24:41Z + + A to the P site + + + + DISCUSS + paragraph + 66924 + Our study provides new insights into the structural understanding of tRNA translocation. First, we propose that tRNA and IRES translocations occur via the same general trajectory. This is evident from the fact that ribosome rearrangements in translocation are inherent to the ribosome and likely occur in similar ways in both cases. Furthermore, the step-wise coupling of ribosome dynamics with IRES translocation is overall consistent with that observed for 2tRNA•mRNA translocation in solution. For example, fluorescence and biochemical studies revealed that the early pre-translocation EF-G-bound ribosomes are fully rotated and translocation of the tRNA-mRNA complex occurs during reverse rotation of the small subunit, coupled with head swivel. The sequence of ribosome rearrangements during IRES translocation also agrees with that inferred from 70S•EF-G structures, including those in which the A-to-P-site translocating tRNA was not present. Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7–9°) body and a partly rotated head (5–7.5°), in agreement with the conformation of our Structure I. The most swiveled head (18–21°) was observed in a mid-rotated ribosome (3–5°) of a later translocation intermediate TIpost, similar to the conformation of our Structure III. Overall, these correlations suggest that the intermediate locations of the elusive A-to-P-site translocating tRNA are similar to those of PKI in our structures. + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:23Z + + tRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:23Z + + tRNA + + + 0.41266745 + site + cleaner0 + 2023-07-14T09:21:07Z + SO: + + IRES + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:32:57Z + + ribosome + + + 0.9977036 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:32:57Z + + ribosome + + + 0.51458466 + site + cleaner0 + 2023-07-14T09:21:07Z + SO: + + IRES + + + 0.9976878 + complex_assembly + cleaner0 + 2023-07-14T09:36:40Z + GO: + + 2tRNA•mRNA + + + 0.9995383 + experimental_method + cleaner0 + 2023-07-17T08:44:22Z + MESH: + + fluorescence and biochemical studies + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:24:43Z + + pre-translocation + + + 0.99949586 + protein_state + cleaner0 + 2023-07-14T10:09:43Z + DUMMY: + + EF-G-bound + + + 0.99894613 + complex_assembly + cleaner0 + 2023-07-19T09:51:52Z + GO: + + ribosomes + + + 0.9995048 + protein_state + cleaner0 + 2023-07-19T12:53:38Z + DUMMY: + + fully rotated + + + 0.9993949 + complex_assembly + cleaner0 + 2023-07-14T09:36:32Z + GO: + + tRNA-mRNA + + + 0.8160001 + structure_element + cleaner0 + 2023-07-14T09:39:03Z + SO: + + small subunit + + + 0.9767209 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + complex_assembly + GO: + cleaner0 + 2023-07-14T09:32:57Z + + ribosome + + + 0.34823525 + site + cleaner0 + 2023-07-14T09:21:07Z + SO: + + IRES + + + 0.9996809 + complex_assembly + cleaner0 + 2023-07-14T09:39:50Z + GO: + + 70S•EF-G + + + 0.99909186 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + 0.9993857 + site + cleaner0 + 2023-07-19T10:25:07Z + SO: + + A-to-P-site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:23Z + + tRNA + + + 0.99565923 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.99942833 + protein_state + cleaner0 + 2023-07-19T12:53:47Z + DUMMY: + + rotated + + + 0.99641526 + structure_element + cleaner0 + 2023-07-18T14:09:35Z + SO: + + body + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T12:54:04Z + + partly rotated + + + 0.9986665 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:24:12Z + + Structure I + + + 0.98800033 + protein_state + cleaner0 + 2023-07-19T12:54:12Z + DUMMY: + + most swiveled + + + 0.99805826 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + 0.9991572 + protein_state + cleaner0 + 2023-07-18T13:57:54Z + DUMMY: + + mid-rotated + + + 0.9985965 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:24:58Z + + Structure III + + + 0.99937236 + site + cleaner0 + 2023-07-19T10:25:10Z + SO: + + A-to-P-site + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:23Z + + tRNA + + + 0.99927706 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.9983277 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + + DISCUSS + paragraph + 68429 + Second, the structures clarify the structural basis of the often-used but structurally undefined terms 'locking' and 'unlocking' with respect to the pre-translocation complex (Figure 6f). We deem the pre-translocation complex locked, because the A-site bound ASL-mRNA is stabilized by interactions with the decoding center. These interactions are maintained for the classical- and hybrid-state tRNAs in the spontaneously sampled non-rotated and rotated ribosomes, respectively. Unlocking involves separation of the codon-anticodon helix from the decoding center residues by the protruding tip of eEF2/EF-G (Figure 7), occurring in the fully rotated ribosome at an early pre-translocation step. This unlatches the head, allowing creation of hitherto elusive intermediate tRNA positions during spontaneous reverse body rotation. + + 0.99842167 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:24:43Z + + pre-translocation + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:24:43Z + + pre-translocation + + + 0.9958444 + protein_state + cleaner0 + 2023-07-19T12:54:17Z + DUMMY: + + locked + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:32:21Z + + A-site bound + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:04Z + + mRNA + + + 0.9978288 + site + cleaner0 + 2023-07-18T14:50:02Z + SO: + + decoding center + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T12:54:51Z + + classical + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T12:55:07Z + + hybrid + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:06Z + + tRNAs + + + 0.9993889 + protein_state + cleaner0 + 2023-07-19T12:29:42Z + DUMMY: + + non-rotated + + + 0.9976907 + protein_state + cleaner0 + 2023-07-19T12:55:14Z + DUMMY: + + rotated + + + 0.99939656 + complex_assembly + cleaner0 + 2023-07-19T09:53:39Z + GO: + + ribosomes + + + 0.9990625 + structure_element + cleaner0 + 2023-07-19T14:26:26Z + SO: + + codon-anticodon helix + + + 0.98965186 + site + cleaner0 + 2023-07-18T14:50:02Z + SO: + + decoding center + + + 0.9982627 + protein + cleaner0 + 2023-07-14T09:30:50Z + PR: + + eEF2 + + + 0.9979115 + protein + cleaner0 + 2023-07-14T09:36:13Z + PR: + + EF-G + + + 0.9995489 + protein_state + cleaner0 + 2023-07-19T12:55:18Z + DUMMY: + + fully rotated + + + 0.99942243 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:24:43Z + + pre-translocation + + + 0.9252438 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:24Z + + tRNA + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:35Z + + body + + + + DISCUSS + paragraph + 69256 + Third, our findings uncover a new role of the head swivel. Previous studies showed that this movement widens the constriction ('gate') between the P and E sites, thus allowing the P-tRNA passage to the E site. In addition to the 'gate-opening' role, we now show that the head swivel brings the head A site to the body P site, allowing a step-wise conveying of the codon-anticodon helix between the A and P sites. + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + 0.9981598 + site + cleaner0 + 2023-07-19T10:25:24Z + SO: + + constriction + + + 0.9984458 + site + cleaner0 + 2023-07-19T10:25:34Z + SO: + + gate + + + 0.9995731 + site + cleaner0 + 2023-07-17T08:57:44Z + SO: + + P and E sites + + + site + SO: + cleaner0 + 2023-07-19T13:56:02Z + + P + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:24Z + + tRNA + + + 0.99951637 + site + cleaner0 + 2023-07-14T09:35:33Z + SO: + + E site + + + 0.9943797 + site + cleaner0 + 2023-07-19T10:25:36Z + SO: + + gate + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + 0.99003184 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + 0.99938923 + site + cleaner0 + 2023-07-14T09:28:53Z + SO: + + A site + + + 0.99493647 + structure_element + cleaner0 + 2023-07-18T14:09:35Z + SO: + + body + + + 0.8478176 + site + cleaner0 + 2023-07-19T10:25:47Z + SO: + + P site + + + structure_element + SO: + cleaner0 + 2023-07-19T14:26:26Z + + codon-anticodon helix + + + 0.99881953 + site + cleaner0 + 2023-07-19T10:27:12Z + SO: + + A and P sites + + + + DISCUSS + paragraph + 69669 + Finally, the similar populations of particles (within a 2X range) in our 80S•IRES•eEF2 reconstructions (Figure 1—figure supplement 2) suggest that the intermediate translocation states sample several energetically similar and interconverting conformations. This is consistent with the idea of a rather flat energy landscape of translocation, suggested by recent work that measured mechanical work produced by the ribosome during translocation. Our findings implicate, however, that the energy landscape is not completely flat and contains local minima for transient positions of the codon-anticodon helix between the A and P sites. The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4 Å each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2—source data 1). Movement of PKI relative to the head occurs during the subsequent reverse swivel in three 3–7 Å sub-steps (II to III to IV to V). It is possible that additional meta-stable but less populated states exist between the conformations we observe. We note that four of our near-atomic resolution maps comprised ~30,000 particles each, the minimum number required for a near-atomic-resolution reconstruction of the ribosome. A larger data set will therefore be necessary to reveal additional sub-states. + + 0.9912579 + experimental_method + cleaner0 + 2023-07-19T14:09:58Z + MESH: + + particles + + + 0.99972755 + complex_assembly + cleaner0 + 2023-07-14T09:44:49Z + GO: + + 80S•IRES•eEF2 + + + 0.99929357 + evidence + cleaner0 + 2023-07-19T14:12:26Z + DUMMY: + + reconstructions + + + 0.99936384 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.9955378 + structure_element + cleaner0 + 2023-07-19T14:26:26Z + SO: + + codon-anticodon helix + + + 0.99943036 + site + cleaner0 + 2023-07-19T10:27:17Z + SO: + + A and P sites + + + 0.9037336 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:35Z + + body + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + complex_assembly + GO: + cleaner0 + 2023-07-19T12:25:15Z + + initiation complex + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:24:46Z + + I + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:24:55Z + + I + + + evidence + DUMMY: + cleaner0 + 2023-07-19T12:24:59Z + + II + + + 0.9491358 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:35Z + + body + + + 0.99934816 + site + cleaner0 + 2023-07-19T10:27:21Z + SO: + + P site + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:26:03Z + + Structures III, IV and V + + + 0.96706176 + structure_element + cleaner0 + 2023-07-14T09:27:42Z + SO: + + PKI + + + 0.9563458 + structure_element + cleaner0 + 2023-07-17T08:56:49Z + SO: + + head + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:26:48Z + + II to III to IV to V + + + 0.99950814 + evidence + cleaner0 + 2023-07-19T14:12:30Z + DUMMY: + + maps + + + 0.9990263 + experimental_method + cleaner0 + 2023-07-19T14:09:58Z + MESH: + + particles + + + evidence + DUMMY: + cleaner0 + 2023-07-19T10:27:02Z + + near-atomic-resolution reconstruction + + + 0.999496 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + + DISCUSS + title_2 + 71093 + Concluding remarks + + + DISCUSS + title_3 + 71112 + Translation of viral mRNA + + 0.9714399 + taxonomy_domain + cleaner0 + 2023-07-14T09:20:22Z + DUMMY: + + viral + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:14:04Z + + mRNA + + + + DISCUSS + paragraph + 71138 + Our work sheds light on the dynamic mechanism of cap-independent translation by IGR IRESs, tightly coupled with the universally conserved dynamic properties of the ribosome. The cryo-EM structures demonstrate that the TSV IRES structurally and dynamically represents a chimera of the 2tRNA•mRNA translocating complex (A/P-tRNA • P/E-tRNA • mRNA). Like in the 2tRNA•mRNA translocating complex in which the two tRNAs move independently of each other, the PKI domain moves relative to the 5´-domain, causing the IRES to undergo an inchworm-walk translocation. A large structural difference between the IRES and the 2tRNA•mRNA complex exists, however, in that the IRES lacks three out of six tRNA-like domains involved in tRNA translocation. This difference likely accounts for the inefficient translocation of the IRES, which is difficult to stabilize in the post-translocation state and therefore is prone to reverse translocation. Although structurally handicapped, the TSV IRES manages to translocate by employing ribosome dynamics that are remarkably similar to that in 2tRNA•mRNA translocation. The uniformity of ribosome dynamics underscores the idea that translocation is an inherent and structurally-optimized property of the ribosome, supported also by translocation activity in the absence of the elongation factor. This property is rendered by the relative mobility of the three major building blocks, the 60S subunit and the 40S head and body, assisted by ligand-interacting extensions including the L1 stalk and the P stalk. Intergenic IRESs, in turn, represent a striking example of convergent molecular evolution. Viral mRNAs have evolved to adopt an atypical structure to employ the inherent ribosome dynamics, to be able to hijack the host translational machinery in a simple fashion. + + 0.6832388 + structure_element + cleaner0 + 2023-07-14T09:26:12Z + SO: + + IGR + + + 0.98576397 + site + cleaner0 + 2023-07-14T09:20:11Z + SO: + + IRESs + + + 0.82903767 + protein_state + cleaner0 + 2023-07-19T12:55:23Z + DUMMY: + + universally conserved + + + 0.99871266 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.99941874 + experimental_method + cleaner0 + 2023-07-17T08:27:36Z + MESH: + + cryo-EM + + + 0.9982248 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + 0.94002247 + species + cleaner0 + 2023-07-14T09:24:20Z + MESH: + + TSV + + + 0.99803907 + site + cleaner0 + 2023-07-14T09:21:07Z + SO: + + IRES + + + 0.9996104 + complex_assembly + cleaner0 + 2023-07-14T09:36:40Z + GO: + + 2tRNA•mRNA + + + complex_assembly + GO: + cleaner0 + 2023-07-19T09:54:13Z + + A/P-tRNA • P/E-tRNA • mRNA + + + 0.9996125 + complex_assembly + cleaner0 + 2023-07-14T09:36:40Z + GO: + + 2tRNA•mRNA + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:06Z + + tRNAs + + + structure_element + SO: + cleaner0 + 2023-07-14T09:27:43Z + + PKI + + + 0.9996648 + structure_element + cleaner0 + 2023-07-19T12:25:40Z + SO: + + 5´-domain + + + 0.99878865 + site + cleaner0 + 2023-07-14T09:21:07Z + SO: + + IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-19T10:13:02Z + + inchworm + + + 0.99404573 + site + cleaner0 + 2023-07-14T09:21:07Z + SO: + + IRES + + + 0.9996314 + complex_assembly + cleaner0 + 2023-07-14T09:36:40Z + GO: + + 2tRNA•mRNA + + + 0.9996086 + site + cleaner0 + 2023-07-14T09:21:07Z + SO: + + IRES + + + 0.9981736 + protein_state + cleaner0 + 2023-07-19T12:55:39Z + DUMMY: + + lacks + + + structure_element + SO: + cleaner0 + 2023-07-19T13:57:07Z + + tRNA-like domains + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:15:24Z + + tRNA + + + 0.9989812 + site + cleaner0 + 2023-07-14T09:21:07Z + SO: + + IRES + + + protein_state + DUMMY: + cleaner0 + 2023-07-14T15:27:20Z + + post-translocation + + + 0.9350405 + species + cleaner0 + 2023-07-14T09:24:20Z + MESH: + + TSV + + + 0.9974492 + site + cleaner0 + 2023-07-14T09:21:07Z + SO: + + IRES + + + 0.8572012 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.9994712 + complex_assembly + cleaner0 + 2023-07-14T09:36:40Z + GO: + + 2tRNA•mRNA + + + 0.63648415 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.9990694 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.9995005 + protein_state + cleaner0 + 2023-07-14T09:55:35Z + DUMMY: + + absence of + + + 0.99564236 + protein_type + cleaner0 + 2023-07-19T09:17:41Z + MESH: + + elongation factor + + + complex_assembly + GO: + cleaner0 + 2023-07-18T13:49:59Z + + 60S + + + structure_element + SO: + cleaner0 + 2023-07-18T13:50:14Z + + subunit + + + 0.99936193 + complex_assembly + cleaner0 + 2023-07-17T09:02:39Z + GO: + + 40S + + + structure_element + SO: + cleaner0 + 2023-07-17T08:56:49Z + + head + + + structure_element + SO: + cleaner0 + 2023-07-18T14:09:35Z + + body + + + 0.9994944 + structure_element + cleaner0 + 2023-07-19T14:34:09Z + SO: + + ligand-interacting extensions + + + 0.99963784 + structure_element + cleaner0 + 2023-07-19T12:21:14Z + SO: + + L1 stalk + + + 0.9996475 + structure_element + cleaner0 + 2023-07-19T12:12:00Z + SO: + + P stalk + + + 0.9801083 + site + cleaner0 + 2023-07-14T09:20:11Z + SO: + + IRESs + + + 0.9983944 + taxonomy_domain + cleaner0 + 2023-07-14T09:20:22Z + DUMMY: + + Viral + + + chemical + CHEBI: + cleaner0 + 2023-07-19T13:13:31Z + + mRNAs + + + evidence + DUMMY: + cleaner0 + 2023-07-14T16:19:14Z + + structure + + + 0.9749979 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + + DISCUSS + title_3 + 72950 + Ensemble cryo-EM + + 0.99953604 + experimental_method + cleaner0 + 2023-07-17T08:27:36Z + MESH: + + cryo-EM + + + + DISCUSS + paragraph + 72967 + Our current understanding of macromolecular machines, such as the ribosome, is often limited by a gap between biophysical/biochemical studies and structural studies. For example, Förster resonance energy transfer can provide insight into the macromolecular dynamics of an assembly at the single-molecule level but is limited to specifically labeled locations within the assembly. High-resolution crystal structures, on the other hand, can provide static images of an assembly, and the structural dynamics can only be inferred by comparing structures that are usually obtained in different experiments and under different, often non-native, conditions. Cryo-EM offers the possibility of obtaining integrated information of both structure and dynamics as demonstrated in lower-resolution studies of bacterial ribosome complexes. Recent technical advances, including direct electron detectors and image processing software, have significantly improved the resolution at which such studies can be performed. The increased resolution, need for larger datasets and more sophisticated algorithms have also led to a massive increase in the computational power required to process the data. The available computing infrastructure and computational efficiency have therefore become deciding factors in how many different structural states can be resolved. This is presumably one of the reasons why most recent studies of ribosome complexes have focused on a single high-resolution structure despite the non-uniform local resolution of the maps that likely reflects structural heterogeneity. The computational efficiency of FREALIGN has allowed us to classify a relatively large dataset (1.1 million particles) into 15 classes (Figure 1—figure supplement 2) and obtain eight near-atomic-resolution structures from it. The classification, which followed an initial alignment of all particles to a single reference, required about 130,000 CPU hours or about five to six full days on a 1000-CPU cluster. While it would clearly be impractical to perform this type of analysis on a desktop computer, one may extrapolate using Moore’s law that such practice will become routine in less than ten years. Therefore, cryo-EM has the potential to become a standard tool for uncovering detailed dynamic pathways of complex macromolecular machines. A particularly exciting application will be to infer the high-resolution temporal trajectory of a pathway from an ensemble of equilibrium states in a single sample, as described in our work, together with an analysis of samples quenched at different time points of the reaction. + + 0.9990188 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + experimental_method + MESH: + cleaner0 + 2023-07-17T08:45:06Z + + biophysical/biochemical studies + + + 0.9179076 + experimental_method + cleaner0 + 2023-07-17T08:39:36Z + MESH: + + structural studies + + + 0.9986306 + experimental_method + cleaner0 + 2023-07-14T09:41:01Z + MESH: + + Förster resonance energy transfer + + + 0.9995963 + evidence + cleaner0 + 2023-07-17T08:45:16Z + DUMMY: + + crystal structures + + + 0.998577 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + 0.99955434 + experimental_method + cleaner0 + 2023-07-17T08:27:36Z + MESH: + + Cryo-EM + + + evidence + DUMMY: + cleaner0 + 2023-07-14T16:19:14Z + + structure + + + 0.99946684 + taxonomy_domain + cleaner0 + 2023-07-14T09:36:04Z + DUMMY: + + bacterial + + + 0.98313105 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.96320486 + complex_assembly + cleaner0 + 2023-07-14T09:32:57Z + GO: + + ribosome + + + 0.99937564 + evidence + cleaner0 + 2023-07-14T16:19:14Z + DUMMY: + + structure + + + 0.9996226 + evidence + cleaner0 + 2023-07-19T14:12:35Z + DUMMY: + + maps + + + 0.99935824 + experimental_method + cleaner0 + 2023-07-17T08:45:43Z + MESH: + + FREALIGN + + + 0.99879444 + experimental_method + cleaner0 + 2023-07-19T14:09:58Z + MESH: + + particles + + + 0.99942577 + evidence + cleaner0 + 2023-07-14T16:19:26Z + DUMMY: + + structures + + + 0.9948547 + experimental_method + cleaner0 + 2023-07-19T14:09:58Z + MESH: + + particles + + + 0.99955434 + experimental_method + cleaner0 + 2023-07-17T08:27:36Z + MESH: + + cryo-EM + + + + METHODS + title_1 + 75576 + Materials and methods + + + METHODS + title_2 + 75598 + S. cerevisiae 80S ribosome preparation + + + METHODS + paragraph + 75637 + 80S ribosomes used in this study were prepared from Saccharomyces cerevisiae strain W303 as described previously. To obtain ribosomal subunits, purified 80S was incubated in dissociation buffer (20 mM HEPES·KOH (pH 7.5), 0.5 M KCl, 2.5 mM magnesium acetate, 2 mM dithiothreitol (DTT), and 0.5 U/μl RNasin) for 1 hr at 4°C. The dissociated subunits were then layered on sucrose gradients (10% to 30% sucrose) in the dissociation buffer and centrifuged for 15 hr at 22,000 rpm in an SW32 rotor. Fractions corresponding to 40S and 60S subunits were pooled and buffer-exchanged to subunit storage buffer containing 50 mM Tris (pH7.5), 20 mM MgCl2, 100 mM KCl, and 2 mM DTT. Purified subunits were flash-frozen in liquid nitrogen and stored in aliquots at –80°C. + + + METHODS + title_2 + 76423 + Taura syndrome virus IRES preparation + + + METHODS + paragraph + 76461 + Plasmid pUC57 (Genscript) containing the synthetic DNA encoding for nucleotides 6741–6990 of the TSV mRNA sequence was used to amplify the 250-nucleotide fragment by PCR. This DNA fragment (TSV IRES RNA) served as a template for in vitro transcription. The transcription reaction was incubated for 4 hr at 37°C, and the resulting transcription product was treated with DNase I for 30 mins at 37°C. The RNA was then extracted with acidic phenol/chloroform, gel-purified, and then ethanol precipitated with 100% ethanol, followed by an 80% ethanol wash. The resulting RNA pellet was air-dried at room temperature and suspended in RNase-free water. The TSV IRES transcription product was folded in 1X IRES refolding buffer (20 mM Potassium acetate pH 7.5 and 5 mM MgCl2), incubated at 65°C for 10 min and cooled down gradually at room temperature, prior to complex preparation for cryo-EM study. + + + METHODS + title_2 + 77366 + S. cerevisiae eEF2 purification + + + METHODS + paragraph + 77398 + C-terminally His6-tagged eEF2 was produced in yeast TKY675 cells obtained from Terri Goss Kinzy. Yeast cells were grown in 4 liters of YPD media at 27°C and 160 rpm, to A600=1.5. Yeast cell pellet (~5 g) was obtained by centrifugation and re-suspended in 20 ml of the lysis buffer (50 mM potassium phosphate pH 7.6, 1 M KCl, 1% Tween 20, 10% Glycerol, 10 mM imidazole, 0.2 mM PMSF, 1 mM DTT, and 1 tablet of Roche miniComplete protease inhibitor). The suspension was lysed with microfluidizer at 25,000 psi at 4°C, and then clarified by centrifugation twice at 30,000 × g for 20 min. The supernatant was subjected to Ni-NTA affinity chromatography using the AKTAexplorer 100 system (GE Healthcare). After lysate application onto the column, the column was washed with a five-column volume of wash buffer (50 mM potassium phosphate pH 7.6, 1 M KCl, 1% Tween 20, 10% Glycerol, 20 mM imidazole, 0.2 mM PMSF and 1 mM DTT). A gradient elution method was used to reach the final imidazole concentration of 250 mM. Eluted fractions were buffer-exchanged into buffer A (30 mM HEPES·KOH (pH 7.5), 5% glycerol, 65 mM ammonium chloride, 7 mM β–mercaptoethanol and 1 tablet of miniComplete protease inhibitor) for HiTrap SP Sepharose High Performance cation-exchange chromatography (GE Healthcare). A gradient elution method was used to reach the final salt concentration of 1 M KCl in buffer A over the 20-column volume (100 ml). The peak fraction was concentrated and buffer-exchanged into buffer A, which is also the buffer used for the subsequent size-exclusion chromatography employing Superdex 200 (GE Healthcare). Fractions corresponding to the eEF2 peak were concentrated and stored in aliquots at -20°C. + + + METHODS + title_2 + 79145 + 80S•TSV IRES•eEF2•GTP•sordarin complex preparation + + + METHODS + paragraph + 79204 + The IRES-eEF2-ribosome complex was assembled in two steps. First, refolded TSV IRES RNA (8 μM - all concentrations are specified for the final complex) was incubated with the yeast 40S small subunit (0.8 μM) for 15 min at 30°C, in the buffer containing 45 mM HEPES·KOH (pH 7.5), 10 mM MgCl2, 100 mM KCl, 2.5 mM spermine and 2 mM β–mercaptoethanol. The 60S subunit (0.8 μM) was then added and incubated for 15 min at 30°C. Subsequently, eEF2 (5 μM), sordarin (800 μM) and GTP (1 mM) were added and incubated for 15 min at 30°C. The solution was then incubated on ice for 10 min and flash-frozen in liquid nitrogen. + + + METHODS + title_2 + 79852 + Cryo-EM specimen preparation + + + METHODS + paragraph + 79881 + Quantifoil Cu 200 mesh grids (SPI Supplies, West Chester, PA) were coated with a thin layer of carbon and glow discharged for 45 s at 25 mA. 3 µL of sample with a concentration of ~0.1 µM was applied to the grid, incubated for 30 s and plunged into liquid ethane using an FEI Vitrobot Mark 2 (FEI Company, Hillsboro, OR) after blotting for 3 s at 4°C and ~85% relative humidity. + + + METHODS + title_2 + 80269 + Electron microscopy + + + METHODS + paragraph + 80289 + Cryo-EM data were collected in movie mode on an FEI Krios microscope (FEI Company, Hillsboro, OR) operating at 300 kV and equipped with a K2 Summit direct detector (Gatan Inc., Pleasanton, CA) operating in super-resolution mode with pixel size of 0.82 Å per super-resolution pixel. Each movie consisted of 50 frames collected over 18.8 s with an exposure per frame of 1.4 e-/Å2 as shown by Digital Micrograph (Gatan Inc., Pleasanton, CA), giving a total exposure of 70 e-/Å2. The defocus ranged between ~0.7 to ~2.5 µm underfocus. + + + METHODS + title_2 + 80828 + Image processing + + + METHODS + paragraph + 80845 + The gain-corrected super-resolution movie frames were corrected for anisotropic magnification using bilinear interpolation. The frames were downsampled by Fourier cropping to a pixel size of 1.64 Å. The downsampled frames were then motion-corrected and exposure filtered using Unblur. The image defocus was determined using CTFFIND4 on non-exposure-filtered images and images with excessive motion, low contrast, ice contamination or poor power spectra were removed based on visual inspection using TIGRIS (http://tigris.sourceforge.net/). 50 particles were picked manually using TIGRIS, summed and rotationally averaged to serve as a reference for correlation-based particle picking in IMAGIC. 1,105,737 two-dimensional images of ribosomes (termed 'particles') were picked automatically, extracted into 256 x 256 boxes and converted to MRC/CCP4 format with a corresponding list of micrograph numbers and defocus values for input to FREALIGN v9. + + + METHODS + paragraph + 81794 + The summary of procedures resulting in 3D cryo-EM maps is presented on Figure 1—figure supplement 2. FREALIGN v9 was used for refinement, classification and 3D reconstruction of all ribosome structures. Initial particle alignments were obtained by performing an angular grid search (FREALIGN mode 3) with a density map calculated from the atomic model of the non-rotated 80S ribosome bound with 2 tRNAs (PDB: 3J78). For this search, the resolution was limited to 20 Å and the resolution of the resulting reconstruction was 3.6 Å, as determined by the FSC = 0.143 threshold criterion. Four additional rounds of mode 3 with the resolution limited to 7 Å improved the resolution of the reconstruction to 3.5 Å. + + + METHODS + paragraph + 82516 + Starting with cycle 6, particles were classified into six classes using 21 rounds of mode 1 refinement. Inspection of the six classes suggested that several represented mixed conformations. The alignment parameters of the class containing the largest number of particles (25%) were therefore used to initialize classification into 15 classes. For this classification, particle images were downsampled by Fourier cropping to a pixel size of 3.28 Å to accelerate processing. 99 rounds of refinement and classification were performed using mode 1 with a resolution limit of 7 Å. To help separate different conformations affecting small subunit, IRES and eEF2, we used a 3D mask that included density belonging to these parts of the structure. This mask was applied in every cycle to the 3D reference structures prior to refinement and classification in 42 additional cycles. The mask was then changed to include only the head of the small subunit, IRES and eEF2, and a final 18 cycles of refinement and classification were run. + + + METHODS + paragraph + 83547 + We selected six out of the 15 final classes based on clear density present for IRES and eEF2 and continued all further processing with this subset of the data (312,698 particles). The six classes were grouped into three groups based on the rotational state of the small subunit, and each group was further refined and classified using between six and 36 cycles of mode 1 and particles downsampled to 1.64 Å pixel size. For this classification, FREALIGN’s focused mask feature was used to select either the region of IRES PKI (for classes showing intermediate rotation of the small subunit) or a region containing both IRES PKI and eEF2 domain 4 (for classes showing no rotation of the small subunit). This refinement and sub-classification produced eight new classes with more distinct features in the regions selected by the focused masks. These eight classes were used as starting references for a final 33 cycles of refinement and classification using mode 1 and focused mask with the radius of 45 Å covering the vicinity of the ribosomal A site. The first 26 cycles were performed using particles downsampled to 3.28 Å pixel size, followed by two cycles at a pixel size of 1.64 Å, and five cycles at a pixel size of 0.82 Å. The resolution limit for the final cycles was set at 5 Å. The resulting eight reconstructions were used for further analyses, model building and structural refinements, as described below. In parallel, to enhance resolution of the IRES 5´ domain, we performed classification and refinement of the eight classes using a mask with the radius of 50 Å covering the vicinity of the E site and L1 stalk; these maps were used for model building and confirmation of the IRES 5´ domain structure, but not for structure refinements. + + + METHODS + paragraph + 85323 + Among the resulting eight reconstructions, four reconstructions contained well defined PKI and eEF2 densities (I, II, IV and V) (Figure 1—figure supplement 1). PKI was poorly resolved in reconstruction III. Reconstruction VI represents the previously reported 80S•TSV IRES initiation complex in the least rotated conformation. Reconstructions VII and VIII correspond to ribosomes adopting intermediate rotational states, similar to that of Structure III, with weak density in the region of the 5’ domain of the IRES and no density for the PKI domain. To resolve heterogeneity of PKI in reconstruction III, we performed additional sub-classification of all eight classes into two or three classes each. This sub-classification did not result into different structures for the four classes of interest (I, II, IV and V), suggesting a high degree of homogeneity in the masked regions of PKI and eEF2 domain IV. Sub-classification of reconstruction III helped improve the PKI density, resulting in a 4.2 Å reconstruction. All maps were subsequently B-factor-filtered by bfactor.exe, using the B-factors of -50 to -120 Å2, as suggested by bfactor.exe for individual maps, and used for real-space structure refinements. FSC curves (Figure 1—figure supplement 3) were calculated by FREALIGN for even and odd particles half-sets. Blocres was used to calculate the local resolution of unfiltered and unmasked volumes using a box size 60 pixel, step size of 3 pixels and FSC resolution criterion (threshold 0.143). The output volumes were then colored according to the local resolution of the final reconstructions (Figure 1—figure supplement 3) using the Surface Color tool of Chimera + + + METHODS + title_2 + 87014 + Model building and refinement + + + METHODS + paragraph + 87044 + The starting structural models were assembled using the high-resolution crystal structure of S. cerevisiae 80S ribosome, the cryo-EM structure of the 80S•TSV IRES complex and the crystal structure of the isolated eEF2•sordarin complex. The structure of the diphthamide residue of eEF2 (699) was adopted from PDB: 1ZM4. Initial domain fitting into the cryo-EM maps was performed using Chimera, followed by manual modeling of local regions into the density using Pymol and Coot. Parts of several ribosomal proteins were modeled using I-TASSER  and Phyre2 . The structural models were refined by real-space simulated-annealing refinement using atomic electron scattering factors, employing RSRef as described. Secondary-structure restrains for ribosomal proteins and base-pairing restraints for RNA molecules were employed, as described. The refined structural models closely agree with the corresponding maps, as shown by low real-space R-factors of ~0.2 to 0.27, and they have good stereochemical parameters, characterized by low deviation from ideal bond lengths and angles (Figure 1—source data 1). The maps revealed regions, which are differently resolved in Structures I to V. The most prominent difference is in the platform subdomain of the 40S subunit, which is well resolved in Structures I, IV and V but poorly resolved in Structures II and III. The following components of the 40S platform in Structures II and III lacked resolution: proteins eS1, uS11, eS26 and eL41, 18S rRNA nt 892–900, 900–918 and the 3´ end beyond nt 1792. These and other regions of low density were modeled as protein or RNA backbone. + + + METHODS + paragraph + 88677 + For structural comparisons, the distances and angles were calculated in Pymol and Chimera, respectively. To calculate an angle of the 40S subunit rotation between two 80S structures, the 25S rRNAs were aligned using Pymol, and the angle between 18S rRNAs was measured in Chimera. To calculate an angle of the 40S-head rotation (swivel) between two 80S structures, the 18S rRNAs of the bulk of the 40S body (18S nucleotides excluding nt 1150–1620) were aligned using Pymol, and the angle between the 18S rRNA residues 1150–1620 was measured in Chimera. Figures were prepared in Pymol and Chimera. + + + ACK_FUND + title_1 + 89277 + Funding Information + + + ACK_FUND + paragraph + 89297 + This paper was supported by the following grants: + + + ACK_FUND + paragraph + 89347 + to Nikolaus Grigorieff. + + + ACK_FUND + paragraph + 89373 + to Nikolaus Grigorieff. + + + ACK_FUND + paragraph + 89398 + to Andrei A Korostelev. + + + ACK_FUND + paragraph + 89424 + to Andrei A Korostelev. + + + ACK_FUND + title_1 + 89450 + Additional information + + + COMP_INT + title_1 + 89473 + Competing interests + + + COMP_INT + footnote + 89493 + NG: Reviewing editor, eLife. + + + COMP_INT + footnote + 89522 + The other authors declare that no competing interests exist. + + + AUTH_CONT + title_1 + 89583 + Author contributions + + + AUTH_CONT + footnote + 89604 + PDA, Collected and analyzed cryo-EM data, Drafting or revising the article. + + + AUTH_CONT + footnote + 89680 + CSK, Prepared the ribosome•IRES•eEF2 complex, Built and refined structural models, Analysis and interpretation of data, Drafting or revising the article. + + + AUTH_CONT + footnote + 89838 + TG, Assisted with cryo-EM data processing and analyses, Drafting or revising the article. + + + AUTH_CONT + footnote + 89928 + NG, Designed the project, Assisted with cryo-EM data processing and analyses, Drafting or revising the article. + + + AUTH_CONT + footnote + 90040 + AAK, Designed the project, Built and refined structural models, Analysis and interpretation of data, Drafting or revising the article. + + + AUTH_CONT + title_1 + 90175 + Additional files + + + AUTH_CONT + title_2 + 90192 + Major datasets + + + AUTH_CONT + paragraph + 90207 + The following datasets were generated: + + + REF + title + 90246 + References + + + 3669 + 3677 + surname:AEvarsson;given-names:A + surname:Brazhnikov;given-names:E + surname:Garber;given-names:M + surname:Zheltonosova;given-names:J + surname:Chirgadze;given-names:Y + surname:al-Karadaghi;given-names:S + surname:Svensson;given-names:LA + surname:Liljas;given-names:A + 8070397 + REF + The EMBO Journal + ref + 13 + 1994 + 90257 + Three-dimensional structure of the ribosomal translocase: elongation factor G from Thermus thermophilus + + + 190 + 197 + surname:Agirrezabala;given-names:X + surname:Lei;given-names:J + surname:Brunelle;given-names:JL + surname:Ortiz-Meoz;given-names:RF + surname:Green;given-names:R + surname:Frank;given-names:J + 10.1016/j.molcel.2008.10.001 + 18951087 + REF + Molecular Cell + ref + 32 + 2008 + 90361 + Visualization of the hybrid state of trna binding promoted by spontaneous ratcheting of the ribosome + + + 9866 + 9895 + surname:Agirrezabala;given-names:X + surname:Valle;given-names:M + 10.3390/ijms16059866 + 25941930 + REF + International Journal of Molecular Sciences + ref + 16 + 2015 + 90462 + Structural insights into trna dynamics on the ribosome + + + e14874 + surname:Au;given-names:HH + surname:Jan;given-names:E + 10.1371/journal.pone.0051477 + REF + PloS One + ref + 7 + 2012 + 90517 + Insights into factorless translational initiation by the trna-like pseudoknot domain of a viral IRES + + + e14874 + surname:Bai;given-names:XC + surname:Fernandez;given-names:IS + surname:McMullan;given-names:G + surname:Scheres;given-names:SH + 10.7554/eLife.00461 + REF + eLife + ref + 2 + 2013 + 90618 + Ribosome structures to near-atomic resolution from thirty thousand cryo-em particles + + + 165 + 169 + surname:Ban;given-names:N + surname:Beckmann;given-names:R + surname:McAlear;given-names:MA + surname:Moore;given-names:PB + surname:Noller;given-names:HF + surname:Ortega;given-names:J + surname:Panse;given-names:VG + surname:Ramakrishnan;given-names:V + surname:Spahn;given-names:CM + surname:Steitz;given-names:TA + surname:Tchorzewski;given-names:M + surname:Tollervey;given-names:D + surname:Cate;given-names:JH + surname:Warren;given-names:AJ + surname:Williamson;given-names:JR + surname:Wilson;given-names:D + surname:Yonath;given-names:A + surname:Yusupov;given-names:M + surname:Dinman;given-names:JD + surname:Dragon;given-names:F + surname:Ellis;given-names:SR + surname:Lafontaine;given-names:DL + surname:Lindahl;given-names:L + surname:Liljas;given-names:A + surname:Lipton;given-names:JM + 10.1016/j.sbi.2014.01.002 + 24524803 + REF + Current Opinion in Structural Biology + ref + 24 + 2014 + 90703 + A new system for naming ribosomal proteins + + + 1524 + 1529 + surname:Ben-Shem;given-names:A + surname:Garreau de Loubresse;given-names:N + surname:Melnikov;given-names:S + surname:Jenner;given-names:L + surname:Yusupova;given-names:G + surname:Yusupov;given-names:M + 10.1126/science.1212642 + 22096102 + REF + Science + ref + 334 + 2011 + 90746 + The structure of the eukaryotic ribosome at 3.0 A resolution + + + 6544 + 6549 + surname:Bermek;given-names:E + 789367 + REF + The Journal of Biological Chemistry + ref + 251 + 1976 + 90807 + Interactions of adenosine diphosphate-ribosylated elongation factor 2 with ribosomes + + + 12893 + 12898 + surname:Blanchard;given-names:SC + surname:Kim;given-names:HD + surname:Gonzalez;given-names:RL + surname:Puglisi;given-names:JD + surname:Chu;given-names:S + 10.1073/pnas.0403884101 + 15317937 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 101 + 2004 + 90892 + tRNA dynamics on the ribosome during translation + + + 675 + 677 + surname:Bretscher;given-names:MS + 10.1038/218675a0 + 5655957 + REF + Nature + ref + 218 + 1968 + 90941 + Translocation in protein synthesis: A hybrid structure model + + + surname:Brilot;given-names:AF + surname:Korostelev;given-names:AA + surname:Ermolenko;given-names:DN + surname:Grigorieff;given-names:N + 10.1073/pnas.1311423110 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 110 + 2013 + 91002 + Structure of the ribosome with elongation factor G trapped in the pretranslocation state + + + 214 + 224 + surname:Budkevich;given-names:T + surname:Giesebrecht;given-names:J + surname:Altman;given-names:RB + surname:Munro;given-names:JB + surname:Mielke;given-names:T + surname:Nierhaus;given-names:KH + surname:Blanchard;given-names:SC + surname:Spahn;given-names:CM + 10.1016/j.molcel.2011.07.040 + 22017870 + REF + Molecular Cell + ref + 44 + 2011 + 91091 + Structure and dynamics of the mammalian ribosomal pretranslocation complex + + + 226 + 236 + surname:Cardone;given-names:G + surname:Heymann;given-names:JB + surname:Steven;given-names:AC + 10.1016/j.jsb.2013.08.002 + 23954653 + REF + Journal of Structural Biology + ref + 184 + 2013 + 91166 + One number does not fit all: Mapping local variations in resolution in cryo-em reconstructions + + + 677 + 683 + surname:Cevallos;given-names:RC + surname:Sarnow;given-names:P + 10.1128/JVI.79.2.677-683.2005 + 15613295 + REF + Journal of Virology + ref + 79 + 2005 + 91261 + Factor-independent assembly of elongation-competent ribosomes by an internal ribosome entry site located in an RNA virus that infects penaeid shrimp + + + 69 + 80 + surname:Chapman;given-names:MS + 10.1107/S0108767394007130 + REF + Acta Crystallographica Section a Foundations of Crystallography + ref + 51 + 1995 + 91410 + Restrained real-space macromolecular atomic refinement using a new resolution-dependent electron-density function + + + 1097 + 1105 + surname:Chen;given-names:B + surname:Kaledhonkar;given-names:S + surname:Sun;given-names:M + surname:Shen;given-names:B + surname:Lu;given-names:Z + surname:Barnard;given-names:D + surname:Lu;given-names:TM + surname:Gonzalez;given-names:RL + surname:Frank;given-names:J + 10.1016/j.str.2015.04.007 + 26004440 + REF + Structure + ref + 23 + 2015 + 91524 + Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy + + + 718 + 727 + surname:Chen;given-names:J + surname:Petrov;given-names:A + surname:Tsai;given-names:A + surname:O'Leary;given-names:SE + surname:Puglisi;given-names:JD + 10.1038/nsmb.2567 + REF + Nature Structural & Molecular Biology + ref + 20 + 2013a + 91647 + Coordinated conformational and compositional dynamics drive ribosome translocation + + + 1077 + 1084 + surname:Chen;given-names:Y + surname:Feng;given-names:S + surname:Kumar;given-names:V + surname:Ero;given-names:R + surname:Gao;given-names:Y-G + 10.1038/nsmb.2645 + REF + Nature Structural & Molecular Biology + ref + 20 + 2013b + 91730 + Structure of ef-g–ribosome complex in a pretranslocation state + + + 438 + 449 + surname:Cheng;given-names:Y + surname:Grigorieff;given-names:N + surname:Penczek;given-names:PA + surname:Walz;given-names:T + 10.1016/j.cell.2015.03.050 + 25910204 + REF + Cell + ref + 161 + 2015 + 91795 + A primer to single-particle cryo-electron microscopy + + + 1793 + 1803 + surname:Collier;given-names:RJ + 10.1016/S0041-0101(01)00165-9 + 11595641 + REF + Toxicon : Official Journal of the International Society on Toxinology + ref + 39 + 2001 + 91848 + Understanding the mode of action of diphtheria toxin: A perspective on progress during the 20th century + + + 110 + 113 + surname:Colussi;given-names:TM + surname:Costantino;given-names:DA + surname:Zhu;given-names:J + surname:Donohue;given-names:JP + surname:Korostelev;given-names:AA + surname:Jaafar;given-names:ZA + surname:Plank;given-names:T-DM + surname:Noller;given-names:HF + surname:Kieft;given-names:JS + 10.1038/nature14219 + 25652826 + REF + Nature + ref + 519 + 2015 + 91952 + Initiation of translation in bacteria by a structured eukaryotic IRES RNA + + + 578 + 588 + surname:Cornish;given-names:PV + surname:Ermolenko;given-names:DN + surname:Noller;given-names:HF + surname:Ha;given-names:T + 10.1016/j.molcel.2008.05.004 + 18538656 + REF + Molecular Cell + ref + 30 + 2008 + 92026 + Spontaneous intersubunit rotation in single ribosomes + + + 57 + 64 + surname:Costantino;given-names:DA + surname:Pfingsten;given-names:JS + surname:Rambo;given-names:RP + surname:Kieft;given-names:JS + 10.1038/nsmb1351 + REF + Nature Structural & Molecular Biology + ref + 15 + 2008 + 92080 + tRNA-mrna mimicry drives translation initiation from a viral IRES + + + 321 + 328 + surname:Cukras;given-names:AR + surname:Southworth;given-names:DR + surname:Brunelle;given-names:JL + surname:Culver;given-names:GM + surname:Green;given-names:R + 10.1016/S1097-2765(03)00275-2 + 14536072 + REF + Molecular Cell + ref + 12 + 2003 + 92146 + Ribosomal proteins S12 and S13 function as control elements for translocation of the mrna:trna complex + + + e14874 + surname:Cunha;given-names:CE + surname:Belardinelli;given-names:R + surname:Peske;given-names:F + surname:Holtkamp;given-names:W + surname:Wintermeyer;given-names:W + surname:Rodnina;given-names:MV + 10.4161/trla.24315 + REF + Translation + ref + 1 + 2013 + 92249 + Dual use of GTP hydrolysis by elongation factor G on the ribosome + + + 3661 + 3668 + surname:Czworkowski;given-names:J + surname:Wang;given-names:J + surname:Steitz;given-names:TA + surname:Moore;given-names:PB + 8070396 + REF + The EMBO Journal + ref + 13 + 1994 + 92315 + The crystal structure of elongation factor G complexed with GDP, at 2.7 A resolution + + + 350 + 352 + surname:Davydova;given-names:EK + surname:Ovchinnikov;given-names:LP + 10.1016/0014-5793(90)80589-B + 2311763 + REF + FEBS Letters + ref + 261 + 1990 + 92400 + ADP-ribosylated elongation factor 2 (adp-ribosyl-ef-2) is unable to promote translocation within the ribosome + + + 48 + 57 + surname:Deforges;given-names:J + surname:Locker;given-names:N + surname:Sargueil;given-names:B + 10.1016/j.biochi.2014.12.008 + 25530261 + REF + Biochimie + ref + 114 + 2015 + 92510 + mRNAs that specifically interact with eukaryotic ribosomal subunits + + + surname:DeLano;given-names:WL + REF + The Pymol Molecular Graphics System + ref + 2002 + 92578 + + + 256 + 259 + surname:Demeshkina;given-names:N + surname:Jenner;given-names:L + surname:Westhof;given-names:E + surname:Yusupov;given-names:M + surname:Yusupova;given-names:G + 10.1038/nature10913 + 22437501 + REF + Nature + ref + 484 + 2012 + 92579 + A new understanding of the decoding principle on the ribosome + + + 22423 + 22427 + surname:Domínguez;given-names:JM + surname:Gómez-Lorenzo;given-names:MG + surname:Martín;given-names:JJ + 10.1074/jbc.274.32.22423 + 10428815 + REF + The Journal of Biological Chemistry + ref + 274 + 1999 + 92641 + Sordarin inhibits fungal protein synthesis by blocking translocation differently to fusidic acid + + + 227 + 244 + surname:Dunkle;given-names:JA + surname:Cate;given-names:JH + 10.1146/annurev.biophys.37.032807.125954 + REF + Annual Review of Biophysics + ref + 39 + 2010 + 92738 + Ribosome structure and dynamics during translocation and termination + + + 2126 + 2132 + surname:Emsley;given-names:P + surname:Cowtan;given-names:K + 10.1107/S0907444904019158 + 15572765 + REF + Acta Crystallographica. Section D, Biological Crystallography + ref + 60 + 2004 + 92807 + Coot: Model-building tools for molecular graphics + + + 158 + 166 + surname:Ermolenko;given-names:DN + surname:Cornish;given-names:PV + surname:Ha;given-names:T + surname:Noller;given-names:HF + 10.1261/rna.035964.112 + 23249745 + REF + RNA + ref + 19 + 2013 + 92857 + Antibiotics that bind to the A site of the large ribosomal subunit can induce mrna translocation + + + 530 + 540 + surname:Ermolenko;given-names:DN + surname:Majumdar;given-names:ZK + surname:Hickerson;given-names:RP + surname:Spiegel;given-names:PC + surname:Clegg;given-names:RM + surname:Noller;given-names:HF + 10.1016/j.jmb.2007.04.042 + 17512008 + REF + Journal of Molecular Biology + ref + 370 + 2007 + 92954 + Observation of intersubunit movement of the ribosome in solution using FRET + + + 457 + 462 + surname:Ermolenko;given-names:DN + surname:Noller;given-names:HF + 10.1038/nsmb.2011 + REF + Nature Structural & Molecular Biology + ref + 18 + 2011 + 93030 + mRNA translocation occurs during the second step of ribosomal intersubunit rotation + + + 348 + 359 + surname:Fei;given-names:J + surname:Kosuri;given-names:P + surname:MacDougall;given-names:DD + surname:Gonzalez;given-names:RL + 10.1016/j.molcel.2008.03.012 + 18471980 + REF + Molecular Cell + ref + 30 + 2008 + 93114 + Coupling of ribosomal L1 stalk and trna dynamics during translation elongation + + + 823 + 831 + surname:Fernández;given-names:IS + surname:Bai;given-names:XC + surname:Murshudov;given-names:G + surname:Scheres;given-names:SH + surname:Ramakrishnan;given-names:V + 10.1016/j.cell.2014.04.015 + 24792965 + REF + Cell + ref + 157 + 2014 + 93193 + Initiation of translation by cricket paralysis virus IRES requires its translocation in the ribosome + + + 329 + 333 + surname:Fischer;given-names:N + surname:Konevega;given-names:AL + surname:Wintermeyer;given-names:W + surname:Rodnina;given-names:MV + surname:Stark;given-names:H + 10.1038/nature09206 + 20631791 + REF + Nature + ref + 466 + 2010 + 93294 + Ribosome dynamics and trna movement by time-resolved electron cryomicroscopy + + + 318 + 322 + surname:Frank;given-names:J + surname:Agrawal;given-names:RK + 10.1038/35018597 + 10917535 + REF + Nature + ref + 406 + 2000 + 93371 + A ratchet-like inter-subunit reorganization of the ribosome during translocation + + + 694 + 699 + surname:Gao;given-names:Y-G + surname:Selmer;given-names:M + surname:Dunham;given-names:CM + surname:Weixlbaumer;given-names:A + surname:Kelley;given-names:AC + surname:Ramakrishnan;given-names:V + 10.1126/science.1179709 + 19833919 + REF + Science + ref + 326 + 2009 + 93452 + The structure of the ribosome with elongation factor G trapped in the posttranslocational state + + + 537 + 552 + surname:Gavrilova;given-names:LP + surname:Kostiashkina;given-names:OE + surname:Koteliansky;given-names:VE + surname:Rutkevitch;given-names:NM + surname:Spirin;given-names:AS + 10.1016/0022-2836(76)90243-6 + 772221 + REF + Journal of Molecular Biology + ref + 101 + 1976 + 93548 + Factor-free ("non-enzymic") and factor-dependent systems of translation of polyuridylic acid by escherichia coli ribosomes + + + 248 + 254 + surname:Gavrilova;given-names:LP + surname:Spirin;given-names:AS + 4566546 + REF + Molecular Biology + ref + 6 + 1972 + 93671 + Mechanism of translocation in ribosomes. II. Activation of spontaneous (nonenzymic) translocation in ribosomes of Escherichia coli by p-chloromercuribenzoate + + + 1087 + 1184 + surname:Givaty;given-names:O + surname:Levy;given-names:Y + 10.1016/j.jmb.2008.11.016 + 19059266 + REF + Journal of Molecular Biology + ref + 385 + 2009 + 93829 + Protein sliding along DNA: dynamics and structural characterization + + + 633 + 638 + surname:Gonen;given-names:T + surname:Cheng;given-names:Y + surname:Sliz;given-names:P + surname:Hiroaki;given-names:Y + surname:Fujiyoshi;given-names:Y + surname:Harrison;given-names:SC + surname:Walz;given-names:T + 10.1038/nature04321 + 16319884 + REF + Nature + ref + 438 + 2005 + 93897 + Lipid–protein interactions in double-layered two-dimensional AQP0 crystals + + + 932 + 940 + surname:Gorman;given-names:J + surname:Plys;given-names:AJ + surname:Visnapuu;given-names:ML + surname:Alani;given-names:E + surname:Greene;given-names:EC + 10.1038/nsmb.1858 + REF + Nature Structural & Molecular Biology + ref + 17 + 2010 + 93974 + Visualizing one-dimensional diffusion of eukaryotic DNA repair factors along a chromatin lattice + + + 204 + 208 + surname:Grant;given-names:T + surname:Grigorieff;given-names:N + 10.1016/j.jsb.2015.08.006 + 26278979 + REF + Journal of Structural Biology + ref + 192 + 2015a + 94071 + Automatic estimation and correction of anisotropic magnification distortion in electron microscopes + + + e14874 + surname:Grant;given-names:T + surname:Grigorieff;given-names:N + 10.7554/eLife.06980 + REF + eLife + ref + 4 + 2015b + 94171 + Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6 + + + 20391 + 20394 + surname:Guo;given-names:Z + surname:Noller;given-names:HF + 10.1073/pnas.1218999109 + 23188795 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 109 + 2012 + 94277 + Rotation of the head of the 30S ribosomal subunit during mrna translocation + + + 343 + 351 + surname:Halford;given-names:SE + 10.1042/BST0370343 + 19290859 + REF + Biochemical Society Transactions + ref + 37 + 2009 + 94353 + An end to 40 years of mistakes in DNA-protein association kinetics? + + + 779 + 786 + surname:Hatakeyama;given-names:Y + surname:Shibuya;given-names:N + surname:Nishiyama;given-names:T + surname:Nakashima;given-names:N + 10.1261/rna.5208104 + 15100433 + REF + RNA + ref + 10 + 2004 + 94421 + Structural variant of the intergenic internal ribosome entry site elements in dicistroviruses and computational search for their counterparts + + + 1073 + 1085 + surname:Holtkamp;given-names:W + surname:Cunha;given-names:CE + surname:Peske;given-names:F + surname:Konevega;given-names:AL + surname:Wintermeyer;given-names:W + surname:Rodnina;given-names:MV + 10.1002/embj.201387465 + 24614227 + REF + The EMBO Journal + ref + 33 + 2014 + 94563 + GTP hydrolysis by EF-G synchronizes trna movement on small and large ribosomal subunits + + + 4881 + 4885 + surname:Horan;given-names:LH + surname:Noller;given-names:HF + 10.1073/pnas.0700762104 + 17360328 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 104 + 2007 + 94651 + Intersubunit movement is required for ribosomal translocation + + + 113 + 127 + surname:Jackson;given-names:RJ + surname:Hellen;given-names:CU + surname:Pestova;given-names:TV + 10.1038/nrm2838 + 20094052 + REF + Nature Reviews. Molecular Cell Biology + ref + 11 + 2010 + 94713 + The mechanism of eukaryotic translation initiation and principles of its regulation + + + 15410 + 15415 + surname:Jan;given-names:E + surname:Kinzy;given-names:TG + surname:Sarnow;given-names:P + 10.1073/pnas.2535183100 + 14673072 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 100 + 2003 + 94797 + Divergent trna-like element supports initiation, elongation, and termination of protein biosynthesis + + + 889 + 902 + surname:Jan;given-names:E + surname:Sarnow;given-names:P + 10.1016/S0022-2836(02)01099-9 + 12470947 + REF + Journal of Molecular Biology + ref + 324 + 2002 + 94898 + Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus + + + 285 + 292 + surname:Jan;given-names:E + surname:Thompson;given-names:SR + surname:Wilson;given-names:JE + surname:Pestova;given-names:TV + surname:Hellen;given-names:CU + surname:Sarnow;given-names:P + 10.1101/sqb.2001.66.285 + 12762030 + REF + Cold Spring Harbor Symposia on Quantitative Biology + ref + 66 + 2001 + 94990 + Initiator met-trna-independent translation mediated by an internal ribosome entry site element in cricket paralysis virus-like insect viruses + + + 1072 + 1078 + surname:Jenner;given-names:L + surname:Demeshkina;given-names:N + surname:Yusupova;given-names:G + surname:Yusupov;given-names:M + 10.1038/nsmb.1880 + REF + Nature Structural & Molecular Biology + ref + 17 + 2010 + 95132 + Structural rearrangements of the ribosome at the trna proofreading step + + + 979 + 984 + surname:Jørgensen;given-names:R + surname:Merrill;given-names:AR + surname:Yates;given-names:SP + surname:Marquez;given-names:VE + surname:Schwan;given-names:AL + surname:Boesen;given-names:T + surname:Andersen;given-names:GR + 10.1038/nature03871 + 16107839 + REF + Nature + ref + 436 + 2005 + 95204 + Exotoxin a–eef2 complex structure indicates ADP ribosylation by ribosome mimicry + + + 3478 + 3483 + surname:Joseph;given-names:S + surname:Noller;given-names:HF + 10.1093/emboj/17.12.3478 + 9628883 + REF + The EMBO Journal + ref + 17 + 1998 + 95287 + EF-g-catalyzed translocation of anticodon stem-loop analogs of transfer RNA in the ribosome + + + 160 + 164 + surname:Joseph;given-names:S + 10.1261/rna.2163103 + 12554856 + REF + RNA + ref + 9 + 2003 + 95379 + After the ribosome structure: How does translocation work? + + + 16924 + 16927 + surname:Julián;given-names:P + surname:Konevega;given-names:AL + surname:Scheres;given-names:SH + surname:Lázaro;given-names:M + surname:Gil;given-names:D + surname:Wintermeyer;given-names:W + surname:Rodnina;given-names:MV + surname:Valle;given-names:M + 10.1073/pnas.0809587105 + 18971332 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 105 + 2008 + 95438 + Structure of ratcheted ribosomes with trnas in hybrid states + + + 3148 + 3151 + surname:Justice;given-names:MC + surname:Hsu;given-names:MJ + surname:Tse;given-names:B + surname:Ku;given-names:T + surname:Balkovec;given-names:J + surname:Schmatz;given-names:D + surname:Nielsen;given-names:J + 10.1074/jbc.273.6.3148 + 9452424 + REF + The Journal of Biological Chemistry + ref + 273 + 1998 + 95499 + Elongation factor 2 as a novel target for selective inhibition of fungal protein synthesis + + + 379 + 385 + surname:Jørgensen;given-names:R + surname:Ortiz;given-names:PA + surname:Carr-Schmid;given-names:A + surname:Nissen;given-names:P + surname:Kinzy;given-names:TG + surname:Andersen;given-names:GR + 10.1038/nsb923 + 12692531 + REF + Nature Structural Biology + ref + 10 + 2003 + 95590 + Two crystal structures demonstrate large conformational changes in the eukaryotic ribosomal translocase + + + 845 + 903 + surname:Kelley;given-names:LA + surname:Mezulis;given-names:S + surname:Yates;given-names:CM + surname:Wass;given-names:MN + surname:Sternberg;given-names:MJ + 10.1038/nprot.2015.053 + 25950237 + REF + Nature Protocols + ref + 10 + 2015 + 95694 + The Phyre2 web portal for protein modeling, prediction and analysis + + + 9139 + 9144 + surname:Koh;given-names:CS + surname:Brilot;given-names:AF + surname:Grigorieff;given-names:N + surname:Korostelev;given-names:AA + 10.1073/pnas.1406335111 + 24927574 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 111 + 2014 + 95762 + Taura syndrome virus IRES initiates translation by binding its trna-mrna-like structural element in the ribosomal decoding center + + + 436 + 479 + surname:Korennykh;given-names:AV + surname:Piccirilli;given-names:JA + surname:Correll;given-names:CC + 10.1038/nsmb1082 + REF + Nature Structural & Molecular Biology + ref + 13 + 2006 + 95892 + The electrostatic character of the ribosomal surface enables extraordinarily rapid target location by ribotoxins + + + surname:Koripella;given-names:RK + surname:Holm;given-names:M + surname:Dourado;given-names:D + surname:Mandava;given-names:CS + surname:Flores;given-names:S + surname:Sanyal;given-names:S + 10.1038/srep12970 + REF + Scientific Reports + ref + 5 + 2015 + 96005 + A conserved histidine in switch-ii of EF-G moderates release of inorganic phosphate + + + 761 + 767 + surname:Korostelev;given-names:A + surname:Bertram;given-names:R + surname:Chapman;given-names:MS + 10.1107/S0907444902003402 + 11976486 + REF + Acta Crystallographica. Section D, Biological Crystallography + ref + 58 + 2002 + 96089 + Simulated-annealing real-space refinement as a tool in model building + + + 674 + 683 + surname:Korostelev;given-names:A + surname:Ermolenko;given-names:DN + surname:Noller;given-names:HF + 10.1016/j.cbpa.2008.08.037 + 18848900 + REF + Current Opinion in Chemical Biology + ref + 12 + 2008 + 96159 + Structural dynamics of the ribosome + + + 1065 + 1077 + surname:Korostelev;given-names:A + surname:Trakhanov;given-names:S + surname:Laurberg;given-names:M + surname:Noller;given-names:HF + 10.1016/j.cell.2006.08.032 + 16962654 + REF + Cell + ref + 126 + 2006 + 96195 + Crystal structure of a 70S ribosome-trna complex reveals functional interactions and rearrangements + + + 852 + 857 + surname:Laurberg;given-names:M + surname:Asahara;given-names:H + surname:Korostelev;given-names:A + surname:Zhu;given-names:J + surname:Trakhanov;given-names:S + surname:Noller;given-names:HF + 10.1038/nature07115 + 18596689 + REF + Nature + ref + 454 + 2008 + 96295 + Structural basis for translation termination on the 70S ribosome + + + 219 + 246 + surname:Lin;given-names:J + surname:Gagnon;given-names:MG + surname:Bulkley;given-names:D + surname:Steitz;given-names:TA + 10.1016/j.cell.2014.11.049 + 25594181 + REF + Cell + ref + 160 + 2015 + 96360 + Conformational changes of elongation factor G on the ribosome during tRNA translocation + + + surname:Ling;given-names:C + surname:Ermolenko;given-names:DN + 10.1002/wrna.1354 + REF + Wiley Interdisciplinary Reviews. RNA + ref + 2016 + 96448 + Structural insights into ribosome translocation + + + e14874 + surname:Liu;given-names:T + surname:Kaplan;given-names:A + surname:Tinoco;given-names:I + surname:Bustamante;given-names:CJ + surname:Alexander;given-names:L + surname:Yan;given-names:S + surname:Wen;given-names:JD + surname:Lancaster;given-names:L + surname:Wickersham;given-names:CE + surname:Fredrick;given-names:K + surname:Fredrik;given-names:K + surname:Noller;given-names:H + 10.7554/eLife.03406 + REF + eLife + ref + 3 + 2014 + 96496 + Direct measurement of the mechanical work during translocation by the ribosome + + + 113 + 120 + surname:Lozano;given-names:G + surname:Martínez-Salas;given-names:E + 10.1016/j.coviro.2015.04.008 + 26004307 + REF + Current Opinion in Virology + ref + 12 + 2015 + 96575 + Structural insights into viral ires-dependent translation mechanisms + + + 377 + 388 + surname:Lyumkis;given-names:D + surname:Brilot;given-names:AF + surname:Theobald;given-names:DL + surname:Grigorieff;given-names:N + 10.1016/j.jsb.2013.07.005 + 23872434 + REF + Journal of Structural Biology + ref + 183 + 2013 + 96644 + Likelihood-based classification of cryo-em images using FREALIGN + + + 155 + 159 + surname:Martemyanov;given-names:KA + surname:Gudkov;given-names:AT + 10.1016/S0014-5793(99)00635-3 + 10386581 + REF + FEBS Letters + ref + 452 + 1999 + 96709 + Domain IV of elongation factor G from Thermus thermophilus is strictly required for translocation + + + 142 + 148 + surname:Moazed;given-names:D + surname:Noller;given-names:HF + 10.1038/342142a0 + 2682263 + REF + Nature + ref + 342 + 1989 + 96807 + Intermediate states in the movement of transfer RNA in the ribosome + + + 114 + 117 + surname:Moore;given-names:GE + REF + Electronics + ref + 86 + 1965 + 96875 + Cramming more components onto integrated circuits + + + 422 + 432 + surname:Muhs;given-names:M + surname:Hilal;given-names:T + surname:Mielke;given-names:T + surname:Skabkin;given-names:MA + surname:Sanbonmatsu;given-names:KY + surname:Pestova;given-names:TV + surname:Spahn;given-names:CM + 10.1016/j.molcel.2014.12.016 + 25601755 + REF + Molecular Cell + ref + 57 + 2015 + 96925 + Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated cricket paralysis virus IRES + + + 2434 + 2442 + surname:Nishiyama;given-names:T + surname:Yamamoto;given-names:H + surname:Shibuya;given-names:N + surname:Hatakeyama;given-names:Y + surname:Hachimori;given-names:A + surname:Uchiumi;given-names:T + surname:Nakashima;given-names:N + 10.1093/nar/gkg336 + 12711689 + REF + Nucleic Acids Research + ref + 31 + 2003 + 97039 + Structural elements in the internal ribosome entry site of plautia stali intestine virus responsible for binding with ribosomes + + + 6030 + 6034 + surname:Nygård;given-names:O + surname:Nilsson;given-names:L + 2318846 + REF + The Journal of Biological Chemistry + ref + 265 + 1990 + 97167 + Kinetic determination of the effects of ADP-ribosylation on the interaction of eukaryotic elongation factor 2 with ribosomes + + + 897 + 902 + surname:Ogle;given-names:JM + surname:Brodersen;given-names:DE + surname:Clemons;given-names:WM + surname:Tarry;given-names:MJ + surname:Carter;given-names:AP + surname:Ramakrishnan;given-names:V + 10.1126/science.1060612 + 11340196 + REF + Science + ref + 292 + 2001 + 97292 + Recognition of cognate transfer RNA by the 30S ribosomal subunit + + + 519 + 529 + surname:Pan;given-names:D + surname:Kirillov;given-names:SV + surname:Cooperman;given-names:BS + 10.1016/j.molcel.2007.01.014 + 17317625 + REF + Molecular Cell + ref + 25 + 2007 + 97357 + Kinetically competent intermediates in the translocation step of protein synthesis + + + 726 + 733 + surname:Pestka;given-names:S + 10.1073/pnas.61.2.726 + 5246003 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 61 + 1968 + 97440 + Studies on the formation of trensfer ribonucleic acid-ribosome complexes. synthesis + + + 181 + 186 + surname:Pestova;given-names:TV + surname:Hellen;given-names:CU + 10.1101/gad.1040803 + 12533507 + REF + Genes & Development + ref + 17 + 2003 + 97524 + Translation elongation after assembly of ribosomes on the cricket paralysis virus internal ribosomal entry site without initiation factors or initiator trna + + + 1605 + 1612 + surname:Pettersen;given-names:EF + surname:Goddard;given-names:TD + surname:Huang;given-names:CC + surname:Couch;given-names:GS + surname:Greenblatt;given-names:DM + surname:Meng;given-names:EC + surname:Ferrin;given-names:TE + 10.1002/jcc.20084 + 15264254 + REF + Journal of Computational Chemistry + ref + 25 + 2004 + 97681 + UCSF chimera--a visualization system for exploratory research and analysis + + + 205 + 217 + surname:Pfingsten;given-names:JS + surname:Castile;given-names:AE + surname:Kieft;given-names:JS + 10.1016/j.jmb.2009.10.047 + 19878683 + REF + Journal of Molecular Biology + ref + 395 + 2010 + 97756 + Mechanistic role of structurally dynamic regions in dicistroviridae IGR iress + + + 1235970 + surname:Pulk;given-names:A + surname:Cate;given-names:JHD + 10.1126/science.1235970 + 23812721 + REF + Science + ref + 340 + 2013 + 97834 + Control of ribosomal subunit rotation by elongation factor G + + + 20964 + 20969 + surname:Ramrath;given-names:DJ + surname:Lancaster;given-names:L + surname:Sprink;given-names:T + surname:Mielke;given-names:T + surname:Loerke;given-names:J + surname:Noller;given-names:HF + surname:Spahn;given-names:CM + 10.1073/pnas.1320387110 + 24324168 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 110 + 2013 + 97895 + Visualization of two transfer rnas trapped in transit during elongation factor g-mediated translocation + + + 713 + 716 + surname:Ratje;given-names:AH + surname:Loerke;given-names:J + surname:Whitford;given-names:PC + surname:Onuchic;given-names:JN + surname:Yu;given-names:Y + surname:Sanbonmatsu;given-names:KY + surname:Hartmann;given-names:RK + surname:Penczek;given-names:PA + surname:Wilson;given-names:DN + surname:Spahn;given-names:CMT + surname:Mikolajka;given-names:A + surname:Brünner;given-names:M + surname:Hildebrand;given-names:PW + surname:Starosta;given-names:AL + surname:Dönhöfer;given-names:A + surname:Connell;given-names:SR + surname:Fucini;given-names:P + surname:Mielke;given-names:T + 10.1038/nature09547 + 21124459 + REF + Nature + ref + 468 + 2010 + 97999 + Head swivel on the ribosome facilitates translocation by means of intra-subunit trna hybrid sites + + + 9366 + 9382 + surname:Ren;given-names:Q + surname:Au;given-names:HH + surname:Wang;given-names:QS + surname:Lee;given-names:S + surname:Jan;given-names:E + 10.1093/nar/gku622 + 25038250 + REF + Nucleic Acids Research + ref + 42 + 2014 + 98097 + Structural determinants of an internal ribosome entry site that direct translational reading frame selection + + + e14874 + 639 + surname:Ren;given-names:Q + surname:Wang;given-names:QS + surname:Firth;given-names:AE + surname:Chan;given-names:MM + surname:Gouw;given-names:JW + surname:Guarna;given-names:MM + surname:Foster;given-names:LJ + surname:Atkins;given-names:JF + surname:Jan;given-names:E + 10.1073/pnas.1111303109 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 109 + 2012 + 98206 + Alternative reading frame selection mediated by a trna-like domain of an internal ribosome entry site + + + 37 + 41 + surname:Rodnina;given-names:MV + surname:Savelsbergh;given-names:A + surname:Katunin;given-names:VI + surname:Wintermeyer;given-names:W + 10.1038/385037a0 + 8985244 + REF + Nature + ref + 385 + 1997 + 98308 + Hydrolysis of GTP by elongation factor G drives trna movement on the ribosome + + + 216 + 221 + surname:Rohou;given-names:A + surname:Grigorieff;given-names:N + 10.1016/j.jsb.2015.08.008 + 26278980 + REF + Journal of Structural Biology + ref + 192 + 2015 + 98386 + CTFFIND4: Fast and accurate defocus estimation from electron micrographs + + + 721 + 745 + surname:Rosenthal;given-names:PB + surname:Henderson;given-names:R + 10.1016/j.jmb.2003.07.013 + 14568533 + REF + Journal of Molecular Biology + ref + 333 + 2003 + 98459 + Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy + + + 725 + 763 + surname:Roy;given-names:A + surname:Kucukural;given-names:A + surname:Zhang;given-names:Y + 10.1038/nprot.2010.5 + 20360767 + REF + Nature Protocols + ref + 5 + 2010 + 98582 + I-TASSER: a unified platform for automated protein structure and function prediction + + + surname:Ruehle;given-names:MD + surname:Zhang;given-names:H + surname:Sheridan;given-names:RM + surname:Mitra;given-names:S + surname:Chen;given-names:Y + surname:Gonzalez;given-names:RL + surname:Cooperman;given-names:BS + surname:Kieft;given-names:JS + 10.7554/eLife.08146 + REF + eLife + ref + 4 + 2015 + 98667 + A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation + + + 7618 + 7626 + surname:Sahu;given-names:B + surname:Khade;given-names:PK + surname:Joseph;given-names:S + 10.1021/bi300930r + 22938718 + REF + Biochemistry + ref + 51 + 2012 + 98773 + Functional replacement of two highly conserved tetraloops in the bacterial ribosome + + + 15060 + 15065 + surname:Salsi;given-names:E + surname:Farah;given-names:E + surname:Dann;given-names:J + surname:Ermolenko;given-names:DN + 10.1073/pnas.1410873111 + 25288752 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 111 + 2014 + 98857 + Following movement of domain IV of elongation factor G during ribosomal translocation + + + 1219 + 1226 + surname:Sasaki;given-names:J + surname:Nakashima;given-names:N + 9882324 + REF + Journal of Virology + ref + 73 + 1999 + 98943 + Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro + + + 1517 + 1523 + surname:Savelsbergh;given-names:A + surname:Katunin;given-names:VI + surname:Mohr;given-names:D + surname:Peske;given-names:F + surname:Rodnina;given-names:MV + surname:Wintermeyer;given-names:W + 10.1016/S1097-2765(03)00230-2 + 12820965 + REF + Molecular Cell + ref + 11 + 2003 + 99072 + An elongation factor g-induced ribosome rearrangement precedes trna-mrna translocation + + + 827 + 834 + surname:Schuwirth;given-names:BS + surname:Borovinskaya;given-names:MA + surname:Hau;given-names:CW + surname:Zhang;given-names:W + surname:Vila-Sanjurjo;given-names:A + surname:Holton;given-names:JM + surname:Cate;given-names:JH + 10.1126/science.1117230 + 16272117 + REF + Science + ref + 310 + 2005 + 99159 + Structures of the bacterial ribosome at 3.5 A resolution + + + 1092 + 1096 + surname:Schüler;given-names:M + surname:Connell;given-names:SR + surname:Lescoute;given-names:A + surname:Giesebrecht;given-names:J + surname:Dabrowski;given-names:M + surname:Schroeer;given-names:B + surname:Mielke;given-names:T + surname:Penczek;given-names:PA + surname:Westhof;given-names:E + surname:Spahn;given-names:CM + 10.1038/nsmb1177 + REF + Nature Structural & Molecular Biology + ref + 13 + 2006 + 99216 + Structure of the ribosome-bound cricket paralysis virus IRES RNA + + + 1935 + 1942 + surname:Selmer;given-names:M + surname:Dunham;given-names:CM + surname:Murphy;given-names:FV + surname:Weixlbaumer;given-names:A + surname:Petry;given-names:S + surname:Kelley;given-names:AC + surname:Weir;given-names:JR + surname:Ramakrishnan;given-names:V + 10.1126/science.1131127 + 16959973 + REF + Science + ref + 313 + 2006 + 99281 + Structure of the 70S ribosome complexed with mrna and trna + + + 179 + 187 + surname:Sengupta;given-names:J + surname:Nilsson;given-names:J + surname:Gursky;given-names:R + surname:Kjeldgaard;given-names:M + surname:Nissen;given-names:P + surname:Frank;given-names:J + 10.1016/j.jmb.2008.07.004 + 18644383 + REF + Journal of Molecular Biology + ref + 382 + 2008 + 99340 + Visualization of the eef2-80s ribosome transition-state complex by cryo-electron microscopy + + + 9822 + 9827 + surname:Shaikh;given-names:TR + surname:Yassin;given-names:AS + surname:Lu;given-names:Z + surname:Barnard;given-names:D + surname:Meng;given-names:X + surname:Lu;given-names:TM + surname:Wagenknecht;given-names:T + surname:Agrawal;given-names:RK + 10.1073/pnas.1406744111 + 24958863 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 111 + 2014 + 99432 + Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-em + + + 1008 + 1019 + surname:Spahn;given-names:CMT + surname:Gomez-Lorenzo;given-names:MG + surname:Grassucci;given-names:RA + surname:Jørgensen;given-names:R + surname:Andersen;given-names:GR + surname:Beckmann;given-names:R + surname:Penczek;given-names:PA + surname:Ballesta;given-names:JPG + surname:Frank;given-names:J + 10.1038/sj.emboj.7600102 + 14976550 + REF + The EMBO Journal + ref + 23 + 2004a + 99551 + Domain movements of elongation factor eef2 and the eukaryotic 80S ribosome facilitate trna translocation + + + 465 + 475 + surname:Spahn;given-names:CMT + surname:Jan;given-names:E + surname:Mulder;given-names:A + surname:Grassucci;given-names:RA + surname:Sarnow;given-names:P + surname:Frank;given-names:J + 10.1016/j.cell.2004.08.001 + 15315759 + REF + Cell + ref + 118 + 2004b + 99656 + Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes + + + 1473 + 1482 + surname:Spiegel;given-names:PC + surname:Ermolenko;given-names:DN + surname:Noller;given-names:HF + 10.1261/rna.601507 + 17630323 + REF + RNA + ref + 13 + 2007 + 99743 + Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome + + + , 197 + -207 + surname:Spirin;given-names:AS + 10.1101/SQB.1969.034.01.026 + 4909498 + REF + Cold Spring Harbor Symposia on Quantitative Biology + ref + 34 + 1969 + 99824 + A model of the functioning ribosome: Locking and unlocking of the ribosome subparticles + + + 369 + 381 + surname:Studer;given-names:SM + surname:Feinberg;given-names:JS + surname:Joseph;given-names:S + 10.1016/S0022-2836(03)00146-3 + 12628244 + REF + Journal of Molecular Biology + ref + 327 + 2003 + 99912 + Rapid kinetic analysis of ef-g-dependent mrna translocation in the ribosome + + + 1210 + 1218 + surname:Svidritskiy;given-names:E + surname:Brilot;given-names:AF + surname:Koh;given-names:CS + surname:Grigorieff;given-names:N + surname:Korostelev;given-names:AA + 10.1016/j.str.2014.06.003 + 25043550 + REF + Structure + ref + 22 + 2014 + 99988 + Structures of yeast 80S ribosome-trna complexes in the rotated and nonrotated conformations + + + 12283 + 12288 + surname:Svidritskiy;given-names:E + surname:Ling;given-names:C + surname:Ermolenko;given-names:DN + surname:Korostelev;given-names:AA + 10.1073/pnas.1304922110 + 23824292 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 110 + 2013 + 100080 + Blasticidin S inhibits translation by trapping deformed trna on the ribosome + + + 18413 + 18418 + surname:Taylor;given-names:D + surname:Unbehaun;given-names:A + surname:Li;given-names:W + surname:Das;given-names:S + surname:Lei;given-names:J + surname:Liao;given-names:HY + surname:Grassucci;given-names:RA + surname:Pestova;given-names:TV + surname:Frank;given-names:J + 10.1073/pnas.1216730109 + 23091004 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 109 + 2012 + 100157 + Cryo-EM structure of the mammalian eukaryotic release factor erf1-erf3-associated termination complex + + + 2421 + 2431 + surname:Taylor;given-names:DJ + surname:Nilsson;given-names:J + surname:Merrill;given-names:AR + surname:Andersen;given-names:GR + surname:Nissen;given-names:P + surname:Frank;given-names:J + 10.1038/sj.emboj.7601677 + 17446867 + REF + The EMBO Journal + ref + 26 + 2007 + 100259 + Structures of modified eef2 80S ribosome complexes reveal the role of GTP hydrolysis in translocation + + + 1235490 + surname:Tourigny;given-names:DS + surname:Fernandez;given-names:IS + surname:Kelley;given-names:AC + surname:Ramakrishnan;given-names:V + 10.1126/science.1235490 + 23812720 + REF + Science + ref + 340 + 2013 + 100361 + Elongation factor G bound to the ribosome in an intermediate state of translocation + + + 123 + 134 + surname:Valle;given-names:M + surname:Zavialov;given-names:A + surname:Sengupta;given-names:J + surname:Rawat;given-names:U + surname:Ehrenberg;given-names:M + surname:Frank;given-names:J + 10.1016/S0092-8674(03)00476-8 + 12859903 + REF + Cell + ref + 114 + 2003 + 100445 + Locking and unlocking of ribosomal motions + + + 17 + 24 + surname:van Heel;given-names:M + surname:Harauz;given-names:G + surname:Orlova;given-names:EV + surname:Schmidt;given-names:R + surname:Schatz;given-names:M + 10.1006/jsbi.1996.0004 + 8742718 + REF + Journal of Structural Biology + ref + 116 + 1996 + 100488 + A new generation of the IMAGIC image processing system + + + 203 + 236 + surname:Voorhees;given-names:RM + surname:Ramakrishnan;given-names:V + 10.1146/annurev-biochem-113009-092313 + REF + Annual Review of Biochemistry + ref + 82 + 2013 + 100543 + Structural basis of the translational elongation cycle + + + 835 + 838 + surname:Voorhees;given-names:RM + surname:Schmeing;given-names:TM + surname:Kelley;given-names:AC + surname:Ramakrishnan;given-names:V + 10.1126/science.1194460 + 21051640 + REF + Science + ref + 330 + 2010 + 100598 + The mechanism for activation of GTP hydrolysis on the ribosome + + + e14874 + surname:Wang;given-names:QS + surname:Jan;given-names:E + 10.1371/journal.pone.0103601 + REF + PloS One + ref + 9 + 2014 + 100661 + Switch from cap- to factorless ires-dependent 0 and +1 frame translation during cellular stress and dicistrovirus infection + + + 511 + 520 + surname:Wilson;given-names:JE + surname:Pestova;given-names:TV + surname:Hellen;given-names:CU + surname:Sarnow;given-names:P + 10.1016/S0092-8674(00)00055-6 + 10966112 + REF + Cell + ref + 102 + 2000 + 100785 + Initiation of protein synthesis from the A site of the ribosome + + + 7770 + 7776 + surname:Yamamoto;given-names:H + surname:Nakashima;given-names:N + surname:Ikeda;given-names:Y + surname:Uchiumi;given-names:T + 10.1074/jbc.M610887200 + 17209036 + REF + The Journal of Biological Chemistry + ref + 282 + 2007 + 100849 + Binding mode of the first aminoacyl-trna in translation initiation mediated by plautia stali intestine virus internal ribosome entry site + + + 7 + 8 + surname:Yang;given-names:J + surname:Yan;given-names:R + surname:Roy;given-names:A + surname:Xu;given-names:D + surname:Poisson;given-names:J + surname:Zhang;given-names:Y + 10.1038/nmeth.3213 + 25549265 + REF + Nature Methods + ref + 12 + 2015 + 100987 + The I-TASSER Suite: protein structure and function prediction + + + 1236086 + surname:Zhou;given-names:J + surname:Lancaster;given-names:L + surname:Donohue;given-names:JP + surname:Noller;given-names:HF + 10.1126/science.1236086 + 23812722 + REF + Science + ref + 340 + 2013 + 101049 + Crystal structures of ef-g-ribosome complexes trapped in intermediate states of translocation + + + 1188 + 1191 + surname:Zhou;given-names:J + surname:Lancaster;given-names:L + surname:Donohue;given-names:JP + surname:Noller;given-names:HF + 10.1126/science.1255030 + 25190797 + REF + Science + ref + 345 + 2014 + 101143 + How the ribosome hands the a-site trna to the P site during ef-g-catalyzed translocation + + + 1839 + 1844 + surname:Zhu;given-names:J + surname:Korostelev;given-names:A + surname:Costantino;given-names:DA + surname:Donohue;given-names:JP + surname:Noller;given-names:HF + surname:Kieft;given-names:JS + 10.1073/pnas.1018582108 + 21245352 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 108 + 2011 + 101232 + Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) rnas bound to the 70S ribosome + + + REF + paragraph + 101365 + 10.7554/eLife.14874.059 + + + REVIEW_INFO + title + 101389 + Decision letter + + + REVIEW_INFO + paragraph + 101405 + Subramaniam + + + REVIEW_INFO + paragraph + 101417 + Sriram + + + REVIEW_INFO + paragraph + 101424 + In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. + + + REVIEW_INFO + paragraph + 101708 + Thank you for submitting your article "Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome" for consideration by eLife. Your article has been favorably evaluated by John Kuriyan (Senior editor) and three reviewers, one of whom, Sriram Subramaniam, is a member of our Board of Reviewing Editors. + + + REVIEW_INFO + paragraph + 102042 + The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. + + + REVIEW_INFO + paragraph + 102193 + Summary: + + + REVIEW_INFO + paragraph + 102202 + Type IV internal ribosome entry sites (IRESs) initiate translation without using any of the canonical eukaryotic translation initiation factors. Thus, they represent the most streamlined mode of eukaryotic translation initiation discovered. They have been studied biochemically and structurally. The current prevailing model is that these IRESs fold into a compact 2-domain structure that bind to the 40S subunit through multiple contacts. Critical interactions occur between two IRES RNA stem-loops and the head of the 40S subunit. This positions one of the IRES domains into the decoding groove. This domain (a pseudoknot, PKI) mimics the tRNA-mRNA anticodon-codon interaction, apparently first docking in the A site. The 60S subunit then joins in a GTP hydrolysis-independent step. This complex is then recognized by elongation factor 2, which catalyzes translocation of the PKI domain into the P site, allowing tRNA delivery to the A-site. Another round of translocation brings this tRNA to the P site. The mechanisms of these IRESs suggests that they can be powerful tools for understand translation in general (a feature the authors of this manuscript exploit). + + + REVIEW_INFO + paragraph + 103370 + A rich set of biochemical and functional data have established that different parts of the IRES affect different steps, have shown similarities and differences between different type IV IRESs, and have established some of the key differences between the canonical and IRES-driven initiation mechanisms. In addition, various structures of the IRES alone or bound to the ribosome have been published, using both crystallography and cryo-EM. Until recently, the cryo-EM structures were of low-or mid-resolution. However, when combined with the higher resolution data from crystal structures and the many functional and biochemical studies, the models that resulted have been very informative and have allowed many predictions to be made. + + + REVIEW_INFO + paragraph + 104105 + In this manuscript, the authors attack the question of IRES translocation. They present a series of cryo-EM structures of a Taura Syndrome Virus (TSV) IRES bound to 80S and eEF2, using the antibiotic sordarin. The authors interpret the set of structures as showing the trajectory of the mRNA-tRNA-mimicking PKI moving from the A to the P site. Overall, this is an impressive piece of work. It appears technically well done, the description is rich and detailed, and the conclusions are well supported and discussed. As such, it represents an important addition to the IRES field. In many ways, the mechanism that is presented is not surprising; an "inchworm"- like mechanism has been predicted in the literature (although never referred to as such!). However, to see it and to have detailed structures along the trajectory is very important. I will say that as the paper is written, it probably speaks as much to the mechanism of eEF2 and translocation in general as it does to IRES-specific function. + + + REVIEW_INFO + paragraph + 105107 + Essential revisions: + + + REVIEW_INFO + paragraph + 105128 + 1) There are few recent discoveries regarding IRES function that are not mentioned in the manuscript. As these discoveries relate directly to the interactions that the authors visualize and discuss, they should add a bit of discussion or analysis: + + + REVIEW_INFO + paragraph + 105376 + • First, some type IV IRESs to can initiate in an alternate reading frame. Do their structures suggest how this might occur? This effect appears to relate to a base adjacent to the codon-anticodon mimic, which they have good density for. References: Au et al. (2015) PNAS 112:E6446-55, Wang et al. (2014) PloS One 9:e103601, Ren et al. (2012) PNAS 109:E630-9, Ren et al. (2014) Nucleic Acids Res. 42:9366-82. + + + REVIEW_INFO + paragraph + 105787 + • Recent work implicates the VLR loop/loop 3 in PKI as having a role in eEF2 function: Ruehle et al., (2015) eLife), and it has been explored in manuscripts from the Jan lab. This is not mentioned or discussed. Can the authors comment on what this loop is doing and contacting and does it explain this previous work? Also, the Ruehle et al. presents biochemical data in favor of their spontaneous forward and reverse translocation that the authors allude to. + + + REVIEW_INFO + paragraph + 106248 + 2) The interactions between the highly conserved apical loops of SL4 and 5 make critical interactions with eS5 and eS25. In addition, the IRES makes critical interaction with the L1 stalk. These regions of the type IV IRESs are very highly conserved, but no high-resolution information is known for these interactions. Was the local resolution good enough to say how binding these mysterious interactions are achieved, and perhaps how it relates to ribosome conformation, IRES conformation, etc.? + + + REVIEW_INFO + paragraph + 106745 + 3) Related to the above, it would be interesting to see some more details of how the IRES changes conformation; not just globally, but internally. Is the resolution sufficient to see this? Any mechanistic insight? + + + REVIEW_INFO + paragraph + 106959 + 4) By the very nature of this work, in which 5 structures at near atomic resolution are dissected, the figures are quite dense in information content and individual panels are generally quite small. In addition, the paper is quite long because of the high information content. The general reader can of course skip the detailed sections in the middle and read the Discussion, which is very clear. What seems to be missing for a general reader who wishes to dive into the "information forest" is an outline figure at the beginning that shows the ribosome translation cycle with various subunit motions and tRNA movements indicated. This would certainly help those who do not work in the ribosome field. + + + REVIEW_INFO + paragraph + 107661 + 10.7554/eLife.14874.060 + + + REVIEW_INFO + title + 107685 + Author response + + + REVIEW_INFO + paragraph + 107701 + 1) There are few recent discoveries regarding IRES function that are not mentioned in the manuscript. As these discoveries relate directly to the interactions that the authors visualize and discuss, they should add a bit of discussion or analysis: + + + REVIEW_INFO + paragraph + 107949 + First, some type IV IRESs to can initiate in an alternate reading frame. Do their structures suggest how this might occur? This effect appears to relate to a base adjacent to the codon-anticodon mimic, which they have good density for. References: Au et al. (2015) PNAS 112:E6446-55, Wang et al. (2014) PloS One 9:e103601, Ren et al. (2012) PNAS 109:E630-9, Ren et al. (2014) Nucleic Acids Res. 42:9366-82. + + + REVIEW_INFO + paragraph + 108356 + We agree that alternative frame selection is an interesting phenomenon andhave added a paragraph to discuss this in “IRES translocation mechanism” (third paragraph). Our structures do not directly suggest how alternate reading frame selection could occur because our data did not reveal a frame-shifted conformation of the IRES. The observation of IRES dynamics in our study indirectly suggests that an alternate (frame-shifted) codon may be transiently placed in the A site following eEF2 release, and this sampling may allow binding of an aminoacyl-tRNA to the off-frame codon. + + + REVIEW_INFO + paragraph + 108940 + Recent work implicates the VLR loop/loop 3 in PKI as having a role in eEF2 function: Ruehle et al., (2015) eLife), and it has been explored in manuscripts from the Jan lab. This is not mentioned or discussed. Can the authors comment on what this loop is doing and contacting and does it explain this previous work? Also, the Ruehle et al. presents biochemical data in favor of their spontaneous forward and reverse translocation that the authors allude to. + + + REVIEW_INFO + paragraph + 109397 + We have revised our Results section to address this important comment. Loop 3 connects the ASL-like and the mRNA-like regions of the PKI domain. Loop 3 of the post-translocated state (Structure V) is stabilized by interactions with the β-hairpin loop of uS7 and helix 23 of 18S rRNA in the E site, in a manner reminiscent of that for the E-site tRNA in the 80S*2tRNA*mRNA structure. In the pre-translocation states, however, loop 3 is poorly resolved in density maps. This implies conformational flexibility of loop 3, also reported by biochemical studies of unbound IGR IRESs (Jan and Sarnow, 2002; Pfingsten et al., 2007). Our structures therefore suggest that loop 3 contributes to stabilization of the post-translocation IRES, rationalizing the recent detailed biochemical study (Ruehle et al., (2015) eLife), which reported that IGR IRES mutated constructs with shortened loop 3 are defective in eEF2-catalyzed translocation. + + + REVIEW_INFO + paragraph + 110331 + 2) The interactions between the highly conserved apical loops of SL4 and 5 make critical interactions with eS5 and eS25. In addition, the IRES makes critical interaction with the L1 stalk. These regions of the type IV IRESs are very highly conserved, but no high-resolution information is known for these interactions. Was the local resolution good enough to say how binding these mysterious interactions are achieved, and perhaps how it relates to ribosome conformation, IRES conformation, etc.? + + + REVIEW_INFO + paragraph + 110828 + We find that the phosphate backbone of SL4 and 5 interact with the positively charged and aromatic residues of eS5 (uS7) and eS25. We have added a description of these interactions in the main text and also Supplementary Figures (Figure 3—figure supplement 2, Figure 3—figure supplement 4) to demonstrate the interactions. In addition, we find that interactions of SL4 and SL5 with the small subunit are somewhat similar to those of the L1 stalk with the small subunit of the hybrid-state 80S*tRNA structure. We have added Figure 3—figure supplement 3 to illustrate this similarity. The interactions between the IRES and the L1 stalk are less well resolved – although the density is strong, the resolution is insufficient to interpret the interactions unambiguously. We therefore refrain from detailed interpretation of L1 stalk interactions. + + + REVIEW_INFO + paragraph + 111679 + 3) Related to the above, it would be interesting to see some more details of how the IRES changes conformation; not just globally, but internally. Is the resolution sufficient to see this? Any mechanistic insight? + + + REVIEW_INFO + paragraph + 111893 + We now provide a more extensive discussion of IRES local interactions and conformational changes, supplemented by additional illustrations. We report the rearrangements of stem loop 3 (conserved in TSV-like IRESs of group 2: Aparavirus), which resembles a tRNA elbow, as we reported previously. Our current structures indicate that SL3 undergoes rearrangements similar to those of the translocating A-site tRNA (Figure 1—figure supplement 6). In addition, we demonstrate local rearrangements of the “bridge” between the 5’ domain and PKI domain (Figure 3—figure supplement 7). This region interacts with protein uL5 in the two most compact IRES conformations (III and IV), but not in other states. This reveals the stabilizing role of protein uL5 at the intermediate stages of IRES translocation. + + + REVIEW_INFO + paragraph + 112700 + 4) By the very nature of this work, in which 5 structures at near atomic resolution are dissected, the figures are quite dense in information content and individual panels are generally quite small. In addition, the paper is quite long because of the high information content. The general reader can of course skip the detailed sections in the middle and read the Discussion, which is very clear. What seems to be missing for a general reader who wishes to dive into the "information forest" is an outline figure at the beginning that shows the ribosome translation cycle with various subunit motions and tRNA movements indicated. This would certainly help those who do not work in the ribosome field. + + + REVIEW_INFO + paragraph + 113402 + We have reorganized the panels in most figures to make the figures less dense and increase the sizes of individual panels. We agree that a figure showing ribosome-2tRNA-mRNA and summarizing conformational differences between structures representing various translocation states would be helpful. We now include a supplementary figure, showing ribosome-2tRNAs-mRNA structures (Figure 1—figure supplement 1), which we refer to in the manuscript. We also include the views of tRNA-bound structures in the supplementary figure showing interactions of the A-site finger with the tRNAs and the IRES (Figure 3—figure supplement 6). + + + diff --git a/BioC_XML/4918759_v0.xml b/BioC_XML/4918759_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..b43bec9ded04abf70548e61ad3e3ad5e46c977d1 --- /dev/null +++ b/BioC_XML/4918759_v0.xml @@ -0,0 +1,8253 @@ + + + + PMC + 20201218 + pmc.key + + 4918759 + NO-CC CODE + no + 0 + 0 + + 10.1038/nsmb.3203 + NIHMS771539 + 4918759 + 27065196 + 426 + 5 + Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: + 433 + surname:Matthews;given-names:Melissa M. + surname:Thomas;given-names:Justin M. + surname:Zheng;given-names:Yuxuan + surname:Tran;given-names:Kiet + surname:Phelps;given-names:Kelly J. + surname:Scott;given-names:Anna I. + surname:Havel;given-names:Jocelyn + surname:Fisher;given-names:Andrew J. + surname:Beal;given-names:Peter A. + TITLE + front + 23 + 2016 + 0 + Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity + + 0.9985019 + evidence + cleaner0 + 2023-07-25T13:52:34Z + DUMMY: + + Structures + + + 0.99872285 + species + cleaner0 + 2023-07-25T09:56:21Z + MESH: + + human + + + 0.9990864 + protein + cleaner0 + 2023-07-25T09:55:24Z + PR: + + ADAR2 + + + 0.998929 + protein_state + cleaner0 + 2023-07-25T13:01:17Z + DUMMY: + + bound to + + + 0.99852717 + chemical + cleaner0 + 2023-07-25T09:56:06Z + CHEBI: + + dsRNA + + + + ABSTRACT + abstract + 103 + ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function. Our understanding of the ADAR reaction mechanism, origin of editing site selectivity and effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. Here we describe four crystal structures of the deaminase domain of human ADAR2 bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis for ADAR deaminase domain’s dsRNA specificity, its base-flipping mechanism, and nearest neighbor preferences. In addition, an ADAR2-specific RNA-binding loop was identified near the enzyme active site rationalizing differences in selectivity observed between different ADARs. Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease. + + 0.99897975 + protein_type + cleaner0 + 2023-07-25T09:55:34Z + MESH: + + ADARs + + + 0.9843998 + protein_type + cleaner0 + 2023-07-25T09:57:13Z + MESH: + + adenosine deaminases acting on RNA + + + 0.95987874 + protein_type + cleaner0 + 2023-07-25T13:51:57Z + MESH: + + editing enzymes + + + 0.94079924 + residue_name + cleaner0 + 2023-07-25T10:01:02Z + SO: + + adenosine + + + 0.83345747 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + A + + + 0.88204086 + residue_name + cleaner0 + 2023-07-25T10:01:31Z + SO: + + inosine + + + 0.8659339 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + I + + + structure_element + SO: + cleaner0 + 2023-07-25T13:31:35Z + + duplex RNA + + + chemical + CHEBI: + cleaner0 + 2023-07-25T09:56:53Z + + RNA + + + 0.9989807 + protein_type + cleaner0 + 2023-07-25T09:55:42Z + MESH: + + ADAR + + + site + SO: + cleaner0 + 2023-07-25T10:14:37Z + + editing site + + + 0.99105096 + evidence + cleaner0 + 2023-07-25T13:52:53Z + DUMMY: + + structural data + + + 0.9989266 + protein_type + cleaner0 + 2023-07-25T09:55:35Z + MESH: + + ADARs + + + 0.9990577 + protein_state + cleaner0 + 2023-07-25T13:01:17Z + DUMMY: + + bound to + + + 0.99584216 + chemical + cleaner0 + 2023-07-25T09:56:00Z + CHEBI: + + RNAs + + + 0.9986969 + evidence + cleaner0 + 2023-07-25T13:52:45Z + DUMMY: + + crystal structures + + + 0.9977211 + structure_element + cleaner0 + 2023-07-25T09:57:46Z + SO: + + deaminase domain + + + 0.9984072 + species + cleaner0 + 2023-07-25T09:56:20Z + MESH: + + human + + + 0.99940026 + protein + cleaner0 + 2023-07-25T09:55:24Z + PR: + + ADAR2 + + + 0.9990988 + protein_state + cleaner0 + 2023-07-25T13:01:17Z + DUMMY: + + bound to + + + 0.83250415 + structure_element + cleaner0 + 2023-07-25T13:32:33Z + SO: + + RNA duplexes + + + 0.9985316 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + 0.99882245 + experimental_method + cleaner0 + 2023-07-25T13:22:41Z + MESH: + + structure-guided mutagenesis + + + 0.9937575 + experimental_method + cleaner0 + 2023-07-25T13:22:45Z + MESH: + + RNA-modification experiments + + + 0.99831885 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + structure_element + SO: + cleaner0 + 2023-07-25T09:57:47Z + + deaminase domain + + + 0.99779785 + chemical + cleaner0 + 2023-07-25T09:56:05Z + CHEBI: + + dsRNA + + + 0.9993356 + protein + cleaner0 + 2023-07-25T09:55:25Z + PR: + + ADAR2 + + + 0.99576366 + structure_element + cleaner0 + 2023-07-25T09:57:37Z + SO: + + RNA-binding loop + + + 0.9980329 + site + cleaner0 + 2023-07-25T10:14:49Z + SO: + + active site + + + 0.998869 + protein_type + cleaner0 + 2023-07-25T09:55:35Z + MESH: + + ADARs + + + 0.998367 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.98297024 + species + cleaner0 + 2023-07-25T09:56:21Z + MESH: + + human + + + + INTRO + title_1 + 1192 + Introduction + + + INTRO + paragraph + 1205 + RNA editing reactions alter a transcript’s genomically encoded sequence by inserting, deleting or modifying nucleotides. Deamination of adenosine (A), the most common form of RNA editing in humans, generates inosine (I) at the corresponding nucleotide position. Since I base pairs with cytidine (C), it functions like guanosine (G) in cellular processes such as splicing, translation and reverse transcription. A to I editing has wide-ranging consequences on RNA function including altering miRNA recognition sites, redirecting splicing and changing the meaning of specific codons. Two different enzymes carry out A to I editing in humans; ADAR1 and ADAR2. ADAR activity is required for nervous system function and altered editing has been linked to neurological disorders such as epilepsy and Prader Willi Syndrome. In addition, mutations in the ADAR1 gene are known to cause the autoimmune disease Aicardi-Goutieres Syndrome (AGS) and the skin disorder Dyschromatosis Symmetrica Hereditaria (DSH). Hyper editing has been observed at certain sites in cancer cells, such as in the mRNA for AZIN1 (antizyme inhibitor 1). However, hypo editing also occurs in cancer-derived cell lines exemplified by reduced editing observed in the message for glioma-associated oncogene 1 (Gli1). + + chemical + CHEBI: + cleaner0 + 2023-07-25T09:56:53Z + + RNA + + + 0.72157687 + residue_name + cleaner0 + 2023-07-25T10:01:01Z + SO: + + adenosine + + + 0.75308394 + residue_name + cleaner0 + 2023-07-25T13:41:26Z + SO: + + A + + + chemical + CHEBI: + cleaner0 + 2023-07-25T09:56:53Z + + RNA + + + 0.99827206 + species + cleaner0 + 2023-07-25T10:04:12Z + MESH: + + humans + + + 0.8841903 + residue_name + cleaner0 + 2023-07-25T10:01:30Z + SO: + + inosine + + + 0.9217293 + residue_name + cleaner0 + 2023-07-25T13:41:31Z + SO: + + I + + + 0.9431153 + residue_name + cleaner0 + 2023-07-25T13:41:38Z + SO: + + I + + + 0.7785633 + residue_name + cleaner0 + 2023-07-25T10:01:15Z + SO: + + cytidine + + + 0.91272897 + residue_name + cleaner0 + 2023-07-25T13:41:43Z + SO: + + C + + + 0.7327572 + residue_name + cleaner0 + 2023-07-25T10:01:20Z + SO: + + guanosine + + + 0.825363 + residue_name + cleaner0 + 2023-07-25T13:41:48Z + SO: + + G + + + chemical + CHEBI: + cleaner0 + 2023-07-25T09:56:53Z + + RNA + + + 0.99877626 + site + cleaner0 + 2023-07-25T10:15:41Z + SO: + + miRNA recognition sites + + + 0.99836284 + species + cleaner0 + 2023-07-25T10:04:11Z + MESH: + + humans + + + 0.9990941 + protein + cleaner0 + 2023-07-25T10:03:04Z + PR: + + ADAR1 + + + 0.9991165 + protein + cleaner0 + 2023-07-25T09:55:25Z + PR: + + ADAR2 + + + 0.99190277 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.99904984 + protein + cleaner0 + 2023-07-25T10:03:05Z + PR: + + ADAR1 + + + 0.9681562 + chemical + cleaner0 + 2023-07-25T10:03:38Z + CHEBI: + + mRNA + + + 0.99926287 + protein + cleaner0 + 2023-07-25T10:03:47Z + PR: + + AZIN1 + + + 0.9986138 + protein + cleaner0 + 2023-07-25T10:03:54Z + PR: + + antizyme inhibitor 1 + + + 0.9641166 + protein + cleaner0 + 2023-07-25T10:04:01Z + PR: + + glioma-associated oncogene 1 + + + 0.99926215 + protein + cleaner0 + 2023-07-25T10:04:06Z + PR: + + Gli1 + + + + INTRO + paragraph + 2486 + The ADAR proteins have a modular structure with double stranded RNA binding domains (dsRBDs) and a C-terminal deaminase domain (see Fig. 1a for hADAR2 domains). ADARs efficiently deaminate specific adenosines in duplex RNA while leaving most adenosines unmodified. The mechanism of adenosine deamination requires ADAR to flip the reactive base out of an RNA double helix to access its active site. How an enzyme could accomplish this task with a duplex RNA substrate is not known. Furthermore, how an ADAR deaminase domain contributes to editing site selectivity is also unknown, since no structures of ADAR deaminase domain-RNA complexes have been reported. To address these knowledge gaps, we set out to trap the human ADAR2 deaminase domain (aa299–701, hADAR2d) bound to different duplex RNAs and solve structures for the resulting complexes using x-ray crystallography. We then evaluated the importance of protein-RNA contacts using structure-guided mutagenesis and RNA-modification experiments coupled with adenosine deamination kinetics. + + protein_type + MESH: + cleaner0 + 2023-07-25T13:52:20Z + + ADAR + + + 0.9992841 + structure_element + cleaner0 + 2023-07-25T10:01:41Z + SO: + + double stranded RNA binding domains + + + 0.9994134 + structure_element + cleaner0 + 2023-07-25T10:01:53Z + SO: + + dsRBDs + + + 0.99909276 + structure_element + cleaner0 + 2023-07-25T09:57:47Z + SO: + + deaminase domain + + + 0.91872007 + protein + cleaner0 + 2023-07-25T12:46:38Z + PR: + + hADAR2 + + + 0.99810696 + protein_type + cleaner0 + 2023-07-25T09:55:35Z + MESH: + + ADARs + + + 0.77721 + residue_name + cleaner0 + 2023-07-25T10:02:00Z + SO: + + adenosines + + + structure_element + SO: + cleaner0 + 2023-07-25T13:31:35Z + + duplex RNA + + + 0.5794063 + residue_name + cleaner0 + 2023-07-25T10:02:01Z + SO: + + adenosines + + + 0.9443331 + residue_name + cleaner0 + 2023-07-25T10:01:02Z + SO: + + adenosine + + + 0.9877467 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + chemical + CHEBI: + cleaner0 + 2023-07-25T13:27:36Z + + RNA double helix + + + 0.99897516 + site + cleaner0 + 2023-07-25T10:14:49Z + SO: + + active site + + + structure_element + SO: + cleaner0 + 2023-07-25T13:31:35Z + + duplex RNA + + + 0.709389 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.9973562 + structure_element + cleaner0 + 2023-07-25T09:57:47Z + SO: + + deaminase domain + + + site + SO: + cleaner0 + 2023-07-25T10:14:37Z + + editing site + + + 0.99661 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-07-25T10:04:44Z + + ADAR deaminase domain-RNA + + + 0.9986897 + species + cleaner0 + 2023-07-25T09:56:21Z + MESH: + + human + + + 0.99944955 + protein + cleaner0 + 2023-07-25T09:55:25Z + PR: + + ADAR2 + + + 0.9989267 + structure_element + cleaner0 + 2023-07-25T09:57:47Z + SO: + + deaminase domain + + + residue_range + DUMMY: + cleaner0 + 2023-07-25T10:02:27Z + + 299–701 + + + 0.7764029 + mutant + cleaner0 + 2023-07-25T10:05:12Z + MESH: + + hADAR2d + + + 0.99901843 + protein_state + cleaner0 + 2023-07-25T13:01:17Z + DUMMY: + + bound to + + + structure_element + SO: + cleaner0 + 2023-07-25T13:32:49Z + + duplex RNAs + + + 0.987329 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + 0.9989038 + experimental_method + cleaner0 + 2023-07-25T13:23:03Z + MESH: + + x-ray crystallography + + + chemical + CHEBI: + cleaner0 + 2023-07-25T09:56:53Z + + RNA + + + 0.9989031 + experimental_method + cleaner0 + 2023-07-25T13:23:06Z + MESH: + + structure-guided mutagenesis + + + 0.9958802 + experimental_method + cleaner0 + 2023-07-25T13:23:10Z + MESH: + + RNA-modification experiments + + + experimental_method + MESH: + cleaner0 + 2023-07-25T10:00:22Z + + adenosine deamination kinetics + + + + RESULTS + title_1 + 3532 + Results + + + RESULTS + title_2 + 3540 + Trapping the flipped conformation + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:06:31Z + + flipped + + + + RESULTS + paragraph + 3574 + The ADAR reaction involves the formation of a hydrated intermediate that loses ammonia to generate the inosine-containing product RNA (for reaction scheme see Fig. 1b). The covalent hydrate of the nucleoside analog 8-azanebularine (N) mimics the proposed high-energy intermediate (for reaction scheme see Fig. 1b). For trapping hADAR2d bound to RNA for crystallography, we incorporated 8-azanebularine into duplex RNAs shown recently to be excellent substrates for deamination by hADAR2d (for duplex sequence see Fig. 1c) (for characterization of protein–RNA complex see Supplementary Fig. 1). In addition, for one of these duplexes (Bdf2), we positioned the 8-azanebularine opposite either uridine or cytidine to mimic an A-U pair or A-C mismatch at the editing site creating a total of three different RNA substrates for structural studies (Fig. 1c). The hADAR2d protein (without RNA bound) has been previously crystallized and structurally characterized revealing features of the active site including the presence of zinc. In addition, an inositol hexakisphosphate (IHP) molecule was found buried in the core of the protein hydrogen bonded to numerous conserved polar residues. For crystallization of hADAR2d-RNA complexes, we used both the wild type (WT) deaminase domain and a mutant (E488Q) that has enhanced catalytic activity. A description of the crystallization conditions, X-ray diffraction data collection and solution of the structures can be found in Online Methods. + + 0.94199526 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.96332026 + residue_name + cleaner0 + 2023-07-25T10:01:31Z + SO: + + inosine + + + 0.99866235 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.9991477 + chemical + cleaner0 + 2023-07-25T13:47:48Z + CHEBI: + + 8-azanebularine + + + 0.99645823 + chemical + cleaner0 + 2023-07-25T13:47:51Z + CHEBI: + + N + + + 0.99907047 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.99884796 + protein_state + cleaner0 + 2023-07-25T13:01:17Z + DUMMY: + + bound to + + + 0.8323808 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.98299843 + experimental_method + cleaner0 + 2023-07-25T13:23:14Z + MESH: + + crystallography + + + 0.99916464 + chemical + cleaner0 + 2023-07-25T13:47:57Z + CHEBI: + + 8-azanebularine + + + structure_element + SO: + cleaner0 + 2023-07-25T13:32:49Z + + duplex RNAs + + + 0.99929583 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.7811063 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.7475698 + chemical + cleaner0 + 2023-07-25T12:51:54Z + CHEBI: + + Bdf2 + + + 0.9990158 + chemical + cleaner0 + 2023-07-25T13:47:57Z + CHEBI: + + 8-azanebularine + + + 0.8530418 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + uridine + + + 0.8605422 + residue_name + cleaner0 + 2023-07-25T10:01:16Z + SO: + + cytidine + + + residue_name + SO: + cleaner0 + 2023-07-25T13:45:49Z + + A + + + 0.5155651 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + U + + + residue_name + SO: + cleaner0 + 2023-07-25T13:45:49Z + + A + + + residue_name + SO: + cleaner0 + 2023-07-25T13:45:49Z + + C + + + 0.99084514 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.9929611 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.99910283 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.97947806 + protein_state + cleaner0 + 2023-07-25T13:02:39Z + DUMMY: + + without RNA bound + + + 0.9955842 + experimental_method + cleaner0 + 2023-07-25T13:23:23Z + MESH: + + crystallized + + + 0.99900174 + site + cleaner0 + 2023-07-25T10:14:49Z + SO: + + active site + + + 0.9987931 + chemical + cleaner0 + 2023-07-25T10:15:30Z + CHEBI: + + zinc + + + 0.9990891 + chemical + cleaner0 + 2023-07-25T10:07:31Z + CHEBI: + + inositol hexakisphosphate + + + 0.9992798 + chemical + cleaner0 + 2023-07-25T10:07:36Z + CHEBI: + + IHP + + + 0.99433273 + bond_interaction + cleaner0 + 2023-07-25T10:07:47Z + MESH: + + hydrogen bonded + + + 0.9975631 + experimental_method + cleaner0 + 2023-07-25T13:23:29Z + MESH: + + crystallization + + + 0.991581 + complex_assembly + cleaner0 + 2023-07-25T10:05:49Z + GO: + + hADAR2d-RNA + + + 0.99913406 + protein_state + cleaner0 + 2023-07-25T10:05:59Z + DUMMY: + + wild type + + + 0.9992747 + protein_state + cleaner0 + 2023-07-25T10:06:04Z + DUMMY: + + WT + + + 0.9838567 + structure_element + cleaner0 + 2023-07-25T09:57:47Z + SO: + + deaminase domain + + + 0.99909234 + protein_state + cleaner0 + 2023-07-25T10:07:53Z + DUMMY: + + mutant + + + 0.9990478 + mutant + cleaner0 + 2023-07-25T10:08:00Z + MESH: + + E488Q + + + experimental_method + MESH: + cleaner0 + 2023-07-25T13:23:59Z + + X-ray diffraction data collection and solution + + + 0.9910028 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + + RESULTS + paragraph + 5058 + Four protein-RNA combinations generated diffracting crystals that resulted in high-resolution structures (hADAR2d WT–Bdf2-U, hADAR2d WT–Bdf2-C, hADAR2d E488Q–Bdf2-C, hADAR2d E488Q–Gli1) (Table 1). In each of these complexes, the protein binds the RNA on one face of the duplex over ~ 20 bp using a positively charged surface near the zinc-containing active site (Fig. 2, Supplementary Fig. 2a). The large binding site (1493 Å2 RNA surface area and 1277 Å2 protein surface area buried) observed for hADAR2d is consistent with recent footprinting studies. Both strands of the RNA contact the protein with the majority of these interactions mediated through the phosphodiester-ribose backbone near the editing site (Fig. 2c, Supplementary Fig. 2 b–d). + + 0.96222466 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.9980579 + evidence + cleaner0 + 2023-07-25T13:52:59Z + DUMMY: + + crystals + + + 0.9982065 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-07-25T10:10:06Z + + hADAR2d WT–Bdf2-U + + + complex_assembly + GO: + cleaner0 + 2023-07-25T10:10:29Z + + hADAR2d WT–Bdf2-C + + + complex_assembly + GO: + cleaner0 + 2023-07-25T10:10:48Z + + hADAR2d E488Q–Bdf2-C + + + complex_assembly + GO: + cleaner0 + 2023-07-25T10:11:08Z + + hADAR2d E488Q–Gli1 + + + 0.9987073 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.9284509 + site + cleaner0 + 2023-07-25T10:14:59Z + SO: + + zinc-containing active site + + + 0.9984596 + site + cleaner0 + 2023-07-25T13:36:20Z + SO: + + binding site + + + 0.9986247 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.9866793 + experimental_method + cleaner0 + 2023-07-25T13:24:05Z + MESH: + + footprinting studies + + + 0.99813 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.99800706 + site + cleaner0 + 2023-07-25T10:14:36Z + SO: + + editing site + + + + RESULTS + paragraph + 5819 + The structures show a large deviation from A-form RNA conformation at the editing site (Fig. 2, Fig. 3, Supplementary Video 1). The 8-azanebularine is flipped out of the helix and bound into the active site as its covalent hydrate where it interacts with several amino acids including V351, T375, K376, E396 and R455 (Fig. 3a, Supplementary Fig. 3a). The side chain of E396 H-bonds to purine N1 and O6. This interaction was expected given the proposed role of E396 in mediating proton transfers to and from N1 of the substrate adenosine. The 2’-hydroxyl of 8-azanebularine H-bonds to the backbone carbonyl of T375 while the T375 side chain contacts its 3’-phosphodiester. R455 and K376 help position the flipped nucleotide in the active site by fastening the phosphate backbone flanking the editing site. The R455 side chain ion pairs with the 5’-phosphodiester of 8-azanebularine while the K376 side chain contacts its 3’-phosphodiester. Lastly, the side chain of V351 provides a hydrophobic surface for interaction with the nucleobase of the edited nucleotide. RNA binding does not alter IHP binding or the H-bonding network linking IHP to the active site. + + 0.99818987 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + 0.9982555 + structure_element + cleaner0 + 2023-07-25T13:02:57Z + SO: + + A-form + + + 0.99887973 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.99887246 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.99917585 + chemical + cleaner0 + 2023-07-25T13:47:57Z + CHEBI: + + 8-azanebularine + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:07:08Z + + flipped out + + + 0.99900454 + structure_element + cleaner0 + 2023-07-25T13:27:53Z + SO: + + helix + + + 0.9155072 + protein_state + cleaner0 + 2023-07-25T13:03:08Z + DUMMY: + + bound into + + + 0.9986167 + site + cleaner0 + 2023-07-25T10:14:48Z + SO: + + active site + + + 0.99922204 + residue_name_number + cleaner0 + 2023-07-25T10:17:49Z + DUMMY: + + V351 + + + 0.99917525 + residue_name_number + cleaner0 + 2023-07-25T10:17:54Z + DUMMY: + + T375 + + + 0.99903846 + residue_name_number + cleaner0 + 2023-07-25T10:17:59Z + DUMMY: + + K376 + + + 0.99884415 + residue_name_number + cleaner0 + 2023-07-25T10:18:04Z + DUMMY: + + E396 + + + 0.9987985 + residue_name_number + cleaner0 + 2023-07-25T10:18:09Z + DUMMY: + + R455 + + + 0.998769 + residue_name_number + cleaner0 + 2023-07-25T10:18:04Z + DUMMY: + + E396 + + + bond_interaction + MESH: + cleaner0 + 2023-07-25T10:59:14Z + + H-bonds + + + 0.9934475 + chemical + cleaner0 + 2023-07-25T13:48:08Z + CHEBI: + + purine + + + 0.9988231 + residue_name_number + cleaner0 + 2023-07-25T10:18:04Z + DUMMY: + + E396 + + + 0.9879749 + residue_name + cleaner0 + 2023-07-25T10:01:02Z + SO: + + adenosine + + + 0.99917847 + chemical + cleaner0 + 2023-07-25T13:47:57Z + CHEBI: + + 8-azanebularine + + + bond_interaction + MESH: + cleaner0 + 2023-07-25T10:59:14Z + + H-bonds + + + 0.9989279 + residue_name_number + cleaner0 + 2023-07-25T10:17:55Z + DUMMY: + + T375 + + + 0.99882776 + residue_name_number + cleaner0 + 2023-07-25T10:17:55Z + DUMMY: + + T375 + + + 0.9987136 + residue_name_number + cleaner0 + 2023-07-25T10:18:10Z + DUMMY: + + R455 + + + 0.99870133 + residue_name_number + cleaner0 + 2023-07-25T10:18:00Z + DUMMY: + + K376 + + + 0.9843885 + protein_state + cleaner0 + 2023-07-25T13:06:30Z + DUMMY: + + flipped + + + 0.99821293 + chemical + cleaner0 + 2023-07-25T13:48:13Z + CHEBI: + + nucleotide + + + 0.99880415 + site + cleaner0 + 2023-07-25T10:14:49Z + SO: + + active site + + + 0.9986296 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.99860805 + residue_name_number + cleaner0 + 2023-07-25T10:18:10Z + DUMMY: + + R455 + + + bond_interaction + MESH: + cleaner0 + 2023-07-25T10:59:14Z + + ion pairs + + + 0.99918294 + chemical + cleaner0 + 2023-07-25T13:47:57Z + CHEBI: + + 8-azanebularine + + + 0.9986051 + residue_name_number + cleaner0 + 2023-07-25T10:18:00Z + DUMMY: + + K376 + + + 0.9990908 + residue_name_number + cleaner0 + 2023-07-25T10:17:50Z + DUMMY: + + V351 + + + 0.99874854 + site + cleaner0 + 2023-07-25T13:36:57Z + SO: + + hydrophobic surface + + + 0.98942393 + protein_state + cleaner0 + 2023-07-25T13:46:23Z + DUMMY: + + edited + + + 0.9981103 + chemical + cleaner0 + 2023-07-25T13:48:16Z + CHEBI: + + nucleotide + + + 0.995777 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.3729107 + chemical + cleaner0 + 2023-07-25T10:07:37Z + CHEBI: + + IHP + + + 0.9979783 + site + cleaner0 + 2023-07-25T10:16:04Z + SO: + + H-bonding network + + + 0.5119935 + chemical + cleaner0 + 2023-07-25T10:07:37Z + CHEBI: + + IHP + + + 0.9989294 + site + cleaner0 + 2023-07-25T10:14:49Z + SO: + + active site + + + + RESULTS + title_2 + 6986 + ADARs use a unique mechanism to modify duplex RNA + + 0.9981047 + protein_type + cleaner0 + 2023-07-25T09:55:35Z + MESH: + + ADARs + + + structure_element + SO: + cleaner0 + 2023-07-25T13:31:35Z + + duplex RNA + + + + RESULTS + paragraph + 7036 + The ADAR2 base-flipping loop, bearing residue 488, approaches the RNA duplex from the minor groove side at the editing site. The side chain of this amino acid penetrates the helix where it occupies the space vacated by the flipped out base and H-bonds to the complementary strand orphaned base and to the 2’ hydroxyl of the nucleotide immediately 5’ to the editing site (Figs. 3b, 3c). In the four structures reported here, three different combinations of helix-penetrating residue and orphan base are observed (i.e. E488 + U, E488 + C and Q488 + C) and all three combinations show the same side chain and base positions (Figs. 3b, 3c, Supplementary Fig. 4a for overlay of all three). For instance, in the complex with hADAR2d E488Q and the Bdf2-C duplex, the protein recognizes an orphaned C by donating H-bonds from Nε2 to cytosine N3 and from its backbone NH to cytosine O2 (Fig. 3b). In the complex with hADAR2d WT and the Bdf2-U duplex, a similar interaction is observed with the E488 backbone NH hydrogen bonded to the uracil O2 and the E488 side chain H-bonded to the uracil N3H (Fig. 3c). Interestingly, the E488Q mutant was discovered in a screen for highly active ADAR2 mutants and this residue was suggested to be involved in base flipping given its effect on editing substrates with a fluorescent nucleobase at the editing site. ADARs react preferentially with adenosines in A•C mismatches and A-U pairs over A•A and A•G mismatches. A purine at the orphan base position (in its anti conformation) would clash with the 488 residue explaining the preference for pyrimidines here. + + 0.9994417 + protein + cleaner0 + 2023-07-25T09:55:25Z + PR: + + ADAR2 + + + 0.99918264 + structure_element + cleaner0 + 2023-07-25T13:27:58Z + SO: + + base-flipping loop + + + 0.99751234 + residue_number + cleaner0 + 2023-07-25T10:17:32Z + DUMMY: + + 488 + + + structure_element + SO: + cleaner0 + 2023-07-25T13:30:55Z + + RNA duplex + + + 0.99722815 + site + cleaner0 + 2023-07-25T10:16:51Z + SO: + + minor groove + + + 0.9979775 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.9921565 + protein_state + cleaner0 + 2023-07-25T13:07:08Z + DUMMY: + + flipped out + + + chemical + CHEBI: + cleaner0 + 2023-07-25T13:07:18Z + + base + + + 0.98789185 + bond_interaction + cleaner0 + 2023-07-25T10:59:14Z + MESH: + + H-bonds + + + 0.9989936 + protein_state + cleaner0 + 2023-07-25T13:06:38Z + DUMMY: + + orphaned + + + 0.6947453 + chemical + cleaner0 + 2023-07-25T13:48:22Z + CHEBI: + + base + + + 0.9976995 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.99824595 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + 0.9381093 + protein_state + cleaner0 + 2023-07-25T13:06:47Z + DUMMY: + + orphan + + + 0.60220456 + chemical + cleaner0 + 2023-07-25T13:48:26Z + CHEBI: + + base + + + 0.999363 + residue_name_number + cleaner0 + 2023-07-25T10:17:10Z + DUMMY: + + E488 + + + 0.9889213 + residue_name + cleaner0 + 2023-07-25T13:41:56Z + SO: + + U + + + 0.9993616 + residue_name_number + cleaner0 + 2023-07-25T10:17:10Z + DUMMY: + + E488 + + + 0.99251854 + residue_name + cleaner0 + 2023-07-25T13:42:00Z + SO: + + C + + + 0.99935347 + residue_name_number + cleaner0 + 2023-07-25T10:17:42Z + DUMMY: + + Q488 + + + 0.9916214 + residue_name + cleaner0 + 2023-07-25T13:42:04Z + SO: + + C + + + 0.9828668 + experimental_method + cleaner0 + 2023-07-25T13:24:16Z + MESH: + + overlay + + + 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2023-07-25T13:48:58Z + CHEBI: + + pyrimidines + + + + RESULTS + paragraph + 8637 + The interaction of the 488 residue with the orphaned base is reminiscent of an interaction observed for Hha I DNA methyltransfersase (MTase), a duplex DNA modifying enzyme that also uses a base flipping mechanism to access 2’-deoxycytidine (dC) for methylation. For that enzyme, Q237 H-bonds to an orphaned dG while it fills the void left by the flipped out dC (Supplementary Fig. 4b). In addition, two glycine residues flank Q237 allowing the loop to adopt the conformation necessary for penetration into the helix. The flipping loop in ADAR2 (i.e. aa487–489) also has the helix-penetrating residue flanked by glycines. However, unlike the case of the DNA MTase that approaches the DNA from the major groove, the ADAR2 loop approaches the duplex from the minor groove side. Such an approach requires deeper penetration of the intercalating residue to access the H-bonding sites on the orphaned base, necessitating an additional conformational change in the RNA duplex. This change includes shifting of the base pairs immediately 5’ to the editing site toward the helical axis and a widening of the major groove opposite the editing site (Figs. 4a, 4b, Supplementary Video 1). In the case of the hADAR2d WT–Bdf2-U RNA, this shift is accompanied by a shearing of the U11-A13' base pair with U11 shifted further in the direction of the major groove creating an unusual U-A "wobble" interaction with adenine N6 and N1 within H-bonding distance to uracil N3H and O2, respectively (Fig. 4c, Supplementary Fig. 3b). This type of wobble pair has been observed before and requires either the imino tautomer of adenine or the enol tautomer of uracil. The ADAR-induced distortion in RNA conformation results in a kink in the RNA strand opposite the editing site (Fig. 4b). This kink is stabilized by interactions of the side chains of R510 and S495 with phosphodiesters in the RNA backbone of the unedited strand (Fig. 4a). Interestingly, ADAR2’s flipping loop approach from the minor groove side is like that seen with certain DNA repair glycosylases (e.g. UDG, HOGG1, and AAG) that also project intercalating residues from loops bound in the minor groove (Supplementary Fig. 5a). However, these enzymes typically bend the DNA duplex at the site of modification to allow for penetration of intercalating residues and damage recognition. While hADAR2d clearly alters the duplex conformation to gain access to the modification site from the minor groove, it does not bend the RNA duplex (Figs. 2a, 2b, 4b). Furthermore, ADARs do not modify duplex DNA. The DNA B-form helix has groove widths and depths that would prevent productive interactions with ADAR. For instance, ADAR can readily penetrate an A-form helix from the minor groove side and place the helix-penetrating residue in the space occupied by the editing site base (Supplementary Fig. 6). However, this residue cannot penetrate the minor groove enough to occupy the base position in a B-form helix (Supplementary Fig. 6). Furthermore, DNA lacks the 2’ hydroxyls that are used by ADAR for substrate recognition (Fig. 2c). Indeed, in each of the four complexes reported here, the protein contacts at least five ribose 2’ hydroxyl groups (Fig. 2c, Supplementary Fig. 2 b–d). Thus, hADAR2d uses a substrate recognition and base flipping mechanism with similarities to other known nucleic acid-modifying enzymes but uniquely suited for reaction with adenosine in the context of duplex RNA. + + 0.9974469 + residue_number + cleaner0 + 2023-07-25T10:17:32Z + DUMMY: + + 488 + + + 0.9990959 + protein_state + cleaner0 + 2023-07-25T13:06:38Z + DUMMY: + + orphaned + + + 0.9591489 + chemical + cleaner0 + 2023-07-25T13:49:01Z + CHEBI: + + base + + + 0.9977617 + protein_type + cleaner0 + 2023-07-25T10:24:59Z + MESH: + + Hha I DNA methyltransfersase + + + 0.9968746 + protein_type + cleaner0 + 2023-07-25T10:25:04Z + MESH: + + MTase + + + structure_element + SO: + cleaner0 + 2023-07-25T13:49:21Z + + duplex DNA + + + 0.99842787 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + 2’-deoxycytidine + + + 0.97770005 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + dC + + + 0.9995128 + residue_name_number + cleaner0 + 2023-07-25T10:18:48Z + DUMMY: + + Q237 + + + 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2023-07-25T13:21:38Z + MESH: + + H-bonding + + + 0.8015464 + residue_name + cleaner0 + 2023-07-25T10:19:01Z + SO: + + uracil + + + 0.6237521 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + adenine + + + 0.6437778 + residue_name + cleaner0 + 2023-07-25T10:19:00Z + SO: + + uracil + + + 0.9985373 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.99536455 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.94565856 + structure_element + cleaner0 + 2023-07-25T13:28:44Z + SO: + + kink + + + 0.9887573 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.99767315 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.9601849 + structure_element + cleaner0 + 2023-07-25T13:28:49Z + SO: + + kink + + + 0.9994771 + residue_name_number + cleaner0 + 2023-07-25T10:19:21Z + DUMMY: + + R510 + + + 0.99949586 + residue_name_number + cleaner0 + 2023-07-25T10:19:26Z + DUMMY: + + S495 + + + 0.9960375 + chemical + cleaner0 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DNA + + + protein_type + MESH: + cleaner0 + 2023-07-25T09:55:43Z + + ADAR + + + mutant + MESH: + cleaner0 + 2023-07-25T10:05:13Z + + hADAR2d + + + protein_type + MESH: + cleaner0 + 2023-07-25T10:25:50Z + + nucleic acid-modifying enzymes + + + residue_name + SO: + cleaner0 + 2023-07-25T10:01:02Z + + adenosine + + + structure_element + SO: + cleaner0 + 2023-07-25T13:31:35Z + + duplex RNA + + + + RESULTS + title_2 + 12089 + Structures explain nearest neighbor preferences + + 0.99768806 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + Structures + + + + RESULTS + paragraph + 12137 + ADARs have a preference for editing adenosines with 5’ nearest neighbor U (or A) and 3’ nearest neighbor G. The ADAR2 flipping loop occupies the minor groove spanning the three base pairs that include the nearest neighbor nucleotides flanking the edited base (Figs. 3b, 3c). As described above, the base pair including the 5’ nearest neighbor U (U11-A13’ in the Bdf2 duplex) is shifted from the position it would occupy in a typical A-form helix to accommodate the loop (Fig. 4a). Also, the minor groove edge of this pair is juxtaposed to the protein backbone at G489. Modeling a G-C or C-G pair at this position (i.e. 5’ G or 5’ C) suggests a 2-amino group in the minor groove would clash with the protein at G489 (Fig. 5a, Supplementary Fig. 7c). Indeed, replacing the U-A pair adjacent to the editing site with a C-G pair in the Gli1 duplex substrate resulted in an 80% reduction in the rate of hADAR2d-catalyzed deamination (Figs. 5b, 5c). To determine whether this effect arises from an increase in local duplex stability from the C-G for U-A substitution or from the presence of the 2-amino group, we replaced the U-A pair with a U-2-aminopurine (2AP) pair. 2AP is an adenosine analog that forms a base pair with uridine of similar stability to a U-A pair, but places an amino group in the minor groove (Fig. 5b). Importantly, this substitution also resulted in an 80% reduction in rate, illustrating the detrimental effect of the amino group in the minor groove at this location. These observations suggest that hADAR2’s 5’ nearest neighbor preference for U (or A) is due to a destabilizing clash with the protein backbone at G489 that results from the presence of an amino group in the minor groove at this location for sequences with 5’ nearest neighbor G or C. However, the observed clash is not severe and the enzyme would be able to accommodate G or C 5’ nearest neighbors by slight structural perturbations, explaining why this sequence preference is not an absolute requirement. + + 0.99847287 + protein_type + cleaner0 + 2023-07-25T09:55:35Z + MESH: + + ADARs + + + 0.61509526 + residue_name + cleaner0 + 2023-07-25T10:02:01Z + SO: + + adenosines + + + 0.9750131 + residue_name + cleaner0 + 2023-07-25T13:42:34Z + SO: + + U + + + 0.97945625 + residue_name + cleaner0 + 2023-07-25T13:42:38Z + SO: + + A + + + 0.99179685 + residue_name + cleaner0 + 2023-07-25T13:42:41Z + SO: + + G + + + 0.9993987 + protein + cleaner0 + 2023-07-25T09:55:25Z + PR: + + ADAR2 + + + 0.99877775 + structure_element + cleaner0 + 2023-07-25T13:29:06Z + SO: + + flipping loop + + + 0.9863656 + site + cleaner0 + 2023-07-25T10:16:52Z + SO: + + minor groove + + + 0.96959674 + residue_name + cleaner0 + 2023-07-25T13:42:45Z + SO: + + U + + + 0.9284168 + residue_name_number + cleaner0 + 2023-07-25T10:19:07Z + DUMMY: + + U11 + + + 0.6566469 + residue_name_number + cleaner0 + 2023-07-25T10:19:15Z + DUMMY: + + A13 + + + 0.9246292 + chemical + cleaner0 + 2023-07-25T12:51:55Z + CHEBI: + + Bdf2 + + + structure_element + SO: + cleaner0 + 2023-07-25T10:27:08Z + + A-form helix + + + 0.9993144 + structure_element + cleaner0 + 2023-07-25T13:29:11Z + SO: + + loop + + + 0.97895014 + site + cleaner0 + 2023-07-25T10:16:52Z + SO: + + minor groove + + + 0.9993475 + residue_name_number + cleaner0 + 2023-07-25T13:21:15Z + DUMMY: + + G489 + + + structure_element + SO: + cleaner0 + 2023-07-25T10:28:27Z + + G-C or C-G pair + + + 0.7440572 + residue_name + cleaner0 + 2023-07-25T13:42:49Z + SO: + + G + + + 0.88882107 + residue_name + cleaner0 + 2023-07-25T13:42:53Z + SO: + + C + + + site + SO: + cleaner0 + 2023-07-25T10:16:52Z + + minor groove + + + 0.9992286 + residue_name_number + cleaner0 + 2023-07-25T13:21:16Z + DUMMY: + + G489 + + + structure_element + SO: + cleaner0 + 2023-07-25T10:29:06Z + + U-A pair + + + 0.9971872 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + structure_element + SO: + cleaner0 + 2023-07-25T10:28:45Z + + C-G pair + + + 0.8445411 + protein + cleaner0 + 2023-07-25T10:04:07Z + PR: + + Gli1 + + + 0.9993086 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + structure_element + SO: + cleaner0 + 2023-07-25T10:29:05Z + + U-A pair + + + structure_element + SO: + cleaner0 + 2023-07-25T10:29:45Z + + U-2-aminopurine (2AP) pair + + + 0.99904865 + structure_element + cleaner0 + 2023-07-25T10:29:58Z + SO: + + 2AP + + + 0.9955627 + residue_name + cleaner0 + 2023-07-25T10:01:02Z + SO: + + adenosine + + + 0.8317101 + residue_name + cleaner0 + 2023-07-25T13:42:57Z + SO: + + uridine + + + structure_element + SO: + cleaner0 + 2023-07-25T10:29:06Z + + U-A pair + + + 0.6579745 + site + cleaner0 + 2023-07-25T10:16:52Z + SO: + + minor groove + + + site + SO: + cleaner0 + 2023-07-25T10:16:52Z + + minor groove + + + 0.9993399 + protein + cleaner0 + 2023-07-25T12:46:38Z + PR: + + hADAR2 + + + 0.9527736 + residue_name + cleaner0 + 2023-07-25T13:43:02Z + SO: + + U + + + 0.97002554 + residue_name + cleaner0 + 2023-07-25T13:43:05Z + SO: + + A + + + 0.99912757 + residue_name_number + cleaner0 + 2023-07-25T13:21:16Z + DUMMY: + + G489 + + + 0.6631111 + site + cleaner0 + 2023-07-25T10:16:52Z + SO: + + minor groove + + + 0.9823569 + residue_name + cleaner0 + 2023-07-25T13:43:09Z + SO: + + G + + + 0.98902214 + residue_name + cleaner0 + 2023-07-25T13:43:14Z + SO: + + C + + + 0.985767 + residue_name + cleaner0 + 2023-07-25T13:43:19Z + SO: + + G + + + 0.98667836 + residue_name + cleaner0 + 2023-07-25T13:43:22Z + SO: + + C + + + + RESULTS + paragraph + 14150 + In each of the hADAR2d-RNA structures reported here, the backbone carbonyl oxygen at S486 accepts an H-bond from the 2-amino group of the G on the 3’ side of the edited nucleotide (Fig. 5d). Guanine is the only common nucleobase that presents an H-bond donor in the RNA minor groove suggesting that other nucleotides in this position would reduce editing efficiency. Indeed, mutating this base to A, C or U, while maintaining base pairing at this position, reduced the rate of deamination by hADAR2d in Gli1 mRNA model substrates (Supplementary Fig. 7 a–b). To test the importance of the amino group on the 3’ G in the hADAR2d reaction, we prepared RNA duplex substrates with purine analogs on the 3’ side of the edited A (Fig. 5e). We tested a G analog that lacks the 2-amino group (inosine, I) and one that blocks access to this amino group (N2-methylguanosine (N2MeG). In addition, we compared a 3’ A to a 3’ 2AP since 2AP could form the H-bonding interaction observed with S486. We found the substrate with a 3’ N2MeG to be unreactive to hADAR2d-catalyzed deamination confirming the importance of the observed close approach by the protein to the 3’ G 2-amino group (Fig. 5f). In addition, the substrate with a 3’ I displayed a reduced deamination rate compared to the substrate with a 3’ G suggesting the observed H-bond to the 2-amino group contributes to the 3’ nearest neighbor selectivity (Fig. 5f). This conclusion is further supported by the observation that deamination in the substrate with a 3’ 2AP is faster than in the substrate with a 3’ A (Fig. 5f). + + 0.99904656 + complex_assembly + cleaner0 + 2023-07-25T10:05:50Z + GO: + + hADAR2d-RNA + + + 0.9972102 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + 0.9994696 + residue_name_number + cleaner0 + 2023-07-25T10:36:39Z + DUMMY: + + S486 + + + 0.9939011 + bond_interaction + cleaner0 + 2023-07-25T10:59:14Z + MESH: + + H-bond + + + 0.9806425 + residue_name + cleaner0 + 2023-07-25T13:43:27Z + SO: + + G + + + 0.7793279 + residue_name + cleaner0 + 2023-07-25T13:43:30Z + SO: + + Guanine + + + 0.8778884 + bond_interaction + cleaner0 + 2023-07-25T10:59:14Z + MESH: + + H-bond + + + site + SO: + cleaner0 + 2023-07-25T13:30:41Z + + RNA minor groove + + + 0.9876578 + experimental_method + cleaner0 + 2023-07-25T13:24:43Z + MESH: + + mutating + + + 0.9561622 + residue_name + cleaner0 + 2023-07-25T13:43:41Z + SO: + + A + + + 0.9668023 + residue_name + cleaner0 + 2023-07-25T13:43:45Z + SO: + + C + + + 0.9219839 + residue_name + cleaner0 + 2023-07-25T13:43:49Z + SO: + + U + + + 0.9993303 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.35575464 + protein + cleaner0 + 2023-07-25T10:04:07Z + PR: + + Gli1 + + + 0.99058235 + chemical + cleaner0 + 2023-07-25T10:03:39Z + CHEBI: + + mRNA + + + 0.95881605 + residue_name + cleaner0 + 2023-07-25T13:43:53Z + SO: + + G + + + 0.9977976 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + structure_element + SO: + cleaner0 + 2023-07-25T13:30:55Z + + RNA duplex + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:44:11Z + + edited + + + 0.9810914 + residue_name + cleaner0 + 2023-07-25T13:44:00Z + SO: + + A + + + 0.98723465 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + G + + + 0.9829754 + residue_name + cleaner0 + 2023-07-25T10:01:31Z + SO: + + inosine + + + 0.92541546 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + I + + + 0.5322165 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + A + + + 0.9744812 + structure_element + cleaner0 + 2023-07-25T10:29:59Z + SO: + + 2AP + + + 0.9918903 + structure_element + cleaner0 + 2023-07-25T10:29:59Z + SO: + + 2AP + + + 0.989429 + bond_interaction + cleaner0 + 2023-07-25T13:21:43Z + MESH: + + H-bonding interaction + + + 0.9993993 + residue_name_number + cleaner0 + 2023-07-25T10:36:40Z + DUMMY: + + S486 + + + 0.99861157 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.69805527 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + G + + + 0.7320845 + residue_name + cleaner0 + 2023-07-25T13:44:28Z + SO: + + I + + + evidence + DUMMY: + cleaner0 + 2023-07-25T13:53:26Z + + reduced deamination rate + + + 0.74582076 + residue_name + cleaner0 + 2023-07-25T13:44:38Z + SO: + + G + + + 0.9892564 + bond_interaction + cleaner0 + 2023-07-25T10:59:14Z + MESH: + + H-bond + + + 0.9920855 + structure_element + cleaner0 + 2023-07-25T10:29:59Z + SO: + + 2AP + + + 0.5132235 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + A + + + + RESULTS + title_2 + 15744 + RNA-binding loops of the ADAR catalytic domain + + 0.91373694 + structure_element + cleaner0 + 2023-07-25T10:37:23Z + SO: + + RNA-binding loops + + + 0.97370166 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.99916327 + structure_element + cleaner0 + 2023-07-25T10:49:04Z + SO: + + catalytic domain + + + + RESULTS + paragraph + 15791 + The structures reported here identify RNA-binding loops of the ADAR catalytic domain and suggest roles for several amino acids not previously known to be important for editing, either substrate binding or catalysis (Fig. 6). The side chain for R510 ion-pairs with the 3’ phosphodiester of the orphaned nucleotide (Figs. 3a, 3c). This residue is conserved in ADAR2s and ADAR1s, but is glutamine in the editing-inactive ADAR3s (Supplementary Table 1). Mutation of hADAR2d at this site to either glutamine (R510Q) or to alanine (R510A) reduced the measured deamination rate constant by approximately an order of magnitude (Fig. 6c). In addition, the contact point near the 5’ end of the unedited strand involves G593, K594 and R348, residues completely conserved in the family of ADAR2s (Fig. 2c, Supplementary Table 1). Mutation of any of these residues to alanine (G593A, K594A, R348A) substantially reduces editing activity (Fig. 6c). In addition, mutation of G593 to glutamic acid (G593E) resulted in a nearly two orders of magnitude reduction in rate, consistent with proximity of this residue to the negatively charged phosphodiester backbone of the RNA (Fig. 6c). + + 0.99837226 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-07-25T10:37:23Z + + RNA-binding loops + + + 0.9976526 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.9970461 + structure_element + cleaner0 + 2023-07-25T10:49:04Z + SO: + + catalytic domain + + + 0.9994267 + residue_name_number + cleaner0 + 2023-07-25T10:19:22Z + DUMMY: + + R510 + + + bond_interaction + MESH: + cleaner0 + 2023-07-25T10:59:14Z + + ion-pairs + + + 0.99920183 + protein_state + cleaner0 + 2023-07-25T13:06:38Z + DUMMY: + + orphaned + + + 0.99883956 + chemical + cleaner0 + 2023-07-25T13:49:50Z + CHEBI: + + nucleotide + + + 0.99890375 + protein_state + cleaner0 + 2023-07-25T13:14:34Z + DUMMY: + + conserved + + + 0.9977901 + protein_type + cleaner0 + 2023-07-25T10:39:28Z + MESH: + + ADAR2s + + + 0.99760556 + protein_type + cleaner0 + 2023-07-25T13:00:29Z + MESH: + + ADAR1s + + + 0.99579865 + residue_name + cleaner0 + 2023-07-25T10:39:08Z + SO: + + glutamine + + + 0.9983585 + protein_state + cleaner0 + 2023-07-25T13:14:40Z + DUMMY: + + editing-inactive + + + 0.9725391 + protein_type + cleaner0 + 2023-07-25T13:22:15Z + MESH: + + ADAR3s + + + 0.99792516 + experimental_method + cleaner0 + 2023-07-25T13:24:49Z + MESH: + + Mutation + + + 0.9440897 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.9937645 + residue_name + cleaner0 + 2023-07-25T10:39:09Z + SO: + + glutamine + + + 0.99827445 + mutant + cleaner0 + 2023-07-25T13:22:23Z + MESH: + + R510Q + + + 0.99398625 + residue_name + cleaner0 + 2023-07-25T10:39:14Z + SO: + + alanine + + + 0.99841607 + mutant + cleaner0 + 2023-07-25T13:22:27Z + MESH: + + R510A + + + 0.9965238 + evidence + cleaner0 + 2023-07-25T13:53:31Z + DUMMY: + + deamination rate constant + + + 0.99949515 + residue_name_number + cleaner0 + 2023-07-25T10:38:30Z + DUMMY: + + G593 + + + 0.9994505 + residue_name_number + cleaner0 + 2023-07-25T10:38:36Z + DUMMY: + + K594 + + + 0.99943036 + residue_name_number + cleaner0 + 2023-07-25T10:38:41Z + DUMMY: + + R348 + + + 0.9988131 + protein_state + cleaner0 + 2023-07-25T13:14:31Z + DUMMY: + + completely conserved + + + 0.9438753 + protein_type + cleaner0 + 2023-07-25T10:39:28Z + MESH: + + ADAR2s + + + 0.99778724 + experimental_method + cleaner0 + 2023-07-25T13:24:58Z + MESH: + + Mutation + + + 0.99302965 + residue_name + cleaner0 + 2023-07-25T10:39:14Z + SO: + + alanine + + + 0.99885595 + mutant + cleaner0 + 2023-07-25T10:38:47Z + MESH: + + G593A + + + 0.9989629 + mutant + cleaner0 + 2023-07-25T10:38:53Z + MESH: + + K594A + + + 0.9989385 + mutant + cleaner0 + 2023-07-25T10:38:58Z + MESH: + + R348A + + + 0.9981035 + experimental_method + cleaner0 + 2023-07-25T13:25:00Z + MESH: + + mutation + + + 0.9993979 + residue_name_number + cleaner0 + 2023-07-25T10:38:32Z + DUMMY: + + G593 + + + residue_name + SO: + cleaner0 + 2023-07-25T13:45:49Z + + glutamic acid + + + 0.99825233 + mutant + cleaner0 + 2023-07-25T10:39:03Z + MESH: + + G593E + + + 0.9984108 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + + RESULTS + paragraph + 16963 + RNA binding leads to an ordering of the 454–477 loop, which was disordered in the RNA-free hADAR2d structure (Fig. 1d, green) (Supplementary Video 2). This loop binds the RNA duplex contacting the minor groove near the editing site and inserting into the adjacent major groove (Fig. 6e). This loop sequence is conserved in ADAR2s but different in the family of ADAR1s (Fig. 6d). The substantial difference in sequence between the ADARs in this RNA-binding loop suggests differences in editing site selectivity between the two ADARs arise, at least in part, from differences in how this loop binds RNA substrates. + + 0.6001128 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + residue_range + DUMMY: + cleaner0 + 2023-07-25T10:40:19Z + + 454–477 + + + 0.9937284 + structure_element + cleaner0 + 2023-07-25T13:29:18Z + SO: + + loop + + + 0.9932376 + protein_state + cleaner0 + 2023-07-25T13:14:50Z + DUMMY: + + disordered + + + 0.9989104 + protein_state + cleaner0 + 2023-07-25T10:39:49Z + DUMMY: + + RNA-free + + + 0.99718505 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.9979814 + evidence + cleaner0 + 2023-07-25T13:53:34Z + DUMMY: + + structure + + + 0.998911 + structure_element + cleaner0 + 2023-07-25T13:29:23Z + SO: + + loop + + + structure_element + SO: + cleaner0 + 2023-07-25T13:30:55Z + + RNA duplex + + + 0.99427783 + site + cleaner0 + 2023-07-25T10:16:52Z + SO: + + minor groove + + + 0.99578905 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.99090636 + site + cleaner0 + 2023-07-25T10:40:25Z + SO: + + major groove + + + 0.9991843 + structure_element + cleaner0 + 2023-07-25T13:29:26Z + SO: + + loop + + + 0.99838054 + protein_state + cleaner0 + 2023-07-25T13:14:53Z + DUMMY: + + conserved + + + 0.6705302 + protein_type + cleaner0 + 2023-07-25T10:39:29Z + MESH: + + ADAR2s + + + 0.6655557 + protein_type + cleaner0 + 2023-07-25T13:00:56Z + MESH: + + ADAR1s + + + 0.999097 + protein_type + cleaner0 + 2023-07-25T09:55:35Z + MESH: + + ADARs + + + 0.99858767 + structure_element + cleaner0 + 2023-07-25T09:57:38Z + SO: + + RNA-binding loop + + + site + SO: + cleaner0 + 2023-07-25T10:14:37Z + + editing site + + + 0.9991285 + protein_type + cleaner0 + 2023-07-25T09:55:35Z + MESH: + + ADARs + + + 0.99679714 + structure_element + cleaner0 + 2023-07-25T13:29:31Z + SO: + + loop + + + 0.98521477 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + + DISCUSS + title_1 + 17578 + Discussion + + + DISCUSS + paragraph + 17589 + Base flipping is a well-characterized mechanism by which nucleic acid modifying enzymes gain access to sites of reaction that are otherwise buried in base-paired structures. DNA methylases, DNA repair glycosylases and RNA loop modifying enzymes are known that flip a nucleotide out of a base pair. However, none of the structurally characterized base-flipping enzymes access their reactive sites from within a normal base-paired RNA duplex. We are aware of one other protein-induced nucleotide flipping from an RNA duplex region. Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b). However, these nucleotides are located in a highly distorted and dynamic duplex region containing several mismatches and are predisposed to undergo this conformational change. Thus, this system is not illustrative of base flipping from a normal duplex and does not involve an enzyme that must carryout a chemical reaction on the flipped out nucleotide. Other RNA modification enzymes are known that flip nucleotides out of loops, even from base pairs in loop regions (pseudoU synthetase, tRNA transglycosylase, and restrictocin bound to sarcin/ricin loop of 28S rRNA) (Supplementary Fig. 5b). Because the modification sites are not flanked on both sides by normal duplex, these enzymes do not experience the same limits in approach to the substrate that ADARs do. The fact that ADARs must induce flipping from a normal duplex has implications on its preference for adenosines flanked by certain base pairs, a phenomenon that was not well understood prior to this work. + + protein_type + MESH: + cleaner0 + 2023-07-25T10:41:18Z + + nucleic acid modifying enzymes + + + evidence + DUMMY: + cleaner0 + 2023-07-25T13:52:35Z + + structures + + + 0.997954 + protein_type + cleaner0 + 2023-07-25T10:40:47Z + MESH: + + DNA methylases + + + 0.9979725 + protein_type + cleaner0 + 2023-07-25T10:22:35Z + MESH: + + DNA repair glycosylases + + + 0.9971772 + protein_type + cleaner0 + 2023-07-25T10:40:53Z + MESH: + + RNA loop modifying enzymes + + + 0.9964095 + chemical + cleaner0 + 2023-07-25T13:50:00Z + CHEBI: + + nucleotide + + + protein_type + MESH: + cleaner0 + 2023-07-25T10:41:38Z + + base-flipping enzymes + + + 0.919165 + site + cleaner0 + 2023-07-25T13:39:46Z + SO: + + reactive sites + + + 0.94549394 + protein_state + cleaner0 + 2023-07-25T13:40:25Z + DUMMY: + + normal base-paired + + + structure_element + SO: + cleaner0 + 2023-07-25T13:30:55Z + + RNA duplex + + + structure_element + SO: + cleaner0 + 2023-07-25T13:30:55Z + + RNA duplex + + + 0.99814737 + taxonomy_domain + cleaner0 + 2023-07-25T10:41:59Z + DUMMY: + + Bacterial + + + 0.99891967 + protein + cleaner0 + 2023-07-25T10:42:05Z + PR: + + initiation factor 1 + + + 0.9992797 + protein + cleaner0 + 2023-07-25T10:42:10Z + PR: + + IF1 + + + 0.9169075 + complex_assembly + cleaner0 + 2023-07-25T13:22:34Z + GO: + + 30S ribosomal subunit + + + 0.9989307 + structure_element + cleaner0 + 2023-07-25T10:45:14Z + SO: + + helix 44 + + + 0.9986292 + chemical + cleaner0 + 2023-07-25T10:41:48Z + CHEBI: + + 16S RNA + + + 0.99948263 + residue_name_number + cleaner0 + 2023-07-25T13:21:21Z + DUMMY: + + A1492 + + + 0.99949086 + residue_name_number + cleaner0 + 2023-07-25T13:21:24Z + DUMMY: + + A1493 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:07:08Z + + flipped out + + + 0.98667866 + protein_state + cleaner0 + 2023-07-25T13:03:09Z + DUMMY: + + bound into + + + 0.9971239 + site + cleaner0 + 2023-07-25T13:39:57Z + SO: + + protein pockets + + + 0.9980407 + protein_state + cleaner0 + 2023-07-25T13:46:58Z + DUMMY: + + highly distorted + + + 0.96871644 + protein_state + cleaner0 + 2023-07-25T13:47:05Z + DUMMY: + + dynamic + + + 0.7489393 + structure_element + cleaner0 + 2023-07-25T13:29:49Z + SO: + + duplex region + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:40:16Z + + normal + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:07:08Z + + flipped out + + + 0.9982912 + chemical + cleaner0 + 2023-07-25T13:50:08Z + CHEBI: + + nucleotide + + + 0.99167943 + protein_type + cleaner0 + 2023-07-25T10:42:22Z + MESH: + + RNA modification enzymes + + + 0.998663 + protein_type + cleaner0 + 2023-07-25T10:42:37Z + MESH: + + pseudoU synthetase + + + 0.9987348 + protein_type + cleaner0 + 2023-07-25T10:42:42Z + MESH: + + tRNA transglycosylase + + + 0.98716146 + protein + cleaner0 + 2023-07-25T10:43:47Z + PR: + + restrictocin + + + 0.99900997 + protein_state + cleaner0 + 2023-07-25T13:01:17Z + DUMMY: + + bound to + + + 0.9991325 + structure_element + cleaner0 + 2023-07-25T10:43:38Z + SO: + + sarcin/ricin loop + + + 0.99810815 + chemical + cleaner0 + 2023-07-25T10:42:28Z + CHEBI: + + 28S rRNA + + + 0.9044156 + site + cleaner0 + 2023-07-25T13:40:01Z + SO: + + modification sites + + + 0.9764273 + protein_state + cleaner0 + 2023-07-25T13:40:15Z + DUMMY: + + normal + + + structure_element + SO: + cleaner0 + 2023-07-25T13:40:35Z + + duplex + + + 0.985137 + protein_type + cleaner0 + 2023-07-25T09:55:35Z + MESH: + + ADARs + + + 0.9747775 + protein_type + cleaner0 + 2023-07-25T09:55:35Z + MESH: + + ADARs + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:40:16Z + + normal + + + structure_element + SO: + cleaner0 + 2023-07-25T13:40:45Z + + duplex + + + 0.9428024 + residue_name + cleaner0 + 2023-07-25T10:02:01Z + SO: + + adenosines + + + + DISCUSS + paragraph + 19284 + In our structures, the flipped out 8-azanebularine is hydrated, mimicking the tetrahedral intermediate predicted for deamination of adenosine (Figs. 1b, 3a, Supplementary Fig. 3 a–b). Our use of 8-azanebularine, with its high propensity to form a covalent hydrate, allowed us to capture a true mimic of the tetrahedral intermediate and reveal the interactions between the deaminase active site and the reactive nucleotide. In addition, 8-azanebularine was found to adopt a 2’-endo sugar pucker with its 2’-hydroxyl H-bonded to the protein backbone carbonyl at T375. The 2’ endo conformation appears to facilitate access of the nucleobase to the zinc-bound water for nucleophilic attack at C6. + + 0.99846375 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + 0.7341455 + protein_state + cleaner0 + 2023-07-25T13:07:08Z + DUMMY: + + flipped out + + + 0.9991346 + chemical + cleaner0 + 2023-07-25T13:47:57Z + CHEBI: + + 8-azanebularine + + + 0.99516124 + residue_name + cleaner0 + 2023-07-25T10:01:02Z + SO: + + adenosine + + + 0.99915075 + chemical + cleaner0 + 2023-07-25T13:47:57Z + CHEBI: + + 8-azanebularine + + + 0.99889934 + protein_type + cleaner0 + 2023-07-25T13:52:26Z + MESH: + + deaminase + + + 0.9989484 + site + cleaner0 + 2023-07-25T10:14:49Z + SO: + + active site + + + 0.99914354 + chemical + cleaner0 + 2023-07-25T13:47:57Z + CHEBI: + + 8-azanebularine + + + bond_interaction + MESH: + cleaner0 + 2023-07-25T10:59:14Z + + H-bonded + + + 0.99943453 + residue_name_number + cleaner0 + 2023-07-25T10:17:55Z + DUMMY: + + T375 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T10:15:31Z + + zinc + + + 0.99898225 + chemical + cleaner0 + 2023-07-25T13:50:47Z + CHEBI: + + water + + + + DISCUSS + paragraph + 19985 + Several other base-flipping enzymes stabilize the altered nucleic acid conformation by intercalation of an amino acid side chain into the space vacated by the flipped out base. For hADAR2, E488 serves this role. In the two structures with wild type hADAR2, the E488 residue and orphan base are in nearly identical positions (see Supplementary Fig. 4a for overlay). Thus, the E488 side chain directly contacts each orphan base, likely by accepting an H-bond from uracil N3H or by donating an H-bond to cytidine N3. The latter interaction requires E488 to be protonated. The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation. + + 0.9445512 + protein_type + cleaner0 + 2023-07-25T10:41:38Z + MESH: + + base-flipping enzymes + + + 0.9972215 + protein_state + cleaner0 + 2023-07-25T13:07:08Z + DUMMY: + + flipped out + + + chemical + CHEBI: + cleaner0 + 2023-07-25T13:16:10Z + + base + + + 0.99940264 + protein + cleaner0 + 2023-07-25T12:46:38Z + PR: + + hADAR2 + + + 0.99926263 + residue_name_number + cleaner0 + 2023-07-25T10:17:10Z + DUMMY: + + E488 + + + 0.99782324 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + 0.9990086 + protein_state + cleaner0 + 2023-07-25T10:05:59Z + DUMMY: + + wild type + + + 0.9993981 + protein + cleaner0 + 2023-07-25T12:46:38Z + PR: + + hADAR2 + + + 0.99897194 + residue_name_number + cleaner0 + 2023-07-25T10:17:10Z + DUMMY: + + E488 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:06:48Z + + orphan + + + chemical + CHEBI: + cleaner0 + 2023-07-25T13:16:18Z + + base + + + 0.6052293 + experimental_method + cleaner0 + 2023-07-25T13:25:11Z + MESH: + + overlay + + + 0.9988833 + residue_name_number + cleaner0 + 2023-07-25T10:17:10Z + DUMMY: + + E488 + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:06:48Z + + orphan + + + chemical + CHEBI: + cleaner0 + 2023-07-25T13:16:26Z + + base + + + bond_interaction + MESH: + cleaner0 + 2023-07-25T10:59:14Z + + H-bond + + + 0.59853625 + residue_name + cleaner0 + 2023-07-25T10:19:01Z + SO: + + uracil + + + bond_interaction + MESH: + cleaner0 + 2023-07-25T10:59:14Z + + H-bond + + + 0.51537895 + residue_name + cleaner0 + 2023-07-25T10:01:16Z + SO: + + cytidine + + + 0.9992355 + residue_name_number + cleaner0 + 2023-07-25T10:17:10Z + DUMMY: + + E488 + + + 0.99447674 + protein_state + cleaner0 + 2023-07-25T13:16:32Z + DUMMY: + + protonated + + + evidence + DUMMY: + cleaner0 + 2023-07-25T10:45:48Z + + pKa + + + 0.9993344 + residue_name_number + cleaner0 + 2023-07-25T10:17:10Z + DUMMY: + + E488 + + + 0.9990718 + complex_assembly + cleaner0 + 2023-07-25T10:45:29Z + GO: + + ADAR-RNA + + + bond_interaction + MESH: + cleaner0 + 2023-07-25T10:59:14Z + + H-bond + + + 0.7995467 + residue_name + cleaner0 + 2023-07-25T10:01:16Z + SO: + + cytidine + + + 0.99874926 + protein_state + cleaner0 + 2023-07-25T13:16:35Z + DUMMY: + + protonated + + + 0.99577385 + residue_name + cleaner0 + 2023-07-25T10:39:09Z + SO: + + glutamine + + + 0.99860775 + protein_state + cleaner0 + 2023-07-25T13:16:36Z + DUMMY: + + fully protonated + + + 0.99905044 + mutant + cleaner0 + 2023-07-25T10:08:01Z + MESH: + + E488Q + + + 0.99912184 + protein_state + cleaner0 + 2023-07-25T10:07:54Z + DUMMY: + + mutant + + + 0.99913186 + residue_name_number + cleaner0 + 2023-07-25T10:17:10Z + DUMMY: + + E488 + + + + DISCUSS + paragraph + 21045 + The interactions of hADAR2d with base pairs adjacent to the editing site adenosine explain the known 5’ and 3’ nearest neighbor preferences (Fig. 5). While these studies indicate the ADAR2 catalytic domain makes an important contact to the 3’ nearest neighbor G, Stefl et al. suggested the 3’ G preference arises from dsRBD binding selectivity for ADAR2. These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. They describe an interaction wherein the 3’ G 2-amino group H-bonds to the backbone carbonyl of S258 found in the β1-β2 loop of ADAR2’s dsRBDII. It is not possible for the S486-3’G interaction we describe here and the S258-3’G interaction reported by Stefl et al. to exist in the same complex since both involve protein loops bound in the RNA minor groove at the same location. Because our structures have captured the edited nucleotide in the conformation required to access the active site, the interactions observed here are highly likely to occur during the deamination reaction at the editing site. However, since dsRBDs are known to bind promiscuously with duplex RNA, it is possible that the S258-3’G interaction found in a complex lacking the deaminase domain is not relevant to catalysis at the editing site. It is also possible that ADAR dsRBD and catalytic domain binding are sequential, with release of the dsRBD from the RNA taking place prior to catalytic domain engagement and base flipping. + + 0.9993193 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.9967619 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.76166403 + residue_name + cleaner0 + 2023-07-25T10:01:02Z + SO: + + adenosine + + + 0.9994361 + protein + cleaner0 + 2023-07-25T09:55:25Z + PR: + + ADAR2 + + + 0.9991903 + structure_element + cleaner0 + 2023-07-25T10:49:04Z + SO: + + catalytic domain + + + 0.98009014 + residue_name + cleaner0 + 2023-07-25T13:44:47Z + SO: + + G + + + 0.51092875 + residue_name + cleaner0 + 2023-07-25T13:44:51Z + SO: + + G + + + 0.9994537 + structure_element + cleaner0 + 2023-07-25T10:47:55Z + SO: + + dsRBD + + + 0.99942076 + protein + cleaner0 + 2023-07-25T09:55:25Z + PR: + + ADAR2 + + + 0.9994153 + protein + cleaner0 + 2023-07-25T09:55:25Z + PR: + + ADAR2 + + + 0.9995172 + structure_element + cleaner0 + 2023-07-25T10:01:54Z + SO: + + dsRBDs + + + 0.99904764 + protein_state + cleaner0 + 2023-07-25T13:01:17Z + DUMMY: + + bound to + + + 0.9986879 + experimental_method + cleaner0 + 2023-07-25T13:25:23Z + MESH: + + NMR + + + 0.96731144 + protein_state + cleaner0 + 2023-07-25T13:47:11Z + DUMMY: + + isolated + + + 0.9992256 + structure_element + cleaner0 + 2023-07-25T10:01:54Z + SO: + + dsRBDs + + + 0.9985185 + protein_state + cleaner0 + 2023-07-25T13:16:51Z + DUMMY: + + lacking + + + 0.9970796 + structure_element + cleaner0 + 2023-07-25T09:57:47Z + SO: + + deaminase domain + + + 0.9971438 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.9595557 + protein + cleaner0 + 2023-07-25T13:00:38Z + PR: + + GluR-B + + + 0.98169994 + site + cleaner0 + 2023-07-25T13:41:00Z + SO: + + R/G site + + + 0.9967219 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.906457 + residue_name + cleaner0 + 2023-07-25T13:44:54Z + SO: + + G + + + bond_interaction + MESH: + cleaner0 + 2023-07-25T10:59:14Z + + H-bonds + + + 0.9995441 + residue_name_number + cleaner0 + 2023-07-25T13:21:31Z + DUMMY: + + S258 + + + 0.9991859 + structure_element + cleaner0 + 2023-07-25T13:34:22Z + SO: + + β1-β2 loop + + + 0.9994288 + protein + cleaner0 + 2023-07-25T09:55:25Z + PR: + + ADAR2 + + + 0.99953234 + structure_element + cleaner0 + 2023-07-25T10:48:02Z + SO: + + dsRBDII + + + 0.99939764 + residue_name_number + cleaner0 + 2023-07-25T10:36:40Z + DUMMY: + + S486 + + + residue_name + SO: + cleaner0 + 2023-07-25T13:45:49Z + + G + + + 0.9991021 + residue_name_number + cleaner0 + 2023-07-25T13:21:31Z + DUMMY: + + S258 + + + residue_name + SO: + cleaner0 + 2023-07-25T13:45:49Z + + G + + + 0.9948474 + protein_state + cleaner0 + 2023-07-25T13:11:43Z + DUMMY: + + bound in + + + 0.9951953 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.991618 + site + cleaner0 + 2023-07-25T10:16:52Z + SO: + + minor groove + + + 0.9984658 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + 0.99736243 + protein_state + cleaner0 + 2023-07-25T13:47:24Z + DUMMY: + + edited + + + 0.99850154 + chemical + cleaner0 + 2023-07-25T13:50:53Z + CHEBI: + + nucleotide + + + 0.99837446 + site + cleaner0 + 2023-07-25T10:14:49Z + SO: + + active site + + + 0.9976063 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.9995388 + structure_element + cleaner0 + 2023-07-25T10:01:54Z + SO: + + dsRBDs + + + structure_element + SO: + cleaner0 + 2023-07-25T13:31:35Z + + duplex RNA + + + 0.9989403 + residue_name_number + cleaner0 + 2023-07-25T13:21:31Z + DUMMY: + + S258 + + + residue_name + SO: + cleaner0 + 2023-07-25T13:45:49Z + + G + + + 0.97664535 + protein_state + cleaner0 + 2023-07-25T13:16:56Z + DUMMY: + + lacking the + + + 0.99540865 + structure_element + cleaner0 + 2023-07-25T09:57:47Z + SO: + + deaminase domain + + + 0.9973092 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.99937254 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.9995197 + structure_element + cleaner0 + 2023-07-25T10:47:55Z + SO: + + dsRBD + + + 0.99380386 + structure_element + cleaner0 + 2023-07-25T10:49:04Z + SO: + + catalytic domain + + + 0.9995154 + structure_element + cleaner0 + 2023-07-25T10:47:54Z + SO: + + dsRBD + + + 0.99624735 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.9874256 + structure_element + cleaner0 + 2023-07-25T10:49:04Z + SO: + + catalytic domain + + + + DISCUSS + paragraph + 22645 + Aicardi-Goutieres Syndrome (AGS) and Dyschromatosis Symmetrica Hereditaria (DSH) are human diseases caused by mutations in the human ADAR1 gene and several of the disease-associated mutations are found in the deaminase domain. Given the conservation in RNA binding surface and active site residues, we expect the hADAR1 catalytic domain to bind RNA with a similar orientation of the helix found in our hADAR2d-RNA structures. When one maps the locations of the AGS-associated mutations onto the hADAR2d-RNA complex, two mutations involve residues in close proximity to the RNA (< 4 Å) (Supplementary Fig. 8a). G487 of hADAR2 is found on the flipping loop near the RNA (Fig. 3b). Sequence in this loop is highly conserved among ADARs and corresponds to G1007 in hADAR1 (Supplementary Table 2). An arginine at this position would preclude close approach of the flipping loop to the RNA, preventing E1008 insertion and base flipping into the active site (Supplementary Fig. 8b). This is consistent with the observation that the G1007R mutation in hADAR1 inhibits RNA editing activity. Also, K376 forms salt bridges with both the 5’ and 3’ phosphodiesters of the guanosine on the 3’ side of the editing site (Fig. 2). The corresponding residue in hADAR1 (R892) could form similar contacts and the R892H mutation would likely alter this interaction. + + 0.9938544 + species + cleaner0 + 2023-07-25T09:56:21Z + MESH: + + human + + + 0.9985089 + species + cleaner0 + 2023-07-25T09:56:21Z + MESH: + + human + + + 0.999252 + protein + cleaner0 + 2023-07-25T10:03:05Z + PR: + + ADAR1 + + + 0.99922824 + structure_element + cleaner0 + 2023-07-25T09:57:47Z + SO: + + deaminase domain + + + 0.9987702 + site + cleaner0 + 2023-07-25T13:41:07Z + SO: + + RNA binding surface + + + site + SO: + cleaner0 + 2023-07-25T10:14:49Z + + active site + + + 0.9994324 + protein + cleaner0 + 2023-07-25T10:49:20Z + PR: + + hADAR1 + + + 0.9993752 + structure_element + cleaner0 + 2023-07-25T10:49:03Z + SO: + + catalytic domain + + + 0.9968593 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.99922734 + complex_assembly + cleaner0 + 2023-07-25T10:05:50Z + GO: + + hADAR2d-RNA + + + 0.9981173 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + 0.9991992 + complex_assembly + cleaner0 + 2023-07-25T10:05:50Z + GO: + + hADAR2d-RNA + + + 0.9861281 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.9995925 + residue_name_number + cleaner0 + 2023-07-25T10:49:40Z + DUMMY: + + G487 + + + 0.9994024 + protein + cleaner0 + 2023-07-25T12:46:38Z + PR: + + hADAR2 + + + 0.9993557 + structure_element + cleaner0 + 2023-07-25T13:35:12Z + SO: + + flipping loop + + + 0.9945998 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.99882966 + structure_element + cleaner0 + 2023-07-25T13:35:16Z + SO: + + loop + + + 0.99887 + protein_state + cleaner0 + 2023-07-25T13:17:13Z + DUMMY: + + highly conserved + + + 0.998966 + protein_type + cleaner0 + 2023-07-25T09:55:35Z + MESH: + + ADARs + + + 0.9995789 + residue_name_number + cleaner0 + 2023-07-25T10:49:48Z + DUMMY: + + G1007 + + + 0.99942446 + protein + cleaner0 + 2023-07-25T10:49:20Z + PR: + + hADAR1 + + + 0.99708956 + residue_name + cleaner0 + 2023-07-25T13:45:03Z + SO: + + arginine + + + 0.9993628 + structure_element + cleaner0 + 2023-07-25T13:35:20Z + SO: + + flipping loop + + + 0.9951999 + chemical + cleaner0 + 2023-07-25T09:56:53Z + CHEBI: + + RNA + + + 0.9995913 + residue_name_number + cleaner0 + 2023-07-25T10:49:54Z + DUMMY: + + E1008 + + + 0.99899435 + site + cleaner0 + 2023-07-25T10:14:49Z + SO: + + active site + + + 0.99878937 + mutant + cleaner0 + 2023-07-25T10:49:59Z + MESH: + + G1007R + + + 0.9994253 + protein + cleaner0 + 2023-07-25T10:49:20Z + PR: + + hADAR1 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T09:56:53Z + + RNA + + + 0.99956375 + residue_name_number + cleaner0 + 2023-07-25T10:18:00Z + DUMMY: + + K376 + + + 0.9936708 + bond_interaction + cleaner0 + 2023-07-25T13:21:50Z + MESH: + + salt bridges + + + 0.96985614 + residue_name + cleaner0 + 2023-07-25T10:01:21Z + SO: + + guanosine + + + 0.998822 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.9994412 + protein + cleaner0 + 2023-07-25T10:49:20Z + PR: + + hADAR1 + + + 0.9995346 + residue_name_number + cleaner0 + 2023-07-25T10:50:09Z + DUMMY: + + R892 + + + 0.99895656 + mutant + cleaner0 + 2023-07-25T10:50:15Z + MESH: + + R892H + + + + DISCUSS + paragraph + 23997 + In summary, the structures described here establish human ADAR2 as a base-flipping enzyme that uses a unique mechanism well suited for modifying duplex RNA. In addition, this work provides a basis for understanding the role of the ADAR catalytic domain in determining editing site selectivity and additional structural context to evaluate the impact of ADAR mutations associated with human disease. + + 0.99809986 + evidence + cleaner0 + 2023-07-25T13:52:35Z + DUMMY: + + structures + + + 0.9985241 + species + cleaner0 + 2023-07-25T09:56:21Z + MESH: + + human + + + 0.99930704 + protein + cleaner0 + 2023-07-25T09:55:25Z + PR: + + ADAR2 + + + structure_element + SO: + cleaner0 + 2023-07-25T13:31:34Z + + duplex RNA + + + 0.9993051 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.99936724 + structure_element + cleaner0 + 2023-07-25T10:49:04Z + SO: + + catalytic domain + + + site + SO: + cleaner0 + 2023-07-25T10:14:37Z + + editing site + + + 0.9992137 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.99801874 + species + cleaner0 + 2023-07-25T09:56:21Z + MESH: + + human + + + + METHODS + title_1 + 24396 + Online Methods + + + METHODS + paragraph + 24411 + Unless otherwise stated, reagents were purchased from Fisher Scientific, Sigma-Aldrich, or Life Technologies. T4 polynucleotide kinase, T4 DNA ligase, molecular biology grade bovine serum albumin (BSA), and RNase inhibitor were purchased from New England Biolabs. γ-[32P] ATP was purchased from Perkin-Elmer Life Sciences. The Avian Myeloblastosis Virus (AMV) reverse transcriptase, deoxynucleotide triphosphate (dNTP) mix and RQ1 RNase free DNase were purchased from Promega. Pfu Ultra II was purchased from Stratagene. Dpn 1 was purchased from Invitrogen. Quickchange XL II mutagenesis kit was purchased from Agilent Technologies. RNA oligonucleotides were synthesized at the University of Utah DNA/Peptide Core Facility or purchased from GE Healthcare Dharmacon, Inc. or Sigma Aldrich. DNA oligonucleotides were purchased from Integrated DNA Technologies. Storage phosphor imaging plates from Molecular Dynamics were imaged using Molecular Dynamics 9400 Typhoon phosphorimager. Data were analyzed using Molecular Dynamics ImageQuant 5.2 software. Electrospray Ionization (ESI) mass spectrometry of oligonucleotide samples was carried out at the Campus Mass Spectrometry Facilities, UC Davis. Oligonucleotide masses were determined using Mongo Oligo Mass Calculator v2.06. + + + METHODS + title_2 + 25690 + Expression and purification of hADAR2 deaminase domain (hADAR2d) for crystallography + + + METHODS + paragraph + 25775 + Protein expression and purification were carried out by modifying a previously reported protocol. In brief, BCY123 cells were transformed with a pSc-ADAR construct encoding either hADAR2d-WT or hADAR2d-E488Q (corresponding to the deaminase domain; residues 299–701). Cells were streaked on yeast minimal media minus uracil (CM-ura) plates. A single colony was used to inoculate a 15 mL CM-ura starter culture. After shaking at 300 rpm and 30 °C overnight, 10 mL of starter culture was used to inoculate each liter of yeast growth media. After 24 h, cells were induced with the addition of 110 mL of sterile 30% galactose per liter, and protein was expressed for 5 h. Cells were collected by centrifugation and stored at −80 °C. Cells were lysed in Buffer A (20 mM Tris-HCl pH 8.0, 5% glycerol, 35 mM imidazole, 1mM BME, 0.01% Triton × 100) with 750 mM NaCl using a microfluidizer and cell lysate clarified by centrifugation (39,000 × g for 25 min). Lysate was passed over a 5 mL Ni-NTA column; washed in three steps with 20–50mL of Lysis Buffer, Wash I buffer (Buffer A + 300 mM NaCl), and Wash II buffer (Buffer A + 100 mM NaCl); and protein eluted by a 35–300 mM imidazole gradient in Wash II over 80 min at a flow rate of 1 ml/min. Fractions containing the target protein were pooled and further purified on a 2 mL GE Healthcare Lifesciences Hi-Trap Heparin HP column in the absence of BME. The 10xHis fusion protein was cleaved with an optimized ratio of 1 mg of TEV protease for each 1 mg of protein. Cleavage was carried out for 1–2 h before passing the product over another Ni-NTA column at 0.5 mL/min. The flow-through and wash were collected; dialyzed against 20 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol, and 1 mM BME; and concentrated to just under 1 mL for gel filtration on a GE Healthcare HiLoad 16/600 Superdex 200 PG column. Fractions containing purified protein were pooled and concentrated to 5–7 mg/mL for crystallography trials. + + + METHODS + title_2 + 27739 + Purification of RNAs for crystallography + + + METHODS + paragraph + 27780 + The 8-azanebularine (N) phosphoramidite was synthesized as previously described and RNAs were synthesized as previously described. Single-stranded RNAs (See Supplementary Table 2 for sequences) were purified by denaturing polyacrylamide gel electrophoresis and visualized using UV shadowing. Bands were excised from the gel, crushed and soaked overnight at 4 °C in 0.5 M NH4OAc, 0.1% sodium dodecyl sulfate (SDS) and 0.1 mM EDTA. Polyacrylamide fragments were removed using a 0.2 µm filter followed by desalting on C18 Sep-Pak column. The RNA solutions were lyophilized to dryness, re-suspended in nuclease-free water, quantified by absorbance at 260 nm and stored at −70 °C. Oligonucleotide mass was confirmed by electrospray ionization mass spectrometry. Unmodified RNA stands were purchased from Dharmacon-GE Life Sciences and purified as described above. Duplex RNA was hybridized in a 1:1 ratio by heating to 95 °C for 5 min and slowly cooling to 30 °C. + + + METHODS + title_2 + 28746 + hADAR2d-RNA complex crystallization + + + METHODS + paragraph + 28782 + Crystals of hADAR2d E488Q+Bdf2-C RNA complex were grown at room temperature by the sitting drop vapor diffusion method. A solution of 0.5 µL volume containing 4.5 mg/mL protein and 70 µM of Bdf2-C 23mer RNA (1:0.7 ADAR2:RNA molar ratio) were mixed with 0.5 µL of 0.1 M MES:NaOH pH 6.5, 9% (w/v) PEG 3350, 13% glycerol, and 0.015M NAD, which was added to improve crystal growth. Crystals took several weeks to grow. A single, cube-shaped crystal about 120 µm in size was soaked briefly in a solution of mother liquor plus 30% glycerol before flash-cooling in liquid nitrogen. Data were collected via fine-phi slicing using 0.2° oscillations on beamline 24-ID-C at the Advanced Photon Source at Argonne National Laboratories in Chicago. To obtain crystals of the hADAR2d+WT:Bdf2-C RNA, an identical procedure was used as above; however, the crystallization conditions had slightly different concentrations of reagents (10% PEG 3350, 15% glycerol, 0.1 M MES:NaOH pH 6.5, no NAD). For the hADAR2d+WT:Bdf2-U construct, hanging drop vapor diffusion using 200 nL of a mixture containing 4.5 mg/mL protein and 70 µM of Bdf2-U (1:0.7 molar ratio) and 200 nL of a mother liquor (0.1 M ammonium acetate, 0.1M Bis-tris pH 5.5, 17% PEG 10,000) yielded several crystals with a morphology similar to the one described above. All wild type crystals were soaked briefly in a solution of mother liquor plus 30% glycerol before flash-cooling in liquid nitrogen. Data were collected via fine-phi slicing using 0.2° oscillations on beamline 12-2 at the Stanford Synchrotron Radiation Lightsource. Crystals of the hADAR2d E488Q+Gli1 RNA complex were grown using hanging drop vapor diffusion. A solution of volume 200 nL containing 4.5 mg/mL protein and 100 µM of Gli1 23mer RNA (1:1 ADAR2:RNA molar ratio) were mixed with 200 nL of 0.1 M MES:NaOH pH 6.5 and 12% PEG 20,000. At room temperature, a single diamond-shaped crystal about 150 µm long and 50 µm wide was observed about a week later. This crystal was soaked briefly in a solution of mother liquor plus 30% glycerol before flash-cooling in liquid nitrogen. Data were collected on beamline 12-2 at the Stanford Synchrotron Radiation Lightsource using the fine-phi splicing described above. + + + METHODS + title_2 + 31039 + Processing and refinement of crystallographic data + + + METHODS + paragraph + 31090 + Data for the E488Q Bdf2-C-bound and Gli1-bound structures were processed using XDS and scaled with Aimless (CCP4 1994). Diffraction data for hADAR2d wild type structures were processed with XDS and scaled with SCALA (Kabsch, 2010). The RNA-free hADAR2d crystal structure (PDB ID: 1ZY7) was used as a model for molecular replacement using PHENIX. The structures were refined using PHENIX including TLS parameters and Zn coordination restraints. Ideal Zn-ligand distances were determined using average distances found for similar coordination models in the PDB database. Table 1 gives the statistics in data processing and model refinement. The asymmetric unit for Gli1-bound hADAR2d E488Q includes two complexes of protein:RNA. In each of these complexes, the first 17 residues of the deaminase domain (residues 299–316) as well as a C-terminal proline (Pro701) are disordered and were therefore not included in the model. However, although the RNA-free ADAR2 structure (PDB ID: 1ZY7) lacked electron density for residues 457–475, we observed density for the backbone atoms of these residues. These residues were initially modeled as polyalanine. After a several rounds of refinement, electron density revealed the location of some side chains. Residues whose basic side chains interact with the RNA backbone are clearly defined in the final density map. Although some non-RNA-binding side chains show only weak density, backbone density is strong. As observed in the original hADAR2d RNA-free structure, inositol hexakisphosphate (IHP) was buried in the enzyme core. The asymmetric units for Bdf2-bound ADARs contain one ADAR2d:RNA complex (protein chain A) and one RNA-free ADAR2d monomer (chain D). The N-terminus of the Bdf2-bound structures include more residues than Gli1-bound, beginning at Pro305 in chain A and Thr304 for chain D in the mutant structure, and beginning at Arg307 in chain A and Thr304 or Pro305 in chain D in the wild type structures. The first few residues (in structures in which the specified residues are modeled) had weak side chain density, including residues 305 and 307 in chain A, and residues 304–307 in chain D, and are modeled in the structure as alanine. The last residue of E488Q+Bdf2-C, Pro701, had very weak electron density for both protein subunits in the asymmetric unit. Unlike the E488Q+Gli1 structure, electron density was defined better in the originally disordered loop (residues 457–475) for most residues in the Bdf2-bound structures. With the exception of Glu466 in the wildtype structures, we were able to model-build in main chain and side chain atoms for all residues of this loop in the ADAR subunit complexed to the Bdf2 RNA duplexes. In the RNA-free subunit (chain D) of E488Q+Bdf2-C, a crystal contact stabilized this flexible loop so that we were able to model in the backbone for residues 457–475, but residues 465–475 were modeled as alanine because of poorly defined side chain density. An identical crystal contact was observed in the wildtype structures. In the wt+Bdf2-C complex, density for residues 467–470 was strong enough for side chains to be included in these structures; however, side chain density was not strong for residues 465, 466, 471, 473–475, and 477. Therefore, these side chains were not included in the model. In wt+Bdf2-U, density for side chains 465, 466, 470, 471, and 473–475 was too weak to model. IHP was observed in all ADAR deaminase domains in the asymmetric unit. To model the hydrated 8-azanebularine nucleotide in all RNAs, a CCP4 dictionary file for adenosine (A) was modified to replace the 6-amino group with hydrogen, to change atom 8 to nitrogen and to include an additional hydroxyl group off carbon 6. Additionally, an energy minimization calculated idealized structure was used to determine ideal bond angles and distances for the modified base of the hydrated 8-azanebularine (unpublished data, Professor Dean Tantillo, University of California- Davis). The refinement restraint dictionary file was edited to match these parameters. + + + METHODS + title_2 + 35146 + Expression and purification of hADAR2d for in vitro deamination kinetics + + + METHODS + paragraph + 35219 + Histidine-tagged human ADAR2 deaminase domain (hADAR2d) and hADAR2d mutant proteins were expressed in S. cerevisiae strain BCY123 and purified as described above with the following modifications. Cell lysate was 0.45 µm filtered after centrifugation and loaded 3 times through 5 mL Ni-NTA Superflow (Qiagen) at 3 mL/min. Washes of 50 ml with buffer 1, 2 and 3 at 4 mL/min followed by elution with 35 mL gradient from Buffer 3 to elution buffer. Selected elution fractions from the Ni NTA column were pooled and loaded at 0.5 mL/min on 1 mL HiTrap Heparin HP column from GE. The column was washed with 10 mL of Heparin 1 buffer at 0.5 mL/min and eluted with a 12 mL gradient from Heparin 1 to Heparin 2 buffer. Selected elution fractions from the Heparin column were pooled and concentrated to <300 µL in 10,000 MWCO Amicon Ultra 4 centrifugal filter at 6500 RCF and 4 °C. TEV protease cleavage and gel filtration steps were omitted. Buffer exchange was accomplished via 3 rounds of concentration to <300 µL followed by addition of 3 mL of Storage buffer. After final concentration, protein concentrations were determined using BSA standards visualized by SYPRO Orange staining on SDS-polyacrylamide gels and the purified proteins were stored at −70 °C. + + + METHODS + title_2 + 36484 + Site-directed mutagenesis + + + METHODS + paragraph + 36510 + Mutagenesis of hADAR2 catalytic domain was carried out via PCR site directed mutagenesis using the primers listed in Supplementary Table 2. All primers were purchased from IDT and PAGE purified as described above but were desalted by phenol chloroform extraction, ethanol precipitation and 70% ethanol wash instead of C18 Sep-Pack. Sequences for mutant plasmids were confirmed by Sanger sequencing. + + + METHODS + title_2 + 36909 + Preparation of hGLI1 splint-ligated RNA + + + METHODS + paragraph + 36949 + Oligonucleotides were purified as described above but were desalted by phenol chloroform extraction, ethanol precipitation and 70% ethanol wash. The 3’ GLI1 top strand 12 mer RNAs were radiolabeled with [γ-32P] at the 5’ end with T4 PNK as described previously. Labeled 3’ GLI1 top strand 12 mer RNAs were ligated as previously described to give an internally labeled RNA. The splint ligation products were PAGE purified as described above. Labeled RNAs were hybridized with the complementary GLI1 bottom strand 24 mer RNA (Y is chosen based on the identity of X, see Fig. 3b) in 10 mM Tris-HCl, 0.1 mM EDTA pH 7.5 and 100 mM NaCl. See Supplementary Table 2 for RNA sequences. + + + METHODS + title_2 + 37633 + In vitro deamination kinetics with internally32P labeled substrates + + + METHODS + paragraph + 37701 + Deamination kinetics of analog containing RNAs were carried out as previously described but with the following modifications. The final reaction volume was 10 µL. Final enzyme concentration was 300 nM. Final RNA concentration was 10 nM. Final reaction conditions were: 16 mM Tris HCl pH 7.4, 3.3% glycerol, 1.6 mM EDTA, 0.003% Nonidet NP-40, 60 mM KCl, 7.1 mM NaCl, 0.5 mM DTT, 160 units/mL Rnasin, 1 µg/mL yeast tRNA. Reactions were quenched by adding 190 µL 95 °C nuclease-free water followed by incubation at 95 °C for 5 min or by 10 µL 0.5% SDS at 95 °C followed by incubation at 95 °C for 5 min. Each experiment was carried out in triplicate, and the rate constants reported in the text are average values ± standard deviations. Sequences of RNAs used to prepare internally labeled substrates are shown in Supplementary Table 2. For comparison of hADAR2-D mutants, deamination kinetics were carried out as described above with the following modifications. Final reaction conditions were 300 nM hADAR2d, 10 nM RNA, 16 mM Tris HCl pH 7.4, 3.6% glycerol, 1.6 mM EDTA, 0.003% Nonidet NP-40, 60 mM KCl, 8.6 mM NaCl, 0.5 mM DTT, 160 units/mL Rnasin, 1 µg/mL yeast tRNA. + + + METHODS + title_2 + 38891 + EMSA analysis of radioactively labelled RNA + + + METHODS + paragraph + 38935 + Duplex RNAs containing 8-azanebularine and32P labeled were prepared as previously described. Samples containing 0.25 nM RNA and different concentrations of hADAR2d E488Q (128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0 nM) were equilibrated in 20 mM Tris-HCl, pH 7, 6% glycerol, 0.5 mM DTT, 60 mM KCl, 20 mM NaCl, 0.1 mM BME, 1.5 mM EDTA, 0.003% NP-40, 160 units/ml RNasin, 100 µg/ml BSA and 1.0 µg/ml yeast tRNA for 30 min at 30 °C. Assay and data analysis were carried out as previously described. See Supplementary Table 2 for RNA sequences. + + + METHODS + title_2 + 39485 + In vitro transcription of RNA + + + METHODS + paragraph + 39515 + A truncation of hGLI1 mRNA incorporating 81 nucleotides upstream and 65 nucleotides downstream of the edited site was transcribed and purified as previously described. 3’ Nearest neighbor mutants of hGLI1 RNA were generated by site directed mutagenesis to generate G to A, G to C and G to U nearest neighbor mutants. A second site −32 bases from the edit site was mutated to maintain the original secondary structure of the RNA. See Supplementary Table 2 for primers used for mutagenesis. + + + METHODS + title_2 + 40008 + Deamination kinetics of transcribed hGLI1 RNAs + + + METHODS + paragraph + 40055 + Deamination kinetics of transcribed RNAs were carried out as previously described but with the following modifications. Final reaction volume was 20 µL. Final enzyme concentrations was 10 nM. Final RNA concentration was 2 nM. Final reaction conditions were: 17 mM Tris HCl pH 7.4, 5.0% glycerol, 1.6 mM EDTA, 0.003% Nonidet NP-40, 60 mM KCl, 15.6 mM NaCl, 0.5 mM DTT, 160 units/mL RNasin, 1 µg/mL yeast tRNA. Reactions were quenched by adding 10 µL 0.5% SDS at 95 °C followed by incubation at 95 °C for 5 min. cDNA was generated from RNA via RT-PCR, Sanger sequenced and quantified using SeqScanner 2 software from Applied Biosystems. The kobs (min−1) of each assay was calculated as described previously. + + + SUPPL + title_1 + 40773 + Supplementary Material + + + SUPPL + footnote + 40796 + Accession codes. Coordinates for the hADAR2d-RNA complexes have been deposited in the Protein Data Bank (PDB) with PDB IDs of 5ED1 and 5ED2 for the hADAR2d-E488Q–Bdf2-C RNA and hADAR2d-E488Q–Gli1 RNA complexes, respectively, and 5HP2 and 5HP3 for the hADAR2d-WT–Bdf2-U RNA and hADAR2d-WT–Bdf2-C RNA complexes, respectively. + + + SUPPL + footnote + 41128 + Author Contributions + + + SUPPL + footnote + 41149 + J.M.T., M.M.M., A.I.S. and Y.Z. purified protein. K.J.P and J.M.T. designed and purified RNA for crystallography and characterized protein/RNA binding. M.M.M. and A.I.S. conducted crystallization trials. M.M.M and A.J.F collected diffraction data and solved/refined crystal structure. J.M.T., Y.Z. and J.H. measured enzyme reaction rates. K.T. synthesized 8-azanebularane phosphoramidite. J.M.T and A.I.S conducted mutagenesis. J.M.T., M.M.M., P.A.B. and A.J.F. analyzed the structures. P.A.B. wrote the initial manuscript draft. J.M.T, M.M.M, P.A.B and A.J.F edited the manuscript. + + + REF + title + 41732 + References + + + surname:Grosjean;given-names:H + REF + Fine-Tuning of RNA Functions by Modification and Editing + ref + 2005 + 41743 + + + 817 + 846 + surname:Bass;given-names:BL + 12045112 + REF + Ann. Rev. Biochem + ref + 71 + 2002 + 41744 + RNA editing by adenosine deaminases that act on RNA + + + 321 + 349 + surname:Nishikura;given-names:K + 20192758 + REF + Ann. Rev. Biochem + ref + 79 + 2010 + 41796 + Functions and regulation of RNA editing by ADAR deaminases + + + 1525 + 1536 + surname:Wang;given-names:Q + 24067935 + REF + RNA + ref + 19 + 2013 + 41855 + ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting miR-30b-3p and miR-573 binding + + + 75 + 80 + surname:Rueter;given-names:SM + surname:Dawson;given-names:TR + surname:Emeson;given-names:RB + 10331393 + REF + Nature + ref + 399 + 1999 + 41961 + Regulation of alternative splicing by RNA editing + + + 20715 + 20719 + surname:Yeo;given-names:J + surname:Goodman;given-names:RA + surname:Schirle;given-names:NT + surname:David;given-names:SS + surname:Beal;given-names:PA + 21068368 + REF + Proc. Natl. Acad. Sci. USA + ref + 107 + 2010 + 42011 + RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1 + + + 947 + 949 + surname:Bass;given-names:BL + 9292492 + REF + RNA + ref + 3 + 1997 + 42086 + A standardized nomenclature for adenosine deaminases that act on RNA + + + 1 + 9 + surname:Maas;given-names:S + surname:Kawahara;given-names:Y + surname:Tamburro;given-names:KM + surname:Nishikura;given-names:K + 17114938 + REF + RNA Biology + ref + 3 + 2006 + 42155 + A to I RNA Editing and Human Disease + + + 105 + surname:Slotkin;given-names:W + surname:Nishikura;given-names:K + 24289319 + REF + Genome Med + ref + 5 + 2013 + 42192 + Adenosine-to-inosine RNA editing and human disease + + + 169 + 180 + surname:Morabito;given-names:MV + 20394819 + REF + Neurobiol. Dis + ref + 39 + 2010 + 42243 + Mice with altered serotonin 2C receptor RNA editing display characteristics of Prader-Willi syndrome + + + 1243 + 1248 + surname:Rice;given-names:GI + 23001123 + REF + Nat. Genetics + ref + 44 + 2012 + 42344 + Mutations in ADAR1 cause Aicardi-Goutieres syndrome associated with a type I interferon signature + + + 693 + 699 + surname:Miyamura;given-names:Y + 12916015 + REF + Am. J. Hum. Genet + ref + 73 + 2003 + 42442 + Mutations of the RNA-specific adenosine deaminase gene (DSRAD) are involved in dyschromatosis symmetrica hereditaria + + + 629 + 630 + surname:Zhang;given-names:XJ + 15146470 + REF + Hum. Mutat + ref + 23 + 2004 + 42559 + Seven novel mutations of the ADAR gene in Chinese families and sporadic patients with disychromatosis symmetrica hereditaria (DSH) + + + 209 + 216 + surname:Chen;given-names:L + 23291631 + REF + Nat. Med + ref + 19 + 2013 + 42690 + Recoding RNA editing of AZIN1 predisposes to hepatocellular carcinoma + + + 130 + 131 + surname:Gallo;given-names:A + 23389604 + REF + Nat. Med + ref + 19 + 2013 + 42760 + RNA editing enters the limelight in cancer + + + 321 + 333 + surname:Shimokawa;given-names:T + 23324600 + REF + RNA Biology + ref + 10 + 2013 + 42803 + RNA editing of the GLI1 transcription factor modulates the output of Hedgehog signaling + + + 1 + 33 + surname:Goodman;given-names:RA + surname:Macbeth;given-names:MR + surname:Beal;given-names:PA + 21769729 + REF + Curr. Top. Microbiol. Immunol + ref + 352 + 2012 + 42891 + ADAR proteins: Structure and Catalytic Mechanism + + + 1210 + 1213 + surname:Li;given-names:JB + 19478186 + REF + Science + ref + 324 + 2009 + 42940 + Genome-wide identification of human RNA editing sites by parallel DNA capturing and sequencing + + + 11213 + 11219 + surname:Haudenschild;given-names:BL + 15355102 + REF + J. Am. Chem. Soc + ref + 126 + 2004 + 43035 + A transition state analogue for an RNA-editing reaction + + + 1123 + 1132 + surname:Phelps;given-names:KJ + 25564529 + REF + Nucleic Acids Res + ref + 43 + 2015 + 43091 + Recognition of Duplex RNA by the Deaminase Domain of the RNA editing Enyzme ADAR2 + + + 1534 + 1539 + surname:Macbeth;given-names:MR + 16141067 + REF + Science + ref + 309 + 2005 + 43173 + Inositol Hexakisphosphate Is Bound in the ADAR2 Core and Required for RNA editing + + + E3295 + E3304 + surname:Kuttan;given-names:A + surname:Bass;given-names:BL + 23129636 + REF + Proc. Natl. Acad. Sci. USA + ref + 109 + 2012 + 43255 + Mechanistic Insights into edtiing-site specificity of ADARs + + + 7857 + 7869 + surname:Eifler;given-names:T + surname:Pokharel;given-names:S + surname:Beal;given-names:PA + 24124932 + REF + Biochemistry + ref + 52 + 2013 + 43315 + RNA-Seq Analysis Identifies A Novel Set of Editing Substrates for Human ADAR2 Present in Saccharomyces cerevisiae + + + 846 + 858 + surname:Wong;given-names:SK + surname:Sato;given-names:S + surname:Lazinski;given-names:DW + 11421361 + REF + RNA + ref + 7 + 2001 + 43429 + Substrate recognition by ADAR1 and ADAR2 + + + 357 + 369 + surname:Klimasauskas;given-names:S + surname:Kumar;given-names:P + surname:Roberts;given-names:RJ + surname:Cheng;given-names:X + 8293469 + REF + Cell + ref + 76 + 1994 + 43470 + HhaI methyltransferase flips its target base out of the DNA helix + + + 1047 + 1055 + surname:Daujotyte;given-names:D + 15274924 + REF + Structure + ref + 12 + 2004 + 43536 + HhaI DNA Methyltransferase Uses the Protruding Gln237 for Active Flipping of Its Target Cytosine + + + 5945 + 5953 + surname:Thiyagarajan;given-names:S + surname:Rajan;given-names:SS + surname:Gautham;given-names:N + 15534365 + REF + Nucleic Acids Res + ref + 32 + 2004 + 43633 + Cobalt hexamine induced tautomeric shift in Z-DNA: the structure of d(CGCGCA).d(TGCGCG) in two crystal forms + + + 87 + 92 + surname:Slupphaug;given-names:G + 8900285 + REF + Nature + ref + 384 + 1996 + 43742 + A nucleotide-flipping mechanism from the structure of human uracil-DNA glycosylase boudn to DNA + + + 859 + 866 + surname:Bruner;given-names:SD + surname:Norman;given-names:DP + surname:Verdine;given-names:GL + 10706276 + REF + Nature + ref + 403 + 2000 + 43838 + Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA + + + 249 + 258 + surname:Lau;given-names:AY + surname:Scharer;given-names:OD + surname:Samson;given-names:L + surname:Verdine;given-names:GL + surname:Ellenberger;given-names:T + 9790531 + REF + Cell + ref + 95 + 1998 + 43928 + Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide flipping and base excision + + + 247 + 271 + surname:Brooks;given-names:SC + surname:Adhikary;given-names:S + surname:Rubinson;given-names:EH + surname:Eichman;given-names:BF + 23076011 + REF + Biochim. Biophys. Acta + ref + 1834 + 2013 + 44056 + Recent advances in the structural mechanisms of DNA glycosylases + + + 1 + 9 + surname:Eggington;given-names:JM + surname:Greene;given-names:T + surname:Bass;given-names:BL + REF + Nat. Commun + ref + 2 + 2011 + 44121 + Predicting sites of ADAR editing in double stranded RNA + + + 1044 + 1047 + surname:Peacock;given-names:H + surname:Maydanovych;given-names:O + surname:Beal;given-names:PA + 20108910 + REF + Org. Lett + ref + 12 + 2010 + 44177 + N2-Modified 2-aminopurine ribonucleosides as minor-groove-modulating adenosine replacements in duplex RNA + + + 181 + 198 + surname:Roberts;given-names:RJ + surname:Cheng;given-names:X + 9759487 + REF + Ann. Rev. Biochem + ref + 67 + 1998 + 44283 + Base flipping + + + 929 + 939 + surname:Hoang;given-names:C + surname:Ferre-D'Amare;given-names:AR + 11779468 + REF + Cell + ref + 107 + 2001 + 44297 + Cocrystal Structure of a tRNA psi55 Pseuduridine Synthase: Nucleotide Flipping by an RNA-modifying Enyzme + + + 5214 + 5226 + surname:Parikh;given-names:SS + 9724657 + REF + EMBO J + ref + 17 + 1998 + 44403 + Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA + + + 968 + 973 + surname:Yang;given-names:X + surname:Gerczei;given-names:T + surname:Glover;given-names:L + surname:Correll;given-names:CC + 11685244 + REF + Nat. Struct. Biol + ref + 8 + 2001 + 44528 + Crystal structures of restrictocin-inhibitor complexes with implications for RNA recognition and base flipping + + + 498 + 501 + surname:Carter;given-names:AP + 11228145 + REF + Science + ref + 291 + 2001 + 44639 + Crystal Structure of an Initiation Factor Bound to the 30S Ribosomal Subunit + + + 504 + 520 + surname:Reblova;given-names:K + 16538608 + REF + Biopolymers + ref + 82 + 2006 + 44716 + Structure, Dynamics, and Elasticity of Free 16S rRNA Helix 44 Studied by Molecular Dynamics Simulations + + + 781 + 788 + surname:Xie;given-names:W + surname:Liu;given-names:X + surname:Huang;given-names:RH + 12949492 + REF + Nat. Struct. Biol + ref + 10 + 2003 + 44820 + Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate + + + 3295 + 3304 + surname:Erion;given-names:MD + surname:Reddy;given-names:MR + REF + J. Am. Chem. Soc + ref + 120 + 1998 + 44927 + Calculation of Relative Hydration Free Energy Differences for Heteroaromatic Compounds: Use in the Design of Adenosine Deaminase and Cytidine Deaminase Inhibitors + + + 225 + 237 + surname:Stefl;given-names:R + 20946981 + REF + Cell + ref + 143 + 2010 + 45090 + The Solution Structure of the ADAR2 dsRBM-RNA Complex Reveals a Sequence-Specific Readout of the Minor Groove + + + 241 + 246 + surname:Fierro-Monti;given-names:I + surname:Mathews;given-names:MB + 10782096 + REF + Trends Biochem. Sci + ref + 25 + 2000 + 45200 + Proteins binding to duplexed RNA: one motif, multiple functions + + + 1482 + 1494 + surname:Mannion;given-names:NM + 25456137 + REF + Cell Rep + ref + 9 + 2014 + 45264 + The RNA-editing enzyme ADAR1 controls innate immune responses to RNA + + + METHODS + title + 45333 + Online methods references + + + 319 + 331 + surname:Macbeth;given-names:MR + surname:Bass;given-names:BL + 17662848 + REF + Methods Enzymol + ref + 424 + 2007 + 45359 + Large-Scale Overexpression and Purification of ADARs from Saccharomyces cerevisiae for Biophysical and Biochemical Studies + + + 11882 + 11891 + surname:Pokharel;given-names:S + 19642681 + REF + J. Am. Chem. Soc + ref + 131 + 2009 + 45482 + Matching Active Site Structure to Substrate Analog for an RNA Editing Reaction + + + 125 + 132 + surname:Kabsch;given-names:W + 20124692 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 45561 + XDS + + + 658 + 674 + surname:McCoy;given-names:AJ + 19461840 + REF + J. Appl. Crystallogr + ref + 40 + 2007 + 45565 + Phaser crystallographic software + + + 352 + 367 + surname:Afonine;given-names:PV + 22505256 + REF + Acta Crystallogr D Biol Crystallogr + ref + 68 + 2012 + 45598 + Towards automated crystallographic structure refinement with phenix.refine + + + 1780 + 1787 + surname:Phelps;given-names:K + surname:Ibarra-Sosa;given-names:J + surname:Tran;given-names:K + surname:Fisher;given-names:AJ + surname:Beal;given-names:PA + 24896732 + REF + ACS Chem. Biol + ref + 9 + 2014 + 45673 + Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA + + + nihms771539f1.jpg + F1 + FIG + fig_title_caption + 45798 + Human ADAR2 and modified RNAs for crystallography + + 0.9986065 + species + cleaner0 + 2023-07-25T09:56:21Z + MESH: + + Human + + + 0.99896836 + protein + cleaner0 + 2023-07-25T09:55:25Z + PR: + + ADAR2 + + + 0.99857223 + chemical + cleaner0 + 2023-07-25T09:56:01Z + CHEBI: + + RNAs + + + 0.9975631 + experimental_method + cleaner0 + 2023-07-25T13:25:39Z + MESH: + + crystallography + + + + nihms771539f1.jpg + F1 + FIG + fig_caption + 45848 + a, Domain map for human ADAR2 b, ADAR reaction showing intermediate and 8-azanebularine (N) hydrate that mimics this structure c, Duplex RNAs used for crystallization. Bdf2 duplex sequence is derived from an editing site found in S. cerevisiae Bdf2 mRNA and Gli1 duplex has sequence surrounding the human Gli1 mRNA editing site. Italics indicate nucleotides added for duplex stability. + + 0.9989446 + species + cleaner0 + 2023-07-25T09:56:21Z + MESH: + + human + + + 0.9990251 + protein + cleaner0 + 2023-07-25T09:55:25Z + PR: + + ADAR2 + + + 0.9266272 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.9857567 + chemical + cleaner0 + 2023-07-25T13:50:59Z + CHEBI: + + 8-azanebularine (N) hydrate + + + 0.96221715 + evidence + cleaner0 + 2023-07-25T13:53:40Z + DUMMY: + + structure + + + structure_element + SO: + cleaner0 + 2023-07-25T13:32:49Z + + Duplex RNAs + + + experimental_method + MESH: + cleaner0 + 2023-07-25T13:18:16Z + + crystallization + + + chemical + CHEBI: + cleaner0 + 2023-07-25T12:56:56Z + + Bdf2 duplex + + + 0.9983212 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.9985363 + species + cleaner0 + 2023-07-25T13:00:09Z + MESH: + + S. cerevisiae + + + chemical + CHEBI: + cleaner0 + 2023-07-25T13:18:38Z + + Bdf2 mRNA + + + 0.9987307 + protein + cleaner0 + 2023-07-25T10:04:07Z + PR: + + Gli1 + + + 0.9989792 + species + cleaner0 + 2023-07-25T09:56:21Z + MESH: + + human + + + 0.9988563 + protein + cleaner0 + 2023-07-25T10:04:07Z + PR: + + Gli1 + + + 0.9985222 + chemical + cleaner0 + 2023-07-25T10:03:39Z + CHEBI: + + mRNA + + + 0.9907459 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + + nihms771539f2.jpg + F2 + FIG + fig_title_caption + 46234 + Structure of hADAR2d E488Q bound to the Bdf2-C RNA duplex at 2.75 Å resolution + + 0.9970282 + evidence + cleaner0 + 2023-07-25T13:53:43Z + DUMMY: + + Structure + + + 0.99791294 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.998582 + mutant + cleaner0 + 2023-07-25T10:08:01Z + MESH: + + E488Q + + + 0.9990499 + protein_state + cleaner0 + 2023-07-25T13:01:18Z + DUMMY: + + bound to + + + chemical + CHEBI: + cleaner0 + 2023-07-25T12:57:17Z + + Bdf2-C RNA duplex + + + + nihms771539f2.jpg + F2 + FIG + fig_caption + 46314 + a, View of structure perpendicular to the dsRNA helical axis. Colors correspond to those in Figs. 1a and 1c; flipped out base N is highlighted red, zinc in grey space-filling sphere, Q488 in yellow, previously disordered aa454–477 loop in green and inositol hexakisphosphate (IHP) in space filling. A transparent surface is shown for the hADAR2d protein. b, View of structure along the dsRNA helical axis. c, Summary of the contacts between hADAR2d E488Q and the Bdf2-C RNA duplex. + + 0.99907035 + chemical + cleaner0 + 2023-07-25T09:56:06Z + CHEBI: + + dsRNA + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:07:08Z + + flipped out + + + 0.999035 + chemical + cleaner0 + 2023-07-25T10:15:31Z + CHEBI: + + zinc + + + 0.99951625 + residue_name_number + cleaner0 + 2023-07-25T10:17:43Z + DUMMY: + + Q488 + + + 0.5178192 + protein_state + cleaner0 + 2023-07-25T13:47:29Z + DUMMY: + + disordered + + + residue_range + DUMMY: + cleaner0 + 2023-07-25T12:57:38Z + + 454–477 + + + 0.9989749 + structure_element + cleaner0 + 2023-07-25T13:35:57Z + SO: + + loop + + + 0.9988637 + chemical + cleaner0 + 2023-07-25T10:07:32Z + CHEBI: + + inositol hexakisphosphate + + + 0.9992667 + chemical + cleaner0 + 2023-07-25T10:07:37Z + CHEBI: + + IHP + + + 0.9985935 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.9990765 + chemical + cleaner0 + 2023-07-25T09:56:06Z + CHEBI: + + dsRNA + + + 0.9896716 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.998769 + mutant + cleaner0 + 2023-07-25T10:08:01Z + MESH: + + E488Q + + + chemical + CHEBI: + cleaner0 + 2023-07-25T12:57:53Z + + Bdf2-C RNA duplex + + + + nihms771539f3.jpg + F3 + FIG + fig_title_caption + 46798 + ADAR recognition of the flipped out and orphaned nucleotides + + 0.998789 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.9985868 + protein_state + cleaner0 + 2023-07-25T13:07:08Z + DUMMY: + + flipped out + + + 0.9991916 + protein_state + cleaner0 + 2023-07-25T13:06:38Z + DUMMY: + + orphaned + + + 0.99902225 + chemical + cleaner0 + 2023-07-25T13:19:09Z + CHEBI: + + nucleotides + + + + nihms771539f3.jpg + F3 + FIG + fig_caption + 46859 + a, Contacts to the editing site nucleotide (N) in the active site. Colors correspond to those in Figs. 1 and 2. b, Orphan nucleotide recognition in the hADAR2d E488Q–Bdf2-C complex. c, Orphan nucleotide recognition in the hADAR2d WT–Bdf2-U complex. + + 0.998191 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.99055076 + chemical + cleaner0 + 2023-07-25T13:51:43Z + CHEBI: + + nucleotide + + + 0.9990679 + site + cleaner0 + 2023-07-25T10:14:49Z + SO: + + active site + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:06:48Z + + Orphan + + + 0.8251463 + chemical + cleaner0 + 2023-07-25T13:19:07Z + CHEBI: + + nucleotide + + + complex_assembly + GO: + cleaner0 + 2023-07-25T10:10:49Z + + hADAR2d E488Q–Bdf2-C + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:06:48Z + + Orphan + + + 0.83549184 + chemical + cleaner0 + 2023-07-25T13:19:03Z + CHEBI: + + nucleotide + + + complex_assembly + GO: + cleaner0 + 2023-07-25T10:10:07Z + + hADAR2d WT–Bdf2-U + + + + nihms771539f4.jpg + F4 + FIG + fig_title_caption + 47112 + Other ADAR-induced changes in RNA conformation + + 0.672993 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.99880266 + chemical + cleaner0 + 2023-07-25T09:56:55Z + CHEBI: + + RNA + + + + nihms771539f4.jpg + F4 + FIG + fig_caption + 47159 + a, hADAR2d shifts the position of U11-A13’ base pair from ideal A-form RNA helix (yellow). b, Overlay of Bdf2 duplex RNA and idealized A form duplex of same sequence (yellow) illustrating kink in strand and widening of major groove opposite editing site induced by hADAR2d. c, Unusual “wobble” A13’-U11 interaction in the hADAR2d WT–Bdf2-U complex shown in stick with H-bond indicated with yellow dashes and distances shown in Å. The position of this base pair in the hADAR2d E488Q–Bdf2-C duplex is shown in wire with H-bonds shown with gray dashes. + + 0.9941765 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.99901235 + residue_name_number + cleaner0 + 2023-07-25T10:19:07Z + DUMMY: + + U11 + + + 0.99883956 + residue_name_number + cleaner0 + 2023-07-25T10:19:16Z + DUMMY: + + A13 + + + structure_element + SO: + cleaner0 + 2023-07-25T13:20:34Z + + A-form RNA helix + + + 0.99814093 + experimental_method + cleaner0 + 2023-07-25T13:25:45Z + MESH: + + Overlay + + + chemical + CHEBI: + cleaner0 + 2023-07-25T13:19:30Z + + Bdf2 duplex RNA + + + structure_element + SO: + cleaner0 + 2023-07-25T13:20:49Z + + A form duplex + + + 0.93573886 + site + cleaner0 + 2023-07-25T10:40:26Z + SO: + + major groove + + + 0.9937397 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.9929726 + mutant + cleaner0 + 2023-07-25T10:05:13Z + MESH: + + hADAR2d + + + 0.9968669 + residue_name_number + cleaner0 + 2023-07-25T10:19:16Z + DUMMY: + + A13 + + + 0.9984688 + residue_name_number + cleaner0 + 2023-07-25T10:19:07Z + DUMMY: + + U11 + + + complex_assembly + GO: + cleaner0 + 2023-07-25T10:10:07Z + + hADAR2d WT–Bdf2-U + + + 0.9863496 + bond_interaction + cleaner0 + 2023-07-25T10:59:14Z + MESH: + + H-bond + + + complex_assembly + GO: + cleaner0 + 2023-07-25T10:10:49Z + + hADAR2d E488Q–Bdf2-C + + + 0.9943462 + bond_interaction + cleaner0 + 2023-07-25T10:59:14Z + MESH: + + H-bonds + + + + nihms771539f5.jpg + F5 + FIG + fig_title_caption + 47722 + Interactions with editing site nearest neighbor nucleotides + + 0.99873394 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.9519506 + chemical + cleaner0 + 2023-07-25T13:51:50Z + CHEBI: + + nucleotides + + + + nihms771539f5.jpg + F5 + FIG + fig_caption + 47782 + a, The minor groove edge of the U11-A13’ base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5’ nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine). c, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 5’ nearest neighbors; krel = kobs/(kobs for unmodified RNA). Error bars, s.d (n=3 technical replicates). d, hADAR2 S486 backbone H-bond with 3’ G 2-amino group; e, RNA duplex substrates prepared with different 3’ nearest neighbor nucleotides adjacent to editing site indicated in red (I = inosine, N2MeG = N2-methylguanosine, 2AP = 2-aminopurine). f, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 3’ nearest neighbors. krel = kobs/(kobs for unmodified RNA). Error bars, s.d (n=3 technical replicates). * indicates no reaction product observed. + + 0.9869756 + site + cleaner0 + 2023-07-25T10:16:52Z + SO: + + minor groove + + + 0.9958093 + residue_name_number + cleaner0 + 2023-07-25T10:19:07Z + DUMMY: + + U11 + + + 0.99367744 + residue_name_number + cleaner0 + 2023-07-25T10:19:16Z + DUMMY: + + A13 + + + chemical + CHEBI: + cleaner0 + 2023-07-25T12:58:57Z + + Bdf2 duplex + + + 0.9993899 + residue_name_number + cleaner0 + 2023-07-25T13:21:16Z + DUMMY: + + G489 + + + structure_element + SO: + cleaner0 + 2023-07-25T10:28:46Z + + C-G pair + + + 0.9566321 + residue_name + cleaner0 + 2023-07-25T13:45:09Z + SO: + + G + + + structure_element + SO: + cleaner0 + 2023-07-25T13:30:56Z + + RNA duplex + + + 0.7529088 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.9990361 + structure_element + cleaner0 + 2023-07-25T10:29:59Z + SO: + + 2AP + + + 0.99899167 + structure_element + cleaner0 + 2023-07-25T12:59:43Z + SO: + + 2-aminopurine + + + 0.9970023 + evidence + cleaner0 + 2023-07-25T13:53:48Z + DUMMY: + + deamination rate constants + + + 0.9987447 + mutant + cleaner0 + 2023-07-25T10:05:14Z + MESH: + + hADAR2d + + + 0.7024581 + site + cleaner0 + 2023-07-25T10:14:37Z + SO: + + editing site + + + 0.55011916 + residue_name + cleaner0 + 2023-07-25T10:01:02Z + SO: + + adenosine + + + 0.97258997 + evidence + cleaner0 + 2023-07-25T13:53:52Z + DUMMY: + + krel + + + 0.94377023 + evidence + cleaner0 + 2023-07-25T13:53:54Z + DUMMY: + + kobs + + + 0.8698986 + evidence + cleaner0 + 2023-07-25T13:53:57Z + DUMMY: + + kobs + + + 0.98879784 + protein_state + cleaner0 + 2023-07-25T13:47:37Z + DUMMY: + + unmodified + + + 0.9937929 + chemical + cleaner0 + 2023-07-25T09:56:55Z + CHEBI: + + RNA + + + 0.9985972 + protein + cleaner0 + 2023-07-25T12:46:38Z + PR: + + hADAR2 + + + 0.99932015 + residue_name_number + cleaner0 + 2023-07-25T10:36:40Z + DUMMY: + + S486 + + + bond_interaction + MESH: + cleaner0 + 2023-07-25T10:59:14Z + + H-bond + + + 0.6379478 + residue_name + cleaner0 + 2023-07-25T13:45:13Z + SO: + + G + + + structure_element + SO: + cleaner0 + 2023-07-25T13:30:56Z + + RNA duplex + + + site + SO: + cleaner0 + 2023-07-25T10:14:37Z + + editing site + + + 0.99500763 + residue_name + cleaner0 + 2023-07-25T13:45:49Z + SO: + + I + + + 0.9854151 + residue_name + cleaner0 + 2023-07-25T10:01:31Z + SO: + + inosine + + + 0.9990403 + structure_element + cleaner0 + 2023-07-25T10:29:59Z + SO: + + 2AP + + + 0.9989734 + structure_element + cleaner0 + 2023-07-25T12:59:43Z + SO: + + 2-aminopurine + + + 0.9968157 + evidence + cleaner0 + 2023-07-25T13:54:01Z + DUMMY: + + deamination rate constants + + + 0.9987704 + mutant + cleaner0 + 2023-07-25T10:05:14Z + MESH: + + hADAR2d + + + site + SO: + cleaner0 + 2023-07-25T10:14:37Z + + editing site + + + 0.65553534 + residue_name + cleaner0 + 2023-07-25T10:01:02Z + SO: + + adenosine + + + 0.9769455 + evidence + cleaner0 + 2023-07-25T13:54:04Z + DUMMY: + + krel + + + 0.9269417 + evidence + cleaner0 + 2023-07-25T13:54:06Z + DUMMY: + + kobs + + + 0.88645303 + evidence + cleaner0 + 2023-07-25T13:54:09Z + DUMMY: + + kobs + + + 0.987695 + protein_state + cleaner0 + 2023-07-25T13:47:40Z + DUMMY: + + unmodified + + + 0.99183536 + chemical + cleaner0 + 2023-07-25T09:56:55Z + CHEBI: + + RNA + + + + nihms771539f6.jpg + F6 + FIG + fig_title_caption + 48849 + RNA-binding loops in the ADAR catalytic domain + + structure_element + SO: + cleaner0 + 2023-07-25T10:37:23Z + + RNA-binding loops + + + 0.9925573 + protein_type + cleaner0 + 2023-07-25T09:55:43Z + MESH: + + ADAR + + + 0.9990459 + structure_element + cleaner0 + 2023-07-25T10:49:04Z + SO: + + catalytic domain + + + + nihms771539f6.jpg + F6 + FIG + fig_caption + 48896 + a, hADAR2 residues that contact phosphodiester backbone near 5’ end of unedited strand. b, Location of mutations introduced at protein-RNA interface. c, Comparison of deamination rate constants of the different hADAR2d mutants (Log scale). krel = kobs for mutant/kobs for WT. Error bars, s.d (n=3 technical replicates). d, Sequence alignment of ADAR2s (A2) and ADAR1s (A1) from different organisms with different levels of conservation colored (Yellow: conserved in all ADAR1s and ADAR2s, red: conserved in ADAR2s, blue: conserved in ADAR1s. e, Interaction of the ADAR-specific RNA-binding loop near the 5’ end of the edited strand. Colors as in d, white: not conserved, flipped out base is shown in pink. + + 0.9970124 + protein + cleaner0 + 2023-07-25T12:46:38Z + PR: + + hADAR2 + + + 0.99850833 + site + cleaner0 + 2023-07-25T13:41:15Z + SO: + + protein-RNA interface + + + 0.99788064 + evidence + cleaner0 + 2023-07-25T13:54:14Z + DUMMY: + + deamination rate constants + + + 0.99785066 + mutant + cleaner0 + 2023-07-25T10:05:14Z + MESH: + + hADAR2d + + + 0.9954659 + evidence + cleaner0 + 2023-07-25T13:54:18Z + DUMMY: + + krel + + + 0.9914875 + evidence + cleaner0 + 2023-07-25T13:54:21Z + DUMMY: + + kobs + + + 0.99773955 + protein_state + cleaner0 + 2023-07-25T10:07:54Z + DUMMY: + + mutant + + + 0.99239796 + evidence + cleaner0 + 2023-07-25T13:54:24Z + DUMMY: + + kobs + + + 0.99920267 + protein_state + cleaner0 + 2023-07-25T10:06:05Z + DUMMY: + + WT + + + 0.9984065 + experimental_method + cleaner0 + 2023-07-25T13:26:06Z + MESH: + + Sequence alignment + + + 0.99840504 + protein_type + cleaner0 + 2023-07-25T10:39:29Z + MESH: + + ADAR2s + + + 0.9985267 + protein_type + cleaner0 + 2023-07-25T13:00:55Z + MESH: + + ADAR1s + + + 0.9959396 + protein_state + cleaner0 + 2023-07-25T13:26:34Z + DUMMY: + + conserved + + + 0.9972862 + protein_type + cleaner0 + 2023-07-25T13:00:56Z + MESH: + + ADAR1s + + + 0.99812835 + protein_type + cleaner0 + 2023-07-25T10:39:29Z + MESH: + + ADAR2s + + + 0.9985238 + protein_state + cleaner0 + 2023-07-25T13:26:31Z + DUMMY: + + conserved + + + 0.9984205 + protein_type + cleaner0 + 2023-07-25T10:39:29Z + MESH: + + ADAR2s + + + 0.99872893 + protein_state + cleaner0 + 2023-07-25T13:26:29Z + DUMMY: + + conserved + + + 0.99857724 + protein_type + cleaner0 + 2023-07-25T13:00:56Z + MESH: + + ADAR1s + + + structure_element + SO: + cleaner0 + 2023-07-25T12:45:44Z + + ADAR-specific RNA-binding loop + + + 0.9985478 + protein_state + cleaner0 + 2023-07-25T13:26:26Z + DUMMY: + + not conserved + + + protein_state + DUMMY: + cleaner0 + 2023-07-25T13:07:08Z + + flipped out + + + chemical + CHEBI: + cleaner0 + 2023-07-25T13:26:21Z + + base + + + + T1.xml + T1 + TABLE + table_caption + 49606 + Data Processing and Refinement Statistics. + + + T1.xml + T1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><thead><tr><th align="left" valign="top" rowspan="1" colspan="1">Complex</th><th align="left" rowspan="1" colspan="1">ADAR2-D_E488Q:BDF2-C<break/>23mer</th><th align="left" rowspan="1" colspan="1">ADAR2-D_E488Q:GLI1<break/>23mer</th><th align="left" rowspan="1" colspan="1">ADAR2-D_wt:BDF2-U<break/>23mer</th><th align="left" rowspan="1" colspan="1">ADAR2-D_wt:BDF2-C<break/>23mer</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><bold>Data Collection</bold></td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">Synchrotron (Beamline)</td><td align="left" rowspan="1" colspan="1">APS (24-ID-C)</td><td align="left" rowspan="1" colspan="1">SSRL (12-2)</td><td align="left" rowspan="1" colspan="1">SSRL (12-2)</td><td align="left" rowspan="1" colspan="1">SSRL (12-2)</td></tr><tr><td align="left" rowspan="1" colspan="1">Wavelength (Å)</td><td align="left" rowspan="1" colspan="1">0.9792</td><td align="left" rowspan="1" colspan="1">0.9795</td><td align="left" rowspan="1" colspan="1">0.9795</td><td align="left" rowspan="1" colspan="1">0.9795</td></tr><tr><td align="left" rowspan="1" colspan="1">Space Group</td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td></tr><tr><td align="left" rowspan="1" colspan="1">Cell dimensions</td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"><italic>a</italic>, <italic>b</italic>, <italic>c</italic>, (Å)</td><td align="left" rowspan="1" colspan="1">82.36, 107.50, 121.10</td><td align="left" rowspan="1" colspan="1">79.13, 81.61, 256.62</td><td align="left" rowspan="1" colspan="1">81.32, 106.68, 120.49</td><td align="left" rowspan="1" colspan="1">81.51, 107.21, 120.62</td></tr><tr><td align="left" rowspan="1" colspan="1">α, β, γ (°)</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="left" rowspan="1" colspan="1">Resolution (Å)</td><td align="left" rowspan="1" colspan="1">100 – 2.75 (2.82 – 2.75)</td><td align="left" rowspan="1" colspan="1">50.0 – 2.95 (3.03 – 2.95)</td><td align="left" rowspan="1" colspan="1">100 – 2.98 (3.06 – 2.98)</td><td align="left" rowspan="1" colspan="1">100 – 3.09 (3.17 – 3.09)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>R<sub>merge</sub></italic> (%)</td><td align="left" rowspan="1" colspan="1">7.0 (68.5)</td><td align="left" rowspan="1" colspan="1">9.6 (135.1)</td><td align="left" rowspan="1" colspan="1">14.4 (86.3)</td><td align="left" rowspan="1" colspan="1">11.6 (68.7)</td></tr><tr><td align="left" rowspan="1" colspan="1">CC<sub>1/2</sub></td><td align="left" rowspan="1" colspan="1">99.6 (66.3)</td><td align="left" rowspan="1" colspan="1">99.7 (47.6)</td><td align="left" rowspan="1" colspan="1">99.1 (75.3)</td><td align="left" rowspan="1" colspan="1">99.3 (77.1)</td></tr><tr><td align="left" rowspan="1" colspan="1">I/σ (I)</td><td align="left" rowspan="1" colspan="1">11.57 (1.52)</td><td align="left" rowspan="1" colspan="1">12.27 (1.16)</td><td align="left" rowspan="1" colspan="1">10.00 (1.88)</td><td align="left" rowspan="1" colspan="1">10.56 (1.75)</td></tr><tr><td align="left" rowspan="1" colspan="1">Completeness (%)</td><td align="left" rowspan="1" colspan="1">96.5 (98.8)</td><td align="left" rowspan="1" colspan="1">98.1 (98.9)</td><td align="left" rowspan="1" colspan="1">97.3 (90.0)</td><td align="left" rowspan="1" colspan="1">96.8 (89.1)</td></tr><tr><td align="left" rowspan="1" colspan="1">Redundancy</td><td align="left" rowspan="1" colspan="1">2.93 (3.00)</td><td align="left" rowspan="1" colspan="1">5.19 (5.13)</td><td align="left" rowspan="1" colspan="1">4.79 (4.56)</td><td align="left" rowspan="1" colspan="1">3.31 (2.84)</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Refinement</bold></td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">  Resolution (Å)</td><td align="left" rowspan="1" colspan="1">2.75</td><td align="left" rowspan="1" colspan="1">2.95</td><td align="left" rowspan="1" colspan="1">2.98</td><td align="left" rowspan="1" colspan="1">3.09</td></tr><tr><td align="left" rowspan="1" colspan="1">  No of reflections (F&gt;0)</td><td align="left" rowspan="1" colspan="1">27,153</td><td align="left" rowspan="1" colspan="1">35,727</td><td align="left" rowspan="1" colspan="1">21,376</td><td align="left" rowspan="1" colspan="1">19,325</td></tr><tr><td align="left" rowspan="1" colspan="1">  <italic>R<sub>work</sub></italic>/ <italic>R<sub>free</sub></italic></td><td align="left" rowspan="1" colspan="1">16.27/22.34</td><td align="left" rowspan="1" colspan="1">18.79/20.75</td><td align="left" rowspan="1" colspan="1">16.67 / 24.67</td><td align="left" rowspan="1" colspan="1">16.29 / 23.79</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>No. of atoms</bold></td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">  Protein</td><td align="left" rowspan="1" colspan="1">6197</td><td align="left" rowspan="1" colspan="1">6038</td><td align="left" rowspan="1" colspan="1">6168</td><td align="left" rowspan="1" colspan="1">6157</td></tr><tr><td align="left" rowspan="1" colspan="1">  RNA</td><td align="left" rowspan="1" colspan="1">973</td><td align="left" rowspan="1" colspan="1">1950</td><td align="left" rowspan="1" colspan="1">973</td><td align="left" rowspan="1" colspan="1">973</td></tr><tr><td align="left" rowspan="1" colspan="1">  Inositol Hexakisphosphate<break/>  (IHP)</td><td align="left" valign="top" rowspan="1" colspan="1">72</td><td align="left" valign="top" rowspan="1" colspan="1">72</td><td align="left" valign="top" rowspan="1" colspan="1">72</td><td align="left" valign="top" rowspan="1" colspan="1">72</td></tr><tr><td align="left" rowspan="1" colspan="1">  Zn</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">2</td></tr><tr><td align="left" rowspan="1" colspan="1">  Waters</td><td align="left" rowspan="1" colspan="1">33</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold><italic>B</italic> factors</bold></td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">  Protein</td><td align="left" rowspan="1" colspan="1">68.46</td><td align="left" rowspan="1" colspan="1">90.65</td><td align="left" rowspan="1" colspan="1">63.63</td><td align="left" rowspan="1" colspan="1">67.92</td></tr><tr><td align="left" rowspan="1" colspan="1">  RNA</td><td align="left" rowspan="1" colspan="1">88.24</td><td align="left" rowspan="1" colspan="1">108.8</td><td align="left" rowspan="1" colspan="1">69.70</td><td align="left" rowspan="1" colspan="1">77.49</td></tr><tr><td align="left" rowspan="1" colspan="1">  Inositol Hexakisphosphate<break/>  (IHP)</td><td align="left" valign="top" rowspan="1" colspan="1">47.10</td><td align="left" valign="top" rowspan="1" colspan="1">65.57</td><td align="left" valign="top" rowspan="1" colspan="1">44.23</td><td align="left" valign="top" rowspan="1" colspan="1">43.77</td></tr><tr><td align="left" rowspan="1" colspan="1">  Zn</td><td align="left" rowspan="1" colspan="1">48.47</td><td align="left" rowspan="1" colspan="1">64.38</td><td align="left" rowspan="1" colspan="1">38.25</td><td align="left" rowspan="1" colspan="1">49.04</td></tr><tr><td align="left" rowspan="1" colspan="1">  Waters</td><td align="left" rowspan="1" colspan="1">48.11</td><td align="left" rowspan="1" colspan="1">N/A</td><td align="left" rowspan="1" colspan="1">43.66</td><td align="left" rowspan="1" colspan="1">50.20</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>r.m.s deviations</bold></td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">  Bond lengths (Å)</td><td align="left" rowspan="1" colspan="1">0.010</td><td align="left" rowspan="1" colspan="1">0.007</td><td align="left" rowspan="1" colspan="1">0.009</td><td align="left" rowspan="1" colspan="1">0.010</td></tr><tr><td align="left" rowspan="1" colspan="1">  Bond angles (°)</td><td align="left" rowspan="1" colspan="1">1.342</td><td align="left" rowspan="1" colspan="1">0.885</td><td align="left" rowspan="1" colspan="1">1.359</td><td align="left" rowspan="1" colspan="1">1.365</td></tr></tbody></table> + + 49649 + Complex ADAR2-D_E488Q:BDF2-C23mer ADAR2-D_E488Q:GLI123mer ADAR2-D_wt:BDF2-U23mer ADAR2-D_wt:BDF2-C23mer Data Collection Synchrotron (Beamline) APS (24-ID-C) SSRL (12-2) SSRL (12-2) SSRL (12-2) Wavelength (Å) 0.9792 0.9795 0.9795 0.9795 Space Group P212121 P212121 P212121 P212121 Cell dimensions a, b, c, (Å) 82.36, 107.50, 121.10 79.13, 81.61, 256.62 81.32, 106.68, 120.49 81.51, 107.21, 120.62 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) 100 – 2.75 (2.82 – 2.75) 50.0 – 2.95 (3.03 – 2.95) 100 – 2.98 (3.06 – 2.98) 100 – 3.09 (3.17 – 3.09) Rmerge (%) 7.0 (68.5) 9.6 (135.1) 14.4 (86.3) 11.6 (68.7) CC1/2 99.6 (66.3) 99.7 (47.6) 99.1 (75.3) 99.3 (77.1) I/σ (I) 11.57 (1.52) 12.27 (1.16) 10.00 (1.88) 10.56 (1.75) Completeness (%) 96.5 (98.8) 98.1 (98.9) 97.3 (90.0) 96.8 (89.1) Redundancy 2.93 (3.00) 5.19 (5.13) 4.79 (4.56) 3.31 (2.84) Refinement   Resolution (Å) 2.75 2.95 2.98 3.09   No of reflections (F>0) 27,153 35,727 21,376 19,325   Rwork/ Rfree 16.27/22.34 18.79/20.75 16.67 / 24.67 16.29 / 23.79 No. of atoms   Protein 6197 6038 6168 6157   RNA 973 1950 973 973   Inositol Hexakisphosphate  (IHP) 72 72 72 72   Zn 2 2 2 2   Waters 33 0 1 1 B factors   Protein 68.46 90.65 63.63 67.92   RNA 88.24 108.8 69.70 77.49   Inositol Hexakisphosphate  (IHP) 47.10 65.57 44.23 43.77   Zn 48.47 64.38 38.25 49.04   Waters 48.11 N/A 43.66 50.20 r.m.s deviations   Bond lengths (Å) 0.010 0.007 0.009 0.010   Bond angles (°) 1.342 0.885 1.359 1.365 + + + diff --git a/BioC_XML/4918766_v0.xml b/BioC_XML/4918766_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..965b33d6777ec3a931dad23fca04c7227191544c --- /dev/null +++ b/BioC_XML/4918766_v0.xml @@ -0,0 +1,10482 @@ + + + + PMC + 20201223 + pmc.key + + 4918766 + NO-CC CODE + no + 0 + 0 + + 10.1038/nsmb.3230 + NIHMS779827 + 4918766 + 27183196 + 590 + 6 + Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: + 599 + surname:Liao;given-names:Jun + surname:Marinelli;given-names:Fabrizio + surname:Lee;given-names:ChangKeun + surname:Huang;given-names:Yihe + surname:Faraldo-Gómez;given-names:José D. + surname:Jiang;given-names:Youxing + TITLE + front + 23 + 2016 + 0 + Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger + + 0.99964213 + site + cleaner0 + 2023-07-12T19:32:26Z + SO: + + binding-site + + + 0.9995489 + protein_type + cleaner0 + 2023-07-12T16:55:52Z + MESH: + + sodium-calcium exchanger + + + + ABSTRACT + abstract + 99 + Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. + + 0.97095776 + protein_type + cleaner0 + 2023-07-12T15:31:56Z + MESH: + + Na+/Ca2+ exchangers + + + 0.9996849 + chemical + cleaner0 + 2023-07-12T15:31:21Z + CHEBI: + + Na+ + + + 0.9996843 + chemical + cleaner0 + 2023-07-12T15:31:36Z + CHEBI: + + Ca2+ + + + 0.9996164 + chemical + cleaner0 + 2023-07-12T15:31:38Z + CHEBI: + + Ca2+ + + + 0.9994154 + experimental_method + cleaner0 + 2023-07-12T16:59:23Z + MESH: + + structural analysis + + + 0.9996061 + protein_type + cleaner0 + 2023-07-12T16:55:56Z + MESH: + + exchanger + + + 0.99922407 + species + cleaner0 + 2023-07-12T15:32:42Z + MESH: + + Methanococcus jannaschii + + + protein + PR: + cleaner0 + 2023-07-12T15:33:37Z + + NCX_Mj + + + 0.9994753 + protein_state + cleaner0 + 2023-07-12T19:41:51Z + DUMMY: + + bound to + + + 0.9996884 + chemical + cleaner0 + 2023-07-12T15:31:28Z + CHEBI: + + Na+ + + + 0.99972093 + chemical + cleaner0 + 2023-07-12T15:31:30Z + CHEBI: + + Ca2+ + + + 0.99971426 + chemical + cleaner0 + 2023-07-12T15:31:33Z + CHEBI: + + Sr2+ + + + 0.99966574 + protein_state + cleaner0 + 2023-07-12T15:48:06Z + DUMMY: + + apo + + + chemical + CHEBI: + cleaner0 + 2023-07-12T15:30:34Z + + Na+ + + + chemical + CHEBI: + cleaner0 + 2023-07-12T15:30:58Z + + Ca2+ + + + 0.9962363 + evidence + cleaner0 + 2023-07-12T19:18:10Z + DUMMY: + + conformational free-energy landscape + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:42:15Z + + ion-occupancy + + + 0.99939805 + experimental_method + cleaner0 + 2023-07-12T16:59:30Z + MESH: + + enhanced-sampling molecular-dynamics simulations + + + 0.99956983 + evidence + cleaner0 + 2023-07-12T19:18:15Z + DUMMY: + + crystal structures + + + 0.9994036 + experimental_method + cleaner0 + 2023-07-12T16:59:40Z + MESH: + + calculations + + + 0.99183875 + protein_state + cleaner0 + 2023-07-12T19:42:18Z + DUMMY: + + outward + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:42:39Z + + inward + + + 0.99943435 + chemical + cleaner0 + 2023-07-12T15:31:40Z + CHEBI: + + Na+ + + + 0.9923915 + chemical + cleaner0 + 2023-07-12T15:31:42Z + CHEBI: + + Ca2+ + + + + INTRO + title_1 + 1257 + Introduction + + + INTRO + paragraph + 1270 + Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis. NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry. This reaction is, however, inherently reversible, its direction being dictated only by the transmembrane electrochemical ion gradients . The mechanism of NCX proteins is therefore highly likely to be consistent with the alternating-access model of secondary-active transport. The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves. Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). + + 0.99797577 + protein_type + cleaner0 + 2023-07-12T15:31:57Z + MESH: + + Na+/Ca2+ exchangers + + + 0.9995409 + protein_type + cleaner0 + 2023-07-12T15:33:12Z + MESH: + + NCX + + + 0.9995762 + chemical + cleaner0 + 2023-07-12T16:53:44Z + CHEBI: + + Ca2+ + + + 0.99959725 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + 0.9996245 + chemical + cleaner0 + 2023-07-12T16:53:47Z + CHEBI: + + Ca2+ + + + 0.9996516 + chemical + cleaner0 + 2023-07-12T16:53:50Z + CHEBI: + + Na+ + + + 0.9993729 + chemical + cleaner0 + 2023-07-12T16:53:53Z + CHEBI: + + Na+ + + + 0.9993702 + chemical + cleaner0 + 2023-07-12T16:53:56Z + CHEBI: + + Ca2+ + + + protein_type + MESH: + cleaner0 + 2023-07-12T15:33:13Z + + NCX + + + 0.99957067 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + 0.9994636 + structure_element + cleaner0 + 2023-07-12T19:36:18Z + SO: + + membrane-spanning segments + + + 0.99272573 + structure_element + cleaner0 + 2023-07-12T19:36:35Z + SO: + + halves + + + 0.9981975 + structure_element + cleaner0 + 2023-07-12T19:36:37Z + SO: + + halves + + + 0.9995371 + protein_state + cleaner0 + 2023-07-12T19:42:43Z + DUMMY: + + highly conserved + + + 0.99968433 + structure_element + cleaner0 + 2023-07-12T19:36:51Z + SO: + + α-repeat + + + 0.99947935 + taxonomy_domain + cleaner0 + 2023-07-12T15:32:26Z + DUMMY: + + eukaryotic + + + 0.99958783 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + 0.99172944 + structure_element + cleaner0 + 2023-07-12T19:36:48Z + SO: + + halves + + + structure_element + SO: + cleaner0 + 2023-07-12T19:37:14Z + + intracellular regulatory domain + + + 0.9465155 + protein_state + cleaner0 + 2023-07-12T19:42:48Z + DUMMY: + + absent + + + 0.9995036 + taxonomy_domain + cleaner0 + 2023-07-12T15:32:31Z + DUMMY: + + microbial + + + 0.9995828 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + + INTRO + paragraph + 2260 + Despite a long history of physiological and functional studies, the molecular mechanism of NCX has been elusive, owing to the lack of structural information. Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein. This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). With similar ion exchange properties to those of its eukaryotic counterparts, NCX_Mj provides a compelling model system to investigate the structural basis for the specificity, stoichiometry and mechanism of the ion-exchange reaction catalyzed by NCX. In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. These structures reveal the mode of recognition of these ions, their relative affinities, and the mechanism of extracellular ion exchange, for a well-defined, functional conformation in a membrane-like environment. An independent analysis based on molecular-dynamics simulations demonstrates that the structures capture mechanistically relevant states. These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism. + + 0.99948573 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + 0.9964077 + evidence + cleaner0 + 2023-07-12T19:18:28Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99919784 + species + cleaner0 + 2023-07-12T15:32:44Z + MESH: + + Methanococcus jannaschii + + + 0.99958605 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + 0.99796706 + evidence + cleaner0 + 2023-07-12T19:18:31Z + DUMMY: + + structure + + + 0.9991055 + protein_type + cleaner0 + 2023-07-12T16:56:01Z + MESH: + + exchanger + + + 0.9994218 + protein_state + cleaner0 + 2023-07-12T15:36:59Z + DUMMY: + + outward-facing + + + 0.9995973 + site + cleaner0 + 2023-07-12T19:32:32Z + SO: + + ion-binding sites + + + site + SO: + cleaner0 + 2023-07-12T15:35:34Z + + internal + + + 0.9518084 + site + cleaner0 + 2023-07-12T15:35:41Z + SO: + + Sint + + + site + SO: + cleaner0 + 2023-07-12T15:35:54Z + + external + + + 0.9600976 + site + cleaner0 + 2023-07-12T15:36:02Z + SO: + + Sext + + + 0.99758303 + site + cleaner0 + 2023-07-12T15:36:15Z + SO: + + Ca2+-binding + + + 0.9913696 + site + cleaner0 + 2023-07-12T15:36:28Z + SO: + + SCa + + + site + SO: + cleaner0 + 2023-07-12T15:36:41Z + + middle + + + 0.99600405 + site + cleaner0 + 2023-07-12T15:36:47Z + SO: + + Smid + + + 0.8781682 + protein_state + cleaner0 + 2023-07-12T19:43:07Z + DUMMY: + + occluded from + + + 0.99951184 + taxonomy_domain + cleaner0 + 2023-07-12T15:32:27Z + DUMMY: + + eukaryotic + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99919754 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + 0.9993592 + evidence + cleaner0 + 2023-07-12T19:18:33Z + DUMMY: + + structures + + + 0.9994232 + protein_state + cleaner0 + 2023-07-12T15:37:00Z + DUMMY: + + outward-facing + + + 0.9995788 + protein_state + cleaner0 + 2023-07-12T15:37:05Z + DUMMY: + + wild-type + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.9991128 + protein_state + cleaner0 + 2023-07-12T15:37:10Z + DUMMY: + + in complex with + + + 0.99974465 + chemical + cleaner0 + 2023-07-12T15:37:15Z + CHEBI: + + Na+ + + + 0.9997424 + chemical + cleaner0 + 2023-07-12T15:37:18Z + CHEBI: + + Ca2+ + + + 0.99974084 + chemical + cleaner0 + 2023-07-12T15:37:20Z + CHEBI: + + Sr2+ + + + 0.9977774 + evidence + cleaner0 + 2023-07-12T19:18:36Z + DUMMY: + + structures + + + 0.9994773 + experimental_method + cleaner0 + 2023-07-12T16:59:50Z + MESH: + + molecular-dynamics simulations + + + 0.9992453 + evidence + cleaner0 + 2023-07-12T19:18:38Z + DUMMY: + + structures + + + 0.997436 + experimental_method + cleaner0 + 2023-07-12T16:59:53Z + MESH: + + calculations + + + 0.9993314 + protein_state + cleaner0 + 2023-07-12T15:37:00Z + DUMMY: + + outward-facing + + + 0.9994099 + protein_type + cleaner0 + 2023-07-12T16:56:05Z + MESH: + + exchanger + + + 0.9994784 + protein_state + cleaner0 + 2023-07-12T15:37:25Z + DUMMY: + + inward-facing + + + + RESULTS + title_1 + 3934 + Results + + + RESULTS + title_2 + 3942 + Extracellular Na+ binding + + 0.9997164 + chemical + cleaner0 + 2023-07-12T15:37:33Z + CHEBI: + + Na+ + + + + RESULTS + paragraph + 3968 + The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. To conclusively clarify this assignment, we first set out to examine the Na+ occupancy of these sites without Ca2+. Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique. The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods). X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated. Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study. First, the electron density at Smid does not depend significantly on the Na+ concentration. Second, the protein coordination geometry at Smid is clearly suboptimal for Na+ (Supplementary Fig. 1d). The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. + + 0.99960774 + site + cleaner0 + 2023-07-12T19:32:39Z + SO: + + central binding sites + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.999382 + evidence + cleaner0 + 2023-07-12T19:18:44Z + DUMMY: + + structure + + + 0.9995949 + chemical + cleaner0 + 2023-07-12T15:37:44Z + CHEBI: + + Na+ + + + 0.999593 + chemical + cleaner0 + 2023-07-12T15:37:47Z + CHEBI: + + Ca2+ + + + 0.9987638 + evidence + cleaner0 + 2023-07-12T19:18:48Z + DUMMY: + + crystals + + + 0.9990246 + chemical + cleaner0 + 2023-07-12T15:37:52Z + CHEBI: + + Na+ + + + 0.9995868 + chemical + cleaner0 + 2023-07-12T15:37:54Z + CHEBI: + + Ca2+ + + + 0.73599255 + evidence + cleaner0 + 2023-07-12T17:00:39Z + DUMMY: + + Crystals + + + 0.9994336 + chemical + cleaner0 + 2023-07-12T15:37:49Z 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chemical + cleaner0 + 2023-07-12T15:38:09Z + CHEBI: + + Na+ + + + 0.97880304 + chemical + cleaner0 + 2023-07-12T15:38:11Z + CHEBI: + + Na+ + + + 0.97307587 + site + cleaner0 + 2023-07-12T15:36:29Z + SO: + + SCa + + + chemical + CHEBI: + cleaner0 + 2023-07-12T16:52:45Z + + Na+ + + + chemical + CHEBI: + cleaner0 + 2023-07-12T16:52:57Z + + Ca2+ + + + site + SO: + cleaner0 + 2023-07-12T15:36:47Z + + Smid + + + 0.9997707 + chemical + cleaner0 + 2023-07-12T15:38:52Z + CHEBI: + + water + + + 0.99955773 + chemical + cleaner0 + 2023-07-12T15:38:47Z + CHEBI: + + Na+ + + + 0.70465773 + site + cleaner0 + 2023-07-12T15:36:47Z + SO: + + Smid + + + 0.94124186 + experimental_method + cleaner0 + 2023-07-12T17:01:10Z + MESH: + + simulation + + + 0.9995507 + evidence + cleaner0 + 2023-07-12T19:19:03Z + DUMMY: + + electron density + + + 0.5928501 + site + cleaner0 + 2023-07-12T15:36:47Z + SO: + + Smid + + + 0.99941397 + chemical + cleaner0 + 2023-07-12T15:38:49Z + CHEBI: + + Na+ + + + 0.5664469 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.9995431 + chemical + cleaner0 + 2023-07-12T15:38:37Z + CHEBI: + + Na+ + + + 0.99977714 + chemical + cleaner0 + 2023-07-12T15:38:54Z + CHEBI: + + water + + + 0.49353516 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:39Z + + hydrogen-bonds + + + 0.9993975 + protein_state + cleaner0 + 2023-07-12T19:43:19Z + DUMMY: + + highly conserved + + + 0.99989235 + residue_name_number + cleaner0 + 2023-07-12T15:40:29Z + DUMMY: + + Glu54 + + + 0.9998932 + residue_name_number + cleaner0 + 2023-07-12T15:40:34Z + DUMMY: + + Glu213 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:39Z + + coordinate + + + 0.99929106 + chemical + cleaner0 + 2023-07-12T16:54:02Z + CHEBI: + + Na+ + + + 0.92876875 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.98581064 + site + cleaner0 + 2023-07-12T15:36:29Z + SO: + + SCa + + + 0.9932243 + site + cleaner0 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+ 6017 + The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2). When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d). However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). TM7a also forms hydrophobic contacts with the C-terminal half of TM6. These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b). This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation. The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa. Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes. The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99961394 + evidence + cleaner0 + 2023-07-12T19:19:09Z + DUMMY: + + structures + + + 0.9994112 + chemical + cleaner0 + 2023-07-12T15:41:06Z + CHEBI: + + Na+ + + + 0.9996445 + chemical + cleaner0 + 2023-07-12T15:41:09Z + CHEBI: + + Na+ + + + 0.8975322 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.9996025 + chemical + cleaner0 + 2023-07-12T15:41:11Z + CHEBI: + + Na+ + + + 0.97438496 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.63517 + protein_state + cleaner0 + 2023-07-12T19:43:51Z + DUMMY: + + high + + + 0.99961746 + structure_element + cleaner0 + 2023-07-12T19:37:23Z + SO: + + N-terminal half + + + 0.9997969 + structure_element + cleaner0 + 2023-07-12T19:37:26Z + SO: + + TM7 + + + 0.9983444 + structure_element + cleaner0 + 2023-07-12T19:37:29Z + SO: + + short helices + + + 0.9997923 + structure_element + cleaner0 + 2023-07-12T15:41:22Z + SO: + + TM7a + + + 0.999793 + structure_element + cleaner0 + 2023-07-12T15:41:27Z + SO: + + TM7b + + + 0.9997937 + structure_element + cleaner0 + 2023-07-12T15:41:27Z + SO: + + TM7b + + + 0.99953514 + site + cleaner0 + 2023-07-12T19:32:45Z + SO: + + central binding sites + + + 0.9999001 + residue_name_number + cleaner0 + 2023-07-12T15:41:02Z + DUMMY: + + Ala206 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:39Z + + coordinating + + + 0.99943876 + chemical + cleaner0 + 2023-07-12T15:41:14Z + CHEBI: + + Na+ + + + 0.5882469 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.9966037 + protein_state + cleaner0 + 2023-07-12T19:44:00Z + DUMMY: + + empty + + + 0.9698598 + protein_state + cleaner0 + 2023-07-12T19:44:04Z + DUMMY: + + low + + + 0.99899304 + chemical + cleaner0 + 2023-07-12T15:41:17Z + CHEBI: + + Na+ + + + 0.9997869 + structure_element + cleaner0 + 2023-07-12T15:41:21Z + SO: + + TM7a + + + 0.9997874 + structure_element + cleaner0 + 2023-07-12T15:41:26Z + SO: + + TM7b + + + 0.7902682 + structure_element + cleaner0 + 2023-07-12T19:37:33Z + SO: + + helix + + + 0.9999031 + residue_name_number + cleaner0 + 2023-07-12T15:41:03Z + DUMMY: + + Ala206 + + + 0.99979156 + structure_element + cleaner0 + 2023-07-12T15:41:22Z + SO: + + TM7a + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:39Z + + hydrophobic contacts + + + 0.9996227 + structure_element + cleaner0 + 2023-07-12T19:37:42Z + SO: + + C-terminal half + + + 0.99979216 + structure_element + cleaner0 + 2023-07-12T15:41:32Z + SO: + + TM6 + + + 0.99959534 + evidence + cleaner0 + 2023-07-12T19:19:13Z + DUMMY: + + structure + + + 0.99957615 + chemical + cleaner0 + 2023-07-12T15:41:53Z + CHEBI: + + Na+ + + + 0.988373 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: 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experiment + + + 0.9962109 + evidence + cleaner0 + 2023-07-12T19:19:46Z + DUMMY: + + K1/2 + + + 0.9995229 + chemical + cleaner0 + 2023-07-12T15:42:03Z + CHEBI: + + Na+ + + + 0.5956819 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:45:09Z + + partially occupied + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.9996499 + evidence + cleaner0 + 2023-07-12T19:19:49Z + DUMMY: + + crystal + + + 0.9996519 + protein_state + cleaner0 + 2023-07-12T19:45:13Z + DUMMY: + + occluded + + + 0.75721264 + protein_state + cleaner0 + 2023-07-12T19:45:16Z + DUMMY: + + partially open + + + 0.972502 + evidence + cleaner0 + 2023-07-12T19:19:52Z + DUMMY: + + Na+ affinity + + + 0.99967176 + chemical + cleaner0 + 2023-07-12T15:42:10Z + CHEBI: + + Na+ + + + 0.999126 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + 0.9995185 + taxonomy_domain + cleaner0 + 2023-07-12T16:53:35Z + DUMMY: + + eukaryotes + + + 0.99950695 + chemical + cleaner0 + 2023-07-12T15:42:06Z + CHEBI: + + Na+ + + + 0.99931836 + protein_type + cleaner0 + 2023-07-12T16:56:14Z + MESH: + + transporter + + + 0.9992928 + evidence + cleaner0 + 2023-07-12T19:19:54Z + DUMMY: + + Hill coefficient + + + 0.9994451 + chemical + cleaner0 + 2023-07-12T15:42:08Z + CHEBI: + + Na+ + + + 0.99952483 + taxonomy_domain + cleaner0 + 2023-07-12T15:32:27Z + DUMMY: + + eukaryotic + + + 0.9994234 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + + RESULTS + title_2 + 8043 + Extracellular Ca2+ and Sr2+ binding and their competition with Na+ + + 0.9997628 + chemical + cleaner0 + 2023-07-12T15:42:21Z + CHEBI: + + Ca2+ + + + 0.9997602 + chemical + cleaner0 + 2023-07-12T15:42:24Z + CHEBI: + + Sr2+ + + + 0.9997529 + chemical + cleaner0 + 2023-07-12T15:42:26Z + CHEBI: + + Na+ + + + + RESULTS + paragraph + 8110 + To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods). Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity. Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations. Binding of Sr2+, however, excludes Na+ entirely. Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. + + 0.9996861 + chemical + cleaner0 + 2023-07-12T15:42:29Z + CHEBI: + + Ca2+ + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.9995922 + chemical + cleaner0 + 2023-07-12T15:42:41Z + CHEBI: + + Na+ + + + experimental_method + MESH: + cleaner0 + 2023-07-12T17:01:45Z + + titrated the crystals + + + 0.9996915 + chemical + cleaner0 + 2023-07-12T15:42:43Z + CHEBI: + + Sr2+ + + + 0.999686 + chemical + cleaner0 + 2023-07-12T15:42:46Z + CHEBI: + + Sr2+ + + + 0.9997907 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + 0.9996725 + chemical + cleaner0 + 2023-07-12T15:42:54Z + CHEBI: + + Ca2+ + + + 0.9996459 + chemical + cleaner0 + 2023-07-12T15:42:52Z + CHEBI: + + Na+ + + + 0.9994124 + evidence + cleaner0 + 2023-07-12T19:20:00Z + DUMMY: + + electron-density intensity + + + experimental_method + MESH: + cleaner0 + 2023-07-12T17:02:07Z + + Protein crystals soaked + + + 0.9996846 + chemical + cleaner0 + 2023-07-12T15:42:50Z + CHEBI: + + Sr2+ + + + 0.9996888 + chemical + cleaner0 + 2023-07-12T15:42:48Z + CHEBI: + + Na+ + + + 0.99926555 + evidence + cleaner0 + 2023-07-12T19:20:03Z + DUMMY: + + electron-density peak + + + 0.5260014 + site + cleaner0 + 2023-07-12T15:36:29Z + SO: + + SCa + + + 0.999609 + chemical + cleaner0 + 2023-07-12T15:42:57Z + CHEBI: + + Sr2+ + + + 0.99643576 + protein_state + cleaner0 + 2023-07-12T15:43:47Z + DUMMY: + + Sr2+-loaded + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99959177 + evidence + cleaner0 + 2023-07-12T19:20:05Z + DUMMY: + + structure + + + 0.9993584 + protein_state + cleaner0 + 2023-07-12T19:45:23Z + DUMMY: + + partially open + + + 0.9996485 + chemical + cleaner0 + 2023-07-12T15:43:09Z + CHEBI: + + Na+ + + + 0.99964035 + chemical + cleaner0 + 2023-07-12T15:43:11Z + CHEBI: + + Sr2+ + + + 0.9994898 + chemical + cleaner0 + 2023-07-12T15:43:13Z + CHEBI: + + Na+ + + + 0.99958897 + experimental_method + cleaner0 + 2023-07-12T19:13:20Z + MESH: + + Crystal titrations + + + 0.84637284 + experimental_method + cleaner0 + 2023-07-12T19:13:24Z + MESH: + + decreasing + + + 0.99964565 + chemical + cleaner0 + 2023-07-12T15:43:15Z + CHEBI: + + Sr2+ + + + 0.95324624 + experimental_method + cleaner0 + 2023-07-12T19:13:26Z + MESH: + + increasing + + + 0.9996711 + chemical + cleaner0 + 2023-07-12T15:43:17Z + CHEBI: + + Na+ + + + 0.99968195 + chemical + cleaner0 + 2023-07-12T15:43:33Z + CHEBI: + + Sr2+ + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T15:37:00Z + + outward-facing + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99965817 + chemical + cleaner0 + 2023-07-12T15:43:36Z + CHEBI: + + Na+ + + + 0.999688 + chemical + cleaner0 + 2023-07-12T15:44:05Z + CHEBI: + + Na+ + + + 0.9996972 + chemical + cleaner0 + 2023-07-12T16:54:20Z + CHEBI: + + Sr2+ + + + 0.9996567 + chemical + cleaner0 + 2023-07-12T15:44:03Z + CHEBI: + + Na+ + + + 0.99961853 + chemical + cleaner0 + 2023-07-12T15:44:01Z + CHEBI: + + Sr2+ + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99837416 + protein_state + cleaner0 + 2023-07-12T15:43:52Z + DUMMY: + + Na+-loaded + + + 0.99951965 + protein_state + cleaner0 + 2023-07-12T15:43:57Z + DUMMY: + + fully occluded + + + + RESULTS + paragraph + 9050 + Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). Binding of Ca2+ to both sites simultaneously is highly improbable due to their close proximity, and at least one water molecule can be discerned coordinating the ion (Fig. 3b). The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport. Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. SCa is therefore the functional Ca2+ site. Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side. Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). + + 0.83672285 + experimental_method + cleaner0 + 2023-07-12T19:13:30Z + MESH: + + titration experiments + + + 0.99967515 + chemical + cleaner0 + 2023-07-12T15:44:35Z + CHEBI: + + Ca2+ + + + 0.9996866 + chemical + cleaner0 + 2023-07-12T15:44:38Z + CHEBI: + + Sr2+ + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.9995885 + evidence + cleaner0 + 2023-07-12T19:20:08Z + DUMMY: + + electron density distribution + + + 0.99763757 + experimental_method + cleaner0 + 2023-07-12T19:13:41Z + MESH: + + crystals soaked in + + + 0.79457146 + protein_state + cleaner0 + 2023-07-12T19:45:41Z + DUMMY: + + high + + + 0.99966097 + chemical + cleaner0 + 2023-07-12T15:44:41Z + CHEBI: + + Ca2+ + + + 0.90524435 + protein_state + cleaner0 + 2023-07-12T19:45:44Z + DUMMY: + + low + + + 0.9996723 + chemical + cleaner0 + 2023-07-12T15:44:44Z + CHEBI: + + Na+ + + + 0.9996927 + chemical + cleaner0 + 2023-07-12T15:44:46Z + CHEBI: + + Ca2+ + + + 0.7841132 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.8154444 + site + cleaner0 + 2023-07-12T15:36:29Z + SO: + + SCa + + + 0.8182291 + site + cleaner0 + 2023-07-12T15:36:29Z + SO: + + SCa + + + 0.99968505 + chemical + cleaner0 + 2023-07-12T15:44:49Z + CHEBI: + + Ca2+ + + + 0.99975723 + chemical + cleaner0 + 2023-07-12T15:45:03Z + CHEBI: + + water + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:39Z + + coordinating + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:45:57Z + + partial + + + 0.99937546 + chemical + cleaner0 + 2023-07-12T15:44:59Z + CHEBI: + + Ca2+ + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:46:08Z + + occupancy + + + 0.611224 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.9999026 + residue_name_number + cleaner0 + 2023-07-12T15:40:41Z + DUMMY: + + Asp240 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:39Z + + coordinate + + + 0.99966395 + chemical + cleaner0 + 2023-07-12T15:45:05Z + CHEBI: + + Ca2+ + + + 0.9973443 + experimental_method + cleaner0 + 2023-07-12T19:13:46Z + MESH: + + functional and computational studies + + + 0.9998989 + residue_name_number + cleaner0 + 2023-07-12T15:40:41Z + DUMMY: + + Asp240 + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:46:31Z + + protonated + + + protein_type + MESH: + cleaner0 + 2023-07-12T15:33:13Z + + NCX + + + 0.9998833 + residue_name_number + cleaner0 + 2023-07-12T15:40:41Z + DUMMY: + + Asp240 + + + experimental_method + MESH: + cleaner0 + 2023-07-12T19:46:42Z + + substituted + + + 0.9993026 + residue_name + cleaner0 + 2023-07-12T16:52:19Z + SO: + + Asn + + + 0.99957514 + chemical + cleaner0 + 2023-07-12T15:45:14Z + CHEBI: + + Ca2+ + + + 0.81406647 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.580456 + site + cleaner0 + 2023-07-12T15:36:29Z + SO: + + SCa + + + 0.9995466 + site + cleaner0 + 2023-07-12T19:32:50Z + SO: + + Ca2+ site + + + 0.9997176 + chemical + cleaner0 + 2023-07-12T15:45:36Z + CHEBI: + + Sr2+ + + + 0.9997066 + chemical + cleaner0 + 2023-07-12T15:45:34Z + CHEBI: + + Ca2+ + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:20:37Z + + affinity + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T15:37:00Z + + outward-facing + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99970555 + chemical + cleaner0 + 2023-07-12T15:45:17Z + CHEBI: + + Na+ + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:47:02Z + + physiological and biochemical data + + + 0.99922514 + taxonomy_domain + cleaner0 + 2023-07-12T15:32:27Z + DUMMY: + + eukaryotic + + + 0.99955577 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + evidence + DUMMY: + cleaner0 + 2023-07-12T15:46:08Z + + Ca2+ affinity + + + 0.99957705 + experimental_method + cleaner0 + 2023-07-12T19:13:51Z + MESH: + + crystallographic titration assay + + + 0.99969375 + chemical + cleaner0 + 2023-07-12T16:54:26Z + CHEBI: + + Ca2+ + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:21:05Z + + affinity + + + evidence + DUMMY: + cleaner0 + 2023-07-12T15:46:28Z + + Ca2+ affinities + + + 0.99935716 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + 0.9995334 + evidence + cleaner0 + 2023-07-12T19:20:17Z + DUMMY: + + Km + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99948776 + evidence + cleaner0 + 2023-07-12T19:20:20Z + DUMMY: + + Km + + + + RESULTS + paragraph + 10498 + Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. + + 0.9993302 + experimental_method + cleaner0 + 2023-07-12T19:14:09Z + MESH: + + crystal titration experiments + + + 0.8698213 + site + cleaner0 + 2023-07-12T19:32:57Z + SO: + + binding sites + + + 0.9966097 + protein_state + cleaner0 + 2023-07-12T15:37:00Z + DUMMY: + + outward-facing + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99553925 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.9974414 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.99956393 + chemical + cleaner0 + 2023-07-12T15:47:05Z + CHEBI: + + Na+ + + + 0.99726665 + site + cleaner0 + 2023-07-12T15:36:29Z + SO: + + SCa + + + 0.99862236 + chemical + cleaner0 + 2023-07-12T15:47:08Z + CHEBI: + + Ca2+ + + + 0.9997227 + chemical + cleaner0 + 2023-07-12T15:47:10Z + CHEBI: + + Na+ + + + 0.9992256 + experimental_method + cleaner0 + 2023-07-12T19:14:14Z + MESH: + + simulation + + + 0.99973094 + chemical + cleaner0 + 2023-07-12T15:47:12Z + CHEBI: + + Sr2+ + + + 0.9943116 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.9997344 + chemical + cleaner0 + 2023-07-12T15:47:17Z + CHEBI: + + Ca2+ + + + 0.99609 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.99974436 + chemical + cleaner0 + 2023-07-12T15:47:15Z + CHEBI: + + water + + + 0.9996326 + site + cleaner0 + 2023-07-12T19:33:00Z + SO: + + ion-binding sites + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99972224 + chemical + cleaner0 + 2023-07-12T15:47:19Z + CHEBI: + + Na+ + + + 0.99973035 + chemical + cleaner0 + 2023-07-12T15:47:21Z + CHEBI: + + Na+ + + + 0.9997362 + chemical + cleaner0 + 2023-07-12T15:47:24Z + CHEBI: + + Ca2+ + + + 0.9997355 + chemical + cleaner0 + 2023-07-12T15:47:26Z + CHEBI: + + Sr2+ + + + 0.99949324 + taxonomy_domain + cleaner0 + 2023-07-12T15:32:27Z + DUMMY: + + eukaryotic + + + 0.99963903 + protein_type + cleaner0 + 2023-07-12T16:56:19Z + MESH: + + exchangers + + + + RESULTS + title_2 + 11126 + A structure of NCX_Mj without Na+ or Ca2+ bound + + 0.99962306 + evidence + cleaner0 + 2023-07-12T19:23:58Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99943596 + protein_state + cleaner0 + 2023-07-12T19:47:20Z + DUMMY: + + without + + + 0.99964356 + chemical + cleaner0 + 2023-07-12T15:47:49Z + CHEBI: + + Na+ + + + 0.99968123 + chemical + cleaner0 + 2023-07-12T15:47:51Z + CHEBI: + + Ca2+ + + + 0.5086934 + protein_state + cleaner0 + 2023-07-12T19:47:22Z + DUMMY: + + bound + + + + RESULTS + paragraph + 11174 + An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM). We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites. Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). Although the binding sites are thus fully accessible to the external solution (Fig. 3e), the lack of electron density therein indicates no ions or ordered solvent molecules. This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind. Such interpretation would be consistent with the computer simulations reported below. Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. + + 0.99966514 + protein_state + cleaner0 + 2023-07-12T15:48:05Z + DUMMY: + + apo + + + 0.9870203 + protein_state + cleaner0 + 2023-07-12T15:37:00Z + DUMMY: + + outward-facing + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.9996991 + chemical + cleaner0 + 2023-07-12T15:48:28Z + CHEBI: + + Na+ + + + 0.9996948 + chemical + cleaner0 + 2023-07-12T15:48:30Z + CHEBI: + + Ca2+ + + + 0.99965405 + protein_state + cleaner0 + 2023-07-12T15:48:06Z + DUMMY: + + apo + + + 0.9995609 + evidence + cleaner0 + 2023-07-12T19:24:30Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99966407 + experimental_method + cleaner0 + 2023-07-12T19:14:19Z + MESH: + + crystallizing + + + 0.6531592 + protein_state + cleaner0 + 2023-07-12T19:47:30Z + DUMMY: + + lower pH + + + 0.9994112 + protein_state + cleaner0 + 2023-07-12T19:47:33Z + DUMMY: + + absence of + + + 0.99970734 + chemical + cleaner0 + 2023-07-12T15:48:33Z + CHEBI: + + Na+ + + + 0.9994924 + evidence + cleaner0 + 2023-07-12T19:24:26Z + DUMMY: + + structure + + + 0.9994571 + protein_state + cleaner0 + 2023-07-12T19:47:37Z + DUMMY: + + partially open + + + 0.99934405 + evidence + cleaner0 + 2023-07-12T19:24:28Z + DUMMY: + + structure + + + 0.99962556 + chemical + cleaner0 + 2023-07-12T15:48:36Z + CHEBI: + + Na+ + + + 0.9996591 + chemical + cleaner0 + 2023-07-12T15:48:38Z + CHEBI: + + Ca2+ + + + 0.9996749 + chemical + cleaner0 + 2023-07-12T15:48:41Z + CHEBI: + + Sr2+ + + + 0.9998122 + structure_element + cleaner0 + 2023-07-12T15:41:47Z + SO: + + TM7ab + + + 0.9994032 + structure_element + cleaner0 + 2023-07-12T19:38:06Z + SO: + + extracellular half + + + 0.99981755 + structure_element + cleaner0 + 2023-07-12T15:41:33Z + SO: + + TM6 + + + 0.999819 + structure_element + cleaner0 + 2023-07-12T15:41:41Z + SO: + + TM1 + + + 0.9996153 + site 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2023-07-12T15:48:06Z + DUMMY: + + apo + + + 0.9995679 + evidence + cleaner0 + 2023-07-12T19:24:18Z + DUMMY: + + structure + + + 0.99966276 + protein_state + cleaner0 + 2023-07-12T19:48:46Z + DUMMY: + + unloaded + + + 0.9996495 + protein_state + cleaner0 + 2023-07-12T19:48:49Z + DUMMY: + + open + + + 0.99581814 + protein_state + cleaner0 + 2023-07-12T15:37:00Z + DUMMY: + + outward-facing + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99950194 + evidence + cleaner0 + 2023-07-12T19:24:20Z + DUMMY: + + structure + + + 0.99955976 + protein_state + cleaner0 + 2023-07-12T19:48:53Z + DUMMY: + + fully protonated + + + 0.9995828 + protein_type + cleaner0 + 2023-07-12T16:56:22Z + MESH: + + transporter + + + 0.999714 + chemical + cleaner0 + 2023-07-12T15:49:00Z + CHEBI: + + Na+ + + + 0.99970317 + chemical + cleaner0 + 2023-07-12T15:49:02Z + CHEBI: + + Ca2+ + + + 0.99953717 + experimental_method + cleaner0 + 2023-07-12T19:14:23Z + MESH: + + computer simulations + + + 0.99950814 + experimental_method + cleaner0 + 2023-07-12T19:14:26Z + MESH: + + transport assays + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.98821247 + protein_state + cleaner0 + 2023-07-12T19:48:56Z + DUMMY: + + presence of + + + 0.9997245 + chemical + cleaner0 + 2023-07-12T15:49:04Z + CHEBI: + + Na+ + + + 0.99972236 + chemical + cleaner0 + 2023-07-12T15:49:07Z + CHEBI: + + Ca2+ + + + 0.94008267 + protein_state + cleaner0 + 2023-07-12T19:48:59Z + DUMMY: + + low pH + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:49:11Z + + inactivates + + + + RESULTS + title_2 + 12609 + Ion occupancy determines the free-energy landscape of NCX_Mj + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + + RESULTS + paragraph + 12670 + That secondary-active transporters are able to harness an electrochemical gradient of one substrate to power the uphill transport of another relies on a seemingly simple principle: they must not transition between outward- and inward-open conformations unless in two precise substrate occupancy states. NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. The reason why only specific occupancy states permit this transition in a given system, thereby determining its biological function, remains unclear. To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations. This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above. As it happens, the results confirm that the structures now available are representing interconverting states of the functional cycle of NCX_Mj, while revealing how the alternating-access mechanism is controlled by the ion-occupancy state. + + 0.9995427 + protein_type + cleaner0 + 2023-07-12T16:56:30Z + MESH: + + secondary-active transporters + + + 0.99890316 + protein_state + cleaner0 + 2023-07-12T19:49:25Z + DUMMY: + + outward- + + + 0.9992698 + protein_state + cleaner0 + 2023-07-12T19:49:28Z + DUMMY: + + inward-open + + + 0.99877745 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + 0.9996822 + chemical + cleaner0 + 2023-07-12T16:54:39Z + CHEBI: + + Na+ + + + 0.99968994 + chemical + cleaner0 + 2023-07-12T16:54:43Z + CHEBI: + + Ca2+ + + + 0.9989229 + protein_type + cleaner0 + 2023-07-12T16:56:33Z + MESH: + + antiporter + + + 0.9994962 + protein_type + cleaner0 + 2023-07-12T16:56:36Z + MESH: + + symporters + + + 0.5239756 + protein_state + cleaner0 + 2023-07-12T19:49:39Z + DUMMY: + + bound + + + 0.9996517 + protein_state + cleaner0 + 2023-07-12T15:48:06Z + DUMMY: + + apo + + + 0.7642773 + evidence + cleaner0 + 2023-07-12T19:18:11Z + DUMMY: + + conformational free-energy landscape + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99953496 + experimental_method + cleaner0 + 2023-07-12T19:14:31Z + MESH: + + molecular dynamics + + + 0.9996784 + experimental_method + cleaner0 + 2023-07-12T19:14:33Z + MESH: + + MD + + + 0.99583435 + experimental_method + cleaner0 + 2023-07-12T19:14:36Z + MESH: + + simulations + + + 0.99943846 + evidence + cleaner0 + 2023-07-12T19:25:12Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.9952549 + experimental_method + cleaner0 + 2023-07-12T19:14:38Z + MESH: + + crystallographic studies + + + 0.9989248 + evidence + cleaner0 + 2023-07-12T19:25:14Z + DUMMY: + + structures + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + + RESULTS + paragraph + 13965 + A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy. Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b). That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. The Na+ ion at Sext was then relocated from the site to the bulk solution (Methods), and this system was then allowed to evolve freely in time. The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out. These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures. The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a). When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). This distortion occludes Sext from the exterior (Fig. 4d, 4h-i) and appears to be induced by the Na+ ion itself, which pulls the carbonyl group of A206 into its coordination sphere (Fig. 4g). With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c). The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e). TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c). Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i). The transporter thus becomes fully outward-open. + + experimental_method + MESH: + cleaner0 + 2023-07-12T15:50:21Z + + MD simulations + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.9995326 + evidence + cleaner0 + 2023-07-12T19:25:16Z + DUMMY: + + structure + + + 0.9996343 + site + cleaner0 + 2023-07-12T19:33:14Z + SO: + + ion-binding sites + + + 0.9993299 + experimental_method + cleaner0 + 2023-07-12T19:14:45Z + MESH: + + simulated + + + 0.99959403 + protein_state + cleaner0 + 2023-07-12T15:51:40Z + DUMMY: + + outward-occluded + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:25:43Z + + high-Na+ + + + 0.99953187 + evidence + cleaner0 + 2023-07-12T19:25:19Z + DUMMY: + + crystal structure + + + 0.99958265 + chemical + cleaner0 + 2023-07-12T15:50:41Z + CHEBI: + + Na+ + + + 0.9526997 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.9144155 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.9161612 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.99989283 + residue_name_number + cleaner0 + 2023-07-12T15:50:33Z + DUMMY: + + D240 + + + 0.99949884 + protein_state + cleaner0 + 2023-07-12T19:49:45Z + DUMMY: + + protonated + + + 0.9997693 + chemical + cleaner0 + 2023-07-12T15:50:46Z + CHEBI: + + water + + + 0.9149831 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.9996277 + chemical + cleaner0 + 2023-07-12T15:50:48Z + CHEBI: + + Na+ + + + 0.92753726 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.9996302 + chemical + cleaner0 + 2023-07-12T15:50:50Z + CHEBI: + + Na+ + + + 0.940987 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.93242574 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.99955755 + experimental_method + cleaner0 + 2023-07-12T19:14:51Z + MESH: + + simulation + + + 0.9996125 + experimental_method + cleaner0 + 2023-07-12T19:14:54Z + MESH: + + simulations + + + 0.99883944 + protein_type + cleaner0 + 2023-07-12T16:56:40Z 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residue_name_number + cleaner0 + 2023-07-12T15:51:11Z + DUMMY: + + T203 + + + 0.999905 + residue_name_number + cleaner0 + 2023-07-12T15:51:16Z + DUMMY: + + F207 + + + 0.7172006 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.9996556 + chemical + cleaner0 + 2023-07-12T15:51:24Z + CHEBI: + + Na+ + + + 0.99990416 + residue_name_number + cleaner0 + 2023-07-12T15:51:07Z + DUMMY: + + A206 + + + 0.9238429 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.99892056 + protein_state + cleaner0 + 2023-07-12T19:50:09Z + DUMMY: + + empty + + + 0.999783 + structure_element + cleaner0 + 2023-07-12T15:41:47Z + SO: + + TM7ab + + + 0.9995744 + structure_element + cleaner0 + 2023-07-12T19:38:15Z + SO: + + α-helix + + + 0.9997913 + structure_element + cleaner0 + 2023-07-12T15:54:55Z + SO: + + TM3 + + + 0.99978286 + structure_element + cleaner0 + 2023-07-12T19:38:20Z + SO: + + TM7 + + + 0.9997751 + chemical + cleaner0 + 2023-07-12T15:51:22Z + CHEBI: + + water + + + 0.9077475 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.99258596 + protein_type + cleaner0 + 2023-07-12T16:56:45Z + MESH: + + transporter + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:50:41Z + + no longer occluded + + + 0.9996489 + chemical + cleaner0 + 2023-07-12T15:51:27Z + CHEBI: + + Na+ + + + 0.92651975 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.9055643 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.9997913 + structure_element + cleaner0 + 2023-07-12T19:38:22Z + SO: + + TM7 + + + 0.999793 + structure_element + cleaner0 + 2023-07-12T19:38:26Z + SO: + + TM2 + + + 0.99963284 + chemical + cleaner0 + 2023-07-12T15:51:31Z + CHEBI: + + Na+ + + + 0.84332246 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.9997956 + structure_element + cleaner0 + 2023-07-12T15:41:41Z + SO: + + TM1 + + + 0.9997918 + structure_element + cleaner0 + 2023-07-12T15:41:33Z + SO: + + TM6 + + + 0.9996016 + protein_state + cleaner0 + 2023-07-12T15:51:39Z + DUMMY: + + outward-occluded + + + 0.993948 + site + cleaner0 + 2023-07-12T19:33:18Z + SO: + + aqueous channel + + + 0.94592416 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.93095547 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.9969367 + protein_type + cleaner0 + 2023-07-12T16:56:48Z + MESH: + + transporter + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T15:52:01Z + + fully outward-open + + + + RESULTS + paragraph + 16116 + To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods). As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. + + 0.9995615 + protein_type + cleaner0 + 2023-07-12T16:56:51Z + MESH: + + exchanger + + + 0.7485032 + experimental_method + cleaner0 + 2023-07-12T19:15:10Z + MESH: + + MD calculations + + + 0.99957466 + protein_state + cleaner0 + 2023-07-12T15:51:40Z + DUMMY: + + outward-occluded + + + 0.9760635 + protein_state + cleaner0 + 2023-07-12T15:52:00Z + DUMMY: + + fully outward-open + + + 0.83825004 + evidence + cleaner0 + 2023-07-12T19:25:54Z + DUMMY: + + free-energy landscape + + + 0.9996829 + chemical + cleaner0 + 2023-07-12T15:52:17Z + CHEBI: + + Na+ + + + site + SO: + cleaner0 + 2023-07-12T15:36:03Z + + Sext + + + 0.97782946 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.96249205 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.99969685 + chemical + cleaner0 + 2023-07-12T15:52:20Z + CHEBI: + + Na+ + + + 0.9897099 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.98150855 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.91960526 + protein_state + cleaner0 + 2023-07-12T19:50:56Z + DUMMY: + + without + + + 0.9996451 + chemical + cleaner0 + 2023-07-12T15:52:15Z + CHEBI: + + Na+ + + + + RESULTS + paragraph + 16630 + These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape. When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c). This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a). Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). The transition to the occluded state in this apo state is again energetically unfeasible. + + 0.99476874 + experimental_method + cleaner0 + 2023-07-12T19:15:21Z + MESH: + + calculations + + + 0.9994221 + chemical + cleaner0 + 2023-07-12T15:52:29Z + CHEBI: + + Na+ + + + 0.99880683 + protein_type + cleaner0 + 2023-07-12T16:56:55Z + MESH: + + transporter + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:18:11Z + + conformational free-energy landscape + + + 0.9963434 + site + cleaner0 + 2023-07-12T19:33:22Z + SO: + + Na+ sites + + + 0.9136924 + evidence + cleaner0 + 2023-07-12T19:26:02Z + DUMMY: + + free-energy minimum + + + 0.99976987 + structure_element + cleaner0 + 2023-07-12T15:41:47Z + SO: + + TM7ab + + + 0.99978584 + structure_element + cleaner0 + 2023-07-12T19:38:32Z + SO: + + TM2 + + + 0.999782 + structure_element + cleaner0 + 2023-07-12T15:54:55Z + SO: + + TM3 + + + 0.99960315 + site + cleaner0 + 2023-07-12T19:33:26Z + SO: + + binding sites + + + 0.9988123 + protein_type + cleaner0 + 2023-07-12T16:56:59Z + MESH: + + transporter + + + 0.9926402 + protein_state + cleaner0 + 2023-07-12T19:51:05Z + DUMMY: + + metastable + + + 0.99951047 + protein_state + cleaner0 + 2023-07-12T19:51:08Z + DUMMY: + + half-open + + + 0.9997415 + structure_element + cleaner0 + 2023-07-12T15:41:47Z + SO: + + TM7ab + + + 0.99897313 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.9991191 + protein_state + cleaner0 + 2023-07-12T19:51:15Z + DUMMY: + + open + + + 0.9996369 + chemical + cleaner0 + 2023-07-12T15:52:39Z + CHEBI: + + Na+ + + + 0.9821666 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:51:28Z + + fully coordinated + + + 0.99978906 + chemical + cleaner0 + 2023-07-12T15:52:36Z + CHEBI: + + water + + + 0.9998319 + residue_name_number + cleaner0 + 2023-07-12T15:51:07Z + DUMMY: + + A206 + + + 0.9997695 + residue_name_number + cleaner0 + 2023-07-12T15:51:03Z + DUMMY: + + F202 + + + 0.99910873 + residue_name_number + cleaner0 + 2023-07-12T15:51:07Z + DUMMY: + + A206 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:39Z + + hydrogen-bond + + + 0.9994727 + protein_state + cleaner0 + 2023-07-12T19:51:40Z + DUMMY: + + semi-open + + + 0.9996475 + chemical + cleaner0 + 2023-07-12T15:52:44Z + CHEBI: + + Na+ + + + 0.9954594 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.99499726 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.99963343 + chemical + cleaner0 + 2023-07-12T15:52:42Z + CHEBI: + + Na+ + + + 0.9996406 + chemical + cleaner0 + 2023-07-12T15:52:47Z + CHEBI: + + Na+ + + + 0.9855064 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.98678976 + protein_state + cleaner0 + 2023-07-12T19:51:48Z + DUMMY: + + metastable + + + 0.954656 + site + cleaner0 + 2023-07-12T19:33:31Z + SO: + + aqueous channel + + + 0.9846718 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.99977773 + structure_element + cleaner0 + 2023-07-12T19:38:37Z + SO: + + TM7 + + + 0.999772 + structure_element + cleaner0 + 2023-07-12T19:38:39Z + SO: + + TM2 + + + 0.99951386 + structure_element + cleaner0 + 2023-07-12T19:38:43Z + SO: + + gating helices + + + 0.99977654 + structure_element + cleaner0 + 2023-07-12T15:41:41Z + SO: + + TM1 + + + 0.9997745 + structure_element + cleaner0 + 2023-07-12T15:41:33Z + SO: + + TM6 + + + 0.999376 + protein_state + cleaner0 + 2023-07-12T19:52:05Z + DUMMY: + + open + + + 0.9921392 + evidence + cleaner0 + 2023-07-12T19:26:05Z + DUMMY: + + free-energy landscape + + + 0.9701071 + protein_state + cleaner0 + 2023-07-12T19:52:10Z + DUMMY: + + partially occupied + + + 0.99963486 + protein_state + cleaner0 + 2023-07-12T19:52:13Z + DUMMY: + + occluded + + + 0.99961114 + chemical + cleaner0 + 2023-07-12T15:52:50Z + CHEBI: + + Na+ + + + 0.9882245 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.9884404 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:52:30Z + + free-energy landscape + + + 0.9990018 + protein_type + cleaner0 + 2023-07-12T16:57:02Z + MESH: + + transporter + + + 0.9993917 + protein_state + cleaner0 + 2023-07-12T19:52:37Z + DUMMY: + + fully open + + + 0.99727106 + site + cleaner0 + 2023-07-12T19:33:33Z + SO: + + aqueous channels + + + 0.99966455 + protein_state + cleaner0 + 2023-07-12T19:52:43Z + DUMMY: + + occluded + + + 0.99969745 + protein_state + cleaner0 + 2023-07-12T15:48:06Z + DUMMY: + + apo + + + + RESULTS + paragraph + 18408 + From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. By contrast, occupancy by H+, which as mentioned are not transported, might be compatible with a semi-open state as well as with the fully open conformation, but should not be conducive to occlusion. + + 0.9996661 + protein_state + cleaner0 + 2023-07-12T19:52:48Z + DUMMY: + + open + + + 0.99954325 + protein_state + cleaner0 + 2023-07-12T19:52:51Z + DUMMY: + + semi-open + + + 0.99965084 + chemical + cleaner0 + 2023-07-12T15:53:08Z + CHEBI: + + Na+ + + + 0.99968684 + chemical + cleaner0 + 2023-07-12T15:52:56Z + CHEBI: + + Na+ + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:53:04Z + + free-energy landscape + + + 0.99940515 + protein_type + cleaner0 + 2023-07-12T16:57:04Z + MESH: + + transporter + + + 0.99961746 + protein_state + cleaner0 + 2023-07-12T19:54:05Z + DUMMY: + + occluded + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:53:49Z + + Na+ occupancy is complete + + + 0.99970305 + chemical + cleaner0 + 2023-07-12T15:53:06Z + CHEBI: + + Na+ + + + 0.99950075 + protein_state + cleaner0 + 2023-07-12T15:53:45Z + DUMMY: + + Ca2+-bound + + + 0.99932635 + protein_type + cleaner0 + 2023-07-12T16:57:06Z + MESH: + + transporter + + + 0.9995554 + protein_state + cleaner0 + 2023-07-12T15:51:48Z + DUMMY: + + outward-open + + + 0.9997084 + chemical + cleaner0 + 2023-07-12T15:53:00Z + CHEBI: + + Ca2+ + + + 0.99964285 + protein_state + cleaner0 + 2023-07-12T19:54:10Z + DUMMY: + + occluded + + + 0.9995422 + protein_state + cleaner0 + 2023-07-12T19:54:24Z + DUMMY: + + semi-open + + + 0.9993727 + protein_state + cleaner0 + 2023-07-12T19:54:29Z + DUMMY: + + inward-open + + + 0.9997597 + chemical + cleaner0 + 2023-07-12T15:53:03Z + CHEBI: + + H+ + + + 0.99954563 + protein_state + cleaner0 + 2023-07-12T19:54:33Z + DUMMY: + + semi-open + + + 0.9995364 + protein_state + cleaner0 + 2023-07-12T19:54:37Z + DUMMY: + + fully open + + + + RESULTS + paragraph + 19431 + To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). + + 0.9993738 + experimental_method + cleaner0 + 2023-07-12T19:15:28Z + MESH: + + enhanced-sampling simulations + + + 0.99746 + protein_state + cleaner0 + 2023-07-12T19:54:46Z + DUMMY: + + Ca2+ + + + 0.9994981 + protein_state + cleaner0 + 2023-07-12T15:53:39Z + DUMMY: + + H+-bound + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T15:37:00Z + + outward-facing + + + protein + PR: + cleaner0 + 2023-07-12T15:33:38Z + + NCX_Mj + + + 0.99955744 + chemical + cleaner0 + 2023-07-12T16:54:48Z + CHEBI: + + Na+ + + + 0.9979315 + evidence + cleaner0 + 2023-07-12T19:26:11Z + DUMMY: + + structures + + + 0.99948627 + protein_state + cleaner0 + 2023-07-12T15:53:45Z + DUMMY: + + Ca2+-bound + + + 0.7329998 + experimental_method + cleaner0 + 2023-07-12T19:15:35Z + MESH: + + calculated + + + 0.99619526 + evidence + cleaner0 + 2023-07-12T19:26:14Z + DUMMY: + + free-energy landscape + + + 0.9994836 + protein_state + cleaner0 + 2023-07-12T15:53:45Z + DUMMY: + + Ca2+-bound + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.99962956 + chemical + cleaner0 + 2023-07-12T16:54:51Z + CHEBI: + + Ca2+ + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.99964595 + chemical + cleaner0 + 2023-07-12T16:54:54Z + CHEBI: + + Ca2+ + + + 0.99966574 + protein_state + cleaner0 + 2023-07-12T19:54:56Z + DUMMY: + + occluded + + + 0.99966586 + protein_state + cleaner0 + 2023-07-12T19:54:59Z + DUMMY: + + dehydrated + + + 0.99929 + protein_type + cleaner0 + 2023-07-12T16:57:11Z + MESH: + + exchanger + + + 0.9995785 + protein_state + cleaner0 + 2023-07-12T19:55:09Z + DUMMY: + + semi-open + + + 0.9996244 + protein_state + cleaner0 + 2023-07-12T19:55:13Z + DUMMY: + + open + + + 0.9996137 + chemical + cleaner0 + 2023-07-12T16:54:58Z + CHEBI: + + Ca2+ + + + 0.99942195 + chemical + cleaner0 + 2023-07-12T16:55:02Z + CHEBI: + + Na+ + + + 0.9995045 + site + cleaner0 + 2023-07-12T19:33:38Z + SO: + + aqueous access channels + + + site + SO: + cleaner0 + 2023-07-12T15:36:03Z + + Sext + + + site + SO: + cleaner0 + 2023-07-12T15:36:30Z + + SCa + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:55:27Z + + protonation + + + 0.9998983 + residue_name_number + cleaner0 + 2023-07-12T15:40:30Z + DUMMY: + + Glu54 + + + 0.9998963 + residue_name_number + cleaner0 + 2023-07-12T15:40:34Z + DUMMY: + + Glu213 + + + 0.999652 + protein_state + cleaner0 + 2023-07-12T19:55:49Z + DUMMY: + + occluded + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + chemical + CHEBI: + cleaner0 + 2023-07-12T19:55:44Z + + protons + + + 0.99950355 + protein_state + cleaner0 + 2023-07-12T15:53:40Z + DUMMY: + + H+-bound + + + 0.99924624 + protein_type + cleaner0 + 2023-07-12T16:57:14Z + MESH: + + exchanger + + + 0.99956053 + protein_state + cleaner0 + 2023-07-12T19:55:53Z + DUMMY: + + semi-open + + + 0.9990244 + protein_state + cleaner0 + 2023-07-12T19:55:59Z + DUMMY: + + low pH + + + 0.9996755 + protein_state + cleaner0 + 2023-07-12T15:48:06Z + DUMMY: + + apo + + + 0.99960977 + evidence + cleaner0 + 2023-07-12T19:26:16Z + DUMMY: + + crystal structure + + + 0.9995214 + chemical + cleaner0 + 2023-07-12T16:55:18Z + CHEBI: + + H+ + + + + RESULTS + paragraph + 20516 + Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. + + 0.9936283 + experimental_method + cleaner0 + 2023-07-12T19:15:38Z + MESH: + + systematic computational analysis + + + 0.99354166 + protein_state + cleaner0 + 2023-07-12T15:37:00Z + DUMMY: + + outward-facing + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.99871165 + protein_type + cleaner0 + 2023-07-12T15:54:34Z + MESH: + + Na+/Ca2+ exchanger + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:56:12Z + + free-energy landscape + + + 0.9996339 + protein_state + cleaner0 + 2023-07-12T19:56:03Z + DUMMY: + + occluded + + + 0.99963164 + protein_state + cleaner0 + 2023-07-12T19:56:51Z + DUMMY: + + occluded + + + 0.9996307 + protein_state + cleaner0 + 2023-07-12T19:56:54Z + DUMMY: + + outward + + + 0.99335724 + protein_state + cleaner0 + 2023-07-12T19:56:56Z + DUMMY: + + inward-open + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:56:26Z + + dehydration + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:56:38Z + + complete occupancy + + + 0.99958426 + site + cleaner0 + 2023-07-12T19:33:42Z + SO: + + binding sites + + + + DISCUSS + title_1 + 21130 + Discussion + + + DISCUSS + paragraph + 21141 + The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized. This control mechanism is functionally crucial, as it precludes the backflow of the species that is transported uphill, and also prevents the dissipation of the driving electrochemical gradients. It is however also non-trivial: antiporters, for example, do not undergo the alternating-access transition without a cargo, but this is precisely how membrane symporters reset their transport cycles. Similarly puzzling is that a given antiporter will undergo this transition upon recognition of substrates of different charge, size and number. Yet, when multiple species are to be co-translocated, by either an antiporter or a symporter, partial occupancies must not be conducive to the alternating-access switch. Here, we have provided novel insights into this intriguing mechanism of conformational control through structural studies and quantitative molecular simulations of a Na+/Ca2+ exchanger. + + 0.9995727 + protein_state + cleaner0 + 2023-07-12T19:57:01Z + DUMMY: + + outward + + + 0.98898214 + protein_state + cleaner0 + 2023-07-12T19:57:04Z + DUMMY: + + inward-open + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T19:57:20Z + + secondary-active + + + 0.9989353 + protein_type + cleaner0 + 2023-07-12T16:57:17Z + MESH: + + transporter + + + 0.9996537 + protein_type + cleaner0 + 2023-07-12T16:57:21Z + MESH: + + antiporters + + + protein_type + MESH: + cleaner0 + 2023-07-12T16:57:35Z + + membrane symporters + + + 0.9996426 + protein_type + cleaner0 + 2023-07-12T16:58:16Z + MESH: + + antiporter + + + 0.99962723 + protein_type + cleaner0 + 2023-07-12T16:58:19Z + MESH: + + antiporter + + + 0.99869835 + protein_type + cleaner0 + 2023-07-12T16:58:21Z + MESH: + + symporter + + + 0.9563345 + site + cleaner0 + 2023-07-12T19:33:47Z + SO: + + alternating-access switch + + + 0.9995649 + experimental_method + cleaner0 + 2023-07-12T19:15:44Z + MESH: + + structural studies + + + 0.99950796 + experimental_method + cleaner0 + 2023-07-12T19:15:46Z + MESH: + + quantitative molecular simulations + + + 0.99925137 + protein_type + cleaner0 + 2023-07-12T15:54:33Z + MESH: + + Na+/Ca2+ exchanger + + + + DISCUSS + paragraph + 22278 + Specifically, our studies of NCX_Mj reveal the mechanism of forward ion exchange (Fig. 7). The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. Here, we demonstrate that conformational changes in the extracellular region of the TM2-TM3 and TM7-TM8 bundle precede and are necessary for the transition, and are associated with ion recognition and/or release. The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. We anticipate that the intracellular ion-exchange process involves analogous conformational changes. + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.9728699 + protein_state + cleaner0 + 2023-07-12T15:37:00Z + DUMMY: + + outward-facing + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.99804515 + protein_state + cleaner0 + 2023-07-12T15:37:26Z + DUMMY: + + inward-facing + + + 0.9996087 + evidence + cleaner0 + 2023-07-12T19:26:22Z + DUMMY: + + crystal structures + + + 0.999475 + protein_type + cleaner0 + 2023-07-12T16:58:26Z + MESH: + + Ca2+/H+ exchangers + + + protein_type + MESH: + cleaner0 + 2023-07-12T15:33:13Z + + NCX + + + 0.9997992 + structure_element + cleaner0 + 2023-07-12T15:41:41Z + SO: + + TM1 + + + 0.99979264 + structure_element + cleaner0 + 2023-07-12T15:41:33Z + SO: + + TM6 + + + 0.9995315 + protein_type + cleaner0 + 2023-07-12T16:58:30Z + MESH: + + transporter + + + 0.6976584 + structure_element + cleaner0 + 2023-07-12T19:38:48Z + SO: + + extracellular region + + + 0.9995459 + structure_element + cleaner0 + 2023-07-12T16:58:38Z + SO: + + TM2-TM3 + + + 0.99947095 + structure_element + cleaner0 + 2023-07-12T19:38:52Z + SO: + + TM7-TM8 bundle + + + 0.9995149 + structure_element + cleaner0 + 2023-07-12T19:38:55Z + SO: + + N-terminal half + + + 0.999793 + structure_element + cleaner0 + 2023-07-12T19:38:59Z + SO: + + TM7 + + + 0.9997991 + structure_element + cleaner0 + 2023-07-12T15:41:47Z + SO: + + TM7ab + + + 0.99979347 + structure_element + cleaner0 + 2023-07-12T19:39:02Z + SO: + + TM2 + + + 0.9997917 + structure_element + cleaner0 + 2023-07-12T15:54:54Z + SO: + + TM3 + + + 0.9997948 + structure_element + cleaner0 + 2023-07-12T15:41:47Z + SO: + + TM7ab + + + 0.9987703 + site + cleaner0 + 2023-07-12T19:33:51Z + SO: + + aqueous channels + + + 0.99962413 + site + cleaner0 + 2023-07-12T19:33:55Z + SO: + + ion-binding sites + + + 0.9997807 + structure_element + cleaner0 + 2023-07-12T19:39:04Z + SO: + + TM7 + + + 0.9996387 + site + cleaner0 + 2023-07-12T19:33:59Z + SO: + + ion-binding sites + + + 0.99978393 + structure_element + cleaner0 + 2023-07-12T15:41:33Z + SO: + + TM6 + + + 0.9997807 + structure_element + cleaner0 + 2023-07-12T15:41:33Z + SO: + + TM6 + + + 0.99978846 + structure_element + cleaner0 + 2023-07-12T15:41:41Z + SO: + + TM1 + + + 0.9989407 + protein_state + cleaner0 + 2023-07-12T15:37:26Z + DUMMY: + + inward-facing + + + + DISCUSS + paragraph + 23451 + The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. These states can only represent a subset among all possible, but they ought to reflect inherent preferences of the transporter, modulated by the experimental conditions. For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). Nonetheless, the calculated free-energy landscapes, derived without knowledge of the experimental data, reassuringly confirm that the crystallized structures correspond to mechanistically relevant, interconverting states. The simulations also demonstrate how this landscape is drastically re-shaped upon each ion-binding event. Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry. We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms. + + 0.99962485 + evidence + cleaner0 + 2023-07-12T19:26:25Z + DUMMY: + + crystal structures + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.9406617 + chemical + cleaner0 + 2023-07-12T15:55:01Z + CHEBI: + + Na+, + + + 0.9671022 + chemical + cleaner0 + 2023-07-12T15:55:03Z + CHEBI: + + Ca2+, + + + 0.9996768 + chemical + cleaner0 + 2023-07-12T16:55:22Z + CHEBI: + + Sr2+ + + + 0.9996743 + chemical + cleaner0 + 2023-07-12T15:55:06Z + CHEBI: + + H+ + + + 0.9984199 + protein_state + cleaner0 + 2023-07-12T19:57:59Z + DUMMY: + + bound + + + 0.9995522 + protein_type + cleaner0 + 2023-07-12T16:58:43Z + MESH: + + exchanger + + + 0.99961483 + protein_type + cleaner0 + 2023-07-12T16:58:46Z + MESH: + + transporter + + + 0.99953866 + evidence + cleaner0 + 2023-07-12T19:26:28Z + DUMMY: + + crystal + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.59371054 + experimental_method + cleaner0 + 2023-07-12T15:55:36Z + MESH: + + LCP + + + 0.9996481 + structure_element + cleaner0 + 2023-07-12T19:39:08Z + SO: + + extracellular half + + + 0.9996382 + structure_element + cleaner0 + 2023-07-12T19:39:10Z + SO: + + gating helices + + + 0.99981445 + structure_element + cleaner0 + 2023-07-12T15:41:33Z + SO: + + TM6 + + + 0.99981624 + structure_element + cleaner0 + 2023-07-12T15:41:41Z + SO: + + TM1 + + + 0.999647 + site + cleaner0 + 2023-07-12T19:34:03Z + SO: + + ion-binding sites + + + 0.9996816 + protein_state + cleaner0 + 2023-07-12T15:48:06Z + DUMMY: + + apo + + + 0.9993656 + protein_state + cleaner0 + 2023-07-12T19:58:06Z + DUMMY: + + low pH + + + 0.9993604 + evidence + cleaner0 + 2023-07-12T19:26:31Z + DUMMY: + + structure + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:28:04Z + + calculated free-energy landscapes + + + 0.7967147 + evidence + cleaner0 + 2023-07-12T19:26:59Z + DUMMY: + + crystallized structures + + + 0.9994253 + experimental_method + cleaner0 + 2023-07-12T19:15:55Z + MESH: + + simulations + + + 0.9996013 + protein_state + cleaner0 + 2023-07-12T19:58:15Z + DUMMY: + + occluded + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + chemical + CHEBI: + cleaner0 + 2023-07-12T16:53:16Z + + Na+ + + + chemical + CHEBI: + cleaner0 + 2023-07-12T16:53:28Z + + Ca2+ + + + 0.999537 + protein_type + cleaner0 + 2023-07-12T16:58:51Z + MESH: + + transporters + + + 0.9981036 + protein_type + cleaner0 + 2023-07-12T16:58:55Z + MESH: + + symporters + + + 0.99957913 + protein_type + cleaner0 + 2023-07-12T16:58:58Z + MESH: + + antiporters + + + 0.99960095 + protein_state + cleaner0 + 2023-07-12T19:58:17Z + DUMMY: + + occluded + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:27:18Z + + protein conformational free-energy landscape + + + + DISCUSS + paragraph + 24901 + In multiple ways, our findings provide an explanation for, existing functional, biochemical and biophysical data for both NCX_Mj and its eukaryotic homologues. The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above. Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d). The crystallographic data also provides the long-sought structural basis for the ‘two-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger. Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. Interestingly, binding of Ca2+ to Smid appears to be also possible, but available evidence indicates that this event transiently blocks the exchange cycle. Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.99939287 + taxonomy_domain + cleaner0 + 2023-07-12T15:32:27Z + DUMMY: + + eukaryotic + + + 0.99909866 + evidence + cleaner0 + 2023-07-12T19:27:22Z + DUMMY: + + ion-binding affinities + + + 0.9746627 + experimental_method + cleaner0 + 2023-07-12T19:15:58Z + MESH: + + crystallographic titrations + + + 0.99950063 + evidence + cleaner0 + 2023-07-12T19:27:24Z + DUMMY: + + Km + + + 0.9809807 + evidence + cleaner0 + 2023-07-12T19:27:27Z + DUMMY: + + K1/2 values + + + experimental_method + MESH: + cleaner0 + 2023-07-12T19:16:21Z + + functional assays + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.99977225 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.99919945 + evidence + cleaner0 + 2023-07-12T19:27:30Z + DUMMY: + + structures + + + 0.9939589 + evidence + cleaner0 + 2023-07-12T19:27:32Z + DUMMY: + + crystallographic data + + + 0.9993855 + taxonomy_domain + cleaner0 + 2023-07-12T15:32:27Z + DUMMY: + + eukaryotic + + + 0.997542 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.9985592 + protein_type + cleaner0 + 2023-07-12T15:54:34Z + MESH: + + Na+/Ca2+ exchanger + + + 0.8415257 + experimental_method + cleaner0 + 2023-07-12T19:16:25Z + MESH: + + crystal titrations + + + 0.82438564 + chemical + cleaner0 + 2023-07-12T15:56:26Z + CHEBI: + + Na+ + + + 0.9428204 + chemical + cleaner0 + 2023-07-12T15:56:28Z + CHEBI: + + Ca2+ + + + 0.79499984 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.7342383 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.9997239 + chemical + cleaner0 + 2023-07-12T15:56:31Z + CHEBI: + + Ca2+ + + + 0.99970806 + chemical + cleaner0 + 2023-07-12T15:56:33Z + CHEBI: + + Na+ + + + 0.9997103 + chemical + cleaner0 + 2023-07-12T15:56:35Z + CHEBI: + + Na+ + + + 0.9997045 + chemical + cleaner0 + 2023-07-12T15:56:37Z + CHEBI: + + Na+ + + + 0.99971414 + chemical + cleaner0 + 2023-07-12T15:56:40Z + CHEBI: + + Na+ + + + 0.9997874 + chemical + cleaner0 + 2023-07-12T15:56:42Z + CHEBI: + + water + + + 0.9237341 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.99967635 + chemical + cleaner0 + 2023-07-12T15:56:45Z + CHEBI: + + Ca2+ + + + 0.9980756 + evidence + cleaner0 + 2023-07-12T19:27:37Z + DUMMY: + + Hill coefficient + + + 0.99949723 + chemical + cleaner0 + 2023-07-12T15:56:47Z + CHEBI: + + Na+ + + + 0.99964225 + chemical + cleaner0 + 2023-07-12T15:56:49Z + CHEBI: + + Ca2+ + + + site + SO: + cleaner0 + 2023-07-12T15:36:03Z + + Sext + + + 0.9995297 + site + cleaner0 + 2023-07-12T19:35:00Z + SO: + + activation site + + + 0.999643 + chemical + cleaner0 + 2023-07-12T15:56:57Z + CHEBI: + + Na+ + + + 0.9996819 + chemical + cleaner0 + 2023-07-12T15:56:54Z + CHEBI: + + Na+ + + + 0.9996492 + chemical + cleaner0 + 2023-07-12T15:56:52Z + CHEBI: + + Na+ + + + 0.99957913 + protein_state + cleaner0 + 2023-07-12T19:58:34Z + DUMMY: + + occluded + + + 0.9996916 + chemical + cleaner0 + 2023-07-12T15:57:00Z + CHEBI: + + Ca2+ + + + 0.970009 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.99948275 + evidence + cleaner0 + 2023-07-12T19:27:39Z + DUMMY: + + structures + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.9992466 + protein_state + cleaner0 + 2023-07-12T19:58:37Z + DUMMY: + + bound to + + + 0.999743 + chemical + cleaner0 + 2023-07-12T15:57:02Z + CHEBI: + + Cd2+ + + + 0.99974525 + chemical + cleaner0 + 2023-07-12T15:57:04Z + CHEBI: + + Mn2+ + + + 0.9707498 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.99974155 + chemical + cleaner0 + 2023-07-12T15:57:07Z + CHEBI: + + Sr2+ + + + 0.9855223 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.9986142 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + 0.99973387 + chemical + cleaner0 + 2023-07-12T15:57:09Z + CHEBI: + + calcium + + + + DISCUSS + paragraph + 26753 + Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices. We interpret these observations as reflecting that the solvent accessibility of the protein interior is diminished upon ion recognition, consistent with our finding that opening and closing of extracellular aqueous pathways to the ion-binding sites depend on ion occupancy state. In addition, the increased compactness of the protein tertiary structure in the occluded state would also slow down the dynamics of the secondary-structure elements, and thus further reduce the HDX rate. Our data would also explain the observation that the reduction in the HDX rate is comparable for Na+ and Ca2+, as well as the finding that the degree of deuterium incorporation remains non-negligible even under saturating ion concentrations. As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.9997053 + chemical + cleaner0 + 2023-07-12T15:57:45Z + CHEBI: + + Ca2+ + + + 0.9996995 + chemical + cleaner0 + 2023-07-12T15:57:47Z + CHEBI: + + Na+ + + + 0.98209786 + experimental_method + cleaner0 + 2023-07-12T19:16:30Z + MESH: + + hydrogen-deuterium exchange + + + 0.97860384 + experimental_method + cleaner0 + 2023-07-12T19:16:34Z + MESH: + + HDX + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.83848304 + protein_state + cleaner0 + 2023-07-12T19:58:42Z + DUMMY: + + presence + + + 0.9987177 + protein_state + cleaner0 + 2023-07-12T19:58:44Z + DUMMY: + + absence of + + + 0.9976303 + protein_state + cleaner0 + 2023-07-12T15:37:00Z + DUMMY: + + outward-facing + + + 0.999722 + chemical + cleaner0 + 2023-07-12T15:57:50Z + CHEBI: + + Ca2+ + + + 0.9997134 + chemical + cleaner0 + 2023-07-12T15:57:53Z + CHEBI: + + Na+ + + + 0.7904098 + evidence + cleaner0 + 2023-07-12T15:58:26Z + DUMMY: + + HDX rate + + + 0.99959975 + structure_element + cleaner0 + 2023-07-12T19:39:14Z + SO: + + α-repeat helices + + + 0.9996377 + site + cleaner0 + 2023-07-12T19:35:08Z + SO: + + ion-binding sites + + + 0.9996598 + protein_state + cleaner0 + 2023-07-12T19:58:54Z + DUMMY: + + occluded + + + evidence + DUMMY: + cleaner0 + 2023-07-12T15:58:25Z + + HDX rate + + + 0.9785595 + evidence + cleaner0 + 2023-07-12T15:58:26Z + DUMMY: + + HDX rate + + + 0.999727 + chemical + cleaner0 + 2023-07-12T15:57:55Z + CHEBI: + + Na+ + + + 0.99972725 + chemical + cleaner0 + 2023-07-12T15:57:57Z + CHEBI: + + Ca2+ + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:28:03Z + + calculated free-energy landscapes + + + 0.99972034 + chemical + cleaner0 + 2023-07-12T15:57:59Z + CHEBI: + + Na+ + + + 0.9997157 + chemical + cleaner0 + 2023-07-12T15:58:02Z + CHEBI: + + Ca2+ + + + 0.99941885 + protein_type + cleaner0 + 2023-07-12T16:59:05Z + MESH: + + transporter + + + 0.999541 + protein_state + cleaner0 + 2023-07-12T19:59:00Z + DUMMY: + + ion-bound + + + 0.977703 + protein_state + cleaner0 + 2023-07-12T19:59:10Z + DUMMY: + + fully open + + + + METHODS + title_1 + 28248 + Methods + + + METHODS + title_2 + 28256 + Protein expression, purification and crystallization + + + METHODS + paragraph + 28309 + NCX_Mj was expressed, purified and crystallized as previously described. Briefly, the NCX_Mj gene with a C-terminal hexa-histidine tag was subcloned into the pQE60 vector and expressed in Escherichia coli BL21(DE3)plysS. Harvested cells were homogenized and incubated in buffer containing 50 mM HEPES pH 7.2, 50 mM NaCl, 12 mM KCl, 10 mM CaCl2, 40 mM DDM. After incubation at room temperature (RT) for 3.5 hours, the supernatant was collected by centrifugation and loaded onto a Talon Co2+ affinity column (Clontech). The non-specifically bound contaminates on the column were washed with buffer containing 50 mM HEPES pH 7.2, 50 mM NaCl, 12 mM KCl, 10 mM CaCl2, 15 mM imidazole, and 1 mM DDM. The bound NCX_Mj was eluted by increasing the imidazole concentration to 300 mM. The eluate was treated with thrombin to remove the hexa-histidine tag and dialyzed against 20 mM HEPES pH 7.2, 50 mM NaCl, 12 mM KCl, 10 mM CaCl2, and 1 mM DDM at RT overnight. After overnight digestion the sample was loaded onto a second Co2+ affinity column to remove any free hexa-histidine tag and contaminant proteins. NCX_Mj in the flow-through was collected and further purified by gel filtration using a Superdex-200 (10/300) column (GE Healthcare) in 20 mM HEPES pH 7.2, 50 mM NaCl, 12 mM KCl, 10 mM CaCl2 and 0.5 mM DDM. The purified protein was then concentrated to 40 mg/ml for crystallization. + + + METHODS + paragraph + 29691 + Native NCX_Mj was crystallized using the lipidic cubic phase (LCP) technique, as previously described. Concentrated NCX_Mj was first reconstituted into 1-oleoyl-rac-glycerol (Sigma) in a protein:lipid weight ratio of 1:1.5, using the two-syringe method. Protein-laden LCP droplets of 35 nL were dispensed onto Corning 96-well protein-crystallization plates and overlaid with 5 μL of precipitant solution containing 40-42% PEG 400, 100 mM MES pH 6.5, 100 mM NaAc. Crystals were observed after 48 hours and grew to full size after 2 weeks. The native crystals belong to space group P212121 with a cell dimension of a=49.5Å, b=72.9Å and c=96.2Å, and contain one subunit per asymmetric unit. As the LCP droplet accounts for less than 1% of the total crystallization volume, the salt composition in the crystallization condition was determined mainly by the overlaying solution, and estimated to have 150 mM Na+ (from MES buffer and NaAc) and 30 μM Ca2+ (from LCP droplet). In these concentration conditions (high Na+ and low Ca2+) Ca2+ does not bind to NCX_Mj (as shown in our crystallographic titration experiments) and thus this native crystal structure represents NCX_Mj in 150 mM Na+. The native crystals were used in all subsequent titration experiments to define low-Na+, Ca2+ and Sr2+-loaded structures. + + + METHODS + paragraph + 31003 + To obtain the apo crystal form, the protein was first purified in a solution containing 20 mM Hepes-Tris pH 7.2, 100 mM NMDG, 10 mM CaCl2 and 0.5 mM DDM. The crystals were obtained in LCP with crystallization solution containing 200 mM KAc, pH 4.0, 35% PEG400. The apo NCX_Mj crystals belong to space group C2 with a cell dimension of a=164.2Å, b=46.8Å, c=97.0 Å and β=106.2°, and contain two protein subunits per asymmetric unit. + + + METHODS + title_2 + 31439 + Crystal titrations + + + METHODS + paragraph + 31458 + Once the native crystals reached their full size, the crystallization solutions overlaying lipid/protein droplets were gradually replaced by titration solutions through multiple steps of solution exchange. In general, 2-3 μL of existing crystallization solutions (normally in 5 μL) were replaced by the same volume of titration solutions, followed by overnight equilibration. The same procedures were repeated 6-10 times until the ion components in the crystal drops reached the targeted concentrations. For titration experiments to define concentration-dependent Na+ binding, the titration solutions contained 100 mM MES-Tris pH 6.5, 44% PEG400, 10 mM EGTA and a 100 mM mixture of NaAc and CsAc, in the following proportions: 100 mM CsAc; 90 mM CsAc and 10 mM NaAc; 80 mM CsAc and 20 mM NaAc; and 100 mM NaAc. Note that Cs+ does not bind NCX proteins and is commonly used as a Na+ substituent to maintain the ionic strength of the solutions. As complete removal of Na+ would deteriorate the crystals, we had to maintain a minimum Na+ concentration of about 2.5 mM in the crystal drops. The final Na+ concentrations in this set of titration experiments were about 2.5, 10, 20 and 100 mM, respectively. It is worth noting that the observed Na+-dependent conformational change occurs while the proteins are in crystal form and embedded in lipid. + + + METHODS + paragraph + 32804 + In the titration experiments carried out to define the mode of divalent cation binding and competition with Na+, the soaking solutions contained 100 mM MES-Tris pH 6.5, 44% PEG400, 100 mM mixture of CsAc and NaAc and various concentrations of XCl2, where X=Ca2+ or Sr2+, in the following proportions: 100 mM CsAc and 10 mM XCl2; 100 mM CsAc and 1 mM XCl2; 100 mM CsAc and 0.1 mM XCl2; 90 mM CsAc, 10mM NaAc and 10mM XCl2; and 100 mM NaAc and 10 mM XCl2. After multiple steps of solution exchanges, the final soaking conditions contained 0.1, 1, or 10 mM of X2+ together with 2.5 mM Na+; or 10 mM X2+ together with 2.5, 10 or 100 mM Na+. + + + METHODS + title_2 + 33441 + Data collection and structure determination + + + METHODS + paragraph + 33485 + After soaking crystals were mounted on 100-μm Mitegen Microloops and frozen in liquid nitrogen. All diffraction data were collected at the Advanced Photon Source (APS) GM/CA-CAT beamlines 23ID-B or 23ID-D using a beam size of 35 μm × 50 μm. Data were processed and scaled using HKL2000 and the structures were determined by molecular replacement in PHASER using our previously published NCX_Mj structure (PDB code 3V5U) as a search model. Model building was completed using COOT and structure refinement was performed with PHENIX. The data sets from crystals soaked in solutions containing 2.5 to 100 mM Na+ were collected using an X-ray wavelength of 1.033Å; the crystal grown with 150 mM Na+, and those soaked with Ca2+ and Sr2+ solutions, were obtained with a wavelength of 0.9793 Å. Lastly, the data from the crystal grown at low pH with no Na+ or Ca2+ were collected with a 2.0-Å wavelength beam. The resulting statistics for data collection and refinement are shown in Tables 2-4. All structure figures were prepared in PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.). The ion passageways in low- and high-Na+ structures as well as the apo state were analyzed using the program CAVER. Due to the variation in diffraction resolution and intensity among crystals, ion-occupancy comparisons were made on the basis of the diffraction data obtained in the titration experiments scaled against a common reference data before map calculation. The NCX_Mj crystal obtained with 2.5 mm Na+ only was used as the reference. + + + METHODS + title_2 + 35042 + Conventional molecular dynamics (MD) simulations + + + METHODS + paragraph + 35091 + Conventional (i.e. not enhanced) MD simulations were carried out with NAMD 2.7-2.9 at constant temperature (298 K), pressure (1 atm), and membrane surface area (~69 Å2 per lipid),and with periodic boundaries in all directions. All calculations used the standard CHARMM27/CMAP force field, except for NBFIX corrections for the interaction between carboxylate-oxygens and Na+ interaction or Ca2+ (Supplementary Note 3, Supplementary Fig. 5). Electrostatic interactions were calculated using PME with a real-space cut-off of 12 Å; the same cut-off distance was used for all van der Waals interactions. + + + METHODS + paragraph + 35693 + Five ion-occupancy states of the transporter were considered, namely with 3 Na+, 2 Na+, 2 H+ or 1 Ca2+, and with no ions bound; in all cases Asp240 is protonated. For the 3×Na+ state, we reanalyzed a 200-ns trajectory of NCX_Mj reported previously. NCX_Mj had been embedded in a POPC lipid membrane using GRIFFIN. The initial configuration of the 2×Na+ state was generated from an equilibrated configuration of 3×Na+ state, from which the Na+ ion at Sext was displaced by means of a slow alchemical transformation that annihilates the bound ion and recreates it in the bulk solution (in the same simulation box). The resulting 2×Na+ state was simulated for 250 ns. Similarly, the state with no Na+ bound was generated from an equilibrated configuration of the 2×Na+ state, from which the remaining Na+ ions were displaced; this state was again simulated for 250 ns. For the 2×H+ state, an initial configuration was generated from an equilibrated configuration of the 3×Na+ state, by gradually annihilating the Na+ ions from the binding sites and creating protonated E54 and E213 side chains; concurrently, acetic acid molecules in the bulk solution (in the same simulation box) were deprotonated and Na+ ions introduced. A second initial configuration of the 2×H+ state was obtained from an equilibrated configuration of the simulation with no ions bound, by slowly transforming deprotonated E54 and E213 into their protonated form, while doing the opposite to acetic acid molecules in the bulk solution. These two initial configurations of the 2×H+ state were then equilibrated for 800 ns. All annihilation/creation simulations were carried out using the FEP module of NAMD; and comprised 32-50 intermediate simulations of 400 ps each, for each transformation. A soft-core van der Waals potential with a radius-shifting coefficient of 2 Å2 was used. The annihilated Na+ ions were confined within their corresponding binding sites using flat-bottom distance restraints. Specifically, the Na+ in Sext was concurrently maintained within 4 Å of E54:Cδ, A206:C, S77:Cβ, T209:Cβ and S210:Cβ. The Na+ ions in SCa and Sint were concurrently kept within 4 Å of the E213:Cδ and A47:C, respectively. The Na+ ions and acetic acid molecules in the bulk solution were kept at a distance greater than 37 Å from the membrane center. Finally, the initial configuration of the Ca2+ state was generated on the basis of the published NCX_Mj X-ray structure by placing Ca2+ in the SCa site and two water molecules coordinating Ca2+ at and near the Smid site, so as to satisfy the expected coordination geometry (see Supplementary Note 2, Supplementary Fig. 3-4). This configuration was initially equilibrated through a series of simulations in which RMSD-based restraints of gradually diminishing strength were applied to the protein Cα atoms as well the side-chains involved in Ca2+ coordination. A 250 ns equilibration was then carried without any restraints. + + + METHODS + title_2 + 38656 + Enhanced-sampling MD simulations + + + METHODS + paragraph + 38689 + Free-energy landscapes were calculated using Bias-Exchange Well-Tempered Metadynamics (BE-WT-MetaD), using GROMACS4.5.5/PLUMED. The force-field and simulation conditions were equivalent to those employed in the unbiased MD simulations. The accumulated simulation time for each of the ion-occupancy states studied was 1.6 μs. Each of these calculations consisted of 16 concurrent, interdependent simulations (or replicas); in 15 of these replicas, a WT-MetaD biasing potential was applied on different subsets of collective variables, as specified below, while the remaining replica was unbiased. Attempts to exchange coordinate configurations among replicas were made every 2-5 ps, using the Metropolis criterion. The inputs for each calculation were equilibrated configurations extracted from the unbiased MD simulations. + + + METHODS + paragraph + 39513 + The choice of collective variables to be biased in the BE-WT-MetaD simulations was also based on analysis of the unbiased MD trajectories (Fig. 4g-i). Specifically, to enhance the reversible opening and closing of the water channels reaching from the extracellular bulk solution into either the Sext or the SCa binding sites, we employed the following time-dependent collective variable (Fig. 4h-i): where ri denotes the distance between the oxygen atom of each water molecule in the system i and the center of the binding site considered (V1 for Sext,V2 for SCa), and β is 10-100 nm. When the binding site was occupied, the ion was used to define its center. If the site was empty, its center was defined as the center-of-mass of the oxygen atoms coordinating the ion if bound. Bound water molecules at or near Smid (coordinating the Na+ or Ca2+ ions) were not considered. + + + METHODS + paragraph + 40391 + To enhance the reversible formation and disruption of selected backbone hydrogen bonds in TM7ab (Fig. 4g), we used an analogous collective variable: In this case, the index i denotes atoms P202:O and T203:O, while the index j denotes atoms A206:N and P207:N. To preclude the artificial unraveling of TM7ab driven by this bias, an upper-bound V3max equal to 0.7 nm was imposed with a boundary potential of the form k (V3(t) – V3max) if V3(t) > V3max, where k = 105 nm−4 kJ/mol. In addition, to control the bending and straightening of TM7ab more globally, we used the following path-collective variables: where d1 and d2 denote the mean-square-differences between the conformation of TM7ab and either the straight or bent conformations, respectively, and λ = 100 nm−2. Note that V 4 is by definition confined between a lower (V4 ~ 1) and upper bound (V4 ~ 2); to confine the exploration of V5, an upper value V5max of 0.020-0.025 nm2 was imposed with boundary potential of the form k (V5(t) – V5max) if V5(t) > V5max, where k = 1011 nm−8 kJ/mol. The mean-square-differences d1 and d2 comprise the backbone atoms of residues 198-211 as well as the side chain carbon atoms of residues P207 and L211. + + + METHODS + paragraph + 41601 + A boundary potential was also applied to confine the ions and water molecule bound to Sext, SCa, Sint and Smid to their corresponding binding sites. Specifically, the variable confined was: where ri denotes the distance between the ion and each of its coordinating oxygen atoms; r0 was set to 0.24 nm for the Na+ ions, and to 0.30 nm for Ca2+. Note that the upper-bound value of Vc is, by definition, approximately the coordination number in the bound state, whereas Vc becomes 0 as the ion becomes unbound. For the Na+ ion bound to Sext and Sint, therefore, a lower bound value Vcmin = 4.3 was imposed with potential of the form k (Vc(t) – Vcmin) when Vc(t) < Vcmin, where k = 2500 kJ/mol. An analogous restraint was used for the Na+ ion at SCa, with Vcmin = 4.75. Similarly, for the Ca2+ ion at SCa a lower bound value Vcmin = 7.4 was imposed with a potential of the form k (Vc(t) – Vcmin) when Vc(t) < Vcmin, with k = 400 kJ/mol. Note that these restraints do not perturb the chemical structure of the ion-coordination sphere when the ion is bound, i.e. Vc(t) > Vcmin. The displacement of the bound water molecules in the ion-coordination sphere by equivalent water molecules in the solvent was prevented similarly. + + + METHODS + paragraph + 42825 + The specific sets of collective variables biased in each of the replica WT-MetaD simulations, as well as further details on the biasing potentials introduced, are specified in Supplementary Table 1. + + + METHODS + title_2 + 43024 + Derivation of conformational free-energy landscapes + + + METHODS + paragraph + 43076 + To translate the data gathered in the BE-WT-MetaD simulations into conformational free-energy landscapes, we sought to identify a low-dimensional representation of the data that is nevertheless also intuitive and representative. We ultimately settled on two structure-based descriptors of the degree of opening of the each of the aqueous channels leading to the ion-binding sites (Fig. 4), defined as: where rij denotes a set of pairwise distances for specific Cα atoms in the protein, for a given simulation snapshot. For S1, index i refers to the Cα of residues 198 to 209 in TM7, while index j refers to those in residues 66 to 80 and 290 to 297, in TM3 and TM10, respectively. For S2, index i refers to the Cα of residues 51 to 64 in TM2, while index j refers to those in residues 177 to 193 and 198 to 209, in TM6 and TM7, respectively. Therefore, S1 describes the effective separation between TM7 and TM3/TM10, on the extracellular half of the protein, and thus reports on the accessibility to the Sext site. Analogously, S2 measures the separation between TM2 and TM6-TM7, also on the extracellular side, and thus reports on the accessibility to SCa. + + + METHODS + paragraph + 44247 + The conformational free energy of NCX_Mj as a function of S1 and S2 was then computed for each ion-occupancy state separately (Figs. 5a, 6a). These landscapes were obtained through reweighting of the biased probability distribution from the BE-WT-MetaD sampling, using the WHAM method; through this approach we combine the statistics gathered in all replicas, and can consider alternate free-energy projections. + + + METHODS + paragraph + 44659 + To correct the landscape calculated for the Ca2+-bound state (Fig. 6a) on account of the excess amount of charge transferred from the ion to the protein (Supplementary Note 4, Supplementary Fig. 6), we reprocessed all the sampling obtained during the original BE-WT-MetaD simulations, introducing in the WHAM equations a re-weighting factor w for each configuration X: where U denotes the ‘uncorrected’ CHARMM27/NBFIX potential-energy function, and Uc denotes the corrected function. To calculate Uc (X), the charge of Ca2+ was reduced to +1.8e from its standard value of +2e, and the difference was distributed among the surrounding protein residues (as specified in Supplementary Note 4). To minimally alter the charge-distribution used in the original CHARMM27 force-field, the charge added to each protein atom was proportional to the absolute value of its uncorrected charge. + + + METHODS + paragraph + 45545 + The statistical errors for all free-energy landscapes are provided in Supplementary Fig. 7. + + + METHODS + title_2 + 45637 + Derivation of representative structures and water-density maps + + + METHODS + paragraph + 45700 + Representative structures and water-density iso-surfaces (Fig. 5b-c, Fig. 6b-c) were derived for each BE-WT-MetaD simulation by clustering all sampling in the multi-dimensional space of V1, V2, and V4 (Eq. 1-3), plus a descriptor S3 of the proximity between TM6-TM7 and TM2-TM3/TM10, on the extracellular side of the protein. More precisely: where r0 = 7.5 Å, and rij denotes a specific set of pairwise Cα distances, for a given simulation snapshot. Similarly to S1 and S2 (Eq. 6), index i refers to Cα atoms in the extracellular halves of TM6 and TM7, while index j refers to Cα atoms in the extracellular halves of TM2, TM3, and TM10. We thus obtained ~2,000 clusters for each of the simulation systems (using RMSD cut-off values of 1.3 Å for V1 and V2, 0.1 Å for V4; and 6.25 Å for S3). Using the WHAM equations, we calculated the relative free energy of each of these clusters, and then identified the major basins in this space with the MCL method (with p = 1.4.) Water occupancy maps were calculated for each of these major free-energy basins, only using the sampling gathered by the unbiased replicas. + + + SUPPL + title_1 + 46822 + Supplementary Material + + + SUPPL + footnote + 46845 + Author Contributions + + + SUPPL + footnote + 46866 + J.L. and Y.J. designed the experimental studies and analyzed the resulting data. J.L., C.L. and Y.H. performed the experimental research. F.M and J.D.F.G. designed the computational research and analyzed the corresponding data. F.M. performed the computational work. J.L., Y.J. F.M. and J.D.F.G. wrote the paper. The authors declare no competing financial interests. + + + SUPPL + footnote + 47233 + Accession codes + + + SUPPL + footnote + 47249 + Atomic coordinates and structural factors have been deposited in the Protein Data Bank, with accession numbers 5HWX, 5HWY, 5HXC, 5HXE, 5HYA, 5HXH, 5HXS and 5HXR, as specified in Tables 2-4. + + + REF + title + 47439 + References + + + 763 + 854 + surname:Blaustein;given-names:MP + surname:Lederer;given-names:WJ + 10390518 + REF + Physiol Rev + ref + 79 + 1999 + 47450 + Sodium/calcium exchange: its physiological implications. + + + 155 + 203 + surname:DiPolo;given-names:R + surname:Beauge;given-names:L + 16371597 + REF + Physiol Rev + ref + 86 + 2006 + 47507 + Sodium/calcium exchanger: influence of metabolic regulation on ion carrier interactions. + + + 1047 + 1058 + surname:Clapham;given-names:DE + 18083096 + REF + Cell + ref + 131 + 2007 + 47596 + Calcium signaling. + + + 517 + 529 + surname:Berridge;given-names:MJ + surname:Bootman;given-names:MD + surname:Roderick;given-names:HL + 12838335 + REF + Nat Rev Mol Cell Biol + ref + 4 + 2003 + 47615 + Calcium signalling: dynamics, homeostasis and remodelling. + + + 759 + 768 + surname:Hilgemann;given-names:DW + 8842214 + REF + Biophys J + ref + 71 + 1996 + 47674 + Unitary cardiac Na+, Ca2+ exchange current magnitudes determined from channel- like noise and charge movements of ion transport. + + + 715 + 718 + surname:Hilgemann;given-names:DW + surname:Nicoll;given-names:DA + surname:Philipson;given-names:KD + 1876186 + REF + Nature + ref + 352 + 1991 + 47803 + Charge movement during Na+ translocation by native and cloned cardiac Na+/Ca2+ exchanger. + + + 7733 + 7739 + surname:Reeves;given-names:JP + surname:Hale;given-names:CC + 6736024 + REF + J Biol Chem + ref + 259 + 1984 + 47893 + The stoichiometry of the cardiac sodium-calcium exchange system. + + + 285 + 312 + surname:Blaustein;given-names:MP + surname:Russell;given-names:JM + 1159780 + REF + J Membr Biol + ref + 22 + 1975 + 47958 + Sodium-calcium exchange and calcium-calcium exchange in internally dialyzed squid giant axons. + + + C499 + 504 + surname:Rasgado-Flores;given-names:H + surname:Blaustein;given-names:MP + 3578502 + REF + Am J Physiol + ref + 252 + 1987 + 48053 + Na/Ca exchange in barnacle muscle cells has a stoichiometry of 3 Na+/1 Ca2+. + + + 596 + 597 + surname:Kimura;given-names:J + surname:Noma;given-names:A + surname:Irisawa;given-names:H + 3945347 + REF + Nature + ref + 319 + 1986 + 48130 + Na-Ca exchange current in mammalian heart cells. + + + 544 + 548 + surname:Kang;given-names:TM + surname:Hilgemann;given-names:DW + 14765196 + REF + Nature + ref + 427 + 2004 + 48179 + Multiple transport modes of the cardiac Na+/Ca2+ exchanger. + + + 969 + 970 + surname:Jardetzky;given-names:O + 5968307 + REF + Nature + ref + 211 + 1966 + 48239 + Simple allosteric model for membrane pumps. + + + 242 + 245 + surname:Hilgemann;given-names:DW + 2314460 + REF + Nature + ref + 344 + 1990 + 48283 + Regulation and deregulation of cardiac Na+-Ca2+ exchange in giant excised sarcolemmal membrane patches. + + + 3870 + 3874 + surname:Matsuoka;given-names:S + surname:Nicoll;given-names:DA + surname:Reilly;given-names:RF + surname:Hilgemann;given-names:DW + surname:Philipson;given-names:KD + 8483905 + REF + Proc Natl Acad Sci USA + ref + 90 + 1993 + 48387 + Initial localization of regulatory regions of the cardiac sarcolemmal Na+-Ca2+ exchanger. + + + 111 + 133 + surname:Philipson;given-names:KD + surname:Nicoll;given-names:DA + 10845086 + REF + Annu Rev Physiol + ref + 62 + 2000 + 48477 + Sodium-calcium exchange: a molecular perspective. + + + 13385 + 13391 + surname:Nicoll;given-names:DA + surname:Hryshko;given-names:LV + surname:Matsuoka;given-names:S + surname:Frank;given-names:JS + surname:Philipson;given-names:KD + 8662775 + REF + J Biol Chem + ref + 271 + 1996 + 48527 + Mutation of amino acid residues in the putative transmembrane segments of the cardiac sarcolemmal Na+-Ca2+ exchanger. + + + 11 + 18 + surname:Nicoll;given-names:DA + surname:Ottolia;given-names:M + surname:Philipson;given-names:KD + 12502529 + REF + Ann N Y Acad Sci + ref + 976 + 2002 + 48645 + Toward a topological model of the NCX1 exchanger. + + + 8585 + 8591 + surname:Ren;given-names:X + surname:Nicoll;given-names:DA + surname:Xu;given-names:L + surname:Qu;given-names:Z + surname:Philipson;given-names:KD + 20735122 + REF + Biochemistry + ref + 49 + 2010 + 48695 + Transmembrane segment packing of the Na+/Ca2+ exchanger investigated with chemical cross-linkers. + + + 1692 + 1703 + surname:Cai;given-names:X + surname:Lytton;given-names:J + 15163769 + REF + Mol Biol Evol + ref + 21 + 2004 + 48793 + The cation/Ca2+ exchanger superfamily: phylogenetic analysis and structural implications. + + + 686 + 690 + surname:Liao;given-names:J + 22323814 + REF + Science + ref + 335 + 2012 + 48883 + Structural insight into the ion-exchange mechanism of the sodium/calcium exchanger. + + + 276 + 284 + surname:Almagor;given-names:L + 25218934 + REF + Cell Calcium + ref + 56 + 2014 + 48967 + Functional asymmetry of bidirectional Ca2+-movements in an archaeal sodium- calcium exchanger (NCX_Mj). + + + E5354 + 5362 + surname:Marinelli;given-names:F + 25468964 + REF + Proc Natl Acad Sci USA + ref + 111 + 2014 + 49071 + Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation. + + + 79 + 111 + surname:Blaustein;given-names:MP + surname:Santiago;given-names:EM + 901903 + REF + Biophys J + ref + 20 + 1977 + 49152 + Effects of internal and external cations and of ATP on sodium- calcium and calcium-calcium exchange in squid axons. + + + 63 + 68 + surname:Trosper;given-names:TL + surname:Philipson;given-names:KD + 6849912 + REF + Biochim Biophys Acta + ref + 731 + 1983 + 49268 + Effects of divalent and trivalent cations on Na+-Ca2+ exchange in cardiac sarcolemmal vesicles. + + + 819 + 835 + surname:DiPolo;given-names:R + surname:Beauge;given-names:L + 2362183 + REF + J Gen Physiol + ref + 95 + 1990 + 49364 + Asymmetrical properties of the Na-Ca exchanger in voltage-clamped, internally dialyzed squid axons under symmetrical ionic conditions. + + + 963 + 1001 + surname:Matsuoka;given-names:S + surname:Hilgemann;given-names:DW + 1336540 + REF + J Gen Physiol + ref + 100 + 1992 + 49499 + Steady-state and dynamic properties of cardiac sodium-calcium exchange. Ion and voltage dependencies of the transport cycle. + + + 1129 + 1145 + surname:Miura;given-names:Y + surname:Kimura;given-names:J + 2549177 + REF + J Gen Physiol + ref + 93 + 1989 + 49624 + Sodium-calcium exchange current. Dependence on internal Ca2+ and Na+ and competitive binding of external Na+ and Ca2+. + + + 107 + 110 + surname:Waight;given-names:AB + 23685453 + REF + Nature + ref + 499 + 2013 + 49743 + Structural basis for alternating access of a eukaryotic calcium/proton exchanger. + + + 168 + 172 + surname:Nishizawa;given-names:T + 23704374 + REF + Science + ref + 341 + 2013 + 49825 + Structural basis for the counter-transport mechanism of a H+/Ca2+ exchanger. + + + 11367 + 11372 + surname:Wu;given-names:M + 23798403 + REF + Proc Natl Acad Sci USA + ref + 110 + 2013 + 49902 + Crystal structure of Ca2+/H+ antiporter protein YfkE reveals the mechanisms of Ca2+ efflux and its pH regulation. + + + 20753 + surname:Giladi;given-names:M + 26876271 + REF + Sci. Rep + ref + 6 + 2016 + 50016 + Asymmetric preorganization of inverted pair residues in the sodium-calcium exchanger. + + + 38571 + 38580 + surname:Iwamoto;given-names:T + surname:Uehara;given-names:A + surname:Imanaga;given-names:I + surname:Shigekawa;given-names:M + 10967097 + REF + J. Biol. Chem + ref + 275 + 2000 + 50102 + The Na+/Ca2+ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca2+ affinity. + + + 7500 + 7505 + surname:John;given-names:SA + surname:Liao;given-names:J + surname:Jiang;given-names:Y + surname:Ottolia;given-names:M + 23589872 + REF + Proc. Natl. Acad. Sci. USA + ref + 110 + 2013 + 50265 + The cardiac Na+-Ca2+ exchanger has two cytoplasmic ion permeation pathways. + + + 1061 + 1069 + surname:Ottolia;given-names:M + surname:Nicoll;given-names:DA + surname:Philipson;given-names:KD + 15519995 + REF + J. Biol. Chem + ref + 280 + 2005 + 50341 + Mutational analysis of the alpha-1 repeat of the cardiac Na+-Ca2+ exchanger. + + + 3178 + 3182 + surname:Reeves;given-names:JP + surname:Sutko;given-names:JL + 6826556 + REF + J Biol Chem + ref + 258 + 1983 + 50418 + Competitive interactions of sodium and calcium with the sodium-calcium exchange system of cardiac sarcolemmal vesicles. + + + 706 + 31 + surname:Caffrey;given-names:M + surname:Cherezov;given-names:V + 19390528 + REF + Nat Protoc + ref + 4 + 2009 + 50538 + Crystallizing membrane proteins using lipidic mesophases. + + + 307 + 326 + surname:Otwinowski;given-names:Z + surname:Minor;given-names:W + REF + Methods Enzymol + ref + 276 + 1997 + 50596 + Processing of X-ray diffraction data collected in oscillation mode. + + + 658 + 674 + surname:McCoy;given-names:AJ + 19461840 + REF + J Appl Crystallogr + ref + 40 + 2007 + 50664 + Phaser crystallographic software. + + + 2126 + 32 + surname:Emsley;given-names:P + surname:Cowtan;given-names:K + 15572765 + REF + Acta Crystallogr D Biol Crystallogr + ref + 60 + 2004 + 50698 + Coot: model-building tools for molecular graphics. + + + 213 + 21 + surname:Adams;given-names:PD + 20124702 + REF + Acta Crystallogr D Biol Crystallogr + ref + 66 + 2010 + 50749 + PHENIX: a comprehensive Python-based system for macromolecular structure solution. + + + 316 + surname:Petrek;given-names:M + 16792811 + REF + BMC Bioinformatics + ref + 7 + 2006 + 50832 + CAVER: a new tool to explore routes from protein clefts, pockets and cavities. + + + 1781 + 1802 + surname:Phillips;given-names:JC + 16222654 + REF + J. Comp. Chem + ref + 26 + 2005 + 50911 + Scalable molecular dynamics with NAMD. + + + 3586 + 3616 + surname:MacKerell;given-names:AD + 24889800 + REF + J. Phys. Chem. B + ref + 102 + 1998 + 50950 + All-atom empirical potential for molecular modeling and dynamics studies of proteins. + + + 1400 + 1415 + surname:Mackerell;given-names:AD + surname:Feig;given-names:M + surname:Brooks;given-names:CL + 15185334 + REF + J. Comp. Chem + ref + 25 + 2004 + 51036 + Extending the treatment of backbone energetics in protein force fields: Limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. + + + 1167 + 1176 + surname:Staritzbichler;given-names:R + surname:Anselmi;given-names:C + surname:Forrest;given-names:LR + surname:Faraldo-Gomez;given-names:JD + REF + J. Chem. Theor. Comp + ref + 7 + 2011 + 51238 + GRIFFIN: A versatile methodology for optimization of protein-lipid interfaces for membrane protein simulations. + + + 4553 + 9 + surname:Piana;given-names:S + surname:Laio;given-names:A + 17419610 + REF + J. Phys. Chem. B + ref + 111 + 2007 + 51350 + A bias-exchange approach to protein folding. + + + 2247 + 2254 + surname:Branduardi;given-names:D + surname:Bussi;given-names:G + surname:Parrinello;given-names:M + REF + J. Chem. Theor. Comp + ref + 8 + 2012 + 51395 + Metadynamics with adaptive gaussians. + + + 435 + 447 + surname:Hess;given-names:B + surname:Kutzner;given-names:C + surname:van der Spoel;given-names:D + surname:Lindahl;given-names:E + REF + J. Chem. Theor. Comp + ref + 4 + 2008 + 51433 + GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. + + + 1961 + 1972 + surname:Bonomi;given-names:M + REF + Comp. Phys. Commun + ref + 180 + 2009 + 51527 + PLUMED: A portable plugin for free-energy calculations with molecular dynamics. + + + 054103 + surname:Branduardi;given-names:D + surname:Gervasio;given-names:FL + surname:Parrinello;given-names:M + 17302470 + REF + J. Chem. Phys + ref + 126 + 2007 + 51607 + From A to B in free energy space. + + + 1011 + 1021 + surname:Kumar;given-names:S + surname:Bouzida;given-names:D + surname:Swendsen;given-names:RH + surname:Kollman;given-names:PA + surname:Rosenberg;given-names:JM + REF + J. Comp. Chem + ref + 13 + 1992 + 51641 + The Weighted Histogram Analysis Method for free-energy calculations on biomolecules .1. The method. + + + 203 + 211 + surname:Biarnes;given-names:X + surname:Pietrucci;given-names:F + surname:Marinelli;given-names:F + surname:Laio;given-names:A + REF + Comp. Phys. Commun + ref + 183 + 2012 + 51741 + METAGUI: A VMD interface for analyzing metadynamics and molecular dynamics simulations. + + + E3372 + E3380 + surname:Corbi-Verge;given-names:C + 23959873 + REF + Proc. Natl. Acad. Sci. USA + ref + 110 + 2013 + 51829 + Two-state dynamics of the SH3-SH2 tandem of Abl kinase and the allosteric role of the N-cap. + + + 236 + 240 + surname:Daura;given-names:X + REF + Angew. Chem. Int. Edit + ref + 38 + 1999 + 51922 + Peptide folding: When simulation meets experiment. + + + in press + surname:Branduardi;given-names:D + surname:Marinelli;given-names:F + REF + J. Comput. Chem + ref + 2015 + 51973 + Faraldo-Gómez, J.D. Atomic-resolution dissection of the energetics and mechanism of isomerization of hydrated ATP-Mg through the SOMA string method. + + + 1575 + 1584 + surname:Enright;given-names:AJ + surname:Van Dongen;given-names:S + surname:Ouzounis;given-names:CA + 11917018 + REF + Nucleic Acids Res + ref + 30 + 2002 + 52123 + An efficient algorithm for large-scale detection of protein families. + + + nihms-779827-f0001.jpg + F1 + FIG + fig_caption + 52193 + Na+ binding to outward-facing NCX_Mj. (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. N- and C-terminal halves are colored yellow and cyan, respectively. Colored spheres represent the bound Na+ (green) and water (red). (b) Structural details and definition of the four central binding sites. Only residues flanking these sites are shown for clarity (same for all other figures). The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). Further details are shown in Supplementary Fig. 1. (c) Concentration-dependent change in Na+ occupancy (see also Table 1). All Fo – Fc ion-omit maps are calculated to 2.4 Å and contoured at 3σ for comparison. The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively). At 20 mM Na+, both conformations co-exist. No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites. + + 0.99970114 + chemical + cleaner0 + 2023-07-12T16:07:44Z + CHEBI: + + Na+ + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T15:37:00Z + + outward-facing + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.9735729 + evidence + cleaner0 + 2023-07-12T19:28:28Z + DUMMY: + + structure + + + 0.99929404 + protein_state + cleaner0 + 2023-07-12T19:59:19Z + DUMMY: + + native + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T15:37:00Z + + outward-facing + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.99713457 + experimental_method + cleaner0 + 2023-07-12T19:16:50Z + MESH: + + crystals grown + + + 0.9996817 + chemical + cleaner0 + 2023-07-12T16:07:46Z + CHEBI: + + Na+ + + + 0.9996953 + chemical + cleaner0 + 2023-07-12T16:07:48Z + CHEBI: + + Na+ + + + 0.9995254 + chemical + cleaner0 + 2023-07-12T16:07:50Z + CHEBI: + + water + + + 0.99958056 + site + cleaner0 + 2023-07-12T19:35:13Z + SO: + + central binding sites + + + 0.99955356 + evidence + cleaner0 + 2023-07-12T19:28:30Z + DUMMY: + + electron density + + + 0.9991879 + evidence + cleaner0 + 2023-07-12T19:28:33Z + DUMMY: + + Fo-Fc ion omit map + + + 0.7673661 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.9964263 + chemical + cleaner0 + 2023-07-12T16:07:52Z + CHEBI: + + water + + + 0.9494199 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.9231161 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.8831704 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.99964094 + chemical + cleaner0 + 2023-07-12T16:07:54Z + CHEBI: + + Na+ + + + 0.9990984 + chemical + cleaner0 + 2023-07-12T16:07:57Z + CHEBI: + + Na+ + + + 0.999241 + evidence + cleaner0 + 2023-07-12T19:28:36Z + DUMMY: + + Fo – Fc ion-omit maps + + + 0.9998784 + residue_name_number + cleaner0 + 2023-07-12T15:51:07Z + DUMMY: + + A206 + + + 0.999323 + chemical + cleaner0 + 2023-07-12T16:07:59Z + CHEBI: + + Na+ + + + 0.9931774 + protein_state + cleaner0 + 2023-07-12T19:59:23Z + DUMMY: + + partially open + + + 0.99960274 + protein_state + cleaner0 + 2023-07-12T19:59:25Z + DUMMY: + + occluded + + + 0.9996083 + chemical + cleaner0 + 2023-07-12T16:08:03Z + CHEBI: + + Na+ + + + 0.9996876 + chemical + cleaner0 + 2023-07-12T16:08:01Z + CHEBI: + + Na+ + + + 0.99939406 + chemical + cleaner0 + 2023-07-12T16:08:05Z + CHEBI: + + water + + + 0.7865566 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.6614779 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.6314445 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + + nihms-779827-f0002.jpg + F2 + FIG + fig_caption + 53361 + Na+-occupancy dependent conformational change in NCX_Mj. (a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations. (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail. (c) Close-up view of the Na+-binding sites. The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere. Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen. (d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+]. Putative solvent channels are represented as light-purple surfaces. + + 0.9972726 + chemical + cleaner0 + 2023-07-12T16:09:12Z + CHEBI: + + Na+ + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.9996729 + experimental_method + cleaner0 + 2023-07-12T19:16:58Z + MESH: + + Superimposition + + + protein + PR: + cleaner0 + 2023-07-12T15:33:39Z + + NCX_Mj + + + 0.9995926 + evidence + cleaner0 + 2023-07-12T19:28:38Z + DUMMY: + + crystal structures + + + 0.99920523 + chemical + cleaner0 + 2023-07-12T16:09:15Z + CHEBI: + + Na+ + + + 0.9993622 + site + cleaner0 + 2023-07-12T19:35:19Z + SO: + + interface + + + 0.99981266 + structure_element + cleaner0 + 2023-07-12T15:41:33Z + SO: + + TM6 + + + 0.99981123 + structure_element + cleaner0 + 2023-07-12T15:41:47Z + SO: + + TM7ab + + + protein + PR: + cleaner0 + 2023-07-12T15:33:40Z + + NCX_Mj + + + 0.99962425 + evidence + cleaner0 + 2023-07-12T19:28:40Z + DUMMY: + + structures + + + 0.9994711 + chemical + cleaner0 + 2023-07-12T16:09:20Z + CHEBI: + + Na+ + + + 0.99940884 + evidence + cleaner0 + 2023-07-12T19:28:43Z + DUMMY: + + structure + + + 0.8585934 + protein_state + cleaner0 + 2023-07-12T19:59:42Z + DUMMY: + + low + + + 0.999156 + chemical + cleaner0 + 2023-07-12T16:09:17Z + CHEBI: + + Na+ + + + 0.9996513 + site + cleaner0 + 2023-07-12T19:35:22Z + SO: + + Na+-binding sites + + + site + SO: + cleaner0 + 2023-07-12T15:36:03Z + + Sext + + + 0.9996259 + evidence + cleaner0 + 2023-07-12T19:28:45Z + DUMMY: + + structure + + + 0.9406699 + protein_state + cleaner0 + 2023-07-12T19:59:50Z + DUMMY: + + low + + + 0.9995061 + chemical + cleaner0 + 2023-07-12T16:09:22Z + CHEBI: + + Na+ + + + 0.99989617 + residue_name_number + cleaner0 + 2023-07-12T15:51:07Z + DUMMY: + + A206 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:39Z + + coordinates + + + 0.99944687 + chemical + cleaner0 + 2023-07-12T16:09:24Z + CHEBI: + + Na+ + + + site + SO: + cleaner0 + 2023-07-12T15:36:03Z + + Sext + + + 0.9996469 + site + cleaner0 + 2023-07-12T19:35:26Z + SO: + + ion binding sites + + + 0.9995679 + evidence + cleaner0 + 2023-07-12T19:28:48Z + DUMMY: + + structures + + + 0.7218978 + protein_state + cleaner0 + 2023-07-12T19:59:46Z + DUMMY: + + high + + + 0.56791896 + protein_state + cleaner0 + 2023-07-12T19:59:48Z + DUMMY: + + low + + + 0.99950194 + chemical + cleaner0 + 2023-07-12T16:09:26Z + CHEBI: + + Na+ + + + 0.99904966 + site + cleaner0 + 2023-07-12T19:35:28Z + SO: + + solvent channels + + + + nihms-779827-f0003.jpg + F3 + FIG + fig_caption + 54284 + Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). Residues involved in Sr2+ coordination are labeled. There are no significant changes in the side-chains involved in ion coordination, relative to the Na+-bound state. T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms. High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel). The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison. White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. + + 0.9996723 + protein_state + cleaner0 + 2023-07-12T15:48:06Z + DUMMY: + + apo + + + 0.9992555 + evidence + cleaner0 + 2023-07-12T19:28:51Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-07-12T15:33:40Z + + NCX_Mj + + + 0.999681 + chemical + cleaner0 + 2023-07-12T16:42:37Z + CHEBI: + + Sr2+ + + + 0.38059822 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.94249403 + experimental_method + cleaner0 + 2023-07-12T19:17:06Z + MESH: + + crystals titrated + + + 0.9996698 + chemical + cleaner0 + 2023-07-12T16:42:40Z + CHEBI: + + Sr2+ + + + 0.9996922 + chemical + cleaner0 + 2023-07-12T16:42:42Z + CHEBI: + + Na+ + + + 0.9994567 + chemical + cleaner0 + 2023-07-12T16:42:44Z + CHEBI: + + Sr2+ + + + 0.9994848 + protein_state + cleaner0 + 2023-07-12T20:00:04Z + DUMMY: + + Na+-bound + + + 0.99990046 + residue_name_number + cleaner0 + 2023-07-12T16:44:39Z + DUMMY: + + T50 + + + 0.9999018 + residue_name_number + cleaner0 + 2023-07-12T16:44:44Z + DUMMY: + + T209 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:39Z + + coordinate + + + 0.9995192 + chemical + cleaner0 + 2023-07-12T16:42:49Z + CHEBI: + + Sr2+ + + + 0.9996927 + chemical + cleaner0 + 2023-07-12T16:55:27Z + CHEBI: + + Na+ + + + 0.9996083 + chemical + cleaner0 + 2023-07-12T16:42:52Z + CHEBI: + + Sr2+ + + + protein + PR: + cleaner0 + 2023-07-12T15:33:40Z + + NCX_Mj + + + 0.9996699 + protein_state + cleaner0 + 2023-07-12T20:00:08Z + DUMMY: + + occluded + + + 0.9992844 + evidence + cleaner0 + 2023-07-12T19:28:57Z + DUMMY: + + Fo – Fc omit map + + + 0.9996213 + evidence + cleaner0 + 2023-07-12T19:28:59Z + DUMMY: + + structure + + + 0.9996326 + chemical + cleaner0 + 2023-07-12T16:43:01Z + CHEBI: + + Na+ + + + 0.9992766 + evidence + cleaner0 + 2023-07-12T19:29:03Z + DUMMY: + + density + + + 0.99966663 + chemical + cleaner0 + 2023-07-12T16:42:54Z + CHEBI: + + Na+ + + + 0.9991315 + chemical + cleaner0 + 2023-07-12T16:42:56Z + CHEBI: + + H2O + + + 0.9997431 + chemical + cleaner0 + 2023-07-12T16:42:59Z + CHEBI: + + Ca2+ + + + 0.82117546 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.96461064 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.8262321 + experimental_method + cleaner0 + 2023-07-12T19:17:09Z + MESH: + + crystals titrated + + + 0.99973714 + chemical + cleaner0 + 2023-07-12T16:43:03Z + CHEBI: + + Ca2+ + + + 0.99970067 + chemical + cleaner0 + 2023-07-12T16:43:06Z + CHEBI: + + Na+ + + + 0.9996834 + experimental_method + cleaner0 + 2023-07-12T19:17:12Z + MESH: + + Superimposition + + + protein + PR: + cleaner0 + 2023-07-12T15:33:40Z + + NCX_Mj + + + 0.9996061 + evidence + cleaner0 + 2023-07-12T19:29:09Z + DUMMY: + + structures + + + 0.9995919 + chemical + cleaner0 + 2023-07-12T16:43:09Z + CHEBI: + + Na+ + + + 0.999273 + protein_state + cleaner0 + 2023-07-12T20:00:13Z + DUMMY: + + absence of + + + 0.9996879 + chemical + cleaner0 + 2023-07-12T16:43:13Z + CHEBI: + + Na+ + + + 0.85081136 + protein_state + cleaner0 + 2023-07-12T20:00:19Z + DUMMY: + + pH 4 + + + 0.99968123 + protein_state + cleaner0 + 2023-07-12T15:48:06Z + DUMMY: + + apo + + + 0.99961984 + site + cleaner0 + 2023-07-12T19:35:34Z + SO: + + ion-binding sites + + + 0.9996793 + protein_state + cleaner0 + 2023-07-12T15:48:06Z + DUMMY: + + apo + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T16:43:44Z + + high H+ + + + 0.99989545 + residue_name_number + cleaner0 + 2023-07-12T16:44:30Z + DUMMY: + + E54 + + + 0.99989486 + residue_name_number + cleaner0 + 2023-07-12T16:44:35Z + DUMMY: + + E213 + + + 0.8679526 + protein_state + cleaner0 + 2023-07-12T16:43:47Z + DUMMY: + + low Na+ + + + 0.9995432 + evidence + cleaner0 + 2023-07-12T19:29:06Z + DUMMY: + + structure + + + 0.69025 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.52867126 + site + cleaner0 + 2023-07-12T15:36:48Z + SO: + + Smid + + + 0.6491887 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.9996259 + site + cleaner0 + 2023-07-12T19:35:37Z + SO: + + ion-binding sites + + + 0.99967813 + protein_state + cleaner0 + 2023-07-12T15:48:06Z + DUMMY: + + apo + + + protein + PR: + cleaner0 + 2023-07-12T15:33:40Z + + NCX_Mj + + + + nihms-779827-f0004.jpg + F4 + FIG + fig_caption + 55691 + Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+. (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). Black lines indicate (i, i + 4) carbonyl-amide pairs along the helix; specific O-N distances are indicated, in Å (magenta). (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). (d) Close-up of the ion-binding region in the fully Na+-occupied state. Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state. (f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f). These descriptors were employed as collective variables in the Bias-Exchange Metadynamics simulations (Methods). (g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1). + + 0.99937314 + evidence + cleaner0 + 2023-07-12T19:29:16Z + DUMMY: + + structure + + + 0.9995842 + protein_state + cleaner0 + 2023-07-12T15:51:40Z + DUMMY: + + outward-occluded + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T16:46:18Z + + fully Na+-occupied + + + protein + PR: + cleaner0 + 2023-07-12T15:33:40Z + + NCX_Mj + + + 0.9996201 + chemical + cleaner0 + 2023-07-12T16:45:25Z + CHEBI: + + Na+ + + + 0.9994692 + experimental_method + cleaner0 + 2023-07-12T19:17:22Z + MESH: + + simulation + + + protein + PR: + cleaner0 + 2023-07-12T15:33:40Z + + NCX_Mj + + + 0.9996111 + chemical + cleaner0 + 2023-07-12T16:45:30Z + CHEBI: + + Na+ + + + 0.9904822 + protein_state + cleaner0 + 2023-07-12T20:00:30Z + DUMMY: + + bound at + + + 0.98990685 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + 0.9873886 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.98048145 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.99961627 + chemical + cleaner0 + 2023-07-12T16:45:27Z + CHEBI: + + Na+ + + + 0.9982833 + protein_state + cleaner0 + 2023-07-12T20:00:34Z + DUMMY: + + bound only at + + + 0.98023087 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.9603746 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.99787515 + structure_element + cleaner0 + 2023-07-12T19:39:19Z + SO: + + N-terminal half + + + 0.99968886 + structure_element + cleaner0 + 2023-07-12T19:39:22Z + SO: + + TM7 + + + 0.9995776 + structure_element + cleaner0 + 2023-07-12T15:41:47Z + SO: + + TM7ab + + + chemical + CHEBI: + cleaner0 + 2023-07-12T16:45:46Z + + Na+ + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:29:41Z + + simulation snapshots + + + 0.9996201 + chemical + cleaner0 + 2023-07-12T16:45:14Z + CHEBI: + + Na+ + + + 0.9945813 + protein_state + cleaner0 + 2023-07-12T20:00:38Z + DUMMY: + + bound at + + + 0.98243904 + site + cleaner0 + 2023-07-12T15:36:30Z + SO: + + SCa + + + 0.9753448 + site + cleaner0 + 2023-07-12T15:35:42Z + SO: + + Sint + + + 0.8014012 + protein_state + cleaner0 + 2023-07-12T20:00:41Z + DUMMY: + + without + + + 0.9995755 + chemical + cleaner0 + 2023-07-12T16:55:34Z + CHEBI: + + Na+ + + + 0.99646664 + protein_state + cleaner0 + 2023-07-12T20:00:45Z + DUMMY: + + bound + + + 0.999531 + site + cleaner0 + 2023-07-12T19:35:45Z + SO: + + ion-binding region + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T16:46:17Z + + fully Na+-occupied + + + 0.99973565 + structure_element + cleaner0 + 2023-07-12T19:39:25Z + SO: + + TM2 + + + 0.99972385 + structure_element + cleaner0 + 2023-07-12T15:54:55Z + SO: + + TM3 + + + 0.9997049 + structure_element + cleaner0 + 2023-07-12T19:39:27Z + SO: + + TM7 + + + 0.9995299 + site + cleaner0 + 2023-07-12T19:35:49Z + SO: + + ion-binding region + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T16:46:46Z + + partially Na+-occupied + + + 0.9995466 + site + cleaner0 + 2023-07-12T19:35:52Z + SO: + + ion-binding region + + + 0.9994396 + protein_state + cleaner0 + 2023-07-12T16:45:52Z + DUMMY: + + Na+-free + + + 0.9995689 + experimental_method + cleaner0 + 2023-07-12T19:17:30Z + MESH: + + simulations + + + 0.7984423 + protein_state + cleaner0 + 2023-07-12T20:00:49Z + DUMMY: + + Na+-occupancy + + + experimental_method + MESH: + cleaner0 + 2023-07-12T19:17:49Z + + Bias-Exchange Metadynamics simulations + + + 0.9994446 + evidence + cleaner0 + 2023-07-12T19:29:48Z + DUMMY: + + Probability distributions + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:39Z + + hydrogen-bonding + + + 0.9996165 + structure_element + cleaner0 + 2023-07-12T15:41:47Z + SO: + + TM7ab + + + site + SO: + cleaner0 + 2023-07-12T15:36:03Z + + Sext + + + site + SO: + cleaner0 + 2023-07-12T15:36:30Z + + SCa + + + + nihms-779827-f0005.jpg + F5 + FIG + fig_caption + 57342 + Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods). Contours correspond to 1 kcal/mol intervals. Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. (b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state. Na+ ions are shown as green spheres. The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine). (c) Close-up views of the ion-binding region in the same conformational free-energy minima. Key residues involved in Na+ and water coordination (W) are highlighted (sticks, black lines). The water-density maps in (b) is shown here as a grey mesh. Note D240 is protonated, while E54 and E213 are ionized. + + 0.9983607 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:30:24Z + + Calculated conformational free-energy landscapes + + + 0.9370653 + protein_state + cleaner0 + 2023-07-12T15:37:00Z + DUMMY: + + outward-facing + + + protein + PR: + cleaner0 + 2023-07-12T15:33:40Z + + NCX_Mj + + + 0.94464195 + chemical + cleaner0 + 2023-07-12T16:47:30Z + CHEBI: + + Na+ + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T20:01:13Z + + no ions bound + + + 0.99926555 + evidence + cleaner0 + 2023-07-12T19:30:31Z + DUMMY: + + free energy + + + 0.9853114 + site + cleaner0 + 2023-07-12T19:35:57Z + SO: + + aqueous channels + + + 0.99879116 + site + cleaner0 + 2023-07-12T15:36:03Z + SO: + + Sext + + + site + SO: + cleaner0 + 2023-07-12T15:36:30Z + + SCa + + + 0.9995549 + evidence + cleaner0 + 2023-07-12T19:30:34Z + DUMMY: + + X-ray structures + + + protein + PR: + cleaner0 + 2023-07-12T15:33:40Z + + NCX_Mj + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T20:01:38Z + + high + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T20:01:47Z + + low + + + 0.9995905 + chemical + cleaner0 + 2023-07-12T16:47:28Z + CHEBI: + + Na+ + + + 0.9859681 + protein_state + cleaner0 + 2023-07-12T20:01:51Z + DUMMY: + + low pH + + + 0.99949825 + evidence + cleaner0 + 2023-07-12T19:30:38Z + DUMMY: + + Density isosurfaces + + + 0.9997478 + chemical + cleaner0 + 2023-07-12T16:47:44Z + CHEBI: + + water + + + 0.99945337 + site + cleaner0 + 2023-07-12T19:35:59Z + SO: + + ion-binding region + + + 0.93207103 + evidence + cleaner0 + 2023-07-12T19:30:42Z + DUMMY: + + conformational free-energy minima + + + 0.9996631 + chemical + cleaner0 + 2023-07-12T16:47:46Z + CHEBI: + + Na+ + + + 0.9994018 + structure_element + cleaner0 + 2023-07-12T19:39:33Z + SO: + + inverted-topology repeats + + + 0.9994667 + protein_type + cleaner0 + 2023-07-12T16:59:10Z + MESH: + + transporter + + + 0.9994167 + evidence + cleaner0 + 2023-07-12T19:30:59Z + DUMMY: + + structure + + + 0.999639 + structure_element + cleaner0 + 2023-07-12T16:48:04Z + SO: + + TM1-5 + + + 0.99954796 + structure_element + cleaner0 + 2023-07-12T16:48:07Z + SO: + + TM6-10 + + + 0.9993889 + site + cleaner0 + 2023-07-12T19:36:03Z + SO: + + ion-binding region + + + 0.8357873 + evidence + cleaner0 + 2023-07-12T19:30:52Z + DUMMY: + + conformational free-energy minima + + + 0.99955535 + chemical + cleaner0 + 2023-07-12T16:47:48Z + CHEBI: + + Na+ + + + 0.9997657 + chemical + cleaner0 + 2023-07-12T16:47:51Z + CHEBI: + + water + + + 0.99945056 + evidence + cleaner0 + 2023-07-12T19:30:55Z + DUMMY: + + water-density maps + + + 0.99990237 + residue_name_number + cleaner0 + 2023-07-12T15:50:34Z + DUMMY: + + D240 + + + 0.9999039 + residue_name_number + cleaner0 + 2023-07-12T16:44:31Z + DUMMY: + + E54 + + + 0.9999013 + residue_name_number + cleaner0 + 2023-07-12T16:44:36Z + DUMMY: + + E213 + + + + nihms-779827-f0006.jpg + F6 + FIG + fig_caption + 58654 + Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. The free energy is plotted as in Fig. 5. For Ca2+, a map is shown in which a correction for the charge-transfer between the ion and the protein is introduced, alongside the uncorrected map (see Supplementary Notes 3-4 and Supplementary Fig. 5-6). The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study. (b) Water-density isosurfaces analogous to those in Fig. 5 are shown for each of the major conformational free-energy minima in the free-energy maps. The Ca2+ ion is shown as a red sphere; the protein is shown as in Fig. 5. (c) Close-up views of the ion-binding region in the same conformational free-energy minima. Key residues involved in Ca2+ and water coordination (W) are highlighted (sticks, black lines). The water-density maps in (b) are shown here as a grey mesh. In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. + + 0.797733 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:28:04Z + + Calculated free-energy landscapes + + + 0.94660574 + protein_state + cleaner0 + 2023-07-12T15:37:00Z + DUMMY: + + outward-facing + + + protein + PR: + cleaner0 + 2023-07-12T15:33:40Z + + NCX_Mj + + + 0.9996346 + chemical + cleaner0 + 2023-07-12T16:48:25Z + CHEBI: + + Ca2+ + + + 0.99955344 + protein_state + cleaner0 + 2023-07-12T20:02:03Z + DUMMY: + + fully protonated + + + 0.9993578 + evidence + cleaner0 + 2023-07-12T19:31:13Z + DUMMY: + + free energy + + + 0.9996518 + chemical + cleaner0 + 2023-07-12T16:48:30Z + CHEBI: + + Ca2+ + + + 0.9995228 + evidence + cleaner0 + 2023-07-12T19:31:18Z + DUMMY: + + map + + + 0.9993074 + evidence + cleaner0 + 2023-07-12T19:31:20Z + DUMMY: + + map + + + 0.9995147 + evidence + cleaner0 + 2023-07-12T19:31:23Z + DUMMY: + + map + + + 0.99963975 + protein_state + cleaner0 + 2023-07-12T20:02:09Z + DUMMY: + + open + + + 0.9994335 + protein_state + cleaner0 + 2023-07-12T20:02:12Z + DUMMY: + + semi-open + + + 0.9996573 + protein_state + cleaner0 + 2023-07-12T20:02:14Z + DUMMY: + + occluded + + + 0.99516165 + protein_state + cleaner0 + 2023-07-12T20:02:26Z + DUMMY: + + bound to + + + 0.9997256 + chemical + cleaner0 + 2023-07-12T16:48:28Z + CHEBI: + + Na+ + + + 0.99962234 + evidence + cleaner0 + 2023-07-12T19:31:26Z + DUMMY: + + crystal structures + + + 0.9996813 + chemical + cleaner0 + 2023-07-12T16:48:35Z + CHEBI: + + Ca2+ + + + 0.9968871 + protein_state + cleaner0 + 2023-07-12T20:02:29Z + DUMMY: + + low pH + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T16:49:28Z + + high H+ + + + 0.9993633 + evidence + cleaner0 + 2023-07-12T19:31:29Z + DUMMY: + + Water-density isosurfaces + + + 0.6663834 + evidence + cleaner0 + 2023-07-12T19:31:32Z + DUMMY: + + free-energy minima + + + 0.9995218 + evidence + cleaner0 + 2023-07-12T19:31:34Z + DUMMY: + + free-energy maps + + + 0.9996765 + chemical + cleaner0 + 2023-07-12T16:49:32Z + CHEBI: + + Ca2+ + + + 0.99904287 + site + cleaner0 + 2023-07-12T19:36:07Z + SO: + + ion-binding region + + + evidence + DUMMY: + cleaner0 + 2023-07-12T19:31:50Z + + conformational free-energy minima + + + 0.9996734 + chemical + cleaner0 + 2023-07-12T16:49:34Z + CHEBI: + + Ca2+ + + + 0.9997689 + chemical + cleaner0 + 2023-07-12T16:49:36Z + CHEBI: + + water + + + 0.9995133 + evidence + cleaner0 + 2023-07-12T19:31:53Z + DUMMY: + + water-density maps + + + 0.99966085 + protein_state + cleaner0 + 2023-07-12T20:02:44Z + DUMMY: + + occluded + + + 0.99963033 + chemical + cleaner0 + 2023-07-12T16:49:41Z + CHEBI: + + Ca2+ + + + 0.98349607 + protein_state + cleaner0 + 2023-07-12T20:02:48Z + DUMMY: + + bound + + + 0.99950075 + structure_element + cleaner0 + 2023-07-12T19:40:58Z + SO: + + helix + + + 0.9997874 + structure_element + cleaner0 + 2023-07-12T15:41:47Z + SO: + + TM7ab + + + 0.9995513 + protein_state + cleaner0 + 2023-07-12T20:02:52Z + DUMMY: + + fully occupied + + + 0.99937093 + chemical + cleaner0 + 2023-07-12T16:49:43Z + CHEBI: + + Na+ + + + 0.99990904 + residue_name_number + cleaner0 + 2023-07-12T15:41:03Z + DUMMY: + + Ala206 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:17:39Z + + hydrogen-bonding interaction + + + 0.99990845 + residue_name_number + cleaner0 + 2023-07-12T16:52:06Z + DUMMY: + + Ser210 + + + + nihms-779827-f0007.jpg + F7 + FIG + fig_caption + 60134 + Structural mechanism of extracellular forward ion exchange in NCX. The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. The green open cylinders represent the gating helices TM1 and TM6. Asterisks mark the states whose crystal structures have been determined in this study. These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. + + 0.61637443 + protein_type + cleaner0 + 2023-07-12T15:33:13Z + MESH: + + NCX + + + 0.9999052 + residue_name_number + cleaner0 + 2023-07-12T16:52:12Z + DUMMY: + + Ala47 + + + 0.9998196 + structure_element + cleaner0 + 2023-07-12T19:41:05Z + SO: + + TM2b + + + 0.9999049 + residue_name_number + cleaner0 + 2023-07-12T15:41:03Z + DUMMY: + + Ala206 + + + 0.9998172 + structure_element + cleaner0 + 2023-07-12T15:41:27Z + SO: + + TM7b + + + 0.9999027 + residue_name_number + cleaner0 + 2023-07-12T15:40:30Z + DUMMY: + + Glu54 + + + 0.9998171 + structure_element + cleaner0 + 2023-07-12T19:41:08Z + SO: + + TM2c + + + 0.99990034 + residue_name_number + cleaner0 + 2023-07-12T15:40:34Z + DUMMY: + + Glu213 + + + 0.99981946 + structure_element + cleaner0 + 2023-07-12T19:41:11Z + SO: + + TM7c + + + 0.9995922 + structure_element + cleaner0 + 2023-07-12T19:41:13Z + SO: + + gating helices + + + 0.999819 + structure_element + cleaner0 + 2023-07-12T15:41:41Z + SO: + + TM1 + + + 0.9998191 + structure_element + cleaner0 + 2023-07-12T15:41:33Z + SO: + + TM6 + + + 0.99955064 + evidence + cleaner0 + 2023-07-12T19:32:00Z + DUMMY: + + crystal structures + + + 0.9958951 + evidence + cleaner0 + 2023-07-12T19:28:04Z + DUMMY: + + calculated free-energy landscapes + + + protein_state + DUMMY: + cleaner0 + 2023-07-12T16:50:06Z + + Ca2+-loaded + + + 0.94450617 + protein_state + cleaner0 + 2023-07-12T15:37:00Z + DUMMY: + + outward-facing + + + 0.99857485 + protein_state + cleaner0 + 2023-07-12T20:03:00Z + DUMMY: + + occluded + + + 0.99964225 + protein_state + cleaner0 + 2023-07-12T20:03:03Z + DUMMY: + + unloaded + + + 0.9994731 + protein_state + cleaner0 + 2023-07-12T20:03:06Z + DUMMY: + + fully open + + + + T1.xml + T1 + TABLE + table_caption + 60772 + Concentration-dependent Na+ occupancy of outward-facing NCX_Mj + + + T1.xml + T1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><thead><tr><th align="left" valign="top" rowspan="1" colspan="1">Sites \ [Na<sup>+</sup>]</th><th align="center" valign="top" rowspan="1" colspan="1">2.5 mM</th><th align="center" valign="top" rowspan="1" colspan="1">10 mM</th><th align="center" valign="top" rowspan="1" colspan="1">20 mM</th><th align="center" valign="top" rowspan="1" colspan="1">100 mM</th><th align="center" valign="top" rowspan="1" colspan="1">150 mM</th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1">S<sub>int</sub></td><td align="center" valign="top" rowspan="1" colspan="1">0.84</td><td align="center" valign="top" rowspan="1" colspan="1">0.92</td><td align="center" valign="top" rowspan="1" colspan="1">0.99</td><td align="center" valign="top" rowspan="1" colspan="1">0.96</td><td align="center" valign="top" rowspan="1" colspan="1">1.00</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">S<sub>Ca</sub></td><td align="center" valign="top" rowspan="1" colspan="1">0.75</td><td align="center" valign="top" rowspan="1" colspan="1">0.87</td><td align="center" valign="top" rowspan="1" colspan="1">0.97</td><td align="center" valign="top" rowspan="1" colspan="1">1.00</td><td align="center" valign="top" rowspan="1" colspan="1">1.00</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">S<sub>ext</sub></td><td align="center" valign="top" rowspan="1" colspan="1">-</td><td align="center" valign="top" rowspan="1" colspan="1">-</td><td align="center" valign="top" rowspan="1" colspan="1">0.59</td><td align="center" valign="top" rowspan="1" colspan="1">0.79</td><td align="center" valign="top" rowspan="1" colspan="1">0.93</td></tr><tr><td colspan="6" align="left" valign="top" rowspan="1"><hr/></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Total</td><td align="center" valign="top" rowspan="1" colspan="1">1.58</td><td align="center" valign="top" rowspan="1" colspan="1">1.79</td><td align="center" valign="top" rowspan="1" colspan="1">2.55</td><td align="center" valign="top" rowspan="1" colspan="1">2.75</td><td align="center" valign="top" rowspan="1" colspan="1">2.93</td></tr><tr><td colspan="6" align="left" valign="top" rowspan="1"><hr/></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Conformation</td><td align="center" valign="top" rowspan="1" colspan="1">Partially open</td><td align="center" valign="top" rowspan="1" colspan="1">Partially open</td><td align="center" valign="top" rowspan="1" colspan="1">Mixed</td><td align="center" valign="top" rowspan="1" colspan="1">Occluded</td><td align="center" valign="top" rowspan="1" colspan="1">Occluded</td></tr></tbody></table> + + 60835 + Sites \ [Na+] 2.5 mM 10 mM 20 mM 100 mM 150 mM Sint 0.84 0.92 0.99 0.96 1.00 SCa 0.75 0.87 0.97 1.00 1.00 Sext - - 0.59 0.79 0.93 Total 1.58 1.79 2.55 2.75 2.93 Conformation Partially open Partially open Mixed Occluded Occluded + + + T2.xml + T2 + TABLE + table_caption + 61082 + Data collection and refinement statistics for the NCX_Mj structures obtained from crystals soaked with varying amounts of Na+, and no Ca2+, and at low pH and no Na+ or Ca2+. + + + T2.xml + T2 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="box" rules="all"><thead><tr><th align="center" valign="top" rowspan="1" colspan="1">[Na<sup>+</sup>]</th><th align="center" valign="top" rowspan="1" colspan="1">2.5 mM PDB 5HWX</th><th align="center" valign="top" rowspan="1" colspan="1">10 mM PDB 5HWY</th><th align="center" valign="top" rowspan="1" colspan="1">20 mM PDB 5HXC</th><th align="center" valign="top" rowspan="1" colspan="1">100 mM PDB 5HXE</th><th align="center" valign="top" rowspan="1" colspan="1">150 mM PDB 5HYA</th><th align="center" valign="top" rowspan="1" colspan="1">0 mM PDB 5HXH</th></tr></thead><tbody><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>Data collection</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Space group</td><td colspan="5" align="center" valign="top" rowspan="1">P212121</td><td align="center" valign="top" rowspan="1" colspan="1">C2</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Cell dimensions</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">a, b, c (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">49.70, 72.28, 95.78</td><td align="center" valign="top" rowspan="1" colspan="1">46.21, 71.97, 95.63</td><td align="center" valign="top" rowspan="1" colspan="1">49.75, 72.56, 95.78</td><td align="center" valign="top" rowspan="1" colspan="1">49.77, 72.85, 96.36</td><td align="center" valign="top" rowspan="1" colspan="1">49.49, 72.88, 96.21</td><td align="center" valign="top" rowspan="1" colspan="1">164.18, 46.83, 96.96</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">α, β, γ (°)</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 106.20, 90</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">2.40 (2.44-2.40)</td><td align="center" valign="top" rowspan="1" colspan="1">2.10 (2.14-2.10)</td><td align="center" valign="top" rowspan="1" colspan="1">2.10 (2.14-2.10)</td><td align="center" valign="top" rowspan="1" colspan="1">2.28 (2.32-2.28)</td><td align="center" valign="top" rowspan="1" colspan="1">1.90 (1.93-1.90)</td><td align="center" valign="top" rowspan="1" colspan="1">2.80 (2.85-2.80)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">R<sub>sym</sub> (%)</td><td align="center" valign="top" rowspan="1" colspan="1">9.4 (99.4)</td><td align="center" valign="top" rowspan="1" colspan="1">9.3 (69.8)</td><td align="center" valign="top" rowspan="1" colspan="1">10.5 (99.6)</td><td align="center" valign="top" rowspan="1" colspan="1">9.9 (56.3)</td><td align="center" valign="top" rowspan="1" colspan="1">8.6 (88.0)</td><td align="center" valign="top" rowspan="1" colspan="1">10.5 (94.3)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">I/σI</td><td align="center" valign="top" rowspan="1" colspan="1">24.2 (1.8)</td><td align="center" valign="top" rowspan="1" colspan="1">24.3 (2.7)</td><td align="center" valign="top" rowspan="1" colspan="1">20.6 (1.9)</td><td align="center" valign="top" rowspan="1" colspan="1">15.9 (2.8)</td><td align="center" valign="top" rowspan="1" colspan="1">32.9 (2.4)</td><td align="center" valign="top" rowspan="1" colspan="1">17.7 (1.2)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">CC<sub>1/2</sub></td><td align="center" valign="top" rowspan="1" colspan="1">(0.625)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.819)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.602)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.722)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.786)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.561)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Completeness (%)</td><td align="center" valign="top" rowspan="1" colspan="1">99.8 (99.1)</td><td align="center" valign="top" rowspan="1" colspan="1">99.9 (100)</td><td align="center" valign="top" rowspan="1" colspan="1">99.9 (99.8)</td><td align="center" valign="top" rowspan="1" colspan="1">96.4 (98.6)</td><td align="center" valign="top" rowspan="1" colspan="1">98.9 (97.8)</td><td align="center" valign="top" rowspan="1" colspan="1">99.9 (99.9)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Redundancy</td><td align="center" valign="top" rowspan="1" colspan="1">6.9 (6.7)</td><td align="center" valign="top" rowspan="1" colspan="1">6.1 (6.0)</td><td align="center" valign="top" rowspan="1" colspan="1">7.1 (7.0)</td><td align="center" valign="top" rowspan="1" colspan="1">3.4 (3.3)</td><td align="center" valign="top" rowspan="1" colspan="1">9.3 (8.9)</td><td align="center" valign="top" rowspan="1" colspan="1">7.1 (5.7)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>Refinement</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.4</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.1</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.1</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.3</td><td align="center" valign="top" rowspan="1" colspan="1">50-1.9</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.80</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">No. reflections</td><td align="center" valign="top" rowspan="1" colspan="1">13977</td><td align="center" valign="top" rowspan="1" colspan="1">19254</td><td align="center" valign="top" rowspan="1" colspan="1">20739</td><td align="center" valign="top" rowspan="1" colspan="1">15767</td><td align="center" valign="top" rowspan="1" colspan="1">27923</td><td align="center" valign="top" rowspan="1" colspan="1">21489</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">R<sub>work</sub>/R<sub>free</sub></td><td align="center" valign="top" rowspan="1" colspan="1">0.21/0.25</td><td align="center" valign="top" rowspan="1" colspan="1">0.19/0.22</td><td align="center" valign="top" rowspan="1" colspan="1">0.19/0.23</td><td align="center" valign="top" rowspan="1" colspan="1">0.19/0.24</td><td align="center" valign="top" rowspan="1" colspan="1">0.179/0.207</td><td align="center" valign="top" rowspan="1" colspan="1">0.20/0.26</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>No. atoms</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Protein</td><td align="center" valign="top" rowspan="1" colspan="1">2206</td><td align="center" valign="top" rowspan="1" colspan="1">2274</td><td align="center" valign="top" rowspan="1" colspan="1">2366</td><td align="center" valign="top" rowspan="1" colspan="1">2229</td><td align="center" valign="top" rowspan="1" colspan="1">2229</td><td align="center" valign="top" rowspan="1" colspan="1">4410</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Ligand/Ion</td><td align="center" valign="top" rowspan="1" colspan="1">56/3</td><td align="center" valign="top" rowspan="1" colspan="1">154/2</td><td align="center" valign="top" rowspan="1" colspan="1">161/5</td><td align="center" valign="top" rowspan="1" colspan="1">162/6</td><td align="center" valign="top" rowspan="1" colspan="1">257/4</td><td align="center" valign="top" rowspan="1" colspan="1">121/2</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Water</td><td align="center" valign="top" rowspan="1" colspan="1">18</td><td align="center" valign="top" rowspan="1" colspan="1">36</td><td align="center" valign="top" rowspan="1" colspan="1">67</td><td align="center" valign="top" rowspan="1" colspan="1">67</td><td align="center" valign="top" rowspan="1" colspan="1">100</td><td align="center" valign="top" rowspan="1" colspan="1">39</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>B-factors</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Protein</td><td align="center" valign="top" rowspan="1" colspan="1">53.25</td><td align="center" valign="top" rowspan="1" colspan="1">34.28</td><td align="center" valign="top" rowspan="1" colspan="1">34.91</td><td align="center" valign="top" rowspan="1" colspan="1">39.75</td><td align="center" valign="top" rowspan="1" colspan="1">26.05</td><td align="center" valign="top" rowspan="1" colspan="1">42.98</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Ligand/Ion</td><td align="center" valign="top" rowspan="1" colspan="1">62.94/47.70</td><td align="center" valign="top" rowspan="1" colspan="1">55.86/31.75</td><td align="center" valign="top" rowspan="1" colspan="1">55.25/36.47</td><td align="center" valign="top" rowspan="1" colspan="1">58.24/38.93</td><td align="center" valign="top" rowspan="1" colspan="1">46.11/22.13</td><td align="center" valign="top" rowspan="1" colspan="1">54.29/63.47</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Water</td><td align="center" valign="top" rowspan="1" colspan="1">58.12</td><td align="center" valign="top" rowspan="1" colspan="1">41.16</td><td align="center" valign="top" rowspan="1" colspan="1">46.97</td><td align="center" valign="top" rowspan="1" colspan="1">49.74</td><td align="center" valign="top" rowspan="1" colspan="1">37.80</td><td align="center" valign="top" rowspan="1" colspan="1">33.17</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>R.m.s deviations</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Bond lengths (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">0.004</td><td align="center" valign="top" rowspan="1" colspan="1">0.006</td><td align="center" valign="top" rowspan="1" colspan="1">0.008</td><td align="center" valign="top" rowspan="1" colspan="1">0.003</td><td align="center" valign="top" rowspan="1" colspan="1">0.006</td><td align="center" valign="top" rowspan="1" colspan="1">0.003</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Bond angles (°)</td><td align="center" valign="top" rowspan="1" colspan="1">0.819</td><td align="center" valign="top" rowspan="1" colspan="1">0.915</td><td align="center" valign="top" rowspan="1" colspan="1">1.269</td><td align="center" valign="top" rowspan="1" colspan="1">1.024</td><td align="center" valign="top" rowspan="1" colspan="1">0.966</td><td align="center" valign="top" rowspan="1" colspan="1">0.705</td></tr></tbody></table> + + 61256 + [Na+] 2.5 mM PDB 5HWX 10 mM PDB 5HWY 20 mM PDB 5HXC 100 mM PDB 5HXE 150 mM PDB 5HYA 0 mM PDB 5HXH Data collection Space group P212121 C2 Cell dimensions a, b, c (Å) 49.70, 72.28, 95.78 46.21, 71.97, 95.63 49.75, 72.56, 95.78 49.77, 72.85, 96.36 49.49, 72.88, 96.21 164.18, 46.83, 96.96 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 106.20, 90 Resolution (Å) 2.40 (2.44-2.40) 2.10 (2.14-2.10) 2.10 (2.14-2.10) 2.28 (2.32-2.28) 1.90 (1.93-1.90) 2.80 (2.85-2.80) Rsym (%) 9.4 (99.4) 9.3 (69.8) 10.5 (99.6) 9.9 (56.3) 8.6 (88.0) 10.5 (94.3) I/σI 24.2 (1.8) 24.3 (2.7) 20.6 (1.9) 15.9 (2.8) 32.9 (2.4) 17.7 (1.2) CC1/2 (0.625) (0.819) (0.602) (0.722) (0.786) (0.561) Completeness (%) 99.8 (99.1) 99.9 (100) 99.9 (99.8) 96.4 (98.6) 98.9 (97.8) 99.9 (99.9) Redundancy 6.9 (6.7) 6.1 (6.0) 7.1 (7.0) 3.4 (3.3) 9.3 (8.9) 7.1 (5.7) Refinement Resolution (Å) 50-2.4 50-2.1 50-2.1 50-2.3 50-1.9 50-2.80 No. reflections 13977 19254 20739 15767 27923 21489 Rwork/Rfree 0.21/0.25 0.19/0.22 0.19/0.23 0.19/0.24 0.179/0.207 0.20/0.26 No. atoms Protein 2206 2274 2366 2229 2229 4410 Ligand/Ion 56/3 154/2 161/5 162/6 257/4 121/2 Water 18 36 67 67 100 39 B-factors Protein 53.25 34.28 34.91 39.75 26.05 42.98 Ligand/Ion 62.94/47.70 55.86/31.75 55.25/36.47 58.24/38.93 46.11/22.13 54.29/63.47 Water 58.12 41.16 46.97 49.74 37.80 33.17 R.m.s deviations Bond lengths (Å) 0.004 0.006 0.008 0.003 0.006 0.003 Bond angles (°) 0.819 0.915 1.269 1.024 0.966 0.705 + + + T2.xml + T2 + TABLE + table_foot + 62823 + Values in parenthesis are for highest resolution shell. 5% of the data was used in the Rfree calculation. ‘Ligand’ atoms are from lipids, PEG400 and acetates. + + + T3.xml + T3 + TABLE + table_caption + 62986 + Data collection and refinement statistics for the NCX_Mj structures obtained from crystals soaked with varying amounts of Na+ and Sr2+. + + + T3.xml + T3 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="box" rules="all"><thead><tr><th align="center" valign="top" rowspan="1" colspan="1">[Sr<sup>2+</sup>] / [Na<sup>+</sup>]</th><th align="center" valign="top" rowspan="1" colspan="1">10 mM / 2.5 mM PDB 5HXS</th><th align="center" valign="top" rowspan="1" colspan="1">1 mM / 2.5 mM N/A<sup><xref ref-type="table-fn" rid="TFN1">*</xref></sup></th><th align="center" valign="top" rowspan="1" colspan="1">0.1 mM / 2.5 mM N/A<sup><xref ref-type="table-fn" rid="TFN1">*</xref></sup></th><th align="center" valign="top" rowspan="1" colspan="1">10 mM / 10 mM N/A<sup><xref ref-type="table-fn" rid="TFN1">*</xref></sup></th><th align="center" valign="top" rowspan="1" colspan="1">10 mM / 100 mM N/A<sup><xref ref-type="table-fn" rid="TFN2">#</xref></sup></th></tr></thead><tbody><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>Data collection</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Space group</td><td colspan="5" align="center" valign="top" rowspan="1">P212121</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Cell dimensions</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">a, b, c (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">49.52, 72.35, 96.00</td><td align="center" valign="top" rowspan="1" colspan="1">49.76, 72.62, 95.62</td><td align="center" valign="top" rowspan="1" colspan="1">49.80, 72.27, 94.84</td><td align="center" valign="top" rowspan="1" colspan="1">49.67, 72.46, 96.43</td><td align="center" valign="top" rowspan="1" colspan="1">49.88, 72.43, 95.91</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">α, β, γ (°)</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">2.80 (2.85-2.80)</td><td align="center" valign="top" rowspan="1" colspan="1">2.90 (2.95-2.90)</td><td align="center" valign="top" rowspan="1" colspan="1">2.54 (2.58-2.54)</td><td align="center" valign="top" rowspan="1" colspan="1">2.30 (2.34-2.30)</td><td align="center" valign="top" rowspan="1" colspan="1">2.50 (2.54-2.50)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">R<sub>sym</sub> (%)</td><td align="center" valign="top" rowspan="1" colspan="1">12.3 (88.0)</td><td align="center" valign="top" rowspan="1" colspan="1">12.3 (91.0)</td><td align="center" valign="top" rowspan="1" colspan="1">11.9 (88.5)</td><td align="center" valign="top" rowspan="1" colspan="1">13.7 (95.8)</td><td align="center" valign="top" rowspan="1" colspan="1">10.9 (96.8)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">I/σI</td><td align="center" valign="top" rowspan="1" colspan="1">19.0 (1.8)</td><td align="center" valign="top" rowspan="1" colspan="1">19.2 (1.6)</td><td align="center" valign="top" rowspan="1" colspan="1">21.5 (1.9)</td><td align="center" valign="top" rowspan="1" colspan="1">18.4 (1.6)</td><td align="center" valign="top" rowspan="1" colspan="1">19.7 (1.8)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">CC<sub>1/2</sub></td><td align="center" valign="top" rowspan="1" colspan="1">(0.674)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.686)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.768)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.420)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.767)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Completeness (%)</td><td align="center" valign="top" rowspan="1" colspan="1">98.8 (90.4)</td><td align="center" valign="top" rowspan="1" colspan="1">94.9 (96.1)</td><td align="center" valign="top" rowspan="1" colspan="1">99.9 (100.0)</td><td align="center" valign="top" rowspan="1" colspan="1">99.8 (99.9)</td><td align="center" valign="top" rowspan="1" colspan="1">99.6 (99.7)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Redundancy</td><td align="center" valign="top" rowspan="1" colspan="1">6.5 (5.4)</td><td align="center" valign="top" rowspan="1" colspan="1">6.7 (6.9)</td><td align="center" valign="top" rowspan="1" colspan="1">7.1 (6.8)</td><td align="center" valign="top" rowspan="1" colspan="1">6.2 (4.5)</td><td align="center" valign="top" rowspan="1" colspan="1">5.9 (6.0)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>Refinement</bold> +</td><td colspan="5" align="center" valign="top" rowspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.80</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.90</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.54</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.3</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.5</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">No. reflections</td><td align="center" valign="top" rowspan="1" colspan="1">8927</td><td align="center" valign="top" rowspan="1" colspan="1">7611</td><td align="center" valign="top" rowspan="1" colspan="1">11483</td><td align="center" valign="top" rowspan="1" colspan="1">16488</td><td align="center" valign="top" rowspan="1" colspan="1">12665</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">R<sub>work</sub>/R<sub>free</sub></td><td align="center" valign="top" rowspan="1" colspan="1">0.22/0.27</td><td align="center" valign="top" rowspan="1" colspan="1">0.22/0.27</td><td align="center" valign="top" rowspan="1" colspan="1">0.23/0.26</td><td align="center" valign="top" rowspan="1" colspan="1">0.20/0.25</td><td align="center" valign="top" rowspan="1" colspan="1">0.21/0.24</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>No. atoms</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Protein</td><td align="center" valign="top" rowspan="1" colspan="1">2227</td><td align="center" valign="top" rowspan="1" colspan="1">2249</td><td align="center" valign="top" rowspan="1" colspan="1">2217</td><td align="center" valign="top" rowspan="1" colspan="1">2271</td><td align="center" valign="top" rowspan="1" colspan="1">2223</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Ligand/Ion</td><td align="center" valign="top" rowspan="1" colspan="1">110/2</td><td align="center" valign="top" rowspan="1" colspan="1">133/3</td><td align="center" valign="top" rowspan="1" colspan="1">155/3</td><td align="center" valign="top" rowspan="1" colspan="1">101/3</td><td align="center" valign="top" rowspan="1" colspan="1">104/6</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Water</td><td align="center" valign="top" rowspan="1" colspan="1">10</td><td align="center" valign="top" rowspan="1" colspan="1">2</td><td align="center" valign="top" rowspan="1" colspan="1">5</td><td align="center" valign="top" rowspan="1" colspan="1">34</td><td align="center" valign="top" rowspan="1" colspan="1">17</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>B-factors</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Protein</td><td align="center" valign="top" rowspan="1" colspan="1">61.55</td><td align="center" valign="top" rowspan="1" colspan="1">67.82</td><td align="center" valign="top" rowspan="1" colspan="1">60.83</td><td align="center" valign="top" rowspan="1" colspan="1">46.42</td><td align="center" valign="top" rowspan="1" colspan="1">56.75</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Ligand/Ion</td><td align="center" valign="top" rowspan="1" colspan="1">74.56/63.32</td><td align="center" valign="top" rowspan="1" colspan="1">85.56/70.47</td><td align="center" valign="top" rowspan="1" colspan="1">80.33/90.41</td><td align="center" valign="top" rowspan="1" colspan="1">64.79/38.25</td><td align="center" valign="top" rowspan="1" colspan="1">71.27/53.50</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Water</td><td align="center" valign="top" rowspan="1" colspan="1">59.22</td><td align="center" valign="top" rowspan="1" colspan="1">61.31</td><td align="center" valign="top" rowspan="1" colspan="1">68.58</td><td align="center" valign="top" rowspan="1" colspan="1">51.39</td><td align="center" valign="top" rowspan="1" colspan="1">55.07</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>R.m.s deviations</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Bond lengths (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">0.004</td><td align="center" valign="top" rowspan="1" colspan="1">0.005</td><td align="center" valign="top" rowspan="1" colspan="1">0.005</td><td align="center" valign="top" rowspan="1" colspan="1">0.007</td><td align="center" valign="top" rowspan="1" colspan="1">0.003</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Bond angles (°)</td><td align="center" valign="top" rowspan="1" colspan="1">0.887</td><td align="center" valign="top" rowspan="1" colspan="1">1.076</td><td align="center" valign="top" rowspan="1" colspan="1">0.963</td><td align="center" valign="top" rowspan="1" colspan="1">1.057</td><td align="center" valign="top" rowspan="1" colspan="1">0.733</td></tr></tbody></table> + + 63122 + [Sr2+] / [Na+] 10 mM / 2.5 mM PDB 5HXS 1 mM / 2.5 mM N/A* 0.1 mM / 2.5 mM N/A* 10 mM / 10 mM N/A* 10 mM / 100 mM N/A# Data collection Space group P212121 Cell dimensions a, b, c (Å) 49.52, 72.35, 96.00 49.76, 72.62, 95.62 49.80, 72.27, 94.84 49.67, 72.46, 96.43 49.88, 72.43, 95.91 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) 2.80 (2.85-2.80) 2.90 (2.95-2.90) 2.54 (2.58-2.54) 2.30 (2.34-2.30) 2.50 (2.54-2.50) Rsym (%) 12.3 (88.0) 12.3 (91.0) 11.9 (88.5) 13.7 (95.8) 10.9 (96.8) I/σI 19.0 (1.8) 19.2 (1.6) 21.5 (1.9) 18.4 (1.6) 19.7 (1.8) CC1/2 (0.674) (0.686) (0.768) (0.420) (0.767) Completeness (%) 98.8 (90.4) 94.9 (96.1) 99.9 (100.0) 99.8 (99.9) 99.6 (99.7) Redundancy 6.5 (5.4) 6.7 (6.9) 7.1 (6.8) 6.2 (4.5) 5.9 (6.0) Refinement Resolution (Å) 50-2.80 50-2.90 50-2.54 50-2.3 50-2.5 No. reflections 8927 7611 11483 16488 12665 Rwork/Rfree 0.22/0.27 0.22/0.27 0.23/0.26 0.20/0.25 0.21/0.24 No. atoms Protein 2227 2249 2217 2271 2223 Ligand/Ion 110/2 133/3 155/3 101/3 104/6 Water 10 2 5 34 17 B-factors Protein 61.55 67.82 60.83 46.42 56.75 Ligand/Ion 74.56/63.32 85.56/70.47 80.33/90.41 64.79/38.25 71.27/53.50 Water 59.22 61.31 68.58 51.39 55.07 R.m.s deviations Bond lengths (Å) 0.004 0.005 0.005 0.007 0.003 Bond angles (°) 0.887 1.076 0.963 1.057 0.733 + + + T3.xml + T3 + TABLE + table_foot + 64519 + Values in parenthesis are for highest resolution shell. 5% of the data was used in the Rfree calculation. ‘Ligand’ atoms are from lipids, PEG400 and acetates. + + + T3.xml + T3 + TABLE + table_footnote + 64682 + These structures are virtually identical to that resolved with 10 mM Sr2+ (PDB 5HXS), except for the weakened electron-density signal for the divalent ion, and were therefore not deposited in the PDB. + + + T3.xml + T3 + TABLE + table_footnote + 64883 + This structure is virtually identical to that resolved with 100 mM Na+ without Ca2+ or Sr2+ (Table 1, PDB 5HXE) and was therefore not deposited in the PDB. + + + T4.xml + T4 + TABLE + table_caption + 65039 + Data collection and refinement statistics for the NCX_Mj structures obtained from crystals soaked with varying amounts of Na+ and Ca2+. + + + T4.xml + T4 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="box" rules="all"><thead><tr><th align="center" valign="top" rowspan="1" colspan="1">[Ca<sup>2+</sup>] / [Na<sup>+</sup>]</th><th align="center" valign="top" rowspan="1" colspan="1">10 mM / 2.5 mM PDB 5HXR</th><th align="center" valign="top" rowspan="1" colspan="1">1 mM / 2.5 mM N/A<sup><xref ref-type="table-fn" rid="TFN3">*</xref></sup></th><th align="center" valign="top" rowspan="1" colspan="1">0.1 mM / 2.5 mM N/A<sup><xref ref-type="table-fn" rid="TFN3">*</xref></sup></th><th align="center" valign="top" rowspan="1" colspan="1">10 mM / 10 mM N/A<sup><xref ref-type="table-fn" rid="TFN3">*</xref></sup></th></tr></thead><tbody><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>Data collection</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Space group</td><td colspan="4" align="center" valign="top" rowspan="1">P212121</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Cell dimensions</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">a, b, c (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">49.70, 72.52, 96.94</td><td align="center" valign="top" rowspan="1" colspan="1">49.80, 72.26, 95.80</td><td align="center" valign="top" rowspan="1" colspan="1">49.48, 72.47, 96.30</td><td align="center" valign="top" rowspan="1" colspan="1">49.88, 72.22, 96.10</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">α, β, γ (°)</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">2.45 (2.49-2.45)</td><td align="center" valign="top" rowspan="1" colspan="1">2.65 (2.70-2.65)</td><td align="center" valign="top" rowspan="1" colspan="1">2.40 (2.44-2.40)</td><td align="center" valign="top" rowspan="1" colspan="1">2.20 (2.24-2.20)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">R<sub>sym</sub> (%)</td><td align="center" valign="top" rowspan="1" colspan="1">11.8 (94.2)</td><td align="center" valign="top" rowspan="1" colspan="1">11.1 (92.1)</td><td align="center" valign="top" rowspan="1" colspan="1">11.2 (91.3)</td><td align="center" valign="top" rowspan="1" colspan="1">10.3 (99.1)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">I/σI</td><td align="center" valign="top" rowspan="1" colspan="1">24.4 (1.6)</td><td align="center" valign="top" rowspan="1" colspan="1">20.9 (1.6)</td><td align="center" valign="top" rowspan="1" colspan="1">20.4 (1.6)</td><td align="center" valign="top" rowspan="1" colspan="1">22.4 (1.8)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">CC<sub>1/2</sub></td><td align="center" valign="top" rowspan="1" colspan="1">(0.611)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.696)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.632)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.549)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Completeness (%)</td><td align="center" valign="top" rowspan="1" colspan="1">99.7 (100.0)</td><td align="center" valign="top" rowspan="1" colspan="1">99.9 (100.0)</td><td align="center" valign="top" rowspan="1" colspan="1">99.2 (100.0)</td><td align="center" valign="top" rowspan="1" colspan="1">99.7 (100.0)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Redundancy</td><td align="center" valign="top" rowspan="1" colspan="1">7.8 (7.8)</td><td align="center" valign="top" rowspan="1" colspan="1">7.0 (6.5)</td><td align="center" valign="top" rowspan="1" colspan="1">6.9 (7.0)</td><td align="center" valign="top" rowspan="1" colspan="1">7.1 (7.1)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>Refinement</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.45</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.65</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.40</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.2</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">No. reflections</td><td align="center" valign="top" rowspan="1" colspan="1">12996</td><td align="center" valign="top" rowspan="1" colspan="1">10548</td><td align="center" valign="top" rowspan="1" colspan="1">13736</td><td align="center" valign="top" rowspan="1" colspan="1">18080</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">R<sub>work</sub>/R<sub>free</sub></td><td align="center" valign="top" rowspan="1" colspan="1">0.22/0.26</td><td align="center" valign="top" rowspan="1" colspan="1">0.22/0.28</td><td align="center" valign="top" rowspan="1" colspan="1">0.20/0.26</td><td align="center" valign="top" rowspan="1" colspan="1">0.19/0.24</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>No. atoms</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Protein</td><td align="center" valign="top" rowspan="1" colspan="1">2211</td><td align="center" valign="top" rowspan="1" colspan="1">2225</td><td align="center" valign="top" rowspan="1" colspan="1">2228</td><td align="center" valign="top" rowspan="1" colspan="1">2284</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Ligand/Ion</td><td align="center" valign="top" rowspan="1" colspan="1">114/3</td><td align="center" valign="top" rowspan="1" colspan="1">130/3</td><td align="center" valign="top" rowspan="1" colspan="1">162/3</td><td align="center" valign="top" rowspan="1" colspan="1">164/4</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Water</td><td align="center" valign="top" rowspan="1" colspan="1">16</td><td align="center" valign="top" rowspan="1" colspan="1">20</td><td align="center" valign="top" rowspan="1" colspan="1">42</td><td align="center" valign="top" rowspan="1" colspan="1">57</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>B-factors</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Protein</td><td align="center" valign="top" rowspan="1" colspan="1">64.97</td><td align="center" valign="top" rowspan="1" colspan="1">71.22</td><td align="center" valign="top" rowspan="1" colspan="1">53.50</td><td align="center" valign="top" rowspan="1" colspan="1">45.16</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Ligand/Ion</td><td align="center" valign="top" rowspan="1" colspan="1">78.98/60.53</td><td align="center" valign="top" rowspan="1" colspan="1">90.03/72.90</td><td align="center" valign="top" rowspan="1" colspan="1">70.67/45.53</td><td align="center" valign="top" rowspan="1" colspan="1">69.09/42.92</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Water</td><td align="center" valign="top" rowspan="1" colspan="1">68.57</td><td align="center" valign="top" rowspan="1" colspan="1">81.02</td><td align="center" valign="top" rowspan="1" colspan="1">64.12</td><td align="center" valign="top" rowspan="1" colspan="1">55.45</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> +<bold>R.m.s deviations</bold> +</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Bond lengths (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">0.002</td><td align="center" valign="top" rowspan="1" colspan="1">0.004</td><td align="center" valign="top" rowspan="1" colspan="1">0.002</td><td align="center" valign="top" rowspan="1" colspan="1">0.004</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Bond angles (°)</td><td align="center" valign="top" rowspan="1" colspan="1">0.630</td><td align="center" valign="top" rowspan="1" colspan="1">1.143</td><td align="center" valign="top" rowspan="1" colspan="1">0.686</td><td align="center" valign="top" rowspan="1" colspan="1">0.650</td></tr></tbody></table> + + 65175 + [Ca2+] / [Na+] 10 mM / 2.5 mM PDB 5HXR 1 mM / 2.5 mM N/A* 0.1 mM / 2.5 mM N/A* 10 mM / 10 mM N/A* Data collection Space group P212121 Cell dimensions a, b, c (Å) 49.70, 72.52, 96.94 49.80, 72.26, 95.80 49.48, 72.47, 96.30 49.88, 72.22, 96.10 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) 2.45 (2.49-2.45) 2.65 (2.70-2.65) 2.40 (2.44-2.40) 2.20 (2.24-2.20) Rsym (%) 11.8 (94.2) 11.1 (92.1) 11.2 (91.3) 10.3 (99.1) I/σI 24.4 (1.6) 20.9 (1.6) 20.4 (1.6) 22.4 (1.8) CC1/2 (0.611) (0.696) (0.632) (0.549) Completeness (%) 99.7 (100.0) 99.9 (100.0) 99.2 (100.0) 99.7 (100.0) Redundancy 7.8 (7.8) 7.0 (6.5) 6.9 (7.0) 7.1 (7.1) Refinement Resolution (Å) 50-2.45 50-2.65 50-2.40 50-2.2 No. reflections 12996 10548 13736 18080 Rwork/Rfree 0.22/0.26 0.22/0.28 0.20/0.26 0.19/0.24 No. atoms Protein 2211 2225 2228 2284 Ligand/Ion 114/3 130/3 162/3 164/4 Water 16 20 42 57 B-factors Protein 64.97 71.22 53.50 45.16 Ligand/Ion 78.98/60.53 90.03/72.90 70.67/45.53 69.09/42.92 Water 68.57 81.02 64.12 55.45 R.m.s deviations Bond lengths (Å) 0.002 0.004 0.002 0.004 Bond angles (°) 0.630 1.143 0.686 0.650 + + + T4.xml + T4 + TABLE + table_foot + 66383 + Values in parenthesis are for highest resolution shell. 5% of the data was used in the Rfree calculation. ‘Ligand’ atoms are from lipids, PEG400 and acetates. + + + T4.xml + T4 + TABLE + table_footnote + 66546 + These structures are virtually identical to that resolved with 10 mM Ca2+ and 2.5 mM Na+ (PDB 5HXR), except for the weakened electron-density signal for the divalent ion, and were therefore not deposited in the PDB. + + + diff --git a/BioC_XML/4919469_v0.xml b/BioC_XML/4919469_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..4fa80828d28e7f77839d5b7ca5d7c0016bf55685 --- /dev/null +++ b/BioC_XML/4919469_v0.xml @@ -0,0 +1,16639 @@ + + + + PMC + 20201223 + pmc.key + + 4919469 + CC BY + no + 0 + 0 + + Investigation of the TOCA1-Cdc42 Interaction + 10.1074/jbc.M116.724294 + 4919469 + 27129201 + M116.724294 + 13875 + 26 + actin CDC42 endocytosis nuclear magnetic resonance (NMR) protein-protein interaction BAR domain CIP4 FBP17 TOCA1 WASP + Author's Choice—Final version free via Creative Commons CC-BY license. + 13890 + surname:Watson;given-names:Joanna R. + surname:Fox;given-names:Helen M. + surname:Nietlispach;given-names:Daniel + surname:Gallop;given-names:Jennifer L. + surname:Owen;given-names:Darerca + surname:Mott;given-names:Helen R. + TITLE + HANDOVER OF Cdc42 TO THE ACTIN REGULATOR N-WASP IS FACILITATED BY DIFFERENTIAL BINDING AFFINITIES* + front + 291 + 2016 + 0 + Investigation of the Interaction between Cdc42 and Its Effector TOCA1 + + 0.8210846 + protein + cleaner0 + 2023-06-30T08:42:49Z + PR: + + Cdc42 + + + 0.9986349 + protein + cleaner0 + 2023-06-30T08:43:34Z + PR: + + TOCA1 + + + + ABSTRACT + abstract + 70 + Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. + + protein + PR: + cleaner0 + 2023-06-30T08:45:55Z + + Transducer of Cdc42-dependent actin assembly protein 1 + + + 0.9983543 + protein + cleaner0 + 2023-06-30T08:43:34Z + PR: + + TOCA1 + + + 0.85511017 + protein_type + cleaner0 + 2023-07-03T10:12:30Z + MESH: + + Rho family small G protein + + + 0.74458474 + protein + cleaner0 + 2023-06-30T08:42:44Z + PR: + + Cdc42 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + 0.9879572 + structure_element + cleaner0 + 2023-07-03T10:19:48Z + SO: + + Src homology 3 + + + 0.99650854 + structure_element + cleaner0 + 2023-07-03T10:19:51Z + SO: + + SH3 + + + 0.99472725 + structure_element + cleaner0 + 2023-07-03T10:19:55Z + SO: + + G protein-binding homology region 1 + + + 0.9979664 + structure_element + cleaner0 + 2023-06-30T08:45:24Z + SO: + + HR1 + + + 0.99841833 + protein + cleaner0 + 2023-06-30T08:43:34Z + PR: + + TOCA1 + + + 0.955355 + protein + cleaner0 + 2023-06-30T08:42:49Z + PR: + + Cdc42 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-07-21T09:53:05Z + + actin + + + 0.886836 + experimental_method + cleaner0 + 2023-07-03T11:42:50Z + MESH: + + solved + + + 0.9950669 + evidence + cleaner0 + 2023-07-03T11:56:18Z + DUMMY: + + structure + + + 0.99860483 + structure_element + cleaner0 + 2023-06-30T08:45:22Z + SO: + + HR1 + + + 0.9988238 + protein + cleaner0 + 2023-06-30T08:43:34Z + PR: + + TOCA1 + + + 0.960793 + evidence + cleaner0 + 2023-07-03T11:56:21Z + DUMMY: + + structural data + + + 0.99884087 + protein + cleaner0 + 2023-06-30T08:43:34Z + PR: + + TOCA1 + + + 0.9984865 + structure_element + cleaner0 + 2023-06-30T08:45:24Z + SO: + + HR1 + + + 0.99869126 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.9985139 + structure_element + cleaner0 + 2023-06-30T08:45:24Z + SO: + + HR1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:24Z + + HR1 + + + 0.99875283 + protein + cleaner0 + 2023-07-03T10:01:15Z + PR: + + TOCA + + + 0.9985581 + structure_element + cleaner0 + 2023-06-30T08:45:24Z + SO: + + HR1 + + + 0.99559635 + protein + cleaner0 + 2023-06-30T08:42:49Z + PR: + + Cdc42 + + + 0.9930555 + protein + cleaner0 + 2023-06-30T08:42:49Z + PR: + + Cdc42 + + + 0.8609198 + site + cleaner0 + 2023-07-03T08:16:02Z + SO: + + G protein-binding regions + + + 0.9987367 + protein + cleaner0 + 2023-06-30T08:43:34Z + PR: + + TOCA1 + + + protein_type + MESH: + cleaner0 + 2023-06-30T08:44:27Z + + Wiskott-Aldrich syndrome protein family + + + 0.9749348 + protein + cleaner0 + 2023-06-30T08:44:49Z + PR: + + N-WASP + + + 0.998451 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.9961826 + protein + cleaner0 + 2023-06-30T08:42:49Z + PR: + + Cdc42 + + + 0.9564533 + protein + cleaner0 + 2023-06-30T08:44:50Z + PR: + + N-WASP + + + 0.82657814 + site + cleaner0 + 2023-07-03T08:16:16Z + SO: + + G protein-binding region + + + 0.9965149 + protein + cleaner0 + 2023-06-30T08:42:49Z + PR: + + Cdc42 + + + 0.98908406 + experimental_method + cleaner0 + 2023-07-03T11:42:54Z + MESH: + + NMR + + + 0.7975464 + site + cleaner0 + 2023-07-03T08:16:23Z + SO: + + Cdc42-binding domain + + + 0.969029 + protein + cleaner0 + 2023-06-30T08:44:50Z + PR: + + N-WASP + + + 0.9986004 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.9983797 + structure_element + cleaner0 + 2023-06-30T08:45:24Z + SO: + + HR1 + + + 0.9954324 + protein + cleaner0 + 2023-06-30T08:42:49Z + PR: + + Cdc42 + + + protein + PR: + cleaner0 + 2023-06-30T08:44:50Z + + N-WASP + + + 0.9983676 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.9985555 + structure_element + cleaner0 + 2023-06-30T08:45:24Z + SO: + + HR1 + + + 0.9982083 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.99187285 + protein + cleaner0 + 2023-06-30T08:42:49Z + PR: + + Cdc42 + + + 0.8817688 + protein + cleaner0 + 2023-06-30T08:42:49Z + PR: + + Cdc42 + + + 0.7142731 + evidence + cleaner0 + 2023-07-03T11:56:24Z + DUMMY: + + binding affinities + + + 0.99870765 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.9540443 + protein + cleaner0 + 2023-06-30T08:44:50Z + PR: + + N-WASP + + + + INTRO + title_1 + 1594 + Introduction + + + INTRO + paragraph + 1607 + The Ras superfamily of small GTPases comprises over 150 members that regulate a multitude of cellular processes in eukaryotes. The superfamily can be divided into five families based on structural and functional similarities: Ras, Rho, Rab, Arf, and Ran. All members share a well defined core structure of ∼20 kDa known as the G domain, which is responsible for guanine nucleotide binding. It is this guanine nucleotide binding that underlies their function as molecular switches, controlling a vast array of signaling pathways. These molecular switches cycle between active, GTP-bound, and inactive, GDP-bound, states with the help of auxiliary proteins. The guanine nucleotide exchange factors mediate formation of the active state by promoting the dissociation of GDP, allowing GTP to bind. The GTPase-activating proteins stimulate the rate of intrinsic GTP hydrolysis, mediating the return to the inactive state (reviewed in Ref.). + + 0.9878229 + protein_type + cleaner0 + 2023-07-03T10:12:35Z + MESH: + + Ras superfamily + + + 0.9795511 + protein_type + cleaner0 + 2023-07-03T10:12:38Z + MESH: + + small GTPases + + + 0.9944252 + taxonomy_domain + cleaner0 + 2023-07-03T10:42:09Z + DUMMY: + + eukaryotes + + + 0.99181974 + protein_type + cleaner0 + 2023-07-03T10:12:48Z + MESH: + + Ras + + + 0.98704046 + protein_type + cleaner0 + 2023-07-03T10:12:51Z + MESH: + + Rho + + + 0.990781 + protein_type + cleaner0 + 2023-07-03T10:12:53Z + MESH: + + Rab + + + 0.9855112 + protein_type + cleaner0 + 2023-07-03T10:12:55Z + MESH: + + Arf + + + 0.93829954 + protein_type + cleaner0 + 2023-07-03T10:12:57Z + MESH: + + Ran + + + 0.9947895 + structure_element + cleaner0 + 2023-07-03T10:20:05Z + SO: + + G domain + + + chemical + CHEBI: + cleaner0 + 2023-07-03T10:20:17Z + + guanine nucleotide + + + 0.997357 + protein_state + cleaner0 + 2023-07-03T10:54:29Z + DUMMY: + + active + + + 0.99498653 + protein_state + cleaner0 + 2023-07-03T08:18:41Z + DUMMY: + + GTP-bound + + + 0.9957853 + protein_state + cleaner0 + 2023-07-03T13:25:57Z + DUMMY: + + inactive + + + 0.9956725 + protein_state + cleaner0 + 2023-07-03T08:18:46Z + DUMMY: + + GDP-bound + + + 0.9952165 + protein_type + cleaner0 + 2023-07-03T10:13:14Z + MESH: + + guanine nucleotide exchange factors + + + 0.9958912 + protein_state + cleaner0 + 2023-07-03T13:26:02Z + DUMMY: + + active + + + 0.9906346 + chemical + cleaner0 + 2023-07-03T08:18:13Z + CHEBI: + + GDP + + + 0.9917891 + chemical + cleaner0 + 2023-07-03T08:18:27Z + CHEBI: + + GTP + + + 0.9971633 + protein_type + cleaner0 + 2023-07-03T10:13:18Z + MESH: + + GTPase-activating proteins + + + chemical + CHEBI: + cleaner0 + 2023-07-03T08:18:28Z + + GTP + + + 0.99240416 + protein_state + cleaner0 + 2023-07-03T13:26:05Z + DUMMY: + + inactive + + + + INTRO + paragraph + 2545 + The overall conformation of small G proteins in the active and inactive states is similar, but they differ significantly in two main regions known as switch I and switch II. These regions are responsible for “sensing” the nucleotide state, with the GTP-bound state showing greater rigidity and the GDP-bound state adopting a more relaxed conformation (reviewed in Ref.). In the active state, G proteins bind to an array of downstream effectors, through which they exert their extensive roles within the cell. The structures of more than 60 small G protein·effector complexes have been solved, and, not surprisingly, the switch regions have been implicated in a large proportion of the G protein-effector interactions (reviewed in Ref.). However, because each of the 150 members of the superfamily interacts with multiple effectors, there are still a huge number of known G protein-effector interactions that have not yet been studied structurally. + + 0.9710317 + protein_type + cleaner0 + 2023-07-03T10:13:22Z + MESH: + + small G proteins + + + 0.9971058 + protein_state + cleaner0 + 2023-07-03T13:27:31Z + DUMMY: + + active + + + 0.9968178 + protein_state + cleaner0 + 2023-07-03T13:27:34Z + DUMMY: + + inactive + + + 0.9972334 + site + cleaner0 + 2023-07-03T09:34:36Z + SO: + + switch I + + + 0.997305 + site + cleaner0 + 2023-07-03T09:26:23Z + SO: + + switch II + + + 0.9949434 + protein_state + cleaner0 + 2023-07-03T08:18:42Z + DUMMY: + + GTP-bound + + + 0.9952473 + protein_state + cleaner0 + 2023-07-03T08:18:47Z + DUMMY: + + GDP-bound + + + 0.99743396 + protein_state + cleaner0 + 2023-07-03T13:27:37Z + DUMMY: + + active + + + 0.986418 + protein_type + cleaner0 + 2023-07-03T08:23:00Z + MESH: + + G proteins + + + 0.9969451 + evidence + cleaner0 + 2023-07-03T11:56:29Z + DUMMY: + + structures + + + 0.685185 + protein_type + cleaner0 + 2023-07-03T08:23:06Z + MESH: + + G protein + + + 0.6945974 + experimental_method + cleaner0 + 2023-07-03T11:43:03Z + MESH: + + solved + + + 0.9977436 + site + cleaner0 + 2023-07-03T09:34:22Z + SO: + + switch regions + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:07Z + + G protein + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:07Z + + G protein + + + + INTRO + paragraph + 3498 + The Rho family comprises 20 members, of which three, RhoA, Rac1, and Cdc42, have been relatively well studied. The role of these three proteins in the coordination of the actin cytoskeleton has been examined extensively. RhoA acts to rearrange existing actin structures to form stress fibers, whereas Rac1 and Cdc42 promote de novo actin polymerization to form lamellipodia and filopodia, respectively. A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. + + 0.830895 + protein_type + cleaner0 + 2023-07-03T10:13:32Z + MESH: + + Rho family + + + 0.7184562 + protein + cleaner0 + 2023-06-30T08:46:55Z + PR: + + RhoA + + + 0.7119027 + protein + cleaner0 + 2023-06-30T08:47:05Z + PR: + + Rac1 + + + 0.78280514 + protein + cleaner0 + 2023-06-30T08:42:49Z + PR: + + Cdc42 + + + 0.78088295 + protein + cleaner0 + 2023-06-30T08:46:55Z + PR: + + RhoA + + + 0.69492334 + protein + cleaner0 + 2023-06-30T08:47:06Z + PR: + + Rac1 + + + 0.68728393 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.6052976 + protein + cleaner0 + 2023-06-30T08:46:55Z + PR: + + RhoA + + + 0.5806924 + protein + cleaner0 + 2023-06-30T08:47:06Z + PR: + + Rac1 + + + 0.8666725 + protein_type + cleaner0 + 2023-07-03T10:13:59Z + MESH: + + protein kinase C-related kinase + + + 0.9031287 + protein_type + cleaner0 + 2023-07-03T10:14:03Z + MESH: + + PRK + + + 0.9225908 + protein_type + cleaner0 + 2023-07-03T10:14:05Z + MESH: + + 6 + + + 0.8432732 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + protein_type + MESH: + cleaner0 + 2023-06-30T08:47:37Z + + Wiskott-Aldrich syndrome + + + 0.6893535 + protein_type + cleaner0 + 2023-07-03T10:14:10Z + MESH: + + WASP + + + 0.600192 + protein_type + cleaner0 + 2023-07-03T08:19:56Z + MESH: + + Cdc42-dependent actin assembly + + + 0.56222975 + protein_type + cleaner0 + 2023-07-03T10:14:16Z + MESH: + + TOCA + + + + INTRO + paragraph + 4256 + Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). N-WASP exists in an autoinhibited conformation, which is released upon PI(4,5)P2 and Cdc42 binding or by other factors, such as phosphorylation. Following their release, the C-terminal regions of N-WASP are free to interact with G-actin and a known nucleator of actin assembly, the Arp2/3 complex. The importance of TOCA1 in actin polymerization has been demonstrated in a range of in vitro and in vivo studies, but the exact role of TOCA1 in the many pathways involving actin assembly remains unclear. The most widely studied role of TOCA1 is in membrane invagination and endocytosis, although it has also been implicated in filopodia formation, neurite elongation, transcriptional reprogramming via nuclear actin, and interaction with ZO-1 at tight junctions. A role in cell motility and invasion has also been established. + + 0.88614994 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.9972018 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.87142515 + protein_type + cleaner0 + 2023-07-03T10:14:22Z + MESH: + + WASP family + + + 0.9864705 + protein + cleaner0 + 2023-06-30T08:44:50Z + PR: + + N-WASP + + + 0.73301053 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.9973161 + chemical + cleaner0 + 2023-07-03T10:42:15Z + CHEBI: + + phosphatidylinositol 4,5-bisphosphate + + + 0.9972431 + chemical + cleaner0 + 2023-07-03T09:41:52Z + CHEBI: + + PI(4,5)P2 + + + 0.9875979 + protein + cleaner0 + 2023-06-30T08:44:50Z + PR: + + N-WASP + + + 0.8615777 + protein_state + cleaner0 + 2023-07-03T13:27:44Z + DUMMY: + + autoinhibited conformation + + + 0.9969345 + chemical + cleaner0 + 2023-07-03T09:41:52Z + CHEBI: + + PI(4,5)P2 + + + 0.8908446 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.99487746 + structure_element + cleaner0 + 2023-07-03T10:20:45Z + SO: + + C-terminal regions + + + 0.98714715 + protein + cleaner0 + 2023-06-30T08:44:50Z + PR: + + N-WASP + + + 0.99644774 + protein_type + cleaner0 + 2023-07-03T13:19:34Z + MESH: + + G-actin + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:20:42Z + + Arp2/3 + + + 0.998018 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.99811685 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.9979778 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-07-21T09:48:20Z + + actin + + + 0.9499348 + protein + cleaner0 + 2023-07-03T10:01:30Z + PR: + + ZO-1 + + + + INTRO + paragraph + 5309 + TOCA1 comprises an N-terminal F-BAR domain, a central homology region 1 (HR1) domain, and a C-terminal SH3 domain. The F-BAR domain is a known dimerization, membrane-binding, and membrane-deforming module found in a number of cell signaling proteins. The TOCA1 SH3 domain has many known binding partners, including N-WASP and dynamin. The HR1 domain has been directly implicated in the interaction between TOCA1 and Cdc42, representing the first Cdc42-HR1 domain interaction to be identified. + + 0.99863905 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + 0.9966971 + structure_element + cleaner0 + 2023-07-03T10:21:05Z + SO: + + central homology region 1 + + + 0.9981489 + structure_element + cleaner0 + 2023-06-30T08:45:24Z + SO: + + HR1 + + + 0.9985121 + structure_element + cleaner0 + 2023-07-03T10:21:09Z + SO: + + SH3 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + 0.99870265 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-07-03T10:21:01Z + + SH3 + + + 0.9708707 + protein + cleaner0 + 2023-06-30T08:44:50Z + PR: + + N-WASP + + + 0.99252945 + protein + cleaner0 + 2023-07-03T10:02:37Z + PR: + + dynamin + + + 0.99865294 + structure_element + cleaner0 + 2023-06-30T08:45:24Z + SO: + + HR1 + + + 0.9986656 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.52810615 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.53813136 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.48928082 + structure_element + cleaner0 + 2023-06-30T08:45:24Z + SO: + + HR1 + + + + INTRO + paragraph + 5802 + Other HR1 domains studied so far, including those from the PRK family, have been found to bind their cognate Rho family G protein-binding partner with high specificity and affinities in the nanomolar range. The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. Both of the G protein switch regions are involved in the interaction. The coiled-coil fold is shared by the HR1 domain of the TOCA family protein, CIP4, and, based on sequence homology, by TOCA1 itself. These HR1 domains, however, show specificity for Cdc42, rather than RhoA or Rac1. How different HR1 domain proteins distinguish their specific G protein partners remains only partially understood, and structural characterization of a novel G protein-HR1 domain interaction would add to the growing body of information pertaining to these protein complexes. Furthermore, the biological function of the interaction between TOCA1 and Cdc42 remains poorly understood, and so far there has been no biophysical or structural insight. + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:24Z + + HR1 + + + 0.89815104 + protein_type + cleaner0 + 2023-07-03T10:14:49Z + MESH: + + PRK family + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:07Z + + G protein + + + 0.99019694 + evidence + cleaner0 + 2023-07-03T11:56:35Z + DUMMY: + + affinities + + + 0.99401087 + evidence + cleaner0 + 2023-07-03T11:56:38Z + DUMMY: + + structures + + + 0.9980007 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.99824715 + structure_element + cleaner0 + 2023-07-03T10:21:14Z + SO: + + HR1a + + + 0.94389236 + protein_state + cleaner0 + 2023-07-03T13:27:49Z + DUMMY: + + complex with + + + 0.9936864 + protein + cleaner0 + 2023-06-30T08:46:56Z + PR: + + RhoA + + + structure_element + SO: + cleaner0 + 2023-07-03T10:21:30Z + + HR1b + + + 0.9635602 + protein_state + cleaner0 + 2023-07-03T13:27:52Z + DUMMY: + + complex with + + + 0.9928364 + protein + cleaner0 + 2023-06-30T08:47:06Z + PR: + + Rac1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:24Z + + HR1 + + + 0.99282736 + structure_element + cleaner0 + 2023-07-03T10:21:38Z + SO: + + anti-parallel coiled-coil + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:07Z + + G protein + + + 0.70623326 + structure_element + cleaner0 + 2023-07-03T10:21:41Z + SO: + + helices + + + site + SO: + cleaner0 + 2023-07-03T10:02:22Z + + G protein switch regions + + + 0.996804 + structure_element + cleaner0 + 2023-07-03T10:21:44Z + SO: + + coiled-coil fold + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:24Z + + HR1 + + + 0.9743039 + protein_type + cleaner0 + 2023-07-03T10:14:55Z + MESH: + + TOCA family protein + + + 0.99742043 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.99869365 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:24Z + + HR1 + + + 0.9809748 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.97429186 + protein + cleaner0 + 2023-06-30T08:46:56Z + PR: + + RhoA + + + 0.97543836 + protein + cleaner0 + 2023-06-30T08:47:06Z + PR: + + Rac1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:24Z + + HR1 + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:07Z + + G protein + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:07Z + + G protein + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:24Z + + HR1 + + + 0.99876165 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.9816049 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + + INTRO + paragraph + 6973 + The interactions of TOCA1 and N-WASP with Cdc42 as well as with each other have raised questions as to whether the two Cdc42 effectors can interact with a single molecule of Cdc42 simultaneously. There is some evidence for a ternary complex between Cdc42, N-WASP, and TOCA1, but there was no direct demonstration of simultaneous contacts between the two effectors and a single molecule of Cdc42. Nonetheless, the substantial difference between the structures of the G protein-binding regions of the two effectors is intriguing and implies that they bind to Cdc42 quite differently, providing motivation for investigating the possibility that Cdc42 can bind both effectors concurrently. WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1. + + 0.99872357 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.98440695 + protein + cleaner0 + 2023-06-30T08:44:50Z + PR: + + N-WASP + + + 0.9968567 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.87137836 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.99684143 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.9507448 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.97352785 + protein + cleaner0 + 2023-06-30T08:44:50Z + PR: + + N-WASP + + + 0.998511 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.99682486 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.9932863 + evidence + cleaner0 + 2023-07-03T11:56:46Z + DUMMY: + + structures + + + 0.8868308 + site + cleaner0 + 2023-07-03T08:16:05Z + SO: + + G protein-binding regions + + + 0.99732095 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.9966552 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.9979085 + protein_type + cleaner0 + 2023-07-03T10:22:06Z + MESH: + + WASP + + + 0.9968953 + protein + cleaner0 + 2023-06-30T08:42:50Z + PR: + + Cdc42 + + + 0.99366945 + protein_state + cleaner0 + 2023-07-03T13:28:04Z + DUMMY: + + conserved + + + 0.9845956 + structure_element + cleaner0 + 2023-07-03T10:21:52Z + SO: + + unstructured binding motif + + + structure_element + SO: + cleaner0 + 2023-07-03T08:24:50Z + + Cdc42- and Rac-interactive binding region + + + 0.99738663 + structure_element + cleaner0 + 2023-07-03T10:22:31Z + SO: + + CRIB + + + 0.99092513 + structure_element + cleaner0 + 2023-07-03T10:22:34Z + SO: + + intermolecular β-sheet + + + 0.8364334 + structure_element + cleaner0 + 2023-07-03T10:22:37Z + SO: + + β2 and β3 strands + + + 0.9955338 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.9845709 + protein_type + cleaner0 + 2023-07-03T10:15:21Z + MESH: + + TOCA family proteins + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:24Z + + HR1 + + + 0.9933011 + structure_element + cleaner0 + 2023-07-03T10:22:40Z + SO: + + triple coiled-coil + + + 0.9933278 + site + cleaner0 + 2023-07-03T09:26:23Z + SO: + + switch II + + + 0.97717255 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + structure_element + SO: + cleaner0 + 2023-07-03T10:22:53Z + + HR1b + + + 0.99831796 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + + INTRO + paragraph + 8068 + Here, we present the solution NMR structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We also present data pertaining to binding of the TOCA HR1 domain to Cdc42, which is the first biophysical description of an HR1 domain binding this particular Rho family small G protein. Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics. + + 0.9930488 + experimental_method + cleaner0 + 2023-07-03T11:43:09Z + MESH: + + solution NMR + + + 0.9892476 + evidence + cleaner0 + 2023-07-03T11:56:51Z + DUMMY: + + structure + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:24Z + + HR1 + + + 0.99888533 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.93522906 + evidence + cleaner0 + 2023-07-03T11:56:53Z + DUMMY: + + structural data + + + 0.99885 + protein_type + cleaner0 + 2023-07-03T10:22:23Z + MESH: + + TOCA + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:24Z + + HR1 + + + 0.986479 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.9983109 + structure_element + cleaner0 + 2023-06-30T08:45:25Z + SO: + + HR1 + + + 0.9928767 + protein_type + cleaner0 + 2023-07-03T10:15:27Z + MESH: + + Rho family small G protein + + + 0.99433196 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.9940287 + site + cleaner0 + 2023-07-03T08:16:05Z + SO: + + G protein-binding regions + + + 0.9988475 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + 0.96599275 + protein + cleaner0 + 2023-06-30T08:44:50Z + PR: + + N-WASP + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:07Z + + G protein + + + 0.9955519 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.94650984 + protein + cleaner0 + 2023-06-30T08:44:50Z + PR: + + N-WASP + + + 0.9988159 + protein + cleaner0 + 2023-06-30T08:43:35Z + PR: + + TOCA1 + + + protein_type + MESH: + melaniev@ebi.ac.uk + 2023-07-21T09:54:37Z + + actin + + + + METHODS + title_1 + 8667 + Experimental Procedures + + + METHODS + title_4 + 8691 + Expression Constructs + + + METHODS + paragraph + 8713 + The Xenopus tropicalis TOCA1 HR1 domain (residues 330–426 and N-terminally extended constructs as indicated) were amplified from cDNA (TOCA1 accession number NM_001005148) and cloned into pGEX-6P-1 (GE Healthcare) or pGEX-HisP. The HR1 domain of human CIP4 (residues 388–481) was amplified from IMAGE clone 3532036, the Xenopus laevis FBP17 HR1 domain (residues 385–486) from IMAGE clone 5514481, and the X. tropicalis N-WASP G protein-binding domain (GBD) (residues 197–255) from IMAGE clone 5379332, and all were cloned into pGEX-6P-1. The resulting constructs express the proteins as N-terminal GST fusions with a 3C protease-cleavable tag, with pGEX-HisP expressing an additional C-terminal His6 tag. Human Cdc42Δ7Q61L and full-length Cdc42 were cloned into pGEX-2T (GE Healthcare) and pGEX-6P-1, respectively. A C-terminally extended construct of TOCA1 comprising residues 330–545 was cloned into pMAT10-P.7 The resulting construct expresses TOCA1 330–545 as an N-terminal His-MBP fusion protein with a 3C protease-cleavable tag. Full-length X. tropicalis TOCA1, TOCA1 F-BAR (residues 1–287), and TOCA1 ΔSH3 (residues 1–480) were PCR-amplified from a cDNA clone (IMAGE 5157175) and cloned into pET-His6-SNAP using FseI and AscI sites that had been incorporated into the primers to create His-SNAP-TOCA1 proteins. + + + METHODS + title_4 + 10049 + Protein Expression + + + METHODS + paragraph + 10068 + GST fusion proteins (HR1 domains and Cdc42) were expressed in E. coli BL21 cells (Invitrogen). Stationary cultures were diluted 1:10 and grown at 37 °C until an A600 of ∼0.8 was reached and then induced with 0.1 mm isopropyl-β-d-thiogalactopyranoside for 20 h at 20 °C. The GST-N-WASP GBD construct was expressed in E. coli BL21-CodonPlus®-RIL (Agilent Technologies). The proteins were purified using glutathione-agarose beads (Sigma) and eluted from the beads by cleavage of the GST tag with 3C protease (HR1 domains, N-WASP GBD, and full-length Cdc42Q61L) or thrombin (Novagen, Cdc42Δ7Q61L) prior to gel filtration on a 16/60 S75 column (GE Healthcare). His-MBP-HR1-SH3 was purified using nickel-nitrilotriacetic acid-agarose beads (Life Technologies) prior to cleavage with 3C protease and gel filtration. Full-length TOCA1, TOCA1 F-BAR, and TOCA1 ΔSH3 were expressed from pET-His6-SNAP in BL21 pLysS, grown at 37 °C until an A600 of ∼0.6 was reached, and induced with 0.3 mm isopropyl-β-d-thiogalactopyranoside overnight at 19 °C. Proteins were coupled to nickel-nitrilotriacetic acid-agarose (Qiagen), eluted using increasing concentrations of imidazole, and further purified by gel filtration using a 16/60 S200 column (GE Healthcare). All protein concentrations were determined by amino acid analysis (Protein and Nucleic Acid Chemistry Facility, Department of Biochemistry, University of Cambridge). + + + METHODS + title_4 + 11490 + Nucleotide Exchange + + + METHODS + paragraph + 11510 + For NMR experiments, Cdc42 was nucleotide-exchanged for the non-hydrolyzable GTP analogue GMPPNP (Sigma) as described previously. For scintillation proximity assays (SPAs), Cdc42 was loaded with [3H]GTP using [8-3H]GTP (PerkinElmer Life Sciences), as described previously. The protein was confirmed as full-length using mass spectrometry (PNAC facility, Department of Biochemistry, University of Cambridge). + + + METHODS + title_4 + 11918 + SPAs + + + METHODS + paragraph + 11923 + For direct assays, GST-PAK, GST-ACK, or His-tagged TOCA1 constructs were attached to a fluoromicrosphere via an anti-GST or anti-His antibody in the presence of Cdc42Δ7Q61L·[3H]GTP. Binding curves were fitted using a direct binding isotherm to obtain Kd values and their curve-fitting errors for the G protein-effector interactions. For competition assays, free ACK GBD, TOCA1 HR1, TOCA1 HR1SH3, or N-WASP GBD was titrated into a mixture of 30 nm Cdc42Δ7Q61L·[3H]GTP and 30 nm GST-ACK immobilized on a fluoromicrosphere as above. Data were fitted to competition binding isotherms to obtain Kd values and curve-fitting errors, as described previously. + + + METHODS + title_4 + 12582 + NMR Spectroscopy + + + METHODS + paragraph + 12599 + The NMR experiments and resonance assignments of the HR1 domain are described. The NMR experiments were carried out with 0.9 mm 13C/15N-labeled HR1 domain in 20 mm sodium phosphate, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 5 mm DTT, 10% D2O. Distance restraints were derived from a 15N-separated NOESY (100-ms mixing time) recorded on a Bruker DRX500 and a 13C-separated NOESY (100-ms mixing time) recorded on an Avance AV600. NMR data were processed using AZARA (W. Boucher, University of Cambridge) and analyzed using ANALYSIS. + + + METHODS + title_4 + 13121 + Structure Calculation + + + METHODS + paragraph + 13143 + Structures were calculated iteratively using CNS version 1.0 interfaced to Aria version 2.3.1. The PROSLQ force field was used for non-bonded parameters. Backbone torsion angles were estimated from CA, CO, CB, N, and HA chemical shifts using TALOS-N. The “strong” φ and ψ restraints were included with an error of ±2 S.D. values of the averaged TALOS-N predictions. Dihedral angle predictions for residues 323–340 were weak, so no restraints were included for this region. + + + METHODS + title_4 + 13625 + NMR Titrations + + + METHODS + paragraph + 13640 + All of the 15N and 13C HSQCs were recorded at 25 °C in 50 mm sodium phosphate, pH 5.5, 25 mm NaCl, 5 mm MgCl2, 5 mm DTT, 10% D2O on a Bruker DRX500. 15N-HR1 HSQC experiments were recorded on 0.2 mm 15N-HR1 domain with HR1/Cdc42·GMPPNP ratios of 1:0, 1:0.25, 1:0.5, 1:1, and 1:4. Experiments were recorded on 0.27 mm 15N-Cdc42·GMPPNP at Cdc42/HR1 ratios of 1:0, 1:0.25, 1:0.5, and 1:2.2. The 15N HSQC titrations with N-WASP were recorded on 0.6 mm 15N-HR1 domain or 0.15 mm 15N-Cdc42 at the ratios indicated in the figures. + + + METHODS + title_4 + 14166 + Chemical Shift Mapping + + + METHODS + paragraph + 14189 + The chemical shift changes, δ, were calculated using the equation, where δ(1H) and δ(15N) are the chemical shift changes for the 1H and 15N dimensions, respectively. Residues that had disappeared were assigned a δ value larger than the maximum calculated δ for the data set, and residues that were too overlapped to be reliably assigned in the complex spectra were assigned δ = 0. The residues that had shifted more than the mean chemical shift change across the spectrum were classed as significant and were filtered for solvent accessibility using NACCESS. Residues with <50% solvent accessibility were considered to be buried and unavailable for binding. + + + METHODS + title_4 + 14872 + Pyrene Actin Assays + + + METHODS + paragraph + 14892 + Pyrene actin assays were carried out as described previously. Xenopus high speed supernatant was used at 5 mg/ml and supplemented with 0.12 mg/ml pyrene actin as described previously. TOCA1 HR1 domain or N-WASP CRIB domain was added at the concentrations indicated. Liposomes were made, using methods described previously, from 60% phosphatidylcholine, 30% phosphatidylserine, and 10% PI(4,5)P2 to 2 mm final lipid concentration. All of the lipids used were natural brain or liver lipids from Avanti Polar Lipids. The assays were initiated by the addition of 5 μl of liposomes per 200 μl of reaction mix. + + + RESULTS + title_1 + 15499 + Results + + + RESULTS + title_4 + 15507 + Cdc42-TOCA1 Binding + + 0.5092524 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.9295319 + protein + cleaner0 + 2023-06-30T08:43:36Z + PR: + + TOCA1 + + + + RESULTS + paragraph + 15527 + TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. The C. elegans TOCA1 orthologues also bind to Cdc42 via their consensus HR1 domain. The HR1 domains from the PRK family bind their G protein partners with a high affinity, exhibiting a range of submicromolar dissociation constants (Kd) as low as 26 nm. A Kd in the nanomolar range was therefore expected for the interaction of the TOCA1 HR1 domain with Cdc42. + + 0.9986526 + protein + cleaner0 + 2023-06-30T08:43:36Z + PR: + + TOCA1 + + + 0.7888495 + taxonomy_domain + cleaner0 + 2023-07-03T09:39:16Z + DUMMY: + + Xenopus + + + 0.991585 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:32:07Z + + Cdc42·GTPγS + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:32:28Z + + Cdc42·GDP + + + 0.9952996 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.99546283 + protein + cleaner0 + 2023-06-30T08:46:56Z + PR: + + RhoA + + + 0.9461143 + site + cleaner0 + 2023-07-03T12:07:22Z + SO: + + Rho family binding modules + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:25Z + + HR1 + + + 0.9988255 + protein + cleaner0 + 2023-06-30T08:43:36Z + PR: + + TOCA1 + + + 0.9971808 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.9944839 + species + cleaner0 + 2023-07-03T10:50:17Z + MESH: + + C. elegans + + + 0.9773745 + protein + cleaner0 + 2023-06-30T08:43:36Z + PR: + + TOCA1 + + + 0.99759334 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:26Z + + HR1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:26Z + + HR1 + + + 0.9276004 + protein_type + cleaner0 + 2023-07-03T10:15:34Z + MESH: + + PRK family + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:07Z + + G protein + + + 0.9576243 + evidence + cleaner0 + 2023-07-03T11:56:58Z + DUMMY: + + dissociation constants + + + 0.96872586 + evidence + cleaner0 + 2023-07-03T09:00:18Z + DUMMY: + + Kd + + + 0.9735395 + evidence + cleaner0 + 2023-07-03T09:00:18Z + DUMMY: + + Kd + + + 0.9988011 + protein + cleaner0 + 2023-06-30T08:43:36Z + PR: + + TOCA1 + + + 0.9985483 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + 0.9972486 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + + RESULTS + paragraph + 16196 + We generated an X. tropicalis TOCA1 HR1 domain construct encompassing residues 330–426. This region comprises the complete HR1 domain based on secondary structure predictions and sequence alignments with another TOCA family member, CIP4, whose structure has been determined. The interaction between [3H]GTP·Cdc42 and a C-terminally His-tagged TOCA1 HR1 domain construct was investigated using SPA. The binding isotherm for the interaction is shown in Fig. 1A, together with the Cdc42-PAK interaction as a positive control. The binding of TOCA1 HR1 to Cdc42 was unexpectedly weak, with a Kd of >1 μm. It was not possible to estimate the Kd more accurately using direct SPA experiments, because saturation could not be reached due to nonspecific signal at higher protein concentrations. + + 0.99386984 + species + cleaner0 + 2023-07-03T10:50:22Z + MESH: + + X. tropicalis + + + 0.9987664 + protein + cleaner0 + 2023-06-30T08:43:36Z + PR: + + TOCA1 + + + 0.9366215 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + 0.98613757 + residue_range + cleaner0 + 2023-07-03T10:50:33Z + DUMMY: + + 330–426 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:26Z + + HR1 + + + 0.9026214 + experimental_method + cleaner0 + 2023-07-03T11:43:14Z + MESH: + + sequence alignments + + + 0.9625473 + protein_type + cleaner0 + 2023-07-03T10:15:39Z + MESH: + + TOCA family + + + 0.9986644 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.9560438 + evidence + cleaner0 + 2023-07-03T11:57:02Z + DUMMY: + + structure + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:32:59Z + + [3H]GTP·Cdc42 + + + 0.7794807 + protein_state + cleaner0 + 2023-07-03T10:16:04Z + DUMMY: + + His-tagged + + + 0.9986418 + protein + cleaner0 + 2023-06-30T08:43:36Z + PR: + + TOCA1 + + + 0.9242694 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + 0.99000335 + experimental_method + cleaner0 + 2023-07-03T08:37:43Z + MESH: + + SPA + + + 0.99487644 + evidence + cleaner0 + 2023-07-03T11:57:06Z + DUMMY: + + binding isotherm + + + protein + PR: + cleaner0 + 2023-06-30T08:42:51Z + + Cdc42 + + + 0.59618574 + protein + cleaner0 + 2023-07-03T10:48:35Z + PR: + + PAK + + + 0.9986374 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + 0.9962793 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + 0.9975297 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.9945845 + evidence + cleaner0 + 2023-07-03T09:00:18Z + DUMMY: + + Kd + + + 0.99462295 + evidence + cleaner0 + 2023-07-03T09:00:18Z + DUMMY: + + Kd + + + 0.988311 + experimental_method + cleaner0 + 2023-07-03T08:37:42Z + MESH: + + SPA + + + + zbc0281646060001.jpg + F1 + FIG + fig_caption + 16985 + The TOCA1 HR1-Cdc42 interaction is low affinity. +A, curves derived from direct binding assays in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-PAK or HR1-His6 in SPAs. The SPA signal was corrected by subtraction of control data with no GST-PAK or HR1-His6. The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. The Kd values derived for the ACK GBD with Cdc42Δ7 and full-length Cdc42 were 0.032 ± 0.01 and 0.011 ± 0.01 μm, respectively. The Kd values derived for the TOCA1 HR1 with Cdc42Δ7 and full-length Cdc42 were 6.05 ± 1.96 and 5.39 ± 1.69 μm, respectively. + + 0.9968162 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + 0.97470653 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + protein + PR: + cleaner0 + 2023-06-30T08:42:51Z + + Cdc42 + + + 0.9902583 + experimental_method + cleaner0 + 2023-07-03T11:43:21Z + MESH: + + direct binding assays + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:34:49Z + + Cdc42Δ7Q61L·[3H]GTP + + + 0.8137186 + experimental_method + cleaner0 + 2023-07-03T11:43:31Z + MESH: + + incubated + + + mutant + MESH: + cleaner0 + 2023-07-03T10:03:34Z + + GST-PAK + + + 0.7914784 + mutant + cleaner0 + 2023-07-03T10:51:18Z + MESH: + + HR1-His6 + + + 0.9534834 + experimental_method + cleaner0 + 2023-07-03T11:43:36Z + MESH: + + SPAs + + + experimental_method + MESH: + cleaner0 + 2023-07-03T08:37:43Z + + SPA + + + mutant + MESH: + cleaner0 + 2023-07-03T10:03:58Z + + GST-PAK + + + 0.9089138 + mutant + cleaner0 + 2023-07-03T10:51:21Z + MESH: + + HR1-His6 + + + 0.67325974 + evidence + cleaner0 + 2023-07-03T11:57:20Z + DUMMY: + + binding isotherm + + + 0.9919743 + evidence + cleaner0 + 2023-07-03T09:00:18Z + DUMMY: + + Kd + + + experimental_method + MESH: + cleaner0 + 2023-07-03T08:39:34Z + + competition SPA + + + 0.85910517 + protein + cleaner0 + 2023-07-03T09:29:57Z + PR: + + ACK + + + 0.9981895 + structure_element + cleaner0 + 2023-07-03T10:23:06Z + SO: + + GBD + + + 0.9983625 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + 0.8750449 + experimental_method + cleaner0 + 2023-07-03T11:43:40Z + MESH: + + titrated + + + mutant + MESH: + cleaner0 + 2023-07-03T08:42:00Z + + GST-ACK + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:34:50Z + + Cdc42Δ7Q61L·[3H]GTP + + + 0.9967753 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + full-length + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:35:25Z + + Cdc42Q61L·[3H]GTP + + + 0.99215734 + evidence + cleaner0 + 2023-07-03T09:00:18Z + DUMMY: + + Kd + + + 0.62932396 + protein + cleaner0 + 2023-07-03T09:29:58Z + PR: + + ACK + + + 0.9980578 + structure_element + cleaner0 + 2023-07-03T10:23:10Z + SO: + + GBD + + + 0.9941896 + mutant + cleaner0 + 2023-07-03T08:36:03Z + MESH: + + Cdc42Δ7 + + + 0.9959187 + protein_state + cleaner0 + 2023-07-03T08:36:10Z + DUMMY: + + full-length + + + 0.99390113 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.9919451 + evidence + cleaner0 + 2023-07-03T09:00:18Z + DUMMY: + + Kd + + + 0.99818856 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + 0.9983367 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + 0.9938573 + mutant + cleaner0 + 2023-07-03T08:36:04Z + MESH: + + Cdc42Δ7 + + + 0.99614626 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + full-length + + + 0.9942028 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + + RESULTS + paragraph + 17901 + It was possible that the low affinity observed was due to negative effects of immobilization of the HR1 domain, so other methods were employed, which utilized untagged proteins. Isothermal titration calorimetry was carried out, but no heat changes were observed at a range of concentrations and temperatures (data not shown), suggesting that the interaction is predominantly entropically driven. Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK. + + 0.998464 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + 0.9960897 + protein_state + cleaner0 + 2023-07-03T13:28:12Z + DUMMY: + + untagged + + + 0.9961185 + experimental_method + cleaner0 + 2023-07-03T11:43:44Z + MESH: + + Isothermal titration calorimetry + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:07Z + + G protein + + + 0.49651623 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + 0.9960615 + experimental_method + cleaner0 + 2023-07-03T11:43:49Z + MESH: + + Infrared interferometry + + + 0.88655823 + protein_state + cleaner0 + 2023-07-03T13:28:15Z + DUMMY: + + immobilized + + + 0.891008 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.99855715 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + 0.9980393 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + 0.9915769 + protein + cleaner0 + 2023-07-03T09:29:58Z + PR: + + ACK + + + + RESULTS + paragraph + 18531 + The affinity was therefore determined using competition SPAs. A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. The data were fitted to a binding isotherm describing competition. Free ACK competed with itself with an affinity of 32 nm, similar to the value obtained by direct binding of 23 nm. The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 μm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments. + + 0.9201407 + evidence + cleaner0 + 2023-07-03T11:57:28Z + DUMMY: + + affinity + + + 0.9952146 + experimental_method + cleaner0 + 2023-07-03T11:43:55Z + MESH: + + competition SPAs + + + 0.77118975 + experimental_method + cleaner0 + 2023-07-03T11:47:18Z + MESH: + + GST fusion + + + 0.9981933 + structure_element + cleaner0 + 2023-07-03T10:23:23Z + SO: + + GBD + + + 0.99730754 + protein + cleaner0 + 2023-07-03T09:29:58Z + PR: + + ACK + + + 0.99683124 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:36:48Z + + [3H]GTP·Cdc42 + + + experimental_method + MESH: + cleaner0 + 2023-07-03T11:46:22Z + + increasing concentrations + + + 0.9938798 + protein_state + cleaner0 + 2023-07-03T13:28:19Z + DUMMY: + + untagged + + + 0.998295 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:26Z + + HR1 + + + mutant + MESH: + cleaner0 + 2023-07-03T08:42:00Z + + GST-ACK + + + 0.9519166 + structure_element + cleaner0 + 2023-07-03T10:23:41Z + SO: + + GBD + + + 0.9709375 + protein_state + cleaner0 + 2023-07-03T13:28:24Z + DUMMY: + + bound to + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:37:09Z + + [3H]GTP·Cdc42 + + + 0.99735993 + protein_state + cleaner0 + 2023-07-03T13:28:28Z + DUMMY: + + free + + + 0.99731404 + protein + cleaner0 + 2023-07-03T09:29:58Z + PR: + + ACK + + + 0.99670666 + structure_element + cleaner0 + 2023-07-03T10:23:26Z + SO: + + GBD + + + 0.9976126 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.68896246 + evidence + cleaner0 + 2023-07-03T11:58:31Z + DUMMY: + + binding isotherm + + + 0.9973116 + protein_state + cleaner0 + 2023-07-03T13:28:39Z + DUMMY: + + Free + + + 0.9951958 + protein + cleaner0 + 2023-07-03T09:29:58Z + PR: + + ACK + + + 0.8252186 + evidence + cleaner0 + 2023-07-03T11:57:32Z + DUMMY: + + affinity + + + experimental_method + MESH: + cleaner0 + 2023-07-03T11:46:35Z + + direct binding + + + 0.9981353 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + 0.9980393 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + mutant + MESH: + cleaner0 + 2023-07-03T08:42:00Z + + GST-ACK + + + 0.5593172 + protein_state + cleaner0 + 2023-07-03T13:28:44Z + DUMMY: + + bound + + + evidence + DUMMY: + cleaner0 + 2023-07-03T13:28:53Z + + affinity + + + evidence + DUMMY: + cleaner0 + 2023-07-03T11:57:51Z + + affinity + + + experimental_method + MESH: + cleaner0 + 2023-07-03T11:46:56Z + + direct binding experiments + + + + RESULTS + paragraph + 19358 + The Cdc42 construct used in the binding assays has seven residues deleted from the C terminus to facilitate purification. These residues are not generally required for G protein-effector interactions, including the interaction between RhoA and the PRK1 HR1a domain. In contrast, the C terminus of Rac1 contains a polybasic sequence, which is crucial for Rac1 binding to the HR1b domain from PRK1. As the observed affinity between TOCA1 HR1 and Cdc42 was much lower than expected, we reasoned that the C terminus of Cdc42 might be necessary for a high affinity interaction. The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). Thus, the C-terminal region of Cdc42 is not required for maximal binding of TOCA1 HR1. + + 0.8992194 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.9828191 + experimental_method + cleaner0 + 2023-07-03T11:47:01Z + MESH: + + binding assays + + + 0.6498607 + residue_range + cleaner0 + 2023-07-03T10:50:48Z + DUMMY: + + seven residues + + + 0.8628736 + experimental_method + cleaner0 + 2023-07-03T11:47:21Z + MESH: + + deleted + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:07Z + + G protein + + + 0.99057966 + protein + cleaner0 + 2023-06-30T08:46:56Z + PR: + + RhoA + + + 0.9981152 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.9985483 + structure_element + cleaner0 + 2023-07-03T10:23:49Z + SO: + + HR1a + + + 0.98936355 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.5294273 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.99855596 + structure_element + cleaner0 + 2023-07-03T10:24:03Z + SO: + + HR1b + + + 0.9981103 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.9897843 + evidence + cleaner0 + 2023-07-03T11:57:57Z + DUMMY: + + affinity + + + 0.99851173 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + 0.99845076 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + 0.97940904 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.8183707 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + evidence + DUMMY: + cleaner0 + 2023-07-03T11:58:15Z + + affinity + + + 0.97323084 + experimental_method + cleaner0 + 2023-07-03T11:48:27Z + MESH: + + binding experiments + + + 0.9973202 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + full-length + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:38:23Z + + [3H]GTP·Cdc42 + + + 0.984329 + evidence + cleaner0 + 2023-07-03T11:58:00Z + DUMMY: + + affinity + + + 0.99854726 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + 0.9974193 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + full-length + + + 0.95086724 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.81067705 + evidence + cleaner0 + 2023-07-03T11:58:02Z + DUMMY: + + affinity + + + 0.99599564 + protein_state + cleaner0 + 2023-07-03T13:29:01Z + DUMMY: + + truncated + + + 0.87006044 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + evidence + DUMMY: + cleaner0 + 2023-07-03T09:00:18Z + + Kd + + + structure_element + SO: + cleaner0 + 2023-07-03T10:24:26Z + + C-terminal region + + + 0.6816385 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.99867505 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + 0.998273 + structure_element + cleaner0 + 2023-06-30T08:45:26Z + SO: + + HR1 + + + + RESULTS + paragraph + 20219 + Another possible explanation for the low affinities observed was that the HR1 domain alone is not sufficient for maximal binding of the TOCA proteins to Cdc42 and that the other domains are required. Indeed, GST pull-downs performed with in vitro translated human TOCA1 fragments had suggested that residues N-terminal to the HR1 domain may be required to stabilize the HR1 domain structure. Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. TOCA1 dimerizes via its F-BAR domain, which could also affect Cdc42 binding, for example by presenting two HR1 domains for Cdc42 interactions. Various TOCA1 fragments (Fig. 2A) were therefore assessed for binding to full-length Cdc42 by direct SPA. The isolated F-BAR domain showed no binding to full-length Cdc42 (Fig. 2B). Full-length TOCA1 and ΔSH3 TOCA1 bound with micromolar affinity (Fig. 2B), in a similar manner to the isolated HR1 domain (Fig. 1A). The HR1-SH3 protein could not be purified to homogeneity as a fusion protein, so it was assayed in competition assays after cleavage of the His tag. This construct competed with GST-ACK GBD to give a similar affinity to the HR1 domain alone (Kd = 4.6 ± 4 μm; Fig. 2C). Taken together, these data suggest that the TOCA1 HR1 domain is sufficient for maximal binding and that this binding is of a relatively low affinity compared with many other Cdc42·effector complexes. + + 0.4559165 + evidence + cleaner0 + 2023-07-03T11:58:36Z + DUMMY: + + affinities + + + 0.9986058 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.9021469 + protein_state + cleaner0 + 2023-07-03T13:29:07Z + DUMMY: + + alone + + + protein_type + MESH: + cleaner0 + 2023-07-03T10:04:59Z + + TOCA proteins + + + 0.99680364 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.9955291 + experimental_method + cleaner0 + 2023-07-03T11:48:33Z + MESH: + + GST pull-downs + + + 0.99461246 + species + cleaner0 + 2023-07-03T10:50:27Z + MESH: + + human + + + 0.9986971 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:27Z + + HR1 + + + 0.99834037 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.99793243 + structure_element + cleaner0 + 2023-07-03T10:24:32Z + SO: + + BAR + + + 0.99475163 + structure_element + cleaner0 + 2023-07-03T10:24:35Z + SO: + + SH3 + + + 0.80330014 + protein_type + cleaner0 + 2023-07-03T08:23:02Z + MESH: + + G proteins + + + structure_element + SO: + cleaner0 + 2023-07-03T10:04:39Z + + BAR + + + 0.9988439 + protein + cleaner0 + 2023-07-03T10:04:11Z + PR: + + Arfaptin2 + + + 0.991657 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.9981688 + protein + cleaner0 + 2023-07-03T10:05:05Z + PR: + + Arl1 + + + 0.9980222 + structure_element + cleaner0 + 2023-07-03T10:24:40Z + SO: + + SH3 + + + 0.98025334 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.9829663 + protein + cleaner0 + 2023-07-03T10:05:39Z + PR: + + guanine nucleotide exchange factor + + + 0.9755714 + protein + cleaner0 + 2023-07-03T10:05:13Z + PR: + + β-PIX + + + 0.9987124 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-03T11:59:00Z + + dimer + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + 0.7265375 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.99834585 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + protein + PR: + cleaner0 + 2023-06-30T08:42:51Z + + Cdc42 + + + 0.99605787 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + 0.99578553 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + full-length + + + 0.9968912 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.9564046 + experimental_method + cleaner0 + 2023-07-03T08:37:43Z + MESH: + + SPA + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + 0.99623495 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + full-length + + + 0.99716526 + protein + cleaner0 + 2023-06-30T08:42:51Z + PR: + + Cdc42 + + + 0.9969497 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + Full-length + + + 0.998749 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + 0.9977174 + mutant + cleaner0 + 2023-07-03T10:51:26Z + MESH: + + ΔSH3 + + + 0.9916163 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + 0.8478406 + protein_state + cleaner0 + 2023-07-03T13:29:13Z + DUMMY: + + bound + + + 0.9983576 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.6989486 + mutant + cleaner0 + 2023-07-03T10:26:50Z + MESH: + + HR1-SH3 + + + 0.96284235 + experimental_method + cleaner0 + 2023-07-03T11:48:38Z + MESH: + + competition assays + + + mutant + MESH: + cleaner0 + 2023-07-03T08:42:00Z + + GST-ACK + + + 0.5497209 + structure_element + cleaner0 + 2023-07-03T10:30:05Z + SO: + + GBD + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:27Z + + HR1 + + + 0.5491047 + protein_state + cleaner0 + 2023-07-03T13:29:17Z + DUMMY: + + alone + + + evidence + DUMMY: + cleaner0 + 2023-07-03T09:00:18Z + + Kd + + + 0.99868995 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + 0.99857676 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + protein + PR: + cleaner0 + 2023-06-30T08:42:52Z + + Cdc42 + + + + zbc0281646060002.jpg + F2 + FIG + fig_caption + 21812 + The Cdc42-HR1 interaction is of low affinity in the context of full-length protein and in TOCA1 paralogues. +A, diagram illustrating the TOCA1 constructs assayed for Cdc42 binding. Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs. The SPA signal was corrected by subtraction of control data with no fusion protein. The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal. C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:40:57Z + + Cdc42-HR1 + + + 0.9972544 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + full-length + + + 0.6960263 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + 0.9876616 + protein + cleaner0 + 2023-06-30T08:43:37Z + PR: + + TOCA1 + + + protein + PR: + cleaner0 + 2023-06-30T08:42:52Z + + Cdc42 + + + 0.9627325 + evidence + cleaner0 + 2023-07-03T11:59:08Z + DUMMY: + + binding curves + + + 0.97862166 + experimental_method + cleaner0 + 2023-07-03T11:48:56Z + MESH: + + direct binding assays + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:34:50Z + + Cdc42Δ7Q61L·[3H]GTP + + + 0.8606415 + experimental_method + cleaner0 + 2023-07-03T11:49:01Z + MESH: + + incubated + + + mutant + MESH: + cleaner0 + 2023-07-03T08:41:54Z + + GST-ACK + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T10:27:27Z + + His-tagged + + + 0.99304646 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.8894759 + experimental_method + cleaner0 + 2023-07-03T11:49:04Z + MESH: + + SPAs + + + 0.575186 + experimental_method + cleaner0 + 2023-07-03T08:37:43Z + MESH: + + SPA + + + 0.90803355 + evidence + cleaner0 + 2023-07-03T11:59:15Z + DUMMY: + + binding isotherm + + + 0.9921481 + evidence + cleaner0 + 2023-07-03T09:00:18Z + DUMMY: + + Kd + + + 0.98921967 + experimental_method + cleaner0 + 2023-07-03T08:39:33Z + MESH: + + competition SPA + + + 0.99752015 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.9798131 + mutant + cleaner0 + 2023-07-03T10:27:06Z + MESH: + + HR1-SH3 + + + 0.9126827 + experimental_method + cleaner0 + 2023-07-03T11:49:08Z + MESH: + + titrated + + + mutant + MESH: + cleaner0 + 2023-07-03T08:42:00Z + + GST-ACK + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:34:50Z + + Cdc42Δ7Q61L·[3H]GTP + + + structure_element + SO: + cleaner0 + 2023-07-03T10:29:13Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T10:29:21Z + + CIP4 + + + structure_element + SO: + cleaner0 + 2023-07-03T10:29:37Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T10:29:46Z + + FBP17 + + + 0.6758686 + experimental_method + cleaner0 + 2023-07-03T11:49:11Z + MESH: + + titrated + + + mutant + MESH: + cleaner0 + 2023-07-03T08:42:00Z + + GST-ACK + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:40:09Z + + Cdc42FLQ61L·[3H]GTP + + + + RESULTS + paragraph + 22768 + The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly. The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain. The affinities of both the FBP17 and CIP4 HR1 domains were also in the low micromolar range (10 and 5 μm, respectively) (Fig. 2, D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family. + + 0.9988821 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.9969554 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + protein + PR: + cleaner0 + 2023-06-30T08:42:52Z + + Cdc42 + + + protein + PR: + cleaner0 + 2023-06-30T08:42:52Z + + Cdc42 + + + 0.92827624 + protein_type + cleaner0 + 2023-07-03T10:16:12Z + MESH: + + TOCA family proteins + + + 0.99891806 + protein + cleaner0 + 2023-07-03T09:39:11Z + PR: + + FBP17 + + + 0.9988306 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.99804926 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.9988697 + protein + cleaner0 + 2023-07-03T09:39:11Z + PR: + + FBP17 + + + 0.9987895 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.65796494 + experimental_method + cleaner0 + 2023-07-03T11:49:16Z + MESH: + + purified + + + protein + PR: + cleaner0 + 2023-06-30T08:42:52Z + + Cdc42 + + + 0.98995584 + experimental_method + cleaner0 + 2023-07-03T11:49:22Z + MESH: + + competition SPAs + + + 0.9988158 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.99836105 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.98984015 + evidence + cleaner0 + 2023-07-03T11:59:19Z + DUMMY: + + affinities + + + 0.99884534 + protein + cleaner0 + 2023-07-03T09:39:11Z + PR: + + FBP17 + + + 0.9987373 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.9981744 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.9975121 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + 0.9836099 + protein_type + cleaner0 + 2023-07-03T10:16:29Z + MESH: + + TOCA family + + + + RESULTS + title_4 + 23334 + Structure of the TOCA1 HR1 Domain + + 0.98409134 + evidence + cleaner0 + 2023-07-03T11:59:27Z + DUMMY: + + Structure + + + 0.99884063 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.9984864 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + + RESULTS + paragraph + 23368 + Because the TOCA1 HR1 domain was sufficient for maximal Cdc42-binding, we used this construct for structural studies. Initial experiments were performed with TOCA1 residues 324–426, but we observed that the N terminus was cleaved during purification to yield a new N terminus at residue 330 (data not shown). We therefore engineered a construct comprising residues 330–426 to produce the minimal, stable HR1 domain. Backbone and side chain resonances were assigned as described. 2,778 non-degenerate NOE restraints were used in initial structure calculations (1,791 unambiguous and 987 ambiguous), derived from three-dimensional 15N-separated NOESY and 13C-separated NOESY experiments. There were 1,845 unambiguous NOEs and 757 ambiguous NOEs after eight iterations. 100 structures were calculated in the final iteration; the 50 lowest energy structures were water-refined; and of these, the 35 lowest energy structures were analyzed. Table 1 indicates that the HR1 domain structure is well defined by the NMR data. + + 0.99864227 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.99835 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + protein + PR: + cleaner0 + 2023-06-30T08:42:52Z + + Cdc42 + + + 0.9986461 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.9822894 + residue_range + cleaner0 + 2023-07-03T10:50:54Z + DUMMY: + + 324–426 + + + 0.6586194 + residue_number + cleaner0 + 2023-07-03T10:52:02Z + DUMMY: + + 330 + + + 0.9837437 + residue_range + cleaner0 + 2023-07-03T10:50:59Z + DUMMY: + + 330–426 + + + 0.99568766 + protein_state + cleaner0 + 2023-07-03T13:29:23Z + DUMMY: + + minimal + + + 0.8597201 + protein_state + cleaner0 + 2023-07-03T13:29:26Z + DUMMY: + + stable + + + 0.99823916 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.86151886 + evidence + cleaner0 + 2023-07-03T09:32:12Z + DUMMY: + + NOE restraints + + + 0.8851528 + experimental_method + cleaner0 + 2023-07-03T11:49:28Z + MESH: + + structure calculations + + + 0.9527896 + experimental_method + cleaner0 + 2023-07-03T11:49:40Z + MESH: + + 15N-separated NOESY + + + 0.9854063 + experimental_method + cleaner0 + 2023-07-03T11:49:43Z + MESH: + + 13C-separated NOESY + + + 0.45354816 + evidence + cleaner0 + 2023-07-03T11:59:34Z + DUMMY: + + NOEs + + + 0.42897236 + evidence + cleaner0 + 2023-07-03T11:59:37Z + DUMMY: + + NOEs + + + 0.9530915 + evidence + cleaner0 + 2023-07-03T11:59:40Z + DUMMY: + + structures + + + 0.5917383 + experimental_method + cleaner0 + 2023-07-03T11:49:50Z + MESH: + + calculated + + + 0.93783134 + evidence + cleaner0 + 2023-07-03T11:59:42Z + DUMMY: + + structures + + + 0.98379946 + evidence + cleaner0 + 2023-07-03T11:59:44Z + DUMMY: + + structures + + + 0.9981406 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.9937012 + evidence + cleaner0 + 2023-07-03T11:59:47Z + DUMMY: + + structure + + + 0.93590724 + experimental_method + cleaner0 + 2023-07-03T11:50:02Z + MESH: + + NMR + + + + + T1 + TABLE + table_caption + 24388 + Experimental restraints and structural statistics + + + + T1 + TABLE + table_footnote + 24438 + a <SA>, the average root mean square deviations for the ensemble ± S.D. + + evidence + DUMMY: + cleaner0 + 2023-07-03T12:00:51Z + + average root mean square deviations + + + + + T1 + TABLE + table_footnote + 24511 + b <SA>c, values for the structure that is closest to the mean. + + 0.96417356 + evidence + cleaner0 + 2023-07-03T12:00:56Z + DUMMY: + + structure + + + + RESULTS + paragraph + 24574 + The structure closest to the mean is shown in Fig. 3A. The two α-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (PDB code 2KE4) and PRK1 (PDB codes 1CXZ and 1URF). A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B. + + 0.99404585 + evidence + cleaner0 + 2023-07-03T12:00:58Z + DUMMY: + + structure + + + 0.8651283 + structure_element + cleaner0 + 2023-07-03T10:30:14Z + SO: + + α-helices + + + 0.99835324 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.9731526 + structure_element + cleaner0 + 2023-07-03T10:30:19Z + SO: + + anti-parallel coiled-coil + + + 0.99825734 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.99867105 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.9985677 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.98215437 + experimental_method + cleaner0 + 2023-07-03T11:50:07Z + MESH: + + sequence alignment + + + 0.99869126 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.9982533 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.9983543 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.9982765 + structure_element + cleaner0 + 2023-07-03T10:30:23Z + SO: + + HR1a + + + 0.9973948 + structure_element + cleaner0 + 2023-07-03T10:30:26Z + SO: + + HR1b + + + 0.9979417 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + + zbc0281646060003.jpg + F3 + FIG + fig_caption + 25001 + The structure of the TOCA1 HR1 domain. +A, the backbone trace of the 35 lowest energy structures of the HR1 domain overlaid with the structure closest to the mean is shown alongside a schematic representation of the structure closest to the mean. Flexible regions at the N and C termini (residues 330–333 and 421–426) are omitted for clarity. B, a sequence alignment of the HR1 domains from TOCA1, CIP4, and PRK1. The secondary structure was deduced using Stride, based on the Ramachandran angles, and is indicated as follows: gray, turn; yellow, α-helix; blue, 310 helix; white, coil. C, a close-up of the N-terminal region of TOCA1 HR1, indicating some of the NOEs defining its position with respect to the two α-helices. Dotted lines, NOE restraints. D, a close-up of the interhelix loop region showing some of the contacts between the loop and helix 1. NOEs are indicated with dotted lines. All structural figures were generated using PyMOL. + + 0.9917618 + evidence + cleaner0 + 2023-07-03T12:01:03Z + DUMMY: + + structure + + + 0.99836546 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.99838996 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.46982583 + evidence + cleaner0 + 2023-07-03T12:01:07Z + DUMMY: + + trace + + + 0.98632556 + evidence + cleaner0 + 2023-07-03T12:01:14Z + DUMMY: + + structures + + + 0.9983033 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.95813525 + evidence + cleaner0 + 2023-07-03T12:01:17Z + DUMMY: + + structure + + + 0.95095694 + evidence + cleaner0 + 2023-07-03T12:01:19Z + DUMMY: + + structure + + + 0.9852314 + residue_range + cleaner0 + 2023-07-03T10:51:04Z + DUMMY: + + 330–333 + + + 0.9833619 + residue_range + cleaner0 + 2023-07-03T10:51:07Z + DUMMY: + + 421–426 + + + 0.9557089 + experimental_method + cleaner0 + 2023-07-03T11:50:11Z + MESH: + + sequence alignment + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:27Z + + HR1 + + + 0.9984187 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.9982509 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.9978508 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.8259468 + experimental_method + cleaner0 + 2023-07-03T11:50:20Z + MESH: + + Stride + + + 0.98253715 + evidence + cleaner0 + 2023-07-03T12:01:22Z + DUMMY: + + Ramachandran angles + + + 0.97842044 + structure_element + cleaner0 + 2023-07-03T10:30:31Z + SO: + + α-helix + + + 0.97030413 + structure_element + cleaner0 + 2023-07-03T10:30:34Z + SO: + + 310 helix + + + 0.99778396 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.9985317 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + evidence + DUMMY: + cleaner0 + 2023-07-03T10:30:57Z + + NOEs + + + 0.9908063 + structure_element + cleaner0 + 2023-07-03T10:30:37Z + SO: + + α-helices + + + evidence + DUMMY: + cleaner0 + 2023-07-03T09:32:13Z + + NOE restraints + + + 0.9965214 + structure_element + cleaner0 + 2023-07-03T10:30:40Z + SO: + + interhelix loop + + + 0.99083734 + structure_element + cleaner0 + 2023-07-03T10:30:43Z + SO: + + loop + + + 0.9737114 + structure_element + cleaner0 + 2023-07-03T10:30:45Z + SO: + + helix 1 + + + evidence + DUMMY: + cleaner0 + 2023-07-03T10:31:06Z + + NOEs + + + + RESULTS + paragraph + 25954 + In the HR1a domain of PRK1, a region N-terminal to helix 1 forms a short α-helix, which packs against both helices of the HR1 domain. This region of TOCA1 HR1 (residues 334–340) is well defined in the family of structures (Fig. 3A) but does not form an α-helix. It instead forms a series of turns, defined by NOE restraints observed between residues separated by one (residues 332–334, 333–335, etc.) or two (residues 337–340) residues in the sequence and the φ and ψ angles, assessed using Stride. These turns cause the chain to reverse direction, allowing the N-terminal segment (residues 334–340) to contact both helices of the HR1 domain. Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. These contacts are summarized in Fig. 3C. + + 0.9984731 + structure_element + cleaner0 + 2023-07-03T10:31:13Z + SO: + + HR1a + + + 0.997982 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.9921024 + structure_element + cleaner0 + 2023-07-03T10:31:16Z + SO: + + helix 1 + + + 0.99745536 + structure_element + cleaner0 + 2023-07-03T10:31:18Z + SO: + + short α-helix + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:27Z + + HR1 + + + 0.99764353 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.99843866 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.9797867 + residue_range + cleaner0 + 2023-07-03T10:31:57Z + DUMMY: + + 334–340 + + + 0.9959418 + evidence + cleaner0 + 2023-07-03T12:01:31Z + DUMMY: + + structures + + + 0.9889684 + structure_element + cleaner0 + 2023-07-03T10:31:26Z + SO: + + α-helix + + + 0.9776318 + evidence + cleaner0 + 2023-07-03T09:32:13Z + DUMMY: + + NOE restraints + + + 0.9790006 + residue_range + cleaner0 + 2023-07-03T10:31:52Z + DUMMY: + + 332–334 + + + 0.9800062 + residue_range + cleaner0 + 2023-07-03T10:31:54Z + DUMMY: + + 333–335 + + + 0.97902375 + residue_range + cleaner0 + 2023-07-03T10:31:50Z + DUMMY: + + 337–340 + + + 0.9578368 + evidence + cleaner0 + 2023-07-03T12:01:38Z + DUMMY: + + φ and ψ angles + + + 0.5262887 + experimental_method + cleaner0 + 2023-07-03T11:50:25Z + MESH: + + Stride + + + 0.9672632 + residue_range + cleaner0 + 2023-07-03T10:31:48Z + DUMMY: + + 334–340 + + + 0.9985348 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.73902595 + evidence + cleaner0 + 2023-07-03T12:01:56Z + DUMMY: + + NOEs + + + 0.8344713 + residue_name_number + cleaner0 + 2023-07-03T10:52:15Z + DUMMY: + + Leu-334 + + + 0.88838047 + residue_name_number + cleaner0 + 2023-07-03T10:52:18Z + DUMMY: + + Glu-335 + + + 0.90254325 + residue_name_number + cleaner0 + 2023-07-03T10:52:20Z + DUMMY: + + Asp-336 + + + 0.90361804 + residue_name_number + cleaner0 + 2023-07-03T10:52:22Z + DUMMY: + + Trp-413 + + + 0.8858938 + structure_element + cleaner0 + 2023-07-03T10:31:31Z + SO: + + helix 2 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:47:33Z + + Leu-334 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:47:33Z + + Lys-409 + + + 0.8401229 + structure_element + cleaner0 + 2023-07-03T10:31:34Z + SO: + + helix 2 + + + 0.93479484 + residue_name_number + cleaner0 + 2023-07-03T10:52:29Z + DUMMY: + + Phe-337 + + + 0.9307402 + residue_name_number + cleaner0 + 2023-07-03T10:52:32Z + DUMMY: + + Ser-338 + + + 0.8267959 + residue_name_number + cleaner0 + 2023-07-03T10:52:34Z + DUMMY: + + Arg-345 + + + 0.8390352 + residue_name_number + cleaner0 + 2023-07-03T10:52:37Z + DUMMY: + + Arg-348 + + + 0.8806424 + residue_name_number + cleaner0 + 2023-07-03T10:52:39Z + DUMMY: + + Leu-349 + + + 0.93881685 + structure_element + cleaner0 + 2023-07-03T10:31:37Z + SO: + + helix 1 + + + + RESULTS + paragraph + 26850 + The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 310 helices (residues 381–383 and 386–389). Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D). The backbone NH and CHα groups of Gly-384 and Asp-385 also show NOEs with the side chain of Tyr-377. + + 0.982701 + structure_element + cleaner0 + 2023-07-03T10:31:39Z + SO: + + α-helices + + + 0.9983334 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.99819154 + structure_element + cleaner0 + 2023-06-30T08:45:27Z + SO: + + HR1 + + + 0.9698297 + structure_element + cleaner0 + 2023-07-03T10:31:42Z + SO: + + loop + + + 0.97348136 + residue_range + cleaner0 + 2023-07-03T10:31:45Z + DUMMY: + + 380–389 + + + 0.9480012 + structure_element + cleaner0 + 2023-07-03T10:32:07Z + SO: + + short 310 helices + + + 0.97844666 + residue_range + cleaner0 + 2023-07-03T10:32:01Z + DUMMY: + + 381–383 + + + 0.9779238 + residue_range + cleaner0 + 2023-07-03T10:31:59Z + DUMMY: + + 386–389 + + + 0.9881967 + structure_element + cleaner0 + 2023-07-03T10:32:13Z + SO: + + loop region + + + 0.99350333 + structure_element + cleaner0 + 2023-07-03T10:32:15Z + SO: + + helix 1 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:47:33Z + + Asn-380 + + + 0.9134205 + residue_name_number + cleaner0 + 2023-07-03T10:52:43Z + DUMMY: + + Met-383 + + + 0.9575105 + structure_element + cleaner0 + 2023-07-03T10:32:18Z + SO: + + loop region + + + 0.90971285 + residue_name_number + cleaner0 + 2023-07-03T10:52:46Z + DUMMY: + + Tyr-377 + + + 0.8663984 + residue_name_number + cleaner0 + 2023-07-03T10:52:48Z + DUMMY: + + Val-376 + + + 0.9936434 + structure_element + cleaner0 + 2023-07-03T10:32:20Z + SO: + + helix 1 + + + 0.8596821 + residue_name_number + cleaner0 + 2023-07-03T10:52:50Z + DUMMY: + + Gly-384 + + + 0.8184116 + residue_name_number + cleaner0 + 2023-07-03T10:52:52Z + DUMMY: + + Asp-385 + + + 0.85514146 + residue_name_number + cleaner0 + 2023-07-03T10:52:55Z + DUMMY: + + Tyr-377 + + + + RESULTS + title_4 + 27368 + Mapping the TOCA1 and Cdc42 Binding Interfaces + + 0.9871063 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.9959323 + site + cleaner0 + 2023-07-03T08:44:28Z + SO: + + Cdc42 Binding Interfaces + + + + RESULTS + paragraph + 27415 + The HR1TOCA1-Cdc42 interface was investigated using NMR spectroscopy. A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A). This behavior cannot be explained by the increase in molecular mass (from 12 to 33 kDa) when Cdc42 binds and is more likely to be due to conformational exchange. This leads to broadening of the peaks so that they are not detectable. Overall chemical shift perturbations (CSPs) were calculated for each residue, whereas those that had disappeared were assigned a shift change of 0.2 (Fig. 4B). A peak that disappeared or had a CSP above the mean CSP for the spectrum was considered to be significantly affected. + + 0.9960294 + site + cleaner0 + 2023-07-03T08:44:31Z + SO: + + HR1TOCA1-Cdc42 interface + + + 0.98970586 + experimental_method + cleaner0 + 2023-07-03T11:50:30Z + MESH: + + NMR spectroscopy + + + 0.97077554 + experimental_method + cleaner0 + 2023-07-03T11:50:34Z + MESH: + + 15N HSQC + + + chemical + CHEBI: + cleaner0 + 2023-07-03T13:31:28Z + + 15N + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T13:31:19Z + + labeled + + + 0.99717796 + protein + cleaner0 + 2023-06-30T08:43:38Z + PR: + + TOCA1 + + + 0.9970541 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T08:55:29Z + + presence of + + + 0.74030936 + experimental_method + cleaner0 + 2023-07-03T11:51:00Z + MESH: + + increasing concentrations + + + 0.9769086 + protein_state + cleaner0 + 2023-07-03T09:35:21Z + DUMMY: + + unlabeled + + + 0.7965565 + complex_assembly + cleaner0 + 2023-07-03T08:48:08Z + GO: + + Cdc42Δ7Q61L·GMPPNP + + + 0.9976872 + site + cleaner0 + 2023-07-03T08:44:35Z + SO: + + Cdc42-binding surface + + + 0.91865736 + experimental_method + cleaner0 + 2023-07-03T11:51:06Z + MESH: + + 15N HSQC + + + 0.8572031 + evidence + cleaner0 + 2023-07-03T12:02:03Z + DUMMY: + + spectra + + + 0.9965006 + protein_state + cleaner0 + 2023-07-03T13:29:39Z + DUMMY: + + free + + + 0.9915951 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.9891768 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.6970707 + protein_state + cleaner0 + 2023-07-03T08:55:29Z + DUMMY: + + presence of + + + 0.99477273 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + 0.9363838 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + 0.932084 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + chemical shift perturbations + + + 0.9159061 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSPs + + + 0.9407353 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSP + + + 0.8318418 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSP + + + + zbc0281646060004.jpg + F4 + FIG + fig_caption + 28418 + Mapping the binding surface of Cdc42 onto the TOCA1 HR1 domain. +A, the 15N HSQC of 200 μm TOCA1 HR1 domain is shown in the free form (black) and in the presence of a 4-fold molar excess of Cdc42Δ7Q61L·GMPPNP (red). Expansions of two regions are shown with peak assignments, showing backbone amides in fast or intermediate exchange. B, CSPs were calculated as described under “Experimental Procedures” and are shown for backbone and side chain NH groups. The mean CSP is marked with a red line. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.2 but were excluded when calculating the mean CSP and are indicated with open bars. Those that were not traceable due to spectral overlap were assigned a CSP of zero and are marked with an asterisk below the bar. Residues with affected side chain CSPs derived from 13C HSQCs are marked with green asterisks above the bars. Secondary structure elements are shown below the graph. C, a schematic representation of the HR1 domain. Residues with significantly affected backbone or side chain chemical shifts when Cdc42 bound and that are buried are colored dark blue, whereas those that are solvent-accessible are colored yellow. Residues with significantly affected backbone and side chain groups that are solvent-accessible are colored red. A close-up of the binding region is shown, with affected side chain heavy atoms shown as sticks. D, the G protein-binding region is marked in red onto structures of the HR1 domains as indicated. + + 0.9903181 + site + cleaner0 + 2023-07-03T12:07:28Z + SO: + + binding surface + + + 0.84945166 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + 0.99849176 + protein + cleaner0 + 2023-06-30T08:43:39Z + PR: + + TOCA1 + + + 0.9983016 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.76035815 + experimental_method + cleaner0 + 2023-07-03T11:51:13Z + MESH: + + 15N HSQC + + + 0.99810374 + protein + cleaner0 + 2023-06-30T08:43:39Z + PR: + + TOCA1 + + + 0.9980843 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.97064435 + protein_state + cleaner0 + 2023-07-03T13:29:57Z + DUMMY: + + free form + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T08:55:29Z + + presence of + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:46:25Z + + Cdc42Δ7Q61L·GMPPNP + + + 0.9844333 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSPs + + + 0.8512195 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSP + + + 0.9545903 + protein_state + cleaner0 + 2023-07-03T08:55:29Z + DUMMY: + + presence of + + + 0.9684184 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + 0.98246807 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSP + + + 0.6897554 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSP + + + 0.96462184 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSP + + + 0.9118489 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSPs + + + 0.8412275 + experimental_method + cleaner0 + 2023-07-03T11:51:17Z + MESH: + + 13C HSQCs + + + 0.9983872 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.96418285 + protein_state + cleaner0 + 2023-07-03T13:30:33Z + DUMMY: + + Cdc42 bound + + + 0.8325718 + protein_state + cleaner0 + 2023-07-03T13:30:37Z + DUMMY: + + solvent-accessible + + + 0.85676384 + protein_state + cleaner0 + 2023-07-03T13:30:48Z + DUMMY: + + solvent-accessible + + + 0.99203086 + site + cleaner0 + 2023-07-03T12:07:31Z + SO: + + binding region + + + 0.99327934 + site + cleaner0 + 2023-07-03T08:16:18Z + SO: + + G protein-binding region + + + 0.8936646 + evidence + cleaner0 + 2023-07-03T12:02:19Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:28Z + + HR1 + + + + RESULTS + paragraph + 29929 + 15N HSQC shift mapping experiments report on changes to amide groups, which are mainly inaccessible because they are buried inside the helices and are involved in hydrogen bonds. Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement. The affected CH groups underwent significant line broadening, similarly to the NH peaks. Side chains whose CH groups disappeared in the presence of Cdc42 are marked on the graph in Fig. 4B with green asterisks. + + 0.9546366 + experimental_method + cleaner0 + 2023-07-03T11:51:23Z + MESH: + + 15N HSQC shift mapping + + + 0.96178645 + structure_element + cleaner0 + 2023-07-03T10:32:26Z + SO: + + helices + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:06Z + + hydrogen bonds + + + 0.98837936 + experimental_method + cleaner0 + 2023-07-03T11:51:27Z + MESH: + + 13C HSQC + + + 0.99327534 + experimental_method + cleaner0 + 2023-07-03T11:51:30Z + MESH: + + methyl-selective SOFAST-HMQC + + + 0.3927528 + chemical + cleaner0 + 2023-07-03T13:19:45Z + CHEBI: + + 15N + + + 0.8210875 + chemical + cleaner0 + 2023-07-03T13:19:48Z + CHEBI: + + 13C + + + 0.5613502 + protein_state + cleaner0 + 2023-07-03T13:30:56Z + DUMMY: + + labeled + + + 0.9974596 + protein + cleaner0 + 2023-06-30T08:43:39Z + PR: + + TOCA1 + + + 0.99560153 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.92838216 + protein_state + cleaner0 + 2023-07-03T08:55:29Z + DUMMY: + + presence of + + + 0.90058 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + + RESULTS + paragraph + 30485 + TOCA1 residues whose signals were affected by Cdc42 binding were mapped onto the structure of TOCA1 HR1 (Fig. 4C). The changes were localized to one end of the coiled-coil, and the binding site appeared to include residues from both α-helices and the loop region that joins them. Residues outside of this region were not significantly affected, indicating that there was no widespread conformational change. The residues in the interhelical loop and helix 1 that contact each other (Fig. 3D) show shift changes in their backbone NH and side chains in the presence of Cdc42. For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D). + + 0.99861026 + protein + cleaner0 + 2023-06-30T08:43:39Z + PR: + + TOCA1 + + + 0.94039685 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + 0.9960568 + evidence + cleaner0 + 2023-07-03T12:02:23Z + DUMMY: + + structure + + + 0.99862313 + protein + cleaner0 + 2023-06-30T08:43:39Z + PR: + + TOCA1 + + + 0.99835455 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.99796206 + structure_element + cleaner0 + 2023-07-03T10:32:31Z + SO: + + coiled-coil + + + 0.9978348 + site + cleaner0 + 2023-07-03T12:07:36Z + SO: + + binding site + + + 0.99615437 + structure_element + cleaner0 + 2023-07-03T10:32:34Z + SO: + + α-helices + + + 0.74406147 + structure_element + cleaner0 + 2023-07-03T10:32:36Z + SO: + + loop region + + + 0.9945371 + structure_element + cleaner0 + 2023-07-03T10:32:38Z + SO: + + interhelical loop + + + 0.9971713 + structure_element + cleaner0 + 2023-07-03T10:32:41Z + SO: + + helix 1 + + + 0.9901208 + protein_state + cleaner0 + 2023-07-03T08:55:29Z + DUMMY: + + presence of + + + 0.98446804 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:47:08Z + + Asn-380 + + + 0.90919566 + residue_name_number + cleaner0 + 2023-07-03T10:53:00Z + DUMMY: + + Val-376 + + + 0.9255726 + residue_name_number + cleaner0 + 2023-07-03T10:53:03Z + DUMMY: + + Tyr-377 + + + 0.99773324 + protein_state + cleaner0 + 2023-07-03T13:31:34Z + DUMMY: + + free + + + 0.99823344 + protein + cleaner0 + 2023-06-30T08:43:39Z + PR: + + TOCA1 + + + 0.9959603 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.99719596 + evidence + cleaner0 + 2023-07-03T12:02:26Z + DUMMY: + + structure + + + 0.9835715 + structure_element + cleaner0 + 2023-07-03T10:32:50Z + SO: + + loop + + + 0.947562 + experimental_method + cleaner0 + 2023-07-03T11:51:33Z + MESH: + + chemical shift mapping + + + 0.9967858 + site + cleaner0 + 2023-07-03T08:16:18Z + SO: + + G protein-binding region + + + 0.99871266 + protein + cleaner0 + 2023-06-30T08:43:39Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:28Z + + HR1 + + + 0.99850225 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.9983553 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.998538 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + + RESULTS + paragraph + 31492 + The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. The overall CSP was calculated for each residue. As was the case when labeled HR1 was observed, several peaks were shifted in the complex, but many disappeared, indicating exchange on an unfavorable, millisecond time scale (Fig. 5A). Detailed side chain data could not be obtained for all residues due to spectral overlap, but constant time 13C HSQC and methyl-selective SOFAST-HMQC experiments provided further information on certain well resolved side chains (marked with green asterisks in Fig. 5B). + + 0.94884044 + experimental_method + cleaner0 + 2023-07-03T11:51:39Z + MESH: + + 15N + + + 0.83301735 + experimental_method + cleaner0 + 2023-07-03T11:51:41Z + MESH: + + 13C NMR + + + 0.67671585 + chemical + cleaner0 + 2023-07-03T13:19:52Z + CHEBI: + + 15N + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:48:08Z + + Cdc42Δ7Q61L·GMPPNP + + + 0.7961572 + chemical + cleaner0 + 2023-07-03T13:19:55Z + CHEBI: + + 15N + + + 0.81509006 + chemical + cleaner0 + 2023-07-03T13:19:58Z + CHEBI: + + 13C + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:48:08Z + + Cdc42Δ7Q61L·GMPPNP + + + 0.66658735 + protein_state + cleaner0 + 2023-07-03T08:55:29Z + DUMMY: + + presence of + + + 0.99382895 + protein_state + cleaner0 + 2023-07-03T09:35:21Z + DUMMY: + + unlabeled + + + 0.99755055 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.9804678 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSP + + + 0.99102825 + protein_state + cleaner0 + 2023-07-03T13:31:42Z + DUMMY: + + labeled + + + 0.9974668 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.98695385 + experimental_method + cleaner0 + 2023-07-03T11:52:04Z + MESH: + + constant time 13C HSQC + + + 0.99371284 + experimental_method + cleaner0 + 2023-07-03T11:52:07Z + MESH: + + methyl-selective SOFAST-HMQC + + + + zbc0281646060005.jpg + F5 + FIG + fig_caption + 32166 + Mapping the binding surface of the HR1 domain onto Cdc42. +A, the 15N HSQC of Cdc42Δ7Q61L·GMPPNP is shown in its free form (black) and in the presence of excess TOCA1 HR1 domain (1:2.2, red). Expansions of two regions are shown, with most peaks in fast or intermediate exchange. B, CSPs are shown for backbone NH groups. The red line indicates the mean CSP, plus one S.D. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.1 and are indicated with open bars. Those that were not traceable due to overlap are marked with an asterisk. Residues with disappeared peaks in 13C HSQC experiments are marked on the chart with green asterisks. Secondary structure elements are indicated below the graph. C, the residues with significantly affected backbone and side chain groups are highlighted on an NMR structure of free Cdc42Δ7Q61L·GMPPNP; those that are buried are colored dark blue, whereas those that are solvent-accessible are colored red. Residues with either side chain or backbone groups affected are colored blue if buried and yellow if solvent-accessible. Residues without information from shift mapping are colored gray. The flexible switch regions are circled. + + 0.9814966 + site + cleaner0 + 2023-07-03T12:07:43Z + SO: + + binding surface + + + 0.99824286 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.96610725 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + 0.84818137 + experimental_method + cleaner0 + 2023-07-03T11:52:11Z + MESH: + + 15N HSQC + + + 0.78093916 + complex_assembly + cleaner0 + 2023-07-03T08:48:07Z + GO: + + Cdc42Δ7Q61L·GMPPNP + + + 0.983827 + protein_state + cleaner0 + 2023-07-03T13:31:48Z + DUMMY: + + free form + + + 0.9001876 + protein_state + cleaner0 + 2023-07-03T08:55:30Z + DUMMY: + + presence of + + + 0.9961718 + protein + cleaner0 + 2023-06-30T08:43:39Z + PR: + + TOCA1 + + + 0.99764293 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.98125 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSPs + + + 0.9674589 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSP + + + 0.9740281 + protein_state + cleaner0 + 2023-07-03T08:55:30Z + DUMMY: + + presence of + + + 0.5233301 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + 0.98254895 + experimental_method + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + MESH: + + CSP + + + 0.87693703 + experimental_method + cleaner0 + 2023-07-03T11:52:16Z + MESH: + + 13C HSQC + + + 0.8213444 + experimental_method + cleaner0 + 2023-07-03T11:52:19Z + MESH: + + NMR + + + 0.99046105 + evidence + cleaner0 + 2023-07-03T12:02:32Z + DUMMY: + + structure + + + 0.99755764 + protein_state + cleaner0 + 2023-07-03T13:31:53Z + DUMMY: + + free + + + 0.82752824 + complex_assembly + cleaner0 + 2023-07-03T08:48:08Z + GO: + + Cdc42Δ7Q61L·GMPPNP + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T13:32:16Z + + solvent-accessible + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T13:32:34Z + + solvent-accessible + + + experimental_method + MESH: + cleaner0 + 2023-07-03T11:53:19Z + + shift mapping + + + 0.71962774 + protein_state + cleaner0 + 2023-07-03T13:32:38Z + DUMMY: + + flexible + + + 0.99757636 + site + cleaner0 + 2023-07-03T09:34:22Z + SO: + + switch regions + + + + RESULTS + paragraph + 33364 + As many of the peaks disappeared, the mean chemical shift change was relatively low, so a threshold of the mean plus one S.D. value was used to define a significant CSP. Residues that disappeared were also classed as significantly affected. Parts of the switch regions (Fig. 5, B and C) are invisible in NMR spectra recorded on free Cdc42 due to conformational exchange. These switch regions become visible in Cdc42 and other small G protein·effector complexes due to a decrease in conformational freedom upon complex formation. The switch regions of Cdc42 did not, however, become visible in the presence of the TOCA1 HR1 domain. Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. This suggests that the switch regions are not rigidified in the HR1 complex and are still in conformational exchange. Nevertheless, mapping of the affected residues onto the NMR structure of free Cdc42Δ7Q61L·GMPPNP (Fig. 5C)8 shows that, although they are relatively widespread compared with changes in the HR1 domain, in general, they are on the face of the protein that includes the switches. Although the binding interface may be overestimated, this suggests that the switch regions are involved in binding to TOCA1. + + 0.8175822 + evidence + cleaner0 + 2023-07-03T12:02:35Z + DUMMY: + + mean chemical shift change + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-07-06T15:22:48Z + + CSP + + + 0.9972377 + site + cleaner0 + 2023-07-03T09:34:22Z + SO: + + switch regions + + + 0.8591063 + experimental_method + cleaner0 + 2023-07-03T11:52:32Z + MESH: + + NMR + + + 0.8200651 + evidence + cleaner0 + 2023-07-03T12:02:50Z + DUMMY: + + spectra + + + 0.99719834 + protein_state + cleaner0 + 2023-07-03T13:32:45Z + DUMMY: + + free + + + 0.9924085 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + 0.9964552 + site + cleaner0 + 2023-07-03T09:34:23Z + SO: + + switch regions + + + 0.9869178 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:08Z + + G protein + + + 0.996436 + site + cleaner0 + 2023-07-03T09:34:23Z + SO: + + switch regions + + + 0.9908557 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + 0.9667624 + protein_state + cleaner0 + 2023-07-03T08:55:30Z + DUMMY: + + presence of + + + 0.9985393 + protein + cleaner0 + 2023-06-30T08:43:39Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:28Z + + HR1 + + + 0.78033185 + residue_name_number + cleaner0 + 2023-07-03T10:53:08Z + DUMMY: + + Ser-30 + + + 0.99427783 + site + cleaner0 + 2023-07-03T09:34:35Z + SO: + + switch I + + + 0.81245255 + residue_name_number + cleaner0 + 2023-07-03T10:53:11Z + DUMMY: + + Arg-66 + + + 0.8686878 + residue_name_number + cleaner0 + 2023-07-03T10:53:13Z + DUMMY: + + Arg-68 + + + 0.85998726 + residue_name_number + cleaner0 + 2023-07-03T10:53:15Z + DUMMY: + + Leu-70 + + + 0.83775693 + residue_name_number + cleaner0 + 2023-07-03T10:53:17Z + DUMMY: + + Ser-71 + + + 0.99408966 + site + cleaner0 + 2023-07-03T09:26:23Z + SO: + + switch II + + + 0.99761605 + protein_state + cleaner0 + 2023-07-03T13:32:49Z + DUMMY: + + free + + + 0.9889713 + protein + cleaner0 + 2023-06-30T08:42:52Z + PR: + + Cdc42 + + + 0.9672233 + protein_state + cleaner0 + 2023-07-03T08:55:30Z + DUMMY: + + presence of + + + 0.9982116 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.99595547 + site + cleaner0 + 2023-07-03T09:34:23Z + SO: + + switch regions + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:28Z + + HR1 + + + 0.7635831 + experimental_method + cleaner0 + 2023-07-03T12:03:00Z + MESH: + + NMR + + + 0.9886608 + evidence + cleaner0 + 2023-07-03T12:03:05Z + DUMMY: + + structure + + + 0.99748886 + protein_state + cleaner0 + 2023-07-03T13:32:52Z + DUMMY: + + free + + + 0.711201 + complex_assembly + cleaner0 + 2023-07-03T08:48:08Z + GO: + + Cdc42Δ7Q61L·GMPPNP + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:28Z + + HR1 + + + 0.99592566 + site + cleaner0 + 2023-07-03T09:34:44Z + SO: + + switches + + + 0.99736357 + site + cleaner0 + 2023-07-03T12:07:49Z + SO: + + binding interface + + + 0.99577093 + site + cleaner0 + 2023-07-03T09:34:21Z + SO: + + switch regions + + + 0.99841404 + protein + cleaner0 + 2023-06-30T08:43:39Z + PR: + + TOCA1 + + + + RESULTS + title_4 + 34673 + Modeling the Cdc42·TOCA1 HR1 Complex + + 0.9399527 + complex_assembly + cleaner0 + 2023-07-03T10:48:47Z + GO: + + Cdc42·TOCA1 HR1 + + + + RESULTS + paragraph + 34711 + The Cdc42·HR1TOCA1 complex was not amenable to full structural analysis due to the weak interaction and the extensive exchange broadening seen in the NMR experiments. HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above. Residues with significantly affected resonances and more than 50% solvent accessibility were defined as active. Passive residues were defined automatically as those neighboring active residues. + + 0.8960567 + complex_assembly + cleaner0 + 2023-07-03T10:48:51Z + GO: + + Cdc42·HR1TOCA1 + + + 0.9907706 + experimental_method + cleaner0 + 2023-07-03T11:52:37Z + MESH: + + NMR + + + 0.98815465 + experimental_method + cleaner0 + 2023-07-03T08:50:25Z + MESH: + + HADDOCK + + + experimental_method + MESH: + melaniev@ebi.ac.uk + 2023-07-21T09:49:54Z + + rigid body docking + + + 0.9960586 + evidence + cleaner0 + 2023-07-03T12:03:11Z + DUMMY: + + structures + + + 0.9974323 + protein_state + cleaner0 + 2023-07-03T13:32:56Z + DUMMY: + + free + + + 0.99764234 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.93101466 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + 0.70206016 + experimental_method + cleaner0 + 2023-07-03T11:52:44Z + MESH: + + titration experiments + + + + RESULTS + paragraph + 35276 + The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42. The cluster with the lowest root mean square deviation from the lowest energy structure is assumed to be the best model. By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. A representative model from this cluster is shown in Fig. 6A alongside the Rac1-HR1b structure (PDB code 2RMK) in Fig. 6B. + + 0.9981976 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.99376076 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + 0.98814255 + experimental_method + cleaner0 + 2023-07-03T08:50:25Z + MESH: + + HADDOCK + + + 0.99816895 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.9891117 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + 0.9906749 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + 0.97768056 + evidence + cleaner0 + 2023-07-03T12:03:20Z + DUMMY: + + root mean square deviation + + + 0.9844689 + evidence + cleaner0 + 2023-07-03T12:03:23Z + DUMMY: + + structure + + + 0.9981712 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.9973526 + structure_element + cleaner0 + 2023-07-03T10:32:55Z + SO: + + HR1a + + + 0.99825805 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.9890354 + protein_state + cleaner0 + 2023-07-03T13:33:26Z + DUMMY: + + bound to + + + 0.93857586 + protein + cleaner0 + 2023-06-30T08:46:56Z + PR: + + RhoA + + + 0.997436 + structure_element + cleaner0 + 2023-07-03T10:34:33Z + SO: + + HR1b + + + 0.98779166 + protein_state + cleaner0 + 2023-07-03T13:33:30Z + DUMMY: + + bound to + + + 0.71836126 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + complex_assembly + GO: + cleaner0 + 2023-07-03T10:33:52Z + + Rac1-HR1b + + + 0.992141 + evidence + cleaner0 + 2023-07-03T12:03:25Z + DUMMY: + + structure + + + + zbc0281646060006.jpg + F6 + FIG + fig_caption + 35970 + Model of Cdc42·HR1 complex. +A, a representative model of the Cdc42·HR1 complex from the cluster closest to the lowest energy model produced using HADDOCK. Residues of Cdc42 that are affected in the presence of the HR1 domain but are not in close proximity to it are colored in red and labeled. B, structure of Rac1 in complex with the HR1b domain of PRK1 (PDB code 2RMK). C, sequence alignment of RhoA, Cdc42 and Rac1. Contact residues of RhoA and Rac1 to PRK1 HR1a and HR1b, respectively, are colored cyan. Residues of Cdc42 that disappear or show chemical shift changes in the presence of TOCA1 are colored cyan if also identified as contacts in RhoA and Rac1 and yellow if they are not. Residues equivalent to Rac1 and RhoA contact sites but that are invisible in free Cdc42 are gray. D, regions of interest of the Cdc42·HR1 domain model. The four lowest energy structures in the chosen HADDOCK cluster are shown overlaid, with the residues of interest shown as sticks and labeled. Cdc42 is shown in cyan, and TOCA1 is shown in purple. + + 0.9912746 + complex_assembly + cleaner0 + 2023-07-03T10:48:56Z + GO: + + Cdc42·HR1 + + + 0.9905351 + complex_assembly + cleaner0 + 2023-07-03T10:48:59Z + GO: + + Cdc42·HR1 + + + 0.9757337 + experimental_method + cleaner0 + 2023-07-03T08:50:24Z + MESH: + + HADDOCK + + + 0.77116495 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T08:55:30Z + + presence of + + + 0.9980514 + structure_element + cleaner0 + 2023-06-30T08:45:28Z + SO: + + HR1 + + + 0.93390775 + evidence + cleaner0 + 2023-07-03T12:03:31Z + DUMMY: + + structure + + + 0.68168455 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.8599246 + protein_state + cleaner0 + 2023-07-03T13:33:34Z + DUMMY: + + in complex with + + + 0.99765766 + structure_element + cleaner0 + 2023-07-03T10:34:37Z + SO: + + HR1b + + + 0.9963977 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.9856179 + experimental_method + cleaner0 + 2023-07-03T11:52:49Z + MESH: + + sequence alignment + + + 0.8277353 + protein + cleaner0 + 2023-06-30T08:46:56Z + PR: + + RhoA + + + 0.7358657 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + 0.86080074 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.77970403 + protein + cleaner0 + 2023-06-30T08:46:56Z + PR: + + RhoA + + + 0.77326703 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.99702734 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.996442 + structure_element + cleaner0 + 2023-07-03T10:34:40Z + SO: + + HR1a + + + 0.99660885 + structure_element + cleaner0 + 2023-07-03T10:34:43Z + SO: + + HR1b + + + 0.5916415 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + 0.95339227 + protein_state + cleaner0 + 2023-07-03T08:55:30Z + DUMMY: + + presence of + + + 0.99708956 + protein + cleaner0 + 2023-06-30T08:43:39Z + PR: + + TOCA1 + + + 0.874548 + protein + cleaner0 + 2023-06-30T08:46:56Z + PR: + + RhoA + + + 0.84730613 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.61742294 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.4232686 + protein + cleaner0 + 2023-06-30T08:46:56Z + PR: + + RhoA + + + 0.9848926 + site + cleaner0 + 2023-07-03T12:08:00Z + SO: + + contact sites + + + 0.9961755 + protein_state + cleaner0 + 2023-07-03T13:33:39Z + DUMMY: + + free + + + 0.8306941 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:50:14Z + + Cdc42·HR1 + + + 0.94688374 + evidence + cleaner0 + 2023-07-03T12:03:38Z + DUMMY: + + structures + + + experimental_method + MESH: + cleaner0 + 2023-07-03T08:50:25Z + + HADDOCK + + + 0.96888226 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + 0.99732983 + protein + cleaner0 + 2023-06-30T08:43:39Z + PR: + + TOCA1 + + + + RESULTS + paragraph + 37012 + A sequence alignment of RhoA, Cdc42, and Rac1 is shown in Fig. 6C. The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins. Several Cdc42 residues identified by chemical shift mapping are not in close contact in the Cdc42·TOCA1 model (Fig. 6A). Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. Lys-16Cdc42 is unlikely to be a contact residue because it is involved in nucleotide binding, but the others may represent specific Cdc42-TOCA1 contacts. In the model, these side chains are involved in direct contacts (Fig. 6D). + + 0.9871194 + experimental_method + cleaner0 + 2023-07-03T11:52:53Z + MESH: + + sequence alignment + + + 0.9734803 + protein + cleaner0 + 2023-06-30T08:46:57Z + PR: + + RhoA + + + 0.97490567 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + 0.9736101 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.8214345 + protein + cleaner0 + 2023-06-30T08:46:57Z + PR: + + RhoA + + + 0.81268275 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.9973299 + site + cleaner0 + 2023-07-03T09:34:23Z + SO: + + switch regions + + + 0.98021424 + evidence + cleaner0 + 2023-07-03T12:03:41Z + DUMMY: + + spectra + + + 0.95324767 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + 0.9873949 + protein_state + cleaner0 + 2023-07-03T13:33:46Z + DUMMY: + + conserved + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:02Z + + G proteins + + + 0.33471435 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + 0.9574032 + experimental_method + cleaner0 + 2023-07-03T11:52:56Z + MESH: + + chemical shift mapping + + + 0.9928748 + complex_assembly + cleaner0 + 2023-07-03T08:50:51Z + GO: + + Cdc42·TOCA1 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:51:40Z + + Thr-24 + + + protein + PR: + cleaner0 + 2023-07-03T08:57:58Z + + Cdc42 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:52:03Z + + Leu-160 + + + protein + PR: + cleaner0 + 2023-07-03T08:58:02Z + + Cdc42 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:52:24Z + + Lys-163 + + + protein + PR: + cleaner0 + 2023-07-03T08:58:05Z + + Cdc42 + + + 0.99660486 + site + cleaner0 + 2023-07-03T09:34:36Z + SO: + + switch I + + + 0.9976216 + site + cleaner0 + 2023-07-03T10:34:56Z + SO: + + switch + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:52:50Z + + Glu-95 + + + protein + PR: + cleaner0 + 2023-07-03T08:58:08Z + + Cdc42 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:53:11Z + + Lys-96 + + + protein + PR: + cleaner0 + 2023-07-03T08:58:11Z + + Cdc42 + + + 0.9833968 + structure_element + cleaner0 + 2023-07-03T10:34:59Z + SO: + + helix + + + 0.99711096 + site + cleaner0 + 2023-07-03T09:26:23Z + SO: + + switch II + + + 0.9944163 + complex_assembly + cleaner0 + 2023-07-03T08:50:52Z + GO: + + Cdc42·TOCA1 + + + 0.9544345 + protein + cleaner0 + 2023-06-30T08:46:57Z + PR: + + RhoA + + + 0.9498586 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:53:33Z + + Gln-2 + + + protein + PR: + cleaner0 + 2023-07-03T08:58:14Z + + Cdc42 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:53:53Z + + Lys-16 + + + protein + PR: + cleaner0 + 2023-07-03T08:58:17Z + + Cdc42 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:54:15Z + + Thr-52 + + + protein + PR: + cleaner0 + 2023-07-03T08:58:20Z + + Cdc42 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:54:36Z + + Arg-68 + + + protein + PR: + cleaner0 + 2023-07-03T08:58:23Z + + Cdc42 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T08:54:56Z + + Lys-16 + + + protein + PR: + cleaner0 + 2023-07-03T08:58:28Z + + Cdc42 + + + 0.9658783 + complex_assembly + cleaner0 + 2023-07-03T10:49:05Z + GO: + + Cdc42-TOCA1 + + + + RESULTS + title_4 + 38047 + Competition between N-WASP and TOCA1 + + 0.97834396 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.9982578 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + + RESULTS + paragraph + 38084 + From the known interactions and effects of the proteins in biological systems, it has been suggested that TOCA1 and N-WASP could bind Cdc42 simultaneously. Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. A basic region in WASP including three lysines (residues 230–232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A). + + 0.99870217 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + 0.97666854 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.9955902 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + 0.9863807 + complex_assembly + cleaner0 + 2023-07-03T10:49:16Z + GO: + + Cdc42·N-WASP·TOCA1 + + + 0.4309258 + evidence + cleaner0 + 2023-07-03T12:03:47Z + DUMMY: + + FRET + + + 0.21124144 + chemical + cleaner0 + 2023-07-03T13:20:15Z + CHEBI: + + RFP + + + 0.99814546 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + 0.2754772 + chemical + cleaner0 + 2023-07-03T13:20:18Z + CHEBI: + + GFP + + + 0.94308394 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.9076988 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.6556517 + protein_state + cleaner0 + 2023-07-03T10:51:40Z + DUMMY: + + mutant + + + 0.9952695 + protein + cleaner0 + 2023-06-30T08:42:53Z + PR: + + Cdc42 + + + 0.9898616 + experimental_method + cleaner0 + 2023-07-03T11:53:25Z + MESH: + + overlay + + + 0.69386333 + experimental_method + cleaner0 + 2023-07-03T08:50:26Z + MESH: + + HADDOCK + + + 0.8598288 + evidence + cleaner0 + 2023-07-03T12:03:53Z + DUMMY: + + model + + + 0.7695202 + complex_assembly + cleaner0 + 2023-07-03T10:49:19Z + GO: + + Cdc42·HR1TOCA1 + + + 0.993002 + evidence + cleaner0 + 2023-07-03T12:03:50Z + DUMMY: + + structure + + + 0.9959825 + protein + cleaner0 + 2023-06-30T08:42:54Z + PR: + + Cdc42 + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T13:34:09Z + + in complex with + + + 0.9987808 + structure_element + cleaner0 + 2023-07-03T10:35:03Z + SO: + + GBD + + + 0.9786554 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.9971476 + protein + cleaner0 + 2023-07-03T10:05:52Z + PR: + + WASP + + + 0.9967649 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.9980094 + site + cleaner0 + 2023-07-03T12:08:05Z + SO: + + GBD binding sites + + + 0.96977437 + protein_state + cleaner0 + 2023-07-03T08:55:27Z + DUMMY: + + presence of + + + 0.9988593 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + 0.99852246 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.9982327 + structure_element + cleaner0 + 2023-07-03T10:35:07Z + SO: + + CRIB + + + 0.99749184 + protein + cleaner0 + 2023-07-03T10:07:14Z + PR: + + WASP + + + 0.99687827 + protein + cleaner0 + 2023-06-30T08:42:54Z + PR: + + Cdc42 + + + 0.9986425 + structure_element + cleaner0 + 2023-07-03T10:35:11Z + SO: + + CRIB + + + 0.9971403 + protein + cleaner0 + 2023-07-03T10:07:21Z + PR: + + WASP + + + 0.9988255 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + 0.998401 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.99724376 + protein + cleaner0 + 2023-07-03T10:07:25Z + PR: + + WASP + + + 0.98834026 + residue_name + cleaner0 + 2023-07-03T10:07:28Z + SO: + + lysines + + + 0.96353865 + residue_range + cleaner0 + 2023-07-03T10:07:31Z + DUMMY: + + 230–232 + + + 0.9983254 + structure_element + cleaner0 + 2023-07-03T10:35:14Z + SO: + + CRIB + + + 0.9870566 + protein_state + cleaner0 + 2023-07-03T08:55:30Z + DUMMY: + + presence of + + + 0.9988324 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + 0.99829537 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + + zbc0281646060007.jpg + F7 + FIG + fig_caption + 39243 + The N-WASP GBD displaces the TOCA1 HR1 domain. +A, the model of the Cdc42·TOCA1 HR1 domain complex overlaid with the Cdc42-WASP structure. Cdc42 is shown in green, and TOCA1 is shown in purple. The core CRIB region of WASP is shown in red, whereas its basic region is shown in orange and the C-terminal region required for maximal affinity is shown in cyan. A semitransparent surface representation of Cdc42 and WASP is shown overlaid with the schematic. B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42Δ7Q61L·[3H]GTP. C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. + + 0.95112664 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.997769 + structure_element + cleaner0 + 2023-07-03T10:35:19Z + SO: + + GBD + + + 0.99819857 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + 0.99853647 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.9385584 + complex_assembly + cleaner0 + 2023-07-03T08:50:52Z + GO: + + Cdc42·TOCA1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:29Z + + HR1 + + + 0.97159547 + complex_assembly + cleaner0 + 2023-07-03T10:49:25Z + GO: + + Cdc42-WASP + + + 0.98695135 + evidence + cleaner0 + 2023-07-03T12:04:19Z + DUMMY: + + structure + + + 0.74240917 + protein + cleaner0 + 2023-06-30T08:42:54Z + PR: + + Cdc42 + + + 0.99751484 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-07-03T10:07:59Z + + CRIB + + + 0.97892684 + protein + cleaner0 + 2023-07-03T10:08:06Z + PR: + + WASP + + + 0.70855325 + protein + cleaner0 + 2023-06-30T08:42:54Z + PR: + + Cdc42 + + + 0.98284966 + protein + cleaner0 + 2023-07-03T10:08:28Z + PR: + + WASP + + + 0.9904779 + experimental_method + cleaner0 + 2023-07-03T08:39:34Z + MESH: + + competition SPA + + + 0.8967762 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.9722796 + structure_element + cleaner0 + 2023-07-03T10:35:24Z + SO: + + GBD + + + 0.9190314 + experimental_method + cleaner0 + 2023-07-03T11:53:33Z + MESH: + + titrated + + + mutant + MESH: + cleaner0 + 2023-07-03T08:42:00Z + + GST-ACK + + + mutant + MESH: + cleaner0 + 2023-07-03T08:57:47Z + + GST-WASP + + + 0.9404615 + structure_element + cleaner0 + 2023-07-03T10:35:29Z + SO: + + GBD + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:34:50Z + + Cdc42Δ7Q61L·[3H]GTP + + + 0.9067199 + experimental_method + cleaner0 + 2023-07-03T12:04:44Z + MESH: + + 15N HSQC + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:48:08Z + + Cdc42Δ7Q61L·GMPPNP + + + 0.9981357 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + 0.99832815 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.94294506 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.9961972 + structure_element + cleaner0 + 2023-07-03T10:35:34Z + SO: + + GBD + + + 0.99766076 + protein + cleaner0 + 2023-07-03T10:08:35Z + PR: + + TOCA + + + 0.99845433 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.94999 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.9978635 + structure_element + cleaner0 + 2023-07-03T10:35:37Z + SO: + + GBD + + + 0.9153466 + experimental_method + cleaner0 + 2023-07-03T12:04:29Z + MESH: + + 15N HSQC + + + 0.99819165 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + 0.9979571 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + protein_state + DUMMY: + melaniev@ebi.ac.uk + 2023-07-06T15:23:26Z + + in complex with + + + 0.99709153 + protein + cleaner0 + 2023-06-30T08:42:54Z + PR: + + Cdc42 + + + 0.9785906 + protein_state + cleaner0 + 2023-07-03T13:35:00Z + DUMMY: + + absence + + + 0.6804149 + protein_state + cleaner0 + 2023-07-03T08:55:30Z + DUMMY: + + presence of + + + 0.9550614 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.99571025 + structure_element + cleaner0 + 2023-07-03T10:35:40Z + SO: + + GBD + + + 0.9975139 + protein + cleaner0 + 2023-06-30T08:42:54Z + PR: + + Cdc42 + + + 0.9984224 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.96989036 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + + RESULTS + paragraph + 40284 + An N-WASP GBD construct was produced, and its affinity for Cdc42 was measured by competition SPA (Fig. 7B). The Kd that was determined (37 nm) is consistent with the previously reported affinity. Unlabeled N-WASP GBD was titrated into 15N-Cdc42Δ7Q61L·GMPPNP, and the backbone NH groups were monitored using HSQCs (Fig. 7C). Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). + + 0.9396019 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.66169256 + structure_element + cleaner0 + 2023-07-03T10:35:45Z + SO: + + GBD + + + 0.9258649 + evidence + cleaner0 + 2023-07-03T12:04:52Z + DUMMY: + + affinity + + + 0.99747723 + protein + cleaner0 + 2023-06-30T08:42:54Z + PR: + + Cdc42 + + + 0.989219 + experimental_method + cleaner0 + 2023-07-03T08:39:34Z + MESH: + + competition SPA + + + 0.9949738 + evidence + cleaner0 + 2023-07-03T09:00:18Z + DUMMY: + + Kd + + + 0.95968497 + evidence + cleaner0 + 2023-07-03T12:04:58Z + DUMMY: + + affinity + + + 0.9111927 + protein_state + cleaner0 + 2023-07-03T09:35:21Z + DUMMY: + + Unlabeled + + + 0.9536869 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.8714688 + structure_element + cleaner0 + 2023-07-03T10:36:05Z + SO: + + GBD + + + 0.89591146 + experimental_method + cleaner0 + 2023-07-03T11:53:37Z + MESH: + + titrated + + + 0.8920911 + chemical + cleaner0 + 2023-07-03T13:20:28Z + CHEBI: + + 15N + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:48:08Z + + Cdc42Δ7Q61L·GMPPNP + + + 0.9824735 + experimental_method + cleaner0 + 2023-07-03T11:53:42Z + MESH: + + HSQCs + + + 0.9628933 + protein_state + cleaner0 + 2023-07-03T09:35:20Z + DUMMY: + + Unlabeled + + + structure_element + SO: + cleaner0 + 2023-07-03T08:59:26Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T08:59:34Z + + TOCA1 + + + 0.9708315 + complex_assembly + cleaner0 + 2023-07-03T10:49:31Z + GO: + + Cdc42·N-WASP + + + 0.95585173 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.9966275 + structure_element + cleaner0 + 2023-07-03T10:36:29Z + SO: + + GBD + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T08:55:30Z + + presence of + + + structure_element + SO: + cleaner0 + 2023-07-03T08:59:50Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T08:59:57Z + + TOCA1 + + + 0.9978908 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.94267553 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.46139213 + structure_element + cleaner0 + 2023-07-03T10:36:16Z + SO: + + GBD + + + 0.99755055 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + 0.9964689 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + evidence + DUMMY: + cleaner0 + 2023-07-03T09:00:11Z + + Kd + + + 0.99711514 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + evidence + DUMMY: + cleaner0 + 2023-07-03T09:00:18Z + + Kd + + + 0.96482223 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + evidence + DUMMY: + cleaner0 + 2023-07-03T09:00:18Z + + Kd + + + 0.97246563 + experimental_method + cleaner0 + 2023-07-03T11:53:47Z + MESH: + + HSQC + + + 0.96047896 + chemical + cleaner0 + 2023-07-03T13:20:32Z + CHEBI: + + 15N + + + 0.9969433 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.97844476 + protein_state + cleaner0 + 2023-07-03T13:35:08Z + DUMMY: + + alone + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T08:55:30Z + + presence of + + + 0.99561125 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + 0.98355776 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.9025988 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.94289047 + structure_element + cleaner0 + 2023-07-03T10:36:25Z + SO: + + GBD + + + 0.97607386 + evidence + cleaner0 + 2023-07-03T12:05:01Z + DUMMY: + + spectra + + + 0.77785087 + protein_state + cleaner0 + 2023-07-03T08:55:30Z + DUMMY: + + presence of + + + 0.9605486 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T08:55:30Z + + presence of + + + 0.95731276 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.9966145 + protein + cleaner0 + 2023-06-30T08:43:40Z + PR: + + TOCA1 + + + 0.98916936 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + + RESULTS + paragraph + 41289 + Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain. Hence, a third, intermediate state that includes all three proteins is unlikely. Again, the experiments were recorded on protein samples far in excess of the individual Kd values (600 μm each protein). These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. Taken together, the data in Fig. 7, C and D, indicate unidirectional competition for Cdc42 binding in which the N-WASP GBD displaces TOCA1 HR1 but not vice versa. + + 0.9332237 + chemical + cleaner0 + 2023-07-03T13:23:00Z + CHEBI: + + 15N + + + 0.9982925 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9974394 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T08:55:30Z + + presence of + + + 0.95294714 + protein_state + cleaner0 + 2023-07-03T09:35:21Z + DUMMY: + + unlabeled + + + complex_assembly + GO: + cleaner0 + 2023-07-03T08:48:08Z + + Cdc42Δ7Q61L·GMPPNP + + + 0.8684736 + protein_state + cleaner0 + 2023-07-03T09:35:21Z + DUMMY: + + unlabeled + + + 0.97671705 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.9954027 + structure_element + cleaner0 + 2023-07-03T10:36:33Z + SO: + + GBD + + + 0.9841208 + evidence + cleaner0 + 2023-07-03T12:05:17Z + DUMMY: + + spectrum + + + 0.9683415 + protein + cleaner0 + 2023-06-30T08:44:51Z + PR: + + N-WASP + + + 0.99813795 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.99704844 + protein_state + cleaner0 + 2023-07-03T13:35:25Z + DUMMY: + + free + + + 0.9981317 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.9801952 + evidence + cleaner0 + 2023-07-03T12:05:19Z + DUMMY: + + spectrum + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T08:55:30Z + + presence of + + + 0.9631322 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.99390113 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.84866285 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.99701947 + protein_state + cleaner0 + 2023-07-03T13:35:32Z + DUMMY: + + free + + + 0.9362432 + protein_state + cleaner0 + 2023-07-03T13:35:35Z + DUMMY: + + complex + + + 0.8787579 + evidence + cleaner0 + 2023-07-03T12:05:22Z + DUMMY: + + spectra + + + 0.94687283 + experimental_method + cleaner0 + 2023-07-03T11:53:49Z + MESH: + + NMR + + + 0.99748236 + protein_state + cleaner0 + 2023-07-03T13:35:46Z + DUMMY: + + free + + + 0.98963296 + protein_state + cleaner0 + 2023-07-03T13:35:49Z + DUMMY: + + bound + + + 0.8823185 + evidence + cleaner0 + 2023-07-03T12:05:25Z + DUMMY: + + spectrum + + + 0.9976744 + protein_state + cleaner0 + 2023-07-03T13:35:53Z + DUMMY: + + free + + + 0.99598557 + protein_state + cleaner0 + 2023-07-03T13:35:56Z + DUMMY: + + bound + + + 0.9983581 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.71245635 + evidence + cleaner0 + 2023-07-03T09:00:18Z + DUMMY: + + Kd + + + 0.9983242 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.99732745 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.9735255 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.99852884 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.997738 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.971786 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9977214 + structure_element + cleaner0 + 2023-07-03T10:36:55Z + SO: + + GBD + + + 0.9973015 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.9388516 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.9646563 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9982608 + structure_element + cleaner0 + 2023-07-03T10:36:57Z + SO: + + GBD + + + 0.9985617 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.99745125 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + + RESULTS + paragraph + 42436 + To extend these studies to a more complex system and to assess the ability of TOCA1 HR1 to compete with full-length N-WASP, pyrene actin assays were employed. These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization. Actin polymerization in all cases was initiated by the addition of PI(4,5)P2-containing liposomes. Actin polymerization triggered by the addition of PI(4,5)P2-containing liposomes has previously been shown to depend on TOCA1 and N-WASP. Endogenous N-WASP is present at ∼100 nm in Xenopus extracts, whereas TOCA1 is present at a 10-fold lower concentration than N-WASP. + + 0.9985815 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9963374 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.9972822 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + full-length + + + 0.98861784 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9900012 + experimental_method + cleaner0 + 2023-07-03T11:53:55Z + MESH: + + pyrene actin assays + + + 0.7194145 + chemical + cleaner0 + 2023-07-03T13:22:25Z + CHEBI: + + pyrene actin + + + 0.71539295 + taxonomy_domain + cleaner0 + 2023-07-03T09:39:16Z + DUMMY: + + Xenopus + + + 0.99879575 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9967769 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.9877314 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9883178 + structure_element + cleaner0 + 2023-07-03T10:37:03Z + SO: + + GBD + + + protein_type + MESH: + cleaner0 + 2023-07-03T13:23:19Z + + actin + + + protein_type + MESH: + cleaner0 + 2023-07-03T13:23:10Z + + Actin + + + 0.99500054 + chemical + cleaner0 + 2023-07-03T09:41:52Z + CHEBI: + + PI(4,5)P2 + + + protein_type + MESH: + cleaner0 + 2023-07-03T13:21:12Z + + Actin + + + 0.9945503 + chemical + cleaner0 + 2023-07-03T09:41:52Z + CHEBI: + + PI(4,5)P2 + + + 0.99870133 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9839406 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9804876 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.51483136 + taxonomy_domain + cleaner0 + 2023-07-03T09:39:16Z + DUMMY: + + Xenopus + + + 0.9981664 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9842655 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + + RESULTS + paragraph + 43189 + The addition of the isolated N-WASP GBD significantly inhibited the polymerization of actin at concentrations as low as 100 nm and completely abolished polymerization at higher concentrations (Fig. 8). The GBD presumably acts as a dominant negative, sequestering endogenous Cdc42 and preventing endogenous full-length N-WASP from binding and becoming activated. The addition of the TOCA1 HR1 domain to 100 μm had no significant effect on the rate of actin polymerization or maximum fluorescence. This is consistent with endogenous N-WASP, activated by other components of the assay, outcompeting the TOCA1 HR1 domain for Cdc42 binding. + + experimental_method + MESH: + cleaner0 + 2023-07-03T11:55:01Z + + addition + + + 0.99054986 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.99848133 + structure_element + cleaner0 + 2023-07-03T10:37:08Z + SO: + + GBD + + + 0.97212505 + protein_type + cleaner0 + 2023-07-03T13:22:34Z + MESH: + + actin + + + 0.9984763 + structure_element + cleaner0 + 2023-07-03T10:37:11Z + SO: + + GBD + + + 0.99606305 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.5580302 + protein_state + cleaner0 + 2023-07-03T13:36:05Z + DUMMY: + + endogenous + + + 0.99722224 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + full-length + + + 0.9901004 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.90093863 + experimental_method + cleaner0 + 2023-07-03T11:55:06Z + MESH: + + addition + + + 0.99855965 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.99839646 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + protein_type + MESH: + cleaner0 + 2023-07-03T13:22:44Z + + actin + + + 0.93018484 + evidence + cleaner0 + 2023-07-03T12:05:31Z + DUMMY: + + maximum fluorescence + + + 0.9937324 + protein_state + cleaner0 + 2023-07-03T13:36:15Z + DUMMY: + + endogenous + + + 0.98900014 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9986438 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.998454 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.99387544 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + + zbc0281646060008.jpg + F8 + FIG + fig_caption + 43826 + Actin polymerization downstream of Cdc42·N-WASP·TOCA1 is inhibited by excess N-WASP GBD but not by the TOCA1 HR1 domain. Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated. Maximal rates of actin polymerization derived from the linear region of the curves are represented in bar charts below. Error bars, S.E. + + complex_assembly + GO: + cleaner0 + 2023-07-03T09:01:02Z + + Cdc42·N-WASP·TOCA1 + + + 0.5454533 + protein_state + cleaner0 + 2023-07-03T13:36:33Z + DUMMY: + + inhibited + + + 0.9489117 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9776324 + structure_element + cleaner0 + 2023-07-03T10:37:17Z + SO: + + GBD + + + 0.9981376 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9981976 + structure_element + cleaner0 + 2023-06-30T08:45:29Z + SO: + + HR1 + + + 0.9425819 + evidence + cleaner0 + 2023-07-03T12:05:34Z + DUMMY: + + Fluorescence curves + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T08:55:30Z + + presence of + + + 0.7197472 + experimental_method + cleaner0 + 2023-07-03T11:55:10Z + MESH: + + increasing concentrations + + + 0.9465313 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9019326 + structure_element + cleaner0 + 2023-07-03T10:37:21Z + SO: + + GBD + + + 0.9979225 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9975677 + structure_element + cleaner0 + 2023-06-30T08:45:30Z + SO: + + HR1 + + + + DISCUSS + title_1 + 44225 + Discussion + + + DISCUSS + title_4 + 44236 + The Cdc42-TOCA1 Interaction + + protein + PR: + cleaner0 + 2023-07-03T13:36:53Z + + Cdc42 + + + protein + PR: + cleaner0 + 2023-07-03T13:36:59Z + + TOCA1 + + + + DISCUSS + paragraph + 44264 + The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). This is over 100 times lower than that of the N-WASP GBD (Kd = 37 nm) and considerably lower than other known G protein-HR1 domain interactions. The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. A single binding interface on both the HR1 domain and Cdc42 can be concluded from the data presented here. Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions (Fig. 4), and the lowest energy model produced in rigid body docking resembles previously studied G protein·HR1 complexes (Fig. 6). It seems, therefore, that the interaction, despite its relatively low affinity, is specific and sterically similar to other HR1 domain-G protein interactions. + + 0.99874794 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + 0.9605068 + protein_state + cleaner0 + 2023-07-03T13:37:03Z + DUMMY: + + alone + + + 0.9922667 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.98681575 + evidence + cleaner0 + 2023-07-03T12:05:40Z + DUMMY: + + affinity + + + 0.9987087 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + 0.996276 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.9067749 + evidence + cleaner0 + 2023-07-03T09:00:18Z + DUMMY: + + Kd + + + 0.95897865 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9980869 + structure_element + cleaner0 + 2023-07-03T10:37:27Z + SO: + + GBD + + + evidence + DUMMY: + cleaner0 + 2023-07-03T09:00:18Z + + Kd + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:08Z + + G protein + + + 0.41392598 + structure_element + cleaner0 + 2023-06-30T08:45:30Z + SO: + + HR1 + + + 0.91059816 + structure_element + cleaner0 + 2023-07-03T10:37:40Z + SO: + + C-terminal region + + + 0.6926602 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.99878424 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9494978 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.99840575 + structure_element + cleaner0 + 2023-07-03T10:37:44Z + SO: + + HR1b + + + 0.9986798 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.9982907 + protein + cleaner0 + 2023-07-03T09:35:52Z + PR: + + PRK1 + + + 0.98214185 + structure_element + cleaner0 + 2023-07-03T10:37:47Z + SO: + + HR1a + + + protein + PR: + cleaner0 + 2023-06-30T08:46:57Z + + RhoA + + + 0.9923845 + site + cleaner0 + 2023-07-03T12:08:10Z + SO: + + binding interface + + + 0.9986285 + structure_element + cleaner0 + 2023-06-30T08:45:30Z + SO: + + HR1 + + + 0.9925438 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.96070576 + site + cleaner0 + 2023-07-03T12:08:13Z + SO: + + interfaces + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:08Z + + G protein + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + evidence + DUMMY: + cleaner0 + 2023-07-03T12:05:58Z + + model + + + 0.9632737 + experimental_method + cleaner0 + 2023-07-03T11:55:15Z + MESH: + + rigid body docking + + + complex_assembly + GO: + cleaner0 + 2023-07-03T09:02:19Z + + G protein·HR1 + + + 0.62017566 + structure_element + cleaner0 + 2023-06-30T08:45:30Z + SO: + + HR1 + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:08Z + + G protein + + + + DISCUSS + paragraph + 45296 + The TOCA1 HR1 domain is a left-handed coiled-coil comparable with other known HR1 domains. A short region N-terminal to the coiled-coil exhibits a series of turns and contacts residues of both helices of the coiled-coil (Fig. 3). The corresponding sequence in CIP4 also includes a series of turns but is flexible, whereas in the HR1a domain of PRK1, the equivalent region adopts an α-helical structure that packs against the coiled-coil. The contacts between the N-terminal region and the coiled-coil are predominantly hydrophobic in both cases, but sequence-specific contacts do not appear to be conserved. This region is distant from the G protein-binding interface of the HR1 domains, so the structural differences may relate to the structure and regulation of these domains rather than their G protein interactions. + + 0.9981933 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9984641 + structure_element + cleaner0 + 2023-06-30T08:45:30Z + SO: + + HR1 + + + 0.99416256 + structure_element + cleaner0 + 2023-07-03T10:37:52Z + SO: + + coiled-coil + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + 0.99510807 + structure_element + cleaner0 + 2023-07-03T10:37:57Z + SO: + + coiled-coil + + + 0.9957609 + structure_element + cleaner0 + 2023-07-03T10:37:59Z + SO: + + coiled-coil + + + 0.99836725 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.99846256 + structure_element + cleaner0 + 2023-07-03T10:38:02Z + SO: + + HR1a + + + 0.99815816 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.8583672 + structure_element + cleaner0 + 2023-07-03T10:38:04Z + SO: + + α-helical structure + + + 0.99529916 + structure_element + cleaner0 + 2023-07-03T10:38:06Z + SO: + + coiled-coil + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:06Z + + contacts + + + 0.992884 + structure_element + cleaner0 + 2023-07-03T10:38:09Z + SO: + + coiled-coil + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:06Z + + hydrophobic + + + 0.9960044 + site + cleaner0 + 2023-07-03T12:08:18Z + SO: + + G protein-binding interface + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:08Z + + G protein + + + + DISCUSS + paragraph + 46120 + The interhelical loops of TOCA1 and CIP4 differ from the same region in the HR1 domains of PRK1 in that they are longer and contain two short stretches of 310-helix. This region lies within the G protein-binding surface of all of the HR1 domains (Fig. 4D). TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. The structural features shared by TOCA1 and CIP4 may therefore be related to Cdc42 binding specificity and the low affinities. In free TOCA1, the side chains of the interhelical region make extensive contacts with residues in helix 1. Many of these residues are significantly affected in the presence of Cdc42, so it is likely that the conformation of this loop is altered in the Cdc42 complex. These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42. + + 0.99714184 + structure_element + cleaner0 + 2023-07-03T10:38:13Z + SO: + + interhelical loops + + + 0.9987884 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.99845755 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + 0.9985311 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.9948606 + structure_element + cleaner0 + 2023-07-03T10:38:17Z + SO: + + 310-helix + + + 0.99405587 + site + cleaner0 + 2023-07-03T12:08:21Z + SO: + + G protein-binding surface + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + 0.9987643 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.99849117 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.99687356 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.9984357 + structure_element + cleaner0 + 2023-07-03T10:38:21Z + SO: + + HR1a + + + 0.9983943 + protein + cleaner0 + 2023-07-03T09:35:53Z + PR: + + PRK1 + + + 0.9965754 + protein + cleaner0 + 2023-06-30T08:46:57Z + PR: + + RhoA + + + 0.99643517 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.99843675 + structure_element + cleaner0 + 2023-07-03T10:38:25Z + SO: + + HR1b + + + 0.9964896 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.9988292 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9986552 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + protein + PR: + cleaner0 + 2023-06-30T08:42:55Z + + Cdc42 + + + 0.9972121 + protein_state + cleaner0 + 2023-07-03T13:37:08Z + DUMMY: + + free + + + 0.99870944 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9975205 + structure_element + cleaner0 + 2023-07-03T10:38:29Z + SO: + + interhelical region + + + 0.996721 + structure_element + cleaner0 + 2023-07-03T10:38:32Z + SO: + + helix 1 + + + 0.99045527 + protein_state + cleaner0 + 2023-07-03T08:55:30Z + DUMMY: + + presence of + + + 0.9958423 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.6028027 + structure_element + cleaner0 + 2023-07-03T10:38:34Z + SO: + + loop + + + 0.9048001 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.9841298 + experimental_method + cleaner0 + 2023-07-03T11:55:20Z + MESH: + + mutation + + + 0.9799066 + residue_name_number + cleaner0 + 2023-07-03T10:53:22Z + DUMMY: + + Met383 + + + 0.87663805 + residue_name_number + cleaner0 + 2023-07-03T10:53:24Z + DUMMY: + + Gly384 + + + 0.95014256 + residue_name_number + cleaner0 + 2023-07-03T10:53:27Z + DUMMY: + + Asp385 + + + 0.98716545 + residue_name_number + cleaner0 + 2023-07-03T10:53:32Z + DUMMY: + + Ile383 + + + 0.96805173 + residue_name_number + cleaner0 + 2023-07-03T10:53:34Z + DUMMY: + + Ser384 + + + 0.9810523 + residue_name_number + cleaner0 + 2023-07-03T10:53:36Z + DUMMY: + + Thr385 + + + 0.9977755 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9953963 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + + DISCUSS + paragraph + 47071 + The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. Some speculative conclusions can be made based on this model. For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42·HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A). Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation. This residue has also been identified as important for Cdc42-WASP binding. Phe-56Cdc42 is therefore likely to be involved in the Cdc42-TOCA1 interaction, probably by stabilizing the position of switch I. + + evidence + DUMMY: + cleaner0 + 2023-07-03T12:06:21Z + + model + + + 0.9877753 + experimental_method + cleaner0 + 2023-07-03T08:50:26Z + MESH: + + HADDOCK + + + 0.34142372 + evidence + cleaner0 + 2023-07-03T12:06:26Z + DUMMY: + + titration + + + 0.99043894 + experimental_method + cleaner0 + 2023-07-03T11:55:24Z + MESH: + + NMR + + + 0.9946361 + evidence + cleaner0 + 2023-07-03T12:06:29Z + DUMMY: + + structures + + + 0.7614925 + protein + cleaner0 + 2023-06-30T08:46:57Z + PR: + + RhoA + + + 0.8085813 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.9361132 + protein_state + cleaner0 + 2023-07-03T13:37:13Z + DUMMY: + + in complex with + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T09:03:45Z + + Phe-56 + + + protein + PR: + cleaner0 + 2023-07-03T09:03:54Z + + Cdc42 + + + 0.9960108 + protein_state + cleaner0 + 2023-07-03T13:37:16Z + DUMMY: + + free + + + 0.5524952 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + complex_assembly + GO: + cleaner0 + 2023-07-03T09:04:18Z + + Cdc42·HR1TOCA1 + + + 0.99825543 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.99869174 + structure_element + cleaner0 + 2023-06-30T08:45:30Z + SO: + + HR1 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T09:36:28Z + + Phe-56 + + + protein + PR: + cleaner0 + 2023-07-03T09:36:37Z + + Cdc42 + + + 0.9886572 + residue_name + cleaner0 + 2023-07-03T10:52:09Z + SO: + + Trp + + + 0.6558546 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.692283 + protein + cleaner0 + 2023-06-30T08:46:57Z + PR: + + RhoA + + + 0.9735813 + site + cleaner0 + 2023-07-03T09:34:36Z + SO: + + switch I + + + 0.9794703 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.98919964 + protein + cleaner0 + 2023-07-03T09:29:56Z + PR: + + ACK + + + protein + PR: + cleaner0 + 2023-06-30T08:42:55Z + + Cdc42 + + + protein + PR: + cleaner0 + 2023-07-03T09:05:04Z + + WASP + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T09:04:42Z + + Phe-56 + + + protein + PR: + cleaner0 + 2023-07-03T09:04:49Z + + Cdc42 + + + 0.53643703 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + 0.5590447 + protein + cleaner0 + 2023-06-30T08:43:41Z + PR: + + TOCA1 + + + 0.9758419 + site + cleaner0 + 2023-07-03T09:34:36Z + SO: + + switch I + + + + DISCUSS + paragraph + 47837 + Some residues that are affected in the Cdc42·HR1TOCA1 complex but do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) may contact HR1TOCA1 directly (Fig. 6D). Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. N52T is one of a combination of seven residues found to confer ACK binding on Rac1 and so may represent a specific Cdc42-effector contact residue. The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b. Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact. Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. The equivalent Arg in Rac1 and RhoA is pointing away from the HR1 domains of PRK1. The importance of this residue in the Cdc42-TOCA1 interaction remains unclear, although its mutation reduces binding to RhoGAP, suggesting that it can be involved in Cdc42 interactions. + + 0.99348646 + complex_assembly + cleaner0 + 2023-07-03T10:49:42Z + GO: + + Cdc42·HR1TOCA1 + + + 0.9682967 + protein + cleaner0 + 2023-06-30T08:46:58Z + PR: + + RhoA + + + 0.9662049 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:28:38Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T09:28:46Z + + TOCA1 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T10:53:56Z + + Gln-2 + + + protein + PR: + cleaner0 + 2023-07-03T10:54:06Z + + Cdc42 + + + 0.9904862 + complex_assembly + cleaner0 + 2023-07-03T10:49:46Z + GO: + + Cdc42·ACK + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T09:20:31Z + + Val-376 + + + protein + PR: + cleaner0 + 2023-07-03T09:20:40Z + + TOCA1 + + + residue_name_number + DUMMY: + cleaner0 + 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structure_element + SO: + cleaner0 + 2023-07-03T09:24:02Z + + HR1b + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T09:24:18Z + + Thr-52 + + + protein + PR: + cleaner0 + 2023-07-03T09:24:32Z + + Cdc42 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T09:24:48Z + + Lys-372 + + + protein + PR: + cleaner0 + 2023-07-03T09:24:55Z + + TOCA1 + + + 0.8249155 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:29:27Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T09:29:37Z + + TOCA1 + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T09:25:15Z + + Arg-68 + + + protein + PR: + cleaner0 + 2023-07-03T09:25:24Z + + Cdc42 + + + 0.98761934 + site + cleaner0 + 2023-07-03T09:26:14Z + SO: + + switch II + + + residue_name_number + DUMMY: + cleaner0 + 2023-07-03T09:25:43Z + + Glu-395 + + + protein + PR: + cleaner0 + 2023-07-03T09:25:50Z + + TOCA1 + + + 0.9952789 + site + cleaner0 + 2023-07-03T09:26:23Z + SO: + 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DISCUSS + paragraph + 49213 + The solution structure of the TOCA1 HR1 domain presented here, along with the model of the HR1TOCA1·Cdc42 complex is consistent with a conserved mode of binding across the known HR1 domain-Rho family interactions, despite their differing affinities. The weak binding prevented detailed structural and thermodynamic studies of the complex. Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. + + 0.8974091 + evidence + cleaner0 + 2023-07-03T12:06:34Z + DUMMY: + + solution structure + + + 0.99868196 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.99838424 + structure_element + cleaner0 + 2023-06-30T08:45:30Z + SO: + + HR1 + + + complex_assembly + GO: + cleaner0 + 2023-07-03T09:27:40Z + + HR1TOCA1·Cdc42 + + + 0.9820432 + structure_element + cleaner0 + 2023-06-30T08:45:30Z + SO: + + HR1 + + + 0.92416924 + experimental_method + cleaner0 + 2023-07-03T11:55:47Z + MESH: + + structural and thermodynamic studies + + + 0.97563076 + experimental_method + cleaner0 + 2023-07-03T11:55:50Z + MESH: + + structural studies + + + 0.9985752 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.99842036 + structure_element + cleaner0 + 2023-06-30T08:45:30Z + SO: + + HR1 + + + 0.97121984 + experimental_method + cleaner0 + 2023-07-03T11:55:52Z + MESH: + + chemical shift mapping + + + 0.9524198 + protein + cleaner0 + 2023-06-30T08:42:55Z + PR: + + Cdc42 + + + structure_element + SO: + cleaner0 + 2023-07-03T10:09:15Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T10:09:21Z + + TOCA1 + + + 0.9924744 + protein + cleaner0 + 2023-06-30T08:46:58Z + PR: + + RhoA + + + 0.98837924 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + structure_element + SO: + cleaner0 + 2023-07-03T10:08:54Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T10:09:01Z + + PRK1 + + + + DISCUSS + paragraph + 49760 + We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures (Tm) and Rac binders being more rigid. The Tm of the TOCA1 HR1 domain is 61.9 °C (data not shown), which is the highest Tm that we have measured for an HR1 domain thus far. As such, the ability of the TOCA1 HR1 domain to bind to Cdc42 (a close relative of Rac1 rather than RhoA) fits this trend. An investigation into the local motions, particularly in the G protein-binding regions, may offer further insight into the differential specificities and affinities of G protein-HR1 domain interactions. + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + 0.8696185 + protein_type + cleaner0 + 2023-07-03T10:17:13Z + MESH: + + Rho family G proteins + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + 0.9898145 + evidence + cleaner0 + 2023-07-03T12:06:39Z + DUMMY: + + melting temperatures + + + 0.9740339 + evidence + cleaner0 + 2023-07-03T12:06:42Z + DUMMY: + + Tm + + + protein_type + MESH: + cleaner0 + 2023-07-03T13:37:43Z + + Rac + + + 0.9928907 + evidence + cleaner0 + 2023-07-03T12:06:45Z + DUMMY: + + Tm + + + 0.9961492 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + 0.98497283 + evidence + cleaner0 + 2023-07-03T12:06:47Z + DUMMY: + + Tm + + + 0.9982868 + structure_element + cleaner0 + 2023-06-30T08:45:30Z + SO: + + HR1 + + + 0.99699247 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + 0.6991982 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9253086 + protein + cleaner0 + 2023-06-30T08:47:07Z + PR: + + Rac1 + + + 0.8978462 + protein + cleaner0 + 2023-06-30T08:46:58Z + PR: + + RhoA + + + 0.9958286 + site + cleaner0 + 2023-07-03T08:16:05Z + SO: + + G protein-binding regions + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:08Z + + G protein + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + + DISCUSS + title_4 + 50517 + Significance of a Weak, Transient Interaction + + + DISCUSS + paragraph + 50563 + The low affinity of the Cdc42-HR1TOCA1 interaction is consistent with a tightly spatially and temporally regulated pathway, requiring combinatorial signals leading to a series of coincident weak interactions that elicit full activation. The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family. Weak, transient protein-protein interactions are functionally significant in several systems; for example, the binding of adaptor proteins to protein cargo during the formation of clathrin-coated vesicles in endocytosis involves multiple interactions of micromolar affinity. + + protein + PR: + cleaner0 + 2023-06-30T08:42:56Z + + Cdc42 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:38:29Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T09:38:37Z + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:30Z + + HR1 + + + 0.90049523 + protein_type + cleaner0 + 2023-07-03T09:38:55Z + MESH: + + TOCA family members + + + 0.9987978 + protein + cleaner0 + 2023-07-03T09:39:04Z + PR: + + CIP4 + + + 0.9988563 + protein + cleaner0 + 2023-07-03T09:39:10Z + PR: + + FBP17 + + + 0.61347103 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9585526 + protein_type + cleaner0 + 2023-07-03T09:38:57Z + MESH: + + HR1 domain proteins + + + 0.84394807 + protein_type + cleaner0 + 2023-07-03T09:39:00Z + MESH: + + PRK family + + + + DISCUSS + paragraph + 51319 + The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. Evidence suggests that the TOCA family of proteins are recruited to the membrane via an interaction between their F-BAR domain and specific signaling lipids. For example, electrostatic interactions between the F-BAR domain and the membrane are required for TOCA1 recruitment to membrane vesicles and tubules, and TOCA1-dependent actin polymerization is known to depend specifically on PI(4,5)P2. Furthermore, the isolated F-BAR domain of FBP17 has been shown to induce membrane tubulation of brain liposomes and BAR domain proteins that promote tubulation cluster on membranes at high densities. Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. Cdc42-HR1TOCA1 binding would then be favorable, as long as coincident activation of Cdc42 had occurred, leading to stabilization of TOCA1 at the membrane and downstream activation of N-WASP. + + 0.4805834 + structure_element + cleaner0 + 2023-07-03T09:19:55Z + SO: + + HR1TOCA1 + + + protein + PR: + cleaner0 + 2023-06-30T08:42:56Z + + Cdc42 + + + 0.99852484 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.8006676 + taxonomy_domain + cleaner0 + 2023-07-03T09:39:15Z + DUMMY: + + Xenopus + + + 0.99829346 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.9548792 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9982255 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.99125063 + protein_state + cleaner0 + 2023-07-03T13:38:08Z + DUMMY: + + activated + + + 0.986712 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.7707484 + protein_type + cleaner0 + 2023-07-03T10:17:19Z + MESH: + + TOCA family + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:40Z + + F-BAR + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:06Z + + electrostatic interactions + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + 0.99763405 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.48940825 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.9973473 + chemical + cleaner0 + 2023-07-03T09:41:52Z + CHEBI: + + PI(4,5)P2 + + + 0.48511603 + experimental_method + cleaner0 + 2023-07-03T11:55:58Z + MESH: + + isolated + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + 0.99882823 + protein + cleaner0 + 2023-07-03T09:39:11Z + PR: + + FBP17 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:41:27Z + + BAR + + + 0.99782467 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.8462519 + evidence + cleaner0 + 2023-07-03T09:00:18Z + DUMMY: + + Kd + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-03T10:10:53Z + + dimer + + + 0.9982218 + protein + cleaner0 + 2023-07-03T10:09:37Z + PR: + + FCHo2 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + protein + PR: + cleaner0 + 2023-06-30T08:42:56Z + + Cdc42 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:39:45Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T09:39:53Z + + TOCA1 + + + protein + PR: + cleaner0 + 2023-06-30T08:42:56Z + + Cdc42 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:10Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T09:40:18Z + + TOCA1 + + + 0.97662354 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.99832636 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.9216368 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + + DISCUSS + paragraph + 52659 + It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. The unfolded, high affinity state of WASP is represented by a short peptide, the GBD, which binds with a low nanomolar affinity to Cdc42. In contrast, the best estimate of the affinity of full-length WASP for Cdc42 is low micromolar. In the inactive state of WASP, the actin- and Arp2/3-binding VCA domain contacts the GBD, competing for Cdc42 binding. The high affinity of Cdc42 for the unfolded, active form pushes the equilibrium in favor of (N-)WASP activation. Binding of PI(4,5)P2 to the basic region just N-terminal to the GBD further favors the active conformation. A substantial body of data has illuminated the complex regulation of WASP/N-WASP proteins, and current evidence suggests that these allosteric activation mechanisms and oligomerization combine to regulate WASP activity, allowing the synchronization and integration of multiple potential activation signals (reviewed in Ref.). Our data are easily reconciled with this model. + + 0.9956033 + protein_type + cleaner0 + 2023-07-03T10:18:34Z + MESH: + + WASP + + + 0.9431486 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9977189 + protein_state + cleaner0 + 2023-07-03T09:43:40Z + DUMMY: + + folded + + + 0.99728596 + protein_state + cleaner0 + 2023-07-03T13:38:13Z + DUMMY: + + inactive + + + 0.9969168 + protein_state + cleaner0 + 2023-07-03T09:43:31Z + DUMMY: + + unfolded + + + 0.9972771 + protein_state + cleaner0 + 2023-07-03T13:38:17Z + DUMMY: + + active + + + 0.96331924 + evidence + cleaner0 + 2023-07-03T12:06:55Z + DUMMY: + + affinity + + + 0.997189 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.99731463 + protein_state + cleaner0 + 2023-07-03T09:43:31Z + DUMMY: + + unfolded + + + 0.8940232 + protein_type + cleaner0 + 2023-07-03T10:17:30Z + MESH: + + WASP + + + 0.9970182 + protein_state + cleaner0 + 2023-07-03T09:43:31Z + DUMMY: + + unfolded + + + 0.9939663 + protein_type + cleaner0 + 2023-07-03T10:18:44Z + MESH: + + WASP + + + 0.40008605 + chemical + cleaner0 + 2023-07-03T13:25:51Z + CHEBI: + + peptide + + + 0.9983557 + structure_element + cleaner0 + 2023-07-03T10:40:03Z + SO: + + GBD + + + 0.99671304 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.96908647 + evidence + cleaner0 + 2023-07-03T12:06:58Z + DUMMY: + + affinity + + + 0.99755406 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + full-length + + + 0.99496436 + protein_type + cleaner0 + 2023-07-03T10:19:01Z + MESH: + + WASP + + + 0.99721116 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9978344 + protein_state + cleaner0 + 2023-07-03T13:38:21Z + DUMMY: + + inactive + + + 0.9945903 + protein_type + cleaner0 + 2023-07-03T10:18:53Z + MESH: + + WASP + + + complex_assembly + GO: + cleaner0 + 2023-07-03T10:10:09Z + + Arp2/3 + + + structure_element + SO: + cleaner0 + 2023-07-03T10:40:19Z + + VCA + + + 0.99843746 + structure_element + cleaner0 + 2023-07-03T10:40:22Z + SO: + + GBD + + + 0.995122 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9971827 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9971264 + protein_state + cleaner0 + 2023-07-03T09:43:31Z + DUMMY: + + unfolded + + + 0.99629635 + protein_state + cleaner0 + 2023-07-03T13:38:25Z + DUMMY: + + active + + + protein + PR: + cleaner0 + 2023-07-03T09:42:29Z + + (N-)WASP + + + 0.99715185 + chemical + cleaner0 + 2023-07-03T09:41:51Z + CHEBI: + + PI(4,5)P2 + + + 0.9984573 + structure_element + cleaner0 + 2023-07-03T10:40:24Z + SO: + + GBD + + + 0.99588937 + protein_state + cleaner0 + 2023-07-03T13:38:28Z + DUMMY: + + active + + + 0.9502268 + protein_type + cleaner0 + 2023-07-03T09:42:01Z + MESH: + + WASP/N-WASP proteins + + + protein_type + MESH: + cleaner0 + 2023-07-03T10:19:10Z + + WASP + + + + DISCUSS + paragraph + 53811 + We envisage that TOCA1 is first recruited to the appropriate membrane in response to PI(4,5)P2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1. Cdc42 is activated in response to co-incident signals and can then bind to TOCA1, further stabilizing TOCA1 at the membrane. TOCA1 can then recruit N-WASP via an interaction between its SH3 domain and the N-WASP proline-rich region. The recruitment of N-WASP alone and of the N-WASP·WIP complex by TOCA1 and FBP17 has been demonstrated. WIP inhibits the activation of N-WASP by Cdc42, an effect that is reversed by TOCA1. It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42. TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state. + + 0.99835396 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.99706554 + chemical + cleaner0 + 2023-07-03T09:41:52Z + CHEBI: + + PI(4,5)P2 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + 0.9961631 + structure_element + cleaner0 + 2023-07-03T09:40:41Z + SO: + + F-BAR + + + oligomeric_state + DUMMY: + cleaner0 + 2023-07-03T10:11:07Z + + dimer + + + 0.99844754 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.9230027 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9984131 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.99826837 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.9982101 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.96607906 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + structure_element + SO: + cleaner0 + 2023-07-03T09:43:10Z + + SH3 + + + 0.9393429 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.996428 + structure_element + cleaner0 + 2023-07-03T10:40:30Z + SO: + + proline-rich region + + + 0.97201604 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.54663813 + protein_state + cleaner0 + 2023-07-03T13:38:32Z + DUMMY: + + alone + + + 0.9858632 + complex_assembly + cleaner0 + 2023-07-03T10:49:52Z + GO: + + N-WASP·WIP + + + 0.9984554 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.9982558 + protein + cleaner0 + 2023-07-03T09:39:11Z + PR: + + FBP17 + + + 0.99213284 + protein + cleaner0 + 2023-07-03T10:11:26Z + PR: + + WIP + + + 0.9729282 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9949456 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9963007 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.9971215 + protein + cleaner0 + 2023-07-03T10:11:30Z + PR: + + WIP + + + 0.99797446 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.9702464 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9983481 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.99529207 + protein_state + cleaner0 + 2023-07-03T09:43:31Z + DUMMY: + + unfolded + + + 0.9926866 + protein_state + cleaner0 + 2023-07-03T13:38:37Z + DUMMY: + + active + + + 0.96953297 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9952303 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9981888 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.97341126 + protein + cleaner0 + 2023-06-30T08:44:52Z + PR: + + N-WASP + + + 0.9983552 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.99681854 + chemical + cleaner0 + 2023-07-03T09:41:52Z + CHEBI: + + PI(4,5)P2 + + + 0.9909847 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.94290686 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.99534625 + protein_state + cleaner0 + 2023-07-03T09:43:31Z + DUMMY: + + unfolded + + + 0.99433976 + protein_state + cleaner0 + 2023-07-03T13:38:40Z + DUMMY: + + active + + + + DISCUSS + paragraph + 54916 + In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization. + + 0.9974664 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + full-length + + + 0.9987618 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.9171439 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.84216446 + evidence + cleaner0 + 2023-07-03T12:07:03Z + DUMMY: + + affinities + + + 0.99756587 + protein_state + cleaner0 + 2023-07-03T13:38:44Z + DUMMY: + + active + + + 0.98924595 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9969163 + protein_state + cleaner0 + 2023-07-03T09:43:30Z + DUMMY: + + unfolded + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T13:39:05Z + + active + + + 0.9685903 + evidence + cleaner0 + 2023-07-03T12:07:06Z + DUMMY: + + affinity + + + 0.93593836 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.99408674 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.94578844 + evidence + cleaner0 + 2023-07-03T12:07:10Z + DUMMY: + + binding data + + + 0.9986324 + protein + cleaner0 + 2023-06-30T08:43:42Z + PR: + + TOCA1 + + + 0.96670634 + structure_element + cleaner0 + 2023-06-30T08:45:30Z + SO: + + HR1 + + + 0.977499 + experimental_method + cleaner0 + 2023-07-03T11:56:07Z + MESH: + + NMR + + + 0.9285208 + protein_state + cleaner0 + 2023-07-03T13:39:16Z + DUMMY: + + stability + + + 0.998789 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.9984781 + structure_element + cleaner0 + 2023-06-30T08:45:30Z + SO: + + HR1 + + + 0.9854224 + protein_state + cleaner0 + 2023-07-03T08:55:30Z + DUMMY: + + presence of + + + 0.9940201 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9972457 + protein_state + cleaner0 + 2023-07-03T08:36:11Z + DUMMY: + + full-length + + + 0.998825 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.4800219 + protein_state + cleaner0 + 2023-07-03T13:39:23Z + DUMMY: + + isolated + + + 0.9981285 + structure_element + cleaner0 + 2023-06-30T08:45:31Z + SO: + + HR1 + + + 0.99625045 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + protein + PR: + cleaner0 + 2023-06-30T08:44:53Z + + N-WASP + + + protein + PR: + cleaner0 + 2023-06-30T08:42:56Z + + Cdc42 + + + 0.91420364 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.9985499 + structure_element + cleaner0 + 2023-07-03T10:40:36Z + SO: + + GBD + + + 0.9988022 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.9985341 + structure_element + cleaner0 + 2023-06-30T08:45:31Z + SO: + + HR1 + + + 0.99631435 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9966834 + chemical + cleaner0 + 2023-07-03T09:41:52Z + CHEBI: + + PI(4,5)P2 + + + 0.9967855 + protein_state + cleaner0 + 2023-07-03T13:39:29Z + DUMMY: + + activated + + + 0.9756092 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9987207 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.99872017 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.75998104 + protein_state + cleaner0 + 2023-07-03T13:39:37Z + DUMMY: + + full activation + + + 0.93770427 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + + DISCUSS + paragraph + 55754 + In such an array of molecules localized to a discrete region of the membrane, it is plausible that WASP could bind to a second Cdc42 molecule rather than displacing TOCA1 from its cognate Cdc42. Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD. Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP·WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding. The commonly used MGD → IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP·WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP. + + 0.99237895 + protein + cleaner0 + 2023-07-03T10:11:38Z + PR: + + WASP + + + 0.9970381 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.99847573 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.9959544 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.8592806 + experimental_method + cleaner0 + 2023-07-03T11:56:11Z + MESH: + + NMR + + + 0.70388633 + evidence + cleaner0 + 2023-07-03T12:07:14Z + DUMMY: + + affinity data + + + 0.9987017 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.9983523 + structure_element + cleaner0 + 2023-06-30T08:45:31Z + SO: + + HR1 + + + 0.9263594 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.99852484 + structure_element + cleaner0 + 2023-07-03T10:40:40Z + SO: + + GBD + + + 0.99824226 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.835499 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.95640403 + complex_assembly + cleaner0 + 2023-07-03T10:49:55Z + GO: + + N-WASP·WIP + + + 0.9964194 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.9571094 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.9974758 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.8687983 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.99370867 + mutant + cleaner0 + 2023-07-03T10:51:53Z + MESH: + + MGD → IST + + + 0.9943775 + protein_state + cleaner0 + 2023-07-03T13:39:42Z + DUMMY: + + Cdc42-binding deficient + + + 0.9986313 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.9150546 + complex_assembly + cleaner0 + 2023-07-03T10:49:57Z + GO: + + N-WASP·WIP + + + 0.7008063 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:44:33Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T09:44:40Z + + TOCA1 + + + 0.95238847 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + + DISCUSS + paragraph + 56477 + In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. Potentially, the TOCA1-Cdc42 interaction functions to position N-WASP and Cdc42 such that they are poised to interact with high affinity. The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP·WIP-induced actin polymerization. There is an advantage to such an effector handover, in that N-WASP would only be robustly recruited when F-BAR domains are already present. Hence, actin polymerization cannot occur until F-BAR domains are poised for membrane distortion. + + 0.9968759 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.98500466 + protein + cleaner0 + 2023-06-30T08:42:56Z + PR: + + Cdc42 + + + 0.91776663 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.8839886 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.9966192 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.8415559 + protein_state + cleaner0 + 2023-07-03T13:40:00Z + DUMMY: + + bound + + + 0.9226735 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.9524078 + protein_state + cleaner0 + 2023-07-03T13:40:03Z + DUMMY: + + fully activated + + + 0.9962457 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.7135997 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + protein + PR: + cleaner0 + 2023-06-30T08:42:57Z + + Cdc42 + + + 0.94013363 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.98841375 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.9968265 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.7197061 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + protein_state + DUMMY: + cleaner0 + 2023-07-03T13:40:28Z + + bound to + + + 0.9319956 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.9976781 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + complex_assembly + GO: + cleaner0 + 2023-07-03T09:45:24Z + + N-WASP·WIP + + + 0.9111567 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + + DISCUSS + paragraph + 57283 + Our model of the Cdc42·HR1TOCA1 complex indicates a mechanism by which such a handover could take place (Fig. 9) because it shows that the effector binding sites only partially overlap on Cdc42. The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound. It has been postulated that the initial interactions between this basic region and Cdc42 could stabilize the active conformation of WASP, leading to high affinity binding between the core CRIB and Cdc42. The region C-terminal to the core CRIB, required for maximal affinity binding, would then fully displace the TOCA1 HR1. + + 0.8920825 + complex_assembly + cleaner0 + 2023-07-03T10:50:04Z + GO: + + Cdc42·HR1TOCA1 + + + 0.99800205 + site + cleaner0 + 2023-07-03T12:08:45Z + SO: + + effector binding sites + + + 0.99696857 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.98572934 + residue_name + cleaner0 + 2023-07-03T10:40:48Z + SO: + + lysine + + + protein + PR: + cleaner0 + 2023-07-03T10:12:04Z + + WASP + + + protein + PR: + cleaner0 + 2023-06-30T08:42:57Z + + Cdc42 + + + 0.98391724 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.9976672 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.998718 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.99860877 + structure_element + cleaner0 + 2023-06-30T08:45:31Z + SO: + + HR1 + + + 0.9362426 + protein_state + cleaner0 + 2023-07-03T13:40:40Z + DUMMY: + + bound + + + 0.99707055 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.99639326 + protein_state + cleaner0 + 2023-07-03T13:40:42Z + DUMMY: + + active + + + 0.9975865 + protein + cleaner0 + 2023-07-03T10:11:54Z + PR: + + WASP + + + 0.99788386 + structure_element + cleaner0 + 2023-07-03T10:40:51Z + SO: + + CRIB + + + 0.9972886 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.9981187 + structure_element + cleaner0 + 2023-07-03T10:40:54Z + SO: + + CRIB + + + 0.9987233 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.99854016 + structure_element + cleaner0 + 2023-06-30T08:45:31Z + SO: + + HR1 + + + + zbc0281646060009.jpg + F9 + FIG + fig_caption + 58021 + A simplified model of the early stages of Cdc42·N-WASP·TOCA1-dependent actin polymerization. +Step 1, TOCA1 is recruited to the membrane via its F-BAR domain and/or Cdc42 interactions. F-BAR oligomerization is expected to occur following membrane binding, but a single monomer is shown for clarity. Step 2, N-WASP exists in an inactive, folded conformation. The TOCA1 SH3 domain interacts with N-WASP, causing an activatory allosteric effect. The HR1TOCA1-Cdc42 and SH3TOCA1-N-WASP interactions position Cdc42 and N-WASP for binding. Step 3, electrostatic interactions between Cdc42 and the basic region upstream of the CRIB initiate Cdc42·N-WASP binding. Step 4, the core CRIB binds with high affinity while the region C-terminal to the CRIB displaces the TOCA1 HR1 domain and increases the affinity of the N-WASP-Cdc42 interaction further. The VCA domain is released for downstream interactions, and actin polymerization proceeds. WH1, WASP homology 1 domain; PP, proline-rich region; VCA, verprolin homology, cofilin homology, acidic region. + + complex_assembly + GO: + cleaner0 + 2023-07-03T09:45:49Z + + Cdc42·N-WASP·TOCA1 + + + 0.99840397 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + 0.93579847 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.9775112 + structure_element + cleaner0 + 2023-07-03T09:40:41Z + SO: + + F-BAR + + + 0.9951125 + oligomeric_state + cleaner0 + 2023-07-03T11:42:11Z + DUMMY: + + monomer + + + 0.947602 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.99736327 + protein_state + cleaner0 + 2023-07-03T13:40:54Z + DUMMY: + + inactive + + + 0.994083 + protein_state + cleaner0 + 2023-07-03T09:43:41Z + DUMMY: + + folded + + + 0.9987005 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-07-03T10:41:09Z + + SH3 + + + 0.9564964 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + structure_element + SO: + cleaner0 + 2023-07-03T09:46:33Z + + HR1 + + + protein + PR: + cleaner0 + 2023-07-03T09:46:41Z + + TOCA1 + + + 0.62519926 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:46:55Z + + SH3 + + + protein + PR: + cleaner0 + 2023-07-03T09:47:03Z + + TOCA1 + + + protein + PR: + cleaner0 + 2023-06-30T08:44:53Z + + N-WASP + + + 0.8279639 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.91391045 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:20:06Z + + electrostatic interactions + + + 0.9968832 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.99876785 + structure_element + cleaner0 + 2023-07-03T10:41:14Z + SO: + + CRIB + + + complex_assembly + GO: + cleaner0 + 2023-07-03T09:46:15Z + + Cdc42·N-WASP + + + 0.99859744 + structure_element + cleaner0 + 2023-07-03T10:41:20Z + SO: + + CRIB + + + 0.9987232 + structure_element + cleaner0 + 2023-07-03T10:41:18Z + SO: + + CRIB + + + 0.9986406 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.99863064 + structure_element + cleaner0 + 2023-06-30T08:45:31Z + SO: + + HR1 + + + 0.57654375 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + protein + PR: + cleaner0 + 2023-06-30T08:42:57Z + + Cdc42 + + + 0.9983918 + structure_element + cleaner0 + 2023-07-03T10:41:23Z + SO: + + VCA + + + 0.9984913 + structure_element + cleaner0 + 2023-07-03T10:41:26Z + SO: + + WH1 + + + 0.99590874 + structure_element + cleaner0 + 2023-07-03T10:41:28Z + SO: + + WASP homology 1 domain + + + 0.99861336 + structure_element + cleaner0 + 2023-07-03T10:41:30Z + SO: + + PP + + + 0.9971271 + structure_element + cleaner0 + 2023-07-03T10:41:32Z + SO: + + proline-rich region + + + 0.99818355 + structure_element + cleaner0 + 2023-07-03T10:41:35Z + SO: + + VCA + + + 0.9958004 + structure_element + cleaner0 + 2023-07-03T10:41:37Z + SO: + + verprolin homology, cofilin homology, acidic region + + + + DISCUSS + paragraph + 59068 + In conclusion, the data presented here show that the TOCA1 HR1 domain is sufficient for Cdc42 binding in vitro and that the interaction is of micromolar affinity, lower than that of other G protein-HR1 domain interactions. The analogous HR1 domains from other TOCA1 family members, FBP17 and CIP4, also exhibit micromolar affinity for Cdc42. A role for the TOCA1-, FBP17-, and CIP4-Cdc42 interactions in the recruitment of these proteins to the membrane therefore appears unlikely. Instead, our findings agree with earlier suggestions that the F-BAR domain is responsible for membrane recruitment. The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway. Our data are therefore easily reconciled with the dynamic instability models described in relation to the formation of endocytic vesicles and with the current data pertaining to the complex activation of WASP/N-WASP pathways by allosteric and oligomeric effects. + + 0.99871993 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:31Z + + HR1 + + + 0.93090767 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + protein_type + MESH: + cleaner0 + 2023-07-03T08:23:08Z + + G protein + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:31Z + + HR1 + + + structure_element + SO: + cleaner0 + 2023-06-30T08:45:31Z + + HR1 + + + 0.95327765 + protein_type + cleaner0 + 2023-07-03T10:19:18Z + MESH: + + TOCA1 family + + + 0.99848145 + protein + cleaner0 + 2023-07-03T09:39:11Z + PR: + + FBP17 + + + 0.9982632 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.99616635 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.94887155 + protein + cleaner0 + 2023-06-30T08:43:43Z + PR: + + TOCA1 + + + 0.9608345 + protein + cleaner0 + 2023-07-03T09:39:11Z + PR: + + FBP17 + + + 0.9575774 + protein + cleaner0 + 2023-07-03T09:39:06Z + PR: + + CIP4 + + + 0.6942188 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + structure_element + SO: + cleaner0 + 2023-07-03T09:40:41Z + + F-BAR + + + protein + PR: + cleaner0 + 2023-06-30T08:42:57Z + + Cdc42 + + + protein + PR: + cleaner0 + 2023-06-30T08:43:43Z + + TOCA1 + + + 0.99709356 + protein_state + cleaner0 + 2023-07-03T13:41:05Z + DUMMY: + + activated + + + 0.5552928 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.935388 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.98660195 + protein_state + cleaner0 + 2023-07-03T13:41:09Z + DUMMY: + + full activation + + + 0.9621456 + protein + cleaner0 + 2023-06-30T08:44:53Z + PR: + + N-WASP + + + 0.9905291 + structure_element + cleaner0 + 2023-07-03T09:40:41Z + SO: + + F-BAR + + + 0.9283731 + protein + cleaner0 + 2023-06-30T08:44:54Z + PR: + + N-WASP + + + 0.99688065 + protein_state + cleaner0 + 2023-07-03T13:41:12Z + DUMMY: + + free + + + 0.9517934 + protein_state + cleaner0 + 2023-07-03T13:41:14Z + DUMMY: + + bound + + + 0.9954417 + protein_state + cleaner0 + 2023-07-03T13:41:17Z + DUMMY: + + active + + + 0.9683954 + protein_state + cleaner0 + 2023-07-03T13:41:21Z + DUMMY: + + inactive + + + 0.6535516 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.84625226 + protein_type + cleaner0 + 2023-07-03T10:19:26Z + MESH: + + TOCA family + + + 0.8193172 + protein_type + cleaner0 + 2023-07-03T10:19:29Z + MESH: + + WASP + + + 0.8569883 + protein_type + cleaner0 + 2023-07-03T10:19:32Z + MESH: + + WASP + + + protein + PR: + cleaner0 + 2023-07-03T10:19:42Z + + N-WASP + + + + DISCUSS + paragraph + 60496 + It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding. We therefore postulate an effector handover mechanism based on current evidence surrounding WASP/N-WASP activation and our model of the Cdc42·HR1TOCA1 complex. The displacement of the TOCA1 HR1 domain from Cdc42 by N-WASP may represent a unidirectional step in the pathway of Cdc42·N-WASP·TOCA1-dependent actin assembly. + + 0.9985322 + protein + cleaner0 + 2023-06-30T08:43:44Z + PR: + + TOCA1 + + + 0.945636 + protein + cleaner0 + 2023-06-30T08:44:54Z + PR: + + N-WASP + + + 0.9955771 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.9167795 + protein + cleaner0 + 2023-06-30T08:44:54Z + PR: + + N-WASP + + + 0.99846774 + protein + cleaner0 + 2023-06-30T08:43:44Z + PR: + + TOCA1 + + + 0.9865458 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.90799123 + protein + cleaner0 + 2023-07-03T10:12:21Z + PR: + + WASP + + + 0.7165899 + protein + cleaner0 + 2023-06-30T08:44:54Z + PR: + + N-WASP + + + 0.9785838 + complex_assembly + cleaner0 + 2023-07-03T10:50:09Z + GO: + + Cdc42·HR1TOCA1 + + + 0.99869764 + protein + cleaner0 + 2023-06-30T08:43:44Z + PR: + + TOCA1 + + + 0.99861073 + structure_element + cleaner0 + 2023-06-30T08:45:31Z + SO: + + HR1 + + + 0.9908448 + protein + cleaner0 + 2023-06-30T08:42:57Z + PR: + + Cdc42 + + + 0.928042 + protein + cleaner0 + 2023-06-30T08:44:54Z + PR: + + N-WASP + + + complex_assembly + GO: + cleaner0 + 2023-07-03T09:49:21Z + + Cdc42·N-WASP·TOCA1 + + + + AUTH_CONT + title_1 + 60981 + Author Contributions + + + AUTH_CONT + paragraph + 61002 + J. R. W. generated constructs and proteins, set up NMR experiments, analyzed NMR data, and performed binding experiments; D. N. set up NMR experiments; H. M. F. generated longer TOCA clones and proteins; J. L. G. supervised the pyrene actin assays; D. O. supervised the protein binding assays; and H. R. M. performed NMR experiments and analyzed NMR data. J. R. W., D. O., and H. R. M. wrote the paper with input from all authors. + + + AUTH_CONT + footnote + 61433 + The authors declare that they have no conflicts of interest with the contents of this article. + + + AUTH_CONT + footnote + 61528 + The atomic coordinates and structure factors (code 5FRG) have been deposited in the Protein Data Bank (http://wwpdb.org/). + + + AUTH_CONT + footnote + 61651 + D. Owen, unpublished data. + + + AUTH_CONT + footnote + 61678 + H. R. Mott and D. Owen, unpublished data. + + + AUTH_CONT + footnote + 61720 + PRK + + + AUTH_CONT + footnote + 61724 + protein kinase C related kinase + + + AUTH_CONT + footnote + 61756 + WASP + + + AUTH_CONT + footnote + 61761 + Wiskott-Aldrich syndrome protein + + + AUTH_CONT + footnote + 61794 + TOCA + + + AUTH_CONT + footnote + 61799 + transducer of Cdc42-dependent actin assembly protein + + + AUTH_CONT + footnote + 61852 + N-WASP + + + AUTH_CONT + footnote + 61859 + neural Wiskott-Aldrich syndrome protein + + + AUTH_CONT + footnote + 61899 + PI(4,5)P2 + + + AUTH_CONT + footnote + 61909 + phosphatidylinositol 4,5-bisphosphate + + + AUTH_CONT + footnote + 61947 + HR1 + + + AUTH_CONT + footnote + 61951 + homology region 1 + + + AUTH_CONT + footnote + 61969 + F-BAR + + + AUTH_CONT + footnote + 61975 + Fes/CIP4 homology BAR + + + AUTH_CONT + footnote + 61997 + SH3 + + + AUTH_CONT + footnote + 62001 + Src homology 3 + + + AUTH_CONT + footnote + 62016 + CRIB + + + AUTH_CONT + footnote + 62021 + Cdc42- and Rac-interactive binding + + + AUTH_CONT + footnote + 62056 + CIP4 + + + AUTH_CONT + footnote + 62061 + Cdc42-interacting protein 4 + + + AUTH_CONT + footnote + 62089 + MBP + + + AUTH_CONT + footnote + 62093 + maltose-binding protein + + + AUTH_CONT + footnote + 62117 + GBD + + + AUTH_CONT + footnote + 62121 + G protein binding domain + + + AUTH_CONT + footnote + 62146 + SPA + + + AUTH_CONT + footnote + 62150 + scintillation proximity assay + + + AUTH_CONT + footnote + 62180 + PAK + + + AUTH_CONT + footnote + 62184 + p21-activated kinase + + + AUTH_CONT + footnote + 62205 + ACK + + + AUTH_CONT + footnote + 62209 + activated Cdc42-associated kinase + + + AUTH_CONT + footnote + 62243 + HSQC + + + AUTH_CONT + footnote + 62248 + heteronuclear single quantum correlation + + + AUTH_CONT + footnote + 62289 + GMPPNP + + + AUTH_CONT + footnote + 62296 + guanosine 5′-[β,γ-imido] triphosphate + + + AUTH_CONT + footnote + 62341 + GTPγS + + + AUTH_CONT + footnote + 62351 + guanosine 5′-3-O-(thio)triphosphate + + + AUTH_CONT + footnote + 62389 + GBD + + + AUTH_CONT + footnote + 62393 + G protein-binding domain + + + AUTH_CONT + footnote + 62418 + CSP + + + AUTH_CONT + footnote + 62422 + chemical shift perturbation + + + AUTH_CONT + footnote + 62450 + PDB + + + AUTH_CONT + footnote + 62454 + Protein Data Bank. + + + AUTH_CONT + footnote + 62473 + The abbreviations used are: + + + REF + title + 62502 + References + + + 117 + 127 + surname:Bourne;given-names:H. R. + surname:Sanders;given-names:D. A. + surname:McCormick;given-names:F. + 1898771 + REF + Nature + ref + 349 + 1991 + 62513 + The GTPase superfamily: conserved structure and molecular mechanism + + + 269 + 309 + surname:Cherfils;given-names:J. + surname:Zeghouf;given-names:M. + 23303910 + REF + Physiol. Rev + ref + 93 + 2013 + 62581 + Regulation of small GTPases by GEFs, GAPs, and GDIs + + + 1299 + 1304 + surname:Vetter;given-names:I. R. + surname:Wittinghofer;given-names:A. + 11701921 + REF + Science + ref + 294 + 2001 + 62633 + The guanine nucleotide-binding switch in three dimensions + + + 85 + 133 + surname:Mott;given-names:H. R. + surname:Owen;given-names:D. + 25830673 + REF + Crit. Rev. Biochem. Mol. Biol + ref + 50 + 2015 + 62691 + Structures of Ras superfamily effector complexes: what have we learnt in two decades? + + + 304 + 310 + surname:Machesky;given-names:L. M. + surname:Hall;given-names:A. + 15157438 + REF + Trends Cell Biol + ref + 6 + 1996 + 62777 + Rho: a connection between membrane receptor signalling and the cytoskeleton + + + 389 + 399 + surname:Ridley;given-names:A. J. + surname:Hall;given-names:A. + 1643657 + REF + Cell + ref + 70 + 1992 + 62853 + The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors + + + 401 + 410 + surname:Ridley;given-names:A. J. + surname:Paterson;given-names:H. F. + surname:Johnston;given-names:C. L. + surname:Diekmann;given-names:D. + surname:Hall;given-names:A. + 1643658 + REF + Cell + ref + 70 + 1992 + 62983 + The small GTP-binding protein rac regulates growth factor-induced membrane ruffling + + + 1256 + 1264 + surname:Ridley;given-names:A. J. + 8939567 + REF + Curr. Biol + ref + 6 + 1996 + 63067 + Rho: theme and variations + + + 53 + 62 + surname:Nobes;given-names:C. D. + surname:Hall;given-names:A. + 7536630 + REF + Cell + ref + 81 + 1995 + 63093 + Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia + + + 509 + 514 + surname:Hall;given-names:A. + 9438836 + REF + Science + ref + 279 + 1998 + 63242 + Rho GTPases and the actin cytoskeleton + + + 267 + 272 + surname:Machesky;given-names:L. M. + surname:Insall;given-names:R. H. + 10427083 + REF + J. Cell Biol + ref + 146 + 1999 + 63281 + Signaling to actin dynamics + + + 1942 + 1952 + surname:Kozma;given-names:R. + surname:Ahmed;given-names:S. + surname:Best;given-names:A. + surname:Lim;given-names:L. + 7891688 + REF + Mol. Cell. Biol + ref + 15 + 1995 + 63309 + The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts + + + e29513 + surname:Kühn;given-names:S. + surname:Geyer;given-names:M. + 24914801 + REF + Small GTPases + ref + 5 + 2014 + 63445 + Formins as effector proteins of Rho GTPases + + + 645 + 648 + surname:Watanabe;given-names:G. + surname:Saito;given-names:Y. + surname:Madaule;given-names:P. + surname:Ishizaki;given-names:T. + surname:Fujisawa;given-names:K. + surname:Morii;given-names:N. + surname:Mukai;given-names:H. + surname:Ono;given-names:Y. + surname:Kakizuka;given-names:A. + surname:Narumiya;given-names:S. + 8571126 + REF + Science + ref + 271 + 1996 + 63489 + Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho + + + 723 + 734 + surname:Symons;given-names:M. + surname:Derry;given-names:J. M. . + surname:Karlak;given-names:B. + surname:Jiang;given-names:S. + surname:Lemahieu;given-names:V. + surname:Mccormick;given-names:F. + surname:Francke;given-names:U. + surname:Abo;given-names:A. + 8625410 + REF + Cell + ref + 84 + 1996 + 63577 + Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization + + + 203 + 216 + surname:Ho;given-names:H. Y. + surname:Rohatgi;given-names:R. + surname:Lebensohn;given-names:A. M. + surname:Le;given-names:Ma + surname:Li;given-names:J. + surname:Gygi;given-names:S. P. + surname:Kirschner;given-names:M. W. + 15260990 + REF + Cell + ref + 118 + 2004 + 63690 + Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex + + + 479 + 487 + surname:Aspenström;given-names:P. + 9210375 + REF + Curr. Biol + ref + 7 + 1997 + 63776 + A Cdc42 target protein with homology to the non-kinase domain of FER has a potential role in regulating the actin cytoskeleton + + + 1339 + 1344 + surname:Wu;given-names:M. + surname:Wu;given-names:X. + surname:De Camilli;given-names:P. + 23297209 + REF + Proc. Natl. Acad. Sci. U.S.A + ref + 110 + 2013 + 63903 + Calcium oscillations-coupled conversion of actin travelling waves to standing oscillations + + + 6932 + 6941 + surname:Miki;given-names:H. + surname:Suetsugu;given-names:S. + surname:Takenawa;given-names:T. + 9843499 + REF + EMBO J + ref + 17 + 1998 + 63994 + WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac + + + 221 + 231 + surname:Rohatgi;given-names:R. + surname:Ma;given-names:L. + surname:Miki;given-names:H. + surname:Lopez;given-names:M. + surname:Kirchhausen;given-names:T. + surname:Takenawa;given-names:T. + surname:Kirschner;given-names:M. W. + 10219243 + REF + Cell + ref + 97 + 1999 + 64076 + The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly + + + 1299 + 1310 + surname:Rohatgi;given-names:R. + surname:Ho;given-names:H. Y. + surname:Kirschner;given-names:M. W. + 10995436 + REF + J. Cell Biol + ref + 150 + 2000 + 64178 + Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4,5-bisphosphate + + + 1125 + 1136 + surname:Ma;given-names:L. + surname:Cantley;given-names:L. C. + surname:Janmey;given-names:P. A. + surname:Kirschner;given-names:M. W. + 9490725 + REF + J. Cell Biol + ref + 140 + 1998 + 64260 + Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly in Xenopus egg extracts + + + 151 + 158 + surname:Kim;given-names:A. S. + surname:Kakalis;given-names:L. T. + surname:Abdul-Manan;given-names:N. + surname:Liu;given-names:G. A. + surname:Rosen;given-names:M. K. + 10724160 + REF + Nature + ref + 404 + 2000 + 64391 + Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein + + + 707 + 735 + surname:Padrick;given-names:S. B. + surname:Rosen;given-names:M. K. + 20533885 + REF + Annu. Rev. Biochem + ref + 79 + 2010 + 64472 + Physical mechanisms of signal integration by WASP family proteins + + + 15362 + 15367 + surname:Ma;given-names:L. + surname:Rohatgi;given-names:R. + surname:Kirschner;given-names:M. W. + 9860974 + REF + Proc. Natl. Acad. Sci. U.S.A + ref + 95 + 1998 + 64538 + The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42 + + + 2817 + 2828 + surname:Takano;given-names:K. + surname:Takano;given-names:K. + surname:Toyooka;given-names:K. + surname:Suetsugu;given-names:S. + 18923421 + REF + EMBO J + ref + 27 + 2008 + 64634 + EFC/F-BAR proteins and the N-WASP-WIP complex induce membrane curvature-dependent actin polymerization + + + 11622 + 11636 + surname:Bu;given-names:W. + surname:Chou;given-names:A. M. + surname:Lim;given-names:K. B. + surname:Sudhaharan;given-names:T. + surname:Ahmed;given-names:S. + 19213734 + REF + J. Biol. Chem + ref + 284 + 2009 + 64737 + The Toca-1-N-WASP complex links filopodial formation to endocytosis + + + 1429 + 1437 + surname:Fricke;given-names:R. + surname:Gohl;given-names:C. + surname:Dharmalingam;given-names:E. + surname:Grevelhörster;given-names:A. + surname:Zahedi;given-names:B. + surname:Harden;given-names:N. + surname:Kessels;given-names:M. + surname:Qualmann;given-names:B. + surname:Bogdan;given-names:S. + 19716703 + REF + Curr. Biol + ref + 19 + 2009 + 64805 + Drosophila Cip4/Toca-1 integrates membrane trafficking and actin dynamics through WASP and SCAR/WAVE + + + e1000675 + surname:Giuliani;given-names:C. + surname:Troglio;given-names:F. + surname:Hardin;given-names:J. D. + surname:Soto;given-names:M. C. + surname:Grant;given-names:B. D. + surname:Scita;given-names:G. + surname:Bai;given-names:Z. + surname:Patel;given-names:F. B. + surname:Zucconi;given-names:A. + surname:Malabarba;given-names:M. G. + surname:Disanza;given-names:A. + surname:Stradal;given-names:T. B. + surname:Cassata;given-names:G. + surname:Confalonieri;given-names:S. + 19798448 + REF + PLoS Genet + ref + 5 + 2009 + 64906 + Requirements for F-BAR proteins TOCA-1 and TOCA-2 in actin dynamics and membrane trafficking during Caenorhabditis elegans oocyte growth and embryonic epidermal morphogenesis + + + 1 + 16 + surname:Bu;given-names:W. + surname:Lim;given-names:K. B. + surname:Yu;given-names:Y. H. + surname:Chou;given-names:A. M. + surname:Sudhaharan;given-names:T. + surname:Ahmed;given-names:S. + REF + PLoS ONE + ref + 5 + 2010 + 65081 + Cdc42 interaction with N-WASP and Toca-1 regulates membrane tubulation, vesicle formation and vesicle motility: implications for endocytosis + + + 1341 + 1345 + surname:Lee;given-names:K. + surname:Gallop;given-names:J. L. + surname:Rambani;given-names:K. + surname:Kirschner;given-names:M. W. + 20829485 + REF + Science + ref + 329 + 2010 + 65222 + Self-assembly of filopodia-like structures on supported lipid bilayers + + + 7193 + 7198 + surname:Gallop;given-names:J. L. + surname:Walrant;given-names:A. + surname:Cantley;given-names:L. C. + surname:Kirschner;given-names:M. W. + 23589871 + REF + Proc. Natl. Acad. Sci. U.S.A + ref + 110 + 2013 + 65293 + Phosphoinositides and membrane curvature switch the mode of actin polymerization via selective recruitment of toca-1 and Snx9 + + + 269 + 279 + surname:Tsujita;given-names:K. + surname:Suetsugu;given-names:S. + surname:Sasaki;given-names:N. + surname:Furutani;given-names:M. + surname:Oikawa;given-names:T. + surname:Takenawa;given-names:T. + 16418535 + REF + J. Cell Biol + ref + 172 + 2006 + 65419 + Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis + + + E1443 + E1452 + surname:Bai;given-names:Z. + surname:Grant;given-names:B. D. + 25775511 + REF + Proc. Natl. Acad. Sci. U.S.A + ref + 112 + 2015 + 65569 + A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling + + + 29042 + 29053 + surname:Kakimoto;given-names:T. + surname:Katoh;given-names:H. + surname:Negishi;given-names:M. + 16885158 + REF + J. Biol. Chem + ref + 281 + 2006 + 65654 + Regulation of neuronal morphology by Toca-1, an F-BAR/EFC protein that induces plasma membrane invagination + + + 946 + 958 + surname:Miyamoto;given-names:K. + surname:Pasque;given-names:V. + surname:Jullien;given-names:J. + surname:Gurdon;given-names:J. B. + 21536734 + REF + Genes Dev + ref + 25 + 2011 + 65762 + Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes + + + 2769 + 2787 + surname:Van Itallie;given-names:C. M. + surname:Tietgens;given-names:A. J. + surname:Krystofiak;given-names:E. + surname:Kachar;given-names:B. + surname:Anderson;given-names:J. M. + 26063734 + REF + Mol. Biol. Cell + ref + 26 + 2015 + 65856 + A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction + + + 2261 + 2272 + surname:Hu;given-names:J. + surname:Mukhopadhyay;given-names:A. + surname:Craig;given-names:A. W. B. + 21062739 + REF + J. Biol. Chem + ref + 286 + 2011 + 65955 + Transducer of Cdc42-dependent actin assembly promotes epidermal growth factor-induced cell motility and invasiveness + + + 3080 + 3090 + surname:Chander;given-names:H. + surname:Truesdell;given-names:P. + surname:Meens;given-names:J. + surname:Craig;given-names:A. W. B. + 22824798 + REF + Oncogene + ref + 32 + 2013 + 66072 + Transducer of Cdc42-dependent actin assembly promotes breast cancer invasion and metastasis + + + 791 + 804 + surname:Itoh;given-names:T. + surname:Erdmann;given-names:K. S. + surname:Roux;given-names:A. + surname:Habermann;given-names:B. + surname:Werner;given-names:H. + surname:De Camilli;given-names:P. + 16326391 + REF + Dev. Cell + ref + 9 + 2005 + 66164 + Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins + + + 839 + 852 + surname:Henne;given-names:W. M. + surname:Kent;given-names:H. M. + surname:Ford;given-names:M. G. J. + surname:Hegde;given-names:B. G. + surname:Daumke;given-names:O. + surname:Butler;given-names:P. J. G. + surname:Mittal;given-names:R. + surname:Langen;given-names:R. + surname:Evans;given-names:P. R. + surname:McMahon;given-names:H. T. + 17540576 + REF + Structure + ref + 15 + 2007 + 66277 + Structure and analysis of FCHo2 F-BAR domain: a dimerizing and membrane recruitment module that effects membrane curvature + + + 793 + 803 + surname:Maesaki;given-names:R. + surname:Ihara;given-names:K. + surname:Shimizu;given-names:T. + surname:Kuroda;given-names:S. + surname:Kaibuchi;given-names:K. + surname:Hakoshima;given-names:T. + 10619026 + REF + Mol. Cell + ref + 4 + 1999 + 66400 + The structural basis of Rho effector recognition revealed by the crystal structure of human RhoA complexed with the effector domain of PKN/PRK1 + + + 50578 + 50587 + surname:Owen;given-names:D. + surname:Lowe;given-names:P. N. + surname:Nietlispach;given-names:D. + surname:Brosnan;given-names:C. E. + surname:Chirgadze;given-names:D. Y. + surname:Parker;given-names:P. J. + surname:Blundell;given-names:T. L. + surname:Mott;given-names:H. R. + 14514689 + REF + J. Biol. Chem + ref + 278 + 2003 + 66544 + Molecular dissection of the interaction between the small G proteins Rac1 and RhoA and protein kinase C-related kinase 1 (PRK1) + + + 2860 + 2869 + surname:Hutchinson;given-names:C. L. + surname:Lowe;given-names:P. N. + surname:McLaughlin;given-names:S. H. + surname:Mott;given-names:H. R. + surname:Owen;given-names:D. + 21351730 + REF + Biochemistry + ref + 50 + 2011 + 66672 + Mutational analysis reveals a single binding interface between RhoA and its effector, PRK1 + + + 7999 + 8011 + surname:Hutchinson;given-names:C. L. + surname:Lowe;given-names:P. N. + surname:McLaughlin;given-names:S. H. + surname:Mott;given-names:H. R. + surname:Owen;given-names:D. + 24128008 + REF + Biochemistry + ref + 52 + 2013 + 66763 + Differential binding of RhoA, RhoB, and RhoC to protein kinase C-related kinase (PRK) isoforms PRK1, PRK2, and PRK3: PRKs have the highest affinity for RhoB + + + 1492 + 1500 + surname:Modha;given-names:R. + surname:Campbell;given-names:L. J. + surname:Nietlispach;given-names:D. + surname:Buhecha;given-names:H. R. + surname:Owen;given-names:D. + surname:Mott;given-names:H. R. + 18006505 + REF + J. Biol. Chem + ref + 283 + 2008 + 66920 + The Rac1 polybasic region is required for interaction with its effector PRK1 + + + 113 + 118 + surname:Kobashigawa;given-names:Y. + surname:Kumeta;given-names:H. + surname:Kanoh;given-names:D. + surname:Inagaki;given-names:F. + 19387844 + REF + J. Biomol. NMR + ref + 44 + 2009 + 66997 + The NMR structure of the TC10- and Cdc42-interacting domain of CIP4 + + + 29071 + 29074 + surname:Burbelo;given-names:P. D. + surname:Drechsel;given-names:D. + surname:Hall;given-names:A. + 7493928 + REF + J. Biol. Chem + ref + 270 + 1995 + 67065 + A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases + + + 379 + 383 + surname:Abdul-Manan;given-names:N. + surname:Aghazadeh;given-names:B. + surname:Liu;given-names:G. A. + surname:Majumdar;given-names:A. + surname:Ouerfelli;given-names:O. + surname:Siminovitch;given-names:K. A. + surname:Rosen;given-names:M. K. + 10360578 + REF + Nature + ref + 399 + 1999 + 67165 + Structure of Cdc42 in complex with the GTPase-binding domain of the “Wiskott-Aldrich syndrome” protein + + + 1243 + 1250 + surname:Owen;given-names:D. + surname:Mott;given-names:H. R. + surname:Laue;given-names:E. D. + surname:Lowe;given-names:P. N. + 10684602 + REF + Biochemistry + ref + 39 + 2000 + 67272 + Residues in Cdc42 that specify binding to individual CRIB effector proteins + + + 992 + 999 + surname:Bailey;given-names:L. K. + surname:Campbell;given-names:L. J. + surname:Evetts;given-names:K. A. + surname:Littlefield;given-names:K. + surname:Rajendra;given-names:E. + surname:Nietlispach;given-names:D. + surname:Owen;given-names:D. + surname:Mott;given-names:H. R. + 18981177 + REF + J. Biol. Chem + ref + 284 + 2009 + 67348 + The structure of binder of Arl2 (BART) reveals a novel G protein binding domain: implications for function + + + 7885 + 7891 + surname:Thompson;given-names:G. + surname:Owen;given-names:D. + surname:Chalk;given-names:P. A. + surname:Lowe;given-names:P. N. + 9601050 + REF + Biochemistry + ref + 37 + 1998 + 67455 + Delineation of the Cdc42/Rac-binding domain of p21-activated kinase + + + 1692 + 1704 + surname:Owen;given-names:D. + surname:Campbell;given-names:L. J. + surname:Littlefield;given-names:K. + surname:Evetts;given-names:K. A. + surname:Li;given-names:Z. + surname:Sacks;given-names:D. B. + surname:Lowe;given-names:P. N. + surname:Mott;given-names:H. R. + 17984089 + REF + J. Biol. Chem + ref + 283 + 2008 + 67523 + The IQGAP1-Rac1 and IQGAP1-Cdc42 interactions: interfaces differ between the complexes + + + surname:Watson;given-names:J. R. + surname:Nietlispach;given-names:D. + surname:Owen;given-names:D. + surname:Mott;given-names:H. R. + 10.1007/s12104-016-9677-8 + REF + Biomol. NMR Assign + ref + 2016 + 67610 + 1H, 13C and 15N resonance assignments of the Cdc42-binding domain of TOCA1 + + + 687 + 696 + surname:Vranken;given-names:W. F. + surname:Boucher;given-names:W. + surname:Stevens;given-names:T. J. + surname:Fogh;given-names:R. H. + surname:Pajon;given-names:A. + surname:Llinas;given-names:M. + surname:Ulrich;given-names:E. L. + surname:Markley;given-names:J. L. + surname:Ionides;given-names:J. + surname:Laue;given-names:E. D. + 15815974 + REF + Proteins + ref + 59 + 2005 + 67685 + The CCPN data model for NMR spectroscopy: development of a software pipeline + + + 381 + 382 + surname:Rieping;given-names:W. + surname:Habeck;given-names:M. + surname:Bardiaux;given-names:B. + surname:Bernard;given-names:A. + surname:Malliavin;given-names:T. E. + surname:Nilges;given-names:M. + 17121777 + REF + Bioinformatics + ref + 23 + 2007 + 67762 + ARIA2: automated NOE assignment and data integration in NMR structure calculation + + + 227 + 241 + surname:Shen;given-names:Y. + surname:Bax;given-names:A. + 23728592 + REF + J. Biomol. NMR + ref + 56 + 2013 + 67844 + Protein backbone and side chain torsion angles predicted from NMR chemical shifts using artificial neural networks + + + surname:Hubbard;given-names:S. + surname:Thornton;given-names:J. + REF + NACCESS + ref + 1993 + 67959 + + + 125 + 147 + surname:Walrant;given-names:A. + surname:Saxton;given-names:D. S. + surname:Correia;given-names:G. P. + surname:Gallop;given-names:J. L. + 25997346 + REF + Methods Cell Biol + ref + 128 + 2015 + 67960 + Triggering actin polymerization in Xenopus egg extracts from phosphoinositide-containing lipid bilayers + + + 512 + 524 + surname:Lebensohn;given-names:A. M. + surname:Kirschner;given-names:M. W. + 19917258 + REF + Mol. Cell + ref + 36 + 2009 + 68064 + Activation of the WAVE complex by coincident signals controls actin assembly + + + 384 + 388 + surname:Mott;given-names:H. R. + surname:Owen;given-names:D. + surname:Nietlispach;given-names:D. + surname:Lowe;given-names:P. N. + surname:Manser;given-names:E. + surname:Lim;given-names:L. + surname:Laue;given-names:E. D. + 10360579 + REF + Nature + ref + 399 + 1999 + 68141 + Structure of the small G protein Cdc42 bound to the GTPase-binding domain of ACK + + + 25478 + 25489 + surname:Nakamura;given-names:K. + surname:Man;given-names:Z. + surname:Xie;given-names:Y. + surname:Hanai;given-names:A. + surname:Makyio;given-names:H. + surname:Kawasaki;given-names:M. + surname:Kato;given-names:R. + surname:Shin;given-names:H.-W. + surname:Nakayama;given-names:K. + surname:Wakatsuki;given-names:S. + 22679020 + REF + J. Biol. Chem + ref + 287 + 2012 + 68222 + Structural basis for membrane binding specificity of the Bin/Amphiphysin/Rvs (BAR) domain of Arfaptin-2 determined by Arl1 GTPase + + + 759 + 769 + surname:ten Klooster;given-names:J. P. + surname:Jaffer;given-names:Z. M. + surname:Chernoff;given-names:J. + surname:Hordijk;given-names:P. L. + 16492808 + REF + J. Cell Biol + ref + 172 + 2006 + 68352 + Targeting and activation of Rac1 are mediated by the exchange factor β-Pix + + + W500 + W502 + surname:Heinig;given-names:M. + surname:Frishman;given-names:D. + 15215436 + REF + Nucleic Acids Res + ref + 32 + 2004 + 68430 + STRIDE: a web server for secondary structure assignment from known atomic coordinates of proteins + + + 199 + 211 + surname:Schanda;given-names:P. + surname:Kupce;given-names:E. + surname:Brutscher;given-names:B. + 16341750 + REF + J. Biomol. NMR + ref + 33 + 2005 + 68528 + SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds + + + 985 + 995 + surname:Fenwick;given-names:R. B. + surname:Campbell;given-names:L. J. + surname:Rajasekar;given-names:K. + surname:Prasannan;given-names:S. + surname:Nietlispach;given-names:D. + surname:Camonis;given-names:J. + surname:Owen;given-names:D. + surname:Mott;given-names:H. R. + 20696399 + REF + Structure + ref + 18 + 2010 + 68649 + The RalB-RLIP76 complex reveals a novel mode of Ral-effector interaction + + + 883 + 897 + surname:de Vries;given-names:S. J. + surname:van Dijk;given-names:M. + surname:Bonvin;given-names:A. M. J. J. + 20431534 + REF + Nat. Protoc + ref + 5 + 2010 + 68722 + The HADDOCK web server for data-driven biomolecular docking + + + 18067 + 18076 + surname:Rudolph;given-names:M. G. + surname:Bayer;given-names:P. + surname:Abo;given-names:A. + surname:Kuhlmann;given-names:J. + surname:Vetter;given-names:I. R. + surname:Wittinghofer;given-names:A. + 9660763 + REF + J. Biol. Chem + ref + 273 + 1998 + 68782 + The Cdc42/Rac interactive binding region motif of the Wiskott Aldrich syndrome protein (WASP) is necessary but not sufficient for tight binding to Cdc42 and structure formation + + + 313 + 324 + surname:Hemsath;given-names:L. + surname:Dvorsky;given-names:R. + surname:Fiegen;given-names:D. + surname:Carlier;given-names:M.-F. + surname:Ahmadian;given-names:M. R. + 16246732 + REF + Mol. Cell + ref + 20 + 2005 + 68959 + An electrostatic steering mechanism of Cdc42 recognition by Wiskott-Aldrich syndrome proteins + + + 14087 + 14099 + surname:Elliot-Smith;given-names:A. E. + surname:Owen;given-names:D. + surname:Mott;given-names:H. R. + surname:Lowe;given-names:P. N. + 17999470 + REF + Biochemistry + ref + 46 + 2007 + 69053 + Double mutant cycle thermodynamic analysis of the hydrophobic Cdc42-ACK protein-protein interaction + + + e78 + surname:Wang;given-names:J. + surname:Lu;given-names:Q. + surname:Lu;given-names:H. P. + 16839193 + REF + PLoS Comput. Biol + ref + 2 + 2006 + 69153 + Single-molecule dynamics reveals cooperative binding-folding in protein recognition + + + 12373 + 12383 + surname:Elliot-Smith;given-names:A. E. + surname:Mott;given-names:H. R. + surname:Lowe;given-names:P. N. + surname:Laue;given-names:E. D. + surname:Owen;given-names:D. + 16156650 + REF + Biochemistry + ref + 44 + 2005 + 69237 + Specificity determinants on Cdc42 for binding its effector protein ACK + + + 1233 + 1243 + surname:Perkins;given-names:J. R. + surname:Diboun;given-names:I. + surname:Dessailly;given-names:B. H. + surname:Lees;given-names:J. G. + surname:Orengo;given-names:C. + 20947012 + REF + Structure + ref + 18 + 2010 + 69308 + Transient protein-protein interactions: structural, functional, and network properties + + + 635 + 648 + surname:Acuner Ozbabacan;given-names:S. E. + surname:Engin;given-names:H. B. + surname:Gursoy;given-names:A. + surname:Keskin;given-names:O. + 21676899 + REF + Protein Eng. Des. Sel + ref + 24 + 2011 + 69395 + Transient protein-protein interactions + + + 883 + 888 + surname:Schmid;given-names:E. M. + surname:McMahon;given-names:H. T. + 17713526 + REF + Nature + ref + 448 + 2007 + 69434 + Integrating molecular and network biology to decode endocytosis + + + 4371 + 4383 + surname:Praefcke;given-names:G. J. K. + surname:Ford;given-names:M. G. J. + surname:Schmid;given-names:E. M. + surname:Olesen;given-names:L. E. + surname:Gallop;given-names:J. L. + surname:Peak-Chew;given-names:S.-Y. + surname:Vallis;given-names:Y. + surname:Babu;given-names:M. M. + surname:Mills;given-names:I. G. + surname:McMahon;given-names:H. T. + 15496985 + REF + EMBO J + ref + 23 + 2004 + 69498 + Evolving nature of the AP2 α-appendage hub during clathrin-coated vesicle endocytosis + + + 519 + 531 + surname:Höning;given-names:S. + surname:Ricotta;given-names:D. + surname:Krauss;given-names:M. + surname:Späte;given-names:K. + surname:Spolaore;given-names:B. + surname:Motley;given-names:A. + surname:Robinson;given-names:M. + surname:Robinson;given-names:C. + surname:Haucke;given-names:V. + surname:Owen;given-names:D. J. + 15916959 + REF + Mol. Cell + ref + 18 + 2005 + 69588 + Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2 + + + 5685 + 5690 + surname:Leung;given-names:D. W. + surname:Rosen;given-names:M. K. + 15821030 + REF + Proc. Natl. Acad. Sci. U.S.A + ref + 102 + 2005 + 69712 + The nucleotide switch in Cdc42 modulates coupling between the GTPase-binding and allosteric equilibria of Wiskott-Aldrich syndrome protein + + + 271 + 285 + surname:Buck;given-names:M. + surname:Xu;given-names:W. + surname:Rosen;given-names:M. K. + 15066431 + REF + J. Mol. Biol + ref + 338 + 2004 + 69851 + A two-state allosteric model for autoinhibition rationalizes WASP signal integration and targeting + + + 73 + 78 + surname:Miki;given-names:H. + surname:Takenawa;given-names:T. + 9473482 + REF + Biochem. Biophys. Res. Commun + ref + 243 + 1998 + 69950 + Direct binding of the verprolin-homology domain in N-WASP to actin is essential for cytoskeletal reorganization + + + diff --git a/BioC_XML/4937829_v0.xml b/BioC_XML/4937829_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..d94e1799401293a468f0d9a6d0036e7a94089c76 --- /dev/null +++ b/BioC_XML/4937829_v0.xml @@ -0,0 +1,6677 @@ + + + + PMC + 20201220 + pmc.key + + 4937829 + NO-CC CODE + no + 0 + 0 + + 10.1038/nsmb.3237 + NIHMS785109 + 4937829 + 27239796 + 691 + 7 + Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: + + 697 + surname:Horowitz;given-names:Scott + surname:Salmon;given-names:Loïc + surname:Trievel;given-names:Raymond C. + surname:Brooks;given-names:Charles L.;suffix:III + surname:Bardwell;given-names:James CA + surname:Koldewey;given-names:Philipp + surname:Ahlstrom;given-names:Logan S. + surname:Martin;given-names:Raoul + surname:Quan;given-names:Shu + surname:Afonine;given-names:Pavel V. + surname:van den Bedem;given-names:Henry + surname:Wang;given-names:Lili + surname:Xu;given-names:Qingping + TITLE + front + 23 + 2016 + 0 + Visualizing chaperone-assisted protein folding + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + + ABSTRACT + abstract + 47 + Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone. + + 0.98602355 + evidence + cleaner0 + 2023-06-29T11:53:47Z + DUMMY: + + structures + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:59:04Z + + chaperone + + + protein_type + MESH: + cleaner0 + 2023-06-29T10:01:05Z + + chaperones + + + 0.9951129 + experimental_method + cleaner0 + 2023-06-29T09:58:23Z + MESH: + + X-ray crystallography + + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:52:13Z + + Residual Electron and Anomalous Density + + + experimental_method + MESH: + cleaner0 + 2023-06-29T12:00:22Z + + READ + + + 0.9784461 + experimental_method + cleaner0 + 2023-06-29T09:51:47Z + MESH: + + READ + + + 0.9859038 + protein + cleaner0 + 2023-06-29T09:56:57Z + PR: + + immunity protein 7 + + + 0.9981862 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.8496159 + protein_state + cleaner0 + 2023-06-29T12:12:05Z + DUMMY: + + in complex with + + + 0.9949854 + species + cleaner0 + 2023-06-29T09:49:55Z + MESH: + + E. coli + + + 0.5430761 + protein_type + cleaner0 + 2023-06-29T09:57:10Z + MESH: + + chaperone + + + 0.99860436 + protein + cleaner0 + 2023-06-29T09:57:24Z + PR: + + Spy + + + 0.9972229 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.9905349 + protein_state + cleaner0 + 2023-06-29T12:12:11Z + DUMMY: + + bound to + + + 0.9983197 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9952988 + protein_state + cleaner0 + 2023-06-29T12:12:15Z + DUMMY: + + Spy-associated + + + 0.99446774 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.97574776 + protein_state + cleaner0 + 2023-06-29T12:12:18Z + DUMMY: + + unfolded + + + 0.7940124 + protein_state + cleaner0 + 2023-06-29T12:12:20Z + DUMMY: + + folded + + + 0.8012317 + protein_state + cleaner0 + 2023-06-29T12:12:23Z + DUMMY: + + native + + + 0.9822854 + protein_state + cleaner0 + 2023-06-29T12:12:25Z + DUMMY: + + bound to + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + + INTRO + paragraph + 1106 + High-resolution structural models of protein-protein interactions are critical for obtaining mechanistic insights into biological processes. However, many protein-protein interactions are highly dynamic, making it difficult to obtain high-resolution data. Particularly challenging are interactions of intrinsically or conditionally disordered sections of proteins with their partner proteins. Recent advances in X-ray crystallography and NMR spectroscopy continue to improve our ability to analyze biomolecules that exist in multiple conformations. X-ray crystallography has historically provided valuable information on small-scale conformational changes, but observing large-amplitude heterogeneous conformational changes often falls beyond the reach of current crystallographic techniques. NMR can theoretically be used to determine heterogeneous ensembles, but in practice, this proves to be very challenging. + + evidence + DUMMY: + cleaner0 + 2023-06-29T09:59:29Z + + structural models + + + 0.83736765 + protein_state + cleaner0 + 2023-06-29T12:12:29Z + DUMMY: + + highly dynamic + + + 0.9667534 + protein_state + cleaner0 + 2023-06-29T12:12:32Z + DUMMY: + + intrinsically or conditionally disordered + + + 0.99611044 + experimental_method + cleaner0 + 2023-06-29T09:58:23Z + MESH: + + X-ray crystallography + + + 0.98872364 + experimental_method + cleaner0 + 2023-06-29T09:59:38Z + MESH: + + NMR spectroscopy + + + 0.99599904 + experimental_method + cleaner0 + 2023-06-29T09:58:23Z + MESH: + + X-ray crystallography + + + 0.99427587 + experimental_method + cleaner0 + 2023-06-29T09:59:43Z + MESH: + + NMR + + + + INTRO + paragraph + 2020 + Despite the importance of understanding how proteins fold into their native state within the cell, our knowledge about this critical process remains limited. It is clear that molecular chaperones aid in protein folding. However, exactly how they facilitate the folding process is still being debated. Structural characterization of chaperone-assisted protein folding likely would help bring clarity to this question. Structural models of chaperone-substrate complexes have recently begun to provide information as to how a chaperone can recognize its substrate. However, the impact that chaperones have on their substrates, and how these interactions affect the folding process remain largely unknown. For most chaperones, it is still unclear whether the chaperone actively participates in and affects the folding of the substrate proteins, or merely provides a suitable microenvironment enabling the substrate to fold on its own. This is a truly fundamental question in the chaperone field, and one that has eluded the community largely because of the highly dynamic nature of the chaperone-substrate complexes. + + protein_type + MESH: + cleaner0 + 2023-06-29T10:01:05Z + + chaperones + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.9913229 + evidence + cleaner0 + 2023-06-29T11:53:52Z + DUMMY: + + Structural models + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + protein_type + MESH: + cleaner0 + 2023-06-29T10:01:05Z + + chaperones + + + protein_type + MESH: + cleaner0 + 2023-06-29T10:01:05Z + + chaperones + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.9763981 + protein_state + cleaner0 + 2023-06-29T12:12:37Z + DUMMY: + + highly dynamic + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + + INTRO + paragraph + 3133 + To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy. Spy prevents protein aggregation and aids in protein folding under various stress conditions, including treatment with tannin and butanol. We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy. The crystal structure of Spy revealed that it forms a thin α-helical homodimeric cradle. Crosslinking and genetic experiments suggested that Spy interacts with substrates somewhere on its concave side. By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex. This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones. + + 0.99309033 + protein_state + cleaner0 + 2023-06-29T12:12:41Z + DUMMY: + + ATP-independent + + + 0.99527353 + species + cleaner0 + 2023-06-29T09:50:02Z + MESH: + + Escherichia coli + + + 0.88843155 + protein_type + cleaner0 + 2023-06-29T09:57:11Z + MESH: + + chaperone + + + 0.9985643 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9985098 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9877836 + chemical + cleaner0 + 2023-06-29T12:04:53Z + CHEBI: + + tannin + + + 0.9702978 + chemical + cleaner0 + 2023-06-29T12:04:57Z + CHEBI: + + butanol + + + 0.9984925 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9975439 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.9958406 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.99794847 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9975363 + evidence + cleaner0 + 2023-06-29T11:53:59Z + DUMMY: + + crystal structure + + + 0.9985667 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.7065689 + oligomeric_state + cleaner0 + 2023-06-29T12:18:00Z + DUMMY: + + homodimeric + + + 0.97890526 + site + cleaner0 + 2023-06-29T10:23:09Z + SO: + + cradle + + + 0.98890847 + experimental_method + cleaner0 + 2023-06-29T12:00:37Z + MESH: + + Crosslinking and genetic experiments + + + 0.9981775 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:58:23Z + + X-ray crystallography + + + 0.9977272 + evidence + cleaner0 + 2023-06-29T11:54:04Z + DUMMY: + + crystal structures + + + 0.77756804 + evidence + cleaner0 + 2023-06-29T11:54:08Z + DUMMY: + + ensemble + + + 0.99241126 + protein_state + cleaner0 + 2023-06-29T12:12:45Z + DUMMY: + + dynamic + + + 0.9943983 + complex_assembly + cleaner0 + 2023-06-29T12:05:48Z + GO: + + Spy:Im7 + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.99670714 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.9471477 + protein_state + cleaner0 + 2023-06-29T12:12:48Z + DUMMY: + + bound to + + + protein_type + MESH: + cleaner0 + 2023-06-29T10:01:04Z + + chaperones + + + + RESULTS + title_1 + 4089 + RESULTS + + + RESULTS + title_2 + 4097 + Crystallizing the Spy:Im7 complex + + 0.97671187 + experimental_method + cleaner0 + 2023-06-29T12:00:41Z + MESH: + + Crystallizing + + + 0.990619 + complex_assembly + cleaner0 + 2023-06-29T12:05:55Z + GO: + + Spy:Im7 + + + + RESULTS + paragraph + 4131 + We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence. We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals. Subsequent crystal washing and dissolution experiments confirmed the presence of the substrates in the co-crystals (Supplementary Fig. 2). The crystals diffracted to ~1.8 Å resolution. We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes. However, modeling of the substrate in the complex proved to be a substantial challenge, as the electron density of the substrate was discontinuous and fragmented. Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a). We hypothesized that the fragmented density was due to multiple, partially occupied conformations of the substrate bound within the crystal. Such residual density is typically not considered usable by traditional X-ray crystallography methods. Thus, we developed a new approach to interpret the chaperone-bound substrate in multiple conformations. + + 0.96328425 + evidence + cleaner0 + 2023-06-29T09:51:24Z + DUMMY: + + crystals + + + 0.9971807 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.5414726 + experimental_method + cleaner0 + 2023-06-29T10:01:47Z + MESH: + + crystallization conditions + + + 0.9924412 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.98916316 + evidence + cleaner0 + 2023-06-29T09:51:24Z + DUMMY: + + crystals + + + 0.8674296 + protein_state + cleaner0 + 2023-06-29T12:12:53Z + DUMMY: + + presence of + + + 0.6865464 + protein_state + cleaner0 + 2023-06-29T12:12:56Z + DUMMY: + + absence + + + 0.59963125 + experimental_method + cleaner0 + 2023-06-29T12:00:53Z + MESH: + + screened + + + 0.8132744 + experimental_method + cleaner0 + 2023-06-29T10:01:47Z + MESH: + + crystallization conditions + + + 0.99615026 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.47748753 + protein_state + cleaner0 + 2023-06-29T12:12:58Z + DUMMY: + + unfolded + + + 0.88614196 + taxonomy_domain + cleaner0 + 2023-06-29T09:50:27Z + DUMMY: + + bovine + + + 0.9140386 + chemical + cleaner0 + 2023-06-29T12:05:02Z + CHEBI: + + α-casein + + + 0.9974244 + protein_state + cleaner0 + 2023-06-29T12:13:00Z + DUMMY: + + wild-type + + + 0.99624205 + protein_state + cleaner0 + 2023-06-29T12:13:02Z + DUMMY: + + WT + + + 0.9939699 + species + cleaner0 + 2023-06-29T09:50:35Z + MESH: + + E. coli + + + 0.9985669 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.99383175 + protein_state + cleaner0 + 2023-06-29T12:13:05Z + DUMMY: + + unfolded + + + 0.9981888 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + mutant + MESH: + cleaner0 + 2023-06-29T10:03:28Z + + L18A + + + mutant + MESH: + cleaner0 + 2023-06-29T10:03:39Z + + L19A + + + mutant + MESH: + cleaner0 + 2023-06-29T10:03:48Z + + L37A + + + 0.9933337 + structure_element + cleaner0 + 2023-06-29T12:18:23Z + SO: + + N-terminal half + + + 0.99848396 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.9150071 + mutant + cleaner0 + 2023-06-29T09:56:05Z + MESH: + + Im76-45 + + + structure_element + SO: + cleaner0 + 2023-06-29T10:14:38Z + + Spy-binding portion + + + 0.9982128 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.98329085 + experimental_method + cleaner0 + 2023-06-29T12:00:58Z + MESH: + + co-crystallized + + + 0.71110064 + protein_state + cleaner0 + 2023-06-29T12:13:09Z + DUMMY: + + with + + + 0.9975012 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.99698585 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.8080074 + protein_state + cleaner0 + 2023-06-29T12:13:12Z + DUMMY: + + alone + + + 0.94558287 + evidence + cleaner0 + 2023-06-29T09:51:24Z + DUMMY: + + crystals + + + 0.8897016 + experimental_method + cleaner0 + 2023-06-29T12:01:05Z + MESH: + + crystal washing and dissolution + + + 0.8521347 + experimental_method + cleaner0 + 2023-06-29T12:01:09Z + MESH: + + co-crystals + + + 0.9779488 + evidence + cleaner0 + 2023-06-29T09:51:23Z + DUMMY: + + crystals + + + complex_assembly + GO: + cleaner0 + 2023-06-29T09:55:24Z + + Spy:Im76-45 + + + 0.9705391 + chemical + cleaner0 + 2023-06-29T10:05:46Z + CHEBI: + + selenomethionine + + + 0.98479927 + evidence + cleaner0 + 2023-06-29T09:51:24Z + DUMMY: + + crystals + + + 0.9901266 + experimental_method + cleaner0 + 2023-06-29T10:04:01Z + MESH: + + single-wavelength anomalous diffraction + + + 0.957922 + experimental_method + cleaner0 + 2023-06-29T10:04:05Z + MESH: + + SAD + + + 0.99743783 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.99567246 + evidence + cleaner0 + 2023-06-29T09:50:58Z + DUMMY: + + electron density + + + structure_element + SO: + cleaner0 + 2023-06-29T10:14:56Z + + minimal binding portion + + + 0.99820125 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + mutant + MESH: + cleaner0 + 2023-06-29T09:56:05Z + + Im76-45 + + + 0.9937921 + evidence + cleaner0 + 2023-06-29T09:50:59Z + DUMMY: + + electron density + + + 0.88117105 + evidence + cleaner0 + 2023-06-29T11:54:22Z + DUMMY: + + density + + + 0.6665854 + evidence + cleaner0 + 2023-06-29T09:51:13Z + DUMMY: + + crystal + + + 0.9937469 + experimental_method + cleaner0 + 2023-06-29T09:58:23Z + MESH: + + X-ray crystallography + + + 0.99554175 + protein_state + cleaner0 + 2023-06-29T12:13:15Z + DUMMY: + + chaperone-bound + + + + RESULTS + title_2 + 5875 + READ: a strategy to visualize heterogeneous and dynamic biomolecules + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:51:47Z + + READ + + + + RESULTS + paragraph + 5944 + To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density. We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate. (2) We then labeled individual residues in the flexible regions of the substrate with the strong anomalous scatterer iodine, which serves to locate these residues in three-dimensional space using their anomalous density. (3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density. (5) Finally, we validated the ensemble using multiple statistical tests. Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues. These two forms of data are therefore complementary: by labeling individual residues, one can locate them to specific points in space. The electron density then allowed us to connect the labeled residues of the substrate by confining the protein chain within regions of detectable density. In this way, the two forms of data together were able to describe multiple conformations of the substrate within the crystal. As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy. However, we believe that READ will prove generally applicable to visualizing heterogeneous and dynamic complexes that have previously escaped detailed structural analysis. + + 0.9949292 + evidence + cleaner0 + 2023-06-29T11:54:25Z + DUMMY: + + structure + + + protein + PR: + cleaner0 + 2023-06-29T09:57:25Z + + Spy + + + 0.5212672 + experimental_method + cleaner0 + 2023-06-29T09:51:47Z + MESH: + + READ + + + 0.9872874 + experimental_method + cleaner0 + 2023-06-29T09:52:08Z + MESH: + + Residual Electron and Anomalous Density + + + protein + PR: + cleaner0 + 2023-06-29T09:57:25Z + + Spy + + + 0.92912316 + evidence + cleaner0 + 2023-06-29T11:54:31Z + DUMMY: + + co-crystal + + + 0.9963336 + evidence + cleaner0 + 2023-06-29T11:54:37Z + DUMMY: + + structure + + + 0.6307563 + protein_state + cleaner0 + 2023-06-29T12:13:19Z + DUMMY: + + folded + + + 0.572075 + structure_element + cleaner0 + 2023-06-29T12:18:35Z + SO: + + domain + + + 0.9985532 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9903908 + evidence + cleaner0 + 2023-06-29T09:52:41Z + DUMMY: + + residual electron density + + + 0.97443974 + protein_state + cleaner0 + 2023-06-29T12:13:22Z + DUMMY: + + dynamic + + + 0.93532926 + protein_state + cleaner0 + 2023-06-29T12:13:25Z + DUMMY: + + flexible + + + 0.99564326 + chemical + cleaner0 + 2023-06-29T12:05:09Z + CHEBI: + + iodine + + + 0.9940871 + evidence + cleaner0 + 2023-06-29T09:51:30Z + DUMMY: + + anomalous density + + + 0.98526114 + experimental_method + cleaner0 + 2023-06-29T09:52:19Z + MESH: + + molecular dynamics + + + 0.9845114 + experimental_method + cleaner0 + 2023-06-29T09:52:24Z + MESH: + + MD + + + 0.76252395 + experimental_method + cleaner0 + 2023-06-29T10:19:31Z + MESH: + + simulations + + + 0.991301 + protein_state + cleaner0 + 2023-06-29T12:13:29Z + DUMMY: + + dynamic + + + 0.9059882 + experimental_method + cleaner0 + 2023-06-29T12:01:15Z + MESH: + + sample-and-select algorithm + + + evidence + DUMMY: + cleaner0 + 2023-06-29T11:55:18Z + + residual and anomalous density + + + 0.97203445 + protein_state + cleaner0 + 2023-06-29T12:13:31Z + DUMMY: + + flexible + + + 0.9963092 + chemical + cleaner0 + 2023-06-29T12:05:09Z + CHEBI: + + iodine + + + 0.9915202 + evidence + cleaner0 + 2023-06-29T09:52:42Z + DUMMY: + + residual electron density + + + 0.76906615 + protein_state + cleaner0 + 2023-06-29T12:13:33Z + DUMMY: + + flexible + + + 0.99548495 + evidence + cleaner0 + 2023-06-29T09:50:59Z + DUMMY: + + electron density + + + 0.91406024 + evidence + cleaner0 + 2023-06-29T11:55:28Z + DUMMY: + + density + + + 0.99207217 + evidence + cleaner0 + 2023-06-29T09:51:14Z + DUMMY: + + crystal + + + 0.92841744 + experimental_method + cleaner0 + 2023-06-29T09:51:47Z + MESH: + + READ + + + structure_element + SO: + cleaner0 + 2023-06-29T10:16:14Z + + Spy-binding domain + + + 0.9985055 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + mutant + MESH: + cleaner0 + 2023-06-29T09:56:05Z + + Im76-45 + + + 0.9920535 + protein_state + cleaner0 + 2023-06-29T12:13:35Z + DUMMY: + + bound to + + + 0.99868625 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.87583953 + experimental_method + cleaner0 + 2023-06-29T09:51:47Z + MESH: + + READ + + + + RESULTS + title_2 + 7950 + Collecting READ data for the Spy:Im76-45 complex + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:51:47Z + + READ + + + 0.9432124 + complex_assembly + cleaner0 + 2023-06-29T09:55:24Z + GO: + + Spy:Im76-45 + + + + RESULTS + paragraph + 7999 + To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3). Moreover, binding experiments indicated that Im76-45 comprises the entire Spy-binding region. To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe). Its strong anomalous scattering allowed us to track the positions of these individual Im76-45 residues one at a time, potentially even if the residue was found in several locations in the same crystal. We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2). Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site. Consistent with the fragmented density, however, we observed multiple iodine positions for seven of the eight substituted residues. Together, these results indicated that the Im7 substrate binds Spy in multiple conformations. + + 0.6954702 + experimental_method + cleaner0 + 2023-06-29T12:01:19Z + MESH: + + READ technique + + + 0.90497404 + protein_type + cleaner0 + 2023-06-29T09:57:11Z + MESH: + + chaperone + + + 0.9988024 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.95279837 + mutant + cleaner0 + 2023-06-29T09:56:05Z + MESH: + + Im76-45 + + + 0.9865262 + experimental_method + cleaner0 + 2023-06-29T12:01:23Z + MESH: + + NMR + + + 0.92164403 + mutant + cleaner0 + 2023-06-29T09:56:05Z + MESH: + + Im76-45 + + + 0.9502464 + protein_state + cleaner0 + 2023-06-29T12:13:41Z + DUMMY: + + unfolded + + + 0.5735778 + protein_state + cleaner0 + 2023-06-29T12:13:43Z + DUMMY: + + folded + + + 0.9982692 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.9560363 + experimental_method + cleaner0 + 2023-06-29T12:01:26Z + MESH: + + binding experiments + + + mutant + MESH: + cleaner0 + 2023-06-29T09:56:05Z + + Im76-45 + + + 0.9808938 + site + cleaner0 + 2023-06-29T12:07:12Z + SO: + + Spy-binding region + + + 0.99265575 + chemical + cleaner0 + 2023-06-29T12:05:09Z + CHEBI: + + iodine + + + 0.99013966 + experimental_method + cleaner0 + 2023-06-29T12:01:31Z + MESH: + + replaced + + + mutant + MESH: + cleaner0 + 2023-06-29T09:56:05Z + + Im76-45 + + + 0.9939632 + chemical + cleaner0 + 2023-06-29T12:05:22Z + CHEBI: + + 4-iodophenylalanine + + + 0.99561006 + chemical + cleaner0 + 2023-06-29T10:18:17Z + CHEBI: + + pI-Phe + + + 0.82237387 + evidence + cleaner0 + 2023-06-29T09:53:13Z + DUMMY: + + anomalous scattering + + + mutant + MESH: + cleaner0 + 2023-06-29T09:56:05Z + + Im76-45 + + + 0.9948049 + evidence + cleaner0 + 2023-06-29T09:51:14Z + DUMMY: + + crystal + + + 0.9945221 + experimental_method + cleaner0 + 2023-06-29T12:01:36Z + MESH: + + co-crystallized + + + 0.9980811 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9786275 + mutant + cleaner0 + 2023-06-29T09:56:05Z + MESH: + + Im76-45 + + + 0.91171074 + chemical + cleaner0 + 2023-06-29T10:18:17Z + CHEBI: + + pI-Phe + + + 0.44063374 + experimental_method + cleaner0 + 2023-06-29T12:01:40Z + MESH: + + substitution + + + 0.6336847 + experimental_method + cleaner0 + 2023-06-29T12:01:44Z + MESH: + + collected + + + 0.70439875 + evidence + cleaner0 + 2023-06-29T09:53:27Z + DUMMY: + + anomalous data + + + 0.99490327 + complex_assembly + cleaner0 + 2023-06-29T09:55:24Z + GO: + + Spy:Im76-45 + + + 0.99621254 + evidence + cleaner0 + 2023-06-29T09:53:23Z + DUMMY: + + electron density map + + + 0.81312895 + evidence + cleaner0 + 2023-06-29T09:53:18Z + DUMMY: + + anomalous signals + + + 0.96556896 + site + cleaner0 + 2023-06-29T10:23:09Z + SO: + + cradle + + + 0.99820566 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.43931144 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.99788827 + site + cleaner0 + 2023-06-29T12:07:15Z + SO: + + substrate binding site + + + 0.8927793 + evidence + cleaner0 + 2023-06-29T11:55:34Z + DUMMY: + + density + + + 0.52766275 + chemical + cleaner0 + 2023-06-29T09:53:41Z + CHEBI: + + iodine + + + 0.9940791 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.99801326 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + + RESULTS + title_2 + 9410 + READ sample-and-select procedure + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:51:47Z + + READ + + + 0.7578166 + experimental_method + cleaner0 + 2023-06-29T12:01:48Z + MESH: + + sample-and-select + + + + RESULTS + paragraph + 9443 + To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes. To generate an accurate depiction of the chaperone-substrate interactions, we devised a selection protocol based on a sample-and-select procedure employed in NMR spectroscopy. This procedure iteratively constructs structural ensembles and then compares them to the experimental data. During each round of the selection, a genetic algorithm alters the ensemble and its agreement to the experimental data is re-evaluated. If successful, the selection identifies the smallest group of specific conformations that best fits the residual electron density and anomalous signals. The READ sample-and-select algorithm is diagrammed in Fig. 2. + + 0.99216205 + mutant + cleaner0 + 2023-06-29T09:55:51Z + MESH: + + Im76-45 + + + 0.99342895 + protein_state + cleaner0 + 2023-06-29T12:13:45Z + DUMMY: + + bound to + + + 0.9970419 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9833829 + evidence + cleaner0 + 2023-06-29T09:52:42Z + DUMMY: + + residual electron density + + + 0.93260133 + evidence + cleaner0 + 2023-06-29T09:53:19Z + DUMMY: + + anomalous signals + + + chemical + CHEBI: + cleaner0 + 2023-06-29T10:18:17Z + + pI-Phe + + + complex_assembly + GO: + cleaner0 + 2023-06-29T09:55:24Z + + Spy:Im76-45 + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.928301 + experimental_method + cleaner0 + 2023-06-29T12:01:52Z + MESH: + + sample-and-select + + + 0.9818716 + experimental_method + cleaner0 + 2023-06-29T09:59:39Z + MESH: + + NMR spectroscopy + + + 0.8969105 + experimental_method + cleaner0 + 2023-06-29T12:02:02Z + MESH: + + genetic algorithm + + + 0.8385226 + evidence + cleaner0 + 2023-06-29T09:52:42Z + DUMMY: + + residual electron density + + + 0.7360922 + evidence + cleaner0 + 2023-06-29T09:53:19Z + DUMMY: + + anomalous signals + + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:51:47Z + + READ + + + 0.93616086 + experimental_method + cleaner0 + 2023-06-29T12:02:05Z + MESH: + + sample-and-select algorithm + + + + RESULTS + paragraph + 10274 + Prior to performing the selection, we generated a large and diverse pool of chaperone-substrate complexes using coarse-grained MD simulations in a pseudo-crystal environment (Fig. 2 and Supplementary Fig. 4). The coarse-grained simulations are based on a single-residue resolution model for protein folding and were extended here to describe Spy-Im76-45 binding events (Online Methods). The initial conditions of the binding simulations are not biased toward a particular conformation of the substrate or any specific chaperone-substrate interaction (Online Methods). Im76-45 binds and unbinds to Spy throughout the simulations. This strategy allows a wide range of substrate conformations to interact with the chaperone. From the MD simulations, we extracted ~10,000 diverse Spy:Im76-45 complexes to be used by the ensuing selection. Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate. This pool was then used by the selection algorithm to identify the minimal ensemble that best satisfies both the residual electron and anomalous crystallographic data. + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.88118553 + experimental_method + cleaner0 + 2023-06-29T12:02:08Z + MESH: + + coarse-grained MD simulations + + + 0.6608005 + experimental_method + cleaner0 + 2023-06-29T12:02:11Z + MESH: + + pseudo-crystal environment + + + 0.9891684 + experimental_method + cleaner0 + 2023-06-29T12:02:13Z + MESH: + + coarse-grained simulations + + + 0.9837824 + complex_assembly + cleaner0 + 2023-06-29T12:06:04Z + GO: + + Spy-Im76-45 + + + 0.9687606 + experimental_method + cleaner0 + 2023-06-29T12:02:17Z + MESH: + + binding simulations + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.9812668 + mutant + cleaner0 + 2023-06-29T09:56:05Z + MESH: + + Im76-45 + + + 0.99846727 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9011532 + experimental_method + cleaner0 + 2023-06-29T10:19:30Z + MESH: + + simulations + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.9936941 + experimental_method + cleaner0 + 2023-06-29T09:52:24Z + MESH: + + MD + + + 0.97938156 + experimental_method + cleaner0 + 2023-06-29T10:19:31Z + MESH: + + simulations + + + 0.99521255 + complex_assembly + cleaner0 + 2023-06-29T09:55:24Z + GO: + + Spy:Im76-45 + + + 0.99863607 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9957451 + oligomeric_state + cleaner0 + 2023-06-29T12:10:19Z + DUMMY: + + dimer + + + 0.9443531 + protein_state + cleaner0 + 2023-06-29T12:13:49Z + DUMMY: + + bound to + + + 0.9847695 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9121185 + evidence + cleaner0 + 2023-06-29T11:56:15Z + DUMMY: + + residual electron and anomalous crystallographic data + + + + RESULTS + paragraph + 11368 + The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals. Simultaneously, it uses the residual electron density to help choose ensembles. To make the electron density selection practical, we needed to develop a method to rapidly evaluate the agreement between the selected sub-ensembles and the experimental electron density on-the-fly during the selection procedure. To accomplish this task, we generated a compressed version of the experimental 2mFo−DFc electron density map for use in the selection. This process provided us with a target map that the ensuing selection tried to recapitulate. To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations. For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods). These individual electron density maps from the separate conformers could then be combined (Fig. 2) and compared to the averaged experimental electron density map as part of the selection algorithm. + + 0.5931571 + evidence + cleaner0 + 2023-06-29T09:53:14Z + DUMMY: + + anomalous scattering + + + chemical + CHEBI: + cleaner0 + 2023-06-29T10:18:17Z + + pI-Phe + + + 0.9956863 + chemical + cleaner0 + 2023-06-29T12:05:09Z + CHEBI: + + iodine + + + 0.9964419 + chemical + cleaner0 + 2023-06-29T12:05:09Z + CHEBI: + + iodine + + + evidence + DUMMY: + cleaner0 + 2023-06-29T09:53:19Z + + anomalous signals + + + evidence + DUMMY: + cleaner0 + 2023-06-29T09:52:42Z + + residual electron density + + + 0.9872704 + experimental_method + cleaner0 + 2023-06-29T12:02:22Z + MESH: + + electron density selection + + + 0.98625016 + evidence + cleaner0 + 2023-06-29T09:50:59Z + DUMMY: + + electron density + + + evidence + DUMMY: + cleaner0 + 2023-06-29T10:20:35Z + + 2mFo−DFc electron density map + + + 0.9862191 + evidence + cleaner0 + 2023-06-29T11:56:29Z + DUMMY: + + map + + + 0.9359912 + evidence + cleaner0 + 2023-06-29T11:56:31Z + DUMMY: + + map + + + 0.9649724 + evidence + cleaner0 + 2023-06-29T11:56:36Z + DUMMY: + + density + + + 0.98811775 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9733921 + complex_assembly + cleaner0 + 2023-06-29T09:55:24Z + GO: + + Spy:Im76-45 + + + 0.9643926 + experimental_method + cleaner0 + 2023-06-29T09:52:24Z + MESH: + + MD + + + experimental_method + MESH: + cleaner0 + 2023-06-29T10:19:31Z + + simulations + + + 0.54676366 + experimental_method + cleaner0 + 2023-06-29T12:02:51Z + MESH: + + coarse-grained + + + 0.5649066 + experimental_method + cleaner0 + 2023-06-29T09:52:24Z + MESH: + + MD + + + 0.99560875 + evidence + cleaner0 + 2023-06-29T09:50:59Z + DUMMY: + + electron density + + + 0.9872942 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9960478 + evidence + cleaner0 + 2023-06-29T09:53:23Z + DUMMY: + + electron density map + + + 0.9960303 + evidence + cleaner0 + 2023-06-29T10:21:36Z + DUMMY: + + electron density maps + + + 0.9947624 + evidence + cleaner0 + 2023-06-29T09:53:23Z + DUMMY: + + electron density map + + + + RESULTS + paragraph + 12790 + This approach allowed us to simultaneously use both the iodine anomalous signals and the residual electron density in the selection procedure. The selection resulted in small ensembles from the MD pool that best fit the READ data (Fig. 1c,d). Before analyzing the details of the Spy:Im76-45 complex, we first engaged in a series of validation tests to verify the ensemble and selection procedure (Supplementary Note 1, Figures 1c,d, Supplemental Figures 5-7). Combined, these validation tests confirmed that the selection procedure and selected six-member ensemble recapitulate the experimental data. Of note, the final six-membered ensemble was the largest ensemble that could simultaneously decrease the RFree and pass the 10-fold cross-validation test. This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1). + + 0.98970246 + chemical + cleaner0 + 2023-06-29T12:05:09Z + CHEBI: + + iodine + + + 0.83912575 + evidence + cleaner0 + 2023-06-29T09:53:19Z + DUMMY: + + anomalous signals + + + evidence + DUMMY: + cleaner0 + 2023-06-29T09:52:42Z + + residual electron density + + + 0.49437568 + experimental_method + cleaner0 + 2023-06-29T09:52:24Z + MESH: + + MD + + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:51:47Z + + READ + + + 0.99502945 + complex_assembly + cleaner0 + 2023-06-29T09:55:24Z + GO: + + Spy:Im76-45 + + + 0.99570507 + evidence + cleaner0 + 2023-06-29T11:56:51Z + DUMMY: + + RFree + + + experimental_method + MESH: + cleaner0 + 2023-06-29T10:22:23Z + + 10-fold cross-validation test + + + 0.9134777 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.98730475 + protein_state + cleaner0 + 2023-06-29T12:14:07Z + DUMMY: + + bound to + + + 0.4701963 + protein_type + cleaner0 + 2023-06-29T09:57:11Z + MESH: + + chaperone + + + 0.99870205 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + + RESULTS + title_2 + 13698 + Folding and interactions of Im7 while bound to Spy + + 0.99858224 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.9928488 + protein_state + cleaner0 + 2023-06-29T12:14:14Z + DUMMY: + + bound to + + + 0.98990685 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + + RESULTS + paragraph + 13749 + Our results showed that by using this novel READ approach, we were able to obtain structural information about the dynamic interaction of a chaperone with its substrate protein. We were particularly interested in finding answers to one of the most fundamental questions in chaperone biology—how does chaperone binding affect substrate structure and vice versa. By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound. We found these conformations to be highly heterogeneous and to include unfolded, partially folded, and native-like states (Fig. 3). The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a). + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:51:47Z + + READ + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.9957247 + evidence + cleaner0 + 2023-06-29T11:57:00Z + DUMMY: + + structures + + + 0.99441284 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9951389 + protein_state + cleaner0 + 2023-06-29T12:14:10Z + DUMMY: + + bound to + + + 0.98687464 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.99474734 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.99560237 + protein_state + cleaner0 + 2023-06-29T12:14:18Z + DUMMY: + + bound + + + 0.9946273 + protein_state + cleaner0 + 2023-06-29T12:14:21Z + DUMMY: + + unfolded + + + 0.8663503 + protein_state + cleaner0 + 2023-06-29T12:14:23Z + DUMMY: + + partially folded + + + 0.9229308 + protein_state + cleaner0 + 2023-06-29T12:14:24Z + DUMMY: + + native-like + + + 0.9941633 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.8029603 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.92596716 + site + cleaner0 + 2023-06-29T10:23:09Z + SO: + + cradle + + + 0.93264157 + site + cleaner0 + 2023-06-29T10:23:09Z + SO: + + cradle + + + + RESULTS + paragraph + 14640 + We constructed a contact map of the complex, which shows the frequency of interactions for chaperone-substrate residue pairs (Fig. 4). We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). The Spy-contacting residues comprise a mixture of charged, polar, and hydrophobic residues. Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle. This mixture suggests the importance of both electrostatic and hydrophobic components in binding the Im76-45 ensemble. With respect to the substrate, we observed that nearly every residue in Im76-45 is in contact with Spy (Fig. 4a). However, we did notice that despite this uniformity, regions of Im76-45 preferentially interact with different regions in Spy (Fig. 4b). For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface. + + 0.99227893 + evidence + cleaner0 + 2023-06-29T11:57:04Z + DUMMY: + + contact map + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.8847004 + site + cleaner0 + 2023-06-29T12:07:23Z + SO: + + interaction sites + + + 0.9974826 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.99912685 + residue_name_number + cleaner0 + 2023-06-29T12:10:42Z + DUMMY: + + Arg122 + + + 0.99911207 + residue_name_number + cleaner0 + 2023-06-29T12:10:44Z + DUMMY: + + Thr124 + + + 0.99908876 + residue_name_number + cleaner0 + 2023-06-29T12:10:47Z + DUMMY: + + Phe29 + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.99911886 + residue_name_number + cleaner0 + 2023-06-29T12:10:49Z + DUMMY: + + Arg61 + + + 0.9991086 + residue_name_number + cleaner0 + 2023-06-29T12:10:51Z + DUMMY: + + Arg43 + + + 0.9991142 + residue_name_number + cleaner0 + 2023-06-29T12:10:54Z + DUMMY: + + Lys47 + + + 0.99912184 + residue_name_number + cleaner0 + 2023-06-29T12:10:56Z + DUMMY: + + His96 + + + 0.99911183 + residue_name_number + cleaner0 + 2023-06-29T12:10:59Z + DUMMY: + + Met46 + + + 0.88955724 + site + cleaner0 + 2023-06-29T12:08:02Z + SO: + + Spy-contacting residues + + + 0.99504805 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.99063617 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.7740819 + site + cleaner0 + 2023-06-29T10:23:09Z + SO: + + cradle + + + 0.5764325 + site + cleaner0 + 2023-06-29T10:23:09Z + SO: + + cradle + + + 0.98738986 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9951992 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9969289 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9949441 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.997964 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.98398495 + structure_element + cleaner0 + 2023-06-29T12:18:39Z + SO: + + N-terminal half + + + 0.99496967 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.8808872 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.70677155 + site + cleaner0 + 2023-06-29T10:23:09Z + SO: + + cradle + + + 0.97302437 + structure_element + cleaner0 + 2023-06-29T12:18:42Z + SO: + + C-terminal half + + + 0.9961086 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + site + SO: + cleaner0 + 2023-06-29T12:08:44Z + + concave surface + + + + RESULTS + paragraph + 15807 + Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly. Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3). This shift in contacts is likely due to hydrophobic residues of Im76-45 preferentially forming intra-molecular contacts upon folding (i.e., hydrophobic collapse), effectively removing themselves from the interaction sites. The diversity of conformations and binding sites observed here emphasizes the dynamic and heterogeneous nature of the chaperone-substrate ensemble. Although we do not yet have time resolution data of these various snapshots of Im76-45, this ensemble illustrates how a substrate samples its folding landscape while bound to a chaperone. + + 0.9955931 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9971462 + protein_state + cleaner0 + 2023-06-29T12:14:29Z + DUMMY: + + unfolded + + + 0.99407166 + protein_state + cleaner0 + 2023-06-29T12:14:32Z + DUMMY: + + native + + + 0.99749655 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9956022 + protein_state + cleaner0 + 2023-06-29T12:14:35Z + DUMMY: + + least-folded + + + 0.9922649 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9970746 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9937761 + protein_state + cleaner0 + 2023-06-29T12:14:38Z + DUMMY: + + most-folded + + + 0.99426556 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.99790394 + site + cleaner0 + 2023-06-29T12:08:19Z + SO: + + interaction sites + + + 0.9976718 + site + cleaner0 + 2023-06-29T12:08:22Z + SO: + + binding sites + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.99526626 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.981724 + protein_state + cleaner0 + 2023-06-29T12:14:41Z + DUMMY: + + bound to + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + + RESULTS + title_2 + 16694 + Spy changes conformation upon substrate binding + + 0.9074146 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + + RESULTS + paragraph + 16742 + Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2). Upon substrate binding, the Spy dimer twists 9° about its center relative to its apo form. This twist yields asymmetry and results in substantially different interaction patterns in the two Spy monomers (Fig. 4b). It is possible that this twist serves to increase heterogeneity in Spy by providing more binding poses. Additionally, we observed that the linker region (residues 47–57) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate. This increased disorder might explain how Spy is able to recognize and bind different substrates and/or differing conformations of the same substrate. Importantly, we observed the same structural changes in Spy regardless of which of the four substrates was bound (Fig. 5b, Table 1). The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site. + + 0.99717087 + evidence + cleaner0 + 2023-06-29T11:57:09Z + DUMMY: + + structure + + + 0.9987859 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.99704045 + protein_state + cleaner0 + 2023-06-29T12:14:44Z + DUMMY: + + substrate-bound + + + 0.9966618 + protein_state + cleaner0 + 2023-06-29T12:14:47Z + DUMMY: + + apo + + + 0.9987929 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9964521 + oligomeric_state + cleaner0 + 2023-06-29T12:10:23Z + DUMMY: + + dimer + + + 0.99871683 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9962998 + oligomeric_state + cleaner0 + 2023-06-29T12:10:26Z + DUMMY: + + dimer + + + 0.9975248 + protein_state + cleaner0 + 2023-06-29T12:14:49Z + DUMMY: + + apo + + + 0.99865746 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.99570364 + oligomeric_state + cleaner0 + 2023-06-29T12:10:28Z + DUMMY: + + monomers + + + 0.99836415 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.99685514 + structure_element + cleaner0 + 2023-06-29T12:18:49Z + SO: + + linker region + + + 0.97453624 + residue_range + cleaner0 + 2023-06-29T12:11:54Z + DUMMY: + + 47–57 + + + 0.9987594 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.8976868 + protein_state + cleaner0 + 2023-06-29T12:14:53Z + DUMMY: + + disordered + + + 0.99830097 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.99859184 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.99523723 + evidence + cleaner0 + 2023-06-29T11:57:12Z + DUMMY: + + RMSD + + + 0.95354253 + protein_state + cleaner0 + 2023-06-29T12:14:55Z + DUMMY: + + well-folded + + + 0.9984742 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.78872776 + experimental_method + cleaner0 + 2023-06-29T12:02:59Z + MESH: + + competition experiments + + + protein + PR: + cleaner0 + 2023-06-29T09:57:25Z + + Spy + + + 0.9981789 + site + cleaner0 + 2023-06-29T12:08:29Z + SO: + + Spy binding site + + + + DISCUSS + title_1 + 18074 + DISCUSSION + + + DISCUSS + paragraph + 18085 + To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate. As a substrate, we used Im76-45, the chaperone-interacting portion of the protein-folding model protein Im7. In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states. The ensemble provides an unprecedented description of the conformations that a substrate assumes while exploring its chaperone-associated folding landscape. This substrate-chaperone ensemble helps accomplish the longstanding goal of obtaining a detailed view of how a chaperone aids protein folding. + + protein_type + MESH: + cleaner0 + 2023-06-29T10:01:05Z + + chaperones + + + 0.87664104 + experimental_method + cleaner0 + 2023-06-29T09:51:47Z + MESH: + + READ + + + 0.9228798 + evidence + cleaner0 + 2023-06-29T11:57:16Z + DUMMY: + + conformational ensemble + + + 0.88223135 + protein_type + cleaner0 + 2023-06-29T09:57:11Z + MESH: + + chaperone + + + 0.998847 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9225119 + protein_state + cleaner0 + 2023-06-29T12:15:00Z + DUMMY: + + bound to substrate + + + 0.914196 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.7164347 + structure_element + cleaner0 + 2023-06-29T12:18:53Z + SO: + + chaperone-interacting portion + + + 0.9980854 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.9964735 + protein_state + cleaner0 + 2023-06-29T12:15:03Z + DUMMY: + + chaperone-bound + + + 0.8293746 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.99512637 + protein_state + cleaner0 + 2023-06-29T12:15:05Z + DUMMY: + + unfolded + + + 0.96388763 + protein_state + cleaner0 + 2023-06-29T12:15:08Z + DUMMY: + + folded + + + 0.9537819 + protein_state + cleaner0 + 2023-06-29T12:15:10Z + DUMMY: + + native + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + + DISCUSS + paragraph + 18808 + We recently showed that Im7 can fold while remaining continuously bound to Spy. The high-resolution ensemble obtained here now provides insight into exactly how this occurs. The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy. The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone. This model is consistent with previous studies postulating that the flexible binding of chaperones allows for substrate protein folding. The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates. + + 0.99826306 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.9076788 + protein_state + cleaner0 + 2023-06-29T12:15:13Z + DUMMY: + + continuously bound to + + + 0.99854445 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9263354 + evidence + cleaner0 + 2023-06-29T11:57:54Z + DUMMY: + + ensemble + + + 0.996702 + evidence + cleaner0 + 2023-06-29T11:57:58Z + DUMMY: + + structures + + + 0.79658026 + evidence + cleaner0 + 2023-06-29T11:58:00Z + DUMMY: + + ensemble + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.71427774 + site + cleaner0 + 2023-06-29T12:08:44Z + SO: + + concave surface + + + 0.9980413 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.61538374 + evidence + cleaner0 + 2023-06-29T11:58:03Z + DUMMY: + + ensemble + + + 0.9962405 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.96033084 + site + cleaner0 + 2023-06-29T12:08:54Z + SO: + + amphipathic surface + + + 0.98483825 + protein_state + cleaner0 + 2023-06-29T12:15:15Z + DUMMY: + + bound to + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + protein_type + MESH: + cleaner0 + 2023-06-29T10:01:05Z + + chaperones + + + 0.7650008 + site + cleaner0 + 2023-06-29T12:08:44Z + SO: + + concave surface + + + 0.9985177 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9986675 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + protein_type + MESH: + cleaner0 + 2023-06-29T10:01:05Z + + chaperones + + + + DISCUSS + paragraph + 19698 + In contrast to Spy’s binding hotspots, Im76-45 displays substantially less specificity in its binding sites. Nearly all Im76-45 residues come in contact with Spy. Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic. This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate. Once the substrate begins to fold within this protected environment, it progressively buries its own hydrophobic residues, and its interactions with the chaperone shift towards becoming more electrostatic. Notably, the most frequent contacts between Spy and Im76-45 are charge-charge interactions. The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states. Residues Asp32 and Asp35 are close to each other in the folded state of Im7. This proximity likely causes electrostatic repulsion that destabilizes Im7’s native state. Interaction with Spy’s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation. As inter-molecular hydrophobic interactions between Spy and the substrate become progressively replaced by intra-molecular interactions within the substrate, the affinity between chaperone and substrates could decrease, eventually leading to release of the folded client protein. + + 0.97845733 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.997659 + site + cleaner0 + 2023-06-29T12:08:58Z + SO: + + binding hotspots + + + mutant + MESH: + cleaner0 + 2023-06-29T09:56:06Z + + Im76-45 + + + 0.9979149 + site + cleaner0 + 2023-06-29T12:09:01Z + SO: + + binding sites + + + mutant + MESH: + cleaner0 + 2023-06-29T09:56:06Z + + Im76-45 + + + 0.99680966 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.99753666 + protein_state + cleaner0 + 2023-06-29T12:15:19Z + DUMMY: + + Unfolded + + + 0.99649507 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:54Z + + hydrophobic and hydrophilic interactions + + + 0.98912925 + protein_state + cleaner0 + 2023-06-29T12:15:21Z + DUMMY: + + native-like + + + 0.98963326 + protein_state + cleaner0 + 2023-06-29T12:15:35Z + DUMMY: + + ATP-independent + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.9956173 + protein_state + cleaner0 + 2023-06-29T12:15:38Z + DUMMY: + + unfolded + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:54Z + + hydrophobic interactions + + + 0.7040446 + site + cleaner0 + 2023-06-29T12:09:05Z + SO: + + hydrophobic regions + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.9965184 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + mutant + MESH: + cleaner0 + 2023-06-29T09:56:06Z + + Im76-45 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:54Z + + charge-charge interactions + + + 0.99789584 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.9990938 + residue_name_number + cleaner0 + 2023-06-29T12:11:05Z + DUMMY: + + Glu21 + + + 0.9990645 + residue_name_number + cleaner0 + 2023-06-29T12:11:08Z + DUMMY: + + Asp32 + + + 0.9990693 + residue_name_number + cleaner0 + 2023-06-29T12:11:10Z + DUMMY: + + Asp35 + + + 0.9972257 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.99615 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.9320808 + site + cleaner0 + 2023-06-29T10:23:09Z + SO: + + cradle + + + 0.9975612 + protein_state + cleaner0 + 2023-06-29T12:15:42Z + DUMMY: + + unfolded + + + 0.993737 + protein_state + cleaner0 + 2023-06-29T12:15:44Z + DUMMY: + + native-like + + + 0.9990429 + residue_name_number + cleaner0 + 2023-06-29T12:11:12Z + DUMMY: + + Asp32 + + + 0.9990433 + residue_name_number + cleaner0 + 2023-06-29T12:11:16Z + DUMMY: + + Asp35 + + + 0.9975516 + protein_state + cleaner0 + 2023-06-29T12:15:46Z + DUMMY: + + folded + + + 0.9979709 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.99788946 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.82028353 + protein_state + cleaner0 + 2023-06-29T12:15:48Z + DUMMY: + + native + + + 0.99727935 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.999037 + residue_name_number + cleaner0 + 2023-06-29T12:11:19Z + DUMMY: + + Asp32 + + + 0.99904805 + residue_name_number + cleaner0 + 2023-06-29T12:11:21Z + DUMMY: + + Asp35 + + + 0.7348036 + protein_state + cleaner0 + 2023-06-29T12:15:55Z + DUMMY: + + helical conformation + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:54Z + + hydrophobic interactions + + + 0.9968719 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.7177229 + protein_state + cleaner0 + 2023-06-29T12:15:57Z + DUMMY: + + folded + + + + DISCUSS + paragraph + 21394 + Recently, we employed a genetic selection system to improve the chaperone activity of Spy. This selection resulted in “Super Spy” variants that were more effective at both preventing aggregation and promoting protein folding. In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function. Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both. Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a). Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker. This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a). This expansion would increase the structural plasticity for substrate binding. By sampling multiple conformations, this linker region may allow diverse substrate conformations to be accommodated. + + 0.95396966 + experimental_method + cleaner0 + 2023-06-29T12:03:16Z + MESH: + + genetic selection system + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.9985115 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + 0.27333757 + protein + cleaner0 + 2023-06-29T09:57:25Z + PR: + + Spy + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T11:52:25Z + + variants + + + 0.9951461 + protein_state + cleaner0 + 2023-06-29T12:16:04Z + DUMMY: + + bound + + + mutant + MESH: + cleaner0 + 2023-06-29T09:56:06Z + + Im76-45 + + + 0.9421 + evidence + cleaner0 + 2023-06-29T11:58:11Z + DUMMY: + + ensemble + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.31445134 + protein + cleaner0 + 2023-06-29T09:57:26Z + PR: + + Spy + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T11:52:24Z + + variants + + + 0.9420128 + protein_state + cleaner0 + 2023-06-29T12:16:08Z + DUMMY: + + bound + + + 0.53971404 + protein + cleaner0 + 2023-06-29T09:57:02Z + PR: + + Im7 + + + 0.99709225 + protein_state + cleaner0 + 2023-06-29T12:16:10Z + DUMMY: + + WT + + + 0.9983999 + protein + cleaner0 + 2023-06-29T09:57:26Z + PR: + + Spy + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.9924574 + experimental_method + cleaner0 + 2023-06-29T12:03:23Z + MESH: + + H/D exchange + + + 0.76765364 + evidence + cleaner0 + 2023-06-29T12:03:40Z + DUMMY: + + ensemble + + + protein + PR: + cleaner0 + 2023-06-29T09:57:26Z + + Spy + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T11:52:57Z + + mutations + + + 0.9960479 + mutant + cleaner0 + 2023-06-29T12:09:16Z + MESH: + + H96L + + + 0.996639 + mutant + cleaner0 + 2023-06-29T12:09:19Z + MESH: + + Q100L + + + 0.931379 + site + cleaner0 + 2023-06-29T11:51:25Z + SO: + + chaperone contact surface + + + 0.6865122 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9421161 + evidence + cleaner0 + 2023-06-29T11:58:15Z + DUMMY: + + co-structure + + + 0.99530506 + mutant + cleaner0 + 2023-06-29T12:09:22Z + MESH: + + L32P + + + 0.99812347 + protein + cleaner0 + 2023-06-29T09:57:26Z + PR: + + Spy + + + structure_element + SO: + cleaner0 + 2023-06-29T12:19:27Z + + N-terminal helix + + + 0.82643086 + protein_state + cleaner0 + 2023-06-29T12:16:13Z + DUMMY: + + disordered + + + 0.994686 + structure_element + cleaner0 + 2023-06-29T12:19:03Z + SO: + + linker + + + protein + PR: + cleaner0 + 2023-06-29T09:57:26Z + + Spy + + + 0.91852707 + evidence + cleaner0 + 2023-06-29T11:58:18Z + DUMMY: + + structures + + + 0.99480677 + structure_element + cleaner0 + 2023-06-29T12:19:05Z + SO: + + linker region + + + 0.99762577 + protein_state + cleaner0 + 2023-06-29T12:16:15Z + DUMMY: + + apo + + + 0.9925647 + structure_element + cleaner0 + 2023-06-29T12:19:08Z + SO: + + linker region + + + + DISCUSS + paragraph + 22639 + Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding. This residue does not directly contact Im76-45 in our READ-derived ensemble. Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b). This interaction presumably reduces the mobility of the C-terminal helix. The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence. As a result, the C-terminus, and possibly also the flexible linker, is likely to become more flexible and thus more accommodating of different conformations of substrates. Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions. + + 0.5755169 + protein + cleaner0 + 2023-06-29T09:57:26Z + PR: + + Spy + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T11:52:58Z + + mutations + + + 0.9984133 + mutant + cleaner0 + 2023-06-29T12:09:24Z + MESH: + + F115I + + + 0.99841094 + mutant + cleaner0 + 2023-06-29T12:09:27Z + MESH: + + F115L + + + 0.95090145 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:51:47Z + + READ + + + 0.9659466 + evidence + cleaner0 + 2023-06-29T11:58:21Z + DUMMY: + + ensemble + + + 0.99572515 + protein + cleaner0 + 2023-06-29T09:57:26Z + PR: + + Spy + + + 0.9169791 + protein_state + cleaner0 + 2023-06-29T12:16:20Z + DUMMY: + + bound to + + + 0.999062 + residue_name_number + cleaner0 + 2023-06-29T12:11:27Z + DUMMY: + + F115 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:54Z + + hydrogen bonds + + + 0.9991068 + residue_name_number + cleaner0 + 2023-06-29T12:11:30Z + DUMMY: + + Tyr104 + + + 0.9650637 + structure_element + cleaner0 + 2023-06-29T12:19:12Z + SO: + + C-terminal helix + + + 0.99830073 + mutant + cleaner0 + 2023-06-29T12:09:31Z + MESH: + + F115I + + + 0.95239127 + mutant + cleaner0 + 2023-06-29T12:09:35Z + MESH: + + L + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:54Z + + hydrogen bonds + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:54Z + + hydrophobic interactions + + + 0.8771667 + protein_state + cleaner0 + 2023-06-29T12:16:23Z + DUMMY: + + flexible + + + 0.98513585 + structure_element + cleaner0 + 2023-06-29T12:19:32Z + SO: + + linker + + + 0.9851317 + evidence + cleaner0 + 2023-06-29T11:58:24Z + DUMMY: + + ensemble + + + 0.28606677 + protein + cleaner0 + 2023-06-29T09:57:26Z + PR: + + Spy + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T11:52:25Z + + variants + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + + DISCUSS + paragraph + 23493 + Despite extensive studies, exactly how complex chaperone machines help proteins fold remains controversial. Our study indicates that the chaperone Spy employs a simple surface binding approach that allows the substrate to explore various conformations and form transiently favorable interactions while being protected from aggregation. We speculate that many other chaperones could utilize a similar strategy. ATP and co-chaperone dependencies may have emerged later through evolution to better modulate and control chaperone action. + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.9588935 + protein_type + cleaner0 + 2023-06-29T09:57:11Z + MESH: + + chaperone + + + 0.9988973 + protein + cleaner0 + 2023-06-29T09:57:26Z + PR: + + Spy + + + protein_type + MESH: + cleaner0 + 2023-06-29T10:01:05Z + + chaperones + + + 0.9926553 + chemical + cleaner0 + 2023-06-29T12:05:31Z + CHEBI: + + ATP + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + + DISCUSS + paragraph + 24027 + In addition to insights into chaperone function, this work presents a new method for determining heterogeneous structural ensembles via a hybrid methodology of X-ray crystallography and computational modeling. Heterogeneous dynamic complexes or disordered regions of single proteins, once considered solely approachable by NMR spectroscopy, can now be visualized through X-ray crystallography. Consequently, this technique could enable structural characterization of many important dynamic and heterogeneous biomolecular systems. + + protein_type + MESH: + cleaner0 + 2023-06-29T09:57:11Z + + chaperone + + + 0.9962828 + experimental_method + cleaner0 + 2023-06-29T09:58:23Z + MESH: + + X-ray crystallography + + + 0.9899347 + experimental_method + cleaner0 + 2023-06-29T12:03:45Z + MESH: + + computational modeling + + + 0.93940705 + protein_state + cleaner0 + 2023-06-29T12:16:27Z + DUMMY: + + disordered + + + 0.98850334 + experimental_method + cleaner0 + 2023-06-29T09:59:39Z + MESH: + + NMR spectroscopy + + + 0.9961585 + experimental_method + cleaner0 + 2023-06-29T09:58:23Z + MESH: + + X-ray crystallography + + + + METHODS + title_1 + 24557 + ONLINE METHODS + + + METHODS + paragraph + 24572 + For computational methods, including simulations of Spy-substrate interactions, binning the residual Im7 electron density, ensemble selection, validation tests, and contact map generation, please see Supplementary Note 1. + + + METHODS + title_2 + 24794 + Spy truncation mutants’ construction and in vitro and in vivo activity measurements + + + METHODS + paragraph + 24880 + To facilitate crystallization, we used Spy 29-124, a truncated Spy version that removes the unstructured N- and C-terminal tails (full length Spy is 138 amino acids). To determine if these alterations impact Spy’s chaperone activity in vitro, we performed in vitro chaperone activity assays and found that they had no significant effect; these deletions also had only a minor effect on Spy’s ability to stabilize Im7 in vivo (Supplementary Fig. 1). The in vitro activity of Spy 29-124 was assessed using the aldolase refolding assay as previously described. Briefly, in the denaturing step, 100 μM aldolase was denatured in buffer containing 6.6 M GdmCl, 40 mM HEPES pH 7.5, and 50 mM NaCl overnight at 22 °C (room temperature). In the refolding step, denatured aldolase was diluted to 3 μM in refolding buffer (40 mM HEPES, 150 mM NaCl, 5 mM DTT pH 7.5) in the presence of 6 μM WT Spy or Spy 29-124 (Spy:aldolase = 2:1). As a control, an identical experiment without Spy added was also performed. The refolding temperature was 37 °C with continuous shaking. The refolding status was monitored at different time points (1 min, 4 min, 10 min, and 20 min) and tested by diluting the refolding sample by 15-fold into the reaction buffer (0.15 mM NADH, 2 mM F1,6-DP, 1.8 U/ml GDH/TPI, 40 mM HEPES, and 150 mM NaCl pH 7.5) at 28 °C. The absorbance was monitored for 1.5 min at 340 nm. The percentage refolding was calculated and averaged over three repeats. + + + METHODS + paragraph + 26343 + To determine the in vivo activity of the Spy mutants, the quantity of the unstable Im7 variant L53A I54A expressed in the periplasm was compared during Spy variant co-expression as previously described. Plasmid Spy (pTrc-spy) was used as the template for the construction of the variant plasmids of Spy for in vivo chaperone activity measurement (Supplementary Table 3). To use the native signal sequence of spy for the periplasmic export of the Spy variants, an NheI site was first introduced between the signal sequence and the mature protein coding region of Spy. The vector was then digested with NheI and BamHI, purified, and ligated with the linear fragments corresponding to truncated sequences (21–130, 24–130, 27–130, 30–130, and 33–130) of Spy. + + + METHODS + paragraph + 27108 + Cells containing a strain that expressed the unstable Im7 mutant IL53A I54A (pCDFTrc-ssIm7L53A I54A) were transformed with plasmids that expressed either WT or one of the five truncated Spy mutants and grown to mid-log phase in LB medium at 37 °C. Im7 L53A I54A and Spy expression were induced with various concentrations of IPTG for 2 h to compare the in vivo chaperone activity of WT Spy and the truncated Spy mutants at similar expression levels. Periplasmic fractions were prepared as previously described and were separated on 16% Tricine gel (Life Technologies Inc.). The bands corresponding to Spy and the C-terminal His-tagged Im7 were either directly visualized on Coomassie stained gels or determined by western blot using anti-His antibody (Abcam ab1187; validation provided on manufacturer’s website). + + + METHODS + title_2 + 27925 + Protein expression and purification + + + METHODS + paragraph + 27961 + The gene for spy 29-124 was amplified from plasmid pET28sumo-spy with primer 1 (5′-CGC GGG ATC CTT CAA AGA CCT GAA CCT GAC CG-3′) and primer 2 (5′-CGC GCT CGA GTT ATG TCA GAC GCT TCT CAA AAT TAG C-3′), and was cloned into pET28sumo via BamHI and XhoI sites. The H96L variant was made by Phusion site-directed mutagenesis (New England Biolabs). WT and H96L Spy 29-124 were expressed and purified as described previously with the exception that Ni-HisTrap columns (GE Healthcare) were utilized instead of the Ni-NTA beads and mini-chromatography column. ULP1 cleavage occurred following elution from the Ni-HisTrap column overnight at 4 °C while dialyzing to 40 mM Tris, 300 mM NaCl, pH 8.0. After dialysis, Spy was passed over the HisTrap column to remove the cleaved SUMO tag (20 mM imidazole was left over from the dialysis). Cleavage of the SUMO tag leaves a single serine in position 28 of Spy. The flow-through was then concentrated and diluted 5 times with 20 mM Tris, pH 8 for further purification on a HiTrap Q column. Spy has an isoelectric point of 9.5 and therefore was collected in the flow-through. The flow-through containing Spy was concentrated and diluted 5-fold with 50 mM sodium phosphate at pH 6.5 and passed over a HiTrap SP column. Spy was then eluted with a gradient from 0 M to 1 M NaCl. Re-buffering to the final reaction buffer was accomplished by gel filtration, passing the pooled and concentrated fractions containing Spy over a HiLoad 75 column in 40 mM HEPES, 100 mM NaCl, pH 7.5. Fractions containing Spy were then concentrated, frozen in liquid nitrogen, and stored at −80 °C. WT Im7, Im7 L18A L19A L37A H40W, and Im7 L18A L19A L37A were purified by the same protocol as Spy, but without the SP column step. In addition to WT Im7 and these various Im7 mutants, co-crystallization experiments extensively utilized Im76-45, a minimal Spy-binding segment that encompasses the first two helices of Im7 and contains 46% of the total Im7 sequence. It displays partial helicity when free in solution (Supplementary Fig. 3). The 6-45 portion of Im7 (H2N-SISDYTEAEFVQLLKEIEKENVAATDDVLD VLLEHFVKIT-OH), 4-iodophenlyalanine variants, and a peptide corresponding to a portion of bovine alpha casein S1 148-177 (Ac-ELFRQFYQLDAYPSGAWYYVPLGTQYTDAP-amide) were obtained from New England Peptide at ≥ 95% purity. Anomalous signals for residues E12, E14, L19, and E21 substitutions were determined using a peptide containing Im7 6–26, which was also obtained from New England Peptide at ≥ 95% purity. + + + METHODS + title_2 + 30494 + Protein crystallization + + + METHODS + paragraph + 30518 + Co-crystals of WT Spy 29-124 and Spy H96L 29-124 in complex with Im7 variants and casein were grown by vapor diffusion. 25–130 mg/ml dimer Spy was incubated with various Im7 or casein substrates at concentrations ranging from equimolar to three-fold excess substrate in 22%–33% PEG 3000, 0.88–1.0 M imidazole pH 8.0, and 40–310 mM zinc acetate at 20 °C. Crystals were flash frozen in liquid nitrogen using 35% PEG 3000 as a cryo-protectant. It is worthwhile to note that the flash freezing could somewhat bias the conformations observed in the crystal structure. However, we chose to freeze the crystal to provide us with the maximum capability to identify and interpret the iodine anomalous signals. + + + METHODS + title_2 + 31228 + Assessing presence of substrate in crystals + + + METHODS + paragraph + 31272 + Crystals were washed by sequential transfer between three to six 2 μl drops of mother liquor, incubating in each wash solution for 2–10 s in an effort to remove all surface bound and precipitated substrate protein before being dissolved for visualization by SDS-PAGE. Before loading, samples were boiled for 10 min in reducing loading buffer, and then loaded onto 16% Tricine gels. Wash samples and dissolved crystal samples were analyzed by Lumetein staining (Biotium) and Flamingo staining (Bio-Rad) per manufacturer’s instructions, and imaged using a FluorChem M Imager (ProteinSimple). + + + METHODS + title_2 + 31867 + X-ray crystallography + + + METHODS + paragraph + 31889 + Data were collected at the LS-CAT beamlines at the Advanced Photon Source at 100 K. SeMet and native Spy:Im76-45 crystals were collected at 12.7 keV and 9.7 keV, respectively. Spy:Casein 148-177, and Spy H96L:WT Im7 crystals were collected also collected at 12.7 keV. Data integration and scaling were performed with iMosflm and AIMLESS, respectively. As molecular replacement attempts using the previously published apo Spy structures (PDB IDs: 3O39 and 3OEO) were unsuccessful, the Spy:Im76-45 complex was solved using Se-SAD phasing with SeMet-Spy, followed by density modification and initial model building by AutoSol in Phenix. The initial model was completed and refined using the native Spy:Im76-45 complex data. The rest of the structures were built using the native Spy:Im76-45 structure as a molecular replacement search model. Refinements, including TLS refinement, were performed using COOT and Phenix. All refined structures were validated using the Molprobity server, with Clashscores ranking better than the 90th percentile for all structures. Structural figures were rendered using PyMOL and UCSF Chimera, and movies generated using UCSF Chimera. Several partially occupied zinc atoms were observed in the crystal structure. Although some of these zinc atoms could also potentially modelled as water molecules, doing so resulted in an increase in the RFree. Additionally, a section of density near His A96 that is potentially partially occupied by a combination of water, Spy linker region, and possibly zinc, was modelled as containing water molecules. Spy H96L:Im76-45 was employed for iodine anomalous scattering experiments due to increased robustness and reproducibility of the crystals. + + + METHODS + paragraph + 33599 + The expected anomalous scatterers in the structures were S in methionine residues of Spy, Zn from the crystallization buffer, and I in the single pI-Phe residue of each synthetic Im76-45 peptide. Each I site is expected to be partially occupied as Im76-45 had diffuse density corresponding to multiple, partially occupied conformers; the Zn sites also may be partially occupied. To identify I, S, and Zn atomic positions using anomalous scattering, datasets were collected at 6.5 keV and 14.0 keV at 100 K using the ID-D beamline at LS-CAT. Anomalous difference maps for initial anomalous signal screening were calculated with phases from a molecular replacement search using the native Spy:Im76-45 (with no Im76-45 built in) complex as the search model. + + + METHODS + paragraph + 34354 + Anomalous difference maps calculated with the 14.0 keV data were used as controls to distinguish iodine from zinc atoms, as the iodine and zinc anomalous scattering factors are comparable at 14.0 keV, whereas at 6.5 keV, f″ is ~9-fold greater for iodine than for zinc. Anomalous differences were also collected and analyzed for a crystal of WT Spy 29-124:Im76-45 containing no iodine. The resulting anomalous difference map was inspected for peaks corresponding to sulfur, which were then excluded when selecting iodine peaks. Also, peaks that overlapped with Spy in the crystal lattice were excluded from analysis. + + + METHODS + paragraph + 34972 + As an initial screen for placing iodine atoms in the 6.5 keV anomalous difference maps, the median methionine sulfur signal was used as a cutoff for each individual map to control for varying data quality between crystals. Then, all anomalous atoms were refined in Phenix using anomalous group refinement. Refined B-factor of placed iodine ions was then used to estimate the positional fluctuation of the anomalous signals. This positional fluctuation was used as estimated error in the ensuing selection. A summary of all the anomalous signal heights (Supplementary Table 1) and anomalous difference maps (Supplemental Dataset 1) are displayed at varying contour levels for maximum clarity of iodine and methionine peak heights. + + + METHODS + title_2 + 35702 + Substrate binding to Spy + + + METHODS + paragraph + 35727 + The dissociation constant of Im76-45 was determined via a fluorescence-based competition experiment with Im7 L18A L19A L37A H40W, and its ability to compete with casein 148-177 for Spy binding was tested. Im7 L18A L19A L37A H40W was chosen for competition experiments due to its tight binding (Supplementary Fig. 8) and substantial fluorescence change upon binding. This mutant binds to Spy tighter than Im7 L18A L19A L37A. 10 μM Spy 29-124 dimer was mixed with 10 μM Im7 L18A L19A L37A H40W or casein 148-177 to form a 1:1 complex in a buffer containing 40 mM HEPES pH 7.5 and 100 mM NaCl at 22 °C. Complex formation was monitored with a QuantaMaster 400 (Photon Technology International) using the tryptophan fluorescence of Im7 L18A L19A L37A H40W. Naturally tryptophan-free Im76-45 was then titrated into the complex to compete with Im7 L18A L19A L37A H40W for Spy binding. The observed fluorescence intensity at 350 nm was plotted as a function of the logarithm of the Im76-45 or casein 148-177 concentration. The data were fit for a one-site-binding competition model (OriginLab 9.1): where A1 and A2 are the maximum and minimum asymptotes, respectively, and x is the concentration of Im76-45. x0 is the apparent KD for Im76-45 based on its ability to compete with Im7 L18A L19A L37A H40W. Using the KD of Im7 L18A L19A L37A H40W binding to Spy 29-124, we then calculated the KD for Im76-45 binding to Spy 29-124 using the Cheng-Prusoff equation: where L is the concentration of Im7 L18A L19A L37A H40W and KD is the dissociation constant for Im7 L18A L19A L37A H40W binding to Spy. Due to interaction between higher oligomer states of Im76-45 and casein 148-177 (Supplementary Fig. 8), the competition curve was unable to be fit for casein 148-177 competing with Im76-45. + + + METHODS + paragraph + 37513 + The stoichiometry of binding of casein 148-177 and Spy was determined by tryptophan fluorescence of the casein upon Spy 29-124 addition. Increasing concentrations of Spy 29-124 were titrated to 20 μM of casein 148-177 in 40 mM HEPES (pH 7.5), 100 mM NaCl, at 22 °C. Complex formation was monitored with a QuantaMaster 400 (Photon Technology International) using the tryptophan fluorescence of casein 148-177. The observed fluorescence intensity at 339 nm was plotted as a function of the Spy 29-124 dimer concentration and fit with a quadratic equation using Origin 9.1 (OriginLab). + + + METHODS + paragraph + 38098 + To determine the dissociation constant, increasing concentrations of Spy 29-124 were titrated to 0.25 μM of casein in 40 mM HEPES (pH 7.5), 100 mM NaCl, at 22 °C. Complex formation was monitored with a QuantaMaster 400 (Photon Technology International) using the tryptophan fluorescence of casein 148-177. The observed fluorescence intensity at 339 nm was corrected for dilution due to the titration and then plotted as a function of the Spy 29-124 dimer concentration. The data were fit using a square hyperbola function in Origin 9.1 (OriginLab): where F is the recorded fluorescence signal, Fmax is the maximum fluorescence reached upon saturation of the complex, L is the concentration of free Spy in solution, KD is the dissociation constant, and C is a parameter for the offset. The calculated KD is an average of three independent repetitions. The measured dissociation constants for the different substrates ranged from 0.1 to 1 μM. + + + METHODS + title_2 + 39044 + Isothermal titration calorimetry (ITC) + + + METHODS + paragraph + 39083 + Spy 29-124 and Im7-L18A L19A L37A H40W were dialyzed overnight against 40 mM HEPES, 100 mM NaCl, pH 7.5. 165 μM Spy dimer was loaded into a syringe and titrated into a cell containing 15 μM Im7 L18A L19 AL37A H40W at 25 °C in an iTC200 (Malvern Instruments) with an injection interval of 120 s and an initial delay time of 60 s. The solution was stirred at 1000 rpm, and the reference power was set to 6 μcal s−1 in high feedback mode. Data analysis was conducted using a plugin for Origin 7 (OriginLab), the software provided by the manufacturer. + + + METHODS + title_2 + 39637 + Analytical ultracentrifugation + + + METHODS + paragraph + 39668 + Sedimentation velocity experiments for the Im76-45 and the bovine α-S1-casein peptide were performed using a Beckman Proteome Lab XL-I analytical ultracentrifuge (Beckman Coulter). Both peptides were first dialyzed against 40 mM HEPES, 100 mM NaCl, pH 7.5, then diluted to a concentration of 10 μM using the dialysis buffer. Samples were loaded into cells containing standard sector shaped 2-channel Epon centerpieces with 1.2 cm path-length (Beckman Coulter) and equilibrated to 22 °C for at least 1 h prior to sedimentation. All samples were spun at 48,000 rpm in a Beckman AN-50 Ti rotor, and the sedimentation of the protein was monitored continuously using interference optics, since the Im76-45 does not absorb strongly at 280 nm. Data analysis was conducted with SEDFIT (version 14.1), using the continuous c(s) distribution model. The confidence level for the maximum entropy (ME) regularization was set to 0.95. Buffer density and viscosity were calculated using SEDNTERP (http://sednterp.unh.edu/). + + + SUPPL + title_1 + 40682 + Supplementary Material + + + SUPPL + footnote + 40705 + ACCESSION CODES + + + SUPPL + footnote + 40721 + Structures and datasets in this work have been deposited in the PDB under the IDs 5INA, 5IOG, 5IOE, and 5IOA. + + + SUPPL + footnote + 40831 + AUTHOR CONTRIBUTIONS + + + SUPPL + footnote + 40852 + Overall concept was conceived by S.H. and J.B. Experiments were designed by S.H., S.Q., J.B., R.T., H.B., and P.K. Experiments were performed by S.H., S.Q., P.K., R.M., and L.W. Analysis and computational modeling was designed by C.B., L.S., P.A., L.A., H.B., and S.H. Computational analysis was carried out by Q.X., S.H., L.S., L.A., P.A., P.K., and R.M. The manuscript was written primarily by S.H. and J.B., with assistance from L.S., L.A. and all other authors. + + + SUPPL + footnote + 41318 + COMPETING FINANCIAL INTERESTS + + + SUPPL + footnote + 41348 + The authors declare no competing financial interests. + + + 1225 + 44 + surname:Keskin;given-names:O + surname:Gursoy;given-names:A + surname:Ma;given-names:B + surname:Nussinov;given-names:R + 18355092 + REF + Chemical Reviews + ref + 108 + 2008 + 41402 + Principles of protein-protein interactions: what are the preferred ways for proteins to interact? + + + 16247 + 16252 + surname:Fraser;given-names:JS + 21918110 + REF + Proceedings of the National Academy of Sciences of the United States of America + ref + 108 + 2011 + 41500 + Accessing protein conformational ensembles using room-temperature X-ray crystallography + + + 323 + 31 + surname:Kay;given-names:LE + 26707200 + REF + J Mol Biol + ref + 428 + 2016 + 41588 + New Views of Functionally Dynamic Proteins by Solution NMR Spectroscopy + + + 126601 + surname:Salmon;given-names:L + surname:Blackledge;given-names:M + 26517337 + REF + Rep Prog Phys + ref + 78 + 2015 + 41660 + Investigating protein conformational energy landscapes and atomic resolution dynamics from NMR dipolar couplings: a review + + + 10960 + 10974 + surname:Blackledge;given-names:MJ + 8218162 + REF + Biochemistry + ref + 32 + 1993 + 41783 + Conformational Backbone Dynamics of the Cyclic Decapeptide Antamanide - Application of a New Multiconformational Search Algorithm-Based on Nmr Data + + + 3181 + 3185 + surname:Guerry;given-names:P + REF + Angewandte Chemie-International Edition + ref + 52 + 2013 + 41931 + Mapping the Population of Protein Conformational Energy Sub-States from NMR Dipolar Couplings + + + 255 + 76 + surname:Jewett;given-names:AI + surname:Shea;given-names:JE + 19851829 + REF + Cell Mol Life Sci + ref + 67 + 2010 + 42025 + Reconciling theories of chaperonin accelerated folding with experimental evidence + + + 98 + 101 + surname:Mashaghi;given-names:A + 23831649 + REF + Nature + ref + 500 + 2013 + 42107 + Reshaping of the conformational search of a protein by the chaperone trigger factor + + + 3571 + 5 + surname:Buckle;given-names:AM + surname:Zahn;given-names:R + surname:Fersht;given-names:AR + 9108017 + REF + Proc Natl Acad Sci U S A + ref + 94 + 1997 + 42191 + A structural model for GroEL-polypeptide recognition + + + 923 + 34 + surname:Martinez-Hackert;given-names:E + surname:Hendrickson;given-names:WA + 19737520 + REF + Cell + ref + 138 + 2009 + 42244 + Promiscuous substrate recognition in folding and assembly activities of the trigger factor chaperone + + + 1250494 + surname:Saio;given-names:T + surname:Guan;given-names:X + surname:Rossi;given-names:P + surname:Economou;given-names:A + surname:Kalodimos;given-names:CG + 24812405 + REF + Science + ref + 344 + 2014 + 42345 + Structural basis for protein antiaggregation activity of the trigger factor chaperone + + + 1042 + 55 + surname:Joachimiak;given-names:LA + surname:Walzthoeni;given-names:T + surname:Liu;given-names:CW + surname:Aebersold;given-names:R + surname:Frydman;given-names:J + 25416944 + REF + Cell + ref + 159 + 2014 + 42431 + The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT + + + 1354 + 65 + surname:Chen;given-names:DH + 23746846 + REF + Cell + ref + 153 + 2013 + 42515 + Visualizing GroEL/ES in the act of encapsulating a folding protein + + + 963 + 74 + surname:Karagoz;given-names:GE + 24581495 + REF + Cell + ref + 156 + 2014 + 42582 + Hsp90-Tau complex reveals molecular basis for specificity in chaperone action + + + 3078 + 90 + surname:Dekker;given-names:C + 21701561 + REF + EMBO J + ref + 30 + 2011 + 42660 + The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins + + + 14 + 9 + surname:Munoz;given-names:IG + 21151115 + REF + Nat Struct Mol Biol + ref + 18 + 2011 + 42759 + Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin + + + 415 + 26 + surname:Elad;given-names:N + 17499047 + REF + Mol Cell + ref + 26 + 2007 + 42858 + Topologies of a substrate protein bound to the chaperonin GroEL + + + 6371 + 6 + surname:Albert;given-names:A + 20056599 + REF + J Biol Chem + ref + 285 + 2010 + 42922 + Structure of GroEL in complex with an early folding intermediate of alanine glyoxylate aminotransferase + + + 262 + 9 + surname:Quan;given-names:S + 21317898 + REF + Nat Struct Mol Biol + ref + 18 + 2011 + 43026 + Genetic selection designed to stabilize proteins uncovers a chaperone called Spy + + + 318 + 24 + surname:Friel;given-names:CT + surname:Smith;given-names:DA + surname:Vendruscolo;given-names:M + surname:Gsponer;given-names:J + surname:Radford;given-names:SE + 19252485 + REF + Nat Struct Mol Biol + ref + 16 + 2009 + 43107 + The mechanism of folding of Im7 reveals competition between functional and kinetic evolutionary constraints + + + 1722 + 38 + surname:Figueiredo;given-names:AM + surname:Whittaker;given-names:SB + surname:Knowling;given-names:SE + surname:Radford;given-names:SE + surname:Moore;given-names:GR + 24123274 + REF + Protein Sci + ref + 22 + 2013 + 43215 + Conformational dynamics is more important than helical propensity for the folding of the all alpha-helical protein Im7 + + + 53 + 8 + surname:Stull;given-names:F + surname:Koldewey;given-names:P + surname:Humes;given-names:JR + surname:Radford;given-names:SE + surname:Bardwell;given-names:JC + 26619265 + REF + Nat Struct Mol Biol + ref + 23 + 2016 + 43334 + Substrate protein folds while it is bound to the ATP-independent chaperone Spy + + + 2252 + 2259 + surname:Kwon;given-names:E + surname:Kim;given-names:DY + surname:Gross;given-names:CA + surname:Gross;given-names:JD + surname:Kim;given-names:KK + 20799348 + REF + Protein Science + ref + 19 + 2010 + 43413 + The crystal structure Escherichia coli Spy + + + e01584 + surname:Quan;given-names:S + 24497545 + REF + Elife + ref + 3 + 2014 + 43456 + Super Spy variants implicate flexibility in chaperone action + + + 689 + 96 + surname:Creamer;given-names:LK + surname:Richardson;given-names:T + surname:Parry;given-names:DA + 7305393 + REF + Arch Biochem Biophys + ref + 211 + 1981 + 43517 + Secondary structure of bovine alpha s1- and beta-casein in solution + + + 6437 + 6442 + surname:Chak;given-names:KF + surname:Safo;given-names:MK + surname:Ku;given-names:WY + surname:Hsieh;given-names:SY + surname:Yuan;given-names:HS + REF + Proceedings of the National Academy of Sciences + ref + 93 + 1996 + 43585 + The crystal structure of the immunity protein of colicin E7 suggests a possible colicin-interacting surface + + + 300 + 318 + surname:Pashley;given-names:CL + 22226836 + REF + Journal of Molecular Biology + ref + 416 + 2012 + 43693 + Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions + + + 72 + 77 + surname:Burling;given-names:FT + surname:Weis;given-names:WI + surname:Flaherty;given-names:KM + surname:Brunger;given-names:AT + 8539602 + REF + Science + ref + 271 + 1996 + 43776 + Direct observation of protein solvation and discrete disorder with experimental crystallographic phases + + + 1107 + 1117 + surname:van den Bedem;given-names:H + surname:Dhanik;given-names:A + surname:Latombe;given-names:JC + surname:Deacon;given-names:AM + REF + Acta Crystallographica Section D-Biological Crystallography + ref + 65 + 2009 + 43880 + Modeling discrete heterogeneity in X-ray diffraction data by fitting multi-conformers + + + Under Review + surname:Salmon;given-names:L + REF + Journal of the American Chemical Society + ref + 2015 + 43966 + Capturing a dynamic chaperone-substrate interaction using NMR-informed molecular modeling + + + 850 + 853 + surname:Brennan;given-names:S + surname:Cowan;given-names:PL + REF + Review of Scientific Instruments + ref + 63 + 1992 + 44056 + A Suite of Programs for Calculating X-Ray Absorption, Reflection, and Diffraction Performance for a Variety of Materials at Arbitrary Wavelengths + + + 2351 + 2361 + surname:Karanicolas;given-names:J + surname:Brooks;given-names:CL;suffix:III + 12237457 + REF + Protein Science + ref + 11 + 2002 + 44202 + The origins of asymmetry in the folding transition states of protein L and protein G + + + 945 + 957 + surname:Jewett;given-names:AI + surname:Shea;given-names:JE + 16987526 + REF + Journal of Molecular Biology + ref + 363 + 2006 + 44287 + Folding on the chaperone: Yield enhancement through loose binding + + + 517 + 25 + surname:Bardwell;given-names:JC + surname:Jakob;given-names:U + 23018052 + REF + Trends Biochem Sci + ref + 37 + 2012 + 44353 + Conditional disorder in chaperone action + + + 359 + 66 + surname:Quan;given-names:S + surname:Hiniker;given-names:A + surname:Collet;given-names:JF + surname:Bardwell;given-names:JC + 23299746 + REF + Methods Mol Biol + ref + 966 + 2013 + 44394 + Isolation of bacteria envelope proteins + + + 1560 + 4 + surname:Fischer;given-names:M + surname:Shoichet;given-names:BK + surname:Fraser;given-names:JS + 26032594 + REF + Chembiochem + ref + 16 + 2015 + 44434 + One Crystal, Two Temperatures: Cryocooling Penalties Alter Ligand Binding to Transient Protein Sites + + + 271 + 81 + surname:Battye;given-names:TG + surname:Kontogiannis;given-names:L + surname:Johnson;given-names:O + surname:Powell;given-names:HR + surname:Leslie;given-names:AG + 21460445 + REF + Acta Crystallogr D Biol Crystallogr + ref + 67 + 2011 + 44535 + iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM + + + 235 + 42 + surname:Winn;given-names:MD + 21460441 + REF + Acta Crystallogr D Biol Crystallogr + ref + 67 + 2011 + 44615 + Overview of the CCP4 suite and current developments + + + 213 + 221 + surname:Adams;given-names:PD + REF + Acta Crystallographica Section D-Biological Crystallography + ref + 66 + 2010 + 44667 + PHENIX: a comprehensive Python-based system for macromolecular structure solution + + + 486 + 501 + surname:Emsley;given-names:P + surname:Lohkamp;given-names:B + surname:Scott;given-names:WG + surname:Cowtan;given-names:K + REF + Acta Crystallographica Section D-Biological Crystallography + ref + 66 + 2010 + 44749 + Features and development of Coot + + + 12 + 21 + surname:Chen;given-names:VB + REF + Acta Crystallographica Section D-Biological Crystallography + ref + 66 + 2010 + 44782 + MolProbity: all-atom structure validation for macromolecular crystallography + + + REF + The PyMOL Molecular Graphics System, Version 1.3r1 + ref + 2010 + 44859 + + + 1605 + 1612 + surname:Pettersen;given-names:EF + 15264254 + REF + Journal of Computational Chemistry + ref + 25 + 2004 + 44860 + UCSF chimera - A visualization system for exploratory research and analysis + + + 352 + 367 + surname:Afonine;given-names:PV + REF + Acta Crystallographica Section D-Biological Crystallography + ref + D68 + 2012 + 44936 + Towards automated crystallographic structure refinement with phenix.refine + + + 1606 + 1619 + surname:Schuck;given-names:P + 10692345 + REF + Biophysical Journal + ref + 78 + 2000 + 45011 + Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling + + + nihms785109f1.jpg + F1 + FIG + fig_caption + 45129 + Crystallographic data and ensemble selection. (a) 2mFo−DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 σ. This is the residual density that is used in the READ selection. (b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence. Multiple iodine positions were detected for most residues. Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black). The agreement from back-calculating a subset of data excluded from the selection procedure is shown by the red curve (cross-validation). The cost function, χ2, decreases as the agreement to the experimental data increases and is defined in the Online Methods. + + evidence + DUMMY: + cleaner0 + 2023-06-29T11:46:25Z + + 2mFo−DFc omit map + + + 0.9824786 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.87016684 + structure_element + cleaner0 + 2023-06-29T12:19:36Z + SO: + + flexible linker + + + 0.7679258 + evidence + cleaner0 + 2023-06-29T09:50:59Z + DUMMY: + + electron density + + + 0.5860164 + evidence + cleaner0 + 2023-06-29T11:58:30Z + DUMMY: + + density + + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:51:48Z + + READ + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:05:10Z + + iodine + + + evidence + DUMMY: + cleaner0 + 2023-06-29T09:53:19Z + + anomalous signals + + + chemical + CHEBI: + cleaner0 + 2023-06-29T10:18:17Z + + pI-Phe + + + 0.8892446 + experimental_method + cleaner0 + 2023-06-29T12:03:53Z + MESH: + + substitutions + + + chemical + CHEBI: + cleaner0 + 2023-06-29T12:05:10Z + + iodine + + + 0.9806015 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9915528 + evidence + cleaner0 + 2023-06-29T09:50:59Z + DUMMY: + + electron density + + + evidence + DUMMY: + cleaner0 + 2023-06-29T11:59:03Z + + anomalous iodine signals + + + 0.50510055 + experimental_method + cleaner0 + 2023-06-29T09:52:24Z + MESH: + + MD + + + 0.8905258 + evidence + cleaner0 + 2023-06-29T11:59:11Z + DUMMY: + + cost function + + + 0.970643 + evidence + cleaner0 + 2023-06-29T11:59:15Z + DUMMY: + + χ2 + + + + nihms785109f2.jpg + F2 + FIG + fig_caption + 46008 + Flowchart of the READ sample-and-select process. + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:51:48Z + + READ + + + 0.827946 + experimental_method + cleaner0 + 2023-06-29T12:04:08Z + MESH: + + sample-and-select + + + + nihms785109f3.jpg + F3 + FIG + fig_caption + 46057 + Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here. Spy is depicted as a gray surface and the Im76-45 conformer is shown as orange balls. Atoms that were either not directly selected in the READ procedure, or whose position could not be justified based on agreement with the residual electron density were removed, leading to non-contiguous sections. Dashed lines connect non-contiguous segments of the Im76-45 substrate. Residues of the Spy flexible linker region that fit the residual electron density are shown as larger gray spheres. Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic. + + complex_assembly + GO: + cleaner0 + 2023-06-29T09:55:24Z + + Spy:Im76-45 + + + 0.9943816 + evidence + cleaner0 + 2023-06-29T11:59:19Z + DUMMY: + + RMSD + + + 0.57364076 + protein_state + cleaner0 + 2023-06-29T12:16:32Z + DUMMY: + + native + + + 0.9697895 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:51:48Z + + READ + + + 0.99273866 + protein + cleaner0 + 2023-06-29T09:57:26Z + PR: + + Spy + + + 0.73834866 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:51:48Z + + READ + + + evidence + DUMMY: + cleaner0 + 2023-06-29T09:52:42Z + + residual electron density + + + 0.9689309 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9660642 + protein + cleaner0 + 2023-06-29T09:57:26Z + PR: + + Spy + + + 0.7810079 + structure_element + cleaner0 + 2023-06-29T12:19:40Z + SO: + + linker region + + + evidence + DUMMY: + cleaner0 + 2023-06-29T09:52:42Z + + residual electron density + + + 0.99446154 + evidence + cleaner0 + 2023-06-29T11:59:23Z + DUMMY: + + RMSD + + + 0.7643502 + protein_state + cleaner0 + 2023-06-29T12:16:36Z + DUMMY: + + native + + + 0.9704957 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.95991987 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.963916 + protein + cleaner0 + 2023-06-29T09:57:26Z + PR: + + Spy + + + + nihms785109f4.jpg + F4 + FIG + fig_caption + 46956 + Contact maps of Spy:Im76-45 complex. (a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures. For clarity, Im76-45 is represented with a single conformation. The frequency plotted is calculated as the average contact frequency from Spy to every residue of Im76-45 and vice-versa. As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45. Contacts to the two Spy monomers are depicted separately. Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps. + + 0.99464214 + evidence + cleaner0 + 2023-06-29T11:59:28Z + DUMMY: + + Contact maps + + + 0.9935497 + complex_assembly + cleaner0 + 2023-06-29T09:55:24Z + GO: + + Spy:Im76-45 + + + 0.9907131 + complex_assembly + cleaner0 + 2023-06-29T09:55:24Z + GO: + + Spy:Im76-45 + + + 0.994877 + evidence + cleaner0 + 2023-06-29T11:59:31Z + DUMMY: + + contact map + + + 0.99700975 + protein_state + cleaner0 + 2023-06-29T12:16:41Z + DUMMY: + + bound + + + 0.99881434 + protein + cleaner0 + 2023-06-29T09:57:26Z + PR: + + Spy + + + 0.9960406 + oligomeric_state + cleaner0 + 2023-06-29T12:10:33Z + DUMMY: + + dimer + + + 0.9831422 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9948394 + evidence + cleaner0 + 2023-06-29T11:59:34Z + DUMMY: + + structures + + + 0.98120135 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.990936 + evidence + cleaner0 + 2023-06-29T11:59:37Z + DUMMY: + + contact frequency + + + 0.99847955 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.99095154 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9880468 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9985318 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.9932604 + evidence + cleaner0 + 2023-06-29T11:59:41Z + DUMMY: + + contact map + + + 0.9946891 + evidence + cleaner0 + 2023-06-29T11:59:44Z + DUMMY: + + contact maps + + + 0.97827303 + complex_assembly + cleaner0 + 2023-06-29T09:55:24Z + GO: + + Spy:Im76-45 + + + 0.9984988 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.99316674 + oligomeric_state + cleaner0 + 2023-06-29T12:10:36Z + DUMMY: + + monomers + + + 0.78499204 + protein_state + cleaner0 + 2023-06-29T12:16:43Z + DUMMY: + + flexible + + + 0.9960276 + structure_element + cleaner0 + 2023-06-29T12:19:44Z + SO: + + linker region + + + 0.99871933 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.9779859 + residue_range + cleaner0 + 2023-06-29T12:11:59Z + DUMMY: + + 47–57 + + + 0.99175453 + evidence + cleaner0 + 2023-06-29T11:59:47Z + DUMMY: + + contact maps + + + + nihms785109f5.jpg + F5 + FIG + fig_caption + 47612 + Spy conformation changes upon substrate binding. (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). Error bars depict standard deviations of n=3 technical replicates. + + 0.9942 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.99299896 + experimental_method + cleaner0 + 2023-06-29T12:04:13Z + MESH: + + Overlay + + + 0.99786407 + protein_state + cleaner0 + 2023-06-29T12:16:48Z + DUMMY: + + apo + + + 0.99821895 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.99747664 + protein_state + cleaner0 + 2023-06-29T12:16:50Z + DUMMY: + + bound + + + 0.9980399 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.98867375 + experimental_method + cleaner0 + 2023-06-29T12:04:15Z + MESH: + + Overlay + + + 0.99659353 + protein_state + cleaner0 + 2023-06-29T12:16:53Z + DUMMY: + + WT + + + 0.9979012 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.988338 + protein_state + cleaner0 + 2023-06-29T12:16:55Z + DUMMY: + + bound to + + + 0.63883996 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9976901 + mutant + cleaner0 + 2023-06-29T12:09:41Z + MESH: + + H96L + + + 0.99283606 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.94561666 + protein_state + cleaner0 + 2023-06-29T12:16:57Z + DUMMY: + + bound to + + + 0.996292 + protein + cleaner0 + 2023-06-29T09:57:03Z + PR: + + Im7 + + + mutant + MESH: + cleaner0 + 2023-06-29T11:48:36Z + + L18A + + + mutant + MESH: + cleaner0 + 2023-06-29T11:49:02Z + + L19 A + + + mutant + MESH: + cleaner0 + 2023-06-29T11:49:14Z + + L13A + + + 0.9974648 + mutant + cleaner0 + 2023-06-29T12:09:50Z + MESH: + + H96L + + + 0.99459994 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.9761287 + protein_state + cleaner0 + 2023-06-29T12:17:00Z + DUMMY: + + bound to + + + 0.99709237 + protein_state + cleaner0 + 2023-06-29T12:17:03Z + DUMMY: + + WT + + + 0.99832183 + protein + cleaner0 + 2023-06-29T09:57:03Z + PR: + + Im7 + + + 0.9970504 + protein_state + cleaner0 + 2023-06-29T12:17:05Z + DUMMY: + + WT + + + 0.99810314 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.9752623 + protein_state + cleaner0 + 2023-06-29T12:17:08Z + DUMMY: + + bound to + + + 0.99773103 + chemical + cleaner0 + 2023-06-29T12:05:38Z + CHEBI: + + casein + + + 0.9873357 + experimental_method + cleaner0 + 2023-06-29T12:04:19Z + MESH: + + Competition assay + + + 0.6766577 + mutant + cleaner0 + 2023-06-29T09:56:06Z + MESH: + + Im76-45 + + + 0.9897697 + protein + cleaner0 + 2023-06-29T09:57:03Z + PR: + + Im7 + + + mutant + MESH: + cleaner0 + 2023-06-29T11:49:29Z + + L18A + + + mutant + MESH: + cleaner0 + 2023-06-29T11:49:38Z + + L19A + + + mutant + MESH: + cleaner0 + 2023-06-29T11:49:46Z + + L37A + + + mutant + MESH: + cleaner0 + 2023-06-29T11:49:57Z + + H40W + + + 0.9944518 + site + cleaner0 + 2023-06-29T12:09:10Z + SO: + + binding site + + + 0.99822253 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.98650044 + experimental_method + cleaner0 + 2023-06-29T12:04:22Z + MESH: + + substrate competition assays + + + + nihms785109f6.jpg + F6 + FIG + fig_caption + 48143 + Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green). (b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility. The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region. L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes. + + 0.59827995 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.9926208 + structure_element + cleaner0 + 2023-06-29T12:19:50Z + SO: + + linker region + + + 0.19467086 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.80332744 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.99444485 + structure_element + cleaner0 + 2023-06-29T12:19:52Z + SO: + + linker region + + + 0.99736905 + protein_state + cleaner0 + 2023-06-29T12:17:14Z + DUMMY: + + apo + + + 0.9945273 + protein_state + cleaner0 + 2023-06-29T12:17:17Z + DUMMY: + + bound + + + 0.9987766 + residue_name_number + cleaner0 + 2023-06-29T12:11:36Z + DUMMY: + + F115 + + + 0.99887234 + residue_name_number + cleaner0 + 2023-06-29T12:11:39Z + DUMMY: + + L32 + + + 0.99102753 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.99522245 + structure_element + cleaner0 + 2023-06-29T12:19:55Z + SO: + + linker region + + + 0.57751876 + site + cleaner0 + 2023-06-29T10:23:09Z + SO: + + cradle + + + protein + PR: + cleaner0 + 2023-06-29T09:57:27Z + + Spy + + + 0.8755758 + structure_element + cleaner0 + 2023-06-29T12:19:57Z + SO: + + linker region + + + 0.21077903 + protein + cleaner0 + 2023-06-29T09:57:27Z + PR: + + Spy + + + 0.99865603 + mutant + cleaner0 + 2023-06-29T12:09:59Z + MESH: + + F115L + + + 0.9986588 + mutant + cleaner0 + 2023-06-29T12:10:02Z + MESH: + + F115I + + + 0.9984819 + mutant + cleaner0 + 2023-06-29T12:10:05Z + MESH: + + L32P + + + 0.9920492 + structure_element + cleaner0 + 2023-06-29T12:19:59Z + SO: + + linker region + + + 0.99873227 + residue_name_number + cleaner0 + 2023-06-29T12:11:42Z + DUMMY: + + L32 + + + 0.9988135 + residue_name_number + cleaner0 + 2023-06-29T12:11:43Z + DUMMY: + + F115 + + + 0.9988507 + residue_name_number + cleaner0 + 2023-06-29T12:11:46Z + DUMMY: + + Y104 + + + protein + PR: + cleaner0 + 2023-06-29T09:57:27Z + + Spy + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T11:52:58Z + + mutations + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:54Z + + hydrogen bonds + + + + T1.xml + T1 + TABLE + table_caption + 48804 + Crystallography Statistics + + + T1.xml + T1 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="void" rules="none"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1"/><th valign="top" align="left" rowspan="1" colspan="1">SeMet Spy:Im7<sub>6-45</sub></th><th valign="top" align="left" rowspan="1" colspan="1">Spy:Im7<sub>6-45</sub></th><th valign="top" align="left" rowspan="1" colspan="1">Spy:Casein 148-177, substrate not modeled</th><th valign="top" align="left" rowspan="1" colspan="1">Spy H96L:Im7 L18A L19A L37A, substrate not modeled</th><th valign="top" align="left" rowspan="1" colspan="1">Spy H96L:WT Im7, substrate not modeled</th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1">PDB ID</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">5INA</td><td align="left" valign="top" rowspan="1" colspan="1">5IOG</td><td align="left" valign="top" rowspan="1" colspan="1">5IOE</td><td align="left" valign="top" rowspan="1" colspan="1">5IOA</td></tr><tr><td colspan="6" align="left" valign="top" rowspan="1"><bold>Data collection</bold></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Space group</td><td align="left" valign="top" rowspan="1" colspan="1">P4<sub>1</sub>22</td><td align="left" valign="top" rowspan="1" colspan="1">P4<sub>1</sub>22</td><td align="left" valign="top" rowspan="1" colspan="1">P4<sub>1</sub>22</td><td align="left" valign="top" rowspan="1" colspan="1">P4<sub>1</sub>22</td><td align="left" valign="top" rowspan="1" colspan="1">P4<sub>1</sub>22</td></tr><tr><td colspan="6" align="left" valign="top" rowspan="1">Cell dimensions</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td align="left" valign="top" rowspan="1" colspan="1">42.9, 42.9, 259.3</td><td align="left" valign="top" rowspan="1" colspan="1">42.9, 42.9, 260.2</td><td align="left" valign="top" rowspan="1" colspan="1">43.0, 43.0, 258.2</td><td align="left" valign="top" rowspan="1" colspan="1">43.1, 43.1, 258.7</td><td align="left" valign="top" rowspan="1" colspan="1">43.1, 43.14, 260.2</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> <italic>α</italic>, <italic>β</italic>, <italic>γ</italic> (°)</td><td align="left" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="left" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="left" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="left" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="left" valign="top" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="left" valign="top" rowspan="1" colspan="1">64.82–2.44(2.53–2.44)</td><td align="left" valign="top" rowspan="1" colspan="1">30.50–1.79(1.83–1.79)</td><td align="left" valign="top" rowspan="1" colspan="1">36.88–1.77 (1.80–1.77)</td><td align="left" valign="top" rowspan="1" colspan="1">30.48–1.87(1.91–1.87)</td><td align="left" valign="top" rowspan="1" colspan="1">33.21–1.87(1.91–1.87)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><italic>R</italic><sub>merge</sub> (%)</td><td align="left" valign="top" rowspan="1" colspan="1">10.6(36)</td><td align="left" valign="top" rowspan="1" colspan="1">8.2(108)</td><td align="left" valign="top" rowspan="1" colspan="1">6.2(134)</td><td align="left" valign="top" rowspan="1" colspan="1">8.4(152)</td><td align="left" valign="top" rowspan="1" colspan="1">9.6(249)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><italic>I</italic>/σ(<italic>I)</italic></td><td align="left" valign="top" rowspan="1" colspan="1">15.1(6.8)</td><td align="left" valign="top" rowspan="1" colspan="1">7.0(1.1)</td><td align="left" valign="top" rowspan="1" colspan="1">15.3(1.6)</td><td align="left" valign="top" rowspan="1" colspan="1">13.8(1.8)</td><td align="left" valign="top" rowspan="1" colspan="1">13.2(1.3)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Completeness (%)</td><td align="left" valign="top" rowspan="1" colspan="1">100(100)</td><td align="left" valign="top" rowspan="1" colspan="1">94.0(90.1)</td><td align="left" valign="top" rowspan="1" colspan="1">99.9(99.5)</td><td align="left" valign="top" rowspan="1" colspan="1">100(100)</td><td align="left" valign="top" rowspan="1" colspan="1">96.8(93.1)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Redundancy</td><td align="left" valign="top" rowspan="1" colspan="1">15.6(15.6)</td><td align="left" valign="top" rowspan="1" colspan="1">4.3(4.2)</td><td align="left" valign="top" rowspan="1" colspan="1">8.7(8.2)</td><td align="left" valign="top" rowspan="1" colspan="1">9.6(9.4)</td><td align="left" valign="top" rowspan="1" colspan="1">8.2(8.2)</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">CC1/2</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">0.998(0.689)</td><td align="left" valign="top" rowspan="1" colspan="1">0.999(0.745)</td><td align="left" valign="top" rowspan="1" colspan="1">0.999(0.676)</td><td align="left" valign="top" rowspan="1" colspan="1">0.998(0.606)</td></tr><tr><td colspan="6" align="left" valign="top" rowspan="1"><bold>Refinement</bold></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">1.79</td><td align="left" valign="top" rowspan="1" colspan="1">1.77</td><td align="left" valign="top" rowspan="1" colspan="1">1.87</td><td align="left" valign="top" rowspan="1" colspan="1">1.87</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">No. of Reflections</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">22583</td><td align="left" valign="top" rowspan="1" colspan="1">25052</td><td align="left" valign="top" rowspan="1" colspan="1">21505</td><td align="left" valign="top" rowspan="1" colspan="1">20838</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"><italic>R</italic><sub>work/</sub><italic>R</italic><sub>free</sub></td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">0.22/0.23</td><td align="left" valign="top" rowspan="1" colspan="1">0.21/0.24</td><td align="left" valign="top" rowspan="1" colspan="1">0.22/0.24</td><td align="left" valign="top" rowspan="1" colspan="1">0.21/0.25</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">No. of Atoms</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">1765</td><td align="left" valign="top" rowspan="1" colspan="1">1669</td><td align="left" valign="top" rowspan="1" colspan="1">1715</td><td align="left" valign="top" rowspan="1" colspan="1">1653</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Protein</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">1586</td><td align="left" valign="top" rowspan="1" colspan="1">1493</td><td align="left" valign="top" rowspan="1" colspan="1">1541</td><td align="left" valign="top" rowspan="1" colspan="1">1444</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Ligand/ion</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">30</td><td align="left" valign="top" rowspan="1" colspan="1">56</td><td align="left" valign="top" rowspan="1" colspan="1">60</td><td align="left" valign="top" rowspan="1" colspan="1">30</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Water</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">149</td><td align="left" valign="top" rowspan="1" colspan="1">120</td><td align="left" valign="top" rowspan="1" colspan="1">114</td><td align="left" valign="top" rowspan="1" colspan="1">179</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">B-factors</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">49.4</td><td align="left" valign="top" rowspan="1" colspan="1">48.5</td><td align="left" valign="top" rowspan="1" colspan="1">47.4</td><td align="left" valign="top" rowspan="1" colspan="1">39.2</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Protein</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">49.0</td><td align="left" valign="top" rowspan="1" colspan="1">47.5</td><td align="left" valign="top" rowspan="1" colspan="1">46.3</td><td align="left" valign="top" rowspan="1" colspan="1">38.3</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Ligand/ion</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">48.6</td><td align="left" valign="top" rowspan="1" colspan="1">65.9</td><td align="left" valign="top" rowspan="1" colspan="1">80.4</td><td align="left" valign="top" rowspan="1" colspan="1">62.9</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Water</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">54.2</td><td align="left" valign="top" rowspan="1" colspan="1">51.9</td><td align="left" valign="top" rowspan="1" colspan="1">44.5</td><td align="left" valign="top" rowspan="1" colspan="1">42.1</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">r.m.s. Deviations</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Bond lengths (Å)</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">0.013</td><td align="left" valign="top" rowspan="1" colspan="1">0.013</td><td align="left" valign="top" rowspan="1" colspan="1">0.013</td><td align="left" valign="top" rowspan="1" colspan="1">0.014</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"> Bond angles (º)</td><td align="left" valign="top" rowspan="1" colspan="1"/><td align="left" valign="top" rowspan="1" colspan="1">1.24</td><td align="left" valign="top" rowspan="1" colspan="1">1.30</td><td align="left" valign="top" rowspan="1" colspan="1">1.24</td><td align="left" valign="top" rowspan="1" colspan="1">1.39</td></tr></tbody></table> + + 48831 + SeMet Spy:Im76-45 Spy:Im76-45 Spy:Casein 148-177, substrate not modeled Spy H96L:Im7 L18A L19A L37A, substrate not modeled Spy H96L:WT Im7, substrate not modeled PDB ID 5INA 5IOG 5IOE 5IOA Data collection Space group P4122 P4122 P4122 P4122 P4122 Cell dimensions  a, b, c (Å) 42.9, 42.9, 259.3 42.9, 42.9, 260.2 43.0, 43.0, 258.2 43.1, 43.1, 258.7 43.1, 43.14, 260.2  α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) 64.82–2.44(2.53–2.44) 30.50–1.79(1.83–1.79) 36.88–1.77 (1.80–1.77) 30.48–1.87(1.91–1.87) 33.21–1.87(1.91–1.87) Rmerge (%) 10.6(36) 8.2(108) 6.2(134) 8.4(152) 9.6(249) I/σ(I) 15.1(6.8) 7.0(1.1) 15.3(1.6) 13.8(1.8) 13.2(1.3) Completeness (%) 100(100) 94.0(90.1) 99.9(99.5) 100(100) 96.8(93.1) Redundancy 15.6(15.6) 4.3(4.2) 8.7(8.2) 9.6(9.4) 8.2(8.2) CC1/2 0.998(0.689) 0.999(0.745) 0.999(0.676) 0.998(0.606) Refinement Resolution (Å) 1.79 1.77 1.87 1.87 No. of Reflections 22583 25052 21505 20838 Rwork/Rfree 0.22/0.23 0.21/0.24 0.22/0.24 0.21/0.25 No. of Atoms 1765 1669 1715 1653  Protein 1586 1493 1541 1444  Ligand/ion 30 56 60 30  Water 149 120 114 179 B-factors 49.4 48.5 47.4 39.2  Protein 49.0 47.5 46.3 38.3  Ligand/ion 48.6 65.9 80.4 62.9  Water 54.2 51.9 44.5 42.1 r.m.s. Deviations  Bond lengths (Å) 0.013 0.013 0.013 0.014  Bond angles (º) 1.24 1.30 1.24 1.39 + + + diff --git a/BioC_XML/4968113_v0.xml b/BioC_XML/4968113_v0.xml new file mode 100644 index 0000000000000000000000000000000000000000..da9f8e354b67c6dcea27c9967fe73c86b1b172fb --- /dev/null +++ b/BioC_XML/4968113_v0.xml @@ -0,0 +1,12421 @@ + + + + PMC + 20201216 + pmc.key + + 4968113 + CC BY-NC + no + 0 + 0 + + A. TEPLYAKOV ET AL. MABS + 10.1080/19420862.2016.1190060 + 4968113 + 27210805 + 1190060 + 1045 + 6 + Antibody structure CDR canonical structure CDR H3 phage library VH:VL packing + This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. + 1063 + surname:Teplyakov;given-names:Alexey + surname:Obmolova;given-names:Galina + surname:Malia;given-names:Thomas J. + surname:Luo;given-names:Jinquan + surname:Muzammil;given-names:Salman + surname:Sweet;given-names:Raymond + surname:Almagro;given-names:Juan Carlos + surname:Gilliland;given-names:Gary L. + TITLE + KEYWORDS + front + 8 + 2016 + 0 + Structural diversity in a human antibody germline library + + 0.9945056 + species + cleaner0 + 2023-06-28T16:03:59Z + MESH: + + human + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:47Z + + antibody + + + + ABSTRACT + abstract_title_1 + 58 + ABSTRACT + + + ABSTRACT + abstract + 67 + To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined. All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. The CDR conformations for the most part are tightly clustered, especially for the ones with shorter lengths. The longer CDRs with tandem glycines or serines have more conformational diversity than the others. CDR H3, despite having the same amino acid sequence, exhibits the largest conformational diversity. About half of the structures have CDR H3 conformations similar to that of the parent; the others diverge significantly. One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing. The stem regions of 14 of the variant pairs are in the ‘kinked’ conformation, and only 2 are in the extended conformation. The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees. Two of 16 structures showed particularly large variations in the tilt angles when compared with the other pairings. The structures and their analyses provide a rich foundation for future antibody modeling and engineering efforts. + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.9971195 + evidence + cleaner0 + 2023-06-28T19:10:19Z + DUMMY: + + crystal structures + + + structure_element + SO: + cleaner0 + 2023-06-28T15:55:50Z + + kappa light chains + + + structure_element + SO: + cleaner0 + 2023-06-28T15:56:14Z + + heavy chains + + + structure_element + SO: + cleaner0 + 2023-06-28T15:56:24Z + + heavy chains + + + structure_element + SO: + cleaner0 + 2023-06-28T15:58:19Z + + antigen-binding fragments + + + 0.29639018 + structure_element + cleaner0 + 2023-06-28T15:58:45Z + SO: + + Fabs + + + 0.9439749 + structure_element + cleaner0 + 2023-06-29T08:49:24Z + SO: + + complementarity-determining region + + + 0.99692184 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.93109226 + structure_element + cleaner0 + 2023-06-28T19:05:14Z + SO: + + H3 + + + 0.37466994 + structure_element + cleaner0 + 2023-06-28T15:59:03Z + SO: + + Fab + + + 0.9929575 + evidence + cleaner0 + 2023-06-28T19:10:54Z + DUMMY: + + structure + + + 0.553402 + experimental_method + cleaner0 + 2023-06-29T09:05:49Z + MESH: + + structure analyses + + + 0.9943771 + evidence + cleaner0 + 2023-06-28T19:10:57Z + DUMMY: + + structures + + + 0.80576843 + evidence + cleaner0 + 2023-06-28T19:11:00Z + DUMMY: + + structures + + + 0.99496186 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-07-21T09:37:10Z + + VH:VL + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:44Z + + packing interactions + + + 0.97404987 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.67458373 + protein_state + cleaner0 + 2023-06-29T09:26:48Z + DUMMY: + + longer + + + 0.9963917 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.95799243 + residue_name + cleaner0 + 2023-06-29T09:11:47Z + SO: + + glycines + + + 0.98402774 + residue_name + cleaner0 + 2023-06-29T09:11:50Z + SO: + + serines + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:14Z + + H3 + + + 0.99673104 + evidence + cleaner0 + 2023-06-28T19:11:02Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:14Z + + H3 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:14Z + + H3 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:16:30Z + + heavy + + + structure_element + SO: + cleaner0 + 2023-06-28T16:13:38Z + + light chain + + + 0.9969053 + structure_element + cleaner0 + 2023-06-29T08:49:40Z + SO: + + stem regions + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T07:42:04Z + + kinked + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T07:41:57Z + + extended + + + 0.99790895 + structure_element + cleaner0 + 2023-06-29T08:49:44Z + SO: + + VH + + + 0.9977047 + structure_element + cleaner0 + 2023-06-28T19:10:07Z + SO: + + VL + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.9943786 + evidence + cleaner0 + 2023-06-28T19:11:05Z + DUMMY: + + structure + + + evidence + DUMMY: + cleaner0 + 2023-06-28T16:02:50Z + + tilt angles + + + 0.9967739 + evidence + cleaner0 + 2023-06-28T19:11:08Z + DUMMY: + + structures + + + evidence + DUMMY: + cleaner0 + 2023-06-28T16:02:51Z + + tilt angles + + + 0.9965509 + evidence + cleaner0 + 2023-06-28T19:11:13Z + DUMMY: + + structures + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + + INTRO + title_1 + 1705 + Introduction + + + INTRO + paragraph + 1718 + At present, therapeutic antibodies are the largest class of biotherapeutic proteins that are in clinical trials. The use of monoclonal antibodies as therapeutics began in the early 1980s, and their composition has transitioned from murine antibodies to generally less immunogenic humanized and human antibodies. The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies. Once a candidate antibody is identified, protein engineering is usually required to produce a molecule with the right biophysical and functional properties. All engineering efforts are guided by our understanding of the atomic structures of antibodies. In such efforts, the crystal structure of the specific antibody may not be available, but modeling can be used to guide the engineering efforts. Today's antibody modeling approaches, which normally focus on the variable region, are being developed by the application of structural principles and insights that are evolving as our knowledge of antibody structures continues to expand. + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:37Z + + antibodies + + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:38Z + + antibodies + + + 0.37784785 + taxonomy_domain + cleaner0 + 2023-06-28T16:06:23Z + DUMMY: + + murine + + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:38Z + + antibodies + + + 0.52457434 + species + cleaner0 + 2023-06-28T16:03:59Z + MESH: + + human + + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:38Z + + antibodies + + + 0.85534525 + species + cleaner0 + 2023-06-28T16:03:58Z + MESH: + + human + + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:38Z + + antibodies + + + 0.36052313 + taxonomy_domain + cleaner0 + 2023-06-28T16:07:17Z + DUMMY: + + mice + + + 0.7995312 + species + cleaner0 + 2023-06-28T16:03:59Z + MESH: + + human + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.9800163 + species + cleaner0 + 2023-06-28T16:03:59Z + MESH: + + human + + + experimental_method + MESH: + cleaner0 + 2023-06-28T16:07:56Z + + in vitro selection + + + 0.68230623 + experimental_method + cleaner0 + 2023-06-29T09:06:00Z + MESH: + + antibody libraries + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + experimental_method + MESH: + cleaner0 + 2023-06-28T16:08:31Z + + protein engineering + + + 0.96321344 + evidence + cleaner0 + 2023-06-28T16:08:57Z + DUMMY: + + atomic structures + + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:38Z + + antibodies + + + 0.99743086 + evidence + cleaner0 + 2023-06-28T16:09:05Z + DUMMY: + + crystal structure + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.9954084 + structure_element + cleaner0 + 2023-06-28T16:09:22Z + SO: + + variable region + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.9851259 + evidence + cleaner0 + 2023-06-28T19:11:18Z + DUMMY: + + structures + + + + INTRO + paragraph + 2909 + Our current structural knowledge of antibodies is based on a multitude of studies that used many techniques to gain insight into the functional and structural properties of this class of macromolecule. Five different antibody isotypes occur, IgG, IgD, IgE, IgA and IgM, and each isotype has a unique role in the adaptive immune system. IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. These multimeric forms are linked with an additional J chain. The LCs that associate with the HCs are divided into 2 functionally indistinguishable classes, κ and λ. Both κ and λ polypeptide chains are composed of a single V domain and a single C domain. + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:38Z + + antibodies + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.5808187 + protein + cleaner0 + 2023-06-28T16:10:07Z + PR: + + IgG + + + 0.579378 + protein + cleaner0 + 2023-06-28T16:10:20Z + PR: + + IgD + + + 0.4901906 + protein + cleaner0 + 2023-06-28T16:10:34Z + PR: + + IgE + + + 0.48674142 + protein + cleaner0 + 2023-06-28T16:10:53Z + PR: + + IgA + + + 0.5469069 + protein + cleaner0 + 2023-06-28T16:11:01Z + PR: + + IgM + + + 0.4615833 + protein + cleaner0 + 2023-06-28T16:10:08Z + PR: + + IgG + + + 0.5355316 + protein + cleaner0 + 2023-06-28T16:10:46Z + PR: + + IgD + + + 0.45904422 + protein + cleaner0 + 2023-06-28T16:10:35Z + PR: + + IgE + + + 0.91602886 + structure_element + cleaner0 + 2023-06-28T15:56:24Z + SO: + + heavy chains + + + 0.99714833 + structure_element + cleaner0 + 2023-06-28T16:11:31Z + SO: + + HCs + + + 0.8479442 + structure_element + cleaner0 + 2023-06-28T16:14:07Z + SO: + + light chains + + + 0.99629045 + structure_element + cleaner0 + 2023-06-28T16:11:38Z + SO: + + LCs + + + ptm + MESH: + melaniev@ebi.ac.uk + 2023-07-06T15:17:35Z + + disulfide bonds + + + 0.58396065 + protein + cleaner0 + 2023-06-28T16:10:55Z + PR: + + IgA + + + 0.6218557 + protein + cleaner0 + 2023-06-28T16:11:02Z + PR: + + IgM + + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:38Z + + antibodies + + + 0.5306497 + protein + cleaner0 + 2023-06-28T16:10:08Z + PR: + + IgG + + + 0.51756364 + protein + cleaner0 + 2023-06-28T16:10:47Z + PR: + + IgD + + + 0.6528544 + protein + cleaner0 + 2023-06-28T16:10:55Z + PR: + + IgA + + + 0.99655485 + structure_element + cleaner0 + 2023-06-29T08:49:51Z + SO: + + variable + + + 0.9962674 + structure_element + cleaner0 + 2023-06-28T16:12:17Z + SO: + + V + + + 0.995513 + structure_element + cleaner0 + 2023-06-29T08:49:58Z + SO: + + constant + + + 0.9924636 + structure_element + cleaner0 + 2023-06-28T16:12:08Z + SO: + + C + + + 0.6833741 + protein + cleaner0 + 2023-06-28T16:10:35Z + PR: + + IgE + + + 0.7602459 + protein + cleaner0 + 2023-06-28T16:11:02Z + PR: + + IgM + + + structure_element + SO: + cleaner0 + 2023-06-29T08:13:46Z + + C domain + + + 0.9844195 + structure_element + cleaner0 + 2023-06-29T08:50:04Z + SO: + + J + + + 0.99804425 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + 0.9981816 + structure_element + cleaner0 + 2023-06-28T16:11:32Z + SO: + + HCs + + + 0.91732925 + structure_element + cleaner0 + 2023-06-28T16:15:20Z + SO: + + κ + + + 0.809085 + structure_element + cleaner0 + 2023-06-28T16:15:35Z + SO: + + λ + + + 0.923577 + structure_element + cleaner0 + 2023-06-28T16:15:21Z + SO: + + κ + + + 0.8177861 + structure_element + cleaner0 + 2023-06-28T16:15:36Z + SO: + + λ + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-06T15:14:51Z + + V domain + + + structure_element + SO: + cleaner0 + 2023-06-29T08:13:25Z + + C domain + + + + INTRO + paragraph + 3893 + The heavy and light chains are composed of structural domains that have ∼110 amino acid residues. These domains have a common folding pattern often referred to as the “immunoglobulin fold,” formed by the packing together of 2 anti-parallel β-sheets. All immunoglobulin chains have an N-terminal V domain followed by 1 to 4 C domains, depending upon the chain type. In antibodies, the heavy and light chain V domains pack together forming the antigen combining site. This site, which interacts with the antigen (or target), is the focus of current antibody modeling efforts. This interaction site is composed of 6 complementarity-determining regions (CDRs) that were identified in early antibody amino acid sequence analyses to be hypervariable in nature, and thus are responsible for the sequence and structural diversity of our antibody repertoire. + + 0.28037655 + structure_element + cleaner0 + 2023-06-29T08:50:09Z + SO: + + heavy + + + structure_element + SO: + cleaner0 + 2023-06-28T16:14:08Z + + light chains + + + 0.8056854 + structure_element + cleaner0 + 2023-06-29T08:50:22Z + SO: + + structural domains + + + residue_range + DUMMY: + cleaner0 + 2023-06-29T09:14:08Z + + ∼110 amino acid residues + + + 0.9660743 + structure_element + cleaner0 + 2023-06-29T08:50:29Z + SO: + + immunoglobulin fold + + + 0.98161864 + structure_element + cleaner0 + 2023-06-29T08:50:36Z + SO: + + anti-parallel β-sheets + + + 0.9119513 + protein_type + cleaner0 + 2023-06-28T19:09:00Z + MESH: + + immunoglobulin chains + + + structure_element + SO: + melaniev@ebi.ac.uk + 2023-07-06T15:15:03Z + + V domain + + + structure_element + SO: + cleaner0 + 2023-06-28T16:21:31Z + + C domains + + + 0.5746893 + protein_type + cleaner0 + 2023-06-28T16:03:38Z + MESH: + + antibodies + + + structure_element + SO: + cleaner0 + 2023-06-28T20:43:25Z + + heavy and light chain + + + structure_element + SO: + cleaner0 + 2023-06-28T16:21:51Z + + V domains + + + 0.9981706 + site + cleaner0 + 2023-06-29T09:10:03Z + SO: + + antigen combining site + + + 0.774341 + protein_type + cleaner0 + 2023-06-28T15:45:48Z + MESH: + + antibody + + + 0.99761343 + site + cleaner0 + 2023-06-29T09:10:11Z + SO: + + interaction site + + + 0.9165287 + structure_element + cleaner0 + 2023-06-29T08:50:44Z + SO: + + complementarity-determining regions + + + 0.99350005 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.92376745 + experimental_method + cleaner0 + 2023-06-29T09:06:06Z + MESH: + + antibody amino acid sequence analyses + + + 0.99413955 + protein_state + cleaner0 + 2023-06-29T09:26:56Z + DUMMY: + + hypervariable + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + + INTRO + paragraph + 4750 + The sequence diversity of the CDR regions presents a substantial challenge to antibody modeling. However, an initial structural analysis of the combining sites of the small set of structures of immunoglobulin fragments available in the 1980s found that 5 of the 6 hypervariable loops or CDRs had canonical structures (a limited set of main-chain conformations). A CDR canonical structure is defined by its length and conserved residues located in the hypervariable loop and framework residues (V-region residues that are not part of the CDRs). Furthermore, studies of antibody sequences revealed that the total number of canonical structures are limited for each CDR, indicating possibly that antigen recognition may be affected by structural restrictions at the antigen-binding site. Later studies found that the CDR loop length is the primary determining factor of antigen-binding site topography because it is the primary factor for determining a canonical structure. Additional efforts have led to our current understanding that the LC CDRs L1, L2, and L3 have preferred sets of canonical structures based on length and amino acid sequence composition. This was also found to be the case for the H1 and H2 CDRs. Classification schemes for the canonical structures of these 5 CDRs have emerged and evolved as the number of depositions in the Protein Data Bank of Fab fragments of antibodies grow. Recently, a comprehensive CDR classification scheme was reported identifying 72 clusters of conformations observed in antibody structures. The knowledge and predictability of these CDR canonical structures have greatly advanced antibody modeling efforts. + + 0.9892019 + structure_element + cleaner0 + 2023-06-29T08:50:50Z + SO: + + CDR regions + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.86392236 + experimental_method + cleaner0 + 2023-06-29T09:06:10Z + MESH: + + structural analysis + + + 0.9967412 + site + cleaner0 + 2023-06-29T09:10:17Z + SO: + + combining sites + + + 0.99583673 + evidence + cleaner0 + 2023-06-28T19:11:25Z + DUMMY: + + structures + + + 0.97308254 + structure_element + cleaner0 + 2023-06-29T08:50:55Z + SO: + + hypervariable loops + + + 0.9967535 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.79579794 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.96811986 + structure_element + cleaner0 + 2023-06-29T08:51:00Z + SO: + + hypervariable loop + + + 0.79810786 + structure_element + cleaner0 + 2023-06-29T08:51:45Z + SO: + + framework residues + + + 0.93935466 + structure_element + cleaner0 + 2023-06-29T08:51:53Z + SO: + + V-region + + + 0.99704546 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.9926368 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.99790645 + site + cleaner0 + 2023-06-29T09:10:21Z + SO: + + antigen-binding site + + + 0.954146 + structure_element + cleaner0 + 2023-06-29T08:51:56Z + SO: + + CDR loop + + + 0.9959662 + site + cleaner0 + 2023-06-29T09:10:24Z + SO: + + antigen-binding site + + + 0.6664975 + structure_element + cleaner0 + 2023-06-28T16:24:54Z + SO: + + LC + + + 0.9848006 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.9951461 + structure_element + cleaner0 + 2023-06-28T16:26:29Z + SO: + + L1 + + + 0.9955865 + structure_element + cleaner0 + 2023-06-28T20:09:53Z + SO: + + L2 + + + 0.99560404 + structure_element + cleaner0 + 2023-06-29T08:52:03Z + SO: + + L3 + + + 0.90607065 + structure_element + cleaner0 + 2023-06-28T19:04:09Z + SO: + + H1 + + + 0.6061986 + structure_element + cleaner0 + 2023-06-28T16:26:20Z + SO: + + H2 + + + 0.97096324 + structure_element + cleaner0 + 2023-06-28T19:04:37Z + SO: + + CDRs + + + 0.9894225 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.4258705 + structure_element + cleaner0 + 2023-06-28T15:59:04Z + SO: + + Fab + + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:38Z + + antibodies + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.99535346 + evidence + cleaner0 + 2023-06-28T19:11:30Z + DUMMY: + + structures + + + 0.95264316 + structure_element + cleaner0 + 2023-06-28T16:25:19Z + SO: + + CDR + + + 0.65532506 + evidence + cleaner0 + 2023-06-28T19:11:33Z + DUMMY: + + structures + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + + INTRO + paragraph + 6405 + In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence. Some clustering of conformations was observed for the shortest lengths; however, for the longer loops, only the portions nearest the framework (torso, stem or anchor region) were found to have defined conformations. In the torso region, 2 primary groups could be identified, which led to sequence-based rules that can predict with some degree of reliability the conformation of the stem region. The “kinked” or “bulged” conformation is the most prevalent, but an “extended” or “non-bulged” conformation is also, but less frequently, observed. The cataloging and development of the rules for predicting the conformation of the anchor region of CDR H3 continue to be refined, producing new insight into the CDR H3 conformations and new tools for antibody engineering. + + 0.9609944 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.8862208 + structure_element + cleaner0 + 2023-06-28T16:26:30Z + SO: + + L1 + + + 0.9949463 + structure_element + cleaner0 + 2023-06-28T20:09:53Z + SO: + + L2 + + + 0.99527967 + structure_element + cleaner0 + 2023-06-29T08:52:14Z + SO: + + L3 + + + 0.9794373 + structure_element + cleaner0 + 2023-06-28T19:04:10Z + SO: + + H1 + + + 0.9499178 + structure_element + cleaner0 + 2023-06-28T16:26:21Z + SO: + + H2 + + + evidence + DUMMY: + cleaner0 + 2023-06-28T20:44:12Z + + structures + + + structure_element + SO: + cleaner0 + 2023-06-28T19:06:45Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:14Z + + H3 + + + 0.9731328 + structure_element + cleaner0 + 2023-06-29T08:52:18Z + SO: + + loops + + + 0.9971501 + structure_element + cleaner0 + 2023-06-29T08:52:25Z + SO: + + framework + + + 0.99759847 + structure_element + cleaner0 + 2023-06-29T08:52:29Z + SO: + + torso + + + 0.9975243 + structure_element + cleaner0 + 2023-06-29T08:52:32Z + SO: + + stem + + + 0.9965924 + structure_element + cleaner0 + 2023-06-29T08:52:35Z + SO: + + anchor region + + + 0.99673957 + structure_element + cleaner0 + 2023-06-29T08:52:42Z + SO: + + torso region + + + 0.99730873 + structure_element + cleaner0 + 2023-06-29T08:52:49Z + SO: + + stem region + + + 0.9651129 + protein_state + cleaner0 + 2023-06-29T07:42:05Z + DUMMY: + + kinked + + + 0.9021778 + protein_state + cleaner0 + 2023-06-29T09:27:20Z + DUMMY: + + bulged + + + 0.9788072 + protein_state + cleaner0 + 2023-06-29T07:41:57Z + DUMMY: + + extended + + + 0.9845164 + protein_state + cleaner0 + 2023-06-29T09:27:24Z + DUMMY: + + non-bulged + + + 0.9973566 + structure_element + cleaner0 + 2023-06-29T08:52:56Z + SO: + + anchor region + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:13Z + + H3 + + + 0.8594353 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.6657651 + structure_element + cleaner0 + 2023-06-28T19:05:14Z + SO: + + H3 + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + + INTRO + paragraph + 7347 + Current antibody modeling approaches take advantage of the most recent advances in homology modeling, the evolving understanding of the CDR canonical structures, the emerging rules for CDR H3 modeling and the growing body of antibody structural data available from the PDB. Recent antibody modeling assessments show continued improvement in the quality of the models being generated by a variety of modeling methods. Although antibody modeling is improving, the latest assessment revealed a number of challenges that need to be overcome to provide accurate 3-dimensional models of antibody V regions, including accuracies in the modeling of CDR H3. The need for improvement in this area was also highlighted in a recent study reporting an approach and results that may influence future antibody modeling efforts. One important finding of the antibody modeling assessments was that errors in the structural templates that are used as the basis for homology models can propagate into the final models, producing inaccuracies that may negatively influence the predictive nature of the V region model. + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.9942754 + experimental_method + cleaner0 + 2023-06-28T19:08:24Z + MESH: + + homology modeling + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + 0.46883506 + evidence + cleaner0 + 2023-06-28T19:11:39Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:14Z + + H3 + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.76543283 + experimental_method + cleaner0 + 2023-06-28T19:06:07Z + MESH: + + antibody modeling assessments + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.9220402 + structure_element + cleaner0 + 2023-06-28T19:05:50Z + SO: + + V regions + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:14Z + + H3 + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.8185734 + experimental_method + cleaner0 + 2023-06-28T19:06:09Z + MESH: + + antibody modeling assessments + + + 0.99114794 + experimental_method + cleaner0 + 2023-06-29T09:06:15Z + MESH: + + homology models + + + 0.94426894 + structure_element + cleaner0 + 2023-06-29T08:53:04Z + SO: + + V region + + + + INTRO + paragraph + 8445 + To support antibody engineering and therapeutic development efforts, a phage library was designed and constructed based on a limited number of scaffolds built with frequently used human germ-line IGV and IGJ gene segments that encode antigen combining sites suitable for recognition of peptides and proteins. This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). Selection of these genes was based on the high frequency of their use and their cognate canonical structures that were found binding to peptides and proteins, as well as their ability to be expressed in bacteria and displayed on filamentous phage. The implementation of the library involves the diversification of the human germline genes to mimic that found in natural human libraries. + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.8285667 + experimental_method + cleaner0 + 2023-06-29T09:06:19Z + MESH: + + phage library + + + 0.99304044 + species + cleaner0 + 2023-06-28T16:03:59Z + MESH: + + human + + + 0.5477985 + structure_element + cleaner0 + 2023-06-29T09:37:42Z + SO: + + IGV + + + 0.37411508 + structure_element + cleaner0 + 2023-06-29T09:37:44Z + SO: + + IGJ + + + 0.9979419 + site + cleaner0 + 2023-06-29T09:10:29Z + SO: + + antigen combining sites + + + 0.22796038 + structure_element + cleaner0 + 2023-06-28T15:59:04Z + SO: + + Fab + + + 0.3749512 + structure_element + cleaner0 + 2023-06-29T08:55:32Z + SO: + + HC + + + mutant + MESH: + cleaner0 + 2023-06-28T21:02:43Z + + IGHV1-69 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:03:12Z + + H1-69 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:03:44Z + + IGHV3-23 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:04:11Z + + H3-23 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:04:40Z + + IGHV5-51 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:05:04Z + + H5-51 + + + 0.29352522 + structure_element + cleaner0 + 2023-06-28T16:24:55Z + SO: + + LC + + + 0.68899107 + structure_element + cleaner0 + 2023-06-28T16:15:21Z + SO: + + κ + + + mutant + MESH: + cleaner0 + 2023-06-28T21:05:26Z + + IGKV1-39 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:05:49Z + + L1-39 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:06:12Z + + IGKV3-11 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:06:33Z + + L3-11 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:06:55Z + + IGKV3-20 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:07:14Z + + L3-20 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:07:38Z + + IGKV4-1 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:07:59Z + + L4-1 + + + evidence + DUMMY: + cleaner0 + 2023-06-28T20:53:28Z + + structures + + + experimental_method + MESH: + cleaner0 + 2023-06-28T21:02:06Z + + expressed in bacteria + + + experimental_method + MESH: + cleaner0 + 2023-06-28T21:02:13Z + + displayed on filamentous phage + + + 0.9926621 + species + cleaner0 + 2023-06-28T16:03:59Z + MESH: + + human + + + 0.9930802 + species + cleaner0 + 2023-06-28T16:03:59Z + MESH: + + human + + + + INTRO + paragraph + 9350 + The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development. All 16 HCs of the Fabs have the same CDR H3 that was reported in an earlier Fab structure. This is the first systematic study of the same VH and VL structures in the context of different pairings. The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. The structures and their analyses provide a foundation for future antibody engineering and structure determination efforts. + + 0.9365645 + experimental_method + cleaner0 + 2023-06-29T09:06:25Z + MESH: + + crystal structure determinations + + + 0.9690031 + experimental_method + cleaner0 + 2023-06-29T09:06:28Z + MESH: + + structural analyses + + + 0.18112527 + structure_element + cleaner0 + 2023-06-28T15:58:46Z + SO: + + Fabs + + + 0.99385357 + evidence + cleaner0 + 2023-06-28T19:11:45Z + DUMMY: + + structures + + + 0.4604112 + structure_element + cleaner0 + 2023-06-29T08:55:41Z + SO: + + HC + + + 0.9870631 + mutant + cleaner0 + 2023-06-29T09:25:45Z + MESH: + + IGHV3-53 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:08:31Z + + H3-53 + + + 0.99305177 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.9857884 + structure_element + cleaner0 + 2023-06-28T16:11:32Z + SO: + + HCs + + + 0.23558842 + structure_element + cleaner0 + 2023-06-28T15:58:46Z + SO: + + Fabs + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:14Z + + H3 + + + 0.24147022 + structure_element + cleaner0 + 2023-06-28T15:59:04Z + SO: + + Fab + + + 0.9950139 + evidence + cleaner0 + 2023-06-28T19:11:56Z + DUMMY: + + structure + + + 0.9822842 + structure_element + cleaner0 + 2023-06-29T08:55:44Z + SO: + + VH + + + 0.956339 + structure_element + cleaner0 + 2023-06-28T19:10:07Z + SO: + + VL + + + 0.9961636 + evidence + cleaner0 + 2023-06-28T19:11:59Z + DUMMY: + + structures + + + 0.99412364 + evidence + cleaner0 + 2023-06-28T19:12:02Z + DUMMY: + + structures + + + 0.97373337 + evidence + cleaner0 + 2023-06-28T19:12:04Z + DUMMY: + + structures + + + 0.9947988 + structure_element + cleaner0 + 2023-06-28T16:26:30Z + SO: + + L1 + + + 0.9907031 + structure_element + cleaner0 + 2023-06-28T20:09:53Z + SO: + + L2 + + + 0.9922057 + structure_element + cleaner0 + 2023-06-29T08:55:50Z + SO: + + L3 + + + 0.9905301 + structure_element + cleaner0 + 2023-06-28T19:04:10Z + SO: + + H1 + + + 0.9891143 + structure_element + cleaner0 + 2023-06-28T16:26:21Z + SO: + + H2 + + + 0.9166478 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.98723364 + evidence + cleaner0 + 2023-06-28T19:12:07Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:07:47Z + + H3s + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-07-21T09:37:57Z + + VH:VL + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:44Z + + packing interactions + + + 0.98796034 + evidence + cleaner0 + 2023-06-28T19:12:10Z + DUMMY: + + structures + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + + RESULTS + title_1 + 10150 + Results + + + RESULTS + title_2 + 10158 + Crystal structures + + 0.9969361 + evidence + cleaner0 + 2023-06-28T19:12:15Z + DUMMY: + + Crystal structures + + + + d37e422.xml + t0001 + TABLE + table_caption + 10177 + Crystal data, X-ray data, and refinement statistics. + + 0.99486244 + evidence + cleaner0 + 2023-06-28T19:12:18Z + DUMMY: + + Crystal data + + + 0.99513537 + evidence + cleaner0 + 2023-06-28T19:12:21Z + DUMMY: + + X-ray data + + + evidence + DUMMY: + cleaner0 + 2023-06-28T19:12:33Z + + refinement statistics + + + + d37e422.xml + t0001 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/></colgroup><thead><tr><th align="left">Fab<hr/></th><th align="center">H1-69:L1-39<hr/></th><th align="center">H1-69:L3-11<hr/></th><th align="center">H1-69:L3-20<hr/></th><th align="center">H1-69:L4-1<hr/></th></tr><tr><th align="left"><italic>PDB identifier</italic></th><th align="center">5I15</th><th align="center">5I16</th><th align="center">5I17</th><th align="center">5I18</th></tr></thead><tbody><tr><td align="left">Crystal Data</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left">Crystallization Solution</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Buffer, pH</td><td align="center">0.1 M MES- pH 6.5</td><td align="center">0.1 M MES pH 6.5</td><td align="center">0.1 M MES, pH 6.5</td><td align="center">0.1 M HEPES, pH 7.5</td></tr><tr><td align="left"> Precipitant<xref rid="t1fn0001" ref-type="fn"><sup>1</sup></xref></td><td align="center">5 M Na Formate</td><td align="center">25% PEG 3350</td><td align="center">2.0 M Amm Sulfate</td><td align="center">10% PEG 8000</td></tr><tr><td align="left"> Additive<xref rid="t1fn0001" ref-type="fn"><sup>1</sup></xref></td><td align="left"> </td><td align="center">0.2 M Na Formate</td><td align="center">5% MPD</td><td align="center">8% EG</td></tr><tr><td align="left"> Space Group</td><td align="center">P3<sub>1</sub>21</td><td align="center">C2</td><td align="center">P422</td><td align="center">P42<sub>1</sub>2</td></tr><tr><td align="left"> Molecules/AU</td><td align="center">1</td><td align="center">2</td><td align="center">2</td><td align="center">1</td></tr><tr><td align="left"> Unit Cell</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> a(Å)</td><td align="center">129.2</td><td align="center">212.0</td><td align="center">152.5</td><td align="center">120.0</td></tr><tr><td align="left"> b(Å)</td><td align="center">129.2</td><td align="center">55.1</td><td align="center">152.5</td><td align="center">120.0</td></tr><tr><td align="left"> c(Å)</td><td align="center">91.8</td><td align="center">80.3</td><td align="center">123.4</td><td align="center">64.2</td></tr><tr><td align="left"> β(°)</td><td align="center">90.0</td><td align="center">97.8</td><td align="center">90.0</td><td align="center">90.0</td></tr><tr><td align="left"> γ(°)</td><td align="center">120.0</td><td align="center">90.0</td><td align="center">90.0</td><td align="center">90.0</td></tr><tr><td align="left"> V<sub>m</sub> (Å<sup>3</sup>/Da)</td><td align="center">4.67</td><td align="center">2.44</td><td align="center">3.77</td><td align="center">2.39</td></tr><tr><td align="left"> Solvent Content (%)</td><td align="center">74</td><td align="center">50</td><td align="center">67</td><td align="center">48</td></tr><tr><td align="left">X-Ray Data<xref rid="t1fn0002" ref-type="fn"><sup>2</sup></xref></td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Resolution (Å)</td><td align="center">30-2.6 (2.7-2.6)</td><td align="center">30.0-1.9 (1.95-1.9)</td><td align="center">30.0-3.3 (3.4-3.3)</td><td align="center">30-1.9 (2.0-1.9)</td></tr><tr><td align="left"> Measured Reflections</td><td align="center">136,745 (8,650)</td><td align="center">241,145 (16,580)</td><td align="center">237,504 (15,007)</td><td align="center">801,080 (19,309)</td></tr><tr><td align="left"> Unique Reflections</td><td align="center">27,349 (1,730)</td><td align="center">71,932 (5,198)</td><td align="center">22,379 (1,590)</td><td align="center">35,965 (2,194)</td></tr><tr><td align="left"> Completeness (%)</td><td align="center">99.3 (98.7)</td><td align="center">99.0 (97.3)</td><td align="center">99.5 (96.8)</td><td align="center">98.5 (82.8)</td></tr><tr><td align="left"> Redundancy</td><td align="center">5.0 (5.0)</td><td align="center">3.4 (3.2)</td><td align="center">10.6 (9.4)</td><td align="center">22.3 (8.8)</td></tr><tr><td align="left"> R<sub>merge</sub></td><td align="center">0.048 (0.522)</td><td align="center">0.044 (0.245)</td><td align="center">0.086 (0.536)</td><td align="center">0.093 (0.231)</td></tr><tr><td align="left"> &lt; I/σ &gt;</td><td align="center">21.2 (3.9)</td><td align="center">17.8 (4.7)</td><td align="center">25.5 (4.5)</td><td align="center">29.2 (8.1)</td></tr><tr><td align="left"> B-factor (Å<sup>2</sup>)</td><td align="center">60.5</td><td align="center">33.2</td><td align="center">61.0</td><td align="center">19.6</td></tr><tr><td align="left">Refinement</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Resolution (Å)</td><td align="center">15-2.6</td><td align="center">15-1.9</td><td align="center">15-3.3</td><td align="center">15-1.9</td></tr><tr><td align="left"> Number of Reflections</td><td align="center">26,238</td><td align="center">70,346</td><td align="center">21,197</td><td align="center">34,850</td></tr><tr><td align="left"> Number of All Atoms</td><td align="center">3,224</td><td align="center">6,975</td><td align="center">6,398</td><td align="center">3,695</td></tr><tr><td align="left"> Number of Waters</td><td align="center">2</td><td align="center">472</td><td align="center">0</td><td align="center">399</td></tr><tr><td align="left"> R-factor (%)</td><td align="center">20.5</td><td align="center">19.2</td><td align="center">20.2</td><td align="center">16.7</td></tr><tr><td align="left"> R-free (%)</td><td align="center">24.1</td><td align="center">22.2</td><td align="center">24.7</td><td align="center">21.3</td></tr><tr><td align="left">RMSD</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Bond Lengths (Å)</td><td align="center">0.006</td><td align="center">0.005</td><td align="center">0.005</td><td align="center">0.008</td></tr><tr><td align="left"> Bond Angles (°)</td><td align="center">1.2</td><td align="center">1.1</td><td align="center">1.0</td><td align="center">1.1</td></tr><tr><td align="left"> Mean B-factor (Å<sup>2</sup>)</td><td align="center">65.3</td><td align="center">34.4</td><td align="center">80.1</td><td align="center">20.0</td></tr><tr><td align="left">Ramachandran Plot (%)</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Outliers</td><td align="center">0.0</td><td align="center">0.0</td><td align="center">0.9</td><td align="center">0.0</td></tr><tr><td align="left"> Favored</td><td align="center">92.3</td><td align="center">96.9</td><td align="center">93.1</td><td align="center">96.9</td></tr></tbody></table> + + 10230 + Fab H1-69:L1-39 H1-69:L3-11 H1-69:L3-20 H1-69:L4-1 PDB identifier 5I15 5I16 5I17 5I18 Crystal Data         Crystallization Solution          Buffer, pH 0.1 M MES- pH 6.5 0.1 M MES pH 6.5 0.1 M MES, pH 6.5 0.1 M HEPES, pH 7.5  Precipitant1 5 M Na Formate 25% PEG 3350 2.0 M Amm Sulfate 10% PEG 8000  Additive1   0.2 M Na Formate 5% MPD 8% EG  Space Group P3121 C2 P422 P4212  Molecules/AU 1 2 2 1  Unit Cell          a(Å) 129.2 212.0 152.5 120.0  b(Å) 129.2 55.1 152.5 120.0  c(Å) 91.8 80.3 123.4 64.2  β(°) 90.0 97.8 90.0 90.0  γ(°) 120.0 90.0 90.0 90.0  Vm (Å3/Da) 4.67 2.44 3.77 2.39  Solvent Content (%) 74 50 67 48 X-Ray Data2          Resolution (Å) 30-2.6 (2.7-2.6) 30.0-1.9 (1.95-1.9) 30.0-3.3 (3.4-3.3) 30-1.9 (2.0-1.9)  Measured Reflections 136,745 (8,650) 241,145 (16,580) 237,504 (15,007) 801,080 (19,309)  Unique Reflections 27,349 (1,730) 71,932 (5,198) 22,379 (1,590) 35,965 (2,194)  Completeness (%) 99.3 (98.7) 99.0 (97.3) 99.5 (96.8) 98.5 (82.8)  Redundancy 5.0 (5.0) 3.4 (3.2) 10.6 (9.4) 22.3 (8.8)  Rmerge 0.048 (0.522) 0.044 (0.245) 0.086 (0.536) 0.093 (0.231)  < I/σ > 21.2 (3.9) 17.8 (4.7) 25.5 (4.5) 29.2 (8.1)  B-factor (Å2) 60.5 33.2 61.0 19.6 Refinement          Resolution (Å) 15-2.6 15-1.9 15-3.3 15-1.9  Number of Reflections 26,238 70,346 21,197 34,850  Number of All Atoms 3,224 6,975 6,398 3,695  Number of Waters 2 472 0 399  R-factor (%) 20.5 19.2 20.2 16.7  R-free (%) 24.1 22.2 24.7 21.3 RMSD          Bond Lengths (Å) 0.006 0.005 0.005 0.008  Bond Angles (°) 1.2 1.1 1.0 1.1  Mean B-factor (Å2) 65.3 34.4 80.1 20.0 Ramachandran Plot (%)          Outliers 0.0 0.0 0.9 0.0  Favored 92.3 96.9 93.1 96.9 + + + d37e422.xml + t0001 + TABLE + table_footnote + 12072 + Abbreviations: Amm, ammonium;EG, ethylene glycol; PEG, polyethylene glycol. + + + d37e422.xml + t0001 + TABLE + table_footnote + 12148 + Values for high-resolution shell are in parentheses. + + + d37e892.xml + t0001 + TABLE + table_caption + 12201 + (Continued) Crystal data, X-ray data, and refinement statistics. + + 0.9933119 + evidence + cleaner0 + 2023-06-28T19:14:53Z + DUMMY: + + Crystal data + + + 0.98985547 + evidence + cleaner0 + 2023-06-28T19:14:56Z + DUMMY: + + X-ray data + + + evidence + DUMMY: + cleaner0 + 2023-06-28T19:15:10Z + + refinement statistics + + + + d37e892.xml + t0001 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/></colgroup><thead><tr><th align="left">Fab<hr/></th><th align="center">H3-23:L1-39<hr/></th><th align="center">H3-23:L3-11<hr/></th><th align="center">H3-23:L3-20<hr/></th><th align="center">H3-23:L4-1<hr/></th></tr><tr><th align="left"><italic> PDB identifier</italic></th><th align="center">5I19</th><th align="center">5I1A</th><th align="center">5I1C</th><th align="center">5I1D</th></tr></thead><tbody><tr><td align="left">Crystal Data</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left">Crystallization Solution</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Buffer, pH</td><td align="center">No Buffer</td><td align="center">0.1 M Na Acetate, pH 4.5</td><td align="center">0.1 M MES, pH 6.5</td><td align="center">0.1 M HEPES, pH 7.5</td></tr><tr><td align="left"> Precipitant<xref rid="t1fn0003" ref-type="fn"><sup>1</sup></xref></td><td align="center">20% PEG 3350</td><td align="center">2.0 M Amm Sulfate</td><td align="center">16% PEG 3350</td><td align="center">2.0 M Amm Sulfate</td></tr><tr><td align="left"> Additive<xref rid="t1fn0003" ref-type="fn"><sup>1</sup></xref></td><td align="center">0.2 M Li Citrate</td><td align="center">5% PEG 400</td><td align="center">0.2 M Amm Acetate</td><td align="center">2% PEG 400</td></tr><tr><td align="left"> Space Group</td><td align="center">P4<sub>1</sub>2<sub>1</sub>2</td><td align="center">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="center">P6<sub>2</sub>22</td><td align="center">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td></tr><tr><td align="left"> Molecules/AU</td><td align="center">1</td><td align="center">2</td><td align="center">1</td><td align="center">2</td></tr><tr><td align="left">Unit Cell</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> a(Å)</td><td align="center">96.6</td><td align="center">60.9</td><td align="center">121.5</td><td align="center">62.7</td></tr><tr><td align="left"> b(Å)</td><td align="center">96.6</td><td align="center">110.6</td><td align="center">121.5</td><td align="center">111.0</td></tr><tr><td align="left"> c(Å)</td><td align="center">105.4</td><td align="center">158.9</td><td align="center">160.4</td><td align="center">160.0</td></tr><tr><td align="left"> β(°)</td><td align="center">90</td><td align="center">90</td><td align="center">90</td><td align="center">90</td></tr><tr><td align="left"> V<sub>m</sub> (Å<sup>3</sup>/Da)</td><td align="center">2.60</td><td align="center">2.82</td><td align="center">3.60</td><td align="center">2.90</td></tr><tr><td align="left"> Solvent Content (%)</td><td align="center">53</td><td align="center">56</td><td align="center">66</td><td align="center">57</td></tr><tr><td align="left">X-Ray Data<xref rid="t1fn0004" ref-type="fn"><sup>2</sup></xref></td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Resolution (Å)</td><td align="center">30-2.8 (2.9-2.8)</td><td align="center">30-2.0 (2.1-2.0)</td><td align="center">30-2.25 (2.3-2.25)</td><td align="center">30-2.0 (2.1-2.0)</td></tr><tr><td align="left"> Measured Reflections</td><td align="center">177,681 (12,072)</td><td align="center">351,312 (8,634)</td><td align="center">887,349 (59,919)</td><td align="center">873,523 (49,118)</td></tr><tr><td align="left"> Unique Reflections</td><td align="center">12,678 (899)</td><td align="center">58,989 (2,870)</td><td align="center">32,572 (2,300)</td><td align="center">75,540 (5,343)</td></tr><tr><td align="left"> Completeness (%)</td><td align="center">99.5 (97.4)</td><td align="center">80.9 (54.2)</td><td align="center">96.9 (94.8)</td><td align="center">99.7 (96.9)</td></tr><tr><td align="left"> Redundancy</td><td align="center">14.0 (13.4)</td><td align="center">6.0 (3.0)</td><td align="center">27.2 (26.1)</td><td align="center">11.6 (9.2)</td></tr><tr><td align="left"> R<sub>merge</sub></td><td align="center">0.091 (0.594)</td><td align="center">0.066 (0.204)</td><td align="center">0.086 (0.478)</td><td align="center">0.094 (0.488)</td></tr><tr><td align="left"> &lt; I/σ &gt;</td><td align="center">31.2 (5.1)</td><td align="center">20.4 (4.6)</td><td align="center">37.0 (10.4)</td><td align="center">21.6 (5.0)</td></tr><tr><td align="left"> B-factor (Å<sup>2</sup>)</td><td align="center">42.8</td><td align="center">27.1</td><td align="center">33.7</td><td align="center">29.4</td></tr><tr><td align="left">Refinement</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Resolution (Å)</td><td align="center">15-2.8</td><td align="center">15-2.0</td><td align="center">15-2.25</td><td align="center">15-2.0</td></tr><tr><td align="left"> Number of Reflections</td><td align="center">11,972</td><td align="center">57,599</td><td align="center">31,411</td><td align="center">74,238</td></tr><tr><td align="left"> Number of All Atoms</td><td align="center">3,234</td><td align="center">6,948</td><td align="center">3,472</td><td align="center">7,210</td></tr><tr><td align="left"> Number of Waters</td><td align="center">0</td><td align="center">416</td><td align="center">222</td><td align="center">635</td></tr><tr><td align="left"> R-factor (%)</td><td align="center">23.9</td><td align="center">20.5</td><td align="center">22.0</td><td align="center">21.6</td></tr><tr><td align="left"> R-free (%)</td><td align="center">31.5</td><td align="center">25.5</td><td align="center">26.6</td><td align="center">25.1</td></tr><tr><td align="left">RMSD</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Bond Lengths (Å)</td><td align="center">0.009</td><td align="center">0.010</td><td align="center">0.005</td><td align="center">0.008</td></tr><tr><td align="left"> Bond Angles (°)</td><td align="center">1.3</td><td align="center">1.3</td><td align="center">1.0</td><td align="center">1.1</td></tr><tr><td align="left"> Mean B-factor (Å<sup>2</sup>)</td><td align="center">48.4</td><td align="center">36.7</td><td align="center">47.7</td><td align="center">46.4</td></tr><tr><td align="left">Ramachandran Plot (%)</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Outliers</td><td align="center">0.0</td><td align="center">0.0</td><td align="center">0.0</td><td align="center">0.0</td></tr><tr><td align="left"> Favored</td><td align="center">92.3</td><td align="center">96.8</td><td align="center">97.5</td><td align="center">97.6</td></tr></tbody></table> + + 12266 + Fab H3-23:L1-39 H3-23:L3-11 H3-23:L3-20 H3-23:L4-1  PDB identifier 5I19 5I1A 5I1C 5I1D Crystal Data         Crystallization Solution          Buffer, pH No Buffer 0.1 M Na Acetate, pH 4.5 0.1 M MES, pH 6.5 0.1 M HEPES, pH 7.5  Precipitant1 20% PEG 3350 2.0 M Amm Sulfate 16% PEG 3350 2.0 M Amm Sulfate  Additive1 0.2 M Li Citrate 5% PEG 400 0.2 M Amm Acetate 2% PEG 400  Space Group P41212 P212121 P6222 P212121  Molecules/AU 1 2 1 2 Unit Cell          a(Å) 96.6 60.9 121.5 62.7  b(Å) 96.6 110.6 121.5 111.0  c(Å) 105.4 158.9 160.4 160.0  β(°) 90 90 90 90  Vm (Å3/Da) 2.60 2.82 3.60 2.90  Solvent Content (%) 53 56 66 57 X-Ray Data2          Resolution (Å) 30-2.8 (2.9-2.8) 30-2.0 (2.1-2.0) 30-2.25 (2.3-2.25) 30-2.0 (2.1-2.0)  Measured Reflections 177,681 (12,072) 351,312 (8,634) 887,349 (59,919) 873,523 (49,118)  Unique Reflections 12,678 (899) 58,989 (2,870) 32,572 (2,300) 75,540 (5,343)  Completeness (%) 99.5 (97.4) 80.9 (54.2) 96.9 (94.8) 99.7 (96.9)  Redundancy 14.0 (13.4) 6.0 (3.0) 27.2 (26.1) 11.6 (9.2)  Rmerge 0.091 (0.594) 0.066 (0.204) 0.086 (0.478) 0.094 (0.488)  < I/σ > 31.2 (5.1) 20.4 (4.6) 37.0 (10.4) 21.6 (5.0)  B-factor (Å2) 42.8 27.1 33.7 29.4 Refinement          Resolution (Å) 15-2.8 15-2.0 15-2.25 15-2.0  Number of Reflections 11,972 57,599 31,411 74,238  Number of All Atoms 3,234 6,948 3,472 7,210  Number of Waters 0 416 222 635  R-factor (%) 23.9 20.5 22.0 21.6  R-free (%) 31.5 25.5 26.6 25.1 RMSD          Bond Lengths (Å) 0.009 0.010 0.005 0.008  Bond Angles (°) 1.3 1.3 1.0 1.1  Mean B-factor (Å2) 48.4 36.7 47.7 46.4 Ramachandran Plot (%)          Outliers 0.0 0.0 0.0 0.0  Favored 92.3 96.8 97.5 97.6 + + + d37e892.xml + t0001 + TABLE + table_footnote + 14105 + Abbreviations: Amm, ammonium; PEG, polyethylene glycol. + + + d37e892.xml + t0001 + TABLE + table_footnote + 14161 + Values for high-resolution shell are in parentheses. + + + d37e1371.xml + t0001 + TABLE + table_caption + 14214 + (Continued) Crystal data, X-ray data, and refinement statistics. + + 0.9933119 + evidence + cleaner0 + 2023-06-28T19:16:41Z + DUMMY: + + Crystal data + + + 0.98985547 + evidence + cleaner0 + 2023-06-28T19:16:44Z + DUMMY: + + X-ray data + + + evidence + DUMMY: + cleaner0 + 2023-06-28T19:15:41Z + + refinement statistics + + + + d37e1371.xml + t0001 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/></colgroup><thead><tr><th align="left">Fab</th><th align="center">H3-53:L1-39</th><th align="center">H3-53:L3-11</th><th align="center">H3-53:L3-20</th><th align="center">H3-53:L4-1</th></tr></thead><tbody><tr><td align="left"><italic>PDB indentifier</italic></td><td align="center">5I1E</td><td align="center">5I1G</td><td align="center">5I1H</td><td align="center">5I1I</td></tr><tr><td align="left">Crystal Data</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left">Crystallization Solution</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Buffer, pH</td><td align="center">No buffer</td><td align="center">0.1 M Na Acetate pH 4.5</td><td align="center">0.1 M Na Acetate pH 4.5</td><td align="center">0.1M MES, pH 6.5</td></tr><tr><td align="left"> Precipitant<xref rid="t1fn0005" ref-type="fn"><sup>1</sup></xref></td><td align="center">16% PEG 3350</td><td align="center">25% PEG 3350</td><td align="center">19% PEG 4000</td><td align="center">17% PEG 3350</td></tr><tr><td align="left"> Additive<xref rid="t1fn0005" ref-type="fn"><sup>1</sup></xref></td><td align="center">0.2 M Amm Sulfate 5% Dioxane</td><td align="center">0.2 M Li<sub>2</sub>SO<sub>4</sub></td><td align="center">0.2 M Amm Sulfate</td><td align="center">0.2 M Na Formate, 5% MPD</td></tr><tr><td align="left"> Space Group</td><td align="center">P6<sub>5</sub>22</td><td align="center">P6<sub>5</sub>22</td><td align="center">P6<sub>5</sub>22</td><td align="center">P3<sub>1</sub></td></tr><tr><td align="left"> Molecules/AU</td><td align="center">1</td><td align="center">1</td><td align="center">1</td><td align="center">1</td></tr><tr><td align="left">Unit Cell</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> a(Å)</td><td align="center">89.4</td><td align="center">88.1</td><td align="center">89.4</td><td align="center">68.1</td></tr><tr><td align="left"> b(Å)</td><td align="center">89.4</td><td align="center">88.1</td><td align="center">89.4</td><td align="center">68.1</td></tr><tr><td align="left"> c(Å)</td><td align="center">212.4</td><td align="center">219.6</td><td align="center">211.7</td><td align="center">95.6</td></tr><tr><td align="left"> β(°)</td><td align="center">90</td><td align="center">90</td><td align="center">90</td><td align="center">90</td></tr><tr><td align="left"> γ(°)</td><td align="center">120</td><td align="center">120</td><td align="center">120</td><td align="center">120</td></tr><tr><td align="left"> V<sub>m</sub> (Å<sup>3</sup>/Da)</td><td align="center">2.57</td><td align="center">2.64</td><td align="center">2.57</td><td align="center">2.64</td></tr><tr><td align="left"> Solvent Content (%)</td><td align="center">52</td><td align="center">53</td><td align="center">52</td><td align="center">53</td></tr><tr><td align="left">X-Ray Data<xref rid="t1fn0006" ref-type="fn"><sup>2</sup></xref></td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Resolution (Å)</td><td align="center">30-2.7 (2.8-2.7)</td><td align="center">30-2.3 (2.4-2.3)</td><td align="center">30-2.2 (2.3-2.0)</td><td align="center">30-2.5 (2.6-2.5)</td></tr><tr><td align="left"> Measured Reflections</td><td align="center">297,367 (19,369)</td><td align="center">333,739 (8,008)</td><td align="center">381,125 (1,591)</td><td align="center">137,992 (9,883)</td></tr><tr><td align="left"> Unique Reflections</td><td align="center">14,402 (1,003)</td><td align="center">21,683 (1,135)</td><td align="center">24,323 (964)</td><td align="center">16,727 (1,227)</td></tr><tr><td align="left"> Completeness (%)</td><td align="center">99.6 (96.8)</td><td align="center">93.8 (68.4)</td><td align="center">95.3 (52.0)</td><td align="center">98.6 (98.1)</td></tr><tr><td align="left"> Redundancy</td><td align="center">20.6 (19.3)</td><td align="center">15.4 (7.1)</td><td align="center">15.7 (1.7)</td><td align="center">8.2 (8.1)</td></tr><tr><td align="left"> R<sub>merge</sub></td><td align="center">0.095 (0.451)</td><td align="center">0.057 (0.324)</td><td align="center">0.062 (0.406)</td><td align="center">0.047 (0.445)</td></tr><tr><td align="left"> &lt; I/σ &gt;</td><td align="center">38.3 (8.1)</td><td align="center">36.7 (5.5)</td><td align="center">36.2 (1.6)</td><td align="center">31.6 (5.6)</td></tr><tr><td align="left"> B-factor (Å<sup>2</sup>)</td><td align="center">33.2</td><td align="center">37.3</td><td align="center">33.7</td><td align="center">54.8</td></tr><tr><td align="left">Refinement</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Resolution (Å)</td><td align="center">15-2.7</td><td align="center">15-2.3</td><td align="center">15-2.2</td><td align="center">15-2.5</td></tr><tr><td align="left"> Number of Reflections</td><td align="center">13,583</td><td align="center">20,255</td><td align="center">24,962</td><td align="center">15,811</td></tr><tr><td align="left"> Number of All Atoms</td><td align="center">3,335</td><td align="center">3,271</td><td align="center">3,298</td><td align="center">3,239</td></tr><tr><td align="left"> Number of Waters</td><td align="center">88</td><td align="center">70</td><td align="center">71</td><td align="center">21</td></tr><tr><td align="left"> R-factor (%)</td><td align="center">19.1</td><td align="center">29.8</td><td align="center">22.8</td><td align="center">25.0</td></tr><tr><td align="left"> R-free (%)</td><td align="center">26.4</td><td align="center">38.3</td><td align="center">26.6</td><td align="center">33.7</td></tr><tr><td align="left">RMSD</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Bond Lengths (Å)</td><td align="center">0.008</td><td align="center">0.005</td><td align="center">0.005</td><td align="center">0.006</td></tr><tr><td align="left"> Bond Angles (°)</td><td align="center">1.2</td><td align="center">1.0</td><td align="center">1.0</td><td align="center">1.1</td></tr><tr><td align="left"> Mean B-factor (Å<sup>2</sup>)</td><td align="center">49.1</td><td align="center">46.3</td><td align="center">51.7</td><td align="center">88.9</td></tr><tr><td align="left">Ramachandran Plot (%)</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Outliers</td><td align="center">0.2</td><td align="center">0.2</td><td align="center">0.2</td><td align="center">1.2</td></tr><tr><td align="left"> Favored</td><td align="center">96.7</td><td align="center">97.1</td><td align="center">96.5</td><td align="center">90.9</td></tr></tbody></table> + + 14279 + Fab H3-53:L1-39 H3-53:L3-11 H3-53:L3-20 H3-53:L4-1 PDB indentifier 5I1E 5I1G 5I1H 5I1I Crystal Data         Crystallization Solution          Buffer, pH No buffer 0.1 M Na Acetate pH 4.5 0.1 M Na Acetate pH 4.5 0.1M MES, pH 6.5  Precipitant1 16% PEG 3350 25% PEG 3350 19% PEG 4000 17% PEG 3350  Additive1 0.2 M Amm Sulfate 5% Dioxane 0.2 M Li2SO4 0.2 M Amm Sulfate 0.2 M Na Formate, 5% MPD  Space Group P6522 P6522 P6522 P31  Molecules/AU 1 1 1 1 Unit Cell          a(Å) 89.4 88.1 89.4 68.1  b(Å) 89.4 88.1 89.4 68.1  c(Å) 212.4 219.6 211.7 95.6  β(°) 90 90 90 90  γ(°) 120 120 120 120  Vm (Å3/Da) 2.57 2.64 2.57 2.64  Solvent Content (%) 52 53 52 53 X-Ray Data2          Resolution (Å) 30-2.7 (2.8-2.7) 30-2.3 (2.4-2.3) 30-2.2 (2.3-2.0) 30-2.5 (2.6-2.5)  Measured Reflections 297,367 (19,369) 333,739 (8,008) 381,125 (1,591) 137,992 (9,883)  Unique Reflections 14,402 (1,003) 21,683 (1,135) 24,323 (964) 16,727 (1,227)  Completeness (%) 99.6 (96.8) 93.8 (68.4) 95.3 (52.0) 98.6 (98.1)  Redundancy 20.6 (19.3) 15.4 (7.1) 15.7 (1.7) 8.2 (8.1)  Rmerge 0.095 (0.451) 0.057 (0.324) 0.062 (0.406) 0.047 (0.445)  < I/σ > 38.3 (8.1) 36.7 (5.5) 36.2 (1.6) 31.6 (5.6)  B-factor (Å2) 33.2 37.3 33.7 54.8 Refinement          Resolution (Å) 15-2.7 15-2.3 15-2.2 15-2.5  Number of Reflections 13,583 20,255 24,962 15,811  Number of All Atoms 3,335 3,271 3,298 3,239  Number of Waters 88 70 71 21  R-factor (%) 19.1 29.8 22.8 25.0  R-free (%) 26.4 38.3 26.6 33.7 RMSD          Bond Lengths (Å) 0.008 0.005 0.005 0.006  Bond Angles (°) 1.2 1.0 1.0 1.1  Mean B-factor (Å2) 49.1 46.3 51.7 88.9 Ramachandran Plot (%)          Outliers 0.2 0.2 0.2 1.2  Favored 96.7 97.1 96.5 90.9 + + + d37e1371.xml + t0001 + TABLE + table_footnote + 16142 + Abbreviations: Amm, ammonium; PEG, polyethylene glycol. + + + d37e1371.xml + t0001 + TABLE + table_footnote + 16198 + Values for high-resolution shell are in parentheses. + + + d37e1852.xml + t0001 + TABLE + table_caption + 16251 + (Continued) Crystal data, X-ray data, and refinement statistics. + + 0.9933119 + evidence + cleaner0 + 2023-06-28T19:16:05Z + DUMMY: + + Crystal data + + + 0.98985547 + evidence + cleaner0 + 2023-06-28T19:16:08Z + DUMMY: + + X-ray data + + + evidence + DUMMY: + cleaner0 + 2023-06-28T19:16:20Z + + refinement statistics + + + + d37e1852.xml + t0001 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/></colgroup><thead><tr><th align="left">Fab<hr/></th><th align="center">H5-51:L1-39<hr/></th><th align="center">H5-51:L3-11<hr/></th><th align="center">H5-51:L3-20<hr/></th><th align="center">H5-51:L4-1<hr/></th></tr><tr><th align="left"><italic> PDB identifier</italic></th><th align="center">4KMT</th><th align="center">5I1J</th><th align="center">5I1K</th><th align="center">5I1L</th></tr></thead><tbody><tr><td align="left">Crystal Data</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left">Crystallization Solution</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Buffer, pH</td><td align="center">0.1 M CHES, pH 9.5</td><td align="center">0.1 M Tris, pH 8.5</td><td align="center">0.1 M CHES, pH 9.5</td><td align="center">0.1 M Tris, pH 8.5</td></tr><tr><td align="left"> Precipitant<xref rid="t1fn0007" ref-type="fn"><sup>1</sup></xref></td><td align="center">1.8 M Amm Sulfate</td><td align="center">25% PEG 3350</td><td align="center">1.0 M Amm Sulfate</td><td align="center">24% PEG 3350</td></tr><tr><td align="left"> Additive<xref rid="t1fn0007" ref-type="fn"><sup>1</sup></xref></td><td align="center">5% dioxane</td><td align="center">0.2 M MgCl<sub>2</sub></td><td align="left"> </td><td align="center">0.2 M Amm Sulfate</td></tr><tr><td align="left"> Space Group</td><td align="center">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="center">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="center">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="center">P2<sub>1</sub></td></tr><tr><td align="left"> Molecules/AU</td><td align="center">1</td><td align="center">1</td><td align="center">1</td><td align="center">2</td></tr><tr><td align="left">Unit Cell</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> a(Å)</td><td align="center">63.7</td><td align="center">64.1</td><td align="center">63.8</td><td align="center">106.0</td></tr><tr><td align="left"> b(Å)</td><td align="center">73.8</td><td align="center">73.8</td><td align="center">74.1</td><td align="center">38.0</td></tr><tr><td align="left"> c(Å)</td><td align="center">103.1</td><td align="center">103.0</td><td align="center">103.0</td><td align="center">112.3</td></tr><tr><td align="left"> β(°)</td><td align="center">90</td><td align="center">90</td><td align="center">90</td><td align="center">100.4</td></tr><tr><td align="left"> V<sub>m</sub> (Å<sup>3</sup>/Da)</td><td align="center">2.53</td><td align="center">2.56</td><td align="center">2.54</td><td align="center">2.28</td></tr><tr><td align="left"> Solvent Content (%)</td><td align="center">51</td><td align="center">52</td><td align="center">51</td><td align="center">46</td></tr><tr><td align="left">X-Ray Data<xref rid="t1fn0008" ref-type="fn"><sup>2</sup></xref></td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Resolution (Å)</td><td align="center">30-2.1 (2.2-2.1)</td><td align="center">30-2.5 (2.6-2.5)</td><td align="center">30-1.65 (1.7-1.65)</td><td align="center">30-1.95 (2.0-1.95)</td></tr><tr><td align="left"> Measured Reflections</td><td align="center">131,839 (6,655)</td><td align="center">120,521 (7,988)</td><td align="center">246,750 (4,142)</td><td align="center">320,324 (12,119)</td></tr><tr><td align="left"> Unique Reflections</td><td align="center">27,026 (1,885)</td><td align="center">17,286 (1,236)</td><td align="center">53,058 (2,141)</td><td align="center">61,554 (3,243)</td></tr><tr><td align="left"> Completeness (%)</td><td align="center">93.6 (89.8)</td><td align="center">99.7 (97.3)</td><td align="center">89.8 (49.8)</td><td align="center">94.4 (67.1)</td></tr><tr><td align="left"> Redundancy</td><td align="center">4.9 (3.5)</td><td align="center">7.0 (6.5)</td><td align="center">4.7 (1.9)</td><td align="center">5.2 (3.7)</td></tr><tr><td align="left"> R<sub>merge</sub></td><td align="center">0.079 (0.278)</td><td align="center">0.080 (0.281)</td><td align="center">0.034 (0.131)</td><td align="center">0.060 (0.395)</td></tr><tr><td align="left"> &lt; I/σ &gt;</td><td align="center">16.8 (5.7)</td><td align="center">21.1(6.9)</td><td align="center">27.5 (5.8)</td><td align="center">19.7 (3.1)</td></tr><tr><td align="left"> B-factor (Å<sup>2</sup>)</td><td align="center">26.0</td><td align="center">27.0</td><td align="center">21.6</td><td align="center">31.4</td></tr><tr><td align="left">Refinement</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Resolution (Å)</td><td align="center">15-2.1</td><td align="center">15-2.5</td><td align="center">15-1.65</td><td align="center">15-1.95</td></tr><tr><td align="left"> Number of Reflections</td><td align="center">25,857</td><td align="center">16,328</td><td align="center">51,882</td><td align="center">60,181</td></tr><tr><td align="left"> Number of All Atoms</td><td align="center">3,676</td><td align="center">3,454</td><td align="center">3,814</td><td align="center">7,175</td></tr><tr><td align="left"> Number of Waters</td><td align="center">302</td><td align="center">196</td><td align="center">527</td><td align="center">445</td></tr><tr><td align="left"> R-factor (%)</td><td align="center">17.1</td><td align="center">17.7</td><td align="center">17.2</td><td align="center">19.4</td></tr><tr><td align="left"> R-free (%)</td><td align="center">22.0</td><td align="center">25.8</td><td align="center">19.7</td><td align="center">25.8</td></tr><tr><td align="left">RMSD</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Bond Lengths (Å)</td><td align="center">0.006</td><td align="center">0.009</td><td align="center">0.005</td><td align="center">0.009</td></tr><tr><td align="left"> Bond Angles (°)</td><td align="center">1.0</td><td align="center">1.3</td><td align="center">1.3</td><td align="center">1.3</td></tr><tr><td align="left"> Mean B-factor (Å<sup>2</sup>)</td><td align="center">25.2</td><td align="center">38.2</td><td align="center">20.0</td><td align="center">19.5</td></tr><tr><td align="left">Ramachandran Plot (%)</td><td align="left"> </td><td align="left"> </td><td align="left"> </td><td align="left"> </td></tr><tr><td align="left"> Outliers</td><td align="center">0.0</td><td align="center">0.0</td><td align="center">0.0</td><td align="center">0.0</td></tr><tr><td align="left"> Favored</td><td align="center">98.4</td><td align="center">97.9</td><td align="center">98.1</td><td align="center">98.0</td></tr></tbody></table> + + 16316 + Fab H5-51:L1-39 H5-51:L3-11 H5-51:L3-20 H5-51:L4-1  PDB identifier 4KMT 5I1J 5I1K 5I1L Crystal Data         Crystallization Solution          Buffer, pH 0.1 M CHES, pH 9.5 0.1 M Tris, pH 8.5 0.1 M CHES, pH 9.5 0.1 M Tris, pH 8.5  Precipitant1 1.8 M Amm Sulfate 25% PEG 3350 1.0 M Amm Sulfate 24% PEG 3350  Additive1 5% dioxane 0.2 M MgCl2   0.2 M Amm Sulfate  Space Group P212121 P212121 P212121 P21  Molecules/AU 1 1 1 2 Unit Cell          a(Å) 63.7 64.1 63.8 106.0  b(Å) 73.8 73.8 74.1 38.0  c(Å) 103.1 103.0 103.0 112.3  β(°) 90 90 90 100.4  Vm (Å3/Da) 2.53 2.56 2.54 2.28  Solvent Content (%) 51 52 51 46 X-Ray Data2          Resolution (Å) 30-2.1 (2.2-2.1) 30-2.5 (2.6-2.5) 30-1.65 (1.7-1.65) 30-1.95 (2.0-1.95)  Measured Reflections 131,839 (6,655) 120,521 (7,988) 246,750 (4,142) 320,324 (12,119)  Unique Reflections 27,026 (1,885) 17,286 (1,236) 53,058 (2,141) 61,554 (3,243)  Completeness (%) 93.6 (89.8) 99.7 (97.3) 89.8 (49.8) 94.4 (67.1)  Redundancy 4.9 (3.5) 7.0 (6.5) 4.7 (1.9) 5.2 (3.7)  Rmerge 0.079 (0.278) 0.080 (0.281) 0.034 (0.131) 0.060 (0.395)  < I/σ > 16.8 (5.7) 21.1(6.9) 27.5 (5.8) 19.7 (3.1)  B-factor (Å2) 26.0 27.0 21.6 31.4 Refinement          Resolution (Å) 15-2.1 15-2.5 15-1.65 15-1.95  Number of Reflections 25,857 16,328 51,882 60,181  Number of All Atoms 3,676 3,454 3,814 7,175  Number of Waters 302 196 527 445  R-factor (%) 17.1 17.7 17.2 19.4  R-free (%) 22.0 25.8 19.7 25.8 RMSD          Bond Lengths (Å) 0.006 0.009 0.005 0.009  Bond Angles (°) 1.0 1.3 1.3 1.3  Mean B-factor (Å2) 25.2 38.2 20.0 19.5 Ramachandran Plot (%)          Outliers 0.0 0.0 0.0 0.0  Favored 98.4 97.9 98.1 98.0 + + + d37e1852.xml + t0001 + TABLE + table_footnote + 18144 + Abbreviations: Amm, ammonium; PEG, polyethylene glycol. + + + d37e1852.xml + t0001 + TABLE + table_footnote + 18200 + Values for high-resolution shell are in parentheses. + + + RESULTS + paragraph + 18253 + The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. The Fab heavy and light chain sequences for the variants numbered according to Chothia are shown in Fig. S1. The four different HCs all have the same CDR H3 sequence, ARYDGIYGELDF. Crystallization of the 16 Fabs was previously reported. Three sets of the crystals were isomorphous with nearly identical unit cells (Table 1). These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121. Crystallization conditions for the 3 groups are also similar, but not identical (Table 1). Variations occur in the pH (buffer) and the additives, and, in group 3, PEG 3350 is the precipitant for one variants while ammonium sulfate is the precipitant for the other two. The similarity in the crystal forms is attributed in part to cross-seeding using the microseed matrix screening for groups 2 and 3. + + 0.9973289 + evidence + cleaner0 + 2023-06-28T19:16:52Z + DUMMY: + + crystal structures + + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:07:12Z + + germline library + + + 0.15716633 + structure_element + cleaner0 + 2023-06-28T15:58:46Z + SO: + + Fabs + + + 0.9913346 + structure_element + cleaner0 + 2023-06-28T16:11:32Z + SO: + + HCs + + + mutant + MESH: + cleaner0 + 2023-06-28T21:03:19Z + + H1-69 + + + 0.54475325 + mutant + cleaner0 + 2023-06-28T21:04:17Z + MESH: + + H3-23 + + + 0.56800383 + mutant + cleaner0 + 2023-06-28T21:08:32Z + MESH: + + H3-53 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:05:05Z + + H5-51 + + + 0.99582016 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + 0.78147143 + mutant + cleaner0 + 2023-06-28T21:05:50Z + MESH: + + L1-39 + + + 0.8090814 + mutant + cleaner0 + 2023-06-28T21:06:34Z + MESH: + + L3-11 + + + 0.78590775 + mutant + cleaner0 + 2023-06-28T21:07:15Z + MESH: + + L3-20 + + + 0.4724779 + mutant + cleaner0 + 2023-06-28T21:08:00Z + MESH: + + L4-1 + + + structure_element + SO: + cleaner0 + 2023-06-28T15:59:04Z + + Fab + + + structure_element + SO: + cleaner0 + 2023-06-28T16:13:49Z + + light chain + + + 0.97605056 + structure_element + cleaner0 + 2023-06-28T16:11:32Z + SO: + + HCs + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:14Z + + H3 + + + 0.3840933 + structure_element + cleaner0 + 2023-06-29T08:56:41Z + SO: + + ARYDGIYGELDF + + + 0.7428561 + experimental_method + cleaner0 + 2023-06-29T09:07:21Z + MESH: + + Crystallization + + + 0.16106084 + structure_element + cleaner0 + 2023-06-28T15:58:46Z + SO: + + Fabs + + + 0.990274 + evidence + cleaner0 + 2023-06-28T19:17:00Z + DUMMY: + + crystals + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:12:09Z + + H3-23:L3-11 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:12:33Z + + H3-23:L4-1 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:13:03Z + + H3-53:L1-39 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:13:26Z + + H3-53:L3-11 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:13:46Z + + H3-53:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:10Z + + H5-51:L1-39 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:30Z + + H5-51:L3-11 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:52Z + + H5-51:L3-20 + + + 0.93274474 + chemical + cleaner0 + 2023-06-29T09:24:13Z + CHEBI: + + PEG 3350 + + + 0.9821999 + chemical + cleaner0 + 2023-06-29T09:24:17Z + CHEBI: + + ammonium sulfate + + + 0.94161546 + evidence + cleaner0 + 2023-06-28T19:17:05Z + DUMMY: + + crystal forms + + + 0.979331 + experimental_method + cleaner0 + 2023-06-29T09:07:27Z + MESH: + + microseed matrix screening + + + + RESULTS + paragraph + 19344 + The crystal structures of the 16 Fabs have been determined at resolutions ranging from 3.3 Å to 1.65 Å (Table 1). The number of Fab molecules in the crystallographic asymmetric unit varies from 1 (for 12 Fabs) to 2 (for 4 Fabs). Overall the structures are fairly complete, and, as can be expected, the models for the higher resolution structures are more complete than those for the lower resolution structures (Table S1). Invariably, the HCs have more disorder than the LCs. For the LC, the disorder is observed at 2 of the C-terminal residues with few exceptions. Apart from the C-terminus, only a few surface residues in LC are disordered. + + 0.9972463 + evidence + cleaner0 + 2023-06-28T19:16:53Z + DUMMY: + + crystal structures + + + 0.26109496 + structure_element + cleaner0 + 2023-06-28T15:58:46Z + SO: + + Fabs + + + 0.19023296 + structure_element + cleaner0 + 2023-06-28T15:59:04Z + SO: + + Fab + + + 0.30332693 + structure_element + cleaner0 + 2023-06-28T15:58:46Z + SO: + + Fabs + + + 0.28805926 + structure_element + cleaner0 + 2023-06-28T15:58:46Z + SO: + + Fabs + + + 0.9964563 + evidence + cleaner0 + 2023-06-28T19:17:25Z + DUMMY: + + structures + + + 0.9963349 + evidence + cleaner0 + 2023-06-28T19:17:28Z + DUMMY: + + structures + + + 0.996055 + evidence + cleaner0 + 2023-06-28T19:17:31Z + DUMMY: + + structures + + + 0.9984939 + structure_element + cleaner0 + 2023-06-28T16:11:32Z + SO: + + HCs + + + 0.92722005 + protein_state + cleaner0 + 2023-06-29T09:27:42Z + DUMMY: + + disorder + + + 0.9984347 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + 0.9984855 + structure_element + cleaner0 + 2023-06-28T16:24:55Z + SO: + + LC + + + 0.8974196 + protein_state + cleaner0 + 2023-06-29T09:27:45Z + DUMMY: + + disorder + + + 0.9983936 + structure_element + cleaner0 + 2023-06-28T16:24:55Z + SO: + + LC + + + 0.9923855 + protein_state + cleaner0 + 2023-06-29T09:27:48Z + DUMMY: + + disordered + + + + RESULTS + paragraph + 19993 + The HCs feature the largest number of disordered residues, with the lower resolution structures having the most. The C-terminal residues including the 6xHis tags are disordered in all 16 structures. In addition to these, 2 primary disordered stretches of residues are observed in a number of structures (Table S1). One involves the loop connecting the first 2 β-strands of the constant domain (in all Fabs except H3-23:L1-39, H3-23:L3-11 and H3-53:L1-39). The other is located in CDR H3 (in H5-51:L3-11, H5-51:L3-20 and in one of 2 copies of H3-23:L4-1). CDR H1 and CDR H2 also show some degree of disorder, but to a lesser extent. + + 0.9985374 + structure_element + cleaner0 + 2023-06-28T16:11:32Z + SO: + + HCs + + + 0.9899644 + protein_state + cleaner0 + 2023-06-29T09:27:51Z + DUMMY: + + disordered + + + 0.99663144 + evidence + cleaner0 + 2023-06-28T19:17:33Z + DUMMY: + + structures + + + 0.99440306 + protein_state + cleaner0 + 2023-06-29T09:27:55Z + DUMMY: + + disordered + + + 0.99707234 + evidence + cleaner0 + 2023-06-28T19:17:36Z + DUMMY: + + structures + + + 0.9967458 + evidence + cleaner0 + 2023-06-28T19:17:39Z + DUMMY: + + structures + + + 0.97793114 + structure_element + cleaner0 + 2023-06-29T08:56:00Z + SO: + + loop + + + 0.9568026 + structure_element + cleaner0 + 2023-06-29T08:56:03Z + SO: + + β-strands + + + 0.9960027 + structure_element + cleaner0 + 2023-06-29T08:56:05Z + SO: + + constant domain + + + 0.46980876 + structure_element + cleaner0 + 2023-06-28T15:58:46Z + SO: + + Fabs + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:15:27Z + + H3-23:L1-39 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:12:10Z + + H3-23:L3-11 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:13:04Z + + H3-53:L1-39 + + + 0.9980945 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.8540226 + structure_element + cleaner0 + 2023-06-28T19:05:15Z + SO: + + H3 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:32Z + + H5-51:L3-11 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:53Z + + H5-51:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:12:34Z + + H3-23:L4-1 + + + 0.99790764 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.97477245 + structure_element + cleaner0 + 2023-06-28T19:04:10Z + SO: + + H1 + + + 0.99792707 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.9856117 + structure_element + cleaner0 + 2023-06-28T16:26:21Z + SO: + + H2 + + + 0.9067931 + protein_state + cleaner0 + 2023-06-29T09:27:58Z + DUMMY: + + disorder + + + + RESULTS + title_2 + 20628 + CDR canonical structures + + 0.9833983 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + evidence + DUMMY: + cleaner0 + 2023-06-28T20:50:47Z + + structures + + + + RESULTS + paragraph + 20653 + Several CDR definitions have evolved over decades of antibody research. Depending on the focus of the study, the CDR boundaries differ slightly between various definitions. In this work, we use the CDR definition of North et al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr. + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + 0.9945135 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.9963715 + structure_element + cleaner0 + 2023-06-28T19:04:10Z + SO: + + H1 + + + 0.9967776 + structure_element + cleaner0 + 2023-06-28T19:05:15Z + SO: + + H3 + + + 0.9913113 + residue_name + cleaner0 + 2023-06-29T09:11:56Z + SO: + + Cys + + + 0.996126 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.99719477 + structure_element + cleaner0 + 2023-06-28T20:09:53Z + SO: + + L2 + + + 0.9898846 + residue_name + cleaner0 + 2023-06-29T09:11:59Z + SO: + + Tyr + + + + RESULTS + title_2 + 21085 + CDR H1 + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:04:11Z + + H1 + + + + kmab-08-06-1190060-g001.jpg + f0001 + FIG + fig_caption + 21092 + The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. + + 0.99592435 + experimental_method + cleaner0 + 2023-06-29T09:07:33Z + MESH: + + superposition + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:04:11Z + + H1 + + + 0.9562733 + complex_assembly + cleaner0 + 2023-06-29T09:23:30Z + GO: + + HC:LC + + + structure_element + SO: + cleaner0 + 2023-06-28T15:56:24Z + + heavy chains + + + 0.89844674 + mutant + cleaner0 + 2023-06-28T21:03:19Z + MESH: + + H1-69 + + + 0.9519102 + mutant + cleaner0 + 2023-06-28T21:04:17Z + MESH: + + H3-23 + + + 0.91820097 + mutant + cleaner0 + 2023-06-28T21:08:32Z + MESH: + + H3-53 + + + 0.93086475 + mutant + cleaner0 + 2023-06-28T21:05:05Z + MESH: + + H5-51 + + + + t0002.xml + t0002 + TABLE + table_caption + 21216 + Canonical structures. + + + t0002.xml + t0002 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/></colgroup><thead><tr><th align="left">Pairs</th><th align="center">PDB</th><th align="center">CDR H1</th><th align="center">CDR H2</th><th align="center">CDR H3</th></tr></thead><tbody><tr><td align="left"><bold>H1-69</bold></td><td align="left"> </td><td align="left">KASGGTFSSYAIS</td><td align="left">GIIPIFGTAN</td><td align="left">ARYDGIYGELDF</td></tr><tr><td align="left"><bold>H1-69</bold>:L1-39</td><td align="center">5I15</td><td align="left">H1-13-4</td><td align="left">H2-10-1</td><td align="left">H3-12-1</td></tr><tr><td align="left"><bold>H1-69</bold>:L3-11</td><td align="center">5I16</td><td align="left">H1-13-1/H1-13-1</td><td align="left">H2-10-1/H2-10-1</td><td align="left">H3-12-1/H3-12-1</td></tr><tr><td align="left"><bold>H1-69</bold>:L3-20</td><td align="center">5I17</td><td align="left">H1-13-3/H1-13-6</td><td align="left">H2-10-1/NA</td><td align="left">H3-12-1/H3-12-1</td></tr><tr><td align="left"><bold>H1-69</bold>:L4-1</td><td align="center">5I18</td><td align="left">H1-13-10</td><td align="left">H2-10-1</td><td align="left">H3-12-1</td></tr><tr><td align="left"><bold>H3-23</bold></td><td align="left"> </td><td align="left">AASGFTFSSYAMS</td><td align="left">AISGSGGSTY</td><td align="left">AKYDGIYDGIYGELDF</td></tr><tr><td align="left"><bold>H3-23</bold>:L1-39</td><td align="center">5I19</td><td align="left">H1-13-1</td><td align="left">H2-10-2</td><td align="left">H3-12-1</td></tr><tr><td align="left"><bold>H3-23</bold>:L3-11</td><td align="center">5I1A</td><td align="left">H1-13-1/H1-13-1</td><td align="left">H2-10-2/H2-10-2</td><td align="left">H3-12-1/H3-12-1</td></tr><tr><td align="left"><bold>H3-23</bold>:L3-20</td><td align="center">5I1C</td><td align="left">H1-13-1</td><td align="left">H2-10-2</td><td align="left">H3-12-1</td></tr><tr><td align="left"><bold>H3-23</bold>:L4-1</td><td align="center">5I1D</td><td align="left">H1-13-1/H1-13-1</td><td align="left">H2-10-2/H2-10-2</td><td align="left">H3-12-1/NA</td></tr><tr><td align="left"><bold>H3-53</bold></td><td align="left"> </td><td align="left">AASGFTVSSNYMS</td><td align="left">VIYSGGSTY</td><td align="left">ARYDGIYGELDF</td></tr><tr><td align="left"><bold>H3-53</bold>:L1-39</td><td align="center">5I1E</td><td align="left">H1-13-1</td><td align="left">H2-9-3</td><td align="left">H3-12-1</td></tr><tr><td align="left"><bold>H3-53</bold>:L3-11</td><td align="center">5I1G</td><td align="left">H1-13-1</td><td align="left">H2-9-3</td><td align="left">H3-12-1</td></tr><tr><td align="left"><bold>H3-53</bold>:L3-20</td><td align="center">5I1H</td><td align="left">H1-13-1</td><td align="left">H2-9-3</td><td align="left">H3-12-1</td></tr><tr><td align="left"><bold>H3-53</bold>:L4-1</td><td align="center">5I1I</td><td align="left">H1-13-1</td><td align="left">H2-9-3</td><td align="left">NA</td></tr><tr><td align="left"><bold>H5-51</bold></td><td align="left"> </td><td align="left">KGSGYSFTSYWIG</td><td align="left">IIYPGDSDTR</td><td align="left">ARYDGIYGELDF</td></tr><tr><td align="left"><bold>H5-51</bold>:L1-39</td><td align="center">4KMT</td><td align="left">H1-13-1</td><td align="left">H2-10-1</td><td align="left">H3-12-1</td></tr><tr><td align="left"><bold>H5-51</bold>:L3-11</td><td align="center">5I1J</td><td align="left">H1-13-1</td><td align="left">H2-10-1</td><td align="left">NA</td></tr><tr><td align="left"><bold>H5-51</bold>:L3-20</td><td align="center">5I1K</td><td align="left">H1-13-1</td><td align="left">H2-10-1</td><td align="left">NA</td></tr><tr><td align="left"><bold>H5-51</bold>:L4-1</td><td align="center">5I1L</td><td align="left">H1-13-1/H1-13-1</td><td align="left">H2-10-1/H2-10-1</td><td align="left">H3-12-1/H3-12-1</td></tr><tr><td align="left"> </td><td align="left"> </td><td align="left"><bold>CDR L1</bold></td><td align="left"><bold>CDR L2</bold></td><td align="left"><bold>CDR L3</bold></td></tr><tr><td align="left"><bold>L1-39</bold></td><td align="left"> </td><td align="left">RASQSISSYLN</td><td align="left">YAASSLQS</td><td align="left">QQSYSTPLT</td></tr><tr><td align="left">H1-69:<bold>L1-39</bold></td><td align="center">5I15</td><td align="left">L1-11-1</td><td align="left">L2-8-1</td><td align="left">L3-9-cis7-1</td></tr><tr><td align="left">H3-23:<bold>L1-39</bold></td><td align="center">5I19</td><td align="left">L1-11-1</td><td align="left">L2-8-1</td><td align="left">L3-9-cis7-1</td></tr><tr><td align="left">H3-53:<bold>L1-39</bold></td><td align="center">5I1E</td><td align="left">L1-11-1</td><td align="left">L2-8-1</td><td align="left">L3-9-cis7-1</td></tr><tr><td align="left">H5-51:<bold>L1-39</bold></td><td align="center">4KMT</td><td align="left">L1-11-1</td><td align="left">L2-8-1</td><td align="left">L3-9-cis7-1</td></tr><tr><td align="left"><bold>L3-11</bold></td><td align="left"> </td><td align="left">RASQSVSSYLA</td><td align="left">YDASNRAT</td><td align="left">QQRSNWPLT</td></tr><tr><td align="left">H1-69:<bold>L3-11</bold></td><td align="center">5I16</td><td align="left">L1-11-1/L1-11-1</td><td align="left">L2-8-1/L2-8-1</td><td align="left">L3-9-cis7-1/L3-9-cis7-1</td></tr><tr><td align="left">H3-23:<bold>L3-11</bold></td><td align="center">5I1A</td><td align="left">L1-11-1/L1-11-1</td><td align="left">L2-8-1/L2-8-1</td><td align="left">L3-9-cis7-1/L3-9-cis7-1</td></tr><tr><td align="left">H3-53:<bold>L3-11</bold></td><td align="center">5I1G</td><td align="left">L1-11-1</td><td align="left">L2-8-1</td><td align="left">L3-9-cis7-1</td></tr><tr><td align="left">H5-51:<bold>L3-11</bold></td><td align="center">5I1J</td><td align="left">L1-11-1</td><td align="left">L2-8-1</td><td align="left">L3-9-cis7-1</td></tr><tr><td align="left"><bold>L3-20</bold></td><td align="left"> </td><td align="left">RASQSVSSSYLA</td><td align="left">YGASSRAT</td><td align="left">QQYGSSPLT</td></tr><tr><td align="left">H1-69:<bold>L3-20</bold></td><td align="center">5I17</td><td align="left">L1-12-2/L1-12-1</td><td align="left">L2-8-1/L2-8-1</td><td align="left">L3-9-cis7-1/L3-9-cis7-1</td></tr><tr><td align="left">H3-23:<bold>L3-20</bold></td><td align="center">5I1C</td><td align="left">L1-12-2</td><td align="left">L2-8-1</td><td align="left">L3-9-cis7-1</td></tr><tr><td align="left">H3-53:<bold>L3-20</bold></td><td align="center">5I1H</td><td align="left">L1-12-1</td><td align="left">L2-8-1</td><td align="left">L3-9-cis7-1</td></tr><tr><td align="left">H5-51:<bold>L3-20</bold></td><td align="center">5I1K</td><td align="left">L1-12-1</td><td align="left">L2-8-1</td><td align="left">L3-9-cis7-1</td></tr><tr><td align="left"><bold>L4-1</bold></td><td align="left"> </td><td align="left">KSSQSVLYSSNNKNYLA</td><td align="left">YWASTRES</td><td align="left">QQYYSTPLT</td></tr><tr><td align="left">H1-69:<bold>L4-1</bold></td><td align="center">5I18</td><td align="left">L1-17-1</td><td align="left">L2-8-1</td><td align="left">L3-9-cis7-1</td></tr><tr><td align="left">H3-23:<bold>L4-1</bold></td><td align="center">5I1D</td><td align="left">L1-17-1/L1-17-1</td><td align="left">L2-8-1/L2-8-1</td><td align="left">L3-9-cis7-1/L3-9-cis7-1</td></tr><tr><td align="left">H3-53:<bold>L4-1</bold></td><td align="center">5I1I</td><td align="left">L1-17-1</td><td align="left">L2-8-1</td><td align="left">L3-9-cis7-1</td></tr><tr><td align="left">H5-51:<bold>L4-1</bold></td><td align="center">5I1L</td><td align="left">L1-17-1/L1-17-1</td><td align="left">L2-8-1/L2-8-1</td><td align="left">L3-9-cis7-1/L3-9-cis7-1</td></tr></tbody></table> + + 21238 + Pairs PDB CDR H1 CDR H2 CDR H3 H1-69   KASGGTFSSYAIS GIIPIFGTAN ARYDGIYGELDF H1-69:L1-39 5I15 H1-13-4 H2-10-1 H3-12-1 H1-69:L3-11 5I16 H1-13-1/H1-13-1 H2-10-1/H2-10-1 H3-12-1/H3-12-1 H1-69:L3-20 5I17 H1-13-3/H1-13-6 H2-10-1/NA H3-12-1/H3-12-1 H1-69:L4-1 5I18 H1-13-10 H2-10-1 H3-12-1 H3-23   AASGFTFSSYAMS AISGSGGSTY AKYDGIYDGIYGELDF H3-23:L1-39 5I19 H1-13-1 H2-10-2 H3-12-1 H3-23:L3-11 5I1A H1-13-1/H1-13-1 H2-10-2/H2-10-2 H3-12-1/H3-12-1 H3-23:L3-20 5I1C H1-13-1 H2-10-2 H3-12-1 H3-23:L4-1 5I1D H1-13-1/H1-13-1 H2-10-2/H2-10-2 H3-12-1/NA H3-53   AASGFTVSSNYMS VIYSGGSTY ARYDGIYGELDF H3-53:L1-39 5I1E H1-13-1 H2-9-3 H3-12-1 H3-53:L3-11 5I1G H1-13-1 H2-9-3 H3-12-1 H3-53:L3-20 5I1H H1-13-1 H2-9-3 H3-12-1 H3-53:L4-1 5I1I H1-13-1 H2-9-3 NA H5-51   KGSGYSFTSYWIG IIYPGDSDTR ARYDGIYGELDF H5-51:L1-39 4KMT H1-13-1 H2-10-1 H3-12-1 H5-51:L3-11 5I1J H1-13-1 H2-10-1 NA H5-51:L3-20 5I1K H1-13-1 H2-10-1 NA H5-51:L4-1 5I1L H1-13-1/H1-13-1 H2-10-1/H2-10-1 H3-12-1/H3-12-1     CDR L1 CDR L2 CDR L3 L1-39   RASQSISSYLN YAASSLQS QQSYSTPLT H1-69:L1-39 5I15 L1-11-1 L2-8-1 L3-9-cis7-1 H3-23:L1-39 5I19 L1-11-1 L2-8-1 L3-9-cis7-1 H3-53:L1-39 5I1E L1-11-1 L2-8-1 L3-9-cis7-1 H5-51:L1-39 4KMT L1-11-1 L2-8-1 L3-9-cis7-1 L3-11   RASQSVSSYLA YDASNRAT QQRSNWPLT H1-69:L3-11 5I16 L1-11-1/L1-11-1 L2-8-1/L2-8-1 L3-9-cis7-1/L3-9-cis7-1 H3-23:L3-11 5I1A L1-11-1/L1-11-1 L2-8-1/L2-8-1 L3-9-cis7-1/L3-9-cis7-1 H3-53:L3-11 5I1G L1-11-1 L2-8-1 L3-9-cis7-1 H5-51:L3-11 5I1J L1-11-1 L2-8-1 L3-9-cis7-1 L3-20   RASQSVSSSYLA YGASSRAT QQYGSSPLT H1-69:L3-20 5I17 L1-12-2/L1-12-1 L2-8-1/L2-8-1 L3-9-cis7-1/L3-9-cis7-1 H3-23:L3-20 5I1C L1-12-2 L2-8-1 L3-9-cis7-1 H3-53:L3-20 5I1H L1-12-1 L2-8-1 L3-9-cis7-1 H5-51:L3-20 5I1K L1-12-1 L2-8-1 L3-9-cis7-1 L4-1   KSSQSVLYSSNNKNYLA YWASTRES QQYYSTPLT H1-69:L4-1 5I18 L1-17-1 L2-8-1 L3-9-cis7-1 H3-23:L4-1 5I1D L1-17-1/L1-17-1 L2-8-1/L2-8-1 L3-9-cis7-1/L3-9-cis7-1 H3-53:L4-1 5I1I L1-17-1 L2-8-1 L3-9-cis7-1 H5-51:L4-1 5I1L L1-17-1/L1-17-1 L2-8-1/L2-8-1 L3-9-cis7-1/L3-9-cis7-1 + + + t0002.xml + t0002 + TABLE + table_footnote + 23317 + CDRs are defined using the Dunbrack convention [12]. Assignments for 2 copies of the Fab in the asymmetric unit are given for 5 structures. No assignment (NA) for CDRs with missing residues. + + 0.9826041 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.34516945 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.9959 + evidence + cleaner0 + 2023-06-28T20:07:21Z + DUMMY: + + structures + + + 0.97968894 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + + RESULTS + paragraph + 23508 + The four HCs feature CDR H1 of the same length, and their sequences are highly similar (Table 2). The CDR H1 backbone conformations for all variants for each of the HCs are shown in Fig. 1. Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig. 1B-D). Some deviation is observed for H3-53, mostly due to H3-53:L4-1, which exhibits a significant degree of disorder in CDR H1. The electron density for the backbone is weak and discontinuous, and completely missing for several side chains. + + 0.99611866 + structure_element + cleaner0 + 2023-06-28T16:11:33Z + SO: + + HCs + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:04:11Z + + H1 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:04:11Z + + H1 + + + 0.9957355 + structure_element + cleaner0 + 2023-06-28T16:11:33Z + SO: + + HCs + + + 0.9940059 + structure_element + cleaner0 + 2023-06-28T16:11:33Z + SO: + + HCs + + + 0.83604187 + mutant + cleaner0 + 2023-06-28T21:04:17Z + MESH: + + H3-23 + + + 0.8691897 + mutant + cleaner0 + 2023-06-28T21:08:32Z + MESH: + + H3-53 + + + 0.8163323 + mutant + cleaner0 + 2023-06-28T21:05:05Z + MESH: + + H5-51 + + + 0.6929795 + mutant + cleaner0 + 2023-06-28T21:17:48Z + MESH: + + H1-13-1 + + + 0.22198454 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.9957462 + evidence + cleaner0 + 2023-06-28T20:07:16Z + DUMMY: + + structures + + + 0.9691633 + evidence + cleaner0 + 2023-06-28T20:07:24Z + DUMMY: + + rmsd values + + + 0.8695476 + mutant + cleaner0 + 2023-06-28T21:08:32Z + MESH: + + H3-53 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:18:19Z + + H3-53:L4-1 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:04:11Z + + H1 + + + 0.9966054 + evidence + cleaner0 + 2023-06-28T20:07:28Z + DUMMY: + + electron density + + + + RESULTS + paragraph + 24155 + The CDR H1 structures with H1-69 shown in Fig. 1A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20. In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10. A major difference of H1-69 from the other germlines in the experimental data set is the presence of Gly instead of Phe or Tyr at position 27 (residue 5 of 13 in CDR H1). Glycine introduces the possibility of a higher degree of conformational flexibility that undoubtedly translates to the differences observed, and contributes to the elevated thermal parameters for the atoms in the amino acid residues in this region. + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:04:11Z + + H1 + + + 0.99516606 + evidence + cleaner0 + 2023-06-28T20:07:31Z + DUMMY: + + structures + + + mutant + MESH: + cleaner0 + 2023-06-28T21:03:19Z + + H1-69 + + + 0.99432063 + evidence + cleaner0 + 2023-06-28T20:07:34Z + DUMMY: + + structures + + + 0.99687505 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + 0.21805374 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:18:58Z + + H1-69:L3-11 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:20Z + + H1-69:L3-20 + + + 0.36521375 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.99359375 + evidence + cleaner0 + 2023-06-28T20:07:37Z + DUMMY: + + structures + + + 0.982238 + evidence + cleaner0 + 2023-06-28T20:07:40Z + DUMMY: + + structures + + + 0.97110045 + mutant + cleaner0 + 2023-06-29T09:25:57Z + MESH: + + H1-13-1 + + + 0.97449195 + mutant + cleaner0 + 2023-06-29T09:26:01Z + MESH: + + H1-13-3 + + + 0.9718064 + mutant + cleaner0 + 2023-06-29T09:26:04Z + MESH: + + H1-13-4 + + + 0.96956253 + mutant + cleaner0 + 2023-06-29T09:26:07Z + MESH: + + H1-13-6 + + + 0.9710226 + mutant + cleaner0 + 2023-06-29T09:26:10Z + MESH: + + H1-13-10 + + + 0.78516006 + mutant + cleaner0 + 2023-06-28T21:03:20Z + MESH: + + H1-69 + + + 0.9891431 + residue_name + cleaner0 + 2023-06-29T09:12:05Z + SO: + + Gly + + + 0.9896833 + residue_name + cleaner0 + 2023-06-29T09:12:08Z + SO: + + Phe + + + 0.9894337 + residue_name + cleaner0 + 2023-06-29T09:12:10Z + SO: + + Tyr + + + 0.956861 + residue_number + cleaner0 + 2023-06-29T09:19:14Z + DUMMY: + + 27 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:04:11Z + + H1 + + + 0.9914665 + residue_name + cleaner0 + 2023-06-29T09:12:14Z + SO: + + Glycine + + + + RESULTS + title_2 + 24914 + CDR H2 + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T16:26:21Z + + H2 + + + + kmab-08-06-1190060-g002.jpg + f0002 + FIG + fig_caption + 24921 + The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. + + 0.99599445 + experimental_method + cleaner0 + 2023-06-29T09:07:39Z + MESH: + + superposition + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T16:26:21Z + + H2 + + + 0.9441673 + complex_assembly + cleaner0 + 2023-06-29T09:23:36Z + GO: + + HC:LC + + + structure_element + SO: + cleaner0 + 2023-06-28T15:56:24Z + + heavy chains + + + 0.91638017 + mutant + cleaner0 + 2023-06-28T21:03:20Z + MESH: + + H1-69 + + + 0.94655305 + mutant + cleaner0 + 2023-06-28T21:04:17Z + MESH: + + H3-23 + + + 0.9128931 + mutant + cleaner0 + 2023-06-28T21:08:32Z + MESH: + + H3-53 + + + 0.91781205 + mutant + cleaner0 + 2023-06-28T21:05:05Z + MESH: + + H5-51 + + + + RESULTS + paragraph + 25045 + The canonical structures of CDR H2 have fairly consistent conformations (Table 2, Fig. 2). Each of the 4 HCs adopts only one canonical structure regardless of the pairing LC. Germlines H1-69 and H5-51 have the same canonical structure assignment H2-10-1, H3-23 has H2-10-2, and H3-53 has H2-9-3. The conformations for all of these CDR H2s are tightly clustered (Fig. 2). In one case, in the second Fab of H1-69:L3-20, CDR H2 is partially disordered (Δ55-60). + + 0.9974942 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.92632896 + structure_element + cleaner0 + 2023-06-28T16:26:21Z + SO: + + H2 + + + 0.9952141 + structure_element + cleaner0 + 2023-06-28T16:11:33Z + SO: + + HCs + + + 0.8810361 + structure_element + cleaner0 + 2023-06-28T16:24:55Z + SO: + + LC + + + 0.69402623 + mutant + cleaner0 + 2023-06-28T21:03:20Z + MESH: + + H1-69 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:05:05Z + + H5-51 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:22:12Z + + H2-10-1 + + + 0.70856994 + mutant + cleaner0 + 2023-06-28T21:04:17Z + MESH: + + H3-23 + + + 0.70212114 + mutant + cleaner0 + 2023-06-28T21:22:37Z + MESH: + + H2-10-2 + + + 0.7003109 + mutant + cleaner0 + 2023-06-28T21:08:32Z + MESH: + + H3-53 + + + 0.79651135 + mutant + cleaner0 + 2023-06-28T21:22:52Z + MESH: + + H2-9-3 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:21Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T21:23:32Z + + H2s + + + 0.27054203 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:21Z + + H1-69:L3-20 + + + 0.9976173 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.96096814 + structure_element + cleaner0 + 2023-06-28T16:26:21Z + SO: + + H2 + + + 0.70799065 + protein_state + cleaner0 + 2023-06-29T09:29:04Z + DUMMY: + + partially disordered + + + mutant + MESH: + cleaner0 + 2023-06-28T21:24:07Z + + Δ55-60 + + + + RESULTS + paragraph + 25510 + Although three of the germlines have CDR H2 of the same length, 10 residues, they adopt 2 distinctively different conformations depending mostly on the residue at position 71 from the so-called CDR H4. Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site. Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. + + 0.99742055 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.81681776 + structure_element + cleaner0 + 2023-06-28T16:26:21Z + SO: + + H2 + + + 0.62872905 + residue_range + cleaner0 + 2023-06-29T09:14:33Z + DUMMY: + + 10 residues + + + 0.9469733 + residue_number + cleaner0 + 2023-06-29T09:19:32Z + DUMMY: + + 71 + + + 0.9968274 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.9557131 + structure_element + cleaner0 + 2023-06-29T08:58:17Z + SO: + + H4 + + + 0.99837995 + residue_name_number + cleaner0 + 2023-06-29T09:21:11Z + DUMMY: + + Arg71 + + + 0.85665005 + mutant + cleaner0 + 2023-06-28T21:04:17Z + MESH: + + H3-23 + + + 0.9966505 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.99316376 + structure_element + cleaner0 + 2023-06-28T16:26:21Z + SO: + + H2 + + + 0.9950023 + structure_element + cleaner0 + 2023-06-29T08:58:20Z + SO: + + H4 + + + 0.9970299 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.92691565 + structure_element + cleaner0 + 2023-06-28T16:26:21Z + SO: + + H2 + + + 0.94479257 + residue_number + cleaner0 + 2023-06-29T09:19:37Z + DUMMY: + + 54 + + + 0.9975385 + site + cleaner0 + 2023-06-29T09:10:35Z + SO: + + antigen binding site + + + mutant + MESH: + cleaner0 + 2023-06-28T21:03:20Z + + H1-69 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:05:05Z + + H5-51 + + + 0.9952029 + species + cleaner0 + 2023-06-28T16:03:59Z + MESH: + + human + + + 0.98539925 + residue_name + cleaner0 + 2023-06-29T09:12:19Z + SO: + + Ala + + + 0.9392848 + residue_number + cleaner0 + 2023-06-29T09:19:40Z + DUMMY: + + 71 + + + structure_element + SO: + cleaner0 + 2023-06-28T21:25:30Z + + H + + + residue_name_number + DUMMY: + cleaner0 + 2023-06-28T21:25:20Z + + Pro52a + + + 0.9970763 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.9823167 + structure_element + cleaner0 + 2023-06-29T08:58:24Z + SO: + + H4 + + + 0.91533685 + residue_number + cleaner0 + 2023-06-29T09:19:43Z + DUMMY: + + 53 + + + 0.9325063 + residue_number + cleaner0 + 2023-06-29T09:19:46Z + DUMMY: + + 54 + + + + RESULTS + paragraph + 26116 + Conformations of CDR H2 in H1-69 and H5-51, both of which have canonical structure H2-10-1, show little deviation within each set of 4 structures. However, there is a significant shift of the CDR as a rigid body when the 2 sets are superimposed. Most likely this is the result of interaction of CDR H2 with CDR H1, namely with the residue at position 33 (residue 11 of 13 in CDR H1). Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center. + + 0.995761 + structure_element + cleaner0 + 2023-06-28T16:25:21Z + SO: + + CDR + + + 0.66203463 + structure_element + cleaner0 + 2023-06-28T16:26:21Z + SO: + + H2 + + + 0.6642932 + mutant + cleaner0 + 2023-06-28T21:03:20Z + MESH: + + H1-69 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:05:05Z + + H5-51 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:26:12Z + + H2-10-1 + + + 0.99680835 + evidence + cleaner0 + 2023-06-28T20:08:11Z + DUMMY: + + structures + + + 0.9964696 + structure_element + cleaner0 + 2023-06-28T16:25:22Z + SO: + + CDR + + + 0.9532751 + experimental_method + cleaner0 + 2023-06-28T20:08:15Z + MESH: + + superimposed + + + 0.9950335 + structure_element + cleaner0 + 2023-06-28T16:25:22Z + SO: + + CDR + + + 0.615978 + structure_element + cleaner0 + 2023-06-28T16:26:21Z + SO: + + H2 + + + 0.99527454 + structure_element + cleaner0 + 2023-06-28T16:25:22Z + SO: + + CDR + + + 0.874357 + structure_element + cleaner0 + 2023-06-28T19:04:11Z + SO: + + H1 + + + 0.9602763 + residue_number + cleaner0 + 2023-06-29T09:19:49Z + DUMMY: + + 33 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:04:11Z + + H1 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:03:20Z + + H1-69 + + + 0.9874812 + residue_name + cleaner0 + 2023-06-29T09:12:25Z + SO: + + Ala + + + 0.9624215 + residue_number + cleaner0 + 2023-06-29T09:19:54Z + DUMMY: + + 33 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:05:05Z + + H5-51 + + + 0.9566874 + residue_number + cleaner0 + 2023-06-29T09:19:56Z + DUMMY: + + 33 + + + 0.98665047 + residue_name + cleaner0 + 2023-06-29T09:12:28Z + SO: + + Trp + + + structure_element + SO: + cleaner0 + 2023-06-28T21:26:43Z + + H + + + 0.98908526 + residue_name_number + cleaner0 + 2023-06-29T09:21:15Z + DUMMY: + + Tyr52 + + + 0.99542576 + structure_element + cleaner0 + 2023-06-28T16:25:22Z + SO: + + CDR + + + 0.57297033 + structure_element + cleaner0 + 2023-06-28T16:26:21Z + SO: + + H2 + + + + RESULTS + title_2 + 26664 + CDR L1 + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T16:26:30Z + + L1 + + + + kmab-08-06-1190060-g003.jpg + f0003 + FIG + fig_caption + 26671 + The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. + + 0.99590635 + experimental_method + cleaner0 + 2023-06-29T09:07:44Z + MESH: + + superposition + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T16:26:30Z + + L1 + + + 0.951538 + complex_assembly + cleaner0 + 2023-06-29T09:23:41Z + GO: + + HC:LC + + + structure_element + SO: + cleaner0 + 2023-06-28T16:14:08Z + + light chains + + + 0.7258582 + mutant + cleaner0 + 2023-06-28T21:05:50Z + MESH: + + L1-39 + + + 0.78686476 + mutant + cleaner0 + 2023-06-28T21:06:35Z + MESH: + + L3-11 + + + 0.8088053 + mutant + cleaner0 + 2023-06-28T21:07:16Z + MESH: + + L3-20 + + + 0.78025514 + mutant + cleaner0 + 2023-06-28T21:08:00Z + MESH: + + L4-1 + + + + RESULTS + paragraph + 26794 + The four LC CDRs L1 feature 3 different lengths (11, 12 and 17 residues) having a total of 4 different canonical structure assignments. Of these LCs, L1-39 and L3-11 have the same canonical structure, L1-11-1, and superimpose very well (Fig. 3A, B). For the remaining 2, L3-20 has 2 different assignments, L1-12-1 and L1-12-2, while L4-1 has a single assignment, L1-17-1. + + 0.8353525 + structure_element + cleaner0 + 2023-06-28T16:24:55Z + SO: + + LC + + + 0.99589455 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.9921481 + structure_element + cleaner0 + 2023-06-28T16:26:30Z + SO: + + L1 + + + 0.7752541 + residue_range + cleaner0 + 2023-06-29T09:14:46Z + DUMMY: + + 11 + + + 0.60394007 + residue_range + cleaner0 + 2023-06-29T09:14:49Z + DUMMY: + + 12 + + + 0.53114396 + residue_range + cleaner0 + 2023-06-29T09:14:52Z + DUMMY: + + 17 + + + 0.84663665 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + 0.86617535 + mutant + cleaner0 + 2023-06-28T21:05:50Z + MESH: + + L1-39 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:06:35Z + + L3-11 + + + 0.73787403 + mutant + cleaner0 + 2023-06-28T21:28:31Z + MESH: + + L1-11-1 + + + 0.992374 + experimental_method + cleaner0 + 2023-06-29T09:07:49Z + MESH: + + superimpose + + + mutant + MESH: + cleaner0 + 2023-06-28T21:07:16Z + + L3-20 + + + 0.91584176 + mutant + cleaner0 + 2023-06-28T21:28:41Z + MESH: + + L1-12-1 + + + 0.8342571 + mutant + cleaner0 + 2023-06-28T21:28:51Z + MESH: + + L1-12-2 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:08:00Z + + L4-1 + + + 0.9382731 + mutant + cleaner0 + 2023-06-28T21:29:01Z + MESH: + + L1-17-1 + + + + RESULTS + paragraph + 27169 + L4-1 has the longest CDR L1, composed of 17 amino acid residues (Fig. 3D). Despite this, the conformations are tightly clustered (rmsd is 0.20 Å). The backbone conformations of the stem regions superimpose well. Some changes in conformation occur between residues 30a and 30f (residues 8 and 13 of 17 in CDR L1). This is the tip of the loop region, which appears to have similar conformations that fan out the structures because of the slight differences in torsion angles in the backbone near Tyr30a and Lys30f. + + 0.97578 + mutant + cleaner0 + 2023-06-28T21:08:00Z + MESH: + + L4-1 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T16:26:31Z + + L1 + + + residue_range + DUMMY: + cleaner0 + 2023-06-29T09:15:15Z + + 17 amino acid residues + + + 0.99481165 + evidence + cleaner0 + 2023-06-28T20:08:29Z + DUMMY: + + rmsd + + + 0.9976742 + structure_element + cleaner0 + 2023-06-28T20:08:32Z + SO: + + stem regions + + + residue_number + DUMMY: + cleaner0 + 2023-06-29T09:17:33Z + + 30a + + + residue_number + DUMMY: + cleaner0 + 2023-06-29T09:17:47Z + + 30f + + + residue_number + DUMMY: + cleaner0 + 2023-06-29T09:17:59Z + + 8 + + + residue_number + DUMMY: + cleaner0 + 2023-06-29T09:18:10Z + + 13 + + + residue_number + DUMMY: + cleaner0 + 2023-06-29T09:18:21Z + + 17 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T16:26:31Z + + L1 + + + 0.9900733 + structure_element + cleaner0 + 2023-06-28T20:08:36Z + SO: + + loop region + + + 0.99666256 + evidence + cleaner0 + 2023-06-28T20:08:39Z + DUMMY: + + structures + + + 0.99906737 + residue_name_number + cleaner0 + 2023-06-29T09:21:20Z + DUMMY: + + Tyr30a + + + 0.9990594 + residue_name_number + cleaner0 + 2023-06-29T09:21:23Z + DUMMY: + + Lys30f + + + + RESULTS + paragraph + 27685 + L3-20 is the most variable in CDR L1 among the 4 germlines as indicated by an rmsd of 0.54 Å (Fig. 3C). Two structures, H3-53:L3-20 and H5-51:L3-20 are assigned to canonical structure L1-12-1 with virtually identical backbone conformations. The third structure, H3-23:L3-20, has CDR L1 as L1-12-2, which deviates from L1-12-1 at residues 29-32, i.e., at the site of insertion with respect to the 11-residue CDR. The fourth member of the set, H1-69:L3-20, was crystallized with 2 Fabs in the asymmetric unit. The conformation of CDR L1 in these 2 Fabs is slightly different, and both conformations fall somewhere between L1-12-1 and L1-12-2. This reflects the lack of accuracy in the structure due to low resolution of the X-ray data (3.3 Å). + + 0.44628587 + mutant + cleaner0 + 2023-06-28T21:07:16Z + MESH: + + L3-20 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T16:26:31Z + + L1 + + + 0.99455017 + evidence + cleaner0 + 2023-06-28T20:09:20Z + DUMMY: + + rmsd + + + 0.99346644 + evidence + cleaner0 + 2023-06-28T20:09:24Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:13:47Z + + H3-53:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:53Z + + H5-51:L3-20 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:30:26Z + + L1-12-1 + + + 0.7816724 + complex_assembly + cleaner0 + 2023-06-28T21:30:52Z + GO: + + H3-23:L3-20 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T16:26:31Z + + L1 + + + 0.76978254 + mutant + cleaner0 + 2023-06-28T21:30:02Z + MESH: + + L1-12-2 + + + 0.73867476 + mutant + cleaner0 + 2023-06-28T21:31:11Z + MESH: + + L1-12-1 + + + 0.9205847 + residue_range + cleaner0 + 2023-06-29T09:18:29Z + DUMMY: + + 29-32 + + + residue_range + DUMMY: + cleaner0 + 2023-06-29T09:20:29Z + + 11-residue + + + 0.997166 + structure_element + cleaner0 + 2023-06-28T16:25:22Z + SO: + + CDR + + + 0.8665576 + complex_assembly + cleaner0 + 2023-06-28T21:20:21Z + GO: + + H1-69:L3-20 + + + 0.98529875 + experimental_method + cleaner0 + 2023-06-28T20:09:16Z + MESH: + + crystallized + + + 0.48778555 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T16:26:31Z + + L1 + + + 0.28599226 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + mutant + MESH: + cleaner0 + 2023-06-28T21:31:55Z + + L1-12-1 + + + 0.7995807 + mutant + cleaner0 + 2023-06-28T21:32:11Z + MESH: + + L1-12-2 + + + 0.9954146 + evidence + cleaner0 + 2023-06-28T20:09:28Z + DUMMY: + + structure + + + 0.9856711 + evidence + cleaner0 + 2023-06-28T20:09:30Z + DUMMY: + + X-ray data + + + + RESULTS + title_2 + 28432 + CDR L2 + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T20:09:54Z + + L2 + + + + kmab-08-06-1190060-g004.jpg + f0004 + FIG + fig_caption + 28439 + The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. + + 0.99593866 + experimental_method + cleaner0 + 2023-06-29T09:07:59Z + MESH: + + superposition + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T20:09:52Z + + L2 + + + 0.95072794 + complex_assembly + cleaner0 + 2023-06-29T09:23:46Z + GO: + + HC:LC + + + structure_element + SO: + cleaner0 + 2023-06-28T16:14:08Z + + light chains + + + 0.73722893 + mutant + cleaner0 + 2023-06-28T21:05:50Z + MESH: + + L1-39 + + + 0.7577236 + mutant + cleaner0 + 2023-06-28T21:06:35Z + MESH: + + L3-11 + + + 0.81435376 + mutant + cleaner0 + 2023-06-28T21:07:16Z + MESH: + + L3-20 + + + 0.76888275 + mutant + cleaner0 + 2023-06-28T21:08:00Z + MESH: + + L4-1 + + + + RESULTS + paragraph + 28562 + All four LCs have CDR L2 of the same length and canonical structure, L2-8-1 (Table 2). The CDR L2 conformations for each of the LCs paired with the 4 HCs are clustered more tightly than any of the other CDRs (rmsd values are in the range 0.09-0.16 Å), and all 4 sets have virtually the same conformation despite the sequence diversity of the loop. No significant conformation outliers are observed (Fig. 4). + + 0.47755066 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T20:09:54Z + + L2 + + + 0.96698934 + mutant + cleaner0 + 2023-06-28T21:32:49Z + MESH: + + L2-8-1 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T20:09:54Z + + L2 + + + 0.6896999 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + 0.94972163 + structure_element + cleaner0 + 2023-06-28T16:11:33Z + SO: + + HCs + + + 0.9946795 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.99516016 + evidence + cleaner0 + 2023-06-28T20:10:11Z + DUMMY: + + rmsd + + + 0.9969625 + structure_element + cleaner0 + 2023-06-29T08:58:52Z + SO: + + loop + + + + RESULTS + title_2 + 28976 + CDR L3 + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T20:10:26Z + + L3 + + + + kmab-08-06-1190060-g005.jpg + f0005 + FIG + fig_caption + 28983 + The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. + + 0.99597675 + experimental_method + cleaner0 + 2023-06-29T09:08:03Z + MESH: + + superposition + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T20:10:43Z + + L3 + + + 0.9498575 + complex_assembly + cleaner0 + 2023-06-29T09:23:51Z + GO: + + HC:LC + + + structure_element + SO: + cleaner0 + 2023-06-28T16:14:08Z + + light chains + + + 0.770716 + mutant + cleaner0 + 2023-06-28T21:05:50Z + MESH: + + L1-39 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:06:35Z + + L3-11 + + + 0.7661131 + mutant + cleaner0 + 2023-06-28T21:07:16Z + MESH: + + L3-20 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:08:01Z + + L4-1 + + + + RESULTS + paragraph + 29106 + As with CDR L2, all 4 LCs have CDR L3 of the same length and canonical structure, L3-9-cis7-1 (Table 2). The conformations of CDR L3 for L1-39, L3-11, and particularly for L320, are not as tightly clustered as those of L4-1 (Fig. 5). The slight conformational variability occurs in the region of amino acid residues 90-92, which is in contact with CDR H3. + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T20:09:54Z + + L2 + + + 0.7866458 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T20:11:10Z + + L3 + + + 0.9468112 + evidence + cleaner0 + 2023-06-28T20:10:48Z + DUMMY: + + structure + + + 0.9630782 + mutant + cleaner0 + 2023-06-29T09:26:16Z + MESH: + + L3-9-cis7-1 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T20:11:25Z + + L3 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:05:50Z + + L1-39 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:06:35Z + + L3-11 + + + 0.7602481 + mutant + cleaner0 + 2023-06-28T21:08:01Z + MESH: + + L4-1 + + + 0.9677697 + residue_range + cleaner0 + 2023-06-29T09:18:44Z + DUMMY: + + 90-92 + + + 0.9951101 + structure_element + cleaner0 + 2023-06-28T16:25:22Z + SO: + + CDR + + + 0.5268623 + structure_element + cleaner0 + 2023-06-28T19:05:15Z + SO: + + H3 + + + + RESULTS + title_2 + 29466 + CDR H3 conformational diversity + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:15Z + + H3 + + + + RESULTS + paragraph + 29498 + As mentioned earlier, all 16 Fabs have the same CDR H3, for which the amino acid sequence is derived from the anti-CCL2 antibody CNTO 888. The loop and the 2 β-strands of the CDR H3 in this ‘parent’ structure are stabilized by H-bonds between the carbonyl oxygen and peptide nitrogen atoms in the 2 strands. An interesting feature of these CDR H3 structures is the presence of a water molecule that interacts with the peptide nitrogens and carbonyl oxygens near the bridging loop connecting the 2 β-strands. This water is present in both the bound (4DN4) and unbound (4DN3) forms of CNTO 888. The stem region of CDR H3 in the parental Fab is in a ‘kinked’ conformation, in which the indole nitrogen of Trp103 forms a hydrogen bond with the carbonyl oxygen of Leu100b. The carboxyl group of Asp101 forms a salt bridge with Arg94. These interactions are illustrated in Fig. S2. + + 0.28241646 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:15Z + + H3 + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + chemical + CHEBI: + cleaner0 + 2023-06-29T09:24:49Z + + CNTO 888 + + + 0.9973061 + structure_element + cleaner0 + 2023-06-29T08:58:58Z + SO: + + loop + + + 0.8799594 + structure_element + cleaner0 + 2023-06-29T08:59:01Z + SO: + + β-strands + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:15Z + + H3 + + + 0.99561113 + evidence + cleaner0 + 2023-06-28T20:11:37Z + DUMMY: + + structure + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:44Z + + H-bonds + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:15Z + + H3 + + + 0.9962054 + evidence + cleaner0 + 2023-06-28T20:11:40Z + DUMMY: + + structures + + + 0.99835867 + chemical + cleaner0 + 2023-06-29T09:24:59Z + CHEBI: + + water + + + 0.99427104 + structure_element + cleaner0 + 2023-06-29T08:59:04Z + SO: + + loop + + + 0.9647501 + structure_element + cleaner0 + 2023-06-29T08:59:06Z + SO: + + β-strands + + + 0.99842465 + chemical + cleaner0 + 2023-06-29T09:25:01Z + CHEBI: + + water + + + 0.99691653 + protein_state + cleaner0 + 2023-06-29T09:29:33Z + DUMMY: + + bound + + + 0.9966217 + protein_state + cleaner0 + 2023-06-29T09:29:36Z + DUMMY: + + unbound + + + 0.7319946 + chemical + cleaner0 + 2023-06-29T09:24:50Z + CHEBI: + + CNTO 888 + + + 0.9975282 + structure_element + cleaner0 + 2023-06-29T08:59:08Z + SO: + + stem region + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:15Z + + H3 + + + 0.14281757 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.8321708 + protein_state + cleaner0 + 2023-06-29T07:42:05Z + DUMMY: + + kinked + + + 0.99914753 + residue_name_number + cleaner0 + 2023-06-29T09:22:08Z + DUMMY: + + Trp103 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:44Z + + hydrogen bond + + + 0.9991297 + residue_name_number + cleaner0 + 2023-06-29T09:22:11Z + DUMMY: + + Leu100b + + + 0.9991573 + residue_name_number + cleaner0 + 2023-06-29T09:22:14Z + DUMMY: + + Asp101 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:44Z + + salt bridge + + + 0.999151 + residue_name_number + cleaner0 + 2023-06-29T09:22:16Z + DUMMY: + + Arg94 + + + + kmab-08-06-1190060-g006.jpg + f0006 + FIG + fig_caption + 30385 + Ribbon representations of (A) the superposition of all CDR H3s of the structures with complete backbone traces. (B) The CDR H3s rotated 90° about the y axis of the page. The structure of each CDR H3 is represented with a different color. + + 0.99487334 + experimental_method + cleaner0 + 2023-06-28T20:12:34Z + MESH: + + superposition + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T20:13:06Z + + H3s + + + 0.99505204 + evidence + cleaner0 + 2023-06-28T20:12:38Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T20:12:58Z + + H3s + + + 0.9879155 + evidence + cleaner0 + 2023-06-28T20:12:40Z + DUMMY: + + structure + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:15Z + + H3 + + + + RESULTS + paragraph + 30624 + Despite having the same amino acid sequence in all variants, CDR H3 has the highest degree of structural diversity and disorder of all of the CDRs in the experimental set. Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop. Another four of the Fabs, H3-23:L1-39, H3-53:L1-39, H3-53:L3-11 and H3-53:L4-1 have missing side-chain atoms. The variations in CDR H3 conformation are illustrated in Fig. 6 for the 18 Fab structures that have ordered backbone atoms. + + 0.9960975 + structure_element + cleaner0 + 2023-06-28T16:25:22Z + SO: + + CDR + + + 0.5586551 + structure_element + cleaner0 + 2023-06-28T19:05:16Z + SO: + + H3 + + + 0.99367166 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.18316415 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.99480397 + evidence + cleaner0 + 2023-06-28T20:13:14Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:32Z + + H5-51:L3-11 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:34:46Z + + H551:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:12:34Z + + H3-23:L4-1 + + + 0.31031048 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + 0.50068367 + protein_state + cleaner0 + 2023-06-29T09:29:55Z + DUMMY: + + missing + + + 0.76184237 + protein_state + cleaner0 + 2023-06-29T09:29:58Z + DUMMY: + + disordered + + + 0.9971243 + structure_element + cleaner0 + 2023-06-28T20:13:25Z + SO: + + CDR loop + + + 0.26492673 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:15:28Z + + H3-23:L1-39 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:13:04Z + + H3-53:L1-39 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:13:26Z + + H3-53:L3-11 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:18:20Z + + H3-53:L4-1 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + 0.18022169 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.9938689 + evidence + cleaner0 + 2023-06-28T20:13:17Z + DUMMY: + + structures + + + + kmab-08-06-1190060-g007.jpg + f0007 + FIG + fig_caption + 31240 + A comparison of representatives of the “kinked” and “extended” structures. (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. (B) The “extended” CDR H3 of H1-69:L3-20 with green carbon atoms and yellow dashed lines connecting the H-bond pairs for Asp101 OD1 and OD2 and Trp103 NE1. + + 0.9908119 + protein_state + cleaner0 + 2023-06-29T07:42:05Z + DUMMY: + + kinked + + + 0.9959126 + protein_state + cleaner0 + 2023-06-29T07:41:57Z + DUMMY: + + extended + + + 0.99503183 + evidence + cleaner0 + 2023-06-28T20:13:38Z + DUMMY: + + structures + + + 0.9928145 + protein_state + cleaner0 + 2023-06-29T07:42:05Z + DUMMY: + + kinked + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + 0.81869656 + complex_assembly + cleaner0 + 2023-06-28T21:36:23Z + GO: + + H1-69:L3-11 + + + 0.999046 + residue_name_number + cleaner0 + 2023-06-29T09:22:22Z + DUMMY: + + Leu100b + + + 0.99903286 + residue_name_number + cleaner0 + 2023-06-29T09:22:25Z + DUMMY: + + Trp103 + + + 0.99903893 + residue_name_number + cleaner0 + 2023-06-29T09:22:27Z + DUMMY: + + Arg94 + + + 0.9990128 + residue_name_number + cleaner0 + 2023-06-29T09:22:32Z + DUMMY: + + Asp101 + + + 0.99903846 + residue_name_number + cleaner0 + 2023-06-29T09:22:35Z + DUMMY: + + Arg94 + + + 0.9990054 + residue_name_number + cleaner0 + 2023-06-29T09:22:37Z + DUMMY: + + Asp101 + + + 0.99604356 + protein_state + cleaner0 + 2023-06-29T07:41:57Z + DUMMY: + + extended + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:21Z + + H1-69:L3-20 + + + 0.9990422 + residue_name_number + cleaner0 + 2023-06-29T09:22:40Z + DUMMY: + + Asp101 + + + 0.9990233 + residue_name_number + cleaner0 + 2023-06-29T09:22:43Z + DUMMY: + + Trp103 + + + + RESULTS + paragraph + 31688 + In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. The bases of these structures have the ‘kinked’ conformation with the H-bond between Trp103 and Leu100b. A representative CDR H3 structure for H1-69:L1-39 illustrating this is shown in Fig. 7A. The largest backbone conformational deviation for the set is at Tyr99, where the C=O is rotated by 90° relative to that observed in 4DN3. Also, it is worth noting that only one of these structures, H1-69:L4-1, has the conserved water molecule in CDR H3 observed in the 4DN3 and 4DN4 structures. In fact, it is the only Fab in the set that has a water molecule present at this site. The CDR H3 for this structure is shown in Fig. S3. + + 0.23736683 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.99279636 + evidence + cleaner0 + 2023-06-28T20:14:41Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:37:02Z + + H1-69:L1-39 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:37:23Z + + H1-69:L3-11 + + + structure_element + SO: + cleaner0 + 2023-06-28T15:58:47Z + + Fabs + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:37:43Z + + H1-69:L4-1 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:12:10Z + + H3-23:L3-11 + + + structure_element + SO: + cleaner0 + 2023-06-28T15:58:47Z + + Fabs + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:38:15Z + + H3-23:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:13:26Z + + H3-53:L3-11 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:13:47Z + + H3-53:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:11Z + + H5-51:L1-39 + + + 0.99718827 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.9816032 + evidence + cleaner0 + 2023-06-28T20:14:43Z + DUMMY: + + structures + + + 0.755971 + protein_state + cleaner0 + 2023-06-29T07:42:05Z + DUMMY: + + kinked + + + 0.9990682 + residue_name_number + cleaner0 + 2023-06-29T09:22:48Z + DUMMY: + + Trp103 + + + 0.9990772 + residue_name_number + cleaner0 + 2023-06-29T09:22:50Z + DUMMY: + + Leu100b + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + 0.9840614 + evidence + cleaner0 + 2023-06-28T20:14:46Z + DUMMY: + + structure + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:39:19Z + + H1-69:L1-39 + + + 0.9991266 + residue_name_number + cleaner0 + 2023-06-29T09:22:55Z + DUMMY: + + Tyr99 + + + 0.9504556 + evidence + cleaner0 + 2023-06-28T20:14:58Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:39:43Z + + H1-69:L4-1 + + + 0.98424697 + protein_state + cleaner0 + 2023-06-29T09:30:13Z + DUMMY: + + conserved + + + 0.99795204 + chemical + cleaner0 + 2023-06-29T09:25:01Z + CHEBI: + + water + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + 0.9916455 + evidence + cleaner0 + 2023-06-28T20:15:00Z + DUMMY: + + structures + + + 0.17939177 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.9982192 + chemical + cleaner0 + 2023-06-29T09:25:01Z + CHEBI: + + water + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + 0.9559372 + evidence + cleaner0 + 2023-06-28T20:15:04Z + DUMMY: + + structure + + + + RESULTS + paragraph + 32539 + The remaining 8 Fabs can be grouped into 5 different conformational classes. Three of the Fabs, H3-23:L1-39, H3-23:L4-1 and H3-53:L1-39, have distinctive conformations. The stem regions in these 3 cases are in the ‘kinked’ conformation consistent with that observed for 4DN3. The five remaining Fabs, H5-51:L4-1 (2 copies), H1-69:L3-20 (2 copies) and H3-53:L4-1, have 3 different CDR H3 conformations (Fig. S4). The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the ‘kinked’ conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the ‘extended’ conformation (Fig. 7B). + + 0.22445008 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + 0.25196815 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:15:28Z + + H3-23:L1-39 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:12:34Z + + H3-23:L4-1 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:13:04Z + + H3-53:L1-39 + + + 0.9969319 + structure_element + cleaner0 + 2023-06-28T20:15:20Z + SO: + + stem regions + + + 0.9289319 + protein_state + cleaner0 + 2023-06-28T20:15:22Z + DUMMY: + + kinked + + + 0.3686641 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:40:48Z + + H5-51:L4-1 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:21Z + + H1-69:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:18:21Z + + H3-53:L4-1 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:22Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + 0.9966538 + structure_element + cleaner0 + 2023-06-29T08:59:58Z + SO: + + stem regions + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:41:35Z + + H5-51:L4-1 + + + structure_element + SO: + cleaner0 + 2023-06-28T15:58:47Z + + Fabs + + + 0.97828144 + protein_state + cleaner0 + 2023-06-28T20:15:25Z + DUMMY: + + kinked + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:21Z + + H1-69:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:18:21Z + + H3-53:L4-1 + + + 0.99039197 + protein_state + cleaner0 + 2023-06-29T07:41:56Z + DUMMY: + + extended + + + + RESULTS + title_2 + 33161 + VH:VL domain packing + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-07-06T15:18:10Z + + VH:VL + + + + RESULTS + paragraph + 33182 + The VH and VL domains have a β-sandwich structure (also often referred as a Greek key motif) and each is composed of a 4-stranded and a 5-stranded antiparallel β-sheets. The two domains pack together such that the 5-stranded β-sheets, which have hydrophobic surfaces, interact with each other bringing the CDRs from both the VH and VL domains into close proximity. The domain packing of the variants was assessed by computing the domain interface interactions, the VH:VL tilt angles, the buried surface area and surface complementarity. The results of these analyses are shown in Tables 3, 4 and S2. + + 0.9976998 + structure_element + cleaner0 + 2023-06-29T09:00:01Z + SO: + + VH + + + 0.9978058 + structure_element + cleaner0 + 2023-06-28T19:10:07Z + SO: + + VL + + + 0.9875603 + structure_element + cleaner0 + 2023-06-29T09:00:04Z + SO: + + β-sandwich structure + + + 0.99606323 + structure_element + cleaner0 + 2023-06-29T09:00:07Z + SO: + + Greek key motif + + + structure_element + SO: + cleaner0 + 2023-06-29T09:00:27Z + + 4-stranded and a 5-stranded antiparallel β-sheets + + + 0.99740285 + structure_element + cleaner0 + 2023-06-29T09:00:40Z + SO: + + 5-stranded β-sheets + + + 0.997398 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.99764204 + structure_element + cleaner0 + 2023-06-29T09:00:43Z + SO: + + VH + + + 0.99792 + structure_element + cleaner0 + 2023-06-28T19:10:07Z + SO: + + VL + + + site + SO: + cleaner0 + 2023-06-29T09:11:11Z + + domain interface + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-07-06T15:18:10Z + + VH:VL + + + evidence + DUMMY: + cleaner0 + 2023-06-28T16:02:51Z + + tilt angles + + + + RESULTS + title_3 + 33791 + VH:VL interface amino acid residue interactions + + 0.98834515 + site + cleaner0 + 2023-06-28T20:18:37Z + SO: + + VH:VL interface + + + + kmab-08-06-1190060-g008.jpg + f0008 + FIG + fig_caption + 33839 + The conserved VH:VL interactions as viewed along the VH/VL axis. The VH residues are in blue, the VL residues are in orange. + + 0.99727947 + protein_state + cleaner0 + 2023-06-29T09:30:27Z + DUMMY: + + conserved + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-07-06T15:18:10Z + + VH:VL + + + 0.79262924 + structure_element + cleaner0 + 2023-06-29T09:00:50Z + SO: + + VH + + + structure_element + SO: + cleaner0 + 2023-06-28T19:10:07Z + + VL + + + 0.99668914 + structure_element + cleaner0 + 2023-06-29T09:00:53Z + SO: + + VH + + + 0.997074 + structure_element + cleaner0 + 2023-06-28T19:10:07Z + SO: + + VL + + + + RESULTS + paragraph + 33964 + The VH:VL interface is pseudosymmetric, and involves 2 stretches of the polypeptide chain from each domain, namely CDR3 and the framework region between CDRs 1 and 2. These stretches form antiparallel β-hairpins within the internal 5-stranded β-sheet. There are a few principal inter-domain interactions that are conserved not only in the experimental set of 16 Fabs, but in all human antibodies. They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). With the exception of L-Ala43, all other residues are conserved in human germlines. Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved. These core interactions provide enough stability to the VH:VL dimer so that additional VH-VL contacts can tolerate amino acid sequence variations in CDRs H3 and L3 that form part of the VH:VL interface. + + 0.99136263 + site + cleaner0 + 2023-06-28T20:18:37Z + SO: + + VH:VL interface + + + 0.53883225 + protein_state + cleaner0 + 2023-06-29T09:30:34Z + DUMMY: + + pseudosymmetric + + + 0.9983753 + structure_element + cleaner0 + 2023-06-29T09:01:18Z + SO: + + CDR3 + + + 0.9973372 + structure_element + cleaner0 + 2023-06-29T09:00:57Z + SO: + + framework region + + + 0.99737406 + structure_element + cleaner0 + 2023-06-29T09:01:20Z + SO: + + CDRs 1 and 2 + + + 0.993996 + structure_element + cleaner0 + 2023-06-29T09:01:01Z + SO: + + antiparallel β-hairpins + + + 0.9913381 + structure_element + cleaner0 + 2023-06-29T09:01:03Z + SO: + + 5-stranded β-sheet + + + 0.95069045 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + 0.9924118 + species + cleaner0 + 2023-06-28T16:03:59Z + MESH: + + human + + + 0.67610204 + protein_type + cleaner0 + 2023-06-28T16:03:38Z + MESH: + + antibodies + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:44Z + + hydrogen bond + + + structure_element + SO: + cleaner0 + 2023-06-28T20:19:40Z + + L + + + residue_name_number + DUMMY: + cleaner0 + 2023-06-28T20:19:27Z + + Gln38 + + + 0.8804055 + structure_element + cleaner0 + 2023-06-28T20:19:52Z + SO: + + H + + + 0.84093565 + residue_name_number + cleaner0 + 2023-06-29T09:23:04Z + DUMMY: + + Gln39 + + + 0.973559 + structure_element + cleaner0 + 2023-06-28T20:20:04Z + SO: + + H + + + 0.692089 + residue_name_number + cleaner0 + 2023-06-29T09:23:07Z + DUMMY: + + Leu45 + + + 0.99601036 + site + cleaner0 + 2023-06-28T20:18:42Z + SO: + + hydrophobic pocket + + + structure_element + SO: + cleaner0 + 2023-06-28T20:20:25Z + + L + + + 0.6455791 + residue_name_number + cleaner0 + 2023-06-28T20:20:11Z + DUMMY: + + Phe98 + + + structure_element + SO: + cleaner0 + 2023-06-28T20:20:47Z + + L + + + 0.75837576 + residue_name_number + cleaner0 + 2023-06-28T20:20:31Z + DUMMY: + + Tyr87 + + + structure_element + SO: + cleaner0 + 2023-06-28T20:21:11Z + + L + + + 0.7018562 + residue_name_number + cleaner0 + 2023-06-28T20:20:51Z + DUMMY: + + Pro44 + + + structure_element + SO: + cleaner0 + 2023-06-28T20:21:27Z + + L + + + 0.58544266 + residue_name_number + cleaner0 + 2023-06-28T20:21:15Z + DUMMY: + + Pro44 + + + 0.9149529 + structure_element + cleaner0 + 2023-06-28T20:21:36Z + SO: + + H + + + 0.9516314 + residue_name_number + cleaner0 + 2023-06-28T20:21:38Z + DUMMY: + + Trp103 + + + structure_element + SO: + cleaner0 + 2023-06-28T20:22:09Z + + L + + + residue_name_number + DUMMY: + cleaner0 + 2023-06-28T20:21:58Z + + Ala43 + + + 0.9800174 + structure_element + cleaner0 + 2023-06-28T20:22:22Z + SO: + + H + + + 0.5058213 + residue_name_number + cleaner0 + 2023-06-29T09:23:13Z + DUMMY: + + Tyr91 + + + structure_element + SO: + cleaner0 + 2023-06-28T20:22:51Z + + L + + + residue_name_number + DUMMY: + cleaner0 + 2023-06-28T20:22:39Z + + Ala43 + + + 0.993141 + species + cleaner0 + 2023-06-28T16:03:59Z + MESH: + + human + + + 0.9463635 + residue_number + cleaner0 + 2023-06-29T09:20:38Z + DUMMY: + + 43 + + + 0.98263526 + residue_name + cleaner0 + 2023-06-29T09:12:33Z + SO: + + Ser + + + 0.98448217 + residue_name + cleaner0 + 2023-06-29T09:12:38Z + SO: + + Val + + + 0.9818835 + residue_name + cleaner0 + 2023-06-29T09:12:41Z + SO: + + Pro + + + 0.914598 + mutant + cleaner0 + 2023-06-28T21:08:01Z + MESH: + + L4-1 + + + bond_interaction + MESH: + melaniev@ebi.ac.uk + 2023-07-28T14:19:44Z + + hydrophobic interaction + + + 0.9252548 + structure_element + cleaner0 + 2023-06-28T20:23:55Z + SO: + + H + + + 0.58144456 + residue_name_number + cleaner0 + 2023-06-29T09:23:17Z + DUMMY: + + Tyr91 + + + 0.91734785 + complex_assembly + cleaner0 + 2023-06-29T09:23:56Z + GO: + + VH:VL + + + 0.9946301 + oligomeric_state + cleaner0 + 2023-06-29T09:23:22Z + DUMMY: + + dimer + + + site + SO: + cleaner0 + 2023-06-28T20:23:28Z + + VH-VL contacts + + + 0.99753404 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.99678314 + structure_element + cleaner0 + 2023-06-28T19:05:16Z + SO: + + H3 + + + 0.9974498 + structure_element + cleaner0 + 2023-06-29T09:01:23Z + SO: + + L3 + + + 0.99011016 + site + cleaner0 + 2023-06-28T20:18:35Z + SO: + + VH:VL interface + + + + RESULTS + paragraph + 35026 + In total, about 20 residues are involved in the VH:VL interactions on each side (Fig. S5). Half of them are in the framework regions and those residues (except residue 61 in HC, which is actually in CDR2 in Kabat's definition) are conserved in the set of 16 Fabs. The side chain conformations of these conserved residues are also highly similar. One notable exception is H-Trp47, which exhibits 2 conformations of the indole ring. In most of the structures, it has the χ2 angle of ∼80°, while the ring is flipped over (χ2 = −100°) in H5-51:L3:11 and H5-51:L3-20. Interestingly, these are the only 2 structures with residues missing in CDR H3 because of disorder, although both structures are determined at high resolution and the rest of the structure is well defined. Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47. + + residue_range + DUMMY: + cleaner0 + 2023-06-29T09:32:11Z + + 20 residues + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-07-06T15:18:10Z + + VH:VL + + + 0.9973575 + structure_element + cleaner0 + 2023-06-29T09:01:26Z + SO: + + framework regions + + + 0.91451746 + residue_number + cleaner0 + 2023-06-29T09:20:44Z + DUMMY: + + 61 + + + 0.99835485 + structure_element + cleaner0 + 2023-06-29T09:02:52Z + SO: + + HC + + + 0.9977931 + structure_element + cleaner0 + 2023-06-29T09:02:23Z + SO: + + CDR2 + + + 0.793795 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + structure_element + SO: + cleaner0 + 2023-06-28T20:25:28Z + + H + + + residue_name_number + DUMMY: + cleaner0 + 2023-06-28T20:25:18Z + + Trp47 + + + 0.9964148 + evidence + cleaner0 + 2023-06-28T20:24:25Z + DUMMY: + + structures + + + 0.9833544 + evidence + cleaner0 + 2023-06-28T20:24:31Z + DUMMY: + + χ2 + + + 0.9682395 + evidence + cleaner0 + 2023-06-28T20:24:34Z + DUMMY: + + χ2 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:42:48Z + + H5-51:L3:11 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:53Z + + H5-51:L3-20 + + + 0.99607605 + evidence + cleaner0 + 2023-06-28T20:26:34Z + DUMMY: + + structures + + + 0.98517585 + protein_state + cleaner0 + 2023-06-29T09:31:44Z + DUMMY: + + missing + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + 0.9922267 + evidence + cleaner0 + 2023-06-28T20:24:37Z + DUMMY: + + structures + + + 0.9962059 + evidence + cleaner0 + 2023-06-28T20:24:39Z + DUMMY: + + structure + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-07-06T15:18:10Z + + VH:VL + + + 0.965915 + protein_state + cleaner0 + 2023-06-29T09:31:42Z + DUMMY: + + stable + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + structure_element + SO: + cleaner0 + 2023-06-28T20:25:05Z + + H + + + residue_name_number + DUMMY: + cleaner0 + 2023-06-28T20:24:55Z + + Trp47 + + + + RESULTS + title_2 + 36020 + VH:VL tilt angles + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-07-06T15:18:10Z + + VH:VL + + + 0.78827584 + evidence + cleaner0 + 2023-06-28T16:02:51Z + DUMMY: + + tilt angles + + + + RESULTS + paragraph + 36038 + The relative orientation of VH and VL has been measured in a number of different ways. Presented here are the results of 2 different approaches for determining the orientation of one domain relative to the other. + + 0.99675924 + structure_element + cleaner0 + 2023-06-29T09:02:25Z + SO: + + VH + + + 0.99686 + structure_element + cleaner0 + 2023-06-28T19:10:07Z + SO: + + VL + + + + RESULTS + paragraph + 36251 + The first approach uses ABangles, the results of which are shown in Table S2. The four LCs all are classified as Type A because they have a proline at position 44, and the results for each orientation parameter are within the range of values of this type reported by Dunbar and co-workers. In fact, the parameter values for the set of 16 Fabs are in the middle of the distribution observed for 351 non-redundant antibody structures determined at 3.0 Å resolution or better. The only exception is HC1, which is shifted toward smaller angles with the mean value of 70.8° as compared to the distribution centered at 72° for the entire PDB. This probably reflects the invariance of CDR H3 in the current set as opposed to the CDR H3 diversity in the PDB. + + 0.99128175 + experimental_method + cleaner0 + 2023-06-29T09:08:25Z + MESH: + + ABangles + + + 0.52872384 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + 0.9892429 + residue_name + cleaner0 + 2023-06-29T09:12:47Z + SO: + + proline + + + 0.96477294 + residue_number + cleaner0 + 2023-06-29T09:20:49Z + DUMMY: + + 44 + + + 0.76806116 + evidence + cleaner0 + 2023-06-28T20:27:21Z + DUMMY: + + orientation parameter + + + 0.14618859 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.9953317 + evidence + cleaner0 + 2023-06-28T20:27:24Z + DUMMY: + + structures + + + 0.99818486 + structure_element + cleaner0 + 2023-06-29T09:02:55Z + SO: + + HC1 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:16Z + + H3 + + + + RESULTS + paragraph + 37007 + The second approach used for comparing tilt angles involved computing the difference in the tilt angles between all pairs of structures. For structures with 2 copies of the Fab in the asymmetric unit, only one structure was used. The differences between independent Fabs in the same structure are 4.9° for H1-69:L3-20, 1.6° for H1-69:L3-11, 1.4° for H3-23:L4-1, 3.3° for H3-23:L3-11, and 2.5° for H5-51:L4-1. With the exception of H1-69:L3-20, the angles are within the range of 2-3° as are observed in the identical structures in the PDB. In H1-69:L3-20, one of the Fabs is substantially disordered so that part of CDR H2 (the outer β-strand, residues 55-60) is completely missing. This kind of disorder may compromise the integrity of the VH domain and its interaction with the VL. Indeed, this Fab has the largest twist angle HC2 within the experimental set that exceeds the mean value by 2.5 standard deviations (Table S2). + + 0.99319816 + evidence + cleaner0 + 2023-06-28T16:02:51Z + DUMMY: + + tilt angles + + + 0.73657733 + evidence + cleaner0 + 2023-06-28T20:27:39Z + DUMMY: + + difference + + + 0.98982793 + evidence + cleaner0 + 2023-06-28T16:02:51Z + DUMMY: + + tilt angles + + + 0.9951414 + evidence + cleaner0 + 2023-06-28T20:27:45Z + DUMMY: + + structures + + + 0.9731836 + evidence + cleaner0 + 2023-06-28T20:27:48Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-06-28T15:59:05Z + + Fab + + + 0.9942374 + evidence + cleaner0 + 2023-06-28T20:27:51Z + DUMMY: + + structure + + + 0.14642434 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + 0.9922145 + evidence + cleaner0 + 2023-06-28T20:27:53Z + DUMMY: + + structure + + + 0.730512 + complex_assembly + cleaner0 + 2023-06-28T21:20:21Z + GO: + + H1-69:L3-20 + + + 0.7319917 + complex_assembly + cleaner0 + 2023-06-28T21:43:25Z + GO: + + H1-69:L3-11 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:12:34Z + + H3-23:L4-1 + + + 0.678943 + complex_assembly + cleaner0 + 2023-06-28T21:12:10Z + GO: + + H3-23:L3-11 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:44:05Z + + H5-51:L4-1 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:21Z + + H1-69:L3-20 + + + 0.99507105 + evidence + cleaner0 + 2023-06-28T20:28:06Z + DUMMY: + + structures + + + 0.92975956 + complex_assembly + cleaner0 + 2023-06-28T21:20:21Z + GO: + + H1-69:L3-20 + + + 0.19363964 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + 0.76591015 + protein_state + cleaner0 + 2023-06-29T09:33:14Z + DUMMY: + + disordered + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T16:26:21Z + + H2 + + + 0.948555 + structure_element + cleaner0 + 2023-06-29T09:03:03Z + SO: + + β-strand + + + 0.9751329 + residue_range + cleaner0 + 2023-06-29T09:18:50Z + DUMMY: + + 55-60 + + + 0.9974107 + structure_element + cleaner0 + 2023-06-29T09:02:36Z + SO: + + VH + + + 0.9971015 + structure_element + cleaner0 + 2023-06-28T19:10:07Z + SO: + + VL + + + 0.25028703 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.9712751 + evidence + cleaner0 + 2023-06-28T20:28:11Z + DUMMY: + + twist angle + + + structure_element + SO: + cleaner0 + 2023-06-28T20:28:21Z + + HC2 + + + + kmab-08-06-1190060-g009.jpg + f0009 + FIG + fig_caption + 37943 + An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5°) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6°). + + 0.99561775 + experimental_method + cleaner0 + 2023-06-28T20:28:35Z + MESH: + + superposition + + + 0.99801207 + structure_element + cleaner0 + 2023-06-29T09:03:16Z + SO: + + VH + + + 0.93959236 + complex_assembly + cleaner0 + 2023-06-28T21:20:21Z + GO: + + H1-69:L3-20 + + + 0.9412726 + complex_assembly + cleaner0 + 2023-06-28T21:14:11Z + GO: + + H5-51:L1-39 + + + 0.9981108 + structure_element + cleaner0 + 2023-06-28T19:10:07Z + SO: + + VL + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:45:10Z + + H1-69:L4-1 + + + 0.9760524 + complex_assembly + cleaner0 + 2023-06-28T21:14:11Z + GO: + + H5-51:L1-39 + + + 0.9981937 + structure_element + cleaner0 + 2023-06-28T19:10:07Z + SO: + + VL + + + + t0003.xml + t0003 + TABLE + table_caption + 38256 + Differences in VH:VL tilt angles. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-07-06T15:18:10Z + + VH:VL + + + evidence + DUMMY: + cleaner0 + 2023-06-28T16:02:51Z + + tilt angles + + + + t0003.xml + t0003 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/></colgroup><tbody><tr><td align="left"> <hr/></td><td align="center">H1-69:L1-39<hr/></td><td align="center">H1-69:L3-11<hr/></td><td align="center">H1-69:L3-20<hr/></td><td align="center">H1-69:L4-1<hr/></td><td align="center">H3-23:L1-39<hr/></td><td align="center">H3-23:L3-11<hr/></td><td align="center">H3-23:L3-20<hr/></td><td align="center">H3-23:L4-1<hr/></td><td align="center">H3-53:L1-39<hr/></td><td align="center">H3-53:L3-11<hr/></td><td align="center">H3-53:L3-20<hr/></td><td align="center">H3-53:L4-1<hr/></td><td align="center">H5-51:L1-39<hr/></td><td align="center">H5-51:L3-11<hr/></td><td align="center">H5-51:L3-20<hr/></td><td align="center">H5-51:L4-1<hr/></td></tr><tr><td align="center">H1-69:L1-39</td><td align="center"><bold>0</bold></td><td align="char" char="."><bold>2.1</bold></td><td align="center"><bold>8.9</bold></td><td align="char" char="."><bold>1.1</bold></td><td align="char" char="."><bold>4.2</bold></td><td align="center"><bold>3.0</bold></td><td align="center"><bold>9.5</bold></td><td align="char" char="."><bold>1.5</bold></td><td align="center"><bold>3.3</bold></td><td align="char" char="."><bold>3.6</bold></td><td align="char" char="."><bold>3.1</bold></td><td align="char" char="."><bold>1.6</bold></td><td align="center"><bold>1.8</bold></td><td align="char" char="."><bold>2.9</bold></td><td align="char" char="."><bold>2.4</bold></td><td align="char" char="."><bold>5.2</bold></td></tr><tr><td align="center">H1-69:L3-11</td><td align="center"> </td><td align="center"><bold>0</bold></td><td align="center"><bold>7.3</bold></td><td align="char" char="."><bold>2.9</bold></td><td align="char" char="."><bold>2.5</bold></td><td align="char" char="."><bold>2.0</bold></td><td align="center"><bold>8.4</bold></td><td align="char" char="."><bold>1.3</bold></td><td align="center"><bold>2.6</bold></td><td align="char" char="."><bold>2.9</bold></td><td align="char" char="."><bold>3.2</bold></td><td align="char" char="."><bold>1.8</bold></td><td align="char" char="."><bold>3.9</bold></td><td align="char" char="."><bold>4.6</bold></td><td align="char" char="."><bold>4.4</bold></td><td align="char" char="."><bold>5.0</bold></td></tr><tr><td align="center">H1-69:L3-20</td><td align="center"> </td><td align="center"> </td><td align="center"><bold>0</bold></td><td align="center"><bold>9.2</bold></td><td align="center"><bold>5.0</bold></td><td align="center"><bold>8.7</bold></td><td align="center"><bold>7.4</bold></td><td align="center"><bold>7.6</bold></td><td align="center"><bold>8.9</bold></td><td align="center"><bold>8.6</bold></td><td align="center"><bold>9.4</bold></td><td align="center"><bold>7.9</bold></td><td align="center"><bold>10.5</bold></td><td align="center"><bold>10.1</bold></td><td align="center"><bold>11.0</bold></td><td align="center"><bold>9.7</bold></td></tr><tr><td align="center">H1-69:L4-1</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"><bold>0</bold></td><td align="char" char="."><bold>4.6</bold></td><td align="char" char="."><bold>3.9</bold></td><td align="center"><bold>10.1</bold></td><td align="char" char="."><bold>1.8</bold></td><td align="char" char="."><bold>4.4</bold></td><td align="char" char="."><bold>4.7</bold></td><td align="char" char="."><bold>4.1</bold></td><td align="char" char="."><bold>2.3</bold></td><td align="char" char="."><bold>1.6</bold></td><td align="char" char="."><bold>2.5</bold></td><td align="char" char="."><bold>2.3</bold></td><td align="char" char="."><bold>6.2</bold></td></tr><tr><td align="center">H3-23:L1-39</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"><bold>0</bold></td><td align="center"><bold>4.0</bold></td><td align="center"><bold>8.0</bold></td><td align="char" char="."><bold>2.8</bold></td><td align="char" char="."><bold>4.8</bold></td><td align="char" char="."><bold>4.8</bold></td><td align="char" char="."><bold>5.4</bold></td><td align="char" char="."><bold>3.6</bold></td><td align="char" char="."><bold>6.0</bold></td><td align="char" char="."><bold>6.2</bold></td><td align="char" char="."><bold>6.6</bold></td><td align="char" char="."><bold>6.6</bold></td></tr><tr><td align="center">H3-23:L3-11</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"><bold>0</bold></td><td align="center"><bold>9.3</bold></td><td align="char" char="."><bold>3.0</bold></td><td align="char" char="."><bold>2.1</bold></td><td align="char" char="."><bold>2.9</bold></td><td align="char" char="."><bold>3.3</bold></td><td align="char" char="."><bold>3.3</bold></td><td align="char" char="."><bold>4.6</bold></td><td align="char" char="."><bold>5.8</bold></td><td align="char" char="."><bold>5.0</bold></td><td align="char" char="."><bold>5.2</bold></td></tr><tr><td align="center">H3-23:L3-20</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"><bold>0</bold></td><td align="center"><bold>8.9</bold></td><td align="center"><bold>7.9</bold></td><td align="center"><bold>7.0</bold></td><td align="center"><bold>7.6</bold></td><td align="center"><bold>7.9</bold></td><td align="center"><bold>10.5</bold></td><td align="center"><bold>9.7</bold></td><td align="center"><bold>10.7</bold></td><td align="center"><bold>6.2</bold></td></tr><tr><td align="center">H3-23:L4-1</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"><bold>0</bold></td><td align="char" char="."><bold>3.6</bold></td><td align="char" char="."><bold>3.8</bold></td><td align="char" char="."><bold>3.7</bold></td><td align="char" char="."><bold>1.5</bold></td><td align="char" char="."><bold>3.2</bold></td><td align="char" char="."><bold>3.7</bold></td><td align="char" char="."><bold>3.8</bold></td><td align="char" char="."><bold>5.6</bold></td></tr><tr><td align="center">H3-53:L1-39</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="char" char="."><bold>0</bold></td><td align="char" char="."><bold>1.0</bold></td><td align="char" char="."><bold>1.6</bold></td><td align="char" char="."><bold>2.9</bold></td><td align="char" char="."><bold>4.6</bold></td><td align="char" char="."><bold>5.3</bold></td><td align="char" char="."><bold>4.8</bold></td><td align="char" char="."><bold>3.1</bold></td></tr><tr><td align="center">H3-53:L3-11</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"><bold>0</bold></td><td align="char" char="."><bold>1.3</bold></td><td align="char" char="."><bold>2.9</bold></td><td align="char" char="."><bold>4.8</bold></td><td align="char" char="."><bold>5.2</bold></td><td align="char" char="."><bold>5.0</bold></td><td align="char" char="."><bold>2.3</bold></td></tr><tr><td align="center">H3-53:L3-20</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="char" char="."><bold>0</bold></td><td align="char" char="."><bold>2.5</bold></td><td align="char" char="."><bold>3.8</bold></td><td align="char" char="."><bold>4.2</bold></td><td align="char" char="."><bold>3.9</bold></td><td align="char" char="."><bold>2.2</bold></td></tr><tr><td align="center">H3-53:L4-1</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"><bold>0</bold></td><td align="char" char="."><bold>2.9</bold></td><td align="char" char="."><bold>3.0</bold></td><td align="char" char="."><bold>3.3</bold></td><td align="char" char="."><bold>4.2</bold></td></tr><tr><td align="center">H5-51:L1-39</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"><bold>0</bold></td><td align="char" char="."><bold>1.9</bold></td><td align="char" char="."><bold>0.6</bold></td><td align="char" char="."><bold>5.8</bold></td></tr><tr><td align="center">H5-51:L3-11</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"><bold>0</bold></td><td align="center"><bold>1.9</bold></td><td align="char" char="."><bold>5.7</bold></td></tr><tr><td align="center">H5-51:L3-20</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"><bold>0</bold></td><td align="char" char="."><bold>5.8</bold></td></tr><tr><td align="center">H5-51:L4-1</td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"> </td><td align="center"><bold>0</bold></td></tr></tbody></table> + + 38290 +   H1-69:L1-39 H1-69:L3-11 H1-69:L3-20 H1-69:L4-1 H3-23:L1-39 H3-23:L3-11 H3-23:L3-20 H3-23:L4-1 H3-53:L1-39 H3-53:L3-11 H3-53:L3-20 H3-53:L4-1 H5-51:L1-39 H5-51:L3-11 H5-51:L3-20 H5-51:L4-1 H1-69:L1-39 0 2.1 8.9 1.1 4.2 3.0 9.5 1.5 3.3 3.6 3.1 1.6 1.8 2.9 2.4 5.2 H1-69:L3-11   0 7.3 2.9 2.5 2.0 8.4 1.3 2.6 2.9 3.2 1.8 3.9 4.6 4.4 5.0 H1-69:L3-20     0 9.2 5.0 8.7 7.4 7.6 8.9 8.6 9.4 7.9 10.5 10.1 11.0 9.7 H1-69:L4-1       0 4.6 3.9 10.1 1.8 4.4 4.7 4.1 2.3 1.6 2.5 2.3 6.2 H3-23:L1-39         0 4.0 8.0 2.8 4.8 4.8 5.4 3.6 6.0 6.2 6.6 6.6 H3-23:L3-11           0 9.3 3.0 2.1 2.9 3.3 3.3 4.6 5.8 5.0 5.2 H3-23:L3-20             0 8.9 7.9 7.0 7.6 7.9 10.5 9.7 10.7 6.2 H3-23:L4-1               0 3.6 3.8 3.7 1.5 3.2 3.7 3.8 5.6 H3-53:L1-39                 0 1.0 1.6 2.9 4.6 5.3 4.8 3.1 H3-53:L3-11                   0 1.3 2.9 4.8 5.2 5.0 2.3 H3-53:L3-20                     0 2.5 3.8 4.2 3.9 2.2 H3-53:L4-1                       0 2.9 3.0 3.3 4.2 H5-51:L1-39                         0 1.9 0.6 5.8 H5-51:L3-11                           0 1.9 5.7 H5-51:L3-20                             0 5.8 H5-51:L4-1                               0 + + + RESULTS + paragraph + 39582 + The differences in the tilt angle are shown for all pairs of V regions in Table 3. They range from 0.6° to 11.0°. The smallest differences in the tilt angle are between the Fabs in isomorphous crystal forms. The largest deviations in the tilt angle, up to 11.0°, are found for 2 structures, H1-69:L3-20 and H3-23:L3-20, that stand out from the other Fabs. One of the 2 structures, H1-69:L3-20, has its CDR H3 in the ‘extended’ conformation; the other structure has it in the ‘kinked’ conformation. Two examples illustrating large (10.5°) and small (1.6°) differences in the tilt angles are shown in Fig. 9. + + 0.8249283 + evidence + cleaner0 + 2023-06-28T20:29:14Z + DUMMY: + + differences + + + 0.98597944 + evidence + cleaner0 + 2023-06-28T20:29:17Z + DUMMY: + + tilt angle + + + 0.99408334 + structure_element + cleaner0 + 2023-06-28T19:05:51Z + SO: + + V regions + + + 0.98391706 + evidence + cleaner0 + 2023-06-28T20:29:28Z + DUMMY: + + tilt angle + + + 0.26356268 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + 0.885286 + evidence + cleaner0 + 2023-06-28T20:29:31Z + DUMMY: + + crystal forms + + + 0.9822982 + evidence + cleaner0 + 2023-06-28T20:29:34Z + DUMMY: + + tilt angle + + + 0.98341817 + evidence + cleaner0 + 2023-06-28T20:29:37Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:21Z + + H1-69:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:46:07Z + + H3-23:L3-20 + + + 0.33730918 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + 0.9868803 + evidence + cleaner0 + 2023-06-28T20:29:40Z + DUMMY: + + structures + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:21Z + + H1-69:L3-20 + + + 0.9975684 + structure_element + cleaner0 + 2023-06-28T16:25:23Z + SO: + + CDR + + + 0.64334047 + structure_element + cleaner0 + 2023-06-28T19:05:17Z + SO: + + H3 + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T07:41:57Z + + extended + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T07:42:05Z + + kinked + + + 0.97952867 + evidence + cleaner0 + 2023-06-28T16:02:51Z + DUMMY: + + tilt angles + + + + RESULTS + title_2 + 40207 + VH:VL buried surface area and complementarity + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-07-06T15:18:10Z + + VH:VL + + + + t0004.xml + t0004 + TABLE + table_caption + 40253 + VH:VL surface areas and surface complementarity. + + complex_assembly + GO: + melaniev@ebi.ac.uk + 2023-07-06T15:18:10Z + + VH:VL + + + + t0004.xml + t0004 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/></colgroup><thead><tr><th align="left">Chain Pairs</th><th align="center">PDB</th><th align="center">Contact surfaceVH (Å<sup>2</sup>)</th><th align="center">Contact surfaceVL (Å<sup>2</sup>)</th><th align="center">Interface(Å<sup>2</sup>)</th><th align="center">Surface complementarity</th></tr></thead><tbody><tr><td align="center"><bold>H1-69:L1-39</bold></td><td align="center">5I15</td><td align="center">727</td><td align="center">771</td><td align="center">749</td><td align="char" char=".">0.743</td></tr><tr><td align="center"><bold>H1-69:L3-11</bold></td><td align="center">5I16</td><td align="center">802</td><td align="center">870</td><td align="center">836</td><td align="char" char=".">0.762</td></tr><tr><td align="center"><bold>H1-69:L3-20</bold></td><td align="center">5I17</td><td align="center">713</td><td align="center">736</td><td align="center">725</td><td align="char" char=".">0.723</td></tr><tr><td align="center"><bold>H1-69:L4-1</bold></td><td align="center">5I18</td><td align="center">729</td><td align="center">736</td><td align="center">733</td><td align="char" char=".">0.734</td></tr><tr><td align="center">H3-23:L1-39<xref ref-type="bibr" rid="cit0001"><sup><bold>1</bold></sup></xref></td><td align="center">5I19</td><td align="center">795</td><td align="center">817</td><td align="center">806</td><td align="char" char=".">0.722</td></tr><tr><td align="center"><bold>H3-23:L3-11</bold></td><td align="center">5I1A</td><td align="center">822</td><td align="center">834</td><td align="center">828</td><td align="char" char=".">0.725</td></tr><tr><td align="center"><bold>H3-23:L3-20</bold></td><td align="center">5I1C</td><td align="center">670</td><td align="center">698</td><td align="center">684</td><td align="char" char=".">0.676</td></tr><tr><td align="center"><bold>H3-23:L4-1</bold></td><td align="center">5I1D</td><td align="center">743</td><td align="center">770</td><td align="center">757</td><td align="char" char=".">0.708</td></tr><tr><td align="center">H3-53:L1-39<xref ref-type="bibr" rid="cit0001"><sup><bold>1</bold></sup></xref></td><td align="center">5I1E</td><td align="center">698</td><td align="center">719</td><td align="center">709</td><td align="center">0.712</td></tr><tr><td align="center">H3-53:L3-11<xref ref-type="bibr" rid="cit0001"><sup><bold>1</bold></sup></xref></td><td align="center">5I1G</td><td align="center">747</td><td align="center">758</td><td align="center">753</td><td align="center">0.690</td></tr><tr><td align="center"><bold>H3-53:L3-20</bold></td><td align="center">5I1H</td><td align="center">743</td><td align="center">735</td><td align="center">739</td><td align="char" char=".">0.687</td></tr><tr><td align="center"><bold>H3-53:L4-1</bold></td><td align="center">5I1I</td><td align="center">689</td><td align="center">693</td><td align="center">691</td><td align="char" char=".">0.711</td></tr><tr><td align="center"><bold>H5-51:L1-39</bold></td><td align="center">4KMT</td><td align="center">761</td><td align="center">808</td><td align="center">785</td><td align="char" char=".">0.728</td></tr><tr><td align="center">H5-51:L3-11<xref ref-type="bibr" rid="cit0002"><sup><bold>2</bold></sup></xref></td><td align="center">5I1J</td><td align="center">648</td><td align="center">714</td><td align="center">681</td><td align="center">0.717</td></tr><tr><td align="center">H5-51:L3-20<xref ref-type="bibr" rid="cit0002"><sup><bold>2</bold></sup></xref></td><td align="center">5I1K</td><td align="center">622</td><td align="center">643</td><td align="center">633</td><td align="center">0.740</td></tr><tr><td align="center"><bold>H5-51:L4-1</bold></td><td align="center">5I1L</td><td align="center">790</td><td align="center">792</td><td align="center">791</td><td align="char" char=".">0.704</td></tr></tbody></table> + + 40302 + Chain Pairs PDB Contact surfaceVH (Å2) Contact surfaceVL (Å2) Interface(Å2) Surface complementarity H1-69:L1-39 5I15 727 771 749 0.743 H1-69:L3-11 5I16 802 870 836 0.762 H1-69:L3-20 5I17 713 736 725 0.723 H1-69:L4-1 5I18 729 736 733 0.734 H3-23:L1-39 5I19 795 817 806 0.722 H3-23:L3-11 5I1A 822 834 828 0.725 H3-23:L3-20 5I1C 670 698 684 0.676 H3-23:L4-1 5I1D 743 770 757 0.708 H3-53:L1-39 5I1E 698 719 709 0.712 H3-53:L3-11 5I1G 747 758 753 0.690 H3-53:L3-20 5I1H 743 735 739 0.687 H3-53:L4-1 5I1I 689 693 691 0.711 H5-51:L1-39 4KMT 761 808 785 0.728 H5-51:L3-11 5I1J 648 714 681 0.717 H5-51:L3-20 5I1K 622 643 633 0.740 H5-51:L4-1 5I1L 790 792 791 0.704 + + + t0004.xml + t0004 + TABLE + table_footnote + 40999 + Some side chain atoms in CDR H3 are missing. + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + + t0004.xml + t0004 + TABLE + table_footnote + 41044 + Residues in CDR H3 are missing: YGE in H5-51:L3-11, GIY in H5-51:L3-20. + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + 0.9964439 + structure_element + cleaner0 + 2023-06-29T07:43:58Z + SO: + + YGE + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:32Z + + H5-51:L3-11 + + + 0.9953916 + structure_element + cleaner0 + 2023-06-29T07:44:08Z + SO: + + GIY + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:53Z + + H5-51:L3-20 + + + + RESULTS + paragraph + 41116 + The results of the PISA contact surface calculation and surface complementarity calculation are shown in Table 4. The interface areas are calculated as the average of the VH and VL contact surfaces. Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness). Among the complete structures, the interface areas range from 684 to 836 Å2. Interestingly, the 2 structures that have the largest tilt angle differences with the other variants, H3-23:L3-20 and H1-69:L3-20, have the smallest VH:VL interfaces, 684 and 725 Å2, respectively. H3-23:L3-20 is also unique in that it has the lowest value (0.676) of surface complementarity. + + 0.99178493 + experimental_method + cleaner0 + 2023-06-28T20:32:34Z + MESH: + + PISA + + + 0.9650426 + experimental_method + cleaner0 + 2023-06-28T20:32:42Z + MESH: + + contact surface calculation + + + 0.96886826 + experimental_method + cleaner0 + 2023-06-28T20:32:54Z + MESH: + + surface complementarity calculation + + + 0.5817277 + site + cleaner0 + 2023-06-29T08:16:00Z + SO: + + interface + + + site + SO: + cleaner0 + 2023-06-29T08:16:44Z + + VH and VL contact surfaces + + + 0.992105 + evidence + cleaner0 + 2023-06-29T08:41:06Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + 0.6253964 + protein_state + cleaner0 + 2023-06-29T09:33:23Z + DUMMY: + + missing + + + 0.8131186 + site + cleaner0 + 2023-06-29T09:11:20Z + SO: + + interfaces + + + 0.9851922 + evidence + cleaner0 + 2023-06-29T08:41:10Z + DUMMY: + + structures + + + 0.9952638 + protein_state + cleaner0 + 2023-06-29T09:33:29Z + DUMMY: + + complete + + + 0.9972295 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.28258172 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + 0.9950252 + protein_state + cleaner0 + 2023-06-29T09:33:35Z + DUMMY: + + complete + + + 0.9946089 + evidence + cleaner0 + 2023-06-29T08:41:13Z + DUMMY: + + structures + + + 0.49001282 + site + cleaner0 + 2023-06-29T08:41:22Z + SO: + + interface + + + 0.9873541 + evidence + cleaner0 + 2023-06-29T08:41:29Z + DUMMY: + + structures + + + 0.9782131 + evidence + cleaner0 + 2023-06-29T08:41:32Z + DUMMY: + + tilt angle differences + + + 0.9572224 + complex_assembly + cleaner0 + 2023-06-29T07:44:30Z + GO: + + H3-23:L3-20 + + + 0.9546193 + complex_assembly + cleaner0 + 2023-06-28T21:20:21Z + GO: + + H1-69:L3-20 + + + 0.87709194 + site + cleaner0 + 2023-06-29T09:11:25Z + SO: + + VH:VL interfaces + + + 0.98440266 + complex_assembly + cleaner0 + 2023-06-29T07:45:05Z + GO: + + H3-23:L3-20 + + + evidence + DUMMY: + cleaner0 + 2023-06-29T08:42:18Z + + surface complementarity + + + + RESULTS + title_3 + 41927 + Stability of germline pairings + + + t0005.xml + t0005 + TABLE + table_caption + 41958 + Melting temperatures for the 16 Fabs. + + 0.99553233 + evidence + cleaner0 + 2023-06-29T08:42:23Z + DUMMY: + + Melting temperatures + + + 0.5436273 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + + t0005.xml + t0005 + TABLE + table + <?xml version="1.0" encoding="UTF-8"?> +<table frame="hsides" rules="groups"><colgroup><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/><col width="86.4pt" align="left"/></colgroup><thead><tr><th align="left"> </th><th align="center">L3-20</th><th align="center">L4-1</th><th align="center">L3-11</th><th align="center">L1-39</th><th align="center">HC average</th></tr></thead><tbody><tr><td align="char" char=".">H1-69</td><td align="char" char=".">73.6</td><td align="char" char=".">74.8</td><td align="char" char=".">75.6</td><td align="char" char=".">80.3</td><td align="char" char=".">76.1</td></tr><tr><td align="left">H3-23</td><td align="char" char=".">74.8</td><td align="char" char=".">75.2</td><td align="char" char=".">4.8</td><td align="char" char=".">81.5</td><td align="char" char=".">76.6</td></tr><tr><td align="left">H3-53</td><td align="char" char=".">68.4</td><td align="char" char=".">68.0</td><td align="char" char=".">71.5</td><td align="char" char=".">73.9</td><td align="char" char=".">70.5</td></tr><tr><td align="left">H5-51</td><td align="char" char=".">68.4</td><td align="char" char=".">68.4</td><td align="char" char=".">71.9</td><td align="char" char=".">77.0</td><td align="char" char=".">71.4</td></tr><tr><td align="left">LC average</td><td align="center">71.3</td><td align="char" char=".">71.6</td><td align="char" char=".">73.5</td><td align="char" char=".">78.2</td><td align="center"> </td></tr></tbody></table> + + 41996 +   L3-20 L4-1 L3-11 L1-39 HC average H1-69 73.6 74.8 75.6 80.3 76.1 H3-23 74.8 75.2 4.8 81.5 76.6 H3-53 68.4 68.0 71.5 73.9 70.5 H5-51 68.4 68.4 71.9 77.0 71.4 LC average 71.3 71.6 73.5 78.2   + + + t0005.xml + t0005 + TABLE + table_footnote + 42203 + Colors: blue (Tm < 70°C), green (70°C < Tm < 73°C), yellow (73°C < Tm < 78°C), orange (Tm > 78°C). + + 0.78301543 + evidence + cleaner0 + 2023-06-29T08:42:28Z + DUMMY: + + Tm + + + 0.8056168 + evidence + cleaner0 + 2023-06-29T08:42:31Z + DUMMY: + + Tm + + + 0.90112174 + evidence + cleaner0 + 2023-06-29T08:42:33Z + DUMMY: + + Tm + + + 0.54672956 + evidence + cleaner0 + 2023-06-29T08:42:36Z + DUMMY: + + Tm + + + + RESULTS + paragraph + 42308 + Melting temperatures (Tm) were measured for all Fabs using differential scanning calorimetry (Table 5). It appears that for each given LC, the Fabs with germlines H1-69 and H3-23 are substantially more stable than those with germlines H3-53 and H5-51. In addition, L1-39 provides a much higher degree of stabilization than the other 3 LC germlines when combined with any of the HCs. As a result, the Tm for pairs H1-69:L1-39 and H3-23:L1-39 is 12-13° higher than for pairs H3-53:L3-20, H3-53:L4-1, H5-51:L3-20 and H5-51:L4-1. + + 0.9944316 + evidence + cleaner0 + 2023-06-29T08:42:40Z + DUMMY: + + Melting temperatures + + + 0.9929166 + evidence + cleaner0 + 2023-06-29T08:42:43Z + DUMMY: + + Tm + + + 0.22407825 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + 0.9954069 + experimental_method + cleaner0 + 2023-06-29T09:08:34Z + MESH: + + differential scanning calorimetry + + + 0.93906766 + structure_element + cleaner0 + 2023-06-28T16:24:55Z + SO: + + LC + + + 0.17053662 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + mutant + MESH: + cleaner0 + 2023-06-28T21:03:20Z + + H1-69 + + + 0.49335834 + mutant + cleaner0 + 2023-06-28T21:04:18Z + MESH: + + H3-23 + + + 0.74506783 + protein_state + cleaner0 + 2023-06-29T09:33:43Z + DUMMY: + + stable + + + mutant + MESH: + cleaner0 + 2023-06-28T21:08:33Z + + H3-53 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:05:06Z + + H5-51 + + + 0.8035386 + mutant + cleaner0 + 2023-06-28T21:05:50Z + MESH: + + L1-39 + + + 0.69415265 + structure_element + cleaner0 + 2023-06-28T16:24:55Z + SO: + + LC + + + 0.99619627 + structure_element + cleaner0 + 2023-06-28T16:11:33Z + SO: + + HCs + + + 0.9950289 + evidence + cleaner0 + 2023-06-29T08:42:56Z + DUMMY: + + Tm + + + complex_assembly + GO: + cleaner0 + 2023-06-29T08:43:14Z + + H1-69:L1-39 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:15:28Z + + H3-23:L1-39 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:13:47Z + + H3-53:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:18:21Z + + H3-53:L4-1 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:53Z + + H5-51:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-29T07:47:40Z + + H5-51:L4-1 + + + + RESULTS + paragraph + 42838 + These findings correlate well with the degree of conformational disorder observed in the crystal structures. Parts of CDR H3 main chain are completely disordered, and were not modeled in Fabs H5-51:L3-20 and H5-51:L3-11 that have the lowest Tms in the set. No electron density is observed for a number of side chains in CDRs H3 and L3 in all Fabs with germline H3-53, which indicates loose packing of the variable domains. All those molecules are relatively unstable, as is reflected in their low Tms. + + 0.9975747 + evidence + cleaner0 + 2023-06-28T19:16:53Z + DUMMY: + + crystal structures + + + 0.997758 + structure_element + cleaner0 + 2023-06-28T16:25:23Z + SO: + + CDR + + + 0.84807676 + structure_element + cleaner0 + 2023-06-28T19:05:17Z + SO: + + H3 + + + 0.68790114 + protein_state + cleaner0 + 2023-06-29T09:33:49Z + DUMMY: + + disordered + + + structure_element + SO: + cleaner0 + 2023-06-28T15:58:47Z + + Fabs + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:53Z + + H5-51:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:14:32Z + + H5-51:L3-11 + + + 0.6941904 + evidence + cleaner0 + 2023-06-29T08:43:41Z + DUMMY: + + Tms + + + 0.9930948 + evidence + cleaner0 + 2023-06-29T08:43:46Z + DUMMY: + + electron density + + + 0.9978542 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.9923321 + structure_element + cleaner0 + 2023-06-28T19:05:17Z + SO: + + H3 + + + 0.99722135 + structure_element + cleaner0 + 2023-06-29T09:03:45Z + SO: + + L3 + + + 0.55399996 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + mutant + MESH: + cleaner0 + 2023-06-28T21:08:33Z + + H3-53 + + + 0.6801788 + structure_element + cleaner0 + 2023-06-29T09:04:01Z + SO: + + variable domains + + + 0.5376354 + evidence + cleaner0 + 2023-06-29T07:48:27Z + DUMMY: + + Tms + + + + DISCUSS + title_1 + 43340 + Discussion + + + DISCUSS + paragraph + 43351 + This is the first report of a systematic structural investigation of a phage germline library. The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. The structural data set taken as a whole provides insight into how the backbone conformations of the CDRs of a specific heavy or light chain vary when it is paired with 4 different light or heavy chains, respectively. A large variability in the CDR conformations for the sets of HCs and LCs is observed. In some cases the CDR conformations for all members of a set are virtually identical, for others subtle changes occur in a few members of a set, and in some cases larger deviations are observed within a set. The five variants that crystallized with 2 copies of the Fab in the asymmetric unit serve somewhat as controls for the influence of crystal packing on the conformations of the CDRs. In four of the 5 structures the CDR conformations are consistent. In only one case, that of H1-69:L3-20 (the lowest resolution structure), do we see differences in the conformations of the 2 copies of CDRs H1 and L1. This variability is likely a result of 2 factors, crystal packing interactions and internal instability of the variable domain. + + experimental_method + MESH: + cleaner0 + 2023-06-29T09:14:19Z + + systematic structural investigation + + + 0.9177982 + experimental_method + cleaner0 + 2023-06-29T09:09:11Z + MESH: + + phage germline library + + + 0.16469127 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.9961118 + evidence + cleaner0 + 2023-06-29T08:43:56Z + DUMMY: + + structures + + + 0.99849904 + structure_element + cleaner0 + 2023-06-28T16:11:33Z + SO: + + HCs + + + 0.8344044 + mutant + cleaner0 + 2023-06-28T21:03:20Z + MESH: + + H1-69 + + + 0.90730447 + mutant + cleaner0 + 2023-06-28T21:04:18Z + MESH: + + H3-23 + + + 0.9297971 + mutant + cleaner0 + 2023-06-28T21:08:33Z + MESH: + + H3-53 + + + 0.9016702 + mutant + cleaner0 + 2023-06-28T21:05:06Z + MESH: + + H5-51 + + + 0.9983071 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + 0.64202046 + mutant + cleaner0 + 2023-06-28T21:05:50Z + MESH: + + L1-39 + + + 0.7273175 + mutant + cleaner0 + 2023-06-28T21:06:35Z + MESH: + + L3-11 + + + 0.7565879 + mutant + cleaner0 + 2023-06-28T21:07:16Z + MESH: + + L3-20 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:08:01Z + + L4-1 + + + 0.99818146 + structure_element + cleaner0 + 2023-06-28T16:25:23Z + SO: + + CDR + + + 0.96377325 + structure_element + cleaner0 + 2023-06-28T19:05:17Z + SO: + + H3 + + + 0.9447263 + evidence + cleaner0 + 2023-06-29T08:44:01Z + DUMMY: + + structural data + + + 0.9982551 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + structure_element + SO: + cleaner0 + 2023-06-28T16:13:49Z + + light chain + + + structure_element + SO: + cleaner0 + 2023-06-28T15:56:24Z + + heavy chains + + + 0.9916883 + structure_element + cleaner0 + 2023-06-28T16:25:23Z + SO: + + CDR + + + 0.9959287 + structure_element + cleaner0 + 2023-06-28T16:11:33Z + SO: + + HCs + + + 0.9971118 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + 0.9891389 + structure_element + cleaner0 + 2023-06-28T16:25:23Z + SO: + + CDR + + + 0.98784024 + experimental_method + cleaner0 + 2023-06-29T09:09:15Z + MESH: + + crystallized + + + 0.1982733 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.9961863 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.99503726 + evidence + cleaner0 + 2023-06-29T08:44:06Z + DUMMY: + + structures + + + 0.96922016 + structure_element + cleaner0 + 2023-06-28T16:25:23Z + SO: + + CDR + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:21Z + + H1-69:L3-20 + + + 0.9952447 + evidence + cleaner0 + 2023-06-29T08:44:10Z + DUMMY: + + structure + + + 0.99505025 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.9711269 + structure_element + cleaner0 + 2023-06-28T19:04:12Z + SO: + + H1 + + + 0.9512453 + structure_element + cleaner0 + 2023-06-28T16:26:32Z + SO: + + L1 + + + 0.9851179 + structure_element + cleaner0 + 2023-06-29T09:03:48Z + SO: + + variable domain + + + + DISCUSS + paragraph + 44673 + For the CDRs with canonical structures, the largest changes in conformation occur for CDR H1 of H1-69 and H3-53. The other 2 HCs, H3-23 and H5-51, have canonical structures that are remarkably well conserved (Fig. 1). Of the 4 HCs, H1-69 has the greatest number of canonical structure assignments (Table 2). H1-69 is unique in having a pair of glycine residues at positions 26 and 27, which provide more conformational freedom in CDR H1. Besides IGHV1-69, only the germlines of the VH4 family possess double glycines in CDR H1, and it will be interesting to see if they are also conformationally unstable. + + 0.99440986 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.99743253 + structure_element + cleaner0 + 2023-06-28T16:25:23Z + SO: + + CDR + + + 0.6280479 + structure_element + cleaner0 + 2023-06-28T19:04:12Z + SO: + + H1 + + + 0.89499897 + mutant + cleaner0 + 2023-06-28T21:03:20Z + MESH: + + H1-69 + + + 0.92543626 + mutant + cleaner0 + 2023-06-28T21:08:33Z + MESH: + + H3-53 + + + 0.9889471 + structure_element + cleaner0 + 2023-06-28T16:11:33Z + SO: + + HCs + + + 0.8808372 + mutant + cleaner0 + 2023-06-28T21:04:18Z + MESH: + + H3-23 + + + 0.90260917 + mutant + cleaner0 + 2023-06-28T21:05:06Z + MESH: + + H5-51 + + + 0.9224551 + protein_state + cleaner0 + 2023-06-29T09:34:13Z + DUMMY: + + remarkably well conserved + + + 0.95445126 + structure_element + cleaner0 + 2023-06-28T16:11:33Z + SO: + + HCs + + + 0.803112 + mutant + cleaner0 + 2023-06-28T21:03:20Z + MESH: + + H1-69 + + + 0.8641219 + mutant + cleaner0 + 2023-06-28T21:03:20Z + MESH: + + H1-69 + + + 0.98897785 + residue_name + cleaner0 + 2023-06-29T09:12:53Z + SO: + + glycine + + + 0.9578594 + residue_number + cleaner0 + 2023-06-29T09:20:55Z + DUMMY: + + 26 + + + 0.91396666 + residue_number + cleaner0 + 2023-06-29T09:20:57Z + DUMMY: + + 27 + + + protein_state + DUMMY: + cleaner0 + 2023-06-29T09:34:53Z + + conformational freedom + + + 0.99736977 + structure_element + cleaner0 + 2023-06-28T16:25:23Z + SO: + + CDR + + + 0.67056143 + structure_element + cleaner0 + 2023-06-28T19:04:12Z + SO: + + H1 + + + 0.9806967 + mutant + cleaner0 + 2023-06-28T21:02:50Z + MESH: + + IGHV1-69 + + + 0.61210406 + structure_element + cleaner0 + 2023-06-29T08:14:53Z + SO: + + VH4 + + + 0.9653481 + residue_name + cleaner0 + 2023-06-29T09:12:56Z + SO: + + glycines + + + 0.99733585 + structure_element + cleaner0 + 2023-06-28T16:25:23Z + SO: + + CDR + + + 0.5672671 + structure_element + cleaner0 + 2023-06-28T19:04:12Z + SO: + + H1 + + + 0.8510997 + protein_state + cleaner0 + 2023-06-29T09:34:57Z + DUMMY: + + conformationally unstable + + + + DISCUSS + paragraph + 45281 + Having all 16 VH:VL pairs with the same CDR H3 provides some insights into why molecular modeling efforts of CDR H3 have proven so difficult. As mentioned in the Results section, this data set is composed of 21 Fabs, since 5 of the 16 variants have 2 Fab copies in the asymmetric unit. For the 18 Fabs with complete backbone atoms for CDR H3, 10 have conformations similar to that of the parent, while the others have significantly different conformations (Fig. 6). Thus, it is likely that the CDR H3 conformation is dependent upon 2 dominating factors: 1) amino acid sequence; and 2) VH and VL context. More than half of the variants retain the conformation of the parent despite having differences in the VH:VL pairing. This subset includes 2 structures with 2 copies of the Fab in the asymmetric unit, all of which are nearly identical in conformation. This provides an internal control showing a consistency in the conformations. The remaining 8 structures exhibit “non-parental” conformations, indicating that the VH and VL context can also be a dominating factor influencing CDR H3. Importantly, there are 5 distinctive conformations in this subset. This subset also has 2 structures with 2 Fab copies in the asymmetric unit. Each pair has nearly identical conformations providing an internal check on the consistency of the conformations. Interestingly, as described earlier, these 2 pairs differ in the stem regions with the H1-69:L3-20 pair in the ‘extended’ conformation and H5-51:L4-1 pair in the ‘kinked’ conformation. The conformations are different from each other, as well as from the parent. + + 0.901212 + complex_assembly + cleaner0 + 2023-06-29T09:24:05Z + GO: + + VH:VL + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + 0.19667532 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + 0.2617504 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.36789358 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + structure_element + SO: + cleaner0 + 2023-06-29T07:50:58Z + + VH + + + structure_element + SO: + cleaner0 + 2023-06-28T19:10:07Z + + VL + + + complex_assembly + GO: + cleaner0 + 2023-06-29T07:50:34Z + + VH:VL + + + 0.98431927 + evidence + cleaner0 + 2023-06-29T08:44:18Z + DUMMY: + + structures + + + 0.18366018 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.91228855 + evidence + cleaner0 + 2023-06-29T08:44:24Z + DUMMY: + + structures + + + structure_element + SO: + cleaner0 + 2023-06-29T07:50:49Z + + VH + + + structure_element + SO: + cleaner0 + 2023-06-28T19:10:07Z + + VL + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + 0.96874285 + evidence + cleaner0 + 2023-06-29T08:44:29Z + DUMMY: + + structures + + + 0.16393337 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.99679464 + structure_element + cleaner0 + 2023-06-29T09:04:12Z + SO: + + stem regions + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:21Z + + H1-69:L3-20 + + + 0.9944728 + protein_state + cleaner0 + 2023-06-29T07:41:57Z + DUMMY: + + extended + + + complex_assembly + GO: + cleaner0 + 2023-06-29T07:51:40Z + + H5-51:L4-1 + + + 0.9884319 + protein_state + cleaner0 + 2023-06-29T07:42:05Z + DUMMY: + + kinked + + + + DISCUSS + paragraph + 46902 + The CDR H3 conformational analysis shows that, for each set of variants of one HC paired with the 4 different LCs, both “parental” and “non-parental” conformations are observed. The same variability is observed for the sets of variants composed of one LC paired with each of the 4 HCs. Thus, no patterns of conformational preference for a particular HC or LC emerge to shed any direct light on what drives the conformational differences. This finding supports the hypothesis of Weitzner et al. that the H3 conformation is controlled both by its sequence and its environment. + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:23Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + 0.7433255 + experimental_method + cleaner0 + 2023-06-29T09:09:20Z + MESH: + + conformational analysis + + + 0.9436985 + structure_element + cleaner0 + 2023-06-29T09:04:34Z + SO: + + HC + + + 0.93943304 + structure_element + cleaner0 + 2023-06-28T16:11:39Z + SO: + + LCs + + + 0.93057585 + structure_element + cleaner0 + 2023-06-28T16:24:55Z + SO: + + LC + + + 0.9718633 + structure_element + cleaner0 + 2023-06-28T16:11:33Z + SO: + + HCs + + + 0.8819633 + structure_element + cleaner0 + 2023-06-29T09:04:20Z + SO: + + HC + + + 0.8892341 + structure_element + cleaner0 + 2023-06-28T16:24:55Z + SO: + + LC + + + 0.9856926 + structure_element + cleaner0 + 2023-06-28T19:05:17Z + SO: + + H3 + + + + DISCUSS + paragraph + 47486 + In looking at a possible correlation between the tilt angle and the conformation of CDR H3, no clear trends are observed. Two variants, H1-69:L3-20 and H3-23:L3-20, have the largest differences in the tilt angles compared to other variants as seen in Table 3. The absolute VH:VL orientation parameters for the 2 Fabs (Table S2) show significant deviation in HL, LC1 and HC2 values (2-3 standard deviations from the mean). One of the variants, H3-23:L3-20, has the CDR H3 conformation similar to the parent, but the other, H1-69:L3-20, is different. + + 0.9763776 + evidence + cleaner0 + 2023-06-29T08:44:51Z + DUMMY: + + tilt angle + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + 0.9966311 + complex_assembly + cleaner0 + 2023-06-28T21:20:21Z + GO: + + H1-69:L3-20 + + + 0.9964395 + complex_assembly + cleaner0 + 2023-06-29T07:52:58Z + GO: + + H3-23:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-29T07:53:33Z + + VH:VL + + + 0.9644147 + evidence + cleaner0 + 2023-06-29T08:47:57Z + DUMMY: + + orientation parameters + + + structure_element + SO: + cleaner0 + 2023-06-28T15:58:47Z + + Fabs + + + evidence + DUMMY: + cleaner0 + 2023-06-29T08:47:49Z + + deviation + + + 0.9223454 + structure_element + cleaner0 + 2023-06-29T08:47:12Z + SO: + + HL + + + 0.9130852 + structure_element + cleaner0 + 2023-06-29T08:47:20Z + SO: + + LC1 + + + 0.92378676 + structure_element + cleaner0 + 2023-06-29T08:47:31Z + SO: + + HC2 + + + 0.996636 + complex_assembly + cleaner0 + 2023-06-29T07:53:07Z + GO: + + H3-23:L3-20 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + 0.99652 + complex_assembly + cleaner0 + 2023-06-28T21:20:21Z + GO: + + H1-69:L3-20 + + + + DISCUSS + paragraph + 48037 + As noted in the Results section, the 2 variants, H1-69:L3-20 and H3-23:L3-20, are outliers in terms of the tilt angle; at the same time, both have the smallest VH:VL interface. These smaller interfaces may perhaps translate to a significant deviation in how VH is oriented relative to VL than the other variants. These deviations from the other variants can also be seen to some extent in VH:VL orientation parameters in Table S2, as well as in the smaller number of residues involved in the VH:VL interfaces of these 2 variants (Fig. S5). These differences undoubtedly influence the conformation of the CDRs, in particular CDR H1 (Fig. 1A) and CDR L1 (Fig. 3C), especially with the tandem glycines and multiple serines present, respectively. + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:21Z + + H1-69:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-29T07:53:56Z + + H3-23:L3-20 + + + 0.65877026 + evidence + cleaner0 + 2023-06-29T08:48:01Z + DUMMY: + + tilt angle + + + 0.9951705 + site + cleaner0 + 2023-06-28T20:18:37Z + SO: + + VH:VL interface + + + 0.92253155 + site + cleaner0 + 2023-06-29T09:11:30Z + SO: + + interfaces + + + 0.9880365 + structure_element + cleaner0 + 2023-06-29T09:04:46Z + SO: + + VH + + + 0.9885372 + structure_element + cleaner0 + 2023-06-28T19:10:08Z + SO: + + VL + + + complex_assembly + GO: + cleaner0 + 2023-06-29T07:54:20Z + + VH:VL + + + 0.99509436 + site + cleaner0 + 2023-06-29T09:11:34Z + SO: + + VH:VL interfaces + + + 0.9969213 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.9970937 + structure_element + cleaner0 + 2023-06-28T16:25:24Z + SO: + + CDR + + + 0.9948553 + structure_element + cleaner0 + 2023-06-28T19:04:12Z + SO: + + H1 + + + 0.9974147 + structure_element + cleaner0 + 2023-06-28T16:25:24Z + SO: + + CDR + + + 0.9947161 + structure_element + cleaner0 + 2023-06-28T16:26:32Z + SO: + + L1 + + + 0.9838372 + residue_name + cleaner0 + 2023-06-29T09:13:01Z + SO: + + glycines + + + 0.986471 + residue_name + cleaner0 + 2023-06-29T09:13:04Z + SO: + + serines + + + + DISCUSS + paragraph + 48784 + Pairing of different germlines yields antibodies with various degrees of stability. As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set. Structural determinants of the differential stability are not always easy to decipher. One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3. Other germlines have bulky residues, Tyr, Arg and Trp, at these positions, whereas L1-39 has Ser and Thr. Various combinations of germline sequences for VL and VH impose certain constraints on CDR H3, which has to adapt to the environment. A more compact CDR L3 may be beneficial in this situation. + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:38Z + + antibodies + + + 0.99466765 + evidence + cleaner0 + 2023-06-29T08:48:10Z + DUMMY: + + melting temperatures + + + 0.75309616 + mutant + cleaner0 + 2023-06-28T21:03:21Z + MESH: + + H1-69 + + + 0.8418204 + mutant + cleaner0 + 2023-06-28T21:04:18Z + MESH: + + H3-23 + + + 0.9949491 + structure_element + cleaner0 + 2023-06-29T09:05:01Z + SO: + + HC + + + 0.4990733 + mutant + cleaner0 + 2023-06-28T21:05:51Z + MESH: + + L1-39 + + + 0.9483518 + structure_element + cleaner0 + 2023-06-28T16:24:55Z + SO: + + LC + + + 0.48471507 + protein_state + cleaner0 + 2023-06-29T09:35:47Z + DUMMY: + + stable + + + 0.24393126 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + 0.9337159 + structure_element + cleaner0 + 2023-06-28T16:24:55Z + SO: + + LC + + + mutant + MESH: + cleaner0 + 2023-06-28T21:05:51Z + + L1-39 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-29T07:55:30Z + + L3 + + + 0.907399 + residue_number + cleaner0 + 2023-06-29T09:21:03Z + DUMMY: + + 91 + + + 0.9088576 + residue_number + cleaner0 + 2023-06-29T09:21:06Z + DUMMY: + + 94 + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + 0.99063313 + residue_name + cleaner0 + 2023-06-29T09:13:07Z + SO: + + Tyr + + + 0.9902063 + residue_name + cleaner0 + 2023-06-29T09:13:09Z + SO: + + Arg + + + 0.9905239 + residue_name + cleaner0 + 2023-06-29T09:13:11Z + SO: + + Trp + + + mutant + MESH: + cleaner0 + 2023-06-28T21:05:51Z + + L1-39 + + + 0.9903556 + residue_name + cleaner0 + 2023-06-29T09:13:14Z + SO: + + Ser + + + 0.99004596 + residue_name + cleaner0 + 2023-06-29T09:13:16Z + SO: + + Thr + + + 0.8680984 + structure_element + cleaner0 + 2023-06-28T19:10:08Z + SO: + + VL + + + 0.82958955 + structure_element + cleaner0 + 2023-06-29T09:05:03Z + SO: + + VH + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + 0.93768626 + protein_state + cleaner0 + 2023-06-29T09:35:55Z + DUMMY: + + compact + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-29T07:55:42Z + + L3 + + + + DISCUSS + paragraph + 49613 + At the other end of the stability range is LC germline L3-20, which yields antibodies with the lowest Tms. While pairings with H3-53 and H5-51 may be safely called a mismatch, those with H1-69 and H3-23 have Tms about 5-6° higher. Curiously, the 2 Fabs, H1-69:L3-20 and H3-23:L3-20, deviate markedly in their tilt angles from the rest of the panel. It is possible that by adopting extreme tilt angles the structure modulates CDR H3 and its environment, which apparently cannot be achieved solely by conformational rearrangement of the CDR. Note that most of the VH:VL interface residues are invariant; therefore, significant change of the tilt angle must come with a penalty in free energy. Yet, for the 2 antibodies, the total gain in stability merits the domain repacking. + + structure_element + SO: + cleaner0 + 2023-06-28T16:24:55Z + + LC + + + 0.9399285 + mutant + cleaner0 + 2023-06-28T21:07:17Z + MESH: + + L3-20 + + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:38Z + + antibodies + + + 0.826334 + evidence + cleaner0 + 2023-06-29T08:48:15Z + DUMMY: + + Tms + + + 0.85578394 + mutant + cleaner0 + 2023-06-28T21:08:33Z + MESH: + + H3-53 + + + 0.909995 + mutant + cleaner0 + 2023-06-28T21:05:06Z + MESH: + + H5-51 + + + mutant + MESH: + cleaner0 + 2023-06-28T21:03:21Z + + H1-69 + + + 0.83195966 + mutant + cleaner0 + 2023-06-28T21:04:18Z + MESH: + + H3-23 + + + 0.72107553 + evidence + cleaner0 + 2023-06-29T08:48:19Z + DUMMY: + + Tms + + + 0.25456816 + structure_element + cleaner0 + 2023-06-28T15:58:47Z + SO: + + Fabs + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:21Z + + H1-69:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-29T07:57:01Z + + H3-23:L3-20 + + + 0.7603384 + evidence + cleaner0 + 2023-06-28T16:02:51Z + DUMMY: + + tilt angles + + + evidence + DUMMY: + cleaner0 + 2023-06-28T16:02:51Z + + tilt angles + + + 0.9946995 + evidence + cleaner0 + 2023-06-29T08:48:28Z + DUMMY: + + structure + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + 0.99783033 + structure_element + cleaner0 + 2023-06-28T16:25:24Z + SO: + + CDR + + + 0.9948602 + site + cleaner0 + 2023-06-28T20:18:37Z + SO: + + VH:VL interface + + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:38Z + + antibodies + + + + DISCUSS + paragraph + 50389 + Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft. The final conformation represents an energetic minimum; however, in most cases it is very shallow, so that a single mutation can cause a dramatic rearrangement of the structure. + + 0.21189184 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.99219584 + evidence + cleaner0 + 2023-06-29T08:48:36Z + DUMMY: + + Tm + + + 0.99844164 + structure_element + cleaner0 + 2023-06-29T09:05:09Z + SO: + + HC + + + 0.99835795 + structure_element + cleaner0 + 2023-06-28T16:24:55Z + SO: + + LC + + + 0.97715676 + structure_element + cleaner0 + 2023-06-29T09:05:17Z + SO: + + variable domains + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + 0.9936478 + site + cleaner0 + 2023-06-29T09:11:40Z + SO: + + VH:VL cleft + + + 0.99620557 + evidence + cleaner0 + 2023-06-29T08:48:39Z + DUMMY: + + structure + + + + DISCUSS + paragraph + 50737 + In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. Comparison of the CDR H3s reveals a large set of variants with conformations similar to the parent, while a second set has significant conformational variability, indicating that both the sequence and the structural context define the CDR H3 conformation. Quite unexpectedly, 2 of the variants, H1-69:L3-20 and H3-53:L4-1, have the ‘extended’ stem region differing from the other 14 that have a ‘kinked’ stem region. Why this is the case is unclear at present. These data reveal the difficulty of modeling CDR H3 accurately, as shown again in Antibody Modeling Assessment II. Furthermore, antibody CDRs, H3 in particular, may go through conformational changes upon binding their targets, making structural prediction for docking purposes an even more difficult task. Fortunately, for most applications of antibody modeling, such as engineering affinity and biophysical properties, an accurate CDR H3 structure is not always necessary. For those applications where accurate CDR structures are essential, such as docking, the results in this work demonstrate the importance of experimental structures. With the recent advances in expression and crystallization methods, Fab structures can be obtained rapidly. + + 0.8765336 + experimental_method + cleaner0 + 2023-06-29T09:09:28Z + MESH: + + structural library + + + 0.934844 + structure_element + cleaner0 + 2023-06-28T16:11:33Z + SO: + + HCs + + + structure_element + SO: + cleaner0 + 2023-06-29T07:57:39Z + + LCs + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.8813323 + evidence + cleaner0 + 2023-06-29T08:48:42Z + DUMMY: + + structure + + + 0.9966403 + structure_element + cleaner0 + 2023-06-28T16:26:32Z + SO: + + L1 + + + 0.99470454 + structure_element + cleaner0 + 2023-06-28T20:09:54Z + SO: + + L2 + + + 0.9959739 + structure_element + cleaner0 + 2023-06-29T09:05:21Z + SO: + + L3 + + + 0.99525684 + structure_element + cleaner0 + 2023-06-28T19:04:13Z + SO: + + H1 + + + 0.9918994 + structure_element + cleaner0 + 2023-06-28T16:26:22Z + SO: + + H2 + + + 0.9839631 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-29T07:58:01Z + + H3s + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:20:21Z + + H1-69:L3-20 + + + complex_assembly + GO: + cleaner0 + 2023-06-28T21:18:21Z + + H3-53:L4-1 + + + 0.87856656 + protein_state + cleaner0 + 2023-06-29T07:41:57Z + DUMMY: + + extended + + + 0.9895041 + structure_element + cleaner0 + 2023-06-29T09:05:30Z + SO: + + stem region + + + 0.5306472 + protein_state + cleaner0 + 2023-06-29T07:42:05Z + DUMMY: + + kinked + + + 0.98894227 + structure_element + cleaner0 + 2023-06-29T09:05:32Z + SO: + + stem region + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.9935402 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + 0.9950309 + structure_element + cleaner0 + 2023-06-28T19:05:17Z + SO: + + H3 + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + structure_element + SO: + cleaner0 + 2023-06-28T16:25:24Z + + CDR + + + structure_element + SO: + cleaner0 + 2023-06-28T19:05:17Z + + H3 + + + 0.98669493 + evidence + cleaner0 + 2023-06-29T08:48:51Z + DUMMY: + + structure + + + 0.991574 + structure_element + cleaner0 + 2023-06-28T16:25:24Z + SO: + + CDR + + + 0.70661396 + evidence + cleaner0 + 2023-06-29T08:48:54Z + DUMMY: + + structures + + + 0.9209164 + evidence + cleaner0 + 2023-06-29T08:48:57Z + DUMMY: + + structures + + + 0.95461094 + experimental_method + cleaner0 + 2023-06-29T09:09:54Z + MESH: + + expression and crystallization methods + + + 0.17987959 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.9961416 + evidence + cleaner0 + 2023-06-29T08:49:00Z + DUMMY: + + structures + + + + DISCUSS + paragraph + 52257 + The set of 16 germline Fab structures offers a unique dataset to facilitate software development for antibody modeling. The results essentially support the underlying idea of canonical structures, indicating that most CDRs with germline sequences tend to adopt predefined conformations. From this point of view, a novel approach to design combinatorial antibody libraries would be to cover the range of CDR conformations that may not necessarily coincide with the germline usage in the human repertoire. This would insure more structural diversity, leading to a more diverse panel of antibodies that would bind to a broad spectrum of targets. + + 0.23744409 + structure_element + cleaner0 + 2023-06-28T15:59:05Z + SO: + + Fab + + + 0.99531835 + evidence + cleaner0 + 2023-06-29T08:49:04Z + DUMMY: + + structures + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.9604034 + evidence + cleaner0 + 2023-06-29T08:49:07Z + DUMMY: + + structures + + + 0.99709547 + structure_element + cleaner0 + 2023-06-28T19:04:38Z + SO: + + CDRs + + + protein_type + MESH: + cleaner0 + 2023-06-28T15:45:48Z + + antibody + + + 0.993736 + structure_element + cleaner0 + 2023-06-28T16:25:24Z + SO: + + CDR + + + 0.99389875 + species + cleaner0 + 2023-06-28T16:04:00Z + MESH: + + human + + + protein_type + MESH: + cleaner0 + 2023-06-28T16:03:38Z + + antibodies + + + + METHODS + title_1 + 52900 + Materials and methods + + + METHODS + title_2 + 52922 + Fab production, purification and crystallization + + + METHODS + paragraph + 52971 + The production, purification and crystallization of the Fabs reported in this article were described previously. Briefly, the 16 Fabs were produced by combining 4 different HC and 4 different LC germline constructs. The human HC germlines were IGHV1-69 (H1-69), IGHV3-23 (H3-23), IGHV3-53 (H3-53) and IGHV5-51 (H5-51) in the IMGT nomenclature. The human LC germlines were IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1) corresponding to O12, L6, A27 and B3 in the V-BASE nomenclature. CDR H3 of the anti-CCL2 antibody CNTO 888 with the amino acid sequence ARYDGIYGELDF was used in all Fab constructs. The J region genes were IGHJ1 for the HC and IGKJ1 for the LC for all Fabs. Human IgG1 and κ constant regions were used in all Fab constructs. A 6xHis tag was added to the C-terminus of the HC to facilitate purification. + + + METHODS + paragraph + 53822 + The Fabs were expressed in HEK 293E cells and purified by affinity and size-exclusion chromatography. For crystallization, the Fabs were dialyzed into 20 mM Tris buffer, pH 7.4, with 50 mM NaCl and concentrated to 12-18 mg/mL. Automated crystallization screening was carried out using the vapor diffusion method at 20°C with an Oryx4 (Douglas Instruments) or a Mosquito (TTP Labtech) crystallization robot in a sitting drop format using Corning 3550 plates. Initial screening was carried out with an in-house 192-well screen optimized for Fab crystallization and the Hampton 96-well Crystal Screen HT (Hampton Research). For the majority of the Fabs, the crystallization protocol employed microseed matrix screening using self-seeding or cross-seeding approaches. A summary of the final crystallization conditions for each of the Fabs is presented in Table 1. + + + METHODS + title_2 + 54687 + X-ray data collection + + + METHODS + paragraph + 54709 + For 13 of the Fab crystals, X-ray data collection was carried out at Janssen Research and Development, LLC using a Rigaku MicroMax™-007HF microfocus X-ray generator equipped with a Saturn 944 CCD detector and an X-stream™ 2000 cryocooling system (Rigaku), and for the remaining 3, X-ray data collection was carried out at the Advanced Photon Source (APS) synchrotron at Argonne National Laboratory using the IMCA 17-ID beamline with a Pilatus 6M detector. For X-ray data collection, the Fab crystals were soaked for a few seconds in a cryo-protectant solution containing the corresponding mother liquor supplemented with 17-25% glycerol (Table S1). The crystals for which data were collected in-house were flash cooled in the stream of nitrogen at 100 K. Crystals sent to the APS were flash cooled in liquid nitrogen prior to shipping them to the synchrotron. Diffraction data for all variants were processed with the program XDS. X-ray data statistics are given in Table 1. + + + METHODS + title_2 + 55691 + Structure determination + + + METHODS + paragraph + 55715 + A summary of the methods used in the structure solution and refinement of the 16 Fabs is presented in Table S1. Twelve of the structures were solved by molecular replacement with Phaser using different combinations of search models for the VH, VL and constant domains. Four of the structures, H3-53:L1-39, H3-53:L3-11, H5-51:L1-39 and H5-51:L3-11, were solved by direct replacement followed by rigid body refinement with REFMAC. All structures were refined using REFMAC. Model adjustments were carried out using the program Coot. The refinement statistics are given in Table 1. Other crystallographic calculations were performed with the CCP4 suite of programs. The structural figures were prepared using the PyMOL Molecular Graphics System, Version 1.0 (Schrödinger, LLC). + + + METHODS + title_2 + 56492 + Structural analysis + + + METHODS + paragraph + 56512 + The canonical structure assignments (Table 2) were made using PyIgClassify, an online canonical structure classification tool (http://dunbrack2.fccc.edu/pyigclassify/) that uses the rules set forth by Dunbrack and coworkers. + + + METHODS + paragraph + 56738 + The conformational variability within the CDRs was assessed by calculating the root-mean-square deviation (rmsd) from the average structure that was generated after superposition of all structures of the set using the main-chain atoms of the CDR in question. The rmsd was calculated for all main-chain atoms (N, CA, C, O) of the CDR. + + + METHODS + paragraph + 57072 + The contact surface areas of the VH and VL domains at the VH:VL inteface were computed with the CCP4 program PISA. The surface complementarity of the VH and VL domains was computed using the CCP4 program SC. + + + METHODS + title_2 + 57280 + VH:VL tilt angles + + + METHODS + paragraph + 57298 + The orientation of the VH domain with respect to the VL domain was assessed using 2 different approaches. The first approach calculates the 6 VH:VL orientation parameters that describe the VH:VL relationship according to Dunbar and co-workers using a script downloaded from the website (http://opig.stats.ox.ac.uk/webapps/abangle). The six parameters include 5 angles, HL, H1, H2, L1 and L2, and a distance, dc. These parameters are derived by first defining 2 planes, one for each domain, based on core residues in the domains. The distance between the planes, dc, is determined along a vector between the planes that is used to establish a consistent coordinate system. The torsion angle between the domains, HL, is much like the VH:VL packing angle defined by Abhinandan and Martin. The tilt of one domain relative to the other is defined by the HC1 and LC1 angles, and the twist of one domain relative to the other is defined by the HC2 and LC2 angles. + + + METHODS + paragraph + 58255 + The second approach calculates the difference in the tilt angle between pairs of Fvs, which reflects the relative orientation between the VH and VL domains. The difference with respect to the reference structure is calculated by sequential root-mean-square superposition of the VL and VH domains using β-sheet core Cα positions (Chothia numbering scheme): 3–13, 18–25, 33–38, 43–49, 61–67, 70–76, 85–90, 97–103 for VL; 3–7, 18–24, 34–40, 44–51, 56–59, 67–72, 77–82a, 87–94, 102–110 for VH. The κ angle in the spherical polar angular system (ω, ϕ, κ) of the latter transformation is the difference in the tilt angle. + + + METHODS + title_2 + 58911 + Differential scanning calorimetry + + + METHODS + paragraph + 58945 + DSC experiments were performed on a VP-capillary DSC system (MicroCal Inc., Northampton, MA) in which temperature differences between the reference and sample cell are continuously measured and calibrated to power units. Samples were heated from 10°C to 95°C at a heating rate of 60°C/hour. The pre-scan time was 15 minutes and the filtering period was 10 seconds. The concentration used in the DSC experiments was about 0.4 mg/mL in phosphate-buffered saline. Analysis of the resulting thermograms was performed using MicroCal Origin 7 software. Melting temperature of proteins was determined by deconvolution of the DSC scans using non-2 state model in the MicroCal Origin 7 software. Scans were deconvoluted using a non-2 state model with either 1-step transition or 2-step transition depending on the number of resolved peaks observed in a scan. + + + METHODS + title_2 + 59799 + Accession numbers + + + METHODS + paragraph + 59817 + Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 4KMT, 5I15, 5I16, 5I17, 5I18, 5I19, 5I1A, 5I1C, 5I1D, 5I1E, 5I1G, 5I1H, 5I1I, 5I1J, 5I1K and 5I1L. + + + SUPPL + title_1 + 60025 + Supplementary Material + + + COMP_INT + title_1 + 60048 + Disclosure of potential conflicts of interest + + + COMP_INT + paragraph + 60094 + No potential conflicts of interest were disclosed. + + + REF + title + 60145 + References + + + 197 + 204 + surname:Reichert;given-names:JM + 10.1080/19420862.2015.1125583 + 26651519 + REF + MAbs + ref + 8 + 2016 + 60156 + Antibodies to watch in 2016 + + + 211 + 250 + surname:Wu;given-names:TT + surname:Kabat;given-names:EA + 10.1084/jem.132.2.211 + 5508247 + REF + J Exp Med + ref + 132 + 1970 + 60184 + An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity + + + 901 + 917 + surname:Chothia;given-names:C + surname:Lesk;given-names:AM + 10.1016/0022-2836(87)90412-8 + 3681981 + REF + J Mol Biol + ref + 196 + 1987 + 60338 + Canonical structures for the hypervariable regions of immunoglobulins + + + 877 + 883 + surname:Chothia;given-names:C + surname:Lesk;given-names:AM + surname:Tramontano;given-names:A + surname:Levitt;given-names:M + surname:Smith-Gill;given-names:SJ + surname:Air;given-names:G + surname:Sheriff;given-names:S + surname:Padlan;given-names:EA + surname:Davies;given-names:D + surname:Tulip;given-names:WR + 10.1038/342877a0 + 2687698 + REF + Nature + ref + 342 + 1989 + 60408 + Conformations of immunoglobulin hypervariable regions + + + 799 + 817 + surname:Chothia;given-names:C + surname:Lesk;given-names:AM + surname:Gherardi;given-names:E + surname:Tomlinson;given-names:IM + surname:Walter;given-names:G + surname:Marks;given-names:JD + surname:Llewelyn;given-names:MB + surname:Winter;given-names:G + 10.1016/0022-2836(92)90224-8 + 1404389 + REF + J Mol Biol + ref + 227 + 1992 + 60462 + Structural repertoire of the human VH segments + + + 4628 + 4638 + surname:Tomlinson;given-names:IM + surname:Cox;given-names:JP + surname:Herardi;given-names:GE + surname:Lesk;given-names:AM + surname:Chothia;given-names:C + 7556106 + REF + EMBO J + ref + 14 + 1995 + 60509 + The structural repertoire of the human V kappa domain + + + 497 + 504 + surname:Vargas-Madrazo;given-names:E + surname:Lara-Ochoa;given-names:F + surname:Almagro;given-names:JC + 10.1006/jmbi.1995.0633 + 7490765 + REF + J Mol Biol + ref + 254 + 1995 + 60563 + Canonical structure repertoire of the antigen-binding site of immunoglobulins suggests strong geometrical restrictions associated to the mechanism of immune recognition + + + 927 + 948 + surname:Al-Lazikani;given-names:B + surname:Lesk;given-names:AM + surname:Chothia;given-names:C + 10.1006/jmbi.1997.1354 + 9367782 + REF + J Mol Biol + ref + 273 + 1997 + 60732 + Standard conformations for the canonical structures of immunoglobulins + + + 337 + 354 + surname:Collis;given-names:AV + surname:Brouwer;given-names:AP + surname:Martin;given-names:AC + 10.1016/S0022-2836(02)01222-6 + 12488099 + REF + J Mol Biol + ref + 325 + 2003 + 60803 + Analysis of the antigen combining site: correlations between length and sequence composition of the hypervariable loops and the nature of the antigen + + + 235 + 242 + surname:Berman;given-names:HM + surname:Westbrook;given-names:J + surname:Feng;given-names:Z + surname:Gilliland;given-names:G + surname:Bhat;given-names:TN + surname:Weissig;given-names:H + surname:Shindyalov;given-names:IN + surname:Bourne;given-names:PE + 10.1093/nar/28.1.235 + 10592235 + REF + Nucleic Acids Res + ref + 28 + 2000 + 60953 + The Protein Data Bank + + + 800 + 815 + surname:Martin;given-names:AC + surname:Thornton;given-names:JM + 10.1006/jmbi.1996.0617 + 8947577 + REF + J Mol Biol + ref + 263 + 1996 + 60975 + Structural families in loops of homologous proteins: automatic classification, modelling and application to antibodies + + + 228 + 256 + surname:North;given-names:B + surname:Lehmann;given-names:A + surname:Dunbrack;given-names:RL + 10.1016/j.jmb.2010.10.030 + 21035459 + REF + J Mol Biol + ref + 406 + 2011 + 61094 + A new clustering of antibody CDR loop conformations + + + D432 + D438 + surname:Adolf-Bryfogle;given-names:J + surname:Xu;given-names:Q + surname:North;given-names:B + surname:Lehmann;given-names:A + surname:Dunbrack;given-names:RL + 10.1093/nar/gku1106 + 25392411 + REF + Nucleic Acids Res + ref + 43 + 2015 + 61146 + PyIgClassify: a database of antibody CDR structural classifications + + + 1 + 8 + surname:Shirai;given-names:H + surname:Kidera;given-names:A + surname:Nakamura;given-names:H + 10.1016/S0014-5793(96)01252-5 + 8980108 + REF + FEBS Lett + ref + 399 + 1996 + 61214 + Structural classification of CDR-H3 in antibodies + + + 9 + 16 + surname:Morea;given-names:V + surname:Tramontano;given-names:A + surname:Rustici;given-names:M + surname:Chothia;given-names:C + surname:Lesk;given-names:AM + 10.1016/S0301-4622(96)02266-1 + 9468606 + REF + Biophys Chem + ref + 68 + 1997 + 61264 + Antibody structure, prediction and redesign + + + 269 + 294 + surname:Morea;given-names:V + surname:Tramontano;given-names:A + surname:Rustici;given-names:M + surname:Chothia;given-names:C + surname:Lesk;given-names:AM + 10.1006/jmbi.1997.1442 + 9466909 + REF + J Mol Biol + ref + 275 + 1998 + 61308 + Conformations of the third hypervariable region in the VH domain of immunoglobulins + + + 608 + 620 + surname:Kuroda;given-names:D + surname:Shirai;given-names:H + surname:Kobori;given-names:M + surname:Nakamura;given-names:H + 10.1002/prot.22087 + 18473362 + REF + Proteins + ref + 73 + 2008 + 61392 + Structural classification of CDR-H3 revisited: a lesson in antibody modeling + + + 302 + 311 + surname:Weitzner;given-names:BD + surname:Dunbrack;given-names:RL + surname:Gray;given-names:JJ + 10.1016/j.str.2014.11.010 + 25579815 + REF + Structure + ref + 23 + 2015 + 61469 + The origin of CDR H3 structural diversity + + + 3050 + 3066 + surname:Almagro;given-names:JC + surname:Beavers;given-names:MP + surname:Hernandez-Guzman;given-names:F + surname:Maier;given-names:J + surname:Shaulsky;given-names:J + surname:Butenhof;given-names:K + surname:Labute;given-names:P + surname:Thorsteinson;given-names:N + surname:Kelly;given-names:K + surname:Teplyakov;given-names:A + 10.1002/prot.23130 + 21935986 + REF + Proteins + ref + 79 + 2011 + 61511 + Antibody modeling assessment + + + 1553 + 1562 + surname:Almagro;given-names:JC + surname:Teplyakov;given-names:A + surname:Luo;given-names:J + surname:Sweet;given-names:R + surname:Kodangattil;given-names:S + surname:Hernadez-Guzman;given-names:F + surname:Gilliland;given-names:GL + 10.1002/prot.24567 + 24668560 + REF + Proteins + ref + 82 + 2014 + 61540 + Second Antibody Modeling Assessment (AMA-II) + + + 1563 + 1582 + surname:Teplyakov;given-names:A + surname:Luo;given-names:J + surname:Obmolova;given-names:G + surname:Malia;given-names:TJ + surname:Sweet;given-names:R + surname:Stanfield;given-names:RL + surname:Kodangattil;given-names:S + surname:Almagro;given-names:JC + surname:Gilliland;given-names:GL + 10.1002/prot.24554 + 24633955 + REF + Proteins + ref + 82 + 2014 + 61585 + Antibody modeling assessment II. Structures and models + + + 385 + 396 + surname:Shi;given-names:L + surname:Wheeler;given-names:JC + surname:Sweet;given-names:RW + surname:Lu;given-names:J + surname:Luo;given-names:J + surname:Tornetta;given-names:M + surname:Whitaker;given-names:B + surname:Reddy;given-names:R + surname:Brittingham;given-names:R + surname:Borozdina;given-names:L + 10.1016/j.jmb.2010.01.034 + 20114051 + REF + J Mol Biol + ref + 397 + 2010 + 61640 + De novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pIX fusion proteins + + + 895 + 901 + surname:de Wildt;given-names:RM + surname:Hoet;given-names:RM + surname:van Venrooij;given-names:WJ + surname:Tomlinson;given-names:IM + surname:Winter;given-names:G + 10.1006/jmbi.1998.2396 + 9887257 + REF + J Mol Biol + ref + 285 + 1999 + 61757 + Analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire + + + 227 + 233 + surname:Obmolova;given-names:G + surname:Teplyakov;given-names:A + surname:Malia;given-names:TJ + surname:Grygiel;given-names:TL + surname:Sweet;given-names:R + surname:Snyder;given-names:LA + surname:Gilliland;given-names:GL + 10.1016/j.molimm.2012.03.022 + 22487721 + REF + Mol Immunol + ref + 51 + 2012 + 61869 + Structural basis for high selectivity of anti-CCL2 neutralizing antibody CNTO 888 + + + 1107 + 1115 + surname:Obmolova;given-names:G + surname:Malia;given-names:TJ + surname:Teplyakov;given-names:A + surname:Sweet;given-names:R + surname:Gilliland;given-names:GL + 10.1107/S2053230X14012552 + 25084393 + REF + Acta Crystallogr + ref + F70 + 2014 + 61951 + Protein crystallization with microseed matrix screening: application to human germline antibody Fabs + + + 175 + 182 + surname:Tramontano;given-names:A + surname:Chothia;given-names:C + surname:Lesk;given-names:AM + 10.1016/S0022-2836(05)80102-0 + 2118959 + REF + J Mol Biol + ref + 215 + 1990 + 62052 + Framework residue-71 is a major determinant of the position and conformation of the 2nd hypervariable region in the VH domains of immunoglobulins + + + 483 + 498 + surname:Mas;given-names:MT + surname:Smith;given-names:KC + surname:Yarmush;given-names:DL + surname:Aisaka;given-names:K + surname:Fine;given-names:RM + 10.1002/prot.340140409 + 1438186 + REF + Proteins + ref + 14 + 1992 + 62198 + Modeling the anti-CEA antibody combining site by homology and conformational search + + + 83 + 93 + surname:Stanfield;given-names:RL + surname:Takimoto-Kamimura;given-names:M + surname:Rini;given-names:JM + surname:Profy;given-names:AT + surname:Wilson;given-names:IA + 10.1016/0969-2126(93)90024-B + 8069628 + REF + Structure + ref + 1 + 1993 + 62282 + Major antigen-induced domain rearrangements in an antibody + + + 941 + 953 + surname:Narayanan;given-names:A + surname:Sellers;given-names:BD + surname:Jacobson;given-names:MP + 10.1016/j.jmb.2009.03.043 + 19324053 + REF + J Mol Biol + ref + 388 + 2009 + 62341 + Energy-based analysis and prediction of the orientation between light- and heavy-chain antibody variable domains + + + 689 + 697 + surname:Abhinandan;given-names:KR + surname:Martin;given-names:AC + 10.1093/protein/gzq043 + 20591902 + REF + Protein Eng Des Sel + ref + 23 + 2010 + 62454 + Analysis and prediction of VH/VL packing in antibodies + + + 611 + 620 + surname:Dunbar;given-names:J + surname:Fuchs;given-names:A + surname:Shi;given-names:J + surname:Deane;given-names:CM + 10.1093/protein/gzt020 + 23708320 + REF + Protein Eng Des Sel + ref + 26 + 2013 + 62509 + ABangle: characterising the VH-VL orientation in antibodies + + + 774 + 797 + surname:Krissinel;given-names:E + surname:Henrick;given-names:K + 10.1016/j.jmb.2007.05.022 + 17681537 + REF + J Mol Biol + ref + 372 + 2007 + 62569 + Inference of macromolecular assemblies from crystalline state + + + 946 + 950 + surname:Lawrence;given-names:MC + surname:Colman;given-names:PM + 10.1006/jmbi.1993.1648 + 8263940 + REF + J Mol Biol + ref + 234 + 1993 + 62631 + Shape complementarity at protein/protein interfaces + + + 959 + 965 + surname:Rini;given-names:JM + surname:Schulze-Gahmen;given-names:U + surname:Wilson;given-names:IA + 10.1126/science.1546293 + 1546293 + REF + Science + ref + 255 + 1992 + 62683 + Structural evidence for induced fit as a mechanism for antibody-antigen recognition + + + Appendix 1P + surname:Lefranc;given-names:MP + 10.1002/0471142735.ima01ps40 + 18432650 + REF + Curr Protoc Immunol + ref + 2001 + 62767 + Nomenclature of the human immunoglobulin genes + + + surname:Tomlinson;given-names:IM + surname:Williams;given-names:SC + surname:Ignatovitch;given-names:O + surname:Corbett;given-names:SJ + surname:Winter;given-names:G + REF + V BASE Sequence Directory, MRC Centre for Protein Engineering + ref + 1998 + 62814 + + + 182 + 189 + surname:Zhao;given-names:Y + surname:Gutshall;given-names:L + surname:Jiang;given-names:H + surname:Baker;given-names:A + surname:Beil;given-names:E + surname:Obmolova;given-names:G + surname:Carton;given-names:J + surname:Taudte;given-names:S + surname:Amegadzie;given-names:B + 10.1016/j.pep.2009.04.012 + 19442740 + REF + Protein Expr Purif + ref + 67 + 2009 + 62815 + Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: papain digestion of mAb and transient expression in mammalian cells + + + 550 + 554 + surname:D'Arcy;given-names:A + surname:Villard;given-names:F + surname:Marsh;given-names:M + 10.1107/S0907444907007652 + 17372361 + REF + Acta Crystallogr + ref + D63 + 2007 + 62984 + An automated microseed matrix-screening method for protein crystallization + + + 927 + 933 + surname:Obmolova;given-names:G + surname:Malia;given-names:TJ + surname:Teplyakov;given-names:A + surname:Sweet;given-names:R + surname:Gilliland;given-names:GL + 10.1107/S0907444910026041 + 20693692 + REF + Acta Crystallogr + ref + D66 + 2010 + 63059 + Promoting crystallization of antibody-antigen complexes via microseed matrix screening + + + 125 + 132 + surname:Kabsch;given-names:W + 10.1107/S0907444909047337 + 20124692 + REF + Acta Crystallogr + ref + D66 + 2010 + 63146 + XDS + + + 658 + 674 + surname:McCoy;given-names:AJ + surname:Grosse-Kunstleve;given-names:RW + surname:Adams;given-names:PD + surname:Winn;given-names:MD + surname:Storoni;given-names:LC + surname:Read;given-names:RJ + 10.1107/S0021889807021206 + 19461840 + REF + J Appl Crystallogr + ref + 40 + 2007 + 63150 + Phaser crystallographic software + + + 355 + 367 + surname:Murshudov;given-names:GN + surname:Skubak;given-names:P + surname:Lebedev;given-names:AA + surname:Pannu;given-names:NS + surname:Steiner;given-names:RA + surname:Nicholls;given-names:RA + surname:Winn;given-names:MD + surname:Long;given-names:F + surname:Vagin;given-names:AA + 10.1107/S0907444911001314 + 21460454 + REF + Acta Crystallogr + ref + D67 + 2011 + 63183 + REFMAC5 for the refinement of macromolecular crystal structures + + + 486 + 501 + surname:Emsley;given-names:P + surname:Lohkamp;given-names:B + surname:Scott;given-names:WG + surname:Cowtan;given-names:K + 10.1107/S0907444910007493 + 20383002 + REF + Acta Crystallogr + ref + D66 + 2010 + 63247 + Features and development of Coot + + + 235 + 242 + surname:Winn;given-names:MD + surname:Ballard;given-names:CC + surname:Cowtan;given-names:KD + surname:Dodson;given-names:EJ + surname:Emsley;given-names:P + surname:Evans;given-names:PR + surname:Keegan;given-names:RM + surname:Krissinel;given-names:EB + surname:Leslie;given-names:AG + surname:McCoy;given-names:A + 10.1107/S0907444910045749 + 21460441 + REF + Acta Crystallogr + ref + D67 + 2011 + 63280 + Overview of the CCP4 suite and current developments + + + diff --git a/annotation_CSV/PMC4746701.csv b/annotation_CSV/PMC4746701.csv new file mode 100644 index 0000000000000000000000000000000000000000..733cc5b57c6e034e4f902fdb704d498c4ffba9d3 --- /dev/null +++ b/annotation_CSV/PMC4746701.csv @@ -0,0 +1,1131 @@ +anno_start anno_end anno_text entity_type sentence section +0 17 Crystal structure evidence Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway TITLE +21 26 SEL1L protein Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway TITLE +54 57 SLR structure_element Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway TITLE +0 5 SEL1L protein SEL1L, a component of the ERAD machinery, plays an important role in selecting and transporting ERAD substrates for degradation. ABSTRACT +23 40 crystal structure evidence We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). ABSTRACT +48 53 mouse taxonomy_domain We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). ABSTRACT +54 59 SEL1L protein We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). ABSTRACT +60 74 central domain structure_element We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). ABSTRACT +91 108 Sel1-Like Repeats structure_element We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). ABSTRACT +110 127 SLR motifs 5 to 9 structure_element We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). ABSTRACT +146 155 SEL1Lcent structure_element We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). ABSTRACT +12 21 SEL1Lcent structure_element Strikingly, SEL1Lcent forms a homodimer with two-fold symmetry in a head-to-tail manner. ABSTRACT +30 39 homodimer oligomeric_state Strikingly, SEL1Lcent forms a homodimer with two-fold symmetry in a head-to-tail manner. ABSTRACT +68 80 head-to-tail protein_state Strikingly, SEL1Lcent forms a homodimer with two-fold symmetry in a head-to-tail manner. ABSTRACT +18 29 SLR motif 9 structure_element Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. ABSTRACT +57 62 dimer oligomeric_state Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. ABSTRACT +87 101 domain-swapped protein_state Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. ABSTRACT +139 156 dimeric interface site Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. ABSTRACT +23 34 full-length protein_state We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. ABSTRACT +35 40 SEL1L protein We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. ABSTRACT +49 62 self-oligomer oligomeric_state We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. ABSTRACT +75 84 SEL1Lcent structure_element We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. ABSTRACT +95 104 mammalian taxonomy_domain We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. ABSTRACT +36 41 SLR-C structure_element Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. ABSTRACT +54 74 SLR motifs 10 and 11 structure_element Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. ABSTRACT +79 84 SEL1L protein Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. ABSTRACT +124 137 luminal loops structure_element Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. ABSTRACT +141 145 HRD1 protein Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. ABSTRACT +35 38 SLR structure_element Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery. ABSTRACT +49 54 SEL1L protein Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery. ABSTRACT +114 124 eukaryotes taxonomy_domain Protein quality control in the endoplasmic reticulum (ER) is essential for maintenance of cellular homeostasis in eukaryotes and is implicated in many severe diseases. INTRO +105 122 polyubiquitinated protein_state Terminally misfolded proteins in the lumen or membrane of the ER are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. INTRO +144 154 proteasome complex_assembly Terminally misfolded proteins in the lumen or membrane of the ER are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. INTRO +70 79 conserved protein_state The process is called ER-associated protein degradation (ERAD) and is conserved in all eukaryotes. INTRO +87 97 eukaryotes taxonomy_domain The process is called ER-associated protein degradation (ERAD) and is conserved in all eukaryotes. INTRO +72 76 HRD1 protein Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +78 83 SEL1L protein Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +85 90 Hrd3p protein Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +93 109 Derlin-1, -2, -3 protein Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +111 116 Der1p protein Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +119 129 HERP-1, -2 protein Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +131 136 Usa1p protein Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +139 142 OS9 protein Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +144 148 Yos9 protein Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +151 156 XTP-B protein Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +162 167 Grp94 protein Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +258 263 yeast taxonomy_domain Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +268 277 metazoans taxonomy_domain Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. INTRO +0 5 Yeast taxonomy_domain Yeast ERAD components, which have been extensively characterized through genetic and biochemical studies, are comparable with mammalian ERAD components, sharing similar molecular functions and structural composition. INTRO +73 104 genetic and biochemical studies experimental_method Yeast ERAD components, which have been extensively characterized through genetic and biochemical studies, are comparable with mammalian ERAD components, sharing similar molecular functions and structural composition. INTRO +126 135 mammalian taxonomy_domain Yeast ERAD components, which have been extensively characterized through genetic and biochemical studies, are comparable with mammalian ERAD components, sharing similar molecular functions and structural composition. INTRO +4 8 HRD1 protein The HRD1 E3 ubiquitin ligase, which is embedded in the ER membrane, is involved in translocating ERAD substrates across the ER membrane and catalyzing substrate ubiquitination via its cytosolic RING finger domain. INTRO +9 28 E3 ubiquitin ligase protein_type The HRD1 E3 ubiquitin ligase, which is embedded in the ER membrane, is involved in translocating ERAD substrates across the ER membrane and catalyzing substrate ubiquitination via its cytosolic RING finger domain. INTRO +194 212 RING finger domain structure_element The HRD1 E3 ubiquitin ligase, which is embedded in the ER membrane, is involved in translocating ERAD substrates across the ER membrane and catalyzing substrate ubiquitination via its cytosolic RING finger domain. INTRO +0 5 SEL1L protein SEL1L, the mammalian homolog of Hrd3p, associates with HRD1, mediates HRD1 interactions with the ER luminal lectin OS9, and recognizes substrates to be degraded. INTRO +11 20 mammalian taxonomy_domain SEL1L, the mammalian homolog of Hrd3p, associates with HRD1, mediates HRD1 interactions with the ER luminal lectin OS9, and recognizes substrates to be degraded. INTRO +32 37 Hrd3p protein SEL1L, the mammalian homolog of Hrd3p, associates with HRD1, mediates HRD1 interactions with the ER luminal lectin OS9, and recognizes substrates to be degraded. INTRO +55 59 HRD1 protein SEL1L, the mammalian homolog of Hrd3p, associates with HRD1, mediates HRD1 interactions with the ER luminal lectin OS9, and recognizes substrates to be degraded. INTRO +70 74 HRD1 protein SEL1L, the mammalian homolog of Hrd3p, associates with HRD1, mediates HRD1 interactions with the ER luminal lectin OS9, and recognizes substrates to be degraded. INTRO +108 114 lectin protein_type SEL1L, the mammalian homolog of Hrd3p, associates with HRD1, mediates HRD1 interactions with the ER luminal lectin OS9, and recognizes substrates to be degraded. INTRO +115 118 OS9 protein SEL1L, the mammalian homolog of Hrd3p, associates with HRD1, mediates HRD1 interactions with the ER luminal lectin OS9, and recognizes substrates to be degraded. INTRO +15 20 SEL1L protein In particular, SEL1L is crucial for translocation of Class I major histocompatibility complex (MHC) heavy chains (HCs). INTRO +53 93 Class I major histocompatibility complex complex_assembly In particular, SEL1L is crucial for translocation of Class I major histocompatibility complex (MHC) heavy chains (HCs). INTRO +95 98 MHC complex_assembly In particular, SEL1L is crucial for translocation of Class I major histocompatibility complex (MHC) heavy chains (HCs). INTRO +100 112 heavy chains protein_type In particular, SEL1L is crucial for translocation of Class I major histocompatibility complex (MHC) heavy chains (HCs). INTRO +114 117 HCs protein_type In particular, SEL1L is crucial for translocation of Class I major histocompatibility complex (MHC) heavy chains (HCs). INTRO +39 44 Sel1l gene Recent research based on the inducible Sel1l knockout mouse model highlights the physiological functions of SEL1L. INTRO +45 59 knockout mouse experimental_method Recent research based on the inducible Sel1l knockout mouse model highlights the physiological functions of SEL1L. INTRO +108 113 SEL1L protein Recent research based on the inducible Sel1l knockout mouse model highlights the physiological functions of SEL1L. INTRO +0 5 SEL1L protein SEL1L is required for ER homeostasis, which is essential for protein translation, pancreatic function, and cellular and organismal survival. INTRO +46 51 SEL1L protein However, despite the functional importance of SEL1L, the molecular structure of SEL1L has not been solved. INTRO +67 76 structure evidence However, despite the functional importance of SEL1L, the molecular structure of SEL1L has not been solved. INTRO +80 85 SEL1L protein However, despite the functional importance of SEL1L, the molecular structure of SEL1L has not been solved. INTRO +9 28 biochemical studies experimental_method Previous biochemical studies reveal that SEL1L is a type I transmembrane protein and has a large luminal domain comprising sets of repeated Sel1-like (SLR) motifs. INTRO +41 46 SEL1L protein Previous biochemical studies reveal that SEL1L is a type I transmembrane protein and has a large luminal domain comprising sets of repeated Sel1-like (SLR) motifs. INTRO +52 80 type I transmembrane protein protein_type Previous biochemical studies reveal that SEL1L is a type I transmembrane protein and has a large luminal domain comprising sets of repeated Sel1-like (SLR) motifs. INTRO +97 111 luminal domain structure_element Previous biochemical studies reveal that SEL1L is a type I transmembrane protein and has a large luminal domain comprising sets of repeated Sel1-like (SLR) motifs. INTRO +131 149 repeated Sel1-like structure_element Previous biochemical studies reveal that SEL1L is a type I transmembrane protein and has a large luminal domain comprising sets of repeated Sel1-like (SLR) motifs. INTRO +151 154 SLR structure_element Previous biochemical studies reveal that SEL1L is a type I transmembrane protein and has a large luminal domain comprising sets of repeated Sel1-like (SLR) motifs. INTRO +4 7 SLR structure_element The SLR motif is a structural motif that closely resembles the tetratricopeptide-repeat (TPR) motif, which is a protein-protein interaction module. INTRO +63 87 tetratricopeptide-repeat structure_element The SLR motif is a structural motif that closely resembles the tetratricopeptide-repeat (TPR) motif, which is a protein-protein interaction module. INTRO +89 92 TPR structure_element The SLR motif is a structural motif that closely resembles the tetratricopeptide-repeat (TPR) motif, which is a protein-protein interaction module. INTRO +36 50 luminal domain structure_element Although there is evidence that the luminal domain of SEL1L is involved in substrate recognition or in forming complexes with chaperones, it is not known how the unique structure of the repeated SLR motifs contributes to the molecular function of the HRD1-SEL1L E3 ligase complex and affects ERAD at the molecular level. INTRO +54 59 SEL1L protein Although there is evidence that the luminal domain of SEL1L is involved in substrate recognition or in forming complexes with chaperones, it is not known how the unique structure of the repeated SLR motifs contributes to the molecular function of the HRD1-SEL1L E3 ligase complex and affects ERAD at the molecular level. INTRO +126 136 chaperones protein_type Although there is evidence that the luminal domain of SEL1L is involved in substrate recognition or in forming complexes with chaperones, it is not known how the unique structure of the repeated SLR motifs contributes to the molecular function of the HRD1-SEL1L E3 ligase complex and affects ERAD at the molecular level. INTRO +195 198 SLR structure_element Although there is evidence that the luminal domain of SEL1L is involved in substrate recognition or in forming complexes with chaperones, it is not known how the unique structure of the repeated SLR motifs contributes to the molecular function of the HRD1-SEL1L E3 ligase complex and affects ERAD at the molecular level. INTRO +251 261 HRD1-SEL1L complex_assembly Although there is evidence that the luminal domain of SEL1L is involved in substrate recognition or in forming complexes with chaperones, it is not known how the unique structure of the repeated SLR motifs contributes to the molecular function of the HRD1-SEL1L E3 ligase complex and affects ERAD at the molecular level. INTRO +262 271 E3 ligase protein_type Although there is evidence that the luminal domain of SEL1L is involved in substrate recognition or in forming complexes with chaperones, it is not known how the unique structure of the repeated SLR motifs contributes to the molecular function of the HRD1-SEL1L E3 ligase complex and affects ERAD at the molecular level. INTRO +28 31 SLR structure_element Furthermore, while repeated SLR motifs are commonly found in tandem arrays, the SLR motifs in SEL1L are, according to the primary structure prediction of SEL1L, interspersed among other sequences in the luminal domain and form three SLR domain clusters. INTRO +80 83 SLR structure_element Furthermore, while repeated SLR motifs are commonly found in tandem arrays, the SLR motifs in SEL1L are, according to the primary structure prediction of SEL1L, interspersed among other sequences in the luminal domain and form three SLR domain clusters. INTRO +94 99 SEL1L protein Furthermore, while repeated SLR motifs are commonly found in tandem arrays, the SLR motifs in SEL1L are, according to the primary structure prediction of SEL1L, interspersed among other sequences in the luminal domain and form three SLR domain clusters. INTRO +154 159 SEL1L protein Furthermore, while repeated SLR motifs are commonly found in tandem arrays, the SLR motifs in SEL1L are, according to the primary structure prediction of SEL1L, interspersed among other sequences in the luminal domain and form three SLR domain clusters. INTRO +203 217 luminal domain structure_element Furthermore, while repeated SLR motifs are commonly found in tandem arrays, the SLR motifs in SEL1L are, according to the primary structure prediction of SEL1L, interspersed among other sequences in the luminal domain and form three SLR domain clusters. INTRO +233 236 SLR structure_element Furthermore, while repeated SLR motifs are commonly found in tandem arrays, the SLR motifs in SEL1L are, according to the primary structure prediction of SEL1L, interspersed among other sequences in the luminal domain and form three SLR domain clusters. INTRO +64 69 SEL1L protein Therefore, the way in which these unique structural features of SEL1L are related to its critical function in ERAD remains to be elucidated. INTRO +50 53 SLR structure_element To clearly understand the biochemical role of the SLR domains of SEL1L in ERAD, we determined the crystal structure of the central SLR domain of SEL1L. INTRO +65 70 SEL1L protein To clearly understand the biochemical role of the SLR domains of SEL1L in ERAD, we determined the crystal structure of the central SLR domain of SEL1L. INTRO +98 115 crystal structure evidence To clearly understand the biochemical role of the SLR domains of SEL1L in ERAD, we determined the crystal structure of the central SLR domain of SEL1L. INTRO +131 134 SLR structure_element To clearly understand the biochemical role of the SLR domains of SEL1L in ERAD, we determined the crystal structure of the central SLR domain of SEL1L. INTRO +145 150 SEL1L protein To clearly understand the biochemical role of the SLR domains of SEL1L in ERAD, we determined the crystal structure of the central SLR domain of SEL1L. INTRO +18 32 central domain structure_element We found that the central domain of SEL1L, comprising SLR motifs 5 through 9 (SEL1Lcent), forms a tight dimer with two-fold symmetry due to domain swapping of the SLR motif 9. INTRO +36 41 SEL1L protein We found that the central domain of SEL1L, comprising SLR motifs 5 through 9 (SEL1Lcent), forms a tight dimer with two-fold symmetry due to domain swapping of the SLR motif 9. INTRO +54 76 SLR motifs 5 through 9 structure_element We found that the central domain of SEL1L, comprising SLR motifs 5 through 9 (SEL1Lcent), forms a tight dimer with two-fold symmetry due to domain swapping of the SLR motif 9. INTRO +78 87 SEL1Lcent structure_element We found that the central domain of SEL1L, comprising SLR motifs 5 through 9 (SEL1Lcent), forms a tight dimer with two-fold symmetry due to domain swapping of the SLR motif 9. INTRO +104 109 dimer oligomeric_state We found that the central domain of SEL1L, comprising SLR motifs 5 through 9 (SEL1Lcent), forms a tight dimer with two-fold symmetry due to domain swapping of the SLR motif 9. INTRO +163 174 SLR motif 9 structure_element We found that the central domain of SEL1L, comprising SLR motifs 5 through 9 (SEL1Lcent), forms a tight dimer with two-fold symmetry due to domain swapping of the SLR motif 9. INTRO +19 24 SLR-C structure_element We also found that SLR-C, consisting of SLR motifs 10 and 11, directly interacts with the N-terminus luminal loop of HRD1. INTRO +40 60 SLR motifs 10 and 11 structure_element We also found that SLR-C, consisting of SLR motifs 10 and 11, directly interacts with the N-terminus luminal loop of HRD1. INTRO +101 113 luminal loop structure_element We also found that SLR-C, consisting of SLR motifs 10 and 11, directly interacts with the N-terminus luminal loop of HRD1. INTRO +117 121 HRD1 protein We also found that SLR-C, consisting of SLR motifs 10 and 11, directly interacts with the N-terminus luminal loop of HRD1. INTRO +60 63 SLR structure_element Based on these observations, we propose a model wherein the SLR domains of SEL1L contribute to the formation of stable oligomers of the ERAD translocation machinery, which is indispensable for ERAD. INTRO +75 80 SEL1L protein Based on these observations, we propose a model wherein the SLR domains of SEL1L contribute to the formation of stable oligomers of the ERAD translocation machinery, which is indispensable for ERAD. INTRO +112 118 stable protein_state Based on these observations, we propose a model wherein the SLR domains of SEL1L contribute to the formation of stable oligomers of the ERAD translocation machinery, which is indispensable for ERAD. INTRO +119 128 oligomers oligomeric_state Based on these observations, we propose a model wherein the SLR domains of SEL1L contribute to the formation of stable oligomers of the ERAD translocation machinery, which is indispensable for ERAD. INTRO +0 23 Structure Determination experimental_method Structure Determination of SEL1Lcent RESULTS +27 36 SEL1Lcent structure_element Structure Determination of SEL1Lcent RESULTS +4 16 Mus musculus species The Mus musculus SEL1L protein contains 790 amino acids and has 17% sequence identity to its yeast homolog, Hrd3p. RESULTS +17 22 SEL1L protein The Mus musculus SEL1L protein contains 790 amino acids and has 17% sequence identity to its yeast homolog, Hrd3p. RESULTS +93 98 yeast taxonomy_domain The Mus musculus SEL1L protein contains 790 amino acids and has 17% sequence identity to its yeast homolog, Hrd3p. RESULTS +108 113 Hrd3p protein The Mus musculus SEL1L protein contains 790 amino acids and has 17% sequence identity to its yeast homolog, Hrd3p. RESULTS +0 5 Mouse taxonomy_domain Mouse SEL1L contains a fibronectin type II domain at the N-terminus, followed by 11 SLR motifs and a single transmembrane domain at the C-terminus (Fig. 1A). RESULTS +6 11 SEL1L protein Mouse SEL1L contains a fibronectin type II domain at the N-terminus, followed by 11 SLR motifs and a single transmembrane domain at the C-terminus (Fig. 1A). RESULTS +23 49 fibronectin type II domain structure_element Mouse SEL1L contains a fibronectin type II domain at the N-terminus, followed by 11 SLR motifs and a single transmembrane domain at the C-terminus (Fig. 1A). RESULTS +84 87 SLR structure_element Mouse SEL1L contains a fibronectin type II domain at the N-terminus, followed by 11 SLR motifs and a single transmembrane domain at the C-terminus (Fig. 1A). RESULTS +108 128 transmembrane domain structure_element Mouse SEL1L contains a fibronectin type II domain at the N-terminus, followed by 11 SLR motifs and a single transmembrane domain at the C-terminus (Fig. 1A). RESULTS +7 10 SLR structure_element The 11 SLR motifs are located in the ER lumen and account for more than two thirds of the mass of full-length SEL1L. RESULTS +98 109 full-length protein_state The 11 SLR motifs are located in the ER lumen and account for more than two thirds of the mass of full-length SEL1L. RESULTS +110 115 SEL1L protein The 11 SLR motifs are located in the ER lumen and account for more than two thirds of the mass of full-length SEL1L. RESULTS +4 7 SLR structure_element The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). RESULTS +72 88 linker sequences structure_element The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). RESULTS +109 112 SLR structure_element The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). RESULTS +121 126 SLR-N structure_element The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). RESULTS +128 145 SLR motifs 1 to 4 structure_element The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). RESULTS +148 153 SLR-M structure_element The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). RESULTS +155 172 SLR motifs 5 to 9 structure_element The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). RESULTS +179 184 SLR-C structure_element The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). RESULTS +186 205 SLR motifs 10 to 11 structure_element The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). RESULTS +0 18 Sequence alignment experimental_method Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). RESULTS +26 29 SLR structure_element Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). RESULTS +68 83 linker sequence structure_element Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). RESULTS +94 101 336–345 residue_range Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). RESULTS +111 116 SLR-N structure_element Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). RESULTS +121 126 SLR-M structure_element Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). RESULTS +138 153 linker sequence structure_element Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). RESULTS +164 171 528–635 residue_range Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). RESULTS +181 186 SLR-M structure_element Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). RESULTS +191 196 SLR-C structure_element Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). RESULTS +30 41 full-length protein_state We first tried to prepare the full-length mouse SEL1L protein, excluding the transmembrane domain at the C-terminus (residues 735–755), by expression in bacteria. RESULTS +42 47 mouse taxonomy_domain We first tried to prepare the full-length mouse SEL1L protein, excluding the transmembrane domain at the C-terminus (residues 735–755), by expression in bacteria. RESULTS +48 53 SEL1L protein We first tried to prepare the full-length mouse SEL1L protein, excluding the transmembrane domain at the C-terminus (residues 735–755), by expression in bacteria. RESULTS +77 97 transmembrane domain structure_element We first tried to prepare the full-length mouse SEL1L protein, excluding the transmembrane domain at the C-terminus (residues 735–755), by expression in bacteria. RESULTS +126 133 735–755 residue_range We first tried to prepare the full-length mouse SEL1L protein, excluding the transmembrane domain at the C-terminus (residues 735–755), by expression in bacteria. RESULTS +139 161 expression in bacteria experimental_method We first tried to prepare the full-length mouse SEL1L protein, excluding the transmembrane domain at the C-terminus (residues 735–755), by expression in bacteria. RESULTS +13 24 full-length protein_state However, the full-length SEL1L protein aggregated in solution and produced no soluble protein. RESULTS +25 30 SEL1L protein However, the full-length SEL1L protein aggregated in solution and produced no soluble protein. RESULTS +30 35 SEL1L protein To identify a soluble form of SEL1L, we generated serial truncation constructs of SEL1L based on the predicted SLR motifs and highly conserved regions across several different species. RESULTS +50 78 serial truncation constructs experimental_method To identify a soluble form of SEL1L, we generated serial truncation constructs of SEL1L based on the predicted SLR motifs and highly conserved regions across several different species. RESULTS +82 87 SEL1L protein To identify a soluble form of SEL1L, we generated serial truncation constructs of SEL1L based on the predicted SLR motifs and highly conserved regions across several different species. RESULTS +111 114 SLR structure_element To identify a soluble form of SEL1L, we generated serial truncation constructs of SEL1L based on the predicted SLR motifs and highly conserved regions across several different species. RESULTS +126 142 highly conserved protein_state To identify a soluble form of SEL1L, we generated serial truncation constructs of SEL1L based on the predicted SLR motifs and highly conserved regions across several different species. RESULTS +5 10 SLR-N structure_element Both SLR-N (residues 194–343) and SLR-C (residues 639–719) alone could be solubilized with an MBP tag at the N-terminus, but appeared to be polydisperse when analyzed by size-exclusion chromatography. RESULTS +21 28 194–343 residue_range Both SLR-N (residues 194–343) and SLR-C (residues 639–719) alone could be solubilized with an MBP tag at the N-terminus, but appeared to be polydisperse when analyzed by size-exclusion chromatography. RESULTS +34 39 SLR-C structure_element Both SLR-N (residues 194–343) and SLR-C (residues 639–719) alone could be solubilized with an MBP tag at the N-terminus, but appeared to be polydisperse when analyzed by size-exclusion chromatography. RESULTS +50 57 639–719 residue_range Both SLR-N (residues 194–343) and SLR-C (residues 639–719) alone could be solubilized with an MBP tag at the N-terminus, but appeared to be polydisperse when analyzed by size-exclusion chromatography. RESULTS +94 119 MBP tag at the N-terminus experimental_method Both SLR-N (residues 194–343) and SLR-C (residues 639–719) alone could be solubilized with an MBP tag at the N-terminus, but appeared to be polydisperse when analyzed by size-exclusion chromatography. RESULTS +170 199 size-exclusion chromatography experimental_method Both SLR-N (residues 194–343) and SLR-C (residues 639–719) alone could be solubilized with an MBP tag at the N-terminus, but appeared to be polydisperse when analyzed by size-exclusion chromatography. RESULTS +13 27 central region structure_element However, the central region of SEL1L, comprising residues 337–554, was soluble and homogenous in size, as determined by size-exclusion chromatography. RESULTS +31 36 SEL1L protein However, the central region of SEL1L, comprising residues 337–554, was soluble and homogenous in size, as determined by size-exclusion chromatography. RESULTS +58 65 337–554 residue_range However, the central region of SEL1L, comprising residues 337–554, was soluble and homogenous in size, as determined by size-exclusion chromatography. RESULTS +120 149 size-exclusion chromatography experimental_method However, the central region of SEL1L, comprising residues 337–554, was soluble and homogenous in size, as determined by size-exclusion chromatography. RESULTS +44 58 central region structure_element To define compact domain boundaries for the central region of SEL1L, we digested the protein with trypsin and analyzed the proteolysis products by SDS-PAGE and N-terminal sequencing. RESULTS +62 67 SEL1L protein To define compact domain boundaries for the central region of SEL1L, we digested the protein with trypsin and analyzed the proteolysis products by SDS-PAGE and N-terminal sequencing. RESULTS +72 105 digested the protein with trypsin experimental_method To define compact domain boundaries for the central region of SEL1L, we digested the protein with trypsin and analyzed the proteolysis products by SDS-PAGE and N-terminal sequencing. RESULTS +147 155 SDS-PAGE experimental_method To define compact domain boundaries for the central region of SEL1L, we digested the protein with trypsin and analyzed the proteolysis products by SDS-PAGE and N-terminal sequencing. RESULTS +160 181 N-terminal sequencing experimental_method To define compact domain boundaries for the central region of SEL1L, we digested the protein with trypsin and analyzed the proteolysis products by SDS-PAGE and N-terminal sequencing. RESULTS +68 73 SEL1L protein The results of this preliminary biochemical analysis suggested that SEL1L residues 348–533 (SEL1Lcent) would be amenable to structural analysis (Fig. 1A). RESULTS +83 90 348–533 residue_range The results of this preliminary biochemical analysis suggested that SEL1L residues 348–533 (SEL1Lcent) would be amenable to structural analysis (Fig. 1A). RESULTS +92 101 SEL1Lcent structure_element The results of this preliminary biochemical analysis suggested that SEL1L residues 348–533 (SEL1Lcent) would be amenable to structural analysis (Fig. 1A). RESULTS +124 143 structural analysis experimental_method The results of this preliminary biochemical analysis suggested that SEL1L residues 348–533 (SEL1Lcent) would be amenable to structural analysis (Fig. 1A). RESULTS +0 8 Crystals evidence Crystals of SEL1Lcent grew in space group P21 with four copies of SEL1Lcent (a total of 82 kDa) in the asymmetric unit. RESULTS +12 21 SEL1Lcent structure_element Crystals of SEL1Lcent grew in space group P21 with four copies of SEL1Lcent (a total of 82 kDa) in the asymmetric unit. RESULTS +66 75 SEL1Lcent structure_element Crystals of SEL1Lcent grew in space group P21 with four copies of SEL1Lcent (a total of 82 kDa) in the asymmetric unit. RESULTS +4 13 structure evidence The structure was determined by the single-wavelength anomalous diffraction (SAD) method using selenium as the anomalous scatterer (Table 1 and Methods). RESULTS +36 75 single-wavelength anomalous diffraction experimental_method The structure was determined by the single-wavelength anomalous diffraction (SAD) method using selenium as the anomalous scatterer (Table 1 and Methods). RESULTS +77 80 SAD experimental_method The structure was determined by the single-wavelength anomalous diffraction (SAD) method using selenium as the anomalous scatterer (Table 1 and Methods). RESULTS +95 103 selenium chemical The structure was determined by the single-wavelength anomalous diffraction (SAD) method using selenium as the anomalous scatterer (Table 1 and Methods). RESULTS +66 74 selenium chemical The assignment of residues during model building was aided by the selenium atom positions, and the structure was refined with native data to 2.6 Å resolution with Rwork/Rfree values of 20.7/27.7%. RESULTS +99 108 structure evidence The assignment of residues during model building was aided by the selenium atom positions, and the structure was refined with native data to 2.6 Å resolution with Rwork/Rfree values of 20.7/27.7%. RESULTS +163 174 Rwork/Rfree evidence The assignment of residues during model building was aided by the selenium atom positions, and the structure was refined with native data to 2.6 Å resolution with Rwork/Rfree values of 20.7/27.7%. RESULTS +8 17 Structure evidence Overall Structure of SEL1Lcent RESULTS +21 30 SEL1Lcent structure_element Overall Structure of SEL1Lcent RESULTS +4 9 mouse taxonomy_domain The mouse SEL1Lcent crystallized as a homodimer, and there were two homodimers in the crystal asymmetric unit (Fig. 1B,C, Supplementary Fig. 1). RESULTS +10 19 SEL1Lcent structure_element The mouse SEL1Lcent crystallized as a homodimer, and there were two homodimers in the crystal asymmetric unit (Fig. 1B,C, Supplementary Fig. 1). RESULTS +20 32 crystallized experimental_method The mouse SEL1Lcent crystallized as a homodimer, and there were two homodimers in the crystal asymmetric unit (Fig. 1B,C, Supplementary Fig. 1). RESULTS +38 47 homodimer oligomeric_state The mouse SEL1Lcent crystallized as a homodimer, and there were two homodimers in the crystal asymmetric unit (Fig. 1B,C, Supplementary Fig. 1). RESULTS +68 78 homodimers oligomeric_state The mouse SEL1Lcent crystallized as a homodimer, and there were two homodimers in the crystal asymmetric unit (Fig. 1B,C, Supplementary Fig. 1). RESULTS +8 17 SEL1Lcent structure_element The two SEL1Lcent molecules dimerize in a head-to-tail manner through a two-fold symmetry interface resulting in a cosmos-like shaped structure (Fig. 1B). RESULTS +28 36 dimerize oligomeric_state The two SEL1Lcent molecules dimerize in a head-to-tail manner through a two-fold symmetry interface resulting in a cosmos-like shaped structure (Fig. 1B). RESULTS +42 54 head-to-tail protein_state The two SEL1Lcent molecules dimerize in a head-to-tail manner through a two-fold symmetry interface resulting in a cosmos-like shaped structure (Fig. 1B). RESULTS +72 99 two-fold symmetry interface site The two SEL1Lcent molecules dimerize in a head-to-tail manner through a two-fold symmetry interface resulting in a cosmos-like shaped structure (Fig. 1B). RESULTS +134 143 structure evidence The two SEL1Lcent molecules dimerize in a head-to-tail manner through a two-fold symmetry interface resulting in a cosmos-like shaped structure (Fig. 1B). RESULTS +14 23 structure evidence The resulting structure resembles the yin-yang symbol with overall dimensions of 60 × 60 × 25 Å, where a SEL1Lcent monomer corresponds to half the symbol. RESULTS +105 114 SEL1Lcent structure_element The resulting structure resembles the yin-yang symbol with overall dimensions of 60 × 60 × 25 Å, where a SEL1Lcent monomer corresponds to half the symbol. RESULTS +115 122 monomer oligomeric_state The resulting structure resembles the yin-yang symbol with overall dimensions of 60 × 60 × 25 Å, where a SEL1Lcent monomer corresponds to half the symbol. RESULTS +4 9 dimer oligomeric_state The dimer formation buries a surface area of 1670 Å2 for each monomer, and no significant differences between the protomers were displayed (final root mean square deviation (RMSD) of 0.6 Å for all Cα atoms). RESULTS +62 69 monomer oligomeric_state The dimer formation buries a surface area of 1670 Å2 for each monomer, and no significant differences between the protomers were displayed (final root mean square deviation (RMSD) of 0.6 Å for all Cα atoms). RESULTS +114 123 protomers oligomeric_state The dimer formation buries a surface area of 1670 Å2 for each monomer, and no significant differences between the protomers were displayed (final root mean square deviation (RMSD) of 0.6 Å for all Cα atoms). RESULTS +146 172 root mean square deviation evidence The dimer formation buries a surface area of 1670 Å2 for each monomer, and no significant differences between the protomers were displayed (final root mean square deviation (RMSD) of 0.6 Å for all Cα atoms). RESULTS +174 178 RMSD evidence The dimer formation buries a surface area of 1670 Å2 for each monomer, and no significant differences between the protomers were displayed (final root mean square deviation (RMSD) of 0.6 Å for all Cα atoms). RESULTS +5 13 protomer oligomeric_state Each protomer is composed of ten α-helices, which form the five SLRs, resulting in an elongated curved structure, confirming the primary structure prediction (Fig. 1D). RESULTS +33 42 α-helices structure_element Each protomer is composed of ten α-helices, which form the five SLRs, resulting in an elongated curved structure, confirming the primary structure prediction (Fig. 1D). RESULTS +64 68 SLRs structure_element Each protomer is composed of ten α-helices, which form the five SLRs, resulting in an elongated curved structure, confirming the primary structure prediction (Fig. 1D). RESULTS +4 13 α-helices structure_element The α-helices subdivide the structure into five pairs (A and B) as shown in a number of TPRs and SLRs. RESULTS +28 37 structure evidence The α-helices subdivide the structure into five pairs (A and B) as shown in a number of TPRs and SLRs. RESULTS +55 56 A structure_element The α-helices subdivide the structure into five pairs (A and B) as shown in a number of TPRs and SLRs. RESULTS +61 62 B structure_element The α-helices subdivide the structure into five pairs (A and B) as shown in a number of TPRs and SLRs. RESULTS +88 92 TPRs structure_element The α-helices subdivide the structure into five pairs (A and B) as shown in a number of TPRs and SLRs. RESULTS +97 101 SLRs structure_element The α-helices subdivide the structure into five pairs (A and B) as shown in a number of TPRs and SLRs. RESULTS +0 15 Helices A and B structure_element Helices A and B are 14 and 13 residues long, respectively, and the two helices are connected by a short turn and loop (Fig. 1D). RESULTS +71 78 helices structure_element Helices A and B are 14 and 13 residues long, respectively, and the two helices are connected by a short turn and loop (Fig. 1D). RESULTS +104 108 turn structure_element Helices A and B are 14 and 13 residues long, respectively, and the two helices are connected by a short turn and loop (Fig. 1D). RESULTS +113 117 loop structure_element Helices A and B are 14 and 13 residues long, respectively, and the two helices are connected by a short turn and loop (Fig. 1D). RESULTS +22 26 loop structure_element In addition, a longer loop, consisting of approximately eight amino acids, is inserted between helix B of one SLR and helix A of the next SLR. RESULTS +95 102 helix B structure_element In addition, a longer loop, consisting of approximately eight amino acids, is inserted between helix B of one SLR and helix A of the next SLR. RESULTS +110 113 SLR structure_element In addition, a longer loop, consisting of approximately eight amino acids, is inserted between helix B of one SLR and helix A of the next SLR. RESULTS +118 125 helix A structure_element In addition, a longer loop, consisting of approximately eight amino acids, is inserted between helix B of one SLR and helix A of the next SLR. RESULTS +138 141 SLR structure_element In addition, a longer loop, consisting of approximately eight amino acids, is inserted between helix B of one SLR and helix A of the next SLR. RESULTS +41 45 SLRs structure_element This arrangement is a unique feature for SLRs among the major classes of repeats containing an α-solenoid. RESULTS +95 105 α-solenoid structure_element This arrangement is a unique feature for SLRs among the major classes of repeats containing an α-solenoid. RESULTS +34 44 α-solenoid structure_element Starting from its N-terminus, the α-solenoid of SEL1L extends across a semi-circle in a right-handed superhelix fashion along the rotation axis of the yin-yang circle. RESULTS +48 53 SEL1L protein Starting from its N-terminus, the α-solenoid of SEL1L extends across a semi-circle in a right-handed superhelix fashion along the rotation axis of the yin-yang circle. RESULTS +151 166 yin-yang circle structure_element Starting from its N-terminus, the α-solenoid of SEL1L extends across a semi-circle in a right-handed superhelix fashion along the rotation axis of the yin-yang circle. RESULTS +25 27 9B structure_element However, the last helix, 9B, at the C-terminus adopts a different conformation, lying parallel to the long axis of helix 9A instead of forming an antiparallel SLR. RESULTS +115 123 helix 9A structure_element However, the last helix, 9B, at the C-terminus adopts a different conformation, lying parallel to the long axis of helix 9A instead of forming an antiparallel SLR. RESULTS +159 162 SLR structure_element However, the last helix, 9B, at the C-terminus adopts a different conformation, lying parallel to the long axis of helix 9A instead of forming an antiparallel SLR. RESULTS +28 36 helix 9B structure_element This unique conformation of helix 9B most likely contributes to formation of the dimer structure of SEL1Lcent, as detailed below. RESULTS +81 86 dimer oligomeric_state This unique conformation of helix 9B most likely contributes to formation of the dimer structure of SEL1Lcent, as detailed below. RESULTS +100 109 SEL1Lcent structure_element This unique conformation of helix 9B most likely contributes to formation of the dimer structure of SEL1Lcent, as detailed below. RESULTS +31 34 SLR structure_element With the exception of the last SLR, the four α-helix pairs possess similar conformations, with RMSD values of 0.7 Å for all Cα atoms. RESULTS +45 52 α-helix structure_element With the exception of the last SLR, the four α-helix pairs possess similar conformations, with RMSD values of 0.7 Å for all Cα atoms. RESULTS +95 99 RMSD evidence With the exception of the last SLR, the four α-helix pairs possess similar conformations, with RMSD values of 0.7 Å for all Cα atoms. RESULTS +41 60 pairwise alignments experimental_method Although the sequence similarity for the pairwise alignments varies between 25% and 35%, all the residues present in the SLR motifs are conserved among the five pairs. RESULTS +121 124 SLR structure_element Although the sequence similarity for the pairwise alignments varies between 25% and 35%, all the residues present in the SLR motifs are conserved among the five pairs. RESULTS +136 145 conserved protein_state Although the sequence similarity for the pairwise alignments varies between 25% and 35%, all the residues present in the SLR motifs are conserved among the five pairs. RESULTS +4 7 SLR structure_element The SLR domain of SLR-M ends at residue 524, and C-terminal amino acids 525–533 of the protein are not visible in the electron density map, suggesting that this region is highly flexible. RESULTS +18 23 SLR-M structure_element The SLR domain of SLR-M ends at residue 524, and C-terminal amino acids 525–533 of the protein are not visible in the electron density map, suggesting that this region is highly flexible. RESULTS +40 43 524 residue_number The SLR domain of SLR-M ends at residue 524, and C-terminal amino acids 525–533 of the protein are not visible in the electron density map, suggesting that this region is highly flexible. RESULTS +72 79 525–533 residue_range The SLR domain of SLR-M ends at residue 524, and C-terminal amino acids 525–533 of the protein are not visible in the electron density map, suggesting that this region is highly flexible. RESULTS +118 138 electron density map evidence The SLR domain of SLR-M ends at residue 524, and C-terminal amino acids 525–533 of the protein are not visible in the electron density map, suggesting that this region is highly flexible. RESULTS +171 186 highly flexible protein_state The SLR domain of SLR-M ends at residue 524, and C-terminal amino acids 525–533 of the protein are not visible in the electron density map, suggesting that this region is highly flexible. RESULTS +31 36 dimer oligomeric_state Since no information regarding dimer formation by SEL1L through its SLR motifs is available, we tested whether the SEL1Lcent dimer shown in our crystal structure is a biological unit. RESULTS +50 55 SEL1L protein Since no information regarding dimer formation by SEL1L through its SLR motifs is available, we tested whether the SEL1Lcent dimer shown in our crystal structure is a biological unit. RESULTS +68 71 SLR structure_element Since no information regarding dimer formation by SEL1L through its SLR motifs is available, we tested whether the SEL1Lcent dimer shown in our crystal structure is a biological unit. RESULTS +115 124 SEL1Lcent structure_element Since no information regarding dimer formation by SEL1L through its SLR motifs is available, we tested whether the SEL1Lcent dimer shown in our crystal structure is a biological unit. RESULTS +125 130 dimer oligomeric_state Since no information regarding dimer formation by SEL1L through its SLR motifs is available, we tested whether the SEL1Lcent dimer shown in our crystal structure is a biological unit. RESULTS +144 161 crystal structure evidence Since no information regarding dimer formation by SEL1L through its SLR motifs is available, we tested whether the SEL1Lcent dimer shown in our crystal structure is a biological unit. RESULTS +10 22 cross-linked experimental_method First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. RESULTS +23 32 SEL1Lcent structure_element First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. RESULTS +58 63 SEL1L protein First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. RESULTS +65 74 SEL1Llong mutant First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. RESULTS +85 92 337–554 residue_range First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. RESULTS +126 140 glutaraldehyde chemical First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. RESULTS +142 144 GA chemical First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. RESULTS +149 170 dimethyl suberimidate chemical First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. RESULTS +172 175 DMS chemical First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. RESULTS +206 214 SDS-PAGE experimental_method First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. RESULTS +35 40 dimer oligomeric_state We detected bands at the mass of a dimer for both SEL1Lcent and SEL1Llong when cross-linked with low concentrations of GA (0.005%) or DMS (0.3 mM) (Supplementary Fig. 2A,B). RESULTS +50 59 SEL1Lcent structure_element We detected bands at the mass of a dimer for both SEL1Lcent and SEL1Llong when cross-linked with low concentrations of GA (0.005%) or DMS (0.3 mM) (Supplementary Fig. 2A,B). RESULTS +64 73 SEL1Llong mutant We detected bands at the mass of a dimer for both SEL1Lcent and SEL1Llong when cross-linked with low concentrations of GA (0.005%) or DMS (0.3 mM) (Supplementary Fig. 2A,B). RESULTS +79 91 cross-linked experimental_method We detected bands at the mass of a dimer for both SEL1Lcent and SEL1Llong when cross-linked with low concentrations of GA (0.005%) or DMS (0.3 mM) (Supplementary Fig. 2A,B). RESULTS +119 121 GA chemical We detected bands at the mass of a dimer for both SEL1Lcent and SEL1Llong when cross-linked with low concentrations of GA (0.005%) or DMS (0.3 mM) (Supplementary Fig. 2A,B). RESULTS +134 137 DMS chemical We detected bands at the mass of a dimer for both SEL1Lcent and SEL1Llong when cross-linked with low concentrations of GA (0.005%) or DMS (0.3 mM) (Supplementary Fig. 2A,B). RESULTS +19 49 analytical ultracentrifugation experimental_method Next, we conducted analytical ultracentrifugation of SEL1Lcent. RESULTS +53 62 SEL1Lcent structure_element Next, we conducted analytical ultracentrifugation of SEL1Lcent. RESULTS +20 33 cross-linking experimental_method Consistent with the cross-linking data, analytical ultracentrifugation revealed that the molecular weight of SEL1Lcent corresponds to a dimer (Supplementary Fig. 2C). RESULTS +40 70 analytical ultracentrifugation experimental_method Consistent with the cross-linking data, analytical ultracentrifugation revealed that the molecular weight of SEL1Lcent corresponds to a dimer (Supplementary Fig. 2C). RESULTS +89 105 molecular weight evidence Consistent with the cross-linking data, analytical ultracentrifugation revealed that the molecular weight of SEL1Lcent corresponds to a dimer (Supplementary Fig. 2C). RESULTS +109 118 SEL1Lcent structure_element Consistent with the cross-linking data, analytical ultracentrifugation revealed that the molecular weight of SEL1Lcent corresponds to a dimer (Supplementary Fig. 2C). RESULTS +136 141 dimer oligomeric_state Consistent with the cross-linking data, analytical ultracentrifugation revealed that the molecular weight of SEL1Lcent corresponds to a dimer (Supplementary Fig. 2C). RESULTS +54 59 dimer oligomeric_state Taken together, these data indicate that some kind of dimer is formed in solution. RESULTS +0 15 Dimer Interface site Dimer Interface of SEL1Lcent RESULTS +19 28 SEL1Lcent structure_element Dimer Interface of SEL1Lcent RESULTS +38 67 SLR motif containing proteins protein_type In contrast to a previously described SLR motif containing proteins that exist as monomers in solution, SEL1Lcent forms an intimate two-fold homotypic dimer interface (Figs 1B and 2A). RESULTS +82 90 monomers oligomeric_state In contrast to a previously described SLR motif containing proteins that exist as monomers in solution, SEL1Lcent forms an intimate two-fold homotypic dimer interface (Figs 1B and 2A). RESULTS +104 113 SEL1Lcent structure_element In contrast to a previously described SLR motif containing proteins that exist as monomers in solution, SEL1Lcent forms an intimate two-fold homotypic dimer interface (Figs 1B and 2A). RESULTS +132 166 two-fold homotypic dimer interface site In contrast to a previously described SLR motif containing proteins that exist as monomers in solution, SEL1Lcent forms an intimate two-fold homotypic dimer interface (Figs 1B and 2A). RESULTS +4 19 concave surface site The concave surface of each SEL1L domain comprising helix 5A to 9A encircles its dimer counterpart in an interlocking clasp-like arrangement. RESULTS +28 33 SEL1L protein The concave surface of each SEL1L domain comprising helix 5A to 9A encircles its dimer counterpart in an interlocking clasp-like arrangement. RESULTS +52 66 helix 5A to 9A structure_element The concave surface of each SEL1L domain comprising helix 5A to 9A encircles its dimer counterpart in an interlocking clasp-like arrangement. RESULTS +81 86 dimer oligomeric_state The concave surface of each SEL1L domain comprising helix 5A to 9A encircles its dimer counterpart in an interlocking clasp-like arrangement. RESULTS +64 73 protomers oligomeric_state However, no interactions were seen between the two-fold-related protomers through the concave inner surfaces themselves. RESULTS +86 108 concave inner surfaces site However, no interactions were seen between the two-fold-related protomers through the concave inner surfaces themselves. RESULTS +32 43 SLR motif 9 structure_element Rather, the unique structure of SLR motif 9, consisting of two parallel helices (9A and 9B), is located in the space generated by the concave surface and provides an extensive dimerization interface between the two-fold-related molecules (Fig. 2A). RESULTS +81 83 9A structure_element Rather, the unique structure of SLR motif 9, consisting of two parallel helices (9A and 9B), is located in the space generated by the concave surface and provides an extensive dimerization interface between the two-fold-related molecules (Fig. 2A). RESULTS +88 90 9B structure_element Rather, the unique structure of SLR motif 9, consisting of two parallel helices (9A and 9B), is located in the space generated by the concave surface and provides an extensive dimerization interface between the two-fold-related molecules (Fig. 2A). RESULTS +134 149 concave surface site Rather, the unique structure of SLR motif 9, consisting of two parallel helices (9A and 9B), is located in the space generated by the concave surface and provides an extensive dimerization interface between the two-fold-related molecules (Fig. 2A). RESULTS +176 198 dimerization interface site Rather, the unique structure of SLR motif 9, consisting of two parallel helices (9A and 9B), is located in the space generated by the concave surface and provides an extensive dimerization interface between the two-fold-related molecules (Fig. 2A). RESULTS +0 8 Helix 9B structure_element Helix 9B from one protomer inserts into the empty space surrounded by the concave region in the other monomer, forming a domain-swapped conformation. RESULTS +18 26 protomer oligomeric_state Helix 9B from one protomer inserts into the empty space surrounded by the concave region in the other monomer, forming a domain-swapped conformation. RESULTS +74 88 concave region site Helix 9B from one protomer inserts into the empty space surrounded by the concave region in the other monomer, forming a domain-swapped conformation. RESULTS +102 109 monomer oligomeric_state Helix 9B from one protomer inserts into the empty space surrounded by the concave region in the other monomer, forming a domain-swapped conformation. RESULTS +121 135 domain-swapped protein_state Helix 9B from one protomer inserts into the empty space surrounded by the concave region in the other monomer, forming a domain-swapped conformation. RESULTS +12 30 contact interfaces site Three major contact interfaces are involved in the interactions, and all interfaces are symmetrically related between the dimer subunits (Fig. 2A). RESULTS +73 83 interfaces site Three major contact interfaces are involved in the interactions, and all interfaces are symmetrically related between the dimer subunits (Fig. 2A). RESULTS +122 127 dimer oligomeric_state Three major contact interfaces are involved in the interactions, and all interfaces are symmetrically related between the dimer subunits (Fig. 2A). RESULTS +0 34 Structure-based sequence alignment experimental_method Structure-based sequence alignment of 135 SEL1L phylogenetic sequences using a ConSurf server revealed that the surface residues in the dimer interfaces were highly conserved among the SEL1L orthologs (Fig. 1E). RESULTS +42 47 SEL1L protein Structure-based sequence alignment of 135 SEL1L phylogenetic sequences using a ConSurf server revealed that the surface residues in the dimer interfaces were highly conserved among the SEL1L orthologs (Fig. 1E). RESULTS +79 93 ConSurf server experimental_method Structure-based sequence alignment of 135 SEL1L phylogenetic sequences using a ConSurf server revealed that the surface residues in the dimer interfaces were highly conserved among the SEL1L orthologs (Fig. 1E). RESULTS +136 152 dimer interfaces site Structure-based sequence alignment of 135 SEL1L phylogenetic sequences using a ConSurf server revealed that the surface residues in the dimer interfaces were highly conserved among the SEL1L orthologs (Fig. 1E). RESULTS +158 174 highly conserved protein_state Structure-based sequence alignment of 135 SEL1L phylogenetic sequences using a ConSurf server revealed that the surface residues in the dimer interfaces were highly conserved among the SEL1L orthologs (Fig. 1E). RESULTS +185 190 SEL1L protein Structure-based sequence alignment of 135 SEL1L phylogenetic sequences using a ConSurf server revealed that the surface residues in the dimer interfaces were highly conserved among the SEL1L orthologs (Fig. 1E). RESULTS +7 15 helix 9B structure_element First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR α-helices (5B, 6B, 7B, and 8B) from its counterpart. RESULTS +24 33 SEL1Lcent structure_element First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR α-helices (5B, 6B, 7B, and 8B) from its counterpart. RESULTS +77 89 inner groove site First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR α-helices (5B, 6B, 7B, and 8B) from its counterpart. RESULTS +99 102 SLR structure_element First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR α-helices (5B, 6B, 7B, and 8B) from its counterpart. RESULTS +103 112 α-helices structure_element First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR α-helices (5B, 6B, 7B, and 8B) from its counterpart. RESULTS +114 116 5B structure_element First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR α-helices (5B, 6B, 7B, and 8B) from its counterpart. RESULTS +118 120 6B structure_element First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR α-helices (5B, 6B, 7B, and 8B) from its counterpart. RESULTS +122 124 7B structure_element First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR α-helices (5B, 6B, 7B, and 8B) from its counterpart. RESULTS +130 132 8B structure_element First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR α-helices (5B, 6B, 7B, and 8B) from its counterpart. RESULTS +8 17 interface site In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +19 26 Leu 516 residue_name_number In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +31 38 Tyr 519 residue_name_number In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +42 50 helix 9B structure_element In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +85 109 hydrophobic interactions bond_interaction In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +115 122 Trp 478 residue_name_number In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +126 134 helix 8B structure_element In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +136 143 Val 444 residue_name_number In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +147 155 helix 7B structure_element In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +157 164 Phe 411 residue_name_number In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +168 176 helix 6B structure_element In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +182 189 Leu 380 residue_name_number In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +193 201 helix 5B structure_element In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +211 220 SEL1Lcent structure_element In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +243 254 Interface 1 site In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). RESULTS +15 39 hydrophobic interactions bond_interaction In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. RESULTS +74 81 Tyr 519 residue_name_number In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. RESULTS +111 118 Ile 515 residue_name_number In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. RESULTS +124 131 H-bonds bond_interaction In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. RESULTS +157 166 conserved protein_state In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. RESULTS +167 174 Gln 377 residue_name_number In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. RESULTS +179 186 His 381 residue_name_number In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. RESULTS +190 198 helix 5B structure_element In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. RESULTS +223 231 protomer oligomeric_state In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. RESULTS +18 25 Gln 523 residue_name_number The side chain of Gln 523 forms an H-bond to the side chain of Asp 480 on the two-fold-related protomer (Fig. 2A, Interface 1 detail). RESULTS +35 41 H-bond bond_interaction The side chain of Gln 523 forms an H-bond to the side chain of Asp 480 on the two-fold-related protomer (Fig. 2A, Interface 1 detail). RESULTS +63 70 Asp 480 residue_name_number The side chain of Gln 523 forms an H-bond to the side chain of Asp 480 on the two-fold-related protomer (Fig. 2A, Interface 1 detail). RESULTS +95 103 protomer oligomeric_state The side chain of Gln 523 forms an H-bond to the side chain of Asp 480 on the two-fold-related protomer (Fig. 2A, Interface 1 detail). RESULTS +114 125 Interface 1 site The side chain of Gln 523 forms an H-bond to the side chain of Asp 480 on the two-fold-related protomer (Fig. 2A, Interface 1 detail). RESULTS +26 34 helix 9A structure_element Second, the residues from helix 9A interact with the residues from helix 5A of its counterpart in a head-to-tail orientation. RESULTS +67 75 helix 5A structure_element Second, the residues from helix 9A interact with the residues from helix 5A of its counterpart in a head-to-tail orientation. RESULTS +100 112 head-to-tail protein_state Second, the residues from helix 9A interact with the residues from helix 5A of its counterpart in a head-to-tail orientation. RESULTS +8 17 interface site In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. RESULTS +47 55 helix 9A structure_element In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. RESULTS +67 74 Leu 503 residue_name_number In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. RESULTS +76 83 Tyr 499 residue_name_number In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. RESULTS +117 124 Lys 500 residue_name_number In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. RESULTS +155 177 van der Waals contacts bond_interaction In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. RESULTS +227 235 helix 5A structure_element In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. RESULTS +247 254 Tyr 360 residue_name_number In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. RESULTS +256 263 Leu 356 residue_name_number In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. RESULTS +265 272 Tyr 359 residue_name_number In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. RESULTS +278 285 Leu 363 residue_name_number In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. RESULTS +15 39 hydrophobic interactions bond_interaction In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). RESULTS +60 67 Asn 507 residue_name_number In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). RESULTS +72 79 Ser 510 residue_name_number In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). RESULTS +83 91 helix 9A structure_element In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). RESULTS +97 104 H-bonds bond_interaction In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). RESULTS +110 126 highly conserved protein_state In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). RESULTS +127 134 Arg 384 residue_name_number In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). RESULTS +142 146 loop structure_element In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). RESULTS +155 163 helix 5B structure_element In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). RESULTS +168 170 6A structure_element In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). RESULTS +197 205 protomer oligomeric_state In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). RESULTS +11 19 helix 9B structure_element Third, the helix 9B from each protomer is involved in the dimer interaction by forming a two-fold antiparallel symmetry. RESULTS +30 38 protomer oligomeric_state Third, the helix 9B from each protomer is involved in the dimer interaction by forming a two-fold antiparallel symmetry. RESULTS +58 63 dimer oligomeric_state Third, the helix 9B from each protomer is involved in the dimer interaction by forming a two-fold antiparallel symmetry. RESULTS +66 73 Phe 518 residue_name_number In particular, the side chains of hydrophobic residues, including Phe 518, Leu 521, and Met 524, are directed toward each other, where they make both inter- and intramolecular contacts (Fig. 2A, Interface 3 detail). RESULTS +75 82 Leu 521 residue_name_number In particular, the side chains of hydrophobic residues, including Phe 518, Leu 521, and Met 524, are directed toward each other, where they make both inter- and intramolecular contacts (Fig. 2A, Interface 3 detail). RESULTS +88 95 Met 524 residue_name_number In particular, the side chains of hydrophobic residues, including Phe 518, Leu 521, and Met 524, are directed toward each other, where they make both inter- and intramolecular contacts (Fig. 2A, Interface 3 detail). RESULTS +195 206 Interface 3 site In particular, the side chains of hydrophobic residues, including Phe 518, Leu 521, and Met 524, are directed toward each other, where they make both inter- and intramolecular contacts (Fig. 2A, Interface 3 detail). RESULTS +56 73 crystal structure evidence To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L348–497) lacking SLR motif 9 (helix 9A and 9B) from SEL1Lcent for comparative analysis. RESULTS +101 116 deletion mutant protein_state To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L348–497) lacking SLR motif 9 (helix 9A and 9B) from SEL1Lcent for comparative analysis. RESULTS +118 130 SEL1L348–497 mutant To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L348–497) lacking SLR motif 9 (helix 9A and 9B) from SEL1Lcent for comparative analysis. RESULTS +132 139 lacking protein_state To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L348–497) lacking SLR motif 9 (helix 9A and 9B) from SEL1Lcent for comparative analysis. RESULTS +140 151 SLR motif 9 structure_element To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L348–497) lacking SLR motif 9 (helix 9A and 9B) from SEL1Lcent for comparative analysis. RESULTS +153 161 helix 9A structure_element To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L348–497) lacking SLR motif 9 (helix 9A and 9B) from SEL1Lcent for comparative analysis. RESULTS +166 168 9B structure_element To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L348–497) lacking SLR motif 9 (helix 9A and 9B) from SEL1Lcent for comparative analysis. RESULTS +175 184 SEL1Lcent structure_element To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L348–497) lacking SLR motif 9 (helix 9A and 9B) from SEL1Lcent for comparative analysis. RESULTS +4 19 deletion mutant protein_state The deletion mutant and the wild-type SEL1Lcent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1Lcent (Supplementary Fig. 3). RESULTS +28 37 wild-type protein_state The deletion mutant and the wild-type SEL1Lcent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1Lcent (Supplementary Fig. 3). RESULTS +38 47 SEL1Lcent structure_element The deletion mutant and the wild-type SEL1Lcent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1Lcent (Supplementary Fig. 3). RESULTS +72 79 spectra evidence The deletion mutant and the wild-type SEL1Lcent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1Lcent (Supplementary Fig. 3). RESULTS +83 98 CD spectroscopy experimental_method The deletion mutant and the wild-type SEL1Lcent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1Lcent (Supplementary Fig. 3). RESULTS +120 128 deletion experimental_method The deletion mutant and the wild-type SEL1Lcent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1Lcent (Supplementary Fig. 3). RESULTS +136 147 SLR motif 9 structure_element The deletion mutant and the wild-type SEL1Lcent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1Lcent (Supplementary Fig. 3). RESULTS +190 199 SEL1Lcent structure_element The deletion mutant and the wild-type SEL1Lcent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1Lcent (Supplementary Fig. 3). RESULTS +13 19 mutant protein_state However, the mutant behaved as a monomer in size-exclusion chromatography and analytical ultracentrifugation experiments (Fig. 2B, Supplementary Fig. 2C). RESULTS +33 40 monomer oligomeric_state However, the mutant behaved as a monomer in size-exclusion chromatography and analytical ultracentrifugation experiments (Fig. 2B, Supplementary Fig. 2C). RESULTS +44 73 size-exclusion chromatography experimental_method However, the mutant behaved as a monomer in size-exclusion chromatography and analytical ultracentrifugation experiments (Fig. 2B, Supplementary Fig. 2C). RESULTS +78 108 analytical ultracentrifugation experimental_method However, the mutant behaved as a monomer in size-exclusion chromatography and analytical ultracentrifugation experiments (Fig. 2B, Supplementary Fig. 2C). RESULTS +63 68 dimer oligomeric_state Additionally, to further validate the key residues involved in dimer formation, we generated a triple point mutant (Interface 1, I515A, L516A, and Y519A) of the hydrophobic residues that are involved in dimerization. RESULTS +95 114 triple point mutant protein_state Additionally, to further validate the key residues involved in dimer formation, we generated a triple point mutant (Interface 1, I515A, L516A, and Y519A) of the hydrophobic residues that are involved in dimerization. RESULTS +116 127 Interface 1 site Additionally, to further validate the key residues involved in dimer formation, we generated a triple point mutant (Interface 1, I515A, L516A, and Y519A) of the hydrophobic residues that are involved in dimerization. RESULTS +129 134 I515A mutant Additionally, to further validate the key residues involved in dimer formation, we generated a triple point mutant (Interface 1, I515A, L516A, and Y519A) of the hydrophobic residues that are involved in dimerization. RESULTS +136 141 L516A mutant Additionally, to further validate the key residues involved in dimer formation, we generated a triple point mutant (Interface 1, I515A, L516A, and Y519A) of the hydrophobic residues that are involved in dimerization. RESULTS +147 152 Y519A mutant Additionally, to further validate the key residues involved in dimer formation, we generated a triple point mutant (Interface 1, I515A, L516A, and Y519A) of the hydrophobic residues that are involved in dimerization. RESULTS +203 215 dimerization oligomeric_state Additionally, to further validate the key residues involved in dimer formation, we generated a triple point mutant (Interface 1, I515A, L516A, and Y519A) of the hydrophobic residues that are involved in dimerization. RESULTS +4 23 triple point mutant protein_state The triple point mutant eluted at the monomer position upon size-exclusion chromatography, while the negative control point mutant (Q460A) eluted at the same position as the wild-type. RESULTS +38 45 monomer oligomeric_state The triple point mutant eluted at the monomer position upon size-exclusion chromatography, while the negative control point mutant (Q460A) eluted at the same position as the wild-type. RESULTS +60 89 size-exclusion chromatography experimental_method The triple point mutant eluted at the monomer position upon size-exclusion chromatography, while the negative control point mutant (Q460A) eluted at the same position as the wild-type. RESULTS +118 130 point mutant protein_state The triple point mutant eluted at the monomer position upon size-exclusion chromatography, while the negative control point mutant (Q460A) eluted at the same position as the wild-type. RESULTS +132 137 Q460A mutant The triple point mutant eluted at the monomer position upon size-exclusion chromatography, while the negative control point mutant (Q460A) eluted at the same position as the wild-type. RESULTS +174 183 wild-type protein_state The triple point mutant eluted at the monomer position upon size-exclusion chromatography, while the negative control point mutant (Q460A) eluted at the same position as the wild-type. RESULTS +11 34 single-residue mutation experimental_method Notably, a single-residue mutation (L521A in interface 3) abolished the dimerization of SEL1Lcent (Fig. 2B). RESULTS +36 41 L521A mutant Notably, a single-residue mutation (L521A in interface 3) abolished the dimerization of SEL1Lcent (Fig. 2B). RESULTS +45 56 interface 3 site Notably, a single-residue mutation (L521A in interface 3) abolished the dimerization of SEL1Lcent (Fig. 2B). RESULTS +58 84 abolished the dimerization protein_state Notably, a single-residue mutation (L521A in interface 3) abolished the dimerization of SEL1Lcent (Fig. 2B). RESULTS +88 97 SEL1Lcent structure_element Notably, a single-residue mutation (L521A in interface 3) abolished the dimerization of SEL1Lcent (Fig. 2B). RESULTS +0 7 Leu 521 residue_name_number Leu 521 is located in the dimerization center of the antiparallel 9B helices in the SEL1Lcent dimer. RESULTS +26 45 dimerization center site Leu 521 is located in the dimerization center of the antiparallel 9B helices in the SEL1Lcent dimer. RESULTS +66 76 9B helices structure_element Leu 521 is located in the dimerization center of the antiparallel 9B helices in the SEL1Lcent dimer. RESULTS +84 93 SEL1Lcent structure_element Leu 521 is located in the dimerization center of the antiparallel 9B helices in the SEL1Lcent dimer. RESULTS +94 99 dimer oligomeric_state Leu 521 is located in the dimerization center of the antiparallel 9B helices in the SEL1Lcent dimer. RESULTS +22 53 structural and biochemical data evidence Taken together, these structural and biochemical data demonstrate that SEL1Lcent exists as a dimer in solution and that SLR motif 9 in SEL1Lcent plays an important role in generating a two-fold dimerization interface. RESULTS +71 80 SEL1Lcent structure_element Taken together, these structural and biochemical data demonstrate that SEL1Lcent exists as a dimer in solution and that SLR motif 9 in SEL1Lcent plays an important role in generating a two-fold dimerization interface. RESULTS +93 98 dimer oligomeric_state Taken together, these structural and biochemical data demonstrate that SEL1Lcent exists as a dimer in solution and that SLR motif 9 in SEL1Lcent plays an important role in generating a two-fold dimerization interface. RESULTS +120 131 SLR motif 9 structure_element Taken together, these structural and biochemical data demonstrate that SEL1Lcent exists as a dimer in solution and that SLR motif 9 in SEL1Lcent plays an important role in generating a two-fold dimerization interface. RESULTS +135 144 SEL1Lcent structure_element Taken together, these structural and biochemical data demonstrate that SEL1Lcent exists as a dimer in solution and that SLR motif 9 in SEL1Lcent plays an important role in generating a two-fold dimerization interface. RESULTS +194 216 dimerization interface site Taken together, these structural and biochemical data demonstrate that SEL1Lcent exists as a dimer in solution and that SLR motif 9 in SEL1Lcent plays an important role in generating a two-fold dimerization interface. RESULTS +8 15 Glycine residue_name The Two Glycine Residues (G512 and G513) Create a Hinge for Domain Swapping of SLR Motif 9 RESULTS +26 30 G512 residue_name_number The Two Glycine Residues (G512 and G513) Create a Hinge for Domain Swapping of SLR Motif 9 RESULTS +35 39 G513 residue_name_number The Two Glycine Residues (G512 and G513) Create a Hinge for Domain Swapping of SLR Motif 9 RESULTS +50 55 Hinge structure_element The Two Glycine Residues (G512 and G513) Create a Hinge for Domain Swapping of SLR Motif 9 RESULTS +79 90 SLR Motif 9 structure_element The Two Glycine Residues (G512 and G513) Create a Hinge for Domain Swapping of SLR Motif 9 RESULTS +0 4 SLRs structure_element SLRs of mouse SEL1L were predicted using the TPRpred server. RESULTS +8 13 mouse taxonomy_domain SLRs of mouse SEL1L were predicted using the TPRpred server. RESULTS +14 19 SEL1L protein SLRs of mouse SEL1L were predicted using the TPRpred server. RESULTS +45 59 TPRpred server experimental_method SLRs of mouse SEL1L were predicted using the TPRpred server. RESULTS +25 36 full-length protein_state Based on the prediction, full-length SEL1L contains a total of 11 SLR motifs, and our construct corresponds to SLR motifs 5 through 9. RESULTS +37 42 SEL1L protein Based on the prediction, full-length SEL1L contains a total of 11 SLR motifs, and our construct corresponds to SLR motifs 5 through 9. RESULTS +66 69 SLR structure_element Based on the prediction, full-length SEL1L contains a total of 11 SLR motifs, and our construct corresponds to SLR motifs 5 through 9. RESULTS +111 133 SLR motifs 5 through 9 structure_element Based on the prediction, full-length SEL1L contains a total of 11 SLR motifs, and our construct corresponds to SLR motifs 5 through 9. RESULTS +35 43 helix 9A structure_element Although amino acid sequences from helix 9A and 9B correctly aligned with the regular SLR repeats and corresponded to SLR motif 9 (Fig. 3A), the structural arrangement of the two helices deviated from the common structure for the SLR motif. RESULTS +48 50 9B structure_element Although amino acid sequences from helix 9A and 9B correctly aligned with the regular SLR repeats and corresponded to SLR motif 9 (Fig. 3A), the structural arrangement of the two helices deviated from the common structure for the SLR motif. RESULTS +86 97 SLR repeats structure_element Although amino acid sequences from helix 9A and 9B correctly aligned with the regular SLR repeats and corresponded to SLR motif 9 (Fig. 3A), the structural arrangement of the two helices deviated from the common structure for the SLR motif. RESULTS +118 129 SLR motif 9 structure_element Although amino acid sequences from helix 9A and 9B correctly aligned with the regular SLR repeats and corresponded to SLR motif 9 (Fig. 3A), the structural arrangement of the two helices deviated from the common structure for the SLR motif. RESULTS +179 186 helices structure_element Although amino acid sequences from helix 9A and 9B correctly aligned with the regular SLR repeats and corresponded to SLR motif 9 (Fig. 3A), the structural arrangement of the two helices deviated from the common structure for the SLR motif. RESULTS +230 233 SLR structure_element Although amino acid sequences from helix 9A and 9B correctly aligned with the regular SLR repeats and corresponded to SLR motif 9 (Fig. 3A), the structural arrangement of the two helices deviated from the common structure for the SLR motif. RESULTS +17 34 crystal structure evidence According to our crystal structure, the central axis of helix 9B is almost parallel to that of helix 9A (Fig. 3B). RESULTS +56 64 helix 9B structure_element According to our crystal structure, the central axis of helix 9B is almost parallel to that of helix 9A (Fig. 3B). RESULTS +95 103 helix 9A structure_element According to our crystal structure, the central axis of helix 9B is almost parallel to that of helix 9A (Fig. 3B). RESULTS +38 49 SLR motif 9 structure_element However, this unusual conformation of SLR motif 9 seems to be essential for dimer formation, as described earlier. RESULTS +76 81 dimer oligomeric_state However, this unusual conformation of SLR motif 9 seems to be essential for dimer formation, as described earlier. RESULTS +53 60 Gly 512 residue_name_number For this structural geometry, two adjacent residues, Gly 512 and Gly 513, in SEL1L confer flexibility at this position by adopting main-chain dihedral angles that are disallowed for non-glycine residues. RESULTS +65 72 Gly 513 residue_name_number For this structural geometry, two adjacent residues, Gly 512 and Gly 513, in SEL1L confer flexibility at this position by adopting main-chain dihedral angles that are disallowed for non-glycine residues. RESULTS +77 82 SEL1L protein For this structural geometry, two adjacent residues, Gly 512 and Gly 513, in SEL1L confer flexibility at this position by adopting main-chain dihedral angles that are disallowed for non-glycine residues. RESULTS +47 54 Gly 512 residue_name_number The phi and psi dihedrals are 100° and 20° for Gly 512, and 110° and −20° for Gly 513, respectively (Fig. 3C). RESULTS +78 85 Gly 513 residue_name_number The phi and psi dihedrals are 100° and 20° for Gly 512, and 110° and −20° for Gly 513, respectively (Fig. 3C). RESULTS +0 7 Gly 513 residue_name_number Gly 513 is conserved among other SLR motifs in the SEL1Lcent, but Gly 512 is present only in the SLR motif 9 of SEL1Lcent (Fig. 3A). RESULTS +11 20 conserved protein_state Gly 513 is conserved among other SLR motifs in the SEL1Lcent, but Gly 512 is present only in the SLR motif 9 of SEL1Lcent (Fig. 3A). RESULTS +33 36 SLR structure_element Gly 513 is conserved among other SLR motifs in the SEL1Lcent, but Gly 512 is present only in the SLR motif 9 of SEL1Lcent (Fig. 3A). RESULTS +51 60 SEL1Lcent structure_element Gly 513 is conserved among other SLR motifs in the SEL1Lcent, but Gly 512 is present only in the SLR motif 9 of SEL1Lcent (Fig. 3A). RESULTS +66 73 Gly 512 residue_name_number Gly 513 is conserved among other SLR motifs in the SEL1Lcent, but Gly 512 is present only in the SLR motif 9 of SEL1Lcent (Fig. 3A). RESULTS +97 108 SLR motif 9 structure_element Gly 513 is conserved among other SLR motifs in the SEL1Lcent, but Gly 512 is present only in the SLR motif 9 of SEL1Lcent (Fig. 3A). RESULTS +112 121 SEL1Lcent structure_element Gly 513 is conserved among other SLR motifs in the SEL1Lcent, but Gly 512 is present only in the SLR motif 9 of SEL1Lcent (Fig. 3A). RESULTS +10 17 Gly-Gly structure_element Thus, the Gly-Gly residues generate an unusual sharp bend at the C-terminal SLR motif 9. RESULTS +76 87 SLR motif 9 structure_element Thus, the Gly-Gly residues generate an unusual sharp bend at the C-terminal SLR motif 9. RESULTS +21 28 glycine residue_name The involvement of a glycine residue in forming a hinge for domain swapping has been reported previously. RESULTS +50 55 hinge structure_element The involvement of a glycine residue in forming a hinge for domain swapping has been reported previously. RESULTS +20 27 Gly 513 residue_name_number The significance of Gly 513 is further highlighted by its absolute conservation among different species, including the budding yeast homolog Hrd3p. RESULTS +58 79 absolute conservation protein_state The significance of Gly 513 is further highlighted by its absolute conservation among different species, including the budding yeast homolog Hrd3p. RESULTS +119 132 budding yeast taxonomy_domain The significance of Gly 513 is further highlighted by its absolute conservation among different species, including the budding yeast homolog Hrd3p. RESULTS +141 146 Hrd3p protein The significance of Gly 513 is further highlighted by its absolute conservation among different species, including the budding yeast homolog Hrd3p. RESULTS +41 48 Gly 512 residue_name_number To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). RESULTS +53 60 Gly 513 residue_name_number To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). RESULTS +76 79 SLR structure_element To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). RESULTS +111 125 point mutation experimental_method To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). RESULTS +127 137 Gly to Ala mutant To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). RESULTS +186 193 Gly 512 residue_name_number To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). RESULTS +198 205 Gly 513 residue_name_number To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). RESULTS +241 249 helix 9B structure_element To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). RESULTS +267 275 protomer oligomeric_state To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). RESULTS +321 328 alanine residue_name To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). RESULTS +389 397 mutation experimental_method To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). RESULTS +34 42 mutation experimental_method This means that the effect of the mutation is mainly to generate a more restricted geometry at the hinge region. RESULTS +99 104 hinge structure_element This means that the effect of the mutation is mainly to generate a more restricted geometry at the hinge region. RESULTS +0 5 G512A mutant G512A or G513A alone showed no differences from wild-type in terms of the size-exclusion chromatography elution profile (Fig. 3D), suggesting that the restriction for single glycine flexibility would not be enough to break the swapped structure of helix 9B. RESULTS +9 14 G513A mutant G512A or G513A alone showed no differences from wild-type in terms of the size-exclusion chromatography elution profile (Fig. 3D), suggesting that the restriction for single glycine flexibility would not be enough to break the swapped structure of helix 9B. RESULTS +48 57 wild-type protein_state G512A or G513A alone showed no differences from wild-type in terms of the size-exclusion chromatography elution profile (Fig. 3D), suggesting that the restriction for single glycine flexibility would not be enough to break the swapped structure of helix 9B. RESULTS +74 103 size-exclusion chromatography experimental_method G512A or G513A alone showed no differences from wild-type in terms of the size-exclusion chromatography elution profile (Fig. 3D), suggesting that the restriction for single glycine flexibility would not be enough to break the swapped structure of helix 9B. RESULTS +174 181 glycine residue_name G512A or G513A alone showed no differences from wild-type in terms of the size-exclusion chromatography elution profile (Fig. 3D), suggesting that the restriction for single glycine flexibility would not be enough to break the swapped structure of helix 9B. RESULTS +248 256 helix 9B structure_element G512A or G513A alone showed no differences from wild-type in terms of the size-exclusion chromatography elution profile (Fig. 3D), suggesting that the restriction for single glycine flexibility would not be enough to break the swapped structure of helix 9B. RESULTS +13 26 double mutant protein_state However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). RESULTS +28 33 G512A mutant However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). RESULTS +34 39 G513A mutant However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). RESULTS +93 102 wild-type protein_state However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). RESULTS +120 128 mutation experimental_method However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). RESULTS +161 166 hinge structure_element However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). RESULTS +175 183 helix 9A structure_element However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). RESULTS +188 190 9B structure_element However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). RESULTS +230 238 helix 9B structure_element However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). RESULTS +325 334 SEL1Lcent structure_element However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). RESULTS +23 33 mutated to experimental_method When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely. RESULTS +34 40 lysine residue_name When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely. RESULTS +42 47 G512K mutant When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely. RESULTS +48 53 G513K mutant When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely. RESULTS +60 66 mutant protein_state When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely. RESULTS +119 124 hinge structure_element When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely. RESULTS +197 205 protomer oligomeric_state When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely. RESULTS +209 218 SEL1Lcent structure_element When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely. RESULTS +259 268 SEL1Lcent structure_element When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely. RESULTS +4 9 G512K mutant The G512K/G513K double mutant eluted at the monomer position in size-exclusion chromatography (Fig. 3D). RESULTS +10 15 G513K mutant The G512K/G513K double mutant eluted at the monomer position in size-exclusion chromatography (Fig. 3D). RESULTS +16 29 double mutant protein_state The G512K/G513K double mutant eluted at the monomer position in size-exclusion chromatography (Fig. 3D). RESULTS +44 51 monomer oligomeric_state The G512K/G513K double mutant eluted at the monomer position in size-exclusion chromatography (Fig. 3D). RESULTS +64 93 size-exclusion chromatography experimental_method The G512K/G513K double mutant eluted at the monomer position in size-exclusion chromatography (Fig. 3D). RESULTS +61 69 mutation experimental_method A previous study shows that induction of steric hindrance by mutation destabilizes the dimerization interface of a different protein, ClC transporter. RESULTS +87 109 dimerization interface site A previous study shows that induction of steric hindrance by mutation destabilizes the dimerization interface of a different protein, ClC transporter. RESULTS +134 149 ClC transporter protein_type A previous study shows that induction of steric hindrance by mutation destabilizes the dimerization interface of a different protein, ClC transporter. RESULTS +42 49 Gly 512 residue_name_number Collectively, these data suggest that the Gly 512 and Gly 513 at the connection between helix 9A and 9B play a crucial role in forming the domain-swapped conformation that enables dimer formation. RESULTS +54 61 Gly 513 residue_name_number Collectively, these data suggest that the Gly 512 and Gly 513 at the connection between helix 9A and 9B play a crucial role in forming the domain-swapped conformation that enables dimer formation. RESULTS +88 96 helix 9A structure_element Collectively, these data suggest that the Gly 512 and Gly 513 at the connection between helix 9A and 9B play a crucial role in forming the domain-swapped conformation that enables dimer formation. RESULTS +101 103 9B structure_element Collectively, these data suggest that the Gly 512 and Gly 513 at the connection between helix 9A and 9B play a crucial role in forming the domain-swapped conformation that enables dimer formation. RESULTS +139 153 domain-swapped protein_state Collectively, these data suggest that the Gly 512 and Gly 513 at the connection between helix 9A and 9B play a crucial role in forming the domain-swapped conformation that enables dimer formation. RESULTS +180 185 dimer oligomeric_state Collectively, these data suggest that the Gly 512 and Gly 513 at the connection between helix 9A and 9B play a crucial role in forming the domain-swapped conformation that enables dimer formation. RESULTS +0 5 SEL1L protein SEL1L Forms Self-oligomers through SEL1Lcent domain in vivo RESULTS +12 26 Self-oligomers oligomeric_state SEL1L Forms Self-oligomers through SEL1Lcent domain in vivo RESULTS +35 44 SEL1Lcent structure_element SEL1L Forms Self-oligomers through SEL1Lcent domain in vivo RESULTS +21 26 SEL1L protein Next, we examined if SEL1L also forms self-oligomers in vivo using HEK293T cells. RESULTS +38 52 self-oligomers oligomeric_state Next, we examined if SEL1L also forms self-oligomers in vivo using HEK293T cells. RESULTS +13 24 full-length protein_state We generated full-length SEL1L-HA and SEL1L-FLAG fusion constructs and co-transfected the constructs into HEK293T cells. RESULTS +25 30 SEL1L protein We generated full-length SEL1L-HA and SEL1L-FLAG fusion constructs and co-transfected the constructs into HEK293T cells. RESULTS +31 33 HA experimental_method We generated full-length SEL1L-HA and SEL1L-FLAG fusion constructs and co-transfected the constructs into HEK293T cells. RESULTS +38 43 SEL1L protein We generated full-length SEL1L-HA and SEL1L-FLAG fusion constructs and co-transfected the constructs into HEK293T cells. RESULTS +44 48 FLAG experimental_method We generated full-length SEL1L-HA and SEL1L-FLAG fusion constructs and co-transfected the constructs into HEK293T cells. RESULTS +49 66 fusion constructs experimental_method We generated full-length SEL1L-HA and SEL1L-FLAG fusion constructs and co-transfected the constructs into HEK293T cells. RESULTS +71 85 co-transfected experimental_method We generated full-length SEL1L-HA and SEL1L-FLAG fusion constructs and co-transfected the constructs into HEK293T cells. RESULTS +2 30 co-immunoprecipitation assay experimental_method A co-immunoprecipitation assay using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody showed that full-length SEL1L forms self-oligomers in vivo (Fig. 4A). RESULTS +45 49 FLAG experimental_method A co-immunoprecipitation assay using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody showed that full-length SEL1L forms self-oligomers in vivo (Fig. 4A). RESULTS +71 83 Western blot experimental_method A co-immunoprecipitation assay using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody showed that full-length SEL1L forms self-oligomers in vivo (Fig. 4A). RESULTS +107 109 HA experimental_method A co-immunoprecipitation assay using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody showed that full-length SEL1L forms self-oligomers in vivo (Fig. 4A). RESULTS +131 142 full-length protein_state A co-immunoprecipitation assay using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody showed that full-length SEL1L forms self-oligomers in vivo (Fig. 4A). RESULTS +143 148 SEL1L protein A co-immunoprecipitation assay using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody showed that full-length SEL1L forms self-oligomers in vivo (Fig. 4A). RESULTS +155 169 self-oligomers oligomeric_state A co-immunoprecipitation assay using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody showed that full-length SEL1L forms self-oligomers in vivo (Fig. 4A). RESULTS +31 40 SEL1Lcent structure_element To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. RESULTS +90 101 full-length protein_state To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. RESULTS +102 107 SEL1L protein To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. RESULTS +122 131 SEL1Lcent structure_element To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. RESULTS +136 147 SLR motif 9 structure_element To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. RESULTS +148 156 deletion experimental_method To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. RESULTS +158 170 SEL1L348–497 mutant To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. RESULTS +194 202 fused to experimental_method To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. RESULTS +221 226 SEL1L protein To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. RESULTS +227 242 signal peptides structure_element To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. RESULTS +0 31 Co-immunoprecipitation analysis experimental_method Co-immunoprecipitation analysis showed that the SEL1Lcent was sufficient to physically interact with the full-length SEL1L, while SEL1L348–497 failed to do so (Fig. 4A). RESULTS +48 57 SEL1Lcent structure_element Co-immunoprecipitation analysis showed that the SEL1Lcent was sufficient to physically interact with the full-length SEL1L, while SEL1L348–497 failed to do so (Fig. 4A). RESULTS +105 116 full-length protein_state Co-immunoprecipitation analysis showed that the SEL1Lcent was sufficient to physically interact with the full-length SEL1L, while SEL1L348–497 failed to do so (Fig. 4A). RESULTS +117 122 SEL1L protein Co-immunoprecipitation analysis showed that the SEL1Lcent was sufficient to physically interact with the full-length SEL1L, while SEL1L348–497 failed to do so (Fig. 4A). RESULTS +130 142 SEL1L348–497 mutant Co-immunoprecipitation analysis showed that the SEL1Lcent was sufficient to physically interact with the full-length SEL1L, while SEL1L348–497 failed to do so (Fig. 4A). RESULTS +48 60 SEL1L348–497 mutant Interestingly, however, the expression level of SEL1L348–497 was consistently lower than that of SEL1Lcent (Fig. 4A,B). RESULTS +97 106 SEL1Lcent structure_element Interestingly, however, the expression level of SEL1L348–497 was consistently lower than that of SEL1Lcent (Fig. 4A,B). RESULTS +0 24 Semi-quantitative RT-PCR experimental_method Semi-quantitative RT-PCR revealed no significant difference in transcriptional levels of the two constructs (data not shown). RESULTS +19 31 SEL1L348–497 mutant We speculated that SEL1L348–497 could be secreted while the SEL1Lcent is retained in the ER by association with the endogenous ERAD complex. RESULTS +60 69 SEL1Lcent structure_element We speculated that SEL1L348–497 could be secreted while the SEL1Lcent is retained in the ER by association with the endogenous ERAD complex. RESULTS +8 27 immunoprecipitation experimental_method Indeed, immunoprecipitation followed by western blot analysis using the culture medium detected secreted SEL1L348–497 fragment, but not SEL1Lcent (Fig. 4B). RESULTS +40 52 western blot experimental_method Indeed, immunoprecipitation followed by western blot analysis using the culture medium detected secreted SEL1L348–497 fragment, but not SEL1Lcent (Fig. 4B). RESULTS +105 117 SEL1L348–497 mutant Indeed, immunoprecipitation followed by western blot analysis using the culture medium detected secreted SEL1L348–497 fragment, but not SEL1Lcent (Fig. 4B). RESULTS +136 145 SEL1Lcent structure_element Indeed, immunoprecipitation followed by western blot analysis using the culture medium detected secreted SEL1L348–497 fragment, but not SEL1Lcent (Fig. 4B). RESULTS +35 47 SEL1L348–497 mutant We next examined if the reason why SEL1L348–497 failed to bind to the full-length SEL1L may be because of the lower level of SEL1L348–497 in the ER lumen compared to SEL1Lcent fragment. RESULTS +70 81 full-length protein_state We next examined if the reason why SEL1L348–497 failed to bind to the full-length SEL1L may be because of the lower level of SEL1L348–497 in the ER lumen compared to SEL1Lcent fragment. RESULTS +82 87 SEL1L protein We next examined if the reason why SEL1L348–497 failed to bind to the full-length SEL1L may be because of the lower level of SEL1L348–497 in the ER lumen compared to SEL1Lcent fragment. RESULTS +125 137 SEL1L348–497 mutant We next examined if the reason why SEL1L348–497 failed to bind to the full-length SEL1L may be because of the lower level of SEL1L348–497 in the ER lumen compared to SEL1Lcent fragment. RESULTS +166 175 SEL1Lcent structure_element We next examined if the reason why SEL1L348–497 failed to bind to the full-length SEL1L may be because of the lower level of SEL1L348–497 in the ER lumen compared to SEL1Lcent fragment. RESULTS +23 28 SEL1L protein In order to retain two SEL1L fragments in the ER lumen, we added KDEL ER retention sequence to the C-terminus of both fragments. RESULTS +65 69 KDEL structure_element In order to retain two SEL1L fragments in the ER lumen, we added KDEL ER retention sequence to the C-terminus of both fragments. RESULTS +70 91 ER retention sequence structure_element In order to retain two SEL1L fragments in the ER lumen, we added KDEL ER retention sequence to the C-terminus of both fragments. RESULTS +24 28 KDEL structure_element Indeed, the addition of KDEL peptide increased the level of SEL1L348–497 in the ER lumen (Fig. 4D,E) and the immunostaining analysis showed both constructs were well localized to the ER (Fig. 4C). RESULTS +60 72 SEL1L348–497 mutant Indeed, the addition of KDEL peptide increased the level of SEL1L348–497 in the ER lumen (Fig. 4D,E) and the immunostaining analysis showed both constructs were well localized to the ER (Fig. 4C). RESULTS +109 123 immunostaining experimental_method Indeed, the addition of KDEL peptide increased the level of SEL1L348–497 in the ER lumen (Fig. 4D,E) and the immunostaining analysis showed both constructs were well localized to the ER (Fig. 4C). RESULTS +28 37 SEL1Lcent structure_element We further analyzed whether SEL1Lcent may competitively inhibit the self-oligomerization of SEL1L in vivo. RESULTS +92 97 SEL1L protein We further analyzed whether SEL1Lcent may competitively inhibit the self-oligomerization of SEL1L in vivo. RESULTS +16 30 co-transfected experimental_method To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. RESULTS +50 56 tagged protein_state To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. RESULTS +57 68 full-length protein_state To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. RESULTS +69 74 SEL1L protein To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. RESULTS +76 81 SEL1L protein To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. RESULTS +82 84 HA experimental_method To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. RESULTS +89 94 SEL1L protein To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. RESULTS +95 99 FLAG experimental_method To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. RESULTS +105 121 increasing doses experimental_method To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. RESULTS +125 139 SEL1Lcent-KDEL mutant To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. RESULTS +141 158 SEL1L348–497-KDEL mutant To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. RESULTS +162 184 SEL1Lcent (L521A)-KDEL mutant To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. RESULTS +0 28 Co-immunoprecipitation assay experimental_method Co-immunoprecipitation assay revealed that wild-type SEL1Lcent-KDEL, indeed, competitively disrupted the self-association of the full-length SEL1L (Fig. 4E). RESULTS +43 52 wild-type protein_state Co-immunoprecipitation assay revealed that wild-type SEL1Lcent-KDEL, indeed, competitively disrupted the self-association of the full-length SEL1L (Fig. 4E). RESULTS +53 67 SEL1Lcent-KDEL mutant Co-immunoprecipitation assay revealed that wild-type SEL1Lcent-KDEL, indeed, competitively disrupted the self-association of the full-length SEL1L (Fig. 4E). RESULTS +129 140 full-length protein_state Co-immunoprecipitation assay revealed that wild-type SEL1Lcent-KDEL, indeed, competitively disrupted the self-association of the full-length SEL1L (Fig. 4E). RESULTS +13 30 SEL1L348–497-KDEL mutant In contrast, SEL1L348–497-KDEL and the single-residue mutation L521A in SEL1Lcent did not competitively inhibit the self-association of full-length SEL1L (Fig. 4E,F). RESULTS +63 68 L521A mutant In contrast, SEL1L348–497-KDEL and the single-residue mutation L521A in SEL1Lcent did not competitively inhibit the self-association of full-length SEL1L (Fig. 4E,F). RESULTS +72 81 SEL1Lcent structure_element In contrast, SEL1L348–497-KDEL and the single-residue mutation L521A in SEL1Lcent did not competitively inhibit the self-association of full-length SEL1L (Fig. 4E,F). RESULTS +136 147 full-length protein_state In contrast, SEL1L348–497-KDEL and the single-residue mutation L521A in SEL1Lcent did not competitively inhibit the self-association of full-length SEL1L (Fig. 4E,F). RESULTS +148 153 SEL1L protein In contrast, SEL1L348–497-KDEL and the single-residue mutation L521A in SEL1Lcent did not competitively inhibit the self-association of full-length SEL1L (Fig. 4E,F). RESULTS +28 33 SEL1L protein These data suggest that the SEL1L forms self-oligomers and the oligomerization is mediated by the SEL1Lcent domain in vivo. RESULTS +40 54 self-oligomers oligomeric_state These data suggest that the SEL1L forms self-oligomers and the oligomerization is mediated by the SEL1Lcent domain in vivo. RESULTS +98 107 SEL1Lcent structure_element These data suggest that the SEL1L forms self-oligomers and the oligomerization is mediated by the SEL1Lcent domain in vivo. RESULTS +0 21 Structural Comparison experimental_method Structural Comparison of SEL1L SLRs with TPRs or SLRs of Other Proteins RESULTS +25 30 SEL1L protein Structural Comparison of SEL1L SLRs with TPRs or SLRs of Other Proteins RESULTS +31 35 SLRs structure_element Structural Comparison of SEL1L SLRs with TPRs or SLRs of Other Proteins RESULTS +41 45 TPRs structure_element Structural Comparison of SEL1L SLRs with TPRs or SLRs of Other Proteins RESULTS +49 53 SLRs structure_element Structural Comparison of SEL1L SLRs with TPRs or SLRs of Other Proteins RESULTS +29 33 TPRs structure_element Previous studies reveal that TPRs and SLRs have similar consensus sequences, suggesting that their three-dimensional structures are also similar. RESULTS +38 42 SLRs structure_element Previous studies reveal that TPRs and SLRs have similar consensus sequences, suggesting that their three-dimensional structures are also similar. RESULTS +4 17 superposition experimental_method The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar. RESULTS +30 34 TPRs structure_element The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar. RESULTS +40 45 Cdc23 protein The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar. RESULTS +47 55 S. pombe species The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar. RESULTS +57 79 cell division cycle 23 protein The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar. RESULTS +108 112 SLRs structure_element The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar. RESULTS +118 122 HcpC protein The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar. RESULTS +124 160 Helicobacter Cysteine-rich Protein C protein The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar. RESULTS +184 189 RMSDs evidence The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar. RESULTS +20 23 SLR structure_element This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). RESULTS +34 39 SEL1L protein This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). RESULTS +53 56 SLR structure_element This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). RESULTS +69 78 SEL1Lcent structure_element This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). RESULTS +91 111 structural alignment experimental_method This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). RESULTS +126 130 TPRs structure_element This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). RESULTS +132 136 RMSD evidence This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). RESULTS +167 172 Cdc23 protein This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). RESULTS +183 187 SLRs structure_element This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). RESULTS +189 193 RMSD evidence This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). RESULTS +224 228 HcpC protein This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). RESULTS +9 22 superimposing experimental_method However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +27 36 structure evidence However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +40 57 SLR motifs 5 to 9 structure_element However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +63 72 SEL1Lcent structure_element However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +90 95 Cdc23 protein However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +105 116 full-length protein_state However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +117 121 HcpC protein However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +122 132 structures evidence However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +147 164 SLR motifs 5 to 9 structure_element However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +168 177 SEL1Lcent structure_element However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +230 235 Cdc23 protein However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +239 243 HcpC protein However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +245 249 RMSD evidence However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5 Å for Cα atoms) (Fig. 5B). RESULTS +73 78 loops structure_element The differences may result from the differing numbers of residues in the loops and differences in antiparallel helix packing. RESULTS +98 116 antiparallel helix structure_element The differences may result from the differing numbers of residues in the loops and differences in antiparallel helix packing. RESULTS +20 29 conserved protein_state Moreover, there are conserved disulfide bonds in the SLR motifs of HcpC and HcpB, but no such bonds are observed in SEL1Lcent. RESULTS +30 45 disulfide bonds ptm Moreover, there are conserved disulfide bonds in the SLR motifs of HcpC and HcpB, but no such bonds are observed in SEL1Lcent. RESULTS +53 56 SLR structure_element Moreover, there are conserved disulfide bonds in the SLR motifs of HcpC and HcpB, but no such bonds are observed in SEL1Lcent. RESULTS +67 71 HcpC protein Moreover, there are conserved disulfide bonds in the SLR motifs of HcpC and HcpB, but no such bonds are observed in SEL1Lcent. RESULTS +76 80 HcpB protein Moreover, there are conserved disulfide bonds in the SLR motifs of HcpC and HcpB, but no such bonds are observed in SEL1Lcent. RESULTS +116 125 SEL1Lcent structure_element Moreover, there are conserved disulfide bonds in the SLR motifs of HcpC and HcpB, but no such bonds are observed in SEL1Lcent. RESULTS +79 82 SLR structure_element These factors contribute to the differences in the overall conformation of the SLR motifs in SEL1L and other SLR or TPR motif-containing proteins. RESULTS +93 98 SEL1L protein These factors contribute to the differences in the overall conformation of the SLR motifs in SEL1L and other SLR or TPR motif-containing proteins. RESULTS +109 145 SLR or TPR motif-containing proteins protein_type These factors contribute to the differences in the overall conformation of the SLR motifs in SEL1L and other SLR or TPR motif-containing proteins. RESULTS +32 41 structure evidence Another major difference in the structure of SLR motifs between SEL1L and HcpC is the oligomeric state of proteins. RESULTS +45 48 SLR structure_element Another major difference in the structure of SLR motifs between SEL1L and HcpC is the oligomeric state of proteins. RESULTS +64 69 SEL1L protein Another major difference in the structure of SLR motifs between SEL1L and HcpC is the oligomeric state of proteins. RESULTS +74 78 HcpC protein Another major difference in the structure of SLR motifs between SEL1L and HcpC is the oligomeric state of proteins. RESULTS +4 7 TPR structure_element The TPR motif is involved in the dimerization of proteins such as Cdc23, Cdc16, and Cdc27. RESULTS +33 45 dimerization oligomeric_state The TPR motif is involved in the dimerization of proteins such as Cdc23, Cdc16, and Cdc27. RESULTS +66 71 Cdc23 protein The TPR motif is involved in the dimerization of proteins such as Cdc23, Cdc16, and Cdc27. RESULTS +73 78 Cdc16 protein The TPR motif is involved in the dimerization of proteins such as Cdc23, Cdc16, and Cdc27. RESULTS +84 89 Cdc27 protein The TPR motif is involved in the dimerization of proteins such as Cdc23, Cdc16, and Cdc27. RESULTS +40 45 Cdc23 protein In particular, the N-terminal domain of Cdc23 (Cdc23N-term) has a TPR-motif organization similar to that of the SLR motif in SEL1Lcent. RESULTS +47 52 Cdc23 protein In particular, the N-terminal domain of Cdc23 (Cdc23N-term) has a TPR-motif organization similar to that of the SLR motif in SEL1Lcent. RESULTS +66 69 TPR structure_element In particular, the N-terminal domain of Cdc23 (Cdc23N-term) has a TPR-motif organization similar to that of the SLR motif in SEL1Lcent. RESULTS +112 115 SLR structure_element In particular, the N-terminal domain of Cdc23 (Cdc23N-term) has a TPR-motif organization similar to that of the SLR motif in SEL1Lcent. RESULTS +125 134 SEL1Lcent structure_element In particular, the N-terminal domain of Cdc23 (Cdc23N-term) has a TPR-motif organization similar to that of the SLR motif in SEL1Lcent. RESULTS +10 13 TPR structure_element The seven TPR motifs of Cdc23N-term are assembled into a superhelical structure, generating a hollow surface and encircling its dimer counterpart in an interlocking clasp-like arrangement (Fig. 5C). RESULTS +24 29 Cdc23 protein The seven TPR motifs of Cdc23N-term are assembled into a superhelical structure, generating a hollow surface and encircling its dimer counterpart in an interlocking clasp-like arrangement (Fig. 5C). RESULTS +57 79 superhelical structure structure_element The seven TPR motifs of Cdc23N-term are assembled into a superhelical structure, generating a hollow surface and encircling its dimer counterpart in an interlocking clasp-like arrangement (Fig. 5C). RESULTS +128 133 dimer oligomeric_state The seven TPR motifs of Cdc23N-term are assembled into a superhelical structure, generating a hollow surface and encircling its dimer counterpart in an interlocking clasp-like arrangement (Fig. 5C). RESULTS +4 15 TPR motif 1 structure_element The TPR motif 1 (TPR1) of each Cdc23N-term subunit is located in the hollow surface of the counter subunit and interacts with residues lining the inner groove TPR α-helices, generating two-fold symmetry homotype interactions. RESULTS +17 21 TPR1 structure_element The TPR motif 1 (TPR1) of each Cdc23N-term subunit is located in the hollow surface of the counter subunit and interacts with residues lining the inner groove TPR α-helices, generating two-fold symmetry homotype interactions. RESULTS +31 36 Cdc23 protein The TPR motif 1 (TPR1) of each Cdc23N-term subunit is located in the hollow surface of the counter subunit and interacts with residues lining the inner groove TPR α-helices, generating two-fold symmetry homotype interactions. RESULTS +146 158 inner groove site The TPR motif 1 (TPR1) of each Cdc23N-term subunit is located in the hollow surface of the counter subunit and interacts with residues lining the inner groove TPR α-helices, generating two-fold symmetry homotype interactions. RESULTS +159 162 TPR structure_element The TPR motif 1 (TPR1) of each Cdc23N-term subunit is located in the hollow surface of the counter subunit and interacts with residues lining the inner groove TPR α-helices, generating two-fold symmetry homotype interactions. RESULTS +163 172 α-helices structure_element The TPR motif 1 (TPR1) of each Cdc23N-term subunit is located in the hollow surface of the counter subunit and interacts with residues lining the inner groove TPR α-helices, generating two-fold symmetry homotype interactions. RESULTS +17 26 structure evidence However, in this structure, a conformational change in the TPR motif itself is not observed. RESULTS +59 62 TPR structure_element However, in this structure, a conformational change in the TPR motif itself is not observed. RESULTS +20 24 HcpC protein Self-association of HcpC has not been reported, and there is no domain-swapped structure in the SLR motifs of HcpC, in contrast to that observed in SEL1Lcent. RESULTS +64 78 domain-swapped protein_state Self-association of HcpC has not been reported, and there is no domain-swapped structure in the SLR motifs of HcpC, in contrast to that observed in SEL1Lcent. RESULTS +96 99 SLR structure_element Self-association of HcpC has not been reported, and there is no domain-swapped structure in the SLR motifs of HcpC, in contrast to that observed in SEL1Lcent. RESULTS +110 114 HcpC protein Self-association of HcpC has not been reported, and there is no domain-swapped structure in the SLR motifs of HcpC, in contrast to that observed in SEL1Lcent. RESULTS +148 157 SEL1Lcent structure_element Self-association of HcpC has not been reported, and there is no domain-swapped structure in the SLR motifs of HcpC, in contrast to that observed in SEL1Lcent. RESULTS +9 14 SEL1L protein Although SEL1L contains a number of SLR motifs comparable to HcpC, the SLR motifs in SEL1L are interrupted by other sequences, making three SLR motif clusters (Fig. 1A). RESULTS +36 39 SLR structure_element Although SEL1L contains a number of SLR motifs comparable to HcpC, the SLR motifs in SEL1L are interrupted by other sequences, making three SLR motif clusters (Fig. 1A). RESULTS +61 65 HcpC protein Although SEL1L contains a number of SLR motifs comparable to HcpC, the SLR motifs in SEL1L are interrupted by other sequences, making three SLR motif clusters (Fig. 1A). RESULTS +71 74 SLR structure_element Although SEL1L contains a number of SLR motifs comparable to HcpC, the SLR motifs in SEL1L are interrupted by other sequences, making three SLR motif clusters (Fig. 1A). RESULTS +85 90 SEL1L protein Although SEL1L contains a number of SLR motifs comparable to HcpC, the SLR motifs in SEL1L are interrupted by other sequences, making three SLR motif clusters (Fig. 1A). RESULTS +140 143 SLR structure_element Although SEL1L contains a number of SLR motifs comparable to HcpC, the SLR motifs in SEL1L are interrupted by other sequences, making three SLR motif clusters (Fig. 1A). RESULTS +16 19 SLR structure_element The interrupted SLR motifs may be required for dimerization of SEL1Lcent, as five SLR motifs are more than enough to form the semicircle of the yin-yang symbol (Fig. 1B). RESULTS +47 59 dimerization oligomeric_state The interrupted SLR motifs may be required for dimerization of SEL1Lcent, as five SLR motifs are more than enough to form the semicircle of the yin-yang symbol (Fig. 1B). RESULTS +63 72 SEL1Lcent structure_element The interrupted SLR motifs may be required for dimerization of SEL1Lcent, as five SLR motifs are more than enough to form the semicircle of the yin-yang symbol (Fig. 1B). RESULTS +82 85 SLR structure_element The interrupted SLR motifs may be required for dimerization of SEL1Lcent, as five SLR motifs are more than enough to form the semicircle of the yin-yang symbol (Fig. 1B). RESULTS +126 152 semicircle of the yin-yang structure_element The interrupted SLR motifs may be required for dimerization of SEL1Lcent, as five SLR motifs are more than enough to form the semicircle of the yin-yang symbol (Fig. 1B). RESULTS +0 8 Helix 5A structure_element Helix 5A from SLR motif 5 meets helix 9A from SLR motif 9 of the counterpart SEL1L. RESULTS +14 25 SLR motif 5 structure_element Helix 5A from SLR motif 5 meets helix 9A from SLR motif 9 of the counterpart SEL1L. RESULTS +32 40 helix 9A structure_element Helix 5A from SLR motif 5 meets helix 9A from SLR motif 9 of the counterpart SEL1L. RESULTS +46 57 SLR motif 9 structure_element Helix 5A from SLR motif 5 meets helix 9A from SLR motif 9 of the counterpart SEL1L. RESULTS +77 82 SEL1L protein Helix 5A from SLR motif 5 meets helix 9A from SLR motif 9 of the counterpart SEL1L. RESULTS +7 24 SLR motifs 5 to 9 structure_element If the SLR motifs 5 to 9 were not isolated from other SLR motifs, steric hindrance could interfere with dimerization of SEL1L. RESULTS +54 57 SLR structure_element If the SLR motifs 5 to 9 were not isolated from other SLR motifs, steric hindrance could interfere with dimerization of SEL1L. RESULTS +104 116 dimerization oligomeric_state If the SLR motifs 5 to 9 were not isolated from other SLR motifs, steric hindrance could interfere with dimerization of SEL1L. RESULTS +120 125 SEL1L protein If the SLR motifs 5 to 9 were not isolated from other SLR motifs, steric hindrance could interfere with dimerization of SEL1L. RESULTS +44 48 TPRs structure_element This is one of the biggest differences from TPRs in Cdc23 and from the SLRs in HcpC, where the motifs exist in tandem. RESULTS +52 57 Cdc23 protein This is one of the biggest differences from TPRs in Cdc23 and from the SLRs in HcpC, where the motifs exist in tandem. RESULTS +71 75 SLRs structure_element This is one of the biggest differences from TPRs in Cdc23 and from the SLRs in HcpC, where the motifs exist in tandem. RESULTS +79 83 HcpC protein This is one of the biggest differences from TPRs in Cdc23 and from the SLRs in HcpC, where the motifs exist in tandem. RESULTS +0 3 TPR structure_element TPR and SLR motifs are generally involved in protein-protein interaction modules, and the sequences between the SLR motifs of SEL1L might actually facilitate the self-association of this protein. RESULTS +8 11 SLR structure_element TPR and SLR motifs are generally involved in protein-protein interaction modules, and the sequences between the SLR motifs of SEL1L might actually facilitate the self-association of this protein. RESULTS +112 115 SLR structure_element TPR and SLR motifs are generally involved in protein-protein interaction modules, and the sequences between the SLR motifs of SEL1L might actually facilitate the self-association of this protein. RESULTS +126 131 SEL1L protein TPR and SLR motifs are generally involved in protein-protein interaction modules, and the sequences between the SLR motifs of SEL1L might actually facilitate the self-association of this protein. RESULTS +0 5 SLR-C structure_element SLR-C of SEL1L Binds HRD1 N-terminus Luminal Loop RESULTS +9 14 SEL1L protein SLR-C of SEL1L Binds HRD1 N-terminus Luminal Loop RESULTS +21 25 HRD1 protein SLR-C of SEL1L Binds HRD1 N-terminus Luminal Loop RESULTS +37 49 Luminal Loop structure_element SLR-C of SEL1L Binds HRD1 N-terminus Luminal Loop RESULTS +13 28 structural data evidence Based on the structural data presented herein, a possible arrangement of membrane-associated ERAD components in mammals, highlighting the molecular functions of SLR domains in SEL1L, is shown in Fig. 6C. RESULTS +112 119 mammals taxonomy_domain Based on the structural data presented herein, a possible arrangement of membrane-associated ERAD components in mammals, highlighting the molecular functions of SLR domains in SEL1L, is shown in Fig. 6C. RESULTS +161 164 SLR structure_element Based on the structural data presented herein, a possible arrangement of membrane-associated ERAD components in mammals, highlighting the molecular functions of SLR domains in SEL1L, is shown in Fig. 6C. RESULTS +176 181 SEL1L protein Based on the structural data presented herein, a possible arrangement of membrane-associated ERAD components in mammals, highlighting the molecular functions of SLR domains in SEL1L, is shown in Fig. 6C. RESULTS +27 30 SLR structure_element We suggest that the middle SLR domains are involved in the dimerization of SEL1L based on the crystal structure and biochemical data. RESULTS +59 71 dimerization oligomeric_state We suggest that the middle SLR domains are involved in the dimerization of SEL1L based on the crystal structure and biochemical data. RESULTS +75 80 SEL1L protein We suggest that the middle SLR domains are involved in the dimerization of SEL1L based on the crystal structure and biochemical data. RESULTS +94 111 crystal structure evidence We suggest that the middle SLR domains are involved in the dimerization of SEL1L based on the crystal structure and biochemical data. RESULTS +0 5 SLR-C structure_element SLR-C, which contains SLR motifs 10 to 11, might be involved in the interaction with HRD1. RESULTS +22 41 SLR motifs 10 to 11 structure_element SLR-C, which contains SLR motifs 10 to 11, might be involved in the interaction with HRD1. RESULTS +85 89 HRD1 protein SLR-C, which contains SLR motifs 10 to 11, might be involved in the interaction with HRD1. RESULTS +34 39 yeast taxonomy_domain Indirect evidence from a previous yeast study shows that the circumscribed region of C-terminal Hrd3p, specifically residues 664–695, forms contacts with the Hrd1 luminal loops. RESULTS +96 101 Hrd3p protein Indirect evidence from a previous yeast study shows that the circumscribed region of C-terminal Hrd3p, specifically residues 664–695, forms contacts with the Hrd1 luminal loops. RESULTS +125 132 664–695 residue_range Indirect evidence from a previous yeast study shows that the circumscribed region of C-terminal Hrd3p, specifically residues 664–695, forms contacts with the Hrd1 luminal loops. RESULTS +158 162 Hrd1 protein Indirect evidence from a previous yeast study shows that the circumscribed region of C-terminal Hrd3p, specifically residues 664–695, forms contacts with the Hrd1 luminal loops. RESULTS +163 176 luminal loops structure_element Indirect evidence from a previous yeast study shows that the circumscribed region of C-terminal Hrd3p, specifically residues 664–695, forms contacts with the Hrd1 luminal loops. RESULTS +4 9 Hrd3p protein The Hrd3p residues 664–695 correspond to mouse SEL1L residues 696–727, which include the entire helix 11B (residue 697–709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4). RESULTS +19 26 664–695 residue_range The Hrd3p residues 664–695 correspond to mouse SEL1L residues 696–727, which include the entire helix 11B (residue 697–709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4). RESULTS +41 46 mouse taxonomy_domain The Hrd3p residues 664–695 correspond to mouse SEL1L residues 696–727, which include the entire helix 11B (residue 697–709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4). RESULTS +47 52 SEL1L protein The Hrd3p residues 664–695 correspond to mouse SEL1L residues 696–727, which include the entire helix 11B (residue 697–709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4). RESULTS +62 69 696–727 residue_range The Hrd3p residues 664–695 correspond to mouse SEL1L residues 696–727, which include the entire helix 11B (residue 697–709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4). RESULTS +96 105 helix 11B structure_element The Hrd3p residues 664–695 correspond to mouse SEL1L residues 696–727, which include the entire helix 11B (residue 697–709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4). RESULTS +115 122 697–709 residue_range The Hrd3p residues 664–695 correspond to mouse SEL1L residues 696–727, which include the entire helix 11B (residue 697–709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4). RESULTS +127 139 SLR motif 11 structure_element The Hrd3p residues 664–695 correspond to mouse SEL1L residues 696–727, which include the entire helix 11B (residue 697–709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4). RESULTS +146 160 well-conserved protein_state The Hrd3p residues 664–695 correspond to mouse SEL1L residues 696–727, which include the entire helix 11B (residue 697–709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4). RESULTS +76 94 SLR motif 10 to 11 structure_element This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). RESULTS +123 159 structure-guided SLR motif alignment experimental_method This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). RESULTS +182 197 structure study experimental_method This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). RESULTS +227 248 sequence conservation protein_state This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). RESULTS +257 266 mammalian taxonomy_domain This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). RESULTS +267 272 SEL1L protein This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). RESULTS +277 282 yeast taxonomy_domain This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). RESULTS +283 288 Hrd3p protein This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). RESULTS +296 315 SLR motifs 10 to 11 structure_element This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). RESULTS +352 356 HRD1 protein This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). RESULTS +358 363 Hrd1p protein This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). RESULTS +60 65 mouse taxonomy_domain To address this hypothesis, we prepared constructs encoding mouse HRD1 luminal fragments fused to GST as shown in Fig. 6A, and tested their ability to bind certain SLR motifs in SEL1L. RESULTS +66 70 HRD1 protein To address this hypothesis, we prepared constructs encoding mouse HRD1 luminal fragments fused to GST as shown in Fig. 6A, and tested their ability to bind certain SLR motifs in SEL1L. RESULTS +89 101 fused to GST experimental_method To address this hypothesis, we prepared constructs encoding mouse HRD1 luminal fragments fused to GST as shown in Fig. 6A, and tested their ability to bind certain SLR motifs in SEL1L. RESULTS +164 167 SLR structure_element To address this hypothesis, we prepared constructs encoding mouse HRD1 luminal fragments fused to GST as shown in Fig. 6A, and tested their ability to bind certain SLR motifs in SEL1L. RESULTS +178 183 SEL1L protein To address this hypothesis, we prepared constructs encoding mouse HRD1 luminal fragments fused to GST as shown in Fig. 6A, and tested their ability to bind certain SLR motifs in SEL1L. RESULTS +94 99 SLR-N structure_element The fusion proteins were immobilized on glutathione-Sepharose beads and probed for binding to SLR-N, SLR-M, SLR-C, and monomer form of SLR-M (SLR-ML521A). RESULTS +101 106 SLR-M structure_element The fusion proteins were immobilized on glutathione-Sepharose beads and probed for binding to SLR-N, SLR-M, SLR-C, and monomer form of SLR-M (SLR-ML521A). RESULTS +108 113 SLR-C structure_element The fusion proteins were immobilized on glutathione-Sepharose beads and probed for binding to SLR-N, SLR-M, SLR-C, and monomer form of SLR-M (SLR-ML521A). RESULTS +119 126 monomer oligomeric_state The fusion proteins were immobilized on glutathione-Sepharose beads and probed for binding to SLR-N, SLR-M, SLR-C, and monomer form of SLR-M (SLR-ML521A). RESULTS +135 140 SLR-M structure_element The fusion proteins were immobilized on glutathione-Sepharose beads and probed for binding to SLR-N, SLR-M, SLR-C, and monomer form of SLR-M (SLR-ML521A). RESULTS +142 152 SLR-ML521A mutant The fusion proteins were immobilized on glutathione-Sepharose beads and probed for binding to SLR-N, SLR-M, SLR-C, and monomer form of SLR-M (SLR-ML521A). RESULTS +25 30 SLR-C structure_element Figure 6B shows that the SLR-C, consisting of SLR motifs 10 and 11, exclusively interacts with N-terminal luminal loop (residues 21–42) of HRD1. RESULTS +46 66 SLR motifs 10 and 11 structure_element Figure 6B shows that the SLR-C, consisting of SLR motifs 10 and 11, exclusively interacts with N-terminal luminal loop (residues 21–42) of HRD1. RESULTS +106 118 luminal loop structure_element Figure 6B shows that the SLR-C, consisting of SLR motifs 10 and 11, exclusively interacts with N-terminal luminal loop (residues 21–42) of HRD1. RESULTS +129 134 21–42 residue_range Figure 6B shows that the SLR-C, consisting of SLR motifs 10 and 11, exclusively interacts with N-terminal luminal loop (residues 21–42) of HRD1. RESULTS +139 143 HRD1 protein Figure 6B shows that the SLR-C, consisting of SLR motifs 10 and 11, exclusively interacts with N-terminal luminal loop (residues 21–42) of HRD1. RESULTS +27 32 SLR-N structure_element The molecular functions of SLR-N are unclear. RESULTS +24 29 SLR-N structure_element One possibility is that SLR-N contributes to substrate recognition of proteins to be degraded because there are a couple of putative glycosylation sites within the SLR-N domain (Fig. 1A). RESULTS +133 152 glycosylation sites site One possibility is that SLR-N contributes to substrate recognition of proteins to be degraded because there are a couple of putative glycosylation sites within the SLR-N domain (Fig. 1A). RESULTS +164 169 SLR-N structure_element One possibility is that SLR-N contributes to substrate recognition of proteins to be degraded because there are a couple of putative glycosylation sites within the SLR-N domain (Fig. 1A). RESULTS +0 9 SEL1Lcent structure_element SEL1Lcent contains a putative N-glycosylation site, Asn 427, which is highly conserved among different species and structurally exposed to the surface of the SEL1L dimer according to the crystal structure (Fig. 6C). RESULTS +30 50 N-glycosylation site site SEL1Lcent contains a putative N-glycosylation site, Asn 427, which is highly conserved among different species and structurally exposed to the surface of the SEL1L dimer according to the crystal structure (Fig. 6C). RESULTS +52 59 Asn 427 residue_name_number SEL1Lcent contains a putative N-glycosylation site, Asn 427, which is highly conserved among different species and structurally exposed to the surface of the SEL1L dimer according to the crystal structure (Fig. 6C). RESULTS +70 86 highly conserved protein_state SEL1Lcent contains a putative N-glycosylation site, Asn 427, which is highly conserved among different species and structurally exposed to the surface of the SEL1L dimer according to the crystal structure (Fig. 6C). RESULTS +158 163 SEL1L protein SEL1Lcent contains a putative N-glycosylation site, Asn 427, which is highly conserved among different species and structurally exposed to the surface of the SEL1L dimer according to the crystal structure (Fig. 6C). RESULTS +164 169 dimer oligomeric_state SEL1Lcent contains a putative N-glycosylation site, Asn 427, which is highly conserved among different species and structurally exposed to the surface of the SEL1L dimer according to the crystal structure (Fig. 6C). RESULTS +187 204 crystal structure evidence SEL1Lcent contains a putative N-glycosylation site, Asn 427, which is highly conserved among different species and structurally exposed to the surface of the SEL1L dimer according to the crystal structure (Fig. 6C). RESULTS +72 77 yeast taxonomy_domain Many reports demonstrate that membrane-bound ERAD machinery proteins in yeast, such as Hrd1p, Der1p, and Usa1p, are involved in oligomerization of ERAD components. DISCUSS +87 92 Hrd1p protein Many reports demonstrate that membrane-bound ERAD machinery proteins in yeast, such as Hrd1p, Der1p, and Usa1p, are involved in oligomerization of ERAD components. DISCUSS +94 99 Der1p protein Many reports demonstrate that membrane-bound ERAD machinery proteins in yeast, such as Hrd1p, Der1p, and Usa1p, are involved in oligomerization of ERAD components. DISCUSS +105 110 Usa1p protein Many reports demonstrate that membrane-bound ERAD machinery proteins in yeast, such as Hrd1p, Der1p, and Usa1p, are involved in oligomerization of ERAD components. DISCUSS +4 9 Hrd1p protein The Hrd1p complex forms dimers upon sucrose gradient sedimentation and size-exclusion chromatography. DISCUSS +24 30 dimers oligomeric_state The Hrd1p complex forms dimers upon sucrose gradient sedimentation and size-exclusion chromatography. DISCUSS +36 66 sucrose gradient sedimentation experimental_method The Hrd1p complex forms dimers upon sucrose gradient sedimentation and size-exclusion chromatography. DISCUSS +71 100 size-exclusion chromatography experimental_method The Hrd1p complex forms dimers upon sucrose gradient sedimentation and size-exclusion chromatography. DISCUSS +24 41 HA-epitope-tagged protein_state Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. DISCUSS +42 47 Hrd3p protein Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. DISCUSS +51 56 Hrd1p protein Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. DISCUSS +89 99 unmodified protein_state Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. DISCUSS +100 105 Hrd3p protein Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. DISCUSS +110 115 Hrd1p protein Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. DISCUSS +152 157 Hrd1p protein Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. DISCUSS +162 167 Hrd3p protein Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. DISCUSS +168 178 homodimers oligomeric_state Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. DISCUSS +219 222 Hrd complex_assembly Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. DISCUSS +100 105 yeast taxonomy_domain Considering that the functional and structural composition of ERAD components are conserved in both yeast and mammals, we propose that the mammalian ERAD components also form self-associating oligomers. DISCUSS +110 117 mammals taxonomy_domain Considering that the functional and structural composition of ERAD components are conserved in both yeast and mammals, we propose that the mammalian ERAD components also form self-associating oligomers. DISCUSS +139 148 mammalian taxonomy_domain Considering that the functional and structural composition of ERAD components are conserved in both yeast and mammals, we propose that the mammalian ERAD components also form self-associating oligomers. DISCUSS +192 201 oligomers oligomeric_state Considering that the functional and structural composition of ERAD components are conserved in both yeast and mammals, we propose that the mammalian ERAD components also form self-associating oligomers. DISCUSS +32 50 cross-linking data experimental_method This hypothesis is supported by cross-linking data suggesting that human HRD1 forms a homodimer. DISCUSS +67 72 human species This hypothesis is supported by cross-linking data suggesting that human HRD1 forms a homodimer. DISCUSS +73 77 HRD1 protein This hypothesis is supported by cross-linking data suggesting that human HRD1 forms a homodimer. DISCUSS +86 95 homodimer oligomeric_state This hypothesis is supported by cross-linking data suggesting that human HRD1 forms a homodimer. DISCUSS +39 56 crystal structure evidence Consistent with the previous data, our crystal structure and biochemical data demonstrate that mouse SEL1Lcent exists as a homodimer in the ER lumen via domain swapping of SLR motif 9. DISCUSS +61 77 biochemical data evidence Consistent with the previous data, our crystal structure and biochemical data demonstrate that mouse SEL1Lcent exists as a homodimer in the ER lumen via domain swapping of SLR motif 9. DISCUSS +95 100 mouse taxonomy_domain Consistent with the previous data, our crystal structure and biochemical data demonstrate that mouse SEL1Lcent exists as a homodimer in the ER lumen via domain swapping of SLR motif 9. DISCUSS +101 110 SEL1Lcent structure_element Consistent with the previous data, our crystal structure and biochemical data demonstrate that mouse SEL1Lcent exists as a homodimer in the ER lumen via domain swapping of SLR motif 9. DISCUSS +123 132 homodimer oligomeric_state Consistent with the previous data, our crystal structure and biochemical data demonstrate that mouse SEL1Lcent exists as a homodimer in the ER lumen via domain swapping of SLR motif 9. DISCUSS +172 183 SLR motif 9 structure_element Consistent with the previous data, our crystal structure and biochemical data demonstrate that mouse SEL1Lcent exists as a homodimer in the ER lumen via domain swapping of SLR motif 9. DISCUSS +63 68 dimer oligomeric_state We need to further test whether there are contacts involved in dimer formation in SEL1L in addition to those in the SLR-M region. DISCUSS +82 87 SEL1L protein We need to further test whether there are contacts involved in dimer formation in SEL1L in addition to those in the SLR-M region. DISCUSS +116 121 SLR-M structure_element We need to further test whether there are contacts involved in dimer formation in SEL1L in addition to those in the SLR-M region. DISCUSS +3 8 yeast taxonomy_domain In yeast, Usa1p acts as a scaffold for Hrd1p and Der1p, in which the N-terminus of Usa1p interacts with the C-terminal 34 amino acids of Hrd1p in the cytosol to induce oligomerization of Hrd1p, which is essential for its activity. DISCUSS +10 15 Usa1p protein In yeast, Usa1p acts as a scaffold for Hrd1p and Der1p, in which the N-terminus of Usa1p interacts with the C-terminal 34 amino acids of Hrd1p in the cytosol to induce oligomerization of Hrd1p, which is essential for its activity. DISCUSS +39 44 Hrd1p protein In yeast, Usa1p acts as a scaffold for Hrd1p and Der1p, in which the N-terminus of Usa1p interacts with the C-terminal 34 amino acids of Hrd1p in the cytosol to induce oligomerization of Hrd1p, which is essential for its activity. DISCUSS +49 54 Der1p protein In yeast, Usa1p acts as a scaffold for Hrd1p and Der1p, in which the N-terminus of Usa1p interacts with the C-terminal 34 amino acids of Hrd1p in the cytosol to induce oligomerization of Hrd1p, which is essential for its activity. DISCUSS +83 88 Usa1p protein In yeast, Usa1p acts as a scaffold for Hrd1p and Der1p, in which the N-terminus of Usa1p interacts with the C-terminal 34 amino acids of Hrd1p in the cytosol to induce oligomerization of Hrd1p, which is essential for its activity. DISCUSS +137 142 Hrd1p protein In yeast, Usa1p acts as a scaffold for Hrd1p and Der1p, in which the N-terminus of Usa1p interacts with the C-terminal 34 amino acids of Hrd1p in the cytosol to induce oligomerization of Hrd1p, which is essential for its activity. DISCUSS +187 192 Hrd1p protein In yeast, Usa1p acts as a scaffold for Hrd1p and Der1p, in which the N-terminus of Usa1p interacts with the C-terminal 34 amino acids of Hrd1p in the cytosol to induce oligomerization of Hrd1p, which is essential for its activity. DISCUSS +9 18 metazoans taxonomy_domain However, metazoans lack a clear Usa1p homolog. DISCUSS +32 37 Usa1p protein However, metazoans lack a clear Usa1p homolog. DISCUSS +9 18 mammalian taxonomy_domain Although mammalian HERP has sequences and domains that are conserved in Usa1p, the molecular function of HERP is not clearly related to that of Usa1p. DISCUSS +19 23 HERP protein_type Although mammalian HERP has sequences and domains that are conserved in Usa1p, the molecular function of HERP is not clearly related to that of Usa1p. DISCUSS +59 71 conserved in protein_state Although mammalian HERP has sequences and domains that are conserved in Usa1p, the molecular function of HERP is not clearly related to that of Usa1p. DISCUSS +72 77 Usa1p protein Although mammalian HERP has sequences and domains that are conserved in Usa1p, the molecular function of HERP is not clearly related to that of Usa1p. DISCUSS +105 109 HERP protein_type Although mammalian HERP has sequences and domains that are conserved in Usa1p, the molecular function of HERP is not clearly related to that of Usa1p. DISCUSS +144 149 Usa1p protein Although mammalian HERP has sequences and domains that are conserved in Usa1p, the molecular function of HERP is not clearly related to that of Usa1p. DISCUSS +37 58 transiently expressed protein_state Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. DISCUSS +59 69 HRD1-SEL1L complex_assembly Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. DISCUSS +109 116 lectins protein_type Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. DISCUSS +117 120 OS9 protein Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. DISCUSS +124 129 XTP-B protein Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. DISCUSS +229 257 α-antitrypsin null Hong-Kong protein Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. DISCUSS +259 262 NHK protein Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. DISCUSS +281 288 NHK-QQQ mutant Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. DISCUSS +296 301 lacks protein_state Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. DISCUSS +306 327 N-glycosylation sites site Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. DISCUSS +117 126 homodimer oligomeric_state Assuming that the correct oligomerization of ERAD components may be critical for their function, we hypothesize that homodimer formation of SEL1L in the ER lumen may stabilize oligomerization of the HRD complex, given that SEL1L forms a stoichiometric complex with HRD1. DISCUSS +140 145 SEL1L protein Assuming that the correct oligomerization of ERAD components may be critical for their function, we hypothesize that homodimer formation of SEL1L in the ER lumen may stabilize oligomerization of the HRD complex, given that SEL1L forms a stoichiometric complex with HRD1. DISCUSS +199 202 HRD complex_assembly Assuming that the correct oligomerization of ERAD components may be critical for their function, we hypothesize that homodimer formation of SEL1L in the ER lumen may stabilize oligomerization of the HRD complex, given that SEL1L forms a stoichiometric complex with HRD1. DISCUSS +223 228 SEL1L protein Assuming that the correct oligomerization of ERAD components may be critical for their function, we hypothesize that homodimer formation of SEL1L in the ER lumen may stabilize oligomerization of the HRD complex, given that SEL1L forms a stoichiometric complex with HRD1. DISCUSS +252 264 complex with protein_state Assuming that the correct oligomerization of ERAD components may be critical for their function, we hypothesize that homodimer formation of SEL1L in the ER lumen may stabilize oligomerization of the HRD complex, given that SEL1L forms a stoichiometric complex with HRD1. DISCUSS +265 269 HRD1 protein Assuming that the correct oligomerization of ERAD components may be critical for their function, we hypothesize that homodimer formation of SEL1L in the ER lumen may stabilize oligomerization of the HRD complex, given that SEL1L forms a stoichiometric complex with HRD1. DISCUSS +55 60 SLR-C structure_element This is further supported by our data showing that the SLR-C of SEL1L directly interacts with the luminal fragment of HRD1 in the ER lumen. DISCUSS +64 69 SEL1L protein This is further supported by our data showing that the SLR-C of SEL1L directly interacts with the luminal fragment of HRD1 in the ER lumen. DISCUSS +118 122 HRD1 protein This is further supported by our data showing that the SLR-C of SEL1L directly interacts with the luminal fragment of HRD1 in the ER lumen. DISCUSS +44 47 HRD complex_assembly Although the organization of membrane-bound HRD complex components may be very similar between metazoans and yeast, the molecular details of interactions between the components may not necessarily be conserved. DISCUSS +95 104 metazoans taxonomy_domain Although the organization of membrane-bound HRD complex components may be very similar between metazoans and yeast, the molecular details of interactions between the components may not necessarily be conserved. DISCUSS +109 114 yeast taxonomy_domain Although the organization of membrane-bound HRD complex components may be very similar between metazoans and yeast, the molecular details of interactions between the components may not necessarily be conserved. DISCUSS +3 8 yeast taxonomy_domain In yeast, it is unclear whether self-association of Hrd3p is due to SLR motifs because the sequence of Hrd3p does not align precisely with the SLR motifs in SEL1L. DISCUSS +52 57 Hrd3p protein In yeast, it is unclear whether self-association of Hrd3p is due to SLR motifs because the sequence of Hrd3p does not align precisely with the SLR motifs in SEL1L. DISCUSS +68 71 SLR structure_element In yeast, it is unclear whether self-association of Hrd3p is due to SLR motifs because the sequence of Hrd3p does not align precisely with the SLR motifs in SEL1L. DISCUSS +103 108 Hrd3p protein In yeast, it is unclear whether self-association of Hrd3p is due to SLR motifs because the sequence of Hrd3p does not align precisely with the SLR motifs in SEL1L. DISCUSS +143 146 SLR structure_element In yeast, it is unclear whether self-association of Hrd3p is due to SLR motifs because the sequence of Hrd3p does not align precisely with the SLR motifs in SEL1L. DISCUSS +157 162 SEL1L protein In yeast, it is unclear whether self-association of Hrd3p is due to SLR motifs because the sequence of Hrd3p does not align precisely with the SLR motifs in SEL1L. DISCUSS +58 63 Hrd3p protein Furthermore, we are uncertain whether self-association of Hrd3p contributes to formation of the active form of the Hrd1p complex. DISCUSS +96 102 active protein_state Furthermore, we are uncertain whether self-association of Hrd3p contributes to formation of the active form of the Hrd1p complex. DISCUSS +115 120 Hrd1p protein Furthermore, we are uncertain whether self-association of Hrd3p contributes to formation of the active form of the Hrd1p complex. DISCUSS +12 21 truncated protein_state Recently, a truncated version of Yos9 was shown to form a dimer in the ER lumen and to contribute to the dimeric state of the Hrd1p complex. DISCUSS +33 37 Yos9 protein Recently, a truncated version of Yos9 was shown to form a dimer in the ER lumen and to contribute to the dimeric state of the Hrd1p complex. DISCUSS +58 63 dimer oligomeric_state Recently, a truncated version of Yos9 was shown to form a dimer in the ER lumen and to contribute to the dimeric state of the Hrd1p complex. DISCUSS +105 112 dimeric oligomeric_state Recently, a truncated version of Yos9 was shown to form a dimer in the ER lumen and to contribute to the dimeric state of the Hrd1p complex. DISCUSS +126 131 Hrd1p protein Recently, a truncated version of Yos9 was shown to form a dimer in the ER lumen and to contribute to the dimeric state of the Hrd1p complex. DISCUSS +49 53 Yos9 protein This interaction seems to be weak because direct Yos9-Yos9 interactions were not detected in immunoprecipitation experiments from yeast cell extracts containing different epitope-tagged variants of Yos9. DISCUSS +54 58 Yos9 protein This interaction seems to be weak because direct Yos9-Yos9 interactions were not detected in immunoprecipitation experiments from yeast cell extracts containing different epitope-tagged variants of Yos9. DISCUSS +93 124 immunoprecipitation experiments experimental_method This interaction seems to be weak because direct Yos9-Yos9 interactions were not detected in immunoprecipitation experiments from yeast cell extracts containing different epitope-tagged variants of Yos9. DISCUSS +130 135 yeast taxonomy_domain This interaction seems to be weak because direct Yos9-Yos9 interactions were not detected in immunoprecipitation experiments from yeast cell extracts containing different epitope-tagged variants of Yos9. DISCUSS +171 185 epitope-tagged protein_state This interaction seems to be weak because direct Yos9-Yos9 interactions were not detected in immunoprecipitation experiments from yeast cell extracts containing different epitope-tagged variants of Yos9. DISCUSS +198 202 Yos9 protein This interaction seems to be weak because direct Yos9-Yos9 interactions were not detected in immunoprecipitation experiments from yeast cell extracts containing different epitope-tagged variants of Yos9. DISCUSS +13 25 dimerization oligomeric_state However, the dimerization of Yos9 could provide a higher stability for the Hrd1p complex oligomer. DISCUSS +29 33 Yos9 protein However, the dimerization of Yos9 could provide a higher stability for the Hrd1p complex oligomer. DISCUSS +75 80 Hrd1p protein However, the dimerization of Yos9 could provide a higher stability for the Hrd1p complex oligomer. DISCUSS +89 97 oligomer oligomeric_state However, the dimerization of Yos9 could provide a higher stability for the Hrd1p complex oligomer. DISCUSS +14 26 dimerization oligomeric_state Likewise, the dimerization of SEL1L might provide stability for the mammalian HRD oligomer complex. DISCUSS +30 35 SEL1L protein Likewise, the dimerization of SEL1L might provide stability for the mammalian HRD oligomer complex. DISCUSS +68 77 mammalian taxonomy_domain Likewise, the dimerization of SEL1L might provide stability for the mammalian HRD oligomer complex. DISCUSS +78 81 HRD complex_assembly Likewise, the dimerization of SEL1L might provide stability for the mammalian HRD oligomer complex. DISCUSS +82 90 oligomer oligomeric_state Likewise, the dimerization of SEL1L might provide stability for the mammalian HRD oligomer complex. DISCUSS +64 69 SEL1L protein Further cell biological studies are required to clarify whether SEL1L (Hrd3p) dimerization could be cooperative with the oligomerization of the HRD complex. DISCUSS +71 76 Hrd3p protein Further cell biological studies are required to clarify whether SEL1L (Hrd3p) dimerization could be cooperative with the oligomerization of the HRD complex. DISCUSS +78 90 dimerization oligomeric_state Further cell biological studies are required to clarify whether SEL1L (Hrd3p) dimerization could be cooperative with the oligomerization of the HRD complex. DISCUSS +144 147 HRD complex_assembly Further cell biological studies are required to clarify whether SEL1L (Hrd3p) dimerization could be cooperative with the oligomerization of the HRD complex. DISCUSS +62 65 HRD complex_assembly Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans. DISCUSS +106 115 oligomers oligomeric_state Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans. DISCUSS +157 162 SEL1L protein Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans. DISCUSS +198 204 active protein_state Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans. DISCUSS +218 221 HRD complex_assembly Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans. DISCUSS +243 253 absence of protein_state Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans. DISCUSS +254 259 Usa1p protein Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans. DISCUSS +264 273 metazoans taxonomy_domain Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans. DISCUSS +109 112 HRD complex_assembly These findings should provide a foundation for molecular-level studies to understand the membrane-associated HRD complex assembly in ERAD. DISCUSS +0 17 Crystal Structure evidence Crystal Structure of SEL1Lcent. FIG +21 30 SEL1Lcent structure_element Crystal Structure of SEL1Lcent. FIG +46 58 Mus musculus species (A) The diagram shows the domain structure of Mus musculus SEL1L, as defined by proteolytic mapping and sequence/structure analysis. FIG +59 64 SEL1L protein (A) The diagram shows the domain structure of Mus musculus SEL1L, as defined by proteolytic mapping and sequence/structure analysis. FIG +80 99 proteolytic mapping experimental_method (A) The diagram shows the domain structure of Mus musculus SEL1L, as defined by proteolytic mapping and sequence/structure analysis. FIG +104 131 sequence/structure analysis experimental_method (A) The diagram shows the domain structure of Mus musculus SEL1L, as defined by proteolytic mapping and sequence/structure analysis. FIG +7 10 SLR structure_element The 11 SLR motifs were divided into three groups (SLR-N, SLR-M, and SLR-C) due to the presence of linker sequences that are not predicted SLR motifs. FIG +50 55 SLR-N structure_element The 11 SLR motifs were divided into three groups (SLR-N, SLR-M, and SLR-C) due to the presence of linker sequences that are not predicted SLR motifs. FIG +57 62 SLR-M structure_element The 11 SLR motifs were divided into three groups (SLR-N, SLR-M, and SLR-C) due to the presence of linker sequences that are not predicted SLR motifs. FIG +68 73 SLR-C structure_element The 11 SLR motifs were divided into three groups (SLR-N, SLR-M, and SLR-C) due to the presence of linker sequences that are not predicted SLR motifs. FIG +98 114 linker sequences structure_element The 11 SLR motifs were divided into three groups (SLR-N, SLR-M, and SLR-C) due to the presence of linker sequences that are not predicted SLR motifs. FIG +138 141 SLR structure_element The 11 SLR motifs were divided into three groups (SLR-N, SLR-M, and SLR-C) due to the presence of linker sequences that are not predicted SLR motifs. FIG +9 30 N-glycosylation sites site Putative N-glycosylation sites are indicated by black triangles. FIG +18 35 crystal structure evidence We determined the crystal structure of the SLR-M, residues 348–533. (B) Ribbon diagram of the biological unit of the SEL1Lcent, viewed along the two-fold NCS axis. FIG +43 48 SLR-M structure_element We determined the crystal structure of the SLR-M, residues 348–533. (B) Ribbon diagram of the biological unit of the SEL1Lcent, viewed along the two-fold NCS axis. FIG +59 66 348–533 residue_range We determined the crystal structure of the SLR-M, residues 348–533. (B) Ribbon diagram of the biological unit of the SEL1Lcent, viewed along the two-fold NCS axis. FIG +117 126 SEL1Lcent structure_element We determined the crystal structure of the SLR-M, residues 348–533. (B) Ribbon diagram of the biological unit of the SEL1Lcent, viewed along the two-fold NCS axis. FIG +4 21 crystal structure evidence The crystal structure was determined by SAD phasing using selenium as the anomalous scatterer and refined to 2.6 Å resolution (Table 1). FIG +40 51 SAD phasing experimental_method The crystal structure was determined by SAD phasing using selenium as the anomalous scatterer and refined to 2.6 Å resolution (Table 1). FIG +58 66 selenium chemical The crystal structure was determined by SAD phasing using selenium as the anomalous scatterer and refined to 2.6 Å resolution (Table 1). FIG +4 13 SEL1Lcent structure_element (C) SEL1Lcent ribbon diagram rotated 90° around a horizontal axis relative to (B). FIG +8 16 protomer oligomeric_state (D) One protomer of the SEL1Lcent dimer. FIG +24 33 SEL1Lcent structure_element (D) One protomer of the SEL1Lcent dimer. FIG +34 39 dimer oligomeric_state (D) One protomer of the SEL1Lcent dimer. FIG +30 39 SEL1Lcent structure_element Starting from the N-terminus, SEL1Lcent has five SLR motifs comprising ten α helices. FIG +49 52 SLR structure_element Starting from the N-terminus, SEL1Lcent has five SLR motifs comprising ten α helices. FIG +75 84 α helices structure_element Starting from the N-terminus, SEL1Lcent has five SLR motifs comprising ten α helices. FIG +5 8 SLR structure_element Each SLR motif (from 5 to 9) is indicated in a different color. (E) Evolutionary conservation of surface residues in SEL1Lcent, calculated using ConSurf, from a structure-based alignment of 135 SEL1L sequences. FIG +117 126 SEL1Lcent structure_element Each SLR motif (from 5 to 9) is indicated in a different color. (E) Evolutionary conservation of surface residues in SEL1Lcent, calculated using ConSurf, from a structure-based alignment of 135 SEL1L sequences. FIG +145 152 ConSurf experimental_method Each SLR motif (from 5 to 9) is indicated in a different color. (E) Evolutionary conservation of surface residues in SEL1Lcent, calculated using ConSurf, from a structure-based alignment of 135 SEL1L sequences. FIG +161 186 structure-based alignment experimental_method Each SLR motif (from 5 to 9) is indicated in a different color. (E) Evolutionary conservation of surface residues in SEL1Lcent, calculated using ConSurf, from a structure-based alignment of 135 SEL1L sequences. FIG +194 199 SEL1L protein Each SLR motif (from 5 to 9) is indicated in a different color. (E) Evolutionary conservation of surface residues in SEL1Lcent, calculated using ConSurf, from a structure-based alignment of 135 SEL1L sequences. FIG +102 107 SEL1L protein The surface is colored from red (high) to white (poor) according to the degree of conservation in the SEL1L phylogenetic orthologs. FIG +38 46 protomer oligomeric_state The ribbon diagram of the counterpart protomer is drawn to show the orientation of the SEL1Lcent dimer. FIG +87 96 SEL1Lcent structure_element The ribbon diagram of the counterpart protomer is drawn to show the orientation of the SEL1Lcent dimer. FIG +97 102 dimer oligomeric_state The ribbon diagram of the counterpart protomer is drawn to show the orientation of the SEL1Lcent dimer. FIG +0 15 Dimer Interface site Dimer Interface of SEL1Lcent. FIG +19 28 SEL1Lcent structure_element Dimer Interface of SEL1Lcent. FIG +38 47 SEL1Lcent structure_element (A) The diagram on the left shows the SEL1Lcent dimer viewed along the two-fold symmetry axis. FIG +48 53 dimer oligomeric_state (A) The diagram on the left shows the SEL1Lcent dimer viewed along the two-fold symmetry axis. FIG +15 30 contact regions site Three distinct contact regions are indicated with labeled boxes. FIG +53 62 SEL1Lcent structure_element The close-up view on the right shows the residues of SEL1Lcent that contribute to dimer formation via the three contact interfaces. FIG +82 87 dimer oligomeric_state The close-up view on the right shows the residues of SEL1Lcent that contribute to dimer formation via the three contact interfaces. FIG +112 130 contact interfaces site The close-up view on the right shows the residues of SEL1Lcent that contribute to dimer formation via the three contact interfaces. FIG +48 62 hydrogen bonds bond_interaction The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins. FIG +75 84 protomers oligomeric_state The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins. FIG +88 97 SEL1Lcent structure_element The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins. FIG +103 132 Size-exclusion chromatography experimental_method The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins. FIG +134 137 SEC experimental_method The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins. FIG +155 164 wild-type protein_state The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins. FIG +169 186 dimeric interface site The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins. FIG +187 196 SEL1Lcent structure_element The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins. FIG +197 204 mutants protein_state The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins. FIG +38 41 SEC experimental_method The standard molecular masses for the SEC experiments (top) were obtained from the following proteins: aldolase, 158 kDa; cobalbumin, 75 kDa; ovalbumin, 44 kDa; and carbonic anhydrase, 29 kDa. FIG +66 74 SDS-PAGE experimental_method The elution fractions, indicated by the gray shading, were run on SDS-PAGE and are shown below the gel-filtration elution profile. FIG +99 129 gel-filtration elution profile evidence The elution fractions, indicated by the gray shading, were run on SDS-PAGE and are shown below the gel-filtration elution profile. FIG +71 74 SEC experimental_method The schematic diagrams representing the protein constructs used in the SEC are shown on the left of each SDS-PAGE profile. FIG +105 113 SDS-PAGE experimental_method The schematic diagrams representing the protein constructs used in the SEC are shown on the left of each SDS-PAGE profile. FIG +20 32 Dimerization oligomeric_state Domain Swapping for Dimerization of SEL1Lcent. FIG +36 45 SEL1Lcent structure_element Domain Swapping for Dimerization of SEL1Lcent. FIG +4 22 Sequence alignment experimental_method (A) Sequence alignment of the SLR motifs in SEL1L. FIG +30 33 SLR structure_element (A) Sequence alignment of the SLR motifs in SEL1L. FIG +44 49 SEL1L protein (A) Sequence alignment of the SLR motifs in SEL1L. FIG +7 10 SLR structure_element The 11 SLR motifs were aligned based on the present crystal structure of SEL1Lcent. FIG +23 30 aligned experimental_method The 11 SLR motifs were aligned based on the present crystal structure of SEL1Lcent. FIG +52 69 crystal structure evidence The 11 SLR motifs were aligned based on the present crystal structure of SEL1Lcent. FIG +73 82 SEL1Lcent structure_element The 11 SLR motifs were aligned based on the present crystal structure of SEL1Lcent. FIG +17 26 SEL1Lcent structure_element The sequences of SEL1Lcent included in the crystal structure are highlighted by the blue box. FIG +43 60 crystal structure evidence The sequences of SEL1Lcent included in the crystal structure are highlighted by the blue box. FIG +73 80 helices structure_element The secondary structure elements are indicated above the sequences, with helices depicted as cylinders. FIG +4 6 GG structure_element The GG sequence in SLR motif 9, which creates the hinge for domain swapping (see text), is shaded yellow. FIG +19 30 SLR motif 9 structure_element The GG sequence in SLR motif 9, which creates the hinge for domain swapping (see text), is shaded yellow. FIG +50 55 hinge structure_element The GG sequence in SLR motif 9, which creates the hinge for domain swapping (see text), is shaded yellow. FIG +81 85 SLRs structure_element Stars below the sequences indicate the specific residues that commonly appear in SLRs. FIG +4 23 Structure alignment experimental_method (B) Structure alignment of five SLR motifs in SEL1Lcent is shown to highlight the unusual geometry of SLR motif 9. FIG +32 35 SLR structure_element (B) Structure alignment of five SLR motifs in SEL1Lcent is shown to highlight the unusual geometry of SLR motif 9. FIG +46 55 SEL1Lcent structure_element (B) Structure alignment of five SLR motifs in SEL1Lcent is shown to highlight the unusual geometry of SLR motif 9. FIG +102 113 SLR motif 9 structure_element (B) Structure alignment of five SLR motifs in SEL1Lcent is shown to highlight the unusual geometry of SLR motif 9. FIG +5 8 SLR structure_element Each SLR motif is shown in a different color. FIG +3 14 SLR motif 9 structure_element In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an α-hairpin structure. (C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer. FIG +37 44 helices structure_element In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an α-hairpin structure. (C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer. FIG +82 85 SLR structure_element In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an α-hairpin structure. (C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer. FIG +102 111 α-hairpin structure_element In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an α-hairpin structure. (C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer. FIG +154 161 Gly 512 residue_name_number In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an α-hairpin structure. (C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer. FIG +166 173 Gly 513 residue_name_number In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an α-hairpin structure. (C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer. FIG +227 235 helix 9B structure_element In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an α-hairpin structure. (C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer. FIG +257 262 dimer oligomeric_state In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an α-hairpin structure. (C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer. FIG +4 11 Gly 512 residue_name_number The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +16 23 Gly 513 residue_name_number The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +92 107 point mutations experimental_method The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +150 157 Gly 512 residue_name_number The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +162 169 Gly 513 residue_name_number The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +206 211 hinge structure_element The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +215 226 SLR motif 9 structure_element The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +228 233 G512A mutant The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +235 240 G513A mutant The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +242 247 G512A mutant The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +248 253 G513A mutant The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +259 264 G512K mutant The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +265 270 G513K mutant The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. FIG +0 29 Size-exclusion chromatography experimental_method Size-exclusion chromatography was conducted as described in Fig. 2B. FIG +0 5 SEL1L protein SEL1L forms self-oligomer mediated by the SEL1Lcent domain in vivo. FIG +12 25 self-oligomer oligomeric_state SEL1L forms self-oligomer mediated by the SEL1Lcent domain in vivo. FIG +42 51 SEL1Lcent structure_element SEL1L forms self-oligomer mediated by the SEL1Lcent domain in vivo. FIG +94 112 immunoprecipitated experimental_method (A) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. FIG +126 130 FLAG experimental_method (A) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. FIG +152 164 western blot experimental_method (A) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. FIG +188 190 HA experimental_method (A) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. FIG +4 15 full-length protein_state The full-length SEL1L-FLAG was co-immunoprecipitated with the full-length SEL1L-HA. FIG +16 21 SEL1L protein The full-length SEL1L-FLAG was co-immunoprecipitated with the full-length SEL1L-HA. FIG +22 26 FLAG experimental_method The full-length SEL1L-FLAG was co-immunoprecipitated with the full-length SEL1L-HA. FIG +31 52 co-immunoprecipitated experimental_method The full-length SEL1L-FLAG was co-immunoprecipitated with the full-length SEL1L-HA. FIG +62 73 full-length protein_state The full-length SEL1L-FLAG was co-immunoprecipitated with the full-length SEL1L-HA. FIG +74 79 SEL1L protein The full-length SEL1L-FLAG was co-immunoprecipitated with the full-length SEL1L-HA. FIG +80 82 HA experimental_method The full-length SEL1L-FLAG was co-immunoprecipitated with the full-length SEL1L-HA. FIG +6 15 SEL1Lcent structure_element Also, SEL1Lcent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively. FIG +20 41 co-immunoprecipitated experimental_method Also, SEL1Lcent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively. FIG +51 62 full-length protein_state Also, SEL1Lcent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively. FIG +63 68 SEL1L protein Also, SEL1Lcent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively. FIG +79 90 SLR motif 9 structure_element Also, SEL1Lcent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively. FIG +91 99 deletion experimental_method Also, SEL1Lcent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively. FIG +249 261 western blot experimental_method Also, SEL1Lcent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively. FIG +275 294 immunoprecipitation experimental_method Also, SEL1Lcent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively. FIG +4 16 SEL1L348–497 mutant The SEL1L348–497 fragment was secreted to the culture media but the SEL1Lcent was retained in the ER. (C) SEL1Lcent-FLAG-KDEL and SEL1L348–497-FLAG-KDEL localized to the ER. FIG +68 77 SEL1Lcent structure_element The SEL1L348–497 fragment was secreted to the culture media but the SEL1Lcent was retained in the ER. (C) SEL1Lcent-FLAG-KDEL and SEL1L348–497-FLAG-KDEL localized to the ER. FIG +106 125 SEL1Lcent-FLAG-KDEL mutant The SEL1L348–497 fragment was secreted to the culture media but the SEL1Lcent was retained in the ER. (C) SEL1Lcent-FLAG-KDEL and SEL1L348–497-FLAG-KDEL localized to the ER. FIG +130 152 SEL1L348–497-FLAG-KDEL mutant The SEL1L348–497 fragment was secreted to the culture media but the SEL1Lcent was retained in the ER. (C) SEL1Lcent-FLAG-KDEL and SEL1L348–497-FLAG-KDEL localized to the ER. FIG +4 9 SEL1L protein The SEL1L fragments were stained in red. (D) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-HA antibody followed by Western blot analysis using an anti-FLAG antibody. FIG +135 153 immunoprecipitated experimental_method The SEL1L fragments were stained in red. (D) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-HA antibody followed by Western blot analysis using an anti-FLAG antibody. FIG +167 169 HA experimental_method The SEL1L fragments were stained in red. (D) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-HA antibody followed by Western blot analysis using an anti-FLAG antibody. FIG +191 203 Western blot experimental_method The SEL1L fragments were stained in red. (D) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-HA antibody followed by Western blot analysis using an anti-FLAG antibody. FIG +227 231 FLAG experimental_method The SEL1L fragments were stained in red. (D) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-HA antibody followed by Western blot analysis using an anti-FLAG antibody. FIG +4 15 full-length protein_state The full-length SEL1L forms self-oligomers and the SEL1Lcent-FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA. FIG +16 21 SEL1L protein The full-length SEL1L forms self-oligomers and the SEL1Lcent-FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA. FIG +28 42 self-oligomers oligomeric_state The full-length SEL1L forms self-oligomers and the SEL1Lcent-FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA. FIG +51 70 SEL1Lcent-FLAG-KDEL mutant The full-length SEL1L forms self-oligomers and the SEL1Lcent-FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA. FIG +75 96 co-immunoprecipitated experimental_method The full-length SEL1L forms self-oligomers and the SEL1Lcent-FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA. FIG +102 113 full-length protein_state The full-length SEL1L forms self-oligomers and the SEL1Lcent-FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA. FIG +114 119 SEL1L protein The full-length SEL1L forms self-oligomers and the SEL1Lcent-FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA. FIG +120 122 HA experimental_method The full-length SEL1L forms self-oligomers and the SEL1Lcent-FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA. FIG +51 73 SEL1L348–497-FLAG-KDEL mutant The red asterisk indicates the expected signal for SEL1L348–497-FLAG-KDEL. FIG +0 22 SEL1L348–497-FLAG-KDEL mutant SEL1L348–497-FLAG-KDEL did not co-immunoprecipitate with full-length SEL1L-HA. FIG +57 68 full-length protein_state SEL1L348–497-FLAG-KDEL did not co-immunoprecipitate with full-length SEL1L-HA. FIG +69 74 SEL1L protein SEL1L348–497-FLAG-KDEL did not co-immunoprecipitate with full-length SEL1L-HA. FIG +75 77 HA experimental_method SEL1L348–497-FLAG-KDEL did not co-immunoprecipitate with full-length SEL1L-HA. FIG +53 70 SEL1Lcent-HA-KDEL mutant The white asterisks indicate non-specific bands. (E) SEL1Lcent-HA-KDEL competitively inhibited self-oligomerization of full-length SEL1L. FIG +119 130 full-length protein_state The white asterisks indicate non-specific bands. (E) SEL1Lcent-HA-KDEL competitively inhibited self-oligomerization of full-length SEL1L. FIG +131 136 SEL1L protein The white asterisks indicate non-specific bands. (E) SEL1Lcent-HA-KDEL competitively inhibited self-oligomerization of full-length SEL1L. FIG +54 79 immunoprecipitation assay experimental_method The indicated plasmid constructs were transfected and immunoprecipitation assay was performed using an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. FIG +108 112 FLAG experimental_method The indicated plasmid constructs were transfected and immunoprecipitation assay was performed using an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. FIG +134 146 western blot experimental_method The indicated plasmid constructs were transfected and immunoprecipitation assay was performed using an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. FIG +170 172 HA experimental_method The indicated plasmid constructs were transfected and immunoprecipitation assay was performed using an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. FIG +52 57 SEL1L protein The red rectangle indicates competitively inhibited SEL1L self-oligomer formation by the increasing doses of SEL1Lcent-HA-KDEL. (F) L521A point mutant in SEL1Lcent did not inhibit the self-association of SEL1L. FIG +63 71 oligomer oligomeric_state The red rectangle indicates competitively inhibited SEL1L self-oligomer formation by the increasing doses of SEL1Lcent-HA-KDEL. (F) L521A point mutant in SEL1Lcent did not inhibit the self-association of SEL1L. FIG +109 126 SEL1Lcent-HA-KDEL mutant The red rectangle indicates competitively inhibited SEL1L self-oligomer formation by the increasing doses of SEL1Lcent-HA-KDEL. (F) L521A point mutant in SEL1Lcent did not inhibit the self-association of SEL1L. FIG +132 137 L521A mutant The red rectangle indicates competitively inhibited SEL1L self-oligomer formation by the increasing doses of SEL1Lcent-HA-KDEL. (F) L521A point mutant in SEL1Lcent did not inhibit the self-association of SEL1L. FIG +138 150 point mutant protein_state The red rectangle indicates competitively inhibited SEL1L self-oligomer formation by the increasing doses of SEL1Lcent-HA-KDEL. (F) L521A point mutant in SEL1Lcent did not inhibit the self-association of SEL1L. FIG +154 163 SEL1Lcent structure_element The red rectangle indicates competitively inhibited SEL1L self-oligomer formation by the increasing doses of SEL1Lcent-HA-KDEL. (F) L521A point mutant in SEL1Lcent did not inhibit the self-association of SEL1L. FIG +204 209 SEL1L protein The red rectangle indicates competitively inhibited SEL1L self-oligomer formation by the increasing doses of SEL1Lcent-HA-KDEL. (F) L521A point mutant in SEL1Lcent did not inhibit the self-association of SEL1L. FIG +14 17 SLR structure_element Comparison of SLR in SEL1L with TPR or Other SLR-Containing Proteins. FIG +21 26 SEL1L protein Comparison of SLR in SEL1L with TPR or Other SLR-Containing Proteins. FIG +32 35 TPR structure_element Comparison of SLR in SEL1L with TPR or Other SLR-Containing Proteins. FIG +45 68 SLR-Containing Proteins protein_type Comparison of SLR in SEL1L with TPR or Other SLR-Containing Proteins. FIG +27 42 superimposition experimental_method (A) Ribbon diagram showing superimposition of an isolated TPR motif from Cdc23 and an SLR motif from SEL1Lcent (left), and SLR motifs in HcpC and SEL1Lcent (right). FIG +58 61 TPR structure_element (A) Ribbon diagram showing superimposition of an isolated TPR motif from Cdc23 and an SLR motif from SEL1Lcent (left), and SLR motifs in HcpC and SEL1Lcent (right). FIG +73 78 Cdc23 protein (A) Ribbon diagram showing superimposition of an isolated TPR motif from Cdc23 and an SLR motif from SEL1Lcent (left), and SLR motifs in HcpC and SEL1Lcent (right). FIG +86 89 SLR structure_element (A) Ribbon diagram showing superimposition of an isolated TPR motif from Cdc23 and an SLR motif from SEL1Lcent (left), and SLR motifs in HcpC and SEL1Lcent (right). FIG +101 110 SEL1Lcent structure_element (A) Ribbon diagram showing superimposition of an isolated TPR motif from Cdc23 and an SLR motif from SEL1Lcent (left), and SLR motifs in HcpC and SEL1Lcent (right). FIG +123 126 SLR structure_element (A) Ribbon diagram showing superimposition of an isolated TPR motif from Cdc23 and an SLR motif from SEL1Lcent (left), and SLR motifs in HcpC and SEL1Lcent (right). FIG +137 141 HcpC protein (A) Ribbon diagram showing superimposition of an isolated TPR motif from Cdc23 and an SLR motif from SEL1Lcent (left), and SLR motifs in HcpC and SEL1Lcent (right). FIG +146 155 SEL1Lcent structure_element (A) Ribbon diagram showing superimposition of an isolated TPR motif from Cdc23 and an SLR motif from SEL1Lcent (left), and SLR motifs in HcpC and SEL1Lcent (right). FIG +4 9 SEL1L protein The SEL1L, Cdc23, and HcpC are colored magenta, green and cyan, respectively. FIG +11 16 Cdc23 protein The SEL1L, Cdc23, and HcpC are colored magenta, green and cyan, respectively. FIG +22 26 HcpC protein The SEL1L, Cdc23, and HcpC are colored magenta, green and cyan, respectively. FIG +24 39 disulfide bonds ptm The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. FIG +47 51 HcpC protein The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. FIG +57 60 Cys residue_name The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. FIG +82 99 disulfide bonding ptm The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. FIG +162 177 superimposition experimental_method The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. FIG +181 186 Cdc23 protein The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. FIG +191 200 SEL1Lcent structure_element The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. FIG +211 215 HcpC protein The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. FIG +220 229 SEL1Lcent structure_element The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. FIG +281 298 α-solenoid domain structure_element The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. FIG +5 14 SEL1Lcent structure_element Both SEL1Lcent schematics are identically oriented for comparison. FIG +37 54 α-solenoid domain structure_element The Cα atoms of the residues in each α-solenoid domain are superimposed with a root-mean-squared deviation of 3.3 Å for Cdc23 and SEL1Lcent (left), and 2.5 Å for HcpC and SEL1Lcent (right). FIG +59 71 superimposed experimental_method The Cα atoms of the residues in each α-solenoid domain are superimposed with a root-mean-squared deviation of 3.3 Å for Cdc23 and SEL1Lcent (left), and 2.5 Å for HcpC and SEL1Lcent (right). FIG +79 106 root-mean-squared deviation evidence The Cα atoms of the residues in each α-solenoid domain are superimposed with a root-mean-squared deviation of 3.3 Å for Cdc23 and SEL1Lcent (left), and 2.5 Å for HcpC and SEL1Lcent (right). FIG +120 125 Cdc23 protein The Cα atoms of the residues in each α-solenoid domain are superimposed with a root-mean-squared deviation of 3.3 Å for Cdc23 and SEL1Lcent (left), and 2.5 Å for HcpC and SEL1Lcent (right). FIG +130 139 SEL1Lcent structure_element The Cα atoms of the residues in each α-solenoid domain are superimposed with a root-mean-squared deviation of 3.3 Å for Cdc23 and SEL1Lcent (left), and 2.5 Å for HcpC and SEL1Lcent (right). FIG +162 166 HcpC protein The Cα atoms of the residues in each α-solenoid domain are superimposed with a root-mean-squared deviation of 3.3 Å for Cdc23 and SEL1Lcent (left), and 2.5 Å for HcpC and SEL1Lcent (right). FIG +171 180 SEL1Lcent structure_element The Cα atoms of the residues in each α-solenoid domain are superimposed with a root-mean-squared deviation of 3.3 Å for Cdc23 and SEL1Lcent (left), and 2.5 Å for HcpC and SEL1Lcent (right). FIG +0 9 SEL1Lcent structure_element SEL1Lcent, Cdc23, and HcpC are colored as in (A). (C) Ribbon diagram showing the overall structure of Cdc23N-term (left) and SEL1Lcent (right) to compare their similarities regarding dimer formation through domain swapping. FIG +11 16 Cdc23 protein SEL1Lcent, Cdc23, and HcpC are colored as in (A). (C) Ribbon diagram showing the overall structure of Cdc23N-term (left) and SEL1Lcent (right) to compare their similarities regarding dimer formation through domain swapping. FIG +22 26 HcpC protein SEL1Lcent, Cdc23, and HcpC are colored as in (A). (C) Ribbon diagram showing the overall structure of Cdc23N-term (left) and SEL1Lcent (right) to compare their similarities regarding dimer formation through domain swapping. FIG +89 98 structure evidence SEL1Lcent, Cdc23, and HcpC are colored as in (A). (C) Ribbon diagram showing the overall structure of Cdc23N-term (left) and SEL1Lcent (right) to compare their similarities regarding dimer formation through domain swapping. FIG +102 107 Cdc23 protein SEL1Lcent, Cdc23, and HcpC are colored as in (A). (C) Ribbon diagram showing the overall structure of Cdc23N-term (left) and SEL1Lcent (right) to compare their similarities regarding dimer formation through domain swapping. FIG +125 134 SEL1Lcent structure_element SEL1Lcent, Cdc23, and HcpC are colored as in (A). (C) Ribbon diagram showing the overall structure of Cdc23N-term (left) and SEL1Lcent (right) to compare their similarities regarding dimer formation through domain swapping. FIG +183 188 dimer oligomeric_state SEL1Lcent, Cdc23, and HcpC are colored as in (A). (C) Ribbon diagram showing the overall structure of Cdc23N-term (left) and SEL1Lcent (right) to compare their similarities regarding dimer formation through domain swapping. FIG +12 17 SLR-C structure_element The Role of SLR-C in ERAD machinery and Model for the Organization of Proteins in Membrane-Associated ERAD Components. FIG +34 38 HRD1 protein (A) Schematic diagram shows three HRD1 fragment constructs used in the GST pull-down experiment. (B) Pull-down experiments to examine the interactions between HRD luminal loops and certain SLR motifs of SEL1L. FIG +71 84 GST pull-down experimental_method (A) Schematic diagram shows three HRD1 fragment constructs used in the GST pull-down experiment. (B) Pull-down experiments to examine the interactions between HRD luminal loops and certain SLR motifs of SEL1L. FIG +101 122 Pull-down experiments experimental_method (A) Schematic diagram shows three HRD1 fragment constructs used in the GST pull-down experiment. (B) Pull-down experiments to examine the interactions between HRD luminal loops and certain SLR motifs of SEL1L. FIG +159 162 HRD complex_assembly (A) Schematic diagram shows three HRD1 fragment constructs used in the GST pull-down experiment. (B) Pull-down experiments to examine the interactions between HRD luminal loops and certain SLR motifs of SEL1L. FIG +163 176 luminal loops structure_element (A) Schematic diagram shows three HRD1 fragment constructs used in the GST pull-down experiment. (B) Pull-down experiments to examine the interactions between HRD luminal loops and certain SLR motifs of SEL1L. FIG +189 192 SLR structure_element (A) Schematic diagram shows three HRD1 fragment constructs used in the GST pull-down experiment. (B) Pull-down experiments to examine the interactions between HRD luminal loops and certain SLR motifs of SEL1L. FIG +203 208 SEL1L protein (A) Schematic diagram shows three HRD1 fragment constructs used in the GST pull-down experiment. (B) Pull-down experiments to examine the interactions between HRD luminal loops and certain SLR motifs of SEL1L. FIG +17 29 luminal loop structure_element Fragments of the luminal loop of HRD1 fused to GST were immobilized on glutathione sepharose beads and incubated with purified three clusters of SLR motifs and monomer form of SLR-M (SLR-ML521A, right panel) in SEL1L. FIG +33 37 HRD1 protein Fragments of the luminal loop of HRD1 fused to GST were immobilized on glutathione sepharose beads and incubated with purified three clusters of SLR motifs and monomer form of SLR-M (SLR-ML521A, right panel) in SEL1L. FIG +47 50 GST chemical Fragments of the luminal loop of HRD1 fused to GST were immobilized on glutathione sepharose beads and incubated with purified three clusters of SLR motifs and monomer form of SLR-M (SLR-ML521A, right panel) in SEL1L. FIG +145 148 SLR structure_element Fragments of the luminal loop of HRD1 fused to GST were immobilized on glutathione sepharose beads and incubated with purified three clusters of SLR motifs and monomer form of SLR-M (SLR-ML521A, right panel) in SEL1L. FIG +160 167 monomer oligomeric_state Fragments of the luminal loop of HRD1 fused to GST were immobilized on glutathione sepharose beads and incubated with purified three clusters of SLR motifs and monomer form of SLR-M (SLR-ML521A, right panel) in SEL1L. FIG +176 181 SLR-M structure_element Fragments of the luminal loop of HRD1 fused to GST were immobilized on glutathione sepharose beads and incubated with purified three clusters of SLR motifs and monomer form of SLR-M (SLR-ML521A, right panel) in SEL1L. FIG +183 193 SLR-ML521A mutant Fragments of the luminal loop of HRD1 fused to GST were immobilized on glutathione sepharose beads and incubated with purified three clusters of SLR motifs and monomer form of SLR-M (SLR-ML521A, right panel) in SEL1L. FIG +211 216 SEL1L protein Fragments of the luminal loop of HRD1 fused to GST were immobilized on glutathione sepharose beads and incubated with purified three clusters of SLR motifs and monomer form of SLR-M (SLR-ML521A, right panel) in SEL1L. FIG +30 38 SDS-PAGE experimental_method Proteins were analyzed by 12% SDS-PAGE and Coomassie blue staining. FIG +52 60 metazoan taxonomy_domain (C) Schematic representation of the organization of metazoan ERAD components in the ER membrane. FIG +7 10 SLR structure_element The 11 SLR motifs of SEL1L were expressed with red cylinders and grouped into three parts (SLR-N, SLR-M, and SLR-C) based on the sequence alignment across the motifs and the crystal structure presented herein. FIG +21 26 SEL1L protein The 11 SLR motifs of SEL1L were expressed with red cylinders and grouped into three parts (SLR-N, SLR-M, and SLR-C) based on the sequence alignment across the motifs and the crystal structure presented herein. FIG +91 96 SLR-N structure_element The 11 SLR motifs of SEL1L were expressed with red cylinders and grouped into three parts (SLR-N, SLR-M, and SLR-C) based on the sequence alignment across the motifs and the crystal structure presented herein. FIG +98 103 SLR-M structure_element The 11 SLR motifs of SEL1L were expressed with red cylinders and grouped into three parts (SLR-N, SLR-M, and SLR-C) based on the sequence alignment across the motifs and the crystal structure presented herein. FIG +109 114 SLR-C structure_element The 11 SLR motifs of SEL1L were expressed with red cylinders and grouped into three parts (SLR-N, SLR-M, and SLR-C) based on the sequence alignment across the motifs and the crystal structure presented herein. FIG +129 147 sequence alignment experimental_method The 11 SLR motifs of SEL1L were expressed with red cylinders and grouped into three parts (SLR-N, SLR-M, and SLR-C) based on the sequence alignment across the motifs and the crystal structure presented herein. FIG +174 191 crystal structure evidence The 11 SLR motifs of SEL1L were expressed with red cylinders and grouped into three parts (SLR-N, SLR-M, and SLR-C) based on the sequence alignment across the motifs and the crystal structure presented herein. FIG +37 40 SLR structure_element We hypothesized that the interrupted SLR motifs of SEL1L have distinct functions such that the SLR-M is important for dimer formation of the protein, and SLR-C is involved in the interaction with HRD1 in the ER lumen. FIG +51 56 SEL1L protein We hypothesized that the interrupted SLR motifs of SEL1L have distinct functions such that the SLR-M is important for dimer formation of the protein, and SLR-C is involved in the interaction with HRD1 in the ER lumen. FIG +95 100 SLR-M structure_element We hypothesized that the interrupted SLR motifs of SEL1L have distinct functions such that the SLR-M is important for dimer formation of the protein, and SLR-C is involved in the interaction with HRD1 in the ER lumen. FIG +118 123 dimer oligomeric_state We hypothesized that the interrupted SLR motifs of SEL1L have distinct functions such that the SLR-M is important for dimer formation of the protein, and SLR-C is involved in the interaction with HRD1 in the ER lumen. FIG +154 159 SLR-C structure_element We hypothesized that the interrupted SLR motifs of SEL1L have distinct functions such that the SLR-M is important for dimer formation of the protein, and SLR-C is involved in the interaction with HRD1 in the ER lumen. FIG +196 200 HRD1 protein We hypothesized that the interrupted SLR motifs of SEL1L have distinct functions such that the SLR-M is important for dimer formation of the protein, and SLR-C is involved in the interaction with HRD1 in the ER lumen. FIG +30 39 SEL1Lcent structure_element The surface representation of SEL1Lcent is placed in the same orientation as that shown in the schematic model to show that the putative N-glycosylation site, residue N427 (indicated in yellow), is exposed on the surface of the protein. FIG +137 157 N-glycosylation site site The surface representation of SEL1Lcent is placed in the same orientation as that shown in the schematic model to show that the putative N-glycosylation site, residue N427 (indicated in yellow), is exposed on the surface of the protein. FIG +167 171 N427 residue_name_number The surface representation of SEL1Lcent is placed in the same orientation as that shown in the schematic model to show that the putative N-glycosylation site, residue N427 (indicated in yellow), is exposed on the surface of the protein. FIG diff --git a/annotation_CSV/PMC4772114.csv b/annotation_CSV/PMC4772114.csv new file mode 100644 index 0000000000000000000000000000000000000000..7c74d9a93b19d1b8cfe1ea404ef1102167fcc4c7 --- /dev/null +++ b/annotation_CSV/PMC4772114.csv @@ -0,0 +1,825 @@ +anno_start anno_end anno_text entity_type sentence section +61 70 Regnase-1 protein Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions TITLE +0 9 Regnase-1 protein Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. ABSTRACT +16 21 RNase protein_type Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. ABSTRACT +44 49 mRNAs chemical Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. ABSTRACT +80 84 IL-6 protein_type Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. ABSTRACT +89 97 IL-12p40 protein_type Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. ABSTRACT +20 30 structures evidence Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). ABSTRACT +50 59 Regnase-1 protein Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). ABSTRACT +65 77 Mus musculus species Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). ABSTRACT +82 99 N-terminal domain structure_element Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). ABSTRACT +101 104 NTD structure_element Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). ABSTRACT +107 127 PilT N-terminus like structure_element Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). ABSTRACT +129 132 PIN structure_element Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). ABSTRACT +142 153 zinc finger structure_element Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). ABSTRACT +155 157 ZF structure_element Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). ABSTRACT +170 187 C-terminal domain structure_element Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). ABSTRACT +189 192 CTD structure_element Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). ABSTRACT +4 7 PIN structure_element The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. ABSTRACT +27 32 RNase protein_type The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. ABSTRACT +33 49 catalytic center site The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. ABSTRACT +18 21 NTD structure_element We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. ABSTRACT +42 45 PIN structure_element We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. ABSTRACT +84 89 RNase protein_type We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. ABSTRACT +4 7 PIN structure_element The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. ABSTRACT +23 35 head-to-tail protein_state The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. ABSTRACT +36 44 oligomer oligomeric_state The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. ABSTRACT +53 68 dimer interface site The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. ABSTRACT +87 103 NTD binding site site The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. ABSTRACT +15 24 mutations experimental_method Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. ABSTRACT +34 37 PIN structure_element Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. ABSTRACT +61 66 RNase protein_type Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. ABSTRACT +118 121 NTD structure_element Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. ABSTRACT +146 151 RNase protein_type Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. ABSTRACT +27 36 Regnase-1 protein These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions. ABSTRACT +37 42 RNase protein_type These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions. ABSTRACT +98 101 NTD structure_element These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions. ABSTRACT +102 105 PIN structure_element These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions. ABSTRACT +127 130 PIN structure_element These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions. ABSTRACT +131 134 PIN structure_element These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions. ABSTRACT +57 86 pattern-recognition receptors protein_type The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. INTRO +88 92 PRRs protein_type The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. INTRO +99 118 Toll-like receptors protein_type The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. INTRO +120 124 TLRs protein_type The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. INTRO +180 184 TLRs protein_type The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. INTRO +0 9 Regnase-1 protein Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. INTRO +25 32 Zc3h12a protein Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. INTRO +37 43 MCPIP1 protein Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. INTRO +51 56 RNase protein_type Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. INTRO +97 116 lipopolysaccharides chemical Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. INTRO +191 196 mRNAs chemical Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. INTRO +239 245 (IL)-6 protein_type Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. INTRO +247 252 IL-1β protein_type Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. INTRO +254 258 IL-2 protein_type Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. INTRO +264 272 IL-12p40 protein_type Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. INTRO +0 9 Regnase-1 protein Regnase-1 accelerates target mRNA degradation via their 3′-terminal untranslated region (3′UTR), and also degrades its own mRNA. INTRO +29 33 mRNA chemical Regnase-1 accelerates target mRNA degradation via their 3′-terminal untranslated region (3′UTR), and also degrades its own mRNA. INTRO +56 87 3′-terminal untranslated region structure_element Regnase-1 accelerates target mRNA degradation via their 3′-terminal untranslated region (3′UTR), and also degrades its own mRNA. INTRO +89 94 3′UTR structure_element Regnase-1 accelerates target mRNA degradation via their 3′-terminal untranslated region (3′UTR), and also degrades its own mRNA. INTRO +123 127 mRNA chemical Regnase-1 accelerates target mRNA degradation via their 3′-terminal untranslated region (3′UTR), and also degrades its own mRNA. INTRO +0 9 Regnase-1 protein Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. INTRO +25 39 Regnase family protein_type Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. INTRO +61 81 PilT N-terminus like structure_element Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. INTRO +83 86 PIN structure_element Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. INTRO +109 130 CCCH-type zinc–finger structure_element Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. INTRO +132 134 ZF structure_element Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. INTRO +154 163 conserved protein_state Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. INTRO +170 192 Regnase family members protein_type Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. INTRO +14 31 crystal structure evidence Recently, the crystal structure of the Regnase-1 PIN domain derived from Homo sapiens was reported. INTRO +39 48 Regnase-1 protein Recently, the crystal structure of the Regnase-1 PIN domain derived from Homo sapiens was reported. INTRO +49 52 PIN structure_element Recently, the crystal structure of the Regnase-1 PIN domain derived from Homo sapiens was reported. INTRO +73 85 Homo sapiens species Recently, the crystal structure of the Regnase-1 PIN domain derived from Homo sapiens was reported. INTRO +4 13 structure evidence The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity. INTRO +91 94 Asp residue_name The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity. INTRO +113 129 catalytic center site The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity. INTRO +144 148 Mg2+ chemical The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity. INTRO +177 182 RNase protein_type The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity. INTRO +8 27 CCCH-type ZF motifs structure_element Several CCCH-type ZF motifs in RNA-binding proteins have been reported to directly bind RNA. INTRO +31 51 RNA-binding proteins protein_type Several CCCH-type ZF motifs in RNA-binding proteins have been reported to directly bind RNA. INTRO +88 91 RNA chemical Several CCCH-type ZF motifs in RNA-binding proteins have been reported to directly bind RNA. INTRO +13 22 Regnase-1 protein In addition, Regnase-1 has been predicted to possess other domains in the N- and C- terminal regions. INTRO +74 100 N- and C- terminal regions structure_element In addition, Regnase-1 has been predicted to possess other domains in the N- and C- terminal regions. INTRO +13 22 structure evidence However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved. INTRO +43 45 ZF structure_element However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved. INTRO +54 71 N-terminal domain structure_element However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved. INTRO +73 76 NTD structure_element However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved. INTRO +82 99 C-terminal domain structure_element However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved. INTRO +101 104 CTD structure_element However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved. INTRO +109 118 Regnase-1 protein However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved. INTRO +19 53 structural and functional analyses experimental_method Here, we performed structural and functional analyses of individual domains of Regnase-1 derived from Mus musculus in order to understand the catalytic activity in vitro. INTRO +79 88 Regnase-1 protein Here, we performed structural and functional analyses of individual domains of Regnase-1 derived from Mus musculus in order to understand the catalytic activity in vitro. INTRO +102 114 Mus musculus species Here, we performed structural and functional analyses of individual domains of Regnase-1 derived from Mus musculus in order to understand the catalytic activity in vitro. INTRO +49 58 Regnase-1 protein Our data revealed that the catalytic activity of Regnase-1 is regulated through both intra and intermolecular domain interactions in vitro. INTRO +4 7 NTD structure_element The NTD plays a crucial role in efficient cleavage of target mRNA, through intramolecular NTD-PIN interactions. INTRO +61 65 mRNA chemical The NTD plays a crucial role in efficient cleavage of target mRNA, through intramolecular NTD-PIN interactions. INTRO +90 93 NTD structure_element The NTD plays a crucial role in efficient cleavage of target mRNA, through intramolecular NTD-PIN interactions. INTRO +94 97 PIN structure_element The NTD plays a crucial role in efficient cleavage of target mRNA, through intramolecular NTD-PIN interactions. INTRO +10 19 Regnase-1 protein Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA. INTRO +35 40 dimer oligomeric_state Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA. INTRO +64 67 PIN structure_element Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA. INTRO +68 71 PIN structure_element Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA. INTRO +111 115 mRNA chemical Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA. INTRO +26 35 Regnase-1 protein Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. INTRO +55 59 mRNA chemical Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. INTRO +66 79 NTD-activated protein_state Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. INTRO +80 90 functional protein_state Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. INTRO +91 94 PIN structure_element Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. INTRO +95 100 dimer oligomeric_state Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. INTRO +112 114 ZF structure_element Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. INTRO +125 128 RNA chemical Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. INTRO +161 164 PIN structure_element Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. INTRO +165 170 dimer oligomeric_state Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer. INTRO +7 17 structures evidence Domain structures of Regnase-1 RESULTS +21 30 Regnase-1 protein Domain structures of Regnase-1 RESULTS +12 21 Rengase-1 protein We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). RESULTS +35 47 Mus musculus species We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). RESULTS +52 58 solved experimental_method We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). RESULTS +63 73 structures evidence We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). RESULTS +95 98 NTD structure_element We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). RESULTS +100 103 PIN structure_element We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). RESULTS +105 107 ZF structure_element We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). RESULTS +113 116 CTD structure_element We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). RESULTS +133 154 X-ray crystallography experimental_method We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). RESULTS +158 161 NMR experimental_method We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). RESULTS +0 21 X-ray crystallography experimental_method X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. RESULTS +73 76 PIN structure_element X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. RESULTS +81 83 ZF structure_element X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. RESULTS +102 118 electron density evidence X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. RESULTS +145 148 PIN structure_element X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. RESULTS +204 213 Regnase-1 protein X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. RESULTS +227 239 Homo sapiens species X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. RESULTS +23 26 PIN structure_element This suggests that the PIN and ZF domains exist independently without interacting with each other. RESULTS +31 33 ZF structure_element This suggests that the PIN and ZF domains exist independently without interacting with each other. RESULTS +11 21 structures evidence The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e). RESULTS +25 28 NTD structure_element The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e). RESULTS +30 32 ZF structure_element The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e). RESULTS +38 41 CTD structure_element The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e). RESULTS +61 64 NMR experimental_method The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e). RESULTS +4 7 NTD structure_element The NTD and CTD are both composed of three α helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server. RESULTS +12 15 CTD structure_element The NTD and CTD are both composed of three α helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server. RESULTS +43 52 α helices structure_element The NTD and CTD are both composed of three α helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server. RESULTS +80 113 ubiquitin conjugating enzyme E2 K protein The NTD and CTD are both composed of three α helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server. RESULTS +133 163 ubiquitin associated protein 1 protein The NTD and CTD are both composed of three α helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server. RESULTS +211 222 Dali server experimental_method The NTD and CTD are both composed of three α helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server. RESULTS +31 40 Regnase-1 protein Contribution of each domain of Regnase-1 to the mRNA binding activity RESULTS +48 52 mRNA chemical Contribution of each domain of Regnase-1 to the mRNA binding activity RESULTS +13 16 PIN structure_element Although the PIN domain is responsible for the catalytic activity of Regnase-1, the roles of the other domains are largely unknown. RESULTS +69 78 Regnase-1 protein Although the PIN domain is responsible for the catalytic activity of Regnase-1, the roles of the other domains are largely unknown. RESULTS +34 37 NTD structure_element First, we evaluated a role of the NTD and ZF domains for mRNA binding by an in vitro gel shift assay (Fig. 1f). RESULTS +42 44 ZF structure_element First, we evaluated a role of the NTD and ZF domains for mRNA binding by an in vitro gel shift assay (Fig. 1f). RESULTS +57 61 mRNA chemical First, we evaluated a role of the NTD and ZF domains for mRNA binding by an in vitro gel shift assay (Fig. 1f). RESULTS +76 100 in vitro gel shift assay experimental_method First, we evaluated a role of the NTD and ZF domains for mRNA binding by an in vitro gel shift assay (Fig. 1f). RESULTS +0 24 Fluorescently 5′-labeled protein_state Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. RESULTS +25 28 RNA chemical Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. RESULTS +72 76 IL-6 protein_type Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. RESULTS +77 81 mRNA chemical Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. RESULTS +82 87 3′UTR structure_element Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. RESULTS +110 118 inactive protein_state Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. RESULTS +119 125 mutant protein_state Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. RESULTS +127 132 D226N mutant Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. RESULTS +137 142 D244N mutant Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. RESULTS +147 156 Regnase-1 protein Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. RESULTS +186 190 DDNN mutant Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. RESULTS +191 197 mutant protein_state Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. RESULTS +36 45 Regnase-1 protein Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. RESULTS +51 63 fluorescence evidence Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. RESULTS +67 71 free protein_state Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. RESULTS +72 75 RNA chemical Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. RESULTS +103 112 Regnase-1 protein Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. RESULTS +113 121 bound to protein_state Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. RESULTS +126 129 RNA chemical Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. RESULTS +34 37 RNA chemical Based on the decrease in the free RNA fluorescence band, we evaluated the contribution of each domain of Regnase-1 to RNA binding. RESULTS +105 114 Regnase-1 protein Based on the decrease in the free RNA fluorescence band, we evaluated the contribution of each domain of Regnase-1 to RNA binding. RESULTS +118 121 RNA chemical Based on the decrease in the free RNA fluorescence band, we evaluated the contribution of each domain of Regnase-1 to RNA binding. RESULTS +10 13 RNA chemical While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1). RESULTS +67 78 presence of protein_state While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1). RESULTS +79 82 NTD structure_element While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1). RESULTS +104 115 presence of protein_state While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1). RESULTS +120 122 ZF structure_element While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1). RESULTS +22 24 ZF structure_element Direct binding of the ZF domain and RNA were confirmed by NMR spectral changes. RESULTS +36 39 RNA chemical Direct binding of the ZF domain and RNA were confirmed by NMR spectral changes. RESULTS +58 61 NMR experimental_method Direct binding of the ZF domain and RNA were confirmed by NMR spectral changes. RESULTS +62 78 spectral changes evidence Direct binding of the ZF domain and RNA were confirmed by NMR spectral changes. RESULTS +19 34 titration curve evidence The fitting of the titration curve of Y314 resulted in an apparent dissociation constant (Kd) of 10 ± 1.1 μM (Supplementary Fig. 2). RESULTS +38 42 Y314 residue_name_number The fitting of the titration curve of Y314 resulted in an apparent dissociation constant (Kd) of 10 ± 1.1 μM (Supplementary Fig. 2). RESULTS +67 88 dissociation constant evidence The fitting of the titration curve of Y314 resulted in an apparent dissociation constant (Kd) of 10 ± 1.1 μM (Supplementary Fig. 2). RESULTS +90 92 Kd evidence The fitting of the titration curve of Y314 resulted in an apparent dissociation constant (Kd) of 10 ± 1.1 μM (Supplementary Fig. 2). RESULTS +41 44 PIN structure_element These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA. RESULTS +58 60 ZF structure_element These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA. RESULTS +82 85 RNA chemical These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA. RESULTS +105 108 NTD structure_element These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA. RESULTS +165 168 RNA chemical These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA. RESULTS +31 40 Regnase-1 protein Contribution of each domain of Regnase-1 to RNase activity RESULTS +44 49 RNase protein_type Contribution of each domain of Regnase-1 to RNase activity RESULTS +56 61 RNase protein_type In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). RESULTS +74 83 Regnase-1 protein In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). RESULTS +101 124 in vitro cleavage assay experimental_method In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). RESULTS +131 155 fluorescently 5′-labeled protein_state In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). RESULTS +156 159 RNA chemical In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). RESULTS +203 207 IL-6 protein_type In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). RESULTS +208 212 mRNA chemical In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). RESULTS +213 218 3′UTR structure_element In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). RESULTS +0 9 Regnase-1 protein Regnase-1 constructs consisting of NTD-PIN-ZF completely cleaved the target mRNA and generated the cleaved products. RESULTS +35 45 NTD-PIN-ZF mutant Regnase-1 constructs consisting of NTD-PIN-ZF completely cleaved the target mRNA and generated the cleaved products. RESULTS +76 80 mRNA chemical Regnase-1 constructs consisting of NTD-PIN-ZF completely cleaved the target mRNA and generated the cleaved products. RESULTS +37 42 RNase protein_type The apparent half-life (T1/2) of the RNase activity was about 20 minutes. RESULTS +0 9 Regnase-1 protein Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2 ~ 70 minutes), while those lacking the NTD did not generate cleaved products (T1/2 > 90 minutes). RESULTS +10 17 lacking protein_state Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2 ~ 70 minutes), while those lacking the NTD did not generate cleaved products (T1/2 > 90 minutes). RESULTS +22 24 ZF structure_element Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2 ~ 70 minutes), while those lacking the NTD did not generate cleaved products (T1/2 > 90 minutes). RESULTS +127 134 lacking protein_state Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2 ~ 70 minutes), while those lacking the NTD did not generate cleaved products (T1/2 > 90 minutes). RESULTS +139 142 NTD structure_element Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2 ~ 70 minutes), while those lacking the NTD did not generate cleaved products (T1/2 > 90 minutes). RESULTS +24 40 NTD-PIN(DDNN)-ZF mutant It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. RESULTS +62 65 NTD structure_element It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. RESULTS +70 75 lacks protein_state It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. RESULTS +80 98 catalytic residues site It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. RESULTS +102 105 PIN structure_element It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. RESULTS +127 132 RNase protein_type It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. RESULTS +199 204 RNase protein_type It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. RESULTS +205 221 catalytic center site It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. RESULTS +240 243 PIN structure_element It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. RESULTS +78 81 NTD structure_element Taken together with the results in the previous section, we conclude that the NTD is crucial for the RNase activity of Regnase-1 in vitro, although it does not contribute to the direct mRNA binding. RESULTS +101 106 RNase protein_type Taken together with the results in the previous section, we conclude that the NTD is crucial for the RNase activity of Regnase-1 in vitro, although it does not contribute to the direct mRNA binding. RESULTS +119 128 Regnase-1 protein Taken together with the results in the previous section, we conclude that the NTD is crucial for the RNase activity of Regnase-1 in vitro, although it does not contribute to the direct mRNA binding. RESULTS +185 189 mRNA chemical Taken together with the results in the previous section, we conclude that the NTD is crucial for the RNase activity of Regnase-1 in vitro, although it does not contribute to the direct mRNA binding. RESULTS +0 5 Dimer oligomeric_state Dimer formation of the PIN domains RESULTS +23 26 PIN structure_element Dimer formation of the PIN domains RESULTS +7 19 purification experimental_method During purification by gel filtration, the PIN domain exhibited extremely asymmetric elution peaks in a concentration dependent manner (Fig. 2a). RESULTS +23 37 gel filtration experimental_method During purification by gel filtration, the PIN domain exhibited extremely asymmetric elution peaks in a concentration dependent manner (Fig. 2a). RESULTS +43 46 PIN structure_element During purification by gel filtration, the PIN domain exhibited extremely asymmetric elution peaks in a concentration dependent manner (Fig. 2a). RESULTS +3 65 comparison with the elution volume of standard marker proteins experimental_method By comparison with the elution volume of standard marker proteins, the PIN domain was assumed to be in equilibrium between a monomer and a dimer in solution at concentrations in the 20–200 μM range. RESULTS +71 74 PIN structure_element By comparison with the elution volume of standard marker proteins, the PIN domain was assumed to be in equilibrium between a monomer and a dimer in solution at concentrations in the 20–200 μM range. RESULTS +125 132 monomer oligomeric_state By comparison with the elution volume of standard marker proteins, the PIN domain was assumed to be in equilibrium between a monomer and a dimer in solution at concentrations in the 20–200 μM range. RESULTS +139 144 dimer oligomeric_state By comparison with the elution volume of standard marker proteins, the PIN domain was assumed to be in equilibrium between a monomer and a dimer in solution at concentrations in the 20–200 μM range. RESULTS +4 21 crystal structure evidence The crystal structure of the PIN domain has been determined in three distinct crystal forms with a space group of P3121 (form I in this study and PDB ID 3V33), P3221 (form II in this study), and P41 (PDB ID 3V32 and 3V34), respectively. RESULTS +29 32 PIN structure_element The crystal structure of the PIN domain has been determined in three distinct crystal forms with a space group of P3121 (form I in this study and PDB ID 3V33), P3221 (form II in this study), and P41 (PDB ID 3V32 and 3V34), respectively. RESULTS +78 91 crystal forms evidence The crystal structure of the PIN domain has been determined in three distinct crystal forms with a space group of P3121 (form I in this study and PDB ID 3V33), P3221 (form II in this study), and P41 (PDB ID 3V32 and 3V34), respectively. RESULTS +18 21 PIN structure_element We found that the PIN domain formed a head-to-tail oligomer that was commonly observed in all three crystal forms in spite of the different crystallization conditions (Supplementary Fig. 3). RESULTS +38 50 head-to-tail protein_state We found that the PIN domain formed a head-to-tail oligomer that was commonly observed in all three crystal forms in spite of the different crystallization conditions (Supplementary Fig. 3). RESULTS +51 59 oligomer oligomeric_state We found that the PIN domain formed a head-to-tail oligomer that was commonly observed in all three crystal forms in spite of the different crystallization conditions (Supplementary Fig. 3). RESULTS +100 113 crystal forms evidence We found that the PIN domain formed a head-to-tail oligomer that was commonly observed in all three crystal forms in spite of the different crystallization conditions (Supplementary Fig. 3). RESULTS +0 8 Mutation experimental_method Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. RESULTS +12 18 Arg215 residue_name_number Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. RESULTS +71 89 oligomeric surface site Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. RESULTS +94 97 Glu residue_name Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. RESULTS +112 119 monomer oligomeric_state Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. RESULTS +120 125 dimer oligomeric_state Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. RESULTS +154 163 wild type protein_state Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. RESULTS +19 35 single mutations experimental_method On the other hand, single mutations of side chains involved in the PIN–PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b). RESULTS +67 70 PIN structure_element On the other hand, single mutations of side chains involved in the PIN–PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b). RESULTS +71 74 PIN structure_element On the other hand, single mutations of side chains involved in the PIN–PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b). RESULTS +110 117 monomer oligomeric_state On the other hand, single mutations of side chains involved in the PIN–PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b). RESULTS +142 156 gel filtration experimental_method On the other hand, single mutations of side chains involved in the PIN–PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b). RESULTS +0 9 Wild type protein_state Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. RESULTS +14 23 monomeric oligomeric_state Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. RESULTS +24 27 PIN structure_element Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. RESULTS +28 35 mutants protein_state Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. RESULTS +37 42 P212A mutant Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. RESULTS +47 52 D278R mutant Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. RESULTS +76 79 NMR experimental_method Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. RESULTS +4 11 spectra evidence The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4). RESULTS +30 45 dimer interface site The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4). RESULTS +53 62 wild type protein_state The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4). RESULTS +63 66 PIN structure_element The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4). RESULTS +119 128 monomeric oligomeric_state The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4). RESULTS +129 136 mutants protein_state The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4). RESULTS +32 35 PIN structure_element These results indicate that the PIN domain forms a head-to-tail oligomer in solution similar to the crystal structure. RESULTS +51 63 head-to-tail protein_state These results indicate that the PIN domain forms a head-to-tail oligomer in solution similar to the crystal structure. RESULTS +64 72 oligomer oligomeric_state These results indicate that the PIN domain forms a head-to-tail oligomer in solution similar to the crystal structure. RESULTS +100 117 crystal structure evidence These results indicate that the PIN domain forms a head-to-tail oligomer in solution similar to the crystal structure. RESULTS +19 28 monomeric oligomeric_state Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). RESULTS +29 32 PIN structure_element Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). RESULTS +33 40 mutants protein_state Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). RESULTS +41 46 P212A mutant Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). RESULTS +48 53 R214A mutant Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). RESULTS +59 64 D278R mutant Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). RESULTS +84 89 RNase protein_type Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). RESULTS +103 107 IL-6 protein_type Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). RESULTS +108 112 mRNA chemical Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). RESULTS +54 70 catalytic center site The side chains of these residues point away from the catalytic center on the same molecule (Fig. 2b). RESULTS +29 41 head-to-tail protein_state Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro. RESULTS +42 45 PIN structure_element Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro. RESULTS +78 81 NTD structure_element Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro. RESULTS +100 109 Regnase-1 protein Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro. RESULTS +110 115 RNase protein_type Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro. RESULTS +38 41 NTD structure_element Domain-domain interaction between the NTD and the PIN domain RESULTS +50 53 PIN structure_element Domain-domain interaction between the NTD and the PIN domain RESULTS +10 13 NTD structure_element While the NTD does not contribute to RNA binding (Fig. 1f,g, and Supplementary Fig. 1), it increases the RNase activity of Regnase-1 (Fig. 1h). RESULTS +37 40 RNA chemical While the NTD does not contribute to RNA binding (Fig. 1f,g, and Supplementary Fig. 1), it increases the RNase activity of Regnase-1 (Fig. 1h). RESULTS +105 110 RNase protein_type While the NTD does not contribute to RNA binding (Fig. 1f,g, and Supplementary Fig. 1), it increases the RNase activity of Regnase-1 (Fig. 1h). RESULTS +123 132 Regnase-1 protein While the NTD does not contribute to RNA binding (Fig. 1f,g, and Supplementary Fig. 1), it increases the RNase activity of Regnase-1 (Fig. 1h). RESULTS +61 64 NTD structure_element In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR. RESULTS +89 98 Regnase-1 protein In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR. RESULTS +99 104 RNase protein_type In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR. RESULTS +181 184 NTD structure_element In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR. RESULTS +193 196 PIN structure_element In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR. RESULTS +210 213 NMR experimental_method In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR. RESULTS +12 34 catalytically inactive protein_state We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. RESULTS +35 44 monomeric oligomeric_state We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. RESULTS +45 48 PIN structure_element We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. RESULTS +49 55 mutant protein_state We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. RESULTS +76 80 DDNN mutant We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. RESULTS +85 90 D278R mutant We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. RESULTS +110 115 dimer oligomeric_state We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. RESULTS +133 136 PIN structure_element We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. RESULTS +4 7 NMR experimental_method The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). RESULTS +25 28 PIN structure_element The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). RESULTS +46 50 V177 residue_name_number The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). RESULTS +52 61 F210-T211 residue_range The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). RESULTS +63 67 R214 residue_name_number The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). RESULTS +69 78 F228-L232 residue_range The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). RESULTS +84 93 F234-S236 residue_range The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). RESULTS +145 156 addition of experimental_method The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). RESULTS +161 164 NTD structure_element The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). RESULTS +15 26 addition of experimental_method Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +31 34 PIN structure_element Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +43 46 NMR experimental_method Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +68 71 R56 residue_name_number Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +73 80 L58-G59 residue_range Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +86 93 V86-H88 residue_range Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +101 104 NTD structure_element Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +157 160 D53 residue_name_number Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +162 165 F55 residue_name_number Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +167 170 K57 residue_name_number Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +172 179 Y60-S61 residue_range Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +181 184 V68 residue_name_number Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +186 193 T80-G83 residue_range Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +195 198 L85 residue_name_number Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +204 207 G89 residue_name_number Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +215 218 NTD structure_element Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +258 261 N79 residue_name_number Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). RESULTS +64 67 PIN structure_element These results clearly indicate a direct interaction between the PIN domain and the NTD. RESULTS +83 86 NTD structure_element These results clearly indicate a direct interaction between the PIN domain and the NTD. RESULTS +13 28 titration curve evidence Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. RESULTS +37 59 chemical shift changes evidence Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. RESULTS +63 66 L58 residue_name_number Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. RESULTS +81 83 Kd evidence Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. RESULTS +105 108 NTD structure_element Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. RESULTS +113 116 PIN structure_element Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. RESULTS +181 184 NTD structure_element Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. RESULTS +189 192 PIN structure_element Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. RESULTS +219 225 linker structure_element Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. RESULTS +238 254 binding affinity evidence Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. RESULTS +286 292 native protein_state Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. RESULTS +70 87 PIN/NTD interface site Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a). RESULTS +106 111 helix structure_element Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a). RESULTS +144 148 D225 residue_name_number Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a). RESULTS +153 157 D226 residue_name_number Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a). RESULTS +165 168 PIN structure_element Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a). RESULTS +28 40 binding site site Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can “terminate” PIN-PIN oligomerization (Fig. 2b). RESULTS +49 52 NTD structure_element Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can “terminate” PIN-PIN oligomerization (Fig. 2b). RESULTS +71 94 PIN-PIN dimer interface site Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can “terminate” PIN-PIN oligomerization (Fig. 2b). RESULTS +110 113 NTD structure_element Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can “terminate” PIN-PIN oligomerization (Fig. 2b). RESULTS +138 141 PIN structure_element Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can “terminate” PIN-PIN oligomerization (Fig. 2b). RESULTS +142 145 PIN structure_element Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can “terminate” PIN-PIN oligomerization (Fig. 2b). RESULTS +3 20 in silico docking experimental_method An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c). RESULTS +28 31 NTD structure_element An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c). RESULTS +36 39 PIN structure_element An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c). RESULTS +54 79 chemical shift restraints evidence An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c). RESULTS +117 120 NMR experimental_method An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c). RESULTS +22 31 Regnase-1 protein Residues critical for Regnase-1 RNase activity RESULTS +32 37 RNase protein_type Residues critical for Regnase-1 RNase activity RESULTS +47 56 Regnase-1 protein To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). RESULTS +57 62 RNase protein_type To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). RESULTS +123 137 catalytic site site To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). RESULTS +145 148 PIN structure_element To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). RESULTS +149 157 oligomer oligomeric_state To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). RESULTS +162 172 mutated to experimental_method To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). RESULTS +173 180 alanine residue_name To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). RESULTS +210 215 RNase protein_type To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). RESULTS +9 30 gel filtration assays experimental_method From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. RESULTS +36 43 mutants protein_state From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. RESULTS +51 56 R214A mutant From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. RESULTS +64 70 dimers oligomeric_state From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. RESULTS +100 105 RNase protein_type From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. RESULTS +122 129 mutants protein_state From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. RESULTS +138 143 R214A mutant From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. RESULTS +234 239 dimer oligomeric_state From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. RESULTS +4 9 W182A mutant The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. RESULTS +11 16 R183A mutant The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. RESULTS +22 27 R214A mutant The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. RESULTS +28 35 mutants protein_state The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. RESULTS +72 76 IL-6 protein_type The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. RESULTS +77 81 mRNA chemical The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. RESULTS +97 106 Regnase-1 protein The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. RESULTS +107 111 mRNA chemical The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. RESULTS +4 9 K184A mutant The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs. RESULTS +11 16 R215A mutant The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs. RESULTS +22 27 R220A mutant The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs. RESULTS +28 35 mutants protein_state The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs. RESULTS +113 118 mRNAs chemical The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs. RESULTS +18 22 K219 residue_name_number The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. RESULTS +27 31 R247 residue_name_number The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. RESULTS +59 63 IL-6 protein_type The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. RESULTS +68 77 Regnase-1 protein The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. RESULTS +78 82 mRNA chemical The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. RESULTS +89 93 K219 residue_name_number The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. RESULTS +98 102 R247 residue_name_number The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. RESULTS +142 146 IL-6 protein_type The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. RESULTS +147 151 mRNA chemical The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. RESULTS +161 170 Regnase-1 protein The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. RESULTS +171 175 mRNA chemical The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. RESULTS +27 31 K152 residue_name_number The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. RESULTS +33 37 R158 residue_name_number The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. RESULTS +39 43 R188 residue_name_number The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. RESULTS +45 49 R200 residue_name_number The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. RESULTS +51 55 K204 residue_name_number The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. RESULTS +57 61 K206 residue_name_number The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. RESULTS +63 67 K257 residue_name_number The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. RESULTS +73 77 R258 residue_name_number The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. RESULTS +100 105 RNase protein_type The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. RESULTS +27 31 W182 residue_name_number The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +36 40 R183 residue_name_number The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +83 92 monomeric oligomeric_state The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +93 96 PIN structure_element The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +97 106 structure evidence The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +139 144 RNase protein_type The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +145 159 catalytic site site The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +196 200 K184 residue_name_number The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +229 240 active site site The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +296 305 structure evidence The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +348 352 K184 residue_name_number The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +377 385 primary” protein_state The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +394 408 catalytic site site The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). RESULTS +13 17 R214 residue_name_number In contrast, R214 is important for oligomerization of the PIN domain and the “secondary” chain’s residue R214 is also positioned near the “primary” chain’s active site within the dimer interface. RESULTS +58 61 PIN structure_element In contrast, R214 is important for oligomerization of the PIN domain and the “secondary” chain’s residue R214 is also positioned near the “primary” chain’s active site within the dimer interface. RESULTS +105 109 R214 residue_name_number In contrast, R214 is important for oligomerization of the PIN domain and the “secondary” chain’s residue R214 is also positioned near the “primary” chain’s active site within the dimer interface. RESULTS +139 146 primary protein_state In contrast, R214 is important for oligomerization of the PIN domain and the “secondary” chain’s residue R214 is also positioned near the “primary” chain’s active site within the dimer interface. RESULTS +156 167 active site site In contrast, R214 is important for oligomerization of the PIN domain and the “secondary” chain’s residue R214 is also positioned near the “primary” chain’s active site within the dimer interface. RESULTS +179 194 dimer interface site In contrast, R214 is important for oligomerization of the PIN domain and the “secondary” chain’s residue R214 is also positioned near the “primary” chain’s active site within the dimer interface. RESULTS +28 57 putative-RNA binding residues site It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains. RESULTS +58 62 K184 residue_name_number It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains. RESULTS +67 71 R214 residue_name_number It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains. RESULTS +86 95 Regnase-1 protein It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains. RESULTS +102 105 PIN structure_element It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains. RESULTS +30 34 mRNA chemical Molecular mechanism of target mRNA cleavage by the PIN dimer RESULTS +51 54 PIN structure_element Molecular mechanism of target mRNA cleavage by the PIN dimer RESULTS +55 60 dimer oligomeric_state Molecular mechanism of target mRNA cleavage by the PIN dimer RESULTS +4 26 mutational experiments experimental_method Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. RESULTS +55 60 dimer oligomeric_state Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. RESULTS +100 109 secondary protein_state Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. RESULTS +110 113 PIN structure_element Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. RESULTS +136 145 Regnase-1 protein Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. RESULTS +153 173 RNA binding residues site Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. RESULTS +183 194 active site site Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. RESULTS +202 209 primary protein_state Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. RESULTS +210 213 PIN structure_element Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. RESULTS +50 72 catalytically inactive protein_state If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. RESULTS +73 76 PIN structure_element If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. RESULTS +83 86 PIN structure_element If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. RESULTS +87 94 lacking protein_state If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. RESULTS +108 128 RNA-binding residues site If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. RESULTS +141 149 inactive protein_state If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. RESULTS +174 180 active protein_state If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. RESULTS +47 71 in vitro cleavage assays experimental_method In order to test this hypothesis, we performed in vitro cleavage assays using combinations of Regnase-1 mutants that had no or decreased RNase activities by themselves (Fig. 5). RESULTS +94 103 Regnase-1 protein In order to test this hypothesis, we performed in vitro cleavage assays using combinations of Regnase-1 mutants that had no or decreased RNase activities by themselves (Fig. 5). RESULTS +104 111 mutants protein_state In order to test this hypothesis, we performed in vitro cleavage assays using combinations of Regnase-1 mutants that had no or decreased RNase activities by themselves (Fig. 5). RESULTS +137 142 RNase protein_type In order to test this hypothesis, we performed in vitro cleavage assays using combinations of Regnase-1 mutants that had no or decreased RNase activities by themselves (Fig. 5). RESULTS +23 43 catalytically active protein_state One group consisted of catalytically active PIN domains with mutation of basic residues found in the previous section to confer decreased RNase activity (Fig. 4). RESULTS +44 47 PIN structure_element One group consisted of catalytically active PIN domains with mutation of basic residues found in the previous section to confer decreased RNase activity (Fig. 4). RESULTS +61 72 mutation of experimental_method One group consisted of catalytically active PIN domains with mutation of basic residues found in the previous section to confer decreased RNase activity (Fig. 4). RESULTS +138 143 RNase protein_type One group consisted of catalytically active PIN domains with mutation of basic residues found in the previous section to confer decreased RNase activity (Fig. 4). RESULTS +25 29 DDNN mutant These were paired with a DDNN mutant that had no RNase activity by itself. RESULTS +30 36 mutant protein_state These were paired with a DDNN mutant that had no RNase activity by itself. RESULTS +49 54 RNase protein_type These were paired with a DDNN mutant that had no RNase activity by itself. RESULTS +59 71 heterodimers oligomeric_state When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +108 112 DDNN mutant When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +113 120 primary protein_state When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +121 124 PIN structure_element When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +145 151 mutant protein_state When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +152 161 secondary protein_state When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +162 165 PIN structure_element When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +196 201 RNase protein_type When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +253 259 mutant protein_state When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +260 267 primary protein_state When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +268 271 PIN structure_element When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +278 282 DDNN mutant When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +283 292 secondary protein_state When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +293 296 PIN structure_element When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +324 329 RNase protein_type When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). RESULTS +21 43 fluorescence intensity evidence When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). RESULTS +47 56 uncleaved protein_state When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). RESULTS +57 61 IL-6 protein_type When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). RESULTS +62 66 mRNA chemical When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). RESULTS +82 89 mutants protein_state When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). RESULTS +90 95 W182A mutant When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). RESULTS +97 102 K184A mutant When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). RESULTS +104 109 R214A mutant When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). RESULTS +115 120 R220A mutant When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). RESULTS +155 159 DDNN mutant When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). RESULTS +160 166 mutant protein_state When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). RESULTS +35 57 fluorescence intensity evidence Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). RESULTS +65 74 uncleaved protein_state Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). RESULTS +75 84 Regnase-1 protein Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). RESULTS +85 89 mRNA chemical Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). RESULTS +105 112 mutants protein_state Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). RESULTS +113 118 K184A mutant Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). RESULTS +123 128 R214A mutant Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). RESULTS +163 167 DDNN mutant Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). RESULTS +168 174 mutant protein_state Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). RESULTS +10 15 K184A mutant Rescue of K184A and R214A by the DDNN mutant was also confirmed by a significant increase in the cleaved products. RESULTS +20 25 R214A mutant Rescue of K184A and R214A by the DDNN mutant was also confirmed by a significant increase in the cleaved products. RESULTS +33 37 DDNN mutant Rescue of K184A and R214A by the DDNN mutant was also confirmed by a significant increase in the cleaved products. RESULTS +38 44 mutant protein_state Rescue of K184A and R214A by the DDNN mutant was also confirmed by a significant increase in the cleaved products. RESULTS +60 64 K184 residue_name_number This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. RESULTS +69 73 R214 residue_name_number This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. RESULTS +81 88 primary protein_state This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. RESULTS +89 92 PIN structure_element This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. RESULTS +126 142 catalytic center site This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. RESULTS +163 172 secondary protein_state This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. RESULTS +173 176 PIN structure_element This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. RESULTS +193 209 catalytic center site This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. RESULTS +217 224 primary protein_state This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. RESULTS +225 228 PIN structure_element This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. RESULTS +0 4 R214 residue_name_number R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize. RESULTS +33 38 dimer oligomeric_state R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize. RESULTS +80 85 R214A mutant R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize. RESULTS +93 102 secondary protein_state R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize. RESULTS +103 106 PIN structure_element R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize. RESULTS +36 41 R214A mutant According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. RESULTS +42 45 PIN structure_element According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. RESULTS +69 74 dimer oligomeric_state According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. RESULTS +84 88 DDNN mutant According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. RESULTS +89 92 PIN structure_element According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. RESULTS +105 114 secondary protein_state According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. RESULTS +115 118 PIN structure_element According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. RESULTS +85 97 head-to-tail protein_state Taken together, the rescue experiments above support the proposed model in which the head-to-tail dimer is functional in vitro. RESULTS +98 103 dimer oligomeric_state Taken together, the rescue experiments above support the proposed model in which the head-to-tail dimer is functional in vitro. RESULTS +36 46 structures evidence We determined the individual domain structures of Regnase-1 by NMR and X-ray crystallography. DISCUSS +50 59 Regnase-1 protein We determined the individual domain structures of Regnase-1 by NMR and X-ray crystallography. DISCUSS +63 66 NMR experimental_method We determined the individual domain structures of Regnase-1 by NMR and X-ray crystallography. DISCUSS +71 92 X-ray crystallography experimental_method We determined the individual domain structures of Regnase-1 by NMR and X-ray crystallography. DISCUSS +29 32 CTD structure_element Although the function of the CTD remains elusive, we revealed the functions of the NTD, PIN, and ZF domains. DISCUSS +83 86 NTD structure_element Although the function of the CTD remains elusive, we revealed the functions of the NTD, PIN, and ZF domains. DISCUSS +88 91 PIN structure_element Although the function of the CTD remains elusive, we revealed the functions of the NTD, PIN, and ZF domains. DISCUSS +97 99 ZF structure_element Although the function of the CTD remains elusive, we revealed the functions of the NTD, PIN, and ZF domains. DISCUSS +2 11 Regnase-1 protein A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. DISCUSS +36 39 PIN structure_element A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. DISCUSS +44 46 ZF structure_element A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. DISCUSS +68 80 Mus musculus species A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. DISCUSS +85 97 crystallized experimental_method A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. DISCUSS +112 128 electron density evidence A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. DISCUSS +136 138 ZF structure_element A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. DISCUSS +175 177 ZF structure_element A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. DISCUSS +188 201 highly mobile protein_state A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. DISCUSS +209 219 absence of protein_state A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. DISCUSS +227 231 mRNA chemical A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. DISCUSS +4 7 NMR experimental_method Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. DISCUSS +52 54 ZF structure_element Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. DISCUSS +65 69 IL-6 protein_type Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. DISCUSS +70 74 mRNA chemical Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. DISCUSS +82 84 Kd evidence Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. DISCUSS +117 141 in vitro gel shift assay experimental_method Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. DISCUSS +157 166 Regnase-1 protein Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. DISCUSS +182 184 ZF structure_element Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. DISCUSS +208 212 mRNA chemical Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. DISCUSS +238 241 RNA chemical Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. DISCUSS +28 37 Regnase-1 protein These results indicate that Regnase-1 directly binds to RNA and precipitates under such experimental conditions. DISCUSS +56 59 RNA chemical These results indicate that Regnase-1 directly binds to RNA and precipitates under such experimental conditions. DISCUSS +59 78 structural analyses experimental_method Due to this limitation, it is difficult to perform further structural analyses of mRNA-Regnase-1 complexes by X-ray crystallography or NMR. DISCUSS +82 96 mRNA-Regnase-1 complex_assembly Due to this limitation, it is difficult to perform further structural analyses of mRNA-Regnase-1 complexes by X-ray crystallography or NMR. DISCUSS +110 131 X-ray crystallography experimental_method Due to this limitation, it is difficult to perform further structural analyses of mRNA-Regnase-1 complexes by X-ray crystallography or NMR. DISCUSS +135 138 NMR experimental_method Due to this limitation, it is difficult to perform further structural analyses of mRNA-Regnase-1 complexes by X-ray crystallography or NMR. DISCUSS +24 41 crystal structure evidence The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. DISCUSS +49 58 Regnase-1 protein The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. DISCUSS +59 62 PIN structure_element The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. DISCUSS +83 95 Homo sapiens species The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. DISCUSS +140 152 Mus musculus species The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. DISCUSS +184 188 RMSD evidence The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. DISCUSS +241 244 PIN structure_element The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. DISCUSS +255 262 134–295 residue_range The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. DISCUSS +9 14 mouse taxonomy_domain Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms. DISCUSS +19 24 human species Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms. DISCUSS +25 28 PIN structure_element Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms. DISCUSS +42 54 head-to-tail protein_state Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms. DISCUSS +55 64 oligomers oligomeric_state Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms. DISCUSS +83 96 crystal forms evidence Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms. DISCUSS +42 45 PIN structure_element Rao and co-workers previously argued that PIN dimerization is likely to be a crystallographic artifact with no physiological significance, since monomers were dominant in their analytical ultra-centrifugation experiments. DISCUSS +145 153 monomers oligomeric_state Rao and co-workers previously argued that PIN dimerization is likely to be a crystallographic artifact with no physiological significance, since monomers were dominant in their analytical ultra-centrifugation experiments. DISCUSS +177 208 analytical ultra-centrifugation experimental_method Rao and co-workers previously argued that PIN dimerization is likely to be a crystallographic artifact with no physiological significance, since monomers were dominant in their analytical ultra-centrifugation experiments. DISCUSS +17 31 gel filtration experimental_method In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. DISCUSS +38 57 mutational analyses experimental_method In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. DISCUSS +63 66 NMR experimental_method In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. DISCUSS +67 74 spectra evidence In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. DISCUSS +97 100 PIN structure_element In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. DISCUSS +116 128 head-to-tail protein_state In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. DISCUSS +129 134 dimer oligomeric_state In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. DISCUSS +174 191 crystal structure evidence In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. DISCUSS +137 145 oligomer oligomeric_state This inconsistency might be due to difference in the analytical methods and/or protein concentrations used in each experiment, since the oligomer formation of PIN was dependent on the protein concentration in our study. DISCUSS +159 162 PIN structure_element This inconsistency might be due to difference in the analytical methods and/or protein concentrations used in each experiment, since the oligomer formation of PIN was dependent on the protein concentration in our study. DISCUSS +0 16 Single mutations experimental_method Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well. DISCUSS +80 83 PIN structure_element Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well. DISCUSS +84 95 monomerized oligomeric_state Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well. DISCUSS +118 125 mutants protein_state Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well. DISCUSS +137 142 RNase protein_type Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well. DISCUSS +10 13 NMR experimental_method Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c). DISCUSS +14 21 spectra evidence Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c). DISCUSS +25 34 monomeric oligomeric_state Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c). DISCUSS +35 42 mutants protein_state Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c). DISCUSS +138 147 monomeric oligomeric_state Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c). DISCUSS +148 155 mutants protein_state Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c). DISCUSS +47 50 PIN structure_element Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro. DISCUSS +51 54 PIN structure_element Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro. DISCUSS +55 60 dimer oligomeric_state Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro. DISCUSS +87 96 Regnase-1 protein Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro. DISCUSS +97 102 RNase protein_type Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro. DISCUSS +11 28 crystal structure evidence Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). DISCUSS +36 39 PIN structure_element Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). DISCUSS +40 45 dimer oligomeric_state Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). DISCUSS +51 60 Regnase-1 protein Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). DISCUSS +97 104 primary protein_state Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). DISCUSS +111 120 secondary protein_state Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). DISCUSS +122 126 PINs structure_element Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). DISCUSS +150 164 catalytic site site Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). DISCUSS +180 183 PIN structure_element Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). DISCUSS +14 49 structure-based mutational analyses experimental_method Moreover, our structure-based mutational analyses showed these two Regnase-1 specific basic regions were essential for target mRNA cleavage in vitro. DISCUSS +67 76 Regnase-1 protein Moreover, our structure-based mutational analyses showed these two Regnase-1 specific basic regions were essential for target mRNA cleavage in vitro. DISCUSS +126 130 mRNA chemical Moreover, our structure-based mutational analyses showed these two Regnase-1 specific basic regions were essential for target mRNA cleavage in vitro. DISCUSS +4 18 cleavage assay experimental_method The cleavage assay also showed that the NTD is crucial for efficient mRNA cleavage. DISCUSS +40 43 NTD structure_element The cleavage assay also showed that the NTD is crucial for efficient mRNA cleavage. DISCUSS +69 73 mRNA chemical The cleavage assay also showed that the NTD is crucial for efficient mRNA cleavage. DISCUSS +28 31 NTD structure_element Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a). DISCUSS +52 70 oligomeric surface site Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a). DISCUSS +78 85 primary protein_state Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a). DISCUSS +86 89 PIN structure_element Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a). DISCUSS +104 109 helix structure_element Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a). DISCUSS +127 145 catalytic residues site Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a). DISCUSS +39 42 NTD structure_element Taken together, this suggests that the NTD and the PIN domain compete for a common binding site. DISCUSS +51 54 PIN structure_element Taken together, this suggests that the NTD and the PIN domain compete for a common binding site. DISCUSS +76 95 common binding site site Taken together, this suggests that the NTD and the PIN domain compete for a common binding site. DISCUSS +4 12 affinity evidence The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. DISCUSS +58 61 PIN structure_element The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. DISCUSS +71 73 Kd evidence The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. DISCUSS +111 114 NTD structure_element The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. DISCUSS +115 118 PIN structure_element The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. DISCUSS +120 122 Kd evidence The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. DISCUSS +212 218 90–133 residue_range The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. DISCUSS +231 234 NTD structure_element The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. DISCUSS +243 250 primary protein_state The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. DISCUSS +251 254 PIN structure_element The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. DISCUSS +329 340 full-length protein_state The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. DISCUSS +341 350 Regnase-1 protein The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. DISCUSS +74 116 docking and molecular dynamics simulations experimental_method While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+. DISCUSS +131 134 NTD structure_element While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+. DISCUSS +158 176 catalytic residues site While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+. DISCUSS +184 187 PIN structure_element While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+. DISCUSS +205 211 active protein_state While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+. DISCUSS +246 250 Mg2+ chemical While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+. DISCUSS +73 78 Malt1 protein In this context, it is interesting that, in response to TCR stimulation, Malt1 cleaves Regnase-1 at R111 to control immune responses in vivo. DISCUSS +87 96 Regnase-1 protein In this context, it is interesting that, in response to TCR stimulation, Malt1 cleaves Regnase-1 at R111 to control immune responses in vivo. DISCUSS +100 104 R111 residue_name_number In this context, it is interesting that, in response to TCR stimulation, Malt1 cleaves Regnase-1 at R111 to control immune responses in vivo. DISCUSS +52 55 NTD structure_element This result is consistent with a model in which the NTD acts as an enhancer, and cleavage of the linker lowers enzymatic activity dramatically. DISCUSS +97 103 linker structure_element This result is consistent with a model in which the NTD acts as an enhancer, and cleavage of the linker lowers enzymatic activity dramatically. DISCUSS +15 49 structural and functional analyses experimental_method Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. DISCUSS +53 62 Regnase-1 protein Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. DISCUSS +104 123 docking simulations experimental_method Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. DISCUSS +131 134 NTD structure_element Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. DISCUSS +136 139 PIN structure_element Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. DISCUSS +140 145 dimer oligomeric_state Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. DISCUSS +151 155 IL-6 protein_type Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. DISCUSS +156 160 mRNA chemical Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. DISCUSS +37 50 cleavage site site We incorporated information from the cleavage site of IL-6 mRNA in vitro is indicated by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 7a,b). DISCUSS +54 58 IL-6 protein_type We incorporated information from the cleavage site of IL-6 mRNA in vitro is indicated by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 7a,b). DISCUSS +59 63 mRNA chemical We incorporated information from the cleavage site of IL-6 mRNA in vitro is indicated by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 7a,b). DISCUSS +100 134 polyacrylamide gel electrophoresis experimental_method We incorporated information from the cleavage site of IL-6 mRNA in vitro is indicated by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 7a,b). DISCUSS +4 11 docking experimental_method The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1. DISCUSS +37 40 RNA chemical The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1. DISCUSS +207 227 RNA-binding proteins protein_type The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1. DISCUSS +247 251 loop structure_element The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1. DISCUSS +277 286 Regnase-1 protein The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1. DISCUSS +35 44 Regnase-1 protein The overall model of regulation of Regnase-1 RNase activity through domain-domain interactions in vitro is summarized in Fig. 6. DISCUSS +45 50 RNase protein_type The overall model of regulation of Regnase-1 RNase activity through domain-domain interactions in vitro is summarized in Fig. 6. DISCUSS +7 17 absence of protein_state In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration. DISCUSS +25 29 mRNA chemical In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration. DISCUSS +35 38 PIN structure_element In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration. DISCUSS +52 64 head-to-tail protein_state In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration. DISCUSS +65 74 oligomers oligomeric_state In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration. DISCUSS +2 14 fully active protein_state A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +15 31 catalytic center site A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +60 63 NTD structure_element A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +84 92 oligomer oligomeric_state A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +108 111 PIN structure_element A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +141 153 head-to-tail protein_state A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +154 162 oligomer oligomeric_state A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +191 198 primary protein_state A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +199 202 PIN structure_element A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +217 227 functional protein_state A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +228 233 dimer oligomeric_state A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +264 267 PIN structure_element A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +269 278 secondary protein_state A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +279 282 PIN structure_element A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). DISCUSS +66 75 Regnase-1 protein While further investigations on the domain-domain interactions of Regnase-1 in vivo are necessary, these intramolecular and intermolecular domain interactions of Regnase-1 appear to structurally constrain Regnase-1activity, which, in turn, enables tight regulation of immune responses. DISCUSS +162 171 Regnase-1 protein While further investigations on the domain-domain interactions of Regnase-1 in vivo are necessary, these intramolecular and intermolecular domain interactions of Regnase-1 appear to structurally constrain Regnase-1activity, which, in turn, enables tight regulation of immune responses. DISCUSS +205 214 Regnase-1 protein While further investigations on the domain-domain interactions of Regnase-1 in vivo are necessary, these intramolecular and intermolecular domain interactions of Regnase-1 appear to structurally constrain Regnase-1activity, which, in turn, enables tight regulation of immune responses. DISCUSS +0 34 Structural and functional analyses experimental_method Structural and functional analyses of Regnase-1. FIG +38 47 Regnase-1 protein Structural and functional analyses of Regnase-1. FIG +27 36 Regnase-1 protein (a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain. FIG +42 60 Solution structure evidence (a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain. FIG +68 71 NTD structure_element (a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain. FIG +77 94 Crystal structure evidence (a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain. FIG +102 105 PIN structure_element (a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain. FIG +0 9 Catalytic protein_state Catalytic Asp residues were shown in sticks. FIG +10 13 Asp residue_name Catalytic Asp residues were shown in sticks. FIG +4 22 Solution structure evidence (d) Solution structure of the ZF domain. FIG +30 32 ZF structure_element (d) Solution structure of the ZF domain. FIG +6 9 Cys residue_name Three Cys residues and one His residue responsible for Zn2+-binding were shown in sticks. FIG +27 30 His residue_name Three Cys residues and one His residue responsible for Zn2+-binding were shown in sticks. FIG +4 22 Solution structure evidence (e) Solution structure of the CTD. FIG +30 33 CTD structure_element (e) Solution structure of the CTD. FIG +8 18 structures evidence All the structures were colored in rainbow from N-terminus (blue) to C-terminus (red). FIG +4 36 In vitro gel shift binding assay experimental_method (f) In vitro gel shift binding assay between Regnase-1 and IL-6 mRNA. FIG +45 54 Regnase-1 protein (f) In vitro gel shift binding assay between Regnase-1 and IL-6 mRNA. FIG +59 63 IL-6 protein_type (f) In vitro gel shift binding assay between Regnase-1 and IL-6 mRNA. FIG +64 68 mRNA chemical (f) In vitro gel shift binding assay between Regnase-1 and IL-6 mRNA. FIG +0 22 Fluorescence intensity evidence Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1. FIG +30 34 free protein_state Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1. FIG +35 39 IL-6 protein_type Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1. FIG +107 117 absence of protein_state Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1. FIG +118 127 Regnase-1 protein Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1. FIG +15 24 Regnase-1 protein (g) Binding of Regnase-1 and IL-6 mRNA was plotted. FIG +29 33 IL-6 protein_type (g) Binding of Regnase-1 and IL-6 mRNA was plotted. FIG +34 38 mRNA chemical (g) Binding of Regnase-1 and IL-6 mRNA was plotted. FIG +28 32 IL-6 protein_type The percentage of the bound IL-6 was calculated based on the fluorescence intensities of the free IL-6 quantified in (f). FIG +61 85 fluorescence intensities evidence The percentage of the bound IL-6 was calculated based on the fluorescence intensities of the free IL-6 quantified in (f). FIG +98 102 IL-6 protein_type The percentage of the bound IL-6 was calculated based on the fluorescence intensities of the free IL-6 quantified in (f). FIG +4 27 In vitro cleavage assay experimental_method (h) In vitro cleavage assay of Regnase-1 to IL-6 mRNA. FIG +31 40 Regnase-1 protein (h) In vitro cleavage assay of Regnase-1 to IL-6 mRNA. FIG +44 48 IL-6 protein_type (h) In vitro cleavage assay of Regnase-1 to IL-6 mRNA. FIG +49 53 mRNA chemical (h) In vitro cleavage assay of Regnase-1 to IL-6 mRNA. FIG +0 22 Fluorescence intensity evidence Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1. FIG +30 39 uncleaved protein_state Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1. FIG +40 44 IL-6 protein_type Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1. FIG +45 49 mRNA chemical Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1. FIG +102 112 absence of protein_state Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1. FIG +113 122 Regnase-1 protein Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1. FIG +0 12 Head-to-tail protein_state Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1. FIG +13 21 oligomer oligomeric_state Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1. FIG +39 42 PIN structure_element Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1. FIG +69 74 RNase protein_type Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1. FIG +87 96 Regnase-1 protein Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1. FIG +4 27 Gel filtration analyses experimental_method (a) Gel filtration analyses of the PIN domain. FIG +35 38 PIN structure_element (a) Gel filtration analyses of the PIN domain. FIG +4 9 Dimer oligomeric_state (b) Dimer structure of the PIN domain. FIG +10 19 structure evidence (b) Dimer structure of the PIN domain. FIG +27 30 PIN structure_element (b) Dimer structure of the PIN domain. FIG +4 7 PIN structure_element Two PIN molecules in the crystal were colored white and green, respectively. FIG +25 32 crystal evidence Two PIN molecules in the crystal were colored white and green, respectively. FIG +0 18 Catalytic residues site Catalytic residues and mutated residues were shown in sticks. FIG +74 78 R215 residue_name_number Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed. FIG +153 158 RNase protein_type Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed. FIG +171 180 monomeric oligomeric_state Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed. FIG +181 188 mutants protein_state Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed. FIG +193 197 IL-6 protein_type Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed. FIG +198 202 mRNA chemical Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed. FIG +38 41 NTD structure_element Domain-domain interaction between the NTD and the PIN domain. FIG +50 53 PIN structure_element Domain-domain interaction between the NTD and the PIN domain. FIG +4 16 NMR analyses experimental_method (a) NMR analyses of the NTD-binding to the PIN domain. FIG +24 27 NTD structure_element (a) NMR analyses of the NTD-binding to the PIN domain. FIG +43 46 PIN structure_element (a) NMR analyses of the NTD-binding to the PIN domain. FIG +73 81 overlaid experimental_method The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red on the surface and ribbon structure of the PIN domain (right). FIG +82 89 spectra evidence The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red on the surface and ribbon structure of the PIN domain (right). FIG +156 159 PIN structure_element The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red on the surface and ribbon structure of the PIN domain (right). FIG +0 3 Pro residue_name Pro and the residues without analysis were colored black and gray, respectively. FIG +4 16 NMR analyses experimental_method (b) NMR analyses of the PIN-binding to the NTD. FIG +24 27 PIN structure_element (b) NMR analyses of the PIN-binding to the NTD. FIG +43 46 NTD structure_element (b) NMR analyses of the PIN-binding to the NTD. FIG +18 52 significant chemical shift changes evidence The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red, yellow, or green on the surface and ribbon structure of the NTD. FIG +73 81 overlaid experimental_method The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red, yellow, or green on the surface and ribbon structure of the NTD. FIG +82 89 spectra evidence The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red, yellow, or green on the surface and ribbon structure of the NTD. FIG +174 177 NTD structure_element The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red, yellow, or green on the surface and ribbon structure of the NTD. FIG +0 3 S62 residue_name_number S62 was colored gray and excluded from the analysis, due to low signal intensity. FIG +25 28 NTD structure_element (c) Docking model of the NTD and the PIN domain. FIG +37 40 PIN structure_element (c) Docking model of the NTD and the PIN domain. FIG +4 7 NTD structure_element The NTD and the PIN domain are shown in cyan and white, respectively. FIG +16 19 PIN structure_element The NTD and the PIN domain are shown in cyan and white, respectively. FIG +56 73 docking structure evidence Residues in close proximity (<5 Å) to each other in the docking structure were colored yellow. FIG +0 18 Catalytic residues site Catalytic residues of the PIN domain are shown in sticks, and the residues that exhibited significant chemical shift changes in (a,b) were labeled. FIG +26 29 PIN structure_element Catalytic residues of the PIN domain are shown in sticks, and the residues that exhibited significant chemical shift changes in (a,b) were labeled. FIG +90 124 significant chemical shift changes evidence Catalytic residues of the PIN domain are shown in sticks, and the residues that exhibited significant chemical shift changes in (a,b) were labeled. FIG +25 28 PIN structure_element Critical residues in the PIN domain for the RNase activity of Regnase-1. FIG +44 49 RNase protein_type Critical residues in the PIN domain for the RNase activity of Regnase-1. FIG +62 71 Regnase-1 protein Critical residues in the PIN domain for the RNase activity of Regnase-1. FIG +4 27 In vitro cleavage assay experimental_method (a) In vitro cleavage assay of basic residue mutants for IL-6 mRNA. FIG +45 52 mutants protein_state (a) In vitro cleavage assay of basic residue mutants for IL-6 mRNA. FIG +57 61 IL-6 protein_type (a) In vitro cleavage assay of basic residue mutants for IL-6 mRNA. FIG +62 66 mRNA chemical (a) In vitro cleavage assay of basic residue mutants for IL-6 mRNA. FIG +4 27 In vitro cleavage assay experimental_method (b) In vitro cleavage assay of basic residue mutants for Regnase-1 mRNA. FIG +45 52 mutants protein_state (b) In vitro cleavage assay of basic residue mutants for Regnase-1 mRNA. FIG +57 66 Regnase-1 protein (b) In vitro cleavage assay of basic residue mutants for Regnase-1 mRNA. FIG +67 71 mRNA chemical (b) In vitro cleavage assay of basic residue mutants for Regnase-1 mRNA. FIG +4 26 fluorescence intensity evidence The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure. FIG +34 43 uncleaved protein_state The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure. FIG +44 48 mRNA chemical The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure. FIG +99 102 PIN structure_element The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure. FIG +103 108 dimer oligomeric_state The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure. FIG +109 118 structure evidence The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure. FIG +81 86 RNase protein_type Mutated basic residues were shown in sticks and those with significantly reduced RNase activities were colored red or yellow. FIG +44 53 Regnase-1 protein Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity. FIG +68 75 mutants protein_state Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity. FIG +84 88 DDNN mutant Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity. FIG +89 95 mutant protein_state Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity. FIG +109 114 RNase protein_type Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity. FIG +65 88 In vitro cleavage assay experimental_method (a) Cartoon representation of the concept of the experiment. (b) In vitro cleavage assay of Regnase-1 for IL-6 mRNA. FIG +92 101 Regnase-1 protein (a) Cartoon representation of the concept of the experiment. (b) In vitro cleavage assay of Regnase-1 for IL-6 mRNA. FIG +106 110 IL-6 protein_type (a) Cartoon representation of the concept of the experiment. (b) In vitro cleavage assay of Regnase-1 for IL-6 mRNA. FIG +111 115 mRNA chemical (a) Cartoon representation of the concept of the experiment. (b) In vitro cleavage assay of Regnase-1 for IL-6 mRNA. FIG +4 27 In vitro cleavage assay experimental_method (c) In vitro cleavage assay of Regnase-1 for Regnase-1 mRNA. FIG +31 40 Regnase-1 protein (c) In vitro cleavage assay of Regnase-1 for Regnase-1 mRNA. FIG +45 54 Regnase-1 protein (c) In vitro cleavage assay of Regnase-1 for Regnase-1 mRNA. FIG +55 59 mRNA chemical (c) In vitro cleavage assay of Regnase-1 for Regnase-1 mRNA. FIG +4 26 fluorescence intensity evidence The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer. FIG +34 43 uncleaved protein_state The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer. FIG +44 48 mRNA chemical The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer. FIG +99 102 PIN structure_element The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer. FIG +103 108 dimer oligomeric_state The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer. FIG +20 25 RNase protein_type The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN. FIG +63 74 presence of protein_state The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN. FIG +75 79 DDNN mutant The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN. FIG +80 86 mutant protein_state The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN. FIG +123 126 PIN structure_element The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN. FIG +20 25 RNase protein_type The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN. FIG +58 69 presence of protein_state The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN. FIG +70 74 DDNN mutant The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN. FIG +75 81 mutant protein_state The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN. FIG +127 130 PIN structure_element The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN. FIG +46 55 Regnase-1 protein Schematic representation of regulation of the Regnase-1 catalytic activity through the domain-domain interactions. FIG diff --git a/annotation_CSV/PMC4773095.csv b/annotation_CSV/PMC4773095.csv new file mode 100644 index 0000000000000000000000000000000000000000..963c3e1374a22ea2472405642a914ebfca5d59d4 --- /dev/null +++ b/annotation_CSV/PMC4773095.csv @@ -0,0 +1,1324 @@ +anno_start anno_end anno_text entity_type sentence section +0 9 Structure evidence Structure of the Response Regulator NsrR from Streptococcus agalactiae, Which Is Involved in Lantibiotic Resistance TITLE +17 35 Response Regulator protein_type Structure of the Response Regulator NsrR from Streptococcus agalactiae, Which Is Involved in Lantibiotic Resistance TITLE +36 40 NsrR protein Structure of the Response Regulator NsrR from Streptococcus agalactiae, Which Is Involved in Lantibiotic Resistance TITLE +46 70 Streptococcus agalactiae species Structure of the Response Regulator NsrR from Streptococcus agalactiae, Which Is Involved in Lantibiotic Resistance TITLE +93 104 Lantibiotic chemical Structure of the Response Regulator NsrR from Streptococcus agalactiae, Which Is Involved in Lantibiotic Resistance TITLE +0 12 Lantibiotics chemical Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria. ABSTRACT +17 39 antimicrobial peptides chemical Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria. ABSTRACT +52 74 Gram-positive bacteria taxonomy_domain Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria. ABSTRACT +47 52 human species Interestingly, several clinically relevant and human pathogenic strains are inherently resistant towards lantibiotics. ABSTRACT +105 117 lantibiotics chemical Interestingly, several clinically relevant and human pathogenic strains are inherently resistant towards lantibiotics. ABSTRACT +44 55 lantibiotic chemical The expression of the genes responsible for lantibiotic resistance is regulated by a specific two-component system consisting of a histidine kinase and a response regulator. ABSTRACT +94 114 two-component system complex_assembly The expression of the genes responsible for lantibiotic resistance is regulated by a specific two-component system consisting of a histidine kinase and a response regulator. ABSTRACT +131 147 histidine kinase protein_type The expression of the genes responsible for lantibiotic resistance is regulated by a specific two-component system consisting of a histidine kinase and a response regulator. ABSTRACT +154 172 response regulator protein_type The expression of the genes responsible for lantibiotic resistance is regulated by a specific two-component system consisting of a histidine kinase and a response regulator. ABSTRACT +22 40 response regulator protein_type Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. ABSTRACT +53 64 lantibiotic chemical Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. ABSTRACT +77 81 NsrR protein Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. ABSTRACT +87 111 Streptococcus agalactiae species Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. ABSTRACT +132 150 crystal structures evidence Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. ABSTRACT +169 184 receiver domain structure_element Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. ABSTRACT +200 227 DNA-binding effector domain structure_element Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. ABSTRACT +4 21 C-terminal domain structure_element The C-terminal domain exhibits a fold that classifies NsrR as a member of the OmpR/PhoB subfamily of regulators. ABSTRACT +54 58 NsrR protein The C-terminal domain exhibits a fold that classifies NsrR as a member of the OmpR/PhoB subfamily of regulators. ABSTRACT +78 97 OmpR/PhoB subfamily protein_type The C-terminal domain exhibits a fold that classifies NsrR as a member of the OmpR/PhoB subfamily of regulators. ABSTRACT +24 39 phosphorylation ptm Amino acids involved in phosphorylation, dimerization, and DNA-binding were identified and demonstrated to be conserved in lantibiotic resistance regulators. ABSTRACT +59 62 DNA chemical Amino acids involved in phosphorylation, dimerization, and DNA-binding were identified and demonstrated to be conserved in lantibiotic resistance regulators. ABSTRACT +110 119 conserved protein_state Amino acids involved in phosphorylation, dimerization, and DNA-binding were identified and demonstrated to be conserved in lantibiotic resistance regulators. ABSTRACT +123 156 lantibiotic resistance regulators protein_type Amino acids involved in phosphorylation, dimerization, and DNA-binding were identified and demonstrated to be conserved in lantibiotic resistance regulators. ABSTRACT +24 35 full-length protein_state Finally, a model of the full-length NsrR in the active and inactive state provides insights into protein dimerization and DNA-binding. ABSTRACT +36 40 NsrR protein Finally, a model of the full-length NsrR in the active and inactive state provides insights into protein dimerization and DNA-binding. ABSTRACT +48 54 active protein_state Finally, a model of the full-length NsrR in the active and inactive state provides insights into protein dimerization and DNA-binding. ABSTRACT +59 67 inactive protein_state Finally, a model of the full-length NsrR in the active and inactive state provides insights into protein dimerization and DNA-binding. ABSTRACT +122 125 DNA chemical Finally, a model of the full-length NsrR in the active and inactive state provides insights into protein dimerization and DNA-binding. ABSTRACT +93 98 human species This has led to the search for novel antibiotics that can be used as pharmaceuticals against human pathogenic bacteria. INTRO +110 118 bacteria taxonomy_domain This has led to the search for novel antibiotics that can be used as pharmaceuticals against human pathogenic bacteria. INTRO +49 61 lantibiotics chemical One of the potential antibiotic alternatives are lantibiotics. INTRO +0 12 Lantibiotics chemical Lantibiotics are small antimicrobial peptides (30–50 amino acids in length), which are produced by several Gram-positive bacterial strains. INTRO +23 45 antimicrobial peptides chemical Lantibiotics are small antimicrobial peptides (30–50 amino acids in length), which are produced by several Gram-positive bacterial strains. INTRO +107 130 Gram-positive bacterial taxonomy_domain Lantibiotics are small antimicrobial peptides (30–50 amino acids in length), which are produced by several Gram-positive bacterial strains. INTRO +60 71 lanthionine chemical They are post-translationally modified and contain specific lanthionine/methyl-lanthionine rings, which are crucial for their high antimicrobial activity. INTRO +72 90 methyl-lanthionine chemical They are post-translationally modified and contain specific lanthionine/methyl-lanthionine rings, which are crucial for their high antimicrobial activity. INTRO +0 12 Lantibiotics chemical Lantibiotics are for example highly effective against various Gram-positive, human pathogenic bacteria including Streptococcus pneumoniae and several methicillin-resistant Staphylococcus aureus (MRSA) strains. INTRO +62 75 Gram-positive taxonomy_domain Lantibiotics are for example highly effective against various Gram-positive, human pathogenic bacteria including Streptococcus pneumoniae and several methicillin-resistant Staphylococcus aureus (MRSA) strains. INTRO +77 82 human species Lantibiotics are for example highly effective against various Gram-positive, human pathogenic bacteria including Streptococcus pneumoniae and several methicillin-resistant Staphylococcus aureus (MRSA) strains. INTRO +94 102 bacteria taxonomy_domain Lantibiotics are for example highly effective against various Gram-positive, human pathogenic bacteria including Streptococcus pneumoniae and several methicillin-resistant Staphylococcus aureus (MRSA) strains. INTRO +113 137 Streptococcus pneumoniae species Lantibiotics are for example highly effective against various Gram-positive, human pathogenic bacteria including Streptococcus pneumoniae and several methicillin-resistant Staphylococcus aureus (MRSA) strains. INTRO +150 193 methicillin-resistant Staphylococcus aureus species Lantibiotics are for example highly effective against various Gram-positive, human pathogenic bacteria including Streptococcus pneumoniae and several methicillin-resistant Staphylococcus aureus (MRSA) strains. INTRO +195 199 MRSA species Lantibiotics are for example highly effective against various Gram-positive, human pathogenic bacteria including Streptococcus pneumoniae and several methicillin-resistant Staphylococcus aureus (MRSA) strains. INTRO +20 32 lantibiotics chemical The high potency of lantibiotics for medical usage has already been noticed, and several lantibiotics are already included in clinical trials. INTRO +89 101 lantibiotics chemical The high potency of lantibiotics for medical usage has already been noticed, and several lantibiotics are already included in clinical trials. INTRO +0 5 Nisin chemical Nisin is the most prominent member of the lantibiotic family and is able to inhibit cell growth, penetrates the membranes of various Gram-positive bacteria, and is characterized by five specific (methyl-)lanthionine rings, which are crucial for stability and activity in the nanomolar range. INTRO +42 53 lantibiotic chemical Nisin is the most prominent member of the lantibiotic family and is able to inhibit cell growth, penetrates the membranes of various Gram-positive bacteria, and is characterized by five specific (methyl-)lanthionine rings, which are crucial for stability and activity in the nanomolar range. INTRO +133 155 Gram-positive bacteria taxonomy_domain Nisin is the most prominent member of the lantibiotic family and is able to inhibit cell growth, penetrates the membranes of various Gram-positive bacteria, and is characterized by five specific (methyl-)lanthionine rings, which are crucial for stability and activity in the nanomolar range. INTRO +10 21 lantibiotic chemical Thus, the lantibiotic producer strains have an inbuilt self-protection mechanism (immunity) to prevent cell death caused due to the action of its cognate lantibiotic. INTRO +154 165 lantibiotic chemical Thus, the lantibiotic producer strains have an inbuilt self-protection mechanism (immunity) to prevent cell death caused due to the action of its cognate lantibiotic. INTRO +35 66 membrane–associated lipoprotein protein_type This immunity system consists of a membrane–associated lipoprotein (usually referred to as LanI) and/or an ABC transporter (termed as LanFEG and comprising three subunits). INTRO +91 95 LanI protein_type This immunity system consists of a membrane–associated lipoprotein (usually referred to as LanI) and/or an ABC transporter (termed as LanFEG and comprising three subunits). INTRO +107 122 ABC transporter protein_type This immunity system consists of a membrane–associated lipoprotein (usually referred to as LanI) and/or an ABC transporter (termed as LanFEG and comprising three subunits). INTRO +134 140 LanFEG protein_type This immunity system consists of a membrane–associated lipoprotein (usually referred to as LanI) and/or an ABC transporter (termed as LanFEG and comprising three subunits). INTRO +14 26 lantibiotics chemical Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +35 39 Pep5 chemical Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +41 49 epicidin chemical Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +51 60 epilancin chemical Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +66 76 lactocin S chemical Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +90 94 LanI protein_type Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +115 127 lantibiotics chemical Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +209 214 nisin chemical Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +216 224 subtilin chemical Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +226 235 epidermin chemical Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +237 248 gallidermin chemical Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +254 267 lacticin 3147 chemical Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +305 311 LanFEG protein_type Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG. INTRO +13 19 LanFEG protein_type Examples for LanFEG are NisI and NisFEG of the nisin system, SpaI and SpaFEG conferring immunity towards subtilin, and PepI constituting the immunity system of Pep5 producing strains. INTRO +24 28 NisI protein Examples for LanFEG are NisI and NisFEG of the nisin system, SpaI and SpaFEG conferring immunity towards subtilin, and PepI constituting the immunity system of Pep5 producing strains. INTRO +33 39 NisFEG protein Examples for LanFEG are NisI and NisFEG of the nisin system, SpaI and SpaFEG conferring immunity towards subtilin, and PepI constituting the immunity system of Pep5 producing strains. INTRO +47 52 nisin chemical Examples for LanFEG are NisI and NisFEG of the nisin system, SpaI and SpaFEG conferring immunity towards subtilin, and PepI constituting the immunity system of Pep5 producing strains. INTRO +61 65 SpaI protein Examples for LanFEG are NisI and NisFEG of the nisin system, SpaI and SpaFEG conferring immunity towards subtilin, and PepI constituting the immunity system of Pep5 producing strains. INTRO +70 76 SpaFEG protein Examples for LanFEG are NisI and NisFEG of the nisin system, SpaI and SpaFEG conferring immunity towards subtilin, and PepI constituting the immunity system of Pep5 producing strains. INTRO +105 113 subtilin chemical Examples for LanFEG are NisI and NisFEG of the nisin system, SpaI and SpaFEG conferring immunity towards subtilin, and PepI constituting the immunity system of Pep5 producing strains. INTRO +119 123 PepI protein Examples for LanFEG are NisI and NisFEG of the nisin system, SpaI and SpaFEG conferring immunity towards subtilin, and PepI constituting the immunity system of Pep5 producing strains. INTRO +160 164 Pep5 chemical Examples for LanFEG are NisI and NisFEG of the nisin system, SpaI and SpaFEG conferring immunity towards subtilin, and PepI constituting the immunity system of Pep5 producing strains. INTRO +0 15 Structural data evidence Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024. INTRO +37 54 immunity proteins protein_type Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024. INTRO +55 59 NisI protein Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024. INTRO +65 83 Lactococcus lactis species Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024. INTRO +85 89 SpaI protein Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024. INTRO +95 112 Bacillus subtilis species Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024. INTRO +117 121 MlbQ protein Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024. INTRO +131 142 lantibiotic chemical Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024. INTRO +143 150 NAI-107 chemical Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024. INTRO +167 193 Microbispora ATCC PTA-5024 species Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024. INTRO +71 76 human species Recently, gene clusters were identified in certain clinically relevant human pathogenic strains such as Streptococcus agalactiae, S. aureus, and others that confer inherent resistance against specific lantibiotics such as nisin and resemble the genetic architecture of the lantibiotic immunity genes found in the producing strains. INTRO +104 128 Streptococcus agalactiae species Recently, gene clusters were identified in certain clinically relevant human pathogenic strains such as Streptococcus agalactiae, S. aureus, and others that confer inherent resistance against specific lantibiotics such as nisin and resemble the genetic architecture of the lantibiotic immunity genes found in the producing strains. INTRO +130 139 S. aureus species Recently, gene clusters were identified in certain clinically relevant human pathogenic strains such as Streptococcus agalactiae, S. aureus, and others that confer inherent resistance against specific lantibiotics such as nisin and resemble the genetic architecture of the lantibiotic immunity genes found in the producing strains. INTRO +201 213 lantibiotics chemical Recently, gene clusters were identified in certain clinically relevant human pathogenic strains such as Streptococcus agalactiae, S. aureus, and others that confer inherent resistance against specific lantibiotics such as nisin and resemble the genetic architecture of the lantibiotic immunity genes found in the producing strains. INTRO +222 227 nisin chemical Recently, gene clusters were identified in certain clinically relevant human pathogenic strains such as Streptococcus agalactiae, S. aureus, and others that confer inherent resistance against specific lantibiotics such as nisin and resemble the genetic architecture of the lantibiotic immunity genes found in the producing strains. INTRO +54 82 membrane-associated protease protein_type Within these resistance operons, genes encoding for a membrane-associated protease and an ABC transporter were identified. INTRO +90 105 ABC transporter protein_type Within these resistance operons, genes encoding for a membrane-associated protease and an ABC transporter were identified. INTRO +57 69 lantibiotics chemical Expression of these proteins provides resistance against lantibiotics. INTRO +14 23 structure evidence Recently, the structure of SaNSR from S. agalactiae was solved which provides resistance against nisin by a protease activity. INTRO +27 32 SaNSR protein Recently, the structure of SaNSR from S. agalactiae was solved which provides resistance against nisin by a protease activity. INTRO +38 51 S. agalactiae species Recently, the structure of SaNSR from S. agalactiae was solved which provides resistance against nisin by a protease activity. INTRO +97 102 nisin chemical Recently, the structure of SaNSR from S. agalactiae was solved which provides resistance against nisin by a protease activity. INTRO +71 91 two-component system complex_assembly Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +93 96 TCS complex_assembly Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +126 137 lantibiotic chemical Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +180 196 histidine kinase protein_type Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +198 200 HK protein_type Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +208 226 response regulator protein_type Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +228 230 RR protein_type Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +295 297 HK protein_type Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +318 329 lantibiotic chemical Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +363 382 auto-phosphorylates ptm Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +388 397 conserved protein_state Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +398 407 histidine residue_name Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +511 513 RR protein_type Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +574 576 RR protein_type Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response. INTRO +0 8 Bacteria taxonomy_domain Bacteria have the ability to sense and survive various environmental stimuli through adaptive responses, which are regulated by TCSs. INTRO +128 132 TCSs complex_assembly Bacteria have the ability to sense and survive various environmental stimuli through adaptive responses, which are regulated by TCSs. INTRO +4 14 absence of protein_state The absence of TCSs within mammals makes them unique targets for novel antimicrobial drugs. INTRO +15 19 TCSs complex_assembly The absence of TCSs within mammals makes them unique targets for novel antimicrobial drugs. INTRO +27 34 mammals taxonomy_domain The absence of TCSs within mammals makes them unique targets for novel antimicrobial drugs. INTRO +22 33 lantibiotic chemical The expression of the lantibiotic-resistance genes via TCS is generally regulated by microorganism-specific lantibiotics, which act via external stimuli. INTRO +55 58 TCS complex_assembly The expression of the lantibiotic-resistance genes via TCS is generally regulated by microorganism-specific lantibiotics, which act via external stimuli. INTRO +108 120 lantibiotics chemical The expression of the lantibiotic-resistance genes via TCS is generally regulated by microorganism-specific lantibiotics, which act via external stimuli. INTRO +17 20 TCS complex_assembly Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +26 31 BraRS protein Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +35 44 S. aureus species Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +65 75 bacitracin chemical Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +77 82 nisin chemical Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +87 100 nukacin-ISK-1 chemical Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +113 118 BceRS protein Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +122 134 Bacillus spp taxonomy_domain Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +155 166 actagardine chemical Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +171 181 mersacidin chemical Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +194 199 LcrRS protein Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +203 223 Streptococcus mutans species Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +235 248 nukacin-ISK-1 chemical Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +253 265 lacticin 481 chemical Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +270 275 LisRK protein Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +279 301 Listeria monocytogenes species Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +313 318 nisin chemical Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance. INTRO +22 34 lantibiotics chemical Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples. INTRO +50 53 TCS complex_assembly Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples. INTRO +54 59 CprRK protein Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples. INTRO +65 86 Clostridium difficile species Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples. INTRO +144 147 cpr gene Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples. INTRO +196 208 lantibiotics chemical Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples. INTRO +218 223 nisin chemical Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples. INTRO +225 236 gallidermin chemical Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples. INTRO +238 246 subtilin chemical Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples. INTRO +252 264 mutacin 1140 chemical Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples. INTRO +19 35 histidine kinase protein_type Interestingly, the histidine kinase contains two-transmembrane helices but lacks an extracellular sensory domain, and are therefore known as ‘intramembrane-sensing’ histidine kinases. INTRO +45 70 two-transmembrane helices structure_element Interestingly, the histidine kinase contains two-transmembrane helices but lacks an extracellular sensory domain, and are therefore known as ‘intramembrane-sensing’ histidine kinases. INTRO +84 112 extracellular sensory domain structure_element Interestingly, the histidine kinase contains two-transmembrane helices but lacks an extracellular sensory domain, and are therefore known as ‘intramembrane-sensing’ histidine kinases. INTRO +141 182 ‘intramembrane-sensing’ histidine kinases protein_type Interestingly, the histidine kinase contains two-transmembrane helices but lacks an extracellular sensory domain, and are therefore known as ‘intramembrane-sensing’ histidine kinases. INTRO +80 92 lantibiotics chemical It has been suggested that in addition to conferring general resistance against lantibiotics, the BceAB-type transporters assist in signalling as via the presence of a large extracellular domain within the transmembrane segment indicated by experimental evidence from various systems. INTRO +98 121 BceAB-type transporters protein_type It has been suggested that in addition to conferring general resistance against lantibiotics, the BceAB-type transporters assist in signalling as via the presence of a large extracellular domain within the transmembrane segment indicated by experimental evidence from various systems. INTRO +174 194 extracellular domain structure_element It has been suggested that in addition to conferring general resistance against lantibiotics, the BceAB-type transporters assist in signalling as via the presence of a large extracellular domain within the transmembrane segment indicated by experimental evidence from various systems. INTRO +206 227 transmembrane segment structure_element It has been suggested that in addition to conferring general resistance against lantibiotics, the BceAB-type transporters assist in signalling as via the presence of a large extracellular domain within the transmembrane segment indicated by experimental evidence from various systems. INTRO +24 27 nsr gene The recently discovered nsr gene cluster of the human pathogen S. agalactiae encodes for the resistance protein NSR and the ABC transporter NsrFP, both conferring resistance against nisin. INTRO +48 53 human species The recently discovered nsr gene cluster of the human pathogen S. agalactiae encodes for the resistance protein NSR and the ABC transporter NsrFP, both conferring resistance against nisin. INTRO +63 76 S. agalactiae species The recently discovered nsr gene cluster of the human pathogen S. agalactiae encodes for the resistance protein NSR and the ABC transporter NsrFP, both conferring resistance against nisin. INTRO +93 111 resistance protein protein_type The recently discovered nsr gene cluster of the human pathogen S. agalactiae encodes for the resistance protein NSR and the ABC transporter NsrFP, both conferring resistance against nisin. INTRO +112 115 NSR protein The recently discovered nsr gene cluster of the human pathogen S. agalactiae encodes for the resistance protein NSR and the ABC transporter NsrFP, both conferring resistance against nisin. INTRO +124 139 ABC transporter protein_type The recently discovered nsr gene cluster of the human pathogen S. agalactiae encodes for the resistance protein NSR and the ABC transporter NsrFP, both conferring resistance against nisin. INTRO +140 145 NsrFP protein The recently discovered nsr gene cluster of the human pathogen S. agalactiae encodes for the resistance protein NSR and the ABC transporter NsrFP, both conferring resistance against nisin. INTRO +182 187 nisin chemical The recently discovered nsr gene cluster of the human pathogen S. agalactiae encodes for the resistance protein NSR and the ABC transporter NsrFP, both conferring resistance against nisin. INTRO +51 56 human species Homologous operons have been identified in various human pathogenic strains such as Staphylococcus epidermis and Streptococcus ictaluri based on the high sequence identity of NSR and NsrFP. INTRO +84 108 Staphylococcus epidermis species Homologous operons have been identified in various human pathogenic strains such as Staphylococcus epidermis and Streptococcus ictaluri based on the high sequence identity of NSR and NsrFP. INTRO +113 135 Streptococcus ictaluri species Homologous operons have been identified in various human pathogenic strains such as Staphylococcus epidermis and Streptococcus ictaluri based on the high sequence identity of NSR and NsrFP. INTRO +175 178 NSR protein Homologous operons have been identified in various human pathogenic strains such as Staphylococcus epidermis and Streptococcus ictaluri based on the high sequence identity of NSR and NsrFP. INTRO +183 188 NsrFP protein Homologous operons have been identified in various human pathogenic strains such as Staphylococcus epidermis and Streptococcus ictaluri based on the high sequence identity of NSR and NsrFP. INTRO +26 29 TCS complex_assembly In this gene cluster, the TCS NsrRK is responsible for the expression of the nsr and nsrFP genes. INTRO +30 35 NsrRK protein In this gene cluster, the TCS NsrRK is responsible for the expression of the nsr and nsrFP genes. INTRO +77 80 nsr gene In this gene cluster, the TCS NsrRK is responsible for the expression of the nsr and nsrFP genes. INTRO +85 90 nsrFP gene In this gene cluster, the TCS NsrRK is responsible for the expression of the nsr and nsrFP genes. INTRO +22 25 TCS complex_assembly The similarity of the TCS within all the described nisin resistance operons suggests an expression specifically induced by nisin. INTRO +51 56 nisin chemical The similarity of the TCS within all the described nisin resistance operons suggests an expression specifically induced by nisin. INTRO +123 128 nisin chemical The similarity of the TCS within all the described nisin resistance operons suggests an expression specifically induced by nisin. INTRO +6 11 NsrRK protein Thus, NsrRK might be a useful target to combat inherently pathogenic lantibiotic-resistant strains. INTRO +69 80 lantibiotic chemical Thus, NsrRK might be a useful target to combat inherently pathogenic lantibiotic-resistant strains. INTRO +11 14 RRs protein_type Generally, RRs consist of two distinct structural domains, a receiver domain (RD) and an effector domain (ED), that are separated from each other by a flexible linker. INTRO +61 76 receiver domain structure_element Generally, RRs consist of two distinct structural domains, a receiver domain (RD) and an effector domain (ED), that are separated from each other by a flexible linker. INTRO +78 80 RD structure_element Generally, RRs consist of two distinct structural domains, a receiver domain (RD) and an effector domain (ED), that are separated from each other by a flexible linker. INTRO +89 104 effector domain structure_element Generally, RRs consist of two distinct structural domains, a receiver domain (RD) and an effector domain (ED), that are separated from each other by a flexible linker. INTRO +106 108 ED structure_element Generally, RRs consist of two distinct structural domains, a receiver domain (RD) and an effector domain (ED), that are separated from each other by a flexible linker. INTRO +151 159 flexible protein_state Generally, RRs consist of two distinct structural domains, a receiver domain (RD) and an effector domain (ED), that are separated from each other by a flexible linker. INTRO +160 166 linker structure_element Generally, RRs consist of two distinct structural domains, a receiver domain (RD) and an effector domain (ED), that are separated from each other by a flexible linker. INTRO +0 3 RDs structure_element RDs contain a highly conserved aspartate residue, which acts as a phosphoryl acceptor that becomes phosphorylated by the kinase domain of the histidine kinase upon reception of an external signal. INTRO +14 30 highly conserved protein_state RDs contain a highly conserved aspartate residue, which acts as a phosphoryl acceptor that becomes phosphorylated by the kinase domain of the histidine kinase upon reception of an external signal. INTRO +31 40 aspartate residue_name RDs contain a highly conserved aspartate residue, which acts as a phosphoryl acceptor that becomes phosphorylated by the kinase domain of the histidine kinase upon reception of an external signal. INTRO +99 113 phosphorylated protein_state RDs contain a highly conserved aspartate residue, which acts as a phosphoryl acceptor that becomes phosphorylated by the kinase domain of the histidine kinase upon reception of an external signal. INTRO +121 134 kinase domain structure_element RDs contain a highly conserved aspartate residue, which acts as a phosphoryl acceptor that becomes phosphorylated by the kinase domain of the histidine kinase upon reception of an external signal. INTRO +142 158 histidine kinase protein_type RDs contain a highly conserved aspartate residue, which acts as a phosphoryl acceptor that becomes phosphorylated by the kinase domain of the histidine kinase upon reception of an external signal. INTRO +4 6 ED structure_element The ED is thereby activated and binds to the designated promoters, thus initiating transcription of the target genes. INTRO +4 7 RRs protein_type The RRs are classified into different subfamilies depending on the three-dimensional structure of their EDs. INTRO +104 107 EDs structure_element The RRs are classified into different subfamilies depending on the three-dimensional structure of their EDs. INTRO +4 23 OmpR/PhoB subfamily protein_type The OmpR/PhoB subfamily is the largest subgroup of RRs and comprises approximately 40% of all response regulators in bacteria. INTRO +51 54 RRs protein_type The OmpR/PhoB subfamily is the largest subgroup of RRs and comprises approximately 40% of all response regulators in bacteria. INTRO +94 113 response regulators protein_type The OmpR/PhoB subfamily is the largest subgroup of RRs and comprises approximately 40% of all response regulators in bacteria. INTRO +117 125 bacteria taxonomy_domain The OmpR/PhoB subfamily is the largest subgroup of RRs and comprises approximately 40% of all response regulators in bacteria. INTRO +41 64 winged helix-turn-helix structure_element All their members are characterized by a winged helix-turn-helix (wHTH) motif. INTRO +66 70 wHTH structure_element All their members are characterized by a winged helix-turn-helix (wHTH) motif. INTRO +18 28 structures evidence Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +73 83 structures evidence Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +87 98 full-length protein_state Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +99 117 OmpR/PhoB-type RRs protein_type Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +140 145 RegX3 protein Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +164 168 MtrA protein Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +187 191 PrrA protein Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +213 217 PhoP protein Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +240 266 Mycobacterium tuberculosis species Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +268 272 DrrB protein Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +294 298 DrrD protein Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +321 340 Thermotoga maritima species Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +346 350 KdpE protein Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +356 372 Escherichia coli species Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY). INTRO +12 22 structures evidence The various structures of RRs reveal that in addition to being in either “inactive” or “active” state, the RRs can also exist in two distinct conformations: “open” and “closed”. INTRO +26 29 RRs protein_type The various structures of RRs reveal that in addition to being in either “inactive” or “active” state, the RRs can also exist in two distinct conformations: “open” and “closed”. INTRO +74 82 inactive protein_state The various structures of RRs reveal that in addition to being in either “inactive” or “active” state, the RRs can also exist in two distinct conformations: “open” and “closed”. INTRO +88 94 active protein_state The various structures of RRs reveal that in addition to being in either “inactive” or “active” state, the RRs can also exist in two distinct conformations: “open” and “closed”. INTRO +107 110 RRs protein_type The various structures of RRs reveal that in addition to being in either “inactive” or “active” state, the RRs can also exist in two distinct conformations: “open” and “closed”. INTRO +158 162 open protein_state The various structures of RRs reveal that in addition to being in either “inactive” or “active” state, the RRs can also exist in two distinct conformations: “open” and “closed”. INTRO +169 175 closed protein_state The various structures of RRs reveal that in addition to being in either “inactive” or “active” state, the RRs can also exist in two distinct conformations: “open” and “closed”. INTRO +0 4 MtrA protein MtrA and PrrA exhibit a very compact, closed structure with the DNA-binding sequence, called recognition helix, of the ED being inaccessible to DNA. INTRO +9 13 PrrA protein MtrA and PrrA exhibit a very compact, closed structure with the DNA-binding sequence, called recognition helix, of the ED being inaccessible to DNA. INTRO +24 36 very compact protein_state MtrA and PrrA exhibit a very compact, closed structure with the DNA-binding sequence, called recognition helix, of the ED being inaccessible to DNA. INTRO +38 44 closed protein_state MtrA and PrrA exhibit a very compact, closed structure with the DNA-binding sequence, called recognition helix, of the ED being inaccessible to DNA. INTRO +45 54 structure evidence MtrA and PrrA exhibit a very compact, closed structure with the DNA-binding sequence, called recognition helix, of the ED being inaccessible to DNA. INTRO +64 84 DNA-binding sequence structure_element MtrA and PrrA exhibit a very compact, closed structure with the DNA-binding sequence, called recognition helix, of the ED being inaccessible to DNA. INTRO +93 110 recognition helix structure_element MtrA and PrrA exhibit a very compact, closed structure with the DNA-binding sequence, called recognition helix, of the ED being inaccessible to DNA. INTRO +119 121 ED structure_element MtrA and PrrA exhibit a very compact, closed structure with the DNA-binding sequence, called recognition helix, of the ED being inaccessible to DNA. INTRO +144 147 DNA chemical MtrA and PrrA exhibit a very compact, closed structure with the DNA-binding sequence, called recognition helix, of the ED being inaccessible to DNA. INTRO +4 14 structures evidence The structures of DrrD and DrrB exist in an open conformation, here the recognition helix is fully exposed, suggesting that RRs are flexible in solution and can adopt multiple conformations. INTRO +18 22 DrrD protein The structures of DrrD and DrrB exist in an open conformation, here the recognition helix is fully exposed, suggesting that RRs are flexible in solution and can adopt multiple conformations. INTRO +27 31 DrrB protein The structures of DrrD and DrrB exist in an open conformation, here the recognition helix is fully exposed, suggesting that RRs are flexible in solution and can adopt multiple conformations. INTRO +44 48 open protein_state The structures of DrrD and DrrB exist in an open conformation, here the recognition helix is fully exposed, suggesting that RRs are flexible in solution and can adopt multiple conformations. INTRO +72 89 recognition helix structure_element The structures of DrrD and DrrB exist in an open conformation, here the recognition helix is fully exposed, suggesting that RRs are flexible in solution and can adopt multiple conformations. INTRO +93 106 fully exposed protein_state The structures of DrrD and DrrB exist in an open conformation, here the recognition helix is fully exposed, suggesting that RRs are flexible in solution and can adopt multiple conformations. INTRO +124 127 RRs protein_type The structures of DrrD and DrrB exist in an open conformation, here the recognition helix is fully exposed, suggesting that RRs are flexible in solution and can adopt multiple conformations. INTRO +132 140 flexible protein_state The structures of DrrD and DrrB exist in an open conformation, here the recognition helix is fully exposed, suggesting that RRs are flexible in solution and can adopt multiple conformations. INTRO +22 40 crystal structures evidence Here, we describe the crystal structures of the N-terminal RD and the C-terminal ED of the lantibiotic resistance-associated RR NsrR from S. agalactiae. INTRO +59 61 RD structure_element Here, we describe the crystal structures of the N-terminal RD and the C-terminal ED of the lantibiotic resistance-associated RR NsrR from S. agalactiae. INTRO +81 83 ED structure_element Here, we describe the crystal structures of the N-terminal RD and the C-terminal ED of the lantibiotic resistance-associated RR NsrR from S. agalactiae. INTRO +91 127 lantibiotic resistance-associated RR protein_type Here, we describe the crystal structures of the N-terminal RD and the C-terminal ED of the lantibiotic resistance-associated RR NsrR from S. agalactiae. INTRO +128 132 NsrR protein Here, we describe the crystal structures of the N-terminal RD and the C-terminal ED of the lantibiotic resistance-associated RR NsrR from S. agalactiae. INTRO +138 151 S. agalactiae species Here, we describe the crystal structures of the N-terminal RD and the C-terminal ED of the lantibiotic resistance-associated RR NsrR from S. agalactiae. INTRO +0 4 NsrR protein NsrR is part of the nisin resistance operon. INTRO +20 25 nisin chemical NsrR is part of the nisin resistance operon. INTRO +59 62 TCS complex_assembly The expression of the genes of this operon is induced by a TCS consisting of the HK NsrK and the RR NsrR. Based on the crystal structures of both the domains, modeling was employed to shed light on the putative DNA-bound state of full-length NsrR. INTRO +81 83 HK protein_type The expression of the genes of this operon is induced by a TCS consisting of the HK NsrK and the RR NsrR. Based on the crystal structures of both the domains, modeling was employed to shed light on the putative DNA-bound state of full-length NsrR. INTRO +84 88 NsrK protein The expression of the genes of this operon is induced by a TCS consisting of the HK NsrK and the RR NsrR. Based on the crystal structures of both the domains, modeling was employed to shed light on the putative DNA-bound state of full-length NsrR. INTRO +97 99 RR protein_type The expression of the genes of this operon is induced by a TCS consisting of the HK NsrK and the RR NsrR. Based on the crystal structures of both the domains, modeling was employed to shed light on the putative DNA-bound state of full-length NsrR. INTRO +100 104 NsrR protein The expression of the genes of this operon is induced by a TCS consisting of the HK NsrK and the RR NsrR. Based on the crystal structures of both the domains, modeling was employed to shed light on the putative DNA-bound state of full-length NsrR. INTRO +119 137 crystal structures evidence The expression of the genes of this operon is induced by a TCS consisting of the HK NsrK and the RR NsrR. Based on the crystal structures of both the domains, modeling was employed to shed light on the putative DNA-bound state of full-length NsrR. INTRO +211 220 DNA-bound protein_state The expression of the genes of this operon is induced by a TCS consisting of the HK NsrK and the RR NsrR. Based on the crystal structures of both the domains, modeling was employed to shed light on the putative DNA-bound state of full-length NsrR. INTRO +230 241 full-length protein_state The expression of the genes of this operon is induced by a TCS consisting of the HK NsrK and the RR NsrR. Based on the crystal structures of both the domains, modeling was employed to shed light on the putative DNA-bound state of full-length NsrR. INTRO +242 246 NsrR protein The expression of the genes of this operon is induced by a TCS consisting of the HK NsrK and the RR NsrR. Based on the crystal structures of both the domains, modeling was employed to shed light on the putative DNA-bound state of full-length NsrR. INTRO +0 4 NsrR protein NsrR was expressed and purified as described, resulting in a homogenous protein as observed by size exclusion chromatography (Fig 1A), with a yield of 2 mg per liter of cell culture. RESULTS +9 31 expressed and purified experimental_method NsrR was expressed and purified as described, resulting in a homogenous protein as observed by size exclusion chromatography (Fig 1A), with a yield of 2 mg per liter of cell culture. RESULTS +95 124 size exclusion chromatography experimental_method NsrR was expressed and purified as described, resulting in a homogenous protein as observed by size exclusion chromatography (Fig 1A), with a yield of 2 mg per liter of cell culture. RESULTS +70 74 NsrR protein By calibrating the column with proteins of known molecular weight the NsrR full length protein elutes as a dimer. RESULTS +75 86 full length protein_state By calibrating the column with proteins of known molecular weight the NsrR full length protein elutes as a dimer. RESULTS +107 112 dimer oligomeric_state By calibrating the column with proteins of known molecular weight the NsrR full length protein elutes as a dimer. RESULTS +13 17 NsrR protein The purified NsrR protein has a theoretical molecular mass of 27.7 kDa and was >98% pure as assessed by SDS-PAGE (Fig 1B, indicated by *). RESULTS +44 58 molecular mass evidence The purified NsrR protein has a theoretical molecular mass of 27.7 kDa and was >98% pure as assessed by SDS-PAGE (Fig 1B, indicated by *). RESULTS +104 112 SDS-PAGE experimental_method The purified NsrR protein has a theoretical molecular mass of 27.7 kDa and was >98% pure as assessed by SDS-PAGE (Fig 1B, indicated by *). RESULTS +24 28 NsrR protein Surprisingly, over time NsrR degraded into two distinct fragments as visible on SDS-PAGE analysis using the same purified protein sample after one week (Fig 1C, indicated by ** and ***, respectively). RESULTS +80 88 SDS-PAGE experimental_method Surprisingly, over time NsrR degraded into two distinct fragments as visible on SDS-PAGE analysis using the same purified protein sample after one week (Fig 1C, indicated by ** and ***, respectively). RESULTS +26 55 size exclusion chromatography experimental_method This was also observed by size exclusion chromatography where a peak at an elution time of 18 min appeared (Fig 1A). RESULTS +29 55 mass spectrometry analysis experimental_method Both bands were subjected to mass spectrometry analysis. RESULTS +78 93 receiver domain structure_element The analysis revealed that the larger fragment (**) represents the N-terminal receiver domain (residues 1–119; referred to as NsrR-RD) whereas the smaller fragment (***) contained the C-terminal DNA-binding effector domain of NsrR (residues 129–243 including 21 amino acids derived from the expression tag; referred to as NsrR-ED) (Fig 1C). RESULTS +104 109 1–119 residue_range The analysis revealed that the larger fragment (**) represents the N-terminal receiver domain (residues 1–119; referred to as NsrR-RD) whereas the smaller fragment (***) contained the C-terminal DNA-binding effector domain of NsrR (residues 129–243 including 21 amino acids derived from the expression tag; referred to as NsrR-ED) (Fig 1C). RESULTS +126 130 NsrR protein The analysis revealed that the larger fragment (**) represents the N-terminal receiver domain (residues 1–119; referred to as NsrR-RD) whereas the smaller fragment (***) contained the C-terminal DNA-binding effector domain of NsrR (residues 129–243 including 21 amino acids derived from the expression tag; referred to as NsrR-ED) (Fig 1C). RESULTS +131 133 RD structure_element The analysis revealed that the larger fragment (**) represents the N-terminal receiver domain (residues 1–119; referred to as NsrR-RD) whereas the smaller fragment (***) contained the C-terminal DNA-binding effector domain of NsrR (residues 129–243 including 21 amino acids derived from the expression tag; referred to as NsrR-ED) (Fig 1C). RESULTS +195 222 DNA-binding effector domain structure_element The analysis revealed that the larger fragment (**) represents the N-terminal receiver domain (residues 1–119; referred to as NsrR-RD) whereas the smaller fragment (***) contained the C-terminal DNA-binding effector domain of NsrR (residues 129–243 including 21 amino acids derived from the expression tag; referred to as NsrR-ED) (Fig 1C). RESULTS +226 230 NsrR protein The analysis revealed that the larger fragment (**) represents the N-terminal receiver domain (residues 1–119; referred to as NsrR-RD) whereas the smaller fragment (***) contained the C-terminal DNA-binding effector domain of NsrR (residues 129–243 including 21 amino acids derived from the expression tag; referred to as NsrR-ED) (Fig 1C). RESULTS +241 248 129–243 residue_range The analysis revealed that the larger fragment (**) represents the N-terminal receiver domain (residues 1–119; referred to as NsrR-RD) whereas the smaller fragment (***) contained the C-terminal DNA-binding effector domain of NsrR (residues 129–243 including 21 amino acids derived from the expression tag; referred to as NsrR-ED) (Fig 1C). RESULTS +322 326 NsrR protein The analysis revealed that the larger fragment (**) represents the N-terminal receiver domain (residues 1–119; referred to as NsrR-RD) whereas the smaller fragment (***) contained the C-terminal DNA-binding effector domain of NsrR (residues 129–243 including 21 amino acids derived from the expression tag; referred to as NsrR-ED) (Fig 1C). RESULTS +327 329 ED structure_element The analysis revealed that the larger fragment (**) represents the N-terminal receiver domain (residues 1–119; referred to as NsrR-RD) whereas the smaller fragment (***) contained the C-terminal DNA-binding effector domain of NsrR (residues 129–243 including 21 amino acids derived from the expression tag; referred to as NsrR-ED) (Fig 1C). RESULTS +9 16 120–128 residue_range Residues 120–128 form the linker connecting the RD and ED. RESULTS +26 32 linker structure_element Residues 120–128 form the linker connecting the RD and ED. RESULTS +48 50 RD structure_element Residues 120–128 form the linker connecting the RD and ED. RESULTS +55 57 ED structure_element Residues 120–128 form the linker connecting the RD and ED. RESULTS +23 34 full-length protein_state Such a cleavage of the full-length RR into two specific domains is not unusual and has been previously reported for other RRs as well. RESULTS +35 37 RR protein_type Such a cleavage of the full-length RR into two specific domains is not unusual and has been previously reported for other RRs as well. RESULTS +122 125 RRs protein_type Such a cleavage of the full-length RR into two specific domains is not unusual and has been previously reported for other RRs as well. RESULTS +0 26 Mass spectrometry analysis experimental_method Mass spectrometry analysis did not reveal the presence of any specific protease in the purified NsrR sample. RESULTS +96 100 NsrR protein Mass spectrometry analysis did not reveal the presence of any specific protease in the purified NsrR sample. RESULTS +55 59 PMSF chemical Furthermore, addition of a protease inhibitor, such as PMSF (Phenylmethylsulfonyl fluoride) and AEBSF {4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride}, even at high concentrations, did not inhibit proteolysis (data not shown). RESULTS +61 90 Phenylmethylsulfonyl fluoride chemical Furthermore, addition of a protease inhibitor, such as PMSF (Phenylmethylsulfonyl fluoride) and AEBSF {4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride}, even at high concentrations, did not inhibit proteolysis (data not shown). RESULTS +96 101 AEBSF chemical Furthermore, addition of a protease inhibitor, such as PMSF (Phenylmethylsulfonyl fluoride) and AEBSF {4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride}, even at high concentrations, did not inhibit proteolysis (data not shown). RESULTS +103 158 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride chemical Furthermore, addition of a protease inhibitor, such as PMSF (Phenylmethylsulfonyl fluoride) and AEBSF {4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride}, even at high concentrations, did not inhibit proteolysis (data not shown). RESULTS +0 12 Purification experimental_method Purification of NsrR and SDS PAGE analysis of purified NsrR directly and one week after purification. FIG +16 20 NsrR protein Purification of NsrR and SDS PAGE analysis of purified NsrR directly and one week after purification. FIG +25 33 SDS PAGE experimental_method Purification of NsrR and SDS PAGE analysis of purified NsrR directly and one week after purification. FIG +55 59 NsrR protein Purification of NsrR and SDS PAGE analysis of purified NsrR directly and one week after purification. FIG +4 19 Elution profile evidence (a) Elution profile of size-exclusion chromatography step of NsrR. The y-axis represents the UV absorption of the protein at 280 nm, while the x-axis represents the elution volume. FIG +23 52 size-exclusion chromatography experimental_method (a) Elution profile of size-exclusion chromatography step of NsrR. The y-axis represents the UV absorption of the protein at 280 nm, while the x-axis represents the elution volume. FIG +61 65 NsrR protein (a) Elution profile of size-exclusion chromatography step of NsrR. The y-axis represents the UV absorption of the protein at 280 nm, while the x-axis represents the elution volume. FIG +29 41 chromatogram evidence The bold line represents the chromatogram of freshly purified NsrR while the dashed line shows the chromatogram of the same NsrR protein after one week. FIG +62 66 NsrR protein The bold line represents the chromatogram of freshly purified NsrR while the dashed line shows the chromatogram of the same NsrR protein after one week. FIG +99 111 chromatogram evidence The bold line represents the chromatogram of freshly purified NsrR while the dashed line shows the chromatogram of the same NsrR protein after one week. FIG +124 128 NsrR protein The bold line represents the chromatogram of freshly purified NsrR while the dashed line shows the chromatogram of the same NsrR protein after one week. FIG +21 25 NsrR protein (b) Freshly purified NsrR protein, and (c) NsrR protein after one week. FIG +43 47 NsrR protein (b) Freshly purified NsrR protein, and (c) NsrR protein after one week. FIG +56 60 NsrR protein Lanes: M represents the PAGE Ruler Unstained Ladder; 1: NsrR after a two-step purification; 2: NsrR one week after purification. FIG +95 99 NsrR protein Lanes: M represents the PAGE Ruler Unstained Ladder; 1: NsrR after a two-step purification; 2: NsrR one week after purification. FIG +17 28 full-length protein_state * corresponds to full-length NsrR protein at 27 kDa, while ** and *** correspond to the NsrR-RD and NsrR-ED domain at around 13 kDa, respectively. FIG +29 33 NsrR protein * corresponds to full-length NsrR protein at 27 kDa, while ** and *** correspond to the NsrR-RD and NsrR-ED domain at around 13 kDa, respectively. FIG +88 92 NsrR protein * corresponds to full-length NsrR protein at 27 kDa, while ** and *** correspond to the NsrR-RD and NsrR-ED domain at around 13 kDa, respectively. FIG +93 95 RD structure_element * corresponds to full-length NsrR protein at 27 kDa, while ** and *** correspond to the NsrR-RD and NsrR-ED domain at around 13 kDa, respectively. FIG +100 104 NsrR protein * corresponds to full-length NsrR protein at 27 kDa, while ** and *** correspond to the NsrR-RD and NsrR-ED domain at around 13 kDa, respectively. FIG +105 107 ED structure_element * corresponds to full-length NsrR protein at 27 kDa, while ** and *** correspond to the NsrR-RD and NsrR-ED domain at around 13 kDa, respectively. FIG +23 31 crystals evidence Since formation of the crystals took around one month, it is not surprising that this cleavage also occurred in the crystallization drop. RESULTS +0 4 NsrR protein NsrR was crystallized yielding two crystal forms, which were distinguishable by visual inspection. RESULTS +9 21 crystallized experimental_method NsrR was crystallized yielding two crystal forms, which were distinguishable by visual inspection. RESULTS +33 42 structure evidence Initially, we tried to solve the structure of NsrR by molecular replacement, which was not successful. RESULTS +46 50 NsrR protein Initially, we tried to solve the structure of NsrR by molecular replacement, which was not successful. RESULTS +54 75 molecular replacement experimental_method Initially, we tried to solve the structure of NsrR by molecular replacement, which was not successful. RESULTS +20 38 heavy atom phasing experimental_method Therefore, we tried heavy atom phasing using a platinum compound. RESULTS +47 55 platinum chemical Therefore, we tried heavy atom phasing using a platinum compound. RESULTS +48 56 crystals evidence This succeeded for the rectangular plate-shaped crystals. RESULTS +10 19 structure evidence After the structure was solved, it became evident that these crystals contained two monomers of the ED of NsrR in the asymmetric unit. RESULTS +61 69 crystals evidence After the structure was solved, it became evident that these crystals contained two monomers of the ED of NsrR in the asymmetric unit. RESULTS +84 92 monomers oligomeric_state After the structure was solved, it became evident that these crystals contained two monomers of the ED of NsrR in the asymmetric unit. RESULTS +100 102 ED structure_element After the structure was solved, it became evident that these crystals contained two monomers of the ED of NsrR in the asymmetric unit. RESULTS +106 110 NsrR protein After the structure was solved, it became evident that these crystals contained two monomers of the ED of NsrR in the asymmetric unit. RESULTS +27 36 structure evidence We also tried to solve the structure of the thin plate-shaped crystals with this template, but the resulting model generated was not sufficient. RESULTS +62 70 crystals evidence We also tried to solve the structure of the thin plate-shaped crystals with this template, but the resulting model generated was not sufficient. RESULTS +33 41 crystals evidence Therefore, we thought that these crystals contained the N-terminal domain of NsrR and successfully phased this dataset using molecular replacement with the N-terminal domain of PhoB (PDB code: 1B00; as a template. RESULTS +56 73 N-terminal domain structure_element Therefore, we thought that these crystals contained the N-terminal domain of NsrR and successfully phased this dataset using molecular replacement with the N-terminal domain of PhoB (PDB code: 1B00; as a template. RESULTS +77 81 NsrR protein Therefore, we thought that these crystals contained the N-terminal domain of NsrR and successfully phased this dataset using molecular replacement with the N-terminal domain of PhoB (PDB code: 1B00; as a template. RESULTS +125 146 molecular replacement experimental_method Therefore, we thought that these crystals contained the N-terminal domain of NsrR and successfully phased this dataset using molecular replacement with the N-terminal domain of PhoB (PDB code: 1B00; as a template. RESULTS +156 173 N-terminal domain structure_element Therefore, we thought that these crystals contained the N-terminal domain of NsrR and successfully phased this dataset using molecular replacement with the N-terminal domain of PhoB (PDB code: 1B00; as a template. RESULTS +177 181 PhoB protein Therefore, we thought that these crystals contained the N-terminal domain of NsrR and successfully phased this dataset using molecular replacement with the N-terminal domain of PhoB (PDB code: 1B00; as a template. RESULTS +67 75 monomers oligomeric_state This approach revealed that this crystal form indeed contained two monomers of the RD of NsrR in the asymmetric unit. RESULTS +83 85 RD structure_element This approach revealed that this crystal form indeed contained two monomers of the RD of NsrR in the asymmetric unit. RESULTS +89 93 NsrR protein This approach revealed that this crystal form indeed contained two monomers of the RD of NsrR in the asymmetric unit. RESULTS +108 116 crystals evidence Since both crystals forms were obtained in the same drop it is not surprising that, when dissolving several crystals and performing subsequent mass-spectrometry to identify the protein in the crystals, it yielded peptide fragments throughout the NsrR sequence. RESULTS +143 160 mass-spectrometry experimental_method Since both crystals forms were obtained in the same drop it is not surprising that, when dissolving several crystals and performing subsequent mass-spectrometry to identify the protein in the crystals, it yielded peptide fragments throughout the NsrR sequence. RESULTS +192 200 crystals evidence Since both crystals forms were obtained in the same drop it is not surprising that, when dissolving several crystals and performing subsequent mass-spectrometry to identify the protein in the crystals, it yielded peptide fragments throughout the NsrR sequence. RESULTS +246 250 NsrR protein Since both crystals forms were obtained in the same drop it is not surprising that, when dissolving several crystals and performing subsequent mass-spectrometry to identify the protein in the crystals, it yielded peptide fragments throughout the NsrR sequence. RESULTS +20 33 crystal forms evidence In summary, the two crystal forms contained one of the two domains, respectively, such that both domains were successfully crystallized. RESULTS +123 135 crystallized experimental_method In summary, the two crystal forms contained one of the two domains, respectively, such that both domains were successfully crystallized. RESULTS +18 36 crystal structures evidence We determined the crystal structures of NsrR-RD and NsrR-ED separately. RESULTS +40 44 NsrR protein We determined the crystal structures of NsrR-RD and NsrR-ED separately. RESULTS +45 47 RD structure_element We determined the crystal structures of NsrR-RD and NsrR-ED separately. RESULTS +52 56 NsrR protein We determined the crystal structures of NsrR-RD and NsrR-ED separately. RESULTS +57 59 ED structure_element We determined the crystal structures of NsrR-RD and NsrR-ED separately. RESULTS +23 36 linker region structure_element However, a part of the linker region (residues 120–128; 120RRSQQFIQQ128; underlined are the amino acid residues not visible in either domain) could not be traced in the electron density. RESULTS +47 54 120–128 residue_range However, a part of the linker region (residues 120–128; 120RRSQQFIQQ128; underlined are the amino acid residues not visible in either domain) could not be traced in the electron density. RESULTS +56 71 120RRSQQFIQQ128 structure_element However, a part of the linker region (residues 120–128; 120RRSQQFIQQ128; underlined are the amino acid residues not visible in either domain) could not be traced in the electron density. RESULTS +169 185 electron density evidence However, a part of the linker region (residues 120–128; 120RRSQQFIQQ128; underlined are the amino acid residues not visible in either domain) could not be traced in the electron density. RESULTS +8 17 structure evidence Overall structure of the N-terminal NsrR receiver domain (NsrR-RD) RESULTS +36 40 NsrR protein Overall structure of the N-terminal NsrR receiver domain (NsrR-RD) RESULTS +41 56 receiver domain structure_element Overall structure of the N-terminal NsrR receiver domain (NsrR-RD) RESULTS +58 62 NsrR protein Overall structure of the N-terminal NsrR receiver domain (NsrR-RD) RESULTS +63 65 RD structure_element Overall structure of the N-terminal NsrR receiver domain (NsrR-RD) RESULTS +4 13 structure evidence The structure of the NsrR-RD was determined at a resolution of 1.8 Å (Table 1). RESULTS +21 25 NsrR protein The structure of the NsrR-RD was determined at a resolution of 1.8 Å (Table 1). RESULTS +26 28 RD structure_element The structure of the NsrR-RD was determined at a resolution of 1.8 Å (Table 1). RESULTS +4 9 Rwork evidence The Rwork and Rfree values after refinement were 0.17 and 0.22, respectively. RESULTS +14 19 Rfree evidence The Rwork and Rfree values after refinement were 0.17 and 0.22, respectively. RESULTS +0 23 Ramachandran validation evidence Ramachandran validation revealed that all residues (100%, 236 amino acids) were in the preferred or allowed regions. RESULTS +4 13 structure evidence The structure contained many ethylene glycol molecules arising from the cryo-protecting procedure. RESULTS +29 44 ethylene glycol chemical The structure contained many ethylene glycol molecules arising from the cryo-protecting procedure. RESULTS +43 47 NsrR protein The asymmetric unit contains two copies of NsrR-RD. RESULTS +48 50 RD structure_element The asymmetric unit contains two copies of NsrR-RD. RESULTS +31 46 receiver domain structure_element Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +71 82 Met1-Leu119 residue_range Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +98 112 Asn4 to Arg121 residue_range Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +116 123 chain A structure_element Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +144 150 Arg120 residue_name_number Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +155 161 Arg121 residue_name_number Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +169 175 linker structure_element Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +181 195 Gln5 to Ser122 residue_range Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +199 206 chain B structure_element Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +227 246 Arg120 until Ser122 residue_range Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +254 260 linker structure_element Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +285 301 electron density evidence Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +305 309 NsrR protein Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +310 312 RD structure_element Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD. RESULTS +4 9 Asn85 residue_name_number For Asn85, Asp86, and Glu87 of chain A, poor electron density was observed for the side chains and, thus, these side chains were deleted during refinement and are not present in the final structure. RESULTS +11 16 Asp86 residue_name_number For Asn85, Asp86, and Glu87 of chain A, poor electron density was observed for the side chains and, thus, these side chains were deleted during refinement and are not present in the final structure. RESULTS +22 27 Glu87 residue_name_number For Asn85, Asp86, and Glu87 of chain A, poor electron density was observed for the side chains and, thus, these side chains were deleted during refinement and are not present in the final structure. RESULTS +31 38 chain A structure_element For Asn85, Asp86, and Glu87 of chain A, poor electron density was observed for the side chains and, thus, these side chains were deleted during refinement and are not present in the final structure. RESULTS +45 61 electron density evidence For Asn85, Asp86, and Glu87 of chain A, poor electron density was observed for the side chains and, thus, these side chains were deleted during refinement and are not present in the final structure. RESULTS +188 197 structure evidence For Asn85, Asp86, and Glu87 of chain A, poor electron density was observed for the side chains and, thus, these side chains were deleted during refinement and are not present in the final structure. RESULTS +14 22 monomers oligomeric_state Since the two monomers of NsrR-RD were virtually identical (rmsd of 0.6 Å over 116 Cα atoms for the two monomers). RESULTS +26 30 NsrR protein Since the two monomers of NsrR-RD were virtually identical (rmsd of 0.6 Å over 116 Cα atoms for the two monomers). RESULTS +31 33 RD structure_element Since the two monomers of NsrR-RD were virtually identical (rmsd of 0.6 Å over 116 Cα atoms for the two monomers). RESULTS +60 64 rmsd evidence Since the two monomers of NsrR-RD were virtually identical (rmsd of 0.6 Å over 116 Cα atoms for the two monomers). RESULTS +104 112 monomers oligomeric_state Since the two monomers of NsrR-RD were virtually identical (rmsd of 0.6 Å over 116 Cα atoms for the two monomers). RESULTS +23 32 structure evidence Therefore, the overall structure is described for monomer A only. RESULTS +50 57 monomer oligomeric_state Therefore, the overall structure is described for monomer A only. RESULTS +58 59 A structure_element Therefore, the overall structure is described for monomer A only. RESULTS +0 4 NsrR protein NsrR-RD structurally adopts a αβ doubly-wound fold previously observed in OmpR/PhoB type regulators. RESULTS +5 7 RD structure_element NsrR-RD structurally adopts a αβ doubly-wound fold previously observed in OmpR/PhoB type regulators. RESULTS +30 50 αβ doubly-wound fold structure_element NsrR-RD structurally adopts a αβ doubly-wound fold previously observed in OmpR/PhoB type regulators. RESULTS +74 99 OmpR/PhoB type regulators protein_type NsrR-RD structurally adopts a αβ doubly-wound fold previously observed in OmpR/PhoB type regulators. RESULTS +5 14 β-strands structure_element Five β-strands (β1-β5) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two α-helices (α1 and α5) on one and three helices (α2, α3, α4) on the other side (Fig 2). RESULTS +16 21 β1-β5 structure_element Five β-strands (β1-β5) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two α-helices (α1 and α5) on one and three helices (α2, α3, α4) on the other side (Fig 2). RESULTS +95 104 structure evidence Five β-strands (β1-β5) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two α-helices (α1 and α5) on one and three helices (α2, α3, α4) on the other side (Fig 2). RESULTS +133 142 α-helices structure_element Five β-strands (β1-β5) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two α-helices (α1 and α5) on one and three helices (α2, α3, α4) on the other side (Fig 2). RESULTS +144 146 α1 structure_element Five β-strands (β1-β5) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two α-helices (α1 and α5) on one and three helices (α2, α3, α4) on the other side (Fig 2). RESULTS +151 153 α5 structure_element Five β-strands (β1-β5) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two α-helices (α1 and α5) on one and three helices (α2, α3, α4) on the other side (Fig 2). RESULTS +172 179 helices structure_element Five β-strands (β1-β5) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two α-helices (α1 and α5) on one and three helices (α2, α3, α4) on the other side (Fig 2). RESULTS +181 183 α2 structure_element Five β-strands (β1-β5) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two α-helices (α1 and α5) on one and three helices (α2, α3, α4) on the other side (Fig 2). RESULTS +185 187 α3 structure_element Five β-strands (β1-β5) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two α-helices (α1 and α5) on one and three helices (α2, α3, α4) on the other side (Fig 2). RESULTS +189 191 α4 structure_element Five β-strands (β1-β5) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two α-helices (α1 and α5) on one and three helices (α2, α3, α4) on the other side (Fig 2). RESULTS +4 8 NsrR protein The NsrR-RD structure shows a β1-α1-β2-α2-β3-α3-β4-α4-β5-α5 topology as also observed for other RRs. RESULTS +9 11 RD structure_element The NsrR-RD structure shows a β1-α1-β2-α2-β3-α3-β4-α4-β5-α5 topology as also observed for other RRs. RESULTS +12 21 structure evidence The NsrR-RD structure shows a β1-α1-β2-α2-β3-α3-β4-α4-β5-α5 topology as also observed for other RRs. RESULTS +30 59 β1-α1-β2-α2-β3-α3-β4-α4-β5-α5 structure_element The NsrR-RD structure shows a β1-α1-β2-α2-β3-α3-β4-α4-β5-α5 topology as also observed for other RRs. RESULTS +96 99 RRs protein_type The NsrR-RD structure shows a β1-α1-β2-α2-β3-α3-β4-α4-β5-α5 topology as also observed for other RRs. RESULTS +0 9 Structure evidence Structure of NsrR-RD. FIG +13 17 NsrR protein Structure of NsrR-RD. FIG +18 20 RD structure_element Structure of NsrR-RD. FIG +30 37 helices structure_element Cartoon representation of the helices (α1 – α5) and β-sheets (β1 - β5). FIG +39 46 α1 – α5 structure_element Cartoon representation of the helices (α1 – α5) and β-sheets (β1 - β5). FIG +52 60 β-sheets structure_element Cartoon representation of the helices (α1 – α5) and β-sheets (β1 - β5). FIG +62 69 β1 - β5 structure_element Cartoon representation of the helices (α1 – α5) and β-sheets (β1 - β5). FIG +52 68 receiver domains structure_element Structural areas with the highest variations to the receiver domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1B00) are marked in separate boxes. FIG +72 76 DrrB protein Structural areas with the highest variations to the receiver domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1B00) are marked in separate boxes. FIG +91 95 MtrA protein Structural areas with the highest variations to the receiver domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1B00) are marked in separate boxes. FIG +114 118 PhoB protein Structural areas with the highest variations to the receiver domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1B00) are marked in separate boxes. FIG +0 10 Comparison experimental_method Comparison with structures of other receiver domains RESULTS +16 26 structures evidence Comparison with structures of other receiver domains RESULTS +36 52 receiver domains structure_element Comparison with structures of other receiver domains RESULTS +0 4 NsrR protein NsrR belongs to the OmpR/PhoB family of RRs. RESULTS +20 36 OmpR/PhoB family protein_type NsrR belongs to the OmpR/PhoB family of RRs. RESULTS +40 43 RRs protein_type NsrR belongs to the OmpR/PhoB family of RRs. RESULTS +4 19 receiver domain structure_element The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +23 27 NsrR protein The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +32 44 superimposed experimental_method The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +83 99 receiver domains structure_element The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +109 125 OmpR/PhoB family protein_type The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +135 139 DrrB protein The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +141 145 KdpE protein The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +147 151 MtrA protein The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +161 178 crystal structure evidence The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +191 206 receiver domain structure_element The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +210 214 PhoB protein The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +220 224 rmsd evidence The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +232 240 overlays experimental_method The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2. RESULTS +0 15 Superimposition experimental_method Superimposition of the structures revealed that helix α4 is slightly rotated outward in NsrR-RD (Fig 2). RESULTS +23 33 structures evidence Superimposition of the structures revealed that helix α4 is slightly rotated outward in NsrR-RD (Fig 2). RESULTS +48 53 helix structure_element Superimposition of the structures revealed that helix α4 is slightly rotated outward in NsrR-RD (Fig 2). RESULTS +54 56 α4 structure_element Superimposition of the structures revealed that helix α4 is slightly rotated outward in NsrR-RD (Fig 2). RESULTS +88 92 NsrR protein Superimposition of the structures revealed that helix α4 is slightly rotated outward in NsrR-RD (Fig 2). RESULTS +93 95 RD structure_element Superimposition of the structures revealed that helix α4 is slightly rotated outward in NsrR-RD (Fig 2). RESULTS +3 19 receiver domains structure_element In receiver domains of response regulators, helix α4 has been shown to be a crucial part of the dimerization interface. RESULTS +23 42 response regulators protein_type In receiver domains of response regulators, helix α4 has been shown to be a crucial part of the dimerization interface. RESULTS +44 49 helix structure_element In receiver domains of response regulators, helix α4 has been shown to be a crucial part of the dimerization interface. RESULTS +50 52 α4 structure_element In receiver domains of response regulators, helix α4 has been shown to be a crucial part of the dimerization interface. RESULTS +96 118 dimerization interface site In receiver domains of response regulators, helix α4 has been shown to be a crucial part of the dimerization interface. RESULTS +13 18 helix structure_element Furthermore, helix α4 in NsrR is shorter than in other RRs. RESULTS +19 21 α4 structure_element Furthermore, helix α4 in NsrR is shorter than in other RRs. RESULTS +25 29 NsrR protein Furthermore, helix α4 in NsrR is shorter than in other RRs. RESULTS +55 58 RRs protein_type Furthermore, helix α4 in NsrR is shorter than in other RRs. RESULTS +4 22 first helical turn structure_element The first helical turn is unwound and adopts an unstructured region (see Fig 2). RESULTS +26 33 unwound protein_state The first helical turn is unwound and adopts an unstructured region (see Fig 2). RESULTS +48 60 unstructured protein_state The first helical turn is unwound and adopts an unstructured region (see Fig 2). RESULTS +44 49 helix structure_element A slightly outward rotation or unwinding of helix α4 has been observed in the structures of other RD of regulators. RESULTS +50 52 α4 structure_element A slightly outward rotation or unwinding of helix α4 has been observed in the structures of other RD of regulators. RESULTS +78 88 structures evidence A slightly outward rotation or unwinding of helix α4 has been observed in the structures of other RD of regulators. RESULTS +98 100 RD structure_element A slightly outward rotation or unwinding of helix α4 has been observed in the structures of other RD of regulators. RESULTS +17 26 structure evidence For example, the structure of BaeR and RegX3 displayed a completely unwound helix α4. RESULTS +30 34 BaeR protein For example, the structure of BaeR and RegX3 displayed a completely unwound helix α4. RESULTS +39 44 RegX3 protein For example, the structure of BaeR and RegX3 displayed a completely unwound helix α4. RESULTS +68 75 unwound protein_state For example, the structure of BaeR and RegX3 displayed a completely unwound helix α4. RESULTS +76 81 helix structure_element For example, the structure of BaeR and RegX3 displayed a completely unwound helix α4. RESULTS +82 84 α4 structure_element For example, the structure of BaeR and RegX3 displayed a completely unwound helix α4. RESULTS +7 16 structure evidence In the structure of DrrD, helix α4 is only partially displaced. RESULTS +20 24 DrrD protein In the structure of DrrD, helix α4 is only partially displaced. RESULTS +26 31 helix structure_element In the structure of DrrD, helix α4 is only partially displaced. RESULTS +32 34 α4 structure_element In the structure of DrrD, helix α4 is only partially displaced. RESULTS +7 22 receiver domain structure_element In the receiver domain of NsrR, helix α4 is also partially displaced but in a different direction (S1 Fig). RESULTS +26 30 NsrR protein In the receiver domain of NsrR, helix α4 is also partially displaced but in a different direction (S1 Fig). RESULTS +32 37 helix structure_element In the receiver domain of NsrR, helix α4 is also partially displaced but in a different direction (S1 Fig). RESULTS +38 40 α4 structure_element In the receiver domain of NsrR, helix α4 is also partially displaced but in a different direction (S1 Fig). RESULTS +127 132 helix structure_element Inspection of the crystal contacts revealed no major interactions in this region that could have influenced the orientation of helix α4. RESULTS +133 135 α4 structure_element Inspection of the crystal contacts revealed no major interactions in this region that could have influenced the orientation of helix α4. RESULTS +13 17 NsrR protein Furthermore, NsrR is crystallized as a monomer, and investigation of the symmetry-related molecules did not reveal a functional dimer within the crystal. RESULTS +21 33 crystallized experimental_method Furthermore, NsrR is crystallized as a monomer, and investigation of the symmetry-related molecules did not reveal a functional dimer within the crystal. RESULTS +39 46 monomer oligomeric_state Furthermore, NsrR is crystallized as a monomer, and investigation of the symmetry-related molecules did not reveal a functional dimer within the crystal. RESULTS +128 133 dimer oligomeric_state Furthermore, NsrR is crystallized as a monomer, and investigation of the symmetry-related molecules did not reveal a functional dimer within the crystal. RESULTS +145 152 crystal evidence Furthermore, NsrR is crystallized as a monomer, and investigation of the symmetry-related molecules did not reveal a functional dimer within the crystal. RESULTS +76 81 helix structure_element This could explain the flexibility and thereby the different orientation of helix α4 in NsrR. RESULTS +82 84 α4 structure_element This could explain the flexibility and thereby the different orientation of helix α4 in NsrR. RESULTS +88 92 NsrR protein This could explain the flexibility and thereby the different orientation of helix α4 in NsrR. RESULTS +4 14 structures evidence The structures of the RD and ED domains of NsrR aligned to other response regulators. TABLE +22 24 RD structure_element The structures of the RD and ED domains of NsrR aligned to other response regulators. TABLE +29 31 ED structure_element The structures of the RD and ED domains of NsrR aligned to other response regulators. TABLE +43 47 NsrR protein The structures of the RD and ED domains of NsrR aligned to other response regulators. TABLE +48 55 aligned experimental_method The structures of the RD and ED domains of NsrR aligned to other response regulators. TABLE +65 84 response regulators protein_type The structures of the RD and ED domains of NsrR aligned to other response regulators. TABLE +4 8 rmsd evidence The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence. TABLE +23 39 superimpositions experimental_method The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence. TABLE +47 57 structures evidence The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence. TABLE +61 65 NsrR protein The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence. TABLE +66 68 RD structure_element The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence. TABLE +73 77 NsrR protein The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence. TABLE +78 80 ED structure_element The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence. TABLE +100 110 structures evidence The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence. TABLE +129 148 OmpR/PhoB subfamily protein_type The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence. TABLE +197 208 full-length protein_state The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence. TABLE +13 24 Dali server experimental_method Based on the Dali server, the NsrR-RD domain is structurally closely related to KdpE (PDB code: 4KNY) from E. coli, displaying a sequence identity of 28%. RESULTS +30 34 NsrR protein Based on the Dali server, the NsrR-RD domain is structurally closely related to KdpE (PDB code: 4KNY) from E. coli, displaying a sequence identity of 28%. RESULTS +35 37 RD structure_element Based on the Dali server, the NsrR-RD domain is structurally closely related to KdpE (PDB code: 4KNY) from E. coli, displaying a sequence identity of 28%. RESULTS +80 84 KdpE protein Based on the Dali server, the NsrR-RD domain is structurally closely related to KdpE (PDB code: 4KNY) from E. coli, displaying a sequence identity of 28%. RESULTS +107 114 E. coli species Based on the Dali server, the NsrR-RD domain is structurally closely related to KdpE (PDB code: 4KNY) from E. coli, displaying a sequence identity of 28%. RESULTS +54 58 rmsd evidence This structural homology is also reflected by the low rmsd of 1.9 Å over 117 Cα atoms after superimposition of the receiver domains of NsrR and KdpE (Table 2). RESULTS +92 107 superimposition experimental_method This structural homology is also reflected by the low rmsd of 1.9 Å over 117 Cα atoms after superimposition of the receiver domains of NsrR and KdpE (Table 2). RESULTS +115 131 receiver domains structure_element This structural homology is also reflected by the low rmsd of 1.9 Å over 117 Cα atoms after superimposition of the receiver domains of NsrR and KdpE (Table 2). RESULTS +135 139 NsrR protein This structural homology is also reflected by the low rmsd of 1.9 Å over 117 Cα atoms after superimposition of the receiver domains of NsrR and KdpE (Table 2). RESULTS +144 148 KdpE protein This structural homology is also reflected by the low rmsd of 1.9 Å over 117 Cα atoms after superimposition of the receiver domains of NsrR and KdpE (Table 2). RESULTS +36 41 helix structure_element Furthermore, the orientation of the helix α4 in NsrR is close to that present in KdpE (S1 Fig). RESULTS +42 44 α4 structure_element Furthermore, the orientation of the helix α4 in NsrR is close to that present in KdpE (S1 Fig). RESULTS +48 52 NsrR protein Furthermore, the orientation of the helix α4 in NsrR is close to that present in KdpE (S1 Fig). RESULTS +81 85 KdpE protein Furthermore, the orientation of the helix α4 in NsrR is close to that present in KdpE (S1 Fig). RESULTS +0 11 Active site site Active site residues and dimerization RESULTS +4 7 RRs protein_type All RRs contain a highly conserved aspartate residue in the active site (Fig 3; shown in red). RESULTS +18 34 highly conserved protein_state All RRs contain a highly conserved aspartate residue in the active site (Fig 3; shown in red). RESULTS +35 44 aspartate residue_name All RRs contain a highly conserved aspartate residue in the active site (Fig 3; shown in red). RESULTS +60 71 active site site All RRs contain a highly conserved aspartate residue in the active site (Fig 3; shown in red). RESULTS +0 15 Phosphorylation ptm Phosphorylation of this aspartate residue induces a conformational change leading to the activation of the effector domain that binds DNA and regulates the transcription of target genes. RESULTS +24 33 aspartate residue_name Phosphorylation of this aspartate residue induces a conformational change leading to the activation of the effector domain that binds DNA and regulates the transcription of target genes. RESULTS +107 122 effector domain structure_element Phosphorylation of this aspartate residue induces a conformational change leading to the activation of the effector domain that binds DNA and regulates the transcription of target genes. RESULTS +134 137 DNA chemical Phosphorylation of this aspartate residue induces a conformational change leading to the activation of the effector domain that binds DNA and regulates the transcription of target genes. RESULTS +13 28 phosphorylation ptm This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +32 41 conserved protein_state This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +67 86 response regulators protein_type This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +102 139 lantibiotic resistance-associated RRs protein_type This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +148 152 BraR protein This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +158 174 L. monocytogenes species This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +176 180 BceR protein This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +186 203 Bacillus subtilis species This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +205 209 CprR protein This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +215 227 C. difficile species This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +229 233 GraR protein This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +239 248 S. aureus species This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +250 254 LcrR protein This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +260 269 S. mutans species This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +271 275 LisR protein This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +281 285 VirR protein This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +291 307 L. monocytogenes species This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3). RESULTS +0 18 Sequence alignment experimental_method Sequence alignment of NsrR protein with other response regulators. FIG +22 26 NsrR protein Sequence alignment of NsrR protein with other response regulators. FIG +46 65 response regulators protein_type Sequence alignment of NsrR protein with other response regulators. FIG +2 20 sequence alignment experimental_method A sequence alignment of NsrR with RRs belonging to the OmpR/PhoB subfamily (marked in grey) and RRs involved in lantibiotic resistance (black) is shown. FIG +24 28 NsrR protein A sequence alignment of NsrR with RRs belonging to the OmpR/PhoB subfamily (marked in grey) and RRs involved in lantibiotic resistance (black) is shown. FIG +34 37 RRs protein_type A sequence alignment of NsrR with RRs belonging to the OmpR/PhoB subfamily (marked in grey) and RRs involved in lantibiotic resistance (black) is shown. FIG +55 74 OmpR/PhoB subfamily protein_type A sequence alignment of NsrR with RRs belonging to the OmpR/PhoB subfamily (marked in grey) and RRs involved in lantibiotic resistance (black) is shown. FIG +96 99 RRs protein_type A sequence alignment of NsrR with RRs belonging to the OmpR/PhoB subfamily (marked in grey) and RRs involved in lantibiotic resistance (black) is shown. FIG +112 123 lantibiotic chemical A sequence alignment of NsrR with RRs belonging to the OmpR/PhoB subfamily (marked in grey) and RRs involved in lantibiotic resistance (black) is shown. FIG +4 15 active site site The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. FIG +16 25 aspartate residue_name The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. FIG +81 94 acidic pocket site The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. FIG +137 152 switch residues site The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. FIG +180 189 conserved protein_state The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. FIG +190 196 lysine residue_name The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. FIG +233 249 highly conserved protein_state The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. FIG +266 279 linker region structure_element The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. FIG +326 341 dimer interface site The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. FIG +345 360 receiver domain structure_element The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. FIG +514 517 DNA chemical The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown. FIG +4 17 linker region structure_element The linker region of the known structures is underlined within the sequence. FIG +31 41 structures evidence The linker region of the known structures is underlined within the sequence. FIG +13 33 phosphorylation site site The putative phosphorylation site of NsrR is Asp55, which is localized at the end of strand β3 (Fig 3, shown in red; Fig 4) and lies within an acidic environment composed of the side chains of Glu12 and Asp13 (Fig 3, highlighted in pink). RESULTS +37 41 NsrR protein The putative phosphorylation site of NsrR is Asp55, which is localized at the end of strand β3 (Fig 3, shown in red; Fig 4) and lies within an acidic environment composed of the side chains of Glu12 and Asp13 (Fig 3, highlighted in pink). RESULTS +45 50 Asp55 residue_name_number The putative phosphorylation site of NsrR is Asp55, which is localized at the end of strand β3 (Fig 3, shown in red; Fig 4) and lies within an acidic environment composed of the side chains of Glu12 and Asp13 (Fig 3, highlighted in pink). RESULTS +85 91 strand structure_element The putative phosphorylation site of NsrR is Asp55, which is localized at the end of strand β3 (Fig 3, shown in red; Fig 4) and lies within an acidic environment composed of the side chains of Glu12 and Asp13 (Fig 3, highlighted in pink). RESULTS +92 94 β3 structure_element The putative phosphorylation site of NsrR is Asp55, which is localized at the end of strand β3 (Fig 3, shown in red; Fig 4) and lies within an acidic environment composed of the side chains of Glu12 and Asp13 (Fig 3, highlighted in pink). RESULTS +193 198 Glu12 residue_name_number The putative phosphorylation site of NsrR is Asp55, which is localized at the end of strand β3 (Fig 3, shown in red; Fig 4) and lies within an acidic environment composed of the side chains of Glu12 and Asp13 (Fig 3, highlighted in pink). RESULTS +203 208 Asp13 residue_name_number The putative phosphorylation site of NsrR is Asp55, which is localized at the end of strand β3 (Fig 3, shown in red; Fig 4) and lies within an acidic environment composed of the side chains of Glu12 and Asp13 (Fig 3, highlighted in pink). RESULTS +5 11 pocket site This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. RESULTS +30 36 acidic protein_state This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. RESULTS +37 48 active site site This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. RESULTS +70 80 structures evidence This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. RESULTS +84 87 RRs protein_type This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. RESULTS +96 100 PhoB protein This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. RESULTS +106 113 E. coli species This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. RESULTS +115 119 PhoP protein This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. RESULTS +125 140 M. tuberculosis species This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. RESULTS +146 150 DivK protein This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. RESULTS +156 178 Caulobacter crescentus species This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus. RESULTS +3 7 NsrR protein In NsrR, Glu12, Asp13, and Asp55 are in close proximity of a highly conserved Lys104 residue (highlighted in green in Fig 3). RESULTS +9 14 Glu12 residue_name_number In NsrR, Glu12, Asp13, and Asp55 are in close proximity of a highly conserved Lys104 residue (highlighted in green in Fig 3). RESULTS +16 21 Asp13 residue_name_number In NsrR, Glu12, Asp13, and Asp55 are in close proximity of a highly conserved Lys104 residue (highlighted in green in Fig 3). RESULTS +27 32 Asp55 residue_name_number In NsrR, Glu12, Asp13, and Asp55 are in close proximity of a highly conserved Lys104 residue (highlighted in green in Fig 3). RESULTS +61 77 highly conserved protein_state In NsrR, Glu12, Asp13, and Asp55 are in close proximity of a highly conserved Lys104 residue (highlighted in green in Fig 3). RESULTS +78 84 Lys104 residue_name_number In NsrR, Glu12, Asp13, and Asp55 are in close proximity of a highly conserved Lys104 residue (highlighted in green in Fig 3). RESULTS +16 32 highly conserved protein_state Location of the highly conserved Asp55 and inactive state conformation of the key switch residues, Ser82 and Phe101 in NsrR-RD. FIG +33 38 Asp55 residue_name_number Location of the highly conserved Asp55 and inactive state conformation of the key switch residues, Ser82 and Phe101 in NsrR-RD. FIG +43 51 inactive protein_state Location of the highly conserved Asp55 and inactive state conformation of the key switch residues, Ser82 and Phe101 in NsrR-RD. FIG +82 97 switch residues site Location of the highly conserved Asp55 and inactive state conformation of the key switch residues, Ser82 and Phe101 in NsrR-RD. FIG +99 104 Ser82 residue_name_number Location of the highly conserved Asp55 and inactive state conformation of the key switch residues, Ser82 and Phe101 in NsrR-RD. FIG +109 115 Phe101 residue_name_number Location of the highly conserved Asp55 and inactive state conformation of the key switch residues, Ser82 and Phe101 in NsrR-RD. FIG +119 123 NsrR protein Location of the highly conserved Asp55 and inactive state conformation of the key switch residues, Ser82 and Phe101 in NsrR-RD. FIG +124 126 RD structure_element Location of the highly conserved Asp55 and inactive state conformation of the key switch residues, Ser82 and Phe101 in NsrR-RD. FIG +0 4 NsrR protein NsrR (represented in yellow) displays a geometry representing the inactive state as deduced from the inactive state structure of PhoB (shown in brown, PDB code 1B00) (a). FIG +66 74 inactive protein_state NsrR (represented in yellow) displays a geometry representing the inactive state as deduced from the inactive state structure of PhoB (shown in brown, PDB code 1B00) (a). FIG +101 109 inactive protein_state NsrR (represented in yellow) displays a geometry representing the inactive state as deduced from the inactive state structure of PhoB (shown in brown, PDB code 1B00) (a). FIG +116 125 structure evidence NsrR (represented in yellow) displays a geometry representing the inactive state as deduced from the inactive state structure of PhoB (shown in brown, PDB code 1B00) (a). FIG +129 133 PhoB protein NsrR (represented in yellow) displays a geometry representing the inactive state as deduced from the inactive state structure of PhoB (shown in brown, PDB code 1B00) (a). FIG +4 12 inactive protein_state The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). FIG +29 33 NsrR protein The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). FIG +51 57 active protein_state The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). FIG +64 73 structure evidence The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). FIG +77 81 PhoB protein The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). FIG +154 169 switch residues site The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). FIG +171 176 Ser82 residue_name_number The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). FIG +181 187 Phe101 residue_name_number The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). FIG +239 250 active site site The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). FIG +252 257 Asp55 residue_name_number The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). FIG +261 265 NsrR protein The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR). FIG +86 101 phosphorylation ptm A divalent metal ion is usually bound in this acidic environment and is essential for phosphorylation and de-phosphorylation of RRs. RESULTS +106 124 de-phosphorylation ptm A divalent metal ion is usually bound in this acidic environment and is essential for phosphorylation and de-phosphorylation of RRs. RESULTS +128 131 RRs protein_type A divalent metal ion is usually bound in this acidic environment and is essential for phosphorylation and de-phosphorylation of RRs. RESULTS +8 11 RRs protein_type In some RRs like CheY, Mg2+ is observed in the structure, bound near the phosphorylation site. RESULTS +17 21 CheY protein In some RRs like CheY, Mg2+ is observed in the structure, bound near the phosphorylation site. RESULTS +23 27 Mg2+ chemical In some RRs like CheY, Mg2+ is observed in the structure, bound near the phosphorylation site. RESULTS +47 56 structure evidence In some RRs like CheY, Mg2+ is observed in the structure, bound near the phosphorylation site. RESULTS +58 63 bound protein_state In some RRs like CheY, Mg2+ is observed in the structure, bound near the phosphorylation site. RESULTS +73 93 phosphorylation site site In some RRs like CheY, Mg2+ is observed in the structure, bound near the phosphorylation site. RESULTS +7 11 KdpE protein In the KdpE regulator from E. coli that is involved in osmoregulation, a divalent calcium ion is present. RESULTS +12 21 regulator protein_type In the KdpE regulator from E. coli that is involved in osmoregulation, a divalent calcium ion is present. RESULTS +27 34 E. coli species In the KdpE regulator from E. coli that is involved in osmoregulation, a divalent calcium ion is present. RESULTS +82 89 calcium chemical In the KdpE regulator from E. coli that is involved in osmoregulation, a divalent calcium ion is present. RESULTS +13 22 structure evidence However, the structure of NsrR-RD did not contain any divalent ion. RESULTS +26 30 NsrR protein However, the structure of NsrR-RD did not contain any divalent ion. RESULTS +31 33 RD structure_element However, the structure of NsrR-RD did not contain any divalent ion. RESULTS +11 16 water chemical Instead, a water molecule is present, which interacts with Glu12 of the acidic pocket, Lys104, and another water molecule in the vicinity. RESULTS +59 64 Glu12 residue_name_number Instead, a water molecule is present, which interacts with Glu12 of the acidic pocket, Lys104, and another water molecule in the vicinity. RESULTS +72 85 acidic pocket site Instead, a water molecule is present, which interacts with Glu12 of the acidic pocket, Lys104, and another water molecule in the vicinity. RESULTS +87 93 Lys104 residue_name_number Instead, a water molecule is present, which interacts with Glu12 of the acidic pocket, Lys104, and another water molecule in the vicinity. RESULTS +107 112 water chemical Instead, a water molecule is present, which interacts with Glu12 of the acidic pocket, Lys104, and another water molecule in the vicinity. RESULTS +11 21 β4-α4 loop structure_element Within the β4-α4 loop and in β5 of the RD of RRs, specific amino acids are crucial for signal transduction from the RD to the ED via conformational changes that are a consequence of phosphorylation of the RD. RESULTS +29 31 β5 structure_element Within the β4-α4 loop and in β5 of the RD of RRs, specific amino acids are crucial for signal transduction from the RD to the ED via conformational changes that are a consequence of phosphorylation of the RD. RESULTS +39 41 RD structure_element Within the β4-α4 loop and in β5 of the RD of RRs, specific amino acids are crucial for signal transduction from the RD to the ED via conformational changes that are a consequence of phosphorylation of the RD. RESULTS +45 48 RRs protein_type Within the β4-α4 loop and in β5 of the RD of RRs, specific amino acids are crucial for signal transduction from the RD to the ED via conformational changes that are a consequence of phosphorylation of the RD. RESULTS +116 118 RD structure_element Within the β4-α4 loop and in β5 of the RD of RRs, specific amino acids are crucial for signal transduction from the RD to the ED via conformational changes that are a consequence of phosphorylation of the RD. RESULTS +126 128 ED structure_element Within the β4-α4 loop and in β5 of the RD of RRs, specific amino acids are crucial for signal transduction from the RD to the ED via conformational changes that are a consequence of phosphorylation of the RD. RESULTS +182 197 phosphorylation ptm Within the β4-α4 loop and in β5 of the RD of RRs, specific amino acids are crucial for signal transduction from the RD to the ED via conformational changes that are a consequence of phosphorylation of the RD. RESULTS +205 207 RD structure_element Within the β4-α4 loop and in β5 of the RD of RRs, specific amino acids are crucial for signal transduction from the RD to the ED via conformational changes that are a consequence of phosphorylation of the RD. RESULTS +22 25 Ser residue_name These amino acids are Ser/Thr and Phe/Tyr located at the end of β4 and before β5, respectively, and designated as “signature switch residues”. RESULTS +26 29 Thr residue_name These amino acids are Ser/Thr and Phe/Tyr located at the end of β4 and before β5, respectively, and designated as “signature switch residues”. RESULTS +34 37 Phe residue_name These amino acids are Ser/Thr and Phe/Tyr located at the end of β4 and before β5, respectively, and designated as “signature switch residues”. RESULTS +38 41 Tyr residue_name These amino acids are Ser/Thr and Phe/Tyr located at the end of β4 and before β5, respectively, and designated as “signature switch residues”. RESULTS +64 66 β4 structure_element These amino acids are Ser/Thr and Phe/Tyr located at the end of β4 and before β5, respectively, and designated as “signature switch residues”. RESULTS +78 80 β5 structure_element These amino acids are Ser/Thr and Phe/Tyr located at the end of β4 and before β5, respectively, and designated as “signature switch residues”. RESULTS +115 140 signature switch residues site These amino acids are Ser/Thr and Phe/Tyr located at the end of β4 and before β5, respectively, and designated as “signature switch residues”. RESULTS +15 24 alignment experimental_method As seen in the alignment (Fig 3, highlighted in blue), these signature residues (Ser/Thr and Phe/Tyr) are highly conserved in the lantibiotic resistance-associated RRs. RESULTS +81 84 Ser residue_name As seen in the alignment (Fig 3, highlighted in blue), these signature residues (Ser/Thr and Phe/Tyr) are highly conserved in the lantibiotic resistance-associated RRs. RESULTS +85 88 Thr residue_name As seen in the alignment (Fig 3, highlighted in blue), these signature residues (Ser/Thr and Phe/Tyr) are highly conserved in the lantibiotic resistance-associated RRs. RESULTS +93 96 Phe residue_name As seen in the alignment (Fig 3, highlighted in blue), these signature residues (Ser/Thr and Phe/Tyr) are highly conserved in the lantibiotic resistance-associated RRs. RESULTS +97 100 Tyr residue_name As seen in the alignment (Fig 3, highlighted in blue), these signature residues (Ser/Thr and Phe/Tyr) are highly conserved in the lantibiotic resistance-associated RRs. RESULTS +106 122 highly conserved protein_state As seen in the alignment (Fig 3, highlighted in blue), these signature residues (Ser/Thr and Phe/Tyr) are highly conserved in the lantibiotic resistance-associated RRs. RESULTS +130 167 lantibiotic resistance-associated RRs protein_type As seen in the alignment (Fig 3, highlighted in blue), these signature residues (Ser/Thr and Phe/Tyr) are highly conserved in the lantibiotic resistance-associated RRs. RESULTS +76 78 RD structure_element The orientation of the side chains of these residues determines whether the RD is in an active or inactive state. RESULTS +88 94 active protein_state The orientation of the side chains of these residues determines whether the RD is in an active or inactive state. RESULTS +98 106 inactive protein_state The orientation of the side chains of these residues determines whether the RD is in an active or inactive state. RESULTS +7 15 inactive protein_state In the inactive state, the phenylalanine or tyrosine residue faces away from the active site, and the corresponding serine or threonine residue adopts an outward-facing conformation as well (Fig 4A). RESULTS +27 40 phenylalanine residue_name In the inactive state, the phenylalanine or tyrosine residue faces away from the active site, and the corresponding serine or threonine residue adopts an outward-facing conformation as well (Fig 4A). RESULTS +44 52 tyrosine residue_name In the inactive state, the phenylalanine or tyrosine residue faces away from the active site, and the corresponding serine or threonine residue adopts an outward-facing conformation as well (Fig 4A). RESULTS +81 92 active site site In the inactive state, the phenylalanine or tyrosine residue faces away from the active site, and the corresponding serine or threonine residue adopts an outward-facing conformation as well (Fig 4A). RESULTS +116 122 serine residue_name In the inactive state, the phenylalanine or tyrosine residue faces away from the active site, and the corresponding serine or threonine residue adopts an outward-facing conformation as well (Fig 4A). RESULTS +126 135 threonine residue_name In the inactive state, the phenylalanine or tyrosine residue faces away from the active site, and the corresponding serine or threonine residue adopts an outward-facing conformation as well (Fig 4A). RESULTS +154 168 outward-facing protein_state In the inactive state, the phenylalanine or tyrosine residue faces away from the active site, and the corresponding serine or threonine residue adopts an outward-facing conformation as well (Fig 4A). RESULTS +17 32 switch residues site In contrast, the switch residues face towards the active site in the active state conformation (Fig 4B). RESULTS +50 61 active site site In contrast, the switch residues face towards the active site in the active state conformation (Fig 4B). RESULTS +69 75 active protein_state In contrast, the switch residues face towards the active site in the active state conformation (Fig 4B). RESULTS +3 21 sequence alignment experimental_method By sequence alignment with other lantibiotic resistance-associated RRs, these “signature switch residues” are identified as Ser82 and Phe101 in NsrR (see above). RESULTS +33 70 lantibiotic resistance-associated RRs protein_type By sequence alignment with other lantibiotic resistance-associated RRs, these “signature switch residues” are identified as Ser82 and Phe101 in NsrR (see above). RESULTS +79 104 signature switch residues site By sequence alignment with other lantibiotic resistance-associated RRs, these “signature switch residues” are identified as Ser82 and Phe101 in NsrR (see above). RESULTS +124 129 Ser82 residue_name_number By sequence alignment with other lantibiotic resistance-associated RRs, these “signature switch residues” are identified as Ser82 and Phe101 in NsrR (see above). RESULTS +134 140 Phe101 residue_name_number By sequence alignment with other lantibiotic resistance-associated RRs, these “signature switch residues” are identified as Ser82 and Phe101 in NsrR (see above). RESULTS +144 148 NsrR protein By sequence alignment with other lantibiotic resistance-associated RRs, these “signature switch residues” are identified as Ser82 and Phe101 in NsrR (see above). RESULTS +14 17 RRs protein_type Although some RRs such as KdpE, BraR, BceR, GraR, and VirR contain a serine residue as the first switch residue, the others possess a threonine instead. RESULTS +26 30 KdpE protein Although some RRs such as KdpE, BraR, BceR, GraR, and VirR contain a serine residue as the first switch residue, the others possess a threonine instead. RESULTS +32 36 BraR protein Although some RRs such as KdpE, BraR, BceR, GraR, and VirR contain a serine residue as the first switch residue, the others possess a threonine instead. RESULTS +38 42 BceR protein Although some RRs such as KdpE, BraR, BceR, GraR, and VirR contain a serine residue as the first switch residue, the others possess a threonine instead. RESULTS +44 48 GraR protein Although some RRs such as KdpE, BraR, BceR, GraR, and VirR contain a serine residue as the first switch residue, the others possess a threonine instead. RESULTS +54 58 VirR protein Although some RRs such as KdpE, BraR, BceR, GraR, and VirR contain a serine residue as the first switch residue, the others possess a threonine instead. RESULTS +69 75 serine residue_name Although some RRs such as KdpE, BraR, BceR, GraR, and VirR contain a serine residue as the first switch residue, the others possess a threonine instead. RESULTS +91 111 first switch residue site Although some RRs such as KdpE, BraR, BceR, GraR, and VirR contain a serine residue as the first switch residue, the others possess a threonine instead. RESULTS +134 143 threonine residue_name Although some RRs such as KdpE, BraR, BceR, GraR, and VirR contain a serine residue as the first switch residue, the others possess a threonine instead. RESULTS +17 38 second switch residue site Furthermore, the second switch residue is mostly a tyrosine, with NsrR, BraR, and BceR being the only exceptions containing a phenylalanine at that position. RESULTS +51 59 tyrosine residue_name Furthermore, the second switch residue is mostly a tyrosine, with NsrR, BraR, and BceR being the only exceptions containing a phenylalanine at that position. RESULTS +66 70 NsrR protein Furthermore, the second switch residue is mostly a tyrosine, with NsrR, BraR, and BceR being the only exceptions containing a phenylalanine at that position. RESULTS +72 76 BraR protein Furthermore, the second switch residue is mostly a tyrosine, with NsrR, BraR, and BceR being the only exceptions containing a phenylalanine at that position. RESULTS +82 86 BceR protein Furthermore, the second switch residue is mostly a tyrosine, with NsrR, BraR, and BceR being the only exceptions containing a phenylalanine at that position. RESULTS +126 139 phenylalanine residue_name Furthermore, the second switch residue is mostly a tyrosine, with NsrR, BraR, and BceR being the only exceptions containing a phenylalanine at that position. RESULTS +20 24 NsrR protein A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +25 27 RD structure_element A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +28 37 structure evidence A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +57 67 structures evidence A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +71 75 PhoB protein A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +91 97 active protein_state A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +119 127 inactive protein_state A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +170 175 Ser82 residue_name_number A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +177 181 NsrR protein A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +182 184 RD structure_element A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +212 223 active site site A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +224 229 Asp55 residue_name_number A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +240 246 Phe101 residue_name_number A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +261 268 outward protein_state A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +296 304 inactive protein_state A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +318 322 NsrR protein A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +323 325 RD structure_element A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A). RESULTS +20 23 RRs protein_type As mentioned above, RRs contain a phosphorylation-activated switch and normally exist in equilibrium between the active and inactive conformations. RESULTS +34 59 phosphorylation-activated protein_state As mentioned above, RRs contain a phosphorylation-activated switch and normally exist in equilibrium between the active and inactive conformations. RESULTS +60 66 switch site As mentioned above, RRs contain a phosphorylation-activated switch and normally exist in equilibrium between the active and inactive conformations. RESULTS +113 119 active protein_state As mentioned above, RRs contain a phosphorylation-activated switch and normally exist in equilibrium between the active and inactive conformations. RESULTS +124 132 inactive protein_state As mentioned above, RRs contain a phosphorylation-activated switch and normally exist in equilibrium between the active and inactive conformations. RESULTS +0 15 Phosphorylation ptm Phosphorylation shifts the equilibrium towards the active conformation and induces the formation of rotationally symmetric dimers on the α4-β5-α5 interface of RDs. RESULTS +51 57 active protein_state Phosphorylation shifts the equilibrium towards the active conformation and induces the formation of rotationally symmetric dimers on the α4-β5-α5 interface of RDs. RESULTS +123 129 dimers oligomeric_state Phosphorylation shifts the equilibrium towards the active conformation and induces the formation of rotationally symmetric dimers on the α4-β5-α5 interface of RDs. RESULTS +137 155 α4-β5-α5 interface site Phosphorylation shifts the equilibrium towards the active conformation and induces the formation of rotationally symmetric dimers on the α4-β5-α5 interface of RDs. RESULTS +159 162 RDs structure_element Phosphorylation shifts the equilibrium towards the active conformation and induces the formation of rotationally symmetric dimers on the α4-β5-α5 interface of RDs. RESULTS +55 58 DNA chemical It has been suggested that dimerization is crucial for DNA-binding of RRs of the OmpR/PhoB subfamily. RESULTS +70 73 RRs protein_type It has been suggested that dimerization is crucial for DNA-binding of RRs of the OmpR/PhoB subfamily. RESULTS +81 100 OmpR/PhoB subfamily protein_type It has been suggested that dimerization is crucial for DNA-binding of RRs of the OmpR/PhoB subfamily. RESULTS +4 6 RD structure_element The RD domain of NsrR was crystallized with two separate monomers in the asymmetric unit. RESULTS +17 21 NsrR protein The RD domain of NsrR was crystallized with two separate monomers in the asymmetric unit. RESULTS +26 38 crystallized experimental_method The RD domain of NsrR was crystallized with two separate monomers in the asymmetric unit. RESULTS +57 65 monomers oligomeric_state The RD domain of NsrR was crystallized with two separate monomers in the asymmetric unit. RESULTS +26 37 DALI search experimental_method Therefore, we performed a DALI search and focused on RD domains that were structurally determined as functional dimers. RESULTS +53 55 RD structure_element Therefore, we performed a DALI search and focused on RD domains that were structurally determined as functional dimers. RESULTS +101 111 functional protein_state Therefore, we performed a DALI search and focused on RD domains that were structurally determined as functional dimers. RESULTS +112 118 dimers oligomeric_state Therefore, we performed a DALI search and focused on RD domains that were structurally determined as functional dimers. RESULTS +21 26 dimer oligomeric_state In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +30 41 full-length protein_state In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +42 46 KdpE protein In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +52 59 E. coli species In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +61 68 Z-score evidence In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +75 79 rmsd evidence In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +130 139 structure evidence In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +147 157 functional protein_state In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +158 163 dimer oligomeric_state In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +171 173 RD structure_element In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +177 181 KdpE protein In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +187 194 E. coli species In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +252 262 structures evidence In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 Å over 117 Cα atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures. RESULTS +3 10 aligned experimental_method We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +11 15 NsrR protein We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +16 18 RD structure_element We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +27 35 monomers oligomeric_state We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +43 45 RD structure_element We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +49 53 KdpE protein We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +61 66 helix structure_element We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +67 69 α4 structure_element We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +73 77 NsrR protein We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +78 80 RD structure_element We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +139 149 structures evidence We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +153 156 RDs structure_element We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +166 171 helix structure_element We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +172 174 α4 structure_element We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +194 198 loop structure_element We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +206 213 monomer oligomeric_state We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +244 251 monomer oligomeric_state We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix α4 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix α4 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig). RESULTS +11 16 helix structure_element Therefore, helix α4 and the N-terminal loop were shifted to the position of KdpE by primarily modifying backbone torsion angles in the region immediately C-terminal to helix α4. RESULTS +17 19 α4 structure_element Therefore, helix α4 and the N-terminal loop were shifted to the position of KdpE by primarily modifying backbone torsion angles in the region immediately C-terminal to helix α4. RESULTS +39 43 loop structure_element Therefore, helix α4 and the N-terminal loop were shifted to the position of KdpE by primarily modifying backbone torsion angles in the region immediately C-terminal to helix α4. RESULTS +76 80 KdpE protein Therefore, helix α4 and the N-terminal loop were shifted to the position of KdpE by primarily modifying backbone torsion angles in the region immediately C-terminal to helix α4. RESULTS +168 173 helix structure_element Therefore, helix α4 and the N-terminal loop were shifted to the position of KdpE by primarily modifying backbone torsion angles in the region immediately C-terminal to helix α4. RESULTS +174 176 α4 structure_element Therefore, helix α4 and the N-terminal loop were shifted to the position of KdpE by primarily modifying backbone torsion angles in the region immediately C-terminal to helix α4. RESULTS +12 17 helix structure_element Afterwards, helix α4 and the adjacent loops were energy minimized with the MAB force field as implemented in the program Moloc; all other atoms of NsrR-RD were kept fixed. RESULTS +18 20 α4 structure_element Afterwards, helix α4 and the adjacent loops were energy minimized with the MAB force field as implemented in the program Moloc; all other atoms of NsrR-RD were kept fixed. RESULTS +38 43 loops structure_element Afterwards, helix α4 and the adjacent loops were energy minimized with the MAB force field as implemented in the program Moloc; all other atoms of NsrR-RD were kept fixed. RESULTS +49 90 energy minimized with the MAB force field experimental_method Afterwards, helix α4 and the adjacent loops were energy minimized with the MAB force field as implemented in the program Moloc; all other atoms of NsrR-RD were kept fixed. RESULTS +147 151 NsrR protein Afterwards, helix α4 and the adjacent loops were energy minimized with the MAB force field as implemented in the program Moloc; all other atoms of NsrR-RD were kept fixed. RESULTS +152 154 RD structure_element Afterwards, helix α4 and the adjacent loops were energy minimized with the MAB force field as implemented in the program Moloc; all other atoms of NsrR-RD were kept fixed. RESULTS +42 58 energy minimized protein_state The result is highlighted in S2B Fig. The energy minimized structure of NsrR-RD was then superimposed on the dimeric structure of KdpE. RESULTS +59 68 structure evidence The result is highlighted in S2B Fig. The energy minimized structure of NsrR-RD was then superimposed on the dimeric structure of KdpE. RESULTS +72 76 NsrR protein The result is highlighted in S2B Fig. The energy minimized structure of NsrR-RD was then superimposed on the dimeric structure of KdpE. RESULTS +77 79 RD structure_element The result is highlighted in S2B Fig. The energy minimized structure of NsrR-RD was then superimposed on the dimeric structure of KdpE. RESULTS +89 101 superimposed experimental_method The result is highlighted in S2B Fig. The energy minimized structure of NsrR-RD was then superimposed on the dimeric structure of KdpE. RESULTS +109 116 dimeric oligomeric_state The result is highlighted in S2B Fig. The energy minimized structure of NsrR-RD was then superimposed on the dimeric structure of KdpE. RESULTS +117 126 structure evidence The result is highlighted in S2B Fig. The energy minimized structure of NsrR-RD was then superimposed on the dimeric structure of KdpE. RESULTS +130 134 KdpE protein The result is highlighted in S2B Fig. The energy minimized structure of NsrR-RD was then superimposed on the dimeric structure of KdpE. RESULTS +24 29 dimer oligomeric_state The putative functional dimer of NsrR-RD is depicted in Fig 5. RESULTS +33 37 NsrR protein The putative functional dimer of NsrR-RD is depicted in Fig 5. RESULTS +38 40 RD structure_element The putative functional dimer of NsrR-RD is depicted in Fig 5. RESULTS +4 21 dimeric interface site The dimeric interface is formed by α4-β5-α5 of RD (Fig 5A), as previously observed in other RRs. RESULTS +35 43 α4-β5-α5 structure_element The dimeric interface is formed by α4-β5-α5 of RD (Fig 5A), as previously observed in other RRs. RESULTS +47 49 RD structure_element The dimeric interface is formed by α4-β5-α5 of RD (Fig 5A), as previously observed in other RRs. RESULTS +92 95 RRs protein_type The dimeric interface is formed by α4-β5-α5 of RD (Fig 5A), as previously observed in other RRs. RESULTS +3 7 KdpE protein In KdpE, a network of salt bridges and other electrostatic interactions stabilize the interface within a single monomer as well as between the monomers. RESULTS +22 34 salt bridges bond_interaction In KdpE, a network of salt bridges and other electrostatic interactions stabilize the interface within a single monomer as well as between the monomers. RESULTS +45 71 electrostatic interactions bond_interaction In KdpE, a network of salt bridges and other electrostatic interactions stabilize the interface within a single monomer as well as between the monomers. RESULTS +86 95 interface site In KdpE, a network of salt bridges and other electrostatic interactions stabilize the interface within a single monomer as well as between the monomers. RESULTS +112 119 monomer oligomeric_state In KdpE, a network of salt bridges and other electrostatic interactions stabilize the interface within a single monomer as well as between the monomers. RESULTS +143 151 monomers oligomeric_state In KdpE, a network of salt bridges and other electrostatic interactions stabilize the interface within a single monomer as well as between the monomers. RESULTS +57 73 highly conserved protein_state Majority of these interactions involve residues that are highly conserved within the OmpR/PhoB subfamily of RRs. RESULTS +85 104 OmpR/PhoB subfamily protein_type Majority of these interactions involve residues that are highly conserved within the OmpR/PhoB subfamily of RRs. RESULTS +108 111 RRs protein_type Majority of these interactions involve residues that are highly conserved within the OmpR/PhoB subfamily of RRs. RESULTS +17 34 dimeric interface site In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). RESULTS +38 42 KdpE protein In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). RESULTS +63 80 hydrophobic patch site In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). RESULTS +100 105 Ile88 residue_name_number In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). RESULTS +107 109 α4 structure_element In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). RESULTS +112 117 Leu91 residue_name_number In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). RESULTS +119 121 α4 structure_element In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). RESULTS +124 130 Ala110 residue_name_number In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). RESULTS +132 134 α5 structure_element In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). RESULTS +141 147 Val114 residue_name_number In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). RESULTS +149 151 α5 structure_element In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (α4), Leu91 (α4), Ala110 (α5), and Val114 (α5). RESULTS +57 61 NsrR protein Structurally, a similar set of residues is also found in NsrR: Leu94 (α4), Val110 (α5) and Ala113 (α5), respectively (depicted as spheres in Fig 5B), which are conserved to some extent on sequence level (highlighted in yellow; Fig 3). RESULTS +63 68 Leu94 residue_name_number Structurally, a similar set of residues is also found in NsrR: Leu94 (α4), Val110 (α5) and Ala113 (α5), respectively (depicted as spheres in Fig 5B), which are conserved to some extent on sequence level (highlighted in yellow; Fig 3). RESULTS +70 72 α4 structure_element Structurally, a similar set of residues is also found in NsrR: Leu94 (α4), Val110 (α5) and Ala113 (α5), respectively (depicted as spheres in Fig 5B), which are conserved to some extent on sequence level (highlighted in yellow; Fig 3). RESULTS +75 81 Val110 residue_name_number Structurally, a similar set of residues is also found in NsrR: Leu94 (α4), Val110 (α5) and Ala113 (α5), respectively (depicted as spheres in Fig 5B), which are conserved to some extent on sequence level (highlighted in yellow; Fig 3). RESULTS +83 85 α5 structure_element Structurally, a similar set of residues is also found in NsrR: Leu94 (α4), Val110 (α5) and Ala113 (α5), respectively (depicted as spheres in Fig 5B), which are conserved to some extent on sequence level (highlighted in yellow; Fig 3). RESULTS +91 97 Ala113 residue_name_number Structurally, a similar set of residues is also found in NsrR: Leu94 (α4), Val110 (α5) and Ala113 (α5), respectively (depicted as spheres in Fig 5B), which are conserved to some extent on sequence level (highlighted in yellow; Fig 3). RESULTS +99 101 α5 structure_element Structurally, a similar set of residues is also found in NsrR: Leu94 (α4), Val110 (α5) and Ala113 (α5), respectively (depicted as spheres in Fig 5B), which are conserved to some extent on sequence level (highlighted in yellow; Fig 3). RESULTS +160 169 conserved protein_state Structurally, a similar set of residues is also found in NsrR: Leu94 (α4), Val110 (α5) and Ala113 (α5), respectively (depicted as spheres in Fig 5B), which are conserved to some extent on sequence level (highlighted in yellow; Fig 3). RESULTS +11 16 dimer oligomeric_state Functional dimer orientation of the RDs of NsrR. FIG +36 39 RDs structure_element Functional dimer orientation of the RDs of NsrR. FIG +43 47 NsrR protein Functional dimer orientation of the RDs of NsrR. FIG +0 7 Dimeric oligomeric_state Dimeric structure of the RD of NsrR aligned to the structure of KdpE (PDB code 1ZH2, not shown). FIG +8 17 structure evidence Dimeric structure of the RD of NsrR aligned to the structure of KdpE (PDB code 1ZH2, not shown). FIG +25 27 RD structure_element Dimeric structure of the RD of NsrR aligned to the structure of KdpE (PDB code 1ZH2, not shown). FIG +31 35 NsrR protein Dimeric structure of the RD of NsrR aligned to the structure of KdpE (PDB code 1ZH2, not shown). FIG +51 60 structure evidence Dimeric structure of the RD of NsrR aligned to the structure of KdpE (PDB code 1ZH2, not shown). FIG +64 68 KdpE protein Dimeric structure of the RD of NsrR aligned to the structure of KdpE (PDB code 1ZH2, not shown). FIG +12 20 monomers oligomeric_state (a) The two monomers of NsrR as functional dimers are represented in a cartoon representation displayed in cyan and yellow colors. FIG +24 28 NsrR protein (a) The two monomers of NsrR as functional dimers are represented in a cartoon representation displayed in cyan and yellow colors. FIG +43 49 dimers oligomeric_state (a) The two monomers of NsrR as functional dimers are represented in a cartoon representation displayed in cyan and yellow colors. FIG +19 36 dimeric interface site (b) Zoom-in of the dimeric interface mediated by α4-β5-α5. FIG +49 57 α4-β5-α5 structure_element (b) Zoom-in of the dimeric interface mediated by α4-β5-α5. FIG +4 11 monomer oligomeric_state The monomer-monomer interactions are facilitated by hydrophobic residues (displayed as spheres), inter- and intra-domain interactions (displayed as sticks). FIG +12 19 monomer oligomeric_state The monomer-monomer interactions are facilitated by hydrophobic residues (displayed as spheres), inter- and intra-domain interactions (displayed as sticks). FIG +25 51 electrostatic interactions bond_interaction Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +74 81 monomer oligomeric_state Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +82 89 monomer oligomeric_state Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +105 109 KdpE protein Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +133 138 Asp97 residue_name_number Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +140 142 β5 structure_element Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +148 154 Arg111 residue_name_number Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +156 158 α5 structure_element Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +161 166 Asp96 residue_name_number Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +168 178 α4–β5 loop structure_element Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +184 190 Arg118 residue_name_number Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +192 194 α5 structure_element Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +201 206 Asp92 residue_name_number Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +208 210 α4 structure_element Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +216 222 Arg113 residue_name_number Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +224 226 α5 structure_element Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (β5) and Arg111 (α5), Asp96 (α4–β5 loop) and Arg118 (α5), and Asp92 (α4) and Arg113 (α5). RESULTS +57 64 dimeric oligomeric_state Some of these interactions can also be identified in the dimeric model of NsrR-RD. RESULTS +74 78 NsrR protein Some of these interactions can also be identified in the dimeric model of NsrR-RD. RESULTS +79 81 RD structure_element Some of these interactions can also be identified in the dimeric model of NsrR-RD. RESULTS +6 12 Asp100 residue_name_number Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). RESULTS +14 16 β5 structure_element Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). RESULTS +22 28 Lys114 residue_name_number Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). RESULTS +30 32 α5 structure_element Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). RESULTS +65 72 monomer oligomeric_state Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). RESULTS +132 137 Asn95 residue_name_number Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). RESULTS +139 141 α4 structure_element Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). RESULTS +150 157 monomer oligomeric_state Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). RESULTS +163 169 Thr116 residue_name_number Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). RESULTS +171 173 α5 structure_element Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). RESULTS +188 195 monomer oligomeric_state Here, Asp100 (β5) and Lys114 (α5) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (α4) of one monomer with Thr116 (α5) of the other monomer (Fig 3, shown in cyan). RESULTS +0 5 Asp99 residue_name_number Asp99 (α4–β5 loop; Fig 3, shown in cyan) points toward the side chain of Arg121. RESULTS +7 17 α4–β5 loop structure_element Asp99 (α4–β5 loop; Fig 3, shown in cyan) points toward the side chain of Arg121. RESULTS +73 79 Arg121 residue_name_number Asp99 (α4–β5 loop; Fig 3, shown in cyan) points toward the side chain of Arg121. RESULTS +37 41 KdpE protein This interaction is also observed in KdpE (Asp96 (α4–β5 loop) and Arg118 (α5)). RESULTS +43 48 Asp96 residue_name_number This interaction is also observed in KdpE (Asp96 (α4–β5 loop) and Arg118 (α5)). RESULTS +50 60 α4–β5 loop structure_element This interaction is also observed in KdpE (Asp96 (α4–β5 loop) and Arg118 (α5)). RESULTS +66 72 Arg118 residue_name_number This interaction is also observed in KdpE (Asp96 (α4–β5 loop) and Arg118 (α5)). RESULTS +74 76 α5 structure_element This interaction is also observed in KdpE (Asp96 (α4–β5 loop) and Arg118 (α5)). RESULTS +3 7 KdpE protein In KdpE, Arg111 is additionally stabilized by another intra-molecular salt bridge with Glu107 (α5). RESULTS +9 15 Arg111 residue_name_number In KdpE, Arg111 is additionally stabilized by another intra-molecular salt bridge with Glu107 (α5). RESULTS +70 81 salt bridge bond_interaction In KdpE, Arg111 is additionally stabilized by another intra-molecular salt bridge with Glu107 (α5). RESULTS +87 93 Glu107 residue_name_number In KdpE, Arg111 is additionally stabilized by another intra-molecular salt bridge with Glu107 (α5). RESULTS +95 97 α5 structure_element In KdpE, Arg111 is additionally stabilized by another intra-molecular salt bridge with Glu107 (α5). RESULTS +18 22 NsrR protein Interestingly, in NsrR-RD this amino acid corresponds to Val110 (highlighted in yellow in Fig 3). RESULTS +23 25 RD structure_element Interestingly, in NsrR-RD this amino acid corresponds to Val110 (highlighted in yellow in Fig 3). RESULTS +57 63 Val110 residue_name_number Interestingly, in NsrR-RD this amino acid corresponds to Val110 (highlighted in yellow in Fig 3). RESULTS +20 29 alignment experimental_method As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan). RESULTS +51 59 arginine residue_name As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan). RESULTS +69 75 Arg111 residue_name_number As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan). RESULTS +79 83 KdpE protein As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan). RESULTS +98 106 arginine residue_name As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan). RESULTS +112 118 lysine residue_name As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan). RESULTS +128 134 Lys114 residue_name_number As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan). RESULTS +138 142 NsrR protein As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan). RESULTS +151 154 RRs protein_type As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan). RESULTS +167 176 alignment experimental_method As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan). RESULTS +27 35 arginine residue_name Interestingly, whenever an arginine is present at this position (Arg111 in KdpE), a glutamate (Glu107 in KdpE) is present as well, presumably stabilizing the arginine side chain. RESULTS +65 71 Arg111 residue_name_number Interestingly, whenever an arginine is present at this position (Arg111 in KdpE), a glutamate (Glu107 in KdpE) is present as well, presumably stabilizing the arginine side chain. RESULTS +75 79 KdpE protein Interestingly, whenever an arginine is present at this position (Arg111 in KdpE), a glutamate (Glu107 in KdpE) is present as well, presumably stabilizing the arginine side chain. RESULTS +84 93 glutamate residue_name Interestingly, whenever an arginine is present at this position (Arg111 in KdpE), a glutamate (Glu107 in KdpE) is present as well, presumably stabilizing the arginine side chain. RESULTS +95 101 Glu107 residue_name_number Interestingly, whenever an arginine is present at this position (Arg111 in KdpE), a glutamate (Glu107 in KdpE) is present as well, presumably stabilizing the arginine side chain. RESULTS +105 109 KdpE protein Interestingly, whenever an arginine is present at this position (Arg111 in KdpE), a glutamate (Glu107 in KdpE) is present as well, presumably stabilizing the arginine side chain. RESULTS +158 166 arginine residue_name Interestingly, whenever an arginine is present at this position (Arg111 in KdpE), a glutamate (Glu107 in KdpE) is present as well, presumably stabilizing the arginine side chain. RESULTS +16 22 lysine residue_name However, when a lysine is present at this position, the glutamate is exchanged to a hydrophobic residue contributing to the hydrophobic patch described above. RESULTS +56 65 glutamate residue_name However, when a lysine is present at this position, the glutamate is exchanged to a hydrophobic residue contributing to the hydrophobic patch described above. RESULTS +124 141 hydrophobic patch site However, when a lysine is present at this position, the glutamate is exchanged to a hydrophobic residue contributing to the hydrophobic patch described above. RESULTS +36 40 PhoB protein Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +46 53 E. coli species Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +58 62 PhoP protein Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +68 79 B. subtilis species Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +85 93 mutating experimental_method Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +156 162 Asp100 residue_name_number Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +164 170 Val110 residue_name_number Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +175 181 Lys114 residue_name_number Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +185 189 NsrR protein Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +202 211 monomeric oligomeric_state Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +220 238 response regulator protein_type Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +249 277 lost the ability to dimerize protein_state Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +305 308 DNA chemical Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities. RESULTS +8 17 Structure evidence Overall Structure of C-terminal DNA-binding effector domain of NsrR RESULTS +32 59 DNA-binding effector domain structure_element Overall Structure of C-terminal DNA-binding effector domain of NsrR RESULTS +63 67 NsrR protein Overall Structure of C-terminal DNA-binding effector domain of NsrR RESULTS +4 13 structure evidence The structure of NsrR-ED from S. agalactiae was determined using experimental phases from a single-wavelength anomalous dispersion dataset from the rectangular plate-shaped crystal derivatized with platinum at a resolution of 1.6 Å in space group P21212. RESULTS +17 21 NsrR protein The structure of NsrR-ED from S. agalactiae was determined using experimental phases from a single-wavelength anomalous dispersion dataset from the rectangular plate-shaped crystal derivatized with platinum at a resolution of 1.6 Å in space group P21212. RESULTS +22 24 ED structure_element The structure of NsrR-ED from S. agalactiae was determined using experimental phases from a single-wavelength anomalous dispersion dataset from the rectangular plate-shaped crystal derivatized with platinum at a resolution of 1.6 Å in space group P21212. RESULTS +30 43 S. agalactiae species The structure of NsrR-ED from S. agalactiae was determined using experimental phases from a single-wavelength anomalous dispersion dataset from the rectangular plate-shaped crystal derivatized with platinum at a resolution of 1.6 Å in space group P21212. RESULTS +92 138 single-wavelength anomalous dispersion dataset experimental_method The structure of NsrR-ED from S. agalactiae was determined using experimental phases from a single-wavelength anomalous dispersion dataset from the rectangular plate-shaped crystal derivatized with platinum at a resolution of 1.6 Å in space group P21212. RESULTS +198 206 platinum chemical The structure of NsrR-ED from S. agalactiae was determined using experimental phases from a single-wavelength anomalous dispersion dataset from the rectangular plate-shaped crystal derivatized with platinum at a resolution of 1.6 Å in space group P21212. RESULTS +4 9 Rwork evidence The Rwork and Rfree values after refinement were 0.18 and 0.22, respectively. RESULTS +14 19 Rfree evidence The Rwork and Rfree values after refinement were 0.18 and 0.22, respectively. RESULTS +0 23 Ramachandran validation evidence Ramachandran validation was done using MolProbity. RESULTS +14 20 Glu128 residue_name_number The latter is Glu128 (last residue of the linker region) of chain B that is involved in crystal contacts and, therefore, likely adopts an unfavorable conformation. RESULTS +42 55 linker region structure_element The latter is Glu128 (last residue of the linker region) of chain B that is involved in crystal contacts and, therefore, likely adopts an unfavorable conformation. RESULTS +60 67 chain B structure_element The latter is Glu128 (last residue of the linker region) of chain B that is involved in crystal contacts and, therefore, likely adopts an unfavorable conformation. RESULTS +4 13 structure evidence The structure contained a few ethylene glycol molecules introduced by the cryo-protecting procedure. RESULTS +30 45 ethylene glycol chemical The structure contained a few ethylene glycol molecules introduced by the cryo-protecting procedure. RESULTS +15 42 effector DNA-binding domain structure_element The C-terminal effector DNA-binding domain of NsrR is about 13 kDa in size and consists of residues 129–243 (including 21 amino acid residues of the expression tag). RESULTS +46 50 NsrR protein The C-terminal effector DNA-binding domain of NsrR is about 13 kDa in size and consists of residues 129–243 (including 21 amino acid residues of the expression tag). RESULTS +100 107 129–243 residue_range The C-terminal effector DNA-binding domain of NsrR is about 13 kDa in size and consists of residues 129–243 (including 21 amino acid residues of the expression tag). RESULTS +0 7 Monomer oligomeric_state Monomer A contains residue 129–224 and monomer B contain residues 128–225. RESULTS +8 9 A structure_element Monomer A contains residue 129–224 and monomer B contain residues 128–225. RESULTS +27 34 129–224 residue_range Monomer A contains residue 129–224 and monomer B contain residues 128–225. RESULTS +39 46 monomer oligomeric_state Monomer A contains residue 129–224 and monomer B contain residues 128–225. RESULTS +47 48 B structure_element Monomer A contains residue 129–224 and monomer B contain residues 128–225. RESULTS +66 73 128–225 residue_range Monomer A contains residue 129–224 and monomer B contain residues 128–225. RESULTS +4 10 Asp147 residue_name_number For Asp147 of chain A and Glu174 of chain B, poor electron density was observed for the side chains and, thus, these side chains were removed during refinement. RESULTS +14 21 chain A structure_element For Asp147 of chain A and Glu174 of chain B, poor electron density was observed for the side chains and, thus, these side chains were removed during refinement. RESULTS +26 32 Glu174 residue_name_number For Asp147 of chain A and Glu174 of chain B, poor electron density was observed for the side chains and, thus, these side chains were removed during refinement. RESULTS +36 43 chain B structure_element For Asp147 of chain A and Glu174 of chain B, poor electron density was observed for the side chains and, thus, these side chains were removed during refinement. RESULTS +50 66 electron density evidence For Asp147 of chain A and Glu174 of chain B, poor electron density was observed for the side chains and, thus, these side chains were removed during refinement. RESULTS +43 47 NsrR protein The asymmetric unit contains two copies of NsrR-ED related by two-fold rotational symmetry. RESULTS +48 50 ED structure_element The asymmetric unit contains two copies of NsrR-ED related by two-fold rotational symmetry. RESULTS +3 10 overlay experimental_method An overlay revealed that both monomers display high similarity in their overall structure with an rmsd of 0.5 Å over 95 Cα atoms. RESULTS +30 38 monomers oligomeric_state An overlay revealed that both monomers display high similarity in their overall structure with an rmsd of 0.5 Å over 95 Cα atoms. RESULTS +80 89 structure evidence An overlay revealed that both monomers display high similarity in their overall structure with an rmsd of 0.5 Å over 95 Cα atoms. RESULTS +98 102 rmsd evidence An overlay revealed that both monomers display high similarity in their overall structure with an rmsd of 0.5 Å over 95 Cα atoms. RESULTS +38 47 structure evidence We therefore describe for the overall structure only monomer A. RESULTS +53 60 monomer oligomeric_state We therefore describe for the overall structure only monomer A. RESULTS +61 62 A structure_element We therefore describe for the overall structure only monomer A. RESULTS +4 6 ED structure_element The ED domain of NsrR consists of six β-strands and three α-helices in a β6-β7-β8-β9-α6-α7-α8-β10-β11 topology (the secondary structure elements are counted in continuation of those of the RD). RESULTS +17 21 NsrR protein The ED domain of NsrR consists of six β-strands and three α-helices in a β6-β7-β8-β9-α6-α7-α8-β10-β11 topology (the secondary structure elements are counted in continuation of those of the RD). RESULTS +38 47 β-strands structure_element The ED domain of NsrR consists of six β-strands and three α-helices in a β6-β7-β8-β9-α6-α7-α8-β10-β11 topology (the secondary structure elements are counted in continuation of those of the RD). RESULTS +58 67 α-helices structure_element The ED domain of NsrR consists of six β-strands and three α-helices in a β6-β7-β8-β9-α6-α7-α8-β10-β11 topology (the secondary structure elements are counted in continuation of those of the RD). RESULTS +73 101 β6-β7-β8-β9-α6-α7-α8-β10-β11 structure_element The ED domain of NsrR consists of six β-strands and three α-helices in a β6-β7-β8-β9-α6-α7-α8-β10-β11 topology (the secondary structure elements are counted in continuation of those of the RD). RESULTS +189 191 RD structure_element The ED domain of NsrR consists of six β-strands and three α-helices in a β6-β7-β8-β9-α6-α7-α8-β10-β11 topology (the secondary structure elements are counted in continuation of those of the RD). RESULTS +4 19 effector domain structure_element The effector domain starts with a 4-stranded antiparallel β-sheet, followed by three α-helices and eventually ends in a C-terminal β-hairpin (Fig 6). RESULTS +34 65 4-stranded antiparallel β-sheet structure_element The effector domain starts with a 4-stranded antiparallel β-sheet, followed by three α-helices and eventually ends in a C-terminal β-hairpin (Fig 6). RESULTS +85 94 α-helices structure_element The effector domain starts with a 4-stranded antiparallel β-sheet, followed by three α-helices and eventually ends in a C-terminal β-hairpin (Fig 6). RESULTS +131 140 β-hairpin structure_element The effector domain starts with a 4-stranded antiparallel β-sheet, followed by three α-helices and eventually ends in a C-terminal β-hairpin (Fig 6). RESULTS +8 16 β-sheets structure_element The two β-sheets sandwich the three α-helices. RESULTS +36 45 α-helices structure_element The two β-sheets sandwich the three α-helices. RESULTS +0 9 Structure evidence Structure of the C-terminal effector domain of NsrR. FIG +28 43 effector domain structure_element Structure of the C-terminal effector domain of NsrR. FIG +47 51 NsrR protein Structure of the C-terminal effector domain of NsrR. FIG +41 56 effector domain structure_element Cartoon representation of the C-terminal effector domain of NsrR (green; recognition helix in cyan). FIG +60 64 NsrR protein Cartoon representation of the C-terminal effector domain of NsrR (green; recognition helix in cyan). FIG +73 90 recognition helix structure_element Cartoon representation of the C-terminal effector domain of NsrR (green; recognition helix in cyan). FIG +65 81 effector domains structure_element The structural areas with the highest variations compared to the effector domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1GXQ) are marked. FIG +85 89 DrrB protein The structural areas with the highest variations compared to the effector domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1GXQ) are marked. FIG +104 108 MtrA protein The structural areas with the highest variations compared to the effector domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1GXQ) are marked. FIG +127 131 PhoB protein The structural areas with the highest variations compared to the effector domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1GXQ) are marked. FIG +4 24 transactivation loop structure_element The transactivation loop of MtrA is missing in the structure, therefore, the two termini are connected by a dashed line. FIG +28 32 MtrA protein The transactivation loop of MtrA is missing in the structure, therefore, the two termini are connected by a dashed line. FIG +36 43 missing protein_state The transactivation loop of MtrA is missing in the structure, therefore, the two termini are connected by a dashed line. FIG +51 60 structure evidence The transactivation loop of MtrA is missing in the structure, therefore, the two termini are connected by a dashed line. FIG +34 53 OmpR/PhoB subfamily protein_type The characteristic feature of the OmpR/PhoB subfamily of RRs is a winged helix-turn-helix (wHTH) fold that is adopted by the α7-loop-α8 segment in full-length and single effector domain structures of RRs of this subfamily. RESULTS +57 60 RRs protein_type The characteristic feature of the OmpR/PhoB subfamily of RRs is a winged helix-turn-helix (wHTH) fold that is adopted by the α7-loop-α8 segment in full-length and single effector domain structures of RRs of this subfamily. RESULTS +66 89 winged helix-turn-helix structure_element The characteristic feature of the OmpR/PhoB subfamily of RRs is a winged helix-turn-helix (wHTH) fold that is adopted by the α7-loop-α8 segment in full-length and single effector domain structures of RRs of this subfamily. RESULTS +91 95 wHTH structure_element The characteristic feature of the OmpR/PhoB subfamily of RRs is a winged helix-turn-helix (wHTH) fold that is adopted by the α7-loop-α8 segment in full-length and single effector domain structures of RRs of this subfamily. RESULTS +125 143 α7-loop-α8 segment structure_element The characteristic feature of the OmpR/PhoB subfamily of RRs is a winged helix-turn-helix (wHTH) fold that is adopted by the α7-loop-α8 segment in full-length and single effector domain structures of RRs of this subfamily. RESULTS +147 158 full-length protein_state The characteristic feature of the OmpR/PhoB subfamily of RRs is a winged helix-turn-helix (wHTH) fold that is adopted by the α7-loop-α8 segment in full-length and single effector domain structures of RRs of this subfamily. RESULTS +170 185 effector domain structure_element The characteristic feature of the OmpR/PhoB subfamily of RRs is a winged helix-turn-helix (wHTH) fold that is adopted by the α7-loop-α8 segment in full-length and single effector domain structures of RRs of this subfamily. RESULTS +186 196 structures evidence The characteristic feature of the OmpR/PhoB subfamily of RRs is a winged helix-turn-helix (wHTH) fold that is adopted by the α7-loop-α8 segment in full-length and single effector domain structures of RRs of this subfamily. RESULTS +200 203 RRs protein_type The characteristic feature of the OmpR/PhoB subfamily of RRs is a winged helix-turn-helix (wHTH) fold that is adopted by the α7-loop-α8 segment in full-length and single effector domain structures of RRs of this subfamily. RESULTS +4 13 structure evidence The structure of NsrR-ED also contains such a wHTH motif built up by helices α7 and α8 (Fig 6). RESULTS +17 21 NsrR protein The structure of NsrR-ED also contains such a wHTH motif built up by helices α7 and α8 (Fig 6). RESULTS +22 24 ED structure_element The structure of NsrR-ED also contains such a wHTH motif built up by helices α7 and α8 (Fig 6). RESULTS +46 50 wHTH structure_element The structure of NsrR-ED also contains such a wHTH motif built up by helices α7 and α8 (Fig 6). RESULTS +69 76 helices structure_element The structure of NsrR-ED also contains such a wHTH motif built up by helices α7 and α8 (Fig 6). RESULTS +77 79 α7 structure_element The structure of NsrR-ED also contains such a wHTH motif built up by helices α7 and α8 (Fig 6). RESULTS +84 86 α8 structure_element The structure of NsrR-ED also contains such a wHTH motif built up by helices α7 and α8 (Fig 6). RESULTS +11 16 helix structure_element The second helix of the wHTH motif is important for DNA-binding and, therefore, is termed “recognition helix” (shown in cyan in Fig 6). RESULTS +24 28 wHTH structure_element The second helix of the wHTH motif is important for DNA-binding and, therefore, is termed “recognition helix” (shown in cyan in Fig 6). RESULTS +52 55 DNA chemical The second helix of the wHTH motif is important for DNA-binding and, therefore, is termed “recognition helix” (shown in cyan in Fig 6). RESULTS +91 108 recognition helix structure_element The second helix of the wHTH motif is important for DNA-binding and, therefore, is termed “recognition helix” (shown in cyan in Fig 6). RESULTS +15 20 helix structure_element Furthermore, a helix within the HTH motif, named “positioning helix”, is important for proper orientation and positioning of the loop between these two helices and is referred to as “transactivation loop” (also called α-loop; Fig 6). RESULTS +32 35 HTH structure_element Furthermore, a helix within the HTH motif, named “positioning helix”, is important for proper orientation and positioning of the loop between these two helices and is referred to as “transactivation loop” (also called α-loop; Fig 6). RESULTS +50 67 positioning helix structure_element Furthermore, a helix within the HTH motif, named “positioning helix”, is important for proper orientation and positioning of the loop between these two helices and is referred to as “transactivation loop” (also called α-loop; Fig 6). RESULTS +129 133 loop structure_element Furthermore, a helix within the HTH motif, named “positioning helix”, is important for proper orientation and positioning of the loop between these two helices and is referred to as “transactivation loop” (also called α-loop; Fig 6). RESULTS +183 203 transactivation loop structure_element Furthermore, a helix within the HTH motif, named “positioning helix”, is important for proper orientation and positioning of the loop between these two helices and is referred to as “transactivation loop” (also called α-loop; Fig 6). RESULTS +218 224 α-loop structure_element Furthermore, a helix within the HTH motif, named “positioning helix”, is important for proper orientation and positioning of the loop between these two helices and is referred to as “transactivation loop” (also called α-loop; Fig 6). RESULTS +7 16 structure evidence In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). RESULTS +20 24 NsrR protein In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). RESULTS +25 27 ED structure_element In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). RESULTS +29 34 helix structure_element In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). RESULTS +35 37 α8 structure_element In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). RESULTS +59 76 recognition helix structure_element In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). RESULTS +78 80 α7 structure_element In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). RESULTS +88 105 positioning helix structure_element In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). RESULTS +115 126 loop region structure_element In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). RESULTS +143 148 α7-α8 structure_element In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). RESULTS +152 172 transactivation loop structure_element In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). RESULTS +194 197 RRs protein_type In the structure of NsrR-ED, helix α8 is identified as the recognition helix, α7 as the positioning helix, and the loop region between helices α7-α8 as transactivation loop as observed in other RRs (Fig 6). RESULTS +21 36 solvent-exposed protein_state The 16-residue long, solvent-exposed recognition helix α8 of NsrR-ED contains four positively charged residues that can potentially interact with DNA. RESULTS +37 54 recognition helix structure_element The 16-residue long, solvent-exposed recognition helix α8 of NsrR-ED contains four positively charged residues that can potentially interact with DNA. RESULTS +55 57 α8 structure_element The 16-residue long, solvent-exposed recognition helix α8 of NsrR-ED contains four positively charged residues that can potentially interact with DNA. RESULTS +61 65 NsrR protein The 16-residue long, solvent-exposed recognition helix α8 of NsrR-ED contains four positively charged residues that can potentially interact with DNA. RESULTS +66 68 ED structure_element The 16-residue long, solvent-exposed recognition helix α8 of NsrR-ED contains four positively charged residues that can potentially interact with DNA. RESULTS +146 149 DNA chemical The 16-residue long, solvent-exposed recognition helix α8 of NsrR-ED contains four positively charged residues that can potentially interact with DNA. RESULTS +10 16 Arg198 residue_name_number These are Arg198, Arg200, Lys201, and Lys202. RESULTS +18 24 Arg200 residue_name_number These are Arg198, Arg200, Lys201, and Lys202. RESULTS +26 32 Lys201 residue_name_number These are Arg198, Arg200, Lys201, and Lys202. RESULTS +38 44 Lys202 residue_name_number These are Arg198, Arg200, Lys201, and Lys202. RESULTS +31 35 NsrR protein When comparing the sequence of NsrR with PhoB, KdpE, and MtrA, the alignment (Fig 3, colored in blue) emphasizes the variations at these positions, except for Arg200, which is conserved throughout the lantibiotic resistance RRs. RESULTS +41 45 PhoB protein When comparing the sequence of NsrR with PhoB, KdpE, and MtrA, the alignment (Fig 3, colored in blue) emphasizes the variations at these positions, except for Arg200, which is conserved throughout the lantibiotic resistance RRs. RESULTS +47 51 KdpE protein When comparing the sequence of NsrR with PhoB, KdpE, and MtrA, the alignment (Fig 3, colored in blue) emphasizes the variations at these positions, except for Arg200, which is conserved throughout the lantibiotic resistance RRs. RESULTS +57 61 MtrA protein When comparing the sequence of NsrR with PhoB, KdpE, and MtrA, the alignment (Fig 3, colored in blue) emphasizes the variations at these positions, except for Arg200, which is conserved throughout the lantibiotic resistance RRs. RESULTS +67 76 alignment experimental_method When comparing the sequence of NsrR with PhoB, KdpE, and MtrA, the alignment (Fig 3, colored in blue) emphasizes the variations at these positions, except for Arg200, which is conserved throughout the lantibiotic resistance RRs. RESULTS +159 165 Arg200 residue_name_number When comparing the sequence of NsrR with PhoB, KdpE, and MtrA, the alignment (Fig 3, colored in blue) emphasizes the variations at these positions, except for Arg200, which is conserved throughout the lantibiotic resistance RRs. RESULTS +176 185 conserved protein_state When comparing the sequence of NsrR with PhoB, KdpE, and MtrA, the alignment (Fig 3, colored in blue) emphasizes the variations at these positions, except for Arg200, which is conserved throughout the lantibiotic resistance RRs. RESULTS +201 227 lantibiotic resistance RRs protein_type When comparing the sequence of NsrR with PhoB, KdpE, and MtrA, the alignment (Fig 3, colored in blue) emphasizes the variations at these positions, except for Arg200, which is conserved throughout the lantibiotic resistance RRs. RESULTS +14 20 Lys202 residue_name_number Additionally, Lys202 is also highly conserved throughout the family of RRs except PhoB, clearly reflecting differences in the sequences of DNA to be bound. RESULTS +29 45 highly conserved protein_state Additionally, Lys202 is also highly conserved throughout the family of RRs except PhoB, clearly reflecting differences in the sequences of DNA to be bound. RESULTS +71 74 RRs protein_type Additionally, Lys202 is also highly conserved throughout the family of RRs except PhoB, clearly reflecting differences in the sequences of DNA to be bound. RESULTS +82 86 PhoB protein Additionally, Lys202 is also highly conserved throughout the family of RRs except PhoB, clearly reflecting differences in the sequences of DNA to be bound. RESULTS +139 142 DNA chemical Additionally, Lys202 is also highly conserved throughout the family of RRs except PhoB, clearly reflecting differences in the sequences of DNA to be bound. RESULTS +16 26 structures evidence Comparison with structures of other effector domains RESULTS +36 52 effector domains structure_element Comparison with structures of other effector domains RESULTS +15 26 DALI search experimental_method We performed a DALI search to identify structurally related proteins to NsrR-ED. RESULTS +72 76 NsrR protein We performed a DALI search to identify structurally related proteins to NsrR-ED. RESULTS +77 79 ED structure_element We performed a DALI search to identify structurally related proteins to NsrR-ED. RESULTS +9 18 structure evidence Here the structure of the effector domain of PhoB from E. coli (PDB code: 1GXQ) (Z-score of 13.7) is structurally the most closely related. RESULTS +26 41 effector domain structure_element Here the structure of the effector domain of PhoB from E. coli (PDB code: 1GXQ) (Z-score of 13.7) is structurally the most closely related. RESULTS +45 49 PhoB protein Here the structure of the effector domain of PhoB from E. coli (PDB code: 1GXQ) (Z-score of 13.7) is structurally the most closely related. RESULTS +55 62 E. coli species Here the structure of the effector domain of PhoB from E. coli (PDB code: 1GXQ) (Z-score of 13.7) is structurally the most closely related. RESULTS +81 88 Z-score evidence Here the structure of the effector domain of PhoB from E. coli (PDB code: 1GXQ) (Z-score of 13.7) is structurally the most closely related. RESULTS +15 19 PhoB protein Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182–189) is also present in the structure of NsrR-ED that connects helices α7 and α8. RESULTS +20 35 effector domain structure_element Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182–189) is also present in the structure of NsrR-ED that connects helices α7 and α8. RESULTS +55 59 loop structure_element Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182–189) is also present in the structure of NsrR-ED that connects helices α7 and α8. RESULTS +72 79 182–189 residue_range Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182–189) is also present in the structure of NsrR-ED that connects helices α7 and α8. RESULTS +104 113 structure evidence Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182–189) is also present in the structure of NsrR-ED that connects helices α7 and α8. RESULTS +117 121 NsrR protein Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182–189) is also present in the structure of NsrR-ED that connects helices α7 and α8. RESULTS +122 124 ED structure_element Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182–189) is also present in the structure of NsrR-ED that connects helices α7 and α8. RESULTS +139 146 helices structure_element Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182–189) is also present in the structure of NsrR-ED that connects helices α7 and α8. RESULTS +147 149 α7 structure_element Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182–189) is also present in the structure of NsrR-ED that connects helices α7 and α8. RESULTS +154 156 α8 structure_element Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182–189) is also present in the structure of NsrR-ED that connects helices α7 and α8. RESULTS +4 8 rmsd evidence The rmsd between the three helices of the effector domain (including the two helices forming the wHTH motif) of PhoB and NsrR-ED is 1.6 Å over 47 Cα atoms, clearly indicating that NsrR belongs to the OmpR/PhoB family of RRs. RESULTS +42 57 effector domain structure_element The rmsd between the three helices of the effector domain (including the two helices forming the wHTH motif) of PhoB and NsrR-ED is 1.6 Å over 47 Cα atoms, clearly indicating that NsrR belongs to the OmpR/PhoB family of RRs. RESULTS +77 84 helices structure_element The rmsd between the three helices of the effector domain (including the two helices forming the wHTH motif) of PhoB and NsrR-ED is 1.6 Å over 47 Cα atoms, clearly indicating that NsrR belongs to the OmpR/PhoB family of RRs. RESULTS +97 101 wHTH structure_element The rmsd between the three helices of the effector domain (including the two helices forming the wHTH motif) of PhoB and NsrR-ED is 1.6 Å over 47 Cα atoms, clearly indicating that NsrR belongs to the OmpR/PhoB family of RRs. RESULTS +112 116 PhoB protein The rmsd between the three helices of the effector domain (including the two helices forming the wHTH motif) of PhoB and NsrR-ED is 1.6 Å over 47 Cα atoms, clearly indicating that NsrR belongs to the OmpR/PhoB family of RRs. RESULTS +121 125 NsrR protein The rmsd between the three helices of the effector domain (including the two helices forming the wHTH motif) of PhoB and NsrR-ED is 1.6 Å over 47 Cα atoms, clearly indicating that NsrR belongs to the OmpR/PhoB family of RRs. RESULTS +126 128 ED structure_element The rmsd between the three helices of the effector domain (including the two helices forming the wHTH motif) of PhoB and NsrR-ED is 1.6 Å over 47 Cα atoms, clearly indicating that NsrR belongs to the OmpR/PhoB family of RRs. RESULTS +180 184 NsrR protein The rmsd between the three helices of the effector domain (including the two helices forming the wHTH motif) of PhoB and NsrR-ED is 1.6 Å over 47 Cα atoms, clearly indicating that NsrR belongs to the OmpR/PhoB family of RRs. RESULTS +200 223 OmpR/PhoB family of RRs protein_type The rmsd between the three helices of the effector domain (including the two helices forming the wHTH motif) of PhoB and NsrR-ED is 1.6 Å over 47 Cα atoms, clearly indicating that NsrR belongs to the OmpR/PhoB family of RRs. RESULTS +14 26 superimposed experimental_method Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +48 63 effector domain structure_element Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +67 71 NsrR protein Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +73 77 NsrR protein Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +78 80 ED structure_element Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +115 131 effector domains structure_element Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +141 157 OmpR/PhoB family protein_type Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +166 170 DrrB protein Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +172 176 MtrA protein Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +193 208 effector domain structure_element Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +209 218 structure evidence Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +222 226 PhoB protein Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +232 239 E. coli species Therefore, we superimposed the Cα traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli. RESULTS +13 23 structures evidence Overall, the structures are very similar with rmsd’s ranging from 1.7 to 2.6 Å (Table 2). RESULTS +46 50 rmsd evidence Overall, the structures are very similar with rmsd’s ranging from 1.7 to 2.6 Å (Table 2). RESULTS +53 57 loop structure_element The highest variations (Fig 6) are visible in in the loop regions α7-α8, which corresponds to the transactivation loop. RESULTS +66 71 α7-α8 structure_element The highest variations (Fig 6) are visible in in the loop regions α7-α8, which corresponds to the transactivation loop. RESULTS +98 118 transactivation loop structure_element The highest variations (Fig 6) are visible in in the loop regions α7-α8, which corresponds to the transactivation loop. RESULTS +8 11 RRs protein_type In many RRs this transactivation loop along with the recognition helix α8, form inter-domain contacts in the inactive state and are only exposed upon activation of the RRs via a conformational change where the N- and C-terminal domains move away from each other. RESULTS +17 37 transactivation loop structure_element In many RRs this transactivation loop along with the recognition helix α8, form inter-domain contacts in the inactive state and are only exposed upon activation of the RRs via a conformational change where the N- and C-terminal domains move away from each other. RESULTS +53 70 recognition helix structure_element In many RRs this transactivation loop along with the recognition helix α8, form inter-domain contacts in the inactive state and are only exposed upon activation of the RRs via a conformational change where the N- and C-terminal domains move away from each other. RESULTS +71 73 α8 structure_element In many RRs this transactivation loop along with the recognition helix α8, form inter-domain contacts in the inactive state and are only exposed upon activation of the RRs via a conformational change where the N- and C-terminal domains move away from each other. RESULTS +109 117 inactive protein_state In many RRs this transactivation loop along with the recognition helix α8, form inter-domain contacts in the inactive state and are only exposed upon activation of the RRs via a conformational change where the N- and C-terminal domains move away from each other. RESULTS +168 171 RRs protein_type In many RRs this transactivation loop along with the recognition helix α8, form inter-domain contacts in the inactive state and are only exposed upon activation of the RRs via a conformational change where the N- and C-terminal domains move away from each other. RESULTS +0 13 Linker region structure_element Linker region RESULTS +4 11 linkers structure_element The linkers that connect the RDs and EDs in response regulators are highly variable with respect to both length and sequence. RESULTS +29 32 RDs structure_element The linkers that connect the RDs and EDs in response regulators are highly variable with respect to both length and sequence. RESULTS +37 40 EDs structure_element The linkers that connect the RDs and EDs in response regulators are highly variable with respect to both length and sequence. RESULTS +44 63 response regulators protein_type The linkers that connect the RDs and EDs in response regulators are highly variable with respect to both length and sequence. RESULTS +68 83 highly variable protein_state The linkers that connect the RDs and EDs in response regulators are highly variable with respect to both length and sequence. RESULTS +30 37 linkers structure_element The exact boundaries of these linkers are difficult to predict from sequence alignments in the absence of structural information of the distinct RR. RESULTS +68 87 sequence alignments experimental_method The exact boundaries of these linkers are difficult to predict from sequence alignments in the absence of structural information of the distinct RR. RESULTS +95 105 absence of protein_state The exact boundaries of these linkers are difficult to predict from sequence alignments in the absence of structural information of the distinct RR. RESULTS +145 147 RR protein_type The exact boundaries of these linkers are difficult to predict from sequence alignments in the absence of structural information of the distinct RR. RESULTS +0 6 Linker structure_element Linker lengths in OmpR/PhoB proteins of unknown structure have been estimated by comparing the number of residues between conserved landmark residues in the regulatory and effector domains to those from structurally characterized family members. RESULTS +18 36 OmpR/PhoB proteins protein_type Linker lengths in OmpR/PhoB proteins of unknown structure have been estimated by comparing the number of residues between conserved landmark residues in the regulatory and effector domains to those from structurally characterized family members. RESULTS +157 188 regulatory and effector domains structure_element Linker lengths in OmpR/PhoB proteins of unknown structure have been estimated by comparing the number of residues between conserved landmark residues in the regulatory and effector domains to those from structurally characterized family members. RESULTS +15 31 OmpR/PhoB family protein_type Similar to the OmpR/PhoB family, the lantibiotic resistance-associated family of response regulators also displays diverse linker regions, which are recognized in sequence alignments by the introduction of gaps (Fig 3). RESULTS +37 100 lantibiotic resistance-associated family of response regulators protein_type Similar to the OmpR/PhoB family, the lantibiotic resistance-associated family of response regulators also displays diverse linker regions, which are recognized in sequence alignments by the introduction of gaps (Fig 3). RESULTS +123 137 linker regions structure_element Similar to the OmpR/PhoB family, the lantibiotic resistance-associated family of response regulators also displays diverse linker regions, which are recognized in sequence alignments by the introduction of gaps (Fig 3). RESULTS +163 182 sequence alignments experimental_method Similar to the OmpR/PhoB family, the lantibiotic resistance-associated family of response regulators also displays diverse linker regions, which are recognized in sequence alignments by the introduction of gaps (Fig 3). RESULTS +19 27 arginine residue_name Interestingly, two arginine residues (Arg120 and Arg121 in NsrR; Fig 3, shown in purple) at the end of the RDs seem to be strictly conserved throughout the family of response regulators in both the OmpR/PhoB and lantibiotic resistance-associated RRs, indicating a conserved similarity. RESULTS +38 44 Arg120 residue_name_number Interestingly, two arginine residues (Arg120 and Arg121 in NsrR; Fig 3, shown in purple) at the end of the RDs seem to be strictly conserved throughout the family of response regulators in both the OmpR/PhoB and lantibiotic resistance-associated RRs, indicating a conserved similarity. RESULTS +49 55 Arg121 residue_name_number Interestingly, two arginine residues (Arg120 and Arg121 in NsrR; Fig 3, shown in purple) at the end of the RDs seem to be strictly conserved throughout the family of response regulators in both the OmpR/PhoB and lantibiotic resistance-associated RRs, indicating a conserved similarity. RESULTS +59 63 NsrR protein Interestingly, two arginine residues (Arg120 and Arg121 in NsrR; Fig 3, shown in purple) at the end of the RDs seem to be strictly conserved throughout the family of response regulators in both the OmpR/PhoB and lantibiotic resistance-associated RRs, indicating a conserved similarity. RESULTS +107 110 RDs structure_element Interestingly, two arginine residues (Arg120 and Arg121 in NsrR; Fig 3, shown in purple) at the end of the RDs seem to be strictly conserved throughout the family of response regulators in both the OmpR/PhoB and lantibiotic resistance-associated RRs, indicating a conserved similarity. RESULTS +122 140 strictly conserved protein_state Interestingly, two arginine residues (Arg120 and Arg121 in NsrR; Fig 3, shown in purple) at the end of the RDs seem to be strictly conserved throughout the family of response regulators in both the OmpR/PhoB and lantibiotic resistance-associated RRs, indicating a conserved similarity. RESULTS +166 185 response regulators protein_type Interestingly, two arginine residues (Arg120 and Arg121 in NsrR; Fig 3, shown in purple) at the end of the RDs seem to be strictly conserved throughout the family of response regulators in both the OmpR/PhoB and lantibiotic resistance-associated RRs, indicating a conserved similarity. RESULTS +198 249 OmpR/PhoB and lantibiotic resistance-associated RRs protein_type Interestingly, two arginine residues (Arg120 and Arg121 in NsrR; Fig 3, shown in purple) at the end of the RDs seem to be strictly conserved throughout the family of response regulators in both the OmpR/PhoB and lantibiotic resistance-associated RRs, indicating a conserved similarity. RESULTS +264 273 conserved protein_state Interestingly, two arginine residues (Arg120 and Arg121 in NsrR; Fig 3, shown in purple) at the end of the RDs seem to be strictly conserved throughout the family of response regulators in both the OmpR/PhoB and lantibiotic resistance-associated RRs, indicating a conserved similarity. RESULTS +15 25 structures evidence As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of α5 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue. RESULTS +29 33 MtrA protein As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of α5 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue. RESULTS +38 42 KdpE protein As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of α5 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue. RESULTS +49 57 arginine residue_name As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of α5 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue. RESULTS +89 91 α5 structure_element As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of α5 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue. RESULTS +112 118 active protein_state As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of α5 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue. RESULTS +125 140 dimer interface site As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of α5 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue. RESULTS +148 150 RD structure_element As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of α5 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue. RESULTS +161 172 salt bridge bond_interaction As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of α5 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue. RESULTS +193 202 aspartate residue_name As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of α5 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue. RESULTS +5 14 aspartate residue_name This aspartate residue is identified in NsrR as Asp99 (see above). RESULTS +40 44 NsrR protein This aspartate residue is identified in NsrR as Asp99 (see above). RESULTS +48 53 Asp99 residue_name_number This aspartate residue is identified in NsrR as Asp99 (see above). RESULTS +0 12 Arginine 121 residue_name_number Arginine 121 of NsrR points towards this Asp99 residue however, the distance for a salt bridge interaction is too large. RESULTS +16 20 NsrR protein Arginine 121 of NsrR points towards this Asp99 residue however, the distance for a salt bridge interaction is too large. RESULTS +41 46 Asp99 residue_name_number Arginine 121 of NsrR points towards this Asp99 residue however, the distance for a salt bridge interaction is too large. RESULTS +83 94 salt bridge bond_interaction Arginine 121 of NsrR points towards this Asp99 residue however, the distance for a salt bridge interaction is too large. RESULTS +21 34 crystallizing experimental_method Although we aimed at crystallizing full-length NsrR, this endeavor failed due to proteolytic cleavage within the linker region during the time period of crystallization. RESULTS +35 46 full-length protein_state Although we aimed at crystallizing full-length NsrR, this endeavor failed due to proteolytic cleavage within the linker region during the time period of crystallization. RESULTS +47 51 NsrR protein Although we aimed at crystallizing full-length NsrR, this endeavor failed due to proteolytic cleavage within the linker region during the time period of crystallization. RESULTS +113 126 linker region structure_element Although we aimed at crystallizing full-length NsrR, this endeavor failed due to proteolytic cleavage within the linker region during the time period of crystallization. RESULTS +153 168 crystallization experimental_method Although we aimed at crystallizing full-length NsrR, this endeavor failed due to proteolytic cleavage within the linker region during the time period of crystallization. RESULTS +17 27 structures evidence Nonetheless, the structures of NsrR-RD and NsrR-ED together provide the required structural knowledge to predict the linker region that joins the receiver and effector domains. RESULTS +31 35 NsrR protein Nonetheless, the structures of NsrR-RD and NsrR-ED together provide the required structural knowledge to predict the linker region that joins the receiver and effector domains. RESULTS +36 38 RD structure_element Nonetheless, the structures of NsrR-RD and NsrR-ED together provide the required structural knowledge to predict the linker region that joins the receiver and effector domains. RESULTS +43 47 NsrR protein Nonetheless, the structures of NsrR-RD and NsrR-ED together provide the required structural knowledge to predict the linker region that joins the receiver and effector domains. RESULTS +48 50 ED structure_element Nonetheless, the structures of NsrR-RD and NsrR-ED together provide the required structural knowledge to predict the linker region that joins the receiver and effector domains. RESULTS +117 130 linker region structure_element Nonetheless, the structures of NsrR-RD and NsrR-ED together provide the required structural knowledge to predict the linker region that joins the receiver and effector domains. RESULTS +146 175 receiver and effector domains structure_element Nonetheless, the structures of NsrR-RD and NsrR-ED together provide the required structural knowledge to predict the linker region that joins the receiver and effector domains. RESULTS +4 17 linker region structure_element The linker region of NsrR consists of approximately nine residues (Fig 3), comprising 120RRSQQFIQQ128 (underlined residues are neither present in the structure of RD nor in ED of NsrR) and contains two positively charged amino acids. RESULTS +21 25 NsrR protein The linker region of NsrR consists of approximately nine residues (Fig 3), comprising 120RRSQQFIQQ128 (underlined residues are neither present in the structure of RD nor in ED of NsrR) and contains two positively charged amino acids. RESULTS +86 101 120RRSQQFIQQ128 structure_element The linker region of NsrR consists of approximately nine residues (Fig 3), comprising 120RRSQQFIQQ128 (underlined residues are neither present in the structure of RD nor in ED of NsrR) and contains two positively charged amino acids. RESULTS +150 159 structure evidence The linker region of NsrR consists of approximately nine residues (Fig 3), comprising 120RRSQQFIQQ128 (underlined residues are neither present in the structure of RD nor in ED of NsrR) and contains two positively charged amino acids. RESULTS +163 165 RD structure_element The linker region of NsrR consists of approximately nine residues (Fig 3), comprising 120RRSQQFIQQ128 (underlined residues are neither present in the structure of RD nor in ED of NsrR) and contains two positively charged amino acids. RESULTS +173 175 ED structure_element The linker region of NsrR consists of approximately nine residues (Fig 3), comprising 120RRSQQFIQQ128 (underlined residues are neither present in the structure of RD nor in ED of NsrR) and contains two positively charged amino acids. RESULTS +179 183 NsrR protein The linker region of NsrR consists of approximately nine residues (Fig 3), comprising 120RRSQQFIQQ128 (underlined residues are neither present in the structure of RD nor in ED of NsrR) and contains two positively charged amino acids. RESULTS +0 3 DNA chemical DNA-binding mode of NsrR using a full-length model RESULTS +20 24 NsrR protein DNA-binding mode of NsrR using a full-length model RESULTS +33 44 full-length protein_state DNA-binding mode of NsrR using a full-length model RESULTS +10 20 structures evidence Since the structures of both domains of NsrR were determined, we used this structural information together with the available crystal structures of related proteins to create a model of the full-length NsrR in its active and inactive state. RESULTS +40 44 NsrR protein Since the structures of both domains of NsrR were determined, we used this structural information together with the available crystal structures of related proteins to create a model of the full-length NsrR in its active and inactive state. RESULTS +75 97 structural information evidence Since the structures of both domains of NsrR were determined, we used this structural information together with the available crystal structures of related proteins to create a model of the full-length NsrR in its active and inactive state. RESULTS +126 144 crystal structures evidence Since the structures of both domains of NsrR were determined, we used this structural information together with the available crystal structures of related proteins to create a model of the full-length NsrR in its active and inactive state. RESULTS +190 201 full-length protein_state Since the structures of both domains of NsrR were determined, we used this structural information together with the available crystal structures of related proteins to create a model of the full-length NsrR in its active and inactive state. RESULTS +202 206 NsrR protein Since the structures of both domains of NsrR were determined, we used this structural information together with the available crystal structures of related proteins to create a model of the full-length NsrR in its active and inactive state. RESULTS +214 220 active protein_state Since the structures of both domains of NsrR were determined, we used this structural information together with the available crystal structures of related proteins to create a model of the full-length NsrR in its active and inactive state. RESULTS +225 233 inactive protein_state Since the structures of both domains of NsrR were determined, we used this structural information together with the available crystal structures of related proteins to create a model of the full-length NsrR in its active and inactive state. RESULTS +64 75 Dali search experimental_method To achieve this, we first carefully analyzed the outcome of the Dali search for each domain and identified structurally highly similar proteins (based on Z-scores and rmsd values) and choose the full-length structures previously reported. RESULTS +154 162 Z-scores evidence To achieve this, we first carefully analyzed the outcome of the Dali search for each domain and identified structurally highly similar proteins (based on Z-scores and rmsd values) and choose the full-length structures previously reported. RESULTS +167 171 rmsd evidence To achieve this, we first carefully analyzed the outcome of the Dali search for each domain and identified structurally highly similar proteins (based on Z-scores and rmsd values) and choose the full-length structures previously reported. RESULTS +195 206 full-length protein_state To achieve this, we first carefully analyzed the outcome of the Dali search for each domain and identified structurally highly similar proteins (based on Z-scores and rmsd values) and choose the full-length structures previously reported. RESULTS +207 217 structures evidence To achieve this, we first carefully analyzed the outcome of the Dali search for each domain and identified structurally highly similar proteins (based on Z-scores and rmsd values) and choose the full-length structures previously reported. RESULTS +63 74 full-length protein_state This resulted in a list of possible templates for modeling the full-length structure of NsrR (Table 2). RESULTS +75 84 structure evidence This resulted in a list of possible templates for modeling the full-length structure of NsrR (Table 2). RESULTS +88 92 NsrR protein This resulted in a list of possible templates for modeling the full-length structure of NsrR (Table 2). RESULTS +13 16 RRs protein_type In solution, RRs exist in equilibrium between the active and inactive state, which is shifted towards the active state upon phosphorylation of the ED. RESULTS +50 56 active protein_state In solution, RRs exist in equilibrium between the active and inactive state, which is shifted towards the active state upon phosphorylation of the ED. RESULTS +61 69 inactive protein_state In solution, RRs exist in equilibrium between the active and inactive state, which is shifted towards the active state upon phosphorylation of the ED. RESULTS +106 112 active protein_state In solution, RRs exist in equilibrium between the active and inactive state, which is shifted towards the active state upon phosphorylation of the ED. RESULTS +124 139 phosphorylation ptm In solution, RRs exist in equilibrium between the active and inactive state, which is shifted towards the active state upon phosphorylation of the ED. RESULTS +147 149 ED structure_element In solution, RRs exist in equilibrium between the active and inactive state, which is shifted towards the active state upon phosphorylation of the ED. RESULTS +39 41 RR protein_type This results in oligomerization of the RR and a higher affinity towards DNA. RESULTS +72 75 DNA chemical This results in oligomerization of the RR and a higher affinity towards DNA. RESULTS +43 52 structure evidence Based on the above-mentioned criteria, the structure of MtrA from M. tuberculosis, crystallized in an inactive and non-phosphorylated state, seemed best suited for modeling purposes. RESULTS +56 60 MtrA protein Based on the above-mentioned criteria, the structure of MtrA from M. tuberculosis, crystallized in an inactive and non-phosphorylated state, seemed best suited for modeling purposes. RESULTS +66 81 M. tuberculosis species Based on the above-mentioned criteria, the structure of MtrA from M. tuberculosis, crystallized in an inactive and non-phosphorylated state, seemed best suited for modeling purposes. RESULTS +83 95 crystallized experimental_method Based on the above-mentioned criteria, the structure of MtrA from M. tuberculosis, crystallized in an inactive and non-phosphorylated state, seemed best suited for modeling purposes. RESULTS +102 110 inactive protein_state Based on the above-mentioned criteria, the structure of MtrA from M. tuberculosis, crystallized in an inactive and non-phosphorylated state, seemed best suited for modeling purposes. RESULTS +115 133 non-phosphorylated protein_state Based on the above-mentioned criteria, the structure of MtrA from M. tuberculosis, crystallized in an inactive and non-phosphorylated state, seemed best suited for modeling purposes. RESULTS +17 23 linker structure_element Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure. RESULTS +51 55 MtrA protein Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure. RESULTS +114 120 linker structure_element Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure. RESULTS +124 128 NsrR protein Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure. RESULTS +133 140 aligned experimental_method Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure. RESULTS +145 149 NsrR protein Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure. RESULTS +150 152 RD structure_element Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure. RESULTS +158 160 ED structure_element Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure. RESULTS +182 186 MtrA protein Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure. RESULTS +213 222 structure evidence Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure. RESULTS +16 22 closed protein_state This mimics the closed inactive conformation of NsrR (Fig 7A; the missing linker is represented as dotted line). RESULTS +23 31 inactive protein_state This mimics the closed inactive conformation of NsrR (Fig 7A; the missing linker is represented as dotted line). RESULTS +48 52 NsrR protein This mimics the closed inactive conformation of NsrR (Fig 7A; the missing linker is represented as dotted line). RESULTS +66 73 missing protein_state This mimics the closed inactive conformation of NsrR (Fig 7A; the missing linker is represented as dotted line). RESULTS +74 80 linker structure_element This mimics the closed inactive conformation of NsrR (Fig 7A; the missing linker is represented as dotted line). RESULTS +9 20 full-length protein_state Model of full-length NsrR in its inactive state and active state. FIG +21 25 NsrR protein Model of full-length NsrR in its inactive state and active state. FIG +33 41 inactive protein_state Model of full-length NsrR in its inactive state and active state. FIG +52 58 active protein_state Model of full-length NsrR in its inactive state and active state. FIG +4 6 RD structure_element The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +17 21 NsrR protein The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +55 57 ED structure_element The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +84 101 recognition helix structure_element The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +124 132 Inactive protein_state The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +169 173 NsrR protein The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +179 186 aligned experimental_method The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +194 203 structure evidence The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +207 211 MtrA protein The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +240 246 closed protein_state The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +247 255 inactive protein_state The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +291 302 full-length protein_state The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +303 307 NsrR protein The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +309 315 Phe101 residue_name_number The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +320 326 Asp187 residue_name_number The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +342 348 closed protein_state The RD domain of NsrR is highlighted in yellow and the ED domain in green with the “recognition helix” colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation. FIG +4 11 missing protein_state The missing linker is represented by a dotted line. FIG +12 18 linker structure_element The missing linker is represented by a dotted line. FIG +4 10 Active protein_state (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +42 53 full-length protein_state (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +54 58 NsrR protein (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +62 68 active protein_state (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +95 104 alignment experimental_method (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +128 132 NsrR protein (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +140 149 structure evidence (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +153 162 DNA bound protein_state (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +163 172 structure evidence (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +176 180 KdpE protein (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +211 217 active protein_state (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +218 222 open protein_state (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +265 269 NsrR protein (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +306 323 recognition helix structure_element (b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green. FIG +3 7 MtrA protein In MtrA, the two domains interact via the α4-β5-α5 interface of the receiver domain and the end of α7, α7-α8 loop and α8 of the effector domain. RESULTS +42 60 α4-β5-α5 interface site In MtrA, the two domains interact via the α4-β5-α5 interface of the receiver domain and the end of α7, α7-α8 loop and α8 of the effector domain. RESULTS +68 83 receiver domain structure_element In MtrA, the two domains interact via the α4-β5-α5 interface of the receiver domain and the end of α7, α7-α8 loop and α8 of the effector domain. RESULTS +99 101 α7 structure_element In MtrA, the two domains interact via the α4-β5-α5 interface of the receiver domain and the end of α7, α7-α8 loop and α8 of the effector domain. RESULTS +103 113 α7-α8 loop structure_element In MtrA, the two domains interact via the α4-β5-α5 interface of the receiver domain and the end of α7, α7-α8 loop and α8 of the effector domain. RESULTS +118 120 α8 structure_element In MtrA, the two domains interact via the α4-β5-α5 interface of the receiver domain and the end of α7, α7-α8 loop and α8 of the effector domain. RESULTS +128 143 effector domain structure_element In MtrA, the two domains interact via the α4-β5-α5 interface of the receiver domain and the end of α7, α7-α8 loop and α8 of the effector domain. RESULTS +5 15 interfaces site Both interfaces have been shown to form functionally important contact areas in the active state within members of the OmpR/PhoB subfamily. RESULTS +84 90 active protein_state Both interfaces have been shown to form functionally important contact areas in the active state within members of the OmpR/PhoB subfamily. RESULTS +119 138 OmpR/PhoB subfamily protein_type Both interfaces have been shown to form functionally important contact areas in the active state within members of the OmpR/PhoB subfamily. RESULTS +16 27 full-length protein_state In our model of full-length NsrR, a similar orientation between the domains is observed, contributing to the inter-domain interactions. RESULTS +28 32 NsrR protein In our model of full-length NsrR, a similar orientation between the domains is observed, contributing to the inter-domain interactions. RESULTS +4 12 inactive protein_state The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation. RESULTS +29 33 MtrA protein The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation. RESULTS +87 93 Tyr102 residue_name_number The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation. RESULTS +122 128 active protein_state The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation. RESULTS +129 134 Asp56 residue_name_number The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation. RESULTS +168 174 Tyr102 residue_name_number The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation. RESULTS +190 196 Asp190 residue_name_number The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation. RESULTS +204 206 RD structure_element The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation. RESULTS +210 214 MtrA protein The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation. RESULTS +240 246 closed protein_state The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation. RESULTS +16 20 NsrR protein In the model of NsrR, similar amino acids are present, Phe101 (switch residue) and Asp188 (Fig 3, represented by orange boxes) forming a likewise similar network of interaction. RESULTS +55 61 Phe101 residue_name_number In the model of NsrR, similar amino acids are present, Phe101 (switch residue) and Asp188 (Fig 3, represented by orange boxes) forming a likewise similar network of interaction. RESULTS +63 77 switch residue site In the model of NsrR, similar amino acids are present, Phe101 (switch residue) and Asp188 (Fig 3, represented by orange boxes) forming a likewise similar network of interaction. RESULTS +83 89 Asp188 residue_name_number In the model of NsrR, similar amino acids are present, Phe101 (switch residue) and Asp188 (Fig 3, represented by orange boxes) forming a likewise similar network of interaction. RESULTS +32 38 active protein_state Next, we were interested in the active conformation of the NsrR protein adopting an active “open” conformation in the dimeric state. RESULTS +59 63 NsrR protein Next, we were interested in the active conformation of the NsrR protein adopting an active “open” conformation in the dimeric state. RESULTS +84 90 active protein_state Next, we were interested in the active conformation of the NsrR protein adopting an active “open” conformation in the dimeric state. RESULTS +92 96 open protein_state Next, we were interested in the active conformation of the NsrR protein adopting an active “open” conformation in the dimeric state. RESULTS +118 125 dimeric oligomeric_state Next, we were interested in the active conformation of the NsrR protein adopting an active “open” conformation in the dimeric state. RESULTS +3 23 compared and aligned experimental_method We compared and aligned the NsrR-RD and ED on the dimeric structure of KdpE that was solved in the DNA-bound state (Fig 7B). RESULTS +28 32 NsrR protein We compared and aligned the NsrR-RD and ED on the dimeric structure of KdpE that was solved in the DNA-bound state (Fig 7B). RESULTS +33 35 RD structure_element We compared and aligned the NsrR-RD and ED on the dimeric structure of KdpE that was solved in the DNA-bound state (Fig 7B). RESULTS +40 42 ED structure_element We compared and aligned the NsrR-RD and ED on the dimeric structure of KdpE that was solved in the DNA-bound state (Fig 7B). RESULTS +50 57 dimeric oligomeric_state We compared and aligned the NsrR-RD and ED on the dimeric structure of KdpE that was solved in the DNA-bound state (Fig 7B). RESULTS +58 67 structure evidence We compared and aligned the NsrR-RD and ED on the dimeric structure of KdpE that was solved in the DNA-bound state (Fig 7B). RESULTS +71 75 KdpE protein We compared and aligned the NsrR-RD and ED on the dimeric structure of KdpE that was solved in the DNA-bound state (Fig 7B). RESULTS +85 91 solved experimental_method We compared and aligned the NsrR-RD and ED on the dimeric structure of KdpE that was solved in the DNA-bound state (Fig 7B). RESULTS +99 108 DNA-bound protein_state We compared and aligned the NsrR-RD and ED on the dimeric structure of KdpE that was solved in the DNA-bound state (Fig 7B). RESULTS +9 22 linker region structure_element Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer. RESULTS +26 30 KdpE protein Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer. RESULTS +58 62 NsrR protein Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer. RESULTS +104 113 DNA-bound protein_state Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer. RESULTS +132 134 RD structure_element Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer. RESULTS +139 141 ED structure_element Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer. RESULTS +145 149 NsrR protein Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer. RESULTS +181 185 KdpE protein Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer. RESULTS +186 192 active protein_state Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer. RESULTS +193 198 dimer oligomeric_state Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer. RESULTS +3 15 superimposed experimental_method We superimposed the ED of NsrR with the DNA-binding domain of KdpE resulting in a reasonably well-aligned structure (rmsd of 2.6Å over 86 Cα atoms; Table 2). RESULTS +20 22 ED structure_element We superimposed the ED of NsrR with the DNA-binding domain of KdpE resulting in a reasonably well-aligned structure (rmsd of 2.6Å over 86 Cα atoms; Table 2). RESULTS +26 30 NsrR protein We superimposed the ED of NsrR with the DNA-binding domain of KdpE resulting in a reasonably well-aligned structure (rmsd of 2.6Å over 86 Cα atoms; Table 2). RESULTS +40 58 DNA-binding domain structure_element We superimposed the ED of NsrR with the DNA-binding domain of KdpE resulting in a reasonably well-aligned structure (rmsd of 2.6Å over 86 Cα atoms; Table 2). RESULTS +62 66 KdpE protein We superimposed the ED of NsrR with the DNA-binding domain of KdpE resulting in a reasonably well-aligned structure (rmsd of 2.6Å over 86 Cα atoms; Table 2). RESULTS +106 115 structure evidence We superimposed the ED of NsrR with the DNA-binding domain of KdpE resulting in a reasonably well-aligned structure (rmsd of 2.6Å over 86 Cα atoms; Table 2). RESULTS +117 121 rmsd evidence We superimposed the ED of NsrR with the DNA-binding domain of KdpE resulting in a reasonably well-aligned structure (rmsd of 2.6Å over 86 Cα atoms; Table 2). RESULTS +15 37 highly positive groove site As a result, a highly positive groove is created by the two ED domains of NsrR which likely represents the DNA binding site as observed in KdpE. A prediction of the putative promoter sequence that NsrR binds via the BPROM online server was performed (S3 Fig). RESULTS +60 62 ED structure_element As a result, a highly positive groove is created by the two ED domains of NsrR which likely represents the DNA binding site as observed in KdpE. A prediction of the putative promoter sequence that NsrR binds via the BPROM online server was performed (S3 Fig). RESULTS +74 78 NsrR protein As a result, a highly positive groove is created by the two ED domains of NsrR which likely represents the DNA binding site as observed in KdpE. A prediction of the putative promoter sequence that NsrR binds via the BPROM online server was performed (S3 Fig). RESULTS +107 123 DNA binding site site As a result, a highly positive groove is created by the two ED domains of NsrR which likely represents the DNA binding site as observed in KdpE. A prediction of the putative promoter sequence that NsrR binds via the BPROM online server was performed (S3 Fig). RESULTS +139 143 KdpE protein As a result, a highly positive groove is created by the two ED domains of NsrR which likely represents the DNA binding site as observed in KdpE. A prediction of the putative promoter sequence that NsrR binds via the BPROM online server was performed (S3 Fig). RESULTS +197 201 NsrR protein As a result, a highly positive groove is created by the two ED domains of NsrR which likely represents the DNA binding site as observed in KdpE. A prediction of the putative promoter sequence that NsrR binds via the BPROM online server was performed (S3 Fig). RESULTS +49 52 nsr gene A promoter region was identified upstream of the nsr operon. RESULTS +58 61 DNA chemical However, the regulation of the predicted promoter and the DNA binding by NsrR has to be confirmed. RESULTS +73 77 NsrR protein However, the regulation of the predicted promoter and the DNA binding by NsrR has to be confirmed. RESULTS +23 31 bacteria taxonomy_domain In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. RESULTS +40 53 S. agalactiae species In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. RESULTS +55 64 S. aureus species In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. RESULTS +70 82 C. difficile species In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. RESULTS +116 127 lantibiotic chemical In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. RESULTS +185 197 lantibiotics chemical In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. RESULTS +206 211 nisin chemical In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. RESULTS +213 226 nukacin ISK-1 chemical In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. RESULTS +228 240 lacticin 481 chemical In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. RESULTS +241 252 gallidermin chemical In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. RESULTS +254 265 actagardine chemical In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. RESULTS +270 280 mersacidin chemical In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin. RESULTS +4 13 structure evidence The structure of the response regulator NsrR from S. agalactiae presented in this study is the first structural information available for the subgroup of lantibiotic resistance-associated RRs. RESULTS +21 39 response regulator protein_type The structure of the response regulator NsrR from S. agalactiae presented in this study is the first structural information available for the subgroup of lantibiotic resistance-associated RRs. RESULTS +40 44 NsrR protein The structure of the response regulator NsrR from S. agalactiae presented in this study is the first structural information available for the subgroup of lantibiotic resistance-associated RRs. RESULTS +50 63 S. agalactiae species The structure of the response regulator NsrR from S. agalactiae presented in this study is the first structural information available for the subgroup of lantibiotic resistance-associated RRs. RESULTS +154 191 lantibiotic resistance-associated RRs protein_type The structure of the response regulator NsrR from S. agalactiae presented in this study is the first structural information available for the subgroup of lantibiotic resistance-associated RRs. RESULTS diff --git a/annotation_CSV/PMC4774019.csv b/annotation_CSV/PMC4774019.csv new file mode 100644 index 0000000000000000000000000000000000000000..26aca2f35000c86104e41b0c23909d4fd5520a63 --- /dev/null +++ b/annotation_CSV/PMC4774019.csv @@ -0,0 +1,531 @@ +anno_start anno_end anno_text entity_type sentence section +4 27 immunity-related GTPase protein_type The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion TITLE +28 33 Irga6 protein The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion TITLE +34 43 dimerizes oligomeric_state The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion TITLE +49 70 parallel head-to-head protein_state The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion TITLE +4 28 immunity-related GTPases protein_type The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens. ABSTRACT +30 34 IRGs protein_type The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens. ABSTRACT +0 5 Irga6 protein Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. ABSTRACT +11 31 dynamin-like protein protein_type Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. ABSTRACT +100 117 Toxoplasma gondii species Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. ABSTRACT +20 40 biochemical analysis experimental_method Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization. ABSTRACT +72 86 GTPase domains structure_element Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization. ABSTRACT +90 95 Irga6 protein Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization. ABSTRACT +96 104 dimerize oligomeric_state Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization. ABSTRACT +111 123 antiparallel protein_state Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization. ABSTRACT +3 13 determined experimental_method We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. ABSTRACT +18 35 crystal structure evidence We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. ABSTRACT +42 66 oligomerization-impaired protein_state We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. ABSTRACT +67 72 Irga6 protein We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. ABSTRACT +73 79 mutant protein_state We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. ABSTRACT +80 88 bound to protein_state We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. ABSTRACT +108 111 GTP chemical We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. ABSTRACT +36 45 structure evidence Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion. ABSTRACT +61 75 GTPase domains structure_element Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion. ABSTRACT +76 84 dimerize oligomeric_state Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion. ABSTRACT +90 98 parallel protein_state Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion. ABSTRACT +4 15 nucleotides chemical The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule. ABSTRACT +37 46 interface site The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule. ABSTRACT +134 142 switch I site The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule. ABSTRACT +166 171 Irga6 protein The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule. ABSTRACT +39 42 GTP chemical The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. ABSTRACT +89 98 catalytic protein_state The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. ABSTRACT +99 112 glutamate 106 residue_name_number The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. ABSTRACT +116 124 switch I site The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. ABSTRACT +138 149 active site site The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. ABSTRACT +38 47 switch II site Further dimerization contacts involve switch II, the G4 helix and the trans stabilizing loop. ABSTRACT +53 61 G4 helix structure_element Further dimerization contacts involve switch II, the G4 helix and the trans stabilizing loop. ABSTRACT +70 92 trans stabilizing loop structure_element Further dimerization contacts involve switch II, the G4 helix and the trans stabilizing loop. ABSTRACT +4 9 Irga6 protein The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. ABSTRACT +10 19 structure evidence The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. ABSTRACT +31 39 parallel protein_state The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. ABSTRACT +40 53 GTPase domain structure_element The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. ABSTRACT +54 59 dimer oligomeric_state The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. ABSTRACT +107 114 dynamin protein_type The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. ABSTRACT +119 125 septin protein_type The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. ABSTRACT +88 91 IRG protein_type This study contributes important insights into the assembly and catalytic mechanisms of IRG proteins as prerequisite to understand their anti-microbial action. ABSTRACT +0 24 Immunity-related GTPases protein_type Immunity-related GTPases (IRGs) comprise a family of dynamin-related cell-autonomous resistance proteins targeting intracellular pathogens, such as Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Trypanosoma cruzi, and Toxoplasma gondii. INTRO +26 30 IRGs protein_type Immunity-related GTPases (IRGs) comprise a family of dynamin-related cell-autonomous resistance proteins targeting intracellular pathogens, such as Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Trypanosoma cruzi, and Toxoplasma gondii. INTRO +53 104 dynamin-related cell-autonomous resistance proteins protein_type Immunity-related GTPases (IRGs) comprise a family of dynamin-related cell-autonomous resistance proteins targeting intracellular pathogens, such as Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Trypanosoma cruzi, and Toxoplasma gondii. INTRO +148 174 Mycobacterium tuberculosis species Immunity-related GTPases (IRGs) comprise a family of dynamin-related cell-autonomous resistance proteins targeting intracellular pathogens, such as Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Trypanosoma cruzi, and Toxoplasma gondii. INTRO +176 195 Mycobacterium avium species Immunity-related GTPases (IRGs) comprise a family of dynamin-related cell-autonomous resistance proteins targeting intracellular pathogens, such as Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Trypanosoma cruzi, and Toxoplasma gondii. INTRO +197 219 Listeria monocytogenes species Immunity-related GTPases (IRGs) comprise a family of dynamin-related cell-autonomous resistance proteins targeting intracellular pathogens, such as Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Trypanosoma cruzi, and Toxoplasma gondii. INTRO +221 238 Trypanosoma cruzi species Immunity-related GTPases (IRGs) comprise a family of dynamin-related cell-autonomous resistance proteins targeting intracellular pathogens, such as Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Trypanosoma cruzi, and Toxoplasma gondii. INTRO +244 261 Toxoplasma gondii species Immunity-related GTPases (IRGs) comprise a family of dynamin-related cell-autonomous resistance proteins targeting intracellular pathogens, such as Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Trypanosoma cruzi, and Toxoplasma gondii. INTRO +3 7 mice taxonomy_domain In mice, the 23 IRG members are induced by interferons, whereas the single human homologue is constitutively expressed in some tissues, especially in testis. INTRO +16 19 IRG protein_type In mice, the 23 IRG members are induced by interferons, whereas the single human homologue is constitutively expressed in some tissues, especially in testis. INTRO +43 54 interferons protein_type In mice, the 23 IRG members are induced by interferons, whereas the single human homologue is constitutively expressed in some tissues, especially in testis. INTRO +75 80 human species In mice, the 23 IRG members are induced by interferons, whereas the single human homologue is constitutively expressed in some tissues, especially in testis. INTRO +28 32 IRGs protein_type In non-infected cells, most IRGs are largely cytosolic. INTRO +13 17 IRGs protein_type In this way, IRGs contribute to the release of the pathogen into the cytoplasm and its subsequent destruction. INTRO +0 5 Irga6 protein Irga6, one of the effector IRG proteins, localizes to the intact parasitophorous vacuole membrane (PVM) and, after disruption of the PVM, is found associated with vesicular accumulations, presumably derived from the PVM. INTRO +27 30 IRG protein_type Irga6, one of the effector IRG proteins, localizes to the intact parasitophorous vacuole membrane (PVM) and, after disruption of the PVM, is found associated with vesicular accumulations, presumably derived from the PVM. INTRO +2 21 myristoylation site site A myristoylation site at Gly2 is necessary for the recruitment to the PVM but not for the weak constitutive binding to the ER membrane. INTRO +25 29 Gly2 residue_name_number A myristoylation site at Gly2 is necessary for the recruitment to the PVM but not for the weak constitutive binding to the ER membrane. INTRO +43 50 helix A structure_element An internally oriented antibody epitope on helix A between positions 20 and 24 was demonstrated to be accessible in the GTP-, but not in the GDP-bound state. INTRO +69 78 20 and 24 residue_range An internally oriented antibody epitope on helix A between positions 20 and 24 was demonstrated to be accessible in the GTP-, but not in the GDP-bound state. INTRO +120 125 GTP-, protein_state An internally oriented antibody epitope on helix A between positions 20 and 24 was demonstrated to be accessible in the GTP-, but not in the GDP-bound state. INTRO +141 150 GDP-bound protein_state An internally oriented antibody epitope on helix A between positions 20 and 24 was demonstrated to be accessible in the GTP-, but not in the GDP-bound state. INTRO +51 54 GTP chemical This indicates large-scale structural changes upon GTP binding that probably include exposure of the myristoyl group, enhancing binding to the PVM. INTRO +0 19 Biochemical studies experimental_method Biochemical studies indicated that Irga6 hydrolyses GTP in a cooperative manner and forms GTP-dependent oligomers in vitro and in vivo. INTRO +35 40 Irga6 protein Biochemical studies indicated that Irga6 hydrolyses GTP in a cooperative manner and forms GTP-dependent oligomers in vitro and in vivo. INTRO +52 55 GTP chemical Biochemical studies indicated that Irga6 hydrolyses GTP in a cooperative manner and forms GTP-dependent oligomers in vitro and in vivo. INTRO +90 103 GTP-dependent protein_state Biochemical studies indicated that Irga6 hydrolyses GTP in a cooperative manner and forms GTP-dependent oligomers in vitro and in vivo. INTRO +104 113 oligomers oligomeric_state Biochemical studies indicated that Irga6 hydrolyses GTP in a cooperative manner and forms GTP-dependent oligomers in vitro and in vivo. INTRO +0 18 Crystal structures evidence Crystal structures of Irga6 in various nucleotide-loaded states revealed the basic architecture of IRG proteins, including a GTPase domain and a composite helical domain. INTRO +22 27 Irga6 protein Crystal structures of Irga6 in various nucleotide-loaded states revealed the basic architecture of IRG proteins, including a GTPase domain and a composite helical domain. INTRO +39 56 nucleotide-loaded protein_state Crystal structures of Irga6 in various nucleotide-loaded states revealed the basic architecture of IRG proteins, including a GTPase domain and a composite helical domain. INTRO +99 102 IRG protein_type Crystal structures of Irga6 in various nucleotide-loaded states revealed the basic architecture of IRG proteins, including a GTPase domain and a composite helical domain. INTRO +125 138 GTPase domain structure_element Crystal structures of Irga6 in various nucleotide-loaded states revealed the basic architecture of IRG proteins, including a GTPase domain and a composite helical domain. INTRO +145 169 composite helical domain structure_element Crystal structures of Irga6 in various nucleotide-loaded states revealed the basic architecture of IRG proteins, including a GTPase domain and a composite helical domain. INTRO +36 58 dimerization interface site These studies additionally showed a dimerization interface in the nucleotide-free protein as well as in all nucleotide-bound states. INTRO +66 81 nucleotide-free protein_state These studies additionally showed a dimerization interface in the nucleotide-free protein as well as in all nucleotide-bound states. INTRO +108 124 nucleotide-bound protein_state These studies additionally showed a dimerization interface in the nucleotide-free protein as well as in all nucleotide-bound states. INTRO +14 35 GTPase domain surface site It involves a GTPase domain surface, which is located at the opposite side of the nucleotide, and an interface in the helical domain, with a water-filled gap between the two contact surfaces. INTRO +101 110 interface site It involves a GTPase domain surface, which is located at the opposite side of the nucleotide, and an interface in the helical domain, with a water-filled gap between the two contact surfaces. INTRO +118 132 helical domain structure_element It involves a GTPase domain surface, which is located at the opposite side of the nucleotide, and an interface in the helical domain, with a water-filled gap between the two contact surfaces. INTRO +141 146 water chemical It involves a GTPase domain surface, which is located at the opposite side of the nucleotide, and an interface in the helical domain, with a water-filled gap between the two contact surfaces. INTRO +174 190 contact surfaces site It involves a GTPase domain surface, which is located at the opposite side of the nucleotide, and an interface in the helical domain, with a water-filled gap between the two contact surfaces. INTRO +0 11 Mutagenesis experimental_method "Mutagenesis of the contact surfaces suggests that this ""backside"" interface is not required for GTP-dependent oligomerization or cooperative hydrolysis, despite an earlier suggestion to the contrary." INTRO +19 35 contact surfaces site "Mutagenesis of the contact surfaces suggests that this ""backside"" interface is not required for GTP-dependent oligomerization or cooperative hydrolysis, despite an earlier suggestion to the contrary." INTRO +56 64 backside site "Mutagenesis of the contact surfaces suggests that this ""backside"" interface is not required for GTP-dependent oligomerization or cooperative hydrolysis, despite an earlier suggestion to the contrary." INTRO +66 75 interface site "Mutagenesis of the contact surfaces suggests that this ""backside"" interface is not required for GTP-dependent oligomerization or cooperative hydrolysis, despite an earlier suggestion to the contrary." INTRO +96 99 GTP chemical "Mutagenesis of the contact surfaces suggests that this ""backside"" interface is not required for GTP-dependent oligomerization or cooperative hydrolysis, despite an earlier suggestion to the contrary." INTRO +10 29 biochemical studies experimental_method Extensive biochemical studies suggested that GTP-induced oligomerization of Irga6 requires an interface in the GTPase domain across the nucleotide-binding site. INTRO +45 48 GTP chemical Extensive biochemical studies suggested that GTP-induced oligomerization of Irga6 requires an interface in the GTPase domain across the nucleotide-binding site. INTRO +76 81 Irga6 protein Extensive biochemical studies suggested that GTP-induced oligomerization of Irga6 requires an interface in the GTPase domain across the nucleotide-binding site. INTRO +94 103 interface site Extensive biochemical studies suggested that GTP-induced oligomerization of Irga6 requires an interface in the GTPase domain across the nucleotide-binding site. INTRO +111 124 GTPase domain structure_element Extensive biochemical studies suggested that GTP-induced oligomerization of Irga6 requires an interface in the GTPase domain across the nucleotide-binding site. INTRO +136 159 nucleotide-binding site site Extensive biochemical studies suggested that GTP-induced oligomerization of Irga6 requires an interface in the GTPase domain across the nucleotide-binding site. INTRO +7 25 structural studies experimental_method Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). INTRO +44 55 G interface site Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). INTRO +71 78 dynamin protein_type Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). INTRO +108 115 dynamin protein_type Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). INTRO +117 120 MxA protein Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). INTRO +126 153 guanylate binding protein-1 protein Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). INTRO +155 160 GBP-1 protein Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). INTRO +163 171 atlastin protein_type Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). INTRO +180 189 bacterial taxonomy_domain Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). INTRO +190 211 dynamin-like proteins protein_type Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). INTRO +213 217 BDLP protein_type Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). INTRO +48 59 G interface site For several of these proteins, formation of the G interface was shown to trigger GTP hydrolysis by inducing rearrangements of catalytic residues in cis. INTRO +81 84 GTP chemical For several of these proteins, formation of the G interface was shown to trigger GTP hydrolysis by inducing rearrangements of catalytic residues in cis. INTRO +3 10 dynamin protein_type In dynamin, the G interface includes residues in the phosphate binding loop, the two switch regions, the 'trans stabilizing loop' and the 'G4 loop'. INTRO +16 27 G interface site In dynamin, the G interface includes residues in the phosphate binding loop, the two switch regions, the 'trans stabilizing loop' and the 'G4 loop'. INTRO +53 75 phosphate binding loop structure_element In dynamin, the G interface includes residues in the phosphate binding loop, the two switch regions, the 'trans stabilizing loop' and the 'G4 loop'. INTRO +85 99 switch regions site In dynamin, the G interface includes residues in the phosphate binding loop, the two switch regions, the 'trans stabilizing loop' and the 'G4 loop'. INTRO +106 128 trans stabilizing loop structure_element In dynamin, the G interface includes residues in the phosphate binding loop, the two switch regions, the 'trans stabilizing loop' and the 'G4 loop'. INTRO +139 146 G4 loop structure_element In dynamin, the G interface includes residues in the phosphate binding loop, the two switch regions, the 'trans stabilizing loop' and the 'G4 loop'. INTRO +4 9 Irga6 protein For Irga6, it was demonstrated that besides residues in the switch I and switch II regions, the 3'-OH group of the ribose participates in this interface. INTRO +60 68 switch I site For Irga6, it was demonstrated that besides residues in the switch I and switch II regions, the 3'-OH group of the ribose participates in this interface. INTRO +73 82 switch II site For Irga6, it was demonstrated that besides residues in the switch I and switch II regions, the 3'-OH group of the ribose participates in this interface. INTRO +143 152 interface site For Irga6, it was demonstrated that besides residues in the switch I and switch II regions, the 3'-OH group of the ribose participates in this interface. INTRO +10 44 signal recognition particle GTPase protein_type Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6. INTRO +64 72 receptor protein_type Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6. INTRO +81 84 FfH protein Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6. INTRO +89 93 FtsY protein Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6. INTRO +97 105 bacteria taxonomy_domain Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6. INTRO +145 153 dimerize oligomeric_state Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6. INTRO +160 173 anti-parallel protein_state Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6. INTRO +211 217 GTPase protein_type Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6. INTRO +268 273 Irga6 protein Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6. INTRO +13 30 crystal structure evidence However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. INTRO +34 39 Irga6 protein However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. INTRO +47 58 presence of protein_state However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. INTRO +80 83 GTP chemical However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. INTRO +93 121 5'-guanylyl imidodiphosphate chemical However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. INTRO +123 129 GMPPNP chemical However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. INTRO +178 181 apo protein_state However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. INTRO +185 194 GDP-bound protein_state However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. INTRO +228 243 dimer interface site However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. INTRO +264 277 GTPase domain structure_element However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. INTRO +5 14 structure evidence This structure was obtained by soaking GMPPNP in nucleotide-free crystals of Irga6, an approach which may have interfered with nucleotide-induced domain rearrangements. INTRO +31 38 soaking experimental_method This structure was obtained by soaking GMPPNP in nucleotide-free crystals of Irga6, an approach which may have interfered with nucleotide-induced domain rearrangements. INTRO +39 45 GMPPNP chemical This structure was obtained by soaking GMPPNP in nucleotide-free crystals of Irga6, an approach which may have interfered with nucleotide-induced domain rearrangements. INTRO +49 64 nucleotide-free protein_state This structure was obtained by soaking GMPPNP in nucleotide-free crystals of Irga6, an approach which may have interfered with nucleotide-induced domain rearrangements. INTRO +65 73 crystals evidence This structure was obtained by soaking GMPPNP in nucleotide-free crystals of Irga6, an approach which may have interfered with nucleotide-induced domain rearrangements. INTRO +77 82 Irga6 protein This structure was obtained by soaking GMPPNP in nucleotide-free crystals of Irga6, an approach which may have interfered with nucleotide-induced domain rearrangements. INTRO +41 52 G interface site To clarify the dimerization mode via the G interface, we determined the GMPPNP-bound crystal structure of a non-oligomerizing Irga6 variant. INTRO +57 67 determined experimental_method To clarify the dimerization mode via the G interface, we determined the GMPPNP-bound crystal structure of a non-oligomerizing Irga6 variant. INTRO +72 84 GMPPNP-bound protein_state To clarify the dimerization mode via the G interface, we determined the GMPPNP-bound crystal structure of a non-oligomerizing Irga6 variant. INTRO +85 102 crystal structure evidence To clarify the dimerization mode via the G interface, we determined the GMPPNP-bound crystal structure of a non-oligomerizing Irga6 variant. INTRO +108 125 non-oligomerizing protein_state To clarify the dimerization mode via the G interface, we determined the GMPPNP-bound crystal structure of a non-oligomerizing Irga6 variant. INTRO +126 131 Irga6 protein To clarify the dimerization mode via the G interface, we determined the GMPPNP-bound crystal structure of a non-oligomerizing Irga6 variant. INTRO +132 139 variant protein_state To clarify the dimerization mode via the G interface, we determined the GMPPNP-bound crystal structure of a non-oligomerizing Irga6 variant. INTRO +4 13 structure evidence The structure revealed that Irga6 can dimerize via the G interface in a parallel head-to-head fashion. INTRO +28 33 Irga6 protein The structure revealed that Irga6 can dimerize via the G interface in a parallel head-to-head fashion. INTRO +38 46 dimerize oligomeric_state The structure revealed that Irga6 can dimerize via the G interface in a parallel head-to-head fashion. INTRO +55 66 G interface site The structure revealed that Irga6 can dimerize via the G interface in a parallel head-to-head fashion. INTRO +72 93 parallel head-to-head protein_state The structure revealed that Irga6 can dimerize via the G interface in a parallel head-to-head fashion. INTRO +24 32 parallel protein_state Our data suggest that a parallel dimerization mode may be a unifying feature in all dynamin and septin superfamily proteins. INTRO +84 91 dynamin protein_type Our data suggest that a parallel dimerization mode may be a unifying feature in all dynamin and septin superfamily proteins. INTRO +96 102 septin protein_type Our data suggest that a parallel dimerization mode may be a unifying feature in all dynamin and septin superfamily proteins. INTRO +32 37 Irga6 protein "Previous results indicated that Irga6 mutations in a loosely defined surface region (the ""secondary patch""), which is distant from the G-interface and only slightly overlapping with the backside interface (see below), individually reduced GTP-dependent oligomerization." RESULTS +38 47 mutations experimental_method "Previous results indicated that Irga6 mutations in a loosely defined surface region (the ""secondary patch""), which is distant from the G-interface and only slightly overlapping with the backside interface (see below), individually reduced GTP-dependent oligomerization." RESULTS +69 83 surface region site "Previous results indicated that Irga6 mutations in a loosely defined surface region (the ""secondary patch""), which is distant from the G-interface and only slightly overlapping with the backside interface (see below), individually reduced GTP-dependent oligomerization." RESULTS +90 105 secondary patch site "Previous results indicated that Irga6 mutations in a loosely defined surface region (the ""secondary patch""), which is distant from the G-interface and only slightly overlapping with the backside interface (see below), individually reduced GTP-dependent oligomerization." RESULTS +135 146 G-interface site "Previous results indicated that Irga6 mutations in a loosely defined surface region (the ""secondary patch""), which is distant from the G-interface and only slightly overlapping with the backside interface (see below), individually reduced GTP-dependent oligomerization." RESULTS +186 204 backside interface site "Previous results indicated that Irga6 mutations in a loosely defined surface region (the ""secondary patch""), which is distant from the G-interface and only slightly overlapping with the backside interface (see below), individually reduced GTP-dependent oligomerization." RESULTS +239 242 GTP chemical "Previous results indicated that Irga6 mutations in a loosely defined surface region (the ""secondary patch""), which is distant from the G-interface and only slightly overlapping with the backside interface (see below), individually reduced GTP-dependent oligomerization." RESULTS +2 40 combination of four of these mutations experimental_method A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. RESULTS +42 46 R31E mutant A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. RESULTS +48 52 K32E mutant A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. RESULTS +54 59 K176E mutant A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. RESULTS +65 70 K246E mutant A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. RESULTS +84 94 eliminated protein_state A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. RESULTS +95 98 GTP chemical A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. RESULTS +161 176 crystallization experimental_method A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. RESULTS +180 185 Irga6 protein A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. RESULTS +193 204 presence of protein_state A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. RESULTS +205 211 GMPPNP chemical A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. RESULTS +0 8 Crystals evidence Crystals diffracted to 3.2 Å resolution and displayed one exceptionally long unit cell axis of 1289 Å (Additional file 1: Table S1). RESULTS +4 13 structure evidence The structure was solved by molecular replacement and refined to Rwork/Rfree of 29.7 %/31.7 % (Additional file 1: Table S2). RESULTS +28 49 molecular replacement experimental_method The structure was solved by molecular replacement and refined to Rwork/Rfree of 29.7 %/31.7 % (Additional file 1: Table S2). RESULTS +65 70 Rwork evidence The structure was solved by molecular replacement and refined to Rwork/Rfree of 29.7 %/31.7 % (Additional file 1: Table S2). RESULTS +71 76 Rfree evidence The structure was solved by molecular replacement and refined to Rwork/Rfree of 29.7 %/31.7 % (Additional file 1: Table S2). RESULTS +36 41 Irga6 protein The asymmetric unit contained seven Irga6 molecules that were arranged in a helical pattern along the long cell axis (Additional file 1: Figure S2). RESULTS +0 9 Structure evidence Structure of the Irga6 dimer. FIG +17 22 Irga6 protein Structure of the Irga6 dimer. FIG +23 28 dimer oligomeric_state Structure of the Irga6 dimer. FIG +47 52 mouse taxonomy_domain a Schematic view of the domain architecture of mouse Irga6. FIG +53 58 Irga6 protein a Schematic view of the domain architecture of mouse Irga6. FIG +36 41 Irga6 protein b Ribbon-type representation of the Irga6 dimer. FIG +42 47 dimer oligomeric_state b Ribbon-type representation of the Irga6 dimer. FIG +19 23 Mg2+ chemical The nucleotide and Mg2+ ion (green) are shown in sphere representation. FIG +4 17 GTPase domain structure_element The GTPase domain dimer is boxed. FIG +18 23 dimer oligomeric_state The GTPase domain dimer is boxed. FIG +70 83 GTPase domain structure_element Secondary structure was numbered according to ref.. c Top view on the GTPase domain dimer. FIG +84 89 dimer oligomeric_state Secondary structure was numbered according to ref.. c Top view on the GTPase domain dimer. FIG +23 36 contact sites site d Magnification of the contact sites. FIG +2 15 Superposition experimental_method e Superposition of different switch I conformations in the asymmetric unit; the same colors as in Additional file 1: Figure S2 are used for the switch I regions of the individual subunits. FIG +29 37 switch I site e Superposition of different switch I conformations in the asymmetric unit; the same colors as in Additional file 1: Figure S2 are used for the switch I regions of the individual subunits. FIG +144 152 switch I site e Superposition of different switch I conformations in the asymmetric unit; the same colors as in Additional file 1: Figure S2 are used for the switch I regions of the individual subunits. FIG +0 8 Switch I site Switch I residues of subunit A (yellow) involved in ribose binding are labelled and shown in stick representation. FIG +29 30 A structure_element Switch I residues of subunit A (yellow) involved in ribose binding are labelled and shown in stick representation. FIG +0 5 Irga6 protein Irga6 immunity-related GTPase 6 FIG +6 31 immunity-related GTPase 6 protein Irga6 immunity-related GTPase 6 FIG +11 18 dynamin protein_type Like other dynamin superfamily members, the GTPase domain of Irga6 comprises a canonical GTPase domain fold, with a central β-sheet surrounded by helices on both sides (Fig. 1a-c). RESULTS +44 57 GTPase domain structure_element Like other dynamin superfamily members, the GTPase domain of Irga6 comprises a canonical GTPase domain fold, with a central β-sheet surrounded by helices on both sides (Fig. 1a-c). RESULTS +61 66 Irga6 protein Like other dynamin superfamily members, the GTPase domain of Irga6 comprises a canonical GTPase domain fold, with a central β-sheet surrounded by helices on both sides (Fig. 1a-c). RESULTS +89 102 GTPase domain structure_element Like other dynamin superfamily members, the GTPase domain of Irga6 comprises a canonical GTPase domain fold, with a central β-sheet surrounded by helices on both sides (Fig. 1a-c). RESULTS +124 131 β-sheet structure_element Like other dynamin superfamily members, the GTPase domain of Irga6 comprises a canonical GTPase domain fold, with a central β-sheet surrounded by helices on both sides (Fig. 1a-c). RESULTS +146 153 helices structure_element Like other dynamin superfamily members, the GTPase domain of Irga6 comprises a canonical GTPase domain fold, with a central β-sheet surrounded by helices on both sides (Fig. 1a-c). RESULTS +4 18 helical domain structure_element The helical domain is a bipartite structure composed of helices αA-C at the N-terminus and helix αF-L at the C-terminus of the GTPase domain. RESULTS +56 63 helices structure_element The helical domain is a bipartite structure composed of helices αA-C at the N-terminus and helix αF-L at the C-terminus of the GTPase domain. RESULTS +64 68 αA-C structure_element The helical domain is a bipartite structure composed of helices αA-C at the N-terminus and helix αF-L at the C-terminus of the GTPase domain. RESULTS +91 96 helix structure_element The helical domain is a bipartite structure composed of helices αA-C at the N-terminus and helix αF-L at the C-terminus of the GTPase domain. RESULTS +97 101 αF-L structure_element The helical domain is a bipartite structure composed of helices αA-C at the N-terminus and helix αF-L at the C-terminus of the GTPase domain. RESULTS +127 140 GTPase domain structure_element The helical domain is a bipartite structure composed of helices αA-C at the N-terminus and helix αF-L at the C-terminus of the GTPase domain. RESULTS +89 116 root mean square deviations evidence Overall, the seven molecules in the asymmetric unit are very similar to each other, with root mean square deviations (rmsd) ranging from 0.32 – 0.45 Å over all Cα atoms. RESULTS +118 122 rmsd evidence Overall, the seven molecules in the asymmetric unit are very similar to each other, with root mean square deviations (rmsd) ranging from 0.32 – 0.45 Å over all Cα atoms. RESULTS +4 14 structures evidence The structures of the seven molecules also agree well with the previously determined structure of native GMPPNP-bound Irga6 (PDB: 1TQ6; rmsd of 1.00-1.13 Å over all Cα atoms). RESULTS +85 94 structure evidence The structures of the seven molecules also agree well with the previously determined structure of native GMPPNP-bound Irga6 (PDB: 1TQ6; rmsd of 1.00-1.13 Å over all Cα atoms). RESULTS +98 104 native protein_state The structures of the seven molecules also agree well with the previously determined structure of native GMPPNP-bound Irga6 (PDB: 1TQ6; rmsd of 1.00-1.13 Å over all Cα atoms). RESULTS +105 117 GMPPNP-bound protein_state The structures of the seven molecules also agree well with the previously determined structure of native GMPPNP-bound Irga6 (PDB: 1TQ6; rmsd of 1.00-1.13 Å over all Cα atoms). RESULTS +118 123 Irga6 protein The structures of the seven molecules also agree well with the previously determined structure of native GMPPNP-bound Irga6 (PDB: 1TQ6; rmsd of 1.00-1.13 Å over all Cα atoms). RESULTS +136 140 rmsd evidence The structures of the seven molecules also agree well with the previously determined structure of native GMPPNP-bound Irga6 (PDB: 1TQ6; rmsd of 1.00-1.13 Å over all Cα atoms). RESULTS +10 15 Irga6 protein The seven Irga6 molecules in the asymmetric unit form various higher order contacts in the crystals. RESULTS +91 99 crystals evidence The seven Irga6 molecules in the asymmetric unit form various higher order contacts in the crystals. RESULTS +42 50 dimerize oligomeric_state Within the asymmetric unit, six molecules dimerize via the symmetric backside dimer interface (buried surface area 930 Å2), and the remaining seventh molecule forms the same type of interaction with its symmetry mate of the adjacent asymmetric unit (Additional file 1: Figure S2a, b, Figure S3). RESULTS +69 93 backside dimer interface site Within the asymmetric unit, six molecules dimerize via the symmetric backside dimer interface (buried surface area 930 Å2), and the remaining seventh molecule forms the same type of interaction with its symmetry mate of the adjacent asymmetric unit (Additional file 1: Figure S2a, b, Figure S3). RESULTS +35 44 mutations experimental_method This indicates that the introduced mutations in the secondary patch, from which only Lys176 is part of the backside interface, do, in fact, not prevent this interaction. RESULTS +52 67 secondary patch site This indicates that the introduced mutations in the secondary patch, from which only Lys176 is part of the backside interface, do, in fact, not prevent this interaction. RESULTS +85 91 Lys176 residue_name_number This indicates that the introduced mutations in the secondary patch, from which only Lys176 is part of the backside interface, do, in fact, not prevent this interaction. RESULTS +107 125 backside interface site This indicates that the introduced mutations in the secondary patch, from which only Lys176 is part of the backside interface, do, in fact, not prevent this interaction. RESULTS +8 26 assembly interface site Another assembly interface with a buried surface area of 450 Å2, which we call the “tertiary patch”, was formed via two interaction sites in the helical domains (Additional file 1: Figure S2c, d, S3). RESULTS +84 98 tertiary patch site Another assembly interface with a buried surface area of 450 Å2, which we call the “tertiary patch”, was formed via two interaction sites in the helical domains (Additional file 1: Figure S2c, d, S3). RESULTS +120 137 interaction sites site Another assembly interface with a buried surface area of 450 Å2, which we call the “tertiary patch”, was formed via two interaction sites in the helical domains (Additional file 1: Figure S2c, d, S3). RESULTS +145 160 helical domains structure_element Another assembly interface with a buried surface area of 450 Å2, which we call the “tertiary patch”, was formed via two interaction sites in the helical domains (Additional file 1: Figure S2c, d, S3). RESULTS +8 17 interface site In this interface, helices αK from two adjacent molecules form a hydrogen bonding network involving residues 373-376. RESULTS +19 26 helices structure_element In this interface, helices αK from two adjacent molecules form a hydrogen bonding network involving residues 373-376. RESULTS +27 29 αK structure_element In this interface, helices αK from two adjacent molecules form a hydrogen bonding network involving residues 373-376. RESULTS +65 89 hydrogen bonding network bond_interaction In this interface, helices αK from two adjacent molecules form a hydrogen bonding network involving residues 373-376. RESULTS +109 116 373-376 residue_range In this interface, helices αK from two adjacent molecules form a hydrogen bonding network involving residues 373-376. RESULTS +26 33 helices structure_element Furthermore, two adjacent helices αA form hydrophobic contacts. RESULTS +34 36 αA structure_element Furthermore, two adjacent helices αA form hydrophobic contacts. RESULTS +42 62 hydrophobic contacts bond_interaction Furthermore, two adjacent helices αA form hydrophobic contacts. RESULTS +33 48 double mutation protein_state It was previously shown that the double mutation L372R/A373R did not prevent GTP-induced assembly, so there is currently no evidence supporting an involvement of this interface in higher-order oligomerization. RESULTS +49 54 L372R mutant It was previously shown that the double mutation L372R/A373R did not prevent GTP-induced assembly, so there is currently no evidence supporting an involvement of this interface in higher-order oligomerization. RESULTS +55 60 A373R mutant It was previously shown that the double mutation L372R/A373R did not prevent GTP-induced assembly, so there is currently no evidence supporting an involvement of this interface in higher-order oligomerization. RESULTS +77 80 GTP chemical It was previously shown that the double mutation L372R/A373R did not prevent GTP-induced assembly, so there is currently no evidence supporting an involvement of this interface in higher-order oligomerization. RESULTS +167 176 interface site It was previously shown that the double mutation L372R/A373R did not prevent GTP-induced assembly, so there is currently no evidence supporting an involvement of this interface in higher-order oligomerization. RESULTS +21 22 A structure_element Strikingly, molecule A of one asymmetric unit assembled with an equivalent molecule of the adjacent asymmetric unit via the G-interface in a symmetric parallel fashion via a 470 Å2 interface. RESULTS +124 135 G-interface site Strikingly, molecule A of one asymmetric unit assembled with an equivalent molecule of the adjacent asymmetric unit via the G-interface in a symmetric parallel fashion via a 470 Å2 interface. RESULTS +151 159 parallel protein_state Strikingly, molecule A of one asymmetric unit assembled with an equivalent molecule of the adjacent asymmetric unit via the G-interface in a symmetric parallel fashion via a 470 Å2 interface. RESULTS +27 43 butterfly-shaped protein_state This assembly results in a butterfly-shaped Irga6 dimer in which the helical domains protrude in parallel orientations (Fig. 1b, Additional file 1: Figure S3). RESULTS +44 49 Irga6 protein This assembly results in a butterfly-shaped Irga6 dimer in which the helical domains protrude in parallel orientations (Fig. 1b, Additional file 1: Figure S3). RESULTS +50 55 dimer oligomeric_state This assembly results in a butterfly-shaped Irga6 dimer in which the helical domains protrude in parallel orientations (Fig. 1b, Additional file 1: Figure S3). RESULTS +69 84 helical domains structure_element This assembly results in a butterfly-shaped Irga6 dimer in which the helical domains protrude in parallel orientations (Fig. 1b, Additional file 1: Figure S3). RESULTS +97 105 parallel protein_state This assembly results in a butterfly-shaped Irga6 dimer in which the helical domains protrude in parallel orientations (Fig. 1b, Additional file 1: Figure S3). RESULTS +84 95 G interface site In contrast, the other six molecules in the asymmetric unit do not assemble via the G interface. RESULTS +4 15 G interface site The G interface in molecule A can be subdivided into three distinct contact sites (Fig. 1c, d). RESULTS +68 81 contact sites site The G interface in molecule A can be subdivided into three distinct contact sites (Fig. 1c, d). RESULTS +0 14 Contact site I site Contact site I is formed between R159 and K161 in the trans stabilizing loops, and S132 in the switch II regions of the opposing molecules. RESULTS +33 37 R159 residue_name_number Contact site I is formed between R159 and K161 in the trans stabilizing loops, and S132 in the switch II regions of the opposing molecules. RESULTS +42 46 K161 residue_name_number Contact site I is formed between R159 and K161 in the trans stabilizing loops, and S132 in the switch II regions of the opposing molecules. RESULTS +54 77 trans stabilizing loops structure_element Contact site I is formed between R159 and K161 in the trans stabilizing loops, and S132 in the switch II regions of the opposing molecules. RESULTS +83 87 S132 residue_name_number Contact site I is formed between R159 and K161 in the trans stabilizing loops, and S132 in the switch II regions of the opposing molecules. RESULTS +95 104 switch II site Contact site I is formed between R159 and K161 in the trans stabilizing loops, and S132 in the switch II regions of the opposing molecules. RESULTS +0 15 Contact site II site Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +25 59 polar and hydrophobic interactions bond_interaction Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +70 78 switch I site Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +80 84 V104 residue_name_number Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +86 90 V107 residue_name_number Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +99 104 helix structure_element Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +119 144 guanine specificity motif structure_element Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +146 154 G4 helix structure_element Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +156 160 K184 residue_name_number Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +165 169 S187 residue_name_number Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +179 201 trans stabilizing loop structure_element Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +203 207 T158 residue_name_number Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +225 238 GTPase domain structure_element Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. RESULTS +3 19 contact site III site In contact site III, G103 of switch I interacts via its main chain nitrogen with the exocyclic 2’-OH and 3’-OH groups of the opposing ribose in trans, whereas the two opposing exocyclic 3’-OH group of the ribose form hydrogen bonds with each other. RESULTS +21 25 G103 residue_name_number In contact site III, G103 of switch I interacts via its main chain nitrogen with the exocyclic 2’-OH and 3’-OH groups of the opposing ribose in trans, whereas the two opposing exocyclic 3’-OH group of the ribose form hydrogen bonds with each other. RESULTS +29 37 switch I site In contact site III, G103 of switch I interacts via its main chain nitrogen with the exocyclic 2’-OH and 3’-OH groups of the opposing ribose in trans, whereas the two opposing exocyclic 3’-OH group of the ribose form hydrogen bonds with each other. RESULTS +134 140 ribose chemical In contact site III, G103 of switch I interacts via its main chain nitrogen with the exocyclic 2’-OH and 3’-OH groups of the opposing ribose in trans, whereas the two opposing exocyclic 3’-OH group of the ribose form hydrogen bonds with each other. RESULTS +205 211 ribose chemical In contact site III, G103 of switch I interacts via its main chain nitrogen with the exocyclic 2’-OH and 3’-OH groups of the opposing ribose in trans, whereas the two opposing exocyclic 3’-OH group of the ribose form hydrogen bonds with each other. RESULTS +217 231 hydrogen bonds bond_interaction In contact site III, G103 of switch I interacts via its main chain nitrogen with the exocyclic 2’-OH and 3’-OH groups of the opposing ribose in trans, whereas the two opposing exocyclic 3’-OH group of the ribose form hydrogen bonds with each other. RESULTS +8 14 ribose chemical Via the ribose contact, switch I is pulled towards the opposing nucleotide (Fig. 1e). RESULTS +24 32 switch I site Via the ribose contact, switch I is pulled towards the opposing nucleotide (Fig. 1e). RESULTS +64 74 nucleotide chemical Via the ribose contact, switch I is pulled towards the opposing nucleotide (Fig. 1e). RESULTS +9 13 E106 residue_name_number In turn, E106 of switch I reorients towards the nucleotide and now participates in the coordination of the Mg2+ ion (Fig. 1e, Additional file 1: Figure S4). RESULTS +17 25 switch I site In turn, E106 of switch I reorients towards the nucleotide and now participates in the coordination of the Mg2+ ion (Fig. 1e, Additional file 1: Figure S4). RESULTS +48 58 nucleotide chemical In turn, E106 of switch I reorients towards the nucleotide and now participates in the coordination of the Mg2+ ion (Fig. 1e, Additional file 1: Figure S4). RESULTS +87 102 coordination of bond_interaction In turn, E106 of switch I reorients towards the nucleotide and now participates in the coordination of the Mg2+ ion (Fig. 1e, Additional file 1: Figure S4). RESULTS +107 111 Mg2+ chemical In turn, E106 of switch I reorients towards the nucleotide and now participates in the coordination of the Mg2+ ion (Fig. 1e, Additional file 1: Figure S4). RESULTS +0 4 E106 residue_name_number E106 was previously shown to be essential for catalysis, and the observed interactions in contact site III explain how dimerization via the ribose is directly coupled to the activation of GTP hydrolysis. RESULTS +90 106 contact site III site E106 was previously shown to be essential for catalysis, and the observed interactions in contact site III explain how dimerization via the ribose is directly coupled to the activation of GTP hydrolysis. RESULTS +140 146 ribose chemical E106 was previously shown to be essential for catalysis, and the observed interactions in contact site III explain how dimerization via the ribose is directly coupled to the activation of GTP hydrolysis. RESULTS +188 191 GTP chemical E106 was previously shown to be essential for catalysis, and the observed interactions in contact site III explain how dimerization via the ribose is directly coupled to the activation of GTP hydrolysis. RESULTS +4 15 G interface site The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. RESULTS +111 114 E77 residue_name_number The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. RESULTS +116 120 G103 residue_name_number The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. RESULTS +122 126 E106 residue_name_number The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. RESULTS +128 132 S132 residue_name_number The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. RESULTS +134 138 R159 residue_name_number The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. RESULTS +140 144 K161 residue_name_number The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. RESULTS +146 150 K162 residue_name_number The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. RESULTS +152 156 D164 residue_name_number The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. RESULTS +158 162 N191 residue_name_number The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. RESULTS +168 172 K196 residue_name_number The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. RESULTS +221 224 GTP chemical The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. RESULTS +56 60 G103 residue_name_number All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). RESULTS +62 66 S132 residue_name_number All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). RESULTS +68 72 R159 residue_name_number All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). RESULTS +78 82 K161 residue_name_number All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). RESULTS +117 126 interface site All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). RESULTS +128 131 E77 residue_name_number All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). RESULTS +133 137 E106 residue_name_number All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). RESULTS +139 143 K162 residue_name_number All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). RESULTS +145 149 D164 residue_name_number All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). RESULTS +155 159 N191 residue_name_number All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). RESULTS +9 12 E77 residue_name_number Residues E77, K162, and D164 appear to orient the trans stabilizing loop which is involved in interface formation in contact site II. RESULTS +14 18 K162 residue_name_number Residues E77, K162, and D164 appear to orient the trans stabilizing loop which is involved in interface formation in contact site II. RESULTS +24 28 D164 residue_name_number Residues E77, K162, and D164 appear to orient the trans stabilizing loop which is involved in interface formation in contact site II. RESULTS +50 72 trans stabilizing loop structure_element Residues E77, K162, and D164 appear to orient the trans stabilizing loop which is involved in interface formation in contact site II. RESULTS +94 103 interface site Residues E77, K162, and D164 appear to orient the trans stabilizing loop which is involved in interface formation in contact site II. RESULTS +117 132 contact site II site Residues E77, K162, and D164 appear to orient the trans stabilizing loop which is involved in interface formation in contact site II. RESULTS +27 40 anti-parallel protein_state In the earlier model of an anti-parallel G interface, it was not possible to position the side chain of R159 to avoid steric conflict. RESULTS +41 52 G interface site In the earlier model of an anti-parallel G interface, it was not possible to position the side chain of R159 to avoid steric conflict. RESULTS +104 108 R159 residue_name_number In the earlier model of an anti-parallel G interface, it was not possible to position the side chain of R159 to avoid steric conflict. RESULTS +15 24 structure evidence In the present structure, the side-chain of R159 projects laterally along the G interface and, therefore, does not cause a steric conflict. RESULTS +44 48 R159 residue_name_number In the present structure, the side-chain of R159 projects laterally along the G interface and, therefore, does not cause a steric conflict. RESULTS +78 89 G interface site In the present structure, the side-chain of R159 projects laterally along the G interface and, therefore, does not cause a steric conflict. RESULTS +38 49 G interface site A conserved dimerization mode via the G interface in dynamin and septin GTPases. FIG +53 60 dynamin protein_type A conserved dimerization mode via the G interface in dynamin and septin GTPases. FIG +65 79 septin GTPases protein_type A conserved dimerization mode via the G interface in dynamin and septin GTPases. FIG +32 40 parallel protein_state The overall architecture of the parallel GTPase domain dimer of Irga6 is related to that of other dynamin and septin superfamily proteins. FIG +41 54 GTPase domain structure_element The overall architecture of the parallel GTPase domain dimer of Irga6 is related to that of other dynamin and septin superfamily proteins. FIG +55 60 dimer oligomeric_state The overall architecture of the parallel GTPase domain dimer of Irga6 is related to that of other dynamin and septin superfamily proteins. FIG +64 69 Irga6 protein The overall architecture of the parallel GTPase domain dimer of Irga6 is related to that of other dynamin and septin superfamily proteins. FIG +98 105 dynamin protein_type The overall architecture of the parallel GTPase domain dimer of Irga6 is related to that of other dynamin and septin superfamily proteins. FIG +110 116 septin protein_type The overall architecture of the parallel GTPase domain dimer of Irga6 is related to that of other dynamin and septin superfamily proteins. FIG +14 24 structures evidence The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +97 111 GTPase domains structure_element The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +119 131 GMPPNP-bound protein_state The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +132 137 Irga6 protein The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +138 143 dimer oligomeric_state The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +168 177 dynamin 1 protein The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +178 192 GTPase-minimal structure_element The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +225 234 GDP-bound protein_state The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +235 245 atlastin 1 protein The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +246 251 dimer oligomeric_state The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +270 285 GDP-AlF3- bound protein_state The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +286 290 GBP1 protein The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +291 304 GTPase domain structure_element The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +305 310 dimer oligomeric_state The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +329 333 BDLP protein_type The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +334 339 dimer oligomeric_state The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +340 348 bound to protein_state The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +349 352 GDP chemical The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +374 383 GTP-bound protein_state The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +384 390 GIMAP2 protein The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +391 396 dimer oligomeric_state The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. FIG +4 18 GTPase domains structure_element The GTPase domains of the left molecules are shown in orange, helical domains or extensions in blue. FIG +62 77 helical domains structure_element The GTPase domains of the left molecules are shown in orange, helical domains or extensions in blue. FIG +12 16 Mg2+ chemical Nucleotide, Mg2+ (green) and AlF4 - are shown in sphere representation, the buried interface sizes per molecule are indicated on the right. FIG +83 92 interface site Nucleotide, Mg2+ (green) and AlF4 - are shown in sphere representation, the buried interface sizes per molecule are indicated on the right. FIG +0 5 Irga6 protein Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 FIG +6 31 immunity-related GTPase 6 protein Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 FIG +33 39 GMPPNP chemical Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 FIG +40 68 5'-guanylyl imidodiphosphate chemical Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 FIG +70 73 GTP chemical Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 FIG +74 96 guanosine-triphosphate chemical Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 FIG +98 102 BDLP protein_type Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 FIG +103 112 bacterial taxonomy_domain Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 FIG +113 133 dynamin like protein protein_type Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 FIG +135 141 GIMAP2 protein Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 FIG +143 182 GTPase of immunity associated protein 2 protein Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2 FIG +50 61 G interface site The buried surface area per molecule (BSA) of the G interface in Irga6 is relatively small (470 Å2) compared to that of other dynamin superfamily members, such as dynamin (BSA: 1400 Å2), atlastin (BSA: 820 Å2), GBP-1 (BSA: 2060 Å2), BDLP (BSA: 2300 Å2) or the septin-related GTPase of immunity associated protein 2 (GIMAP2) (BSA: 590 Å2) (Fig. 2). RESULTS +65 70 Irga6 protein The buried surface area per molecule (BSA) of the G interface in Irga6 is relatively small (470 Å2) compared to that of other dynamin superfamily members, such as dynamin (BSA: 1400 Å2), atlastin (BSA: 820 Å2), GBP-1 (BSA: 2060 Å2), BDLP (BSA: 2300 Å2) or the septin-related GTPase of immunity associated protein 2 (GIMAP2) (BSA: 590 Å2) (Fig. 2). RESULTS +126 133 dynamin protein_type The buried surface area per molecule (BSA) of the G interface in Irga6 is relatively small (470 Å2) compared to that of other dynamin superfamily members, such as dynamin (BSA: 1400 Å2), atlastin (BSA: 820 Å2), GBP-1 (BSA: 2060 Å2), BDLP (BSA: 2300 Å2) or the septin-related GTPase of immunity associated protein 2 (GIMAP2) (BSA: 590 Å2) (Fig. 2). RESULTS +163 170 dynamin protein_type The buried surface area per molecule (BSA) of the G interface in Irga6 is relatively small (470 Å2) compared to that of other dynamin superfamily members, such as dynamin (BSA: 1400 Å2), atlastin (BSA: 820 Å2), GBP-1 (BSA: 2060 Å2), BDLP (BSA: 2300 Å2) or the septin-related GTPase of immunity associated protein 2 (GIMAP2) (BSA: 590 Å2) (Fig. 2). RESULTS +187 195 atlastin protein_type The buried surface area per molecule (BSA) of the G interface in Irga6 is relatively small (470 Å2) compared to that of other dynamin superfamily members, such as dynamin (BSA: 1400 Å2), atlastin (BSA: 820 Å2), GBP-1 (BSA: 2060 Å2), BDLP (BSA: 2300 Å2) or the septin-related GTPase of immunity associated protein 2 (GIMAP2) (BSA: 590 Å2) (Fig. 2). RESULTS +211 216 GBP-1 protein The buried surface area per molecule (BSA) of the G interface in Irga6 is relatively small (470 Å2) compared to that of other dynamin superfamily members, such as dynamin (BSA: 1400 Å2), atlastin (BSA: 820 Å2), GBP-1 (BSA: 2060 Å2), BDLP (BSA: 2300 Å2) or the septin-related GTPase of immunity associated protein 2 (GIMAP2) (BSA: 590 Å2) (Fig. 2). RESULTS +233 237 BDLP protein_type The buried surface area per molecule (BSA) of the G interface in Irga6 is relatively small (470 Å2) compared to that of other dynamin superfamily members, such as dynamin (BSA: 1400 Å2), atlastin (BSA: 820 Å2), GBP-1 (BSA: 2060 Å2), BDLP (BSA: 2300 Å2) or the septin-related GTPase of immunity associated protein 2 (GIMAP2) (BSA: 590 Å2) (Fig. 2). RESULTS +260 314 septin-related GTPase of immunity associated protein 2 protein The buried surface area per molecule (BSA) of the G interface in Irga6 is relatively small (470 Å2) compared to that of other dynamin superfamily members, such as dynamin (BSA: 1400 Å2), atlastin (BSA: 820 Å2), GBP-1 (BSA: 2060 Å2), BDLP (BSA: 2300 Å2) or the septin-related GTPase of immunity associated protein 2 (GIMAP2) (BSA: 590 Å2) (Fig. 2). RESULTS +316 322 GIMAP2 protein The buried surface area per molecule (BSA) of the G interface in Irga6 is relatively small (470 Å2) compared to that of other dynamin superfamily members, such as dynamin (BSA: 1400 Å2), atlastin (BSA: 820 Å2), GBP-1 (BSA: 2060 Å2), BDLP (BSA: 2300 Å2) or the septin-related GTPase of immunity associated protein 2 (GIMAP2) (BSA: 590 Å2) (Fig. 2). RESULTS +42 56 GTPase domains structure_element However, the relative orientations of the GTPase domains in these dimers are strikingly similar, and the same elements, such as switch I, switch II, the trans activating and G4 loops are involved in the parallel dimerization mode in all of these GTPase families. RESULTS +66 72 dimers oligomeric_state However, the relative orientations of the GTPase domains in these dimers are strikingly similar, and the same elements, such as switch I, switch II, the trans activating and G4 loops are involved in the parallel dimerization mode in all of these GTPase families. RESULTS +128 136 switch I site However, the relative orientations of the GTPase domains in these dimers are strikingly similar, and the same elements, such as switch I, switch II, the trans activating and G4 loops are involved in the parallel dimerization mode in all of these GTPase families. RESULTS +138 147 switch II site However, the relative orientations of the GTPase domains in these dimers are strikingly similar, and the same elements, such as switch I, switch II, the trans activating and G4 loops are involved in the parallel dimerization mode in all of these GTPase families. RESULTS +153 182 trans activating and G4 loops structure_element However, the relative orientations of the GTPase domains in these dimers are strikingly similar, and the same elements, such as switch I, switch II, the trans activating and G4 loops are involved in the parallel dimerization mode in all of these GTPase families. RESULTS +203 211 parallel protein_state However, the relative orientations of the GTPase domains in these dimers are strikingly similar, and the same elements, such as switch I, switch II, the trans activating and G4 loops are involved in the parallel dimerization mode in all of these GTPase families. RESULTS +246 252 GTPase protein_type However, the relative orientations of the GTPase domains in these dimers are strikingly similar, and the same elements, such as switch I, switch II, the trans activating and G4 loops are involved in the parallel dimerization mode in all of these GTPase families. RESULTS +0 3 IRG protein_type IRG proteins are crucial mediators of the innate immune response in mice against a specific subset of intracellular pathogens, all of which enter the cell to form a membrane-bounded vacuole without engagement of the phagocytic machinery. DISCUSS +68 72 mice taxonomy_domain IRG proteins are crucial mediators of the innate immune response in mice against a specific subset of intracellular pathogens, all of which enter the cell to form a membrane-bounded vacuole without engagement of the phagocytic machinery. DISCUSS +18 25 dynamin protein_type As members of the dynamin superfamily, IRGs oligomerize at cellular membranes in response to GTP binding. DISCUSS +39 43 IRGs protein_type As members of the dynamin superfamily, IRGs oligomerize at cellular membranes in response to GTP binding. DISCUSS +93 96 GTP chemical As members of the dynamin superfamily, IRGs oligomerize at cellular membranes in response to GTP binding. DISCUSS +44 47 GTP chemical Oligomerization and oligomerization-induced GTP hydrolysis are thought to induce membrane remodeling events ultimately leading to disruption of the PVM. DISCUSS +7 42 structural and mechanistic analyses experimental_method Recent structural and mechanistic analyses have begun to unravel the molecular basis for the membrane-remodeling activity and mechano-chemical function of some members (reviewed in). DISCUSS +17 24 dynamin protein_type For example, for dynamin and atlastin, it was shown that GTP binding and/or hydrolysis leads to dimerization of the GTPase domains and to the reorientation of the adjacent helical domains. DISCUSS +29 37 atlastin protein_type For example, for dynamin and atlastin, it was shown that GTP binding and/or hydrolysis leads to dimerization of the GTPase domains and to the reorientation of the adjacent helical domains. DISCUSS +57 60 GTP chemical For example, for dynamin and atlastin, it was shown that GTP binding and/or hydrolysis leads to dimerization of the GTPase domains and to the reorientation of the adjacent helical domains. DISCUSS +116 130 GTPase domains structure_element For example, for dynamin and atlastin, it was shown that GTP binding and/or hydrolysis leads to dimerization of the GTPase domains and to the reorientation of the adjacent helical domains. DISCUSS +172 187 helical domains structure_element For example, for dynamin and atlastin, it was shown that GTP binding and/or hydrolysis leads to dimerization of the GTPase domains and to the reorientation of the adjacent helical domains. DISCUSS +19 26 dynamin protein_type However, for other dynamin superfamily members such as IRGs, the molecular basis for GTP hydrolysis and the exact role of the mechano-chemical function are still unclear. DISCUSS +55 59 IRGs protein_type However, for other dynamin superfamily members such as IRGs, the molecular basis for GTP hydrolysis and the exact role of the mechano-chemical function are still unclear. DISCUSS +85 88 GTP chemical However, for other dynamin superfamily members such as IRGs, the molecular basis for GTP hydrolysis and the exact role of the mechano-chemical function are still unclear. DISCUSS +4 23 structural analysis experimental_method Our structural analysis of an oligomerization- and GTPase-defective Irga6 mutant indicates that Irga6 dimerizes via the G interface in a parallel orientation. DISCUSS +30 67 oligomerization- and GTPase-defective protein_state Our structural analysis of an oligomerization- and GTPase-defective Irga6 mutant indicates that Irga6 dimerizes via the G interface in a parallel orientation. DISCUSS +68 73 Irga6 protein Our structural analysis of an oligomerization- and GTPase-defective Irga6 mutant indicates that Irga6 dimerizes via the G interface in a parallel orientation. DISCUSS +74 80 mutant protein_state Our structural analysis of an oligomerization- and GTPase-defective Irga6 mutant indicates that Irga6 dimerizes via the G interface in a parallel orientation. DISCUSS +96 101 Irga6 protein Our structural analysis of an oligomerization- and GTPase-defective Irga6 mutant indicates that Irga6 dimerizes via the G interface in a parallel orientation. DISCUSS +102 111 dimerizes oligomeric_state Our structural analysis of an oligomerization- and GTPase-defective Irga6 mutant indicates that Irga6 dimerizes via the G interface in a parallel orientation. DISCUSS +120 131 G interface site Our structural analysis of an oligomerization- and GTPase-defective Irga6 mutant indicates that Irga6 dimerizes via the G interface in a parallel orientation. DISCUSS +137 145 parallel protein_state Our structural analysis of an oligomerization- and GTPase-defective Irga6 mutant indicates that Irga6 dimerizes via the G interface in a parallel orientation. DISCUSS +22 27 Irga6 protein Only one of the seven Irga6 molecules in the asymmetric unit formed this contact pointing to a low affinity interaction via the G interface, which is in agreement with its small size. DISCUSS +128 139 G interface site Only one of the seven Irga6 molecules in the asymmetric unit formed this contact pointing to a low affinity interaction via the G interface, which is in agreement with its small size. DISCUSS +7 15 crystals evidence In the crystals, dimerization via the G interface is promoted by the high protein concentrations which may mimic a situation when Irga6 oligomerizes on a membrane surface. DISCUSS +38 49 G interface site In the crystals, dimerization via the G interface is promoted by the high protein concentrations which may mimic a situation when Irga6 oligomerizes on a membrane surface. DISCUSS +130 135 Irga6 protein In the crystals, dimerization via the G interface is promoted by the high protein concentrations which may mimic a situation when Irga6 oligomerizes on a membrane surface. DISCUSS +90 93 GTP chemical Such a low affinity interaction mode may allow reversibility of oligomerization following GTP hydrolysis. DISCUSS +21 32 G interface site Similar low affinity G interface interactions were reported for dynamin and MxA. DISCUSS +64 71 dynamin protein_type Similar low affinity G interface interactions were reported for dynamin and MxA. DISCUSS +76 79 MxA protein Similar low affinity G interface interactions were reported for dynamin and MxA. DISCUSS +75 88 anti-parallel protein_state The dimerization mode is strikingly different from the previously proposed anti-parallel model that was based on the crystal structure of the signal recognition particle GTPase, SRP54 and its homologous receptor. DISCUSS +117 134 crystal structure evidence The dimerization mode is strikingly different from the previously proposed anti-parallel model that was based on the crystal structure of the signal recognition particle GTPase, SRP54 and its homologous receptor. DISCUSS +142 176 signal recognition particle GTPase protein_type The dimerization mode is strikingly different from the previously proposed anti-parallel model that was based on the crystal structure of the signal recognition particle GTPase, SRP54 and its homologous receptor. DISCUSS +178 183 SRP54 protein The dimerization mode is strikingly different from the previously proposed anti-parallel model that was based on the crystal structure of the signal recognition particle GTPase, SRP54 and its homologous receptor. DISCUSS +13 30 G dimer interface site However, the G dimer interface is reminiscent of the GTPase domain dimers observed for several other dynamin superfamily members, such as dynamin, GBP1, atlastin, and BDLP. DISCUSS +53 66 GTPase domain structure_element However, the G dimer interface is reminiscent of the GTPase domain dimers observed for several other dynamin superfamily members, such as dynamin, GBP1, atlastin, and BDLP. DISCUSS +67 73 dimers oligomeric_state However, the G dimer interface is reminiscent of the GTPase domain dimers observed for several other dynamin superfamily members, such as dynamin, GBP1, atlastin, and BDLP. DISCUSS +101 108 dynamin protein_type However, the G dimer interface is reminiscent of the GTPase domain dimers observed for several other dynamin superfamily members, such as dynamin, GBP1, atlastin, and BDLP. DISCUSS +138 145 dynamin protein_type However, the G dimer interface is reminiscent of the GTPase domain dimers observed for several other dynamin superfamily members, such as dynamin, GBP1, atlastin, and BDLP. DISCUSS +147 151 GBP1 protein However, the G dimer interface is reminiscent of the GTPase domain dimers observed for several other dynamin superfamily members, such as dynamin, GBP1, atlastin, and BDLP. DISCUSS +153 161 atlastin protein_type However, the G dimer interface is reminiscent of the GTPase domain dimers observed for several other dynamin superfamily members, such as dynamin, GBP1, atlastin, and BDLP. DISCUSS +167 171 BDLP protein_type However, the G dimer interface is reminiscent of the GTPase domain dimers observed for several other dynamin superfamily members, such as dynamin, GBP1, atlastin, and BDLP. DISCUSS +27 33 septin protein_type It was recently shown that septin and septin-related GTPases, such as the Tocs GTPases or GTPases of immunity related proteins (GIMAPs), also employ a GTP-dependent parallel dimerization mode. DISCUSS +38 60 septin-related GTPases protein_type It was recently shown that septin and septin-related GTPases, such as the Tocs GTPases or GTPases of immunity related proteins (GIMAPs), also employ a GTP-dependent parallel dimerization mode. DISCUSS +74 86 Tocs GTPases protein_type It was recently shown that septin and septin-related GTPases, such as the Tocs GTPases or GTPases of immunity related proteins (GIMAPs), also employ a GTP-dependent parallel dimerization mode. DISCUSS +90 126 GTPases of immunity related proteins protein_type It was recently shown that septin and septin-related GTPases, such as the Tocs GTPases or GTPases of immunity related proteins (GIMAPs), also employ a GTP-dependent parallel dimerization mode. DISCUSS +128 134 GIMAPs protein_type It was recently shown that septin and septin-related GTPases, such as the Tocs GTPases or GTPases of immunity related proteins (GIMAPs), also employ a GTP-dependent parallel dimerization mode. DISCUSS +151 154 GTP chemical It was recently shown that septin and septin-related GTPases, such as the Tocs GTPases or GTPases of immunity related proteins (GIMAPs), also employ a GTP-dependent parallel dimerization mode. DISCUSS +165 173 parallel protein_state It was recently shown that septin and septin-related GTPases, such as the Tocs GTPases or GTPases of immunity related proteins (GIMAPs), also employ a GTP-dependent parallel dimerization mode. DISCUSS +9 45 phylogenetic and structural analysis experimental_method Based on phylogenetic and structural analysis, these observations suggest that dynamin and septin superfamilies are derived from a common ancestral membrane-associated GTPase that featured a GTP-dependent parallel dimerization mode. DISCUSS +79 86 dynamin protein_type Based on phylogenetic and structural analysis, these observations suggest that dynamin and septin superfamilies are derived from a common ancestral membrane-associated GTPase that featured a GTP-dependent parallel dimerization mode. DISCUSS +91 97 septin protein_type Based on phylogenetic and structural analysis, these observations suggest that dynamin and septin superfamilies are derived from a common ancestral membrane-associated GTPase that featured a GTP-dependent parallel dimerization mode. DISCUSS +148 174 membrane-associated GTPase protein_type Based on phylogenetic and structural analysis, these observations suggest that dynamin and septin superfamilies are derived from a common ancestral membrane-associated GTPase that featured a GTP-dependent parallel dimerization mode. DISCUSS +191 194 GTP chemical Based on phylogenetic and structural analysis, these observations suggest that dynamin and septin superfamilies are derived from a common ancestral membrane-associated GTPase that featured a GTP-dependent parallel dimerization mode. DISCUSS +205 213 parallel protein_state Based on phylogenetic and structural analysis, these observations suggest that dynamin and septin superfamilies are derived from a common ancestral membrane-associated GTPase that featured a GTP-dependent parallel dimerization mode. DISCUSS +41 45 IRGs protein_type Importantly, our analysis indicates that IRGs are not outliers, but bona-fide representatives of the dynamin superfamily. DISCUSS +101 108 dynamin protein_type Importantly, our analysis indicates that IRGs are not outliers, but bona-fide representatives of the dynamin superfamily. DISCUSS +52 58 septin protein_type Whereas the overall dimerization mode is similar in septin and dynamin GTPases, family-specific differences in the G interface and the oligomerization interfaces exist. DISCUSS +63 78 dynamin GTPases protein_type Whereas the overall dimerization mode is similar in septin and dynamin GTPases, family-specific differences in the G interface and the oligomerization interfaces exist. DISCUSS +115 126 G interface site Whereas the overall dimerization mode is similar in septin and dynamin GTPases, family-specific differences in the G interface and the oligomerization interfaces exist. DISCUSS +135 161 oligomerization interfaces site Whereas the overall dimerization mode is similar in septin and dynamin GTPases, family-specific differences in the G interface and the oligomerization interfaces exist. DISCUSS +63 69 ribose chemical For example, the involvement of the 2’ and 3’-OH groups of the ribose in the dimerization interface of Irga6 has not been observed for other dynamin and septin superfamily members. DISCUSS +77 99 dimerization interface site For example, the involvement of the 2’ and 3’-OH groups of the ribose in the dimerization interface of Irga6 has not been observed for other dynamin and septin superfamily members. DISCUSS +103 108 Irga6 protein For example, the involvement of the 2’ and 3’-OH groups of the ribose in the dimerization interface of Irga6 has not been observed for other dynamin and septin superfamily members. DISCUSS +141 148 dynamin protein_type For example, the involvement of the 2’ and 3’-OH groups of the ribose in the dimerization interface of Irga6 has not been observed for other dynamin and septin superfamily members. DISCUSS +153 159 septin protein_type For example, the involvement of the 2’ and 3’-OH groups of the ribose in the dimerization interface of Irga6 has not been observed for other dynamin and septin superfamily members. DISCUSS +50 55 Irga6 protein The surface-exposed location of the ribose in the Irga6 structure, with a wide-open nucleotide-binding pocket, facilitates its engagement in the dimerization interface. DISCUSS +56 65 structure evidence The surface-exposed location of the ribose in the Irga6 structure, with a wide-open nucleotide-binding pocket, facilitates its engagement in the dimerization interface. DISCUSS +74 83 wide-open protein_state The surface-exposed location of the ribose in the Irga6 structure, with a wide-open nucleotide-binding pocket, facilitates its engagement in the dimerization interface. DISCUSS +84 109 nucleotide-binding pocket site The surface-exposed location of the ribose in the Irga6 structure, with a wide-open nucleotide-binding pocket, facilitates its engagement in the dimerization interface. DISCUSS +145 167 dimerization interface site The surface-exposed location of the ribose in the Irga6 structure, with a wide-open nucleotide-binding pocket, facilitates its engagement in the dimerization interface. DISCUSS +43 46 GTP chemical This contact, in turn, appears to activate GTP hydrolysis by inducing rearrangements in switch I and the positioning of the catalytic E106. DISCUSS +88 96 switch I site This contact, in turn, appears to activate GTP hydrolysis by inducing rearrangements in switch I and the positioning of the catalytic E106. DISCUSS +124 133 catalytic protein_state This contact, in turn, appears to activate GTP hydrolysis by inducing rearrangements in switch I and the positioning of the catalytic E106. DISCUSS +134 138 E106 residue_name_number This contact, in turn, appears to activate GTP hydrolysis by inducing rearrangements in switch I and the positioning of the catalytic E106. DISCUSS +23 27 GBP1 protein During dimerization of GBP1, an arginine finger from the P loop reorients towards the nucleotide in cis to trigger GTP hydrolysis. DISCUSS +32 47 arginine finger structure_element During dimerization of GBP1, an arginine finger from the P loop reorients towards the nucleotide in cis to trigger GTP hydrolysis. DISCUSS +57 63 P loop structure_element During dimerization of GBP1, an arginine finger from the P loop reorients towards the nucleotide in cis to trigger GTP hydrolysis. DISCUSS +86 96 nucleotide chemical During dimerization of GBP1, an arginine finger from the P loop reorients towards the nucleotide in cis to trigger GTP hydrolysis. DISCUSS +115 118 GTP chemical During dimerization of GBP1, an arginine finger from the P loop reorients towards the nucleotide in cis to trigger GTP hydrolysis. DISCUSS +3 10 dynamin protein_type In dynamin, the corresponding serine residue coordinates a sodium ion that is crucial for GTP hydrolysis. DISCUSS +30 36 serine residue_name In dynamin, the corresponding serine residue coordinates a sodium ion that is crucial for GTP hydrolysis. DISCUSS +59 65 sodium chemical In dynamin, the corresponding serine residue coordinates a sodium ion that is crucial for GTP hydrolysis. DISCUSS +90 93 GTP chemical In dynamin, the corresponding serine residue coordinates a sodium ion that is crucial for GTP hydrolysis. DISCUSS +0 5 Irga6 protein Irga6 bears Gly79 at this position, which in the dimerizing molecule A appears to approach the bridging imido group of GMPPNP via a main chain hydrogen bond. DISCUSS +12 17 Gly79 residue_name_number Irga6 bears Gly79 at this position, which in the dimerizing molecule A appears to approach the bridging imido group of GMPPNP via a main chain hydrogen bond. DISCUSS +49 59 dimerizing oligomeric_state Irga6 bears Gly79 at this position, which in the dimerizing molecule A appears to approach the bridging imido group of GMPPNP via a main chain hydrogen bond. DISCUSS +69 70 A structure_element Irga6 bears Gly79 at this position, which in the dimerizing molecule A appears to approach the bridging imido group of GMPPNP via a main chain hydrogen bond. DISCUSS +119 125 GMPPNP chemical Irga6 bears Gly79 at this position, which in the dimerizing molecule A appears to approach the bridging imido group of GMPPNP via a main chain hydrogen bond. DISCUSS +143 156 hydrogen bond bond_interaction Irga6 bears Gly79 at this position, which in the dimerizing molecule A appears to approach the bridging imido group of GMPPNP via a main chain hydrogen bond. DISCUSS +18 28 structures evidence Higher resolution structures of the Irga6 dimer in the presence of a transition state analogue are required to show whether Gly79 directly participates in GTP hydrolysis or whether it may also position a catalytic cation. DISCUSS +36 41 Irga6 protein Higher resolution structures of the Irga6 dimer in the presence of a transition state analogue are required to show whether Gly79 directly participates in GTP hydrolysis or whether it may also position a catalytic cation. DISCUSS +42 47 dimer oligomeric_state Higher resolution structures of the Irga6 dimer in the presence of a transition state analogue are required to show whether Gly79 directly participates in GTP hydrolysis or whether it may also position a catalytic cation. DISCUSS +55 66 presence of protein_state Higher resolution structures of the Irga6 dimer in the presence of a transition state analogue are required to show whether Gly79 directly participates in GTP hydrolysis or whether it may also position a catalytic cation. DISCUSS +124 129 Gly79 residue_name_number Higher resolution structures of the Irga6 dimer in the presence of a transition state analogue are required to show whether Gly79 directly participates in GTP hydrolysis or whether it may also position a catalytic cation. DISCUSS +155 158 GTP chemical Higher resolution structures of the Irga6 dimer in the presence of a transition state analogue are required to show whether Gly79 directly participates in GTP hydrolysis or whether it may also position a catalytic cation. DISCUSS +3 10 dynamin protein_type In dynamin, further assembly sites are provided by the helical domains which assemble in a criss-cross fashion to form a helical filament. DISCUSS +20 34 assembly sites site In dynamin, further assembly sites are provided by the helical domains which assemble in a criss-cross fashion to form a helical filament. DISCUSS +55 70 helical domains structure_element In dynamin, further assembly sites are provided by the helical domains which assemble in a criss-cross fashion to form a helical filament. DISCUSS +121 137 helical filament structure_element In dynamin, further assembly sites are provided by the helical domains which assemble in a criss-cross fashion to form a helical filament. DISCUSS +3 60 dynamin-related Eps15 homology domain containing proteins protein_type In dynamin-related Eps15 homology domain containing proteins (EHDs), a second assembly interface is present in the GTPase domain. DISCUSS +62 66 EHDs protein_type In dynamin-related Eps15 homology domain containing proteins (EHDs), a second assembly interface is present in the GTPase domain. DISCUSS +71 96 second assembly interface site In dynamin-related Eps15 homology domain containing proteins (EHDs), a second assembly interface is present in the GTPase domain. DISCUSS +115 128 GTPase domain structure_element In dynamin-related Eps15 homology domain containing proteins (EHDs), a second assembly interface is present in the GTPase domain. DISCUSS +4 9 Irga6 protein For Irga6, additional interfaces in the helical domain are presumably involved in oligomerization, such as the secondary patch residues whose mutation prevented oligomerization in the crystallized mutant. DISCUSS +22 32 interfaces site For Irga6, additional interfaces in the helical domain are presumably involved in oligomerization, such as the secondary patch residues whose mutation prevented oligomerization in the crystallized mutant. DISCUSS +40 54 helical domain structure_element For Irga6, additional interfaces in the helical domain are presumably involved in oligomerization, such as the secondary patch residues whose mutation prevented oligomerization in the crystallized mutant. DISCUSS +111 126 secondary patch site For Irga6, additional interfaces in the helical domain are presumably involved in oligomerization, such as the secondary patch residues whose mutation prevented oligomerization in the crystallized mutant. DISCUSS +142 150 mutation experimental_method For Irga6, additional interfaces in the helical domain are presumably involved in oligomerization, such as the secondary patch residues whose mutation prevented oligomerization in the crystallized mutant. DISCUSS +184 196 crystallized evidence For Irga6, additional interfaces in the helical domain are presumably involved in oligomerization, such as the secondary patch residues whose mutation prevented oligomerization in the crystallized mutant. DISCUSS +197 203 mutant protein_state For Irga6, additional interfaces in the helical domain are presumably involved in oligomerization, such as the secondary patch residues whose mutation prevented oligomerization in the crystallized mutant. DISCUSS +8 26 structural studies experimental_method Further structural studies, especially electron microscopy analysis of the Irga6 oligomers, are required to clarify the assembly mode via the helical domains and to show how these interfaces cooperate with the G interface to mediate the regulated assembly on a membrane surface. DISCUSS +39 67 electron microscopy analysis experimental_method Further structural studies, especially electron microscopy analysis of the Irga6 oligomers, are required to clarify the assembly mode via the helical domains and to show how these interfaces cooperate with the G interface to mediate the regulated assembly on a membrane surface. DISCUSS +75 80 Irga6 protein Further structural studies, especially electron microscopy analysis of the Irga6 oligomers, are required to clarify the assembly mode via the helical domains and to show how these interfaces cooperate with the G interface to mediate the regulated assembly on a membrane surface. DISCUSS +81 90 oligomers oligomeric_state Further structural studies, especially electron microscopy analysis of the Irga6 oligomers, are required to clarify the assembly mode via the helical domains and to show how these interfaces cooperate with the G interface to mediate the regulated assembly on a membrane surface. DISCUSS +142 157 helical domains structure_element Further structural studies, especially electron microscopy analysis of the Irga6 oligomers, are required to clarify the assembly mode via the helical domains and to show how these interfaces cooperate with the G interface to mediate the regulated assembly on a membrane surface. DISCUSS +180 190 interfaces site Further structural studies, especially electron microscopy analysis of the Irga6 oligomers, are required to clarify the assembly mode via the helical domains and to show how these interfaces cooperate with the G interface to mediate the regulated assembly on a membrane surface. DISCUSS +210 221 G interface site Further structural studies, especially electron microscopy analysis of the Irga6 oligomers, are required to clarify the assembly mode via the helical domains and to show how these interfaces cooperate with the G interface to mediate the regulated assembly on a membrane surface. DISCUSS +56 70 helical domain structure_element Notably, we did not observe major rearrangements of the helical domain versus the GTPase domain in the Irga6 molecules that dimerized via the G interface. DISCUSS +82 95 GTPase domain structure_element Notably, we did not observe major rearrangements of the helical domain versus the GTPase domain in the Irga6 molecules that dimerized via the G interface. DISCUSS +103 108 Irga6 protein Notably, we did not observe major rearrangements of the helical domain versus the GTPase domain in the Irga6 molecules that dimerized via the G interface. DISCUSS +124 133 dimerized protein_state Notably, we did not observe major rearrangements of the helical domain versus the GTPase domain in the Irga6 molecules that dimerized via the G interface. DISCUSS +142 153 G interface site Notably, we did not observe major rearrangements of the helical domain versus the GTPase domain in the Irga6 molecules that dimerized via the G interface. DISCUSS +23 27 BDLP protein_type In a manner similar to BDLP, such large-scale conformational changes may be induced by membrane binding. DISCUSS +4 23 structural analysis experimental_method Our structural analysis and the identification of the G-interface paves the way for determining the specific assembly of Irga6 into a membrane-associated scaffold as the prerequisite to understand its action as an anti-parasitic machine. DISCUSS +54 65 G-interface site Our structural analysis and the identification of the G-interface paves the way for determining the specific assembly of Irga6 into a membrane-associated scaffold as the prerequisite to understand its action as an anti-parasitic machine. DISCUSS +121 126 Irga6 protein Our structural analysis and the identification of the G-interface paves the way for determining the specific assembly of Irga6 into a membrane-associated scaffold as the prerequisite to understand its action as an anti-parasitic machine. DISCUSS +25 28 Irg protein_type Our study indicates that Irg proteins dimerize via the G interface in a parallel head-to-head fashion thereby facilitating GTPase activation. CONCL +38 46 dimerize oligomeric_state Our study indicates that Irg proteins dimerize via the G interface in a parallel head-to-head fashion thereby facilitating GTPase activation. CONCL +55 66 G interface site Our study indicates that Irg proteins dimerize via the G interface in a parallel head-to-head fashion thereby facilitating GTPase activation. CONCL +72 93 parallel head-to-head protein_state Our study indicates that Irg proteins dimerize via the G interface in a parallel head-to-head fashion thereby facilitating GTPase activation. CONCL +123 129 GTPase protein_type Our study indicates that Irg proteins dimerize via the G interface in a parallel head-to-head fashion thereby facilitating GTPase activation. CONCL +91 94 Irg protein_type These findings contribute to a molecular understanding of the anti-parasitic action of the Irg protein family and suggest that Irgs are bona-fide members of the dynamin superfamily. CONCL +127 131 Irgs protein_type These findings contribute to a molecular understanding of the anti-parasitic action of the Irg protein family and suggest that Irgs are bona-fide members of the dynamin superfamily. CONCL +161 168 dynamin protein_type These findings contribute to a molecular understanding of the anti-parasitic action of the Irg protein family and suggest that Irgs are bona-fide members of the dynamin superfamily. CONCL diff --git a/annotation_CSV/PMC4781976.csv b/annotation_CSV/PMC4781976.csv new file mode 100644 index 0000000000000000000000000000000000000000..40bd4df74c8f0d7e9e1fd3b0ac7578e30ca63d90 --- /dev/null +++ b/annotation_CSV/PMC4781976.csv @@ -0,0 +1,114 @@ +anno_start anno_end anno_text entity_type sentence section +0 9 Structure evidence Structure of the GAT domain of the endosomal adapter protein Tom1 TITLE +17 20 GAT structure_element Structure of the GAT domain of the endosomal adapter protein Tom1 TITLE +45 60 adapter protein protein_type Structure of the GAT domain of the endosomal adapter protein Tom1 TITLE +61 65 Tom1 protein Structure of the GAT domain of the endosomal adapter protein Tom1 TITLE +50 71 cell-surface receptor protein_type Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments. ABSTRACT +4 20 adapter proteins protein_type The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. ABSTRACT +21 25 Tom1 protein The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. ABSTRACT +30 36 Tollip protein The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. ABSTRACT +64 77 ubiquitinated ptm The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. ABSTRACT +15 19 Tom1 protein Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD). ABSTRACT +69 72 GAT structure_element Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD). ABSTRACT +97 103 Tollip protein Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD). ABSTRACT +106 125 Tom1-binding domain structure_element Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD). ABSTRACT +127 130 TBD structure_element Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD). ABSTRACT +36 48 solution NMR experimental_method In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. ABSTRACT +57 66 structure evidence In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. ABSTRACT +74 78 Tom1 protein In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. ABSTRACT +79 82 GAT structure_element In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. ABSTRACT +22 31 structure evidence The estimated protein structure exhibits a bundle of three helical elements. ABSTRACT +3 10 compare experimental_method We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states. ABSTRACT +15 19 Tom1 protein We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states. ABSTRACT +20 23 GAT structure_element We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states. ABSTRACT +24 33 structure evidence We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states. ABSTRACT +45 55 structures evidence We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states. ABSTRACT +77 83 Tollip protein We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states. ABSTRACT +84 88 TBD- protein_state We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states. ABSTRACT +93 108 ubiquitin-bound protein_state We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states. ABSTRACT +141 159 Circular dichroism experimental_method "Subject area Biology More specific subject area Structural biology Type of data Table, text file, graph, figures How data was acquired Circular dichroism and NMR." TABLE +164 167 NMR experimental_method "Subject area Biology More specific subject area Structural biology Type of data Table, text file, graph, figures How data was acquired Circular dichroism and NMR." TABLE +0 3 NMR experimental_method "NMR data was recorded using a Bruker 800 MHz Data format PDB format text file." TABLE +12 22 CS-Rosetta experimental_method "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +24 59 Protein Structure Validation Server experimental_method "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +61 65 PSVS experimental_method "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +68 75 NMRPipe experimental_method "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +77 84 NMRDraw experimental_method "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +131 136 human species "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +137 141 Tom1 protein "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +142 145 GAT structure_element "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +216 234 Solution structure evidence "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +238 242 Tom1 protein "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +243 246 GAT structure_element "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +267 270 NMR experimental_method "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +271 285 chemical shift evidence "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol Experimental factors Recombinant human Tom1 GAT domain was purified to homogeneity before use Experimental features Solution structure of Tom1 GAT was determined from NMR chemical shift data Data source location Virginia and Colorado, United States." TABLE +5 8 GAT structure_element "Tom1 GAT structural data is publicly available in the RCSB Protein Data Bank (http://www.rscb.org/) under the accession number PDB: 2n9d " TABLE +4 8 Tom1 protein The Tom1 GAT domain solution structure will provide additional tools for modulating its biological function. TABLE +9 12 GAT structure_element The Tom1 GAT domain solution structure will provide additional tools for modulating its biological function. TABLE +20 38 solution structure evidence The Tom1 GAT domain solution structure will provide additional tools for modulating its biological function. TABLE +0 4 Tom1 protein Tom1 GAT can adopt distinct conformations upon ligand binding. TABLE +5 8 GAT structure_element Tom1 GAT can adopt distinct conformations upon ligand binding. TABLE +33 37 Tom1 protein A conformational response of the Tom1 GAT domain upon Tollip TBD binding can serve as an example to explain mutually exclusive ligand binding events. TABLE +38 41 GAT structure_element A conformational response of the Tom1 GAT domain upon Tollip TBD binding can serve as an example to explain mutually exclusive ligand binding events. TABLE +54 60 Tollip protein A conformational response of the Tom1 GAT domain upon Tollip TBD binding can serve as an example to explain mutually exclusive ligand binding events. TABLE +61 64 TBD structure_element A conformational response of the Tom1 GAT domain upon Tollip TBD binding can serve as an example to explain mutually exclusive ligand binding events. TABLE +16 41 far-UV circular dichroism experimental_method Analysis of the far-UV circular dichroism (CD) spectrum of the Tom 1 GAT domain (Fig. 1) predicts 58.7% α-helix, 3% β-strand, 15.5% turn, and 22.8% disordered regions. TABLE +43 45 CD experimental_method Analysis of the far-UV circular dichroism (CD) spectrum of the Tom 1 GAT domain (Fig. 1) predicts 58.7% α-helix, 3% β-strand, 15.5% turn, and 22.8% disordered regions. TABLE +47 55 spectrum evidence Analysis of the far-UV circular dichroism (CD) spectrum of the Tom 1 GAT domain (Fig. 1) predicts 58.7% α-helix, 3% β-strand, 15.5% turn, and 22.8% disordered regions. TABLE +63 68 Tom 1 protein Analysis of the far-UV circular dichroism (CD) spectrum of the Tom 1 GAT domain (Fig. 1) predicts 58.7% α-helix, 3% β-strand, 15.5% turn, and 22.8% disordered regions. TABLE +69 72 GAT structure_element Analysis of the far-UV circular dichroism (CD) spectrum of the Tom 1 GAT domain (Fig. 1) predicts 58.7% α-helix, 3% β-strand, 15.5% turn, and 22.8% disordered regions. TABLE +104 111 α-helix structure_element Analysis of the far-UV circular dichroism (CD) spectrum of the Tom 1 GAT domain (Fig. 1) predicts 58.7% α-helix, 3% β-strand, 15.5% turn, and 22.8% disordered regions. TABLE +116 124 β-strand structure_element Analysis of the far-UV circular dichroism (CD) spectrum of the Tom 1 GAT domain (Fig. 1) predicts 58.7% α-helix, 3% β-strand, 15.5% turn, and 22.8% disordered regions. TABLE +4 8 Tom1 protein The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). TABLE +9 12 GAT structure_element The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). TABLE +13 34 structural restraints evidence The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). TABLE +55 65 structures evidence The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). TABLE +85 111 root mean square deviation evidence The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). TABLE +113 117 RMSD evidence The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). TABLE +243 252 Q216-E240 residue_range The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). TABLE +254 263 α-helix 1 structure_element The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). TABLE +266 275 P248-Q274 residue_range The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). TABLE +277 286 α-helix 2 structure_element The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). TABLE +293 302 E278-T306 residue_range The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). TABLE +304 313 α-helix 3 structure_element The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). TABLE +7 16 ubiquitin chemical Unlike ubiquitin binding, data suggest that conformational changes of the Tom1 GAT α-helices 1 and 2 occur upon Tollip TBD binding (Fig. 3A,B). TABLE +74 78 Tom1 protein Unlike ubiquitin binding, data suggest that conformational changes of the Tom1 GAT α-helices 1 and 2 occur upon Tollip TBD binding (Fig. 3A,B). TABLE +79 82 GAT structure_element Unlike ubiquitin binding, data suggest that conformational changes of the Tom1 GAT α-helices 1 and 2 occur upon Tollip TBD binding (Fig. 3A,B). TABLE +83 100 α-helices 1 and 2 structure_element Unlike ubiquitin binding, data suggest that conformational changes of the Tom1 GAT α-helices 1 and 2 occur upon Tollip TBD binding (Fig. 3A,B). TABLE +112 118 Tollip protein Unlike ubiquitin binding, data suggest that conformational changes of the Tom1 GAT α-helices 1 and 2 occur upon Tollip TBD binding (Fig. 3A,B). TABLE +119 122 TBD structure_element Unlike ubiquitin binding, data suggest that conformational changes of the Tom1 GAT α-helices 1 and 2 occur upon Tollip TBD binding (Fig. 3A,B). TABLE +15 24 far-UV CD experimental_method Representative far-UV CD spectrum of the His-Tom1 GAT domain. FIG +25 33 spectrum evidence Representative far-UV CD spectrum of the His-Tom1 GAT domain. FIG +41 45 His- experimental_method Representative far-UV CD spectrum of the His-Tom1 GAT domain. FIG +45 49 Tom1 protein Representative far-UV CD spectrum of the His-Tom1 GAT domain. FIG +50 53 GAT structure_element Representative far-UV CD spectrum of the His-Tom1 GAT domain. FIG +40 62 backbone superposition experimental_method (A) Stereo view displaying the best-fit backbone superposition of the refined structures for the Tom1 GAT domain. FIG +78 88 structures evidence (A) Stereo view displaying the best-fit backbone superposition of the refined structures for the Tom1 GAT domain. FIG +97 101 Tom1 protein (A) Stereo view displaying the best-fit backbone superposition of the refined structures for the Tom1 GAT domain. FIG +102 105 GAT structure_element (A) Stereo view displaying the best-fit backbone superposition of the refined structures for the Tom1 GAT domain. FIG +96 100 Tom1 protein Helices are shown in orange, whereas loops are colored in green. (B) Ribbon illustration of the Tom1 GAT domain. FIG +101 104 GAT structure_element Helices are shown in orange, whereas loops are colored in green. (B) Ribbon illustration of the Tom1 GAT domain. FIG +21 44 superimposed structures experimental_method (A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green). FIG +52 56 Tom1 protein (A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green). FIG +57 60 GAT structure_element (A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green). FIG +75 79 free protein_state (A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green). FIG +110 116 Tollip protein (A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green). FIG +117 126 TBD-bound protein_state (A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green). FIG +161 184 superimposed structures experimental_method (A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green). FIG +192 196 Tom1 protein (A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green). FIG +197 200 GAT structure_element (A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green). FIG +232 240 Ub-bound protein_state (A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green). FIG +0 3 NMR experimental_method NMR and refinement statistics for the Tom1 GAT domain. TABLE +8 29 refinement statistics evidence NMR and refinement statistics for the Tom1 GAT domain. TABLE +38 42 Tom1 protein NMR and refinement statistics for the Tom1 GAT domain. TABLE +43 46 GAT structure_element NMR and refinement statistics for the Tom1 GAT domain. TABLE +0 3 NMR experimental_method NMR structural statistics for lowest energy conformers of Tom1 GAT using PSVS. TABLE +4 25 structural statistics evidence NMR structural statistics for lowest energy conformers of Tom1 GAT using PSVS. TABLE +58 62 Tom1 protein NMR structural statistics for lowest energy conformers of Tom1 GAT using PSVS. TABLE +63 66 GAT structure_element NMR structural statistics for lowest energy conformers of Tom1 GAT using PSVS. TABLE +73 77 PSVS experimental_method NMR structural statistics for lowest energy conformers of Tom1 GAT using PSVS. TABLE +28 41 superimposing experimental_method deviations were obtained by superimposing residues 215–309 of Tom1 GAT among 10 lowest energy refined structures. TABLE +51 58 215–309 residue_range deviations were obtained by superimposing residues 215–309 of Tom1 GAT among 10 lowest energy refined structures. TABLE +62 66 Tom1 protein deviations were obtained by superimposing residues 215–309 of Tom1 GAT among 10 lowest energy refined structures. TABLE +67 70 GAT structure_element deviations were obtained by superimposing residues 215–309 of Tom1 GAT among 10 lowest energy refined structures. TABLE +102 112 structures evidence deviations were obtained by superimposing residues 215–309 of Tom1 GAT among 10 lowest energy refined structures. TABLE diff --git a/annotation_CSV/PMC4784909.csv b/annotation_CSV/PMC4784909.csv new file mode 100644 index 0000000000000000000000000000000000000000..35c8b1769177cc975b4ab30f38a7cced87f53b98 --- /dev/null +++ b/annotation_CSV/PMC4784909.csv @@ -0,0 +1,869 @@ +anno_start anno_end anno_text entity_type sentence section +24 34 Coenzyme A chemical The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle TITLE +50 59 Bacterial taxonomy_domain The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle TITLE +0 9 Bacterial taxonomy_domain Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. ABSTRACT +10 27 Microcompartments complex_assembly Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. ABSTRACT +29 33 BMCs complex_assembly Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. ABSTRACT +16 25 catabolic protein_state The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. ABSTRACT +26 30 BMCs complex_assembly The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. ABSTRACT +32 45 metabolosomes complex_assembly The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. ABSTRACT +141 149 aldehyde chemical The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. ABSTRACT +16 35 phosphotransacylase protein_type The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. ABSTRACT +37 41 PTAC protein_type The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. ABSTRACT +52 62 Coenzyme A chemical The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. ABSTRACT +80 94 acyl phosphate chemical The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. ABSTRACT +4 8 PTAC protein_type The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). ABSTRACT +39 52 metabolosomes complex_assembly The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). ABSTRACT +54 58 PduL protein_type The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). ABSTRACT +92 96 PTAC protein_type The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). ABSTRACT +114 135 fermentative bacteria taxonomy_domain The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). ABSTRACT +137 140 Pta protein_type The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). ABSTRACT +36 40 PduL protein_type Here, we report two high-resolution PduL crystal structures with bound substrates. ABSTRACT +41 59 crystal structures evidence Here, we report two high-resolution PduL crystal structures with bound substrates. ABSTRACT +60 81 with bound substrates protein_state Here, we report two high-resolution PduL crystal structures with bound substrates. ABSTRACT +4 8 PduL protein_type The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. ABSTRACT +9 13 fold structure_element The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. ABSTRACT +30 34 that structure_element The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. ABSTRACT +38 41 Pta protein_type The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. ABSTRACT +57 76 dimetal active site site The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. ABSTRACT +137 149 housekeeping protein_state The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. ABSTRACT +150 154 PTAC protein_type The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. ABSTRACT +13 17 PduL protein_type Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. ABSTRACT +22 25 Pta protein_type Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. ABSTRACT +4 8 PduL protein_type The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. ABSTRACT +9 18 structure evidence The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. ABSTRACT +139 151 metabolosome complex_assembly The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. ABSTRACT +25 34 structure evidence This study describes the structure of a novel phosphotransacylase enzyme that facilitates the recycling of the essential cofactor acetyl-CoA within a bacterial organelle and discusses the properties of the enzyme's active site and how it is packaged into the organelle. ABSTRACT +46 65 phosphotransacylase protein_type This study describes the structure of a novel phosphotransacylase enzyme that facilitates the recycling of the essential cofactor acetyl-CoA within a bacterial organelle and discusses the properties of the enzyme's active site and how it is packaged into the organelle. ABSTRACT +130 140 acetyl-CoA chemical This study describes the structure of a novel phosphotransacylase enzyme that facilitates the recycling of the essential cofactor acetyl-CoA within a bacterial organelle and discusses the properties of the enzyme's active site and how it is packaged into the organelle. ABSTRACT +150 159 bacterial taxonomy_domain This study describes the structure of a novel phosphotransacylase enzyme that facilitates the recycling of the essential cofactor acetyl-CoA within a bacterial organelle and discusses the properties of the enzyme's active site and how it is packaged into the organelle. ABSTRACT +215 226 active site site This study describes the structure of a novel phosphotransacylase enzyme that facilitates the recycling of the essential cofactor acetyl-CoA within a bacterial organelle and discusses the properties of the enzyme's active site and how it is packaged into the organelle. ABSTRACT +57 60 ATP chemical In metabolism, molecules with “high-energy” bonds (e.g., ATP and Acetyl~CoA) are critical for both catabolic and anabolic processes. ABSTRACT +65 75 Acetyl~CoA chemical In metabolism, molecules with “high-energy” bonds (e.g., ATP and Acetyl~CoA) are critical for both catabolic and anabolic processes. ABSTRACT +4 23 phosphotransacylase protein_type The phosphotransacylase (Pta) enzyme catalyzes the conversion between acyl-CoA and acyl-phosphate. ABSTRACT +25 28 Pta protein_type The phosphotransacylase (Pta) enzyme catalyzes the conversion between acyl-CoA and acyl-phosphate. ABSTRACT +70 78 acyl-CoA chemical The phosphotransacylase (Pta) enzyme catalyzes the conversion between acyl-CoA and acyl-phosphate. ABSTRACT +83 97 acyl-phosphate chemical The phosphotransacylase (Pta) enzyme catalyzes the conversion between acyl-CoA and acyl-phosphate. ABSTRACT +32 40 acyl-CoA chemical This reaction directly links an acyl-CoA with ATP generation via substrate-level phosphorylation, producing short-chain fatty acids (e.g., acetate), and also provides a path for short-chain fatty acids to enter central metabolism. ABSTRACT +46 49 ATP chemical This reaction directly links an acyl-CoA with ATP generation via substrate-level phosphorylation, producing short-chain fatty acids (e.g., acetate), and also provides a path for short-chain fatty acids to enter central metabolism. ABSTRACT +108 131 short-chain fatty acids chemical This reaction directly links an acyl-CoA with ATP generation via substrate-level phosphorylation, producing short-chain fatty acids (e.g., acetate), and also provides a path for short-chain fatty acids to enter central metabolism. ABSTRACT +139 146 acetate chemical This reaction directly links an acyl-CoA with ATP generation via substrate-level phosphorylation, producing short-chain fatty acids (e.g., acetate), and also provides a path for short-chain fatty acids to enter central metabolism. ABSTRACT +178 201 short-chain fatty acids chemical This reaction directly links an acyl-CoA with ATP generation via substrate-level phosphorylation, producing short-chain fatty acids (e.g., acetate), and also provides a path for short-chain fatty acids to enter central metabolism. ABSTRACT +33 42 conserved protein_state Due to this key function, Pta is conserved across the bacterial kingdom. ABSTRACT +54 71 bacterial kingdom taxonomy_domain Due to this key function, Pta is conserved across the bacterial kingdom. ABSTRACT +24 43 phosphotransacylase protein_type Recently, a new type of phosphotransacylase was described that shares no evolutionary relation to Pta. ABSTRACT +98 101 Pta protein_type Recently, a new type of phosphotransacylase was described that shares no evolutionary relation to Pta. ABSTRACT +13 17 PduL protein_type This enzyme, PduL, is exclusively associated with organelles called bacterial microcompartments, which are used to catabolize various compounds. ABSTRACT +22 33 exclusively protein_state This enzyme, PduL, is exclusively associated with organelles called bacterial microcompartments, which are used to catabolize various compounds. ABSTRACT +68 77 bacterial taxonomy_domain This enzyme, PduL, is exclusively associated with organelles called bacterial microcompartments, which are used to catabolize various compounds. ABSTRACT +78 95 microcompartments complex_assembly This enzyme, PduL, is exclusively associated with organelles called bacterial microcompartments, which are used to catabolize various compounds. ABSTRACT +14 18 PduL protein_type Not only does PduL facilitate substrate level phosphorylation, but it also is critical for cofactor recycling within, and product efflux from, the organelle. ABSTRACT +3 9 solved experimental_method We solved the structure of this convergent phosphotransacylase and show that it is completely structurally different from Pta, including its active site architecture. ABSTRACT +14 23 structure evidence We solved the structure of this convergent phosphotransacylase and show that it is completely structurally different from Pta, including its active site architecture. ABSTRACT +32 42 convergent protein_state We solved the structure of this convergent phosphotransacylase and show that it is completely structurally different from Pta, including its active site architecture. ABSTRACT +43 62 phosphotransacylase protein_type We solved the structure of this convergent phosphotransacylase and show that it is completely structurally different from Pta, including its active site architecture. ABSTRACT +122 125 Pta protein_type We solved the structure of this convergent phosphotransacylase and show that it is completely structurally different from Pta, including its active site architecture. ABSTRACT +141 152 active site site We solved the structure of this convergent phosphotransacylase and show that it is completely structurally different from Pta, including its active site architecture. ABSTRACT +0 9 Bacterial taxonomy_domain Bacterial Microcompartments (BMCs) are organelles that encapsulate enzymes for sequential biochemical reactions within a protein shell. INTRO +10 27 Microcompartments complex_assembly Bacterial Microcompartments (BMCs) are organelles that encapsulate enzymes for sequential biochemical reactions within a protein shell. INTRO +29 33 BMCs complex_assembly Bacterial Microcompartments (BMCs) are organelles that encapsulate enzymes for sequential biochemical reactions within a protein shell. INTRO +129 134 shell structure_element Bacterial Microcompartments (BMCs) are organelles that encapsulate enzymes for sequential biochemical reactions within a protein shell. INTRO +4 9 shell structure_element The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell. INTRO +86 95 hexagonal protein_state The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell. INTRO +97 102 BMC-H complex_assembly The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell. INTRO +107 112 BMC-T complex_assembly The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell. INTRO +117 127 pentagonal protein_state The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell. INTRO +129 134 BMC-P complex_assembly The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell. INTRO +163 173 polyhedral protein_state The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell. INTRO +174 179 shell structure_element The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell. INTRO +18 23 shell structure_element The facets of the shell are composed primarily of hexamers that are typically perforated by pores lined with highly conserved, polar residues that presumably function as the conduits for metabolites into and out of the shell. INTRO +50 58 hexamers oligomeric_state The facets of the shell are composed primarily of hexamers that are typically perforated by pores lined with highly conserved, polar residues that presumably function as the conduits for metabolites into and out of the shell. INTRO +92 97 pores site The facets of the shell are composed primarily of hexamers that are typically perforated by pores lined with highly conserved, polar residues that presumably function as the conduits for metabolites into and out of the shell. INTRO +109 125 highly conserved protein_state The facets of the shell are composed primarily of hexamers that are typically perforated by pores lined with highly conserved, polar residues that presumably function as the conduits for metabolites into and out of the shell. INTRO +127 132 polar protein_state The facets of the shell are composed primarily of hexamers that are typically perforated by pores lined with highly conserved, polar residues that presumably function as the conduits for metabolites into and out of the shell. INTRO +133 141 residues structure_element The facets of the shell are composed primarily of hexamers that are typically perforated by pores lined with highly conserved, polar residues that presumably function as the conduits for metabolites into and out of the shell. INTRO +219 224 shell structure_element The facets of the shell are composed primarily of hexamers that are typically perforated by pores lined with highly conserved, polar residues that presumably function as the conduits for metabolites into and out of the shell. INTRO +4 57 vitamin B12-dependent propanediol-utilizing (PDU) BMC complex_assembly The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1). INTRO +106 115 catabolic protein_state The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1). INTRO +116 120 BMCs complex_assembly The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1). INTRO +191 203 ethanolamine chemical The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1). INTRO +205 212 choline chemical The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1). INTRO +214 220 fucose chemical The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1). INTRO +222 230 rhamnose chemical The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1). INTRO +236 243 ethanol chemical The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1). INTRO +276 284 aldehyde chemical The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1). INTRO +15 36 bioinformatic studies experimental_method More recently, bioinformatic studies have demonstrated the widespread distribution of BMCs among diverse bacterial phyla and grouped them into 23 different functional types. INTRO +86 90 BMCs complex_assembly More recently, bioinformatic studies have demonstrated the widespread distribution of BMCs among diverse bacterial phyla and grouped them into 23 different functional types. INTRO +105 120 bacterial phyla taxonomy_domain More recently, bioinformatic studies have demonstrated the widespread distribution of BMCs among diverse bacterial phyla and grouped them into 23 different functional types. INTRO +45 54 catabolic protein_state The reactions carried out in the majority of catabolic BMCs (also known as metabolosomes) fit a generalized biochemical paradigm for the oxidation of aldehydes (Fig 1). INTRO +55 59 BMCs complex_assembly The reactions carried out in the majority of catabolic BMCs (also known as metabolosomes) fit a generalized biochemical paradigm for the oxidation of aldehydes (Fig 1). INTRO +75 88 metabolosomes complex_assembly The reactions carried out in the majority of catabolic BMCs (also known as metabolosomes) fit a generalized biochemical paradigm for the oxidation of aldehydes (Fig 1). INTRO +150 159 aldehydes chemical The reactions carried out in the majority of catabolic BMCs (also known as metabolosomes) fit a generalized biochemical paradigm for the oxidation of aldehydes (Fig 1). INTRO +16 19 BMC complex_assembly This involves a BMC-encapsulated signature enzyme that generates a toxic and/or volatile aldehyde that the BMC shell sequesters from the cytosol. INTRO +89 97 aldehyde chemical This involves a BMC-encapsulated signature enzyme that generates a toxic and/or volatile aldehyde that the BMC shell sequesters from the cytosol. INTRO +107 110 BMC complex_assembly This involves a BMC-encapsulated signature enzyme that generates a toxic and/or volatile aldehyde that the BMC shell sequesters from the cytosol. INTRO +111 116 shell structure_element This involves a BMC-encapsulated signature enzyme that generates a toxic and/or volatile aldehyde that the BMC shell sequesters from the cytosol. INTRO +4 12 aldehyde chemical The aldehyde is subsequently converted into an acyl-CoA by aldehyde dehydrogenase, which uses NAD+ and CoA as cofactors. INTRO +47 55 acyl-CoA chemical The aldehyde is subsequently converted into an acyl-CoA by aldehyde dehydrogenase, which uses NAD+ and CoA as cofactors. INTRO +59 81 aldehyde dehydrogenase protein_type The aldehyde is subsequently converted into an acyl-CoA by aldehyde dehydrogenase, which uses NAD+ and CoA as cofactors. INTRO +94 98 NAD+ chemical The aldehyde is subsequently converted into an acyl-CoA by aldehyde dehydrogenase, which uses NAD+ and CoA as cofactors. INTRO +103 106 CoA chemical The aldehyde is subsequently converted into an acyl-CoA by aldehyde dehydrogenase, which uses NAD+ and CoA as cofactors. INTRO +73 86 protein shell structure_element These two cofactors are relatively large, and their diffusion across the protein shell is thought to be restricted, necessitating their regeneration within the BMC lumen. INTRO +160 163 BMC complex_assembly These two cofactors are relatively large, and their diffusion across the protein shell is thought to be restricted, necessitating their regeneration within the BMC lumen. INTRO +0 4 NAD+ chemical NAD+ is recycled via alcohol dehydrogenase, and CoA is recycled via phosphotransacetylase (PTAC) (Fig 1). INTRO +21 42 alcohol dehydrogenase protein_type NAD+ is recycled via alcohol dehydrogenase, and CoA is recycled via phosphotransacetylase (PTAC) (Fig 1). INTRO +48 51 CoA chemical NAD+ is recycled via alcohol dehydrogenase, and CoA is recycled via phosphotransacetylase (PTAC) (Fig 1). INTRO +68 89 phosphotransacetylase protein_type NAD+ is recycled via alcohol dehydrogenase, and CoA is recycled via phosphotransacetylase (PTAC) (Fig 1). INTRO +91 95 PTAC protein_type NAD+ is recycled via alcohol dehydrogenase, and CoA is recycled via phosphotransacetylase (PTAC) (Fig 1). INTRO +25 28 BMC complex_assembly The final product of the BMC, an acyl-phosphate, can then be used to generate ATP via acyl kinase, or revert back to acyl-CoA by Pta for biosynthesis. INTRO +33 47 acyl-phosphate chemical The final product of the BMC, an acyl-phosphate, can then be used to generate ATP via acyl kinase, or revert back to acyl-CoA by Pta for biosynthesis. INTRO +78 81 ATP chemical The final product of the BMC, an acyl-phosphate, can then be used to generate ATP via acyl kinase, or revert back to acyl-CoA by Pta for biosynthesis. INTRO +86 97 acyl kinase protein_type The final product of the BMC, an acyl-phosphate, can then be used to generate ATP via acyl kinase, or revert back to acyl-CoA by Pta for biosynthesis. INTRO +117 125 acyl-CoA chemical The final product of the BMC, an acyl-phosphate, can then be used to generate ATP via acyl kinase, or revert back to acyl-CoA by Pta for biosynthesis. INTRO +129 132 Pta protein_type The final product of the BMC, an acyl-phosphate, can then be used to generate ATP via acyl kinase, or revert back to acyl-CoA by Pta for biosynthesis. INTRO +18 53 aldehyde and alcohol dehydrogenases protein_type Collectively, the aldehyde and alcohol dehydrogenases, as well as the PTAC, constitute the common metabolosome core. INTRO +70 74 PTAC protein_type Collectively, the aldehyde and alcohol dehydrogenases, as well as the PTAC, constitute the common metabolosome core. INTRO +98 110 metabolosome complex_assembly Collectively, the aldehyde and alcohol dehydrogenases, as well as the PTAC, constitute the common metabolosome core. INTRO +29 47 aldehyde-degrading protein_state General biochemical model of aldehyde-degrading BMCs (metabolosomes) illustrating the common metabolosome core enzymes and reactions. FIG +48 52 BMCs complex_assembly General biochemical model of aldehyde-degrading BMCs (metabolosomes) illustrating the common metabolosome core enzymes and reactions. FIG +54 67 metabolosomes complex_assembly General biochemical model of aldehyde-degrading BMCs (metabolosomes) illustrating the common metabolosome core enzymes and reactions. FIG +93 105 metabolosome complex_assembly General biochemical model of aldehyde-degrading BMCs (metabolosomes) illustrating the common metabolosome core enzymes and reactions. FIG +39 43 PTAC protein_type Substrates and cofactors involving the PTAC reaction are shown in red; other substrates and enzymes are shown in black, and other cofactors are shown in gray. FIG +28 37 catabolic protein_state Characterized and predicted catabolic BMC (metabolosome) types that represent the aldehyde-degrading paradigm (for definition of types see Kerfeld and Erbilgin). TABLE +38 41 BMC complex_assembly Characterized and predicted catabolic BMC (metabolosome) types that represent the aldehyde-degrading paradigm (for definition of types see Kerfeld and Erbilgin). TABLE +43 55 metabolosome complex_assembly Characterized and predicted catabolic BMC (metabolosome) types that represent the aldehyde-degrading paradigm (for definition of types see Kerfeld and Erbilgin). TABLE +82 90 aldehyde chemical Characterized and predicted catabolic BMC (metabolosome) types that represent the aldehyde-degrading paradigm (for definition of types see Kerfeld and Erbilgin). TABLE +5 9 PTAC protein_type "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +27 35 Aldehyde chemical "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +38 41 PDU complex_assembly "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +43 47 PduL protein_type "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +48 63 propionaldehyde chemical "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +66 70 EUT1 complex_assembly "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +71 78 PTA_PTB protein_type "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +79 91 acetaldehyde chemical "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +94 98 EUT2 complex_assembly "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +99 103 PduL protein_type "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +104 116 acetaldehyde chemical "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +119 122 ETU complex_assembly "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +128 140 acetaldehyde chemical "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +143 151 GRM1/CUT complex_assembly "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +152 156 PduL protein_type "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +157 169 acetaldehyde chemical "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +172 176 GRM2 complex_assembly "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +177 181 PduL protein_type "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +182 194 acetaldehyde chemical "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +197 204 GRM3*,4 complex_assembly "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +205 209 PduL protein_type "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +210 225 propionaldehyde chemical "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +228 236 GRM5/GRP complex_assembly "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +237 241 PduL protein_type "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +242 257 propionaldehyde chemical "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +260 263 PVM complex_assembly "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +265 269 PduL protein_type "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +270 282 lactaldehyde chemical "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +285 291 RMM1,2 complex_assembly "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +307 310 SPU complex_assembly "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +311 315 PduL protein_type "Name PTAC Type Sequestered Aldehyde PDU* PduL propionaldehyde EUT1 PTA_PTB acetaldehyde EUT2 PduL acetaldehyde ETU None acetaldehyde GRM1/CUT PduL acetaldehyde GRM2 PduL acetaldehyde GRM3*,4 PduL propionaldehyde GRM5/GRP PduL propionaldehyde PVM* PduL lactaldehyde RMM1,2 None unknown SPU PduL unknown " TABLE +2 6 PduL protein_type * PduL from these functional types of metabolosomes were purified in this study. TABLE +38 51 metabolosomes complex_assembly * PduL from these functional types of metabolosomes were purified in this study. TABLE +51 54 BMC complex_assembly The activities of core enzymes are not confined to BMC-associated functions: aldehyde and alcohol dehydrogenases are utilized in diverse metabolic reactions, and PTAC catalyzes a key biochemical reaction in the process of obtaining energy during fermentation. INTRO +77 112 aldehyde and alcohol dehydrogenases protein_type The activities of core enzymes are not confined to BMC-associated functions: aldehyde and alcohol dehydrogenases are utilized in diverse metabolic reactions, and PTAC catalyzes a key biochemical reaction in the process of obtaining energy during fermentation. INTRO +162 166 PTAC protein_type The activities of core enzymes are not confined to BMC-associated functions: aldehyde and alcohol dehydrogenases are utilized in diverse metabolic reactions, and PTAC catalyzes a key biochemical reaction in the process of obtaining energy during fermentation. INTRO +31 35 PTAC protein_type The concerted functioning of a PTAC and an acetate kinase (Ack) is crucial for ATP generation in the fermentation of pyruvate to acetate (see Reactions 1 and 2). INTRO +43 57 acetate kinase protein_type The concerted functioning of a PTAC and an acetate kinase (Ack) is crucial for ATP generation in the fermentation of pyruvate to acetate (see Reactions 1 and 2). INTRO +59 62 Ack protein_type The concerted functioning of a PTAC and an acetate kinase (Ack) is crucial for ATP generation in the fermentation of pyruvate to acetate (see Reactions 1 and 2). INTRO +79 82 ATP chemical The concerted functioning of a PTAC and an acetate kinase (Ack) is crucial for ATP generation in the fermentation of pyruvate to acetate (see Reactions 1 and 2). INTRO +117 125 pyruvate chemical The concerted functioning of a PTAC and an acetate kinase (Ack) is crucial for ATP generation in the fermentation of pyruvate to acetate (see Reactions 1 and 2). INTRO +129 136 acetate chemical The concerted functioning of a PTAC and an acetate kinase (Ack) is crucial for ATP generation in the fermentation of pyruvate to acetate (see Reactions 1 and 2). INTRO +45 67 fermentative organisms taxonomy_domain Both enzymes are, however, not restricted to fermentative organisms. INTRO +56 63 acetate chemical They can also work in the reverse direction to activate acetate to the CoA-thioester. INTRO +71 84 CoA-thioester chemical They can also work in the reverse direction to activate acetate to the CoA-thioester. INTRO +68 76 archaeal taxonomy_domain This occurs, for example, during acetoclastic methanogenesis in the archaeal Methanosarcina species. INTRO +77 99 Methanosarcina species taxonomy_domain This occurs, for example, during acetoclastic methanogenesis in the archaeal Methanosarcina species. INTRO +13 25 acetyl-S-CoA chemical Reaction 1: acetyl-S-CoA + Pi ←→ acetyl phosphate + CoA-SH (PTAC) INTRO +28 30 Pi chemical Reaction 1: acetyl-S-CoA + Pi ←→ acetyl phosphate + CoA-SH (PTAC) INTRO +34 50 acetyl phosphate chemical Reaction 1: acetyl-S-CoA + Pi ←→ acetyl phosphate + CoA-SH (PTAC) INTRO +53 59 CoA-SH chemical Reaction 1: acetyl-S-CoA + Pi ←→ acetyl phosphate + CoA-SH (PTAC) INTRO +61 65 PTAC protein_type Reaction 1: acetyl-S-CoA + Pi ←→ acetyl phosphate + CoA-SH (PTAC) INTRO +13 29 acetyl phosphate chemical Reaction 2: acetyl phosphate + ADP ←→ acetate + ATP (Ack) INTRO +32 35 ADP chemical Reaction 2: acetyl phosphate + ADP ←→ acetate + ATP (Ack) INTRO +39 46 acetate chemical Reaction 2: acetyl phosphate + ADP ←→ acetate + ATP (Ack) INTRO +49 52 ATP chemical Reaction 2: acetyl phosphate + ADP ←→ acetate + ATP (Ack) INTRO +54 57 Ack protein_type Reaction 2: acetyl phosphate + ADP ←→ acetate + ATP (Ack) INTRO +14 18 PTAC protein_type The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515). INTRO +20 23 Pta protein_type The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515). INTRO +60 70 eukaryotes taxonomy_domain The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515). INTRO +75 82 archaea taxonomy_domain The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515). INTRO +109 117 bacteria taxonomy_domain The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515). INTRO +130 139 bacterial taxonomy_domain The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515). INTRO +221 248 PTA_PTB phosphotransacylase protein_type The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515). INTRO +262 269 PF01515 structure_element The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515). INTRO +0 3 Pta protein_type Pta has been extensively characterized due to its key role in fermentation. INTRO +32 36 PTAC protein_type More recently, a second type of PTAC without any sequence homology to Pta was identified. INTRO +70 73 Pta protein_type More recently, a second type of PTAC without any sequence homology to Pta was identified. INTRO +14 18 PduL protein_type This protein, PduL (Pfam domain PF06130), was shown to catalyze the conversion of propionyl-CoA to propionyl-phosphate and is associated with a BMC involved in propanediol utilization, the PDU BMC. INTRO +32 39 PF06130 structure_element This protein, PduL (Pfam domain PF06130), was shown to catalyze the conversion of propionyl-CoA to propionyl-phosphate and is associated with a BMC involved in propanediol utilization, the PDU BMC. INTRO +82 95 propionyl-CoA chemical This protein, PduL (Pfam domain PF06130), was shown to catalyze the conversion of propionyl-CoA to propionyl-phosphate and is associated with a BMC involved in propanediol utilization, the PDU BMC. INTRO +99 118 propionyl-phosphate chemical This protein, PduL (Pfam domain PF06130), was shown to catalyze the conversion of propionyl-CoA to propionyl-phosphate and is associated with a BMC involved in propanediol utilization, the PDU BMC. INTRO +144 147 BMC complex_assembly This protein, PduL (Pfam domain PF06130), was shown to catalyze the conversion of propionyl-CoA to propionyl-phosphate and is associated with a BMC involved in propanediol utilization, the PDU BMC. INTRO +189 196 PDU BMC complex_assembly This protein, PduL (Pfam domain PF06130), was shown to catalyze the conversion of propionyl-CoA to propionyl-phosphate and is associated with a BMC involved in propanediol utilization, the PDU BMC. INTRO +5 9 pduL gene Both pduL and pta genes can be found in genetic loci of functionally distinct BMCs, although the PduL type is much more prevalent, being found in all but one type of metabolosome locus: EUT1 (Table 1). INTRO +14 17 pta gene Both pduL and pta genes can be found in genetic loci of functionally distinct BMCs, although the PduL type is much more prevalent, being found in all but one type of metabolosome locus: EUT1 (Table 1). INTRO +78 82 BMCs complex_assembly Both pduL and pta genes can be found in genetic loci of functionally distinct BMCs, although the PduL type is much more prevalent, being found in all but one type of metabolosome locus: EUT1 (Table 1). INTRO +97 101 PduL protein_type Both pduL and pta genes can be found in genetic loci of functionally distinct BMCs, although the PduL type is much more prevalent, being found in all but one type of metabolosome locus: EUT1 (Table 1). INTRO +166 184 metabolosome locus gene Both pduL and pta genes can be found in genetic loci of functionally distinct BMCs, although the PduL type is much more prevalent, being found in all but one type of metabolosome locus: EUT1 (Table 1). INTRO +186 190 EUT1 gene Both pduL and pta genes can be found in genetic loci of functionally distinct BMCs, although the PduL type is much more prevalent, being found in all but one type of metabolosome locus: EUT1 (Table 1). INTRO +86 93 PF06130 structure_element Furthermore, in the Integrated Microbial Genomes Database, 91% of genomes that encode PF06130 also encode genes for shell proteins. INTRO +70 88 aldehyde-oxidizing protein_state As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +89 102 metabolosomes complex_assembly As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +104 108 PduL protein_type As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +197 200 Pta protein_type As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +246 256 acetyl-CoA chemical As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +258 262 PduL protein_type As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +272 279 PDU BMC complex_assembly As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +283 302 Salmonella enterica species As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +310 323 propionyl-CoA chemical As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +329 339 acetyl-CoA chemical As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +363 367 PduL protein_type As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +402 406 BMCs complex_assembly As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +450 453 CoA chemical As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives. INTRO +31 45 BMC-associated protein_state Another distinctive feature of BMC-associated PduL homologs is an N-terminal encapsulation peptide (EP) that is thought to “target” proteins for encapsulation by the BMC shell. INTRO +46 50 PduL protein_type Another distinctive feature of BMC-associated PduL homologs is an N-terminal encapsulation peptide (EP) that is thought to “target” proteins for encapsulation by the BMC shell. INTRO +77 98 encapsulation peptide structure_element Another distinctive feature of BMC-associated PduL homologs is an N-terminal encapsulation peptide (EP) that is thought to “target” proteins for encapsulation by the BMC shell. INTRO +100 102 EP structure_element Another distinctive feature of BMC-associated PduL homologs is an N-terminal encapsulation peptide (EP) that is thought to “target” proteins for encapsulation by the BMC shell. INTRO +166 169 BMC complex_assembly Another distinctive feature of BMC-associated PduL homologs is an N-terminal encapsulation peptide (EP) that is thought to “target” proteins for encapsulation by the BMC shell. INTRO +170 175 shell structure_element Another distinctive feature of BMC-associated PduL homologs is an N-terminal encapsulation peptide (EP) that is thought to “target” proteins for encapsulation by the BMC shell. INTRO +0 3 EPs structure_element EPs are frequently found on BMC-associated proteins and have been shown to interact with shell proteins. INTRO +28 51 BMC-associated proteins protein_type EPs are frequently found on BMC-associated proteins and have been shown to interact with shell proteins. INTRO +0 3 EPs structure_element EPs have also been observed to cause proteins to aggregate, and this has recently been suggested to be functionally relevant as an initial step in metabolosome assembly, in which a multifunctional protein core is formed, around which the shell assembles. INTRO +147 159 metabolosome complex_assembly EPs have also been observed to cause proteins to aggregate, and this has recently been suggested to be functionally relevant as an initial step in metabolosome assembly, in which a multifunctional protein core is formed, around which the shell assembles. INTRO +238 243 shell structure_element EPs have also been observed to cause proteins to aggregate, and this has recently been suggested to be functionally relevant as an initial step in metabolosome assembly, in which a multifunctional protein core is formed, around which the shell assembles. INTRO +20 32 metabolosome complex_assembly Of the three common metabolosome core enzymes, crystal structures are available for both the alcohol and aldehyde dehydrogenases. INTRO +47 65 crystal structures evidence Of the three common metabolosome core enzymes, crystal structures are available for both the alcohol and aldehyde dehydrogenases. INTRO +93 128 alcohol and aldehyde dehydrogenases protein_type Of the three common metabolosome core enzymes, crystal structures are available for both the alcohol and aldehyde dehydrogenases. INTRO +17 26 structure evidence In contrast, the structure of PduL, the PTAC found in the vast majority of catabolic BMCs, has not been determined. INTRO +30 34 PduL protein_type In contrast, the structure of PduL, the PTAC found in the vast majority of catabolic BMCs, has not been determined. INTRO +40 44 PTAC protein_type In contrast, the structure of PduL, the PTAC found in the vast majority of catabolic BMCs, has not been determined. INTRO +75 84 catabolic protein_state In contrast, the structure of PduL, the PTAC found in the vast majority of catabolic BMCs, has not been determined. INTRO +85 89 BMCs complex_assembly In contrast, the structure of PduL, the PTAC found in the vast majority of catabolic BMCs, has not been determined. INTRO +44 56 metabolosome complex_assembly This is a major gap in our understanding of metabolosome-encapsulated biochemistry and cofactor recycling. INTRO +66 69 BMC complex_assembly Moreover, it will be useful for guiding efforts to engineer novel BMC cores for biotechnological applications. INTRO +25 29 PduL protein_type The primary structure of PduL homologs is subdivided into two PF06130 domains, each roughly 80 residues in length. INTRO +62 69 PF06130 structure_element The primary structure of PduL homologs is subdivided into two PF06130 domains, each roughly 80 residues in length. INTRO +92 113 80 residues in length residue_range The primary structure of PduL homologs is subdivided into two PF06130 domains, each roughly 80 residues in length. INTRO +44 51 PF06130 structure_element No available protein structures contain the PF06130 domain, and homology searches using the primary structure of PduL do not return any significant results that would allow prediction of the structure. INTRO +64 81 homology searches experimental_method No available protein structures contain the PF06130 domain, and homology searches using the primary structure of PduL do not return any significant results that would allow prediction of the structure. INTRO +113 117 PduL protein_type No available protein structures contain the PF06130 domain, and homology searches using the primary structure of PduL do not return any significant results that would allow prediction of the structure. INTRO +191 200 structure evidence No available protein structures contain the PF06130 domain, and homology searches using the primary structure of PduL do not return any significant results that would allow prediction of the structure. INTRO +33 37 PduL protein_type Moreover, the evident novelty of PduL makes its structure interesting in the context of convergent evolution of PTAC function; to-date, only the Pta active site and catalytic mechanism is known. INTRO +48 57 structure evidence Moreover, the evident novelty of PduL makes its structure interesting in the context of convergent evolution of PTAC function; to-date, only the Pta active site and catalytic mechanism is known. INTRO +112 116 PTAC protein_type Moreover, the evident novelty of PduL makes its structure interesting in the context of convergent evolution of PTAC function; to-date, only the Pta active site and catalytic mechanism is known. INTRO +145 148 Pta protein_type Moreover, the evident novelty of PduL makes its structure interesting in the context of convergent evolution of PTAC function; to-date, only the Pta active site and catalytic mechanism is known. INTRO +149 160 active site site Moreover, the evident novelty of PduL makes its structure interesting in the context of convergent evolution of PTAC function; to-date, only the Pta active site and catalytic mechanism is known. INTRO +31 49 crystal structures evidence Here we report high-resolution crystal structures of a PduL-type PTAC in both CoA- and phosphate-bound forms, completing our understanding of the structural basis of catalysis by the metabolosome common core enzymes. INTRO +55 69 PduL-type PTAC protein_type Here we report high-resolution crystal structures of a PduL-type PTAC in both CoA- and phosphate-bound forms, completing our understanding of the structural basis of catalysis by the metabolosome common core enzymes. INTRO +78 82 CoA- protein_state Here we report high-resolution crystal structures of a PduL-type PTAC in both CoA- and phosphate-bound forms, completing our understanding of the structural basis of catalysis by the metabolosome common core enzymes. INTRO +87 102 phosphate-bound protein_state Here we report high-resolution crystal structures of a PduL-type PTAC in both CoA- and phosphate-bound forms, completing our understanding of the structural basis of catalysis by the metabolosome common core enzymes. INTRO +183 195 metabolosome complex_assembly Here we report high-resolution crystal structures of a PduL-type PTAC in both CoA- and phosphate-bound forms, completing our understanding of the structural basis of catalysis by the metabolosome common core enzymes. INTRO +80 83 Pta protein_type We propose a catalytic mechanism analogous but yet distinct from the ubiquitous Pta enzyme, highlighting the functional convergence of two enzymes with completely different structures and metal requirements. INTRO +65 69 PduL protein_type We also investigate the quaternary structures of three different PduL homologs and situate our findings in the context of organelle biogenesis in functionally diverse BMCs. INTRO +167 171 BMCs complex_assembly We also investigate the quaternary structures of three different PduL homologs and situate our findings in the context of organelle biogenesis in functionally diverse BMCs. INTRO +0 23 Structure Determination experimental_method Structure Determination of PduL RESULTS +27 31 PduL protein_type Structure Determination of PduL RESULTS +3 34 cloned, expressed, and purified experimental_method We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). RESULTS +51 55 PduL protein_type We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). RESULTS +92 96 BMCs complex_assembly We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). RESULTS +130 139 pdu locus gene We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). RESULTS +143 170 S. enterica Typhimurium LT2 species We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). RESULTS +172 177 sPduL protein We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). RESULTS +212 221 pvm locus gene We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). RESULTS +225 249 Planctomyces limnophilus species We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). RESULTS +251 256 pPduL protein We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). RESULTS +272 282 grm3 locus gene We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). RESULTS +286 319 Rhodopseudomonas palustris BisB18 species We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). RESULTS +321 326 rPduL protein We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL). RESULTS +16 27 full-length protein_state While purifying full-length sPduL, we observed a tendency to aggregation as described previously, with a large fraction of the expressed protein found in the insoluble fraction in a white, cake-like pellet. RESULTS +28 33 sPduL protein While purifying full-length sPduL, we observed a tendency to aggregation as described previously, with a large fraction of the expressed protein found in the insoluble fraction in a white, cake-like pellet. RESULTS +18 26 removing experimental_method Remarkably, after removing the N-terminal putative EP (27 amino acids), most of the sPduLΔEP protein was in the soluble fraction upon cell lysis. RESULTS +51 53 EP structure_element Remarkably, after removing the N-terminal putative EP (27 amino acids), most of the sPduLΔEP protein was in the soluble fraction upon cell lysis. RESULTS +55 69 27 amino acids residue_range Remarkably, after removing the N-terminal putative EP (27 amino acids), most of the sPduLΔEP protein was in the soluble fraction upon cell lysis. RESULTS +84 92 sPduLΔEP mutant Remarkably, after removing the N-terminal putative EP (27 amino acids), most of the sPduLΔEP protein was in the soluble fraction upon cell lysis. RESULTS +52 57 pPduL protein Similar differences in solubility were observed for pPduL and rPduL when comparing EP-truncated forms to the full-length protein, but none were quite as dramatic as for sPduL. We confirmed that all homologs were active (S1a and S1b Fig). RESULTS +62 67 rPduL protein Similar differences in solubility were observed for pPduL and rPduL when comparing EP-truncated forms to the full-length protein, but none were quite as dramatic as for sPduL. We confirmed that all homologs were active (S1a and S1b Fig). RESULTS +83 95 EP-truncated protein_state Similar differences in solubility were observed for pPduL and rPduL when comparing EP-truncated forms to the full-length protein, but none were quite as dramatic as for sPduL. We confirmed that all homologs were active (S1a and S1b Fig). RESULTS +109 120 full-length protein_state Similar differences in solubility were observed for pPduL and rPduL when comparing EP-truncated forms to the full-length protein, but none were quite as dramatic as for sPduL. We confirmed that all homologs were active (S1a and S1b Fig). RESULTS +169 174 sPduL protein Similar differences in solubility were observed for pPduL and rPduL when comparing EP-truncated forms to the full-length protein, but none were quite as dramatic as for sPduL. We confirmed that all homologs were active (S1a and S1b Fig). RESULTS +212 218 active protein_state Similar differences in solubility were observed for pPduL and rPduL when comparing EP-truncated forms to the full-length protein, but none were quite as dramatic as for sPduL. We confirmed that all homologs were active (S1a and S1b Fig). RESULTS +41 69 diffraction-quality crystals evidence Among these, we were only able to obtain diffraction-quality crystals of rPduL after removing the N-terminal putative EP (33 amino acids, also see Fig 2a) (rPduLΔEP). RESULTS +73 78 rPduL protein Among these, we were only able to obtain diffraction-quality crystals of rPduL after removing the N-terminal putative EP (33 amino acids, also see Fig 2a) (rPduLΔEP). RESULTS +85 93 removing experimental_method Among these, we were only able to obtain diffraction-quality crystals of rPduL after removing the N-terminal putative EP (33 amino acids, also see Fig 2a) (rPduLΔEP). RESULTS +118 120 EP structure_element Among these, we were only able to obtain diffraction-quality crystals of rPduL after removing the N-terminal putative EP (33 amino acids, also see Fig 2a) (rPduLΔEP). RESULTS +122 136 33 amino acids residue_range Among these, we were only able to obtain diffraction-quality crystals of rPduL after removing the N-terminal putative EP (33 amino acids, also see Fig 2a) (rPduLΔEP). RESULTS +156 164 rPduLΔEP mutant Among these, we were only able to obtain diffraction-quality crystals of rPduL after removing the N-terminal putative EP (33 amino acids, also see Fig 2a) (rPduLΔEP). RESULTS +0 9 Truncated protein_state Truncated rPduLΔEP had comparable enzymatic activity to the full-length enzyme (S1a Fig). RESULTS +10 18 rPduLΔEP mutant Truncated rPduLΔEP had comparable enzymatic activity to the full-length enzyme (S1a Fig). RESULTS +60 71 full-length protein_state Truncated rPduLΔEP had comparable enzymatic activity to the full-length enzyme (S1a Fig). RESULTS +23 35 R. palustris species Structural overview of R. palustris PduL from the grm3 locus. FIG +36 40 PduL protein_type Structural overview of R. palustris PduL from the grm3 locus. FIG +50 60 grm3 locus gene Structural overview of R. palustris PduL from the grm3 locus. FIG +39 44 rPduL protein (a) Primary and secondary structure of rPduL (tubes represent α-helices, arrows β-sheets and dashed line residues disordered in the structure. FIG +62 71 α-helices structure_element (a) Primary and secondary structure of rPduL (tubes represent α-helices, arrows β-sheets and dashed line residues disordered in the structure. FIG +80 88 β-sheets structure_element (a) Primary and secondary structure of rPduL (tubes represent α-helices, arrows β-sheets and dashed line residues disordered in the structure. FIG +132 141 structure evidence (a) Primary and secondary structure of rPduL (tubes represent α-helices, arrows β-sheets and dashed line residues disordered in the structure. FIG +4 24 first 33 amino acids residue_range The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +95 97 EP structure_element The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +98 109 alpha helix structure_element The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +111 113 α0 structure_element The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +120 129 truncated protein_state The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +130 138 rPduLΔEP mutant The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +148 160 crystallized experimental_method The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +173 174 M residue_name The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +175 176 G residue_name The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +177 178 V residue_name The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +225 233 domain 1 structure_element The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +234 241 D36-N46 residue_range The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +242 251 Q155-C224 residue_range The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +259 273 loop insertion structure_element The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +274 281 G61-E81 residue_range The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +289 297 domain 2 structure_element The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +298 305 R47-F60 residue_range The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +306 314 E82-A154 residue_range The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, α0); the truncated rPduLΔEP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red). FIG +0 27 Metal coordination residues site Metal coordination residues are highlighted in light blue and CoA contacting residues in magenta, residues contacting the CoA of the other chain are also outlined. FIG +62 85 CoA contacting residues site Metal coordination residues are highlighted in light blue and CoA contacting residues in magenta, residues contacting the CoA of the other chain are also outlined. FIG +122 125 CoA chemical Metal coordination residues are highlighted in light blue and CoA contacting residues in magenta, residues contacting the CoA of the other chain are also outlined. FIG +34 43 structure evidence (b) Cartoon representation of the structure colored by domains and including secondary structure numbering. FIG +87 96 structure evidence (b) Cartoon representation of the structure colored by domains and including secondary structure numbering. FIG +0 10 Coenzyme A chemical Coenzyme A is shown in magenta sticks and Zinc (grey) as spheres. FIG +42 46 Zinc chemical Coenzyme A is shown in magenta sticks and Zinc (grey) as spheres. FIG +3 29 collected a native dataset experimental_method We collected a native dataset from rPduLΔEP crystals diffracting to a resolution of 1.54 Å (Table 2). RESULTS +35 43 rPduLΔEP mutant We collected a native dataset from rPduLΔEP crystals diffracting to a resolution of 1.54 Å (Table 2). RESULTS +44 52 crystals evidence We collected a native dataset from rPduLΔEP crystals diffracting to a resolution of 1.54 Å (Table 2). RESULTS +8 34 mercury-derivative crystal experimental_method Using a mercury-derivative crystal form diffracting to 1.99 Å (Table 2), we obtained high quality electron density for model building and used the initial model to refine against the native data to Rwork/Rfree values of 18.9/22.1%. RESULTS +98 114 electron density evidence Using a mercury-derivative crystal form diffracting to 1.99 Å (Table 2), we obtained high quality electron density for model building and used the initial model to refine against the native data to Rwork/Rfree values of 18.9/22.1%. RESULTS +198 203 Rwork evidence Using a mercury-derivative crystal form diffracting to 1.99 Å (Table 2), we obtained high quality electron density for model building and used the initial model to refine against the native data to Rwork/Rfree values of 18.9/22.1%. RESULTS +204 209 Rfree evidence Using a mercury-derivative crystal form diffracting to 1.99 Å (Table 2), we obtained high quality electron density for model building and used the initial model to refine against the native data to Rwork/Rfree values of 18.9/22.1%. RESULTS +14 18 PduL protein_type There are two PduL molecules in the asymmetric unit of the P212121 unit cell. RESULTS +52 59 PduLΔEP mutant We were able to fit all of the primary structure of PduLΔEP into the electron density with the exception of three amino acids at the N-terminus and two amino acids at the C-terminus (Fig 2a); the model is of excellent quality (Table 2). RESULTS +69 85 electron density evidence We were able to fit all of the primary structure of PduLΔEP into the electron density with the exception of three amino acids at the N-terminus and two amino acids at the C-terminus (Fig 2a); the model is of excellent quality (Table 2). RESULTS +2 5 CoA chemical A CoA cofactor as well as two metal ions are clearly resolved in the density (for omit maps of CoA see S2 Fig). RESULTS +69 76 density evidence A CoA cofactor as well as two metal ions are clearly resolved in the density (for omit maps of CoA see S2 Fig). RESULTS +82 91 omit maps evidence A CoA cofactor as well as two metal ions are clearly resolved in the density (for omit maps of CoA see S2 Fig). RESULTS +95 98 CoA chemical A CoA cofactor as well as two metal ions are clearly resolved in the density (for omit maps of CoA see S2 Fig). RESULTS +14 18 PduL protein_type Structurally, PduL consists of two domains (Fig 2, blue/red), each a beta-barrel that is capped on both ends by short α-helices. RESULTS +35 42 domains structure_element Structurally, PduL consists of two domains (Fig 2, blue/red), each a beta-barrel that is capped on both ends by short α-helices. RESULTS +69 80 beta-barrel structure_element Structurally, PduL consists of two domains (Fig 2, blue/red), each a beta-barrel that is capped on both ends by short α-helices. RESULTS +118 127 α-helices structure_element Structurally, PduL consists of two domains (Fig 2, blue/red), each a beta-barrel that is capped on both ends by short α-helices. RESULTS +0 10 β-Barrel 1 structure_element β-Barrel 1 consists of the N-terminal β strand and β strands from the C-terminal half of the polypeptide chain (β1, β10-β14; residues 37–46 and 155–224). RESULTS +38 46 β strand structure_element β-Barrel 1 consists of the N-terminal β strand and β strands from the C-terminal half of the polypeptide chain (β1, β10-β14; residues 37–46 and 155–224). RESULTS +51 60 β strands structure_element β-Barrel 1 consists of the N-terminal β strand and β strands from the C-terminal half of the polypeptide chain (β1, β10-β14; residues 37–46 and 155–224). RESULTS +70 85 C-terminal half structure_element β-Barrel 1 consists of the N-terminal β strand and β strands from the C-terminal half of the polypeptide chain (β1, β10-β14; residues 37–46 and 155–224). RESULTS +112 114 β1 structure_element β-Barrel 1 consists of the N-terminal β strand and β strands from the C-terminal half of the polypeptide chain (β1, β10-β14; residues 37–46 and 155–224). RESULTS +116 123 β10-β14 structure_element β-Barrel 1 consists of the N-terminal β strand and β strands from the C-terminal half of the polypeptide chain (β1, β10-β14; residues 37–46 and 155–224). RESULTS +134 139 37–46 residue_range β-Barrel 1 consists of the N-terminal β strand and β strands from the C-terminal half of the polypeptide chain (β1, β10-β14; residues 37–46 and 155–224). RESULTS +144 151 155–224 residue_range β-Barrel 1 consists of the N-terminal β strand and β strands from the C-terminal half of the polypeptide chain (β1, β10-β14; residues 37–46 and 155–224). RESULTS +0 10 β-Barrel 2 structure_element β-Barrel 2 consists mainly of the central segment of primary structure (β2, β5–β9; residues 47–60 and 82–154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (β3-β4, residues 61–81). RESULTS +72 74 β2 structure_element β-Barrel 2 consists mainly of the central segment of primary structure (β2, β5–β9; residues 47–60 and 82–154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (β3-β4, residues 61–81). RESULTS +76 81 β5–β9 structure_element β-Barrel 2 consists mainly of the central segment of primary structure (β2, β5–β9; residues 47–60 and 82–154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (β3-β4, residues 61–81). RESULTS +92 97 47–60 residue_range β-Barrel 2 consists mainly of the central segment of primary structure (β2, β5–β9; residues 47–60 and 82–154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (β3-β4, residues 61–81). RESULTS +102 108 82–154 residue_range β-Barrel 2 consists mainly of the central segment of primary structure (β2, β5–β9; residues 47–60 and 82–154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (β3-β4, residues 61–81). RESULTS +148 175 short two-strand beta sheet structure_element β-Barrel 2 consists mainly of the central segment of primary structure (β2, β5–β9; residues 47–60 and 82–154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (β3-β4, residues 61–81). RESULTS +177 182 β3-β4 structure_element β-Barrel 2 consists mainly of the central segment of primary structure (β2, β5–β9; residues 47–60 and 82–154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (β3-β4, residues 61–81). RESULTS +193 198 61–81 residue_range β-Barrel 2 consists mainly of the central segment of primary structure (β2, β5–β9; residues 47–60 and 82–154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (β3-β4, residues 61–81). RESULTS +5 12 β-sheet structure_element This β-sheet is involved in contacts between the two domains and forms a lid over the active site. RESULTS +86 97 active site site This β-sheet is involved in contacts between the two domains and forms a lid over the active site. RESULTS +25 30 Gln42 residue_name_number Residues in this region (Gln42, Pro43, Gly44), covering the active site, are strongly conserved (Fig 3). RESULTS +32 37 Pro43 residue_name_number Residues in this region (Gln42, Pro43, Gly44), covering the active site, are strongly conserved (Fig 3). RESULTS +39 44 Gly44 residue_name_number Residues in this region (Gln42, Pro43, Gly44), covering the active site, are strongly conserved (Fig 3). RESULTS +60 71 active site site Residues in this region (Gln42, Pro43, Gly44), covering the active site, are strongly conserved (Fig 3). RESULTS +77 95 strongly conserved protein_state Residues in this region (Gln42, Pro43, Gly44), covering the active site, are strongly conserved (Fig 3). RESULTS +82 85 Pta protein_type This structural arrangement is completely different from the functionally related Pta, which is composed of two domains, each consisting of a central flat beta sheet with alpha-helices on the top and bottom. RESULTS +112 119 domains structure_element This structural arrangement is completely different from the functionally related Pta, which is composed of two domains, each consisting of a central flat beta sheet with alpha-helices on the top and bottom. RESULTS +155 165 beta sheet structure_element This structural arrangement is completely different from the functionally related Pta, which is composed of two domains, each consisting of a central flat beta sheet with alpha-helices on the top and bottom. RESULTS +171 184 alpha-helices structure_element This structural arrangement is completely different from the functionally related Pta, which is composed of two domains, each consisting of a central flat beta sheet with alpha-helices on the top and bottom. RESULTS +38 42 PduL protein_type Primary structure conservation of the PduL protein family. FIG +34 61 multiple sequence alignment experimental_method Sequence logo calculated from the multiple sequence alignment of PduL homologs (see Materials and Methods), but not including putative EP sequences. FIG +65 69 PduL protein_type Sequence logo calculated from the multiple sequence alignment of PduL homologs (see Materials and Methods), but not including putative EP sequences. FIG +112 125 not including protein_state Sequence logo calculated from the multiple sequence alignment of PduL homologs (see Materials and Methods), but not including putative EP sequences. FIG +135 137 EP structure_element Sequence logo calculated from the multiple sequence alignment of PduL homologs (see Materials and Methods), but not including putative EP sequences. FIG +35 39 PduL protein_type Residues 100% conserved across all PduL homologs in our dataset are noted with an asterisk, and residues conserved in over 90% of sequences are noted with a colon. FIG +30 37 PF06130 structure_element The sequences aligning to the PF06130 domain (determined by BLAST) are highlighted in red and blue. FIG +66 71 rPduL protein The position numbers shown correspond to the residue numbering of rPduL; note that some positions in the logo represent gaps in the rPduL sequence. FIG +132 137 rPduL protein The position numbers shown correspond to the residue numbering of rPduL; note that some positions in the logo represent gaps in the rPduL sequence. FIG +14 18 PduL protein_type There are two PduL molecules in the asymmetric unit forming a butterfly-shaped dimer (Fig 4c). RESULTS +62 78 butterfly-shaped protein_state There are two PduL molecules in the asymmetric unit forming a butterfly-shaped dimer (Fig 4c). RESULTS +79 84 dimer oligomeric_state There are two PduL molecules in the asymmetric unit forming a butterfly-shaped dimer (Fig 4c). RESULTS +35 64 size exclusion chromatography experimental_method Consistent with this, results from size exclusion chromatography of rPduLΔEP suggest that it is a dimer in solution (Fig 5e). RESULTS +68 76 rPduLΔEP mutant Consistent with this, results from size exclusion chromatography of rPduLΔEP suggest that it is a dimer in solution (Fig 5e). RESULTS +98 103 dimer oligomeric_state Consistent with this, results from size exclusion chromatography of rPduLΔEP suggest that it is a dimer in solution (Fig 5e). RESULTS +4 13 interface site The interface between the two chains buries 882 Å2 per monomer and is mainly formed by α-helices 2 and 4 and parts of β-sheets 12 and 14, as well as a π–π stacking of the adenine moiety of CoA with Phe116 of the adjacent chain (Fig 4c). RESULTS +55 62 monomer oligomeric_state The interface between the two chains buries 882 Å2 per monomer and is mainly formed by α-helices 2 and 4 and parts of β-sheets 12 and 14, as well as a π–π stacking of the adenine moiety of CoA with Phe116 of the adjacent chain (Fig 4c). RESULTS +87 104 α-helices 2 and 4 structure_element The interface between the two chains buries 882 Å2 per monomer and is mainly formed by α-helices 2 and 4 and parts of β-sheets 12 and 14, as well as a π–π stacking of the adenine moiety of CoA with Phe116 of the adjacent chain (Fig 4c). RESULTS +118 136 β-sheets 12 and 14 structure_element The interface between the two chains buries 882 Å2 per monomer and is mainly formed by α-helices 2 and 4 and parts of β-sheets 12 and 14, as well as a π–π stacking of the adenine moiety of CoA with Phe116 of the adjacent chain (Fig 4c). RESULTS +171 178 adenine chemical The interface between the two chains buries 882 Å2 per monomer and is mainly formed by α-helices 2 and 4 and parts of β-sheets 12 and 14, as well as a π–π stacking of the adenine moiety of CoA with Phe116 of the adjacent chain (Fig 4c). RESULTS +189 192 CoA chemical The interface between the two chains buries 882 Å2 per monomer and is mainly formed by α-helices 2 and 4 and parts of β-sheets 12 and 14, as well as a π–π stacking of the adenine moiety of CoA with Phe116 of the adjacent chain (Fig 4c). RESULTS +198 204 Phe116 residue_name_number The interface between the two chains buries 882 Å2 per monomer and is mainly formed by α-helices 2 and 4 and parts of β-sheets 12 and 14, as well as a π–π stacking of the adenine moiety of CoA with Phe116 of the adjacent chain (Fig 4c). RESULTS +69 82 superimposing experimental_method The folds of the two chains in the asymmetric unit are very similar, superimposing with a rmsd of 0.16 Å over 2,306 aligned atom pairs. RESULTS +90 94 rmsd evidence The folds of the two chains in the asymmetric unit are very similar, superimposing with a rmsd of 0.16 Å over 2,306 aligned atom pairs. RESULTS +15 22 helices structure_element The peripheral helices and the short antiparallel β3–4 sheet mediate most of the crystal contacts. RESULTS +31 60 short antiparallel β3–4 sheet structure_element The peripheral helices and the short antiparallel β3–4 sheet mediate most of the crystal contacts. RESULTS +11 22 active site site Details of active site, dimeric assembly, and sequence conservation of PduL. FIG +24 31 dimeric oligomeric_state Details of active site, dimeric assembly, and sequence conservation of PduL. FIG +71 75 PduL protein_type Details of active site, dimeric assembly, and sequence conservation of PduL. FIG +15 26 active site site (a,b) Proposed active site of PduL with relevant residues shown as sticks in atom coloring (nitrogen blue, oxygen red, sulfur yellow), zinc as grey colored spheres and coordinating ordered water molecules in red. FIG +30 34 PduL protein_type (a,b) Proposed active site of PduL with relevant residues shown as sticks in atom coloring (nitrogen blue, oxygen red, sulfur yellow), zinc as grey colored spheres and coordinating ordered water molecules in red. FIG +92 100 nitrogen chemical (a,b) Proposed active site of PduL with relevant residues shown as sticks in atom coloring (nitrogen blue, oxygen red, sulfur yellow), zinc as grey colored spheres and coordinating ordered water molecules in red. FIG +107 113 oxygen chemical (a,b) Proposed active site of PduL with relevant residues shown as sticks in atom coloring (nitrogen blue, oxygen red, sulfur yellow), zinc as grey colored spheres and coordinating ordered water molecules in red. FIG +119 125 sulfur chemical (a,b) Proposed active site of PduL with relevant residues shown as sticks in atom coloring (nitrogen blue, oxygen red, sulfur yellow), zinc as grey colored spheres and coordinating ordered water molecules in red. FIG +135 139 zinc chemical (a,b) Proposed active site of PduL with relevant residues shown as sticks in atom coloring (nitrogen blue, oxygen red, sulfur yellow), zinc as grey colored spheres and coordinating ordered water molecules in red. FIG +189 194 water chemical (a,b) Proposed active site of PduL with relevant residues shown as sticks in atom coloring (nitrogen blue, oxygen red, sulfur yellow), zinc as grey colored spheres and coordinating ordered water molecules in red. FIG +55 65 Coenzyme A chemical Distances between atom centers are indicated in Å. (a) Coenzyme A containing, (b) phosphate-bound structure. FIG +82 97 phosphate-bound protein_state Distances between atom centers are indicated in Å. (a) Coenzyme A containing, (b) phosphate-bound structure. FIG +98 107 structure evidence Distances between atom centers are indicated in Å. (a) Coenzyme A containing, (b) phosphate-bound structure. FIG +16 21 dimer oligomeric_state (c) View of the dimer in the asymmetric unit from the side, domains 1 and 2 colored as in Fig 2 and the two chains differentiated by blue/red versus slate/firebrick. FIG +60 75 domains 1 and 2 structure_element (c) View of the dimer in the asymmetric unit from the side, domains 1 and 2 colored as in Fig 2 and the two chains differentiated by blue/red versus slate/firebrick. FIG +56 71 π–π interaction bond_interaction The asterisk and double arrow marks the location of the π–π interaction between F116 and the CoA base of the other dimer chain. FIG +80 84 F116 residue_name_number The asterisk and double arrow marks the location of the π–π interaction between F116 and the CoA base of the other dimer chain. FIG +93 96 CoA chemical The asterisk and double arrow marks the location of the π–π interaction between F116 and the CoA base of the other dimer chain. FIG +115 120 dimer oligomeric_state The asterisk and double arrow marks the location of the π–π interaction between F116 and the CoA base of the other dimer chain. FIG +34 43 structure evidence (d) Surface representation of the structure with indicated conservation (red: high, white: intermediate, yellow: low). FIG +0 29 Size exclusion chromatography experimental_method Size exclusion chromatography of PduL homologs. FIG +33 37 PduL protein_type Size exclusion chromatography of PduL homologs. FIG +9 22 Chromatograms evidence (a)–(c): Chromatograms of sPduL (a), rPduL (b), and pPduL (c) with (orange) or without (blue) the predicted EP, post-nickel affinity purification, applied over a preparative size exclusion column (see Materials and Methods). FIG +26 31 sPduL protein (a)–(c): Chromatograms of sPduL (a), rPduL (b), and pPduL (c) with (orange) or without (blue) the predicted EP, post-nickel affinity purification, applied over a preparative size exclusion column (see Materials and Methods). FIG +37 42 rPduL protein (a)–(c): Chromatograms of sPduL (a), rPduL (b), and pPduL (c) with (orange) or without (blue) the predicted EP, post-nickel affinity purification, applied over a preparative size exclusion column (see Materials and Methods). FIG +52 57 pPduL protein (a)–(c): Chromatograms of sPduL (a), rPduL (b), and pPduL (c) with (orange) or without (blue) the predicted EP, post-nickel affinity purification, applied over a preparative size exclusion column (see Materials and Methods). FIG +108 110 EP structure_element (a)–(c): Chromatograms of sPduL (a), rPduL (b), and pPduL (c) with (orange) or without (blue) the predicted EP, post-nickel affinity purification, applied over a preparative size exclusion column (see Materials and Methods). FIG +117 145 nickel affinity purification experimental_method (a)–(c): Chromatograms of sPduL (a), rPduL (b), and pPduL (c) with (orange) or without (blue) the predicted EP, post-nickel affinity purification, applied over a preparative size exclusion column (see Materials and Methods). FIG +9 22 Chromatograms evidence (d)–(f): Chromatograms of sPduL (d), rPduL (e), and pPduL (f) post-preparative size exclusion chromatography with different size fractions separated, applied over an analytical size exclusion column (see Materials and Methods). FIG +26 31 sPduL protein (d)–(f): Chromatograms of sPduL (d), rPduL (e), and pPduL (f) post-preparative size exclusion chromatography with different size fractions separated, applied over an analytical size exclusion column (see Materials and Methods). FIG +37 42 rPduL protein (d)–(f): Chromatograms of sPduL (d), rPduL (e), and pPduL (f) post-preparative size exclusion chromatography with different size fractions separated, applied over an analytical size exclusion column (see Materials and Methods). FIG +52 57 pPduL protein (d)–(f): Chromatograms of sPduL (d), rPduL (e), and pPduL (f) post-preparative size exclusion chromatography with different size fractions separated, applied over an analytical size exclusion column (see Materials and Methods). FIG +79 108 size exclusion chromatography experimental_method (d)–(f): Chromatograms of sPduL (d), rPduL (e), and pPduL (f) post-preparative size exclusion chromatography with different size fractions separated, applied over an analytical size exclusion column (see Materials and Methods). FIG +4 17 chromatograms evidence All chromatograms are cropped to show only the linear range of separation based on standard runs, shown in black squares with a dashed linear trend line. FIG +0 11 Active Site site Active Site Properties RESULTS +0 3 CoA chemical CoA and the metal ions bind between the two domains, presumably in the active site (Figs 2b and 4a). RESULTS +71 82 active site site CoA and the metal ions bind between the two domains, presumably in the active site (Figs 2b and 4a). RESULTS +46 69 X-ray fluorescence scan experimental_method To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn). RESULTS +77 85 crystals evidence To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn). RESULTS +142 144 Mn chemical To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn). RESULTS +146 148 Fe chemical To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn). RESULTS +150 152 Co chemical To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn). RESULTS +154 156 Ni chemical To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn). RESULTS +158 160 Cu chemical To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn). RESULTS +166 168 Zn chemical To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn). RESULTS +77 81 zinc chemical There was a large signal at the zinc edge, and we tested for the presence of zinc by collecting full data sets before and after the Zn K-edge (1.2861 and 1.2822 Å, respectively). RESULTS +85 162 collecting full data sets before and after the Zn K-edge (1.2861 and 1.2822 Å experimental_method There was a large signal at the zinc edge, and we tested for the presence of zinc by collecting full data sets before and after the Zn K-edge (1.2861 and 1.2822 Å, respectively). RESULTS +76 80 zinc chemical The large differences between the anomalous signals confirm the presence of zinc at both metal sites (S3 Fig). RESULTS +10 14 zinc chemical The first zinc ion (Zn1) is in a tetrahedral coordination state with His48, His50, Glu109, and the CoA sulfur (Fig 4a). RESULTS +20 23 Zn1 chemical The first zinc ion (Zn1) is in a tetrahedral coordination state with His48, His50, Glu109, and the CoA sulfur (Fig 4a). RESULTS +69 74 His48 residue_name_number The first zinc ion (Zn1) is in a tetrahedral coordination state with His48, His50, Glu109, and the CoA sulfur (Fig 4a). RESULTS +76 81 His50 residue_name_number The first zinc ion (Zn1) is in a tetrahedral coordination state with His48, His50, Glu109, and the CoA sulfur (Fig 4a). RESULTS +83 89 Glu109 residue_name_number The first zinc ion (Zn1) is in a tetrahedral coordination state with His48, His50, Glu109, and the CoA sulfur (Fig 4a). RESULTS +99 102 CoA chemical The first zinc ion (Zn1) is in a tetrahedral coordination state with His48, His50, Glu109, and the CoA sulfur (Fig 4a). RESULTS +103 109 sulfur chemical The first zinc ion (Zn1) is in a tetrahedral coordination state with His48, His50, Glu109, and the CoA sulfur (Fig 4a). RESULTS +12 15 Zn2 chemical The second (Zn2) is in octahedral coordination by three conserved histidine residues (His157, His159 and His204) as well as three water molecules (Fig 4a). RESULTS +56 65 conserved protein_state The second (Zn2) is in octahedral coordination by three conserved histidine residues (His157, His159 and His204) as well as three water molecules (Fig 4a). RESULTS +66 75 histidine residue_name The second (Zn2) is in octahedral coordination by three conserved histidine residues (His157, His159 and His204) as well as three water molecules (Fig 4a). RESULTS +86 92 His157 residue_name_number The second (Zn2) is in octahedral coordination by three conserved histidine residues (His157, His159 and His204) as well as three water molecules (Fig 4a). RESULTS +94 100 His159 residue_name_number The second (Zn2) is in octahedral coordination by three conserved histidine residues (His157, His159 and His204) as well as three water molecules (Fig 4a). RESULTS +105 111 His204 residue_name_number The second (Zn2) is in octahedral coordination by three conserved histidine residues (His157, His159 and His204) as well as three water molecules (Fig 4a). RESULTS +130 135 water chemical The second (Zn2) is in octahedral coordination by three conserved histidine residues (His157, His159 and His204) as well as three water molecules (Fig 4a). RESULTS +35 39 zinc chemical The nitrogen atom coordinating the zinc is the Nε in each histidine residue, as is typical for this interaction. RESULTS +58 67 histidine residue_name The nitrogen atom coordinating the zinc is the Nε in each histidine residue, as is typical for this interaction. RESULTS +9 29 crystals were soaked experimental_method When the crystals were soaked in a sodium phosphate solution for 2 d prior to data collection, the CoA dissociates, and density for a phosphate molecule is visible at the active site (Table 2, Fig 4b). RESULTS +35 51 sodium phosphate chemical When the crystals were soaked in a sodium phosphate solution for 2 d prior to data collection, the CoA dissociates, and density for a phosphate molecule is visible at the active site (Table 2, Fig 4b). RESULTS +99 102 CoA chemical When the crystals were soaked in a sodium phosphate solution for 2 d prior to data collection, the CoA dissociates, and density for a phosphate molecule is visible at the active site (Table 2, Fig 4b). RESULTS +120 127 density evidence When the crystals were soaked in a sodium phosphate solution for 2 d prior to data collection, the CoA dissociates, and density for a phosphate molecule is visible at the active site (Table 2, Fig 4b). RESULTS +134 143 phosphate chemical When the crystals were soaked in a sodium phosphate solution for 2 d prior to data collection, the CoA dissociates, and density for a phosphate molecule is visible at the active site (Table 2, Fig 4b). RESULTS +171 182 active site site When the crystals were soaked in a sodium phosphate solution for 2 d prior to data collection, the CoA dissociates, and density for a phosphate molecule is visible at the active site (Table 2, Fig 4b). RESULTS +4 19 phosphate-bound protein_state The phosphate-bound structure aligns well with the CoA-bound structure (0.43 Å rmsd over 2,361 atoms for the monomer, 0.83 Å over 5,259 aligned atoms for the dimer). RESULTS +20 29 structure evidence The phosphate-bound structure aligns well with the CoA-bound structure (0.43 Å rmsd over 2,361 atoms for the monomer, 0.83 Å over 5,259 aligned atoms for the dimer). RESULTS +30 36 aligns experimental_method The phosphate-bound structure aligns well with the CoA-bound structure (0.43 Å rmsd over 2,361 atoms for the monomer, 0.83 Å over 5,259 aligned atoms for the dimer). RESULTS +51 60 CoA-bound protein_state The phosphate-bound structure aligns well with the CoA-bound structure (0.43 Å rmsd over 2,361 atoms for the monomer, 0.83 Å over 5,259 aligned atoms for the dimer). RESULTS +61 70 structure evidence The phosphate-bound structure aligns well with the CoA-bound structure (0.43 Å rmsd over 2,361 atoms for the monomer, 0.83 Å over 5,259 aligned atoms for the dimer). RESULTS +79 83 rmsd evidence The phosphate-bound structure aligns well with the CoA-bound structure (0.43 Å rmsd over 2,361 atoms for the monomer, 0.83 Å over 5,259 aligned atoms for the dimer). RESULTS +109 116 monomer oligomeric_state The phosphate-bound structure aligns well with the CoA-bound structure (0.43 Å rmsd over 2,361 atoms for the monomer, 0.83 Å over 5,259 aligned atoms for the dimer). RESULTS +158 163 dimer oligomeric_state The phosphate-bound structure aligns well with the CoA-bound structure (0.43 Å rmsd over 2,361 atoms for the monomer, 0.83 Å over 5,259 aligned atoms for the dimer). RESULTS +4 13 phosphate chemical The phosphate contacts both zinc atoms (Fig 4b) and replaces the coordination by CoA at Zn1; the coordination for Zn2 changes from octahedral with three bound waters to tetrahedral with a phosphate ion as one of the ligands (Fig 4b). RESULTS +28 32 zinc chemical The phosphate contacts both zinc atoms (Fig 4b) and replaces the coordination by CoA at Zn1; the coordination for Zn2 changes from octahedral with three bound waters to tetrahedral with a phosphate ion as one of the ligands (Fig 4b). RESULTS +81 84 CoA chemical The phosphate contacts both zinc atoms (Fig 4b) and replaces the coordination by CoA at Zn1; the coordination for Zn2 changes from octahedral with three bound waters to tetrahedral with a phosphate ion as one of the ligands (Fig 4b). RESULTS +88 91 Zn1 chemical The phosphate contacts both zinc atoms (Fig 4b) and replaces the coordination by CoA at Zn1; the coordination for Zn2 changes from octahedral with three bound waters to tetrahedral with a phosphate ion as one of the ligands (Fig 4b). RESULTS +114 117 Zn2 chemical The phosphate contacts both zinc atoms (Fig 4b) and replaces the coordination by CoA at Zn1; the coordination for Zn2 changes from octahedral with three bound waters to tetrahedral with a phosphate ion as one of the ligands (Fig 4b). RESULTS +159 165 waters chemical The phosphate contacts both zinc atoms (Fig 4b) and replaces the coordination by CoA at Zn1; the coordination for Zn2 changes from octahedral with three bound waters to tetrahedral with a phosphate ion as one of the ligands (Fig 4b). RESULTS +188 197 phosphate chemical The phosphate contacts both zinc atoms (Fig 4b) and replaces the coordination by CoA at Zn1; the coordination for Zn2 changes from octahedral with three bound waters to tetrahedral with a phosphate ion as one of the ligands (Fig 4b). RESULTS +0 9 Conserved protein_state Conserved Arg103 seems to be involved in maintaining the phosphate in that position. RESULTS +10 16 Arg103 residue_name_number Conserved Arg103 seems to be involved in maintaining the phosphate in that position. RESULTS +57 66 phosphate chemical Conserved Arg103 seems to be involved in maintaining the phosphate in that position. RESULTS +8 12 zinc chemical The two zinc atoms are slightly closer together in the phosphate-bound form (5.8 Å vs 6.3 Å), possibly due to the bridging effect of the phosphate. RESULTS +55 70 phosphate-bound protein_state The two zinc atoms are slightly closer together in the phosphate-bound form (5.8 Å vs 6.3 Å), possibly due to the bridging effect of the phosphate. RESULTS +137 146 phosphate chemical The two zinc atoms are slightly closer together in the phosphate-bound form (5.8 Å vs 6.3 Å), possibly due to the bridging effect of the phosphate. RESULTS +14 23 phosphate chemical An additional phosphate molecule is bound at a crystal contact interface, perhaps accounting for the 14 Å shorter c-axis in the phosphate-bound crystal form (Table 2). RESULTS +128 143 phosphate-bound protein_state An additional phosphate molecule is bound at a crystal contact interface, perhaps accounting for the 14 Å shorter c-axis in the phosphate-bound crystal form (Table 2). RESULTS +21 25 PduL protein_type Oligomeric States of PduL Orthologs Are Influenced by the EP RESULTS +58 60 EP structure_element Oligomeric States of PduL Orthologs Are Influenced by the EP RESULTS +66 71 rPduL protein Interestingly, some of the residues important for dimerization of rPduL, particularly Phe116, are poorly conserved across PduL homologs associated with functionally diverse BMCs (Figs 4c and 3), suggesting that they may have alternative oligomeric states. RESULTS +86 92 Phe116 residue_name_number Interestingly, some of the residues important for dimerization of rPduL, particularly Phe116, are poorly conserved across PduL homologs associated with functionally diverse BMCs (Figs 4c and 3), suggesting that they may have alternative oligomeric states. RESULTS +98 114 poorly conserved protein_state Interestingly, some of the residues important for dimerization of rPduL, particularly Phe116, are poorly conserved across PduL homologs associated with functionally diverse BMCs (Figs 4c and 3), suggesting that they may have alternative oligomeric states. RESULTS +122 126 PduL protein_type Interestingly, some of the residues important for dimerization of rPduL, particularly Phe116, are poorly conserved across PduL homologs associated with functionally diverse BMCs (Figs 4c and 3), suggesting that they may have alternative oligomeric states. RESULTS +173 177 BMCs complex_assembly Interestingly, some of the residues important for dimerization of rPduL, particularly Phe116, are poorly conserved across PduL homologs associated with functionally diverse BMCs (Figs 4c and 3), suggesting that they may have alternative oligomeric states. RESULTS +40 69 size exclusion chromatography experimental_method We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, ΔEP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1). RESULTS +78 89 full-length protein_state We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, ΔEP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1). RESULTS +114 121 lacking protein_state We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, ΔEP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1). RESULTS +126 128 EP structure_element We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, ΔEP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1). RESULTS +130 133 ΔEP mutant We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, ΔEP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1). RESULTS +138 143 sPduL protein We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, ΔEP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1). RESULTS +145 150 rPduL protein We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, ΔEP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1). RESULTS +156 161 pPduL protein We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, ΔEP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1). RESULTS +219 223 BMCs complex_assembly We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, ΔEP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1). RESULTS +30 39 catabolic protein_state It has been proposed that the catabolic BMCs may assemble in a core-first manner, with the luminal enzymes (signature enzyme, aldehyde, and alcohol dehydrogenases and the BMC PTAC) forming an initial bolus, or prometabolosome, around which a shell assembles. RESULTS +40 44 BMCs complex_assembly It has been proposed that the catabolic BMCs may assemble in a core-first manner, with the luminal enzymes (signature enzyme, aldehyde, and alcohol dehydrogenases and the BMC PTAC) forming an initial bolus, or prometabolosome, around which a shell assembles. RESULTS +126 162 aldehyde, and alcohol dehydrogenases protein_type It has been proposed that the catabolic BMCs may assemble in a core-first manner, with the luminal enzymes (signature enzyme, aldehyde, and alcohol dehydrogenases and the BMC PTAC) forming an initial bolus, or prometabolosome, around which a shell assembles. RESULTS +171 174 BMC complex_assembly It has been proposed that the catabolic BMCs may assemble in a core-first manner, with the luminal enzymes (signature enzyme, aldehyde, and alcohol dehydrogenases and the BMC PTAC) forming an initial bolus, or prometabolosome, around which a shell assembles. RESULTS +175 179 PTAC protein_type It has been proposed that the catabolic BMCs may assemble in a core-first manner, with the luminal enzymes (signature enzyme, aldehyde, and alcohol dehydrogenases and the BMC PTAC) forming an initial bolus, or prometabolosome, around which a shell assembles. RESULTS +242 247 shell structure_element It has been proposed that the catabolic BMCs may assemble in a core-first manner, with the luminal enzymes (signature enzyme, aldehyde, and alcohol dehydrogenases and the BMC PTAC) forming an initial bolus, or prometabolosome, around which a shell assembles. RESULTS +73 77 PduL protein_type Given the diversity of signature enzymes (Table 1), it is plausible that PduL orthologs may adopt different oligomeric states that reflect the differences in the proteins being packaged with them in the organelle lumen. RESULTS +170 172 EP structure_element We found that not only did the different orthologs appear to assemble into different oligomeric states, but that quaternary structure was dependent on whether or not the EP was present. RESULTS +0 11 Full-length protein_state Full-length sPduL was unstable in solution—precipitating over time—and eluted throughout the entire volume of a size exclusion column, indicating it was nonspecifically aggregating. RESULTS +12 17 sPduL protein Full-length sPduL was unstable in solution—precipitating over time—and eluted throughout the entire volume of a size exclusion column, indicating it was nonspecifically aggregating. RESULTS +27 29 EP structure_element However, when the putative EP (residues 1–27) was removed (sPduL ΔEP), the truncated protein was stable and eluted as a single peak (Fig 5a) consistent with the size of a monomer (Fig 5d, blue curve). RESULTS +40 44 1–27 residue_range However, when the putative EP (residues 1–27) was removed (sPduL ΔEP), the truncated protein was stable and eluted as a single peak (Fig 5a) consistent with the size of a monomer (Fig 5d, blue curve). RESULTS +50 57 removed experimental_method However, when the putative EP (residues 1–27) was removed (sPduL ΔEP), the truncated protein was stable and eluted as a single peak (Fig 5a) consistent with the size of a monomer (Fig 5d, blue curve). RESULTS +59 68 sPduL ΔEP mutant However, when the putative EP (residues 1–27) was removed (sPduL ΔEP), the truncated protein was stable and eluted as a single peak (Fig 5a) consistent with the size of a monomer (Fig 5d, blue curve). RESULTS +75 84 truncated protein_state However, when the putative EP (residues 1–27) was removed (sPduL ΔEP), the truncated protein was stable and eluted as a single peak (Fig 5a) consistent with the size of a monomer (Fig 5d, blue curve). RESULTS +171 178 monomer oligomeric_state However, when the putative EP (residues 1–27) was removed (sPduL ΔEP), the truncated protein was stable and eluted as a single peak (Fig 5a) consistent with the size of a monomer (Fig 5d, blue curve). RESULTS +18 29 full-length protein_state In contrast, both full-length rPduL and pPduL appeared to exist in two distinct oligomeric states (Fig 5b and 5c respectively, orange curves), one form of the approximate size of a dimer and the second, a higher molecular weight oligomer (~150 kDa). RESULTS +30 35 rPduL protein In contrast, both full-length rPduL and pPduL appeared to exist in two distinct oligomeric states (Fig 5b and 5c respectively, orange curves), one form of the approximate size of a dimer and the second, a higher molecular weight oligomer (~150 kDa). RESULTS +40 45 pPduL protein In contrast, both full-length rPduL and pPduL appeared to exist in two distinct oligomeric states (Fig 5b and 5c respectively, orange curves), one form of the approximate size of a dimer and the second, a higher molecular weight oligomer (~150 kDa). RESULTS +181 186 dimer oligomeric_state In contrast, both full-length rPduL and pPduL appeared to exist in two distinct oligomeric states (Fig 5b and 5c respectively, orange curves), one form of the approximate size of a dimer and the second, a higher molecular weight oligomer (~150 kDa). RESULTS +229 237 oligomer oligomeric_state In contrast, both full-length rPduL and pPduL appeared to exist in two distinct oligomeric states (Fig 5b and 5c respectively, orange curves), one form of the approximate size of a dimer and the second, a higher molecular weight oligomer (~150 kDa). RESULTS +5 13 deletion experimental_method Upon deletion of the putative EP (residues 1–47 for rPduL, and 1–20 for pPduL), there was a distinct change in the elution profiles (Fig 5b and 5c respectively, blue curves). RESULTS +30 32 EP structure_element Upon deletion of the putative EP (residues 1–47 for rPduL, and 1–20 for pPduL), there was a distinct change in the elution profiles (Fig 5b and 5c respectively, blue curves). RESULTS +43 47 1–47 residue_range Upon deletion of the putative EP (residues 1–47 for rPduL, and 1–20 for pPduL), there was a distinct change in the elution profiles (Fig 5b and 5c respectively, blue curves). RESULTS +52 57 rPduL protein Upon deletion of the putative EP (residues 1–47 for rPduL, and 1–20 for pPduL), there was a distinct change in the elution profiles (Fig 5b and 5c respectively, blue curves). RESULTS +63 67 1–20 residue_range Upon deletion of the putative EP (residues 1–47 for rPduL, and 1–20 for pPduL), there was a distinct change in the elution profiles (Fig 5b and 5c respectively, blue curves). RESULTS +72 77 pPduL protein Upon deletion of the putative EP (residues 1–47 for rPduL, and 1–20 for pPduL), there was a distinct change in the elution profiles (Fig 5b and 5c respectively, blue curves). RESULTS +0 8 pPduLΔEP mutant pPduLΔEP eluted as two smaller forms, possibly corresponding to a trimer and a monomer. RESULTS +66 72 trimer oligomeric_state pPduLΔEP eluted as two smaller forms, possibly corresponding to a trimer and a monomer. RESULTS +79 86 monomer oligomeric_state pPduLΔEP eluted as two smaller forms, possibly corresponding to a trimer and a monomer. RESULTS +13 21 rPduLΔEP mutant In contrast, rPduLΔEP eluted as one smaller oligomer, possibly a dimer. RESULTS +65 70 dimer oligomeric_state In contrast, rPduLΔEP eluted as one smaller oligomer, possibly a dimer. RESULTS +26 31 rPduL protein We also analyzed purified rPduL and rPduLΔEP by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) for a complementary approach to assessing oligomeric state. RESULTS +36 44 rPduLΔEP mutant We also analyzed purified rPduL and rPduLΔEP by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) for a complementary approach to assessing oligomeric state. RESULTS +48 77 size exclusion chromatography experimental_method We also analyzed purified rPduL and rPduLΔEP by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) for a complementary approach to assessing oligomeric state. RESULTS +91 118 multiangle light scattering experimental_method We also analyzed purified rPduL and rPduLΔEP by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) for a complementary approach to assessing oligomeric state. RESULTS +120 128 SEC-MALS experimental_method We also analyzed purified rPduL and rPduLΔEP by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) for a complementary approach to assessing oligomeric state. RESULTS +0 8 SEC-MALS experimental_method SEC-MALS analysis of rPdulΔEP is consistent with a dimer (as observed in the crystal structure) with a weighted average (Mw) and number average (Mn) of the molar mass of 58.4 kDa +/− 11.2% and 58.8 kDa +/− 10.9%, respectively (S4a Fig). RESULTS +21 29 rPdulΔEP mutant SEC-MALS analysis of rPdulΔEP is consistent with a dimer (as observed in the crystal structure) with a weighted average (Mw) and number average (Mn) of the molar mass of 58.4 kDa +/− 11.2% and 58.8 kDa +/− 10.9%, respectively (S4a Fig). RESULTS +51 56 dimer oligomeric_state SEC-MALS analysis of rPdulΔEP is consistent with a dimer (as observed in the crystal structure) with a weighted average (Mw) and number average (Mn) of the molar mass of 58.4 kDa +/− 11.2% and 58.8 kDa +/− 10.9%, respectively (S4a Fig). RESULTS +77 94 crystal structure evidence SEC-MALS analysis of rPdulΔEP is consistent with a dimer (as observed in the crystal structure) with a weighted average (Mw) and number average (Mn) of the molar mass of 58.4 kDa +/− 11.2% and 58.8 kDa +/− 10.9%, respectively (S4a Fig). RESULTS +103 166 weighted average (Mw) and number average (Mn) of the molar mass evidence SEC-MALS analysis of rPdulΔEP is consistent with a dimer (as observed in the crystal structure) with a weighted average (Mw) and number average (Mn) of the molar mass of 58.4 kDa +/− 11.2% and 58.8 kDa +/− 10.9%, respectively (S4a Fig). RESULTS +0 5 rPduL protein rPduL full length runs as Mw = 140.3 kDa +/− 1.2% and Mn = 140.5 kDa +/− 1.2%. RESULTS +6 17 full length protein_state rPduL full length runs as Mw = 140.3 kDa +/− 1.2% and Mn = 140.5 kDa +/− 1.2%. RESULTS +26 28 Mw evidence rPduL full length runs as Mw = 140.3 kDa +/− 1.2% and Mn = 140.5 kDa +/− 1.2%. RESULTS +54 56 Mn evidence rPduL full length runs as Mw = 140.3 kDa +/− 1.2% and Mn = 140.5 kDa +/− 1.2%. RESULTS +43 55 six subunits oligomeric_state This corresponds to an oligomeric state of six subunits (calculated molecular weight of 144 kDa). RESULTS +68 84 molecular weight evidence This corresponds to an oligomeric state of six subunits (calculated molecular weight of 144 kDa). RESULTS +62 64 EP structure_element Collectively, these data strongly suggest that the N-terminal EP of PduL plays a role in defining the quaternary structure of the protein. RESULTS +68 72 PduL protein_type Collectively, these data strongly suggest that the N-terminal EP of PduL plays a role in defining the quaternary structure of the protein. RESULTS +4 7 BMC complex_assembly The BMC shell not only sequesters specific enzymes but also their cofactors, thereby establishing a private cofactor pool dedicated to the encapsulated reactions. DISCUSS +8 13 shell structure_element The BMC shell not only sequesters specific enzymes but also their cofactors, thereby establishing a private cofactor pool dedicated to the encapsulated reactions. DISCUSS +3 12 catabolic protein_state In catabolic BMCs, CoA and NAD+ must be continually recycled within the organelle (Fig 1). DISCUSS +13 17 BMCs complex_assembly In catabolic BMCs, CoA and NAD+ must be continually recycled within the organelle (Fig 1). DISCUSS +19 22 CoA chemical In catabolic BMCs, CoA and NAD+ must be continually recycled within the organelle (Fig 1). DISCUSS +27 31 NAD+ chemical In catabolic BMCs, CoA and NAD+ must be continually recycled within the organelle (Fig 1). DISCUSS +47 69 aldehyde dehydrogenase protein_type Homologs of the predominant cofactor utilizer (aldehyde dehydrogenase) and NAD+ regenerator (alcohol dehydrogenase) have been structurally characterized, but until now structural information was lacking for PduL, which recycles CoA in the organelle lumen. DISCUSS +75 79 NAD+ chemical Homologs of the predominant cofactor utilizer (aldehyde dehydrogenase) and NAD+ regenerator (alcohol dehydrogenase) have been structurally characterized, but until now structural information was lacking for PduL, which recycles CoA in the organelle lumen. DISCUSS +93 114 alcohol dehydrogenase protein_type Homologs of the predominant cofactor utilizer (aldehyde dehydrogenase) and NAD+ regenerator (alcohol dehydrogenase) have been structurally characterized, but until now structural information was lacking for PduL, which recycles CoA in the organelle lumen. DISCUSS +207 211 PduL protein_type Homologs of the predominant cofactor utilizer (aldehyde dehydrogenase) and NAD+ regenerator (alcohol dehydrogenase) have been structurally characterized, but until now structural information was lacking for PduL, which recycles CoA in the organelle lumen. DISCUSS +228 231 CoA chemical Homologs of the predominant cofactor utilizer (aldehyde dehydrogenase) and NAD+ regenerator (alcohol dehydrogenase) have been structurally characterized, but until now structural information was lacking for PduL, which recycles CoA in the organelle lumen. DISCUSS +21 33 housekeeping protein_state Curiously, while the housekeeping Pta could provide this function, and indeed does so in the case of one type of ethanolamine-utilizing (EUT) BMC, the evolutionarily unrelated PduL fulfills this function for the majority of metabolosomes using a novel structure and active site for convergent evolution of function. DISCUSS +34 37 Pta protein_type Curiously, while the housekeeping Pta could provide this function, and indeed does so in the case of one type of ethanolamine-utilizing (EUT) BMC, the evolutionarily unrelated PduL fulfills this function for the majority of metabolosomes using a novel structure and active site for convergent evolution of function. DISCUSS +113 145 ethanolamine-utilizing (EUT) BMC complex_assembly Curiously, while the housekeeping Pta could provide this function, and indeed does so in the case of one type of ethanolamine-utilizing (EUT) BMC, the evolutionarily unrelated PduL fulfills this function for the majority of metabolosomes using a novel structure and active site for convergent evolution of function. DISCUSS +176 180 PduL protein_type Curiously, while the housekeeping Pta could provide this function, and indeed does so in the case of one type of ethanolamine-utilizing (EUT) BMC, the evolutionarily unrelated PduL fulfills this function for the majority of metabolosomes using a novel structure and active site for convergent evolution of function. DISCUSS +224 237 metabolosomes complex_assembly Curiously, while the housekeeping Pta could provide this function, and indeed does so in the case of one type of ethanolamine-utilizing (EUT) BMC, the evolutionarily unrelated PduL fulfills this function for the majority of metabolosomes using a novel structure and active site for convergent evolution of function. DISCUSS +252 261 structure evidence Curiously, while the housekeeping Pta could provide this function, and indeed does so in the case of one type of ethanolamine-utilizing (EUT) BMC, the evolutionarily unrelated PduL fulfills this function for the majority of metabolosomes using a novel structure and active site for convergent evolution of function. DISCUSS +266 277 active site site Curiously, while the housekeeping Pta could provide this function, and indeed does so in the case of one type of ethanolamine-utilizing (EUT) BMC, the evolutionarily unrelated PduL fulfills this function for the majority of metabolosomes using a novel structure and active site for convergent evolution of function. DISCUSS +26 30 PduL protein_type The Tertiary Structure of PduL Is Formed by Discontinuous Segments of Primary Structure DISCUSS +4 13 structure evidence The structure of PduL consists of two β-barrel domains capped by short alpha helical segments (Fig 2b). DISCUSS +17 21 PduL protein_type The structure of PduL consists of two β-barrel domains capped by short alpha helical segments (Fig 2b). DISCUSS +34 54 two β-barrel domains structure_element The structure of PduL consists of two β-barrel domains capped by short alpha helical segments (Fig 2b). DISCUSS +65 93 short alpha helical segments structure_element The structure of PduL consists of two β-barrel domains capped by short alpha helical segments (Fig 2b). DISCUSS +47 60 superimposing experimental_method The two domains are structurally very similar (superimposing with a rmsd of 1.34 Å (over 123 out of 320/348 aligned backbone atoms, S5a Fig). DISCUSS +68 72 rmsd evidence The two domains are structurally very similar (superimposing with a rmsd of 1.34 Å (over 123 out of 320/348 aligned backbone atoms, S5a Fig). DISCUSS +88 98 RHxH motif structure_element However, the amino acid sequences of the two domains are only 16% identical (mainly the RHxH motif, β2 and β10), and 34% similar. DISCUSS +100 102 β2 structure_element However, the amino acid sequences of the two domains are only 16% identical (mainly the RHxH motif, β2 and β10), and 34% similar. DISCUSS +107 110 β10 structure_element However, the amino acid sequences of the two domains are only 16% identical (mainly the RHxH motif, β2 and β10), and 34% similar. DISCUSS +4 13 structure evidence Our structure reveals that the two assigned PF06130 domains (Fig 3) do not form structurally discrete units; this reduces the apparent sequence conservation at the level of primary structure. DISCUSS +44 51 PF06130 structure_element Our structure reveals that the two assigned PF06130 domains (Fig 3) do not form structurally discrete units; this reduces the apparent sequence conservation at the level of primary structure. DISCUSS +4 10 strand structure_element One strand of the domain 1 beta barrel (shown in blue in Fig 2) is contributed by the N-terminus, while the rest of the domain is formed by the residues from the C-terminal half of the protein. DISCUSS +18 26 domain 1 structure_element One strand of the domain 1 beta barrel (shown in blue in Fig 2) is contributed by the N-terminus, while the rest of the domain is formed by the residues from the C-terminal half of the protein. DISCUSS +27 38 beta barrel structure_element One strand of the domain 1 beta barrel (shown in blue in Fig 2) is contributed by the N-terminus, while the rest of the domain is formed by the residues from the C-terminal half of the protein. DISCUSS +162 177 C-terminal half structure_element One strand of the domain 1 beta barrel (shown in blue in Fig 2) is contributed by the N-terminus, while the rest of the domain is formed by the residues from the C-terminal half of the protein. DISCUSS +185 192 protein protein_type One strand of the domain 1 beta barrel (shown in blue in Fig 2) is contributed by the N-terminus, while the rest of the domain is formed by the residues from the C-terminal half of the protein. DISCUSS +5 12 aligned experimental_method When aligned by structure, the β1 strand of the first domain (Fig 2a and 2b, blue) corresponds to the final strand of the second domain (β9), effectively making the domains continuous if the first strand was transplanted to the C-terminus. DISCUSS +16 25 structure evidence When aligned by structure, the β1 strand of the first domain (Fig 2a and 2b, blue) corresponds to the final strand of the second domain (β9), effectively making the domains continuous if the first strand was transplanted to the C-terminus. DISCUSS +31 40 β1 strand structure_element When aligned by structure, the β1 strand of the first domain (Fig 2a and 2b, blue) corresponds to the final strand of the second domain (β9), effectively making the domains continuous if the first strand was transplanted to the C-terminus. DISCUSS +48 60 first domain structure_element When aligned by structure, the β1 strand of the first domain (Fig 2a and 2b, blue) corresponds to the final strand of the second domain (β9), effectively making the domains continuous if the first strand was transplanted to the C-terminus. DISCUSS +102 114 final strand structure_element When aligned by structure, the β1 strand of the first domain (Fig 2a and 2b, blue) corresponds to the final strand of the second domain (β9), effectively making the domains continuous if the first strand was transplanted to the C-terminus. DISCUSS +122 135 second domain structure_element When aligned by structure, the β1 strand of the first domain (Fig 2a and 2b, blue) corresponds to the final strand of the second domain (β9), effectively making the domains continuous if the first strand was transplanted to the C-terminus. DISCUSS +137 139 β9 structure_element When aligned by structure, the β1 strand of the first domain (Fig 2a and 2b, blue) corresponds to the final strand of the second domain (β9), effectively making the domains continuous if the first strand was transplanted to the C-terminus. DISCUSS +39 48 structure evidence Refined domain assignment based on our structure should be able to predict domains of PF06130 homologs much more accurately. DISCUSS +86 93 PF06130 structure_element Refined domain assignment based on our structure should be able to predict domains of PF06130 homologs much more accurately. DISCUSS +38 42 PduL protein_type The closest structural homolog of the PduL barrel domain is a subdomain of a multienzyme complex, the alpha subunit of ethylbenzene dehydrogenase (S5b Fig, rmsd of 2.26 Å over 226 aligned atoms consisting of one beta barrel and one capping helix). DISCUSS +43 56 barrel domain structure_element The closest structural homolog of the PduL barrel domain is a subdomain of a multienzyme complex, the alpha subunit of ethylbenzene dehydrogenase (S5b Fig, rmsd of 2.26 Å over 226 aligned atoms consisting of one beta barrel and one capping helix). DISCUSS +102 115 alpha subunit structure_element The closest structural homolog of the PduL barrel domain is a subdomain of a multienzyme complex, the alpha subunit of ethylbenzene dehydrogenase (S5b Fig, rmsd of 2.26 Å over 226 aligned atoms consisting of one beta barrel and one capping helix). DISCUSS +119 145 ethylbenzene dehydrogenase protein_type The closest structural homolog of the PduL barrel domain is a subdomain of a multienzyme complex, the alpha subunit of ethylbenzene dehydrogenase (S5b Fig, rmsd of 2.26 Å over 226 aligned atoms consisting of one beta barrel and one capping helix). DISCUSS +156 160 rmsd evidence The closest structural homolog of the PduL barrel domain is a subdomain of a multienzyme complex, the alpha subunit of ethylbenzene dehydrogenase (S5b Fig, rmsd of 2.26 Å over 226 aligned atoms consisting of one beta barrel and one capping helix). DISCUSS +212 223 beta barrel structure_element The closest structural homolog of the PduL barrel domain is a subdomain of a multienzyme complex, the alpha subunit of ethylbenzene dehydrogenase (S5b Fig, rmsd of 2.26 Å over 226 aligned atoms consisting of one beta barrel and one capping helix). DISCUSS +232 245 capping helix structure_element The closest structural homolog of the PduL barrel domain is a subdomain of a multienzyme complex, the alpha subunit of ethylbenzene dehydrogenase (S5b Fig, rmsd of 2.26 Å over 226 aligned atoms consisting of one beta barrel and one capping helix). DISCUSS +15 19 PduL protein_type In contrast to PduL, there is only one barrel present in ethylbenzene dehydrogenase, and there is no comparable active site arrangement. DISCUSS +39 45 barrel structure_element In contrast to PduL, there is only one barrel present in ethylbenzene dehydrogenase, and there is no comparable active site arrangement. DISCUSS +57 83 ethylbenzene dehydrogenase protein_type In contrast to PduL, there is only one barrel present in ethylbenzene dehydrogenase, and there is no comparable active site arrangement. DISCUSS +112 123 active site site In contrast to PduL, there is only one barrel present in ethylbenzene dehydrogenase, and there is no comparable active site arrangement. DISCUSS +4 8 PduL protein_type The PduL signature primary structure, two PF06130 domains, occurs in some multidomain proteins, most of them annotated as Acks, suggesting that PduL may also replace Pta in variants of the phosphotransacetylase-Ack pathway. DISCUSS +42 49 PF06130 structure_element The PduL signature primary structure, two PF06130 domains, occurs in some multidomain proteins, most of them annotated as Acks, suggesting that PduL may also replace Pta in variants of the phosphotransacetylase-Ack pathway. DISCUSS +122 126 Acks protein_type The PduL signature primary structure, two PF06130 domains, occurs in some multidomain proteins, most of them annotated as Acks, suggesting that PduL may also replace Pta in variants of the phosphotransacetylase-Ack pathway. DISCUSS +144 148 PduL protein_type The PduL signature primary structure, two PF06130 domains, occurs in some multidomain proteins, most of them annotated as Acks, suggesting that PduL may also replace Pta in variants of the phosphotransacetylase-Ack pathway. DISCUSS +166 169 Pta protein_type The PduL signature primary structure, two PF06130 domains, occurs in some multidomain proteins, most of them annotated as Acks, suggesting that PduL may also replace Pta in variants of the phosphotransacetylase-Ack pathway. DISCUSS +189 210 phosphotransacetylase protein_type The PduL signature primary structure, two PF06130 domains, occurs in some multidomain proteins, most of them annotated as Acks, suggesting that PduL may also replace Pta in variants of the phosphotransacetylase-Ack pathway. DISCUSS +211 214 Ack protein_type The PduL signature primary structure, two PF06130 domains, occurs in some multidomain proteins, most of them annotated as Acks, suggesting that PduL may also replace Pta in variants of the phosphotransacetylase-Ack pathway. DISCUSS +6 10 PduL protein_type These PduL homologs lack EPs, and their fusion to Ack may have evolved as a way to facilitate substrate channeling between the two enzymes. DISCUSS +20 24 lack protein_state These PduL homologs lack EPs, and their fusion to Ack may have evolved as a way to facilitate substrate channeling between the two enzymes. DISCUSS +25 28 EPs structure_element These PduL homologs lack EPs, and their fusion to Ack may have evolved as a way to facilitate substrate channeling between the two enzymes. DISCUSS +34 39 their protein_type These PduL homologs lack EPs, and their fusion to Ack may have evolved as a way to facilitate substrate channeling between the two enzymes. DISCUSS +50 53 Ack protein_type These PduL homologs lack EPs, and their fusion to Ack may have evolved as a way to facilitate substrate channeling between the two enzymes. DISCUSS +17 29 Metabolosome complex_assembly Implications for Metabolosome Core Assembly DISCUSS +4 7 BMC complex_assembly For BMC-encapsulated proteins to properly function together, they must be targeted to the lumen and assemble into an organization that facilitates substrate/product channeling among the different catalytic sites of the signature and core enzymes. DISCUSS +196 211 catalytic sites site For BMC-encapsulated proteins to properly function together, they must be targeted to the lumen and assemble into an organization that facilitates substrate/product channeling among the different catalytic sites of the signature and core enzymes. DISCUSS +4 24 N-terminal extension structure_element The N-terminal extension on PduL homologs may serve both of these functions. DISCUSS +28 32 PduL protein_type The N-terminal extension on PduL homologs may serve both of these functions. DISCUSS +0 13 The extension structure_element The extension shares many features with previously characterized EPs: it is present only in homologs associated with BMC loci, and it is predicted to form an amphipathic α-helix. DISCUSS +65 68 EPs structure_element The extension shares many features with previously characterized EPs: it is present only in homologs associated with BMC loci, and it is predicted to form an amphipathic α-helix. DISCUSS +117 125 BMC loci gene The extension shares many features with previously characterized EPs: it is present only in homologs associated with BMC loci, and it is predicted to form an amphipathic α-helix. DISCUSS +158 169 amphipathic protein_state The extension shares many features with previously characterized EPs: it is present only in homologs associated with BMC loci, and it is predicted to form an amphipathic α-helix. DISCUSS +170 177 α-helix structure_element The extension shares many features with previously characterized EPs: it is present only in homologs associated with BMC loci, and it is predicted to form an amphipathic α-helix. DISCUSS +14 21 removal experimental_method Moreover, its removal affects the oligomeric state of the protein. DISCUSS +0 2 EP structure_element EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP. DISCUSS +73 76 BMC complex_assembly EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP. DISCUSS +99 110 full-length protein_state EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP. DISCUSS +111 134 propanediol dehydratase protein_type EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP. DISCUSS +139 165 ethanolamine ammonia-lyase protein_type EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP. DISCUSS +189 192 PDU complex_assembly EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP. DISCUSS +197 205 EUT BMCs complex_assembly EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP. DISCUSS +285 287 EP structure_element EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP. DISCUSS +0 5 sPduL protein sPduL has also previously been reported to localize to inclusion bodies when overexpressed; we show here that this is dependent on the presence of the EP. DISCUSS +77 90 overexpressed experimental_method sPduL has also previously been reported to localize to inclusion bodies when overexpressed; we show here that this is dependent on the presence of the EP. DISCUSS +151 153 EP structure_element sPduL has also previously been reported to localize to inclusion bodies when overexpressed; we show here that this is dependent on the presence of the EP. DISCUSS +23 25 EP structure_element This propensity of the EP to cause proteins to form complexes (Fig 5) might not be a coincidence, but could be a necessary step in the assembly of BMCs. DISCUSS +147 151 BMCs complex_assembly This propensity of the EP to cause proteins to form complexes (Fig 5) might not be a coincidence, but could be a necessary step in the assembly of BMCs. DISCUSS +87 99 metabolosome complex_assembly Structured aggregation of the core enzymes has been proposed to be the initial step in metabolosome assembly and is known to be the first step of β-carboxysome biogenesis, where the core enzyme Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) is aggregated by the CcmM protein. DISCUSS +194 237 Ribulose Bisphosphate Carboxylase/Oxygenase protein_type Structured aggregation of the core enzymes has been proposed to be the initial step in metabolosome assembly and is known to be the first step of β-carboxysome biogenesis, where the core enzyme Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) is aggregated by the CcmM protein. DISCUSS +239 246 RuBisCO protein_type Structured aggregation of the core enzymes has been proposed to be the initial step in metabolosome assembly and is known to be the first step of β-carboxysome biogenesis, where the core enzyme Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) is aggregated by the CcmM protein. DISCUSS +269 273 CcmM protein_type Structured aggregation of the core enzymes has been proposed to be the initial step in metabolosome assembly and is known to be the first step of β-carboxysome biogenesis, where the core enzyme Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) is aggregated by the CcmM protein. DISCUSS +10 15 CsoS2 protein_type Likewise, CsoS2, a protein in the α-carboxysome core, also aggregates when purified and is proposed to facilitate the nucleation and encapsulation of RuBisCO molecules in the lumen of the organelle. DISCUSS +34 47 α-carboxysome complex_assembly Likewise, CsoS2, a protein in the α-carboxysome core, also aggregates when purified and is proposed to facilitate the nucleation and encapsulation of RuBisCO molecules in the lumen of the organelle. DISCUSS +150 157 RuBisCO protein_type Likewise, CsoS2, a protein in the α-carboxysome core, also aggregates when purified and is proposed to facilitate the nucleation and encapsulation of RuBisCO molecules in the lumen of the organelle. DISCUSS +14 17 EPs structure_element This role for EPs in BMC assembly is in addition to their interaction with shell proteins. DISCUSS +21 24 BMC complex_assembly This role for EPs in BMC assembly is in addition to their interaction with shell proteins. DISCUSS +14 18 PduL protein_type Moreover, the PduL crystal structures offer a clue as to how required cofactors enter the BMC lumen during assembly. DISCUSS +19 37 crystal structures evidence Moreover, the PduL crystal structures offer a clue as to how required cofactors enter the BMC lumen during assembly. DISCUSS +90 93 BMC complex_assembly Moreover, the PduL crystal structures offer a clue as to how required cofactors enter the BMC lumen during assembly. DISCUSS +5 8 CoA chemical Free CoA and NAD+/H could potentially be bound to the enzymes as the core assembles and is encapsulated. DISCUSS +13 17 NAD+ chemical Free CoA and NAD+/H could potentially be bound to the enzymes as the core assembles and is encapsulated. DISCUSS +18 19 H chemical Free CoA and NAD+/H could potentially be bound to the enzymes as the core assembles and is encapsulated. DISCUSS +4 8 PduL protein_type Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the “ground state” of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen. DISCUSS +9 17 crystals evidence Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the “ground state” of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen. DISCUSS +28 31 CoA chemical Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the “ground state” of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen. DISCUSS +59 75 Escherichia coli species Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the “ground state” of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen. DISCUSS +123 127 PduL protein_type Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the “ground state” of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen. DISCUSS +138 147 CoA-bound protein_state Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the “ground state” of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen. DISCUSS +230 238 CoA:PduL complex_assembly Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the “ground state” of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen. DISCUSS +250 262 metabolosome complex_assembly Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the “ground state” of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen. DISCUSS +0 11 Active Site site Active Site Identification and Structural Insights into Catalysis DISCUSS +4 15 active site site The active site of PduL is formed at the interface of the two structural domains (Fig 2b). DISCUSS +19 23 PduL protein_type The active site of PduL is formed at the interface of the two structural domains (Fig 2b). DISCUSS +41 50 interface site The active site of PduL is formed at the interface of the two structural domains (Fig 2b). DISCUSS +73 80 domains structure_element The active site of PduL is formed at the interface of the two structural domains (Fig 2b). DISCUSS +95 106 active site site As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211). DISCUSS +117 133 highly conserved protein_state As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211). DISCUSS +164 167 CoA chemical As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211). DISCUSS +201 206 Ser45 residue_name_number As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211). DISCUSS +208 213 Lys70 residue_name_number As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211). DISCUSS +215 220 Arg97 residue_name_number As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211). DISCUSS +222 227 Leu99 residue_name_number As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211). DISCUSS +229 235 His204 residue_name_number As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211). DISCUSS +237 243 Asn211 residue_name_number As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211). DISCUSS +11 38 metal-coordinating residues site All of the metal-coordinating residues (Fig 2a) are absolutely conserved, implicating them in catalysis or the correct spatial orientation of the substrates. DISCUSS +52 72 absolutely conserved protein_state All of the metal-coordinating residues (Fig 2a) are absolutely conserved, implicating them in catalysis or the correct spatial orientation of the substrates. DISCUSS +0 6 Arg103 residue_name_number Arg103, which contacts the phosphate (Fig 4b), is present in all PduL homologs. DISCUSS +27 36 phosphate chemical Arg103, which contacts the phosphate (Fig 4b), is present in all PduL homologs. DISCUSS +65 69 PduL protein_type Arg103, which contacts the phosphate (Fig 4b), is present in all PduL homologs. DISCUSS +53 56 CoA chemical The close resemblance between the structures binding CoA and phosphate likely indicates that no large changes in protein conformation are involved in catalysis, and that our crystal structures are representative of the active form. DISCUSS +61 70 phosphate chemical The close resemblance between the structures binding CoA and phosphate likely indicates that no large changes in protein conformation are involved in catalysis, and that our crystal structures are representative of the active form. DISCUSS +174 192 crystal structures evidence The close resemblance between the structures binding CoA and phosphate likely indicates that no large changes in protein conformation are involved in catalysis, and that our crystal structures are representative of the active form. DISCUSS +219 225 active protein_state The close resemblance between the structures binding CoA and phosphate likely indicates that no large changes in protein conformation are involved in catalysis, and that our crystal structures are representative of the active form. DISCUSS +49 54 rPduL protein The native substrate for the forward reaction of rPduL and pPduL, propionyl-CoA, most likely binds to the enzyme in the same way at the observed nucleotide and pantothenic acid moiety, but the propionyl group in the CoA-thioester might point in a different direction. DISCUSS +59 64 pPduL protein The native substrate for the forward reaction of rPduL and pPduL, propionyl-CoA, most likely binds to the enzyme in the same way at the observed nucleotide and pantothenic acid moiety, but the propionyl group in the CoA-thioester might point in a different direction. DISCUSS +66 79 propionyl-CoA chemical The native substrate for the forward reaction of rPduL and pPduL, propionyl-CoA, most likely binds to the enzyme in the same way at the observed nucleotide and pantothenic acid moiety, but the propionyl group in the CoA-thioester might point in a different direction. DISCUSS +145 155 nucleotide chemical The native substrate for the forward reaction of rPduL and pPduL, propionyl-CoA, most likely binds to the enzyme in the same way at the observed nucleotide and pantothenic acid moiety, but the propionyl group in the CoA-thioester might point in a different direction. DISCUSS +160 176 pantothenic acid chemical The native substrate for the forward reaction of rPduL and pPduL, propionyl-CoA, most likely binds to the enzyme in the same way at the observed nucleotide and pantothenic acid moiety, but the propionyl group in the CoA-thioester might point in a different direction. DISCUSS +216 229 CoA-thioester chemical The native substrate for the forward reaction of rPduL and pPduL, propionyl-CoA, most likely binds to the enzyme in the same way at the observed nucleotide and pantothenic acid moiety, but the propionyl group in the CoA-thioester might point in a different direction. DISCUSS +11 17 pocket site There is a pocket nearby the active site between the well-conserved residues Ser45 and Ala154, which could accommodate the propionyl group (S6 Fig). DISCUSS +29 40 active site site There is a pocket nearby the active site between the well-conserved residues Ser45 and Ala154, which could accommodate the propionyl group (S6 Fig). DISCUSS +53 67 well-conserved protein_state There is a pocket nearby the active site between the well-conserved residues Ser45 and Ala154, which could accommodate the propionyl group (S6 Fig). DISCUSS +77 82 Ser45 residue_name_number There is a pocket nearby the active site between the well-conserved residues Ser45 and Ala154, which could accommodate the propionyl group (S6 Fig). DISCUSS +87 93 Ala154 residue_name_number There is a pocket nearby the active site between the well-conserved residues Ser45 and Ala154, which could accommodate the propionyl group (S6 Fig). DISCUSS +2 16 homology model experimental_method A homology model of sPduL indicates that the residues making up this pocket and the surrounding active site region are identical to that of rPduL, which is not surprising, because these two homologs presumably have the same propionyl-CoA substrate. DISCUSS +20 25 sPduL protein A homology model of sPduL indicates that the residues making up this pocket and the surrounding active site region are identical to that of rPduL, which is not surprising, because these two homologs presumably have the same propionyl-CoA substrate. DISCUSS +69 75 pocket site A homology model of sPduL indicates that the residues making up this pocket and the surrounding active site region are identical to that of rPduL, which is not surprising, because these two homologs presumably have the same propionyl-CoA substrate. DISCUSS +96 107 active site site A homology model of sPduL indicates that the residues making up this pocket and the surrounding active site region are identical to that of rPduL, which is not surprising, because these two homologs presumably have the same propionyl-CoA substrate. DISCUSS +140 145 rPduL protein A homology model of sPduL indicates that the residues making up this pocket and the surrounding active site region are identical to that of rPduL, which is not surprising, because these two homologs presumably have the same propionyl-CoA substrate. DISCUSS +224 237 propionyl-CoA chemical A homology model of sPduL indicates that the residues making up this pocket and the surrounding active site region are identical to that of rPduL, which is not surprising, because these two homologs presumably have the same propionyl-CoA substrate. DISCUSS +4 18 homology model experimental_method The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +22 27 pPduL protein The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +70 76 pocket site The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +127 138 active site site The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +140 145 Gln77 residue_name_number The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +149 154 rPduL protein The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +172 180 tyrosine residue_name The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +182 187 Tyr77 residue_name_number The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +192 197 pPduL protein The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +230 235 pPduL protein The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +263 273 lactyl-CoA chemical The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +331 344 propionyl-CoA chemical The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +365 373 aromatic protein_state The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +374 381 residue structure_element The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +444 448 PduL protein_type The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +465 474 pvm locus gene The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus. DISCUSS +27 32 PduLs protein_type Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +44 52 pvm loci gene Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +54 59 Gln77 residue_name_number Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +87 90 Tyr residue_name Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +94 97 Phe residue_name Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +125 128 Gln residue_name Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +132 135 Glu residue_name Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +139 144 PduLs protein_type Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +158 161 BMC complex_assembly Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +181 188 acetyl- chemical Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +192 205 propionyl-CoA chemical Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +209 219 comparison experimental_method Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +227 231 PduL protein_type Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +232 243 active site site Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +282 285 Pta protein_type Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms. DISCUSS +27 30 Pta protein_type The catalytic mechanism of Pta involves the abstraction of a thiol hydrogen by an aspartate residue, resulting in the nucleophilic attack of thiolate upon the carbonyl carbon of acetyl-phosphate, oriented by an arginine and stabilized by a serine —there are no metals involved. DISCUSS +82 91 aspartate residue_name The catalytic mechanism of Pta involves the abstraction of a thiol hydrogen by an aspartate residue, resulting in the nucleophilic attack of thiolate upon the carbonyl carbon of acetyl-phosphate, oriented by an arginine and stabilized by a serine —there are no metals involved. DISCUSS +178 194 acetyl-phosphate chemical The catalytic mechanism of Pta involves the abstraction of a thiol hydrogen by an aspartate residue, resulting in the nucleophilic attack of thiolate upon the carbonyl carbon of acetyl-phosphate, oriented by an arginine and stabilized by a serine —there are no metals involved. DISCUSS +211 219 arginine residue_name The catalytic mechanism of Pta involves the abstraction of a thiol hydrogen by an aspartate residue, resulting in the nucleophilic attack of thiolate upon the carbonyl carbon of acetyl-phosphate, oriented by an arginine and stabilized by a serine —there are no metals involved. DISCUSS +240 246 serine residue_name The catalytic mechanism of Pta involves the abstraction of a thiol hydrogen by an aspartate residue, resulting in the nucleophilic attack of thiolate upon the carbonyl carbon of acetyl-phosphate, oriented by an arginine and stabilized by a serine —there are no metals involved. DISCUSS +20 25 rPduL protein In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions. DISCUSS +26 35 structure evidence In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions. DISCUSS +60 69 aspartate residue_name In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions. DISCUSS +96 107 active site site In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions. DISCUSS +122 136 well-conserved protein_state In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions. DISCUSS +137 146 glutamate residue_name In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions. DISCUSS +162 173 active site site In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions. DISCUSS +189 201 coordinating bond_interaction In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions. DISCUSS +44 50 acidic protein_state These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site. DISCUSS +51 58 residue structure_element These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site. DISCUSS +100 104 PduL protein_type These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site. DISCUSS +119 138 dimetal active site site These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site. DISCUSS +142 146 PduL protein_type These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site. DISCUSS +212 221 phosphate chemical These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site. DISCUSS +280 288 acyl-CoA chemical These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site. DISCUSS +412 426 acyl-phosphate chemical These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site. DISCUSS +471 483 phosphatases protein_type These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site. DISCUSS +564 583 dimetal active site site These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site. DISCUSS +4 14 structures evidence Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. DISCUSS +105 109 PduL protein_type Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. DISCUSS +111 120 Conserved protein_state Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. DISCUSS +137 148 active site site Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. DISCUSS +236 242 Ser127 residue_name_number Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. DISCUSS +244 250 Arg103 residue_name_number Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. DISCUSS +252 258 Arg194 residue_name_number Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. DISCUSS +260 266 Gln107 residue_name_number Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. DISCUSS +268 273 Gln74 residue_name_number Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. DISCUSS +279 282 Gln residue_name_number Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. DISCUSS +283 288 Glu77 residue_name_number Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77. DISCUSS +7 22 phosphate-bound protein_state In the phosphate-bound crystal structure, Ser127 and Arg103 appear to position the phosphate (Fig 4b). DISCUSS +23 40 crystal structure evidence In the phosphate-bound crystal structure, Ser127 and Arg103 appear to position the phosphate (Fig 4b). DISCUSS +42 48 Ser127 residue_name_number In the phosphate-bound crystal structure, Ser127 and Arg103 appear to position the phosphate (Fig 4b). DISCUSS +53 59 Arg103 residue_name_number In the phosphate-bound crystal structure, Ser127 and Arg103 appear to position the phosphate (Fig 4b). DISCUSS +83 92 phosphate chemical In the phosphate-bound crystal structure, Ser127 and Arg103 appear to position the phosphate (Fig 4b). DISCUSS +15 21 Arg103 residue_name_number Alternatively, Arg103 might act as a base to render the phosphate more nucleophilic. DISCUSS +56 65 phosphate chemical Alternatively, Arg103 might act as a base to render the phosphate more nucleophilic. DISCUSS +25 30 Gln74 residue_name_number The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate. DISCUSS +32 35 Gln residue_name_number The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate. DISCUSS +36 41 Glu77 residue_name_number The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate. DISCUSS +47 53 Arg194 residue_name_number The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate. DISCUSS +81 92 active site site The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate. DISCUSS +101 104 CoA protein_state The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate. DISCUSS +109 124 phosphate-bound protein_state The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate. DISCUSS +125 143 crystal structures evidence The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate. DISCUSS +180 196 hydrogen bonding bond_interaction The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate. DISCUSS +272 286 acyl-phosphate chemical The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate. DISCUSS +9 18 CoA-bound protein_state The free CoA-bound form is presumably poised for attack upon an acyl-phosphate, indicating that the enzyme initially binds CoA as opposed to acyl-phosphate. DISCUSS +64 78 acyl-phosphate chemical The free CoA-bound form is presumably poised for attack upon an acyl-phosphate, indicating that the enzyme initially binds CoA as opposed to acyl-phosphate. DISCUSS +123 126 CoA chemical The free CoA-bound form is presumably poised for attack upon an acyl-phosphate, indicating that the enzyme initially binds CoA as opposed to acyl-phosphate. DISCUSS +141 155 acyl-phosphate chemical The free CoA-bound form is presumably poised for attack upon an acyl-phosphate, indicating that the enzyme initially binds CoA as opposed to acyl-phosphate. DISCUSS +53 62 CoA-bound protein_state This hypothesis is strengthened by the fact that the CoA-bound crystals were obtained without added CoA, indicating that the protein bound CoA from the E. coli expression strain and retained it throughout purification and crystallization. DISCUSS +63 71 crystals evidence This hypothesis is strengthened by the fact that the CoA-bound crystals were obtained without added CoA, indicating that the protein bound CoA from the E. coli expression strain and retained it throughout purification and crystallization. DISCUSS +100 103 CoA chemical This hypothesis is strengthened by the fact that the CoA-bound crystals were obtained without added CoA, indicating that the protein bound CoA from the E. coli expression strain and retained it throughout purification and crystallization. DISCUSS +133 138 bound protein_state This hypothesis is strengthened by the fact that the CoA-bound crystals were obtained without added CoA, indicating that the protein bound CoA from the E. coli expression strain and retained it throughout purification and crystallization. DISCUSS +139 142 CoA chemical This hypothesis is strengthened by the fact that the CoA-bound crystals were obtained without added CoA, indicating that the protein bound CoA from the E. coli expression strain and retained it throughout purification and crystallization. DISCUSS +152 159 E. coli species This hypothesis is strengthened by the fact that the CoA-bound crystals were obtained without added CoA, indicating that the protein bound CoA from the E. coli expression strain and retained it throughout purification and crystallization. DISCUSS +4 19 phosphate-bound protein_state The phosphate-bound structure indicates that in the opposite reaction direction phosphate is bound first, and then an acyl-CoA enters. DISCUSS +20 29 structure evidence The phosphate-bound structure indicates that in the opposite reaction direction phosphate is bound first, and then an acyl-CoA enters. DISCUSS +80 89 phosphate chemical The phosphate-bound structure indicates that in the opposite reaction direction phosphate is bound first, and then an acyl-CoA enters. DISCUSS +118 126 acyl-CoA chemical The phosphate-bound structure indicates that in the opposite reaction direction phosphate is bound first, and then an acyl-CoA enters. DISCUSS +24 42 crystal structures evidence The two high-resolution crystal structures presented here will serve as the foundation for mechanistic studies on this noncanonical PTAC enzyme to determine how the dimetal active site functions to catalyze both forward and reverse reactions. DISCUSS +132 136 PTAC protein_type The two high-resolution crystal structures presented here will serve as the foundation for mechanistic studies on this noncanonical PTAC enzyme to determine how the dimetal active site functions to catalyze both forward and reverse reactions. DISCUSS +165 184 dimetal active site site The two high-resolution crystal structures presented here will serve as the foundation for mechanistic studies on this noncanonical PTAC enzyme to determine how the dimetal active site functions to catalyze both forward and reverse reactions. DISCUSS +47 51 PduL protein_type Functional, but Not Structural, Convergence of PduL and Pta DISCUSS +56 59 Pta protein_type Functional, but Not Structural, Convergence of PduL and Pta DISCUSS +0 4 PduL protein_type PduL and Pta are mechanistically and structurally distinct enzymes that catalyze the same reaction, a prime example of evolutionary convergence upon a function. DISCUSS +9 12 Pta protein_type PduL and Pta are mechanistically and structurally distinct enzymes that catalyze the same reaction, a prime example of evolutionary convergence upon a function. DISCUSS +155 167 active sites site There are several examples of such functional convergence of enzymes, although typically the enzymes have independently evolved similar, or even identical active sites; for example, the carbonic anhydrase family. DISCUSS +186 204 carbonic anhydrase protein_type There are several examples of such functional convergence of enzymes, although typically the enzymes have independently evolved similar, or even identical active sites; for example, the carbonic anhydrase family. DISCUSS +102 114 active sites site However, apparently less frequent is functional convergence that is supported by distinctly different active sites and accordingly catalytic mechanism, as revealed by comparison of the structures of Pta and PduL. One well-studied example of this is the β-lactamase family of enzymes, in which the active site of Class A and Class C enzymes involve serine-based catalysis, but Class B enzymes are metalloproteins. DISCUSS +199 202 Pta protein_type However, apparently less frequent is functional convergence that is supported by distinctly different active sites and accordingly catalytic mechanism, as revealed by comparison of the structures of Pta and PduL. One well-studied example of this is the β-lactamase family of enzymes, in which the active site of Class A and Class C enzymes involve serine-based catalysis, but Class B enzymes are metalloproteins. DISCUSS +207 211 PduL protein_type However, apparently less frequent is functional convergence that is supported by distinctly different active sites and accordingly catalytic mechanism, as revealed by comparison of the structures of Pta and PduL. One well-studied example of this is the β-lactamase family of enzymes, in which the active site of Class A and Class C enzymes involve serine-based catalysis, but Class B enzymes are metalloproteins. DISCUSS +253 264 β-lactamase protein_type However, apparently less frequent is functional convergence that is supported by distinctly different active sites and accordingly catalytic mechanism, as revealed by comparison of the structures of Pta and PduL. One well-studied example of this is the β-lactamase family of enzymes, in which the active site of Class A and Class C enzymes involve serine-based catalysis, but Class B enzymes are metalloproteins. DISCUSS +297 308 active site site However, apparently less frequent is functional convergence that is supported by distinctly different active sites and accordingly catalytic mechanism, as revealed by comparison of the structures of Pta and PduL. One well-studied example of this is the β-lactamase family of enzymes, in which the active site of Class A and Class C enzymes involve serine-based catalysis, but Class B enzymes are metalloproteins. DISCUSS +396 411 metalloproteins protein_type However, apparently less frequent is functional convergence that is supported by distinctly different active sites and accordingly catalytic mechanism, as revealed by comparison of the structures of Pta and PduL. One well-studied example of this is the β-lactamase family of enzymes, in which the active site of Class A and Class C enzymes involve serine-based catalysis, but Class B enzymes are metalloproteins. DISCUSS +27 39 β-lactamases protein_type This is not surprising, as β-lactamases are not so widespread among bacteria and therefore would be expected to have evolved independently several times as a defense mechanism against β-lactam antibiotics. DISCUSS +68 76 bacteria taxonomy_domain This is not surprising, as β-lactamases are not so widespread among bacteria and therefore would be expected to have evolved independently several times as a defense mechanism against β-lactam antibiotics. DISCUSS +20 28 bacteria taxonomy_domain However, nearly all bacteria encode Pta, and it is not immediately clear why the Pta/PduL functional convergence should have evolved: it would seem to be evolutionarily more resourceful for the Pta-encoding gene to be duplicated and repurposed for BMCs, as is apparently the case in one type of BMC—EUT1 (Table 1). DISCUSS +36 39 Pta protein_type However, nearly all bacteria encode Pta, and it is not immediately clear why the Pta/PduL functional convergence should have evolved: it would seem to be evolutionarily more resourceful for the Pta-encoding gene to be duplicated and repurposed for BMCs, as is apparently the case in one type of BMC—EUT1 (Table 1). DISCUSS +81 84 Pta protein_type However, nearly all bacteria encode Pta, and it is not immediately clear why the Pta/PduL functional convergence should have evolved: it would seem to be evolutionarily more resourceful for the Pta-encoding gene to be duplicated and repurposed for BMCs, as is apparently the case in one type of BMC—EUT1 (Table 1). DISCUSS +85 89 PduL protein_type However, nearly all bacteria encode Pta, and it is not immediately clear why the Pta/PduL functional convergence should have evolved: it would seem to be evolutionarily more resourceful for the Pta-encoding gene to be duplicated and repurposed for BMCs, as is apparently the case in one type of BMC—EUT1 (Table 1). DISCUSS +194 211 Pta-encoding gene gene However, nearly all bacteria encode Pta, and it is not immediately clear why the Pta/PduL functional convergence should have evolved: it would seem to be evolutionarily more resourceful for the Pta-encoding gene to be duplicated and repurposed for BMCs, as is apparently the case in one type of BMC—EUT1 (Table 1). DISCUSS +248 252 BMCs complex_assembly However, nearly all bacteria encode Pta, and it is not immediately clear why the Pta/PduL functional convergence should have evolved: it would seem to be evolutionarily more resourceful for the Pta-encoding gene to be duplicated and repurposed for BMCs, as is apparently the case in one type of BMC—EUT1 (Table 1). DISCUSS +295 303 BMC—EUT1 complex_assembly However, nearly all bacteria encode Pta, and it is not immediately clear why the Pta/PduL functional convergence should have evolved: it would seem to be evolutionarily more resourceful for the Pta-encoding gene to be duplicated and repurposed for BMCs, as is apparently the case in one type of BMC—EUT1 (Table 1). DISCUSS +90 94 PduL protein_type There could be some intrinsic biochemical difference between the two enzymes that renders PduL a more attractive candidate for encapsulation in a BMC—for example, PduL might be more amenable to tight packaging, or is better suited for the chemical microenvironment formed within the lumen of the BMC, which can be quite different from the cytosol. DISCUSS +146 149 BMC complex_assembly There could be some intrinsic biochemical difference between the two enzymes that renders PduL a more attractive candidate for encapsulation in a BMC—for example, PduL might be more amenable to tight packaging, or is better suited for the chemical microenvironment formed within the lumen of the BMC, which can be quite different from the cytosol. DISCUSS +163 167 PduL protein_type There could be some intrinsic biochemical difference between the two enzymes that renders PduL a more attractive candidate for encapsulation in a BMC—for example, PduL might be more amenable to tight packaging, or is better suited for the chemical microenvironment formed within the lumen of the BMC, which can be quite different from the cytosol. DISCUSS +296 299 BMC complex_assembly There could be some intrinsic biochemical difference between the two enzymes that renders PduL a more attractive candidate for encapsulation in a BMC—for example, PduL might be more amenable to tight packaging, or is better suited for the chemical microenvironment formed within the lumen of the BMC, which can be quite different from the cytosol. DISCUSS +47 52 PTACs protein_type Further biochemical comparison between the two PTACs will likely yield exciting results that could answer this evolutionary question. DISCUSS +0 4 BMCs complex_assembly BMCs are now known to be widespread among the bacteria and are involved in critical segments of both autotrophic and heterotrophic biochemical pathways that confer to the host organism a competitive (metabolic) advantage in select niches. DISCUSS +46 54 bacteria taxonomy_domain BMCs are now known to be widespread among the bacteria and are involved in critical segments of both autotrophic and heterotrophic biochemical pathways that confer to the host organism a competitive (metabolic) advantage in select niches. DISCUSS +27 39 metabolosome complex_assembly As one of the three common metabolosome core enzymes, the structure of PduL provides a key missing piece to our structural picture of the shared core biochemistry (Fig 1) of functionally diverse catabolic BMCs. DISCUSS +58 67 structure evidence As one of the three common metabolosome core enzymes, the structure of PduL provides a key missing piece to our structural picture of the shared core biochemistry (Fig 1) of functionally diverse catabolic BMCs. DISCUSS +71 75 PduL protein_type As one of the three common metabolosome core enzymes, the structure of PduL provides a key missing piece to our structural picture of the shared core biochemistry (Fig 1) of functionally diverse catabolic BMCs. DISCUSS +195 204 catabolic protein_state As one of the three common metabolosome core enzymes, the structure of PduL provides a key missing piece to our structural picture of the shared core biochemistry (Fig 1) of functionally diverse catabolic BMCs. DISCUSS +205 209 BMCs complex_assembly As one of the three common metabolosome core enzymes, the structure of PduL provides a key missing piece to our structural picture of the shared core biochemistry (Fig 1) of functionally diverse catabolic BMCs. DISCUSS +53 57 PduL protein_type We have observed the oligomeric state differences of PduL to correlate with the presence of an EP, providing new insight into the function of this sequence extension in BMC assembly. DISCUSS +95 97 EP structure_element We have observed the oligomeric state differences of PduL to correlate with the presence of an EP, providing new insight into the function of this sequence extension in BMC assembly. DISCUSS +169 172 BMC complex_assembly We have observed the oligomeric state differences of PduL to correlate with the presence of an EP, providing new insight into the function of this sequence extension in BMC assembly. DISCUSS +42 52 Coenzyme A chemical Moreover, our results suggest a means for Coenzyme A incorporation during metabolosome biogenesis. DISCUSS +74 86 metabolosome complex_assembly Moreover, our results suggest a means for Coenzyme A incorporation during metabolosome biogenesis. DISCUSS +117 121 BMCs complex_assembly A detailed understanding of the underlying principles governing the assembly and internal structural organization of BMCs is a requisite for synthetic biologists to design custom nanoreactors that use BMC architectures as a template. DISCUSS +201 204 BMC complex_assembly A detailed understanding of the underlying principles governing the assembly and internal structural organization of BMCs is a requisite for synthetic biologists to design custom nanoreactors that use BMC architectures as a template. DISCUSS +41 54 metabolosomes complex_assembly Furthermore, given the growing number of metabolosomes implicated in pathogenesis, the PduL structure will be useful in the development of therapeutics. DISCUSS +87 91 PduL protein_type Furthermore, given the growing number of metabolosomes implicated in pathogenesis, the PduL structure will be useful in the development of therapeutics. DISCUSS +92 101 structure evidence Furthermore, given the growing number of metabolosomes implicated in pathogenesis, the PduL structure will be useful in the development of therapeutics. DISCUSS +14 18 PduL protein_type The fact that PduL is confined almost exclusively to metabolosomes can be used to develop an inhibitor that blocks only PduL and not Pta as a way to selectively disrupt BMC-based metabolism, while not affecting most commensal organisms that require PTAC activity. DISCUSS +53 66 metabolosomes complex_assembly The fact that PduL is confined almost exclusively to metabolosomes can be used to develop an inhibitor that blocks only PduL and not Pta as a way to selectively disrupt BMC-based metabolism, while not affecting most commensal organisms that require PTAC activity. DISCUSS +120 124 PduL protein_type The fact that PduL is confined almost exclusively to metabolosomes can be used to develop an inhibitor that blocks only PduL and not Pta as a way to selectively disrupt BMC-based metabolism, while not affecting most commensal organisms that require PTAC activity. DISCUSS +133 136 Pta protein_type The fact that PduL is confined almost exclusively to metabolosomes can be used to develop an inhibitor that blocks only PduL and not Pta as a way to selectively disrupt BMC-based metabolism, while not affecting most commensal organisms that require PTAC activity. DISCUSS +169 172 BMC complex_assembly The fact that PduL is confined almost exclusively to metabolosomes can be used to develop an inhibitor that blocks only PduL and not Pta as a way to selectively disrupt BMC-based metabolism, while not affecting most commensal organisms that require PTAC activity. DISCUSS +249 253 PTAC protein_type The fact that PduL is confined almost exclusively to metabolosomes can be used to develop an inhibitor that blocks only PduL and not Pta as a way to selectively disrupt BMC-based metabolism, while not affecting most commensal organisms that require PTAC activity. DISCUSS diff --git a/annotation_CSV/PMC4786784.csv b/annotation_CSV/PMC4786784.csv new file mode 100644 index 0000000000000000000000000000000000000000..ac63adc39e0018004890bb0cd4486ceb429dd4f5 --- /dev/null +++ b/annotation_CSV/PMC4786784.csv @@ -0,0 +1,1574 @@ +anno_start anno_end anno_text entity_type sentence section +3 11 extended protein_state An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal TITLE +12 37 U2AF65–RNA-binding domain structure_element An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal TITLE +53 67 3′ splice site site An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal TITLE +18 42 pre-mRNA splicing factor protein_type How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. ABSTRACT +43 49 U2AF65 protein How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. ABSTRACT +65 79 polypyrimidine chemical How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. ABSTRACT +81 83 Py chemical How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. ABSTRACT +115 130 3′ splice sites site How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. ABSTRACT +134 139 human species How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. ABSTRACT +3 29 determined four structures experimental_method We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. ABSTRACT +36 44 extended protein_state We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. ABSTRACT +45 70 U2AF65–RNA-binding domain structure_element We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. ABSTRACT +71 79 bound to protein_state We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. ABSTRACT +80 105 Py-tract oligonucleotides chemical We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. ABSTRACT +150 160 structures evidence We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. ABSTRACT +175 206 RNA binding and splicing assays experimental_method We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. ABSTRACT +235 241 U2AF65 protein We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. ABSTRACT +242 263 inter-domain residues site We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. ABSTRACT +281 291 contiguous structure_element We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. ABSTRACT +298 308 nucleotide chemical We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. ABSTRACT +309 317 Py tract chemical We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. ABSTRACT +4 10 U2AF65 protein The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. ABSTRACT +11 17 linker structure_element The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. ABSTRACT +44 66 RNA recognition motifs structure_element The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. ABSTRACT +68 72 RRMs structure_element The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. ABSTRACT +96 106 nucleotide chemical The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. ABSTRACT +138 152 RRM extensions structure_element The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. ABSTRACT +167 178 3′ terminus site The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. ABSTRACT +183 188 third residue_number The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. ABSTRACT +189 199 nucleotide chemical The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. ABSTRACT +0 20 Single-molecule FRET experimental_method Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. ABSTRACT +94 100 U2AF65 protein Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. ABSTRACT +101 105 RRMs structure_element Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. ABSTRACT +139 147 Py-tract chemical Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. ABSTRACT +110 121 splice site site Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals. ABSTRACT +5 29 pre-mRNA splicing factor protein_type The pre-mRNA splicing factor U2AF65 recognizes 3′ splice sites in human gene transcripts, but the details are not fully understood. ABSTRACT +30 36 U2AF65 protein The pre-mRNA splicing factor U2AF65 recognizes 3′ splice sites in human gene transcripts, but the details are not fully understood. ABSTRACT +48 63 3′ splice sites site The pre-mRNA splicing factor U2AF65 recognizes 3′ splice sites in human gene transcripts, but the details are not fully understood. ABSTRACT +67 72 human species The pre-mRNA splicing factor U2AF65 recognizes 3′ splice sites in human gene transcripts, but the details are not fully understood. ABSTRACT +25 31 U2AF65 protein Here, the authors report U2AF65 structures and single molecule FRET that reveal mechanistic insights into splice site recognition. ABSTRACT +32 42 structures evidence Here, the authors report U2AF65 structures and single molecule FRET that reveal mechanistic insights into splice site recognition. ABSTRACT +47 67 single molecule FRET experimental_method Here, the authors report U2AF65 structures and single molecule FRET that reveal mechanistic insights into splice site recognition. ABSTRACT +106 117 splice site site Here, the authors report U2AF65 structures and single molecule FRET that reveal mechanistic insights into splice site recognition. ABSTRACT +64 80 pre-mRNA regions structure_element The differential skipping or inclusion of alternatively spliced pre-mRNA regions is a major source of diversity for nearly all human gene transcripts. INTRO +127 132 human species The differential skipping or inclusion of alternatively spliced pre-mRNA regions is a major source of diversity for nearly all human gene transcripts. INTRO +4 16 splice sites site The splice sites are marked by relatively short consensus sequences and are regulated by additional pre-mRNA motifs (reviewed in ref.). INTRO +42 67 short consensus sequences structure_element The splice sites are marked by relatively short consensus sequences and are regulated by additional pre-mRNA motifs (reviewed in ref.). INTRO +100 115 pre-mRNA motifs structure_element The splice sites are marked by relatively short consensus sequences and are regulated by additional pre-mRNA motifs (reviewed in ref.). INTRO +7 21 3′ splice site site At the 3′ splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3′ splice site junction. INTRO +65 90 polypyrimidine (Py) tract chemical At the 3′ splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3′ splice site junction. INTRO +112 113 U residue_name At the 3′ splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3′ splice site junction. INTRO +118 119 C residue_name At the 3′ splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3′ splice site junction. INTRO +145 166 branch point sequence site At the 3′ splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3′ splice site junction. INTRO +168 171 BPS site At the 3′ splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3′ splice site junction. INTRO +247 262 AG-dinucleotide chemical At the 3′ splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3′ splice site junction. INTRO +270 284 3′ splice site site At the 3′ splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3′ splice site junction. INTRO +43 51 pre-mRNA chemical Disease-causing mutations often compromise pre-mRNA splicing (reviewed in refs), yet a priori predictions of splice sites and the consequences of their mutations are challenged by the brevity and degeneracy of known splice site sequences. INTRO +109 121 splice sites site Disease-causing mutations often compromise pre-mRNA splicing (reviewed in refs), yet a priori predictions of splice sites and the consequences of their mutations are challenged by the brevity and degeneracy of known splice site sequences. INTRO +216 227 splice site site Disease-causing mutations often compromise pre-mRNA splicing (reviewed in refs), yet a priori predictions of splice sites and the consequences of their mutations are challenged by the brevity and degeneracy of known splice site sequences. INTRO +16 26 structures evidence High-resolution structures of intact splicing factor–RNA complexes would offer key insights regarding the juxtaposition of the distinct splice site consensus sequences and their relationship to disease-causing point mutations. INTRO +30 36 intact protein_state High-resolution structures of intact splicing factor–RNA complexes would offer key insights regarding the juxtaposition of the distinct splice site consensus sequences and their relationship to disease-causing point mutations. INTRO +37 56 splicing factor–RNA complex_assembly High-resolution structures of intact splicing factor–RNA complexes would offer key insights regarding the juxtaposition of the distinct splice site consensus sequences and their relationship to disease-causing point mutations. INTRO +136 147 splice site site High-resolution structures of intact splicing factor–RNA complexes would offer key insights regarding the juxtaposition of the distinct splice site consensus sequences and their relationship to disease-causing point mutations. INTRO +16 40 pre-mRNA splicing factor protein_type The early-stage pre-mRNA splicing factor U2AF65 is essential for viability in vertebrates and other model organisms (for example, ref.). INTRO +41 47 U2AF65 protein The early-stage pre-mRNA splicing factor U2AF65 is essential for viability in vertebrates and other model organisms (for example, ref.). INTRO +78 89 vertebrates taxonomy_domain The early-stage pre-mRNA splicing factor U2AF65 is essential for viability in vertebrates and other model organisms (for example, ref.). INTRO +21 29 assembly complex_assembly A tightly controlled assembly among U2AF65, the pre-mRNA, and partner proteins sequentially identifies the 3′ splice site and promotes association of the spliceosome, which ultimately accomplishes the task of splicing. INTRO +36 42 U2AF65 protein A tightly controlled assembly among U2AF65, the pre-mRNA, and partner proteins sequentially identifies the 3′ splice site and promotes association of the spliceosome, which ultimately accomplishes the task of splicing. INTRO +48 56 pre-mRNA chemical A tightly controlled assembly among U2AF65, the pre-mRNA, and partner proteins sequentially identifies the 3′ splice site and promotes association of the spliceosome, which ultimately accomplishes the task of splicing. INTRO +107 121 3′ splice site site A tightly controlled assembly among U2AF65, the pre-mRNA, and partner proteins sequentially identifies the 3′ splice site and promotes association of the spliceosome, which ultimately accomplishes the task of splicing. INTRO +154 165 spliceosome complex_assembly A tightly controlled assembly among U2AF65, the pre-mRNA, and partner proteins sequentially identifies the 3′ splice site and promotes association of the spliceosome, which ultimately accomplishes the task of splicing. INTRO +10 16 U2AF65 protein Initially U2AF65 recognizes the Py-tract splice site signal. INTRO +32 40 Py-tract chemical Initially U2AF65 recognizes the Py-tract splice site signal. INTRO +41 52 splice site site Initially U2AF65 recognizes the Py-tract splice site signal. INTRO +13 28 ternary complex complex_assembly In turn, the ternary complex of U2AF65 with SF1 and U2AF35 identifies the surrounding BPS and 3′ splice site junctions. INTRO +32 38 U2AF65 protein In turn, the ternary complex of U2AF65 with SF1 and U2AF35 identifies the surrounding BPS and 3′ splice site junctions. INTRO +44 47 SF1 protein In turn, the ternary complex of U2AF65 with SF1 and U2AF35 identifies the surrounding BPS and 3′ splice site junctions. INTRO +52 58 U2AF35 protein In turn, the ternary complex of U2AF65 with SF1 and U2AF35 identifies the surrounding BPS and 3′ splice site junctions. INTRO +86 89 BPS site In turn, the ternary complex of U2AF65 with SF1 and U2AF35 identifies the surrounding BPS and 3′ splice site junctions. INTRO +94 108 3′ splice site site In turn, the ternary complex of U2AF65 with SF1 and U2AF35 identifies the surrounding BPS and 3′ splice site junctions. INTRO +13 19 U2AF65 protein Subsequently U2AF65 recruits the U2 small nuclear ribonucleoprotein particle (snRNP) and ultimately dissociates from the active spliceosome. INTRO +33 76 U2 small nuclear ribonucleoprotein particle complex_assembly Subsequently U2AF65 recruits the U2 small nuclear ribonucleoprotein particle (snRNP) and ultimately dissociates from the active spliceosome. INTRO +78 83 snRNP complex_assembly Subsequently U2AF65 recruits the U2 small nuclear ribonucleoprotein particle (snRNP) and ultimately dissociates from the active spliceosome. INTRO +121 127 active protein_state Subsequently U2AF65 recruits the U2 small nuclear ribonucleoprotein particle (snRNP) and ultimately dissociates from the active spliceosome. INTRO +128 139 spliceosome complex_assembly Subsequently U2AF65 recruits the U2 small nuclear ribonucleoprotein particle (snRNP) and ultimately dissociates from the active spliceosome. INTRO +0 29 Biochemical characterizations experimental_method Biochemical characterizations of U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract (Fig. 1a). INTRO +33 39 U2AF65 protein Biochemical characterizations of U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract (Fig. 1a). INTRO +65 87 RNA recognition motifs structure_element Biochemical characterizations of U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract (Fig. 1a). INTRO +89 93 RRM1 structure_element Biochemical characterizations of U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract (Fig. 1a). INTRO +98 102 RRM2 structure_element Biochemical characterizations of U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract (Fig. 1a). INTRO +118 126 Py tract chemical Biochemical characterizations of U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract (Fig. 1a). INTRO +10 28 crystal structures evidence Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. INTRO +36 40 core protein_state Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. INTRO +41 47 U2AF65 protein Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. INTRO +48 52 RRM1 structure_element Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. INTRO +57 61 RRM2 structure_element Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. INTRO +77 86 shortened protein_state Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. INTRO +87 103 inter-RRM linker structure_element Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. INTRO +105 115 dU2AF651,2 mutant Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. INTRO +182 188 U2AF65 protein Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. INTRO +189 193 RRMs structure_element Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. INTRO +13 16 NMR experimental_method A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +17 26 structure evidence A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +45 57 side-by-side protein_state A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +77 84 minimal protein_state A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +85 91 U2AF65 protein A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +92 96 RRM1 structure_element A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +101 105 RRM2 structure_element A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +121 127 linker structure_element A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +131 145 natural length protein_state A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +147 156 U2AF651,2 mutant A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +179 189 dU2AF651,2 mutant A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +190 208 crystal structures evidence A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +213 216 RNA chemical A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +261 277 inter-RRM linker structure_element A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. INTRO +38 46 Py-tract chemical As such, the molecular mechanisms for Py-tract recognition by the intact U2AF65–RNA-binding domain remained unknown. INTRO +66 72 intact protein_state As such, the molecular mechanisms for Py-tract recognition by the intact U2AF65–RNA-binding domain remained unknown. INTRO +73 98 U2AF65–RNA-binding domain structure_element As such, the molecular mechanisms for Py-tract recognition by the intact U2AF65–RNA-binding domain remained unknown. INTRO +13 34 X-ray crystallography experimental_method Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs. INTRO +39 58 biochemical studies experimental_method Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs. INTRO +82 90 Py-tract chemical Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs. INTRO +111 127 inter-RRM linker structure_element Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs. INTRO +161 165 core protein_state Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs. INTRO +166 172 U2AF65 protein Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs. INTRO +173 177 RRMs structure_element Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs. INTRO +7 56 single-molecule Förster resonance energy transfer experimental_method We use single-molecule Förster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65–RNA-binding domain during Py-tract recognition. INTRO +58 64 smFRET experimental_method We use single-molecule Förster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65–RNA-binding domain during Py-tract recognition. INTRO +86 109 conformational dynamics evidence We use single-molecule Förster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65–RNA-binding domain during Py-tract recognition. INTRO +118 126 extended protein_state We use single-molecule Förster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65–RNA-binding domain during Py-tract recognition. INTRO +127 152 U2AF65–RNA-binding domain structure_element We use single-molecule Förster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65–RNA-binding domain during Py-tract recognition. INTRO +160 168 Py-tract chemical We use single-molecule Förster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65–RNA-binding domain during Py-tract recognition. INTRO +8 14 U2AF65 protein Cognate U2AF65–Py-tract recognition requires RRM extensions RESULTS +15 23 Py-tract chemical Cognate U2AF65–Py-tract recognition requires RRM extensions RESULTS +45 59 RRM extensions structure_element Cognate U2AF65–Py-tract recognition requires RRM extensions RESULTS +4 16 RNA affinity evidence The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). RESULTS +24 31 minimal protein_state The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). RESULTS +32 41 U2AF651,2 mutant The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). RESULTS +64 68 core protein_state The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). RESULTS +69 73 RRM1 structure_element The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). RESULTS +74 78 RRM2 structure_element The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). RESULTS +79 84 folds structure_element The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). RESULTS +86 95 U2AF651,2 mutant The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). RESULTS +106 113 148–336 residue_range The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). RESULTS +148 159 full-length protein_state The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). RESULTS +160 166 U2AF65 protein The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). RESULTS +52 58 U2AF65 protein Historically, this difference was attributed to the U2AF65 arginine–serine rich domain, which contacts pre-mRNA–U2 snRNA duplexes outside of the Py tract. RESULTS +59 86 arginine–serine rich domain structure_element Historically, this difference was attributed to the U2AF65 arginine–serine rich domain, which contacts pre-mRNA–U2 snRNA duplexes outside of the Py tract. RESULTS +103 129 pre-mRNA–U2 snRNA duplexes complex_assembly Historically, this difference was attributed to the U2AF65 arginine–serine rich domain, which contacts pre-mRNA–U2 snRNA duplexes outside of the Py tract. RESULTS +145 153 Py tract chemical Historically, this difference was attributed to the U2AF65 arginine–serine rich domain, which contacts pre-mRNA–U2 snRNA duplexes outside of the Py tract. RESULTS +20 40 RNA-binding affinity evidence We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141–342; Fig. 1a). RESULTS +48 57 U2AF651,2 mutant We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141–342; Fig. 1a). RESULTS +93 127 addition of seven and six residues experimental_method We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141–342; Fig. 1a). RESULTS +169 176 minimal protein_state We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141–342; Fig. 1a). RESULTS +177 181 RRM1 structure_element We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141–342; Fig. 1a). RESULTS +186 190 RRM2 structure_element We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141–342; Fig. 1a). RESULTS +192 202 U2AF651,2L mutant We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141–342; Fig. 1a). RESULTS +213 220 141–342 residue_range We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141–342; Fig. 1a). RESULTS +5 34 fluorescence anisotropy assay experimental_method In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). RESULTS +64 72 Py tract chemical In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). RESULTS +109 120 splice site site In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). RESULTS +128 158 adenovirus major late promoter gene In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). RESULTS +160 164 AdML gene In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). RESULTS +171 183 RNA affinity evidence In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). RESULTS +187 197 U2AF651,2L mutant In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). RESULTS +232 241 U2AF651,2 mutant In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). RESULTS +266 277 full-length protein_state In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). RESULTS +278 284 U2AF65 protein In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). RESULTS +15 25 U2AF651,2L mutant Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h). RESULTS +30 41 full-length protein_state Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h). RESULTS +42 48 U2AF65 protein Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h). RESULTS +64 84 sequence specificity evidence Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h). RESULTS +89 105 U-rich stretches structure_element Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h). RESULTS +113 122 5′-region site Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h). RESULTS +130 138 Py tract chemical Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h). RESULTS +159 173 C-rich regions structure_element Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h). RESULTS +181 190 3′-region site Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h). RESULTS +0 12 U2AF65-bound protein_state U2AF65-bound Py tract comprises nine contiguous nucleotides RESULTS +13 21 Py tract chemical U2AF65-bound Py tract comprises nine contiguous nucleotides RESULTS +37 47 contiguous structure_element U2AF65-bound Py tract comprises nine contiguous nucleotides RESULTS +48 59 nucleotides chemical U2AF65-bound Py tract comprises nine contiguous nucleotides RESULTS +48 54 U2AF65 protein To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1). RESULTS +72 82 contiguous structure_element To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1). RESULTS +83 91 Py tract chemical To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1). RESULTS +96 106 determined experimental_method To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1). RESULTS +112 130 crystal structures evidence To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1). RESULTS +134 144 U2AF651,2L mutant To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1). RESULTS +145 153 bound to protein_state To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1). RESULTS +154 179 Py-tract oligonucleotides chemical To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1). RESULTS +3 28 sequential boot strapping experimental_method By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. RESULTS +57 72 oligonucleotide chemical By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. RESULTS +99 104 Br-dU chemical By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. RESULTS +139 149 nucleotide chemical By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. RESULTS +151 153 rU residue_name By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. RESULTS +155 157 dU residue_name By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. RESULTS +162 164 rC residue_name By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. RESULTS +191 201 U2AF651,2L mutant By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. RESULTS +202 210 bound to protein_state By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. RESULTS +211 221 contiguous structure_element By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. RESULTS +222 231 Py tracts chemical By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. RESULTS +16 31 oligonucleotide chemical The protein and oligonucleotide conformations are nearly identical among the four new U2AF651,2L structures (Supplementary Fig. 2a). RESULTS +86 96 U2AF651,2L mutant The protein and oligonucleotide conformations are nearly identical among the four new U2AF651,2L structures (Supplementary Fig. 2a). RESULTS +97 107 structures evidence The protein and oligonucleotide conformations are nearly identical among the four new U2AF651,2L structures (Supplementary Fig. 2a). RESULTS +4 14 U2AF651,2L mutant The U2AF651,2L RRM1 and RRM2 associate with the Py tract in a parallel, side-by-side arrangement (shown for representative structure iv in Fig. 2b,c; Supplementary Movie 1). RESULTS +15 19 RRM1 structure_element The U2AF651,2L RRM1 and RRM2 associate with the Py tract in a parallel, side-by-side arrangement (shown for representative structure iv in Fig. 2b,c; Supplementary Movie 1). RESULTS +24 28 RRM2 structure_element The U2AF651,2L RRM1 and RRM2 associate with the Py tract in a parallel, side-by-side arrangement (shown for representative structure iv in Fig. 2b,c; Supplementary Movie 1). RESULTS +48 56 Py tract chemical The U2AF651,2L RRM1 and RRM2 associate with the Py tract in a parallel, side-by-side arrangement (shown for representative structure iv in Fig. 2b,c; Supplementary Movie 1). RESULTS +62 70 parallel protein_state The U2AF651,2L RRM1 and RRM2 associate with the Py tract in a parallel, side-by-side arrangement (shown for representative structure iv in Fig. 2b,c; Supplementary Movie 1). RESULTS +72 84 side-by-side protein_state The U2AF651,2L RRM1 and RRM2 associate with the Py tract in a parallel, side-by-side arrangement (shown for representative structure iv in Fig. 2b,c; Supplementary Movie 1). RESULTS +3 24 extended conformation protein_state An extended conformation of the U2AF65 inter-RRM linker traverses across the α-helical surface of RRM1 and the central β-strands of RRM2 and is well defined in the electron density (Fig. 2b). RESULTS +32 38 U2AF65 protein An extended conformation of the U2AF65 inter-RRM linker traverses across the α-helical surface of RRM1 and the central β-strands of RRM2 and is well defined in the electron density (Fig. 2b). RESULTS +39 55 inter-RRM linker structure_element An extended conformation of the U2AF65 inter-RRM linker traverses across the α-helical surface of RRM1 and the central β-strands of RRM2 and is well defined in the electron density (Fig. 2b). RESULTS +77 94 α-helical surface structure_element An extended conformation of the U2AF65 inter-RRM linker traverses across the α-helical surface of RRM1 and the central β-strands of RRM2 and is well defined in the electron density (Fig. 2b). RESULTS +98 102 RRM1 structure_element An extended conformation of the U2AF65 inter-RRM linker traverses across the α-helical surface of RRM1 and the central β-strands of RRM2 and is well defined in the electron density (Fig. 2b). RESULTS +119 128 β-strands structure_element An extended conformation of the U2AF65 inter-RRM linker traverses across the α-helical surface of RRM1 and the central β-strands of RRM2 and is well defined in the electron density (Fig. 2b). RESULTS +132 136 RRM2 structure_element An extended conformation of the U2AF65 inter-RRM linker traverses across the α-helical surface of RRM1 and the central β-strands of RRM2 and is well defined in the electron density (Fig. 2b). RESULTS +164 180 electron density evidence An extended conformation of the U2AF65 inter-RRM linker traverses across the α-helical surface of RRM1 and the central β-strands of RRM2 and is well defined in the electron density (Fig. 2b). RESULTS +4 14 extensions structure_element The extensions at the N terminus of RRM1 and C terminus of RRM2 adopt well-ordered α-helices. RESULTS +36 40 RRM1 structure_element The extensions at the N terminus of RRM1 and C terminus of RRM2 adopt well-ordered α-helices. RESULTS +59 63 RRM2 structure_element The extensions at the N terminus of RRM1 and C terminus of RRM2 adopt well-ordered α-helices. RESULTS +83 92 α-helices structure_element The extensions at the N terminus of RRM1 and C terminus of RRM2 adopt well-ordered α-helices. RESULTS +5 9 RRM1 structure_element Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide. RESULTS +10 14 RRM2 structure_element Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide. RESULTS +15 25 extensions structure_element Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide. RESULTS +34 50 inter-RRM linker structure_element Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide. RESULTS +54 64 U2AF651,2L mutant Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide. RESULTS +88 93 bound protein_state Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide. RESULTS +94 109 oligonucleotide chemical Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide. RESULTS +42 52 U2AF651,2L mutant We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2. RESULTS +53 63 structures evidence We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2. RESULTS +79 89 dU2AF651,2 mutant We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2. RESULTS +90 107 crystal structure evidence We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2. RESULTS +112 121 U2AF651,2 mutant We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2. RESULTS +122 125 NMR experimental_method We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2. RESULTS +126 135 structure evidence We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2. RESULTS +22 42 U2AF65-binding sites site The discovery of nine U2AF65-binding sites for contiguous Py-tract nucleotides was unexpected. RESULTS +47 57 contiguous structure_element The discovery of nine U2AF65-binding sites for contiguous Py-tract nucleotides was unexpected. RESULTS +58 78 Py-tract nucleotides chemical The discovery of nine U2AF65-binding sites for contiguous Py-tract nucleotides was unexpected. RESULTS +9 19 dU2AF651,2 mutant Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures. RESULTS +20 30 structures evidence Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures. RESULTS +68 74 U2AF65 protein Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures. RESULTS +75 79 RRMs structure_element Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures. RESULTS +95 102 minimal protein_state Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures. RESULTS +109 120 nucleotides chemical Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures. RESULTS +139 149 structures evidence Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures. RESULTS +18 32 RRM2 extension structure_element Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3′-terminal nucleotide (Fig. 2c; Supplementary Movie 1). RESULTS +33 49 inter-RRM linker structure_element Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3′-terminal nucleotide (Fig. 2c; Supplementary Movie 1). RESULTS +73 97 nucleotide-binding sites site Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3′-terminal nucleotide (Fig. 2c; Supplementary Movie 1). RESULTS +107 125 RRM1/RRM2 junction site Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3′-terminal nucleotide (Fig. 2c; Supplementary Movie 1). RESULTS +134 148 RRM1 extension structure_element Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3′-terminal nucleotide (Fig. 2c; Supplementary Movie 1). RESULTS +176 186 nucleotide chemical Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3′-terminal nucleotide (Fig. 2c; Supplementary Movie 1). RESULTS +4 14 U2AF651,2L mutant The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. RESULTS +15 25 structures evidence The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. RESULTS +39 45 ribose chemical The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. RESULTS +47 48 r chemical The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. RESULTS +50 61 nucleotides chemical The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. RESULTS +76 89 binding sites site The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. RESULTS +101 108 seventh residue_number The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. RESULTS +113 119 eighth residue_number The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. RESULTS +120 130 deoxy-(d)U chemical The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. RESULTS +191 200 RNA-bound protein_state The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. RESULTS +201 211 dU2AF651,2 mutant The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. RESULTS +212 221 structure evidence The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. RESULTS +31 66 U2AF651,2L-nucleotide-binding sites site Qualitatively, a subset of the U2AF651,2L-nucleotide-binding sites (sites 1–3 and 7–9) share similar locations to those of the dU2AF651,2 structures (Supplementary Figs 2c,d and 3). RESULTS +68 77 sites 1–3 site Qualitatively, a subset of the U2AF651,2L-nucleotide-binding sites (sites 1–3 and 7–9) share similar locations to those of the dU2AF651,2 structures (Supplementary Figs 2c,d and 3). RESULTS +82 85 7–9 site Qualitatively, a subset of the U2AF651,2L-nucleotide-binding sites (sites 1–3 and 7–9) share similar locations to those of the dU2AF651,2 structures (Supplementary Figs 2c,d and 3). RESULTS +127 137 dU2AF651,2 mutant Qualitatively, a subset of the U2AF651,2L-nucleotide-binding sites (sites 1–3 and 7–9) share similar locations to those of the dU2AF651,2 structures (Supplementary Figs 2c,d and 3). RESULTS +138 148 structures evidence Qualitatively, a subset of the U2AF651,2L-nucleotide-binding sites (sites 1–3 and 7–9) share similar locations to those of the dU2AF651,2 structures (Supplementary Figs 2c,d and 3). RESULTS +14 24 U2AF651,2L mutant Yet, only the U2AF651,2L interactions at sites 1 and 7 are nearly identical to those of the dU2AF651,2 structures (Supplementary Fig. 3a,f). RESULTS +41 54 sites 1 and 7 site Yet, only the U2AF651,2L interactions at sites 1 and 7 are nearly identical to those of the dU2AF651,2 structures (Supplementary Fig. 3a,f). RESULTS +92 102 dU2AF651,2 mutant Yet, only the U2AF651,2L interactions at sites 1 and 7 are nearly identical to those of the dU2AF651,2 structures (Supplementary Fig. 3a,f). RESULTS +103 113 structures evidence Yet, only the U2AF651,2L interactions at sites 1 and 7 are nearly identical to those of the dU2AF651,2 structures (Supplementary Fig. 3a,f). RESULTS +53 63 U2AF651,2L mutant In striking departures from prior partial views, the U2AF651,2L structures reveal three unanticipated nucleotide-binding sites at the centre of the Py tract, as well as numerous new interactions that underlie cognate recognition of the Py tract (Fig. 3a–h). RESULTS +64 74 structures evidence In striking departures from prior partial views, the U2AF651,2L structures reveal three unanticipated nucleotide-binding sites at the centre of the Py tract, as well as numerous new interactions that underlie cognate recognition of the Py tract (Fig. 3a–h). RESULTS +102 126 nucleotide-binding sites site In striking departures from prior partial views, the U2AF651,2L structures reveal three unanticipated nucleotide-binding sites at the centre of the Py tract, as well as numerous new interactions that underlie cognate recognition of the Py tract (Fig. 3a–h). RESULTS +148 156 Py tract chemical In striking departures from prior partial views, the U2AF651,2L structures reveal three unanticipated nucleotide-binding sites at the centre of the Py tract, as well as numerous new interactions that underlie cognate recognition of the Py tract (Fig. 3a–h). RESULTS +236 244 Py tract chemical In striking departures from prior partial views, the U2AF651,2L structures reveal three unanticipated nucleotide-binding sites at the centre of the Py tract, as well as numerous new interactions that underlie cognate recognition of the Py tract (Fig. 3a–h). RESULTS +0 6 U2AF65 protein U2AF65 inter-RRM linker interacts with the Py tract RESULTS +7 23 inter-RRM linker structure_element U2AF65 inter-RRM linker interacts with the Py tract RESULTS +43 51 Py tract chemical U2AF65 inter-RRM linker interacts with the Py tract RESULTS +4 14 U2AF651,2L mutant The U2AF651,2L RRM2, the inter-RRM linker and RRM1 concomitantly recognize the three central nucleotides of the Py tract, which are likely to coordinate the conformational arrangement of these disparate portions of the protein. RESULTS +15 19 RRM2 structure_element The U2AF651,2L RRM2, the inter-RRM linker and RRM1 concomitantly recognize the three central nucleotides of the Py tract, which are likely to coordinate the conformational arrangement of these disparate portions of the protein. RESULTS +25 41 inter-RRM linker structure_element The U2AF651,2L RRM2, the inter-RRM linker and RRM1 concomitantly recognize the three central nucleotides of the Py tract, which are likely to coordinate the conformational arrangement of these disparate portions of the protein. RESULTS +46 50 RRM1 structure_element The U2AF651,2L RRM2, the inter-RRM linker and RRM1 concomitantly recognize the three central nucleotides of the Py tract, which are likely to coordinate the conformational arrangement of these disparate portions of the protein. RESULTS +93 104 nucleotides chemical The U2AF651,2L RRM2, the inter-RRM linker and RRM1 concomitantly recognize the three central nucleotides of the Py tract, which are likely to coordinate the conformational arrangement of these disparate portions of the protein. RESULTS +112 120 Py tract chemical The U2AF651,2L RRM2, the inter-RRM linker and RRM1 concomitantly recognize the three central nucleotides of the Py tract, which are likely to coordinate the conformational arrangement of these disparate portions of the protein. RESULTS +16 33 C-terminal region structure_element Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d). RESULTS +41 47 U2AF65 protein Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d). RESULTS +48 64 inter-RRM linker structure_element Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d). RESULTS +94 106 binding site site Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d). RESULTS +115 120 fifth residue_number Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d). RESULTS +121 131 nucleotide chemical Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d). RESULTS +139 151 RRM2 surface site Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d). RESULTS +169 188 RRM1/RRM2 interface site Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d). RESULTS +26 32 linker structure_element The backbone amide of the linker V254 and the carbonyl of T252 engage in hydrogen bonds with the rU5-O4 and -N3H atoms. RESULTS +33 37 V254 residue_name_number The backbone amide of the linker V254 and the carbonyl of T252 engage in hydrogen bonds with the rU5-O4 and -N3H atoms. RESULTS +58 62 T252 residue_name_number The backbone amide of the linker V254 and the carbonyl of T252 engage in hydrogen bonds with the rU5-O4 and -N3H atoms. RESULTS +73 87 hydrogen bonds bond_interaction The backbone amide of the linker V254 and the carbonyl of T252 engage in hydrogen bonds with the rU5-O4 and -N3H atoms. RESULTS +97 100 rU5 residue_name_number The backbone amide of the linker V254 and the carbonyl of T252 engage in hydrogen bonds with the rU5-O4 and -N3H atoms. RESULTS +18 26 β-strand structure_element In the C-terminal β-strand of RRM1, the side chains of K225 and R227 donate additional hydrogen bonds to the rU5-O2 lone pair electrons. RESULTS +30 34 RRM1 structure_element In the C-terminal β-strand of RRM1, the side chains of K225 and R227 donate additional hydrogen bonds to the rU5-O2 lone pair electrons. RESULTS +55 59 K225 residue_name_number In the C-terminal β-strand of RRM1, the side chains of K225 and R227 donate additional hydrogen bonds to the rU5-O2 lone pair electrons. RESULTS +64 68 R227 residue_name_number In the C-terminal β-strand of RRM1, the side chains of K225 and R227 donate additional hydrogen bonds to the rU5-O2 lone pair electrons. RESULTS +87 101 hydrogen bonds bond_interaction In the C-terminal β-strand of RRM1, the side chains of K225 and R227 donate additional hydrogen bonds to the rU5-O2 lone pair electrons. RESULTS +109 112 rU5 residue_name_number In the C-terminal β-strand of RRM1, the side chains of K225 and R227 donate additional hydrogen bonds to the rU5-O2 lone pair electrons. RESULTS +4 21 C-terminal region structure_element The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c). RESULTS +29 45 inter-RRM linker structure_element The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c). RESULTS +81 97 rU4-binding site site The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c). RESULTS +109 113 V254 residue_name_number The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c). RESULTS +136 140 D256 residue_name_number The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c). RESULTS +166 170 K260 residue_name_number The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c). RESULTS +185 198 hydrogen bond bond_interaction The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c). RESULTS +208 211 rU4 residue_name_number The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c). RESULTS +15 18 rU4 residue_name_number Otherwise, the rU4 nucleotide packs against F304 in the signature ribonucleoprotein consensus motif (RNP)-2 of RRM2. RESULTS +19 29 nucleotide chemical Otherwise, the rU4 nucleotide packs against F304 in the signature ribonucleoprotein consensus motif (RNP)-2 of RRM2. RESULTS +44 48 F304 residue_name_number Otherwise, the rU4 nucleotide packs against F304 in the signature ribonucleoprotein consensus motif (RNP)-2 of RRM2. RESULTS +66 107 ribonucleoprotein consensus motif (RNP)-2 structure_element Otherwise, the rU4 nucleotide packs against F304 in the signature ribonucleoprotein consensus motif (RNP)-2 of RRM2. RESULTS +111 115 RRM2 structure_element Otherwise, the rU4 nucleotide packs against F304 in the signature ribonucleoprotein consensus motif (RNP)-2 of RRM2. RESULTS +36 41 fifth residue_number At the opposite side of the central fifth nucleotide, the sixth rU6 nucleotide is located at the inter-RRM1/RRM2 interface (Fig. 3e; Supplementary Movie 1). RESULTS +42 52 nucleotide chemical At the opposite side of the central fifth nucleotide, the sixth rU6 nucleotide is located at the inter-RRM1/RRM2 interface (Fig. 3e; Supplementary Movie 1). RESULTS +58 63 sixth residue_number At the opposite side of the central fifth nucleotide, the sixth rU6 nucleotide is located at the inter-RRM1/RRM2 interface (Fig. 3e; Supplementary Movie 1). RESULTS +64 67 rU6 residue_name_number At the opposite side of the central fifth nucleotide, the sixth rU6 nucleotide is located at the inter-RRM1/RRM2 interface (Fig. 3e; Supplementary Movie 1). RESULTS +68 78 nucleotide chemical At the opposite side of the central fifth nucleotide, the sixth rU6 nucleotide is located at the inter-RRM1/RRM2 interface (Fig. 3e; Supplementary Movie 1). RESULTS +97 122 inter-RRM1/RRM2 interface site At the opposite side of the central fifth nucleotide, the sixth rU6 nucleotide is located at the inter-RRM1/RRM2 interface (Fig. 3e; Supplementary Movie 1). RESULTS +5 15 nucleotide chemical This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the β2/β3 loops of RRM1 and RRM2. RESULTS +45 51 U2AF65 protein This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the β2/β3 loops of RRM1 and RRM2. RESULTS +52 58 linker structure_element This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the β2/β3 loops of RRM1 and RRM2. RESULTS +83 86 rU6 residue_name_number This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the β2/β3 loops of RRM1 and RRM2. RESULTS +87 93 uracil residue_name This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the β2/β3 loops of RRM1 and RRM2. RESULTS +122 133 β2/β3 loops structure_element This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the β2/β3 loops of RRM1 and RRM2. RESULTS +137 141 RRM1 structure_element This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the β2/β3 loops of RRM1 and RRM2. RESULTS +146 150 RRM2 structure_element This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the β2/β3 loops of RRM1 and RRM2. RESULTS +4 7 rU6 residue_name_number The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain. RESULTS +32 47 solvent exposed protein_state The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain. RESULTS +66 69 rU6 residue_name_number The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain. RESULTS +70 84 hydrogen bonds bond_interaction The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain. RESULTS +90 96 U2AF65 protein The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain. RESULTS +101 106 water chemical The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain. RESULTS +162 166 RRM1 structure_element The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain. RESULTS +167 171 N196 residue_name_number The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain. RESULTS +3 26 tested the contribution experimental_method We tested the contribution of the U2AF651,2L interactions with the new central nucleotide to Py-tract affinity (Fig. 3i; Supplementary Fig. 4a,b). RESULTS +34 44 U2AF651,2L mutant We tested the contribution of the U2AF651,2L interactions with the new central nucleotide to Py-tract affinity (Fig. 3i; Supplementary Fig. 4a,b). RESULTS +79 89 nucleotide chemical We tested the contribution of the U2AF651,2L interactions with the new central nucleotide to Py-tract affinity (Fig. 3i; Supplementary Fig. 4a,b). RESULTS +93 110 Py-tract affinity evidence We tested the contribution of the U2AF651,2L interactions with the new central nucleotide to Py-tract affinity (Fig. 3i; Supplementary Fig. 4a,b). RESULTS +0 11 Mutagenesis experimental_method Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +22 26 V254 residue_name_number Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +34 40 U2AF65 protein Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +41 57 inter-RRM linker structure_element Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +61 68 proline residue_name Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +72 76 RRM1 structure_element Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +77 81 R227 residue_name_number Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +85 92 alanine residue_name Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +111 124 hydrogen bond bond_interaction Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +134 139 fifth residue_number Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +140 146 uracil residue_name Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +170 180 affinities evidence Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +184 194 U2AF651,2L mutant Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +218 222 AdML gene Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +223 231 Py tract chemical Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. RESULTS +48 51 ΔΔG evidence The energetic penalties due to these mutations (ΔΔG 0.8–0.9 kcal mol−1) are consistent with the loss of each hydrogen bond with the rU5 base and support the relevance of the central nucleotide interactions observed in the U2AF651,2L structures. RESULTS +109 122 hydrogen bond bond_interaction The energetic penalties due to these mutations (ΔΔG 0.8–0.9 kcal mol−1) are consistent with the loss of each hydrogen bond with the rU5 base and support the relevance of the central nucleotide interactions observed in the U2AF651,2L structures. RESULTS +132 135 rU5 residue_name_number The energetic penalties due to these mutations (ΔΔG 0.8–0.9 kcal mol−1) are consistent with the loss of each hydrogen bond with the rU5 base and support the relevance of the central nucleotide interactions observed in the U2AF651,2L structures. RESULTS +222 232 U2AF651,2L mutant The energetic penalties due to these mutations (ΔΔG 0.8–0.9 kcal mol−1) are consistent with the loss of each hydrogen bond with the rU5 base and support the relevance of the central nucleotide interactions observed in the U2AF651,2L structures. RESULTS +233 243 structures evidence The energetic penalties due to these mutations (ΔΔG 0.8–0.9 kcal mol−1) are consistent with the loss of each hydrogen bond with the rU5 base and support the relevance of the central nucleotide interactions observed in the U2AF651,2L structures. RESULTS +0 6 U2AF65 protein U2AF65 RRM extensions interact with the Py tract RESULTS +7 21 RRM extensions structure_element U2AF65 RRM extensions interact with the Py tract RESULTS +40 48 Py tract chemical U2AF65 RRM extensions interact with the Py tract RESULTS +4 32 N- and C-terminal extensions structure_element The N- and C-terminal extensions of the U2AF65 RRM1 and RRM2 directly contact the bound Py tract. RESULTS +40 46 U2AF65 protein The N- and C-terminal extensions of the U2AF65 RRM1 and RRM2 directly contact the bound Py tract. RESULTS +47 51 RRM1 structure_element The N- and C-terminal extensions of the U2AF65 RRM1 and RRM2 directly contact the bound Py tract. RESULTS +56 60 RRM2 structure_element The N- and C-terminal extensions of the U2AF65 RRM1 and RRM2 directly contact the bound Py tract. RESULTS +82 87 bound protein_state The N- and C-terminal extensions of the U2AF65 RRM1 and RRM2 directly contact the bound Py tract. RESULTS +88 96 Py tract chemical The N- and C-terminal extensions of the U2AF65 RRM1 and RRM2 directly contact the bound Py tract. RESULTS +47 57 nucleotide chemical Rather than interacting with a new 5′-terminal nucleotide as we had hypothesized, the C-terminal α-helix of RRM2 instead folds across one surface of rU3 in the third binding site (Fig. 3b). RESULTS +97 104 α-helix structure_element Rather than interacting with a new 5′-terminal nucleotide as we had hypothesized, the C-terminal α-helix of RRM2 instead folds across one surface of rU3 in the third binding site (Fig. 3b). RESULTS +108 112 RRM2 structure_element Rather than interacting with a new 5′-terminal nucleotide as we had hypothesized, the C-terminal α-helix of RRM2 instead folds across one surface of rU3 in the third binding site (Fig. 3b). RESULTS +149 152 rU3 residue_name_number Rather than interacting with a new 5′-terminal nucleotide as we had hypothesized, the C-terminal α-helix of RRM2 instead folds across one surface of rU3 in the third binding site (Fig. 3b). RESULTS +160 178 third binding site site Rather than interacting with a new 5′-terminal nucleotide as we had hypothesized, the C-terminal α-helix of RRM2 instead folds across one surface of rU3 in the third binding site (Fig. 3b). RESULTS +9 20 salt bridge bond_interaction There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. RESULTS +33 37 K340 residue_name_number There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. RESULTS +53 63 nucleotide chemical There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. RESULTS +86 90 G338 residue_name_number There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. RESULTS +96 104 stacking bond_interaction There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. RESULTS +111 124 hydrogen bond bond_interaction There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. RESULTS +155 159 G338 residue_name_number There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. RESULTS +168 171 rU3 residue_name_number There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. RESULTS +187 201 RRM2 extension structure_element There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. RESULTS +45 50 third residue_number Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal β-strand of RRM2. RESULTS +51 61 nucleotide chemical Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal β-strand of RRM2. RESULTS +72 75 rU2 residue_name_number Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal β-strand of RRM2. RESULTS +76 86 nucleotide chemical Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal β-strand of RRM2. RESULTS +94 113 second binding site site Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal β-strand of RRM2. RESULTS +139 147 β-strand structure_element Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal β-strand of RRM2. RESULTS +151 155 RRM2 structure_element Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal β-strand of RRM2. RESULTS +18 34 U2AF651,2L-bound protein_state Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b). RESULTS +35 38 rU2 residue_name_number Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b). RESULTS +61 75 hydrogen bonds bond_interaction Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b). RESULTS +85 89 K329 residue_name_number Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b). RESULTS +137 150 hydrogen bond bond_interaction Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b). RESULTS +160 164 K329 residue_name_number Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b). RESULTS +192 202 dU2AF651,2 mutant Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b). RESULTS +203 212 structure evidence Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b). RESULTS +23 42 α-helical extension structure_element At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). RESULTS +46 52 U2AF65 protein At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). RESULTS +53 57 RRM1 structure_element At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). RESULTS +72 76 Q147 residue_name_number At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). RESULTS +102 108 eighth residue_number At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). RESULTS +113 118 ninth residue_number At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). RESULTS +119 130 nucleotides chemical At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). RESULTS +138 149 3′ terminus site At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). RESULTS +157 165 Py tract chemical At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). RESULTS +4 8 Q147 residue_name_number The Q147 residue participates in hydrogen bonds with the -N3H of the eighth uracil and -O2 of the ninth pyrimidine. RESULTS +33 47 hydrogen bonds bond_interaction The Q147 residue participates in hydrogen bonds with the -N3H of the eighth uracil and -O2 of the ninth pyrimidine. RESULTS +69 75 eighth residue_number The Q147 residue participates in hydrogen bonds with the -N3H of the eighth uracil and -O2 of the ninth pyrimidine. RESULTS +76 82 uracil residue_name The Q147 residue participates in hydrogen bonds with the -N3H of the eighth uracil and -O2 of the ninth pyrimidine. RESULTS +98 103 ninth residue_number The Q147 residue participates in hydrogen bonds with the -N3H of the eighth uracil and -O2 of the ninth pyrimidine. RESULTS +104 114 pyrimidine chemical The Q147 residue participates in hydrogen bonds with the -N3H of the eighth uracil and -O2 of the ninth pyrimidine. RESULTS +13 17 R146 residue_name_number The adjacent R146 guanidinium group donates hydrogen bonds to the 3′-terminal ribose-O2′ and O3′ atoms, where it could form a salt bridge with a phospho-diester group in the context of a longer pre-mRNA. RESULTS +44 58 hydrogen bonds bond_interaction The adjacent R146 guanidinium group donates hydrogen bonds to the 3′-terminal ribose-O2′ and O3′ atoms, where it could form a salt bridge with a phospho-diester group in the context of a longer pre-mRNA. RESULTS +78 84 ribose chemical The adjacent R146 guanidinium group donates hydrogen bonds to the 3′-terminal ribose-O2′ and O3′ atoms, where it could form a salt bridge with a phospho-diester group in the context of a longer pre-mRNA. RESULTS +126 137 salt bridge bond_interaction The adjacent R146 guanidinium group donates hydrogen bonds to the 3′-terminal ribose-O2′ and O3′ atoms, where it could form a salt bridge with a phospho-diester group in the context of a longer pre-mRNA. RESULTS +194 202 pre-mRNA chemical The adjacent R146 guanidinium group donates hydrogen bonds to the 3′-terminal ribose-O2′ and O3′ atoms, where it could form a salt bridge with a phospho-diester group in the context of a longer pre-mRNA. RESULTS +26 39 hydrogen bond bond_interaction Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). RESULTS +49 54 ninth residue_number Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). RESULTS +55 65 pyrimidine chemical Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). RESULTS +70 73 ΔΔG evidence Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). RESULTS +91 99 mutation experimental_method Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). RESULTS +107 111 Q147 residue_name_number Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). RESULTS +118 125 alanine residue_name Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). RESULTS +134 153 U2AF651,2L affinity evidence Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). RESULTS +162 166 AdML gene Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). RESULTS +167 175 Py tract chemical Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). RESULTS +3 10 compare experimental_method We compare U2AF65 interactions with uracil relative to cytosine pyrimidines at the ninth binding site in Fig. 3g,h and the Supplementary Discussion. RESULTS +11 17 U2AF65 protein We compare U2AF65 interactions with uracil relative to cytosine pyrimidines at the ninth binding site in Fig. 3g,h and the Supplementary Discussion. RESULTS +36 42 uracil residue_name We compare U2AF65 interactions with uracil relative to cytosine pyrimidines at the ninth binding site in Fig. 3g,h and the Supplementary Discussion. RESULTS +55 63 cytosine residue_name We compare U2AF65 interactions with uracil relative to cytosine pyrimidines at the ninth binding site in Fig. 3g,h and the Supplementary Discussion. RESULTS +64 75 pyrimidines chemical We compare U2AF65 interactions with uracil relative to cytosine pyrimidines at the ninth binding site in Fig. 3g,h and the Supplementary Discussion. RESULTS +83 101 ninth binding site site We compare U2AF65 interactions with uracil relative to cytosine pyrimidines at the ninth binding site in Fig. 3g,h and the Supplementary Discussion. RESULTS +34 40 U2AF65 protein Versatile primary sequence of the U2AF65 inter-RRM linker RESULTS +41 57 inter-RRM linker structure_element Versatile primary sequence of the U2AF65 inter-RRM linker RESULTS +4 14 U2AF651,2L mutant The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. RESULTS +15 25 structures evidence The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. RESULTS +42 58 inter-RRM linker structure_element The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. RESULTS +71 90 extensive interface site The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. RESULTS +107 114 α-helix structure_element The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. RESULTS +118 122 RRM1 structure_element The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. RESULTS +128 141 β2/β3 strands structure_element The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. RESULTS +145 149 RRM2 structure_element The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. RESULTS +169 188 α-helical extension structure_element The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. RESULTS +192 196 RRM1 structure_element The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. RESULTS +16 22 U2AF65 protein Altogether, the U2AF65 inter-RRM linker residues (R228–K260) bury 2,800 Å2 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900 Å2 for a typical protein–protein complex. RESULTS +23 39 inter-RRM linker structure_element Altogether, the U2AF65 inter-RRM linker residues (R228–K260) bury 2,800 Å2 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900 Å2 for a typical protein–protein complex. RESULTS +50 59 R228–K260 residue_range Altogether, the U2AF65 inter-RRM linker residues (R228–K260) bury 2,800 Å2 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900 Å2 for a typical protein–protein complex. RESULTS +98 108 U2AF651,2L mutant Altogether, the U2AF65 inter-RRM linker residues (R228–K260) bury 2,800 Å2 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900 Å2 for a typical protein–protein complex. RESULTS +109 121 holo-protein protein_state Altogether, the U2AF65 inter-RRM linker residues (R228–K260) bury 2,800 Å2 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900 Å2 for a typical protein–protein complex. RESULTS +139 156 cognate interface site Altogether, the U2AF65 inter-RRM linker residues (R228–K260) bury 2,800 Å2 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900 Å2 for a typical protein–protein complex. RESULTS +16 22 linker structure_element The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). RESULTS +36 40 P229 residue_name_number The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). RESULTS +55 59 core protein_state The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). RESULTS +60 64 RRM1 structure_element The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). RESULTS +65 73 β-strand structure_element The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). RESULTS +80 84 kink structure_element The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). RESULTS +107 131 intra-molecular stacking bond_interaction The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). RESULTS +154 158 R228 residue_name_number The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). RESULTS +160 164 Y232 residue_name_number The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). RESULTS +169 173 P234 residue_name_number The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). RESULTS +2 13 second kink structure_element A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. RESULTS +17 21 P236 residue_name_number A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. RESULTS +62 66 L235 residue_name_number A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. RESULTS +71 75 M238 residue_name_number A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. RESULTS +106 130 α-helical RRM1 extension structure_element A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. RESULTS +139 143 core protein_state A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. RESULTS +144 148 RRM1 structure_element A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. RESULTS +149 157 α2-helix structure_element A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. RESULTS +189 205 inter-RRM linker structure_element A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. RESULTS +218 237 RRM1/RRM2 interface site A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. RESULTS +256 272 RNA-binding site site A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. RESULTS +41 47 linker structure_element In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two α-helices of the core RRM1. RESULTS +53 57 V244 residue_name_number In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two α-helices of the core RRM1. RESULTS +62 66 V246 residue_name_number In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two α-helices of the core RRM1. RESULTS +89 107 hydrophobic pocket site In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two α-helices of the core RRM1. RESULTS +120 129 α-helices structure_element In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two α-helices of the core RRM1. RESULTS +137 141 core protein_state In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two α-helices of the core RRM1. RESULTS +142 146 RRM1 structure_element In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two α-helices of the core RRM1. RESULTS +13 17 V249 residue_name_number The adjacent V249 and V250 are notable for their respective interactions that connect RRM1 and RRM2 at this distal interface from the RNA-binding site (Fig. 4a, top). RESULTS +22 26 V250 residue_name_number The adjacent V249 and V250 are notable for their respective interactions that connect RRM1 and RRM2 at this distal interface from the RNA-binding site (Fig. 4a, top). RESULTS +86 90 RRM1 structure_element The adjacent V249 and V250 are notable for their respective interactions that connect RRM1 and RRM2 at this distal interface from the RNA-binding site (Fig. 4a, top). RESULTS +95 99 RRM2 structure_element The adjacent V249 and V250 are notable for their respective interactions that connect RRM1 and RRM2 at this distal interface from the RNA-binding site (Fig. 4a, top). RESULTS +115 124 interface site The adjacent V249 and V250 are notable for their respective interactions that connect RRM1 and RRM2 at this distal interface from the RNA-binding site (Fig. 4a, top). RESULTS +134 150 RNA-binding site site The adjacent V249 and V250 are notable for their respective interactions that connect RRM1 and RRM2 at this distal interface from the RNA-binding site (Fig. 4a, top). RESULTS +2 12 third kink structure_element A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. RESULTS +13 19 stacks bond_interaction A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. RESULTS +20 24 P247 residue_name_number A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. RESULTS +29 33 G248 residue_name_number A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. RESULTS +39 43 Y245 residue_name_number A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. RESULTS +63 80 C-terminal region structure_element A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. RESULTS +88 94 linker structure_element A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. RESULTS +107 111 RRM2 structure_element A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. RESULTS +116 121 bound protein_state A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. RESULTS +122 125 RNA chemical A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. RESULTS +7 10 RNA chemical At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). RESULTS +28 32 V254 residue_name_number At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). RESULTS +53 58 fifth residue_number At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). RESULTS +59 65 uracil residue_name At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). RESULTS +81 101 hydrophobic contacts bond_interaction At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). RESULTS +133 148 β-sheet surface structure_element At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). RESULTS +152 156 RRM2 structure_element At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). RESULTS +180 184 RNP1 structure_element At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). RESULTS +185 189 F304 residue_name_number At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). RESULTS +203 209 stacks bond_interaction At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). RESULTS +219 225 fourth residue_number At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). RESULTS +226 232 uracil residue_name At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). RESULTS +67 73 linker structure_element Few direct contacts are made between the remaining residues of the linker and the U2AF65 RRM2; instead, the C-terminal conformation of the linker appears primarily RNA mediated (Fig. 3c,d). RESULTS +82 88 U2AF65 protein Few direct contacts are made between the remaining residues of the linker and the U2AF65 RRM2; instead, the C-terminal conformation of the linker appears primarily RNA mediated (Fig. 3c,d). RESULTS +89 93 RRM2 structure_element Few direct contacts are made between the remaining residues of the linker and the U2AF65 RRM2; instead, the C-terminal conformation of the linker appears primarily RNA mediated (Fig. 3c,d). RESULTS +139 145 linker structure_element Few direct contacts are made between the remaining residues of the linker and the U2AF65 RRM2; instead, the C-terminal conformation of the linker appears primarily RNA mediated (Fig. 3c,d). RESULTS +164 167 RNA chemical Few direct contacts are made between the remaining residues of the linker and the U2AF65 RRM2; instead, the C-terminal conformation of the linker appears primarily RNA mediated (Fig. 3c,d). RESULTS +58 62 RRMs structure_element We investigated whether the observed contacts between the RRMs and linker were critical for RNA binding by structure-guided mutagenesis (Fig. 4b). RESULTS +67 73 linker structure_element We investigated whether the observed contacts between the RRMs and linker were critical for RNA binding by structure-guided mutagenesis (Fig. 4b). RESULTS +107 135 structure-guided mutagenesis experimental_method We investigated whether the observed contacts between the RRMs and linker were critical for RNA binding by structure-guided mutagenesis (Fig. 4b). RESULTS +3 11 titrated experimental_method We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d–h). RESULTS +18 24 mutant protein_state We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d–h). RESULTS +25 35 U2AF651,2L mutant We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d–h). RESULTS +50 61 fluorescein chemical We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d–h). RESULTS +71 75 AdML gene We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d–h). RESULTS +76 88 Py-tract RNA chemical We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d–h). RESULTS +101 132 fluorescence anisotropy changes evidence We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d–h). RESULTS +156 178 equilibrium affinities evidence We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d–h). RESULTS +14 21 glycine residue_name We introduced glycine substitutions to maximally reduce the buried surface area without directly interfering with its hydrogen bonds between backbone atoms and the base. RESULTS +22 35 substitutions experimental_method We introduced glycine substitutions to maximally reduce the buried surface area without directly interfering with its hydrogen bonds between backbone atoms and the base. RESULTS +118 132 hydrogen bonds bond_interaction We introduced glycine substitutions to maximally reduce the buried surface area without directly interfering with its hydrogen bonds between backbone atoms and the base. RESULTS +10 18 replaced experimental_method First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). RESULTS +19 23 V249 residue_name_number First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). RESULTS +28 32 V250 residue_name_number First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). RESULTS +40 59 RRM1/RRM2 interface site First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). RESULTS +64 68 V254 residue_name_number First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). RESULTS +76 81 bound protein_state First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). RESULTS +82 85 RNA chemical First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). RESULTS +96 103 glycine residue_name First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). RESULTS +105 109 3Gly mutant First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). RESULTS +39 43 AdML gene However, the resulting decrease in the AdML RNA affinity of the U2AF651,2L-3Gly mutant relative to wild-type protein was not significant (Fig. 4b). RESULTS +44 56 RNA affinity evidence However, the resulting decrease in the AdML RNA affinity of the U2AF651,2L-3Gly mutant relative to wild-type protein was not significant (Fig. 4b). RESULTS +64 79 U2AF651,2L-3Gly mutant However, the resulting decrease in the AdML RNA affinity of the U2AF651,2L-3Gly mutant relative to wild-type protein was not significant (Fig. 4b). RESULTS +80 86 mutant protein_state However, the resulting decrease in the AdML RNA affinity of the U2AF651,2L-3Gly mutant relative to wild-type protein was not significant (Fig. 4b). RESULTS +99 108 wild-type protein_state However, the resulting decrease in the AdML RNA affinity of the U2AF651,2L-3Gly mutant relative to wild-type protein was not significant (Fig. 4b). RESULTS +109 116 protein protein However, the resulting decrease in the AdML RNA affinity of the U2AF651,2L-3Gly mutant relative to wild-type protein was not significant (Fig. 4b). RESULTS +16 24 replaced experimental_method In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +30 45 linker residues structure_element In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +47 51 S251 residue_name_number In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +53 57 T252 residue_name_number In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +59 63 V253 residue_name_number In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +65 69 V254 residue_name_number In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +74 78 P255 residue_name_number In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +87 116 fifth nucleotide-binding site site In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +122 130 glycines residue_name In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +132 136 5Gly mutant In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +162 174 RNA affinity evidence In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +182 197 U2AF651,2L-5Gly mutant In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +198 204 mutant protein_state In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +250 259 wild-type protein_state In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +260 267 protein protein In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. RESULTS +7 32 conservative substitution experimental_method A more conservative substitution of these five residues (251–255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. RESULTS +57 64 251–255 residue_range A more conservative substitution of these five residues (251–255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. RESULTS +122 136 hydrogen bonds bond_interaction A more conservative substitution of these five residues (251–255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. RESULTS +138 149 STVVP>NLALA mutant A more conservative substitution of these five residues (251–255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. RESULTS +197 215 inter-RRM sequence structure_element A more conservative substitution of these five residues (251–255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. RESULTS +219 227 affinity evidence A more conservative substitution of these five residues (251–255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. RESULTS +236 240 AdML gene A more conservative substitution of these five residues (251–255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. RESULTS +241 249 Py tract chemical A more conservative substitution of these five residues (251–255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. RESULTS +81 87 linker structure_element Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +88 91 RRM structure_element Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +105 116 substituted experimental_method Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +117 124 glycine residue_name Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +180 196 inter-RRM linker structure_element Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +208 212 M144 residue_name_number Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +214 218 L235 residue_name_number Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +220 224 M238 residue_name_number Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +226 230 V244 residue_name_number Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +232 236 V246 residue_name_number Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +238 242 V249 residue_name_number Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +244 248 V250 residue_name_number Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +250 254 S251 residue_name_number Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +256 260 T252 residue_name_number Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +262 266 V253 residue_name_number Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +268 272 V254 residue_name_number Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +274 278 P255 residue_name_number Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +287 292 12Gly mutant Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). RESULTS +8 31 12 concurrent mutations experimental_method Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). RESULTS +37 41 AdML gene Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). RESULTS +42 54 RNA affinity evidence Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). RESULTS +62 78 U2AF651,2L-12Gly mutant Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). RESULTS +79 86 variant protein_state Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). RESULTS +134 144 unmodified protein_state Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). RESULTS +145 152 protein protein Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). RESULTS +202 207 V254P mutant Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). RESULTS +235 238 rU5 residue_name_number Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). RESULTS +239 252 hydrogen bond bond_interaction Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). RESULTS +29 35 U2AF65 protein To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). RESULTS +36 52 inter-RRM linker structure_element To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). RESULTS +80 94 RRM extensions structure_element To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). RESULTS +99 110 constructed experimental_method To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). RESULTS +123 138 linker deletion experimental_method To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). RESULTS +142 153 20-residues residue_range To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). RESULTS +165 173 extended protein_state To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). RESULTS +174 192 RNA-binding domain structure_element To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). RESULTS +194 205 dU2AF651,2L mutant To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). RESULTS +18 26 affinity evidence We found that the affinity of dU2AF651,2L for the AdML RNA was significantly reduced relative to U2AF651,2L (four-fold, Figs 1b and 4b; Supplementary Fig. 4i). RESULTS +30 41 dU2AF651,2L mutant We found that the affinity of dU2AF651,2L for the AdML RNA was significantly reduced relative to U2AF651,2L (four-fold, Figs 1b and 4b; Supplementary Fig. 4i). RESULTS +50 54 AdML gene We found that the affinity of dU2AF651,2L for the AdML RNA was significantly reduced relative to U2AF651,2L (four-fold, Figs 1b and 4b; Supplementary Fig. 4i). RESULTS +55 58 RNA chemical We found that the affinity of dU2AF651,2L for the AdML RNA was significantly reduced relative to U2AF651,2L (four-fold, Figs 1b and 4b; Supplementary Fig. 4i). RESULTS +97 107 U2AF651,2L mutant We found that the affinity of dU2AF651,2L for the AdML RNA was significantly reduced relative to U2AF651,2L (four-fold, Figs 1b and 4b; Supplementary Fig. 4i). RESULTS +31 46 linker deletion experimental_method Yet, it is well known that the linker deletion in the context of the minimal RRM1–RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j). RESULTS +69 76 minimal protein_state Yet, it is well known that the linker deletion in the context of the minimal RRM1–RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j). RESULTS +77 81 RRM1 structure_element Yet, it is well known that the linker deletion in the context of the minimal RRM1–RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j). RESULTS +82 86 RRM2 structure_element Yet, it is well known that the linker deletion in the context of the minimal RRM1–RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j). RESULTS +130 144 RNA affinities evidence Yet, it is well known that the linker deletion in the context of the minimal RRM1–RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j). RESULTS +148 158 dU2AF651,2 mutant Yet, it is well known that the linker deletion in the context of the minimal RRM1–RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j). RESULTS +173 182 U2AF651,2 mutant Yet, it is well known that the linker deletion in the context of the minimal RRM1–RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j). RESULTS +4 14 U2AF651,2L mutant The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +15 25 structures evidence The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +42 63 extended conformation protein_state The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +71 80 truncated protein_state The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +81 91 dU2AF651,2 mutant The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +92 108 inter-RRM linker structure_element The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +138 148 U2AF651,2L mutant The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +149 153 RRM1 structure_element The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +186 190 RRM2 structure_element The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +214 224 U2AF651,2L mutant The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +225 229 R227 residue_name_number The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +233 237 H259 residue_name_number The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +279 293 RNA affinities evidence The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +297 307 dU2AF651,2 mutant The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +312 321 U2AF651,2 mutant The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +322 326 dual protein_state The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +327 331 RRMs structure_element The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +350 360 individual protein_state The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +361 367 U2AF65 protein The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +368 372 RRMs structure_element The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. RESULTS +27 36 truncated protein_state However, stretching of the truncated dU2AF651,2L linker to connect the RRM termini is expected to disrupt its nucleotide interactions. RESULTS +37 48 dU2AF651,2L mutant However, stretching of the truncated dU2AF651,2L linker to connect the RRM termini is expected to disrupt its nucleotide interactions. RESULTS +49 55 linker structure_element However, stretching of the truncated dU2AF651,2L linker to connect the RRM termini is expected to disrupt its nucleotide interactions. RESULTS +71 82 RRM termini structure_element However, stretching of the truncated dU2AF651,2L linker to connect the RRM termini is expected to disrupt its nucleotide interactions. RESULTS +10 18 deletion experimental_method Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. RESULTS +37 51 RRM1 extension structure_element Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. RESULTS +59 68 shortened protein_state Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. RESULTS +132 138 linker structure_element Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. RESULTS +144 155 kinked turn structure_element Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. RESULTS +166 170 P229 residue_name_number Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. RESULTS +208 222 RNA affinities evidence Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. RESULTS +226 237 dU2AF651,2L mutant Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. RESULTS +239 249 dU2AF651,2 mutant Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. RESULTS +254 263 U2AF651,2 mutant Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. RESULTS +278 288 U2AF651,2L mutant Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. RESULTS +38 44 U2AF65 protein To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d). RESULTS +45 59 RRM extensions structure_element To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d). RESULTS +64 80 inter-RRM linker structure_element To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d). RESULTS +135 140 Q147A mutant To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d). RESULTS +141 146 V254P mutant To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d). RESULTS +147 152 R227A mutant To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d). RESULTS +153 161 mutation experimental_method To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d). RESULTS +163 178 U2AF651,2L-3Mut mutant To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d). RESULTS +13 18 Q147A mutant Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the ΔΔG's for the single mutations. RESULTS +19 24 V254P mutant Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the ΔΔG's for the single mutations. RESULTS +25 30 R227A mutant Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the ΔΔG's for the single mutations. RESULTS +31 39 mutation experimental_method Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the ΔΔG's for the single mutations. RESULTS +52 64 RNA affinity evidence Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the ΔΔG's for the single mutations. RESULTS +72 87 U2AF651,2L-3Mut mutant Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the ΔΔG's for the single mutations. RESULTS +167 170 ΔΔG evidence Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the ΔΔG's for the single mutations. RESULTS +35 51 linearly distant protein_state This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. RESULTS +52 59 regions structure_element This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. RESULTS +67 73 U2AF65 protein This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. RESULTS +102 106 Q147 residue_name_number This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. RESULTS +125 139 RRM1 extension structure_element This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. RESULTS +144 148 R227 residue_name_number This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. RESULTS +149 153 V254 residue_name_number This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. RESULTS +175 189 linker regions structure_element This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. RESULTS +197 218 fifth nucleotide site site This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. RESULTS +248 256 Py tract chemical This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. RESULTS +53 59 U2AF65 protein Altogether, we conclude that the conformation of the U2AF65 inter-RRM linker is key for recognizing RNA and is positioned by the RRM extension but otherwise relatively independent of the side chain composition. RESULTS +60 76 inter-RRM linker structure_element Altogether, we conclude that the conformation of the U2AF65 inter-RRM linker is key for recognizing RNA and is positioned by the RRM extension but otherwise relatively independent of the side chain composition. RESULTS +100 103 RNA chemical Altogether, we conclude that the conformation of the U2AF65 inter-RRM linker is key for recognizing RNA and is positioned by the RRM extension but otherwise relatively independent of the side chain composition. RESULTS +129 142 RRM extension structure_element Altogether, we conclude that the conformation of the U2AF65 inter-RRM linker is key for recognizing RNA and is positioned by the RRM extension but otherwise relatively independent of the side chain composition. RESULTS +32 37 Q147A mutant The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. RESULTS +38 43 V254P mutant The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. RESULTS +44 49 R227A mutant The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. RESULTS +50 65 triple mutation experimental_method The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. RESULTS +127 133 U2AF65 protein The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. RESULTS +134 149 linker deletion experimental_method The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. RESULTS +215 221 U2AF65 protein The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. RESULTS +222 228 linker structure_element The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. RESULTS +237 265 N- and C-terminal extensions structure_element The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. RESULTS +279 283 core protein_state The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. RESULTS +284 288 RRMs structure_element The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. RESULTS +14 24 U2AF65–RNA complex_assembly Importance of U2AF65–RNA contacts for pre-mRNA splicing RESULTS +38 46 pre-mRNA chemical Importance of U2AF65–RNA contacts for pre-mRNA splicing RESULTS +43 58 U2AF65–Py-tract complex_assembly We proceeded to test the importance of new U2AF65–Py-tract interactions for splicing of a model pre-mRNA substrate in a human cell line (Fig. 5; Supplementary Fig. 5). RESULTS +96 104 pre-mRNA chemical We proceeded to test the importance of new U2AF65–Py-tract interactions for splicing of a model pre-mRNA substrate in a human cell line (Fig. 5; Supplementary Fig. 5). RESULTS +120 125 human species We proceeded to test the importance of new U2AF65–Py-tract interactions for splicing of a model pre-mRNA substrate in a human cell line (Fig. 5; Supplementary Fig. 5). RESULTS +73 99 minigene splicing reporter chemical As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a). RESULTS +108 112 pyPY chemical As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a). RESULTS +154 156 py chemical As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a). RESULTS +179 185 U-rich structure_element As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a). RESULTS +187 189 PY chemical As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a). RESULTS +191 212 polypyrimidine tracts chemical As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a). RESULTS +239 251 splice sites site As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a). RESULTS +5 16 transfected experimental_method When transfected into HEK293T cells containing only endogenous U2AF65, the PY splice site is used and the remaining transcript remains unspliced. RESULTS +52 62 endogenous protein_state When transfected into HEK293T cells containing only endogenous U2AF65, the PY splice site is used and the remaining transcript remains unspliced. RESULTS +63 69 U2AF65 protein When transfected into HEK293T cells containing only endogenous U2AF65, the PY splice site is used and the remaining transcript remains unspliced. RESULTS +75 89 PY splice site site When transfected into HEK293T cells containing only endogenous U2AF65, the PY splice site is used and the remaining transcript remains unspliced. RESULTS +5 19 co-transfected experimental_method When co-transfected with an expression plasmid for wild-type U2AF65, use of the py splice site significantly increases (by more than five-fold) and as documented converts a fraction of the unspliced to spliced transcript. RESULTS +28 46 expression plasmid experimental_method When co-transfected with an expression plasmid for wild-type U2AF65, use of the py splice site significantly increases (by more than five-fold) and as documented converts a fraction of the unspliced to spliced transcript. RESULTS +51 60 wild-type protein_state When co-transfected with an expression plasmid for wild-type U2AF65, use of the py splice site significantly increases (by more than five-fold) and as documented converts a fraction of the unspliced to spliced transcript. RESULTS +61 67 U2AF65 protein When co-transfected with an expression plasmid for wild-type U2AF65, use of the py splice site significantly increases (by more than five-fold) and as documented converts a fraction of the unspliced to spliced transcript. RESULTS +80 94 py splice site site When co-transfected with an expression plasmid for wild-type U2AF65, use of the py splice site significantly increases (by more than five-fold) and as documented converts a fraction of the unspliced to spliced transcript. RESULTS +11 25 PY splice site site The strong PY splice site is insensitive to added U2AF65, suggesting that endogenous U2AF65 levels are sufficient to saturate this site (Supplementary Fig. 5b). RESULTS +50 56 U2AF65 protein The strong PY splice site is insensitive to added U2AF65, suggesting that endogenous U2AF65 levels are sufficient to saturate this site (Supplementary Fig. 5b). RESULTS +74 84 endogenous protein_state The strong PY splice site is insensitive to added U2AF65, suggesting that endogenous U2AF65 levels are sufficient to saturate this site (Supplementary Fig. 5b). RESULTS +85 91 U2AF65 protein The strong PY splice site is insensitive to added U2AF65, suggesting that endogenous U2AF65 levels are sufficient to saturate this site (Supplementary Fig. 5b). RESULTS +18 33 triple mutation experimental_method We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). RESULTS +35 40 V254P mutant We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). RESULTS +41 46 R227A mutant We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). RESULTS +47 52 Q147A mutant We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). RESULTS +81 91 U2AF651,2L mutant We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). RESULTS +113 121 Py tract chemical We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). RESULTS +150 161 full-length protein_state We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). RESULTS +162 168 U2AF65 protein We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). RESULTS +170 181 U2AF65-3Mut mutant We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). RESULTS +0 15 Co-transfection experimental_method Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak ‘py' splice site relative to wild-type U2AF65 (Fig. 5b,c). RESULTS +23 34 U2AF65-3Mut mutant Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak ‘py' splice site relative to wild-type U2AF65 (Fig. 5b,c). RESULTS +44 48 pyPY chemical Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak ‘py' splice site relative to wild-type U2AF65 (Fig. 5b,c). RESULTS +111 127 ‘py' splice site site Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak ‘py' splice site relative to wild-type U2AF65 (Fig. 5b,c). RESULTS +140 149 wild-type protein_state Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak ‘py' splice site relative to wild-type U2AF65 (Fig. 5b,c). RESULTS +150 156 U2AF65 protein Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak ‘py' splice site relative to wild-type U2AF65 (Fig. 5b,c). RESULTS +21 29 Py-tract chemical We conclude that the Py-tract interactions with these residues of the U2AF65 inter-RRM linker and RRM extensions are important for splicing as well as for binding a representative of the major U2-class of splice sites. RESULTS +70 76 U2AF65 protein We conclude that the Py-tract interactions with these residues of the U2AF65 inter-RRM linker and RRM extensions are important for splicing as well as for binding a representative of the major U2-class of splice sites. RESULTS +77 93 inter-RRM linker structure_element We conclude that the Py-tract interactions with these residues of the U2AF65 inter-RRM linker and RRM extensions are important for splicing as well as for binding a representative of the major U2-class of splice sites. RESULTS +98 112 RRM extensions structure_element We conclude that the Py-tract interactions with these residues of the U2AF65 inter-RRM linker and RRM extensions are important for splicing as well as for binding a representative of the major U2-class of splice sites. RESULTS +187 217 major U2-class of splice sites structure_element We conclude that the Py-tract interactions with these residues of the U2AF65 inter-RRM linker and RRM extensions are important for splicing as well as for binding a representative of the major U2-class of splice sites. RESULTS +7 16 inter-RRM structure_element Sparse inter-RRM contacts underlie apo-U2AF65 dynamics RESULTS +35 38 apo protein_state Sparse inter-RRM contacts underlie apo-U2AF65 dynamics RESULTS +39 45 U2AF65 protein Sparse inter-RRM contacts underlie apo-U2AF65 dynamics RESULTS +11 20 interface site The direct interface between U2AF651,2L RRM1 and RRM2 is minor, burying 265 Å2 of solvent accessible surface area compared with 570 Å2 on average for a crystal packing interface. RESULTS +29 39 U2AF651,2L mutant The direct interface between U2AF651,2L RRM1 and RRM2 is minor, burying 265 Å2 of solvent accessible surface area compared with 570 Å2 on average for a crystal packing interface. RESULTS +40 44 RRM1 structure_element The direct interface between U2AF651,2L RRM1 and RRM2 is minor, burying 265 Å2 of solvent accessible surface area compared with 570 Å2 on average for a crystal packing interface. RESULTS +49 53 RRM2 structure_element The direct interface between U2AF651,2L RRM1 and RRM2 is minor, burying 265 Å2 of solvent accessible surface area compared with 570 Å2 on average for a crystal packing interface. RESULTS +13 22 inter-RRM structure_element A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +23 37 hydrogen bonds bond_interaction A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +78 82 RRM1 structure_element A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +83 87 N155 residue_name_number A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +92 96 RRM2 structure_element A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +97 101 K292 residue_name_number A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +103 107 RRM1 structure_element A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +108 112 N155 residue_name_number A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +117 121 RRM2 structure_element A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +122 126 D272 residue_name_number A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +160 164 RRM1 structure_element A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +165 169 G221 residue_name_number A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +174 178 RRM2 structure_element A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +179 183 D273 residue_name_number A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). RESULTS +11 17 U2AF65 protein This minor U2AF65 RRM1/RRM2 interface, coupled with the versatile sequence of the inter-RRM linker, highlighted the potential role for inter-RRM conformational dynamics in U2AF65-splice site recognition. RESULTS +18 37 RRM1/RRM2 interface site This minor U2AF65 RRM1/RRM2 interface, coupled with the versatile sequence of the inter-RRM linker, highlighted the potential role for inter-RRM conformational dynamics in U2AF65-splice site recognition. RESULTS +82 98 inter-RRM linker structure_element This minor U2AF65 RRM1/RRM2 interface, coupled with the versatile sequence of the inter-RRM linker, highlighted the potential role for inter-RRM conformational dynamics in U2AF65-splice site recognition. RESULTS +135 144 inter-RRM structure_element This minor U2AF65 RRM1/RRM2 interface, coupled with the versatile sequence of the inter-RRM linker, highlighted the potential role for inter-RRM conformational dynamics in U2AF65-splice site recognition. RESULTS +172 178 U2AF65 protein This minor U2AF65 RRM1/RRM2 interface, coupled with the versatile sequence of the inter-RRM linker, highlighted the potential role for inter-RRM conformational dynamics in U2AF65-splice site recognition. RESULTS +0 34 Paramagnetic resonance enhancement experimental_method Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. RESULTS +36 39 PRE experimental_method Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. RESULTS +93 105 back-to-back protein_state Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. RESULTS +111 117 closed protein_state Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. RESULTS +139 142 apo protein_state Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. RESULTS +143 152 U2AF651,2 mutant Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. RESULTS +153 157 RRM1 structure_element Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. RESULTS +162 166 RRM2 structure_element Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. RESULTS +196 200 open protein_state Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. RESULTS +230 239 RNA-bound protein_state Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. RESULTS +240 249 inter-RRM structure_element Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. RESULTS +5 33 small-angle X-ray scattering experimental_method Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. RESULTS +35 39 SAXS experimental_method Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. RESULTS +70 77 minimal protein_state Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. RESULTS +78 87 U2AF651,2 mutant Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. RESULTS +121 162 highly diverse continuum of conformations protein_state Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. RESULTS +170 180 absence of protein_state Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. RESULTS +181 184 RNA chemical Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. RESULTS +204 210 closed protein_state Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. RESULTS +217 221 open protein_state Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. RESULTS +38 48 U2AF651,2L mutant To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution. RESULTS +49 58 structure evidence To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution. RESULTS +85 106 X-ray crystallography experimental_method To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution. RESULTS +116 122 smFRET experimental_method To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution. RESULTS +143 177 probability distribution functions evidence To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution. RESULTS +201 207 U2AF65 protein To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution. RESULTS +208 217 inter-RRM structure_element To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution. RESULTS +4 13 inter-RRM structure_element The inter-RRM dynamics of U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2 (Fig. 6a,b, Methods). RESULTS +26 32 U2AF65 protein The inter-RRM dynamics of U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2 (Fig. 6a,b, Methods). RESULTS +53 57 FRET experimental_method The inter-RRM dynamics of U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2 (Fig. 6a,b, Methods). RESULTS +66 78 fluorophores chemical The inter-RRM dynamics of U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2 (Fig. 6a,b, Methods). RESULTS +91 95 RRM1 structure_element The inter-RRM dynamics of U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2 (Fig. 6a,b, Methods). RESULTS +100 104 RRM2 structure_element The inter-RRM dynamics of U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2 (Fig. 6a,b, Methods). RESULTS +24 32 cysteine residue_name The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. RESULTS +33 42 mutations experimental_method The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. RESULTS +47 58 fluorophore chemical The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. RESULTS +71 76 A181C mutant The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. RESULTS +80 84 RRM1 structure_element The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. RESULTS +89 94 Q324C mutant The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. RESULTS +98 102 RRM2 structure_element The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. RESULTS +143 153 U2AF651,2L mutant The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. RESULTS +154 164 structures evidence The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. RESULTS +174 180 closed protein_state The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. RESULTS +191 194 apo protein_state The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. RESULTS +195 204 U2AF651,2 mutant The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. RESULTS +107 114 RRM/RNA complex_assembly Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +118 138 inter-RRM interfaces site Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +175 194 U2AF651,2L–Py tract complex_assembly Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +212 218 closed protein_state Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +219 222 apo protein_state Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +263 282 Förster radius (Ro) experimental_method Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +291 294 Cy3 chemical Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +295 298 Cy5 chemical Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +405 422 FRET efficiencies evidence Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +480 486 closed protein_state Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +488 491 apo protein_state Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +517 521 open protein_state Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +523 532 RNA-bound protein_state Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +533 543 structures evidence Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). RESULTS +4 21 FRET efficiencies evidence The FRET efficiencies of either of these structurally characterized conformations also are expected to be significantly greater than elongated U2AF65 conformations that lack inter-RRM contacts. RESULTS +133 142 elongated protein_state The FRET efficiencies of either of these structurally characterized conformations also are expected to be significantly greater than elongated U2AF65 conformations that lack inter-RRM contacts. RESULTS +143 149 U2AF65 protein The FRET efficiencies of either of these structurally characterized conformations also are expected to be significantly greater than elongated U2AF65 conformations that lack inter-RRM contacts. RESULTS +169 173 lack protein_state The FRET efficiencies of either of these structurally characterized conformations also are expected to be significantly greater than elongated U2AF65 conformations that lack inter-RRM contacts. RESULTS +180 183 RRM structure_element The FRET efficiencies of either of these structurally characterized conformations also are expected to be significantly greater than elongated U2AF65 conformations that lack inter-RRM contacts. RESULTS +7 15 cysteine residue_name Double-cysteine variant of U2AF651,2 was modified with equimolar amount of Cy3 and Cy5. RESULTS +16 23 variant protein_state Double-cysteine variant of U2AF651,2 was modified with equimolar amount of Cy3 and Cy5. RESULTS +27 36 U2AF651,2 mutant Double-cysteine variant of U2AF651,2 was modified with equimolar amount of Cy3 and Cy5. RESULTS +41 49 modified experimental_method Double-cysteine variant of U2AF651,2 was modified with equimolar amount of Cy3 and Cy5. RESULTS +75 78 Cy3 chemical Double-cysteine variant of U2AF651,2 was modified with equimolar amount of Cy3 and Cy5. RESULTS +83 86 Cy5 chemical Double-cysteine variant of U2AF651,2 was modified with equimolar amount of Cy3 and Cy5. RESULTS +5 11 traces evidence Only traces that showed single photobleaching events for both donor and acceptor dyes and anti-correlated changes in acceptor and donor fluorescence were included in smFRET data analysis. RESULTS +166 172 smFRET experimental_method Only traces that showed single photobleaching events for both donor and acceptor dyes and anti-correlated changes in acceptor and donor fluorescence were included in smFRET data analysis. RESULTS +63 69 U2AF65 protein We first characterized the conformational dynamics spectrum of U2AF65 in the absence of RNA (Fig. 6c,d; Supplementary Fig. 7a,b). RESULTS +77 87 absence of protein_state We first characterized the conformational dynamics spectrum of U2AF65 in the absence of RNA (Fig. 6c,d; Supplementary Fig. 7a,b). RESULTS +88 91 RNA chemical We first characterized the conformational dynamics spectrum of U2AF65 in the absence of RNA (Fig. 6c,d; Supplementary Fig. 7a,b). RESULTS +20 34 U2AF651,2LFRET mutant The double-labelled U2AF651,2LFRET(Cy3/Cy5) protein was tethered to a slide via biotin-NTA/Ni+2 resin. RESULTS +35 38 Cy3 chemical The double-labelled U2AF651,2LFRET(Cy3/Cy5) protein was tethered to a slide via biotin-NTA/Ni+2 resin. RESULTS +39 42 Cy5 chemical The double-labelled U2AF651,2LFRET(Cy3/Cy5) protein was tethered to a slide via biotin-NTA/Ni+2 resin. RESULTS +56 64 tethered protein_state The double-labelled U2AF651,2LFRET(Cy3/Cy5) protein was tethered to a slide via biotin-NTA/Ni+2 resin. RESULTS +80 101 biotin-NTA/Ni+2 resin chemical The double-labelled U2AF651,2LFRET(Cy3/Cy5) protein was tethered to a slide via biotin-NTA/Ni+2 resin. RESULTS +56 66 absence of protein_state Virtually no fluorescent molecules were detected in the absence of biotin-NTA/Ni+2, which demonstrates the absence of detectable non-specific binding of U2AF651,2LFRET to the slide. RESULTS +67 82 biotin-NTA/Ni+2 chemical Virtually no fluorescent molecules were detected in the absence of biotin-NTA/Ni+2, which demonstrates the absence of detectable non-specific binding of U2AF651,2LFRET to the slide. RESULTS +107 117 absence of protein_state Virtually no fluorescent molecules were detected in the absence of biotin-NTA/Ni+2, which demonstrates the absence of detectable non-specific binding of U2AF651,2LFRET to the slide. RESULTS +153 167 U2AF651,2LFRET mutant Virtually no fluorescent molecules were detected in the absence of biotin-NTA/Ni+2, which demonstrates the absence of detectable non-specific binding of U2AF651,2LFRET to the slide. RESULTS +4 31 FRET distribution histogram evidence The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4 (Fig. 6d). RESULTS +64 70 traces evidence The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4 (Fig. 6d). RESULTS +74 88 U2AF651,2LFRET mutant The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4 (Fig. 6d). RESULTS +89 92 Cy3 chemical The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4 (Fig. 6d). RESULTS +93 96 Cy5 chemical The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4 (Fig. 6d). RESULTS +105 115 absence of protein_state The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4 (Fig. 6d). RESULTS +116 122 ligand chemical The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4 (Fig. 6d). RESULTS +175 190 FRET efficiency evidence The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4 (Fig. 6d). RESULTS +25 31 smFRET experimental_method Approximately 40% of the smFRET traces showed apparent transitions between multiple FRET values (for example, Fig. 6c). RESULTS +32 38 traces evidence Approximately 40% of the smFRET traces showed apparent transitions between multiple FRET values (for example, Fig. 6c). RESULTS +84 95 FRET values evidence Approximately 40% of the smFRET traces showed apparent transitions between multiple FRET values (for example, Fig. 6c). RESULTS +31 58 FRET-distribution histogram evidence Despite the large width of the FRET-distribution histogram, the majority (80%) of traces that showed fluctuations sampled only two distinct FRET states (for example, Supplementary Fig. 7a). RESULTS +82 88 traces evidence Despite the large width of the FRET-distribution histogram, the majority (80%) of traces that showed fluctuations sampled only two distinct FRET states (for example, Supplementary Fig. 7a). RESULTS +140 151 FRET states evidence Despite the large width of the FRET-distribution histogram, the majority (80%) of traces that showed fluctuations sampled only two distinct FRET states (for example, Supplementary Fig. 7a). RESULTS +89 100 FRET values evidence Approximately 70% of observed fluctuations were interchanges between the ∼0.65 and ∼0.45 FRET values (Supplementary Fig. 7b). RESULTS +50 64 U2AF651,2LFRET mutant We cannot exclude a possibility that tethering of U2AF651,2LFRET(Cy3/Cy5) to the microscope slide introduces structural heterogeneity into the protein and, thus, contributes to the breadth of the FRET distribution histogram. RESULTS +65 68 Cy3 chemical We cannot exclude a possibility that tethering of U2AF651,2LFRET(Cy3/Cy5) to the microscope slide introduces structural heterogeneity into the protein and, thus, contributes to the breadth of the FRET distribution histogram. RESULTS +69 72 Cy5 chemical We cannot exclude a possibility that tethering of U2AF651,2LFRET(Cy3/Cy5) to the microscope slide introduces structural heterogeneity into the protein and, thus, contributes to the breadth of the FRET distribution histogram. RESULTS +196 223 FRET distribution histogram evidence We cannot exclude a possibility that tethering of U2AF651,2LFRET(Cy3/Cy5) to the microscope slide introduces structural heterogeneity into the protein and, thus, contributes to the breadth of the FRET distribution histogram. RESULTS +68 79 FRET values evidence However, the presence of repetitive fluctuations between particular FRET values supports the hypothesis that RNA-free U2AF65 samples several distinct conformations. RESULTS +109 117 RNA-free protein_state However, the presence of repetitive fluctuations between particular FRET values supports the hypothesis that RNA-free U2AF65 samples several distinct conformations. RESULTS +118 124 U2AF65 protein However, the presence of repetitive fluctuations between particular FRET values supports the hypothesis that RNA-free U2AF65 samples several distinct conformations. RESULTS +54 62 extended protein_state This result is consistent with the broad ensembles of extended solution conformations that best fit the SAXS data collected for U2AF651,2 as well as for a longer construct (residues 136–347). RESULTS +104 108 SAXS experimental_method This result is consistent with the broad ensembles of extended solution conformations that best fit the SAXS data collected for U2AF651,2 as well as for a longer construct (residues 136–347). RESULTS +128 137 U2AF651,2 mutant This result is consistent with the broad ensembles of extended solution conformations that best fit the SAXS data collected for U2AF651,2 as well as for a longer construct (residues 136–347). RESULTS +182 189 136–347 residue_range This result is consistent with the broad ensembles of extended solution conformations that best fit the SAXS data collected for U2AF651,2 as well as for a longer construct (residues 136–347). RESULTS +43 49 U2AF65 protein We conclude that weak contacts between the U2AF65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA. RESULTS +50 54 RRM1 structure_element We conclude that weak contacts between the U2AF65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA. RESULTS +59 63 RRM2 structure_element We conclude that weak contacts between the U2AF65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA. RESULTS +93 97 RRMs structure_element We conclude that weak contacts between the U2AF65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA. RESULTS +105 115 absence of protein_state We conclude that weak contacts between the U2AF65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA. RESULTS +116 119 RNA chemical We conclude that weak contacts between the U2AF65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA. RESULTS +0 6 U2AF65 protein U2AF65 conformational selection and induced fit by bound RNA RESULTS +51 56 bound protein_state U2AF65 conformational selection and induced fit by bound RNA RESULTS +57 60 RNA chemical U2AF65 conformational selection and induced fit by bound RNA RESULTS +13 19 smFRET experimental_method We next used smFRET to probe the conformational selection of distinct inter-RRM arrangements following association of U2AF65 with the AdML Py-tract prototype. RESULTS +70 79 inter-RRM structure_element We next used smFRET to probe the conformational selection of distinct inter-RRM arrangements following association of U2AF65 with the AdML Py-tract prototype. RESULTS +118 124 U2AF65 protein We next used smFRET to probe the conformational selection of distinct inter-RRM arrangements following association of U2AF65 with the AdML Py-tract prototype. RESULTS +134 138 AdML gene We next used smFRET to probe the conformational selection of distinct inter-RRM arrangements following association of U2AF65 with the AdML Py-tract prototype. RESULTS +139 147 Py-tract chemical We next used smFRET to probe the conformational selection of distinct inter-RRM arrangements following association of U2AF65 with the AdML Py-tract prototype. RESULTS +16 20 AdML gene Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f). RESULTS +21 24 RNA chemical Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f). RESULTS +28 36 tethered protein_state Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f). RESULTS +37 51 U2AF651,2LFRET mutant Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f). RESULTS +52 55 Cy3 chemical Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f). RESULTS +56 59 Cy5 chemical Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f). RESULTS +133 148 FRET efficiency evidence Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f). RESULTS +242 252 FRET state evidence Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f). RESULTS +40 48 RNA-free protein_state To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. RESULTS +66 72 U2AF65 protein To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. RESULTS +119 128 tethering experimental_method To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. RESULTS +132 146 U2AF651,2LFRET mutant To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. RESULTS +147 150 Cy3 chemical To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. RESULTS +151 154 Cy5 chemical To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. RESULTS +185 212 distribution of FRET values evidence To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. RESULTS +217 251 reversed the immobilization scheme experimental_method To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. RESULTS +3 11 tethered protein_state We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). RESULTS +16 20 AdML gene We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). RESULTS +21 24 RNA chemical We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). RESULTS +44 76 biotinylated oligonucleotide DNA chemical We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). RESULTS +88 93 added experimental_method We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). RESULTS +94 108 U2AF651,2LFRET mutant We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). RESULTS +109 112 Cy3 chemical We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). RESULTS +113 116 Cy5 chemical We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). RESULTS +125 135 absence of protein_state We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). RESULTS +136 152 biotin-NTA resin chemical We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). RESULTS +7 17 FRET value evidence A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5). RESULTS +62 71 RNA-bound protein_state A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5). RESULTS +117 127 untethered protein_state A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5). RESULTS +132 140 tethered protein_state A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5). RESULTS +141 155 U2AF651,2LFRET mutant A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5). RESULTS +156 159 Cy3 chemical A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5). RESULTS +160 163 Cy5 chemical A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5). RESULTS +26 36 U2AF651,2L mutant We examined the effect on U2AF651,2L conformations of purine interruptions that often occur in relatively degenerate human Py tracts. RESULTS +54 74 purine interruptions experimental_method We examined the effect on U2AF651,2L conformations of purine interruptions that often occur in relatively degenerate human Py tracts. RESULTS +117 122 human species We examined the effect on U2AF651,2L conformations of purine interruptions that often occur in relatively degenerate human Py tracts. RESULTS +123 132 Py tracts chemical We examined the effect on U2AF651,2L conformations of purine interruptions that often occur in relatively degenerate human Py tracts. RESULTS +3 13 introduced experimental_method We introduced an rArA purine dinucleotide within a variant of the AdML Py tract (detailed in Methods). RESULTS +17 21 rArA chemical We introduced an rArA purine dinucleotide within a variant of the AdML Py tract (detailed in Methods). RESULTS +22 41 purine dinucleotide chemical We introduced an rArA purine dinucleotide within a variant of the AdML Py tract (detailed in Methods). RESULTS +66 70 AdML gene We introduced an rArA purine dinucleotide within a variant of the AdML Py tract (detailed in Methods). RESULTS +71 79 Py tract chemical We introduced an rArA purine dinucleotide within a variant of the AdML Py tract (detailed in Methods). RESULTS +0 9 Insertion experimental_method Insertion of adenine nucleotides decreased binding affinity of U2AF65 to RNA by approximately five-fold. RESULTS +13 32 adenine nucleotides chemical Insertion of adenine nucleotides decreased binding affinity of U2AF65 to RNA by approximately five-fold. RESULTS +43 59 binding affinity evidence Insertion of adenine nucleotides decreased binding affinity of U2AF65 to RNA by approximately five-fold. RESULTS +63 69 U2AF65 protein Insertion of adenine nucleotides decreased binding affinity of U2AF65 to RNA by approximately five-fold. RESULTS +73 76 RNA chemical Insertion of adenine nucleotides decreased binding affinity of U2AF65 to RNA by approximately five-fold. RESULTS +62 66 rArA chemical Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent ∼0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract. RESULTS +79 82 RNA chemical Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent ∼0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract. RESULTS +83 97 slide-tethered protein_state Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent ∼0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract. RESULTS +98 112 U2AF651,2LFRET mutant Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent ∼0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract. RESULTS +113 116 Cy3 chemical Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent ∼0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract. RESULTS +117 120 Cy5 chemical Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent ∼0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract. RESULTS +156 166 FRET value evidence Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent ∼0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract. RESULTS +237 245 Py tract chemical Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent ∼0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract. RESULTS +11 15 RRM1 structure_element Therefore, RRM1-to-RRM2 distance remains similar regardless of whether U2AF65 is bound to interrupted or continuous Py tract. RESULTS +19 23 RRM2 structure_element Therefore, RRM1-to-RRM2 distance remains similar regardless of whether U2AF65 is bound to interrupted or continuous Py tract. RESULTS +71 77 U2AF65 protein Therefore, RRM1-to-RRM2 distance remains similar regardless of whether U2AF65 is bound to interrupted or continuous Py tract. RESULTS +81 89 bound to protein_state Therefore, RRM1-to-RRM2 distance remains similar regardless of whether U2AF65 is bound to interrupted or continuous Py tract. RESULTS +116 124 Py tract chemical Therefore, RRM1-to-RRM2 distance remains similar regardless of whether U2AF65 is bound to interrupted or continuous Py tract. RESULTS +4 31 inter-fluorophore distances evidence The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the α-carbon atoms of the respective residues in the crystal structures of U2AF651,2L bound to Py-tract oligonucleotides. RESULTS +63 73 FRET state evidence The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the α-carbon atoms of the respective residues in the crystal structures of U2AF651,2L bound to Py-tract oligonucleotides. RESULTS +160 178 crystal structures evidence The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the α-carbon atoms of the respective residues in the crystal structures of U2AF651,2L bound to Py-tract oligonucleotides. RESULTS +182 192 U2AF651,2L mutant The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the α-carbon atoms of the respective residues in the crystal structures of U2AF651,2L bound to Py-tract oligonucleotides. RESULTS +193 201 bound to protein_state The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the α-carbon atoms of the respective residues in the crystal structures of U2AF651,2L bound to Py-tract oligonucleotides. RESULTS +202 227 Py-tract oligonucleotides chemical The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the α-carbon atoms of the respective residues in the crystal structures of U2AF651,2L bound to Py-tract oligonucleotides. RESULTS +49 60 FRET values evidence It should be noted that inferring distances from FRET values is prone to significant error because of uncertainties in the determination of fluorophore orientation factor κ2 and Förster radius R0, the parameters used in distance calculations. RESULTS +35 45 FRET state evidence Nevertheless, the predominant 0.45 FRET state in the presence of RNA agrees with the Py-tract-bound crystal structure of U2AF651,2L. RESULTS +65 68 RNA chemical Nevertheless, the predominant 0.45 FRET state in the presence of RNA agrees with the Py-tract-bound crystal structure of U2AF651,2L. RESULTS +85 99 Py-tract-bound protein_state Nevertheless, the predominant 0.45 FRET state in the presence of RNA agrees with the Py-tract-bound crystal structure of U2AF651,2L. RESULTS +100 117 crystal structure evidence Nevertheless, the predominant 0.45 FRET state in the presence of RNA agrees with the Py-tract-bound crystal structure of U2AF651,2L. RESULTS +121 131 U2AF651,2L mutant Nevertheless, the predominant 0.45 FRET state in the presence of RNA agrees with the Py-tract-bound crystal structure of U2AF651,2L. RESULTS +29 35 traces evidence Importantly, the majority of traces (∼70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a ∼0.45 FRET value (for example, Fig. 6g). RESULTS +46 60 U2AF651,2LFRET mutant Importantly, the majority of traces (∼70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a ∼0.45 FRET value (for example, Fig. 6g). RESULTS +61 64 Cy3 chemical Importantly, the majority of traces (∼70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a ∼0.45 FRET value (for example, Fig. 6g). RESULTS +65 68 Cy5 chemical Importantly, the majority of traces (∼70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a ∼0.45 FRET value (for example, Fig. 6g). RESULTS +70 78 bound to protein_state Importantly, the majority of traces (∼70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a ∼0.45 FRET value (for example, Fig. 6g). RESULTS +98 101 RNA chemical Importantly, the majority of traces (∼70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a ∼0.45 FRET value (for example, Fig. 6g). RESULTS +163 173 FRET value evidence Importantly, the majority of traces (∼70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a ∼0.45 FRET value (for example, Fig. 6g). RESULTS +22 28 traces evidence The remaining ∼30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values. RESULTS +33 47 U2AF651,2LFRET mutant The remaining ∼30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values. RESULTS +48 51 Cy3 chemical The remaining ∼30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values. RESULTS +52 55 Cy5 chemical The remaining ∼30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values. RESULTS +57 65 bound to protein_state The remaining ∼30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values. RESULTS +85 88 RNA chemical The remaining ∼30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values. RESULTS +126 137 FRET values evidence The remaining ∼30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values. RESULTS +16 22 traces evidence The majority of traces that show fluctuations began at high (0.65–0.8) FRET value and transitioned to a ∼0.45 FRET value (Supplementary Fig. 7c–g). RESULTS +71 81 FRET value evidence The majority of traces that show fluctuations began at high (0.65–0.8) FRET value and transitioned to a ∼0.45 FRET value (Supplementary Fig. 7c–g). RESULTS +110 120 FRET value evidence The majority of traces that show fluctuations began at high (0.65–0.8) FRET value and transitioned to a ∼0.45 FRET value (Supplementary Fig. 7c–g). RESULTS +0 32 Hidden Markov modelling analysis experimental_method Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to ∼0.7–0.8 and ∼0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state. RESULTS +36 42 smFRET experimental_method Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to ∼0.7–0.8 and ∼0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state. RESULTS +43 49 traces evidence Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to ∼0.7–0.8 and ∼0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state. RESULTS +64 73 RNA-bound protein_state Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to ∼0.7–0.8 and ∼0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state. RESULTS +74 84 U2AF651,2L mutant Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to ∼0.7–0.8 and ∼0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state. RESULTS +164 175 FRET values evidence Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to ∼0.7–0.8 and ∼0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state. RESULTS +246 256 FRET state evidence Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to ∼0.7–0.8 and ∼0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state. RESULTS +63 73 U2AF651,2L mutant Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. RESULTS +100 111 FRET values evidence Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. RESULTS +121 124 RNA chemical Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. RESULTS +129 132 RNA chemical Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. RESULTS +148 155 compact protein_state Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. RESULTS +173 183 U2AF651,2L mutant Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. RESULTS +257 267 FRET value evidence Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. RESULTS +297 309 side-by-side protein_state Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. RESULTS +310 319 inter-RRM structure_element Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. RESULTS +339 349 U2AF651,2L mutant Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. RESULTS +350 368 crystal structures evidence Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. RESULTS +51 57 U2AF65 protein Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below. RESULTS +70 76 smFRET experimental_method Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below. RESULTS +77 83 traces evidence Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below. RESULTS +166 174 Py-tract chemical Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below. RESULTS +227 241 pre-configured protein_state Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below. RESULTS +242 248 U2AF65 protein Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below. RESULTS +339 345 U2AF65 protein Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below. RESULTS +346 350 RRMs structure_element Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below. RESULTS +373 385 side-by-side protein_state Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below. RESULTS +4 10 U2AF65 protein The U2AF65 structures and analyses presented here represent a successful step towards defining a molecular map of the 3′ splice site. DISCUSS +11 21 structures evidence The U2AF65 structures and analyses presented here represent a successful step towards defining a molecular map of the 3′ splice site. DISCUSS +26 34 analyses evidence The U2AF65 structures and analyses presented here represent a successful step towards defining a molecular map of the 3′ splice site. DISCUSS +118 132 3′ splice site site The U2AF65 structures and analyses presented here represent a successful step towards defining a molecular map of the 3′ splice site. DISCUSS +97 113 inter-RRM linker structure_element Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65–Py-tract recognition. DISCUSS +118 122 RRM1 structure_element Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65–Py-tract recognition. DISCUSS +124 128 RRM2 structure_element Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65–Py-tract recognition. DISCUSS +149 163 RRM1 extension structure_element Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65–Py-tract recognition. DISCUSS +192 198 U2AF65 protein Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65–Py-tract recognition. DISCUSS +199 207 Py-tract chemical Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65–Py-tract recognition. DISCUSS +0 10 Truncation experimental_method Truncation of U2AF65 to the core RRM1–RRM2 region reduces its RNA affinity by 100-fold. DISCUSS +14 20 U2AF65 protein Truncation of U2AF65 to the core RRM1–RRM2 region reduces its RNA affinity by 100-fold. DISCUSS +28 32 core protein_state Truncation of U2AF65 to the core RRM1–RRM2 region reduces its RNA affinity by 100-fold. DISCUSS +33 49 RRM1–RRM2 region structure_element Truncation of U2AF65 to the core RRM1–RRM2 region reduces its RNA affinity by 100-fold. DISCUSS +62 74 RNA affinity evidence Truncation of U2AF65 to the core RRM1–RRM2 region reduces its RNA affinity by 100-fold. DISCUSS +10 18 deletion experimental_method Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. DISCUSS +22 24 20 residue_range Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. DISCUSS +25 50 inter-RRM linker residues structure_element Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. DISCUSS +73 79 U2AF65 protein Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. DISCUSS +80 83 RNA chemical Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. DISCUSS +135 141 longer protein_state Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. DISCUSS +142 152 U2AF651,2L mutant Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. DISCUSS +178 192 RRM extensions structure_element Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. DISCUSS +221 227 linker structure_element Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. DISCUSS +232 235 RNA chemical Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. DISCUSS +11 26 triple mutation protein_state Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold. DISCUSS +46 51 V254P mutant Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold. DISCUSS +53 58 Q147A mutant Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold. DISCUSS +63 68 R227A mutant Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold. DISCUSS +88 104 inter-RRM linker structure_element Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold. DISCUSS +106 134 N- and C-terminal extensions structure_element Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold. DISCUSS +157 168 RNA binding evidence Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold. DISCUSS +60 66 U2AF65 protein Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition. DISCUSS +67 71 RRM1 structure_element Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition. DISCUSS +72 76 RRM2 structure_element Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition. DISCUSS +78 94 inter-RRM linker structure_element Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition. DISCUSS +96 123 N-and C-terminal extensions structure_element Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition. DISCUSS +165 173 Py-tract chemical Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition. DISCUSS +37 43 U2AF65 protein The implications of this finding for U2AF65 conservation and Py-tract recognition are detailed in the Supplementary Discussion. DISCUSS +61 69 Py-tract chemical The implications of this finding for U2AF65 conservation and Py-tract recognition are detailed in the Supplementary Discussion. DISCUSS +10 44 high-throughput sequencing studies experimental_method Recently, high-throughput sequencing studies have shown that somatic mutations in pre-mRNA splicing factors occur in the majority of patients with myelodysplastic syndrome (MDS). DISCUSS +82 107 pre-mRNA splicing factors protein_type Recently, high-throughput sequencing studies have shown that somatic mutations in pre-mRNA splicing factors occur in the majority of patients with myelodysplastic syndrome (MDS). DISCUSS +41 46 small protein_state MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)—possibly due to the selective versus nearly universal requirements of these factors for splicing. DISCUSS +47 59 U2AF subunit protein_type MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)—possibly due to the selective versus nearly universal requirements of these factors for splicing. DISCUSS +61 67 U2AF35 protein MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)—possibly due to the selective versus nearly universal requirements of these factors for splicing. DISCUSS +72 77 U2AF1 protein MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)—possibly due to the selective versus nearly universal requirements of these factors for splicing. DISCUSS +115 120 large protein_state MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)—possibly due to the selective versus nearly universal requirements of these factors for splicing. DISCUSS +121 127 U2AF65 protein MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)—possibly due to the selective versus nearly universal requirements of these factors for splicing. DISCUSS +149 154 U2AF2 protein MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)—possibly due to the selective versus nearly universal requirements of these factors for splicing. DISCUSS +32 38 U2AF65 protein A confirmed somatic mutation of U2AF65 in patients with MDS, L187V, is located on a solvent-exposed surface of RRM1 that is distinct from the RNA interface (Fig. 7a). DISCUSS +61 66 L187V mutant A confirmed somatic mutation of U2AF65 in patients with MDS, L187V, is located on a solvent-exposed surface of RRM1 that is distinct from the RNA interface (Fig. 7a). DISCUSS +84 107 solvent-exposed surface site A confirmed somatic mutation of U2AF65 in patients with MDS, L187V, is located on a solvent-exposed surface of RRM1 that is distinct from the RNA interface (Fig. 7a). DISCUSS +111 115 RRM1 structure_element A confirmed somatic mutation of U2AF65 in patients with MDS, L187V, is located on a solvent-exposed surface of RRM1 that is distinct from the RNA interface (Fig. 7a). DISCUSS +142 155 RNA interface site A confirmed somatic mutation of U2AF65 in patients with MDS, L187V, is located on a solvent-exposed surface of RRM1 that is distinct from the RNA interface (Fig. 7a). DISCUSS +5 9 L187 residue_name_number This L187 surface is oriented towards the N terminus of the U2AF651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer. DISCUSS +60 70 U2AF651,2L mutant This L187 surface is oriented towards the N terminus of the U2AF651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer. DISCUSS +115 134 U2AF35-binding site site This L187 surface is oriented towards the N terminus of the U2AF651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer. DISCUSS +157 168 full-length protein_state This L187 surface is oriented towards the N terminus of the U2AF651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer. DISCUSS +169 173 U2AF protein This L187 surface is oriented towards the N terminus of the U2AF651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer. DISCUSS +174 185 heterodimer oligomeric_state This L187 surface is oriented towards the N terminus of the U2AF651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer. DISCUSS +25 30 M144I mutant Likewise, an unconfirmed M144I mutation reported by the same group corresponds to the N-terminal residue of U2AF651,2L, which is separated by only ∼20 residues from the U2AF35-binding site. DISCUSS +108 118 U2AF651,2L mutant Likewise, an unconfirmed M144I mutation reported by the same group corresponds to the N-terminal residue of U2AF651,2L, which is separated by only ∼20 residues from the U2AF35-binding site. DISCUSS +169 188 U2AF35-binding site site Likewise, an unconfirmed M144I mutation reported by the same group corresponds to the N-terminal residue of U2AF651,2L, which is separated by only ∼20 residues from the U2AF35-binding site. DISCUSS +42 48 U2AF65 protein As such, we suggest that the MDS-relevant U2AF65 mutations contribute to MDS progression indirectly, by destabilizing a relevant conformation of the conjoined U2AF35 subunit rather than affecting U2AF65 functions in RNA binding or spliceosome recruitment per se. DISCUSS +159 165 U2AF35 protein As such, we suggest that the MDS-relevant U2AF65 mutations contribute to MDS progression indirectly, by destabilizing a relevant conformation of the conjoined U2AF35 subunit rather than affecting U2AF65 functions in RNA binding or spliceosome recruitment per se. DISCUSS +196 202 U2AF65 protein As such, we suggest that the MDS-relevant U2AF65 mutations contribute to MDS progression indirectly, by destabilizing a relevant conformation of the conjoined U2AF35 subunit rather than affecting U2AF65 functions in RNA binding or spliceosome recruitment per se. DISCUSS +216 219 RNA chemical As such, we suggest that the MDS-relevant U2AF65 mutations contribute to MDS progression indirectly, by destabilizing a relevant conformation of the conjoined U2AF35 subunit rather than affecting U2AF65 functions in RNA binding or spliceosome recruitment per se. DISCUSS +231 242 spliceosome complex_assembly As such, we suggest that the MDS-relevant U2AF65 mutations contribute to MDS progression indirectly, by destabilizing a relevant conformation of the conjoined U2AF35 subunit rather than affecting U2AF65 functions in RNA binding or spliceosome recruitment per se. DISCUSS +4 10 smFRET experimental_method Our smFRET results agree with prior NMR/PRE evidence for multi-domain conformational selection as one mechanistic basis for U2AF65–RNA association (Fig. 7b). DISCUSS +36 39 NMR experimental_method Our smFRET results agree with prior NMR/PRE evidence for multi-domain conformational selection as one mechanistic basis for U2AF65–RNA association (Fig. 7b). DISCUSS +40 43 PRE experimental_method Our smFRET results agree with prior NMR/PRE evidence for multi-domain conformational selection as one mechanistic basis for U2AF65–RNA association (Fig. 7b). DISCUSS +124 130 U2AF65 protein Our smFRET results agree with prior NMR/PRE evidence for multi-domain conformational selection as one mechanistic basis for U2AF65–RNA association (Fig. 7b). DISCUSS +131 134 RNA chemical Our smFRET results agree with prior NMR/PRE evidence for multi-domain conformational selection as one mechanistic basis for U2AF65–RNA association (Fig. 7b). DISCUSS +9 19 FRET value evidence An ∼0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide. DISCUSS +51 57 U2AF65 protein An ∼0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide. DISCUSS +89 99 U2AF651,2L mutant An ∼0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide. DISCUSS +100 118 crystal structures evidence An ∼0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide. DISCUSS +133 137 RRM1 structure_element An ∼0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide. DISCUSS +142 146 RRM2 structure_element An ∼0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide. DISCUSS +152 164 side-by-side protein_state An ∼0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide. DISCUSS +172 196 Py-tract oligonucleotide chemical An ∼0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide. DISCUSS +32 43 FRET values evidence The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +51 61 untethered protein_state The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +62 76 U2AF651,2LFRET mutant The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +77 80 Cy3 chemical The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +81 84 Cy5 chemical The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +145 151 closed protein_state The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +154 166 back-to-back protein_state The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +167 173 U2AF65 protein The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +205 208 NMR experimental_method The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +209 212 PRE experimental_method The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +225 233 extended protein_state The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +234 240 U2AF65 protein The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +284 288 RRM1 structure_element The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +289 293 RRM2 structure_element The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +328 335 protein protein The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +339 347 bound to protein_state The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +348 351 RNA chemical The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +356 362 single protein_state The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +363 367 RRMs structure_element The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. DISCUSS +37 47 FRET value evidence An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value. DISCUSS +58 64 U2AF65 protein An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value. DISCUSS +65 68 RNA chemical An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value. DISCUSS +104 114 absence of protein_state An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value. DISCUSS +163 169 traces evidence An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value. DISCUSS +231 234 RNA chemical An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value. DISCUSS +282 296 pre-configured protein_state An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value. DISCUSS +297 306 inter-RRM structure_element An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value. DISCUSS +358 368 FRET value evidence An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value. DISCUSS +13 19 smFRET experimental_method Notably, our smFRET results reveal that U2AF65–Py-tract recognition can be characterized by an ‘extended conformational selection' model (Fig. 7b). DISCUSS +40 46 U2AF65 protein Notably, our smFRET results reveal that U2AF65–Py-tract recognition can be characterized by an ‘extended conformational selection' model (Fig. 7b). DISCUSS +47 55 Py-tract chemical Notably, our smFRET results reveal that U2AF65–Py-tract recognition can be characterized by an ‘extended conformational selection' model (Fig. 7b). DISCUSS +13 21 extended protein_state Examples of ‘extended conformational selection' during ligand binding have been characterized for a growing number of macromolecules (for example, adenylate kinase, LAO-binding protein, poly-ubiquitin, maltose-binding protein and the preQ1 riboswitch, among others). DISCUSS +147 163 adenylate kinase protein_type Examples of ‘extended conformational selection' during ligand binding have been characterized for a growing number of macromolecules (for example, adenylate kinase, LAO-binding protein, poly-ubiquitin, maltose-binding protein and the preQ1 riboswitch, among others). DISCUSS +165 184 LAO-binding protein protein_type Examples of ‘extended conformational selection' during ligand binding have been characterized for a growing number of macromolecules (for example, adenylate kinase, LAO-binding protein, poly-ubiquitin, maltose-binding protein and the preQ1 riboswitch, among others). DISCUSS +186 200 poly-ubiquitin protein_type Examples of ‘extended conformational selection' during ligand binding have been characterized for a growing number of macromolecules (for example, adenylate kinase, LAO-binding protein, poly-ubiquitin, maltose-binding protein and the preQ1 riboswitch, among others). DISCUSS +202 225 maltose-binding protein protein_type Examples of ‘extended conformational selection' during ligand binding have been characterized for a growing number of macromolecules (for example, adenylate kinase, LAO-binding protein, poly-ubiquitin, maltose-binding protein and the preQ1 riboswitch, among others). DISCUSS +234 250 preQ1 riboswitch protein_type Examples of ‘extended conformational selection' during ligand binding have been characterized for a growing number of macromolecules (for example, adenylate kinase, LAO-binding protein, poly-ubiquitin, maltose-binding protein and the preQ1 riboswitch, among others). DISCUSS +33 39 smFRET experimental_method Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). DISCUSS +40 46 traces evidence Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). DISCUSS +51 65 U2AF651,2LFRET mutant Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). DISCUSS +66 69 Cy3 chemical Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). DISCUSS +70 73 Cy5 chemical Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). DISCUSS +75 83 bound to protein_state Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). DISCUSS +99 102 RNA chemical Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). DISCUSS +128 138 FRET value evidence Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). DISCUSS +178 188 FRET value evidence Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). DISCUSS +62 68 closed protein_state These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. DISCUSS +70 73 NMR experimental_method These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. DISCUSS +74 77 PRE experimental_method These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. DISCUSS +84 90 U2AF65 protein These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. DISCUSS +117 136 RNA-binding surface site These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. DISCUSS +147 153 single protein_state These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. DISCUSS +154 157 RRM structure_element These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. DISCUSS +236 248 side-by-side protein_state These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. DISCUSS +250 259 RNA-bound protein_state These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. DISCUSS +260 277 crystal structure evidence These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. DISCUSS +13 19 smFRET experimental_method As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein. DISCUSS +116 119 NMR experimental_method As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein. DISCUSS +120 123 PRE experimental_method As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein. DISCUSS +168 177 inter-RRM structure_element As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein. DISCUSS +204 208 SAXS experimental_method As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein. DISCUSS +222 225 apo protein_state As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein. DISCUSS +226 233 protein protein As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein. DISCUSS +176 179 RRM structure_element Similar interdisciplinary structural approaches are likely to illuminate whether similar mechanistic bases for RNA binding are widespread among other members of the vast multi-RRM family. DISCUSS +17 23 U2AF65 protein The finding that U2AF65 recognizes a nine base pair Py tract contributes to an elusive ‘code' for predicting splicing patterns from primary sequences in the post-genomic era (reviewed in ref.). DISCUSS +52 60 Py tract chemical The finding that U2AF65 recognizes a nine base pair Py tract contributes to an elusive ‘code' for predicting splicing patterns from primary sequences in the post-genomic era (reviewed in ref.). DISCUSS +21 35 RNA affinities evidence Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +39 45 U2AF65 protein Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +50 60 U2AF651,2L mutant Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +110 120 U2AF651,2L mutant Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +121 131 structures evidence Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +188 197 penalties evidence Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +201 227 structure-guided mutations experimental_method Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +231 262 RNA binding and splicing assays experimental_method Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +284 292 extended protein_state Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +293 310 inter-RRM regions structure_element Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +318 328 U2AF651,2L mutant Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +329 339 structures evidence Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +357 365 Py-tract chemical Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +385 396 full-length protein_state Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +397 403 U2AF65 protein Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. DISCUSS +59 62 SF1 protein Further research will be needed to understand the roles of SF1 and U2AF35 subunits in the conformational equilibria underlying U2AF65 association with Py tracts. DISCUSS +67 73 U2AF35 protein Further research will be needed to understand the roles of SF1 and U2AF35 subunits in the conformational equilibria underlying U2AF65 association with Py tracts. DISCUSS +127 133 U2AF65 protein Further research will be needed to understand the roles of SF1 and U2AF35 subunits in the conformational equilibria underlying U2AF65 association with Py tracts. DISCUSS +151 160 Py tracts chemical Further research will be needed to understand the roles of SF1 and U2AF35 subunits in the conformational equilibria underlying U2AF65 association with Py tracts. DISCUSS +39 45 U2AF65 protein Moreover, structural differences among U2AF65 homologues and paralogues may regulate splice site selection. DISCUSS +85 96 splice site site Moreover, structural differences among U2AF65 homologues and paralogues may regulate splice site selection. DISCUSS +63 78 3′ splice sites site Ultimately, these guidelines will assist the identification of 3′ splice sites and the relationship of disease-causing mutations to penalties for U2AF65 association. DISCUSS +146 152 U2AF65 protein Ultimately, these guidelines will assist the identification of 3′ splice sites and the relationship of disease-causing mutations to penalties for U2AF65 association. DISCUSS +4 10 intact protein_state The intact U2AF65 RRM1/RRM2-containing domain and flanking residues are required for binding contiguous Py tracts. FIG +11 17 U2AF65 protein The intact U2AF65 RRM1/RRM2-containing domain and flanking residues are required for binding contiguous Py tracts. FIG +18 22 RRM1 structure_element The intact U2AF65 RRM1/RRM2-containing domain and flanking residues are required for binding contiguous Py tracts. FIG +23 27 RRM2 structure_element The intact U2AF65 RRM1/RRM2-containing domain and flanking residues are required for binding contiguous Py tracts. FIG +93 103 contiguous structure_element The intact U2AF65 RRM1/RRM2-containing domain and flanking residues are required for binding contiguous Py tracts. FIG +104 113 Py tracts chemical The intact U2AF65 RRM1/RRM2-containing domain and flanking residues are required for binding contiguous Py tracts. FIG +27 38 full-length protein_state (a) Domain organization of full-length (fl) U2AF65 and constructs used for RNA binding and structural experiments. FIG +40 42 fl protein_state (a) Domain organization of full-length (fl) U2AF65 and constructs used for RNA binding and structural experiments. FIG +44 50 U2AF65 protein (a) Domain organization of full-length (fl) U2AF65 and constructs used for RNA binding and structural experiments. FIG +75 78 RNA chemical (a) Domain organization of full-length (fl) U2AF65 and constructs used for RNA binding and structural experiments. FIG +22 23 d mutant An internal deletion (d, Δ) of residues 238–257 removes a portion of the inter-RRM linker from the dU2AF651,2 and dU2AF651,2L constructs. FIG +25 26 Δ mutant An internal deletion (d, Δ) of residues 238–257 removes a portion of the inter-RRM linker from the dU2AF651,2 and dU2AF651,2L constructs. FIG +40 47 238–257 residue_range An internal deletion (d, Δ) of residues 238–257 removes a portion of the inter-RRM linker from the dU2AF651,2 and dU2AF651,2L constructs. FIG +73 89 inter-RRM linker structure_element An internal deletion (d, Δ) of residues 238–257 removes a portion of the inter-RRM linker from the dU2AF651,2 and dU2AF651,2L constructs. FIG +99 109 dU2AF651,2 mutant An internal deletion (d, Δ) of residues 238–257 removes a portion of the inter-RRM linker from the dU2AF651,2 and dU2AF651,2L constructs. FIG +114 125 dU2AF651,2L mutant An internal deletion (d, Δ) of residues 238–257 removes a portion of the inter-RRM linker from the dU2AF651,2 and dU2AF651,2L constructs. FIG +31 53 equilibrium affinities evidence (b) Comparison of the apparent equilibrium affinities of various U2AF65 constructs for binding the prototypical AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +65 71 U2AF65 protein (b) Comparison of the apparent equilibrium affinities of various U2AF65 constructs for binding the prototypical AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +112 116 AdML gene (b) Comparison of the apparent equilibrium affinities of various U2AF65 constructs for binding the prototypical AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +117 125 Py tract chemical (b) Comparison of the apparent equilibrium affinities of various U2AF65 constructs for binding the prototypical AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +127 146 5′-CCCUUUUUUUUCC-3′ chemical (b) Comparison of the apparent equilibrium affinities of various U2AF65 constructs for binding the prototypical AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +4 12 flU2AF65 protein The flU2AF65 protein includes a heterodimerization domain of the U2AF35 subunit to promote solubility and folding. FIG +32 57 heterodimerization domain structure_element The flU2AF65 protein includes a heterodimerization domain of the U2AF35 subunit to promote solubility and folding. FIG +65 71 U2AF35 protein The flU2AF65 protein includes a heterodimerization domain of the U2AF35 subunit to promote solubility and folding. FIG +13 47 equilibrium dissociation constants evidence The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. FIG +49 51 KD evidence The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. FIG +69 73 AdML gene The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. FIG +96 104 flU2AF65 protein The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. FIG +115 125 U2AF651,2L mutant The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. FIG +136 145 U2AF651,2 mutant The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. FIG +183 209 RNA sequence specificities evidence The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. FIG +213 221 flU2AF65 protein The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. FIG +226 236 U2AF651,2L mutant The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. FIG +256 262 C-rich structure_element The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. FIG +263 272 Py tracts chemical The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. FIG +4 6 KD evidence The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. FIG +21 40 5′-CCUUUUCCCCCCC-3′ chemical The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. FIG +46 54 flU2AF65 protein The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. FIG +65 75 U2AF651,2L mutant The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. FIG +90 92 KD evidence The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. FIG +107 126 5′-CCCCCCCUUUUCC-3′ chemical The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. FIG +132 140 flU2AF65 protein The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. FIG +153 163 U2AF651,2L mutant The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. FIG +239 276 average apparent equilibrium affinity evidence The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. FIG +278 280 KA evidence The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. FIG +25 82 average fitted fluorescence anisotropy RNA-binding curves evidence The purified protein and average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 1. FIG +0 3 RRM structure_element RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif. FIG +5 26 RNA recognition motif structure_element RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif. FIG +28 30 RS structure_element RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif. FIG +32 52 arginine-serine rich structure_element RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif. FIG +54 57 UHM structure_element RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif. FIG +59 78 U2AF homology motif structure_element RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif. FIG +80 83 ULM structure_element RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif. FIG +85 102 U2AF ligand motif structure_element RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif. FIG +0 10 Structures evidence Structures of U2AF651,2L recognizing a contiguous Py tract. FIG +14 24 U2AF651,2L mutant Structures of U2AF651,2L recognizing a contiguous Py tract. FIG +39 49 contiguous structure_element Structures of U2AF651,2L recognizing a contiguous Py tract. FIG +50 58 Py tract chemical Structures of U2AF651,2L recognizing a contiguous Py tract. FIG +4 13 Alignment experimental_method (a) Alignment of oligonucleotide sequences that were co-crystallized in the indicated U2AF651,2L structures. FIG +17 32 oligonucleotide chemical (a) Alignment of oligonucleotide sequences that were co-crystallized in the indicated U2AF651,2L structures. FIG +53 68 co-crystallized experimental_method (a) Alignment of oligonucleotide sequences that were co-crystallized in the indicated U2AF651,2L structures. FIG +86 96 U2AF651,2L mutant (a) Alignment of oligonucleotide sequences that were co-crystallized in the indicated U2AF651,2L structures. FIG +97 107 structures evidence (a) Alignment of oligonucleotide sequences that were co-crystallized in the indicated U2AF651,2L structures. FIG +15 19 RRM1 structure_element The regions of RRM1, RRM2 and linker contacts are indicated above by respective black and blue arrows from N- to C-terminus. FIG +21 25 RRM2 structure_element The regions of RRM1, RRM2 and linker contacts are indicated above by respective black and blue arrows from N- to C-terminus. FIG +30 36 linker structure_element The regions of RRM1, RRM2 and linker contacts are indicated above by respective black and blue arrows from N- to C-terminus. FIG +40 50 U2AF651,2L mutant For clarity, we consistently number the U2AF651,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty. FIG +51 75 nucleotide-binding sites site For clarity, we consistently number the U2AF651,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty. FIG +121 136 co-crystallized experimental_method For clarity, we consistently number the U2AF651,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty. FIG +137 152 oligonucleotide chemical For clarity, we consistently number the U2AF651,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty. FIG +169 180 nucleotides chemical For clarity, we consistently number the U2AF651,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty. FIG +204 222 first binding site site For clarity, we consistently number the U2AF651,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty. FIG +10 20 dU2AF651,2 mutant The prior dU2AF651,2 nucleotide-binding sites are given in parentheses (site 4' interacts with dU2AF65 RRM1 and RRM2 by crystallographic symmetry). FIG +21 45 nucleotide-binding sites site The prior dU2AF651,2 nucleotide-binding sites are given in parentheses (site 4' interacts with dU2AF65 RRM1 and RRM2 by crystallographic symmetry). FIG +95 102 dU2AF65 mutant The prior dU2AF651,2 nucleotide-binding sites are given in parentheses (site 4' interacts with dU2AF65 RRM1 and RRM2 by crystallographic symmetry). FIG +103 107 RRM1 structure_element The prior dU2AF651,2 nucleotide-binding sites are given in parentheses (site 4' interacts with dU2AF65 RRM1 and RRM2 by crystallographic symmetry). FIG +112 116 RRM2 structure_element The prior dU2AF651,2 nucleotide-binding sites are given in parentheses (site 4' interacts with dU2AF65 RRM1 and RRM2 by crystallographic symmetry). FIG +31 62 2|Fo|−|Fc| electron density map evidence (b) Stereo views of a ‘kicked' 2|Fo|−|Fc| electron density map contoured at 1σ for the inter-RRM linker, N- and C-terminal residues (blue) or bound oligonucleotide of a representative U2AF651,2L structure (structure iv, bound to 5′-(P)rUrUrUdUrUrU(BrdU)dUrC) (magenta). FIG +87 103 inter-RRM linker structure_element (b) Stereo views of a ‘kicked' 2|Fo|−|Fc| electron density map contoured at 1σ for the inter-RRM linker, N- and C-terminal residues (blue) or bound oligonucleotide of a representative U2AF651,2L structure (structure iv, bound to 5′-(P)rUrUrUdUrUrU(BrdU)dUrC) (magenta). FIG +148 163 oligonucleotide chemical (b) Stereo views of a ‘kicked' 2|Fo|−|Fc| electron density map contoured at 1σ for the inter-RRM linker, N- and C-terminal residues (blue) or bound oligonucleotide of a representative U2AF651,2L structure (structure iv, bound to 5′-(P)rUrUrUdUrUrU(BrdU)dUrC) (magenta). FIG +184 194 U2AF651,2L mutant (b) Stereo views of a ‘kicked' 2|Fo|−|Fc| electron density map contoured at 1σ for the inter-RRM linker, N- and C-terminal residues (blue) or bound oligonucleotide of a representative U2AF651,2L structure (structure iv, bound to 5′-(P)rUrUrUdUrUrU(BrdU)dUrC) (magenta). FIG +220 228 bound to protein_state (b) Stereo views of a ‘kicked' 2|Fo|−|Fc| electron density map contoured at 1σ for the inter-RRM linker, N- and C-terminal residues (blue) or bound oligonucleotide of a representative U2AF651,2L structure (structure iv, bound to 5′-(P)rUrUrUdUrUrU(BrdU)dUrC) (magenta). FIG +82 92 U2AF651,2L mutant Crystallographic statistics are given in Table 1 and the overall conformations of U2AF651,2L and prior dU2AF651,2/U2AF651,2 structures are compared in Supplementary Fig. 2. FIG +103 113 dU2AF651,2 mutant Crystallographic statistics are given in Table 1 and the overall conformations of U2AF651,2L and prior dU2AF651,2/U2AF651,2 structures are compared in Supplementary Fig. 2. FIG +114 123 U2AF651,2 mutant Crystallographic statistics are given in Table 1 and the overall conformations of U2AF651,2L and prior dU2AF651,2/U2AF651,2 structures are compared in Supplementary Fig. 2. FIG +124 134 structures evidence Crystallographic statistics are given in Table 1 and the overall conformations of U2AF651,2L and prior dU2AF651,2/U2AF651,2 structures are compared in Supplementary Fig. 2. FIG +0 4 BrdU chemical BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose. FIG +6 27 5-bromo-deoxy-uridine chemical BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose. FIG +29 30 d chemical BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose. FIG +32 44 deoxy-ribose chemical BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose. FIG +46 48 P- chemical BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose. FIG +50 68 5′-phosphorylation chemical BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose. FIG +70 71 r chemical BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose. FIG +73 79 ribose chemical BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose. FIG +28 38 U2AF651,2L mutant Representative views of the U2AF651,2L interactions with each new nucleotide of the bound Py tract. FIG +66 76 nucleotide chemical Representative views of the U2AF651,2L interactions with each new nucleotide of the bound Py tract. FIG +84 89 bound protein_state Representative views of the U2AF651,2L interactions with each new nucleotide of the bound Py tract. FIG +90 98 Py tract chemical Representative views of the U2AF651,2L interactions with each new nucleotide of the bound Py tract. FIG +20 30 U2AF651,2L mutant New residues of the U2AF651,2L structures are coloured a darker shade of blue, apart from residues that were tested by site-directed mutagenesis, which are coloured yellow. FIG +31 41 structures evidence New residues of the U2AF651,2L structures are coloured a darker shade of blue, apart from residues that were tested by site-directed mutagenesis, which are coloured yellow. FIG +119 144 site-directed mutagenesis experimental_method New residues of the U2AF651,2L structures are coloured a darker shade of blue, apart from residues that were tested by site-directed mutagenesis, which are coloured yellow. FIG +4 28 nucleotide-binding sites site The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +36 46 U2AF651,2L mutant The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +57 67 dU2AF651,2 mutant The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +68 77 structure evidence The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +123 165 first and seventh U2AF651,2L-binding sites site The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +195 209 dU2AF651,2–RNA complex_assembly The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +210 219 structure evidence The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +275 285 U2AF651,2L mutant The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +286 296 structures evidence The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +362 372 ninth site site The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +438 448 U2AF651,2L mutant The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +449 458 structure evidence The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +499 516 ribose nucleotide chemical The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +540 543 rU2 residue_name_number The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +565 568 rU3 residue_name_number The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +591 594 rU4 residue_name_number The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +615 618 rU5 residue_name_number The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +641 644 rU6 residue_name_number The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +666 669 dU8 residue_name_number The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +692 695 dU9 residue_name_number The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +718 721 rC9 residue_name_number The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. FIG +26 48 equilibrium affinities evidence (i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +50 52 KA evidence (i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +61 70 wild type protein_state (i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +96 102 mutant protein_state (i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +112 122 U2AF651,2L mutant (i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +144 148 AdML gene (i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +149 157 Py tract chemical (i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +159 178 5′-CCCUUUUUUUUCC-3′ chemical (i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). FIG +13 47 equilibrium dissociation constants evidence The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +49 51 KD evidence The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +60 70 U2AF651,2L mutant The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +71 77 mutant protein_state The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +92 101 wild type protein_state The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +103 105 WT protein_state The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +117 122 R227A mutant The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +134 139 V254P mutant The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +152 157 Q147A mutant The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +182 184 KA evidence The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +43 61 RNA-binding curves evidence The average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4a–c. FIG +4 10 U2AF65 protein The U2AF65 linker/RRM and inter-RRM interactions. FIG +11 17 linker structure_element The U2AF65 linker/RRM and inter-RRM interactions. FIG +18 21 RRM structure_element The U2AF65 linker/RRM and inter-RRM interactions. FIG +32 35 RRM structure_element The U2AF65 linker/RRM and inter-RRM interactions. FIG +20 26 U2AF65 protein (a) Contacts of the U2AF65 inter-RRM linker with the RRMs. FIG +27 43 inter-RRM linker structure_element (a) Contacts of the U2AF65 inter-RRM linker with the RRMs. FIG +53 57 RRMs structure_element (a) Contacts of the U2AF65 inter-RRM linker with the RRMs. FIG +58 62 RRM1 structure_element A semi-transparent space-filling surface is shown for the RRM1 (green) and RRM2 (light blue). FIG +75 79 RRM2 structure_element A semi-transparent space-filling surface is shown for the RRM1 (green) and RRM2 (light blue). FIG +9 13 V249 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +15 19 V250 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +21 25 V254 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +39 46 mutated experimental_method Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +50 55 V249G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +56 61 V250G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +62 67 V254G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +75 86 3Gly mutant mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +97 101 S251 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +103 107 T252 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +109 113 V253 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +115 119 P255 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +137 141 V254 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +146 153 mutated experimental_method Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +157 162 S251G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +163 168 T252G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +169 174 V253G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +175 180 V254G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +181 186 P255G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +194 205 5Gly mutant mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +212 217 S251N mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +218 223 T252L mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +224 229 V253A mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +230 235 V254L mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +236 241 P255A mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +249 261 NLALA mutant mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +272 276 M144 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +278 282 L235 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +284 288 M238 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +290 294 V244 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +296 300 V246 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +321 325 V249 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +327 331 V250 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +333 337 S251 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +339 343 T252 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +345 349 V253 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +351 355 V254 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +357 361 P255 residue_name_number Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +366 373 mutated experimental_method Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +377 382 M144G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +383 388 L235G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +389 394 M238G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +395 400 V244G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +401 406 V246G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +407 412 V249G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +414 419 V250G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +420 425 S251G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +426 431 T252G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +432 437 V253G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +438 443 V254G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +444 449 P255G mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +457 469 12Gly mutant mutant Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. FIG +6 12 linker structure_element Other linker residues are coloured either dark blue for new residues in the U2AF651,2L structure or light blue for the remaining inter-RRM residues. FIG +76 86 U2AF651,2L mutant Other linker residues are coloured either dark blue for new residues in the U2AF651,2L structure or light blue for the remaining inter-RRM residues. FIG +129 138 inter-RRM structure_element Other linker residues are coloured either dark blue for new residues in the U2AF651,2L structure or light blue for the remaining inter-RRM residues. FIG +150 172 central linker regions structure_element The central panel shows an overall view with stick diagrams for mutated residues; boxed regions are expanded to show the C-terminal (bottom left) and central linker regions (top) at the inter-RRM interfaces, and N-terminal linker region contacts with RRM1 (bottom right). FIG +186 206 inter-RRM interfaces structure_element The central panel shows an overall view with stick diagrams for mutated residues; boxed regions are expanded to show the C-terminal (bottom left) and central linker regions (top) at the inter-RRM interfaces, and N-terminal linker region contacts with RRM1 (bottom right). FIG +251 255 RRM1 structure_element The central panel shows an overall view with stick diagrams for mutated residues; boxed regions are expanded to show the C-terminal (bottom left) and central linker regions (top) at the inter-RRM interfaces, and N-terminal linker region contacts with RRM1 (bottom right). FIG +26 48 equilibrium affinities evidence (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +50 52 KA evidence (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +62 66 AdML gene (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +67 75 Py tract chemical (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +77 96 5′-CCCUUUUUUUUCC-3′ chemical (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +105 114 wild-type protein_state (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +122 132 U2AF651,2L mutant (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +193 197 3Gly mutant (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +208 212 5Gly mutant (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +220 225 NLALA mutant (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +241 246 12Gly mutant (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +264 280 linker deletions experimental_method (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +281 291 dU2AF651,2 mutant (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +299 306 minimal protein_state (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +307 323 RRM1–RRM2 region structure_element (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +334 341 148–237 residue_range (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +343 350 258–336 residue_range (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +355 366 dU2AF651,2L mutant (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +377 384 141–237 residue_range (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +386 393 258–342 residue_range (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). FIG +13 47 equilibrium dissociation constants evidence The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +49 51 KD evidence The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +60 70 U2AF651,2L mutant The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +71 77 mutant protein_state The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +92 101 wild type protein_state The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +103 105 WT protein_state The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +117 121 3Gly mutant The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +132 136 5Gly mutant The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +147 152 12Gly mutant The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +164 169 NLALA mutant The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +180 191 dU2AF651,2L mutant The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +203 213 dU2AF651,2 mutant The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +228 232 3Mut mutant The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +258 260 KA evidence The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. FIG +35 53 RNA-binding curves evidence The fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4d–j. (c) Close view of the U2AF65 RRM1/RRM2 interface following a two-fold rotation about the x-axis relative to a. FIG +114 120 U2AF65 protein The fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4d–j. (c) Close view of the U2AF65 RRM1/RRM2 interface following a two-fold rotation about the x-axis relative to a. FIG +121 140 RRM1/RRM2 interface site The fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4d–j. (c) Close view of the U2AF65 RRM1/RRM2 interface following a two-fold rotation about the x-axis relative to a. FIG +0 6 U2AF65 protein U2AF65 inter-domain residues are important for splicing a representative pre-mRNA substrate in human cells. FIG +73 81 pre-mRNA chemical U2AF65 inter-domain residues are important for splicing a representative pre-mRNA substrate in human cells. FIG +95 100 human species U2AF65 inter-domain residues are important for splicing a representative pre-mRNA substrate in human cells. FIG +29 33 pyPY chemical (a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset). FIG +89 101 splice sites site (a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset). FIG +134 142 Py tract chemical (a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset). FIG +144 146 py chemical (a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset). FIG +162 166 AdML gene (a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset). FIG +167 175 Py tract chemical (a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset). FIG +177 179 PY chemical (a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset). FIG +19 25 RT-PCR experimental_method (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +29 33 pyPY chemical (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +65 79 co-transfected experimental_method (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +109 113 pyPY chemical (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +134 143 wild-type protein_state (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +145 147 WT protein_state (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +149 155 U2AF65 protein (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +168 174 U2AF65 protein (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +175 181 mutant protein_state (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +183 187 3Mut mutant (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +192 197 Q147A mutant (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +199 204 R227A mutant (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +209 214 V254P mutant (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +274 276 py chemical (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +285 289 mRNA chemical (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +317 321 pyPY chemical (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +401 407 U2AF65 protein (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +422 424 WT protein_state (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +425 431 U2AF65 protein (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +439 443 3Mut mutant (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +444 450 U2AF65 protein (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). FIG +0 22 Protein overexpression experimental_method Protein overexpression and qRT-PCR results are shown in Supplementary Fig. 5. FIG +27 34 qRT-PCR experimental_method Protein overexpression and qRT-PCR results are shown in Supplementary Fig. 5. FIG +27 39 side-by-side protein_state RNA binding stabilizes the side-by-side conformation of U2AF65 RRMs. FIG +56 62 U2AF65 protein RNA binding stabilizes the side-by-side conformation of U2AF65 RRMs. FIG +63 67 RRMs structure_element RNA binding stabilizes the side-by-side conformation of U2AF65 RRMs. FIG +15 19 FRET experimental_method (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +68 72 RRM1 structure_element (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +77 81 RRM2 structure_element (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +89 106 crystal structure evidence (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +111 123 side-by-side protein_state (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +125 135 U2AF651,2L mutant (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +136 140 RRMs structure_element (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +141 149 bound to protein_state (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +152 176 Py-tract oligonucleotide chemical (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +214 220 closed protein_state (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +222 225 NMR experimental_method (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +226 229 PRE experimental_method (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +245 254 U2AF651,2 mutant (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +301 305 RRM2 structure_element (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. FIG +4 18 U2AF651,2LFRET mutant The U2AF651,2LFRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site. FIG +52 57 A181C mutant The U2AF651,2LFRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site. FIG +58 63 Q324C mutant The U2AF651,2LFRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site. FIG +87 90 Cy3 chemical The U2AF651,2LFRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site. FIG +91 94 Cy5 chemical The U2AF651,2LFRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site. FIG +95 107 fluorophores chemical The U2AF651,2LFRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site. FIG +14 28 U2AF651,2LFRET mutant (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). FIG +29 32 Cy3 chemical (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). FIG +33 36 Cy5 chemical (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). FIG +90 105 biotin-NTA/Ni+2 chemical (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). FIG +217 227 absence of protein_state (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). FIG +228 235 ligands chemical (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). FIG +267 271 AdML gene (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). FIG +272 284 Py-tract RNA chemical (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). FIG +286 304 5′-CCUUUUUUUUCC-3′ chemical (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). FIG +341 350 adenosine residue_name (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). FIG +371 374 RNA chemical (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). FIG +376 396 5′-CUUUUUAAUUUCCA-3′ chemical (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). FIG +4 14 untethered protein_state The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1 nM) was added to AdML RNA–polyethylene-glycol-linker–DNA oligonucleotide (10 nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line). FIG +15 29 U2AF651,2LFRET mutant The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1 nM) was added to AdML RNA–polyethylene-glycol-linker–DNA oligonucleotide (10 nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line). FIG +30 33 Cy3 chemical The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1 nM) was added to AdML RNA–polyethylene-glycol-linker–DNA oligonucleotide (10 nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line). FIG +34 37 Cy5 chemical The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1 nM) was added to AdML RNA–polyethylene-glycol-linker–DNA oligonucleotide (10 nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line). FIG +67 71 AdML gene The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1 nM) was added to AdML RNA–polyethylene-glycol-linker–DNA oligonucleotide (10 nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line). FIG +72 122 RNA–polyethylene-glycol-linker–DNA oligonucleotide chemical The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1 nM) was added to AdML RNA–polyethylene-glycol-linker–DNA oligonucleotide (10 nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line). FIG +212 240 biotinyl-DNA oligonucleotide chemical The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1 nM) was added to AdML RNA–polyethylene-glycol-linker–DNA oligonucleotide (10 nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line). FIG +8 28 single-molecule FRET experimental_method Typical single-molecule FRET traces (c,e,g,i) show fluorescence intensities from Cy3 (green) and Cy5 (red) and the calculated apparent FRET efficiency (blue). FIG +29 35 traces evidence Typical single-molecule FRET traces (c,e,g,i) show fluorescence intensities from Cy3 (green) and Cy5 (red) and the calculated apparent FRET efficiency (blue). FIG +81 84 Cy3 chemical Typical single-molecule FRET traces (c,e,g,i) show fluorescence intensities from Cy3 (green) and Cy5 (red) and the calculated apparent FRET efficiency (blue). FIG +97 100 Cy5 chemical Typical single-molecule FRET traces (c,e,g,i) show fluorescence intensities from Cy3 (green) and Cy5 (red) and the calculated apparent FRET efficiency (blue). FIG +115 150 calculated apparent FRET efficiency evidence Typical single-molecule FRET traces (c,e,g,i) show fluorescence intensities from Cy3 (green) and Cy5 (red) and the calculated apparent FRET efficiency (blue). FIG +11 17 traces evidence Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +22 32 untethered protein_state Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +34 43 RNA-bound protein_state Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +44 58 U2AF651,2LFRET mutant Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +59 62 Cy3 chemical Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +63 66 Cy5 chemical Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +106 116 Histograms evidence Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +136 163 distribution of FRET values evidence Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +167 175 RNA-free protein_state Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +177 191 slide-tethered protein_state Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +192 206 U2AF651,2LFRET mutant Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +207 210 Cy3 chemical Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +211 214 Cy5 chemical Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +221 225 AdML gene Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +226 235 RNA-bound protein_state Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +237 251 slide-tethered protein_state Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +252 266 U2AF651,2LFRET mutant Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +267 270 Cy3 chemical Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +271 274 Cy5 chemical Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +281 285 AdML gene Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +286 295 RNA-bound protein_state Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +297 307 untethered protein_state Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +308 322 U2AF651,2LFRET mutant Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +323 326 Cy3 chemical Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +327 330 Cy5 chemical Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +362 371 RNA-bound protein_state Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +373 387 slide-tethered protein_state Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +388 402 U2AF651,2LFRET mutant Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +403 406 Cy3 chemical Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +407 410 Cy5 chemical Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). FIG +35 41 traces evidence N is the number of single-molecule traces compiled for each histogram. FIG +60 69 histogram evidence N is the number of single-molecule traces compiled for each histogram. FIG +20 26 U2AF65 protein Schematic models of U2AF65 recognizing the Py tract. FIG +43 51 Py tract chemical Schematic models of U2AF65 recognizing the Py tract. FIG +19 25 U2AF65 protein (a) Diagram of the U2AF65, SF1 and U2AF35 splicing factors bound to the consensus elements of the 3′ splice site. FIG +27 30 SF1 protein (a) Diagram of the U2AF65, SF1 and U2AF35 splicing factors bound to the consensus elements of the 3′ splice site. FIG +35 41 U2AF35 protein (a) Diagram of the U2AF65, SF1 and U2AF35 splicing factors bound to the consensus elements of the 3′ splice site. FIG +59 67 bound to protein_state (a) Diagram of the U2AF65, SF1 and U2AF35 splicing factors bound to the consensus elements of the 3′ splice site. FIG +98 112 3′ splice site site (a) Diagram of the U2AF65, SF1 and U2AF35 splicing factors bound to the consensus elements of the 3′ splice site. FIG +28 38 U2AF651,2L mutant A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3′ splice site are unknown. FIG +48 56 bound to protein_state A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3′ splice site are unknown. FIG +62 73 nucleotides chemical A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3′ splice site are unknown. FIG +128 149 branch point sequence site A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3′ splice site are unknown. FIG +151 154 BPS site A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3′ splice site are unknown. FIG +170 185 AG dinucleotide chemical A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3′ splice site are unknown. FIG +193 207 3′ splice site site A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3′ splice site are unknown. FIG +33 39 U2AF65 protein MDS-relevant mutated residues of U2AF65 are shown as yellow spheres (L187 and M144). FIG +69 73 L187 residue_name_number MDS-relevant mutated residues of U2AF65 are shown as yellow spheres (L187 and M144). FIG +78 82 M144 residue_name_number MDS-relevant mutated residues of U2AF65 are shown as yellow spheres (L187 and M144). FIG +29 41 Py-tract RNA chemical (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +75 84 high FRET evidence (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +110 116 closed protein_state (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +119 131 back-to-back protein_state (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +132 135 apo protein_state (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +136 142 U2AF65 protein (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +164 167 PRE experimental_method (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +168 171 NMR experimental_method (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +262 272 FRET value evidence (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +300 304 open protein_state (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +307 319 side-by-side protein_state (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +320 324 RRMs structure_element (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +337 347 U2AF651,2L mutant (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +348 366 crystal structures evidence (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. FIG +33 39 U2AF65 protein Alternatively, a conformation of U2AF65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF65 population towards the ∼0.45 FRET value. FIG +63 73 FRET value evidence Alternatively, a conformation of U2AF65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF65 population towards the ∼0.45 FRET value. FIG +95 98 RNA chemical Alternatively, a conformation of U2AF65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF65 population towards the ∼0.45 FRET value. FIG +100 103 RNA chemical Alternatively, a conformation of U2AF65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF65 population towards the ∼0.45 FRET value. FIG +128 132 open protein_state Alternatively, a conformation of U2AF65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF65 population towards the ∼0.45 FRET value. FIG +135 147 side-by-side protein_state Alternatively, a conformation of U2AF65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF65 population towards the ∼0.45 FRET value. FIG +181 187 U2AF65 protein Alternatively, a conformation of U2AF65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF65 population towards the ∼0.45 FRET value. FIG +217 227 FRET value evidence Alternatively, a conformation of U2AF65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF65 population towards the ∼0.45 FRET value. FIG +0 4 RRM1 structure_element RRM1, green; RRM2, pale blue; RRM extensions/linker, blue. FIG +13 17 RRM2 structure_element RRM1, green; RRM2, pale blue; RRM extensions/linker, blue. FIG +30 44 RRM extensions structure_element RRM1, green; RRM2, pale blue; RRM extensions/linker, blue. FIG +45 51 linker structure_element RRM1, green; RRM2, pale blue; RRM extensions/linker, blue. FIG diff --git a/annotation_CSV/PMC4792962.csv b/annotation_CSV/PMC4792962.csv new file mode 100644 index 0000000000000000000000000000000000000000..a1f8dc60c62a93a94c6998f382889e51ac3d7370 --- /dev/null +++ b/annotation_CSV/PMC4792962.csv @@ -0,0 +1,1298 @@ +anno_start anno_end anno_text entity_type sentence section +40 64 autocatalytic activation ptm A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome TITLE +72 86 20S proteasome complex_assembly A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome TITLE +18 32 20S proteasome complex_assembly Biogenesis of the 20S proteasome is tightly regulated. ABSTRACT +15 26 propeptides structure_element The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. ABSTRACT +42 53 active-site site The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. ABSTRACT +54 64 threonines residue_name The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. ABSTRACT +69 86 autocatalytically ptm The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. ABSTRACT +68 87 strict conservation protein_state However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. ABSTRACT +91 100 threonine residue_name However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. ABSTRACT +12 23 mutagenesis experimental_method Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT +25 46 X-ray crystallography experimental_method Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT +51 69 biochemical assays experimental_method Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT +86 91 Lys33 residue_name_number Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT +129 139 propeptide structure_element Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT +161 165 Thr1 residue_name_number Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT +218 223 Asp17 residue_name_number Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT +238 253 catalytic triad site Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. ABSTRACT +0 12 Substitution experimental_method Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. ABSTRACT +16 20 Thr1 residue_name_number Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. ABSTRACT +24 27 Cys residue_name Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. ABSTRACT +58 63 Lys33 residue_name_number Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. ABSTRACT +68 79 inactivates protein_state Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. ABSTRACT +84 94 proteasome complex_assembly Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. ABSTRACT +11 18 Thr1Ser mutant Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. ABSTRACT +19 25 mutant protein_state Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. ABSTRACT +29 35 active protein_state Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. ABSTRACT +72 81 wild type protein_state Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. ABSTRACT +125 129 Ser1 residue_name_number Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. ABSTRACT +72 92 propeptide autolysis ptm This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease. ABSTRACT +147 157 proteasome complex_assembly This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease. ABSTRACT +170 188 threonine protease protein_type This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease. ABSTRACT +5 15 proteasome complex_assembly The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear. ABSTRACT +54 72 threonine protease protein_type The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear. ABSTRACT +85 95 proteasome complex_assembly Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity. ABSTRACT +96 107 active site site Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity. ABSTRACT +4 32 20S proteasome core particle complex_assembly The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells. INTRO +34 36 CP complex_assembly The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells. INTRO +49 71 non-lysosomal protease protein_type The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells. INTRO +75 85 eukaryotic taxonomy_domain The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells. INTRO +20 21 α protein Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. INTRO +42 52 β subunits protein Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. INTRO +72 82 heptameric oligomeric_state Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. INTRO +83 88 rings structure_element Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. INTRO +130 145 hollow cylinder structure_element Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder. INTRO +10 18 inactive protein_state While the inactive α subunits build the two outer rings, the β subunits form the inner rings. INTRO +19 29 α subunits protein While the inactive α subunits build the two outer rings, the β subunits form the inner rings. INTRO +50 55 rings structure_element While the inactive α subunits build the two outer rings, the β subunits form the inner rings. INTRO +61 71 β subunits protein While the inactive α subunits build the two outer rings, the β subunits form the inner rings. INTRO +87 92 rings structure_element While the inactive α subunits build the two outer rings, the β subunits form the inner rings. INTRO +38 48 β subunits protein Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO +57 59 β1 protein Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO +61 63 β2 protein Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO +68 70 β5 protein Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO +88 114 proteolytic active centres site Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO +127 129 CP complex_assembly Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO +164 175 propeptides structure_element Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides. INTRO +21 23 CP complex_assembly In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). INTRO +40 51 prosegments structure_element In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). INTRO +56 81 autocatalytically removed ptm In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). INTRO +117 136 active site residue site In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). INTRO +137 141 Thr1 residue_name_number In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). INTRO +182 189 Gly(-1) residue_name_number In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1). INTRO +15 26 propeptides structure_element Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides. INTRO +50 56 active protein_state Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides. INTRO +57 59 CP complex_assembly Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides. INTRO +36 61 substrate-binding channel site Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds. INTRO +129 135 active protein_state Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds. INTRO +136 146 β subunits protein Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds. INTRO +152 164 active sites site Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds. INTRO +23 27 Thr1 residue_name_number Nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of the scissile peptide bond creates a first cleavage product and a covalent acyl-enzyme intermediate. INTRO +19 26 complex complex_assembly Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment. INTRO +61 66 water chemical Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment. INTRO +92 98 enzyme complex_assembly Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment. INTRO +123 130 peptide chemical Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment. INTRO +4 14 proteasome complex_assembly The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. INTRO +40 80 N-terminal nucleophilic (Ntn) hydrolases protein_type The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. INTRO +90 94 free protein_state The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. INTRO +121 125 Thr1 residue_name_number The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. INTRO +158 162 Thr1 residue_name_number The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. INTRO +205 209 Thr1 residue_name_number The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage. INTRO +40 74 autocatalytic precursor processing ptm This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. INTRO +90 98 immature protein_state This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. INTRO +99 111 active sites site This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. INTRO +113 117 Thr1 residue_name_number This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. INTRO +152 159 Gly(-1) residue_name_number This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed. INTRO +47 51 Thr1 residue_name_number An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å). INTRO +88 93 Lys33 residue_name_number An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å). INTRO +110 126 hydrogen-bonding bond_interaction An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å). INTRO +139 143 Thr1 residue_name_number An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å). INTRO +63 84 autocatalytic removal ptm In principle it could function as the general base during both autocatalytic removal of the propeptide and protein substrate cleavage. INTRO +92 102 propeptide structure_element In principle it could function as the general base during both autocatalytic removal of the propeptide and protein substrate cleavage. INTRO +69 79 proteasome complex_assembly Here we provide experimental evidences for this distinct view of the proteasome active-site mechanism. INTRO +80 91 active-site site Here we provide experimental evidences for this distinct view of the proteasome active-site mechanism. INTRO +10 45 biochemical and structural analyses experimental_method Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. INTRO +91 93 β5 protein Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. INTRO +94 104 propeptide structure_element Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. INTRO +113 124 active site site Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. INTRO +228 252 autocatalytic activation ptm Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. INTRO +260 270 proteasome complex_assembly Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome. INTRO +44 47 Thr residue_name Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays. INTRO +53 56 Cys residue_name Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays. INTRO +60 63 Ser residue_name Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays. INTRO +101 122 X-ray crystallography experimental_method Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays. INTRO +137 167 activity and inhibition assays experimental_method Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays. INTRO +16 26 proteasome complex_assembly Inactivation of proteasome subunits by T1A mutations RESULTS +27 35 subunits protein Inactivation of proteasome subunits by T1A mutations RESULTS +39 42 T1A mutant Inactivation of proteasome subunits by T1A mutations RESULTS +43 52 mutations experimental_method Inactivation of proteasome subunits by T1A mutations RESULTS +0 10 Proteasome complex_assembly Proteasome-mediated degradation of cell-cycle regulators and potentially toxic misfolded proteins is required for the viability of eukaryotic cells. RESULTS +131 141 eukaryotic taxonomy_domain Proteasome-mediated degradation of cell-cycle regulators and potentially toxic misfolded proteins is required for the viability of eukaryotic cells. RESULTS +20 31 active site site Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. RESULTS +32 36 Thr1 residue_name_number Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. RESULTS +40 51 mutation to experimental_method Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. RESULTS +52 55 Ala residue_name Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. RESULTS +126 136 proteasome complex_assembly Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. RESULTS +137 149 active sites site Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites. RESULTS +0 5 Yeast taxonomy_domain Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS +44 50 β1-T1A mutant Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS +54 60 β2-T1A mutant Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS +128 137 catalytic protein_state Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS +138 148 β subunits protein Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS +153 161 disabled protein_state Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS +166 183 carry remnants of protein_state Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS +201 212 propeptides structure_element Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1). RESULTS +32 34 β1 protein These results indicate that the β1 and β2 proteolytic activities are not essential for cell survival. RESULTS +39 41 β2 protein These results indicate that the β1 and β2 proteolytic activities are not essential for cell survival. RESULTS +17 20 T1A mutant By contrast, the T1A mutation in subunit β5 has been reported to be lethal or nearly so. RESULTS +41 43 β5 protein By contrast, the T1A mutation in subunit β5 has been reported to be lethal or nearly so. RESULTS +29 35 β5-T1A mutant Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS +52 62 propeptide structure_element Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS +64 66 pp chemical Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS +68 100 deleted but expressed separately experimental_method Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS +104 109 trans protein_state Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS +111 117 β5-T1A mutant Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS +118 120 pp chemical Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS +121 126 trans protein_state Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1). RESULTS +12 37 crystallographic analysis experimental_method Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +45 51 β5-T1A mutant Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +52 54 pp chemical Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +55 60 trans protein_state Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +61 67 mutant protein_state Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +90 98 mutation experimental_method Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +138 159 catalytic active site site Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +173 188 trans-expressed experimental_method Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +189 191 β5 protein Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +192 202 propeptide structure_element Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +206 215 not bound protein_state Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +223 225 β5 protein Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +226 251 substrate-binding channel site Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a). RESULTS +33 39 β5-T1A mutant The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy. RESULTS +40 46 mutant protein_state The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy. RESULTS +47 49 pp chemical The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy. RESULTS +50 53 cis protein_state The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy. RESULTS +0 26 Sequencing of the plasmids experimental_method Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS +59 64 yeast taxonomy_domain Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS +88 113 site-directed mutagenesis experimental_method Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS +132 138 β5-T1A mutant Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS +139 145 mutant protein_state Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS +146 148 pp chemical Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS +149 152 cis protein_state Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods). RESULTS +48 52 K81R mutant We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS +64 66 β5 protein We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS +112 143 This single amino-acid exchange experimental_method We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS +162 171 interface site We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS +188 190 α4 protein We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS +192 194 β4 protein We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS +199 201 β5 protein We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS +251 253 CP complex_assembly We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts. RESULTS +34 45 β5-T1A-K81R mutant The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS +46 52 mutant protein_state The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS +77 94 crystal structure evidence The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS +100 105 yeast taxonomy_domain The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS +106 116 proteasome complex_assembly The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS +118 121 yCP complex_assembly The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS +132 138 β5-T1A mutant The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1). RESULTS +0 10 Propeptide structure_element Propeptide conformation and triggering of autolysis RESULTS +42 51 autolysis ptm Propeptide conformation and triggering of autolysis RESULTS +22 32 proteasome complex_assembly In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. RESULTS +49 60 propeptides structure_element In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. RESULTS +65 90 autocatalytically cleaved ptm In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. RESULTS +100 106 mature protein_state In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. RESULTS +107 124 β-subunit domains protein In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains. RESULTS +12 14 β1 protein For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). RESULTS +73 83 propeptide structure_element For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). RESULTS +104 108 (-2) residue_number For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). RESULTS +147 168 S1 specificity pocket site For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). RESULTS +176 201 substrate-binding channel site For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). RESULTS +223 225 45 residue_number For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2). RESULTS +38 48 prosegment structure_element Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS +58 78 antiparallel β-sheet structure_element Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS +86 97 active site site Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS +108 115 Gly(-1) residue_name_number Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS +125 144 γ-turn conformation structure_element Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS +188 201 hydrogen bond bond_interaction Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS +210 217 Leu(-2) residue_name_number Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS +223 227 Thr1 residue_name_number Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.). RESULTS +27 33 β1-T1A mutant Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). RESULTS +34 40 mutant protein_state Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). RESULTS +41 61 crystallographically experimental_method Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). RESULTS +93 103 structures evidence Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). RESULTS +111 117 β2-T1A mutant Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). RESULTS +129 142 β1-T1A-β2-T1A mutant Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1). RESULTS +11 13 β1 protein In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a). RESULTS +29 36 Gly(-1) residue_name_number In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a). RESULTS +52 62 sharp turn structure_element In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a). RESULTS +81 100 prosegment cleavage ptm In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a). RESULTS +13 32 γ-turn conformation structure_element However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1. RESULTS +52 65 hydrogen bond bond_interaction However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1. RESULTS +198 205 Gly(-1) residue_name_number However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1. RESULTS +233 237 Thr1 residue_name_number However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1. RESULTS +14 16 β2 protein Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS +17 27 propeptide structure_element Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS +29 36 Thr(-2) residue_name_number Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS +50 59 S1 pocket site Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS +102 109 Leu(-2) residue_name_number Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS +113 115 β1 protein Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS +156 158 β2 protein Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS +159 168 S1 pocket site Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS +180 185 Gly45 residue_name_number Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45. RESULTS +0 7 Thr(-2) residue_name_number Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b). RESULTS +18 25 Gly(-1) residue_name_number Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b). RESULTS +31 47 hydrogen bonding bond_interaction Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b). RESULTS +112 116 Thr1 residue_name_number Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b). RESULTS +38 40 β5 protein Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant. RESULTS +41 51 propeptide structure_element Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant. RESULTS +59 70 β5-T1A-K81R mutant Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant. RESULTS +71 77 mutant protein_state Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant. RESULTS +14 21 Gly(-1) residue_name_number Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. RESULTS +60 69 histidine residue_name Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. RESULTS +93 97 (-2) residue_number Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. RESULTS +112 114 S2 site Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. RESULTS +130 139 S1 pocket site Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. RESULTS +164 184 antiparallel β-sheet structure_element Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet. RESULTS +36 43 Gly(-1) residue_name_number Nonetheless, the carbonyl carbon of Gly(-1) would be ideally placed for nucleophilic attack by Thr1Oγ (Fig. 1c and Supplementary Fig. 2c,d). RESULTS +95 99 Thr1 residue_name_number Nonetheless, the carbonyl carbon of Gly(-1) would be ideally placed for nucleophilic attack by Thr1Oγ (Fig. 1c and Supplementary Fig. 2c,d). RESULTS +7 11 K81R mutant As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. RESULTS +45 56 active site site As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. RESULTS +58 62 Thr1 residue_name_number As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. RESULTS +65 70 Arg81 residue_name_number As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. RESULTS +98 108 propeptide structure_element As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded. RESULTS +31 33 β5 protein Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. RESULTS +34 43 S1 pocket site Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. RESULTS +84 89 Met45 residue_name_number Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. RESULTS +128 135 His(-2) residue_name_number Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. RESULTS +143 150 S1 site site Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. RESULTS +201 210 autolysis ptm Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis. RESULTS +61 65 Thr1 residue_name_number Processing of β-subunit precursors requires deprotonation of Thr1OH; however, the general base initiating autolysis is unknown. RESULTS +106 115 autolysis ptm Processing of β-subunit precursors requires deprotonation of Thr1OH; however, the general base initiating autolysis is unknown. RESULTS +12 22 eukaryotic taxonomy_domain Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). RESULTS +35 37 β5 protein Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). RESULTS +54 57 His residue_name Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). RESULTS +78 82 (-2) residue_number Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). RESULTS +90 100 propeptide structure_element Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a). RESULTS +3 12 histidine residue_name As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +59 75 catalytic triads site As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +79 95 serine proteases protein_type As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +125 132 His(-2) residue_name_number As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +154 156 β5 protein As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +157 167 propeptide structure_element As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +171 188 exchanging it for experimental_method As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +189 192 Asn residue_name As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +194 197 Lys residue_name As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +199 202 Phe residue_name As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +207 210 Ala residue_name As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +216 221 yeast taxonomy_domain As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +303 309 H(-2)N mutant As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +314 320 H(-2)F mutant As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1). RESULTS +56 62 H(-2)N mutant In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c). RESULTS +67 73 H(-2)F mutant In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c). RESULTS +74 80 mutant protein_state In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c). RESULTS +81 85 yCPs complex_assembly In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c). RESULTS +0 19 Structural analyses experimental_method Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. RESULTS +38 49 propeptides structure_element Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. RESULTS +57 63 mutant protein_state Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. RESULTS +64 68 yCPs complex_assembly Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. RESULTS +85 110 2FO–FC electron densities evidence Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities. RESULTS +0 7 Gly(-1) residue_name_number Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. RESULTS +12 15 Phe residue_name Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. RESULTS +16 23 Lys(-2) residue_name_number Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. RESULTS +64 67 Ala residue_name Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. RESULTS +68 75 Asn(-2) residue_name_number Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned. RESULTS +40 49 processed protein_state This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base. RESULTS +54 65 unprocessed protein_state This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base. RESULTS +66 68 β5 protein This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base. RESULTS +101 110 autolysis ptm This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base. RESULTS +160 164 (-2) residue_number This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base. RESULTS +40 44 (-2) residue_number Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS +71 81 propeptide structure_element Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS +85 114 creating mutants that combine experimental_method Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS +119 122 T1A mutant Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS +124 128 K81R mutant Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS +130 141 mutation(s) experimental_method Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS +147 153 H(-2)L mutant Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS +155 161 H(-2)T mutant Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS +165 171 H(-2)A mutant Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS +172 185 substitutions experimental_method Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions. RESULTS +0 7 Leu(-2) residue_name_number Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS +26 31 yeast taxonomy_domain Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS +32 34 β1 protein Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS +78 85 Thr(-2) residue_name_number Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS +107 109 β2 protein Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS +110 121 propeptides structure_element Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS +151 158 Ala(-2) residue_name_number Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS +183 185 β5 protein Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS +186 195 S1 pocket site Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS +239 244 Met45 residue_name_number Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning. RESULTS +17 23 β5-T1A mutant As expected from β5-T1A mutants, the yeasts show severe growth phenotypes, with minor variations (Supplementary Fig. 4a and Table 1). RESULTS +37 43 yeasts taxonomy_domain As expected from β5-T1A mutants, the yeasts show severe growth phenotypes, with minor variations (Supplementary Fig. 4a and Table 1). RESULTS +14 32 crystal structures evidence We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1). RESULTS +40 53 β5-H(-2)L-T1A mutant We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1). RESULTS +55 68 β5-H(-2)T-T1A mutant We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1). RESULTS +77 95 β5-H(-2)A-T1A-K81R mutant We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1). RESULTS +8 26 β5-H(-2)A-T1A-K81R mutant For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). RESULTS +54 61 Gly(-1) residue_name_number For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). RESULTS +66 73 Ala(-2) residue_name_number For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). RESULTS +111 118 Ala(-2) residue_name_number For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). RESULTS +162 172 propeptide structure_element For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). RESULTS +180 205 substrate-binding channel site For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d). RESULTS +17 28 prosegments structure_element By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. RESULTS +36 49 β5-H(-2)L-T1A mutant By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. RESULTS +58 71 β5-H(-2)T-T1A mutant By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. RESULTS +122 150 2FO–FC electron-density maps evidence By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. RESULTS +258 268 propeptide structure_element By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. RESULTS +277 284 His(-2) residue_name_number By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable. RESULTS +19 26 Leu(-2) residue_name_number Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h). RESULTS +31 38 Thr(-2) residue_name_number Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h). RESULTS +64 85 S1 specificity pocket site Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h). RESULTS +96 101 Met45 residue_name_number Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h). RESULTS +48 55 His(-2) residue_name_number This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site. RESULTS +63 65 β5 protein This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site. RESULTS +66 76 propeptide structure_element This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site. RESULTS +106 113 S1 site site This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site. RESULTS +6 13 Gly(-1) residue_name_number Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS +47 56 wild-type protein_state Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS +58 60 WT protein_state Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS +66 72 mutant protein_state Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS +73 75 β5 protein Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS +76 87 propeptides structure_element Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS +179 183 Thr1 residue_name_number Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS +240 244 (-2) residue_number Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS +252 261 S1 pocket site Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS +283 303 antiparallel β-sheet structure_element Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS +321 330 autolysis ptm Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS +338 348 propeptide structure_element Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide. RESULTS +24 41 crystal structure evidence Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS +47 55 chimeric protein_state Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS +56 59 yCP complex_assembly Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS +71 76 yeast taxonomy_domain Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS +77 79 β1 protein Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS +80 90 propeptide structure_element Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS +91 102 replaced by experimental_method Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS +107 109 β5 protein Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS +110 121 counterpart structure_element Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart. RESULTS +34 57 2FO–FC electron density evidence Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable. RESULTS +65 67 β1 protein Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable. RESULTS +68 79 active site site Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable. RESULTS +36 43 Thr(-2) residue_name_number Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS +47 49 β2 protein Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS +51 58 Leu(-2) residue_name_number Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS +70 72 β1 protein Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS +76 89 not conserved protein_state Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS +132 139 created experimental_method Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS +142 151 β2-T(-2)V mutant Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS +152 162 proteasome complex_assembly Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS +163 169 mutant protein_state Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant. RESULTS +17 23 β2-T1A mutant As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS +24 42 crystal structures evidence As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS +44 51 Thr(-2) residue_name_number As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS +52 66 hydrogen bonds bond_interaction As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS +70 77 Gly(-1) residue_name_number As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS +131 144 β5-H(-2)T-T1A mutant As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS +145 151 mutant protein_state As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS +191 199 exchange experimental_method As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS +203 210 Thr(-2) residue_name_number As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS +214 217 Val residue_name As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS +221 223 β2 protein As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j). RESULTS +13 40 2FO–FC electron-density map evidence Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. RESULTS +82 84 β2 protein Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. RESULTS +85 95 propeptide structure_element Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. RESULTS +127 133 β2-T1A mutant Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. RESULTS +134 144 proteasome complex_assembly Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome. RESULTS +15 22 Val(-2) residue_name_number In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k). RESULTS +45 52 S1 site site In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k). RESULTS +57 64 Gly(-1) residue_name_number In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k). RESULTS +176 180 Thr1 residue_name_number In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k). RESULTS +62 82 active-site residues site These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome. RESULTS +87 94 Gly(-1) residue_name_number These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome. RESULTS +133 143 proteasome complex_assembly These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome. RESULTS +4 15 active site site The active site of the proteasome RESULTS +23 33 proteasome complex_assembly The active site of the proteasome RESULTS +38 49 active site site Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis. RESULTS +50 54 Thr1 residue_name_number Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis. RESULTS +60 64 Thr1 residue_name_number Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis. RESULTS +87 92 water chemical Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis. RESULTS +16 24 immature protein_state However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. RESULTS +25 33 particle complex_assembly However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. RESULTS +34 38 Thr1 residue_name_number However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. RESULTS +60 70 propeptide structure_element However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. RESULTS +91 95 Thr1 residue_name_number However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ. RESULTS +9 14 Lys33 residue_name_number Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. RESULTS +31 47 hydrogen-bonding bond_interaction Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. RESULTS +60 64 Thr1 residue_name_number Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. RESULTS +82 102 catalytically active protein_state Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. RESULTS +103 113 β subunits protein Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor. RESULTS +11 27 catalytic tetrad site A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS +44 48 Thr1 residue_name_number A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS +52 56 Thr1 residue_name_number A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS +61 66 Lys33 residue_name_number A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS +74 79 Asp17 residue_name_number A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS +109 114 water chemical A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS +208 219 active site site A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site. RESULTS +39 42 yCP complex_assembly Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base. RESULTS +43 59 X-ray structures evidence Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base. RESULTS +98 109 active site site Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base. RESULTS +117 127 proteasome complex_assembly Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base. RESULTS +0 8 Mutation experimental_method Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). RESULTS +12 14 β5 protein Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). RESULTS +15 20 Lys33 residue_name_number Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). RESULTS +24 27 Ala residue_name Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). RESULTS +82 117 structural and biochemical analyses experimental_method Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). RESULTS +162 181 propeptide cleavage ptm Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1). RESULTS +21 28 β5-K33A mutant The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a). RESULTS +29 35 mutant protein_state The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a). RESULTS +77 88 β5-T1A-K81R mutant The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a). RESULTS +89 94 yeast taxonomy_domain The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a). RESULTS +70 77 L(-49)S mutant This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). RESULTS +85 87 β5 protein This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). RESULTS +88 98 propeptide structure_element This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). RESULTS +106 113 β5-K33A mutant This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). RESULTS +114 120 mutant protein_state This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1). RESULTS +0 21 Structural comparison experimental_method Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS +29 44 β5-L(-49)S-K33A mutant Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS +49 60 β5-T1A-K81R mutant Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS +61 73 active sites site Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS +88 96 mutation experimental_method Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS +100 105 Lys33 residue_name_number Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS +109 112 Ala residue_name Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS +150 154 Thr1 residue_name_number Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS +171 181 propeptide structure_element Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide. RESULTS +36 47 active-site site This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a). RESULTS +98 100 β5 protein This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a). RESULTS +101 112 active site site This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a). RESULTS +41 46 Lys33 residue_name_number Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS +69 76 β5-K33A mutant Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS +77 83 mutant protein_state Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS +94 104 propeptide structure_element Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS +105 125 expressed separately experimental_method Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS +149 151 pp chemical Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS +152 157 trans protein_state Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans). RESULTS +4 8 Thr1 residue_name_number The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b). RESULTS +28 34 mutant protein_state The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b). RESULTS +57 67 propeptide structure_element The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b). RESULTS +26 43 crystal structure evidence Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS +51 58 β5-K33A mutant Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS +59 61 pp chemical Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS +62 67 trans protein_state Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS +68 74 mutant protein_state Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS +75 90 in complex with protein_state Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS +91 102 carfilzomib chemical Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS +154 156 β5 protein Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS +157 169 active sites site Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1). RESULTS +9 20 acetylation ptm Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS +28 32 Thr1 residue_name_number Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS +65 72 β5-K33A mutant Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS +73 75 pp chemical Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS +76 81 trans protein_state Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS +82 85 apo protein_state Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS +86 103 crystal structure evidence Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS +177 182 Lys33 residue_name_number Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS +199 203 Thr1 residue_name_number Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS +235 239 Thr1 residue_name_number Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH. RESULTS +17 34 crystal structure evidence Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS +42 49 β5-K33A mutant Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS +50 52 pp chemical Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS +53 58 trans protein_state Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS +59 65 mutant protein_state Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS +66 83 without inhibitor protein_state Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS +98 102 Thr1 residue_name_number Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS +114 125 coordinates bond_interaction Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS +141 146 water chemical Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d). RESULTS +5 10 water chemical This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. RESULTS +11 25 hydrogen bonds bond_interaction This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. RESULTS +34 39 Arg19 residue_name_number This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. RESULTS +54 59 Asp17 residue_name_number This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. RESULTS +128 134 mutant protein_state This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant. RESULTS +73 78 Lys33 residue_name_number Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. RESULTS +89 91 WT protein_state Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. RESULTS +92 102 proteasome complex_assembly Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. RESULTS +103 112 structure evidence Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. RESULTS +168 173 Lys33 residue_name_number Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. RESULTS +198 207 autolysis ptm Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis. RESULTS +0 25 Conservative substitution experimental_method Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). RESULTS +29 34 Lys33 residue_name_number Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). RESULTS +38 41 Arg residue_name Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). RESULTS +49 58 autolysis ptm Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). RESULTS +66 68 β5 protein Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). RESULTS +91 96 yeast taxonomy_domain Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1). RESULTS +6 10 Thr1 residue_name_number While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS +44 46 WT protein_state While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS +47 51 yCPs complex_assembly While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS +53 58 Arg33 residue_name_number While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS +72 85 hydrogen bond bond_interaction While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS +89 94 Asp17 residue_name_number While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS +121 123 β5 protein While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS +124 135 active site site While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e). RESULTS +4 25 conservative mutation experimental_method The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). RESULTS +29 34 Asp17 residue_name_number The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). RESULTS +38 41 Asn residue_name The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). RESULTS +53 55 β5 protein The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). RESULTS +63 66 yCP complex_assembly The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1). RESULTS +49 56 L(-49)S mutant Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. RESULTS +72 74 β5 protein Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. RESULTS +75 85 propeptide structure_element Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. RESULTS +124 131 β5-D17N mutant Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. RESULTS +132 138 mutant protein_state Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. RESULTS +139 142 yCP complex_assembly Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP. RESULTS +17 42 crystallographic analysis experimental_method As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS +49 55 mutant protein_state As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS +56 58 β5 protein As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS +71 90 partially processed protein_state As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS +147 157 proteasome complex_assembly As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS +168 179 carfilzomib chemical As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS +207 209 β1 protein As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS +214 216 β2 protein As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS +227 229 WT protein_state As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS +230 232 β5 protein As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a). RESULTS +19 22 cis protein_state In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS +34 44 expression experimental_method In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS +52 54 β5 protein In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS +55 65 propeptide structure_element In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS +69 74 trans protein_state In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS +99 108 isolation experimental_method In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS +113 128 crystallization experimental_method In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS +136 140 D17N mutant In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS +141 147 mutant protein_state In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS +148 158 proteasome complex_assembly In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome. RESULTS +26 33 β5-D17N mutant The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS +34 36 pp chemical The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS +40 45 trans protein_state The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS +46 48 CP complex_assembly The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS +71 73 β5 protein The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS +91 142 N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin chemical The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS +144 156 Suc-LLVY-AMC chemical The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS +162 205 carboxybenzyl-Gly-Gly-Leu-para-nitroanilide chemical The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS +207 216 Z-GGL-pNA chemical The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS +280 285 Asp17 residue_name_number The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS +349 355 mature protein_state The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS +356 366 proteasome complex_assembly The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome. RESULTS +16 23 β5-D17N mutant Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS +24 26 pp chemical Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS +27 32 trans protein_state Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS +33 36 yCP complex_assembly Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS +37 54 crystal structure evidence Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS +81 83 WT protein_state Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS +84 87 yCP complex_assembly Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS +117 137 co-crystal structure evidence Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS +147 165 α′, β′ epoxyketone chemical Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS +176 187 carfilzomib chemical Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS +243 245 β5 protein Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS +246 257 active site site Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a). RESULTS +69 71 β5 protein This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS +72 76 Thr1 residue_name_number This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS +85 102 crystal structure evidence This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS +110 117 β5-D17N mutant This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS +118 120 pp chemical This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS +121 124 cis protein_state This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS +125 131 mutant protein_state This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS +132 147 in complex with protein_state This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS +148 159 carfilzomib chemical This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib. RESULTS +0 9 Autolysis ptm Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. RESULTS +49 56 β5-D17N mutant Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. RESULTS +106 111 Asn17 residue_name_number Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. RESULTS +163 168 Lys33 residue_name_number Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. RESULTS +191 195 Thr1 residue_name_number Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1. RESULTS +17 21 E17A mutant In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. RESULTS +22 28 mutant protein_state In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. RESULTS +48 57 β-subunit protein In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. RESULTS +65 73 archaeon taxonomy_domain In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. RESULTS +74 98 Thermoplasma acidophilum species In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. RESULTS +108 117 autolysis ptm In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis. RESULTS +25 35 X-ray data evidence Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS +43 50 β5-D17N mutant Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS +51 57 mutant protein_state Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS +67 77 propeptide structure_element Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS +78 87 expressed experimental_method Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS +91 94 cis protein_state Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS +102 107 trans protein_state Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b). RESULTS +47 50 CPs complex_assembly On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS +82 97 catalytic triad site On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS +112 116 Thr1 residue_name_number On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS +118 123 Lys33 residue_name_number On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS +128 131 Asp residue_name On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS +132 137 Glu17 residue_name_number On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS +147 181 autocatalytic precursor processing ptm On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d). RESULTS +60 65 water chemical This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. RESULTS +94 100 mature protein_state This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. RESULTS +101 103 WT protein_state This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. RESULTS +116 127 active site site This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. RESULTS +163 167 Thr1 residue_name_number This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. RESULTS +173 177 Thr1 residue_name_number This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2. RESULTS +16 27 active-site site To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. RESULTS +46 69 exchanged the conserved experimental_method To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. RESULTS +70 76 Asp166 residue_name_number To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. RESULTS +89 92 Asn residue_name To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. RESULTS +100 105 yeast taxonomy_domain To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. RESULTS +106 108 β5 protein To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit. RESULTS +0 6 Asp166 residue_name_number Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. RESULTS +12 27 hydrogen-bonded bond_interaction Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. RESULTS +31 35 Thr1 residue_name_number Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. RESULTS +43 49 Ser129 residue_name_number Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. RESULTS +56 62 Ser169 residue_name_number Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis. RESULTS +4 12 β5-D166N mutant The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). RESULTS +13 15 pp chemical The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). RESULTS +16 19 cis protein_state The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). RESULTS +20 25 yeast taxonomy_domain The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). RESULTS +26 32 mutant protein_state The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1). RESULTS +0 10 X-ray data evidence X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS +18 26 β5-D166N mutant X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS +27 33 mutant protein_state X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS +52 54 β5 protein X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS +55 65 propeptide structure_element X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS +109 115 Ser129 residue_name_number X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS +140 146 Asn166 residue_name_number X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a). RESULTS +11 16 water chemical Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. RESULTS +29 37 bound to protein_state Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. RESULTS +38 44 Ser129 residue_name_number Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. RESULTS +51 55 Thr1 residue_name_number Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. RESULTS +101 121 precursor processing ptm Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing. RESULTS +4 18 hydrogen bonds bond_interaction The hydrogen bonds involving Ser169OH are intact and may account for residual substrate turnover. RESULTS +29 35 Ser169 residue_name_number The hydrogen bonds involving Ser169OH are intact and may account for residual substrate turnover. RESULTS +0 7 Soaking experimental_method Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). RESULTS +12 20 β5-D166N mutant Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). RESULTS +21 29 crystals experimental_method Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). RESULTS +35 46 carfilzomib chemical Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). RESULTS +51 56 MG132 chemical Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). RESULTS +94 98 Thr1 residue_name_number Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c). RESULTS +7 26 carfilzomib complex complex_assembly In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. RESULTS +27 36 structure evidence In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. RESULTS +38 42 Thr1 residue_name_number In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. RESULTS +49 53 Thr1 residue_name_number In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. RESULTS +104 110 Ser129 residue_name_number In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. RESULTS +122 124 WT protein_state In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation. RESULTS +7 24 MG132-bound state protein_state In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS +26 30 Thr1 residue_name_number In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS +35 45 unmodified protein_state In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS +73 79 Ser129 residue_name_number In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS +83 98 hydrogen-bonded bond_interaction In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS +104 109 water chemical In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS +130 136 Asn166 residue_name_number In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166. RESULTS +8 11 Asn residue_name Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis. RESULTS +39 45 Asp166 residue_name_number Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis. RESULTS +91 94 Ala residue_name Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis. RESULTS +138 147 autolysis ptm Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis. RESULTS +27 33 Asp166 residue_name_number These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis. RESULTS +38 44 Ser129 residue_name_number These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis. RESULTS +110 114 Thr1 residue_name_number These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis. RESULTS +123 132 autolysis ptm These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis. RESULTS +0 12 Substitution experimental_method Substitution of the active-site Thr1 by Cys RESULTS +20 31 active-site site Substitution of the active-site Thr1 by Cys RESULTS +32 36 Thr1 residue_name_number Substitution of the active-site Thr1 by Cys RESULTS +40 43 Cys residue_name Substitution of the active-site Thr1 by Cys RESULTS +0 8 Mutation experimental_method Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. RESULTS +12 16 Thr1 residue_name_number Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. RESULTS +20 23 Cys residue_name Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. RESULTS +40 54 20S proteasome complex_assembly Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. RESULTS +64 72 archaeon taxonomy_domain Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. RESULTS +73 87 T. acidophilum species Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum. RESULTS +3 8 yeast taxonomy_domain In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure. RESULTS +15 23 mutation experimental_method In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure. RESULTS +90 100 propeptide structure_element In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure. RESULTS +137 152 X-ray structure evidence In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure. RESULTS +18 20 β5 protein In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). RESULTS +53 60 cleaved protein_state In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). RESULTS +61 71 propeptide structure_element In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). RESULTS +72 83 still bound protein_state In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). RESULTS +91 116 substrate-binding channel site In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c). RESULTS +0 7 His(-2) residue_name_number His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS +21 30 S2 pocket site His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS +53 64 β5-T1A-K81R mutant His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS +65 71 mutant protein_state His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS +108 118 propeptide structure_element His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS +126 129 T1C mutant His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS +130 136 mutant protein_state His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS +147 167 antiparallel β-sheet structure_element His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS +211 216 MG132 chemical His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b). RESULTS +37 40 T1C mutant On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. RESULTS +41 47 mutant protein_state On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. RESULTS +56 66 propeptide structure_element On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. RESULTS +93 104 active site site On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. RESULTS +122 131 autolysis ptm On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. RESULTS +183 198 crystallization experimental_method On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization. RESULTS +42 44 β5 protein Owing to the unequal positions of the two β5 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits. RESULTS +65 67 CP complex_assembly Owing to the unequal positions of the two β5 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits. RESULTS +107 117 propeptide structure_element Owing to the unequal positions of the two β5 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits. RESULTS +8 29 propeptide hydrolysis ptm Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a). RESULTS +35 41 β5-T1C mutant Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a). RESULTS +42 53 active site site Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a). RESULTS +57 79 catalytically inactive protein_state Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a). RESULTS +14 30 soaking crystals experimental_method In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS +40 42 CP complex_assembly In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS +54 64 bortezomib chemical In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS +68 79 carfilzomib chemical In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS +98 100 β1 protein In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS +105 107 β2 protein In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS +108 120 active sites site In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS +140 146 β5-T1C mutant In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS +147 166 proteolytic centres site In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS +167 177 unmodified protein_state In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS +230 237 cleaved protein_state In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS +238 248 propeptide structure_element In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant. RESULTS +14 29 structural data evidence Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74° with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b). RESULTS +61 65 Cys1 residue_name_number Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74° with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b). RESULTS +127 131 Thr1 residue_name_number Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74° with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b). RESULTS +18 31 hydrogen bond bond_interaction Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. RESULTS +73 78 Lys33 residue_name_number Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. RESULTS +82 84 WT protein_state Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. RESULTS +85 88 CPs complex_assembly Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. RESULTS +104 108 Cys1 residue_name_number Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1. RESULTS +13 40 2FO–FC electron-density map evidence Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. RESULTS +48 51 T1C mutant Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. RESULTS +52 58 mutant protein_state Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. RESULTS +79 84 Lys33 residue_name_number Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. RESULTS +91 101 disordered protein_state Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered. RESULTS +90 95 Lys33 residue_name_number Together, these observations suggest that efficient peptide-bond hydrolysis requires that Lys33NH2 hydrogen bonds to the active site nucleophile. RESULTS +99 113 hydrogen bonds bond_interaction Together, these observations suggest that efficient peptide-bond hydrolysis requires that Lys33NH2 hydrogen bonds to the active site nucleophile. RESULTS +15 18 Thr residue_name The benefit of Thr over Ser as the active-site nucleophile RESULTS +24 27 Ser residue_name The benefit of Thr over Ser as the active-site nucleophile RESULTS +4 15 proteasomes complex_assembly All proteasomes strictly employ threonine as the active-site residue instead of serine. RESULTS +16 31 strictly employ protein_state All proteasomes strictly employ threonine as the active-site residue instead of serine. RESULTS +32 41 threonine residue_name All proteasomes strictly employ threonine as the active-site residue instead of serine. RESULTS +49 68 active-site residue site All proteasomes strictly employ threonine as the active-site residue instead of serine. RESULTS +80 86 serine residue_name All proteasomes strictly employ threonine as the active-site residue instead of serine. RESULTS +62 68 β5-T1S mutant To investigate the reason for this singularity, we analysed a β5-T1S mutant, which is viable but suffers from growth defects (Fig. 4a and Table 1). RESULTS +69 75 mutant protein_state To investigate the reason for this singularity, we analysed a β5-T1S mutant, which is viable but suffers from growth defects (Fig. 4a and Table 1). RESULTS +0 15 Activity assays experimental_method Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS +25 27 β5 protein Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS +47 59 Suc-LLVY-AMC chemical Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS +104 107 T1S mutant Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS +108 114 mutant protein_state Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS +150 152 WT protein_state Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS +153 164 proteasomes complex_assembly Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c). RESULTS +39 48 Z-GGL-pNA chemical By contrast, turnover of the substrate Z-GGL-pNA, used to monitor ChT-L activity in situ but in a less quantitative fashion, is not detectably impaired (Supplementary Fig. 9a). RESULTS +0 17 Crystal structure evidence Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS +34 40 β5-T1S mutant Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS +41 47 mutant protein_state Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS +58 78 precursor processing ptm Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS +94 108 ligand-complex complex_assembly Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS +109 119 structures evidence Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS +125 135 bortezomib chemical Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS +140 151 carfilzomib chemical Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS +197 201 Ser1 residue_name_number Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5). RESULTS +13 16 apo protein_state However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g). RESULTS +17 34 crystal structure evidence However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g). RESULTS +49 53 Ser1 residue_name_number However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g). RESULTS +80 105 substrate-binding channel site However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g). RESULTS +14 18 Thr1 residue_name_number Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. RESULTS +24 26 WT protein_state Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. RESULTS +27 29 CP complex_assembly Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. RESULTS +30 40 structures evidence Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. RESULTS +42 46 Ser1 residue_name_number Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°. RESULTS +30 34 Ser1 residue_name_number Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1. RESULTS +41 56 hydrogen-bonded bond_interaction Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1. RESULTS +60 65 Lys33 residue_name_number Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1. RESULTS +179 183 Thr1 residue_name_number Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1. RESULTS +4 23 active-site residue site The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). RESULTS +24 28 Thr1 residue_name_number The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). RESULTS +89 113 hydrophobic interactions bond_interaction The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). RESULTS +119 123 Thr3 residue_name_number The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). RESULTS +128 133 Ala46 residue_name_number The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h). RESULTS +36 40 Thr1 residue_name_number Consequently, the hydroxyl group of Thr1 requires no reorientation before substrate cleavage and is thus more catalytically efficient than Ser1. RESULTS +139 143 Ser1 residue_name_number Consequently, the hydroxyl group of Thr1 requires no reorientation before substrate cleavage and is thus more catalytically efficient than Ser1. RESULTS +62 65 T1S mutant In agreement, at an elevated growing temperature of 37 °C the T1S mutant is unable to grow (Fig. 4a). RESULTS +66 72 mutant protein_state In agreement, at an elevated growing temperature of 37 °C the T1S mutant is unable to grow (Fig. 4a). RESULTS +14 20 mutant protein_state In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). RESULTS +21 31 proteasome complex_assembly In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). RESULTS +55 65 proteasome complex_assembly In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). RESULTS +80 90 bortezomib chemical In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). RESULTS +106 117 carfilzomib chemical In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5). RESULTS +14 31 inhibitor complex complex_assembly Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. RESULTS +32 42 structures evidence Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. RESULTS +94 96 WT protein_state Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. RESULTS +97 100 yCP complex_assembly Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. RESULTS +101 111 structures evidence Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. RESULTS +113 137 with the same inhibitors protein_state Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors. RESULTS +13 21 affinity evidence Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein. RESULTS +42 53 carfilzomib chemical Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein. RESULTS +106 131 substrate-binding channel site Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein. RESULTS +151 161 bortezomib chemical Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein. RESULTS +11 30 mean residence time evidence Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased. RESULTS +34 45 carfilzomib chemical Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased. RESULTS +53 64 active site site Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased. RESULTS +123 127 Ser1 residue_name_number Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased. RESULTS +89 98 threonine residue_name Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.). RESULTS +133 144 proteasomes complex_assembly Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.). RESULTS +189 214 proteasome-like proteases protein_type Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.). RESULTS +223 227 HslV protein Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.). RESULTS +4 18 20S proteasome complex_assembly The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized. DISCUSS +19 21 CP complex_assembly The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized. DISCUSS +35 57 non-lysosomal protease protein_type The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized. DISCUSS +61 71 eukaryotic taxonomy_domain The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized. DISCUSS +4 13 β-subunit protein The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex. DISCUSS +14 25 propeptides structure_element The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex. DISCUSS +48 50 β5 protein The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex. DISCUSS +107 109 CP complex_assembly The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex. DISCUSS +59 63 Thr1 residue_name_number In addition, they prevent irreversible inactivation of the Thr1 N terminus by N-acetylation. DISCUSS +78 91 N-acetylation ptm In addition, they prevent irreversible inactivation of the Thr1 N terminus by N-acetylation. DISCUSS +17 28 prosegments structure_element By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. DISCUSS +32 42 β subunits protein By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. DISCUSS +63 71 archaeal taxonomy_domain By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. DISCUSS +72 82 proteasome complex_assembly By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. DISCUSS +107 131 heterologously expressed experimental_method By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. DISCUSS +135 151 Escherichia coli species By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli. DISCUSS +3 13 eukaryotes taxonomy_domain In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated. DISCUSS +52 54 β1 protein In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated. DISCUSS +59 61 β2 protein In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated. DISCUSS +62 73 propeptides structure_element In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated. DISCUSS +9 19 removal of experimental_method However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects. DISCUSS +24 26 β5 protein However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects. DISCUSS +27 37 prosegment structure_element However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects. DISCUSS +71 73 β5 protein These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. DISCUSS +74 84 propeptide structure_element These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. DISCUSS +100 102 β5 protein These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. DISCUSS +103 114 active site site These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. DISCUSS +150 160 eukaryotic taxonomy_domain These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. DISCUSS +161 163 CP complex_assembly These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP. DISCUSS +27 44 atomic structures evidence Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide. DISCUSS +56 62 β5-T1A mutant Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide. DISCUSS +135 137 β5 protein Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide. DISCUSS +138 148 propeptide structure_element Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide. DISCUSS +17 21 (-2) residue_number Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS +50 60 propeptide structure_element Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS +80 87 Gly(-1) residue_name_number Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS +98 108 structures evidence Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS +158 162 Thr1 residue_name_number Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS +197 207 tight turn structure_element Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS +225 235 prosegment structure_element Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS +247 249 β1 protein Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1. DISCUSS +57 64 Gly(-1) residue_name_number From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis. DISCUSS +69 73 Thr1 residue_name_number From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis. DISCUSS +118 129 active site site From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis. DISCUSS +147 156 autolysis ptm From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis. DISCUSS +30 43 N-acetylation ptm In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1Oγ due to the rotational freedom and flexibility of the acetyl group. DISCUSS +51 55 Thr1 residue_name_number In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1Oγ due to the rotational freedom and flexibility of the acetyl group. DISCUSS +88 92 Thr1 residue_name_number In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1Oγ due to the rotational freedom and flexibility of the acetyl group. DISCUSS +4 14 propeptide structure_element The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2). DISCUSS +43 68 substrate-binding channel site The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2). DISCUSS +90 97 Gly(-1) residue_name_number The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2). DISCUSS +162 166 (-2) residue_number The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2). DISCUSS +28 30 CP complex_assembly Autolytic activation of the CP constitutes one of the final steps of proteasome biogenesis, but the trigger for propeptide cleavage had remained enigmatic. DISCUSS +112 131 propeptide cleavage ptm Autolytic activation of the CP constitutes one of the final steps of proteasome biogenesis, but the trigger for propeptide cleavage had remained enigmatic. DISCUSS +29 38 CP:ligand complex_assembly On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture. DISCUSS +146 156 proteasome complex_assembly On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture. DISCUSS +157 181 active-site architecture site On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture. DISCUSS +13 28 catalytic triad site We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS +37 48 active site site We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS +56 58 CP complex_assembly We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS +82 86 Thr1 residue_name_number We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS +88 93 Lys33 residue_name_number We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS +98 101 Asp residue_name We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS +102 107 Glu17 residue_name_number We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS +162 172 eukaryotic taxonomy_domain We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS +174 183 bacterial taxonomy_domain We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS +188 196 archaeal taxonomy_domain We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS +197 207 proteasome complex_assembly We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits. DISCUSS +0 5 Lys33 residue_name_number Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS +58 79 autocatalytic removal ptm Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS +87 98 propeptides structure_element Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS +147 152 Asp17 residue_name_number Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS +163 168 Lys33 residue_name_number Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS +231 244 hydrogen bond bond_interaction Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS +255 260 Lys33 residue_name_number Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS +265 270 Asp17 residue_name_number Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å). DISCUSS +19 29 proteasome complex_assembly Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase. DISCUSS +33 50 Thr–Lys–Asp triad site Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase. DISCUSS +68 82 L-asparaginase protein_type Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase. DISCUSS +107 110 Lys residue_name Thus, specific protein surroundings can significantly alter the chemical properties of amino acids such as Lys to function as an acid–base catalyst. DISCUSS +36 47 active site site In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). DISCUSS +72 76 Thr1 residue_name_number In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). DISCUSS +90 104 hydrogen bonds bond_interaction In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). DISCUSS +207 231 endoproteolytic cleavage ptm In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). DISCUSS +235 239 Thr1 residue_name_number In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d). DISCUSS +51 55 Thr1 residue_name_number Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). DISCUSS +81 95 hydrogen bonds bond_interaction Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). DISCUSS +127 140 fellutamide B chemical Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). DISCUSS +149 161 α-ketoamides chemical Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). DISCUSS +163 179 homobelactosin C chemical Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). DISCUSS +191 208 salinosporamide A chemical Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.). DISCUSS +47 56 omuralide chemical Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+. DISCUSS +60 64 Thr1 residue_name_number Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+. DISCUSS +123 127 Thr1 residue_name_number Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+. DISCUSS +24 28 Thr1 residue_name_number The resulting uncharged Thr1NH2 is hydrogen-bridged to the C3-OH group. DISCUSS +35 51 hydrogen-bridged bond_interaction The resulting uncharged Thr1NH2 is hydrogen-bridged to the C3-OH group. DISCUSS +14 25 acetylation ptm In agreement, acetylation of the Thr1 N terminus irreversibly blocks hydrolytic activity, and binding of substrates is prevented for steric reasons. DISCUSS +33 37 Thr1 residue_name_number In agreement, acetylation of the Thr1 N terminus irreversibly blocks hydrolytic activity, and binding of substrates is prevented for steric reasons. DISCUSS +50 54 Thr1 residue_name_number By acting as a proton donor during catalysis, the Thr1 N terminus may also favour cleavage of substrate peptide bonds (Fig. 3d). DISCUSS +98 108 proteasome complex_assembly Cleavage of the scissile peptide bond requires protonation of the emerging free amine, and in the proteasome, the Thr1 amine group is likely to assume this function. DISCUSS +114 118 Thr1 residue_name_number Cleavage of the scissile peptide bond requires protonation of the emerging free amine, and in the proteasome, the Thr1 amine group is likely to assume this function. DISCUSS +13 17 Thr1 residue_name_number Analogously, Thr1NH3+ might promote the bivalent reaction mode of epoxyketone inhibitors by protonating the epoxide moiety to create a positively charged trivalent oxygen atom that is subsequently nucleophilically attacked by Thr1NH2. DISCUSS +226 230 Thr1 residue_name_number Analogously, Thr1NH3+ might promote the bivalent reaction mode of epoxyketone inhibitors by protonating the epoxide moiety to create a positively charged trivalent oxygen atom that is subsequently nucleophilically attacked by Thr1NH2. DISCUSS +7 16 autolysis ptm During autolysis the Thr1 N terminus is engaged in a hydroxyoxazolidine ring intermediate (Fig. 3d), which is unstable and short-lived. DISCUSS +21 25 Thr1 residue_name_number During autolysis the Thr1 N terminus is engaged in a hydroxyoxazolidine ring intermediate (Fig. 3d), which is unstable and short-lived. DISCUSS +60 64 Thr1 residue_name_number Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+. DISCUSS +98 115 aspartic acid 166 residue_name_number Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+. DISCUSS +120 126 Ser129 residue_name_number Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+. DISCUSS +138 142 Thr1 residue_name_number Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+. DISCUSS +13 19 Ser129 residue_name_number The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state. DISCUSS +24 30 Asp166 residue_name_number The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state. DISCUSS +73 77 Thr1 residue_name_number The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state. DISCUSS +67 75 mutation experimental_method Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS +76 81 D166A mutant Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS +91 100 autolysis ptm Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS +108 116 archaeal taxonomy_domain Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS +117 119 CP complex_assembly Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS +128 136 exchange experimental_method Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS +137 142 D166N mutant Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS +177 182 yeast taxonomy_domain Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS +183 185 CP complex_assembly Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%. DISCUSS +4 12 mutation experimental_method The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2). DISCUSS +13 18 D166N mutant The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2). DISCUSS +37 41 Thr1 residue_name_number The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2). DISCUSS +116 120 Thr1 residue_name_number The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2). DISCUSS +79 87 β5-D166N mutant This interpretation agrees with the strongly reduced catalytic activity of the β5-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other. DISCUSS +88 94 mutant protein_state This interpretation agrees with the strongly reduced catalytic activity of the β5-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other. DISCUSS +150 161 carfilzomib chemical This interpretation agrees with the strongly reduced catalytic activity of the β5-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other. DISCUSS +11 21 proteasome complex_assembly Hence, the proteasome can be viewed as having a second triad that is essential for efficient proteolysis. DISCUSS +48 60 second triad site Hence, the proteasome can be viewed as having a second triad that is essential for efficient proteolysis. DISCUSS +6 11 Lys33 residue_name_number While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. DISCUSS +19 24 Asp17 residue_name_number While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. DISCUSS +59 63 Thr1 residue_name_number While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. DISCUSS +85 91 Ser129 residue_name_number While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. DISCUSS +98 104 Asp166 residue_name_number While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. DISCUSS +156 160 Thr1 residue_name_number While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1. DISCUSS +28 32 Thr1 residue_name_number In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS +33 38 Lys33 residue_name_number In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS +39 44 Asp17 residue_name_number In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS +45 60 catalytic triad site In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS +62 83 crystallographic data evidence In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS +91 115 proteolytically inactive protein_state In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS +116 122 β5-T1C mutant In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS +123 129 mutant protein_state In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS +166 171 Lys33 residue_name_number In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS +179 183 Cys1 residue_name_number In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken. DISCUSS +18 21 Cys residue_name However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time. DISCUSS +54 64 propeptide structure_element However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time. DISCUSS +78 85 cleaved protein_state However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time. DISCUSS +48 57 autolysis ptm While only one single turnover is necessary for autolysis, continuous enzymatic activity is required for significant and detectable substrate hydrolysis. DISCUSS +16 29 Ntn hydrolase protein_type Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS +30 48 penicillin acylase protein_type Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS +50 62 substitution experimental_method Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS +70 79 catalytic protein_state Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS +91 94 Ser residue_name Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS +106 109 Cys residue_name Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS +115 126 inactivates protein_state Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS +131 137 enzyme protein_type Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS +156 176 precursor processing ptm Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing. DISCUSS +23 25 CP complex_assembly To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS +47 56 threonine residue_name To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS +64 83 active-site residue site To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS +95 101 β5-T1S mutant To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS +102 108 mutant protein_state To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS +116 119 yCP complex_assembly To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS +141 171 biochemically and structurally experimental_method To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally. DISCUSS +0 15 Activity assays experimental_method Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC. DISCUSS +25 31 β5-T1S mutant Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC. DISCUSS +32 38 mutant protein_state Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC. DISCUSS +68 80 Suc-LLVY-AMC chemical Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC. DISCUSS +48 54 β5-T1S mutant We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. DISCUSS +55 61 mutant protein_state We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. DISCUSS +62 65 yCP complex_assembly We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. DISCUSS +112 122 proteasome complex_assembly We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. DISCUSS +134 144 bortezomib chemical We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. DISCUSS +149 160 carfilzomib chemical We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib. DISCUSS +0 19 Structural analyses evidence Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. DISCUSS +52 55 T1S mutant Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. DISCUSS +56 62 mutant protein_state Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. DISCUSS +98 111 strict use of protein_state Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. DISCUSS +112 115 Thr residue_name Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. DISCUSS +128 139 proteasomes complex_assembly Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes. DISCUSS +0 4 Thr1 residue_name_number Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). DISCUSS +29 40 active site site Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). DISCUSS +44 68 hydrophobic interactions bond_interaction Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). DISCUSS +97 102 Ala46 residue_name_number Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). DISCUSS +109 114 Lys33 residue_name_number Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). DISCUSS +139 143 Thr3 residue_name_number Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ). DISCUSS +9 31 proteolytically active protein_state Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS +32 42 proteasome complex_assembly Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS +57 64 archaea taxonomy_domain Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS +66 71 yeast taxonomy_domain Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS +76 83 mammals taxonomy_domain Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS +161 164 Thr residue_name Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS +168 171 Ile residue_name Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS +184 185 3 residue_number Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS +267 271 Thr1 residue_name_number Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1. DISCUSS +15 19 Thr1 residue_name_number In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). DISCUSS +43 47 Ser1 residue_name_number In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). DISCUSS +77 81 Thr1 residue_name_number In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). DISCUSS +107 109 WT protein_state In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). DISCUSS +110 116 enzyme complex_assembly In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h). DISCUSS +16 35 threonine aspartase protein_type Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS +36 44 Taspase1 protein Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS +46 54 mutation experimental_method Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS +62 73 active-site site Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS +74 80 Thr234 residue_name_number Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS +84 87 Ser residue_name Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS +154 160 Thr234 residue_name_number Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS +168 170 WT protein_state Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity. DISCUSS +24 30 serine residue_name Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. DISCUSS +31 37 mutant protein_state Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. DISCUSS +41 47 active protein_state Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. DISCUSS +49 58 threonine residue_name Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. DISCUSS +99 109 proteasome complex_assembly Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. DISCUSS +110 121 active site site Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site. DISCUSS +27 36 threonine residue_name The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS +45 55 proteasome complex_assembly The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS +56 67 active site site The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS +172 184 conservation protein_state The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS +192 196 Thr1 residue_name_number The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS +212 223 proteasomes complex_assembly The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS +229 237 bacteria taxonomy_domain The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS +241 251 eukaryotes taxonomy_domain The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes. DISCUSS +28 39 propeptides structure_element Conformation of proteasomal propeptides. FIG +4 28 Structural superposition experimental_method (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG +36 42 β1-T1A mutant (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG +43 53 propeptide structure_element (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG +62 69 matured protein_state (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG +70 72 WT protein_state (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG +73 75 β1 protein (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG +76 87 active-site site (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG +88 92 Thr1 residue_name_number (a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1. FIG +18 30 (-5) to (-1) residue_range Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed. FIG +38 44 β1-T1A mutant Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed. FIG +45 55 propeptide structure_element Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed. FIG +29 50 S1 specificity pocket site The major determinant of the S1 specificity pocket, residue 45, is depicted. FIG +60 62 45 residue_number The major determinant of the S1 specificity pocket, residue 45, is depicted. FIG +29 50 S1 specificity pocket site The major determinant of the S1 specificity pocket, residue 45, is depicted. FIG +60 62 45 residue_number The major determinant of the S1 specificity pocket, residue 45, is depicted. FIG +31 38 Gly(-1) residue_name_number Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG +43 47 Ala1 residue_name_number Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG +55 65 propeptide structure_element Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG +75 80 G(-1) residue_name_number Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG +133 142 processed protein_state Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG +143 145 WT protein_state Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG +146 157 active-site site Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG +158 162 Thr1 residue_name_number Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow). FIG +40 44 Thr1 residue_name_number The black arrow indicates the attack of Thr1Oγ onto the carbonyl carbon atom of Gly(-1). FIG +80 87 Gly(-1) residue_name_number The black arrow indicates the attack of Thr1Oγ onto the carbonyl carbon atom of Gly(-1). FIG +4 28 Structural superposition experimental_method (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG +36 42 β1-T1A mutant (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG +43 53 propeptide structure_element (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG +62 68 β2-T1A mutant (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG +69 79 propeptide structure_element (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG +155 159 Ala1 residue_name_number (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG +164 171 Gly(-1) residue_name_number (b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well. FIG +0 7 Thr(-2) residue_name_number Thr(-2)OH is hydrogen-bonded to Gly(-1)O (∼2.8 Å; black dashed line). FIG +13 28 hydrogen-bonded bond_interaction Thr(-2)OH is hydrogen-bonded to Gly(-1)O (∼2.8 Å; black dashed line). FIG +32 39 Gly(-1) residue_name_number Thr(-2)OH is hydrogen-bonded to Gly(-1)O (∼2.8 Å; black dashed line). FIG +4 28 Structural superposition experimental_method (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. FIG +36 42 β1-T1A mutant (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. FIG +48 54 β2-T1A mutant (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. FIG +63 74 β5-T1A-K81R mutant (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. FIG +75 85 propeptide structure_element (c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation. FIG +14 18 (-2) residue_number While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG +26 28 β1 protein While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG +33 35 β2 protein While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG +36 47 prosegments structure_element While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG +56 65 S1 pocket site While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG +67 74 His(-2) residue_name_number While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG +82 84 β5 protein While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG +85 95 propeptide structure_element While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG +109 118 S2 pocket site While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket. FIG +56 63 Gly(-1) residue_name_number Nonetheless, in all mutants the carbonyl carbon atom of Gly(-1) is ideally placed for the nucleophilic attack by Thr1Oγ. FIG +113 117 Thr1 residue_name_number Nonetheless, in all mutants the carbonyl carbon atom of Gly(-1) is ideally placed for the nucleophilic attack by Thr1Oγ. FIG +4 17 hydrogen bond bond_interaction The hydrogen bond between Thr(-2)OH and Gly(-1)O (∼2.8 Å) is indicated by a black dashed line. FIG +26 33 Thr(-2) residue_name_number The hydrogen bond between Thr(-2)OH and Gly(-1)O (∼2.8 Å) is indicated by a black dashed line. FIG +40 47 Gly(-1) residue_name_number The hydrogen bond between Thr(-2)OH and Gly(-1)O (∼2.8 Å) is indicated by a black dashed line. FIG +0 9 Mutations experimental_method Mutations of residue (-2) and their influence on propeptide conformation and autolysis. FIG +21 25 (-2) residue_number Mutations of residue (-2) and their influence on propeptide conformation and autolysis. FIG +49 59 propeptide structure_element Mutations of residue (-2) and their influence on propeptide conformation and autolysis. FIG +77 86 autolysis ptm Mutations of residue (-2) and their influence on propeptide conformation and autolysis. FIG +4 28 Structural superposition experimental_method (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. FIG +36 42 β1-T1A mutant (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. FIG +43 53 propeptide structure_element (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. FIG +62 75 β5-H(-2)L-T1A mutant (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. FIG +76 82 mutant protein_state (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. FIG +83 93 propeptide structure_element (a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide. FIG +4 8 (-2) residue_number The (-2) residues of both prosegments point into the S1 pocket. FIG +26 37 prosegments structure_element The (-2) residues of both prosegments point into the S1 pocket. FIG +53 62 S1 pocket site The (-2) residues of both prosegments point into the S1 pocket. FIG +4 28 Structural superposition experimental_method (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG +36 38 β5 protein (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG +39 50 propeptides structure_element (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG +58 71 β5-H(-2)L-T1A mutant (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG +73 86 β5-H(-2)T-T1A mutant (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG +88 106 β5-(H-2)A-T1A-K81R mutant (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG +111 122 β5-T1A-K81R mutant (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG +123 129 mutant protein_state (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG +130 141 proteasomes complex_assembly (b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes. FIG +19 31 (-2) to (-4) residue_range While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions. FIG +60 67 Gly(-1) residue_name_number While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions. FIG +72 76 Ala1 residue_name_number While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions. FIG +96 106 structures evidence While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions. FIG +4 28 Structural superposition experimental_method (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. FIG +36 42 β2-T1A mutant (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. FIG +43 53 propeptide structure_element (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. FIG +62 75 β5-H(-2)T-T1A mutant (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. FIG +76 82 mutant protein_state (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. FIG +83 93 propeptide structure_element (c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide. FIG +4 8 (-2) residue_number The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG +26 37 prosegments structure_element The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG +53 62 S1 pocket site The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG +73 80 Thr(-2) residue_name_number The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG +86 88 β2 protein The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG +97 112 hydrogen bridge bond_interaction The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG +116 123 Gly(-1) residue_name_number The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line). FIG +4 28 Structural superposition experimental_method (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG +36 43 matured protein_state (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG +44 46 β2 protein (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG +47 58 active site site (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG +64 66 WT protein_state (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG +67 73 β2-T1A mutant (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG +74 84 propeptide structure_element (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG +93 102 β2-T(-2)V mutant (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG +103 109 mutant protein_state (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG +110 120 propeptide structure_element (d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide. FIG +9 16 Val(-2) residue_name_number Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). FIG +51 60 S1 pocket site Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). FIG +98 105 Gly(-1) residue_name_number Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). FIG +144 148 Thr1 residue_name_number Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). FIG +182 189 Gly(-1) residue_name_number Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1). FIG +64 75 active site site Architecture and proposed reaction mechanism of the proteasomal active site. FIG +4 28 Hydrogen-bonding network site (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). FIG +36 42 mature protein_state (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). FIG +43 45 WT protein_state (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). FIG +46 48 β5 protein (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). FIG +61 72 active site site (a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). FIG +0 4 Thr1 residue_name_number Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ. FIG +10 25 hydrogen-bonded bond_interaction Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ. FIG +29 34 Lys33 residue_name_number Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ. FIG +76 81 Asp17 residue_name_number Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ. FIG +4 8 Thr1 residue_name_number The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG +34 48 hydrogen bonds bond_interaction The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG +54 60 Ser129 residue_name_number The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG +95 98 168 residue_number The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG +100 106 Ser169 residue_name_number The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG +113 119 Asp166 residue_name_number The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG +151 171 active-site residues site The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG +184 200 hydrogen bonding bond_interaction The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG +205 223 strictly conserved protein_state The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG +232 250 proteolytic centre site The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG +264 277 superposition experimental_method The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG +285 295 β subunits protein The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. FIG +4 28 Structural superposition experimental_method (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG +36 38 WT protein_state (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG +39 41 β5 protein (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG +50 57 β5-K33A mutant (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG +58 60 pp chemical (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG +61 66 trans protein_state (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG +67 73 mutant protein_state (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG +74 85 active site site (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. FIG +17 22 water chemical In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located. FIG +83 85 WT protein_state In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located. FIG +126 131 Lys33 residue_name_number In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located. FIG +13 18 Lys33 residue_name_number Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. FIG +24 29 water chemical Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. FIG +39 53 hydrogen bonds bond_interaction Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. FIG +57 62 Arg19 residue_name_number Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. FIG +65 70 Asp17 residue_name_number Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. FIG +77 81 Thr1 residue_name_number Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH. FIG +38 43 water chemical Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place. FIG +77 81 Thr1 residue_name_number Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place. FIG +99 119 active-site residues site Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place. FIG +45 79 autocatalytic precursor processing ptm (d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome. FIG +103 113 proteasome complex_assembly (d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome. FIG +4 15 active-site site The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red. FIG +16 20 Thr1 residue_name_number The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red. FIG +46 56 propeptide structure_element The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red. FIG +0 9 Autolysis ptm Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state. FIG +68 72 Thr1 residue_name_number Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state. FIG +79 84 Lys33 residue_name_number Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state. FIG +4 22 strictly conserved protein_state The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle. FIG +37 42 Gly47 residue_name_number The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle. FIG +43 47 Thr1 residue_name_number Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH. FIG +102 112 propeptide structure_element Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH. FIG +151 157 Asp166 residue_name_number Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH. FIG +164 170 Ser129 residue_name_number Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH. FIG +6 10 Thr1 residue_name_number Next, Thr1NH2 polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate. FIG +26 31 water chemical Next, Thr1NH2 polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate. FIG +33 44 active-site site On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures). FIG +45 49 Thr1 residue_name_number On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures). FIG +12 16 Thr1 residue_name_number The charged Thr1 N terminus may engage in the orientation of the amide moiety and donate a proton to the emerging N terminus of the C-terminal cleavage product. FIG +27 31 Thr1 residue_name_number The resulting deprotonated Thr1NH2 finally activates a water molecule for hydrolysis of the acyl-enzyme. FIG +55 60 water chemical The resulting deprotonated Thr1NH2 finally activates a water molecule for hydrolysis of the acyl-enzyme. FIG +4 14 proteasome complex_assembly The proteasome favours threonine as the active-site nucleophile. FIG +23 32 threonine residue_name The proteasome favours threonine as the active-site nucleophile. FIG +4 35 Growth tests by serial dilution experimental_method (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG +39 41 WT protein_state (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG +52 54 β5 protein (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG +56 62 mutant protein_state (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG +63 68 yeast taxonomy_domain (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG +107 118 active-site site (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG +119 126 mutants experimental_method (a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation. FIG +13 15 WT protein_state (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. FIG +20 26 mutant protein_state (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. FIG +27 38 proteasomes complex_assembly (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. FIG +89 91 β5 protein (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. FIG +113 125 Suc-LLVY-AMC chemical (b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC. FIG +24 51 2FO–FC electron-density map evidence (c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment. FIG +88 94 β5-T1C mutant (c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment. FIG +95 105 propeptide structure_element (c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment. FIG +4 14 prosegment structure_element The prosegment is cleaved but still bound in the substrate-binding channel. FIG +18 25 cleaved protein_state The prosegment is cleaved but still bound in the substrate-binding channel. FIG +30 41 still bound protein_state The prosegment is cleaved but still bound in the substrate-binding channel. FIG +49 74 substrate-binding channel site The prosegment is cleaved but still bound in the substrate-binding channel. FIG +9 16 His(-2) residue_name_number Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. FIG +37 46 S1 pocket site Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. FIG +57 62 Met45 residue_name_number Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. FIG +101 112 β5-T1A-K81R mutant Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. FIG +113 119 mutant protein_state Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant. FIG +4 28 Structural superposition experimental_method (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +36 47 β5-T1A-K81R mutant (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +56 62 β5-T1C mutant (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +63 69 mutant protein_state (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +88 90 WT protein_state (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +91 93 β5 protein (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +107 131 Structural superposition experimental_method (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +139 145 β5-T1C mutant (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +146 156 propeptide structure_element (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +166 172 β1-T1A mutant (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +173 184 active site site (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +200 202 WT protein_state (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +203 205 β5 protein (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +206 217 active site site (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +218 233 in complex with protein_state (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +238 248 proteasome complex_assembly (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +259 264 MG132 chemical (d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.). FIG +4 13 inhibitor chemical The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel. FIG +29 40 propeptides structure_element The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel. FIG +76 101 substrate-binding channel site The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel. FIG +4 28 Structural superposition experimental_method (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG +36 38 WT protein_state (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG +39 41 β5 protein (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG +46 52 β5-T1C mutant (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG +53 59 mutant protein_state (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG +60 72 active sites site (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG +137 141 Thr1 residue_name_number (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG +170 174 Cys1 residue_name_number (f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1. FIG +4 28 Structural superposition experimental_method (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG +36 38 WT protein_state (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG +39 41 β5 protein (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG +46 52 β5-T1S mutant (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG +53 59 mutant protein_state (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG +60 72 active sites site (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG +130 134 Thr1 residue_name_number (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG +139 143 Ser1 residue_name_number (g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively. FIG +4 31 2FO–FC electron-density map evidence The 2FO–FC electron-density map for Ser1 (blue mesh contoured at 1σ) is illustrated. FIG +36 40 Ser1 residue_name_number The 2FO–FC electron-density map for Ser1 (blue mesh contoured at 1σ) is illustrated. FIG +24 28 Thr1 residue_name_number (h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ. FIG +44 68 hydrophobic interactions bond_interaction (h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ. FIG +74 79 Ala46 residue_name_number (h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ. FIG +86 90 Thr3 residue_name_number (h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ. FIG +0 4 Ser1 residue_name_number Ser1 lacks this stabilization and is therefore rotated by 60°. FIG +5 10 lacks protein_state Ser1 lacks this stabilization and is therefore rotated by 60°. FIG +14 16 WT protein_state Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. FIG +21 27 mutant protein_state Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. FIG +28 34 β5-T1S mutant Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. FIG +35 46 proteasomes complex_assembly Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. FIG +50 60 bortezomib chemical Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. FIG +65 76 carfilzomib chemical Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib. FIG +0 17 Inhibition assays experimental_method Inhibition assays (left panel). FIG +9 14 yeast taxonomy_domain Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. FIG +15 26 proteasomes complex_assembly Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. FIG +78 80 β5 protein Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. FIG +108 118 bortezomib chemical Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. FIG +123 134 carfilzomib chemical Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. FIG +155 167 Suc-LLVY-AMC chemical Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC. FIG +0 11 IC50 values evidence IC50 values were determined in triplicate; s.d.'s are indicated by error bars. FIG +10 21 IC50 values evidence Note that IC50 values depend on time and enzyme concentration. FIG +0 11 Proteasomes complex_assembly Proteasomes (final concentration: 66 nM) were incubated with inhibitor for 45 min before substrate addition (final concentration: 200 μM). FIG +0 10 Structures evidence Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). FIG +18 24 β5-T1S mutant Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). FIG +25 31 mutant protein_state Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). FIG +35 60 complex with both ligands complex_assembly Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). FIG +93 97 Ser1 residue_name_number Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel). FIG +4 32 2FO–FC electron-density maps evidence The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ. FIG +49 53 Ser1 residue_name_number The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ. FIG +112 119 P1 site site The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ. FIG +121 125 Leu1 residue_name_number The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ. FIG +4 6 WT protein_state The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG +7 35 proteasome:inhibitor complex complex_assembly The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG +36 46 structures evidence The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG +67 71 Thr1 residue_name_number The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG +86 98 superimposed experimental_method The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG +120 128 mutation experimental_method The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG +132 136 Thr1 residue_name_number The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG +140 143 Ser residue_name The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG +180 190 bortezomib chemical The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG +194 205 carfilzomib chemical The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib. FIG diff --git a/annotation_CSV/PMC4795551.csv b/annotation_CSV/PMC4795551.csv new file mode 100644 index 0000000000000000000000000000000000000000..fcffdd8ff0bedc6a2e5136041e1722b6967e66e1 --- /dev/null +++ b/annotation_CSV/PMC4795551.csv @@ -0,0 +1,1476 @@ +anno_start anno_end anno_text entity_type sentence section +17 34 Crystal Structure evidence Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily TITLE +38 54 Ectoine Synthase protein_type Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily TITLE +58 74 Metal-Containing protein_state Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily TITLE +89 106 Cupin Superfamily protein_type Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily TITLE +0 7 Ectoine chemical Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. ABSTRACT +84 92 Bacteria taxonomy_domain Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. ABSTRACT +103 110 Archaea taxonomy_domain Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. ABSTRACT +0 16 Ectoine synthase protein_type Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. ABSTRACT +18 22 EctC protein_type Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. ABSTRACT +51 58 ectoine chemical Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. ABSTRACT +117 157 N-gamma-acetyl-L-2,4-diaminobutyric acid chemical Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. ABSTRACT +168 173 water chemical Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. ABSTRACT +13 30 crystal structure evidence However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. ABSTRACT +34 50 ectoine synthase protein_type However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. ABSTRACT +10 26 ectoine synthase protein_type Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. ABSTRACT +49 65 marine bacterium taxonomy_domain Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. ABSTRACT +66 89 Sphingopyxis alaskensis species Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. ABSTRACT +91 93 Sa species Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. ABSTRACT +163 167 EctC protein Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. ABSTRACT +199 216 crystal structure evidence Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. ABSTRACT +224 227 apo protein_state Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. ABSTRACT +0 19 Structural analysis experimental_method Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. ABSTRACT +36 38 Sa species Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. ABSTRACT +39 43 EctC protein Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. ABSTRACT +71 88 cupin superfamily protein_type Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. ABSTRACT +0 4 EctC protein EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. ABSTRACT +13 18 dimer oligomeric_state EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. ABSTRACT +26 38 head-to-tail protein_state EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. ABSTRACT +80 97 crystal structure evidence EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. ABSTRACT +4 13 interface site The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. ABSTRACT +21 26 dimer oligomeric_state The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. ABSTRACT +81 105 hydrophobic interactions bond_interaction The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. ABSTRACT +122 133 beta-sheets structure_element The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. ABSTRACT +146 153 monomer oligomeric_state The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. ABSTRACT +32 48 ectoine synthase protein_type We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. ABSTRACT +83 88 metal chemical We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. ABSTRACT +100 146 metal depletion and reconstitution experiments experimental_method We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. ABSTRACT +160 164 EctC protein We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. ABSTRACT +180 194 iron-dependent protein_state We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. ABSTRACT +14 18 EctC protein We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. ABSTRACT +71 111 N-gamma-acetyl-L-2,4-diaminobutyric acid chemical We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. ABSTRACT +117 124 ectoine chemical We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. ABSTRACT +199 239 N-alpha-acetyl-L-2,4-diaminobutyric acid chemical We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. ABSTRACT +292 312 catalytic efficiency evidence We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. ABSTRACT +0 42 Structure-guided site-directed mutagenesis experimental_method Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. ABSTRACT +94 125 evolutionarily highly conserved protein_state Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. ABSTRACT +145 164 EctC protein family protein_type Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. ABSTRACT +206 223 iron-binding site site Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. ABSTRACT +296 300 EctC protein Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. ABSTRACT +37 41 iron chemical An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel. ABSTRACT +130 141 active site site An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel. ABSTRACT +176 180 EctC protein An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel. ABSTRACT +181 193 cupin barrel structure_element An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel. ABSTRACT +0 7 Ectoine chemical Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes. INTRO +9 68 (S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid chemical Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes. INTRO +89 105 5-hydroxyectoine chemical Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes. INTRO +107 180 (4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid chemical Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes. INTRO +5 42 marine and terrestrial microorganisms taxonomy_domain Both marine and terrestrial microorganisms produce them widely in response to osmotic or temperature stress. INTRO +13 20 ectoine chemical Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1). INTRO +61 87 L-aspartate-ß-semialdehyde chemical Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1). INTRO +146 180 L-2,4-diaminobutyrate transaminase protein_type Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1). INTRO +182 186 EctB protein_type Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1). INTRO +202 239 2,4-diaminobutyrate acetyltransferase protein_type Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1). INTRO +241 245 EctA protein_type Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1). INTRO +266 282 ectoine synthase protein_type Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1). INTRO +284 288 EctC protein_type Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1). INTRO +4 11 ectoine chemical The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers. INTRO +23 39 5-hydroxyectoine chemical The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers. INTRO +144 151 ectoine chemical The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers. INTRO +45 52 ectoine chemical This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. INTRO +81 100 ectoine hydroxylase protein_type This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. INTRO +102 106 EctD protein_type This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. INTRO +138 219 non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily protein_type This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. INTRO +46 54 ectoines chemical The remarkable function preserving effects of ectoines for macromolecules and cells, frequently also addressed as chemical chaperones, led to a substantial interest in exploiting these compounds for biotechnological purposes and medical applications. INTRO +24 31 ectoine chemical Biosynthetic routes for ectoine and 5-hydroxyectoine. FIG +36 52 5-hydroxyectoine chemical Biosynthetic routes for ectoine and 5-hydroxyectoine. FIG +14 21 ectoine chemical Scheme of the ectoine and 5-hydroxyectoine biosynthetic pathway. FIG +26 42 5-hydroxyectoine chemical Scheme of the ectoine and 5-hydroxyectoine biosynthetic pathway. FIG +17 33 ectoine synthase protein_type Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1). INTRO +35 39 EctC protein Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1). INTRO +64 71 ectoine chemical Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1). INTRO +12 29 characterizations experimental_method Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out. INTRO +33 50 ectoine synthases protein_type Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out. INTRO +60 73 extremophiles taxonomy_domain Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out. INTRO +74 92 Halomonas elongata species Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out. INTRO +94 123 Methylomicrobium alcaliphilum species Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out. INTRO +129 149 Acidiphilium cryptum species Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out. INTRO +164 183 nitrifying archaeon taxonomy_domain Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out. INTRO +184 208 Nitrosopumilus maritimus species Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out. INTRO +74 110 N-γ-acetyl-L-2,4-diaminobutyric acid chemical Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1). INTRO +112 121 N-γ-ADABA chemical Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1). INTRO +152 189 2,4-diaminobutyrate acetyltransferase protein_type Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1). INTRO +191 195 EctA protein_type Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1). INTRO +201 208 ectoine chemical Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1). INTRO +243 248 water chemical Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1). INTRO +19 23 EctC protein In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes. INTRO +85 129 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate chemical In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes. INTRO +131 135 ADPC chemical In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes. INTRO +176 185 glutamine chemical In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes. INTRO +250 257 ectoine chemical In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes. INTRO +272 279 ectoine chemical In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes. INTRO +84 100 ectoine synthase protein_type Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa). INTRO +135 144 structure evidence Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa). INTRO +248 275 biochemically characterized experimental_method Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa). INTRO +280 296 ectoine synthase protein_type Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa). INTRO +319 335 marine bacterium taxonomy_domain Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa). INTRO +336 359 Sphingopyxis alaskensis species Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa). INTRO +361 363 Sa species Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa). INTRO +48 64 ectoine synthase protein_type We demonstrate here for the first time that the ectoine synthase is a metal-dependent enzyme, with iron as the most likely physiologically relevant co-factor. INTRO +70 75 metal chemical We demonstrate here for the first time that the ectoine synthase is a metal-dependent enzyme, with iron as the most likely physiologically relevant co-factor. INTRO +99 103 iron chemical We demonstrate here for the first time that the ectoine synthase is a metal-dependent enzyme, with iron as the most likely physiologically relevant co-factor. INTRO +4 8 EctC protein The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily. INTRO +25 30 dimer oligomeric_state The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily. INTRO +51 70 structural analysis experimental_method The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily. INTRO +104 121 cupin superfamily protein_type The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily. INTRO +8 26 crystal structures evidence The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase. INTRO +56 58 Sa species The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase. INTRO +59 63 EctC protein The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase. INTRO +157 182 site-directed mutagenesis experimental_method The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase. INTRO +205 236 evolutionarily highly conserved protein_state The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase. INTRO +266 278 EctC protein protein_type The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase. INTRO +337 351 catalytic core site The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase. INTRO +359 375 ectoine synthase protein_type The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase. INTRO +0 14 Overproduction experimental_method Overproduction, purification and oligomeric state of the ectoine synthase in solution RESULTS +16 28 purification experimental_method Overproduction, purification and oligomeric state of the ectoine synthase in solution RESULTS +57 73 ectoine synthase protein_type Overproduction, purification and oligomeric state of the ectoine synthase in solution RESULTS +15 49 biochemical and structural studies experimental_method We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product. RESULTS +57 73 ectoine synthase protein_type We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product. RESULTS +79 92 S. alaskensis species We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product. RESULTS +95 97 Sa species We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product. RESULTS +98 102 EctC protein We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product. RESULTS +120 147 marine ultra-microbacterium taxonomy_domain We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product. RESULTS +192 209 crystal structure evidence We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product. RESULTS +217 236 ectoine hydroxylase protein_type We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product. RESULTS +238 242 EctD protein_type We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product. RESULTS +244 259 in complex with protein_state We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product. RESULTS +46 59 S. alaskensis species We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography. RESULTS +60 64 ectC gene We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography. RESULTS +73 80 E. coli species We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography. RESULTS +149 178 Strep-tag II affinity peptide experimental_method We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography. RESULTS +209 211 Sa species We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography. RESULTS +212 216 EctC protein We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography. RESULTS +217 229 Strep-Tag-II experimental_method We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography. RESULTS +241 264 affinity chromatography experimental_method We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography. RESULTS +5 7 Sa species The (Sa)EctC protein was overproduced and isolated with good yields (30–40 mg L-1 of culture) and purity (S2a Fig). RESULTS +8 12 EctC protein The (Sa)EctC protein was overproduced and isolated with good yields (30–40 mg L-1 of culture) and purity (S2a Fig). RESULTS +13 42 size-exclusion chromatography experimental_method Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution. RESULTS +44 47 SEC experimental_method Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution. RESULTS +73 75 Sa species Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution. RESULTS +76 80 EctC protein Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution. RESULTS +162 168 dimers oligomeric_state Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution. RESULTS +0 38 High performance liquid chromatography experimental_method High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig). RESULTS +52 90 multi-angle light-scattering detection experimental_method High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig). RESULTS +92 101 HPLC-MALS experimental_method High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig). RESULTS +161 163 Sa species High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig). RESULTS +164 168 EctC protein High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig). RESULTS +89 91 Sa species This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa). RESULTS +92 96 EctC protein This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa). RESULTS +97 102 dimer oligomeric_state This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa). RESULTS +126 133 monomer oligomeric_state This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa). RESULTS +149 178 Strep-tag II affinity peptide experimental_method This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa). RESULTS +30 35 dimer oligomeric_state Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus. RESULTS +67 80 EctC proteins protein_type Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus. RESULTS +86 97 H. elongata species Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus. RESULTS +102 114 N. maritimus species Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus. RESULTS +30 46 ectoine synthase protein_type Biochemical properties of the ectoine synthase RESULTS +4 8 EctA protein The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available. RESULTS +35 51 ectoine synthase protein_type The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available. RESULTS +53 89 N-γ-acetyl-L-2,4-diaminobutyric acid chemical The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available. RESULTS +91 100 N-γ-ADABA chemical The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available. RESULTS +31 38 ectoine chemical We used alkaline hydrolysis of ectoine and subsequent chromatography on silica gel columns to obtain N-γ-ADABA in chemically highly purified form (S1a Fig). RESULTS +101 110 N-γ-ADABA chemical We used alkaline hydrolysis of ectoine and subsequent chromatography on silica gel columns to obtain N-γ-ADABA in chemically highly purified form (S1a Fig). RESULTS +42 51 N-γ-ADABA chemical This procedure also yielded the isomer of N-γ-ADABA, N-α-acetyl-L-2,4-diaminobutyric acid (N-α-ADABA) (S1b Fig). RESULTS +53 89 N-α-acetyl-L-2,4-diaminobutyric acid chemical This procedure also yielded the isomer of N-γ-ADABA, N-α-acetyl-L-2,4-diaminobutyric acid (N-α-ADABA) (S1b Fig). RESULTS +91 100 N-α-ADABA chemical This procedure also yielded the isomer of N-γ-ADABA, N-α-acetyl-L-2,4-diaminobutyric acid (N-α-ADABA) (S1b Fig). RESULTS +0 9 N-α-ADABA chemical N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism. RESULTS +60 64 EctC protein N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism. RESULTS +70 84 microorganisms taxonomy_domain N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism. RESULTS +94 101 ectoine chemical N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism. RESULTS +6 15 N-γ-ADABA chemical Using N-γ-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein. RESULTS +109 111 Sa species Using N-γ-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein. RESULTS +112 116 EctC protein Using N-γ-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein. RESULTS +0 13 S. alaskensis species S. alaskensis, from which the studied ectoine synthase was originally derived, is a microorganism that is well-adapted to a life in permanently cold ocean waters. RESULTS +38 54 ectoine synthase protein_type S. alaskensis, from which the studied ectoine synthase was originally derived, is a microorganism that is well-adapted to a life in permanently cold ocean waters. RESULTS +84 97 microorganism taxonomy_domain S. alaskensis, from which the studied ectoine synthase was originally derived, is a microorganism that is well-adapted to a life in permanently cold ocean waters. RESULTS +69 71 Sa species Consistent with the physicochemical attributes of this habitat, the (Sa)EctC protein was already enzymatically active at 5°C, had a temperature optimum of 15°C and was able to function over a broad range of temperatures (S3a Fig). RESULTS +72 76 EctC protein Consistent with the physicochemical attributes of this habitat, the (Sa)EctC protein was already enzymatically active at 5°C, had a temperature optimum of 15°C and was able to function over a broad range of temperatures (S3a Fig). RESULTS +97 117 enzymatically active protein_state Consistent with the physicochemical attributes of this habitat, the (Sa)EctC protein was already enzymatically active at 5°C, had a temperature optimum of 15°C and was able to function over a broad range of temperatures (S3a Fig). RESULTS +16 24 alkaline protein_state It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0). RESULTS +77 94 ectoine synthases protein_type It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0). RESULTS +104 117 halo-tolerant protein_state It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0). RESULTS +118 129 H. elongata species It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0). RESULTS +162 173 alkaliphile taxonomy_domain It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0). RESULTS +174 189 M. alcaliphilum species It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0). RESULTS +219 229 acidophile taxonomy_domain It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0). RESULTS +230 250 Acidiphilium cryptum species It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0). RESULTS +291 295 EctC protein It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0). RESULTS +309 321 N. maritimus species It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0). RESULTS +328 338 neutral pH protein_state It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0). RESULTS +100 102 Sa species The salinity of the assay buffer had a significant influence on the maximal enzyme activity of the (Sa)EctC protein. RESULTS +103 107 EctC protein The salinity of the assay buffer had a significant influence on the maximal enzyme activity of the (Sa)EctC protein. RESULTS +26 30 NaCl chemical An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity. RESULTS +38 41 KCl chemical An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity. RESULTS +106 122 ectoine synthase protein_type An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity. RESULTS +32 34 Sa species The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively. RESULTS +35 39 EctC protein The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively. RESULTS +63 67 NaCl chemical The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively. RESULTS +71 74 KCl chemical The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively. RESULTS +1 3 Sa species (Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig). RESULTS +4 8 EctC protein (Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig). RESULTS +97 101 NaCl chemical (Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig). RESULTS +106 109 KCl chemical (Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig). RESULTS +19 23 EctC protein The stimulation of EctC enzyme activity by salts has previously also been observed for other ectoine synthases. RESULTS +93 110 ectoine synthases protein_type The stimulation of EctC enzyme activity by salts has previously also been observed for other ectoine synthases. RESULTS +4 20 ectoine synthase protein_type The ectoine synthase is a metal-containing protein RESULTS +26 50 metal-containing protein protein_type The ectoine synthase is a metal-containing protein RESULTS +53 57 EctC protein Considerations based on bioinformatics suggests that EctC belongs to the cupin superfamily. RESULTS +73 90 cupin superfamily protein_type Considerations based on bioinformatics suggests that EctC belongs to the cupin superfamily. RESULTS +87 91 iron chemical Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel. RESULTS +93 99 copper chemical Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel. RESULTS +101 105 zinc chemical Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel. RESULTS +107 116 manganese chemical Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel. RESULTS +118 124 cobalt chemical Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel. RESULTS +129 135 nickel chemical Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel. RESULTS +0 6 Cupins protein_type Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal). RESULTS +19 28 conserved protein_state Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal). RESULTS +37 57 G(X)5HXH(X)3,4E(X)6G structure_element Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal). RESULTS +62 80 G(X)5PXG(X)2H(X)3N structure_element Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal). RESULTS +153 158 metal chemical Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal). RESULTS +25 62 alignment of the amino acid sequences experimental_method Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family. RESULTS +70 88 EctC-type proteins protein_type Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family. RESULTS +117 136 metal-binding motif structure_element Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family. RESULTS +143 162 cupin-type proteins protein_type Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family. RESULTS +166 179 not conserved protein_state Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family. RESULTS +210 241 ectoine synthase protein family protein_type Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family. RESULTS +15 51 alignment of the amino acid sequence experimental_method An abbreviated alignment of the amino acid sequence of EctC-type proteins is shown in Fig 2. RESULTS +55 73 EctC-type proteins protein_type An abbreviated alignment of the amino acid sequence of EctC-type proteins is shown in Fig 2. RESULTS +12 21 alignment experimental_method Abbreviated alignment of EctC-type proteins. FIG +25 43 EctC-type proteins protein_type Abbreviated alignment of EctC-type proteins. FIG +40 58 EctC-type proteins protein_type The amino acid sequences of 20 selected EctC-type proteins are compared. FIG +0 18 Strictly conserved protein_state Strictly conserved amino acid residues are shown in yellow. FIG +22 24 Sa species Dots shown above the (Sa)EctC protein sequence indicate residues likely to be involved in iron-binding (red), ligand-binding (green) and stabilization of the loop-architecture (blue). FIG +25 29 EctC protein Dots shown above the (Sa)EctC protein sequence indicate residues likely to be involved in iron-binding (red), ligand-binding (green) and stabilization of the loop-architecture (blue). FIG +90 94 iron chemical Dots shown above the (Sa)EctC protein sequence indicate residues likely to be involved in iron-binding (red), ligand-binding (green) and stabilization of the loop-architecture (blue). FIG +4 13 conserved protein_state The conserved residue Tyr-52 with so-far undefined functions is indicated by a green dot circled in red. FIG +22 28 Tyr-52 residue_name_number The conserved residue Tyr-52 with so-far undefined functions is indicated by a green dot circled in red. FIG +31 40 α-helices structure_element Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins. FIG +45 53 β-sheets structure_element Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins. FIG +69 71 Sa species Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins. FIG +72 76 EctC protein Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins. FIG +77 94 crystal structure evidence Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins. FIG +142 160 EctC-type proteins protein_type Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins. FIG +40 59 metal-binding motif structure_element Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS). RESULTS +140 145 metal chemical Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS). RESULTS +178 180 Sa species Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS). RESULTS +181 185 EctC protein Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS). RESULTS +197 239 inductive-coupled plasma mass spectrometry experimental_method Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS). RESULTS +241 247 ICP-MS experimental_method Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS). RESULTS +39 41 Sa species For this analysis we used recombinant (Sa)EctC preparations from three independent protein overproduction and purification experiments. RESULTS +42 46 EctC protein For this analysis we used recombinant (Sa)EctC preparations from three independent protein overproduction and purification experiments. RESULTS +4 10 ICP-MS experimental_method The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein). RESULTS +31 35 iron chemical The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein). RESULTS +63 67 iron chemical The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein). RESULTS +101 103 Sa species The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein). RESULTS +104 108 EctC protein The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein). RESULTS +163 167 zinc chemical The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein). RESULTS +178 182 zinc chemical The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein). RESULTS +26 32 copper chemical All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively). RESULTS +37 43 nickel chemical All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively). RESULTS +90 95 metal chemical All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively). RESULTS +16 20 iron chemical The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein. RESULTS +31 33 Sa species The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein. RESULTS +34 38 EctC protein The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein. RESULTS +87 106 colorimetric method experimental_method The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein. RESULTS +127 131 iron chemical The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein. RESULTS +178 182 iron chemical The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein. RESULTS +222 224 Sa species The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein. RESULTS +225 229 EctC protein The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein. RESULTS +12 18 ICP-MS experimental_method Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein. RESULTS +27 46 colorimetric method experimental_method Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein. RESULTS +99 115 ectoine synthase protein_type Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein. RESULTS +121 134 S. alaskensis species Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein. RESULTS +141 145 iron chemical Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein. RESULTS +58 62 iron chemical We note in this context, that the values obtained for the iron content of the (Sa)EctC proteins varied by approximately 10 to 20% between the two methods. RESULTS +79 81 Sa species We note in this context, that the values obtained for the iron content of the (Sa)EctC proteins varied by approximately 10 to 20% between the two methods. RESULTS +82 86 EctC protein We note in this context, that the values obtained for the iron content of the (Sa)EctC proteins varied by approximately 10 to 20% between the two methods. RESULTS +85 103 colorimetric assay experimental_method The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree. RESULTS +131 135 iron chemical The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree. RESULTS +148 150 Sa species The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree. RESULTS +151 155 EctC protein The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree. RESULTS +2 7 metal chemical A metal cofactor is important for the catalytic activity of EctC RESULTS +60 64 EctC protein A metal cofactor is important for the catalytic activity of EctC RESULTS +4 8 iron chemical The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily. RESULTS +26 28 Sa species The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily. RESULTS +29 33 EctC protein The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily. RESULTS +180 197 cupin superfamily protein_type The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily. RESULTS +31 40 incubated experimental_method To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity. RESULTS +46 48 Sa species To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity. RESULTS +49 53 EctC protein To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity. RESULTS +61 91 with increasing concentrations experimental_method To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity. RESULTS +99 104 metal chemical To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity. RESULTS +114 147 ethylene-diamine-tetraacetic-acid chemical To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity. RESULTS +149 153 EDTA chemical To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity. RESULTS +180 196 ectoine synthase protein_type To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity. RESULTS +43 47 EDTA chemical The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a). RESULTS +65 69 EctC protein The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a). RESULTS +123 139 ectoine synthase protein_type The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a). RESULTS +174 178 EDTA chemical The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a). RESULTS +18 34 ectoine synthase protein_type Dependency of the ectoine synthase activity on metals. FIG +18 22 iron chemical (a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein. FIG +32 36 EDTA chemical (a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein. FIG +77 79 Sa species (a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein. FIG +80 84 EctC protein (a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein. FIG +0 46 Metal depletion and reconstitution experiments experimental_method Metal depletion and reconstitution experiments with (b) stoichiometric and (c) excess amounts of metals. FIG +5 7 Sa species The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added. FIG +8 12 EctC protein The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added. FIG +139 155 ectoine synthase protein_type The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added. FIG +156 169 enzyme assays experimental_method The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added. FIG +184 189 FeCl2 chemical The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added. FIG +21 32 inactivated protein_state We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme. RESULTS +65 69 EDTA chemical We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme. RESULTS +73 81 dialysis experimental_method We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme. RESULTS +150 152 Sa species We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme. RESULTS +153 157 EctC protein We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme. RESULTS +16 21 FeCl2 chemical The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively. RESULTS +29 41 enzyme assay experimental_method The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively. RESULTS +105 110 ZnCl2 chemical The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively. RESULTS +114 119 CoCl2 chemical The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively. RESULTS +129 131 Sa species The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively. RESULTS +132 136 EctC protein The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively. RESULTS +35 39 Fe3+ chemical All other tested metals, including Fe3+, were unable to restore activity (Fig 3b). RESULTS +52 64 enzyme assay experimental_method When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c). RESULTS +89 93 Fe2+ chemical When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c). RESULTS +233 235 Sa species When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c). RESULTS +236 240 EctC protein When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c). RESULTS +300 304 EDTA chemical When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c). RESULTS +64 68 zinc chemical However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c). RESULTS +70 76 cobalt chemical However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c). RESULTS +78 84 nickel chemical However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c). RESULTS +86 92 copper chemical However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c). RESULTS +98 107 manganese chemical However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c). RESULTS +143 160 cupin superfamily protein_type However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c). RESULTS +191 207 ectoine synthase protein_type However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c). RESULTS +50 68 cupin-type enzymes protein_type This is in line with literature data showing that cupin-type enzymes are often promiscuous with respect to the use of the catalytically important metal. RESULTS +146 151 metal chemical This is in line with literature data showing that cupin-type enzymes are often promiscuous with respect to the use of the catalytically important metal. RESULTS +22 26 EctC protein Kinetic parameters of EctC for N-γ-ADABA and N-α-ADABA RESULTS +31 40 N-γ-ADABA chemical Kinetic parameters of EctC for N-γ-ADABA and N-α-ADABA RESULTS +45 54 N-α-ADABA chemical Kinetic parameters of EctC for N-γ-ADABA and N-α-ADABA RESULTS +66 80 activity assay experimental_method Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA. RESULTS +89 105 ectoine synthase protein_type Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA. RESULTS +109 122 S. alaskensis species Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA. RESULTS +181 183 Sa species Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA. RESULTS +184 188 EctC protein Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA. RESULTS +227 236 N-γ-ADABA chemical Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA. RESULTS +252 261 N-α-ADABA chemical Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA. RESULTS +4 8 EctC protein The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig). RESULTS +35 44 N-γ-ADABA chemical The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig). RESULTS +53 60 ectoine chemical The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig). RESULTS +71 96 Michaelis-Menten-kinetics experimental_method The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig). RESULTS +114 116 Km evidence The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig). RESULTS +136 140 vmax evidence The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig). RESULTS +166 170 kcat evidence The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig). RESULTS +34 43 N-α-ADABA chemical Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine. RESULTS +70 79 N-γ-ADABA chemical Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine. RESULTS +88 104 ectoine synthase protein_type Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine. RESULTS +145 147 Sa species Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine. RESULTS +148 152 EctC protein Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine. RESULTS +168 177 N-α-ADABA chemical Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine. RESULTS +189 196 ectoine chemical Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine. RESULTS +1 3 Sa species (Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig). RESULTS +4 8 EctC protein (Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig). RESULTS +38 63 Michaelis-Menten-kinetics experimental_method (Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig). RESULTS +87 89 Km evidence (Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig). RESULTS +110 114 vmax evidence (Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig). RESULTS +140 144 kcat evidence (Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig). RESULTS +7 16 N-α-ADABA chemical Hence, N-α-ADABA is a newly recognized substrate for ectoine synthase. RESULTS +53 69 ectoine synthase protein_type Hence, N-α-ADABA is a newly recognized substrate for ectoine synthase. RESULTS +18 26 affinity evidence However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA. RESULTS +28 30 Km evidence However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA. RESULTS +40 42 Sa species However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA. RESULTS +43 47 EctC protein However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA. RESULTS +64 84 catalytic efficiency evidence However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA. RESULTS +86 93 kcat/Km evidence However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA. RESULTS +136 145 N-γ-ADABA chemical However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA. RESULTS +4 6 Km evidence The Km dropped fife-fold from 4.9 ± 0.5 mM to 25.4 ± 2.9 mM, and the catalytic efficiency was reduced from 1.47 mM-1 s-1 to 0.02 mM-1 s-1, a 73-fold decrease. RESULTS +69 89 catalytic efficiency evidence The Km dropped fife-fold from 4.9 ± 0.5 mM to 25.4 ± 2.9 mM, and the catalytic efficiency was reduced from 1.47 mM-1 s-1 to 0.02 mM-1 s-1, a 73-fold decrease. RESULTS +5 14 N-γ-ADABA chemical Both N-γ-ADABA and N-α-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism. RESULTS +19 28 N-α-ADABA chemical Both N-γ-ADABA and N-α-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism. RESULTS +93 100 ectoine chemical Both N-γ-ADABA and N-α-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism. RESULTS +17 26 N-α-ADABA chemical Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine. RESULTS +46 62 ectoine synthase protein_type Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine. RESULTS +124 138 microorganisms taxonomy_domain Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine. RESULTS +179 186 ectoine chemical Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine. RESULTS +24 38 microorganisms taxonomy_domain However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig). RESULTS +102 110 affinity evidence However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig). RESULTS +114 130 ectoine synthase protein_type However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig). RESULTS +135 144 N-γ-ADABA chemical However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig). RESULTS +149 158 N-α-ADABA chemical However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig). RESULTS +168 188 catalytic efficiency evidence However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig). RESULTS +0 15 Crystallization experimental_method Crystallization of the (Sa)EctC protein RESULTS +24 26 Sa species Crystallization of the (Sa)EctC protein RESULTS +27 31 EctC protein Crystallization of the (Sa)EctC protein RESULTS +9 26 crystal structure evidence Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein. RESULTS +30 46 ectoine synthase protein_type Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein. RESULTS +80 91 crystallize experimental_method Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein. RESULTS +97 99 Sa species Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein. RESULTS +100 104 EctC protein Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein. RESULTS +19 27 crystals evidence Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful. RESULTS +32 34 Sa species Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful. RESULTS +35 39 EctC protein Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful. RESULTS +40 50 in complex protein_state Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful. RESULTS +77 86 N-γ-ADABA chemical Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful. RESULTS +111 118 ectoine chemical Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful. RESULTS +13 26 crystal forms evidence However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained. RESULTS +35 37 Sa species However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained. RESULTS +38 42 EctC protein However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained. RESULTS +58 68 absence of protein_state However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained. RESULTS +22 39 crystal structure evidence Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed. RESULTS +48 50 Sa species Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed. RESULTS +51 55 EctC protein Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed. RESULTS +67 88 molecular replacement experimental_method Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed. RESULTS +32 40 crystals evidence However, we were able to obtain crystals of form B that were derivatized with mercury and these diffracted up to 2.8 Å (S1 Table). RESULTS +78 85 mercury chemical However, we were able to obtain crystals of form B that were derivatized with mercury and these diffracted up to 2.8 Å (S1 Table). RESULTS +43 59 structural model evidence This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table). RESULTS +68 70 Sa species This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table). RESULTS +71 75 EctC protein This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table). RESULTS +130 151 molecular replacement experimental_method This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table). RESULTS +274 282 monomers oligomeric_state This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table). RESULTS +287 289 Sa species This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table). RESULTS +290 294 EctC protein This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table). RESULTS +319 336 crystal structure evidence This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table). RESULTS +360 366 Rcryst evidence This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table). RESULTS +383 388 Rfree evidence This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table). RESULTS +11 18 monomer oligomeric_state Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table). RESULTS +27 36 structure evidence Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table). RESULTS +64 85 molecular replacement experimental_method Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table). RESULTS +192 198 Rcryst evidence Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table). RESULTS +215 220 Rfree evidence Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table). RESULTS +21 23 Sa species Overall fold of the (Sa)EctC protein RESULTS +24 28 EctC protein Overall fold of the (Sa)EctC protein RESULTS +8 12 EctC protein The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c). RESULTS +13 23 structures evidence The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c). RESULTS +61 77 ectoine synthase protein_type The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c). RESULTS +93 110 cupin superfamily protein_type The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c). RESULTS +52 67 137 amino acids residue_range However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2). RESULTS +80 82 Sa species However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2). RESULTS +83 87 EctC protein However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2). RESULTS +17 26 structure evidence First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein. RESULTS +69 71 Sa species First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein. RESULTS +72 76 EctC protein First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein. RESULTS +109 125 Met-1 to Glu-115 residue_range First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein. RESULTS +137 142 lacks protein_state First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein. RESULTS +143 157 22 amino acids residue_range First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein. RESULTS +165 181 carboxy-terminus structure_element First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein. RESULTS +200 202 Sa species First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein. RESULTS +203 207 EctC protein First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein. RESULTS +5 14 structure evidence This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b). RESULTS +25 29 open protein_state This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b). RESULTS +79 92 cupin barrels structure_element This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b). RESULTS +139 143 open protein_state This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b). RESULTS +146 148 Sa species This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b). RESULTS +149 153 EctC protein This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b). RESULTS +154 163 structure evidence This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b). RESULTS +8 17 structure evidence In this structure no metal co-factor was identified. RESULTS +21 26 metal chemical In this structure no metal co-factor was identified. RESULTS +11 28 crystal structure evidence The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation. RESULTS +37 39 Sa species The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation. RESULTS +40 44 EctC protein The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation. RESULTS +57 63 solved experimental_method The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation. RESULTS +165 173 protomer oligomeric_state The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation. RESULTS +174 175 A structure_element The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation. RESULTS +197 213 Met-1 to Gly-121 residue_range The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation. RESULTS +227 233 closed protein_state The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation. RESULTS +16 21 lacks protein_state Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2). RESULTS +22 35 16 amino acid residue_range Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2). RESULTS +52 68 carboxy-terminus structure_element Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2). RESULTS +86 101 137 amino acids residue_range Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2). RESULTS +114 116 Sa species Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2). RESULTS +117 121 EctC protein Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2). RESULTS +38 55 crystal structure evidence We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure. RESULTS +79 91 fully closed protein_state We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure. RESULTS +105 121 ectoine synthase protein_type We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure. RESULTS +167 178 semi-closed protein_state We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure. RESULTS +181 183 Sa species We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure. RESULTS +184 188 EctC protein We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure. RESULTS +189 198 structure evidence We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure. RESULTS +31 39 monomers oligomeric_state Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure. RESULTS +86 102 Met-1 to Glu-115 residue_range Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure. RESULTS +144 148 open protein_state Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure. RESULTS +150 154 EctC protein Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure. RESULTS +155 164 structure evidence Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure. RESULTS +8 17 structure evidence Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC. FIG +26 30 open protein_state Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC. FIG +37 48 semi-closed protein_state Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC. FIG +50 68 crystal structures evidence Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC. FIG +73 75 Sa species Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC. FIG +76 80 EctC protein Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC. FIG +16 25 structure evidence (a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation. FIG +34 45 semi-closed protein_state (a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation. FIG +48 50 Sa species (a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation. FIG +51 55 EctC protein (a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation. FIG +4 13 β-strands structure_element The β-strands are numbered β1-β11 and the helices α-I to α-II. FIG +27 33 β1-β11 structure_element The β-strands are numbered β1-β11 and the helices α-I to α-II. FIG +42 49 helices structure_element The β-strands are numbered β1-β11 and the helices α-I to α-II. FIG +50 61 α-I to α-II structure_element The β-strands are numbered β1-β11 and the helices α-I to α-II. FIG +16 25 structure evidence (b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation. FIG +34 38 open protein_state (b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation. FIG +41 43 Sa species (b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation. FIG +44 48 EctC protein (b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation. FIG +20 31 active site site The entrance to the active site of the ectoine synthase is marked. FIG +39 55 ectoine synthase protein_type The entrance to the active site of the ectoine synthase is marked. FIG +4 11 Overlay experimental_method (c) Overlay of the “semi-closed” and “open” (Sa)EctC structures. FIG +20 31 semi-closed protein_state (c) Overlay of the “semi-closed” and “open” (Sa)EctC structures. FIG +38 42 open protein_state (c) Overlay of the “semi-closed” and “open” (Sa)EctC structures. FIG +45 47 Sa species (c) Overlay of the “semi-closed” and “open” (Sa)EctC structures. FIG +48 52 EctC protein (c) Overlay of the “semi-closed” and “open” (Sa)EctC structures. FIG +53 63 structures evidence (c) Overlay of the “semi-closed” and “open” (Sa)EctC structures. FIG +12 21 structure evidence The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure. RESULTS +26 28 Sa species The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure. RESULTS +29 33 EctC protein The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure. RESULTS +64 72 crystals evidence The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure. RESULTS +88 104 carboxy-terminus structure_element The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure. RESULTS +148 160 cupin barrel structure_element The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure. RESULTS +186 193 monomer oligomeric_state The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure. RESULTS +194 195 A structure_element The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure. RESULTS +204 215 semi-closed protein_state The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure. RESULTS +217 226 structure evidence The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure. RESULTS +36 62 root mean square deviation evidence This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other. RESULTS +64 68 RMSD evidence This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other. RESULTS +143 147 open protein_state This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other. RESULTS +149 157 monomers oligomeric_state This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other. RESULTS +14 25 semi-closed protein_state However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure. RESULTS +27 34 monomer oligomeric_state However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure. RESULTS +57 61 RMSD evidence However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure. RESULTS +115 119 open protein_state However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure. RESULTS +127 136 structure evidence However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure. RESULTS +52 61 structure evidence Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail. RESULTS +71 82 semi-closed protein_state Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail. RESULTS +97 99 Sa species Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail. RESULTS +100 104 EctC protein Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail. RESULTS +180 184 open protein_state Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail. RESULTS +191 202 semi-closed protein_state Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail. RESULTS +4 13 structure evidence The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a). RESULTS +22 33 semi-closed protein_state The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a). RESULTS +36 38 Sa species The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a). RESULTS +39 43 EctC protein The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a). RESULTS +67 76 β-strands structure_element The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a). RESULTS +78 84 β1-β11 structure_element The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a). RESULTS +94 103 α-helices structure_element The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a). RESULTS +105 108 α-I structure_element The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a). RESULTS +113 117 α-II structure_element The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a). RESULTS +4 13 β-strands structure_element The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. RESULTS +23 45 anti-parallel β-sheets structure_element The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. RESULTS +47 49 β2 structure_element The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. RESULTS +50 52 β3 structure_element The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. RESULTS +54 56 β4 structure_element The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. RESULTS +58 61 β11 structure_element The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. RESULTS +63 65 β6 structure_element The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. RESULTS +71 73 β9 structure_element The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. RESULTS +89 111 three-stranded β-sheet structure_element The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. RESULTS +113 115 β7 structure_element The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. RESULTS +117 119 β8 structure_element The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. RESULTS +125 128 β10 structure_element The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively. RESULTS +10 18 β-sheets structure_element These two β-sheets pack against each other, forming a cup-shaped β-sandwich with a topology characteristic for the cupin-fold. RESULTS +54 75 cup-shaped β-sandwich structure_element These two β-sheets pack against each other, forming a cup-shaped β-sandwich with a topology characteristic for the cupin-fold. RESULTS +115 125 cupin-fold structure_element These two β-sheets pack against each other, forming a cup-shaped β-sandwich with a topology characteristic for the cupin-fold. RESULTS +8 10 Sa species Hence, (Sa)EctC adopts an overall bowl shape in which one side is opened towards the solvent (Fig 4a to 4c). RESULTS +11 15 EctC protein Hence, (Sa)EctC adopts an overall bowl shape in which one side is opened towards the solvent (Fig 4a to 4c). RESULTS +8 19 semi-closed protein_state In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a). RESULTS +21 30 structure evidence In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a). RESULTS +41 62 carboxy-terminal tail structure_element In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a). RESULTS +81 97 electron density evidence In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a). RESULTS +114 125 small helix structure_element In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a). RESULTS +127 131 α-II structure_element In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a). RESULTS +149 160 active site site In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a). RESULTS +169 171 Sa species In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a). RESULTS +172 176 EctC protein In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a). RESULTS +22 32 α-II helix structure_element The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +77 89 unstructured protein_state The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +90 94 loop structure_element The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +116 120 open protein_state The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +122 131 structure evidence The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +144 146 β4 structure_element The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +151 153 β6 structure_element The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +189 195 stable protein_state The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +196 204 β-strand structure_element The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +205 207 β5 structure_element The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +228 239 semi-closed protein_state The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +254 256 Sa species The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +257 261 EctC protein The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a). RESULTS +0 30 Structural comparison analyses experimental_method Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily. RESULTS +41 52 DALI server experimental_method Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily. RESULTS +68 70 Sa species Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily. RESULTS +71 75 EctC protein Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily. RESULTS +122 139 cupin superfamily protein_type Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily. RESULTS +57 96 Cupin 2 conserved barrel domain protein protein The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +98 109 YP_751781.1 protein The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +116 140 Shewanella frigidimarina species The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +175 182 Z-score evidence The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +198 202 RMSD evidence The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +332 341 structure evidence The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +369 395 manganese-containing cupin protein The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +397 403 TM1459 protein The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +410 429 Thermotoga maritima species The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +464 471 Z-score evidence The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +487 491 RMSD evidence The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +524 531 cyclase protein_type The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +532 536 RemF protein The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +542 571 Streptomyces resistomycificus species The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +605 612 Z-score evidence The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +628 632 RMSD evidence The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +669 692 auxin-binding protein 1 protein The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +698 706 Zea mays species The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +742 749 Z-score evidence The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +765 769 RMSD evidence The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms). RESULTS +18 22 EctC protein Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction. RESULTS +43 61 polyketide cyclase protein_type Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction. RESULTS +62 66 RemF protein Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction. RESULTS +88 101 cupin-related protein_type Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction. RESULTS +8 12 RemF protein Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily. RESULTS +21 46 aldos-2-ulose dehydratase protein_type Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily. RESULTS +47 56 isomerase protein_type Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily. RESULTS +62 78 ectoine synthase protein_type Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily. RESULTS +111 122 dehydratase protein_type Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily. RESULTS +134 151 cupin superfamily protein_type Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily. RESULTS +16 20 EctC protein Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure RESULTS +21 36 dimer interface site Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure RESULTS +57 59 Sa species Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure RESULTS +60 64 EctC protein Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure RESULTS +65 82 crystal structure evidence Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure RESULTS +9 12 SEC experimental_method Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution. RESULTS +30 39 HPLC-MALS experimental_method Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution. RESULTS +82 98 ectoine synthase protein_type Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution. RESULTS +104 117 S. alaskensis species Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution. RESULTS +123 128 dimer oligomeric_state Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution. RESULTS +4 21 crystal structure evidence The crystal structure of this protein reflects this quaternary arrangement. RESULTS +8 19 semi-closed protein_state In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit. RESULTS +21 38 crystal structure evidence In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit. RESULTS +41 43 Sa species In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit. RESULTS +44 48 EctC protein In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit. RESULTS +53 65 crystallized experimental_method In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit. RESULTS +71 76 dimer oligomeric_state In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit. RESULTS +80 86 dimers oligomeric_state In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit. RESULTS +5 10 dimer oligomeric_state This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +46 54 monomers oligomeric_state This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +69 81 head-to-tail protein_state This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +152 174 antiparallel β-strands structure_element This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +176 184 β-strand structure_element This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +185 187 β1 structure_element This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +198 205 1MIVRN5 structure_element This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +212 219 monomer oligomeric_state This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +220 221 A structure_element This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +226 234 β-strand structure_element This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +235 237 β8 structure_element This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +243 250 monomer oligomeric_state This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +251 252 B structure_element This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +263 273 82GVMYAL87 structure_element This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c). RESULTS +38 47 β-strands structure_element The strong interactions between these β-strands rely primarily on backbone contacts. RESULTS +47 71 hydrophobic interactions bond_interaction In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands. RESULTS +106 114 monomers oligomeric_state In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands. RESULTS +123 128 loops structure_element In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands. RESULTS +144 153 β-strands structure_element In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands. RESULTS +19 27 PDBePISA experimental_method As calculated with PDBePISA, the surface area buried upon dimer formation is 1462 Å2, which is 20.5% of the total accessible surface of a monomer of this protein. RESULTS +58 63 dimer oligomeric_state As calculated with PDBePISA, the surface area buried upon dimer formation is 1462 Å2, which is 20.5% of the total accessible surface of a monomer of this protein. RESULTS +138 145 monomer oligomeric_state As calculated with PDBePISA, the surface area buried upon dimer formation is 1462 Å2, which is 20.5% of the total accessible surface of a monomer of this protein. RESULTS +55 61 dimers oligomeric_state Both values fall within the range for known functional dimers. RESULTS +0 17 Crystal structure evidence Crystal structure of (Sa)EctC. FIG +22 24 Sa species Crystal structure of (Sa)EctC. FIG +25 29 EctC protein Crystal structure of (Sa)EctC. FIG +20 25 dimer oligomeric_state (a) Top-view of the dimer of the (Sa)EctC protein. FIG +34 36 Sa species (a) Top-view of the dimer of the (Sa)EctC protein. FIG +37 41 EctC protein (a) Top-view of the dimer of the (Sa)EctC protein. FIG +20 25 water chemical The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer. FIG +92 100 monomers oligomeric_state The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer. FIG +142 144 Sa species The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer. FIG +145 149 EctC protein The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer. FIG +150 155 dimer oligomeric_state The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer. FIG +186 201 dimer interface site The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer. FIG +216 225 β-strands structure_element The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer. FIG +234 241 monomer oligomeric_state The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer. FIG +35 50 dimer interface site (c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6. FIG +63 74 beta-strand structure_element (c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6. FIG +75 77 β1 structure_element (c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6. FIG +82 84 β6 structure_element (c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6. FIG +8 12 open protein_state In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit. RESULTS +15 17 Sa species In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit. RESULTS +18 22 EctC protein In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit. RESULTS +23 32 structure evidence In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit. RESULTS +38 45 monomer oligomeric_state In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit. RESULTS +35 42 packing experimental_method We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well. RESULTS +60 67 monomer oligomeric_state We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well. RESULTS +68 75 monomer oligomeric_state We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well. RESULTS +169 174 dimer oligomeric_state We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well. RESULTS +202 214 crystal form evidence We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well. RESULTS +18 23 dimer oligomeric_state Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets. RESULTS +68 79 semi-closed protein_state Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets. RESULTS +82 84 Sa species Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets. RESULTS +85 89 EctC protein Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets. RESULTS +90 99 structure evidence Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets. RESULTS +126 133 monomer oligomeric_state Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets. RESULTS +134 141 monomer oligomeric_state Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets. RESULTS +175 183 β-sheets structure_element Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets. RESULTS +109 117 monomers oligomeric_state The crystallographic two-fold axis present within the crystal symmetry is located exactly in between the two monomers, resulting in a monomer within the asymmetric unit. RESULTS +134 141 monomer oligomeric_state The crystallographic two-fold axis present within the crystal symmetry is located exactly in between the two monomers, resulting in a monomer within the asymmetric unit. RESULTS +16 21 dimer oligomeric_state Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure. RESULTS +39 50 semi-closed protein_state Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure. RESULTS +52 61 structure evidence Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure. RESULTS +66 68 Sa species Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure. RESULTS +69 73 EctC protein Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure. RESULTS +103 107 open protein_state Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure. RESULTS +109 118 structure evidence Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure. RESULTS +62 73 DALI search experimental_method Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5). RESULTS +105 109 EctC protein Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5). RESULTS +135 141 dimers oligomeric_state Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5). RESULTS +161 168 monomer oligomeric_state Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5). RESULTS +169 176 monomer oligomeric_state Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5). RESULTS +201 206 dimer oligomeric_state Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5). RESULTS +33 41 flexible protein_state Structural rearrangements of the flexible (Sa)EctC carboxy-terminus RESULTS +43 45 Sa species Structural rearrangements of the flexible (Sa)EctC carboxy-terminus RESULTS +46 50 EctC protein Structural rearrangements of the flexible (Sa)EctC carboxy-terminus RESULTS +51 67 carboxy-terminus structure_element Structural rearrangements of the flexible (Sa)EctC carboxy-terminus RESULTS +54 70 ectoine synthase protein_type The cupin core represents the structural framework of ectoine synthase (Figs 4 and 5). RESULTS +32 50 crystal structures evidence The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus. RESULTS +59 61 Sa species The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus. RESULTS +62 66 EctC protein The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus. RESULTS +115 131 carboxy-terminus structure_element The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus. RESULTS +32 55 carboxy-terminal region structure_element Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family. RESULTS +63 78 137 amino acids residue_range Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family. RESULTS +91 93 Sa species Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family. RESULTS +94 98 EctC protein Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family. RESULTS +111 127 highly conserved protein_state Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family. RESULTS +147 155 extended protein_state Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family. RESULTS +156 168 EctC protein protein_type Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family. RESULTS +14 22 β-strand structure_element At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein. RESULTS +23 26 β11 structure_element At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein. RESULTS +44 53 conserved protein_state At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein. RESULTS +54 61 proline residue_name At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein. RESULTS +72 79 Pro-109 residue_name_number At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein. RESULTS +84 91 Pro-110 residue_name_number At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein. RESULTS +163 165 Sa species At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein. RESULTS +166 170 EctC protein At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein. RESULTS +8 19 semi-closed protein_state In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121. RESULTS +22 24 Sa species In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121. RESULTS +25 29 EctC protein In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121. RESULTS +30 39 structure evidence In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121. RESULTS +53 69 electron density evidence In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121. RESULTS +77 93 carboxy-terminus structure_element In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121. RESULTS +109 130 7 amino acid residues residue_range In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121. RESULTS +152 159 Gly-121 residue_name_number In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121. RESULTS +41 52 small helix structure_element These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a). RESULTS +70 74 open protein_state These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a). RESULTS +75 81 cavity site These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a). RESULTS +89 99 cupin-fold structure_element These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a). RESULTS +108 110 Sa species These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a). RESULTS +111 115 EctC protein These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a). RESULTS +18 23 helix structure_element Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement. RESULTS +64 75 loop region structure_element Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement. RESULTS +84 93 β-strands structure_element Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement. RESULTS +94 96 β4 structure_element Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement. RESULTS +101 103 β6 structure_element Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement. RESULTS +30 38 β-strand structure_element This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure. RESULTS +39 41 β5 structure_element This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure. RESULTS +73 95 small C-terminal helix structure_element This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure. RESULTS +99 105 absent protein_state This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure. RESULTS +126 130 open protein_state This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure. RESULTS +133 135 Sa species This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure. RESULTS +136 140 EctC protein This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure. RESULTS +141 150 structure evidence This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure. RESULTS +30 38 β-strand structure_element As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c). RESULTS +39 41 β5 structure_element As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c). RESULTS +87 98 semi-closed protein_state As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c). RESULTS +101 103 Sa species As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c). RESULTS +104 108 EctC protein As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c). RESULTS +109 118 structure evidence As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c). RESULTS +28 36 β-strand structure_element It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below). RESULTS +37 39 β5 structure_element It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below). RESULTS +59 65 His-93 residue_name_number It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below). RESULTS +103 108 metal chemical It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below). RESULTS +21 24 His residue_name The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5. RESULTS +62 64 Sa species The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5. RESULTS +65 69 EctC protein The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5. RESULTS +70 80 structures evidence The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5. RESULTS +120 128 β-strand structure_element The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5. RESULTS +129 131 β5 structure_element The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5. RESULTS +29 39 cupin fold structure_element Therefore the sealing of the cupin fold, as described above, seem to have an indirect influence on the architecture of the postulated iron-binding site. RESULTS +134 151 iron-binding site site Therefore the sealing of the cupin fold, as described above, seem to have an indirect influence on the architecture of the postulated iron-binding site. RESULTS +16 23 Pro-109 residue_name_number The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2). RESULTS +28 35 Pro-110 residue_name_number The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2). RESULTS +65 73 β-strand structure_element The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2). RESULTS +74 77 β11 structure_element The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2). RESULTS +81 97 highly conserved protein_state The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2). RESULTS +101 119 EctC-type proteins protein_type The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2). RESULTS +69 85 carboxy-terminus structure_element They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold. RESULTS +87 109 27 amino acid residues residue_range They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold. RESULTS +115 117 Sa species They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold. RESULTS +118 122 EctC protein They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold. RESULTS +136 146 cupin fold structure_element They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold. RESULTS +8 19 semi-closed protein_state In the “semi-closed” structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c). RESULTS +21 30 structure evidence In the “semi-closed” structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c). RESULTS +86 98 cupin barrel structure_element In the “semi-closed” structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c). RESULTS +8 12 open protein_state In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus. RESULTS +15 17 Sa species In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus. RESULTS +18 22 EctC protein In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus. RESULTS +23 32 structure evidence In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus. RESULTS +39 46 proline residue_name In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus. RESULTS +75 91 electron density evidence In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus. RESULTS +124 131 Pro-110 residue_name_number In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus. RESULTS +137 153 electron density evidence In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus. RESULTS +213 229 carboxy-terminus structure_element In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus. RESULTS +39 46 Pro-109 residue_name_number A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures. RESULTS +121 127 His-55 residue_name_number A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures. RESULTS +152 156 open protein_state A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures. RESULTS +163 174 semi-closed protein_state A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures. RESULTS +177 179 Sa species A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures. RESULTS +180 184 EctC protein A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures. RESULTS +185 195 structures evidence A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures. RESULTS +4 11 Pro-109 residue_name_number The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11. RESULTS +12 18 His-55 residue_name_number The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11. RESULTS +43 49 stable protein_state The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11. RESULTS +70 77 proline residue_name The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11. RESULTS +101 109 β-strand structure_element The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11. RESULTS +110 113 β11 structure_element The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11. RESULTS +12 19 proline residue_name Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein. RESULTS +49 72 carboxy-terminal region structure_element Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein. RESULTS +81 83 Sa species Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein. RESULTS +84 88 EctC protein Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein. RESULTS +117 123 His-55 residue_name_number Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein. RESULTS +129 136 Pro-109 residue_name_number Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein. RESULTS +40 47 Pro-109 residue_name_number In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further. RESULTS +52 58 His-55 residue_name_number In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further. RESULTS +64 87 carboxy-terminal region structure_element In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further. RESULTS +92 94 Sa species In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further. RESULTS +95 99 EctC protein In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further. RESULTS +142 149 Glu-115 residue_name_number In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further. RESULTS +155 161 His-55 residue_name_number In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further. RESULTS +204 215 small helix structure_element In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further. RESULTS +223 239 carboxy-terminus structure_element In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further. RESULTS +24 31 Glu-115 residue_name_number The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density. RESULTS +36 42 His-55 residue_name_number The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density. RESULTS +67 78 semi-closed protein_state The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density. RESULTS +80 89 structure evidence The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density. RESULTS +100 118 partially extended protein_state The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density. RESULTS +119 135 carboxy-terminus structure_element The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density. RESULTS +155 171 electron density evidence The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density. RESULTS +8 12 open protein_state In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b). RESULTS +14 23 structure evidence In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b). RESULTS +32 34 Sa species In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b). RESULTS +35 39 EctC protein In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b). RESULTS +87 94 Glu-115 residue_name_number In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b). RESULTS +105 121 carboxy-terminus structure_element Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region. RESULTS +130 134 open protein_state Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region. RESULTS +137 139 Sa species Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region. RESULTS +140 144 EctC protein Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region. RESULTS +145 154 structure evidence Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region. RESULTS +201 217 electron density evidence Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region. RESULTS +29 47 metal-binding site site Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus. FIG +56 58 Sa species Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus. FIG +59 63 EctC protein Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus. FIG +80 88 flexible protein_state Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus. FIG +89 105 carboxy-terminus structure_element Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus. FIG +18 23 water chemical (a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93. FIG +111 117 Glu-57 residue_name_number (a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93. FIG +119 125 Tyr-85 residue_name_number (a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93. FIG +131 137 His-93 residue_name_number (a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93. FIG +30 35 water chemical The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase. FIG +85 89 Fe2+ chemical The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase. FIG +106 117 active side site The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase. FIG +125 141 ectoine synthase protein_type The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase. FIG +0 6 His-55 residue_name_number His-55 interacts with the double proline motif (Pro-109 and Pro-110). FIG +26 46 double proline motif structure_element His-55 interacts with the double proline motif (Pro-109 and Pro-110). FIG +48 55 Pro-109 residue_name_number His-55 interacts with the double proline motif (Pro-109 and Pro-110). FIG +60 67 Pro-110 residue_name_number His-55 interacts with the double proline motif (Pro-109 and Pro-110). FIG +67 74 Glu-115 residue_name_number It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure. FIG +101 109 flexible protein_state It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure. FIG +110 126 carboxy-terminus structure_element It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure. FIG +151 153 Sa species It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure. FIG +154 158 EctC protein It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure. FIG +183 194 semi-closed protein_state It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure. FIG +197 199 Sa species It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure. FIG +200 204 EctC protein It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure. FIG +205 214 structure evidence It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure. FIG +7 14 overlay experimental_method (b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein. FIG +23 27 open protein_state (b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein. FIG +62 73 semi-closed protein_state (b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein. FIG +94 103 structure evidence (b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein. FIG +112 114 Sa species (b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein. FIG +115 119 EctC protein (b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein. FIG +13 30 iron binding site site The putative iron binding site of (Sa)EctC RESULTS +35 37 Sa species The putative iron binding site of (Sa)EctC RESULTS +38 42 EctC protein The putative iron binding site of (Sa)EctC RESULTS +8 19 semi-closed protein_state In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel. RESULTS +21 30 structure evidence In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel. RESULTS +35 37 Sa species In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel. RESULTS +38 42 EctC protein In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel. RESULTS +61 69 monomers oligomeric_state In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel. RESULTS +120 136 electron density evidence In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel. RESULTS +159 171 cupin barrel structure_element In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel. RESULTS +7 9 Sa species Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy. RESULTS +10 14 EctC protein Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy. RESULTS +20 25 metal chemical Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy. RESULTS +77 81 Fe2+ chemical Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy. RESULTS +86 90 Zn2+ chemical Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy. RESULTS +106 113 density evidence Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy. RESULTS +123 140 refined occupancy experimental_method Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy. RESULTS +23 27 Fe2+ chemical Only the refinement of Fe2+ resulted in a visibly improved electron density, however with a low degree of occupancy. RESULTS +59 75 electron density evidence Only the refinement of Fe2+ resulted in a visibly improved electron density, however with a low degree of occupancy. RESULTS +14 18 iron chemical This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b). RESULTS +59 65 Glu-57 residue_name_number This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b). RESULTS +67 73 Tyr-85 residue_name_number This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b). RESULTS +78 84 His-93 residue_name_number This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b). RESULTS +74 78 iron chemical The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively. RESULTS +102 108 Glu-57 residue_name_number The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively. RESULTS +120 126 Tyr-85 residue_name_number The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively. RESULTS +142 148 His-93 residue_name_number The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively. RESULTS +51 69 iron binding sites site These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure. RESULTS +106 116 absence of protein_state These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure. RESULTS +146 148 Sa species These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure. RESULTS +149 153 EctC protein These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure. RESULTS +154 171 crystal structure evidence These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure. RESULTS +71 75 iron chemical Since both the refinement and the distance did not clearly identify an iron molecule, we decided to conservatively place a water molecule at this position. RESULTS +123 128 water chemical Since both the refinement and the distance did not clearly identify an iron molecule, we decided to conservatively place a water molecule at this position. RESULTS +21 26 water chemical The position of this water molecule is described in more detail below and is highlighted in Figs 5a and 5b and 6a and 6b as a sphere. RESULTS +55 60 water chemical Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2). RESULTS +74 92 strictly conserved protein_state Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2). RESULTS +103 112 alignment experimental_method Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2). RESULTS +135 147 EctC protein protein_type Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2). RESULTS +188 206 EctC-type proteins protein_type Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2). RESULTS +8 12 open protein_state In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure. RESULTS +14 23 structure evidence In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure. RESULTS +32 34 Sa species In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure. RESULTS +35 39 EctC protein In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure. RESULTS +49 65 electron density evidence In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure. RESULTS +99 103 iron chemical In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure. RESULTS +126 137 semi-closed protein_state In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure. RESULTS +139 148 structure evidence In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure. RESULTS +14 30 electron density evidence However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure. RESULTS +51 56 water chemical However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure. RESULTS +80 84 iron chemical However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure. RESULTS +94 99 water chemical However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure. RESULTS +190 194 open protein_state However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure. RESULTS +197 199 Sa species However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure. RESULTS +200 204 EctC protein However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure. RESULTS +205 214 structure evidence However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure. RESULTS +5 20 superimposition experimental_method In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +30 32 Sa species In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +33 37 EctC protein In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +38 56 crystal structures evidence In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +128 134 Glu-57 residue_name_number In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +136 142 Tyr-85 residue_name_number In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +148 154 His-93 residue_name_number In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +178 182 iron chemical In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +191 202 semi-closed protein_state In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +204 213 structure evidence In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +276 285 iron-free protein_state In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +288 292 open protein_state In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +294 303 structure evidence In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b). RESULTS +5 11 His-93 residue_name_number Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above. RESULTS +48 59 semi-closed protein_state Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above. RESULTS +61 70 structure evidence Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above. RESULTS +104 112 β-strand structure_element Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above. RESULTS +113 115 β5 structure_element Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above. RESULTS +85 102 iron-binding site site Taken together, this observations indicate, that the architecture of the presumptive iron-binding site is pre-set for the binding of the catalytically important metal by the ectoine synthase. RESULTS +161 166 metal chemical Taken together, this observations indicate, that the architecture of the presumptive iron-binding site is pre-set for the binding of the catalytically important metal by the ectoine synthase. RESULTS +174 190 ectoine synthase protein_type Taken together, this observations indicate, that the architecture of the presumptive iron-binding site is pre-set for the binding of the catalytically important metal by the ectoine synthase. RESULTS +66 72 Tyr-52 residue_name_number Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures. RESULTS +87 91 loop structure_element Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures. RESULTS +109 117 β-strand structure_element Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures. RESULTS +118 120 β5 structure_element Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures. RESULTS +130 134 open protein_state Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures. RESULTS +141 152 semi-closed protein_state Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures. RESULTS +155 157 Sa species Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures. RESULTS +158 162 EctC protein Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures. RESULTS +163 173 structures evidence Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures. RESULTS +8 19 semi-closed protein_state In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding. RESULTS +21 30 structure evidence In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding. RESULTS +72 78 Tyr-52 residue_name_number In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding. RESULTS +98 102 iron chemical In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding. RESULTS +189 195 Tyr-52 residue_name_number In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding. RESULTS +220 225 metal chemical In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding. RESULTS +18 30 substitution experimental_method Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1). RESULTS +37 40 Ala residue_name Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1). RESULTS +77 81 iron chemical Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1). RESULTS +117 123 mutant protein_state Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1). RESULTS +28 35 overlay experimental_method It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). RESULTS +44 48 open protein_state It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). RESULTS +55 66 semi-closed protein_state It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). RESULTS +69 71 Sa species It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). RESULTS +72 76 EctC protein It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). RESULTS +77 95 crystal structures evidence It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). RESULTS +119 125 Tyr-52 residue_name_number It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). RESULTS +176 180 iron chemical It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). RESULTS +253 258 metal chemical It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). RESULTS +269 275 Glu-57 residue_name_number It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). RESULTS +277 283 Tyr-85 residue_name_number It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). RESULTS +289 295 His-93 residue_name_number It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b). RESULTS +6 12 Tyr-52 residue_name_number Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility. RESULTS +16 34 strictly conserved protein_state Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility. RESULTS +41 50 alignment experimental_method Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility. RESULTS +58 76 EctC-type proteins protein_type Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility. RESULTS +160 176 ectoine synthase protein_type Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility. RESULTS +190 200 absence of protein_state Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility. RESULTS +201 210 N-γ-ADABA chemical Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility. RESULTS +219 221 Sa species Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility. RESULTS +222 226 EctC protein Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility. RESULTS +227 245 crystal structures evidence Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility. RESULTS +276 282 Tyr-52 residue_name_number Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility. RESULTS +32 49 iron binding site site To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity. RESULTS +73 115 structure-guided site-directed mutagenesis experimental_method To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity. RESULTS +144 146 Sa species To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity. RESULTS +147 151 EctC protein To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity. RESULTS +171 175 iron chemical To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity. RESULTS +27 33 Glu-57 residue_name_number When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1). RESULTS +35 41 Tyr-85 residue_name_number When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1). RESULTS +43 49 His-93 residue_name_number When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1). RESULTS +72 96 mono-nuclear iron center site When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1). RESULTS +105 107 Sa species When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1). RESULTS +108 112 EctC protein When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1). RESULTS +113 130 crystal structure evidence When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1). RESULTS +149 157 replaced experimental_method When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1). RESULTS +164 167 Ala residue_name When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1). RESULTS +213 217 iron chemical When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1). RESULTS +233 239 mutant protein_state When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1). RESULTS +28 54 iron-coordinating residues site For some of the presumptive iron-coordinating residues, additional site-directed mutagenesis experiments were carried out. RESULTS +67 92 site-directed mutagenesis experimental_method For some of the presumptive iron-coordinating residues, additional site-directed mutagenesis experiments were carried out. RESULTS +67 73 Glu-57 residue_name_number To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant. RESULTS +89 92 Asp residue_name To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant. RESULTS +93 100 variant protein_state To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant. RESULTS +5 11 mutant protein_state This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1). RESULTS +52 56 iron chemical This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1). RESULTS +72 75 Ala residue_name This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1). RESULTS +76 88 substitution experimental_method This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1). RESULTS +8 16 replaced experimental_method We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative. RESULTS +17 23 Tyr-85 residue_name_number We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative. RESULTS +38 41 Phe residue_name We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative. RESULTS +47 50 Trp residue_name We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative. RESULTS +68 74 mutant protein_state We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative. RESULTS +126 130 iron chemical We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative. RESULTS +64 70 Tyr-85 residue_name_number Collectively, these data suggest that the hydroxyl group of the Tyr-85 side chain is needed for the binding of the iron (Fig 6a). RESULTS +115 119 iron chemical Collectively, these data suggest that the hydroxyl group of the Tyr-85 side chain is needed for the binding of the iron (Fig 6a). RESULTS +8 16 replaced experimental_method We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1). RESULTS +33 53 iron-binding residue site We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1). RESULTS +54 60 His-93 residue_name_number We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1). RESULTS +67 70 Asn residue_name We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1). RESULTS +92 94 Sa species We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1). RESULTS +95 99 EctC protein We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1). RESULTS +161 165 iron chemical We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1). RESULTS +210 219 wild-type protein_state We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1). RESULTS +68 94 iron-coordinating residues site Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance. RESULTS +96 102 Glu-57 residue_name_number Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance. RESULTS +104 110 Tyr-85 residue_name_number Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance. RESULTS +112 118 His-93 residue_name_number Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance. RESULTS +149 153 Fe2+ chemical Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance. RESULTS +170 172 Sa species Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance. RESULTS +173 177 EctC protein Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance. RESULTS +38 40 Sa species A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate RESULTS +41 45 EctC protein A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate RESULTS +46 55 structure evidence A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate RESULTS +94 103 N-γ-ADABA chemical A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate RESULTS +47 65 co-crystallization experimental_method Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1). RESULTS +69 88 soaking experiments experimental_method Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1). RESULTS +120 122 Sa species Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1). RESULTS +123 127 EctC protein Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1). RESULTS +128 146 crystal structures evidence Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1). RESULTS +183 192 N-γ-ADABA chemical Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1). RESULTS +197 204 ectoine chemical Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1). RESULTS +230 246 ectoine synthase protein_type Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1). RESULTS +17 28 semi-closed protein_state However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement. RESULTS +31 33 Sa species However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement. RESULTS +34 38 EctC protein However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement. RESULTS +39 48 structure evidence However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement. RESULTS +59 80 carboxy-terminal loop structure_element However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement. RESULTS +119 135 electron density evidence However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement. RESULTS +174 185 active site site However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement. RESULTS +227 254 crystallographic refinement experimental_method However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement. RESULTS +44 48 open protein_state This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement. RESULTS +50 59 structure evidence This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement. RESULTS +68 70 Sa species This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement. RESULTS +71 75 EctC protein This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement. RESULTS +104 120 electron density evidence This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement. RESULTS +57 69 purification experimental_method We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched. RESULTS +74 89 crystallization experimental_method We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched. RESULTS +108 124 electron density evidence We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched. RESULTS +91 93 Sa species This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization. RESULTS +94 98 EctC protein This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization. RESULTS +145 152 E. coli species This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization. RESULTS +163 178 crystallization experimental_method This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization. RESULTS +14 17 PEG chemical Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments. RESULTS +76 83 density evidence Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments. RESULTS +121 124 PEG chemical Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments. RESULTS +159 162 PEG chemical Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments. RESULTS +207 210 PEG chemical Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments. RESULTS +38 62 electron density feature evidence Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density. RESULTS +125 127 Sa species Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density. RESULTS +128 132 EctC protein Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density. RESULTS +146 161 hexane-1,6-diol chemical Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density. RESULTS +218 234 electron density evidence Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density. RESULTS +39 54 hexane-1,6-diol chemical However, to the best of our knowledge, hexane-1,6-diol is not part of the E. coli metabolome. RESULTS +74 81 E. coli species However, to the best of our knowledge, hexane-1,6-diol is not part of the E. coli metabolome. RESULTS +179 183 EctC protein Despite these notable limitations, we considered the serendipitously trapped compound as a mock ligand that might provide useful insights into the spatial positioning of the true EctC substrate and those residues that coordinate it within the ectoine synthase active site. RESULTS +243 259 ectoine synthase protein_type Despite these notable limitations, we considered the serendipitously trapped compound as a mock ligand that might provide useful insights into the spatial positioning of the true EctC substrate and those residues that coordinate it within the ectoine synthase active site. RESULTS +260 271 active site site Despite these notable limitations, we considered the serendipitously trapped compound as a mock ligand that might provide useful insights into the spatial positioning of the true EctC substrate and those residues that coordinate it within the ectoine synthase active site. RESULTS +18 27 N-γ-ADABA chemical We note that both N-γ-ADABA and hexane-1,6-diol are both C6-compounds and display similar length (Fig 7a). RESULTS +32 47 hexane-1,6-diol chemical We note that both N-γ-ADABA and hexane-1,6-diol are both C6-compounds and display similar length (Fig 7a). RESULTS +49 60 active site site A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure. FIG +69 80 semi-closed protein_state A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure. FIG +83 85 Sa species A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure. FIG +86 90 EctC protein A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure. FIG +91 108 crystal structure evidence A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure. FIG +17 33 electron density evidence (a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds. FIG +41 52 active site site (a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds. FIG +61 72 semi-closed protein_state (a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds. FIG +74 83 structure evidence (a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds. FIG +88 90 Sa species (a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds. FIG +91 95 EctC protein (a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds. FIG +112 127 hexane-1,6-diol chemical (a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds. FIG +159 175 electron density evidence (a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds. FIG +183 192 N-γ-ADABA chemical (a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds. FIG +210 226 ectoine synthase protein_type (a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds. FIG +19 31 binding site site (b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted. FIG +39 43 iron chemical (b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted. FIG +73 88 hexane-1,6-diol chemical (b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted. FIG +39 43 iron chemical The amino acid side chains involved in iron-ligand binding are colored in blue and those involved in the binding of the chemically undefined ligand are colored in green using a ball and stick representation. FIG +4 12 flexible protein_state The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange. FIG +13 34 carboxy-terminal loop structure_element The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange. FIG +39 41 Sa species The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange. FIG +42 46 EctC protein The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange. FIG +4 20 electron density evidence The electron density was calculated as an omit map and contoured at 1.0 σ. FIG +42 50 omit map evidence The electron density was calculated as an omit map and contoured at 1.0 σ. FIG +3 10 refined experimental_method We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%. RESULTS +16 18 Sa species We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%. RESULTS +19 23 EctC protein We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%. RESULTS +24 33 structure evidence We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%. RESULTS +116 135 R- and Rfree-factor evidence We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%. RESULTS +22 30 omit map evidence We also calculated an omit map and the electron density reappeared (Fig 7b). RESULTS +39 55 electron density evidence We also calculated an omit map and the electron density reappeared (Fig 7b). RESULTS +61 63 Sa species When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +64 68 EctC protein When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +98 103 bound protein_state When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +126 132 Trp-21 residue_name_number When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +137 143 Ser-23 residue_name_number When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +147 154 β-sheet structure_element When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +155 157 β3 structure_element When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +159 165 Thr-40 residue_name_number When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +177 184 β-sheet structure_element When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +185 187 β4 structure_element When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +193 200 Cys-105 residue_name_number When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +205 212 Phe-107 residue_name_number When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +237 244 β-sheet structure_element When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +245 248 β11 structure_element When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11. RESULTS +38 54 highly conserved protein_state Remarkably, all of these residues are highly conserved throughout the extended EctC protein family (Fig 2). RESULTS +79 91 EctC protein protein_type Remarkably, all of these residues are highly conserved throughout the extended EctC protein family (Fig 2). RESULTS +0 42 Structure-guided site-directed mutagenesis experimental_method Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase RESULTS +50 64 catalytic core site Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase RESULTS +72 88 ectoine synthase protein_type Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase RESULTS +14 51 alignment of the amino acid sequences experimental_method In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues. RESULTS +59 77 EctC-type proteins protein_type In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues. RESULTS +113 131 strictly conserved protein_state In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues. RESULTS +32 38 Thr-40 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +40 46 Tyr-52 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +48 54 His-55 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +56 62 Glu-57 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +64 70 Gly-64 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +72 78 Tyr-85 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +80 86 Leu-87 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +88 94 His-93 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +96 103 Phe-107 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +105 112 Pro-109 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +114 121 Gly-113 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +123 130 Glu-115 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +136 143 His-117 residue_name_number These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +152 154 Sa species These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +155 159 EctC protein These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2). RESULTS +20 26 Gly-64 residue_name_number Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices. RESULTS +28 35 Pro-109 residue_name_number Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices. RESULTS +41 48 Gly-113 residue_name_number Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices. RESULTS +148 157 β-strands structure_element Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices. RESULTS +162 171 α-helices structure_element Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices. RESULTS +147 155 flexible protein_state We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein. RESULTS +156 172 carboxy-terminus structure_element We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein. RESULTS +181 183 Sa species We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein. RESULTS +184 188 EctC protein We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein. RESULTS +39 45 Glu-57 residue_name_number As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a). RESULTS +47 53 Tyr-85 residue_name_number As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a). RESULTS +59 65 His-93 residue_name_number As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a). RESULTS +91 95 iron chemical As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a). RESULTS +16 18 Sa species In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1). RESULTS +19 23 EctC protein In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1). RESULTS +24 33 structure evidence In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1). RESULTS +172 214 structure-guided site-directed mutagenesis experimental_method In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1). RESULTS +14 20 mutant protein_state Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations. RESULTS +22 24 Sa species Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations. RESULTS +25 29 EctC protein Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations. RESULTS +59 66 E. coli species Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations. RESULTS +83 106 affinity chromatography experimental_method Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations. RESULTS +36 38 Sa species We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min. RESULTS +39 43 EctC protein We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min. RESULTS +58 88 single time-point enzyme assay experimental_method We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min. RESULTS +125 134 wild-type protein_state We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min. RESULTS +136 138 Sa species We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min. RESULTS +139 143 EctC protein We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min. RESULTS +199 208 N-γ-ADABA chemical We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min. RESULTS +230 237 ectoine chemical We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min. RESULTS +31 35 iron chemical In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1). RESULTS +59 65 mutant protein_state In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1). RESULTS +67 69 Sa species In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1). RESULTS +70 74 EctC protein In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1). RESULTS +88 106 colorimetric assay experimental_method In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1). RESULTS +23 47 evolutionarily conserved protein_state The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b). RESULTS +48 54 Trp-21 residue_name_number The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b). RESULTS +56 62 Ser-23 residue_name_number The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b). RESULTS +64 70 Thr-40 residue_name_number The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b). RESULTS +72 79 Cys-105 residue_name_number The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b). RESULTS +85 92 Phe-107 residue_name_number The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b). RESULTS +186 197 semi-closed protein_state The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b). RESULTS +200 202 Sa species The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b). RESULTS +203 207 EctC protein The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b). RESULTS +208 217 structure evidence The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b). RESULTS +3 11 replaced experimental_method We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins. RESULTS +43 46 Ala residue_name We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins. RESULTS +107 111 iron chemical We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins. RESULTS +127 133 mutant protein_state We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins. RESULTS +0 6 Thr-40 residue_name_number Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b). RESULTS +24 32 β-strand structure_element Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b). RESULTS +33 35 β5 structure_element Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b). RESULTS +87 99 cupin barrel structure_element Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b). RESULTS +115 117 Sa species Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b). RESULTS +118 122 EctC protein Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b). RESULTS +8 16 replaced experimental_method We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. RESULTS +17 24 Phe-107 residue_name_number We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. RESULTS +40 43 Tyr residue_name We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. RESULTS +50 53 Trp residue_name We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. RESULTS +67 78 Phe-107/Tyr mutant We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. RESULTS +79 91 substitution experimental_method We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. RESULTS +107 116 wild-type protein_state We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. RESULTS +158 162 iron chemical We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. RESULTS +180 191 Phe-107/Trp mutant We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. RESULTS +192 204 substitution experimental_method We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. RESULTS +248 252 iron chemical We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. RESULTS +277 286 wild-type protein_state We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein. RESULTS +24 30 mutant protein_state The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding. RESULTS +90 93 107 residue_number The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding. RESULTS +98 100 Sa species The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding. RESULTS +101 105 EctC protein The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding. RESULTS +134 146 substitution experimental_method The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding. RESULTS +260 264 iron chemical The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding. RESULTS +0 11 Replacement experimental_method Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +24 27 Cys residue_name Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +40 42 Sa species Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +43 47 EctC protein Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +49 56 Cys-105 residue_name_number Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +70 73 Ser residue_name Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +130 143 EctC proteins protein_type Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +197 199 Sa species Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +200 204 EctC protein Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +205 212 variant protein_state Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +222 231 wild-type protein_state Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +248 252 iron chemical Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +284 293 wild-type protein_state Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein. RESULTS +13 24 Cys-105/Ala mutant However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1). RESULTS +25 32 variant protein_state However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1). RESULTS +49 71 catalytically inactive protein_state However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1). RESULTS +102 106 iron chemical However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1). RESULTS +25 28 Cys residue_name Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase. RESULTS +105 112 Cys-105 residue_name_number Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase. RESULTS +117 124 Ser-105 residue_name_number Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase. RESULTS +154 170 ectoine synthase protein_type Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase. RESULTS +16 40 amino acid substitutions experimental_method We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1). RESULTS +99 103 iron chemical We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1). RESULTS +128 138 Tyr-52/Ala mutant We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1). RESULTS +147 157 His-55/Ala mutant We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1). RESULTS +159 161 Sa species We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1). RESULTS +162 166 EctC protein We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1). RESULTS +14 16 Sa species Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1). RESULTS +17 21 EctC protein Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1). RESULTS +22 40 crystal structures evidence Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1). RESULTS +110 121 replacement experimental_method Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1). RESULTS +125 131 Tyr-52 residue_name_number Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1). RESULTS +135 138 Ala residue_name Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1). RESULTS +167 171 iron chemical Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1). RESULTS +26 36 His-55/Ala mutant This is different for the His-55/Ala substitution. RESULTS +4 27 carboxy-terminal region structure_element The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b). RESULTS +36 38 Sa species The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b). RESULTS +39 43 EctC protein The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b). RESULTS +98 105 Glu-115 residue_name_number The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b). RESULTS +111 117 His-55 residue_name_number The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b). RESULTS +125 131 His-55 residue_name_number The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b). RESULTS +155 162 Pro-110 residue_name_number The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b). RESULTS +26 57 evolutionarily highly conserved protein_state Each of these residues is evolutionarily highly conserved. RESULTS +15 27 substitution experimental_method The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity. RESULTS +38 45 Glu-115 residue_name_number The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity. RESULTS +49 55 His-55 residue_name_number The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity. RESULTS +62 65 Ala residue_name The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity. RESULTS +103 122 interactive network site The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity. RESULTS +12 23 Glu-115/Ala mutant Indeed, the Glu-115/Ala and the His-55/Ala substitutions possessed only 21% and 16% activity of the wild-type protein, respectively (Table 1). RESULTS +32 42 His-55/Ala mutant Indeed, the Glu-115/Ala and the His-55/Ala substitutions possessed only 21% and 16% activity of the wild-type protein, respectively (Table 1). RESULTS +100 109 wild-type protein_state Indeed, the Glu-115/Ala and the His-55/Ala substitutions possessed only 21% and 16% activity of the wild-type protein, respectively (Table 1). RESULTS +4 15 Glu-115/Ala mutant The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1). RESULTS +16 22 mutant protein_state The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1). RESULTS +33 42 wild-type protein_state The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1). RESULTS +53 57 iron chemical The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1). RESULTS +71 75 iron chemical The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1). RESULTS +91 101 His-55/Ala mutant The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1). RESULTS +138 147 wild-type protein_state The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1). RESULTS +8 16 replaced experimental_method We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity. RESULTS +17 24 Glu-115 residue_name_number We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity. RESULTS +60 63 Asp residue_name We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity. RESULTS +72 74 Sa species We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity. RESULTS +75 79 EctC protein We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity. RESULTS +98 107 wild-type protein_state We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity. RESULTS +118 122 iron chemical We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity. RESULTS +150 159 wild-type protein_state We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity. RESULTS +69 85 carboxy-terminus structure_element Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase. RESULTS +94 96 Sa species Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase. RESULTS +97 101 EctC protein Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase. RESULTS +177 193 ectoine synthase protein_type Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase. RESULTS +9 15 Leu-87 residue_name_number Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family. RESULTS +20 26 Asp-91 residue_name_number Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family. RESULTS +31 47 highly conserved protein_state Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family. RESULTS +55 71 ectoine synthase protein_type Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family. RESULTS +4 15 replacement experimental_method The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1). RESULTS +19 25 Leu-87 residue_name_number The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1). RESULTS +29 32 Ala residue_name The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1). RESULTS +16 27 replacement experimental_method Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1). RESULTS +31 37 Asp-91 residue_name_number Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1). RESULTS +41 44 Ala residue_name Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1). RESULTS +49 52 Glu residue_name Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1). RESULTS +67 69 Sa species Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1). RESULTS +70 74 EctC protein Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1). RESULTS +56 62 Asp-91 residue_name_number We currently cannot comment on possible functional role Asp-91. RESULTS +9 15 Leu-87 residue_name_number However, Leu-87 is positioned at the end of one of the β-sheets that form the dimer interface (Fig 5c) and it might therefore possess a structural role. RESULTS +55 63 β-sheets structure_element However, Leu-87 is positioned at the end of one of the β-sheets that form the dimer interface (Fig 5c) and it might therefore possess a structural role. RESULTS +78 93 dimer interface site However, Leu-87 is positioned at the end of one of the β-sheets that form the dimer interface (Fig 5c) and it might therefore possess a structural role. RESULTS +24 30 Tyr-85 residue_name_number It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects. RESULTS +81 85 iron chemical It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects. RESULTS +108 110 Sa species It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects. RESULTS +111 115 EctC protein It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects. RESULTS +116 127 active site site It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects. RESULTS +0 7 His-117 residue_name_number His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1). RESULTS +13 31 strictly conserved protein_state His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1). RESULTS +48 60 substitution experimental_method His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1). RESULTS +67 70 Ala residue_name His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1). RESULTS +137 141 iron chemical His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1). RESULTS +13 20 His-117 residue_name_number We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase. RESULTS +81 83 Sa species We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase. RESULTS +84 88 EctC protein We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase. RESULTS +89 98 structure evidence We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase. RESULTS +178 194 ectoine synthase protein_type We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase. RESULTS +31 54 mutagenesis experiments experimental_method As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues. RESULTS +64 75 substituted experimental_method As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues. RESULTS +76 82 Thr-41 residue_name_number As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues. RESULTS +87 93 His-51 residue_name_number As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues. RESULTS +117 145 not evolutionarily conserved protein_state As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues. RESULTS +149 167 EctC-type proteins protein_type As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues. RESULTS +173 176 Ala residue_name As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues. RESULTS +6 8 Sa species Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1). RESULTS +9 13 EctC protein Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1). RESULTS +41 50 wild-type protein_state Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1). RESULTS +91 95 iron chemical Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1). RESULTS +125 134 wild-type protein_state Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1). RESULTS +64 66 Sa species This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content. RESULTS +67 71 EctC protein This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content. RESULTS +141 145 iron chemical This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content. RESULTS +4 25 crystallographic data evidence The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily. DISCUSS +57 73 ectoine synthase protein_type The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily. DISCUSS +75 79 EctC protein The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily. DISCUSS +127 136 microbial taxonomy_domain The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily. DISCUSS +175 182 ectoine chemical The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily. DISCUSS +207 224 cupin superfamily protein_type The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily. DISCUSS +40 42 Sa species The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins. DISCUSS +43 47 EctC protein The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins. DISCUSS +83 92 β-strands structure_element The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins. DISCUSS +94 100 β1-β11 structure_element The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins. DISCUSS +110 119 α-helices structure_element The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins. DISCUSS +121 124 α-I structure_element The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins. DISCUSS +129 133 α-II structure_element The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins. DISCUSS +195 213 crystal structures evidence The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins. DISCUSS +217 223 cupins protein_type The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins. DISCUSS +19 35 ectoine synthase protein_type In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different. DISCUSS +41 59 polyketide cyclase protein_type In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different. DISCUSS +60 64 RemF protein In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different. DISCUSS +99 112 cupin-related protein_type In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different. DISCUSS +190 194 EctC protein In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different. DISCUSS +199 203 RemF protein In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different. DISCUSS +50 54 EctC protein As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins. DISCUSS +59 63 RemF protein As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins. DISCUSS +162 180 EctC-type proteins protein_type As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins. DISCUSS +215 224 microbial taxonomy_domain As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins. DISCUSS +246 255 RemF-like protein_type As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins. DISCUSS +4 8 pro- taxonomy_domain The pro- and eukaryotic members of the cupin superfamily perform a variety of both enzymatic and non-enzymatic functions that are built upon a common structural scaffold. DISCUSS +13 23 eukaryotic taxonomy_domain The pro- and eukaryotic members of the cupin superfamily perform a variety of both enzymatic and non-enzymatic functions that are built upon a common structural scaffold. DISCUSS +39 56 cupin superfamily protein_type The pro- and eukaryotic members of the cupin superfamily perform a variety of both enzymatic and non-enzymatic functions that are built upon a common structural scaffold. DISCUSS +5 11 cupins protein_type Most cupins contain transition state metals that can promote different types of chemical reactions. DISCUSS +16 38 cupin-related proteins protein_type Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites. DISCUSS +64 82 metallo-chaperones protein_type Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites. DISCUSS +88 93 bound protein_state Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites. DISCUSS +94 99 metal chemical Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites. DISCUSS +138 150 active sites site Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites. DISCUSS +43 59 ectoine synthase protein_type We report here for the first time that the ectoine synthase is a metal-dependent enzyme. DISCUSS +65 70 metal chemical We report here for the first time that the ectoine synthase is a metal-dependent enzyme. DISCUSS +0 6 ICP-MS experimental_method ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction. DISCUSS +8 54 metal-depletion and reconstitution experiments experimental_method ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction. DISCUSS +85 89 iron chemical ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction. DISCUSS +124 129 metal chemical ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction. DISCUSS +138 142 EctC protein ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction. DISCUSS +32 38 cupins protein_type However, as observed with other cupins, EctC is a somewhat promiscuous enzyme as far as the catalytically important metal is concerned when they are provided in large molar excess (Fig 3c). DISCUSS +40 44 EctC protein However, as observed with other cupins, EctC is a somewhat promiscuous enzyme as far as the catalytically important metal is concerned when they are provided in large molar excess (Fig 3c). DISCUSS +116 121 metal chemical However, as observed with other cupins, EctC is a somewhat promiscuous enzyme as far as the catalytically important metal is concerned when they are provided in large molar excess (Fig 3c). DISCUSS +114 119 metal chemical Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1). DISCUSS +132 134 Sa species Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1). DISCUSS +135 139 EctC protein Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1). DISCUSS +145 187 structure-guided site-directed mutagenesis experimental_method Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1). DISCUSS +226 247 iron-binding residues site Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1). DISCUSS +24 36 metal center site The architecture of the metal center of ectoine synthase seems to be subjected to considerable evolutionary constraints. DISCUSS +40 56 ectoine synthase protein_type The architecture of the metal center of ectoine synthase seems to be subjected to considerable evolutionary constraints. DISCUSS +20 26 Glu-57 residue_name_number The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2). DISCUSS +28 34 Tyr-85 residue_name_number The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2). DISCUSS +36 42 His-93 residue_name_number The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2). DISCUSS +88 106 strictly conserved protein_state The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2). DISCUSS +132 150 EctC-type proteins protein_type The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2). DISCUSS +171 180 bacterial taxonomy_domain The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2). DISCUSS +191 199 archaeal taxonomy_domain The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2). DISCUSS +80 89 N-γ-ADABA chemical We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig). DISCUSS +91 95 EctC protein We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig). DISCUSS +121 130 N-α-ADABA chemical We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig). DISCUSS +136 143 ectoine chemical We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig). DISCUSS +175 195 catalytic efficiency evidence We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig). DISCUSS +11 22 active site site Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules. DISCUSS +26 42 ectoine synthase protein_type Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules. DISCUSS +147 151 EctC protein Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules. DISCUSS +207 211 ADPC chemical Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules. DISCUSS +251 260 glutamine chemical Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules. DISCUSS +17 26 N-α-ADABA chemical Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route. DISCUSS +53 69 ectoine synthase protein_type Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route. DISCUSS +123 137 microorganisms taxonomy_domain Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route. DISCUSS +178 185 ectoine chemical Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route. DISCUSS +263 272 N-α-ADABA chemical Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route. DISCUSS +60 62 C6 chemical Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a). DISCUSS +97 99 Sa species Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a). DISCUSS +100 104 EctC protein Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a). DISCUSS +105 114 structure evidence Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a). DISCUSS +204 220 ectoine synthase protein_type Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a). DISCUSS +122 131 N-γ-ADABA chemical We assumed that its location and mode of binding gives, in all likelihood, clues as to the position of the true substrate N-γ-ADABA within the EctC active site. DISCUSS +143 147 EctC protein We assumed that its location and mode of binding gives, in all likelihood, clues as to the position of the true substrate N-γ-ADABA within the EctC active site. DISCUSS +148 159 active site site We assumed that its location and mode of binding gives, in all likelihood, clues as to the position of the true substrate N-γ-ADABA within the EctC active site. DISCUSS +8 33 site-directed mutagenesis experimental_method Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1). DISCUSS +112 114 Sa species Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1). DISCUSS +115 119 EctC protein Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1). DISCUSS +177 186 wild-type protein_state Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1). DISCUSS +197 201 iron chemical Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1). DISCUSS +61 79 strongly conserved protein_state This set of data and the fact that the targeted residues are strongly conserved among EctC-type proteins (Fig 2) is consistent with their potential role in N-γ-ADABA binding or enzyme catalysis. DISCUSS +86 104 EctC-type proteins protein_type This set of data and the fact that the targeted residues are strongly conserved among EctC-type proteins (Fig 2) is consistent with their potential role in N-γ-ADABA binding or enzyme catalysis. DISCUSS +156 165 N-γ-ADABA chemical This set of data and the fact that the targeted residues are strongly conserved among EctC-type proteins (Fig 2) is consistent with their potential role in N-γ-ADABA binding or enzyme catalysis. DISCUSS +30 51 crystallographic data evidence We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b). DISCUSS +60 91 site-directed mutagenesis study experimental_method We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b). DISCUSS +176 180 EctC protein We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b). DISCUSS +181 192 active site site We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b). DISCUSS +4 20 ectoine synthase protein_type The ectoine synthase from the cold-adapted marine bacterium S. alaskensis can be considered as a psychrophilic enzyme (S3a Fig), types of proteins with a considerable structural flexibility. DISCUSS +43 59 marine bacterium taxonomy_domain The ectoine synthase from the cold-adapted marine bacterium S. alaskensis can be considered as a psychrophilic enzyme (S3a Fig), types of proteins with a considerable structural flexibility. DISCUSS +60 73 S. alaskensis species The ectoine synthase from the cold-adapted marine bacterium S. alaskensis can be considered as a psychrophilic enzyme (S3a Fig), types of proteins with a considerable structural flexibility. DISCUSS +64 82 crystal structures evidence This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine. DISCUSS +90 101 full-length protein_state This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine. DISCUSS +103 105 Sa species This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine. DISCUSS +106 110 EctC protein This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine. DISCUSS +119 134 in complex with protein_state This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine. DISCUSS +142 151 N-γ-ADABA chemical This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine. DISCUSS +155 162 ectoine chemical This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine. DISCUSS +8 17 microbial taxonomy_domain Because microbial ectoine producers can colonize ecological niches with rather different physicochemical attributes, it seems promising to exploit this considerable biodiversity to identify EctC proteins with enhanced protein stability. DISCUSS +18 25 ectoine chemical Because microbial ectoine producers can colonize ecological niches with rather different physicochemical attributes, it seems promising to exploit this considerable biodiversity to identify EctC proteins with enhanced protein stability. DISCUSS +190 203 EctC proteins protein_type Because microbial ectoine producers can colonize ecological niches with rather different physicochemical attributes, it seems promising to exploit this considerable biodiversity to identify EctC proteins with enhanced protein stability. DISCUSS +57 61 EctC protein It is hoped that these can be further employed to obtain EctC crystal structures with either the substrate or the reaction product. DISCUSS +62 80 crystal structures evidence It is hoped that these can be further employed to obtain EctC crystal structures with either the substrate or the reaction product. DISCUSS +31 47 ectoine synthase protein_type Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction. DISCUSS +51 66 metal dependent protein_state Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction. DISCUSS +74 92 crystal structures evidence Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction. DISCUSS +168 172 EctC protein Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction. DISCUSS diff --git a/annotation_CSV/PMC4802042.csv b/annotation_CSV/PMC4802042.csv new file mode 100644 index 0000000000000000000000000000000000000000..8ff624c05dc3ffc94ba87e2ed30dd43693ec2a56 --- /dev/null +++ b/annotation_CSV/PMC4802042.csv @@ -0,0 +1,1442 @@ +anno_start anno_end anno_text entity_type sentence section +21 55 JNK/p38-specific MAPK phosphatases protein_type A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation TITLE +77 81 JNK1 protein A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation TITLE +0 33 Mitogen-activated protein kinases protein_type Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs). ABSTRACT +35 40 MAPKs protein_type Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs). ABSTRACT +133 148 protein kinases protein_type Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs). ABSTRACT +156 173 MAPK phosphatases protein_type Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs). ABSTRACT +175 179 MKPs protein_type Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs). ABSTRACT +0 4 MAPK protein_type MAPK signalling efficiency and specificity is modulated by protein–protein interactions between individual MAPKs and the docking motifs in cognate binding partners. ABSTRACT +107 112 MAPKs protein_type MAPK signalling efficiency and specificity is modulated by protein–protein interactions between individual MAPKs and the docking motifs in cognate binding partners. ABSTRACT +121 135 docking motifs structure_element MAPK signalling efficiency and specificity is modulated by protein–protein interactions between individual MAPKs and the docking motifs in cognate binding partners. ABSTRACT +56 63 D-motif structure_element Two types of docking interactions have been identified: D-motif-mediated interaction and FXF-docking interaction. ABSTRACT +89 112 FXF-docking interaction site Two types of docking interactions have been identified: D-motif-mediated interaction and FXF-docking interaction. ABSTRACT +19 36 crystal structure evidence Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-Å resolution, providing high-resolution structural insight into the FXF-docking interaction. ABSTRACT +40 44 JNK1 protein Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-Å resolution, providing high-resolution structural insight into the FXF-docking interaction. ABSTRACT +45 53 bound to protein_state Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-Å resolution, providing high-resolution structural insight into the FXF-docking interaction. ABSTRACT +58 74 catalytic domain structure_element Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-Å resolution, providing high-resolution structural insight into the FXF-docking interaction. ABSTRACT +78 82 MKP7 protein Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-Å resolution, providing high-resolution structural insight into the FXF-docking interaction. ABSTRACT +158 181 FXF-docking interaction site Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-Å resolution, providing high-resolution structural insight into the FXF-docking interaction. ABSTRACT +4 22 285FNFL288 segment structure_element The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +26 30 MKP7 protein The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +51 67 hydrophobic site site The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +71 75 JNK1 protein The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +93 97 MAPK protein_type The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +112 117 helix structure_element The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +118 120 αG structure_element The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +122 141 Biochemical studies experimental_method The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +167 183 highly conserved protein_state The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +184 200 structural motif structure_element The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +234 244 MKP family protein_type The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +305 308 MKP protein_type The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +309 313 MAPK protein_type The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. ABSTRACT +15 26 MAPK family protein_type The important MAPK family of signalling proteins is controlled by MAPK phosphatases (MKPs). ABSTRACT +67 84 MAPK phosphatases protein_type The important MAPK family of signalling proteins is controlled by MAPK phosphatases (MKPs). ABSTRACT +86 90 MKPs protein_type The important MAPK family of signalling proteins is controlled by MAPK phosphatases (MKPs). ABSTRACT +29 38 structure evidence Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction. ABSTRACT +42 46 MKP7 protein Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction. ABSTRACT +47 55 bound to protein_state Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction. ABSTRACT +56 60 JNK1 protein Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction. ABSTRACT +82 91 conserved protein_state Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction. ABSTRACT +92 95 MKP protein_type Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction. ABSTRACT +96 100 MAPK protein_type Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction. ABSTRACT +4 37 mitogen-activated protein kinases protein_type The mitogen-activated protein kinases (MAPKs) are central components of the signal-transduction pathways, which mediate the cellular response to a variety of extracellular stimuli, ranging from growth factors to environmental stresses. INTRO +39 44 MAPKs protein_type The mitogen-activated protein kinases (MAPKs) are central components of the signal-transduction pathways, which mediate the cellular response to a variety of extracellular stimuli, ranging from growth factors to environmental stresses. INTRO +4 8 MAPK protein_type The MAPK signalling pathways are evolutionally highly conserved. INTRO +22 26 MAPK protein_type The basic assembly of MAPK pathways is a three-tier kinase module that establishes a sequential activation cascade: a MAPK kinase kinase activates a MAPK kinase, which in turn activates a MAPK. INTRO +52 58 kinase protein_type The basic assembly of MAPK pathways is a three-tier kinase module that establishes a sequential activation cascade: a MAPK kinase kinase activates a MAPK kinase, which in turn activates a MAPK. INTRO +118 136 MAPK kinase kinase protein_type The basic assembly of MAPK pathways is a three-tier kinase module that establishes a sequential activation cascade: a MAPK kinase kinase activates a MAPK kinase, which in turn activates a MAPK. INTRO +149 160 MAPK kinase protein_type The basic assembly of MAPK pathways is a three-tier kinase module that establishes a sequential activation cascade: a MAPK kinase kinase activates a MAPK kinase, which in turn activates a MAPK. INTRO +188 192 MAPK protein_type The basic assembly of MAPK pathways is a three-tier kinase module that establishes a sequential activation cascade: a MAPK kinase kinase activates a MAPK kinase, which in turn activates a MAPK. INTRO +29 33 MAPK protein_type The three best-characterized MAPK signalling pathways are mediated by the kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. INTRO +74 81 kinases protein_type The three best-characterized MAPK signalling pathways are mediated by the kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. INTRO +82 119 extracellular signal-regulated kinase protein_type The three best-characterized MAPK signalling pathways are mediated by the kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. INTRO +121 124 ERK protein_type The three best-characterized MAPK signalling pathways are mediated by the kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. INTRO +127 150 c-Jun N-terminal kinase protein_type The three best-characterized MAPK signalling pathways are mediated by the kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. INTRO +152 155 JNK protein_type The three best-characterized MAPK signalling pathways are mediated by the kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. INTRO +161 164 p38 protein_type The three best-characterized MAPK signalling pathways are mediated by the kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. INTRO +4 7 ERK protein_type The ERK pathway is activated by various mitogens and phorbol esters, whereas the JNK and p38 pathways are stimulated mainly by environmental stress and inflammatory cytokines. INTRO +81 84 JNK protein_type The ERK pathway is activated by various mitogens and phorbol esters, whereas the JNK and p38 pathways are stimulated mainly by environmental stress and inflammatory cytokines. INTRO +89 92 p38 protein_type The ERK pathway is activated by various mitogens and phorbol esters, whereas the JNK and p38 pathways are stimulated mainly by environmental stress and inflammatory cytokines. INTRO +165 174 cytokines protein_type The ERK pathway is activated by various mitogens and phorbol esters, whereas the JNK and p38 pathways are stimulated mainly by environmental stress and inflammatory cytokines. INTRO +4 9 MAPKs protein_type The MAPKs are activated by MAPK kinases that phosphorylate the MAPKs at conserved threonine and tyrosine residues within their activation loop. INTRO +27 39 MAPK kinases protein_type The MAPKs are activated by MAPK kinases that phosphorylate the MAPKs at conserved threonine and tyrosine residues within their activation loop. INTRO +63 68 MAPKs protein_type The MAPKs are activated by MAPK kinases that phosphorylate the MAPKs at conserved threonine and tyrosine residues within their activation loop. INTRO +72 81 conserved protein_state The MAPKs are activated by MAPK kinases that phosphorylate the MAPKs at conserved threonine and tyrosine residues within their activation loop. INTRO +82 91 threonine residue_name The MAPKs are activated by MAPK kinases that phosphorylate the MAPKs at conserved threonine and tyrosine residues within their activation loop. INTRO +96 104 tyrosine residue_name The MAPKs are activated by MAPK kinases that phosphorylate the MAPKs at conserved threonine and tyrosine residues within their activation loop. INTRO +127 142 activation loop structure_element The MAPKs are activated by MAPK kinases that phosphorylate the MAPKs at conserved threonine and tyrosine residues within their activation loop. INTRO +23 27 MAPK protein_type After activation, each MAPK phosphorylates a distinct set of protein substrates, which act as the critical effectors that enable cells to mount the appropriate responses to varied stimuli. INTRO +0 5 MAPKs protein_type MAPKs lie at the bottom of conserved three-component phosphorylation cascades and utilize docking interactions to link module components and bind substrates. INTRO +13 27 docking motifs structure_element Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF). INTRO +52 56 MAPK protein_type Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF). INTRO +90 114 kinase-interacting motif structure_element Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF). INTRO +116 123 D-motif structure_element Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF). INTRO +129 138 FXF-motif structure_element Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF). INTRO +152 161 DEF motif structure_element Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF). INTRO +163 175 docking site site Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF). INTRO +180 183 ERK protein_type Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF). INTRO +184 187 FXF structure_element Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF). INTRO +56 67 MAP kinases protein_type The best-studied docking interactions are those between MAP kinases and ‘D-motifs', which consists of two or more basic residues followed by a short linker and a cluster of hydrophobic residues. INTRO +73 81 D-motifs structure_element The best-studied docking interactions are those between MAP kinases and ‘D-motifs', which consists of two or more basic residues followed by a short linker and a cluster of hydrophobic residues. INTRO +143 155 short linker structure_element The best-studied docking interactions are those between MAP kinases and ‘D-motifs', which consists of two or more basic residues followed by a short linker and a cluster of hydrophobic residues. INTRO +4 24 D-motif-docking site site The D-motif-docking site (D-site) in MAPKs is situated in a noncatalytic region opposite of the kinase catalytic pocket and is comprised of a highly acidic patch and a hydrophobic groove. INTRO +26 32 D-site site The D-motif-docking site (D-site) in MAPKs is situated in a noncatalytic region opposite of the kinase catalytic pocket and is comprised of a highly acidic patch and a hydrophobic groove. INTRO +37 42 MAPKs protein_type The D-motif-docking site (D-site) in MAPKs is situated in a noncatalytic region opposite of the kinase catalytic pocket and is comprised of a highly acidic patch and a hydrophobic groove. INTRO +60 79 noncatalytic region site The D-motif-docking site (D-site) in MAPKs is situated in a noncatalytic region opposite of the kinase catalytic pocket and is comprised of a highly acidic patch and a hydrophobic groove. INTRO +96 102 kinase protein_type The D-motif-docking site (D-site) in MAPKs is situated in a noncatalytic region opposite of the kinase catalytic pocket and is comprised of a highly acidic patch and a hydrophobic groove. INTRO +103 119 catalytic pocket site The D-motif-docking site (D-site) in MAPKs is situated in a noncatalytic region opposite of the kinase catalytic pocket and is comprised of a highly acidic patch and a hydrophobic groove. INTRO +142 161 highly acidic patch site The D-motif-docking site (D-site) in MAPKs is situated in a noncatalytic region opposite of the kinase catalytic pocket and is comprised of a highly acidic patch and a hydrophobic groove. INTRO +168 186 hydrophobic groove site The D-motif-docking site (D-site) in MAPKs is situated in a noncatalytic region opposite of the kinase catalytic pocket and is comprised of a highly acidic patch and a hydrophobic groove. INTRO +0 8 D-motifs structure_element D-motifs are found in many MAPK-interacting proteins, including substrates, activating kinases and inactivating phosphatases, as well as scaffolding proteins. INTRO +27 52 MAPK-interacting proteins protein_type D-motifs are found in many MAPK-interacting proteins, including substrates, activating kinases and inactivating phosphatases, as well as scaffolding proteins. INTRO +87 94 kinases protein_type D-motifs are found in many MAPK-interacting proteins, including substrates, activating kinases and inactivating phosphatases, as well as scaffolding proteins. INTRO +112 124 phosphatases protein_type D-motifs are found in many MAPK-interacting proteins, including substrates, activating kinases and inactivating phosphatases, as well as scaffolding proteins. INTRO +2 22 second docking motif structure_element A second docking motif for MAPKs consists of two Phe residues separated by one residue (FXF-motif). INTRO +27 32 MAPKs protein_type A second docking motif for MAPKs consists of two Phe residues separated by one residue (FXF-motif). INTRO +49 52 Phe residue_name A second docking motif for MAPKs consists of two Phe residues separated by one residue (FXF-motif). INTRO +88 97 FXF-motif structure_element A second docking motif for MAPKs consists of two Phe residues separated by one residue (FXF-motif). INTRO +40 44 MAPK protein_type This motif has been observed in several MAPK substrates. INTRO +4 26 FXF-motif-binding site site The FXF-motif-binding site of ERK2 has been mapped to a hydrophobic pocket formed between the P+1 site, αG helix and the MAPK insert. INTRO +30 34 ERK2 protein The FXF-motif-binding site of ERK2 has been mapped to a hydrophobic pocket formed between the P+1 site, αG helix and the MAPK insert. INTRO +56 74 hydrophobic pocket site The FXF-motif-binding site of ERK2 has been mapped to a hydrophobic pocket formed between the P+1 site, αG helix and the MAPK insert. INTRO +94 102 P+1 site site The FXF-motif-binding site of ERK2 has been mapped to a hydrophobic pocket formed between the P+1 site, αG helix and the MAPK insert. INTRO +104 112 αG helix structure_element The FXF-motif-binding site of ERK2 has been mapped to a hydrophobic pocket formed between the P+1 site, αG helix and the MAPK insert. INTRO +121 132 MAPK insert structure_element The FXF-motif-binding site of ERK2 has been mapped to a hydrophobic pocket formed between the P+1 site, αG helix and the MAPK insert. INTRO +45 48 FXF structure_element However, the generality and mechanism of the FXF-mediated interaction is unclear. INTRO +29 33 MAPK protein_type The physiological outcome of MAPK signalling depends on both the magnitude and the duration of kinase activation. INTRO +18 22 MAPK protein_type Downregulation of MAPK activity can be achieved through direct dephosphorylation of the phospho-threonine and/or tyrosine residues by various serine/threonine phosphatases, tyrosine phosphatases and dual-specificity phosphatases (DUSPs) termed MKPs. INTRO +88 121 phospho-threonine and/or tyrosine residue_name Downregulation of MAPK activity can be achieved through direct dephosphorylation of the phospho-threonine and/or tyrosine residues by various serine/threonine phosphatases, tyrosine phosphatases and dual-specificity phosphatases (DUSPs) termed MKPs. INTRO +142 171 serine/threonine phosphatases protein_type Downregulation of MAPK activity can be achieved through direct dephosphorylation of the phospho-threonine and/or tyrosine residues by various serine/threonine phosphatases, tyrosine phosphatases and dual-specificity phosphatases (DUSPs) termed MKPs. INTRO +173 194 tyrosine phosphatases protein_type Downregulation of MAPK activity can be achieved through direct dephosphorylation of the phospho-threonine and/or tyrosine residues by various serine/threonine phosphatases, tyrosine phosphatases and dual-specificity phosphatases (DUSPs) termed MKPs. INTRO +199 228 dual-specificity phosphatases protein_type Downregulation of MAPK activity can be achieved through direct dephosphorylation of the phospho-threonine and/or tyrosine residues by various serine/threonine phosphatases, tyrosine phosphatases and dual-specificity phosphatases (DUSPs) termed MKPs. INTRO +230 235 DUSPs protein_type Downregulation of MAPK activity can be achieved through direct dephosphorylation of the phospho-threonine and/or tyrosine residues by various serine/threonine phosphatases, tyrosine phosphatases and dual-specificity phosphatases (DUSPs) termed MKPs. INTRO +244 248 MKPs protein_type Downregulation of MAPK activity can be achieved through direct dephosphorylation of the phospho-threonine and/or tyrosine residues by various serine/threonine phosphatases, tyrosine phosphatases and dual-specificity phosphatases (DUSPs) termed MKPs. INTRO +0 4 MKPs protein_type MKPs constitute a group of DUSPs that are characterized by their ability to dephosphorylate both phosphotyrosine and phosphoserine/phospho-threonine residues within a substrate. INTRO +27 32 DUSPs protein_type MKPs constitute a group of DUSPs that are characterized by their ability to dephosphorylate both phosphotyrosine and phosphoserine/phospho-threonine residues within a substrate. INTRO +97 112 phosphotyrosine residue_name MKPs constitute a group of DUSPs that are characterized by their ability to dephosphorylate both phosphotyrosine and phosphoserine/phospho-threonine residues within a substrate. INTRO +117 130 phosphoserine residue_name MKPs constitute a group of DUSPs that are characterized by their ability to dephosphorylate both phosphotyrosine and phosphoserine/phospho-threonine residues within a substrate. INTRO +131 148 phospho-threonine residue_name MKPs constitute a group of DUSPs that are characterized by their ability to dephosphorylate both phosphotyrosine and phosphoserine/phospho-threonine residues within a substrate. INTRO +27 31 MKPs protein_type Dysregulated expression of MKPs has been associated with pathogenesis of various diseases, and understanding their precise recognition mechanism presents an important challenge and opportunity for drug development. INTRO +21 38 crystal structure evidence Here, we present the crystal structure of JNK1 in complex with the catalytic domain of MKP7. INTRO +42 46 JNK1 protein Here, we present the crystal structure of JNK1 in complex with the catalytic domain of MKP7. INTRO +47 62 in complex with protein_state Here, we present the crystal structure of JNK1 in complex with the catalytic domain of MKP7. INTRO +67 83 catalytic domain structure_element Here, we present the crystal structure of JNK1 in complex with the catalytic domain of MKP7. INTRO +87 91 MKP7 protein Here, we present the crystal structure of JNK1 in complex with the catalytic domain of MKP7. INTRO +5 14 structure evidence This structure reveals the molecular mechanism underlying the docking interaction between MKP7 and JNK1. INTRO +90 94 MKP7 protein This structure reveals the molecular mechanism underlying the docking interaction between MKP7 and JNK1. INTRO +99 103 JNK1 protein This structure reveals the molecular mechanism underlying the docking interaction between MKP7 and JNK1. INTRO +7 16 JNK1–MKP7 complex_assembly In the JNK1–MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix α5 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction. INTRO +28 45 hydrophobic motif structure_element In the JNK1–MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix α5 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction. INTRO +47 57 285FNFL288 structure_element In the JNK1–MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix α5 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction. INTRO +78 83 helix structure_element In the JNK1–MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix α5 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction. INTRO +84 86 α5 structure_element In the JNK1–MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix α5 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction. INTRO +94 98 MKP7 protein In the JNK1–MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix α5 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction. INTRO +99 115 catalytic domain structure_element In the JNK1–MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix α5 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction. INTRO +138 160 FXF-motif-binding site site In the JNK1–MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix α5 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction. INTRO +164 168 JNK1 protein In the JNK1–MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix α5 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction. INTRO +220 248 FXF-type docking interaction site In the JNK1–MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix α5 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction. INTRO +0 33 Biochemical and modelling studies experimental_method Biochemical and modelling studies further demonstrate that the molecular interactions mediate this key element for substrate recognition are highly conserved among all MKP-family members. INTRO +168 186 MKP-family members protein_type Biochemical and modelling studies further demonstrate that the molecular interactions mediate this key element for substrate recognition are highly conserved among all MKP-family members. INTRO +111 124 MAPK isoforms protein_type Thus, our study reveals a hitherto unrecognized interaction mode for encoding complex target specificity among MAPK isoforms. INTRO +15 19 JNK1 protein Interaction of JNK1 with the MKP7 catalytic domain RESULTS +29 33 MKP7 protein Interaction of JNK1 with the MKP7 catalytic domain RESULTS +34 50 catalytic domain structure_element Interaction of JNK1 with the MKP7 catalytic domain RESULTS +0 5 DUSPs protein_type DUSPs belong to the protein-tyrosine phosphatases (PTPase) superfamily, which is defined by the PTPase-signature motif CXXGXXR. RESULTS +20 49 protein-tyrosine phosphatases protein_type DUSPs belong to the protein-tyrosine phosphatases (PTPase) superfamily, which is defined by the PTPase-signature motif CXXGXXR. RESULTS +51 57 PTPase protein_type DUSPs belong to the protein-tyrosine phosphatases (PTPase) superfamily, which is defined by the PTPase-signature motif CXXGXXR. RESULTS +96 102 PTPase protein_type DUSPs belong to the protein-tyrosine phosphatases (PTPase) superfamily, which is defined by the PTPase-signature motif CXXGXXR. RESULTS +119 126 CXXGXXR structure_element DUSPs belong to the protein-tyrosine phosphatases (PTPase) superfamily, which is defined by the PTPase-signature motif CXXGXXR. RESULTS +0 4 MKPs protein_type MKPs represent a distinct subfamily within a larger group of DUSPs. RESULTS +61 66 DUSPs protein_type MKPs represent a distinct subfamily within a larger group of DUSPs. RESULTS +3 12 mammalian taxonomy_domain In mammalian cells, the MKP subfamily includes 10 distinct catalytically active MKPs. RESULTS +24 37 MKP subfamily protein_type In mammalian cells, the MKP subfamily includes 10 distinct catalytically active MKPs. RESULTS +59 79 catalytically active protein_state In mammalian cells, the MKP subfamily includes 10 distinct catalytically active MKPs. RESULTS +80 84 MKPs protein_type In mammalian cells, the MKP subfamily includes 10 distinct catalytically active MKPs. RESULTS +4 8 MKPs protein_type All MKPs contain a highly conserved C-terminal catalytic domain (CD) and an N-terminal kinase-binding domain (KBD). RESULTS +19 35 highly conserved protein_state All MKPs contain a highly conserved C-terminal catalytic domain (CD) and an N-terminal kinase-binding domain (KBD). RESULTS +47 63 catalytic domain structure_element All MKPs contain a highly conserved C-terminal catalytic domain (CD) and an N-terminal kinase-binding domain (KBD). RESULTS +65 67 CD structure_element All MKPs contain a highly conserved C-terminal catalytic domain (CD) and an N-terminal kinase-binding domain (KBD). RESULTS +87 108 kinase-binding domain structure_element All MKPs contain a highly conserved C-terminal catalytic domain (CD) and an N-terminal kinase-binding domain (KBD). RESULTS +110 113 KBD structure_element All MKPs contain a highly conserved C-terminal catalytic domain (CD) and an N-terminal kinase-binding domain (KBD). RESULTS +4 7 KBD structure_element The KBD is homologous to the rhodanese family and contains an intervening cluster of basic amino acids, which has been suggested to be important for interacting with the target MAPKs. RESULTS +29 45 rhodanese family protein_type The KBD is homologous to the rhodanese family and contains an intervening cluster of basic amino acids, which has been suggested to be important for interacting with the target MAPKs. RESULTS +177 182 MAPKs protein_type The KBD is homologous to the rhodanese family and contains an intervening cluster of basic amino acids, which has been suggested to be important for interacting with the target MAPKs. RESULTS +105 115 MKP family protein_type On the basis of sequence similarity, substrate specificity and predominant subcellular localization, the MKP family can be further divided into three groups (Fig. 1). RESULTS +0 34 Biochemical and structural studies experimental_method Biochemical and structural studies have revealed that the KBD of MKPs is critical for MKP3 docking to ERK2, and MKP5 binding to p38α, although their binding mechanisms are completely different. RESULTS +58 61 KBD structure_element Biochemical and structural studies have revealed that the KBD of MKPs is critical for MKP3 docking to ERK2, and MKP5 binding to p38α, although their binding mechanisms are completely different. RESULTS +65 69 MKPs protein_type Biochemical and structural studies have revealed that the KBD of MKPs is critical for MKP3 docking to ERK2, and MKP5 binding to p38α, although their binding mechanisms are completely different. RESULTS +86 90 MKP3 protein Biochemical and structural studies have revealed that the KBD of MKPs is critical for MKP3 docking to ERK2, and MKP5 binding to p38α, although their binding mechanisms are completely different. RESULTS +102 106 ERK2 protein Biochemical and structural studies have revealed that the KBD of MKPs is critical for MKP3 docking to ERK2, and MKP5 binding to p38α, although their binding mechanisms are completely different. RESULTS +112 116 MKP5 protein Biochemical and structural studies have revealed that the KBD of MKPs is critical for MKP3 docking to ERK2, and MKP5 binding to p38α, although their binding mechanisms are completely different. RESULTS +128 132 p38α protein Biochemical and structural studies have revealed that the KBD of MKPs is critical for MKP3 docking to ERK2, and MKP5 binding to p38α, although their binding mechanisms are completely different. RESULTS +32 37 MAPKs protein_type However, it is unknown if other MAPKs can interact with the KBD of their cognate phosphatases in the same manner as observed for recognition of ERK2 and p38α by their MKPs, or whether they recognize distinct docking motifs of MKPs. RESULTS +60 63 KBD structure_element However, it is unknown if other MAPKs can interact with the KBD of their cognate phosphatases in the same manner as observed for recognition of ERK2 and p38α by their MKPs, or whether they recognize distinct docking motifs of MKPs. RESULTS +81 93 phosphatases protein_type However, it is unknown if other MAPKs can interact with the KBD of their cognate phosphatases in the same manner as observed for recognition of ERK2 and p38α by their MKPs, or whether they recognize distinct docking motifs of MKPs. RESULTS +144 148 ERK2 protein However, it is unknown if other MAPKs can interact with the KBD of their cognate phosphatases in the same manner as observed for recognition of ERK2 and p38α by their MKPs, or whether they recognize distinct docking motifs of MKPs. RESULTS +153 157 p38α protein However, it is unknown if other MAPKs can interact with the KBD of their cognate phosphatases in the same manner as observed for recognition of ERK2 and p38α by their MKPs, or whether they recognize distinct docking motifs of MKPs. RESULTS +167 171 MKPs protein_type However, it is unknown if other MAPKs can interact with the KBD of their cognate phosphatases in the same manner as observed for recognition of ERK2 and p38α by their MKPs, or whether they recognize distinct docking motifs of MKPs. RESULTS +208 222 docking motifs structure_element However, it is unknown if other MAPKs can interact with the KBD of their cognate phosphatases in the same manner as observed for recognition of ERK2 and p38α by their MKPs, or whether they recognize distinct docking motifs of MKPs. RESULTS +226 230 MKPs protein_type However, it is unknown if other MAPKs can interact with the KBD of their cognate phosphatases in the same manner as observed for recognition of ERK2 and p38α by their MKPs, or whether they recognize distinct docking motifs of MKPs. RESULTS +0 4 MKP7 protein MKP7, the biggest molecule in the MKP family, selectively inactivates JNK and p38 following stress activation. RESULTS +34 44 MKP family protein_type MKP7, the biggest molecule in the MKP family, selectively inactivates JNK and p38 following stress activation. RESULTS +70 73 JNK protein_type MKP7, the biggest molecule in the MKP family, selectively inactivates JNK and p38 following stress activation. RESULTS +78 81 p38 protein_type MKP7, the biggest molecule in the MKP family, selectively inactivates JNK and p38 following stress activation. RESULTS +19 21 CD structure_element In addition to the CD and KBD, MKP7 has a long C-terminal region that contains both nuclear localization and export sequences by which MKP7 shuttles between the nucleus and the cytoplasm (Fig. 2a). RESULTS +26 29 KBD structure_element In addition to the CD and KBD, MKP7 has a long C-terminal region that contains both nuclear localization and export sequences by which MKP7 shuttles between the nucleus and the cytoplasm (Fig. 2a). RESULTS +31 35 MKP7 protein In addition to the CD and KBD, MKP7 has a long C-terminal region that contains both nuclear localization and export sequences by which MKP7 shuttles between the nucleus and the cytoplasm (Fig. 2a). RESULTS +47 64 C-terminal region structure_element In addition to the CD and KBD, MKP7 has a long C-terminal region that contains both nuclear localization and export sequences by which MKP7 shuttles between the nucleus and the cytoplasm (Fig. 2a). RESULTS +135 139 MKP7 protein In addition to the CD and KBD, MKP7 has a long C-terminal region that contains both nuclear localization and export sequences by which MKP7 shuttles between the nucleus and the cytoplasm (Fig. 2a). RESULTS +49 66 N-terminal domain structure_element To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +74 78 MKP7 protein To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +89 93 JNK1 protein To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +94 111 dephosphorylation ptm To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +135 142 kinetic evidence To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +172 182 truncation experimental_method To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +186 190 MKP7 protein To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +192 201 MKP7ΔC304 mutant To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +212 217 5–303 residue_range To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +223 227 MKP7 protein To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +228 230 CD structure_element To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +241 248 156–301 residue_range To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +258 272 phosphorylated protein_state To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +273 277 JNK1 protein To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +279 280 p protein_state To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +280 284 JNK1 protein To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7ΔC304, residues 5–303) and MKP7-CD (residues 156–301) towards phosphorylated JNK1 (pJNK1). RESULTS +20 46 variation of initial rates evidence Figure 2b shows the variation of initial rates of the MKP7ΔC304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid. RESULTS +54 63 MKP7ΔC304 mutant Figure 2b shows the variation of initial rates of the MKP7ΔC304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid. RESULTS +68 72 MKP7 protein Figure 2b shows the variation of initial rates of the MKP7ΔC304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid. RESULTS +73 75 CD structure_element Figure 2b shows the variation of initial rates of the MKP7ΔC304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid. RESULTS +121 128 phospho protein_state Figure 2b shows the variation of initial rates of the MKP7ΔC304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid. RESULTS +129 133 JNK1 protein Figure 2b shows the variation of initial rates of the MKP7ΔC304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid. RESULTS +165 169 MKP7 protein Figure 2b shows the variation of initial rates of the MKP7ΔC304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid. RESULTS +174 175 p protein_state Figure 2b shows the variation of initial rates of the MKP7ΔC304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid. RESULTS +175 179 JNK1 protein Figure 2b shows the variation of initial rates of the MKP7ΔC304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid. RESULTS +10 22 kinetic data evidence Thus, the kinetic data were analysed using the general initial velocity equation, taking substrate depletion into account: RESULTS +55 80 initial velocity equation evidence Thus, the kinetic data were analysed using the general initial velocity equation, taking substrate depletion into account: RESULTS +4 8 kcat evidence The kcat and Km of the MKP7-CD (0.028 s−1 and 0.26 μM) so determined were nearly identical to those of MKP7ΔC304 (0.029 s−1 and 0.27 μM), indicating that the MKP7-KBD has no effect on enzyme catalysis. RESULTS +13 15 Km evidence The kcat and Km of the MKP7-CD (0.028 s−1 and 0.26 μM) so determined were nearly identical to those of MKP7ΔC304 (0.029 s−1 and 0.27 μM), indicating that the MKP7-KBD has no effect on enzyme catalysis. RESULTS +23 27 MKP7 protein The kcat and Km of the MKP7-CD (0.028 s−1 and 0.26 μM) so determined were nearly identical to those of MKP7ΔC304 (0.029 s−1 and 0.27 μM), indicating that the MKP7-KBD has no effect on enzyme catalysis. RESULTS +28 30 CD structure_element The kcat and Km of the MKP7-CD (0.028 s−1 and 0.26 μM) so determined were nearly identical to those of MKP7ΔC304 (0.029 s−1 and 0.27 μM), indicating that the MKP7-KBD has no effect on enzyme catalysis. RESULTS +103 112 MKP7ΔC304 mutant The kcat and Km of the MKP7-CD (0.028 s−1 and 0.26 μM) so determined were nearly identical to those of MKP7ΔC304 (0.029 s−1 and 0.27 μM), indicating that the MKP7-KBD has no effect on enzyme catalysis. RESULTS +158 162 MKP7 protein The kcat and Km of the MKP7-CD (0.028 s−1 and 0.26 μM) so determined were nearly identical to those of MKP7ΔC304 (0.029 s−1 and 0.27 μM), indicating that the MKP7-KBD has no effect on enzyme catalysis. RESULTS +163 166 KBD structure_element The kcat and Km of the MKP7-CD (0.028 s−1 and 0.26 μM) so determined were nearly identical to those of MKP7ΔC304 (0.029 s−1 and 0.27 μM), indicating that the MKP7-KBD has no effect on enzyme catalysis. RESULTS +36 40 JNK1 protein We next examined the interaction of JNK1 with the CD and KBD of MKP7 by gel filtration analysis. RESULTS +50 52 CD structure_element We next examined the interaction of JNK1 with the CD and KBD of MKP7 by gel filtration analysis. RESULTS +57 60 KBD structure_element We next examined the interaction of JNK1 with the CD and KBD of MKP7 by gel filtration analysis. RESULTS +64 68 MKP7 protein We next examined the interaction of JNK1 with the CD and KBD of MKP7 by gel filtration analysis. RESULTS +72 95 gel filtration analysis experimental_method We next examined the interaction of JNK1 with the CD and KBD of MKP7 by gel filtration analysis. RESULTS +28 30 CD structure_element When 3 molar equivalents of CD were mixed with 1 molar equivalent of JNK1, a significant amount of CD co-migrated with JNK1 to earlier fractions, and the excess amount of CD was eluted from the size exclusion column as a monomer, indicating stable complex formation (Fig. 2c). RESULTS +69 73 JNK1 protein When 3 molar equivalents of CD were mixed with 1 molar equivalent of JNK1, a significant amount of CD co-migrated with JNK1 to earlier fractions, and the excess amount of CD was eluted from the size exclusion column as a monomer, indicating stable complex formation (Fig. 2c). RESULTS +99 101 CD structure_element When 3 molar equivalents of CD were mixed with 1 molar equivalent of JNK1, a significant amount of CD co-migrated with JNK1 to earlier fractions, and the excess amount of CD was eluted from the size exclusion column as a monomer, indicating stable complex formation (Fig. 2c). RESULTS +119 123 JNK1 protein When 3 molar equivalents of CD were mixed with 1 molar equivalent of JNK1, a significant amount of CD co-migrated with JNK1 to earlier fractions, and the excess amount of CD was eluted from the size exclusion column as a monomer, indicating stable complex formation (Fig. 2c). RESULTS +171 173 CD structure_element When 3 molar equivalents of CD were mixed with 1 molar equivalent of JNK1, a significant amount of CD co-migrated with JNK1 to earlier fractions, and the excess amount of CD was eluted from the size exclusion column as a monomer, indicating stable complex formation (Fig. 2c). RESULTS +221 228 monomer oligomeric_state When 3 molar equivalents of CD were mixed with 1 molar equivalent of JNK1, a significant amount of CD co-migrated with JNK1 to earlier fractions, and the excess amount of CD was eluted from the size exclusion column as a monomer, indicating stable complex formation (Fig. 2c). RESULTS +16 24 KBD–JNK1 complex_assembly In contrast, no KBD–JNK1 complex was detected when 3 molar equivalents of KBD were mixed with 1 molar equivalent of JNK1. RESULTS +74 77 KBD structure_element In contrast, no KBD–JNK1 complex was detected when 3 molar equivalents of KBD were mixed with 1 molar equivalent of JNK1. RESULTS +116 120 JNK1 protein In contrast, no KBD–JNK1 complex was detected when 3 molar equivalents of KBD were mixed with 1 molar equivalent of JNK1. RESULTS +23 35 JNK1–MKP7-CD complex_assembly To further confirm the JNK1–MKP7-CD interaction, we performed a pull-down assay using the purified proteins. RESULTS +64 79 pull-down assay experimental_method To further confirm the JNK1–MKP7-CD interaction, we performed a pull-down assay using the purified proteins. RESULTS +25 27 CD structure_element As shown in Fig. 2d, the CD of MKP7 can be pulled down by JNK1, while the KBD failed to bind to the counterpart protein. RESULTS +31 35 MKP7 protein As shown in Fig. 2d, the CD of MKP7 can be pulled down by JNK1, while the KBD failed to bind to the counterpart protein. RESULTS +58 62 JNK1 protein As shown in Fig. 2d, the CD of MKP7 can be pulled down by JNK1, while the KBD failed to bind to the counterpart protein. RESULTS +74 77 KBD structure_element As shown in Fig. 2d, the CD of MKP7 can be pulled down by JNK1, while the KBD failed to bind to the counterpart protein. RESULTS +43 45 CD structure_element Taken together, our data indicate that the CD of MKP7, but not the KBD domain, is responsible for JNK substrate-binding and enzymatic specificity. RESULTS +49 53 MKP7 protein Taken together, our data indicate that the CD of MKP7, but not the KBD domain, is responsible for JNK substrate-binding and enzymatic specificity. RESULTS +67 70 KBD structure_element Taken together, our data indicate that the CD of MKP7, but not the KBD domain, is responsible for JNK substrate-binding and enzymatic specificity. RESULTS +98 101 JNK protein_type Taken together, our data indicate that the CD of MKP7, but not the KBD domain, is responsible for JNK substrate-binding and enzymatic specificity. RESULTS +0 17 Crystal structure evidence Crystal structure of JNK1 in complex with the MKP7-CD RESULTS +21 25 JNK1 protein Crystal structure of JNK1 in complex with the MKP7-CD RESULTS +26 41 in complex with protein_state Crystal structure of JNK1 in complex with the MKP7-CD RESULTS +46 50 MKP7 protein Crystal structure of JNK1 in complex with the MKP7-CD RESULTS +51 53 CD structure_element Crystal structure of JNK1 in complex with the MKP7-CD RESULTS +37 41 JNK1 protein To understand the molecular basis of JNK1 recognition by MKP7, we determined the crystal structure of unphosphorylated JNK1 in complex with the MKP7-CD (Fig. 3a, Supplementary Fig. 1a and Table 1). RESULTS +57 61 MKP7 protein To understand the molecular basis of JNK1 recognition by MKP7, we determined the crystal structure of unphosphorylated JNK1 in complex with the MKP7-CD (Fig. 3a, Supplementary Fig. 1a and Table 1). RESULTS +81 98 crystal structure evidence To understand the molecular basis of JNK1 recognition by MKP7, we determined the crystal structure of unphosphorylated JNK1 in complex with the MKP7-CD (Fig. 3a, Supplementary Fig. 1a and Table 1). RESULTS +102 118 unphosphorylated protein_state To understand the molecular basis of JNK1 recognition by MKP7, we determined the crystal structure of unphosphorylated JNK1 in complex with the MKP7-CD (Fig. 3a, Supplementary Fig. 1a and Table 1). RESULTS +119 123 JNK1 protein To understand the molecular basis of JNK1 recognition by MKP7, we determined the crystal structure of unphosphorylated JNK1 in complex with the MKP7-CD (Fig. 3a, Supplementary Fig. 1a and Table 1). RESULTS +124 139 in complex with protein_state To understand the molecular basis of JNK1 recognition by MKP7, we determined the crystal structure of unphosphorylated JNK1 in complex with the MKP7-CD (Fig. 3a, Supplementary Fig. 1a and Table 1). RESULTS +144 148 MKP7 protein To understand the molecular basis of JNK1 recognition by MKP7, we determined the crystal structure of unphosphorylated JNK1 in complex with the MKP7-CD (Fig. 3a, Supplementary Fig. 1a and Table 1). RESULTS +149 151 CD structure_element To understand the molecular basis of JNK1 recognition by MKP7, we determined the crystal structure of unphosphorylated JNK1 in complex with the MKP7-CD (Fig. 3a, Supplementary Fig. 1a and Table 1). RESULTS +16 20 JNK1 protein In the complex, JNK1 has its characteristic bilobal structure comprising an N-terminal lobe rich in β-sheet and a C-terminal lobe that is mostly α-helical. RESULTS +76 91 N-terminal lobe structure_element In the complex, JNK1 has its characteristic bilobal structure comprising an N-terminal lobe rich in β-sheet and a C-terminal lobe that is mostly α-helical. RESULTS +100 107 β-sheet structure_element In the complex, JNK1 has its characteristic bilobal structure comprising an N-terminal lobe rich in β-sheet and a C-terminal lobe that is mostly α-helical. RESULTS +114 129 C-terminal lobe structure_element In the complex, JNK1 has its characteristic bilobal structure comprising an N-terminal lobe rich in β-sheet and a C-terminal lobe that is mostly α-helical. RESULTS +145 154 α-helical structure_element In the complex, JNK1 has its characteristic bilobal structure comprising an N-terminal lobe rich in β-sheet and a C-terminal lobe that is mostly α-helical. RESULTS +23 27 MKP7 protein The overall folding of MKP7-CD is typical of DUSPs, with a central twisted five-stranded β-sheet surrounded by six α-helices. RESULTS +28 30 CD structure_element The overall folding of MKP7-CD is typical of DUSPs, with a central twisted five-stranded β-sheet surrounded by six α-helices. RESULTS +45 50 DUSPs protein_type The overall folding of MKP7-CD is typical of DUSPs, with a central twisted five-stranded β-sheet surrounded by six α-helices. RESULTS +67 96 twisted five-stranded β-sheet structure_element The overall folding of MKP7-CD is typical of DUSPs, with a central twisted five-stranded β-sheet surrounded by six α-helices. RESULTS +115 124 α-helices structure_element The overall folding of MKP7-CD is typical of DUSPs, with a central twisted five-stranded β-sheet surrounded by six α-helices. RESULTS +16 23 β-sheet structure_element One side of the β-sheet is covered with two α-helices and the other is covered with four α-helices (Fig. 3b). RESULTS +44 53 α-helices structure_element One side of the β-sheet is covered with two α-helices and the other is covered with four α-helices (Fig. 3b). RESULTS +89 98 α-helices structure_element One side of the β-sheet is covered with two α-helices and the other is covered with four α-helices (Fig. 3b). RESULTS +4 20 catalytic domain structure_element The catalytic domain of MKP7 interacts with JNK1 through a contiguous surface area that is remote from the active site. RESULTS +24 28 MKP7 protein The catalytic domain of MKP7 interacts with JNK1 through a contiguous surface area that is remote from the active site. RESULTS +44 48 JNK1 protein The catalytic domain of MKP7 interacts with JNK1 through a contiguous surface area that is remote from the active site. RESULTS +107 118 active site site The catalytic domain of MKP7 interacts with JNK1 through a contiguous surface area that is remote from the active site. RESULTS +0 4 MKP7 protein MKP7-CD is positioned onto the JNK1 molecule so that the active site of the phosphatase faces towards the activation segment. RESULTS +5 7 CD structure_element MKP7-CD is positioned onto the JNK1 molecule so that the active site of the phosphatase faces towards the activation segment. RESULTS +31 35 JNK1 protein MKP7-CD is positioned onto the JNK1 molecule so that the active site of the phosphatase faces towards the activation segment. RESULTS +57 68 active site site MKP7-CD is positioned onto the JNK1 molecule so that the active site of the phosphatase faces towards the activation segment. RESULTS +76 87 phosphatase protein_type MKP7-CD is positioned onto the JNK1 molecule so that the active site of the phosphatase faces towards the activation segment. RESULTS +106 124 activation segment structure_element MKP7-CD is positioned onto the JNK1 molecule so that the active site of the phosphatase faces towards the activation segment. RESULTS +6 15 alignment experimental_method In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). RESULTS +23 32 structure evidence In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). RESULTS +36 40 MKP7 protein In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). RESULTS +41 43 CD structure_element In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). RESULTS +57 60 VHR protein In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). RESULTS +75 78 MKP protein_type In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). RESULTS +101 117 catalytic domain structure_element In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). RESULTS +130 134 MKP7 protein In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). RESULTS +135 137 CD structure_element In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). RESULTS +156 168 superimposed experimental_method In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). RESULTS +176 184 r.m.s.d. evidence In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). RESULTS +186 213 root mean squared deviation evidence In an alignment of the structure of MKP7-CD with that of VHR, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05 Å (Fig. 3c). RESULTS +37 42 helix structure_element The most striking difference is that helix α0 and loop α0–β1 of VHR are absent in MKP7-CD. RESULTS +43 45 α0 structure_element The most striking difference is that helix α0 and loop α0–β1 of VHR are absent in MKP7-CD. RESULTS +50 54 loop structure_element The most striking difference is that helix α0 and loop α0–β1 of VHR are absent in MKP7-CD. RESULTS +55 60 α0–β1 structure_element The most striking difference is that helix α0 and loop α0–β1 of VHR are absent in MKP7-CD. RESULTS +64 67 VHR protein The most striking difference is that helix α0 and loop α0–β1 of VHR are absent in MKP7-CD. RESULTS +82 86 MKP7 protein The most striking difference is that helix α0 and loop α0–β1 of VHR are absent in MKP7-CD. RESULTS +87 89 CD structure_element The most striking difference is that helix α0 and loop α0–β1 of VHR are absent in MKP7-CD. RESULTS +43 46 VHR protein Another region that cannot be aligned with VHR is found in loop β3–β4. RESULTS +59 63 loop structure_element Another region that cannot be aligned with VHR is found in loop β3–β4. RESULTS +64 69 β3–β4 structure_element Another region that cannot be aligned with VHR is found in loop β3–β4. RESULTS +5 9 loop structure_element This loop is shortened by nine residues in MKP7-CD compared with that in VHR. RESULTS +43 47 MKP7 protein This loop is shortened by nine residues in MKP7-CD compared with that in VHR. RESULTS +48 50 CD structure_element This loop is shortened by nine residues in MKP7-CD compared with that in VHR. RESULTS +73 76 VHR protein This loop is shortened by nine residues in MKP7-CD compared with that in VHR. RESULTS +6 11 helix structure_element Since helix α0 and the following loop α0–β1 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7. RESULTS +12 14 α0 structure_element Since helix α0 and the following loop α0–β1 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7. RESULTS +33 37 loop structure_element Since helix α0 and the following loop α0–β1 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7. RESULTS +38 43 α0–β1 structure_element Since helix α0 and the following loop α0–β1 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7. RESULTS +60 87 substrate-recognition motif site Since helix α0 and the following loop α0–β1 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7. RESULTS +91 94 VHR protein Since helix α0 and the following loop α0–β1 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7. RESULTS +105 117 phosphatases protein_type Since helix α0 and the following loop α0–β1 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7. RESULTS +198 202 MKP7 protein Since helix α0 and the following loop α0–β1 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7. RESULTS +4 15 active site site The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +19 23 MKP7 protein The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +40 62 phosphate-binding loop structure_element The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +64 70 P-loop structure_element The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +72 78 Cys244 residue_name_number The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +79 85 Leu245 residue_name_number The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +86 92 Ala246 residue_name_number The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +93 99 Gly247 residue_name_number The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +100 106 Ile248 residue_name_number The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +107 113 Ser249 residue_name_number The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +114 120 Arg250 residue_name_number The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +127 133 Asp213 residue_name_number The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +141 158 general acid loop structure_element The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b). RESULTS +4 8 MKP7 protein The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs. RESULTS +9 11 CD structure_element The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs. RESULTS +12 21 structure evidence The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs. RESULTS +31 42 active site site The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs. RESULTS +62 81 active conformation protein_state The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs. RESULTS +94 97 VHR protein The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs. RESULTS +108 112 PTPs protein_type The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs. RESULTS +4 21 catalytic residue site The catalytic residue, Cys244, is located just after strand β5 and optimally positioned for nucleophilic attack. RESULTS +23 29 Cys244 residue_name_number The catalytic residue, Cys244, is located just after strand β5 and optimally positioned for nucleophilic attack. RESULTS +53 59 strand structure_element The catalytic residue, Cys244, is located just after strand β5 and optimally positioned for nucleophilic attack. RESULTS +60 62 β5 structure_element The catalytic residue, Cys244, is located just after strand β5 and optimally positioned for nucleophilic attack. RESULTS +0 6 Asp213 residue_name_number Asp213 in MKP7 also adopts a position similar to that of Asp92 in VHR (Supplementary Fig. 1c), indicating that Asp213 is likely to function as the general acid in MKP7. RESULTS +10 14 MKP7 protein Asp213 in MKP7 also adopts a position similar to that of Asp92 in VHR (Supplementary Fig. 1c), indicating that Asp213 is likely to function as the general acid in MKP7. RESULTS +57 62 Asp92 residue_name_number Asp213 in MKP7 also adopts a position similar to that of Asp92 in VHR (Supplementary Fig. 1c), indicating that Asp213 is likely to function as the general acid in MKP7. RESULTS +66 69 VHR protein Asp213 in MKP7 also adopts a position similar to that of Asp92 in VHR (Supplementary Fig. 1c), indicating that Asp213 is likely to function as the general acid in MKP7. RESULTS +111 117 Asp213 residue_name_number Asp213 in MKP7 also adopts a position similar to that of Asp92 in VHR (Supplementary Fig. 1c), indicating that Asp213 is likely to function as the general acid in MKP7. RESULTS +163 167 MKP7 protein Asp213 in MKP7 also adopts a position similar to that of Asp92 in VHR (Supplementary Fig. 1c), indicating that Asp213 is likely to function as the general acid in MKP7. RESULTS +34 42 chloride chemical We also observed the binding of a chloride ion in the active site of MKP7-CD. RESULTS +54 65 active site site We also observed the binding of a chloride ion in the active site of MKP7-CD. RESULTS +69 73 MKP7 protein We also observed the binding of a chloride ion in the active site of MKP7-CD. RESULTS +74 76 CD structure_element We also observed the binding of a chloride ion in the active site of MKP7-CD. RESULTS +30 36 Cys244 residue_name_number It is located 3.36 Å from the Cys244 side chain and makes electrostatic interactions with the dipole moment of helix α3 and with several main-chain amide groups. RESULTS +58 84 electrostatic interactions bond_interaction It is located 3.36 Å from the Cys244 side chain and makes electrostatic interactions with the dipole moment of helix α3 and with several main-chain amide groups. RESULTS +111 116 helix structure_element It is located 3.36 Å from the Cys244 side chain and makes electrostatic interactions with the dipole moment of helix α3 and with several main-chain amide groups. RESULTS +117 119 α3 structure_element It is located 3.36 Å from the Cys244 side chain and makes electrostatic interactions with the dipole moment of helix α3 and with several main-chain amide groups. RESULTS +18 36 strictly conserved protein_state The side chain of strictly conserved Arg250 is oriented towards the negatively charged chloride, similar to the canonical phosphate-coordinating conformation. RESULTS +37 43 Arg250 residue_name_number The side chain of strictly conserved Arg250 is oriented towards the negatively charged chloride, similar to the canonical phosphate-coordinating conformation. RESULTS +87 95 chloride chemical The side chain of strictly conserved Arg250 is oriented towards the negatively charged chloride, similar to the canonical phosphate-coordinating conformation. RESULTS +122 157 phosphate-coordinating conformation structure_element The side chain of strictly conserved Arg250 is oriented towards the negatively charged chloride, similar to the canonical phosphate-coordinating conformation. RESULTS +10 18 chloride chemical Thus this chloride ion is a mimic for the phosphate group of the substrate, as revealed by a comparison with the structure of PTP1B in complex with phosphotyrosine (Supplementary Fig. 1d). RESULTS +42 51 phosphate chemical Thus this chloride ion is a mimic for the phosphate group of the substrate, as revealed by a comparison with the structure of PTP1B in complex with phosphotyrosine (Supplementary Fig. 1d). RESULTS +113 122 structure evidence Thus this chloride ion is a mimic for the phosphate group of the substrate, as revealed by a comparison with the structure of PTP1B in complex with phosphotyrosine (Supplementary Fig. 1d). RESULTS +126 131 PTP1B protein Thus this chloride ion is a mimic for the phosphate group of the substrate, as revealed by a comparison with the structure of PTP1B in complex with phosphotyrosine (Supplementary Fig. 1d). RESULTS +132 147 in complex with protein_state Thus this chloride ion is a mimic for the phosphate group of the substrate, as revealed by a comparison with the structure of PTP1B in complex with phosphotyrosine (Supplementary Fig. 1d). RESULTS +148 163 phosphotyrosine residue_name Thus this chloride ion is a mimic for the phosphate group of the substrate, as revealed by a comparison with the structure of PTP1B in complex with phosphotyrosine (Supplementary Fig. 1d). RESULTS +49 53 MKP7 protein Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +54 56 CD structure_element Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +88 91 VHR protein Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +113 119 P-loop structure_element Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +123 127 MKP7 protein Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +192 195 VHR protein Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +197 203 Cys124 residue_name_number Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +204 210 Arg125 residue_name_number Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +211 217 Glu126 residue_name_number Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +218 224 Gly127 residue_name_number Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +225 231 Tyr128 residue_name_number Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +232 238 Gly129 residue_name_number Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +239 245 Arg130 residue_name_number Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c). RESULTS +162 174 phosphatases protein_type The difference in the polarity/hydrophobicity of the surface may also point to the origin of the differences in the substrate-recognition mechanism for these two phosphatases (Supplementary Fig. 1e,f). RESULTS +16 20 MKP7 protein In the complex, MKP7-CD and JNK1 form extensive protein–protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e). RESULTS +21 23 CD structure_element In the complex, MKP7-CD and JNK1 form extensive protein–protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e). RESULTS +28 32 JNK1 protein In the complex, MKP7-CD and JNK1 form extensive protein–protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e). RESULTS +91 109 C-terminal helices structure_element In the complex, MKP7-CD and JNK1 form extensive protein–protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e). RESULTS +113 117 MKP7 protein In the complex, MKP7-CD and JNK1 form extensive protein–protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e). RESULTS +118 120 CD structure_element In the complex, MKP7-CD and JNK1 form extensive protein–protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e). RESULTS +125 131 C-lobe structure_element In the complex, MKP7-CD and JNK1 form extensive protein–protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e). RESULTS +135 139 JNK1 protein In the complex, MKP7-CD and JNK1 form extensive protein–protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e). RESULTS +76 93 C-terminal domain structure_element As a result, the buried solvent-accessible surface area is ∼1,315 Å. In the C-terminal domain, JNK1 has an insertion after the helix αG. This insertion consists of two helices (α1L14 and α2L14) that are common to all members of the MAPK family. RESULTS +95 99 JNK1 protein As a result, the buried solvent-accessible surface area is ∼1,315 Å. In the C-terminal domain, JNK1 has an insertion after the helix αG. This insertion consists of two helices (α1L14 and α2L14) that are common to all members of the MAPK family. RESULTS +127 132 helix structure_element As a result, the buried solvent-accessible surface area is ∼1,315 Å. In the C-terminal domain, JNK1 has an insertion after the helix αG. This insertion consists of two helices (α1L14 and α2L14) that are common to all members of the MAPK family. RESULTS +133 135 αG structure_element As a result, the buried solvent-accessible surface area is ∼1,315 Å. In the C-terminal domain, JNK1 has an insertion after the helix αG. This insertion consists of two helices (α1L14 and α2L14) that are common to all members of the MAPK family. RESULTS +168 175 helices structure_element As a result, the buried solvent-accessible surface area is ∼1,315 Å. In the C-terminal domain, JNK1 has an insertion after the helix αG. This insertion consists of two helices (α1L14 and α2L14) that are common to all members of the MAPK family. RESULTS +177 182 α1L14 structure_element As a result, the buried solvent-accessible surface area is ∼1,315 Å. In the C-terminal domain, JNK1 has an insertion after the helix αG. This insertion consists of two helices (α1L14 and α2L14) that are common to all members of the MAPK family. RESULTS +187 192 α2L14 structure_element As a result, the buried solvent-accessible surface area is ∼1,315 Å. In the C-terminal domain, JNK1 has an insertion after the helix αG. This insertion consists of two helices (α1L14 and α2L14) that are common to all members of the MAPK family. RESULTS +232 243 MAPK family protein_type As a result, the buried solvent-accessible surface area is ∼1,315 Å. In the C-terminal domain, JNK1 has an insertion after the helix αG. This insertion consists of two helices (α1L14 and α2L14) that are common to all members of the MAPK family. RESULTS +4 23 interactive surface site The interactive surface in JNK1, formed by the helices αG and α2L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d). RESULTS +27 31 JNK1 protein The interactive surface in JNK1, formed by the helices αG and α2L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d). RESULTS +47 54 helices structure_element The interactive surface in JNK1, formed by the helices αG and α2L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d). RESULTS +55 57 αG structure_element The interactive surface in JNK1, formed by the helices αG and α2L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d). RESULTS +62 67 α2L14 structure_element The interactive surface in JNK1, formed by the helices αG and α2L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d). RESULTS +80 98 hydrophobic region site The interactive surface in JNK1, formed by the helices αG and α2L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d). RESULTS +111 117 Trp234 residue_name_number The interactive surface in JNK1, formed by the helices αG and α2L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d). RESULTS +4 23 MKP7-docking region site The MKP7-docking region includes two helices, α4 and α5, and the general acid loop. RESULTS +37 44 helices structure_element The MKP7-docking region includes two helices, α4 and α5, and the general acid loop. RESULTS +46 48 α4 structure_element The MKP7-docking region includes two helices, α4 and α5, and the general acid loop. RESULTS +53 55 α5 structure_element The MKP7-docking region includes two helices, α4 and α5, and the general acid loop. RESULTS +65 82 general acid loop structure_element The MKP7-docking region includes two helices, α4 and α5, and the general acid loop. RESULTS +21 27 Phe285 residue_name_number The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +31 35 MKP7 protein The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +36 44 α5-helix structure_element The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +61 79 hydrophobic pocket site The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +83 87 JNK1 protein The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +114 120 Ile197 residue_name_number The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +122 128 Leu198 residue_name_number The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +130 136 Ile231 residue_name_number The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +138 144 Trp234 residue_name_number The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +146 152 Val256 residue_name_number The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +154 160 Tyr259 residue_name_number The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +162 168 Val260 residue_name_number The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +198 204 His230 residue_name_number The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g). RESULTS +23 37 hydrogen bonds bond_interaction In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +46 52 Ser282 residue_name_number In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +57 63 Asn286 residue_name_number In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +67 71 MKP7 protein In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +76 82 His230 residue_name_number In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +87 93 Thr255 residue_name_number In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +97 101 JNK1 protein In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +125 131 Phe215 residue_name_number In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +139 156 general acid loop structure_element In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +160 164 MKP7 protein In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +168 183 hydrogen-bonded bond_interaction In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +205 211 Gln253 residue_name_number In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +215 219 JNK1 protein In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1. RESULTS +4 27 second interactive area site The second interactive area involves the α4 helix of MKP7 and charged/polar residues of JNK1 (Fig. 3e). RESULTS +41 49 α4 helix structure_element The second interactive area involves the α4 helix of MKP7 and charged/polar residues of JNK1 (Fig. 3e). RESULTS +53 57 MKP7 protein The second interactive area involves the α4 helix of MKP7 and charged/polar residues of JNK1 (Fig. 3e). RESULTS +88 92 JNK1 protein The second interactive area involves the α4 helix of MKP7 and charged/polar residues of JNK1 (Fig. 3e). RESULTS +19 25 Asp268 residue_name_number The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. RESULTS +29 33 MKP7 protein The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. RESULTS +42 53 salt bridge bond_interaction The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. RESULTS +73 79 Arg263 residue_name_number The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. RESULTS +83 87 JNK1 protein The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. RESULTS +93 99 Lys275 residue_name_number The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. RESULTS +103 107 MKP7 protein The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. RESULTS +116 129 hydrogen bond bond_interaction The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. RESULTS +136 147 salt bridge bond_interaction The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. RESULTS +153 159 Thr228 residue_name_number The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. RESULTS +164 170 Asp229 residue_name_number The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. RESULTS +174 178 JNK1 protein The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively. RESULTS +0 19 Mutational analysis experimental_method Mutational analysis of the JNK1–MKP7 docking interface RESULTS +27 54 JNK1–MKP7 docking interface site Mutational analysis of the JNK1–MKP7 docking interface RESULTS +86 101 point mutations experimental_method To assess the importance of the aforementioned interactions, we generated a series of point mutations on the MKP7-CD and examined their effect on the MKP7-catalysed JNK1 dephosphorylation (Fig. 4a). RESULTS +109 113 MKP7 protein To assess the importance of the aforementioned interactions, we generated a series of point mutations on the MKP7-CD and examined their effect on the MKP7-catalysed JNK1 dephosphorylation (Fig. 4a). RESULTS +114 116 CD structure_element To assess the importance of the aforementioned interactions, we generated a series of point mutations on the MKP7-CD and examined their effect on the MKP7-catalysed JNK1 dephosphorylation (Fig. 4a). RESULTS +150 154 MKP7 protein To assess the importance of the aforementioned interactions, we generated a series of point mutations on the MKP7-CD and examined their effect on the MKP7-catalysed JNK1 dephosphorylation (Fig. 4a). RESULTS +165 169 JNK1 protein To assess the importance of the aforementioned interactions, we generated a series of point mutations on the MKP7-CD and examined their effect on the MKP7-catalysed JNK1 dephosphorylation (Fig. 4a). RESULTS +170 187 dephosphorylation ptm To assess the importance of the aforementioned interactions, we generated a series of point mutations on the MKP7-CD and examined their effect on the MKP7-catalysed JNK1 dephosphorylation (Fig. 4a). RESULTS +30 36 Phe285 residue_name_number When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold. RESULTS +41 47 Phe287 residue_name_number When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold. RESULTS +55 57 α5 structure_element When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold. RESULTS +61 65 MKP7 protein When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold. RESULTS +66 68 CD structure_element When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold. RESULTS +74 82 replaced experimental_method When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold. RESULTS +86 89 Asp residue_name When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold. RESULTS +93 96 Ala residue_name When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold. RESULTS +131 135 JNK1 protein When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold. RESULTS +136 153 dephosphorylation ptm When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold. RESULTS +15 26 replacement experimental_method In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP. RESULTS +50 56 Phe215 residue_name_number In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP. RESULTS +58 64 Asp268 residue_name_number In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP. RESULTS +66 72 Lys275 residue_name_number In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP. RESULTS +74 80 Ser282 residue_name_number In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP. RESULTS +82 88 Asn286 residue_name_number In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP. RESULTS +93 99 Leu292 residue_name_number In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP. RESULTS +109 112 Ala residue_name In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP. RESULTS +116 119 Asp residue_name In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP. RESULTS +251 254 MKP protein_type In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP. RESULTS +0 8 Mutation experimental_method Mutation of Leu288 markedly reduced its solubility when expressed in Escherichia coli, resulting in the insoluble aggregation of the mutant protein. RESULTS +12 18 Leu288 residue_name_number Mutation of Leu288 markedly reduced its solubility when expressed in Escherichia coli, resulting in the insoluble aggregation of the mutant protein. RESULTS +69 85 Escherichia coli species Mutation of Leu288 markedly reduced its solubility when expressed in Escherichia coli, resulting in the insoluble aggregation of the mutant protein. RESULTS +133 139 mutant protein_state Mutation of Leu288 markedly reduced its solubility when expressed in Escherichia coli, resulting in the insoluble aggregation of the mutant protein. RESULTS +0 23 Gel filtration analysis experimental_method Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b). RESULTS +58 64 Phe285 residue_name_number Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b). RESULTS +72 76 MKP7 protein Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b). RESULTS +77 81 JNK1 protein Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b). RESULTS +98 108 F285D–JNK1 complex_assembly Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b). RESULTS +158 162 MKP7 protein Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b). RESULTS +163 165 CD structure_element Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b). RESULTS +167 172 F285D mutant Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b). RESULTS +212 216 JNK1 protein Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b). RESULTS +15 23 mutation experimental_method Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a). RESULTS +27 33 Phe287 residue_name_number Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a). RESULTS +85 86 p protein_state Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a). RESULTS +86 90 JNK1 protein Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a). RESULTS +112 120 affinity evidence Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a). RESULTS +124 128 MKP7 protein Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a). RESULTS +129 131 CD structure_element Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a). RESULTS +136 140 JNK1 protein Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a). RESULTS +30 45 point mutations experimental_method We also generated a series of point mutations in the JNK1 and assessed the effect on JNK1 binding using the GST pull-down assay (Fig. 4c). RESULTS +53 57 JNK1 protein We also generated a series of point mutations in the JNK1 and assessed the effect on JNK1 binding using the GST pull-down assay (Fig. 4c). RESULTS +85 89 JNK1 protein We also generated a series of point mutations in the JNK1 and assessed the effect on JNK1 binding using the GST pull-down assay (Fig. 4c). RESULTS +108 127 GST pull-down assay experimental_method We also generated a series of point mutations in the JNK1 and assessed the effect on JNK1 binding using the GST pull-down assay (Fig. 4c). RESULTS +0 12 Substitution experimental_method Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. RESULTS +16 22 Asp229 residue_name_number Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. RESULTS +24 30 Trp234 residue_name_number Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. RESULTS +32 38 Thr255 residue_name_number Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. RESULTS +40 46 Val256 residue_name_number Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. RESULTS +48 54 Tyr259 residue_name_number Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. RESULTS +59 65 Val260 residue_name_number Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. RESULTS +92 108 binding affinity evidence Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. RESULTS +112 116 MKP7 protein Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. RESULTS +117 119 CD structure_element Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. RESULTS +124 127 JNK protein_type Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK. RESULTS +154 160 mutant protein_state To determine whether the deficiencies in their abilities to bind partner proteins or carry out catalytic function are owing to misfolding of the purified mutant proteins, we also examined the folding properties of the JNK1 and MKP7 mutants with circular dichroism. RESULTS +218 222 JNK1 protein To determine whether the deficiencies in their abilities to bind partner proteins or carry out catalytic function are owing to misfolding of the purified mutant proteins, we also examined the folding properties of the JNK1 and MKP7 mutants with circular dichroism. RESULTS +227 231 MKP7 protein To determine whether the deficiencies in their abilities to bind partner proteins or carry out catalytic function are owing to misfolding of the purified mutant proteins, we also examined the folding properties of the JNK1 and MKP7 mutants with circular dichroism. RESULTS +232 239 mutants protein_state To determine whether the deficiencies in their abilities to bind partner proteins or carry out catalytic function are owing to misfolding of the purified mutant proteins, we also examined the folding properties of the JNK1 and MKP7 mutants with circular dichroism. RESULTS +245 263 circular dichroism experimental_method To determine whether the deficiencies in their abilities to bind partner proteins or carry out catalytic function are owing to misfolding of the purified mutant proteins, we also examined the folding properties of the JNK1 and MKP7 mutants with circular dichroism. RESULTS +4 11 spectra evidence The spectra of these mutants are similar to the wild-type proteins, indicating that these mutants fold as well as the wild-type proteins (Fig. 4d,e). RESULTS +21 28 mutants protein_state The spectra of these mutants are similar to the wild-type proteins, indicating that these mutants fold as well as the wild-type proteins (Fig. 4d,e). RESULTS +48 57 wild-type protein_state The spectra of these mutants are similar to the wild-type proteins, indicating that these mutants fold as well as the wild-type proteins (Fig. 4d,e). RESULTS +90 97 mutants protein_state The spectra of these mutants are similar to the wild-type proteins, indicating that these mutants fold as well as the wild-type proteins (Fig. 4d,e). RESULTS +118 127 wild-type protein_state The spectra of these mutants are similar to the wild-type proteins, indicating that these mutants fold as well as the wild-type proteins (Fig. 4d,e). RESULTS +62 84 crystallographic model evidence Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1. RESULTS +103 123 hydrophobic contacts bond_interaction Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1. RESULTS +136 140 MKP7 protein Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1. RESULTS +141 157 catalytic domain structure_element Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1. RESULTS +162 166 JNK1 protein Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1. RESULTS +252 258 Phe285 residue_name_number Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1. RESULTS +266 270 MKP7 protein Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1. RESULTS +271 273 CD structure_element Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1. RESULTS +324 328 JNK1 protein Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1. RESULTS +77 81 MKPs protein_type It has previously been reported that several cytosolic and inducible nuclear MKPs undergo catalytic activation upon interaction with the MAPK substrates. RESULTS +137 141 MAPK protein_type It has previously been reported that several cytosolic and inducible nuclear MKPs undergo catalytic activation upon interaction with the MAPK substrates. RESULTS +30 34 MKP3 protein This allosteric activation of MKP3 has been well-documented in vitro using pNPP, a small-molecule phosphotyrosine analogue of its normal substrate. RESULTS +75 79 pNPP chemical This allosteric activation of MKP3 has been well-documented in vitro using pNPP, a small-molecule phosphotyrosine analogue of its normal substrate. RESULTS +98 113 phosphotyrosine residue_name This allosteric activation of MKP3 has been well-documented in vitro using pNPP, a small-molecule phosphotyrosine analogue of its normal substrate. RESULTS +16 23 pNPPase protein_type We then assayed pNPPase activities of MKP7ΔC304 and MKP7-CD in the presence of JNK1. RESULTS +38 47 MKP7ΔC304 mutant We then assayed pNPPase activities of MKP7ΔC304 and MKP7-CD in the presence of JNK1. RESULTS +52 56 MKP7 protein We then assayed pNPPase activities of MKP7ΔC304 and MKP7-CD in the presence of JNK1. RESULTS +57 59 CD structure_element We then assayed pNPPase activities of MKP7ΔC304 and MKP7-CD in the presence of JNK1. RESULTS +67 78 presence of protein_state We then assayed pNPPase activities of MKP7ΔC304 and MKP7-CD in the presence of JNK1. RESULTS +79 83 JNK1 protein We then assayed pNPPase activities of MKP7ΔC304 and MKP7-CD in the presence of JNK1. RESULTS +0 10 Incubation experimental_method Incubation of MKP7 with JNK1 did not markedly stimulate the phosphatase activity, which is consistent with previous results that MKP7 solely possesses the intrinsic activity (Supplementary Fig. 2b). RESULTS +14 18 MKP7 protein Incubation of MKP7 with JNK1 did not markedly stimulate the phosphatase activity, which is consistent with previous results that MKP7 solely possesses the intrinsic activity (Supplementary Fig. 2b). RESULTS +24 28 JNK1 protein Incubation of MKP7 with JNK1 did not markedly stimulate the phosphatase activity, which is consistent with previous results that MKP7 solely possesses the intrinsic activity (Supplementary Fig. 2b). RESULTS +60 71 phosphatase protein_type Incubation of MKP7 with JNK1 did not markedly stimulate the phosphatase activity, which is consistent with previous results that MKP7 solely possesses the intrinsic activity (Supplementary Fig. 2b). RESULTS +129 133 MKP7 protein Incubation of MKP7 with JNK1 did not markedly stimulate the phosphatase activity, which is consistent with previous results that MKP7 solely possesses the intrinsic activity (Supplementary Fig. 2b). RESULTS +10 14 pNPP chemical The small pNPP molecule binds directly at the enzyme active site and can be used to probe the reaction mechanism of protein phosphatases. RESULTS +53 64 active site site The small pNPP molecule binds directly at the enzyme active site and can be used to probe the reaction mechanism of protein phosphatases. RESULTS +116 136 protein phosphatases protein_type The small pNPP molecule binds directly at the enzyme active site and can be used to probe the reaction mechanism of protein phosphatases. RESULTS +41 45 MKP7 protein We therefore examined the effects of the MKP7-CD mutants on their pNPPase activities. RESULTS +46 48 CD structure_element We therefore examined the effects of the MKP7-CD mutants on their pNPPase activities. RESULTS +49 56 mutants protein_state We therefore examined the effects of the MKP7-CD mutants on their pNPPase activities. RESULTS +66 73 pNPPase protein_type We therefore examined the effects of the MKP7-CD mutants on their pNPPase activities. RESULTS +29 36 mutants protein_state As shown in Fig. 4f, all the mutants, except F287D/A, showed little or no activity change compared with the wild-type MKP7-CD. RESULTS +45 52 F287D/A mutant As shown in Fig. 4f, all the mutants, except F287D/A, showed little or no activity change compared with the wild-type MKP7-CD. RESULTS +108 117 wild-type protein_state As shown in Fig. 4f, all the mutants, except F287D/A, showed little or no activity change compared with the wild-type MKP7-CD. RESULTS +118 122 MKP7 protein As shown in Fig. 4f, all the mutants, except F287D/A, showed little or no activity change compared with the wild-type MKP7-CD. RESULTS +123 125 CD structure_element As shown in Fig. 4f, all the mutants, except F287D/A, showed little or no activity change compared with the wild-type MKP7-CD. RESULTS +7 19 JNK1/MKP7-CD complex_assembly In the JNK1/MKP7-CD complex structure, Phe287 of MKP7 does not make contacts with JNK1 substrate. RESULTS +28 37 structure evidence In the JNK1/MKP7-CD complex structure, Phe287 of MKP7 does not make contacts with JNK1 substrate. RESULTS +39 45 Phe287 residue_name_number In the JNK1/MKP7-CD complex structure, Phe287 of MKP7 does not make contacts with JNK1 substrate. RESULTS +49 53 MKP7 protein In the JNK1/MKP7-CD complex structure, Phe287 of MKP7 does not make contacts with JNK1 substrate. RESULTS +82 86 JNK1 protein In the JNK1/MKP7-CD complex structure, Phe287 of MKP7 does not make contacts with JNK1 substrate. RESULTS +21 27 pocket site It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +56 62 P-loop structure_element It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +67 84 general acid loop structure_element It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +95 115 hydrophobic contacts bond_interaction It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +162 168 Arg250 residue_name_number It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +170 176 Glu217 residue_name_number It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +181 187 Ile219 residue_name_number It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +205 211 Phe287 residue_name_number It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +215 219 MKP7 protein It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +291 295 PTPs protein_type It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +297 303 Gln266 residue_name_number It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +307 312 PTP1B protein It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +318 321 VHR protein It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +323 329 Phe166 residue_name_number It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +333 336 VHR protein It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +366 386 active-site residues site It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +390 394 MKP7 protein It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c). RESULTS +0 29 Kinase-associated phosphatase protein Kinase-associated phosphatase (KAP), a member of the DUSP family, plays a crucial role in cell cycle regulation by dephosphorylating the pThr160 residue of CDK2 (cyclin-dependent kinase 2). RESULTS +31 34 KAP protein Kinase-associated phosphatase (KAP), a member of the DUSP family, plays a crucial role in cell cycle regulation by dephosphorylating the pThr160 residue of CDK2 (cyclin-dependent kinase 2). RESULTS +53 64 DUSP family protein_type Kinase-associated phosphatase (KAP), a member of the DUSP family, plays a crucial role in cell cycle regulation by dephosphorylating the pThr160 residue of CDK2 (cyclin-dependent kinase 2). RESULTS +137 144 pThr160 ptm Kinase-associated phosphatase (KAP), a member of the DUSP family, plays a crucial role in cell cycle regulation by dephosphorylating the pThr160 residue of CDK2 (cyclin-dependent kinase 2). RESULTS +156 160 CDK2 protein Kinase-associated phosphatase (KAP), a member of the DUSP family, plays a crucial role in cell cycle regulation by dephosphorylating the pThr160 residue of CDK2 (cyclin-dependent kinase 2). RESULTS +162 187 cyclin-dependent kinase 2 protein Kinase-associated phosphatase (KAP), a member of the DUSP family, plays a crucial role in cell cycle regulation by dephosphorylating the pThr160 residue of CDK2 (cyclin-dependent kinase 2). RESULTS +4 21 crystal structure evidence The crystal structure of the CDK2/KAP complex has been determined at 3.0 Å (Fig. 5a). RESULTS +29 37 CDK2/KAP complex_assembly The crystal structure of the CDK2/KAP complex has been determined at 3.0 Å (Fig. 5a). RESULTS +4 13 interface site The interface between these two proteins consists of three discontinuous contact regions. RESULTS +72 75 KAP protein Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the αG helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b). RESULTS +80 84 CDK2 protein Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the αG helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b). RESULTS +102 118 recognition site site Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the αG helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b). RESULTS +130 134 CDK2 protein Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the αG helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b). RESULTS +153 161 αG helix structure_element Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the αG helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b). RESULTS +166 174 L14 loop structure_element Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the αG helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b). RESULTS +183 208 N-terminal helical region structure_element Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the αG helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b). RESULTS +212 215 KAP protein Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the αG helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b). RESULTS +11 24 hydrogen bond bond_interaction There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2. RESULTS +60 66 Ile183 residue_name_number There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2. RESULTS +68 71 KAP protein There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2. RESULTS +98 104 Glu208 residue_name_number There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2. RESULTS +106 110 CDK2 protein There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2. RESULTS +138 144 Lys184 residue_name_number There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2. RESULTS +148 151 KAP protein There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2. RESULTS +156 162 Asp235 residue_name_number There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2. RESULTS +166 170 CDK2 protein There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2. RESULTS +0 19 Structural analysis experimental_method Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). RESULTS +24 42 sequence alignment experimental_method Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). RESULTS +90 94 MKP7 protein Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). RESULTS +95 97 CD structure_element Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). RESULTS +102 105 KAP protein Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). RESULTS +113 137 substrate-binding region site Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). RESULTS +167 171 FNFL structure_element Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). RESULTS +175 179 MKP7 protein Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). RESULTS +180 182 CD structure_element Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). RESULTS +205 209 IKQY structure_element Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). RESULTS +213 216 KAP protein Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c). RESULTS +4 16 substitution experimental_method The substitution of the two hydrophobic residues with charged/polar residues (F285I/N286K) seriously disrupts the hydrophobic interaction required for MKP7 binding on JNK1 (Fig. 4a). RESULTS +78 83 F285I mutant The substitution of the two hydrophobic residues with charged/polar residues (F285I/N286K) seriously disrupts the hydrophobic interaction required for MKP7 binding on JNK1 (Fig. 4a). RESULTS +84 89 N286K mutant The substitution of the two hydrophobic residues with charged/polar residues (F285I/N286K) seriously disrupts the hydrophobic interaction required for MKP7 binding on JNK1 (Fig. 4a). RESULTS +114 137 hydrophobic interaction bond_interaction The substitution of the two hydrophobic residues with charged/polar residues (F285I/N286K) seriously disrupts the hydrophobic interaction required for MKP7 binding on JNK1 (Fig. 4a). RESULTS +151 155 MKP7 protein The substitution of the two hydrophobic residues with charged/polar residues (F285I/N286K) seriously disrupts the hydrophobic interaction required for MKP7 binding on JNK1 (Fig. 4a). RESULTS +167 171 JNK1 protein The substitution of the two hydrophobic residues with charged/polar residues (F285I/N286K) seriously disrupts the hydrophobic interaction required for MKP7 binding on JNK1 (Fig. 4a). RESULTS +13 19 His230 residue_name_number In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK. RESULTS +24 30 Val256 residue_name_number In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK. RESULTS +34 38 JNK1 protein In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK. RESULTS +87 93 Glu208 residue_name_number In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK. RESULTS +98 104 Asp235 residue_name_number In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK. RESULTS +108 112 CDK2 protein In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK. RESULTS +159 163 CDK2 protein In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK. RESULTS +164 183 interactive surface site In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK. RESULTS +216 219 JNK protein_type In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK. RESULTS +35 53 hydrophobic pocket site These data indicated that a unique hydrophobic pocket formed between the MAPK insert and αG helix plays a major role in the substrate recognition by MKPs. RESULTS +73 84 MAPK insert structure_element These data indicated that a unique hydrophobic pocket formed between the MAPK insert and αG helix plays a major role in the substrate recognition by MKPs. RESULTS +89 97 αG helix structure_element These data indicated that a unique hydrophobic pocket formed between the MAPK insert and αG helix plays a major role in the substrate recognition by MKPs. RESULTS +149 153 MKPs protein_type These data indicated that a unique hydrophobic pocket formed between the MAPK insert and αG helix plays a major role in the substrate recognition by MKPs. RESULTS +0 6 F-site site F-site interaction is crucial for JNK1 inactivation in vivo RESULTS +34 38 JNK1 protein F-site interaction is crucial for JNK1 inactivation in vivo RESULTS +0 3 JNK protein_type JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol). RESULTS +82 92 anisomycin chemical JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol). RESULTS +94 98 H2O2 chemical JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol). RESULTS +119 127 sorbitol chemical JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol). RESULTS +188 197 etoposide chemical JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol). RESULTS +199 208 cisplatin chemical JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol). RESULTS +213 218 taxol chemical JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol). RESULTS +25 29 MKP7 protein To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. RESULTS +38 45 mutants protein_state To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. RESULTS +78 81 JNK protein_type To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. RESULTS +148 157 HA-tagged protein_state To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. RESULTS +173 184 full-length protein_state To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. RESULTS +185 189 MKP7 protein To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. RESULTS +191 200 MKP7ΔC304 mutant To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. RESULTS +205 209 MKP7 protein To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. RESULTS +210 212 CD structure_element To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. RESULTS +216 220 MKP7 protein To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. RESULTS +221 228 mutants protein_state To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. RESULTS +265 274 etoposide chemical To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment. RESULTS +23 36 immunobloting experimental_method As shown in Fig. 6a–c, immunobloting showed similar expression levels for the different MKP7 constructs in all the cells. RESULTS +88 92 MKP7 protein As shown in Fig. 6a–c, immunobloting showed similar expression levels for the different MKP7 constructs in all the cells. RESULTS +0 13 Overexpressed experimental_method Overexpressed full-length MKP7, MKP7ΔC304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells. RESULTS +14 25 full-length protein_state Overexpressed full-length MKP7, MKP7ΔC304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells. RESULTS +26 30 MKP7 protein Overexpressed full-length MKP7, MKP7ΔC304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells. RESULTS +32 41 MKP7ΔC304 mutant Overexpressed full-length MKP7, MKP7ΔC304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells. RESULTS +46 50 MKP7 protein Overexpressed full-length MKP7, MKP7ΔC304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells. RESULTS +51 53 CD structure_element Overexpressed full-length MKP7, MKP7ΔC304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells. RESULTS +100 114 phosphorylated protein_state Overexpressed full-length MKP7, MKP7ΔC304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells. RESULTS +115 118 JNK protein_type Overexpressed full-length MKP7, MKP7ΔC304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells. RESULTS +45 52 D-motif structure_element Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. RESULTS +53 60 mutants protein_state Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. RESULTS +62 66 R56A mutant Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. RESULTS +67 71 R57A mutant Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. RESULTS +76 80 V63A mutant Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. RESULTS +81 85 I65A mutant Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. RESULTS +87 103 dephosphorylated protein_state Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. RESULTS +104 107 JNK protein_type Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. RESULTS +119 128 wild type protein_state Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. RESULTS +184 188 MKP7 protein Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. RESULTS +189 192 KBD structure_element Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. RESULTS +217 220 JNK protein_type Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo. RESULTS +48 62 phosphorylated protein_state Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +63 66 JNK protein_type Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +96 100 MKP7 protein Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +101 110 FXF-motif structure_element Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +111 118 mutants protein_state Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +120 125 F285D mutant Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +127 132 F287D mutant Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +137 142 L288D mutant Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +171 175 MKP7 protein Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +176 181 D268A mutant Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +186 191 N286A mutant Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +192 199 mutants protein_state Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +261 264 JNK protein_type Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK. RESULTS +44 48 JNK1 protein We next tested in vivo interactions between JNK1 mutants and full-length MKP7 by coimmunoprecipitation experiments under unstimulated conditions. RESULTS +49 56 mutants protein_state We next tested in vivo interactions between JNK1 mutants and full-length MKP7 by coimmunoprecipitation experiments under unstimulated conditions. RESULTS +61 72 full-length protein_state We next tested in vivo interactions between JNK1 mutants and full-length MKP7 by coimmunoprecipitation experiments under unstimulated conditions. RESULTS +73 77 MKP7 protein We next tested in vivo interactions between JNK1 mutants and full-length MKP7 by coimmunoprecipitation experiments under unstimulated conditions. RESULTS +81 114 coimmunoprecipitation experiments experimental_method We next tested in vivo interactions between JNK1 mutants and full-length MKP7 by coimmunoprecipitation experiments under unstimulated conditions. RESULTS +5 17 co-expressed experimental_method When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo. RESULTS +36 45 wild-type protein_state When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo. RESULTS +51 55 JNK1 protein When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo. RESULTS +92 96 MKP7 protein When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo. RESULTS +124 128 MKP7 protein When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo. RESULTS +135 151 dephosphorylated protein_state When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo. RESULTS +152 156 JNK1 protein When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo. RESULTS +22 40 in vitro pull-down experimental_method In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a). RESULTS +54 61 mutants protein_state In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a). RESULTS +62 67 D229A mutant In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a). RESULTS +69 74 W234D mutant In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a). RESULTS +79 84 Y259D mutant In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a). RESULTS +115 119 MKP7 protein In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a). RESULTS +129 134 I231D mutant In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a). RESULTS +135 141 mutant protein_state In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a). RESULTS +172 181 JNK1–MKP7 complex_assembly In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a). RESULTS +18 21 JNK protein_type Activation of the JNK signalling pathway is frequently associated with apoptotic cell death, and inhibition of JNK can prevent apoptotic death of multiple cells. RESULTS +111 114 JNK protein_type Activation of the JNK signalling pathway is frequently associated with apoptotic cell death, and inhibition of JNK can prevent apoptotic death of multiple cells. RESULTS +37 40 JNK protein_type To examine whether the inhibition of JNK activity by MKP7 would provide protections against the apoptosis, we analysed the rate of apoptosis in ultraviolet-irradiated cells transfected with MKP7 (wild type or mutants) by flow cytometry. RESULTS +53 57 MKP7 protein To examine whether the inhibition of JNK activity by MKP7 would provide protections against the apoptosis, we analysed the rate of apoptosis in ultraviolet-irradiated cells transfected with MKP7 (wild type or mutants) by flow cytometry. RESULTS +190 194 MKP7 protein To examine whether the inhibition of JNK activity by MKP7 would provide protections against the apoptosis, we analysed the rate of apoptosis in ultraviolet-irradiated cells transfected with MKP7 (wild type or mutants) by flow cytometry. RESULTS +196 205 wild type protein_state To examine whether the inhibition of JNK activity by MKP7 would provide protections against the apoptosis, we analysed the rate of apoptosis in ultraviolet-irradiated cells transfected with MKP7 (wild type or mutants) by flow cytometry. RESULTS +209 216 mutants protein_state To examine whether the inhibition of JNK activity by MKP7 would provide protections against the apoptosis, we analysed the rate of apoptosis in ultraviolet-irradiated cells transfected with MKP7 (wild type or mutants) by flow cytometry. RESULTS +221 235 flow cytometry experimental_method To examine whether the inhibition of JNK activity by MKP7 would provide protections against the apoptosis, we analysed the rate of apoptosis in ultraviolet-irradiated cells transfected with MKP7 (wild type or mutants) by flow cytometry. RESULTS +95 99 MKP7 protein The results showed similar apoptotic rates between cells transfected with blank vector or with MKP7 (wild type or mutants) under unstimulated conditions (Supplementary Fig. 3b), while ultraviolet-irradiation significantly increased apoptotic rate in cells transfected with blank vector (Fig. 6e). RESULTS +101 110 wild type protein_state The results showed similar apoptotic rates between cells transfected with blank vector or with MKP7 (wild type or mutants) under unstimulated conditions (Supplementary Fig. 3b), while ultraviolet-irradiation significantly increased apoptotic rate in cells transfected with blank vector (Fig. 6e). RESULTS +114 121 mutants protein_state The results showed similar apoptotic rates between cells transfected with blank vector or with MKP7 (wild type or mutants) under unstimulated conditions (Supplementary Fig. 3b), while ultraviolet-irradiation significantly increased apoptotic rate in cells transfected with blank vector (Fig. 6e). RESULTS +0 11 Expressions experimental_method Expressions of wild-type MKP7, MKP7ΔC304 and MKP7-CD significantly decreased the proportion of apoptotic cells after ultraviolet treatment. RESULTS +15 24 wild-type protein_state Expressions of wild-type MKP7, MKP7ΔC304 and MKP7-CD significantly decreased the proportion of apoptotic cells after ultraviolet treatment. RESULTS +25 29 MKP7 protein Expressions of wild-type MKP7, MKP7ΔC304 and MKP7-CD significantly decreased the proportion of apoptotic cells after ultraviolet treatment. RESULTS +31 40 MKP7ΔC304 mutant Expressions of wild-type MKP7, MKP7ΔC304 and MKP7-CD significantly decreased the proportion of apoptotic cells after ultraviolet treatment. RESULTS +45 49 MKP7 protein Expressions of wild-type MKP7, MKP7ΔC304 and MKP7-CD significantly decreased the proportion of apoptotic cells after ultraviolet treatment. RESULTS +50 52 CD structure_element Expressions of wild-type MKP7, MKP7ΔC304 and MKP7-CD significantly decreased the proportion of apoptotic cells after ultraviolet treatment. RESULTS +40 44 MKP7 protein Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did. RESULTS +45 48 KBD structure_element Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did. RESULTS +49 56 mutants protein_state Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did. RESULTS +58 62 R56A mutant Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did. RESULTS +63 67 R57A mutant Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did. RESULTS +72 76 V63A mutant Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did. RESULTS +77 81 I65A mutant Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did. RESULTS +136 140 MKP7 protein Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did. RESULTS +141 150 wild type protein_state Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did. RESULTS +40 44 MKP7 protein In contrast, cells transfected with the MKP7 FXF-motif mutants (F285D, F287D and L288D) showed little protective effect after ultraviolet treatment and similar levels of apoptosis rates were detected to cells transfected with control vectors (Fig. 6e,f). RESULTS +45 54 FXF-motif structure_element In contrast, cells transfected with the MKP7 FXF-motif mutants (F285D, F287D and L288D) showed little protective effect after ultraviolet treatment and similar levels of apoptosis rates were detected to cells transfected with control vectors (Fig. 6e,f). RESULTS +55 62 mutants protein_state In contrast, cells transfected with the MKP7 FXF-motif mutants (F285D, F287D and L288D) showed little protective effect after ultraviolet treatment and similar levels of apoptosis rates were detected to cells transfected with control vectors (Fig. 6e,f). RESULTS +64 69 F285D mutant In contrast, cells transfected with the MKP7 FXF-motif mutants (F285D, F287D and L288D) showed little protective effect after ultraviolet treatment and similar levels of apoptosis rates were detected to cells transfected with control vectors (Fig. 6e,f). RESULTS +71 76 F287D mutant In contrast, cells transfected with the MKP7 FXF-motif mutants (F285D, F287D and L288D) showed little protective effect after ultraviolet treatment and similar levels of apoptosis rates were detected to cells transfected with control vectors (Fig. 6e,f). RESULTS +81 86 L288D mutant In contrast, cells transfected with the MKP7 FXF-motif mutants (F285D, F287D and L288D) showed little protective effect after ultraviolet treatment and similar levels of apoptosis rates were detected to cells transfected with control vectors (Fig. 6e,f). RESULTS +43 52 FXF-motif structure_element Taken together, our results suggested that FXF-motif-mediated, rather than KBD-mediated, interaction is essential for MKP7 to block ultraviolet-induced apoptosis. RESULTS +75 78 KBD structure_element Taken together, our results suggested that FXF-motif-mediated, rather than KBD-mediated, interaction is essential for MKP7 to block ultraviolet-induced apoptosis. RESULTS +118 122 MKP7 protein Taken together, our results suggested that FXF-motif-mediated, rather than KBD-mediated, interaction is essential for MKP7 to block ultraviolet-induced apoptosis. RESULTS +32 36 JNK1 protein A similar docking mechanism for JNK1 recognition by MKP5 RESULTS +52 56 MKP5 protein A similar docking mechanism for JNK1 recognition by MKP5 RESULTS +0 4 MKP5 protein MKP5 belongs to the same subfamily as MKP7. RESULTS +38 42 MKP7 protein MKP5 belongs to the same subfamily as MKP7. RESULTS +0 4 MKP5 protein MKP5 is unique among the MKPs in possessing an additional domain of unknown function at the N-terminus (Fig. 7a). RESULTS +25 29 MKPs protein_type MKP5 is unique among the MKPs in possessing an additional domain of unknown function at the N-terminus (Fig. 7a). RESULTS +4 7 KBD structure_element The KBD of MKP5 interacts with the D-site of p38α to mediate the enzyme–substrate interaction. RESULTS +11 15 MKP5 protein The KBD of MKP5 interacts with the D-site of p38α to mediate the enzyme–substrate interaction. RESULTS +35 41 D-site site The KBD of MKP5 interacts with the D-site of p38α to mediate the enzyme–substrate interaction. RESULTS +45 49 p38α protein The KBD of MKP5 interacts with the D-site of p38α to mediate the enzyme–substrate interaction. RESULTS +0 11 Deletion of experimental_method Deletion of the KBD in MKP5 leads to a 280-fold increase in Km for p38α substrate. RESULTS +16 19 KBD structure_element Deletion of the KBD in MKP5 leads to a 280-fold increase in Km for p38α substrate. RESULTS +23 27 MKP5 protein Deletion of the KBD in MKP5 leads to a 280-fold increase in Km for p38α substrate. RESULTS +60 62 Km evidence Deletion of the KBD in MKP5 leads to a 280-fold increase in Km for p38α substrate. RESULTS +67 71 p38α protein Deletion of the KBD in MKP5 leads to a 280-fold increase in Km for p38α substrate. RESULTS +15 19 p38α protein In contrast to p38α substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b). RESULTS +31 42 deletion of experimental_method In contrast to p38α substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b). RESULTS +47 51 MKP5 protein In contrast to p38α substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b). RESULTS +52 55 KBD structure_element In contrast to p38α substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b). RESULTS +109 113 JNK1 protein In contrast to p38α substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b). RESULTS +153 156 KBD structure_element In contrast to p38α substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b). RESULTS +160 164 MKP5 protein In contrast to p38α substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b). RESULTS +189 193 JNK1 protein In contrast to p38α substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b). RESULTS +4 34 substrate specificity constant evidence The substrate specificity constant kcat /Km value for MKP5-CD was calculated as 1.0 × 105 M−1 s−1, which is very close to that of MKP7-CD (1.07 × 105 M−1 s−1). RESULTS +35 43 kcat /Km evidence The substrate specificity constant kcat /Km value for MKP5-CD was calculated as 1.0 × 105 M−1 s−1, which is very close to that of MKP7-CD (1.07 × 105 M−1 s−1). RESULTS +54 58 MKP5 protein The substrate specificity constant kcat /Km value for MKP5-CD was calculated as 1.0 × 105 M−1 s−1, which is very close to that of MKP7-CD (1.07 × 105 M−1 s−1). RESULTS +59 61 CD structure_element The substrate specificity constant kcat /Km value for MKP5-CD was calculated as 1.0 × 105 M−1 s−1, which is very close to that of MKP7-CD (1.07 × 105 M−1 s−1). RESULTS +130 134 MKP7 protein The substrate specificity constant kcat /Km value for MKP5-CD was calculated as 1.0 × 105 M−1 s−1, which is very close to that of MKP7-CD (1.07 × 105 M−1 s−1). RESULTS +135 137 CD structure_element The substrate specificity constant kcat /Km value for MKP5-CD was calculated as 1.0 × 105 M−1 s−1, which is very close to that of MKP7-CD (1.07 × 105 M−1 s−1). RESULTS +4 21 crystal structure evidence The crystal structure of human MKP5-CD has been determined. RESULTS +25 30 human species The crystal structure of human MKP5-CD has been determined. RESULTS +31 35 MKP5 protein The crystal structure of human MKP5-CD has been determined. RESULTS +36 38 CD structure_element The crystal structure of human MKP5-CD has been determined. RESULTS +20 37 catalytic domains structure_element Comparisons between catalytic domains structures of MKP5 and MKP7 reveal that the overall folds of the two proteins are highly similar, with only a few regions exhibiting small deviations (r.m.s.d. of 0.79 Å; Fig. 7c). RESULTS +38 48 structures evidence Comparisons between catalytic domains structures of MKP5 and MKP7 reveal that the overall folds of the two proteins are highly similar, with only a few regions exhibiting small deviations (r.m.s.d. of 0.79 Å; Fig. 7c). RESULTS +52 56 MKP5 protein Comparisons between catalytic domains structures of MKP5 and MKP7 reveal that the overall folds of the two proteins are highly similar, with only a few regions exhibiting small deviations (r.m.s.d. of 0.79 Å; Fig. 7c). RESULTS +61 65 MKP7 protein Comparisons between catalytic domains structures of MKP5 and MKP7 reveal that the overall folds of the two proteins are highly similar, with only a few regions exhibiting small deviations (r.m.s.d. of 0.79 Å; Fig. 7c). RESULTS +189 197 r.m.s.d. evidence Comparisons between catalytic domains structures of MKP5 and MKP7 reveal that the overall folds of the two proteins are highly similar, with only a few regions exhibiting small deviations (r.m.s.d. of 0.79 Å; Fig. 7c). RESULTS +52 69 crystal structure evidence Given the distinct interaction mode revealed by the crystal structure of JNK1–MKP7-CD, one obvious question is whether this is a general mechanism used by all members of the JNK-specific MKPs. RESULTS +73 85 JNK1–MKP7-CD complex_assembly Given the distinct interaction mode revealed by the crystal structure of JNK1–MKP7-CD, one obvious question is whether this is a general mechanism used by all members of the JNK-specific MKPs. RESULTS +174 191 JNK-specific MKPs protein_type Given the distinct interaction mode revealed by the crystal structure of JNK1–MKP7-CD, one obvious question is whether this is a general mechanism used by all members of the JNK-specific MKPs. RESULTS +64 68 JNK1 protein To address this issue, we first examined the docking ability of JNK1 to the KBD and CD of MKP5 using gel filtration analysis and pull-down assays. RESULTS +76 79 KBD structure_element To address this issue, we first examined the docking ability of JNK1 to the KBD and CD of MKP5 using gel filtration analysis and pull-down assays. RESULTS +84 86 CD structure_element To address this issue, we first examined the docking ability of JNK1 to the KBD and CD of MKP5 using gel filtration analysis and pull-down assays. RESULTS +90 94 MKP5 protein To address this issue, we first examined the docking ability of JNK1 to the KBD and CD of MKP5 using gel filtration analysis and pull-down assays. RESULTS +101 124 gel filtration analysis experimental_method To address this issue, we first examined the docking ability of JNK1 to the KBD and CD of MKP5 using gel filtration analysis and pull-down assays. RESULTS +129 145 pull-down assays experimental_method To address this issue, we first examined the docking ability of JNK1 to the KBD and CD of MKP5 using gel filtration analysis and pull-down assays. RESULTS +20 46 gel filtration experiments experimental_method It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d). RESULTS +52 56 JNK1 protein It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d). RESULTS +69 75 stable protein_state It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d). RESULTS +76 87 heterodimer oligomeric_state It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d). RESULTS +93 97 MKP5 protein It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d). RESULTS +98 100 CD structure_element It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d). RESULTS +163 166 KBD structure_element It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d). RESULTS +0 16 Pull-down assays experimental_method Pull-down assays also confirmed the protein–protein interactions observed above. RESULTS +4 20 catalytic domain structure_element The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs. RESULTS +24 28 MKP5 protein The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs. RESULTS +42 45 KBD structure_element The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs. RESULTS +92 96 JNK1 protein The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs. RESULTS +169 172 p38 protein_type The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs. RESULTS +177 180 JNK protein_type The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs. RESULTS +181 186 MAPKs protein_type The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs. RESULTS +54 58 MKP5 protein To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity. RESULTS +59 61 CD structure_element To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity. RESULTS +70 79 mutations experimental_method To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity. RESULTS +120 124 MKP7 protein To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity. RESULTS +125 127 CD structure_element To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity. RESULTS +144 177 sequence and structural alignment experimental_method To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity. RESULTS +212 223 phosphatase protein_type To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity. RESULTS +25 30 T432A mutant As shown in Fig. 7f, the T432A and L449F MKP5 mutant showed little or no difference in phosphatase activity, whereas the other mutants showed reduced specific activities of MKP5. RESULTS +35 40 L449F mutant As shown in Fig. 7f, the T432A and L449F MKP5 mutant showed little or no difference in phosphatase activity, whereas the other mutants showed reduced specific activities of MKP5. RESULTS +41 45 MKP5 protein As shown in Fig. 7f, the T432A and L449F MKP5 mutant showed little or no difference in phosphatase activity, whereas the other mutants showed reduced specific activities of MKP5. RESULTS +46 52 mutant protein_state As shown in Fig. 7f, the T432A and L449F MKP5 mutant showed little or no difference in phosphatase activity, whereas the other mutants showed reduced specific activities of MKP5. RESULTS +127 134 mutants protein_state As shown in Fig. 7f, the T432A and L449F MKP5 mutant showed little or no difference in phosphatase activity, whereas the other mutants showed reduced specific activities of MKP5. RESULTS +173 177 MKP5 protein As shown in Fig. 7f, the T432A and L449F MKP5 mutant showed little or no difference in phosphatase activity, whereas the other mutants showed reduced specific activities of MKP5. RESULTS +18 22 MKP7 protein As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +32 39 mutants protein_state As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +48 55 F451D/A mutant As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +67 74 pNPPase protein_type As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +110 119 wild-type protein_state As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +120 124 MKP5 protein As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +125 127 CD structure_element As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +147 162 point mutations experimental_method As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +166 170 JNK1 protein As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +188 204 binding affinity evidence As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +208 212 MKP5 protein As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +213 215 CD structure_element As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +220 224 JNK1 protein As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h). RESULTS +58 68 CD spectra evidence In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a). RESULTS +77 86 wild-type protein_state In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a). RESULTS +91 97 mutant protein_state In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a). RESULTS +136 146 structures evidence In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a). RESULTS +156 163 mutants protein_state In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a). RESULTS +206 215 wild-type protein_state In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a). RESULTS +216 220 MKP5 protein In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a). RESULTS +41 45 MKP5 protein Taken together, our results suggest that MKP5 binds JNK1 in a docking mode similar to that in the JNK1–MKP7 complex, and the detailed interaction model can be generated using molecular dynamics simulation based on the structure of JNK1–MKP7-CD complex (Supplementary Fig. 4b,c). RESULTS +52 56 JNK1 protein Taken together, our results suggest that MKP5 binds JNK1 in a docking mode similar to that in the JNK1–MKP7 complex, and the detailed interaction model can be generated using molecular dynamics simulation based on the structure of JNK1–MKP7-CD complex (Supplementary Fig. 4b,c). RESULTS +98 107 JNK1–MKP7 complex_assembly Taken together, our results suggest that MKP5 binds JNK1 in a docking mode similar to that in the JNK1–MKP7 complex, and the detailed interaction model can be generated using molecular dynamics simulation based on the structure of JNK1–MKP7-CD complex (Supplementary Fig. 4b,c). RESULTS +175 204 molecular dynamics simulation experimental_method Taken together, our results suggest that MKP5 binds JNK1 in a docking mode similar to that in the JNK1–MKP7 complex, and the detailed interaction model can be generated using molecular dynamics simulation based on the structure of JNK1–MKP7-CD complex (Supplementary Fig. 4b,c). RESULTS +218 227 structure evidence Taken together, our results suggest that MKP5 binds JNK1 in a docking mode similar to that in the JNK1–MKP7 complex, and the detailed interaction model can be generated using molecular dynamics simulation based on the structure of JNK1–MKP7-CD complex (Supplementary Fig. 4b,c). RESULTS +231 243 JNK1–MKP7-CD complex_assembly Taken together, our results suggest that MKP5 binds JNK1 in a docking mode similar to that in the JNK1–MKP7 complex, and the detailed interaction model can be generated using molecular dynamics simulation based on the structure of JNK1–MKP7-CD complex (Supplementary Fig. 4b,c). RESULTS +19 23 MKP5 protein In this model, the MKP5-CD adopts a conformation nearly identical to that in its unbound form, suggesting that the conformation of the catalytic domain undergoes little change, if any at all, upon JNK1 binding. RESULTS +24 26 CD structure_element In this model, the MKP5-CD adopts a conformation nearly identical to that in its unbound form, suggesting that the conformation of the catalytic domain undergoes little change, if any at all, upon JNK1 binding. RESULTS +81 88 unbound protein_state In this model, the MKP5-CD adopts a conformation nearly identical to that in its unbound form, suggesting that the conformation of the catalytic domain undergoes little change, if any at all, upon JNK1 binding. RESULTS +135 151 catalytic domain structure_element In this model, the MKP5-CD adopts a conformation nearly identical to that in its unbound form, suggesting that the conformation of the catalytic domain undergoes little change, if any at all, upon JNK1 binding. RESULTS +197 201 JNK1 protein In this model, the MKP5-CD adopts a conformation nearly identical to that in its unbound form, suggesting that the conformation of the catalytic domain undergoes little change, if any at all, upon JNK1 binding. RESULTS +15 21 Leu449 residue_name_number In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1–MKP7-CD complex (Supplementary Fig. 4d). RESULTS +25 29 MKP5 protein In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1–MKP7-CD complex (Supplementary Fig. 4d). RESULTS +70 76 Phe285 residue_name_number In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1–MKP7-CD complex (Supplementary Fig. 4d). RESULTS +80 84 MKP7 protein In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1–MKP7-CD complex (Supplementary Fig. 4d). RESULTS +111 129 hydrophobic pocket site In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1–MKP7-CD complex (Supplementary Fig. 4d). RESULTS +133 137 JNK1 protein In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1–MKP7-CD complex (Supplementary Fig. 4d). RESULTS +157 163 Phe285 residue_name_number In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1–MKP7-CD complex (Supplementary Fig. 4d). RESULTS +171 183 JNK1–MKP7-CD complex_assembly In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1–MKP7-CD complex (Supplementary Fig. 4d). RESULTS +40 44 JNK1 protein Despite the strong similarities between JNK1–MKP5-CD and JNK1–MKP7-CD, however, there are differences. RESULTS +45 49 MKP5 protein Despite the strong similarities between JNK1–MKP5-CD and JNK1–MKP7-CD, however, there are differences. RESULTS +50 52 CD structure_element Despite the strong similarities between JNK1–MKP5-CD and JNK1–MKP7-CD, however, there are differences. RESULTS +57 69 JNK1–MKP7-CD complex_assembly Despite the strong similarities between JNK1–MKP5-CD and JNK1–MKP7-CD, however, there are differences. RESULTS +4 16 JNK1–MKP7-CD complex_assembly The JNK1–MKP7-CD interaction is better and more extensive. RESULTS +0 6 Asp268 residue_name_number Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1. RESULTS +10 14 MKP7 protein Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1. RESULTS +15 17 CD structure_element Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1. RESULTS +24 35 salt bridge bond_interaction Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1. RESULTS +41 45 JNK1 protein Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1. RESULTS +46 52 Arg263 residue_name_number Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1. RESULTS +88 94 Thr432 residue_name_number Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1. RESULTS +98 102 MKP5 protein Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1. RESULTS +103 105 CD structure_element Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1. RESULTS +128 132 JNK1 protein Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1. RESULTS +45 49 MKP7 protein In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +50 52 CD structure_element In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +54 60 Phe215 residue_name_number In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +62 68 Leu267 residue_name_number In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +73 79 Leu288 residue_name_number In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +124 130 Asn379 residue_name_number In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +132 138 Met431 residue_name_number In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +143 149 Met452 residue_name_number In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +153 157 MKP5 protein In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +158 160 CD structure_element In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +213 237 hydrophobic interactions bond_interaction In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +246 250 MKP5 protein In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +251 253 CD structure_element In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +258 262 JNK1 protein In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1. RESULTS +66 70 JNK1 protein This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). RESULTS +80 84 MKP7 protein This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). RESULTS +85 87 CD structure_element This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). RESULTS +111 115 MKP5 protein This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). RESULTS +116 118 CD structure_element This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). RESULTS +132 136 MKP5 protein This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). RESULTS +137 139 CD structure_element This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). RESULTS +144 145 p protein_state This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). RESULTS +145 149 JNK1 protein This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). RESULTS +192 196 MKP7 protein This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). RESULTS +197 199 CD structure_element This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD). RESULTS +4 9 MAPKs protein_type The MAPKs p38, ERK and JNK, are central to evolutionarily conserved signalling pathways that are present in all eukaryotic cells. DISCUSS +10 13 p38 protein_type The MAPKs p38, ERK and JNK, are central to evolutionarily conserved signalling pathways that are present in all eukaryotic cells. DISCUSS +15 18 ERK protein_type The MAPKs p38, ERK and JNK, are central to evolutionarily conserved signalling pathways that are present in all eukaryotic cells. DISCUSS +23 26 JNK protein_type The MAPKs p38, ERK and JNK, are central to evolutionarily conserved signalling pathways that are present in all eukaryotic cells. DISCUSS +112 122 eukaryotic taxonomy_domain The MAPKs p38, ERK and JNK, are central to evolutionarily conserved signalling pathways that are present in all eukaryotic cells. DISCUSS +5 9 MAPK protein_type Each MAPK cascade is activated in response to a diverse array of extracellular signals and culminates in the dual-phosphorylation of a threonine and a tyrosine residue in the MAPK-activation loop. DISCUSS +109 129 dual-phosphorylation ptm Each MAPK cascade is activated in response to a diverse array of extracellular signals and culminates in the dual-phosphorylation of a threonine and a tyrosine residue in the MAPK-activation loop. DISCUSS +135 144 threonine residue_name Each MAPK cascade is activated in response to a diverse array of extracellular signals and culminates in the dual-phosphorylation of a threonine and a tyrosine residue in the MAPK-activation loop. DISCUSS +151 159 tyrosine residue_name Each MAPK cascade is activated in response to a diverse array of extracellular signals and culminates in the dual-phosphorylation of a threonine and a tyrosine residue in the MAPK-activation loop. DISCUSS +175 195 MAPK-activation loop structure_element Each MAPK cascade is activated in response to a diverse array of extracellular signals and culminates in the dual-phosphorylation of a threonine and a tyrosine residue in the MAPK-activation loop. DISCUSS +19 23 MAPK protein_type The propagation of MAPK signals is attenuated through the actions of the MKPs. DISCUSS +73 77 MKPs protein_type The propagation of MAPK signals is attenuated through the actions of the MKPs. DISCUSS +54 59 MAPKs protein_type Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the ‘kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL). DISCUSS +63 75 phosphatases protein_type Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the ‘kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL). DISCUSS +92 116 kinase-interaction motif structure_element Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the ‘kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL). DISCUSS +120 127 D-motif structure_element Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the ‘kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL). DISCUSS +151 156 DUSPs protein_type Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the ‘kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL). DISCUSS +158 162 MKPs protein_type Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the ‘kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL). DISCUSS +192 213 tyrosine phosphatases protein_type Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the ‘kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL). DISCUSS +215 220 HePTP protein Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the ‘kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL). DISCUSS +222 226 STEP protein Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the ‘kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL). DISCUSS +231 237 PTP-SL protein Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the ‘kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL). DISCUSS +0 18 Crystal structures evidence Crystal structures of ERK2 bound with the D-motif sequences derived from MKP3 and HePTP have been reported. DISCUSS +22 26 ERK2 protein Crystal structures of ERK2 bound with the D-motif sequences derived from MKP3 and HePTP have been reported. DISCUSS +27 37 bound with protein_state Crystal structures of ERK2 bound with the D-motif sequences derived from MKP3 and HePTP have been reported. DISCUSS +42 49 D-motif structure_element Crystal structures of ERK2 bound with the D-motif sequences derived from MKP3 and HePTP have been reported. DISCUSS +73 77 MKP3 protein Crystal structures of ERK2 bound with the D-motif sequences derived from MKP3 and HePTP have been reported. DISCUSS +82 87 HePTP protein Crystal structures of ERK2 bound with the D-motif sequences derived from MKP3 and HePTP have been reported. DISCUSS +6 16 structures evidence These structures revealed that linear docking motifs in interacting proteins bind to a common docking site on MAPKs outside the kinase active site. DISCUSS +31 52 linear docking motifs structure_element These structures revealed that linear docking motifs in interacting proteins bind to a common docking site on MAPKs outside the kinase active site. DISCUSS +94 106 docking site site These structures revealed that linear docking motifs in interacting proteins bind to a common docking site on MAPKs outside the kinase active site. DISCUSS +110 115 MAPKs protein_type These structures revealed that linear docking motifs in interacting proteins bind to a common docking site on MAPKs outside the kinase active site. DISCUSS +128 134 kinase protein_type These structures revealed that linear docking motifs in interacting proteins bind to a common docking site on MAPKs outside the kinase active site. DISCUSS +135 146 active site site These structures revealed that linear docking motifs in interacting proteins bind to a common docking site on MAPKs outside the kinase active site. DISCUSS +52 59 D-motif structure_element The particular amino acids and their spacing within D-motif sequences and amino acid composition of the docking sites on MAPKs appear to determine the specificity of D-motifs for individual MAPKs. DISCUSS +104 117 docking sites site The particular amino acids and their spacing within D-motif sequences and amino acid composition of the docking sites on MAPKs appear to determine the specificity of D-motifs for individual MAPKs. DISCUSS +121 126 MAPKs protein_type The particular amino acids and their spacing within D-motif sequences and amino acid composition of the docking sites on MAPKs appear to determine the specificity of D-motifs for individual MAPKs. DISCUSS +166 174 D-motifs structure_element The particular amino acids and their spacing within D-motif sequences and amino acid composition of the docking sites on MAPKs appear to determine the specificity of D-motifs for individual MAPKs. DISCUSS +190 195 MAPKs protein_type The particular amino acids and their spacing within D-motif sequences and amino acid composition of the docking sites on MAPKs appear to determine the specificity of D-motifs for individual MAPKs. DISCUSS +14 31 crystal structure evidence Recently, the crystal structure of a complex between the KBD of MKP5 and p38α has been obtained. DISCUSS +57 60 KBD structure_element Recently, the crystal structure of a complex between the KBD of MKP5 and p38α has been obtained. DISCUSS +64 68 MKP5 protein Recently, the crystal structure of a complex between the KBD of MKP5 and p38α has been obtained. DISCUSS +73 77 p38α protein Recently, the crystal structure of a complex between the KBD of MKP5 and p38α has been obtained. DISCUSS +58 62 MKP5 protein This complex has revealed a distinct interaction mode for MKP5. DISCUSS +4 7 KBD structure_element The KBD of MKP5 binds to p38α in the opposite polypeptide direction compared with how the D-motif of MKP3 binds to ERK2. DISCUSS +11 15 MKP5 protein The KBD of MKP5 binds to p38α in the opposite polypeptide direction compared with how the D-motif of MKP3 binds to ERK2. DISCUSS +25 29 p38α protein The KBD of MKP5 binds to p38α in the opposite polypeptide direction compared with how the D-motif of MKP3 binds to ERK2. DISCUSS +90 97 D-motif structure_element The KBD of MKP5 binds to p38α in the opposite polypeptide direction compared with how the D-motif of MKP3 binds to ERK2. DISCUSS +101 105 MKP3 protein The KBD of MKP5 binds to p38α in the opposite polypeptide direction compared with how the D-motif of MKP3 binds to ERK2. DISCUSS +115 119 ERK2 protein The KBD of MKP5 binds to p38α in the opposite polypeptide direction compared with how the D-motif of MKP3 binds to ERK2. DISCUSS +29 49 D-motif-binding mode site In contrast to the canonical D-motif-binding mode, separate helices, α2 and α3′, in the KBD of MKP5 engage the p38α-docking site. DISCUSS +60 67 helices structure_element In contrast to the canonical D-motif-binding mode, separate helices, α2 and α3′, in the KBD of MKP5 engage the p38α-docking site. DISCUSS +69 71 α2 structure_element In contrast to the canonical D-motif-binding mode, separate helices, α2 and α3′, in the KBD of MKP5 engage the p38α-docking site. DISCUSS +76 79 α3′ structure_element In contrast to the canonical D-motif-binding mode, separate helices, α2 and α3′, in the KBD of MKP5 engage the p38α-docking site. DISCUSS +88 91 KBD structure_element In contrast to the canonical D-motif-binding mode, separate helices, α2 and α3′, in the KBD of MKP5 engage the p38α-docking site. DISCUSS +95 99 MKP5 protein In contrast to the canonical D-motif-binding mode, separate helices, α2 and α3′, in the KBD of MKP5 engage the p38α-docking site. DISCUSS +111 128 p38α-docking site site In contrast to the canonical D-motif-binding mode, separate helices, α2 and α3′, in the KBD of MKP5 engage the p38α-docking site. DISCUSS +8 42 structural and biochemical studies experimental_method Further structural and biochemical studies indicate that KBD of MKP7 may interact with p38α in a similar manner to that of MKP5. DISCUSS +57 60 KBD structure_element Further structural and biochemical studies indicate that KBD of MKP7 may interact with p38α in a similar manner to that of MKP5. DISCUSS +64 68 MKP7 protein Further structural and biochemical studies indicate that KBD of MKP7 may interact with p38α in a similar manner to that of MKP5. DISCUSS +87 91 p38α protein Further structural and biochemical studies indicate that KBD of MKP7 may interact with p38α in a similar manner to that of MKP5. DISCUSS +123 127 MKP5 protein Further structural and biochemical studies indicate that KBD of MKP7 may interact with p38α in a similar manner to that of MKP5. DISCUSS +15 19 MKP5 protein In contrast to MKP5, removal of the KBD domain from MKP7 does not drastically affect enzyme catalysis, and the kinetic parameters of MKP7-CD for p38α substrate are very similar to those for JNK1 substrate. DISCUSS +21 31 removal of experimental_method In contrast to MKP5, removal of the KBD domain from MKP7 does not drastically affect enzyme catalysis, and the kinetic parameters of MKP7-CD for p38α substrate are very similar to those for JNK1 substrate. DISCUSS +36 39 KBD structure_element In contrast to MKP5, removal of the KBD domain from MKP7 does not drastically affect enzyme catalysis, and the kinetic parameters of MKP7-CD for p38α substrate are very similar to those for JNK1 substrate. DISCUSS +52 56 MKP7 protein In contrast to MKP5, removal of the KBD domain from MKP7 does not drastically affect enzyme catalysis, and the kinetic parameters of MKP7-CD for p38α substrate are very similar to those for JNK1 substrate. DISCUSS +133 137 MKP7 protein In contrast to MKP5, removal of the KBD domain from MKP7 does not drastically affect enzyme catalysis, and the kinetic parameters of MKP7-CD for p38α substrate are very similar to those for JNK1 substrate. DISCUSS +138 140 CD structure_element In contrast to MKP5, removal of the KBD domain from MKP7 does not drastically affect enzyme catalysis, and the kinetic parameters of MKP7-CD for p38α substrate are very similar to those for JNK1 substrate. DISCUSS +145 149 p38α protein In contrast to MKP5, removal of the KBD domain from MKP7 does not drastically affect enzyme catalysis, and the kinetic parameters of MKP7-CD for p38α substrate are very similar to those for JNK1 substrate. DISCUSS +190 194 JNK1 protein In contrast to MKP5, removal of the KBD domain from MKP7 does not drastically affect enzyme catalysis, and the kinetic parameters of MKP7-CD for p38α substrate are very similar to those for JNK1 substrate. DISCUSS +43 47 MKP7 protein Taken together, these results suggest that MKP7 utilizes a bipartite recognition mechanism to achieve the efficiency and fidelity of p38α signalling. DISCUSS +133 137 p38α protein Taken together, these results suggest that MKP7 utilizes a bipartite recognition mechanism to achieve the efficiency and fidelity of p38α signalling. DISCUSS +4 8 MKP7 protein The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. DISCUSS +9 12 KBD structure_element The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. DISCUSS +26 32 D-site site The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. DISCUSS +65 69 p38α protein The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. DISCUSS +70 86 catalytic pocket site The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. DISCUSS +149 153 MKP7 protein The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. DISCUSS +154 156 CD structure_element The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. DISCUSS +170 174 p38α protein The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. DISCUSS +216 231 activation loop structure_element The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. DISCUSS +317 321 MKP7 protein The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. DISCUSS +322 333 active site site The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. DISCUSS +377 381 p38α protein The MKP7-KBD docks to the D-site located on the back side of the p38α catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38α structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38α substrate for efficient dephosphorylation. DISCUSS +29 35 D-site site In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. DISCUSS +41 45 MAPK protein_type In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. DISCUSS +46 50 ERK2 protein In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. DISCUSS +62 81 second binding site site In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. DISCUSS +130 142 phosphatases protein_type In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. DISCUSS +148 170 FXF-motif-binding site site In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. DISCUSS +184 190 F-site site In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. DISCUSS +212 218 active protein_state In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. DISCUSS +219 223 ERK2 protein In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. DISCUSS +232 239 D-motif structure_element In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. DISCUSS +272 277 MAPKs protein_type In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs. DISCUSS +5 21 hydrophobic site site This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). DISCUSS +46 84 changes in deuterium exchange profiles evidence This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). DISCUSS +102 116 MAPK insertion structure_element This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). DISCUSS +121 126 helix structure_element This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). DISCUSS +127 129 αG structure_element This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). DISCUSS +181 185 JNK1 protein This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). DISCUSS +201 205 MKP7 protein This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). DISCUSS +206 208 CD structure_element This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). DISCUSS +261 265 ERK2 protein This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). DISCUSS +323 331 DEF-site site This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). DISCUSS +357 361 ERK2 protein This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix αG. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d). DISCUSS +0 4 MKP3 protein MKP3 is highly specific in dephosphorylating and inactivating ERK2, and the phosphatase activity of the MKP3-catalysed pNPP reaction can be markedly increased in the presence of ERK2 (refs). DISCUSS +62 66 ERK2 protein MKP3 is highly specific in dephosphorylating and inactivating ERK2, and the phosphatase activity of the MKP3-catalysed pNPP reaction can be markedly increased in the presence of ERK2 (refs). DISCUSS +104 108 MKP3 protein MKP3 is highly specific in dephosphorylating and inactivating ERK2, and the phosphatase activity of the MKP3-catalysed pNPP reaction can be markedly increased in the presence of ERK2 (refs). DISCUSS +119 123 pNPP chemical MKP3 is highly specific in dephosphorylating and inactivating ERK2, and the phosphatase activity of the MKP3-catalysed pNPP reaction can be markedly increased in the presence of ERK2 (refs). DISCUSS +166 177 presence of protein_state MKP3 is highly specific in dephosphorylating and inactivating ERK2, and the phosphatase activity of the MKP3-catalysed pNPP reaction can be markedly increased in the presence of ERK2 (refs). DISCUSS +178 182 ERK2 protein MKP3 is highly specific in dephosphorylating and inactivating ERK2, and the phosphatase activity of the MKP3-catalysed pNPP reaction can be markedly increased in the presence of ERK2 (refs). DISCUSS +0 18 Sequence alignment experimental_method Sequence alignment of all MKPs reveals a high degree of conservation of residues surrounding the interacting region observed in JNK1–MKP7-CD complex (Supplementary Fig. 5). DISCUSS +26 30 MKPs protein_type Sequence alignment of all MKPs reveals a high degree of conservation of residues surrounding the interacting region observed in JNK1–MKP7-CD complex (Supplementary Fig. 5). DISCUSS +97 115 interacting region site Sequence alignment of all MKPs reveals a high degree of conservation of residues surrounding the interacting region observed in JNK1–MKP7-CD complex (Supplementary Fig. 5). DISCUSS +128 140 JNK1–MKP7-CD complex_assembly Sequence alignment of all MKPs reveals a high degree of conservation of residues surrounding the interacting region observed in JNK1–MKP7-CD complex (Supplementary Fig. 5). DISCUSS +48 64 catalytic domain structure_element Therefore, it is tempting to speculate that the catalytic domain of MKP3 may bind to ERK2 in a manner analogous to the way by which MKP7-CD binds to JNK1. DISCUSS +68 72 MKP3 protein Therefore, it is tempting to speculate that the catalytic domain of MKP3 may bind to ERK2 in a manner analogous to the way by which MKP7-CD binds to JNK1. DISCUSS +85 89 ERK2 protein Therefore, it is tempting to speculate that the catalytic domain of MKP3 may bind to ERK2 in a manner analogous to the way by which MKP7-CD binds to JNK1. DISCUSS +132 136 MKP7 protein Therefore, it is tempting to speculate that the catalytic domain of MKP3 may bind to ERK2 in a manner analogous to the way by which MKP7-CD binds to JNK1. DISCUSS +137 139 CD structure_element Therefore, it is tempting to speculate that the catalytic domain of MKP3 may bind to ERK2 in a manner analogous to the way by which MKP7-CD binds to JNK1. DISCUSS +149 153 JNK1 protein Therefore, it is tempting to speculate that the catalytic domain of MKP3 may bind to ERK2 in a manner analogous to the way by which MKP7-CD binds to JNK1. DISCUSS +67 71 ERK2 protein A comprehensive examination of the molecular basis of the specific ERK2 recognition by MKP3 is underway. DISCUSS +87 91 MKP3 protein A comprehensive examination of the molecular basis of the specific ERK2 recognition by MKP3 is underway. DISCUSS +98 110 JNK1–MKP7-CD complex_assembly The ongoing work demonstrates that although the overall interaction modes are similar between the JNK1–MKP7-CD and ERK2–MKP3-CD complexes, the ERK2–MKP3-CD interaction is less extensive and helix α4 from MKP3-CD does not interact directly with ERK2. DISCUSS +115 127 ERK2–MKP3-CD complex_assembly The ongoing work demonstrates that although the overall interaction modes are similar between the JNK1–MKP7-CD and ERK2–MKP3-CD complexes, the ERK2–MKP3-CD interaction is less extensive and helix α4 from MKP3-CD does not interact directly with ERK2. DISCUSS +143 155 ERK2–MKP3-CD complex_assembly The ongoing work demonstrates that although the overall interaction modes are similar between the JNK1–MKP7-CD and ERK2–MKP3-CD complexes, the ERK2–MKP3-CD interaction is less extensive and helix α4 from MKP3-CD does not interact directly with ERK2. DISCUSS +190 195 helix structure_element The ongoing work demonstrates that although the overall interaction modes are similar between the JNK1–MKP7-CD and ERK2–MKP3-CD complexes, the ERK2–MKP3-CD interaction is less extensive and helix α4 from MKP3-CD does not interact directly with ERK2. DISCUSS +196 198 α4 structure_element The ongoing work demonstrates that although the overall interaction modes are similar between the JNK1–MKP7-CD and ERK2–MKP3-CD complexes, the ERK2–MKP3-CD interaction is less extensive and helix α4 from MKP3-CD does not interact directly with ERK2. DISCUSS +204 208 MKP3 protein The ongoing work demonstrates that although the overall interaction modes are similar between the JNK1–MKP7-CD and ERK2–MKP3-CD complexes, the ERK2–MKP3-CD interaction is less extensive and helix α4 from MKP3-CD does not interact directly with ERK2. DISCUSS +209 211 CD structure_element The ongoing work demonstrates that although the overall interaction modes are similar between the JNK1–MKP7-CD and ERK2–MKP3-CD complexes, the ERK2–MKP3-CD interaction is less extensive and helix α4 from MKP3-CD does not interact directly with ERK2. DISCUSS +244 248 ERK2 protein The ongoing work demonstrates that although the overall interaction modes are similar between the JNK1–MKP7-CD and ERK2–MKP3-CD complexes, the ERK2–MKP3-CD interaction is less extensive and helix α4 from MKP3-CD does not interact directly with ERK2. DISCUSS +4 13 FXF-motif structure_element The FXF-motif-mediated interaction is critical for both pERK2 inactivation and ERK2-induced MKP3 activation (manuscript in preparation). DISCUSS +56 57 p protein_state The FXF-motif-mediated interaction is critical for both pERK2 inactivation and ERK2-induced MKP3 activation (manuscript in preparation). DISCUSS +57 61 ERK2 protein The FXF-motif-mediated interaction is critical for both pERK2 inactivation and ERK2-induced MKP3 activation (manuscript in preparation). DISCUSS +79 83 ERK2 protein The FXF-motif-mediated interaction is critical for both pERK2 inactivation and ERK2-induced MKP3 activation (manuscript in preparation). DISCUSS +92 96 MKP3 protein The FXF-motif-mediated interaction is critical for both pERK2 inactivation and ERK2-induced MKP3 activation (manuscript in preparation). DISCUSS +33 42 structure evidence In summary, we have resolved the structure of JNK1 in complex with the catalytic domain of MKP7. DISCUSS +46 50 JNK1 protein In summary, we have resolved the structure of JNK1 in complex with the catalytic domain of MKP7. DISCUSS +51 66 in complex with protein_state In summary, we have resolved the structure of JNK1 in complex with the catalytic domain of MKP7. DISCUSS +71 87 catalytic domain structure_element In summary, we have resolved the structure of JNK1 in complex with the catalytic domain of MKP7. DISCUSS +91 95 MKP7 protein In summary, we have resolved the structure of JNK1 in complex with the catalytic domain of MKP7. DISCUSS +5 14 structure evidence This structure reveals an FXF-docking interaction mode between MAPK and MKP. DISCUSS +26 54 FXF-docking interaction mode site This structure reveals an FXF-docking interaction mode between MAPK and MKP. DISCUSS +63 67 MAPK protein_type This structure reveals an FXF-docking interaction mode between MAPK and MKP. DISCUSS +72 75 MKP protein_type This structure reveals an FXF-docking interaction mode between MAPK and MKP. DISCUSS +13 41 biochemical characterization experimental_method Results from biochemical characterization of the Phe285 and Phe287 MKP7 mutants combined with structural information support the conclusion that the two Phe residues serve different roles in the catalytic reaction. DISCUSS +49 55 Phe285 residue_name_number Results from biochemical characterization of the Phe285 and Phe287 MKP7 mutants combined with structural information support the conclusion that the two Phe residues serve different roles in the catalytic reaction. DISCUSS +60 66 Phe287 residue_name_number Results from biochemical characterization of the Phe285 and Phe287 MKP7 mutants combined with structural information support the conclusion that the two Phe residues serve different roles in the catalytic reaction. DISCUSS +67 71 MKP7 protein Results from biochemical characterization of the Phe285 and Phe287 MKP7 mutants combined with structural information support the conclusion that the two Phe residues serve different roles in the catalytic reaction. DISCUSS +72 79 mutants protein_state Results from biochemical characterization of the Phe285 and Phe287 MKP7 mutants combined with structural information support the conclusion that the two Phe residues serve different roles in the catalytic reaction. DISCUSS +94 116 structural information evidence Results from biochemical characterization of the Phe285 and Phe287 MKP7 mutants combined with structural information support the conclusion that the two Phe residues serve different roles in the catalytic reaction. DISCUSS +153 156 Phe residue_name Results from biochemical characterization of the Phe285 and Phe287 MKP7 mutants combined with structural information support the conclusion that the two Phe residues serve different roles in the catalytic reaction. DISCUSS +0 6 Phe285 residue_name_number Phe285 is essential for JNK1 substrate binding, whereas Phe287 plays a role for the precise alignment of active-site residues, which are important for transition-state stabilization. DISCUSS +24 28 JNK1 protein Phe285 is essential for JNK1 substrate binding, whereas Phe287 plays a role for the precise alignment of active-site residues, which are important for transition-state stabilization. DISCUSS +56 62 Phe287 residue_name_number Phe285 is essential for JNK1 substrate binding, whereas Phe287 plays a role for the precise alignment of active-site residues, which are important for transition-state stabilization. DISCUSS +105 125 active-site residues site Phe285 is essential for JNK1 substrate binding, whereas Phe287 plays a role for the precise alignment of active-site residues, which are important for transition-state stabilization. DISCUSS +22 36 FXF-type motif structure_element This newly identified FXF-type motif is present in all MKPs, except that the residue at the first position in MKP5 is an equivalent hydrophobic leucine residue (see also Fig. 7f,g), suggesting that these two Phe residues would play a similar role in facilitating substrate recognition and catalysis, respectively. DISCUSS +55 59 MKPs protein_type This newly identified FXF-type motif is present in all MKPs, except that the residue at the first position in MKP5 is an equivalent hydrophobic leucine residue (see also Fig. 7f,g), suggesting that these two Phe residues would play a similar role in facilitating substrate recognition and catalysis, respectively. DISCUSS +110 114 MKP5 protein This newly identified FXF-type motif is present in all MKPs, except that the residue at the first position in MKP5 is an equivalent hydrophobic leucine residue (see also Fig. 7f,g), suggesting that these two Phe residues would play a similar role in facilitating substrate recognition and catalysis, respectively. DISCUSS +144 151 leucine residue_name This newly identified FXF-type motif is present in all MKPs, except that the residue at the first position in MKP5 is an equivalent hydrophobic leucine residue (see also Fig. 7f,g), suggesting that these two Phe residues would play a similar role in facilitating substrate recognition and catalysis, respectively. DISCUSS +208 211 Phe residue_name This newly identified FXF-type motif is present in all MKPs, except that the residue at the first position in MKP5 is an equivalent hydrophobic leucine residue (see also Fig. 7f,g), suggesting that these two Phe residues would play a similar role in facilitating substrate recognition and catalysis, respectively. DISCUSS +24 27 MKP protein_type An important feature of MKP–JNK1 interactions is that MKP7 or MKP5 only interact with the F-site of JNK1. DISCUSS +28 32 JNK1 protein An important feature of MKP–JNK1 interactions is that MKP7 or MKP5 only interact with the F-site of JNK1. DISCUSS +54 58 MKP7 protein An important feature of MKP–JNK1 interactions is that MKP7 or MKP5 only interact with the F-site of JNK1. DISCUSS +62 66 MKP5 protein An important feature of MKP–JNK1 interactions is that MKP7 or MKP5 only interact with the F-site of JNK1. DISCUSS +90 96 F-site site An important feature of MKP–JNK1 interactions is that MKP7 or MKP5 only interact with the F-site of JNK1. DISCUSS +100 104 JNK1 protein An important feature of MKP–JNK1 interactions is that MKP7 or MKP5 only interact with the F-site of JNK1. DISCUSS +33 37 JNK1 protein One possible explanation is that JNK1 needs to use the D-site to interact with JIP-1, a scaffold protein for JNK signalling. DISCUSS +55 61 D-site site One possible explanation is that JNK1 needs to use the D-site to interact with JIP-1, a scaffold protein for JNK signalling. DISCUSS +79 84 JIP-1 protein One possible explanation is that JNK1 needs to use the D-site to interact with JIP-1, a scaffold protein for JNK signalling. DISCUSS +109 112 JNK protein_type One possible explanation is that JNK1 needs to use the D-site to interact with JIP-1, a scaffold protein for JNK signalling. DISCUSS +15 33 JNK-binding domain structure_element The N-terminal JNK-binding domain of JIP-1 interacts with the D-site on JNK and this interaction is required for JIP-1-mediated enhancement of JNK activation. DISCUSS +37 42 JIP-1 protein The N-terminal JNK-binding domain of JIP-1 interacts with the D-site on JNK and this interaction is required for JIP-1-mediated enhancement of JNK activation. DISCUSS +62 68 D-site site The N-terminal JNK-binding domain of JIP-1 interacts with the D-site on JNK and this interaction is required for JIP-1-mediated enhancement of JNK activation. DISCUSS +72 75 JNK protein_type The N-terminal JNK-binding domain of JIP-1 interacts with the D-site on JNK and this interaction is required for JIP-1-mediated enhancement of JNK activation. DISCUSS +113 118 JIP-1 protein The N-terminal JNK-binding domain of JIP-1 interacts with the D-site on JNK and this interaction is required for JIP-1-mediated enhancement of JNK activation. DISCUSS +143 146 JNK protein_type The N-terminal JNK-binding domain of JIP-1 interacts with the D-site on JNK and this interaction is required for JIP-1-mediated enhancement of JNK activation. DISCUSS +13 18 JIP-1 protein In addition, JIP-1 can also associate with MKP7 via the C-terminal region of MKP7 (ref.). DISCUSS +43 47 MKP7 protein In addition, JIP-1 can also associate with MKP7 via the C-terminal region of MKP7 (ref.). DISCUSS +56 73 C-terminal region structure_element In addition, JIP-1 can also associate with MKP7 via the C-terminal region of MKP7 (ref.). DISCUSS +77 81 MKP7 protein In addition, JIP-1 can also associate with MKP7 via the C-terminal region of MKP7 (ref.). DISCUSS +5 9 MKP7 protein When MKP7 is bound to JIP-1, it reduces JNK activation, leading to reduced phosphorylation of the JNK target c-Jun. DISCUSS +13 21 bound to protein_state When MKP7 is bound to JIP-1, it reduces JNK activation, leading to reduced phosphorylation of the JNK target c-Jun. DISCUSS +22 27 JIP-1 protein When MKP7 is bound to JIP-1, it reduces JNK activation, leading to reduced phosphorylation of the JNK target c-Jun. DISCUSS +40 43 JNK protein_type When MKP7 is bound to JIP-1, it reduces JNK activation, leading to reduced phosphorylation of the JNK target c-Jun. DISCUSS +98 101 JNK protein_type When MKP7 is bound to JIP-1, it reduces JNK activation, leading to reduced phosphorylation of the JNK target c-Jun. DISCUSS +109 114 c-Jun protein_type When MKP7 is bound to JIP-1, it reduces JNK activation, leading to reduced phosphorylation of the JNK target c-Jun. DISCUSS +10 41 biochemical and structural data evidence Thus, our biochemical and structural data allow us to present a model for the JNK1–JIP-1–MKP7 ternary complex and provide an important insight into the assembly and function of JNK signalling modules (Supplementary Fig. 6). DISCUSS +78 93 JNK1–JIP-1–MKP7 complex_assembly Thus, our biochemical and structural data allow us to present a model for the JNK1–JIP-1–MKP7 ternary complex and provide an important insight into the assembly and function of JNK signalling modules (Supplementary Fig. 6). DISCUSS +177 180 JNK protein_type Thus, our biochemical and structural data allow us to present a model for the JNK1–JIP-1–MKP7 ternary complex and provide an important insight into the assembly and function of JNK signalling modules (Supplementary Fig. 6). DISCUSS +7 17 structures evidence Domain structures of ten human MKPs and the atypical VHR. FIG +25 30 human species Domain structures of ten human MKPs and the atypical VHR. FIG +31 35 MKPs protein_type Domain structures of ten human MKPs and the atypical VHR. FIG +53 56 VHR protein Domain structures of ten human MKPs and the atypical VHR. FIG +45 54 structure evidence On the basis of sequence similarity, protein structure, substrate specificity and subcellular localization, the ten members of MKP family can be divided into three groups. FIG +127 137 MKP family protein_type On the basis of sequence similarity, protein structure, substrate specificity and subcellular localization, the ten members of MKP family can be divided into three groups. FIG +30 34 MKP1 protein The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs. FIG +36 40 MKP2 protein The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs. FIG +42 46 PAC1 protein The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs. FIG +51 55 hVH3 protein The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs. FIG +67 76 inducible protein_state The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs. FIG +77 97 nuclear phosphatases protein_type The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs. FIG +122 125 ERK protein_type The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs. FIG +131 134 JNK protein_type The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs. FIG +136 139 p38 protein_type The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs. FIG +141 146 MAPKs protein_type The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs. FIG +30 34 MKP3 protein The second subfamily contains MKP3, MKP4 and MKPX, which are cytoplasmic ERK-specific MKPs. FIG +36 40 MKP4 protein The second subfamily contains MKP3, MKP4 and MKPX, which are cytoplasmic ERK-specific MKPs. FIG +45 49 MKPX protein The second subfamily contains MKP3, MKP4 and MKPX, which are cytoplasmic ERK-specific MKPs. FIG +73 90 ERK-specific MKPs protein_type The second subfamily contains MKP3, MKP4 and MKPX, which are cytoplasmic ERK-specific MKPs. FIG +30 34 MKP5 protein The third subfamily comprises MKP5, MKP7 and hVH5, which were located in both nucleus and cytoplasm, and selectively inactivate JNK and p38. FIG +36 40 MKP7 protein The third subfamily comprises MKP5, MKP7 and hVH5, which were located in both nucleus and cytoplasm, and selectively inactivate JNK and p38. FIG +45 49 hVH5 protein The third subfamily comprises MKP5, MKP7 and hVH5, which were located in both nucleus and cytoplasm, and selectively inactivate JNK and p38. FIG +128 131 JNK protein_type The third subfamily comprises MKP5, MKP7 and hVH5, which were located in both nucleus and cytoplasm, and selectively inactivate JNK and p38. FIG +136 139 p38 protein_type The third subfamily comprises MKP5, MKP7 and hVH5, which were located in both nucleus and cytoplasm, and selectively inactivate JNK and p38. FIG +4 8 MKPs protein_type All MKPs contain both the CD and KBD domains, whereas VHR, an atypical MKP, only contains a highly conserved catalytic domain. FIG +26 28 CD structure_element All MKPs contain both the CD and KBD domains, whereas VHR, an atypical MKP, only contains a highly conserved catalytic domain. FIG +33 36 KBD structure_element All MKPs contain both the CD and KBD domains, whereas VHR, an atypical MKP, only contains a highly conserved catalytic domain. FIG +54 57 VHR protein All MKPs contain both the CD and KBD domains, whereas VHR, an atypical MKP, only contains a highly conserved catalytic domain. FIG +71 74 MKP protein_type All MKPs contain both the CD and KBD domains, whereas VHR, an atypical MKP, only contains a highly conserved catalytic domain. FIG +92 108 highly conserved protein_state All MKPs contain both the CD and KBD domains, whereas VHR, an atypical MKP, only contains a highly conserved catalytic domain. FIG +109 125 catalytic domain structure_element All MKPs contain both the CD and KBD domains, whereas VHR, an atypical MKP, only contains a highly conserved catalytic domain. FIG +19 21 CD structure_element In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs. FIG +26 29 KBD structure_element In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs. FIG +31 35 MKP7 protein In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs. FIG +59 76 C-terminal region structure_element In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs. FIG +91 94 NES structure_element In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs. FIG +96 99 NLS structure_element In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs. FIG +104 115 PEST motifs structure_element In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs. FIG +188 192 MKP7 protein In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs. FIG +200 205 MAPKs protein_type In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs. FIG +0 3 NES structure_element NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines. FIG +5 26 nuclear export signal structure_element NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines. FIG +28 31 NLS structure_element NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines. FIG +33 60 nuclear localization signal structure_element NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines. FIG +62 66 PEST structure_element NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines. FIG +68 92 C-terminal sequence rich structure_element NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines. FIG +96 104 prolines residue_name NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines. FIG +106 116 glutamates residue_name NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines. FIG +118 125 serines residue_name NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines. FIG +130 140 threonines residue_name NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines. FIG +0 4 MKP7 protein MKP7-CD is crucial for JNK1 binding and enzyme catalysis. FIG +5 7 CD structure_element MKP7-CD is crucial for JNK1 binding and enzyme catalysis. FIG +23 27 JNK1 protein MKP7-CD is crucial for JNK1 binding and enzyme catalysis. FIG +27 32 human species (a) Domain organization of human MKP7 and JNK1. FIG +33 37 MKP7 protein (a) Domain organization of human MKP7 and JNK1. FIG +42 46 JNK1 protein (a) Domain organization of human MKP7 and JNK1. FIG +4 7 KBD structure_element The KBD and CD of MKP7 are shown in green and blue, and the N-lobe and C-lobe of JNK1 are coloured in lemon and yellow, respectively. FIG +12 14 CD structure_element The KBD and CD of MKP7 are shown in green and blue, and the N-lobe and C-lobe of JNK1 are coloured in lemon and yellow, respectively. FIG +18 22 MKP7 protein The KBD and CD of MKP7 are shown in green and blue, and the N-lobe and C-lobe of JNK1 are coloured in lemon and yellow, respectively. FIG +60 66 N-lobe structure_element The KBD and CD of MKP7 are shown in green and blue, and the N-lobe and C-lobe of JNK1 are coloured in lemon and yellow, respectively. FIG +71 77 C-lobe structure_element The KBD and CD of MKP7 are shown in green and blue, and the N-lobe and C-lobe of JNK1 are coloured in lemon and yellow, respectively. FIG +81 85 JNK1 protein The KBD and CD of MKP7 are shown in green and blue, and the N-lobe and C-lobe of JNK1 are coloured in lemon and yellow, respectively. FIG +87 112 Plots of initial velocity evidence The colour scheme is the same in the following figures unless indicated otherwise. (b) Plots of initial velocity of the MKP7-catalysed reaction versus phospho-JNK1 concentration. FIG +120 124 MKP7 protein The colour scheme is the same in the following figures unless indicated otherwise. (b) Plots of initial velocity of the MKP7-catalysed reaction versus phospho-JNK1 concentration. FIG +151 158 phospho ptm The colour scheme is the same in the following figures unless indicated otherwise. (b) Plots of initial velocity of the MKP7-catalysed reaction versus phospho-JNK1 concentration. FIG +159 163 JNK1 protein The colour scheme is the same in the following figures unless indicated otherwise. (b) Plots of initial velocity of the MKP7-catalysed reaction versus phospho-JNK1 concentration. FIG +36 59 Gel filtration analysis experimental_method The error bars represent s.e.m. (c) Gel filtration analysis for interaction of JNK1 with MKP7-CD and MKP7-KBD. FIG +79 83 JNK1 protein The error bars represent s.e.m. (c) Gel filtration analysis for interaction of JNK1 with MKP7-CD and MKP7-KBD. FIG +89 93 MKP7 protein The error bars represent s.e.m. (c) Gel filtration analysis for interaction of JNK1 with MKP7-CD and MKP7-KBD. FIG +94 96 CD structure_element The error bars represent s.e.m. (c) Gel filtration analysis for interaction of JNK1 with MKP7-CD and MKP7-KBD. FIG +101 105 MKP7 protein The error bars represent s.e.m. (c) Gel filtration analysis for interaction of JNK1 with MKP7-CD and MKP7-KBD. FIG +106 109 KBD structure_element The error bars represent s.e.m. (c) Gel filtration analysis for interaction of JNK1 with MKP7-CD and MKP7-KBD. FIG +4 32 GST-mediated pull-down assay experimental_method (d) GST-mediated pull-down assay for interaction of JNK1 with MKP7-CD and MKP7-KBD. FIG +52 56 JNK1 protein (d) GST-mediated pull-down assay for interaction of JNK1 with MKP7-CD and MKP7-KBD. FIG +62 66 MKP7 protein (d) GST-mediated pull-down assay for interaction of JNK1 with MKP7-CD and MKP7-KBD. FIG +67 69 CD structure_element (d) GST-mediated pull-down assay for interaction of JNK1 with MKP7-CD and MKP7-KBD. FIG +74 78 MKP7 protein (d) GST-mediated pull-down assay for interaction of JNK1 with MKP7-CD and MKP7-KBD. FIG +79 82 KBD structure_element (d) GST-mediated pull-down assay for interaction of JNK1 with MKP7-CD and MKP7-KBD. FIG +33 43 affinities evidence The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. FIG +47 51 MKP7 protein The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. FIG +52 54 CD structure_element The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. FIG +59 63 MKP7 protein The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. FIG +64 67 KBD structure_element The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. FIG +71 75 JNK1 protein The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. FIG +86 94 affinity evidence The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. FIG +98 102 MKP7 protein The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. FIG +103 105 CD structure_element The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. FIG +174 178 MKP7 protein The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. FIG +183 187 JNK1 protein The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. FIG +194 214 GST pull-down assays experimental_method The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. FIG +23 27 MKP7 protein The protein amounts of MKP7 used are shown at the bottom. FIG +0 9 Structure evidence Structure of JNK1 in complex with MKP7-CD. FIG +13 17 JNK1 protein Structure of JNK1 in complex with MKP7-CD. FIG +18 33 in complex with protein_state Structure of JNK1 in complex with MKP7-CD. FIG +34 38 MKP7 protein Structure of JNK1 in complex with MKP7-CD. FIG +39 41 CD structure_element Structure of JNK1 in complex with MKP7-CD. FIG +22 34 JNK1–MKP7-CD complex_assembly (a) Ribbon diagram of JNK1–MKP7-CD complex in two views related by a 45° rotation around a vertical axis. (b) Structure of MKP7-CD with its active site highlight in cyan. FIG +110 119 Structure evidence (a) Ribbon diagram of JNK1–MKP7-CD complex in two views related by a 45° rotation around a vertical axis. (b) Structure of MKP7-CD with its active site highlight in cyan. FIG +123 127 MKP7 protein (a) Ribbon diagram of JNK1–MKP7-CD complex in two views related by a 45° rotation around a vertical axis. (b) Structure of MKP7-CD with its active site highlight in cyan. FIG +128 130 CD structure_element (a) Ribbon diagram of JNK1–MKP7-CD complex in two views related by a 45° rotation around a vertical axis. (b) Structure of MKP7-CD with its active site highlight in cyan. FIG +140 151 active site site (a) Ribbon diagram of JNK1–MKP7-CD complex in two views related by a 45° rotation around a vertical axis. (b) Structure of MKP7-CD with its active site highlight in cyan. FIG +4 19 2Fo−Fc omit map evidence The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). FIG +48 54 P-loop structure_element The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). FIG +58 62 MKP7 protein The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). FIG +63 65 CD structure_element The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). FIG +94 103 Structure evidence The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). FIG +107 110 VHR protein The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). FIG +120 131 active site site The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). FIG +185 204 JNK1–MKP7 interface site The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). FIG +240 244 JNK1 protein The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). FIG +258 262 MKP7 protein The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). FIG +263 265 CD structure_element The 2Fo−Fc omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). FIG +4 8 JNK1 protein The JNK1 is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). FIG +4 24 Interaction networks site (e) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. FIG +42 49 helices structure_element (e) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. FIG +50 52 α4 structure_element (e) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. FIG +57 59 α5 structure_element (e) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. FIG +65 69 MKP7 protein (e) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. FIG +70 72 CD structure_element (e) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. FIG +78 80 αG structure_element (e) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. FIG +85 90 α2L14 structure_element (e) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. FIG +94 98 JNK1 protein (e) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. FIG +0 4 MKP7 protein MKP7-CD is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). FIG +5 7 CD structure_element MKP7-CD is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). FIG +28 46 polar interactions bond_interaction Blue dashed lines represent polar interactions. FIG +28 46 polar interactions bond_interaction Blue dashed lines represent polar interactions. FIG +8 23 2Fo−Fc omit map evidence (f) The 2Fo−Fc omit map (contoured at 1.5σ) clearly shows electron density for the 285FNFL288 segment of MKP7-CD. FIG +58 74 electron density evidence (f) The 2Fo−Fc omit map (contoured at 1.5σ) clearly shows electron density for the 285FNFL288 segment of MKP7-CD. FIG +83 101 285FNFL288 segment structure_element (f) The 2Fo−Fc omit map (contoured at 1.5σ) clearly shows electron density for the 285FNFL288 segment of MKP7-CD. FIG +105 109 MKP7 protein (f) The 2Fo−Fc omit map (contoured at 1.5σ) clearly shows electron density for the 285FNFL288 segment of MKP7-CD. FIG +110 112 CD structure_element (f) The 2Fo−Fc omit map (contoured at 1.5σ) clearly shows electron density for the 285FNFL288 segment of MKP7-CD. FIG +0 19 Mutational analysis experimental_method Mutational analysis on interactions between MKP7-CD and JNK1. FIG +44 48 MKP7 protein Mutational analysis on interactions between MKP7-CD and JNK1. FIG +49 51 CD structure_element Mutational analysis on interactions between MKP7-CD and JNK1. FIG +56 60 JNK1 protein Mutational analysis on interactions between MKP7-CD and JNK1. FIG +28 32 MKP7 protein (a) Effects of mutations in MKP7-CD on the JNK1 dephosphorylation (mean±s.e.m., n=3). FIG +33 35 CD structure_element (a) Effects of mutations in MKP7-CD on the JNK1 dephosphorylation (mean±s.e.m., n=3). FIG +43 47 JNK1 protein (a) Effects of mutations in MKP7-CD on the JNK1 dephosphorylation (mean±s.e.m., n=3). FIG +48 65 dephosphorylation ptm (a) Effects of mutations in MKP7-CD on the JNK1 dephosphorylation (mean±s.e.m., n=3). FIG +21 57 hydrophobic and hydrophilic contacts bond_interaction Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. (b) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D. FIG +106 129 Gel filtration analysis experimental_method Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. (b) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D. FIG +149 153 JNK1 protein Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. (b) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D. FIG +159 163 MKP7 protein Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. (b) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D. FIG +164 166 CD structure_element Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. (b) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D. FIG +167 173 mutant protein_state Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. (b) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D. FIG +174 179 F285D mutant Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. (b) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D. FIG +0 6 Mutant protein_state Mutant F285D and JNK1 were eluted as monomers, with the molecular masses of ∼17 and 44 kDa, respectively. FIG +7 12 F285D mutant Mutant F285D and JNK1 were eluted as monomers, with the molecular masses of ∼17 and 44 kDa, respectively. FIG +17 21 JNK1 protein Mutant F285D and JNK1 were eluted as monomers, with the molecular masses of ∼17 and 44 kDa, respectively. FIG +37 45 monomers oligomeric_state Mutant F285D and JNK1 were eluted as monomers, with the molecular masses of ∼17 and 44 kDa, respectively. FIG +28 37 wild-type protein_state However, in contrast to the wild-type MKP7-CD, mutant F285D did not co-migrate with JNK1. FIG +38 42 MKP7 protein However, in contrast to the wild-type MKP7-CD, mutant F285D did not co-migrate with JNK1. FIG +43 45 CD structure_element However, in contrast to the wild-type MKP7-CD, mutant F285D did not co-migrate with JNK1. FIG +47 53 mutant protein_state However, in contrast to the wild-type MKP7-CD, mutant F285D did not co-migrate with JNK1. FIG +54 59 F285D mutant However, in contrast to the wild-type MKP7-CD, mutant F285D did not co-migrate with JNK1. FIG +84 88 JNK1 protein However, in contrast to the wild-type MKP7-CD, mutant F285D did not co-migrate with JNK1. FIG +4 20 Pull-down assays experimental_method (c) Pull-down assays of MKP7-CD by GST-tagged JNK1 mutants. FIG +24 28 MKP7 protein (c) Pull-down assays of MKP7-CD by GST-tagged JNK1 mutants. FIG +29 31 CD structure_element (c) Pull-down assays of MKP7-CD by GST-tagged JNK1 mutants. FIG +35 45 GST-tagged protein_state (c) Pull-down assays of MKP7-CD by GST-tagged JNK1 mutants. FIG +46 50 JNK1 protein (c) Pull-down assays of MKP7-CD by GST-tagged JNK1 mutants. FIG +51 58 mutants protein_state (c) Pull-down assays of MKP7-CD by GST-tagged JNK1 mutants. FIG +33 43 affinities evidence The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +47 51 MKP7 protein The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +52 54 CD structure_element The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +58 62 JNK1 protein The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +63 70 mutants protein_state The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +81 89 affinity evidence The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +93 102 wild-type protein_state The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +103 107 JNK1 protein The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +176 180 MKP7 protein The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +181 183 CD structure_element The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +188 192 JNK1 protein The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +193 200 mutants protein_state The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +207 227 GST pull-down assays experimental_method The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. FIG +23 27 MKP7 protein The protein amounts of MKP7-CD used are shown at the bottom. (d) Circular dichroism spectra for MKP7-CD wild type and mutants. FIG +28 30 CD structure_element The protein amounts of MKP7-CD used are shown at the bottom. (d) Circular dichroism spectra for MKP7-CD wild type and mutants. FIG +65 83 Circular dichroism experimental_method The protein amounts of MKP7-CD used are shown at the bottom. (d) Circular dichroism spectra for MKP7-CD wild type and mutants. FIG +84 91 spectra evidence The protein amounts of MKP7-CD used are shown at the bottom. (d) Circular dichroism spectra for MKP7-CD wild type and mutants. FIG +96 100 MKP7 protein The protein amounts of MKP7-CD used are shown at the bottom. (d) Circular dichroism spectra for MKP7-CD wild type and mutants. FIG +101 103 CD structure_element The protein amounts of MKP7-CD used are shown at the bottom. (d) Circular dichroism spectra for MKP7-CD wild type and mutants. FIG +104 113 wild type protein_state The protein amounts of MKP7-CD used are shown at the bottom. (d) Circular dichroism spectra for MKP7-CD wild type and mutants. FIG +118 125 mutants protein_state The protein amounts of MKP7-CD used are shown at the bottom. (d) Circular dichroism spectra for MKP7-CD wild type and mutants. FIG +48 66 Circular dichroism experimental_method Measurements were averaged for three scans. (e) Circular dichroism spectra for JNK1 wild type and mutants. FIG +67 74 spectra evidence Measurements were averaged for three scans. (e) Circular dichroism spectra for JNK1 wild type and mutants. FIG +79 83 JNK1 protein Measurements were averaged for three scans. (e) Circular dichroism spectra for JNK1 wild type and mutants. FIG +84 93 wild type protein_state Measurements were averaged for three scans. (e) Circular dichroism spectra for JNK1 wild type and mutants. FIG +98 105 mutants protein_state Measurements were averaged for three scans. (e) Circular dichroism spectra for JNK1 wild type and mutants. FIG +15 24 mutations experimental_method (f) Effects of mutations in MKP7-CD on the pNPP hydrolysis reaction (mean±s.e.m., n=3). FIG +28 32 MKP7 protein (f) Effects of mutations in MKP7-CD on the pNPP hydrolysis reaction (mean±s.e.m., n=3). FIG +33 35 CD structure_element (f) Effects of mutations in MKP7-CD on the pNPP hydrolysis reaction (mean±s.e.m., n=3). FIG +43 47 pNPP chemical (f) Effects of mutations in MKP7-CD on the pNPP hydrolysis reaction (mean±s.e.m., n=3). FIG +14 22 CDK2-KAP complex_assembly Comparison of CDK2-KAP and JNK1–MKP7-CD. FIG +27 39 JNK1–MKP7-CD complex_assembly Comparison of CDK2-KAP and JNK1–MKP7-CD. FIG +4 17 Superposition experimental_method (a) Superposition of the complex structures of CDK2-KAP (PDB 1FQ1) and JNK1–MKP7-CD. FIG +33 43 structures evidence (a) Superposition of the complex structures of CDK2-KAP (PDB 1FQ1) and JNK1–MKP7-CD. FIG +47 55 CDK2-KAP complex_assembly (a) Superposition of the complex structures of CDK2-KAP (PDB 1FQ1) and JNK1–MKP7-CD. FIG +71 83 JNK1–MKP7-CD complex_assembly (a) Superposition of the complex structures of CDK2-KAP (PDB 1FQ1) and JNK1–MKP7-CD. FIG +4 10 N-lobe structure_element The N-lobe and C-lobe of CDK2 are coloured in grey and pink, respectively, and KAP is coloured in green. FIG +15 21 C-lobe structure_element The N-lobe and C-lobe of CDK2 are coloured in grey and pink, respectively, and KAP is coloured in green. FIG +25 29 CDK2 protein The N-lobe and C-lobe of CDK2 are coloured in grey and pink, respectively, and KAP is coloured in green. FIG +79 82 KAP protein The N-lobe and C-lobe of CDK2 are coloured in grey and pink, respectively, and KAP is coloured in green. FIG +75 90 contact regions site The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I). FIG +116 130 hydrogen bonds bond_interaction The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I). FIG +143 150 pThr160 ptm The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I). FIG +154 158 CDK2 protein The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I). FIG +167 178 active site site The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I). FIG +182 185 KAP protein The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I). FIG +187 195 region I structure_element The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I). FIG +34 38 CDK2 protein Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II). FIG +42 45 KAP protein Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II). FIG +72 81 interface site Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II). FIG +117 121 JNK1 protein Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II). FIG +126 130 MKP7 protein Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II). FIG +131 133 CD structure_element Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II). FIG +135 144 region II structure_element Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II). FIG +33 52 auxiliary region II structure_element (b) Interactions networks at the auxiliary region II mainly involving helix α7 from KAP and the αG helix and following L14 loop of CDK2. FIG +70 75 helix structure_element (b) Interactions networks at the auxiliary region II mainly involving helix α7 from KAP and the αG helix and following L14 loop of CDK2. FIG +76 78 α7 structure_element (b) Interactions networks at the auxiliary region II mainly involving helix α7 from KAP and the αG helix and following L14 loop of CDK2. FIG +84 87 KAP protein (b) Interactions networks at the auxiliary region II mainly involving helix α7 from KAP and the αG helix and following L14 loop of CDK2. FIG +96 104 αG helix structure_element (b) Interactions networks at the auxiliary region II mainly involving helix α7 from KAP and the αG helix and following L14 loop of CDK2. FIG +119 127 L14 loop structure_element (b) Interactions networks at the auxiliary region II mainly involving helix α7 from KAP and the αG helix and following L14 loop of CDK2. FIG +131 135 CDK2 protein (b) Interactions networks at the auxiliary region II mainly involving helix α7 from KAP and the αG helix and following L14 loop of CDK2. FIG +4 8 CDK2 protein The CDK2 is shown in surface representation coloured according to the electrostatic potential (positive, blue; negative, red). FIG +12 15 KAP protein Residues of KAP and CDK2 are highlighted as green and red sticks, respectively. FIG +20 24 CDK2 protein Residues of KAP and CDK2 are highlighted as green and red sticks, respectively. FIG +81 86 helix structure_element One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +87 89 α6 structure_element One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +93 96 KAP protein One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +115 120 helix structure_element One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +121 123 α4 structure_element One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +127 131 MKP7 protein One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +132 134 CD structure_element One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +185 191 stable protein_state One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +192 203 heterodimer oligomeric_state One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +207 211 CDK2 protein One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +216 219 KAP protein One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +225 243 Sequence alignment experimental_method One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +251 274 JNK-interacting regions site One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +278 282 MKPs protein_type One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs. FIG +12 16 MKP7 protein Residues of MKP7-CD involved in JNK1 recognition are indicated by cyan asterisks, and the conserved FXF-motif is highlighted in cyan. FIG +17 19 CD structure_element Residues of MKP7-CD involved in JNK1 recognition are indicated by cyan asterisks, and the conserved FXF-motif is highlighted in cyan. FIG +32 36 JNK1 protein Residues of MKP7-CD involved in JNK1 recognition are indicated by cyan asterisks, and the conserved FXF-motif is highlighted in cyan. FIG +90 99 conserved protein_state Residues of MKP7-CD involved in JNK1 recognition are indicated by cyan asterisks, and the conserved FXF-motif is highlighted in cyan. FIG +100 109 FXF-motif structure_element Residues of MKP7-CD involved in JNK1 recognition are indicated by cyan asterisks, and the conserved FXF-motif is highlighted in cyan. FIG +39 43 MKP7 protein The secondary structure assignments of MKP7-CD and KAP are shown above and below each sequence. FIG +44 46 CD structure_element The secondary structure assignments of MKP7-CD and KAP are shown above and below each sequence. FIG +51 54 KAP protein The secondary structure assignments of MKP7-CD and KAP are shown above and below each sequence. FIG +4 22 Sequence alignment experimental_method (d) Sequence alignment of the F-site regions on MAPKs. FIG +30 44 F-site regions structure_element (d) Sequence alignment of the F-site regions on MAPKs. FIG +48 53 MAPKs protein_type (d) Sequence alignment of the F-site regions on MAPKs. FIG +12 16 JNK1 protein Residues of JNK1 involved in recognition of MKP7 are indicated by orange asterisks, and those forming the F-site are highlighted in yellow. FIG +44 48 MKP7 protein Residues of JNK1 involved in recognition of MKP7 are indicated by orange asterisks, and those forming the F-site are highlighted in yellow. FIG +106 112 F-site site Residues of JNK1 involved in recognition of MKP7 are indicated by orange asterisks, and those forming the F-site are highlighted in yellow. FIG +0 9 FXF-motif structure_element FXF-motif is critical for controlling the phosphorylation of JNK and ultraviolet-induced apoptosis. FIG +42 57 phosphorylation ptm FXF-motif is critical for controlling the phosphorylation of JNK and ultraviolet-induced apoptosis. FIG +61 64 JNK protein_type FXF-motif is critical for controlling the phosphorylation of JNK and ultraviolet-induced apoptosis. FIG +6 15 FXF-motif structure_element (a–c) FXF-motif is essential for the dephosphorylation of JNK by MKP7. FIG +58 61 JNK protein_type (a–c) FXF-motif is essential for the dephosphorylation of JNK by MKP7. FIG +65 69 MKP7 protein (a–c) FXF-motif is essential for the dephosphorylation of JNK by MKP7. FIG +33 45 lentiviruses taxonomy_domain HEK293T cells were infected with lentiviruses expressing MKP7 and its mutants (1.0 μg). FIG +57 61 MKP7 protein HEK293T cells were infected with lentiviruses expressing MKP7 and its mutants (1.0 μg). FIG +70 77 mutants protein_state HEK293T cells were infected with lentiviruses expressing MKP7 and its mutants (1.0 μg). FIG +71 80 etoposide chemical After 36 h infection, cells were untreated in a, stimulated with 30 μM etoposide for 3 h in b or irradiated with 25 J m−2 ultraviolet light at 30 min before lysis in c. Whole-cell extracts were then immunoblotted with antibody indicated. FIG +34 48 phosphorylated protein_state Shown is a typical immunoblot for phosphorylated JNK from three independent experiments. FIG +49 52 JNK protein_type Shown is a typical immunoblot for phosphorylated JNK from three independent experiments. FIG +4 10 F-site site (d) F-site is required for JNK1 to interact with MKP7. FIG +27 31 JNK1 protein (d) F-site is required for JNK1 to interact with MKP7. FIG +49 53 MKP7 protein (d) F-site is required for JNK1 to interact with MKP7. FIG +19 33 co-transfected experimental_method HEK293T cells were co-transfected with MKP7 full-length (1.0 μg) and JNK1 (wild type or mutants as indicated, 1.0 μg). FIG +39 43 MKP7 protein HEK293T cells were co-transfected with MKP7 full-length (1.0 μg) and JNK1 (wild type or mutants as indicated, 1.0 μg). FIG +44 55 full-length protein_state HEK293T cells were co-transfected with MKP7 full-length (1.0 μg) and JNK1 (wild type or mutants as indicated, 1.0 μg). FIG +69 73 JNK1 protein HEK293T cells were co-transfected with MKP7 full-length (1.0 μg) and JNK1 (wild type or mutants as indicated, 1.0 μg). FIG +75 84 wild type protein_state HEK293T cells were co-transfected with MKP7 full-length (1.0 μg) and JNK1 (wild type or mutants as indicated, 1.0 μg). FIG +88 95 mutants protein_state HEK293T cells were co-transfected with MKP7 full-length (1.0 μg) and JNK1 (wild type or mutants as indicated, 1.0 μg). FIG +30 48 immunoprecipitated experimental_method Whole-cell extracts were then immunoprecipitated with antibody against Myc for MKP7; immunobloting was carried out with antibodies indicated. FIG +79 83 MKP7 protein Whole-cell extracts were then immunoprecipitated with antibody against Myc for MKP7; immunobloting was carried out with antibodies indicated. FIG +0 2 IP experimental_method IP, immunoprecipitation; TCL, total cell lysate. FIG +4 23 immunoprecipitation experimental_method IP, immunoprecipitation; TCL, total cell lysate. FIG +14 18 MKP7 protein (e) Effect of MKP7 (wild type or mutants) expression on ultraviolet-induced apoptosis. FIG +20 29 wild type protein_state (e) Effect of MKP7 (wild type or mutants) expression on ultraviolet-induced apoptosis. FIG +33 40 mutants protein_state (e) Effect of MKP7 (wild type or mutants) expression on ultraviolet-induced apoptosis. FIG +30 42 lentiviruses taxonomy_domain HeLa cells were infected with lentiviruses expressing MKP7 full-length and its mutants. FIG +54 58 MKP7 protein HeLa cells were infected with lentiviruses expressing MKP7 full-length and its mutants. FIG +59 70 full-length protein_state HeLa cells were infected with lentiviruses expressing MKP7 full-length and its mutants. FIG +79 86 mutants protein_state HeLa cells were infected with lentiviruses expressing MKP7 full-length and its mutants. FIG +29 43 flow cytometry experimental_method Cells were then subjected to flow cytometry analysis. FIG +35 48 Annexin-V-APC chemical Apoptotic cells were determined by Annexin-V-APC/PI staining. FIG +49 51 PI chemical Apoptotic cells were determined by Annexin-V-APC/PI staining. FIG +18 27 Annexin-V chemical The results using Annexin-V stain for membrane phosphatidylserine eversion, combined with propidium iodide (PI) uptake to evaluate cells whose membranes had been compromised. FIG +90 106 propidium iodide chemical The results using Annexin-V stain for membrane phosphatidylserine eversion, combined with propidium iodide (PI) uptake to evaluate cells whose membranes had been compromised. FIG +108 110 PI chemical The results using Annexin-V stain for membrane phosphatidylserine eversion, combined with propidium iodide (PI) uptake to evaluate cells whose membranes had been compromised. FIG +19 28 Annexin-V chemical Staining with both Annexin-V and PI indicate apoptosis (upper right quadrant). FIG +33 35 PI chemical Staining with both Annexin-V and PI indicate apoptosis (upper right quadrant). FIG +64 66 *P evidence (f) Statistical analysis of apoptotic cells (mean±s.e.m., n=3), *P<0.05, ***P<0.001 (ANOVA followed by Tukey's test). FIG +73 77 ***P evidence (f) Statistical analysis of apoptotic cells (mean±s.e.m., n=3), *P<0.05, ***P<0.001 (ANOVA followed by Tukey's test). FIG +85 90 ANOVA experimental_method (f) Statistical analysis of apoptotic cells (mean±s.e.m., n=3), *P<0.05, ***P<0.001 (ANOVA followed by Tukey's test). FIG +103 115 Tukey's test experimental_method (f) Statistical analysis of apoptotic cells (mean±s.e.m., n=3), *P<0.05, ***P<0.001 (ANOVA followed by Tukey's test). FIG +0 4 MKP5 protein MKP5-CD is crucial for JNK1 binding and enzyme catalysis. FIG +5 7 CD structure_element MKP5-CD is crucial for JNK1 binding and enzyme catalysis. FIG +23 27 JNK1 protein MKP5-CD is crucial for JNK1 binding and enzyme catalysis. FIG +27 32 human species (a) Domain organization of human MKP5. FIG +33 37 MKP5 protein (a) Domain organization of human MKP5. FIG +4 7 KBD structure_element The KBD and CD of MKP5 are shown in brown and grey, respectively. (b) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration. FIG +12 14 CD structure_element The KBD and CD of MKP5 are shown in brown and grey, respectively. (b) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration. FIG +18 22 MKP5 protein The KBD and CD of MKP5 are shown in brown and grey, respectively. (b) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration. FIG +70 95 Plots of initial velocity evidence The KBD and CD of MKP5 are shown in brown and grey, respectively. (b) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration. FIG +103 107 MKP5 protein The KBD and CD of MKP5 are shown in brown and grey, respectively. (b) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration. FIG +134 141 phospho protein_state The KBD and CD of MKP5 are shown in brown and grey, respectively. (b) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration. FIG +142 146 JNK1 protein The KBD and CD of MKP5 are shown in brown and grey, respectively. (b) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration. FIG +89 91 Km evidence The solid lines are best-fitting results according to the Michaelis–Menten equation with Km and kcat values indicated. FIG +96 100 kcat evidence The solid lines are best-fitting results according to the Michaelis–Menten equation with Km and kcat values indicated. FIG +36 57 Structural comparison experimental_method The error bars represent s.e.m. (c) Structural comparison of the JNK-interacting residues on MKP5-CD (PDB 1ZZW) and MKP7-CD. FIG +65 89 JNK-interacting residues site The error bars represent s.e.m. (c) Structural comparison of the JNK-interacting residues on MKP5-CD (PDB 1ZZW) and MKP7-CD. FIG +93 97 MKP5 protein The error bars represent s.e.m. (c) Structural comparison of the JNK-interacting residues on MKP5-CD (PDB 1ZZW) and MKP7-CD. FIG +98 100 CD structure_element The error bars represent s.e.m. (c) Structural comparison of the JNK-interacting residues on MKP5-CD (PDB 1ZZW) and MKP7-CD. FIG +116 120 MKP7 protein The error bars represent s.e.m. (c) Structural comparison of the JNK-interacting residues on MKP5-CD (PDB 1ZZW) and MKP7-CD. FIG +121 123 CD structure_element The error bars represent s.e.m. (c) Structural comparison of the JNK-interacting residues on MKP5-CD (PDB 1ZZW) and MKP7-CD. FIG +30 34 MKP5 protein The corresponding residues on MKP5 are depicted as orange sticks, and MKP5 residues numbers are in parentheses. FIG +70 74 MKP5 protein The corresponding residues on MKP5 are depicted as orange sticks, and MKP5 residues numbers are in parentheses. FIG +4 27 Gel filtration analysis experimental_method (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. FIG +47 51 JNK1 protein (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. FIG +57 61 MKP5 protein (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. FIG +62 64 CD structure_element (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. FIG +69 73 MKP5 protein (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. FIG +74 77 KBD structure_element (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. FIG +83 112 GST-mediated pull-down assays experimental_method (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. FIG +132 136 JNK1 protein (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. FIG +142 146 MKP5 protein (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. FIG +147 149 CD structure_element (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. FIG +154 158 MKP5 protein (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. FIG +159 162 KBD structure_element (d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. FIG +63 72 mutations experimental_method The panels are arranged the same as in Fig. 2d. (f) Effects of mutations in MKP5-CD on the JNK1 dephosphorylation (mean±s.e.m., n=3). FIG +76 80 MKP5 protein The panels are arranged the same as in Fig. 2d. (f) Effects of mutations in MKP5-CD on the JNK1 dephosphorylation (mean±s.e.m., n=3). FIG +81 83 CD structure_element The panels are arranged the same as in Fig. 2d. (f) Effects of mutations in MKP5-CD on the JNK1 dephosphorylation (mean±s.e.m., n=3). FIG +91 95 JNK1 protein The panels are arranged the same as in Fig. 2d. (f) Effects of mutations in MKP5-CD on the JNK1 dephosphorylation (mean±s.e.m., n=3). FIG +15 24 mutations experimental_method (g) Effects of mutations in MKP5-CD on the pNPP hydrolysis reaction (mean±s.e.m., n=3). FIG +28 32 MKP5 protein (g) Effects of mutations in MKP5-CD on the pNPP hydrolysis reaction (mean±s.e.m., n=3). FIG +33 35 CD structure_element (g) Effects of mutations in MKP5-CD on the pNPP hydrolysis reaction (mean±s.e.m., n=3). FIG +43 47 pNPP chemical (g) Effects of mutations in MKP5-CD on the pNPP hydrolysis reaction (mean±s.e.m., n=3). FIG +4 20 Pull-down assays experimental_method (h) Pull-down assays of MKP5-CD by GST-tagged JNK1 mutants. FIG +24 28 MKP5 protein (h) Pull-down assays of MKP5-CD by GST-tagged JNK1 mutants. FIG +29 31 CD structure_element (h) Pull-down assays of MKP5-CD by GST-tagged JNK1 mutants. FIG +35 45 GST-tagged protein_state (h) Pull-down assays of MKP5-CD by GST-tagged JNK1 mutants. FIG +46 50 JNK1 protein (h) Pull-down assays of MKP5-CD by GST-tagged JNK1 mutants. FIG +51 58 mutants protein_state (h) Pull-down assays of MKP5-CD by GST-tagged JNK1 mutants. FIG diff --git a/annotation_CSV/PMC4802085.csv b/annotation_CSV/PMC4802085.csv new file mode 100644 index 0000000000000000000000000000000000000000..7482f0a7e6b8562d8a6c74924f3de9138dcaef26 --- /dev/null +++ b/annotation_CSV/PMC4802085.csv @@ -0,0 +1,984 @@ +anno_start anno_end anno_text entity_type sentence section +0 4 Haem chemical Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance TITLE +15 27 dimerization oligomeric_state Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance TITLE +31 37 PGRMC1 protein Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance TITLE +38 45 Sigma-2 protein Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance TITLE +0 42 Progesterone-receptor membrane component 1 protein Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. ABSTRACT +44 50 PGRMC1 protein Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. ABSTRACT +51 67 Sigma-2 receptor protein Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. ABSTRACT +74 97 haem-containing protein protein_type Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. ABSTRACT +118 150 epidermal growth factor receptor protein_type Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. ABSTRACT +152 156 EGFR protein_type Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. ABSTRACT +162 178 cytochromes P450 protein_type Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. ABSTRACT +5 30 crystallographic analyses experimental_method Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. ABSTRACT +38 44 PGRMC1 protein Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. ABSTRACT +45 61 cytosolic domain structure_element Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. ABSTRACT +106 112 stable protein_state Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. ABSTRACT +113 118 dimer oligomeric_state Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. ABSTRACT +127 148 stacking interactions bond_interaction Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. ABSTRACT +167 171 haem chemical Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. ABSTRACT +4 8 haem chemical The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. ABSTRACT +9 13 iron chemical The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. ABSTRACT +17 36 five-coordinated by bond_interaction The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. ABSTRACT +37 43 Tyr113 residue_name_number The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. ABSTRACT +58 65 surface site The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. ABSTRACT +73 77 haem chemical The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. ABSTRACT +87 99 dimerization oligomeric_state The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. ABSTRACT +0 15 Carbon monoxide chemical Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. ABSTRACT +17 19 CO chemical Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. ABSTRACT +37 43 PGRMC1 protein Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. ABSTRACT +44 56 dimerization oligomeric_state Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. ABSTRACT +75 98 sixth coordination site site Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. ABSTRACT +106 110 haem chemical Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. ABSTRACT +0 4 Haem chemical Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. ABSTRACT +14 20 PGRMC1 protein Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. ABSTRACT +21 33 dimerization oligomeric_state Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. ABSTRACT +68 72 EGFR protein_type Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. ABSTRACT +77 93 cytochromes P450 protein_type Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. ABSTRACT +201 203 CO chemical Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. ABSTRACT +207 211 haem chemical Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. ABSTRACT +32 44 dimerization oligomeric_state This study demonstrates protein dimerization via haem–haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer. ABSTRACT +49 67 haem–haem stacking bond_interaction This study demonstrates protein dimerization via haem–haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer. ABSTRACT +96 106 eukaryotes taxonomy_domain This study demonstrates protein dimerization via haem–haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer. ABSTRACT +1 7 PGRMC1 protein PGRMC1 binds to EGFR and cytochromes P450, and is known to be involved in cancer proliferation and in drug resistance. ABSTRACT +17 21 EGFR protein_type PGRMC1 binds to EGFR and cytochromes P450, and is known to be involved in cancer proliferation and in drug resistance. ABSTRACT +26 42 cytochromes P450 protein_type PGRMC1 binds to EGFR and cytochromes P450, and is known to be involved in cancer proliferation and in drug resistance. ABSTRACT +32 41 structure evidence Here, the authors determine the structure of the cytosolic domain of PGRMC1, which forms a dimer via haem–haem stacking, and propose how this interaction could be involved in its function. ABSTRACT +49 65 cytosolic domain structure_element Here, the authors determine the structure of the cytosolic domain of PGRMC1, which forms a dimer via haem–haem stacking, and propose how this interaction could be involved in its function. ABSTRACT +69 75 PGRMC1 protein Here, the authors determine the structure of the cytosolic domain of PGRMC1, which forms a dimer via haem–haem stacking, and propose how this interaction could be involved in its function. ABSTRACT +91 96 dimer oligomeric_state Here, the authors determine the structure of the cytosolic domain of PGRMC1, which forms a dimer via haem–haem stacking, and propose how this interaction could be involved in its function. ABSTRACT +101 119 haem–haem stacking bond_interaction Here, the authors determine the structure of the cytosolic domain of PGRMC1, which forms a dimer via haem–haem stacking, and propose how this interaction could be involved in its function. ABSTRACT +45 49 haem chemical Much attention has been paid to the roles of haem-iron in cancer development. INTRO +50 54 iron chemical Much attention has been paid to the roles of haem-iron in cancer development. INTRO +28 32 haem chemical Increased dietary intake of haem is a risk factor for several types of cancer. INTRO +29 43 deprivation of protein_state Previous studies showed that deprivation of iron or haem suppresses tumourigenesis. INTRO +44 48 iron chemical Previous studies showed that deprivation of iron or haem suppresses tumourigenesis. INTRO +52 56 haem chemical Previous studies showed that deprivation of iron or haem suppresses tumourigenesis. INTRO +19 34 carbon monoxide chemical On the other hand, carbon monoxide (CO), the gaseous mediator generated by oxidative degradation of haem via haem oxygenase (HO), inhibits tumour growth. INTRO +36 38 CO chemical On the other hand, carbon monoxide (CO), the gaseous mediator generated by oxidative degradation of haem via haem oxygenase (HO), inhibits tumour growth. INTRO +100 104 haem chemical On the other hand, carbon monoxide (CO), the gaseous mediator generated by oxidative degradation of haem via haem oxygenase (HO), inhibits tumour growth. INTRO +109 123 haem oxygenase protein_type On the other hand, carbon monoxide (CO), the gaseous mediator generated by oxidative degradation of haem via haem oxygenase (HO), inhibits tumour growth. INTRO +125 127 HO protein_type On the other hand, carbon monoxide (CO), the gaseous mediator generated by oxidative degradation of haem via haem oxygenase (HO), inhibits tumour growth. INTRO +37 41 haem chemical Thus, a tenuous balance between free haem and CO plays key roles in cancer development and chemoresistance, although the underlying mechanisms are not fully understood. INTRO +46 48 CO chemical Thus, a tenuous balance between free haem and CO plays key roles in cancer development and chemoresistance, although the underlying mechanisms are not fully understood. INTRO +93 111 affinity nanobeads experimental_method To gain insight into the underlying mechanisms, we took chemical biological approaches using affinity nanobeads carrying haem and identified progesterone-receptor membrane component 1 (PGRMC1) as a haem-binding protein from mouse liver extracts (Supplementary Fig. 1). INTRO +121 125 haem chemical To gain insight into the underlying mechanisms, we took chemical biological approaches using affinity nanobeads carrying haem and identified progesterone-receptor membrane component 1 (PGRMC1) as a haem-binding protein from mouse liver extracts (Supplementary Fig. 1). INTRO +141 183 progesterone-receptor membrane component 1 protein To gain insight into the underlying mechanisms, we took chemical biological approaches using affinity nanobeads carrying haem and identified progesterone-receptor membrane component 1 (PGRMC1) as a haem-binding protein from mouse liver extracts (Supplementary Fig. 1). INTRO +185 191 PGRMC1 protein To gain insight into the underlying mechanisms, we took chemical biological approaches using affinity nanobeads carrying haem and identified progesterone-receptor membrane component 1 (PGRMC1) as a haem-binding protein from mouse liver extracts (Supplementary Fig. 1). INTRO +198 202 haem chemical To gain insight into the underlying mechanisms, we took chemical biological approaches using affinity nanobeads carrying haem and identified progesterone-receptor membrane component 1 (PGRMC1) as a haem-binding protein from mouse liver extracts (Supplementary Fig. 1). INTRO +224 229 mouse taxonomy_domain To gain insight into the underlying mechanisms, we took chemical biological approaches using affinity nanobeads carrying haem and identified progesterone-receptor membrane component 1 (PGRMC1) as a haem-binding protein from mouse liver extracts (Supplementary Fig. 1). INTRO +0 6 PGRMC1 protein PGRMC1 is a member of the membrane-associated progesterone receptor (MAPR) family with a cytochrome b5-like haem-binding region, and is known to be highly expressed in various types of cancers. INTRO +26 67 membrane-associated progesterone receptor protein_type PGRMC1 is a member of the membrane-associated progesterone receptor (MAPR) family with a cytochrome b5-like haem-binding region, and is known to be highly expressed in various types of cancers. INTRO +69 73 MAPR protein_type PGRMC1 is a member of the membrane-associated progesterone receptor (MAPR) family with a cytochrome b5-like haem-binding region, and is known to be highly expressed in various types of cancers. INTRO +89 107 cytochrome b5-like structure_element PGRMC1 is a member of the membrane-associated progesterone receptor (MAPR) family with a cytochrome b5-like haem-binding region, and is known to be highly expressed in various types of cancers. INTRO +108 127 haem-binding region site PGRMC1 is a member of the membrane-associated progesterone receptor (MAPR) family with a cytochrome b5-like haem-binding region, and is known to be highly expressed in various types of cancers. INTRO +148 164 highly expressed protein_state PGRMC1 is a member of the membrane-associated progesterone receptor (MAPR) family with a cytochrome b5-like haem-binding region, and is known to be highly expressed in various types of cancers. INTRO +0 6 PGRMC1 protein PGRMC1 is anchored to the cell membrane through the N-terminal transmembrane helix and interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 (ref). INTRO +63 82 transmembrane helix structure_element PGRMC1 is anchored to the cell membrane through the N-terminal transmembrane helix and interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 (ref). INTRO +102 134 epidermal growth factor receptor protein_type PGRMC1 is anchored to the cell membrane through the N-terminal transmembrane helix and interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 (ref). INTRO +136 140 EGFR protein_type PGRMC1 is anchored to the cell membrane through the N-terminal transmembrane helix and interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 (ref). INTRO +146 162 cytochromes P450 protein_type PGRMC1 is anchored to the cell membrane through the N-terminal transmembrane helix and interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 (ref). INTRO +6 12 PGRMC1 protein While PGRMC1 is implicated in cell proliferation and cholesterol biosynthesis, the structural basis on which PGRMC1 exerts its function remains largely unknown. INTRO +109 115 PGRMC1 protein While PGRMC1 is implicated in cell proliferation and cholesterol biosynthesis, the structural basis on which PGRMC1 exerts its function remains largely unknown. INTRO +18 24 PGRMC1 protein Here we show that PGRMC1 exhibits a unique haem-dependent dimerization. INTRO +43 47 haem chemical Here we show that PGRMC1 exhibits a unique haem-dependent dimerization. INTRO +58 70 dimerization oligomeric_state Here we show that PGRMC1 exhibits a unique haem-dependent dimerization. INTRO +4 9 dimer oligomeric_state The dimer binds to EGFR and cytochromes P450 to enhance tumour cell proliferation and chemoresistance. INTRO +19 23 EGFR protein_type The dimer binds to EGFR and cytochromes P450 to enhance tumour cell proliferation and chemoresistance. INTRO +28 44 cytochromes P450 protein_type The dimer binds to EGFR and cytochromes P450 to enhance tumour cell proliferation and chemoresistance. INTRO +4 9 dimer oligomeric_state The dimer is dissociated to monomers by physiological levels of CO, suggesting that PGRMC1 serves as a CO-sensitive molecular switch regulating cancer cell proliferation. INTRO +28 36 monomers oligomeric_state The dimer is dissociated to monomers by physiological levels of CO, suggesting that PGRMC1 serves as a CO-sensitive molecular switch regulating cancer cell proliferation. INTRO +64 66 CO chemical The dimer is dissociated to monomers by physiological levels of CO, suggesting that PGRMC1 serves as a CO-sensitive molecular switch regulating cancer cell proliferation. INTRO +84 90 PGRMC1 protein The dimer is dissociated to monomers by physiological levels of CO, suggesting that PGRMC1 serves as a CO-sensitive molecular switch regulating cancer cell proliferation. INTRO +103 105 CO chemical The dimer is dissociated to monomers by physiological levels of CO, suggesting that PGRMC1 serves as a CO-sensitive molecular switch regulating cancer cell proliferation. INTRO +0 23 X-ray crystal structure evidence X-ray crystal structure of PGRMC1 RESULTS +27 33 PGRMC1 protein X-ray crystal structure of PGRMC1 RESULTS +3 9 solved experimental_method We solved the crystal structure of the haem-bound PGRMC1 cytosolic domain (a.a.72–195) at 1.95 Å resolution (Supplementary Fig. 2). RESULTS +14 31 crystal structure evidence We solved the crystal structure of the haem-bound PGRMC1 cytosolic domain (a.a.72–195) at 1.95 Å resolution (Supplementary Fig. 2). RESULTS +39 49 haem-bound protein_state We solved the crystal structure of the haem-bound PGRMC1 cytosolic domain (a.a.72–195) at 1.95 Å resolution (Supplementary Fig. 2). RESULTS +50 56 PGRMC1 protein We solved the crystal structure of the haem-bound PGRMC1 cytosolic domain (a.a.72–195) at 1.95 Å resolution (Supplementary Fig. 2). RESULTS +57 73 cytosolic domain structure_element We solved the crystal structure of the haem-bound PGRMC1 cytosolic domain (a.a.72–195) at 1.95 Å resolution (Supplementary Fig. 2). RESULTS +79 85 72–195 residue_range We solved the crystal structure of the haem-bound PGRMC1 cytosolic domain (a.a.72–195) at 1.95 Å resolution (Supplementary Fig. 2). RESULTS +7 18 presence of protein_state In the presence of haem, PGRMC1 forms a dimeric structure largely through hydrophobic interactions between the haem moieties of two monomers (Fig. 1a, Table 1 and Supplementary Fig. 3; a stereo-structural image is shown in Supplementary Fig 4). RESULTS +19 23 haem chemical In the presence of haem, PGRMC1 forms a dimeric structure largely through hydrophobic interactions between the haem moieties of two monomers (Fig. 1a, Table 1 and Supplementary Fig. 3; a stereo-structural image is shown in Supplementary Fig 4). RESULTS +25 31 PGRMC1 protein In the presence of haem, PGRMC1 forms a dimeric structure largely through hydrophobic interactions between the haem moieties of two monomers (Fig. 1a, Table 1 and Supplementary Fig. 3; a stereo-structural image is shown in Supplementary Fig 4). RESULTS +40 47 dimeric oligomeric_state In the presence of haem, PGRMC1 forms a dimeric structure largely through hydrophobic interactions between the haem moieties of two monomers (Fig. 1a, Table 1 and Supplementary Fig. 3; a stereo-structural image is shown in Supplementary Fig 4). RESULTS +74 98 hydrophobic interactions bond_interaction In the presence of haem, PGRMC1 forms a dimeric structure largely through hydrophobic interactions between the haem moieties of two monomers (Fig. 1a, Table 1 and Supplementary Fig. 3; a stereo-structural image is shown in Supplementary Fig 4). RESULTS +111 115 haem chemical In the presence of haem, PGRMC1 forms a dimeric structure largely through hydrophobic interactions between the haem moieties of two monomers (Fig. 1a, Table 1 and Supplementary Fig. 3; a stereo-structural image is shown in Supplementary Fig 4). RESULTS +132 140 monomers oligomeric_state In the presence of haem, PGRMC1 forms a dimeric structure largely through hydrophobic interactions between the haem moieties of two monomers (Fig. 1a, Table 1 and Supplementary Fig. 3; a stereo-structural image is shown in Supplementary Fig 4). RESULTS +26 32 PGRMC1 protein While the overall fold of PGRMC1 is similar to that of canonical cytochrome b5, their haem irons are coordinated differently. RESULTS +65 78 cytochrome b5 protein_type While the overall fold of PGRMC1 is similar to that of canonical cytochrome b5, their haem irons are coordinated differently. RESULTS +86 90 haem chemical While the overall fold of PGRMC1 is similar to that of canonical cytochrome b5, their haem irons are coordinated differently. RESULTS +3 16 cytochrome b5 protein_type In cytochrome b5, the haem iron is six-coordinated by two axial histidine residues. RESULTS +22 26 haem chemical In cytochrome b5, the haem iron is six-coordinated by two axial histidine residues. RESULTS +27 31 iron chemical In cytochrome b5, the haem iron is six-coordinated by two axial histidine residues. RESULTS +35 53 six-coordinated by bond_interaction In cytochrome b5, the haem iron is six-coordinated by two axial histidine residues. RESULTS +64 73 histidine residue_name In cytochrome b5, the haem iron is six-coordinated by two axial histidine residues. RESULTS +6 16 histidines residue_name These histidines are missing in PGRMC1, and the haem iron is five-coordinated by Tyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3). RESULTS +21 28 missing protein_state These histidines are missing in PGRMC1, and the haem iron is five-coordinated by Tyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3). RESULTS +32 38 PGRMC1 protein These histidines are missing in PGRMC1, and the haem iron is five-coordinated by Tyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3). RESULTS +48 52 haem chemical These histidines are missing in PGRMC1, and the haem iron is five-coordinated by Tyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3). RESULTS +53 57 iron chemical These histidines are missing in PGRMC1, and the haem iron is five-coordinated by Tyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3). RESULTS +61 80 five-coordinated by bond_interaction These histidines are missing in PGRMC1, and the haem iron is five-coordinated by Tyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3). RESULTS +81 87 Tyr113 residue_name_number These histidines are missing in PGRMC1, and the haem iron is five-coordinated by Tyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3). RESULTS +89 93 Y113 residue_name_number These histidines are missing in PGRMC1, and the haem iron is five-coordinated by Tyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3). RESULTS +95 100 alone protein_state These histidines are missing in PGRMC1, and the haem iron is five-coordinated by Tyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3). RESULTS +2 18 homologous helix structure_element A homologous helix that holds haem in cytochrome b5 is longer, shifts away from haem, and does not form a coordinate bond in PGRMC1 (Fig. 1c). RESULTS +30 34 haem chemical A homologous helix that holds haem in cytochrome b5 is longer, shifts away from haem, and does not form a coordinate bond in PGRMC1 (Fig. 1c). RESULTS +38 51 cytochrome b5 protein_type A homologous helix that holds haem in cytochrome b5 is longer, shifts away from haem, and does not form a coordinate bond in PGRMC1 (Fig. 1c). RESULTS +80 84 haem chemical A homologous helix that holds haem in cytochrome b5 is longer, shifts away from haem, and does not form a coordinate bond in PGRMC1 (Fig. 1c). RESULTS +125 131 PGRMC1 protein A homologous helix that holds haem in cytochrome b5 is longer, shifts away from haem, and does not form a coordinate bond in PGRMC1 (Fig. 1c). RESULTS +35 39 haem chemical Consequently, the five-coordinated haem of PGRMC1 has an open surface that allows its dimerization through hydrophobic haem–haem stacking. RESULTS +43 49 PGRMC1 protein Consequently, the five-coordinated haem of PGRMC1 has an open surface that allows its dimerization through hydrophobic haem–haem stacking. RESULTS +62 69 surface site Consequently, the five-coordinated haem of PGRMC1 has an open surface that allows its dimerization through hydrophobic haem–haem stacking. RESULTS +86 98 dimerization oligomeric_state Consequently, the five-coordinated haem of PGRMC1 has an open surface that allows its dimerization through hydrophobic haem–haem stacking. RESULTS +107 137 hydrophobic haem–haem stacking bond_interaction Consequently, the five-coordinated haem of PGRMC1 has an open surface that allows its dimerization through hydrophobic haem–haem stacking. RESULTS +62 68 Tyr164 residue_name_number Contrary to our finding, Kaluka et al. recently reported that Tyr164 of PGRMC1 is the axial ligand of haem because mutation of this residue impairs haem binding. RESULTS +72 78 PGRMC1 protein Contrary to our finding, Kaluka et al. recently reported that Tyr164 of PGRMC1 is the axial ligand of haem because mutation of this residue impairs haem binding. RESULTS +102 106 haem chemical Contrary to our finding, Kaluka et al. recently reported that Tyr164 of PGRMC1 is the axial ligand of haem because mutation of this residue impairs haem binding. RESULTS +115 123 mutation experimental_method Contrary to our finding, Kaluka et al. recently reported that Tyr164 of PGRMC1 is the axial ligand of haem because mutation of this residue impairs haem binding. RESULTS +148 152 haem chemical Contrary to our finding, Kaluka et al. recently reported that Tyr164 of PGRMC1 is the axial ligand of haem because mutation of this residue impairs haem binding. RESULTS +4 19 structural data evidence Our structural data revealed that Tyr164 and a few other residues such as Tyr107 and Lys163 are in fact hydrogen-bonded to haem propionates. RESULTS +34 40 Tyr164 residue_name_number Our structural data revealed that Tyr164 and a few other residues such as Tyr107 and Lys163 are in fact hydrogen-bonded to haem propionates. RESULTS +74 80 Tyr107 residue_name_number Our structural data revealed that Tyr164 and a few other residues such as Tyr107 and Lys163 are in fact hydrogen-bonded to haem propionates. RESULTS +85 91 Lys163 residue_name_number Our structural data revealed that Tyr164 and a few other residues such as Tyr107 and Lys163 are in fact hydrogen-bonded to haem propionates. RESULTS +104 119 hydrogen-bonded bond_interaction Our structural data revealed that Tyr164 and a few other residues such as Tyr107 and Lys163 are in fact hydrogen-bonded to haem propionates. RESULTS +123 127 haem chemical Our structural data revealed that Tyr164 and a few other residues such as Tyr107 and Lys163 are in fact hydrogen-bonded to haem propionates. RESULTS +56 63 Tyr 107 residue_name_number This is consistent with observations by Min et al. that Tyr 107 and Tyr113 of PGRMC1 are involved in binding with haem. RESULTS +68 74 Tyr113 residue_name_number This is consistent with observations by Min et al. that Tyr 107 and Tyr113 of PGRMC1 are involved in binding with haem. RESULTS +78 84 PGRMC1 protein This is consistent with observations by Min et al. that Tyr 107 and Tyr113 of PGRMC1 are involved in binding with haem. RESULTS +114 118 haem chemical This is consistent with observations by Min et al. that Tyr 107 and Tyr113 of PGRMC1 are involved in binding with haem. RESULTS +30 39 conserved protein_state These amino acid residues are conserved among MAPR family members (Supplementary Fig. 5a), suggesting that these proteins share the ability to exhibit haem-dependent dimerization. RESULTS +46 50 MAPR protein_type These amino acid residues are conserved among MAPR family members (Supplementary Fig. 5a), suggesting that these proteins share the ability to exhibit haem-dependent dimerization. RESULTS +151 155 haem chemical These amino acid residues are conserved among MAPR family members (Supplementary Fig. 5a), suggesting that these proteins share the ability to exhibit haem-dependent dimerization. RESULTS +166 178 dimerization oligomeric_state These amino acid residues are conserved among MAPR family members (Supplementary Fig. 5a), suggesting that these proteins share the ability to exhibit haem-dependent dimerization. RESULTS +0 6 PGRMC1 protein PGRMC1 exhibits haem-dependent dimerization in solution RESULTS +16 20 haem chemical PGRMC1 exhibits haem-dependent dimerization in solution RESULTS +31 43 dimerization oligomeric_state PGRMC1 exhibits haem-dependent dimerization in solution RESULTS +7 13 PGRMC1 protein In the PGRMC1 crystal, two different types of crystal contacts (chain A–A″ and A–B) were observed in addition to the haem-mediated dimer (chain A–A′) (Supplementary Figs 3 and 6a). RESULTS +14 21 crystal evidence In the PGRMC1 crystal, two different types of crystal contacts (chain A–A″ and A–B) were observed in addition to the haem-mediated dimer (chain A–A′) (Supplementary Figs 3 and 6a). RESULTS +117 121 haem chemical In the PGRMC1 crystal, two different types of crystal contacts (chain A–A″ and A–B) were observed in addition to the haem-mediated dimer (chain A–A′) (Supplementary Figs 3 and 6a). RESULTS +131 136 dimer oligomeric_state In the PGRMC1 crystal, two different types of crystal contacts (chain A–A″ and A–B) were observed in addition to the haem-mediated dimer (chain A–A′) (Supplementary Figs 3 and 6a). RESULTS +16 20 haem chemical To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7). RESULTS +30 42 dimerization oligomeric_state To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7). RESULTS +46 52 PGRMC1 protein To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7). RESULTS +89 98 structure evidence To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7). RESULTS +102 105 apo protein_state To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7). RESULTS +111 121 haem-bound protein_state To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7). RESULTS +122 128 PGMRC1 protein To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7). RESULTS +132 174 two-dimensional nuclear magnetic resonance experimental_method To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7). RESULTS +176 179 NMR experimental_method To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7). RESULTS +187 274 heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy experimental_method To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7). RESULTS +0 3 NMR experimental_method NMR signals from some amino acid residues of PGRMC1 disappeared due to the paramagnetic relaxation effect of haem (Supplementary Figs 6b); these residues were located in the haem-binding region. RESULTS +45 51 PGRMC1 protein NMR signals from some amino acid residues of PGRMC1 disappeared due to the paramagnetic relaxation effect of haem (Supplementary Figs 6b); these residues were located in the haem-binding region. RESULTS +109 113 haem chemical NMR signals from some amino acid residues of PGRMC1 disappeared due to the paramagnetic relaxation effect of haem (Supplementary Figs 6b); these residues were located in the haem-binding region. RESULTS +174 193 haem-binding region site NMR signals from some amino acid residues of PGRMC1 disappeared due to the paramagnetic relaxation effect of haem (Supplementary Figs 6b); these residues were located in the haem-binding region. RESULTS +5 20 chemical shifts evidence When chemical shifts of apo- and haem-bound forms of PGMRC1 were compared, some amino acid residues close to those which disappeared because of the paramagnetic relaxation effect of haem exhibit notable chemical shifts (Supplementary Fig. 6a,b; dark yellow). RESULTS +24 27 apo protein_state When chemical shifts of apo- and haem-bound forms of PGMRC1 were compared, some amino acid residues close to those which disappeared because of the paramagnetic relaxation effect of haem exhibit notable chemical shifts (Supplementary Fig. 6a,b; dark yellow). RESULTS +33 43 haem-bound protein_state When chemical shifts of apo- and haem-bound forms of PGMRC1 were compared, some amino acid residues close to those which disappeared because of the paramagnetic relaxation effect of haem exhibit notable chemical shifts (Supplementary Fig. 6a,b; dark yellow). RESULTS +53 59 PGMRC1 protein When chemical shifts of apo- and haem-bound forms of PGMRC1 were compared, some amino acid residues close to those which disappeared because of the paramagnetic relaxation effect of haem exhibit notable chemical shifts (Supplementary Fig. 6a,b; dark yellow). RESULTS +182 186 haem chemical When chemical shifts of apo- and haem-bound forms of PGMRC1 were compared, some amino acid residues close to those which disappeared because of the paramagnetic relaxation effect of haem exhibit notable chemical shifts (Supplementary Fig. 6a,b; dark yellow). RESULTS +16 26 interfaces site However, at the interfaces of the other possible dimeric structures (Supplementary Fig. 6a, chain A–A″; cyan and chain A–B; violet), no significant difference was observed. RESULTS +49 56 dimeric oligomeric_state However, at the interfaces of the other possible dimeric structures (Supplementary Fig. 6a, chain A–A″; cyan and chain A–B; violet), no significant difference was observed. RESULTS +57 67 structures evidence However, at the interfaces of the other possible dimeric structures (Supplementary Fig. 6a, chain A–A″; cyan and chain A–B; violet), no significant difference was observed. RESULTS +13 40 free energy of dissociation evidence Furthermore, free energy of dissociation predicted by PISA suggested that the haem-mediated dimer is stable in solution while the other potential interactions are not. RESULTS +54 58 PISA experimental_method Furthermore, free energy of dissociation predicted by PISA suggested that the haem-mediated dimer is stable in solution while the other potential interactions are not. RESULTS +78 82 haem chemical Furthermore, free energy of dissociation predicted by PISA suggested that the haem-mediated dimer is stable in solution while the other potential interactions are not. RESULTS +92 97 dimer oligomeric_state Furthermore, free energy of dissociation predicted by PISA suggested that the haem-mediated dimer is stable in solution while the other potential interactions are not. RESULTS +101 107 stable protein_state Furthermore, free energy of dissociation predicted by PISA suggested that the haem-mediated dimer is stable in solution while the other potential interactions are not. RESULTS +56 62 PGRMC1 protein We also attempted to predict the secondary structure of PGRMC1 through NMR data by calculating with TALOS+ program (Supplementary Fig. 8); the prediction suggested that the overall secondary structure is comparable between apo- and haem-bound forms of PGRMC1 in solution. RESULTS +71 74 NMR experimental_method We also attempted to predict the secondary structure of PGRMC1 through NMR data by calculating with TALOS+ program (Supplementary Fig. 8); the prediction suggested that the overall secondary structure is comparable between apo- and haem-bound forms of PGRMC1 in solution. RESULTS +100 114 TALOS+ program experimental_method We also attempted to predict the secondary structure of PGRMC1 through NMR data by calculating with TALOS+ program (Supplementary Fig. 8); the prediction suggested that the overall secondary structure is comparable between apo- and haem-bound forms of PGRMC1 in solution. RESULTS +223 226 apo protein_state We also attempted to predict the secondary structure of PGRMC1 through NMR data by calculating with TALOS+ program (Supplementary Fig. 8); the prediction suggested that the overall secondary structure is comparable between apo- and haem-bound forms of PGRMC1 in solution. RESULTS +232 242 haem-bound protein_state We also attempted to predict the secondary structure of PGRMC1 through NMR data by calculating with TALOS+ program (Supplementary Fig. 8); the prediction suggested that the overall secondary structure is comparable between apo- and haem-bound forms of PGRMC1 in solution. RESULTS +252 258 PGRMC1 protein We also attempted to predict the secondary structure of PGRMC1 through NMR data by calculating with TALOS+ program (Supplementary Fig. 8); the prediction suggested that the overall secondary structure is comparable between apo- and haem-bound forms of PGRMC1 in solution. RESULTS +16 20 haem chemical We analysed the haem-dependent dimerization of the PGRMC1 cytosolic domain (a.a.44–195) in solution (Fig. 2 and Table 2). RESULTS +31 43 dimerization oligomeric_state We analysed the haem-dependent dimerization of the PGRMC1 cytosolic domain (a.a.44–195) in solution (Fig. 2 and Table 2). RESULTS +51 57 PGRMC1 protein We analysed the haem-dependent dimerization of the PGRMC1 cytosolic domain (a.a.44–195) in solution (Fig. 2 and Table 2). RESULTS +58 74 cytosolic domain structure_element We analysed the haem-dependent dimerization of the PGRMC1 cytosolic domain (a.a.44–195) in solution (Fig. 2 and Table 2). RESULTS +80 86 44–195 residue_range We analysed the haem-dependent dimerization of the PGRMC1 cytosolic domain (a.a.44–195) in solution (Fig. 2 and Table 2). RESULTS +0 17 Mass spectrometry experimental_method Mass spectrometry (MS) analyses under non-denaturing condition demonstrated that the apo-monomer PGRMC1 resulted in dimerization by binding with haem (Fig. 2a). RESULTS +19 21 MS experimental_method Mass spectrometry (MS) analyses under non-denaturing condition demonstrated that the apo-monomer PGRMC1 resulted in dimerization by binding with haem (Fig. 2a). RESULTS +38 62 non-denaturing condition experimental_method Mass spectrometry (MS) analyses under non-denaturing condition demonstrated that the apo-monomer PGRMC1 resulted in dimerization by binding with haem (Fig. 2a). RESULTS +85 88 apo protein_state Mass spectrometry (MS) analyses under non-denaturing condition demonstrated that the apo-monomer PGRMC1 resulted in dimerization by binding with haem (Fig. 2a). RESULTS +89 96 monomer oligomeric_state Mass spectrometry (MS) analyses under non-denaturing condition demonstrated that the apo-monomer PGRMC1 resulted in dimerization by binding with haem (Fig. 2a). RESULTS +97 103 PGRMC1 protein Mass spectrometry (MS) analyses under non-denaturing condition demonstrated that the apo-monomer PGRMC1 resulted in dimerization by binding with haem (Fig. 2a). RESULTS +116 128 dimerization oligomeric_state Mass spectrometry (MS) analyses under non-denaturing condition demonstrated that the apo-monomer PGRMC1 resulted in dimerization by binding with haem (Fig. 2a). RESULTS +145 149 haem chemical Mass spectrometry (MS) analyses under non-denaturing condition demonstrated that the apo-monomer PGRMC1 resulted in dimerization by binding with haem (Fig. 2a). RESULTS +26 40 disulfide bond ptm It should be noted that a disulfide bond between two Cys129 residues is observed in the crystal of PGRMC1 (Fig. 1a), while Cys129 is not conserved among the MAPR family proteins (Supplementary Fig. 5a). RESULTS +53 59 Cys129 residue_name_number It should be noted that a disulfide bond between two Cys129 residues is observed in the crystal of PGRMC1 (Fig. 1a), while Cys129 is not conserved among the MAPR family proteins (Supplementary Fig. 5a). RESULTS +88 95 crystal evidence It should be noted that a disulfide bond between two Cys129 residues is observed in the crystal of PGRMC1 (Fig. 1a), while Cys129 is not conserved among the MAPR family proteins (Supplementary Fig. 5a). RESULTS +99 105 PGRMC1 protein It should be noted that a disulfide bond between two Cys129 residues is observed in the crystal of PGRMC1 (Fig. 1a), while Cys129 is not conserved among the MAPR family proteins (Supplementary Fig. 5a). RESULTS +123 129 Cys129 residue_name_number It should be noted that a disulfide bond between two Cys129 residues is observed in the crystal of PGRMC1 (Fig. 1a), while Cys129 is not conserved among the MAPR family proteins (Supplementary Fig. 5a). RESULTS +133 146 not conserved protein_state It should be noted that a disulfide bond between two Cys129 residues is observed in the crystal of PGRMC1 (Fig. 1a), while Cys129 is not conserved among the MAPR family proteins (Supplementary Fig. 5a). RESULTS +157 161 MAPR protein_type It should be noted that a disulfide bond between two Cys129 residues is observed in the crystal of PGRMC1 (Fig. 1a), while Cys129 is not conserved among the MAPR family proteins (Supplementary Fig. 5a). RESULTS +54 68 disulfide bond ptm This observation led us to examine whether or not the disulfide bond contributes to PGRMC1 dimerization. RESULTS +84 90 PGRMC1 protein This observation led us to examine whether or not the disulfide bond contributes to PGRMC1 dimerization. RESULTS +91 103 dimerization oligomeric_state This observation led us to examine whether or not the disulfide bond contributes to PGRMC1 dimerization. RESULTS +0 2 MS experimental_method MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +18 43 non-denaturing conditions experimental_method MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +68 77 Cys129Ser mutant MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +79 84 C129S mutant MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +86 92 mutant protein_state MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +96 105 dimerized protein_state MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +113 124 presence of protein_state MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +125 129 haem chemical MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +151 155 haem chemical MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +165 177 dimerization oligomeric_state MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +181 187 PGRMC1 protein MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +216 230 disulfide bond ptm MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +245 251 Cys129 residue_name_number MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a). RESULTS +17 19 MS experimental_method Supporting this, MS analyses under denaturing conditions showed that haem-mediated PGRMC1 dimer is completely dissociated into monomer, indicating that dimerization of this kind is not mediated by any covalent bond such as disulfide bond (Supplementary Fig. 9). RESULTS +35 56 denaturing conditions experimental_method Supporting this, MS analyses under denaturing conditions showed that haem-mediated PGRMC1 dimer is completely dissociated into monomer, indicating that dimerization of this kind is not mediated by any covalent bond such as disulfide bond (Supplementary Fig. 9). RESULTS +69 73 haem chemical Supporting this, MS analyses under denaturing conditions showed that haem-mediated PGRMC1 dimer is completely dissociated into monomer, indicating that dimerization of this kind is not mediated by any covalent bond such as disulfide bond (Supplementary Fig. 9). RESULTS +83 89 PGRMC1 protein Supporting this, MS analyses under denaturing conditions showed that haem-mediated PGRMC1 dimer is completely dissociated into monomer, indicating that dimerization of this kind is not mediated by any covalent bond such as disulfide bond (Supplementary Fig. 9). RESULTS +90 95 dimer oligomeric_state Supporting this, MS analyses under denaturing conditions showed that haem-mediated PGRMC1 dimer is completely dissociated into monomer, indicating that dimerization of this kind is not mediated by any covalent bond such as disulfide bond (Supplementary Fig. 9). RESULTS +127 134 monomer oligomeric_state Supporting this, MS analyses under denaturing conditions showed that haem-mediated PGRMC1 dimer is completely dissociated into monomer, indicating that dimerization of this kind is not mediated by any covalent bond such as disulfide bond (Supplementary Fig. 9). RESULTS +152 164 dimerization oligomeric_state Supporting this, MS analyses under denaturing conditions showed that haem-mediated PGRMC1 dimer is completely dissociated into monomer, indicating that dimerization of this kind is not mediated by any covalent bond such as disulfide bond (Supplementary Fig. 9). RESULTS +223 237 disulfide bond ptm Supporting this, MS analyses under denaturing conditions showed that haem-mediated PGRMC1 dimer is completely dissociated into monomer, indicating that dimerization of this kind is not mediated by any covalent bond such as disulfide bond (Supplementary Fig. 9). RESULTS +21 25 haem chemical We also analysed the haem-dependent dimerization of PGRMC1 by diffusion-ordered NMR spectroscopy (DOSY) analyses (Table 2, Supplementary Fig. 10). RESULTS +36 48 dimerization oligomeric_state We also analysed the haem-dependent dimerization of PGRMC1 by diffusion-ordered NMR spectroscopy (DOSY) analyses (Table 2, Supplementary Fig. 10). RESULTS +52 58 PGRMC1 protein We also analysed the haem-dependent dimerization of PGRMC1 by diffusion-ordered NMR spectroscopy (DOSY) analyses (Table 2, Supplementary Fig. 10). RESULTS +62 96 diffusion-ordered NMR spectroscopy experimental_method We also analysed the haem-dependent dimerization of PGRMC1 by diffusion-ordered NMR spectroscopy (DOSY) analyses (Table 2, Supplementary Fig. 10). RESULTS +98 102 DOSY experimental_method We also analysed the haem-dependent dimerization of PGRMC1 by diffusion-ordered NMR spectroscopy (DOSY) analyses (Table 2, Supplementary Fig. 10). RESULTS +31 50 hydrodynamic radius evidence The results suggested that the hydrodynamic radius of haem-bound PGRMC1 is larger than that of apo-PGRMC1. RESULTS +54 64 haem-bound protein_state The results suggested that the hydrodynamic radius of haem-bound PGRMC1 is larger than that of apo-PGRMC1. RESULTS +65 71 PGRMC1 protein The results suggested that the hydrodynamic radius of haem-bound PGRMC1 is larger than that of apo-PGRMC1. RESULTS +95 98 apo protein_state The results suggested that the hydrodynamic radius of haem-bound PGRMC1 is larger than that of apo-PGRMC1. RESULTS +99 105 PGRMC1 protein The results suggested that the hydrodynamic radius of haem-bound PGRMC1 is larger than that of apo-PGRMC1. RESULTS +52 64 dimerization oligomeric_state To further evaluate changes in molecular weights in dimerization of PGRMC1, sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis was carried out. RESULTS +68 74 PGRMC1 protein To further evaluate changes in molecular weights in dimerization of PGRMC1, sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis was carried out. RESULTS +76 129 sedimentation velocity analytical ultracentrifugation experimental_method To further evaluate changes in molecular weights in dimerization of PGRMC1, sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis was carried out. RESULTS +131 137 SV-AUC experimental_method To further evaluate changes in molecular weights in dimerization of PGRMC1, sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis was carried out. RESULTS +12 21 wild-type protein_state Whereas the wild-type (wt) apo-PGRMC1 appeared at a 1.9 S peak as monomer, the haem-binding PGRMC1 was converted into dimer at a 3.1 S peak (Fig. 2b). RESULTS +23 25 wt protein_state Whereas the wild-type (wt) apo-PGRMC1 appeared at a 1.9 S peak as monomer, the haem-binding PGRMC1 was converted into dimer at a 3.1 S peak (Fig. 2b). RESULTS +27 30 apo protein_state Whereas the wild-type (wt) apo-PGRMC1 appeared at a 1.9 S peak as monomer, the haem-binding PGRMC1 was converted into dimer at a 3.1 S peak (Fig. 2b). RESULTS +31 37 PGRMC1 protein Whereas the wild-type (wt) apo-PGRMC1 appeared at a 1.9 S peak as monomer, the haem-binding PGRMC1 was converted into dimer at a 3.1 S peak (Fig. 2b). RESULTS +66 73 monomer oligomeric_state Whereas the wild-type (wt) apo-PGRMC1 appeared at a 1.9 S peak as monomer, the haem-binding PGRMC1 was converted into dimer at a 3.1 S peak (Fig. 2b). RESULTS +79 83 haem chemical Whereas the wild-type (wt) apo-PGRMC1 appeared at a 1.9 S peak as monomer, the haem-binding PGRMC1 was converted into dimer at a 3.1 S peak (Fig. 2b). RESULTS +92 98 PGRMC1 protein Whereas the wild-type (wt) apo-PGRMC1 appeared at a 1.9 S peak as monomer, the haem-binding PGRMC1 was converted into dimer at a 3.1 S peak (Fig. 2b). RESULTS +118 123 dimer oligomeric_state Whereas the wild-type (wt) apo-PGRMC1 appeared at a 1.9 S peak as monomer, the haem-binding PGRMC1 was converted into dimer at a 3.1 S peak (Fig. 2b). RESULTS +15 20 C129S mutant Similarly, the C129S mutant of PGRMC1 converted from monomer to dimer by binding to haem (Fig. 2b). RESULTS +21 27 mutant protein_state Similarly, the C129S mutant of PGRMC1 converted from monomer to dimer by binding to haem (Fig. 2b). RESULTS +31 37 PGRMC1 protein Similarly, the C129S mutant of PGRMC1 converted from monomer to dimer by binding to haem (Fig. 2b). RESULTS +53 60 monomer oligomeric_state Similarly, the C129S mutant of PGRMC1 converted from monomer to dimer by binding to haem (Fig. 2b). RESULTS +64 69 dimer oligomeric_state Similarly, the C129S mutant of PGRMC1 converted from monomer to dimer by binding to haem (Fig. 2b). RESULTS +84 88 haem chemical Similarly, the C129S mutant of PGRMC1 converted from monomer to dimer by binding to haem (Fig. 2b). RESULTS +0 6 SV-AUC experimental_method SV-AUC analyses also allowed us to examine the stability of haem/PGRMC1 dimer. RESULTS +60 64 haem chemical SV-AUC analyses also allowed us to examine the stability of haem/PGRMC1 dimer. RESULTS +65 71 PGRMC1 protein SV-AUC analyses also allowed us to examine the stability of haem/PGRMC1 dimer. RESULTS +72 77 dimer oligomeric_state SV-AUC analyses also allowed us to examine the stability of haem/PGRMC1 dimer. RESULTS +68 78 haem-bound protein_state To this end, we used different concentrations (3.5–147 μmol l−1) of haem-bound PGRMC1 protein (a.a. 72–195), which were identical to that used in the crystallographic analysis. RESULTS +79 85 PGRMC1 protein To this end, we used different concentrations (3.5–147 μmol l−1) of haem-bound PGRMC1 protein (a.a. 72–195), which were identical to that used in the crystallographic analysis. RESULTS +100 106 72–195 residue_range To this end, we used different concentrations (3.5–147 μmol l−1) of haem-bound PGRMC1 protein (a.a. 72–195), which were identical to that used in the crystallographic analysis. RESULTS +150 175 crystallographic analysis experimental_method To this end, we used different concentrations (3.5–147 μmol l−1) of haem-bound PGRMC1 protein (a.a. 72–195), which were identical to that used in the crystallographic analysis. RESULTS +4 30 sedimentation coefficients evidence The sedimentation coefficients calculated on the basis of the crystal structure were 1.71 S for monomer and 2.56 S for dimer (Supplementary Fig. 11, upper panel). RESULTS +62 79 crystal structure evidence The sedimentation coefficients calculated on the basis of the crystal structure were 1.71 S for monomer and 2.56 S for dimer (Supplementary Fig. 11, upper panel). RESULTS +96 103 monomer oligomeric_state The sedimentation coefficients calculated on the basis of the crystal structure were 1.71 S for monomer and 2.56 S for dimer (Supplementary Fig. 11, upper panel). RESULTS +119 124 dimer oligomeric_state The sedimentation coefficients calculated on the basis of the crystal structure were 1.71 S for monomer and 2.56 S for dimer (Supplementary Fig. 11, upper panel). RESULTS +28 34 PGRMC1 protein The results showed that the PGRMC1 dimer is not dissociated into monomer at all concentrations examined (Supplementary Fig. 11, lower panel), suggesting that the Kd value of haem-mediated dimer of PGRMC1 is under 3.5 μmol l−1. RESULTS +35 40 dimer oligomeric_state The results showed that the PGRMC1 dimer is not dissociated into monomer at all concentrations examined (Supplementary Fig. 11, lower panel), suggesting that the Kd value of haem-mediated dimer of PGRMC1 is under 3.5 μmol l−1. RESULTS +65 72 monomer oligomeric_state The results showed that the PGRMC1 dimer is not dissociated into monomer at all concentrations examined (Supplementary Fig. 11, lower panel), suggesting that the Kd value of haem-mediated dimer of PGRMC1 is under 3.5 μmol l−1. RESULTS +162 164 Kd evidence The results showed that the PGRMC1 dimer is not dissociated into monomer at all concentrations examined (Supplementary Fig. 11, lower panel), suggesting that the Kd value of haem-mediated dimer of PGRMC1 is under 3.5 μmol l−1. RESULTS +174 178 haem chemical The results showed that the PGRMC1 dimer is not dissociated into monomer at all concentrations examined (Supplementary Fig. 11, lower panel), suggesting that the Kd value of haem-mediated dimer of PGRMC1 is under 3.5 μmol l−1. RESULTS +188 193 dimer oligomeric_state The results showed that the PGRMC1 dimer is not dissociated into monomer at all concentrations examined (Supplementary Fig. 11, lower panel), suggesting that the Kd value of haem-mediated dimer of PGRMC1 is under 3.5 μmol l−1. RESULTS +197 203 PGRMC1 protein The results showed that the PGRMC1 dimer is not dissociated into monomer at all concentrations examined (Supplementary Fig. 11, lower panel), suggesting that the Kd value of haem-mediated dimer of PGRMC1 is under 3.5 μmol l−1. RESULTS +38 44 PGRMC1 protein A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20 μmol l−1) (ref.) and plexin A2 receptor (dimerization Kd higher than 300 μmol l−1) (ref.). RESULTS +45 50 dimer oligomeric_state A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20 μmol l−1) (ref.) and plexin A2 receptor (dimerization Kd higher than 300 μmol l−1) (ref.). RESULTS +77 83 dimers oligomeric_state A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20 μmol l−1) (ref.) and plexin A2 receptor (dimerization Kd higher than 300 μmol l−1) (ref.). RESULTS +87 107 extracellular domain structure_element A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20 μmol l−1) (ref.) and plexin A2 receptor (dimerization Kd higher than 300 μmol l−1) (ref.). RESULTS +111 128 membrane proteins protein_type A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20 μmol l−1) (ref.) and plexin A2 receptor (dimerization Kd higher than 300 μmol l−1) (ref.). RESULTS +137 157 Toll like receptor 9 protein A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20 μmol l−1) (ref.) and plexin A2 receptor (dimerization Kd higher than 300 μmol l−1) (ref.). RESULTS +159 171 dimerization oligomeric_state A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20 μmol l−1) (ref.) and plexin A2 receptor (dimerization Kd higher than 300 μmol l−1) (ref.). RESULTS +172 174 Kd evidence A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20 μmol l−1) (ref.) and plexin A2 receptor (dimerization Kd higher than 300 μmol l−1) (ref.). RESULTS +202 220 plexin A2 receptor protein A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20 μmol l−1) (ref.) and plexin A2 receptor (dimerization Kd higher than 300 μmol l−1) (ref.). RESULTS +222 234 dimerization oligomeric_state A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20 μmol l−1) (ref.) and plexin A2 receptor (dimerization Kd higher than 300 μmol l−1) (ref.). RESULTS +235 237 Kd evidence A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20 μmol l−1) (ref.) and plexin A2 receptor (dimerization Kd higher than 300 μmol l−1) (ref.). RESULTS +43 46 apo protein_state The current analytical data confirmed that apo-PGRMC1 monomer converts into dimer by binding to haem in solution (Table 2). RESULTS +47 53 PGRMC1 protein The current analytical data confirmed that apo-PGRMC1 monomer converts into dimer by binding to haem in solution (Table 2). RESULTS +54 61 monomer oligomeric_state The current analytical data confirmed that apo-PGRMC1 monomer converts into dimer by binding to haem in solution (Table 2). RESULTS +76 81 dimer oligomeric_state The current analytical data confirmed that apo-PGRMC1 monomer converts into dimer by binding to haem in solution (Table 2). RESULTS +96 100 haem chemical The current analytical data confirmed that apo-PGRMC1 monomer converts into dimer by binding to haem in solution (Table 2). RESULTS +18 44 haem titration experiments experimental_method We also showed by haem titration experiments that haem binding to PGRMC1 was of low affinity with a Kd value of 50 nmol l−1; this is comparable with that of iron regulatory protein 2, which is known to be regulated by intracellular levels of haem (Fig. 2c and Supplementary Table 1). RESULTS +50 54 haem chemical We also showed by haem titration experiments that haem binding to PGRMC1 was of low affinity with a Kd value of 50 nmol l−1; this is comparable with that of iron regulatory protein 2, which is known to be regulated by intracellular levels of haem (Fig. 2c and Supplementary Table 1). RESULTS +66 72 PGRMC1 protein We also showed by haem titration experiments that haem binding to PGRMC1 was of low affinity with a Kd value of 50 nmol l−1; this is comparable with that of iron regulatory protein 2, which is known to be regulated by intracellular levels of haem (Fig. 2c and Supplementary Table 1). RESULTS +100 102 Kd evidence We also showed by haem titration experiments that haem binding to PGRMC1 was of low affinity with a Kd value of 50 nmol l−1; this is comparable with that of iron regulatory protein 2, which is known to be regulated by intracellular levels of haem (Fig. 2c and Supplementary Table 1). RESULTS +157 182 iron regulatory protein 2 protein We also showed by haem titration experiments that haem binding to PGRMC1 was of low affinity with a Kd value of 50 nmol l−1; this is comparable with that of iron regulatory protein 2, which is known to be regulated by intracellular levels of haem (Fig. 2c and Supplementary Table 1). RESULTS +242 246 haem chemical We also showed by haem titration experiments that haem binding to PGRMC1 was of low affinity with a Kd value of 50 nmol l−1; this is comparable with that of iron regulatory protein 2, which is known to be regulated by intracellular levels of haem (Fig. 2c and Supplementary Table 1). RESULTS +58 64 PGRMC1 protein These results raised the possibility that the function of PGRMC1 is regulated by intracellular haem concentrations. RESULTS +95 99 haem chemical These results raised the possibility that the function of PGRMC1 is regulated by intracellular haem concentrations. RESULTS +0 2 CO chemical CO inhibits haem-dependent dimerization of PGRMC1 RESULTS +12 16 haem chemical CO inhibits haem-dependent dimerization of PGRMC1 RESULTS +27 39 dimerization oligomeric_state CO inhibits haem-dependent dimerization of PGRMC1 RESULTS +43 49 PGRMC1 protein CO inhibits haem-dependent dimerization of PGRMC1 RESULTS +0 25 Crystallographic analyses experimental_method Crystallographic analyses revealed that Tyr113 of PGRMC1 is an axial ligand for haem and contributes to haem-dependent dimerization (Fig. 1a). RESULTS +40 46 Tyr113 residue_name_number Crystallographic analyses revealed that Tyr113 of PGRMC1 is an axial ligand for haem and contributes to haem-dependent dimerization (Fig. 1a). RESULTS +50 56 PGRMC1 protein Crystallographic analyses revealed that Tyr113 of PGRMC1 is an axial ligand for haem and contributes to haem-dependent dimerization (Fig. 1a). RESULTS +80 84 haem chemical Crystallographic analyses revealed that Tyr113 of PGRMC1 is an axial ligand for haem and contributes to haem-dependent dimerization (Fig. 1a). RESULTS +104 108 haem chemical Crystallographic analyses revealed that Tyr113 of PGRMC1 is an axial ligand for haem and contributes to haem-dependent dimerization (Fig. 1a). RESULTS +119 131 dimerization oligomeric_state Crystallographic analyses revealed that Tyr113 of PGRMC1 is an axial ligand for haem and contributes to haem-dependent dimerization (Fig. 1a). RESULTS +12 30 UV-visible spectra evidence Analysis of UV-visible spectra revealed that the heme of PGRMC1 is reducible from ferric to ferrous state, thus allowing CO binding (Fig. 3a). RESULTS +49 53 heme chemical Analysis of UV-visible spectra revealed that the heme of PGRMC1 is reducible from ferric to ferrous state, thus allowing CO binding (Fig. 3a). RESULTS +57 63 PGRMC1 protein Analysis of UV-visible spectra revealed that the heme of PGRMC1 is reducible from ferric to ferrous state, thus allowing CO binding (Fig. 3a). RESULTS +82 88 ferric protein_state Analysis of UV-visible spectra revealed that the heme of PGRMC1 is reducible from ferric to ferrous state, thus allowing CO binding (Fig. 3a). RESULTS +92 99 ferrous protein_state Analysis of UV-visible spectra revealed that the heme of PGRMC1 is reducible from ferric to ferrous state, thus allowing CO binding (Fig. 3a). RESULTS +121 123 CO chemical Analysis of UV-visible spectra revealed that the heme of PGRMC1 is reducible from ferric to ferrous state, thus allowing CO binding (Fig. 3a). RESULTS +17 36 UV-visible spectrum evidence Furthermore, the UV-visible spectrum of the wild type PGRMC1 was the same as that of the C129S mutant of PGRMC1, and the R/Z ratio determined by the intensities between the Soret band (394 nm) peak and the 274-nm peak showed that these proteins were fully loaded with haem (Supplementary Fig. 12). RESULTS +44 53 wild type protein_state Furthermore, the UV-visible spectrum of the wild type PGRMC1 was the same as that of the C129S mutant of PGRMC1, and the R/Z ratio determined by the intensities between the Soret band (394 nm) peak and the 274-nm peak showed that these proteins were fully loaded with haem (Supplementary Fig. 12). RESULTS +54 60 PGRMC1 protein Furthermore, the UV-visible spectrum of the wild type PGRMC1 was the same as that of the C129S mutant of PGRMC1, and the R/Z ratio determined by the intensities between the Soret band (394 nm) peak and the 274-nm peak showed that these proteins were fully loaded with haem (Supplementary Fig. 12). RESULTS +89 94 C129S mutant Furthermore, the UV-visible spectrum of the wild type PGRMC1 was the same as that of the C129S mutant of PGRMC1, and the R/Z ratio determined by the intensities between the Soret band (394 nm) peak and the 274-nm peak showed that these proteins were fully loaded with haem (Supplementary Fig. 12). RESULTS +95 101 mutant protein_state Furthermore, the UV-visible spectrum of the wild type PGRMC1 was the same as that of the C129S mutant of PGRMC1, and the R/Z ratio determined by the intensities between the Soret band (394 nm) peak and the 274-nm peak showed that these proteins were fully loaded with haem (Supplementary Fig. 12). RESULTS +105 111 PGRMC1 protein Furthermore, the UV-visible spectrum of the wild type PGRMC1 was the same as that of the C129S mutant of PGRMC1, and the R/Z ratio determined by the intensities between the Soret band (394 nm) peak and the 274-nm peak showed that these proteins were fully loaded with haem (Supplementary Fig. 12). RESULTS +121 130 R/Z ratio evidence Furthermore, the UV-visible spectrum of the wild type PGRMC1 was the same as that of the C129S mutant of PGRMC1, and the R/Z ratio determined by the intensities between the Soret band (394 nm) peak and the 274-nm peak showed that these proteins were fully loaded with haem (Supplementary Fig. 12). RESULTS +250 267 fully loaded with protein_state Furthermore, the UV-visible spectrum of the wild type PGRMC1 was the same as that of the C129S mutant of PGRMC1, and the R/Z ratio determined by the intensities between the Soret band (394 nm) peak and the 274-nm peak showed that these proteins were fully loaded with haem (Supplementary Fig. 12). RESULTS +268 272 haem chemical Furthermore, the UV-visible spectrum of the wild type PGRMC1 was the same as that of the C129S mutant of PGRMC1, and the R/Z ratio determined by the intensities between the Soret band (394 nm) peak and the 274-nm peak showed that these proteins were fully loaded with haem (Supplementary Fig. 12). RESULTS +16 22 ferric protein_state Analysis of the ferric form of PGRMC1 using resonance Raman spectroscopy (Supplementary Fig. 13) showed that the relative intensity of oxidation and spin state marker bands (ν4 and ν3) is close to 1.0, which is consistent with it being a haem protein with a proximal Tyr coordination. RESULTS +31 37 PGRMC1 protein Analysis of the ferric form of PGRMC1 using resonance Raman spectroscopy (Supplementary Fig. 13) showed that the relative intensity of oxidation and spin state marker bands (ν4 and ν3) is close to 1.0, which is consistent with it being a haem protein with a proximal Tyr coordination. RESULTS +44 72 resonance Raman spectroscopy experimental_method Analysis of the ferric form of PGRMC1 using resonance Raman spectroscopy (Supplementary Fig. 13) showed that the relative intensity of oxidation and spin state marker bands (ν4 and ν3) is close to 1.0, which is consistent with it being a haem protein with a proximal Tyr coordination. RESULTS +238 242 haem chemical Analysis of the ferric form of PGRMC1 using resonance Raman spectroscopy (Supplementary Fig. 13) showed that the relative intensity of oxidation and spin state marker bands (ν4 and ν3) is close to 1.0, which is consistent with it being a haem protein with a proximal Tyr coordination. RESULTS +267 270 Tyr residue_name Analysis of the ferric form of PGRMC1 using resonance Raman spectroscopy (Supplementary Fig. 13) showed that the relative intensity of oxidation and spin state marker bands (ν4 and ν3) is close to 1.0, which is consistent with it being a haem protein with a proximal Tyr coordination. RESULTS +11 22 Raman shift evidence A specific Raman shift peaking at vFe–CO=500 cm−1 demonstrated that the CO-bound haem of PGRMC1 is six-coordinated (Supplementary Fig. 13). RESULTS +72 80 CO-bound protein_state A specific Raman shift peaking at vFe–CO=500 cm−1 demonstrated that the CO-bound haem of PGRMC1 is six-coordinated (Supplementary Fig. 13). RESULTS +81 85 haem chemical A specific Raman shift peaking at vFe–CO=500 cm−1 demonstrated that the CO-bound haem of PGRMC1 is six-coordinated (Supplementary Fig. 13). RESULTS +89 95 PGRMC1 protein A specific Raman shift peaking at vFe–CO=500 cm−1 demonstrated that the CO-bound haem of PGRMC1 is six-coordinated (Supplementary Fig. 13). RESULTS +6 12 PGRMC1 protein Since PGRMC1 dimerization involves the open surface of haem on the opposite side of the axial Tyr113, no space for CO binding is available in the dimeric structure (Fig. 3b). RESULTS +13 25 dimerization oligomeric_state Since PGRMC1 dimerization involves the open surface of haem on the opposite side of the axial Tyr113, no space for CO binding is available in the dimeric structure (Fig. 3b). RESULTS +44 51 surface site Since PGRMC1 dimerization involves the open surface of haem on the opposite side of the axial Tyr113, no space for CO binding is available in the dimeric structure (Fig. 3b). RESULTS +55 59 haem chemical Since PGRMC1 dimerization involves the open surface of haem on the opposite side of the axial Tyr113, no space for CO binding is available in the dimeric structure (Fig. 3b). RESULTS +94 100 Tyr113 residue_name_number Since PGRMC1 dimerization involves the open surface of haem on the opposite side of the axial Tyr113, no space for CO binding is available in the dimeric structure (Fig. 3b). RESULTS +115 117 CO chemical Since PGRMC1 dimerization involves the open surface of haem on the opposite side of the axial Tyr113, no space for CO binding is available in the dimeric structure (Fig. 3b). RESULTS +146 153 dimeric oligomeric_state Since PGRMC1 dimerization involves the open surface of haem on the opposite side of the axial Tyr113, no space for CO binding is available in the dimeric structure (Fig. 3b). RESULTS +154 163 structure evidence Since PGRMC1 dimerization involves the open surface of haem on the opposite side of the axial Tyr113, no space for CO binding is available in the dimeric structure (Fig. 3b). RESULTS +27 29 CO chemical This prompted us to ask if CO binding to haem causes dissociation of the PGRMC1 dimer. RESULTS +41 45 haem chemical This prompted us to ask if CO binding to haem causes dissociation of the PGRMC1 dimer. RESULTS +73 79 PGRMC1 protein This prompted us to ask if CO binding to haem causes dissociation of the PGRMC1 dimer. RESULTS +80 85 dimer oligomeric_state This prompted us to ask if CO binding to haem causes dissociation of the PGRMC1 dimer. RESULTS +12 41 gel filtration chromatography experimental_method Analysis by gel filtration chromatography revealed that the relative molecular sizes of the wild-type and the C129S mutant of PGRMC1 are increased by adding haem to apo-PGRMC1 regardless of the oxidation state of the iron (Fig. 3c), which is in agreement with the results in Table 1. RESULTS +92 101 wild-type protein_state Analysis by gel filtration chromatography revealed that the relative molecular sizes of the wild-type and the C129S mutant of PGRMC1 are increased by adding haem to apo-PGRMC1 regardless of the oxidation state of the iron (Fig. 3c), which is in agreement with the results in Table 1. RESULTS +110 115 C129S mutant Analysis by gel filtration chromatography revealed that the relative molecular sizes of the wild-type and the C129S mutant of PGRMC1 are increased by adding haem to apo-PGRMC1 regardless of the oxidation state of the iron (Fig. 3c), which is in agreement with the results in Table 1. RESULTS +116 122 mutant protein_state Analysis by gel filtration chromatography revealed that the relative molecular sizes of the wild-type and the C129S mutant of PGRMC1 are increased by adding haem to apo-PGRMC1 regardless of the oxidation state of the iron (Fig. 3c), which is in agreement with the results in Table 1. RESULTS +126 132 PGRMC1 protein Analysis by gel filtration chromatography revealed that the relative molecular sizes of the wild-type and the C129S mutant of PGRMC1 are increased by adding haem to apo-PGRMC1 regardless of the oxidation state of the iron (Fig. 3c), which is in agreement with the results in Table 1. RESULTS +157 161 haem chemical Analysis by gel filtration chromatography revealed that the relative molecular sizes of the wild-type and the C129S mutant of PGRMC1 are increased by adding haem to apo-PGRMC1 regardless of the oxidation state of the iron (Fig. 3c), which is in agreement with the results in Table 1. RESULTS +165 168 apo protein_state Analysis by gel filtration chromatography revealed that the relative molecular sizes of the wild-type and the C129S mutant of PGRMC1 are increased by adding haem to apo-PGRMC1 regardless of the oxidation state of the iron (Fig. 3c), which is in agreement with the results in Table 1. RESULTS +169 175 PGRMC1 protein Analysis by gel filtration chromatography revealed that the relative molecular sizes of the wild-type and the C129S mutant of PGRMC1 are increased by adding haem to apo-PGRMC1 regardless of the oxidation state of the iron (Fig. 3c), which is in agreement with the results in Table 1. RESULTS +217 221 iron chemical Analysis by gel filtration chromatography revealed that the relative molecular sizes of the wild-type and the C129S mutant of PGRMC1 are increased by adding haem to apo-PGRMC1 regardless of the oxidation state of the iron (Fig. 3c), which is in agreement with the results in Table 1. RESULTS +0 2 CO chemical CO application to ferrous PGRMC1 abolished the haem-dependent increase in its molecular size. RESULTS +18 25 ferrous protein_state CO application to ferrous PGRMC1 abolished the haem-dependent increase in its molecular size. RESULTS +26 32 PGRMC1 protein CO application to ferrous PGRMC1 abolished the haem-dependent increase in its molecular size. RESULTS +47 51 haem chemical CO application to ferrous PGRMC1 abolished the haem-dependent increase in its molecular size. RESULTS +37 48 presence of protein_state Under this reducing condition in the presence of dithionite, analyses of UV-visible spectra indicated that CO-binding with haem-PGRMC1 is stable, showing only 20% reduction of the absorbance at 412 nm within 2 h (Supplementary Fig. 14). RESULTS +49 59 dithionite chemical Under this reducing condition in the presence of dithionite, analyses of UV-visible spectra indicated that CO-binding with haem-PGRMC1 is stable, showing only 20% reduction of the absorbance at 412 nm within 2 h (Supplementary Fig. 14). RESULTS +73 91 UV-visible spectra evidence Under this reducing condition in the presence of dithionite, analyses of UV-visible spectra indicated that CO-binding with haem-PGRMC1 is stable, showing only 20% reduction of the absorbance at 412 nm within 2 h (Supplementary Fig. 14). RESULTS +107 109 CO chemical Under this reducing condition in the presence of dithionite, analyses of UV-visible spectra indicated that CO-binding with haem-PGRMC1 is stable, showing only 20% reduction of the absorbance at 412 nm within 2 h (Supplementary Fig. 14). RESULTS +123 134 haem-PGRMC1 complex_assembly Under this reducing condition in the presence of dithionite, analyses of UV-visible spectra indicated that CO-binding with haem-PGRMC1 is stable, showing only 20% reduction of the absorbance at 412 nm within 2 h (Supplementary Fig. 14). RESULTS +138 144 stable protein_state Under this reducing condition in the presence of dithionite, analyses of UV-visible spectra indicated that CO-binding with haem-PGRMC1 is stable, showing only 20% reduction of the absorbance at 412 nm within 2 h (Supplementary Fig. 14). RESULTS +17 26 Tyr113Phe mutant Furthermore, the Tyr113Phe (Y113F) mutant of PGRMC1 was not responsive to haem. RESULTS +28 33 Y113F mutant Furthermore, the Tyr113Phe (Y113F) mutant of PGRMC1 was not responsive to haem. RESULTS +35 41 mutant protein_state Furthermore, the Tyr113Phe (Y113F) mutant of PGRMC1 was not responsive to haem. RESULTS +45 51 PGRMC1 protein Furthermore, the Tyr113Phe (Y113F) mutant of PGRMC1 was not responsive to haem. RESULTS +74 78 haem chemical Furthermore, the Tyr113Phe (Y113F) mutant of PGRMC1 was not responsive to haem. RESULTS +27 29 CO chemical These results suggest that CO favours the six-coordinate form of haem and interferes with the haem-mediated dimerization of PGRMC1. RESULTS +65 69 haem chemical These results suggest that CO favours the six-coordinate form of haem and interferes with the haem-mediated dimerization of PGRMC1. RESULTS +94 98 haem chemical These results suggest that CO favours the six-coordinate form of haem and interferes with the haem-mediated dimerization of PGRMC1. RESULTS +108 120 dimerization oligomeric_state These results suggest that CO favours the six-coordinate form of haem and interferes with the haem-mediated dimerization of PGRMC1. RESULTS +124 130 PGRMC1 protein These results suggest that CO favours the six-coordinate form of haem and interferes with the haem-mediated dimerization of PGRMC1. RESULTS +37 39 CO chemical To examine the inhibitory effects of CO on haem-mediated PGRMC1 dimerization, SV-AUC analysis was carried out. RESULTS +43 47 haem chemical To examine the inhibitory effects of CO on haem-mediated PGRMC1 dimerization, SV-AUC analysis was carried out. RESULTS +57 63 PGRMC1 protein To examine the inhibitory effects of CO on haem-mediated PGRMC1 dimerization, SV-AUC analysis was carried out. RESULTS +64 76 dimerization oligomeric_state To examine the inhibitory effects of CO on haem-mediated PGRMC1 dimerization, SV-AUC analysis was carried out. RESULTS +78 84 SV-AUC experimental_method To examine the inhibitory effects of CO on haem-mediated PGRMC1 dimerization, SV-AUC analysis was carried out. RESULTS +30 34 haem chemical The peak corresponding to the haem/PGRMC1 dimer was detected under reducing conditions in the presence of dithionite (Supplementary Fig. 15, middle panel). RESULTS +35 41 PGRMC1 protein The peak corresponding to the haem/PGRMC1 dimer was detected under reducing conditions in the presence of dithionite (Supplementary Fig. 15, middle panel). RESULTS +42 47 dimer oligomeric_state The peak corresponding to the haem/PGRMC1 dimer was detected under reducing conditions in the presence of dithionite (Supplementary Fig. 15, middle panel). RESULTS +94 105 presence of protein_state The peak corresponding to the haem/PGRMC1 dimer was detected under reducing conditions in the presence of dithionite (Supplementary Fig. 15, middle panel). RESULTS +106 116 dithionite chemical The peak corresponding to the haem/PGRMC1 dimer was detected under reducing conditions in the presence of dithionite (Supplementary Fig. 15, middle panel). RESULTS +27 29 CO chemical Under these circumstances, CO application induced dissociation of the haem-mediated dimers of PGRMC1 to generate a peak of monomers (Supplementary Fig. 15, lower panel). RESULTS +70 74 haem chemical Under these circumstances, CO application induced dissociation of the haem-mediated dimers of PGRMC1 to generate a peak of monomers (Supplementary Fig. 15, lower panel). RESULTS +84 90 dimers oligomeric_state Under these circumstances, CO application induced dissociation of the haem-mediated dimers of PGRMC1 to generate a peak of monomers (Supplementary Fig. 15, lower panel). RESULTS +94 100 PGRMC1 protein Under these circumstances, CO application induced dissociation of the haem-mediated dimers of PGRMC1 to generate a peak of monomers (Supplementary Fig. 15, lower panel). RESULTS +123 131 monomers oligomeric_state Under these circumstances, CO application induced dissociation of the haem-mediated dimers of PGRMC1 to generate a peak of monomers (Supplementary Fig. 15, lower panel). RESULTS +76 82 PGRMC1 protein These observations raised the transition model for structural regulation of PGRMC1 in response to haem (Fig. 3d). RESULTS +98 102 haem chemical These observations raised the transition model for structural regulation of PGRMC1 in response to haem (Fig. 3d). RESULTS +20 23 apo protein_state As mentioned above, apo-PGRMC1 exists as monomer. RESULTS +24 30 PGRMC1 protein As mentioned above, apo-PGRMC1 exists as monomer. RESULTS +41 48 monomer oligomeric_state As mentioned above, apo-PGRMC1 exists as monomer. RESULTS +16 20 haem chemical By binding with haem (binding Kd=50 nmol l−1), PGRMC1 forms a stable dimer (dimerization Kd<<3.5 μmol l−1) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer. RESULTS +30 32 Kd evidence By binding with haem (binding Kd=50 nmol l−1), PGRMC1 forms a stable dimer (dimerization Kd<<3.5 μmol l−1) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer. RESULTS +47 53 PGRMC1 protein By binding with haem (binding Kd=50 nmol l−1), PGRMC1 forms a stable dimer (dimerization Kd<<3.5 μmol l−1) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer. RESULTS +62 68 stable protein_state By binding with haem (binding Kd=50 nmol l−1), PGRMC1 forms a stable dimer (dimerization Kd<<3.5 μmol l−1) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer. RESULTS +69 74 dimer oligomeric_state By binding with haem (binding Kd=50 nmol l−1), PGRMC1 forms a stable dimer (dimerization Kd<<3.5 μmol l−1) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer. RESULTS +76 88 dimerization oligomeric_state By binding with haem (binding Kd=50 nmol l−1), PGRMC1 forms a stable dimer (dimerization Kd<<3.5 μmol l−1) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer. RESULTS +89 91 Kd evidence By binding with haem (binding Kd=50 nmol l−1), PGRMC1 forms a stable dimer (dimerization Kd<<3.5 μmol l−1) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer. RESULTS +115 123 stacking bond_interaction By binding with haem (binding Kd=50 nmol l−1), PGRMC1 forms a stable dimer (dimerization Kd<<3.5 μmol l−1) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer. RESULTS +140 148 surfaces site By binding with haem (binding Kd=50 nmol l−1), PGRMC1 forms a stable dimer (dimerization Kd<<3.5 μmol l−1) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer. RESULTS +173 177 haem chemical By binding with haem (binding Kd=50 nmol l−1), PGRMC1 forms a stable dimer (dimerization Kd<<3.5 μmol l−1) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer. RESULTS +196 203 monomer oligomeric_state By binding with haem (binding Kd=50 nmol l−1), PGRMC1 forms a stable dimer (dimerization Kd<<3.5 μmol l−1) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer. RESULTS +0 2 CO chemical CO induces the dissociation of the haem-mediated dimer of PGRMC1 by interfering with the haem-stacking interface via formation of the six-coordinated CO-haem-PGRMC1 complex. RESULTS +35 39 haem chemical CO induces the dissociation of the haem-mediated dimer of PGRMC1 by interfering with the haem-stacking interface via formation of the six-coordinated CO-haem-PGRMC1 complex. RESULTS +49 54 dimer oligomeric_state CO induces the dissociation of the haem-mediated dimer of PGRMC1 by interfering with the haem-stacking interface via formation of the six-coordinated CO-haem-PGRMC1 complex. RESULTS +58 64 PGRMC1 protein CO induces the dissociation of the haem-mediated dimer of PGRMC1 by interfering with the haem-stacking interface via formation of the six-coordinated CO-haem-PGRMC1 complex. RESULTS +89 112 haem-stacking interface site CO induces the dissociation of the haem-mediated dimer of PGRMC1 by interfering with the haem-stacking interface via formation of the six-coordinated CO-haem-PGRMC1 complex. RESULTS +150 164 CO-haem-PGRMC1 complex_assembly CO induces the dissociation of the haem-mediated dimer of PGRMC1 by interfering with the haem-stacking interface via formation of the six-coordinated CO-haem-PGRMC1 complex. RESULTS +81 87 PGRMC1 protein Such a dynamic structural regulation led us to further examine the regulation of PGRMC1 functions in cancer cells. RESULTS +0 6 PGRMC1 protein PGRMC1 dimerization is required for binding to EGFR RESULTS +7 19 dimerization oligomeric_state PGRMC1 dimerization is required for binding to EGFR RESULTS +47 51 EGFR protein_type PGRMC1 dimerization is required for binding to EGFR RESULTS +8 14 PGRMC1 protein Because PGRMC1 is known to interact with EGFR and to accelerate tumour progression, we examined the effect of haem-dependent dimerization of PGRMC1 on its interaction with EGFR by using purified proteins. RESULTS +41 45 EGFR protein_type Because PGRMC1 is known to interact with EGFR and to accelerate tumour progression, we examined the effect of haem-dependent dimerization of PGRMC1 on its interaction with EGFR by using purified proteins. RESULTS +110 114 haem chemical Because PGRMC1 is known to interact with EGFR and to accelerate tumour progression, we examined the effect of haem-dependent dimerization of PGRMC1 on its interaction with EGFR by using purified proteins. RESULTS +125 137 dimerization oligomeric_state Because PGRMC1 is known to interact with EGFR and to accelerate tumour progression, we examined the effect of haem-dependent dimerization of PGRMC1 on its interaction with EGFR by using purified proteins. RESULTS +141 147 PGRMC1 protein Because PGRMC1 is known to interact with EGFR and to accelerate tumour progression, we examined the effect of haem-dependent dimerization of PGRMC1 on its interaction with EGFR by using purified proteins. RESULTS +172 176 EGFR protein_type Because PGRMC1 is known to interact with EGFR and to accelerate tumour progression, we examined the effect of haem-dependent dimerization of PGRMC1 on its interaction with EGFR by using purified proteins. RESULTS +25 41 cytosolic domain structure_element As shown in Fig. 4a, the cytosolic domain of wild-type PGRMC1, but not the Y113F mutant, interacted with purified EGFR in a haem-dependent manner. RESULTS +45 54 wild-type protein_state As shown in Fig. 4a, the cytosolic domain of wild-type PGRMC1, but not the Y113F mutant, interacted with purified EGFR in a haem-dependent manner. RESULTS +55 61 PGRMC1 protein As shown in Fig. 4a, the cytosolic domain of wild-type PGRMC1, but not the Y113F mutant, interacted with purified EGFR in a haem-dependent manner. RESULTS +75 80 Y113F mutant As shown in Fig. 4a, the cytosolic domain of wild-type PGRMC1, but not the Y113F mutant, interacted with purified EGFR in a haem-dependent manner. RESULTS +81 87 mutant protein_state As shown in Fig. 4a, the cytosolic domain of wild-type PGRMC1, but not the Y113F mutant, interacted with purified EGFR in a haem-dependent manner. RESULTS +114 118 EGFR protein_type As shown in Fig. 4a, the cytosolic domain of wild-type PGRMC1, but not the Y113F mutant, interacted with purified EGFR in a haem-dependent manner. RESULTS +124 128 haem chemical As shown in Fig. 4a, the cytosolic domain of wild-type PGRMC1, but not the Y113F mutant, interacted with purified EGFR in a haem-dependent manner. RESULTS +38 47 ruthenium chemical This interaction was disrupted by the ruthenium-based CO-releasing molecule, CORM3, but not by RuCl3 as a control reagent (Fig. 4b). RESULTS +54 56 CO chemical This interaction was disrupted by the ruthenium-based CO-releasing molecule, CORM3, but not by RuCl3 as a control reagent (Fig. 4b). RESULTS +77 82 CORM3 chemical This interaction was disrupted by the ruthenium-based CO-releasing molecule, CORM3, but not by RuCl3 as a control reagent (Fig. 4b). RESULTS +95 100 RuCl3 chemical This interaction was disrupted by the ruthenium-based CO-releasing molecule, CORM3, but not by RuCl3 as a control reagent (Fig. 4b). RESULTS +58 64 PGRMC1 protein We further analysed the intracellular interaction between PGRMC1 and EGFR. RESULTS +69 73 EGFR protein_type We further analysed the intracellular interaction between PGRMC1 and EGFR. RESULTS +0 11 FLAG-tagged protein_state FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c). RESULTS +12 18 PGRMC1 protein FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c). RESULTS +19 40 ectopically expressed experimental_method FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c). RESULTS +44 49 human species FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c). RESULTS +80 98 immunoprecipitated experimental_method FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c). RESULTS +128 149 co-immunoprecipitated experimental_method FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c). RESULTS +150 154 EGFR protein_type FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c). RESULTS +159 169 endogenous protein_state FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c). RESULTS +170 176 PGRMC1 protein FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c). RESULTS +193 199 PGRMC1 protein FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c). RESULTS +217 233 Western blotting experimental_method FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c). RESULTS +4 9 C129S mutant The C129S mutant of PGRMC1 also interacted with endogenous PGRMC1 and EGFR (Supplementary Fig. 16). RESULTS +10 16 mutant protein_state The C129S mutant of PGRMC1 also interacted with endogenous PGRMC1 and EGFR (Supplementary Fig. 16). RESULTS +20 26 PGRMC1 protein The C129S mutant of PGRMC1 also interacted with endogenous PGRMC1 and EGFR (Supplementary Fig. 16). RESULTS +48 58 endogenous protein_state The C129S mutant of PGRMC1 also interacted with endogenous PGRMC1 and EGFR (Supplementary Fig. 16). RESULTS +59 65 PGRMC1 protein The C129S mutant of PGRMC1 also interacted with endogenous PGRMC1 and EGFR (Supplementary Fig. 16). RESULTS +70 74 EGFR protein_type The C129S mutant of PGRMC1 also interacted with endogenous PGRMC1 and EGFR (Supplementary Fig. 16). RESULTS +8 19 FLAG-tagged protein_state Whereas FLAG-tagged wild-type PGRMC1 interacted with endogenous PGRMC1 and EGFR, the Y113F mutant did not. RESULTS +20 29 wild-type protein_state Whereas FLAG-tagged wild-type PGRMC1 interacted with endogenous PGRMC1 and EGFR, the Y113F mutant did not. RESULTS +30 36 PGRMC1 protein Whereas FLAG-tagged wild-type PGRMC1 interacted with endogenous PGRMC1 and EGFR, the Y113F mutant did not. RESULTS +53 63 endogenous protein_state Whereas FLAG-tagged wild-type PGRMC1 interacted with endogenous PGRMC1 and EGFR, the Y113F mutant did not. RESULTS +64 70 PGRMC1 protein Whereas FLAG-tagged wild-type PGRMC1 interacted with endogenous PGRMC1 and EGFR, the Y113F mutant did not. RESULTS +75 79 EGFR protein_type Whereas FLAG-tagged wild-type PGRMC1 interacted with endogenous PGRMC1 and EGFR, the Y113F mutant did not. RESULTS +85 90 Y113F mutant Whereas FLAG-tagged wild-type PGRMC1 interacted with endogenous PGRMC1 and EGFR, the Y113F mutant did not. RESULTS +91 97 mutant protein_state Whereas FLAG-tagged wild-type PGRMC1 interacted with endogenous PGRMC1 and EGFR, the Y113F mutant did not. RESULTS +31 46 succinylacetone chemical We also examined the effect of succinylacetone (SA), an inhibitor of haem biosynthesis (Fig. 4d). RESULTS +48 50 SA chemical We also examined the effect of succinylacetone (SA), an inhibitor of haem biosynthesis (Fig. 4d). RESULTS +69 73 haem chemical We also examined the effect of succinylacetone (SA), an inhibitor of haem biosynthesis (Fig. 4d). RESULTS +13 15 SA chemical As expected, SA significantly reduced PGRMC1 dimerization and its interaction with EGFR (Fig. 4e), indicating that haem-mediated dimerization of PGMRC1 is critical for its binding to EGFR. RESULTS +30 37 reduced protein_state As expected, SA significantly reduced PGRMC1 dimerization and its interaction with EGFR (Fig. 4e), indicating that haem-mediated dimerization of PGMRC1 is critical for its binding to EGFR. RESULTS +38 44 PGRMC1 protein As expected, SA significantly reduced PGRMC1 dimerization and its interaction with EGFR (Fig. 4e), indicating that haem-mediated dimerization of PGMRC1 is critical for its binding to EGFR. RESULTS +45 57 dimerization oligomeric_state As expected, SA significantly reduced PGRMC1 dimerization and its interaction with EGFR (Fig. 4e), indicating that haem-mediated dimerization of PGMRC1 is critical for its binding to EGFR. RESULTS +83 87 EGFR protein_type As expected, SA significantly reduced PGRMC1 dimerization and its interaction with EGFR (Fig. 4e), indicating that haem-mediated dimerization of PGMRC1 is critical for its binding to EGFR. RESULTS +115 119 haem chemical As expected, SA significantly reduced PGRMC1 dimerization and its interaction with EGFR (Fig. 4e), indicating that haem-mediated dimerization of PGMRC1 is critical for its binding to EGFR. RESULTS +129 141 dimerization oligomeric_state As expected, SA significantly reduced PGRMC1 dimerization and its interaction with EGFR (Fig. 4e), indicating that haem-mediated dimerization of PGMRC1 is critical for its binding to EGFR. RESULTS +145 151 PGMRC1 protein As expected, SA significantly reduced PGRMC1 dimerization and its interaction with EGFR (Fig. 4e), indicating that haem-mediated dimerization of PGMRC1 is critical for its binding to EGFR. RESULTS +183 187 EGFR protein_type As expected, SA significantly reduced PGRMC1 dimerization and its interaction with EGFR (Fig. 4e), indicating that haem-mediated dimerization of PGMRC1 is critical for its binding to EGFR. RESULTS +0 6 PGRMC1 protein PGRMC1 dimer facilitates EGFR-mediated cancer growth RESULTS +7 12 dimer oligomeric_state PGRMC1 dimer facilitates EGFR-mediated cancer growth RESULTS +25 29 EGFR protein_type PGRMC1 dimer facilitates EGFR-mediated cancer growth RESULTS +53 59 PGRMC1 protein Next, we investigated the functional significance of PGRMC1 dimerization in EGFR signaling. RESULTS +60 72 dimerization oligomeric_state Next, we investigated the functional significance of PGRMC1 dimerization in EGFR signaling. RESULTS +76 80 EGFR protein_type Next, we investigated the functional significance of PGRMC1 dimerization in EGFR signaling. RESULTS +0 3 EGF protein_type EGF-induced phosphorylations of EGFR and its downstream targets AKT and ERK were decreased by PGRMC1 knockdown (PGRMC1-KD) (Fig. 4f). RESULTS +12 28 phosphorylations ptm EGF-induced phosphorylations of EGFR and its downstream targets AKT and ERK were decreased by PGRMC1 knockdown (PGRMC1-KD) (Fig. 4f). RESULTS +32 36 EGFR protein_type EGF-induced phosphorylations of EGFR and its downstream targets AKT and ERK were decreased by PGRMC1 knockdown (PGRMC1-KD) (Fig. 4f). RESULTS +64 67 AKT protein_type EGF-induced phosphorylations of EGFR and its downstream targets AKT and ERK were decreased by PGRMC1 knockdown (PGRMC1-KD) (Fig. 4f). RESULTS +72 75 ERK protein_type EGF-induced phosphorylations of EGFR and its downstream targets AKT and ERK were decreased by PGRMC1 knockdown (PGRMC1-KD) (Fig. 4f). RESULTS +94 100 PGRMC1 protein EGF-induced phosphorylations of EGFR and its downstream targets AKT and ERK were decreased by PGRMC1 knockdown (PGRMC1-KD) (Fig. 4f). RESULTS +101 110 knockdown protein_state EGF-induced phosphorylations of EGFR and its downstream targets AKT and ERK were decreased by PGRMC1 knockdown (PGRMC1-KD) (Fig. 4f). RESULTS +112 121 PGRMC1-KD mutant EGF-induced phosphorylations of EGFR and its downstream targets AKT and ERK were decreased by PGRMC1 knockdown (PGRMC1-KD) (Fig. 4f). RESULTS +11 15 EGFR protein_type Similarly, EGFR signaling was suppressed by treatment of HCT116 cells with SA (Fig. 4g) or CORM3 (Fig. 4h). RESULTS +75 77 SA chemical Similarly, EGFR signaling was suppressed by treatment of HCT116 cells with SA (Fig. 4g) or CORM3 (Fig. 4h). RESULTS +91 96 CORM3 chemical Similarly, EGFR signaling was suppressed by treatment of HCT116 cells with SA (Fig. 4g) or CORM3 (Fig. 4h). RESULTS +29 33 haem chemical These results suggested that haem-mediated dimerization of PGRMC1 is critical for EGFR signaling. RESULTS +43 55 dimerization oligomeric_state These results suggested that haem-mediated dimerization of PGRMC1 is critical for EGFR signaling. RESULTS +59 65 PGRMC1 protein These results suggested that haem-mediated dimerization of PGRMC1 is critical for EGFR signaling. RESULTS +82 86 EGFR protein_type These results suggested that haem-mediated dimerization of PGRMC1 is critical for EGFR signaling. RESULTS +39 48 dimerized protein_state To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). RESULTS +57 63 PGRMC1 protein To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). RESULTS +102 108 PGRMC1 protein To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). RESULTS +109 137 knockdown-rescue experiments experimental_method To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). RESULTS +144 155 FLAG-tagged protein_state To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). RESULTS +156 165 wild-type protein_state To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). RESULTS +170 175 Y113F mutant To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). RESULTS +176 182 PGRMC1 protein To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). RESULTS +183 201 expression vectors experimental_method To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). RESULTS +212 228 silent mutations experimental_method To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). RESULTS +234 244 introduced experimental_method To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). RESULTS +286 291 shRNA chemical To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a). RESULTS +56 69 knocking down experimental_method While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b). RESULTS +70 76 PGRMC1 protein While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b). RESULTS +78 87 PGRMC1-KD mutant While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b). RESULTS +121 125 EGFR protein_type While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b). RESULTS +136 145 erlotinib chemical While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b). RESULTS +214 227 co-expression experimental_method While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b). RESULTS +231 246 shRNA-resistant protein_state While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b). RESULTS +247 256 wild-type protein_state While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b). RESULTS +257 263 PGRMC1 protein While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b). RESULTS +279 284 Y113F mutant While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b). RESULTS +285 291 mutant protein_state While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b). RESULTS +46 52 shRNAs chemical Chemosensitivity enhancement by two different shRNAs to PGRMC1 was seen also in HCT116 cells and human hepatoma HuH7 cells (Supplementary Fig. 17). RESULTS +56 62 PGRMC1 protein Chemosensitivity enhancement by two different shRNAs to PGRMC1 was seen also in HCT116 cells and human hepatoma HuH7 cells (Supplementary Fig. 17). RESULTS +97 102 human species Chemosensitivity enhancement by two different shRNAs to PGRMC1 was seen also in HCT116 cells and human hepatoma HuH7 cells (Supplementary Fig. 17). RESULTS +13 22 PGRMC1-KD mutant Furthermore, PGRMC1-KD inhibited spheroid formation of HCT116 cells in culture, and this inhibition was reversed by co-expression of wild-type PGRMC1 but not of the Y113F mutant (Fig. 5c and Supplementary Fig. 18). RESULTS +116 129 co-expression experimental_method Furthermore, PGRMC1-KD inhibited spheroid formation of HCT116 cells in culture, and this inhibition was reversed by co-expression of wild-type PGRMC1 but not of the Y113F mutant (Fig. 5c and Supplementary Fig. 18). RESULTS +133 142 wild-type protein_state Furthermore, PGRMC1-KD inhibited spheroid formation of HCT116 cells in culture, and this inhibition was reversed by co-expression of wild-type PGRMC1 but not of the Y113F mutant (Fig. 5c and Supplementary Fig. 18). RESULTS +143 149 PGRMC1 protein Furthermore, PGRMC1-KD inhibited spheroid formation of HCT116 cells in culture, and this inhibition was reversed by co-expression of wild-type PGRMC1 but not of the Y113F mutant (Fig. 5c and Supplementary Fig. 18). RESULTS +165 170 Y113F mutant Furthermore, PGRMC1-KD inhibited spheroid formation of HCT116 cells in culture, and this inhibition was reversed by co-expression of wild-type PGRMC1 but not of the Y113F mutant (Fig. 5c and Supplementary Fig. 18). RESULTS +171 177 mutant protein_state Furthermore, PGRMC1-KD inhibited spheroid formation of HCT116 cells in culture, and this inhibition was reversed by co-expression of wild-type PGRMC1 but not of the Y113F mutant (Fig. 5c and Supplementary Fig. 18). RESULTS +6 12 PGRMC1 protein Thus, PGRMC1 dimerization is important for cancer cell proliferation and chemoresistance. RESULTS +13 25 dimerization oligomeric_state Thus, PGRMC1 dimerization is important for cancer cell proliferation and chemoresistance. RESULTS +24 30 PGRMC1 protein We examined the role of PGRMC1 in metastatic progression by xenograft transplantation assays using super-immunodeficient NOD/scid/γnull (NOG) mice. RESULTS +60 92 xenograft transplantation assays experimental_method We examined the role of PGRMC1 in metastatic progression by xenograft transplantation assays using super-immunodeficient NOD/scid/γnull (NOG) mice. RESULTS +15 41 intra-splenic implantation experimental_method Ten days after intra-splenic implantation of HCT116 cells that were genetically tagged with a fluorescent protein Venus, the group implanted with PGRMC1-KD cells showed a significant decrease of liver metastasis in comparison with the control group (Fig. 5d). RESULTS +146 155 PGRMC1-KD mutant Ten days after intra-splenic implantation of HCT116 cells that were genetically tagged with a fluorescent protein Venus, the group implanted with PGRMC1-KD cells showed a significant decrease of liver metastasis in comparison with the control group (Fig. 5d). RESULTS +15 21 PGRMC1 protein Interaction of PGRMC1 dimer with cytochromes P450 RESULTS +22 27 dimer oligomeric_state Interaction of PGRMC1 dimer with cytochromes P450 RESULTS +33 49 cytochromes P450 protein_type Interaction of PGRMC1 dimer with cytochromes P450 RESULTS +6 12 PGRMC1 protein Since PGRMC1 has been shown to interact with cytochromes P450 (ref), we investigated whether the haem-mediated dimerization of PGRMC1 is necessary for their interactions. RESULTS +45 61 cytochromes P450 protein_type Since PGRMC1 has been shown to interact with cytochromes P450 (ref), we investigated whether the haem-mediated dimerization of PGRMC1 is necessary for their interactions. RESULTS +97 101 haem chemical Since PGRMC1 has been shown to interact with cytochromes P450 (ref), we investigated whether the haem-mediated dimerization of PGRMC1 is necessary for their interactions. RESULTS +111 123 dimerization oligomeric_state Since PGRMC1 has been shown to interact with cytochromes P450 (ref), we investigated whether the haem-mediated dimerization of PGRMC1 is necessary for their interactions. RESULTS +127 133 PGRMC1 protein Since PGRMC1 has been shown to interact with cytochromes P450 (ref), we investigated whether the haem-mediated dimerization of PGRMC1 is necessary for their interactions. RESULTS +12 18 CYP1A2 protein Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b). RESULTS +23 29 CYP3A4 protein Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b). RESULTS +76 89 cytochrome b5 protein_type Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b). RESULTS +94 119 cytochrome P450 reductase protein Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b). RESULTS +139 155 cytochromes P450 protein_type Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b). RESULTS +173 182 wild-type protein_state Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b). RESULTS +183 189 PGRMC1 protein Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b). RESULTS +208 213 Y113F mutant Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b). RESULTS +214 220 mutant protein_state Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b). RESULTS +227 231 haem chemical Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b). RESULTS +29 35 PGRMC1 protein Moreover, the interaction of PGRMC1 with CYP1A2 was blocked by CORM3 under reducing conditions (Fig. 6c), indicating that PGRMC1 dimerization is necessary for its interaction with cytochromes P450. RESULTS +41 47 CYP1A2 protein Moreover, the interaction of PGRMC1 with CYP1A2 was blocked by CORM3 under reducing conditions (Fig. 6c), indicating that PGRMC1 dimerization is necessary for its interaction with cytochromes P450. RESULTS +63 68 CORM3 chemical Moreover, the interaction of PGRMC1 with CYP1A2 was blocked by CORM3 under reducing conditions (Fig. 6c), indicating that PGRMC1 dimerization is necessary for its interaction with cytochromes P450. RESULTS +122 128 PGRMC1 protein Moreover, the interaction of PGRMC1 with CYP1A2 was blocked by CORM3 under reducing conditions (Fig. 6c), indicating that PGRMC1 dimerization is necessary for its interaction with cytochromes P450. RESULTS +129 141 dimerization oligomeric_state Moreover, the interaction of PGRMC1 with CYP1A2 was blocked by CORM3 under reducing conditions (Fig. 6c), indicating that PGRMC1 dimerization is necessary for its interaction with cytochromes P450. RESULTS +180 196 cytochromes P450 protein_type Moreover, the interaction of PGRMC1 with CYP1A2 was blocked by CORM3 under reducing conditions (Fig. 6c), indicating that PGRMC1 dimerization is necessary for its interaction with cytochromes P450. RESULTS +0 11 Doxorubicin chemical Doxorubicin is an anti-cancer reagent that is metabolized into inactive doxorubicinol by CYP2D6 and CYP3A4 (Fig. 6d). RESULTS +72 85 doxorubicinol chemical Doxorubicin is an anti-cancer reagent that is metabolized into inactive doxorubicinol by CYP2D6 and CYP3A4 (Fig. 6d). RESULTS +89 95 CYP2D6 protein Doxorubicin is an anti-cancer reagent that is metabolized into inactive doxorubicinol by CYP2D6 and CYP3A4 (Fig. 6d). RESULTS +100 106 CYP3A4 protein Doxorubicin is an anti-cancer reagent that is metabolized into inactive doxorubicinol by CYP2D6 and CYP3A4 (Fig. 6d). RESULTS +0 9 PGRMC1-KD mutant PGRMC1-KD significantly suppressed the conversion of doxorubicin to doxorubicinol (Fig. 6d) and increased sensitivity to doxorubicin (Fig. 6e). RESULTS +53 64 doxorubicin chemical PGRMC1-KD significantly suppressed the conversion of doxorubicin to doxorubicinol (Fig. 6d) and increased sensitivity to doxorubicin (Fig. 6e). RESULTS +68 81 doxorubicinol chemical PGRMC1-KD significantly suppressed the conversion of doxorubicin to doxorubicinol (Fig. 6d) and increased sensitivity to doxorubicin (Fig. 6e). RESULTS +121 132 doxorubicin chemical PGRMC1-KD significantly suppressed the conversion of doxorubicin to doxorubicinol (Fig. 6d) and increased sensitivity to doxorubicin (Fig. 6e). RESULTS +9 20 doxorubicin chemical Enhanced doxorubicin sensitivity was modestly but significantly induced by PGRMC1-KD. RESULTS +75 84 PGRMC1-KD mutant Enhanced doxorubicin sensitivity was modestly but significantly induced by PGRMC1-KD. RESULTS +28 41 co-expression experimental_method This effect was reversed by co-expression of the wild-type PGRMC1 but not of the Y113F mutant, suggesting that PGRMC1 enhances doxorubicin resistance of cancer cells by facilitating its degradation via cytochromes P450. RESULTS +49 58 wild-type protein_state This effect was reversed by co-expression of the wild-type PGRMC1 but not of the Y113F mutant, suggesting that PGRMC1 enhances doxorubicin resistance of cancer cells by facilitating its degradation via cytochromes P450. RESULTS +59 65 PGRMC1 protein This effect was reversed by co-expression of the wild-type PGRMC1 but not of the Y113F mutant, suggesting that PGRMC1 enhances doxorubicin resistance of cancer cells by facilitating its degradation via cytochromes P450. RESULTS +81 86 Y113F mutant This effect was reversed by co-expression of the wild-type PGRMC1 but not of the Y113F mutant, suggesting that PGRMC1 enhances doxorubicin resistance of cancer cells by facilitating its degradation via cytochromes P450. RESULTS +87 93 mutant protein_state This effect was reversed by co-expression of the wild-type PGRMC1 but not of the Y113F mutant, suggesting that PGRMC1 enhances doxorubicin resistance of cancer cells by facilitating its degradation via cytochromes P450. RESULTS +111 117 PGRMC1 protein This effect was reversed by co-expression of the wild-type PGRMC1 but not of the Y113F mutant, suggesting that PGRMC1 enhances doxorubicin resistance of cancer cells by facilitating its degradation via cytochromes P450. RESULTS +127 138 doxorubicin chemical This effect was reversed by co-expression of the wild-type PGRMC1 but not of the Y113F mutant, suggesting that PGRMC1 enhances doxorubicin resistance of cancer cells by facilitating its degradation via cytochromes P450. RESULTS +202 218 cytochromes P450 protein_type This effect was reversed by co-expression of the wild-type PGRMC1 but not of the Y113F mutant, suggesting that PGRMC1 enhances doxorubicin resistance of cancer cells by facilitating its degradation via cytochromes P450. RESULTS +53 59 PGRMC1 protein To gain further insight into the interaction between PGRMC1 and cytochromes P450, surface plasmon resonance analyses were conducted using recombinant CYP51 and PGRMC1. RESULTS +64 80 cytochromes P450 protein_type To gain further insight into the interaction between PGRMC1 and cytochromes P450, surface plasmon resonance analyses were conducted using recombinant CYP51 and PGRMC1. RESULTS +82 116 surface plasmon resonance analyses experimental_method To gain further insight into the interaction between PGRMC1 and cytochromes P450, surface plasmon resonance analyses were conducted using recombinant CYP51 and PGRMC1. RESULTS +150 155 CYP51 protein To gain further insight into the interaction between PGRMC1 and cytochromes P450, surface plasmon resonance analyses were conducted using recombinant CYP51 and PGRMC1. RESULTS +160 166 PGRMC1 protein To gain further insight into the interaction between PGRMC1 and cytochromes P450, surface plasmon resonance analyses were conducted using recombinant CYP51 and PGRMC1. RESULTS +48 54 PGRMC1 protein This was based on a previous study showing that PGRMC1 binds to CYP51 and enhances cholesterol biosynthesis by CYP51 (refs). RESULTS +64 69 CYP51 protein This was based on a previous study showing that PGRMC1 binds to CYP51 and enhances cholesterol biosynthesis by CYP51 (refs). RESULTS +111 116 CYP51 protein This was based on a previous study showing that PGRMC1 binds to CYP51 and enhances cholesterol biosynthesis by CYP51 (refs). RESULTS +0 5 CYP51 protein CYP51 interacted with PGRMC1 in a concentration-dependent manner in the presence of haem, but not in its absence (Supplementary Fig. 19), suggesting the requirement for the haem-dependent dimerization of PGRMC1. RESULTS +22 28 PGRMC1 protein CYP51 interacted with PGRMC1 in a concentration-dependent manner in the presence of haem, but not in its absence (Supplementary Fig. 19), suggesting the requirement for the haem-dependent dimerization of PGRMC1. RESULTS +72 83 presence of protein_state CYP51 interacted with PGRMC1 in a concentration-dependent manner in the presence of haem, but not in its absence (Supplementary Fig. 19), suggesting the requirement for the haem-dependent dimerization of PGRMC1. RESULTS +84 88 haem chemical CYP51 interacted with PGRMC1 in a concentration-dependent manner in the presence of haem, but not in its absence (Supplementary Fig. 19), suggesting the requirement for the haem-dependent dimerization of PGRMC1. RESULTS +105 112 absence protein_state CYP51 interacted with PGRMC1 in a concentration-dependent manner in the presence of haem, but not in its absence (Supplementary Fig. 19), suggesting the requirement for the haem-dependent dimerization of PGRMC1. RESULTS +173 177 haem chemical CYP51 interacted with PGRMC1 in a concentration-dependent manner in the presence of haem, but not in its absence (Supplementary Fig. 19), suggesting the requirement for the haem-dependent dimerization of PGRMC1. RESULTS +188 200 dimerization oligomeric_state CYP51 interacted with PGRMC1 in a concentration-dependent manner in the presence of haem, but not in its absence (Supplementary Fig. 19), suggesting the requirement for the haem-dependent dimerization of PGRMC1. RESULTS +204 210 PGRMC1 protein CYP51 interacted with PGRMC1 in a concentration-dependent manner in the presence of haem, but not in its absence (Supplementary Fig. 19), suggesting the requirement for the haem-dependent dimerization of PGRMC1. RESULTS +4 6 Kd evidence The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/Gαi1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant. RESULTS +16 22 PGRMC1 protein The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/Gαi1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant. RESULTS +34 39 CYP51 protein The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/Gαi1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant. RESULTS +101 105 haem chemical The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/Gαi1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant. RESULTS +124 149 cytochrome P450 reductase protein The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/Gαi1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant. RESULTS +154 165 neuroglobin protein The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/Gαi1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant. RESULTS +166 170 Gαi1 protein The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/Gαi1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant. RESULTS +195 199 haem chemical The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/Gαi1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant. RESULTS +210 216 PGRMC1 protein The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/Gαi1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant. RESULTS +234 239 CYP51 protein The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/Gαi1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant. RESULTS +30 36 PGRMC1 protein In this study, we showed that PGRMC1 dimerizes by stacking interactions of haem molecules from each monomer. DISCUSS +37 46 dimerizes oligomeric_state In this study, we showed that PGRMC1 dimerizes by stacking interactions of haem molecules from each monomer. DISCUSS +50 71 stacking interactions bond_interaction In this study, we showed that PGRMC1 dimerizes by stacking interactions of haem molecules from each monomer. DISCUSS +75 79 haem chemical In this study, we showed that PGRMC1 dimerizes by stacking interactions of haem molecules from each monomer. DISCUSS +100 107 monomer oligomeric_state In this study, we showed that PGRMC1 dimerizes by stacking interactions of haem molecules from each monomer. DISCUSS +37 78 translationally-controlled tumour protein protein_type Recently, Lucas et al. reported that translationally-controlled tumour protein was dimerized by binding with haem, but its structural basis remains unclear. DISCUSS +83 92 dimerized protein_state Recently, Lucas et al. reported that translationally-controlled tumour protein was dimerized by binding with haem, but its structural basis remains unclear. DISCUSS +109 113 haem chemical Recently, Lucas et al. reported that translationally-controlled tumour protein was dimerized by binding with haem, but its structural basis remains unclear. DISCUSS +88 106 haem–haem stacking bond_interaction This is the report showing crystallographic evidence that indicates roles of the direct haem–haem stacking in haem-mediated dimerization in eukaryotes, although a few examples are known in bacteria. DISCUSS +110 114 haem chemical This is the report showing crystallographic evidence that indicates roles of the direct haem–haem stacking in haem-mediated dimerization in eukaryotes, although a few examples are known in bacteria. DISCUSS +124 136 dimerization oligomeric_state This is the report showing crystallographic evidence that indicates roles of the direct haem–haem stacking in haem-mediated dimerization in eukaryotes, although a few examples are known in bacteria. DISCUSS +140 150 eukaryotes taxonomy_domain This is the report showing crystallographic evidence that indicates roles of the direct haem–haem stacking in haem-mediated dimerization in eukaryotes, although a few examples are known in bacteria. DISCUSS +189 197 bacteria taxonomy_domain This is the report showing crystallographic evidence that indicates roles of the direct haem–haem stacking in haem-mediated dimerization in eukaryotes, although a few examples are known in bacteria. DISCUSS +0 19 Sequence alignments experimental_method Sequence alignments show that haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in PGRMC1 are conserved among MAPR proteins (Supplementary Fig. 5). DISCUSS +30 51 haem-binding residues site Sequence alignments show that haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in PGRMC1 are conserved among MAPR proteins (Supplementary Fig. 5). DISCUSS +53 59 Tyr113 residue_name_number Sequence alignments show that haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in PGRMC1 are conserved among MAPR proteins (Supplementary Fig. 5). DISCUSS +61 67 Tyr107 residue_name_number Sequence alignments show that haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in PGRMC1 are conserved among MAPR proteins (Supplementary Fig. 5). DISCUSS +69 75 Lys163 residue_name_number Sequence alignments show that haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in PGRMC1 are conserved among MAPR proteins (Supplementary Fig. 5). DISCUSS +80 86 Tyr164 residue_name_number Sequence alignments show that haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in PGRMC1 are conserved among MAPR proteins (Supplementary Fig. 5). DISCUSS +91 97 PGRMC1 protein Sequence alignments show that haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in PGRMC1 are conserved among MAPR proteins (Supplementary Fig. 5). DISCUSS +102 111 conserved protein_state Sequence alignments show that haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in PGRMC1 are conserved among MAPR proteins (Supplementary Fig. 5). DISCUSS +118 122 MAPR protein_type Sequence alignments show that haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in PGRMC1 are conserved among MAPR proteins (Supplementary Fig. 5). DISCUSS +26 30 Y113 residue_name_number In the current study, the Y113 residue plays a crucial role for the haem-dependent dimerization of PGRMC1 and resultant regulation of cancer proliferation and chemoresistance (Figs 5c and 6e). DISCUSS +68 72 haem chemical In the current study, the Y113 residue plays a crucial role for the haem-dependent dimerization of PGRMC1 and resultant regulation of cancer proliferation and chemoresistance (Figs 5c and 6e). DISCUSS +83 95 dimerization oligomeric_state In the current study, the Y113 residue plays a crucial role for the haem-dependent dimerization of PGRMC1 and resultant regulation of cancer proliferation and chemoresistance (Figs 5c and 6e). DISCUSS +99 105 PGRMC1 protein In the current study, the Y113 residue plays a crucial role for the haem-dependent dimerization of PGRMC1 and resultant regulation of cancer proliferation and chemoresistance (Figs 5c and 6e). DISCUSS +10 14 Y113 residue_name_number Since the Y113 residue is involved in the putative consensus motif of phosphorylation by tyrosine kinases such as Abl and Lck, we investigated whether phosphorylated Y113 is present in HCT116 cells by ESI-MS analysis. DISCUSS +51 66 consensus motif structure_element Since the Y113 residue is involved in the putative consensus motif of phosphorylation by tyrosine kinases such as Abl and Lck, we investigated whether phosphorylated Y113 is present in HCT116 cells by ESI-MS analysis. DISCUSS +70 85 phosphorylation ptm Since the Y113 residue is involved in the putative consensus motif of phosphorylation by tyrosine kinases such as Abl and Lck, we investigated whether phosphorylated Y113 is present in HCT116 cells by ESI-MS analysis. DISCUSS +89 105 tyrosine kinases protein_type Since the Y113 residue is involved in the putative consensus motif of phosphorylation by tyrosine kinases such as Abl and Lck, we investigated whether phosphorylated Y113 is present in HCT116 cells by ESI-MS analysis. DISCUSS +114 117 Abl protein_type Since the Y113 residue is involved in the putative consensus motif of phosphorylation by tyrosine kinases such as Abl and Lck, we investigated whether phosphorylated Y113 is present in HCT116 cells by ESI-MS analysis. DISCUSS +122 125 Lck protein_type Since the Y113 residue is involved in the putative consensus motif of phosphorylation by tyrosine kinases such as Abl and Lck, we investigated whether phosphorylated Y113 is present in HCT116 cells by ESI-MS analysis. DISCUSS +151 165 phosphorylated protein_state Since the Y113 residue is involved in the putative consensus motif of phosphorylation by tyrosine kinases such as Abl and Lck, we investigated whether phosphorylated Y113 is present in HCT116 cells by ESI-MS analysis. DISCUSS +166 170 Y113 residue_name_number Since the Y113 residue is involved in the putative consensus motif of phosphorylation by tyrosine kinases such as Abl and Lck, we investigated whether phosphorylated Y113 is present in HCT116 cells by ESI-MS analysis. DISCUSS +201 207 ESI-MS experimental_method Since the Y113 residue is involved in the putative consensus motif of phosphorylation by tyrosine kinases such as Abl and Lck, we investigated whether phosphorylated Y113 is present in HCT116 cells by ESI-MS analysis. DISCUSS +38 44 PGRMC1 protein Recently, Peluso et al. reported that PGRMC1 binds to PGRMC2, suggesting that MAPR family members may also undergo haem-mediated heterodimerization. DISCUSS +54 60 PGRMC2 protein Recently, Peluso et al. reported that PGRMC1 binds to PGRMC2, suggesting that MAPR family members may also undergo haem-mediated heterodimerization. DISCUSS +78 82 MAPR protein_type Recently, Peluso et al. reported that PGRMC1 binds to PGRMC2, suggesting that MAPR family members may also undergo haem-mediated heterodimerization. DISCUSS +115 119 haem chemical Recently, Peluso et al. reported that PGRMC1 binds to PGRMC2, suggesting that MAPR family members may also undergo haem-mediated heterodimerization. DISCUSS +19 23 haem chemical We showed that the haem-mediated dimer of PGRMC1 enables interaction with different subclasses of cytochromes P450 (CYP) (Fig. 6). DISCUSS +33 38 dimer oligomeric_state We showed that the haem-mediated dimer of PGRMC1 enables interaction with different subclasses of cytochromes P450 (CYP) (Fig. 6). DISCUSS +42 48 PGRMC1 protein We showed that the haem-mediated dimer of PGRMC1 enables interaction with different subclasses of cytochromes P450 (CYP) (Fig. 6). DISCUSS +98 114 cytochromes P450 protein_type We showed that the haem-mediated dimer of PGRMC1 enables interaction with different subclasses of cytochromes P450 (CYP) (Fig. 6). DISCUSS +116 119 CYP protein_type We showed that the haem-mediated dimer of PGRMC1 enables interaction with different subclasses of cytochromes P450 (CYP) (Fig. 6). DISCUSS +21 27 PGRMC1 protein While the effects of PGRMC1 on cholesterol synthesis mediated by CYP51 have been well documented in yeast and human cells, it has not been clear whether drug-metabolizing CYP activities are regulated by PGRMC1. DISCUSS +65 70 CYP51 protein While the effects of PGRMC1 on cholesterol synthesis mediated by CYP51 have been well documented in yeast and human cells, it has not been clear whether drug-metabolizing CYP activities are regulated by PGRMC1. DISCUSS +100 105 yeast taxonomy_domain While the effects of PGRMC1 on cholesterol synthesis mediated by CYP51 have been well documented in yeast and human cells, it has not been clear whether drug-metabolizing CYP activities are regulated by PGRMC1. DISCUSS +110 115 human species While the effects of PGRMC1 on cholesterol synthesis mediated by CYP51 have been well documented in yeast and human cells, it has not been clear whether drug-metabolizing CYP activities are regulated by PGRMC1. DISCUSS +171 174 CYP protein_type While the effects of PGRMC1 on cholesterol synthesis mediated by CYP51 have been well documented in yeast and human cells, it has not been clear whether drug-metabolizing CYP activities are regulated by PGRMC1. DISCUSS +203 209 PGRMC1 protein While the effects of PGRMC1 on cholesterol synthesis mediated by CYP51 have been well documented in yeast and human cells, it has not been clear whether drug-metabolizing CYP activities are regulated by PGRMC1. DISCUSS +42 48 PGRMC1 protein Szczesna-Skorupa and Kemper reported that PGRMC1 exhibited an inhibitory effect on CYP3A4 drug metabolizing activity by competitively binding with cytochrome P450 reductase (CPR) in HEK293 or HepG2 cells. DISCUSS +83 89 CYP3A4 protein Szczesna-Skorupa and Kemper reported that PGRMC1 exhibited an inhibitory effect on CYP3A4 drug metabolizing activity by competitively binding with cytochrome P450 reductase (CPR) in HEK293 or HepG2 cells. DISCUSS +147 172 cytochrome P450 reductase protein Szczesna-Skorupa and Kemper reported that PGRMC1 exhibited an inhibitory effect on CYP3A4 drug metabolizing activity by competitively binding with cytochrome P450 reductase (CPR) in HEK293 or HepG2 cells. DISCUSS +174 177 CPR protein Szczesna-Skorupa and Kemper reported that PGRMC1 exhibited an inhibitory effect on CYP3A4 drug metabolizing activity by competitively binding with cytochrome P450 reductase (CPR) in HEK293 or HepG2 cells. DISCUSS +44 50 PGRMC1 protein On the other hand, Oda et al. reported that PGRMC1 had no effect to CYP2E1 and CYP3A4 activities in HepG2 cell. DISCUSS +68 74 CYP2E1 protein On the other hand, Oda et al. reported that PGRMC1 had no effect to CYP2E1 and CYP3A4 activities in HepG2 cell. DISCUSS +79 85 CYP3A4 protein On the other hand, Oda et al. reported that PGRMC1 had no effect to CYP2E1 and CYP3A4 activities in HepG2 cell. DISCUSS +33 39 PGRMC1 protein Several other groups showed that PGRMC1 enhanced chemoresistance in several cancer cells such as uterine sarcoma, breast cancer, endometrial tumour and ovarian cancer; however, no evidence of PGRMC1-dependent regulation of CYP activity was provided. DISCUSS +192 198 PGRMC1 protein Several other groups showed that PGRMC1 enhanced chemoresistance in several cancer cells such as uterine sarcoma, breast cancer, endometrial tumour and ovarian cancer; however, no evidence of PGRMC1-dependent regulation of CYP activity was provided. DISCUSS +223 226 CYP protein_type Several other groups showed that PGRMC1 enhanced chemoresistance in several cancer cells such as uterine sarcoma, breast cancer, endometrial tumour and ovarian cancer; however, no evidence of PGRMC1-dependent regulation of CYP activity was provided. DISCUSS +24 30 PGRMC1 protein Our results showed that PGRMC1 contributes to enhancement of the doxorubicin metabolism, which is mediated by CYP2D6 or CYP3A4 in human colon cancer HCT116 cells (Fig. 6d). DISCUSS +65 76 doxorubicin chemical Our results showed that PGRMC1 contributes to enhancement of the doxorubicin metabolism, which is mediated by CYP2D6 or CYP3A4 in human colon cancer HCT116 cells (Fig. 6d). DISCUSS +110 116 CYP2D6 protein Our results showed that PGRMC1 contributes to enhancement of the doxorubicin metabolism, which is mediated by CYP2D6 or CYP3A4 in human colon cancer HCT116 cells (Fig. 6d). DISCUSS +120 126 CYP3A4 protein Our results showed that PGRMC1 contributes to enhancement of the doxorubicin metabolism, which is mediated by CYP2D6 or CYP3A4 in human colon cancer HCT116 cells (Fig. 6d). DISCUSS +130 135 human species Our results showed that PGRMC1 contributes to enhancement of the doxorubicin metabolism, which is mediated by CYP2D6 or CYP3A4 in human colon cancer HCT116 cells (Fig. 6d). DISCUSS +45 48 CYP protein_type While the effects of structural diversity of CYP family proteins and interactions with different xenobiotic substrates should further be examined, the current results suggest that the interaction of drug-metabolizing CYPs with the haem-mediated dimer of PGRMC1 plays a crucial role in regulating their activities. DISCUSS +217 221 CYPs protein_type While the effects of structural diversity of CYP family proteins and interactions with different xenobiotic substrates should further be examined, the current results suggest that the interaction of drug-metabolizing CYPs with the haem-mediated dimer of PGRMC1 plays a crucial role in regulating their activities. DISCUSS +231 235 haem chemical While the effects of structural diversity of CYP family proteins and interactions with different xenobiotic substrates should further be examined, the current results suggest that the interaction of drug-metabolizing CYPs with the haem-mediated dimer of PGRMC1 plays a crucial role in regulating their activities. DISCUSS +245 250 dimer oligomeric_state While the effects of structural diversity of CYP family proteins and interactions with different xenobiotic substrates should further be examined, the current results suggest that the interaction of drug-metabolizing CYPs with the haem-mediated dimer of PGRMC1 plays a crucial role in regulating their activities. DISCUSS +254 260 PGRMC1 protein While the effects of structural diversity of CYP family proteins and interactions with different xenobiotic substrates should further be examined, the current results suggest that the interaction of drug-metabolizing CYPs with the haem-mediated dimer of PGRMC1 plays a crucial role in regulating their activities. DISCUSS +15 19 haem chemical We showed that haem-mediated dimerization of PGRMC1 enhances proliferation and chemoresistance of cancer cells through binding to and regulating EGFR and cytochromes P450 (illustrated in Fig. 7). DISCUSS +29 41 dimerization oligomeric_state We showed that haem-mediated dimerization of PGRMC1 enhances proliferation and chemoresistance of cancer cells through binding to and regulating EGFR and cytochromes P450 (illustrated in Fig. 7). DISCUSS +45 51 PGRMC1 protein We showed that haem-mediated dimerization of PGRMC1 enhances proliferation and chemoresistance of cancer cells through binding to and regulating EGFR and cytochromes P450 (illustrated in Fig. 7). DISCUSS +145 149 EGFR protein_type We showed that haem-mediated dimerization of PGRMC1 enhances proliferation and chemoresistance of cancer cells through binding to and regulating EGFR and cytochromes P450 (illustrated in Fig. 7). DISCUSS +154 170 cytochromes P450 protein_type We showed that haem-mediated dimerization of PGRMC1 enhances proliferation and chemoresistance of cancer cells through binding to and regulating EGFR and cytochromes P450 (illustrated in Fig. 7). DISCUSS +10 31 haem-binding affinity evidence Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration. DISCUSS +35 41 PGRMC1 protein Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration. DISCUSS +65 77 constitutive protein_state Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration. DISCUSS +78 99 haem-binding proteins protein_type Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration. DISCUSS +108 117 myoglobin protein Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration. DISCUSS +119 125 PGMRC1 protein Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration. DISCUSS +161 164 apo protein_state Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration. DISCUSS +165 172 monomer oligomeric_state Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration. DISCUSS +177 187 haem-bound protein_state Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration. DISCUSS +188 193 dimer oligomeric_state Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration. DISCUSS +244 248 haem chemical Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration. DISCUSS +67 71 haem chemical Considering microenvironments in and around malignant tumours, the haem concentration in cancer cells is likely to be elevated through multiple mechanisms, such as (i) an increased intake of haem, (ii) mutation of enzymes in TCA cycle (for example, fumarate hydratase) that increases the level of succinyl CoA, a substrate for haem biosynthesis and (iii) metastasis to haem-rich organs such as liver, brain and bone marrow. DISCUSS +191 195 haem chemical Considering microenvironments in and around malignant tumours, the haem concentration in cancer cells is likely to be elevated through multiple mechanisms, such as (i) an increased intake of haem, (ii) mutation of enzymes in TCA cycle (for example, fumarate hydratase) that increases the level of succinyl CoA, a substrate for haem biosynthesis and (iii) metastasis to haem-rich organs such as liver, brain and bone marrow. DISCUSS +249 267 fumarate hydratase protein_type Considering microenvironments in and around malignant tumours, the haem concentration in cancer cells is likely to be elevated through multiple mechanisms, such as (i) an increased intake of haem, (ii) mutation of enzymes in TCA cycle (for example, fumarate hydratase) that increases the level of succinyl CoA, a substrate for haem biosynthesis and (iii) metastasis to haem-rich organs such as liver, brain and bone marrow. DISCUSS +297 309 succinyl CoA chemical Considering microenvironments in and around malignant tumours, the haem concentration in cancer cells is likely to be elevated through multiple mechanisms, such as (i) an increased intake of haem, (ii) mutation of enzymes in TCA cycle (for example, fumarate hydratase) that increases the level of succinyl CoA, a substrate for haem biosynthesis and (iii) metastasis to haem-rich organs such as liver, brain and bone marrow. DISCUSS +327 331 haem chemical Considering microenvironments in and around malignant tumours, the haem concentration in cancer cells is likely to be elevated through multiple mechanisms, such as (i) an increased intake of haem, (ii) mutation of enzymes in TCA cycle (for example, fumarate hydratase) that increases the level of succinyl CoA, a substrate for haem biosynthesis and (iii) metastasis to haem-rich organs such as liver, brain and bone marrow. DISCUSS +369 373 haem chemical Considering microenvironments in and around malignant tumours, the haem concentration in cancer cells is likely to be elevated through multiple mechanisms, such as (i) an increased intake of haem, (ii) mutation of enzymes in TCA cycle (for example, fumarate hydratase) that increases the level of succinyl CoA, a substrate for haem biosynthesis and (iii) metastasis to haem-rich organs such as liver, brain and bone marrow. DISCUSS +220 224 haem chemical Moreover, exposure of cancer cells to stimuli such as hypoxia, radiation and chemotherapy causes cell damages and leads to protein degradation, resulting in increased levels of TCA cycle intermediates and in an enhanced haem biosynthesis. DISCUSS +29 33 haem chemical On the other hand, excessive haem induces HO-1, the enzyme that oxidatively degrades haem and generates CO. DISCUSS +42 46 HO-1 protein On the other hand, excessive haem induces HO-1, the enzyme that oxidatively degrades haem and generates CO. DISCUSS +85 89 haem chemical On the other hand, excessive haem induces HO-1, the enzyme that oxidatively degrades haem and generates CO. DISCUSS +104 106 CO chemical On the other hand, excessive haem induces HO-1, the enzyme that oxidatively degrades haem and generates CO. DISCUSS +6 10 HO-1 protein Thus, HO-1 induction in cancer cells may inhibit the haem-mediated dimerization of PGRMC1 through the production of CO and thereby suppress tumour progression. DISCUSS +53 57 haem chemical Thus, HO-1 induction in cancer cells may inhibit the haem-mediated dimerization of PGRMC1 through the production of CO and thereby suppress tumour progression. DISCUSS +67 79 dimerization oligomeric_state Thus, HO-1 induction in cancer cells may inhibit the haem-mediated dimerization of PGRMC1 through the production of CO and thereby suppress tumour progression. DISCUSS +83 89 PGRMC1 protein Thus, HO-1 induction in cancer cells may inhibit the haem-mediated dimerization of PGRMC1 through the production of CO and thereby suppress tumour progression. DISCUSS +116 118 CO chemical Thus, HO-1 induction in cancer cells may inhibit the haem-mediated dimerization of PGRMC1 through the production of CO and thereby suppress tumour progression. DISCUSS +50 54 HO-1 protein This idea is consistent with the observation that HO-1 induction or CO inhibits tumour growth. DISCUSS +68 70 CO chemical This idea is consistent with the observation that HO-1 induction or CO inhibits tumour growth. DISCUSS +32 38 PGRMC1 protein Besides the regulatory roles of PGRMC1/Sigma-2 receptor in proliferation and chemoresistance in cancer cells (ref.), recent reports show that PGRMC1 is able to bind to amyloid beta oligomer to enhance its neurotoxicity. DISCUSS +39 46 Sigma-2 protein Besides the regulatory roles of PGRMC1/Sigma-2 receptor in proliferation and chemoresistance in cancer cells (ref.), recent reports show that PGRMC1 is able to bind to amyloid beta oligomer to enhance its neurotoxicity. DISCUSS +142 148 PGRMC1 protein Besides the regulatory roles of PGRMC1/Sigma-2 receptor in proliferation and chemoresistance in cancer cells (ref.), recent reports show that PGRMC1 is able to bind to amyloid beta oligomer to enhance its neurotoxicity. DISCUSS +168 180 amyloid beta protein Besides the regulatory roles of PGRMC1/Sigma-2 receptor in proliferation and chemoresistance in cancer cells (ref.), recent reports show that PGRMC1 is able to bind to amyloid beta oligomer to enhance its neurotoxicity. DISCUSS +181 189 oligomer oligomeric_state Besides the regulatory roles of PGRMC1/Sigma-2 receptor in proliferation and chemoresistance in cancer cells (ref.), recent reports show that PGRMC1 is able to bind to amyloid beta oligomer to enhance its neurotoxicity. DISCUSS +13 20 Sigma-2 protein Furthermore, Sigma-2 ligand-binding is decreased in transgenic amyloid beta deposition model APP/PS1 female mice. DISCUSS +48 54 PGRMC1 protein These results suggest a possible involvement of PGRMC1 in Alzheimer's disease. DISCUSS +13 17 haem chemical The roles of haem-dependent dimerization of PGRMC1 in the functional regulation of its target proteins deserve further studies to find evidence that therapeutic interventions to interfere with the function of the dimer may control varied disease conditions. DISCUSS +28 40 dimerization oligomeric_state The roles of haem-dependent dimerization of PGRMC1 in the functional regulation of its target proteins deserve further studies to find evidence that therapeutic interventions to interfere with the function of the dimer may control varied disease conditions. DISCUSS +44 50 PGRMC1 protein The roles of haem-dependent dimerization of PGRMC1 in the functional regulation of its target proteins deserve further studies to find evidence that therapeutic interventions to interfere with the function of the dimer may control varied disease conditions. DISCUSS +213 218 dimer oligomeric_state The roles of haem-dependent dimerization of PGRMC1 in the functional regulation of its target proteins deserve further studies to find evidence that therapeutic interventions to interfere with the function of the dimer may control varied disease conditions. DISCUSS +109 117 oligomer oligomeric_state Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers II: Sigma-2/PGRMC1 receptors mediate Abeta 42 oligomer binding and synaptotoxicity REF +0 23 X-ray crystal structure evidence X-ray crystal structure of PGRMC1. FIG +27 33 PGRMC1 protein X-ray crystal structure of PGRMC1. FIG +21 27 PGRMC1 protein (a) Structure of the PGRMC1 dimer formed through stacked haems. FIG +28 33 dimer oligomeric_state (a) Structure of the PGRMC1 dimer formed through stacked haems. FIG +57 62 haems chemical (a) Structure of the PGRMC1 dimer formed through stacked haems. FIG +4 10 PGRMC1 protein Two PGRMC1 subunits (blue and green ribbons) dimerize via stacking of the haem molecules. FIG +11 19 subunits structure_element Two PGRMC1 subunits (blue and green ribbons) dimerize via stacking of the haem molecules. FIG +45 53 dimerize oligomeric_state Two PGRMC1 subunits (blue and green ribbons) dimerize via stacking of the haem molecules. FIG +58 66 stacking bond_interaction Two PGRMC1 subunits (blue and green ribbons) dimerize via stacking of the haem molecules. FIG +74 78 haem chemical Two PGRMC1 subunits (blue and green ribbons) dimerize via stacking of the haem molecules. FIG +4 8 Haem chemical (b) Haem coordination of PGRMC1 with Tyr113. FIG +9 21 coordination bond_interaction (b) Haem coordination of PGRMC1 with Tyr113. FIG +25 31 PGRMC1 protein (b) Haem coordination of PGRMC1 with Tyr113. FIG +37 43 Tyr113 residue_name_number (b) Haem coordination of PGRMC1 with Tyr113. FIG +14 20 PGRMC1 protein Comparison of PGRMC1 (blue) and cytochrome b5 (yellow, ID: 3NER). (c) PGRMC1 has a longer helix (a.a.147–163), which is shifted away from the haem (arrow). FIG +32 45 cytochrome b5 protein_type Comparison of PGRMC1 (blue) and cytochrome b5 (yellow, ID: 3NER). (c) PGRMC1 has a longer helix (a.a.147–163), which is shifted away from the haem (arrow). FIG +70 76 PGRMC1 protein Comparison of PGRMC1 (blue) and cytochrome b5 (yellow, ID: 3NER). (c) PGRMC1 has a longer helix (a.a.147–163), which is shifted away from the haem (arrow). FIG +90 95 helix structure_element Comparison of PGRMC1 (blue) and cytochrome b5 (yellow, ID: 3NER). (c) PGRMC1 has a longer helix (a.a.147–163), which is shifted away from the haem (arrow). FIG +101 108 147–163 residue_range Comparison of PGRMC1 (blue) and cytochrome b5 (yellow, ID: 3NER). (c) PGRMC1 has a longer helix (a.a.147–163), which is shifted away from the haem (arrow). FIG +142 146 haem chemical Comparison of PGRMC1 (blue) and cytochrome b5 (yellow, ID: 3NER). (c) PGRMC1 has a longer helix (a.a.147–163), which is shifted away from the haem (arrow). FIG +0 6 PGRCM1 protein PGRCM1 is dimerized by binding with haem. FIG +10 19 dimerized protein_state PGRCM1 is dimerized by binding with haem. FIG +36 40 haem chemical PGRCM1 is dimerized by binding with haem. FIG +4 22 Mass spectrometric experimental_method (a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in the presence or absence of haem under non-denaturing condition. FIG +39 48 wild-type protein_state (a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in the presence or absence of haem under non-denaturing condition. FIG +50 52 wt protein_state (a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in the presence or absence of haem under non-denaturing condition. FIG +54 60 PGRMC1 protein (a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in the presence or absence of haem under non-denaturing condition. FIG +68 73 C129S mutant (a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in the presence or absence of haem under non-denaturing condition. FIG +74 80 mutant protein_state (a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in the presence or absence of haem under non-denaturing condition. FIG +88 96 presence protein_state (a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in the presence or absence of haem under non-denaturing condition. FIG +100 110 absence of protein_state (a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in the presence or absence of haem under non-denaturing condition. FIG +111 115 haem chemical (a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in the presence or absence of haem under non-denaturing condition. FIG +41 47 44–195 residue_range Both proteins had identical lengths (a.a.44–195). FIG +4 10 SV-AUC experimental_method (b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44–195) in the presence or absence of haem. FIG +27 29 wt protein_state (b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44–195) in the presence or absence of haem. FIG +30 36 PGRMC1 protein (b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44–195) in the presence or absence of haem. FIG +45 50 C129S mutant (b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44–195) in the presence or absence of haem. FIG +51 57 mutant protein_state (b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44–195) in the presence or absence of haem. FIG +63 69 44–195 residue_range (b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44–195) in the presence or absence of haem. FIG +78 86 presence protein_state (b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44–195) in the presence or absence of haem. FIG +90 100 absence of protein_state (b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44–195) in the presence or absence of haem. FIG +101 105 haem chemical (b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44–195) in the presence or absence of haem. FIG +0 6 SV-AUC experimental_method SV-AUC experiments were performed with 1.5 mg ml−1 of PGRMC1 proteins. FIG +54 60 PGRMC1 protein SV-AUC experiments were performed with 1.5 mg ml−1 of PGRMC1 proteins. FIG +20 45 sedimentation coefficient evidence The major peak with sedimentation coefficient S20,w of 1.9∼2.0 S (monomer) or 3.1 S (dimer) was detected. FIG +46 51 S20,w evidence The major peak with sedimentation coefficient S20,w of 1.9∼2.0 S (monomer) or 3.1 S (dimer) was detected. FIG +66 73 monomer oligomeric_state The major peak with sedimentation coefficient S20,w of 1.9∼2.0 S (monomer) or 3.1 S (dimer) was detected. FIG +85 90 dimer oligomeric_state The major peak with sedimentation coefficient S20,w of 1.9∼2.0 S (monomer) or 3.1 S (dimer) was detected. FIG +4 33 Difference absorption spectra evidence (c) Difference absorption spectra of PGRMC1 (a.a.44–195) titrated with haem (left panel). FIG +37 43 PGRMC1 protein (c) Difference absorption spectra of PGRMC1 (a.a.44–195) titrated with haem (left panel). FIG +49 55 44–195 residue_range (c) Difference absorption spectra of PGRMC1 (a.a.44–195) titrated with haem (left panel). FIG +57 70 titrated with experimental_method (c) Difference absorption spectra of PGRMC1 (a.a.44–195) titrated with haem (left panel). FIG +71 75 haem chemical (c) Difference absorption spectra of PGRMC1 (a.a.44–195) titrated with haem (left panel). FIG +4 19 titration curve evidence The titration curve of haem to PGRMC1 (right panel). FIG +23 27 haem chemical The titration curve of haem to PGRMC1 (right panel). FIG +31 37 PGRMC1 protein The titration curve of haem to PGRMC1 (right panel). FIG +4 25 absorbance difference evidence The absorbance difference at 400 nm is plotted against the haem concentration. FIG +59 63 haem chemical The absorbance difference at 400 nm is plotted against the haem concentration. FIG +0 15 Carbon monoxide chemical Carbon monoxide inhibits haem-dependent PGRMC1 dimerization. FIG +25 29 haem chemical Carbon monoxide inhibits haem-dependent PGRMC1 dimerization. FIG +40 46 PGRMC1 protein Carbon monoxide inhibits haem-dependent PGRMC1 dimerization. FIG +47 59 dimerization oligomeric_state Carbon monoxide inhibits haem-dependent PGRMC1 dimerization. FIG +4 33 UV-visible absorption spectra evidence (a) UV-visible absorption spectra of PGRMC1 (a.a.44–195). FIG +37 43 PGRMC1 protein (a) UV-visible absorption spectra of PGRMC1 (a.a.44–195). FIG +49 55 44–195 residue_range (a) UV-visible absorption spectra of PGRMC1 (a.a.44–195). FIG +35 46 presence of protein_state Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). FIG +51 59 oxidized protein_state Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). FIG +68 72 haem chemical Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). FIG +74 80 ferric protein_state Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). FIG +87 94 reduced protein_state Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). FIG +103 107 haem chemical Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). FIG +109 116 ferrous protein_state Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). FIG +126 133 reduced protein_state Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). FIG +142 146 haem chemical Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). FIG +152 154 CO chemical Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). FIG +160 167 ferrous protein_state Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). FIG +168 170 CO chemical Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO). FIG +21 34 haem stacking bond_interaction (b) Close-up view of haem stacking. FIG +4 33 Gel-filtration chromatography experimental_method (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +46 52 PGRMC1 protein (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +58 64 44–195 residue_range (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +66 75 wild-type protein_state (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +77 79 wt protein_state (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +89 94 Y113F mutant (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +98 103 C129S mutant (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +104 110 mutant protein_state (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +118 126 presence protein_state (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +130 140 absence of protein_state (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +141 145 haem chemical (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +147 157 dithionite chemical (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +165 167 CO chemical (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +219 225 PGRMC1 protein (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +241 245 haem chemical (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +250 252 CO chemical (c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44–195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO. FIG +0 4 Haem chemical Haem-dependent dimerization of PGRMC1 is necessary for tumour proliferation mediated by EGFR signalling. FIG +15 27 dimerization oligomeric_state Haem-dependent dimerization of PGRMC1 is necessary for tumour proliferation mediated by EGFR signalling. FIG +31 37 PGRMC1 protein Haem-dependent dimerization of PGRMC1 is necessary for tumour proliferation mediated by EGFR signalling. FIG +88 92 EGFR protein_type Haem-dependent dimerization of PGRMC1 is necessary for tumour proliferation mediated by EGFR signalling. FIG +9 15 PGRMC1 protein (a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +16 25 wild-type protein_state (a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +27 29 wt protein_state (a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +35 40 Y113F mutant (a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +41 47 mutant protein_state (a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +62 68 44–195 residue_range (a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +81 84 apo protein_state (a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +89 99 haem-bound protein_state (a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +111 120 incubated experimental_method (a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +135 139 EGFR protein_type (a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +144 165 co-immunoprecipitated experimental_method (a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +42 58 Western blotting experimental_method Input and bound proteins were detected by Western blotting. FIG +42 58 Western blotting experimental_method Input and bound proteins were detected by Western blotting. FIG +4 26 In vitro binding assay experimental_method (b) In vitro binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt (a.a.44–195) and purified EGFR with or without treatment of RuCl3 and CORM3. FIG +57 67 haem-bound protein_state (b) In vitro binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt (a.a.44–195) and purified EGFR with or without treatment of RuCl3 and CORM3. FIG +73 79 PGRMC1 protein (b) In vitro binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt (a.a.44–195) and purified EGFR with or without treatment of RuCl3 and CORM3. FIG +80 82 wt protein_state (b) In vitro binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt (a.a.44–195) and purified EGFR with or without treatment of RuCl3 and CORM3. FIG +88 94 44–195 residue_range (b) In vitro binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt (a.a.44–195) and purified EGFR with or without treatment of RuCl3 and CORM3. FIG +109 113 EGFR protein_type (b) In vitro binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt (a.a.44–195) and purified EGFR with or without treatment of RuCl3 and CORM3. FIG +143 148 RuCl3 chemical (b) In vitro binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt (a.a.44–195) and purified EGFR with or without treatment of RuCl3 and CORM3. FIG +153 158 CORM3 chemical (b) In vitro binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt (a.a.44–195) and purified EGFR with or without treatment of RuCl3 and CORM3. FIG +9 15 PGRMC1 protein (c) FLAG-PGRMC1 wt or Y113F (full length) was over-expressed in HCT116 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +16 18 wt protein_state (c) FLAG-PGRMC1 wt or Y113F (full length) was over-expressed in HCT116 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +22 27 Y113F mutant (c) FLAG-PGRMC1 wt or Y113F (full length) was over-expressed in HCT116 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +29 40 full length protein_state (c) FLAG-PGRMC1 wt or Y113F (full length) was over-expressed in HCT116 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +46 60 over-expressed experimental_method (c) FLAG-PGRMC1 wt or Y113F (full length) was over-expressed in HCT116 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +81 99 immunoprecipitated experimental_method (c) FLAG-PGRMC1 wt or Y113F (full length) was over-expressed in HCT116 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +0 21 Co-immunoprecipitated experimental_method Co-immunoprecipitated proteins (FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody. FIG +37 43 PGRMC1 protein Co-immunoprecipitated proteins (FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody. FIG +45 55 endogenous protein_state Co-immunoprecipitated proteins (FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody. FIG +56 62 PGRMC1 protein Co-immunoprecipitated proteins (FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody. FIG +67 71 EGFR protein_type Co-immunoprecipitated proteins (FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody. FIG +92 108 Western blotting experimental_method Co-immunoprecipitated proteins (FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody. FIG +123 129 PGRMC1 protein Co-immunoprecipitated proteins (FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody. FIG +138 142 EGFR protein_type Co-immunoprecipitated proteins (FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody. FIG +62 77 succinylacetone chemical (d) HCT116 cells were treated with or without 250 μmol l−1 of succinylacetone (SA) for 48 h. The intracellular haem was extracted and quantified by reverse-phase HPLC. FIG +79 81 SA chemical (d) HCT116 cells were treated with or without 250 μmol l−1 of succinylacetone (SA) for 48 h. The intracellular haem was extracted and quantified by reverse-phase HPLC. FIG +111 115 haem chemical (d) HCT116 cells were treated with or without 250 μmol l−1 of succinylacetone (SA) for 48 h. The intracellular haem was extracted and quantified by reverse-phase HPLC. FIG +148 166 reverse-phase HPLC experimental_method (d) HCT116 cells were treated with or without 250 μmol l−1 of succinylacetone (SA) for 48 h. The intracellular haem was extracted and quantified by reverse-phase HPLC. FIG +54 70 Student's t-test experimental_method of four separate experiments. **P<0.01 using unpaired Student's t-test. (e) Co-immunoprecipitation assay was performed as in (c) with or without SA treatment in HCT116 cells. FIG +76 104 Co-immunoprecipitation assay experimental_method of four separate experiments. **P<0.01 using unpaired Student's t-test. (e) Co-immunoprecipitation assay was performed as in (c) with or without SA treatment in HCT116 cells. FIG +145 147 SA chemical of four separate experiments. **P<0.01 using unpaired Student's t-test. (e) Co-immunoprecipitation assay was performed as in (c) with or without SA treatment in HCT116 cells. FIG +36 41 shRNA chemical (f) HCT116 cells expressing control shRNA or those knocking down PGRMC1 (PGRMC1-KD) were treated with EGF or left untreated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +51 64 knocking down experimental_method (f) HCT116 cells expressing control shRNA or those knocking down PGRMC1 (PGRMC1-KD) were treated with EGF or left untreated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +65 71 PGRMC1 protein (f) HCT116 cells expressing control shRNA or those knocking down PGRMC1 (PGRMC1-KD) were treated with EGF or left untreated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +73 82 PGRMC1-KD mutant (f) HCT116 cells expressing control shRNA or those knocking down PGRMC1 (PGRMC1-KD) were treated with EGF or left untreated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +102 105 EGF protein_type (f) HCT116 cells expressing control shRNA or those knocking down PGRMC1 (PGRMC1-KD) were treated with EGF or left untreated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +147 151 EGFR protein_type (f) HCT116 cells expressing control shRNA or those knocking down PGRMC1 (PGRMC1-KD) were treated with EGF or left untreated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +187 203 Western blotting experimental_method (f) HCT116 cells expressing control shRNA or those knocking down PGRMC1 (PGRMC1-KD) were treated with EGF or left untreated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +48 51 EGF protein_type (g,h) HCT116 cells were treated with or without EGF, SA, RuCl3 and CORM3 as indicated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +53 55 SA chemical (g,h) HCT116 cells were treated with or without EGF, SA, RuCl3 and CORM3 as indicated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +57 62 RuCl3 chemical (g,h) HCT116 cells were treated with or without EGF, SA, RuCl3 and CORM3 as indicated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +67 72 CORM3 chemical (g,h) HCT116 cells were treated with or without EGF, SA, RuCl3 and CORM3 as indicated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +109 113 EGFR protein_type (g,h) HCT116 cells were treated with or without EGF, SA, RuCl3 and CORM3 as indicated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +149 165 Western blotting experimental_method (g,h) HCT116 cells were treated with or without EGF, SA, RuCl3 and CORM3 as indicated, and components of the EGFR signaling pathway were detected by Western blotting. FIG +0 4 Haem chemical Haem-dependent dimerization of PGRMC1 accelerates tumour growth through the EGFR signaling pathway. FIG +15 27 dimerization oligomeric_state Haem-dependent dimerization of PGRMC1 accelerates tumour growth through the EGFR signaling pathway. FIG +31 37 PGRMC1 protein Haem-dependent dimerization of PGRMC1 accelerates tumour growth through the EGFR signaling pathway. FIG +76 80 EGFR protein_type Haem-dependent dimerization of PGRMC1 accelerates tumour growth through the EGFR signaling pathway. FIG +28 34 PGRMC1 protein (a) Nucleotide sequences of PGRMC1 targeted by shRNA and of the shRNA-resistant full length PGRMC1 expression vector. FIG +47 52 shRNA chemical (a) Nucleotide sequences of PGRMC1 targeted by shRNA and of the shRNA-resistant full length PGRMC1 expression vector. FIG +64 79 shRNA-resistant protein_state (a) Nucleotide sequences of PGRMC1 targeted by shRNA and of the shRNA-resistant full length PGRMC1 expression vector. FIG +80 91 full length protein_state (a) Nucleotide sequences of PGRMC1 targeted by shRNA and of the shRNA-resistant full length PGRMC1 expression vector. FIG +92 98 PGRMC1 protein (a) Nucleotide sequences of PGRMC1 targeted by shRNA and of the shRNA-resistant full length PGRMC1 expression vector. FIG +7 23 PGRMC1-knockdown mutant Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). FIG +25 34 PGRMC1-KD mutant Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). FIG +54 77 transiently transfected experimental_method Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). FIG +87 102 shRNA-resistant protein_state Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). FIG +103 120 expression vector experimental_method Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). FIG +124 133 wild-type protein_state Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). FIG +134 140 PGRMC1 protein Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). FIG +142 144 wt protein_state Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). FIG +153 158 Y113F mutant Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). FIG +159 165 mutant protein_state Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). FIG +167 172 Y113F mutant Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F). FIG +4 13 Erlotinib chemical (b) Erlotinib was added to HCT116 (control) cells, PGRMC1-KD cells or PGRMC1-KD cells expressing shRNA-resistant PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +51 60 PGRMC1-KD mutant (b) Erlotinib was added to HCT116 (control) cells, PGRMC1-KD cells or PGRMC1-KD cells expressing shRNA-resistant PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +70 79 PGRMC1-KD mutant (b) Erlotinib was added to HCT116 (control) cells, PGRMC1-KD cells or PGRMC1-KD cells expressing shRNA-resistant PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +97 112 shRNA-resistant protein_state (b) Erlotinib was added to HCT116 (control) cells, PGRMC1-KD cells or PGRMC1-KD cells expressing shRNA-resistant PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +113 119 PGRMC1 protein (b) Erlotinib was added to HCT116 (control) cells, PGRMC1-KD cells or PGRMC1-KD cells expressing shRNA-resistant PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +120 122 wt protein_state (b) Erlotinib was added to HCT116 (control) cells, PGRMC1-KD cells or PGRMC1-KD cells expressing shRNA-resistant PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +126 131 Y113F mutant (b) Erlotinib was added to HCT116 (control) cells, PGRMC1-KD cells or PGRMC1-KD cells expressing shRNA-resistant PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +168 177 MTT assay experimental_method (b) Erlotinib was added to HCT116 (control) cells, PGRMC1-KD cells or PGRMC1-KD cells expressing shRNA-resistant PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +30 32 *P evidence of four separate experiments. *P<0.01 using ANOVA with Fischer's LSD test. FIG +44 49 ANOVA experimental_method of four separate experiments. *P<0.01 using ANOVA with Fischer's LSD test. FIG +55 73 Fischer's LSD test experimental_method of four separate experiments. *P<0.01 using ANOVA with Fischer's LSD test. FIG +30 32 *P evidence of four separate experiments. *P<0.01 using ANOVA with Fischer's LSD test. FIG +44 49 ANOVA experimental_method of four separate experiments. *P<0.01 using ANOVA with Fischer's LSD test. FIG +55 73 Fischer's LSD test experimental_method of four separate experiments. *P<0.01 using ANOVA with Fischer's LSD test. FIG +38 47 PGRMC1-KD mutant (c) Spheroid formation in control and PGRMC1-KD HCT116 cells. FIG +54 56 *P evidence The graph represents mean±s.e. of each spheroid size. *P<0.01 using ANOVA with Fischer's LSD test. FIG +68 73 ANOVA experimental_method The graph represents mean±s.e. of each spheroid size. *P<0.01 using ANOVA with Fischer's LSD test. FIG +79 97 Fischer's LSD test experimental_method The graph represents mean±s.e. of each spheroid size. *P<0.01 using ANOVA with Fischer's LSD test. FIG +74 96 intrasplenic injection experimental_method Scale bar: 0.1 mm. (d) Tumour-bearing livers of NOG mice at 10 days after intrasplenic injection of HCT116 (control) or PGRMC1-KD cells. FIG +120 129 PGRMC1-KD mutant Scale bar: 0.1 mm. (d) Tumour-bearing livers of NOG mice at 10 days after intrasplenic injection of HCT116 (control) or PGRMC1-KD cells. FIG +28 30 *P evidence of 10 separate experiments. *P<0.05 using unpaired Student's t-test. FIG +51 67 Student's t-test experimental_method of 10 separate experiments. *P<0.05 using unpaired Student's t-test. FIG +0 4 Haem chemical Haem-dependent PGRMC1 dimerization enhances tumour chemoresistance through interaction with cytochromes P450. FIG +15 21 PGRMC1 protein Haem-dependent PGRMC1 dimerization enhances tumour chemoresistance through interaction with cytochromes P450. FIG +22 34 dimerization oligomeric_state Haem-dependent PGRMC1 dimerization enhances tumour chemoresistance through interaction with cytochromes P450. FIG +92 108 cytochromes P450 protein_type Haem-dependent PGRMC1 dimerization enhances tumour chemoresistance through interaction with cytochromes P450. FIG +11 17 PGRMC1 protein (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +18 27 wild-type protein_state (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +29 31 wt protein_state (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +37 42 Y113F mutant (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +43 49 mutant protein_state (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +64 70 44–195 residue_range (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +83 86 apo protein_state (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +90 100 haem-bound protein_state (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +112 121 incubated experimental_method (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +127 133 CYP1A2 protein (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +141 147 CYP3A4 protein (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +156 174 immunoprecipitated experimental_method (a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44–195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads. FIG +4 17 Binding assay experimental_method (c) Binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt and CYP1A2 with or without RuCl3 and CORM3. FIG +48 58 haem-bound protein_state (c) Binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt and CYP1A2 with or without RuCl3 and CORM3. FIG +64 70 PGRMC1 protein (c) Binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt and CYP1A2 with or without RuCl3 and CORM3. FIG +71 73 wt protein_state (c) Binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt and CYP1A2 with or without RuCl3 and CORM3. FIG +78 84 CYP1A2 protein (c) Binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt and CYP1A2 with or without RuCl3 and CORM3. FIG +101 106 RuCl3 chemical (c) Binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt and CYP1A2 with or without RuCl3 and CORM3. FIG +111 116 CORM3 chemical (c) Binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt and CYP1A2 with or without RuCl3 and CORM3. FIG +30 41 doxorubicin chemical (d) Schematic illustration of doxorubicin metabolism is shown on the left. FIG +0 11 Doxorubicin chemical Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD), and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS. FIG +16 25 incubated experimental_method Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD), and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS. FIG +63 68 shRNA chemical Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD), and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS. FIG +72 80 shPGRMC1 chemical Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD), and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS. FIG +82 91 PGRMC1-KD mutant Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD), and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS. FIG +102 115 doxorubicinol chemical Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD), and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS. FIG +116 127 doxorubicin chemical Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD), and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS. FIG +173 178 LC-MS experimental_method Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD), and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS. FIG +31 33 *P evidence of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +69 85 Student's t-test experimental_method of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +112 123 doxorubicin chemical of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +162 171 PGRMC1-KD mutant of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +182 191 PGRMC1-KD mutant of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +209 224 shRNA-resistant protein_state of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +225 236 full-length protein_state of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +237 243 PGRMC1 protein of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +244 246 wt protein_state of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +250 255 Y113F mutant of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +292 301 MTT assay experimental_method of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay. FIG +40 46 PGRMC1 protein Schematic diagram for the regulation of PGRMC1 functions. FIG +0 3 Apo protein_state Apo-PGRMC1 exists as an inactive monomer. FIG +4 10 PGRMC1 protein Apo-PGRMC1 exists as an inactive monomer. FIG +24 32 inactive protein_state Apo-PGRMC1 exists as an inactive monomer. FIG +33 40 monomer oligomeric_state Apo-PGRMC1 exists as an inactive monomer. FIG +3 13 binding to protein_state On binding to haem, PGRMC1 forms a dimer through stacking interactions between the haem moieties, which enables PGRMC1 to interact with EGFR and cytochromes P450, leading to an enhanced proliferation and chemoresistance of cancer cells. FIG +14 18 haem chemical On binding to haem, PGRMC1 forms a dimer through stacking interactions between the haem moieties, which enables PGRMC1 to interact with EGFR and cytochromes P450, leading to an enhanced proliferation and chemoresistance of cancer cells. FIG +20 26 PGRMC1 protein On binding to haem, PGRMC1 forms a dimer through stacking interactions between the haem moieties, which enables PGRMC1 to interact with EGFR and cytochromes P450, leading to an enhanced proliferation and chemoresistance of cancer cells. FIG +35 40 dimer oligomeric_state On binding to haem, PGRMC1 forms a dimer through stacking interactions between the haem moieties, which enables PGRMC1 to interact with EGFR and cytochromes P450, leading to an enhanced proliferation and chemoresistance of cancer cells. FIG +49 70 stacking interactions bond_interaction On binding to haem, PGRMC1 forms a dimer through stacking interactions between the haem moieties, which enables PGRMC1 to interact with EGFR and cytochromes P450, leading to an enhanced proliferation and chemoresistance of cancer cells. FIG +83 87 haem chemical On binding to haem, PGRMC1 forms a dimer through stacking interactions between the haem moieties, which enables PGRMC1 to interact with EGFR and cytochromes P450, leading to an enhanced proliferation and chemoresistance of cancer cells. FIG +112 118 PGRMC1 protein On binding to haem, PGRMC1 forms a dimer through stacking interactions between the haem moieties, which enables PGRMC1 to interact with EGFR and cytochromes P450, leading to an enhanced proliferation and chemoresistance of cancer cells. FIG +136 140 EGFR protein_type On binding to haem, PGRMC1 forms a dimer through stacking interactions between the haem moieties, which enables PGRMC1 to interact with EGFR and cytochromes P450, leading to an enhanced proliferation and chemoresistance of cancer cells. FIG +145 161 cytochromes P450 protein_type On binding to haem, PGRMC1 forms a dimer through stacking interactions between the haem moieties, which enables PGRMC1 to interact with EGFR and cytochromes P450, leading to an enhanced proliferation and chemoresistance of cancer cells. FIG +0 2 CO chemical CO interferes with the stacking interactions of the haems and thereby inhibits PGRMC1 functions. FIG +23 44 stacking interactions bond_interaction CO interferes with the stacking interactions of the haems and thereby inhibits PGRMC1 functions. FIG +52 57 haems chemical CO interferes with the stacking interactions of the haems and thereby inhibits PGRMC1 functions. FIG +79 85 PGRMC1 protein CO interferes with the stacking interactions of the haems and thereby inhibits PGRMC1 functions. FIG +39 51 dimerization oligomeric_state PGRMC1 proteins exhibit haem-dependent dimerization in solution. TABLE +360 365 C129S mutant "  Apo form Haem-bound form     Mass (Da)   Mass (Da) aPGRMC1 wt (a.a.44–195)  ESI-MS — 17,844.14 — 36,920.19  Theoretical   17,843.65   36,918.06   Hydrodynamic radius 10−9 (m) MW (kDa) Hydrodynamic radius 10−9 (m) MW (kDa)  DOSY 2.04–2.15 20 2.94–3.02 42   S20,w (S) MW (kDa) S20,w (S) MW (kDa)  SV-AUC 1.9 17.6 3.1 35.5           bPGRMC1 C129S (a.a.44–195)  ESI-MS — 17,827.91 — 36,887.07  Theoretical   17,827.59   36,885.6   S20,w (S) MW (kDa) S20,w (S) MW (kDa)  SV-AUC 2.0 18.1 3.1 35.8 " TABLE +66 71 C129S mutant Differences in molecular weights of the wild-type (wt; a) and the C129S mutant (b) PGRMC1 proteins in the absence (apo form) or the presence of haem (haem-bound form). TABLE +32 37 C129S mutant The protein sizes of the wt and C129S PGRMC1 cytosolic domains (a.a.44–195) in the presence or absence of haem were estimated by ESI-MS, DOSY and SV-AUC. TABLE diff --git a/annotation_CSV/PMC4820378.csv b/annotation_CSV/PMC4820378.csv new file mode 100644 index 0000000000000000000000000000000000000000..6d004545bae226ca2e922c2c061cca37ac9c05d6 --- /dev/null +++ b/annotation_CSV/PMC4820378.csv @@ -0,0 +1,734 @@ +anno_start anno_end anno_text entity_type sentence section +75 92 Thg1-like protein protein_type Template-dependent nucleotide addition in the reverse (3′-5′) direction by Thg1-like protein TITLE +0 10 Structures evidence Structures of Thg1-like proteins provide insight into the template-dependent nucleotide addition in the reverse (3′-5′) direction. ABSTRACT +14 32 Thg1-like proteins protein_type Structures of Thg1-like proteins provide insight into the template-dependent nucleotide addition in the reverse (3′-5′) direction. ABSTRACT +0 17 Thg1-like protein protein_type Thg1-like protein (TLP) catalyzes the addition of a nucleotide to the 5′-end of truncated transfer RNA (tRNA) species in a Watson-Crick template–dependent manner. ABSTRACT +19 22 TLP protein_type Thg1-like protein (TLP) catalyzes the addition of a nucleotide to the 5′-end of truncated transfer RNA (tRNA) species in a Watson-Crick template–dependent manner. ABSTRACT +90 102 transfer RNA chemical Thg1-like protein (TLP) catalyzes the addition of a nucleotide to the 5′-end of truncated transfer RNA (tRNA) species in a Watson-Crick template–dependent manner. ABSTRACT +104 108 tRNA chemical Thg1-like protein (TLP) catalyzes the addition of a nucleotide to the 5′-end of truncated transfer RNA (tRNA) species in a Watson-Crick template–dependent manner. ABSTRACT +68 93 adenosine 5′-triphosphate chemical The reaction proceeds in two steps: the activation of the 5′-end by adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleotide addition. ABSTRACT +95 98 ATP chemical The reaction proceeds in two steps: the activation of the 5′-end by adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleotide addition. ABSTRACT +100 125 guanosine 5′-triphosphate chemical The reaction proceeds in two steps: the activation of the 5′-end by adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleotide addition. ABSTRACT +127 130 GTP chemical The reaction proceeds in two steps: the activation of the 5′-end by adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleotide addition. ABSTRACT +0 19 Structural analyses experimental_method Structural analyses of the TLP and its reaction intermediates have revealed the atomic detail of the template-dependent elongation reaction in the 3′-5′ direction. ABSTRACT +27 30 TLP protein_type Structural analyses of the TLP and its reaction intermediates have revealed the atomic detail of the template-dependent elongation reaction in the 3′-5′ direction. ABSTRACT +23 46 substrate binding sites site The enzyme creates two substrate binding sites for the first- and second-step reactions in the vicinity of one reaction center consisting of two Mg2+ ions, and the two reactions are executed at the same reaction center in a stepwise fashion. ABSTRACT +111 126 reaction center site The enzyme creates two substrate binding sites for the first- and second-step reactions in the vicinity of one reaction center consisting of two Mg2+ ions, and the two reactions are executed at the same reaction center in a stepwise fashion. ABSTRACT +145 149 Mg2+ chemical The enzyme creates two substrate binding sites for the first- and second-step reactions in the vicinity of one reaction center consisting of two Mg2+ ions, and the two reactions are executed at the same reaction center in a stepwise fashion. ABSTRACT +203 218 reaction center site The enzyme creates two substrate binding sites for the first- and second-step reactions in the vicinity of one reaction center consisting of two Mg2+ ions, and the two reactions are executed at the same reaction center in a stepwise fashion. ABSTRACT +18 28 nucleotide chemical When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3′-OH of the incoming nucleotide and the 5′-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. ABSTRACT +32 40 bound to protein_state When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3′-OH of the incoming nucleotide and the 5′-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. ABSTRACT +45 64 second binding site site When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3′-OH of the incoming nucleotide and the 5′-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. ABSTRACT +70 97 Watson-Crick hydrogen bonds bond_interaction When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3′-OH of the incoming nucleotide and the 5′-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. ABSTRACT +144 159 5′-triphosphate chemical When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3′-OH of the incoming nucleotide and the 5′-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. ABSTRACT +167 171 tRNA chemical When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3′-OH of the incoming nucleotide and the 5′-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. ABSTRACT +189 204 reaction center site When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3′-OH of the incoming nucleotide and the 5′-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. ABSTRACT +9 32 3′-5′ elongation enzyme protein_type That the 3′-5′ elongation enzyme performs this elaborate two-step reaction in one catalytic center suggests that these two reactions have been inseparable throughout the process of protein evolution. ABSTRACT +82 98 catalytic center site That the 3′-5′ elongation enzyme performs this elaborate two-step reaction in one catalytic center suggests that these two reactions have been inseparable throughout the process of protein evolution. ABSTRACT +9 12 TLP protein_type Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNAHis-specific G−1 addition enzyme. ABSTRACT +17 21 Thg1 protein Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNAHis-specific G−1 addition enzyme. ABSTRACT +35 45 tetrameric oligomeric_state Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNAHis-specific G−1 addition enzyme. ABSTRACT +64 68 tRNA chemical Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNAHis-specific G−1 addition enzyme. ABSTRACT +85 88 TLP protein_type Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNAHis-specific G−1 addition enzyme. ABSTRACT +115 119 Thg1 protein Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNAHis-specific G−1 addition enzyme. ABSTRACT +123 159 tRNAHis-specific G−1 addition enzyme protein_type Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNAHis-specific G−1 addition enzyme. ABSTRACT +5 12 tRNAHis chemical Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. ABSTRACT +40 44 Thg1 protein Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. ABSTRACT +45 53 tetramer oligomeric_state Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. ABSTRACT +54 62 subunits structure_element Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. ABSTRACT +75 78 TLP protein_type Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. ABSTRACT +80 84 tRNA chemical Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. ABSTRACT +101 116 dimer interface site Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. ABSTRACT +176 189 accepter stem structure_element Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. ABSTRACT +209 217 flexible protein_state Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. ABSTRACT +218 227 β-hairpin structure_element Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. ABSTRACT +13 32 mutational analyses experimental_method Furthermore, mutational analyses show that tRNAHis is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode. ABSTRACT +43 50 tRNAHis chemical Furthermore, mutational analyses show that tRNAHis is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode. ABSTRACT +54 62 bound to protein_state Furthermore, mutational analyses show that tRNAHis is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode. ABSTRACT +63 66 TLP protein_type Furthermore, mutational analyses show that tRNAHis is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode. ABSTRACT +90 94 Thg1 protein Furthermore, mutational analyses show that tRNAHis is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode. ABSTRACT +117 120 TLP protein_type Furthermore, mutational analyses show that tRNAHis is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode. ABSTRACT +60 63 DNA chemical All polynucleotide chain elongation reactions, whether with DNA or RNA, proceed in the 5′-3′ direction. INTRO +67 70 RNA chemical All polynucleotide chain elongation reactions, whether with DNA or RNA, proceed in the 5′-3′ direction. INTRO +118 127 phosphate chemical This reaction involves the nucleophilic attack of a 3′-OH of the terminal nucleotide in the elongating chain on the α-phosphate of an incoming nucleotide. INTRO +28 31 DNA chemical This elongation reaction of DNA/RNA chains is in clear contrast to the elongation of protein chains in which the high energy of the incoming aminoacyl-tRNA is not used for its own addition but for the addition of the next monomer (termed head polymerization). INTRO +32 35 RNA chemical This elongation reaction of DNA/RNA chains is in clear contrast to the elongation of protein chains in which the high energy of the incoming aminoacyl-tRNA is not used for its own addition but for the addition of the next monomer (termed head polymerization). INTRO +141 155 aminoacyl-tRNA chemical This elongation reaction of DNA/RNA chains is in clear contrast to the elongation of protein chains in which the high energy of the incoming aminoacyl-tRNA is not used for its own addition but for the addition of the next monomer (termed head polymerization). INTRO +222 229 monomer oligomeric_state This elongation reaction of DNA/RNA chains is in clear contrast to the elongation of protein chains in which the high energy of the incoming aminoacyl-tRNA is not used for its own addition but for the addition of the next monomer (termed head polymerization). INTRO +44 48 Thg1 protein However, recent studies have shown that the Thg1/Thg1-like protein (TLP) family of proteins extends tRNA nucleotide chains in the reverse (3′-5′) direction. INTRO +49 66 Thg1-like protein protein_type However, recent studies have shown that the Thg1/Thg1-like protein (TLP) family of proteins extends tRNA nucleotide chains in the reverse (3′-5′) direction. INTRO +68 71 TLP protein_type However, recent studies have shown that the Thg1/Thg1-like protein (TLP) family of proteins extends tRNA nucleotide chains in the reverse (3′-5′) direction. INTRO +100 104 tRNA chemical However, recent studies have shown that the Thg1/Thg1-like protein (TLP) family of proteins extends tRNA nucleotide chains in the reverse (3′-5′) direction. INTRO +28 32 tRNA chemical In this case, the 5′-end of tRNA is first activated using adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleophilic attack of a 3′-OH on the incoming nucleotide [nucleoside 5′-triphosphate (NTP)] to yield pppN-tRNA. INTRO +58 83 adenosine 5′-triphosphate chemical In this case, the 5′-end of tRNA is first activated using adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleophilic attack of a 3′-OH on the incoming nucleotide [nucleoside 5′-triphosphate (NTP)] to yield pppN-tRNA. INTRO +85 88 ATP chemical In this case, the 5′-end of tRNA is first activated using adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleophilic attack of a 3′-OH on the incoming nucleotide [nucleoside 5′-triphosphate (NTP)] to yield pppN-tRNA. INTRO +90 115 guanosine 5′-triphosphate chemical In this case, the 5′-end of tRNA is first activated using adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleophilic attack of a 3′-OH on the incoming nucleotide [nucleoside 5′-triphosphate (NTP)] to yield pppN-tRNA. INTRO +117 120 GTP chemical In this case, the 5′-end of tRNA is first activated using adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleophilic attack of a 3′-OH on the incoming nucleotide [nucleoside 5′-triphosphate (NTP)] to yield pppN-tRNA. INTRO +194 220 nucleoside 5′-triphosphate chemical In this case, the 5′-end of tRNA is first activated using adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleophilic attack of a 3′-OH on the incoming nucleotide [nucleoside 5′-triphosphate (NTP)] to yield pppN-tRNA. INTRO +222 225 NTP chemical In this case, the 5′-end of tRNA is first activated using adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleophilic attack of a 3′-OH on the incoming nucleotide [nucleoside 5′-triphosphate (NTP)] to yield pppN-tRNA. INTRO +237 246 pppN-tRNA chemical In this case, the 5′-end of tRNA is first activated using adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleophilic attack of a 3′-OH on the incoming nucleotide [nucleoside 5′-triphosphate (NTP)] to yield pppN-tRNA. INTRO +24 36 triphosphate chemical Thus, the energy in the triphosphate bond of the incoming nucleotide is not used for its own addition but is reserved for subsequent polymerization (that is, head polymerization) (Fig. 1). INTRO +44 48 Thg1 protein Top: Reaction scheme of 3′-5′ elongation by Thg1/TLP family proteins. FIG +49 52 TLP protein_type Top: Reaction scheme of 3′-5′ elongation by Thg1/TLP family proteins. FIG +47 66 DNA/RNA polymerases protein_type Bottom: Reaction scheme of 5′-3′ elongation by DNA/RNA polymerases. FIG +23 27 Thg1 protein In 3′-5′ elongation by Thg1/TLP family proteins, the 5′-monophosphate of the tRNA is first activated by ATP/GTP, followed by the actual elongation reaction. FIG +28 31 TLP protein_type In 3′-5′ elongation by Thg1/TLP family proteins, the 5′-monophosphate of the tRNA is first activated by ATP/GTP, followed by the actual elongation reaction. FIG +53 69 5′-monophosphate chemical In 3′-5′ elongation by Thg1/TLP family proteins, the 5′-monophosphate of the tRNA is first activated by ATP/GTP, followed by the actual elongation reaction. FIG +77 81 tRNA chemical In 3′-5′ elongation by Thg1/TLP family proteins, the 5′-monophosphate of the tRNA is first activated by ATP/GTP, followed by the actual elongation reaction. FIG +104 107 ATP chemical In 3′-5′ elongation by Thg1/TLP family proteins, the 5′-monophosphate of the tRNA is first activated by ATP/GTP, followed by the actual elongation reaction. FIG +108 111 GTP chemical In 3′-5′ elongation by Thg1/TLP family proteins, the 5′-monophosphate of the tRNA is first activated by ATP/GTP, followed by the actual elongation reaction. FIG +23 42 DNA/RNA polymerases protein_type In 5′-3′ elongation by DNA/RNA polymerases, the energy of the incoming nucleotide is used for its own addition (tail polymerization). FIG +60 70 eukaryotic taxonomy_domain The best-characterized member of this family of proteins is eukaryotic Thg1 (tRNAHis guanylyltransferase), which catalyzes the nontemplated addition of a guanylate to the 5′-end of immature tRNAHis. INTRO +71 75 Thg1 protein The best-characterized member of this family of proteins is eukaryotic Thg1 (tRNAHis guanylyltransferase), which catalyzes the nontemplated addition of a guanylate to the 5′-end of immature tRNAHis. INTRO +77 104 tRNAHis guanylyltransferase protein_type The best-characterized member of this family of proteins is eukaryotic Thg1 (tRNAHis guanylyltransferase), which catalyzes the nontemplated addition of a guanylate to the 5′-end of immature tRNAHis. INTRO +190 197 tRNAHis chemical The best-characterized member of this family of proteins is eukaryotic Thg1 (tRNAHis guanylyltransferase), which catalyzes the nontemplated addition of a guanylate to the 5′-end of immature tRNAHis. INTRO +5 14 guanosine chemical This guanosine at position −1 (G−1) of tRNAHis is a critical identity element for recognition by the histidyl-tRNA synthase. INTRO +27 29 −1 residue_number This guanosine at position −1 (G−1) of tRNAHis is a critical identity element for recognition by the histidyl-tRNA synthase. INTRO +31 34 G−1 residue_name_number This guanosine at position −1 (G−1) of tRNAHis is a critical identity element for recognition by the histidyl-tRNA synthase. INTRO +39 46 tRNAHis chemical This guanosine at position −1 (G−1) of tRNAHis is a critical identity element for recognition by the histidyl-tRNA synthase. INTRO +101 123 histidyl-tRNA synthase protein_type This guanosine at position −1 (G−1) of tRNAHis is a critical identity element for recognition by the histidyl-tRNA synthase. INTRO +11 15 Thg1 protein Therefore, Thg1 is essential to the fidelity of protein synthesis in eukaryotes. INTRO +69 79 eukaryotes taxonomy_domain Therefore, Thg1 is essential to the fidelity of protein synthesis in eukaryotes. INTRO +9 13 Thg1 protein However, Thg1 homologs or TLPs are found in organisms in which G−1 is genetically encoded, and thus, posttranscriptional modification is not required. INTRO +26 30 TLPs protein_type However, Thg1 homologs or TLPs are found in organisms in which G−1 is genetically encoded, and thus, posttranscriptional modification is not required. INTRO +63 66 G−1 residue_name_number However, Thg1 homologs or TLPs are found in organisms in which G−1 is genetically encoded, and thus, posttranscriptional modification is not required. INTRO +27 31 TLPs protein_type This finding suggests that TLPs may have potential functions other than tRNAHis maturation. INTRO +72 79 tRNAHis chemical This finding suggests that TLPs may have potential functions other than tRNAHis maturation. INTRO +0 4 TLPs protein_type TLPs have been shown to catalyze 5′-end nucleotide addition to truncated tRNA species in vitro in a Watson-Crick template–dependent manner. INTRO +73 77 tRNA chemical TLPs have been shown to catalyze 5′-end nucleotide addition to truncated tRNA species in vitro in a Watson-Crick template–dependent manner. INTRO +17 21 TLPs protein_type This function of TLPs is not limited to tRNAHis but occurs efficiently with other tRNAs. INTRO +40 47 tRNAHis chemical This function of TLPs is not limited to tRNAHis but occurs efficiently with other tRNAs. INTRO +82 87 tRNAs chemical This function of TLPs is not limited to tRNAHis but occurs efficiently with other tRNAs. INTRO +17 22 yeast taxonomy_domain Furthermore, the yeast homolog (Thg1p) has been shown to interact with the replication origin recognition complex for DNA replication, and the plant homolog (ICA1) was identified as a protein affecting the capacity to repair DNA damage. INTRO +32 37 Thg1p protein Furthermore, the yeast homolog (Thg1p) has been shown to interact with the replication origin recognition complex for DNA replication, and the plant homolog (ICA1) was identified as a protein affecting the capacity to repair DNA damage. INTRO +118 121 DNA chemical Furthermore, the yeast homolog (Thg1p) has been shown to interact with the replication origin recognition complex for DNA replication, and the plant homolog (ICA1) was identified as a protein affecting the capacity to repair DNA damage. INTRO +143 148 plant taxonomy_domain Furthermore, the yeast homolog (Thg1p) has been shown to interact with the replication origin recognition complex for DNA replication, and the plant homolog (ICA1) was identified as a protein affecting the capacity to repair DNA damage. INTRO +158 162 ICA1 protein Furthermore, the yeast homolog (Thg1p) has been shown to interact with the replication origin recognition complex for DNA replication, and the plant homolog (ICA1) was identified as a protein affecting the capacity to repair DNA damage. INTRO +225 228 DNA chemical Furthermore, the yeast homolog (Thg1p) has been shown to interact with the replication origin recognition complex for DNA replication, and the plant homolog (ICA1) was identified as a protein affecting the capacity to repair DNA damage. INTRO +32 36 TLPs protein_type These observations suggest that TLPs may have more general functions in DNA/RNA repair. INTRO +72 75 DNA chemical These observations suggest that TLPs may have more general functions in DNA/RNA repair. INTRO +76 79 RNA chemical These observations suggest that TLPs may have more general functions in DNA/RNA repair. INTRO +41 45 Thg1 protein The 3′-5′ addition reaction catalyzed by Thg1 occurs through three reaction steps. INTRO +45 52 tRNAHis chemical In the first step, the 5′-monophosphorylated tRNAHis, which is cleaved by ribonuclease P from pre-tRNAHis, is activated by ATP, creating a 5′-adenylylated tRNAHis intermediate. INTRO +74 88 ribonuclease P protein_type In the first step, the 5′-monophosphorylated tRNAHis, which is cleaved by ribonuclease P from pre-tRNAHis, is activated by ATP, creating a 5′-adenylylated tRNAHis intermediate. INTRO +94 105 pre-tRNAHis chemical In the first step, the 5′-monophosphorylated tRNAHis, which is cleaved by ribonuclease P from pre-tRNAHis, is activated by ATP, creating a 5′-adenylylated tRNAHis intermediate. INTRO +123 126 ATP chemical In the first step, the 5′-monophosphorylated tRNAHis, which is cleaved by ribonuclease P from pre-tRNAHis, is activated by ATP, creating a 5′-adenylylated tRNAHis intermediate. INTRO +155 162 tRNAHis chemical In the first step, the 5′-monophosphorylated tRNAHis, which is cleaved by ribonuclease P from pre-tRNAHis, is activated by ATP, creating a 5′-adenylylated tRNAHis intermediate. INTRO +46 49 GTP chemical In the second step, the 3′-OH of the incoming GTP attacks the activated intermediate, yielding pppG−1-tRNAHis. INTRO +95 109 pppG−1-tRNAHis chemical In the second step, the 3′-OH of the incoming GTP attacks the activated intermediate, yielding pppG−1-tRNAHis. INTRO +13 26 pyrophosphate chemical Finally, the pyrophosphate is removed, and mature pG−1-tRNAHis is created. INTRO +50 62 pG−1-tRNAHis chemical Finally, the pyrophosphate is removed, and mature pG−1-tRNAHis is created. INTRO +4 21 crystal structure evidence The crystal structure of human Thg1 (HsThg1) shows that its catalytic core shares structural homology with canonical 5′-3′ nucleotide polymerases, such as T7 DNA/RNA polymerases. INTRO +25 30 human species The crystal structure of human Thg1 (HsThg1) shows that its catalytic core shares structural homology with canonical 5′-3′ nucleotide polymerases, such as T7 DNA/RNA polymerases. INTRO +31 35 Thg1 protein The crystal structure of human Thg1 (HsThg1) shows that its catalytic core shares structural homology with canonical 5′-3′ nucleotide polymerases, such as T7 DNA/RNA polymerases. INTRO +37 43 HsThg1 protein The crystal structure of human Thg1 (HsThg1) shows that its catalytic core shares structural homology with canonical 5′-3′ nucleotide polymerases, such as T7 DNA/RNA polymerases. INTRO +60 74 catalytic core site The crystal structure of human Thg1 (HsThg1) shows that its catalytic core shares structural homology with canonical 5′-3′ nucleotide polymerases, such as T7 DNA/RNA polymerases. INTRO +117 145 5′-3′ nucleotide polymerases protein_type The crystal structure of human Thg1 (HsThg1) shows that its catalytic core shares structural homology with canonical 5′-3′ nucleotide polymerases, such as T7 DNA/RNA polymerases. INTRO +155 177 T7 DNA/RNA polymerases protein_type The crystal structure of human Thg1 (HsThg1) shows that its catalytic core shares structural homology with canonical 5′-3′ nucleotide polymerases, such as T7 DNA/RNA polymerases. INTRO +27 51 3′-5′ elongation enzymes protein_type This finding suggests that 3′-5′ elongation enzymes are related to 5′-3′ polymerases and raises important questions on why 5′-3′ polymerases predominate in nature. INTRO +67 84 5′-3′ polymerases protein_type This finding suggests that 3′-5′ elongation enzymes are related to 5′-3′ polymerases and raises important questions on why 5′-3′ polymerases predominate in nature. INTRO +123 140 5′-3′ polymerases protein_type This finding suggests that 3′-5′ elongation enzymes are related to 5′-3′ polymerases and raises important questions on why 5′-3′ polymerases predominate in nature. INTRO +4 21 crystal structure evidence The crystal structure of TLP from Bacillus thuringiensis shows that it shares a similar tetrameric assembly and active-site architecture with HsThg1. INTRO +25 28 TLP protein_type The crystal structure of TLP from Bacillus thuringiensis shows that it shares a similar tetrameric assembly and active-site architecture with HsThg1. INTRO +34 56 Bacillus thuringiensis species The crystal structure of TLP from Bacillus thuringiensis shows that it shares a similar tetrameric assembly and active-site architecture with HsThg1. INTRO +88 98 tetrameric oligomeric_state The crystal structure of TLP from Bacillus thuringiensis shows that it shares a similar tetrameric assembly and active-site architecture with HsThg1. INTRO +112 123 active-site site The crystal structure of TLP from Bacillus thuringiensis shows that it shares a similar tetrameric assembly and active-site architecture with HsThg1. INTRO +142 148 HsThg1 protein The crystal structure of TLP from Bacillus thuringiensis shows that it shares a similar tetrameric assembly and active-site architecture with HsThg1. INTRO +17 26 structure evidence Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase. INTRO +30 46 Candida albicans species Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase. INTRO +47 51 Thg1 protein Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase. INTRO +53 59 CaThg1 protein Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase. INTRO +61 75 complexed with protein_state Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase. INTRO +76 83 tRNAHis chemical Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase. INTRO +101 105 tRNA chemical Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase. INTRO +129 144 reaction center site Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase. INTRO +192 210 DNA/RNA polymerase protein_type Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase. INTRO +17 36 structural analysis experimental_method However, in this structural analysis, the 5′-end of tRNAHis was not activated and the second substrate (GTP) was not bound. INTRO +52 59 tRNAHis chemical However, in this structural analysis, the 5′-end of tRNAHis was not activated and the second substrate (GTP) was not bound. INTRO +104 107 GTP chemical However, in this structural analysis, the 5′-end of tRNAHis was not activated and the second substrate (GTP) was not bound. INTRO +113 122 not bound protein_state However, in this structural analysis, the 5′-end of tRNAHis was not activated and the second substrate (GTP) was not bound. INTRO +22 28 solved experimental_method Here, we successfully solved the structure of TLP from the methanogenic archaeon Methanosarcina acetivorans (MaTLP) in complex with ppptRNAPheΔ1, which mimics the activated intermediate of the repair substrate. INTRO +33 42 structure evidence Here, we successfully solved the structure of TLP from the methanogenic archaeon Methanosarcina acetivorans (MaTLP) in complex with ppptRNAPheΔ1, which mimics the activated intermediate of the repair substrate. INTRO +46 49 TLP protein_type Here, we successfully solved the structure of TLP from the methanogenic archaeon Methanosarcina acetivorans (MaTLP) in complex with ppptRNAPheΔ1, which mimics the activated intermediate of the repair substrate. INTRO +59 80 methanogenic archaeon taxonomy_domain Here, we successfully solved the structure of TLP from the methanogenic archaeon Methanosarcina acetivorans (MaTLP) in complex with ppptRNAPheΔ1, which mimics the activated intermediate of the repair substrate. INTRO +81 107 Methanosarcina acetivorans species Here, we successfully solved the structure of TLP from the methanogenic archaeon Methanosarcina acetivorans (MaTLP) in complex with ppptRNAPheΔ1, which mimics the activated intermediate of the repair substrate. INTRO +109 114 MaTLP protein Here, we successfully solved the structure of TLP from the methanogenic archaeon Methanosarcina acetivorans (MaTLP) in complex with ppptRNAPheΔ1, which mimics the activated intermediate of the repair substrate. INTRO +116 131 in complex with protein_state Here, we successfully solved the structure of TLP from the methanogenic archaeon Methanosarcina acetivorans (MaTLP) in complex with ppptRNAPheΔ1, which mimics the activated intermediate of the repair substrate. INTRO +132 144 ppptRNAPheΔ1 chemical Here, we successfully solved the structure of TLP from the methanogenic archaeon Methanosarcina acetivorans (MaTLP) in complex with ppptRNAPheΔ1, which mimics the activated intermediate of the repair substrate. INTRO +9 12 TLP protein_type Although TLP and Thg1 have similar tetrameric organization, the mode of tRNA binding is different in TLP. INTRO +17 21 Thg1 protein Although TLP and Thg1 have similar tetrameric organization, the mode of tRNA binding is different in TLP. INTRO +35 45 tetrameric oligomeric_state Although TLP and Thg1 have similar tetrameric organization, the mode of tRNA binding is different in TLP. INTRO +72 76 tRNA chemical Although TLP and Thg1 have similar tetrameric organization, the mode of tRNA binding is different in TLP. INTRO +101 104 TLP protein_type Although TLP and Thg1 have similar tetrameric organization, the mode of tRNA binding is different in TLP. INTRO +29 38 structure evidence Furthermore, we obtained the structure in which the GTP analog (GDPNP) was inserted into this complex to form a Watson-Crick base pair with C72 at the 3′-end region of the tRNA. INTRO +52 55 GTP chemical Furthermore, we obtained the structure in which the GTP analog (GDPNP) was inserted into this complex to form a Watson-Crick base pair with C72 at the 3′-end region of the tRNA. INTRO +64 69 GDPNP chemical Furthermore, we obtained the structure in which the GTP analog (GDPNP) was inserted into this complex to form a Watson-Crick base pair with C72 at the 3′-end region of the tRNA. INTRO +112 134 Watson-Crick base pair bond_interaction Furthermore, we obtained the structure in which the GTP analog (GDPNP) was inserted into this complex to form a Watson-Crick base pair with C72 at the 3′-end region of the tRNA. INTRO +140 143 C72 residue_name_number Furthermore, we obtained the structure in which the GTP analog (GDPNP) was inserted into this complex to form a Watson-Crick base pair with C72 at the 3′-end region of the tRNA. INTRO +172 176 tRNA chemical Furthermore, we obtained the structure in which the GTP analog (GDPNP) was inserted into this complex to form a Watson-Crick base pair with C72 at the 3′-end region of the tRNA. INTRO +22 32 structures evidence On the basis of these structures, we discuss the reaction mechanism of template-dependent reverse (3′-5′) polymerization in comparison with canonical 5′-3′ polymerization. INTRO +33 45 ppptRNAPheΔ1 chemical Anticodon-independent binding of ppptRNAPheΔ1 to MaTLP RESULTS +49 54 MaTLP protein Anticodon-independent binding of ppptRNAPheΔ1 to MaTLP RESULTS +9 32 biochemical experiments experimental_method Previous biochemical experiments have suggested that ppptRNAPheΔ1, in which the 5′-end of tRNAPhe was triphosphorylated and G1 was deleted, can be an efficient substrate for the repair reaction (guanylyl transfer) of Thg1/TLP. RESULTS +53 65 ppptRNAPheΔ1 chemical Previous biochemical experiments have suggested that ppptRNAPheΔ1, in which the 5′-end of tRNAPhe was triphosphorylated and G1 was deleted, can be an efficient substrate for the repair reaction (guanylyl transfer) of Thg1/TLP. RESULTS +90 97 tRNAPhe chemical Previous biochemical experiments have suggested that ppptRNAPheΔ1, in which the 5′-end of tRNAPhe was triphosphorylated and G1 was deleted, can be an efficient substrate for the repair reaction (guanylyl transfer) of Thg1/TLP. RESULTS +124 126 G1 residue_name_number Previous biochemical experiments have suggested that ppptRNAPheΔ1, in which the 5′-end of tRNAPhe was triphosphorylated and G1 was deleted, can be an efficient substrate for the repair reaction (guanylyl transfer) of Thg1/TLP. RESULTS +131 138 deleted experimental_method Previous biochemical experiments have suggested that ppptRNAPheΔ1, in which the 5′-end of tRNAPhe was triphosphorylated and G1 was deleted, can be an efficient substrate for the repair reaction (guanylyl transfer) of Thg1/TLP. RESULTS +217 221 Thg1 protein Previous biochemical experiments have suggested that ppptRNAPheΔ1, in which the 5′-end of tRNAPhe was triphosphorylated and G1 was deleted, can be an efficient substrate for the repair reaction (guanylyl transfer) of Thg1/TLP. RESULTS +222 225 TLP protein_type Previous biochemical experiments have suggested that ppptRNAPheΔ1, in which the 5′-end of tRNAPhe was triphosphorylated and G1 was deleted, can be an efficient substrate for the repair reaction (guanylyl transfer) of Thg1/TLP. RESULTS +25 32 crystal evidence Therefore, we prepared a crystal of MaTLP complexed with ppptRNAPheΔ1 and solved its structure to study the template-directed 3′-5′ elongation reaction by TLP (fig. S1). RESULTS +36 41 MaTLP protein Therefore, we prepared a crystal of MaTLP complexed with ppptRNAPheΔ1 and solved its structure to study the template-directed 3′-5′ elongation reaction by TLP (fig. S1). RESULTS +42 56 complexed with protein_state Therefore, we prepared a crystal of MaTLP complexed with ppptRNAPheΔ1 and solved its structure to study the template-directed 3′-5′ elongation reaction by TLP (fig. S1). RESULTS +57 69 ppptRNAPheΔ1 chemical Therefore, we prepared a crystal of MaTLP complexed with ppptRNAPheΔ1 and solved its structure to study the template-directed 3′-5′ elongation reaction by TLP (fig. S1). RESULTS +74 80 solved experimental_method Therefore, we prepared a crystal of MaTLP complexed with ppptRNAPheΔ1 and solved its structure to study the template-directed 3′-5′ elongation reaction by TLP (fig. S1). RESULTS +85 94 structure evidence Therefore, we prepared a crystal of MaTLP complexed with ppptRNAPheΔ1 and solved its structure to study the template-directed 3′-5′ elongation reaction by TLP (fig. S1). RESULTS +155 158 TLP protein_type Therefore, we prepared a crystal of MaTLP complexed with ppptRNAPheΔ1 and solved its structure to study the template-directed 3′-5′ elongation reaction by TLP (fig. S1). RESULTS +4 11 crystal evidence The crystal contained a dimer of TLP (A and B molecules) and one tRNA in an asymmetric unit. RESULTS +24 29 dimer oligomeric_state The crystal contained a dimer of TLP (A and B molecules) and one tRNA in an asymmetric unit. RESULTS +33 36 TLP protein_type The crystal contained a dimer of TLP (A and B molecules) and one tRNA in an asymmetric unit. RESULTS +38 39 A structure_element The crystal contained a dimer of TLP (A and B molecules) and one tRNA in an asymmetric unit. RESULTS +44 45 B structure_element The crystal contained a dimer of TLP (A and B molecules) and one tRNA in an asymmetric unit. RESULTS +65 69 tRNA chemical The crystal contained a dimer of TLP (A and B molecules) and one tRNA in an asymmetric unit. RESULTS +4 10 dimers oligomeric_state Two dimers in the crystal further assembled as a dimer of dimers by the crystallographic twofold axis (Fig. 2). RESULTS +18 25 crystal evidence Two dimers in the crystal further assembled as a dimer of dimers by the crystallographic twofold axis (Fig. 2). RESULTS +49 54 dimer oligomeric_state Two dimers in the crystal further assembled as a dimer of dimers by the crystallographic twofold axis (Fig. 2). RESULTS +58 64 dimers oligomeric_state Two dimers in the crystal further assembled as a dimer of dimers by the crystallographic twofold axis (Fig. 2). RESULTS +5 15 tetrameric oligomeric_state This tetrameric structure and 4:2 stoichiometry of the TLP-tRNA complex are the same as those of the CaThg1-tRNA complex. RESULTS +16 25 structure evidence This tetrameric structure and 4:2 stoichiometry of the TLP-tRNA complex are the same as those of the CaThg1-tRNA complex. RESULTS +55 63 TLP-tRNA complex_assembly This tetrameric structure and 4:2 stoichiometry of the TLP-tRNA complex are the same as those of the CaThg1-tRNA complex. RESULTS +101 112 CaThg1-tRNA complex_assembly This tetrameric structure and 4:2 stoichiometry of the TLP-tRNA complex are the same as those of the CaThg1-tRNA complex. RESULTS +21 23 AB structure_element However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +28 30 CD structure_element However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +31 37 dimers oligomeric_state However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +41 51 tetrameric oligomeric_state However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +52 58 CaThg1 protein However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +110 123 accepter stem structure_element However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +141 148 tRNAHis chemical However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +154 156 AB structure_element However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +157 162 dimer oligomeric_state However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +186 188 CD structure_element However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +189 194 dimer oligomeric_state However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +199 209 tetrameric oligomeric_state However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +210 215 MaTLP protein However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +251 255 tRNA chemical However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +283 287 tRNA chemical However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +288 301 accepter stem structure_element However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +306 318 elbow region structure_element However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. RESULTS +38 43 MaTLP protein Thus, consistent with the notion that MaTLP is an anticodon-independent repair enzyme, the anticodon was not recognized in the MaTLP-tRNA complex, whereas the binding mode of CaThg1 is for the G−1 addition reaction, therefore the His anticodon has to be recognized (see “Dual binding mode for tRNA repair”). RESULTS +50 85 anticodon-independent repair enzyme protein_type Thus, consistent with the notion that MaTLP is an anticodon-independent repair enzyme, the anticodon was not recognized in the MaTLP-tRNA complex, whereas the binding mode of CaThg1 is for the G−1 addition reaction, therefore the His anticodon has to be recognized (see “Dual binding mode for tRNA repair”). RESULTS +127 137 MaTLP-tRNA complex_assembly Thus, consistent with the notion that MaTLP is an anticodon-independent repair enzyme, the anticodon was not recognized in the MaTLP-tRNA complex, whereas the binding mode of CaThg1 is for the G−1 addition reaction, therefore the His anticodon has to be recognized (see “Dual binding mode for tRNA repair”). RESULTS +175 181 CaThg1 protein Thus, consistent with the notion that MaTLP is an anticodon-independent repair enzyme, the anticodon was not recognized in the MaTLP-tRNA complex, whereas the binding mode of CaThg1 is for the G−1 addition reaction, therefore the His anticodon has to be recognized (see “Dual binding mode for tRNA repair”). RESULTS +193 196 G−1 residue_name_number Thus, consistent with the notion that MaTLP is an anticodon-independent repair enzyme, the anticodon was not recognized in the MaTLP-tRNA complex, whereas the binding mode of CaThg1 is for the G−1 addition reaction, therefore the His anticodon has to be recognized (see “Dual binding mode for tRNA repair”). RESULTS +230 233 His residue_name Thus, consistent with the notion that MaTLP is an anticodon-independent repair enzyme, the anticodon was not recognized in the MaTLP-tRNA complex, whereas the binding mode of CaThg1 is for the G−1 addition reaction, therefore the His anticodon has to be recognized (see “Dual binding mode for tRNA repair”). RESULTS +293 297 tRNA chemical Thus, consistent with the notion that MaTLP is an anticodon-independent repair enzyme, the anticodon was not recognized in the MaTLP-tRNA complex, whereas the binding mode of CaThg1 is for the G−1 addition reaction, therefore the His anticodon has to be recognized (see “Dual binding mode for tRNA repair”). RESULTS +0 9 Structure evidence Structure of the MaTLP complex with ppptRNAPheΔ1. FIG +17 22 MaTLP protein Structure of the MaTLP complex with ppptRNAPheΔ1. FIG +23 35 complex with protein_state Structure of the MaTLP complex with ppptRNAPheΔ1. FIG +36 48 ppptRNAPheΔ1 chemical Structure of the MaTLP complex with ppptRNAPheΔ1. FIG +26 30 tRNA chemical Left: One molecule of the tRNA substrate (ppptRNAPheΔ1) is bound to the MaTLP dimer. FIG +42 54 ppptRNAPheΔ1 chemical Left: One molecule of the tRNA substrate (ppptRNAPheΔ1) is bound to the MaTLP dimer. FIG +59 67 bound to protein_state Left: One molecule of the tRNA substrate (ppptRNAPheΔ1) is bound to the MaTLP dimer. FIG +72 77 MaTLP protein Left: One molecule of the tRNA substrate (ppptRNAPheΔ1) is bound to the MaTLP dimer. FIG +78 83 dimer oligomeric_state Left: One molecule of the tRNA substrate (ppptRNAPheΔ1) is bound to the MaTLP dimer. FIG +4 6 AB structure_element The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers). FIG +11 13 CD structure_element The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers). FIG +14 20 dimers oligomeric_state The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers). FIG +33 42 dimerized oligomeric_state The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers). FIG +90 100 tetrameric oligomeric_state The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers). FIG +101 110 structure evidence The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers). FIG +112 117 dimer oligomeric_state The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers). FIG +121 127 dimers oligomeric_state The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers). FIG +4 6 CD structure_element The CD dimer is omitted for clarity. FIG +7 12 dimer oligomeric_state The CD dimer is omitted for clarity. FIG +4 17 accepter stem structure_element The accepter stem of the tRNA is recognized by molecule A (yellow), and the elbow region by molecule B (blue). FIG +25 29 tRNA chemical The accepter stem of the tRNA is recognized by molecule A (yellow), and the elbow region by molecule B (blue). FIG +76 88 elbow region structure_element The accepter stem of the tRNA is recognized by molecule A (yellow), and the elbow region by molecule B (blue). FIG +4 13 β-hairpin structure_element The β-hairpin region of molecule B is shown in red. FIG +4 16 elbow region structure_element The elbow region of the tRNA substrate was recognized by the β-hairpin of molecule B of MaTLP. RESULTS +24 28 tRNA chemical The elbow region of the tRNA substrate was recognized by the β-hairpin of molecule B of MaTLP. RESULTS +61 70 β-hairpin structure_element The elbow region of the tRNA substrate was recognized by the β-hairpin of molecule B of MaTLP. RESULTS +88 93 MaTLP protein The elbow region of the tRNA substrate was recognized by the β-hairpin of molecule B of MaTLP. RESULTS +33 37 R215 residue_name_number The N atoms in the side chain of R215 in the β-hairpin region of MaTLP were hydrogen-bonded to the phosphate groups of U55 and G57. RESULTS +45 54 β-hairpin structure_element The N atoms in the side chain of R215 in the β-hairpin region of MaTLP were hydrogen-bonded to the phosphate groups of U55 and G57. RESULTS +65 70 MaTLP protein The N atoms in the side chain of R215 in the β-hairpin region of MaTLP were hydrogen-bonded to the phosphate groups of U55 and G57. RESULTS +76 91 hydrogen-bonded bond_interaction The N atoms in the side chain of R215 in the β-hairpin region of MaTLP were hydrogen-bonded to the phosphate groups of U55 and G57. RESULTS +99 108 phosphate chemical The N atoms in the side chain of R215 in the β-hairpin region of MaTLP were hydrogen-bonded to the phosphate groups of U55 and G57. RESULTS +119 122 U55 residue_name_number The N atoms in the side chain of R215 in the β-hairpin region of MaTLP were hydrogen-bonded to the phosphate groups of U55 and G57. RESULTS +127 130 G57 residue_name_number The N atoms in the side chain of R215 in the β-hairpin region of MaTLP were hydrogen-bonded to the phosphate groups of U55 and G57. RESULTS +18 22 S213 residue_name_number The O atom on the S213 side chain was also hydrogen-bonded to the phosphate moiety of G57 of the tRNA (Fig. 2). RESULTS +43 58 hydrogen-bonded bond_interaction The O atom on the S213 side chain was also hydrogen-bonded to the phosphate moiety of G57 of the tRNA (Fig. 2). RESULTS +66 75 phosphate chemical The O atom on the S213 side chain was also hydrogen-bonded to the phosphate moiety of G57 of the tRNA (Fig. 2). RESULTS +86 89 G57 residue_name_number The O atom on the S213 side chain was also hydrogen-bonded to the phosphate moiety of G57 of the tRNA (Fig. 2). RESULTS +97 101 tRNA chemical The O atom on the S213 side chain was also hydrogen-bonded to the phosphate moiety of G57 of the tRNA (Fig. 2). RESULTS +5 14 β-hairpin structure_element This β-hairpin region was disordered in the crystal structure of the CaThg1-tRNA complex. RESULTS +26 36 disordered protein_state This β-hairpin region was disordered in the crystal structure of the CaThg1-tRNA complex. RESULTS +44 61 crystal structure evidence This β-hairpin region was disordered in the crystal structure of the CaThg1-tRNA complex. RESULTS +69 80 CaThg1-tRNA complex_assembly This β-hairpin region was disordered in the crystal structure of the CaThg1-tRNA complex. RESULTS +4 17 accepter stem structure_element The accepter stem of the tRNA substrate was recognized by molecule A of MaTLP. RESULTS +25 29 tRNA chemical The accepter stem of the tRNA substrate was recognized by molecule A of MaTLP. RESULTS +72 77 MaTLP protein The accepter stem of the tRNA substrate was recognized by molecule A of MaTLP. RESULTS +15 17 G2 residue_name_number The N7 atom of G2 at the 5′-end was hydrogen-bonded to the N atom of the R136 side chain, whereas the α-phosphate was bonded to the N137 side chain (Fig. 2). RESULTS +36 51 hydrogen-bonded bond_interaction The N7 atom of G2 at the 5′-end was hydrogen-bonded to the N atom of the R136 side chain, whereas the α-phosphate was bonded to the N137 side chain (Fig. 2). RESULTS +73 77 R136 residue_name_number The N7 atom of G2 at the 5′-end was hydrogen-bonded to the N atom of the R136 side chain, whereas the α-phosphate was bonded to the N137 side chain (Fig. 2). RESULTS +104 113 phosphate chemical The N7 atom of G2 at the 5′-end was hydrogen-bonded to the N atom of the R136 side chain, whereas the α-phosphate was bonded to the N137 side chain (Fig. 2). RESULTS +132 136 N137 residue_name_number The N7 atom of G2 at the 5′-end was hydrogen-bonded to the N atom of the R136 side chain, whereas the α-phosphate was bonded to the N137 side chain (Fig. 2). RESULTS +0 4 R136 residue_name_number R136 was also hydrogen-bonded to the base of C72 (the Watson-Crick bond partner of ΔG1). RESULTS +14 29 hydrogen-bonded bond_interaction R136 was also hydrogen-bonded to the base of C72 (the Watson-Crick bond partner of ΔG1). RESULTS +45 48 C72 residue_name_number R136 was also hydrogen-bonded to the base of C72 (the Watson-Crick bond partner of ΔG1). RESULTS +4 16 triphosphate chemical The triphosphate moiety at the 5′-end of the tRNA was bonded to the D21-K26 region. RESULTS +45 49 tRNA chemical The triphosphate moiety at the 5′-end of the tRNA was bonded to the D21-K26 region. RESULTS +68 75 D21-K26 residue_range The triphosphate moiety at the 5′-end of the tRNA was bonded to the D21-K26 region. RESULTS +6 16 phosphates chemical These phosphates were also coordinated to two metal ions, presumably Mg2+ (Mg2+A and Mg2+B) because they were observed at the same positions (figs. S3 and S4) previously identified by CaThg1 and HsThg1 structures. RESULTS +27 41 coordinated to bond_interaction These phosphates were also coordinated to two metal ions, presumably Mg2+ (Mg2+A and Mg2+B) because they were observed at the same positions (figs. S3 and S4) previously identified by CaThg1 and HsThg1 structures. RESULTS +69 73 Mg2+ chemical These phosphates were also coordinated to two metal ions, presumably Mg2+ (Mg2+A and Mg2+B) because they were observed at the same positions (figs. S3 and S4) previously identified by CaThg1 and HsThg1 structures. RESULTS +75 79 Mg2+ chemical These phosphates were also coordinated to two metal ions, presumably Mg2+ (Mg2+A and Mg2+B) because they were observed at the same positions (figs. S3 and S4) previously identified by CaThg1 and HsThg1 structures. RESULTS +85 89 Mg2+ chemical These phosphates were also coordinated to two metal ions, presumably Mg2+ (Mg2+A and Mg2+B) because they were observed at the same positions (figs. S3 and S4) previously identified by CaThg1 and HsThg1 structures. RESULTS +184 190 CaThg1 protein These phosphates were also coordinated to two metal ions, presumably Mg2+ (Mg2+A and Mg2+B) because they were observed at the same positions (figs. S3 and S4) previously identified by CaThg1 and HsThg1 structures. RESULTS +195 201 HsThg1 protein These phosphates were also coordinated to two metal ions, presumably Mg2+ (Mg2+A and Mg2+B) because they were observed at the same positions (figs. S3 and S4) previously identified by CaThg1 and HsThg1 structures. RESULTS +202 212 structures evidence These phosphates were also coordinated to two metal ions, presumably Mg2+ (Mg2+A and Mg2+B) because they were observed at the same positions (figs. S3 and S4) previously identified by CaThg1 and HsThg1 structures. RESULTS +24 38 coordinated by bond_interaction These ions were in turn coordinated by the O atoms of the side chains of D21 and D69 and the main-chain O of G22 (fig. S3A). RESULTS +73 76 D21 residue_name_number These ions were in turn coordinated by the O atoms of the side chains of D21 and D69 and the main-chain O of G22 (fig. S3A). RESULTS +81 84 D69 residue_name_number These ions were in turn coordinated by the O atoms of the side chains of D21 and D69 and the main-chain O of G22 (fig. S3A). RESULTS +109 112 G22 residue_name_number These ions were in turn coordinated by the O atoms of the side chains of D21 and D69 and the main-chain O of G22 (fig. S3A). RESULTS +0 8 Mutation experimental_method Mutation of D29 and D76 in HsThg1 (corresponding to D21 and D69 of MaTLP) has been shown to markedly decrease G−1 addition activity. RESULTS +12 15 D29 residue_name_number Mutation of D29 and D76 in HsThg1 (corresponding to D21 and D69 of MaTLP) has been shown to markedly decrease G−1 addition activity. RESULTS +20 23 D76 residue_name_number Mutation of D29 and D76 in HsThg1 (corresponding to D21 and D69 of MaTLP) has been shown to markedly decrease G−1 addition activity. RESULTS +27 33 HsThg1 protein Mutation of D29 and D76 in HsThg1 (corresponding to D21 and D69 of MaTLP) has been shown to markedly decrease G−1 addition activity. RESULTS +52 55 D21 residue_name_number Mutation of D29 and D76 in HsThg1 (corresponding to D21 and D69 of MaTLP) has been shown to markedly decrease G−1 addition activity. RESULTS +60 63 D69 residue_name_number Mutation of D29 and D76 in HsThg1 (corresponding to D21 and D69 of MaTLP) has been shown to markedly decrease G−1 addition activity. RESULTS +67 72 MaTLP protein Mutation of D29 and D76 in HsThg1 (corresponding to D21 and D69 of MaTLP) has been shown to markedly decrease G−1 addition activity. RESULTS +110 113 G−1 residue_name_number Mutation of D29 and D76 in HsThg1 (corresponding to D21 and D69 of MaTLP) has been shown to markedly decrease G−1 addition activity. RESULTS +34 37 GTP chemical Template-dependent binding of the GTP analog to the MaTLP-ppptRNAPheΔ1 complex RESULTS +52 70 MaTLP-ppptRNAPheΔ1 complex_assembly Template-dependent binding of the GTP analog to the MaTLP-ppptRNAPheΔ1 complex RESULTS +35 44 structure evidence Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. RESULTS +71 76 MaTLP protein Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. RESULTS +91 95 tRNA chemical Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. RESULTS +97 109 ppptRNAPheΔ1 chemical Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. RESULTS +120 123 GTP chemical Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. RESULTS +132 137 GDPNP chemical Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. RESULTS +163 170 soaking experimental_method Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. RESULTS +175 193 MaTLP-ppptRNAPheΔ1 complex_assembly Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. RESULTS +202 209 crystal evidence Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. RESULTS +235 240 GDPNP chemical Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. RESULTS +13 22 structure evidence The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme. RESULTS +39 46 guanine chemical The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme. RESULTS +68 73 GDPNP chemical The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme. RESULTS +81 108 Watson-Crick hydrogen bonds bond_interaction The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme. RESULTS +114 117 C72 residue_name_number The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme. RESULTS +134 160 base-stacking interactions bond_interaction The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme. RESULTS +166 168 G2 residue_name_number The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme. RESULTS +176 180 tRNA chemical The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme. RESULTS +240 247 guanine chemical The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme. RESULTS +31 35 tRNA chemical The 5′-end (position 2) of the tRNA moved significantly (Fig. 3C) due to the insertion of GDPNP. RESULTS +90 95 GDPNP chemical The 5′-end (position 2) of the tRNA moved significantly (Fig. 3C) due to the insertion of GDPNP. RESULTS +18 33 5′-triphosphate chemical Surprisingly, the 5′-triphosphate moiety after movement occupied the GTP/ATP triphosphate position during the activation step (Fig. 3D). RESULTS +69 72 GTP chemical Surprisingly, the 5′-triphosphate moiety after movement occupied the GTP/ATP triphosphate position during the activation step (Fig. 3D). RESULTS +73 76 ATP chemical Surprisingly, the 5′-triphosphate moiety after movement occupied the GTP/ATP triphosphate position during the activation step (Fig. 3D). RESULTS +77 89 triphosphate chemical Surprisingly, the 5′-triphosphate moiety after movement occupied the GTP/ATP triphosphate position during the activation step (Fig. 3D). RESULTS +61 64 GTP chemical Together with the observation that the 3′-OH of the incoming GTP analog was within coordination distance (2.8 Å) to Mg2+A (fig. S3B) and was able to execute a nucleophilic attack on the α-phosphate of the 5′-end, this structure indicates that the elongation reaction (second reaction) takes place at the same reaction center where the activation reaction (first reaction) occurs. RESULTS +116 120 Mg2+ chemical Together with the observation that the 3′-OH of the incoming GTP analog was within coordination distance (2.8 Å) to Mg2+A (fig. S3B) and was able to execute a nucleophilic attack on the α-phosphate of the 5′-end, this structure indicates that the elongation reaction (second reaction) takes place at the same reaction center where the activation reaction (first reaction) occurs. RESULTS +188 197 phosphate chemical Together with the observation that the 3′-OH of the incoming GTP analog was within coordination distance (2.8 Å) to Mg2+A (fig. S3B) and was able to execute a nucleophilic attack on the α-phosphate of the 5′-end, this structure indicates that the elongation reaction (second reaction) takes place at the same reaction center where the activation reaction (first reaction) occurs. RESULTS +218 227 structure evidence Together with the observation that the 3′-OH of the incoming GTP analog was within coordination distance (2.8 Å) to Mg2+A (fig. S3B) and was able to execute a nucleophilic attack on the α-phosphate of the 5′-end, this structure indicates that the elongation reaction (second reaction) takes place at the same reaction center where the activation reaction (first reaction) occurs. RESULTS +309 324 reaction center site Together with the observation that the 3′-OH of the incoming GTP analog was within coordination distance (2.8 Å) to Mg2+A (fig. S3B) and was able to execute a nucleophilic attack on the α-phosphate of the 5′-end, this structure indicates that the elongation reaction (second reaction) takes place at the same reaction center where the activation reaction (first reaction) occurs. RESULTS +25 29 tRNA chemical Structural change of the tRNA (ppptRNAPheΔ1). FIG +31 43 ppptRNAPheΔ1 chemical Structural change of the tRNA (ppptRNAPheΔ1). FIG +25 29 tRNA chemical Structural change of the tRNA (ppptRNAPheΔ1) accepter stem in MaTLP caused by insertion of GDPNP. (A) Structure before GDPNP binding. FIG +31 43 ppptRNAPheΔ1 chemical Structural change of the tRNA (ppptRNAPheΔ1) accepter stem in MaTLP caused by insertion of GDPNP. (A) Structure before GDPNP binding. FIG +45 58 accepter stem structure_element Structural change of the tRNA (ppptRNAPheΔ1) accepter stem in MaTLP caused by insertion of GDPNP. (A) Structure before GDPNP binding. FIG +62 67 MaTLP protein Structural change of the tRNA (ppptRNAPheΔ1) accepter stem in MaTLP caused by insertion of GDPNP. (A) Structure before GDPNP binding. FIG +91 96 GDPNP chemical Structural change of the tRNA (ppptRNAPheΔ1) accepter stem in MaTLP caused by insertion of GDPNP. (A) Structure before GDPNP binding. FIG +102 111 Structure evidence Structural change of the tRNA (ppptRNAPheΔ1) accepter stem in MaTLP caused by insertion of GDPNP. (A) Structure before GDPNP binding. FIG +119 124 GDPNP chemical Structural change of the tRNA (ppptRNAPheΔ1) accepter stem in MaTLP caused by insertion of GDPNP. (A) Structure before GDPNP binding. FIG +4 13 Structure evidence (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. FIG +20 25 GDPNP chemical (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. FIG +39 52 Superposition experimental_method (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. FIG +64 74 structures evidence (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. FIG +113 117 tRNA chemical (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. FIG +161 166 GDPNP chemical (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. FIG +172 185 Superposition experimental_method (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. FIG +207 211 tRNA chemical (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. FIG +218 223 GDPNP chemical (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. FIG +245 248 GTP chemical (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. FIG +299 311 triphosphate chemical (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. FIG +4 16 triphosphate chemical The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +27 32 GDPNP chemical The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +44 53 interface site The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +72 73 A structure_element The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +78 79 B structure_element The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +147 150 R19 residue_name_number The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +161 162 A structure_element The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +165 168 R83 residue_name_number The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +179 180 B structure_element The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +183 186 K86 residue_name_number The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +197 198 B structure_element The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +205 209 R114 residue_name_number The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +220 221 A structure_element The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). RESULTS +26 40 well conserved protein_state All of these residues are well conserved (fig. S5), and mutation of corresponding residues in ScThg1 (R27, R93, K96, and R133) decreased the catalytic efficiency of G−1 addition. RESULTS +56 64 mutation experimental_method All of these residues are well conserved (fig. S5), and mutation of corresponding residues in ScThg1 (R27, R93, K96, and R133) decreased the catalytic efficiency of G−1 addition. RESULTS +94 100 ScThg1 protein All of these residues are well conserved (fig. S5), and mutation of corresponding residues in ScThg1 (R27, R93, K96, and R133) decreased the catalytic efficiency of G−1 addition. RESULTS +102 105 R27 residue_name_number All of these residues are well conserved (fig. S5), and mutation of corresponding residues in ScThg1 (R27, R93, K96, and R133) decreased the catalytic efficiency of G−1 addition. RESULTS +107 110 R93 residue_name_number All of these residues are well conserved (fig. S5), and mutation of corresponding residues in ScThg1 (R27, R93, K96, and R133) decreased the catalytic efficiency of G−1 addition. RESULTS +112 115 K96 residue_name_number All of these residues are well conserved (fig. S5), and mutation of corresponding residues in ScThg1 (R27, R93, K96, and R133) decreased the catalytic efficiency of G−1 addition. RESULTS +121 125 R133 residue_name_number All of these residues are well conserved (fig. S5), and mutation of corresponding residues in ScThg1 (R27, R93, K96, and R133) decreased the catalytic efficiency of G−1 addition. RESULTS +165 168 G−1 residue_name_number All of these residues are well conserved (fig. S5), and mutation of corresponding residues in ScThg1 (R27, R93, K96, and R133) decreased the catalytic efficiency of G−1 addition. RESULTS +4 16 triphosphate chemical The triphosphate of the GDPNP was also bonded to the third Mg2+ (Mg2+C), which, unlike Mg2+A and Mg2+B, is not coordinated by the TLP molecule (fig. S3B). RESULTS +24 29 GDPNP chemical The triphosphate of the GDPNP was also bonded to the third Mg2+ (Mg2+C), which, unlike Mg2+A and Mg2+B, is not coordinated by the TLP molecule (fig. S3B). RESULTS +59 63 Mg2+ chemical The triphosphate of the GDPNP was also bonded to the third Mg2+ (Mg2+C), which, unlike Mg2+A and Mg2+B, is not coordinated by the TLP molecule (fig. S3B). RESULTS +65 69 Mg2+ chemical The triphosphate of the GDPNP was also bonded to the third Mg2+ (Mg2+C), which, unlike Mg2+A and Mg2+B, is not coordinated by the TLP molecule (fig. S3B). RESULTS +87 91 Mg2+ chemical The triphosphate of the GDPNP was also bonded to the third Mg2+ (Mg2+C), which, unlike Mg2+A and Mg2+B, is not coordinated by the TLP molecule (fig. S3B). RESULTS +97 101 Mg2+ chemical The triphosphate of the GDPNP was also bonded to the third Mg2+ (Mg2+C), which, unlike Mg2+A and Mg2+B, is not coordinated by the TLP molecule (fig. S3B). RESULTS +111 125 coordinated by bond_interaction The triphosphate of the GDPNP was also bonded to the third Mg2+ (Mg2+C), which, unlike Mg2+A and Mg2+B, is not coordinated by the TLP molecule (fig. S3B). RESULTS +130 133 TLP protein_type The triphosphate of the GDPNP was also bonded to the third Mg2+ (Mg2+C), which, unlike Mg2+A and Mg2+B, is not coordinated by the TLP molecule (fig. S3B). RESULTS +5 17 triphosphate chemical This triphosphate binding mode is the same as that for the second nucleotide binding site in Thg1. RESULTS +59 89 second nucleotide binding site site This triphosphate binding mode is the same as that for the second nucleotide binding site in Thg1. RESULTS +93 97 Thg1 protein This triphosphate binding mode is the same as that for the second nucleotide binding site in Thg1. RESULTS +54 65 second site site However, in previous analyses, the base moiety at the second site was either invisible or far beyond the reaction distance of the phosphate, and therefore, flipping of the base was expected to occur. RESULTS +130 139 phosphate chemical However, in previous analyses, the base moiety at the second site was either invisible or far beyond the reaction distance of the phosphate, and therefore, flipping of the base was expected to occur. RESULTS +0 35 tRNA binding and repair experiments experimental_method tRNA binding and repair experiments of the β-hairpin mutants RESULTS +43 52 β-hairpin structure_element tRNA binding and repair experiments of the β-hairpin mutants RESULTS +53 60 mutants protein_state tRNA binding and repair experiments of the β-hairpin mutants RESULTS +11 15 tRNA chemical To confirm tRNA recognition by the β-hairpin, we created mutation variants with altered residues in the β-hairpin region. RESULTS +35 44 β-hairpin structure_element To confirm tRNA recognition by the β-hairpin, we created mutation variants with altered residues in the β-hairpin region. RESULTS +49 74 created mutation variants experimental_method To confirm tRNA recognition by the β-hairpin, we created mutation variants with altered residues in the β-hairpin region. RESULTS +104 113 β-hairpin structure_element To confirm tRNA recognition by the β-hairpin, we created mutation variants with altered residues in the β-hairpin region. RESULTS +6 57 tRNA binding and enzymatic activities were measured experimental_method Then, tRNA binding and enzymatic activities were measured. RESULTS +0 9 β-Hairpin structure_element β-Hairpin deletion variant delR198-R215 almost completely abolished the binding of tRNAPheΔ1 (fig. S6). RESULTS +10 26 deletion variant protein_state β-Hairpin deletion variant delR198-R215 almost completely abolished the binding of tRNAPheΔ1 (fig. S6). RESULTS +27 39 delR198-R215 mutant β-Hairpin deletion variant delR198-R215 almost completely abolished the binding of tRNAPheΔ1 (fig. S6). RESULTS +83 92 tRNAPheΔ1 chemical β-Hairpin deletion variant delR198-R215 almost completely abolished the binding of tRNAPheΔ1 (fig. S6). RESULTS +41 53 delR198-R215 mutant Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). RESULTS +58 70 delG202-E210 mutant Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). RESULTS +130 139 wild type protein_state Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). RESULTS +149 158 mutations experimental_method Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). RESULTS +160 165 N179A mutant Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). RESULTS +170 175 F174A mutant Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). RESULTS +176 181 N179A mutant Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). RESULTS +182 187 R188A mutant Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). RESULTS +196 222 anticodon recognition site site Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). RESULTS +241 253 Thg1-tRNAHis complex_assembly Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). RESULTS +262 271 structure evidence Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). RESULTS +22 31 tRNAHisΔ1 chemical Experiments using the tRNAHisΔ1 substrate gave similar results (Fig. 4A). RESULTS +42 59 crystal structure evidence All these results are consistent with the crystal structure and suggest that the β-hairpin plays an important role in anticodon-independent binding of the tRNA substrate. RESULTS +81 90 β-hairpin structure_element All these results are consistent with the crystal structure and suggest that the β-hairpin plays an important role in anticodon-independent binding of the tRNA substrate. RESULTS +155 159 tRNA chemical All these results are consistent with the crystal structure and suggest that the β-hairpin plays an important role in anticodon-independent binding of the tRNA substrate. RESULTS +16 25 β-hairpin structure_element Residues in the β-hairpin are not well conserved, except for R215 (fig. S5). RESULTS +30 48 not well conserved protein_state Residues in the β-hairpin are not well conserved, except for R215 (fig. S5). RESULTS +61 65 R215 residue_name_number Residues in the β-hairpin are not well conserved, except for R215 (fig. S5). RESULTS +0 7 Mutants protein_state Mutants R215A and R215A/S213A, in which the completely conserved R215 was changed to alanine, showed a moderate effect on the activity (27.3 and 16.3%, respectively). RESULTS +8 13 R215A mutant Mutants R215A and R215A/S213A, in which the completely conserved R215 was changed to alanine, showed a moderate effect on the activity (27.3 and 16.3%, respectively). RESULTS +18 23 R215A mutant Mutants R215A and R215A/S213A, in which the completely conserved R215 was changed to alanine, showed a moderate effect on the activity (27.3 and 16.3%, respectively). RESULTS +24 29 S213A mutant Mutants R215A and R215A/S213A, in which the completely conserved R215 was changed to alanine, showed a moderate effect on the activity (27.3 and 16.3%, respectively). RESULTS +44 64 completely conserved protein_state Mutants R215A and R215A/S213A, in which the completely conserved R215 was changed to alanine, showed a moderate effect on the activity (27.3 and 16.3%, respectively). RESULTS +65 69 R215 residue_name_number Mutants R215A and R215A/S213A, in which the completely conserved R215 was changed to alanine, showed a moderate effect on the activity (27.3 and 16.3%, respectively). RESULTS +74 81 changed experimental_method Mutants R215A and R215A/S213A, in which the completely conserved R215 was changed to alanine, showed a moderate effect on the activity (27.3 and 16.3%, respectively). RESULTS +85 92 alanine residue_name Mutants R215A and R215A/S213A, in which the completely conserved R215 was changed to alanine, showed a moderate effect on the activity (27.3 and 16.3%, respectively). RESULTS +37 46 conserved protein_state Thus, specific interactions with the conserved R215 and van der Waals contacts to residues in the β-hairpin would be important for tRNA recognition. RESULTS +47 51 R215 residue_name_number Thus, specific interactions with the conserved R215 and van der Waals contacts to residues in the β-hairpin would be important for tRNA recognition. RESULTS +56 78 van der Waals contacts bond_interaction Thus, specific interactions with the conserved R215 and van der Waals contacts to residues in the β-hairpin would be important for tRNA recognition. RESULTS +98 107 β-hairpin structure_element Thus, specific interactions with the conserved R215 and van der Waals contacts to residues in the β-hairpin would be important for tRNA recognition. RESULTS +131 135 tRNA chemical Thus, specific interactions with the conserved R215 and van der Waals contacts to residues in the β-hairpin would be important for tRNA recognition. RESULTS +0 19 Mutational analysis experimental_method Mutational analysis of the β-hairpin and anticodon binding region. FIG +27 36 β-hairpin structure_element Mutational analysis of the β-hairpin and anticodon binding region. FIG +41 65 anticodon binding region site Mutational analysis of the β-hairpin and anticodon binding region. FIG +21 33 ppptRNAPheΔ1 chemical (A) Guanylylation of ppptRNAPheΔ1 and ppptRNAHisΔ1 by various TLP mutants. FIG +38 50 ppptRNAHisΔ1 chemical (A) Guanylylation of ppptRNAPheΔ1 and ppptRNAHisΔ1 by various TLP mutants. FIG +62 65 TLP protein_type (A) Guanylylation of ppptRNAPheΔ1 and ppptRNAHisΔ1 by various TLP mutants. FIG +66 73 mutants protein_state (A) Guanylylation of ppptRNAPheΔ1 and ppptRNAHisΔ1 by various TLP mutants. FIG +19 29 [α-32P]GTP chemical The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. FIG +31 40 wild-type protein_state The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. FIG +41 46 MaTLP protein The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. FIG +52 64 ppptRNAPheΔ1 chemical The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. FIG +105 114 tRNAPheΔ1 chemical The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. FIG +116 123 tRNAPhe chemical The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. FIG +129 139 tRNAHisΔ−1 chemical The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. FIG +151 154 TLP protein_type The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. FIG +155 162 mutants protein_state The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. FIG +16 25 tRNAPheΔ1 chemical The activity to tRNAPheΔ1 is about 10% of ppptRNAPheΔ1. FIG +42 54 ppptRNAPheΔ1 chemical The activity to tRNAPheΔ1 is about 10% of ppptRNAPheΔ1. FIG +56 69 accepter stem structure_element Termination of the elongation reaction by measuring the accepter stem RESULTS +0 4 TLPs protein_type TLPs catalyze the Watson-Crick template–dependent elongation or repair reaction for 5′-end truncated tRNAPhe substrates lacking G1 only (tRNAPheΔ1), or lacking both G1 and G2 (tRNAPheΔ1,2), whereas they do not show any activity with intact tRNAPhe (thus, repair is unnecessary). RESULTS +101 108 tRNAPhe chemical TLPs catalyze the Watson-Crick template–dependent elongation or repair reaction for 5′-end truncated tRNAPhe substrates lacking G1 only (tRNAPheΔ1), or lacking both G1 and G2 (tRNAPheΔ1,2), whereas they do not show any activity with intact tRNAPhe (thus, repair is unnecessary). RESULTS +128 130 G1 residue_name_number TLPs catalyze the Watson-Crick template–dependent elongation or repair reaction for 5′-end truncated tRNAPhe substrates lacking G1 only (tRNAPheΔ1), or lacking both G1 and G2 (tRNAPheΔ1,2), whereas they do not show any activity with intact tRNAPhe (thus, repair is unnecessary). RESULTS +137 146 tRNAPheΔ1 chemical TLPs catalyze the Watson-Crick template–dependent elongation or repair reaction for 5′-end truncated tRNAPhe substrates lacking G1 only (tRNAPheΔ1), or lacking both G1 and G2 (tRNAPheΔ1,2), whereas they do not show any activity with intact tRNAPhe (thus, repair is unnecessary). RESULTS +165 167 G1 residue_name_number TLPs catalyze the Watson-Crick template–dependent elongation or repair reaction for 5′-end truncated tRNAPhe substrates lacking G1 only (tRNAPheΔ1), or lacking both G1 and G2 (tRNAPheΔ1,2), whereas they do not show any activity with intact tRNAPhe (thus, repair is unnecessary). RESULTS +172 174 G2 residue_name_number TLPs catalyze the Watson-Crick template–dependent elongation or repair reaction for 5′-end truncated tRNAPhe substrates lacking G1 only (tRNAPheΔ1), or lacking both G1 and G2 (tRNAPheΔ1,2), whereas they do not show any activity with intact tRNAPhe (thus, repair is unnecessary). RESULTS +176 187 tRNAPheΔ1,2 chemical TLPs catalyze the Watson-Crick template–dependent elongation or repair reaction for 5′-end truncated tRNAPhe substrates lacking G1 only (tRNAPheΔ1), or lacking both G1 and G2 (tRNAPheΔ1,2), whereas they do not show any activity with intact tRNAPhe (thus, repair is unnecessary). RESULTS +240 247 tRNAPhe chemical TLPs catalyze the Watson-Crick template–dependent elongation or repair reaction for 5′-end truncated tRNAPhe substrates lacking G1 only (tRNAPheΔ1), or lacking both G1 and G2 (tRNAPheΔ1,2), whereas they do not show any activity with intact tRNAPhe (thus, repair is unnecessary). RESULTS +4 7 TLP protein_type How TLP distinguishes between tRNAs that need 5′-end repair from ones that do not, or in other words, how the elongation reaction is properly terminated, remains unknown. RESULTS +30 35 tRNAs chemical How TLP distinguishes between tRNAs that need 5′-end repair from ones that do not, or in other words, how the elongation reaction is properly terminated, remains unknown. RESULTS +12 21 structure evidence The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +29 47 MaTLP-ppptRNAPheΔ1 complex_assembly The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +75 79 Thg1 protein The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +85 88 TLP protein_type The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +89 94 dimer oligomeric_state The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +117 121 tRNA chemical The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +141 153 elbow region structure_element The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +161 170 β-hairpin structure_element The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +183 184 B structure_element The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +212 213 A structure_element The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +249 257 flexible protein_state The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +272 281 β-hairpin structure_element The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +309 313 tRNA chemical The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +340 353 accepter stem structure_element The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. RESULTS +32 65 used computer graphics to examine experimental_method To confirm this speculation, we used computer graphics to examine whether the β-hairpin region was able to bind tRNA substrates with different accepter stem lengths when the 5′-end was properly placed in the reaction site. RESULTS +78 87 β-hairpin structure_element To confirm this speculation, we used computer graphics to examine whether the β-hairpin region was able to bind tRNA substrates with different accepter stem lengths when the 5′-end was properly placed in the reaction site. RESULTS +112 116 tRNA chemical To confirm this speculation, we used computer graphics to examine whether the β-hairpin region was able to bind tRNA substrates with different accepter stem lengths when the 5′-end was properly placed in the reaction site. RESULTS +143 156 accepter stem structure_element To confirm this speculation, we used computer graphics to examine whether the β-hairpin region was able to bind tRNA substrates with different accepter stem lengths when the 5′-end was properly placed in the reaction site. RESULTS +208 221 reaction site site To confirm this speculation, we used computer graphics to examine whether the β-hairpin region was able to bind tRNA substrates with different accepter stem lengths when the 5′-end was properly placed in the reaction site. RESULTS +65 69 tRNA chemical When the 5′-end was placed in the reaction site, the body of the tRNA molecule shifted in a manner dependent on the accepter stem length. RESULTS +116 129 accepter stem structure_element When the 5′-end was placed in the reaction site, the body of the tRNA molecule shifted in a manner dependent on the accepter stem length. RESULTS +4 8 tRNA chemical The tRNA body also rotated because of the helical nature of the accepter stem (fig. S7). RESULTS +64 77 accepter stem structure_element The tRNA body also rotated because of the helical nature of the accepter stem (fig. S7). RESULTS +11 20 structure evidence This model structure showed that the accepter stem of intact tRNAPhe was too long for the β-hairpin to recognize its elbow region, whereas tRNAPheΔ1 and tRNAPheΔ1,2 were recognized by the β-hairpin region (fig. S7), which is consistent with previous experiments. RESULTS +37 50 accepter stem structure_element This model structure showed that the accepter stem of intact tRNAPhe was too long for the β-hairpin to recognize its elbow region, whereas tRNAPheΔ1 and tRNAPheΔ1,2 were recognized by the β-hairpin region (fig. S7), which is consistent with previous experiments. RESULTS +61 68 tRNAPhe chemical This model structure showed that the accepter stem of intact tRNAPhe was too long for the β-hairpin to recognize its elbow region, whereas tRNAPheΔ1 and tRNAPheΔ1,2 were recognized by the β-hairpin region (fig. S7), which is consistent with previous experiments. RESULTS +90 99 β-hairpin structure_element This model structure showed that the accepter stem of intact tRNAPhe was too long for the β-hairpin to recognize its elbow region, whereas tRNAPheΔ1 and tRNAPheΔ1,2 were recognized by the β-hairpin region (fig. S7), which is consistent with previous experiments. RESULTS +117 129 elbow region structure_element This model structure showed that the accepter stem of intact tRNAPhe was too long for the β-hairpin to recognize its elbow region, whereas tRNAPheΔ1 and tRNAPheΔ1,2 were recognized by the β-hairpin region (fig. S7), which is consistent with previous experiments. RESULTS +139 148 tRNAPheΔ1 chemical This model structure showed that the accepter stem of intact tRNAPhe was too long for the β-hairpin to recognize its elbow region, whereas tRNAPheΔ1 and tRNAPheΔ1,2 were recognized by the β-hairpin region (fig. S7), which is consistent with previous experiments. RESULTS +153 164 tRNAPheΔ1,2 chemical This model structure showed that the accepter stem of intact tRNAPhe was too long for the β-hairpin to recognize its elbow region, whereas tRNAPheΔ1 and tRNAPheΔ1,2 were recognized by the β-hairpin region (fig. S7), which is consistent with previous experiments. RESULTS +188 197 β-hairpin structure_element This model structure showed that the accepter stem of intact tRNAPhe was too long for the β-hairpin to recognize its elbow region, whereas tRNAPheΔ1 and tRNAPheΔ1,2 were recognized by the β-hairpin region (fig. S7), which is consistent with previous experiments. RESULTS +28 38 structures evidence On the basis of these model structures, we concluded that the TLP molecule can properly terminate elongation by measuring the accepter stem length of tRNA substrates. RESULTS +62 65 TLP protein_type On the basis of these model structures, we concluded that the TLP molecule can properly terminate elongation by measuring the accepter stem length of tRNA substrates. RESULTS +126 139 accepter stem structure_element On the basis of these model structures, we concluded that the TLP molecule can properly terminate elongation by measuring the accepter stem length of tRNA substrates. RESULTS +150 154 tRNA chemical On the basis of these model structures, we concluded that the TLP molecule can properly terminate elongation by measuring the accepter stem length of tRNA substrates. RESULTS +22 26 tRNA chemical Dual binding mode for tRNA repair RESULTS +12 31 structural analysis experimental_method The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +55 58 TLP protein_type The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +63 67 Thg1 protein The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +83 93 tetrameric oligomeric_state The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +146 151 tRNAs chemical The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +153 157 Thg1 protein The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +161 169 bound to protein_state The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +170 177 tRNAHis chemical The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +183 191 tetramer oligomeric_state The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +201 204 TLP protein_type The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +208 216 bound to protein_state The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +217 224 tRNAPhe chemical The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +230 235 dimer oligomeric_state The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. RESULTS +23 27 tRNA chemical This difference in the tRNA binding modes is closely related to their enzymatic functions. RESULTS +4 40 tRNAHis-specific G−1 addition enzyme protein_type The tRNAHis-specific G−1 addition enzyme Thg1 needs to recognize both the accepter stem and anticodon of tRNAHis. RESULTS +41 45 Thg1 protein The tRNAHis-specific G−1 addition enzyme Thg1 needs to recognize both the accepter stem and anticodon of tRNAHis. RESULTS +74 87 accepter stem structure_element The tRNAHis-specific G−1 addition enzyme Thg1 needs to recognize both the accepter stem and anticodon of tRNAHis. RESULTS +92 101 anticodon structure_element The tRNAHis-specific G−1 addition enzyme Thg1 needs to recognize both the accepter stem and anticodon of tRNAHis. RESULTS +105 112 tRNAHis chemical The tRNAHis-specific G−1 addition enzyme Thg1 needs to recognize both the accepter stem and anticodon of tRNAHis. RESULTS +4 14 tetrameric oligomeric_state The tetrameric architecture of the Thg1 molecule allows it to access both regions located at the opposite side of the tRNA molecule [the AB dimer recognizes the accepter stem and CD dimer anticodon ]. RESULTS +35 39 Thg1 protein The tetrameric architecture of the Thg1 molecule allows it to access both regions located at the opposite side of the tRNA molecule [the AB dimer recognizes the accepter stem and CD dimer anticodon ]. RESULTS +118 122 tRNA chemical The tetrameric architecture of the Thg1 molecule allows it to access both regions located at the opposite side of the tRNA molecule [the AB dimer recognizes the accepter stem and CD dimer anticodon ]. RESULTS +137 139 AB structure_element The tetrameric architecture of the Thg1 molecule allows it to access both regions located at the opposite side of the tRNA molecule [the AB dimer recognizes the accepter stem and CD dimer anticodon ]. RESULTS +140 145 dimer oligomeric_state The tetrameric architecture of the Thg1 molecule allows it to access both regions located at the opposite side of the tRNA molecule [the AB dimer recognizes the accepter stem and CD dimer anticodon ]. RESULTS +161 174 accepter stem structure_element The tetrameric architecture of the Thg1 molecule allows it to access both regions located at the opposite side of the tRNA molecule [the AB dimer recognizes the accepter stem and CD dimer anticodon ]. RESULTS +179 181 CD structure_element The tetrameric architecture of the Thg1 molecule allows it to access both regions located at the opposite side of the tRNA molecule [the AB dimer recognizes the accepter stem and CD dimer anticodon ]. RESULTS +182 187 dimer oligomeric_state The tetrameric architecture of the Thg1 molecule allows it to access both regions located at the opposite side of the tRNA molecule [the AB dimer recognizes the accepter stem and CD dimer anticodon ]. RESULTS +33 36 TLP protein_type In contrast, the binding mode of TLP corresponds to the anticodon-independent repair reactions of 5′-truncated general tRNAs. RESULTS +119 124 tRNAs chemical In contrast, the binding mode of TLP corresponds to the anticodon-independent repair reactions of 5′-truncated general tRNAs. RESULTS +131 144 accepter stem structure_element This binding mode is also suitable for the correct termination of the elongation or repair reaction by measuring the length of the accepter stem by the flexible β-hairpin. RESULTS +152 160 flexible protein_state This binding mode is also suitable for the correct termination of the elongation or repair reaction by measuring the length of the accepter stem by the flexible β-hairpin. RESULTS +161 170 β-hairpin structure_element This binding mode is also suitable for the correct termination of the elongation or repair reaction by measuring the length of the accepter stem by the flexible β-hairpin. RESULTS +8 15 tRNAHis chemical Because tRNAHis requires an extra guanosine (G−1) at the 5′-end, the repair enzyme has to extend the 5′-end by one more nucleotide than other tRNAs. RESULTS +34 43 guanosine chemical Because tRNAHis requires an extra guanosine (G−1) at the 5′-end, the repair enzyme has to extend the 5′-end by one more nucleotide than other tRNAs. RESULTS +45 48 G−1 residue_name_number Because tRNAHis requires an extra guanosine (G−1) at the 5′-end, the repair enzyme has to extend the 5′-end by one more nucleotide than other tRNAs. RESULTS +142 147 tRNAs chemical Because tRNAHis requires an extra guanosine (G−1) at the 5′-end, the repair enzyme has to extend the 5′-end by one more nucleotide than other tRNAs. RESULTS +0 3 TLP protein_type TLP has been shown to confer such catalytic activity on tRNAHisΔ−1 (Fig. 4B). RESULTS +56 66 tRNAHisΔ−1 chemical TLP has been shown to confer such catalytic activity on tRNAHisΔ−1 (Fig. 4B). RESULTS +25 28 TLP protein_type Here, we showed that the TLP mutants, wherein the β-hairpin is truncated and tRNAPheΔ1 binding ability is lost, can still bind to tRNAPhe (GUG) whose anticodon is changed to that for His (fig. S6, C, H, and I). RESULTS +29 36 mutants protein_state Here, we showed that the TLP mutants, wherein the β-hairpin is truncated and tRNAPheΔ1 binding ability is lost, can still bind to tRNAPhe (GUG) whose anticodon is changed to that for His (fig. S6, C, H, and I). RESULTS +50 59 β-hairpin structure_element Here, we showed that the TLP mutants, wherein the β-hairpin is truncated and tRNAPheΔ1 binding ability is lost, can still bind to tRNAPhe (GUG) whose anticodon is changed to that for His (fig. S6, C, H, and I). RESULTS +63 72 truncated protein_state Here, we showed that the TLP mutants, wherein the β-hairpin is truncated and tRNAPheΔ1 binding ability is lost, can still bind to tRNAPhe (GUG) whose anticodon is changed to that for His (fig. S6, C, H, and I). RESULTS +77 86 tRNAPheΔ1 chemical Here, we showed that the TLP mutants, wherein the β-hairpin is truncated and tRNAPheΔ1 binding ability is lost, can still bind to tRNAPhe (GUG) whose anticodon is changed to that for His (fig. S6, C, H, and I). RESULTS +130 137 tRNAPhe chemical Here, we showed that the TLP mutants, wherein the β-hairpin is truncated and tRNAPheΔ1 binding ability is lost, can still bind to tRNAPhe (GUG) whose anticodon is changed to that for His (fig. S6, C, H, and I). RESULTS +139 142 GUG chemical Here, we showed that the TLP mutants, wherein the β-hairpin is truncated and tRNAPheΔ1 binding ability is lost, can still bind to tRNAPhe (GUG) whose anticodon is changed to that for His (fig. S6, C, H, and I). RESULTS +183 186 His residue_name Here, we showed that the TLP mutants, wherein the β-hairpin is truncated and tRNAPheΔ1 binding ability is lost, can still bind to tRNAPhe (GUG) whose anticodon is changed to that for His (fig. S6, C, H, and I). RESULTS +17 24 tRNAPhe chemical Also, the intact tRNAPhe, which is not recognized by TLP (Fig. 4B and fig. S6E), can be recognized when its anticodon is changed to that for His (fig. S6D). RESULTS +53 56 TLP protein_type Also, the intact tRNAPhe, which is not recognized by TLP (Fig. 4B and fig. S6E), can be recognized when its anticodon is changed to that for His (fig. S6D). RESULTS +141 144 His residue_name Also, the intact tRNAPhe, which is not recognized by TLP (Fig. 4B and fig. S6E), can be recognized when its anticodon is changed to that for His (fig. S6D). RESULTS +17 20 TLP protein_type Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHisΔ−1 (Fig. 4B). RESULTS +21 28 variant protein_state Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHisΔ−1 (Fig. 4B). RESULTS +30 35 F174A mutant Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHisΔ−1 (Fig. 4B). RESULTS +36 41 N179A mutant Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHisΔ−1 (Fig. 4B). RESULTS +42 47 R188A mutant Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHisΔ−1 (Fig. 4B). RESULTS +55 81 anticodon recognition site site Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHisΔ−1 (Fig. 4B). RESULTS +100 112 Thg1-tRNAHis complex_assembly Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHisΔ−1 (Fig. 4B). RESULTS +121 130 structure evidence Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHisΔ−1 (Fig. 4B). RESULTS +201 211 tRNAHisΔ−1 chemical Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHisΔ−1 (Fig. 4B). RESULTS +45 48 TLP protein_type All these experimental results indicate that TLP recognizes and binds tRNAs carrying the His anticodon in the same way that Thg1 recognizes tRNAHis. RESULTS +70 75 tRNAs chemical All these experimental results indicate that TLP recognizes and binds tRNAs carrying the His anticodon in the same way that Thg1 recognizes tRNAHis. RESULTS +89 92 His residue_name All these experimental results indicate that TLP recognizes and binds tRNAs carrying the His anticodon in the same way that Thg1 recognizes tRNAHis. RESULTS +124 128 Thg1 protein All these experimental results indicate that TLP recognizes and binds tRNAs carrying the His anticodon in the same way that Thg1 recognizes tRNAHis. RESULTS +140 147 tRNAHis chemical All these experimental results indicate that TLP recognizes and binds tRNAs carrying the His anticodon in the same way that Thg1 recognizes tRNAHis. RESULTS +24 27 TLP protein_type Thus, we concluded that TLP has two tRNA binding modes that are selectively used, depending on both the length of the accepter stem and the anticodon. RESULTS +36 40 tRNA chemical Thus, we concluded that TLP has two tRNA binding modes that are selectively used, depending on both the length of the accepter stem and the anticodon. RESULTS +118 131 accepter stem structure_element Thus, we concluded that TLP has two tRNA binding modes that are selectively used, depending on both the length of the accepter stem and the anticodon. RESULTS +140 149 anticodon structure_element Thus, we concluded that TLP has two tRNA binding modes that are selectively used, depending on both the length of the accepter stem and the anticodon. RESULTS +103 106 His residue_name The elongation or repair reaction normally terminates when the 5′-end reaches position 1, but when the His anticodon is present, TLP binds the tRNA in the second mode by recognizing the anticodon to execute the G−1 addition reaction. RESULTS +129 132 TLP protein_type The elongation or repair reaction normally terminates when the 5′-end reaches position 1, but when the His anticodon is present, TLP binds the tRNA in the second mode by recognizing the anticodon to execute the G−1 addition reaction. RESULTS +143 147 tRNA chemical The elongation or repair reaction normally terminates when the 5′-end reaches position 1, but when the His anticodon is present, TLP binds the tRNA in the second mode by recognizing the anticodon to execute the G−1 addition reaction. RESULTS +211 214 G−1 residue_name_number The elongation or repair reaction normally terminates when the 5′-end reaches position 1, but when the His anticodon is present, TLP binds the tRNA in the second mode by recognizing the anticodon to execute the G−1 addition reaction. RESULTS +39 42 TLP protein_type By having two different binding modes, TLP can manage this special feature of tRNAHis. RESULTS +78 85 tRNAHis chemical By having two different binding modes, TLP can manage this special feature of tRNAHis. RESULTS +4 8 Thg1 protein The Thg1/TLP family of proteins extends tRNA chains in the 3′-5′ direction. DISCUSS +9 12 TLP protein_type The Thg1/TLP family of proteins extends tRNA chains in the 3′-5′ direction. DISCUSS +40 44 tRNA chemical The Thg1/TLP family of proteins extends tRNA chains in the 3′-5′ direction. DISCUSS +11 23 5′-phosphate chemical First, the 5′-phosphate is activated by GTP/ATP. DISCUSS +40 43 GTP chemical First, the 5′-phosphate is activated by GTP/ATP. DISCUSS +44 47 ATP chemical First, the 5′-phosphate is activated by GTP/ATP. DISCUSS +20 29 phosphate chemical Then, the activated phosphate is attacked by the incoming nucleotide, resulting in an extension by one nucleotide at the 5′-end. DISCUSS +22 28 solved experimental_method Here, we successfully solved for the first time the intermediate structures of the template-dependent 3′-5′ elongation complex of MaTLP. DISCUSS +65 75 structures evidence Here, we successfully solved for the first time the intermediate structures of the template-dependent 3′-5′ elongation complex of MaTLP. DISCUSS +130 135 MaTLP protein Here, we successfully solved for the first time the intermediate structures of the template-dependent 3′-5′ elongation complex of MaTLP. DISCUSS +22 32 structures evidence On the basis of these structures, we will discuss the 3′-5′ addition reaction compared with canonical 5′-3′ elongation by DNA/RNA polymerases. DISCUSS +122 141 DNA/RNA polymerases protein_type On the basis of these structures, we will discuss the 3′-5′ addition reaction compared with canonical 5′-3′ elongation by DNA/RNA polymerases. DISCUSS +66 69 TLP protein_type Figure 5 is a schematic diagram of the 3′-5′ addition reaction of TLP. DISCUSS +20 46 triphosphate binding sites site This enzyme has two triphosphate binding sites and one reaction center at the position overlapping these two binding sites (Fig. 5A). DISCUSS +55 70 reaction center site This enzyme has two triphosphate binding sites and one reaction center at the position overlapping these two binding sites (Fig. 5A). DISCUSS +109 122 binding sites site This enzyme has two triphosphate binding sites and one reaction center at the position overlapping these two binding sites (Fig. 5A). DISCUSS +35 38 GTP chemical In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). DISCUSS +39 42 ATP chemical In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). DISCUSS +46 54 bound to protein_state In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). DISCUSS +55 61 site 1 site In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). DISCUSS +77 89 5′-phosphate chemical In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). DISCUSS +97 101 tRNA chemical In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). DISCUSS +121 125 Mg2+ chemical In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). DISCUSS +145 154 phosphate chemical In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). DISCUSS +162 165 GTP chemical In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). DISCUSS +166 169 ATP chemical In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). DISCUSS +4 13 structure evidence The structure of the MaTLP-ppptRNAPheΔ1 complex, wherein β- and γ-phosphates coordinate with Mg2+A and Mg2+B, respectively (Figs. 3A and 5C′), may represent this activated intermediate. DISCUSS +21 39 MaTLP-ppptRNAPheΔ1 complex_assembly The structure of the MaTLP-ppptRNAPheΔ1 complex, wherein β- and γ-phosphates coordinate with Mg2+A and Mg2+B, respectively (Figs. 3A and 5C′), may represent this activated intermediate. DISCUSS +66 76 phosphates chemical The structure of the MaTLP-ppptRNAPheΔ1 complex, wherein β- and γ-phosphates coordinate with Mg2+A and Mg2+B, respectively (Figs. 3A and 5C′), may represent this activated intermediate. DISCUSS +77 92 coordinate with bond_interaction The structure of the MaTLP-ppptRNAPheΔ1 complex, wherein β- and γ-phosphates coordinate with Mg2+A and Mg2+B, respectively (Figs. 3A and 5C′), may represent this activated intermediate. DISCUSS +93 97 Mg2+ chemical The structure of the MaTLP-ppptRNAPheΔ1 complex, wherein β- and γ-phosphates coordinate with Mg2+A and Mg2+B, respectively (Figs. 3A and 5C′), may represent this activated intermediate. DISCUSS +103 107 Mg2+ chemical The structure of the MaTLP-ppptRNAPheΔ1 complex, wherein β- and γ-phosphates coordinate with Mg2+A and Mg2+B, respectively (Figs. 3A and 5C′), may represent this activated intermediate. DISCUSS +34 44 nucleotide chemical Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3′-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5′-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D). DISCUSS +48 54 site 2 site Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3′-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5′-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D). DISCUSS +84 106 Watson-Crick base pair bond_interaction Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3′-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5′-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D). DISCUSS +114 124 nucleotide chemical Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3′-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5′-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D). DISCUSS +182 192 nucleotide chemical Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3′-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5′-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D). DISCUSS +229 233 Mg2+ chemical Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3′-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5′-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D). DISCUSS +243 258 5′-triphosphate chemical Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3′-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5′-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D). DISCUSS +266 270 tRNA chemical Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3′-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5′-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D). DISCUSS +278 293 reaction center site Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3′-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5′-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D). DISCUSS +18 27 structure evidence Thus, the present structure shows that this 3′-5′ elongation enzyme utilizes a reaction center homologous to that of 5′-3′ elongation enzymes for both activation and elongation in a stepwise fashion. DISCUSS +44 67 3′-5′ elongation enzyme protein_type Thus, the present structure shows that this 3′-5′ elongation enzyme utilizes a reaction center homologous to that of 5′-3′ elongation enzymes for both activation and elongation in a stepwise fashion. DISCUSS +79 94 reaction center site Thus, the present structure shows that this 3′-5′ elongation enzyme utilizes a reaction center homologous to that of 5′-3′ elongation enzymes for both activation and elongation in a stepwise fashion. DISCUSS +117 141 5′-3′ elongation enzymes protein_type Thus, the present structure shows that this 3′-5′ elongation enzyme utilizes a reaction center homologous to that of 5′-3′ elongation enzymes for both activation and elongation in a stepwise fashion. DISCUSS +24 27 TLP protein_type It should be noted that TLP has evolved to allow the occurrence of these two elaborate reaction steps within one reaction center. DISCUSS +113 128 reaction center site It should be noted that TLP has evolved to allow the occurrence of these two elaborate reaction steps within one reaction center. DISCUSS +8 23 reaction center site (A) The reaction center overlapped with two triphosphate binding sites. FIG +44 70 triphosphate binding sites site (A) The reaction center overlapped with two triphosphate binding sites. FIG +33 46 binding sites site A, B, and C (in green) represent binding sites for Mg2+A, Mg2+B, and Mg2+C. P (in blue) represents the phosphate binding sites; O− (in red) is the binding site for the deprotonated OH group. FIG +51 55 Mg2+ chemical A, B, and C (in green) represent binding sites for Mg2+A, Mg2+B, and Mg2+C. P (in blue) represents the phosphate binding sites; O− (in red) is the binding site for the deprotonated OH group. FIG +58 62 Mg2+ chemical A, B, and C (in green) represent binding sites for Mg2+A, Mg2+B, and Mg2+C. P (in blue) represents the phosphate binding sites; O− (in red) is the binding site for the deprotonated OH group. FIG +69 73 Mg2+ chemical A, B, and C (in green) represent binding sites for Mg2+A, Mg2+B, and Mg2+C. P (in blue) represents the phosphate binding sites; O− (in red) is the binding site for the deprotonated OH group. FIG +76 77 P site A, B, and C (in green) represent binding sites for Mg2+A, Mg2+B, and Mg2+C. P (in blue) represents the phosphate binding sites; O− (in red) is the binding site for the deprotonated OH group. FIG +103 126 phosphate binding sites site A, B, and C (in green) represent binding sites for Mg2+A, Mg2+B, and Mg2+C. P (in blue) represents the phosphate binding sites; O− (in red) is the binding site for the deprotonated OH group. FIG +147 159 binding site site A, B, and C (in green) represent binding sites for Mg2+A, Mg2+B, and Mg2+C. P (in blue) represents the phosphate binding sites; O− (in red) is the binding site for the deprotonated OH group. FIG +10 13 TLP protein_type Important TLP residues for tRNA and Mg2+ binding are also shown. (B) Structure of the activation complex (corresponding to fig. S8). FIG +27 31 tRNA chemical Important TLP residues for tRNA and Mg2+ binding are also shown. (B) Structure of the activation complex (corresponding to fig. S8). FIG +36 40 Mg2+ chemical Important TLP residues for tRNA and Mg2+ binding are also shown. (B) Structure of the activation complex (corresponding to fig. S8). FIG +69 78 Structure evidence Important TLP residues for tRNA and Mg2+ binding are also shown. (B) Structure of the activation complex (corresponding to fig. S8). FIG +0 3 GTP chemical GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). FIG +4 7 ATP chemical GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). FIG +17 44 triphosphate binding site 1 site GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). FIG +79 91 5′-phosphate chemical GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). FIG +106 115 phosphate chemical GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). FIG +119 122 GTP chemical GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). FIG +123 126 ATP chemical GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). FIG +132 135 PPi chemical GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). FIG +137 160 inorganic pyrophosphate chemical GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). FIG +188 197 structure evidence GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). FIG +246 255 structure evidence GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). FIG +5 14 Structure evidence (C′) Structure before the elongation reaction (corresponding to Fig. 3A). FIG +4 19 5′-triphosphate chemical The 5′-triphosphate of the tRNA binds to the same site as for activation of the 5′-terminus of the tRNA in (B). FIG +27 31 tRNA chemical The 5′-triphosphate of the tRNA binds to the same site as for activation of the 5′-terminus of the tRNA in (B). FIG +99 103 tRNA chemical The 5′-triphosphate of the tRNA binds to the same site as for activation of the 5′-terminus of the tRNA in (B). FIG +4 13 Structure evidence (D) Structure of initiation of the elongation reaction (corresponding to Fig. 3B). FIG +25 28 GTP chemical The base of the incoming GTP forms a Watson-Crick hydrogen bond with the nucleotide at position 72 in the template chain and a base-stacking interaction with a neighboring base (G2). FIG +37 63 Watson-Crick hydrogen bond bond_interaction The base of the incoming GTP forms a Watson-Crick hydrogen bond with the nucleotide at position 72 in the template chain and a base-stacking interaction with a neighboring base (G2). FIG +73 83 nucleotide chemical The base of the incoming GTP forms a Watson-Crick hydrogen bond with the nucleotide at position 72 in the template chain and a base-stacking interaction with a neighboring base (G2). FIG +96 98 72 residue_number The base of the incoming GTP forms a Watson-Crick hydrogen bond with the nucleotide at position 72 in the template chain and a base-stacking interaction with a neighboring base (G2). FIG +127 152 base-stacking interaction bond_interaction The base of the incoming GTP forms a Watson-Crick hydrogen bond with the nucleotide at position 72 in the template chain and a base-stacking interaction with a neighboring base (G2). FIG +178 180 G2 residue_name_number The base of the incoming GTP forms a Watson-Crick hydrogen bond with the nucleotide at position 72 in the template chain and a base-stacking interaction with a neighboring base (G2). FIG +45 60 5′-triphosphate chemical Movement of the 5′-terminal chain leaves the 5′-triphosphate of the tRNA in the same site as the activation step in (B). FIG +68 72 tRNA chemical Movement of the 5′-terminal chain leaves the 5′-triphosphate of the tRNA in the same site as the activation step in (B). FIG +26 29 GTP chemical The 3′-OH of the incoming GTP is deprotonated by Mg2+A and attacks the α-phosphate to form a covalent bond. (E) After the elongation reaction, the triphosphate of the new nucleotide is placed on site 1, as in (C′), and is ready for the next reaction. FIG +49 53 Mg2+ chemical The 3′-OH of the incoming GTP is deprotonated by Mg2+A and attacks the α-phosphate to form a covalent bond. (E) After the elongation reaction, the triphosphate of the new nucleotide is placed on site 1, as in (C′), and is ready for the next reaction. FIG +73 82 phosphate chemical The 3′-OH of the incoming GTP is deprotonated by Mg2+A and attacks the α-phosphate to form a covalent bond. (E) After the elongation reaction, the triphosphate of the new nucleotide is placed on site 1, as in (C′), and is ready for the next reaction. FIG +147 159 triphosphate chemical The 3′-OH of the incoming GTP is deprotonated by Mg2+A and attacks the α-phosphate to form a covalent bond. (E) After the elongation reaction, the triphosphate of the new nucleotide is placed on site 1, as in (C′), and is ready for the next reaction. FIG +171 181 nucleotide chemical The 3′-OH of the incoming GTP is deprotonated by Mg2+A and attacks the α-phosphate to form a covalent bond. (E) After the elongation reaction, the triphosphate of the new nucleotide is placed on site 1, as in (C′), and is ready for the next reaction. FIG +195 201 site 1 site The 3′-OH of the incoming GTP is deprotonated by Mg2+A and attacks the α-phosphate to form a covalent bond. (E) After the elongation reaction, the triphosphate of the new nucleotide is placed on site 1, as in (C′), and is ready for the next reaction. FIG +137 152 reaction center site Figure 6 compares the 3′-5′ and 5′-3′ elongation mechanisms, showing the symmetrical nature of both elongation reactions using a similar reaction center composed of Mg2+A and Mg2+B in the conserved catalytic core. DISCUSS +165 169 Mg2+ chemical Figure 6 compares the 3′-5′ and 5′-3′ elongation mechanisms, showing the symmetrical nature of both elongation reactions using a similar reaction center composed of Mg2+A and Mg2+B in the conserved catalytic core. DISCUSS +175 179 Mg2+ chemical Figure 6 compares the 3′-5′ and 5′-3′ elongation mechanisms, showing the symmetrical nature of both elongation reactions using a similar reaction center composed of Mg2+A and Mg2+B in the conserved catalytic core. DISCUSS +188 197 conserved protein_state Figure 6 compares the 3′-5′ and 5′-3′ elongation mechanisms, showing the symmetrical nature of both elongation reactions using a similar reaction center composed of Mg2+A and Mg2+B in the conserved catalytic core. DISCUSS +198 212 catalytic core site Figure 6 compares the 3′-5′ and 5′-3′ elongation mechanisms, showing the symmetrical nature of both elongation reactions using a similar reaction center composed of Mg2+A and Mg2+B in the conserved catalytic core. DISCUSS +3 6 TLP protein_type In TLP, which carries out 3′-5′ elongation, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the T7 RNA polymerase, a representative 5′-3′ DNA/RNA polymerase, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. DISCUSS +106 115 phosphate chemical In TLP, which carries out 3′-5′ elongation, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the T7 RNA polymerase, a representative 5′-3′ DNA/RNA polymerase, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. DISCUSS +123 127 tRNA chemical In TLP, which carries out 3′-5′ elongation, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the T7 RNA polymerase, a representative 5′-3′ DNA/RNA polymerase, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. DISCUSS +174 191 T7 RNA polymerase protein In TLP, which carries out 3′-5′ elongation, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the T7 RNA polymerase, a representative 5′-3′ DNA/RNA polymerase, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. DISCUSS +210 234 5′-3′ DNA/RNA polymerase protein_type In TLP, which carries out 3′-5′ elongation, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the T7 RNA polymerase, a representative 5′-3′ DNA/RNA polymerase, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. DISCUSS +283 286 RNA chemical In TLP, which carries out 3′-5′ elongation, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the T7 RNA polymerase, a representative 5′-3′ DNA/RNA polymerase, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. DISCUSS +309 318 phosphate chemical In TLP, which carries out 3′-5′ elongation, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the T7 RNA polymerase, a representative 5′-3′ DNA/RNA polymerase, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. DISCUSS +41 43 Mg chemical In these reactions, the roles of the two Mg ions are identical. DISCUSS +0 4 Mg2+ chemical Mg2+A activates the 3′-OH of the incoming nucleotide in TLP and the 3′-OH of the 3′-end of the RNA chain in T7 RNA polymerase. DISCUSS +56 59 TLP protein_type Mg2+A activates the 3′-OH of the incoming nucleotide in TLP and the 3′-OH of the 3′-end of the RNA chain in T7 RNA polymerase. DISCUSS +95 98 RNA chemical Mg2+A activates the 3′-OH of the incoming nucleotide in TLP and the 3′-OH of the 3′-end of the RNA chain in T7 RNA polymerase. DISCUSS +108 125 T7 RNA polymerase protein Mg2+A activates the 3′-OH of the incoming nucleotide in TLP and the 3′-OH of the 3′-end of the RNA chain in T7 RNA polymerase. DISCUSS +12 16 Mg2+ chemical The role of Mg2+B is to position the 5′-triphosphate of the tRNA in TLP and the incoming nucleotide in T7 RNA polymerase. DISCUSS +37 52 5′-triphosphate chemical The role of Mg2+B is to position the 5′-triphosphate of the tRNA in TLP and the incoming nucleotide in T7 RNA polymerase. DISCUSS +60 64 tRNA chemical The role of Mg2+B is to position the 5′-triphosphate of the tRNA in TLP and the incoming nucleotide in T7 RNA polymerase. DISCUSS +68 71 TLP protein_type The role of Mg2+B is to position the 5′-triphosphate of the tRNA in TLP and the incoming nucleotide in T7 RNA polymerase. DISCUSS +103 120 T7 RNA polymerase protein The role of Mg2+B is to position the 5′-triphosphate of the tRNA in TLP and the incoming nucleotide in T7 RNA polymerase. DISCUSS +10 14 Mg2+ chemical These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core. DISCUSS +24 38 coordinated by bond_interaction These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core. DISCUSS +41 50 conserved protein_state These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core. DISCUSS +51 54 Asp residue_name These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core. DISCUSS +56 59 D21 residue_name_number These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core. DISCUSS +64 67 D69 residue_name_number These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core. DISCUSS +71 74 TLP protein_type These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core. DISCUSS +83 92 conserved protein_state These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core. DISCUSS +93 107 catalytic core site These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core. DISCUSS +0 10 Structures evidence Structures of template-dependent nucleotide elongation in the 3′-5′ and 5′-3′ directions. FIG +53 56 TLP protein_type Symmetrical relationship between 3′-5′ elongation by TLP (this study) (left) and 5′-3′ elongation by T7 RNA polymerase [Protein Data Bank (PDB) ID: 1S76] (right). FIG +101 118 T7 RNA polymerase protein Symmetrical relationship between 3′-5′ elongation by TLP (this study) (left) and 5′-3′ elongation by T7 RNA polymerase [Protein Data Bank (PDB) ID: 1S76] (right). FIG +96 105 phosphate chemical In the 3′-5′ elongation reaction, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the 5′-3′ elongation reaction, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. FIG +113 117 tRNA chemical In the 3′-5′ elongation reaction, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the 5′-3′ elongation reaction, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. FIG +238 241 RNA chemical In the 3′-5′ elongation reaction, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the 5′-3′ elongation reaction, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. FIG +264 273 phosphate chemical In the 3′-5′ elongation reaction, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the 5′-3′ elongation reaction, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. FIG +24 28 Mg2+ chemical Green spheres represent Mg2+ ions. FIG +30 34 tRNA chemical Because the chemical roles of tRNA and the incoming nucleotide are reversed in these two reactions, these two substrates are inserted into a similar reaction center from opposite directions (Fig. 6). DISCUSS +149 164 reaction center site Because the chemical roles of tRNA and the incoming nucleotide are reversed in these two reactions, these two substrates are inserted into a similar reaction center from opposite directions (Fig. 6). DISCUSS +164 183 DNA/RNA polymerases protein_type However, from an energetic viewpoint, these two reactions are clearly different: Whereas the high energy of the incoming nucleotide is used for its own addition in DNA/RNA polymerases, the high energy of the incoming nucleotide is used for subsequent addition in TLP. DISCUSS +263 266 TLP protein_type However, from an energetic viewpoint, these two reactions are clearly different: Whereas the high energy of the incoming nucleotide is used for its own addition in DNA/RNA polymerases, the high energy of the incoming nucleotide is used for subsequent addition in TLP. DISCUSS +17 20 TLP protein_type For this reason, TLP requires a mechanism that activates the 5′-terminus of the tRNA during the initial step of the reaction. DISCUSS +80 84 tRNA chemical For this reason, TLP requires a mechanism that activates the 5′-terminus of the tRNA during the initial step of the reaction. DISCUSS +110 125 reaction center site Our analysis showed that the initial activation and subsequent elongation reactions occur sequentially at one reaction center. DISCUSS +45 68 substrate binding sites site In this case, the enzyme needs to create two substrate binding sites for two different reactions in the vicinities of one reaction center. DISCUSS +122 137 reaction center site In this case, the enzyme needs to create two substrate binding sites for two different reactions in the vicinities of one reaction center. DISCUSS +0 3 TLP protein_type TLP has successfully created such sites by utilizing a conformational change in the tRNA through Watson-Crick base pairing (Fig. 3). DISCUSS +84 88 tRNA chemical TLP has successfully created such sites by utilizing a conformational change in the tRNA through Watson-Crick base pairing (Fig. 3). DISCUSS +97 122 Watson-Crick base pairing bond_interaction TLP has successfully created such sites by utilizing a conformational change in the tRNA through Watson-Crick base pairing (Fig. 3). DISCUSS +33 36 TLP protein_type These structural features of the TLP molecule suggest that development of an activation reaction site is a prerequisite for developing the 3′-5′ elongation enzyme. DISCUSS +77 101 activation reaction site site These structural features of the TLP molecule suggest that development of an activation reaction site is a prerequisite for developing the 3′-5′ elongation enzyme. DISCUSS +139 162 3′-5′ elongation enzyme protein_type These structural features of the TLP molecule suggest that development of an activation reaction site is a prerequisite for developing the 3′-5′ elongation enzyme. DISCUSS +51 74 5′-3′ elongation enzyme protein_type This is clearly more difficult than developing the 5′-3′ elongation enzyme, wherein the activation reaction site is not necessary, and which may be the primary reason why the 5′-3′ elongation enzyme has been exclusively developed. DISCUSS +88 112 activation reaction site site This is clearly more difficult than developing the 5′-3′ elongation enzyme, wherein the activation reaction site is not necessary, and which may be the primary reason why the 5′-3′ elongation enzyme has been exclusively developed. DISCUSS +175 198 5′-3′ elongation enzyme protein_type This is clearly more difficult than developing the 5′-3′ elongation enzyme, wherein the activation reaction site is not necessary, and which may be the primary reason why the 5′-3′ elongation enzyme has been exclusively developed. DISCUSS +88 91 TLP protein_type Here, we established a structural basis for 3′-5′ nucleotide elongation and showed that TLP has evolved to acquire a two-step Watson-Crick template–dependent 3′-5′ elongation reaction using the catalytic center homologous to 5′-3′ elongation enzymes. DISCUSS +194 210 catalytic center site Here, we established a structural basis for 3′-5′ nucleotide elongation and showed that TLP has evolved to acquire a two-step Watson-Crick template–dependent 3′-5′ elongation reaction using the catalytic center homologous to 5′-3′ elongation enzymes. DISCUSS +225 249 5′-3′ elongation enzymes protein_type Here, we established a structural basis for 3′-5′ nucleotide elongation and showed that TLP has evolved to acquire a two-step Watson-Crick template–dependent 3′-5′ elongation reaction using the catalytic center homologous to 5′-3′ elongation enzymes. DISCUSS +4 15 active site site The active site of this enzyme is created at the dimerization interface. DISCUSS +49 71 dimerization interface site The active site of this enzyme is created at the dimerization interface. DISCUSS +88 101 accepter stem structure_element The dimerization also endows this protein with the ability to measure the length of the accepter stem of the tRNA substrate, so that the enzyme can properly terminate the elongation reaction. DISCUSS +109 113 tRNA chemical The dimerization also endows this protein with the ability to measure the length of the accepter stem of the tRNA substrate, so that the enzyme can properly terminate the elongation reaction. DISCUSS +97 100 G−1 residue_name_number Furthermore, the dual binding mode of this protein suggests that it has further evolved to cover G−1 addition of tRNAHis by additional dimerization (dimer of dimers). DISCUSS +113 120 tRNAHis chemical Furthermore, the dual binding mode of this protein suggests that it has further evolved to cover G−1 addition of tRNAHis by additional dimerization (dimer of dimers). DISCUSS +149 154 dimer oligomeric_state Furthermore, the dual binding mode of this protein suggests that it has further evolved to cover G−1 addition of tRNAHis by additional dimerization (dimer of dimers). DISCUSS +158 164 dimers oligomeric_state Furthermore, the dual binding mode of this protein suggests that it has further evolved to cover G−1 addition of tRNAHis by additional dimerization (dimer of dimers). DISCUSS +18 37 structural analysis experimental_method Thus, the present structural analysis is consistent with the scenario in which TLP began as a 5′-end repair enzyme and evolved into a tRNAHis-specific G−1 addition enzyme. DISCUSS +79 82 TLP protein_type Thus, the present structural analysis is consistent with the scenario in which TLP began as a 5′-end repair enzyme and evolved into a tRNAHis-specific G−1 addition enzyme. DISCUSS +134 170 tRNAHis-specific G−1 addition enzyme protein_type Thus, the present structural analysis is consistent with the scenario in which TLP began as a 5′-end repair enzyme and evolved into a tRNAHis-specific G−1 addition enzyme. DISCUSS +40 44 Thg1 protein The detailed molecular mechanism of the Thg1/TLP family established by our analysis will open up new perspectives in our understanding of 3′-5′ versus 5′-3′ polymerization and the molecular evolution of template-dependent polymerases. DISCUSS +45 48 TLP protein_type The detailed molecular mechanism of the Thg1/TLP family established by our analysis will open up new perspectives in our understanding of 3′-5′ versus 5′-3′ polymerization and the molecular evolution of template-dependent polymerases. DISCUSS +203 233 template-dependent polymerases protein_type The detailed molecular mechanism of the Thg1/TLP family established by our analysis will open up new perspectives in our understanding of 3′-5′ versus 5′-3′ polymerization and the molecular evolution of template-dependent polymerases. DISCUSS +12 17 tRNAs chemical Transcribed tRNAs were purified by a HiTrap DEAE FF column (GE Healthcare) as previously described. METHODS +7 12 tRNAs chemical Pooled tRNAs were precipitated with isopropanol and dissolved in buffer E [20 mM Hepes-NaOH (pH 7.5), 100 mM NaCl, and 10 mM MgCl2]. METHODS +49 54 Thg1p protein The highly conserved tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex and is required for the G2/M phase transition in the yeast Saccharomyces cerevisiae REF diff --git a/annotation_CSV/PMC4822050.csv b/annotation_CSV/PMC4822050.csv new file mode 100644 index 0000000000000000000000000000000000000000..02faac125dbc6da6ac5c977bb2b7ba239f95a8c7 --- /dev/null +++ b/annotation_CSV/PMC4822050.csv @@ -0,0 +1,1522 @@ +anno_start anno_end anno_text entity_type sentence section +0 19 Hemi-methylated DNA chemical Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition TITLE +28 34 closed protein_state Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition TITLE +51 56 UHRF1 protein Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition TITLE +75 82 histone protein_type Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition TITLE +0 5 UHRF1 protein UHRF1 is an important epigenetic regulator for maintenance DNA methylation. ABSTRACT +63 74 methylation ptm UHRF1 is an important epigenetic regulator for maintenance DNA methylation. ABSTRACT +0 5 UHRF1 protein UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. ABSTRACT +17 36 hemi-methylated DNA chemical UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. ABSTRACT +38 44 hm-DNA chemical UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. ABSTRACT +50 64 trimethylation ptm UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. ABSTRACT +68 75 histone protein_type UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. ABSTRACT +76 78 H3 protein_type UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. ABSTRACT +78 80 K9 residue_name_number UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. ABSTRACT +82 84 H3 protein_type UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. ABSTRACT +84 89 K9me3 ptm UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. ABSTRACT +18 23 UHRF1 protein Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +33 39 closed protein_state Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +65 82 C-terminal region structure_element Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +84 90 Spacer structure_element Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +92 100 binds to protein_state Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +105 124 tandem Tudor domain structure_element Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +126 129 TTD structure_element Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +144 146 H3 protein_type Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +146 151 K9me3 ptm Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +177 200 SET-and-RING-associated structure_element Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +202 205 SRA structure_element Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +214 222 binds to protein_state Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +227 244 plant homeodomain structure_element Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +246 249 PHD structure_element Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +264 268 H3R2 site Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. ABSTRACT +0 6 Hm-DNA chemical Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD–PHD. ABSTRACT +60 62 H3 protein_type Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD–PHD. ABSTRACT +62 67 K9me3 ptm Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD–PHD. ABSTRACT +83 90 TTD–PHD structure_element Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD–PHD. ABSTRACT +4 10 Spacer structure_element The Spacer also facilitates UHRF1–DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. ABSTRACT +28 39 UHRF1–DNMT1 complex_assembly The Spacer also facilitates UHRF1–DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. ABSTRACT +65 88 hm-DNA-binding affinity evidence The Spacer also facilitates UHRF1–DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. ABSTRACT +96 99 SRA structure_element The Spacer also facilitates UHRF1–DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. ABSTRACT +5 12 TTD–PHD structure_element When TTD–PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. ABSTRACT +13 21 binds to protein_state When TTD–PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. ABSTRACT +22 24 H3 protein_type When TTD–PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. ABSTRACT +24 29 K9me3 ptm When TTD–PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. ABSTRACT +31 41 SRA-Spacer structure_element When TTD–PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. ABSTRACT +96 102 hm-DNA chemical When TTD–PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. ABSTRACT +115 120 DNMT1 protein When TTD–PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. ABSTRACT +50 52 H3 protein_type Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1. ABSTRACT +52 57 K9me3 ptm Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1. ABSTRACT +62 68 hm-DNA chemical Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1. ABSTRACT +84 89 URHF1 protein Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1. ABSTRACT +1 6 UHRF1 protein UHRF1 is involved in the maintenance of DNA methylation, but the regulatory mechanism of this epigenetic regulator is unclear. ABSTRACT +45 56 methylation ptm UHRF1 is involved in the maintenance of DNA methylation, but the regulatory mechanism of this epigenetic regulator is unclear. ABSTRACT +37 43 closed protein_state Here, the authors show that it has a closed conformation and are able to make conclusions about the mechanism of recognition of epigenetic marks. ABSTRACT +4 15 methylation ptm DNA methylation is an important epigenetic modification for gene repression, X-chromosome inactivation, genome imprinting and maintenance of genome stability. INTRO +0 9 Mammalian taxonomy_domain Mammalian DNA methylation is established by de novo DNA methyltransferases DNMT3A/3B, and DNA methylation patterns are maintained by maintenance DNA methyltransferase 1 (DNMT1) during DNA replication. INTRO +14 25 methylation ptm Mammalian DNA methylation is established by de novo DNA methyltransferases DNMT3A/3B, and DNA methylation patterns are maintained by maintenance DNA methyltransferase 1 (DNMT1) during DNA replication. INTRO +52 74 DNA methyltransferases protein_type Mammalian DNA methylation is established by de novo DNA methyltransferases DNMT3A/3B, and DNA methylation patterns are maintained by maintenance DNA methyltransferase 1 (DNMT1) during DNA replication. INTRO +75 84 DNMT3A/3B protein Mammalian DNA methylation is established by de novo DNA methyltransferases DNMT3A/3B, and DNA methylation patterns are maintained by maintenance DNA methyltransferase 1 (DNMT1) during DNA replication. INTRO +94 105 methylation ptm Mammalian DNA methylation is established by de novo DNA methyltransferases DNMT3A/3B, and DNA methylation patterns are maintained by maintenance DNA methyltransferase 1 (DNMT1) during DNA replication. INTRO +145 168 DNA methyltransferase 1 protein Mammalian DNA methylation is established by de novo DNA methyltransferases DNMT3A/3B, and DNA methylation patterns are maintained by maintenance DNA methyltransferase 1 (DNMT1) during DNA replication. INTRO +170 175 DNMT1 protein Mammalian DNA methylation is established by de novo DNA methyltransferases DNMT3A/3B, and DNA methylation patterns are maintained by maintenance DNA methyltransferase 1 (DNMT1) during DNA replication. INTRO +0 58 Ubiquitin-like, containing PHD and RING fingers domains, 1 protein Ubiquitin-like, containing PHD and RING fingers domains, 1 (UHRF1, also known as ICBP90 and NP95 in mouse) was shown to be essential for maintenance DNA methylation through recruiting DNMT1 to replication forks in S phase of the cell cycle. INTRO +60 65 UHRF1 protein Ubiquitin-like, containing PHD and RING fingers domains, 1 (UHRF1, also known as ICBP90 and NP95 in mouse) was shown to be essential for maintenance DNA methylation through recruiting DNMT1 to replication forks in S phase of the cell cycle. INTRO +81 87 ICBP90 protein Ubiquitin-like, containing PHD and RING fingers domains, 1 (UHRF1, also known as ICBP90 and NP95 in mouse) was shown to be essential for maintenance DNA methylation through recruiting DNMT1 to replication forks in S phase of the cell cycle. INTRO +92 96 NP95 protein Ubiquitin-like, containing PHD and RING fingers domains, 1 (UHRF1, also known as ICBP90 and NP95 in mouse) was shown to be essential for maintenance DNA methylation through recruiting DNMT1 to replication forks in S phase of the cell cycle. INTRO +100 105 mouse taxonomy_domain Ubiquitin-like, containing PHD and RING fingers domains, 1 (UHRF1, also known as ICBP90 and NP95 in mouse) was shown to be essential for maintenance DNA methylation through recruiting DNMT1 to replication forks in S phase of the cell cycle. INTRO +153 164 methylation ptm Ubiquitin-like, containing PHD and RING fingers domains, 1 (UHRF1, also known as ICBP90 and NP95 in mouse) was shown to be essential for maintenance DNA methylation through recruiting DNMT1 to replication forks in S phase of the cell cycle. INTRO +184 189 DNMT1 protein Ubiquitin-like, containing PHD and RING fingers domains, 1 (UHRF1, also known as ICBP90 and NP95 in mouse) was shown to be essential for maintenance DNA methylation through recruiting DNMT1 to replication forks in S phase of the cell cycle. INTRO +0 5 UHRF1 protein UHRF1 is essential for S phase entry and is involved in heterochromatin formation. INTRO +0 5 UHRF1 protein UHRF1 also plays an important role in promoting proliferation and is shown to be upregulated in a number of cancers, suggesting that UHRF1 may serve as a potential drug target for therapeutic applications. INTRO +133 138 UHRF1 protein UHRF1 also plays an important role in promoting proliferation and is shown to be upregulated in a number of cancers, suggesting that UHRF1 may serve as a potential drug target for therapeutic applications. INTRO +0 5 UHRF1 protein UHRF1 is a multi-domain containing protein connecting histone modification and DNA methylation. INTRO +54 61 histone protein_type UHRF1 is a multi-domain containing protein connecting histone modification and DNA methylation. INTRO +79 82 DNA chemical UHRF1 is a multi-domain containing protein connecting histone modification and DNA methylation. INTRO +83 94 methylation ptm UHRF1 is a multi-domain containing protein connecting histone modification and DNA methylation. INTRO +21 26 UHRF1 protein As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. INTRO +57 78 ubiquitin-like domain structure_element As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. INTRO +94 113 tandem Tudor domain structure_element As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. INTRO +115 118 TTD structure_element As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. INTRO +130 134 TTDN structure_element As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. INTRO +139 143 TTDC structure_element As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. INTRO +160 177 plant homeodomain structure_element As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. INTRO +179 182 PHD structure_element As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. INTRO +187 210 SET-and-RING-associated structure_element As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. INTRO +212 215 SRA structure_element As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. INTRO +242 269 really interesting new gene structure_element As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. INTRO +271 275 RING structure_element As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain. INTRO +42 45 TTD structure_element We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD. INTRO +54 57 PHD structure_element We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD. INTRO +81 88 histone protein_type We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD. INTRO +89 91 H3 protein_type We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD. INTRO +91 96 K9me3 ptm We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD. INTRO +115 117 R2 residue_name_number We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD. INTRO +139 142 PHD structure_element We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD. INTRO +147 162 tri-methylation ptm We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD. INTRO +174 176 K9 residue_name_number We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD. INTRO +178 183 K9me3 ptm We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD. INTRO +206 209 TTD structure_element We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD. INTRO +4 7 SRA structure_element The SRA preferentially binds to hemi-methylated DNA (hm-DNA). INTRO +23 31 binds to protein_state The SRA preferentially binds to hemi-methylated DNA (hm-DNA). INTRO +32 51 hemi-methylated DNA chemical The SRA preferentially binds to hemi-methylated DNA (hm-DNA). INTRO +53 59 hm-DNA chemical The SRA preferentially binds to hemi-methylated DNA (hm-DNA). INTRO +29 32 SRA structure_element Recent studies show that the SRA directly binds to replication focus targeting sequence (RFTS) of DNMT1 (RFTSDNMT1). INTRO +42 50 binds to protein_state Recent studies show that the SRA directly binds to replication focus targeting sequence (RFTS) of DNMT1 (RFTSDNMT1). INTRO +51 87 replication focus targeting sequence structure_element Recent studies show that the SRA directly binds to replication focus targeting sequence (RFTS) of DNMT1 (RFTSDNMT1). INTRO +89 93 RFTS structure_element Recent studies show that the SRA directly binds to replication focus targeting sequence (RFTS) of DNMT1 (RFTSDNMT1). INTRO +98 103 DNMT1 protein Recent studies show that the SRA directly binds to replication focus targeting sequence (RFTS) of DNMT1 (RFTSDNMT1). INTRO +105 114 RFTSDNMT1 protein Recent studies show that the SRA directly binds to replication focus targeting sequence (RFTS) of DNMT1 (RFTSDNMT1). INTRO +2 15 spacer region structure_element A spacer region (Fig. 1a, designated Spacer hereafter) connecting the SRA and the RING is rich in basic residues and predicted to be unstructured for unknown function. INTRO +37 43 Spacer structure_element A spacer region (Fig. 1a, designated Spacer hereafter) connecting the SRA and the RING is rich in basic residues and predicted to be unstructured for unknown function. INTRO +70 73 SRA structure_element A spacer region (Fig. 1a, designated Spacer hereafter) connecting the SRA and the RING is rich in basic residues and predicted to be unstructured for unknown function. INTRO +82 86 RING structure_element A spacer region (Fig. 1a, designated Spacer hereafter) connecting the SRA and the RING is rich in basic residues and predicted to be unstructured for unknown function. INTRO +133 145 unstructured protein_state A spacer region (Fig. 1a, designated Spacer hereafter) connecting the SRA and the RING is rich in basic residues and predicted to be unstructured for unknown function. INTRO +24 54 phosphatidylinostiol phosphate chemical Recent study shows that phosphatidylinostiol phosphate PI5P binds to the Spacer and induces a conformational change of UHRF1 to allow the TTD to recognize H3K9me3 (ref.). INTRO +55 59 PI5P chemical Recent study shows that phosphatidylinostiol phosphate PI5P binds to the Spacer and induces a conformational change of UHRF1 to allow the TTD to recognize H3K9me3 (ref.). INTRO +60 68 binds to protein_state Recent study shows that phosphatidylinostiol phosphate PI5P binds to the Spacer and induces a conformational change of UHRF1 to allow the TTD to recognize H3K9me3 (ref.). INTRO +73 79 Spacer structure_element Recent study shows that phosphatidylinostiol phosphate PI5P binds to the Spacer and induces a conformational change of UHRF1 to allow the TTD to recognize H3K9me3 (ref.). INTRO +119 124 UHRF1 protein Recent study shows that phosphatidylinostiol phosphate PI5P binds to the Spacer and induces a conformational change of UHRF1 to allow the TTD to recognize H3K9me3 (ref.). INTRO +138 141 TTD structure_element Recent study shows that phosphatidylinostiol phosphate PI5P binds to the Spacer and induces a conformational change of UHRF1 to allow the TTD to recognize H3K9me3 (ref.). INTRO +155 157 H3 protein_type Recent study shows that phosphatidylinostiol phosphate PI5P binds to the Spacer and induces a conformational change of UHRF1 to allow the TTD to recognize H3K9me3 (ref.). INTRO +157 162 K9me3 ptm Recent study shows that phosphatidylinostiol phosphate PI5P binds to the Spacer and induces a conformational change of UHRF1 to allow the TTD to recognize H3K9me3 (ref.). INTRO +28 33 UHRF1 protein These studies indicate that UHRF1 connects dynamic regulation of DNA methylation and H3K9me3, which are positively correlated in human genome. INTRO +65 68 DNA chemical These studies indicate that UHRF1 connects dynamic regulation of DNA methylation and H3K9me3, which are positively correlated in human genome. INTRO +69 80 methylation ptm These studies indicate that UHRF1 connects dynamic regulation of DNA methylation and H3K9me3, which are positively correlated in human genome. INTRO +85 87 H3 protein_type These studies indicate that UHRF1 connects dynamic regulation of DNA methylation and H3K9me3, which are positively correlated in human genome. INTRO +87 92 K9me3 ptm These studies indicate that UHRF1 connects dynamic regulation of DNA methylation and H3K9me3, which are positively correlated in human genome. INTRO +129 134 human species These studies indicate that UHRF1 connects dynamic regulation of DNA methylation and H3K9me3, which are positively correlated in human genome. INTRO +13 18 UHRF1 protein However, how UHRF1 regulates the recognition of these two repressive epigenetic marks and recruits DNMT1 for chromatin localization remain largely unknown. INTRO +99 104 DNMT1 protein However, how UHRF1 regulates the recognition of these two repressive epigenetic marks and recruits DNMT1 for chromatin localization remain largely unknown. INTRO +20 25 UHRF1 protein Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +35 41 closed protein_state Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +80 86 Spacer structure_element Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +87 95 binds to protein_state Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +100 103 TTD structure_element Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +136 138 H3 protein_type Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +138 143 K9me3 ptm Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +157 160 SRA structure_element Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +161 169 binds to protein_state Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +174 177 PHD structure_element Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +210 214 H3R2 site Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +216 228 unmethylated protein_state Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +229 236 histone protein_type Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +237 239 H3 protein_type Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +251 253 R2 residue_name_number Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2). INTRO +5 15 binding to protein_state Upon binding to hm-DNA, UHRF1 impairs the intramolecular interactions and promotes the H3K9me3 recognition by TTD–PHD, which may further enhance its genomic localization. INTRO +16 22 hm-DNA chemical Upon binding to hm-DNA, UHRF1 impairs the intramolecular interactions and promotes the H3K9me3 recognition by TTD–PHD, which may further enhance its genomic localization. INTRO +24 29 UHRF1 protein Upon binding to hm-DNA, UHRF1 impairs the intramolecular interactions and promotes the H3K9me3 recognition by TTD–PHD, which may further enhance its genomic localization. INTRO +87 89 H3 protein_type Upon binding to hm-DNA, UHRF1 impairs the intramolecular interactions and promotes the H3K9me3 recognition by TTD–PHD, which may further enhance its genomic localization. INTRO +89 94 K9me3 ptm Upon binding to hm-DNA, UHRF1 impairs the intramolecular interactions and promotes the H3K9me3 recognition by TTD–PHD, which may further enhance its genomic localization. INTRO +110 117 TTD–PHD structure_element Upon binding to hm-DNA, UHRF1 impairs the intramolecular interactions and promotes the H3K9me3 recognition by TTD–PHD, which may further enhance its genomic localization. INTRO +13 18 UHRF1 protein As a result, UHRF1 is locked in the open conformation by the association of H3K9me3 by TTD–PHD, and thus SRA-Spacer either recognizes hm-DNA or recruits DNMT1 for DNA methylation. INTRO +36 40 open protein_state As a result, UHRF1 is locked in the open conformation by the association of H3K9me3 by TTD–PHD, and thus SRA-Spacer either recognizes hm-DNA or recruits DNMT1 for DNA methylation. INTRO +76 78 H3 protein_type As a result, UHRF1 is locked in the open conformation by the association of H3K9me3 by TTD–PHD, and thus SRA-Spacer either recognizes hm-DNA or recruits DNMT1 for DNA methylation. INTRO +78 83 K9me3 ptm As a result, UHRF1 is locked in the open conformation by the association of H3K9me3 by TTD–PHD, and thus SRA-Spacer either recognizes hm-DNA or recruits DNMT1 for DNA methylation. INTRO +87 94 TTD–PHD structure_element As a result, UHRF1 is locked in the open conformation by the association of H3K9me3 by TTD–PHD, and thus SRA-Spacer either recognizes hm-DNA or recruits DNMT1 for DNA methylation. INTRO +105 115 SRA-Spacer structure_element As a result, UHRF1 is locked in the open conformation by the association of H3K9me3 by TTD–PHD, and thus SRA-Spacer either recognizes hm-DNA or recruits DNMT1 for DNA methylation. INTRO +134 140 hm-DNA chemical As a result, UHRF1 is locked in the open conformation by the association of H3K9me3 by TTD–PHD, and thus SRA-Spacer either recognizes hm-DNA or recruits DNMT1 for DNA methylation. INTRO +153 158 DNMT1 protein As a result, UHRF1 is locked in the open conformation by the association of H3K9me3 by TTD–PHD, and thus SRA-Spacer either recognizes hm-DNA or recruits DNMT1 for DNA methylation. INTRO +163 166 DNA chemical As a result, UHRF1 is locked in the open conformation by the association of H3K9me3 by TTD–PHD, and thus SRA-Spacer either recognizes hm-DNA or recruits DNMT1 for DNA methylation. INTRO +167 178 methylation ptm As a result, UHRF1 is locked in the open conformation by the association of H3K9me3 by TTD–PHD, and thus SRA-Spacer either recognizes hm-DNA or recruits DNMT1 for DNA methylation. INTRO +11 16 UHRF1 protein Therefore, UHRF1 may engage in a sophisticated regulation for its chromatin localization and recruitment of DNMT1 through a mechanism yet to be fully elucidated. INTRO +108 113 DNMT1 protein Therefore, UHRF1 may engage in a sophisticated regulation for its chromatin localization and recruitment of DNMT1 through a mechanism yet to be fully elucidated. INTRO +50 52 H3 protein_type Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by UHRF1. INTRO +52 57 K9me3 ptm Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by UHRF1. INTRO +62 68 hm-DNA chemical Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by UHRF1. INTRO +84 89 UHRF1 protein Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by UHRF1. INTRO +0 6 Hm-DNA chemical Hm-DNA facilitates histone H3K9me3 recognition by UHRF1 RESULTS +19 26 histone protein_type Hm-DNA facilitates histone H3K9me3 recognition by UHRF1 RESULTS +27 29 H3 protein_type Hm-DNA facilitates histone H3K9me3 recognition by UHRF1 RESULTS +29 34 K9me3 ptm Hm-DNA facilitates histone H3K9me3 recognition by UHRF1 RESULTS +50 55 UHRF1 protein Hm-DNA facilitates histone H3K9me3 recognition by UHRF1 RESULTS +19 24 UHRF1 protein To investigate how UHRF1 coordinates the recognition of H3K9me3 and hm-DNA, we purified recombinant UHRF1 (truncations and mutations) proteins from bacteria. RESULTS +56 58 H3 protein_type To investigate how UHRF1 coordinates the recognition of H3K9me3 and hm-DNA, we purified recombinant UHRF1 (truncations and mutations) proteins from bacteria. RESULTS +58 63 K9me3 ptm To investigate how UHRF1 coordinates the recognition of H3K9me3 and hm-DNA, we purified recombinant UHRF1 (truncations and mutations) proteins from bacteria. RESULTS +68 74 hm-DNA chemical To investigate how UHRF1 coordinates the recognition of H3K9me3 and hm-DNA, we purified recombinant UHRF1 (truncations and mutations) proteins from bacteria. RESULTS +100 105 UHRF1 protein To investigate how UHRF1 coordinates the recognition of H3K9me3 and hm-DNA, we purified recombinant UHRF1 (truncations and mutations) proteins from bacteria. RESULTS +22 46 in vitro pull-down assay experimental_method We first performed an in vitro pull-down assay using biotinylated histone H3 peptides and hm-DNA (Supplementary Table 1). RESULTS +53 65 biotinylated protein_state We first performed an in vitro pull-down assay using biotinylated histone H3 peptides and hm-DNA (Supplementary Table 1). RESULTS +66 73 histone protein_type We first performed an in vitro pull-down assay using biotinylated histone H3 peptides and hm-DNA (Supplementary Table 1). RESULTS +74 76 H3 protein_type We first performed an in vitro pull-down assay using biotinylated histone H3 peptides and hm-DNA (Supplementary Table 1). RESULTS +90 96 hm-DNA chemical We first performed an in vitro pull-down assay using biotinylated histone H3 peptides and hm-DNA (Supplementary Table 1). RESULTS +21 27 hm-DNA chemical As shown in Fig. 1b, hm-DNA largely enhanced the interaction between full-length UHRF1 and unmethylated histone H3 (H3K9me0) or H3K9me3 peptide. RESULTS +69 80 full-length protein_state As shown in Fig. 1b, hm-DNA largely enhanced the interaction between full-length UHRF1 and unmethylated histone H3 (H3K9me0) or H3K9me3 peptide. RESULTS +81 86 UHRF1 protein As shown in Fig. 1b, hm-DNA largely enhanced the interaction between full-length UHRF1 and unmethylated histone H3 (H3K9me0) or H3K9me3 peptide. RESULTS +91 103 unmethylated protein_state As shown in Fig. 1b, hm-DNA largely enhanced the interaction between full-length UHRF1 and unmethylated histone H3 (H3K9me0) or H3K9me3 peptide. RESULTS +104 111 histone protein_type As shown in Fig. 1b, hm-DNA largely enhanced the interaction between full-length UHRF1 and unmethylated histone H3 (H3K9me0) or H3K9me3 peptide. RESULTS +112 114 H3 protein_type As shown in Fig. 1b, hm-DNA largely enhanced the interaction between full-length UHRF1 and unmethylated histone H3 (H3K9me0) or H3K9me3 peptide. RESULTS +116 118 H3 protein_type As shown in Fig. 1b, hm-DNA largely enhanced the interaction between full-length UHRF1 and unmethylated histone H3 (H3K9me0) or H3K9me3 peptide. RESULTS +118 123 K9me0 ptm As shown in Fig. 1b, hm-DNA largely enhanced the interaction between full-length UHRF1 and unmethylated histone H3 (H3K9me0) or H3K9me3 peptide. RESULTS +128 130 H3 protein_type As shown in Fig. 1b, hm-DNA largely enhanced the interaction between full-length UHRF1 and unmethylated histone H3 (H3K9me0) or H3K9me3 peptide. RESULTS +130 135 K9me3 ptm As shown in Fig. 1b, hm-DNA largely enhanced the interaction between full-length UHRF1 and unmethylated histone H3 (H3K9me0) or H3K9me3 peptide. RESULTS +14 20 hm-DNA chemical Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a). RESULTS +22 28 um-DNA chemical Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a). RESULTS +30 42 unmethylated protein_state Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a). RESULTS +43 46 DNA chemical Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a). RESULTS +51 57 fm-DNA chemical Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a). RESULTS +59 75 fully methylated protein_state Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a). RESULTS +76 79 DNA chemical Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a). RESULTS +144 149 UHRF1 protein Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a). RESULTS +154 161 histone protein_type Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a). RESULTS +219 224 UHRF1 protein Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a). RESULTS +233 239 hm-DNA chemical Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a). RESULTS +13 20 histone protein_type In contrast, histone peptides showed no enhancement on the interaction between hm-DNA and UHRF1 (Fig. 1c). RESULTS +79 85 hm-DNA chemical In contrast, histone peptides showed no enhancement on the interaction between hm-DNA and UHRF1 (Fig. 1c). RESULTS +90 95 UHRF1 protein In contrast, histone peptides showed no enhancement on the interaction between hm-DNA and UHRF1 (Fig. 1c). RESULTS +27 33 hm-DNA chemical These results suggest that hm-DNA facilitates histone recognition by UHRF1. RESULTS +46 53 histone protein_type These results suggest that hm-DNA facilitates histone recognition by UHRF1. RESULTS +69 74 UHRF1 protein These results suggest that hm-DNA facilitates histone recognition by UHRF1. RESULTS +35 38 PHD structure_element Our previous studies show that the PHD recognizes H3K9me0 and the TTD and the PHD together (TTD–PHD) coordinately recognize H3K9me3 (refs.). RESULTS +50 52 H3 protein_type Our previous studies show that the PHD recognizes H3K9me0 and the TTD and the PHD together (TTD–PHD) coordinately recognize H3K9me3 (refs.). RESULTS +52 57 K9me0 ptm Our previous studies show that the PHD recognizes H3K9me0 and the TTD and the PHD together (TTD–PHD) coordinately recognize H3K9me3 (refs.). RESULTS +66 69 TTD structure_element Our previous studies show that the PHD recognizes H3K9me0 and the TTD and the PHD together (TTD–PHD) coordinately recognize H3K9me3 (refs.). RESULTS +78 81 PHD structure_element Our previous studies show that the PHD recognizes H3K9me0 and the TTD and the PHD together (TTD–PHD) coordinately recognize H3K9me3 (refs.). RESULTS +92 99 TTD–PHD structure_element Our previous studies show that the PHD recognizes H3K9me0 and the TTD and the PHD together (TTD–PHD) coordinately recognize H3K9me3 (refs.). RESULTS +124 126 H3 protein_type Our previous studies show that the PHD recognizes H3K9me0 and the TTD and the PHD together (TTD–PHD) coordinately recognize H3K9me3 (refs.). RESULTS +126 131 K9me3 ptm Our previous studies show that the PHD recognizes H3K9me0 and the TTD and the PHD together (TTD–PHD) coordinately recognize H3K9me3 (refs.). RESULTS +20 28 isolated protein_state We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +29 36 TTD–PHD structure_element We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +67 83 binding affinity evidence We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +87 89 H3 protein_type We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +89 94 K9me3 ptm We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +108 119 full-length protein_state We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +120 125 UHRF1 protein We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +180 183 PHD structure_element We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +214 230 binding affinity evidence We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +234 236 H3 protein_type We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +236 241 K9me0 ptm We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +255 266 full-length protein_state We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +267 272 UHRF1 protein We noticed that the isolated TTD–PHD showed much higher (∼31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (∼34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e). RESULTS +4 27 gel filtration analysis experimental_method The gel filtration analysis showed that UHRF1 is a monomer in solution (Supplementary Fig. 1b), indicating that the intramolecular (not intermolecular) interaction of UHRF1 regulates histone recognition. RESULTS +40 45 UHRF1 protein The gel filtration analysis showed that UHRF1 is a monomer in solution (Supplementary Fig. 1b), indicating that the intramolecular (not intermolecular) interaction of UHRF1 regulates histone recognition. RESULTS +51 58 monomer oligomeric_state The gel filtration analysis showed that UHRF1 is a monomer in solution (Supplementary Fig. 1b), indicating that the intramolecular (not intermolecular) interaction of UHRF1 regulates histone recognition. RESULTS +167 172 UHRF1 protein The gel filtration analysis showed that UHRF1 is a monomer in solution (Supplementary Fig. 1b), indicating that the intramolecular (not intermolecular) interaction of UHRF1 regulates histone recognition. RESULTS +183 190 histone protein_type The gel filtration analysis showed that UHRF1 is a monomer in solution (Supplementary Fig. 1b), indicating that the intramolecular (not intermolecular) interaction of UHRF1 regulates histone recognition. RESULTS +27 32 UHRF1 protein These results suggest that UHRF1 adopts an unfavourable conformation for histone H3 tails recognition, in which TTD–PHD might be blocked by other regions of UHRF1, and hm-DNA impairs this intramolecular interaction to facilitate its recognition of histone H3 tails. RESULTS +73 80 histone protein_type These results suggest that UHRF1 adopts an unfavourable conformation for histone H3 tails recognition, in which TTD–PHD might be blocked by other regions of UHRF1, and hm-DNA impairs this intramolecular interaction to facilitate its recognition of histone H3 tails. RESULTS +81 83 H3 protein_type These results suggest that UHRF1 adopts an unfavourable conformation for histone H3 tails recognition, in which TTD–PHD might be blocked by other regions of UHRF1, and hm-DNA impairs this intramolecular interaction to facilitate its recognition of histone H3 tails. RESULTS +112 119 TTD–PHD structure_element These results suggest that UHRF1 adopts an unfavourable conformation for histone H3 tails recognition, in which TTD–PHD might be blocked by other regions of UHRF1, and hm-DNA impairs this intramolecular interaction to facilitate its recognition of histone H3 tails. RESULTS +157 162 UHRF1 protein These results suggest that UHRF1 adopts an unfavourable conformation for histone H3 tails recognition, in which TTD–PHD might be blocked by other regions of UHRF1, and hm-DNA impairs this intramolecular interaction to facilitate its recognition of histone H3 tails. RESULTS +168 174 hm-DNA chemical These results suggest that UHRF1 adopts an unfavourable conformation for histone H3 tails recognition, in which TTD–PHD might be blocked by other regions of UHRF1, and hm-DNA impairs this intramolecular interaction to facilitate its recognition of histone H3 tails. RESULTS +248 255 histone protein_type These results suggest that UHRF1 adopts an unfavourable conformation for histone H3 tails recognition, in which TTD–PHD might be blocked by other regions of UHRF1, and hm-DNA impairs this intramolecular interaction to facilitate its recognition of histone H3 tails. RESULTS +256 258 H3 protein_type These results suggest that UHRF1 adopts an unfavourable conformation for histone H3 tails recognition, in which TTD–PHD might be blocked by other regions of UHRF1, and hm-DNA impairs this intramolecular interaction to facilitate its recognition of histone H3 tails. RESULTS +34 39 UHRF1 protein Intramolecular interaction within UHRF1 RESULTS +39 86 glutathione S-transferase (GST) pull-down assay experimental_method To test above hypothesis, we performed glutathione S-transferase (GST) pull-down assay using various truncations of UHRF1. RESULTS +101 112 truncations experimental_method To test above hypothesis, we performed glutathione S-transferase (GST) pull-down assay using various truncations of UHRF1. RESULTS +116 121 UHRF1 protein To test above hypothesis, we performed glutathione S-transferase (GST) pull-down assay using various truncations of UHRF1. RESULTS +19 22 TTD structure_element Interestingly, the TTD directly bound to SRA-Spacer but not the SRA, suggesting that the Spacer (residues 587–674) is important for the intramolecular interaction (Fig. 2a). RESULTS +32 40 bound to protein_state Interestingly, the TTD directly bound to SRA-Spacer but not the SRA, suggesting that the Spacer (residues 587–674) is important for the intramolecular interaction (Fig. 2a). RESULTS +41 51 SRA-Spacer structure_element Interestingly, the TTD directly bound to SRA-Spacer but not the SRA, suggesting that the Spacer (residues 587–674) is important for the intramolecular interaction (Fig. 2a). RESULTS +64 67 SRA structure_element Interestingly, the TTD directly bound to SRA-Spacer but not the SRA, suggesting that the Spacer (residues 587–674) is important for the intramolecular interaction (Fig. 2a). RESULTS +89 95 Spacer structure_element Interestingly, the TTD directly bound to SRA-Spacer but not the SRA, suggesting that the Spacer (residues 587–674) is important for the intramolecular interaction (Fig. 2a). RESULTS +106 113 587–674 residue_range Interestingly, the TTD directly bound to SRA-Spacer but not the SRA, suggesting that the Spacer (residues 587–674) is important for the intramolecular interaction (Fig. 2a). RESULTS +4 36 isothermal titration calorimetry experimental_method The isothermal titration calorimetry (ITC) measurements show that the TTD bound to the Spacer (but not the SRA) in a 1:1 stoichiometry with a binding affinity (KD) of 1.59 μM (Fig. 2b). RESULTS +38 41 ITC experimental_method The isothermal titration calorimetry (ITC) measurements show that the TTD bound to the Spacer (but not the SRA) in a 1:1 stoichiometry with a binding affinity (KD) of 1.59 μM (Fig. 2b). RESULTS +70 73 TTD structure_element The isothermal titration calorimetry (ITC) measurements show that the TTD bound to the Spacer (but not the SRA) in a 1:1 stoichiometry with a binding affinity (KD) of 1.59 μM (Fig. 2b). RESULTS +74 82 bound to protein_state The isothermal titration calorimetry (ITC) measurements show that the TTD bound to the Spacer (but not the SRA) in a 1:1 stoichiometry with a binding affinity (KD) of 1.59 μM (Fig. 2b). RESULTS +87 93 Spacer structure_element The isothermal titration calorimetry (ITC) measurements show that the TTD bound to the Spacer (but not the SRA) in a 1:1 stoichiometry with a binding affinity (KD) of 1.59 μM (Fig. 2b). RESULTS +107 110 SRA structure_element The isothermal titration calorimetry (ITC) measurements show that the TTD bound to the Spacer (but not the SRA) in a 1:1 stoichiometry with a binding affinity (KD) of 1.59 μM (Fig. 2b). RESULTS +142 158 binding affinity evidence The isothermal titration calorimetry (ITC) measurements show that the TTD bound to the Spacer (but not the SRA) in a 1:1 stoichiometry with a binding affinity (KD) of 1.59 μM (Fig. 2b). RESULTS +160 162 KD evidence The isothermal titration calorimetry (ITC) measurements show that the TTD bound to the Spacer (but not the SRA) in a 1:1 stoichiometry with a binding affinity (KD) of 1.59 μM (Fig. 2b). RESULTS +4 15 presence of protein_state The presence of the Spacer markedly impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +20 26 Spacer structure_element The presence of the Spacer markedly impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +69 76 TTD–PHD structure_element The presence of the Spacer markedly impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +81 83 H3 protein_type The presence of the Spacer markedly impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +83 88 K9me3 ptm The presence of the Spacer markedly impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +30 36 Spacer structure_element The results indicate that the Spacer directly binds to the TTD and inhibits its interaction with H3K9me3. RESULTS +46 54 binds to protein_state The results indicate that the Spacer directly binds to the TTD and inhibits its interaction with H3K9me3. RESULTS +59 62 TTD structure_element The results indicate that the Spacer directly binds to the TTD and inhibits its interaction with H3K9me3. RESULTS +97 99 H3 protein_type The results indicate that the Spacer directly binds to the TTD and inhibits its interaction with H3K9me3. RESULTS +99 104 K9me3 ptm The results indicate that the Spacer directly binds to the TTD and inhibits its interaction with H3K9me3. RESULTS +4 23 GST pull-down assay experimental_method The GST pull-down assay also shows that the PHD bound to the SRA, which was further confirmed by the ITC measurements (KD=26.7 μM; Fig. 2a,d). RESULTS +44 47 PHD structure_element The GST pull-down assay also shows that the PHD bound to the SRA, which was further confirmed by the ITC measurements (KD=26.7 μM; Fig. 2a,d). RESULTS +48 56 bound to protein_state The GST pull-down assay also shows that the PHD bound to the SRA, which was further confirmed by the ITC measurements (KD=26.7 μM; Fig. 2a,d). RESULTS +61 64 SRA structure_element The GST pull-down assay also shows that the PHD bound to the SRA, which was further confirmed by the ITC measurements (KD=26.7 μM; Fig. 2a,d). RESULTS +101 104 ITC experimental_method The GST pull-down assay also shows that the PHD bound to the SRA, which was further confirmed by the ITC measurements (KD=26.7 μM; Fig. 2a,d). RESULTS +119 121 KD evidence The GST pull-down assay also shows that the PHD bound to the SRA, which was further confirmed by the ITC measurements (KD=26.7 μM; Fig. 2a,d). RESULTS +18 21 PHD structure_element Compared with the PHD alone, PHD-SRA showed decreased binding affinity to H3K9me0 peptide by a factor of eight (Fig. 2e). RESULTS +22 27 alone protein_state Compared with the PHD alone, PHD-SRA showed decreased binding affinity to H3K9me0 peptide by a factor of eight (Fig. 2e). RESULTS +29 36 PHD-SRA structure_element Compared with the PHD alone, PHD-SRA showed decreased binding affinity to H3K9me0 peptide by a factor of eight (Fig. 2e). RESULTS +54 70 binding affinity evidence Compared with the PHD alone, PHD-SRA showed decreased binding affinity to H3K9me0 peptide by a factor of eight (Fig. 2e). RESULTS +74 76 H3 protein_type Compared with the PHD alone, PHD-SRA showed decreased binding affinity to H3K9me0 peptide by a factor of eight (Fig. 2e). RESULTS +76 81 K9me0 ptm Compared with the PHD alone, PHD-SRA showed decreased binding affinity to H3K9me0 peptide by a factor of eight (Fig. 2e). RESULTS +0 14 Pre-incubation experimental_method Pre-incubation of the SRA also modestly impaired PHD–H3K9me0 interaction. RESULTS +22 25 SRA structure_element Pre-incubation of the SRA also modestly impaired PHD–H3K9me0 interaction. RESULTS +49 52 PHD structure_element Pre-incubation of the SRA also modestly impaired PHD–H3K9me0 interaction. RESULTS +53 55 H3 protein_type Pre-incubation of the SRA also modestly impaired PHD–H3K9me0 interaction. RESULTS +55 60 K9me0 ptm Pre-incubation of the SRA also modestly impaired PHD–H3K9me0 interaction. RESULTS +32 35 SRA structure_element These results indicate that the SRA directly binds to the PHD and inhibits its binding affinity to H3K9me0. RESULTS +45 53 binds to protein_state These results indicate that the SRA directly binds to the PHD and inhibits its binding affinity to H3K9me0. RESULTS +58 61 PHD structure_element These results indicate that the SRA directly binds to the PHD and inhibits its binding affinity to H3K9me0. RESULTS +79 95 binding affinity evidence These results indicate that the SRA directly binds to the PHD and inhibits its binding affinity to H3K9me0. RESULTS +99 101 H3 protein_type These results indicate that the SRA directly binds to the PHD and inhibits its binding affinity to H3K9me0. RESULTS +101 106 K9me0 ptm These results indicate that the SRA directly binds to the PHD and inhibits its binding affinity to H3K9me0. RESULTS +16 21 UHRF1 protein Taken together, UHRF1 seems to adopt a closed form through intramolecular interactions (TTD–Spacer and PHD-SRA), which inhibit histone H3 tail recognition by UHRF1. RESULTS +39 45 closed protein_state Taken together, UHRF1 seems to adopt a closed form through intramolecular interactions (TTD–Spacer and PHD-SRA), which inhibit histone H3 tail recognition by UHRF1. RESULTS +88 98 TTD–Spacer structure_element Taken together, UHRF1 seems to adopt a closed form through intramolecular interactions (TTD–Spacer and PHD-SRA), which inhibit histone H3 tail recognition by UHRF1. RESULTS +103 110 PHD-SRA structure_element Taken together, UHRF1 seems to adopt a closed form through intramolecular interactions (TTD–Spacer and PHD-SRA), which inhibit histone H3 tail recognition by UHRF1. RESULTS +127 134 histone protein_type Taken together, UHRF1 seems to adopt a closed form through intramolecular interactions (TTD–Spacer and PHD-SRA), which inhibit histone H3 tail recognition by UHRF1. RESULTS +135 137 H3 protein_type Taken together, UHRF1 seems to adopt a closed form through intramolecular interactions (TTD–Spacer and PHD-SRA), which inhibit histone H3 tail recognition by UHRF1. RESULTS +158 163 UHRF1 protein Taken together, UHRF1 seems to adopt a closed form through intramolecular interactions (TTD–Spacer and PHD-SRA), which inhibit histone H3 tail recognition by UHRF1. RESULTS +8 17 structure evidence Overall structure of TTD–Spacer RESULTS +21 31 TTD–Spacer structure_element Overall structure of TTD–Spacer RESULTS +53 58 UHRF1 protein To investigate the intramolecular interaction within UHRF1, we first mapped the minimal regions within the Spacer for the interaction with the TTD (Supplementary Fig. 2a). RESULTS +107 113 Spacer structure_element To investigate the intramolecular interaction within UHRF1, we first mapped the minimal regions within the Spacer for the interaction with the TTD (Supplementary Fig. 2a). RESULTS +143 146 TTD structure_element To investigate the intramolecular interaction within UHRF1, we first mapped the minimal regions within the Spacer for the interaction with the TTD (Supplementary Fig. 2a). RESULTS +9 18 deletions experimental_method Internal deletions of the Spacer, including SpacerΔ660–664, SpacerΔ665–669, SpacerΔ670–674 and Spacer642–674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587–641 showed no detectable interaction. RESULTS +26 32 Spacer structure_element Internal deletions of the Spacer, including SpacerΔ660–664, SpacerΔ665–669, SpacerΔ670–674 and Spacer642–674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587–641 showed no detectable interaction. RESULTS +44 58 SpacerΔ660–664 mutant Internal deletions of the Spacer, including SpacerΔ660–664, SpacerΔ665–669, SpacerΔ670–674 and Spacer642–674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587–641 showed no detectable interaction. RESULTS +60 74 SpacerΔ665–669 mutant Internal deletions of the Spacer, including SpacerΔ660–664, SpacerΔ665–669, SpacerΔ670–674 and Spacer642–674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587–641 showed no detectable interaction. RESULTS +76 90 SpacerΔ670–674 mutant Internal deletions of the Spacer, including SpacerΔ660–664, SpacerΔ665–669, SpacerΔ670–674 and Spacer642–674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587–641 showed no detectable interaction. RESULTS +95 108 Spacer642–674 mutant Internal deletions of the Spacer, including SpacerΔ660–664, SpacerΔ665–669, SpacerΔ670–674 and Spacer642–674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587–641 showed no detectable interaction. RESULTS +110 118 bound to protein_state Internal deletions of the Spacer, including SpacerΔ660–664, SpacerΔ665–669, SpacerΔ670–674 and Spacer642–674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587–641 showed no detectable interaction. RESULTS +123 126 TTD structure_element Internal deletions of the Spacer, including SpacerΔ660–664, SpacerΔ665–669, SpacerΔ670–674 and Spacer642–674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587–641 showed no detectable interaction. RESULTS +143 161 binding affinities evidence Internal deletions of the Spacer, including SpacerΔ660–664, SpacerΔ665–669, SpacerΔ670–674 and Spacer642–674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587–641 showed no detectable interaction. RESULTS +177 183 Spacer structure_element Internal deletions of the Spacer, including SpacerΔ660–664, SpacerΔ665–669, SpacerΔ670–674 and Spacer642–674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587–641 showed no detectable interaction. RESULTS +193 206 Spacer587–641 mutant Internal deletions of the Spacer, including SpacerΔ660–664, SpacerΔ665–669, SpacerΔ670–674 and Spacer642–674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587–641 showed no detectable interaction. RESULTS +0 14 SpacerΔ642–651 mutant SpacerΔ642–651, SpacerΔ650–654 and SpacerΔ655–659 also decreased binding affinities, indicating that residues 642–674 are important for TTD–Spacer interaction. RESULTS +16 30 SpacerΔ650–654 mutant SpacerΔ642–651, SpacerΔ650–654 and SpacerΔ655–659 also decreased binding affinities, indicating that residues 642–674 are important for TTD–Spacer interaction. RESULTS +35 49 SpacerΔ655–659 mutant SpacerΔ642–651, SpacerΔ650–654 and SpacerΔ655–659 also decreased binding affinities, indicating that residues 642–674 are important for TTD–Spacer interaction. RESULTS +65 83 binding affinities evidence SpacerΔ642–651, SpacerΔ650–654 and SpacerΔ655–659 also decreased binding affinities, indicating that residues 642–674 are important for TTD–Spacer interaction. RESULTS +110 117 642–674 residue_range SpacerΔ642–651, SpacerΔ650–654 and SpacerΔ655–659 also decreased binding affinities, indicating that residues 642–674 are important for TTD–Spacer interaction. RESULTS +136 146 TTD–Spacer structure_element SpacerΔ642–651, SpacerΔ650–654 and SpacerΔ655–659 also decreased binding affinities, indicating that residues 642–674 are important for TTD–Spacer interaction. RESULTS +23 41 solution structure evidence We next determined the solution structure of the TTD (residues 134–285) bound to Spacer627–674 by conventional NMR techniques (Supplementary Table 3 and Supplementary Fig. 3a,b). RESULTS +49 52 TTD structure_element We next determined the solution structure of the TTD (residues 134–285) bound to Spacer627–674 by conventional NMR techniques (Supplementary Table 3 and Supplementary Fig. 3a,b). RESULTS +63 70 134–285 residue_range We next determined the solution structure of the TTD (residues 134–285) bound to Spacer627–674 by conventional NMR techniques (Supplementary Table 3 and Supplementary Fig. 3a,b). RESULTS +72 80 bound to protein_state We next determined the solution structure of the TTD (residues 134–285) bound to Spacer627–674 by conventional NMR techniques (Supplementary Table 3 and Supplementary Fig. 3a,b). RESULTS +81 94 Spacer627–674 residue_range We next determined the solution structure of the TTD (residues 134–285) bound to Spacer627–674 by conventional NMR techniques (Supplementary Table 3 and Supplementary Fig. 3a,b). RESULTS +111 114 NMR experimental_method We next determined the solution structure of the TTD (residues 134–285) bound to Spacer627–674 by conventional NMR techniques (Supplementary Table 3 and Supplementary Fig. 3a,b). RESULTS +7 24 complex structure evidence In the complex structure, each Tudor domain adopts a ‘Royal' fold containing a characteristic five-stranded β-sheet and the two Tudor domains tightly pack against each other with a buried area of 573 Å2 (Fig. 3a). RESULTS +31 43 Tudor domain structure_element In the complex structure, each Tudor domain adopts a ‘Royal' fold containing a characteristic five-stranded β-sheet and the two Tudor domains tightly pack against each other with a buried area of 573 Å2 (Fig. 3a). RESULTS +53 65 ‘Royal' fold structure_element In the complex structure, each Tudor domain adopts a ‘Royal' fold containing a characteristic five-stranded β-sheet and the two Tudor domains tightly pack against each other with a buried area of 573 Å2 (Fig. 3a). RESULTS +94 115 five-stranded β-sheet structure_element In the complex structure, each Tudor domain adopts a ‘Royal' fold containing a characteristic five-stranded β-sheet and the two Tudor domains tightly pack against each other with a buried area of 573 Å2 (Fig. 3a). RESULTS +128 141 Tudor domains structure_element In the complex structure, each Tudor domain adopts a ‘Royal' fold containing a characteristic five-stranded β-sheet and the two Tudor domains tightly pack against each other with a buried area of 573 Å2 (Fig. 3a). RESULTS +4 7 TTD structure_element The TTD adopts similar fold to that in TTD–PHD–H3K9me3 complex structure (PDB: 4GY5) with a root-mean-square deviation of 1.09 Å for 128 Cα atoms, indicating that the Spacer does not result in obvious conformational change of the TTD (Fig. 3b). RESULTS +39 54 TTD–PHD–H3K9me3 complex_assembly The TTD adopts similar fold to that in TTD–PHD–H3K9me3 complex structure (PDB: 4GY5) with a root-mean-square deviation of 1.09 Å for 128 Cα atoms, indicating that the Spacer does not result in obvious conformational change of the TTD (Fig. 3b). RESULTS +63 72 structure evidence The TTD adopts similar fold to that in TTD–PHD–H3K9me3 complex structure (PDB: 4GY5) with a root-mean-square deviation of 1.09 Å for 128 Cα atoms, indicating that the Spacer does not result in obvious conformational change of the TTD (Fig. 3b). RESULTS +92 118 root-mean-square deviation evidence The TTD adopts similar fold to that in TTD–PHD–H3K9me3 complex structure (PDB: 4GY5) with a root-mean-square deviation of 1.09 Å for 128 Cα atoms, indicating that the Spacer does not result in obvious conformational change of the TTD (Fig. 3b). RESULTS +167 173 Spacer structure_element The TTD adopts similar fold to that in TTD–PHD–H3K9me3 complex structure (PDB: 4GY5) with a root-mean-square deviation of 1.09 Å for 128 Cα atoms, indicating that the Spacer does not result in obvious conformational change of the TTD (Fig. 3b). RESULTS +230 233 TTD structure_element The TTD adopts similar fold to that in TTD–PHD–H3K9me3 complex structure (PDB: 4GY5) with a root-mean-square deviation of 1.09 Å for 128 Cα atoms, indicating that the Spacer does not result in obvious conformational change of the TTD (Fig. 3b). RESULTS +4 10 Spacer structure_element The Spacer (residues 643–655 were built in the model) adopts an extended conformation and binds to an acidic groove on the TTD (Fig. 3c). RESULTS +21 28 643–655 residue_range The Spacer (residues 643–655 were built in the model) adopts an extended conformation and binds to an acidic groove on the TTD (Fig. 3c). RESULTS +64 85 extended conformation protein_state The Spacer (residues 643–655 were built in the model) adopts an extended conformation and binds to an acidic groove on the TTD (Fig. 3c). RESULTS +90 98 binds to protein_state The Spacer (residues 643–655 were built in the model) adopts an extended conformation and binds to an acidic groove on the TTD (Fig. 3c). RESULTS +102 115 acidic groove site The Spacer (residues 643–655 were built in the model) adopts an extended conformation and binds to an acidic groove on the TTD (Fig. 3c). RESULTS +123 126 TTD structure_element The Spacer (residues 643–655 were built in the model) adopts an extended conformation and binds to an acidic groove on the TTD (Fig. 3c). RESULTS +4 14 TTD–Spacer structure_element The TTD–Spacer interaction is mediated by a number of hydrogen bonds (Fig. 3d). RESULTS +54 68 hydrogen bonds bond_interaction The TTD–Spacer interaction is mediated by a number of hydrogen bonds (Fig. 3d). RESULTS +26 30 K648 residue_name_number The side chain of residue K648 forms hydrogen bonds with the carbonyl oxygen atom of D189 and side chain of D190 of the TTD. RESULTS +37 51 hydrogen bonds bond_interaction The side chain of residue K648 forms hydrogen bonds with the carbonyl oxygen atom of D189 and side chain of D190 of the TTD. RESULTS +85 89 D189 residue_name_number The side chain of residue K648 forms hydrogen bonds with the carbonyl oxygen atom of D189 and side chain of D190 of the TTD. RESULTS +108 112 D190 residue_name_number The side chain of residue K648 forms hydrogen bonds with the carbonyl oxygen atom of D189 and side chain of D190 of the TTD. RESULTS +120 123 TTD structure_element The side chain of residue K648 forms hydrogen bonds with the carbonyl oxygen atom of D189 and side chain of D190 of the TTD. RESULTS +26 30 R649 residue_name_number The side chain of residue R649 packs against an acidic surface mainly formed by residues D142 and E153. RESULTS +31 44 packs against bond_interaction The side chain of residue R649 packs against an acidic surface mainly formed by residues D142 and E153. RESULTS +89 93 D142 residue_name_number The side chain of residue R649 packs against an acidic surface mainly formed by residues D142 and E153. RESULTS +98 102 E153 residue_name_number The side chain of residue R649 packs against an acidic surface mainly formed by residues D142 and E153. RESULTS +8 12 S651 residue_name_number Residue S651 forms hydrogen bonds with the main chain of residues G236 and W238. RESULTS +19 33 hydrogen bonds bond_interaction Residue S651 forms hydrogen bonds with the main chain of residues G236 and W238. RESULTS +66 70 G236 residue_name_number Residue S651 forms hydrogen bonds with the main chain of residues G236 and W238. RESULTS +75 79 W238 residue_name_number Residue S651 forms hydrogen bonds with the main chain of residues G236 and W238. RESULTS +40 54 hydrogen bonds bond_interaction The interaction is further supported by hydrogen bonds formed between residues K650, A652, G653 and G654 of the Spacer and residues N228, G236 and W238 of the TTD, respectively. RESULTS +79 83 K650 residue_name_number The interaction is further supported by hydrogen bonds formed between residues K650, A652, G653 and G654 of the Spacer and residues N228, G236 and W238 of the TTD, respectively. RESULTS +85 89 A652 residue_name_number The interaction is further supported by hydrogen bonds formed between residues K650, A652, G653 and G654 of the Spacer and residues N228, G236 and W238 of the TTD, respectively. RESULTS +91 95 G653 residue_name_number The interaction is further supported by hydrogen bonds formed between residues K650, A652, G653 and G654 of the Spacer and residues N228, G236 and W238 of the TTD, respectively. RESULTS +100 104 G654 residue_name_number The interaction is further supported by hydrogen bonds formed between residues K650, A652, G653 and G654 of the Spacer and residues N228, G236 and W238 of the TTD, respectively. RESULTS +112 118 Spacer structure_element The interaction is further supported by hydrogen bonds formed between residues K650, A652, G653 and G654 of the Spacer and residues N228, G236 and W238 of the TTD, respectively. RESULTS +132 136 N228 residue_name_number The interaction is further supported by hydrogen bonds formed between residues K650, A652, G653 and G654 of the Spacer and residues N228, G236 and W238 of the TTD, respectively. RESULTS +138 142 G236 residue_name_number The interaction is further supported by hydrogen bonds formed between residues K650, A652, G653 and G654 of the Spacer and residues N228, G236 and W238 of the TTD, respectively. RESULTS +147 151 W238 residue_name_number The interaction is further supported by hydrogen bonds formed between residues K650, A652, G653 and G654 of the Spacer and residues N228, G236 and W238 of the TTD, respectively. RESULTS +159 162 TTD structure_element The interaction is further supported by hydrogen bonds formed between residues K650, A652, G653 and G654 of the Spacer and residues N228, G236 and W238 of the TTD, respectively. RESULTS +20 39 structural analyses experimental_method In support of above structural analyses, mutation D142A/E153A of the TTD abolished its interaction with the Spacer (Fig. 3e). RESULTS +41 49 mutation experimental_method In support of above structural analyses, mutation D142A/E153A of the TTD abolished its interaction with the Spacer (Fig. 3e). RESULTS +50 55 D142A mutant In support of above structural analyses, mutation D142A/E153A of the TTD abolished its interaction with the Spacer (Fig. 3e). RESULTS +56 61 E153A mutant In support of above structural analyses, mutation D142A/E153A of the TTD abolished its interaction with the Spacer (Fig. 3e). RESULTS +69 72 TTD structure_element In support of above structural analyses, mutation D142A/E153A of the TTD abolished its interaction with the Spacer (Fig. 3e). RESULTS +108 114 Spacer structure_element In support of above structural analyses, mutation D142A/E153A of the TTD abolished its interaction with the Spacer (Fig. 3e). RESULTS +0 9 Mutations experimental_method Mutations K648D and S651D of the Spacer decreased their binding affinities to the TTD, and mutation R649A of the Spacer showed more significant decrease (∼13-fold) in the binding affinity (Fig. 3f). RESULTS +10 15 K648D mutant Mutations K648D and S651D of the Spacer decreased their binding affinities to the TTD, and mutation R649A of the Spacer showed more significant decrease (∼13-fold) in the binding affinity (Fig. 3f). RESULTS +20 25 S651D mutant Mutations K648D and S651D of the Spacer decreased their binding affinities to the TTD, and mutation R649A of the Spacer showed more significant decrease (∼13-fold) in the binding affinity (Fig. 3f). RESULTS +33 39 Spacer structure_element Mutations K648D and S651D of the Spacer decreased their binding affinities to the TTD, and mutation R649A of the Spacer showed more significant decrease (∼13-fold) in the binding affinity (Fig. 3f). RESULTS +56 74 binding affinities evidence Mutations K648D and S651D of the Spacer decreased their binding affinities to the TTD, and mutation R649A of the Spacer showed more significant decrease (∼13-fold) in the binding affinity (Fig. 3f). RESULTS +82 85 TTD structure_element Mutations K648D and S651D of the Spacer decreased their binding affinities to the TTD, and mutation R649A of the Spacer showed more significant decrease (∼13-fold) in the binding affinity (Fig. 3f). RESULTS +91 99 mutation experimental_method Mutations K648D and S651D of the Spacer decreased their binding affinities to the TTD, and mutation R649A of the Spacer showed more significant decrease (∼13-fold) in the binding affinity (Fig. 3f). RESULTS +100 105 R649A mutant Mutations K648D and S651D of the Spacer decreased their binding affinities to the TTD, and mutation R649A of the Spacer showed more significant decrease (∼13-fold) in the binding affinity (Fig. 3f). RESULTS +113 119 Spacer structure_element Mutations K648D and S651D of the Spacer decreased their binding affinities to the TTD, and mutation R649A of the Spacer showed more significant decrease (∼13-fold) in the binding affinity (Fig. 3f). RESULTS +171 187 binding affinity evidence Mutations K648D and S651D of the Spacer decreased their binding affinities to the TTD, and mutation R649A of the Spacer showed more significant decrease (∼13-fold) in the binding affinity (Fig. 3f). RESULTS +21 30 mutations experimental_method As negative control, mutations S639D and S666D of the Spacer showed little effect on the interaction. RESULTS +31 36 S639D mutant As negative control, mutations S639D and S666D of the Spacer showed little effect on the interaction. RESULTS +41 46 S666D mutant As negative control, mutations S639D and S666D of the Spacer showed little effect on the interaction. RESULTS +54 60 Spacer structure_element As negative control, mutations S639D and S666D of the Spacer showed little effect on the interaction. RESULTS +15 30 phosphorylation ptm Interestingly, phosphorylation at residue S651 of UHRF1 was observed in previous mass-spectrometry analyses. RESULTS +42 46 S651 residue_name_number Interestingly, phosphorylation at residue S651 of UHRF1 was observed in previous mass-spectrometry analyses. RESULTS +50 55 UHRF1 protein Interestingly, phosphorylation at residue S651 of UHRF1 was observed in previous mass-spectrometry analyses. RESULTS +81 98 mass-spectrometry experimental_method Interestingly, phosphorylation at residue S651 of UHRF1 was observed in previous mass-spectrometry analyses. RESULTS +18 28 unmodified protein_state Compared with the unmodified peptide of Spacer642–664, a phosphorylation at S651 markedly decreased the binding affinity to the TTD (Supplementary Fig. 2b), suggesting that the phosphorylation may regulate the intramolecular interaction within UHRF1. RESULTS +40 53 Spacer642–664 mutant Compared with the unmodified peptide of Spacer642–664, a phosphorylation at S651 markedly decreased the binding affinity to the TTD (Supplementary Fig. 2b), suggesting that the phosphorylation may regulate the intramolecular interaction within UHRF1. RESULTS +57 72 phosphorylation ptm Compared with the unmodified peptide of Spacer642–664, a phosphorylation at S651 markedly decreased the binding affinity to the TTD (Supplementary Fig. 2b), suggesting that the phosphorylation may regulate the intramolecular interaction within UHRF1. RESULTS +76 80 S651 residue_name_number Compared with the unmodified peptide of Spacer642–664, a phosphorylation at S651 markedly decreased the binding affinity to the TTD (Supplementary Fig. 2b), suggesting that the phosphorylation may regulate the intramolecular interaction within UHRF1. RESULTS +104 120 binding affinity evidence Compared with the unmodified peptide of Spacer642–664, a phosphorylation at S651 markedly decreased the binding affinity to the TTD (Supplementary Fig. 2b), suggesting that the phosphorylation may regulate the intramolecular interaction within UHRF1. RESULTS +128 131 TTD structure_element Compared with the unmodified peptide of Spacer642–664, a phosphorylation at S651 markedly decreased the binding affinity to the TTD (Supplementary Fig. 2b), suggesting that the phosphorylation may regulate the intramolecular interaction within UHRF1. RESULTS +177 192 phosphorylation ptm Compared with the unmodified peptide of Spacer642–664, a phosphorylation at S651 markedly decreased the binding affinity to the TTD (Supplementary Fig. 2b), suggesting that the phosphorylation may regulate the intramolecular interaction within UHRF1. RESULTS +244 249 UHRF1 protein Compared with the unmodified peptide of Spacer642–664, a phosphorylation at S651 markedly decreased the binding affinity to the TTD (Supplementary Fig. 2b), suggesting that the phosphorylation may regulate the intramolecular interaction within UHRF1. RESULTS +4 10 spacer structure_element The spacer binds to the TTD by competing with the linker RESULTS +11 19 binds to protein_state The spacer binds to the TTD by competing with the linker RESULTS +24 27 TTD structure_element The spacer binds to the TTD by competing with the linker RESULTS +50 56 linker structure_element The spacer binds to the TTD by competing with the linker RESULTS +35 38 TTD structure_element Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286–306, designated Linker, Fig. 1a), and TTD–Linker interaction is essential for H3K9me3 recognition by TTD–PHD. RESULTS +39 47 binds to protein_state Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286–306, designated Linker, Fig. 1a), and TTD–Linker interaction is essential for H3K9me3 recognition by TTD–PHD. RESULTS +50 63 linker region structure_element Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286–306, designated Linker, Fig. 1a), and TTD–Linker interaction is essential for H3K9me3 recognition by TTD–PHD. RESULTS +79 82 TTD structure_element Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286–306, designated Linker, Fig. 1a), and TTD–Linker interaction is essential for H3K9me3 recognition by TTD–PHD. RESULTS +87 90 PHD structure_element Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286–306, designated Linker, Fig. 1a), and TTD–Linker interaction is essential for H3K9me3 recognition by TTD–PHD. RESULTS +101 108 286–306 residue_range Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286–306, designated Linker, Fig. 1a), and TTD–Linker interaction is essential for H3K9me3 recognition by TTD–PHD. RESULTS +121 127 Linker structure_element Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286–306, designated Linker, Fig. 1a), and TTD–Linker interaction is essential for H3K9me3 recognition by TTD–PHD. RESULTS +143 153 TTD–Linker structure_element Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286–306, designated Linker, Fig. 1a), and TTD–Linker interaction is essential for H3K9me3 recognition by TTD–PHD. RESULTS +183 185 H3 protein_type Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286–306, designated Linker, Fig. 1a), and TTD–Linker interaction is essential for H3K9me3 recognition by TTD–PHD. RESULTS +185 190 K9me3 ptm Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286–306, designated Linker, Fig. 1a), and TTD–Linker interaction is essential for H3K9me3 recognition by TTD–PHD. RESULTS +206 213 TTD–PHD structure_element Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286–306, designated Linker, Fig. 1a), and TTD–Linker interaction is essential for H3K9me3 recognition by TTD–PHD. RESULTS +0 10 Comparison experimental_method Comparison of TTD–Spacer and TTD–PHD–H3K9me3 (PDB: 4GY5) structures indicates that the Spacer and the Linker bind to the TTD in a similar manner in the two complexes (Fig. 3b). RESULTS +14 24 TTD–Spacer structure_element Comparison of TTD–Spacer and TTD–PHD–H3K9me3 (PDB: 4GY5) structures indicates that the Spacer and the Linker bind to the TTD in a similar manner in the two complexes (Fig. 3b). RESULTS +29 44 TTD–PHD–H3K9me3 complex_assembly Comparison of TTD–Spacer and TTD–PHD–H3K9me3 (PDB: 4GY5) structures indicates that the Spacer and the Linker bind to the TTD in a similar manner in the two complexes (Fig. 3b). RESULTS +57 67 structures evidence Comparison of TTD–Spacer and TTD–PHD–H3K9me3 (PDB: 4GY5) structures indicates that the Spacer and the Linker bind to the TTD in a similar manner in the two complexes (Fig. 3b). RESULTS +87 93 Spacer structure_element Comparison of TTD–Spacer and TTD–PHD–H3K9me3 (PDB: 4GY5) structures indicates that the Spacer and the Linker bind to the TTD in a similar manner in the two complexes (Fig. 3b). RESULTS +102 108 Linker structure_element Comparison of TTD–Spacer and TTD–PHD–H3K9me3 (PDB: 4GY5) structures indicates that the Spacer and the Linker bind to the TTD in a similar manner in the two complexes (Fig. 3b). RESULTS +121 124 TTD structure_element Comparison of TTD–Spacer and TTD–PHD–H3K9me3 (PDB: 4GY5) structures indicates that the Spacer and the Linker bind to the TTD in a similar manner in the two complexes (Fig. 3b). RESULTS +3 18 TTD–PHD–H3K9me3 complex_assembly In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. RESULTS +19 28 structure evidence In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. RESULTS +39 43 R295 residue_name_number In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. RESULTS +45 49 R296 residue_name_number In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. RESULTS +54 58 S298 residue_name_number In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. RESULTS +66 72 Linker structure_element In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. RESULTS +121 125 K648 residue_name_number In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. RESULTS +127 131 R649 residue_name_number In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. RESULTS +136 140 S651 residue_name_number In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. RESULTS +148 154 Spacer structure_element In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. RESULTS +158 168 TTD–Spacer structure_element In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. RESULTS +169 178 structure evidence In TTD–PHD–H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD–Spacer structure, respectively. RESULTS +33 43 TTD–Linker structure_element Similar intramolecular contacts (TTD–Linker and TTD–Spacer) were observed in the two structures (Fig. 3b,d and Supplementary Fig. 4a). RESULTS +48 58 TTD–Spacer structure_element Similar intramolecular contacts (TTD–Linker and TTD–Spacer) were observed in the two structures (Fig. 3b,d and Supplementary Fig. 4a). RESULTS +85 95 structures evidence Similar intramolecular contacts (TTD–Linker and TTD–Spacer) were observed in the two structures (Fig. 3b,d and Supplementary Fig. 4a). RESULTS +10 16 Spacer structure_element Thus, the Spacer may disrupt the TTD–Linker interaction and inhibits the recognition of H3K9me3 by TTD–PHD. RESULTS +33 43 TTD–Linker structure_element Thus, the Spacer may disrupt the TTD–Linker interaction and inhibits the recognition of H3K9me3 by TTD–PHD. RESULTS +88 90 H3 protein_type Thus, the Spacer may disrupt the TTD–Linker interaction and inhibits the recognition of H3K9me3 by TTD–PHD. RESULTS +90 95 K9me3 ptm Thus, the Spacer may disrupt the TTD–Linker interaction and inhibits the recognition of H3K9me3 by TTD–PHD. RESULTS +99 106 TTD–PHD structure_element Thus, the Spacer may disrupt the TTD–Linker interaction and inhibits the recognition of H3K9me3 by TTD–PHD. RESULTS +85 91 Linker structure_element To test this hypothesis, we first investigated the potential competition between the Linker and the Spacer for their interaction with the TTD. RESULTS +100 106 Spacer structure_element To test this hypothesis, we first investigated the potential competition between the Linker and the Spacer for their interaction with the TTD. RESULTS +138 141 TTD structure_element To test this hypothesis, we first investigated the potential competition between the Linker and the Spacer for their interaction with the TTD. RESULTS +4 7 ITC experimental_method The ITC experiment shows that the Linker peptide (289–306) bound to the TTD with a binding affinity of 24.04 μM (Supplementary Fig. 4b), ∼15-fold lower than that of the Spacer peptide (KD=1.59 μM, Fig. 3e). RESULTS +34 40 Linker structure_element The ITC experiment shows that the Linker peptide (289–306) bound to the TTD with a binding affinity of 24.04 μM (Supplementary Fig. 4b), ∼15-fold lower than that of the Spacer peptide (KD=1.59 μM, Fig. 3e). RESULTS +50 57 289–306 residue_range The ITC experiment shows that the Linker peptide (289–306) bound to the TTD with a binding affinity of 24.04 μM (Supplementary Fig. 4b), ∼15-fold lower than that of the Spacer peptide (KD=1.59 μM, Fig. 3e). RESULTS +59 67 bound to protein_state The ITC experiment shows that the Linker peptide (289–306) bound to the TTD with a binding affinity of 24.04 μM (Supplementary Fig. 4b), ∼15-fold lower than that of the Spacer peptide (KD=1.59 μM, Fig. 3e). RESULTS +72 75 TTD structure_element The ITC experiment shows that the Linker peptide (289–306) bound to the TTD with a binding affinity of 24.04 μM (Supplementary Fig. 4b), ∼15-fold lower than that of the Spacer peptide (KD=1.59 μM, Fig. 3e). RESULTS +83 99 binding affinity evidence The ITC experiment shows that the Linker peptide (289–306) bound to the TTD with a binding affinity of 24.04 μM (Supplementary Fig. 4b), ∼15-fold lower than that of the Spacer peptide (KD=1.59 μM, Fig. 3e). RESULTS +169 175 Spacer structure_element The ITC experiment shows that the Linker peptide (289–306) bound to the TTD with a binding affinity of 24.04 μM (Supplementary Fig. 4b), ∼15-fold lower than that of the Spacer peptide (KD=1.59 μM, Fig. 3e). RESULTS +185 187 KD evidence The ITC experiment shows that the Linker peptide (289–306) bound to the TTD with a binding affinity of 24.04 μM (Supplementary Fig. 4b), ∼15-fold lower than that of the Spacer peptide (KD=1.59 μM, Fig. 3e). RESULTS +4 19 competitive ITC experimental_method The competitive ITC experiments show that TTD–Spacer binding affinity decreased by a factor of two in the presence of the Linker, whereas TTD–Linker interaction was abolished in the presence of the Spacer (Supplementary Fig. 4c). RESULTS +42 69 TTD–Spacer binding affinity evidence The competitive ITC experiments show that TTD–Spacer binding affinity decreased by a factor of two in the presence of the Linker, whereas TTD–Linker interaction was abolished in the presence of the Spacer (Supplementary Fig. 4c). RESULTS +106 117 presence of protein_state The competitive ITC experiments show that TTD–Spacer binding affinity decreased by a factor of two in the presence of the Linker, whereas TTD–Linker interaction was abolished in the presence of the Spacer (Supplementary Fig. 4c). RESULTS +122 128 Linker structure_element The competitive ITC experiments show that TTD–Spacer binding affinity decreased by a factor of two in the presence of the Linker, whereas TTD–Linker interaction was abolished in the presence of the Spacer (Supplementary Fig. 4c). RESULTS +138 148 TTD–Linker structure_element The competitive ITC experiments show that TTD–Spacer binding affinity decreased by a factor of two in the presence of the Linker, whereas TTD–Linker interaction was abolished in the presence of the Spacer (Supplementary Fig. 4c). RESULTS +182 193 presence of protein_state The competitive ITC experiments show that TTD–Spacer binding affinity decreased by a factor of two in the presence of the Linker, whereas TTD–Linker interaction was abolished in the presence of the Spacer (Supplementary Fig. 4c). RESULTS +198 204 Spacer structure_element The competitive ITC experiments show that TTD–Spacer binding affinity decreased by a factor of two in the presence of the Linker, whereas TTD–Linker interaction was abolished in the presence of the Spacer (Supplementary Fig. 4c). RESULTS +14 24 TTD–Spacer structure_element Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +38 40 KD evidence Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +51 58 TTD–PHD structure_element Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +73 89 binding affinity evidence Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +97 103 Spacer structure_element Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +105 107 KD evidence Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +127 135 mutation experimental_method Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +136 141 R295D mutant Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +142 147 R296D mutant Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +160 166 Linker structure_element Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +185 195 TTD–Linker structure_element Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +212 219 TTD–PHD structure_element Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +249 265 binding affinity evidence Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +267 269 KD evidence Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +326 332 Spacer structure_element Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +341 347 Linker structure_element Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +360 372 binding site site Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +380 383 TTD structure_element Compared with TTD–Spacer interaction (KD=1.48 μM), TTD–PHD decreased the binding affinity to the Spacer (KD=10.68 μM), whereas mutation R295D/R296D (within the Linker and important for TTD–Linker interaction) of TTD–PHD showed minor decrease in the binding affinity (KD=2.69 μM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD. RESULTS +22 28 Linker structure_element Notably, although the Linker (in the context of TTD-PHD) impairs the TTD–Spacer interaction to some extent, the isolated Spacer could still bind to TTD–PHD with moderate binding affinity (KD=10.68 μM), supporting the existence of the intramolecular interaction within UHRF1. RESULTS +48 55 TTD-PHD structure_element Notably, although the Linker (in the context of TTD-PHD) impairs the TTD–Spacer interaction to some extent, the isolated Spacer could still bind to TTD–PHD with moderate binding affinity (KD=10.68 μM), supporting the existence of the intramolecular interaction within UHRF1. RESULTS +69 79 TTD–Spacer structure_element Notably, although the Linker (in the context of TTD-PHD) impairs the TTD–Spacer interaction to some extent, the isolated Spacer could still bind to TTD–PHD with moderate binding affinity (KD=10.68 μM), supporting the existence of the intramolecular interaction within UHRF1. RESULTS +121 127 Spacer structure_element Notably, although the Linker (in the context of TTD-PHD) impairs the TTD–Spacer interaction to some extent, the isolated Spacer could still bind to TTD–PHD with moderate binding affinity (KD=10.68 μM), supporting the existence of the intramolecular interaction within UHRF1. RESULTS +148 155 TTD–PHD structure_element Notably, although the Linker (in the context of TTD-PHD) impairs the TTD–Spacer interaction to some extent, the isolated Spacer could still bind to TTD–PHD with moderate binding affinity (KD=10.68 μM), supporting the existence of the intramolecular interaction within UHRF1. RESULTS +170 186 binding affinity evidence Notably, although the Linker (in the context of TTD-PHD) impairs the TTD–Spacer interaction to some extent, the isolated Spacer could still bind to TTD–PHD with moderate binding affinity (KD=10.68 μM), supporting the existence of the intramolecular interaction within UHRF1. RESULTS +188 190 KD evidence Notably, although the Linker (in the context of TTD-PHD) impairs the TTD–Spacer interaction to some extent, the isolated Spacer could still bind to TTD–PHD with moderate binding affinity (KD=10.68 μM), supporting the existence of the intramolecular interaction within UHRF1. RESULTS +268 273 UHRF1 protein Notably, although the Linker (in the context of TTD-PHD) impairs the TTD–Spacer interaction to some extent, the isolated Spacer could still bind to TTD–PHD with moderate binding affinity (KD=10.68 μM), supporting the existence of the intramolecular interaction within UHRF1. RESULTS +16 26 TTD–Spacer structure_element To test whether TTD–Spacer association exists in the context of full-length UHRF1, we used various truncations of UHRF1 in the GST pull-down assay. RESULTS +64 75 full-length protein_state To test whether TTD–Spacer association exists in the context of full-length UHRF1, we used various truncations of UHRF1 in the GST pull-down assay. RESULTS +76 81 UHRF1 protein To test whether TTD–Spacer association exists in the context of full-length UHRF1, we used various truncations of UHRF1 in the GST pull-down assay. RESULTS +99 110 truncations experimental_method To test whether TTD–Spacer association exists in the context of full-length UHRF1, we used various truncations of UHRF1 in the GST pull-down assay. RESULTS +114 119 UHRF1 protein To test whether TTD–Spacer association exists in the context of full-length UHRF1, we used various truncations of UHRF1 in the GST pull-down assay. RESULTS +127 146 GST pull-down assay experimental_method To test whether TTD–Spacer association exists in the context of full-length UHRF1, we used various truncations of UHRF1 in the GST pull-down assay. RESULTS +25 36 full-length protein_state As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +37 42 UHRF1 protein As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +47 56 UHRF1ΔSRA mutant As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +84 94 GST-tagged protein_state As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +95 98 TTD structure_element As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +100 106 Linker structure_element As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +110 116 Spacer structure_element As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +134 144 TTD–Spacer structure_element As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +157 163 in-cis protein_state As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +171 182 full-length protein_state As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +183 188 UHRF1 protein As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +192 201 UHRF1ΔSRA mutant As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +212 222 TTD–Spacer structure_element As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +241 249 in-trans protein_state As indicated in Fig. 3h, full-length UHRF1 and UHRF1ΔSRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD–Spacer interaction in-cis within full-length UHRF1 or UHRF1ΔSRA prohibits TTD–Spacer complex formation in-trans. RESULTS +13 22 UHRF1ΔTTD mutant In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +23 31 bound to protein_state In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +32 35 GST experimental_method In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +36 39 TTD structure_element In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +45 58 UHRF1Δ627–674 mutant In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +59 67 bound to protein_state In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +68 71 GST experimental_method In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +72 78 Spacer structure_element In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +104 114 TTD–Spacer structure_element In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +127 133 in-cis protein_state In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +135 145 TTD–Spacer structure_element In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +165 173 in-trans protein_state In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +195 198 TTD structure_element In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +199 207 binds to protein_state In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +212 218 Spacer structure_element In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +237 248 full-length protein_state In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +249 254 UHRF1 protein In contrast, UHRF1ΔTTD bound to GST-TTD, and UHRF1Δ627–674 bound to GST-Spacer, indicating that lack of TTD–Spacer interaction in-cis, TTD–Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1. RESULTS +10 13 GST experimental_method Moreover, GST-Linker showed very weak if not undetectable interaction with wild-type or deletions of UHRF1, suggesting that TTD–Linker interaction is much weaker than that of TTD–Spacer. RESULTS +14 20 Linker structure_element Moreover, GST-Linker showed very weak if not undetectable interaction with wild-type or deletions of UHRF1, suggesting that TTD–Linker interaction is much weaker than that of TTD–Spacer. RESULTS +75 84 wild-type protein_state Moreover, GST-Linker showed very weak if not undetectable interaction with wild-type or deletions of UHRF1, suggesting that TTD–Linker interaction is much weaker than that of TTD–Spacer. RESULTS +101 106 UHRF1 protein Moreover, GST-Linker showed very weak if not undetectable interaction with wild-type or deletions of UHRF1, suggesting that TTD–Linker interaction is much weaker than that of TTD–Spacer. RESULTS +124 134 TTD–Linker structure_element Moreover, GST-Linker showed very weak if not undetectable interaction with wild-type or deletions of UHRF1, suggesting that TTD–Linker interaction is much weaker than that of TTD–Spacer. RESULTS +175 185 TTD–Spacer structure_element Moreover, GST-Linker showed very weak if not undetectable interaction with wild-type or deletions of UHRF1, suggesting that TTD–Linker interaction is much weaker than that of TTD–Spacer. RESULTS +16 21 UHRF1 protein Taken together, UHRF1 adopts a closed conformation, in which the Spacer binds to the TTD through competing with the Linker, and therefore inhibits H3K9me3 recognition by UHRF1. RESULTS +31 37 closed protein_state Taken together, UHRF1 adopts a closed conformation, in which the Spacer binds to the TTD through competing with the Linker, and therefore inhibits H3K9me3 recognition by UHRF1. RESULTS +65 71 Spacer structure_element Taken together, UHRF1 adopts a closed conformation, in which the Spacer binds to the TTD through competing with the Linker, and therefore inhibits H3K9me3 recognition by UHRF1. RESULTS +72 80 binds to protein_state Taken together, UHRF1 adopts a closed conformation, in which the Spacer binds to the TTD through competing with the Linker, and therefore inhibits H3K9me3 recognition by UHRF1. RESULTS +85 88 TTD structure_element Taken together, UHRF1 adopts a closed conformation, in which the Spacer binds to the TTD through competing with the Linker, and therefore inhibits H3K9me3 recognition by UHRF1. RESULTS +116 122 Linker structure_element Taken together, UHRF1 adopts a closed conformation, in which the Spacer binds to the TTD through competing with the Linker, and therefore inhibits H3K9me3 recognition by UHRF1. RESULTS +147 149 H3 protein_type Taken together, UHRF1 adopts a closed conformation, in which the Spacer binds to the TTD through competing with the Linker, and therefore inhibits H3K9me3 recognition by UHRF1. RESULTS +149 154 K9me3 ptm Taken together, UHRF1 adopts a closed conformation, in which the Spacer binds to the TTD through competing with the Linker, and therefore inhibits H3K9me3 recognition by UHRF1. RESULTS +170 175 UHRF1 protein Taken together, UHRF1 adopts a closed conformation, in which the Spacer binds to the TTD through competing with the Linker, and therefore inhibits H3K9me3 recognition by UHRF1. RESULTS +4 10 spacer structure_element The spacer inhibits H3K9me3 recognition by the isolated TTD RESULTS +20 22 H3 protein_type The spacer inhibits H3K9me3 recognition by the isolated TTD RESULTS +22 27 K9me3 ptm The spacer inhibits H3K9me3 recognition by the isolated TTD RESULTS +56 59 TTD structure_element The spacer inhibits H3K9me3 recognition by the isolated TTD RESULTS +34 36 H3 protein_type Our previous study indicates that H3K9me3 binds to the TTD in different manner in TTD–PHD–H3K9me3 (ref.) and TTD-H3K9me3 (PDB: 2L3R) structures. RESULTS +36 41 K9me3 ptm Our previous study indicates that H3K9me3 binds to the TTD in different manner in TTD–PHD–H3K9me3 (ref.) and TTD-H3K9me3 (PDB: 2L3R) structures. RESULTS +42 50 binds to protein_state Our previous study indicates that H3K9me3 binds to the TTD in different manner in TTD–PHD–H3K9me3 (ref.) and TTD-H3K9me3 (PDB: 2L3R) structures. RESULTS +55 58 TTD structure_element Our previous study indicates that H3K9me3 binds to the TTD in different manner in TTD–PHD–H3K9me3 (ref.) and TTD-H3K9me3 (PDB: 2L3R) structures. RESULTS +82 97 TTD–PHD–H3K9me3 complex_assembly Our previous study indicates that H3K9me3 binds to the TTD in different manner in TTD–PHD–H3K9me3 (ref.) and TTD-H3K9me3 (PDB: 2L3R) structures. RESULTS +109 120 TTD-H3K9me3 complex_assembly Our previous study indicates that H3K9me3 binds to the TTD in different manner in TTD–PHD–H3K9me3 (ref.) and TTD-H3K9me3 (PDB: 2L3R) structures. RESULTS +133 143 structures evidence Our previous study indicates that H3K9me3 binds to the TTD in different manner in TTD–PHD–H3K9me3 (ref.) and TTD-H3K9me3 (PDB: 2L3R) structures. RESULTS +12 15 TTD structure_element Because the TTD is always associated with the PHD, whether the pattern of TTD–H3K9me3 interaction exists in vivo remains unknown. RESULTS +46 49 PHD structure_element Because the TTD is always associated with the PHD, whether the pattern of TTD–H3K9me3 interaction exists in vivo remains unknown. RESULTS +74 85 TTD–H3K9me3 complex_assembly Because the TTD is always associated with the PHD, whether the pattern of TTD–H3K9me3 interaction exists in vivo remains unknown. RESULTS +14 24 comparison experimental_method Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. RESULTS +28 39 TTD–H3K9me3 complex_assembly Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. RESULTS +44 54 TTD–Spacer structure_element Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. RESULTS +55 65 structures evidence Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. RESULTS +81 83 H3 protein_type Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. RESULTS +83 88 K9me3 ptm Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. RESULTS +97 103 Spacer structure_element Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. RESULTS +134 137 TTD structure_element Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. RESULTS +183 189 Spacer structure_element Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. RESULTS +206 208 H3 protein_type Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. RESULTS +208 213 K9me3 ptm Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. RESULTS +242 245 TTD structure_element Nevertheless, comparison of TTD–H3K9me3 and TTD–Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD. RESULTS +39 45 Spacer structure_element As shown in Supplementary Fig. 4e, the Spacer inhibited TTD–H3K9me3 interaction, whereas its TTD-binding defective mutants of the Spacer or the SRA (a negative control) markedly decreased the inhibition. RESULTS +56 67 TTD–H3K9me3 complex_assembly As shown in Supplementary Fig. 4e, the Spacer inhibited TTD–H3K9me3 interaction, whereas its TTD-binding defective mutants of the Spacer or the SRA (a negative control) markedly decreased the inhibition. RESULTS +93 114 TTD-binding defective protein_state As shown in Supplementary Fig. 4e, the Spacer inhibited TTD–H3K9me3 interaction, whereas its TTD-binding defective mutants of the Spacer or the SRA (a negative control) markedly decreased the inhibition. RESULTS +115 122 mutants protein_state As shown in Supplementary Fig. 4e, the Spacer inhibited TTD–H3K9me3 interaction, whereas its TTD-binding defective mutants of the Spacer or the SRA (a negative control) markedly decreased the inhibition. RESULTS +130 136 Spacer structure_element As shown in Supplementary Fig. 4e, the Spacer inhibited TTD–H3K9me3 interaction, whereas its TTD-binding defective mutants of the Spacer or the SRA (a negative control) markedly decreased the inhibition. RESULTS +144 147 SRA structure_element As shown in Supplementary Fig. 4e, the Spacer inhibited TTD–H3K9me3 interaction, whereas its TTD-binding defective mutants of the Spacer or the SRA (a negative control) markedly decreased the inhibition. RESULTS +69 80 full-length protein_state We next tested whether such inhibition also occurs in the context of full-length UHRF1. RESULTS +81 86 UHRF1 protein We next tested whether such inhibition also occurs in the context of full-length UHRF1. RESULTS +14 25 full-length protein_state Compared with full-length UHRF1, UHRF1Δ627–674 enhanced H3K9me3-binding affinity by a factor of four (Supplementary Fig. 4f). RESULTS +26 31 UHRF1 protein Compared with full-length UHRF1, UHRF1Δ627–674 enhanced H3K9me3-binding affinity by a factor of four (Supplementary Fig. 4f). RESULTS +33 46 UHRF1Δ627–674 mutant Compared with full-length UHRF1, UHRF1Δ627–674 enhanced H3K9me3-binding affinity by a factor of four (Supplementary Fig. 4f). RESULTS +56 80 H3K9me3-binding affinity evidence Compared with full-length UHRF1, UHRF1Δ627–674 enhanced H3K9me3-binding affinity by a factor of four (Supplementary Fig. 4f). RESULTS +19 43 H3K9me3-binding affinity evidence The restoration of H3K9me3-binding affinity is not dramatic because the PHD still binds to histone H3 in both proteins. RESULTS +72 75 PHD structure_element The restoration of H3K9me3-binding affinity is not dramatic because the PHD still binds to histone H3 in both proteins. RESULTS +82 90 binds to protein_state The restoration of H3K9me3-binding affinity is not dramatic because the PHD still binds to histone H3 in both proteins. RESULTS +91 98 histone protein_type The restoration of H3K9me3-binding affinity is not dramatic because the PHD still binds to histone H3 in both proteins. RESULTS +99 101 H3 protein_type The restoration of H3K9me3-binding affinity is not dramatic because the PHD still binds to histone H3 in both proteins. RESULTS +53 63 UHRF1D334A mutant To exclude this effect, we performed the assay using UHRF1D334A, which abolishes H3R2-binding affinity of the PHD. RESULTS +71 80 abolishes protein_state To exclude this effect, we performed the assay using UHRF1D334A, which abolishes H3R2-binding affinity of the PHD. RESULTS +81 102 H3R2-binding affinity evidence To exclude this effect, we performed the assay using UHRF1D334A, which abolishes H3R2-binding affinity of the PHD. RESULTS +110 113 PHD structure_element To exclude this effect, we performed the assay using UHRF1D334A, which abolishes H3R2-binding affinity of the PHD. RESULTS +0 10 UHRF1D334A mutant UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&Δ627–674 dramatically restored its H3K9me3-binding affinity (KD=8.69 μM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD. RESULTS +31 55 H3K9me3-binding affinity evidence UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&Δ627–674 dramatically restored its H3K9me3-binding affinity (KD=8.69 μM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD. RESULTS +65 75 UHRF1D334A mutant UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&Δ627–674 dramatically restored its H3K9me3-binding affinity (KD=8.69 μM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD. RESULTS +76 84 Δ627–674 mutant UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&Δ627–674 dramatically restored its H3K9me3-binding affinity (KD=8.69 μM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD. RESULTS +111 135 H3K9me3-binding affinity evidence UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&Δ627–674 dramatically restored its H3K9me3-binding affinity (KD=8.69 μM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD. RESULTS +137 139 KD evidence UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&Δ627–674 dramatically restored its H3K9me3-binding affinity (KD=8.69 μM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD. RESULTS +189 191 H3 protein_type UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&Δ627–674 dramatically restored its H3K9me3-binding affinity (KD=8.69 μM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD. RESULTS +191 196 K9me3 ptm UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&Δ627–674 dramatically restored its H3K9me3-binding affinity (KD=8.69 μM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD. RESULTS +216 219 TTD structure_element UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&Δ627–674 dramatically restored its H3K9me3-binding affinity (KD=8.69 μM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD. RESULTS +238 244 Spacer structure_element UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&Δ627–674 dramatically restored its H3K9me3-binding affinity (KD=8.69 μM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD. RESULTS +286 289 TTD structure_element UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&Δ627–674 dramatically restored its H3K9me3-binding affinity (KD=8.69 μM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD. RESULTS +14 19 R295D mutant Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +20 25 R296D mutant Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +26 32 mutant protein_state Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +36 47 full-length protein_state Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +48 53 UHRF1 protein Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +71 87 binding affinity evidence Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +91 93 H3 protein_type Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +93 98 K9me3 ptm Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +121 130 wild type protein_state Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +149 157 mutation experimental_method Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +161 166 R295D mutant Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +167 172 R296D mutant Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +181 191 TTD–Spacer structure_element Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +227 232 UHRF1 protein Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +258 264 closed protein_state Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD–Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g). RESULTS +20 26 Spacer structure_element Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +27 35 binds to protein_state Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +40 43 TTD structure_element Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +57 59 H3 protein_type Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +59 64 K9me3 ptm Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +80 85 UHRF1 protein Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +109 119 TTD–Linker structure_element Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +156 158 H3 protein_type Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +158 163 K9me3 ptm Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +179 186 TTD–PHD structure_element Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +205 207 H3 protein_type Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +207 212 K9me3 ptm Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +237 240 TTD structure_element Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD–Linker interaction, which is essential for H3K9me3 recognition by TTD–PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD. RESULTS +0 15 TTD–PHD–H3K9me3 complex_assembly TTD–PHD–H3K9me3 complex inhibits TTD–spacer interaction RESULTS +33 43 TTD–spacer structure_element TTD–PHD–H3K9me3 complex inhibits TTD–spacer interaction RESULTS +15 29 pre-incubation experimental_method Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +33 35 H3 protein_type Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +35 40 K9me3 ptm Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +96 102 Spacer structure_element Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +111 114 TTD structure_element Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +115 120 alone protein_state Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +124 131 TTD–PHD structure_element Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +169 180 presence of protein_state Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +185 191 Spacer structure_element Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +235 242 TTD–PHD structure_element Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +247 249 H3 protein_type Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +249 254 K9me3 ptm Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD–PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD–PHD and H3K9me3 (Fig. 2c). RESULTS +91 98 TTD–PHD structure_element The results are also consistent with the previous observation that the interaction between TTD–PHD and the Spacer is much weaker (KD=10.68 μM, Fig. 3g) than that between TTD–PHD and H3K9me3 (KD=0.15 μM, Fig. 1d). RESULTS +107 113 Spacer structure_element The results are also consistent with the previous observation that the interaction between TTD–PHD and the Spacer is much weaker (KD=10.68 μM, Fig. 3g) than that between TTD–PHD and H3K9me3 (KD=0.15 μM, Fig. 1d). RESULTS +130 132 KD evidence The results are also consistent with the previous observation that the interaction between TTD–PHD and the Spacer is much weaker (KD=10.68 μM, Fig. 3g) than that between TTD–PHD and H3K9me3 (KD=0.15 μM, Fig. 1d). RESULTS +170 177 TTD–PHD structure_element The results are also consistent with the previous observation that the interaction between TTD–PHD and the Spacer is much weaker (KD=10.68 μM, Fig. 3g) than that between TTD–PHD and H3K9me3 (KD=0.15 μM, Fig. 1d). RESULTS +182 184 H3 protein_type The results are also consistent with the previous observation that the interaction between TTD–PHD and the Spacer is much weaker (KD=10.68 μM, Fig. 3g) than that between TTD–PHD and H3K9me3 (KD=0.15 μM, Fig. 1d). RESULTS +184 189 K9me3 ptm The results are also consistent with the previous observation that the interaction between TTD–PHD and the Spacer is much weaker (KD=10.68 μM, Fig. 3g) than that between TTD–PHD and H3K9me3 (KD=0.15 μM, Fig. 1d). RESULTS +191 193 KD evidence The results are also consistent with the previous observation that the interaction between TTD–PHD and the Spacer is much weaker (KD=10.68 μM, Fig. 3g) than that between TTD–PHD and H3K9me3 (KD=0.15 μM, Fig. 1d). RESULTS +32 39 TTD–PHD structure_element These results suggest that once TTD–PHD binds to H3K9me3, UHRF1 will be locked by H3K9me3 and the Spacer is unlikely to fold back for the intramolecular interaction. RESULTS +40 48 binds to protein_state These results suggest that once TTD–PHD binds to H3K9me3, UHRF1 will be locked by H3K9me3 and the Spacer is unlikely to fold back for the intramolecular interaction. RESULTS +49 51 H3 protein_type These results suggest that once TTD–PHD binds to H3K9me3, UHRF1 will be locked by H3K9me3 and the Spacer is unlikely to fold back for the intramolecular interaction. RESULTS +51 56 K9me3 ptm These results suggest that once TTD–PHD binds to H3K9me3, UHRF1 will be locked by H3K9me3 and the Spacer is unlikely to fold back for the intramolecular interaction. RESULTS +58 63 UHRF1 protein These results suggest that once TTD–PHD binds to H3K9me3, UHRF1 will be locked by H3K9me3 and the Spacer is unlikely to fold back for the intramolecular interaction. RESULTS +82 84 H3 protein_type These results suggest that once TTD–PHD binds to H3K9me3, UHRF1 will be locked by H3K9me3 and the Spacer is unlikely to fold back for the intramolecular interaction. RESULTS +84 89 K9me3 ptm These results suggest that once TTD–PHD binds to H3K9me3, UHRF1 will be locked by H3K9me3 and the Spacer is unlikely to fold back for the intramolecular interaction. RESULTS +98 104 Spacer structure_element These results suggest that once TTD–PHD binds to H3K9me3, UHRF1 will be locked by H3K9me3 and the Spacer is unlikely to fold back for the intramolecular interaction. RESULTS +0 6 Hm-DNA chemical Hm-DNA disrupts intramolecular interaction within UHRF1 RESULTS +50 55 UHRF1 protein Hm-DNA disrupts intramolecular interaction within UHRF1 RESULTS +23 29 hm-DNA chemical To investigate whether hm-DNA could open the closed conformation of UHRF1, we first measured the intramolecular interaction using UHRF1 truncations in the presence or absence of hm-DNA. RESULTS +36 40 open protein_state To investigate whether hm-DNA could open the closed conformation of UHRF1, we first measured the intramolecular interaction using UHRF1 truncations in the presence or absence of hm-DNA. RESULTS +45 51 closed protein_state To investigate whether hm-DNA could open the closed conformation of UHRF1, we first measured the intramolecular interaction using UHRF1 truncations in the presence or absence of hm-DNA. RESULTS +68 73 UHRF1 protein To investigate whether hm-DNA could open the closed conformation of UHRF1, we first measured the intramolecular interaction using UHRF1 truncations in the presence or absence of hm-DNA. RESULTS +130 135 UHRF1 protein To investigate whether hm-DNA could open the closed conformation of UHRF1, we first measured the intramolecular interaction using UHRF1 truncations in the presence or absence of hm-DNA. RESULTS +136 147 truncations experimental_method To investigate whether hm-DNA could open the closed conformation of UHRF1, we first measured the intramolecular interaction using UHRF1 truncations in the presence or absence of hm-DNA. RESULTS +155 163 presence protein_state To investigate whether hm-DNA could open the closed conformation of UHRF1, we first measured the intramolecular interaction using UHRF1 truncations in the presence or absence of hm-DNA. RESULTS +167 177 absence of protein_state To investigate whether hm-DNA could open the closed conformation of UHRF1, we first measured the intramolecular interaction using UHRF1 truncations in the presence or absence of hm-DNA. RESULTS +178 184 hm-DNA chemical To investigate whether hm-DNA could open the closed conformation of UHRF1, we first measured the intramolecular interaction using UHRF1 truncations in the presence or absence of hm-DNA. RESULTS +4 24 GST pull-down assays experimental_method The GST pull-down assays show that the PHD bound to the SRA and such interaction was impaired by the addition of hm-DNA (Fig. 4a). RESULTS +39 42 PHD structure_element The GST pull-down assays show that the PHD bound to the SRA and such interaction was impaired by the addition of hm-DNA (Fig. 4a). RESULTS +43 51 bound to protein_state The GST pull-down assays show that the PHD bound to the SRA and such interaction was impaired by the addition of hm-DNA (Fig. 4a). RESULTS +56 59 SRA structure_element The GST pull-down assays show that the PHD bound to the SRA and such interaction was impaired by the addition of hm-DNA (Fig. 4a). RESULTS +113 119 hm-DNA chemical The GST pull-down assays show that the PHD bound to the SRA and such interaction was impaired by the addition of hm-DNA (Fig. 4a). RESULTS +0 27 H3 peptide pull-down assays experimental_method H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1ΔTTD and UHRF1ΔSpacer (Fig. 4b). RESULTS +38 44 hm-DNA chemical H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1ΔTTD and UHRF1ΔSpacer (Fig. 4b). RESULTS +63 89 H3K9me0-binding affinities evidence H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1ΔTTD and UHRF1ΔSpacer (Fig. 4b). RESULTS +93 98 UHRF1 protein H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1ΔTTD and UHRF1ΔSpacer (Fig. 4b). RESULTS +99 110 truncations experimental_method H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1ΔTTD and UHRF1ΔSpacer (Fig. 4b). RESULTS +122 129 PHD-SRA structure_element H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1ΔTTD and UHRF1ΔSpacer (Fig. 4b). RESULTS +139 146 PHD-SRA structure_element H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1ΔTTD and UHRF1ΔSpacer (Fig. 4b). RESULTS +148 159 TTD-PHD-SRA structure_element H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1ΔTTD and UHRF1ΔSpacer (Fig. 4b). RESULTS +161 179 TTD-PHD-SRA-Spacer structure_element H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1ΔTTD and UHRF1ΔSpacer (Fig. 4b). RESULTS +181 190 UHRF1ΔTTD mutant H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1ΔTTD and UHRF1ΔSpacer (Fig. 4b). RESULTS +195 207 UHRF1ΔSpacer mutant H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1ΔTTD and UHRF1ΔSpacer (Fig. 4b). RESULTS +26 32 hm-DNA chemical The result indicates that hm-DNA disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. RESULTS +42 49 PHD–SRA structure_element The result indicates that hm-DNA disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. RESULTS +78 102 H3K9me0-binding affinity evidence The result indicates that hm-DNA disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. RESULTS +110 113 PHD structure_element The result indicates that hm-DNA disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. RESULTS +145 148 TTD structure_element The result indicates that hm-DNA disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. RESULTS +156 162 Spacer structure_element The result indicates that hm-DNA disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. RESULTS +14 17 TTD structure_element Moreover, the TTD or TTD–PHD bound to SRA–Spacer and the interaction was impaired by the addition of hm-DNA (Fig. 4c). RESULTS +21 28 TTD–PHD structure_element Moreover, the TTD or TTD–PHD bound to SRA–Spacer and the interaction was impaired by the addition of hm-DNA (Fig. 4c). RESULTS +29 37 bound to protein_state Moreover, the TTD or TTD–PHD bound to SRA–Spacer and the interaction was impaired by the addition of hm-DNA (Fig. 4c). RESULTS +38 48 SRA–Spacer structure_element Moreover, the TTD or TTD–PHD bound to SRA–Spacer and the interaction was impaired by the addition of hm-DNA (Fig. 4c). RESULTS +101 107 hm-DNA chemical Moreover, the TTD or TTD–PHD bound to SRA–Spacer and the interaction was impaired by the addition of hm-DNA (Fig. 4c). RESULTS +4 7 ITC experimental_method The ITC measurements show that the presence of hm-DNA markedly impaired the interaction between the TTD and SRA–Spacer (Supplementary Fig. 5a). RESULTS +35 46 presence of protein_state The ITC measurements show that the presence of hm-DNA markedly impaired the interaction between the TTD and SRA–Spacer (Supplementary Fig. 5a). RESULTS +47 53 hm-DNA chemical The ITC measurements show that the presence of hm-DNA markedly impaired the interaction between the TTD and SRA–Spacer (Supplementary Fig. 5a). RESULTS +100 103 TTD structure_element The ITC measurements show that the presence of hm-DNA markedly impaired the interaction between the TTD and SRA–Spacer (Supplementary Fig. 5a). RESULTS +108 118 SRA–Spacer structure_element The ITC measurements show that the presence of hm-DNA markedly impaired the interaction between the TTD and SRA–Spacer (Supplementary Fig. 5a). RESULTS +13 23 TTD–Spacer structure_element However, the TTD–Spacer interaction was not affected by the presence of the hm-DNA, indicating that hm-DNA displaces the Spacer from the TTD in a SRA-dependent manner (Supplementary Fig. 5b). RESULTS +60 71 presence of protein_state However, the TTD–Spacer interaction was not affected by the presence of the hm-DNA, indicating that hm-DNA displaces the Spacer from the TTD in a SRA-dependent manner (Supplementary Fig. 5b). RESULTS +76 82 hm-DNA chemical However, the TTD–Spacer interaction was not affected by the presence of the hm-DNA, indicating that hm-DNA displaces the Spacer from the TTD in a SRA-dependent manner (Supplementary Fig. 5b). RESULTS +100 106 hm-DNA chemical However, the TTD–Spacer interaction was not affected by the presence of the hm-DNA, indicating that hm-DNA displaces the Spacer from the TTD in a SRA-dependent manner (Supplementary Fig. 5b). RESULTS +121 127 Spacer structure_element However, the TTD–Spacer interaction was not affected by the presence of the hm-DNA, indicating that hm-DNA displaces the Spacer from the TTD in a SRA-dependent manner (Supplementary Fig. 5b). RESULTS +137 140 TTD structure_element However, the TTD–Spacer interaction was not affected by the presence of the hm-DNA, indicating that hm-DNA displaces the Spacer from the TTD in a SRA-dependent manner (Supplementary Fig. 5b). RESULTS +146 149 SRA structure_element However, the TTD–Spacer interaction was not affected by the presence of the hm-DNA, indicating that hm-DNA displaces the Spacer from the TTD in a SRA-dependent manner (Supplementary Fig. 5b). RESULTS +23 29 hm-DNA chemical To investigate whether hm-DNA disrupts TTD–Spacer interaction in the context of full-length UHRF1, we monitored the conformational changes of UHRF1 using its histone-binding affinity as read-out. RESULTS +39 49 TTD–Spacer structure_element To investigate whether hm-DNA disrupts TTD–Spacer interaction in the context of full-length UHRF1, we monitored the conformational changes of UHRF1 using its histone-binding affinity as read-out. RESULTS +80 91 full-length protein_state To investigate whether hm-DNA disrupts TTD–Spacer interaction in the context of full-length UHRF1, we monitored the conformational changes of UHRF1 using its histone-binding affinity as read-out. RESULTS +92 97 UHRF1 protein To investigate whether hm-DNA disrupts TTD–Spacer interaction in the context of full-length UHRF1, we monitored the conformational changes of UHRF1 using its histone-binding affinity as read-out. RESULTS +142 147 UHRF1 protein To investigate whether hm-DNA disrupts TTD–Spacer interaction in the context of full-length UHRF1, we monitored the conformational changes of UHRF1 using its histone-binding affinity as read-out. RESULTS +158 182 histone-binding affinity evidence To investigate whether hm-DNA disrupts TTD–Spacer interaction in the context of full-length UHRF1, we monitored the conformational changes of UHRF1 using its histone-binding affinity as read-out. RESULTS +0 10 UHRF1D334A mutant UHRF1D334A was used to exclude the effect of H3K9me0 recognition by the PHD. RESULTS +45 47 H3 protein_type UHRF1D334A was used to exclude the effect of H3K9me0 recognition by the PHD. RESULTS +47 52 K9me0 ptm UHRF1D334A was used to exclude the effect of H3K9me0 recognition by the PHD. RESULTS +72 75 PHD structure_element UHRF1D334A was used to exclude the effect of H3K9me0 recognition by the PHD. RESULTS +17 22 D334A mutant As expected, all D334A-containing mutants showed undetectable interaction with H3K9me0 (Fig. 4d). RESULTS +34 41 mutants protein_state As expected, all D334A-containing mutants showed undetectable interaction with H3K9me0 (Fig. 4d). RESULTS +79 81 H3 protein_type As expected, all D334A-containing mutants showed undetectable interaction with H3K9me0 (Fig. 4d). RESULTS +81 86 K9me0 ptm As expected, all D334A-containing mutants showed undetectable interaction with H3K9me0 (Fig. 4d). RESULTS +0 10 UHRF1D334A mutant UHRF1D334A bound to H3K9me3 peptide in the presence of hm-DNA, but showed no interaction in the absence of hm-DNA, which is consistent with the ITC experiments (Supplementary Fig. 4f). RESULTS +11 19 bound to protein_state UHRF1D334A bound to H3K9me3 peptide in the presence of hm-DNA, but showed no interaction in the absence of hm-DNA, which is consistent with the ITC experiments (Supplementary Fig. 4f). RESULTS +20 22 H3 protein_type UHRF1D334A bound to H3K9me3 peptide in the presence of hm-DNA, but showed no interaction in the absence of hm-DNA, which is consistent with the ITC experiments (Supplementary Fig. 4f). RESULTS +22 27 K9me3 ptm UHRF1D334A bound to H3K9me3 peptide in the presence of hm-DNA, but showed no interaction in the absence of hm-DNA, which is consistent with the ITC experiments (Supplementary Fig. 4f). RESULTS +43 54 presence of protein_state UHRF1D334A bound to H3K9me3 peptide in the presence of hm-DNA, but showed no interaction in the absence of hm-DNA, which is consistent with the ITC experiments (Supplementary Fig. 4f). RESULTS +55 61 hm-DNA chemical UHRF1D334A bound to H3K9me3 peptide in the presence of hm-DNA, but showed no interaction in the absence of hm-DNA, which is consistent with the ITC experiments (Supplementary Fig. 4f). RESULTS +96 106 absence of protein_state UHRF1D334A bound to H3K9me3 peptide in the presence of hm-DNA, but showed no interaction in the absence of hm-DNA, which is consistent with the ITC experiments (Supplementary Fig. 4f). RESULTS +107 113 hm-DNA chemical UHRF1D334A bound to H3K9me3 peptide in the presence of hm-DNA, but showed no interaction in the absence of hm-DNA, which is consistent with the ITC experiments (Supplementary Fig. 4f). RESULTS +144 147 ITC experimental_method UHRF1D334A bound to H3K9me3 peptide in the presence of hm-DNA, but showed no interaction in the absence of hm-DNA, which is consistent with the ITC experiments (Supplementary Fig. 4f). RESULTS +13 23 UHRF1D334A mutant In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. RESULTS +24 32 Δ627–674 mutant In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. RESULTS +42 50 bound to protein_state In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. RESULTS +51 53 H3 protein_type In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. RESULTS +53 58 K9me3 ptm In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. RESULTS +71 81 absence of protein_state In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. RESULTS +82 88 hm-DNA chemical In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. RESULTS +120 128 deletion experimental_method In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. RESULTS +136 142 Spacer structure_element In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. RESULTS +170 177 TTD–PHD structure_element In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. RESULTS +182 184 H3 protein_type In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. RESULTS +184 189 K9me3 ptm In contrast, UHRF1D334A&Δ627–674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD–PHD for H3K9me3 recognition. RESULTS +52 58 Spacer structure_element The results further support the conclusion that the Spacer binds to the TTD in the context of full-length UHRF1 and the intramolecular interactions are disrupted by hm-DNA. RESULTS +59 67 binds to protein_state The results further support the conclusion that the Spacer binds to the TTD in the context of full-length UHRF1 and the intramolecular interactions are disrupted by hm-DNA. RESULTS +72 75 TTD structure_element The results further support the conclusion that the Spacer binds to the TTD in the context of full-length UHRF1 and the intramolecular interactions are disrupted by hm-DNA. RESULTS +94 105 full-length protein_state The results further support the conclusion that the Spacer binds to the TTD in the context of full-length UHRF1 and the intramolecular interactions are disrupted by hm-DNA. RESULTS +106 111 UHRF1 protein The results further support the conclusion that the Spacer binds to the TTD in the context of full-length UHRF1 and the intramolecular interactions are disrupted by hm-DNA. RESULTS +165 171 hm-DNA chemical The results further support the conclusion that the Spacer binds to the TTD in the context of full-length UHRF1 and the intramolecular interactions are disrupted by hm-DNA. RESULTS +26 49 peptide pull-down assay experimental_method We next performed similar peptide pull-down assay using two mutants (N228C/G653C and R235C/G654C) generated on UHRF1D334A. RESULTS +60 67 mutants protein_state We next performed similar peptide pull-down assay using two mutants (N228C/G653C and R235C/G654C) generated on UHRF1D334A. RESULTS +69 74 N228C mutant We next performed similar peptide pull-down assay using two mutants (N228C/G653C and R235C/G654C) generated on UHRF1D334A. RESULTS +75 80 G653C mutant We next performed similar peptide pull-down assay using two mutants (N228C/G653C and R235C/G654C) generated on UHRF1D334A. RESULTS +85 90 R235C mutant We next performed similar peptide pull-down assay using two mutants (N228C/G653C and R235C/G654C) generated on UHRF1D334A. RESULTS +91 96 G654C mutant We next performed similar peptide pull-down assay using two mutants (N228C/G653C and R235C/G654C) generated on UHRF1D334A. RESULTS +111 121 UHRF1D334A mutant We next performed similar peptide pull-down assay using two mutants (N228C/G653C and R235C/G654C) generated on UHRF1D334A. RESULTS +9 13 N228 residue_name_number Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +14 18 R235 residue_name_number Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +28 31 TTD structure_element Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +36 40 G653 residue_name_number Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +41 45 G654 residue_name_number Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +55 61 Spacer structure_element Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +91 101 TTD–Spacer structure_element Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +110 119 structure evidence Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +165 173 Cysteine residue_name Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +197 200 TTD structure_element Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +218 224 Spacer structure_element Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +278 293 disulphide bond ptm Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +301 311 absence of protein_state Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +330 344 dithiothreitol chemical Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +346 349 DTT chemical Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD–Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT). RESULTS +21 27 hm-DNA chemical As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +49 75 H3K9me3-binding affinities evidence As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +84 91 mutants protein_state As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +99 110 presence of protein_state As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +111 114 DTT chemical As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +131 141 absence of protein_state As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +142 145 DTT chemical As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +167 182 disulphide bond ptm As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +201 211 absence of protein_state As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +212 215 DTT chemical As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +227 233 hm-DNA chemical As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +245 255 TTD–Spacer structure_element As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +272 274 H3 protein_type As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +274 279 K9me3 ptm As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD–Spacer interaction for H3K9me3 recognition. RESULTS +22 24 H3 protein_type As negative controls, H3K9me3 recognition by UHRF1D334A or UHRF1D334A&Δ627–674 is not affected by DTT. RESULTS +24 29 K9me3 ptm As negative controls, H3K9me3 recognition by UHRF1D334A or UHRF1D334A&Δ627–674 is not affected by DTT. RESULTS +45 55 UHRF1D334A mutant As negative controls, H3K9me3 recognition by UHRF1D334A or UHRF1D334A&Δ627–674 is not affected by DTT. RESULTS +59 69 UHRF1D334A mutant As negative controls, H3K9me3 recognition by UHRF1D334A or UHRF1D334A&Δ627–674 is not affected by DTT. RESULTS +70 78 Δ627–674 mutant As negative controls, H3K9me3 recognition by UHRF1D334A or UHRF1D334A&Δ627–674 is not affected by DTT. RESULTS +98 101 DTT chemical As negative controls, H3K9me3 recognition by UHRF1D334A or UHRF1D334A&Δ627–674 is not affected by DTT. RESULTS +52 63 full-length protein_state The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +64 69 UHRF1 protein The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +79 85 closed protein_state The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +105 111 Spacer structure_element The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +112 120 binds to protein_state The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +125 128 TTD structure_element The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +133 135 H3 protein_type The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +135 140 K9me3 ptm The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +172 178 hm-DNA chemical The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +193 199 Spacer structure_element The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +209 212 TTD structure_element The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +231 242 full-length protein_state The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +243 248 UHRF1 protein The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +284 291 histone protein_type The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +292 294 H3 protein_type The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +294 299 K9me3 ptm The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +348 351 PHD structure_element The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +353 356 SRA structure_element The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required). RESULTS +37 43 hm-DNA chemical We have previously demonstrated that hm-DNA also disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. RESULTS +58 65 PHD–SRA structure_element We have previously demonstrated that hm-DNA also disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. RESULTS +94 118 H3K9me0-binding affinity evidence We have previously demonstrated that hm-DNA also disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. RESULTS +126 129 PHD structure_element We have previously demonstrated that hm-DNA also disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. RESULTS +161 164 TTD structure_element We have previously demonstrated that hm-DNA also disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. RESULTS +172 178 Spacer structure_element We have previously demonstrated that hm-DNA also disrupts PHD–SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer. RESULTS +16 22 hm-DNA chemical Taken together, hm-DNA disrupts the intramolecular interactions within UHRF1, and therefore facilitates the coordinate recognition of H3K9me3 by TTD–PHD. RESULTS +71 76 UHRF1 protein Taken together, hm-DNA disrupts the intramolecular interactions within UHRF1, and therefore facilitates the coordinate recognition of H3K9me3 by TTD–PHD. RESULTS +134 136 H3 protein_type Taken together, hm-DNA disrupts the intramolecular interactions within UHRF1, and therefore facilitates the coordinate recognition of H3K9me3 by TTD–PHD. RESULTS +136 141 K9me3 ptm Taken together, hm-DNA disrupts the intramolecular interactions within UHRF1, and therefore facilitates the coordinate recognition of H3K9me3 by TTD–PHD. RESULTS +145 152 TTD–PHD structure_element Taken together, hm-DNA disrupts the intramolecular interactions within UHRF1, and therefore facilitates the coordinate recognition of H3K9me3 by TTD–PHD. RESULTS +4 10 spacer structure_element The spacer enhances hm-DNA-binding affinity of the SRA RESULTS +20 43 hm-DNA-binding affinity evidence The spacer enhances hm-DNA-binding affinity of the SRA RESULTS +51 54 SRA structure_element The spacer enhances hm-DNA-binding affinity of the SRA RESULTS +19 25 hm-DNA chemical To investigate how hm-DNA impairs TTD–Spacer interaction, we tested whether the Spacer is involved in hm-DNA recognition by the SRA, which is the only known domain for hm-DNA recognition within UHRF1. RESULTS +34 44 TTD–Spacer structure_element To investigate how hm-DNA impairs TTD–Spacer interaction, we tested whether the Spacer is involved in hm-DNA recognition by the SRA, which is the only known domain for hm-DNA recognition within UHRF1. RESULTS +80 86 Spacer structure_element To investigate how hm-DNA impairs TTD–Spacer interaction, we tested whether the Spacer is involved in hm-DNA recognition by the SRA, which is the only known domain for hm-DNA recognition within UHRF1. RESULTS +102 108 hm-DNA chemical To investigate how hm-DNA impairs TTD–Spacer interaction, we tested whether the Spacer is involved in hm-DNA recognition by the SRA, which is the only known domain for hm-DNA recognition within UHRF1. RESULTS +128 131 SRA structure_element To investigate how hm-DNA impairs TTD–Spacer interaction, we tested whether the Spacer is involved in hm-DNA recognition by the SRA, which is the only known domain for hm-DNA recognition within UHRF1. RESULTS +168 174 hm-DNA chemical To investigate how hm-DNA impairs TTD–Spacer interaction, we tested whether the Spacer is involved in hm-DNA recognition by the SRA, which is the only known domain for hm-DNA recognition within UHRF1. RESULTS +194 199 UHRF1 protein To investigate how hm-DNA impairs TTD–Spacer interaction, we tested whether the Spacer is involved in hm-DNA recognition by the SRA, which is the only known domain for hm-DNA recognition within UHRF1. RESULTS +7 43 electrophoretic mobility-shift assay experimental_method In the electrophoretic mobility-shift assay, SRA–Spacer showed higher hm-DNA-binding affinity than the SRA alone (Supplementary Fig. 6a). RESULTS +45 55 SRA–Spacer structure_element In the electrophoretic mobility-shift assay, SRA–Spacer showed higher hm-DNA-binding affinity than the SRA alone (Supplementary Fig. 6a). RESULTS +70 93 hm-DNA-binding affinity evidence In the electrophoretic mobility-shift assay, SRA–Spacer showed higher hm-DNA-binding affinity than the SRA alone (Supplementary Fig. 6a). RESULTS +103 106 SRA structure_element In the electrophoretic mobility-shift assay, SRA–Spacer showed higher hm-DNA-binding affinity than the SRA alone (Supplementary Fig. 6a). RESULTS +107 112 alone protein_state In the electrophoretic mobility-shift assay, SRA–Spacer showed higher hm-DNA-binding affinity than the SRA alone (Supplementary Fig. 6a). RESULTS +0 3 ITC experimental_method ITC measurements show that SRA–Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75 μM) than the SRA (KD=25.12 μM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a). RESULTS +27 37 SRA–Spacer structure_element ITC measurements show that SRA–Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75 μM) than the SRA (KD=25.12 μM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a). RESULTS +38 46 bound to protein_state ITC measurements show that SRA–Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75 μM) than the SRA (KD=25.12 μM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a). RESULTS +47 53 hm-DNA chemical ITC measurements show that SRA–Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75 μM) than the SRA (KD=25.12 μM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a). RESULTS +73 89 binding affinity evidence ITC measurements show that SRA–Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75 μM) than the SRA (KD=25.12 μM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a). RESULTS +91 93 KD evidence ITC measurements show that SRA–Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75 μM) than the SRA (KD=25.12 μM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a). RESULTS +112 115 SRA structure_element ITC measurements show that SRA–Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75 μM) than the SRA (KD=25.12 μM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a). RESULTS +117 119 KD evidence ITC measurements show that SRA–Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75 μM) than the SRA (KD=25.12 μM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a). RESULTS +143 149 Spacer structure_element ITC measurements show that SRA–Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75 μM) than the SRA (KD=25.12 μM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a). RESULTS +150 155 alone protein_state ITC measurements show that SRA–Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75 μM) than the SRA (KD=25.12 μM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a). RESULTS +183 189 hm-DNA chemical ITC measurements show that SRA–Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75 μM) than the SRA (KD=25.12 μM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a). RESULTS +7 36 fluorescence polarization (FP experimental_method In the fluorescence polarization (FP) measurements, SRA–Spacer, full-length UHRF1 and UHRF1ΔTTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not. RESULTS +52 62 SRA–Spacer structure_element In the fluorescence polarization (FP) measurements, SRA–Spacer, full-length UHRF1 and UHRF1ΔTTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not. RESULTS +64 75 full-length protein_state In the fluorescence polarization (FP) measurements, SRA–Spacer, full-length UHRF1 and UHRF1ΔTTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not. RESULTS +76 81 UHRF1 protein In the fluorescence polarization (FP) measurements, SRA–Spacer, full-length UHRF1 and UHRF1ΔTTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not. RESULTS +86 95 UHRF1ΔTTD mutant In the fluorescence polarization (FP) measurements, SRA–Spacer, full-length UHRF1 and UHRF1ΔTTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not. RESULTS +114 139 hm-DNA-binding affinities evidence In the fluorescence polarization (FP) measurements, SRA–Spacer, full-length UHRF1 and UHRF1ΔTTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not. RESULTS +193 198 UHRF1 protein In the fluorescence polarization (FP) measurements, SRA–Spacer, full-length UHRF1 and UHRF1ΔTTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not. RESULTS +199 207 binds to protein_state In the fluorescence polarization (FP) measurements, SRA–Spacer, full-length UHRF1 and UHRF1ΔTTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not. RESULTS +208 214 hm-DNA chemical In the fluorescence polarization (FP) measurements, SRA–Spacer, full-length UHRF1 and UHRF1ΔTTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not. RESULTS +225 230 UHRF1 protein In the fluorescence polarization (FP) measurements, SRA–Spacer, full-length UHRF1 and UHRF1ΔTTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not. RESULTS +240 246 closed protein_state In the fluorescence polarization (FP) measurements, SRA–Spacer, full-length UHRF1 and UHRF1ΔTTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not. RESULTS +13 22 UHRF1ΔSRA mutant In contrast, UHRF1ΔSRA abolished hm-DNA-binding affinity, indicating that the SRA is essential for hm-DNA recognition. RESULTS +33 56 hm-DNA-binding affinity evidence In contrast, UHRF1ΔSRA abolished hm-DNA-binding affinity, indicating that the SRA is essential for hm-DNA recognition. RESULTS +78 81 SRA structure_element In contrast, UHRF1ΔSRA abolished hm-DNA-binding affinity, indicating that the SRA is essential for hm-DNA recognition. RESULTS +99 105 hm-DNA chemical In contrast, UHRF1ΔSRA abolished hm-DNA-binding affinity, indicating that the SRA is essential for hm-DNA recognition. RESULTS +14 25 full-length protein_state Compared with full-length UHRF1, UHRF1Δ627–674 decreased the hm-DNA-binding affinity by a factor of 14 (Fig. 5b), further supporting that the Spacer plays an important role in hm-DNA recognition in the context of full-length UHRF1. RESULTS +26 31 UHRF1 protein Compared with full-length UHRF1, UHRF1Δ627–674 decreased the hm-DNA-binding affinity by a factor of 14 (Fig. 5b), further supporting that the Spacer plays an important role in hm-DNA recognition in the context of full-length UHRF1. RESULTS +33 46 UHRF1Δ627–674 mutant Compared with full-length UHRF1, UHRF1Δ627–674 decreased the hm-DNA-binding affinity by a factor of 14 (Fig. 5b), further supporting that the Spacer plays an important role in hm-DNA recognition in the context of full-length UHRF1. RESULTS +61 84 hm-DNA-binding affinity evidence Compared with full-length UHRF1, UHRF1Δ627–674 decreased the hm-DNA-binding affinity by a factor of 14 (Fig. 5b), further supporting that the Spacer plays an important role in hm-DNA recognition in the context of full-length UHRF1. RESULTS +142 148 Spacer structure_element Compared with full-length UHRF1, UHRF1Δ627–674 decreased the hm-DNA-binding affinity by a factor of 14 (Fig. 5b), further supporting that the Spacer plays an important role in hm-DNA recognition in the context of full-length UHRF1. RESULTS +176 182 hm-DNA chemical Compared with full-length UHRF1, UHRF1Δ627–674 decreased the hm-DNA-binding affinity by a factor of 14 (Fig. 5b), further supporting that the Spacer plays an important role in hm-DNA recognition in the context of full-length UHRF1. RESULTS +213 224 full-length protein_state Compared with full-length UHRF1, UHRF1Δ627–674 decreased the hm-DNA-binding affinity by a factor of 14 (Fig. 5b), further supporting that the Spacer plays an important role in hm-DNA recognition in the context of full-length UHRF1. RESULTS +225 230 UHRF1 protein Compared with full-length UHRF1, UHRF1Δ627–674 decreased the hm-DNA-binding affinity by a factor of 14 (Fig. 5b), further supporting that the Spacer plays an important role in hm-DNA recognition in the context of full-length UHRF1. RESULTS +13 38 hm-DNA-binding affinities evidence In addition, hm-DNA-binding affinities of SRA or SRA–Spacer did not obviously vary upon the change of DNA lengths but did decrease with the increasing salt concentrations (Supplementary Fig. 6b,c and Supplementary Table 5). RESULTS +42 45 SRA structure_element In addition, hm-DNA-binding affinities of SRA or SRA–Spacer did not obviously vary upon the change of DNA lengths but did decrease with the increasing salt concentrations (Supplementary Fig. 6b,c and Supplementary Table 5). RESULTS +49 59 SRA–Spacer structure_element In addition, hm-DNA-binding affinities of SRA or SRA–Spacer did not obviously vary upon the change of DNA lengths but did decrease with the increasing salt concentrations (Supplementary Fig. 6b,c and Supplementary Table 5). RESULTS +32 38 Spacer structure_element These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. RESULTS +48 56 binds to protein_state These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. RESULTS +61 64 TTD structure_element These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. RESULTS +78 80 H3 protein_type These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. RESULTS +80 85 K9me3 ptm These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. RESULTS +103 108 UHRF1 protein These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. RESULTS +116 122 closed protein_state These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. RESULTS +158 164 hm-DNA chemical These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. RESULTS +184 187 SRA structure_element These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. RESULTS +193 198 UHRF1 protein These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. RESULTS +199 207 binds to protein_state These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. RESULTS +208 214 hm-DNA chemical These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA. RESULTS +41 47 Spacer structure_element We next mapped the minimal region of the Spacer for the enhancement of hm-DNA-binding affinity. RESULTS +71 94 hm-DNA-binding affinity evidence We next mapped the minimal region of the Spacer for the enhancement of hm-DNA-binding affinity. RESULTS +0 14 SRA–Spacer-661 mutant SRA–Spacer-661 (residues 414–661) still maintained strong hm-DNA-binding affinity comparable to that of SRA–Spacer (residues 414–674), whereas SRA–Spacer-652 and SRA–Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642–661 are important for enhancing hm-DNA-binding affinity of the SRA. RESULTS +25 32 414–661 residue_range SRA–Spacer-661 (residues 414–661) still maintained strong hm-DNA-binding affinity comparable to that of SRA–Spacer (residues 414–674), whereas SRA–Spacer-652 and SRA–Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642–661 are important for enhancing hm-DNA-binding affinity of the SRA. RESULTS +58 81 hm-DNA-binding affinity evidence SRA–Spacer-661 (residues 414–661) still maintained strong hm-DNA-binding affinity comparable to that of SRA–Spacer (residues 414–674), whereas SRA–Spacer-652 and SRA–Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642–661 are important for enhancing hm-DNA-binding affinity of the SRA. RESULTS +104 114 SRA–Spacer structure_element SRA–Spacer-661 (residues 414–661) still maintained strong hm-DNA-binding affinity comparable to that of SRA–Spacer (residues 414–674), whereas SRA–Spacer-652 and SRA–Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642–661 are important for enhancing hm-DNA-binding affinity of the SRA. RESULTS +125 132 414–674 residue_range SRA–Spacer-661 (residues 414–661) still maintained strong hm-DNA-binding affinity comparable to that of SRA–Spacer (residues 414–674), whereas SRA–Spacer-652 and SRA–Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642–661 are important for enhancing hm-DNA-binding affinity of the SRA. RESULTS +143 157 SRA–Spacer-652 mutant SRA–Spacer-661 (residues 414–661) still maintained strong hm-DNA-binding affinity comparable to that of SRA–Spacer (residues 414–674), whereas SRA–Spacer-652 and SRA–Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642–661 are important for enhancing hm-DNA-binding affinity of the SRA. RESULTS +162 176 SRA–Spacer-642 mutant SRA–Spacer-661 (residues 414–661) still maintained strong hm-DNA-binding affinity comparable to that of SRA–Spacer (residues 414–674), whereas SRA–Spacer-652 and SRA–Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642–661 are important for enhancing hm-DNA-binding affinity of the SRA. RESULTS +202 227 hm-DNA-binding affinities evidence SRA–Spacer-661 (residues 414–661) still maintained strong hm-DNA-binding affinity comparable to that of SRA–Spacer (residues 414–674), whereas SRA–Spacer-652 and SRA–Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642–661 are important for enhancing hm-DNA-binding affinity of the SRA. RESULTS +264 271 642–661 residue_range SRA–Spacer-661 (residues 414–661) still maintained strong hm-DNA-binding affinity comparable to that of SRA–Spacer (residues 414–674), whereas SRA–Spacer-652 and SRA–Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642–661 are important for enhancing hm-DNA-binding affinity of the SRA. RESULTS +300 323 hm-DNA-binding affinity evidence SRA–Spacer-661 (residues 414–661) still maintained strong hm-DNA-binding affinity comparable to that of SRA–Spacer (residues 414–674), whereas SRA–Spacer-652 and SRA–Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642–661 are important for enhancing hm-DNA-binding affinity of the SRA. RESULTS +331 334 SRA structure_element SRA–Spacer-661 (residues 414–661) still maintained strong hm-DNA-binding affinity comparable to that of SRA–Spacer (residues 414–674), whereas SRA–Spacer-652 and SRA–Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642–661 are important for enhancing hm-DNA-binding affinity of the SRA. RESULTS +46 52 Spacer structure_element This minimal region largely overlaps with the Spacer region (643–655) essential for TTD interaction. RESULTS +61 68 643–655 residue_range This minimal region largely overlaps with the Spacer region (643–655) essential for TTD interaction. RESULTS +84 87 TTD structure_element This minimal region largely overlaps with the Spacer region (643–655) essential for TTD interaction. RESULTS +23 40 crystal structure evidence We also determined the crystal structure of SRA–Spacer bound to hm-DNA at 3.15 Å resolution (Supplementary Table 6 and Supplementary Fig. 7a). RESULTS +44 54 SRA–Spacer structure_element We also determined the crystal structure of SRA–Spacer bound to hm-DNA at 3.15 Å resolution (Supplementary Table 6 and Supplementary Fig. 7a). RESULTS +55 63 bound to protein_state We also determined the crystal structure of SRA–Spacer bound to hm-DNA at 3.15 Å resolution (Supplementary Table 6 and Supplementary Fig. 7a). RESULTS +64 70 hm-DNA chemical We also determined the crystal structure of SRA–Spacer bound to hm-DNA at 3.15 Å resolution (Supplementary Table 6 and Supplementary Fig. 7a). RESULTS +4 13 structure evidence The structure shows that the SRA binds to hm-DNA in a manner similar to that observed in the previously reported SRA-hm-DNA structures. RESULTS +29 32 SRA structure_element The structure shows that the SRA binds to hm-DNA in a manner similar to that observed in the previously reported SRA-hm-DNA structures. RESULTS +33 41 binds to protein_state The structure shows that the SRA binds to hm-DNA in a manner similar to that observed in the previously reported SRA-hm-DNA structures. RESULTS +42 48 hm-DNA chemical The structure shows that the SRA binds to hm-DNA in a manner similar to that observed in the previously reported SRA-hm-DNA structures. RESULTS +113 123 SRA-hm-DNA complex_assembly The structure shows that the SRA binds to hm-DNA in a manner similar to that observed in the previously reported SRA-hm-DNA structures. RESULTS +124 134 structures evidence The structure shows that the SRA binds to hm-DNA in a manner similar to that observed in the previously reported SRA-hm-DNA structures. RESULTS +17 33 electron density evidence Intriguingly, no electron density was observed for the Spacer. RESULTS +55 61 Spacer structure_element Intriguingly, no electron density was observed for the Spacer. RESULTS +35 41 Spacer structure_element A possible explanation is that the Spacer facilitates SRA–hm-DNA interaction through nonspecific salt bridge contacts because DNA is rich in acidic groups and the Spacer is rich in basic residues (Supplementary Fig. 7b). RESULTS +54 64 SRA–hm-DNA complex_assembly A possible explanation is that the Spacer facilitates SRA–hm-DNA interaction through nonspecific salt bridge contacts because DNA is rich in acidic groups and the Spacer is rich in basic residues (Supplementary Fig. 7b). RESULTS +97 108 salt bridge bond_interaction A possible explanation is that the Spacer facilitates SRA–hm-DNA interaction through nonspecific salt bridge contacts because DNA is rich in acidic groups and the Spacer is rich in basic residues (Supplementary Fig. 7b). RESULTS +126 129 DNA chemical A possible explanation is that the Spacer facilitates SRA–hm-DNA interaction through nonspecific salt bridge contacts because DNA is rich in acidic groups and the Spacer is rich in basic residues (Supplementary Fig. 7b). RESULTS +163 169 Spacer structure_element A possible explanation is that the Spacer facilitates SRA–hm-DNA interaction through nonspecific salt bridge contacts because DNA is rich in acidic groups and the Spacer is rich in basic residues (Supplementary Fig. 7b). RESULTS +77 82 UHRF1 protein The nonspecific interaction is consistent with the previous observation that UHRF1 has no DNA sequence selectivity besides hm-CpG dinucleotide. RESULTS +90 93 DNA chemical The nonspecific interaction is consistent with the previous observation that UHRF1 has no DNA sequence selectivity besides hm-CpG dinucleotide. RESULTS +123 142 hm-CpG dinucleotide chemical The nonspecific interaction is consistent with the previous observation that UHRF1 has no DNA sequence selectivity besides hm-CpG dinucleotide. RESULTS +4 10 spacer structure_element The spacer is important for PCH localization of UHRF1 RESULTS +48 53 UHRF1 protein The spacer is important for PCH localization of UHRF1 RESULTS +31 37 Spacer structure_element To investigate the role of the Spacer in the regulation of UHRF1 function, we transiently overexpressed GFP-tagged wild type or mutants of UHRF1 in NIH3T3 cells to determine their subcellular localization. RESULTS +59 64 UHRF1 protein To investigate the role of the Spacer in the regulation of UHRF1 function, we transiently overexpressed GFP-tagged wild type or mutants of UHRF1 in NIH3T3 cells to determine their subcellular localization. RESULTS +78 103 transiently overexpressed experimental_method To investigate the role of the Spacer in the regulation of UHRF1 function, we transiently overexpressed GFP-tagged wild type or mutants of UHRF1 in NIH3T3 cells to determine their subcellular localization. RESULTS +104 114 GFP-tagged protein_state To investigate the role of the Spacer in the regulation of UHRF1 function, we transiently overexpressed GFP-tagged wild type or mutants of UHRF1 in NIH3T3 cells to determine their subcellular localization. RESULTS +115 124 wild type protein_state To investigate the role of the Spacer in the regulation of UHRF1 function, we transiently overexpressed GFP-tagged wild type or mutants of UHRF1 in NIH3T3 cells to determine their subcellular localization. RESULTS +128 135 mutants protein_state To investigate the role of the Spacer in the regulation of UHRF1 function, we transiently overexpressed GFP-tagged wild type or mutants of UHRF1 in NIH3T3 cells to determine their subcellular localization. RESULTS +139 144 UHRF1 protein To investigate the role of the Spacer in the regulation of UHRF1 function, we transiently overexpressed GFP-tagged wild type or mutants of UHRF1 in NIH3T3 cells to determine their subcellular localization. RESULTS +32 41 wild-type protein_state For the NIH3T3 cells expressing wild-type UHRF1, most cells (∼74.6%) showed a focal pattern of protein that is co-localized with 4,6-diamidino-2-phenylindole (DAPI) foci (Fig. 5d), whereas the rest cells showed a diffuse nuclear staining pattern. RESULTS +42 47 UHRF1 protein For the NIH3T3 cells expressing wild-type UHRF1, most cells (∼74.6%) showed a focal pattern of protein that is co-localized with 4,6-diamidino-2-phenylindole (DAPI) foci (Fig. 5d), whereas the rest cells showed a diffuse nuclear staining pattern. RESULTS +129 157 4,6-diamidino-2-phenylindole chemical For the NIH3T3 cells expressing wild-type UHRF1, most cells (∼74.6%) showed a focal pattern of protein that is co-localized with 4,6-diamidino-2-phenylindole (DAPI) foci (Fig. 5d), whereas the rest cells showed a diffuse nuclear staining pattern. RESULTS +159 163 DAPI chemical For the NIH3T3 cells expressing wild-type UHRF1, most cells (∼74.6%) showed a focal pattern of protein that is co-localized with 4,6-diamidino-2-phenylindole (DAPI) foci (Fig. 5d), whereas the rest cells showed a diffuse nuclear staining pattern. RESULTS +56 61 UHRF1 protein The result is consistent with the previous studies that UHRF1 is mainly localized to highly methylated pericentromeric heterochromatin (PCH). RESULTS +85 102 highly methylated protein_state The result is consistent with the previous studies that UHRF1 is mainly localized to highly methylated pericentromeric heterochromatin (PCH). RESULTS +38 51 UHRF1Δ627–674 mutant In contrast, for the cells expressing UHRF1Δ627–674, a spacer deletion mutant with decreased hm-DNA-binding affinity (Fig. 5b), only ∼22.1% cells showed co-localization with DAPI. RESULTS +55 77 spacer deletion mutant protein_state In contrast, for the cells expressing UHRF1Δ627–674, a spacer deletion mutant with decreased hm-DNA-binding affinity (Fig. 5b), only ∼22.1% cells showed co-localization with DAPI. RESULTS +93 116 hm-DNA-binding affinity evidence In contrast, for the cells expressing UHRF1Δ627–674, a spacer deletion mutant with decreased hm-DNA-binding affinity (Fig. 5b), only ∼22.1% cells showed co-localization with DAPI. RESULTS +174 178 DAPI chemical In contrast, for the cells expressing UHRF1Δ627–674, a spacer deletion mutant with decreased hm-DNA-binding affinity (Fig. 5b), only ∼22.1% cells showed co-localization with DAPI. RESULTS +37 39 H3 protein_type Previous reports have shown that the H3K9me3 recognition of UHRF1 also plays an important role in its heterochromatin localization. RESULTS +39 44 K9me3 ptm Previous reports have shown that the H3K9me3 recognition of UHRF1 also plays an important role in its heterochromatin localization. RESULTS +60 65 UHRF1 protein Previous reports have shown that the H3K9me3 recognition of UHRF1 also plays an important role in its heterochromatin localization. RESULTS +13 18 UHRF1 protein For example, UHRF1 mutant (within TTD domain) lacking H3K9me3-binding affinity largely reduces its co-localization with heterochromatin. RESULTS +19 25 mutant protein_state For example, UHRF1 mutant (within TTD domain) lacking H3K9me3-binding affinity largely reduces its co-localization with heterochromatin. RESULTS +34 37 TTD structure_element For example, UHRF1 mutant (within TTD domain) lacking H3K9me3-binding affinity largely reduces its co-localization with heterochromatin. RESULTS +46 53 lacking protein_state For example, UHRF1 mutant (within TTD domain) lacking H3K9me3-binding affinity largely reduces its co-localization with heterochromatin. RESULTS +54 78 H3K9me3-binding affinity evidence For example, UHRF1 mutant (within TTD domain) lacking H3K9me3-binding affinity largely reduces its co-localization with heterochromatin. RESULTS +35 41 hm-DNA chemical Because manipulation of endogenous hm-DNA in cells is technically challenging, we used UHRF1ΔSRA (lacks hm-DNA-binding affinity but maintains closed conformation, Figs 3h and 5b) to test whether closed conformation of UHRF1 exists in vivo. RESULTS +87 96 UHRF1ΔSRA mutant Because manipulation of endogenous hm-DNA in cells is technically challenging, we used UHRF1ΔSRA (lacks hm-DNA-binding affinity but maintains closed conformation, Figs 3h and 5b) to test whether closed conformation of UHRF1 exists in vivo. RESULTS +98 103 lacks protein_state Because manipulation of endogenous hm-DNA in cells is technically challenging, we used UHRF1ΔSRA (lacks hm-DNA-binding affinity but maintains closed conformation, Figs 3h and 5b) to test whether closed conformation of UHRF1 exists in vivo. RESULTS +104 127 hm-DNA-binding affinity evidence Because manipulation of endogenous hm-DNA in cells is technically challenging, we used UHRF1ΔSRA (lacks hm-DNA-binding affinity but maintains closed conformation, Figs 3h and 5b) to test whether closed conformation of UHRF1 exists in vivo. RESULTS +142 148 closed protein_state Because manipulation of endogenous hm-DNA in cells is technically challenging, we used UHRF1ΔSRA (lacks hm-DNA-binding affinity but maintains closed conformation, Figs 3h and 5b) to test whether closed conformation of UHRF1 exists in vivo. RESULTS +195 201 closed protein_state Because manipulation of endogenous hm-DNA in cells is technically challenging, we used UHRF1ΔSRA (lacks hm-DNA-binding affinity but maintains closed conformation, Figs 3h and 5b) to test whether closed conformation of UHRF1 exists in vivo. RESULTS +218 223 UHRF1 protein Because manipulation of endogenous hm-DNA in cells is technically challenging, we used UHRF1ΔSRA (lacks hm-DNA-binding affinity but maintains closed conformation, Figs 3h and 5b) to test whether closed conformation of UHRF1 exists in vivo. RESULTS +17 26 UHRF1ΔSRA mutant In NIH3T3 cells, UHRF1ΔSRA largely decreased chromatin association (Fig. 5d). RESULTS +28 37 UHRF1ΔSRA mutant Only ∼4.8% cells expressing UHRF1ΔSRA showed an intermediate enrichment, but not characteristic focal pattern, at DAPI foci, whereas the majority of the cells showed a diffuse nuclear staining pattern. RESULTS +114 118 DAPI chemical Only ∼4.8% cells expressing UHRF1ΔSRA showed an intermediate enrichment, but not characteristic focal pattern, at DAPI foci, whereas the majority of the cells showed a diffuse nuclear staining pattern. RESULTS +25 34 UHRF1ΔSRA mutant The results suggest that UHRF1ΔSRA adopts closed conformation so that H3K9me3 recognition by TTD–PHD is blocked by the intramolecular interaction, and support the regulatory role of the Spacer in PCH localization of UHRF1 in vivo. RESULTS +42 48 closed protein_state The results suggest that UHRF1ΔSRA adopts closed conformation so that H3K9me3 recognition by TTD–PHD is blocked by the intramolecular interaction, and support the regulatory role of the Spacer in PCH localization of UHRF1 in vivo. RESULTS +70 72 H3 protein_type The results suggest that UHRF1ΔSRA adopts closed conformation so that H3K9me3 recognition by TTD–PHD is blocked by the intramolecular interaction, and support the regulatory role of the Spacer in PCH localization of UHRF1 in vivo. RESULTS +72 77 K9me3 ptm The results suggest that UHRF1ΔSRA adopts closed conformation so that H3K9me3 recognition by TTD–PHD is blocked by the intramolecular interaction, and support the regulatory role of the Spacer in PCH localization of UHRF1 in vivo. RESULTS +93 100 TTD–PHD structure_element The results suggest that UHRF1ΔSRA adopts closed conformation so that H3K9me3 recognition by TTD–PHD is blocked by the intramolecular interaction, and support the regulatory role of the Spacer in PCH localization of UHRF1 in vivo. RESULTS +186 192 Spacer structure_element The results suggest that UHRF1ΔSRA adopts closed conformation so that H3K9me3 recognition by TTD–PHD is blocked by the intramolecular interaction, and support the regulatory role of the Spacer in PCH localization of UHRF1 in vivo. RESULTS +216 221 UHRF1 protein The results suggest that UHRF1ΔSRA adopts closed conformation so that H3K9me3 recognition by TTD–PHD is blocked by the intramolecular interaction, and support the regulatory role of the Spacer in PCH localization of UHRF1 in vivo. RESULTS +4 10 spacer structure_element The spacer facilitates UHRF1–DNMT1 interaction RESULTS +23 34 UHRF1–DNMT1 complex_assembly The spacer facilitates UHRF1–DNMT1 interaction RESULTS +27 32 UHRF1 protein Previous studies show that UHRF1 recruits DNMT1 to hm-DNA for maintenance DNA methylation through the interaction between the SRA and RFTSDNMT1 (refs). RESULTS +42 47 DNMT1 protein Previous studies show that UHRF1 recruits DNMT1 to hm-DNA for maintenance DNA methylation through the interaction between the SRA and RFTSDNMT1 (refs). RESULTS +51 57 hm-DNA chemical Previous studies show that UHRF1 recruits DNMT1 to hm-DNA for maintenance DNA methylation through the interaction between the SRA and RFTSDNMT1 (refs). RESULTS +74 77 DNA chemical Previous studies show that UHRF1 recruits DNMT1 to hm-DNA for maintenance DNA methylation through the interaction between the SRA and RFTSDNMT1 (refs). RESULTS +78 89 methylation ptm Previous studies show that UHRF1 recruits DNMT1 to hm-DNA for maintenance DNA methylation through the interaction between the SRA and RFTSDNMT1 (refs). RESULTS +126 129 SRA structure_element Previous studies show that UHRF1 recruits DNMT1 to hm-DNA for maintenance DNA methylation through the interaction between the SRA and RFTSDNMT1 (refs). RESULTS +134 143 RFTSDNMT1 protein Previous studies show that UHRF1 recruits DNMT1 to hm-DNA for maintenance DNA methylation through the interaction between the SRA and RFTSDNMT1 (refs). RESULTS +44 53 RFTSDNMT1 protein We confirmed the direct interaction between RFTSDNMT1 and the SRA in a solution with low salt concentration (50 mM NaCl), but observed weak or undetectable interaction in a solution with higher salt concentrations (100 or 150 mM NaCl) (Supplementary Fig. 8a). RESULTS +62 65 SRA structure_element We confirmed the direct interaction between RFTSDNMT1 and the SRA in a solution with low salt concentration (50 mM NaCl), but observed weak or undetectable interaction in a solution with higher salt concentrations (100 or 150 mM NaCl) (Supplementary Fig. 8a). RESULTS +115 119 NaCl chemical We confirmed the direct interaction between RFTSDNMT1 and the SRA in a solution with low salt concentration (50 mM NaCl), but observed weak or undetectable interaction in a solution with higher salt concentrations (100 or 150 mM NaCl) (Supplementary Fig. 8a). RESULTS +229 233 NaCl chemical We confirmed the direct interaction between RFTSDNMT1 and the SRA in a solution with low salt concentration (50 mM NaCl), but observed weak or undetectable interaction in a solution with higher salt concentrations (100 or 150 mM NaCl) (Supplementary Fig. 8a). RESULTS +18 21 SRA structure_element Compared with the SRA, SRA–Spacer exhibited stronger interaction with RFTSDNMT1. RESULTS +23 33 SRA–Spacer structure_element Compared with the SRA, SRA–Spacer exhibited stronger interaction with RFTSDNMT1. RESULTS +70 79 RFTSDNMT1 protein Compared with the SRA, SRA–Spacer exhibited stronger interaction with RFTSDNMT1. RESULTS +13 22 RFTSDNMT1 protein In addition, RFTSDNMT1 bound to SRA–Spacer with a binding affinity of 7.09 μM, but showed no detectable interaction with the SRA (Supplementary Fig. 8b). RESULTS +23 31 bound to protein_state In addition, RFTSDNMT1 bound to SRA–Spacer with a binding affinity of 7.09 μM, but showed no detectable interaction with the SRA (Supplementary Fig. 8b). RESULTS +32 42 SRA–Spacer structure_element In addition, RFTSDNMT1 bound to SRA–Spacer with a binding affinity of 7.09 μM, but showed no detectable interaction with the SRA (Supplementary Fig. 8b). RESULTS +50 66 binding affinity evidence In addition, RFTSDNMT1 bound to SRA–Spacer with a binding affinity of 7.09 μM, but showed no detectable interaction with the SRA (Supplementary Fig. 8b). RESULTS +125 128 SRA structure_element In addition, RFTSDNMT1 bound to SRA–Spacer with a binding affinity of 7.09 μM, but showed no detectable interaction with the SRA (Supplementary Fig. 8b). RESULTS +31 37 hm-DNA chemical Interestingly, the addition of hm-DNA abolished the interaction between RFTSDNMT1 and SRA–Spacer, suggesting that hm-DNA also regulates UHRF1–DNMT1 interaction (Supplementary Fig. 8c). RESULTS +72 81 RFTSDNMT1 protein Interestingly, the addition of hm-DNA abolished the interaction between RFTSDNMT1 and SRA–Spacer, suggesting that hm-DNA also regulates UHRF1–DNMT1 interaction (Supplementary Fig. 8c). RESULTS +86 96 SRA–Spacer structure_element Interestingly, the addition of hm-DNA abolished the interaction between RFTSDNMT1 and SRA–Spacer, suggesting that hm-DNA also regulates UHRF1–DNMT1 interaction (Supplementary Fig. 8c). RESULTS +114 120 hm-DNA chemical Interestingly, the addition of hm-DNA abolished the interaction between RFTSDNMT1 and SRA–Spacer, suggesting that hm-DNA also regulates UHRF1–DNMT1 interaction (Supplementary Fig. 8c). RESULTS +136 147 UHRF1–DNMT1 complex_assembly Interestingly, the addition of hm-DNA abolished the interaction between RFTSDNMT1 and SRA–Spacer, suggesting that hm-DNA also regulates UHRF1–DNMT1 interaction (Supplementary Fig. 8c). RESULTS +32 38 Spacer structure_element These results indicate that the Spacer facilitates the interaction between RFTSDNMT1 and the SRA, and the interaction is impaired by the presence of hm-DNA. RESULTS +75 84 RFTSDNMT1 protein These results indicate that the Spacer facilitates the interaction between RFTSDNMT1 and the SRA, and the interaction is impaired by the presence of hm-DNA. RESULTS +93 96 SRA structure_element These results indicate that the Spacer facilitates the interaction between RFTSDNMT1 and the SRA, and the interaction is impaired by the presence of hm-DNA. RESULTS +137 148 presence of protein_state These results indicate that the Spacer facilitates the interaction between RFTSDNMT1 and the SRA, and the interaction is impaired by the presence of hm-DNA. RESULTS +149 155 hm-DNA chemical These results indicate that the Spacer facilitates the interaction between RFTSDNMT1 and the SRA, and the interaction is impaired by the presence of hm-DNA. RESULTS +27 38 UHRF1–DNMT1 complex_assembly We next tested whether the UHRF1–DNMT1 interaction is regulated by the conformational change of UHRF1. RESULTS +96 101 UHRF1 protein We next tested whether the UHRF1–DNMT1 interaction is regulated by the conformational change of UHRF1. RESULTS +24 30 hm-DNA chemical Because the addition of hm-DNA disrupts the interaction between the SRA–Spacer and RFTSDNMT1, we used various truncations to mimic open and closed forms of UHRF1. RESULTS +68 78 SRA–Spacer structure_element Because the addition of hm-DNA disrupts the interaction between the SRA–Spacer and RFTSDNMT1, we used various truncations to mimic open and closed forms of UHRF1. RESULTS +83 92 RFTSDNMT1 protein Because the addition of hm-DNA disrupts the interaction between the SRA–Spacer and RFTSDNMT1, we used various truncations to mimic open and closed forms of UHRF1. RESULTS +110 121 truncations experimental_method Because the addition of hm-DNA disrupts the interaction between the SRA–Spacer and RFTSDNMT1, we used various truncations to mimic open and closed forms of UHRF1. RESULTS +131 135 open protein_state Because the addition of hm-DNA disrupts the interaction between the SRA–Spacer and RFTSDNMT1, we used various truncations to mimic open and closed forms of UHRF1. RESULTS +140 146 closed protein_state Because the addition of hm-DNA disrupts the interaction between the SRA–Spacer and RFTSDNMT1, we used various truncations to mimic open and closed forms of UHRF1. RESULTS +156 161 UHRF1 protein Because the addition of hm-DNA disrupts the interaction between the SRA–Spacer and RFTSDNMT1, we used various truncations to mimic open and closed forms of UHRF1. RESULTS +7 17 absence of protein_state In the absence of hm-DNA, only UHRF1ΔTTD bound to RFTSDNMT1, whereas full-length UHRF1, UHRF1ΔSRA and UHRF1Δ627–674 showed undetectable interaction (Fig. 5e). RESULTS +18 24 hm-DNA chemical In the absence of hm-DNA, only UHRF1ΔTTD bound to RFTSDNMT1, whereas full-length UHRF1, UHRF1ΔSRA and UHRF1Δ627–674 showed undetectable interaction (Fig. 5e). RESULTS +31 40 UHRF1ΔTTD mutant In the absence of hm-DNA, only UHRF1ΔTTD bound to RFTSDNMT1, whereas full-length UHRF1, UHRF1ΔSRA and UHRF1Δ627–674 showed undetectable interaction (Fig. 5e). RESULTS +41 49 bound to protein_state In the absence of hm-DNA, only UHRF1ΔTTD bound to RFTSDNMT1, whereas full-length UHRF1, UHRF1ΔSRA and UHRF1Δ627–674 showed undetectable interaction (Fig. 5e). RESULTS +50 59 RFTSDNMT1 protein In the absence of hm-DNA, only UHRF1ΔTTD bound to RFTSDNMT1, whereas full-length UHRF1, UHRF1ΔSRA and UHRF1Δ627–674 showed undetectable interaction (Fig. 5e). RESULTS +69 80 full-length protein_state In the absence of hm-DNA, only UHRF1ΔTTD bound to RFTSDNMT1, whereas full-length UHRF1, UHRF1ΔSRA and UHRF1Δ627–674 showed undetectable interaction (Fig. 5e). RESULTS +81 86 UHRF1 protein In the absence of hm-DNA, only UHRF1ΔTTD bound to RFTSDNMT1, whereas full-length UHRF1, UHRF1ΔSRA and UHRF1Δ627–674 showed undetectable interaction (Fig. 5e). RESULTS +88 97 UHRF1ΔSRA mutant In the absence of hm-DNA, only UHRF1ΔTTD bound to RFTSDNMT1, whereas full-length UHRF1, UHRF1ΔSRA and UHRF1Δ627–674 showed undetectable interaction (Fig. 5e). RESULTS +102 115 UHRF1Δ627–674 mutant In the absence of hm-DNA, only UHRF1ΔTTD bound to RFTSDNMT1, whereas full-length UHRF1, UHRF1ΔSRA and UHRF1Δ627–674 showed undetectable interaction (Fig. 5e). RESULTS +7 18 deletion of experimental_method As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA–Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA. RESULTS +23 26 TTD structure_element As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA–Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA. RESULTS +34 39 UHRF1 protein As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA–Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA. RESULTS +52 56 open protein_state As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA–Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA. RESULTS +96 105 RFTSDNMT1 protein As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA–Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA. RESULTS +106 114 binds to protein_state As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA–Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA. RESULTS +115 125 SRA–Spacer structure_element As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA–Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA. RESULTS +131 136 UHRF1 protein As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA–Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA. RESULTS +147 151 open protein_state As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA–Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA. RESULTS +172 182 absence of protein_state As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA–Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA. RESULTS +183 189 hm-DNA chemical As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA–Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA. RESULTS +38 46 addition experimental_method In support of above observations, the addition of large amount of RFTSDNMT1 impaired the interaction between UHRF1 and hm-DNA (Supplementary Fig. 8d), suggesting an existence of dynamic equilibrium between UHRF1–hm-DNA and UHRF1–DNMT1 complexes. RESULTS +66 75 RFTSDNMT1 protein In support of above observations, the addition of large amount of RFTSDNMT1 impaired the interaction between UHRF1 and hm-DNA (Supplementary Fig. 8d), suggesting an existence of dynamic equilibrium between UHRF1–hm-DNA and UHRF1–DNMT1 complexes. RESULTS +109 114 UHRF1 protein In support of above observations, the addition of large amount of RFTSDNMT1 impaired the interaction between UHRF1 and hm-DNA (Supplementary Fig. 8d), suggesting an existence of dynamic equilibrium between UHRF1–hm-DNA and UHRF1–DNMT1 complexes. RESULTS +119 125 hm-DNA chemical In support of above observations, the addition of large amount of RFTSDNMT1 impaired the interaction between UHRF1 and hm-DNA (Supplementary Fig. 8d), suggesting an existence of dynamic equilibrium between UHRF1–hm-DNA and UHRF1–DNMT1 complexes. RESULTS +206 218 UHRF1–hm-DNA complex_assembly In support of above observations, the addition of large amount of RFTSDNMT1 impaired the interaction between UHRF1 and hm-DNA (Supplementary Fig. 8d), suggesting an existence of dynamic equilibrium between UHRF1–hm-DNA and UHRF1–DNMT1 complexes. RESULTS +223 234 UHRF1–DNMT1 complex_assembly In support of above observations, the addition of large amount of RFTSDNMT1 impaired the interaction between UHRF1 and hm-DNA (Supplementary Fig. 8d), suggesting an existence of dynamic equilibrium between UHRF1–hm-DNA and UHRF1–DNMT1 complexes. RESULTS +69 75 hm-DNA chemical According to the above results, we here proposed a working model for hm-DNA-mediated regulation of UHRF1 conformation (Fig. 5f). DISCUSS +99 104 UHRF1 protein According to the above results, we here proposed a working model for hm-DNA-mediated regulation of UHRF1 conformation (Fig. 5f). DISCUSS +7 17 absence of protein_state In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD. DISCUSS +18 24 hm-DNA chemical In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD. DISCUSS +30 35 UHRF1 protein In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD. DISCUSS +46 52 closed protein_state In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD. DISCUSS +80 86 Spacer structure_element In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD. DISCUSS +87 95 binds to protein_state In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD. DISCUSS +100 103 TTD structure_element In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD. DISCUSS +126 132 Linker structure_element In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD. DISCUSS +141 144 SRA structure_element In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD. DISCUSS +145 153 binds to protein_state In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD. DISCUSS +158 161 PHD structure_element In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD. DISCUSS +32 39 histone protein_type As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA. DISCUSS +40 42 H3 protein_type As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA. DISCUSS +42 47 K9me3 ptm As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA. DISCUSS +55 58 TTD structure_element As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA. DISCUSS +77 83 Spacer structure_element As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA. DISCUSS +104 114 unmodified protein_state As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA. DISCUSS +115 122 histone protein_type As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA. DISCUSS +123 125 H3 protein_type As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA. DISCUSS +127 131 H3R2 site As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA. DISCUSS +140 143 PHD structure_element As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA. DISCUSS +164 167 SRA structure_element As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA. DISCUSS +24 29 UHRF1 protein The interaction between UHRF1 and DNMT1 is also weak because the Spacer is unable to facilitate the intermolecular interaction. DISCUSS +34 39 DNMT1 protein The interaction between UHRF1 and DNMT1 is also weak because the Spacer is unable to facilitate the intermolecular interaction. DISCUSS +65 71 Spacer structure_element The interaction between UHRF1 and DNMT1 is also weak because the Spacer is unable to facilitate the intermolecular interaction. DISCUSS +7 18 presence of protein_state In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +19 25 hm-DNA chemical In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +31 36 UHRF1 protein In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +48 52 open protein_state In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +80 83 SRA structure_element In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +84 92 binds to protein_state In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +97 103 hm-DNA chemical In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +109 115 Spacer structure_element In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +137 140 TTD structure_element In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +185 188 SRA structure_element In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +193 199 hm-DNA chemical In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +205 211 Linker structure_element In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +212 220 binds to protein_state In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +225 228 TTD structure_element In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +240 247 TTD–PHD structure_element In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +261 268 histone protein_type In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +269 271 H3 protein_type In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +271 276 K9me3 ptm In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD–PHD to recognize histone H3K9me3. DISCUSS +5 10 UHRF1 protein When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. DISCUSS +21 25 open protein_state When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. DISCUSS +55 63 bound to protein_state When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. DISCUSS +64 66 H3 protein_type When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. DISCUSS +66 71 K9me3 ptm When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. DISCUSS +101 103 H3 protein_type When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. DISCUSS +103 108 K9me3 ptm When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. DISCUSS +113 120 TTD–PHD structure_element When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. DISCUSS +142 148 Spacer structure_element When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. DISCUSS +188 191 TTD structure_element When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. DISCUSS +213 218 UHRF1 protein When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. DISCUSS +225 229 open protein_state When UHRF1 adopts an open conformation and has already bound to H3K9me3 (B), the interaction between H3K9me3 and TTD–PHD further prevents the Spacer from folding back to interact with the TTD, and therefore locks UHRF1 in an open conformation. DISCUSS +19 24 UHRF1 protein The association of UHRF1 to the histone may facilitate the ubiquitination of histone tail (mediated by RING domain) for DNMT1 targeting. DISCUSS +32 39 histone protein_type The association of UHRF1 to the histone may facilitate the ubiquitination of histone tail (mediated by RING domain) for DNMT1 targeting. DISCUSS +59 73 ubiquitination ptm The association of UHRF1 to the histone may facilitate the ubiquitination of histone tail (mediated by RING domain) for DNMT1 targeting. DISCUSS +77 84 histone protein_type The association of UHRF1 to the histone may facilitate the ubiquitination of histone tail (mediated by RING domain) for DNMT1 targeting. DISCUSS +103 107 RING structure_element The association of UHRF1 to the histone may facilitate the ubiquitination of histone tail (mediated by RING domain) for DNMT1 targeting. DISCUSS +120 125 DNMT1 protein The association of UHRF1 to the histone may facilitate the ubiquitination of histone tail (mediated by RING domain) for DNMT1 targeting. DISCUSS +58 63 DNMT1 protein Moreover, through a mechanism yet to be fully elucidated, DNMT1 targets hm-DNA for maintenance DNA methylation, probably through interaction with the histone ubiquitylation and/or SRA-Spacer. DISCUSS +72 78 hm-DNA chemical Moreover, through a mechanism yet to be fully elucidated, DNMT1 targets hm-DNA for maintenance DNA methylation, probably through interaction with the histone ubiquitylation and/or SRA-Spacer. DISCUSS +99 110 methylation ptm Moreover, through a mechanism yet to be fully elucidated, DNMT1 targets hm-DNA for maintenance DNA methylation, probably through interaction with the histone ubiquitylation and/or SRA-Spacer. DISCUSS +150 157 histone protein_type Moreover, through a mechanism yet to be fully elucidated, DNMT1 targets hm-DNA for maintenance DNA methylation, probably through interaction with the histone ubiquitylation and/or SRA-Spacer. DISCUSS +158 172 ubiquitylation ptm Moreover, through a mechanism yet to be fully elucidated, DNMT1 targets hm-DNA for maintenance DNA methylation, probably through interaction with the histone ubiquitylation and/or SRA-Spacer. DISCUSS +180 190 SRA-Spacer structure_element Moreover, through a mechanism yet to be fully elucidated, DNMT1 targets hm-DNA for maintenance DNA methylation, probably through interaction with the histone ubiquitylation and/or SRA-Spacer. DISCUSS +4 17 P(r) function evidence The P(r) function obtained from small-angle X-ray scattering (SAXS) measurements of TTD–PHD–SRA–Spacer–hm-DNA complex showed a broader distribution than that of the TTD–PHD–SRA–Spacer alone, supporting the proposed model that UHRF1 adopts an open conformation in the presence of hm-DNA (Supplementary Fig. 8e). DISCUSS +32 60 small-angle X-ray scattering experimental_method The P(r) function obtained from small-angle X-ray scattering (SAXS) measurements of TTD–PHD–SRA–Spacer–hm-DNA complex showed a broader distribution than that of the TTD–PHD–SRA–Spacer alone, supporting the proposed model that UHRF1 adopts an open conformation in the presence of hm-DNA (Supplementary Fig. 8e). DISCUSS +62 66 SAXS experimental_method The P(r) function obtained from small-angle X-ray scattering (SAXS) measurements of TTD–PHD–SRA–Spacer–hm-DNA complex showed a broader distribution than that of the TTD–PHD–SRA–Spacer alone, supporting the proposed model that UHRF1 adopts an open conformation in the presence of hm-DNA (Supplementary Fig. 8e). DISCUSS +84 109 TTD–PHD–SRA–Spacer–hm-DNA complex_assembly The P(r) function obtained from small-angle X-ray scattering (SAXS) measurements of TTD–PHD–SRA–Spacer–hm-DNA complex showed a broader distribution than that of the TTD–PHD–SRA–Spacer alone, supporting the proposed model that UHRF1 adopts an open conformation in the presence of hm-DNA (Supplementary Fig. 8e). DISCUSS +165 183 TTD–PHD–SRA–Spacer complex_assembly The P(r) function obtained from small-angle X-ray scattering (SAXS) measurements of TTD–PHD–SRA–Spacer–hm-DNA complex showed a broader distribution than that of the TTD–PHD–SRA–Spacer alone, supporting the proposed model that UHRF1 adopts an open conformation in the presence of hm-DNA (Supplementary Fig. 8e). DISCUSS +226 231 UHRF1 protein The P(r) function obtained from small-angle X-ray scattering (SAXS) measurements of TTD–PHD–SRA–Spacer–hm-DNA complex showed a broader distribution than that of the TTD–PHD–SRA–Spacer alone, supporting the proposed model that UHRF1 adopts an open conformation in the presence of hm-DNA (Supplementary Fig. 8e). DISCUSS +242 246 open protein_state The P(r) function obtained from small-angle X-ray scattering (SAXS) measurements of TTD–PHD–SRA–Spacer–hm-DNA complex showed a broader distribution than that of the TTD–PHD–SRA–Spacer alone, supporting the proposed model that UHRF1 adopts an open conformation in the presence of hm-DNA (Supplementary Fig. 8e). DISCUSS +267 278 presence of protein_state The P(r) function obtained from small-angle X-ray scattering (SAXS) measurements of TTD–PHD–SRA–Spacer–hm-DNA complex showed a broader distribution than that of the TTD–PHD–SRA–Spacer alone, supporting the proposed model that UHRF1 adopts an open conformation in the presence of hm-DNA (Supplementary Fig. 8e). DISCUSS +279 285 hm-DNA chemical The P(r) function obtained from small-angle X-ray scattering (SAXS) measurements of TTD–PHD–SRA–Spacer–hm-DNA complex showed a broader distribution than that of the TTD–PHD–SRA–Spacer alone, supporting the proposed model that UHRF1 adopts an open conformation in the presence of hm-DNA (Supplementary Fig. 8e). DISCUSS +14 27 crystallizing experimental_method We have tried crystallizing more than three sub-constructs with and without DNA across over 1,200 crystallization conditions but failed to determine the structure of TTD–PHD–SRA–Spacer in the absence or presence of hm-DNA. DISCUSS +59 63 with protein_state We have tried crystallizing more than three sub-constructs with and without DNA across over 1,200 crystallization conditions but failed to determine the structure of TTD–PHD–SRA–Spacer in the absence or presence of hm-DNA. DISCUSS +68 75 without protein_state We have tried crystallizing more than three sub-constructs with and without DNA across over 1,200 crystallization conditions but failed to determine the structure of TTD–PHD–SRA–Spacer in the absence or presence of hm-DNA. DISCUSS +76 79 DNA chemical We have tried crystallizing more than three sub-constructs with and without DNA across over 1,200 crystallization conditions but failed to determine the structure of TTD–PHD–SRA–Spacer in the absence or presence of hm-DNA. DISCUSS +153 162 structure evidence We have tried crystallizing more than three sub-constructs with and without DNA across over 1,200 crystallization conditions but failed to determine the structure of TTD–PHD–SRA–Spacer in the absence or presence of hm-DNA. DISCUSS +166 184 TTD–PHD–SRA–Spacer complex_assembly We have tried crystallizing more than three sub-constructs with and without DNA across over 1,200 crystallization conditions but failed to determine the structure of TTD–PHD–SRA–Spacer in the absence or presence of hm-DNA. DISCUSS +192 199 absence protein_state We have tried crystallizing more than three sub-constructs with and without DNA across over 1,200 crystallization conditions but failed to determine the structure of TTD–PHD–SRA–Spacer in the absence or presence of hm-DNA. DISCUSS +203 214 presence of protein_state We have tried crystallizing more than three sub-constructs with and without DNA across over 1,200 crystallization conditions but failed to determine the structure of TTD–PHD–SRA–Spacer in the absence or presence of hm-DNA. DISCUSS +215 221 hm-DNA chemical We have tried crystallizing more than three sub-constructs with and without DNA across over 1,200 crystallization conditions but failed to determine the structure of TTD–PHD–SRA–Spacer in the absence or presence of hm-DNA. DISCUSS +14 24 structures evidence Getting these structures would greatly help for understanding the hm-DNA-mediated regulation of UHRF1. DISCUSS +66 72 hm-DNA chemical Getting these structures would greatly help for understanding the hm-DNA-mediated regulation of UHRF1. DISCUSS +96 101 UHRF1 protein Getting these structures would greatly help for understanding the hm-DNA-mediated regulation of UHRF1. DISCUSS +104 132 single molecular measurement experimental_method In addition, this regulatory process should be further characterized using advanced techniques, such as single molecular measurement. DISCUSS +31 46 phosphorylation ptm Our previous studies show that phosphorylation at S639 within the Spacer disrupts interaction between UHRF1 and deubiquitylase USP7 and decreases UHRF1 stability in the M phase of the cell cycle. DISCUSS +50 54 S639 residue_name_number Our previous studies show that phosphorylation at S639 within the Spacer disrupts interaction between UHRF1 and deubiquitylase USP7 and decreases UHRF1 stability in the M phase of the cell cycle. DISCUSS +66 72 Spacer structure_element Our previous studies show that phosphorylation at S639 within the Spacer disrupts interaction between UHRF1 and deubiquitylase USP7 and decreases UHRF1 stability in the M phase of the cell cycle. DISCUSS +102 107 UHRF1 protein Our previous studies show that phosphorylation at S639 within the Spacer disrupts interaction between UHRF1 and deubiquitylase USP7 and decreases UHRF1 stability in the M phase of the cell cycle. DISCUSS +112 126 deubiquitylase protein_type Our previous studies show that phosphorylation at S639 within the Spacer disrupts interaction between UHRF1 and deubiquitylase USP7 and decreases UHRF1 stability in the M phase of the cell cycle. DISCUSS +127 131 USP7 protein Our previous studies show that phosphorylation at S639 within the Spacer disrupts interaction between UHRF1 and deubiquitylase USP7 and decreases UHRF1 stability in the M phase of the cell cycle. DISCUSS +146 151 UHRF1 protein Our previous studies show that phosphorylation at S639 within the Spacer disrupts interaction between UHRF1 and deubiquitylase USP7 and decreases UHRF1 stability in the M phase of the cell cycle. DISCUSS +4 10 Spacer structure_element The Spacer was predicted to contain two nuclear localization signals, residues 581–600 and 648-670 (ref.). DISCUSS +40 68 nuclear localization signals structure_element The Spacer was predicted to contain two nuclear localization signals, residues 581–600 and 648-670 (ref.). DISCUSS +79 86 581–600 residue_range The Spacer was predicted to contain two nuclear localization signals, residues 581–600 and 648-670 (ref.). DISCUSS +91 98 648-670 residue_range The Spacer was predicted to contain two nuclear localization signals, residues 581–600 and 648-670 (ref.). DISCUSS +34 40 Spacer structure_element In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1. DISCUSS +45 53 binds to protein_state In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1. DISCUSS +58 61 TTD structure_element In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1. DISCUSS +69 75 closed protein_state In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1. DISCUSS +84 89 UHRF1 protein In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1. DISCUSS +124 126 H3 protein_type In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1. DISCUSS +126 131 K9me3 ptm In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1. DISCUSS +150 156 hm-DNA chemical In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1. DISCUSS +176 179 SRA structure_element In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1. DISCUSS +230 233 SRA structure_element In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1. DISCUSS +238 247 RFTSDNMT1 protein In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1. DISCUSS +42 48 Spacer structure_element These findings together indicate that the Spacer plays a very important role in the dynamic regulation of UHRF1. DISCUSS +106 111 UHRF1 protein These findings together indicate that the Spacer plays a very important role in the dynamic regulation of UHRF1. DISCUSS +79 83 PI5P chemical When our manuscript was in preparation, Gelato et al. reported that binding of PI5P to the Spacer opens the closed conformation of UHRF1 and increases H3K9me3-binding affinity of the TTD. DISCUSS +91 97 Spacer structure_element When our manuscript was in preparation, Gelato et al. reported that binding of PI5P to the Spacer opens the closed conformation of UHRF1 and increases H3K9me3-binding affinity of the TTD. DISCUSS +108 114 closed protein_state When our manuscript was in preparation, Gelato et al. reported that binding of PI5P to the Spacer opens the closed conformation of UHRF1 and increases H3K9me3-binding affinity of the TTD. DISCUSS +131 136 UHRF1 protein When our manuscript was in preparation, Gelato et al. reported that binding of PI5P to the Spacer opens the closed conformation of UHRF1 and increases H3K9me3-binding affinity of the TTD. DISCUSS +151 175 H3K9me3-binding affinity evidence When our manuscript was in preparation, Gelato et al. reported that binding of PI5P to the Spacer opens the closed conformation of UHRF1 and increases H3K9me3-binding affinity of the TTD. DISCUSS +183 186 TTD structure_element When our manuscript was in preparation, Gelato et al. reported that binding of PI5P to the Spacer opens the closed conformation of UHRF1 and increases H3K9me3-binding affinity of the TTD. DISCUSS +25 29 PI5P chemical The result suggests that PI5P may facilitate the conformational change of UHRF1 induced by hm-DNA when UHRF1 is recruited to chromatin. DISCUSS +74 79 UHRF1 protein The result suggests that PI5P may facilitate the conformational change of UHRF1 induced by hm-DNA when UHRF1 is recruited to chromatin. DISCUSS +91 97 hm-DNA chemical The result suggests that PI5P may facilitate the conformational change of UHRF1 induced by hm-DNA when UHRF1 is recruited to chromatin. DISCUSS +103 108 UHRF1 protein The result suggests that PI5P may facilitate the conformational change of UHRF1 induced by hm-DNA when UHRF1 is recruited to chromatin. DISCUSS +13 30 mass-spectrometry experimental_method In addition, mass-spectrometry analyses have identified several phosphorylation sites (S639, S651, S661) within the Spacer, suggesting that post-translational modification may add another layer of regulation of UHRF1 (refs). DISCUSS +64 85 phosphorylation sites site In addition, mass-spectrometry analyses have identified several phosphorylation sites (S639, S651, S661) within the Spacer, suggesting that post-translational modification may add another layer of regulation of UHRF1 (refs). DISCUSS +87 91 S639 residue_name_number In addition, mass-spectrometry analyses have identified several phosphorylation sites (S639, S651, S661) within the Spacer, suggesting that post-translational modification may add another layer of regulation of UHRF1 (refs). DISCUSS +93 97 S651 residue_name_number In addition, mass-spectrometry analyses have identified several phosphorylation sites (S639, S651, S661) within the Spacer, suggesting that post-translational modification may add another layer of regulation of UHRF1 (refs). DISCUSS +99 103 S661 residue_name_number In addition, mass-spectrometry analyses have identified several phosphorylation sites (S639, S651, S661) within the Spacer, suggesting that post-translational modification may add another layer of regulation of UHRF1 (refs). DISCUSS +116 122 Spacer structure_element In addition, mass-spectrometry analyses have identified several phosphorylation sites (S639, S651, S661) within the Spacer, suggesting that post-translational modification may add another layer of regulation of UHRF1 (refs). DISCUSS +211 216 UHRF1 protein In addition, mass-spectrometry analyses have identified several phosphorylation sites (S639, S651, S661) within the Spacer, suggesting that post-translational modification may add another layer of regulation of UHRF1 (refs). DISCUSS +40 43 SRA structure_element It has been well characterized that the SRA of UHRF1 preferentially recognizes hm-DNA through a base-flipping mechanism. DISCUSS +47 52 UHRF1 protein It has been well characterized that the SRA of UHRF1 preferentially recognizes hm-DNA through a base-flipping mechanism. DISCUSS +79 85 hm-DNA chemical It has been well characterized that the SRA of UHRF1 preferentially recognizes hm-DNA through a base-flipping mechanism. DISCUSS +32 38 Spacer structure_element Our study demonstrates that the Spacer markedly enhances the hm-DNA-binding affinity of the SRA and the deletion of the Spacer impairs heterochromatin localization of UHRF1, indicating that the Spacer is essential for recognition of hm-DNA in the context of full-length UHRF1. DISCUSS +61 84 hm-DNA-binding affinity evidence Our study demonstrates that the Spacer markedly enhances the hm-DNA-binding affinity of the SRA and the deletion of the Spacer impairs heterochromatin localization of UHRF1, indicating that the Spacer is essential for recognition of hm-DNA in the context of full-length UHRF1. DISCUSS +92 95 SRA structure_element Our study demonstrates that the Spacer markedly enhances the hm-DNA-binding affinity of the SRA and the deletion of the Spacer impairs heterochromatin localization of UHRF1, indicating that the Spacer is essential for recognition of hm-DNA in the context of full-length UHRF1. DISCUSS +104 115 deletion of experimental_method Our study demonstrates that the Spacer markedly enhances the hm-DNA-binding affinity of the SRA and the deletion of the Spacer impairs heterochromatin localization of UHRF1, indicating that the Spacer is essential for recognition of hm-DNA in the context of full-length UHRF1. DISCUSS +120 126 Spacer structure_element Our study demonstrates that the Spacer markedly enhances the hm-DNA-binding affinity of the SRA and the deletion of the Spacer impairs heterochromatin localization of UHRF1, indicating that the Spacer is essential for recognition of hm-DNA in the context of full-length UHRF1. DISCUSS +167 172 UHRF1 protein Our study demonstrates that the Spacer markedly enhances the hm-DNA-binding affinity of the SRA and the deletion of the Spacer impairs heterochromatin localization of UHRF1, indicating that the Spacer is essential for recognition of hm-DNA in the context of full-length UHRF1. DISCUSS +194 200 Spacer structure_element Our study demonstrates that the Spacer markedly enhances the hm-DNA-binding affinity of the SRA and the deletion of the Spacer impairs heterochromatin localization of UHRF1, indicating that the Spacer is essential for recognition of hm-DNA in the context of full-length UHRF1. DISCUSS +233 239 hm-DNA chemical Our study demonstrates that the Spacer markedly enhances the hm-DNA-binding affinity of the SRA and the deletion of the Spacer impairs heterochromatin localization of UHRF1, indicating that the Spacer is essential for recognition of hm-DNA in the context of full-length UHRF1. DISCUSS +258 269 full-length protein_state Our study demonstrates that the Spacer markedly enhances the hm-DNA-binding affinity of the SRA and the deletion of the Spacer impairs heterochromatin localization of UHRF1, indicating that the Spacer is essential for recognition of hm-DNA in the context of full-length UHRF1. DISCUSS +270 275 UHRF1 protein Our study demonstrates that the Spacer markedly enhances the hm-DNA-binding affinity of the SRA and the deletion of the Spacer impairs heterochromatin localization of UHRF1, indicating that the Spacer is essential for recognition of hm-DNA in the context of full-length UHRF1. DISCUSS +15 39 variant in methylation 1 protein Interestingly, variant in methylation 1 (VIM1, a UHRF1 homologue in Arabidopsis) contains an equivalent spacer region, which was shown to be required for hm-DNA recognition by its SRA domain, suggesting a conserved regulatory mechanism in SRA domain-containing proteins. DISCUSS +41 45 VIM1 protein Interestingly, variant in methylation 1 (VIM1, a UHRF1 homologue in Arabidopsis) contains an equivalent spacer region, which was shown to be required for hm-DNA recognition by its SRA domain, suggesting a conserved regulatory mechanism in SRA domain-containing proteins. DISCUSS +49 54 UHRF1 protein Interestingly, variant in methylation 1 (VIM1, a UHRF1 homologue in Arabidopsis) contains an equivalent spacer region, which was shown to be required for hm-DNA recognition by its SRA domain, suggesting a conserved regulatory mechanism in SRA domain-containing proteins. DISCUSS +68 79 Arabidopsis taxonomy_domain Interestingly, variant in methylation 1 (VIM1, a UHRF1 homologue in Arabidopsis) contains an equivalent spacer region, which was shown to be required for hm-DNA recognition by its SRA domain, suggesting a conserved regulatory mechanism in SRA domain-containing proteins. DISCUSS +104 110 spacer structure_element Interestingly, variant in methylation 1 (VIM1, a UHRF1 homologue in Arabidopsis) contains an equivalent spacer region, which was shown to be required for hm-DNA recognition by its SRA domain, suggesting a conserved regulatory mechanism in SRA domain-containing proteins. DISCUSS +154 160 hm-DNA chemical Interestingly, variant in methylation 1 (VIM1, a UHRF1 homologue in Arabidopsis) contains an equivalent spacer region, which was shown to be required for hm-DNA recognition by its SRA domain, suggesting a conserved regulatory mechanism in SRA domain-containing proteins. DISCUSS +180 183 SRA structure_element Interestingly, variant in methylation 1 (VIM1, a UHRF1 homologue in Arabidopsis) contains an equivalent spacer region, which was shown to be required for hm-DNA recognition by its SRA domain, suggesting a conserved regulatory mechanism in SRA domain-containing proteins. DISCUSS +239 242 SRA structure_element Interestingly, variant in methylation 1 (VIM1, a UHRF1 homologue in Arabidopsis) contains an equivalent spacer region, which was shown to be required for hm-DNA recognition by its SRA domain, suggesting a conserved regulatory mechanism in SRA domain-containing proteins. DISCUSS +14 19 UHRF2 protein Intriguingly, UHRF2 (the only mammalian homologue of UHRF1) and UHRF1 show very high sequence similarities for all the domains but very low similarity for the Spacer (Supplementary Fig. 7c). DISCUSS +30 39 mammalian taxonomy_domain Intriguingly, UHRF2 (the only mammalian homologue of UHRF1) and UHRF1 show very high sequence similarities for all the domains but very low similarity for the Spacer (Supplementary Fig. 7c). DISCUSS +53 58 UHRF1 protein Intriguingly, UHRF2 (the only mammalian homologue of UHRF1) and UHRF1 show very high sequence similarities for all the domains but very low similarity for the Spacer (Supplementary Fig. 7c). DISCUSS +64 69 UHRF1 protein Intriguingly, UHRF2 (the only mammalian homologue of UHRF1) and UHRF1 show very high sequence similarities for all the domains but very low similarity for the Spacer (Supplementary Fig. 7c). DISCUSS +159 165 Spacer structure_element Intriguingly, UHRF2 (the only mammalian homologue of UHRF1) and UHRF1 show very high sequence similarities for all the domains but very low similarity for the Spacer (Supplementary Fig. 7c). DISCUSS +15 20 UHRF2 protein Thus, although UHRF2 exhibits the histone- and hm-DNA-binding activities, the difference in the Spacer region may contribute to the functional differences between UHRF1 and UHRF2. DISCUSS +96 102 Spacer structure_element Thus, although UHRF2 exhibits the histone- and hm-DNA-binding activities, the difference in the Spacer region may contribute to the functional differences between UHRF1 and UHRF2. DISCUSS +163 168 UHRF1 protein Thus, although UHRF2 exhibits the histone- and hm-DNA-binding activities, the difference in the Spacer region may contribute to the functional differences between UHRF1 and UHRF2. DISCUSS +173 178 UHRF2 protein Thus, although UHRF2 exhibits the histone- and hm-DNA-binding activities, the difference in the Spacer region may contribute to the functional differences between UHRF1 and UHRF2. DISCUSS +51 56 UHRF2 protein This is also consistent with previous finding that UHRF2 is unable to replace UHRF1 to maintain the DNA methylation. DISCUSS +78 83 UHRF1 protein This is also consistent with previous finding that UHRF2 is unable to replace UHRF1 to maintain the DNA methylation. DISCUSS +100 103 DNA chemical This is also consistent with previous finding that UHRF2 is unable to replace UHRF1 to maintain the DNA methylation. DISCUSS +104 115 methylation ptm This is also consistent with previous finding that UHRF2 is unable to replace UHRF1 to maintain the DNA methylation. DISCUSS +41 44 DNA chemical One of the key questions in the field of DNA methylation is why UHRF1 contains modules recognizing two repressive epigenetic marks: H3K9me3 (by TTD–PHD) and hm-DNA (by the SRA). DISCUSS +45 56 methylation ptm One of the key questions in the field of DNA methylation is why UHRF1 contains modules recognizing two repressive epigenetic marks: H3K9me3 (by TTD–PHD) and hm-DNA (by the SRA). DISCUSS +64 69 UHRF1 protein One of the key questions in the field of DNA methylation is why UHRF1 contains modules recognizing two repressive epigenetic marks: H3K9me3 (by TTD–PHD) and hm-DNA (by the SRA). DISCUSS +132 134 H3 protein_type One of the key questions in the field of DNA methylation is why UHRF1 contains modules recognizing two repressive epigenetic marks: H3K9me3 (by TTD–PHD) and hm-DNA (by the SRA). DISCUSS +134 139 K9me3 ptm One of the key questions in the field of DNA methylation is why UHRF1 contains modules recognizing two repressive epigenetic marks: H3K9me3 (by TTD–PHD) and hm-DNA (by the SRA). DISCUSS +144 151 TTD–PHD structure_element One of the key questions in the field of DNA methylation is why UHRF1 contains modules recognizing two repressive epigenetic marks: H3K9me3 (by TTD–PHD) and hm-DNA (by the SRA). DISCUSS +157 163 hm-DNA chemical One of the key questions in the field of DNA methylation is why UHRF1 contains modules recognizing two repressive epigenetic marks: H3K9me3 (by TTD–PHD) and hm-DNA (by the SRA). DISCUSS +172 175 SRA structure_element One of the key questions in the field of DNA methylation is why UHRF1 contains modules recognizing two repressive epigenetic marks: H3K9me3 (by TTD–PHD) and hm-DNA (by the SRA). DISCUSS +53 58 UHRF1 protein Previous studies show that chromatin localization of UHRF1 is dependent on hm-DNA, whereas other studies indicate that histone H3K9me3 recognition and hm-DNA association are both required for UHRF1-mediated maintenance DNA methylation. DISCUSS +75 81 hm-DNA chemical Previous studies show that chromatin localization of UHRF1 is dependent on hm-DNA, whereas other studies indicate that histone H3K9me3 recognition and hm-DNA association are both required for UHRF1-mediated maintenance DNA methylation. DISCUSS +119 126 histone protein_type Previous studies show that chromatin localization of UHRF1 is dependent on hm-DNA, whereas other studies indicate that histone H3K9me3 recognition and hm-DNA association are both required for UHRF1-mediated maintenance DNA methylation. DISCUSS +127 129 H3 protein_type Previous studies show that chromatin localization of UHRF1 is dependent on hm-DNA, whereas other studies indicate that histone H3K9me3 recognition and hm-DNA association are both required for UHRF1-mediated maintenance DNA methylation. DISCUSS +129 134 K9me3 ptm Previous studies show that chromatin localization of UHRF1 is dependent on hm-DNA, whereas other studies indicate that histone H3K9me3 recognition and hm-DNA association are both required for UHRF1-mediated maintenance DNA methylation. DISCUSS +151 157 hm-DNA chemical Previous studies show that chromatin localization of UHRF1 is dependent on hm-DNA, whereas other studies indicate that histone H3K9me3 recognition and hm-DNA association are both required for UHRF1-mediated maintenance DNA methylation. DISCUSS +192 197 UHRF1 protein Previous studies show that chromatin localization of UHRF1 is dependent on hm-DNA, whereas other studies indicate that histone H3K9me3 recognition and hm-DNA association are both required for UHRF1-mediated maintenance DNA methylation. DISCUSS +219 222 DNA chemical Previous studies show that chromatin localization of UHRF1 is dependent on hm-DNA, whereas other studies indicate that histone H3K9me3 recognition and hm-DNA association are both required for UHRF1-mediated maintenance DNA methylation. DISCUSS +223 234 methylation ptm Previous studies show that chromatin localization of UHRF1 is dependent on hm-DNA, whereas other studies indicate that histone H3K9me3 recognition and hm-DNA association are both required for UHRF1-mediated maintenance DNA methylation. DISCUSS +87 92 UHRF1 protein However, little is known about the crosstalk between these two epigenetic marks within UHRF1. DISCUSS +47 49 H3 protein_type As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61 μM) or H3K9me0 (KD=25.99 μM). DISCUSS +49 54 K9me3 ptm As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61 μM) or H3K9me0 (KD=25.99 μM). DISCUSS +58 69 full-length protein_state As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61 μM) or H3K9me0 (KD=25.99 μM). DISCUSS +70 75 UHRF1 protein As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61 μM) or H3K9me0 (KD=25.99 μM). DISCUSS +121 133 unmethylated protein_state As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61 μM) or H3K9me0 (KD=25.99 μM). DISCUSS +178 180 H3 protein_type As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61 μM) or H3K9me0 (KD=25.99 μM). DISCUSS +180 185 K9me3 ptm As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61 μM) or H3K9me0 (KD=25.99 μM). DISCUSS +187 189 KD evidence As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61 μM) or H3K9me0 (KD=25.99 μM). DISCUSS +202 204 H3 protein_type As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61 μM) or H3K9me0 (KD=25.99 μM). DISCUSS +204 209 K9me0 ptm As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61 μM) or H3K9me0 (KD=25.99 μM). DISCUSS +211 213 KD evidence As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61 μM) or H3K9me0 (KD=25.99 μM). DISCUSS +19 30 full-length protein_state We have shown that full-length UHRF1 and SRA–Spacer strongly bind to hm-DNA (0.35 and 0.49 μM, respectively) and the Spacer plays an important role in PCH localization (Fig. 5d). DISCUSS +31 36 UHRF1 protein We have shown that full-length UHRF1 and SRA–Spacer strongly bind to hm-DNA (0.35 and 0.49 μM, respectively) and the Spacer plays an important role in PCH localization (Fig. 5d). DISCUSS +41 51 SRA–Spacer structure_element We have shown that full-length UHRF1 and SRA–Spacer strongly bind to hm-DNA (0.35 and 0.49 μM, respectively) and the Spacer plays an important role in PCH localization (Fig. 5d). DISCUSS +69 75 hm-DNA chemical We have shown that full-length UHRF1 and SRA–Spacer strongly bind to hm-DNA (0.35 and 0.49 μM, respectively) and the Spacer plays an important role in PCH localization (Fig. 5d). DISCUSS +117 123 Spacer structure_element We have shown that full-length UHRF1 and SRA–Spacer strongly bind to hm-DNA (0.35 and 0.49 μM, respectively) and the Spacer plays an important role in PCH localization (Fig. 5d). DISCUSS +35 40 UHRF1 protein Therefore, genomic localization of UHRF1 is primarily determined by its recognition of hm-DNA, which allows UHRF1 to adopt an open form and promotes its histone tail recognition for proper genomic localization and function. DISCUSS +87 93 hm-DNA chemical Therefore, genomic localization of UHRF1 is primarily determined by its recognition of hm-DNA, which allows UHRF1 to adopt an open form and promotes its histone tail recognition for proper genomic localization and function. DISCUSS +108 113 UHRF1 protein Therefore, genomic localization of UHRF1 is primarily determined by its recognition of hm-DNA, which allows UHRF1 to adopt an open form and promotes its histone tail recognition for proper genomic localization and function. DISCUSS +126 130 open protein_state Therefore, genomic localization of UHRF1 is primarily determined by its recognition of hm-DNA, which allows UHRF1 to adopt an open form and promotes its histone tail recognition for proper genomic localization and function. DISCUSS +153 160 histone protein_type Therefore, genomic localization of UHRF1 is primarily determined by its recognition of hm-DNA, which allows UHRF1 to adopt an open form and promotes its histone tail recognition for proper genomic localization and function. DISCUSS +18 28 SRA–Spacer structure_element As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +46 52 hm-DNA chemical As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +57 65 binds to protein_state As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +66 71 DNMT1 protein As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +108 113 UHRF1 protein As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +193 200 TTD–PHD structure_element As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +205 207 H3 protein_type As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +207 212 K9me3 ptm As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +217 220 PHD structure_element As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +221 223 H3 protein_type As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +251 256 DNMT1 protein As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +274 277 DNA chemical As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +292 303 methylation ptm As a result, when SRA–Spacer dissociates from hm-DNA and binds to DNMT1 with a currently unknown mechanism, UHRF1 may keep the complex associated with chromatin through the interaction between TTD–PHD and H3K9me3 (or PHD-H3), and make it possible for DNMT1 to target proper DNA substrate for methylation. DISCUSS +116 118 H3 protein_type This explanation agrees nicely with previous observations and clarifies the importance of coordinate recognition of H3K9me3 and hm-DNA by UHRF1 for maintenance DNA methylation. DISCUSS +118 123 K9me3 ptm This explanation agrees nicely with previous observations and clarifies the importance of coordinate recognition of H3K9me3 and hm-DNA by UHRF1 for maintenance DNA methylation. DISCUSS +128 134 hm-DNA chemical This explanation agrees nicely with previous observations and clarifies the importance of coordinate recognition of H3K9me3 and hm-DNA by UHRF1 for maintenance DNA methylation. DISCUSS +138 143 UHRF1 protein This explanation agrees nicely with previous observations and clarifies the importance of coordinate recognition of H3K9me3 and hm-DNA by UHRF1 for maintenance DNA methylation. DISCUSS +164 175 methylation ptm This explanation agrees nicely with previous observations and clarifies the importance of coordinate recognition of H3K9me3 and hm-DNA by UHRF1 for maintenance DNA methylation. DISCUSS +0 5 UHRF1 protein UHRF1 is essential for maintenance DNA methylation through recruiting DNMT1 to DNA replication forks during S phase. DISCUSS +35 38 DNA chemical UHRF1 is essential for maintenance DNA methylation through recruiting DNMT1 to DNA replication forks during S phase. DISCUSS +39 50 methylation ptm UHRF1 is essential for maintenance DNA methylation through recruiting DNMT1 to DNA replication forks during S phase. DISCUSS +70 75 DNMT1 protein UHRF1 is essential for maintenance DNA methylation through recruiting DNMT1 to DNA replication forks during S phase. DISCUSS +79 82 DNA chemical UHRF1 is essential for maintenance DNA methylation through recruiting DNMT1 to DNA replication forks during S phase. DISCUSS +70 73 SRA structure_element This function is probably induced by a direct interaction between the SRA and RFTSDNMT1 (refs) or interaction between DNMT1 and ubiquitylation of histione tail. DISCUSS +78 87 RFTSDNMT1 protein This function is probably induced by a direct interaction between the SRA and RFTSDNMT1 (refs) or interaction between DNMT1 and ubiquitylation of histione tail. DISCUSS +118 123 DNMT1 protein This function is probably induced by a direct interaction between the SRA and RFTSDNMT1 (refs) or interaction between DNMT1 and ubiquitylation of histione tail. DISCUSS +128 142 ubiquitylation ptm This function is probably induced by a direct interaction between the SRA and RFTSDNMT1 (refs) or interaction between DNMT1 and ubiquitylation of histione tail. DISCUSS +146 154 histione protein_type This function is probably induced by a direct interaction between the SRA and RFTSDNMT1 (refs) or interaction between DNMT1 and ubiquitylation of histione tail. DISCUSS +28 35 histone protein_type Recent study indicates that histone tail association of UHRF1 (by the PHD domain) is required for histone H3 ubiquitylation, which is dependent on ubiquitin ligase activity of the RING domain of UHRF1 (ref.). DISCUSS +56 61 UHRF1 protein Recent study indicates that histone tail association of UHRF1 (by the PHD domain) is required for histone H3 ubiquitylation, which is dependent on ubiquitin ligase activity of the RING domain of UHRF1 (ref.). DISCUSS +70 73 PHD structure_element Recent study indicates that histone tail association of UHRF1 (by the PHD domain) is required for histone H3 ubiquitylation, which is dependent on ubiquitin ligase activity of the RING domain of UHRF1 (ref.). DISCUSS +98 105 histone protein_type Recent study indicates that histone tail association of UHRF1 (by the PHD domain) is required for histone H3 ubiquitylation, which is dependent on ubiquitin ligase activity of the RING domain of UHRF1 (ref.). DISCUSS +106 108 H3 protein_type Recent study indicates that histone tail association of UHRF1 (by the PHD domain) is required for histone H3 ubiquitylation, which is dependent on ubiquitin ligase activity of the RING domain of UHRF1 (ref.). DISCUSS +109 123 ubiquitylation ptm Recent study indicates that histone tail association of UHRF1 (by the PHD domain) is required for histone H3 ubiquitylation, which is dependent on ubiquitin ligase activity of the RING domain of UHRF1 (ref.). DISCUSS +147 163 ubiquitin ligase protein_type Recent study indicates that histone tail association of UHRF1 (by the PHD domain) is required for histone H3 ubiquitylation, which is dependent on ubiquitin ligase activity of the RING domain of UHRF1 (ref.). DISCUSS +180 184 RING structure_element Recent study indicates that histone tail association of UHRF1 (by the PHD domain) is required for histone H3 ubiquitylation, which is dependent on ubiquitin ligase activity of the RING domain of UHRF1 (ref.). DISCUSS +195 200 UHRF1 protein Recent study indicates that histone tail association of UHRF1 (by the PHD domain) is required for histone H3 ubiquitylation, which is dependent on ubiquitin ligase activity of the RING domain of UHRF1 (ref.). DISCUSS +0 5 DNMT1 protein DNMT1 binds to ubiquitylated histone H3 and ubiquitylation is required for maintenance of DNA methylation in vivo. DISCUSS +6 14 binds to protein_state DNMT1 binds to ubiquitylated histone H3 and ubiquitylation is required for maintenance of DNA methylation in vivo. DISCUSS +15 28 ubiquitylated protein_state DNMT1 binds to ubiquitylated histone H3 and ubiquitylation is required for maintenance of DNA methylation in vivo. DISCUSS +29 36 histone protein_type DNMT1 binds to ubiquitylated histone H3 and ubiquitylation is required for maintenance of DNA methylation in vivo. DISCUSS +37 39 H3 protein_type DNMT1 binds to ubiquitylated histone H3 and ubiquitylation is required for maintenance of DNA methylation in vivo. DISCUSS +44 58 ubiquitylation ptm DNMT1 binds to ubiquitylated histone H3 and ubiquitylation is required for maintenance of DNA methylation in vivo. DISCUSS +90 93 DNA chemical DNMT1 binds to ubiquitylated histone H3 and ubiquitylation is required for maintenance of DNA methylation in vivo. DISCUSS +94 105 methylation ptm DNMT1 binds to ubiquitylated histone H3 and ubiquitylation is required for maintenance of DNA methylation in vivo. DISCUSS +34 37 TTD structure_element In this study, we found that both TTD and PHD are regulated by hm-DNA to recognize histone tail. DISCUSS +42 45 PHD structure_element In this study, we found that both TTD and PHD are regulated by hm-DNA to recognize histone tail. DISCUSS +63 69 hm-DNA chemical In this study, we found that both TTD and PHD are regulated by hm-DNA to recognize histone tail. DISCUSS +83 90 histone protein_type In this study, we found that both TTD and PHD are regulated by hm-DNA to recognize histone tail. DISCUSS +10 16 closed protein_state Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation. DISCUSS +22 27 UHRF1 protein Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation. DISCUSS +61 66 URHF1 protein Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation. DISCUSS +85 90 UHRF1 protein Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation. DISCUSS +94 98 open protein_state Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation. DISCUSS +124 130 hm-DNA chemical Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation. DISCUSS +147 155 binds to protein_state Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation. DISCUSS +156 163 histone protein_type Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation. DISCUSS +173 187 ubiquitylation ptm Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation. DISCUSS +203 206 DNA chemical Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation. DISCUSS +207 218 methylation ptm Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation. DISCUSS +10 29 structural analyses experimental_method Moreover, structural analyses of DNMT1–DNA and SRA–DNA complexes also indicate that it is impossible for DNMT1 to methylate the hm-DNA that UHRF1 binds to because of steric hindrance. DISCUSS +33 42 DNMT1–DNA complex_assembly Moreover, structural analyses of DNMT1–DNA and SRA–DNA complexes also indicate that it is impossible for DNMT1 to methylate the hm-DNA that UHRF1 binds to because of steric hindrance. DISCUSS +47 54 SRA–DNA complex_assembly Moreover, structural analyses of DNMT1–DNA and SRA–DNA complexes also indicate that it is impossible for DNMT1 to methylate the hm-DNA that UHRF1 binds to because of steric hindrance. DISCUSS +105 110 DNMT1 protein Moreover, structural analyses of DNMT1–DNA and SRA–DNA complexes also indicate that it is impossible for DNMT1 to methylate the hm-DNA that UHRF1 binds to because of steric hindrance. DISCUSS +128 134 hm-DNA chemical Moreover, structural analyses of DNMT1–DNA and SRA–DNA complexes also indicate that it is impossible for DNMT1 to methylate the hm-DNA that UHRF1 binds to because of steric hindrance. DISCUSS +140 145 UHRF1 protein Moreover, structural analyses of DNMT1–DNA and SRA–DNA complexes also indicate that it is impossible for DNMT1 to methylate the hm-DNA that UHRF1 binds to because of steric hindrance. DISCUSS +146 154 binds to protein_state Moreover, structural analyses of DNMT1–DNA and SRA–DNA complexes also indicate that it is impossible for DNMT1 to methylate the hm-DNA that UHRF1 binds to because of steric hindrance. DISCUSS +7 22 in vitro assays experimental_method In our in vitro assays, we could detect interaction between SRA–Spacer and RFTSDNMT1, but not the interaction between full-length UHRF1 and RFTSDNMT1 (Supplementary Fig. 8a,b and Fig. 5e). DISCUSS +60 70 SRA–Spacer structure_element In our in vitro assays, we could detect interaction between SRA–Spacer and RFTSDNMT1, but not the interaction between full-length UHRF1 and RFTSDNMT1 (Supplementary Fig. 8a,b and Fig. 5e). DISCUSS +75 84 RFTSDNMT1 protein In our in vitro assays, we could detect interaction between SRA–Spacer and RFTSDNMT1, but not the interaction between full-length UHRF1 and RFTSDNMT1 (Supplementary Fig. 8a,b and Fig. 5e). DISCUSS +118 129 full-length protein_state In our in vitro assays, we could detect interaction between SRA–Spacer and RFTSDNMT1, but not the interaction between full-length UHRF1 and RFTSDNMT1 (Supplementary Fig. 8a,b and Fig. 5e). DISCUSS +130 135 UHRF1 protein In our in vitro assays, we could detect interaction between SRA–Spacer and RFTSDNMT1, but not the interaction between full-length UHRF1 and RFTSDNMT1 (Supplementary Fig. 8a,b and Fig. 5e). DISCUSS +140 149 RFTSDNMT1 protein In our in vitro assays, we could detect interaction between SRA–Spacer and RFTSDNMT1, but not the interaction between full-length UHRF1 and RFTSDNMT1 (Supplementary Fig. 8a,b and Fig. 5e). DISCUSS +25 30 UHRF1 protein The results suggest that UHRF1 adopts multiple conformations. DISCUSS +11 16 UHRF1 protein Binding of UHRF1 to hm-DNA may serve as a switch for its recruitment of DNMT1. DISCUSS +20 26 hm-DNA chemical Binding of UHRF1 to hm-DNA may serve as a switch for its recruitment of DNMT1. DISCUSS +72 77 DNMT1 protein Binding of UHRF1 to hm-DNA may serve as a switch for its recruitment of DNMT1. DISCUSS +42 47 UHRF1 protein The S phase-dependent interaction between UHRF1 and DNMT1 (refs) suggest that DNMT1 may also undergo conformation changes so that RFTSDNMT1 binds to UHRF1 and the catalytic domain of DNMT1 binds to hm-DNA for reaction. DISCUSS +52 57 DNMT1 protein The S phase-dependent interaction between UHRF1 and DNMT1 (refs) suggest that DNMT1 may also undergo conformation changes so that RFTSDNMT1 binds to UHRF1 and the catalytic domain of DNMT1 binds to hm-DNA for reaction. DISCUSS +78 83 DNMT1 protein The S phase-dependent interaction between UHRF1 and DNMT1 (refs) suggest that DNMT1 may also undergo conformation changes so that RFTSDNMT1 binds to UHRF1 and the catalytic domain of DNMT1 binds to hm-DNA for reaction. DISCUSS +130 139 RFTSDNMT1 protein The S phase-dependent interaction between UHRF1 and DNMT1 (refs) suggest that DNMT1 may also undergo conformation changes so that RFTSDNMT1 binds to UHRF1 and the catalytic domain of DNMT1 binds to hm-DNA for reaction. DISCUSS +140 148 binds to protein_state The S phase-dependent interaction between UHRF1 and DNMT1 (refs) suggest that DNMT1 may also undergo conformation changes so that RFTSDNMT1 binds to UHRF1 and the catalytic domain of DNMT1 binds to hm-DNA for reaction. DISCUSS +149 154 UHRF1 protein The S phase-dependent interaction between UHRF1 and DNMT1 (refs) suggest that DNMT1 may also undergo conformation changes so that RFTSDNMT1 binds to UHRF1 and the catalytic domain of DNMT1 binds to hm-DNA for reaction. DISCUSS +163 179 catalytic domain structure_element The S phase-dependent interaction between UHRF1 and DNMT1 (refs) suggest that DNMT1 may also undergo conformation changes so that RFTSDNMT1 binds to UHRF1 and the catalytic domain of DNMT1 binds to hm-DNA for reaction. DISCUSS +183 188 DNMT1 protein The S phase-dependent interaction between UHRF1 and DNMT1 (refs) suggest that DNMT1 may also undergo conformation changes so that RFTSDNMT1 binds to UHRF1 and the catalytic domain of DNMT1 binds to hm-DNA for reaction. DISCUSS +189 197 binds to protein_state The S phase-dependent interaction between UHRF1 and DNMT1 (refs) suggest that DNMT1 may also undergo conformation changes so that RFTSDNMT1 binds to UHRF1 and the catalytic domain of DNMT1 binds to hm-DNA for reaction. DISCUSS +198 204 hm-DNA chemical The S phase-dependent interaction between UHRF1 and DNMT1 (refs) suggest that DNMT1 may also undergo conformation changes so that RFTSDNMT1 binds to UHRF1 and the catalytic domain of DNMT1 binds to hm-DNA for reaction. DISCUSS +0 6 Hm-DNA chemical Hm-DNA facilities histione tails recognition by full-length UHRF1. FIG +18 26 histione protein_type Hm-DNA facilities histione tails recognition by full-length UHRF1. FIG +48 59 full-length protein_state Hm-DNA facilities histione tails recognition by full-length UHRF1. FIG +60 65 UHRF1 protein Hm-DNA facilities histione tails recognition by full-length UHRF1. FIG +37 42 human species (a) Colour-coded domain structure of human UHRF1. FIG +43 48 UHRF1 protein (a) Colour-coded domain structure of human UHRF1. FIG +14 23 conserved protein_state Note that the conserved motif (green background) of the Linker (residues 286–306) and the Spacer (residues 587–674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1. FIG +56 62 Linker structure_element Note that the conserved motif (green background) of the Linker (residues 286–306) and the Spacer (residues 587–674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1. FIG +73 80 286–306 residue_range Note that the conserved motif (green background) of the Linker (residues 286–306) and the Spacer (residues 587–674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1. FIG +90 96 Spacer structure_element Note that the conserved motif (green background) of the Linker (residues 286–306) and the Spacer (residues 587–674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1. FIG +107 114 587–674 residue_range Note that the conserved motif (green background) of the Linker (residues 286–306) and the Spacer (residues 587–674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1. FIG +128 131 TTD structure_element Note that the conserved motif (green background) of the Linker (residues 286–306) and the Spacer (residues 587–674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1. FIG +167 173 Hm-DNA chemical Note that the conserved motif (green background) of the Linker (residues 286–306) and the Spacer (residues 587–674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1. FIG +185 192 histone protein_type Note that the conserved motif (green background) of the Linker (residues 286–306) and the Spacer (residues 587–674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1. FIG +193 195 H3 protein_type Note that the conserved motif (green background) of the Linker (residues 286–306) and the Spacer (residues 587–674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1. FIG +200 202 H3 protein_type Note that the conserved motif (green background) of the Linker (residues 286–306) and the Spacer (residues 587–674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1. FIG +223 228 UHRF1 protein Note that the conserved motif (green background) of the Linker (residues 286–306) and the Spacer (residues 587–674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1. FIG +9 20 full-length protein_state Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). FIG +21 26 UHRF1 protein Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). FIG +46 58 biotinylated protein_state Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). FIG +59 61 H3 protein_type Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). FIG +63 67 1–21 residue_range Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). FIG +72 74 H3 protein_type Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). FIG +74 79 K9me3 ptm Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). FIG +81 85 1–21 residue_range Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). FIG +115 125 absence of protein_state Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). FIG +126 132 hm-DNA chemical Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). FIG +146 151 UHRF1 protein Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). FIG +152 158 hm-DNA chemical Purified full-length UHRF1 was incubated with biotinylated H3 (1–21) or H3K9me3 (1–21) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2). FIG +36 44 SDS–PAGE experimental_method The bound proteins were analysed in SDS–PAGE followed by Coomassie blue staining. FIG +70 77 Histone protein_type Sequences of the peptides are indicated in Supplementary Table 1. (c) Histone peptides do not affect hm-DNA-binding affinity of UHRF1. FIG +101 124 hm-DNA-binding affinity evidence Sequences of the peptides are indicated in Supplementary Table 1. (c) Histone peptides do not affect hm-DNA-binding affinity of UHRF1. FIG +128 133 UHRF1 protein Sequences of the peptides are indicated in Supplementary Table 1. (c) Histone peptides do not affect hm-DNA-binding affinity of UHRF1. FIG +0 11 Full-length protein_state Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +12 17 UHRF1 protein Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +22 36 incubated with experimental_method Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +37 49 biotinylated protein_state Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +50 56 hm-DNA chemical Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +76 86 absence of protein_state Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +87 89 H3 protein_type Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +91 95 1–17 residue_range Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +100 102 H3 protein_type Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +102 107 K9me3 ptm Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +109 113 1–17 residue_range Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +165 168 ITC experimental_method Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +169 183 enthalpy plots evidence Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +199 201 H3 protein_type Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +201 206 K9me3 ptm Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +216 220 1–17 residue_range Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +225 232 TTD–PHD structure_element Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +237 248 full-length protein_state Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +249 254 UHRF1 protein Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +264 266 H3 protein_type Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +276 280 1–17 residue_range Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +289 292 PHD structure_element Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +297 308 full-length protein_state Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +309 314 UHRF1 protein Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1–17) or H3K9me3 (1–17) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1–17) to TTD–PHD and full-length UHRF1 (d), and H3 peptide (1–17) to the PHD and full-length UHRF1 (e). FIG +14 32 binding affinities evidence The estimated binding affinities (KD) are listed. FIG +34 36 KD evidence The estimated binding affinities (KD) are listed. FIG +36 43 histone protein_type Intramolecular interactions inhibit histone recognition by UHRF1. FIG +59 64 UHRF1 protein Intramolecular interactions inhibit histone recognition by UHRF1. FIG +4 24 GST pull-down assays experimental_method (a) GST pull-down assays for the intramolecular interactions. FIG +24 29 UHRF1 protein The isolated domains of UHRF1 were incubated with GST-tagged TTD or PHD immobilized on glutathione resin. FIG +35 44 incubated experimental_method The isolated domains of UHRF1 were incubated with GST-tagged TTD or PHD immobilized on glutathione resin. FIG +50 60 GST-tagged protein_state The isolated domains of UHRF1 were incubated with GST-tagged TTD or PHD immobilized on glutathione resin. FIG +61 64 TTD structure_element The isolated domains of UHRF1 were incubated with GST-tagged TTD or PHD immobilized on glutathione resin. FIG +68 71 PHD structure_element The isolated domains of UHRF1 were incubated with GST-tagged TTD or PHD immobilized on glutathione resin. FIG +36 44 SDS–PAGE experimental_method The bound proteins were analysed by SDS–PAGE and Coomassie blue staining. FIG +36 44 SDS–PAGE experimental_method The bound proteins were analysed by SDS–PAGE and Coomassie blue staining. FIG +19 22 ITC experimental_method (b,d) Superimposed ITC enthalpy plots for the intramolecular interactions of isolated UHRF1 domains. FIG +23 37 enthalpy plots evidence (b,d) Superimposed ITC enthalpy plots for the intramolecular interactions of isolated UHRF1 domains. FIG +86 91 UHRF1 protein (b,d) Superimposed ITC enthalpy plots for the intramolecular interactions of isolated UHRF1 domains. FIG +14 32 binding affinities evidence The estimated binding affinities (KD) were listed. FIG +34 36 KD evidence The estimated binding affinities (KD) were listed. FIG +37 40 ITC experimental_method ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +41 55 enthalpy plots evidence ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +75 77 H3 protein_type ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +77 82 K9me3 ptm ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +86 93 TTD–PHD structure_element ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +101 108 absence protein_state ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +112 123 presence of protein_state ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +128 134 Spacer structure_element ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +148 155 TTD–PHD structure_element ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +156 162 Spacer structure_element ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +186 189 ITC experimental_method ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +190 204 enthalpy plots evidence ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +224 226 H3 protein_type ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +230 237 PHD–SRA structure_element ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +241 244 PHD structure_element ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +252 259 absence protein_state ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +263 274 presence of protein_state ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +279 282 SRA structure_element ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +296 299 PHD structure_element ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +300 303 SRA structure_element ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD–PHD in the absence or presence of the Spacer (molar ratio TTD–PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD–SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2). FIG +0 3 NMR experimental_method NMR structure of the TTD bound to the Spacer. FIG +4 13 structure evidence NMR structure of the TTD bound to the Spacer. FIG +21 24 TTD structure_element NMR structure of the TTD bound to the Spacer. FIG +25 33 bound to protein_state NMR structure of the TTD bound to the Spacer. FIG +38 44 Spacer structure_element NMR structure of the TTD bound to the Spacer. FIG +29 39 TTD–Spacer structure_element (a) Ribbon representation of TTD–Spacer structure. FIG +40 49 structure evidence (a) Ribbon representation of TTD–Spacer structure. FIG +24 30 Spacer structure_element N- and C-termini of the Spacer are indicated. FIG +4 7 TTD structure_element The TTD is coloured in green, and the Spacer is coloured in yellow. FIG +38 44 Spacer structure_element The TTD is coloured in green, and the Spacer is coloured in yellow. FIG +4 19 Superimposition experimental_method (b) Superimposition of TTD–Spacer and TTD–PHD–H3K9me3 (4GY5.PDB) structures shown in ribbon representations. FIG +23 33 TTD–Spacer structure_element (b) Superimposition of TTD–Spacer and TTD–PHD–H3K9me3 (4GY5.PDB) structures shown in ribbon representations. FIG +38 53 TTD–PHD–H3K9me3 complex_assembly (b) Superimposition of TTD–Spacer and TTD–PHD–H3K9me3 (4GY5.PDB) structures shown in ribbon representations. FIG +65 75 structures evidence (b) Superimposition of TTD–Spacer and TTD–PHD–H3K9me3 (4GY5.PDB) structures shown in ribbon representations. FIG +4 7 TTD structure_element The TTD is coloured in green and the Spacer in yellow in TTD–Spacer structure. FIG +37 43 Spacer structure_element The TTD is coloured in green and the Spacer in yellow in TTD–Spacer structure. FIG +57 67 TTD–Spacer structure_element The TTD is coloured in green and the Spacer in yellow in TTD–Spacer structure. FIG +68 77 structure evidence The TTD is coloured in green and the Spacer in yellow in TTD–Spacer structure. FIG +0 15 TTD–PHD–H3K9me3 complex_assembly TTD–PHD–H3K9me3 complex is coloured in grey, and the PHD and H3K9me3 are omitted for simplicity. FIG +53 56 PHD structure_element TTD–PHD–H3K9me3 complex is coloured in grey, and the PHD and H3K9me3 are omitted for simplicity. FIG +61 63 H3 protein_type TTD–PHD–H3K9me3 complex is coloured in grey, and the PHD and H3K9me3 are omitted for simplicity. FIG +63 68 K9me3 ptm TTD–PHD–H3K9me3 complex is coloured in grey, and the PHD and H3K9me3 are omitted for simplicity. FIG +58 61 TTD structure_element (c) Electrostatic potential surface representation of the TTD with the Spacer shown in ribbon representation. FIG +71 77 Spacer structure_element (c) Electrostatic potential surface representation of the TTD with the Spacer shown in ribbon representation. FIG +29 35 Spacer structure_element The critical residues on the Spacer for the interaction are shown in stick representation. FIG +21 31 TTD–Spacer structure_element (d) Close-up view of TTD–Spacer interaction. FIG +0 14 Hydrogen bonds bond_interaction Hydrogen bonds are indicated as dashed lines. FIG +19 22 ITC experimental_method (e–g) Superimposed ITC enthalpy plots for the interaction between the Spacer and the TTD (or TTD–PHD) with the estimated binding affinity (KD) indicated. FIG +23 37 enthalpy plots evidence (e–g) Superimposed ITC enthalpy plots for the interaction between the Spacer and the TTD (or TTD–PHD) with the estimated binding affinity (KD) indicated. FIG +70 76 Spacer structure_element (e–g) Superimposed ITC enthalpy plots for the interaction between the Spacer and the TTD (or TTD–PHD) with the estimated binding affinity (KD) indicated. FIG +85 88 TTD structure_element (e–g) Superimposed ITC enthalpy plots for the interaction between the Spacer and the TTD (or TTD–PHD) with the estimated binding affinity (KD) indicated. FIG +93 100 TTD–PHD structure_element (e–g) Superimposed ITC enthalpy plots for the interaction between the Spacer and the TTD (or TTD–PHD) with the estimated binding affinity (KD) indicated. FIG +121 137 binding affinity evidence (e–g) Superimposed ITC enthalpy plots for the interaction between the Spacer and the TTD (or TTD–PHD) with the estimated binding affinity (KD) indicated. FIG +139 141 KD evidence (e–g) Superimposed ITC enthalpy plots for the interaction between the Spacer and the TTD (or TTD–PHD) with the estimated binding affinity (KD) indicated. FIG +0 9 Wild-type protein_state Wild-type and mutant proteins for the measurements are indicated. FIG +14 20 mutant protein_state Wild-type and mutant proteins for the measurements are indicated. FIG +4 24 GST pull-down assays experimental_method (h) GST pull-down assays for the intramolecular interactions. FIG +4 13 wild-type protein_state The wild-type or indicated truncations of UHRF1 were incubated with GST-tagged TTD, Linker or Spacer. FIG +42 47 UHRF1 protein The wild-type or indicated truncations of UHRF1 were incubated with GST-tagged TTD, Linker or Spacer. FIG +68 78 GST-tagged protein_state The wild-type or indicated truncations of UHRF1 were incubated with GST-tagged TTD, Linker or Spacer. FIG +79 82 TTD structure_element The wild-type or indicated truncations of UHRF1 were incubated with GST-tagged TTD, Linker or Spacer. FIG +84 90 Linker structure_element The wild-type or indicated truncations of UHRF1 were incubated with GST-tagged TTD, Linker or Spacer. FIG +94 100 Spacer structure_element The wild-type or indicated truncations of UHRF1 were incubated with GST-tagged TTD, Linker or Spacer. FIG +0 6 Hm-DNA chemical Hm-DNA impairs the intramolecular interaction of UHRF1 and facilitates its histone recognition. FIG +49 54 UHRF1 protein Hm-DNA impairs the intramolecular interaction of UHRF1 and facilitates its histone recognition. FIG +75 82 histone protein_type Hm-DNA impairs the intramolecular interaction of UHRF1 and facilitates its histone recognition. FIG +4 10 Hm-DNA chemical (a) Hm-DNA impairs the intramolecular interaction of PHD–SRA. FIG +53 60 PHD–SRA structure_element (a) Hm-DNA impairs the intramolecular interaction of PHD–SRA. FIG +4 7 SRA structure_element The SRA was incubated with GST-tagged PHD in the presence of increasing concentrations of hm-DNA and immobilized on glutathione resin. FIG +12 21 incubated experimental_method The SRA was incubated with GST-tagged PHD in the presence of increasing concentrations of hm-DNA and immobilized on glutathione resin. FIG +27 37 GST-tagged protein_state The SRA was incubated with GST-tagged PHD in the presence of increasing concentrations of hm-DNA and immobilized on glutathione resin. FIG +38 41 PHD structure_element The SRA was incubated with GST-tagged PHD in the presence of increasing concentrations of hm-DNA and immobilized on glutathione resin. FIG +49 60 presence of protein_state The SRA was incubated with GST-tagged PHD in the presence of increasing concentrations of hm-DNA and immobilized on glutathione resin. FIG +90 96 hm-DNA chemical The SRA was incubated with GST-tagged PHD in the presence of increasing concentrations of hm-DNA and immobilized on glutathione resin. FIG +36 44 SDS–PAGE experimental_method The bound proteins were analysed in SDS–PAGE and Coomassie blue staining (left) and quantified by band densitometry (right). FIG +49 72 Coomassie blue staining experimental_method The bound proteins were analysed in SDS–PAGE and Coomassie blue staining (left) and quantified by band densitometry (right). FIG +98 115 band densitometry experimental_method The bound proteins were analysed in SDS–PAGE and Coomassie blue staining (left) and quantified by band densitometry (right). FIG +26 31 UHRF1 protein (b) Purified fragments of UHRF1 were analysed by histone peptide (H3K9me0) pull-down assay as described in Fig. 1b. (c) Hm-DNA impairs the intramolecular interaction of TTD–Spacer. FIG +49 64 histone peptide experimental_method (b) Purified fragments of UHRF1 were analysed by histone peptide (H3K9me0) pull-down assay as described in Fig. 1b. (c) Hm-DNA impairs the intramolecular interaction of TTD–Spacer. FIG +66 68 H3 protein_type (b) Purified fragments of UHRF1 were analysed by histone peptide (H3K9me0) pull-down assay as described in Fig. 1b. (c) Hm-DNA impairs the intramolecular interaction of TTD–Spacer. FIG +68 73 K9me0 ptm (b) Purified fragments of UHRF1 were analysed by histone peptide (H3K9me0) pull-down assay as described in Fig. 1b. (c) Hm-DNA impairs the intramolecular interaction of TTD–Spacer. FIG +75 90 pull-down assay experimental_method (b) Purified fragments of UHRF1 were analysed by histone peptide (H3K9me0) pull-down assay as described in Fig. 1b. (c) Hm-DNA impairs the intramolecular interaction of TTD–Spacer. FIG +120 126 Hm-DNA chemical (b) Purified fragments of UHRF1 were analysed by histone peptide (H3K9me0) pull-down assay as described in Fig. 1b. (c) Hm-DNA impairs the intramolecular interaction of TTD–Spacer. FIG +169 179 TTD–Spacer structure_element (b) Purified fragments of UHRF1 were analysed by histone peptide (H3K9me0) pull-down assay as described in Fig. 1b. (c) Hm-DNA impairs the intramolecular interaction of TTD–Spacer. FIG +0 10 SRA–Spacer structure_element SRA–Spacer was incubated with GST-tagged TTD–PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining. FIG +15 24 incubated experimental_method SRA–Spacer was incubated with GST-tagged TTD–PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining. FIG +30 40 GST-tagged protein_state SRA–Spacer was incubated with GST-tagged TTD–PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining. FIG +41 48 TTD–PHD structure_element SRA–Spacer was incubated with GST-tagged TTD–PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining. FIG +52 55 TTD structure_element SRA–Spacer was incubated with GST-tagged TTD–PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining. FIG +63 74 presence of protein_state SRA–Spacer was incubated with GST-tagged TTD–PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining. FIG +104 110 hm-DNA chemical SRA–Spacer was incubated with GST-tagged TTD–PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining. FIG +127 147 pull-down experiment experimental_method SRA–Spacer was incubated with GST-tagged TTD–PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining. FIG +182 201 band densitometries experimental_method SRA–Spacer was incubated with GST-tagged TTD–PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining. FIG +226 249 Coomassie blue staining experimental_method SRA–Spacer was incubated with GST-tagged TTD–PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining. FIG +4 35 Histone peptide pull-down assay experimental_method (d) Histone peptide pull-down assay using UHRF1 mutants as indicated. FIG +42 47 UHRF1 protein (d) Histone peptide pull-down assay using UHRF1 mutants as indicated. FIG +48 55 mutants protein_state (d) Histone peptide pull-down assay using UHRF1 mutants as indicated. FIG +44 47 DTT chemical The assays were performed in the presence (+DTT) or absence (−DTT) of 15 mM DTT. FIG +62 65 DTT chemical The assays were performed in the presence (+DTT) or absence (−DTT) of 15 mM DTT. FIG +76 79 DTT chemical The assays were performed in the presence (+DTT) or absence (−DTT) of 15 mM DTT. FIG +4 10 Spacer structure_element The Spacer facilitates hm-DNA–SRA interaction and DNMT1–UHRF1 interaction. FIG +23 29 hm-DNA chemical The Spacer facilitates hm-DNA–SRA interaction and DNMT1–UHRF1 interaction. FIG +30 33 SRA structure_element The Spacer facilitates hm-DNA–SRA interaction and DNMT1–UHRF1 interaction. FIG +50 61 DNMT1–UHRF1 complex_assembly The Spacer facilitates hm-DNA–SRA interaction and DNMT1–UHRF1 interaction. FIG +17 20 ITC experimental_method (a) Superimposed ITC enthalpy plots for hm-DNA-binding affinities of the SRA, the Spacer and SRA–Spacer. (b,c) Superimposed fluorescence polarization (FP) plots for hm-DNA-binding affinities of truncations or full-length UHRF1. FIG +21 35 enthalpy plots evidence (a) Superimposed ITC enthalpy plots for hm-DNA-binding affinities of the SRA, the Spacer and SRA–Spacer. (b,c) Superimposed fluorescence polarization (FP) plots for hm-DNA-binding affinities of truncations or full-length UHRF1. FIG +40 65 hm-DNA-binding affinities evidence (a) Superimposed ITC enthalpy plots for hm-DNA-binding affinities of the SRA, the Spacer and SRA–Spacer. (b,c) Superimposed fluorescence polarization (FP) plots for hm-DNA-binding affinities of truncations or full-length UHRF1. FIG +73 76 SRA structure_element (a) Superimposed ITC enthalpy plots for hm-DNA-binding affinities of the SRA, the Spacer and SRA–Spacer. (b,c) Superimposed fluorescence polarization (FP) plots for hm-DNA-binding affinities of truncations or full-length UHRF1. FIG +82 88 Spacer structure_element (a) Superimposed ITC enthalpy plots for hm-DNA-binding affinities of the SRA, the Spacer and SRA–Spacer. (b,c) Superimposed fluorescence polarization (FP) plots for hm-DNA-binding affinities of truncations or full-length UHRF1. FIG +93 103 SRA–Spacer structure_element (a) Superimposed ITC enthalpy plots for hm-DNA-binding affinities of the SRA, the Spacer and SRA–Spacer. (b,c) Superimposed fluorescence polarization (FP) plots for hm-DNA-binding affinities of truncations or full-length UHRF1. FIG +124 160 fluorescence polarization (FP) plots evidence (a) Superimposed ITC enthalpy plots for hm-DNA-binding affinities of the SRA, the Spacer and SRA–Spacer. (b,c) Superimposed fluorescence polarization (FP) plots for hm-DNA-binding affinities of truncations or full-length UHRF1. FIG +165 190 hm-DNA-binding affinities evidence (a) Superimposed ITC enthalpy plots for hm-DNA-binding affinities of the SRA, the Spacer and SRA–Spacer. (b,c) Superimposed fluorescence polarization (FP) plots for hm-DNA-binding affinities of truncations or full-length UHRF1. FIG +209 220 full-length protein_state (a) Superimposed ITC enthalpy plots for hm-DNA-binding affinities of the SRA, the Spacer and SRA–Spacer. (b,c) Superimposed fluorescence polarization (FP) plots for hm-DNA-binding affinities of truncations or full-length UHRF1. FIG +221 226 UHRF1 protein (a) Superimposed ITC enthalpy plots for hm-DNA-binding affinities of the SRA, the Spacer and SRA–Spacer. (b,c) Superimposed fluorescence polarization (FP) plots for hm-DNA-binding affinities of truncations or full-length UHRF1. FIG +14 32 binding affinities evidence The estimated binding affinities (KD) are listed above. (d) Subcellular localization of GFP-tagged wild-type or indicated mutants of UHRF1 in NIH3T3 cells. FIG +34 36 KD evidence The estimated binding affinities (KD) are listed above. (d) Subcellular localization of GFP-tagged wild-type or indicated mutants of UHRF1 in NIH3T3 cells. FIG +88 98 GFP-tagged protein_state The estimated binding affinities (KD) are listed above. (d) Subcellular localization of GFP-tagged wild-type or indicated mutants of UHRF1 in NIH3T3 cells. FIG +99 108 wild-type protein_state The estimated binding affinities (KD) are listed above. (d) Subcellular localization of GFP-tagged wild-type or indicated mutants of UHRF1 in NIH3T3 cells. FIG +122 129 mutants protein_state The estimated binding affinities (KD) are listed above. (d) Subcellular localization of GFP-tagged wild-type or indicated mutants of UHRF1 in NIH3T3 cells. FIG +133 138 UHRF1 protein The estimated binding affinities (KD) are listed above. (d) Subcellular localization of GFP-tagged wild-type or indicated mutants of UHRF1 in NIH3T3 cells. FIG +54 58 DAPI chemical The percentages of cells showing co-localization with DAPI foci were counted from at least 100 cells and shown on the left of the corresponding representative confocal microscopy. FIG +159 178 confocal microscopy experimental_method The percentages of cells showing co-localization with DAPI foci were counted from at least 100 cells and shown on the left of the corresponding representative confocal microscopy. FIG +21 45 GST pull-down experiment experimental_method Scale bar, 5 μm. (e) GST pull-down experiment for the interactions between wild-type or truncations of UHRF1 and RFTSDNMT1 as described in Fig. 2a. (f) Working model for hm-DNA-mediated conformational changes of UHRF1, as described in the Discussion. FIG +75 84 wild-type protein_state Scale bar, 5 μm. (e) GST pull-down experiment for the interactions between wild-type or truncations of UHRF1 and RFTSDNMT1 as described in Fig. 2a. (f) Working model for hm-DNA-mediated conformational changes of UHRF1, as described in the Discussion. FIG +88 99 truncations experimental_method Scale bar, 5 μm. (e) GST pull-down experiment for the interactions between wild-type or truncations of UHRF1 and RFTSDNMT1 as described in Fig. 2a. (f) Working model for hm-DNA-mediated conformational changes of UHRF1, as described in the Discussion. FIG +103 108 UHRF1 protein Scale bar, 5 μm. (e) GST pull-down experiment for the interactions between wild-type or truncations of UHRF1 and RFTSDNMT1 as described in Fig. 2a. (f) Working model for hm-DNA-mediated conformational changes of UHRF1, as described in the Discussion. FIG +113 122 RFTSDNMT1 protein Scale bar, 5 μm. (e) GST pull-down experiment for the interactions between wild-type or truncations of UHRF1 and RFTSDNMT1 as described in Fig. 2a. (f) Working model for hm-DNA-mediated conformational changes of UHRF1, as described in the Discussion. FIG +170 176 hm-DNA chemical Scale bar, 5 μm. (e) GST pull-down experiment for the interactions between wild-type or truncations of UHRF1 and RFTSDNMT1 as described in Fig. 2a. (f) Working model for hm-DNA-mediated conformational changes of UHRF1, as described in the Discussion. FIG +212 217 UHRF1 protein Scale bar, 5 μm. (e) GST pull-down experiment for the interactions between wild-type or truncations of UHRF1 and RFTSDNMT1 as described in Fig. 2a. (f) Working model for hm-DNA-mediated conformational changes of UHRF1, as described in the Discussion. FIG diff --git a/annotation_CSV/PMC4822561.csv b/annotation_CSV/PMC4822561.csv new file mode 100644 index 0000000000000000000000000000000000000000..c15b5e086a522304f3ed3a1c02425022dd3cfaa9 --- /dev/null +++ b/annotation_CSV/PMC4822561.csv @@ -0,0 +1,367 @@ +anno_start anno_end anno_text entity_type sentence section +0 9 Structure evidence Structure of a quinolone-stabilized cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex TITLE +56 72 topoisomerase IV complex_assembly Structure of a quinolone-stabilized cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex TITLE +78 99 Klebsiella pneumoniae species Structure of a quinolone-stabilized cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex TITLE +130 154 Streptococcus pneumoniae species Structure of a quinolone-stabilized cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex TITLE +0 18 Crystal structures evidence Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared. ABSTRACT +48 64 topoisomerase IV complex_assembly Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared. ABSTRACT +70 83 Gram-negative taxonomy_domain Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared. ABSTRACT +85 98 K. pneumoniae species Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared. ABSTRACT +104 117 Gram-positive taxonomy_domain Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared. ABSTRACT +119 132 S. pneumoniae species Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared. ABSTRACT +134 143 bacterial taxonomy_domain Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared. ABSTRACT +212 224 levofloxacin chemical Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared. ABSTRACT +4 17 K. pneumoniae species For K. pneumoniae, this is the first high-resolution cleavage complex structure to be reported. ABSTRACT +70 79 structure evidence For K. pneumoniae, this is the first high-resolution cleavage complex structure to be reported. ABSTRACT +1 22 Klebsiella pneumoniae species Klebsiella pneumoniae is a Gram-negative bacterium that is responsible for a range of common infections, including pulmonary pneumonia, bloodstream infections and meningitis. ABSTRACT +28 51 Gram-negative bacterium taxonomy_domain Klebsiella pneumoniae is a Gram-negative bacterium that is responsible for a range of common infections, including pulmonary pneumonia, bloodstream infections and meningitis. ABSTRACT +19 29 Klebsiella taxonomy_domain Certain strains of Klebsiella have become highly resistant to antibiotics. ABSTRACT +65 73 bacteria taxonomy_domain Despite the vast amount of research carried out on this class of bacteria, the molecular structure of its topoisomerase IV, a type II topoisomerase essential for catalysing chromosomal segregation, had remained unknown. ABSTRACT +89 98 structure evidence Despite the vast amount of research carried out on this class of bacteria, the molecular structure of its topoisomerase IV, a type II topoisomerase essential for catalysing chromosomal segregation, had remained unknown. ABSTRACT +106 122 topoisomerase IV complex_assembly Despite the vast amount of research carried out on this class of bacteria, the molecular structure of its topoisomerase IV, a type II topoisomerase essential for catalysing chromosomal segregation, had remained unknown. ABSTRACT +126 147 type II topoisomerase protein_type Despite the vast amount of research carried out on this class of bacteria, the molecular structure of its topoisomerase IV, a type II topoisomerase essential for catalysing chromosomal segregation, had remained unknown. ABSTRACT +19 28 structure evidence In this paper, the structure of its DNA-cleavage complex is reported at 3.35 Å resolution. ABSTRACT +36 39 DNA chemical In this paper, the structure of its DNA-cleavage complex is reported at 3.35 Å resolution. ABSTRACT +28 32 ParC protein The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. ABSTRACT +33 49 breakage-reunion structure_element The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. ABSTRACT +54 58 ParE protein The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. ABSTRACT +59 65 TOPRIM structure_element The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. ABSTRACT +77 90 K. pneumoniae species The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. ABSTRACT +91 107 topoisomerase IV complex_assembly The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. ABSTRACT +113 116 DNA chemical The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. ABSTRACT +131 143 levofloxacin chemical The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. ABSTRACT +162 177 fluoroquinolone chemical The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. ABSTRACT +53 77 Streptococcus pneumoniae species This complex is compared with a similar complex from Streptococcus pneumoniae, which has recently been solved. ABSTRACT +1 11 Klebsiella taxonomy_domain Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. INTRO +40 58 Enterobacteriaceae taxonomy_domain Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. INTRO +69 90 Gram-negative bacilli taxonomy_domain Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. INTRO +162 165 DNA chemical Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. INTRO +176 189 K. pneumoniae species Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. INTRO +191 201 K. ozaenae species Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. INTRO +203 222 K. rhinoscleromatis species Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. INTRO +224 234 K. oxytoca species Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. INTRO +236 249 K. planticola species Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. INTRO +251 263 K. terrigena species Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. INTRO +268 286 K. ornithinolytica species Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. INTRO +0 13 K. pneumoniae species K. pneumoniae is the most medically important species of the genus owing to its high resistance to antibiotics. INTRO +106 119 K. pneumoniae species Significant morbidity and mortality has been associated with an emerging, highly drug-resistant strain of K. pneumoniae characterized as producing the carbapenemase enzyme (KPC-producing bacteria; Nordmann et al., 2009). INTRO +151 164 carbapenemase protein_type Significant morbidity and mortality has been associated with an emerging, highly drug-resistant strain of K. pneumoniae characterized as producing the carbapenemase enzyme (KPC-producing bacteria; Nordmann et al., 2009). INTRO +187 195 bacteria taxonomy_domain Significant morbidity and mortality has been associated with an emerging, highly drug-resistant strain of K. pneumoniae characterized as producing the carbapenemase enzyme (KPC-producing bacteria; Nordmann et al., 2009). INTRO +37 68 in vitro susceptibility testing experimental_method However, common treatments (based on in vitro susceptibility testing) are the polymyxins, tigecycline and, less frequently, aminoglycoside antibiotics (Arnold et al., 2011). INTRO +78 88 polymyxins chemical However, common treatments (based on in vitro susceptibility testing) are the polymyxins, tigecycline and, less frequently, aminoglycoside antibiotics (Arnold et al., 2011). INTRO +90 101 tigecycline chemical However, common treatments (based on in vitro susceptibility testing) are the polymyxins, tigecycline and, less frequently, aminoglycoside antibiotics (Arnold et al., 2011). INTRO +124 138 aminoglycoside chemical However, common treatments (based on in vitro susceptibility testing) are the polymyxins, tigecycline and, less frequently, aminoglycoside antibiotics (Arnold et al., 2011). INTRO +92 108 fluoroquinolones chemical Another effective strategy involves the limited use of certain antimicrobials, specifically fluoroquinolones and cephalo­sporins (Gasink et al., 2009). INTRO +113 128 cephalo­sporins chemical Another effective strategy involves the limited use of certain antimicrobials, specifically fluoroquinolones and cephalo­sporins (Gasink et al., 2009). INTRO +69 80 β-lactamase protein_type These include combinations of existing β-lactam antibiotics with new β-lactamase inhibitors able to circumvent KPC resistance. INTRO +0 13 Neoglycosides chemical Neoglycosides are novel aminoglycosides that have activity against KPC-producing bacteria that are also being developed (Chen et al., 2012). INTRO +24 39 aminoglycosides chemical Neoglycosides are novel aminoglycosides that have activity against KPC-producing bacteria that are also being developed (Chen et al., 2012). INTRO +81 89 bacteria taxonomy_domain Neoglycosides are novel aminoglycosides that have activity against KPC-producing bacteria that are also being developed (Chen et al., 2012). INTRO +0 29 Type II topoisomerase enzymes protein_type Type II topoisomerase enzymes play important roles in prokaryotic and eukaryotic DNA replication, recombination and transcription (Drlica et al., 2008; Laponogov et al., 2013; Lee et al., 2013; Nitiss, 2009a ,b ; Schoeffler & Berger, 2008; Sissi & Palumbo, 2009; Vos et al., 2011; Wendorff et al., 2012; Wu et al., 2011, 2013). INTRO +54 65 prokaryotic taxonomy_domain Type II topoisomerase enzymes play important roles in prokaryotic and eukaryotic DNA replication, recombination and transcription (Drlica et al., 2008; Laponogov et al., 2013; Lee et al., 2013; Nitiss, 2009a ,b ; Schoeffler & Berger, 2008; Sissi & Palumbo, 2009; Vos et al., 2011; Wendorff et al., 2012; Wu et al., 2011, 2013). INTRO +70 80 eukaryotic taxonomy_domain Type II topoisomerase enzymes play important roles in prokaryotic and eukaryotic DNA replication, recombination and transcription (Drlica et al., 2008; Laponogov et al., 2013; Lee et al., 2013; Nitiss, 2009a ,b ; Schoeffler & Berger, 2008; Sissi & Palumbo, 2009; Vos et al., 2011; Wendorff et al., 2012; Wu et al., 2011, 2013). INTRO +81 84 DNA chemical Type II topoisomerase enzymes play important roles in prokaryotic and eukaryotic DNA replication, recombination and transcription (Drlica et al., 2008; Laponogov et al., 2013; Lee et al., 2013; Nitiss, 2009a ,b ; Schoeffler & Berger, 2008; Sissi & Palumbo, 2009; Vos et al., 2011; Wendorff et al., 2012; Wu et al., 2011, 2013). INTRO +3 11 bacteria taxonomy_domain In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks. INTRO +13 29 topoisomerase IV complex_assembly In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks. INTRO +33 41 tetramer oligomeric_state In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks. INTRO +49 53 ParC protein In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks. INTRO +62 66 ParE protein In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks. INTRO +157 163 gyrase protein_type In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks. INTRO +167 177 GyrA2GyrB2 complex_assembly In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks. INTRO +178 186 tetramer oligomeric_state In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks. INTRO +199 202 DNA chemical In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks. INTRO +220 223 DNA chemical In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks. INTRO +37 40 DNA chemical Both enzymes act via a double-strand DNA break involving a cleavage complex and are targets for quinolone antimicrobials that act by trapping these enzymes at the DNA-cleavage stage and preventing strand re-joining (Drlica et al., 2008). INTRO +163 166 DNA chemical Both enzymes act via a double-strand DNA break involving a cleavage complex and are targets for quinolone antimicrobials that act by trapping these enzymes at the DNA-cleavage stage and preventing strand re-joining (Drlica et al., 2008). INTRO +0 12 Levofloxacin chemical Levofloxacin is a broad-spectrum third-generation fluoro­quinolone antibiotic. INTRO +21 34 Gram-positive taxonomy_domain It is active against Gram-positive and Gram-negative bacteria and functions by inhibiting gyrase and topoisomerase IV (Drlica & Zhao, 1997; Laponogov et al., 2010). INTRO +39 61 Gram-negative bacteria taxonomy_domain It is active against Gram-positive and Gram-negative bacteria and functions by inhibiting gyrase and topoisomerase IV (Drlica & Zhao, 1997; Laponogov et al., 2010). INTRO +90 96 gyrase protein_type It is active against Gram-positive and Gram-negative bacteria and functions by inhibiting gyrase and topoisomerase IV (Drlica & Zhao, 1997; Laponogov et al., 2010). INTRO +101 117 topoisomerase IV complex_assembly It is active against Gram-positive and Gram-negative bacteria and functions by inhibiting gyrase and topoisomerase IV (Drlica & Zhao, 1997; Laponogov et al., 2010). INTRO +82 98 fluoroquinolones chemical Acquiring a deep structural and functional understanding of the mode of action of fluoroquinolones (Tomašić & Mašič, 2014) and the development of new drugs targeted against topoisomerase IV and gyrase from a wide range of Gram-positive and Gram-negative pathogenic bacteria are highly active areas of current research directed at overcoming the vexed problem of drug resistance (Bax et al., 2010; Chan et al., 2015; Drlica et al., 2014; Mutsaev et al., 2014; Pommier, 2013; Srikannathasan et al., 2015). INTRO +173 189 topoisomerase IV complex_assembly Acquiring a deep structural and functional understanding of the mode of action of fluoroquinolones (Tomašić & Mašič, 2014) and the development of new drugs targeted against topoisomerase IV and gyrase from a wide range of Gram-positive and Gram-negative pathogenic bacteria are highly active areas of current research directed at overcoming the vexed problem of drug resistance (Bax et al., 2010; Chan et al., 2015; Drlica et al., 2014; Mutsaev et al., 2014; Pommier, 2013; Srikannathasan et al., 2015). INTRO +194 200 gyrase protein_type Acquiring a deep structural and functional understanding of the mode of action of fluoroquinolones (Tomašić & Mašič, 2014) and the development of new drugs targeted against topoisomerase IV and gyrase from a wide range of Gram-positive and Gram-negative pathogenic bacteria are highly active areas of current research directed at overcoming the vexed problem of drug resistance (Bax et al., 2010; Chan et al., 2015; Drlica et al., 2014; Mutsaev et al., 2014; Pommier, 2013; Srikannathasan et al., 2015). INTRO +222 235 Gram-positive taxonomy_domain Acquiring a deep structural and functional understanding of the mode of action of fluoroquinolones (Tomašić & Mašič, 2014) and the development of new drugs targeted against topoisomerase IV and gyrase from a wide range of Gram-positive and Gram-negative pathogenic bacteria are highly active areas of current research directed at overcoming the vexed problem of drug resistance (Bax et al., 2010; Chan et al., 2015; Drlica et al., 2014; Mutsaev et al., 2014; Pommier, 2013; Srikannathasan et al., 2015). INTRO +240 253 Gram-negative taxonomy_domain Acquiring a deep structural and functional understanding of the mode of action of fluoroquinolones (Tomašić & Mašič, 2014) and the development of new drugs targeted against topoisomerase IV and gyrase from a wide range of Gram-positive and Gram-negative pathogenic bacteria are highly active areas of current research directed at overcoming the vexed problem of drug resistance (Bax et al., 2010; Chan et al., 2015; Drlica et al., 2014; Mutsaev et al., 2014; Pommier, 2013; Srikannathasan et al., 2015). INTRO +265 273 bacteria taxonomy_domain Acquiring a deep structural and functional understanding of the mode of action of fluoroquinolones (Tomašić & Mašič, 2014) and the development of new drugs targeted against topoisomerase IV and gyrase from a wide range of Gram-positive and Gram-negative pathogenic bacteria are highly active areas of current research directed at overcoming the vexed problem of drug resistance (Bax et al., 2010; Chan et al., 2015; Drlica et al., 2014; Mutsaev et al., 2014; Pommier, 2013; Srikannathasan et al., 2015). INTRO +44 59 X-ray structure evidence Here, we report the first three-dimensional X-ray structure of a K. pneumoniae topoisomerase IV ParC/ParE cleavage complex with DNA stabilized by levofloxacin. INTRO +65 78 K. pneumoniae species Here, we report the first three-dimensional X-ray structure of a K. pneumoniae topoisomerase IV ParC/ParE cleavage complex with DNA stabilized by levofloxacin. INTRO +79 95 topoisomerase IV complex_assembly Here, we report the first three-dimensional X-ray structure of a K. pneumoniae topoisomerase IV ParC/ParE cleavage complex with DNA stabilized by levofloxacin. INTRO +96 105 ParC/ParE complex_assembly Here, we report the first three-dimensional X-ray structure of a K. pneumoniae topoisomerase IV ParC/ParE cleavage complex with DNA stabilized by levofloxacin. INTRO +128 131 DNA chemical Here, we report the first three-dimensional X-ray structure of a K. pneumoniae topoisomerase IV ParC/ParE cleavage complex with DNA stabilized by levofloxacin. INTRO +146 158 levofloxacin chemical Here, we report the first three-dimensional X-ray structure of a K. pneumoniae topoisomerase IV ParC/ParE cleavage complex with DNA stabilized by levofloxacin. INTRO +4 21 crystal structure evidence The crystal structure provides structural information on topoisomerase IV from K. pneumoniae, a pathogen for which drug resistance is a serious concern. INTRO +57 73 topoisomerase IV complex_assembly The crystal structure provides structural information on topoisomerase IV from K. pneumoniae, a pathogen for which drug resistance is a serious concern. INTRO +79 92 K. pneumoniae species The crystal structure provides structural information on topoisomerase IV from K. pneumoniae, a pathogen for which drug resistance is a serious concern. INTRO +4 13 structure evidence The structure of the ParC/ParE–DNA–levofloxacin binding site highlights the details of the cleavage-complex assembly that are essential for the rational design of Klebsiella topoisomerase inhibitors. INTRO +21 30 ParC/ParE complex_assembly The structure of the ParC/ParE–DNA–levofloxacin binding site highlights the details of the cleavage-complex assembly that are essential for the rational design of Klebsiella topoisomerase inhibitors. INTRO +31 60 DNA–levofloxacin binding site site The structure of the ParC/ParE–DNA–levofloxacin binding site highlights the details of the cleavage-complex assembly that are essential for the rational design of Klebsiella topoisomerase inhibitors. INTRO +163 173 Klebsiella taxonomy_domain The structure of the ParC/ParE–DNA–levofloxacin binding site highlights the details of the cleavage-complex assembly that are essential for the rational design of Klebsiella topoisomerase inhibitors. INTRO +174 187 topoisomerase protein_type The structure of the ParC/ParE–DNA–levofloxacin binding site highlights the details of the cleavage-complex assembly that are essential for the rational design of Klebsiella topoisomerase inhibitors. INTRO +8 23 co-crystallized experimental_method We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +28 41 K. pneumoniae species We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +42 58 topoisomerase IV complex_assembly We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +59 68 ParC/ParE complex_assembly We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +69 85 breakage-reunion structure_element We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +94 100 ParC55 protein We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +111 116 1–490 residue_range We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +122 126 ParE protein We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +127 133 TOPRIM structure_element We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +142 148 ParE30 protein We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +159 166 390–631 residue_range We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +188 191 DNA chemical We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +204 210 E-site site We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +227 239 levofloxacin chemical We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. RESULTS +4 27 X-ray crystal structure evidence The X-ray crystal structure of the complex was determined to 3.35 Å resolution, revealing a closed ParC55 dimer flanked by two ParE30 monomers (Figs. 1 ▸, 2 ▸ and 3 ▸). RESULTS +92 98 closed protein_state The X-ray crystal structure of the complex was determined to 3.35 Å resolution, revealing a closed ParC55 dimer flanked by two ParE30 monomers (Figs. 1 ▸, 2 ▸ and 3 ▸). RESULTS +99 105 ParC55 protein The X-ray crystal structure of the complex was determined to 3.35 Å resolution, revealing a closed ParC55 dimer flanked by two ParE30 monomers (Figs. 1 ▸, 2 ▸ and 3 ▸). RESULTS +106 111 dimer oligomeric_state The X-ray crystal structure of the complex was determined to 3.35 Å resolution, revealing a closed ParC55 dimer flanked by two ParE30 monomers (Figs. 1 ▸, 2 ▸ and 3 ▸). RESULTS +127 133 ParE30 protein The X-ray crystal structure of the complex was determined to 3.35 Å resolution, revealing a closed ParC55 dimer flanked by two ParE30 monomers (Figs. 1 ▸, 2 ▸ and 3 ▸). RESULTS +134 142 monomers oligomeric_state The X-ray crystal structure of the complex was determined to 3.35 Å resolution, revealing a closed ParC55 dimer flanked by two ParE30 monomers (Figs. 1 ▸, 2 ▸ and 3 ▸). RESULTS +70 83 S. pneumoniae species The overall architecture of this complex is similar to that found for S. pneumoniae topoisomerase–DNA–drug complexes (Laponogov et al., 2009, 2010). RESULTS +9 13 6–30 residue_range Residues 6–30 of the N-terminal α-helix α1 of the ParC subunit again embrace the ParE subunit, ‘hugging’ the ParE subunits close to either side of the ParC dimer (Laponogov et al., 2010). RESULTS +32 39 α-helix structure_element Residues 6–30 of the N-terminal α-helix α1 of the ParC subunit again embrace the ParE subunit, ‘hugging’ the ParE subunits close to either side of the ParC dimer (Laponogov et al., 2010). RESULTS +40 42 α1 structure_element Residues 6–30 of the N-terminal α-helix α1 of the ParC subunit again embrace the ParE subunit, ‘hugging’ the ParE subunits close to either side of the ParC dimer (Laponogov et al., 2010). RESULTS +50 54 ParC protein Residues 6–30 of the N-terminal α-helix α1 of the ParC subunit again embrace the ParE subunit, ‘hugging’ the ParE subunits close to either side of the ParC dimer (Laponogov et al., 2010). RESULTS +81 85 ParE protein Residues 6–30 of the N-terminal α-helix α1 of the ParC subunit again embrace the ParE subunit, ‘hugging’ the ParE subunits close to either side of the ParC dimer (Laponogov et al., 2010). RESULTS +109 113 ParE protein Residues 6–30 of the N-terminal α-helix α1 of the ParC subunit again embrace the ParE subunit, ‘hugging’ the ParE subunits close to either side of the ParC dimer (Laponogov et al., 2010). RESULTS +151 155 ParC protein Residues 6–30 of the N-terminal α-helix α1 of the ParC subunit again embrace the ParE subunit, ‘hugging’ the ParE subunits close to either side of the ParC dimer (Laponogov et al., 2010). RESULTS +156 161 dimer oligomeric_state Residues 6–30 of the N-terminal α-helix α1 of the ParC subunit again embrace the ParE subunit, ‘hugging’ the ParE subunits close to either side of the ParC dimer (Laponogov et al., 2010). RESULTS +0 11 Deletion of experimental_method Deletion of this ‘arm’ α1 results in loss of DNA-cleavage activity (Laponogov et al., 2007) and is clearly very important in complex stability (Fig. 3 ▸). RESULTS +18 21 arm structure_element Deletion of this ‘arm’ α1 results in loss of DNA-cleavage activity (Laponogov et al., 2007) and is clearly very important in complex stability (Fig. 3 ▸). RESULTS +23 25 α1 structure_element Deletion of this ‘arm’ α1 results in loss of DNA-cleavage activity (Laponogov et al., 2007) and is clearly very important in complex stability (Fig. 3 ▸). RESULTS +37 66 loss of DNA-cleavage activity protein_state Deletion of this ‘arm’ α1 results in loss of DNA-cleavage activity (Laponogov et al., 2007) and is clearly very important in complex stability (Fig. 3 ▸). RESULTS +51 57 ParC55 protein This structural feature was absent in our original ParC55 structure (Laponogov et al., 2007; Sohi et al., 2008). RESULTS +58 67 structure evidence This structural feature was absent in our original ParC55 structure (Laponogov et al., 2007; Sohi et al., 2008). RESULTS +24 37 topoisomerase protein_type The upper region of the topoisomerase complex consists of the E-subunit TOPRIM metal-binding domain formed of four parallel β-sheets and the surrounding α-helices. RESULTS +62 71 E-subunit protein The upper region of the topoisomerase complex consists of the E-subunit TOPRIM metal-binding domain formed of four parallel β-sheets and the surrounding α-helices. RESULTS +72 99 TOPRIM metal-binding domain structure_element The upper region of the topoisomerase complex consists of the E-subunit TOPRIM metal-binding domain formed of four parallel β-sheets and the surrounding α-helices. RESULTS +115 132 parallel β-sheets structure_element The upper region of the topoisomerase complex consists of the E-subunit TOPRIM metal-binding domain formed of four parallel β-sheets and the surrounding α-helices. RESULTS +153 162 α-helices structure_element The upper region of the topoisomerase complex consists of the E-subunit TOPRIM metal-binding domain formed of four parallel β-sheets and the surrounding α-helices. RESULTS +4 13 C-subunit protein The C-subunit provides the WHD (winged-helix domain; a CAP-like structure; McKay & Steitz, 1981) and the ‘tower’ which form the U groove-shaped protein region into which the G-gate DNA binds with an induced U-shaped bend. RESULTS +27 30 WHD structure_element The C-subunit provides the WHD (winged-helix domain; a CAP-like structure; McKay & Steitz, 1981) and the ‘tower’ which form the U groove-shaped protein region into which the G-gate DNA binds with an induced U-shaped bend. RESULTS +32 51 winged-helix domain structure_element The C-subunit provides the WHD (winged-helix domain; a CAP-like structure; McKay & Steitz, 1981) and the ‘tower’ which form the U groove-shaped protein region into which the G-gate DNA binds with an induced U-shaped bend. RESULTS +55 73 CAP-like structure structure_element The C-subunit provides the WHD (winged-helix domain; a CAP-like structure; McKay & Steitz, 1981) and the ‘tower’ which form the U groove-shaped protein region into which the G-gate DNA binds with an induced U-shaped bend. RESULTS +106 111 tower structure_element The C-subunit provides the WHD (winged-helix domain; a CAP-like structure; McKay & Steitz, 1981) and the ‘tower’ which form the U groove-shaped protein region into which the G-gate DNA binds with an induced U-shaped bend. RESULTS +128 136 U groove structure_element The C-subunit provides the WHD (winged-helix domain; a CAP-like structure; McKay & Steitz, 1981) and the ‘tower’ which form the U groove-shaped protein region into which the G-gate DNA binds with an induced U-shaped bend. RESULTS +174 180 G-gate structure_element The C-subunit provides the WHD (winged-helix domain; a CAP-like structure; McKay & Steitz, 1981) and the ‘tower’ which form the U groove-shaped protein region into which the G-gate DNA binds with an induced U-shaped bend. RESULTS +181 184 DNA chemical The C-subunit provides the WHD (winged-helix domain; a CAP-like structure; McKay & Steitz, 1981) and the ‘tower’ which form the U groove-shaped protein region into which the G-gate DNA binds with an induced U-shaped bend. RESULTS +10 16 C-gate structure_element The lower C-gate region (Fig. 3 ▸) consists of the same disposition of pairs of two long α-helices terminated by a spanning short α-helix forming a 30 Å wide DNA-accommodating cavity through which the T-gate DNA passes as found in the S. pneumoniae complex. RESULTS +84 98 long α-helices structure_element The lower C-gate region (Fig. 3 ▸) consists of the same disposition of pairs of two long α-helices terminated by a spanning short α-helix forming a 30 Å wide DNA-accommodating cavity through which the T-gate DNA passes as found in the S. pneumoniae complex. RESULTS +124 137 short α-helix structure_element The lower C-gate region (Fig. 3 ▸) consists of the same disposition of pairs of two long α-helices terminated by a spanning short α-helix forming a 30 Å wide DNA-accommodating cavity through which the T-gate DNA passes as found in the S. pneumoniae complex. RESULTS +158 182 DNA-accommodating cavity site The lower C-gate region (Fig. 3 ▸) consists of the same disposition of pairs of two long α-helices terminated by a spanning short α-helix forming a 30 Å wide DNA-accommodating cavity through which the T-gate DNA passes as found in the S. pneumoniae complex. RESULTS +201 207 T-gate structure_element The lower C-gate region (Fig. 3 ▸) consists of the same disposition of pairs of two long α-helices terminated by a spanning short α-helix forming a 30 Å wide DNA-accommodating cavity through which the T-gate DNA passes as found in the S. pneumoniae complex. RESULTS +208 211 DNA chemical The lower C-gate region (Fig. 3 ▸) consists of the same disposition of pairs of two long α-helices terminated by a spanning short α-helix forming a 30 Å wide DNA-accommodating cavity through which the T-gate DNA passes as found in the S. pneumoniae complex. RESULTS +235 248 S. pneumoniae species The lower C-gate region (Fig. 3 ▸) consists of the same disposition of pairs of two long α-helices terminated by a spanning short α-helix forming a 30 Å wide DNA-accommodating cavity through which the T-gate DNA passes as found in the S. pneumoniae complex. RESULTS +56 74 topo­isomerases IV complex_assembly Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013). RESULTS +80 93 K. pneumoniae species Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013). RESULTS +98 111 S. pneumoniae species Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013). RESULTS +151 164 topoisomerase protein_type Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013). RESULTS +260 269 structure evidence Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013). RESULTS +277 289 full complex protein_state Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013). RESULTS +295 305 holoenzyme protein_state Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013). RESULTS +315 329 CTD β-pinwheel structure_element Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013). RESULTS +347 360 ATPase domain structure_element Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013). RESULTS +368 372 open protein_state Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013). RESULTS +4 10 G-gate structure_element The G-gate DNA for the S. pneumoniae complex consists of an 18-base-pair E-site sequence (our designation for a DNA site which we first found from DNA-mapping studies; Leo et al., 2005; Arnoldi et al., 2013; Fig. 1 ▸). RESULTS +11 14 DNA chemical The G-gate DNA for the S. pneumoniae complex consists of an 18-base-pair E-site sequence (our designation for a DNA site which we first found from DNA-mapping studies; Leo et al., 2005; Arnoldi et al., 2013; Fig. 1 ▸). RESULTS +23 36 S. pneumoniae species The G-gate DNA for the S. pneumoniae complex consists of an 18-base-pair E-site sequence (our designation for a DNA site which we first found from DNA-mapping studies; Leo et al., 2005; Arnoldi et al., 2013; Fig. 1 ▸). RESULTS +73 79 E-site site The G-gate DNA for the S. pneumoniae complex consists of an 18-base-pair E-site sequence (our designation for a DNA site which we first found from DNA-mapping studies; Leo et al., 2005; Arnoldi et al., 2013; Fig. 1 ▸). RESULTS +112 120 DNA site site The G-gate DNA for the S. pneumoniae complex consists of an 18-base-pair E-site sequence (our designation for a DNA site which we first found from DNA-mapping studies; Leo et al., 2005; Arnoldi et al., 2013; Fig. 1 ▸). RESULTS +147 166 DNA-mapping studies experimental_method The G-gate DNA for the S. pneumoniae complex consists of an 18-base-pair E-site sequence (our designation for a DNA site which we first found from DNA-mapping studies; Leo et al., 2005; Arnoldi et al., 2013; Fig. 1 ▸). RESULTS +4 16 crystallized experimental_method The crystallized complex was formed by turning over the topoisomerase tetramer in the presence of DNA and levofloxacin and crystallizing the product. RESULTS +56 69 topoisomerase protein_type The crystallized complex was formed by turning over the topoisomerase tetramer in the presence of DNA and levofloxacin and crystallizing the product. RESULTS +70 78 tetramer oligomeric_state The crystallized complex was formed by turning over the topoisomerase tetramer in the presence of DNA and levofloxacin and crystallizing the product. RESULTS +86 97 presence of protein_state The crystallized complex was formed by turning over the topoisomerase tetramer in the presence of DNA and levofloxacin and crystallizing the product. RESULTS +98 101 DNA chemical The crystallized complex was formed by turning over the topoisomerase tetramer in the presence of DNA and levofloxacin and crystallizing the product. RESULTS +106 118 levofloxacin chemical The crystallized complex was formed by turning over the topoisomerase tetramer in the presence of DNA and levofloxacin and crystallizing the product. RESULTS +123 136 crystallizing experimental_method The crystallized complex was formed by turning over the topoisomerase tetramer in the presence of DNA and levofloxacin and crystallizing the product. RESULTS +17 30 K. pneumoniae species In contrast, the K. pneumoniae complex was formed by co-crystallizing the topoisomerase tetramer complex in the presence of a 34-base-pair pre-cleaved DNA in the presence of levofloxacin. RESULTS +53 69 co-crystallizing experimental_method In contrast, the K. pneumoniae complex was formed by co-crystallizing the topoisomerase tetramer complex in the presence of a 34-base-pair pre-cleaved DNA in the presence of levofloxacin. RESULTS +74 87 topoisomerase protein_type In contrast, the K. pneumoniae complex was formed by co-crystallizing the topoisomerase tetramer complex in the presence of a 34-base-pair pre-cleaved DNA in the presence of levofloxacin. RESULTS +88 96 tetramer oligomeric_state In contrast, the K. pneumoniae complex was formed by co-crystallizing the topoisomerase tetramer complex in the presence of a 34-base-pair pre-cleaved DNA in the presence of levofloxacin. RESULTS +112 123 presence of protein_state In contrast, the K. pneumoniae complex was formed by co-crystallizing the topoisomerase tetramer complex in the presence of a 34-base-pair pre-cleaved DNA in the presence of levofloxacin. RESULTS +139 150 pre-cleaved protein_state In contrast, the K. pneumoniae complex was formed by co-crystallizing the topoisomerase tetramer complex in the presence of a 34-base-pair pre-cleaved DNA in the presence of levofloxacin. RESULTS +151 154 DNA chemical In contrast, the K. pneumoniae complex was formed by co-crystallizing the topoisomerase tetramer complex in the presence of a 34-base-pair pre-cleaved DNA in the presence of levofloxacin. RESULTS +162 173 presence of protein_state In contrast, the K. pneumoniae complex was formed by co-crystallizing the topoisomerase tetramer complex in the presence of a 34-base-pair pre-cleaved DNA in the presence of levofloxacin. RESULTS +174 186 levofloxacin chemical In contrast, the K. pneumoniae complex was formed by co-crystallizing the topoisomerase tetramer complex in the presence of a 34-base-pair pre-cleaved DNA in the presence of levofloxacin. RESULTS +18 21 DNA chemical In both cases the DNA is bent into a U-form and bound snugly against the protein of the G-gate. RESULTS +37 43 U-form protein_state In both cases the DNA is bent into a U-form and bound snugly against the protein of the G-gate. RESULTS +48 53 bound protein_state In both cases the DNA is bent into a U-form and bound snugly against the protein of the G-gate. RESULTS +88 94 G-gate structure_element In both cases the DNA is bent into a U-form and bound snugly against the protein of the G-gate. RESULTS +48 51 DNA chemical We have been able to unambiguously read off the DNA sequences in the electron-density maps. RESULTS +69 90 electron-density maps evidence We have been able to unambiguously read off the DNA sequences in the electron-density maps. RESULTS +73 77 ParE protein There is 41.6% sequence identity and 54.4% sequence homology between the ParE subunit of K. pneumoniae and that of S. pneumoniae. RESULTS +89 102 K. pneumoniae species There is 41.6% sequence identity and 54.4% sequence homology between the ParE subunit of K. pneumoniae and that of S. pneumoniae. RESULTS +115 128 S. pneumoniae species There is 41.6% sequence identity and 54.4% sequence homology between the ParE subunit of K. pneumoniae and that of S. pneumoniae. RESULTS +8 12 ParC protein For the ParC subunits, the figures are 40.8 identity and 55.6% homology between the two organisms. RESULTS +4 22 sequence alignment experimental_method The sequence alignment is given in Supplementary Fig. S1, with the key metal-binding residues and those which give rise to quinolone resistance highlighted. RESULTS +71 93 metal-binding residues site The sequence alignment is given in Supplementary Fig. S1, with the key metal-binding residues and those which give rise to quinolone resistance highlighted. RESULTS +15 27 levofloxacin chemical The binding of levofloxacin in the K. pneumoniae complex is shown in Figs. 2 ▸, 3 ▸ and 5 ▸ and is hemi-intercalated into the DNA and stacked against the DNA bases at the cleavage site (positions −1 and +1 of the four-base-pair staggered cut in the 34-mer DNA) which is similar to that found for the S. pneumoniae complex. RESULTS +35 48 K. pneumoniae species The binding of levofloxacin in the K. pneumoniae complex is shown in Figs. 2 ▸, 3 ▸ and 5 ▸ and is hemi-intercalated into the DNA and stacked against the DNA bases at the cleavage site (positions −1 and +1 of the four-base-pair staggered cut in the 34-mer DNA) which is similar to that found for the S. pneumoniae complex. RESULTS +126 129 DNA chemical The binding of levofloxacin in the K. pneumoniae complex is shown in Figs. 2 ▸, 3 ▸ and 5 ▸ and is hemi-intercalated into the DNA and stacked against the DNA bases at the cleavage site (positions −1 and +1 of the four-base-pair staggered cut in the 34-mer DNA) which is similar to that found for the S. pneumoniae complex. RESULTS +154 157 DNA chemical The binding of levofloxacin in the K. pneumoniae complex is shown in Figs. 2 ▸, 3 ▸ and 5 ▸ and is hemi-intercalated into the DNA and stacked against the DNA bases at the cleavage site (positions −1 and +1 of the four-base-pair staggered cut in the 34-mer DNA) which is similar to that found for the S. pneumoniae complex. RESULTS +171 184 cleavage site site The binding of levofloxacin in the K. pneumoniae complex is shown in Figs. 2 ▸, 3 ▸ and 5 ▸ and is hemi-intercalated into the DNA and stacked against the DNA bases at the cleavage site (positions −1 and +1 of the four-base-pair staggered cut in the 34-mer DNA) which is similar to that found for the S. pneumoniae complex. RESULTS +196 198 −1 residue_number The binding of levofloxacin in the K. pneumoniae complex is shown in Figs. 2 ▸, 3 ▸ and 5 ▸ and is hemi-intercalated into the DNA and stacked against the DNA bases at the cleavage site (positions −1 and +1 of the four-base-pair staggered cut in the 34-mer DNA) which is similar to that found for the S. pneumoniae complex. RESULTS +203 205 +1 residue_number The binding of levofloxacin in the K. pneumoniae complex is shown in Figs. 2 ▸, 3 ▸ and 5 ▸ and is hemi-intercalated into the DNA and stacked against the DNA bases at the cleavage site (positions −1 and +1 of the four-base-pair staggered cut in the 34-mer DNA) which is similar to that found for the S. pneumoniae complex. RESULTS +256 259 DNA chemical The binding of levofloxacin in the K. pneumoniae complex is shown in Figs. 2 ▸, 3 ▸ and 5 ▸ and is hemi-intercalated into the DNA and stacked against the DNA bases at the cleavage site (positions −1 and +1 of the four-base-pair staggered cut in the 34-mer DNA) which is similar to that found for the S. pneumoniae complex. RESULTS +300 313 S. pneumoniae species The binding of levofloxacin in the K. pneumoniae complex is shown in Figs. 2 ▸, 3 ▸ and 5 ▸ and is hemi-intercalated into the DNA and stacked against the DNA bases at the cleavage site (positions −1 and +1 of the four-base-pair staggered cut in the 34-mer DNA) which is similar to that found for the S. pneumoniae complex. RESULTS +44 57 K. pneumoniae species Fig. 5 ▸ presents side-by-side views of the K. pneumoniae and S. pneumoniae active sites which shows that levofloxacin binds in a very similar manner in these two complexes with extensive π–π stacking interaction between the bases and the drug. RESULTS +62 75 S. pneumoniae species Fig. 5 ▸ presents side-by-side views of the K. pneumoniae and S. pneumoniae active sites which shows that levofloxacin binds in a very similar manner in these two complexes with extensive π–π stacking interaction between the bases and the drug. RESULTS +76 88 active sites site Fig. 5 ▸ presents side-by-side views of the K. pneumoniae and S. pneumoniae active sites which shows that levofloxacin binds in a very similar manner in these two complexes with extensive π–π stacking interaction between the bases and the drug. RESULTS +106 118 levofloxacin chemical Fig. 5 ▸ presents side-by-side views of the K. pneumoniae and S. pneumoniae active sites which shows that levofloxacin binds in a very similar manner in these two complexes with extensive π–π stacking interaction between the bases and the drug. RESULTS +188 212 π–π stacking interaction bond_interaction Fig. 5 ▸ presents side-by-side views of the K. pneumoniae and S. pneumoniae active sites which shows that levofloxacin binds in a very similar manner in these two complexes with extensive π–π stacking interaction between the bases and the drug. RESULTS +4 20 methylpiperazine chemical The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. RESULTS +51 60 quinolone chemical The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. RESULTS +136 142 Glu474 residue_name_number The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. RESULTS +147 153 Glu475 residue_name_number The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. RESULTS +158 171 S. pneumoniae species The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. RESULTS +184 190 Gln460 residue_name_number The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. RESULTS +195 201 Glu461 residue_name_number The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. RESULTS +206 219 K. pneumoniae species The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. RESULTS +231 247 glutamate at 474 residue_name_number The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. RESULTS +268 284 glutamine at 460 residue_name_number The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. RESULTS +292 302 Klebsiella taxonomy_domain The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. RESULTS +19 32 S. pneumoniae species Interestingly, for S. pneumoniae we observe only one possible orientation of the C7 groups in both sub­units, while for K. pneumoniae we can see two: one with the same orientation as in S. pneumoniae and other rotated 180° away. RESULTS +120 133 K. pneumoniae species Interestingly, for S. pneumoniae we observe only one possible orientation of the C7 groups in both sub­units, while for K. pneumoniae we can see two: one with the same orientation as in S. pneumoniae and other rotated 180° away. RESULTS +186 199 S. pneumoniae species Interestingly, for S. pneumoniae we observe only one possible orientation of the C7 groups in both sub­units, while for K. pneumoniae we can see two: one with the same orientation as in S. pneumoniae and other rotated 180° away. RESULTS +32 39 crystal evidence They both exist within the same crystal in the two different dimers in the asymmetric unit. RESULTS +61 67 dimers oligomeric_state They both exist within the same crystal in the two different dimers in the asymmetric unit. RESULTS +36 40 ParE protein The side chains surrounding them in ParE are quite disordered and are more defined in K. pneumoniae (even though this complex is at lower resolution) than in S. pneumoniae. RESULTS +86 99 K. pneumoniae species The side chains surrounding them in ParE are quite disordered and are more defined in K. pneumoniae (even though this complex is at lower resolution) than in S. pneumoniae. RESULTS +158 171 S. pneumoniae species The side chains surrounding them in ParE are quite disordered and are more defined in K. pneumoniae (even though this complex is at lower resolution) than in S. pneumoniae. RESULTS +20 34 hydrogen bonds bond_interaction There are no direct hydrogen bonds from the drug to these residues (although it is possible that some are formed through water, which cannot be observed at this resolution). RESULTS +121 126 water chemical There are no direct hydrogen bonds from the drug to these residues (although it is possible that some are formed through water, which cannot be observed at this resolution). RESULTS +20 24 ParE protein Obviously, the drug–ParE interaction in this region is less strong compared with PD 0305970 binding to the S. pneumoniae DNA complex, where PD 0305970 forms a hydrogen bond to ParE residue Asp475 and can form one to Asp474 if the bond rotates (Laponogov et al., 2010). RESULTS +81 91 PD 0305970 chemical Obviously, the drug–ParE interaction in this region is less strong compared with PD 0305970 binding to the S. pneumoniae DNA complex, where PD 0305970 forms a hydrogen bond to ParE residue Asp475 and can form one to Asp474 if the bond rotates (Laponogov et al., 2010). RESULTS +107 120 S. pneumoniae species Obviously, the drug–ParE interaction in this region is less strong compared with PD 0305970 binding to the S. pneumoniae DNA complex, where PD 0305970 forms a hydrogen bond to ParE residue Asp475 and can form one to Asp474 if the bond rotates (Laponogov et al., 2010). RESULTS +121 124 DNA chemical Obviously, the drug–ParE interaction in this region is less strong compared with PD 0305970 binding to the S. pneumoniae DNA complex, where PD 0305970 forms a hydrogen bond to ParE residue Asp475 and can form one to Asp474 if the bond rotates (Laponogov et al., 2010). RESULTS +140 150 PD 0305970 chemical Obviously, the drug–ParE interaction in this region is less strong compared with PD 0305970 binding to the S. pneumoniae DNA complex, where PD 0305970 forms a hydrogen bond to ParE residue Asp475 and can form one to Asp474 if the bond rotates (Laponogov et al., 2010). RESULTS +159 172 hydrogen bond bond_interaction Obviously, the drug–ParE interaction in this region is less strong compared with PD 0305970 binding to the S. pneumoniae DNA complex, where PD 0305970 forms a hydrogen bond to ParE residue Asp475 and can form one to Asp474 if the bond rotates (Laponogov et al., 2010). RESULTS +176 180 ParE protein Obviously, the drug–ParE interaction in this region is less strong compared with PD 0305970 binding to the S. pneumoniae DNA complex, where PD 0305970 forms a hydrogen bond to ParE residue Asp475 and can form one to Asp474 if the bond rotates (Laponogov et al., 2010). RESULTS +189 195 Asp475 residue_name_number Obviously, the drug–ParE interaction in this region is less strong compared with PD 0305970 binding to the S. pneumoniae DNA complex, where PD 0305970 forms a hydrogen bond to ParE residue Asp475 and can form one to Asp474 if the bond rotates (Laponogov et al., 2010). RESULTS +216 222 Asp474 residue_name_number Obviously, the drug–ParE interaction in this region is less strong compared with PD 0305970 binding to the S. pneumoniae DNA complex, where PD 0305970 forms a hydrogen bond to ParE residue Asp475 and can form one to Asp474 if the bond rotates (Laponogov et al., 2010). RESULTS +51 63 levofloxacin chemical This may explain why drug-resistance mutations for levofloxacin are more likely to form in the ParC subunits rather than in the ParE subunits. RESULTS +95 99 ParC protein This may explain why drug-resistance mutations for levofloxacin are more likely to form in the ParC subunits rather than in the ParE subunits. RESULTS +128 132 ParE protein This may explain why drug-resistance mutations for levofloxacin are more likely to form in the ParC subunits rather than in the ParE subunits. RESULTS +30 34 Mg2+ chemical For both complexes there is a Mg2+ ion bound to levofloxacin between the carbonyl group at position 4 of the quinolone and the carboxyl at position 6 (Figs. 2 ▸ and 5 ▸ and Supplementary Fig. 2 ▸). RESULTS +39 47 bound to protein_state For both complexes there is a Mg2+ ion bound to levofloxacin between the carbonyl group at position 4 of the quinolone and the carboxyl at position 6 (Figs. 2 ▸ and 5 ▸ and Supplementary Fig. 2 ▸). RESULTS +48 60 levofloxacin chemical For both complexes there is a Mg2+ ion bound to levofloxacin between the carbonyl group at position 4 of the quinolone and the carboxyl at position 6 (Figs. 2 ▸ and 5 ▸ and Supplementary Fig. 2 ▸). RESULTS +109 118 quinolone chemical For both complexes there is a Mg2+ ion bound to levofloxacin between the carbonyl group at position 4 of the quinolone and the carboxyl at position 6 (Figs. 2 ▸ and 5 ▸ and Supplementary Fig. 2 ▸). RESULTS +4 17 S. pneumoniae species For S. pneumoniae topoisomerase IV, one of the O atoms of the carboxyl of Asp83 points towards the Mg2+ ion and is within hydrogen-bonding distance (5.04 Å) through an Mg2+-coordinated water. RESULTS +18 34 topoisomerase IV complex_assembly For S. pneumoniae topoisomerase IV, one of the O atoms of the carboxyl of Asp83 points towards the Mg2+ ion and is within hydrogen-bonding distance (5.04 Å) through an Mg2+-coordinated water. RESULTS +74 79 Asp83 residue_name_number For S. pneumoniae topoisomerase IV, one of the O atoms of the carboxyl of Asp83 points towards the Mg2+ ion and is within hydrogen-bonding distance (5.04 Å) through an Mg2+-coordinated water. RESULTS +99 103 Mg2+ chemical For S. pneumoniae topoisomerase IV, one of the O atoms of the carboxyl of Asp83 points towards the Mg2+ ion and is within hydrogen-bonding distance (5.04 Å) through an Mg2+-coordinated water. RESULTS +122 138 hydrogen-bonding bond_interaction For S. pneumoniae topoisomerase IV, one of the O atoms of the carboxyl of Asp83 points towards the Mg2+ ion and is within hydrogen-bonding distance (5.04 Å) through an Mg2+-coordinated water. RESULTS +168 172 Mg2+ chemical For S. pneumoniae topoisomerase IV, one of the O atoms of the carboxyl of Asp83 points towards the Mg2+ ion and is within hydrogen-bonding distance (5.04 Å) through an Mg2+-coordinated water. RESULTS +185 190 water chemical For S. pneumoniae topoisomerase IV, one of the O atoms of the carboxyl of Asp83 points towards the Mg2+ ion and is within hydrogen-bonding distance (5.04 Å) through an Mg2+-coordinated water. RESULTS +4 17 K. pneumoniae species For K. pneumoniae both of the carboxyl O atoms are pointing towards the Mg2+ ion at distances of 4.86 and 4.23 Å. These residues are ordered in only one of the two dimers in the K. pneumoniae crystal (the one in which the C7 group is pointing towards the DNA away from ParE, although the conformations of these two groups on the drug are probably not correlated). RESULTS +72 76 Mg2+ chemical For K. pneumoniae both of the carboxyl O atoms are pointing towards the Mg2+ ion at distances of 4.86 and 4.23 Å. These residues are ordered in only one of the two dimers in the K. pneumoniae crystal (the one in which the C7 group is pointing towards the DNA away from ParE, although the conformations of these two groups on the drug are probably not correlated). RESULTS +164 170 dimers oligomeric_state For K. pneumoniae both of the carboxyl O atoms are pointing towards the Mg2+ ion at distances of 4.86 and 4.23 Å. These residues are ordered in only one of the two dimers in the K. pneumoniae crystal (the one in which the C7 group is pointing towards the DNA away from ParE, although the conformations of these two groups on the drug are probably not correlated). RESULTS +178 191 K. pneumoniae species For K. pneumoniae both of the carboxyl O atoms are pointing towards the Mg2+ ion at distances of 4.86 and 4.23 Å. These residues are ordered in only one of the two dimers in the K. pneumoniae crystal (the one in which the C7 group is pointing towards the DNA away from ParE, although the conformations of these two groups on the drug are probably not correlated). RESULTS +192 199 crystal evidence For K. pneumoniae both of the carboxyl O atoms are pointing towards the Mg2+ ion at distances of 4.86 and 4.23 Å. These residues are ordered in only one of the two dimers in the K. pneumoniae crystal (the one in which the C7 group is pointing towards the DNA away from ParE, although the conformations of these two groups on the drug are probably not correlated). RESULTS +255 258 DNA chemical For K. pneumoniae both of the carboxyl O atoms are pointing towards the Mg2+ ion at distances of 4.86 and 4.23 Å. These residues are ordered in only one of the two dimers in the K. pneumoniae crystal (the one in which the C7 group is pointing towards the DNA away from ParE, although the conformations of these two groups on the drug are probably not correlated). RESULTS +269 273 ParE protein For K. pneumoniae both of the carboxyl O atoms are pointing towards the Mg2+ ion at distances of 4.86 and 4.23 Å. These residues are ordered in only one of the two dimers in the K. pneumoniae crystal (the one in which the C7 group is pointing towards the DNA away from ParE, although the conformations of these two groups on the drug are probably not correlated). RESULTS +4 20 topoisomerase IV complex_assembly The topoisomerase IV ParE27-ParC55 fusion protein from K. pneumoniae was fully active in promoting levofloxacin-mediated cleavage of DNA (Fig. 6 ▸). RESULTS +21 34 ParE27-ParC55 complex_assembly The topoisomerase IV ParE27-ParC55 fusion protein from K. pneumoniae was fully active in promoting levofloxacin-mediated cleavage of DNA (Fig. 6 ▸). RESULTS +55 68 K. pneumoniae species The topoisomerase IV ParE27-ParC55 fusion protein from K. pneumoniae was fully active in promoting levofloxacin-mediated cleavage of DNA (Fig. 6 ▸). RESULTS +99 111 levofloxacin chemical The topoisomerase IV ParE27-ParC55 fusion protein from K. pneumoniae was fully active in promoting levofloxacin-mediated cleavage of DNA (Fig. 6 ▸). RESULTS +133 136 DNA chemical The topoisomerase IV ParE27-ParC55 fusion protein from K. pneumoniae was fully active in promoting levofloxacin-mediated cleavage of DNA (Fig. 6 ▸). RESULTS +7 17 absence of protein_state In the absence of the drug and ATP, the protein converted supercoiled pBR322 into a ladder of bands corresponding to relaxed DNA. RESULTS +22 26 drug chemical In the absence of the drug and ATP, the protein converted supercoiled pBR322 into a ladder of bands corresponding to relaxed DNA. RESULTS +31 34 ATP chemical In the absence of the drug and ATP, the protein converted supercoiled pBR322 into a ladder of bands corresponding to relaxed DNA. RESULTS +125 128 DNA chemical In the absence of the drug and ATP, the protein converted supercoiled pBR322 into a ladder of bands corresponding to relaxed DNA. RESULTS +17 29 levofloxacin chemical The inclusion of levofloxacin produced linear DNA in a dose-dependent and ATP-independent fashion. RESULTS +46 49 DNA chemical The inclusion of levofloxacin produced linear DNA in a dose-dependent and ATP-independent fashion. RESULTS +74 77 ATP chemical The inclusion of levofloxacin produced linear DNA in a dose-dependent and ATP-independent fashion. RESULTS +39 52 S. pneumoniae species Similar behaviour was observed for the S. pneumoniae topo­isomerase IV ParE30-ParC55 fusion protein. RESULTS +53 70 topo­isomerase IV complex_assembly Similar behaviour was observed for the S. pneumoniae topo­isomerase IV ParE30-ParC55 fusion protein. RESULTS +71 84 ParE30-ParC55 complex_assembly Similar behaviour was observed for the S. pneumoniae topo­isomerase IV ParE30-ParC55 fusion protein. RESULTS +4 8 CC25 evidence The CC25 (the drug concentration that converted 25% of the supercoiled DNA substrate to a linear form) was 0.5 µM for the Klebsiella enzyme and 1 µM for the pneumococcal enzyme. RESULTS +71 74 DNA chemical The CC25 (the drug concentration that converted 25% of the supercoiled DNA substrate to a linear form) was 0.5 µM for the Klebsiella enzyme and 1 µM for the pneumococcal enzyme. RESULTS +122 132 Klebsiella taxonomy_domain The CC25 (the drug concentration that converted 25% of the supercoiled DNA substrate to a linear form) was 0.5 µM for the Klebsiella enzyme and 1 µM for the pneumococcal enzyme. RESULTS +157 169 pneumococcal taxonomy_domain The CC25 (the drug concentration that converted 25% of the supercoiled DNA substrate to a linear form) was 0.5 µM for the Klebsiella enzyme and 1 µM for the pneumococcal enzyme. RESULTS +15 28 K. pneumoniae species Interestingly, K. pneumoniae strains are much more susceptible to levofloxacin than S. pneumoniae, with typical MIC values of 0.016 and 1 mg l−1, respectively (Odenholt & Cars, 2006), reflecting differences in multiple factors (in addition to binding affinity) that influence drug responses, including membrane, peptidoglycan structure, drug-uptake and efflux mechanisms. RESULTS +66 78 levofloxacin chemical Interestingly, K. pneumoniae strains are much more susceptible to levofloxacin than S. pneumoniae, with typical MIC values of 0.016 and 1 mg l−1, respectively (Odenholt & Cars, 2006), reflecting differences in multiple factors (in addition to binding affinity) that influence drug responses, including membrane, peptidoglycan structure, drug-uptake and efflux mechanisms. RESULTS +84 97 S. pneumoniae species Interestingly, K. pneumoniae strains are much more susceptible to levofloxacin than S. pneumoniae, with typical MIC values of 0.016 and 1 mg l−1, respectively (Odenholt & Cars, 2006), reflecting differences in multiple factors (in addition to binding affinity) that influence drug responses, including membrane, peptidoglycan structure, drug-uptake and efflux mechanisms. RESULTS +243 259 binding affinity evidence Interestingly, K. pneumoniae strains are much more susceptible to levofloxacin than S. pneumoniae, with typical MIC values of 0.016 and 1 mg l−1, respectively (Odenholt & Cars, 2006), reflecting differences in multiple factors (in addition to binding affinity) that influence drug responses, including membrane, peptidoglycan structure, drug-uptake and efflux mechanisms. RESULTS +19 35 topoisomerase IV complex_assembly Moreover, although topoisomerase IV is primarily the target of levofloxacin in S. pneumoniae, it is likely to be gyrase in the Gram-negative K. pneumoniae. RESULTS +63 75 levofloxacin chemical Moreover, although topoisomerase IV is primarily the target of levofloxacin in S. pneumoniae, it is likely to be gyrase in the Gram-negative K. pneumoniae. RESULTS +79 92 S. pneumoniae species Moreover, although topoisomerase IV is primarily the target of levofloxacin in S. pneumoniae, it is likely to be gyrase in the Gram-negative K. pneumoniae. RESULTS +113 119 gyrase protein_type Moreover, although topoisomerase IV is primarily the target of levofloxacin in S. pneumoniae, it is likely to be gyrase in the Gram-negative K. pneumoniae. RESULTS +127 140 Gram-negative taxonomy_domain Moreover, although topoisomerase IV is primarily the target of levofloxacin in S. pneumoniae, it is likely to be gyrase in the Gram-negative K. pneumoniae. RESULTS +141 154 K. pneumoniae species Moreover, although topoisomerase IV is primarily the target of levofloxacin in S. pneumoniae, it is likely to be gyrase in the Gram-negative K. pneumoniae. RESULTS +41 50 structure evidence In summary, we have determined the first structure of a quinolone–DNA cleavage complex involving a type II topo­isomerase from K. pneumoniae. RESULTS +56 65 quinolone chemical In summary, we have determined the first structure of a quinolone–DNA cleavage complex involving a type II topo­isomerase from K. pneumoniae. RESULTS +66 69 DNA chemical In summary, we have determined the first structure of a quinolone–DNA cleavage complex involving a type II topo­isomerase from K. pneumoniae. RESULTS +99 121 type II topo­isomerase protein_type In summary, we have determined the first structure of a quinolone–DNA cleavage complex involving a type II topo­isomerase from K. pneumoniae. RESULTS +127 140 K. pneumoniae species In summary, we have determined the first structure of a quinolone–DNA cleavage complex involving a type II topo­isomerase from K. pneumoniae. RESULTS +59 69 Klebsiella taxonomy_domain Given the current concerns about drug-resistant strains of Klebsiella, the structure reported here provides key information in understanding the action of currently used quinolones and should aid in the development of other topoisomerase-targeting therapeutics active against this major human pathogen. RESULTS +75 84 structure evidence Given the current concerns about drug-resistant strains of Klebsiella, the structure reported here provides key information in understanding the action of currently used quinolones and should aid in the development of other topoisomerase-targeting therapeutics active against this major human pathogen. RESULTS +170 180 quinolones chemical Given the current concerns about drug-resistant strains of Klebsiella, the structure reported here provides key information in understanding the action of currently used quinolones and should aid in the development of other topoisomerase-targeting therapeutics active against this major human pathogen. RESULTS +224 237 topoisomerase protein_type Given the current concerns about drug-resistant strains of Klebsiella, the structure reported here provides key information in understanding the action of currently used quinolones and should aid in the development of other topoisomerase-targeting therapeutics active against this major human pathogen. RESULTS +287 292 human species Given the current concerns about drug-resistant strains of Klebsiella, the structure reported here provides key information in understanding the action of currently used quinolones and should aid in the development of other topoisomerase-targeting therapeutics active against this major human pathogen. RESULTS +12 15 DNA chemical Protein and DNA used in the co-crystallization experiment. FIG +28 46 co-crystallization experimental_method Protein and DNA used in the co-crystallization experiment. FIG +55 70 crystallization experimental_method (a) Coloured diagram of the protein constructs used in crystallization. FIG +4 7 DNA chemical (b) DNA sequences used in crystallization. FIG +26 41 crystallization experimental_method (b) DNA sequences used in crystallization. FIG +22 34 levofloxacin chemical Chemical structure of levofloxacin (a) and its conformations observed within the active sites of S. pneumoniae topoisomerase IV (b) and K. pneumoniae topoisomerase IV (c, d). FIG +81 93 active sites site Chemical structure of levofloxacin (a) and its conformations observed within the active sites of S. pneumoniae topoisomerase IV (b) and K. pneumoniae topoisomerase IV (c, d). FIG +97 110 S. pneumoniae species Chemical structure of levofloxacin (a) and its conformations observed within the active sites of S. pneumoniae topoisomerase IV (b) and K. pneumoniae topoisomerase IV (c, d). FIG +111 127 topoisomerase IV complex_assembly Chemical structure of levofloxacin (a) and its conformations observed within the active sites of S. pneumoniae topoisomerase IV (b) and K. pneumoniae topoisomerase IV (c, d). FIG +136 149 K. pneumoniae species Chemical structure of levofloxacin (a) and its conformations observed within the active sites of S. pneumoniae topoisomerase IV (b) and K. pneumoniae topoisomerase IV (c, d). FIG +150 166 topoisomerase IV complex_assembly Chemical structure of levofloxacin (a) and its conformations observed within the active sites of S. pneumoniae topoisomerase IV (b) and K. pneumoniae topoisomerase IV (c, d). FIG +0 21 Electron-density maps evidence Electron-density maps (2F obs − F calc) are shown as meshes for the drug molecules contoured at 1.5σ and are limited to a distance of 2.3 Å from the drug atoms. FIG +52 68 topoisomerase IV complex_assembly Overall orthogonal views of the cleavage complex of topoisomerase IV from K. pneumoniae in surface (left) and cartoon (right) representations. FIG +74 87 K. pneumoniae species Overall orthogonal views of the cleavage complex of topoisomerase IV from K. pneumoniae in surface (left) and cartoon (right) representations. FIG +4 8 ParC protein The ParC subunit is in blue, ParE is in yellow and DNA is in cyan. FIG +29 33 ParE protein The ParC subunit is in blue, ParE is in yellow and DNA is in cyan. FIG +51 54 DNA chemical The ParC subunit is in blue, ParE is in yellow and DNA is in cyan. FIG +4 9 bound protein_state The bound quinolone molecules (levofloxacin) are in red and are shown using van der Waals representation. FIG +10 19 quinolone chemical The bound quinolone molecules (levofloxacin) are in red and are shown using van der Waals representation. FIG +31 43 levofloxacin chemical The bound quinolone molecules (levofloxacin) are in red and are shown using van der Waals representation. FIG +51 73 type II topoisomerases protein_type Schematic representation of the catalytic cycle of type II topoisomerases. FIG +4 8 ParC protein The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +28 34 ParC55 protein The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +52 56 ParC protein The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +68 86 β-­pinwheel domain structure_element The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +105 109 ParE protein The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +121 134 ATPase domain structure_element The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +150 154 ParE protein The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +155 172 C-terminal domain structure_element The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +174 180 ParE30 protein The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +200 206 G-gate structure_element The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +207 210 DNA chemical The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +231 240 T-segment structure_element The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +241 244 DNA chemical The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. FIG +0 5 Bound protein_state Bound ATP is indicated by pink circles in the ATPase domains (reproduced with permission from Fig. 1 of Lapanogov et al., 2013). FIG +6 9 ATP chemical Bound ATP is indicated by pink circles in the ATPase domains (reproduced with permission from Fig. 1 of Lapanogov et al., 2013). FIG +46 60 ATPase domains structure_element Bound ATP is indicated by pink circles in the ATPase domains (reproduced with permission from Fig. 1 of Lapanogov et al., 2013). FIG +22 34 active sites site Detailed views of the active sites of topoisomerase IV from S. pneumoniae and K. pneumoniae with quinolone molecules bound. FIG +38 54 topoisomerase IV complex_assembly Detailed views of the active sites of topoisomerase IV from S. pneumoniae and K. pneumoniae with quinolone molecules bound. FIG +60 73 S. pneumoniae species Detailed views of the active sites of topoisomerase IV from S. pneumoniae and K. pneumoniae with quinolone molecules bound. FIG +78 91 K. pneumoniae species Detailed views of the active sites of topoisomerase IV from S. pneumoniae and K. pneumoniae with quinolone molecules bound. FIG +97 106 quinolone chemical Detailed views of the active sites of topoisomerase IV from S. pneumoniae and K. pneumoniae with quinolone molecules bound. FIG +117 122 bound protein_state Detailed views of the active sites of topoisomerase IV from S. pneumoniae and K. pneumoniae with quinolone molecules bound. FIG +4 13 magnesium chemical The magnesium ions and their coordinating amino acids are shown in purple. FIG +4 15 active-site site The active-site tyrosine and arginine are in orange. FIG +16 24 tyrosine residue_name The active-site tyrosine and arginine are in orange. FIG +29 37 arginine residue_name The active-site tyrosine and arginine are in orange. FIG +4 7 DNA chemical The DNA is shown in silver/cyan. FIG +4 8 ParC protein The ParC and ParE backbones are shown in blue and yellow, respectively. FIG +13 17 ParE protein The ParC and ParE backbones are shown in blue and yellow, respectively. FIG +14 17 DNA chemical Comparison of DNA cleavage by topoisomerase IV core ParE-ParC fusion proteins from K. pneumoniae (KP) and S. pneumoniae (SP) promoted by levofloxacin. FIG +30 46 topoisomerase IV complex_assembly Comparison of DNA cleavage by topoisomerase IV core ParE-ParC fusion proteins from K. pneumoniae (KP) and S. pneumoniae (SP) promoted by levofloxacin. FIG +52 61 ParE-ParC complex_assembly Comparison of DNA cleavage by topoisomerase IV core ParE-ParC fusion proteins from K. pneumoniae (KP) and S. pneumoniae (SP) promoted by levofloxacin. FIG +83 96 K. pneumoniae species Comparison of DNA cleavage by topoisomerase IV core ParE-ParC fusion proteins from K. pneumoniae (KP) and S. pneumoniae (SP) promoted by levofloxacin. FIG +98 100 KP species Comparison of DNA cleavage by topoisomerase IV core ParE-ParC fusion proteins from K. pneumoniae (KP) and S. pneumoniae (SP) promoted by levofloxacin. FIG +106 119 S. pneumoniae species Comparison of DNA cleavage by topoisomerase IV core ParE-ParC fusion proteins from K. pneumoniae (KP) and S. pneumoniae (SP) promoted by levofloxacin. FIG +121 123 SP species Comparison of DNA cleavage by topoisomerase IV core ParE-ParC fusion proteins from K. pneumoniae (KP) and S. pneumoniae (SP) promoted by levofloxacin. FIG +137 149 levofloxacin chemical Comparison of DNA cleavage by topoisomerase IV core ParE-ParC fusion proteins from K. pneumoniae (KP) and S. pneumoniae (SP) promoted by levofloxacin. FIG +55 71 topoisomerase IV complex_assembly Supercoiled plasmid pBR322 (400 ng) was incubated with topoisomerase IV proteins (400 ng) in the absence or presence of levofloxacin at the indicated concentrations. FIG +108 119 presence of protein_state Supercoiled plasmid pBR322 (400 ng) was incubated with topoisomerase IV proteins (400 ng) in the absence or presence of levofloxacin at the indicated concentrations. FIG +120 132 levofloxacin chemical Supercoiled plasmid pBR322 (400 ng) was incubated with topoisomerase IV proteins (400 ng) in the absence or presence of levofloxacin at the indicated concentrations. FIG +109 112 DNA chemical After 60 min incubation, samples were treated with SDS and proteinase K to remove proteins covalent bound to DNA, and the DNA products were examined by gel electrophoresis in 1% agarose. FIG +122 125 DNA chemical After 60 min incubation, samples were treated with SDS and proteinase K to remove proteins covalent bound to DNA, and the DNA products were examined by gel electrophoresis in 1% agarose. FIG +27 30 DNA chemical Lane A, supercoiled pBR322 DNA; N, L and S, nicked, linear and supercoiled pBR322, respectively. FIG diff --git a/annotation_CSV/PMC4831588.csv b/annotation_CSV/PMC4831588.csv new file mode 100644 index 0000000000000000000000000000000000000000..d1208fd8fd4f33d3498db6cabe1c49c24ad7c63e --- /dev/null +++ b/annotation_CSV/PMC4831588.csv @@ -0,0 +1,1071 @@ +anno_start anno_end anno_text entity_type sentence section +0 33 X-ray Crystallographic Structures evidence X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an Aβ17–36 β-Hairpin TITLE +39 45 Trimer oligomeric_state X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an Aβ17–36 β-Hairpin TITLE +47 56 Dodecamer oligomeric_state X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an Aβ17–36 β-Hairpin TITLE +62 74 Annular Pore site X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an Aβ17–36 β-Hairpin TITLE +88 90 Aβ protein X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an Aβ17–36 β-Hairpin TITLE +90 95 17–36 residue_range X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an Aβ17–36 β-Hairpin TITLE +96 105 β-Hairpin structure_element X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an Aβ17–36 β-Hairpin TITLE +16 26 structures evidence High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are needed to understand the molecular basis of Alzheimer’s disease and develop therapies. ABSTRACT +30 39 oligomers oligomeric_state High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are needed to understand the molecular basis of Alzheimer’s disease and develop therapies. ABSTRACT +54 71 β-amyloid peptide protein High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are needed to understand the molecular basis of Alzheimer’s disease and develop therapies. ABSTRACT +72 74 Aβ protein High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are needed to understand the molecular basis of Alzheimer’s disease and develop therapies. ABSTRACT +24 57 X-ray crystallographic structures evidence This paper presents the X-ray crystallographic structures of oligomers formed by a 20-residue peptide segment derived from Aβ. ABSTRACT +61 70 oligomers oligomeric_state This paper presents the X-ray crystallographic structures of oligomers formed by a 20-residue peptide segment derived from Aβ. ABSTRACT +83 109 20-residue peptide segment residue_range This paper presents the X-ray crystallographic structures of oligomers formed by a 20-residue peptide segment derived from Aβ. ABSTRACT +123 125 Aβ protein This paper presents the X-ray crystallographic structures of oligomers formed by a 20-residue peptide segment derived from Aβ. ABSTRACT +38 40 Aβ protein The development of a peptide in which Aβ17–36 is stabilized as a β-hairpin is described, and the X-ray crystallographic structures of oligomers it forms are reported. ABSTRACT +40 45 17–36 residue_range The development of a peptide in which Aβ17–36 is stabilized as a β-hairpin is described, and the X-ray crystallographic structures of oligomers it forms are reported. ABSTRACT +65 74 β-hairpin structure_element The development of a peptide in which Aβ17–36 is stabilized as a β-hairpin is described, and the X-ray crystallographic structures of oligomers it forms are reported. ABSTRACT +97 130 X-ray crystallographic structures evidence The development of a peptide in which Aβ17–36 is stabilized as a β-hairpin is described, and the X-ray crystallographic structures of oligomers it forms are reported. ABSTRACT +134 143 oligomers oligomeric_state The development of a peptide in which Aβ17–36 is stabilized as a β-hairpin is described, and the X-ray crystallographic structures of oligomers it forms are reported. ABSTRACT +56 58 Aβ protein Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. ABSTRACT +58 63 17–36 residue_range Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. ABSTRACT +77 84 hairpin structure_element Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. ABSTRACT +101 109 δ-linked protein_state Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. ABSTRACT +110 119 ornithine residue_name Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. ABSTRACT +120 124 turn structure_element Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. ABSTRACT +146 148 17 residue_number Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. ABSTRACT +153 155 36 residue_number Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. ABSTRACT +201 218 disulfide linkage ptm Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. ABSTRACT +237 239 24 residue_number Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. ABSTRACT +244 246 29 residue_number Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. ABSTRACT +30 32 33 residue_number An N-methyl group at position 33 blocks uncontrolled aggregation. ABSTRACT +12 32 readily crystallizes evidence The peptide readily crystallizes as a folded β-hairpin, which assembles hierarchically in the crystal lattice. ABSTRACT +38 44 folded protein_state The peptide readily crystallizes as a folded β-hairpin, which assembles hierarchically in the crystal lattice. ABSTRACT +45 54 β-hairpin structure_element The peptide readily crystallizes as a folded β-hairpin, which assembles hierarchically in the crystal lattice. ABSTRACT +94 109 crystal lattice evidence The peptide readily crystallizes as a folded β-hairpin, which assembles hierarchically in the crystal lattice. ABSTRACT +6 15 β-hairpin structure_element Three β-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore. ABSTRACT +16 24 monomers oligomeric_state Three β-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore. ABSTRACT +44 54 triangular protein_state Three β-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore. ABSTRACT +55 61 trimer oligomeric_state Three β-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore. ABSTRACT +68 75 trimers oligomeric_state Three β-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore. ABSTRACT +124 133 dodecamer oligomeric_state Three β-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore. ABSTRACT +144 154 dodecamers oligomeric_state Three β-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore. ABSTRACT +180 192 annular pore site Three β-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore. ABSTRACT +54 65 full-length protein_state This hierarchical assembly provides a model, in which full-length Aβ transitions from an unfolded monomer to a folded β-hairpin, which assembles to form oligomers that further pack to form an annular pore. ABSTRACT +66 68 Aβ protein This hierarchical assembly provides a model, in which full-length Aβ transitions from an unfolded monomer to a folded β-hairpin, which assembles to form oligomers that further pack to form an annular pore. ABSTRACT +89 97 unfolded protein_state This hierarchical assembly provides a model, in which full-length Aβ transitions from an unfolded monomer to a folded β-hairpin, which assembles to form oligomers that further pack to form an annular pore. ABSTRACT +98 105 monomer oligomeric_state This hierarchical assembly provides a model, in which full-length Aβ transitions from an unfolded monomer to a folded β-hairpin, which assembles to form oligomers that further pack to form an annular pore. ABSTRACT +111 117 folded protein_state This hierarchical assembly provides a model, in which full-length Aβ transitions from an unfolded monomer to a folded β-hairpin, which assembles to form oligomers that further pack to form an annular pore. ABSTRACT +118 127 β-hairpin structure_element This hierarchical assembly provides a model, in which full-length Aβ transitions from an unfolded monomer to a folded β-hairpin, which assembles to form oligomers that further pack to form an annular pore. ABSTRACT +153 162 oligomers oligomeric_state This hierarchical assembly provides a model, in which full-length Aβ transitions from an unfolded monomer to a folded β-hairpin, which assembles to form oligomers that further pack to form an annular pore. ABSTRACT +192 204 annular pore site This hierarchical assembly provides a model, in which full-length Aβ transitions from an unfolded monomer to a folded β-hairpin, which assembles to form oligomers that further pack to form an annular pore. ABSTRACT +16 26 structures evidence High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are desperately needed to understand the molecular basis of Alzheimer’s disease and ultimately develop preventions or treatments. INTRO +30 39 oligomers oligomeric_state High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are desperately needed to understand the molecular basis of Alzheimer’s disease and ultimately develop preventions or treatments. INTRO +54 71 β-amyloid peptide protein High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are desperately needed to understand the molecular basis of Alzheimer’s disease and ultimately develop preventions or treatments. INTRO +72 74 Aβ protein High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are desperately needed to understand the molecular basis of Alzheimer’s disease and ultimately develop preventions or treatments. INTRO +24 33 monomeric oligomeric_state In Alzheimer’s disease, monomeric Aβ aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils. INTRO +34 36 Aβ protein In Alzheimer’s disease, monomeric Aβ aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils. INTRO +85 94 oligomers oligomeric_state In Alzheimer’s disease, monomeric Aβ aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils. INTRO +104 110 dimers oligomeric_state In Alzheimer’s disease, monomeric Aβ aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils. INTRO +112 119 trimers oligomeric_state In Alzheimer’s disease, monomeric Aβ aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils. INTRO +121 130 tetramers oligomeric_state In Alzheimer’s disease, monomeric Aβ aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils. INTRO +132 140 hexamers oligomeric_state In Alzheimer’s disease, monomeric Aβ aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils. INTRO +142 150 nonamers oligomeric_state In Alzheimer’s disease, monomeric Aβ aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils. INTRO +156 166 dodecamers oligomeric_state In Alzheimer’s disease, monomeric Aβ aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils. INTRO +221 241 annular protofibrils complex_assembly In Alzheimer’s disease, monomeric Aβ aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils. INTRO +38 40 Aβ protein Over the last two decades the role of Aβ oligomers in the pathophysiology of Alzheimer’s disease has begun to unfold. INTRO +41 50 oligomers oligomeric_state Over the last two decades the role of Aβ oligomers in the pathophysiology of Alzheimer’s disease has begun to unfold. INTRO +0 5 Mouse taxonomy_domain Mouse models for Alzheimer’s disease have helped shape our current understanding about the Aβ oligomerization that precedes neurodegeneration. INTRO +91 93 Aβ protein Mouse models for Alzheimer’s disease have helped shape our current understanding about the Aβ oligomerization that precedes neurodegeneration. INTRO +0 2 Aβ protein Aβ isolated from the brains of young plaque-free Tg2576 mice forms a mixture of low molecular weight oligomers. INTRO +56 60 mice taxonomy_domain Aβ isolated from the brains of young plaque-free Tg2576 mice forms a mixture of low molecular weight oligomers. INTRO +101 110 oligomers oligomeric_state Aβ isolated from the brains of young plaque-free Tg2576 mice forms a mixture of low molecular weight oligomers. INTRO +17 25 oligomer oligomeric_state A 56 kDa soluble oligomer identified by SDS-PAGE was found to be especially important within this mixture. INTRO +40 48 SDS-PAGE experimental_method A 56 kDa soluble oligomer identified by SDS-PAGE was found to be especially important within this mixture. INTRO +5 13 oligomer oligomeric_state This oligomer was termed Aβ*56 and appears to be a dodecamer of Aβ. INTRO +25 30 Aβ*56 complex_assembly This oligomer was termed Aβ*56 and appears to be a dodecamer of Aβ. INTRO +51 60 dodecamer oligomeric_state This oligomer was termed Aβ*56 and appears to be a dodecamer of Aβ. INTRO +64 66 Aβ protein This oligomer was termed Aβ*56 and appears to be a dodecamer of Aβ. INTRO +9 14 Aβ*56 complex_assembly Purified Aβ*56 injected intercranially into healthy rats was found to impair memory, providing evidence that this Aβ oligomer may cause memory loss in Alzheimer’s disease. INTRO +15 38 injected intercranially experimental_method Purified Aβ*56 injected intercranially into healthy rats was found to impair memory, providing evidence that this Aβ oligomer may cause memory loss in Alzheimer’s disease. INTRO +52 56 rats taxonomy_domain Purified Aβ*56 injected intercranially into healthy rats was found to impair memory, providing evidence that this Aβ oligomer may cause memory loss in Alzheimer’s disease. INTRO +114 116 Aβ protein Purified Aβ*56 injected intercranially into healthy rats was found to impair memory, providing evidence that this Aβ oligomer may cause memory loss in Alzheimer’s disease. INTRO +117 125 oligomer oligomeric_state Purified Aβ*56 injected intercranially into healthy rats was found to impair memory, providing evidence that this Aβ oligomer may cause memory loss in Alzheimer’s disease. INTRO +8 17 oligomers oligomeric_state Smaller oligomers with molecular weights consistent with trimers, hexamers, and nonamers were also identified within the mixture of low molecular weight oligomers. INTRO +57 64 trimers oligomeric_state Smaller oligomers with molecular weights consistent with trimers, hexamers, and nonamers were also identified within the mixture of low molecular weight oligomers. INTRO +66 74 hexamers oligomeric_state Smaller oligomers with molecular weights consistent with trimers, hexamers, and nonamers were also identified within the mixture of low molecular weight oligomers. INTRO +80 88 nonamers oligomeric_state Smaller oligomers with molecular weights consistent with trimers, hexamers, and nonamers were also identified within the mixture of low molecular weight oligomers. INTRO +153 162 oligomers oligomeric_state Smaller oligomers with molecular weights consistent with trimers, hexamers, and nonamers were also identified within the mixture of low molecular weight oligomers. INTRO +49 58 oligomers oligomeric_state Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. INTRO +64 85 hexafluoroisopropanol chemical Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. INTRO +131 141 dodecamers oligomeric_state Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. INTRO +143 151 nonamers oligomeric_state Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. INTRO +157 165 hexamers oligomeric_state Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. INTRO +171 178 trimers oligomeric_state Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. INTRO +183 191 monomers oligomeric_state Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. INTRO +209 216 trimers oligomeric_state Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. INTRO +250 260 dodecamers oligomeric_state Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. INTRO +262 270 nonamers oligomeric_state Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. INTRO +276 284 hexamers oligomeric_state Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. INTRO +10 12 Aβ protein Recently, Aβ trimers and Aβ*56 were identified in the brains of cognitively normal humans and were found to increase with age. INTRO +13 20 trimers oligomeric_state Recently, Aβ trimers and Aβ*56 were identified in the brains of cognitively normal humans and were found to increase with age. INTRO +25 30 Aβ*56 complex_assembly Recently, Aβ trimers and Aβ*56 were identified in the brains of cognitively normal humans and were found to increase with age. INTRO +83 89 humans species Recently, Aβ trimers and Aβ*56 were identified in the brains of cognitively normal humans and were found to increase with age. INTRO +16 25 oligomers oligomeric_state A type of large oligomers called annular protofibrils (APFs) have also been observed in the brains of transgenic mice and isolated from the brains of Alzheimer’s patients. INTRO +33 53 annular protofibrils complex_assembly A type of large oligomers called annular protofibrils (APFs) have also been observed in the brains of transgenic mice and isolated from the brains of Alzheimer’s patients. INTRO +55 59 APFs complex_assembly A type of large oligomers called annular protofibrils (APFs) have also been observed in the brains of transgenic mice and isolated from the brains of Alzheimer’s patients. INTRO +113 117 mice taxonomy_domain A type of large oligomers called annular protofibrils (APFs) have also been observed in the brains of transgenic mice and isolated from the brains of Alzheimer’s patients. INTRO +0 4 APFs complex_assembly APFs were first discovered in vitro using chemically synthesized Aβ that aggregated into porelike structures that could be observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). INTRO +42 64 chemically synthesized protein_state APFs were first discovered in vitro using chemically synthesized Aβ that aggregated into porelike structures that could be observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). INTRO +65 67 Aβ protein APFs were first discovered in vitro using chemically synthesized Aβ that aggregated into porelike structures that could be observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). INTRO +89 108 porelike structures structure_element APFs were first discovered in vitro using chemically synthesized Aβ that aggregated into porelike structures that could be observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). INTRO +135 158 atomic force microscopy experimental_method APFs were first discovered in vitro using chemically synthesized Aβ that aggregated into porelike structures that could be observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). INTRO +160 163 AFM experimental_method APFs were first discovered in vitro using chemically synthesized Aβ that aggregated into porelike structures that could be observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). INTRO +169 201 transmission electron microscopy experimental_method APFs were first discovered in vitro using chemically synthesized Aβ that aggregated into porelike structures that could be observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). INTRO +203 206 TEM experimental_method APFs were first discovered in vitro using chemically synthesized Aβ that aggregated into porelike structures that could be observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). INTRO +13 17 APFs complex_assembly The sizes of APFs prepared in vitro vary among different studies. INTRO +24 28 APFs complex_assembly Lashuel et al. observed APFs with an outer diameter that ranged from 7–10 nm and an inner diameter that ranged from 1.5–2 nm, consistent with molecular weights of 150–250 kDa. INTRO +22 26 APFs complex_assembly Quist et al. observed APFs with an outer diameter of 16 nm embedded in a lipid bilayer. INTRO +22 26 APFs complex_assembly Kayed et al. observed APFs with an outer diameter that ranged from 8–25 nm, which were composed of small spherical Aβ oligomers, 3–5 nm in diameter. INTRO +99 114 small spherical protein_state Kayed et al. observed APFs with an outer diameter that ranged from 8–25 nm, which were composed of small spherical Aβ oligomers, 3–5 nm in diameter. INTRO +115 117 Aβ protein Kayed et al. observed APFs with an outer diameter that ranged from 8–25 nm, which were composed of small spherical Aβ oligomers, 3–5 nm in diameter. INTRO +118 127 oligomers oligomeric_state Kayed et al. observed APFs with an outer diameter that ranged from 8–25 nm, which were composed of small spherical Aβ oligomers, 3–5 nm in diameter. INTRO +13 17 APFs complex_assembly Although the APFs in these studies differ in size, they share a similar annular morphology and appear to be composed of smaller oligomers. INTRO +128 137 oligomers oligomeric_state Although the APFs in these studies differ in size, they share a similar annular morphology and appear to be composed of smaller oligomers. INTRO +0 4 APFs complex_assembly APFs have also been observed in the brains of APP23 transgenic mice by immunofluorescence with an anti-APF antibody and were found to accumulate in neuronal processes and synapses. INTRO +63 67 mice taxonomy_domain APFs have also been observed in the brains of APP23 transgenic mice by immunofluorescence with an anti-APF antibody and were found to accumulate in neuronal processes and synapses. INTRO +71 89 immunofluorescence experimental_method APFs have also been observed in the brains of APP23 transgenic mice by immunofluorescence with an anti-APF antibody and were found to accumulate in neuronal processes and synapses. INTRO +103 106 APF complex_assembly APFs have also been observed in the brains of APP23 transgenic mice by immunofluorescence with an anti-APF antibody and were found to accumulate in neuronal processes and synapses. INTRO +23 27 APFs complex_assembly In a subsequent study, APFs were isolated from the brains of Alzheimer’s patients by immunoprecipitation with an anti-APF antibody. INTRO +85 104 immunoprecipitation experimental_method In a subsequent study, APFs were isolated from the brains of Alzheimer’s patients by immunoprecipitation with an anti-APF antibody. INTRO +118 121 APF complex_assembly In a subsequent study, APFs were isolated from the brains of Alzheimer’s patients by immunoprecipitation with an anti-APF antibody. INTRO +6 10 APFs complex_assembly These APFs had an outer diameter that ranged from 11–14 nm and an inner diameter that ranged from 2.5–4 nm. INTRO +0 6 Dimers oligomeric_state Dimers of Aβ have also been isolated from the brains of Alzheimer’s patients.− Aβ dimers inhibit long-term potentiation in mice and promote hyperphosphorylation of the microtubule-associated protein tau, leading to neuritic damage. INTRO +10 12 Aβ protein Dimers of Aβ have also been isolated from the brains of Alzheimer’s patients.− Aβ dimers inhibit long-term potentiation in mice and promote hyperphosphorylation of the microtubule-associated protein tau, leading to neuritic damage. INTRO +79 81 Aβ protein Dimers of Aβ have also been isolated from the brains of Alzheimer’s patients.− Aβ dimers inhibit long-term potentiation in mice and promote hyperphosphorylation of the microtubule-associated protein tau, leading to neuritic damage. INTRO +82 88 dimers oligomeric_state Dimers of Aβ have also been isolated from the brains of Alzheimer’s patients.− Aβ dimers inhibit long-term potentiation in mice and promote hyperphosphorylation of the microtubule-associated protein tau, leading to neuritic damage. INTRO +123 127 mice taxonomy_domain Dimers of Aβ have also been isolated from the brains of Alzheimer’s patients.− Aβ dimers inhibit long-term potentiation in mice and promote hyperphosphorylation of the microtubule-associated protein tau, leading to neuritic damage. INTRO +140 160 hyperphosphorylation ptm Dimers of Aβ have also been isolated from the brains of Alzheimer’s patients.− Aβ dimers inhibit long-term potentiation in mice and promote hyperphosphorylation of the microtubule-associated protein tau, leading to neuritic damage. INTRO +168 202 microtubule-associated protein tau protein Dimers of Aβ have also been isolated from the brains of Alzheimer’s patients.− Aβ dimers inhibit long-term potentiation in mice and promote hyperphosphorylation of the microtubule-associated protein tau, leading to neuritic damage. INTRO +0 2 Aβ protein Aβ dimers have only been isolated from human or transgenic mouse brains that contain the pathognomonic fibrillar Aβ plaques associated with Alzheimer’s disease. INTRO +3 9 dimers oligomeric_state Aβ dimers have only been isolated from human or transgenic mouse brains that contain the pathognomonic fibrillar Aβ plaques associated with Alzheimer’s disease. INTRO +39 44 human species Aβ dimers have only been isolated from human or transgenic mouse brains that contain the pathognomonic fibrillar Aβ plaques associated with Alzheimer’s disease. INTRO +59 64 mouse taxonomy_domain Aβ dimers have only been isolated from human or transgenic mouse brains that contain the pathognomonic fibrillar Aβ plaques associated with Alzheimer’s disease. INTRO +103 112 fibrillar protein_state Aβ dimers have only been isolated from human or transgenic mouse brains that contain the pathognomonic fibrillar Aβ plaques associated with Alzheimer’s disease. INTRO +113 115 Aβ protein Aβ dimers have only been isolated from human or transgenic mouse brains that contain the pathognomonic fibrillar Aβ plaques associated with Alzheimer’s disease. INTRO +36 38 Aβ protein Furthermore, the endogenous rise of Aβ dimers in the brains of Tg2576 and J20 transgenic mice coincides with the deposition of Aβ plaques. INTRO +39 45 dimers oligomeric_state Furthermore, the endogenous rise of Aβ dimers in the brains of Tg2576 and J20 transgenic mice coincides with the deposition of Aβ plaques. INTRO +89 93 mice taxonomy_domain Furthermore, the endogenous rise of Aβ dimers in the brains of Tg2576 and J20 transgenic mice coincides with the deposition of Aβ plaques. INTRO +127 129 Aβ protein Furthermore, the endogenous rise of Aβ dimers in the brains of Tg2576 and J20 transgenic mice coincides with the deposition of Aβ plaques. INTRO +36 38 Aβ protein These observations suggest that the Aβ trimers, hexamers, dodecamers, and related assemblies may be associated with presymptomatic neurodegeneration, while Aβ dimers are more closely associated with fibril formation and plaque deposition during symptomatic Alzheimer’s disease.− INTRO +39 46 trimers oligomeric_state These observations suggest that the Aβ trimers, hexamers, dodecamers, and related assemblies may be associated with presymptomatic neurodegeneration, while Aβ dimers are more closely associated with fibril formation and plaque deposition during symptomatic Alzheimer’s disease.− INTRO +48 56 hexamers oligomeric_state These observations suggest that the Aβ trimers, hexamers, dodecamers, and related assemblies may be associated with presymptomatic neurodegeneration, while Aβ dimers are more closely associated with fibril formation and plaque deposition during symptomatic Alzheimer’s disease.− INTRO +58 68 dodecamers oligomeric_state These observations suggest that the Aβ trimers, hexamers, dodecamers, and related assemblies may be associated with presymptomatic neurodegeneration, while Aβ dimers are more closely associated with fibril formation and plaque deposition during symptomatic Alzheimer’s disease.− INTRO +156 158 Aβ protein These observations suggest that the Aβ trimers, hexamers, dodecamers, and related assemblies may be associated with presymptomatic neurodegeneration, while Aβ dimers are more closely associated with fibril formation and plaque deposition during symptomatic Alzheimer’s disease.− INTRO +159 165 dimers oligomeric_state These observations suggest that the Aβ trimers, hexamers, dodecamers, and related assemblies may be associated with presymptomatic neurodegeneration, while Aβ dimers are more closely associated with fibril formation and plaque deposition during symptomatic Alzheimer’s disease.− INTRO +45 47 Aβ protein The approach of isolating and characterizing Aβ oligomers has not provided any high-resolution structures of Aβ oligomers. INTRO +48 57 oligomers oligomeric_state The approach of isolating and characterizing Aβ oligomers has not provided any high-resolution structures of Aβ oligomers. INTRO +95 105 structures evidence The approach of isolating and characterizing Aβ oligomers has not provided any high-resolution structures of Aβ oligomers. INTRO +109 111 Aβ protein The approach of isolating and characterizing Aβ oligomers has not provided any high-resolution structures of Aβ oligomers. INTRO +112 121 oligomers oligomeric_state The approach of isolating and characterizing Aβ oligomers has not provided any high-resolution structures of Aβ oligomers. INTRO +19 27 SDS-PAGE experimental_method Techniques such as SDS-PAGE, TEM, and AFM have only provided information about the molecular weights, sizes, morphologies, and stoichiometry of Aβ oligomers. INTRO +29 32 TEM experimental_method Techniques such as SDS-PAGE, TEM, and AFM have only provided information about the molecular weights, sizes, morphologies, and stoichiometry of Aβ oligomers. INTRO +38 41 AFM experimental_method Techniques such as SDS-PAGE, TEM, and AFM have only provided information about the molecular weights, sizes, morphologies, and stoichiometry of Aβ oligomers. INTRO +144 146 Aβ protein Techniques such as SDS-PAGE, TEM, and AFM have only provided information about the molecular weights, sizes, morphologies, and stoichiometry of Aβ oligomers. INTRO +147 156 oligomers oligomeric_state Techniques such as SDS-PAGE, TEM, and AFM have only provided information about the molecular weights, sizes, morphologies, and stoichiometry of Aβ oligomers. INTRO +16 34 structural studies experimental_method High-resolution structural studies of Aβ have primarily focused on Aβ fibrils and Aβ monomers. INTRO +38 40 Aβ protein High-resolution structural studies of Aβ have primarily focused on Aβ fibrils and Aβ monomers. INTRO +67 69 Aβ protein High-resolution structural studies of Aβ have primarily focused on Aβ fibrils and Aβ monomers. INTRO +70 77 fibrils oligomeric_state High-resolution structural studies of Aβ have primarily focused on Aβ fibrils and Aβ monomers. INTRO +82 84 Aβ protein High-resolution structural studies of Aβ have primarily focused on Aβ fibrils and Aβ monomers. INTRO +85 93 monomers oligomeric_state High-resolution structural studies of Aβ have primarily focused on Aβ fibrils and Aβ monomers. INTRO +0 28 Solid-state NMR spectroscopy experimental_method Solid-state NMR spectroscopy studies of Aβ fibrils revealed that Aβ fibrils are generally composed of extended networks of in-register parallel β-sheets.− X-ray crystallographic studies using fragments of Aβ have provided additional information about how Aβ fibrils pack. INTRO +40 42 Aβ protein Solid-state NMR spectroscopy studies of Aβ fibrils revealed that Aβ fibrils are generally composed of extended networks of in-register parallel β-sheets.− X-ray crystallographic studies using fragments of Aβ have provided additional information about how Aβ fibrils pack. INTRO +43 50 fibrils oligomeric_state Solid-state NMR spectroscopy studies of Aβ fibrils revealed that Aβ fibrils are generally composed of extended networks of in-register parallel β-sheets.− X-ray crystallographic studies using fragments of Aβ have provided additional information about how Aβ fibrils pack. INTRO +65 67 Aβ protein Solid-state NMR spectroscopy studies of Aβ fibrils revealed that Aβ fibrils are generally composed of extended networks of in-register parallel β-sheets.− X-ray crystallographic studies using fragments of Aβ have provided additional information about how Aβ fibrils pack. INTRO +68 75 fibrils oligomeric_state Solid-state NMR spectroscopy studies of Aβ fibrils revealed that Aβ fibrils are generally composed of extended networks of in-register parallel β-sheets.− X-ray crystallographic studies using fragments of Aβ have provided additional information about how Aβ fibrils pack. INTRO +123 152 in-register parallel β-sheets structure_element Solid-state NMR spectroscopy studies of Aβ fibrils revealed that Aβ fibrils are generally composed of extended networks of in-register parallel β-sheets.− X-ray crystallographic studies using fragments of Aβ have provided additional information about how Aβ fibrils pack. INTRO +155 185 X-ray crystallographic studies experimental_method Solid-state NMR spectroscopy studies of Aβ fibrils revealed that Aβ fibrils are generally composed of extended networks of in-register parallel β-sheets.− X-ray crystallographic studies using fragments of Aβ have provided additional information about how Aβ fibrils pack. INTRO +205 207 Aβ protein Solid-state NMR spectroscopy studies of Aβ fibrils revealed that Aβ fibrils are generally composed of extended networks of in-register parallel β-sheets.− X-ray crystallographic studies using fragments of Aβ have provided additional information about how Aβ fibrils pack. INTRO +255 257 Aβ protein Solid-state NMR spectroscopy studies of Aβ fibrils revealed that Aβ fibrils are generally composed of extended networks of in-register parallel β-sheets.− X-ray crystallographic studies using fragments of Aβ have provided additional information about how Aβ fibrils pack. INTRO +258 265 fibrils oligomeric_state Solid-state NMR spectroscopy studies of Aβ fibrils revealed that Aβ fibrils are generally composed of extended networks of in-register parallel β-sheets.− X-ray crystallographic studies using fragments of Aβ have provided additional information about how Aβ fibrils pack. INTRO +0 18 Solution-phase NMR experimental_method Solution-phase NMR and solid-state NMR have been used to study the structures of the Aβ monomers within oligomeric assemblies.− A major finding from these studies is that oligomeric assemblies of Aβ are primarily composed of antiparallel β-sheets. INTRO +23 38 solid-state NMR experimental_method Solution-phase NMR and solid-state NMR have been used to study the structures of the Aβ monomers within oligomeric assemblies.− A major finding from these studies is that oligomeric assemblies of Aβ are primarily composed of antiparallel β-sheets. INTRO +67 77 structures evidence Solution-phase NMR and solid-state NMR have been used to study the structures of the Aβ monomers within oligomeric assemblies.− A major finding from these studies is that oligomeric assemblies of Aβ are primarily composed of antiparallel β-sheets. INTRO +85 87 Aβ protein Solution-phase NMR and solid-state NMR have been used to study the structures of the Aβ monomers within oligomeric assemblies.− A major finding from these studies is that oligomeric assemblies of Aβ are primarily composed of antiparallel β-sheets. INTRO +88 96 monomers oligomeric_state Solution-phase NMR and solid-state NMR have been used to study the structures of the Aβ monomers within oligomeric assemblies.− A major finding from these studies is that oligomeric assemblies of Aβ are primarily composed of antiparallel β-sheets. INTRO +196 198 Aβ protein Solution-phase NMR and solid-state NMR have been used to study the structures of the Aβ monomers within oligomeric assemblies.− A major finding from these studies is that oligomeric assemblies of Aβ are primarily composed of antiparallel β-sheets. INTRO +225 246 antiparallel β-sheets structure_element Solution-phase NMR and solid-state NMR have been used to study the structures of the Aβ monomers within oligomeric assemblies.− A major finding from these studies is that oligomeric assemblies of Aβ are primarily composed of antiparallel β-sheets. INTRO +40 47 monomer oligomeric_state Many of these studies have reported the monomer subunit as adopting a β-hairpin conformation, in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. INTRO +48 55 subunit structure_element Many of these studies have reported the monomer subunit as adopting a β-hairpin conformation, in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. INTRO +70 79 β-hairpin structure_element Many of these studies have reported the monomer subunit as adopting a β-hairpin conformation, in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. INTRO +119 126 central structure_element Many of these studies have reported the monomer subunit as adopting a β-hairpin conformation, in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. INTRO +131 149 C-terminal regions structure_element Many of these studies have reported the monomer subunit as adopting a β-hairpin conformation, in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. INTRO +158 178 antiparallel β-sheet structure_element Many of these studies have reported the monomer subunit as adopting a β-hairpin conformation, in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. INTRO +35 38 NMR experimental_method In 2008, Hoyer et al. reported the NMR structure of an Aβ monomer bound to an artificial binding protein called an affibody (PDB 2OTK). INTRO +39 48 structure evidence In 2008, Hoyer et al. reported the NMR structure of an Aβ monomer bound to an artificial binding protein called an affibody (PDB 2OTK). INTRO +55 57 Aβ protein In 2008, Hoyer et al. reported the NMR structure of an Aβ monomer bound to an artificial binding protein called an affibody (PDB 2OTK). INTRO +58 65 monomer oligomeric_state In 2008, Hoyer et al. reported the NMR structure of an Aβ monomer bound to an artificial binding protein called an affibody (PDB 2OTK). INTRO +66 74 bound to protein_state In 2008, Hoyer et al. reported the NMR structure of an Aβ monomer bound to an artificial binding protein called an affibody (PDB 2OTK). INTRO +78 104 artificial binding protein chemical In 2008, Hoyer et al. reported the NMR structure of an Aβ monomer bound to an artificial binding protein called an affibody (PDB 2OTK). INTRO +115 123 affibody chemical In 2008, Hoyer et al. reported the NMR structure of an Aβ monomer bound to an artificial binding protein called an affibody (PDB 2OTK). INTRO +4 13 structure evidence The structure revealed that monomeric Aβ forms a β-hairpin when bound to the affibody. INTRO +28 37 monomeric oligomeric_state The structure revealed that monomeric Aβ forms a β-hairpin when bound to the affibody. INTRO +38 40 Aβ protein The structure revealed that monomeric Aβ forms a β-hairpin when bound to the affibody. INTRO +49 58 β-hairpin structure_element The structure revealed that monomeric Aβ forms a β-hairpin when bound to the affibody. INTRO +64 72 bound to protein_state The structure revealed that monomeric Aβ forms a β-hairpin when bound to the affibody. INTRO +77 85 affibody chemical The structure revealed that monomeric Aβ forms a β-hairpin when bound to the affibody. INTRO +5 7 Aβ protein This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. INTRO +8 17 β-hairpin structure_element This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. INTRO +39 44 17–37 residue_range This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. INTRO +62 71 β-strands structure_element This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. INTRO +83 85 Aβ protein This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. INTRO +85 90 17–24 residue_range This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. INTRO +95 97 Aβ protein This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. INTRO +97 102 30–37 residue_range This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. INTRO +119 121 Aβ protein This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. INTRO +121 126 25–29 residue_range This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. INTRO +127 131 loop structure_element This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. INTRO +13 15 Aβ protein Sequestering Aβ within the affibody prevents its fibrilization and reduces its neurotoxicity, providing evidence that the β-hairpin structure may contribute to the ability of Aβ to form neurotoxic oligomers. INTRO +27 35 affibody chemical Sequestering Aβ within the affibody prevents its fibrilization and reduces its neurotoxicity, providing evidence that the β-hairpin structure may contribute to the ability of Aβ to form neurotoxic oligomers. INTRO +122 131 β-hairpin structure_element Sequestering Aβ within the affibody prevents its fibrilization and reduces its neurotoxicity, providing evidence that the β-hairpin structure may contribute to the ability of Aβ to form neurotoxic oligomers. INTRO +175 177 Aβ protein Sequestering Aβ within the affibody prevents its fibrilization and reduces its neurotoxicity, providing evidence that the β-hairpin structure may contribute to the ability of Aβ to form neurotoxic oligomers. INTRO +197 206 oligomers oligomeric_state Sequestering Aβ within the affibody prevents its fibrilization and reduces its neurotoxicity, providing evidence that the β-hairpin structure may contribute to the ability of Aβ to form neurotoxic oligomers. INTRO +48 50 Aβ protein In a related study, Sandberg et al. constrained Aβ in a β-hairpin conformation by mutating residues A21 and A30 to cysteine and forming an intramolecular disulfide bond. INTRO +56 65 β-hairpin structure_element In a related study, Sandberg et al. constrained Aβ in a β-hairpin conformation by mutating residues A21 and A30 to cysteine and forming an intramolecular disulfide bond. INTRO +82 90 mutating experimental_method In a related study, Sandberg et al. constrained Aβ in a β-hairpin conformation by mutating residues A21 and A30 to cysteine and forming an intramolecular disulfide bond. INTRO +100 103 A21 residue_name_number In a related study, Sandberg et al. constrained Aβ in a β-hairpin conformation by mutating residues A21 and A30 to cysteine and forming an intramolecular disulfide bond. INTRO +108 111 A30 residue_name_number In a related study, Sandberg et al. constrained Aβ in a β-hairpin conformation by mutating residues A21 and A30 to cysteine and forming an intramolecular disulfide bond. INTRO +115 123 cysteine residue_name In a related study, Sandberg et al. constrained Aβ in a β-hairpin conformation by mutating residues A21 and A30 to cysteine and forming an intramolecular disulfide bond. INTRO +154 168 disulfide bond ptm In a related study, Sandberg et al. constrained Aβ in a β-hairpin conformation by mutating residues A21 and A30 to cysteine and forming an intramolecular disulfide bond. INTRO +8 10 Aβ protein Locking Aβ into a β-hairpin structure resulted in the formation Aβ oligomers, which were observed by size exclusion chromatography (SEC) and SDS-PAGE. INTRO +18 27 β-hairpin structure_element Locking Aβ into a β-hairpin structure resulted in the formation Aβ oligomers, which were observed by size exclusion chromatography (SEC) and SDS-PAGE. INTRO +64 66 Aβ protein Locking Aβ into a β-hairpin structure resulted in the formation Aβ oligomers, which were observed by size exclusion chromatography (SEC) and SDS-PAGE. INTRO +67 76 oligomers oligomeric_state Locking Aβ into a β-hairpin structure resulted in the formation Aβ oligomers, which were observed by size exclusion chromatography (SEC) and SDS-PAGE. INTRO +101 130 size exclusion chromatography experimental_method Locking Aβ into a β-hairpin structure resulted in the formation Aβ oligomers, which were observed by size exclusion chromatography (SEC) and SDS-PAGE. INTRO +132 135 SEC experimental_method Locking Aβ into a β-hairpin structure resulted in the formation Aβ oligomers, which were observed by size exclusion chromatography (SEC) and SDS-PAGE. INTRO +141 149 SDS-PAGE experimental_method Locking Aβ into a β-hairpin structure resulted in the formation Aβ oligomers, which were observed by size exclusion chromatography (SEC) and SDS-PAGE. INTRO +4 13 oligomers oligomeric_state The oligomers with a molecular weight of ∼100 kDa that were isolated by SEC were toxic toward neuronally derived SH-SY5Y cells. INTRO +72 75 SEC experimental_method The oligomers with a molecular weight of ∼100 kDa that were isolated by SEC were toxic toward neuronally derived SH-SY5Y cells. INTRO +45 54 β-hairpin structure_element This study provides evidence for the role of β-hairpin structure in Aβ oligomerization and neurotoxicity. INTRO +68 70 Aβ protein This study provides evidence for the role of β-hairpin structure in Aβ oligomerization and neurotoxicity. INTRO +18 27 β-hairpin structure_element Inspired by these β-hairpin structures, our laboratory developed a macrocyclic β-sheet peptide derived from Aβ17–36 designed to mimic an Aβ β-hairpin and reported its X-ray crystallographic structure. INTRO +28 38 structures evidence Inspired by these β-hairpin structures, our laboratory developed a macrocyclic β-sheet peptide derived from Aβ17–36 designed to mimic an Aβ β-hairpin and reported its X-ray crystallographic structure. INTRO +79 86 β-sheet structure_element Inspired by these β-hairpin structures, our laboratory developed a macrocyclic β-sheet peptide derived from Aβ17–36 designed to mimic an Aβ β-hairpin and reported its X-ray crystallographic structure. INTRO +108 110 Aβ protein Inspired by these β-hairpin structures, our laboratory developed a macrocyclic β-sheet peptide derived from Aβ17–36 designed to mimic an Aβ β-hairpin and reported its X-ray crystallographic structure. INTRO +110 115 17–36 residue_range Inspired by these β-hairpin structures, our laboratory developed a macrocyclic β-sheet peptide derived from Aβ17–36 designed to mimic an Aβ β-hairpin and reported its X-ray crystallographic structure. INTRO +137 139 Aβ protein Inspired by these β-hairpin structures, our laboratory developed a macrocyclic β-sheet peptide derived from Aβ17–36 designed to mimic an Aβ β-hairpin and reported its X-ray crystallographic structure. INTRO +140 149 β-hairpin structure_element Inspired by these β-hairpin structures, our laboratory developed a macrocyclic β-sheet peptide derived from Aβ17–36 designed to mimic an Aβ β-hairpin and reported its X-ray crystallographic structure. INTRO +167 199 X-ray crystallographic structure evidence Inspired by these β-hairpin structures, our laboratory developed a macrocyclic β-sheet peptide derived from Aβ17–36 designed to mimic an Aβ β-hairpin and reported its X-ray crystallographic structure. INTRO +14 23 peptide 1 mutant This peptide (peptide 1) consists of two β-strands comprising Aβ17–23 and Aβ30–36 covalently linked by two δ-linked ornithine (δOrn) β-turn mimics. INTRO +41 50 β-strands structure_element This peptide (peptide 1) consists of two β-strands comprising Aβ17–23 and Aβ30–36 covalently linked by two δ-linked ornithine (δOrn) β-turn mimics. INTRO +62 64 Aβ protein This peptide (peptide 1) consists of two β-strands comprising Aβ17–23 and Aβ30–36 covalently linked by two δ-linked ornithine (δOrn) β-turn mimics. INTRO +64 69 17–23 residue_range This peptide (peptide 1) consists of two β-strands comprising Aβ17–23 and Aβ30–36 covalently linked by two δ-linked ornithine (δOrn) β-turn mimics. INTRO +74 76 Aβ protein This peptide (peptide 1) consists of two β-strands comprising Aβ17–23 and Aβ30–36 covalently linked by two δ-linked ornithine (δOrn) β-turn mimics. INTRO +76 81 30–36 residue_range This peptide (peptide 1) consists of two β-strands comprising Aβ17–23 and Aβ30–36 covalently linked by two δ-linked ornithine (δOrn) β-turn mimics. INTRO +107 115 δ-linked protein_state This peptide (peptide 1) consists of two β-strands comprising Aβ17–23 and Aβ30–36 covalently linked by two δ-linked ornithine (δOrn) β-turn mimics. INTRO +116 125 ornithine residue_name This peptide (peptide 1) consists of two β-strands comprising Aβ17–23 and Aβ30–36 covalently linked by two δ-linked ornithine (δOrn) β-turn mimics. INTRO +127 131 δOrn structure_element This peptide (peptide 1) consists of two β-strands comprising Aβ17–23 and Aβ30–36 covalently linked by two δ-linked ornithine (δOrn) β-turn mimics. INTRO +133 139 β-turn structure_element This peptide (peptide 1) consists of two β-strands comprising Aβ17–23 and Aβ30–36 covalently linked by two δ-linked ornithine (δOrn) β-turn mimics. INTRO +4 8 δOrn structure_element The δOrn that connects residues D23 and A30 replaces the Aβ24–29 loop. INTRO +32 35 D23 residue_name_number The δOrn that connects residues D23 and A30 replaces the Aβ24–29 loop. INTRO +40 43 A30 residue_name_number The δOrn that connects residues D23 and A30 replaces the Aβ24–29 loop. INTRO +57 59 Aβ protein The δOrn that connects residues D23 and A30 replaces the Aβ24–29 loop. INTRO +59 64 24–29 residue_range The δOrn that connects residues D23 and A30 replaces the Aβ24–29 loop. INTRO +65 69 loop structure_element The δOrn that connects residues D23 and A30 replaces the Aβ24–29 loop. INTRO +4 8 δOrn structure_element The δOrn that connects residues L17 and V36 enforces β-hairpin structure. INTRO +32 35 L17 residue_name_number The δOrn that connects residues L17 and V36 enforces β-hairpin structure. INTRO +40 43 V36 residue_name_number The δOrn that connects residues L17 and V36 enforces β-hairpin structure. INTRO +53 62 β-hairpin structure_element The δOrn that connects residues L17 and V36 enforces β-hairpin structure. INTRO +46 49 G33 residue_name_number We incorporated an N-methyl group at position G33 to prevent uncontrolled aggregation and precipitation of the peptide. INTRO +44 52 replaced experimental_method To improve the solubility of the peptide we replaced M35 with the hydrophilic isostere of methionine, ornithine (α-linked) (Figure 1B). INTRO +53 56 M35 residue_name_number To improve the solubility of the peptide we replaced M35 with the hydrophilic isostere of methionine, ornithine (α-linked) (Figure 1B). INTRO +90 100 methionine residue_name To improve the solubility of the peptide we replaced M35 with the hydrophilic isostere of methionine, ornithine (α-linked) (Figure 1B). INTRO +102 111 ornithine residue_name To improve the solubility of the peptide we replaced M35 with the hydrophilic isostere of methionine, ornithine (α-linked) (Figure 1B). INTRO +113 121 α-linked protein_state To improve the solubility of the peptide we replaced M35 with the hydrophilic isostere of methionine, ornithine (α-linked) (Figure 1B). INTRO +4 36 X-ray crystallographic structure evidence The X-ray crystallographic structure of peptide 1 reveals that it folds to form a β-hairpin that assembles to form trimers and that the trimers further assemble to form hexamers and dodecamers. INTRO +40 49 peptide 1 mutant The X-ray crystallographic structure of peptide 1 reveals that it folds to form a β-hairpin that assembles to form trimers and that the trimers further assemble to form hexamers and dodecamers. INTRO +82 91 β-hairpin structure_element The X-ray crystallographic structure of peptide 1 reveals that it folds to form a β-hairpin that assembles to form trimers and that the trimers further assemble to form hexamers and dodecamers. INTRO +115 122 trimers oligomeric_state The X-ray crystallographic structure of peptide 1 reveals that it folds to form a β-hairpin that assembles to form trimers and that the trimers further assemble to form hexamers and dodecamers. INTRO +136 143 trimers oligomeric_state The X-ray crystallographic structure of peptide 1 reveals that it folds to form a β-hairpin that assembles to form trimers and that the trimers further assemble to form hexamers and dodecamers. INTRO +169 177 hexamers oligomeric_state The X-ray crystallographic structure of peptide 1 reveals that it folds to form a β-hairpin that assembles to form trimers and that the trimers further assemble to form hexamers and dodecamers. INTRO +182 192 dodecamers oligomeric_state The X-ray crystallographic structure of peptide 1 reveals that it folds to form a β-hairpin that assembles to form trimers and that the trimers further assemble to form hexamers and dodecamers. INTRO +39 55 peptides 1 and 2 chemical (A) Cartoon illustrating the design of peptides 1 and 2 and their relationship to an Aβ17–36 β-hairpin. FIG +85 87 Aβ protein (A) Cartoon illustrating the design of peptides 1 and 2 and their relationship to an Aβ17–36 β-hairpin. FIG +87 92 17–36 residue_range (A) Cartoon illustrating the design of peptides 1 and 2 and their relationship to an Aβ17–36 β-hairpin. FIG +93 102 β-hairpin structure_element (A) Cartoon illustrating the design of peptides 1 and 2 and their relationship to an Aβ17–36 β-hairpin. FIG +27 36 peptide 1 mutant (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +50 52 Aβ protein (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +62 64 Aβ protein (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +107 115 δ-linked protein_state (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +116 125 ornithine residue_name (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +126 131 turns structure_element (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +159 168 peptide 2 mutant (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +182 184 Aβ protein (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +215 229 disulfide bond ptm (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +247 249 24 residue_number (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +254 256 29 residue_number (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +266 274 δ-linked protein_state (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +275 284 ornithine residue_name (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +285 289 turn structure_element (B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating Aβ17–36, the N-methyl group, the disulfide bond across positions 24 and 29, and the δ-linked ornithine turn. FIG +14 23 peptide 1 mutant Our design of peptide 1 omitted the Aβ24–29 loop. INTRO +36 38 Aβ protein Our design of peptide 1 omitted the Aβ24–29 loop. INTRO +38 43 24–29 residue_range Our design of peptide 1 omitted the Aβ24–29 loop. INTRO +44 48 loop structure_element Our design of peptide 1 omitted the Aβ24–29 loop. INTRO +17 19 Aβ protein To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +19 24 24–29 residue_range To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +25 29 loop structure_element To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +44 79 replica-exchange molecular dynamics experimental_method To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +81 85 REMD experimental_method To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +87 98 simulations experimental_method To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +102 104 Aβ protein To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +104 109 17–36 residue_range To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +120 154 X-ray crystallographic coordinates evidence To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +158 160 Aβ protein To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +160 165 17–23 residue_range To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +170 172 Aβ protein To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +172 177 30–36 residue_range To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +183 192 peptide 1 mutant To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. INTRO +45 51 trimer oligomeric_state These studies provided a working model for a trimer of Aβ17–36 β-hairpins and demonstrated that the trimer should be capable of accommodating the Aβ24–29 loop. INTRO +55 57 Aβ protein These studies provided a working model for a trimer of Aβ17–36 β-hairpins and demonstrated that the trimer should be capable of accommodating the Aβ24–29 loop. INTRO +57 62 17–36 residue_range These studies provided a working model for a trimer of Aβ17–36 β-hairpins and demonstrated that the trimer should be capable of accommodating the Aβ24–29 loop. INTRO +63 73 β-hairpins structure_element These studies provided a working model for a trimer of Aβ17–36 β-hairpins and demonstrated that the trimer should be capable of accommodating the Aβ24–29 loop. INTRO +100 106 trimer oligomeric_state These studies provided a working model for a trimer of Aβ17–36 β-hairpins and demonstrated that the trimer should be capable of accommodating the Aβ24–29 loop. INTRO +146 148 Aβ protein These studies provided a working model for a trimer of Aβ17–36 β-hairpins and demonstrated that the trimer should be capable of accommodating the Aβ24–29 loop. INTRO +148 153 24–29 residue_range These studies provided a working model for a trimer of Aβ17–36 β-hairpins and demonstrated that the trimer should be capable of accommodating the Aβ24–29 loop. INTRO +154 158 loop structure_element These studies provided a working model for a trimer of Aβ17–36 β-hairpins and demonstrated that the trimer should be capable of accommodating the Aβ24–29 loop. INTRO +35 42 restore experimental_method In the current study we set out to restore the Aβ24–29 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form. INTRO +47 49 Aβ protein In the current study we set out to restore the Aβ24–29 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form. INTRO +49 54 24–29 residue_range In the current study we set out to restore the Aβ24–29 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form. INTRO +55 59 loop structure_element In the current study we set out to restore the Aβ24–29 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form. INTRO +61 72 reintroduce experimental_method In the current study we set out to restore the Aβ24–29 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form. INTRO +77 87 methionine residue_name In the current study we set out to restore the Aβ24–29 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form. INTRO +108 110 35 residue_number In the current study we set out to restore the Aβ24–29 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form. INTRO +130 163 X-ray crystallographic structures evidence In the current study we set out to restore the Aβ24–29 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form. INTRO +167 176 oligomers oligomeric_state In the current study we set out to restore the Aβ24–29 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form. INTRO +12 21 peptide 2 mutant We designed peptide 2 as a homologue of peptide 1 that embodies these ideas. INTRO +40 49 peptide 1 mutant We designed peptide 2 as a homologue of peptide 1 that embodies these ideas. INTRO +0 9 Peptide 2 mutant Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +21 31 methionine residue_name Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +52 54 35 residue_number Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +62 64 Aβ protein Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +64 69 24–29 residue_range Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +70 74 loop structure_element Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +89 91 24 residue_number Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +96 98 29 residue_number Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +100 103 Val residue_name Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +108 111 Gly residue_name Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +113 120 mutated experimental_method Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +124 132 cysteine residue_name Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +149 163 disulfide bond ptm Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). INTRO +37 46 peptide 2 mutant Here, we describe the development of peptide 2 and report the X-ray crystallographic structures of the trimer, dodecamer, and annular pore observed within the crystal structure. INTRO +62 95 X-ray crystallographic structures evidence Here, we describe the development of peptide 2 and report the X-ray crystallographic structures of the trimer, dodecamer, and annular pore observed within the crystal structure. INTRO +103 109 trimer oligomeric_state Here, we describe the development of peptide 2 and report the X-ray crystallographic structures of the trimer, dodecamer, and annular pore observed within the crystal structure. INTRO +111 120 dodecamer oligomeric_state Here, we describe the development of peptide 2 and report the X-ray crystallographic structures of the trimer, dodecamer, and annular pore observed within the crystal structure. INTRO +126 138 annular pore site Here, we describe the development of peptide 2 and report the X-ray crystallographic structures of the trimer, dodecamer, and annular pore observed within the crystal structure. INTRO +159 176 crystal structure evidence Here, we describe the development of peptide 2 and report the X-ray crystallographic structures of the trimer, dodecamer, and annular pore observed within the crystal structure. INTRO +15 24 Peptide 2 mutant Development of Peptide 2 RESULTS +13 22 peptide 2 mutant We developed peptide 2 from peptide 1 by an iterative process, in which we first attempted to restore the Aβ24–29 loop without a disulfide linkage. RESULTS +28 37 peptide 1 mutant We developed peptide 2 from peptide 1 by an iterative process, in which we first attempted to restore the Aβ24–29 loop without a disulfide linkage. RESULTS +106 108 Aβ protein We developed peptide 2 from peptide 1 by an iterative process, in which we first attempted to restore the Aβ24–29 loop without a disulfide linkage. RESULTS +108 113 24–29 residue_range We developed peptide 2 from peptide 1 by an iterative process, in which we first attempted to restore the Aβ24–29 loop without a disulfide linkage. RESULTS +114 118 loop structure_element We developed peptide 2 from peptide 1 by an iterative process, in which we first attempted to restore the Aβ24–29 loop without a disulfide linkage. RESULTS +129 146 disulfide linkage ptm We developed peptide 2 from peptide 1 by an iterative process, in which we first attempted to restore the Aβ24–29 loop without a disulfide linkage. RESULTS +14 23 peptide 3 mutant We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. RESULTS +42 51 peptide 1 mutant We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. RESULTS +61 63 Aβ protein We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. RESULTS +63 68 24–29 residue_range We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. RESULTS +69 73 loop structure_element We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. RESULTS +90 94 δOrn structure_element We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. RESULTS +109 112 D23 residue_name_number We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. RESULTS +117 120 A30 residue_name_number We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. RESULTS +125 144 p-iodophenylalanine chemical We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. RESULTS +146 148 FI chemical We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. RESULTS +162 165 F19 residue_name_number We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. RESULTS +17 36 p-iodophenylalanine chemical We routinely use p-iodophenylalanine to determine the X-ray crystallographic phases. RESULTS +54 83 X-ray crystallographic phases evidence We routinely use p-iodophenylalanine to determine the X-ray crystallographic phases. RESULTS +22 54 X-ray crystallographic structure evidence After determining the X-ray crystallographic structure of the p-iodophenylalanine variant we attempt to determine the structure of the native phenylalanine compound by isomorphous replacement. RESULTS +62 81 p-iodophenylalanine chemical After determining the X-ray crystallographic structure of the p-iodophenylalanine variant we attempt to determine the structure of the native phenylalanine compound by isomorphous replacement. RESULTS +118 127 structure evidence After determining the X-ray crystallographic structure of the p-iodophenylalanine variant we attempt to determine the structure of the native phenylalanine compound by isomorphous replacement. RESULTS +142 155 phenylalanine residue_name After determining the X-ray crystallographic structure of the p-iodophenylalanine variant we attempt to determine the structure of the native phenylalanine compound by isomorphous replacement. RESULTS +168 191 isomorphous replacement experimental_method After determining the X-ray crystallographic structure of the p-iodophenylalanine variant we attempt to determine the structure of the native phenylalanine compound by isomorphous replacement. RESULTS +18 27 peptide 3 mutant Upon synthesizing peptide 3, we found that it formed an amorphous precipitate in most crystallization conditions screened and failed to afford crystals in any condition. RESULTS +143 151 crystals evidence Upon synthesizing peptide 3, we found that it formed an amorphous precipitate in most crystallization conditions screened and failed to afford crystals in any condition. RESULTS +34 38 δOrn structure_element We postulate that the loss of the δOrn constraint leads to conformational heterogeneity that prevents peptide 3 from crystallizing. RESULTS +102 111 peptide 3 mutant We postulate that the loss of the δOrn constraint leads to conformational heterogeneity that prevents peptide 3 from crystallizing. RESULTS +46 60 disulfide bond ptm To address this issue, we next incorporated a disulfide bond between residues 24 and 29 as a conformational constraint that serves as a surrogate for δOrn. RESULTS +78 80 24 residue_number To address this issue, we next incorporated a disulfide bond between residues 24 and 29 as a conformational constraint that serves as a surrogate for δOrn. RESULTS +85 87 29 residue_number To address this issue, we next incorporated a disulfide bond between residues 24 and 29 as a conformational constraint that serves as a surrogate for δOrn. RESULTS +150 154 δOrn structure_element To address this issue, we next incorporated a disulfide bond between residues 24 and 29 as a conformational constraint that serves as a surrogate for δOrn. RESULTS +12 21 peptide 4 mutant We designed peptide 4 to embody this idea, mutating Val24 and Gly29 to cysteine and forming an interstrand disulfide linkage. RESULTS +43 51 mutating experimental_method We designed peptide 4 to embody this idea, mutating Val24 and Gly29 to cysteine and forming an interstrand disulfide linkage. RESULTS +52 57 Val24 residue_name_number We designed peptide 4 to embody this idea, mutating Val24 and Gly29 to cysteine and forming an interstrand disulfide linkage. RESULTS +62 67 Gly29 residue_name_number We designed peptide 4 to embody this idea, mutating Val24 and Gly29 to cysteine and forming an interstrand disulfide linkage. RESULTS +71 79 cysteine residue_name We designed peptide 4 to embody this idea, mutating Val24 and Gly29 to cysteine and forming an interstrand disulfide linkage. RESULTS +107 124 disulfide linkage ptm We designed peptide 4 to embody this idea, mutating Val24 and Gly29 to cysteine and forming an interstrand disulfide linkage. RESULTS +3 10 mutated experimental_method We mutated these residues because they occupy the same position as the δOrn that connects D23 and A30 in peptide 1. RESULTS +71 75 δOrn structure_element We mutated these residues because they occupy the same position as the δOrn that connects D23 and A30 in peptide 1. RESULTS +90 93 D23 residue_name_number We mutated these residues because they occupy the same position as the δOrn that connects D23 and A30 in peptide 1. RESULTS +98 101 A30 residue_name_number We mutated these residues because they occupy the same position as the δOrn that connects D23 and A30 in peptide 1. RESULTS +105 114 peptide 1 mutant We mutated these residues because they occupy the same position as the δOrn that connects D23 and A30 in peptide 1. RESULTS +9 12 V24 residue_name_number Residues V24 and G29 form a non-hydrogen-bonded pair, which can readily accommodate disulfide linkages in antiparallel β-sheets. RESULTS +17 20 G29 residue_name_number Residues V24 and G29 form a non-hydrogen-bonded pair, which can readily accommodate disulfide linkages in antiparallel β-sheets. RESULTS +28 52 non-hydrogen-bonded pair bond_interaction Residues V24 and G29 form a non-hydrogen-bonded pair, which can readily accommodate disulfide linkages in antiparallel β-sheets. RESULTS +84 102 disulfide linkages ptm Residues V24 and G29 form a non-hydrogen-bonded pair, which can readily accommodate disulfide linkages in antiparallel β-sheets. RESULTS +106 127 antiparallel β-sheets structure_element Residues V24 and G29 form a non-hydrogen-bonded pair, which can readily accommodate disulfide linkages in antiparallel β-sheets. RESULTS +0 15 Disulfide bonds ptm Disulfide bonds across non-hydrogen-bonded pairs stabilize β-hairpins, while disulfide bonds across hydrogen-bonded pairs do not. RESULTS +23 48 non-hydrogen-bonded pairs bond_interaction Disulfide bonds across non-hydrogen-bonded pairs stabilize β-hairpins, while disulfide bonds across hydrogen-bonded pairs do not. RESULTS +59 69 β-hairpins structure_element Disulfide bonds across non-hydrogen-bonded pairs stabilize β-hairpins, while disulfide bonds across hydrogen-bonded pairs do not. RESULTS +77 92 disulfide bonds ptm Disulfide bonds across non-hydrogen-bonded pairs stabilize β-hairpins, while disulfide bonds across hydrogen-bonded pairs do not. RESULTS +100 121 hydrogen-bonded pairs bond_interaction Disulfide bonds across non-hydrogen-bonded pairs stabilize β-hairpins, while disulfide bonds across hydrogen-bonded pairs do not. RESULTS +13 27 disulfide bond ptm Although the disulfide bond between positions 24 and 29 helps stabilize the β-hairpin, it does not alter the charge or substantially change the hydrophobicity of the Aβ17–36 β-hairpin. RESULTS +46 48 24 residue_number Although the disulfide bond between positions 24 and 29 helps stabilize the β-hairpin, it does not alter the charge or substantially change the hydrophobicity of the Aβ17–36 β-hairpin. RESULTS +53 55 29 residue_number Although the disulfide bond between positions 24 and 29 helps stabilize the β-hairpin, it does not alter the charge or substantially change the hydrophobicity of the Aβ17–36 β-hairpin. RESULTS +76 85 β-hairpin structure_element Although the disulfide bond between positions 24 and 29 helps stabilize the β-hairpin, it does not alter the charge or substantially change the hydrophobicity of the Aβ17–36 β-hairpin. RESULTS +166 168 Aβ protein Although the disulfide bond between positions 24 and 29 helps stabilize the β-hairpin, it does not alter the charge or substantially change the hydrophobicity of the Aβ17–36 β-hairpin. RESULTS +168 173 17–36 residue_range Although the disulfide bond between positions 24 and 29 helps stabilize the β-hairpin, it does not alter the charge or substantially change the hydrophobicity of the Aβ17–36 β-hairpin. RESULTS +174 183 β-hairpin structure_element Although the disulfide bond between positions 24 and 29 helps stabilize the β-hairpin, it does not alter the charge or substantially change the hydrophobicity of the Aβ17–36 β-hairpin. RESULTS +31 40 peptide 4 mutant We were gratified to find that peptide 4 afforded crystals suitable for X-ray crystallography. RESULTS +50 58 crystals evidence We were gratified to find that peptide 4 afforded crystals suitable for X-ray crystallography. RESULTS +72 93 X-ray crystallography experimental_method We were gratified to find that peptide 4 afforded crystals suitable for X-ray crystallography. RESULTS +46 56 determined experimental_method As the next step in the iterative process, we determined the X-ray crystallographic structure of this peptide (PDB 5HOW). RESULTS +61 93 X-ray crystallographic structure evidence As the next step in the iterative process, we determined the X-ray crystallographic structure of this peptide (PDB 5HOW). RESULTS +22 54 X-ray crystallographic structure evidence After determining the X-ray crystallographic structure of peptide 4 we reintroduced the native phenylalanine at position 19 and the methionine at position 35 to afford peptide 2. RESULTS +58 67 peptide 4 mutant After determining the X-ray crystallographic structure of peptide 4 we reintroduced the native phenylalanine at position 19 and the methionine at position 35 to afford peptide 2. RESULTS +71 83 reintroduced experimental_method After determining the X-ray crystallographic structure of peptide 4 we reintroduced the native phenylalanine at position 19 and the methionine at position 35 to afford peptide 2. RESULTS +95 108 phenylalanine residue_name After determining the X-ray crystallographic structure of peptide 4 we reintroduced the native phenylalanine at position 19 and the methionine at position 35 to afford peptide 2. RESULTS +121 123 19 residue_number After determining the X-ray crystallographic structure of peptide 4 we reintroduced the native phenylalanine at position 19 and the methionine at position 35 to afford peptide 2. RESULTS +132 142 methionine residue_name After determining the X-ray crystallographic structure of peptide 4 we reintroduced the native phenylalanine at position 19 and the methionine at position 35 to afford peptide 2. RESULTS +155 157 35 residue_number After determining the X-ray crystallographic structure of peptide 4 we reintroduced the native phenylalanine at position 19 and the methionine at position 35 to afford peptide 2. RESULTS +168 177 peptide 2 mutant After determining the X-ray crystallographic structure of peptide 4 we reintroduced the native phenylalanine at position 19 and the methionine at position 35 to afford peptide 2. RESULTS +89 121 X-ray crystallographic structure evidence We completed the iterative process—from 1 to 3 to 4 to 2—by successfully determining the X-ray crystallographic structure of peptide 2 (PDB 5HOX and 5HOY). RESULTS +125 134 peptide 2 mutant We completed the iterative process—from 1 to 3 to 4 to 2—by successfully determining the X-ray crystallographic structure of peptide 2 (PDB 5HOX and 5HOY). RESULTS +49 61 peptides 2–4 mutant The following sections describe the synthesis of peptides 2–4 and the X-ray crystallographic structure of peptide 2. RESULTS +70 102 X-ray crystallographic structure evidence The following sections describe the synthesis of peptides 2–4 and the X-ray crystallographic structure of peptide 2. RESULTS +106 115 peptide 2 mutant The following sections describe the synthesis of peptides 2–4 and the X-ray crystallographic structure of peptide 2. RESULTS +13 25 Peptides 2–4 mutant Synthesis of Peptides 2–4 RESULTS +15 27 peptides 2–4 mutant We synthesized peptides 2–4 by similar procedures to those we have developed for other macrocyclic peptides. RESULTS +16 32 peptides 2 and 4 mutant In synthesizing peptides 2 and 4 we formed the disulfide linkage after macrolactamization and deprotection of the acid-labile side chain protecting groups. RESULTS +47 64 disulfide linkage ptm In synthesizing peptides 2 and 4 we formed the disulfide linkage after macrolactamization and deprotection of the acid-labile side chain protecting groups. RESULTS +8 19 acid-stable protein_state We used acid-stable Acm-protected cysteine residues at positions 24 and 29 and removed the Acm groups by oxidation with I2 in aqueous acetic acid to afford the disulfide linkage. RESULTS +20 33 Acm-protected protein_state We used acid-stable Acm-protected cysteine residues at positions 24 and 29 and removed the Acm groups by oxidation with I2 in aqueous acetic acid to afford the disulfide linkage. RESULTS +34 42 cysteine residue_name We used acid-stable Acm-protected cysteine residues at positions 24 and 29 and removed the Acm groups by oxidation with I2 in aqueous acetic acid to afford the disulfide linkage. RESULTS +65 67 24 residue_number We used acid-stable Acm-protected cysteine residues at positions 24 and 29 and removed the Acm groups by oxidation with I2 in aqueous acetic acid to afford the disulfide linkage. RESULTS +72 74 29 residue_number We used acid-stable Acm-protected cysteine residues at positions 24 and 29 and removed the Acm groups by oxidation with I2 in aqueous acetic acid to afford the disulfide linkage. RESULTS +134 145 acetic acid chemical We used acid-stable Acm-protected cysteine residues at positions 24 and 29 and removed the Acm groups by oxidation with I2 in aqueous acetic acid to afford the disulfide linkage. RESULTS +160 177 disulfide linkage ptm We used acid-stable Acm-protected cysteine residues at positions 24 and 29 and removed the Acm groups by oxidation with I2 in aqueous acetic acid to afford the disulfide linkage. RESULTS +0 12 Peptides 2–4 mutant Peptides 2–4 were purified by RP-HPLC. RESULTS +30 37 RP-HPLC experimental_method Peptides 2–4 were purified by RP-HPLC. RESULTS +0 15 Crystallization experimental_method Crystallization, X-ray Crystallographic Data Collection, Data Processing, and Structure Determination of Peptides 2 and 4 RESULTS +17 55 X-ray Crystallographic Data Collection experimental_method Crystallization, X-ray Crystallographic Data Collection, Data Processing, and Structure Determination of Peptides 2 and 4 RESULTS +78 101 Structure Determination experimental_method Crystallization, X-ray Crystallographic Data Collection, Data Processing, and Structure Determination of Peptides 2 and 4 RESULTS +105 121 Peptides 2 and 4 mutant Crystallization, X-ray Crystallographic Data Collection, Data Processing, and Structure Determination of Peptides 2 and 4 RESULTS +3 38 screened crystallization conditions experimental_method We screened crystallization conditions for peptide 4 in a 96-well-plate format using three different Hampton Research crystallization kits (Crystal Screen, Index, and PEG/Ion) with three ratios of peptide and mother liquor per condition (864 experiments). RESULTS +43 52 peptide 4 mutant We screened crystallization conditions for peptide 4 in a 96-well-plate format using three different Hampton Research crystallization kits (Crystal Screen, Index, and PEG/Ion) with three ratios of peptide and mother liquor per condition (864 experiments). RESULTS +0 9 Peptide 4 mutant Peptide 4 afforded crystals in a single set of conditions containing HEPES buffer and Jeffamine M-600—the same crystallization conditions that afforded crystals of peptide 1. RESULTS +19 27 crystals evidence Peptide 4 afforded crystals in a single set of conditions containing HEPES buffer and Jeffamine M-600—the same crystallization conditions that afforded crystals of peptide 1. RESULTS +86 101 Jeffamine M-600 chemical Peptide 4 afforded crystals in a single set of conditions containing HEPES buffer and Jeffamine M-600—the same crystallization conditions that afforded crystals of peptide 1. RESULTS +152 160 crystals evidence Peptide 4 afforded crystals in a single set of conditions containing HEPES buffer and Jeffamine M-600—the same crystallization conditions that afforded crystals of peptide 1. RESULTS +164 173 peptide 1 mutant Peptide 4 afforded crystals in a single set of conditions containing HEPES buffer and Jeffamine M-600—the same crystallization conditions that afforded crystals of peptide 1. RESULTS +0 9 Peptide 2 mutant Peptide 2 also afforded crystals in these conditions. RESULTS +24 32 crystals evidence Peptide 2 also afforded crystals in these conditions. RESULTS +63 71 crystals evidence We further optimized these conditions to rapidly (∼72 h) yield crystals suitable for X-ray crystallography. RESULTS +85 106 X-ray crystallography experimental_method We further optimized these conditions to rapidly (∼72 h) yield crystals suitable for X-ray crystallography. RESULTS +42 47 HEPES chemical The optimized conditions consist of 0.1 M HEPES at pH 6.4 with 31% Jeffamine M-600 for peptide 4 and 0.1 M HEPES pH 7.1 with 29% Jeffamine M-600 for peptide 2. RESULTS +67 82 Jeffamine M-600 chemical The optimized conditions consist of 0.1 M HEPES at pH 6.4 with 31% Jeffamine M-600 for peptide 4 and 0.1 M HEPES pH 7.1 with 29% Jeffamine M-600 for peptide 2. RESULTS +87 96 peptide 4 mutant The optimized conditions consist of 0.1 M HEPES at pH 6.4 with 31% Jeffamine M-600 for peptide 4 and 0.1 M HEPES pH 7.1 with 29% Jeffamine M-600 for peptide 2. RESULTS +107 112 HEPES chemical The optimized conditions consist of 0.1 M HEPES at pH 6.4 with 31% Jeffamine M-600 for peptide 4 and 0.1 M HEPES pH 7.1 with 29% Jeffamine M-600 for peptide 2. RESULTS +129 144 Jeffamine M-600 chemical The optimized conditions consist of 0.1 M HEPES at pH 6.4 with 31% Jeffamine M-600 for peptide 4 and 0.1 M HEPES pH 7.1 with 29% Jeffamine M-600 for peptide 2. RESULTS +149 158 peptide 2 mutant The optimized conditions consist of 0.1 M HEPES at pH 6.4 with 31% Jeffamine M-600 for peptide 4 and 0.1 M HEPES pH 7.1 with 29% Jeffamine M-600 for peptide 2. RESULTS +0 24 Crystal diffraction data evidence Crystal diffraction data for peptides 4 and 2 were collected in-house with a Rigaku MicroMax 007HF X-ray diffractometer at 1.54 Å wavelength. RESULTS +29 45 peptides 4 and 2 mutant Crystal diffraction data for peptides 4 and 2 were collected in-house with a Rigaku MicroMax 007HF X-ray diffractometer at 1.54 Å wavelength. RESULTS +0 24 Crystal diffraction data evidence Crystal diffraction data for peptide 2 were also collected at the Advanced Light Source at Lawrence Berkeley National Laboratory with a synchrotron source at 1.00 Å wavelength to achieve higher resolution. RESULTS +29 38 peptide 2 mutant Crystal diffraction data for peptide 2 were also collected at the Advanced Light Source at Lawrence Berkeley National Laboratory with a synchrotron source at 1.00 Å wavelength to achieve higher resolution. RESULTS +10 26 peptides 4 and 2 mutant Data from peptides 4 and 2 suitable for refinement at 2.30 Å were obtained from the diffractometer; data from peptide 2 suitable for refinement at 1.90 Å were obtained from the synchrotron. RESULTS +110 119 peptide 2 mutant Data from peptides 4 and 2 suitable for refinement at 2.30 Å were obtained from the diffractometer; data from peptide 2 suitable for refinement at 1.90 Å were obtained from the synchrotron. RESULTS +9 25 peptides 4 and 2 mutant Data for peptides 4 and 2 were scaled and merged using XDS. RESULTS +0 6 Phases evidence Phases for peptide 4 were determined by single-wavelength anomalous diffraction (SAD) phasing by using the coordinates of the iodine anomalous signal from p-iodophenylalanine. RESULTS +11 20 peptide 4 mutant Phases for peptide 4 were determined by single-wavelength anomalous diffraction (SAD) phasing by using the coordinates of the iodine anomalous signal from p-iodophenylalanine. RESULTS +40 79 single-wavelength anomalous diffraction experimental_method Phases for peptide 4 were determined by single-wavelength anomalous diffraction (SAD) phasing by using the coordinates of the iodine anomalous signal from p-iodophenylalanine. RESULTS +81 84 SAD experimental_method Phases for peptide 4 were determined by single-wavelength anomalous diffraction (SAD) phasing by using the coordinates of the iodine anomalous signal from p-iodophenylalanine. RESULTS +86 93 phasing experimental_method Phases for peptide 4 were determined by single-wavelength anomalous diffraction (SAD) phasing by using the coordinates of the iodine anomalous signal from p-iodophenylalanine. RESULTS +126 149 iodine anomalous signal evidence Phases for peptide 4 were determined by single-wavelength anomalous diffraction (SAD) phasing by using the coordinates of the iodine anomalous signal from p-iodophenylalanine. RESULTS +155 174 p-iodophenylalanine chemical Phases for peptide 4 were determined by single-wavelength anomalous diffraction (SAD) phasing by using the coordinates of the iodine anomalous signal from p-iodophenylalanine. RESULTS +0 6 Phases evidence Phases for peptide 2 were determined by isomorphous replacement of peptide 4. RESULTS +11 20 peptide 2 mutant Phases for peptide 2 were determined by isomorphous replacement of peptide 4. RESULTS +40 63 isomorphous replacement experimental_method Phases for peptide 2 were determined by isomorphous replacement of peptide 4. RESULTS +67 76 peptide 4 mutant Phases for peptide 2 were determined by isomorphous replacement of peptide 4. RESULTS +4 14 structures evidence The structures of peptides 2 and 4 were solved and refined in the P6122 space group. RESULTS +18 34 peptides 2 and 4 mutant The structures of peptides 2 and 4 were solved and refined in the P6122 space group. RESULTS +40 46 solved experimental_method The structures of peptides 2 and 4 were solved and refined in the P6122 space group. RESULTS +28 35 peptide chemical The asymmetric unit of each peptide consists of six monomers, arranged as two trimers. RESULTS +52 60 monomers oligomeric_state The asymmetric unit of each peptide consists of six monomers, arranged as two trimers. RESULTS +78 85 trimers oligomeric_state The asymmetric unit of each peptide consists of six monomers, arranged as two trimers. RESULTS +0 16 Peptides 2 and 4 mutant Peptides 2 and 4 form morphologically identical structures and assemblies in the crystal lattice. RESULTS +81 96 crystal lattice evidence Peptides 2 and 4 form morphologically identical structures and assemblies in the crystal lattice. RESULTS +0 32 X-ray Crystallographic Structure evidence X-ray Crystallographic Structure of Peptide 2 and the Oligomers It Forms RESULTS +36 45 Peptide 2 mutant X-ray Crystallographic Structure of Peptide 2 and the Oligomers It Forms RESULTS +54 63 Oligomers oligomeric_state X-ray Crystallographic Structure of Peptide 2 and the Oligomers It Forms RESULTS +4 36 X-ray crystallographic structure evidence The X-ray crystallographic structure of peptide 2 reveals that it folds to form a twisted β-hairpin comprising two β-strands connected by a loop (Figure 2A). RESULTS +40 49 peptide 2 mutant The X-ray crystallographic structure of peptide 2 reveals that it folds to form a twisted β-hairpin comprising two β-strands connected by a loop (Figure 2A). RESULTS +82 99 twisted β-hairpin structure_element The X-ray crystallographic structure of peptide 2 reveals that it folds to form a twisted β-hairpin comprising two β-strands connected by a loop (Figure 2A). RESULTS +115 124 β-strands structure_element The X-ray crystallographic structure of peptide 2 reveals that it folds to form a twisted β-hairpin comprising two β-strands connected by a loop (Figure 2A). RESULTS +140 144 loop structure_element The X-ray crystallographic structure of peptide 2 reveals that it folds to form a twisted β-hairpin comprising two β-strands connected by a loop (Figure 2A). RESULTS +43 52 β-hairpin structure_element Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +54 57 L17 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +59 62 F19 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +64 67 A21 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +69 72 D23 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +74 77 A30 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +79 82 I32 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +84 87 L34 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +93 96 V36 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +118 121 V18 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +123 126 F20 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +128 131 E22 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +133 136 C24 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +138 141 C29 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +143 146 I31 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +148 151 G33 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +157 160 M35 residue_name_number Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. RESULTS +4 13 β-strands structure_element The β-strands of the monomers in the asymmetric unit are virtually identical, differing primarily in rotamers of F20, E22, C24, C29, I31, and M35 (Figure S1). RESULTS +21 29 monomers oligomeric_state The β-strands of the monomers in the asymmetric unit are virtually identical, differing primarily in rotamers of F20, E22, C24, C29, I31, and M35 (Figure S1). RESULTS +113 116 F20 residue_name_number The β-strands of the monomers in the asymmetric unit are virtually identical, differing primarily in rotamers of F20, E22, C24, C29, I31, and M35 (Figure S1). RESULTS +118 121 E22 residue_name_number The β-strands of the monomers in the asymmetric unit are virtually identical, differing primarily in rotamers of F20, E22, C24, C29, I31, and M35 (Figure S1). RESULTS +123 126 C24 residue_name_number The β-strands of the monomers in the asymmetric unit are virtually identical, differing primarily in rotamers of F20, E22, C24, C29, I31, and M35 (Figure S1). RESULTS +128 131 C29 residue_name_number The β-strands of the monomers in the asymmetric unit are virtually identical, differing primarily in rotamers of F20, E22, C24, C29, I31, and M35 (Figure S1). RESULTS +133 136 I31 residue_name_number The β-strands of the monomers in the asymmetric unit are virtually identical, differing primarily in rotamers of F20, E22, C24, C29, I31, and M35 (Figure S1). RESULTS +142 145 M35 residue_name_number The β-strands of the monomers in the asymmetric unit are virtually identical, differing primarily in rotamers of F20, E22, C24, C29, I31, and M35 (Figure S1). RESULTS +4 22 disulfide linkages ptm The disulfide linkages suffered radiation damage under synchrotron radiation. RESULTS +3 10 refined experimental_method We refined three of the β-hairpins with intact disulfide linkages and three with thiols to represent cleaved disulfide linkages in the synchrotron data set (PDB 5HOX). RESULTS +24 34 β-hairpins structure_element We refined three of the β-hairpins with intact disulfide linkages and three with thiols to represent cleaved disulfide linkages in the synchrotron data set (PDB 5HOX). RESULTS +40 46 intact protein_state We refined three of the β-hairpins with intact disulfide linkages and three with thiols to represent cleaved disulfide linkages in the synchrotron data set (PDB 5HOX). RESULTS +47 65 disulfide linkages ptm We refined three of the β-hairpins with intact disulfide linkages and three with thiols to represent cleaved disulfide linkages in the synchrotron data set (PDB 5HOX). RESULTS +101 108 cleaved protein_state We refined three of the β-hairpins with intact disulfide linkages and three with thiols to represent cleaved disulfide linkages in the synchrotron data set (PDB 5HOX). RESULTS +109 127 disulfide linkages ptm We refined three of the β-hairpins with intact disulfide linkages and three with thiols to represent cleaved disulfide linkages in the synchrotron data set (PDB 5HOX). RESULTS +32 42 disulfides ptm No evidence for cleavage of the disulfides was observed in the refinement of the data set collected on the X-ray diffractometer, and we refined all disulfide linkages as intact (PDB 5HOY). RESULTS +63 73 refinement experimental_method No evidence for cleavage of the disulfides was observed in the refinement of the data set collected on the X-ray diffractometer, and we refined all disulfide linkages as intact (PDB 5HOY). RESULTS +136 143 refined experimental_method No evidence for cleavage of the disulfides was observed in the refinement of the data set collected on the X-ray diffractometer, and we refined all disulfide linkages as intact (PDB 5HOY). RESULTS +148 166 disulfide linkages ptm No evidence for cleavage of the disulfides was observed in the refinement of the data set collected on the X-ray diffractometer, and we refined all disulfide linkages as intact (PDB 5HOY). RESULTS +170 176 intact protein_state No evidence for cleavage of the disulfides was observed in the refinement of the data set collected on the X-ray diffractometer, and we refined all disulfide linkages as intact (PDB 5HOY). RESULTS +36 45 peptide 2 mutant X-ray crystallographic structure of peptide 2 (PDB 5HOX, synchrotron data set). (A) X-ray crystallographic structure of a representative β-hairpin monomer formed by peptide 2. (B) Overlay of the six β-hairpin monomers in the asymmetric unit. FIG +84 116 X-ray crystallographic structure evidence X-ray crystallographic structure of peptide 2 (PDB 5HOX, synchrotron data set). (A) X-ray crystallographic structure of a representative β-hairpin monomer formed by peptide 2. (B) Overlay of the six β-hairpin monomers in the asymmetric unit. FIG +137 146 β-hairpin structure_element X-ray crystallographic structure of peptide 2 (PDB 5HOX, synchrotron data set). (A) X-ray crystallographic structure of a representative β-hairpin monomer formed by peptide 2. (B) Overlay of the six β-hairpin monomers in the asymmetric unit. FIG +147 154 monomer oligomeric_state X-ray crystallographic structure of peptide 2 (PDB 5HOX, synchrotron data set). (A) X-ray crystallographic structure of a representative β-hairpin monomer formed by peptide 2. (B) Overlay of the six β-hairpin monomers in the asymmetric unit. FIG +165 174 peptide 2 mutant X-ray crystallographic structure of peptide 2 (PDB 5HOX, synchrotron data set). (A) X-ray crystallographic structure of a representative β-hairpin monomer formed by peptide 2. (B) Overlay of the six β-hairpin monomers in the asymmetric unit. FIG +180 187 Overlay experimental_method X-ray crystallographic structure of peptide 2 (PDB 5HOX, synchrotron data set). (A) X-ray crystallographic structure of a representative β-hairpin monomer formed by peptide 2. (B) Overlay of the six β-hairpin monomers in the asymmetric unit. FIG +199 208 β-hairpin structure_element X-ray crystallographic structure of peptide 2 (PDB 5HOX, synchrotron data set). (A) X-ray crystallographic structure of a representative β-hairpin monomer formed by peptide 2. (B) Overlay of the six β-hairpin monomers in the asymmetric unit. FIG +209 217 monomers oligomeric_state X-ray crystallographic structure of peptide 2 (PDB 5HOX, synchrotron data set). (A) X-ray crystallographic structure of a representative β-hairpin monomer formed by peptide 2. (B) Overlay of the six β-hairpin monomers in the asymmetric unit. FIG +4 14 β-hairpins structure_element The β-hairpins are shown as cartoons to illustrate the differences in the Aβ25–28 loops. FIG +74 76 Aβ protein The β-hairpins are shown as cartoons to illustrate the differences in the Aβ25–28 loops. FIG +76 81 25–28 residue_range The β-hairpins are shown as cartoons to illustrate the differences in the Aβ25–28 loops. FIG +82 87 loops structure_element The β-hairpins are shown as cartoons to illustrate the differences in the Aβ25–28 loops. FIG +4 6 Aβ protein The Aβ25–28 loops of the six monomers within the asymmetric unit vary substantially in backbone geometry and side chain rotamers (Figures 2B and S1). RESULTS +6 11 25–28 residue_range The Aβ25–28 loops of the six monomers within the asymmetric unit vary substantially in backbone geometry and side chain rotamers (Figures 2B and S1). RESULTS +12 17 loops structure_element The Aβ25–28 loops of the six monomers within the asymmetric unit vary substantially in backbone geometry and side chain rotamers (Figures 2B and S1). RESULTS +29 37 monomers oligomeric_state The Aβ25–28 loops of the six monomers within the asymmetric unit vary substantially in backbone geometry and side chain rotamers (Figures 2B and S1). RESULTS +4 20 electron density evidence The electron density for the loops is weak and diffuse compared to the electron density for the β-strands. RESULTS +29 34 loops structure_element The electron density for the loops is weak and diffuse compared to the electron density for the β-strands. RESULTS +71 87 electron density evidence The electron density for the loops is weak and diffuse compared to the electron density for the β-strands. RESULTS +96 105 β-strands structure_element The electron density for the loops is weak and diffuse compared to the electron density for the β-strands. RESULTS +4 12 B values evidence The B values for the loops are large, indicating that the loops are dynamic and not well ordered. RESULTS +21 26 loops structure_element The B values for the loops are large, indicating that the loops are dynamic and not well ordered. RESULTS +58 63 loops structure_element The B values for the loops are large, indicating that the loops are dynamic and not well ordered. RESULTS +77 82 loops structure_element Thus, the differences in backbone geometry and side chain rotamers among the loops are likely of little significance and should be interpreted with caution. RESULTS +0 9 Peptide 2 mutant Peptide 2 assembles into oligomers similar in morphology to those formed by peptide 1. RESULTS +25 34 oligomers oligomeric_state Peptide 2 assembles into oligomers similar in morphology to those formed by peptide 1. RESULTS +76 85 peptide 1 mutant Peptide 2 assembles into oligomers similar in morphology to those formed by peptide 1. RESULTS +5 14 peptide 1 mutant Like peptide 1, peptide 2 forms a triangular trimer, and four trimers assemble to form a dodecamer. RESULTS +16 25 peptide 2 mutant Like peptide 1, peptide 2 forms a triangular trimer, and four trimers assemble to form a dodecamer. RESULTS +34 44 triangular protein_state Like peptide 1, peptide 2 forms a triangular trimer, and four trimers assemble to form a dodecamer. RESULTS +45 51 trimer oligomeric_state Like peptide 1, peptide 2 forms a triangular trimer, and four trimers assemble to form a dodecamer. RESULTS +62 69 trimers oligomeric_state Like peptide 1, peptide 2 forms a triangular trimer, and four trimers assemble to form a dodecamer. RESULTS +89 98 dodecamer oligomeric_state Like peptide 1, peptide 2 forms a triangular trimer, and four trimers assemble to form a dodecamer. RESULTS +36 46 dodecamers oligomeric_state In the higher-order assembly of the dodecamers formed by peptide 2 a new structure emerges, not seen in peptide 1, an annular pore consisting of five dodecamers. RESULTS +57 66 peptide 2 mutant In the higher-order assembly of the dodecamers formed by peptide 2 a new structure emerges, not seen in peptide 1, an annular pore consisting of five dodecamers. RESULTS +73 82 structure evidence In the higher-order assembly of the dodecamers formed by peptide 2 a new structure emerges, not seen in peptide 1, an annular pore consisting of five dodecamers. RESULTS +104 113 peptide 1 mutant In the higher-order assembly of the dodecamers formed by peptide 2 a new structure emerges, not seen in peptide 1, an annular pore consisting of five dodecamers. RESULTS +118 130 annular pore site In the higher-order assembly of the dodecamers formed by peptide 2 a new structure emerges, not seen in peptide 1, an annular pore consisting of five dodecamers. RESULTS +150 160 dodecamers oligomeric_state In the higher-order assembly of the dodecamers formed by peptide 2 a new structure emerges, not seen in peptide 1, an annular pore consisting of five dodecamers. RESULTS +0 6 Trimer oligomeric_state Trimer RESULTS +0 9 Peptide 2 mutant Peptide 2 forms a trimer, much like that which we observed previously for peptide 1, in which three β-hairpins assemble to form an equilateral triangle (Figure 3A). RESULTS +18 24 trimer oligomeric_state Peptide 2 forms a trimer, much like that which we observed previously for peptide 1, in which three β-hairpins assemble to form an equilateral triangle (Figure 3A). RESULTS +74 83 peptide 1 mutant Peptide 2 forms a trimer, much like that which we observed previously for peptide 1, in which three β-hairpins assemble to form an equilateral triangle (Figure 3A). RESULTS +100 110 β-hairpins structure_element Peptide 2 forms a trimer, much like that which we observed previously for peptide 1, in which three β-hairpins assemble to form an equilateral triangle (Figure 3A). RESULTS +131 151 equilateral triangle structure_element Peptide 2 forms a trimer, much like that which we observed previously for peptide 1, in which three β-hairpins assemble to form an equilateral triangle (Figure 3A). RESULTS +4 10 trimer oligomeric_state The trimer maintains all of the same stabilizing contacts as those of peptide 1. RESULTS +70 79 peptide 1 mutant The trimer maintains all of the same stabilizing contacts as those of peptide 1. RESULTS +0 16 Hydrogen bonding bond_interaction Hydrogen bonding and hydrophobic interactions between residues on the β-strands comprising Aβ17–23 and Aβ30–36 stabilize the core of the trimer. RESULTS +21 45 hydrophobic interactions bond_interaction Hydrogen bonding and hydrophobic interactions between residues on the β-strands comprising Aβ17–23 and Aβ30–36 stabilize the core of the trimer. RESULTS +70 79 β-strands structure_element Hydrogen bonding and hydrophobic interactions between residues on the β-strands comprising Aβ17–23 and Aβ30–36 stabilize the core of the trimer. RESULTS +91 93 Aβ protein Hydrogen bonding and hydrophobic interactions between residues on the β-strands comprising Aβ17–23 and Aβ30–36 stabilize the core of the trimer. RESULTS +93 98 17–23 residue_range Hydrogen bonding and hydrophobic interactions between residues on the β-strands comprising Aβ17–23 and Aβ30–36 stabilize the core of the trimer. RESULTS +103 105 Aβ protein Hydrogen bonding and hydrophobic interactions between residues on the β-strands comprising Aβ17–23 and Aβ30–36 stabilize the core of the trimer. RESULTS +105 110 30–36 residue_range Hydrogen bonding and hydrophobic interactions between residues on the β-strands comprising Aβ17–23 and Aβ30–36 stabilize the core of the trimer. RESULTS +125 129 core structure_element Hydrogen bonding and hydrophobic interactions between residues on the β-strands comprising Aβ17–23 and Aβ30–36 stabilize the core of the trimer. RESULTS +137 143 trimer oligomeric_state Hydrogen bonding and hydrophobic interactions between residues on the β-strands comprising Aβ17–23 and Aβ30–36 stabilize the core of the trimer. RESULTS +4 19 disulfide bonds ptm The disulfide bonds between residues 24 and 29 are adjacent to the structural core of the trimer and do not make any substantial intermolecular contacts. RESULTS +37 39 24 residue_number The disulfide bonds between residues 24 and 29 are adjacent to the structural core of the trimer and do not make any substantial intermolecular contacts. RESULTS +44 46 29 residue_number The disulfide bonds between residues 24 and 29 are adjacent to the structural core of the trimer and do not make any substantial intermolecular contacts. RESULTS +67 82 structural core structure_element The disulfide bonds between residues 24 and 29 are adjacent to the structural core of the trimer and do not make any substantial intermolecular contacts. RESULTS +90 96 trimer oligomeric_state The disulfide bonds between residues 24 and 29 are adjacent to the structural core of the trimer and do not make any substantial intermolecular contacts. RESULTS +34 41 trimers oligomeric_state Two crystallographically distinct trimers comprise the peptide portion of the asymmetric unit. RESULTS +55 62 peptide chemical Two crystallographically distinct trimers comprise the peptide portion of the asymmetric unit. RESULTS +8 15 trimers oligomeric_state The two trimers are almost identical in structure, differing slightly among side chain rotamers and loop conformations. RESULTS +100 104 loop structure_element The two trimers are almost identical in structure, differing slightly among side chain rotamers and loop conformations. RESULTS +0 32 X-ray crystallographic structure evidence X-ray crystallographic structure of the trimer formed by peptide 2. (A) Triangular trimer. FIG +40 46 trimer oligomeric_state X-ray crystallographic structure of the trimer formed by peptide 2. (A) Triangular trimer. FIG +57 66 peptide 2 mutant X-ray crystallographic structure of the trimer formed by peptide 2. (A) Triangular trimer. FIG +72 82 Triangular protein_state X-ray crystallographic structure of the trimer formed by peptide 2. (A) Triangular trimer. FIG +83 89 trimer oligomeric_state X-ray crystallographic structure of the trimer formed by peptide 2. (A) Triangular trimer. FIG +10 15 water chemical The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +52 58 trimer oligomeric_state The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +121 135 hydrogen bonds bond_interaction The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +163 166 V18 residue_name_number The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +171 174 E22 residue_name_number The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +179 183 δOrn structure_element The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +188 191 C24 residue_name_number The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +221 231 triangular protein_state The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +232 238 trimer oligomeric_state The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +248 251 F19 residue_name_number The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +264 270 trimer oligomeric_state The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +319 322 F20 residue_name_number The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +335 341 trimer oligomeric_state The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres. FIG +31 45 hydrogen bonds bond_interaction A network of 18 intermolecular hydrogen bonds helps stabilize the trimer. RESULTS +66 72 trimer oligomeric_state A network of 18 intermolecular hydrogen bonds helps stabilize the trimer. RESULTS +22 28 trimer oligomeric_state At the corners of the trimer, the pairs of β-hairpin monomers form four hydrogen bonds: two between the main chains of V18 and E22 and two between δOrn and the main chain of C24 (Figure 3B). RESULTS +43 52 β-hairpin structure_element At the corners of the trimer, the pairs of β-hairpin monomers form four hydrogen bonds: two between the main chains of V18 and E22 and two between δOrn and the main chain of C24 (Figure 3B). RESULTS +53 61 monomers oligomeric_state At the corners of the trimer, the pairs of β-hairpin monomers form four hydrogen bonds: two between the main chains of V18 and E22 and two between δOrn and the main chain of C24 (Figure 3B). RESULTS +72 86 hydrogen bonds bond_interaction At the corners of the trimer, the pairs of β-hairpin monomers form four hydrogen bonds: two between the main chains of V18 and E22 and two between δOrn and the main chain of C24 (Figure 3B). RESULTS +119 122 V18 residue_name_number At the corners of the trimer, the pairs of β-hairpin monomers form four hydrogen bonds: two between the main chains of V18 and E22 and two between δOrn and the main chain of C24 (Figure 3B). RESULTS +127 130 E22 residue_name_number At the corners of the trimer, the pairs of β-hairpin monomers form four hydrogen bonds: two between the main chains of V18 and E22 and two between δOrn and the main chain of C24 (Figure 3B). RESULTS +147 151 δOrn structure_element At the corners of the trimer, the pairs of β-hairpin monomers form four hydrogen bonds: two between the main chains of V18 and E22 and two between δOrn and the main chain of C24 (Figure 3B). RESULTS +174 177 C24 residue_name_number At the corners of the trimer, the pairs of β-hairpin monomers form four hydrogen bonds: two between the main chains of V18 and E22 and two between δOrn and the main chain of C24 (Figure 3B). RESULTS +14 19 water chemical Three ordered water molecules fill the hole in the center of the trimer, hydrogen bonding to each other and to the main chain of F20 (Figure 3A). RESULTS +65 71 trimer oligomeric_state Three ordered water molecules fill the hole in the center of the trimer, hydrogen bonding to each other and to the main chain of F20 (Figure 3A). RESULTS +73 89 hydrogen bonding bond_interaction Three ordered water molecules fill the hole in the center of the trimer, hydrogen bonding to each other and to the main chain of F20 (Figure 3A). RESULTS +129 132 F20 residue_name_number Three ordered water molecules fill the hole in the center of the trimer, hydrogen bonding to each other and to the main chain of F20 (Figure 3A). RESULTS +0 20 Hydrophobic contacts bond_interaction Hydrophobic contacts between residues at the three corners of the trimer, where the β-hairpins meet, further stabilize the trimer. RESULTS +66 72 trimer oligomeric_state Hydrophobic contacts between residues at the three corners of the trimer, where the β-hairpins meet, further stabilize the trimer. RESULTS +84 94 β-hairpins structure_element Hydrophobic contacts between residues at the three corners of the trimer, where the β-hairpins meet, further stabilize the trimer. RESULTS +123 129 trimer oligomeric_state Hydrophobic contacts between residues at the three corners of the trimer, where the β-hairpins meet, further stabilize the trimer. RESULTS +44 47 L17 residue_name_number At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +49 52 F19 residue_name_number At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +58 61 V36 residue_name_number At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +69 78 β-hairpin structure_element At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +120 123 A21 residue_name_number At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +125 128 I32 residue_name_number At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +130 133 L34 residue_name_number At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +144 147 D23 residue_name_number At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +164 173 β-hairpin structure_element At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +186 205 hydrophobic cluster site At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +229 249 hydrophobic clusters site At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +265 284 hydrophobic surface site At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +304 310 trimer oligomeric_state At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. RESULTS +22 28 trimer oligomeric_state The other face of the trimer displays a smaller hydrophobic surface, which includes the side chains of residues V18, F20, and I31 of the three β-hairpins (Figure 3D). RESULTS +48 67 hydrophobic surface site The other face of the trimer displays a smaller hydrophobic surface, which includes the side chains of residues V18, F20, and I31 of the three β-hairpins (Figure 3D). RESULTS +112 115 V18 residue_name_number The other face of the trimer displays a smaller hydrophobic surface, which includes the side chains of residues V18, F20, and I31 of the three β-hairpins (Figure 3D). RESULTS +117 120 F20 residue_name_number The other face of the trimer displays a smaller hydrophobic surface, which includes the side chains of residues V18, F20, and I31 of the three β-hairpins (Figure 3D). RESULTS +126 129 I31 residue_name_number The other face of the trimer displays a smaller hydrophobic surface, which includes the side chains of residues V18, F20, and I31 of the three β-hairpins (Figure 3D). RESULTS +143 153 β-hairpins structure_element The other face of the trimer displays a smaller hydrophobic surface, which includes the side chains of residues V18, F20, and I31 of the three β-hairpins (Figure 3D). RESULTS +63 66 F19 residue_name_number In subsequent discussion, we designate the former surface the “F19 face” and the latter surface the “F20 face”. RESULTS +101 104 F20 residue_name_number In subsequent discussion, we designate the former surface the “F19 face” and the latter surface the “F20 face”. RESULTS +0 9 Dodecamer oligomeric_state Dodecamer RESULTS +5 12 trimers oligomeric_state Four trimers assemble to form a dodecamer. RESULTS +32 41 dodecamer oligomeric_state Four trimers assemble to form a dodecamer. RESULTS +9 16 trimers oligomeric_state The four trimers arrange in a tetrahedral fashion, creating a central cavity inside the dodecamer. Because each trimer is triangular, the resulting arrangement resembles an octahedron. RESULTS +30 41 tetrahedral protein_state The four trimers arrange in a tetrahedral fashion, creating a central cavity inside the dodecamer. Because each trimer is triangular, the resulting arrangement resembles an octahedron. RESULTS +62 76 central cavity site The four trimers arrange in a tetrahedral fashion, creating a central cavity inside the dodecamer. Because each trimer is triangular, the resulting arrangement resembles an octahedron. RESULTS +88 97 dodecamer oligomeric_state The four trimers arrange in a tetrahedral fashion, creating a central cavity inside the dodecamer. Because each trimer is triangular, the resulting arrangement resembles an octahedron. RESULTS +112 118 trimer oligomeric_state The four trimers arrange in a tetrahedral fashion, creating a central cavity inside the dodecamer. Because each trimer is triangular, the resulting arrangement resembles an octahedron. RESULTS +122 132 triangular protein_state The four trimers arrange in a tetrahedral fashion, creating a central cavity inside the dodecamer. Because each trimer is triangular, the resulting arrangement resembles an octahedron. RESULTS +173 183 octahedron protein_state The four trimers arrange in a tetrahedral fashion, creating a central cavity inside the dodecamer. Because each trimer is triangular, the resulting arrangement resembles an octahedron. RESULTS +15 25 β-hairpins structure_element Each of the 12 β-hairpins constitutes an edge of the octahedron, and the triangular trimers occupy four of the eight faces of the octahedron. RESULTS +53 63 octahedron protein_state Each of the 12 β-hairpins constitutes an edge of the octahedron, and the triangular trimers occupy four of the eight faces of the octahedron. RESULTS +73 83 triangular protein_state Each of the 12 β-hairpins constitutes an edge of the octahedron, and the triangular trimers occupy four of the eight faces of the octahedron. RESULTS +84 91 trimers oligomeric_state Each of the 12 β-hairpins constitutes an edge of the octahedron, and the triangular trimers occupy four of the eight faces of the octahedron. RESULTS +130 140 octahedron protein_state Each of the 12 β-hairpins constitutes an edge of the octahedron, and the triangular trimers occupy four of the eight faces of the octahedron. RESULTS +26 36 octahedral protein_state Figure 4A illustrates the octahedral shape of the dodecamer. RESULTS +50 59 dodecamer oligomeric_state Figure 4A illustrates the octahedral shape of the dodecamer. RESULTS +26 37 tetrahedral protein_state Figure 4B illustrates the tetrahedral arrangement of the four trimers. RESULTS +62 69 trimers oligomeric_state Figure 4B illustrates the tetrahedral arrangement of the four trimers. RESULTS +40 49 dodecamer oligomeric_state X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer. FIG +60 69 peptide 2 mutant X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer. FIG +87 96 dodecamer oligomeric_state X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer. FIG +118 128 octahedral protein_state X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer. FIG +152 161 dodecamer oligomeric_state X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer. FIG +183 194 tetrahedral protein_state X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer. FIG +219 226 trimers oligomeric_state X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer. FIG +245 254 dodecamer oligomeric_state X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer. FIG +272 278 trimer oligomeric_state X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer. FIG +279 287 subunits structure_element X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer. FIG +304 310 cavity site X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer. FIG +318 327 dodecamer oligomeric_state X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer. FIG +9 12 L17 residue_name_number Residues L17, L34, and V36 are shown as spheres, illustrating the hydrophobic packing that occurs at the six vertices of the dodecamer. (D) Detailed view of one of the six vertices of the dodecamer. FIG +14 17 L34 residue_name_number Residues L17, L34, and V36 are shown as spheres, illustrating the hydrophobic packing that occurs at the six vertices of the dodecamer. (D) Detailed view of one of the six vertices of the dodecamer. FIG +23 26 V36 residue_name_number Residues L17, L34, and V36 are shown as spheres, illustrating the hydrophobic packing that occurs at the six vertices of the dodecamer. (D) Detailed view of one of the six vertices of the dodecamer. FIG +66 85 hydrophobic packing bond_interaction Residues L17, L34, and V36 are shown as spheres, illustrating the hydrophobic packing that occurs at the six vertices of the dodecamer. (D) Detailed view of one of the six vertices of the dodecamer. FIG +125 134 dodecamer oligomeric_state Residues L17, L34, and V36 are shown as spheres, illustrating the hydrophobic packing that occurs at the six vertices of the dodecamer. (D) Detailed view of one of the six vertices of the dodecamer. FIG +188 197 dodecamer oligomeric_state Residues L17, L34, and V36 are shown as spheres, illustrating the hydrophobic packing that occurs at the six vertices of the dodecamer. (D) Detailed view of one of the six vertices of the dodecamer. FIG +4 7 F19 residue_name_number The F19 faces of the trimers line the interior of the dodecamer. RESULTS +21 28 trimers oligomeric_state The F19 faces of the trimers line the interior of the dodecamer. RESULTS +54 63 dodecamer oligomeric_state The F19 faces of the trimers line the interior of the dodecamer. RESULTS +21 40 hydrophobic packing bond_interaction At the six vertices, hydrophobic packing between the side chains of L17, L34, and V36 helps stabilize the dodecamer (Figures 4C and D). RESULTS +68 71 L17 residue_name_number At the six vertices, hydrophobic packing between the side chains of L17, L34, and V36 helps stabilize the dodecamer (Figures 4C and D). RESULTS +73 76 L34 residue_name_number At the six vertices, hydrophobic packing between the side chains of L17, L34, and V36 helps stabilize the dodecamer (Figures 4C and D). RESULTS +82 85 V36 residue_name_number At the six vertices, hydrophobic packing between the side chains of L17, L34, and V36 helps stabilize the dodecamer (Figures 4C and D). RESULTS +106 115 dodecamer oligomeric_state At the six vertices, hydrophobic packing between the side chains of L17, L34, and V36 helps stabilize the dodecamer (Figures 4C and D). RESULTS +40 43 D23 residue_name_number Salt bridges between the side chains of D23 and δOrn at the vertices further stabilize the dodecamer. RESULTS +48 52 δOrn structure_element Salt bridges between the side chains of D23 and δOrn at the vertices further stabilize the dodecamer. RESULTS +91 100 dodecamer oligomeric_state Salt bridges between the side chains of D23 and δOrn at the vertices further stabilize the dodecamer. RESULTS +38 40 Aβ protein Each of the six vertices includes two Aβ25–28 loops that extend past the core of the dodecamer without making any substantial intermolecular contacts. RESULTS +40 45 25–28 residue_range Each of the six vertices includes two Aβ25–28 loops that extend past the core of the dodecamer without making any substantial intermolecular contacts. RESULTS +46 51 loops structure_element Each of the six vertices includes two Aβ25–28 loops that extend past the core of the dodecamer without making any substantial intermolecular contacts. RESULTS +73 77 core structure_element Each of the six vertices includes two Aβ25–28 loops that extend past the core of the dodecamer without making any substantial intermolecular contacts. RESULTS +85 94 dodecamer oligomeric_state Each of the six vertices includes two Aβ25–28 loops that extend past the core of the dodecamer without making any substantial intermolecular contacts. RESULTS +20 29 dodecamer oligomeric_state The exterior of the dodecamer displays four F20 faces (Figure S3). RESULTS +44 47 F20 residue_name_number The exterior of the dodecamer displays four F20 faces (Figure S3). RESULTS +7 22 crystal lattice evidence In the crystal lattice, each F20 face of one dodecamer packs against an F20 face of another dodecamer. RESULTS +29 32 F20 residue_name_number In the crystal lattice, each F20 face of one dodecamer packs against an F20 face of another dodecamer. RESULTS +45 54 dodecamer oligomeric_state In the crystal lattice, each F20 face of one dodecamer packs against an F20 face of another dodecamer. RESULTS +72 75 F20 residue_name_number In the crystal lattice, each F20 face of one dodecamer packs against an F20 face of another dodecamer. RESULTS +92 101 dodecamer oligomeric_state In the crystal lattice, each F20 face of one dodecamer packs against an F20 face of another dodecamer. RESULTS +46 55 dodecamer oligomeric_state Although the asymmetric unit comprises half a dodecamer, the crystal lattice may be thought of as being built of dodecamers. RESULTS +61 76 crystal lattice evidence Although the asymmetric unit comprises half a dodecamer, the crystal lattice may be thought of as being built of dodecamers. RESULTS +113 123 dodecamers oligomeric_state Although the asymmetric unit comprises half a dodecamer, the crystal lattice may be thought of as being built of dodecamers. RESULTS +4 24 electron density map evidence The electron density map for the X-ray crystallographic structure of peptide 2 has long tubes of electron density inside the central cavity of the dodecamer. RESULTS +33 65 X-ray crystallographic structure evidence The electron density map for the X-ray crystallographic structure of peptide 2 has long tubes of electron density inside the central cavity of the dodecamer. RESULTS +69 78 peptide 2 mutant The electron density map for the X-ray crystallographic structure of peptide 2 has long tubes of electron density inside the central cavity of the dodecamer. RESULTS +97 113 electron density evidence The electron density map for the X-ray crystallographic structure of peptide 2 has long tubes of electron density inside the central cavity of the dodecamer. RESULTS +125 139 central cavity site The electron density map for the X-ray crystallographic structure of peptide 2 has long tubes of electron density inside the central cavity of the dodecamer. RESULTS +147 156 dodecamer oligomeric_state The electron density map for the X-ray crystallographic structure of peptide 2 has long tubes of electron density inside the central cavity of the dodecamer. RESULTS +28 44 electron density evidence The shape and length of the electron density is consistent with the structure of Jeffamine M-600, which is an essential component of the crystallization conditions. RESULTS +68 77 structure evidence The shape and length of the electron density is consistent with the structure of Jeffamine M-600, which is an essential component of the crystallization conditions. RESULTS +81 96 Jeffamine M-600 chemical The shape and length of the electron density is consistent with the structure of Jeffamine M-600, which is an essential component of the crystallization conditions. RESULTS +0 15 Jeffamine M-600 chemical Jeffamine M-600 is a polypropylene glycol derivative with a 2-methoxyethoxy unit at one end and a 2-aminopropyl unit at the other end. RESULTS +9 24 Jeffamine M-600 chemical Although Jeffamine M-600 is a heterogeneous mixture with varying chain lengths and stereochemistry, we modeled a single stereoisomer with nine propylene glycol units (n = 9) to fit the electron density. RESULTS +185 201 electron density evidence Although Jeffamine M-600 is a heterogeneous mixture with varying chain lengths and stereochemistry, we modeled a single stereoisomer with nine propylene glycol units (n = 9) to fit the electron density. RESULTS +4 19 Jeffamine M-600 chemical The Jeffamine M-600 appears to stabilize the dodecamer by occupying the central cavity and making hydrophobic contacts with residues lining the cavity (Figure S3). RESULTS +45 54 dodecamer oligomeric_state The Jeffamine M-600 appears to stabilize the dodecamer by occupying the central cavity and making hydrophobic contacts with residues lining the cavity (Figure S3). RESULTS +72 86 central cavity site The Jeffamine M-600 appears to stabilize the dodecamer by occupying the central cavity and making hydrophobic contacts with residues lining the cavity (Figure S3). RESULTS +98 118 hydrophobic contacts bond_interaction The Jeffamine M-600 appears to stabilize the dodecamer by occupying the central cavity and making hydrophobic contacts with residues lining the cavity (Figure S3). RESULTS +144 150 cavity site The Jeffamine M-600 appears to stabilize the dodecamer by occupying the central cavity and making hydrophobic contacts with residues lining the cavity (Figure S3). RESULTS +5 14 dodecamer oligomeric_state In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer. RESULTS +25 36 full-length protein_state In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer. RESULTS +37 39 Aβ protein In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer. RESULTS +78 80 Aβ protein In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer. RESULTS +80 85 37–40 residue_range In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer. RESULTS +89 91 Aβ protein In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer. RESULTS +91 96 37–42 residue_range In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer. RESULTS +139 148 dodecamer oligomeric_state In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer. RESULTS +174 190 hydrophobic core site In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer. RESULTS +202 216 central cavity site In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer. RESULTS +224 233 dodecamer oligomeric_state In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer. RESULTS +0 12 Annular Pore site Annular Pore RESULTS +5 15 dodecamers oligomeric_state Five dodecamers assemble to form an annular porelike structure (Figure 5A). RESULTS +44 52 porelike structure_element Five dodecamers assemble to form an annular porelike structure (Figure 5A). RESULTS +0 19 Hydrophobic packing bond_interaction Hydrophobic packing between the F20 faces of trimers displayed on the outer surface of each dodecamer stabilizes the porelike assembly. RESULTS +32 35 F20 residue_name_number Hydrophobic packing between the F20 faces of trimers displayed on the outer surface of each dodecamer stabilizes the porelike assembly. RESULTS +45 52 trimers oligomeric_state Hydrophobic packing between the F20 faces of trimers displayed on the outer surface of each dodecamer stabilizes the porelike assembly. RESULTS +92 101 dodecamer oligomeric_state Hydrophobic packing between the F20 faces of trimers displayed on the outer surface of each dodecamer stabilizes the porelike assembly. RESULTS +50 57 trimers oligomeric_state Two morphologically distinct interactions between trimers occur at the interfaces of the five dodecamers: one in which the trimers are eclipsed (Figure 5B), and one in which the trimers are staggered (Figure 5C). RESULTS +71 81 interfaces site Two morphologically distinct interactions between trimers occur at the interfaces of the five dodecamers: one in which the trimers are eclipsed (Figure 5B), and one in which the trimers are staggered (Figure 5C). RESULTS +94 104 dodecamers oligomeric_state Two morphologically distinct interactions between trimers occur at the interfaces of the five dodecamers: one in which the trimers are eclipsed (Figure 5B), and one in which the trimers are staggered (Figure 5C). RESULTS +123 130 trimers oligomeric_state Two morphologically distinct interactions between trimers occur at the interfaces of the five dodecamers: one in which the trimers are eclipsed (Figure 5B), and one in which the trimers are staggered (Figure 5C). RESULTS +135 143 eclipsed protein_state Two morphologically distinct interactions between trimers occur at the interfaces of the five dodecamers: one in which the trimers are eclipsed (Figure 5B), and one in which the trimers are staggered (Figure 5C). RESULTS +178 185 trimers oligomeric_state Two morphologically distinct interactions between trimers occur at the interfaces of the five dodecamers: one in which the trimers are eclipsed (Figure 5B), and one in which the trimers are staggered (Figure 5C). RESULTS +190 199 staggered protein_state Two morphologically distinct interactions between trimers occur at the interfaces of the five dodecamers: one in which the trimers are eclipsed (Figure 5B), and one in which the trimers are staggered (Figure 5C). RESULTS +0 19 Hydrophobic packing bond_interaction Hydrophobic packing between the side chains of F20, I31, and E22 stabilizes these interfaces (Figure 5D and E). RESULTS +47 50 F20 residue_name_number Hydrophobic packing between the side chains of F20, I31, and E22 stabilizes these interfaces (Figure 5D and E). RESULTS +52 55 I31 residue_name_number Hydrophobic packing between the side chains of F20, I31, and E22 stabilizes these interfaces (Figure 5D and E). RESULTS +61 64 E22 residue_name_number Hydrophobic packing between the side chains of F20, I31, and E22 stabilizes these interfaces (Figure 5D and E). RESULTS +82 92 interfaces site Hydrophobic packing between the side chains of F20, I31, and E22 stabilizes these interfaces (Figure 5D and E). RESULTS +4 16 annular pore site The annular pore contains three eclipsed interfaces and two staggered interfaces. RESULTS +32 40 eclipsed protein_state The annular pore contains three eclipsed interfaces and two staggered interfaces. RESULTS +41 51 interfaces site The annular pore contains three eclipsed interfaces and two staggered interfaces. RESULTS +60 69 staggered protein_state The annular pore contains three eclipsed interfaces and two staggered interfaces. RESULTS +70 80 interfaces site The annular pore contains three eclipsed interfaces and two staggered interfaces. RESULTS +4 12 eclipsed protein_state The eclipsed interfaces occur between dodecamers 1 and 2, 1 and 5, and 3 and 4, as shown in Figure 5A. RESULTS +13 23 interfaces site The eclipsed interfaces occur between dodecamers 1 and 2, 1 and 5, and 3 and 4, as shown in Figure 5A. RESULTS +38 56 dodecamers 1 and 2 structure_element The eclipsed interfaces occur between dodecamers 1 and 2, 1 and 5, and 3 and 4, as shown in Figure 5A. RESULTS +58 65 1 and 5 structure_element The eclipsed interfaces occur between dodecamers 1 and 2, 1 and 5, and 3 and 4, as shown in Figure 5A. RESULTS +71 78 3 and 4 structure_element The eclipsed interfaces occur between dodecamers 1 and 2, 1 and 5, and 3 and 4, as shown in Figure 5A. RESULTS +4 13 staggered protein_state The staggered interfaces occur between dodecamers 2 and 3 and 4 and 5. RESULTS +14 24 interfaces site The staggered interfaces occur between dodecamers 2 and 3 and 4 and 5. RESULTS +39 57 dodecamers 2 and 3 structure_element The staggered interfaces occur between dodecamers 2 and 3 and 4 and 5. RESULTS +62 69 4 and 5 structure_element The staggered interfaces occur between dodecamers 2 and 3 and 4 and 5. RESULTS +4 16 annular pore site The annular pore is not completely flat, instead, adopting a slightly puckered shape, which accommodates the eclipsed and staggered interfaces. RESULTS +109 117 eclipsed protein_state The annular pore is not completely flat, instead, adopting a slightly puckered shape, which accommodates the eclipsed and staggered interfaces. RESULTS +122 131 staggered protein_state The annular pore is not completely flat, instead, adopting a slightly puckered shape, which accommodates the eclipsed and staggered interfaces. RESULTS +132 142 interfaces site The annular pore is not completely flat, instead, adopting a slightly puckered shape, which accommodates the eclipsed and staggered interfaces. RESULTS +4 6 Aβ protein Ten Aβ25–28 loops from the vertices of the five dodecamers line the hole in the center of the pore. RESULTS +6 11 25–28 residue_range Ten Aβ25–28 loops from the vertices of the five dodecamers line the hole in the center of the pore. RESULTS +12 17 loops structure_element Ten Aβ25–28 loops from the vertices of the five dodecamers line the hole in the center of the pore. RESULTS +48 58 dodecamers oligomeric_state Ten Aβ25–28 loops from the vertices of the five dodecamers line the hole in the center of the pore. RESULTS +94 98 pore site Ten Aβ25–28 loops from the vertices of the five dodecamers line the hole in the center of the pore. RESULTS +31 34 S26 residue_name_number The hydrophilic side chains of S26, N27, and K28 decorate the hole. RESULTS +36 39 N27 residue_name_number The hydrophilic side chains of S26, N27, and K28 decorate the hole. RESULTS +45 48 K28 residue_name_number The hydrophilic side chains of S26, N27, and K28 decorate the hole. RESULTS +0 32 X-ray crystallographic structure evidence X-ray crystallographic structure of the annular pore formed by peptide 2. (A) Annular porelike structure illustrating the relationship of the five dodecamers that form the pore (top view). FIG +40 52 annular pore site X-ray crystallographic structure of the annular pore formed by peptide 2. (A) Annular porelike structure illustrating the relationship of the five dodecamers that form the pore (top view). FIG +63 72 peptide 2 mutant X-ray crystallographic structure of the annular pore formed by peptide 2. (A) Annular porelike structure illustrating the relationship of the five dodecamers that form the pore (top view). FIG +78 94 Annular porelike structure_element X-ray crystallographic structure of the annular pore formed by peptide 2. (A) Annular porelike structure illustrating the relationship of the five dodecamers that form the pore (top view). FIG +95 104 structure evidence X-ray crystallographic structure of the annular pore formed by peptide 2. (A) Annular porelike structure illustrating the relationship of the five dodecamers that form the pore (top view). FIG +147 157 dodecamers oligomeric_state X-ray crystallographic structure of the annular pore formed by peptide 2. (A) Annular porelike structure illustrating the relationship of the five dodecamers that form the pore (top view). FIG +172 176 pore site X-ray crystallographic structure of the annular pore formed by peptide 2. (A) Annular porelike structure illustrating the relationship of the five dodecamers that form the pore (top view). FIG +5 23 Eclipsed interface site (B) Eclipsed interface between dodecamers 1 and 2 (side view). FIG +32 50 dodecamers 1 and 2 structure_element (B) Eclipsed interface between dodecamers 1 and 2 (side view). FIG +9 27 eclipsed interface site The same eclipsed interface also occurs between dodecamers 1 and 5 and 3 and 4. (C) Staggered interface between dodecamers 2 and 3 (side view). FIG +48 66 dodecamers 1 and 5 structure_element The same eclipsed interface also occurs between dodecamers 1 and 5 and 3 and 4. (C) Staggered interface between dodecamers 2 and 3 (side view). FIG +71 78 3 and 4 structure_element The same eclipsed interface also occurs between dodecamers 1 and 5 and 3 and 4. (C) Staggered interface between dodecamers 2 and 3 (side view). FIG +84 103 Staggered interface site The same eclipsed interface also occurs between dodecamers 1 and 5 and 3 and 4. (C) Staggered interface between dodecamers 2 and 3 (side view). FIG +112 130 dodecamers 2 and 3 structure_element The same eclipsed interface also occurs between dodecamers 1 and 5 and 3 and 4. (C) Staggered interface between dodecamers 2 and 3 (side view). FIG +9 28 staggered interface site The same staggered interface also occurs between dodecamers 4 and 5. (D) Eclipsed interface between dodecamers 1 and 5 (top view). FIG +73 91 Eclipsed interface site The same staggered interface also occurs between dodecamers 4 and 5. (D) Eclipsed interface between dodecamers 1 and 5 (top view). FIG +100 118 dodecamers 1 and 5 structure_element The same staggered interface also occurs between dodecamers 4 and 5. (D) Eclipsed interface between dodecamers 1 and 5 (top view). FIG +4 16 annular pore site The annular pore is comparable in size to other large protein assemblies. RESULTS +46 50 pore site The diameter of the hole in the center of the pore is ∼2 nm. RESULTS +21 25 pore site The thickness of the pore is ∼5 nm, which is comparable to that of a lipid bilayer membrane. RESULTS +33 45 annular pore site It is important to note that the annular pore formed by peptide 2 is not a discrete unit in the crystal lattice. RESULTS +56 65 peptide 2 mutant It is important to note that the annular pore formed by peptide 2 is not a discrete unit in the crystal lattice. RESULTS +96 111 crystal lattice evidence It is important to note that the annular pore formed by peptide 2 is not a discrete unit in the crystal lattice. RESULTS +12 27 crystal lattice evidence Rather, the crystal lattice is composed of conjoined annular pores in which all four F20 faces on the surface of each dodecamer contact F20 faces on other dodecamers (Figure S4). RESULTS +53 66 annular pores site Rather, the crystal lattice is composed of conjoined annular pores in which all four F20 faces on the surface of each dodecamer contact F20 faces on other dodecamers (Figure S4). RESULTS +85 88 F20 residue_name_number Rather, the crystal lattice is composed of conjoined annular pores in which all four F20 faces on the surface of each dodecamer contact F20 faces on other dodecamers (Figure S4). RESULTS +118 127 dodecamer oligomeric_state Rather, the crystal lattice is composed of conjoined annular pores in which all four F20 faces on the surface of each dodecamer contact F20 faces on other dodecamers (Figure S4). RESULTS +136 139 F20 residue_name_number Rather, the crystal lattice is composed of conjoined annular pores in which all four F20 faces on the surface of each dodecamer contact F20 faces on other dodecamers (Figure S4). RESULTS +155 165 dodecamers oligomeric_state Rather, the crystal lattice is composed of conjoined annular pores in which all four F20 faces on the surface of each dodecamer contact F20 faces on other dodecamers (Figure S4). RESULTS +4 19 crystal lattice evidence The crystal lattice shows how the dodecamers can further assemble to form larger structures. RESULTS +34 44 dodecamers oligomeric_state The crystal lattice shows how the dodecamers can further assemble to form larger structures. RESULTS +5 14 dodecamer oligomeric_state Each dodecamer may be thought of as a tetravalent building block with the potential to assemble on all four faces to form higher-order supramolecular assemblies. RESULTS +4 32 X-ray crystallographic study experimental_method The X-ray crystallographic study of peptide 2 described here provides high-resolution structures of oligomers formed by an Aβ17–36 β-hairpin. DISCUSS +36 45 peptide 2 mutant The X-ray crystallographic study of peptide 2 described here provides high-resolution structures of oligomers formed by an Aβ17–36 β-hairpin. DISCUSS +86 96 structures evidence The X-ray crystallographic study of peptide 2 described here provides high-resolution structures of oligomers formed by an Aβ17–36 β-hairpin. DISCUSS +100 109 oligomers oligomeric_state The X-ray crystallographic study of peptide 2 described here provides high-resolution structures of oligomers formed by an Aβ17–36 β-hairpin. DISCUSS +123 125 Aβ protein The X-ray crystallographic study of peptide 2 described here provides high-resolution structures of oligomers formed by an Aβ17–36 β-hairpin. DISCUSS +125 130 17–36 residue_range The X-ray crystallographic study of peptide 2 described here provides high-resolution structures of oligomers formed by an Aβ17–36 β-hairpin. DISCUSS +131 140 β-hairpin structure_element The X-ray crystallographic study of peptide 2 described here provides high-resolution structures of oligomers formed by an Aβ17–36 β-hairpin. DISCUSS +4 29 crystallographic assembly evidence The crystallographic assembly of peptide 2 into a trimer, dodecamer, and annular pore provides a model for the assembly of the full-length Aβ peptide to form oligomers. DISCUSS +33 42 peptide 2 mutant The crystallographic assembly of peptide 2 into a trimer, dodecamer, and annular pore provides a model for the assembly of the full-length Aβ peptide to form oligomers. DISCUSS +50 56 trimer oligomeric_state The crystallographic assembly of peptide 2 into a trimer, dodecamer, and annular pore provides a model for the assembly of the full-length Aβ peptide to form oligomers. DISCUSS +58 67 dodecamer oligomeric_state The crystallographic assembly of peptide 2 into a trimer, dodecamer, and annular pore provides a model for the assembly of the full-length Aβ peptide to form oligomers. DISCUSS +73 85 annular pore site The crystallographic assembly of peptide 2 into a trimer, dodecamer, and annular pore provides a model for the assembly of the full-length Aβ peptide to form oligomers. DISCUSS +127 138 full-length protein_state The crystallographic assembly of peptide 2 into a trimer, dodecamer, and annular pore provides a model for the assembly of the full-length Aβ peptide to form oligomers. DISCUSS +139 141 Aβ protein The crystallographic assembly of peptide 2 into a trimer, dodecamer, and annular pore provides a model for the assembly of the full-length Aβ peptide to form oligomers. DISCUSS +158 167 oligomers oligomeric_state The crystallographic assembly of peptide 2 into a trimer, dodecamer, and annular pore provides a model for the assembly of the full-length Aβ peptide to form oligomers. DISCUSS +14 16 Aβ protein In this model Aβ folds to form a β-hairpin comprising the hydrophobic central and C-terminal regions. DISCUSS +33 42 β-hairpin structure_element In this model Aβ folds to form a β-hairpin comprising the hydrophobic central and C-terminal regions. DISCUSS +70 100 central and C-terminal regions structure_element In this model Aβ folds to form a β-hairpin comprising the hydrophobic central and C-terminal regions. DISCUSS +6 16 β-hairpins structure_element Three β-hairpins assemble to form a trimer, and four trimers assemble to form a dodecamer. DISCUSS +36 42 trimer oligomeric_state Three β-hairpins assemble to form a trimer, and four trimers assemble to form a dodecamer. DISCUSS +53 60 trimers oligomeric_state Three β-hairpins assemble to form a trimer, and four trimers assemble to form a dodecamer. DISCUSS +80 89 dodecamer oligomeric_state Three β-hairpins assemble to form a trimer, and four trimers assemble to form a dodecamer. DISCUSS +4 14 dodecamers oligomeric_state The dodecamers further assemble to form an annular pore (Figure 6). DISCUSS +43 55 annular pore site The dodecamers further assemble to form an annular pore (Figure 6). DISCUSS +42 44 Aβ protein Model for the hierarchical assembly of an Aβ β-hairpin into a trimer, dodecamer, and annular pore based on the crystallographic assembly of peptide 2. FIG +45 54 β-hairpin structure_element Model for the hierarchical assembly of an Aβ β-hairpin into a trimer, dodecamer, and annular pore based on the crystallographic assembly of peptide 2. FIG +62 68 trimer oligomeric_state Model for the hierarchical assembly of an Aβ β-hairpin into a trimer, dodecamer, and annular pore based on the crystallographic assembly of peptide 2. FIG +70 79 dodecamer oligomeric_state Model for the hierarchical assembly of an Aβ β-hairpin into a trimer, dodecamer, and annular pore based on the crystallographic assembly of peptide 2. FIG +85 97 annular pore site Model for the hierarchical assembly of an Aβ β-hairpin into a trimer, dodecamer, and annular pore based on the crystallographic assembly of peptide 2. FIG +140 149 peptide 2 mutant Model for the hierarchical assembly of an Aβ β-hairpin into a trimer, dodecamer, and annular pore based on the crystallographic assembly of peptide 2. FIG +0 9 Monomeric oligomeric_state Monomeric Aβ folds to form a β-hairpin in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. FIG +10 12 Aβ protein Monomeric Aβ folds to form a β-hairpin in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. FIG +29 38 β-hairpin structure_element Monomeric Aβ folds to form a β-hairpin in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. FIG +64 71 central structure_element Monomeric Aβ folds to form a β-hairpin in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. FIG +76 94 C-terminal regions structure_element Monomeric Aβ folds to form a β-hairpin in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. FIG +103 123 antiparallel β-sheet structure_element Monomeric Aβ folds to form a β-hairpin in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. FIG +6 15 β-hairpin structure_element Three β-hairpin monomers assemble to form a triangular trimer. FIG +16 24 monomers oligomeric_state Three β-hairpin monomers assemble to form a triangular trimer. FIG +44 54 triangular protein_state Three β-hairpin monomers assemble to form a triangular trimer. FIG +55 61 trimer oligomeric_state Three β-hairpin monomers assemble to form a triangular trimer. FIG +5 15 triangular protein_state Four triangular trimers assemble to form a dodecamer. FIG +16 23 trimers oligomeric_state Four triangular trimers assemble to form a dodecamer. FIG +43 52 dodecamer oligomeric_state Four triangular trimers assemble to form a dodecamer. FIG +5 15 dodecamers oligomeric_state Five dodecamers assemble to form an annular pore. FIG +36 48 annular pore site Five dodecamers assemble to form an annular pore. FIG +45 49 Aβ42 protein The molecular weights shown correspond to an Aβ42 monomer (∼4.5 kDa), an Aβ42 trimer (∼13.5 kDa), an Aβ42 dodecamer (∼54 kDa), and an Aβ42 annular pore composed of five dodecamers (∼270 kDa). FIG +50 57 monomer oligomeric_state The molecular weights shown correspond to an Aβ42 monomer (∼4.5 kDa), an Aβ42 trimer (∼13.5 kDa), an Aβ42 dodecamer (∼54 kDa), and an Aβ42 annular pore composed of five dodecamers (∼270 kDa). FIG +73 77 Aβ42 protein The molecular weights shown correspond to an Aβ42 monomer (∼4.5 kDa), an Aβ42 trimer (∼13.5 kDa), an Aβ42 dodecamer (∼54 kDa), and an Aβ42 annular pore composed of five dodecamers (∼270 kDa). FIG +78 84 trimer oligomeric_state The molecular weights shown correspond to an Aβ42 monomer (∼4.5 kDa), an Aβ42 trimer (∼13.5 kDa), an Aβ42 dodecamer (∼54 kDa), and an Aβ42 annular pore composed of five dodecamers (∼270 kDa). FIG +101 105 Aβ42 protein The molecular weights shown correspond to an Aβ42 monomer (∼4.5 kDa), an Aβ42 trimer (∼13.5 kDa), an Aβ42 dodecamer (∼54 kDa), and an Aβ42 annular pore composed of five dodecamers (∼270 kDa). FIG +106 115 dodecamer oligomeric_state The molecular weights shown correspond to an Aβ42 monomer (∼4.5 kDa), an Aβ42 trimer (∼13.5 kDa), an Aβ42 dodecamer (∼54 kDa), and an Aβ42 annular pore composed of five dodecamers (∼270 kDa). FIG +134 138 Aβ42 protein The molecular weights shown correspond to an Aβ42 monomer (∼4.5 kDa), an Aβ42 trimer (∼13.5 kDa), an Aβ42 dodecamer (∼54 kDa), and an Aβ42 annular pore composed of five dodecamers (∼270 kDa). FIG +139 151 annular pore site The molecular weights shown correspond to an Aβ42 monomer (∼4.5 kDa), an Aβ42 trimer (∼13.5 kDa), an Aβ42 dodecamer (∼54 kDa), and an Aβ42 annular pore composed of five dodecamers (∼270 kDa). FIG +169 179 dodecamers oligomeric_state The molecular weights shown correspond to an Aβ42 monomer (∼4.5 kDa), an Aβ42 trimer (∼13.5 kDa), an Aβ42 dodecamer (∼54 kDa), and an Aβ42 annular pore composed of five dodecamers (∼270 kDa). FIG +91 93 Aβ protein The model put forth in Figure 6 is consistent with the current understanding of endogenous Aβ oligomerization and explains at atomic resolution many key observations about Aβ oligomers. DISCUSS +172 174 Aβ protein The model put forth in Figure 6 is consistent with the current understanding of endogenous Aβ oligomerization and explains at atomic resolution many key observations about Aβ oligomers. DISCUSS +175 184 oligomers oligomeric_state The model put forth in Figure 6 is consistent with the current understanding of endogenous Aβ oligomerization and explains at atomic resolution many key observations about Aβ oligomers. DISCUSS +32 34 Aβ protein Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +35 44 oligomers oligomeric_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +65 67 Aβ protein Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +68 77 oligomers oligomeric_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +105 112 fibrils oligomeric_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +118 127 fibrillar protein_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +128 137 oligomers oligomeric_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +144 146 Aβ protein Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +147 156 oligomers oligomeric_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +170 179 fibrillar protein_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +190 202 nonfibrillar protein_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +203 212 oligomers oligomeric_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +216 225 Fibrillar protein_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +226 235 oligomers oligomeric_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +281 293 nonfibrillar protein_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +294 303 oligomers oligomeric_state Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. DISCUSS +0 12 Nonfibrillar protein_state Nonfibrillar oligomers accumulate early in Alzheimer’s disease before plaque deposition. DISCUSS +13 22 oligomers oligomeric_state Nonfibrillar oligomers accumulate early in Alzheimer’s disease before plaque deposition. DISCUSS +0 9 Fibrillar protein_state Fibrillar and nonfibrillar oligomers have structurally distinct characteristics, which are reflected in their reactivity with the fibril-specific OC antibody and the oligomer-specific A11 antibody. DISCUSS +14 26 nonfibrillar protein_state Fibrillar and nonfibrillar oligomers have structurally distinct characteristics, which are reflected in their reactivity with the fibril-specific OC antibody and the oligomer-specific A11 antibody. DISCUSS +27 36 oligomers oligomeric_state Fibrillar and nonfibrillar oligomers have structurally distinct characteristics, which are reflected in their reactivity with the fibril-specific OC antibody and the oligomer-specific A11 antibody. DISCUSS +166 174 oligomer oligomeric_state Fibrillar and nonfibrillar oligomers have structurally distinct characteristics, which are reflected in their reactivity with the fibril-specific OC antibody and the oligomer-specific A11 antibody. DISCUSS +0 9 Fibrillar protein_state Fibrillar oligomers are recognized by the OC antibody but not the A11 antibody, whereas nonfibrillar oligomers are recognized by the A11 antibody but not the OC antibody. DISCUSS +10 19 oligomers oligomeric_state Fibrillar oligomers are recognized by the OC antibody but not the A11 antibody, whereas nonfibrillar oligomers are recognized by the A11 antibody but not the OC antibody. DISCUSS +88 100 nonfibrillar protein_state Fibrillar oligomers are recognized by the OC antibody but not the A11 antibody, whereas nonfibrillar oligomers are recognized by the A11 antibody but not the OC antibody. DISCUSS +101 110 oligomers oligomeric_state Fibrillar oligomers are recognized by the OC antibody but not the A11 antibody, whereas nonfibrillar oligomers are recognized by the A11 antibody but not the OC antibody. DISCUSS +46 48 Aβ protein These criteria have been used to classify the Aβ oligomers that accumulate in vivo. DISCUSS +49 58 oligomers oligomeric_state These criteria have been used to classify the Aβ oligomers that accumulate in vivo. DISCUSS +0 2 Aβ protein Aβ dimers have been classified as fibrillar oligomers, whereas Aβ trimers, Aβ*56, and APFs have been classified as nonfibrillar oligomers. DISCUSS +3 9 dimers oligomeric_state Aβ dimers have been classified as fibrillar oligomers, whereas Aβ trimers, Aβ*56, and APFs have been classified as nonfibrillar oligomers. DISCUSS +34 43 fibrillar protein_state Aβ dimers have been classified as fibrillar oligomers, whereas Aβ trimers, Aβ*56, and APFs have been classified as nonfibrillar oligomers. DISCUSS +44 53 oligomers oligomeric_state Aβ dimers have been classified as fibrillar oligomers, whereas Aβ trimers, Aβ*56, and APFs have been classified as nonfibrillar oligomers. DISCUSS +63 65 Aβ protein Aβ dimers have been classified as fibrillar oligomers, whereas Aβ trimers, Aβ*56, and APFs have been classified as nonfibrillar oligomers. DISCUSS +66 73 trimers oligomeric_state Aβ dimers have been classified as fibrillar oligomers, whereas Aβ trimers, Aβ*56, and APFs have been classified as nonfibrillar oligomers. DISCUSS +75 80 Aβ*56 complex_assembly Aβ dimers have been classified as fibrillar oligomers, whereas Aβ trimers, Aβ*56, and APFs have been classified as nonfibrillar oligomers. DISCUSS +86 90 APFs complex_assembly Aβ dimers have been classified as fibrillar oligomers, whereas Aβ trimers, Aβ*56, and APFs have been classified as nonfibrillar oligomers. DISCUSS +115 127 nonfibrillar protein_state Aβ dimers have been classified as fibrillar oligomers, whereas Aβ trimers, Aβ*56, and APFs have been classified as nonfibrillar oligomers. DISCUSS +128 137 oligomers oligomeric_state Aβ dimers have been classified as fibrillar oligomers, whereas Aβ trimers, Aβ*56, and APFs have been classified as nonfibrillar oligomers. DISCUSS +67 79 nonfibrillar protein_state Larson and Lesné proposed a model for the endogenous production of nonfibrillar oligomers that explains these observations. DISCUSS +80 89 oligomers oligomeric_state Larson and Lesné proposed a model for the endogenous production of nonfibrillar oligomers that explains these observations. DISCUSS +15 21 folded protein_state In this model, folded Aβ monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils. DISCUSS +22 24 Aβ protein In this model, folded Aβ monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils. DISCUSS +25 32 monomer oligomeric_state In this model, folded Aβ monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils. DISCUSS +50 56 trimer oligomeric_state In this model, folded Aβ monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils. DISCUSS +62 68 trimer oligomeric_state In this model, folded Aβ monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils. DISCUSS +92 100 hexamers oligomeric_state In this model, folded Aβ monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils. DISCUSS +105 115 dodecamers oligomeric_state In this model, folded Aβ monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils. DISCUSS +125 135 dodecamers oligomeric_state In this model, folded Aβ monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils. DISCUSS +161 181 annular protofibrils complex_assembly In this model, folded Aβ monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils. DISCUSS +29 38 peptide 2 mutant The hierarchical assembly of peptide 2 is consistent with this model; and the trimer, dodecamer, and annular pore formed by peptide 2 may share similarities to the trimers, Aβ*56, and APFs observed in vivo. DISCUSS +78 84 trimer oligomeric_state The hierarchical assembly of peptide 2 is consistent with this model; and the trimer, dodecamer, and annular pore formed by peptide 2 may share similarities to the trimers, Aβ*56, and APFs observed in vivo. DISCUSS +86 95 dodecamer oligomeric_state The hierarchical assembly of peptide 2 is consistent with this model; and the trimer, dodecamer, and annular pore formed by peptide 2 may share similarities to the trimers, Aβ*56, and APFs observed in vivo. DISCUSS +101 113 annular pore site The hierarchical assembly of peptide 2 is consistent with this model; and the trimer, dodecamer, and annular pore formed by peptide 2 may share similarities to the trimers, Aβ*56, and APFs observed in vivo. DISCUSS +124 133 peptide 2 mutant The hierarchical assembly of peptide 2 is consistent with this model; and the trimer, dodecamer, and annular pore formed by peptide 2 may share similarities to the trimers, Aβ*56, and APFs observed in vivo. DISCUSS +164 171 trimers oligomeric_state The hierarchical assembly of peptide 2 is consistent with this model; and the trimer, dodecamer, and annular pore formed by peptide 2 may share similarities to the trimers, Aβ*56, and APFs observed in vivo. DISCUSS +173 178 Aβ*56 complex_assembly The hierarchical assembly of peptide 2 is consistent with this model; and the trimer, dodecamer, and annular pore formed by peptide 2 may share similarities to the trimers, Aβ*56, and APFs observed in vivo. DISCUSS +184 188 APFs complex_assembly The hierarchical assembly of peptide 2 is consistent with this model; and the trimer, dodecamer, and annular pore formed by peptide 2 may share similarities to the trimers, Aβ*56, and APFs observed in vivo. DISCUSS +49 55 trimer oligomeric_state At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to Aβ trimers and Aβ*56, as little is known about the structure of Aβ trimers and Aβ*56. DISCUSS +60 69 dodecamer oligomeric_state At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to Aβ trimers and Aβ*56, as little is known about the structure of Aβ trimers and Aβ*56. DISCUSS +80 89 peptide 2 mutant At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to Aβ trimers and Aβ*56, as little is known about the structure of Aβ trimers and Aβ*56. DISCUSS +123 125 Aβ protein At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to Aβ trimers and Aβ*56, as little is known about the structure of Aβ trimers and Aβ*56. DISCUSS +126 133 trimers oligomeric_state At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to Aβ trimers and Aβ*56, as little is known about the structure of Aβ trimers and Aβ*56. DISCUSS +138 143 Aβ*56 complex_assembly At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to Aβ trimers and Aβ*56, as little is known about the structure of Aβ trimers and Aβ*56. DISCUSS +174 183 structure evidence At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to Aβ trimers and Aβ*56, as little is known about the structure of Aβ trimers and Aβ*56. DISCUSS +187 189 Aβ protein At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to Aβ trimers and Aβ*56, as little is known about the structure of Aβ trimers and Aβ*56. DISCUSS +190 197 trimers oligomeric_state At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to Aβ trimers and Aβ*56, as little is known about the structure of Aβ trimers and Aβ*56. DISCUSS +202 207 Aβ*56 complex_assembly At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to Aβ trimers and Aβ*56, as little is known about the structure of Aβ trimers and Aβ*56. DISCUSS +4 33 crystallographically observed evidence The crystallographically observed annular pore formed by peptide 2 is morphologically similar to the APFs formed by full-length Aβ. DISCUSS +34 46 annular pore site The crystallographically observed annular pore formed by peptide 2 is morphologically similar to the APFs formed by full-length Aβ. DISCUSS +57 66 peptide 2 mutant The crystallographically observed annular pore formed by peptide 2 is morphologically similar to the APFs formed by full-length Aβ. DISCUSS +101 105 APFs complex_assembly The crystallographically observed annular pore formed by peptide 2 is morphologically similar to the APFs formed by full-length Aβ. DISCUSS +116 127 full-length protein_state The crystallographically observed annular pore formed by peptide 2 is morphologically similar to the APFs formed by full-length Aβ. DISCUSS +128 130 Aβ protein The crystallographically observed annular pore formed by peptide 2 is morphologically similar to the APFs formed by full-length Aβ. DISCUSS +4 16 annular pore site The annular pore formed by peptide 2 is comparable in size to the APFs prepared in vitro or isolated from Alzheimer’s brains (Figure 7 and Table 1). DISCUSS +27 36 peptide 2 mutant The annular pore formed by peptide 2 is comparable in size to the APFs prepared in vitro or isolated from Alzheimer’s brains (Figure 7 and Table 1). DISCUSS +66 70 APFs complex_assembly The annular pore formed by peptide 2 is comparable in size to the APFs prepared in vitro or isolated from Alzheimer’s brains (Figure 7 and Table 1). DISCUSS +21 25 APFs complex_assembly The varying sizes of APFs formed by full-length Aβ might result from differences in the number of oligomer subunits comprising each APF. DISCUSS +36 47 full-length protein_state The varying sizes of APFs formed by full-length Aβ might result from differences in the number of oligomer subunits comprising each APF. DISCUSS +48 50 Aβ protein The varying sizes of APFs formed by full-length Aβ might result from differences in the number of oligomer subunits comprising each APF. DISCUSS +98 106 oligomer oligomeric_state The varying sizes of APFs formed by full-length Aβ might result from differences in the number of oligomer subunits comprising each APF. DISCUSS +107 115 subunits structure_element The varying sizes of APFs formed by full-length Aβ might result from differences in the number of oligomer subunits comprising each APF. DISCUSS +132 135 APF complex_assembly The varying sizes of APFs formed by full-length Aβ might result from differences in the number of oligomer subunits comprising each APF. DISCUSS +13 25 annular pore site Although the annular pore formed by peptide 2 contains five dodecamer subunits, pores containing fewer or more subunits can easily be envisioned. DISCUSS +36 45 peptide 2 mutant Although the annular pore formed by peptide 2 contains five dodecamer subunits, pores containing fewer or more subunits can easily be envisioned. DISCUSS +60 69 dodecamer oligomeric_state Although the annular pore formed by peptide 2 contains five dodecamer subunits, pores containing fewer or more subunits can easily be envisioned. DISCUSS +70 78 subunits structure_element Although the annular pore formed by peptide 2 contains five dodecamer subunits, pores containing fewer or more subunits can easily be envisioned. DISCUSS +80 85 pores site Although the annular pore formed by peptide 2 contains five dodecamer subunits, pores containing fewer or more subunits can easily be envisioned. DISCUSS +111 119 subunits structure_element Although the annular pore formed by peptide 2 contains five dodecamer subunits, pores containing fewer or more subunits can easily be envisioned. DISCUSS +4 14 dodecamers oligomeric_state The dodecamers that comprise the annular pore exhibit two modes of assembly—eclipsed interactions and staggered interactions between the F20 faces of trimers within dodecamers. DISCUSS +33 45 annular pore site The dodecamers that comprise the annular pore exhibit two modes of assembly—eclipsed interactions and staggered interactions between the F20 faces of trimers within dodecamers. DISCUSS +76 84 eclipsed protein_state The dodecamers that comprise the annular pore exhibit two modes of assembly—eclipsed interactions and staggered interactions between the F20 faces of trimers within dodecamers. DISCUSS +102 111 staggered protein_state The dodecamers that comprise the annular pore exhibit two modes of assembly—eclipsed interactions and staggered interactions between the F20 faces of trimers within dodecamers. DISCUSS +137 140 F20 residue_name_number The dodecamers that comprise the annular pore exhibit two modes of assembly—eclipsed interactions and staggered interactions between the F20 faces of trimers within dodecamers. DISCUSS +150 157 trimers oligomeric_state The dodecamers that comprise the annular pore exhibit two modes of assembly—eclipsed interactions and staggered interactions between the F20 faces of trimers within dodecamers. DISCUSS +165 175 dodecamers oligomeric_state The dodecamers that comprise the annular pore exhibit two modes of assembly—eclipsed interactions and staggered interactions between the F20 faces of trimers within dodecamers. DISCUSS +72 82 dodecamers oligomeric_state These two modes of assembly might reflect a dynamic interaction between dodecamers, which could permit assemblies of more dodecamers into larger annular pores. DISCUSS +122 132 dodecamers oligomeric_state These two modes of assembly might reflect a dynamic interaction between dodecamers, which could permit assemblies of more dodecamers into larger annular pores. DISCUSS +145 158 annular pores site These two modes of assembly might reflect a dynamic interaction between dodecamers, which could permit assemblies of more dodecamers into larger annular pores. DISCUSS +21 33 annular pore site Surface views of the annular pore formed by peptide 2. (A) Top view. FIG +44 53 peptide 2 mutant Surface views of the annular pore formed by peptide 2. (A) Top view. FIG +0 13 Annular Pores site Annular Pores Formed by Aβ and Peptide 2 TABLE +24 26 Aβ protein Annular Pores Formed by Aβ and Peptide 2 TABLE +31 40 Peptide 2 mutant Annular Pores Formed by Aβ and Peptide 2 TABLE +0 12 annular pore site "annular pore source outer diameter inner diameter observation method peptide 2 ∼11–12 nm ∼2 nm X-ray crystallography synthetic Aβ 7–10 nm 1.5–2 nm TEM synthetic Aβ 16 nm not reported AFM synthetic Aβ 8–25 nm not reported TEM Alzheimer’s brain 11–14 nm 2.5–4 nm TEM " TABLE +75 82 peptide chemical "annular pore source outer diameter inner diameter observation method peptide 2 ∼11–12 nm ∼2 nm X-ray crystallography synthetic Aβ 7–10 nm 1.5–2 nm TEM synthetic Aβ 16 nm not reported AFM synthetic Aβ 8–25 nm not reported TEM Alzheimer’s brain 11–14 nm 2.5–4 nm TEM " TABLE +102 123 X-ray crystallography experimental_method "annular pore source outer diameter inner diameter observation method peptide 2 ∼11–12 nm ∼2 nm X-ray crystallography synthetic Aβ 7–10 nm 1.5–2 nm TEM synthetic Aβ 16 nm not reported AFM synthetic Aβ 8–25 nm not reported TEM Alzheimer’s brain 11–14 nm 2.5–4 nm TEM " TABLE +126 135 synthetic protein_state "annular pore source outer diameter inner diameter observation method peptide 2 ∼11–12 nm ∼2 nm X-ray crystallography synthetic Aβ 7–10 nm 1.5–2 nm TEM synthetic Aβ 16 nm not reported AFM synthetic Aβ 8–25 nm not reported TEM Alzheimer’s brain 11–14 nm 2.5–4 nm TEM " TABLE +136 138 Aβ protein "annular pore source outer diameter inner diameter observation method peptide 2 ∼11–12 nm ∼2 nm X-ray crystallography synthetic Aβ 7–10 nm 1.5–2 nm TEM synthetic Aβ 16 nm not reported AFM synthetic Aβ 8–25 nm not reported TEM Alzheimer’s brain 11–14 nm 2.5–4 nm TEM " TABLE +156 159 TEM experimental_method "annular pore source outer diameter inner diameter observation method peptide 2 ∼11–12 nm ∼2 nm X-ray crystallography synthetic Aβ 7–10 nm 1.5–2 nm TEM synthetic Aβ 16 nm not reported AFM synthetic Aβ 8–25 nm not reported TEM Alzheimer’s brain 11–14 nm 2.5–4 nm TEM " TABLE +172 174 Aβ protein "annular pore source outer diameter inner diameter observation method peptide 2 ∼11–12 nm ∼2 nm X-ray crystallography synthetic Aβ 7–10 nm 1.5–2 nm TEM synthetic Aβ 16 nm not reported AFM synthetic Aβ 8–25 nm not reported TEM Alzheimer’s brain 11–14 nm 2.5–4 nm TEM " TABLE +194 197 AFM experimental_method "annular pore source outer diameter inner diameter observation method peptide 2 ∼11–12 nm ∼2 nm X-ray crystallography synthetic Aβ 7–10 nm 1.5–2 nm TEM synthetic Aβ 16 nm not reported AFM synthetic Aβ 8–25 nm not reported TEM Alzheimer’s brain 11–14 nm 2.5–4 nm TEM " TABLE +210 212 Aβ protein "annular pore source outer diameter inner diameter observation method peptide 2 ∼11–12 nm ∼2 nm X-ray crystallography synthetic Aβ 7–10 nm 1.5–2 nm TEM synthetic Aβ 16 nm not reported AFM synthetic Aβ 8–25 nm not reported TEM Alzheimer’s brain 11–14 nm 2.5–4 nm TEM " TABLE +234 237 TEM experimental_method "annular pore source outer diameter inner diameter observation method peptide 2 ∼11–12 nm ∼2 nm X-ray crystallography synthetic Aβ 7–10 nm 1.5–2 nm TEM synthetic Aβ 16 nm not reported AFM synthetic Aβ 8–25 nm not reported TEM Alzheimer’s brain 11–14 nm 2.5–4 nm TEM " TABLE +276 279 TEM experimental_method "annular pore source outer diameter inner diameter observation method peptide 2 ∼11–12 nm ∼2 nm X-ray crystallography synthetic Aβ 7–10 nm 1.5–2 nm TEM synthetic Aβ 16 nm not reported AFM synthetic Aβ 8–25 nm not reported TEM Alzheimer’s brain 11–14 nm 2.5–4 nm TEM " TABLE +0 8 Dot blot experimental_method Dot blot analysis shows that peptide 2 is reactive toward the A11 antibody (Figure S5). DISCUSS +29 38 peptide 2 mutant Dot blot analysis shows that peptide 2 is reactive toward the A11 antibody (Figure S5). DISCUSS +30 39 peptide 2 mutant This reactivity suggests that peptide 2 forms oligomers in solution that share structural similarities to the nonfibrillar oligomers formed by full-length Aβ. DISCUSS +46 55 oligomers oligomeric_state This reactivity suggests that peptide 2 forms oligomers in solution that share structural similarities to the nonfibrillar oligomers formed by full-length Aβ. DISCUSS +110 122 nonfibrillar protein_state This reactivity suggests that peptide 2 forms oligomers in solution that share structural similarities to the nonfibrillar oligomers formed by full-length Aβ. DISCUSS +123 132 oligomers oligomeric_state This reactivity suggests that peptide 2 forms oligomers in solution that share structural similarities to the nonfibrillar oligomers formed by full-length Aβ. DISCUSS +143 154 full-length protein_state This reactivity suggests that peptide 2 forms oligomers in solution that share structural similarities to the nonfibrillar oligomers formed by full-length Aβ. DISCUSS +155 157 Aβ protein This reactivity suggests that peptide 2 forms oligomers in solution that share structural similarities to the nonfibrillar oligomers formed by full-length Aβ. DISCUSS +57 66 peptide 2 mutant Further studies are needed to elucidate the species that peptide 2 forms in solution and to study their biological properties. DISCUSS +47 50 SEC experimental_method Preliminary attempts to study these species by SEC and SDS-PAGE have not provided a clear measure of the structures formed in solution. DISCUSS +55 63 SDS-PAGE experimental_method Preliminary attempts to study these species by SEC and SDS-PAGE have not provided a clear measure of the structures formed in solution. DISCUSS +105 115 structures evidence Preliminary attempts to study these species by SEC and SDS-PAGE have not provided a clear measure of the structures formed in solution. DISCUSS +31 40 oligomers oligomeric_state The difficulty in studying the oligomers formed in solution may reflect the propensity of the dodecamer to assemble on all four F20 faces. DISCUSS +94 103 dodecamer oligomeric_state The difficulty in studying the oligomers formed in solution may reflect the propensity of the dodecamer to assemble on all four F20 faces. DISCUSS +128 131 F20 residue_name_number The difficulty in studying the oligomers formed in solution may reflect the propensity of the dodecamer to assemble on all four F20 faces. DISCUSS +4 36 X-ray crystallographic structure evidence The X-ray crystallographic structure and A11 reactivity of peptide 2 support the model proposed by Larsen and Lesné and suggest that β-hairpins constitute a fundamental building block for nonfibrillar oligomers. DISCUSS +59 68 peptide 2 mutant The X-ray crystallographic structure and A11 reactivity of peptide 2 support the model proposed by Larsen and Lesné and suggest that β-hairpins constitute a fundamental building block for nonfibrillar oligomers. DISCUSS +133 143 β-hairpins structure_element The X-ray crystallographic structure and A11 reactivity of peptide 2 support the model proposed by Larsen and Lesné and suggest that β-hairpins constitute a fundamental building block for nonfibrillar oligomers. DISCUSS +188 200 nonfibrillar protein_state The X-ray crystallographic structure and A11 reactivity of peptide 2 support the model proposed by Larsen and Lesné and suggest that β-hairpins constitute a fundamental building block for nonfibrillar oligomers. DISCUSS +201 210 oligomers oligomeric_state The X-ray crystallographic structure and A11 reactivity of peptide 2 support the model proposed by Larsen and Lesné and suggest that β-hairpins constitute a fundamental building block for nonfibrillar oligomers. DISCUSS +11 21 β-hairpins structure_element What makes β-hairpins special is that three β-hairpins can nestle together to form trimers, stabilized by a network of hydrogen bonds and hydrophobic interactions. DISCUSS +44 54 β-hairpins structure_element What makes β-hairpins special is that three β-hairpins can nestle together to form trimers, stabilized by a network of hydrogen bonds and hydrophobic interactions. DISCUSS +83 90 trimers oligomeric_state What makes β-hairpins special is that three β-hairpins can nestle together to form trimers, stabilized by a network of hydrogen bonds and hydrophobic interactions. DISCUSS +119 133 hydrogen bonds bond_interaction What makes β-hairpins special is that three β-hairpins can nestle together to form trimers, stabilized by a network of hydrogen bonds and hydrophobic interactions. DISCUSS +138 162 hydrophobic interactions bond_interaction What makes β-hairpins special is that three β-hairpins can nestle together to form trimers, stabilized by a network of hydrogen bonds and hydrophobic interactions. DISCUSS +39 41 Aβ protein This mode of assembly is not unique to Aβ. DISCUSS +4 17 foldon domain structure_element The foldon domain of bacteriophage T4 fibritin is composed of three β-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2. DISCUSS +21 37 bacteriophage T4 species The foldon domain of bacteriophage T4 fibritin is composed of three β-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2. DISCUSS +38 46 fibritin protein The foldon domain of bacteriophage T4 fibritin is composed of three β-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2. DISCUSS +68 78 β-hairpins structure_element The foldon domain of bacteriophage T4 fibritin is composed of three β-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2. DISCUSS +100 110 triangular protein_state The foldon domain of bacteriophage T4 fibritin is composed of three β-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2. DISCUSS +111 117 trimer oligomeric_state The foldon domain of bacteriophage T4 fibritin is composed of three β-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2. DISCUSS +133 143 triangular protein_state The foldon domain of bacteriophage T4 fibritin is composed of three β-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2. DISCUSS +144 150 trimer oligomeric_state The foldon domain of bacteriophage T4 fibritin is composed of three β-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2. DISCUSS +161 170 peptide 2 mutant The foldon domain of bacteriophage T4 fibritin is composed of three β-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2. DISCUSS +70 79 β-hairpin structure_element Additionally, our research group has observed a similar assembly of a β-hairpin peptide derived from β2-microglobulin. DISCUSS +101 117 β2-microglobulin protein Additionally, our research group has observed a similar assembly of a β-hairpin peptide derived from β2-microglobulin. DISCUSS +77 84 trimers oligomeric_state Although we began these studies with a relatively simple hypothesis—that the trimers and dodecamers formed by peptide 1 could accommodate the Aβ24–29 loop—an even more exciting finding has emerged—that the dodecamers can assemble to form annular pores. CONCL +89 99 dodecamers oligomeric_state Although we began these studies with a relatively simple hypothesis—that the trimers and dodecamers formed by peptide 1 could accommodate the Aβ24–29 loop—an even more exciting finding has emerged—that the dodecamers can assemble to form annular pores. CONCL +110 119 peptide 1 mutant Although we began these studies with a relatively simple hypothesis—that the trimers and dodecamers formed by peptide 1 could accommodate the Aβ24–29 loop—an even more exciting finding has emerged—that the dodecamers can assemble to form annular pores. CONCL +142 144 Aβ protein Although we began these studies with a relatively simple hypothesis—that the trimers and dodecamers formed by peptide 1 could accommodate the Aβ24–29 loop—an even more exciting finding has emerged—that the dodecamers can assemble to form annular pores. CONCL +144 149 24–29 residue_range Although we began these studies with a relatively simple hypothesis—that the trimers and dodecamers formed by peptide 1 could accommodate the Aβ24–29 loop—an even more exciting finding has emerged—that the dodecamers can assemble to form annular pores. CONCL +150 154 loop structure_element Although we began these studies with a relatively simple hypothesis—that the trimers and dodecamers formed by peptide 1 could accommodate the Aβ24–29 loop—an even more exciting finding has emerged—that the dodecamers can assemble to form annular pores. CONCL +206 216 dodecamers oligomeric_state Although we began these studies with a relatively simple hypothesis—that the trimers and dodecamers formed by peptide 1 could accommodate the Aβ24–29 loop—an even more exciting finding has emerged—that the dodecamers can assemble to form annular pores. CONCL +238 251 annular pores site Although we began these studies with a relatively simple hypothesis—that the trimers and dodecamers formed by peptide 1 could accommodate the Aβ24–29 loop—an even more exciting finding has emerged—that the dodecamers can assemble to form annular pores. CONCL +54 86 X-ray crystallographic structure evidence This finding could not have been anticipated from the X-ray crystallographic structure of peptide 1 and reveals a new level of hierarchical assembly that recapitulates micrographic observations of annular protofibrils. CONCL +90 99 peptide 1 mutant This finding could not have been anticipated from the X-ray crystallographic structure of peptide 1 and reveals a new level of hierarchical assembly that recapitulates micrographic observations of annular protofibrils. CONCL +197 217 annular protofibrils complex_assembly This finding could not have been anticipated from the X-ray crystallographic structure of peptide 1 and reveals a new level of hierarchical assembly that recapitulates micrographic observations of annular protofibrils. CONCL +4 33 crystallographically observed evidence The crystallographically observed dodecamer, in turn, recapitulates the observation of Aβ*56, which appears to be a dodecamer of Aβ. CONCL +34 43 dodecamer oligomeric_state The crystallographically observed dodecamer, in turn, recapitulates the observation of Aβ*56, which appears to be a dodecamer of Aβ. CONCL +87 92 Aβ*56 complex_assembly The crystallographically observed dodecamer, in turn, recapitulates the observation of Aβ*56, which appears to be a dodecamer of Aβ. CONCL +116 125 dodecamer oligomeric_state The crystallographically observed dodecamer, in turn, recapitulates the observation of Aβ*56, which appears to be a dodecamer of Aβ. CONCL +129 131 Aβ protein The crystallographically observed dodecamer, in turn, recapitulates the observation of Aβ*56, which appears to be a dodecamer of Aβ. CONCL +4 33 crystallographically observed evidence The crystallographically observed trimer recapitulates the Aβ trimers that are observed even before the onset of symptoms in Alzheimer’s disease. CONCL +34 40 trimer oligomeric_state The crystallographically observed trimer recapitulates the Aβ trimers that are observed even before the onset of symptoms in Alzheimer’s disease. CONCL +59 61 Aβ protein The crystallographically observed trimer recapitulates the Aβ trimers that are observed even before the onset of symptoms in Alzheimer’s disease. CONCL +62 69 trimers oligomeric_state The crystallographically observed trimer recapitulates the Aβ trimers that are observed even before the onset of symptoms in Alzheimer’s disease. CONCL +29 31 Aβ protein Our approach of constraining Aβ17–36 into a β-hairpin conformation and blocking aggregation with an N-methyl group has allowed us to crystallize a large fragment of what is generally considered to be an uncrystallizable peptide. CONCL +31 36 17–36 residue_range Our approach of constraining Aβ17–36 into a β-hairpin conformation and blocking aggregation with an N-methyl group has allowed us to crystallize a large fragment of what is generally considered to be an uncrystallizable peptide. CONCL +44 53 β-hairpin structure_element Our approach of constraining Aβ17–36 into a β-hairpin conformation and blocking aggregation with an N-methyl group has allowed us to crystallize a large fragment of what is generally considered to be an uncrystallizable peptide. CONCL +133 144 crystallize experimental_method Our approach of constraining Aβ17–36 into a β-hairpin conformation and blocking aggregation with an N-methyl group has allowed us to crystallize a large fragment of what is generally considered to be an uncrystallizable peptide. CONCL +100 111 crystallize experimental_method We believe this iterative, “bottom up” approach of identifying the minimal modification required to crystallize Aβ peptides will ultimately allow larger fragments of Aβ to be crystallized, thus providing greater insights into the structures of Aβ oligomers. CONCL +112 114 Aβ protein We believe this iterative, “bottom up” approach of identifying the minimal modification required to crystallize Aβ peptides will ultimately allow larger fragments of Aβ to be crystallized, thus providing greater insights into the structures of Aβ oligomers. CONCL +166 168 Aβ protein We believe this iterative, “bottom up” approach of identifying the minimal modification required to crystallize Aβ peptides will ultimately allow larger fragments of Aβ to be crystallized, thus providing greater insights into the structures of Aβ oligomers. CONCL +175 187 crystallized experimental_method We believe this iterative, “bottom up” approach of identifying the minimal modification required to crystallize Aβ peptides will ultimately allow larger fragments of Aβ to be crystallized, thus providing greater insights into the structures of Aβ oligomers. CONCL +230 240 structures evidence We believe this iterative, “bottom up” approach of identifying the minimal modification required to crystallize Aβ peptides will ultimately allow larger fragments of Aβ to be crystallized, thus providing greater insights into the structures of Aβ oligomers. CONCL +244 246 Aβ protein We believe this iterative, “bottom up” approach of identifying the minimal modification required to crystallize Aβ peptides will ultimately allow larger fragments of Aβ to be crystallized, thus providing greater insights into the structures of Aβ oligomers. CONCL +247 256 oligomers oligomeric_state We believe this iterative, “bottom up” approach of identifying the minimal modification required to crystallize Aβ peptides will ultimately allow larger fragments of Aβ to be crystallized, thus providing greater insights into the structures of Aβ oligomers. CONCL diff --git a/annotation_CSV/PMC4832331.csv b/annotation_CSV/PMC4832331.csv new file mode 100644 index 0000000000000000000000000000000000000000..d3da43b5ad9c1b9c520619fc6400702d6d6a0e50 --- /dev/null +++ b/annotation_CSV/PMC4832331.csv @@ -0,0 +1,719 @@ +anno_start anno_end anno_text entity_type sentence section +29 45 Escherichia coli species Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA TITLE +46 67 lysine decarboxylases protein_type Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA TITLE +119 130 AAA+ ATPase protein_type Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA TITLE +131 135 RavA protein Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA TITLE +4 13 inducible protein_state The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. ABSTRACT +14 34 lysine decarboxylase protein_type The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. ABSTRACT +35 39 LdcI protein The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. ABSTRACT +56 71 enterobacterial taxonomy_domain The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. ABSTRACT +72 99 acid stress response enzyme protein_type The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. ABSTRACT +108 112 LdcC protein The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. ABSTRACT +43 51 decamers oligomeric_state A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. ABSTRACT +59 75 Escherichia coli species A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. ABSTRACT +76 80 LdcI protein A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. ABSTRACT +90 98 hexamers oligomeric_state A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. ABSTRACT +106 117 AAA+ ATPase protein_type A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. ABSTRACT +118 122 RavA protein A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. ABSTRACT +26 44 pseudoatomic model evidence Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. ABSTRACT +52 61 LdcI-RavA complex_assembly Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. ABSTRACT +80 104 cryo-electron microscopy experimental_method Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. ABSTRACT +105 108 map evidence Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. ABSTRACT +113 131 crystal structures evidence Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. ABSTRACT +138 146 inactive protein_state Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. ABSTRACT +147 151 LdcI protein Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. ABSTRACT +152 159 decamer oligomeric_state Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. ABSTRACT +166 170 RavA protein Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. ABSTRACT +171 178 monomer oligomeric_state Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. ABSTRACT +15 39 cryo-electron microscopy experimental_method We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. ABSTRACT +40 58 3D reconstructions evidence We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. ABSTRACT +66 73 E. coli species We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. ABSTRACT +74 78 LdcI protein We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. ABSTRACT +83 87 LdcC protein We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. ABSTRACT +96 108 improved map evidence We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. ABSTRACT +116 120 LdcI protein We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. ABSTRACT +121 129 bound to protein_state We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. ABSTRACT +134 145 LARA domain structure_element We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. ABSTRACT +149 153 RavA protein We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. ABSTRACT +158 168 pH optimal protein_state We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. ABSTRACT +0 10 Comparison experimental_method Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. ABSTRACT +46 56 structures evidence Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. ABSTRACT +86 90 LdcI protein Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. ABSTRACT +95 99 LdcC protein Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. ABSTRACT +124 151 acid stress response enzyme protein_type Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. ABSTRACT +174 178 RavA protein Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. ABSTRACT +234 246 pH-dependent protein_state Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. ABSTRACT +278 282 RavA protein Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. ABSTRACT +0 27 Multiple sequence alignment experimental_method Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions. ABSTRACT +41 62 phylogenetic analysis experimental_method Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions. ABSTRACT +84 98 enterobacteria taxonomy_domain Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions. ABSTRACT +134 154 lysine decarboxylase protein_type Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions. ABSTRACT +191 195 RavA protein Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions. ABSTRACT +0 15 Enterobacterial taxonomy_domain Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. INTRO +16 25 inducible protein_state Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. INTRO +26 40 decarboxylases protein_type Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. INTRO +44 49 basic protein_state Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. INTRO +50 61 amino acids chemical Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. INTRO +62 68 lysine residue_name Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. INTRO +70 78 arginine residue_name Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. INTRO +83 92 ornithine residue_name Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. INTRO +145 153 α-family protein_type Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. INTRO +157 179 pyridoxal-5′-phosphate chemical Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. INTRO +181 184 PLP chemical Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. INTRO +47 56 bacterium taxonomy_domain They counteract acid stress experienced by the bacterium in the host digestive and urinary tract, and in particular in the extremely acidic stomach. INTRO +5 18 decarboxylase protein_type Each decarboxylase is induced by an excess of the target amino acid and a specific range of extracellular pH, and works in conjunction with a cognate inner membrane antiporter. INTRO +57 67 amino acid chemical Each decarboxylase is induced by an excess of the target amino acid and a specific range of extracellular pH, and works in conjunction with a cognate inner membrane antiporter. INTRO +150 175 inner membrane antiporter protein_type Each decarboxylase is induced by an excess of the target amino acid and a specific range of extracellular pH, and works in conjunction with a cognate inner membrane antiporter. INTRO +23 33 amino acid chemical Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. INTRO +41 50 polyamine chemical Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. INTRO +69 72 PLP chemical Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. INTRO +134 140 proton chemical Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. INTRO +156 159 CO2 chemical Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. INTRO +216 225 polyamine chemical Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. INTRO +245 255 antiporter protein_type Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. INTRO +278 288 amino acid chemical Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. INTRO +44 53 bacterial taxonomy_domain Consequently, these enzymes buffer both the bacterial cytoplasm and the local extracellular environment. INTRO +6 31 amino acid decarboxylases protein_type These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH. INTRO +65 74 inducible protein_state These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH. INTRO +78 92 biodegradative protein_state These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH. INTRO +124 136 biosynthetic protein_state These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH. INTRO +137 171 lysine and ornithine decarboxylase protein_type These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH. INTRO +234 243 polyamine chemical These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH. INTRO +258 268 neutral pH protein_state These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH. INTRO +0 9 Inducible protein_state Inducible enterobacterial amino acid decarboxylases have been intensively studied since the early 1940 because the ability of bacteria to withstand acid stress can be linked to their pathogenicity in humans. INTRO +10 25 enterobacterial taxonomy_domain Inducible enterobacterial amino acid decarboxylases have been intensively studied since the early 1940 because the ability of bacteria to withstand acid stress can be linked to their pathogenicity in humans. INTRO +26 51 amino acid decarboxylases protein_type Inducible enterobacterial amino acid decarboxylases have been intensively studied since the early 1940 because the ability of bacteria to withstand acid stress can be linked to their pathogenicity in humans. INTRO +126 134 bacteria taxonomy_domain Inducible enterobacterial amino acid decarboxylases have been intensively studied since the early 1940 because the ability of bacteria to withstand acid stress can be linked to their pathogenicity in humans. INTRO +200 206 humans species Inducible enterobacterial amino acid decarboxylases have been intensively studied since the early 1940 because the ability of bacteria to withstand acid stress can be linked to their pathogenicity in humans. INTRO +19 28 inducible protein_state In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. INTRO +29 49 lysine decarboxylase protein_type In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. INTRO +50 54 LdcI protein In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. INTRO +59 63 CadA protein In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. INTRO +95 109 broad pH range protein_state In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. INTRO +184 198 enterobacteria taxonomy_domain In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. INTRO +207 246 Salmonella enterica serovar Typhimurium species In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. INTRO +248 263 Vibrio cholerae species In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. INTRO +268 285 Vibrio vulnificus species In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. INTRO +18 22 LdcI protein Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI). INTRO +31 43 biosynthetic protein_state Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI). INTRO +44 64 lysine decarboxylase protein_type Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI). INTRO +65 69 LdcC protein Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI). INTRO +73 103 uropathogenic Escherichia coli species Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI). INTRO +105 109 UPEC species Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI). INTRO +219 231 nitric oxide chemical Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI). INTRO +29 39 cadaverine chemical This effect is attributed to cadaverine, the diamine produced by decarboxylation of lysine by LdcI and LdcC, that was shown to enhance UPEC colonisation of the bladder. INTRO +84 90 lysine residue_name This effect is attributed to cadaverine, the diamine produced by decarboxylation of lysine by LdcI and LdcC, that was shown to enhance UPEC colonisation of the bladder. INTRO +94 98 LdcI protein This effect is attributed to cadaverine, the diamine produced by decarboxylation of lysine by LdcI and LdcC, that was shown to enhance UPEC colonisation of the bladder. INTRO +103 107 LdcC protein This effect is attributed to cadaverine, the diamine produced by decarboxylation of lysine by LdcI and LdcC, that was shown to enhance UPEC colonisation of the bladder. INTRO +135 139 UPEC species This effect is attributed to cadaverine, the diamine produced by decarboxylation of lysine by LdcI and LdcC, that was shown to enhance UPEC colonisation of the bladder. INTRO +17 29 biosynthetic protein_state In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. INTRO +30 37 E. coli species In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. INTRO +38 58 lysine decarboxylase protein_type In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. INTRO +59 63 LdcC protein In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. INTRO +172 188 fluoroquinolones chemical In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. INTRO +212 216 RpoS protein In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. INTRO +260 270 cadaverine chemical In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. INTRO +293 300 E. coli species In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. INTRO +5 12 acid pH protein_state Both acid pH and cadaverine induce closure of outer membrane porins thereby contributing to bacterial protection from acid stress, but also from certain antibiotics, by reduction in membrane permeability. INTRO +17 27 cadaverine chemical Both acid pH and cadaverine induce closure of outer membrane porins thereby contributing to bacterial protection from acid stress, but also from certain antibiotics, by reduction in membrane permeability. INTRO +61 67 porins protein_type Both acid pH and cadaverine induce closure of outer membrane porins thereby contributing to bacterial protection from acid stress, but also from certain antibiotics, by reduction in membrane permeability. INTRO +92 101 bacterial taxonomy_domain Both acid pH and cadaverine induce closure of outer membrane porins thereby contributing to bacterial protection from acid stress, but also from certain antibiotics, by reduction in membrane permeability. INTRO +4 21 crystal structure evidence The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. INTRO +29 36 E. coli species The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. INTRO +37 41 LdcI protein The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. INTRO +92 111 electron microscopy experimental_method The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. INTRO +113 115 EM experimental_method The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. INTRO +137 144 decamer oligomeric_state The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. INTRO +157 167 pentameric oligomeric_state The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. INTRO +168 173 rings structure_element The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. INTRO +5 12 monomer oligomeric_state Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). INTRO +58 69 wing domain structure_element Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). INTRO +80 85 1–129 residue_range Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). INTRO +90 113 PLP-binding core domain structure_element Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). INTRO +124 131 130–563 residue_range Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). INTRO +140 157 C-terminal domain structure_element Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). INTRO +159 162 CTD structure_element Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). INTRO +173 180 564–715 residue_range Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). INTRO +0 8 Monomers oligomeric_state Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery. INTRO +37 49 core domains structure_element Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery. INTRO +55 73 2-fold symmetrical protein_state Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery. INTRO +74 80 dimers oligomeric_state Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery. INTRO +99 111 active sites site Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery. INTRO +133 166 toroidal D5-symmetrical structure structure_element Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery. INTRO +179 183 wing structure_element Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery. INTRO +188 199 core domain structure_element Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery. INTRO +224 236 central pore structure_element Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery. INTRO +247 251 CTDs structure_element Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery. INTRO +33 40 E. coli species Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. INTRO +41 52 AAA+ ATPase protein_type Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. INTRO +53 57 RavA protein Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. INTRO +130 134 LdcI protein Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. INTRO +169 173 LdcC protein Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. INTRO +203 213 pentameric oligomeric_state Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. INTRO +214 219 rings structure_element Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. INTRO +227 231 LdcI protein Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. INTRO +260 269 hexameric oligomeric_state Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. INTRO +270 275 rings structure_element Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. INTRO +279 283 RavA protein Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. INTRO +341 350 bacterium taxonomy_domain Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. INTRO +25 45 solved the structure experimental_method Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins. INTRO +53 60 E. coli species Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins. INTRO +61 70 LdcI-RavA complex_assembly Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins. INTRO +82 106 cryo-electron microscopy experimental_method Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins. INTRO +108 114 cryoEM experimental_method Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins. INTRO +141 159 crystal structures evidence Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins. INTRO +26 44 pseudoatomic model evidence This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation. INTRO +85 91 cryoEM experimental_method This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation. INTRO +92 95 map evidence This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation. INTRO +103 112 LdcI-LARA complex_assembly This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation. INTRO +127 131 LARA structure_element This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation. INTRO +145 176 LdcI associating domain of RavA structure_element This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation. INTRO +29 38 LdcI-RavA complex_assembly The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal β-sheet of LdcI. INTRO +83 87 loop structure_element The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal β-sheet of LdcI. INTRO +95 106 LARA domain structure_element The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal β-sheet of LdcI. INTRO +110 114 RavA protein The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal β-sheet of LdcI. INTRO +134 141 β-sheet structure_element The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal β-sheet of LdcI. INTRO +145 149 LdcI protein The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal β-sheet of LdcI. INTRO +27 49 structural information evidence In spite of this wealth of structural information, the fact that LdcC does not interact with RavA, although the two lysine decarboxylases are 69% identical and 84% similar, and the physiological significance of the absence of this interaction remained unexplored. INTRO +65 69 LdcC protein In spite of this wealth of structural information, the fact that LdcC does not interact with RavA, although the two lysine decarboxylases are 69% identical and 84% similar, and the physiological significance of the absence of this interaction remained unexplored. INTRO +93 97 RavA protein In spite of this wealth of structural information, the fact that LdcC does not interact with RavA, although the two lysine decarboxylases are 69% identical and 84% similar, and the physiological significance of the absence of this interaction remained unexplored. INTRO +116 137 lysine decarboxylases protein_type In spite of this wealth of structural information, the fact that LdcC does not interact with RavA, although the two lysine decarboxylases are 69% identical and 84% similar, and the physiological significance of the absence of this interaction remained unexplored. INTRO +84 90 cryoEM experimental_method To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures. INTRO +91 105 reconstruction evidence To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures. INTRO +109 113 LdcC protein To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures. INTRO +149 153 LdcI protein To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures. INTRO +158 167 LdcI-RavA complex_assembly To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures. INTRO +168 178 structures evidence To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures. INTRO +15 19 LdcI protein Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +20 38 crystal structures evidence Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +56 63 high pH protein_state Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +84 92 inactive protein_state Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +94 99 LdcIi protein Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +101 107 pH 8.5 protein_state Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +122 128 cryoEM experimental_method Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +129 144 reconstructions evidence Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +148 157 LdcI-RavA complex_assembly Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +162 171 LdcI-LARA complex_assembly Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +185 202 acidic pH optimal protein_state Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +279 296 3D reconstruction evidence Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +304 308 LdcI protein Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +312 321 active pH protein_state Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +323 328 LdcIa protein Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +330 336 pH 6.2 protein_state Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). INTRO +51 65 biodegradative protein_state This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. INTRO +74 86 biosynthetic protein_state This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. INTRO +87 108 lysine decarboxylases protein_type This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. INTRO +166 178 pH-dependent protein_state This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. INTRO +201 205 RavA protein This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. INTRO +240 257 RavA binding site site This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. INTRO +289 296 β-sheet structure_element This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. INTRO +300 304 LdcI protein This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. INTRO +364 382 LdcI-LdcC chimeras mutant This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. INTRO +392 404 interchanged experimental_method This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. INTRO +420 428 β-sheets structure_element This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. INTRO +22 49 multiple sequence alignment experimental_method Finally, we performed multiple sequence alignment of 22 lysine decarboxylases from Enterobacteriaceae containing the ravA-viaA operon in their genome. INTRO +56 77 lysine decarboxylases protein_type Finally, we performed multiple sequence alignment of 22 lysine decarboxylases from Enterobacteriaceae containing the ravA-viaA operon in their genome. INTRO +83 101 Enterobacteriaceae taxonomy_domain Finally, we performed multiple sequence alignment of 22 lysine decarboxylases from Enterobacteriaceae containing the ravA-viaA operon in their genome. INTRO +117 133 ravA-viaA operon gene Finally, we performed multiple sequence alignment of 22 lysine decarboxylases from Enterobacteriaceae containing the ravA-viaA operon in their genome. INTRO +48 65 specific residues structure_element Remarkably, this analysis revealed that several specific residues in the above-mentioned β-sheet, independently of the rest of the protein sequence, are sufficient to define if a particular lysine decarboxylase should be classified as an “LdcC-like” or an “LdcI-like”. INTRO +89 96 β-sheet structure_element Remarkably, this analysis revealed that several specific residues in the above-mentioned β-sheet, independently of the rest of the protein sequence, are sufficient to define if a particular lysine decarboxylase should be classified as an “LdcC-like” or an “LdcI-like”. INTRO +190 210 lysine decarboxylase protein_type Remarkably, this analysis revealed that several specific residues in the above-mentioned β-sheet, independently of the rest of the protein sequence, are sufficient to define if a particular lysine decarboxylase should be classified as an “LdcC-like” or an “LdcI-like”. INTRO +239 248 LdcC-like protein_type Remarkably, this analysis revealed that several specific residues in the above-mentioned β-sheet, independently of the rest of the protein sequence, are sufficient to define if a particular lysine decarboxylase should be classified as an “LdcC-like” or an “LdcI-like”. INTRO +257 266 LdcI-like protein_type Remarkably, this analysis revealed that several specific residues in the above-mentioned β-sheet, independently of the rest of the protein sequence, are sufficient to define if a particular lysine decarboxylase should be classified as an “LdcC-like” or an “LdcI-like”. INTRO +56 60 RavA protein This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA. INTRO +103 118 enterobacterial taxonomy_domain This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA. INTRO +119 139 lysine decarboxylase protein_type This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA. INTRO +156 183 high degree of conservation protein_state This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA. INTRO +192 214 small structural motif structure_element This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA. INTRO +279 293 biodegradative protein_state This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA. INTRO +294 309 enterobacterial taxonomy_domain This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA. INTRO +310 331 lysine decarboxylases protein_type This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA. INTRO +340 351 AAA+ ATPase protein_type This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA. INTRO +352 356 RavA protein This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA. INTRO +0 6 CryoEM experimental_method CryoEM 3D reconstructions of LdcC, LdcIa and LdcI-LARA RESULTS +7 25 3D reconstructions evidence CryoEM 3D reconstructions of LdcC, LdcIa and LdcI-LARA RESULTS +29 33 LdcC protein CryoEM 3D reconstructions of LdcC, LdcIa and LdcI-LARA RESULTS +35 40 LdcIa protein CryoEM 3D reconstructions of LdcC, LdcIa and LdcI-LARA RESULTS +45 54 LdcI-LARA complex_assembly CryoEM 3D reconstructions of LdcC, LdcIa and LdcI-LARA RESULTS +73 79 cryoEM experimental_method In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +80 95 reconstructions evidence In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +103 110 E. coli species In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +111 132 lysine decarboxylases protein_type In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +136 146 pH optimal protein_state In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +197 203 cryoEM experimental_method In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +204 207 map evidence In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +215 219 LdcC protein In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +221 227 pH 7.5 protein_state In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +341 347 cryoEM experimental_method In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +348 351 map evidence In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +359 364 LdcIa protein In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +367 373 pH 6.2 protein_state In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). RESULTS +37 43 cryoEM experimental_method In addition, we improved our earlier cryoEM map of the LdcI-LARA complex from 7.5 Å to 6.2 Å resolution (Figs 1E,F and S3). RESULTS +44 47 map evidence In addition, we improved our earlier cryoEM map of the LdcI-LARA complex from 7.5 Å to 6.2 Å resolution (Figs 1E,F and S3). RESULTS +55 64 LdcI-LARA complex_assembly In addition, we improved our earlier cryoEM map of the LdcI-LARA complex from 7.5 Å to 6.2 Å resolution (Figs 1E,F and S3). RESULTS +15 30 reconstructions evidence Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1). RESULTS +41 60 pseudoatomic models evidence Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1). RESULTS +102 121 flexible fitting of experimental_method Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1). RESULTS +133 150 crystal structure evidence Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1). RESULTS +154 159 LdcIi protein Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1). RESULTS +173 198 structural homology model experimental_method Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1). RESULTS +202 206 LdcC protein Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1). RESULTS +38 57 pseudoatomic models evidence Significant differences between these pseudoatomic models can be interpreted as movements between specific biological states of the proteins as described below. RESULTS +4 16 wing domains structure_element The wing domains as a stable anchor at the center of the double-ring RESULTS +57 68 double-ring structure_element The wing domains as a stable anchor at the center of the double-ring RESULTS +46 58 superimposed experimental_method As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). RESULTS +69 75 cryoEM experimental_method As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). RESULTS +76 91 reconstructions evidence As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). RESULTS +93 98 LdcIa protein As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). RESULTS +100 109 LdcI-LARA complex_assembly As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). RESULTS +114 118 LdcC protein As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). RESULTS +128 145 crystal structure evidence As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). RESULTS +153 158 LdcIi protein As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). RESULTS +159 166 decamer oligomeric_state As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). RESULTS +5 18 superposition experimental_method This superposition reveals that the densities lining the central hole of the toroid are roughly at the same location, while the rest of the structure exhibits noticeable changes. RESULTS +36 45 densities evidence This superposition reveals that the densities lining the central hole of the toroid are roughly at the same location, while the rest of the structure exhibits noticeable changes. RESULTS +57 69 central hole structure_element This superposition reveals that the densities lining the central hole of the toroid are roughly at the same location, while the rest of the structure exhibits noticeable changes. RESULTS +77 83 toroid structure_element This superposition reveals that the densities lining the central hole of the toroid are roughly at the same location, while the rest of the structure exhibits noticeable changes. RESULTS +140 149 structure evidence This superposition reveals that the densities lining the central hole of the toroid are roughly at the same location, while the rest of the structure exhibits noticeable changes. RESULTS +35 46 double-ring structure_element Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). RESULTS +51 63 wing domains structure_element Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). RESULTS +92 101 conserved protein_state Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). RESULTS +134 167 lowest root mean square deviation evidence Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). RESULTS +169 173 RMSD evidence Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). RESULTS +243 247 CTDs structure_element Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). RESULTS +263 285 RavA binding interface site Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). RESULTS +307 311 RMSD evidence Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). RESULTS +17 29 wing domains structure_element In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. RESULTS +37 47 structures evidence In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. RESULTS +75 79 RMSD evidence In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. RESULTS +116 123 RMSDmin evidence In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. RESULTS +185 191 cryoEM experimental_method In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. RESULTS +192 196 maps evidence In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. RESULTS +232 244 wing domains structure_element In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. RESULTS +261 271 structures evidence In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. RESULTS +332 336 RMSD evidence In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. RESULTS +25 37 central part structure_element This preservation of the central part of the double-ring assembly may help the enzymes to maintain their decameric state upon activation and incorporation into the LdcI-RavA cage. RESULTS +105 114 decameric oligomeric_state This preservation of the central part of the double-ring assembly may help the enzymes to maintain their decameric state upon activation and incorporation into the LdcI-RavA cage. RESULTS +164 173 LdcI-RavA complex_assembly This preservation of the central part of the double-ring assembly may help the enzymes to maintain their decameric state upon activation and incorporation into the LdcI-RavA cage. RESULTS +4 15 core domain structure_element The core domain and the active site rearrangements upon pH-dependent enzyme activation and LARA binding RESULTS +24 35 active site site The core domain and the active site rearrangements upon pH-dependent enzyme activation and LARA binding RESULTS +56 68 pH-dependent protein_state The core domain and the active site rearrangements upon pH-dependent enzyme activation and LARA binding RESULTS +5 22 visual inspection experimental_method Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). RESULTS +36 53 RMSD calculations experimental_method Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). RESULTS +94 104 structures evidence Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). RESULTS +108 117 active pH protein_state Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). RESULTS +119 124 LdcIa protein Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). RESULTS +126 135 LdcI-LARA complex_assembly Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). RESULTS +140 144 LdcC protein Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). RESULTS +213 220 high pH protein_state Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). RESULTS +233 238 LdcIi protein Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). RESULTS +4 13 decameric oligomeric_state The decameric enzyme is built of five dimers associating into a 5-fold symmetrical double-ring (two monomers making a dimer are delineated in Fig. 1). RESULTS +38 44 dimers oligomeric_state The decameric enzyme is built of five dimers associating into a 5-fold symmetrical double-ring (two monomers making a dimer are delineated in Fig. 1). RESULTS +64 94 5-fold symmetrical double-ring structure_element The decameric enzyme is built of five dimers associating into a 5-fold symmetrical double-ring (two monomers making a dimer are delineated in Fig. 1). RESULTS +100 108 monomers oligomeric_state The decameric enzyme is built of five dimers associating into a 5-fold symmetrical double-ring (two monomers making a dimer are delineated in Fig. 1). RESULTS +118 123 dimer oligomeric_state The decameric enzyme is built of five dimers associating into a 5-fold symmetrical double-ring (two monomers making a dimer are delineated in Fig. 1). RESULTS +18 26 α family protein_type As common for the α family of the PLP-dependent decarboxylases, dimerization is required for the enzymatic activity because the active site is buried in the dimer interface (Fig. 3A,B). RESULTS +34 62 PLP-dependent decarboxylases protein_type As common for the α family of the PLP-dependent decarboxylases, dimerization is required for the enzymatic activity because the active site is buried in the dimer interface (Fig. 3A,B). RESULTS +128 139 active site site As common for the α family of the PLP-dependent decarboxylases, dimerization is required for the enzymatic activity because the active site is buried in the dimer interface (Fig. 3A,B). RESULTS +157 172 dimer interface site As common for the α family of the PLP-dependent decarboxylases, dimerization is required for the enzymatic activity because the active site is buried in the dimer interface (Fig. 3A,B). RESULTS +5 14 interface site This interface is formed essentially by the core domains with some contribution of the CTDs. RESULTS +44 56 core domains structure_element This interface is formed essentially by the core domains with some contribution of the CTDs. RESULTS +87 91 CTDs structure_element This interface is formed essentially by the core domains with some contribution of the CTDs. RESULTS +4 15 core domain structure_element The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). RESULTS +32 53 PLP-binding subdomain structure_element The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). RESULTS +55 61 PLP-SD structure_element The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). RESULTS +72 79 184–417 residue_range The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). RESULTS +104 114 subdomains structure_element The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). RESULTS +123 140 partly disordered protein_state The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). RESULTS +141 146 loops structure_element The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). RESULTS +153 166 linker region structure_element The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). RESULTS +177 184 130–183 residue_range The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). RESULTS +194 205 subdomain 4 structure_element The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). RESULTS +216 223 418–563 residue_range The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). RESULTS +33 39 PLP-SD structure_element Zooming in the variations in the PLP-SD shows that most of the structural changes concern displacements in the active site (Fig. 3C–F). RESULTS +111 122 active site site Zooming in the variations in the PLP-SD shows that most of the structural changes concern displacements in the active site (Fig. 3C–F). RESULTS +45 52 PLP-SDs structure_element The most conspicuous differences between the PLP-SDs can be linked to the pH-dependent activation of the enzymes. RESULTS +74 86 pH-dependent protein_state The most conspicuous differences between the PLP-SDs can be linked to the pH-dependent activation of the enzymes. RESULTS +22 28 cryoEM experimental_method The resolution of the cryoEM maps does not allow modeling the position of the PLP moiety and calls for caution in detailed mechanistic interpretations in terms of individual amino acids. RESULTS +29 33 maps evidence The resolution of the cryoEM maps does not allow modeling the position of the PLP moiety and calls for caution in detailed mechanistic interpretations in terms of individual amino acids. RESULTS +78 81 PLP chemical The resolution of the cryoEM maps does not allow modeling the position of the PLP moiety and calls for caution in detailed mechanistic interpretations in terms of individual amino acids. RESULTS +174 185 amino acids chemical The resolution of the cryoEM maps does not allow modeling the position of the PLP moiety and calls for caution in detailed mechanistic interpretations in terms of individual amino acids. RESULTS +31 36 LdcIi protein In particular, transition from LdcIi to LdcI-LARA involves ~3.5 Å and ~4.5 Å shifts away from the 5-fold axis in the active site α-helices spanning residues 218–232 and 246–254 respectively (Fig. 3C–E). RESULTS +40 49 LdcI-LARA complex_assembly In particular, transition from LdcIi to LdcI-LARA involves ~3.5 Å and ~4.5 Å shifts away from the 5-fold axis in the active site α-helices spanning residues 218–232 and 246–254 respectively (Fig. 3C–E). RESULTS +117 128 active site site In particular, transition from LdcIi to LdcI-LARA involves ~3.5 Å and ~4.5 Å shifts away from the 5-fold axis in the active site α-helices spanning residues 218–232 and 246–254 respectively (Fig. 3C–E). RESULTS +129 138 α-helices structure_element In particular, transition from LdcIi to LdcI-LARA involves ~3.5 Å and ~4.5 Å shifts away from the 5-fold axis in the active site α-helices spanning residues 218–232 and 246–254 respectively (Fig. 3C–E). RESULTS +157 164 218–232 residue_range In particular, transition from LdcIi to LdcI-LARA involves ~3.5 Å and ~4.5 Å shifts away from the 5-fold axis in the active site α-helices spanning residues 218–232 and 246–254 respectively (Fig. 3C–E). RESULTS +169 176 246–254 residue_range In particular, transition from LdcIi to LdcI-LARA involves ~3.5 Å and ~4.5 Å shifts away from the 5-fold axis in the active site α-helices spanning residues 218–232 and 246–254 respectively (Fig. 3C–E). RESULTS +32 39 PLP-SDs structure_element Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin = 0.9 Å, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity. RESULTS +43 48 LdcIa protein Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin = 0.9 Å, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity. RESULTS +53 57 LdcC protein Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin = 0.9 Å, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity. RESULTS +97 104 decamer oligomeric_state Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin = 0.9 Å, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity. RESULTS +131 138 RMSDmin evidence Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin = 0.9 Å, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity. RESULTS +194 204 optimal pH protein_state Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin = 0.9 Å, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity. RESULTS +212 233 lysine decarboxylases protein_type Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin = 0.9 Å, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity. RESULTS +25 48 biochemical observation experimental_method In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA. RESULTS +80 85 LdcIa protein In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA. RESULTS +103 107 RavA protein In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA. RESULTS +181 192 active site site In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA. RESULTS +214 219 LdcIa protein In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA. RESULTS +223 232 LdcI-LARA complex_assembly In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA. RESULTS +47 64 crystal structure evidence Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). RESULTS +68 73 LdcIi protein Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). RESULTS +91 100 inducible protein_state Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). RESULTS +101 123 arginine decarboxylase protein_type Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). RESULTS +124 128 AdiA protein Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). RESULTS +138 155 high conservation protein_state Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). RESULTS +163 188 PLP-coordinating residues site Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). RESULTS +206 242 patch of negatively charged residues site Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). RESULTS +254 273 active site channel site Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). RESULTS +289 301 binding site site Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). RESULTS +317 327 amino acid chemical Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.). RESULTS +22 42 ppGpp binding pocket site Rearrangements of the ppGpp binding pocket upon pH-dependent enzyme activation and LARA binding RESULTS +48 60 pH-dependent protein_state Rearrangements of the ppGpp binding pocket upon pH-dependent enzyme activation and LARA binding RESULTS +83 87 LARA structure_element Rearrangements of the ppGpp binding pocket upon pH-dependent enzyme activation and LARA binding RESULTS +20 24 LdcI protein An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4). RESULTS +29 33 LdcC protein An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4). RESULTS +48 75 stringent response alarmone chemical An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4). RESULTS +76 81 ppGpp chemical An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4). RESULTS +107 116 interface site An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4). RESULTS +137 145 monomers oligomeric_state An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4). RESULTS +158 162 ring structure_element An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4). RESULTS +4 24 ppGpp binding pocket site The ppGpp binding pocket is made up by residues from all domains and is located approximately 30 Å away from the PLP moiety. RESULTS +113 116 PLP chemical The ppGpp binding pocket is made up by residues from all domains and is located approximately 30 Å away from the PLP moiety. RESULTS +12 29 crystal structure evidence Whereas the crystal structure of the ppGpp-LdcIi was solved to 2 Å resolution, only a 4.1 Å resolution structure of the ppGpp-free LdcIi could be obtained. RESULTS +37 48 ppGpp-LdcIi complex_assembly Whereas the crystal structure of the ppGpp-LdcIi was solved to 2 Å resolution, only a 4.1 Å resolution structure of the ppGpp-free LdcIi could be obtained. RESULTS +53 59 solved experimental_method Whereas the crystal structure of the ppGpp-LdcIi was solved to 2 Å resolution, only a 4.1 Å resolution structure of the ppGpp-free LdcIi could be obtained. RESULTS +103 112 structure evidence Whereas the crystal structure of the ppGpp-LdcIi was solved to 2 Å resolution, only a 4.1 Å resolution structure of the ppGpp-free LdcIi could be obtained. RESULTS +120 130 ppGpp-free protein_state Whereas the crystal structure of the ppGpp-LdcIi was solved to 2 Å resolution, only a 4.1 Å resolution structure of the ppGpp-free LdcIi could be obtained. RESULTS +131 136 LdcIi protein Whereas the crystal structure of the ppGpp-LdcIi was solved to 2 Å resolution, only a 4.1 Å resolution structure of the ppGpp-free LdcIi could be obtained. RESULTS +24 27 apo protein_state At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ). RESULTS +28 33 LdcIi protein At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ). RESULTS +38 49 ppGpp-LdcIi complex_assembly At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ). RESULTS +50 60 structures evidence At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ). RESULTS +77 83 pH 8.5 protein_state At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ). RESULTS +139 144 ppGpp chemical At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ). RESULTS +39 43 LdcI protein Thus, we speculated that inhibition of LdcI by ppGpp would be accompanied by a transduction of subtle structural changes at the level of individual amino acid side chains between the ppGpp binding pocket and the active site of the enzyme. RESULTS +47 52 ppGpp chemical Thus, we speculated that inhibition of LdcI by ppGpp would be accompanied by a transduction of subtle structural changes at the level of individual amino acid side chains between the ppGpp binding pocket and the active site of the enzyme. RESULTS +148 158 amino acid chemical Thus, we speculated that inhibition of LdcI by ppGpp would be accompanied by a transduction of subtle structural changes at the level of individual amino acid side chains between the ppGpp binding pocket and the active site of the enzyme. RESULTS +183 203 ppGpp binding pocket site Thus, we speculated that inhibition of LdcI by ppGpp would be accompanied by a transduction of subtle structural changes at the level of individual amino acid side chains between the ppGpp binding pocket and the active site of the enzyme. RESULTS +212 223 active site site Thus, we speculated that inhibition of LdcI by ppGpp would be accompanied by a transduction of subtle structural changes at the level of individual amino acid side chains between the ppGpp binding pocket and the active site of the enzyme. RESULTS +16 22 cryoEM experimental_method All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study. RESULTS +23 38 reconstructions evidence All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study. RESULTS +46 67 lysine decarboxylases protein_type All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study. RESULTS +89 99 absence of protein_state All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study. RESULTS +100 105 ppGpp chemical All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study. RESULTS +135 141 active protein_state All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study. RESULTS +25 43 ppGpp binding site site While differences in the ppGpp binding site could indeed be visualized (Fig. S4), the level of resolution warns against speculations about their significance. RESULTS +31 35 RavA protein The fact that interaction with RavA reduces the ppGpp affinity for LdcI despite the long distance of ~30 Å between the LARA domain binding site and the closest ppGpp binding pocket (Fig. S5) seems to favor an allosteric regulation mechanism. RESULTS +48 53 ppGpp chemical The fact that interaction with RavA reduces the ppGpp affinity for LdcI despite the long distance of ~30 Å between the LARA domain binding site and the closest ppGpp binding pocket (Fig. S5) seems to favor an allosteric regulation mechanism. RESULTS +67 71 LdcI protein The fact that interaction with RavA reduces the ppGpp affinity for LdcI despite the long distance of ~30 Å between the LARA domain binding site and the closest ppGpp binding pocket (Fig. S5) seems to favor an allosteric regulation mechanism. RESULTS +119 143 LARA domain binding site site The fact that interaction with RavA reduces the ppGpp affinity for LdcI despite the long distance of ~30 Å between the LARA domain binding site and the closest ppGpp binding pocket (Fig. S5) seems to favor an allosteric regulation mechanism. RESULTS +160 180 ppGpp binding pocket site The fact that interaction with RavA reduces the ppGpp affinity for LdcI despite the long distance of ~30 Å between the LARA domain binding site and the closest ppGpp binding pocket (Fig. S5) seems to favor an allosteric regulation mechanism. RESULTS +36 58 ppGpp binding residues site Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated. RESULTS +63 81 strictly conserved protein_state Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated. RESULTS +90 94 LdcI protein Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated. RESULTS +99 103 AdiA protein Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated. RESULTS +120 128 decamers oligomeric_state Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated. RESULTS +132 146 low pH optimal protein_state Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated. RESULTS +155 177 arginine decarboxylase protein_type Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated. RESULTS +191 196 ppGpp chemical Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated. RESULTS +211 215 AdiA protein Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated. RESULTS +31 35 CTDs structure_element Swinging and stretching of the CTDs upon pH-dependent LdcI activation and LARA binding RESULTS +41 53 pH-dependent protein_state Swinging and stretching of the CTDs upon pH-dependent LdcI activation and LARA binding RESULTS +54 58 LdcI protein Swinging and stretching of the CTDs upon pH-dependent LdcI activation and LARA binding RESULTS +74 78 LARA structure_element Swinging and stretching of the CTDs upon pH-dependent LdcI activation and LARA binding RESULTS +18 30 superimposed experimental_method Inspection of the superimposed decameric structures (Figs 2 and S6) suggests a depiction of the wing domains as an anchor around which the peripheral CTDs swing. RESULTS +31 40 decameric oligomeric_state Inspection of the superimposed decameric structures (Figs 2 and S6) suggests a depiction of the wing domains as an anchor around which the peripheral CTDs swing. RESULTS +41 51 structures evidence Inspection of the superimposed decameric structures (Figs 2 and S6) suggests a depiction of the wing domains as an anchor around which the peripheral CTDs swing. RESULTS +96 108 wing domains structure_element Inspection of the superimposed decameric structures (Figs 2 and S6) suggests a depiction of the wing domains as an anchor around which the peripheral CTDs swing. RESULTS +150 154 CTDs structure_element Inspection of the superimposed decameric structures (Figs 2 and S6) suggests a depiction of the wing domains as an anchor around which the peripheral CTDs swing. RESULTS +51 63 core domains structure_element This swinging movement seems to be mediated by the core domains and is accompanied by a stretching of the whole LdcI subunits attracted by the RavA magnets. RESULTS +112 116 LdcI protein This swinging movement seems to be mediated by the core domains and is accompanied by a stretching of the whole LdcI subunits attracted by the RavA magnets. RESULTS +117 125 subunits structure_element This swinging movement seems to be mediated by the core domains and is accompanied by a stretching of the whole LdcI subunits attracted by the RavA magnets. RESULTS +143 147 RavA protein This swinging movement seems to be mediated by the core domains and is accompanied by a stretching of the whole LdcI subunits attracted by the RavA magnets. RESULTS +12 16 CTDs structure_element Indeed, all CTDs have very similar structures (RMSDmin <1 Å). RESULTS +47 54 RMSDmin evidence Indeed, all CTDs have very similar structures (RMSDmin <1 Å). RESULTS +8 21 superposition experimental_method Yet the superposition of the decamers lays bare a progressive movement of the CTD as a whole upon enzyme activation by pH and the binding of LARA. RESULTS +29 37 decamers oligomeric_state Yet the superposition of the decamers lays bare a progressive movement of the CTD as a whole upon enzyme activation by pH and the binding of LARA. RESULTS +78 81 CTD structure_element Yet the superposition of the decamers lays bare a progressive movement of the CTD as a whole upon enzyme activation by pH and the binding of LARA. RESULTS +141 145 LARA structure_element Yet the superposition of the decamers lays bare a progressive movement of the CTD as a whole upon enzyme activation by pH and the binding of LARA. RESULTS +4 9 LdcIi protein The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). RESULTS +10 17 monomer oligomeric_state The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). RESULTS +25 37 most compact protein_state The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). RESULTS +47 52 LdcIa protein The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). RESULTS +68 77 LdcI-LARA complex_assembly The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). RESULTS +78 94 gradually extend protein_state The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). RESULTS +101 105 CTDs structure_element The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). RESULTS +118 129 LARA domain structure_element The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). RESULTS +133 137 RavA protein The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). RESULTS +107 111 LdcI protein These small but noticeable swinging and stretching (up to ~4 Å) may be related to the incorporation of the LdcI decamer into the LdcI-RavA cage. RESULTS +112 119 decamer oligomeric_state These small but noticeable swinging and stretching (up to ~4 Å) may be related to the incorporation of the LdcI decamer into the LdcI-RavA cage. RESULTS +129 138 LdcI-RavA complex_assembly These small but noticeable swinging and stretching (up to ~4 Å) may be related to the incorporation of the LdcI decamer into the LdcI-RavA cage. RESULTS +15 22 β-sheet structure_element The C-terminal β-sheet of a lysine decarboxylase as a major determinant of the interaction with RavA RESULTS +28 48 lysine decarboxylase protein_type The C-terminal β-sheet of a lysine decarboxylase as a major determinant of the interaction with RavA RESULTS +96 100 RavA protein The C-terminal β-sheet of a lysine decarboxylase as a major determinant of the interaction with RavA RESULTS +54 59 LdcIi protein In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +68 72 LARA structure_element In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +73 91 crystal structures evidence In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +101 110 LdcI-LARA complex_assembly In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +111 117 cryoEM experimental_method In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +118 125 density evidence In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +149 158 LdcI-RavA complex_assembly In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +201 221 two-stranded β-sheet structure_element In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +229 233 LdcI protein In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +247 253 cryoEM experimental_method In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +254 258 maps evidence In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +263 282 pseudoatomic models evidence In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +355 364 inducible protein_state In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +373 385 constitutive protein_state In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +386 407 lysine decarboxylases protein_type In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. RESULTS +137 141 RavA protein Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). RESULTS +159 166 swapped experimental_method Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). RESULTS +180 188 β-sheets structure_element Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). RESULTS +228 236 chimeras mutant Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). RESULTS +245 250 LdcIC mutant Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). RESULTS +257 261 LdcI protein Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). RESULTS +282 289 β-sheet structure_element Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). RESULTS +293 297 LdcC protein Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). RESULTS +303 308 LdcCI mutant Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). RESULTS +315 319 LdcC protein Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). RESULTS +340 347 β-sheet structure_element Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). RESULTS +351 355 LdcI protein Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). RESULTS +0 15 Both constructs mutant Both constructs could be purified and could form decamers visually indistinguishable from the wild-type proteins. RESULTS +49 57 decamers oligomeric_state Both constructs could be purified and could form decamers visually indistinguishable from the wild-type proteins. RESULTS +94 103 wild-type protein_state Both constructs could be purified and could form decamers visually indistinguishable from the wild-type proteins. RESULTS +24 28 LdcI protein As expected, binding of LdcI to RavA was completely abolished by this procedure and no LdcIC-RavA complex could be detected. RESULTS +32 36 RavA protein As expected, binding of LdcI to RavA was completely abolished by this procedure and no LdcIC-RavA complex could be detected. RESULTS +87 97 LdcIC-RavA complex_assembly As expected, binding of LdcI to RavA was completely abolished by this procedure and no LdcIC-RavA complex could be detected. RESULTS +17 29 introduction experimental_method On the contrary, introduction of the C-terminal β-sheet of LdcI into LdcC led to an assembly of the LdcCI-RavA complex. RESULTS +48 55 β-sheet structure_element On the contrary, introduction of the C-terminal β-sheet of LdcI into LdcC led to an assembly of the LdcCI-RavA complex. RESULTS +59 63 LdcI protein On the contrary, introduction of the C-terminal β-sheet of LdcI into LdcC led to an assembly of the LdcCI-RavA complex. RESULTS +69 73 LdcC protein On the contrary, introduction of the C-terminal β-sheet of LdcI into LdcC led to an assembly of the LdcCI-RavA complex. RESULTS +100 110 LdcCI-RavA complex_assembly On the contrary, introduction of the C-terminal β-sheet of LdcI into LdcC led to an assembly of the LdcCI-RavA complex. RESULTS +7 29 negative stain EM grid experimental_method On the negative stain EM grid, the chimeric cages appeared less rigid than the native LdcI-RavA, which probably means that the environment of the β-sheet contributes to the efficiency of the interaction and the stability of the entire architecture (Fig. 5D–F). RESULTS +35 43 chimeric protein_state On the negative stain EM grid, the chimeric cages appeared less rigid than the native LdcI-RavA, which probably means that the environment of the β-sheet contributes to the efficiency of the interaction and the stability of the entire architecture (Fig. 5D–F). RESULTS +79 85 native protein_state On the negative stain EM grid, the chimeric cages appeared less rigid than the native LdcI-RavA, which probably means that the environment of the β-sheet contributes to the efficiency of the interaction and the stability of the entire architecture (Fig. 5D–F). RESULTS +86 95 LdcI-RavA complex_assembly On the negative stain EM grid, the chimeric cages appeared less rigid than the native LdcI-RavA, which probably means that the environment of the β-sheet contributes to the efficiency of the interaction and the stability of the entire architecture (Fig. 5D–F). RESULTS +146 153 β-sheet structure_element On the negative stain EM grid, the chimeric cages appeared less rigid than the native LdcI-RavA, which probably means that the environment of the β-sheet contributes to the efficiency of the interaction and the stability of the entire architecture (Fig. 5D–F). RESULTS +15 22 β-sheet structure_element The C-terminal β-sheet of a lysine decarboxylase is a highly conserved signature allowing to distinguish between LdcI and LdcC RESULTS +28 48 lysine decarboxylase protein_type The C-terminal β-sheet of a lysine decarboxylase is a highly conserved signature allowing to distinguish between LdcI and LdcC RESULTS +54 70 highly conserved protein_state The C-terminal β-sheet of a lysine decarboxylase is a highly conserved signature allowing to distinguish between LdcI and LdcC RESULTS +113 117 LdcI protein The C-terminal β-sheet of a lysine decarboxylase is a highly conserved signature allowing to distinguish between LdcI and LdcC RESULTS +122 126 LdcC protein The C-terminal β-sheet of a lysine decarboxylase is a highly conserved signature allowing to distinguish between LdcI and LdcC RESULTS +0 34 Alignment of the primary sequences experimental_method Alignment of the primary sequences of the E. coli LdcI and LdcC shows that some amino acid residues of the C-terminal β-sheet are the same in the two proteins, whereas others are notably different in chemical nature. RESULTS +42 49 E. coli species Alignment of the primary sequences of the E. coli LdcI and LdcC shows that some amino acid residues of the C-terminal β-sheet are the same in the two proteins, whereas others are notably different in chemical nature. RESULTS +50 54 LdcI protein Alignment of the primary sequences of the E. coli LdcI and LdcC shows that some amino acid residues of the C-terminal β-sheet are the same in the two proteins, whereas others are notably different in chemical nature. RESULTS +59 63 LdcC protein Alignment of the primary sequences of the E. coli LdcI and LdcC shows that some amino acid residues of the C-terminal β-sheet are the same in the two proteins, whereas others are notably different in chemical nature. RESULTS +118 125 β-sheet structure_element Alignment of the primary sequences of the E. coli LdcI and LdcC shows that some amino acid residues of the C-terminal β-sheet are the same in the two proteins, whereas others are notably different in chemical nature. RESULTS +92 103 very region structure_element Importantly, most of the amino acid differences between the two enzymes are located in this very region. RESULTS +72 79 β-sheet structure_element Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. RESULTS +135 155 lysine decarboxylase protein_type Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. RESULTS +176 180 RavA protein Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. RESULTS +216 232 certain residues structure_element Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. RESULTS +241 248 β-sheet structure_element Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. RESULTS +253 262 conserved protein_state Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. RESULTS +266 287 lysine decarboxylases protein_type Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. RESULTS +301 315 enterobacteria taxonomy_domain Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. RESULTS +330 346 ravA-viaA operon gene Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. RESULTS +3 36 inspected the genetic environment experimental_method We inspected the genetic environment of lysine decarboxylases from 22 enterobacterial species referenced in the NCBI database, corrected the gene annotation where necessary (Tables S3 and S4), and performed multiple sequence alignment coupled to a phylogenetic analysis (see Methods). RESULTS +40 61 lysine decarboxylases protein_type We inspected the genetic environment of lysine decarboxylases from 22 enterobacterial species referenced in the NCBI database, corrected the gene annotation where necessary (Tables S3 and S4), and performed multiple sequence alignment coupled to a phylogenetic analysis (see Methods). RESULTS +70 85 enterobacterial taxonomy_domain We inspected the genetic environment of lysine decarboxylases from 22 enterobacterial species referenced in the NCBI database, corrected the gene annotation where necessary (Tables S3 and S4), and performed multiple sequence alignment coupled to a phylogenetic analysis (see Methods). RESULTS +207 234 multiple sequence alignment experimental_method We inspected the genetic environment of lysine decarboxylases from 22 enterobacterial species referenced in the NCBI database, corrected the gene annotation where necessary (Tables S3 and S4), and performed multiple sequence alignment coupled to a phylogenetic analysis (see Methods). RESULTS +248 269 phylogenetic analysis experimental_method We inspected the genetic environment of lysine decarboxylases from 22 enterobacterial species referenced in the NCBI database, corrected the gene annotation where necessary (Tables S3 and S4), and performed multiple sequence alignment coupled to a phylogenetic analysis (see Methods). RESULTS +14 32 consensus sequence evidence First of all, consensus sequence for the entire lysine decarboxylase family was derived. RESULTS +48 68 lysine decarboxylase protein_type First of all, consensus sequence for the entire lysine decarboxylase family was derived. RESULTS +12 33 phylogenetic analysis experimental_method Second, the phylogenetic analysis clearly split the lysine decarboxylases into two groups (Fig. 6A). RESULTS +52 73 lysine decarboxylases protein_type Second, the phylogenetic analysis clearly split the lysine decarboxylases into two groups (Fig. 6A). RESULTS +4 25 lysine decarboxylases protein_type All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. RESULTS +43 52 LdcI-like protein_type All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. RESULTS +57 70 biodegradable protein_state All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. RESULTS +179 183 CadB protein All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. RESULTS +184 194 antiporter protein_type All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. RESULTS +247 254 enzymes protein_type All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. RESULTS +269 278 LdcC-like protein_type All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. RESULTS +283 295 biosynthetic protein_state All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. RESULTS +6 25 consensus sequences evidence Thus, consensus sequences could also be determined for each of the two groups (Figs 6B,C and S7). RESULTS +20 39 consensus sequences evidence Inspection of these consensus sequences revealed important differences between the groups regarding charge, size and hydrophobicity of several residues precisely at the level of the C-terminal β-sheet that is responsible for the interaction with RavA (Fig. 6B–D). RESULTS +193 200 β-sheet structure_element Inspection of these consensus sequences revealed important differences between the groups regarding charge, size and hydrophobicity of several residues precisely at the level of the C-terminal β-sheet that is responsible for the interaction with RavA (Fig. 6B–D). RESULTS +246 250 RavA protein Inspection of these consensus sequences revealed important differences between the groups regarding charge, size and hydrophobicity of several residues precisely at the level of the C-terminal β-sheet that is responsible for the interaction with RavA (Fig. 6B–D). RESULTS +36 59 site-directed mutations experimental_method For example, in our previous study, site-directed mutations identified Y697 as critically required for the RavA binding. RESULTS +71 75 Y697 residue_name_number For example, in our previous study, site-directed mutations identified Y697 as critically required for the RavA binding. RESULTS +107 111 RavA protein For example, in our previous study, site-directed mutations identified Y697 as critically required for the RavA binding. RESULTS +32 36 Y697 residue_name_number Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. RESULTS +40 58 strictly conserved protein_state Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. RESULTS +67 76 LdcI-like protein_type Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. RESULTS +97 106 LdcC-like protein_type Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. RESULTS +116 127 always have protein_state Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. RESULTS +130 136 lysine residue_name Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. RESULTS +242 246 RavA protein Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. RESULTS +277 302 site-directed mutagenesis experimental_method Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. RESULTS +81 90 LdcI-like protein_type The third and most remarkable finding was that exactly the same separation into “LdcI-like” and “LdcC”-like groups can be obtained based on a comparison of the C-terminal β-sheets only, without taking the rest of the primary sequence into account. RESULTS +97 107 LdcC”-like protein_type The third and most remarkable finding was that exactly the same separation into “LdcI-like” and “LdcC”-like groups can be obtained based on a comparison of the C-terminal β-sheets only, without taking the rest of the primary sequence into account. RESULTS +171 179 β-sheets structure_element The third and most remarkable finding was that exactly the same separation into “LdcI-like” and “LdcC”-like groups can be obtained based on a comparison of the C-terminal β-sheets only, without taking the rest of the primary sequence into account. RESULTS +25 32 β-sheet structure_element Therefore the C-terminal β-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the “LdcI-like” and “LdcC-like” enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7). RESULTS +52 68 highly conserved protein_state Therefore the C-terminal β-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the “LdcI-like” and “LdcC-like” enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7). RESULTS +69 87 signature sequence structure_element Therefore the C-terminal β-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the “LdcI-like” and “LdcC-like” enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7). RESULTS +143 152 LdcI-like protein_type Therefore the C-terminal β-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the “LdcI-like” and “LdcC-like” enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7). RESULTS +159 168 LdcC-like protein_type Therefore the C-terminal β-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the “LdcI-like” and “LdcC-like” enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7). RESULTS +170 185 enterobacterial taxonomy_domain Therefore the C-terminal β-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the “LdcI-like” and “LdcC-like” enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7). RESULTS +186 207 lysine decarboxylases protein_type Therefore the C-terminal β-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the “LdcI-like” and “LdcC-like” enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7). RESULTS +4 14 structures evidence Our structures show that this motif is not involved in the enzymatic activity or the oligomeric state of the proteins. RESULTS +25 35 this motif structure_element Our structures show that this motif is not involved in the enzymatic activity or the oligomeric state of the proteins. RESULTS +6 20 enterobacteria taxonomy_domain Thus, enterobacteria identified here (Fig. 6, Table S4) appear to exert evolutionary pressure on the biodegradative lysine decarboxylase towards the RavA binding. RESULTS +101 115 biodegradative protein_state Thus, enterobacteria identified here (Fig. 6, Table S4) appear to exert evolutionary pressure on the biodegradative lysine decarboxylase towards the RavA binding. RESULTS +116 136 lysine decarboxylase protein_type Thus, enterobacteria identified here (Fig. 6, Table S4) appear to exert evolutionary pressure on the biodegradative lysine decarboxylase towards the RavA binding. RESULTS +149 153 RavA protein Thus, enterobacteria identified here (Fig. 6, Table S4) appear to exert evolutionary pressure on the biodegradative lysine decarboxylase towards the RavA binding. RESULTS +35 44 LdcI-RavA complex_assembly One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp. RESULTS +65 69 LdcI protein One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp. RESULTS +99 114 enterobacterial taxonomy_domain One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp. RESULTS +140 144 LdcI protein One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp. RESULTS +163 190 stringent response alarmone chemical One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp. RESULTS +191 196 ppGpp chemical One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp. RESULTS +57 61 LdcI protein Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5 MDa assembly. RESULTS +66 70 RavA protein Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5 MDa assembly. RESULTS +85 93 subunits structure_element Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5 MDa assembly. RESULTS +101 122 respiratory complex I protein_type Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5 MDa assembly. RESULTS +169 173 RavA protein Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5 MDa assembly. RESULTS +201 221 iron-sulfur proteins protein_type Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5 MDa assembly. RESULTS +37 41 LdcI protein The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. RESULTS +69 73 RavA protein The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. RESULTS +167 176 LdcI-RavA complex_assembly The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. RESULTS +250 265 enterobacterial taxonomy_domain The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. RESULTS +266 286 lysine decarboxylase protein_type The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. RESULTS +299 308 LdcI-like protein_type The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. RESULTS +314 323 LdcC-like protein_type The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. RESULTS +397 406 LdcI-RavA complex_assembly The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. RESULTS +13 23 structures evidence Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. RESULTS +32 51 pseudoatomic models evidence Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. RESULTS +59 65 active protein_state Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. RESULTS +66 76 ppGpp-free protein_state Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. RESULTS +96 110 biodegradative protein_state Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. RESULTS +119 131 biosynthetic protein_state Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. RESULTS +132 139 E. coli species Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. RESULTS +140 161 lysine decarboxylases protein_type Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. RESULTS +217 221 UPEC species Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. RESULTS +18 21 apo protein_state Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. RESULTS +22 26 LdcI protein Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. RESULTS +31 42 ppGpp-LdcIi complex_assembly Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. RESULTS +43 61 crystal structures evidence Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. RESULTS +67 73 cryoEM experimental_method Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. RESULTS +74 89 reconstructions evidence Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. RESULTS +179 200 lysine decarboxylases protein_type Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. RESULTS +212 226 enterobacteria taxonomy_domain Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. RESULTS +264 282 Enterobacteriaceae taxonomy_domain Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. RESULTS +301 321 lysine decarboxylase protein_type Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. RESULTS +325 344 Eikenella corrodens species Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. RESULTS +9 19 cadaverine chemical Finally, cadaverine being an important platform chemical for the production of industrial polymers such as nylon, structural information is valuable for optimisation of bacterial lysine decarboxylases used for its production in biotechnology. RESULTS +169 178 bacterial taxonomy_domain Finally, cadaverine being an important platform chemical for the production of industrial polymers such as nylon, structural information is valuable for optimisation of bacterial lysine decarboxylases used for its production in biotechnology. RESULTS +179 200 lysine decarboxylases protein_type Finally, cadaverine being an important platform chemical for the production of industrial polymers such as nylon, structural information is valuable for optimisation of bacterial lysine decarboxylases used for its production in biotechnology. RESULTS +3 9 cryoEM experimental_method 3D cryoEM reconstructions of LdcC, LdcI-LARA and LdcIa. FIG +10 25 reconstructions evidence 3D cryoEM reconstructions of LdcC, LdcI-LARA and LdcIa. FIG +29 33 LdcC protein 3D cryoEM reconstructions of LdcC, LdcI-LARA and LdcIa. FIG +35 44 LdcI-LARA complex_assembly 3D cryoEM reconstructions of LdcC, LdcI-LARA and LdcIa. FIG +49 54 LdcIa protein 3D cryoEM reconstructions of LdcC, LdcI-LARA and LdcIa. FIG +8 14 cryoEM experimental_method (A,C,E) cryoEM map of the LdcC (A), LdcIa (C) and LdcI-LARA (E) decamers with one protomer in light grey. FIG +15 18 map evidence (A,C,E) cryoEM map of the LdcC (A), LdcIa (C) and LdcI-LARA (E) decamers with one protomer in light grey. FIG +26 30 LdcC protein (A,C,E) cryoEM map of the LdcC (A), LdcIa (C) and LdcI-LARA (E) decamers with one protomer in light grey. FIG +36 41 LdcIa protein (A,C,E) cryoEM map of the LdcC (A), LdcIa (C) and LdcI-LARA (E) decamers with one protomer in light grey. FIG +50 59 LdcI-LARA complex_assembly (A,C,E) cryoEM map of the LdcC (A), LdcIa (C) and LdcI-LARA (E) decamers with one protomer in light grey. FIG +64 72 decamers oligomeric_state (A,C,E) cryoEM map of the LdcC (A), LdcIa (C) and LdcI-LARA (E) decamers with one protomer in light grey. FIG +82 90 protomer oligomeric_state (A,C,E) cryoEM map of the LdcC (A), LdcIa (C) and LdcI-LARA (E) decamers with one protomer in light grey. FIG +19 28 protomers oligomeric_state In the rest of the protomers, the wing, core and C-terminal domains are colored from light to dark in shades of green for LdcC (A), pink for LdcIa (C) and blue for LdcI in LdcI-LARA (E). FIG +34 38 wing structure_element In the rest of the protomers, the wing, core and C-terminal domains are colored from light to dark in shades of green for LdcC (A), pink for LdcIa (C) and blue for LdcI in LdcI-LARA (E). FIG +40 44 core structure_element In the rest of the protomers, the wing, core and C-terminal domains are colored from light to dark in shades of green for LdcC (A), pink for LdcIa (C) and blue for LdcI in LdcI-LARA (E). FIG +49 67 C-terminal domains structure_element In the rest of the protomers, the wing, core and C-terminal domains are colored from light to dark in shades of green for LdcC (A), pink for LdcIa (C) and blue for LdcI in LdcI-LARA (E). FIG +122 126 LdcC protein In the rest of the protomers, the wing, core and C-terminal domains are colored from light to dark in shades of green for LdcC (A), pink for LdcIa (C) and blue for LdcI in LdcI-LARA (E). FIG +141 146 LdcIa protein In the rest of the protomers, the wing, core and C-terminal domains are colored from light to dark in shades of green for LdcC (A), pink for LdcIa (C) and blue for LdcI in LdcI-LARA (E). FIG +164 168 LdcI protein In the rest of the protomers, the wing, core and C-terminal domains are colored from light to dark in shades of green for LdcC (A), pink for LdcIa (C) and blue for LdcI in LdcI-LARA (E). FIG +172 181 LdcI-LARA complex_assembly In the rest of the protomers, the wing, core and C-terminal domains are colored from light to dark in shades of green for LdcC (A), pink for LdcIa (C) and blue for LdcI in LdcI-LARA (E). FIG +13 24 LARA domain structure_element In (E), the LARA domain density is shown in dark grey. FIG +4 12 monomers oligomeric_state Two monomers making a dimer are delineated. FIG +22 27 dimer oligomeric_state Two monomers making a dimer are delineated. FIG +28 36 protomer oligomeric_state Scale bar 50 Å. (B,D,F) One protomer from the cryoEM map of the LdcC (B), LdcIa (D) and LdcI-LARA (F) in light grey with the pseudoatomic model represented as cartoons and colored as the densities in (A,C,E). FIG +46 52 cryoEM experimental_method Scale bar 50 Å. (B,D,F) One protomer from the cryoEM map of the LdcC (B), LdcIa (D) and LdcI-LARA (F) in light grey with the pseudoatomic model represented as cartoons and colored as the densities in (A,C,E). FIG +53 56 map evidence Scale bar 50 Å. (B,D,F) One protomer from the cryoEM map of the LdcC (B), LdcIa (D) and LdcI-LARA (F) in light grey with the pseudoatomic model represented as cartoons and colored as the densities in (A,C,E). FIG +64 68 LdcC protein Scale bar 50 Å. (B,D,F) One protomer from the cryoEM map of the LdcC (B), LdcIa (D) and LdcI-LARA (F) in light grey with the pseudoatomic model represented as cartoons and colored as the densities in (A,C,E). FIG +74 79 LdcIa protein Scale bar 50 Å. (B,D,F) One protomer from the cryoEM map of the LdcC (B), LdcIa (D) and LdcI-LARA (F) in light grey with the pseudoatomic model represented as cartoons and colored as the densities in (A,C,E). FIG +88 97 LdcI-LARA complex_assembly Scale bar 50 Å. (B,D,F) One protomer from the cryoEM map of the LdcC (B), LdcIa (D) and LdcI-LARA (F) in light grey with the pseudoatomic model represented as cartoons and colored as the densities in (A,C,E). FIG +125 143 pseudoatomic model evidence Scale bar 50 Å. (B,D,F) One protomer from the cryoEM map of the LdcC (B), LdcIa (D) and LdcI-LARA (F) in light grey with the pseudoatomic model represented as cartoons and colored as the densities in (A,C,E). FIG +0 13 Superposition experimental_method Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and LdcIa colored as in Fig. 1, and the crystal structure of LdcIi in shades of yellow. FIG +21 40 pseudoatomic models evidence Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and LdcIa colored as in Fig. 1, and the crystal structure of LdcIi in shades of yellow. FIG +44 48 LdcC protein Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and LdcIa colored as in Fig. 1, and the crystal structure of LdcIi in shades of yellow. FIG +50 54 LdcI protein Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and LdcIa colored as in Fig. 1, and the crystal structure of LdcIi in shades of yellow. FIG +60 69 LdcI-LARA complex_assembly Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and LdcIa colored as in Fig. 1, and the crystal structure of LdcIi in shades of yellow. FIG +74 79 LdcIa protein Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and LdcIa colored as in Fig. 1, and the crystal structure of LdcIi in shades of yellow. FIG +110 127 crystal structure evidence Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and LdcIa colored as in Fig. 1, and the crystal structure of LdcIi in shades of yellow. FIG +131 136 LdcIi protein Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and LdcIa colored as in Fig. 1, and the crystal structure of LdcIi in shades of yellow. FIG +20 25 rings structure_element Only one of the two rings of the double toroid is shown for clarity. FIG +33 46 double toroid structure_element Only one of the two rings of the double toroid is shown for clarity. FIG +40 46 region structure_element The dashed circle indicates the central region that remains virtually unchanged between all the structures, while the periphery undergoes visible movements. FIG +96 106 structures evidence The dashed circle indicates the central region that remains virtually unchanged between all the structures, while the periphery undergoes visible movements. FIG +44 55 active site site Conformational rearrangements in the enzyme active site. FIG +4 9 LdcIi protein (A) LdcIi crystal structure, with one ring represented as a grey surface and the second as a cartoon. FIG +10 27 crystal structure evidence (A) LdcIi crystal structure, with one ring represented as a grey surface and the second as a cartoon. FIG +38 42 ring structure_element (A) LdcIi crystal structure, with one ring represented as a grey surface and the second as a cartoon. FIG +2 9 monomer oligomeric_state A monomer with its PLP cofactor is delineated. FIG +19 22 PLP chemical A monomer with its PLP cofactor is delineated. FIG +4 7 PLP chemical The PLP moieties of the cartoon ring are shown in red. FIG +32 36 ring structure_element The PLP moieties of the cartoon ring are shown in red. FIG +9 14 LdcIi protein (B) The LdcIi dimer extracted from the crystal structure of the decamer. FIG +15 20 dimer oligomeric_state (B) The LdcIi dimer extracted from the crystal structure of the decamer. FIG +40 57 crystal structure evidence (B) The LdcIi dimer extracted from the crystal structure of the decamer. FIG +65 72 decamer oligomeric_state (B) The LdcIi dimer extracted from the crystal structure of the decamer. FIG +4 11 monomer oligomeric_state One monomer is colored in shades of yellow as in Figs 1 and 2, while the monomer related by C2 symmetry is grey. FIG +73 80 monomer oligomeric_state One monomer is colored in shades of yellow as in Figs 1 and 2, while the monomer related by C2 symmetry is grey. FIG +4 7 PLP chemical The PLP is red. FIG +4 15 active site site The active site is boxed. FIG +18 22 LdcI protein Stretching of the LdcI monomer upon pH-dependent enzyme activation and LARA binding. FIG +23 30 monomer oligomeric_state Stretching of the LdcI monomer upon pH-dependent enzyme activation and LARA binding. FIG +36 48 pH-dependent protein_state Stretching of the LdcI monomer upon pH-dependent enzyme activation and LARA binding. FIG +71 75 LARA structure_element Stretching of the LdcI monomer upon pH-dependent enzyme activation and LARA binding. FIG +26 45 pseudoatomic models evidence (A–C) A slice through the pseudoatomic models of the LdcI monomers extracted from the superimposed decamers (Fig. 2) The rectangle indicates the regions enlarged in (D–F). FIG +53 57 LdcI protein (A–C) A slice through the pseudoatomic models of the LdcI monomers extracted from the superimposed decamers (Fig. 2) The rectangle indicates the regions enlarged in (D–F). FIG +58 66 monomers oligomeric_state (A–C) A slice through the pseudoatomic models of the LdcI monomers extracted from the superimposed decamers (Fig. 2) The rectangle indicates the regions enlarged in (D–F). FIG +86 98 superimposed experimental_method (A–C) A slice through the pseudoatomic models of the LdcI monomers extracted from the superimposed decamers (Fig. 2) The rectangle indicates the regions enlarged in (D–F). FIG +99 107 decamers oligomeric_state (A–C) A slice through the pseudoatomic models of the LdcI monomers extracted from the superimposed decamers (Fig. 2) The rectangle indicates the regions enlarged in (D–F). FIG +13 18 LdcIi protein (A) compares LdcIi (yellow) and LdcIa (pink), (B) compares LdcIa (pink) and LdcI-LARA (blue), and (C) compares LdcIi (yellow), LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to show the progressive stretching described in the text. FIG +32 37 LdcIa protein (A) compares LdcIi (yellow) and LdcIa (pink), (B) compares LdcIa (pink) and LdcI-LARA (blue), and (C) compares LdcIi (yellow), LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to show the progressive stretching described in the text. FIG +59 64 LdcIa protein (A) compares LdcIi (yellow) and LdcIa (pink), (B) compares LdcIa (pink) and LdcI-LARA (blue), and (C) compares LdcIi (yellow), LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to show the progressive stretching described in the text. FIG +76 85 LdcI-LARA complex_assembly (A) compares LdcIi (yellow) and LdcIa (pink), (B) compares LdcIa (pink) and LdcI-LARA (blue), and (C) compares LdcIi (yellow), LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to show the progressive stretching described in the text. FIG +111 116 LdcIi protein (A) compares LdcIi (yellow) and LdcIa (pink), (B) compares LdcIa (pink) and LdcI-LARA (blue), and (C) compares LdcIi (yellow), LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to show the progressive stretching described in the text. FIG +127 132 LdcIa protein (A) compares LdcIi (yellow) and LdcIa (pink), (B) compares LdcIa (pink) and LdcI-LARA (blue), and (C) compares LdcIi (yellow), LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to show the progressive stretching described in the text. FIG +144 153 LdcI-LARA complex_assembly (A) compares LdcIi (yellow) and LdcIa (pink), (B) compares LdcIa (pink) and LdcI-LARA (blue), and (C) compares LdcIi (yellow), LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to show the progressive stretching described in the text. FIG +4 10 cryoEM experimental_method The cryoEM density of the LARA domain is represented as a grey surface to show the position of the binding site and the direction of the movement. FIG +11 18 density evidence The cryoEM density of the LARA domain is represented as a grey surface to show the position of the binding site and the direction of the movement. FIG +26 37 LARA domain structure_element The cryoEM density of the LARA domain is represented as a grey surface to show the position of the binding site and the direction of the movement. FIG +99 111 binding site site The cryoEM density of the LARA domain is represented as a grey surface to show the position of the binding site and the direction of the movement. FIG +29 32 CTD structure_element (D–F) Inserts zooming at the CTD part in proximity of the LARA binding site. FIG +58 75 LARA binding site site (D–F) Inserts zooming at the CTD part in proximity of the LARA binding site. FIG +16 21 LdcIC mutant Analysis of the LdcIC and LdcCI chimeras. FIG +26 31 LdcCI mutant Analysis of the LdcIC and LdcCI chimeras. FIG +32 40 chimeras mutant Analysis of the LdcIC and LdcCI chimeras. FIG +24 43 pseudoatomic models evidence (A) A slice through the pseudoatomic models of the LdcIa (purple) and LdcC (green) monomers extracted from the superimposed decamers (Fig. 2). (B) The C-terminal β-sheet in LdcIa and LdcC enlarged from (A,C) Exchanged primary sequences (capital letters) and their immediate vicinity (lower case letters) colored as in (A,B), with the corresponding secondary structure elements and the amino acid numbering shown. FIG +51 56 LdcIa protein (A) A slice through the pseudoatomic models of the LdcIa (purple) and LdcC (green) monomers extracted from the superimposed decamers (Fig. 2). (B) The C-terminal β-sheet in LdcIa and LdcC enlarged from (A,C) Exchanged primary sequences (capital letters) and their immediate vicinity (lower case letters) colored as in (A,B), with the corresponding secondary structure elements and the amino acid numbering shown. FIG +70 74 LdcC protein (A) A slice through the pseudoatomic models of the LdcIa (purple) and LdcC (green) monomers extracted from the superimposed decamers (Fig. 2). (B) The C-terminal β-sheet in LdcIa and LdcC enlarged from (A,C) Exchanged primary sequences (capital letters) and their immediate vicinity (lower case letters) colored as in (A,B), with the corresponding secondary structure elements and the amino acid numbering shown. FIG +83 91 monomers oligomeric_state (A) A slice through the pseudoatomic models of the LdcIa (purple) and LdcC (green) monomers extracted from the superimposed decamers (Fig. 2). (B) The C-terminal β-sheet in LdcIa and LdcC enlarged from (A,C) Exchanged primary sequences (capital letters) and their immediate vicinity (lower case letters) colored as in (A,B), with the corresponding secondary structure elements and the amino acid numbering shown. FIG +111 123 superimposed experimental_method (A) A slice through the pseudoatomic models of the LdcIa (purple) and LdcC (green) monomers extracted from the superimposed decamers (Fig. 2). (B) The C-terminal β-sheet in LdcIa and LdcC enlarged from (A,C) Exchanged primary sequences (capital letters) and their immediate vicinity (lower case letters) colored as in (A,B), with the corresponding secondary structure elements and the amino acid numbering shown. FIG +124 132 decamers oligomeric_state (A) A slice through the pseudoatomic models of the LdcIa (purple) and LdcC (green) monomers extracted from the superimposed decamers (Fig. 2). (B) The C-terminal β-sheet in LdcIa and LdcC enlarged from (A,C) Exchanged primary sequences (capital letters) and their immediate vicinity (lower case letters) colored as in (A,B), with the corresponding secondary structure elements and the amino acid numbering shown. FIG +162 169 β-sheet structure_element (A) A slice through the pseudoatomic models of the LdcIa (purple) and LdcC (green) monomers extracted from the superimposed decamers (Fig. 2). (B) The C-terminal β-sheet in LdcIa and LdcC enlarged from (A,C) Exchanged primary sequences (capital letters) and their immediate vicinity (lower case letters) colored as in (A,B), with the corresponding secondary structure elements and the amino acid numbering shown. FIG +173 178 LdcIa protein (A) A slice through the pseudoatomic models of the LdcIa (purple) and LdcC (green) monomers extracted from the superimposed decamers (Fig. 2). (B) The C-terminal β-sheet in LdcIa and LdcC enlarged from (A,C) Exchanged primary sequences (capital letters) and their immediate vicinity (lower case letters) colored as in (A,B), with the corresponding secondary structure elements and the amino acid numbering shown. FIG +183 187 LdcC protein (A) A slice through the pseudoatomic models of the LdcIa (purple) and LdcC (green) monomers extracted from the superimposed decamers (Fig. 2). (B) The C-terminal β-sheet in LdcIa and LdcC enlarged from (A,C) Exchanged primary sequences (capital letters) and their immediate vicinity (lower case letters) colored as in (A,B), with the corresponding secondary structure elements and the amino acid numbering shown. FIG +55 64 wild type protein_state (D,E) A gallery of negative stain EM images of (D) the wild type LdcI-RavA cage and (E) the LdcCI-RavA cage-like particles. (F) Some representative class averages of the LdcCI-RavA cage-like particles. FIG +65 74 LdcI-RavA complex_assembly (D,E) A gallery of negative stain EM images of (D) the wild type LdcI-RavA cage and (E) the LdcCI-RavA cage-like particles. (F) Some representative class averages of the LdcCI-RavA cage-like particles. FIG +92 122 LdcCI-RavA cage-like particles mutant (D,E) A gallery of negative stain EM images of (D) the wild type LdcI-RavA cage and (E) the LdcCI-RavA cage-like particles. (F) Some representative class averages of the LdcCI-RavA cage-like particles. FIG +170 200 LdcCI-RavA cage-like particles mutant (D,E) A gallery of negative stain EM images of (D) the wild type LdcI-RavA cage and (E) the LdcCI-RavA cage-like particles. (F) Some representative class averages of the LdcCI-RavA cage-like particles. FIG +0 17 Sequence analysis experimental_method Sequence analysis of enterobacterial lysine decarboxylases. FIG +21 36 enterobacterial taxonomy_domain Sequence analysis of enterobacterial lysine decarboxylases. FIG +37 58 lysine decarboxylases protein_type Sequence analysis of enterobacterial lysine decarboxylases. FIG +4 27 Maximum likelihood tree evidence (A) Maximum likelihood tree with the “LdcC-like” and the “LdcI-like” groups highlighted in green and pink, respectively. FIG +38 47 LdcC-like protein_type (A) Maximum likelihood tree with the “LdcC-like” and the “LdcI-like” groups highlighted in green and pink, respectively. FIG +58 67 LdcI-like protein_type (A) Maximum likelihood tree with the “LdcC-like” and the “LdcI-like” groups highlighted in green and pink, respectively. FIG +27 36 LdcI-like protein_type (B) Analysis of consensus “LdcI-like” and “LdcC-like” sequences around the first and second C-terminal β-strands. FIG +43 52 LdcC-like protein_type (B) Analysis of consensus “LdcI-like” and “LdcC-like” sequences around the first and second C-terminal β-strands. FIG +103 112 β-strands structure_element (B) Analysis of consensus “LdcI-like” and “LdcC-like” sequences around the first and second C-terminal β-strands. FIG +16 23 E. coli species Numbering as in E. coli. FIG +28 32 LdcI protein (C) Signature sequences of LdcI and LdcC in the C-terminal β-sheet. FIG +37 41 LdcC protein (C) Signature sequences of LdcI and LdcC in the C-terminal β-sheet. FIG +60 67 β-sheet structure_element (C) Signature sequences of LdcI and LdcC in the C-terminal β-sheet. FIG +116 122 cryoEM experimental_method Polarity differences are highlighted. (D) Position and nature of these differences at the surface of the respective cryoEM maps with the color code as in B. See also Fig. S7 and Tables S3 and S4. FIG +123 127 maps evidence Polarity differences are highlighted. (D) Position and nature of these differences at the surface of the respective cryoEM maps with the color code as in B. See also Fig. S7 and Tables S3 and S4. FIG diff --git a/annotation_CSV/PMC4833862.csv b/annotation_CSV/PMC4833862.csv new file mode 100644 index 0000000000000000000000000000000000000000..f9979213ab9fc3b0cf129d57725bb504d5b24a5c --- /dev/null +++ b/annotation_CSV/PMC4833862.csv @@ -0,0 +1,1045 @@ +anno_start anno_end anno_text entity_type sentence section +4 11 dynamic protein_state The dynamic organization of fungal acetyl-CoA carboxylase TITLE +28 34 fungal taxonomy_domain The dynamic organization of fungal acetyl-CoA carboxylase TITLE +35 57 acetyl-CoA carboxylase protein_type The dynamic organization of fungal acetyl-CoA carboxylase TITLE +0 23 Acetyl-CoA carboxylases protein_type Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. ABSTRACT +25 29 ACCs protein_type Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. ABSTRACT +91 94 ATP chemical Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. ABSTRACT +122 132 acetyl-CoA chemical Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. ABSTRACT +136 147 malonyl-CoA chemical Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. ABSTRACT +0 10 Eukaryotic taxonomy_domain Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. ABSTRACT +11 15 ACCs protein_type Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. ABSTRACT +20 45 single-chain multienzymes protein_type Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. ABSTRACT +72 85 non-catalytic protein_state Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. ABSTRACT +86 100 central domain structure_element Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. ABSTRACT +102 104 CD structure_element Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. ABSTRACT +121 124 ACC protein_type Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. ABSTRACT +19 36 crystal structure evidence Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. ABSTRACT +44 49 yeast taxonomy_domain Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. ABSTRACT +50 53 ACC protein_type Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. ABSTRACT +54 56 CD structure_element Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. ABSTRACT +2 17 regulatory loop structure_element A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. ABSTRACT +28 42 phosphorylated protein_state A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. ABSTRACT +65 85 phosphorylation site site A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. ABSTRACT +89 95 fungal taxonomy_domain A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. ABSTRACT +96 99 ACC protein_type A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. ABSTRACT +146 148 CD structure_element A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. ABSTRACT +14 19 yeast taxonomy_domain Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. ABSTRACT +20 22 CD structure_element Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. ABSTRACT +23 32 structure evidence Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. ABSTRACT +78 94 larger fragments mutant Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. ABSTRACT +101 107 intact protein_state Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. ABSTRACT +108 112 ACCs protein_type Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. ABSTRACT +162 169 dynamic protein_state Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. ABSTRACT +170 176 fungal taxonomy_domain Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. ABSTRACT +177 180 ACC protein_type Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. ABSTRACT +23 35 carboxylases protein_type In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control. ABSTRACT +133 135 CD structure_element In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control. ABSTRACT +142 157 phosphorylation ptm In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control. ABSTRACT +1 24 Acetyl-CoA carboxylases protein_type Acetyl-CoA carboxylases are central regulatory hubs of fatty acid metabolism and are important targets for drug development in obesity and cancer. ABSTRACT +59 73 highly dynamic protein_state Here, the authors demonstrate that the regulation of these highly dynamic enzymes in fungi is governed by a mechanism based on phosphorylation-dependent conformational variability. ABSTRACT +74 81 enzymes protein_type Here, the authors demonstrate that the regulation of these highly dynamic enzymes in fungi is governed by a mechanism based on phosphorylation-dependent conformational variability. ABSTRACT +85 90 fungi taxonomy_domain Here, the authors demonstrate that the regulation of these highly dynamic enzymes in fungi is governed by a mechanism based on phosphorylation-dependent conformational variability. ABSTRACT +127 142 phosphorylation ptm Here, the authors demonstrate that the regulation of these highly dynamic enzymes in fungi is governed by a mechanism based on phosphorylation-dependent conformational variability. ABSTRACT +0 40 Biotin-dependent acetyl-CoA carboxylases protein_type Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase. INTRO +42 46 ACCs protein_type Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase. INTRO +88 91 ATP chemical Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase. INTRO +119 129 acetyl-CoA chemical Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase. INTRO +133 144 malonyl-CoA chemical Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase. INTRO +227 238 fatty acids chemical Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase. INTRO +243 262 fatty-acid synthase protein_type Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase. INTRO +66 69 ACC protein_type By catalysing this rate-limiting step in fatty-acid biosynthesis, ACC plays a key role in anabolic metabolism. INTRO +0 36 ACC inhibition and knock-out studies experimental_method ACC inhibition and knock-out studies show the potential of targeting ACC for treatment of the metabolic syndrome. INTRO +69 72 ACC protein_type ACC inhibition and knock-out studies show the potential of targeting ACC for treatment of the metabolic syndrome. INTRO +22 25 ACC protein_type Furthermore, elevated ACC activity is observed in malignant tumours. INTRO +22 25 ACC protein_type A direct link between ACC and cancer is provided by cancer-associated mutations in the breast cancer susceptibility gene 1 (BRCA1), which relieve inhibitory interactions of BRCA1 with ACC. INTRO +70 79 mutations mutant A direct link between ACC and cancer is provided by cancer-associated mutations in the breast cancer susceptibility gene 1 (BRCA1), which relieve inhibitory interactions of BRCA1 with ACC. INTRO +87 122 breast cancer susceptibility gene 1 protein A direct link between ACC and cancer is provided by cancer-associated mutations in the breast cancer susceptibility gene 1 (BRCA1), which relieve inhibitory interactions of BRCA1 with ACC. INTRO +124 129 BRCA1 protein A direct link between ACC and cancer is provided by cancer-associated mutations in the breast cancer susceptibility gene 1 (BRCA1), which relieve inhibitory interactions of BRCA1 with ACC. INTRO +173 178 BRCA1 protein A direct link between ACC and cancer is provided by cancer-associated mutations in the breast cancer susceptibility gene 1 (BRCA1), which relieve inhibitory interactions of BRCA1 with ACC. INTRO +184 187 ACC protein_type A direct link between ACC and cancer is provided by cancer-associated mutations in the breast cancer susceptibility gene 1 (BRCA1), which relieve inhibitory interactions of BRCA1 with ACC. INTRO +6 9 ACC protein_type Thus, ACC is a relevant drug target for type 2 diabetes and cancer. INTRO +0 9 Microbial taxonomy_domain Microbial ACCs are also the principal target of antifungal and antibiotic compounds, such as Soraphen A. INTRO +10 14 ACCs protein_type Microbial ACCs are also the principal target of antifungal and antibiotic compounds, such as Soraphen A. INTRO +93 103 Soraphen A chemical Microbial ACCs are also the principal target of antifungal and antibiotic compounds, such as Soraphen A. INTRO +47 51 ACCs protein_type The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). INTRO +102 118 Escherichia coli species The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). INTRO +120 127 E. coli species The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). INTRO +129 132 ACC protein_type The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). INTRO +134 152 Biotin carboxylase protein_type The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). INTRO +154 156 BC protein_type The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). INTRO +172 175 ATP chemical The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). INTRO +205 211 biotin chemical The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). INTRO +254 285 biotin carboxyl carrier protein protein_type The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). INTRO +287 291 BCCP protein_type The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). INTRO +0 19 Carboxyltransferase protein_type Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. INTRO +21 23 CT protein_type Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. INTRO +49 57 carboxyl chemical Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. INTRO +69 82 carboxybiotin chemical Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. INTRO +86 96 acetyl-CoA chemical Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. INTRO +106 117 malonyl-CoA chemical Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. INTRO +119 130 Prokaryotic taxonomy_domain Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. INTRO +131 135 ACCs protein_type Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. INTRO +140 149 transient protein_state Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. INTRO +175 177 BC protein_type Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. INTRO +179 181 CT protein_type Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. INTRO +186 190 BCCP protein_type Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. INTRO +0 10 Eukaryotic taxonomy_domain Eukaryotic ACCs, instead, are multienzymes, which integrate all functional components into a single polypeptide chain of ∼2,300 amino acids. INTRO +11 15 ACCs protein_type Eukaryotic ACCs, instead, are multienzymes, which integrate all functional components into a single polypeptide chain of ∼2,300 amino acids. INTRO +30 42 multienzymes protein_type Eukaryotic ACCs, instead, are multienzymes, which integrate all functional components into a single polypeptide chain of ∼2,300 amino acids. INTRO +0 5 Human species Human ACC occurs in two closely related isoforms, ACC1 and 2, located in the cytosol and at the outer mitochondrial membrane, respectively. INTRO +6 9 ACC protein_type Human ACC occurs in two closely related isoforms, ACC1 and 2, located in the cytosol and at the outer mitochondrial membrane, respectively. INTRO +40 48 isoforms protein_state Human ACC occurs in two closely related isoforms, ACC1 and 2, located in the cytosol and at the outer mitochondrial membrane, respectively. INTRO +50 54 ACC1 protein Human ACC occurs in two closely related isoforms, ACC1 and 2, located in the cytosol and at the outer mitochondrial membrane, respectively. INTRO +59 60 2 protein Human ACC occurs in two closely related isoforms, ACC1 and 2, located in the cytosol and at the outer mitochondrial membrane, respectively. INTRO +29 43 ACC components structure_element In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). INTRO +45 55 eukaryotic taxonomy_domain In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). INTRO +56 60 ACCs protein_type In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). INTRO +73 86 non-catalytic protein_state In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). INTRO +87 94 regions structure_element In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). INTRO +106 120 central domain structure_element In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). INTRO +122 124 CD structure_element In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). INTRO +134 158 BC–CT interaction domain structure_element In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). INTRO +160 162 BT structure_element In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). INTRO +4 6 CD structure_element The CD comprises one-third of the protein and is a unique feature of eukaryotic ACCs without homologues in other proteins. INTRO +51 68 unique feature of protein_state The CD comprises one-third of the protein and is a unique feature of eukaryotic ACCs without homologues in other proteins. INTRO +69 79 eukaryotic taxonomy_domain The CD comprises one-third of the protein and is a unique feature of eukaryotic ACCs without homologues in other proteins. INTRO +80 84 ACCs protein_type The CD comprises one-third of the protein and is a unique feature of eukaryotic ACCs without homologues in other proteins. INTRO +67 82 phosphorylation ptm The function of this domain remains poorly characterized, although phosphorylation of several serine residues in the CD regulates ACC activity. INTRO +94 100 serine residue_name The function of this domain remains poorly characterized, although phosphorylation of several serine residues in the CD regulates ACC activity. INTRO +117 119 CD structure_element The function of this domain remains poorly characterized, although phosphorylation of several serine residues in the CD regulates ACC activity. INTRO +130 133 ACC protein_type The function of this domain remains poorly characterized, although phosphorylation of several serine residues in the CD regulates ACC activity. INTRO +4 6 BT structure_element The BT domain has been visualized in bacterial carboxylases, where it mediates contacts between α- and β-subunits. INTRO +37 46 bacterial taxonomy_domain The BT domain has been visualized in bacterial carboxylases, where it mediates contacts between α- and β-subunits. INTRO +47 59 carboxylases protein_type The BT domain has been visualized in bacterial carboxylases, where it mediates contacts between α- and β-subunits. INTRO +96 98 α- structure_element The BT domain has been visualized in bacterial carboxylases, where it mediates contacts between α- and β-subunits. INTRO +103 113 β-subunits structure_element The BT domain has been visualized in bacterial carboxylases, where it mediates contacts between α- and β-subunits. INTRO +0 18 Structural studies experimental_method Structural studies on the functional architecture of intact ACCs have been hindered by their huge size and pronounced dynamics, as well as the transient assembly mode of bacterial ACCs. INTRO +53 59 intact protein_state Structural studies on the functional architecture of intact ACCs have been hindered by their huge size and pronounced dynamics, as well as the transient assembly mode of bacterial ACCs. INTRO +60 64 ACCs protein_type Structural studies on the functional architecture of intact ACCs have been hindered by their huge size and pronounced dynamics, as well as the transient assembly mode of bacterial ACCs. INTRO +143 152 transient protein_state Structural studies on the functional architecture of intact ACCs have been hindered by their huge size and pronounced dynamics, as well as the transient assembly mode of bacterial ACCs. INTRO +170 179 bacterial taxonomy_domain Structural studies on the functional architecture of intact ACCs have been hindered by their huge size and pronounced dynamics, as well as the transient assembly mode of bacterial ACCs. INTRO +180 184 ACCs protein_type Structural studies on the functional architecture of intact ACCs have been hindered by their huge size and pronounced dynamics, as well as the transient assembly mode of bacterial ACCs. INTRO +9 27 crystal structures evidence However, crystal structures of individual components or domains from prokaryotic and eukaryotic ACCs, respectively, have been solved. INTRO +69 80 prokaryotic taxonomy_domain However, crystal structures of individual components or domains from prokaryotic and eukaryotic ACCs, respectively, have been solved. INTRO +85 95 eukaryotic taxonomy_domain However, crystal structures of individual components or domains from prokaryotic and eukaryotic ACCs, respectively, have been solved. INTRO +96 100 ACCs protein_type However, crystal structures of individual components or domains from prokaryotic and eukaryotic ACCs, respectively, have been solved. INTRO +4 27 structure determination experimental_method The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. INTRO +35 46 holoenzymes protein_state The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. INTRO +50 59 bacterial taxonomy_domain The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. INTRO +60 89 biotin-dependent carboxylases protein_type The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. INTRO +97 101 lack protein_state The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. INTRO +121 123 CD structure_element The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. INTRO +137 157 pyruvate carboxylase protein_type The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. INTRO +159 161 PC protein_type The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. INTRO +164 189 propionyl-CoA carboxylase protein_type The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. INTRO +191 224 3-methyl-crotonyl-CoA carboxylase protein_type The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. INTRO +231 262 long-chain acyl-CoA carboxylase protein_type The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. INTRO +9 19 structures evidence In these structures, the BC and CT active sites are at distances between 40 and 80 Å, such that substrate transfer could be mediated solely by the mobility of the flexibly tethered BCCP. INTRO +25 27 BC protein_type In these structures, the BC and CT active sites are at distances between 40 and 80 Å, such that substrate transfer could be mediated solely by the mobility of the flexibly tethered BCCP. INTRO +32 34 CT protein_type In these structures, the BC and CT active sites are at distances between 40 and 80 Å, such that substrate transfer could be mediated solely by the mobility of the flexibly tethered BCCP. INTRO +35 47 active sites site In these structures, the BC and CT active sites are at distances between 40 and 80 Å, such that substrate transfer could be mediated solely by the mobility of the flexibly tethered BCCP. INTRO +163 180 flexibly tethered protein_state In these structures, the BC and CT active sites are at distances between 40 and 80 Å, such that substrate transfer could be mediated solely by the mobility of the flexibly tethered BCCP. INTRO +181 185 BCCP protein_type In these structures, the BC and CT active sites are at distances between 40 and 80 Å, such that substrate transfer could be mediated solely by the mobility of the flexibly tethered BCCP. INTRO +0 5 Human species Human ACC1 is regulated allosterically, via specific protein–protein interactions, and by reversible phosphorylation. INTRO +6 10 ACC1 protein Human ACC1 is regulated allosterically, via specific protein–protein interactions, and by reversible phosphorylation. INTRO +14 38 regulated allosterically protein_state Human ACC1 is regulated allosterically, via specific protein–protein interactions, and by reversible phosphorylation. INTRO +101 116 phosphorylation ptm Human ACC1 is regulated allosterically, via specific protein–protein interactions, and by reversible phosphorylation. INTRO +26 31 human species Dynamic polymerization of human ACC1 is linked to increased activity and is regulated allosterically by the activator citrate and the inhibitor palmitate, or by binding of the small protein MIG-12 (ref.). INTRO +32 36 ACC1 protein Dynamic polymerization of human ACC1 is linked to increased activity and is regulated allosterically by the activator citrate and the inhibitor palmitate, or by binding of the small protein MIG-12 (ref.). INTRO +76 100 regulated allosterically protein_state Dynamic polymerization of human ACC1 is linked to increased activity and is regulated allosterically by the activator citrate and the inhibitor palmitate, or by binding of the small protein MIG-12 (ref.). INTRO +118 125 citrate chemical Dynamic polymerization of human ACC1 is linked to increased activity and is regulated allosterically by the activator citrate and the inhibitor palmitate, or by binding of the small protein MIG-12 (ref.). INTRO +144 153 palmitate chemical Dynamic polymerization of human ACC1 is linked to increased activity and is regulated allosterically by the activator citrate and the inhibitor palmitate, or by binding of the small protein MIG-12 (ref.). INTRO +190 196 MIG-12 protein Dynamic polymerization of human ACC1 is linked to increased activity and is regulated allosterically by the activator citrate and the inhibitor palmitate, or by binding of the small protein MIG-12 (ref.). INTRO +0 5 Human species Human ACC1 is further regulated by specific phosphorylation-dependent binding of BRCA1 to Ser1263 in the CD. INTRO +6 10 ACC1 protein Human ACC1 is further regulated by specific phosphorylation-dependent binding of BRCA1 to Ser1263 in the CD. INTRO +44 59 phosphorylation ptm Human ACC1 is further regulated by specific phosphorylation-dependent binding of BRCA1 to Ser1263 in the CD. INTRO +81 86 BRCA1 protein Human ACC1 is further regulated by specific phosphorylation-dependent binding of BRCA1 to Ser1263 in the CD. INTRO +90 97 Ser1263 residue_name_number Human ACC1 is further regulated by specific phosphorylation-dependent binding of BRCA1 to Ser1263 in the CD. INTRO +105 107 CD structure_element Human ACC1 is further regulated by specific phosphorylation-dependent binding of BRCA1 to Ser1263 in the CD. INTRO +0 5 BRCA1 protein BRCA1 binds only to the phosphorylated form of ACC1 and prevents ACC activation by phosphatase-mediated dephosphorylation. INTRO +24 38 phosphorylated protein_state BRCA1 binds only to the phosphorylated form of ACC1 and prevents ACC activation by phosphatase-mediated dephosphorylation. INTRO +47 51 ACC1 protein BRCA1 binds only to the phosphorylated form of ACC1 and prevents ACC activation by phosphatase-mediated dephosphorylation. INTRO +65 68 ACC protein_type BRCA1 binds only to the phosphorylated form of ACC1 and prevents ACC activation by phosphatase-mediated dephosphorylation. INTRO +83 94 phosphatase protein_type BRCA1 binds only to the phosphorylated form of ACC1 and prevents ACC activation by phosphatase-mediated dephosphorylation. INTRO +13 28 phosphorylation ptm Furthermore, phosphorylation by AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA) leads to a decrease in ACC1 activity. INTRO +32 60 AMP-activated protein kinase protein Furthermore, phosphorylation by AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA) leads to a decrease in ACC1 activity. INTRO +62 66 AMPK protein Furthermore, phosphorylation by AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA) leads to a decrease in ACC1 activity. INTRO +72 101 cAMP-dependent protein kinase protein Furthermore, phosphorylation by AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA) leads to a decrease in ACC1 activity. INTRO +103 106 PKA protein Furthermore, phosphorylation by AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA) leads to a decrease in ACC1 activity. INTRO +131 135 ACC1 protein Furthermore, phosphorylation by AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA) leads to a decrease in ACC1 activity. INTRO +0 4 AMPK protein AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201. INTRO +20 24 ACC1 protein AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201. INTRO +37 42 Ser80 residue_name_number AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201. INTRO +44 51 Ser1201 residue_name_number AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201. INTRO +56 63 Ser1216 residue_name_number AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201. INTRO +68 71 PKA protein AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201. INTRO +75 80 Ser78 residue_name_number AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201. INTRO +85 92 Ser1201 residue_name_number AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201. INTRO +31 35 ACC1 protein However, regulatory effects on ACC1 activity are mainly mediated by phosphorylation of Ser80 and Ser1201 (refs). INTRO +68 83 phosphorylation ptm However, regulatory effects on ACC1 activity are mainly mediated by phosphorylation of Ser80 and Ser1201 (refs). INTRO +87 92 Ser80 residue_name_number However, regulatory effects on ACC1 activity are mainly mediated by phosphorylation of Ser80 and Ser1201 (refs). INTRO +97 104 Ser1201 residue_name_number However, regulatory effects on ACC1 activity are mainly mediated by phosphorylation of Ser80 and Ser1201 (refs). INTRO +0 14 Phosphorylated protein_state Phosphorylated Ser80, which is highly conserved only in higher eukaryotes, presumably binds into the Soraphen A-binding pocket. INTRO +15 20 Ser80 residue_name_number Phosphorylated Ser80, which is highly conserved only in higher eukaryotes, presumably binds into the Soraphen A-binding pocket. INTRO +31 47 highly conserved protein_state Phosphorylated Ser80, which is highly conserved only in higher eukaryotes, presumably binds into the Soraphen A-binding pocket. INTRO +56 73 higher eukaryotes taxonomy_domain Phosphorylated Ser80, which is highly conserved only in higher eukaryotes, presumably binds into the Soraphen A-binding pocket. INTRO +101 126 Soraphen A-binding pocket site Phosphorylated Ser80, which is highly conserved only in higher eukaryotes, presumably binds into the Soraphen A-binding pocket. INTRO +15 22 Ser1201 residue_name_number The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes. INTRO +34 55 moderate conservation protein_state The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes. INTRO +63 80 higher eukaryotes taxonomy_domain The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes. INTRO +92 106 phosphorylated protein_state The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes. INTRO +107 114 Ser1216 residue_name_number The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes. INTRO +118 134 highly conserved protein_state The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes. INTRO +146 156 eukaryotes taxonomy_domain The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes. INTRO +22 29 Ser1216 residue_name_number However, no effect of Ser1216 phosphorylation on ACC activity has been reported in higher eukaryotes. INTRO +30 45 phosphorylation ptm However, no effect of Ser1216 phosphorylation on ACC activity has been reported in higher eukaryotes. INTRO +49 52 ACC protein_type However, no effect of Ser1216 phosphorylation on ACC activity has been reported in higher eukaryotes. INTRO +83 100 higher eukaryotes taxonomy_domain However, no effect of Ser1216 phosphorylation on ACC activity has been reported in higher eukaryotes. INTRO +4 10 fungal taxonomy_domain For fungal ACC, neither spontaneous nor inducible polymerization has been detected despite considerable sequence conservation to human ACC1. INTRO +11 14 ACC protein_type For fungal ACC, neither spontaneous nor inducible polymerization has been detected despite considerable sequence conservation to human ACC1. INTRO +129 134 human species For fungal ACC, neither spontaneous nor inducible polymerization has been detected despite considerable sequence conservation to human ACC1. INTRO +135 139 ACC1 protein For fungal ACC, neither spontaneous nor inducible polymerization has been detected despite considerable sequence conservation to human ACC1. INTRO +4 9 BRCA1 protein The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein–protein interactions of fungal ACC have been described. INTRO +22 35 phosphoserine residue_name The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein–protein interactions of fungal ACC have been described. INTRO +48 61 not conserved protein_state The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein–protein interactions of fungal ACC have been described. INTRO +65 71 fungal taxonomy_domain The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein–protein interactions of fungal ACC have been described. INTRO +72 75 ACC protein_type The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein–protein interactions of fungal ACC have been described. INTRO +140 146 fungal taxonomy_domain The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein–protein interactions of fungal ACC have been described. INTRO +147 150 ACC protein_type The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein–protein interactions of fungal ACC have been described. INTRO +3 8 yeast taxonomy_domain In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.). INTRO +9 12 ACC protein_type In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.). INTRO +14 35 phosphorylation sites site In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.). INTRO +60 64 Ser2 residue_name_number In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.). INTRO +66 72 Ser735 residue_name_number In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.). INTRO +74 81 Ser1148 residue_name_number In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.). INTRO +83 90 Ser1157 residue_name_number In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.). INTRO +95 102 Ser1162 residue_name_number In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.). INTRO +15 22 Ser1157 residue_name_number Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1. INTRO +26 42 highly conserved protein_state Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1. INTRO +46 52 fungal taxonomy_domain Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1. INTRO +53 56 ACC protein_type Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1. INTRO +61 70 aligns to experimental_method Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1. INTRO +71 78 Ser1216 residue_name_number Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1. INTRO +82 87 human species Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1. INTRO +88 92 ACC1 protein Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1. INTRO +4 19 phosphorylation ptm Its phosphorylation by the AMPK homologue SNF1 results in strongly reduced ACC activity. INTRO +27 31 AMPK protein Its phosphorylation by the AMPK homologue SNF1 results in strongly reduced ACC activity. INTRO +42 46 SNF1 protein Its phosphorylation by the AMPK homologue SNF1 results in strongly reduced ACC activity. INTRO +75 78 ACC protein_type Its phosphorylation by the AMPK homologue SNF1 results in strongly reduced ACC activity. INTRO +37 40 ACC protein_type Despite the outstanding relevance of ACC in primary metabolism and disease, the dynamic organization and regulation of the giant eukaryotic, and in particular fungal ACC, remain poorly characterized. INTRO +129 139 eukaryotic taxonomy_domain Despite the outstanding relevance of ACC in primary metabolism and disease, the dynamic organization and regulation of the giant eukaryotic, and in particular fungal ACC, remain poorly characterized. INTRO +159 165 fungal taxonomy_domain Despite the outstanding relevance of ACC in primary metabolism and disease, the dynamic organization and regulation of the giant eukaryotic, and in particular fungal ACC, remain poorly characterized. INTRO +166 169 ACC protein_type Despite the outstanding relevance of ACC in primary metabolism and disease, the dynamic organization and regulation of the giant eukaryotic, and in particular fungal ACC, remain poorly characterized. INTRO +20 29 structure evidence Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +33 57 Saccharomyces cerevisiae species Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +59 62 Sce species Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +64 67 ACC protein_type Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +68 70 CD structure_element Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +105 115 structures evidence Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +119 124 human species Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +126 129 Hsa species Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +131 134 ACC protein_type Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +135 137 CD structure_element Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +142 158 larger fragments mutant Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +162 168 fungal taxonomy_domain Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +169 172 ACC protein_type Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +178 201 Chaetomium thermophilum species Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +203 206 Cth species Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). INTRO +28 56 small-angle X-ray scattering experimental_method Integrating these data with small-angle X-ray scattering (SAXS) and electron microscopy (EM) observations yield a comprehensive representation of the dynamic structure and regulation of fungal ACC. INTRO +58 62 SAXS experimental_method Integrating these data with small-angle X-ray scattering (SAXS) and electron microscopy (EM) observations yield a comprehensive representation of the dynamic structure and regulation of fungal ACC. INTRO +68 87 electron microscopy experimental_method Integrating these data with small-angle X-ray scattering (SAXS) and electron microscopy (EM) observations yield a comprehensive representation of the dynamic structure and regulation of fungal ACC. INTRO +89 91 EM experimental_method Integrating these data with small-angle X-ray scattering (SAXS) and electron microscopy (EM) observations yield a comprehensive representation of the dynamic structure and regulation of fungal ACC. INTRO +186 192 fungal taxonomy_domain Integrating these data with small-angle X-ray scattering (SAXS) and electron microscopy (EM) observations yield a comprehensive representation of the dynamic structure and regulation of fungal ACC. INTRO +193 196 ACC protein_type Integrating these data with small-angle X-ray scattering (SAXS) and electron microscopy (EM) observations yield a comprehensive representation of the dynamic structure and regulation of fungal ACC. INTRO +24 29 yeast taxonomy_domain The organization of the yeast ACC CD RESULTS +30 33 ACC protein_type The organization of the yeast ACC CD RESULTS +34 36 CD structure_element The organization of the yeast ACC CD RESULTS +21 44 structure determination experimental_method First, we focused on structure determination of the 82-kDa CD. RESULTS +59 61 CD structure_element First, we focused on structure determination of the 82-kDa CD. RESULTS +4 21 crystal structure evidence The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). RESULTS +29 31 CD structure_element The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). RESULTS +35 41 SceACC protein The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). RESULTS +43 46 Sce species The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). RESULTS +46 48 CD structure_element The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). RESULTS +88 108 experimental phasing experimental_method The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). RESULTS +113 120 refined experimental_method The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). RESULTS +124 129 Rwork evidence The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). RESULTS +130 135 Rfree evidence The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). RESULTS +26 29 Sce species The overall extent of the SceCD is 70 by 75 Å (Fig. 1b and Supplementary Fig. 1a,b), and the attachment points of the N-terminal 26-residue linker to the BCCP domain and the C-terminal CT domain are separated by 46 Å (the N- and C termini are indicated with spheres in Fig. 1b). RESULTS +29 31 CD structure_element The overall extent of the SceCD is 70 by 75 Å (Fig. 1b and Supplementary Fig. 1a,b), and the attachment points of the N-terminal 26-residue linker to the BCCP domain and the C-terminal CT domain are separated by 46 Å (the N- and C termini are indicated with spheres in Fig. 1b). RESULTS +129 146 26-residue linker structure_element The overall extent of the SceCD is 70 by 75 Å (Fig. 1b and Supplementary Fig. 1a,b), and the attachment points of the N-terminal 26-residue linker to the BCCP domain and the C-terminal CT domain are separated by 46 Å (the N- and C termini are indicated with spheres in Fig. 1b). RESULTS +154 158 BCCP structure_element The overall extent of the SceCD is 70 by 75 Å (Fig. 1b and Supplementary Fig. 1a,b), and the attachment points of the N-terminal 26-residue linker to the BCCP domain and the C-terminal CT domain are separated by 46 Å (the N- and C termini are indicated with spheres in Fig. 1b). RESULTS +185 187 CT structure_element The overall extent of the SceCD is 70 by 75 Å (Fig. 1b and Supplementary Fig. 1a,b), and the attachment points of the N-terminal 26-residue linker to the BCCP domain and the C-terminal CT domain are separated by 46 Å (the N- and C termini are indicated with spheres in Fig. 1b). RESULTS +0 3 Sce species SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). RESULTS +3 5 CD structure_element SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). RESULTS +53 69 α-helical domain structure_element SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). RESULTS +71 74 CDN structure_element SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). RESULTS +91 122 four-helix bundle linker domain structure_element SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). RESULTS +124 127 CDL structure_element SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). RESULTS +146 173 α–β-fold C-terminal domains structure_element SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). RESULTS +175 179 CDC1 structure_element SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). RESULTS +180 184 CDC2 structure_element SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). RESULTS +0 3 CDN structure_element CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). RESULTS +20 27 C shape protein_state CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). RESULTS +56 81 regular four-helix bundle structure_element CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). RESULTS +83 88 Nα3-6 structure_element CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). RESULTS +110 125 helical hairpin structure_element CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). RESULTS +127 132 Nα8,9 structure_element CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). RESULTS +142 157 bridging region structure_element CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). RESULTS +172 179 helices structure_element CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). RESULTS +181 194 Nα1,2,7,10–12 structure_element CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). RESULTS +0 3 CDL structure_element CDL is composed of a small, irregular four-helix bundle (Lα1–4) and tightly interacts with the open face of CDC1 via an interface of 1,300 Å2 involving helices Lα3 and Lα4. RESULTS +21 55 small, irregular four-helix bundle structure_element CDL is composed of a small, irregular four-helix bundle (Lα1–4) and tightly interacts with the open face of CDC1 via an interface of 1,300 Å2 involving helices Lα3 and Lα4. RESULTS +57 62 Lα1–4 structure_element CDL is composed of a small, irregular four-helix bundle (Lα1–4) and tightly interacts with the open face of CDC1 via an interface of 1,300 Å2 involving helices Lα3 and Lα4. RESULTS +108 112 CDC1 structure_element CDL is composed of a small, irregular four-helix bundle (Lα1–4) and tightly interacts with the open face of CDC1 via an interface of 1,300 Å2 involving helices Lα3 and Lα4. RESULTS +120 129 interface site CDL is composed of a small, irregular four-helix bundle (Lα1–4) and tightly interacts with the open face of CDC1 via an interface of 1,300 Å2 involving helices Lα3 and Lα4. RESULTS +152 159 helices structure_element CDL is composed of a small, irregular four-helix bundle (Lα1–4) and tightly interacts with the open face of CDC1 via an interface of 1,300 Å2 involving helices Lα3 and Lα4. RESULTS +160 163 Lα3 structure_element CDL is composed of a small, irregular four-helix bundle (Lα1–4) and tightly interacts with the open face of CDC1 via an interface of 1,300 Å2 involving helices Lα3 and Lα4. RESULTS +168 171 Lα4 structure_element CDL is composed of a small, irregular four-helix bundle (Lα1–4) and tightly interacts with the open face of CDC1 via an interface of 1,300 Å2 involving helices Lα3 and Lα4. RESULTS +0 3 CDL structure_element CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between Lα2/α3 and the N-terminal end of Lα1, with an interface area of 400 Å2. RESULTS +27 30 CDN structure_element CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between Lα2/α3 and the N-terminal end of Lα1, with an interface area of 400 Å2. RESULTS +97 101 CDC2 structure_element CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between Lα2/α3 and the N-terminal end of Lα1, with an interface area of 400 Å2. RESULTS +108 112 loop structure_element CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between Lα2/α3 and the N-terminal end of Lα1, with an interface area of 400 Å2. RESULTS +121 127 Lα2/α3 structure_element CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between Lα2/α3 and the N-terminal end of Lα1, with an interface area of 400 Å2. RESULTS +154 157 Lα1 structure_element CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between Lα2/α3 and the N-terminal end of Lα1, with an interface area of 400 Å2. RESULTS +167 176 interface site CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between Lα2/α3 and the N-terminal end of Lα1, with an interface area of 400 Å2. RESULTS +0 4 CDC1 structure_element CDC1/CDC2 share a common fold; they are composed of six-stranded β-sheets flanked on one side by two long, bent helices inserted between strands β3/β4 and β4/β5. RESULTS +5 9 CDC2 structure_element CDC1/CDC2 share a common fold; they are composed of six-stranded β-sheets flanked on one side by two long, bent helices inserted between strands β3/β4 and β4/β5. RESULTS +52 73 six-stranded β-sheets structure_element CDC1/CDC2 share a common fold; they are composed of six-stranded β-sheets flanked on one side by two long, bent helices inserted between strands β3/β4 and β4/β5. RESULTS +101 119 long, bent helices structure_element CDC1/CDC2 share a common fold; they are composed of six-stranded β-sheets flanked on one side by two long, bent helices inserted between strands β3/β4 and β4/β5. RESULTS +137 144 strands structure_element CDC1/CDC2 share a common fold; they are composed of six-stranded β-sheets flanked on one side by two long, bent helices inserted between strands β3/β4 and β4/β5. RESULTS +145 150 β3/β4 structure_element CDC1/CDC2 share a common fold; they are composed of six-stranded β-sheets flanked on one side by two long, bent helices inserted between strands β3/β4 and β4/β5. RESULTS +155 160 β4/β5 structure_element CDC1/CDC2 share a common fold; they are composed of six-stranded β-sheets flanked on one side by two long, bent helices inserted between strands β3/β4 and β4/β5. RESULTS +0 4 CDC2 structure_element CDC2 is extended at its C terminus by an additional β-strand and an irregular β-hairpin. RESULTS +8 16 extended protein_state CDC2 is extended at its C terminus by an additional β-strand and an irregular β-hairpin. RESULTS +52 60 β-strand structure_element CDC2 is extended at its C terminus by an additional β-strand and an irregular β-hairpin. RESULTS +68 87 irregular β-hairpin structure_element CDC2 is extended at its C terminus by an additional β-strand and an irregular β-hairpin. RESULTS +18 44 root mean square deviation evidence On the basis of a root mean square deviation of main chain atom positions of 2.2 Å, CDC1/CDC2 are structurally more closely related to each other than to any other protein (Fig. 1c); they may thus have evolved by duplication. RESULTS +84 88 CDC1 structure_element On the basis of a root mean square deviation of main chain atom positions of 2.2 Å, CDC1/CDC2 are structurally more closely related to each other than to any other protein (Fig. 1c); they may thus have evolved by duplication. RESULTS +89 93 CDC2 structure_element On the basis of a root mean square deviation of main chain atom positions of 2.2 Å, CDC1/CDC2 are structurally more closely related to each other than to any other protein (Fig. 1c); they may thus have evolved by duplication. RESULTS +55 58 CDN structure_element Close structural homologues could not be found for the CDN or the CDC domains. RESULTS +66 69 CDC structure_element Close structural homologues could not be found for the CDN or the CDC domains. RESULTS +2 17 regulatory loop structure_element A regulatory loop mediates interdomain interactions RESULTS +34 55 insect-cell-expressed experimental_method To define the functional state of insect-cell-expressed ACC variants, we employed mass spectrometry (MS) for phosphorylation site detection. RESULTS +56 59 ACC protein_type To define the functional state of insect-cell-expressed ACC variants, we employed mass spectrometry (MS) for phosphorylation site detection. RESULTS +82 99 mass spectrometry experimental_method To define the functional state of insect-cell-expressed ACC variants, we employed mass spectrometry (MS) for phosphorylation site detection. RESULTS +101 103 MS experimental_method To define the functional state of insect-cell-expressed ACC variants, we employed mass spectrometry (MS) for phosphorylation site detection. RESULTS +109 139 phosphorylation site detection experimental_method To define the functional state of insect-cell-expressed ACC variants, we employed mass spectrometry (MS) for phosphorylation site detection. RESULTS +3 24 insect-cell-expressed experimental_method In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae. RESULTS +25 36 full-length protein_state In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae. RESULTS +37 43 SceACC protein In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae. RESULTS +49 65 highly conserved protein_state In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae. RESULTS +66 73 Ser1157 residue_name_number In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae. RESULTS +86 100 fully occupied protein_state In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae. RESULTS +101 121 phosphorylation site site In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae. RESULTS +151 164 S. cerevisiae species In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae. RESULTS +11 26 phosphorylation ptm Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast. RESULTS +44 51 Ser2101 residue_name_number Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast. RESULTS +56 63 Tyr2179 residue_name_number Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast. RESULTS +90 107 neither conserved protein_state Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast. RESULTS +115 121 fungal taxonomy_domain Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast. RESULTS +122 125 ACC protein_type Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast. RESULTS +126 153 nor natively phosphorylated protein_state Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast. RESULTS +157 162 yeast taxonomy_domain Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast. RESULTS +0 2 MS experimental_method MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals. RESULTS +15 33 dissolved crystals experimental_method MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals. RESULTS +48 62 phosphorylated protein_state MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals. RESULTS +72 79 Ser1157 residue_name_number MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals. RESULTS +88 91 Sce species MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals. RESULTS +91 93 CD structure_element MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals. RESULTS +94 102 crystals evidence MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals. RESULTS +4 7 Sce species The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation. RESULTS +7 9 CD structure_element The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation. RESULTS +10 19 structure evidence The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation. RESULTS +63 69 SceACC protein The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation. RESULTS +81 87 enzyme protein The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation. RESULTS +91 100 inhibited protein_state The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation. RESULTS +104 134 SNF1-dependent phosphorylation ptm The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation. RESULTS +7 10 Sce species In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +10 12 CD structure_element In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +13 30 crystal structure evidence In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +36 50 phosphorylated protein_state In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +51 58 Ser1157 residue_name_number In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +72 101 regulatory 36-amino-acid loop structure_element In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +110 117 strands structure_element In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +118 120 β2 structure_element In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +125 127 β3 structure_element In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +131 135 CDC1 structure_element In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +179 193 less-conserved protein_state In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +194 215 phosphorylation sites site In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +217 224 Ser1148 residue_name_number In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +229 236 Ser1162 residue_name_number In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +251 256 yeast taxonomy_domain In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. RESULTS +5 20 regulatory loop structure_element This regulatory loop wedges between the CDC1 and CDC2 domains and provides the largest contribution to the interdomain interface. RESULTS +40 44 CDC1 structure_element This regulatory loop wedges between the CDC1 and CDC2 domains and provides the largest contribution to the interdomain interface. RESULTS +49 53 CDC2 structure_element This regulatory loop wedges between the CDC1 and CDC2 domains and provides the largest contribution to the interdomain interface. RESULTS +107 128 interdomain interface site This regulatory loop wedges between the CDC1 and CDC2 domains and provides the largest contribution to the interdomain interface. RESULTS +29 44 regulatory loop structure_element The N-terminal region of the regulatory loop also directly contacts the C-terminal region of CDC2 leading into CT. RESULTS +93 97 CDC2 structure_element The N-terminal region of the regulatory loop also directly contacts the C-terminal region of CDC2 leading into CT. RESULTS +111 113 CT structure_element The N-terminal region of the regulatory loop also directly contacts the C-terminal region of CDC2 leading into CT. RESULTS +0 18 Phosphoserine 1157 residue_name_number Phosphoserine 1157 is tightly bound by two highly conserved arginines (Arg1173 and Arg1260) of CDC1 (Fig. 1d). RESULTS +43 59 highly conserved protein_state Phosphoserine 1157 is tightly bound by two highly conserved arginines (Arg1173 and Arg1260) of CDC1 (Fig. 1d). RESULTS +60 69 arginines residue_name Phosphoserine 1157 is tightly bound by two highly conserved arginines (Arg1173 and Arg1260) of CDC1 (Fig. 1d). RESULTS +71 78 Arg1173 residue_name_number Phosphoserine 1157 is tightly bound by two highly conserved arginines (Arg1173 and Arg1260) of CDC1 (Fig. 1d). RESULTS +83 90 Arg1260 residue_name_number Phosphoserine 1157 is tightly bound by two highly conserved arginines (Arg1173 and Arg1260) of CDC1 (Fig. 1d). RESULTS +95 99 CDC1 structure_element Phosphoserine 1157 is tightly bound by two highly conserved arginines (Arg1173 and Arg1260) of CDC1 (Fig. 1d). RESULTS +23 37 phosphorylated protein_state Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. RESULTS +38 45 Ser1157 residue_name_number Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. RESULTS +72 87 regulatory loop structure_element Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. RESULTS +116 137 phosphorylation sites site Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. RESULTS +138 145 Ser1148 residue_name_number Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. RESULTS +150 157 Ser1162 residue_name_number Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. RESULTS +165 174 same loop structure_element Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. RESULTS +236 251 regulatory loop structure_element Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. RESULTS +260 264 CDC1 structure_element Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. RESULTS +269 273 CDC2 structure_element Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. RESULTS +0 15 Phosphorylation ptm Phosphorylation of the regulatory loop thus determines interdomain interactions of CDC1 and CDC2, suggesting that it may exert its regulatory function by modifying the overall structure and dynamics of the CD. RESULTS +23 38 regulatory loop structure_element Phosphorylation of the regulatory loop thus determines interdomain interactions of CDC1 and CDC2, suggesting that it may exert its regulatory function by modifying the overall structure and dynamics of the CD. RESULTS +83 87 CDC1 structure_element Phosphorylation of the regulatory loop thus determines interdomain interactions of CDC1 and CDC2, suggesting that it may exert its regulatory function by modifying the overall structure and dynamics of the CD. RESULTS +92 96 CDC2 structure_element Phosphorylation of the regulatory loop thus determines interdomain interactions of CDC1 and CDC2, suggesting that it may exert its regulatory function by modifying the overall structure and dynamics of the CD. RESULTS +206 208 CD structure_element Phosphorylation of the regulatory loop thus determines interdomain interactions of CDC1 and CDC2, suggesting that it may exert its regulatory function by modifying the overall structure and dynamics of the CD. RESULTS +23 30 Ser1157 residue_name_number The functional role of Ser1157 was confirmed by an activity assay based on the incorporation of radioactive carbonate into acid non-volatile material. RESULTS +51 65 activity assay experimental_method The functional role of Ser1157 was confirmed by an activity assay based on the incorporation of radioactive carbonate into acid non-volatile material. RESULTS +0 14 Phosphorylated protein_state Phosphorylated SceACC shows only residual activity (kcat=0.4±0.2 s−1, s.d. based on five replicate measurements), which increases 16-fold (kcat=6.5±0.3 s−1) after dephosphorylation with λ protein phosphatase. RESULTS +15 21 SceACC protein Phosphorylated SceACC shows only residual activity (kcat=0.4±0.2 s−1, s.d. based on five replicate measurements), which increases 16-fold (kcat=6.5±0.3 s−1) after dephosphorylation with λ protein phosphatase. RESULTS +52 56 kcat evidence Phosphorylated SceACC shows only residual activity (kcat=0.4±0.2 s−1, s.d. based on five replicate measurements), which increases 16-fold (kcat=6.5±0.3 s−1) after dephosphorylation with λ protein phosphatase. RESULTS +139 143 kcat evidence Phosphorylated SceACC shows only residual activity (kcat=0.4±0.2 s−1, s.d. based on five replicate measurements), which increases 16-fold (kcat=6.5±0.3 s−1) after dephosphorylation with λ protein phosphatase. RESULTS +186 207 λ protein phosphatase protein_type Phosphorylated SceACC shows only residual activity (kcat=0.4±0.2 s−1, s.d. based on five replicate measurements), which increases 16-fold (kcat=6.5±0.3 s−1) after dephosphorylation with λ protein phosphatase. RESULTS +24 40 dephosphorylated protein_state The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli. RESULTS +41 47 SceACC protein The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli. RESULTS +90 108 non-phosphorylated protein_state The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli. RESULTS +109 114 yeast taxonomy_domain The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli. RESULTS +115 118 ACC protein_type The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli. RESULTS +119 131 expressed in experimental_method The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli. RESULTS +132 139 E. coli species The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli. RESULTS +13 15 CD structure_element The variable CD is conserved between yeast and human RESULTS +19 28 conserved protein_state The variable CD is conserved between yeast and human RESULTS +37 42 yeast taxonomy_domain The variable CD is conserved between yeast and human RESULTS +47 52 human species The variable CD is conserved between yeast and human RESULTS +31 37 fungal taxonomy_domain To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). RESULTS +42 47 human species To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). RESULTS +48 51 ACC protein_type To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). RESULTS +52 54 CD structure_element To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). RESULTS +59 83 determined the structure experimental_method To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). RESULTS +89 94 human species To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). RESULTS +95 108 ACC1 fragment mutant To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). RESULTS +128 130 BT structure_element To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). RESULTS +135 137 CD structure_element To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). RESULTS +147 155 HsaBT-CD mutant To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). RESULTS +162 167 lacks protein_state To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). RESULTS +179 183 BCCP structure_element To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). RESULTS +3 28 experimentally phased map evidence An experimentally phased map was obtained at 3.7 Å resolution for a cadmium-derivatized crystal and was interpreted by a poly-alanine model (Fig. 1e and Table 1). RESULTS +68 75 cadmium chemical An experimentally phased map was obtained at 3.7 Å resolution for a cadmium-derivatized crystal and was interpreted by a poly-alanine model (Fig. 1e and Table 1). RESULTS +17 19 CD structure_element Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures. RESULTS +31 39 HsaBT-CD mutant Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures. RESULTS +81 84 Sce species Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures. RESULTS +84 86 CD structure_element Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures. RESULTS +104 109 human species Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures. RESULTS +114 119 yeast taxonomy_domain Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures. RESULTS +120 123 CDs structure_element Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures. RESULTS +149 159 structures evidence Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures. RESULTS +45 48 Sce species In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). RESULTS +48 50 CD structure_element In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). RESULTS +88 91 CDL structure_element In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). RESULTS +96 100 CDC1 structure_element In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). RESULTS +117 125 HsaBT-CD mutant In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). RESULTS +135 140 human species In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). RESULTS +141 144 CDL structure_element In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). RESULTS +145 149 CDC1 structure_element In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). RESULTS +187 200 superposition experimental_method In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). RESULTS +204 209 human species In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). RESULTS +214 219 yeast taxonomy_domain In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). RESULTS +220 224 CDC2 structure_element In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). RESULTS +31 34 CDL structure_element As a result, the N terminus of CDL at helix Lα1, which connects to CDN, is shifted by 12 Å. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD. RESULTS +38 43 helix structure_element As a result, the N terminus of CDL at helix Lα1, which connects to CDN, is shifted by 12 Å. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD. RESULTS +44 47 Lα1 structure_element As a result, the N terminus of CDL at helix Lα1, which connects to CDN, is shifted by 12 Å. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD. RESULTS +67 70 CDN structure_element As a result, the N terminus of CDL at helix Lα1, which connects to CDN, is shifted by 12 Å. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD. RESULTS +104 107 CDN structure_element As a result, the N terminus of CDL at helix Lα1, which connects to CDN, is shifted by 12 Å. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD. RESULTS +111 119 HsaBT-CD mutant As a result, the N terminus of CDL at helix Lα1, which connects to CDN, is shifted by 12 Å. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD. RESULTS +176 179 Sce species As a result, the N terminus of CDL at helix Lα1, which connects to CDN, is shifted by 12 Å. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD. RESULTS +179 181 CD structure_element As a result, the N terminus of CDL at helix Lα1, which connects to CDN, is shifted by 12 Å. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD. RESULTS +5 8 CDL structure_element With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160° around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d). RESULTS +9 13 CDC1 structure_element With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160° around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d). RESULTS +14 24 superposed experimental_method With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160° around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d). RESULTS +26 29 CDN structure_element With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160° around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d). RESULTS +33 41 HsaBT-CD mutant With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160° around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d). RESULTS +70 75 hinge structure_element With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160° around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d). RESULTS +97 100 CDN structure_element With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160° around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d). RESULTS +101 104 CDL structure_element With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160° around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d). RESULTS +42 45 CDN structure_element This rotation displaces the N terminus of CDN in HsaBT-CD by 51 Å compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67 Å (the attachment points are indicated with spheres in Fig. 1e). RESULTS +49 57 HsaBT-CD mutant This rotation displaces the N terminus of CDN in HsaBT-CD by 51 Å compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67 Å (the attachment points are indicated with spheres in Fig. 1e). RESULTS +80 83 Sce species This rotation displaces the N terminus of CDN in HsaBT-CD by 51 Å compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67 Å (the attachment points are indicated with spheres in Fig. 1e). RESULTS +83 85 CD structure_element This rotation displaces the N terminus of CDN in HsaBT-CD by 51 Å compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67 Å (the attachment points are indicated with spheres in Fig. 1e). RESULTS +156 162 linker structure_element This rotation displaces the N terminus of CDN in HsaBT-CD by 51 Å compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67 Å (the attachment points are indicated with spheres in Fig. 1e). RESULTS +170 181 BCCP domain structure_element This rotation displaces the N terminus of CDN in HsaBT-CD by 51 Å compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67 Å (the attachment points are indicated with spheres in Fig. 1e). RESULTS +201 203 CT structure_element This rotation displaces the N terminus of CDN in HsaBT-CD by 51 Å compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67 Å (the attachment points are indicated with spheres in Fig. 1e). RESULTS +4 6 BT structure_element The BT domain of HsaBT-CD consists of a helix that is surrounded at its N terminus by an antiparallel eight-stranded β-barrel. RESULTS +17 25 HsaBT-CD mutant The BT domain of HsaBT-CD consists of a helix that is surrounded at its N terminus by an antiparallel eight-stranded β-barrel. RESULTS +40 45 helix structure_element The BT domain of HsaBT-CD consists of a helix that is surrounded at its N terminus by an antiparallel eight-stranded β-barrel. RESULTS +89 125 antiparallel eight-stranded β-barrel structure_element The BT domain of HsaBT-CD consists of a helix that is surrounded at its N terminus by an antiparallel eight-stranded β-barrel. RESULTS +17 19 BT structure_element It resembles the BT of propionyl-CoA carboxylase; only the four C-terminal strands of the β-barrel are slightly tilted. RESULTS +23 48 propionyl-CoA carboxylase protein_type It resembles the BT of propionyl-CoA carboxylase; only the four C-terminal strands of the β-barrel are slightly tilted. RESULTS +75 98 strands of the β-barrel structure_element It resembles the BT of propionyl-CoA carboxylase; only the four C-terminal strands of the β-barrel are slightly tilted. RESULTS +16 18 MS experimental_method On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%). RESULTS +31 52 insect-cell-expressed experimental_method On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%). RESULTS +53 58 human species On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%). RESULTS +59 70 full-length protein_state On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%). RESULTS +71 74 ACC protein_type On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%). RESULTS +76 81 Ser80 residue_name_number On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%). RESULTS +110 125 phosphorylation ptm On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%). RESULTS +0 5 Ser29 residue_name_number Ser29 and Ser1263, implicated in insulin-dependent phosphorylation and BRCA1 binding, respectively, are phosphorylated at intermediate levels (40%). RESULTS +10 17 Ser1263 residue_name_number Ser29 and Ser1263, implicated in insulin-dependent phosphorylation and BRCA1 binding, respectively, are phosphorylated at intermediate levels (40%). RESULTS +33 66 insulin-dependent phosphorylation ptm Ser29 and Ser1263, implicated in insulin-dependent phosphorylation and BRCA1 binding, respectively, are phosphorylated at intermediate levels (40%). RESULTS +71 76 BRCA1 protein Ser29 and Ser1263, implicated in insulin-dependent phosphorylation and BRCA1 binding, respectively, are phosphorylated at intermediate levels (40%). RESULTS +104 118 phosphorylated protein_state Ser29 and Ser1263, implicated in insulin-dependent phosphorylation and BRCA1 binding, respectively, are phosphorylated at intermediate levels (40%). RESULTS +4 20 highly conserved protein_state The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated. RESULTS +21 28 Ser1216 residue_name_number The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated. RESULTS +47 60 S. cerevisiae species The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated. RESULTS +61 68 Ser1157 residue_name_number The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated. RESULTS +82 89 Ser1201 residue_name_number The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated. RESULTS +103 118 regulatory loop structure_element The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated. RESULTS +140 158 not phosphorylated protein_state The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated. RESULTS +18 33 phosphorylation ptm However, residual phosphorylation levels were detected for Ser1204 (7%) and Ser1218 (7%) in the same loop. RESULTS +59 66 Ser1204 residue_name_number However, residual phosphorylation levels were detected for Ser1204 (7%) and Ser1218 (7%) in the same loop. RESULTS +76 83 Ser1218 residue_name_number However, residual phosphorylation levels were detected for Ser1204 (7%) and Ser1218 (7%) in the same loop. RESULTS +96 105 same loop structure_element However, residual phosphorylation levels were detected for Ser1204 (7%) and Ser1218 (7%) in the same loop. RESULTS +0 2 MS experimental_method MS analysis of the HsaBT-CD crystallization sample reveals partial proteolytic digestion of the regulatory loop. RESULTS +19 27 HsaBT-CD mutant MS analysis of the HsaBT-CD crystallization sample reveals partial proteolytic digestion of the regulatory loop. RESULTS +28 50 crystallization sample evidence MS analysis of the HsaBT-CD crystallization sample reveals partial proteolytic digestion of the regulatory loop. RESULTS +96 111 regulatory loop structure_element MS analysis of the HsaBT-CD crystallization sample reveals partial proteolytic digestion of the regulatory loop. RESULTS +21 30 this loop structure_element Accordingly, most of this loop is not represented in the HsaBT-CD crystal structure. RESULTS +57 65 HsaBT-CD mutant Accordingly, most of this loop is not represented in the HsaBT-CD crystal structure. RESULTS +66 83 crystal structure evidence Accordingly, most of this loop is not represented in the HsaBT-CD crystal structure. RESULTS +4 14 absence of protein_state The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains. RESULTS +19 34 regulatory loop structure_element The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains. RESULTS +58 73 less-restrained protein_state The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains. RESULTS +74 83 interface site The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains. RESULTS +87 90 CDL structure_element The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains. RESULTS +91 95 CDC1 structure_element The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains. RESULTS +100 104 CDC2 structure_element The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains. RESULTS +148 155 domains structure_element The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains. RESULTS +12 27 regulatory loop structure_element Besides the regulatory loop, also the phosphopeptide target region for BRCA1 interaction is not resolved presumably because of pronounced flexibility. RESULTS +38 66 phosphopeptide target region site Besides the regulatory loop, also the phosphopeptide target region for BRCA1 interaction is not resolved presumably because of pronounced flexibility. RESULTS +71 76 BRCA1 protein Besides the regulatory loop, also the phosphopeptide target region for BRCA1 interaction is not resolved presumably because of pronounced flexibility. RESULTS +16 24 isolated experimental_method At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. RESULTS +25 30 yeast taxonomy_domain At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. RESULTS +35 40 human species At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. RESULTS +41 43 CD structure_element At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. RESULTS +49 68 structural analysis experimental_method At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. RESULTS +108 114 hinges structure_element At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. RESULTS +156 174 CDN/CDL connection structure_element At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. RESULTS +216 219 CDL structure_element At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. RESULTS +220 224 CDC1 structure_element At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. RESULTS +229 233 CDC2 structure_element At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. RESULTS +257 272 phosphorylation ptm At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. RESULTS +280 295 regulatory loop structure_element At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. RESULTS +19 21 CD structure_element The integration of CD into the fungal ACC multienzyme RESULTS +31 37 fungal taxonomy_domain The integration of CD into the fungal ACC multienzyme RESULTS +38 53 ACC multienzyme protein_type The integration of CD into the fungal ACC multienzyme RESULTS +63 69 fungal taxonomy_domain To further obtain insights into the functional architecture of fungal ACC, we characterized larger multidomain fragments up to the intact enzymes. RESULTS +70 73 ACC protein_type To further obtain insights into the functional architecture of fungal ACC, we characterized larger multidomain fragments up to the intact enzymes. RESULTS +92 120 larger multidomain fragments mutant To further obtain insights into the functional architecture of fungal ACC, we characterized larger multidomain fragments up to the intact enzymes. RESULTS +131 137 intact protein_state To further obtain insights into the functional architecture of fungal ACC, we characterized larger multidomain fragments up to the intact enzymes. RESULTS +138 145 enzymes protein To further obtain insights into the functional architecture of fungal ACC, we characterized larger multidomain fragments up to the intact enzymes. RESULTS +6 27 molecular replacement experimental_method Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +37 43 fungal taxonomy_domain Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +44 47 ACC protein_type Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +48 50 CD structure_element Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +55 57 CT structure_element Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +78 88 structures evidence Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +94 101 variant mutant Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +113 116 Cth species Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +116 118 CT structure_element Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +123 127 CDC1 structure_element Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +128 132 CDC2 structure_element Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +136 153 two crystal forms evidence Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +187 202 CthCD-CTCter1/2 mutant Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +235 238 Cth species Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +238 240 CT structure_element Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +262 264 CD structure_element Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +286 294 CthCD-CT mutant Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). RESULTS +67 97 larger BC-containing fragments mutant No crystals diffracting to sufficient resolution were obtained for larger BC-containing fragments, or for full-length Cth or SceACC. RESULTS +106 117 full-length protein_state No crystals diffracting to sufficient resolution were obtained for larger BC-containing fragments, or for full-length Cth or SceACC. RESULTS +118 121 Cth species No crystals diffracting to sufficient resolution were obtained for larger BC-containing fragments, or for full-length Cth or SceACC. RESULTS +125 131 SceACC protein No crystals diffracting to sufficient resolution were obtained for larger BC-containing fragments, or for full-length Cth or SceACC. RESULTS +3 28 improve crystallizability experimental_method To improve crystallizability, we generated ΔBCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a–c). RESULTS +33 42 generated experimental_method To improve crystallizability, we generated ΔBCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a–c). RESULTS +43 57 ΔBCCP variants mutant To improve crystallizability, we generated ΔBCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a–c). RESULTS +61 72 full-length protein_state To improve crystallizability, we generated ΔBCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a–c). RESULTS +73 76 ACC protein_type To improve crystallizability, we generated ΔBCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a–c). RESULTS +94 107 SAXS analysis experimental_method To improve crystallizability, we generated ΔBCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a–c). RESULTS +132 138 intact protein_state To improve crystallizability, we generated ΔBCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a–c). RESULTS +139 142 ACC protein_type To improve crystallizability, we generated ΔBCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a–c). RESULTS +4 12 CthΔBCCP mutant For CthΔBCCP, crystals diffracting to 8.4 Å resolution were obtained. RESULTS +14 22 crystals evidence For CthΔBCCP, crystals diffracting to 8.4 Å resolution were obtained. RESULTS +9 30 molecular replacement experimental_method However, molecular replacement did not reveal a unique positioning of the BC domain. RESULTS +74 76 BC structure_element However, molecular replacement did not reveal a unique positioning of the BC domain. RESULTS +50 60 structures evidence Owing to the limited resolution the discussion of structures of CthCD-CT and CthΔBCCP is restricted to the analysis of domain localization. RESULTS +64 72 CthCD-CT mutant Owing to the limited resolution the discussion of structures of CthCD-CT and CthΔBCCP is restricted to the analysis of domain localization. RESULTS +77 85 CthΔBCCP mutant Owing to the limited resolution the discussion of structures of CthCD-CT and CthΔBCCP is restricted to the analysis of domain localization. RESULTS +7 23 these structures evidence Still, these structures contribute considerably to the visualization of an intrinsically dynamic fungal ACC. RESULTS +89 96 dynamic protein_state Still, these structures contribute considerably to the visualization of an intrinsically dynamic fungal ACC. RESULTS +97 103 fungal taxonomy_domain Still, these structures contribute considerably to the visualization of an intrinsically dynamic fungal ACC. RESULTS +104 107 ACC protein_type Still, these structures contribute considerably to the visualization of an intrinsically dynamic fungal ACC. RESULTS +13 31 crystal structures evidence In all these crystal structures, the CT domains build a canonical head-to-tail dimer, with active sites formed by contributions from both protomers (Fig. 2 and Supplementary Fig. 3a). RESULTS +37 39 CT structure_element In all these crystal structures, the CT domains build a canonical head-to-tail dimer, with active sites formed by contributions from both protomers (Fig. 2 and Supplementary Fig. 3a). RESULTS +66 78 head-to-tail protein_state In all these crystal structures, the CT domains build a canonical head-to-tail dimer, with active sites formed by contributions from both protomers (Fig. 2 and Supplementary Fig. 3a). RESULTS +79 84 dimer oligomeric_state In all these crystal structures, the CT domains build a canonical head-to-tail dimer, with active sites formed by contributions from both protomers (Fig. 2 and Supplementary Fig. 3a). RESULTS +91 103 active sites site In all these crystal structures, the CT domains build a canonical head-to-tail dimer, with active sites formed by contributions from both protomers (Fig. 2 and Supplementary Fig. 3a). RESULTS +138 147 protomers oligomeric_state In all these crystal structures, the CT domains build a canonical head-to-tail dimer, with active sites formed by contributions from both protomers (Fig. 2 and Supplementary Fig. 3a). RESULTS +4 14 connection structure_element The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular β-hairpin/β-strand extension of CDC2 (Supplementary Fig. 3b). RESULTS +18 20 CD structure_element The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular β-hairpin/β-strand extension of CDC2 (Supplementary Fig. 3b). RESULTS +25 27 CT structure_element The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular β-hairpin/β-strand extension of CDC2 (Supplementary Fig. 3b). RESULTS +45 71 10-residue peptide stretch residue_range The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular β-hairpin/β-strand extension of CDC2 (Supplementary Fig. 3b). RESULTS +103 105 CT structure_element The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular β-hairpin/β-strand extension of CDC2 (Supplementary Fig. 3b). RESULTS +113 151 irregular β-hairpin/β-strand extension structure_element The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular β-hairpin/β-strand extension of CDC2 (Supplementary Fig. 3b). RESULTS +155 159 CDC2 structure_element The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular β-hairpin/β-strand extension of CDC2 (Supplementary Fig. 3b). RESULTS +4 21 connecting region structure_element The connecting region is remarkably similar in isolated CD and CthCD-CTCter structures, indicating inherent conformational stability. RESULTS +47 55 isolated protein_state The connecting region is remarkably similar in isolated CD and CthCD-CTCter structures, indicating inherent conformational stability. RESULTS +56 58 CD structure_element The connecting region is remarkably similar in isolated CD and CthCD-CTCter structures, indicating inherent conformational stability. RESULTS +63 75 CthCD-CTCter mutant The connecting region is remarkably similar in isolated CD and CthCD-CTCter structures, indicating inherent conformational stability. RESULTS +76 86 structures evidence The connecting region is remarkably similar in isolated CD and CthCD-CTCter structures, indicating inherent conformational stability. RESULTS +0 2 CD structure_element CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. RESULTS +3 5 CT structure_element CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. RESULTS +90 109 β-hairpin extension structure_element CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. RESULTS +113 117 CDC2 structure_element CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. RESULTS +133 137 loop structure_element CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. RESULTS +146 159 strands β2/β3 structure_element CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. RESULTS +167 176 CT N-lobe structure_element CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. RESULTS +195 204 conserved protein_state CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. RESULTS +205 217 RxxGxN motif structure_element CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. RESULTS +17 21 loop structure_element The neighbouring loop on the CT side (between CT β1/β2) is displaced by 2.5 Å compared to isolated CT structures (Supplementary Fig. 3c). RESULTS +29 31 CT structure_element The neighbouring loop on the CT side (between CT β1/β2) is displaced by 2.5 Å compared to isolated CT structures (Supplementary Fig. 3c). RESULTS +46 48 CT structure_element The neighbouring loop on the CT side (between CT β1/β2) is displaced by 2.5 Å compared to isolated CT structures (Supplementary Fig. 3c). RESULTS +49 51 β1 structure_element The neighbouring loop on the CT side (between CT β1/β2) is displaced by 2.5 Å compared to isolated CT structures (Supplementary Fig. 3c). RESULTS +52 54 β2 structure_element The neighbouring loop on the CT side (between CT β1/β2) is displaced by 2.5 Å compared to isolated CT structures (Supplementary Fig. 3c). RESULTS +90 98 isolated protein_state The neighbouring loop on the CT side (between CT β1/β2) is displaced by 2.5 Å compared to isolated CT structures (Supplementary Fig. 3c). RESULTS +99 101 CT structure_element The neighbouring loop on the CT side (between CT β1/β2) is displaced by 2.5 Å compared to isolated CT structures (Supplementary Fig. 3c). RESULTS +102 112 structures evidence The neighbouring loop on the CT side (between CT β1/β2) is displaced by 2.5 Å compared to isolated CT structures (Supplementary Fig. 3c). RESULTS +98 107 interface site On the basis of an interface area of ∼600 Å2 and its edge-to-edge connection characteristics, the interface between CT and CD might be classified as conformationally variable. RESULTS +116 118 CT structure_element On the basis of an interface area of ∼600 Å2 and its edge-to-edge connection characteristics, the interface between CT and CD might be classified as conformationally variable. RESULTS +123 125 CD structure_element On the basis of an interface area of ∼600 Å2 and its edge-to-edge connection characteristics, the interface between CT and CD might be classified as conformationally variable. RESULTS +87 89 CD structure_element Indeed, the comparison of the positioning of eight instances of the C-terminal part of CD relative to CT in crystal structures determined here, reveals flexible interdomain linking (Fig. 3a). RESULTS +102 104 CT structure_element Indeed, the comparison of the positioning of eight instances of the C-terminal part of CD relative to CT in crystal structures determined here, reveals flexible interdomain linking (Fig. 3a). RESULTS +108 126 crystal structures evidence Indeed, the comparison of the positioning of eight instances of the C-terminal part of CD relative to CT in crystal structures determined here, reveals flexible interdomain linking (Fig. 3a). RESULTS +127 137 determined experimental_method Indeed, the comparison of the positioning of eight instances of the C-terminal part of CD relative to CT in crystal structures determined here, reveals flexible interdomain linking (Fig. 3a). RESULTS +4 21 CDC2/CT interface site The CDC2/CT interface acts as a true hinge with observed rotation up to 16°, which results in a translocation of the distal end of CDC2 by 8 Å. RESULTS +32 42 true hinge structure_element The CDC2/CT interface acts as a true hinge with observed rotation up to 16°, which results in a translocation of the distal end of CDC2 by 8 Å. RESULTS +131 135 CDC2 structure_element The CDC2/CT interface acts as a true hinge with observed rotation up to 16°, which results in a translocation of the distal end of CDC2 by 8 Å. RESULTS +4 13 interface site The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). RESULTS +22 26 CDC2 structure_element The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). RESULTS +31 34 CDL structure_element The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). RESULTS +35 39 CDC1 structure_element The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). RESULTS +66 80 phosphorylated protein_state The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). RESULTS +81 96 regulatory loop structure_element The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). RESULTS +104 107 Sce species The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). RESULTS +107 109 CD structure_element The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). RESULTS +110 119 structure evidence The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). RESULTS +147 161 CD–CT junction structure_element The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). RESULTS +26 41 phosphorylation ptm Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution. RESULTS +49 58 interface site Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution. RESULTS +67 71 CDC2 structure_element Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution. RESULTS +76 79 CDL structure_element Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution. RESULTS +80 84 CDC1 structure_element Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution. RESULTS +88 102 CthACC variant mutant Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution. RESULTS +103 113 structures evidence Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution. RESULTS +9 11 MS experimental_method However, MS analysis of CthCD-CT and CthΔBCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157). RESULTS +24 32 CthCD-CT mutant However, MS analysis of CthCD-CT and CthΔBCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157). RESULTS +37 45 CthΔBCCP mutant However, MS analysis of CthCD-CT and CthΔBCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157). RESULTS +85 100 phosphorylation ptm However, MS analysis of CthCD-CT and CthΔBCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157). RESULTS +104 111 Ser1170 residue_name_number However, MS analysis of CthCD-CT and CthΔBCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157). RESULTS +130 136 SceACC protein However, MS analysis of CthCD-CT and CthΔBCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157). RESULTS +137 144 Ser1157 residue_name_number However, MS analysis of CthCD-CT and CthΔBCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157). RESULTS +4 7 CDN structure_element The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +39 42 CDL structure_element The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +43 47 CDC1 structure_element The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +112 122 structures evidence The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +126 129 Sce species The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +129 131 CD structure_element The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +140 163 larger CthACC fragments mutant The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +165 168 CDN structure_element The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +257 266 protomers oligomeric_state The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +270 278 CthCD-CT mutant The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +287 295 protomer oligomeric_state The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +299 307 CthΔBCCP mutant The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +320 331 CthCD-CT1/2 mutant The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +336 345 CthΔBCCP1 mutant The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). RESULTS +13 16 CDN structure_element In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. RESULTS +35 41 hinges structure_element In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. RESULTS +68 71 CDN structure_element In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. RESULTS +72 75 CDL structure_element In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. RESULTS +116 124 protomer oligomeric_state In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. RESULTS +128 136 CthΔBCCP mutant In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. RESULTS +149 158 CthΔBCCP2 mutant In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. RESULTS +191 194 Sce species In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. RESULTS +194 196 CD structure_element In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. RESULTS +229 240 anchor site site In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. RESULTS +249 260 BCCP linker structure_element In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. RESULTS +34 36 CD structure_element Conformational variability in the CD thus contributes considerably to variations in the spacing between the BC and CT domains, and may extend to distance variations beyond the mobility range of the flexibly tethered BCCP. RESULTS +108 110 BC structure_element Conformational variability in the CD thus contributes considerably to variations in the spacing between the BC and CT domains, and may extend to distance variations beyond the mobility range of the flexibly tethered BCCP. RESULTS +115 117 CT structure_element Conformational variability in the CD thus contributes considerably to variations in the spacing between the BC and CT domains, and may extend to distance variations beyond the mobility range of the flexibly tethered BCCP. RESULTS +198 215 flexibly tethered protein_state Conformational variability in the CD thus contributes considerably to variations in the spacing between the BC and CT domains, and may extend to distance variations beyond the mobility range of the flexibly tethered BCCP. RESULTS +216 220 BCCP structure_element Conformational variability in the CD thus contributes considerably to variations in the spacing between the BC and CT domains, and may extend to distance variations beyond the mobility range of the flexibly tethered BCCP. RESULTS +73 79 fungal taxonomy_domain On the basis of the occurrence of related conformational changes between fungal and human ACC fragments, the observed set of conformations may well represent general states present in all eukaryotic ACCs. RESULTS +84 89 human species On the basis of the occurrence of related conformational changes between fungal and human ACC fragments, the observed set of conformations may well represent general states present in all eukaryotic ACCs. RESULTS +90 103 ACC fragments mutant On the basis of the occurrence of related conformational changes between fungal and human ACC fragments, the observed set of conformations may well represent general states present in all eukaryotic ACCs. RESULTS +188 198 eukaryotic taxonomy_domain On the basis of the occurrence of related conformational changes between fungal and human ACC fragments, the observed set of conformations may well represent general states present in all eukaryotic ACCs. RESULTS +199 203 ACCs protein_type On the basis of the occurrence of related conformational changes between fungal and human ACC fragments, the observed set of conformations may well represent general states present in all eukaryotic ACCs. RESULTS +42 48 fungal taxonomy_domain Large-scale conformational variability of fungal ACC RESULTS +49 52 ACC protein_type Large-scale conformational variability of fungal ACC RESULTS +34 40 fungal taxonomy_domain To obtain a comprehensive view of fungal ACC dynamics in solution, we employed SAXS and EM. RESULTS +41 44 ACC protein_type To obtain a comprehensive view of fungal ACC dynamics in solution, we employed SAXS and EM. RESULTS +54 65 in solution protein_state To obtain a comprehensive view of fungal ACC dynamics in solution, we employed SAXS and EM. RESULTS +79 83 SAXS experimental_method To obtain a comprehensive view of fungal ACC dynamics in solution, we employed SAXS and EM. RESULTS +88 90 EM experimental_method To obtain a comprehensive view of fungal ACC dynamics in solution, we employed SAXS and EM. RESULTS +0 4 SAXS experimental_method SAXS analysis of CthACC agrees with a dimeric state and an elongated shape with a maximum extent of 350 Å (Supplementary Table 1). RESULTS +17 23 CthACC protein SAXS analysis of CthACC agrees with a dimeric state and an elongated shape with a maximum extent of 350 Å (Supplementary Table 1). RESULTS +38 45 dimeric oligomeric_state SAXS analysis of CthACC agrees with a dimeric state and an elongated shape with a maximum extent of 350 Å (Supplementary Table 1). RESULTS +59 74 elongated shape protein_state SAXS analysis of CthACC agrees with a dimeric state and an elongated shape with a maximum extent of 350 Å (Supplementary Table 1). RESULTS +25 42 scattering curves evidence The smooth appearance of scattering curves and derived distance distributions might indicate substantial interdomain flexibility (Supplementary Fig. 2a–c). RESULTS +47 77 derived distance distributions evidence The smooth appearance of scattering curves and derived distance distributions might indicate substantial interdomain flexibility (Supplementary Fig. 2a–c). RESULTS +33 44 full-length protein_state Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. RESULTS +45 51 CthACC protein Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. RESULTS +52 61 particles evidence Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. RESULTS +76 78 MS experimental_method Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. RESULTS +106 120 phosphorylated protein_state Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. RESULTS +121 139 low-activity state protein_state Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. RESULTS +144 161 negative stain EM experimental_method Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. RESULTS +204 221 rod-like extended protein_state Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. RESULTS +225 233 U-shaped protein_state Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. RESULTS +234 243 particles evidence Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. RESULTS +0 14 Class averages evidence Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. RESULTS +28 88 maximum-likelihood-based two-dimensional (2D) classification experimental_method Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. RESULTS +109 116 dimeric oligomeric_state Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. RESULTS +117 119 CT structure_element Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. RESULTS +135 139 full protein_state Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. RESULTS +140 150 BC–BCCP–CD mutant Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. RESULTS +170 178 protomer oligomeric_state Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. RESULTS +230 232 BC structure_element Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. RESULTS +233 235 CD structure_element Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. RESULTS +260 262 CT structure_element Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. RESULTS +263 268 dimer oligomeric_state Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. RESULTS +38 41 CDN structure_element They identify the connections between CDN/CDL and between CDC2/CT as major contributors to conformational heterogeneity (Supplementary Fig. 4a,b). RESULTS +42 45 CDL structure_element They identify the connections between CDN/CDL and between CDC2/CT as major contributors to conformational heterogeneity (Supplementary Fig. 4a,b). RESULTS +58 62 CDC2 structure_element They identify the connections between CDN/CDL and between CDC2/CT as major contributors to conformational heterogeneity (Supplementary Fig. 4a,b). RESULTS +63 65 CT structure_element They identify the connections between CDN/CDL and between CDC2/CT as major contributors to conformational heterogeneity (Supplementary Fig. 4a,b). RESULTS +23 36 CDC2/CT hinge structure_element The flexibility in the CDC2/CT hinge appears substantially larger than the variations observed in the set of crystal structures. RESULTS +109 127 crystal structures evidence The flexibility in the CDC2/CT hinge appears substantially larger than the variations observed in the set of crystal structures. RESULTS +4 6 BC structure_element The BC domain is not completely disordered, but laterally attached to BT/CDN in a generally conserved position, albeit with increased flexibility. RESULTS +70 72 BT structure_element The BC domain is not completely disordered, but laterally attached to BT/CDN in a generally conserved position, albeit with increased flexibility. RESULTS +73 76 CDN structure_element The BC domain is not completely disordered, but laterally attached to BT/CDN in a generally conserved position, albeit with increased flexibility. RESULTS +82 110 generally conserved position protein_state The BC domain is not completely disordered, but laterally attached to BT/CDN in a generally conserved position, albeit with increased flexibility. RESULTS +26 59 linear and U-shaped conformations protein_state Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110 Å. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase. RESULTS +99 101 BC structure_element Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110 Å. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase. RESULTS +106 108 CT structure_element Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110 Å. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase. RESULTS +109 121 active sites site Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110 Å. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase. RESULTS +211 217 static protein_state Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110 Å. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase. RESULTS +218 228 structures evidence Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110 Å. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase. RESULTS +250 278 biotin-dependent carboxylase protein_type Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110 Å. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase. RESULTS +47 61 BCCP–CD linker structure_element Furthermore, based on an average length of the BCCP–CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT. RESULTS +65 71 fungal taxonomy_domain Furthermore, based on an average length of the BCCP–CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT. RESULTS +72 75 ACC protein_type Furthermore, based on an average length of the BCCP–CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT. RESULTS +79 93 26 amino acids residue_range Furthermore, based on an average length of the BCCP–CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT. RESULTS +111 115 BCCP structure_element Furthermore, based on an average length of the BCCP–CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT. RESULTS +160 172 active sites site Furthermore, based on an average length of the BCCP–CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT. RESULTS +176 178 BC structure_element Furthermore, based on an average length of the BCCP–CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT. RESULTS +183 185 CT structure_element Furthermore, based on an average length of the BCCP–CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT. RESULTS +130 149 CDC1/CDC2 interface site The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure. RESULTS +178 185 Ser1157 residue_name_number The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure. RESULTS +186 200 phosphorylated protein_state The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure. RESULTS +201 216 regulatory loop structure_element The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure. RESULTS +237 240 Sce species The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure. RESULTS +240 242 CD structure_element The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure. RESULTS +243 260 crystal structure evidence The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure. RESULTS +32 38 fungal taxonomy_domain Altogether, the architecture of fungal ACC is based on the central dimeric CT domain (Fig. 4d). DISCUSS +39 42 ACC protein_type Altogether, the architecture of fungal ACC is based on the central dimeric CT domain (Fig. 4d). DISCUSS +67 74 dimeric oligomeric_state Altogether, the architecture of fungal ACC is based on the central dimeric CT domain (Fig. 4d). DISCUSS +75 77 CT structure_element Altogether, the architecture of fungal ACC is based on the central dimeric CT domain (Fig. 4d). DISCUSS +4 6 CD structure_element The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain. DISCUSS +33 43 subdomains structure_element The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain. DISCUSS +74 76 CT structure_element The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain. DISCUSS +84 90 mobile protein_state The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain. DISCUSS +91 95 BCCP structure_element The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain. DISCUSS +103 111 oriented protein_state The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain. DISCUSS +112 114 BC structure_element The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain. DISCUSS +4 6 CD structure_element The CD has no direct role in substrate recognition or catalysis but contributes to the regulation of all eukaryotic ACCs. DISCUSS +105 115 eukaryotic taxonomy_domain The CD has no direct role in substrate recognition or catalysis but contributes to the regulation of all eukaryotic ACCs. DISCUSS +116 120 ACCs protein_type The CD has no direct role in substrate recognition or catalysis but contributes to the regulation of all eukaryotic ACCs. DISCUSS +3 20 higher eukaryotic taxonomy_domain In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263. DISCUSS +21 25 ACCs protein_type In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263. DISCUSS +42 57 phosphorylation ptm In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263. DISCUSS +98 113 phosphorylation ptm In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263. DISCUSS +117 122 Ser80 residue_name_number In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263. DISCUSS +124 131 Ser1201 residue_name_number In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263. DISCUSS +136 143 Ser1263 residue_name_number In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263. DISCUSS +3 9 fungal taxonomy_domain In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity. DISCUSS +10 13 ACC protein_type In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity. DISCUSS +24 31 Ser1157 residue_name_number In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity. DISCUSS +39 54 regulatory loop structure_element In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity. DISCUSS +62 64 CD structure_element In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity. DISCUSS +77 97 phosphorylation site site In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity. DISCUSS +136 150 phosphorylated protein_state In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity. DISCUSS +193 196 ACC protein_type In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity. DISCUSS +7 21 phosphorylated protein_state In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface. DISCUSS +33 48 regulatory loop structure_element In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface. DISCUSS +60 67 Ser1157 residue_name_number In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface. DISCUSS +83 87 CDC1 structure_element In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface. DISCUSS +88 92 CDC2 structure_element In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface. DISCUSS +119 141 conformational freedom protein_state In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface. DISCUSS +150 171 interdomain interface site In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface. DISCUSS +29 34 hinge structure_element However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer. DISCUSS +55 72 full ACC activity protein_state However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer. DISCUSS +103 121 BCCP anchor points structure_element However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer. DISCUSS +130 142 active sites site However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer. DISCUSS +146 148 BC structure_element However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer. DISCUSS +153 155 CT structure_element However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer. DISCUSS +206 210 BCCP structure_element However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer. DISCUSS +49 55 fungal taxonomy_domain The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT. DISCUSS +56 59 ACC protein_type The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT. DISCUSS +107 113 unique protein_state The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT. DISCUSS +114 116 CD structure_element The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT. DISCUSS +175 187 active sites site The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT. DISCUSS +191 193 BC structure_element The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT. DISCUSS +198 200 CT structure_element The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT. DISCUSS +21 27 fungal taxonomy_domain A comparison between fungal and human ACC will help to further discriminate mechanistic differences that contribute to the extended control and polymerization of human ACC. DISCUSS +32 37 human species A comparison between fungal and human ACC will help to further discriminate mechanistic differences that contribute to the extended control and polymerization of human ACC. DISCUSS +38 41 ACC protein_type A comparison between fungal and human ACC will help to further discriminate mechanistic differences that contribute to the extended control and polymerization of human ACC. DISCUSS +162 167 human species A comparison between fungal and human ACC will help to further discriminate mechanistic differences that contribute to the extended control and polymerization of human ACC. DISCUSS +168 171 ACC protein_type A comparison between fungal and human ACC will help to further discriminate mechanistic differences that contribute to the extended control and polymerization of human ACC. DISCUSS +17 34 crystal structure evidence Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong. DISCUSS +38 54 near full-length protein_state Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong. DISCUSS +55 73 non-phosphorylated protein_state Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong. DISCUSS +74 77 ACC protein_type Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong. DISCUSS +83 95 S. cerevisae species Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong. DISCUSS +97 109 lacking only protein_state Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong. DISCUSS +110 112 21 residue_range Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong. DISCUSS +153 158 flACC protein Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong. DISCUSS +3 8 flACC protein In flACC, the ACC dimer obeys twofold symmetry and assembles in a triangular architecture with dimeric BC domains (Supplementary Fig. 5a). DISCUSS +14 17 ACC protein_type In flACC, the ACC dimer obeys twofold symmetry and assembles in a triangular architecture with dimeric BC domains (Supplementary Fig. 5a). DISCUSS +18 23 dimer oligomeric_state In flACC, the ACC dimer obeys twofold symmetry and assembles in a triangular architecture with dimeric BC domains (Supplementary Fig. 5a). DISCUSS +66 89 triangular architecture protein_state In flACC, the ACC dimer obeys twofold symmetry and assembles in a triangular architecture with dimeric BC domains (Supplementary Fig. 5a). DISCUSS +95 102 dimeric oligomeric_state In flACC, the ACC dimer obeys twofold symmetry and assembles in a triangular architecture with dimeric BC domains (Supplementary Fig. 5a). DISCUSS +103 105 BC structure_element In flACC, the ACC dimer obeys twofold symmetry and assembles in a triangular architecture with dimeric BC domains (Supplementary Fig. 5a). DISCUSS +16 31 mutational data experimental_method In their study, mutational data indicate a requirement for BC dimerization for catalytic activity. DISCUSS +24 44 elongated open shape protein_state The transition from the elongated open shape, observed in our experiments, towards a compact triangular shape is based on an intricate interplay of several hinge-bending motions in the CD (Fig. 4d). DISCUSS +85 109 compact triangular shape protein_state The transition from the elongated open shape, observed in our experiments, towards a compact triangular shape is based on an intricate interplay of several hinge-bending motions in the CD (Fig. 4d). DISCUSS +185 187 CD structure_element The transition from the elongated open shape, observed in our experiments, towards a compact triangular shape is based on an intricate interplay of several hinge-bending motions in the CD (Fig. 4d). DISCUSS +0 10 Comparison experimental_method Comparison of flACC with our CthΔBCCP structure reveals the CDC2/CT hinge as a major contributor to conformational flexibility (Supplementary Fig. 5b,c). DISCUSS +14 19 flACC protein Comparison of flACC with our CthΔBCCP structure reveals the CDC2/CT hinge as a major contributor to conformational flexibility (Supplementary Fig. 5b,c). DISCUSS +29 37 CthΔBCCP mutant Comparison of flACC with our CthΔBCCP structure reveals the CDC2/CT hinge as a major contributor to conformational flexibility (Supplementary Fig. 5b,c). DISCUSS +38 47 structure evidence Comparison of flACC with our CthΔBCCP structure reveals the CDC2/CT hinge as a major contributor to conformational flexibility (Supplementary Fig. 5b,c). DISCUSS +60 73 CDC2/CT hinge structure_element Comparison of flACC with our CthΔBCCP structure reveals the CDC2/CT hinge as a major contributor to conformational flexibility (Supplementary Fig. 5b,c). DISCUSS +3 8 flACC protein In flACC, CDC2 rotates ∼120° with respect to the CT domain. DISCUSS +10 14 CDC2 structure_element In flACC, CDC2 rotates ∼120° with respect to the CT domain. DISCUSS +49 51 CT structure_element In flACC, CDC2 rotates ∼120° with respect to the CT domain. DISCUSS +2 14 second hinge structure_element A second hinge can be identified between CDC1/CDC2. DISCUSS +41 45 CDC1 structure_element A second hinge can be identified between CDC1/CDC2. DISCUSS +46 50 CDC2 structure_element A second hinge can be identified between CDC1/CDC2. DISCUSS +18 31 superposition experimental_method On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). DISCUSS +35 39 CDC2 structure_element On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). DISCUSS +41 45 CDC1 structure_element On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). DISCUSS +53 67 phosphorylated protein_state On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). DISCUSS +68 71 Sce species On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). DISCUSS +71 73 CD structure_element On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). DISCUSS +104 108 CDC1 structure_element On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). DISCUSS +116 134 non-phosphorylated protein_state On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). DISCUSS +135 140 flACC protein On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). DISCUSS +207 225 non-phosphorylated protein_state On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). DISCUSS +226 234 HsaBT-CD mutant On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). DISCUSS +5 15 inspecting experimental_method When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations. DISCUSS +31 39 protomer oligomeric_state When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations. DISCUSS +44 52 fragment mutant When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations. DISCUSS +53 63 structures evidence When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations. DISCUSS +111 130 CDN/CDC1 connection structure_element When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations. DISCUSS +136 151 highly flexible protein_state When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations. DISCUSS +152 157 hinge structure_element When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations. DISCUSS +19 29 regulatory protein_state The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation. DISCUSS +30 49 phophorylation site site The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation. DISCUSS +53 59 fungal taxonomy_domain The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation. DISCUSS +60 63 ACC protein_type The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation. DISCUSS +71 86 regulatory loop structure_element The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation. DISCUSS +116 120 CDC1 structure_element The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation. DISCUSS +121 125 CDC2 structure_element The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation. DISCUSS +170 188 hinge conformation structure_element The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation. DISCUSS +3 8 flACC protein In flACC, the regulatory loop is mostly disordered, illustrating the increased flexibility due to the absence of the phosphoryl group. DISCUSS +14 29 regulatory loop structure_element In flACC, the regulatory loop is mostly disordered, illustrating the increased flexibility due to the absence of the phosphoryl group. DISCUSS +33 50 mostly disordered protein_state In flACC, the regulatory loop is mostly disordered, illustrating the increased flexibility due to the absence of the phosphoryl group. DISCUSS +117 127 phosphoryl chemical In flACC, the regulatory loop is mostly disordered, illustrating the increased flexibility due to the absence of the phosphoryl group. DISCUSS +36 45 protomers oligomeric_state Only in three out of eight observed protomers a short peptide stretch (including Ser1157) was modelled. DISCUSS +48 61 short peptide structure_element Only in three out of eight observed protomers a short peptide stretch (including Ser1157) was modelled. DISCUSS +81 88 Ser1157 residue_name_number Only in three out of eight observed protomers a short peptide stretch (including Ser1157) was modelled. DISCUSS +94 102 modelled evidence Only in three out of eight observed protomers a short peptide stretch (including Ser1157) was modelled. DISCUSS +23 30 Ser1157 residue_name_number In those instances the Ser1157 residue is located at a distance of 14–20 Å away from the location of the phosphorylated serine observed here, based on superposition of either CDC1 or CDC2. DISCUSS +105 119 phosphorylated protein_state In those instances the Ser1157 residue is located at a distance of 14–20 Å away from the location of the phosphorylated serine observed here, based on superposition of either CDC1 or CDC2. DISCUSS +120 126 serine residue_name In those instances the Ser1157 residue is located at a distance of 14–20 Å away from the location of the phosphorylated serine observed here, based on superposition of either CDC1 or CDC2. DISCUSS +151 164 superposition experimental_method In those instances the Ser1157 residue is located at a distance of 14–20 Å away from the location of the phosphorylated serine observed here, based on superposition of either CDC1 or CDC2. DISCUSS +175 179 CDC1 structure_element In those instances the Ser1157 residue is located at a distance of 14–20 Å away from the location of the phosphorylated serine observed here, based on superposition of either CDC1 or CDC2. DISCUSS +183 187 CDC2 structure_element In those instances the Ser1157 residue is located at a distance of 14–20 Å away from the location of the phosphorylated serine observed here, based on superposition of either CDC1 or CDC2. DISCUSS +0 8 Applying experimental_method Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). DISCUSS +33 48 CDC1/CDC2 hinge structure_element Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). DISCUSS +61 64 Sce species Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). DISCUSS +64 66 CD structure_element Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). DISCUSS +70 75 flACC protein Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). DISCUSS +85 88 CDN structure_element Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). DISCUSS +114 118 CDC2 structure_element Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). DISCUSS +123 125 BT structure_element Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). DISCUSS +126 129 CDN structure_element Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). DISCUSS +144 146 CT structure_element Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). DISCUSS +53 68 phosphorylation ptm Thus, in accordance with the results presented here, phosphorylation of Ser1157 in SceACC most likely limits flexibility in the CDC1/CDC2 hinge such that activation through BC dimerization is not possible (Fig. 4d), which however does not exclude intermolecular dimerization. DISCUSS +72 79 Ser1157 residue_name_number Thus, in accordance with the results presented here, phosphorylation of Ser1157 in SceACC most likely limits flexibility in the CDC1/CDC2 hinge such that activation through BC dimerization is not possible (Fig. 4d), which however does not exclude intermolecular dimerization. DISCUSS +83 89 SceACC protein Thus, in accordance with the results presented here, phosphorylation of Ser1157 in SceACC most likely limits flexibility in the CDC1/CDC2 hinge such that activation through BC dimerization is not possible (Fig. 4d), which however does not exclude intermolecular dimerization. DISCUSS +128 143 CDC1/CDC2 hinge structure_element Thus, in accordance with the results presented here, phosphorylation of Ser1157 in SceACC most likely limits flexibility in the CDC1/CDC2 hinge such that activation through BC dimerization is not possible (Fig. 4d), which however does not exclude intermolecular dimerization. DISCUSS +173 175 BC structure_element Thus, in accordance with the results presented here, phosphorylation of Ser1157 in SceACC most likely limits flexibility in the CDC1/CDC2 hinge such that activation through BC dimerization is not possible (Fig. 4d), which however does not exclude intermolecular dimerization. DISCUSS +13 15 EM experimental_method In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7). DISCUSS +16 27 micrographs evidence In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7). DISCUSS +31 45 phosphorylated protein_state In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7). DISCUSS +50 66 dephosphorylated protein_state In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7). DISCUSS +67 73 SceACC protein In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7). DISCUSS +106 142 elongated and U-shaped conformations protein_state In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7). DISCUSS +181 209 particle shape distributions evidence In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7). DISCUSS +25 41 triangular shape protein_state This implicates that the triangular shape with dimeric BC domains has a low population also in the active form, even though a biasing influence of grid preparation cannot be excluded completely. DISCUSS +47 54 dimeric oligomeric_state This implicates that the triangular shape with dimeric BC domains has a low population also in the active form, even though a biasing influence of grid preparation cannot be excluded completely. DISCUSS +55 57 BC structure_element This implicates that the triangular shape with dimeric BC domains has a low population also in the active form, even though a biasing influence of grid preparation cannot be excluded completely. DISCUSS +99 110 active form protein_state This implicates that the triangular shape with dimeric BC domains has a low population also in the active form, even though a biasing influence of grid preparation cannot be excluded completely. DISCUSS +76 110 carrier protein-based multienzymes protein_type Large-scale conformational variability has also been observed in most other carrier protein-based multienzymes, including polyketide and fatty-acid synthases (with the exception of fungal-type fatty-acid synthases), non-ribosomal peptide synthetases and the pyruvate dehydrogenase complexes, although based on completely different architectures. DISCUSS +122 157 polyketide and fatty-acid synthases protein_type Large-scale conformational variability has also been observed in most other carrier protein-based multienzymes, including polyketide and fatty-acid synthases (with the exception of fungal-type fatty-acid synthases), non-ribosomal peptide synthetases and the pyruvate dehydrogenase complexes, although based on completely different architectures. DISCUSS +181 213 fungal-type fatty-acid synthases protein_type Large-scale conformational variability has also been observed in most other carrier protein-based multienzymes, including polyketide and fatty-acid synthases (with the exception of fungal-type fatty-acid synthases), non-ribosomal peptide synthetases and the pyruvate dehydrogenase complexes, although based on completely different architectures. DISCUSS +216 249 non-ribosomal peptide synthetases protein_type Large-scale conformational variability has also been observed in most other carrier protein-based multienzymes, including polyketide and fatty-acid synthases (with the exception of fungal-type fatty-acid synthases), non-ribosomal peptide synthetases and the pyruvate dehydrogenase complexes, although based on completely different architectures. DISCUSS +258 290 pyruvate dehydrogenase complexes protein_type Large-scale conformational variability has also been observed in most other carrier protein-based multienzymes, including polyketide and fatty-acid synthases (with the exception of fungal-type fatty-acid synthases), non-ribosomal peptide synthetases and the pyruvate dehydrogenase complexes, although based on completely different architectures. DISCUSS +15 37 structural information evidence Together, this structural information suggests that variable carrier protein tethering is not sufficient for efficient substrate transfer and catalysis in any of these systems. DISCUSS +4 29 determination of a set of experimental_method The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d). DISCUSS +30 48 crystal structures evidence The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d). DISCUSS +52 58 SceACC protein The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d). DISCUSS +74 90 unphosphorylated protein_state The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d). DISCUSS +95 109 phosphorylated protein_state The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d). DISCUSS +117 138 major regulatory site site The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d). DISCUSS +139 146 Ser1157 residue_name_number The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d). DISCUSS +4 18 phosphorylated protein_state The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC. DISCUSS +19 34 regulatory loop structure_element The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC. DISCUSS +47 62 allosteric site site The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC. DISCUSS +70 79 interface site The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC. DISCUSS +87 100 non-catalytic protein_state The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC. DISCUSS +157 163 hinges structure_element The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC. DISCUSS +171 178 dynamic protein_state The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC. DISCUSS +179 182 ACC protein_type The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC. DISCUSS +32 58 rare, compact conformation protein_state It disfavours the adoption of a rare, compact conformation, in which intramolecular dimerization of the BC domains results in catalytic turnover. DISCUSS +104 106 BC structure_element It disfavours the adoption of a rare, compact conformation, in which intramolecular dimerization of the BC domains results in catalytic turnover. DISCUSS +138 159 active site structure site The regulation of activity thus results from restrained large-scale conformational dynamics rather than a direct or indirect influence on active site structure. DISCUSS +23 26 ACC protein_type To our best knowledge, ACC is the first multienzyme for which such a phosphorylation-dependent mechanical control mechanism has been visualized. DISCUSS +40 51 multienzyme protein_type To our best knowledge, ACC is the first multienzyme for which such a phosphorylation-dependent mechanical control mechanism has been visualized. DISCUSS +69 84 phosphorylation ptm To our best knowledge, ACC is the first multienzyme for which such a phosphorylation-dependent mechanical control mechanism has been visualized. DISCUSS +24 27 ACC protein_type However, the example of ACC now demonstrates the possibility of regulating activity by controlled dynamics of non-enzymatic linker regions also in other families of carrier-dependent multienzymes. DISCUSS +110 138 non-enzymatic linker regions structure_element However, the example of ACC now demonstrates the possibility of regulating activity by controlled dynamics of non-enzymatic linker regions also in other families of carrier-dependent multienzymes. DISCUSS +165 195 carrier-dependent multienzymes protein_type However, the example of ACC now demonstrates the possibility of regulating activity by controlled dynamics of non-enzymatic linker regions also in other families of carrier-dependent multienzymes. DISCUSS +4 18 phosphorylated protein_state The phosphorylated central domain of yeast ACC. FIG +19 33 central domain structure_element The phosphorylated central domain of yeast ACC. FIG +37 42 yeast taxonomy_domain The phosphorylated central domain of yeast ACC. FIG +43 46 ACC protein_type The phosphorylated central domain of yeast ACC. FIG +53 63 eukaryotic taxonomy_domain (a) Schematic overview of the domain organization of eukaryotic ACCs. FIG +64 68 ACCs protein_type (a) Schematic overview of the domain organization of eukaryotic ACCs. FIG +0 23 Crystallized constructs evidence Crystallized constructs are indicated. FIG +34 37 Sce species (b) Cartoon representation of the SceCD crystal structure. FIG +37 39 CD structure_element (b) Cartoon representation of the SceCD crystal structure. FIG +40 57 crystal structure evidence (b) Cartoon representation of the SceCD crystal structure. FIG +0 3 CDN structure_element CDN is linked by a four-helix bundle (CDL) to two α–β-fold domains (CDC1 and CDC2). FIG +19 36 four-helix bundle structure_element CDN is linked by a four-helix bundle (CDL) to two α–β-fold domains (CDC1 and CDC2). FIG +38 41 CDL structure_element CDN is linked by a four-helix bundle (CDL) to two α–β-fold domains (CDC1 and CDC2). FIG +46 66 two α–β-fold domains structure_element CDN is linked by a four-helix bundle (CDL) to two α–β-fold domains (CDC1 and CDC2). FIG +68 72 CDC1 structure_element CDN is linked by a four-helix bundle (CDL) to two α–β-fold domains (CDC1 and CDC2). FIG +77 81 CDC2 structure_element CDN is linked by a four-helix bundle (CDL) to two α–β-fold domains (CDC1 and CDC2). FIG +4 19 regulatory loop structure_element The regulatory loop is shown as bold cartoon, and the phosphorylated Ser1157 is marked by a red triangle. FIG +54 68 phosphorylated protein_state The regulatory loop is shown as bold cartoon, and the phosphorylated Ser1157 is marked by a red triangle. FIG +69 76 Ser1157 residue_name_number The regulatory loop is shown as bold cartoon, and the phosphorylated Ser1157 is marked by a red triangle. FIG +4 17 Superposition experimental_method (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +21 25 CDC1 structure_element (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +30 34 CDC2 structure_element (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +43 59 highly conserved protein_state (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +60 65 folds structure_element (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +75 90 regulatory loop structure_element (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +100 114 phosphorylated protein_state (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +115 122 Ser1157 residue_name_number (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +155 159 CDC1 structure_element (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +164 168 CDC2 structure_element (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +174 183 conserved protein_state (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +193 200 Arg1173 residue_name_number (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +205 212 Arg1260 residue_name_number (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +228 238 phosphoryl chemical (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. FIG +27 35 HsaBT-CD mutant (e) Structural overview of HsaBT-CD. FIG +40 44 BCCP structure_element The attachment points to the N-terminal BCCP domain and the C-terminal CT domain are indicated with spheres. FIG +71 73 CT structure_element The attachment points to the N-terminal BCCP domain and the C-terminal CT domain are indicated with spheres. FIG +20 22 CD structure_element Architecture of the CD–CT core of fungal ACC. FIG +23 25 CT structure_element Architecture of the CD–CT core of fungal ACC. FIG +34 40 fungal taxonomy_domain Architecture of the CD–CT core of fungal ACC. FIG +41 44 ACC protein_type Architecture of the CD–CT core of fungal ACC. FIG +26 44 crystal structures evidence Cartoon representation of crystal structures of multidomain constructs of CthACC. FIG +48 70 multidomain constructs mutant Cartoon representation of crystal structures of multidomain constructs of CthACC. FIG +74 80 CthACC protein Cartoon representation of crystal structures of multidomain constructs of CthACC. FIG +4 12 protomer oligomeric_state One protomer is shown in colour and one in grey. FIG +37 48 active site site Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively. FIG +52 54 CT structure_element Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively. FIG +79 88 conserved protein_state Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively. FIG +89 99 regulatory protein_state Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively. FIG +100 118 phosphoserine site site Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively. FIG +128 131 Sce species Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively. FIG +131 133 CD structure_element Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively. FIG +34 38 CDC2 structure_element Variability of the connections of CDC2 to CT and CDC1 in fungal ACC. FIG +42 44 CT structure_element Variability of the connections of CDC2 to CT and CDC1 in fungal ACC. FIG +49 53 CDC1 structure_element Variability of the connections of CDC2 to CT and CDC1 in fungal ACC. FIG +57 63 fungal taxonomy_domain Variability of the connections of CDC2 to CT and CDC1 in fungal ACC. FIG +64 67 ACC protein_type Variability of the connections of CDC2 to CT and CDC1 in fungal ACC. FIG +4 9 Hinge structure_element (a) Hinge properties of the CDC2–CT connection analysed by a CT-based superposition of eight instances of the CDC2-CT segment. FIG +28 46 CDC2–CT connection structure_element (a) Hinge properties of the CDC2–CT connection analysed by a CT-based superposition of eight instances of the CDC2-CT segment. FIG +61 83 CT-based superposition experimental_method (a) Hinge properties of the CDC2–CT connection analysed by a CT-based superposition of eight instances of the CDC2-CT segment. FIG +110 125 CDC2-CT segment mutant (a) Hinge properties of the CDC2–CT connection analysed by a CT-based superposition of eight instances of the CDC2-CT segment. FIG +22 30 protomer oligomeric_state For clarity, only one protomer of CthCD-CTCter1 is shown in full colour as reference. FIG +34 47 CthCD-CTCter1 mutant For clarity, only one protomer of CthCD-CTCter1 is shown in full colour as reference. FIG +21 25 CDC2 structure_element For other instances, CDC2 domains are shown in transparent tube representation with only one helix each highlighted. FIG +74 78 CDC2 structure_element The range of hinge bending is indicated and the connection points between CDC2 and CT (blue) as well as between CDC1 and CDC2 (green and grey) are marked as spheres. FIG +83 85 CT structure_element The range of hinge bending is indicated and the connection points between CDC2 and CT (blue) as well as between CDC1 and CDC2 (green and grey) are marked as spheres. FIG +112 116 CDC1 structure_element The range of hinge bending is indicated and the connection points between CDC2 and CT (blue) as well as between CDC1 and CDC2 (green and grey) are marked as spheres. FIG +121 125 CDC2 structure_element The range of hinge bending is indicated and the connection points between CDC2 and CT (blue) as well as between CDC1 and CDC2 (green and grey) are marked as spheres. FIG +8 29 interdomain interface site (b) The interdomain interface of CDC1 and CDC2 exhibits only limited plasticity. FIG +33 37 CDC1 structure_element (b) The interdomain interface of CDC1 and CDC2 exhibits only limited plasticity. FIG +42 46 CDC2 structure_element (b) The interdomain interface of CDC1 and CDC2 exhibits only limited plasticity. FIG +32 36 CDC1 structure_element Representation as in a, but the CDC1 and CDC2 are superposed based on CDC2. FIG +41 45 CDC2 structure_element Representation as in a, but the CDC1 and CDC2 are superposed based on CDC2. FIG +50 60 superposed experimental_method Representation as in a, but the CDC1 and CDC2 are superposed based on CDC2. FIG +70 74 CDC2 structure_element Representation as in a, but the CDC1 and CDC2 are superposed based on CDC2. FIG +4 12 protomer oligomeric_state One protomer of CthΔBCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle. FIG +16 24 CthΔBCCP mutant One protomer of CthΔBCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle. FIG +49 52 CDL structure_element One protomer of CthΔBCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle. FIG +109 123 phosphorylated protein_state One protomer of CthΔBCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle. FIG +124 130 serine residue_name One protomer of CthΔBCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle. FIG +140 143 Sce species One protomer of CthΔBCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle. FIG +143 145 CD structure_element One protomer of CthΔBCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle. FIG +27 31 CDC1 structure_element The connection points from CDC1 to CDC2 and to CDL are represented by green spheres. FIG +35 39 CDC2 structure_element The connection points from CDC1 to CDC2 and to CDL are represented by green spheres. FIG +47 50 CDL structure_element The connection points from CDC1 to CDC2 and to CDL are represented by green spheres. FIG +31 37 fungal taxonomy_domain The conformational dynamics of fungal ACC. FIG +38 41 ACC protein_type The conformational dynamics of fungal ACC. FIG +52 55 CDN structure_element (a–c) Large-scale conformational variability of the CDN domain relative to the CDL/CDC1 domain. FIG +79 82 CDL structure_element (a–c) Large-scale conformational variability of the CDN domain relative to the CDL/CDC1 domain. FIG +83 87 CDC1 structure_element (a–c) Large-scale conformational variability of the CDN domain relative to the CDL/CDC1 domain. FIG +0 9 CthCD-CT1 mutant CthCD-CT1 (in colour) serves as reference, the compared structures (as indicated, numbers after construct name differentiate between individual protomers) are shown in grey. FIG +47 66 compared structures experimental_method CthCD-CT1 (in colour) serves as reference, the compared structures (as indicated, numbers after construct name differentiate between individual protomers) are shown in grey. FIG +144 153 protomers oligomeric_state CthCD-CT1 (in colour) serves as reference, the compared structures (as indicated, numbers after construct name differentiate between individual protomers) are shown in grey. FIG +19 22 CDN structure_element Domains other than CDN and CDL/CDC1 are omitted for clarity. FIG +27 30 CDL structure_element Domains other than CDN and CDL/CDC1 are omitted for clarity. FIG +31 35 CDC1 structure_element Domains other than CDN and CDL/CDC1 are omitted for clarity. FIG +68 71 CDN structure_element The domains are labelled and the distances between the N termini of CDN (spheres) in the compared structures are indicated. FIG +23 29 fungal taxonomy_domain (d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state. FIG +30 33 ACC protein_type (d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state. FIG +82 84 CD structure_element (d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state. FIG +92 106 phosphorylated protein_state (d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state. FIG +107 116 inhibited protein_state (d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state. FIG +124 142 non-phosphorylated protein_state (d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state. FIG +143 152 activated protein_state (d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state. FIG +19 23 CDC2 structure_element Flexibility of the CDC2/CT and CDN/CDL hinges is illustrated by arrows. FIG +24 26 CT structure_element Flexibility of the CDC2/CT and CDN/CDL hinges is illustrated by arrows. FIG +31 34 CDN structure_element Flexibility of the CDC2/CT and CDN/CDL hinges is illustrated by arrows. FIG +35 38 CDL structure_element Flexibility of the CDC2/CT and CDN/CDL hinges is illustrated by arrows. FIG +39 45 hinges structure_element Flexibility of the CDC2/CT and CDN/CDL hinges is illustrated by arrows. FIG +4 11 Ser1157 residue_name_number The Ser1157 phosphorylation site and the regulatory loop are schematically indicated in magenta. FIG +12 27 phosphorylation ptm The Ser1157 phosphorylation site and the regulatory loop are schematically indicated in magenta. FIG +41 56 regulatory loop structure_element The Ser1157 phosphorylation site and the regulatory loop are schematically indicated in magenta. FIG diff --git a/annotation_CSV/PMC4841544.csv b/annotation_CSV/PMC4841544.csv new file mode 100644 index 0000000000000000000000000000000000000000..2b5f6c5c8fda7f4adcd0798365b2bc003f4cdcd9 --- /dev/null +++ b/annotation_CSV/PMC4841544.csv @@ -0,0 +1,1460 @@ +anno_start anno_end anno_text entity_type sentence section +50 54 NadR protein Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA TITLE +60 85 Transcriptional Repressor protein_type Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA TITLE +89 102 Meningococcal taxonomy_domain Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA TITLE +120 124 NadA protein Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA TITLE +1 20 Neisseria adhesin A protein Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. ABSTRACT +22 26 NadA protein Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. ABSTRACT +46 59 meningococcal taxonomy_domain Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. ABSTRACT +115 120 human species Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. ABSTRACT +0 4 NadA protein NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. ABSTRACT +112 137 serogroup B meningococcus taxonomy_domain NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. ABSTRACT +14 18 NadA protein The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. ABSTRACT +26 35 bacterial taxonomy_domain The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. ABSTRACT +173 178 human species The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. ABSTRACT +231 240 bacterial taxonomy_domain The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. ABSTRACT +70 74 nadA gene It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. ABSTRACT +136 161 transcriptional regulator protein_type It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. ABSTRACT +162 166 NadR protein It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. ABSTRACT +168 197 Neisseria adhesin A Regulator protein It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. ABSTRACT +0 4 NadR protein NadR binds the nadA promoter and represses gene transcription. ABSTRACT +15 19 nadA gene NadR binds the nadA promoter and represses gene transcription. ABSTRACT +0 4 NadR protein NadR binds the nadA promoter and represses gene transcription. ABSTRACT +15 19 nadA gene NadR binds the nadA promoter and represses gene transcription. ABSTRACT +7 18 presence of protein_state In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. ABSTRACT +19 41 4-hydroxyphenylacetate chemical In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. ABSTRACT +43 48 4-HPA chemical In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. ABSTRACT +75 80 human species In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. ABSTRACT +135 144 bacterial taxonomy_domain In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. ABSTRACT +171 175 NadR protein In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. ABSTRACT +183 187 nadA gene In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. ABSTRACT +215 219 nadA gene In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. ABSTRACT +0 4 NadR protein NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. ABSTRACT +61 74 meningococcal taxonomy_domain NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. ABSTRACT +40 44 NadR protein To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. ABSTRACT +57 62 4-HPA chemical To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. ABSTRACT +76 124 structural, biochemical, and mutagenesis studies experimental_method To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. ABSTRACT +23 41 crystal structures evidence In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +45 56 ligand-free protein_state In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +61 73 ligand-bound protein_state In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +74 78 NadR protein In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +172 177 4-HPA chemical In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +199 206 dimeric oligomeric_state In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +207 211 NadR protein In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +329 349 hydroxyphenylacetate chemical In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +369 378 3Cl,4-HPA chemical In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +442 449 leucine residue_name In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +489 498 conserved protein_state In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +507 511 MarR protein_type In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +553 557 His7 residue_name_number In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +559 563 Ser9 residue_name_number In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +565 570 Asn11 residue_name_number In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +575 580 Phe25 residue_name_number In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +613 618 4-HPA chemical In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +697 705 bacteria taxonomy_domain In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. ABSTRACT +120 124 nadA gene Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis. ABSTRACT +153 157 NadR protein Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis. ABSTRACT +259 272 meningococcal taxonomy_domain Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis. ABSTRACT +0 25 Serogroup B meningococcus taxonomy_domain Serogroup B meningococcus (MenB) causes fatal sepsis and invasive meningococcal disease, particularly in young children and adolescents, as highlighted by recent MenB outbreaks in universities of the United States and Canada. ABSTRACT +27 31 MenB species Serogroup B meningococcus (MenB) causes fatal sepsis and invasive meningococcal disease, particularly in young children and adolescents, as highlighted by recent MenB outbreaks in universities of the United States and Canada. ABSTRACT +66 79 meningococcal taxonomy_domain Serogroup B meningococcus (MenB) causes fatal sepsis and invasive meningococcal disease, particularly in young children and adolescents, as highlighted by recent MenB outbreaks in universities of the United States and Canada. ABSTRACT +162 166 MenB species Serogroup B meningococcus (MenB) causes fatal sepsis and invasive meningococcal disease, particularly in young children and adolescents, as highlighted by recent MenB outbreaks in universities of the United States and Canada. ABSTRACT +37 41 MenB species The Bexsero vaccine protects against MenB and has recently been approved in > 35 countries worldwide. ABSTRACT +0 19 Neisseria adhesin A protein Neisseria adhesin A (NadA) present on the meningococcal surface can mediate binding to human cells and is one of the three MenB vaccine protein antigens. ABSTRACT +21 25 NadA protein Neisseria adhesin A (NadA) present on the meningococcal surface can mediate binding to human cells and is one of the three MenB vaccine protein antigens. ABSTRACT +42 55 meningococcal taxonomy_domain Neisseria adhesin A (NadA) present on the meningococcal surface can mediate binding to human cells and is one of the three MenB vaccine protein antigens. ABSTRACT +87 92 human species Neisseria adhesin A (NadA) present on the meningococcal surface can mediate binding to human cells and is one of the three MenB vaccine protein antigens. ABSTRACT +123 127 MenB species Neisseria adhesin A (NadA) present on the meningococcal surface can mediate binding to human cells and is one of the three MenB vaccine protein antigens. ABSTRACT +14 18 NadA protein The amount of NadA exposed on the meningococcal surface also influences the antibody-mediated serum bactericidal response measured in vitro. ABSTRACT +34 47 meningococcal taxonomy_domain The amount of NadA exposed on the meningococcal surface also influences the antibody-mediated serum bactericidal response measured in vitro. ABSTRACT +24 28 nadA gene A deep understanding of nadA expression is therefore important, otherwise the contribution of NadA to vaccine-induced protection against meningococcal meningitis may be underestimated. ABSTRACT +94 98 NadA protein A deep understanding of nadA expression is therefore important, otherwise the contribution of NadA to vaccine-induced protection against meningococcal meningitis may be underestimated. ABSTRACT +137 150 meningococcal taxonomy_domain A deep understanding of nadA expression is therefore important, otherwise the contribution of NadA to vaccine-induced protection against meningococcal meningitis may be underestimated. ABSTRACT +33 37 NadA protein The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. ABSTRACT +58 101 ligand-responsive transcriptional repressor protein_type The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. ABSTRACT +102 106 NadR protein The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. ABSTRACT +125 184 functional, biochemical and high-resolution structural data evidence The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. ABSTRACT +188 192 NadR protein The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. ABSTRACT +273 293 hydroxyphenylacetate chemical The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. ABSTRACT +377 381 NadR protein The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. ABSTRACT +416 424 inactive protein_state The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. ABSTRACT +47 51 NadR protein These findings shed light on the regulation of NadR, a key MarR-family virulence factor of this important human pathogen. ABSTRACT +59 63 MarR protein_type These findings shed light on the regulation of NadR, a key MarR-family virulence factor of this important human pathogen. ABSTRACT +106 111 human species These findings shed light on the regulation of NadR, a key MarR-family virulence factor of this important human pathogen. ABSTRACT +5 24 Reverse Vaccinology experimental_method The ‘Reverse Vaccinology’ approach was pioneered to identify antigens for a protein-based vaccine against serogroup B Neisseria meningitidis (MenB), a human pathogen causing potentially-fatal sepsis and invasive meningococcal disease. INTRO +106 140 serogroup B Neisseria meningitidis species The ‘Reverse Vaccinology’ approach was pioneered to identify antigens for a protein-based vaccine against serogroup B Neisseria meningitidis (MenB), a human pathogen causing potentially-fatal sepsis and invasive meningococcal disease. INTRO +142 146 MenB species The ‘Reverse Vaccinology’ approach was pioneered to identify antigens for a protein-based vaccine against serogroup B Neisseria meningitidis (MenB), a human pathogen causing potentially-fatal sepsis and invasive meningococcal disease. INTRO +151 156 human species The ‘Reverse Vaccinology’ approach was pioneered to identify antigens for a protein-based vaccine against serogroup B Neisseria meningitidis (MenB), a human pathogen causing potentially-fatal sepsis and invasive meningococcal disease. INTRO +212 225 meningococcal taxonomy_domain The ‘Reverse Vaccinology’ approach was pioneered to identify antigens for a protein-based vaccine against serogroup B Neisseria meningitidis (MenB), a human pathogen causing potentially-fatal sepsis and invasive meningococcal disease. INTRO +8 27 Reverse Vaccinology experimental_method Indeed, Reverse Vaccinology identified Neisseria adhesin A (NadA), a surface-exposed protein involved in epithelial cell invasion and found in ~30% of clinical isolates. INTRO +39 58 Neisseria adhesin A protein Indeed, Reverse Vaccinology identified Neisseria adhesin A (NadA), a surface-exposed protein involved in epithelial cell invasion and found in ~30% of clinical isolates. INTRO +60 64 NadA protein Indeed, Reverse Vaccinology identified Neisseria adhesin A (NadA), a surface-exposed protein involved in epithelial cell invasion and found in ~30% of clinical isolates. INTRO +26 43 crystal structure evidence Recently, we reported the crystal structure of NadA, providing insights into its biological and immunological functions. INTRO +47 51 NadA protein Recently, we reported the crystal structure of NadA, providing insights into its biological and immunological functions. INTRO +12 16 NadA protein Recombinant NadA elicits a strong bactericidal immune response and is therefore included in the Bexsero vaccine that protects against MenB and which was recently approved in over 35 countries worldwide. INTRO +134 138 MenB species Recombinant NadA elicits a strong bactericidal immune response and is therefore included in the Bexsero vaccine that protects against MenB and which was recently approved in over 35 countries worldwide. INTRO +31 35 nadA gene Previous studies revealed that nadA expression levels are mainly regulated by the Neisseria adhesin A Regulator (NadR). INTRO +82 111 Neisseria adhesin A Regulator protein Previous studies revealed that nadA expression levels are mainly regulated by the Neisseria adhesin A Regulator (NadR). INTRO +113 117 NadR protein Previous studies revealed that nadA expression levels are mainly regulated by the Neisseria adhesin A Regulator (NadR). INTRO +38 42 nadA gene Although additional factors influence nadA expression, we focused on its regulation by NadR, the major mediator of NadA phase variable expression. INTRO +87 91 NadR protein Although additional factors influence nadA expression, we focused on its regulation by NadR, the major mediator of NadA phase variable expression. INTRO +115 119 NadA protein Although additional factors influence nadA expression, we focused on its regulation by NadR, the major mediator of NadA phase variable expression. INTRO +11 15 NadR protein Studies of NadR also have broader implications, since a genome-wide analysis of MenB wild-type and nadR knock-out strains revealed that NadR influences the regulation of > 30 genes, including maf genes, from the multiple adhesin family. INTRO +80 84 MenB species Studies of NadR also have broader implications, since a genome-wide analysis of MenB wild-type and nadR knock-out strains revealed that NadR influences the regulation of > 30 genes, including maf genes, from the multiple adhesin family. INTRO +85 94 wild-type protein_state Studies of NadR also have broader implications, since a genome-wide analysis of MenB wild-type and nadR knock-out strains revealed that NadR influences the regulation of > 30 genes, including maf genes, from the multiple adhesin family. INTRO +99 103 nadR gene Studies of NadR also have broader implications, since a genome-wide analysis of MenB wild-type and nadR knock-out strains revealed that NadR influences the regulation of > 30 genes, including maf genes, from the multiple adhesin family. INTRO +104 113 knock-out protein_state Studies of NadR also have broader implications, since a genome-wide analysis of MenB wild-type and nadR knock-out strains revealed that NadR influences the regulation of > 30 genes, including maf genes, from the multiple adhesin family. INTRO +136 140 NadR protein Studies of NadR also have broader implications, since a genome-wide analysis of MenB wild-type and nadR knock-out strains revealed that NadR influences the regulation of > 30 genes, including maf genes, from the multiple adhesin family. INTRO +221 228 adhesin protein_type Studies of NadR also have broader implications, since a genome-wide analysis of MenB wild-type and nadR knock-out strains revealed that NadR influences the regulation of > 30 genes, including maf genes, from the multiple adhesin family. INTRO +101 110 bacterial taxonomy_domain These genes encode a wide variety of proteins connected to many biological processes contributing to bacterial survival, adaptation in the host niche, colonization and invasion. INTRO +0 4 NadR protein NadR belongs to the MarR (Multiple Antibiotic Resistance Regulator) family, a group of ligand-responsive transcriptional regulators ubiquitous in bacteria and archaea. INTRO +20 24 MarR protein_type NadR belongs to the MarR (Multiple Antibiotic Resistance Regulator) family, a group of ligand-responsive transcriptional regulators ubiquitous in bacteria and archaea. INTRO +26 66 Multiple Antibiotic Resistance Regulator protein_type NadR belongs to the MarR (Multiple Antibiotic Resistance Regulator) family, a group of ligand-responsive transcriptional regulators ubiquitous in bacteria and archaea. INTRO +87 131 ligand-responsive transcriptional regulators protein_type NadR belongs to the MarR (Multiple Antibiotic Resistance Regulator) family, a group of ligand-responsive transcriptional regulators ubiquitous in bacteria and archaea. INTRO +146 154 bacteria taxonomy_domain NadR belongs to the MarR (Multiple Antibiotic Resistance Regulator) family, a group of ligand-responsive transcriptional regulators ubiquitous in bacteria and archaea. INTRO +159 166 archaea taxonomy_domain NadR belongs to the MarR (Multiple Antibiotic Resistance Regulator) family, a group of ligand-responsive transcriptional regulators ubiquitous in bacteria and archaea. INTRO +0 4 MarR protein_type MarR family proteins can promote bacterial survival in the presence of antibiotics, toxic chemicals, organic solvents or reactive oxygen species and can regulate virulence factor expression. INTRO +33 42 bacterial taxonomy_domain MarR family proteins can promote bacterial survival in the presence of antibiotics, toxic chemicals, organic solvents or reactive oxygen species and can regulate virulence factor expression. INTRO +0 4 MarR protein_type MarR homologues can act either as transcriptional repressors or as activators. INTRO +14 18 MarR protein_type Although > 50 MarR family structures are known, a molecular understanding of their ligand-dependent regulatory mechanisms is still limited, often hampered by lack of identification of their ligands and/or DNA targets. INTRO +26 36 structures evidence Although > 50 MarR family structures are known, a molecular understanding of their ligand-dependent regulatory mechanisms is still limited, often hampered by lack of identification of their ligands and/or DNA targets. INTRO +51 62 ligand-free protein_state A potentially interesting exception comes from the ligand-free and salicylate-bound forms of the Methanobacterium thermoautotrophicum protein MTH313 which revealed that two salicylate molecules bind to one MTH313 dimer and induce large conformational changes, apparently sufficient to prevent DNA binding. INTRO +67 83 salicylate-bound protein_state A potentially interesting exception comes from the ligand-free and salicylate-bound forms of the Methanobacterium thermoautotrophicum protein MTH313 which revealed that two salicylate molecules bind to one MTH313 dimer and induce large conformational changes, apparently sufficient to prevent DNA binding. INTRO +97 133 Methanobacterium thermoautotrophicum species A potentially interesting exception comes from the ligand-free and salicylate-bound forms of the Methanobacterium thermoautotrophicum protein MTH313 which revealed that two salicylate molecules bind to one MTH313 dimer and induce large conformational changes, apparently sufficient to prevent DNA binding. INTRO +142 148 MTH313 protein A potentially interesting exception comes from the ligand-free and salicylate-bound forms of the Methanobacterium thermoautotrophicum protein MTH313 which revealed that two salicylate molecules bind to one MTH313 dimer and induce large conformational changes, apparently sufficient to prevent DNA binding. INTRO +173 183 salicylate chemical A potentially interesting exception comes from the ligand-free and salicylate-bound forms of the Methanobacterium thermoautotrophicum protein MTH313 which revealed that two salicylate molecules bind to one MTH313 dimer and induce large conformational changes, apparently sufficient to prevent DNA binding. INTRO +206 212 MTH313 protein A potentially interesting exception comes from the ligand-free and salicylate-bound forms of the Methanobacterium thermoautotrophicum protein MTH313 which revealed that two salicylate molecules bind to one MTH313 dimer and induce large conformational changes, apparently sufficient to prevent DNA binding. INTRO +213 218 dimer oligomeric_state A potentially interesting exception comes from the ligand-free and salicylate-bound forms of the Methanobacterium thermoautotrophicum protein MTH313 which revealed that two salicylate molecules bind to one MTH313 dimer and induce large conformational changes, apparently sufficient to prevent DNA binding. INTRO +24 31 archeal taxonomy_domain However, the homologous archeal Sulfolobus tokodaii protein ST1710 presented essentially the same structure in ligand-free and salicylate-bound forms, apparently contrasting the mechanism proposed for MTH313. INTRO +32 51 Sulfolobus tokodaii species However, the homologous archeal Sulfolobus tokodaii protein ST1710 presented essentially the same structure in ligand-free and salicylate-bound forms, apparently contrasting the mechanism proposed for MTH313. INTRO +60 66 ST1710 protein However, the homologous archeal Sulfolobus tokodaii protein ST1710 presented essentially the same structure in ligand-free and salicylate-bound forms, apparently contrasting the mechanism proposed for MTH313. INTRO +98 107 structure evidence However, the homologous archeal Sulfolobus tokodaii protein ST1710 presented essentially the same structure in ligand-free and salicylate-bound forms, apparently contrasting the mechanism proposed for MTH313. INTRO +111 122 ligand-free protein_state However, the homologous archeal Sulfolobus tokodaii protein ST1710 presented essentially the same structure in ligand-free and salicylate-bound forms, apparently contrasting the mechanism proposed for MTH313. INTRO +127 143 salicylate-bound protein_state However, the homologous archeal Sulfolobus tokodaii protein ST1710 presented essentially the same structure in ligand-free and salicylate-bound forms, apparently contrasting the mechanism proposed for MTH313. INTRO +201 207 MTH313 protein However, the homologous archeal Sulfolobus tokodaii protein ST1710 presented essentially the same structure in ligand-free and salicylate-bound forms, apparently contrasting the mechanism proposed for MTH313. INTRO +36 42 MTH313 protein Despite these apparent differences, MTH313 and ST1710 bind salicylate in approximately the same site, between their dimerization and DNA-binding domains. INTRO +47 53 ST1710 protein Despite these apparent differences, MTH313 and ST1710 bind salicylate in approximately the same site, between their dimerization and DNA-binding domains. INTRO +59 69 salicylate chemical Despite these apparent differences, MTH313 and ST1710 bind salicylate in approximately the same site, between their dimerization and DNA-binding domains. INTRO +116 152 dimerization and DNA-binding domains structure_element Despite these apparent differences, MTH313 and ST1710 bind salicylate in approximately the same site, between their dimerization and DNA-binding domains. INTRO +31 41 salicylate chemical However, it is unknown whether salicylate is a relevant in vivo ligand of either of these two proteins, which share ~20% sequence identity with NadR, rendering unclear the interpretation of these findings in relation to the regulatory mechanisms of NadR or other MarR family proteins. INTRO +144 148 NadR protein However, it is unknown whether salicylate is a relevant in vivo ligand of either of these two proteins, which share ~20% sequence identity with NadR, rendering unclear the interpretation of these findings in relation to the regulatory mechanisms of NadR or other MarR family proteins. INTRO +249 253 NadR protein However, it is unknown whether salicylate is a relevant in vivo ligand of either of these two proteins, which share ~20% sequence identity with NadR, rendering unclear the interpretation of these findings in relation to the regulatory mechanisms of NadR or other MarR family proteins. INTRO +263 267 MarR protein_type However, it is unknown whether salicylate is a relevant in vivo ligand of either of these two proteins, which share ~20% sequence identity with NadR, rendering unclear the interpretation of these findings in relation to the regulatory mechanisms of NadR or other MarR family proteins. INTRO +0 4 NadR protein NadR binds nadA on three different operators (OpI, OpII and OpIII). INTRO +11 15 nadA gene NadR binds nadA on three different operators (OpI, OpII and OpIII). INTRO +28 32 NadR protein The DNA-binding activity of NadR is attenuated in vitro upon addition of various hydroxyphenylacetate (HPA) derivatives, including 4-HPA. INTRO +81 101 hydroxyphenylacetate chemical The DNA-binding activity of NadR is attenuated in vitro upon addition of various hydroxyphenylacetate (HPA) derivatives, including 4-HPA. INTRO +103 106 HPA chemical The DNA-binding activity of NadR is attenuated in vitro upon addition of various hydroxyphenylacetate (HPA) derivatives, including 4-HPA. INTRO +131 136 4-HPA chemical The DNA-binding activity of NadR is attenuated in vitro upon addition of various hydroxyphenylacetate (HPA) derivatives, including 4-HPA. INTRO +0 5 4-HPA chemical 4-HPA is a small molecule derived from mammalian aromatic amino acid catabolism and is released in human saliva, where it has been detected at micromolar concentration. INTRO +39 48 mammalian taxonomy_domain 4-HPA is a small molecule derived from mammalian aromatic amino acid catabolism and is released in human saliva, where it has been detected at micromolar concentration. INTRO +99 104 human species 4-HPA is a small molecule derived from mammalian aromatic amino acid catabolism and is released in human saliva, where it has been detected at micromolar concentration. INTRO +19 24 4-HPA chemical In the presence of 4-HPA, NadR is unable to bind the nadA promoter and nadA gene expression is induced. INTRO +26 30 NadR protein In the presence of 4-HPA, NadR is unable to bind the nadA promoter and nadA gene expression is induced. INTRO +53 57 nadA gene In the presence of 4-HPA, NadR is unable to bind the nadA promoter and nadA gene expression is induced. INTRO +71 75 nadA gene In the presence of 4-HPA, NadR is unable to bind the nadA promoter and nadA gene expression is induced. INTRO +25 30 4-HPA chemical In vivo, the presence of 4-HPA in the host niche of N. meningitidis serves as an inducer of NadA production, thereby promoting bacterial adhesion to host cells. INTRO +52 67 N. meningitidis species In vivo, the presence of 4-HPA in the host niche of N. meningitidis serves as an inducer of NadA production, thereby promoting bacterial adhesion to host cells. INTRO +92 96 NadA protein In vivo, the presence of 4-HPA in the host niche of N. meningitidis serves as an inducer of NadA production, thereby promoting bacterial adhesion to host cells. INTRO +127 136 bacterial taxonomy_domain In vivo, the presence of 4-HPA in the host niche of N. meningitidis serves as an inducer of NadA production, thereby promoting bacterial adhesion to host cells. INTRO +35 44 3Cl,4-HPA chemical Further, we recently reported that 3Cl,4-HPA, produced during inflammation, is another inducer of nadA expression. INTRO +98 102 nadA gene Further, we recently reported that 3Cl,4-HPA, produced during inflammation, is another inducer of nadA expression. INTRO +40 85 hydrogen-deuterium exchange mass spectrometry experimental_method Extending our previous studies based on hydrogen-deuterium exchange mass spectrometry (HDX-MS), here we sought to reveal the molecular mechanisms and effects of NadR/HPA interactions via X-ray crystallography, NMR spectroscopy and complementary biochemical and in vivo mutagenesis studies. INTRO +87 93 HDX-MS experimental_method Extending our previous studies based on hydrogen-deuterium exchange mass spectrometry (HDX-MS), here we sought to reveal the molecular mechanisms and effects of NadR/HPA interactions via X-ray crystallography, NMR spectroscopy and complementary biochemical and in vivo mutagenesis studies. INTRO +161 165 NadR protein Extending our previous studies based on hydrogen-deuterium exchange mass spectrometry (HDX-MS), here we sought to reveal the molecular mechanisms and effects of NadR/HPA interactions via X-ray crystallography, NMR spectroscopy and complementary biochemical and in vivo mutagenesis studies. INTRO +166 169 HPA chemical Extending our previous studies based on hydrogen-deuterium exchange mass spectrometry (HDX-MS), here we sought to reveal the molecular mechanisms and effects of NadR/HPA interactions via X-ray crystallography, NMR spectroscopy and complementary biochemical and in vivo mutagenesis studies. INTRO +187 208 X-ray crystallography experimental_method Extending our previous studies based on hydrogen-deuterium exchange mass spectrometry (HDX-MS), here we sought to reveal the molecular mechanisms and effects of NadR/HPA interactions via X-ray crystallography, NMR spectroscopy and complementary biochemical and in vivo mutagenesis studies. INTRO +210 226 NMR spectroscopy experimental_method Extending our previous studies based on hydrogen-deuterium exchange mass spectrometry (HDX-MS), here we sought to reveal the molecular mechanisms and effects of NadR/HPA interactions via X-ray crystallography, NMR spectroscopy and complementary biochemical and in vivo mutagenesis studies. INTRO +245 288 biochemical and in vivo mutagenesis studies experimental_method Extending our previous studies based on hydrogen-deuterium exchange mass spectrometry (HDX-MS), here we sought to reveal the molecular mechanisms and effects of NadR/HPA interactions via X-ray crystallography, NMR spectroscopy and complementary biochemical and in vivo mutagenesis studies. INTRO +127 131 NadR protein We obtained detailed new insights into ligand specificity, how the ligand allosterically influences the DNA-binding ability of NadR, and the regulation of nadA expression, thus also providing a deeper structural understanding of the ligand-responsive MarR super-family. INTRO +155 159 nadA gene We obtained detailed new insights into ligand specificity, how the ligand allosterically influences the DNA-binding ability of NadR, and the regulation of nadA expression, thus also providing a deeper structural understanding of the ligand-responsive MarR super-family. INTRO +251 255 MarR protein_type We obtained detailed new insights into ligand specificity, how the ligand allosterically influences the DNA-binding ability of NadR, and the regulation of nadA expression, thus also providing a deeper structural understanding of the ligand-responsive MarR super-family. INTRO +63 67 NadR protein Moreover, these findings are important because the activity of NadR impacts the potential coverage provided by anti-NadA antibodies elicited by the Bexsero vaccine and influences host-bacteria interactions that contribute to meningococcal pathogenesis. INTRO +116 120 NadA protein Moreover, these findings are important because the activity of NadR impacts the potential coverage provided by anti-NadA antibodies elicited by the Bexsero vaccine and influences host-bacteria interactions that contribute to meningococcal pathogenesis. INTRO +184 192 bacteria taxonomy_domain Moreover, these findings are important because the activity of NadR impacts the potential coverage provided by anti-NadA antibodies elicited by the Bexsero vaccine and influences host-bacteria interactions that contribute to meningococcal pathogenesis. INTRO +225 238 meningococcal taxonomy_domain Moreover, these findings are important because the activity of NadR impacts the potential coverage provided by anti-NadA antibodies elicited by the Bexsero vaccine and influences host-bacteria interactions that contribute to meningococcal pathogenesis. INTRO +0 4 NadR protein NadR is dimeric and is stabilized by specific hydroxyphenylacetate ligands RESULTS +8 15 dimeric oligomeric_state NadR is dimeric and is stabilized by specific hydroxyphenylacetate ligands RESULTS +46 66 hydroxyphenylacetate chemical NadR is dimeric and is stabilized by specific hydroxyphenylacetate ligands RESULTS +12 16 NadR protein Recombinant NadR was produced in E. coli using an expression construct prepared from N. meningitidis serogroup B strain MC58. RESULTS +33 40 E. coli species Recombinant NadR was produced in E. coli using an expression construct prepared from N. meningitidis serogroup B strain MC58. RESULTS +50 70 expression construct experimental_method Recombinant NadR was produced in E. coli using an expression construct prepared from N. meningitidis serogroup B strain MC58. RESULTS +85 124 N. meningitidis serogroup B strain MC58 species Recombinant NadR was produced in E. coli using an expression construct prepared from N. meningitidis serogroup B strain MC58. RESULTS +84 88 NadR protein Standard chromatographic techniques were used to obtain a highly purified sample of NadR (see Materials and Methods). RESULTS +3 67 analytical size-exclusion high-performance liquid chromatography experimental_method In analytical size-exclusion high-performance liquid chromatography (SE-HPLC) experiments coupled with multi-angle laser light scattering (MALLS), NadR presented a single species with an absolute molecular mass of 35 kDa (S1 Fig). RESULTS +69 76 SE-HPLC experimental_method In analytical size-exclusion high-performance liquid chromatography (SE-HPLC) experiments coupled with multi-angle laser light scattering (MALLS), NadR presented a single species with an absolute molecular mass of 35 kDa (S1 Fig). RESULTS +103 137 multi-angle laser light scattering experimental_method In analytical size-exclusion high-performance liquid chromatography (SE-HPLC) experiments coupled with multi-angle laser light scattering (MALLS), NadR presented a single species with an absolute molecular mass of 35 kDa (S1 Fig). RESULTS +139 144 MALLS experimental_method In analytical size-exclusion high-performance liquid chromatography (SE-HPLC) experiments coupled with multi-angle laser light scattering (MALLS), NadR presented a single species with an absolute molecular mass of 35 kDa (S1 Fig). RESULTS +147 151 NadR protein In analytical size-exclusion high-performance liquid chromatography (SE-HPLC) experiments coupled with multi-angle laser light scattering (MALLS), NadR presented a single species with an absolute molecular mass of 35 kDa (S1 Fig). RESULTS +23 27 NadR protein These data showed that NadR was dimeric in solution, since the theoretical molecular mass of the NadR dimer is 33.73 kDa; and, there was no change in oligomeric state on addition of 4-HPA. RESULTS +32 39 dimeric oligomeric_state These data showed that NadR was dimeric in solution, since the theoretical molecular mass of the NadR dimer is 33.73 kDa; and, there was no change in oligomeric state on addition of 4-HPA. RESULTS +97 101 NadR protein These data showed that NadR was dimeric in solution, since the theoretical molecular mass of the NadR dimer is 33.73 kDa; and, there was no change in oligomeric state on addition of 4-HPA. RESULTS +102 107 dimer oligomeric_state These data showed that NadR was dimeric in solution, since the theoretical molecular mass of the NadR dimer is 33.73 kDa; and, there was no change in oligomeric state on addition of 4-HPA. RESULTS +182 187 4-HPA chemical These data showed that NadR was dimeric in solution, since the theoretical molecular mass of the NadR dimer is 33.73 kDa; and, there was no change in oligomeric state on addition of 4-HPA. RESULTS +25 29 NadR protein The thermal stability of NadR was examined using differential scanning calorimetry (DSC). RESULTS +49 82 differential scanning calorimetry experimental_method The thermal stability of NadR was examined using differential scanning calorimetry (DSC). RESULTS +84 87 DSC experimental_method The thermal stability of NadR was examined using differential scanning calorimetry (DSC). RESULTS +99 103 HPAs chemical Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313. RESULTS +120 139 melting temperature evidence Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313. RESULTS +141 143 Tm evidence Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313. RESULTS +148 152 NadR protein Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313. RESULTS +198 208 salicylate chemical Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313. RESULTS +233 237 MarR protein_type Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313. RESULTS +283 285 Tm evidence Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313. RESULTS +289 295 ST1710 protein Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313. RESULTS +300 306 MTH313 protein Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313. RESULTS +4 6 Tm evidence The Tm of NadR was 67.4 ± 0.1°C in the absence of ligand, and was unaffected by salicylate. RESULTS +10 14 NadR protein The Tm of NadR was 67.4 ± 0.1°C in the absence of ligand, and was unaffected by salicylate. RESULTS +39 56 absence of ligand protein_state The Tm of NadR was 67.4 ± 0.1°C in the absence of ligand, and was unaffected by salicylate. RESULTS +80 90 salicylate chemical The Tm of NadR was 67.4 ± 0.1°C in the absence of ligand, and was unaffected by salicylate. RESULTS +55 60 4-HPA chemical However, an increased thermal stability was induced by 4-HPA and, to a lesser extent, by 3-HPA. RESULTS +89 94 3-HPA chemical However, an increased thermal stability was induced by 4-HPA and, to a lesser extent, by 3-HPA. RESULTS +15 19 NadR protein Interestingly, NadR displayed the greatest Tm increase upon addition of 3Cl,4-HPA (Table 1 and Fig 1B). RESULTS +43 45 Tm evidence Interestingly, NadR displayed the greatest Tm increase upon addition of 3Cl,4-HPA (Table 1 and Fig 1B). RESULTS +72 81 3Cl,4-HPA chemical Interestingly, NadR displayed the greatest Tm increase upon addition of 3Cl,4-HPA (Table 1 and Fig 1B). RESULTS +13 17 NadR protein Stability of NadR is increased by small molecule ligands. FIG +29 34 3-HPA chemical (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). FIG +47 52 4-HPA chemical (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). FIG +65 74 3Cl,4-HPA chemical (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). FIG +90 104 salicylic acid chemical (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). FIG +121 124 DSC experimental_method (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). FIG +125 133 profiles evidence (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). FIG +155 158 apo protein_state (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). FIG +159 163 NadR protein (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). FIG +174 189 NadR+salicylate complex_assembly (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). FIG +197 207 NadR+3-HPA complex_assembly (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). FIG +217 227 NadR+4-HPA complex_assembly (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). FIG +236 250 NadR+3Cl,4-HPA complex_assembly (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink). FIG +4 7 DSC experimental_method All DSC profiles are representative of triplicate experiments. FIG +8 16 profiles evidence All DSC profiles are representative of triplicate experiments. FIG +0 13 Melting-point evidence Melting-point (Tm) and its ligand-induced increase (ΔTm) derived from DSC thermostability experiments. TABLE +15 17 Tm evidence Melting-point (Tm) and its ligand-induced increase (ΔTm) derived from DSC thermostability experiments. TABLE +52 55 ΔTm evidence Melting-point (Tm) and its ligand-induced increase (ΔTm) derived from DSC thermostability experiments. TABLE +70 73 DSC experimental_method Melting-point (Tm) and its ligand-induced increase (ΔTm) derived from DSC thermostability experiments. TABLE +74 101 thermostability experiments experimental_method Melting-point (Tm) and its ligand-induced increase (ΔTm) derived from DSC thermostability experiments. TABLE +0 22 Dissociation constants evidence Dissociation constants (KD) of the NadR/ligand interactions from SPR steady-state binding experiments. TABLE +24 26 KD evidence Dissociation constants (KD) of the NadR/ligand interactions from SPR steady-state binding experiments. TABLE +35 39 NadR protein Dissociation constants (KD) of the NadR/ligand interactions from SPR steady-state binding experiments. TABLE +65 101 SPR steady-state binding experiments experimental_method Dissociation constants (KD) of the NadR/ligand interactions from SPR steady-state binding experiments. TABLE +7 9 Tm evidence "Ligand Tm (°C) ΔTm (°C) KD (mM) No ligand 67.4 ± 0.1 n.a. n.a." TABLE +15 18 ΔTm evidence "Ligand Tm (°C) ΔTm (°C) KD (mM) No ligand 67.4 ± 0.1 n.a. n.a." TABLE +24 26 KD evidence "Ligand Tm (°C) ΔTm (°C) KD (mM) No ligand 67.4 ± 0.1 n.a. n.a." TABLE +2 7 3-HPA chemical " 3-HPA 70.0 ± 0.1 2.7 2.7 ± 0.1 4-HPA 70.7 ± 0.1 3.3 1.5 ± 0.1 3Cl,4-HPA 71.3 ± 0.2 3.9 1.1 ± 0.1 " TABLE +35 40 4-HPA chemical " 3-HPA 70.0 ± 0.1 2.7 2.7 ± 0.1 4-HPA 70.7 ± 0.1 3.3 1.5 ± 0.1 3Cl,4-HPA 71.3 ± 0.2 3.9 1.1 ± 0.1 " TABLE +68 77 3Cl,4-HPA chemical " 3-HPA 70.0 ± 0.1 2.7 2.7 ± 0.1 4-HPA 70.7 ± 0.1 3.3 1.5 ± 0.1 3Cl,4-HPA 71.3 ± 0.2 3.9 1.1 ± 0.1 " TABLE +0 4 NadR protein NadR displays distinct binding affinities for hydroxyphenylacetate ligands RESULTS +23 41 binding affinities evidence NadR displays distinct binding affinities for hydroxyphenylacetate ligands RESULTS +46 66 hydroxyphenylacetate chemical NadR displays distinct binding affinities for hydroxyphenylacetate ligands RESULTS +38 42 HPAs chemical To further investigate the binding of HPAs to NadR, we used surface plasmon resonance (SPR). RESULTS +46 50 NadR protein To further investigate the binding of HPAs to NadR, we used surface plasmon resonance (SPR). RESULTS +60 85 surface plasmon resonance experimental_method To further investigate the binding of HPAs to NadR, we used surface plasmon resonance (SPR). RESULTS +87 90 SPR experimental_method To further investigate the binding of HPAs to NadR, we used surface plasmon resonance (SPR). RESULTS +4 7 SPR experimental_method The SPR sensorgrams revealed very fast association and dissociation events, typical of small molecule ligands, thus prohibiting a detailed study of binding kinetics. RESULTS +8 19 sensorgrams evidence The SPR sensorgrams revealed very fast association and dissociation events, typical of small molecule ligands, thus prohibiting a detailed study of binding kinetics. RESULTS +9 25 steady-state SPR experimental_method However, steady-state SPR analyses of the NadR-HPA interactions allowed determination of the equilibrium dissociation constants (KD) (Table 1 and S2 Fig). RESULTS +42 50 NadR-HPA complex_assembly However, steady-state SPR analyses of the NadR-HPA interactions allowed determination of the equilibrium dissociation constants (KD) (Table 1 and S2 Fig). RESULTS +93 127 equilibrium dissociation constants evidence However, steady-state SPR analyses of the NadR-HPA interactions allowed determination of the equilibrium dissociation constants (KD) (Table 1 and S2 Fig). RESULTS +129 131 KD evidence However, steady-state SPR analyses of the NadR-HPA interactions allowed determination of the equilibrium dissociation constants (KD) (Table 1 and S2 Fig). RESULTS +20 25 4-HPA chemical The interactions of 4-HPA and 3Cl,4-HPA with NadR exhibited KD values of 1.5 mM and 1.1 mM, respectively. RESULTS +30 39 3Cl,4-HPA chemical The interactions of 4-HPA and 3Cl,4-HPA with NadR exhibited KD values of 1.5 mM and 1.1 mM, respectively. RESULTS +45 49 NadR protein The interactions of 4-HPA and 3Cl,4-HPA with NadR exhibited KD values of 1.5 mM and 1.1 mM, respectively. RESULTS +60 62 KD evidence The interactions of 4-HPA and 3Cl,4-HPA with NadR exhibited KD values of 1.5 mM and 1.1 mM, respectively. RESULTS +0 5 3-HPA chemical 3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments. RESULTS +42 44 KD evidence 3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments. RESULTS +62 72 salicylate chemical 3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments. RESULTS +180 184 NadR protein 3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments. RESULTS +205 207 KD evidence 3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments. RESULTS +227 236 3Cl,4-HPA chemical 3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments. RESULTS +309 311 Tm evidence 3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments. RESULTS +338 341 DSC experimental_method 3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments. RESULTS +15 17 KD evidence Although these KD values indicate rather weak interactions, they are similar to the values reported previously for the MarR/salicylate interaction (KD ~1 mM) and the MTH313/salicylate interaction (KD 2–3 mM), and approximately 20-fold tighter than the ST1710/salicylate interaction (KD ~20 mM). RESULTS +119 123 MarR protein_type Although these KD values indicate rather weak interactions, they are similar to the values reported previously for the MarR/salicylate interaction (KD ~1 mM) and the MTH313/salicylate interaction (KD 2–3 mM), and approximately 20-fold tighter than the ST1710/salicylate interaction (KD ~20 mM). RESULTS +124 134 salicylate chemical Although these KD values indicate rather weak interactions, they are similar to the values reported previously for the MarR/salicylate interaction (KD ~1 mM) and the MTH313/salicylate interaction (KD 2–3 mM), and approximately 20-fold tighter than the ST1710/salicylate interaction (KD ~20 mM). RESULTS +166 172 MTH313 protein Although these KD values indicate rather weak interactions, they are similar to the values reported previously for the MarR/salicylate interaction (KD ~1 mM) and the MTH313/salicylate interaction (KD 2–3 mM), and approximately 20-fold tighter than the ST1710/salicylate interaction (KD ~20 mM). RESULTS +173 183 salicylate chemical Although these KD values indicate rather weak interactions, they are similar to the values reported previously for the MarR/salicylate interaction (KD ~1 mM) and the MTH313/salicylate interaction (KD 2–3 mM), and approximately 20-fold tighter than the ST1710/salicylate interaction (KD ~20 mM). RESULTS +252 258 ST1710 protein Although these KD values indicate rather weak interactions, they are similar to the values reported previously for the MarR/salicylate interaction (KD ~1 mM) and the MTH313/salicylate interaction (KD 2–3 mM), and approximately 20-fold tighter than the ST1710/salicylate interaction (KD ~20 mM). RESULTS +259 269 salicylate chemical Although these KD values indicate rather weak interactions, they are similar to the values reported previously for the MarR/salicylate interaction (KD ~1 mM) and the MTH313/salicylate interaction (KD 2–3 mM), and approximately 20-fold tighter than the ST1710/salicylate interaction (KD ~20 mM). RESULTS +0 18 Crystal structures evidence Crystal structures of holo-NadR and apo-NadR RESULTS +22 26 holo protein_state Crystal structures of holo-NadR and apo-NadR RESULTS +27 31 NadR protein Crystal structures of holo-NadR and apo-NadR RESULTS +36 39 apo protein_state Crystal structures of holo-NadR and apo-NadR RESULTS +40 44 NadR protein Crystal structures of holo-NadR and apo-NadR RESULTS +26 30 NadR protein To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms. RESULTS +31 34 HPA chemical To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms. RESULTS +72 90 crystal structures evidence To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms. RESULTS +94 98 NadR protein To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms. RESULTS +102 114 ligand-bound protein_state To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms. RESULTS +116 120 holo protein_state To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms. RESULTS +126 137 ligand-free protein_state To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms. RESULTS +139 142 apo protein_state To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms. RESULTS +10 22 crystallized experimental_method First, we crystallized NadR (a selenomethionine-labelled derivative) in the presence of a 200-fold molar excess of 4-HPA. RESULTS +23 27 NadR protein First, we crystallized NadR (a selenomethionine-labelled derivative) in the presence of a 200-fold molar excess of 4-HPA. RESULTS +31 67 selenomethionine-labelled derivative experimental_method First, we crystallized NadR (a selenomethionine-labelled derivative) in the presence of a 200-fold molar excess of 4-HPA. RESULTS +115 120 4-HPA chemical First, we crystallized NadR (a selenomethionine-labelled derivative) in the presence of a 200-fold molar excess of 4-HPA. RESULTS +4 13 structure evidence The structure of the NadR/4-HPA complex was determined at 2.3 Å resolution using a combination of the single-wavelength anomalous dispersion (SAD) and molecular replacement (MR) methods, and was refined to R work/R free values of 20.9/26.0% (Table 2). RESULTS +21 31 NadR/4-HPA complex_assembly The structure of the NadR/4-HPA complex was determined at 2.3 Å resolution using a combination of the single-wavelength anomalous dispersion (SAD) and molecular replacement (MR) methods, and was refined to R work/R free values of 20.9/26.0% (Table 2). RESULTS +102 140 single-wavelength anomalous dispersion experimental_method The structure of the NadR/4-HPA complex was determined at 2.3 Å resolution using a combination of the single-wavelength anomalous dispersion (SAD) and molecular replacement (MR) methods, and was refined to R work/R free values of 20.9/26.0% (Table 2). RESULTS +142 145 SAD experimental_method The structure of the NadR/4-HPA complex was determined at 2.3 Å resolution using a combination of the single-wavelength anomalous dispersion (SAD) and molecular replacement (MR) methods, and was refined to R work/R free values of 20.9/26.0% (Table 2). RESULTS +151 172 molecular replacement experimental_method The structure of the NadR/4-HPA complex was determined at 2.3 Å resolution using a combination of the single-wavelength anomalous dispersion (SAD) and molecular replacement (MR) methods, and was refined to R work/R free values of 20.9/26.0% (Table 2). RESULTS +174 176 MR experimental_method The structure of the NadR/4-HPA complex was determined at 2.3 Å resolution using a combination of the single-wavelength anomalous dispersion (SAD) and molecular replacement (MR) methods, and was refined to R work/R free values of 20.9/26.0% (Table 2). RESULTS +206 219 R work/R free evidence The structure of the NadR/4-HPA complex was determined at 2.3 Å resolution using a combination of the single-wavelength anomalous dispersion (SAD) and molecular replacement (MR) methods, and was refined to R work/R free values of 20.9/26.0% (Table 2). RESULTS +65 73 crystals evidence Despite numerous attempts, we were unable to obtain high-quality crystals of NadR complexed with 3Cl,4-HPA, 3,4-HPA, 3-HPA or DNA targets. RESULTS +77 81 NadR protein Despite numerous attempts, we were unable to obtain high-quality crystals of NadR complexed with 3Cl,4-HPA, 3,4-HPA, 3-HPA or DNA targets. RESULTS +82 96 complexed with protein_state Despite numerous attempts, we were unable to obtain high-quality crystals of NadR complexed with 3Cl,4-HPA, 3,4-HPA, 3-HPA or DNA targets. RESULTS +97 106 3Cl,4-HPA chemical Despite numerous attempts, we were unable to obtain high-quality crystals of NadR complexed with 3Cl,4-HPA, 3,4-HPA, 3-HPA or DNA targets. RESULTS +108 115 3,4-HPA chemical Despite numerous attempts, we were unable to obtain high-quality crystals of NadR complexed with 3Cl,4-HPA, 3,4-HPA, 3-HPA or DNA targets. RESULTS +117 122 3-HPA chemical Despite numerous attempts, we were unable to obtain high-quality crystals of NadR complexed with 3Cl,4-HPA, 3,4-HPA, 3-HPA or DNA targets. RESULTS +39 50 crystallize experimental_method However, it was eventually possible to crystallize apo-NadR, and the structure was determined at 2.7 Å resolution by MR methods using the NadR/4-HPA complex as the search model. RESULTS +51 54 apo protein_state However, it was eventually possible to crystallize apo-NadR, and the structure was determined at 2.7 Å resolution by MR methods using the NadR/4-HPA complex as the search model. RESULTS +55 59 NadR protein However, it was eventually possible to crystallize apo-NadR, and the structure was determined at 2.7 Å resolution by MR methods using the NadR/4-HPA complex as the search model. RESULTS +69 78 structure evidence However, it was eventually possible to crystallize apo-NadR, and the structure was determined at 2.7 Å resolution by MR methods using the NadR/4-HPA complex as the search model. RESULTS +117 119 MR experimental_method However, it was eventually possible to crystallize apo-NadR, and the structure was determined at 2.7 Å resolution by MR methods using the NadR/4-HPA complex as the search model. RESULTS +138 148 NadR/4-HPA complex_assembly However, it was eventually possible to crystallize apo-NadR, and the structure was determined at 2.7 Å resolution by MR methods using the NadR/4-HPA complex as the search model. RESULTS +4 7 apo protein_state The apo-NadR structure was refined to R work/R free values of 19.1/26.8% (Table 2). RESULTS +8 12 NadR protein The apo-NadR structure was refined to R work/R free values of 19.1/26.8% (Table 2). RESULTS +13 22 structure evidence The apo-NadR structure was refined to R work/R free values of 19.1/26.8% (Table 2). RESULTS +38 51 R work/R free evidence The apo-NadR structure was refined to R work/R free values of 19.1/26.8% (Table 2). RESULTS +46 50 NadR protein Data collection and refinement statistics for NadR structures. TABLE +51 61 structures evidence Data collection and refinement statistics for NadR structures. TABLE +27 37 NadR/4-HPA complex_assembly The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. RESULTS +38 46 crystals evidence The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. RESULTS +48 52 holo protein_state The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. RESULTS +53 57 NadR protein The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. RESULTS +73 77 NadR protein The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. RESULTS +78 87 homodimer oligomeric_state The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. RESULTS +99 102 apo protein_state The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. RESULTS +103 107 NadR protein The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. RESULTS +108 116 crystals evidence The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. RESULTS +131 141 homodimers oligomeric_state The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers. RESULTS +7 10 apo protein_state In the apo-NadR crystals, the two homodimers were related by a rotation of ~90°; the observed association of the two dimers was presumably merely an effect of crystal packing, since the interface between the two homodimers is small (< 550 Å2 of buried surface area), and is not predicted to be physiologically relevant by the PISA software. RESULTS +11 15 NadR protein In the apo-NadR crystals, the two homodimers were related by a rotation of ~90°; the observed association of the two dimers was presumably merely an effect of crystal packing, since the interface between the two homodimers is small (< 550 Å2 of buried surface area), and is not predicted to be physiologically relevant by the PISA software. RESULTS +16 24 crystals evidence In the apo-NadR crystals, the two homodimers were related by a rotation of ~90°; the observed association of the two dimers was presumably merely an effect of crystal packing, since the interface between the two homodimers is small (< 550 Å2 of buried surface area), and is not predicted to be physiologically relevant by the PISA software. RESULTS +34 44 homodimers oligomeric_state In the apo-NadR crystals, the two homodimers were related by a rotation of ~90°; the observed association of the two dimers was presumably merely an effect of crystal packing, since the interface between the two homodimers is small (< 550 Å2 of buried surface area), and is not predicted to be physiologically relevant by the PISA software. RESULTS +117 123 dimers oligomeric_state In the apo-NadR crystals, the two homodimers were related by a rotation of ~90°; the observed association of the two dimers was presumably merely an effect of crystal packing, since the interface between the two homodimers is small (< 550 Å2 of buried surface area), and is not predicted to be physiologically relevant by the PISA software. RESULTS +186 195 interface site In the apo-NadR crystals, the two homodimers were related by a rotation of ~90°; the observed association of the two dimers was presumably merely an effect of crystal packing, since the interface between the two homodimers is small (< 550 Å2 of buried surface area), and is not predicted to be physiologically relevant by the PISA software. RESULTS +212 222 homodimers oligomeric_state In the apo-NadR crystals, the two homodimers were related by a rotation of ~90°; the observed association of the two dimers was presumably merely an effect of crystal packing, since the interface between the two homodimers is small (< 550 Å2 of buried surface area), and is not predicted to be physiologically relevant by the PISA software. RESULTS +14 27 SE-HPLC/MALLS experimental_method Moreover, our SE-HPLC/MALLS analyses (see above) revealed that in solution NadR is dimeric, and previous studies using native mass spectrometry (MS) revealed dimers, not tetramers. RESULTS +75 79 NadR protein Moreover, our SE-HPLC/MALLS analyses (see above) revealed that in solution NadR is dimeric, and previous studies using native mass spectrometry (MS) revealed dimers, not tetramers. RESULTS +83 90 dimeric oligomeric_state Moreover, our SE-HPLC/MALLS analyses (see above) revealed that in solution NadR is dimeric, and previous studies using native mass spectrometry (MS) revealed dimers, not tetramers. RESULTS +119 143 native mass spectrometry experimental_method Moreover, our SE-HPLC/MALLS analyses (see above) revealed that in solution NadR is dimeric, and previous studies using native mass spectrometry (MS) revealed dimers, not tetramers. RESULTS +145 147 MS experimental_method Moreover, our SE-HPLC/MALLS analyses (see above) revealed that in solution NadR is dimeric, and previous studies using native mass spectrometry (MS) revealed dimers, not tetramers. RESULTS +158 164 dimers oligomeric_state Moreover, our SE-HPLC/MALLS analyses (see above) revealed that in solution NadR is dimeric, and previous studies using native mass spectrometry (MS) revealed dimers, not tetramers. RESULTS +170 179 tetramers oligomeric_state Moreover, our SE-HPLC/MALLS analyses (see above) revealed that in solution NadR is dimeric, and previous studies using native mass spectrometry (MS) revealed dimers, not tetramers. RESULTS +4 8 NadR protein The NadR homodimer bound to 4-HPA has a dimerization interface mostly involving the top of its ‘triangular’ form, while the two DNA-binding domains are located at the base (Fig 2A). RESULTS +9 18 homodimer oligomeric_state The NadR homodimer bound to 4-HPA has a dimerization interface mostly involving the top of its ‘triangular’ form, while the two DNA-binding domains are located at the base (Fig 2A). RESULTS +19 27 bound to protein_state The NadR homodimer bound to 4-HPA has a dimerization interface mostly involving the top of its ‘triangular’ form, while the two DNA-binding domains are located at the base (Fig 2A). RESULTS +28 33 4-HPA chemical The NadR homodimer bound to 4-HPA has a dimerization interface mostly involving the top of its ‘triangular’ form, while the two DNA-binding domains are located at the base (Fig 2A). RESULTS +40 62 dimerization interface site The NadR homodimer bound to 4-HPA has a dimerization interface mostly involving the top of its ‘triangular’ form, while the two DNA-binding domains are located at the base (Fig 2A). RESULTS +96 106 triangular protein_state The NadR homodimer bound to 4-HPA has a dimerization interface mostly involving the top of its ‘triangular’ form, while the two DNA-binding domains are located at the base (Fig 2A). RESULTS +128 147 DNA-binding domains structure_element The NadR homodimer bound to 4-HPA has a dimerization interface mostly involving the top of its ‘triangular’ form, while the two DNA-binding domains are located at the base (Fig 2A). RESULTS +13 34 electron density maps evidence High-quality electron density maps allowed clear identification of the bound ligand, 4-HPA (Fig 2B). RESULTS +71 76 bound protein_state High-quality electron density maps allowed clear identification of the bound ligand, 4-HPA (Fig 2B). RESULTS +85 90 4-HPA chemical High-quality electron density maps allowed clear identification of the bound ligand, 4-HPA (Fig 2B). RESULTS +12 21 structure evidence The overall structure of NadR shows dimensions of ~50 × 65 × 50 Å and a large homodimer interface that buries a total surface area of ~ 4800 Å2. RESULTS +25 29 NadR protein The overall structure of NadR shows dimensions of ~50 × 65 × 50 Å and a large homodimer interface that buries a total surface area of ~ 4800 Å2. RESULTS +78 97 homodimer interface site The overall structure of NadR shows dimensions of ~50 × 65 × 50 Å and a large homodimer interface that buries a total surface area of ~ 4800 Å2. RESULTS +5 9 NadR protein Each NadR monomer consists of six α-helices and two short β-strands, with helices α1, α5, and α6 forming the dimer interface. RESULTS +10 17 monomer oligomeric_state Each NadR monomer consists of six α-helices and two short β-strands, with helices α1, α5, and α6 forming the dimer interface. RESULTS +34 43 α-helices structure_element Each NadR monomer consists of six α-helices and two short β-strands, with helices α1, α5, and α6 forming the dimer interface. RESULTS +52 67 short β-strands structure_element Each NadR monomer consists of six α-helices and two short β-strands, with helices α1, α5, and α6 forming the dimer interface. RESULTS +74 81 helices structure_element Each NadR monomer consists of six α-helices and two short β-strands, with helices α1, α5, and α6 forming the dimer interface. RESULTS +82 84 α1 structure_element Each NadR monomer consists of six α-helices and two short β-strands, with helices α1, α5, and α6 forming the dimer interface. RESULTS +86 88 α5 structure_element Each NadR monomer consists of six α-helices and two short β-strands, with helices α1, α5, and α6 forming the dimer interface. RESULTS +94 96 α6 structure_element Each NadR monomer consists of six α-helices and two short β-strands, with helices α1, α5, and α6 forming the dimer interface. RESULTS +109 124 dimer interface site Each NadR monomer consists of six α-helices and two short β-strands, with helices α1, α5, and α6 forming the dimer interface. RESULTS +0 7 Helices structure_element Helices α3 and α4 form a helix-turn-helix motif, followed by the “wing motif” comprised of two short antiparallel β-strands (β1-β2) linked by a relatively long and flexible loop. RESULTS +8 10 α3 structure_element Helices α3 and α4 form a helix-turn-helix motif, followed by the “wing motif” comprised of two short antiparallel β-strands (β1-β2) linked by a relatively long and flexible loop. RESULTS +15 17 α4 structure_element Helices α3 and α4 form a helix-turn-helix motif, followed by the “wing motif” comprised of two short antiparallel β-strands (β1-β2) linked by a relatively long and flexible loop. RESULTS +25 47 helix-turn-helix motif structure_element Helices α3 and α4 form a helix-turn-helix motif, followed by the “wing motif” comprised of two short antiparallel β-strands (β1-β2) linked by a relatively long and flexible loop. RESULTS +66 76 wing motif structure_element Helices α3 and α4 form a helix-turn-helix motif, followed by the “wing motif” comprised of two short antiparallel β-strands (β1-β2) linked by a relatively long and flexible loop. RESULTS +95 123 short antiparallel β-strands structure_element Helices α3 and α4 form a helix-turn-helix motif, followed by the “wing motif” comprised of two short antiparallel β-strands (β1-β2) linked by a relatively long and flexible loop. RESULTS +125 130 β1-β2 structure_element Helices α3 and α4 form a helix-turn-helix motif, followed by the “wing motif” comprised of two short antiparallel β-strands (β1-β2) linked by a relatively long and flexible loop. RESULTS +173 177 loop structure_element Helices α3 and α4 form a helix-turn-helix motif, followed by the “wing motif” comprised of two short antiparallel β-strands (β1-β2) linked by a relatively long and flexible loop. RESULTS +22 34 α4-β2 region structure_element Interestingly, in the α4-β2 region, the stretch of residues from R64-R91 presents seven positively-charged side chains, all available for potential interactions with DNA. RESULTS +65 72 R64-R91 residue_range Interestingly, in the α4-β2 region, the stretch of residues from R64-R91 presents seven positively-charged side chains, all available for potential interactions with DNA. RESULTS +166 169 DNA chemical Interestingly, in the α4-β2 region, the stretch of residues from R64-R91 presents seven positively-charged side chains, all available for potential interactions with DNA. RESULTS +51 74 winged helix-turn-helix structure_element Together, these structural elements constitute the winged helix-turn-helix (wHTH) DNA-binding domain and, together with the dimeric organization, are the hallmarks of MarR family structures. RESULTS +76 80 wHTH structure_element Together, these structural elements constitute the winged helix-turn-helix (wHTH) DNA-binding domain and, together with the dimeric organization, are the hallmarks of MarR family structures. RESULTS +82 100 DNA-binding domain structure_element Together, these structural elements constitute the winged helix-turn-helix (wHTH) DNA-binding domain and, together with the dimeric organization, are the hallmarks of MarR family structures. RESULTS +124 131 dimeric oligomeric_state Together, these structural elements constitute the winged helix-turn-helix (wHTH) DNA-binding domain and, together with the dimeric organization, are the hallmarks of MarR family structures. RESULTS +167 171 MarR protein_type Together, these structural elements constitute the winged helix-turn-helix (wHTH) DNA-binding domain and, together with the dimeric organization, are the hallmarks of MarR family structures. RESULTS +179 189 structures evidence Together, these structural elements constitute the winged helix-turn-helix (wHTH) DNA-binding domain and, together with the dimeric organization, are the hallmarks of MarR family structures. RESULTS +4 21 crystal structure evidence The crystal structure of NadR in complex with 4-HPA. FIG +25 29 NadR protein The crystal structure of NadR in complex with 4-HPA. FIG +30 45 in complex with protein_state The crystal structure of NadR in complex with 4-HPA. FIG +46 51 4-HPA chemical The crystal structure of NadR in complex with 4-HPA. FIG +9 13 holo protein_state (A) The holo-NadR homodimer is depicted in green and blue for chains A and B respectively, while yellow sticks depict the 4-HPA ligand (labelled). FIG +14 18 NadR protein (A) The holo-NadR homodimer is depicted in green and blue for chains A and B respectively, while yellow sticks depict the 4-HPA ligand (labelled). FIG +19 28 homodimer oligomeric_state (A) The holo-NadR homodimer is depicted in green and blue for chains A and B respectively, while yellow sticks depict the 4-HPA ligand (labelled). FIG +63 77 chains A and B structure_element (A) The holo-NadR homodimer is depicted in green and blue for chains A and B respectively, while yellow sticks depict the 4-HPA ligand (labelled). FIG +123 128 4-HPA chemical (A) The holo-NadR homodimer is depicted in green and blue for chains A and B respectively, while yellow sticks depict the 4-HPA ligand (labelled). FIG +62 69 chain B structure_element For simplicity, secondary structure elements are labelled for chain B only. FIG +42 49 chain B structure_element Red dashes show hypothetical positions of chain B residues 88–90 that were not modeled due to lack of electron density. FIG +59 64 88–90 residue_range Red dashes show hypothetical positions of chain B residues 88–90 that were not modeled due to lack of electron density. FIG +102 118 electron density evidence Red dashes show hypothetical positions of chain B residues 88–90 that were not modeled due to lack of electron density. FIG +20 26 pocket site (B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix. FIG +39 44 4-HPA chemical (B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix. FIG +81 95 chains A and B structure_element (B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix. FIG +113 129 electron density evidence (B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix. FIG +137 142 4-HPA chemical (B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix. FIG +161 179 composite omit map evidence (B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix. FIG +190 195 4-HPA chemical (B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix. FIG +204 210 phenix experimental_method (B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix. FIG +4 7 map evidence The map is contoured at 1σ and the figure was prepared with a density mesh carve factor of 1.7, using Pymol (www.pymol.org). FIG +62 74 density mesh evidence The map is contoured at 1σ and the figure was prepared with a density mesh carve factor of 1.7, using Pymol (www.pymol.org). FIG +9 18 conserved protein_state A single conserved leucine residue (L130) is crucial for dimerization RESULTS +19 26 leucine residue_name A single conserved leucine residue (L130) is crucial for dimerization RESULTS +36 40 L130 residue_name_number A single conserved leucine residue (L130) is crucial for dimerization RESULTS +4 8 NadR protein The NadR dimer interface is formed by at least 32 residues, which establish numerous inter-chain salt bridges or hydrogen bonds, and many hydrophobic packing interactions (Fig 3A and 3B). RESULTS +9 24 dimer interface site The NadR dimer interface is formed by at least 32 residues, which establish numerous inter-chain salt bridges or hydrogen bonds, and many hydrophobic packing interactions (Fig 3A and 3B). RESULTS +97 109 salt bridges bond_interaction The NadR dimer interface is formed by at least 32 residues, which establish numerous inter-chain salt bridges or hydrogen bonds, and many hydrophobic packing interactions (Fig 3A and 3B). RESULTS +113 127 hydrogen bonds bond_interaction The NadR dimer interface is formed by at least 32 residues, which establish numerous inter-chain salt bridges or hydrogen bonds, and many hydrophobic packing interactions (Fig 3A and 3B). RESULTS +138 170 hydrophobic packing interactions bond_interaction The NadR dimer interface is formed by at least 32 residues, which establish numerous inter-chain salt bridges or hydrogen bonds, and many hydrophobic packing interactions (Fig 3A and 3B). RESULTS +81 90 interface site To determine which residues were most important for dimerization, we studied the interface in silico and identified several residues as potential mediators of key stabilizing interactions. RESULTS +6 31 site-directed mutagenesis experimental_method Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +50 56 mutant protein_state Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +57 61 NadR protein Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +105 108 H7A mutant Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +110 113 S9A mutant Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +115 119 N11A mutant Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +121 126 D112A mutant Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +128 133 R114A mutant Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +135 140 Y115A mutant Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +142 147 K126A mutant Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +149 154 L130K mutant Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +159 164 L133K mutant Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +200 215 dimer interface site Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface. RESULTS +5 11 mutant protein_state Each mutant NadR protein was purified, and then its oligomeric state was examined by analytical SE-HPLC. RESULTS +12 16 NadR protein Each mutant NadR protein was purified, and then its oligomeric state was examined by analytical SE-HPLC. RESULTS +85 103 analytical SE-HPLC experimental_method Each mutant NadR protein was purified, and then its oligomeric state was examined by analytical SE-HPLC. RESULTS +62 71 wild-type protein_state Almost all the mutants showed the same elution profile as the wild-type (WT) NadR protein. RESULTS +73 75 WT protein_state Almost all the mutants showed the same elution profile as the wild-type (WT) NadR protein. RESULTS +77 81 NadR protein Almost all the mutants showed the same elution profile as the wild-type (WT) NadR protein. RESULTS +9 14 L130K mutant Only the L130K mutation induced a notable change in the oligomeric state of NadR (Fig 3C). RESULTS +76 80 NadR protein Only the L130K mutation induced a notable change in the oligomeric state of NadR (Fig 3C). RESULTS +12 20 SE-MALLS experimental_method Further, in SE-MALLS analyses, the L130K mutant displayed two distinct species in solution, approximately 80% being monomeric (a 19 kDa species), and only 20% retaining the typical native dimeric state (a 35 kDa species) (Fig 3D), demonstrating that Leu130 is crucial for stable dimerization. RESULTS +35 40 L130K mutant Further, in SE-MALLS analyses, the L130K mutant displayed two distinct species in solution, approximately 80% being monomeric (a 19 kDa species), and only 20% retaining the typical native dimeric state (a 35 kDa species) (Fig 3D), demonstrating that Leu130 is crucial for stable dimerization. RESULTS +41 47 mutant protein_state Further, in SE-MALLS analyses, the L130K mutant displayed two distinct species in solution, approximately 80% being monomeric (a 19 kDa species), and only 20% retaining the typical native dimeric state (a 35 kDa species) (Fig 3D), demonstrating that Leu130 is crucial for stable dimerization. RESULTS +116 125 monomeric oligomeric_state Further, in SE-MALLS analyses, the L130K mutant displayed two distinct species in solution, approximately 80% being monomeric (a 19 kDa species), and only 20% retaining the typical native dimeric state (a 35 kDa species) (Fig 3D), demonstrating that Leu130 is crucial for stable dimerization. RESULTS +188 195 dimeric oligomeric_state Further, in SE-MALLS analyses, the L130K mutant displayed two distinct species in solution, approximately 80% being monomeric (a 19 kDa species), and only 20% retaining the typical native dimeric state (a 35 kDa species) (Fig 3D), demonstrating that Leu130 is crucial for stable dimerization. RESULTS +250 256 Leu130 residue_name_number Further, in SE-MALLS analyses, the L130K mutant displayed two distinct species in solution, approximately 80% being monomeric (a 19 kDa species), and only 20% retaining the typical native dimeric state (a 35 kDa species) (Fig 3D), demonstrating that Leu130 is crucial for stable dimerization. RESULTS +19 23 L130 residue_name_number It is notable that L130 is usually present as Leu, or an alternative bulky hydrophobic amino acid (e.g. Phe, Val), in many MarR family proteins, suggesting a conserved role in stabilizing the dimer interface. RESULTS +46 49 Leu residue_name It is notable that L130 is usually present as Leu, or an alternative bulky hydrophobic amino acid (e.g. Phe, Val), in many MarR family proteins, suggesting a conserved role in stabilizing the dimer interface. RESULTS +104 107 Phe residue_name It is notable that L130 is usually present as Leu, or an alternative bulky hydrophobic amino acid (e.g. Phe, Val), in many MarR family proteins, suggesting a conserved role in stabilizing the dimer interface. RESULTS +109 112 Val residue_name It is notable that L130 is usually present as Leu, or an alternative bulky hydrophobic amino acid (e.g. Phe, Val), in many MarR family proteins, suggesting a conserved role in stabilizing the dimer interface. RESULTS +123 127 MarR protein_type It is notable that L130 is usually present as Leu, or an alternative bulky hydrophobic amino acid (e.g. Phe, Val), in many MarR family proteins, suggesting a conserved role in stabilizing the dimer interface. RESULTS +158 167 conserved protein_state It is notable that L130 is usually present as Leu, or an alternative bulky hydrophobic amino acid (e.g. Phe, Val), in many MarR family proteins, suggesting a conserved role in stabilizing the dimer interface. RESULTS +192 207 dimer interface site It is notable that L130 is usually present as Leu, or an alternative bulky hydrophobic amino acid (e.g. Phe, Val), in many MarR family proteins, suggesting a conserved role in stabilizing the dimer interface. RESULTS +58 62 NadR protein In contrast, most of the other residues identified in the NadR dimer interface were poorly conserved in the MarR family. RESULTS +63 78 dimer interface site In contrast, most of the other residues identified in the NadR dimer interface were poorly conserved in the MarR family. RESULTS +84 100 poorly conserved protein_state In contrast, most of the other residues identified in the NadR dimer interface were poorly conserved in the MarR family. RESULTS +108 112 MarR protein_type In contrast, most of the other residues identified in the NadR dimer interface were poorly conserved in the MarR family. RESULTS +16 20 NadR protein Analysis of the NadR dimer interface. FIG +21 36 dimer interface site Analysis of the NadR dimer interface. FIG +28 35 chain A structure_element (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +145 157 salt bridges bond_interaction (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +161 175 hydrogen bonds bond_interaction (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +186 188 Q4 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +190 192 S5 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +194 196 K6 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +198 200 H7 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +202 204 S9 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +206 209 I10 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +211 214 N11 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +216 219 I15 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +221 224 Q16 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +226 229 R18 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +231 234 D36 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +236 239 R43 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +241 244 A46 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +246 249 Q59 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +251 254 C61 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +256 260 Y104 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +262 266 D112 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +268 272 R114 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +274 278 Y115 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +280 284 D116 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +286 290 E119 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +292 296 K126 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +298 302 E136 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +304 308 E141 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +310 314 N145 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +324 356 hydrophobic packing interactions bond_interaction (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +367 370 I10 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +372 375 I12 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +377 380 L14 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +382 385 I15 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +387 390 R18 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +392 396 Y115 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +398 402 I118 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +404 408 L130 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +410 414 L133 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +416 420 L134 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +425 429 L137 residue_name_number (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137. FIG +0 7 Chain B structure_element Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +70 95 site-directed mutagenesis experimental_method Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +97 101 E136 residue_name_number Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +115 126 salt bridge bond_interaction Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +132 136 K126 residue_name_number Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +178 183 K126A mutant Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +226 243 ionic interaction bond_interaction Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +249 251 H7 residue_name_number Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +277 284 monomer oligomeric_state Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +285 286 A structure_element Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +294 310 electron density evidence Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +327 334 monomer oligomeric_state Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +335 336 B structure_element Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +374 379 helix structure_element Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +380 382 α6 structure_element Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +403 407 L130 residue_name_number Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +408 415 chain B structure_element Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +448 480 hydrophobic packing interactions bond_interaction Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +486 490 L130 residue_name_number Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +492 496 L133 residue_name_number Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +498 502 L134 residue_name_number Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +507 511 L137 residue_name_number Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +515 522 chain A structure_element Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix α6 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains). FIG +4 11 SE-HPLC experimental_method (C) SE-HPLC analyses of all mutant forms of NadR are compared with the wild-type (WT) protein. FIG +28 34 mutant protein_state (C) SE-HPLC analyses of all mutant forms of NadR are compared with the wild-type (WT) protein. FIG +44 48 NadR protein (C) SE-HPLC analyses of all mutant forms of NadR are compared with the wild-type (WT) protein. FIG +71 80 wild-type protein_state (C) SE-HPLC analyses of all mutant forms of NadR are compared with the wild-type (WT) protein. FIG +82 84 WT protein_state (C) SE-HPLC analyses of all mutant forms of NadR are compared with the wild-type (WT) protein. FIG +4 6 WT protein_state The WT and most of the mutants show a single elution peak with an absorbance maximum at 17.5 min. FIG +18 23 L130K mutant Only the mutation L130K has a noteworthy effect on the oligomeric state, inducing a second peak with a longer retention time and a second peak maximum at 18.6 min. FIG +29 34 L133K mutant To a much lesser extent, the L133K mutation also appears to induce a ‘shoulder’ to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer. FIG +142 147 dimer oligomeric_state To a much lesser extent, the L133K mutation also appears to induce a ‘shoulder’ to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer. FIG +153 166 SE-HPLC/MALLS experimental_method To a much lesser extent, the L133K mutation also appears to induce a ‘shoulder’ to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer. FIG +183 188 L130K mutant To a much lesser extent, the L133K mutation also appears to induce a ‘shoulder’ to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer. FIG +189 195 mutant protein_state To a much lesser extent, the L133K mutation also appears to induce a ‘shoulder’ to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer. FIG +207 212 dimer oligomeric_state To a much lesser extent, the L133K mutation also appears to induce a ‘shoulder’ to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer. FIG +221 228 monomer oligomeric_state To a much lesser extent, the L133K mutation also appears to induce a ‘shoulder’ to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer. FIG +4 8 holo protein_state The holo-NadR structure presents only one occupied ligand-binding pocket RESULTS +9 13 NadR protein The holo-NadR structure presents only one occupied ligand-binding pocket RESULTS +14 23 structure evidence The holo-NadR structure presents only one occupied ligand-binding pocket RESULTS +51 72 ligand-binding pocket site The holo-NadR structure presents only one occupied ligand-binding pocket RESULTS +4 14 NadR/4-HPA complex_assembly The NadR/4-HPA structure revealed the ligand-binding site nestled between the dimerization and DNA-binding domains (Fig 2). RESULTS +15 24 structure evidence The NadR/4-HPA structure revealed the ligand-binding site nestled between the dimerization and DNA-binding domains (Fig 2). RESULTS +38 57 ligand-binding site site The NadR/4-HPA structure revealed the ligand-binding site nestled between the dimerization and DNA-binding domains (Fig 2). RESULTS +78 114 dimerization and DNA-binding domains structure_element The NadR/4-HPA structure revealed the ligand-binding site nestled between the dimerization and DNA-binding domains (Fig 2). RESULTS +67 77 salicylate chemical The ligand showed a different position and orientation compared to salicylate complexed with MTH313 and ST1710 (see Discussion). RESULTS +78 92 complexed with protein_state The ligand showed a different position and orientation compared to salicylate complexed with MTH313 and ST1710 (see Discussion). RESULTS +93 99 MTH313 protein The ligand showed a different position and orientation compared to salicylate complexed with MTH313 and ST1710 (see Discussion). RESULTS +104 110 ST1710 protein The ligand showed a different position and orientation compared to salicylate complexed with MTH313 and ST1710 (see Discussion). RESULTS +4 18 binding pocket site The binding pocket was almost entirely filled by 4-HPA and one water molecule, although there also remained a small tunnel 2-4Å in diameter and 5-6Å long leading from the pocket (proximal to the 4-hydroxyl position) to the protein surface. RESULTS +49 54 4-HPA chemical The binding pocket was almost entirely filled by 4-HPA and one water molecule, although there also remained a small tunnel 2-4Å in diameter and 5-6Å long leading from the pocket (proximal to the 4-hydroxyl position) to the protein surface. RESULTS +63 68 water chemical The binding pocket was almost entirely filled by 4-HPA and one water molecule, although there also remained a small tunnel 2-4Å in diameter and 5-6Å long leading from the pocket (proximal to the 4-hydroxyl position) to the protein surface. RESULTS +116 122 tunnel site The binding pocket was almost entirely filled by 4-HPA and one water molecule, although there also remained a small tunnel 2-4Å in diameter and 5-6Å long leading from the pocket (proximal to the 4-hydroxyl position) to the protein surface. RESULTS +171 177 pocket site The binding pocket was almost entirely filled by 4-HPA and one water molecule, although there also remained a small tunnel 2-4Å in diameter and 5-6Å long leading from the pocket (proximal to the 4-hydroxyl position) to the protein surface. RESULTS +4 10 tunnel site The tunnel was lined with rather hydrophobic amino acids, and did not contain water molecules. RESULTS +78 83 water chemical The tunnel was lined with rather hydrophobic amino acids, and did not contain water molecules. RESULTS +23 30 monomer oligomeric_state Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions. RESULTS +38 42 holo protein_state Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions. RESULTS +43 47 NadR protein Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions. RESULTS +48 57 homodimer oligomeric_state Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions. RESULTS +68 73 4-HPA chemical Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions. RESULTS +81 95 binding pocket site Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions. RESULTS +123 129 pocket site Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions. RESULTS +143 150 monomer oligomeric_state Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions. RESULTS +205 210 4-HPA chemical Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions. RESULTS +18 52 protein-ligand interaction network site Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). RESULTS +76 80 NadR protein Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). RESULTS +148 166 hydrogen (H)-bonds bond_interaction Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). RESULTS +171 189 ionic interactions bond_interaction Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). RESULTS +237 242 4-HPA chemical Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). RESULTS +247 251 Ser9 residue_name_number Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). RESULTS +255 262 chain A structure_element Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). RESULTS +264 269 SerA9 residue_name_number Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). RESULTS +276 283 chain B structure_element Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). RESULTS +293 299 TrpB39 residue_name_number Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). RESULTS +301 307 ArgB43 residue_name_number Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). RESULTS +312 319 TyrB115 residue_name_number Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A). RESULTS +71 77 AspB36 residue_name_number At the other ‘end’ of the ligand, the 4-hydroxyl group was proximal to AspB36, with which it may establish an H-bond (see bond distances in Table 3). RESULTS +110 116 H-bond bond_interaction At the other ‘end’ of the ligand, the 4-hydroxyl group was proximal to AspB36, with which it may establish an H-bond (see bond distances in Table 3). RESULTS +4 9 water chemical The water molecule observed in the pocket was bound by the carboxylate group and the side chains of SerA9 and AsnA11. RESULTS +100 105 SerA9 residue_name_number The water molecule observed in the pocket was bound by the carboxylate group and the side chains of SerA9 and AsnA11. RESULTS +110 116 AsnA11 residue_name_number The water molecule observed in the pocket was bound by the carboxylate group and the side chains of SerA9 and AsnA11. RESULTS +18 22 NadR protein Atomic details of NadR/HPA interactions. FIG +23 26 HPA chemical Atomic details of NadR/HPA interactions. FIG +32 46 binding pocket site A) A stereo-view zoom into the binding pocket showing side chain sticks for all interactions between NadR and 4-HPA. FIG +102 106 NadR protein A) A stereo-view zoom into the binding pocket showing side chain sticks for all interactions between NadR and 4-HPA. FIG +111 116 4-HPA chemical A) A stereo-view zoom into the binding pocket showing side chain sticks for all interactions between NadR and 4-HPA. FIG +30 34 NadR protein Green and blue ribbons depict NadR chains A and B, respectively. FIG +35 49 chains A and B structure_element Green and blue ribbons depict NadR chains A and B, respectively. FIG +0 5 4-HPA chemical 4-HPA is shown in yellow sticks, with oxygen atoms in red. FIG +2 7 water chemical A water molecule is shown by the red sphere. FIG +0 7 H-bonds bond_interaction H-bonds up to 3.6Å are shown as dashed lines. FIG +34 41 H-bonds bond_interaction The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +45 64 non-bonded contacts bond_interaction The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +70 75 4-HPA chemical The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +91 96 SerA9 residue_name_number The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +98 104 AsnA11 residue_name_number The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +106 112 LeuB21 residue_name_number The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +114 120 MetB22 residue_name_number The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +122 128 PheB25 residue_name_number The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +130 136 LeuB29 residue_name_number The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +138 144 AspB36 residue_name_number The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +146 152 TrpB39 residue_name_number The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +154 160 ArgB43 residue_name_number The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +162 169 ValB111 residue_name_number The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +174 181 TyrB115 residue_name_number The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +218 224 PDBsum experimental_method The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually). FIG +9 15 AsnA11 residue_name_number Residues AsnA11 and ArgB18 likely make indirect yet local contributions to ligand binding, mainly by stabilizing the position of AspB36. FIG +20 26 ArgB18 residue_name_number Residues AsnA11 and ArgB18 likely make indirect yet local contributions to ligand binding, mainly by stabilizing the position of AspB36. FIG +129 135 AspB36 residue_name_number Residues AsnA11 and ArgB18 likely make indirect yet local contributions to ligand binding, mainly by stabilizing the position of AspB36. FIG +22 46 hydrophobic interactions bond_interaction Side chains mediating hydrophobic interactions are shown in orange. (B) A model was prepared to visualize putative interactions of 3Cl,4-HPA (pink) with NadR, revealing the potential for additional contacts (dashed lines) of the chloro moiety (green stick) with LeuB29 and AspB36. FIG +131 140 3Cl,4-HPA chemical Side chains mediating hydrophobic interactions are shown in orange. (B) A model was prepared to visualize putative interactions of 3Cl,4-HPA (pink) with NadR, revealing the potential for additional contacts (dashed lines) of the chloro moiety (green stick) with LeuB29 and AspB36. FIG +153 157 NadR protein Side chains mediating hydrophobic interactions are shown in orange. (B) A model was prepared to visualize putative interactions of 3Cl,4-HPA (pink) with NadR, revealing the potential for additional contacts (dashed lines) of the chloro moiety (green stick) with LeuB29 and AspB36. FIG +262 268 LeuB29 residue_name_number Side chains mediating hydrophobic interactions are shown in orange. (B) A model was prepared to visualize putative interactions of 3Cl,4-HPA (pink) with NadR, revealing the potential for additional contacts (dashed lines) of the chloro moiety (green stick) with LeuB29 and AspB36. FIG +273 279 AspB36 residue_name_number Side chains mediating hydrophobic interactions are shown in orange. (B) A model was prepared to visualize putative interactions of 3Cl,4-HPA (pink) with NadR, revealing the potential for additional contacts (dashed lines) of the chloro moiety (green stick) with LeuB29 and AspB36. FIG +8 13 4-HPA chemical List of 4-HPA atoms bound to NadR via ionic interactions and/or H-bonds. TABLE +29 33 NadR protein List of 4-HPA atoms bound to NadR via ionic interactions and/or H-bonds. TABLE +38 56 ionic interactions bond_interaction List of 4-HPA atoms bound to NadR via ionic interactions and/or H-bonds. TABLE +64 71 H-bonds bond_interaction List of 4-HPA atoms bound to NadR via ionic interactions and/or H-bonds. TABLE +0 5 4-HPA chemical "4-HPA atom NadR residue/atom Distance (Å) O2 TrpB39/NE1 2.83 O2 ArgB43/NH1 2.76 O1 ArgB43/NH1 3.84 O1 SerA9/OG 2.75 O1 TyrB115/OH 2.50 O2 Water (*Ser9/Asn11) 2.88 OH AspB36/OD1/OD2 3.6/3.7 " TABLE +11 15 NadR protein "4-HPA atom NadR residue/atom Distance (Å) O2 TrpB39/NE1 2.83 O2 ArgB43/NH1 2.76 O1 ArgB43/NH1 3.84 O1 SerA9/OG 2.75 O1 TyrB115/OH 2.50 O2 Water (*Ser9/Asn11) 2.88 OH AspB36/OD1/OD2 3.6/3.7 " TABLE +47 53 TrpB39 residue_name_number "4-HPA atom NadR residue/atom Distance (Å) O2 TrpB39/NE1 2.83 O2 ArgB43/NH1 2.76 O1 ArgB43/NH1 3.84 O1 SerA9/OG 2.75 O1 TyrB115/OH 2.50 O2 Water (*Ser9/Asn11) 2.88 OH AspB36/OD1/OD2 3.6/3.7 " TABLE +68 74 ArgB43 residue_name_number "4-HPA atom NadR residue/atom Distance (Å) O2 TrpB39/NE1 2.83 O2 ArgB43/NH1 2.76 O1 ArgB43/NH1 3.84 O1 SerA9/OG 2.75 O1 TyrB115/OH 2.50 O2 Water (*Ser9/Asn11) 2.88 OH AspB36/OD1/OD2 3.6/3.7 " TABLE +89 95 ArgB43 residue_name_number "4-HPA atom NadR residue/atom Distance (Å) O2 TrpB39/NE1 2.83 O2 ArgB43/NH1 2.76 O1 ArgB43/NH1 3.84 O1 SerA9/OG 2.75 O1 TyrB115/OH 2.50 O2 Water (*Ser9/Asn11) 2.88 OH AspB36/OD1/OD2 3.6/3.7 " TABLE +110 115 SerA9 residue_name_number "4-HPA atom NadR residue/atom Distance (Å) O2 TrpB39/NE1 2.83 O2 ArgB43/NH1 2.76 O1 ArgB43/NH1 3.84 O1 SerA9/OG 2.75 O1 TyrB115/OH 2.50 O2 Water (*Ser9/Asn11) 2.88 OH AspB36/OD1/OD2 3.6/3.7 " TABLE +129 136 TyrB115 residue_name_number "4-HPA atom NadR residue/atom Distance (Å) O2 TrpB39/NE1 2.83 O2 ArgB43/NH1 2.76 O1 ArgB43/NH1 3.84 O1 SerA9/OG 2.75 O1 TyrB115/OH 2.50 O2 Water (*Ser9/Asn11) 2.88 OH AspB36/OD1/OD2 3.6/3.7 " TABLE +150 155 Water chemical "4-HPA atom NadR residue/atom Distance (Å) O2 TrpB39/NE1 2.83 O2 ArgB43/NH1 2.76 O1 ArgB43/NH1 3.84 O1 SerA9/OG 2.75 O1 TyrB115/OH 2.50 O2 Water (*Ser9/Asn11) 2.88 OH AspB36/OD1/OD2 3.6/3.7 " TABLE +158 162 Ser9 residue_name_number "4-HPA atom NadR residue/atom Distance (Å) O2 TrpB39/NE1 2.83 O2 ArgB43/NH1 2.76 O1 ArgB43/NH1 3.84 O1 SerA9/OG 2.75 O1 TyrB115/OH 2.50 O2 Water (*Ser9/Asn11) 2.88 OH AspB36/OD1/OD2 3.6/3.7 " TABLE +163 168 Asn11 residue_name_number "4-HPA atom NadR residue/atom Distance (Å) O2 TrpB39/NE1 2.83 O2 ArgB43/NH1 2.76 O1 ArgB43/NH1 3.84 O1 SerA9/OG 2.75 O1 TyrB115/OH 2.50 O2 Water (*Ser9/Asn11) 2.88 OH AspB36/OD1/OD2 3.6/3.7 " TABLE +180 186 AspB36 residue_name_number "4-HPA atom NadR residue/atom Distance (Å) O2 TrpB39/NE1 2.83 O2 ArgB43/NH1 2.76 O1 ArgB43/NH1 3.84 O1 SerA9/OG 2.75 O1 TyrB115/OH 2.50 O2 Water (*Ser9/Asn11) 2.88 OH AspB36/OD1/OD2 3.6/3.7 " TABLE +61 66 water chemical * Bond distance between the ligand carboxylate group and the water molecule, which in turn makes H-bond to the SerA9 and AsnA11 side chains. TABLE +97 103 H-bond bond_interaction * Bond distance between the ligand carboxylate group and the water molecule, which in turn makes H-bond to the SerA9 and AsnA11 side chains. TABLE +111 116 SerA9 residue_name_number * Bond distance between the ligand carboxylate group and the water molecule, which in turn makes H-bond to the SerA9 and AsnA11 side chains. TABLE +121 127 AsnA11 residue_name_number * Bond distance between the ligand carboxylate group and the water molecule, which in turn makes H-bond to the SerA9 and AsnA11 side chains. TABLE +19 26 H-bonds bond_interaction In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals’ contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A). RESULTS +76 81 4-HPA chemical In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals’ contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A). RESULTS +149 172 van der Waals’ contacts bond_interaction In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals’ contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A). RESULTS +239 245 LeuB21 residue_name_number In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals’ contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A). RESULTS +247 253 MetB22 residue_name_number In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals’ contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A). RESULTS +255 261 PheB25 residue_name_number In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals’ contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A). RESULTS +263 269 LeuB29 residue_name_number In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals’ contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A). RESULTS +274 281 ValB111 residue_name_number In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals’ contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A). RESULTS +28 34 PheB25 residue_name_number Notably, the phenyl ring of PheB25 was positioned parallel to the phenyl ring of 4-HPA, potentially forming π-π parallel-displaced stacking interactions. RESULTS +81 86 4-HPA chemical Notably, the phenyl ring of PheB25 was positioned parallel to the phenyl ring of 4-HPA, potentially forming π-π parallel-displaced stacking interactions. RESULTS +108 152 π-π parallel-displaced stacking interactions bond_interaction Notably, the phenyl ring of PheB25 was positioned parallel to the phenyl ring of 4-HPA, potentially forming π-π parallel-displaced stacking interactions. RESULTS +30 50 4-HPA binding pocket site Consequently, residues in the 4-HPA binding pocket are mostly contributed by NadR chain B, and effectively created a polar ‘floor’ and a hydrophobic ‘ceiling’, which house the ligand. RESULTS +77 81 NadR protein Consequently, residues in the 4-HPA binding pocket are mostly contributed by NadR chain B, and effectively created a polar ‘floor’ and a hydrophobic ‘ceiling’, which house the ligand. RESULTS +82 89 chain B structure_element Consequently, residues in the 4-HPA binding pocket are mostly contributed by NadR chain B, and effectively created a polar ‘floor’ and a hydrophobic ‘ceiling’, which house the ligand. RESULTS +36 70 polar and hydrophobic interactions bond_interaction Collectively, this mixed network of polar and hydrophobic interactions endows NadR with a strong recognition pattern for HPAs, with additional medium-range interactions potentially established with the hydroxyl group at the 4-position. RESULTS +78 82 NadR protein Collectively, this mixed network of polar and hydrophobic interactions endows NadR with a strong recognition pattern for HPAs, with additional medium-range interactions potentially established with the hydroxyl group at the 4-position. RESULTS +121 125 HPAs chemical Collectively, this mixed network of polar and hydrophobic interactions endows NadR with a strong recognition pattern for HPAs, with additional medium-range interactions potentially established with the hydroxyl group at the 4-position. RESULTS +79 88 3Cl,4-HPA chemical Structure-activity relationships: molecular basis of enhanced stabilization by 3Cl,4-HPA RESULTS +3 11 modelled experimental_method We modelled the binding of other HPAs by in silico superposition onto 4-HPA in the holo-NadR structure, and thereby obtained molecular explanations for the binding specificities of diverse ligands. RESULTS +33 37 HPAs chemical We modelled the binding of other HPAs by in silico superposition onto 4-HPA in the holo-NadR structure, and thereby obtained molecular explanations for the binding specificities of diverse ligands. RESULTS +41 64 in silico superposition experimental_method We modelled the binding of other HPAs by in silico superposition onto 4-HPA in the holo-NadR structure, and thereby obtained molecular explanations for the binding specificities of diverse ligands. RESULTS +70 75 4-HPA chemical We modelled the binding of other HPAs by in silico superposition onto 4-HPA in the holo-NadR structure, and thereby obtained molecular explanations for the binding specificities of diverse ligands. RESULTS +83 87 holo protein_state We modelled the binding of other HPAs by in silico superposition onto 4-HPA in the holo-NadR structure, and thereby obtained molecular explanations for the binding specificities of diverse ligands. RESULTS +88 92 NadR protein We modelled the binding of other HPAs by in silico superposition onto 4-HPA in the holo-NadR structure, and thereby obtained molecular explanations for the binding specificities of diverse ligands. RESULTS +93 102 structure evidence We modelled the binding of other HPAs by in silico superposition onto 4-HPA in the holo-NadR structure, and thereby obtained molecular explanations for the binding specificities of diverse ligands. RESULTS +24 29 4-HPA chemical For example, similar to 4-HPA, the binding of 3Cl,4-HPA could involve multiple bonds towards the carboxylate group of the ligand and some to the 4-hydroxyl group. RESULTS +46 55 3Cl,4-HPA chemical For example, similar to 4-HPA, the binding of 3Cl,4-HPA could involve multiple bonds towards the carboxylate group of the ligand and some to the 4-hydroxyl group. RESULTS +33 39 LeuB29 residue_name_number Additionally, the side chains of LeuB29 and AspB36 would be only 2.6–3.5 Å from the chlorine atom, thus providing van der Waals’ interactions or H-bonds to generate the additional binding affinity observed for 3Cl,4-HPA (Fig 4B). RESULTS +44 50 AspB36 residue_name_number Additionally, the side chains of LeuB29 and AspB36 would be only 2.6–3.5 Å from the chlorine atom, thus providing van der Waals’ interactions or H-bonds to generate the additional binding affinity observed for 3Cl,4-HPA (Fig 4B). RESULTS +114 141 van der Waals’ interactions bond_interaction Additionally, the side chains of LeuB29 and AspB36 would be only 2.6–3.5 Å from the chlorine atom, thus providing van der Waals’ interactions or H-bonds to generate the additional binding affinity observed for 3Cl,4-HPA (Fig 4B). RESULTS +145 152 H-bonds bond_interaction Additionally, the side chains of LeuB29 and AspB36 would be only 2.6–3.5 Å from the chlorine atom, thus providing van der Waals’ interactions or H-bonds to generate the additional binding affinity observed for 3Cl,4-HPA (Fig 4B). RESULTS +180 196 binding affinity evidence Additionally, the side chains of LeuB29 and AspB36 would be only 2.6–3.5 Å from the chlorine atom, thus providing van der Waals’ interactions or H-bonds to generate the additional binding affinity observed for 3Cl,4-HPA (Fig 4B). RESULTS +210 219 3Cl,4-HPA chemical Additionally, the side chains of LeuB29 and AspB36 would be only 2.6–3.5 Å from the chlorine atom, thus providing van der Waals’ interactions or H-bonds to generate the additional binding affinity observed for 3Cl,4-HPA (Fig 4B). RESULTS +61 66 2-HPA chemical The presence of a single hydroxyl group at position 2, as in 2-HPA, rather than at position 4, would eliminate the possibility of favorable interactions with AspB36, resulting in the lack of NadR regulation by 2-HPA described previously. RESULTS +158 164 AspB36 residue_name_number The presence of a single hydroxyl group at position 2, as in 2-HPA, rather than at position 4, would eliminate the possibility of favorable interactions with AspB36, resulting in the lack of NadR regulation by 2-HPA described previously. RESULTS +191 195 NadR protein The presence of a single hydroxyl group at position 2, as in 2-HPA, rather than at position 4, would eliminate the possibility of favorable interactions with AspB36, resulting in the lack of NadR regulation by 2-HPA described previously. RESULTS +210 215 2-HPA chemical The presence of a single hydroxyl group at position 2, as in 2-HPA, rather than at position 4, would eliminate the possibility of favorable interactions with AspB36, resulting in the lack of NadR regulation by 2-HPA described previously. RESULTS +9 19 salicylate chemical Finally, salicylate is presumably unable to specifically bind NadR due to the 2-hydroxyl substitution and the shorter aliphatic chain connecting its carboxylate group (Fig 1A): the compound simply seems too small to simultaneously establish the network of beneficial bonds observed in the NadR/HPA interactions. RESULTS +62 66 NadR protein Finally, salicylate is presumably unable to specifically bind NadR due to the 2-hydroxyl substitution and the shorter aliphatic chain connecting its carboxylate group (Fig 1A): the compound simply seems too small to simultaneously establish the network of beneficial bonds observed in the NadR/HPA interactions. RESULTS +289 293 NadR protein Finally, salicylate is presumably unable to specifically bind NadR due to the 2-hydroxyl substitution and the shorter aliphatic chain connecting its carboxylate group (Fig 1A): the compound simply seems too small to simultaneously establish the network of beneficial bonds observed in the NadR/HPA interactions. RESULTS +294 297 HPA chemical Finally, salicylate is presumably unable to specifically bind NadR due to the 2-hydroxyl substitution and the shorter aliphatic chain connecting its carboxylate group (Fig 1A): the compound simply seems too small to simultaneously establish the network of beneficial bonds observed in the NadR/HPA interactions. RESULTS +16 23 pockets site Analysis of the pockets reveals the molecular basis for asymmetric binding and stoichiometry RESULTS +26 49 tryptophan fluorescence experimental_method However, studies based on tryptophan fluorescence were confounded by the fluorescence of the HPA ligands, and isothermal titration calorimetry (ITC) was unfeasible due to the need for very high concentrations of NadR in the ITC chamber (due to the relatively low affinity), which exceeded the solubility limits of the protein. RESULTS +93 96 HPA chemical However, studies based on tryptophan fluorescence were confounded by the fluorescence of the HPA ligands, and isothermal titration calorimetry (ITC) was unfeasible due to the need for very high concentrations of NadR in the ITC chamber (due to the relatively low affinity), which exceeded the solubility limits of the protein. RESULTS +110 142 isothermal titration calorimetry experimental_method However, studies based on tryptophan fluorescence were confounded by the fluorescence of the HPA ligands, and isothermal titration calorimetry (ITC) was unfeasible due to the need for very high concentrations of NadR in the ITC chamber (due to the relatively low affinity), which exceeded the solubility limits of the protein. RESULTS +144 147 ITC experimental_method However, studies based on tryptophan fluorescence were confounded by the fluorescence of the HPA ligands, and isothermal titration calorimetry (ITC) was unfeasible due to the need for very high concentrations of NadR in the ITC chamber (due to the relatively low affinity), which exceeded the solubility limits of the protein. RESULTS +212 216 NadR protein However, studies based on tryptophan fluorescence were confounded by the fluorescence of the HPA ligands, and isothermal titration calorimetry (ITC) was unfeasible due to the need for very high concentrations of NadR in the ITC chamber (due to the relatively low affinity), which exceeded the solubility limits of the protein. RESULTS +224 227 ITC experimental_method However, studies based on tryptophan fluorescence were confounded by the fluorescence of the HPA ligands, and isothermal titration calorimetry (ITC) was unfeasible due to the need for very high concentrations of NadR in the ITC chamber (due to the relatively low affinity), which exceeded the solubility limits of the protein. RESULTS +42 63 binding stoichiometry evidence However, it was possible to calculate the binding stoichiometry of the NadR-HPA interactions using an SPR-based approach. RESULTS +71 79 NadR-HPA complex_assembly However, it was possible to calculate the binding stoichiometry of the NadR-HPA interactions using an SPR-based approach. RESULTS +102 105 SPR experimental_method However, it was possible to calculate the binding stoichiometry of the NadR-HPA interactions using an SPR-based approach. RESULTS +3 6 SPR experimental_method In SPR, the signal measured is proportional to the total molecular mass proximal to the sensor surface; consequently, if the molecular weights of the interactors are known, then the stoichiometry of the resulting complex can be determined. RESULTS +93 96 SPR experimental_method This approach relies on the assumption that the captured protein (‘the ligand’, according to SPR conventions) is 100% active and freely-accessible to potential interactors (‘the analytes’). RESULTS +9 13 NadR protein Firstly, NadR is expected to be covalently immobilized on the sensor chip as a dimer in random orientations, since it is a stable dimer in solution and has sixteen lysines well-distributed around its surface, all able to act as potential sites for amine coupling to the chip, and none of which are close to the ligand-binding pocket. RESULTS +79 84 dimer oligomeric_state Firstly, NadR is expected to be covalently immobilized on the sensor chip as a dimer in random orientations, since it is a stable dimer in solution and has sixteen lysines well-distributed around its surface, all able to act as potential sites for amine coupling to the chip, and none of which are close to the ligand-binding pocket. RESULTS +123 129 stable protein_state Firstly, NadR is expected to be covalently immobilized on the sensor chip as a dimer in random orientations, since it is a stable dimer in solution and has sixteen lysines well-distributed around its surface, all able to act as potential sites for amine coupling to the chip, and none of which are close to the ligand-binding pocket. RESULTS +130 135 dimer oligomeric_state Firstly, NadR is expected to be covalently immobilized on the sensor chip as a dimer in random orientations, since it is a stable dimer in solution and has sixteen lysines well-distributed around its surface, all able to act as potential sites for amine coupling to the chip, and none of which are close to the ligand-binding pocket. RESULTS +164 171 lysines residue_name Firstly, NadR is expected to be covalently immobilized on the sensor chip as a dimer in random orientations, since it is a stable dimer in solution and has sixteen lysines well-distributed around its surface, all able to act as potential sites for amine coupling to the chip, and none of which are close to the ligand-binding pocket. RESULTS +311 332 ligand-binding pocket site Firstly, NadR is expected to be covalently immobilized on the sensor chip as a dimer in random orientations, since it is a stable dimer in solution and has sixteen lysines well-distributed around its surface, all able to act as potential sites for amine coupling to the chip, and none of which are close to the ligand-binding pocket. RESULTS +14 17 HPA chemical Secondly, the HPA analytes are all very small (MW 150–170, Fig 1A) and therefore are expected to be able to diffuse readily into all potential binding sites, irrespective of the random orientations of the immobilized NadR dimers on the chip. RESULTS +143 156 binding sites site Secondly, the HPA analytes are all very small (MW 150–170, Fig 1A) and therefore are expected to be able to diffuse readily into all potential binding sites, irrespective of the random orientations of the immobilized NadR dimers on the chip. RESULTS +217 221 NadR protein Secondly, the HPA analytes are all very small (MW 150–170, Fig 1A) and therefore are expected to be able to diffuse readily into all potential binding sites, irrespective of the random orientations of the immobilized NadR dimers on the chip. RESULTS +222 228 dimers oligomeric_state Secondly, the HPA analytes are all very small (MW 150–170, Fig 1A) and therefore are expected to be able to diffuse readily into all potential binding sites, irrespective of the random orientations of the immobilized NadR dimers on the chip. RESULTS +25 33 NadR-HPA complex_assembly The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule. RESULTS +115 130 stoichiometries evidence The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule. RESULTS +143 148 4-HPA chemical The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule. RESULTS +159 164 3-HPA chemical The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule. RESULTS +179 188 3Cl,4-HPA chemical The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule. RESULTS +219 223 NadR protein The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule. RESULTS +224 229 dimer oligomeric_state The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule. RESULTS +230 238 bound to protein_state The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule. RESULTS +241 244 HPA chemical The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule. RESULTS +4 25 crystallographic data evidence The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins. RESULTS +44 47 SPR experimental_method The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins. RESULTS +59 80 binding stoichiometry evidence The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins. RESULTS +112 117 4-HPA chemical The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins. RESULTS +134 143 homodimer oligomeric_state The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins. RESULTS +223 229 MTH313 protein The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins. RESULTS +230 240 salicylate chemical The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins. RESULTS +267 271 MarR protein_type The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins. RESULTS +47 51 holo protein_state To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B). RESULTS +52 56 NadR protein To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B). RESULTS +61 71 superposed experimental_method To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B). RESULTS +76 87 ligand-free protein_state To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B). RESULTS +88 95 monomer oligomeric_state To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B). RESULTS +97 104 chain A structure_element To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B). RESULTS +115 130 ligand-occupied protein_state To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B). RESULTS +131 138 monomer oligomeric_state To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B). RESULTS +140 147 chain B structure_element To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B). RESULTS +13 26 superposition experimental_method Overall, the superposition revealed a high degree of structural similarity (Cα root mean square deviation (rmsd) of 1.5Å), though on closer inspection a rotational difference of ~9 degrees along the long axis of helix α6 was observed, suggesting that 4-HPA induced a slight conformational change (Fig 5A). RESULTS +79 105 root mean square deviation evidence Overall, the superposition revealed a high degree of structural similarity (Cα root mean square deviation (rmsd) of 1.5Å), though on closer inspection a rotational difference of ~9 degrees along the long axis of helix α6 was observed, suggesting that 4-HPA induced a slight conformational change (Fig 5A). RESULTS +107 111 rmsd evidence Overall, the superposition revealed a high degree of structural similarity (Cα root mean square deviation (rmsd) of 1.5Å), though on closer inspection a rotational difference of ~9 degrees along the long axis of helix α6 was observed, suggesting that 4-HPA induced a slight conformational change (Fig 5A). RESULTS +212 217 helix structure_element Overall, the superposition revealed a high degree of structural similarity (Cα root mean square deviation (rmsd) of 1.5Å), though on closer inspection a rotational difference of ~9 degrees along the long axis of helix α6 was observed, suggesting that 4-HPA induced a slight conformational change (Fig 5A). RESULTS +218 220 α6 structure_element Overall, the superposition revealed a high degree of structural similarity (Cα root mean square deviation (rmsd) of 1.5Å), though on closer inspection a rotational difference of ~9 degrees along the long axis of helix α6 was observed, suggesting that 4-HPA induced a slight conformational change (Fig 5A). RESULTS +251 256 4-HPA chemical Overall, the superposition revealed a high degree of structural similarity (Cα root mean square deviation (rmsd) of 1.5Å), though on closer inspection a rotational difference of ~9 degrees along the long axis of helix α6 was observed, suggesting that 4-HPA induced a slight conformational change (Fig 5A). RESULTS +27 32 helix structure_element However, since residues of helix α6 were not directly involved in ligand binding, an explanation for the lack of 4-HPA in monomer A did not emerge by analyzing only these backbone atom positions, suggesting that a more complex series of allosteric events may occur. RESULTS +33 35 α6 structure_element However, since residues of helix α6 were not directly involved in ligand binding, an explanation for the lack of 4-HPA in monomer A did not emerge by analyzing only these backbone atom positions, suggesting that a more complex series of allosteric events may occur. RESULTS +113 118 4-HPA chemical However, since residues of helix α6 were not directly involved in ligand binding, an explanation for the lack of 4-HPA in monomer A did not emerge by analyzing only these backbone atom positions, suggesting that a more complex series of allosteric events may occur. RESULTS +122 129 monomer oligomeric_state However, since residues of helix α6 were not directly involved in ligand binding, an explanation for the lack of 4-HPA in monomer A did not emerge by analyzing only these backbone atom positions, suggesting that a more complex series of allosteric events may occur. RESULTS +130 131 A structure_element However, since residues of helix α6 were not directly involved in ligand binding, an explanation for the lack of 4-HPA in monomer A did not emerge by analyzing only these backbone atom positions, suggesting that a more complex series of allosteric events may occur. RESULTS +63 68 Met22 residue_name_number Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly. RESULTS +70 75 Phe25 residue_name_number Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly. RESULTS +80 85 Arg43 residue_name_number Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly. RESULTS +96 103 monomer oligomeric_state Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly. RESULTS +104 105 B structure_element Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly. RESULTS +146 153 monomer oligomeric_state Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly. RESULTS +154 155 A structure_element Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly. RESULTS +184 190 pocket site Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly. RESULTS +37 42 CASTp experimental_method Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 Å3, while the pocket in chain A was occupied by these three side chains that adopted ‘inward’ positions and thereby divided the space into a few much smaller pockets, each with volume < 50 Å3, evidently rendering chain A unfavorable for ligand binding. RESULTS +57 63 pocket site Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 Å3, while the pocket in chain A was occupied by these three side chains that adopted ‘inward’ positions and thereby divided the space into a few much smaller pockets, each with volume < 50 Å3, evidently rendering chain A unfavorable for ligand binding. RESULTS +67 74 chain B structure_element Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 Å3, while the pocket in chain A was occupied by these three side chains that adopted ‘inward’ positions and thereby divided the space into a few much smaller pockets, each with volume < 50 Å3, evidently rendering chain A unfavorable for ligand binding. RESULTS +90 95 4-HPA chemical Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 Å3, while the pocket in chain A was occupied by these three side chains that adopted ‘inward’ positions and thereby divided the space into a few much smaller pockets, each with volume < 50 Å3, evidently rendering chain A unfavorable for ligand binding. RESULTS +156 162 pocket site Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 Å3, while the pocket in chain A was occupied by these three side chains that adopted ‘inward’ positions and thereby divided the space into a few much smaller pockets, each with volume < 50 Å3, evidently rendering chain A unfavorable for ligand binding. RESULTS +166 173 chain A structure_element Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 Å3, while the pocket in chain A was occupied by these three side chains that adopted ‘inward’ positions and thereby divided the space into a few much smaller pockets, each with volume < 50 Å3, evidently rendering chain A unfavorable for ligand binding. RESULTS +228 234 inward protein_state Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 Å3, while the pocket in chain A was occupied by these three side chains that adopted ‘inward’ positions and thereby divided the space into a few much smaller pockets, each with volume < 50 Å3, evidently rendering chain A unfavorable for ligand binding. RESULTS +355 362 chain A structure_element Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 Å3, while the pocket in chain A was occupied by these three side chains that adopted ‘inward’ positions and thereby divided the space into a few much smaller pockets, each with volume < 50 Å3, evidently rendering chain A unfavorable for ligand binding. RESULTS +71 77 MetA22 residue_name_number Most notably, atomic clashes between the ligand and the side chains of MetA22, PheA25 and ArgA43 would occur if 4-HPA were present in the monomer A pocket (Fig 5B). RESULTS +79 85 PheA25 residue_name_number Most notably, atomic clashes between the ligand and the side chains of MetA22, PheA25 and ArgA43 would occur if 4-HPA were present in the monomer A pocket (Fig 5B). RESULTS +90 96 ArgA43 residue_name_number Most notably, atomic clashes between the ligand and the side chains of MetA22, PheA25 and ArgA43 would occur if 4-HPA were present in the monomer A pocket (Fig 5B). RESULTS +112 117 4-HPA chemical Most notably, atomic clashes between the ligand and the side chains of MetA22, PheA25 and ArgA43 would occur if 4-HPA were present in the monomer A pocket (Fig 5B). RESULTS +138 145 monomer oligomeric_state Most notably, atomic clashes between the ligand and the side chains of MetA22, PheA25 and ArgA43 would occur if 4-HPA were present in the monomer A pocket (Fig 5B). RESULTS +146 147 A structure_element Most notably, atomic clashes between the ligand and the side chains of MetA22, PheA25 and ArgA43 would occur if 4-HPA were present in the monomer A pocket (Fig 5B). RESULTS +148 154 pocket site Most notably, atomic clashes between the ligand and the side chains of MetA22, PheA25 and ArgA43 would occur if 4-HPA were present in the monomer A pocket (Fig 5B). RESULTS +30 37 pockets site Subsequently, analyses of the pockets in apo-NadR revealed that in the absence of ligand the long Arg43 side chain was always in the open ‘outward’ position compatible with binding to the 4-HPA carboxylate group. RESULTS +41 44 apo protein_state Subsequently, analyses of the pockets in apo-NadR revealed that in the absence of ligand the long Arg43 side chain was always in the open ‘outward’ position compatible with binding to the 4-HPA carboxylate group. RESULTS +45 49 NadR protein Subsequently, analyses of the pockets in apo-NadR revealed that in the absence of ligand the long Arg43 side chain was always in the open ‘outward’ position compatible with binding to the 4-HPA carboxylate group. RESULTS +71 88 absence of ligand protein_state Subsequently, analyses of the pockets in apo-NadR revealed that in the absence of ligand the long Arg43 side chain was always in the open ‘outward’ position compatible with binding to the 4-HPA carboxylate group. RESULTS +98 103 Arg43 residue_name_number Subsequently, analyses of the pockets in apo-NadR revealed that in the absence of ligand the long Arg43 side chain was always in the open ‘outward’ position compatible with binding to the 4-HPA carboxylate group. RESULTS +139 146 outward protein_state Subsequently, analyses of the pockets in apo-NadR revealed that in the absence of ligand the long Arg43 side chain was always in the open ‘outward’ position compatible with binding to the 4-HPA carboxylate group. RESULTS +188 193 4-HPA chemical Subsequently, analyses of the pockets in apo-NadR revealed that in the absence of ligand the long Arg43 side chain was always in the open ‘outward’ position compatible with binding to the 4-HPA carboxylate group. RESULTS +17 20 apo protein_state In contrast, the apo-form Met22 and Phe25 residues were still encroaching the spaces of the 4-hydroxyl group and the phenyl ring of the ligand, respectively (Fig 5C). RESULTS +26 31 Met22 residue_name_number In contrast, the apo-form Met22 and Phe25 residues were still encroaching the spaces of the 4-hydroxyl group and the phenyl ring of the ligand, respectively (Fig 5C). RESULTS +36 41 Phe25 residue_name_number In contrast, the apo-form Met22 and Phe25 residues were still encroaching the spaces of the 4-hydroxyl group and the phenyl ring of the ligand, respectively (Fig 5C). RESULTS +5 12 outward protein_state The ‘outward’ position of Arg43 generated an open apo-form pocket with volume approximately 380Å3. RESULTS +26 31 Arg43 residue_name_number The ‘outward’ position of Arg43 generated an open apo-form pocket with volume approximately 380Å3. RESULTS +45 49 open protein_state The ‘outward’ position of Arg43 generated an open apo-form pocket with volume approximately 380Å3. RESULTS +50 53 apo protein_state The ‘outward’ position of Arg43 generated an open apo-form pocket with volume approximately 380Å3. RESULTS +59 65 pocket site The ‘outward’ position of Arg43 generated an open apo-form pocket with volume approximately 380Å3. RESULTS +48 53 Arg43 residue_name_number Taken together, these observations suggest that Arg43 is a major determinant of ligand binding, and that its ‘inward’ position inhibits the binding of 4-HPA to the empty pocket of holo-NadR. RESULTS +110 116 inward protein_state Taken together, these observations suggest that Arg43 is a major determinant of ligand binding, and that its ‘inward’ position inhibits the binding of 4-HPA to the empty pocket of holo-NadR. RESULTS +151 156 4-HPA chemical Taken together, these observations suggest that Arg43 is a major determinant of ligand binding, and that its ‘inward’ position inhibits the binding of 4-HPA to the empty pocket of holo-NadR. RESULTS +170 176 pocket site Taken together, these observations suggest that Arg43 is a major determinant of ligand binding, and that its ‘inward’ position inhibits the binding of 4-HPA to the empty pocket of holo-NadR. RESULTS +180 184 holo protein_state Taken together, these observations suggest that Arg43 is a major determinant of ligand binding, and that its ‘inward’ position inhibits the binding of 4-HPA to the empty pocket of holo-NadR. RESULTS +185 189 NadR protein Taken together, these observations suggest that Arg43 is a major determinant of ligand binding, and that its ‘inward’ position inhibits the binding of 4-HPA to the empty pocket of holo-NadR. RESULTS +26 30 NadR protein Structural differences of NadR in ligand-bound or free forms. FIG +34 46 ligand-bound protein_state Structural differences of NadR in ligand-bound or free forms. FIG +50 54 free protein_state Structural differences of NadR in ligand-bound or free forms. FIG +5 12 Aligned experimental_method (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +13 21 monomers oligomeric_state (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +25 29 holo protein_state (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +30 34 NadR protein (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +36 43 chain A structure_element (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +52 59 chain B structure_element (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +117 122 helix structure_element (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +123 125 α6 structure_element (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +131 141 Comparison experimental_method (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +153 168 binding pockets site (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +172 176 holo protein_state (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +177 181 NadR protein (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +200 211 ligand-free protein_state (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +212 219 monomer oligomeric_state (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +220 221 A structure_element (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +239 244 Met22 residue_name_number (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +246 251 Phe25 residue_name_number (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +256 261 Arg43 residue_name_number (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +269 275 inward protein_state (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +327 342 ligand-occupied protein_state (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +343 349 pocket site (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +374 380 inward protein_state (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +430 435 4-HPA chemical (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix α6. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt ‘inward’ positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these ‘inward’ conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively. FIG +9 17 crystals evidence In these crystals, the ArgA43 side chain showed two alternate conformations, modelled with 50% occupancy in each state, as indicated by the two ‘mirrored’ arrows. FIG +23 29 ArgA43 residue_name_number In these crystals, the ArgA43 side chain showed two alternate conformations, modelled with 50% occupancy in each state, as indicated by the two ‘mirrored’ arrows. FIG +67 72 4-HPA chemical The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. FIG +115 121 pocket site The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. FIG +127 131 holo protein_state The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. FIG +132 136 NadR protein The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. FIG +174 181 pockets site The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. FIG +185 188 apo protein_state The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. FIG +189 193 NadR protein The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. FIG +229 239 absence of protein_state The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. FIG +240 245 4-HPA chemical The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. FIG +250 255 Arg43 residue_name_number The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. FIG +294 301 outward protein_state The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the ‘outward’ conformation. FIG +20 63 15N heteronuclear solution NMR spectroscopy experimental_method Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). RESULTS +94 99 4-HPA chemical Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). RESULTS +105 108 apo protein_state Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). RESULTS +109 113 NadR protein Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). RESULTS +128 131 NMR experimental_method Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). RESULTS +132 139 spectra evidence Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). RESULTS +143 147 NadR protein Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). RESULTS +148 163 in the presence protein_state Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). RESULTS +168 178 absence of protein_state Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). RESULTS +179 184 4-HPA chemical Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods). RESULTS +4 21 1H-15N TROSY-HSQC experimental_method The 1H-15N TROSY-HSQC spectrum of apo-NadR, acquired at 25°C, displayed approximately 140 distinct peaks (Fig 6A), most of which correspond to backbone amide N-H groups. RESULTS +22 30 spectrum evidence The 1H-15N TROSY-HSQC spectrum of apo-NadR, acquired at 25°C, displayed approximately 140 distinct peaks (Fig 6A), most of which correspond to backbone amide N-H groups. RESULTS +34 37 apo protein_state The 1H-15N TROSY-HSQC spectrum of apo-NadR, acquired at 25°C, displayed approximately 140 distinct peaks (Fig 6A), most of which correspond to backbone amide N-H groups. RESULTS +38 42 NadR protein The 1H-15N TROSY-HSQC spectrum of apo-NadR, acquired at 25°C, displayed approximately 140 distinct peaks (Fig 6A), most of which correspond to backbone amide N-H groups. RESULTS +168 171 apo protein_state The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25°C, two or more distinct conformations of apo-NadR monomers were not readily apparent. RESULTS +172 176 NadR protein The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25°C, two or more distinct conformations of apo-NadR monomers were not readily apparent. RESULTS +180 191 well-folded protein_state The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25°C, two or more distinct conformations of apo-NadR monomers were not readily apparent. RESULTS +264 267 NMR experimental_method The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25°C, two or more distinct conformations of apo-NadR monomers were not readily apparent. RESULTS +291 294 NMR experimental_method The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25°C, two or more distinct conformations of apo-NadR monomers were not readily apparent. RESULTS +354 357 apo protein_state The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25°C, two or more distinct conformations of apo-NadR monomers were not readily apparent. RESULTS +358 362 NadR protein The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25°C, two or more distinct conformations of apo-NadR monomers were not readily apparent. RESULTS +363 371 monomers oligomeric_state The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25°C, two or more distinct conformations of apo-NadR monomers were not readily apparent. RESULTS +21 26 4-HPA chemical Upon the addition of 4-HPA, over 45 peaks showed chemical shift perturbations, i.e. changed position in the spectrum or disappeared, while the remaining peaks remained unchanged. RESULTS +29 34 4-HPA chemical This observation showed that 4-HPA was able to bind NadR and induce notable changes in specific regions of the protein. RESULTS +52 56 NadR protein This observation showed that 4-HPA was able to bind NadR and induce notable changes in specific regions of the protein. RESULTS +0 3 NMR experimental_method NMR spectra of NadR in the presence and absence of 4-HPA. FIG +4 11 spectra evidence NMR spectra of NadR in the presence and absence of 4-HPA. FIG +15 19 NadR protein NMR spectra of NadR in the presence and absence of 4-HPA. FIG +20 35 in the presence protein_state NMR spectra of NadR in the presence and absence of 4-HPA. FIG +40 50 absence of protein_state NMR spectra of NadR in the presence and absence of 4-HPA. FIG +51 56 4-HPA chemical NMR spectra of NadR in the presence and absence of 4-HPA. FIG +5 18 Superposition experimental_method (A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25°C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red). FIG +26 43 1H-15N TROSY-HSQC experimental_method (A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25°C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red). FIG +44 51 spectra evidence (A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25°C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red). FIG +72 75 apo protein_state (A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25°C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red). FIG +76 80 NadR protein (A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25°C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red). FIG +95 99 NadR protein (A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25°C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red). FIG +107 118 presence of protein_state (A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25°C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red). FIG +119 124 4-HPA chemical (A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25°C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red). FIG +6 13 Overlay experimental_method (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). FIG +41 58 1H-15N TROSY-HSQC experimental_method (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). FIG +59 66 spectra evidence (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). FIG +87 90 apo protein_state (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). FIG +91 95 NadR protein (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). FIG +107 117 NadR/4-HPA complex_assembly (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). FIG +124 136 superimposed experimental_method (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). FIG +146 153 spectra evidence (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). FIG +174 177 apo protein_state (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). FIG +178 182 NadR protein (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). FIG +194 204 NadR/4-HPA complex_assembly (B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25°C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10°C of apo-NadR (blue) and NadR/4-HPA (green). FIG +4 11 spectra evidence The spectra acquired at 10°C are excluded from panel A for simplicity. FIG +16 27 presence of protein_state However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable. RESULTS +28 33 4-HPA chemical However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable. RESULTS +39 56 1H-15N TROSY-HSQC experimental_method However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable. RESULTS +57 65 spectrum evidence However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable. RESULTS +69 73 NadR protein However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable. RESULTS +116 119 apo protein_state However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable. RESULTS +120 124 NadR protein However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable. RESULTS +236 256 crystallographically experimental_method However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable. RESULTS +62 78 binding affinity evidence Considering the small size, fast diffusion and relatively low binding affinity of 4-HPA, it would not be surprising if the ligand associates and dissociates rapidly on the NMR time scale, resulting in only one set of peaks whose chemical shifts represent the average environment of the bound and unbound states. RESULTS +82 87 4-HPA chemical Considering the small size, fast diffusion and relatively low binding affinity of 4-HPA, it would not be surprising if the ligand associates and dissociates rapidly on the NMR time scale, resulting in only one set of peaks whose chemical shifts represent the average environment of the bound and unbound states. RESULTS +172 175 NMR experimental_method Considering the small size, fast diffusion and relatively low binding affinity of 4-HPA, it would not be surprising if the ligand associates and dissociates rapidly on the NMR time scale, resulting in only one set of peaks whose chemical shifts represent the average environment of the bound and unbound states. RESULTS +286 291 bound protein_state Considering the small size, fast diffusion and relatively low binding affinity of 4-HPA, it would not be surprising if the ligand associates and dissociates rapidly on the NMR time scale, resulting in only one set of peaks whose chemical shifts represent the average environment of the bound and unbound states. RESULTS +296 303 unbound protein_state Considering the small size, fast diffusion and relatively low binding affinity of 4-HPA, it would not be surprising if the ligand associates and dissociates rapidly on the NMR time scale, resulting in only one set of peaks whose chemical shifts represent the average environment of the bound and unbound states. RESULTS +109 114 4-HPA chemical Interestingly, by cooling the samples to 10°C, we observed that a number of those peaks strongly affected by 4-HPA (and therefore likely to be in the ligand-binding site) demonstrated evidence of peak splitting, i.e. a tendency to become two distinct peaks rather than one single peak (Fig 6B and 6C). RESULTS +150 169 ligand-binding site site Interestingly, by cooling the samples to 10°C, we observed that a number of those peaks strongly affected by 4-HPA (and therefore likely to be in the ligand-binding site) demonstrated evidence of peak splitting, i.e. a tendency to become two distinct peaks rather than one single peak (Fig 6B and 6C). RESULTS +140 148 presence protein_state These doubled peaks may therefore reveal that the cooler temperature partially trapped the existence in solution of two distinct states, in presence or absence of 4-HPA, with minor conformational differences occurring at least in proximity to the binding pocket. RESULTS +152 162 absence of protein_state These doubled peaks may therefore reveal that the cooler temperature partially trapped the existence in solution of two distinct states, in presence or absence of 4-HPA, with minor conformational differences occurring at least in proximity to the binding pocket. RESULTS +163 168 4-HPA chemical These doubled peaks may therefore reveal that the cooler temperature partially trapped the existence in solution of two distinct states, in presence or absence of 4-HPA, with minor conformational differences occurring at least in proximity to the binding pocket. RESULTS +247 261 binding pocket site These doubled peaks may therefore reveal that the cooler temperature partially trapped the existence in solution of two distinct states, in presence or absence of 4-HPA, with minor conformational differences occurring at least in proximity to the binding pocket. RESULTS +28 31 NMR experimental_method Although more comprehensive NMR experiments and full chemical shift assignment of the spectra would be required to precisely define this multi-state behavior, the NMR data clearly demonstrate that NadR exhibits conformational flexibility which is modulated by 4-HPA in solution. RESULTS +86 93 spectra evidence Although more comprehensive NMR experiments and full chemical shift assignment of the spectra would be required to precisely define this multi-state behavior, the NMR data clearly demonstrate that NadR exhibits conformational flexibility which is modulated by 4-HPA in solution. RESULTS +163 166 NMR experimental_method Although more comprehensive NMR experiments and full chemical shift assignment of the spectra would be required to precisely define this multi-state behavior, the NMR data clearly demonstrate that NadR exhibits conformational flexibility which is modulated by 4-HPA in solution. RESULTS +197 201 NadR protein Although more comprehensive NMR experiments and full chemical shift assignment of the spectra would be required to precisely define this multi-state behavior, the NMR data clearly demonstrate that NadR exhibits conformational flexibility which is modulated by 4-HPA in solution. RESULTS +260 265 4-HPA chemical Although more comprehensive NMR experiments and full chemical shift assignment of the spectra would be required to precisely define this multi-state behavior, the NMR data clearly demonstrate that NadR exhibits conformational flexibility which is modulated by 4-HPA in solution. RESULTS +0 3 Apo protein_state Apo-NadR structures reveal intrinsic conformational flexibility RESULTS +4 8 NadR protein Apo-NadR structures reveal intrinsic conformational flexibility RESULTS +9 19 structures evidence Apo-NadR structures reveal intrinsic conformational flexibility RESULTS +4 7 apo protein_state The apo-NadR crystal structure contained two homodimers in the asymmetric unit (chains A+B and chains C+D). RESULTS +8 12 NadR protein The apo-NadR crystal structure contained two homodimers in the asymmetric unit (chains A+B and chains C+D). RESULTS +13 30 crystal structure evidence The apo-NadR crystal structure contained two homodimers in the asymmetric unit (chains A+B and chains C+D). RESULTS +45 55 homodimers oligomeric_state The apo-NadR crystal structure contained two homodimers in the asymmetric unit (chains A+B and chains C+D). RESULTS +80 90 chains A+B structure_element The apo-NadR crystal structure contained two homodimers in the asymmetric unit (chains A+B and chains C+D). RESULTS +95 105 chains C+D structure_element The apo-NadR crystal structure contained two homodimers in the asymmetric unit (chains A+B and chains C+D). RESULTS +13 37 structural superposition experimental_method Upon overall structural superposition, these dimers revealed a few minor differences in the α6 helix (a major component of the dimer interface) and the helices α4-α5 (the DNA binding region), and an rmsd of 1.55Å (Fig 7A). RESULTS +45 51 dimers oligomeric_state Upon overall structural superposition, these dimers revealed a few minor differences in the α6 helix (a major component of the dimer interface) and the helices α4-α5 (the DNA binding region), and an rmsd of 1.55Å (Fig 7A). RESULTS +92 100 α6 helix structure_element Upon overall structural superposition, these dimers revealed a few minor differences in the α6 helix (a major component of the dimer interface) and the helices α4-α5 (the DNA binding region), and an rmsd of 1.55Å (Fig 7A). RESULTS +127 142 dimer interface site Upon overall structural superposition, these dimers revealed a few minor differences in the α6 helix (a major component of the dimer interface) and the helices α4-α5 (the DNA binding region), and an rmsd of 1.55Å (Fig 7A). RESULTS +152 159 helices structure_element Upon overall structural superposition, these dimers revealed a few minor differences in the α6 helix (a major component of the dimer interface) and the helices α4-α5 (the DNA binding region), and an rmsd of 1.55Å (Fig 7A). RESULTS +160 165 α4-α5 structure_element Upon overall structural superposition, these dimers revealed a few minor differences in the α6 helix (a major component of the dimer interface) and the helices α4-α5 (the DNA binding region), and an rmsd of 1.55Å (Fig 7A). RESULTS +171 189 DNA binding region site Upon overall structural superposition, these dimers revealed a few minor differences in the α6 helix (a major component of the dimer interface) and the helices α4-α5 (the DNA binding region), and an rmsd of 1.55Å (Fig 7A). RESULTS +199 203 rmsd evidence Upon overall structural superposition, these dimers revealed a few minor differences in the α6 helix (a major component of the dimer interface) and the helices α4-α5 (the DNA binding region), and an rmsd of 1.55Å (Fig 7A). RESULTS +22 26 holo protein_state Similarly, the entire holo-homodimer could be closely superposed onto each of the apo-homodimers, showing rmsd values of 1.29Å and 1.31Å, and with more notable differences in the α6 helix positions (Fig 7B). RESULTS +27 36 homodimer oligomeric_state Similarly, the entire holo-homodimer could be closely superposed onto each of the apo-homodimers, showing rmsd values of 1.29Å and 1.31Å, and with more notable differences in the α6 helix positions (Fig 7B). RESULTS +46 64 closely superposed experimental_method Similarly, the entire holo-homodimer could be closely superposed onto each of the apo-homodimers, showing rmsd values of 1.29Å and 1.31Å, and with more notable differences in the α6 helix positions (Fig 7B). RESULTS +82 85 apo protein_state Similarly, the entire holo-homodimer could be closely superposed onto each of the apo-homodimers, showing rmsd values of 1.29Å and 1.31Å, and with more notable differences in the α6 helix positions (Fig 7B). RESULTS +86 96 homodimers oligomeric_state Similarly, the entire holo-homodimer could be closely superposed onto each of the apo-homodimers, showing rmsd values of 1.29Å and 1.31Å, and with more notable differences in the α6 helix positions (Fig 7B). RESULTS +106 110 rmsd evidence Similarly, the entire holo-homodimer could be closely superposed onto each of the apo-homodimers, showing rmsd values of 1.29Å and 1.31Å, and with more notable differences in the α6 helix positions (Fig 7B). RESULTS +179 187 α6 helix structure_element Similarly, the entire holo-homodimer could be closely superposed onto each of the apo-homodimers, showing rmsd values of 1.29Å and 1.31Å, and with more notable differences in the α6 helix positions (Fig 7B). RESULTS +20 24 rmsd evidence The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility. RESULTS +41 44 apo protein_state The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility. RESULTS +45 55 homodimers oligomeric_state The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility. RESULTS +77 80 apo protein_state The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility. RESULTS +86 90 holo protein_state The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility. RESULTS +91 101 homodimers oligomeric_state The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility. RESULTS +125 128 apo protein_state The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility. RESULTS +129 133 NadR protein The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility. RESULTS +8 11 apo protein_state Overall apo- and holo-NadR structures are similar. FIG +17 21 holo protein_state Overall apo- and holo-NadR structures are similar. FIG +22 26 NadR protein Overall apo- and holo-NadR structures are similar. FIG +27 37 structures evidence Overall apo- and holo-NadR structures are similar. FIG +5 23 Pairwise alignment experimental_method (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +44 47 apo protein_state (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +48 52 NadR protein (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +53 63 homodimers oligomeric_state (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +65 67 AB structure_element (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +72 74 CD structure_element (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +91 94 apo protein_state (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +95 99 NadR protein (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +100 108 crystals evidence (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +114 123 Alignment experimental_method (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +131 135 holo protein_state (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +136 140 NadR protein (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +141 150 homodimer oligomeric_state (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +184 187 apo protein_state (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +188 192 NadR protein (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +193 203 homodimers oligomeric_state (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers. FIG +45 55 α6 helices structure_element Here, larger differences are observed in the α6 helices (top). FIG +0 5 4-HPA chemical 4-HPA stabilizes concerted conformational changes in NadR that prevent DNA-binding RESULTS +53 57 NadR protein 4-HPA stabilizes concerted conformational changes in NadR that prevent DNA-binding RESULTS +60 64 NadR protein To further investigate the conformational rearrangements of NadR, we performed local structural alignments using only a subset of residues in the DNA-binding helix (α4). RESULTS +79 106 local structural alignments experimental_method To further investigate the conformational rearrangements of NadR, we performed local structural alignments using only a subset of residues in the DNA-binding helix (α4). RESULTS +146 163 DNA-binding helix structure_element To further investigate the conformational rearrangements of NadR, we performed local structural alignments using only a subset of residues in the DNA-binding helix (α4). RESULTS +165 167 α4 structure_element To further investigate the conformational rearrangements of NadR, we performed local structural alignments using only a subset of residues in the DNA-binding helix (α4). RESULTS +3 12 selecting experimental_method By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. RESULTS +17 25 aligning experimental_method By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. RESULTS +35 46 Arg64-Ala77 residue_range By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. RESULTS +54 62 α4 helix structure_element By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. RESULTS +67 72 dimer oligomeric_state By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. RESULTS +74 87 superposition experimental_method By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. RESULTS +95 99 holo protein_state By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. RESULTS +100 109 homodimer oligomeric_state By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. RESULTS +123 126 apo protein_state By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. RESULTS +127 137 homodimers oligomeric_state By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. RESULTS +166 173 monomer oligomeric_state By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. RESULTS +196 205 structure evidence By selecting and aligning residues Arg64-Ala77 of one α4 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure. RESULTS +10 17 monomer oligomeric_state While one monomer from each structure was closely superimposable (Fig 8A, left side), the second monomer displayed quite large differences (Fig 8A, right side). RESULTS +28 37 structure evidence While one monomer from each structure was closely superimposable (Fig 8A, left side), the second monomer displayed quite large differences (Fig 8A, right side). RESULTS +97 104 monomer oligomeric_state While one monomer from each structure was closely superimposable (Fig 8A, left side), the second monomer displayed quite large differences (Fig 8A, right side). RESULTS +34 37 DNA chemical Most notably, the position of the DNA-binding helix α4 shifted by as much as 6 Å (Fig 8B). RESULTS +46 51 helix structure_element Most notably, the position of the DNA-binding helix α4 shifted by as much as 6 Å (Fig 8B). RESULTS +52 54 α4 structure_element Most notably, the position of the DNA-binding helix α4 shifted by as much as 6 Å (Fig 8B). RESULTS +13 18 helix structure_element Accordingly, helix α4 was also found to be one of the most dynamic regions in previous HDX-MS analyses of apo-NadR in solution. RESULTS +19 21 α4 structure_element Accordingly, helix α4 was also found to be one of the most dynamic regions in previous HDX-MS analyses of apo-NadR in solution. RESULTS +87 93 HDX-MS experimental_method Accordingly, helix α4 was also found to be one of the most dynamic regions in previous HDX-MS analyses of apo-NadR in solution. RESULTS +106 109 apo protein_state Accordingly, helix α4 was also found to be one of the most dynamic regions in previous HDX-MS analyses of apo-NadR in solution. RESULTS +110 114 NadR protein Accordingly, helix α4 was also found to be one of the most dynamic regions in previous HDX-MS analyses of apo-NadR in solution. RESULTS +0 22 Structural comparisons experimental_method Structural comparisons of NadR and modelling of interactions with DNA. FIG +26 30 NadR protein Structural comparisons of NadR and modelling of interactions with DNA. FIG +66 69 DNA chemical Structural comparisons of NadR and modelling of interactions with DNA. FIG +9 13 holo protein_state (A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. FIG +14 23 homodimer oligomeric_state (A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. FIG +24 33 structure evidence (A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. FIG +75 88 chain A and B structure_element (A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. FIG +118 128 homodimers oligomeric_state (A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. FIG +132 135 apo protein_state (A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. FIG +136 140 NadR protein (A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. FIG +180 183 A/C structure_element (A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. FIG +188 191 B/D structure_element (A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively. FIG +10 20 homodimers oligomeric_state The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +29 31 AB structure_element The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +32 36 holo protein_state The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +38 40 AB structure_element The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +41 44 apo protein_state The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +50 52 CD structure_element The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +53 56 apo protein_state The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +63 71 overlaid experimental_method The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +75 95 structural alignment experimental_method The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +139 146 R64-A77 residue_range The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +196 197 A structure_element The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +198 202 holo protein_state The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +204 205 A structure_element The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +206 209 apo protein_state The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +215 216 C structure_element The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +217 220 apo protein_state The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +235 240 helix structure_element The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +241 243 α4 structure_element The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix α4 (left). FIG +4 14 α4 helices structure_element The α4 helices aligned closely, Cα rmsd 0.2Å for 14 residues. FIG +35 39 rmsd evidence The α4 helices aligned closely, Cα rmsd 0.2Å for 14 residues. FIG +34 44 α4 helices structure_element (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. FIG +52 63 4-HPA-bound protein_state (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. FIG +64 68 holo protein_state (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. FIG +69 78 homodimer oligomeric_state (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. FIG +79 86 chain B structure_element (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. FIG +102 105 apo protein_state (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. FIG +106 116 homodimers oligomeric_state (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. FIG +117 119 AB structure_element (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. FIG +124 126 CD structure_element (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. FIG +136 150 chains B and D structure_element (B) The relative positions of the α4 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue. FIG +20 25 Ala77 residue_name_number Dashes indicate the Ala77 Cα atoms, in the most highly shifted region of the ‘non-fixed’ α4 helix. FIG +89 97 α4 helix structure_element Dashes indicate the Ala77 Cα atoms, in the most highly shifted region of the ‘non-fixed’ α4 helix. FIG +24 27 DNA chemical (C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR α4 helices in the DNA major grooves. FIG +61 70 OhrR-ohrA complex_assembly (C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR α4 helices in the DNA major grooves. FIG +94 107 superposition experimental_method (C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR α4 helices in the DNA major grooves. FIG +113 117 NadR protein (C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR α4 helices in the DNA major grooves. FIG +162 166 NadR protein (C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR α4 helices in the DNA major grooves. FIG +167 177 α4 helices structure_element (C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR α4 helices in the DNA major grooves. FIG +185 188 DNA chemical (C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR α4 helices in the DNA major grooves. FIG +22 32 α4 helices structure_element For clarity, only the α4 helices are shown in panels (B) and (C). (D) Upon comparison with the experimentally-determined OhrR:ohrA structure (grey), the α4 helix of holo-NadR (blue) is shifted ~8Å out of the major groove. FIG +121 130 OhrR:ohrA complex_assembly For clarity, only the α4 helices are shown in panels (B) and (C). (D) Upon comparison with the experimentally-determined OhrR:ohrA structure (grey), the α4 helix of holo-NadR (blue) is shifted ~8Å out of the major groove. FIG +131 140 structure evidence For clarity, only the α4 helices are shown in panels (B) and (C). (D) Upon comparison with the experimentally-determined OhrR:ohrA structure (grey), the α4 helix of holo-NadR (blue) is shifted ~8Å out of the major groove. FIG +153 161 α4 helix structure_element For clarity, only the α4 helices are shown in panels (B) and (C). (D) Upon comparison with the experimentally-determined OhrR:ohrA structure (grey), the α4 helix of holo-NadR (blue) is shifted ~8Å out of the major groove. FIG +165 169 holo protein_state For clarity, only the α4 helices are shown in panels (B) and (C). (D) Upon comparison with the experimentally-determined OhrR:ohrA structure (grey), the α4 helix of holo-NadR (blue) is shifted ~8Å out of the major groove. FIG +170 174 NadR protein For clarity, only the α4 helices are shown in panels (B) and (C). (D) Upon comparison with the experimentally-determined OhrR:ohrA structure (grey), the α4 helix of holo-NadR (blue) is shifted ~8Å out of the major groove. FIG +9 31 structural comparisons experimental_method However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). RESULTS +59 63 holo protein_state However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). RESULTS +64 68 NadR protein However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). RESULTS +69 74 helix structure_element However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). RESULTS +75 77 α4 structure_element However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). RESULTS +93 104 presence of protein_state However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). RESULTS +105 110 4-HPA chemical However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). RESULTS +158 162 holo protein_state However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). RESULTS +163 178 dimer interface site However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). RESULTS +249 252 apo protein_state However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). RESULTS +253 269 dimer interfaces site However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). RESULTS +311 321 α6 helices structure_element However, structural comparisons revealed that the shift of holo-NadR helix α4 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the α6 helices (S3 Fig). RESULTS +24 41 ligand-stabilized protein_state In summary, compared to ligand-stabilized holo-NadR, apo-NadR displayed an intrinsic flexibility focused in the DNA-binding region. RESULTS +42 46 holo protein_state In summary, compared to ligand-stabilized holo-NadR, apo-NadR displayed an intrinsic flexibility focused in the DNA-binding region. RESULTS +47 51 NadR protein In summary, compared to ligand-stabilized holo-NadR, apo-NadR displayed an intrinsic flexibility focused in the DNA-binding region. RESULTS +53 56 apo protein_state In summary, compared to ligand-stabilized holo-NadR, apo-NadR displayed an intrinsic flexibility focused in the DNA-binding region. RESULTS +57 61 NadR protein In summary, compared to ligand-stabilized holo-NadR, apo-NadR displayed an intrinsic flexibility focused in the DNA-binding region. RESULTS +112 130 DNA-binding region site In summary, compared to ligand-stabilized holo-NadR, apo-NadR displayed an intrinsic flexibility focused in the DNA-binding region. RESULTS +70 86 electron density evidence This was also evident in the greater disorder (i.e. less well-defined electron density) in the β1-β2 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing). RESULTS +95 106 β1-β2 loops structure_element This was also evident in the greater disorder (i.e. less well-defined electron density) in the β1-β2 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing). RESULTS +114 117 apo protein_state This was also evident in the greater disorder (i.e. less well-defined electron density) in the β1-β2 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing). RESULTS +118 124 dimers oligomeric_state This was also evident in the greater disorder (i.e. less well-defined electron density) in the β1-β2 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing). RESULTS +126 133 density evidence This was also evident in the greater disorder (i.e. less well-defined electron density) in the β1-β2 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing). RESULTS +154 159 dimer oligomeric_state This was also evident in the greater disorder (i.e. less well-defined electron density) in the β1-β2 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing). RESULTS +189 193 holo protein_state This was also evident in the greater disorder (i.e. less well-defined electron density) in the β1-β2 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing). RESULTS +194 199 dimer oligomeric_state This was also evident in the greater disorder (i.e. less well-defined electron density) in the β1-β2 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing). RESULTS +201 208 density evidence This was also evident in the greater disorder (i.e. less well-defined electron density) in the β1-β2 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing). RESULTS +3 7 holo protein_state In holo-NadR, the distance separating the two DNA-binding α4 helices was 32 Å, while in apo-NadR it was 29 Å for homodimer AB, and 34 Å for homodimer CD (Fig 8C). RESULTS +8 12 NadR protein In holo-NadR, the distance separating the two DNA-binding α4 helices was 32 Å, while in apo-NadR it was 29 Å for homodimer AB, and 34 Å for homodimer CD (Fig 8C). RESULTS +58 68 α4 helices structure_element In holo-NadR, the distance separating the two DNA-binding α4 helices was 32 Å, while in apo-NadR it was 29 Å for homodimer AB, and 34 Å for homodimer CD (Fig 8C). RESULTS +88 91 apo protein_state In holo-NadR, the distance separating the two DNA-binding α4 helices was 32 Å, while in apo-NadR it was 29 Å for homodimer AB, and 34 Å for homodimer CD (Fig 8C). RESULTS +92 96 NadR protein In holo-NadR, the distance separating the two DNA-binding α4 helices was 32 Å, while in apo-NadR it was 29 Å for homodimer AB, and 34 Å for homodimer CD (Fig 8C). RESULTS +113 122 homodimer oligomeric_state In holo-NadR, the distance separating the two DNA-binding α4 helices was 32 Å, while in apo-NadR it was 29 Å for homodimer AB, and 34 Å for homodimer CD (Fig 8C). RESULTS +123 125 AB structure_element In holo-NadR, the distance separating the two DNA-binding α4 helices was 32 Å, while in apo-NadR it was 29 Å for homodimer AB, and 34 Å for homodimer CD (Fig 8C). RESULTS +140 149 homodimer oligomeric_state In holo-NadR, the distance separating the two DNA-binding α4 helices was 32 Å, while in apo-NadR it was 29 Å for homodimer AB, and 34 Å for homodimer CD (Fig 8C). RESULTS +150 152 CD structure_element In holo-NadR, the distance separating the two DNA-binding α4 helices was 32 Å, while in apo-NadR it was 29 Å for homodimer AB, and 34 Å for homodimer CD (Fig 8C). RESULTS +10 13 apo protein_state Thus, the apo-homodimer AB presented the DNA-binding helices in a conformation similar to that observed in the protein:DNA complex of OhrR:ohrA from Bacillus subtilis (Fig 8C). RESULTS +14 23 homodimer oligomeric_state Thus, the apo-homodimer AB presented the DNA-binding helices in a conformation similar to that observed in the protein:DNA complex of OhrR:ohrA from Bacillus subtilis (Fig 8C). RESULTS +24 26 AB structure_element Thus, the apo-homodimer AB presented the DNA-binding helices in a conformation similar to that observed in the protein:DNA complex of OhrR:ohrA from Bacillus subtilis (Fig 8C). RESULTS +41 60 DNA-binding helices structure_element Thus, the apo-homodimer AB presented the DNA-binding helices in a conformation similar to that observed in the protein:DNA complex of OhrR:ohrA from Bacillus subtilis (Fig 8C). RESULTS +134 143 OhrR:ohrA complex_assembly Thus, the apo-homodimer AB presented the DNA-binding helices in a conformation similar to that observed in the protein:DNA complex of OhrR:ohrA from Bacillus subtilis (Fig 8C). RESULTS +149 166 Bacillus subtilis species Thus, the apo-homodimer AB presented the DNA-binding helices in a conformation similar to that observed in the protein:DNA complex of OhrR:ohrA from Bacillus subtilis (Fig 8C). RESULTS +15 19 OhrR protein Interestingly, OhrR contacts ohrA across 22 base pairs (bp), and similarly the main NadR target sites identified in the nadA promoter (the operators Op I and Op II) both span 22 bp. RESULTS +29 33 ohrA gene Interestingly, OhrR contacts ohrA across 22 base pairs (bp), and similarly the main NadR target sites identified in the nadA promoter (the operators Op I and Op II) both span 22 bp. RESULTS +84 88 NadR protein Interestingly, OhrR contacts ohrA across 22 base pairs (bp), and similarly the main NadR target sites identified in the nadA promoter (the operators Op I and Op II) both span 22 bp. RESULTS +89 101 target sites site Interestingly, OhrR contacts ohrA across 22 base pairs (bp), and similarly the main NadR target sites identified in the nadA promoter (the operators Op I and Op II) both span 22 bp. RESULTS +120 124 nadA gene Interestingly, OhrR contacts ohrA across 22 base pairs (bp), and similarly the main NadR target sites identified in the nadA promoter (the operators Op I and Op II) both span 22 bp. RESULTS +0 23 Pairwise superpositions experimental_method Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). RESULTS +40 44 NadR protein Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). RESULTS +45 48 apo protein_state Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). RESULTS +49 58 homodimer oligomeric_state Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). RESULTS +59 61 AB structure_element Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). RESULTS +86 90 OhrR protein Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). RESULTS +92 96 rmsd evidence Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). RESULTS +115 119 holo protein_state Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). RESULTS +120 129 homodimer oligomeric_state Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). RESULTS +154 158 rmsd evidence Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 Å), while the holo-homodimer was the most divergent (rmsd 3.3 Å) (Fig 8C). RESULTS +18 21 DNA chemical Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. RESULTS +51 55 OhrR protein Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. RESULTS +60 64 NadR protein Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. RESULTS +70 73 apo protein_state Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. RESULTS +74 83 homodimer oligomeric_state Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. RESULTS +84 86 AB structure_element Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. RESULTS +120 123 DNA chemical Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. RESULTS +139 144 4-HPA chemical Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. RESULTS +167 171 holo protein_state Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. RESULTS +172 176 NadR protein Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. RESULTS +213 216 DNA chemical Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding. RESULTS +43 80 inter-helical translational distances evidence Specifically, in addition to the different inter-helical translational distances, the α4 helices in the holo-NadR homodimer were also reoriented, resulting in movement of α4 out of the major groove, by up to 8Å, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D). RESULTS +86 96 α4 helices structure_element Specifically, in addition to the different inter-helical translational distances, the α4 helices in the holo-NadR homodimer were also reoriented, resulting in movement of α4 out of the major groove, by up to 8Å, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D). RESULTS +104 108 holo protein_state Specifically, in addition to the different inter-helical translational distances, the α4 helices in the holo-NadR homodimer were also reoriented, resulting in movement of α4 out of the major groove, by up to 8Å, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D). RESULTS +109 113 NadR protein Specifically, in addition to the different inter-helical translational distances, the α4 helices in the holo-NadR homodimer were also reoriented, resulting in movement of α4 out of the major groove, by up to 8Å, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D). RESULTS +114 123 homodimer oligomeric_state Specifically, in addition to the different inter-helical translational distances, the α4 helices in the holo-NadR homodimer were also reoriented, resulting in movement of α4 out of the major groove, by up to 8Å, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D). RESULTS +171 173 α4 structure_element Specifically, in addition to the different inter-helical translational distances, the α4 helices in the holo-NadR homodimer were also reoriented, resulting in movement of α4 out of the major groove, by up to 8Å, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D). RESULTS +248 251 DNA chemical Specifically, in addition to the different inter-helical translational distances, the α4 helices in the holo-NadR homodimer were also reoriented, resulting in movement of α4 out of the major groove, by up to 8Å, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D). RESULTS +279 284 4-HPA chemical Specifically, in addition to the different inter-helical translational distances, the α4 helices in the holo-NadR homodimer were also reoriented, resulting in movement of α4 out of the major groove, by up to 8Å, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D). RESULTS +5 12 aligned experimental_method When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9Å), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above. RESULTS +18 22 OhrR protein When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9Å), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above. RESULTS +28 31 apo protein_state When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9Å), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above. RESULTS +32 41 homodimer oligomeric_state When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9Å), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above. RESULTS +42 44 CD structure_element When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9Å), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above. RESULTS +104 108 rmsd evidence When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9Å), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above. RESULTS +158 161 DNA chemical When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9Å), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above. RESULTS +226 228 AB structure_element When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9Å), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above. RESULTS +0 4 NadR protein NadR residues His7, Ser9, Asn11 and Phe25 are essential for regulation of NadA expression in vivo RESULTS +14 18 His7 residue_name_number NadR residues His7, Ser9, Asn11 and Phe25 are essential for regulation of NadA expression in vivo RESULTS +20 24 Ser9 residue_name_number NadR residues His7, Ser9, Asn11 and Phe25 are essential for regulation of NadA expression in vivo RESULTS +26 31 Asn11 residue_name_number NadR residues His7, Ser9, Asn11 and Phe25 are essential for regulation of NadA expression in vivo RESULTS +36 41 Phe25 residue_name_number NadR residues His7, Ser9, Asn11 and Phe25 are essential for regulation of NadA expression in vivo RESULTS +74 78 NadA protein NadR residues His7, Ser9, Asn11 and Phe25 are essential for regulation of NadA expression in vivo RESULTS +74 78 NadR protein While previous studies had correctly suggested the involvement of several NadR residues in ligand binding, the crystal structures presented here revealed additional residues with previously unknown roles in dimerization and/or binding to 4-HPA. RESULTS +111 129 crystal structures evidence While previous studies had correctly suggested the involvement of several NadR residues in ligand binding, the crystal structures presented here revealed additional residues with previously unknown roles in dimerization and/or binding to 4-HPA. RESULTS +238 243 4-HPA chemical While previous studies had correctly suggested the involvement of several NadR residues in ligand binding, the crystal structures presented here revealed additional residues with previously unknown roles in dimerization and/or binding to 4-HPA. RESULTS +99 103 NadR protein To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. RESULTS +113 116 H7A mutant To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. RESULTS +118 121 S9A mutant To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. RESULTS +123 127 N11A mutant To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. RESULTS +132 136 F25A mutant To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. RESULTS +189 199 MC58-Δ1843 mutant To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. RESULTS +200 204 nadR gene To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. RESULTS +210 216 mutant protein_state To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. RESULTS +258 267 wild-type protein_state To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. RESULTS +268 272 nadR gene To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. RESULTS +283 287 nadR gene To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. RESULTS +288 295 mutants protein_state To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-Δ1843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants. RESULTS +0 4 NadA protein NadA protein abundance levels were assessed by Western blotting to evaluate the ability of the NadR mutants to repress the nadA promoter, in the presence or absence of 4-HPA. RESULTS +47 63 Western blotting experimental_method NadA protein abundance levels were assessed by Western blotting to evaluate the ability of the NadR mutants to repress the nadA promoter, in the presence or absence of 4-HPA. RESULTS +95 99 NadR protein NadA protein abundance levels were assessed by Western blotting to evaluate the ability of the NadR mutants to repress the nadA promoter, in the presence or absence of 4-HPA. RESULTS +100 107 mutants protein_state NadA protein abundance levels were assessed by Western blotting to evaluate the ability of the NadR mutants to repress the nadA promoter, in the presence or absence of 4-HPA. RESULTS +123 127 nadA gene NadA protein abundance levels were assessed by Western blotting to evaluate the ability of the NadR mutants to repress the nadA promoter, in the presence or absence of 4-HPA. RESULTS +168 173 4-HPA chemical NadA protein abundance levels were assessed by Western blotting to evaluate the ability of the NadR mutants to repress the nadA promoter, in the presence or absence of 4-HPA. RESULTS +4 8 nadR gene The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +9 12 H7A mutant The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +14 17 S9A mutant The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +22 26 F25A mutant The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +75 79 nadA gene The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +129 133 nadA gene The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +160 164 NadR protein The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +165 167 WT protein_state The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +214 219 4-HPA chemical The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +248 257 wild-type protein_state The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +258 262 nadR gene The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +294 298 NadA protein The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +323 328 4-HPA chemical The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9). RESULTS +40 44 nadR gene Interestingly, and on the contrary, the nadR N11A complemented strain showed hypo-repression (i.e. exhibited high expression of nadA both in absence and presence of 4-HPA). RESULTS +45 49 N11A mutant Interestingly, and on the contrary, the nadR N11A complemented strain showed hypo-repression (i.e. exhibited high expression of nadA both in absence and presence of 4-HPA). RESULTS +128 132 nadA gene Interestingly, and on the contrary, the nadR N11A complemented strain showed hypo-repression (i.e. exhibited high expression of nadA both in absence and presence of 4-HPA). RESULTS +165 170 4-HPA chemical Interestingly, and on the contrary, the nadR N11A complemented strain showed hypo-repression (i.e. exhibited high expression of nadA both in absence and presence of 4-HPA). RESULTS +5 16 mutagenesis experimental_method This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo. RESULTS +36 40 NadR protein This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo. RESULTS +50 54 His7 residue_name_number This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo. RESULTS +56 60 Ser9 residue_name_number This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo. RESULTS +62 67 Asn11 residue_name_number This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo. RESULTS +72 77 Phe25 residue_name_number This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo. RESULTS +130 134 NadR protein This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo. RESULTS +198 202 nadA gene This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo. RESULTS +229 233 NadA protein This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo. RESULTS +249 254 4-HPA chemical This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo. RESULTS +0 31 Structure-based point mutations experimental_method Structure-based point mutations shed light on ligand-induced regulation of NadR. FIG +75 79 NadR protein Structure-based point mutations shed light on ligand-induced regulation of NadR. FIG +0 12 Western blot experimental_method Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. FIG +25 34 wild-type protein_state Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. FIG +36 38 WT protein_state Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. FIG +71 75 nadR gene Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. FIG +94 99 ΔNadR mutant Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. FIG +139 143 NadR protein Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. FIG +144 146 WT protein_state Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. FIG +150 156 mutant protein_state Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. FIG +279 284 4-HPA chemical Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. FIG +294 298 NadA protein Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. FIG +303 307 NadR protein Western blot analyses of wild-type (WT) strain (lanes 1–2) or isogenic nadR knockout strains (ΔNadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3–12) or not complemented (lanes 13–14), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression. FIG +19 24 ΔNadR mutant Complementation of ΔNadR with WT NadR enables induction of nadA expression by 4-HPA. FIG +30 32 WT protein_state Complementation of ΔNadR with WT NadR enables induction of nadA expression by 4-HPA. FIG +33 37 NadR protein Complementation of ΔNadR with WT NadR enables induction of nadA expression by 4-HPA. FIG +59 63 nadA gene Complementation of ΔNadR with WT NadR enables induction of nadA expression by 4-HPA. FIG +78 83 4-HPA chemical Complementation of ΔNadR with WT NadR enables induction of nadA expression by 4-HPA. FIG +4 7 H7A mutant The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +9 12 S9A mutant The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +17 21 F25A mutant The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +50 54 nadA gene The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +102 104 WT protein_state The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +105 109 NadR protein The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +115 119 N11A mutant The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +120 126 mutant protein_state The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +156 160 nadA gene The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +205 210 4-HPA chemical The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +249 262 meningococcal taxonomy_domain The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +263 287 factor H binding protein protein The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +289 293 fHbp protein The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control). FIG +0 4 NadA protein NadA is a surface-exposed meningococcal protein contributing to pathogenesis, and is one of three main antigens present in the vaccine Bexsero. DISCUSS +26 39 meningococcal taxonomy_domain NadA is a surface-exposed meningococcal protein contributing to pathogenesis, and is one of three main antigens present in the vaccine Bexsero. DISCUSS +55 59 nadA gene A detailed understanding of the in vitro repression of nadA expression by the transcriptional regulator NadR is important, both because it is a relevant disease-related model of how small-molecule ligands can regulate MarR family proteins and thereby impact bacterial virulence, and because nadA expression levels are linked to the prediction of vaccine coverage. DISCUSS +78 103 transcriptional regulator protein_type A detailed understanding of the in vitro repression of nadA expression by the transcriptional regulator NadR is important, both because it is a relevant disease-related model of how small-molecule ligands can regulate MarR family proteins and thereby impact bacterial virulence, and because nadA expression levels are linked to the prediction of vaccine coverage. DISCUSS +104 108 NadR protein A detailed understanding of the in vitro repression of nadA expression by the transcriptional regulator NadR is important, both because it is a relevant disease-related model of how small-molecule ligands can regulate MarR family proteins and thereby impact bacterial virulence, and because nadA expression levels are linked to the prediction of vaccine coverage. DISCUSS +218 222 MarR protein_type A detailed understanding of the in vitro repression of nadA expression by the transcriptional regulator NadR is important, both because it is a relevant disease-related model of how small-molecule ligands can regulate MarR family proteins and thereby impact bacterial virulence, and because nadA expression levels are linked to the prediction of vaccine coverage. DISCUSS +258 267 bacterial taxonomy_domain A detailed understanding of the in vitro repression of nadA expression by the transcriptional regulator NadR is important, both because it is a relevant disease-related model of how small-molecule ligands can regulate MarR family proteins and thereby impact bacterial virulence, and because nadA expression levels are linked to the prediction of vaccine coverage. DISCUSS +291 295 nadA gene A detailed understanding of the in vitro repression of nadA expression by the transcriptional regulator NadR is important, both because it is a relevant disease-related model of how small-molecule ligands can regulate MarR family proteins and thereby impact bacterial virulence, and because nadA expression levels are linked to the prediction of vaccine coverage. DISCUSS +27 31 NadR protein The repressive activity of NadR can be relieved by hydroxyphenylacetate (HPA) ligands, and HDX-MS studies previously indicated that 4-HPA stabilizes dimeric NadR in a configuration incompatible with DNA binding. DISCUSS +51 71 hydroxyphenylacetate chemical The repressive activity of NadR can be relieved by hydroxyphenylacetate (HPA) ligands, and HDX-MS studies previously indicated that 4-HPA stabilizes dimeric NadR in a configuration incompatible with DNA binding. DISCUSS +73 76 HPA chemical The repressive activity of NadR can be relieved by hydroxyphenylacetate (HPA) ligands, and HDX-MS studies previously indicated that 4-HPA stabilizes dimeric NadR in a configuration incompatible with DNA binding. DISCUSS +91 97 HDX-MS experimental_method The repressive activity of NadR can be relieved by hydroxyphenylacetate (HPA) ligands, and HDX-MS studies previously indicated that 4-HPA stabilizes dimeric NadR in a configuration incompatible with DNA binding. DISCUSS +132 137 4-HPA chemical The repressive activity of NadR can be relieved by hydroxyphenylacetate (HPA) ligands, and HDX-MS studies previously indicated that 4-HPA stabilizes dimeric NadR in a configuration incompatible with DNA binding. DISCUSS +149 156 dimeric oligomeric_state The repressive activity of NadR can be relieved by hydroxyphenylacetate (HPA) ligands, and HDX-MS studies previously indicated that 4-HPA stabilizes dimeric NadR in a configuration incompatible with DNA binding. DISCUSS +157 161 NadR protein The repressive activity of NadR can be relieved by hydroxyphenylacetate (HPA) ligands, and HDX-MS studies previously indicated that 4-HPA stabilizes dimeric NadR in a configuration incompatible with DNA binding. DISCUSS +84 88 MarR protein_type Despite these and other studies, the molecular mechanisms by which ligands regulate MarR family proteins are relatively poorly understood and likely differ depending on the specific ligand. DISCUSS +24 28 NadR protein Given the importance of NadR-mediated regulation of NadA levels in the contexts of meningococcal pathogenesis, we sought to characterize NadR, and its interaction with ligands, at atomic resolution. DISCUSS +52 56 NadA protein Given the importance of NadR-mediated regulation of NadA levels in the contexts of meningococcal pathogenesis, we sought to characterize NadR, and its interaction with ligands, at atomic resolution. DISCUSS +83 96 meningococcal taxonomy_domain Given the importance of NadR-mediated regulation of NadA levels in the contexts of meningococcal pathogenesis, we sought to characterize NadR, and its interaction with ligands, at atomic resolution. DISCUSS +137 141 NadR protein Given the importance of NadR-mediated regulation of NadA levels in the contexts of meningococcal pathogenesis, we sought to characterize NadR, and its interaction with ligands, at atomic resolution. DISCUSS +27 31 NadR protein Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +35 42 dimeric oligomeric_state Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +92 99 dimeric oligomeric_state Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +113 124 presence of protein_state Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +125 130 4-HPA chemical Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +163 172 monomeric oligomeric_state Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +207 212 4-HPA chemical Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +223 227 NadR protein Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +295 299 NadR protein Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +316 319 SEC experimental_method Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +324 341 mass spectrometry experimental_method Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +352 376 crystallographic studies experimental_method Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +398 402 MarR protein_type Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +418 425 dimeric oligomeric_state Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric. DISCUSS +13 55 structure-guided site-directed mutagenesis experimental_method We also used structure-guided site-directed mutagenesis to identify an important conserved residue, Leu130, which stabilizes the NadR dimer interface, knowledge of which may also inform future studies to explore the regulatory mechanisms of other MarR family proteins. DISCUSS +81 90 conserved protein_state We also used structure-guided site-directed mutagenesis to identify an important conserved residue, Leu130, which stabilizes the NadR dimer interface, knowledge of which may also inform future studies to explore the regulatory mechanisms of other MarR family proteins. DISCUSS +100 106 Leu130 residue_name_number We also used structure-guided site-directed mutagenesis to identify an important conserved residue, Leu130, which stabilizes the NadR dimer interface, knowledge of which may also inform future studies to explore the regulatory mechanisms of other MarR family proteins. DISCUSS +129 133 NadR protein We also used structure-guided site-directed mutagenesis to identify an important conserved residue, Leu130, which stabilizes the NadR dimer interface, knowledge of which may also inform future studies to explore the regulatory mechanisms of other MarR family proteins. DISCUSS +134 149 dimer interface site We also used structure-guided site-directed mutagenesis to identify an important conserved residue, Leu130, which stabilizes the NadR dimer interface, knowledge of which may also inform future studies to explore the regulatory mechanisms of other MarR family proteins. DISCUSS +247 251 MarR protein_type We also used structure-guided site-directed mutagenesis to identify an important conserved residue, Leu130, which stabilizes the NadR dimer interface, knowledge of which may also inform future studies to explore the regulatory mechanisms of other MarR family proteins. DISCUSS +13 43 assessed the thermal stability experimental_method Secondly, we assessed the thermal stability and unfolding of NadR in the presence or absence of ligands. DISCUSS +61 65 NadR protein Secondly, we assessed the thermal stability and unfolding of NadR in the presence or absence of ligands. DISCUSS +66 81 in the presence protein_state Secondly, we assessed the thermal stability and unfolding of NadR in the presence or absence of ligands. DISCUSS +85 95 absence of protein_state Secondly, we assessed the thermal stability and unfolding of NadR in the presence or absence of ligands. DISCUSS +4 7 DSC experimental_method All DSC profiles showed a single peak, suggesting that a single unfolding event simultaneously disrupted the dimer and the monomer. DISCUSS +8 16 profiles evidence All DSC profiles showed a single peak, suggesting that a single unfolding event simultaneously disrupted the dimer and the monomer. DISCUSS +109 114 dimer oligomeric_state All DSC profiles showed a single peak, suggesting that a single unfolding event simultaneously disrupted the dimer and the monomer. DISCUSS +123 130 monomer oligomeric_state All DSC profiles showed a single peak, suggesting that a single unfolding event simultaneously disrupted the dimer and the monomer. DISCUSS +52 56 NadR protein HPA ligands specifically increased the stability of NadR. The largest effects were induced by the naturally-occurring compounds 4-HPA and 3Cl,4-HPA, which, in SPR assays, were found to bind NadR with KD values of 1.5 mM and 1.1 mM, respectively. DISCUSS +128 133 4-HPA chemical HPA ligands specifically increased the stability of NadR. The largest effects were induced by the naturally-occurring compounds 4-HPA and 3Cl,4-HPA, which, in SPR assays, were found to bind NadR with KD values of 1.5 mM and 1.1 mM, respectively. DISCUSS +138 147 3Cl,4-HPA chemical HPA ligands specifically increased the stability of NadR. The largest effects were induced by the naturally-occurring compounds 4-HPA and 3Cl,4-HPA, which, in SPR assays, were found to bind NadR with KD values of 1.5 mM and 1.1 mM, respectively. DISCUSS +159 169 SPR assays experimental_method HPA ligands specifically increased the stability of NadR. The largest effects were induced by the naturally-occurring compounds 4-HPA and 3Cl,4-HPA, which, in SPR assays, were found to bind NadR with KD values of 1.5 mM and 1.1 mM, respectively. DISCUSS +190 194 NadR protein HPA ligands specifically increased the stability of NadR. The largest effects were induced by the naturally-occurring compounds 4-HPA and 3Cl,4-HPA, which, in SPR assays, were found to bind NadR with KD values of 1.5 mM and 1.1 mM, respectively. DISCUSS +200 202 KD evidence HPA ligands specifically increased the stability of NadR. The largest effects were induced by the naturally-occurring compounds 4-HPA and 3Cl,4-HPA, which, in SPR assays, were found to bind NadR with KD values of 1.5 mM and 1.1 mM, respectively. DISCUSS +15 19 NadR protein Although these NadR/HPA interactions appeared rather weak, their distinct affinities and specificities matched their in vitro effects and their biological relevance appears similar to previous proposals that certain small molecules, including some antibiotics, in the millimolar concentration range may be broad inhibitors of MarR family proteins. DISCUSS +20 23 HPA chemical Although these NadR/HPA interactions appeared rather weak, their distinct affinities and specificities matched their in vitro effects and their biological relevance appears similar to previous proposals that certain small molecules, including some antibiotics, in the millimolar concentration range may be broad inhibitors of MarR family proteins. DISCUSS +326 330 MarR protein_type Although these NadR/HPA interactions appeared rather weak, their distinct affinities and specificities matched their in vitro effects and their biological relevance appears similar to previous proposals that certain small molecules, including some antibiotics, in the millimolar concentration range may be broad inhibitors of MarR family proteins. DISCUSS +8 13 4-HPA chemical Indeed, 4-HPA is found in human saliva and 3Cl,4-HPA is produced during inflammatory processes, suggesting that these natural ligands are encountered by N. meningitidis in the mucosa of the oropharynx during infections. DISCUSS +26 31 human species Indeed, 4-HPA is found in human saliva and 3Cl,4-HPA is produced during inflammatory processes, suggesting that these natural ligands are encountered by N. meningitidis in the mucosa of the oropharynx during infections. DISCUSS +43 52 3Cl,4-HPA chemical Indeed, 4-HPA is found in human saliva and 3Cl,4-HPA is produced during inflammatory processes, suggesting that these natural ligands are encountered by N. meningitidis in the mucosa of the oropharynx during infections. DISCUSS +153 168 N. meningitidis species Indeed, 4-HPA is found in human saliva and 3Cl,4-HPA is produced during inflammatory processes, suggesting that these natural ligands are encountered by N. meningitidis in the mucosa of the oropharynx during infections. DISCUSS +25 29 NadR protein It is also possible that NadR responds to currently unidentified HPA analogues. DISCUSS +65 68 HPA chemical It is also possible that NadR responds to currently unidentified HPA analogues. DISCUSS +15 25 NadR/4-HPA complex_assembly Indeed, in the NadR/4-HPA complex there was a water molecule close to the carboxylate group and also a small unfilled tunnel ~5Å long, both factors suggesting that alternative larger ligands could occupy the pocket. DISCUSS +46 51 water chemical Indeed, in the NadR/4-HPA complex there was a water molecule close to the carboxylate group and also a small unfilled tunnel ~5Å long, both factors suggesting that alternative larger ligands could occupy the pocket. DISCUSS +118 124 tunnel site Indeed, in the NadR/4-HPA complex there was a water molecule close to the carboxylate group and also a small unfilled tunnel ~5Å long, both factors suggesting that alternative larger ligands could occupy the pocket. DISCUSS +55 59 NadR protein The ability to respond to various ligands might enable NadR in vivo to orchestrate multiple response mechanisms and modulate expression of genes other than nadA. Ultimately, confirmation of the relevance of each ligand will require a deeper understanding of the available concentration in vivo in the host niche during bacterial colonization and inflammation. DISCUSS +156 160 nadA gene The ability to respond to various ligands might enable NadR in vivo to orchestrate multiple response mechanisms and modulate expression of genes other than nadA. Ultimately, confirmation of the relevance of each ligand will require a deeper understanding of the available concentration in vivo in the host niche during bacterial colonization and inflammation. DISCUSS +319 328 bacterial taxonomy_domain The ability to respond to various ligands might enable NadR in vivo to orchestrate multiple response mechanisms and modulate expression of genes other than nadA. Ultimately, confirmation of the relevance of each ligand will require a deeper understanding of the available concentration in vivo in the host niche during bacterial colonization and inflammation. DISCUSS +30 48 crystal structures evidence Here, we determined the first crystal structures of apo-NadR and holo-NadR. These experimentally-determined structures enabled a new detailed characterization of the ligand-binding pocket. DISCUSS +52 55 apo protein_state Here, we determined the first crystal structures of apo-NadR and holo-NadR. These experimentally-determined structures enabled a new detailed characterization of the ligand-binding pocket. DISCUSS +56 60 NadR protein Here, we determined the first crystal structures of apo-NadR and holo-NadR. These experimentally-determined structures enabled a new detailed characterization of the ligand-binding pocket. DISCUSS +65 69 holo protein_state Here, we determined the first crystal structures of apo-NadR and holo-NadR. These experimentally-determined structures enabled a new detailed characterization of the ligand-binding pocket. DISCUSS +70 74 NadR protein Here, we determined the first crystal structures of apo-NadR and holo-NadR. These experimentally-determined structures enabled a new detailed characterization of the ligand-binding pocket. DISCUSS +108 118 structures evidence Here, we determined the first crystal structures of apo-NadR and holo-NadR. These experimentally-determined structures enabled a new detailed characterization of the ligand-binding pocket. DISCUSS +166 187 ligand-binding pocket site Here, we determined the first crystal structures of apo-NadR and holo-NadR. These experimentally-determined structures enabled a new detailed characterization of the ligand-binding pocket. DISCUSS +3 7 holo protein_state In holo-NadR, 4-HPA interacted directly with at least 11 polar and hydrophobic residues. DISCUSS +8 12 NadR protein In holo-NadR, 4-HPA interacted directly with at least 11 polar and hydrophobic residues. DISCUSS +14 19 4-HPA chemical In holo-NadR, 4-HPA interacted directly with at least 11 polar and hydrophobic residues. DISCUSS +73 91 homology modelling experimental_method Several, but not all, of these interactions were predicted previously by homology modelling combined with ligand docking in silico. DISCUSS +106 120 ligand docking experimental_method Several, but not all, of these interactions were predicted previously by homology modelling combined with ligand docking in silico. DISCUSS +58 62 His7 residue_name_number Subsequently, we established the functional importance of His7, Ser9, Asn11 and Phe25 in the in vitro response of meningococcus to 4-HPA, via site-directed mutagenesis. DISCUSS +64 68 Ser9 residue_name_number Subsequently, we established the functional importance of His7, Ser9, Asn11 and Phe25 in the in vitro response of meningococcus to 4-HPA, via site-directed mutagenesis. DISCUSS +70 75 Asn11 residue_name_number Subsequently, we established the functional importance of His7, Ser9, Asn11 and Phe25 in the in vitro response of meningococcus to 4-HPA, via site-directed mutagenesis. DISCUSS +80 85 Phe25 residue_name_number Subsequently, we established the functional importance of His7, Ser9, Asn11 and Phe25 in the in vitro response of meningococcus to 4-HPA, via site-directed mutagenesis. DISCUSS +114 127 meningococcus taxonomy_domain Subsequently, we established the functional importance of His7, Ser9, Asn11 and Phe25 in the in vitro response of meningococcus to 4-HPA, via site-directed mutagenesis. DISCUSS +131 136 4-HPA chemical Subsequently, we established the functional importance of His7, Ser9, Asn11 and Phe25 in the in vitro response of meningococcus to 4-HPA, via site-directed mutagenesis. DISCUSS +142 167 site-directed mutagenesis experimental_method Subsequently, we established the functional importance of His7, Ser9, Asn11 and Phe25 in the in vitro response of meningococcus to 4-HPA, via site-directed mutagenesis. DISCUSS +23 40 crystal structure evidence More unexpectedly, the crystal structure revealed that only one molecule of 4-HPA was bound per NadR dimer. DISCUSS +76 81 4-HPA chemical More unexpectedly, the crystal structure revealed that only one molecule of 4-HPA was bound per NadR dimer. DISCUSS +86 91 bound protein_state More unexpectedly, the crystal structure revealed that only one molecule of 4-HPA was bound per NadR dimer. DISCUSS +96 100 NadR protein More unexpectedly, the crystal structure revealed that only one molecule of 4-HPA was bound per NadR dimer. DISCUSS +101 106 dimer oligomeric_state More unexpectedly, the crystal structure revealed that only one molecule of 4-HPA was bound per NadR dimer. DISCUSS +50 53 SPR experimental_method We confirmed this stoichiometry in solution using SPR methods. DISCUSS +13 43 heteronuclear NMR spectroscopy experimental_method We also used heteronuclear NMR spectroscopy to detect substantial conformational changes of NadR occurring in solution upon addition of 4-HPA. DISCUSS +92 96 NadR protein We also used heteronuclear NMR spectroscopy to detect substantial conformational changes of NadR occurring in solution upon addition of 4-HPA. DISCUSS +136 141 4-HPA chemical We also used heteronuclear NMR spectroscopy to detect substantial conformational changes of NadR occurring in solution upon addition of 4-HPA. DISCUSS +10 13 NMR experimental_method Moreover, NMR spectra at 10°C suggested the existence of two distinct conformations of NadR in the vicinity of the ligand-binding pocket. DISCUSS +14 21 spectra evidence Moreover, NMR spectra at 10°C suggested the existence of two distinct conformations of NadR in the vicinity of the ligand-binding pocket. DISCUSS +87 91 NadR protein Moreover, NMR spectra at 10°C suggested the existence of two distinct conformations of NadR in the vicinity of the ligand-binding pocket. DISCUSS +115 136 ligand-binding pocket site Moreover, NMR spectra at 10°C suggested the existence of two distinct conformations of NadR in the vicinity of the ligand-binding pocket. DISCUSS +28 56 crystallographic observation evidence More powerfully, our unique crystallographic observation of this ‘occupied vs unoccupied site’ asymmetry in the NadR/4-HPA interaction is, to our knowledge, the first example reported for a MarR family protein. DISCUSS +66 74 occupied protein_state More powerfully, our unique crystallographic observation of this ‘occupied vs unoccupied site’ asymmetry in the NadR/4-HPA interaction is, to our knowledge, the first example reported for a MarR family protein. DISCUSS +78 88 unoccupied protein_state More powerfully, our unique crystallographic observation of this ‘occupied vs unoccupied site’ asymmetry in the NadR/4-HPA interaction is, to our knowledge, the first example reported for a MarR family protein. DISCUSS +112 122 NadR/4-HPA complex_assembly More powerfully, our unique crystallographic observation of this ‘occupied vs unoccupied site’ asymmetry in the NadR/4-HPA interaction is, to our knowledge, the first example reported for a MarR family protein. DISCUSS +190 194 MarR protein_type More powerfully, our unique crystallographic observation of this ‘occupied vs unoccupied site’ asymmetry in the NadR/4-HPA interaction is, to our knowledge, the first example reported for a MarR family protein. DISCUSS +0 19 Structural analyses experimental_method Structural analyses suggested that ‘inward’ side chain positions of Met22, Phe25 and especially Arg43 precluded binding of a second ligand molecule. DISCUSS +36 42 inward protein_state Structural analyses suggested that ‘inward’ side chain positions of Met22, Phe25 and especially Arg43 precluded binding of a second ligand molecule. DISCUSS +68 73 Met22 residue_name_number Structural analyses suggested that ‘inward’ side chain positions of Met22, Phe25 and especially Arg43 precluded binding of a second ligand molecule. DISCUSS +75 80 Phe25 residue_name_number Structural analyses suggested that ‘inward’ side chain positions of Met22, Phe25 and especially Arg43 precluded binding of a second ligand molecule. DISCUSS +96 101 Arg43 residue_name_number Structural analyses suggested that ‘inward’ side chain positions of Met22, Phe25 and especially Arg43 precluded binding of a second ligand molecule. DISCUSS +98 102 NadR protein Such a mechanism indicates negative cooperativity, which may enhance the ligand-responsiveness of NadR. DISCUSS +19 29 NadR/4-HPA complex_assembly Comparisons of the NadR/4-HPA complex with available MarR family/salicylate complexes revealed that 4-HPA has a previously unobserved binding mode. DISCUSS +53 57 MarR protein_type Comparisons of the NadR/4-HPA complex with available MarR family/salicylate complexes revealed that 4-HPA has a previously unobserved binding mode. DISCUSS +65 75 salicylate chemical Comparisons of the NadR/4-HPA complex with available MarR family/salicylate complexes revealed that 4-HPA has a previously unobserved binding mode. DISCUSS +100 105 4-HPA chemical Comparisons of the NadR/4-HPA complex with available MarR family/salicylate complexes revealed that 4-HPA has a previously unobserved binding mode. DISCUSS +16 38 M. thermoautotrophicum species Briefly, in the M. thermoautotrophicum MTH313 dimer, one molecule of salicylate binds in the pocket of each monomer, though with two rather different positions and orientations, only one of which (site-1) is thought to be biologically relevant (Fig 10A). DISCUSS +39 45 MTH313 protein Briefly, in the M. thermoautotrophicum MTH313 dimer, one molecule of salicylate binds in the pocket of each monomer, though with two rather different positions and orientations, only one of which (site-1) is thought to be biologically relevant (Fig 10A). DISCUSS +46 51 dimer oligomeric_state Briefly, in the M. thermoautotrophicum MTH313 dimer, one molecule of salicylate binds in the pocket of each monomer, though with two rather different positions and orientations, only one of which (site-1) is thought to be biologically relevant (Fig 10A). DISCUSS +69 79 salicylate chemical Briefly, in the M. thermoautotrophicum MTH313 dimer, one molecule of salicylate binds in the pocket of each monomer, though with two rather different positions and orientations, only one of which (site-1) is thought to be biologically relevant (Fig 10A). DISCUSS +93 99 pocket site Briefly, in the M. thermoautotrophicum MTH313 dimer, one molecule of salicylate binds in the pocket of each monomer, though with two rather different positions and orientations, only one of which (site-1) is thought to be biologically relevant (Fig 10A). DISCUSS +108 115 monomer oligomeric_state Briefly, in the M. thermoautotrophicum MTH313 dimer, one molecule of salicylate binds in the pocket of each monomer, though with two rather different positions and orientations, only one of which (site-1) is thought to be biologically relevant (Fig 10A). DISCUSS +197 203 site-1 site Briefly, in the M. thermoautotrophicum MTH313 dimer, one molecule of salicylate binds in the pocket of each monomer, though with two rather different positions and orientations, only one of which (site-1) is thought to be biologically relevant (Fig 10A). DISCUSS +7 18 S. tokodaii species In the S. tokodaii protein ST1710, salicylate binds to the same position in each monomer of the dimer, in a site equivalent to the putative biologically relevant site of MTH313 (Fig 10B). DISCUSS +27 33 ST1710 protein In the S. tokodaii protein ST1710, salicylate binds to the same position in each monomer of the dimer, in a site equivalent to the putative biologically relevant site of MTH313 (Fig 10B). DISCUSS +35 45 salicylate chemical In the S. tokodaii protein ST1710, salicylate binds to the same position in each monomer of the dimer, in a site equivalent to the putative biologically relevant site of MTH313 (Fig 10B). DISCUSS +81 88 monomer oligomeric_state In the S. tokodaii protein ST1710, salicylate binds to the same position in each monomer of the dimer, in a site equivalent to the putative biologically relevant site of MTH313 (Fig 10B). DISCUSS +96 101 dimer oligomeric_state In the S. tokodaii protein ST1710, salicylate binds to the same position in each monomer of the dimer, in a site equivalent to the putative biologically relevant site of MTH313 (Fig 10B). DISCUSS +170 176 MTH313 protein In the S. tokodaii protein ST1710, salicylate binds to the same position in each monomer of the dimer, in a site equivalent to the putative biologically relevant site of MTH313 (Fig 10B). DISCUSS +13 17 MarR protein_type Unlike other MarR family proteins which revealed multiple ligand binding interactions, we observed only 1 molecule of 4-HPA bound to NadR, suggesting a more specific and less promiscuous interaction. DISCUSS +118 123 4-HPA chemical Unlike other MarR family proteins which revealed multiple ligand binding interactions, we observed only 1 molecule of 4-HPA bound to NadR, suggesting a more specific and less promiscuous interaction. DISCUSS +124 132 bound to protein_state Unlike other MarR family proteins which revealed multiple ligand binding interactions, we observed only 1 molecule of 4-HPA bound to NadR, suggesting a more specific and less promiscuous interaction. DISCUSS +133 137 NadR protein Unlike other MarR family proteins which revealed multiple ligand binding interactions, we observed only 1 molecule of 4-HPA bound to NadR, suggesting a more specific and less promiscuous interaction. DISCUSS +3 7 NadR protein In NadR, the single molecule of 4-HPA binds in a position distinctly different from the salicylate binding site: translated by > 10 Å and with a 180° inverted orientation (Fig 10C). DISCUSS +32 37 4-HPA chemical In NadR, the single molecule of 4-HPA binds in a position distinctly different from the salicylate binding site: translated by > 10 Å and with a 180° inverted orientation (Fig 10C). DISCUSS +88 111 salicylate binding site site In NadR, the single molecule of 4-HPA binds in a position distinctly different from the salicylate binding site: translated by > 10 Å and with a 180° inverted orientation (Fig 10C). DISCUSS +0 4 NadR protein NadR shows a ligand binding site distinct from other MarR homologues. FIG +13 32 ligand binding site site NadR shows a ligand binding site distinct from other MarR homologues. FIG +53 57 MarR protein_type NadR shows a ligand binding site distinct from other MarR homologues. FIG +7 27 structural alignment experimental_method (A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7Å (indicated by black dotted line) and also by ligand orientation. FIG +31 37 MTH313 protein (A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7Å (indicated by black dotted line) and also by ligand orientation. FIG +38 52 chains A and B structure_element (A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7Å (indicated by black dotted line) and also by ligand orientation. FIG +64 74 salicylate chemical (A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7Å (indicated by black dotted line) and also by ligand orientation. FIG +78 83 bound protein_state (A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7Å (indicated by black dotted line) and also by ligand orientation. FIG +114 121 monomer oligomeric_state (A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7Å (indicated by black dotted line) and also by ligand orientation. FIG +123 129 site-1 site (A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7Å (indicated by black dotted line) and also by ligand orientation. FIG +181 187 site-2 site (A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7Å (indicated by black dotted line) and also by ligand orientation. FIG +6 26 structural alignment experimental_method (B) A structural alignment of MTH313 chain A and ST1710 (pink) (Cα rmsd 2.3Å), shows that they bind salicylate in equivalent sites (differing by only ~3Å) and with the same orientation. FIG +30 36 MTH313 protein (B) A structural alignment of MTH313 chain A and ST1710 (pink) (Cα rmsd 2.3Å), shows that they bind salicylate in equivalent sites (differing by only ~3Å) and with the same orientation. FIG +37 44 chain A structure_element (B) A structural alignment of MTH313 chain A and ST1710 (pink) (Cα rmsd 2.3Å), shows that they bind salicylate in equivalent sites (differing by only ~3Å) and with the same orientation. FIG +49 55 ST1710 protein (B) A structural alignment of MTH313 chain A and ST1710 (pink) (Cα rmsd 2.3Å), shows that they bind salicylate in equivalent sites (differing by only ~3Å) and with the same orientation. FIG +67 71 rmsd evidence (B) A structural alignment of MTH313 chain A and ST1710 (pink) (Cα rmsd 2.3Å), shows that they bind salicylate in equivalent sites (differing by only ~3Å) and with the same orientation. FIG +100 110 salicylate chemical (B) A structural alignment of MTH313 chain A and ST1710 (pink) (Cα rmsd 2.3Å), shows that they bind salicylate in equivalent sites (differing by only ~3Å) and with the same orientation. FIG +16 20 holo protein_state (C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by > 10 Å compared to salicylate, and adopts a novel orientation. FIG +21 25 NadR protein (C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by > 10 Å compared to salicylate, and adopts a novel orientation. FIG +27 34 chain B structure_element (C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by > 10 Å compared to salicylate, and adopts a novel orientation. FIG +49 58 alignment experimental_method (C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by > 10 Å compared to salicylate, and adopts a novel orientation. FIG +72 77 bound protein_state (C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by > 10 Å compared to salicylate, and adopts a novel orientation. FIG +78 83 4-HPA chemical (C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by > 10 Å compared to salicylate, and adopts a novel orientation. FIG +126 136 salicylate chemical (C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by > 10 Å compared to salicylate, and adopts a novel orientation. FIG +17 34 crystal structure evidence Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. DISCUSS +94 107 meningococcal taxonomy_domain Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. DISCUSS +121 125 NadR protein Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. DISCUSS +134 141 NMB1585 protein Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. DISCUSS +183 187 NadR protein Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. DISCUSS +197 207 structures evidence Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. DISCUSS +232 236 rmsd evidence Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. DISCUSS +249 256 NMB1585 protein Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. DISCUSS +286 290 HPAs chemical Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. DISCUSS +316 323 ‘pocket site Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 Å), but NMB1585 appears unsuited for binding HPAs, since its corresponding ‘pocket’ region is occupied by several bulky hydrophobic side chains. DISCUSS +26 30 MarR protein_type It can be speculated that MarR family members have evolved separately to engage distinct signaling molecules, thus enabling bacteria to use the overall conserved MarR scaffold to adapt and respond to diverse changing environmental stimuli experienced in their natural niches. DISCUSS +124 132 bacteria taxonomy_domain It can be speculated that MarR family members have evolved separately to engage distinct signaling molecules, thus enabling bacteria to use the overall conserved MarR scaffold to adapt and respond to diverse changing environmental stimuli experienced in their natural niches. DISCUSS +162 166 MarR protein_type It can be speculated that MarR family members have evolved separately to engage distinct signaling molecules, thus enabling bacteria to use the overall conserved MarR scaffold to adapt and respond to diverse changing environmental stimuli experienced in their natural niches. DISCUSS +41 45 MarR protein_type Alternatively, it is possible that other MarR homologues (e.g. NMB1585) may have no extant functional binding pocket and thus may have lost the ability to respond to a ligand, acting instead as constitutive DNA-binding regulatory proteins. DISCUSS +63 70 NMB1585 protein Alternatively, it is possible that other MarR homologues (e.g. NMB1585) may have no extant functional binding pocket and thus may have lost the ability to respond to a ligand, acting instead as constitutive DNA-binding regulatory proteins. DISCUSS +102 116 binding pocket site Alternatively, it is possible that other MarR homologues (e.g. NMB1585) may have no extant functional binding pocket and thus may have lost the ability to respond to a ligand, acting instead as constitutive DNA-binding regulatory proteins. DISCUSS +207 210 DNA chemical Alternatively, it is possible that other MarR homologues (e.g. NMB1585) may have no extant functional binding pocket and thus may have lost the ability to respond to a ligand, acting instead as constitutive DNA-binding regulatory proteins. DISCUSS +4 7 apo protein_state The apo-NadR crystal structures revealed two dimers with slightly different conformations, most divergent in the DNA-binding domain. DISCUSS +8 12 NadR protein The apo-NadR crystal structures revealed two dimers with slightly different conformations, most divergent in the DNA-binding domain. DISCUSS +13 31 crystal structures evidence The apo-NadR crystal structures revealed two dimers with slightly different conformations, most divergent in the DNA-binding domain. DISCUSS +45 51 dimers oligomeric_state The apo-NadR crystal structures revealed two dimers with slightly different conformations, most divergent in the DNA-binding domain. DISCUSS +113 131 DNA-binding domain structure_element The apo-NadR crystal structures revealed two dimers with slightly different conformations, most divergent in the DNA-binding domain. DISCUSS +24 41 crystal structure evidence It is not unusual for a crystal structure to reveal multiple copies of the same protein in very slightly different conformations, which are likely representative of the lowest-energy conformations sampled by the dynamic ensemble of molecular states occurring in solution, and which likely have only small energetic differences, as described previously for MexR (a MarR protein) or more recently for the solute-binding protein FhuD2. DISCUSS +356 360 MexR protein It is not unusual for a crystal structure to reveal multiple copies of the same protein in very slightly different conformations, which are likely representative of the lowest-energy conformations sampled by the dynamic ensemble of molecular states occurring in solution, and which likely have only small energetic differences, as described previously for MexR (a MarR protein) or more recently for the solute-binding protein FhuD2. DISCUSS +364 368 MarR protein_type It is not unusual for a crystal structure to reveal multiple copies of the same protein in very slightly different conformations, which are likely representative of the lowest-energy conformations sampled by the dynamic ensemble of molecular states occurring in solution, and which likely have only small energetic differences, as described previously for MexR (a MarR protein) or more recently for the solute-binding protein FhuD2. DISCUSS +403 425 solute-binding protein protein_type It is not unusual for a crystal structure to reveal multiple copies of the same protein in very slightly different conformations, which are likely representative of the lowest-energy conformations sampled by the dynamic ensemble of molecular states occurring in solution, and which likely have only small energetic differences, as described previously for MexR (a MarR protein) or more recently for the solute-binding protein FhuD2. DISCUSS +426 431 FhuD2 protein It is not unusual for a crystal structure to reveal multiple copies of the same protein in very slightly different conformations, which are likely representative of the lowest-energy conformations sampled by the dynamic ensemble of molecular states occurring in solution, and which likely have only small energetic differences, as described previously for MexR (a MarR protein) or more recently for the solute-binding protein FhuD2. DISCUSS +13 17 holo protein_state Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +18 22 NadR protein Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +23 32 structure evidence Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +73 76 apo protein_state Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +77 81 NadR protein Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +82 92 structures evidence Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +94 98 rmsd evidence Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +193 197 NadR protein Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +231 236 4-HPA chemical Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +357 363 active protein_state Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +368 376 inactive protein_state Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +430 434 NadR protein Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +457 460 DNA chemical Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3Å), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA. DISCUSS +19 22 apo protein_state Comparisons of the apo- and holo-NadR structures revealed that the largest differences occurred in the DNA-binding helix α4. DISCUSS +28 32 holo protein_state Comparisons of the apo- and holo-NadR structures revealed that the largest differences occurred in the DNA-binding helix α4. DISCUSS +33 37 NadR protein Comparisons of the apo- and holo-NadR structures revealed that the largest differences occurred in the DNA-binding helix α4. DISCUSS +38 48 structures evidence Comparisons of the apo- and holo-NadR structures revealed that the largest differences occurred in the DNA-binding helix α4. DISCUSS +103 106 DNA chemical Comparisons of the apo- and holo-NadR structures revealed that the largest differences occurred in the DNA-binding helix α4. DISCUSS +115 120 helix structure_element Comparisons of the apo- and holo-NadR structures revealed that the largest differences occurred in the DNA-binding helix α4. DISCUSS +121 123 α4 structure_element Comparisons of the apo- and holo-NadR structures revealed that the largest differences occurred in the DNA-binding helix α4. DISCUSS +13 18 helix structure_element The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. DISCUSS +19 21 α4 structure_element The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. DISCUSS +25 29 holo protein_state The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. DISCUSS +30 34 NadR protein The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. DISCUSS +81 96 dimer interface site The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. DISCUSS +108 115 helices structure_element The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. DISCUSS +116 118 α1 structure_element The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. DISCUSS +120 122 α5 structure_element The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. DISCUSS +128 130 α6 structure_element The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. DISCUSS +141 145 holo protein_state The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. DISCUSS +178 181 DNA chemical The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. DISCUSS +219 228 OhrR:ohrA complex_assembly The shift of helix α4 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices α1, α5, and α6, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex. DISCUSS +26 31 helix structure_element While some flexibility of helix α4 was also observed in the two apo-structures, concomitant changes in the dimer interfaces were not observed, possibly due to the absence of ligand. DISCUSS +32 34 α4 structure_element While some flexibility of helix α4 was also observed in the two apo-structures, concomitant changes in the dimer interfaces were not observed, possibly due to the absence of ligand. DISCUSS +64 67 apo protein_state While some flexibility of helix α4 was also observed in the two apo-structures, concomitant changes in the dimer interfaces were not observed, possibly due to the absence of ligand. DISCUSS +68 78 structures evidence While some flexibility of helix α4 was also observed in the two apo-structures, concomitant changes in the dimer interfaces were not observed, possibly due to the absence of ligand. DISCUSS +107 123 dimer interfaces site While some flexibility of helix α4 was also observed in the two apo-structures, concomitant changes in the dimer interfaces were not observed, possibly due to the absence of ligand. DISCUSS +163 180 absence of ligand protein_state While some flexibility of helix α4 was also observed in the two apo-structures, concomitant changes in the dimer interfaces were not observed, possibly due to the absence of ligand. DISCUSS +32 35 apo protein_state One of the two conformations of apo-NadR appeared ideally suited for DNA-binding. DISCUSS +36 40 NadR protein One of the two conformations of apo-NadR appeared ideally suited for DNA-binding. DISCUSS +69 72 DNA chemical One of the two conformations of apo-NadR appeared ideally suited for DNA-binding. DISCUSS +41 44 apo protein_state Overall, these analyses suggest that the apo-NadR dimer has a pre-existing equilibrium that samples a variety of conformations, some of which are compatible with DNA binding. DISCUSS +45 49 NadR protein Overall, these analyses suggest that the apo-NadR dimer has a pre-existing equilibrium that samples a variety of conformations, some of which are compatible with DNA binding. DISCUSS +50 55 dimer oligomeric_state Overall, these analyses suggest that the apo-NadR dimer has a pre-existing equilibrium that samples a variety of conformations, some of which are compatible with DNA binding. DISCUSS +162 165 DNA chemical Overall, these analyses suggest that the apo-NadR dimer has a pre-existing equilibrium that samples a variety of conformations, some of which are compatible with DNA binding. DISCUSS +43 47 NadR protein The noted flexibility may also explain how NadR can adapt to bind various DNA target sequences with slightly different structural features. DISCUSS +74 77 DNA chemical The noted flexibility may also explain how NadR can adapt to bind various DNA target sequences with slightly different structural features. DISCUSS +35 39 holo protein_state Subsequently, upon ligand binding, holo-NadR adopts a structure less suited for DNA-binding and this conformation is selected and stabilized by a network of protein-ligand interactions and concomitant rearrangements at the NadR holo dimer interface. DISCUSS +40 44 NadR protein Subsequently, upon ligand binding, holo-NadR adopts a structure less suited for DNA-binding and this conformation is selected and stabilized by a network of protein-ligand interactions and concomitant rearrangements at the NadR holo dimer interface. DISCUSS +80 83 DNA chemical Subsequently, upon ligand binding, holo-NadR adopts a structure less suited for DNA-binding and this conformation is selected and stabilized by a network of protein-ligand interactions and concomitant rearrangements at the NadR holo dimer interface. DISCUSS +223 227 NadR protein Subsequently, upon ligand binding, holo-NadR adopts a structure less suited for DNA-binding and this conformation is selected and stabilized by a network of protein-ligand interactions and concomitant rearrangements at the NadR holo dimer interface. DISCUSS +228 232 holo protein_state Subsequently, upon ligand binding, holo-NadR adopts a structure less suited for DNA-binding and this conformation is selected and stabilized by a network of protein-ligand interactions and concomitant rearrangements at the NadR holo dimer interface. DISCUSS +233 248 dimer interface site Subsequently, upon ligand binding, holo-NadR adopts a structure less suited for DNA-binding and this conformation is selected and stabilized by a network of protein-ligand interactions and concomitant rearrangements at the NadR holo dimer interface. DISCUSS +64 74 salicylate chemical In an alternative and less extensive manner, the binding of two salicylate molecules to the M. thermoautotrophicum protein MTH313 appeared to induce large changes in the wHTH domain, which was associated with reduced DNA-binding activity. DISCUSS +92 114 M. thermoautotrophicum species In an alternative and less extensive manner, the binding of two salicylate molecules to the M. thermoautotrophicum protein MTH313 appeared to induce large changes in the wHTH domain, which was associated with reduced DNA-binding activity. DISCUSS +123 129 MTH313 protein In an alternative and less extensive manner, the binding of two salicylate molecules to the M. thermoautotrophicum protein MTH313 appeared to induce large changes in the wHTH domain, which was associated with reduced DNA-binding activity. DISCUSS +170 181 wHTH domain structure_element In an alternative and less extensive manner, the binding of two salicylate molecules to the M. thermoautotrophicum protein MTH313 appeared to induce large changes in the wHTH domain, which was associated with reduced DNA-binding activity. DISCUSS +31 49 crystal structures evidence Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +57 77 transcription factor protein_type Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +79 83 NadR protein Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +119 132 meningococcal taxonomy_domain Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +187 191 NadA protein Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +202 221 structural analyses experimental_method Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +295 299 NadR protein Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +336 349 meningococcal taxonomy_domain Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +369 373 NadR protein Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +385 389 nadA gene Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +405 409 NadR protein Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +493 497 mafA gene Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +534 538 NadR protein Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +596 601 4-HPA chemical Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family–i.e. NadR represses these genes in the presence but not absence of 4-HPA. DISCUSS +98 114 highly conserved protein_state The latter may influence the surface abundance or secretion of maf proteins, an emerging class of highly conserved meningococcal putative adhesins and toxins with many important roles. DISCUSS +115 128 meningococcal taxonomy_domain The latter may influence the surface abundance or secretion of maf proteins, an emerging class of highly conserved meningococcal putative adhesins and toxins with many important roles. DISCUSS +116 120 NadR protein Further work is required to investigate how the two different promoter types influence the ligand-responsiveness of NadR during bacterial infection and may provide insights into the regulatory mechanisms occurring during these host-pathogen interactions. DISCUSS +128 137 bacterial taxonomy_domain Further work is required to investigate how the two different promoter types influence the ligand-responsiveness of NadR during bacterial infection and may provide insights into the regulatory mechanisms occurring during these host-pathogen interactions. DISCUSS +58 62 NadR protein Ultimately, knowledge of the ligand-dependent activity of NadR will continue to deepen our understanding of nadA expression levels, which influence meningococcal pathogenesis. DISCUSS +108 112 nadA gene Ultimately, knowledge of the ligand-dependent activity of NadR will continue to deepen our understanding of nadA expression levels, which influence meningococcal pathogenesis. DISCUSS +148 161 meningococcal taxonomy_domain Ultimately, knowledge of the ligand-dependent activity of NadR will continue to deepen our understanding of nadA expression levels, which influence meningococcal pathogenesis. DISCUSS +21 25 ohrA gene Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of the MarR family REF +17 24 NMB1585 protein The structure of NMB1585, a MarR-family regulator from Neisseria meningitidis REF diff --git a/annotation_CSV/PMC4848090.csv b/annotation_CSV/PMC4848090.csv new file mode 100644 index 0000000000000000000000000000000000000000..b105314d842f980d81f87a51ada9f9df70e11e1d --- /dev/null +++ b/annotation_CSV/PMC4848090.csv @@ -0,0 +1,999 @@ +anno_start anno_end anno_text entity_type sentence section +27 42 peptide hormone protein_type Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission TITLE +0 6 Plants taxonomy_domain Plants constantly renew during their life cycle and thus require to shed senescent and damaged organs. ABSTRACT +39 74 leucine-rich repeat receptor kinase protein_type Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. ABSTRACT +76 82 LRR-RK protein_type Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. ABSTRACT +84 89 HAESA protein Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. ABSTRACT +98 113 peptide hormone protein_type Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. ABSTRACT +114 117 IDA protein Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. ABSTRACT +32 35 IDA protein It is unknown how expression of IDA in the abscission zone leads to HAESA activation. ABSTRACT +68 73 HAESA protein It is unknown how expression of IDA in the abscission zone leads to HAESA activation. ABSTRACT +18 21 IDA protein Here we show that IDA is sensed directly by the HAESA ectodomain. ABSTRACT +48 53 HAESA protein Here we show that IDA is sensed directly by the HAESA ectodomain. ABSTRACT +54 64 ectodomain structure_element Here we show that IDA is sensed directly by the HAESA ectodomain. ABSTRACT +0 18 Crystal structures evidence Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. ABSTRACT +22 27 HAESA protein Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. ABSTRACT +28 43 in complex with protein_state Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. ABSTRACT +44 47 IDA protein Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. ABSTRACT +57 79 hormone binding pocket site Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. ABSTRACT +101 107 active protein_state Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. ABSTRACT +108 117 dodecamer structure_element Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. ABSTRACT +118 125 peptide chemical Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. ABSTRACT +10 24 hydroxyproline residue_name A central hydroxyproline residue anchors IDA to the receptor. ABSTRACT +41 44 IDA protein A central hydroxyproline residue anchors IDA to the receptor. ABSTRACT +4 9 HAESA protein The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. ABSTRACT +10 21 co-receptor protein_type The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. ABSTRACT +22 27 SERK1 protein The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. ABSTRACT +124 139 peptide hormone protein_type The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. ABSTRACT +157 174 Arg-His-Asn motif structure_element The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. ABSTRACT +178 181 IDA protein The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. ABSTRACT +25 34 conserved protein_state This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism. ABSTRACT +49 54 plant taxonomy_domain This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism. ABSTRACT +55 63 peptides chemical This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism. ABSTRACT +81 86 plant taxonomy_domain This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism. ABSTRACT +87 112 peptide hormone receptors protein_type This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism. ABSTRACT +0 6 Plants taxonomy_domain Plants can shed their leaves, flowers or other organs when they no longer need them. But how does a leaf or a flower know when to let go? A receptor protein called HAESA is found on the surface of the cells that surround a future break point on the plant. When its time to shed an organ, a hormone called IDA instructs HAESA to trigger the shedding process. ABSTRACT +140 156 receptor protein protein_type Plants can shed their leaves, flowers or other organs when they no longer need them. But how does a leaf or a flower know when to let go? A receptor protein called HAESA is found on the surface of the cells that surround a future break point on the plant. When its time to shed an organ, a hormone called IDA instructs HAESA to trigger the shedding process. ABSTRACT +164 169 HAESA protein Plants can shed their leaves, flowers or other organs when they no longer need them. But how does a leaf or a flower know when to let go? A receptor protein called HAESA is found on the surface of the cells that surround a future break point on the plant. When its time to shed an organ, a hormone called IDA instructs HAESA to trigger the shedding process. ABSTRACT +290 297 hormone chemical Plants can shed their leaves, flowers or other organs when they no longer need them. But how does a leaf or a flower know when to let go? A receptor protein called HAESA is found on the surface of the cells that surround a future break point on the plant. When its time to shed an organ, a hormone called IDA instructs HAESA to trigger the shedding process. ABSTRACT +305 308 IDA protein Plants can shed their leaves, flowers or other organs when they no longer need them. But how does a leaf or a flower know when to let go? A receptor protein called HAESA is found on the surface of the cells that surround a future break point on the plant. When its time to shed an organ, a hormone called IDA instructs HAESA to trigger the shedding process. ABSTRACT +319 324 HAESA protein Plants can shed their leaves, flowers or other organs when they no longer need them. But how does a leaf or a flower know when to let go? A receptor protein called HAESA is found on the surface of the cells that surround a future break point on the plant. When its time to shed an organ, a hormone called IDA instructs HAESA to trigger the shedding process. ABSTRACT +38 41 IDA protein However, the molecular details of how IDA triggers organ shedding are not clear. ABSTRACT +75 80 plant taxonomy_domain The shedding of floral organs (or leaves) can be easily studied in a model plant called Arabidopsis. ABSTRACT +88 99 Arabidopsis taxonomy_domain The shedding of floral organs (or leaves) can be easily studied in a model plant called Arabidopsis. ABSTRACT +21 41 protein biochemistry experimental_method Santiago et al. used protein biochemistry, structural biology and genetics to uncover how the IDA hormone activates HAESA. ABSTRACT +43 61 structural biology experimental_method Santiago et al. used protein biochemistry, structural biology and genetics to uncover how the IDA hormone activates HAESA. ABSTRACT +66 74 genetics experimental_method Santiago et al. used protein biochemistry, structural biology and genetics to uncover how the IDA hormone activates HAESA. ABSTRACT +94 97 IDA protein Santiago et al. used protein biochemistry, structural biology and genetics to uncover how the IDA hormone activates HAESA. ABSTRACT +98 105 hormone chemical Santiago et al. used protein biochemistry, structural biology and genetics to uncover how the IDA hormone activates HAESA. ABSTRACT +116 121 HAESA protein Santiago et al. used protein biochemistry, structural biology and genetics to uncover how the IDA hormone activates HAESA. ABSTRACT +26 29 IDA protein The experiments show that IDA binds directly to a canyon shaped pocket in HAESA that extends out from the surface of the cell. ABSTRACT +30 47 binds directly to protein_state The experiments show that IDA binds directly to a canyon shaped pocket in HAESA that extends out from the surface of the cell. ABSTRACT +50 63 canyon shaped protein_state The experiments show that IDA binds directly to a canyon shaped pocket in HAESA that extends out from the surface of the cell. ABSTRACT +64 70 pocket site The experiments show that IDA binds directly to a canyon shaped pocket in HAESA that extends out from the surface of the cell. ABSTRACT +74 79 HAESA protein The experiments show that IDA binds directly to a canyon shaped pocket in HAESA that extends out from the surface of the cell. ABSTRACT +0 3 IDA protein IDA binding to HAESA allows another receptor protein called SERK1 to bind to HAESA, which results in the release of signals inside the cell that trigger the shedding of organs. ABSTRACT +15 20 HAESA protein IDA binding to HAESA allows another receptor protein called SERK1 to bind to HAESA, which results in the release of signals inside the cell that trigger the shedding of organs. ABSTRACT +36 52 receptor protein protein_type IDA binding to HAESA allows another receptor protein called SERK1 to bind to HAESA, which results in the release of signals inside the cell that trigger the shedding of organs. ABSTRACT +60 65 SERK1 protein IDA binding to HAESA allows another receptor protein called SERK1 to bind to HAESA, which results in the release of signals inside the cell that trigger the shedding of organs. ABSTRACT +66 76 to bind to protein_state IDA binding to HAESA allows another receptor protein called SERK1 to bind to HAESA, which results in the release of signals inside the cell that trigger the shedding of organs. ABSTRACT +77 82 HAESA protein IDA binding to HAESA allows another receptor protein called SERK1 to bind to HAESA, which results in the release of signals inside the cell that trigger the shedding of organs. ABSTRACT +90 93 IDA protein The next step following on from this work is to understand what signals are produced when IDA activates HAESA. ABSTRACT +104 109 HAESA protein The next step following on from this work is to understand what signals are produced when IDA activates HAESA. ABSTRACT +44 47 IDA protein Another challenge will be to find out where IDA is produced in the plant and what causes it to accumulate in specific places in preparation for organ shedding. ABSTRACT +67 72 plant taxonomy_domain Another challenge will be to find out where IDA is produced in the plant and what causes it to accumulate in specific places in preparation for organ shedding. ABSTRACT +4 9 HAESA protein The HAESA ectodomain folds into a superhelical assembly of 21 leucine-rich repeats. FIG +10 20 ectodomain structure_element The HAESA ectodomain folds into a superhelical assembly of 21 leucine-rich repeats. FIG +34 55 superhelical assembly structure_element The HAESA ectodomain folds into a superhelical assembly of 21 leucine-rich repeats. FIG +62 82 leucine-rich repeats structure_element The HAESA ectodomain folds into a superhelical assembly of 21 leucine-rich repeats. FIG +4 12 SDS PAGE experimental_method (A) SDS PAGE analysis of the purified Arabidopsis thaliana HAESA ectodomain (residues 20–620) obtained by secreted expression in insect cells. FIG +38 58 Arabidopsis thaliana species (A) SDS PAGE analysis of the purified Arabidopsis thaliana HAESA ectodomain (residues 20–620) obtained by secreted expression in insect cells. FIG +59 64 HAESA protein (A) SDS PAGE analysis of the purified Arabidopsis thaliana HAESA ectodomain (residues 20–620) obtained by secreted expression in insect cells. FIG +65 75 ectodomain structure_element (A) SDS PAGE analysis of the purified Arabidopsis thaliana HAESA ectodomain (residues 20–620) obtained by secreted expression in insect cells. FIG +86 92 20–620 residue_range (A) SDS PAGE analysis of the purified Arabidopsis thaliana HAESA ectodomain (residues 20–620) obtained by secreted expression in insect cells. FIG +106 141 secreted expression in insect cells experimental_method (A) SDS PAGE analysis of the purified Arabidopsis thaliana HAESA ectodomain (residues 20–620) obtained by secreted expression in insect cells. FIG +81 98 mass spectrometry experimental_method The calculated molecular mass is 65.7 kDa, the actual molecular mass obtained by mass spectrometry is 74,896 Da, accounting for the N-glycans. (B) Ribbon diagrams showing front (left panel) and side views (right panel) of the isolated HAESA LRR domain. FIG +132 141 N-glycans chemical The calculated molecular mass is 65.7 kDa, the actual molecular mass obtained by mass spectrometry is 74,896 Da, accounting for the N-glycans. (B) Ribbon diagrams showing front (left panel) and side views (right panel) of the isolated HAESA LRR domain. FIG +235 240 HAESA protein The calculated molecular mass is 65.7 kDa, the actual molecular mass obtained by mass spectrometry is 74,896 Da, accounting for the N-glycans. (B) Ribbon diagrams showing front (left panel) and side views (right panel) of the isolated HAESA LRR domain. FIG +241 251 LRR domain structure_element The calculated molecular mass is 65.7 kDa, the actual molecular mass obtained by mass spectrometry is 74,896 Da, accounting for the N-glycans. (B) Ribbon diagrams showing front (left panel) and side views (right panel) of the isolated HAESA LRR domain. FIG +17 22 20–88 residue_range The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +49 56 593–615 residue_range The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +58 73 capping domains structure_element The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +110 120 LRR motifs structure_element The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +137 153 disulphide bonds ptm The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +210 244 Structure based sequence alignment experimental_method The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +255 275 leucine-rich repeats structure_element The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +279 284 HAESA protein The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +294 299 plant taxonomy_domain The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +300 303 LRR structure_element The N- (residues 20–88) and C-terminal (residues 593–615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +0 9 Conserved protein_state Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. FIG +10 21 hydrophobic protein_state Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. FIG +22 30 residues structure_element Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. FIG +51 72 N-glycosylation sites site Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. FIG +88 98 structures evidence Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. FIG +124 132 cysteine residue_name Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. FIG +154 171 disulphide bridge ptm Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. FIG +196 214 Asn-linked glycans ptm Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. FIG +250 255 HAESA protein Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. FIG +256 266 ectodomain structure_element Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain. FIG +0 12 Oligomannose chemical Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. FIG +45 64 N-actylglucosamines chemical Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. FIG +84 91 mannose chemical Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. FIG +111 126 Trichoplusia ni species Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. FIG +140 146 plants taxonomy_domain Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. FIG +175 194 glycosylation sites site Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. FIG +211 216 HAESA protein Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. FIG +217 227 structures evidence Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. FIG +286 298 carbohydrate chemical Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate. FIG +4 9 HAESA protein The HAESA ectodomain is shown in blue (in surface representation), the glycan structures are shown in yellow. FIG +10 20 ectodomain structure_element The HAESA ectodomain is shown in blue (in surface representation), the glycan structures are shown in yellow. FIG +71 77 glycan chemical The HAESA ectodomain is shown in blue (in surface representation), the glycan structures are shown in yellow. FIG +0 20 Hydrophobic contacts bond_interaction Hydrophobic contacts and a hydrogen-bond network mediate the interaction between HAESA and the peptide hormone IDA. FIG +27 48 hydrogen-bond network site Hydrophobic contacts and a hydrogen-bond network mediate the interaction between HAESA and the peptide hormone IDA. FIG +81 86 HAESA protein Hydrophobic contacts and a hydrogen-bond network mediate the interaction between HAESA and the peptide hormone IDA. FIG +95 110 peptide hormone protein_type Hydrophobic contacts and a hydrogen-bond network mediate the interaction between HAESA and the peptide hormone IDA. FIG +111 114 IDA protein Hydrophobic contacts and a hydrogen-bond network mediate the interaction between HAESA and the peptide hormone IDA. FIG +19 37 IDA binding pocket site (A) Details of the IDA binding pocket. FIG +0 5 HAESA protein HAESA is shown in blue (ribbon diagram), the C-terminal Arg-His-Asn motif (left panel), the central Hyp anchor (center) and the N-terminal Pro-rich motif in IDA (right panel) are shown in yellow (in bonds representation). FIG +56 73 Arg-His-Asn motif structure_element HAESA is shown in blue (ribbon diagram), the C-terminal Arg-His-Asn motif (left panel), the central Hyp anchor (center) and the N-terminal Pro-rich motif in IDA (right panel) are shown in yellow (in bonds representation). FIG +100 110 Hyp anchor structure_element HAESA is shown in blue (ribbon diagram), the C-terminal Arg-His-Asn motif (left panel), the central Hyp anchor (center) and the N-terminal Pro-rich motif in IDA (right panel) are shown in yellow (in bonds representation). FIG +139 153 Pro-rich motif structure_element HAESA is shown in blue (ribbon diagram), the C-terminal Arg-His-Asn motif (left panel), the central Hyp anchor (center) and the N-terminal Pro-rich motif in IDA (right panel) are shown in yellow (in bonds representation). FIG +157 160 IDA protein HAESA is shown in blue (ribbon diagram), the C-terminal Arg-His-Asn motif (left panel), the central Hyp anchor (center) and the N-terminal Pro-rich motif in IDA (right panel) are shown in yellow (in bonds representation). FIG +0 24 HAESA interface residues site HAESA interface residues are shown as sticks, selected hydrogen bond interactions are denoted as dotted lines (in magenta). (B) View of the complete IDA (in bonds representation, in yellow) binding pocket in HAESA (surface view, in blue). FIG +55 81 hydrogen bond interactions bond_interaction HAESA interface residues are shown as sticks, selected hydrogen bond interactions are denoted as dotted lines (in magenta). (B) View of the complete IDA (in bonds representation, in yellow) binding pocket in HAESA (surface view, in blue). FIG +149 152 IDA protein HAESA interface residues are shown as sticks, selected hydrogen bond interactions are denoted as dotted lines (in magenta). (B) View of the complete IDA (in bonds representation, in yellow) binding pocket in HAESA (surface view, in blue). FIG +190 204 binding pocket site HAESA interface residues are shown as sticks, selected hydrogen bond interactions are denoted as dotted lines (in magenta). (B) View of the complete IDA (in bonds representation, in yellow) binding pocket in HAESA (surface view, in blue). FIG +208 213 HAESA protein HAESA interface residues are shown as sticks, selected hydrogen bond interactions are denoted as dotted lines (in magenta). (B) View of the complete IDA (in bonds representation, in yellow) binding pocket in HAESA (surface view, in blue). FIG +27 61 Structure based sequence alignment experimental_method Orientation as in (A). (C) Structure based sequence alignment of leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +65 85 leucine-rich repeats structure_element Orientation as in (A). (C) Structure based sequence alignment of leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +89 94 HAESA protein Orientation as in (A). (C) Structure based sequence alignment of leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +104 109 plant taxonomy_domain Orientation as in (A). (C) Structure based sequence alignment of leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +110 113 LRR structure_element Orientation as in (A). (C) Structure based sequence alignment of leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +114 132 consensus sequence evidence Orientation as in (A). (C) Structure based sequence alignment of leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison. FIG +19 43 hydrophobic interactions bond_interaction Residues mediating hydrophobic interactions with the IDA peptide are highlighted in blue, residues contributing to hydrogen bond interactions and/or salt bridges are shown in red. FIG +53 64 IDA peptide chemical Residues mediating hydrophobic interactions with the IDA peptide are highlighted in blue, residues contributing to hydrogen bond interactions and/or salt bridges are shown in red. FIG +115 141 hydrogen bond interactions bond_interaction Residues mediating hydrophobic interactions with the IDA peptide are highlighted in blue, residues contributing to hydrogen bond interactions and/or salt bridges are shown in red. FIG +149 161 salt bridges bond_interaction Residues mediating hydrophobic interactions with the IDA peptide are highlighted in blue, residues contributing to hydrogen bond interactions and/or salt bridges are shown in red. FIG +4 22 IDA binding pocket site The IDA binding pocket covers LRRs 2–14 and all residues originate from the inner surface of the HAESA superhelix. FIG +30 39 LRRs 2–14 structure_element The IDA binding pocket covers LRRs 2–14 and all residues originate from the inner surface of the HAESA superhelix. FIG +97 102 HAESA protein The IDA binding pocket covers LRRs 2–14 and all residues originate from the inner surface of the HAESA superhelix. FIG +103 113 superhelix structure_element The IDA binding pocket covers LRRs 2–14 and all residues originate from the inner surface of the HAESA superhelix. FIG +4 13 IDA-HAESA complex_assembly The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like proteins from different plant species. FIG +18 29 SERK1-HAESA complex_assembly The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like proteins from different plant species. FIG +38 48 interfaces site The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like proteins from different plant species. FIG +53 62 conserved protein_state The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like proteins from different plant species. FIG +69 74 HAESA protein The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like proteins from different plant species. FIG +79 98 HAESA-like proteins protein_type The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like proteins from different plant species. FIG +114 119 plant taxonomy_domain The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like proteins from different plant species. FIG +0 34 Structure-based sequence alignment experimental_method Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). FIG +42 62 HAESA family members protein_type Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). FIG +64 84 Arabidopsis thaliana species Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). FIG +85 90 HAESA protein Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). FIG +137 157 Arabidopsis thaliana species Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). FIG +158 162 HSL2 protein Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). FIG +184 200 Capsella rubella species Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). FIG +201 206 HAESA protein Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). FIG +228 245 Citrus clementina species Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). FIG +246 250 HSL2 protein Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). FIG +272 286 Vitis vinifera species Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). FIG +287 292 HAESA protein Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39). FIG +151 155 caps structure_element The alignment includes a secondary structure assignment calculated with the program DSSP and colored according to Figure 1, with the N- and C-terminal caps and the 21 LRR motifs indicated in orange and blue, respectively. FIG +167 177 LRR motifs structure_element The alignment includes a secondary structure assignment calculated with the program DSSP and colored according to Figure 1, with the N- and C-terminal caps and the 21 LRR motifs indicated in orange and blue, respectively. FIG +0 8 Cysteine residue_name Cysteine residues engaged in disulphide bonds are depicted in green. FIG +29 45 disulphide bonds ptm Cysteine residues engaged in disulphide bonds are depicted in green. FIG +0 5 HAESA protein HAESA residues interacting with the IDA peptide and/or the SERK1 co-receptor kinase ectodomain are highlighted in blue and orange, respectively. FIG +36 47 IDA peptide chemical HAESA residues interacting with the IDA peptide and/or the SERK1 co-receptor kinase ectodomain are highlighted in blue and orange, respectively. FIG +59 64 SERK1 protein HAESA residues interacting with the IDA peptide and/or the SERK1 co-receptor kinase ectodomain are highlighted in blue and orange, respectively. FIG +65 83 co-receptor kinase protein_type HAESA residues interacting with the IDA peptide and/or the SERK1 co-receptor kinase ectodomain are highlighted in blue and orange, respectively. FIG +84 94 ectodomain structure_element HAESA residues interacting with the IDA peptide and/or the SERK1 co-receptor kinase ectodomain are highlighted in blue and orange, respectively. FIG +4 19 peptide hormone protein_type The peptide hormone IDA binds to the HAESA LRR ectodomain. FIG +20 23 IDA protein The peptide hormone IDA binds to the HAESA LRR ectodomain. FIG +37 42 HAESA protein The peptide hormone IDA binds to the HAESA LRR ectodomain. FIG +43 57 LRR ectodomain structure_element The peptide hormone IDA binds to the HAESA LRR ectodomain. FIG +4 31 Multiple sequence alignment experimental_method (A) Multiple sequence alignment of selected IDA family members. FIG +44 62 IDA family members protein_type (A) Multiple sequence alignment of selected IDA family members. FIG +4 13 conserved protein_state The conserved PIP motif is highlighted in yellow, the central Hyp in blue. FIG +14 23 PIP motif structure_element The conserved PIP motif is highlighted in yellow, the central Hyp in blue. FIG +62 65 Hyp residue_name The conserved PIP motif is highlighted in yellow, the central Hyp in blue. FIG +4 14 PKGV motif structure_element The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. FIG +30 51 N-terminally extended protein_state The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. FIG +52 63 IDA peptide chemical The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. FIG +91 123 Isothermal titration calorimetry experimental_method The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. FIG +131 136 HAESA protein The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. FIG +137 147 ectodomain structure_element The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. FIG +152 155 IDA protein The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. FIG +174 183 synthetic protein_state The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. FIG +184 191 peptide chemical The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence. FIG +21 32 HAESA – IDA complex_assembly (C) Structure of the HAESA – IDA complex with HAESA shown in blue (ribbon diagram). FIG +46 51 HAESA protein (C) Structure of the HAESA – IDA complex with HAESA shown in blue (ribbon diagram). FIG +0 3 IDA protein IDA (in bonds representation, surface view included) is depicted in yellow. FIG +4 26 peptide binding pocket site The peptide binding pocket covers HAESA LRRs 2–14. (D) Close-up view of the entire IDA (in yellow) peptide binding site in HAESA (in blue). FIG +34 39 HAESA protein The peptide binding pocket covers HAESA LRRs 2–14. (D) Close-up view of the entire IDA (in yellow) peptide binding site in HAESA (in blue). FIG +40 49 LRRs 2–14 structure_element The peptide binding pocket covers HAESA LRRs 2–14. (D) Close-up view of the entire IDA (in yellow) peptide binding site in HAESA (in blue). FIG +83 86 IDA protein The peptide binding pocket covers HAESA LRRs 2–14. (D) Close-up view of the entire IDA (in yellow) peptide binding site in HAESA (in blue). FIG +99 119 peptide binding site site The peptide binding pocket covers HAESA LRRs 2–14. (D) Close-up view of the entire IDA (in yellow) peptide binding site in HAESA (in blue). FIG +123 128 HAESA protein The peptide binding pocket covers HAESA LRRs 2–14. (D) Close-up view of the entire IDA (in yellow) peptide binding site in HAESA (in blue). FIG +48 58 Hyp anchor structure_element Details of the interactions between the central Hyp anchor in IDA and the C-terminal Arg-His-Asn motif with HAESA are highlighted in (E) and (F), respectively. FIG +62 65 IDA protein Details of the interactions between the central Hyp anchor in IDA and the C-terminal Arg-His-Asn motif with HAESA are highlighted in (E) and (F), respectively. FIG +85 102 Arg-His-Asn motif structure_element Details of the interactions between the central Hyp anchor in IDA and the C-terminal Arg-His-Asn motif with HAESA are highlighted in (E) and (F), respectively. FIG +108 113 HAESA protein Details of the interactions between the central Hyp anchor in IDA and the C-terminal Arg-His-Asn motif with HAESA are highlighted in (E) and (F), respectively. FIG +61 66 water chemical Hydrogren bonds are depicted as dotted lines (in magenta), a water molecule is shown as a red sphere. FIG +50 56 plants taxonomy_domain During their growth, development and reproduction plants use cell separation processes to detach no-longer required, damaged or senescent organs. INTRO +31 42 Arabidopsis taxonomy_domain Abscission of floral organs in Arabidopsis is a model system to study these cell separation processes in molecular detail. INTRO +4 11 LRR-RKs structure_element The LRR-RKs HAESA (greek: to adhere to) and HAESA-LIKE 2 (HSL2) redundantly control floral abscission. INTRO +12 17 HAESA protein The LRR-RKs HAESA (greek: to adhere to) and HAESA-LIKE 2 (HSL2) redundantly control floral abscission. INTRO +44 56 HAESA-LIKE 2 protein The LRR-RKs HAESA (greek: to adhere to) and HAESA-LIKE 2 (HSL2) redundantly control floral abscission. INTRO +58 62 HSL2 protein The LRR-RKs HAESA (greek: to adhere to) and HAESA-LIKE 2 (HSL2) redundantly control floral abscission. INTRO +47 84 INFLORESCENCE DEFICIENT IN ABSCISSION protein Loss-of-function of the secreted small protein INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) causes floral organs to remain attached while its over-expression leads to premature shedding. INTRO +86 89 IDA protein Loss-of-function of the secreted small protein INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) causes floral organs to remain attached while its over-expression leads to premature shedding. INTRO +0 11 Full-length protein_state Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A). INTRO +12 15 IDA protein Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A). INTRO +19 44 proteolytically processed ptm Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A). INTRO +51 60 conserved protein_state Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A). INTRO +61 86 stretch of 20 amino-acids residue_range Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A). INTRO +95 99 EPIP structure_element Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A). INTRO +116 119 IDA protein Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A). INTRO +32 41 dodecamer structure_element It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and HSL2 in transient assays in tobacco cells. INTRO +42 49 peptide chemical It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and HSL2 in transient assays in tobacco cells. INTRO +57 61 EPIP structure_element It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and HSL2 in transient assays in tobacco cells. INTRO +82 87 HAESA protein It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and HSL2 in transient assays in tobacco cells. INTRO +92 96 HSL2 protein It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and HSL2 in transient assays in tobacco cells. INTRO +100 116 transient assays experimental_method It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and HSL2 in transient assays in tobacco cells. INTRO +120 127 tobacco taxonomy_domain It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and HSL2 in transient assays in tobacco cells. INTRO +0 19 This sequence motif structure_element This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). INTRO +23 39 highly conserved protein_state This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). INTRO +46 64 IDA family members protein_type This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). INTRO +66 83 IDA-LIKE PROTEINS protein_type This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). INTRO +85 89 IDLs protein_type This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). INTRO +114 117 Pro residue_name This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). INTRO +142 171 post-translationally modified protein_state This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). INTRO +175 189 hydroxyproline residue_name This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). INTRO +191 194 Hyp residue_name This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A). INTRO +61 64 IDA protein The available genetic and biochemical evidence suggests that IDA and HAESA together control floral abscission, but it is poorly understood if IDA is directly sensed by the receptor kinase HAESA and how IDA binding at the cell surface would activate the receptor. INTRO +69 74 HAESA protein The available genetic and biochemical evidence suggests that IDA and HAESA together control floral abscission, but it is poorly understood if IDA is directly sensed by the receptor kinase HAESA and how IDA binding at the cell surface would activate the receptor. INTRO +142 145 IDA protein The available genetic and biochemical evidence suggests that IDA and HAESA together control floral abscission, but it is poorly understood if IDA is directly sensed by the receptor kinase HAESA and how IDA binding at the cell surface would activate the receptor. INTRO +172 187 receptor kinase protein_type The available genetic and biochemical evidence suggests that IDA and HAESA together control floral abscission, but it is poorly understood if IDA is directly sensed by the receptor kinase HAESA and how IDA binding at the cell surface would activate the receptor. INTRO +188 193 HAESA protein The available genetic and biochemical evidence suggests that IDA and HAESA together control floral abscission, but it is poorly understood if IDA is directly sensed by the receptor kinase HAESA and how IDA binding at the cell surface would activate the receptor. INTRO +202 205 IDA protein The available genetic and biochemical evidence suggests that IDA and HAESA together control floral abscission, but it is poorly understood if IDA is directly sensed by the receptor kinase HAESA and how IDA binding at the cell surface would activate the receptor. INTRO +0 3 IDA protein IDA directly binds to the LRR domain of HAESA RESULTS +26 36 LRR domain structure_element IDA directly binds to the LRR domain of HAESA RESULTS +40 45 HAESA protein IDA directly binds to the LRR domain of HAESA RESULTS +0 6 Active protein_state Active IDA-family peptide hormones are hydroxyprolinated dodecamers. FIG +7 34 IDA-family peptide hormones protein_type Active IDA-family peptide hormones are hydroxyprolinated dodecamers. FIG +39 56 hydroxyprolinated protein_state Active IDA-family peptide hormones are hydroxyprolinated dodecamers. FIG +57 67 dodecamers structure_element Active IDA-family peptide hormones are hydroxyprolinated dodecamers. FIG +22 25 IDA protein Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. FIG +35 56 N-terminally extended protein_state Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. FIG +57 65 PKGV-IDA mutant Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. FIG +74 78 IDL1 protein Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. FIG +79 87 bound to protein_state Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. FIG +92 97 HAESA protein Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. FIG +98 120 hormone binding pocket site Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. FIG +172 191 simulated annealing experimental_method Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. FIG +192 225 2Fo–Fc omit electron density maps evidence Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo–Fc omit electron density maps contoured at 1.0 σ. FIG +10 15 Pro58 residue_name_number Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +15 18 IDA protein Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +23 28 Leu67 residue_name_number Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +28 31 IDA protein Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +66 82 electron density evidence Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +88 96 bound to protein_state Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +101 106 HAESA protein Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +107 117 ectodomain structure_element Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +143 177 equilibrium dissociation constants evidence Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +179 181 Kd evidence Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +184 202 binding enthalpies evidence Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +204 206 ΔH evidence Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +209 226 binding entropies evidence Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +228 230 ΔS evidence Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +270 282 IDA peptides chemical Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +298 303 HAESA protein Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +304 314 ectodomain structure_element Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (ΔH), binding entropies (ΔS) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( ± fitting errors; n.d. FIG +28 52 Structural superposition experimental_method no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain. FIG +60 66 active protein_state no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain. FIG +67 70 IDA protein no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain. FIG +110 122 IDL1 peptide chemical no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain. FIG +144 152 bound to protein_state no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain. FIG +157 162 HAESA protein no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain. FIG +163 173 ectodomain structure_element no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain. FIG +0 26 Root mean square deviation evidence Root mean square deviation (r.m.s.d.) is 1.0 Å comparing 100 corresponding atoms. FIG +28 36 r.m.s.d. evidence Root mean square deviation (r.m.s.d.) is 1.0 Å comparing 100 corresponding atoms. FIG +4 19 receptor kinase protein_type The receptor kinase SERK1 acts as a HAESA co-receptor and promotes high-affinity IDA sensing. FIG +20 25 SERK1 protein The receptor kinase SERK1 acts as a HAESA co-receptor and promotes high-affinity IDA sensing. FIG +36 53 HAESA co-receptor protein_type The receptor kinase SERK1 acts as a HAESA co-receptor and promotes high-affinity IDA sensing. FIG +81 84 IDA protein The receptor kinase SERK1 acts as a HAESA co-receptor and promotes high-affinity IDA sensing. FIG +4 31 Petal break-strength assays experimental_method (A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. FIG +131 135 serk gene (A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. FIG +136 142 mutant protein_state (A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. FIG +143 149 plants taxonomy_domain (A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. FIG +162 167 haesa gene (A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. FIG +168 172 hsl2 gene (A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. FIG +173 179 mutant protein_state (A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. FIG +190 199 wild-type protein_state (A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers. FIG +104 109 haesa gene Petal break-strength was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. FIG +110 114 hsl2 gene Petal break-strength was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. FIG +119 126 serk1-1 gene Petal break-strength was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. FIG +127 133 mutant protein_state Petal break-strength was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. FIG +134 140 plants taxonomy_domain Petal break-strength was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control. FIG +4 44 Analytical size-exclusion chromatography experimental_method (B) Analytical size-exclusion chromatography. FIG +4 9 HAESA protein The HAESA LRR domain elutes as a monomer (black dotted line), as does the isolated SERK1 ectodomain (blue dotted line). FIG +10 20 LRR domain structure_element The HAESA LRR domain elutes as a monomer (black dotted line), as does the isolated SERK1 ectodomain (blue dotted line). FIG +33 40 monomer oligomeric_state The HAESA LRR domain elutes as a monomer (black dotted line), as does the isolated SERK1 ectodomain (blue dotted line). FIG +83 88 SERK1 protein The HAESA LRR domain elutes as a monomer (black dotted line), as does the isolated SERK1 ectodomain (blue dotted line). FIG +89 99 ectodomain structure_element The HAESA LRR domain elutes as a monomer (black dotted line), as does the isolated SERK1 ectodomain (blue dotted line). FIG +2 21 HAESA – IDA – SERK1 complex_assembly A HAESA – IDA – SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line). FIG +52 63 heterodimer oligomeric_state A HAESA – IDA – SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line). FIG +95 100 HAESA protein A HAESA – IDA – SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line). FIG +105 110 SERK1 protein A HAESA – IDA – SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line). FIG +156 165 monomeric oligomeric_state A HAESA – IDA – SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line). FIG +166 171 HAESA protein A HAESA – IDA – SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line). FIG +176 181 SERK1 protein A HAESA – IDA – SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line). FIG +113 126 Thyroglobulin protein Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). FIG +143 151 Ferritin protein Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). FIG +167 175 Aldolase protein Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). FIG +192 202 Conalbumin protein Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). FIG +218 227 Ovalbumin protein Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). FIG +243 261 Carbonic anhydrase protein Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). FIG +113 126 Thyroglobulin protein Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). FIG +143 151 Ferritin protein Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). FIG +167 175 Aldolase protein Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). FIG +192 202 Conalbumin protein Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). FIG +218 227 Ovalbumin protein Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). FIG +243 261 Carbonic anhydrase protein Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da). FIG +2 10 SDS PAGE experimental_method A SDS PAGE of the peak fractions is shown alongside. FIG +2 10 SDS PAGE experimental_method A SDS PAGE of the peak fractions is shown alongside. FIG +9 14 HAESA protein Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. FIG +19 24 SERK1 protein Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. FIG +64 96 Isothermal titration calorimetry experimental_method Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. FIG +100 109 wild-type protein_state Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. FIG +114 123 Hyp64→Pro ptm Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. FIG +124 127 IDA protein Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. FIG +139 144 HAESA protein Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. FIG +149 154 SERK1 protein Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. FIG +155 166 ectodomains structure_element Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64→Pro IDA versus the HAESA and SERK1 ectodomains. FIG +4 13 titration experimental_method The titration of IDA wild-type versus the isolated HAESA ectodomain from Figure 1B is shown for comparison (red line; n.d. FIG +17 20 IDA protein The titration of IDA wild-type versus the isolated HAESA ectodomain from Figure 1B is shown for comparison (red line; n.d. FIG +21 30 wild-type protein_state The titration of IDA wild-type versus the isolated HAESA ectodomain from Figure 1B is shown for comparison (red line; n.d. FIG +51 56 HAESA protein The titration of IDA wild-type versus the isolated HAESA ectodomain from Figure 1B is shown for comparison (red line; n.d. FIG +57 67 ectodomain structure_element The titration of IDA wild-type versus the isolated HAESA ectodomain from Figure 1B is shown for comparison (red line; n.d. FIG +27 67 Analytical size-exclusion chromatography experimental_method no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. FIG +75 86 presence of protein_state no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. FIG +91 94 IDA protein no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. FIG +95 104 Hyp64→Pro ptm no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. FIG +105 111 mutant protein_state no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. FIG +112 119 peptide chemical no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. FIG +157 162 HAESA protein no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. FIG +167 172 SERK1 protein no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. FIG +173 184 ectodomains structure_element no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64→Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains. FIG +4 26 In vitro kinase assays experimental_method (E) In vitro kinase assays of the HAESA and SERK1 kinase domains. FIG +34 39 HAESA protein (E) In vitro kinase assays of the HAESA and SERK1 kinase domains. FIG +44 49 SERK1 protein (E) In vitro kinase assays of the HAESA and SERK1 kinase domains. FIG +50 64 kinase domains structure_element (E) In vitro kinase assays of the HAESA and SERK1 kinase domains. FIG +0 9 Wild-type protein_state Wild-type HAESA and SERK1 kinase domains (KDs) exhibit auto-phosphorylation activities (lanes 1 + 3). FIG +10 15 HAESA protein Wild-type HAESA and SERK1 kinase domains (KDs) exhibit auto-phosphorylation activities (lanes 1 + 3). FIG +20 25 SERK1 protein Wild-type HAESA and SERK1 kinase domains (KDs) exhibit auto-phosphorylation activities (lanes 1 + 3). FIG +26 40 kinase domains structure_element Wild-type HAESA and SERK1 kinase domains (KDs) exhibit auto-phosphorylation activities (lanes 1 + 3). FIG +42 45 KDs structure_element Wild-type HAESA and SERK1 kinase domains (KDs) exhibit auto-phosphorylation activities (lanes 1 + 3). FIG +0 6 Mutant protein_state Mutant (m) versions, which carry point mutations in their active sites (Asp837HAESA→Asn, Asp447SERK1→Asn) possess no autophosphorylation activity (lanes 2+4). FIG +33 48 point mutations experimental_method Mutant (m) versions, which carry point mutations in their active sites (Asp837HAESA→Asn, Asp447SERK1→Asn) possess no autophosphorylation activity (lanes 2+4). FIG +58 70 active sites site Mutant (m) versions, which carry point mutations in their active sites (Asp837HAESA→Asn, Asp447SERK1→Asn) possess no autophosphorylation activity (lanes 2+4). FIG +72 87 Asp837HAESA→Asn mutant Mutant (m) versions, which carry point mutations in their active sites (Asp837HAESA→Asn, Asp447SERK1→Asn) possess no autophosphorylation activity (lanes 2+4). FIG +89 104 Asp447SERK1→Asn mutant Mutant (m) versions, which carry point mutations in their active sites (Asp837HAESA→Asn, Asp447SERK1→Asn) possess no autophosphorylation activity (lanes 2+4). FIG +39 45 active protein_state Transphosphorylation activity from the active kinase to the mutated form can be observed in both directions (lanes 5+6). FIG +60 67 mutated protein_state Transphosphorylation activity from the active kinase to the mutated form can be observed in both directions (lanes 5+6). FIG +3 11 purified experimental_method We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). RESULTS +16 21 HAESA protein We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). RESULTS +22 32 ectodomain structure_element We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). RESULTS +43 49 20–620 residue_range We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). RESULTS +56 89 baculovirus-infected insect cells experimental_method We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). RESULTS +195 207 glycoprotein protein_type We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). RESULTS +213 222 synthetic protein_state We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). RESULTS +223 235 IDA peptides chemical We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). RESULTS +242 274 isothermal titration calorimetry experimental_method We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). RESULTS +276 279 ITC experimental_method We purified the HAESA ectodomain (residues 20–620) from baculovirus-infected insect cells (Figure 1—figure supplement 1A, see Materials and methods) and quantified the interaction of the ~75 kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC). RESULTS +2 14 Hyp-modified protein_state A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20 μM (Figure 1B). RESULTS +15 24 dodecamer structure_element A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20 μM (Figure 1B). RESULTS +40 56 highly conserved protein_state A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20 μM (Figure 1B). RESULTS +57 66 PIP motif structure_element A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20 μM (Figure 1B). RESULTS +70 73 IDA protein A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20 μM (Figure 1B). RESULTS +101 106 HAESA protein A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20 μM (Figure 1B). RESULTS +145 166 dissociation constant evidence A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20 μM (Figure 1B). RESULTS +168 170 Kd evidence A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20 μM (Figure 1B). RESULTS +19 37 crystal structures evidence We next determined crystal structures of the apo HAESA ectodomain and of a HAESA-IDA complex, at 1.74 and 1.86 Å resolution, respectively (Figure 1C; Figure 1—figure supplement 1B–D; Tables 1,2). RESULTS +45 48 apo protein_state We next determined crystal structures of the apo HAESA ectodomain and of a HAESA-IDA complex, at 1.74 and 1.86 Å resolution, respectively (Figure 1C; Figure 1—figure supplement 1B–D; Tables 1,2). RESULTS +49 54 HAESA protein We next determined crystal structures of the apo HAESA ectodomain and of a HAESA-IDA complex, at 1.74 and 1.86 Å resolution, respectively (Figure 1C; Figure 1—figure supplement 1B–D; Tables 1,2). RESULTS +55 65 ectodomain structure_element We next determined crystal structures of the apo HAESA ectodomain and of a HAESA-IDA complex, at 1.74 and 1.86 Å resolution, respectively (Figure 1C; Figure 1—figure supplement 1B–D; Tables 1,2). RESULTS +75 84 HAESA-IDA complex_assembly We next determined crystal structures of the apo HAESA ectodomain and of a HAESA-IDA complex, at 1.74 and 1.86 Å resolution, respectively (Figure 1C; Figure 1—figure supplement 1B–D; Tables 1,2). RESULTS +0 3 IDA protein IDA binds in a completely extended conformation along the inner surface of the HAESA ectodomain, covering LRRs 2–14 (Figure 1C,D, Figure 1—figure supplement 2). RESULTS +15 47 completely extended conformation protein_state IDA binds in a completely extended conformation along the inner surface of the HAESA ectodomain, covering LRRs 2–14 (Figure 1C,D, Figure 1—figure supplement 2). RESULTS +79 84 HAESA protein IDA binds in a completely extended conformation along the inner surface of the HAESA ectodomain, covering LRRs 2–14 (Figure 1C,D, Figure 1—figure supplement 2). RESULTS +85 95 ectodomain structure_element IDA binds in a completely extended conformation along the inner surface of the HAESA ectodomain, covering LRRs 2–14 (Figure 1C,D, Figure 1—figure supplement 2). RESULTS +106 115 LRRs 2–14 structure_element IDA binds in a completely extended conformation along the inner surface of the HAESA ectodomain, covering LRRs 2–14 (Figure 1C,D, Figure 1—figure supplement 2). RESULTS +12 17 Hyp64 ptm The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +17 20 IDA protein The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +45 51 pocket site The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +62 67 HAESA protein The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +68 77 LRRs 8–10 structure_element The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +116 130 hydrogen bonds bond_interaction The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +140 158 strictly conserved protein_state The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +159 165 Glu266 residue_name_number The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +165 170 HAESA protein The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +182 187 water chemical The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +256 262 Phe289 residue_name_number The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +262 267 HAESA protein The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +272 278 Ser311 residue_name_number The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +278 283 HAESA protein The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8–10, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1—figure supplement 3). RESULTS +27 37 Hyp pocket site The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones. RESULTS +52 55 IDA protein The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones. RESULTS +73 88 arabinosylation ptm The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones. RESULTS +92 97 Hyp64 ptm The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones. RESULTS +97 100 IDA protein The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones. RESULTS +165 168 Hyp residue_name The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones. RESULTS +181 186 plant taxonomy_domain The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones. RESULTS +187 207 CLE peptide hormones protein_type The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones. RESULTS +15 32 Arg-His-Asn motif structure_element The C-terminal Arg-His-Asn motif in IDA maps to a cavity formed by HAESA LRRs 11–14 (Figure 1D,F). RESULTS +36 39 IDA protein The C-terminal Arg-His-Asn motif in IDA maps to a cavity formed by HAESA LRRs 11–14 (Figure 1D,F). RESULTS +50 56 cavity site The C-terminal Arg-His-Asn motif in IDA maps to a cavity formed by HAESA LRRs 11–14 (Figure 1D,F). RESULTS +67 72 HAESA protein The C-terminal Arg-His-Asn motif in IDA maps to a cavity formed by HAESA LRRs 11–14 (Figure 1D,F). RESULTS +73 83 LRRs 11–14 structure_element The C-terminal Arg-His-Asn motif in IDA maps to a cavity formed by HAESA LRRs 11–14 (Figure 1D,F). RESULTS +18 23 Asn69 residue_name_number The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). RESULTS +23 26 IDA protein The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). RESULTS +53 59 Arg407 residue_name_number The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). RESULTS +59 64 HAESA protein The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). RESULTS +69 75 Arg409 residue_name_number The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). RESULTS +75 80 HAESA protein The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). RESULTS +85 90 HAESA protein The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). RESULTS +105 126 C-terminally extended protein_state The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). RESULTS +127 135 IDA-SFVN mutant The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D). RESULTS +23 32 conserved protein_state This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein. RESULTS +33 38 Asn69 residue_name_number This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein. RESULTS +38 41 IDA protein This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein. RESULTS +84 90 mature protein_state This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein. RESULTS +91 102 IDA peptide chemical This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein. RESULTS +106 112 planta taxonomy_domain This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein. RESULTS +122 128 active protein_state This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein. RESULTS +129 132 IDA protein This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein. RESULTS +0 8 Mutation experimental_method Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features. RESULTS +12 18 Arg417 residue_name_number Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features. RESULTS +18 22 HSL2 protein Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features. RESULTS +45 51 Arg409 residue_name_number Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features. RESULTS +51 56 HAESA protein Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features. RESULTS +97 101 HSL2 protein Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features. RESULTS +128 151 peptide binding pockets site Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features. RESULTS +165 180 HAESA receptors protein_type Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features. RESULTS +74 93 IDA binding surface site Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and in other HAESA-type receptors in different plant species (Figure 1—figure supplement 3). RESULTS +97 102 HAESA protein Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and in other HAESA-type receptors in different plant species (Figure 1—figure supplement 3). RESULTS +109 118 conserved protein_state Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and in other HAESA-type receptors in different plant species (Figure 1—figure supplement 3). RESULTS +122 126 HSL2 protein Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and in other HAESA-type receptors in different plant species (Figure 1—figure supplement 3). RESULTS +140 160 HAESA-type receptors protein_type Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and in other HAESA-type receptors in different plant species (Figure 1—figure supplement 3). RESULTS +174 179 plant taxonomy_domain Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and in other HAESA-type receptors in different plant species (Figure 1—figure supplement 3). RESULTS +13 27 Pro-rich motif structure_element A N-terminal Pro-rich motif in IDA makes contacts with LRRs 2–6 of the receptor (Figure 1D, Figure 1—figure supplement 2A–C). RESULTS +31 34 IDA protein A N-terminal Pro-rich motif in IDA makes contacts with LRRs 2–6 of the receptor (Figure 1D, Figure 1—figure supplement 2A–C). RESULTS +55 63 LRRs 2–6 structure_element A N-terminal Pro-rich motif in IDA makes contacts with LRRs 2–6 of the receptor (Figure 1D, Figure 1—figure supplement 2A–C). RESULTS +6 40 hydrophobic and polar interactions bond_interaction Other hydrophobic and polar interactions are mediated by Ser62IDA, Ser65IDA and by backbone atoms along the IDA peptide (Figure 1D, Figure 1—figure supplement 2A–C). RESULTS +57 62 Ser62 residue_name_number Other hydrophobic and polar interactions are mediated by Ser62IDA, Ser65IDA and by backbone atoms along the IDA peptide (Figure 1D, Figure 1—figure supplement 2A–C). RESULTS +62 65 IDA protein Other hydrophobic and polar interactions are mediated by Ser62IDA, Ser65IDA and by backbone atoms along the IDA peptide (Figure 1D, Figure 1—figure supplement 2A–C). RESULTS +67 72 Ser65 residue_name_number Other hydrophobic and polar interactions are mediated by Ser62IDA, Ser65IDA and by backbone atoms along the IDA peptide (Figure 1D, Figure 1—figure supplement 2A–C). RESULTS +72 75 IDA protein Other hydrophobic and polar interactions are mediated by Ser62IDA, Ser65IDA and by backbone atoms along the IDA peptide (Figure 1D, Figure 1—figure supplement 2A–C). RESULTS +108 119 IDA peptide chemical Other hydrophobic and polar interactions are mediated by Ser62IDA, Ser65IDA and by backbone atoms along the IDA peptide (Figure 1D, Figure 1—figure supplement 2A–C). RESULTS +0 5 HAESA protein HAESA specifically senses IDA-family dodecamer peptides RESULTS +26 36 IDA-family protein_type HAESA specifically senses IDA-family dodecamer peptides RESULTS +37 46 dodecamer structure_element HAESA specifically senses IDA-family dodecamer peptides RESULTS +47 55 peptides chemical HAESA specifically senses IDA-family dodecamer peptides RESULTS +29 34 HAESA protein We next investigated whether HAESA binds N-terminally extended versions of IDA. RESULTS +41 62 N-terminally extended protein_state We next investigated whether HAESA binds N-terminally extended versions of IDA. RESULTS +75 78 IDA protein We next investigated whether HAESA binds N-terminally extended versions of IDA. RESULTS +14 23 structure evidence We obtained a structure of HAESA in complex with a PKGV-IDA peptide at 1.94 Å resolution (Table 2). RESULTS +27 32 HAESA protein We obtained a structure of HAESA in complex with a PKGV-IDA peptide at 1.94 Å resolution (Table 2). RESULTS +33 48 in complex with protein_state We obtained a structure of HAESA in complex with a PKGV-IDA peptide at 1.94 Å resolution (Table 2). RESULTS +51 59 PKGV-IDA mutant We obtained a structure of HAESA in complex with a PKGV-IDA peptide at 1.94 Å resolution (Table 2). RESULTS +60 67 peptide chemical We obtained a structure of HAESA in complex with a PKGV-IDA peptide at 1.94 Å resolution (Table 2). RESULTS +8 17 structure evidence In this structure, no additional electron density accounts for the PKGV motif at the IDA N-terminus (Figure 2A,B). RESULTS +33 49 electron density evidence In this structure, no additional electron density accounts for the PKGV motif at the IDA N-terminus (Figure 2A,B). RESULTS +67 77 PKGV motif structure_element In this structure, no additional electron density accounts for the PKGV motif at the IDA N-terminus (Figure 2A,B). RESULTS +85 88 IDA protein In this structure, no additional electron density accounts for the PKGV motif at the IDA N-terminus (Figure 2A,B). RESULTS +14 22 PKGV-IDA mutant Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D). RESULTS +27 30 IDA protein Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D). RESULTS +44 62 binding affinities evidence Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D). RESULTS +70 80 ITC assays experimental_method Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D). RESULTS +106 111 HAESA protein Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D). RESULTS +121 130 dodecamer structure_element Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D). RESULTS +131 138 peptide chemical Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D). RESULTS +159 164 58-69 residue_range Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D). RESULTS +164 167 IDA protein Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D). RESULTS +18 23 HAESA protein We next tested if HAESA binds other IDA peptide family members. RESULTS +36 62 IDA peptide family members chemical We next tested if HAESA binds other IDA peptide family members. RESULTS +0 4 IDL1 protein IDL1, which can rescue IDA loss-of-function mutants when introduced in abscission zone cells, can also be sensed by HAESA, albeit with lower affinity (Figure 2D). RESULTS +23 26 IDA protein IDL1, which can rescue IDA loss-of-function mutants when introduced in abscission zone cells, can also be sensed by HAESA, albeit with lower affinity (Figure 2D). RESULTS +116 121 HAESA protein IDL1, which can rescue IDA loss-of-function mutants when introduced in abscission zone cells, can also be sensed by HAESA, albeit with lower affinity (Figure 2D). RESULTS +141 149 affinity evidence IDL1, which can rescue IDA loss-of-function mutants when introduced in abscission zone cells, can also be sensed by HAESA, albeit with lower affinity (Figure 2D). RESULTS +9 29 co-crystal structure evidence A 2.56 Å co-crystal structure with IDL1 reveals that different IDA family members use a common binding mode to interact with HAESA-type receptors (Figure 2A–C,E, Table 2). RESULTS +35 39 IDL1 protein A 2.56 Å co-crystal structure with IDL1 reveals that different IDA family members use a common binding mode to interact with HAESA-type receptors (Figure 2A–C,E, Table 2). RESULTS +63 81 IDA family members protein_type A 2.56 Å co-crystal structure with IDL1 reveals that different IDA family members use a common binding mode to interact with HAESA-type receptors (Figure 2A–C,E, Table 2). RESULTS +125 145 HAESA-type receptors protein_type A 2.56 Å co-crystal structure with IDL1 reveals that different IDA family members use a common binding mode to interact with HAESA-type receptors (Figure 2A–C,E, Table 2). RESULTS +37 42 HAESA protein We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +49 58 synthetic protein_state We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +59 66 peptide chemical We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +67 89 missing the C-terminal protein_state We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +90 95 Asn69 residue_name_number We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +95 98 IDA protein We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +100 104 ΔN69 mutant We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +142 160 polar interactions bond_interaction We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +173 176 IDA protein We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +198 204 Arg407 residue_name_number We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +204 209 HAESA protein We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +210 216 Arg409 residue_name_number We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +216 221 HAESA protein We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (ΔN69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D). RESULTS +0 9 Replacing experimental_method Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA → Ala double-mutant discussed below (Figure 1A, 2D). RESULTS +10 15 Hyp64 ptm Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA → Ala double-mutant discussed below (Figure 1A, 2D). RESULTS +15 18 IDA protein Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA → Ala double-mutant discussed below (Figure 1A, 2D). RESULTS +43 47 IDLs protein_type Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA → Ala double-mutant discussed below (Figure 1A, 2D). RESULTS +54 61 proline residue_name Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA → Ala double-mutant discussed below (Figure 1A, 2D). RESULTS +117 140 Lys66IDA/Arg67IDA → Ala mutant Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA → Ala double-mutant discussed below (Figure 1A, 2D). RESULTS +141 154 double-mutant protein_state Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA → Ala double-mutant discussed below (Figure 1A, 2D). RESULTS +9 14 HAESA protein Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7). RESULTS +40 44 IDLs protein_type Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7). RESULTS +49 71 functionally unrelated protein_state Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7). RESULTS +72 81 dodecamer structure_element Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7). RESULTS +82 90 peptides chemical Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7). RESULTS +96 113 Hyp modifications ptm Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7). RESULTS +123 127 CLV3 protein Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7). RESULTS +4 22 co-receptor kinase protein_type The co-receptor kinase SERK1 allows for high-affinity IDA sensing RESULTS +23 28 SERK1 protein The co-receptor kinase SERK1 allows for high-affinity IDA sensing RESULTS +4 18 binding assays experimental_method Our binding assays reveal that IDA family peptides are sensed by the isolated HAESA ectodomain with relatively weak binding affinities (Figures 1B, 2A–D). RESULTS +31 50 IDA family peptides chemical Our binding assays reveal that IDA family peptides are sensed by the isolated HAESA ectodomain with relatively weak binding affinities (Figures 1B, 2A–D). RESULTS +69 77 isolated protein_state Our binding assays reveal that IDA family peptides are sensed by the isolated HAESA ectodomain with relatively weak binding affinities (Figures 1B, 2A–D). RESULTS +78 83 HAESA protein Our binding assays reveal that IDA family peptides are sensed by the isolated HAESA ectodomain with relatively weak binding affinities (Figures 1B, 2A–D). RESULTS +84 94 ectodomain structure_element Our binding assays reveal that IDA family peptides are sensed by the isolated HAESA ectodomain with relatively weak binding affinities (Figures 1B, 2A–D). RESULTS +116 134 binding affinities evidence Our binding assays reveal that IDA family peptides are sensed by the isolated HAESA ectodomain with relatively weak binding affinities (Figures 1B, 2A–D). RESULTS +35 73 SOMATIC EMBRYOGENESIS RECEPTOR KINASES protein_type It has been recently reported that SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) are positive regulators of floral abscission and can interact with HAESA and HSL2 in an IDA-dependent manner. RESULTS +75 80 SERKs protein_type It has been recently reported that SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) are positive regulators of floral abscission and can interact with HAESA and HSL2 in an IDA-dependent manner. RESULTS +149 154 HAESA protein It has been recently reported that SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) are positive regulators of floral abscission and can interact with HAESA and HSL2 in an IDA-dependent manner. RESULTS +159 163 HSL2 protein It has been recently reported that SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) are positive regulators of floral abscission and can interact with HAESA and HSL2 in an IDA-dependent manner. RESULTS +12 31 SERK family members protein_type As all five SERK family members appear to be expressed in the Arabidopsis abscission zone, we quantified their relative contribution to floral abscission in Arabidopsis using a petal break-strength assay. RESULTS +62 73 Arabidopsis taxonomy_domain As all five SERK family members appear to be expressed in the Arabidopsis abscission zone, we quantified their relative contribution to floral abscission in Arabidopsis using a petal break-strength assay. RESULTS +157 168 Arabidopsis taxonomy_domain As all five SERK family members appear to be expressed in the Arabidopsis abscission zone, we quantified their relative contribution to floral abscission in Arabidopsis using a petal break-strength assay. RESULTS +177 203 petal break-strength assay experimental_method As all five SERK family members appear to be expressed in the Arabidopsis abscission zone, we quantified their relative contribution to floral abscission in Arabidopsis using a petal break-strength assay. RESULTS +39 58 SERK family members protein_type Our experiments suggest that among the SERK family members, SERK1 is a positive regulator of floral abscission. RESULTS +60 65 SERK1 protein Our experiments suggest that among the SERK family members, SERK1 is a positive regulator of floral abscission. RESULTS +57 64 serk1-1 gene We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A). RESULTS +65 72 mutants protein_state We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A). RESULTS +118 127 wild-type protein_state We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A). RESULTS +128 134 plants taxonomy_domain We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A). RESULTS +163 168 haesa gene We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A). RESULTS +169 173 hsl2 gene We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A). RESULTS +174 181 mutants protein_state We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A). RESULTS +224 231 serk1-1 gene We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A). RESULTS +4 11 serk2-2 gene The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants. RESULTS +13 20 serk3-1 gene The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants. RESULTS +22 29 serk4-1 gene The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants. RESULTS +34 41 serk5-1 gene The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants. RESULTS +42 48 mutant protein_state The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants. RESULTS +126 135 wild-type protein_state The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants. RESULTS +136 142 plants taxonomy_domain The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants. RESULTS +17 22 SERKs protein_type Possibly because SERKs have additional roles in plant development such as in pollen formation and brassinosteroid signaling, we found that higher-order SERK mutants exhibit pleiotropic phenotypes in the flower, rendering their analysis and comparison by quantitative petal break-strength assays difficult. RESULTS +254 294 quantitative petal break-strength assays experimental_method Possibly because SERKs have additional roles in plant development such as in pollen formation and brassinosteroid signaling, we found that higher-order SERK mutants exhibit pleiotropic phenotypes in the flower, rendering their analysis and comparison by quantitative petal break-strength assays difficult. RESULTS +49 54 SERK1 protein We thus focused on analyzing the contribution of SERK1 to HAESA ligand sensing and receptor activation. RESULTS +58 63 HAESA protein We thus focused on analyzing the contribution of SERK1 to HAESA ligand sensing and receptor activation. RESULTS +14 28 LRR ectodomain structure_element In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). RESULTS +32 37 SERK1 protein In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). RESULTS +48 54 24–213 residue_range In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). RESULTS +62 68 stable protein_state In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). RESULTS +70 83 IDA-dependent protein_state In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). RESULTS +84 97 heterodimeric oligomeric_state In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). RESULTS +98 112 complexes with protein_state In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). RESULTS +113 118 HAESA protein In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). RESULTS +122 151 size exclusion chromatography experimental_method In vitro, the LRR ectodomain of SERK1 (residues 24–213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B). RESULTS +39 44 SERK1 protein We next quantified the contribution of SERK1 to IDA recognition by HAESA. RESULTS +48 51 IDA protein We next quantified the contribution of SERK1 to IDA recognition by HAESA. RESULTS +67 72 HAESA protein We next quantified the contribution of SERK1 to IDA recognition by HAESA. RESULTS +14 19 HAESA protein We found that HAESA senses IDA with a ~60 fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C). RESULTS +27 30 IDA protein We found that HAESA senses IDA with a ~60 fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C). RESULTS +54 70 binding affinity evidence We found that HAESA senses IDA with a ~60 fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C). RESULTS +78 89 presence of protein_state We found that HAESA senses IDA with a ~60 fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C). RESULTS +90 95 SERK1 protein We found that HAESA senses IDA with a ~60 fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C). RESULTS +113 118 SERK1 protein We found that HAESA senses IDA with a ~60 fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C). RESULTS +166 181 peptide hormone protein_type We found that HAESA senses IDA with a ~60 fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C). RESULTS +8 16 titrated experimental_method We next titrated SERK1 into a solution containing only the HAESA ectodomain. RESULTS +17 22 SERK1 protein We next titrated SERK1 into a solution containing only the HAESA ectodomain. RESULTS +59 64 HAESA protein We next titrated SERK1 into a solution containing only the HAESA ectodomain. RESULTS +65 75 ectodomain structure_element We next titrated SERK1 into a solution containing only the HAESA ectodomain. RESULTS +97 108 presence of protein_state In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range (Figure 3C). RESULTS +109 112 IDA protein In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range (Figure 3C). RESULTS +114 119 SERK1 protein In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range (Figure 3C). RESULTS +135 140 HAESA protein In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range (Figure 3C). RESULTS +148 169 dissociation constant evidence In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range (Figure 3C). RESULTS +19 22 IDA protein This suggests that IDA itself promotes receptor – co-receptor association, as previously described for the steroid hormone brassinolide and for other LRR-RK complexes. RESULTS +107 122 steroid hormone chemical This suggests that IDA itself promotes receptor – co-receptor association, as previously described for the steroid hormone brassinolide and for other LRR-RK complexes. RESULTS +123 135 brassinolide chemical This suggests that IDA itself promotes receptor – co-receptor association, as previously described for the steroid hormone brassinolide and for other LRR-RK complexes. RESULTS +150 156 LRR-RK complex_assembly This suggests that IDA itself promotes receptor – co-receptor association, as previously described for the steroid hormone brassinolide and for other LRR-RK complexes. RESULTS +13 31 hydroxyprolination ptm Importantly, hydroxyprolination of IDA is critical for HAESA-IDA-SERK1 complex formation (Figure 3C,D). RESULTS +35 38 IDA protein Importantly, hydroxyprolination of IDA is critical for HAESA-IDA-SERK1 complex formation (Figure 3C,D). RESULTS +55 70 HAESA-IDA-SERK1 complex_assembly Importantly, hydroxyprolination of IDA is critical for HAESA-IDA-SERK1 complex formation (Figure 3C,D). RESULTS +4 15 calorimetry experimental_method Our calorimetry experiments now reveal that SERKs may render HAESA, and potentially other receptor kinases, competent for high-affinity sensing of their cognate ligands. RESULTS +44 49 SERKs protein_type Our calorimetry experiments now reveal that SERKs may render HAESA, and potentially other receptor kinases, competent for high-affinity sensing of their cognate ligands. RESULTS +61 66 HAESA protein Our calorimetry experiments now reveal that SERKs may render HAESA, and potentially other receptor kinases, competent for high-affinity sensing of their cognate ligands. RESULTS +90 106 receptor kinases protein_type Our calorimetry experiments now reveal that SERKs may render HAESA, and potentially other receptor kinases, competent for high-affinity sensing of their cognate ligands. RESULTS +5 8 IDA protein Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases, may interact in the cytoplasm to activate each other. RESULTS +42 56 kinase domains structure_element Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases, may interact in the cytoplasm to activate each other. RESULTS +60 65 HAESA protein Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases, may interact in the cytoplasm to activate each other. RESULTS +70 75 SERK1 protein Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases, may interact in the cytoplasm to activate each other. RESULTS +105 111 active protein_state Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases, may interact in the cytoplasm to activate each other. RESULTS +112 127 protein kinases protein_type Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases, may interact in the cytoplasm to activate each other. RESULTS +18 23 HAESA protein Consistently, the HAESA kinase domain can transphosphorylate SERK1 and vice versa in in vitro transphosphorylation assays (Figure 3E). RESULTS +24 37 kinase domain structure_element Consistently, the HAESA kinase domain can transphosphorylate SERK1 and vice versa in in vitro transphosphorylation assays (Figure 3E). RESULTS +61 66 SERK1 protein Consistently, the HAESA kinase domain can transphosphorylate SERK1 and vice versa in in vitro transphosphorylation assays (Figure 3E). RESULTS +94 121 transphosphorylation assays experimental_method Consistently, the HAESA kinase domain can transphosphorylate SERK1 and vice versa in in vitro transphosphorylation assays (Figure 3E). RESULTS +14 49 genetic and biochemical experiments experimental_method Together, our genetic and biochemical experiments implicate SERK1 as a HAESA co-receptor in the Arabidopsis abscission zone. RESULTS +60 65 SERK1 protein Together, our genetic and biochemical experiments implicate SERK1 as a HAESA co-receptor in the Arabidopsis abscission zone. RESULTS +71 88 HAESA co-receptor protein_type Together, our genetic and biochemical experiments implicate SERK1 as a HAESA co-receptor in the Arabidopsis abscission zone. RESULTS +96 107 Arabidopsis taxonomy_domain Together, our genetic and biochemical experiments implicate SERK1 as a HAESA co-receptor in the Arabidopsis abscission zone. RESULTS +0 5 SERK1 protein SERK1 senses a conserved motif in IDA family peptides RESULTS +15 24 conserved protein_state SERK1 senses a conserved motif in IDA family peptides RESULTS +25 30 motif structure_element SERK1 senses a conserved motif in IDA family peptides RESULTS +34 53 IDA family peptides chemical SERK1 senses a conserved motif in IDA family peptides RESULTS +0 17 Crystal structure evidence Crystal structure of a HAESA – IDA – SERK1 signaling complex. FIG +23 42 HAESA – IDA – SERK1 complex_assembly Crystal structure of a HAESA – IDA – SERK1 signaling complex. FIG +41 46 HAESA protein (A) Overview of the ternary complex with HAESA in blue (surface representation), IDA in yellow (bonds representation) and SERK1 in orange (surface view). (B) The HAESA ectodomain undergoes a conformational change upon SERK1 co-receptor binding. FIG +81 84 IDA protein (A) Overview of the ternary complex with HAESA in blue (surface representation), IDA in yellow (bonds representation) and SERK1 in orange (surface view). (B) The HAESA ectodomain undergoes a conformational change upon SERK1 co-receptor binding. FIG +122 127 SERK1 protein (A) Overview of the ternary complex with HAESA in blue (surface representation), IDA in yellow (bonds representation) and SERK1 in orange (surface view). (B) The HAESA ectodomain undergoes a conformational change upon SERK1 co-receptor binding. FIG +162 167 HAESA protein (A) Overview of the ternary complex with HAESA in blue (surface representation), IDA in yellow (bonds representation) and SERK1 in orange (surface view). (B) The HAESA ectodomain undergoes a conformational change upon SERK1 co-receptor binding. FIG +168 178 ectodomain structure_element (A) Overview of the ternary complex with HAESA in blue (surface representation), IDA in yellow (bonds representation) and SERK1 in orange (surface view). (B) The HAESA ectodomain undergoes a conformational change upon SERK1 co-receptor binding. FIG +218 223 SERK1 protein (A) Overview of the ternary complex with HAESA in blue (surface representation), IDA in yellow (bonds representation) and SERK1 in orange (surface view). (B) The HAESA ectodomain undergoes a conformational change upon SERK1 co-receptor binding. FIG +25 49 structural superposition experimental_method Shown are Cα traces of a structural superposition of the unbound (yellow) and SERK1-bound (blue) HAESA ectodomains (r.m.s.d. is 1.5 Å between 572 corresponding Cα atoms). FIG +57 64 unbound protein_state Shown are Cα traces of a structural superposition of the unbound (yellow) and SERK1-bound (blue) HAESA ectodomains (r.m.s.d. is 1.5 Å between 572 corresponding Cα atoms). FIG +78 89 SERK1-bound protein_state Shown are Cα traces of a structural superposition of the unbound (yellow) and SERK1-bound (blue) HAESA ectodomains (r.m.s.d. is 1.5 Å between 572 corresponding Cα atoms). FIG +97 102 HAESA protein Shown are Cα traces of a structural superposition of the unbound (yellow) and SERK1-bound (blue) HAESA ectodomains (r.m.s.d. is 1.5 Å between 572 corresponding Cα atoms). FIG +103 114 ectodomains structure_element Shown are Cα traces of a structural superposition of the unbound (yellow) and SERK1-bound (blue) HAESA ectodomains (r.m.s.d. is 1.5 Å between 572 corresponding Cα atoms). FIG +116 124 r.m.s.d. evidence Shown are Cα traces of a structural superposition of the unbound (yellow) and SERK1-bound (blue) HAESA ectodomains (r.m.s.d. is 1.5 Å between 572 corresponding Cα atoms). FIG +0 5 SERK1 protein SERK1 (in orange) and IDA (in red) are shown alongside. FIG +22 25 IDA protein SERK1 (in orange) and IDA (in red) are shown alongside. FIG +44 48 LRRs structure_element The conformational change in the C-terminal LRRs and capping domain is indicated by an arrow. (C) SERK1 forms an integral part of the receptor's peptide binding pocket. FIG +53 67 capping domain structure_element The conformational change in the C-terminal LRRs and capping domain is indicated by an arrow. (C) SERK1 forms an integral part of the receptor's peptide binding pocket. FIG +98 103 SERK1 protein The conformational change in the C-terminal LRRs and capping domain is indicated by an arrow. (C) SERK1 forms an integral part of the receptor's peptide binding pocket. FIG +145 167 peptide binding pocket site The conformational change in the C-terminal LRRs and capping domain is indicated by an arrow. (C) SERK1 forms an integral part of the receptor's peptide binding pocket. FIG +15 29 capping domain structure_element The N-terminal capping domain of SERK1 (in orange) directly contacts the C-terminal part of IDA (in yellow, in bonds representation) and the receptor HAESA (in blue). FIG +33 38 SERK1 protein The N-terminal capping domain of SERK1 (in orange) directly contacts the C-terminal part of IDA (in yellow, in bonds representation) and the receptor HAESA (in blue). FIG +92 95 IDA protein The N-terminal capping domain of SERK1 (in orange) directly contacts the C-terminal part of IDA (in yellow, in bonds representation) and the receptor HAESA (in blue). FIG +141 149 receptor protein_type The N-terminal capping domain of SERK1 (in orange) directly contacts the C-terminal part of IDA (in yellow, in bonds representation) and the receptor HAESA (in blue). FIG +150 155 HAESA protein The N-terminal capping domain of SERK1 (in orange) directly contacts the C-terminal part of IDA (in yellow, in bonds representation) and the receptor HAESA (in blue). FIG +0 14 Polar contacts bond_interaction Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface. FIG +18 23 SERK1 protein Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface. FIG +29 32 IDA protein Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface. FIG +64 69 HAESA protein Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface. FIG +70 80 LRR domain structure_element Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface. FIG +109 120 zipper-like structure_element Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface. FIG +121 142 SERK1-HAESA interface site Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface. FIG +19 24 HAESA protein Ribbon diagrams of HAESA (in blue) and SERK1 (in orange) are shown with selected interface residues (in bonds representation). FIG +39 44 SERK1 protein Ribbon diagrams of HAESA (in blue) and SERK1 (in orange) are shown with selected interface residues (in bonds representation). FIG +81 99 interface residues site Ribbon diagrams of HAESA (in blue) and SERK1 (in orange) are shown with selected interface residues (in bonds representation). FIG +0 18 Polar interactions bond_interaction Polar interactions are highlighted as dotted lines (in magenta). FIG +37 42 SERK1 protein To understand in molecular terms how SERK1 contributes to high-affinity IDA recognition, we solved a 2.43 Å crystal structure of the ternary HAESA – IDA – SERK1 complex (Figure 4A, Table 2). RESULTS +72 75 IDA protein To understand in molecular terms how SERK1 contributes to high-affinity IDA recognition, we solved a 2.43 Å crystal structure of the ternary HAESA – IDA – SERK1 complex (Figure 4A, Table 2). RESULTS +108 125 crystal structure evidence To understand in molecular terms how SERK1 contributes to high-affinity IDA recognition, we solved a 2.43 Å crystal structure of the ternary HAESA – IDA – SERK1 complex (Figure 4A, Table 2). RESULTS +141 160 HAESA – IDA – SERK1 complex_assembly To understand in molecular terms how SERK1 contributes to high-affinity IDA recognition, we solved a 2.43 Å crystal structure of the ternary HAESA – IDA – SERK1 complex (Figure 4A, Table 2). RESULTS +0 5 HAESA protein HAESA LRRs 16–21 and its C-terminal capping domain undergo a conformational change upon SERK1 binding (Figure 4B). RESULTS +6 16 LRRs 16–21 structure_element HAESA LRRs 16–21 and its C-terminal capping domain undergo a conformational change upon SERK1 binding (Figure 4B). RESULTS +36 50 capping domain structure_element HAESA LRRs 16–21 and its C-terminal capping domain undergo a conformational change upon SERK1 binding (Figure 4B). RESULTS +88 93 SERK1 protein HAESA LRRs 16–21 and its C-terminal capping domain undergo a conformational change upon SERK1 binding (Figure 4B). RESULTS +4 9 SERK1 protein The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C). RESULTS +10 20 ectodomain structure_element The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C). RESULTS +40 64 IDA peptide binding site site The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C). RESULTS +73 84 loop region structure_element The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C). RESULTS +95 100 51-59 residue_range The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C). RESULTS +100 105 SERK1 protein The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C). RESULTS +127 130 cap structure_element The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C). RESULTS +0 5 SERK1 protein SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C). RESULTS +6 10 loop structure_element SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C). RESULTS +39 69 hydrophobic and polar contacts bond_interaction SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C). RESULTS +75 80 Lys66 residue_name_number SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C). RESULTS +80 83 IDA protein SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C). RESULTS +103 120 Arg-His-Asn motif structure_element SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C). RESULTS +124 127 IDA protein SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C). RESULTS +0 5 SERK1 protein SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). RESULTS +6 14 LRRs 1–5 structure_element SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). RESULTS +34 48 capping domain structure_element SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). RESULTS +68 79 zipper-like structure_element SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). RESULTS +80 89 interface site SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). RESULTS +121 126 HAESA protein SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). RESULTS +127 137 LRRs 15–21 structure_element SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). RESULTS +151 156 HAESA protein SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). RESULTS +168 171 cap structure_element SERK1 LRRs 1–5 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15–21 and from the HAESA C-terminal cap (Figure 4D). RESULTS +0 5 SERK1 protein SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1—figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft. RESULTS +12 17 HAESA protein SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1—figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft. RESULTS +43 63 interaction surfaces site SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1—figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft. RESULTS +105 110 N-cap structure_element SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1—figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft. RESULTS +118 123 SERK1 protein SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1—figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft. RESULTS +124 134 LRR domain structure_element SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1—figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft. RESULTS +158 183 IDA peptide binding cleft site SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1—figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft. RESULTS +4 7 IDA protein The IDA C-terminal motif is required for HAESA-SERK1 complex formation and for IDA bioactivity. FIG +8 24 C-terminal motif structure_element The IDA C-terminal motif is required for HAESA-SERK1 complex formation and for IDA bioactivity. FIG +41 52 HAESA-SERK1 complex_assembly The IDA C-terminal motif is required for HAESA-SERK1 complex formation and for IDA bioactivity. FIG +4 33 Size exclusion chromatography experimental_method (A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes. FIG +81 84 IDA protein (A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes. FIG +85 91 mutant protein_state (A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes. FIG +92 100 peptides chemical (A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes. FIG +115 131 C-terminal motif structure_element (A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes. FIG +144 164 biochemically stable protein_state (A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes. FIG +165 180 HAESA-IDA-SERK1 complex_assembly (A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes. FIG +0 8 Deletion experimental_method Deletion of the C-terminal Asn69IDA completely inhibits complex formation. FIG +27 32 Asn69 residue_name_number Deletion of the C-terminal Asn69IDA completely inhibits complex formation. FIG +32 35 IDA protein Deletion of the C-terminal Asn69IDA completely inhibits complex formation. FIG +47 55 inhibits protein_state Deletion of the C-terminal Asn69IDA completely inhibits complex formation. FIG +0 8 Purified experimental_method Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. FIG +9 14 HAESA protein Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. FIG +19 24 SERK1 protein Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. FIG +12 25 IDA K66A/R67A mutant Left panel: IDA K66A/R67A; center: IDA ΔN69, right panel: SDS-PAGE of peak fractions. FIG +35 43 IDA ΔN69 mutant Left panel: IDA K66A/R67A; center: IDA ΔN69, right panel: SDS-PAGE of peak fractions. FIG +58 66 SDS-PAGE experimental_method Left panel: IDA K66A/R67A; center: IDA ΔN69, right panel: SDS-PAGE of peak fractions. FIG +14 19 HAESA protein Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. FIG +24 29 SERK1 protein Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. FIG +84 117 Isothermal titration thermographs evidence Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. FIG +121 130 wild-type protein_state Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. FIG +135 141 mutant protein_state Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. FIG +142 154 IDA peptides chemical Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. FIG +155 163 titrated experimental_method Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. FIG +171 176 HAESA protein Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. FIG +179 184 SERK1 protein Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell. FIG +20 50 calorimetric binding constants evidence Table summaries for calorimetric binding constants and stoichoimetries for different IDA peptides binding to the HAESA – SERK1 ectodomain mixture ( ± fitting errors; n.d. FIG +85 97 IDA peptides chemical Table summaries for calorimetric binding constants and stoichoimetries for different IDA peptides binding to the HAESA – SERK1 ectodomain mixture ( ± fitting errors; n.d. FIG +113 118 HAESA protein Table summaries for calorimetric binding constants and stoichoimetries for different IDA peptides binding to the HAESA – SERK1 ectodomain mixture ( ± fitting errors; n.d. FIG +121 126 SERK1 protein Table summaries for calorimetric binding constants and stoichoimetries for different IDA peptides binding to the HAESA – SERK1 ectodomain mixture ( ± fitting errors; n.d. FIG +127 137 ectodomain structure_element Table summaries for calorimetric binding constants and stoichoimetries for different IDA peptides binding to the HAESA – SERK1 ectodomain mixture ( ± fitting errors; n.d. FIG +17 43 petal break-strength assay experimental_method (C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. FIG +54 63 wild-type protein_state (C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. FIG +76 79 35S gene (C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. FIG +81 84 IDA protein (C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. FIG +85 94 wild-type protein_state (C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. FIG +99 102 35S gene (C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. FIG +104 117 IDA K66A/R67A mutant (C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. FIG +118 124 mutant protein_state (C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers. FIG +0 3 35S gene 35S::IDA plants showed significantly increased abscission compared to Col-0 controls in inflorescence positions 2 and 3 (a). FIG +5 8 IDA protein 35S::IDA plants showed significantly increased abscission compared to Col-0 controls in inflorescence positions 2 and 3 (a). FIG +9 15 plants taxonomy_domain 35S::IDA plants showed significantly increased abscission compared to Col-0 controls in inflorescence positions 2 and 3 (a). FIG +47 50 35S gene Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +52 65 IDA K66A/R67A mutant Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +66 72 mutant protein_state Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +73 79 plants taxonomy_domain Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +139 145 plants taxonomy_domain Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +154 157 35S gene Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +159 162 IDA protein Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +163 169 plants taxonomy_domain Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +283 286 35S gene Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +288 291 IDA protein Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +304 307 35S gene Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +309 322 IDA K66A/R67A mutant Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +339 342 IDA protein Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +343 352 wild-type protein_state Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +357 363 mutant protein_state Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +383 417 35S promoter over-expression lines experimental_method Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +494 497 35S gene Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +499 502 IDA protein Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +510 519 wild-type protein_state Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +524 527 35S gene Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +529 542 IDA K66A/R67A mutant Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +543 556 double-mutant protein_state Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +557 576 T3 transgenic lines experimental_method Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression ± standard error; ida: -0.02 ± 0.001; Col-0: 1 ± 0.11; 35S::IDA 124 ± 0.75; 35S::IDA K66A/R67A: 159 ± 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines. FIG +13 16 35S gene 15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed). FIG +18 21 IDA protein 15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed). FIG +22 28 plants taxonomy_domain 15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed). FIG +48 54 plants taxonomy_domain 15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed). FIG +71 74 35S gene 15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed). FIG +76 89 IDA K66A/R67A mutant 15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed). FIG +90 103 double-mutant protein_state 15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed). FIG +104 110 plants taxonomy_domain 15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed). FIG +32 35 IDA protein The four C-terminal residues in IDA (Lys66IDA-Asn69IDA) are conserved among IDA family members and are in direct contact with SERK1 (Figures 1A, 4C). RESULTS +37 54 Lys66IDA-Asn69IDA residue_range The four C-terminal residues in IDA (Lys66IDA-Asn69IDA) are conserved among IDA family members and are in direct contact with SERK1 (Figures 1A, 4C). RESULTS +60 69 conserved protein_state The four C-terminal residues in IDA (Lys66IDA-Asn69IDA) are conserved among IDA family members and are in direct contact with SERK1 (Figures 1A, 4C). RESULTS +76 94 IDA family members protein_type The four C-terminal residues in IDA (Lys66IDA-Asn69IDA) are conserved among IDA family members and are in direct contact with SERK1 (Figures 1A, 4C). RESULTS +126 131 SERK1 protein The four C-terminal residues in IDA (Lys66IDA-Asn69IDA) are conserved among IDA family members and are in direct contact with SERK1 (Figures 1A, 4C). RESULTS +39 52 HAESA – SERK1 complex_assembly We thus assessed their contribution to HAESA – SERK1 complex formation. RESULTS +0 8 Deletion experimental_method Deletion of the buried Asn69IDA completely inhibits receptor – co-receptor complex formation and HSL2 activation (Figure 5A,B). RESULTS +23 28 Asn69 residue_name_number Deletion of the buried Asn69IDA completely inhibits receptor – co-receptor complex formation and HSL2 activation (Figure 5A,B). RESULTS +28 31 IDA protein Deletion of the buried Asn69IDA completely inhibits receptor – co-receptor complex formation and HSL2 activation (Figure 5A,B). RESULTS +32 51 completely inhibits protein_state Deletion of the buried Asn69IDA completely inhibits receptor – co-receptor complex formation and HSL2 activation (Figure 5A,B). RESULTS +2 11 synthetic protein_state A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). RESULTS +12 35 Lys66IDA/Arg67IDA → Ala mutant A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). RESULTS +36 42 mutant protein_state A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). RESULTS +43 50 peptide chemical A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). RESULTS +52 65 IDA K66A/R66A mutant A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). RESULTS +92 108 binding affinity evidence A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). RESULTS +114 122 titrated experimental_method A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). RESULTS +128 133 HAESA protein A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). RESULTS +134 139 SERK1 protein A synthetic Lys66IDA/Arg67IDA → Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D). RESULTS +3 17 over-expressed experimental_method We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). RESULTS +18 29 full-length protein_state We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). RESULTS +30 39 wild-type protein_state We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). RESULTS +40 43 IDA protein We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). RESULTS +52 75 Lys66IDA/Arg67IDA → Ala mutant We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). RESULTS +76 89 double-mutant protein_state We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). RESULTS +117 128 Arabidopsis taxonomy_domain We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). RESULTS +129 135 plants taxonomy_domain We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA → Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D). RESULTS +14 29 over-expression experimental_method We found that over-expression of wild-type IDA leads to early floral abscission and an enlargement of the abscission zone (Figure 5C–E). RESULTS +33 42 wild-type protein_state We found that over-expression of wild-type IDA leads to early floral abscission and an enlargement of the abscission zone (Figure 5C–E). RESULTS +43 46 IDA protein We found that over-expression of wild-type IDA leads to early floral abscission and an enlargement of the abscission zone (Figure 5C–E). RESULTS +13 28 over-expression experimental_method In contrast, over-expression of the IDA Lys66IDA/Arg67IDA → Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C–E). RESULTS +36 63 IDA Lys66IDA/Arg67IDA → Ala mutant In contrast, over-expression of the IDA Lys66IDA/Arg67IDA → Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C–E). RESULTS +64 77 double mutant protein_state In contrast, over-expression of the IDA Lys66IDA/Arg67IDA → Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C–E). RESULTS +134 143 wild-type protein_state In contrast, over-expression of the IDA Lys66IDA/Arg67IDA → Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C–E). RESULTS +152 158 plants taxonomy_domain In contrast, over-expression of the IDA Lys66IDA/Arg67IDA → Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C–E). RESULTS +180 186 mutant protein_state In contrast, over-expression of the IDA Lys66IDA/Arg67IDA → Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C–E). RESULTS +187 198 IDA peptide chemical In contrast, over-expression of the IDA Lys66IDA/Arg67IDA → Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C–E). RESULTS +223 229 planta taxonomy_domain In contrast, over-expression of the IDA Lys66IDA/Arg67IDA → Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C–E). RESULTS +14 17 35S gene Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA → Ala may cause a weak dominant negative effect (Figure 5C–E). RESULTS +19 22 IDA protein Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA → Ala may cause a weak dominant negative effect (Figure 5C–E). RESULTS +23 32 wild-type protein_state Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA → Ala may cause a weak dominant negative effect (Figure 5C–E). RESULTS +37 43 mutant protein_state Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA → Ala may cause a weak dominant negative effect (Figure 5C–E). RESULTS +44 50 plants taxonomy_domain Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA → Ala may cause a weak dominant negative effect (Figure 5C–E). RESULTS +74 82 mutation experimental_method Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA → Ala may cause a weak dominant negative effect (Figure 5C–E). RESULTS +86 109 Lys66IDA/Arg67IDA → Ala mutant Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA → Ala may cause a weak dominant negative effect (Figure 5C–E). RESULTS +22 32 structures evidence In agreement with our structures and biochemical assays, this experiment suggests a role of the conserved IDA C-terminus in the control of floral abscission. RESULTS +37 55 biochemical assays experimental_method In agreement with our structures and biochemical assays, this experiment suggests a role of the conserved IDA C-terminus in the control of floral abscission. RESULTS +96 105 conserved protein_state In agreement with our structures and biochemical assays, this experiment suggests a role of the conserved IDA C-terminus in the control of floral abscission. RESULTS +106 109 IDA protein In agreement with our structures and biochemical assays, this experiment suggests a role of the conserved IDA C-terminus in the control of floral abscission. RESULTS +15 21 animal taxonomy_domain In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation. DISCUSS +22 35 LRR receptors protein_type In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation. DISCUSS +37 42 plant taxonomy_domain In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation. DISCUSS +43 50 LRR-RKs structure_element In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation. DISCUSS +58 71 spiral-shaped protein_state In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation. DISCUSS +72 83 ectodomains structure_element In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation. DISCUSS +106 125 shape-complementary protein_state In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation. DISCUSS +126 146 co-receptor proteins protein_type In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation. DISCUSS +32 37 plant taxonomy_domain For a rapidly growing number of plant signaling pathways, SERK proteins act as these essential co-receptors (; ). DISCUSS +58 71 SERK proteins protein_type For a rapidly growing number of plant signaling pathways, SERK proteins act as these essential co-receptors (; ). DISCUSS +95 107 co-receptors protein_type For a rapidly growing number of plant signaling pathways, SERK proteins act as these essential co-receptors (; ). DISCUSS +63 68 plant taxonomy_domain  SERK1 has been previously reported as a positive regulator in plant embryogenesis, male sporogenesis, brassinosteroid signaling and in phytosulfokine perception. DISCUSS +84 89 SERK1 protein Recent findings by and our mechanistic studies now also support a positive role for SERK1 in floral abscission. DISCUSS +3 10 serk1-1 gene As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A). DISCUSS +11 17 mutant protein_state As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A). DISCUSS +18 24 plants taxonomy_domain As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A). DISCUSS +82 87 haesa gene As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A). DISCUSS +93 100 mutants protein_state As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A). DISCUSS +102 107 SERK1 protein As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A). DISCUSS +143 148 SERKs protein_type As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A). DISCUSS +38 43 SERK1 protein It has been previously suggested that SERK1 can inhibit cell separation. DISCUSS +30 35 SERK1 protein However our results show that SERK1 also can activate this process upon IDA sensing, indicating that SERKs may fulfill several different functions in the course of the abscission process. DISCUSS +72 75 IDA protein However our results show that SERK1 also can activate this process upon IDA sensing, indicating that SERKs may fulfill several different functions in the course of the abscission process. DISCUSS +101 106 SERKs protein_type However our results show that SERK1 also can activate this process upon IDA sensing, indicating that SERKs may fulfill several different functions in the course of the abscission process. DISCUSS +26 32 mature protein_state While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. DISCUSS +33 44 IDA peptide chemical While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. DISCUSS +87 93 planta taxonomy_domain While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. DISCUSS +99 108 HAESA-IDA complex_assembly While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. DISCUSS +117 127 structures evidence While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. DISCUSS +132 150 calorimetry assays evidence While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. DISCUSS +164 170 active protein_state While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. DISCUSS +171 175 IDLs protein_type While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. DISCUSS +180 197 hydroxyprolinated protein_state While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. DISCUSS +198 208 dodecamers structure_element While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers. DISCUSS +64 75 full-length protein_state It will be thus interesting to see if proteolytic processing of full-length IDA in vivo is regulated in a cell-type or tissue-specific manner. DISCUSS +76 79 IDA protein It will be thus interesting to see if proteolytic processing of full-length IDA in vivo is regulated in a cell-type or tissue-specific manner. DISCUSS +12 15 Hyp residue_name The central Hyp residue in IDA is found buried in the HAESA peptide binding surface and thus this post-translational modification may regulate IDA bioactivity. DISCUSS +27 30 IDA protein The central Hyp residue in IDA is found buried in the HAESA peptide binding surface and thus this post-translational modification may regulate IDA bioactivity. DISCUSS +54 59 HAESA protein The central Hyp residue in IDA is found buried in the HAESA peptide binding surface and thus this post-translational modification may regulate IDA bioactivity. DISCUSS +60 83 peptide binding surface site The central Hyp residue in IDA is found buried in the HAESA peptide binding surface and thus this post-translational modification may regulate IDA bioactivity. DISCUSS +143 146 IDA protein The central Hyp residue in IDA is found buried in the HAESA peptide binding surface and thus this post-translational modification may regulate IDA bioactivity. DISCUSS +4 51 comparative structural and biochemical analysis experimental_method Our comparative structural and biochemical analysis further suggests that IDLs share a common receptor binding mode, but may preferably bind to HAESA, HSL1 or HSL2 in different plant tissues and organs. DISCUSS +74 78 IDLs protein_type Our comparative structural and biochemical analysis further suggests that IDLs share a common receptor binding mode, but may preferably bind to HAESA, HSL1 or HSL2 in different plant tissues and organs. DISCUSS +144 149 HAESA protein Our comparative structural and biochemical analysis further suggests that IDLs share a common receptor binding mode, but may preferably bind to HAESA, HSL1 or HSL2 in different plant tissues and organs. DISCUSS +151 155 HSL1 protein Our comparative structural and biochemical analysis further suggests that IDLs share a common receptor binding mode, but may preferably bind to HAESA, HSL1 or HSL2 in different plant tissues and organs. DISCUSS +159 163 HSL2 protein Our comparative structural and biochemical analysis further suggests that IDLs share a common receptor binding mode, but may preferably bind to HAESA, HSL1 or HSL2 in different plant tissues and organs. DISCUSS +177 182 plant taxonomy_domain Our comparative structural and biochemical analysis further suggests that IDLs share a common receptor binding mode, but may preferably bind to HAESA, HSL1 or HSL2 in different plant tissues and organs. DISCUSS +7 38 quantitative biochemical assays experimental_method In our quantitative biochemical assays, the presence of SERK1 dramatically increases the HAESA binding specificity and affinity for IDA. DISCUSS +44 55 presence of protein_state In our quantitative biochemical assays, the presence of SERK1 dramatically increases the HAESA binding specificity and affinity for IDA. DISCUSS +56 61 SERK1 protein In our quantitative biochemical assays, the presence of SERK1 dramatically increases the HAESA binding specificity and affinity for IDA. DISCUSS +89 94 HAESA protein In our quantitative biochemical assays, the presence of SERK1 dramatically increases the HAESA binding specificity and affinity for IDA. DISCUSS +132 135 IDA protein In our quantitative biochemical assays, the presence of SERK1 dramatically increases the HAESA binding specificity and affinity for IDA. DISCUSS +48 57 structure evidence This observation is consistent with our complex structure in which receptor and co-receptor together form the IDA binding pocket. DISCUSS +110 128 IDA binding pocket site This observation is consistent with our complex structure in which receptor and co-receptor together form the IDA binding pocket. DISCUSS +14 19 SERK1 protein The fact that SERK1 specifically interacts with the very C-terminus of IDLs may allow for the rational design of peptide hormone antagonists, as previously demonstrated for the brassinosteroid pathway. DISCUSS +71 75 IDLs protein_type The fact that SERK1 specifically interacts with the very C-terminus of IDLs may allow for the rational design of peptide hormone antagonists, as previously demonstrated for the brassinosteroid pathway. DISCUSS +113 140 peptide hormone antagonists chemical The fact that SERK1 specifically interacts with the very C-terminus of IDLs may allow for the rational design of peptide hormone antagonists, as previously demonstrated for the brassinosteroid pathway. DISCUSS +17 35 calorimetry assays experimental_method Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C). DISCUSS +52 57 SERK1 protein Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C). DISCUSS +58 68 ectodomain structure_element Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C). DISCUSS +69 74 binds protein_state Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C). DISCUSS +75 80 HAESA protein Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C). DISCUSS +122 133 presence of protein_state Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C). DISCUSS +134 137 IDA protein Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C). DISCUSS +80 85 HAESA protein This ligand-induced formation of a receptor – co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm. DISCUSS +90 95 SERK1 protein This ligand-induced formation of a receptor – co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm. DISCUSS +96 110 kinase domains structure_element This ligand-induced formation of a receptor – co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm. DISCUSS +32 50 binding affinities evidence It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. DISCUSS +55 58 IDA protein It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. DISCUSS +63 68 SERK1 protein It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. DISCUSS +94 103 synthetic protein_state It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. DISCUSS +104 112 peptides chemical It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. DISCUSS +121 129 isolated experimental_method It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. DISCUSS +130 135 HAESA protein It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. DISCUSS +140 145 SERK1 protein It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. DISCUSS +146 157 ectodomains structure_element It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. DISCUSS +203 214 full-length protein_state It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. DISCUSS +216 233 membrane-embedded protein_state It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus might differ in the context of the full-length, membrane-embedded signaling complex. DISCUSS +0 5 SERK1 protein SERK1 uses partially overlapping surface areas to activate different plant signaling receptors. FIG +69 74 plant taxonomy_domain SERK1 uses partially overlapping surface areas to activate different plant signaling receptors. FIG +75 94 signaling receptors protein_type SERK1 uses partially overlapping surface areas to activate different plant signaling receptors. FIG +4 25 Structural comparison experimental_method (A) Structural comparison of plant steroid and peptide hormone membrane signaling complexes. FIG +29 34 plant taxonomy_domain (A) Structural comparison of plant steroid and peptide hormone membrane signaling complexes. FIG +35 42 steroid chemical (A) Structural comparison of plant steroid and peptide hormone membrane signaling complexes. FIG +47 62 peptide hormone protein_type (A) Structural comparison of plant steroid and peptide hormone membrane signaling complexes. FIG +63 91 membrane signaling complexes protein_type (A) Structural comparison of plant steroid and peptide hormone membrane signaling complexes. FIG +30 35 HAESA protein Left panel: Ribbon diagram of HAESA (in blue), SERK1 (in orange) and IDA (in bonds and surface represention). FIG +47 52 SERK1 protein Left panel: Ribbon diagram of HAESA (in blue), SERK1 (in orange) and IDA (in bonds and surface represention). FIG +69 72 IDA protein Left panel: Ribbon diagram of HAESA (in blue), SERK1 (in orange) and IDA (in bonds and surface represention). FIG +35 40 plant taxonomy_domain Right panel: Ribbon diagram of the plant steroid receptor BRI1 (in blue) bound to brassinolide (in gray, in bonds representation) and to SERK1, shown in the same orientation (PDB-ID. 4lsx). FIG +41 57 steroid receptor protein_type Right panel: Ribbon diagram of the plant steroid receptor BRI1 (in blue) bound to brassinolide (in gray, in bonds representation) and to SERK1, shown in the same orientation (PDB-ID. 4lsx). FIG +58 62 BRI1 protein Right panel: Ribbon diagram of the plant steroid receptor BRI1 (in blue) bound to brassinolide (in gray, in bonds representation) and to SERK1, shown in the same orientation (PDB-ID. 4lsx). FIG +73 81 bound to protein_state Right panel: Ribbon diagram of the plant steroid receptor BRI1 (in blue) bound to brassinolide (in gray, in bonds representation) and to SERK1, shown in the same orientation (PDB-ID. 4lsx). FIG +82 94 brassinolide chemical Right panel: Ribbon diagram of the plant steroid receptor BRI1 (in blue) bound to brassinolide (in gray, in bonds representation) and to SERK1, shown in the same orientation (PDB-ID. 4lsx). FIG +137 142 SERK1 protein Right panel: Ribbon diagram of the plant steroid receptor BRI1 (in blue) bound to brassinolide (in gray, in bonds representation) and to SERK1, shown in the same orientation (PDB-ID. 4lsx). FIG +37 42 SERK1 protein (B) View of the inner surface of the SERK1 LRR domain (PDB-ID 4lsc, surface representation, in gray). FIG +43 53 LRR domain structure_element (B) View of the inner surface of the SERK1 LRR domain (PDB-ID 4lsc, surface representation, in gray). FIG +20 25 SERK1 protein A ribbon diagram of SERK1 in the same orientation is shown alongside. FIG +30 35 HAESA protein Residues interacting with the HAESA or BRI1 LRR domains are shown in orange or magenta, respectively. FIG +39 43 BRI1 protein Residues interacting with the HAESA or BRI1 LRR domains are shown in orange or magenta, respectively. FIG +44 55 LRR domains structure_element Residues interacting with the HAESA or BRI1 LRR domains are shown in orange or magenta, respectively. FIG +0 10 Comparison experimental_method Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +18 37 HAESA – IDA – SERK1 complex_assembly Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +38 47 structure evidence Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +107 112 SERK1 protein Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +126 137 co-receptor protein_type Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +158 167 conserved protein_state Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +176 181 SERK1 protein Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +201 223 ligand binding pockets site Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +284 295 LRRs 2 – 14 structure_element Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +297 302 HAESA protein Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +304 316 LRRs 21 – 25 structure_element Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +318 322 BRI1 protein Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +358 362 BRI1 protein Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +372 377 HAESA protein Comparison of our HAESA – IDA – SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 – 14; HAESA; LRRs 21 – 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A). DISCUSS +24 29 SERK1 protein Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +41 55 capping domain structure_element Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +57 62 Thr59 residue_name_number Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +62 67 SERK1 protein Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +69 74 Phe61 residue_name_number Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +74 79 SERK1 protein Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +89 106 LRR inner surface site Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +108 113 Asp75 residue_name_number Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +113 118 SERK1 protein Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +120 126 Tyr101 residue_name_number Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +126 131 SERK1 protein Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +133 139 SER121 residue_name_number Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +139 144 SERK1 protein Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +146 152 Phe145 residue_name_number Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +152 157 SERK1 protein Several residues in the SERK1 N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B). DISCUSS +22 27 53-55 residue_range In addition, residues 53-55SERK1 from the SERK1 N-terminal cap mediate specific interactions with the IDA peptide (Figures 4C, 6B). DISCUSS +27 32 SERK1 protein In addition, residues 53-55SERK1 from the SERK1 N-terminal cap mediate specific interactions with the IDA peptide (Figures 4C, 6B). DISCUSS +42 47 SERK1 protein In addition, residues 53-55SERK1 from the SERK1 N-terminal cap mediate specific interactions with the IDA peptide (Figures 4C, 6B). DISCUSS +59 62 cap structure_element In addition, residues 53-55SERK1 from the SERK1 N-terminal cap mediate specific interactions with the IDA peptide (Figures 4C, 6B). DISCUSS +102 113 IDA peptide chemical In addition, residues 53-55SERK1 from the SERK1 N-terminal cap mediate specific interactions with the IDA peptide (Figures 4C, 6B). DISCUSS +54 69 steroid hormone chemical These residues are not involved in the sensing of the steroid hormone brassinolide. DISCUSS +70 82 brassinolide chemical These residues are not involved in the sensing of the steroid hormone brassinolide. DISCUSS +53 75 hormone binding pocket site In both cases however, the co-receptor completes the hormone binding pocket. DISCUSS +48 70 SERK1 binding surfaces site This fact together with the largely overlapping SERK1 binding surfaces in HAESA and BRI1 allows us to speculate that SERK1 may promote high-affinity peptide hormone and brassinosteroid sensing by simply slowing down dissociation of the ligand from its cognate receptor. DISCUSS +74 79 HAESA protein This fact together with the largely overlapping SERK1 binding surfaces in HAESA and BRI1 allows us to speculate that SERK1 may promote high-affinity peptide hormone and brassinosteroid sensing by simply slowing down dissociation of the ligand from its cognate receptor. DISCUSS +84 88 BRI1 protein This fact together with the largely overlapping SERK1 binding surfaces in HAESA and BRI1 allows us to speculate that SERK1 may promote high-affinity peptide hormone and brassinosteroid sensing by simply slowing down dissociation of the ligand from its cognate receptor. DISCUSS +117 122 SERK1 protein This fact together with the largely overlapping SERK1 binding surfaces in HAESA and BRI1 allows us to speculate that SERK1 may promote high-affinity peptide hormone and brassinosteroid sensing by simply slowing down dissociation of the ligand from its cognate receptor. DISCUSS +149 164 peptide hormone protein_type This fact together with the largely overlapping SERK1 binding surfaces in HAESA and BRI1 allows us to speculate that SERK1 may promote high-affinity peptide hormone and brassinosteroid sensing by simply slowing down dissociation of the ligand from its cognate receptor. DISCUSS +10 15 plant taxonomy_domain Different plant peptide hormone families contain a C-terminal (Arg)-His-Asn motif, which in IDA represents the co-receptor recognition site. FIG +16 40 peptide hormone families protein_type Different plant peptide hormone families contain a C-terminal (Arg)-His-Asn motif, which in IDA represents the co-receptor recognition site. FIG +62 81 (Arg)-His-Asn motif structure_element Different plant peptide hormone families contain a C-terminal (Arg)-His-Asn motif, which in IDA represents the co-receptor recognition site. FIG +92 95 IDA protein Different plant peptide hormone families contain a C-terminal (Arg)-His-Asn motif, which in IDA represents the co-receptor recognition site. FIG +111 139 co-receptor recognition site site Different plant peptide hormone families contain a C-terminal (Arg)-His-Asn motif, which in IDA represents the co-receptor recognition site. FIG +0 44 Structure-guided multiple sequence alignment experimental_method Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. FIG +48 51 IDA protein Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. FIG +56 73 IDA-like peptides chemical Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. FIG +85 90 plant taxonomy_domain Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. FIG +91 115 peptide hormone families protein_type Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. FIG +127 171 CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED protein_type Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. FIG +173 181 CLV3/CLE protein_type Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. FIG +184 211 ROOT GROWTH FACTOR – GOLVEN protein_type Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. FIG +213 220 RGF/GLV protein_type Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. FIG +223 245 PRECURSOR GENE PROPEP1 protein_type Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. FIG +247 251 PEP1 protein_type Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. FIG +258 278 Arabidopsis thaliana species Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 – EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR – GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana. FIG +4 13 conserved protein_state The conserved (Arg)-His-Asn motif is highlighted in red, the central Hyp residue in IDLs and CLEs is marked in blue. FIG +14 33 (Arg)-His-Asn motif structure_element The conserved (Arg)-His-Asn motif is highlighted in red, the central Hyp residue in IDLs and CLEs is marked in blue. FIG +69 72 Hyp residue_name The conserved (Arg)-His-Asn motif is highlighted in red, the central Hyp residue in IDLs and CLEs is marked in blue. FIG +84 88 IDLs protein_type The conserved (Arg)-His-Asn motif is highlighted in red, the central Hyp residue in IDLs and CLEs is marked in blue. FIG +93 97 CLEs protein_type The conserved (Arg)-His-Asn motif is highlighted in red, the central Hyp residue in IDLs and CLEs is marked in blue. FIG +28 33 SERK1 protein Our experiments reveal that SERK1 recognizes a C-terminal Arg-His-Asn motif in IDA. DISCUSS +58 75 Arg-His-Asn motif structure_element Our experiments reveal that SERK1 recognizes a C-terminal Arg-His-Asn motif in IDA. DISCUSS +79 82 IDA protein Our experiments reveal that SERK1 recognizes a C-terminal Arg-His-Asn motif in IDA. DISCUSS +13 23 this motif structure_element Importantly, this motif can also be found in other peptide hormone families (Figure 7). DISCUSS +51 75 peptide hormone families protein_type Importantly, this motif can also be found in other peptide hormone families (Figure 7). DISCUSS +20 32 CLE peptides chemical Among these are the CLE peptides regulating stem cell maintenance in the shoot and the root. DISCUSS +32 36 CLEs protein_type It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA. DISCUSS +46 57 mature form protein_state It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA. DISCUSS +67 84 hydroxyprolinated protein_state It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA. DISCUSS +85 95 dodecamers structure_element It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA. DISCUSS +113 125 surface area site It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA. DISCUSS +133 163 BARELY ANY MERISTEM 1 receptor protein_type It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA. DISCUSS +201 218 IDA binding cleft site It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA. DISCUSS +222 227 HAESA protein It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA. DISCUSS +8 13 plant taxonomy_domain Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation. DISCUSS +14 30 peptide hormones protein_type Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation. DISCUSS +56 72 LRR-RK receptors protein_type Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation. DISCUSS +79 100 extended conformation protein_state Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation. DISCUSS +132 142 LRR domain structure_element Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation. DISCUSS +160 165 small protein_state Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation. DISCUSS +167 186 shape-complementary protein_state Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation. DISCUSS +187 199 co-receptors protein_type Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation. DISCUSS diff --git a/annotation_CSV/PMC4848761.csv b/annotation_CSV/PMC4848761.csv new file mode 100644 index 0000000000000000000000000000000000000000..026a97c51b46886a64c8b4c24e0f1d52f30893e5 --- /dev/null +++ b/annotation_CSV/PMC4848761.csv @@ -0,0 +1,885 @@ +anno_start anno_end anno_text entity_type sentence section +61 78 estrogen receptor protein_type Predictive features of ligand‐specific signaling through the estrogen receptor TITLE +5 24 estrogen receptor‐α protein Some estrogen receptor‐α (ERα)‐targeted breast cancer therapies such as tamoxifen have tissue‐selective or cell‐specific activities, while others have similar activities in different cell types. ABSTRACT +26 29 ERα protein Some estrogen receptor‐α (ERα)‐targeted breast cancer therapies such as tamoxifen have tissue‐selective or cell‐specific activities, while others have similar activities in different cell types. ABSTRACT +72 81 tamoxifen chemical Some estrogen receptor‐α (ERα)‐targeted breast cancer therapies such as tamoxifen have tissue‐selective or cell‐specific activities, while others have similar activities in different cell types. ABSTRACT +105 116 synthesized experimental_method To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. ABSTRACT +121 124 ERα protein To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. ABSTRACT +200 222 quantitative bioassays experimental_method To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. ABSTRACT +237 240 ERα protein To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. ABSTRACT +256 277 X‐ray crystallography experimental_method To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. ABSTRACT +56 80 coactivator‐binding site site Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. ABSTRACT +99 120 ligand‐binding domain structure_element Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. ABSTRACT +129 150 activation function‐2 structure_element Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. ABSTRACT +152 156 AF‐2 structure_element Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. ABSTRACT +134 143 NCOA1/2/3 protein Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. ABSTRACT +165 170 GREB1 protein Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. ABSTRACT +33 54 inter‐atomic distance evidence For some ligand series, a single inter‐atomic distance in the ligand‐binding domain predicted their proliferative effects. ABSTRACT +62 83 ligand‐binding domain structure_element For some ligand series, a single inter‐atomic distance in the ligand‐binding domain predicted their proliferative effects. ABSTRACT +28 52 coactivator‐binding site site In contrast, the N‐terminal coactivator‐binding site, activation function‐1 (AF‐1), determined cell‐specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. ABSTRACT +54 75 activation function‐1 structure_element In contrast, the N‐terminal coactivator‐binding site, activation function‐1 (AF‐1), determined cell‐specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. ABSTRACT +77 81 AF‐1 structure_element In contrast, the N‐terminal coactivator‐binding site, activation function‐1 (AF‐1), determined cell‐specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. ABSTRACT +20 47 systems structural analyses experimental_method Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα. ABSTRACT +53 82 quantitative chemical biology experimental_method Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα. ABSTRACT +162 165 ERα protein Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα. ABSTRACT +82 109 G protein‐coupled receptors protein_type Many drugs are small‐molecule ligands of allosteric signaling proteins, including G protein‐coupled receptors (GPCRs) and nuclear receptors such as ERα. INTRO +111 116 GPCRs protein_type Many drugs are small‐molecule ligands of allosteric signaling proteins, including G protein‐coupled receptors (GPCRs) and nuclear receptors such as ERα. INTRO +122 139 nuclear receptors protein_type Many drugs are small‐molecule ligands of allosteric signaling proteins, including G protein‐coupled receptors (GPCRs) and nuclear receptors such as ERα. INTRO +148 151 ERα protein Many drugs are small‐molecule ligands of allosteric signaling proteins, including G protein‐coupled receptors (GPCRs) and nuclear receptors such as ERα. INTRO +146 165 ligand‐binding site site Small‐molecule ligands control receptor activity by modulating recruitment of effector enzymes to distal regions of the receptor, relative to the ligand‐binding site. INTRO +23 51 estrogen receptor modulators protein_type For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003). INTRO +53 58 SERMs protein_type For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003). INTRO +68 77 tamoxifen chemical For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003). INTRO +79 88 Nolvadex® chemical For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003). INTRO +106 116 raloxifene chemical For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003). INTRO +118 125 Evista® chemical For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003). INTRO +157 160 ERα protein For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003). INTRO +206 214 estrogen chemical For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003). INTRO +216 229 17β‐estradiol chemical For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003). INTRO +231 233 E2 chemical For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003). INTRO +22 25 ERα protein Allosteric control of ERα activity FIG +35 38 ERα protein Chemical structures of some common ERα ligands. FIG +0 2 E2 chemical E2‐rings are numbered A‐D. The E‐ring is the common site of attachment for BSC found in many SERMS. FIG +93 98 SERMS protein_type E2‐rings are numbered A‐D. The E‐ring is the common site of attachment for BSC found in many SERMS. FIG +0 3 ERα protein ERα domain organization lettered, A‐F. DBD, DNA‐binding domain; LBD, ligand‐binding domain; AF, activation function FIG +39 42 DBD structure_element ERα domain organization lettered, A‐F. DBD, DNA‐binding domain; LBD, ligand‐binding domain; AF, activation function FIG +44 62 DNA‐binding domain structure_element ERα domain organization lettered, A‐F. DBD, DNA‐binding domain; LBD, ligand‐binding domain; AF, activation function FIG +64 67 LBD structure_element ERα domain organization lettered, A‐F. DBD, DNA‐binding domain; LBD, ligand‐binding domain; AF, activation function FIG +69 90 ligand‐binding domain structure_element ERα domain organization lettered, A‐F. DBD, DNA‐binding domain; LBD, ligand‐binding domain; AF, activation function FIG +92 94 AF structure_element ERα domain organization lettered, A‐F. DBD, DNA‐binding domain; LBD, ligand‐binding domain; AF, activation function FIG +96 115 activation function structure_element ERα domain organization lettered, A‐F. DBD, DNA‐binding domain; LBD, ligand‐binding domain; AF, activation function FIG +40 43 ERα protein Schematic illustration of the canonical ERα signaling pathway. FIG +27 30 ERα protein Linear causality model for ERα‐mediated cell proliferation. FIG +29 32 ERα protein Branched causality model for ERα‐mediated cell proliferation. FIG +0 3 ERα protein ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +13 35 structurally conserved protein_state ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +36 52 globular domains structure_element ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +60 88 nuclear receptor superfamily protein_type ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +102 120 DNA‐binding domain structure_element ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +122 125 DBD structure_element ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +150 158 flexible protein_state ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +159 171 hinge region structure_element ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +179 200 ligand‐binding domain structure_element ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +202 205 LBD structure_element ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +219 231 unstructured protein_state ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +232 234 AB structure_element ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +239 240 F structure_element ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). INTRO +4 7 LBD structure_element The LBD contains a ligand‐dependent coactivator‐binding site called activation function‐2 (AF‐2). INTRO +36 60 coactivator‐binding site site The LBD contains a ligand‐dependent coactivator‐binding site called activation function‐2 (AF‐2). INTRO +68 89 activation function‐2 structure_element The LBD contains a ligand‐dependent coactivator‐binding site called activation function‐2 (AF‐2). INTRO +91 95 AF‐2 structure_element The LBD contains a ligand‐dependent coactivator‐binding site called activation function‐2 (AF‐2). INTRO +33 38 SERMs protein_type However, the agonist activity of SERMs derives from activation function‐1 (AF‐1)—a coactivator recruitment site located in the AB domain (Berry et al, 1990; Shang & Brown, 2002; Abot et al, 2013). INTRO +52 73 activation function‐1 structure_element However, the agonist activity of SERMs derives from activation function‐1 (AF‐1)—a coactivator recruitment site located in the AB domain (Berry et al, 1990; Shang & Brown, 2002; Abot et al, 2013). INTRO +75 79 AF‐1 structure_element However, the agonist activity of SERMs derives from activation function‐1 (AF‐1)—a coactivator recruitment site located in the AB domain (Berry et al, 1990; Shang & Brown, 2002; Abot et al, 2013). INTRO +83 111 coactivator recruitment site site However, the agonist activity of SERMs derives from activation function‐1 (AF‐1)—a coactivator recruitment site located in the AB domain (Berry et al, 1990; Shang & Brown, 2002; Abot et al, 2013). INTRO +127 129 AB structure_element However, the agonist activity of SERMs derives from activation function‐1 (AF‐1)—a coactivator recruitment site located in the AB domain (Berry et al, 1990; Shang & Brown, 2002; Abot et al, 2013). INTRO +0 4 AF‐1 structure_element AF‐1 and AF‐2 bind distinct but overlapping sets of coregulators (Webb et al, 1998; Endoh et al, 1999; Delage‐Mourroux et al, 2000; Yi et al, 2015). INTRO +9 13 AF‐2 structure_element AF‐1 and AF‐2 bind distinct but overlapping sets of coregulators (Webb et al, 1998; Endoh et al, 1999; Delage‐Mourroux et al, 2000; Yi et al, 2015). INTRO +0 4 AF‐2 structure_element AF‐2 binds the signature LxxLL motif peptides of coactivators such as NCOA1/2/3 (also known as SRC‐1/2/3). INTRO +25 36 LxxLL motif structure_element AF‐2 binds the signature LxxLL motif peptides of coactivators such as NCOA1/2/3 (also known as SRC‐1/2/3). INTRO +70 79 NCOA1/2/3 protein AF‐2 binds the signature LxxLL motif peptides of coactivators such as NCOA1/2/3 (also known as SRC‐1/2/3). INTRO +95 104 SRC‐1/2/3 protein AF‐2 binds the signature LxxLL motif peptides of coactivators such as NCOA1/2/3 (also known as SRC‐1/2/3). INTRO +0 4 AF‐1 structure_element AF‐1 binds a separate surface on these coactivators (Webb et al, 1998; Yi et al, 2015). INTRO +33 36 ERα protein Yet, it is unknown how different ERα ligands control AF‐1 through the LBD, and whether this inter‐domain communication is required for cell‐specific signaling or anti‐proliferative responses. INTRO +53 57 AF‐1 structure_element Yet, it is unknown how different ERα ligands control AF‐1 through the LBD, and whether this inter‐domain communication is required for cell‐specific signaling or anti‐proliferative responses. INTRO +70 73 LBD structure_element Yet, it is unknown how different ERα ligands control AF‐1 through the LBD, and whether this inter‐domain communication is required for cell‐specific signaling or anti‐proliferative responses. INTRO +30 33 ERα protein In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson & O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). INTRO +62 70 E2‐bound protein_state In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson & O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). INTRO +71 74 ERα protein In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson & O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). INTRO +83 92 homodimer oligomeric_state In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson & O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). INTRO +111 137 estrogen‐response elements site In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson & O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). INTRO +139 143 EREs site In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson & O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). INTRO +155 164 NCOA1/2/3 protein In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson & O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). INTRO +233 238 GREB1 protein In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson & O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). INTRO +284 287 ERα protein In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson & O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). INTRO +9 12 ERα protein However, ERα‐mediated proliferative responses vary in a ligand‐dependent manner (Srinivasan et al, 2013); thus, it is not known whether this canonical model is widely applicable across diverse ERα ligands. INTRO +193 196 ERα protein However, ERα‐mediated proliferative responses vary in a ligand‐dependent manner (Srinivasan et al, 2013); thus, it is not known whether this canonical model is widely applicable across diverse ERα ligands. INTRO +131 148 crystal structure evidence Our long‐term goal is to be able to predict proliferative or anti‐proliferative activity of a ligand in different tissues from its crystal structure by identifying different structural perturbations that lead to specific signaling outcomes. INTRO +119 128 NCOA1/2/3 protein The simplest response model for ligand‐specific proliferative effects is a linear causality model, where the degree of NCOA1/2/3 recruitment determines GREB1 expression, which in turn drives ligand‐specific cell proliferation (Fig 1D). INTRO +152 157 GREB1 protein The simplest response model for ligand‐specific proliferative effects is a linear causality model, where the degree of NCOA1/2/3 recruitment determines GREB1 expression, which in turn drives ligand‐specific cell proliferation (Fig 1D). INTRO +126 129 LBD structure_element In this signaling model, multiple coregulator binding events and target genes (Won Jeong et al, 2012; Nwachukwu et al, 2014), LBD conformation, nucleocytoplasmic shuttling, the occupancy and dynamics of DNA binding, and other biophysical features could contribute independently to cell proliferation (Lickwar et al, 2012). INTRO +65 68 ERα protein To test these signaling models, we profiled a diverse library of ERα ligands using systems biology approaches to X‐ray crystallography and chemical biology (Srinivasan et al, 2013), including a series of quantitative bioassays for ERα function that were statistically robust and reproducible, based on the Z’‐statistic (Fig EV1A and B; see Materials and Methods). INTRO +113 134 X‐ray crystallography experimental_method To test these signaling models, we profiled a diverse library of ERα ligands using systems biology approaches to X‐ray crystallography and chemical biology (Srinivasan et al, 2013), including a series of quantitative bioassays for ERα function that were statistically robust and reproducible, based on the Z’‐statistic (Fig EV1A and B; see Materials and Methods). INTRO +139 155 chemical biology experimental_method To test these signaling models, we profiled a diverse library of ERα ligands using systems biology approaches to X‐ray crystallography and chemical biology (Srinivasan et al, 2013), including a series of quantitative bioassays for ERα function that were statistically robust and reproducible, based on the Z’‐statistic (Fig EV1A and B; see Materials and Methods). INTRO +231 234 ERα protein To test these signaling models, we profiled a diverse library of ERα ligands using systems biology approaches to X‐ray crystallography and chemical biology (Srinivasan et al, 2013), including a series of quantitative bioassays for ERα function that were statistically robust and reproducible, based on the Z’‐statistic (Fig EV1A and B; see Materials and Methods). INTRO +306 318 Z’‐statistic evidence To test these signaling models, we profiled a diverse library of ERα ligands using systems biology approaches to X‐ray crystallography and chemical biology (Srinivasan et al, 2013), including a series of quantitative bioassays for ERα function that were statistically robust and reproducible, based on the Z’‐statistic (Fig EV1A and B; see Materials and Methods). INTRO +8 18 determined experimental_method We also determined the structures of 76 distinct ERα LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ERα LBD. INTRO +23 33 structures evidence We also determined the structures of 76 distinct ERα LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ERα LBD. INTRO +49 52 ERα protein We also determined the structures of 76 distinct ERα LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ERα LBD. INTRO +53 56 LBD structure_element We also determined the structures of 76 distinct ERα LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ERα LBD. INTRO +67 75 bound to protein_state We also determined the structures of 76 distinct ERα LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ERα LBD. INTRO +172 178 active protein_state We also determined the structures of 76 distinct ERα LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ERα LBD. INTRO +199 202 ERα protein We also determined the structures of 76 distinct ERα LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ERα LBD. INTRO +203 206 LBD structure_element We also determined the structures of 76 distinct ERα LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ERα LBD. INTRO +297 306 estrogens chemical Our findings here indicate that specific structural perturbations can be tied to ligand‐selective domain usage and signaling patterns, thus providing a framework for structure‐based design of improved breast cancer therapeutics, and understanding the different phenotypic effects of environmental estrogens. INTRO +28 31 ERα protein High‐throughput screens for ERα ligand profiling FIG +11 34 ligand screening assays experimental_method Summary of ligand screening assays used to measure ER‐mediated activities. FIG +0 3 ERE structure_element ERE, estrogen‐response element; Luc, luciferase reporter gene; M2H, mammalian 2‐hybrid; UAS, upstream‐activating sequence. FIG +5 30 estrogen‐response element structure_element ERE, estrogen‐response element; Luc, luciferase reporter gene; M2H, mammalian 2‐hybrid; UAS, upstream‐activating sequence. FIG +32 35 Luc experimental_method ERE, estrogen‐response element; Luc, luciferase reporter gene; M2H, mammalian 2‐hybrid; UAS, upstream‐activating sequence. FIG +37 61 luciferase reporter gene experimental_method ERE, estrogen‐response element; Luc, luciferase reporter gene; M2H, mammalian 2‐hybrid; UAS, upstream‐activating sequence. FIG +63 66 M2H experimental_method ERE, estrogen‐response element; Luc, luciferase reporter gene; M2H, mammalian 2‐hybrid; UAS, upstream‐activating sequence. FIG +68 86 mammalian 2‐hybrid experimental_method ERE, estrogen‐response element; Luc, luciferase reporter gene; M2H, mammalian 2‐hybrid; UAS, upstream‐activating sequence. FIG +88 91 UAS structure_element ERE, estrogen‐response element; Luc, luciferase reporter gene; M2H, mammalian 2‐hybrid; UAS, upstream‐activating sequence. FIG +93 121 upstream‐activating sequence structure_element ERE, estrogen‐response element; Luc, luciferase reporter gene; M2H, mammalian 2‐hybrid; UAS, upstream‐activating sequence. FIG +12 16 AF‐1 structure_element Strength of AF‐1 signaling does not determine cell‐specific signaling RESULTS +11 14 ERα protein To compare ERα signaling induced by diverse ligand types, we synthesized and assayed a library of 241 ERα ligands containing 19 distinct molecular scaffolds. RESULTS +61 84 synthesized and assayed experimental_method To compare ERα signaling induced by diverse ligand types, we synthesized and assayed a library of 241 ERα ligands containing 19 distinct molecular scaffolds. RESULTS +102 105 ERα protein To compare ERα signaling induced by diverse ligand types, we synthesized and assayed a library of 241 ERα ligands containing 19 distinct molecular scaffolds. RESULTS +50 54 lack protein_state These include 15 indirect modulator series, which lack a SERM‐like side chain and modulate coactivator binding indirectly from the ligand‐binding pocket (Fig 2A–E; Dataset EV1) (Zheng et al, 2012) (Zhu et al, 2012) (Muthyala et al, 2003; Seo et al, 2006) (Srinivasan et al, 2013) (Wang et al, 2012) (Liao et al, 2014) (Min et al, 2013). RESULTS +57 66 SERM‐like protein_type These include 15 indirect modulator series, which lack a SERM‐like side chain and modulate coactivator binding indirectly from the ligand‐binding pocket (Fig 2A–E; Dataset EV1) (Zheng et al, 2012) (Zhu et al, 2012) (Muthyala et al, 2003; Seo et al, 2006) (Srinivasan et al, 2013) (Wang et al, 2012) (Liao et al, 2014) (Min et al, 2013). RESULTS +131 152 ligand‐binding pocket site These include 15 indirect modulator series, which lack a SERM‐like side chain and modulate coactivator binding indirectly from the ligand‐binding pocket (Fig 2A–E; Dataset EV1) (Zheng et al, 2012) (Zhu et al, 2012) (Muthyala et al, 2003; Seo et al, 2006) (Srinivasan et al, 2013) (Wang et al, 2012) (Liao et al, 2014) (Min et al, 2013). RESULTS +95 98 h12 structure_element We also generated four direct modulator series with side chains designed to directly dislocate h12 and thereby completely occlude the AF‐2 surface (Fig 2C and E; Dataset EV1) (Kieser et al, 2010). RESULTS +134 146 AF‐2 surface site We also generated four direct modulator series with side chains designed to directly dislocate h12 and thereby completely occlude the AF‐2 surface (Fig 2C and E; Dataset EV1) (Kieser et al, 2010). RESULTS +0 16 Ligand profiling experimental_method Ligand profiling using our quantitative bioassays revealed a wide range of ligand‐induced GREB1 expression, reporter gene activities, ERα‐coactivator interactions, and proliferative effects on MCF‐7 breast cancer cells (Figs EV1 and EV2A–J). RESULTS +27 49 quantitative bioassays experimental_method Ligand profiling using our quantitative bioassays revealed a wide range of ligand‐induced GREB1 expression, reporter gene activities, ERα‐coactivator interactions, and proliferative effects on MCF‐7 breast cancer cells (Figs EV1 and EV2A–J). RESULTS +90 95 GREB1 protein Ligand profiling using our quantitative bioassays revealed a wide range of ligand‐induced GREB1 expression, reporter gene activities, ERα‐coactivator interactions, and proliferative effects on MCF‐7 breast cancer cells (Figs EV1 and EV2A–J). RESULTS +134 137 ERα protein Ligand profiling using our quantitative bioassays revealed a wide range of ligand‐induced GREB1 expression, reporter gene activities, ERα‐coactivator interactions, and proliferative effects on MCF‐7 breast cancer cells (Figs EV1 and EV2A–J). RESULTS +60 63 ERα protein This wide variance enabled us to probe specific features of ERα signaling using ligand class analyses, and identify signaling patterns shared by specific ligand series or scaffolds. RESULTS +80 101 ligand class analyses experimental_method This wide variance enabled us to probe specific features of ERα signaling using ligand class analyses, and identify signaling patterns shared by specific ligand series or scaffolds. RESULTS +28 31 ERα protein Classes of compounds in the ERα ligand library FIG +0 9 Structure evidence Structure of the E2‐bound ERα LBD in complex with an NCOA2 peptide of (PDB 1GWR). FIG +17 25 E2‐bound protein_state Structure of the E2‐bound ERα LBD in complex with an NCOA2 peptide of (PDB 1GWR). FIG +26 29 ERα protein Structure of the E2‐bound ERα LBD in complex with an NCOA2 peptide of (PDB 1GWR). FIG +30 33 LBD structure_element Structure of the E2‐bound ERα LBD in complex with an NCOA2 peptide of (PDB 1GWR). FIG +34 49 in complex with protein_state Structure of the E2‐bound ERα LBD in complex with an NCOA2 peptide of (PDB 1GWR). FIG +53 58 NCOA2 protein Structure of the E2‐bound ERα LBD in complex with an NCOA2 peptide of (PDB 1GWR). FIG +26 29 ERα protein Structural details of the ERα LBD bound to the indicated ligands. FIG +30 33 LBD structure_element Structural details of the ERα LBD bound to the indicated ligands. FIG +34 42 bound to protein_state Structural details of the ERα LBD bound to the indicated ligands. FIG +7 9 E2 chemical Unlike E2 (PDB 1GWR), TAM is a direct modulator with a BSC that dislocates h12 to block the NCOA2‐binding site (PDB 3ERT). FIG +22 25 TAM chemical Unlike E2 (PDB 1GWR), TAM is a direct modulator with a BSC that dislocates h12 to block the NCOA2‐binding site (PDB 3ERT). FIG +75 78 h12 structure_element Unlike E2 (PDB 1GWR), TAM is a direct modulator with a BSC that dislocates h12 to block the NCOA2‐binding site (PDB 3ERT). FIG +92 110 NCOA2‐binding site site Unlike E2 (PDB 1GWR), TAM is a direct modulator with a BSC that dislocates h12 to block the NCOA2‐binding site (PDB 3ERT). FIG +0 4 OBHS chemical OBHS is an indirect modulator that dislocates the h11 C‐terminus to destabilize the h11–h12 interface (PDB 4ZN9). FIG +50 53 h11 structure_element OBHS is an indirect modulator that dislocates the h11 C‐terminus to destabilize the h11–h12 interface (PDB 4ZN9). FIG +84 101 h11–h12 interface site OBHS is an indirect modulator that dislocates the h11 C‐terminus to destabilize the h11–h12 interface (PDB 4ZN9). FIG +4 7 ERα protein The ERα ligand library contains 241 ligands representing 15 indirect modulator scaffolds, plus 4 direct modulator scaffolds. FIG +0 3 ERα protein ERα ligands induced a range of agonist activity profiles FIG +52 57 GREB1 protein To this end, we compared the average ligand‐induced GREB1 mRNA levels in MCF‐7 cells and 3×ERE‐Luc reporter gene activity in Ishikawa endometrial cancer cells (E‐Luc) or in HepG2 cells transfected with wild‐type ERα (L‐Luc ERα‐WT) (Figs 3A and EV2A–C). RESULTS +89 98 3×ERE‐Luc experimental_method To this end, we compared the average ligand‐induced GREB1 mRNA levels in MCF‐7 cells and 3×ERE‐Luc reporter gene activity in Ishikawa endometrial cancer cells (E‐Luc) or in HepG2 cells transfected with wild‐type ERα (L‐Luc ERα‐WT) (Figs 3A and EV2A–C). RESULTS +160 165 E‐Luc experimental_method To this end, we compared the average ligand‐induced GREB1 mRNA levels in MCF‐7 cells and 3×ERE‐Luc reporter gene activity in Ishikawa endometrial cancer cells (E‐Luc) or in HepG2 cells transfected with wild‐type ERα (L‐Luc ERα‐WT) (Figs 3A and EV2A–C). RESULTS +202 211 wild‐type protein_state To this end, we compared the average ligand‐induced GREB1 mRNA levels in MCF‐7 cells and 3×ERE‐Luc reporter gene activity in Ishikawa endometrial cancer cells (E‐Luc) or in HepG2 cells transfected with wild‐type ERα (L‐Luc ERα‐WT) (Figs 3A and EV2A–C). RESULTS +212 215 ERα protein To this end, we compared the average ligand‐induced GREB1 mRNA levels in MCF‐7 cells and 3×ERE‐Luc reporter gene activity in Ishikawa endometrial cancer cells (E‐Luc) or in HepG2 cells transfected with wild‐type ERα (L‐Luc ERα‐WT) (Figs 3A and EV2A–C). RESULTS +217 222 L‐Luc experimental_method To this end, we compared the average ligand‐induced GREB1 mRNA levels in MCF‐7 cells and 3×ERE‐Luc reporter gene activity in Ishikawa endometrial cancer cells (E‐Luc) or in HepG2 cells transfected with wild‐type ERα (L‐Luc ERα‐WT) (Figs 3A and EV2A–C). RESULTS +223 226 ERα protein To this end, we compared the average ligand‐induced GREB1 mRNA levels in MCF‐7 cells and 3×ERE‐Luc reporter gene activity in Ishikawa endometrial cancer cells (E‐Luc) or in HepG2 cells transfected with wild‐type ERα (L‐Luc ERα‐WT) (Figs 3A and EV2A–C). RESULTS +227 229 WT protein_state To this end, we compared the average ligand‐induced GREB1 mRNA levels in MCF‐7 cells and 3×ERE‐Luc reporter gene activity in Ishikawa endometrial cancer cells (E‐Luc) or in HepG2 cells transfected with wild‐type ERα (L‐Luc ERα‐WT) (Figs 3A and EV2A–C). RESULTS +95 103 OBHS‐ASC chemical Direct modulators showed significant differences in average activity between cell types except OBHS‐ASC analogs, which had similar low agonist activities in the three cell types. RESULTS +50 59 tamoxifen chemical While it was known that direct modulators such as tamoxifen drive cell‐specific signaling, these experiments reveal that indirect modulators also drive cell‐specific signaling, since eight of fourteen classes showed significant differences in average activity (Figs 3A and EV2A–C). RESULTS +36 39 ERα protein Ligand‐specific signaling underlies ERα‐mediated cell proliferation FIG +20 23 ERα protein (A) Ligand‐specific ERα activities in HepG2, Ishikawa and MCF‐7 cells. FIG +19 24 L‐Luc experimental_method The ligand‐induced L‐Luc ERα‐WT and E‐Luc activities and GREB1 mRNA levels are shown by scaffold (mean + SD). FIG +25 28 ERα protein The ligand‐induced L‐Luc ERα‐WT and E‐Luc activities and GREB1 mRNA levels are shown by scaffold (mean + SD). FIG +29 31 WT protein_state The ligand‐induced L‐Luc ERα‐WT and E‐Luc activities and GREB1 mRNA levels are shown by scaffold (mean + SD). FIG +36 41 E‐Luc experimental_method The ligand‐induced L‐Luc ERα‐WT and E‐Luc activities and GREB1 mRNA levels are shown by scaffold (mean + SD). FIG +57 62 GREB1 protein The ligand‐induced L‐Luc ERα‐WT and E‐Luc activities and GREB1 mRNA levels are shown by scaffold (mean + SD). FIG +11 25 class analysis experimental_method (B) Ligand class analysis of the L‐Luc ERα‐WT and ERα‐ΔAB activities in HepG2 cells. FIG +33 38 L‐Luc experimental_method (B) Ligand class analysis of the L‐Luc ERα‐WT and ERα‐ΔAB activities in HepG2 cells. FIG +39 42 ERα protein (B) Ligand class analysis of the L‐Luc ERα‐WT and ERα‐ΔAB activities in HepG2 cells. FIG +43 45 WT protein_state (B) Ligand class analysis of the L‐Luc ERα‐WT and ERα‐ΔAB activities in HepG2 cells. FIG +50 57 ERα‐ΔAB mutant (B) Ligand class analysis of the L‐Luc ERα‐WT and ERα‐ΔAB activities in HepG2 cells. FIG +27 29 AB structure_element Significant sensitivity to AB domain deletion was determined by Student's t‐test (n = number of ligands per scaffold in Fig 2). FIG +64 80 Student's t‐test experimental_method Significant sensitivity to AB domain deletion was determined by Student's t‐test (n = number of ligands per scaffold in Fig 2). FIG +0 35 Correlation and regression analyses experimental_method Correlation and regression analyses in a large test set. FIG +91 97 F‐test experimental_method In cluster 1, the first three comparisons (rows) showed significant positive correlations (F‐test for nonzero slope, P ≤ 0.05). FIG +117 118 P evidence In cluster 1, the first three comparisons (rows) showed significant positive correlations (F‐test for nonzero slope, P ≤ 0.05). FIG +182 190 deletion experimental_method In cluster 2, only one of these comparisons revealed a significant positive correlation, while none was significant in cluster 3. +, statistically significant correlations gained by deletion of the AB or F domains. FIG +198 200 AB structure_element In cluster 2, only one of these comparisons revealed a significant positive correlation, while none was significant in cluster 3. +, statistically significant correlations gained by deletion of the AB or F domains. FIG +204 205 F structure_element In cluster 2, only one of these comparisons revealed a significant positive correlation, while none was significant in cluster 3. +, statistically significant correlations gained by deletion of the AB or F domains. FIG +50 52 AB structure_element −, significant correlations lost upon deletion of AB or F domains. FIG +56 57 F structure_element −, significant correlations lost upon deletion of AB or F domains. FIG +0 9 Tamoxifen chemical Tamoxifen depends on AF‐1 for its cell‐specific activity (Sakamoto et al, 2002); therefore, we asked whether cell‐specific signaling observed here is due to a similar dependence on AF‐1 for activity (Fig EV1). RESULTS +21 25 AF‐1 structure_element Tamoxifen depends on AF‐1 for its cell‐specific activity (Sakamoto et al, 2002); therefore, we asked whether cell‐specific signaling observed here is due to a similar dependence on AF‐1 for activity (Fig EV1). RESULTS +181 185 AF‐1 structure_element Tamoxifen depends on AF‐1 for its cell‐specific activity (Sakamoto et al, 2002); therefore, we asked whether cell‐specific signaling observed here is due to a similar dependence on AF‐1 for activity (Fig EV1). RESULTS +35 59 average L‐Luc activities evidence To test this idea, we compared the average L‐Luc activities of each scaffold in HepG2 cells co‐transfected with wild‐type ERα or with ERα lacking the AB domain (Figs 1B and EV1). RESULTS +92 106 co‐transfected experimental_method To test this idea, we compared the average L‐Luc activities of each scaffold in HepG2 cells co‐transfected with wild‐type ERα or with ERα lacking the AB domain (Figs 1B and EV1). RESULTS +112 121 wild‐type protein_state To test this idea, we compared the average L‐Luc activities of each scaffold in HepG2 cells co‐transfected with wild‐type ERα or with ERα lacking the AB domain (Figs 1B and EV1). RESULTS +122 125 ERα protein To test this idea, we compared the average L‐Luc activities of each scaffold in HepG2 cells co‐transfected with wild‐type ERα or with ERα lacking the AB domain (Figs 1B and EV1). RESULTS +134 137 ERα protein To test this idea, we compared the average L‐Luc activities of each scaffold in HepG2 cells co‐transfected with wild‐type ERα or with ERα lacking the AB domain (Figs 1B and EV1). RESULTS +138 149 lacking the protein_state To test this idea, we compared the average L‐Luc activities of each scaffold in HepG2 cells co‐transfected with wild‐type ERα or with ERα lacking the AB domain (Figs 1B and EV1). RESULTS +150 152 AB structure_element To test this idea, we compared the average L‐Luc activities of each scaffold in HepG2 cells co‐transfected with wild‐type ERα or with ERα lacking the AB domain (Figs 1B and EV1). RESULTS +6 8 E2 chemical While E2 showed similar L‐Luc ERα‐WT and ERα‐ΔAB activities, tamoxifen showed complete loss of activity without the AB domain (Fig EV1B). RESULTS +24 29 L‐Luc experimental_method While E2 showed similar L‐Luc ERα‐WT and ERα‐ΔAB activities, tamoxifen showed complete loss of activity without the AB domain (Fig EV1B). RESULTS +30 33 ERα protein While E2 showed similar L‐Luc ERα‐WT and ERα‐ΔAB activities, tamoxifen showed complete loss of activity without the AB domain (Fig EV1B). RESULTS +34 36 WT protein_state While E2 showed similar L‐Luc ERα‐WT and ERα‐ΔAB activities, tamoxifen showed complete loss of activity without the AB domain (Fig EV1B). RESULTS +41 48 ERα‐ΔAB mutant While E2 showed similar L‐Luc ERα‐WT and ERα‐ΔAB activities, tamoxifen showed complete loss of activity without the AB domain (Fig EV1B). RESULTS +61 70 tamoxifen chemical While E2 showed similar L‐Luc ERα‐WT and ERα‐ΔAB activities, tamoxifen showed complete loss of activity without the AB domain (Fig EV1B). RESULTS +104 111 without protein_state While E2 showed similar L‐Luc ERα‐WT and ERα‐ΔAB activities, tamoxifen showed complete loss of activity without the AB domain (Fig EV1B). RESULTS +116 118 AB structure_element While E2 showed similar L‐Luc ERα‐WT and ERα‐ΔAB activities, tamoxifen showed complete loss of activity without the AB domain (Fig EV1B). RESULTS +0 11 Deletion of experimental_method Deletion of the AB domain significantly reduced the average L‐Luc activities of 14 scaffolds (Student's t‐test, P ≤ 0.05) (Fig 3B). RESULTS +16 18 AB structure_element Deletion of the AB domain significantly reduced the average L‐Luc activities of 14 scaffolds (Student's t‐test, P ≤ 0.05) (Fig 3B). RESULTS +52 76 average L‐Luc activities evidence Deletion of the AB domain significantly reduced the average L‐Luc activities of 14 scaffolds (Student's t‐test, P ≤ 0.05) (Fig 3B). RESULTS +94 110 Student's t‐test experimental_method Deletion of the AB domain significantly reduced the average L‐Luc activities of 14 scaffolds (Student's t‐test, P ≤ 0.05) (Fig 3B). RESULTS +112 113 P evidence Deletion of the AB domain significantly reduced the average L‐Luc activities of 14 scaffolds (Student's t‐test, P ≤ 0.05) (Fig 3B). RESULTS +7 11 AF‐1 structure_element These “AF‐1‐sensitive” activities were exhibited by both direct and indirect modulators, and were not limited to scaffolds that showed cell‐specific signaling (Fig 3A and B). RESULTS +22 26 AF‐1 structure_element Thus, the strength of AF‐1 signaling does not determine cell‐specific signaling. RESULTS +48 51 ERα protein Identifying cell‐specific signaling clusters in ERα ligand classes RESULTS +53 86 Pearson's correlation coefficient evidence For each ligand class or scaffold, we calculated the Pearson's correlation coefficient, r, for pairwise comparison of activity profiles in breast (GREB1), liver (L‐Luc), and endometrial cells (E‐Luc). RESULTS +88 89 r evidence For each ligand class or scaffold, we calculated the Pearson's correlation coefficient, r, for pairwise comparison of activity profiles in breast (GREB1), liver (L‐Luc), and endometrial cells (E‐Luc). RESULTS +147 152 GREB1 protein For each ligand class or scaffold, we calculated the Pearson's correlation coefficient, r, for pairwise comparison of activity profiles in breast (GREB1), liver (L‐Luc), and endometrial cells (E‐Luc). RESULTS +162 167 L‐Luc experimental_method For each ligand class or scaffold, we calculated the Pearson's correlation coefficient, r, for pairwise comparison of activity profiles in breast (GREB1), liver (L‐Luc), and endometrial cells (E‐Luc). RESULTS +193 198 E‐Luc experimental_method For each ligand class or scaffold, we calculated the Pearson's correlation coefficient, r, for pairwise comparison of activity profiles in breast (GREB1), liver (L‐Luc), and endometrial cells (E‐Luc). RESULTS +13 14 r evidence The value of r ranges from −1 to 1, and it defines the extent to which the data fit a straight line when compounds show similar agonist/antagonist activity profiles between cell types (Fig EV3A). RESULTS +23 51 coefficient of determination evidence We also calculated the coefficient of determination, r 2, which describes the percentage of variance in a dependent variable such as proliferation that can be predicted by an independent variable such as GREB1 expression. RESULTS +53 56 r 2 evidence We also calculated the coefficient of determination, r 2, which describes the percentage of variance in a dependent variable such as proliferation that can be predicted by an independent variable such as GREB1 expression. RESULTS +204 209 GREB1 protein We also calculated the coefficient of determination, r 2, which describes the percentage of variance in a dependent variable such as proliferation that can be predicted by an independent variable such as GREB1 expression. RESULTS +32 35 r 2 evidence We present both calculations as r 2 to readily compare signaling specificities using a heat map on which the red–yellow palette indicates significant positive correlations (P ≤ 0.05, F‐test for nonzero slope), while the blue palette denotes negative correlations (Fig 3C–F). RESULTS +173 174 P evidence We present both calculations as r 2 to readily compare signaling specificities using a heat map on which the red–yellow palette indicates significant positive correlations (P ≤ 0.05, F‐test for nonzero slope), while the blue palette denotes negative correlations (Fig 3C–F). RESULTS +183 189 F‐test experimental_method We present both calculations as r 2 to readily compare signaling specificities using a heat map on which the red–yellow palette indicates significant positive correlations (P ≤ 0.05, F‐test for nonzero slope), while the blue palette denotes negative correlations (Fig 3C–F). RESULTS +18 26 OBHS‐BSC chemical The side chain of OBHS‐BSC analogs induces cell‐specific signaling FIG +24 28 OBHS chemical Correlation analysis of OBHS versus OBHS‐BSC activity across cell types. FIG +36 44 OBHS‐BSC chemical Correlation analysis of OBHS versus OBHS‐BSC activity across cell types. FIG +24 29 L‐Luc experimental_method Correlation analysis of L‐Luc ERα‐ΔAB activity versus endogenous ERα activity of OBHS analogs. FIG +30 37 ERα‐ΔAB mutant Correlation analysis of L‐Luc ERα‐ΔAB activity versus endogenous ERα activity of OBHS analogs. FIG +65 68 ERα protein Correlation analysis of L‐Luc ERα‐ΔAB activity versus endogenous ERα activity of OBHS analogs. FIG +81 85 OBHS chemical Correlation analysis of L‐Luc ERα‐ΔAB activity versus endogenous ERα activity of OBHS analogs. FIG +14 19 L‐Luc experimental_method In panel (D), L‐Luc ERα‐WT activity from panel (B) is shown for comparison. FIG +20 23 ERα protein In panel (D), L‐Luc ERα‐WT activity from panel (B) is shown for comparison. FIG +24 26 WT protein_state In panel (D), L‐Luc ERα‐WT activity from panel (B) is shown for comparison. FIG +24 29 L‐Luc experimental_method Correlation analysis of L‐Luc ERα‐ΔF activity versus endogenous ERα activities of OBHS analogs. FIG +30 36 ERα‐ΔF mutant Correlation analysis of L‐Luc ERα‐ΔF activity versus endogenous ERα activities of OBHS analogs. FIG +64 67 ERα protein Correlation analysis of L‐Luc ERα‐ΔF activity versus endogenous ERα activities of OBHS analogs. FIG +82 86 OBHS chemical Correlation analysis of L‐Luc ERα‐ΔF activity versus endogenous ERα activities of OBHS analogs. FIG +56 63 NCOA2/3 protein Correlation analysis of MCF‐7 cell proliferation versus NCOA2/3 recruitment or GREB1 levels observed in response to (G) OBHS‐N and (H) OBHS‐BSC analogs. FIG +79 84 GREB1 protein Correlation analysis of MCF‐7 cell proliferation versus NCOA2/3 recruitment or GREB1 levels observed in response to (G) OBHS‐N and (H) OBHS‐BSC analogs. FIG +120 126 OBHS‐N chemical Correlation analysis of MCF‐7 cell proliferation versus NCOA2/3 recruitment or GREB1 levels observed in response to (G) OBHS‐N and (H) OBHS‐BSC analogs. FIG +135 143 OBHS‐BSC chemical Correlation analysis of MCF‐7 cell proliferation versus NCOA2/3 recruitment or GREB1 levels observed in response to (G) OBHS‐N and (H) OBHS‐BSC analogs. FIG +53 58 GREB1 protein Scaffolds in cluster 1 exhibited strongly correlated GREB1 levels, E‐Luc and L‐Luc activity profiles across the three cell types (Fig 3C lanes 1–4), suggesting these ligands use similar ERα signaling pathways in the breast, endometrial, and liver cell types. RESULTS +67 72 E‐Luc experimental_method Scaffolds in cluster 1 exhibited strongly correlated GREB1 levels, E‐Luc and L‐Luc activity profiles across the three cell types (Fig 3C lanes 1–4), suggesting these ligands use similar ERα signaling pathways in the breast, endometrial, and liver cell types. RESULTS +77 82 L‐Luc experimental_method Scaffolds in cluster 1 exhibited strongly correlated GREB1 levels, E‐Luc and L‐Luc activity profiles across the three cell types (Fig 3C lanes 1–4), suggesting these ligands use similar ERα signaling pathways in the breast, endometrial, and liver cell types. RESULTS +186 189 ERα protein Scaffolds in cluster 1 exhibited strongly correlated GREB1 levels, E‐Luc and L‐Luc activity profiles across the three cell types (Fig 3C lanes 1–4), suggesting these ligands use similar ERα signaling pathways in the breast, endometrial, and liver cell types. RESULTS +22 27 WAY‐C chemical This cluster includes WAY‐C, OBHS, OBHS‐N, and triaryl‐ethylene analogs, all of which are indirect modulators. RESULTS +29 33 OBHS chemical This cluster includes WAY‐C, OBHS, OBHS‐N, and triaryl‐ethylene analogs, all of which are indirect modulators. RESULTS +35 41 OBHS‐N chemical This cluster includes WAY‐C, OBHS, OBHS‐N, and triaryl‐ethylene analogs, all of which are indirect modulators. RESULTS +47 63 triaryl‐ethylene chemical This cluster includes WAY‐C, OBHS, OBHS‐N, and triaryl‐ethylene analogs, all of which are indirect modulators. RESULTS +56 70 cyclofenil‐ASC chemical This cluster includes two classes of direct modulators (cyclofenil‐ASC and WAY dimer), and six classes of indirect modulators (2,5‐DTP, 3,4‐DTP, S‐OBHS‐2 and S‐OBHS‐3, furan, and WAY‐D). RESULTS +75 84 WAY dimer chemical This cluster includes two classes of direct modulators (cyclofenil‐ASC and WAY dimer), and six classes of indirect modulators (2,5‐DTP, 3,4‐DTP, S‐OBHS‐2 and S‐OBHS‐3, furan, and WAY‐D). RESULTS +127 134 2,5‐DTP chemical This cluster includes two classes of direct modulators (cyclofenil‐ASC and WAY dimer), and six classes of indirect modulators (2,5‐DTP, 3,4‐DTP, S‐OBHS‐2 and S‐OBHS‐3, furan, and WAY‐D). RESULTS +136 143 3,4‐DTP chemical This cluster includes two classes of direct modulators (cyclofenil‐ASC and WAY dimer), and six classes of indirect modulators (2,5‐DTP, 3,4‐DTP, S‐OBHS‐2 and S‐OBHS‐3, furan, and WAY‐D). RESULTS +145 153 S‐OBHS‐2 chemical This cluster includes two classes of direct modulators (cyclofenil‐ASC and WAY dimer), and six classes of indirect modulators (2,5‐DTP, 3,4‐DTP, S‐OBHS‐2 and S‐OBHS‐3, furan, and WAY‐D). RESULTS +158 166 S‐OBHS‐3 chemical This cluster includes two classes of direct modulators (cyclofenil‐ASC and WAY dimer), and six classes of indirect modulators (2,5‐DTP, 3,4‐DTP, S‐OBHS‐2 and S‐OBHS‐3, furan, and WAY‐D). RESULTS +168 173 furan chemical This cluster includes two classes of direct modulators (cyclofenil‐ASC and WAY dimer), and six classes of indirect modulators (2,5‐DTP, 3,4‐DTP, S‐OBHS‐2 and S‐OBHS‐3, furan, and WAY‐D). RESULTS +179 184 WAY‐D chemical This cluster includes two classes of direct modulators (cyclofenil‐ASC and WAY dimer), and six classes of indirect modulators (2,5‐DTP, 3,4‐DTP, S‐OBHS‐2 and S‐OBHS‐3, furan, and WAY‐D). RESULTS +13 20 3,4‐DTP chemical For example, 3,4‐DTP, furan, and S‐OBHS‐2 drove positively correlated GREB1 levels and E‐Luc but not L‐Luc ERα‐WT activity (Fig 3C lanes 5–7). RESULTS +22 27 furan chemical For example, 3,4‐DTP, furan, and S‐OBHS‐2 drove positively correlated GREB1 levels and E‐Luc but not L‐Luc ERα‐WT activity (Fig 3C lanes 5–7). RESULTS +33 41 S‐OBHS‐2 chemical For example, 3,4‐DTP, furan, and S‐OBHS‐2 drove positively correlated GREB1 levels and E‐Luc but not L‐Luc ERα‐WT activity (Fig 3C lanes 5–7). RESULTS +70 75 GREB1 protein For example, 3,4‐DTP, furan, and S‐OBHS‐2 drove positively correlated GREB1 levels and E‐Luc but not L‐Luc ERα‐WT activity (Fig 3C lanes 5–7). RESULTS +87 92 E‐Luc experimental_method For example, 3,4‐DTP, furan, and S‐OBHS‐2 drove positively correlated GREB1 levels and E‐Luc but not L‐Luc ERα‐WT activity (Fig 3C lanes 5–7). RESULTS +101 106 L‐Luc experimental_method For example, 3,4‐DTP, furan, and S‐OBHS‐2 drove positively correlated GREB1 levels and E‐Luc but not L‐Luc ERα‐WT activity (Fig 3C lanes 5–7). RESULTS +107 110 ERα protein For example, 3,4‐DTP, furan, and S‐OBHS‐2 drove positively correlated GREB1 levels and E‐Luc but not L‐Luc ERα‐WT activity (Fig 3C lanes 5–7). RESULTS +111 113 WT protein_state For example, 3,4‐DTP, furan, and S‐OBHS‐2 drove positively correlated GREB1 levels and E‐Luc but not L‐Luc ERα‐WT activity (Fig 3C lanes 5–7). RESULTS +13 22 WAY dimer chemical In contrast, WAY dimer and WAY‐D analogs drove positively correlated GREB1 levels and L‐Luc ERα‐WT but not E‐Luc activity (Fig 3C lanes 8 and 9). RESULTS +27 32 WAY‐D chemical In contrast, WAY dimer and WAY‐D analogs drove positively correlated GREB1 levels and L‐Luc ERα‐WT but not E‐Luc activity (Fig 3C lanes 8 and 9). RESULTS +69 74 GREB1 protein In contrast, WAY dimer and WAY‐D analogs drove positively correlated GREB1 levels and L‐Luc ERα‐WT but not E‐Luc activity (Fig 3C lanes 8 and 9). RESULTS +86 91 L‐Luc experimental_method In contrast, WAY dimer and WAY‐D analogs drove positively correlated GREB1 levels and L‐Luc ERα‐WT but not E‐Luc activity (Fig 3C lanes 8 and 9). RESULTS +92 95 ERα protein In contrast, WAY dimer and WAY‐D analogs drove positively correlated GREB1 levels and L‐Luc ERα‐WT but not E‐Luc activity (Fig 3C lanes 8 and 9). RESULTS +96 98 WT protein_state In contrast, WAY dimer and WAY‐D analogs drove positively correlated GREB1 levels and L‐Luc ERα‐WT but not E‐Luc activity (Fig 3C lanes 8 and 9). RESULTS +107 112 E‐Luc experimental_method In contrast, WAY dimer and WAY‐D analogs drove positively correlated GREB1 levels and L‐Luc ERα‐WT but not E‐Luc activity (Fig 3C lanes 8 and 9). RESULTS +54 62 OBHS‐ASC chemical This cluster includes two direct modulator scaffolds (OBHS‐ASC and OBHS‐BSC), and five indirect modulator scaffolds (A‐CD, cyclofenil, 3,4‐DTPD, imine, and imidazopyridine). RESULTS +67 75 OBHS‐BSC chemical This cluster includes two direct modulator scaffolds (OBHS‐ASC and OBHS‐BSC), and five indirect modulator scaffolds (A‐CD, cyclofenil, 3,4‐DTPD, imine, and imidazopyridine). RESULTS +117 121 A‐CD chemical This cluster includes two direct modulator scaffolds (OBHS‐ASC and OBHS‐BSC), and five indirect modulator scaffolds (A‐CD, cyclofenil, 3,4‐DTPD, imine, and imidazopyridine). RESULTS +123 133 cyclofenil chemical This cluster includes two direct modulator scaffolds (OBHS‐ASC and OBHS‐BSC), and five indirect modulator scaffolds (A‐CD, cyclofenil, 3,4‐DTPD, imine, and imidazopyridine). RESULTS +135 143 3,4‐DTPD chemical This cluster includes two direct modulator scaffolds (OBHS‐ASC and OBHS‐BSC), and five indirect modulator scaffolds (A‐CD, cyclofenil, 3,4‐DTPD, imine, and imidazopyridine). RESULTS +145 150 imine chemical This cluster includes two direct modulator scaffolds (OBHS‐ASC and OBHS‐BSC), and five indirect modulator scaffolds (A‐CD, cyclofenil, 3,4‐DTPD, imine, and imidazopyridine). RESULTS +156 171 imidazopyridine chemical This cluster includes two direct modulator scaffolds (OBHS‐ASC and OBHS‐BSC), and five indirect modulator scaffolds (A‐CD, cyclofenil, 3,4‐DTPD, imine, and imidazopyridine). RESULTS +68 71 ERα protein These results suggest that addition of an extended side chain to an ERα ligand scaffold is sufficient to induce cell‐specific signaling, where the relative activity profiles of the individual ligands change between cell types. RESULTS +82 86 OBHS chemical This is demonstrated by directly comparing the signaling specificities of matched OBHS (indirect modulator, cluster 1) and OBHS‐BSC analogs (direct modulator, cluster 3), which differ only in the basic side chain (Fig 2E). RESULTS +123 131 OBHS‐BSC chemical This is demonstrated by directly comparing the signaling specificities of matched OBHS (indirect modulator, cluster 1) and OBHS‐BSC analogs (direct modulator, cluster 3), which differ only in the basic side chain (Fig 2E). RESULTS +18 22 OBHS chemical The activities of OBHS analogs were positively correlated across the three cell types, but the side chain of OBHS‐BSC analogs was sufficient to abolish these correlations (Figs 3C lanes 1 and 19, and EV3A–C). RESULTS +109 117 OBHS‐BSC chemical The activities of OBHS analogs were positively correlated across the three cell types, but the side chain of OBHS‐BSC analogs was sufficient to abolish these correlations (Figs 3C lanes 1 and 19, and EV3A–C). RESULTS +43 46 ERα protein Thus, examining the correlated patterns of ERα activity within each scaffold demonstrates that an extended side chain is not required for cell‐specific signaling. RESULTS +39 43 AF‐1 structure_element Modulation of signaling specificity by AF‐1 RESULTS +24 28 AF‐1 structure_element To evaluate the role of AF‐1 and the F domain in ERα signaling specificity, we compared activity of truncated ERα constructs in HepG2 liver cells with endogenous ERα activity in the other cell types. RESULTS +37 38 F structure_element To evaluate the role of AF‐1 and the F domain in ERα signaling specificity, we compared activity of truncated ERα constructs in HepG2 liver cells with endogenous ERα activity in the other cell types. RESULTS +49 52 ERα protein To evaluate the role of AF‐1 and the F domain in ERα signaling specificity, we compared activity of truncated ERα constructs in HepG2 liver cells with endogenous ERα activity in the other cell types. RESULTS +110 113 ERα protein To evaluate the role of AF‐1 and the F domain in ERα signaling specificity, we compared activity of truncated ERα constructs in HepG2 liver cells with endogenous ERα activity in the other cell types. RESULTS +162 165 ERα protein To evaluate the role of AF‐1 and the F domain in ERα signaling specificity, we compared activity of truncated ERα constructs in HepG2 liver cells with endogenous ERα activity in the other cell types. RESULTS +37 42 L‐Luc experimental_method The positive correlation between the L‐Luc and E‐Luc activities or GREB1 levels induced by scaffolds in cluster 1 was generally retained without the AB domain, or the F domain (Fig 3D lanes 1–4). RESULTS +47 52 E‐Luc experimental_method The positive correlation between the L‐Luc and E‐Luc activities or GREB1 levels induced by scaffolds in cluster 1 was generally retained without the AB domain, or the F domain (Fig 3D lanes 1–4). RESULTS +67 72 GREB1 protein The positive correlation between the L‐Luc and E‐Luc activities or GREB1 levels induced by scaffolds in cluster 1 was generally retained without the AB domain, or the F domain (Fig 3D lanes 1–4). RESULTS +149 151 AB structure_element The positive correlation between the L‐Luc and E‐Luc activities or GREB1 levels induced by scaffolds in cluster 1 was generally retained without the AB domain, or the F domain (Fig 3D lanes 1–4). RESULTS +167 168 F structure_element The positive correlation between the L‐Luc and E‐Luc activities or GREB1 levels induced by scaffolds in cluster 1 was generally retained without the AB domain, or the F domain (Fig 3D lanes 1–4). RESULTS +110 114 AF‐1 structure_element This demonstrates that the signaling specificities underlying these positive correlations are not modified by AF‐1. RESULTS +0 4 OBHS chemical OBHS analogs showed an average L‐Luc ERα‐ΔAB activity of 3.2% ± 3 (mean + SEM) relative to E2. RESULTS +31 36 L‐Luc experimental_method OBHS analogs showed an average L‐Luc ERα‐ΔAB activity of 3.2% ± 3 (mean + SEM) relative to E2. RESULTS +37 44 ERα‐ΔAB mutant OBHS analogs showed an average L‐Luc ERα‐ΔAB activity of 3.2% ± 3 (mean + SEM) relative to E2. RESULTS +91 93 E2 chemical OBHS analogs showed an average L‐Luc ERα‐ΔAB activity of 3.2% ± 3 (mean + SEM) relative to E2. RESULTS +62 67 L‐Luc experimental_method Despite this nearly complete lack of activity, the pattern of L‐Luc ERα‐ΔAB activity was still highly correlated with the E‐Luc activity and GREB1 expression (Fig EV3D and E), demonstrating that very small AF‐2 activities can be amplified by AF‐1 to produce robust signals. RESULTS +68 75 ERα‐ΔAB mutant Despite this nearly complete lack of activity, the pattern of L‐Luc ERα‐ΔAB activity was still highly correlated with the E‐Luc activity and GREB1 expression (Fig EV3D and E), demonstrating that very small AF‐2 activities can be amplified by AF‐1 to produce robust signals. RESULTS +122 127 E‐Luc experimental_method Despite this nearly complete lack of activity, the pattern of L‐Luc ERα‐ΔAB activity was still highly correlated with the E‐Luc activity and GREB1 expression (Fig EV3D and E), demonstrating that very small AF‐2 activities can be amplified by AF‐1 to produce robust signals. RESULTS +141 146 GREB1 protein Despite this nearly complete lack of activity, the pattern of L‐Luc ERα‐ΔAB activity was still highly correlated with the E‐Luc activity and GREB1 expression (Fig EV3D and E), demonstrating that very small AF‐2 activities can be amplified by AF‐1 to produce robust signals. RESULTS +206 210 AF‐2 structure_element Despite this nearly complete lack of activity, the pattern of L‐Luc ERα‐ΔAB activity was still highly correlated with the E‐Luc activity and GREB1 expression (Fig EV3D and E), demonstrating that very small AF‐2 activities can be amplified by AF‐1 to produce robust signals. RESULTS +242 246 AF‐1 structure_element Despite this nearly complete lack of activity, the pattern of L‐Luc ERα‐ΔAB activity was still highly correlated with the E‐Luc activity and GREB1 expression (Fig EV3D and E), demonstrating that very small AF‐2 activities can be amplified by AF‐1 to produce robust signals. RESULTS +11 22 deletion of experimental_method Similarly, deletion of the F domain did not abolish correlations between the L‐Luc and E‐Luc or GREB1 levels induced by OBHS analogs (Fig EV3F). RESULTS +27 28 F structure_element Similarly, deletion of the F domain did not abolish correlations between the L‐Luc and E‐Luc or GREB1 levels induced by OBHS analogs (Fig EV3F). RESULTS +77 82 L‐Luc experimental_method Similarly, deletion of the F domain did not abolish correlations between the L‐Luc and E‐Luc or GREB1 levels induced by OBHS analogs (Fig EV3F). RESULTS +87 92 E‐Luc experimental_method Similarly, deletion of the F domain did not abolish correlations between the L‐Luc and E‐Luc or GREB1 levels induced by OBHS analogs (Fig EV3F). RESULTS +96 101 GREB1 protein Similarly, deletion of the F domain did not abolish correlations between the L‐Luc and E‐Luc or GREB1 levels induced by OBHS analogs (Fig EV3F). RESULTS +120 124 OBHS chemical Similarly, deletion of the F domain did not abolish correlations between the L‐Luc and E‐Luc or GREB1 levels induced by OBHS analogs (Fig EV3F). RESULTS +49 58 wild‐type protein_state These similar patterns of ligand activity in the wild‐type and deletion mutants suggest that AF‐1 and the F domain purely amplify the AF‐2 activities of ligands in cluster 1. RESULTS +72 79 mutants protein_state These similar patterns of ligand activity in the wild‐type and deletion mutants suggest that AF‐1 and the F domain purely amplify the AF‐2 activities of ligands in cluster 1. RESULTS +93 97 AF‐1 structure_element These similar patterns of ligand activity in the wild‐type and deletion mutants suggest that AF‐1 and the F domain purely amplify the AF‐2 activities of ligands in cluster 1. RESULTS +106 107 F structure_element These similar patterns of ligand activity in the wild‐type and deletion mutants suggest that AF‐1 and the F domain purely amplify the AF‐2 activities of ligands in cluster 1. RESULTS +134 138 AF‐2 structure_element These similar patterns of ligand activity in the wild‐type and deletion mutants suggest that AF‐1 and the F domain purely amplify the AF‐2 activities of ligands in cluster 1. RESULTS +13 17 AF‐1 structure_element In contrast, AF‐1 was a determinant of signaling specificity for scaffolds in cluster 2. RESULTS +0 11 Deletion of experimental_method Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). RESULTS +16 18 AB structure_element Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). RESULTS +22 23 F structure_element Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). RESULTS +100 107 2,5‐DTP chemical Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). RESULTS +109 116 3,4‐DTP chemical Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). RESULTS +118 126 S‐OBHS‐3 chemical Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). RESULTS +128 133 WAY‐D chemical Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). RESULTS +135 144 WAY dimer chemical Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). RESULTS +150 164 cyclofenil‐ASC chemical Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). RESULTS +61 83 deletion mutant assays experimental_method Comparing Fig 3C and D, the + and − signs indicate where the deletion mutant assays led to a gain or loss of statically significant correlation, respectively. RESULTS +20 24 AF‐1 structure_element Thus, in cluster 2, AF‐1 substantially modulated the specificity of ligands with cell‐specific activity (Fig 3D lanes 5–12). RESULTS +60 64 AF‐1 structure_element For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). RESULTS +183 194 deletion of experimental_method For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). RESULTS +199 201 AB structure_element For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). RESULTS +205 206 F structure_element For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). RESULTS +268 272 A‐CD chemical For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). RESULTS +277 285 OBHS‐ASC chemical For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). RESULTS +301 312 deletion of experimental_method For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). RESULTS +317 319 AB structure_element For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). RESULTS +330 331 F structure_element For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). RESULTS +373 378 E‐Luc experimental_method For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). RESULTS +395 400 GREB1 protein For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). RESULTS +35 39 AF‐1 structure_element Thus, ligands in cluster 2 rely on AF‐1 for both activity (Fig 3B) and signaling specificity (Fig 3D). RESULTS +27 32 GREB1 protein Ligand‐specific control of GREB1 expression RESULTS +65 68 ERα protein To determine whether ligand classes control expression of native ERα target genes through the canonical linear signaling pathway, we performed pairwise linear regression analyses using ERα–NCOA1/2/3 interactions in M2H assay as independent predictors of GREB1 expression (the dependent variable) (Figs EV1 and EV2A, F–H). RESULTS +143 178 pairwise linear regression analyses experimental_method To determine whether ligand classes control expression of native ERα target genes through the canonical linear signaling pathway, we performed pairwise linear regression analyses using ERα–NCOA1/2/3 interactions in M2H assay as independent predictors of GREB1 expression (the dependent variable) (Figs EV1 and EV2A, F–H). RESULTS +185 198 ERα–NCOA1/2/3 complex_assembly To determine whether ligand classes control expression of native ERα target genes through the canonical linear signaling pathway, we performed pairwise linear regression analyses using ERα–NCOA1/2/3 interactions in M2H assay as independent predictors of GREB1 expression (the dependent variable) (Figs EV1 and EV2A, F–H). RESULTS +215 224 M2H assay experimental_method To determine whether ligand classes control expression of native ERα target genes through the canonical linear signaling pathway, we performed pairwise linear regression analyses using ERα–NCOA1/2/3 interactions in M2H assay as independent predictors of GREB1 expression (the dependent variable) (Figs EV1 and EV2A, F–H). RESULTS +254 259 GREB1 protein To determine whether ligand classes control expression of native ERα target genes through the canonical linear signaling pathway, we performed pairwise linear regression analyses using ERα–NCOA1/2/3 interactions in M2H assay as independent predictors of GREB1 expression (the dependent variable) (Figs EV1 and EV2A, F–H). RESULTS +33 38 NCOA1 protein In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). RESULTS +43 48 NCOA2 protein In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). RESULTS +65 70 WAY‐C chemical In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). RESULTS +84 100 triaryl‐ethylene chemical In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). RESULTS +102 108 OBHS‐N chemical In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). RESULTS +114 118 OBHS chemical In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). RESULTS +137 142 NCOA3 protein In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). RESULTS +144 150 OBHS‐N chemical In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). RESULTS +194 198 OBHS chemical In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). RESULTS +25 30 GREB1 protein The average induction of GREB1 by cluster 1 ligands showed greater variance, with a range between ~25 and ~75% for OBHS and a range from full agonist to inverse agonist for the others in cluster 1 (Fig EV2A). RESULTS +115 119 OBHS chemical The average induction of GREB1 by cluster 1 ligands showed greater variance, with a range between ~25 and ~75% for OBHS and a range from full agonist to inverse agonist for the others in cluster 1 (Fig EV2A). RESULTS +0 5 GREB1 protein GREB1 levels induced by OBHS analogs were determined by recruitment of NCOA1 but not NCOA2/3 (Fig 3E lane 1), suggesting that there may be alternate or preferential use of these coactivators by different classes. RESULTS +24 28 OBHS chemical GREB1 levels induced by OBHS analogs were determined by recruitment of NCOA1 but not NCOA2/3 (Fig 3E lane 1), suggesting that there may be alternate or preferential use of these coactivators by different classes. RESULTS +71 76 NCOA1 protein GREB1 levels induced by OBHS analogs were determined by recruitment of NCOA1 but not NCOA2/3 (Fig 3E lane 1), suggesting that there may be alternate or preferential use of these coactivators by different classes. RESULTS +85 92 NCOA2/3 protein GREB1 levels induced by OBHS analogs were determined by recruitment of NCOA1 but not NCOA2/3 (Fig 3E lane 1), suggesting that there may be alternate or preferential use of these coactivators by different classes. RESULTS +23 32 NCOA1/2/3 protein However, in cluster 1, NCOA1/2/3 recruitment generally predicted GREB1 levels (Fig 3E lanes 1–4), consistent with the canonical signaling model (Fig 1D). RESULTS +65 70 GREB1 protein However, in cluster 1, NCOA1/2/3 recruitment generally predicted GREB1 levels (Fig 3E lanes 1–4), consistent with the canonical signaling model (Fig 1D). RESULTS +22 27 GREB1 protein For clusters 2 and 3, GREB1 activity was generally not predicted by NCOA1/2/3 recruitment. RESULTS +68 77 NCOA1/2/3 protein For clusters 2 and 3, GREB1 activity was generally not predicted by NCOA1/2/3 recruitment. RESULTS +29 38 NCOA1/2/3 protein Direct modulators showed low NCOA1/2/3 recruitment (Fig EV2F–H), but only OBHS‐ASC analogs had NCOA2 recruitment profiles that predicted a full range of effects on GREB1 levels (Figs 3E lanes 9, 11, 18–19, and EV2A). RESULTS +74 82 OBHS‐ASC chemical Direct modulators showed low NCOA1/2/3 recruitment (Fig EV2F–H), but only OBHS‐ASC analogs had NCOA2 recruitment profiles that predicted a full range of effects on GREB1 levels (Figs 3E lanes 9, 11, 18–19, and EV2A). RESULTS +95 100 NCOA2 protein Direct modulators showed low NCOA1/2/3 recruitment (Fig EV2F–H), but only OBHS‐ASC analogs had NCOA2 recruitment profiles that predicted a full range of effects on GREB1 levels (Figs 3E lanes 9, 11, 18–19, and EV2A). RESULTS +164 169 GREB1 protein Direct modulators showed low NCOA1/2/3 recruitment (Fig EV2F–H), but only OBHS‐ASC analogs had NCOA2 recruitment profiles that predicted a full range of effects on GREB1 levels (Figs 3E lanes 9, 11, 18–19, and EV2A). RESULTS +55 64 NCOA1/2/3 protein The indirect modulators in clusters 2 and 3 stimulated NCOA1/2/3 recruitment and GREB1 expression with substantial variance (Figs 3A and EV2F–H). RESULTS +81 86 GREB1 protein The indirect modulators in clusters 2 and 3 stimulated NCOA1/2/3 recruitment and GREB1 expression with substantial variance (Figs 3A and EV2F–H). RESULTS +24 29 GREB1 protein However, ligand‐induced GREB1 levels were generally not determined by NCOA1/2/3 recruitment (Fig 3E lanes 5–19), consistent with an alternate causality model (Fig 1E). RESULTS +70 79 NCOA1/2/3 protein However, ligand‐induced GREB1 levels were generally not determined by NCOA1/2/3 recruitment (Fig 3E lanes 5–19), consistent with an alternate causality model (Fig 1E). RESULTS +64 72 S‐OBHS‐3 chemical Out of 11 indirect modulator series in cluster 2 or 3, only the S‐OBHS‐3 class had NCOA1/2/3 recruitment profiles that predicted GREB1 levels (Fig 3E lane 12). RESULTS +83 92 NCOA1/2/3 protein Out of 11 indirect modulator series in cluster 2 or 3, only the S‐OBHS‐3 class had NCOA1/2/3 recruitment profiles that predicted GREB1 levels (Fig 3E lane 12). RESULTS +129 134 GREB1 protein Out of 11 indirect modulator series in cluster 2 or 3, only the S‐OBHS‐3 class had NCOA1/2/3 recruitment profiles that predicted GREB1 levels (Fig 3E lane 12). RESULTS +87 92 GREB1 protein These results suggest that compounds that show cell‐specific signaling do not activate GREB1, or use coactivators other than NCOA1/2/3 to control GREB1 expression (Fig 1E). RESULTS +125 134 NCOA1/2/3 protein These results suggest that compounds that show cell‐specific signaling do not activate GREB1, or use coactivators other than NCOA1/2/3 to control GREB1 expression (Fig 1E). RESULTS +146 151 GREB1 protein These results suggest that compounds that show cell‐specific signaling do not activate GREB1, or use coactivators other than NCOA1/2/3 to control GREB1 expression (Fig 1E). RESULTS +103 129 linear regression analyses experimental_method To determine mechanisms for ligand‐dependent control of breast cancer cell proliferation, we performed linear regression analyses across the 19 scaffolds using MCF‐7 cell proliferation as the dependent variable, and the other activities as independent variables (Fig 3F). RESULTS +14 19 E‐Luc experimental_method In cluster 1, E‐Luc and L‐Luc activities, NCOA1/2/3 recruitment, and GREB1 levels generally predicted the proliferative response (Fig 3F lanes 2–4). RESULTS +24 29 L‐Luc experimental_method In cluster 1, E‐Luc and L‐Luc activities, NCOA1/2/3 recruitment, and GREB1 levels generally predicted the proliferative response (Fig 3F lanes 2–4). RESULTS +42 51 NCOA1/2/3 protein In cluster 1, E‐Luc and L‐Luc activities, NCOA1/2/3 recruitment, and GREB1 levels generally predicted the proliferative response (Fig 3F lanes 2–4). RESULTS +69 74 GREB1 protein In cluster 1, E‐Luc and L‐Luc activities, NCOA1/2/3 recruitment, and GREB1 levels generally predicted the proliferative response (Fig 3F lanes 2–4). RESULTS +9 15 OBHS‐N chemical With the OBHS‐N compounds, NCOA3 and GREB1 showed near perfect prediction of proliferation (Fig EV3G), with unexplained variance similar to the noise in the assays. RESULTS +27 32 NCOA3 protein With the OBHS‐N compounds, NCOA3 and GREB1 showed near perfect prediction of proliferation (Fig EV3G), with unexplained variance similar to the noise in the assays. RESULTS +37 42 GREB1 protein With the OBHS‐N compounds, NCOA3 and GREB1 showed near perfect prediction of proliferation (Fig EV3G), with unexplained variance similar to the noise in the assays. RESULTS +39 43 OBHS chemical The lack of significant predictors for OBHS analogs (Fig 3F lane 1) reflects their small range of proliferative effects on MCF‐7 cells (Fig EV2I). RESULTS +34 39 GREB1 protein The significant correlations with GREB1 expression and NCOA1/2/3 recruitment observed in this cluster are consistent with the canonical signaling model (Fig 1D), where NCOA1/2/3 recruitment determines GREB1 expression, which then drives proliferation. RESULTS +55 64 NCOA1/2/3 protein The significant correlations with GREB1 expression and NCOA1/2/3 recruitment observed in this cluster are consistent with the canonical signaling model (Fig 1D), where NCOA1/2/3 recruitment determines GREB1 expression, which then drives proliferation. RESULTS +168 177 NCOA1/2/3 protein The significant correlations with GREB1 expression and NCOA1/2/3 recruitment observed in this cluster are consistent with the canonical signaling model (Fig 1D), where NCOA1/2/3 recruitment determines GREB1 expression, which then drives proliferation. RESULTS +201 206 GREB1 protein The significant correlations with GREB1 expression and NCOA1/2/3 recruitment observed in this cluster are consistent with the canonical signaling model (Fig 1D), where NCOA1/2/3 recruitment determines GREB1 expression, which then drives proliferation. RESULTS +90 99 NCOA1/2/3 protein Despite this phenotypic variance, proliferation was not generally predicted by correlated NCOA1/2/3 recruitment and GREB1 induction (Figs 3F lanes 5–19, and EV3H). RESULTS +116 121 GREB1 protein Despite this phenotypic variance, proliferation was not generally predicted by correlated NCOA1/2/3 recruitment and GREB1 induction (Figs 3F lanes 5–19, and EV3H). RESULTS +48 55 2,5‐DTP chemical Out of 15 ligand series in these clusters, only 2,5‐DTP analogs induced a proliferative response that was predicted by GREB1 levels, which were not determined by NCOA1/2/3 recruitment (Fig 3E and F lane 10). RESULTS +119 124 GREB1 protein Out of 15 ligand series in these clusters, only 2,5‐DTP analogs induced a proliferative response that was predicted by GREB1 levels, which were not determined by NCOA1/2/3 recruitment (Fig 3E and F lane 10). RESULTS +162 171 NCOA1/2/3 protein Out of 15 ligand series in these clusters, only 2,5‐DTP analogs induced a proliferative response that was predicted by GREB1 levels, which were not determined by NCOA1/2/3 recruitment (Fig 3E and F lane 10). RESULTS +0 7 3,4‐DTP chemical 3,4‐DTP, cyclofenil, 3,4‐DTPD, and imidazopyridine analogs had NCOA1/3 recruitment profiles that predicted their proliferative effects, without determining GREB1 levels (Fig 3E and F, lanes 5 and 14–16). RESULTS +9 19 cyclofenil chemical 3,4‐DTP, cyclofenil, 3,4‐DTPD, and imidazopyridine analogs had NCOA1/3 recruitment profiles that predicted their proliferative effects, without determining GREB1 levels (Fig 3E and F, lanes 5 and 14–16). RESULTS +21 29 3,4‐DTPD chemical 3,4‐DTP, cyclofenil, 3,4‐DTPD, and imidazopyridine analogs had NCOA1/3 recruitment profiles that predicted their proliferative effects, without determining GREB1 levels (Fig 3E and F, lanes 5 and 14–16). RESULTS +35 50 imidazopyridine chemical 3,4‐DTP, cyclofenil, 3,4‐DTPD, and imidazopyridine analogs had NCOA1/3 recruitment profiles that predicted their proliferative effects, without determining GREB1 levels (Fig 3E and F, lanes 5 and 14–16). RESULTS +63 70 NCOA1/3 protein 3,4‐DTP, cyclofenil, 3,4‐DTPD, and imidazopyridine analogs had NCOA1/3 recruitment profiles that predicted their proliferative effects, without determining GREB1 levels (Fig 3E and F, lanes 5 and 14–16). RESULTS +156 161 GREB1 protein 3,4‐DTP, cyclofenil, 3,4‐DTPD, and imidazopyridine analogs had NCOA1/3 recruitment profiles that predicted their proliferative effects, without determining GREB1 levels (Fig 3E and F, lanes 5 and 14–16). RESULTS +11 19 S‐OBHS‐3 chemical Similarly, S‐OBHS‐3, cyclofenil‐ASC, and OBHS‐ASC had positively correlated NCOA1/2/3 recruitment and GREB1 levels, but none of these activities determined their proliferative effects (Fig 3E and F lanes 11–12 and 18). RESULTS +21 35 cyclofenil‐ASC chemical Similarly, S‐OBHS‐3, cyclofenil‐ASC, and OBHS‐ASC had positively correlated NCOA1/2/3 recruitment and GREB1 levels, but none of these activities determined their proliferative effects (Fig 3E and F lanes 11–12 and 18). RESULTS +41 49 OBHS‐ASC chemical Similarly, S‐OBHS‐3, cyclofenil‐ASC, and OBHS‐ASC had positively correlated NCOA1/2/3 recruitment and GREB1 levels, but none of these activities determined their proliferative effects (Fig 3E and F lanes 11–12 and 18). RESULTS +76 85 NCOA1/2/3 protein Similarly, S‐OBHS‐3, cyclofenil‐ASC, and OBHS‐ASC had positively correlated NCOA1/2/3 recruitment and GREB1 levels, but none of these activities determined their proliferative effects (Fig 3E and F lanes 11–12 and 18). RESULTS +102 107 GREB1 protein Similarly, S‐OBHS‐3, cyclofenil‐ASC, and OBHS‐ASC had positively correlated NCOA1/2/3 recruitment and GREB1 levels, but none of these activities determined their proliferative effects (Fig 3E and F lanes 11–12 and 18). RESULTS +47 50 ERα protein For ligands that show cell‐specific signaling, ERα‐mediated recruitment of other coregulators and activation of other target genes likely determine their proliferative effects on MCF‐7 cells. RESULTS +0 5 NCOA3 protein NCOA3 occupancy at GREB1 did not predict the proliferative response RESULTS +19 24 GREB1 protein NCOA3 occupancy at GREB1 did not predict the proliferative response RESULTS +137 199 chromatin immunoprecipitation (ChIP)‐based quantitative assay, experimental_method We also questioned whether promoter occupancy by coactivators is statistically robust and reproducible for ligand class analysis using a chromatin immunoprecipitation (ChIP)‐based quantitative assay, and whether it has a better predictive power than the M2H assay. RESULTS +254 263 M2H assay experimental_method We also questioned whether promoter occupancy by coactivators is statistically robust and reproducible for ligand class analysis using a chromatin immunoprecipitation (ChIP)‐based quantitative assay, and whether it has a better predictive power than the M2H assay. RESULTS +0 3 ERα protein ERα and NCOA3 cycle on and off the GREB1 promoter (Nwachukwu et al, 2014). RESULTS +8 13 NCOA3 protein ERα and NCOA3 cycle on and off the GREB1 promoter (Nwachukwu et al, 2014). RESULTS +35 40 GREB1 protein ERα and NCOA3 cycle on and off the GREB1 promoter (Nwachukwu et al, 2014). RESULTS +32 49 time‐course study experimental_method Therefore, we first performed a time‐course study, and found that E2 and the WAY‐C analog, AAPII‐151‐4, induced recruitment of NCOA3 to the GREB1 promoter in a temporal cycle that peaked after 45 min in MCF‐7 cells (Fig 4A). RESULTS +66 68 E2 chemical Therefore, we first performed a time‐course study, and found that E2 and the WAY‐C analog, AAPII‐151‐4, induced recruitment of NCOA3 to the GREB1 promoter in a temporal cycle that peaked after 45 min in MCF‐7 cells (Fig 4A). RESULTS +77 82 WAY‐C chemical Therefore, we first performed a time‐course study, and found that E2 and the WAY‐C analog, AAPII‐151‐4, induced recruitment of NCOA3 to the GREB1 promoter in a temporal cycle that peaked after 45 min in MCF‐7 cells (Fig 4A). RESULTS +91 102 AAPII‐151‐4 chemical Therefore, we first performed a time‐course study, and found that E2 and the WAY‐C analog, AAPII‐151‐4, induced recruitment of NCOA3 to the GREB1 promoter in a temporal cycle that peaked after 45 min in MCF‐7 cells (Fig 4A). RESULTS +127 132 NCOA3 protein Therefore, we first performed a time‐course study, and found that E2 and the WAY‐C analog, AAPII‐151‐4, induced recruitment of NCOA3 to the GREB1 promoter in a temporal cycle that peaked after 45 min in MCF‐7 cells (Fig 4A). RESULTS +140 145 GREB1 protein Therefore, we first performed a time‐course study, and found that E2 and the WAY‐C analog, AAPII‐151‐4, induced recruitment of NCOA3 to the GREB1 promoter in a temporal cycle that peaked after 45 min in MCF‐7 cells (Fig 4A). RESULTS +26 31 WAY‐C chemical At this time point, other WAY‐C analogs also induced recruitment of NCOA3 at this site to varying degrees (Fig 4B). RESULTS +68 73 NCOA3 protein At this time point, other WAY‐C analogs also induced recruitment of NCOA3 at this site to varying degrees (Fig 4B). RESULTS +4 6 Z’ evidence The Z’ for this assay was 0.6, showing statistical robustness (see Materials and Methods). RESULTS +116 119 r 2 evidence We prepared biological replicates with different cell passage numbers and separately prepared samples, which showed r 2 of 0.81, demonstrating high reproducibility (Fig 4C). RESULTS +1 6 NCOA3 protein NCOA3 occupancy at GREB1 is statistically robust but does not predict transcriptional activity FIG +20 25 GREB1 protein NCOA3 occupancy at GREB1 is statistically robust but does not predict transcriptional activity FIG +0 18 Kinetic ChIP assay experimental_method Kinetic ChIP assay examining recruitment of NCOA3 to the GREB1 gene in MCF‐7 cells stimulated with E2 or the indicated WAY‐C analog. FIG +44 49 NCOA3 protein Kinetic ChIP assay examining recruitment of NCOA3 to the GREB1 gene in MCF‐7 cells stimulated with E2 or the indicated WAY‐C analog. FIG +57 62 GREB1 protein Kinetic ChIP assay examining recruitment of NCOA3 to the GREB1 gene in MCF‐7 cells stimulated with E2 or the indicated WAY‐C analog. FIG +99 101 E2 chemical Kinetic ChIP assay examining recruitment of NCOA3 to the GREB1 gene in MCF‐7 cells stimulated with E2 or the indicated WAY‐C analog. FIG +119 124 WAY‐C chemical Kinetic ChIP assay examining recruitment of NCOA3 to the GREB1 gene in MCF‐7 cells stimulated with E2 or the indicated WAY‐C analog. FIG +0 5 NCOA3 protein NCOA3 occupancy at GREB1 was compared by ChIP assay 45 min after stimulation with vehicle, E2, or the WAY‐C analogs. FIG +19 24 GREB1 protein NCOA3 occupancy at GREB1 was compared by ChIP assay 45 min after stimulation with vehicle, E2, or the WAY‐C analogs. FIG +41 51 ChIP assay experimental_method NCOA3 occupancy at GREB1 was compared by ChIP assay 45 min after stimulation with vehicle, E2, or the WAY‐C analogs. FIG +91 93 E2 chemical NCOA3 occupancy at GREB1 was compared by ChIP assay 45 min after stimulation with vehicle, E2, or the WAY‐C analogs. FIG +102 107 WAY‐C chemical NCOA3 occupancy at GREB1 was compared by ChIP assay 45 min after stimulation with vehicle, E2, or the WAY‐C analogs. FIG +100 107 Z‐score evidence In panel (B), the average recruitment of two biological replicates are shown as mean + SEM, and the Z‐score is indicated. FIG +14 34 correlation analysis experimental_method In panel (C), correlation analysis was performed for two biological replicates. FIG +0 26 Linear regression analyses experimental_method Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY‐C analogs. *Significant positive correlation (F‐test for nonzero slope, P‐value). FIG +52 57 NCOA3 protein Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY‐C analogs. *Significant positive correlation (F‐test for nonzero slope, P‐value). FIG +83 87 ChIP experimental_method Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY‐C analogs. *Significant positive correlation (F‐test for nonzero slope, P‐value). FIG +91 94 M2H experimental_method Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY‐C analogs. *Significant positive correlation (F‐test for nonzero slope, P‐value). FIG +135 140 WAY‐C chemical Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY‐C analogs. *Significant positive correlation (F‐test for nonzero slope, P‐value). FIG +185 191 F‐test experimental_method Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY‐C analogs. *Significant positive correlation (F‐test for nonzero slope, P‐value). FIG +211 218 P‐value evidence Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY‐C analogs. *Significant positive correlation (F‐test for nonzero slope, P‐value). FIG +4 13 M2H assay experimental_method The M2H assay for NCOA3 recruitment broadly correlated with the other assays, and was predictive for GREB1 expression and cell proliferation (Fig 3E). RESULTS +18 23 NCOA3 protein The M2H assay for NCOA3 recruitment broadly correlated with the other assays, and was predictive for GREB1 expression and cell proliferation (Fig 3E). RESULTS +101 106 GREB1 protein The M2H assay for NCOA3 recruitment broadly correlated with the other assays, and was predictive for GREB1 expression and cell proliferation (Fig 3E). RESULTS +13 24 ChIP assays experimental_method However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). RESULTS +29 34 WAY‐C chemical However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). RESULTS +58 63 NCOA3 protein However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). RESULTS +71 76 GREB1 protein However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). RESULTS +126 131 WAY‐C chemical However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). RESULTS +202 213 ChIP assays experimental_method However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). RESULTS +218 223 NCOA3 protein However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). RESULTS +240 249 M2H assay experimental_method However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). RESULTS +290 293 r 2 evidence However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). RESULTS +305 306 P evidence However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). RESULTS +315 321 F‐test experimental_method However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). RESULTS +21 46 coactivator‐binding assay experimental_method Thus, the simplified coactivator‐binding assay showed much greater predictive power than the ChIP assay for ligand‐specific effects on GREB1 expression and cell proliferation. RESULTS +93 103 ChIP assay experimental_method Thus, the simplified coactivator‐binding assay showed much greater predictive power than the ChIP assay for ligand‐specific effects on GREB1 expression and cell proliferation. RESULTS +135 140 GREB1 protein Thus, the simplified coactivator‐binding assay showed much greater predictive power than the ChIP assay for ligand‐specific effects on GREB1 expression and cell proliferation. RESULTS +0 3 ERβ protein ERβ activity is not an independent predictor of cell‐specific activity RESULTS +110 113 ERβ protein One difference between MCF‐7 breast cancer cells and Ishikawa endometrial cancer cells is the contribution of ERβ to estrogenic response, as Ishikawa cells may express ERβ (Bhat & Pezzuto, 2001). RESULTS +168 171 ERβ protein One difference between MCF‐7 breast cancer cells and Ishikawa endometrial cancer cells is the contribution of ERβ to estrogenic response, as Ishikawa cells may express ERβ (Bhat & Pezzuto, 2001). RESULTS +5 18 overexpressed experimental_method When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). RESULTS +35 38 ERβ protein When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). RESULTS +46 48 E2 chemical When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). RESULTS +88 91 ERα protein When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). RESULTS +167 170 ERβ protein When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). RESULTS +198 203 E‐Luc experimental_method When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). RESULTS +270 275 E‐Luc experimental_method When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). RESULTS +280 285 L‐Luc experimental_method When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). RESULTS +286 289 ERα protein When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). RESULTS +290 292 WT protein_state When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). RESULTS +307 312 GREB1 protein When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C). RESULTS +37 42 L‐Luc experimental_method To test this idea, we determined the L‐Luc ERβ activity profiles of the ligands (Fig EV1). RESULTS +59 63 OBHS chemical All direct modulator and two indirect modulator scaffolds (OBHS and S‐OBHS‐3) lacked ERβ agonist activity. RESULTS +68 76 S‐OBHS‐3 chemical All direct modulator and two indirect modulator scaffolds (OBHS and S‐OBHS‐3) lacked ERβ agonist activity. RESULTS +20 25 L‐Luc experimental_method For most scaffolds, L‐Luc ERβ and E‐Luc activities were not correlated, except for 2,5‐DTP and cyclofenil analogs, which showed moderate but significant correlations (Fig EV4A). RESULTS +34 39 E‐Luc experimental_method For most scaffolds, L‐Luc ERβ and E‐Luc activities were not correlated, except for 2,5‐DTP and cyclofenil analogs, which showed moderate but significant correlations (Fig EV4A). RESULTS +83 90 2,5‐DTP chemical For most scaffolds, L‐Luc ERβ and E‐Luc activities were not correlated, except for 2,5‐DTP and cyclofenil analogs, which showed moderate but significant correlations (Fig EV4A). RESULTS +95 105 cyclofenil chemical For most scaffolds, L‐Luc ERβ and E‐Luc activities were not correlated, except for 2,5‐DTP and cyclofenil analogs, which showed moderate but significant correlations (Fig EV4A). RESULTS +18 23 E‐Luc experimental_method Nevertheless, the E‐Luc activities of both 2,5‐DTP and cyclofenil analogs were better predicted by their L‐Luc ERα‐WT than L‐Luc ERβ activities (Fig EV4A and B). RESULTS +43 50 2,5‐DTP chemical Nevertheless, the E‐Luc activities of both 2,5‐DTP and cyclofenil analogs were better predicted by their L‐Luc ERα‐WT than L‐Luc ERβ activities (Fig EV4A and B). RESULTS +55 65 cyclofenil chemical Nevertheless, the E‐Luc activities of both 2,5‐DTP and cyclofenil analogs were better predicted by their L‐Luc ERα‐WT than L‐Luc ERβ activities (Fig EV4A and B). RESULTS +105 110 L‐Luc experimental_method Nevertheless, the E‐Luc activities of both 2,5‐DTP and cyclofenil analogs were better predicted by their L‐Luc ERα‐WT than L‐Luc ERβ activities (Fig EV4A and B). RESULTS +111 114 ERα protein Nevertheless, the E‐Luc activities of both 2,5‐DTP and cyclofenil analogs were better predicted by their L‐Luc ERα‐WT than L‐Luc ERβ activities (Fig EV4A and B). RESULTS +115 117 WT protein_state Nevertheless, the E‐Luc activities of both 2,5‐DTP and cyclofenil analogs were better predicted by their L‐Luc ERα‐WT than L‐Luc ERβ activities (Fig EV4A and B). RESULTS +123 128 L‐Luc experimental_method Nevertheless, the E‐Luc activities of both 2,5‐DTP and cyclofenil analogs were better predicted by their L‐Luc ERα‐WT than L‐Luc ERβ activities (Fig EV4A and B). RESULTS +129 132 ERβ protein Nevertheless, the E‐Luc activities of both 2,5‐DTP and cyclofenil analogs were better predicted by their L‐Luc ERα‐WT than L‐Luc ERβ activities (Fig EV4A and B). RESULTS +0 3 ERβ protein ERβ activity is not an independent predictor of E‐Luc activity FIG +48 53 E‐Luc experimental_method ERβ activity is not an independent predictor of E‐Luc activity FIG +0 3 ERβ protein ERβ activity in HepG2 cells rarely correlates with E‐Luc activity. FIG +51 56 E‐Luc experimental_method ERβ activity in HepG2 cells rarely correlates with E‐Luc activity. FIG +0 3 ERα protein ERα activity of 2,5‐DTP and cyclofenil analogs correlates with E‐Luc activity. FIG +16 23 2,5‐DTP chemical ERα activity of 2,5‐DTP and cyclofenil analogs correlates with E‐Luc activity. FIG +28 38 cyclofenil chemical ERα activity of 2,5‐DTP and cyclofenil analogs correlates with E‐Luc activity. FIG +63 68 E‐Luc experimental_method ERα activity of 2,5‐DTP and cyclofenil analogs correlates with E‐Luc activity. FIG +31 39 P values evidence Data information: The r 2 and P values for the indicated correlations are shown in both panels. *Significant positive correlation (F‐test for nonzero slope, P‐value) FIG +132 138 F‐test experimental_method Data information: The r 2 and P values for the indicated correlations are shown in both panels. *Significant positive correlation (F‐test for nonzero slope, P‐value) FIG +158 165 P‐value evidence Data information: The r 2 and P values for the indicated correlations are shown in both panels. *Significant positive correlation (F‐test for nonzero slope, P‐value) FIG +24 39 crystallization experimental_method To overcome barriers to crystallization of ERα LBD complexes, we developed a conformation‐trapping X‐ray crystallography approach using the ERα‐Y537S mutation (Nettles et al, 2008; Bruning et al, 2010; Srinivasan et al, 2013). RESULTS +43 46 ERα protein To overcome barriers to crystallization of ERα LBD complexes, we developed a conformation‐trapping X‐ray crystallography approach using the ERα‐Y537S mutation (Nettles et al, 2008; Bruning et al, 2010; Srinivasan et al, 2013). RESULTS +47 50 LBD structure_element To overcome barriers to crystallization of ERα LBD complexes, we developed a conformation‐trapping X‐ray crystallography approach using the ERα‐Y537S mutation (Nettles et al, 2008; Bruning et al, 2010; Srinivasan et al, 2013). RESULTS +77 120 conformation‐trapping X‐ray crystallography experimental_method To overcome barriers to crystallization of ERα LBD complexes, we developed a conformation‐trapping X‐ray crystallography approach using the ERα‐Y537S mutation (Nettles et al, 2008; Bruning et al, 2010; Srinivasan et al, 2013). RESULTS +140 149 ERα‐Y537S mutant To overcome barriers to crystallization of ERα LBD complexes, we developed a conformation‐trapping X‐ray crystallography approach using the ERα‐Y537S mutation (Nettles et al, 2008; Bruning et al, 2010; Srinivasan et al, 2013). RESULTS +38 44 solved experimental_method To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). RESULTS +49 58 structure evidence To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). RESULTS +66 75 ERα‐Y537S mutant To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). RESULTS +76 79 LBD structure_element To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). RESULTS +80 95 in complex with protein_state To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). RESULTS +96 114 diethylstilbestrol chemical To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). RESULTS +116 119 DES chemical To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). RESULTS +153 162 wild‐type protein_state To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). RESULTS +167 176 ERα‐Y537S mutant To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). RESULTS +177 181 LBDs structure_element To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). RESULTS +241 244 h12 structure_element To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). RESULTS +24 30 solved experimental_method Using this approach, we solved 76 ERα LBD structures in the active conformation and bound to ligands studied here (Appendix Fig S1C). RESULTS +34 37 ERα protein Using this approach, we solved 76 ERα LBD structures in the active conformation and bound to ligands studied here (Appendix Fig S1C). RESULTS +38 41 LBD structure_element Using this approach, we solved 76 ERα LBD structures in the active conformation and bound to ligands studied here (Appendix Fig S1C). RESULTS +42 52 structures evidence Using this approach, we solved 76 ERα LBD structures in the active conformation and bound to ligands studied here (Appendix Fig S1C). RESULTS +60 79 active conformation protein_state Using this approach, we solved 76 ERα LBD structures in the active conformation and bound to ligands studied here (Appendix Fig S1C). RESULTS +84 100 bound to ligands protein_state Using this approach, we solved 76 ERα LBD structures in the active conformation and bound to ligands studied here (Appendix Fig S1C). RESULTS +16 26 structures evidence Eleven of these structures have been published, while 65 are new, including the DES‐bound ERα‐Y537S LBD. RESULTS +80 89 DES‐bound protein_state Eleven of these structures have been published, while 65 are new, including the DES‐bound ERα‐Y537S LBD. RESULTS +90 99 ERα‐Y537S mutant Eleven of these structures have been published, while 65 are new, including the DES‐bound ERα‐Y537S LBD. RESULTS +100 103 LBD structure_element Eleven of these structures have been published, while 65 are new, including the DES‐bound ERα‐Y537S LBD. RESULTS +27 37 structures evidence We present 57 of these new structures here (Dataset EV2), while the remaining eight new structures bound to OBHS‐N analogs will be published elsewhere (S. Srinivasan et al, in preparation). RESULTS +88 98 structures evidence We present 57 of these new structures here (Dataset EV2), while the remaining eight new structures bound to OBHS‐N analogs will be published elsewhere (S. Srinivasan et al, in preparation). RESULTS +99 107 bound to protein_state We present 57 of these new structures here (Dataset EV2), while the remaining eight new structures bound to OBHS‐N analogs will be published elsewhere (S. Srinivasan et al, in preparation). RESULTS +108 114 OBHS‐N chemical We present 57 of these new structures here (Dataset EV2), while the remaining eight new structures bound to OBHS‐N analogs will be published elsewhere (S. Srinivasan et al, in preparation). RESULTS +31 41 structures evidence Examining many closely related structures allows us to visualize subtle structural differences, in effect using X‐ray crystallography as a systems biology tool. RESULTS +112 133 X‐ray crystallography experimental_method Examining many closely related structures allows us to visualize subtle structural differences, in effect using X‐ray crystallography as a systems biology tool. RESULTS +165 168 h12 structure_element The indirect modulator scaffolds in cluster 1 did not show cell‐specific signaling (Fig 3C), but shared common structural perturbations that we designed to modulate h12 dynamics. RESULTS +22 26 OBHS chemical Based on our original OBHS structure, the OBHS, OBHS‐N, and triaryl‐ethylene compounds were modified with h11‐directed pendant groups (Zheng et al, 2012; Zhu et al, 2012; Liao et al, 2014). RESULTS +27 36 structure evidence Based on our original OBHS structure, the OBHS, OBHS‐N, and triaryl‐ethylene compounds were modified with h11‐directed pendant groups (Zheng et al, 2012; Zhu et al, 2012; Liao et al, 2014). RESULTS +42 46 OBHS chemical Based on our original OBHS structure, the OBHS, OBHS‐N, and triaryl‐ethylene compounds were modified with h11‐directed pendant groups (Zheng et al, 2012; Zhu et al, 2012; Liao et al, 2014). RESULTS +48 54 OBHS‐N chemical Based on our original OBHS structure, the OBHS, OBHS‐N, and triaryl‐ethylene compounds were modified with h11‐directed pendant groups (Zheng et al, 2012; Zhu et al, 2012; Liao et al, 2014). RESULTS +60 76 triaryl‐ethylene chemical Based on our original OBHS structure, the OBHS, OBHS‐N, and triaryl‐ethylene compounds were modified with h11‐directed pendant groups (Zheng et al, 2012; Zhu et al, 2012; Liao et al, 2014). RESULTS +106 109 h11 structure_element Based on our original OBHS structure, the OBHS, OBHS‐N, and triaryl‐ethylene compounds were modified with h11‐directed pendant groups (Zheng et al, 2012; Zhu et al, 2012; Liao et al, 2014). RESULTS +0 11 Superposing experimental_method Superposing the LBDs based on the class of bound ligands provides an ensemble view of the structural variance and clarifies what part of the ligand‐binding pocket is differentially perturbed or targeted. RESULTS +16 20 LBDs structure_element Superposing the LBDs based on the class of bound ligands provides an ensemble view of the structural variance and clarifies what part of the ligand‐binding pocket is differentially perturbed or targeted. RESULTS +141 162 ligand‐binding pocket site Superposing the LBDs based on the class of bound ligands provides an ensemble view of the structural variance and clarifies what part of the ligand‐binding pocket is differentially perturbed or targeted. RESULTS +7 17 structures evidence The 24 structures containing OBHS, OBHS‐N, or triaryl‐ethylene analogs showed structural diversity in the same part of the scaffolds (Figs 5A and EV5A), and the same region of the LBD—the C‐terminal end of h11 (Figs 5B and C, and EV5B), which in turn nudges h12 (Fig 5C and D). RESULTS +29 33 OBHS chemical The 24 structures containing OBHS, OBHS‐N, or triaryl‐ethylene analogs showed structural diversity in the same part of the scaffolds (Figs 5A and EV5A), and the same region of the LBD—the C‐terminal end of h11 (Figs 5B and C, and EV5B), which in turn nudges h12 (Fig 5C and D). RESULTS +35 41 OBHS‐N chemical The 24 structures containing OBHS, OBHS‐N, or triaryl‐ethylene analogs showed structural diversity in the same part of the scaffolds (Figs 5A and EV5A), and the same region of the LBD—the C‐terminal end of h11 (Figs 5B and C, and EV5B), which in turn nudges h12 (Fig 5C and D). RESULTS +46 62 triaryl‐ethylene chemical The 24 structures containing OBHS, OBHS‐N, or triaryl‐ethylene analogs showed structural diversity in the same part of the scaffolds (Figs 5A and EV5A), and the same region of the LBD—the C‐terminal end of h11 (Figs 5B and C, and EV5B), which in turn nudges h12 (Fig 5C and D). RESULTS +180 183 LBD structure_element The 24 structures containing OBHS, OBHS‐N, or triaryl‐ethylene analogs showed structural diversity in the same part of the scaffolds (Figs 5A and EV5A), and the same region of the LBD—the C‐terminal end of h11 (Figs 5B and C, and EV5B), which in turn nudges h12 (Fig 5C and D). RESULTS +206 209 h11 structure_element The 24 structures containing OBHS, OBHS‐N, or triaryl‐ethylene analogs showed structural diversity in the same part of the scaffolds (Figs 5A and EV5A), and the same region of the LBD—the C‐terminal end of h11 (Figs 5B and C, and EV5B), which in turn nudges h12 (Fig 5C and D). RESULTS +258 261 h12 structure_element The 24 structures containing OBHS, OBHS‐N, or triaryl‐ethylene analogs showed structural diversity in the same part of the scaffolds (Figs 5A and EV5A), and the same region of the LBD—the C‐terminal end of h11 (Figs 5B and C, and EV5B), which in turn nudges h12 (Fig 5C and D). RESULTS +21 27 OBHS‐N chemical We observed that the OBHS‐N analogs displaced h11 along a vector away from Leu354 in a region of h3 that is unaffected by the ligands, and toward the dimer interface. RESULTS +46 49 h11 structure_element We observed that the OBHS‐N analogs displaced h11 along a vector away from Leu354 in a region of h3 that is unaffected by the ligands, and toward the dimer interface. RESULTS +75 81 Leu354 residue_name_number We observed that the OBHS‐N analogs displaced h11 along a vector away from Leu354 in a region of h3 that is unaffected by the ligands, and toward the dimer interface. RESULTS +97 99 h3 structure_element We observed that the OBHS‐N analogs displaced h11 along a vector away from Leu354 in a region of h3 that is unaffected by the ligands, and toward the dimer interface. RESULTS +150 165 dimer interface site We observed that the OBHS‐N analogs displaced h11 along a vector away from Leu354 in a region of h3 that is unaffected by the ligands, and toward the dimer interface. RESULTS +8 24 triaryl‐ethylene chemical For the triaryl‐ethylene analogs, the displacement of h11 was in a perpendicular direction, away from Ile424 in h8 and toward h12. RESULTS +54 57 h11 structure_element For the triaryl‐ethylene analogs, the displacement of h11 was in a perpendicular direction, away from Ile424 in h8 and toward h12. RESULTS +102 108 Ile424 residue_name_number For the triaryl‐ethylene analogs, the displacement of h11 was in a perpendicular direction, away from Ile424 in h8 and toward h12. RESULTS +112 114 h8 structure_element For the triaryl‐ethylene analogs, the displacement of h11 was in a perpendicular direction, away from Ile424 in h8 and toward h12. RESULTS +126 129 h12 structure_element For the triaryl‐ethylene analogs, the displacement of h11 was in a perpendicular direction, away from Ile424 in h8 and toward h12. RESULTS +29 51 inter‐atomic distances evidence Remarkably, these individual inter‐atomic distances showed a ligand class‐specific ability to significantly predict proliferative effects (Fig 5E and F), demonstrating the feasibility of developing a minimal set of activity predictors from crystal structures. RESULTS +240 258 crystal structures evidence Remarkably, these individual inter‐atomic distances showed a ligand class‐specific ability to significantly predict proliferative effects (Fig 5E and F), demonstrating the feasibility of developing a minimal set of activity predictors from crystal structures. RESULTS +0 24 Structure‐class analysis experimental_method Structure‐class analysis of triaryl‐ethylene analogs. FIG +28 44 triaryl‐ethylene chemical Structure‐class analysis of triaryl‐ethylene analogs. FIG +0 16 Triaryl‐ethylene chemical Triaryl‐ethylene analogs bound to the superposed crystal structures of the ERα LBD are shown. FIG +25 33 bound to protein_state Triaryl‐ethylene analogs bound to the superposed crystal structures of the ERα LBD are shown. FIG +38 48 superposed experimental_method Triaryl‐ethylene analogs bound to the superposed crystal structures of the ERα LBD are shown. FIG +49 67 crystal structures evidence Triaryl‐ethylene analogs bound to the superposed crystal structures of the ERα LBD are shown. FIG +75 78 ERα protein Triaryl‐ethylene analogs bound to the superposed crystal structures of the ERα LBD are shown. FIG +79 82 LBD structure_element Triaryl‐ethylene analogs bound to the superposed crystal structures of the ERα LBD are shown. FIG +70 73 h11 structure_element Arrows indicate chemical variance in the orientation of the different h11‐directed ligand side groups (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF and 5DP0). FIG +0 16 Triaryl‐ethylene chemical Triaryl‐ethylene analogs induce variance of ERα conformations at the C‐terminal region of h11. FIG +44 47 ERα protein Triaryl‐ethylene analogs induce variance of ERα conformations at the C‐terminal region of h11. FIG +90 93 h11 structure_element Triaryl‐ethylene analogs induce variance of ERα conformations at the C‐terminal region of h11. FIG +20 37 crystal structure evidence Panel (B) shows the crystal structure of a triaryl‐ethylene analog‐bound ERα LBD (PDB 5DLR). FIG +43 59 triaryl‐ethylene chemical Panel (B) shows the crystal structure of a triaryl‐ethylene analog‐bound ERα LBD (PDB 5DLR). FIG +73 76 ERα protein Panel (B) shows the crystal structure of a triaryl‐ethylene analog‐bound ERα LBD (PDB 5DLR). FIG +77 80 LBD structure_element Panel (B) shows the crystal structure of a triaryl‐ethylene analog‐bound ERα LBD (PDB 5DLR). FIG +4 21 h11–h12 interface site The h11–h12 interface (circled) includes the C‐terminal part of h11. FIG +64 67 h11 structure_element The h11–h12 interface (circled) includes the C‐terminal part of h11. FIG +52 68 triaryl‐ethylene chemical This region was expanded in panel (C), where the 10 triaryl‐ethylene analog‐bound ERα LBD structures (see Datasets EV1 and EV2) were superposed to show variations in the h11 C‐terminus (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, and 5DP0). FIG +82 85 ERα protein This region was expanded in panel (C), where the 10 triaryl‐ethylene analog‐bound ERα LBD structures (see Datasets EV1 and EV2) were superposed to show variations in the h11 C‐terminus (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, and 5DP0). FIG +86 89 LBD structure_element This region was expanded in panel (C), where the 10 triaryl‐ethylene analog‐bound ERα LBD structures (see Datasets EV1 and EV2) were superposed to show variations in the h11 C‐terminus (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, and 5DP0). FIG +90 100 structures evidence This region was expanded in panel (C), where the 10 triaryl‐ethylene analog‐bound ERα LBD structures (see Datasets EV1 and EV2) were superposed to show variations in the h11 C‐terminus (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, and 5DP0). FIG +133 143 superposed experimental_method This region was expanded in panel (C), where the 10 triaryl‐ethylene analog‐bound ERα LBD structures (see Datasets EV1 and EV2) were superposed to show variations in the h11 C‐terminus (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, and 5DP0). FIG +170 173 h11 structure_element This region was expanded in panel (C), where the 10 triaryl‐ethylene analog‐bound ERα LBD structures (see Datasets EV1 and EV2) were superposed to show variations in the h11 C‐terminus (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, and 5DP0). FIG +0 3 ERα protein ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC). FIG +4 8 LBDs structure_element ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC). FIG +9 24 in complex with protein_state ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC). FIG +25 43 diethylstilbestrol chemical ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC). FIG +45 48 DES chemical ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC). FIG +55 71 triaryl‐ethylene chemical ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC). FIG +84 94 superposed experimental_method ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC). FIG +141 144 h11 structure_element ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC). FIG +194 197 h12 structure_element ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC). FIG +0 22 Inter‐atomic distances evidence Inter‐atomic distances predict the proliferative effects of specific ligand series. FIG +0 6 Ile424 residue_name_number Ile424–His524 distance measured in the crystal structures correlates with the proliferative effect of triaryl‐ethylene analogs in MCF‐7 cells. FIG +7 13 His524 residue_name_number Ile424–His524 distance measured in the crystal structures correlates with the proliferative effect of triaryl‐ethylene analogs in MCF‐7 cells. FIG +14 22 distance evidence Ile424–His524 distance measured in the crystal structures correlates with the proliferative effect of triaryl‐ethylene analogs in MCF‐7 cells. FIG +39 57 crystal structures evidence Ile424–His524 distance measured in the crystal structures correlates with the proliferative effect of triaryl‐ethylene analogs in MCF‐7 cells. FIG +102 118 triaryl‐ethylene chemical Ile424–His524 distance measured in the crystal structures correlates with the proliferative effect of triaryl‐ethylene analogs in MCF‐7 cells. FIG +17 23 Leu354 residue_name_number In contrast, the Leu354–Leu525 distance correlates with the proliferative effects of OBHS‐N analogs in MCF‐7 cells. FIG +24 30 Leu525 residue_name_number In contrast, the Leu354–Leu525 distance correlates with the proliferative effects of OBHS‐N analogs in MCF‐7 cells. FIG +31 39 distance evidence In contrast, the Leu354–Leu525 distance correlates with the proliferative effects of OBHS‐N analogs in MCF‐7 cells. FIG +85 91 OBHS‐N chemical In contrast, the Leu354–Leu525 distance correlates with the proliferative effects of OBHS‐N analogs in MCF‐7 cells. FIG +0 24 Structure‐class analysis experimental_method Structure‐class analysis of WAY‐C analogs. FIG +28 33 WAY‐C chemical Structure‐class analysis of WAY‐C analogs. FIG +0 5 WAY‐C chemical WAY‐C side groups subtly nudge h12 Leu540. FIG +31 34 h12 structure_element WAY‐C side groups subtly nudge h12 Leu540. FIG +35 41 Leu540 residue_name_number WAY‐C side groups subtly nudge h12 Leu540. FIG +0 3 ERα protein ERα LBD structures bound to 4 distinct WAY‐C analogs were superposed (PDB 4 IU7, 4IV4, 4IVW, 4IW6) (see Datasets EV1 and EV2). FIG +4 7 LBD structure_element ERα LBD structures bound to 4 distinct WAY‐C analogs were superposed (PDB 4 IU7, 4IV4, 4IVW, 4IW6) (see Datasets EV1 and EV2). FIG +8 18 structures evidence ERα LBD structures bound to 4 distinct WAY‐C analogs were superposed (PDB 4 IU7, 4IV4, 4IVW, 4IW6) (see Datasets EV1 and EV2). FIG +19 27 bound to protein_state ERα LBD structures bound to 4 distinct WAY‐C analogs were superposed (PDB 4 IU7, 4IV4, 4IVW, 4IW6) (see Datasets EV1 and EV2). FIG +39 44 WAY‐C chemical ERα LBD structures bound to 4 distinct WAY‐C analogs were superposed (PDB 4 IU7, 4IV4, 4IVW, 4IW6) (see Datasets EV1 and EV2). FIG +58 68 superposed experimental_method ERα LBD structures bound to 4 distinct WAY‐C analogs were superposed (PDB 4 IU7, 4IV4, 4IVW, 4IW6) (see Datasets EV1 and EV2). FIG +0 24 Structure‐class analysis experimental_method Structure‐class analysis of indirect modulators FIG +0 24 Structure‐class analysis experimental_method Structure‐class analysis of indirect modulators in cluster 1. FIG +0 18 Crystal structures evidence Crystal structures of the ERα LBD bound to OBHS and OBHS‐N analogs were superposed. FIG +26 29 ERα protein Crystal structures of the ERα LBD bound to OBHS and OBHS‐N analogs were superposed. FIG +30 33 LBD structure_element Crystal structures of the ERα LBD bound to OBHS and OBHS‐N analogs were superposed. FIG +34 42 bound to protein_state Crystal structures of the ERα LBD bound to OBHS and OBHS‐N analogs were superposed. FIG +43 47 OBHS chemical Crystal structures of the ERα LBD bound to OBHS and OBHS‐N analogs were superposed. FIG +52 58 OBHS‐N chemical Crystal structures of the ERα LBD bound to OBHS and OBHS‐N analogs were superposed. FIG +72 82 superposed experimental_method Crystal structures of the ERα LBD bound to OBHS and OBHS‐N analogs were superposed. FIG +70 73 h11 structure_element Arrows indicate chemical variance in the orientation of the different h11‐directed ligand side groups. FIG +88 91 h11 structure_element Panel (B) shows the ligand‐induced conformational variation at the C‐terminal region of h11 (OBHS: PDB 4ZN9, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, and 4ZNW; OBHS‐N: PDB 4ZUB, 4ZUC, 4ZWH, 4ZWK, 5BNU, 5BP6, 5BPR, and 5BQ4). FIG +93 97 OBHS chemical Panel (B) shows the ligand‐induced conformational variation at the C‐terminal region of h11 (OBHS: PDB 4ZN9, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, and 4ZNW; OBHS‐N: PDB 4ZUB, 4ZUC, 4ZWH, 4ZWK, 5BNU, 5BP6, 5BPR, and 5BQ4). FIG +149 155 OBHS‐N chemical Panel (B) shows the ligand‐induced conformational variation at the C‐terminal region of h11 (OBHS: PDB 4ZN9, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, and 4ZNW; OBHS‐N: PDB 4ZUB, 4ZUC, 4ZWH, 4ZWK, 5BNU, 5BP6, 5BPR, and 5BQ4). FIG +0 24 Structure‐class analysis experimental_method Structure‐class analysis of indirect modulators in clusters 2 and 3. FIG +0 18 Crystal structures evidence Crystal structures of the ERα LBD bound to ligands with cell‐specific activities were superposed. FIG +26 29 ERα protein Crystal structures of the ERα LBD bound to ligands with cell‐specific activities were superposed. FIG +30 33 LBD structure_element Crystal structures of the ERα LBD bound to ligands with cell‐specific activities were superposed. FIG +34 42 bound to protein_state Crystal structures of the ERα LBD bound to ligands with cell‐specific activities were superposed. FIG +86 96 superposed experimental_method Crystal structures of the ERα LBD bound to ligands with cell‐specific activities were superposed. FIG +108 110 h3 structure_element The bound ligands are shown, and arrows indicate considerable variation in the orientation of the different h3‐, h8‐, h11‐, or h12‐directed ligand side groups. FIG +113 115 h8 structure_element The bound ligands are shown, and arrows indicate considerable variation in the orientation of the different h3‐, h8‐, h11‐, or h12‐directed ligand side groups. FIG +118 121 h11 structure_element The bound ligands are shown, and arrows indicate considerable variation in the orientation of the different h3‐, h8‐, h11‐, or h12‐directed ligand side groups. FIG +127 130 h12 structure_element The bound ligands are shown, and arrows indicate considerable variation in the orientation of the different h3‐, h8‐, h11‐, or h12‐directed ligand side groups. FIG +22 25 LBD structure_element As visualized in four LBD structures (Srinivasan et al, 2013), WAY‐C analogs were designed with small substitutions that slightly nudge h12 Leu540, without exiting the ligand‐binding pocket (Fig 5G and H). RESULTS +26 36 structures evidence As visualized in four LBD structures (Srinivasan et al, 2013), WAY‐C analogs were designed with small substitutions that slightly nudge h12 Leu540, without exiting the ligand‐binding pocket (Fig 5G and H). RESULTS +63 68 WAY‐C chemical As visualized in four LBD structures (Srinivasan et al, 2013), WAY‐C analogs were designed with small substitutions that slightly nudge h12 Leu540, without exiting the ligand‐binding pocket (Fig 5G and H). RESULTS +136 139 h12 structure_element As visualized in four LBD structures (Srinivasan et al, 2013), WAY‐C analogs were designed with small substitutions that slightly nudge h12 Leu540, without exiting the ligand‐binding pocket (Fig 5G and H). RESULTS +140 146 Leu540 residue_name_number As visualized in four LBD structures (Srinivasan et al, 2013), WAY‐C analogs were designed with small substitutions that slightly nudge h12 Leu540, without exiting the ligand‐binding pocket (Fig 5G and H). RESULTS +168 189 ligand‐binding pocket site As visualized in four LBD structures (Srinivasan et al, 2013), WAY‐C analogs were designed with small substitutions that slightly nudge h12 Leu540, without exiting the ligand‐binding pocket (Fig 5G and H). RESULTS +20 23 h12 structure_element Therefore, changing h12 dynamics maintains the canonical signaling pathway defined by E2 (Fig 1D) to support AF‐2‐driven signaling and recruit NCOA1/2/3 for GREB1‐stimulated proliferation. RESULTS +86 88 E2 chemical Therefore, changing h12 dynamics maintains the canonical signaling pathway defined by E2 (Fig 1D) to support AF‐2‐driven signaling and recruit NCOA1/2/3 for GREB1‐stimulated proliferation. RESULTS +109 113 AF‐2 structure_element Therefore, changing h12 dynamics maintains the canonical signaling pathway defined by E2 (Fig 1D) to support AF‐2‐driven signaling and recruit NCOA1/2/3 for GREB1‐stimulated proliferation. RESULTS +143 152 NCOA1/2/3 protein Therefore, changing h12 dynamics maintains the canonical signaling pathway defined by E2 (Fig 1D) to support AF‐2‐driven signaling and recruit NCOA1/2/3 for GREB1‐stimulated proliferation. RESULTS +157 162 GREB1 protein Therefore, changing h12 dynamics maintains the canonical signaling pathway defined by E2 (Fig 1D) to support AF‐2‐driven signaling and recruit NCOA1/2/3 for GREB1‐stimulated proliferation. RESULTS +59 71 AF‐2 surface site Ligands with cell‐specific activity alter the shape of the AF‐2 surface RESULTS +23 32 tamoxifen chemical Direct modulators like tamoxifen drive AF‐1‐dependent cell‐specific activity by completely occluding AF‐2, but it is not known how indirect modulators produce cell‐specific ERα activity. RESULTS +39 43 AF‐1 structure_element Direct modulators like tamoxifen drive AF‐1‐dependent cell‐specific activity by completely occluding AF‐2, but it is not known how indirect modulators produce cell‐specific ERα activity. RESULTS +101 105 AF‐2 structure_element Direct modulators like tamoxifen drive AF‐1‐dependent cell‐specific activity by completely occluding AF‐2, but it is not known how indirect modulators produce cell‐specific ERα activity. RESULTS +173 176 ERα protein Direct modulators like tamoxifen drive AF‐1‐dependent cell‐specific activity by completely occluding AF‐2, but it is not known how indirect modulators produce cell‐specific ERα activity. RESULTS +34 37 LBD structure_element Therefore, we examined another 50 LBD structures containing ligands in clusters 2 and 3. RESULTS +38 48 structures evidence Therefore, we examined another 50 LBD structures containing ligands in clusters 2 and 3. RESULTS +6 16 structures evidence These structures demonstrated that cell‐specific activity derived from altering the shape of the AF‐2 surface without an extended side chain. RESULTS +97 109 AF‐2 surface site These structures demonstrated that cell‐specific activity derived from altering the shape of the AF‐2 surface without an extended side chain. RESULTS +165 167 h3 structure_element Ligands in cluster 2 and cluster 3 showed conformational heterogeneity in parts of the scaffold that were directed toward multiple regions of the receptor including h3, h8, h11, h12, and/or the β‐sheets (Fig EV5C–G). RESULTS +169 171 h8 structure_element Ligands in cluster 2 and cluster 3 showed conformational heterogeneity in parts of the scaffold that were directed toward multiple regions of the receptor including h3, h8, h11, h12, and/or the β‐sheets (Fig EV5C–G). RESULTS +173 176 h11 structure_element Ligands in cluster 2 and cluster 3 showed conformational heterogeneity in parts of the scaffold that were directed toward multiple regions of the receptor including h3, h8, h11, h12, and/or the β‐sheets (Fig EV5C–G). RESULTS +178 181 h12 structure_element Ligands in cluster 2 and cluster 3 showed conformational heterogeneity in parts of the scaffold that were directed toward multiple regions of the receptor including h3, h8, h11, h12, and/or the β‐sheets (Fig EV5C–G). RESULTS +194 202 β‐sheets structure_element Ligands in cluster 2 and cluster 3 showed conformational heterogeneity in parts of the scaffold that were directed toward multiple regions of the receptor including h3, h8, h11, h12, and/or the β‐sheets (Fig EV5C–G). RESULTS +14 22 S‐OBHS‐2 chemical For instance, S‐OBHS‐2 and S‐OBHS‐3 analogs (Fig 2) had similar ERα activity profiles in the different cell types (Fig EV2A–C), but the 2‐ versus 3‐methyl substituted phenol rings altered the correlated signaling patterns in different cell types (Fig 3B lanes 7 and 12). RESULTS +27 35 S‐OBHS‐3 chemical For instance, S‐OBHS‐2 and S‐OBHS‐3 analogs (Fig 2) had similar ERα activity profiles in the different cell types (Fig EV2A–C), but the 2‐ versus 3‐methyl substituted phenol rings altered the correlated signaling patterns in different cell types (Fig 3B lanes 7 and 12). RESULTS +64 67 ERα protein For instance, S‐OBHS‐2 and S‐OBHS‐3 analogs (Fig 2) had similar ERα activity profiles in the different cell types (Fig EV2A–C), but the 2‐ versus 3‐methyl substituted phenol rings altered the correlated signaling patterns in different cell types (Fig 3B lanes 7 and 12). RESULTS +50 62 AF‐2 surface site This difference in ligand positioning altered the AF‐2 surface via a shift in the N‐terminus of h12, which directly contacts the coactivator. RESULTS +96 99 h12 structure_element This difference in ligand positioning altered the AF‐2 surface via a shift in the N‐terminus of h12, which directly contacts the coactivator. RESULTS +35 44 structure evidence This effect is evident in a single structure due to its 1 Å magnitude (Fig 6A and B). RESULTS +14 17 h12 structure_element The shifts in h12 residues Asp538 and Leu539 led to rotation of the coactivator peptide (Fig 6C). RESULTS +27 33 Asp538 residue_name_number The shifts in h12 residues Asp538 and Leu539 led to rotation of the coactivator peptide (Fig 6C). RESULTS +38 44 Leu539 residue_name_number The shifts in h12 residues Asp538 and Leu539 led to rotation of the coactivator peptide (Fig 6C). RESULTS +63 75 AF‐2 surface site Thus, cell‐specific activity can stem from perturbation of the AF‐2 surface without an extended side chain, which presumably alters the receptor–coregulator interaction profile. RESULTS +0 10 S‐OBHS‐2/3 chemical S‐OBHS‐2/3 analogs subtly distort the AF‐2 surface. FIG +38 50 AF‐2 surface site S‐OBHS‐2/3 analogs subtly distort the AF‐2 surface. FIG +20 37 crystal structure evidence Panel (A) shows the crystal structure of an S‐OBHS‐3‐bound ERα LBD (PDB 5DUH). FIG +44 58 S‐OBHS‐3‐bound protein_state Panel (A) shows the crystal structure of an S‐OBHS‐3‐bound ERα LBD (PDB 5DUH). FIG +59 62 ERα protein Panel (A) shows the crystal structure of an S‐OBHS‐3‐bound ERα LBD (PDB 5DUH). FIG +63 66 LBD structure_element Panel (A) shows the crystal structure of an S‐OBHS‐3‐bound ERα LBD (PDB 5DUH). FIG +4 20 h3–h12 interface site The h3–h12 interface (circled) at AF‐2 (pink) was expanded in panels (B, C). FIG +34 38 AF‐2 structure_element The h3–h12 interface (circled) at AF‐2 (pink) was expanded in panels (B, C). FIG +4 20 S‐OBHS‐2/3‐bound protein_state The S‐OBHS‐2/3‐bound ERα LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF‐2 surface (panel C). FIG +21 24 ERα protein The S‐OBHS‐2/3‐bound ERα LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF‐2 surface (panel C). FIG +25 29 LBDs structure_element The S‐OBHS‐2/3‐bound ERα LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF‐2 surface (panel C). FIG +35 45 superposed experimental_method The S‐OBHS‐2/3‐bound ERα LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF‐2 surface (panel C). FIG +64 66 h3 structure_element The S‐OBHS‐2/3‐bound ERα LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF‐2 surface (panel C). FIG +85 90 NCOA2 protein The S‐OBHS‐2/3‐bound ERα LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF‐2 surface (panel C). FIG +113 125 AF‐2 surface site The S‐OBHS‐2/3‐bound ERα LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF‐2 surface (panel C). FIG +0 18 Crystal structures evidence Crystal structures show that 2,5‐DTP analogs shift h3 and h11 further apart compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DRM, 5DRJ). FIG +29 36 2,5‐DTP chemical Crystal structures show that 2,5‐DTP analogs shift h3 and h11 further apart compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DRM, 5DRJ). FIG +51 53 h3 structure_element Crystal structures show that 2,5‐DTP analogs shift h3 and h11 further apart compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DRM, 5DRJ). FIG +58 61 h11 structure_element Crystal structures show that 2,5‐DTP analogs shift h3 and h11 further apart compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DRM, 5DRJ). FIG +101 109 estrogen chemical Crystal structures show that 2,5‐DTP analogs shift h3 and h11 further apart compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DRM, 5DRJ). FIG +66 79 2,5‐DTP‐bound protein_state The 2F o‐F c electron density map and F o‐F c difference map of a 2,5‐DTP‐bound structure (PDB 5DRJ) were contoured at 1.0 sigma and ± 3.0 sigma, respectively. FIG +80 89 structure evidence The 2F o‐F c electron density map and F o‐F c difference map of a 2,5‐DTP‐bound structure (PDB 5DRJ) were contoured at 1.0 sigma and ± 3.0 sigma, respectively. FIG +21 38 α‐carbon distance evidence Average (mean + SEM) α‐carbon distance measured from h3 Thr347 to h11 Leu525 of A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound ERα LBDs. FIG +53 55 h3 structure_element Average (mean + SEM) α‐carbon distance measured from h3 Thr347 to h11 Leu525 of A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound ERα LBDs. FIG +56 62 Thr347 residue_name_number Average (mean + SEM) α‐carbon distance measured from h3 Thr347 to h11 Leu525 of A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound ERα LBDs. FIG +66 69 h11 structure_element Average (mean + SEM) α‐carbon distance measured from h3 Thr347 to h11 Leu525 of A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound ERα LBDs. FIG +70 76 Leu525 residue_name_number Average (mean + SEM) α‐carbon distance measured from h3 Thr347 to h11 Leu525 of A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound ERα LBDs. FIG +80 115 A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound protein_state Average (mean + SEM) α‐carbon distance measured from h3 Thr347 to h11 Leu525 of A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound ERα LBDs. FIG +116 119 ERα protein Average (mean + SEM) α‐carbon distance measured from h3 Thr347 to h11 Leu525 of A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound ERα LBDs. FIG +120 124 LBDs structure_element Average (mean + SEM) α‐carbon distance measured from h3 Thr347 to h11 Leu525 of A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound ERα LBDs. FIG +12 28 Student's t‐test experimental_method *Two‐tailed Student's t‐test, P = 0.002 (PDB A‐CD: 5DI7, 5DID, 5DIE, 5DIG, and 4PPS; 2,5‐DTP: 4IWC, 5DRM, and 5DRJ; 3,4‐DTPD: 5DTV and 5DU5). FIG +30 31 P evidence *Two‐tailed Student's t‐test, P = 0.002 (PDB A‐CD: 5DI7, 5DID, 5DIE, 5DIG, and 4PPS; 2,5‐DTP: 4IWC, 5DRM, and 5DRJ; 3,4‐DTPD: 5DTV and 5DU5). FIG +45 49 A‐CD chemical *Two‐tailed Student's t‐test, P = 0.002 (PDB A‐CD: 5DI7, 5DID, 5DIE, 5DIG, and 4PPS; 2,5‐DTP: 4IWC, 5DRM, and 5DRJ; 3,4‐DTPD: 5DTV and 5DU5). FIG +85 92 2,5‐DTP chemical *Two‐tailed Student's t‐test, P = 0.002 (PDB A‐CD: 5DI7, 5DID, 5DIE, 5DIG, and 4PPS; 2,5‐DTP: 4IWC, 5DRM, and 5DRJ; 3,4‐DTPD: 5DTV and 5DU5). FIG +116 124 3,4‐DTPD chemical *Two‐tailed Student's t‐test, P = 0.002 (PDB A‐CD: 5DI7, 5DID, 5DIE, 5DIG, and 4PPS; 2,5‐DTP: 4IWC, 5DRM, and 5DRJ; 3,4‐DTPD: 5DTV and 5DU5). FIG +0 18 Crystal structures evidence Crystal structures show that a 3,4‐DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DTV). FIG +31 39 3,4‐DTPD chemical Crystal structures show that a 3,4‐DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DTV). FIG +54 56 h3 structure_element Crystal structures show that a 3,4‐DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DTV). FIG +58 59 F structure_element Crystal structures show that a 3,4‐DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DTV). FIG +69 74 NCOA2 protein Crystal structures show that a 3,4‐DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DTV). FIG +102 106 A‐CD chemical Crystal structures show that a 3,4‐DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DTV). FIG +112 120 estrogen chemical Crystal structures show that a 3,4‐DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DTV). FIG +0 23 Hierarchical clustering experimental_method Hierarchical clustering of ligand‐specific binding of 154 interacting peptides to the ERα LBD was performed in triplicate by MARCoNI analysis. FIG +86 89 ERα protein Hierarchical clustering of ligand‐specific binding of 154 interacting peptides to the ERα LBD was performed in triplicate by MARCoNI analysis. FIG +90 93 LBD structure_element Hierarchical clustering of ligand‐specific binding of 154 interacting peptides to the ERα LBD was performed in triplicate by MARCoNI analysis. FIG +125 141 MARCoNI analysis experimental_method Hierarchical clustering of ligand‐specific binding of 154 interacting peptides to the ERα LBD was performed in triplicate by MARCoNI analysis. FIG +4 11 2,5‐DTP chemical The 2,5‐DTP analogs showed perturbation of h11, as well as h3, which forms part of the AF‐2 surface. RESULTS +43 46 h11 structure_element The 2,5‐DTP analogs showed perturbation of h11, as well as h3, which forms part of the AF‐2 surface. RESULTS +59 61 h3 structure_element The 2,5‐DTP analogs showed perturbation of h11, as well as h3, which forms part of the AF‐2 surface. RESULTS +87 99 AF‐2 surface site The 2,5‐DTP analogs showed perturbation of h11, as well as h3, which forms part of the AF‐2 surface. RESULTS +25 28 LBD structure_element These compounds bind the LBD in an unusual fashion because they have a phenol‐to‐phenol length of ~12 Å, which is longer than steroids and other prototypical ERα agonists that are ~10 Å in length. RESULTS +158 161 ERα protein These compounds bind the LBD in an unusual fashion because they have a phenol‐to‐phenol length of ~12 Å, which is longer than steroids and other prototypical ERα agonists that are ~10 Å in length. RESULTS +33 35 h3 structure_element One phenol pushed further toward h3 (Fig 6D), while the other phenol pushed toward the C‐terminus of h11 to a greater extent than A‐CD‐ring estrogens (Nwachukwu et al, 2014), which are close structural analogs of E2 that lack a B‐ring (Fig 2). RESULTS +101 104 h11 structure_element One phenol pushed further toward h3 (Fig 6D), while the other phenol pushed toward the C‐terminus of h11 to a greater extent than A‐CD‐ring estrogens (Nwachukwu et al, 2014), which are close structural analogs of E2 that lack a B‐ring (Fig 2). RESULTS +130 134 A‐CD chemical One phenol pushed further toward h3 (Fig 6D), while the other phenol pushed toward the C‐terminus of h11 to a greater extent than A‐CD‐ring estrogens (Nwachukwu et al, 2014), which are close structural analogs of E2 that lack a B‐ring (Fig 2). RESULTS +140 149 estrogens chemical One phenol pushed further toward h3 (Fig 6D), while the other phenol pushed toward the C‐terminus of h11 to a greater extent than A‐CD‐ring estrogens (Nwachukwu et al, 2014), which are close structural analogs of E2 that lack a B‐ring (Fig 2). RESULTS +213 215 E2 chemical One phenol pushed further toward h3 (Fig 6D), while the other phenol pushed toward the C‐terminus of h11 to a greater extent than A‐CD‐ring estrogens (Nwachukwu et al, 2014), which are close structural analogs of E2 that lack a B‐ring (Fig 2). RESULTS +45 53 distance evidence To quantify this difference, we compared the distance between α‐carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5‐DTP analogs (n = 3) or A‐CD‐ring analogs (n = 5) (Fig 6E). RESULTS +75 77 h3 structure_element To quantify this difference, we compared the distance between α‐carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5‐DTP analogs (n = 3) or A‐CD‐ring analogs (n = 5) (Fig 6E). RESULTS +78 84 Thr347 residue_name_number To quantify this difference, we compared the distance between α‐carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5‐DTP analogs (n = 3) or A‐CD‐ring analogs (n = 5) (Fig 6E). RESULTS +89 92 h11 structure_element To quantify this difference, we compared the distance between α‐carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5‐DTP analogs (n = 3) or A‐CD‐ring analogs (n = 5) (Fig 6E). RESULTS +93 99 Leu525 residue_name_number To quantify this difference, we compared the distance between α‐carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5‐DTP analogs (n = 3) or A‐CD‐ring analogs (n = 5) (Fig 6E). RESULTS +114 124 structures evidence To quantify this difference, we compared the distance between α‐carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5‐DTP analogs (n = 3) or A‐CD‐ring analogs (n = 5) (Fig 6E). RESULTS +136 143 2,5‐DTP chemical To quantify this difference, we compared the distance between α‐carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5‐DTP analogs (n = 3) or A‐CD‐ring analogs (n = 5) (Fig 6E). RESULTS +163 167 A‐CD chemical To quantify this difference, we compared the distance between α‐carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5‐DTP analogs (n = 3) or A‐CD‐ring analogs (n = 5) (Fig 6E). RESULTS +67 83 Student's t‐test experimental_method We observed a difference of 0.4 Å that was significant (two‐tailed Student's t‐test, P = 0.002) due to the very tight clustering of the 2,5‐DTP‐induced LBD conformation. RESULTS +85 86 P evidence We observed a difference of 0.4 Å that was significant (two‐tailed Student's t‐test, P = 0.002) due to the very tight clustering of the 2,5‐DTP‐induced LBD conformation. RESULTS +136 143 2,5‐DTP chemical We observed a difference of 0.4 Å that was significant (two‐tailed Student's t‐test, P = 0.002) due to the very tight clustering of the 2,5‐DTP‐induced LBD conformation. RESULTS +152 155 LBD structure_element We observed a difference of 0.4 Å that was significant (two‐tailed Student's t‐test, P = 0.002) due to the very tight clustering of the 2,5‐DTP‐induced LBD conformation. RESULTS +14 16 h3 structure_element The shifts in h3 suggest these compounds are positioned to alter coregulator preferences. RESULTS +4 11 2,5‐DTP chemical The 2,5‐DTP and 3,4‐DTP scaffolds are isomeric, but with aryl groups at obtuse and acute angles, respectively (Fig 2). RESULTS +16 23 3,4‐DTP chemical The 2,5‐DTP and 3,4‐DTP scaffolds are isomeric, but with aryl groups at obtuse and acute angles, respectively (Fig 2). RESULTS +4 21 crystal structure evidence The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). RESULTS +25 28 ERα protein The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). RESULTS +29 44 in complex with protein_state The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). RESULTS +47 54 3,4‐DTP chemical The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). RESULTS +79 85 solved experimental_method The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). RESULTS +90 108 crystal structures evidence The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). RESULTS +112 115 ERα protein The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). RESULTS +116 124 bound to protein_state The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). RESULTS +125 133 3,4‐DTPD chemical The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). RESULTS +150 159 structure evidence The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). RESULTS +173 178 furan chemical The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). RESULTS +9 19 structures evidence In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). RESULTS +47 55 3,4‐DTPD chemical In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). RESULTS +71 73 h3 structure_element In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). RESULTS +74 80 Glu353 residue_name_number In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). RESULTS +130 132 h3 structure_element In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). RESULTS +143 156 hydrogen bond bond_interaction In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). RESULTS +162 168 Thr347 residue_name_number In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). RESULTS +217 220 ERα protein In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). RESULTS +221 242 ligand‐binding pocket site In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). RESULTS +4 12 3,4‐DTPD chemical The 3,4‐DTPD analogs also induced a shift in h3 positioning, which translated again into a shift in the bound coactivator peptide (Fig 6F). RESULTS +45 47 h3 structure_element The 3,4‐DTPD analogs also induced a shift in h3 positioning, which translated again into a shift in the bound coactivator peptide (Fig 6F). RESULTS +48 56 S‐OBHS‐2 chemical Therefore, these indirect modulators, including S‐OBHS‐2, S‐OBHS‐3, 2,5‐DTP, and 3,4‐DTPD analogs—all of which show cell‐specific activity profiles—induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF‐2 surface. RESULTS +58 66 S‐OBHS‐3 chemical Therefore, these indirect modulators, including S‐OBHS‐2, S‐OBHS‐3, 2,5‐DTP, and 3,4‐DTPD analogs—all of which show cell‐specific activity profiles—induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF‐2 surface. RESULTS +68 75 2,5‐DTP chemical Therefore, these indirect modulators, including S‐OBHS‐2, S‐OBHS‐3, 2,5‐DTP, and 3,4‐DTPD analogs—all of which show cell‐specific activity profiles—induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF‐2 surface. RESULTS +81 89 3,4‐DTPD chemical Therefore, these indirect modulators, including S‐OBHS‐2, S‐OBHS‐3, 2,5‐DTP, and 3,4‐DTPD analogs—all of which show cell‐specific activity profiles—induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF‐2 surface. RESULTS +166 168 h3 structure_element Therefore, these indirect modulators, including S‐OBHS‐2, S‐OBHS‐3, 2,5‐DTP, and 3,4‐DTPD analogs—all of which show cell‐specific activity profiles—induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF‐2 surface. RESULTS +173 176 h12 structure_element Therefore, these indirect modulators, including S‐OBHS‐2, S‐OBHS‐3, 2,5‐DTP, and 3,4‐DTPD analogs—all of which show cell‐specific activity profiles—induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF‐2 surface. RESULTS +241 253 AF‐2 surface site Therefore, these indirect modulators, including S‐OBHS‐2, S‐OBHS‐3, 2,5‐DTP, and 3,4‐DTPD analogs—all of which show cell‐specific activity profiles—induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF‐2 surface. RESULTS +20 32 AF‐2 surface site To test whether the AF‐2 surface shows changes in shape in solution, we used the microarray assay for real‐time coregulator–nuclear receptor interaction (MARCoNI) analysis (Aarts et al, 2013). RESULTS +81 152 microarray assay for real‐time coregulator–nuclear receptor interaction experimental_method To test whether the AF‐2 surface shows changes in shape in solution, we used the microarray assay for real‐time coregulator–nuclear receptor interaction (MARCoNI) analysis (Aarts et al, 2013). RESULTS +154 161 MARCoNI experimental_method To test whether the AF‐2 surface shows changes in shape in solution, we used the microarray assay for real‐time coregulator–nuclear receptor interaction (MARCoNI) analysis (Aarts et al, 2013). RESULTS +47 50 ERα protein Here, the ligand‐dependent interactions of the ERα LBD with over 150 distinct LxxLL motif peptides were assayed to define structural fingerprints for the AF‐2 surface, in a manner similar to the use of phage display peptides as structural probes (Connor et al, 2001). RESULTS +51 54 LBD structure_element Here, the ligand‐dependent interactions of the ERα LBD with over 150 distinct LxxLL motif peptides were assayed to define structural fingerprints for the AF‐2 surface, in a manner similar to the use of phage display peptides as structural probes (Connor et al, 2001). RESULTS +78 89 LxxLL motif structure_element Here, the ligand‐dependent interactions of the ERα LBD with over 150 distinct LxxLL motif peptides were assayed to define structural fingerprints for the AF‐2 surface, in a manner similar to the use of phage display peptides as structural probes (Connor et al, 2001). RESULTS +154 166 AF‐2 surface site Here, the ligand‐dependent interactions of the ERα LBD with over 150 distinct LxxLL motif peptides were assayed to define structural fingerprints for the AF‐2 surface, in a manner similar to the use of phage display peptides as structural probes (Connor et al, 2001). RESULTS +202 224 phage display peptides experimental_method Here, the ligand‐dependent interactions of the ERα LBD with over 150 distinct LxxLL motif peptides were assayed to define structural fingerprints for the AF‐2 surface, in a manner similar to the use of phage display peptides as structural probes (Connor et al, 2001). RESULTS +79 86 2,5‐DTP chemical Despite the similar average activities of these ligand classes (Fig 3A and B), 2,5‐DTP and 3,4‐DTP analogs displayed remarkably different peptide recruitment patterns (Fig 6H), consistent with the structural analyses. RESULTS +91 98 3,4‐DTP chemical Despite the similar average activities of these ligand classes (Fig 3A and B), 2,5‐DTP and 3,4‐DTP analogs displayed remarkably different peptide recruitment patterns (Fig 6H), consistent with the structural analyses. RESULTS +197 216 structural analyses experimental_method Despite the similar average activities of these ligand classes (Fig 3A and B), 2,5‐DTP and 3,4‐DTP analogs displayed remarkably different peptide recruitment patterns (Fig 6H), consistent with the structural analyses. RESULTS +0 23 Hierarchical clustering experimental_method Hierarchical clustering revealed that many of the 2,5‐DTP analogs recapitulated most of the peptide recruitment and dismissal patterns observed with E2 (Fig 6H). RESULTS +50 57 2,5‐DTP chemical Hierarchical clustering revealed that many of the 2,5‐DTP analogs recapitulated most of the peptide recruitment and dismissal patterns observed with E2 (Fig 6H). RESULTS +149 151 E2 chemical Hierarchical clustering revealed that many of the 2,5‐DTP analogs recapitulated most of the peptide recruitment and dismissal patterns observed with E2 (Fig 6H). RESULTS +71 73 E2 chemical However, there was a unique cluster of peptides that were recruited by E2 but not the 2,5‐DTP analogs. RESULTS +86 93 2,5‐DTP chemical However, there was a unique cluster of peptides that were recruited by E2 but not the 2,5‐DTP analogs. RESULTS +13 20 3,4‐DTP chemical In contrast, 3,4‐DTP analogs dismissed most of the peptides from the AF‐2 surface (Fig 6H). RESULTS +69 81 AF‐2 surface site In contrast, 3,4‐DTP analogs dismissed most of the peptides from the AF‐2 surface (Fig 6H). RESULTS +54 63 thiophene chemical Thus, the isomeric attachment of diaryl groups to the thiophene core changed the AF‐2 surface from inside the ligand‐binding pocket, as predicted by the crystal structures. RESULTS +81 93 AF‐2 surface site Thus, the isomeric attachment of diaryl groups to the thiophene core changed the AF‐2 surface from inside the ligand‐binding pocket, as predicted by the crystal structures. RESULTS +110 131 ligand‐binding pocket site Thus, the isomeric attachment of diaryl groups to the thiophene core changed the AF‐2 surface from inside the ligand‐binding pocket, as predicted by the crystal structures. RESULTS +153 171 crystal structures evidence Thus, the isomeric attachment of diaryl groups to the thiophene core changed the AF‐2 surface from inside the ligand‐binding pocket, as predicted by the crystal structures. RESULTS +198 210 AF‐2 surface site Together, these findings suggest that without an extended side chain, cell‐specific activity stems from different coregulator recruitment profiles, due to unique ligand‐induced conformations of the AF‐2 surface, in addition to differential usage of AF‐1. RESULTS +249 253 AF‐1 structure_element Together, these findings suggest that without an extended side chain, cell‐specific activity stems from different coregulator recruitment profiles, due to unique ligand‐induced conformations of the AF‐2 surface, in addition to differential usage of AF‐1. RESULTS +62 79 h11–h12 interface site Indirect modulators in cluster 1 avoid this by perturbing the h11–h12 interface, and modulating the dynamics of h12 without changing the shape of AF‐2 when stabilized. RESULTS +112 115 h12 structure_element Indirect modulators in cluster 1 avoid this by perturbing the h11–h12 interface, and modulating the dynamics of h12 without changing the shape of AF‐2 when stabilized. RESULTS +146 150 AF‐2 structure_element Indirect modulators in cluster 1 avoid this by perturbing the h11–h12 interface, and modulating the dynamics of h12 without changing the shape of AF‐2 when stabilized. RESULTS +106 109 ERα protein Our goal was to identify a minimal set of predictors that would link specific structural perturbations to ERα signaling pathways that control cell‐specific signaling and proliferation. DISCUSS +148 151 h12 structure_element We found a very strong set of predictors, where ligands in cluster 1, defined by similar signaling across cell types, showed indirect modulation of h12 dynamics via the h11–12 interface or slight contact with h12. DISCUSS +169 185 h11–12 interface site We found a very strong set of predictors, where ligands in cluster 1, defined by similar signaling across cell types, showed indirect modulation of h12 dynamics via the h11–12 interface or slight contact with h12. DISCUSS +209 212 h12 structure_element We found a very strong set of predictors, where ligands in cluster 1, defined by similar signaling across cell types, showed indirect modulation of h12 dynamics via the h11–12 interface or slight contact with h12. DISCUSS +73 77 AF‐2 structure_element This perturbation determined proliferation that correlated strongly with AF‐2 activity, recruitment of NCOA1/2/3 family members, and induction of the GREB1 gene, consistent with the canonical ERα signaling pathway (Fig 1D). DISCUSS +103 112 NCOA1/2/3 protein This perturbation determined proliferation that correlated strongly with AF‐2 activity, recruitment of NCOA1/2/3 family members, and induction of the GREB1 gene, consistent with the canonical ERα signaling pathway (Fig 1D). DISCUSS +150 155 GREB1 protein This perturbation determined proliferation that correlated strongly with AF‐2 activity, recruitment of NCOA1/2/3 family members, and induction of the GREB1 gene, consistent with the canonical ERα signaling pathway (Fig 1D). DISCUSS +192 195 ERα protein This perturbation determined proliferation that correlated strongly with AF‐2 activity, recruitment of NCOA1/2/3 family members, and induction of the GREB1 gene, consistent with the canonical ERα signaling pathway (Fig 1D). DISCUSS +26 34 deletion experimental_method For ligands in cluster 1, deletion of AF‐1 reduced activity to varying degrees, but did not change the underlying signaling patterns established through AF‐2. DISCUSS +38 42 AF‐1 structure_element For ligands in cluster 1, deletion of AF‐1 reduced activity to varying degrees, but did not change the underlying signaling patterns established through AF‐2. DISCUSS +153 157 AF‐2 structure_element For ligands in cluster 1, deletion of AF‐1 reduced activity to varying degrees, but did not change the underlying signaling patterns established through AF‐2. DISCUSS +68 71 h12 structure_element In contrast, an extended side chain designed to directly reposition h12 and completely disrupt the AF‐2 surface results in cell‐specific signaling. DISCUSS +99 111 AF‐2 surface site In contrast, an extended side chain designed to directly reposition h12 and completely disrupt the AF‐2 surface results in cell‐specific signaling. DISCUSS +137 140 LBD structure_element Compared to cluster 1, the structural rules are less clear in clusters 2 and 3, but a number of indirect modulator classes perturbed the LBD conformation at the intersection of h3, the h12 N‐terminus, and the AF‐2 surface. DISCUSS +177 179 h3 structure_element Compared to cluster 1, the structural rules are less clear in clusters 2 and 3, but a number of indirect modulator classes perturbed the LBD conformation at the intersection of h3, the h12 N‐terminus, and the AF‐2 surface. DISCUSS +185 188 h12 structure_element Compared to cluster 1, the structural rules are less clear in clusters 2 and 3, but a number of indirect modulator classes perturbed the LBD conformation at the intersection of h3, the h12 N‐terminus, and the AF‐2 surface. DISCUSS +209 221 AF‐2 surface site Compared to cluster 1, the structural rules are less clear in clusters 2 and 3, but a number of indirect modulator classes perturbed the LBD conformation at the intersection of h3, the h12 N‐terminus, and the AF‐2 surface. DISCUSS +46 50 AF‐2 structure_element Ligands in these classes altered the shape of AF‐2 to affect coregulator preferences. DISCUSS +68 71 ERα protein For direct and indirect modulators in cluster 2 or 3, the canonical ERα signaling pathway involving recruitment of NCOA1/2/3 and induction of GREB1 did not generally predict their proliferative effects, indicating an alternate causal model (Fig 1E). DISCUSS +115 124 NCOA1/2/3 protein For direct and indirect modulators in cluster 2 or 3, the canonical ERα signaling pathway involving recruitment of NCOA1/2/3 and induction of GREB1 did not generally predict their proliferative effects, indicating an alternate causal model (Fig 1E). DISCUSS +142 147 GREB1 protein For direct and indirect modulators in cluster 2 or 3, the canonical ERα signaling pathway involving recruitment of NCOA1/2/3 and induction of GREB1 did not generally predict their proliferative effects, indicating an alternate causal model (Fig 1E). DISCUSS +71 96 ligand–receptor interface site These principles outlined above provide a structural basis for how the ligand–receptor interface leads to different signaling specificities through AF‐1 and AF‐2. DISCUSS +148 152 AF‐1 structure_element These principles outlined above provide a structural basis for how the ligand–receptor interface leads to different signaling specificities through AF‐1 and AF‐2. DISCUSS +157 161 AF‐2 structure_element These principles outlined above provide a structural basis for how the ligand–receptor interface leads to different signaling specificities through AF‐1 and AF‐2. DISCUSS +36 39 h12 structure_element It is noteworthy that regulation of h12 dynamics indirectly through h11 can virtually abolish AF‐2 activity, and yet still drive robust transcriptional activity through AF‐1, as demonstrated with the OBHS series. DISCUSS +68 71 h11 structure_element It is noteworthy that regulation of h12 dynamics indirectly through h11 can virtually abolish AF‐2 activity, and yet still drive robust transcriptional activity through AF‐1, as demonstrated with the OBHS series. DISCUSS +94 98 AF‐2 structure_element It is noteworthy that regulation of h12 dynamics indirectly through h11 can virtually abolish AF‐2 activity, and yet still drive robust transcriptional activity through AF‐1, as demonstrated with the OBHS series. DISCUSS +169 173 AF‐1 structure_element It is noteworthy that regulation of h12 dynamics indirectly through h11 can virtually abolish AF‐2 activity, and yet still drive robust transcriptional activity through AF‐1, as demonstrated with the OBHS series. DISCUSS +200 204 OBHS chemical It is noteworthy that regulation of h12 dynamics indirectly through h11 can virtually abolish AF‐2 activity, and yet still drive robust transcriptional activity through AF‐1, as demonstrated with the OBHS series. DISCUSS +47 56 NCOA1/2/3 protein This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF‐1 and AF‐2 (McInerney et al, 1996; Webb et al, 1998), which allows ligands to nucleate ERα–NCOA1/2/3 interaction through AF‐2, and reinforce this interaction with additional binding to AF‐1. DISCUSS +74 87 binding sites site This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF‐1 and AF‐2 (McInerney et al, 1996; Webb et al, 1998), which allows ligands to nucleate ERα–NCOA1/2/3 interaction through AF‐2, and reinforce this interaction with additional binding to AF‐1. DISCUSS +109 113 AF‐1 structure_element This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF‐1 and AF‐2 (McInerney et al, 1996; Webb et al, 1998), which allows ligands to nucleate ERα–NCOA1/2/3 interaction through AF‐2, and reinforce this interaction with additional binding to AF‐1. DISCUSS +118 122 AF‐2 structure_element This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF‐1 and AF‐2 (McInerney et al, 1996; Webb et al, 1998), which allows ligands to nucleate ERα–NCOA1/2/3 interaction through AF‐2, and reinforce this interaction with additional binding to AF‐1. DISCUSS +199 212 ERα–NCOA1/2/3 complex_assembly This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF‐1 and AF‐2 (McInerney et al, 1996; Webb et al, 1998), which allows ligands to nucleate ERα–NCOA1/2/3 interaction through AF‐2, and reinforce this interaction with additional binding to AF‐1. DISCUSS +233 237 AF‐2 structure_element This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF‐1 and AF‐2 (McInerney et al, 1996; Webb et al, 1998), which allows ligands to nucleate ERα–NCOA1/2/3 interaction through AF‐2, and reinforce this interaction with additional binding to AF‐1. DISCUSS +297 301 AF‐1 structure_element This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF‐1 and AF‐2 (McInerney et al, 1996; Webb et al, 1998), which allows ligands to nucleate ERα–NCOA1/2/3 interaction through AF‐2, and reinforce this interaction with additional binding to AF‐1. DISCUSS +20 24 AF‐2 structure_element Completely blocking AF‐2 with an extended side chain or altering the shape of AF‐2 changes the preference away from NCOA1/2/3 for determining GREB1 levels and proliferation of breast cancer cells. DISCUSS +78 82 AF‐2 structure_element Completely blocking AF‐2 with an extended side chain or altering the shape of AF‐2 changes the preference away from NCOA1/2/3 for determining GREB1 levels and proliferation of breast cancer cells. DISCUSS +116 125 NCOA1/2/3 protein Completely blocking AF‐2 with an extended side chain or altering the shape of AF‐2 changes the preference away from NCOA1/2/3 for determining GREB1 levels and proliferation of breast cancer cells. DISCUSS +142 147 GREB1 protein Completely blocking AF‐2 with an extended side chain or altering the shape of AF‐2 changes the preference away from NCOA1/2/3 for determining GREB1 levels and proliferation of breast cancer cells. DISCUSS +0 4 AF‐2 structure_element AF‐2 blockade also allows AF‐1 to function independently, which is important since AF‐1 drives tissue‐selective effects in vivo. DISCUSS +26 30 AF‐1 structure_element AF‐2 blockade also allows AF‐1 to function independently, which is important since AF‐1 drives tissue‐selective effects in vivo. DISCUSS +83 87 AF‐1 structure_element AF‐2 blockade also allows AF‐1 to function independently, which is important since AF‐1 drives tissue‐selective effects in vivo. DISCUSS +27 31 AF‐1 structure_element This was demonstrated with AF‐1 knockout mice that show E2‐dependent vascular protection, but not uterine proliferation, thus highlighting the role of AF‐1 in tissue‐selective or cell‐specific signaling (Billon‐Gales et al, 2009; Abot et al, 2013). DISCUSS +56 58 E2 chemical This was demonstrated with AF‐1 knockout mice that show E2‐dependent vascular protection, but not uterine proliferation, thus highlighting the role of AF‐1 in tissue‐selective or cell‐specific signaling (Billon‐Gales et al, 2009; Abot et al, 2013). DISCUSS +151 155 AF‐1 structure_element This was demonstrated with AF‐1 knockout mice that show E2‐dependent vascular protection, but not uterine proliferation, thus highlighting the role of AF‐1 in tissue‐selective or cell‐specific signaling (Billon‐Gales et al, 2009; Abot et al, 2013). DISCUSS +23 26 LBD structure_element Here, we examined many LBD structures and tested several variables that were not predictive, including ERβ activity, the strength of AF‐1 signaling, and NCOA3 occupancy at the GREB1 gene. DISCUSS +27 37 structures evidence Here, we examined many LBD structures and tested several variables that were not predictive, including ERβ activity, the strength of AF‐1 signaling, and NCOA3 occupancy at the GREB1 gene. DISCUSS +103 106 ERβ protein Here, we examined many LBD structures and tested several variables that were not predictive, including ERβ activity, the strength of AF‐1 signaling, and NCOA3 occupancy at the GREB1 gene. DISCUSS +133 137 AF‐1 structure_element Here, we examined many LBD structures and tested several variables that were not predictive, including ERβ activity, the strength of AF‐1 signaling, and NCOA3 occupancy at the GREB1 gene. DISCUSS +153 158 NCOA3 protein Here, we examined many LBD structures and tested several variables that were not predictive, including ERβ activity, the strength of AF‐1 signaling, and NCOA3 occupancy at the GREB1 gene. DISCUSS +176 181 GREB1 protein Here, we examined many LBD structures and tested several variables that were not predictive, including ERβ activity, the strength of AF‐1 signaling, and NCOA3 occupancy at the GREB1 gene. DISCUSS +25 35 structures evidence Similarly, we visualized structures to identify patterns. DISCUSS +13 26 phage display experimental_method For example, phage display was used to identify the androgen receptor interactome, which was cloned into an M2H library and used to identify clusters of ligand‐selective interactions (Norris et al, 2009). DISCUSS +108 111 M2H experimental_method For example, phage display was used to identify the androgen receptor interactome, which was cloned into an M2H library and used to identify clusters of ligand‐selective interactions (Norris et al, 2009). DISCUSS +19 34 siRNA screening experimental_method Also, we have used siRNA screening to identify a number of coregulators required for ERα‐mediated repression of the IL‐6 gene (Nwachukwu et al, 2014). DISCUSS +85 88 ERα protein Also, we have used siRNA screening to identify a number of coregulators required for ERα‐mediated repression of the IL‐6 gene (Nwachukwu et al, 2014). DISCUSS +17 47 inter‐atomic distance matrices evidence If we calculated inter‐atomic distance matrices containing 4,000 atoms per structure × 76 ligand–receptor complexes, we would have 3 × 105 predictions. DISCUSS +19 33 atomic vectors evidence We have identified atomic vectors for the OBHS‐N and triaryl‐ethylene classes that predict ligand response (Fig 5E and F). DISCUSS +42 48 OBHS‐N chemical We have identified atomic vectors for the OBHS‐N and triaryl‐ethylene classes that predict ligand response (Fig 5E and F). DISCUSS +53 69 triaryl‐ethylene chemical We have identified atomic vectors for the OBHS‐N and triaryl‐ethylene classes that predict ligand response (Fig 5E and F). DISCUSS +52 58 OBHS‐N chemical Indeed, the most anti‐proliferative compound in the OBHS‐N series had a fulvestrant‐like profile across a battery of assays (S. Srinivasan et al, in preparation). DISCUSS +27 32 WAY‐C chemical Secondly, our finding that WAY‐C compounds do not rely of AF‐1 for signaling efficacy may derive from the slight contacts with h12 observed in crystal structures (Figs 3B and 5H), unlike other compounds in cluster 1 that dislocate h11 and rely on AF‐1 for signaling efficacy (Figs 3B and 5C, and EV5B). DISCUSS +58 62 AF‐1 structure_element Secondly, our finding that WAY‐C compounds do not rely of AF‐1 for signaling efficacy may derive from the slight contacts with h12 observed in crystal structures (Figs 3B and 5H), unlike other compounds in cluster 1 that dislocate h11 and rely on AF‐1 for signaling efficacy (Figs 3B and 5C, and EV5B). DISCUSS +127 130 h12 structure_element Secondly, our finding that WAY‐C compounds do not rely of AF‐1 for signaling efficacy may derive from the slight contacts with h12 observed in crystal structures (Figs 3B and 5H), unlike other compounds in cluster 1 that dislocate h11 and rely on AF‐1 for signaling efficacy (Figs 3B and 5C, and EV5B). DISCUSS +143 161 crystal structures evidence Secondly, our finding that WAY‐C compounds do not rely of AF‐1 for signaling efficacy may derive from the slight contacts with h12 observed in crystal structures (Figs 3B and 5H), unlike other compounds in cluster 1 that dislocate h11 and rely on AF‐1 for signaling efficacy (Figs 3B and 5C, and EV5B). DISCUSS +231 234 h11 structure_element Secondly, our finding that WAY‐C compounds do not rely of AF‐1 for signaling efficacy may derive from the slight contacts with h12 observed in crystal structures (Figs 3B and 5H), unlike other compounds in cluster 1 that dislocate h11 and rely on AF‐1 for signaling efficacy (Figs 3B and 5C, and EV5B). DISCUSS +247 251 AF‐1 structure_element Secondly, our finding that WAY‐C compounds do not rely of AF‐1 for signaling efficacy may derive from the slight contacts with h12 observed in crystal structures (Figs 3B and 5H), unlike other compounds in cluster 1 that dislocate h11 and rely on AF‐1 for signaling efficacy (Figs 3B and 5C, and EV5B). DISCUSS +47 59 AF‐2 surface site Some of these ligands altered the shape of the AF‐2 surface by perturbing the h3–h12 interface, thus providing a route to new SERM‐like activity profiles by combining indirect and direct modulation of receptor structure. DISCUSS +78 94 h3–h12 interface site Some of these ligands altered the shape of the AF‐2 surface by perturbing the h3–h12 interface, thus providing a route to new SERM‐like activity profiles by combining indirect and direct modulation of receptor structure. DISCUSS +156 159 ERα protein Incorporation of statistical approaches to understand relationships between structure and signaling variables moves us toward predictive models for complex ERα‐mediated responses such as in vivo uterine proliferation or tumor growth, and more generally toward structure‐based design for other allosteric drug targets including GPCRs and other nuclear receptors. DISCUSS +327 332 GPCRs protein_type Incorporation of statistical approaches to understand relationships between structure and signaling variables moves us toward predictive models for complex ERα‐mediated responses such as in vivo uterine proliferation or tumor growth, and more generally toward structure‐based design for other allosteric drug targets including GPCRs and other nuclear receptors. DISCUSS +343 360 nuclear receptors protein_type Incorporation of statistical approaches to understand relationships between structure and signaling variables moves us toward predictive models for complex ERα‐mediated responses such as in vivo uterine proliferation or tumor growth, and more generally toward structure‐based design for other allosteric drug targets including GPCRs and other nuclear receptors. DISCUSS diff --git a/annotation_CSV/PMC4850273.csv b/annotation_CSV/PMC4850273.csv new file mode 100644 index 0000000000000000000000000000000000000000..3cdf7c40be44a719fcf42fb97384d2b058b7e3de --- /dev/null +++ b/annotation_CSV/PMC4850273.csv @@ -0,0 +1,1130 @@ +anno_start anno_end anno_text entity_type sentence section +24 34 Xyloglucan chemical Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont TITLE +62 67 Human species Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont TITLE +0 31 Polysaccharide utilization loci gene Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. ABSTRACT +33 36 PUL gene Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. ABSTRACT +69 74 human species Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. ABSTRACT +79 92 Bacteroidetes taxonomy_domain Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. ABSTRACT +161 174 carbohydrates chemical Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. ABSTRACT +241 244 PUL gene Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. ABSTRACT +339 344 human species Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. ABSTRACT +345 354 bacterial taxonomy_domain Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. ABSTRACT +38 60 complex polysaccharide chemical In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. ABSTRACT +130 142 glycosidases protein_type In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. ABSTRACT +21 82 biochemical, structural, and reverse genetic characterization experimental_method Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. ABSTRACT +97 133 cell surface glycan-binding proteins protein_type Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. ABSTRACT +135 140 SGBPs protein_type Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. ABSTRACT +155 183 xyloglucan utilization locus gene Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. ABSTRACT +185 190 XyGUL gene Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. ABSTRACT +197 215 Bacteroides ovatus species Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. ABSTRACT +266 275 vegetable taxonomy_domain Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. ABSTRACT +276 290 polysaccharide chemical Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. ABSTRACT +0 20 Biochemical analysis experimental_method Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. ABSTRACT +40 72 outer membrane-anchored proteins protein_type Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. ABSTRACT +130 140 xyloglucan chemical Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. ABSTRACT +142 145 XyG chemical Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. ABSTRACT +147 161 polysaccharide chemical Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. ABSTRACT +4 21 crystal structure evidence The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. ABSTRACT +25 31 SGBP-A protein The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. ABSTRACT +35 39 SusD protein The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. ABSTRACT +56 61 bound protein_state The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. ABSTRACT +62 65 XyG chemical The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. ABSTRACT +66 85 tetradecasaccharide chemical The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. ABSTRACT +106 135 carbohydrate-binding platform site The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. ABSTRACT +180 188 β-glucan chemical The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. ABSTRACT +12 25 tetra-modular structure_element The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. ABSTRACT +26 35 structure evidence The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. ABSTRACT +39 45 SGBP-B protein The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. ABSTRACT +62 82 tandem Ig-like folds structure_element The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. ABSTRACT +89 92 XyG chemical The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. ABSTRACT +124 141 C-terminal domain structure_element The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. ABSTRACT +27 37 affinities evidence Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +42 45 XyG chemical Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +47 71 reverse-genetic analysis experimental_method Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +85 91 SGBP-B protein Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +146 162 oligosaccharides chemical Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +188 194 SGBP-A protein Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +221 233 carbohydrate chemical Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +264 267 XyG chemical Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +307 313 SGBP-A protein Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +318 324 SGBP-B protein Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +366 378 carbohydrate chemical Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +390 399 B. ovatus species Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +429 438 vegetable taxonomy_domain Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +439 450 xyloglucans chemical Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +471 484 Bacteroidetes taxonomy_domain Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. ABSTRACT +4 17 Bacteroidetes taxonomy_domain The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. ABSTRACT +31 39 bacteria taxonomy_domain The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. ABSTRACT +47 52 human species The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. ABSTRACT +103 126 complex polysaccharides chemical The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. ABSTRACT +267 280 Bacteroidetes taxonomy_domain The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. ABSTRACT +299 304 plant taxonomy_domain The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. ABSTRACT +315 330 polysaccharides chemical The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. ABSTRACT +27 77 biochemical, crystallographic, and genetic insight experimental_method Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. ABSTRACT +91 122 surface glycan-binding proteins protein_type Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. ABSTRACT +140 158 Bacteroides ovatus species Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. ABSTRACT +159 187 xyloglucan utilization locus gene Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. ABSTRACT +189 194 XyGUL gene Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. ABSTRACT +245 254 vegetable taxonomy_domain Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. ABSTRACT +255 269 polysaccharide chemical Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. ABSTRACT +61 83 complex polysaccharide chemical Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria. ABSTRACT +125 138 Bacteroidetes taxonomy_domain Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria. ABSTRACT +231 239 bacteria taxonomy_domain Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria. ABSTRACT +4 9 human species The human gut microbiota influences the course of human development and health, playing key roles in immune stimulation, intestinal cell proliferation, and metabolic balance. INTRO +14 24 microbiota taxonomy_domain The human gut microbiota influences the course of human development and health, playing key roles in immune stimulation, intestinal cell proliferation, and metabolic balance. INTRO +50 55 human species The human gut microbiota influences the course of human development and health, playing key roles in immune stimulation, intestinal cell proliferation, and metabolic balance. INTRO +5 14 microbial taxonomy_domain This microbial community is largely bacterial, with the Bacteroidetes, Firmicutes, and Actinobacteria comprising the dominant phyla. INTRO +36 45 bacterial taxonomy_domain This microbial community is largely bacterial, with the Bacteroidetes, Firmicutes, and Actinobacteria comprising the dominant phyla. INTRO +56 69 Bacteroidetes taxonomy_domain This microbial community is largely bacterial, with the Bacteroidetes, Firmicutes, and Actinobacteria comprising the dominant phyla. INTRO +71 81 Firmicutes taxonomy_domain This microbial community is largely bacterial, with the Bacteroidetes, Firmicutes, and Actinobacteria comprising the dominant phyla. INTRO +87 101 Actinobacteria taxonomy_domain This microbial community is largely bacterial, with the Bacteroidetes, Firmicutes, and Actinobacteria comprising the dominant phyla. INTRO +35 48 carbohydrates chemical The ability to acquire energy from carbohydrates of dietary or host origin is central to the adaptation of human gut bacterial species to their niche. INTRO +107 112 human species The ability to acquire energy from carbohydrates of dietary or host origin is central to the adaptation of human gut bacterial species to their niche. INTRO +117 126 bacterial taxonomy_domain The ability to acquire energy from carbohydrates of dietary or host origin is central to the adaptation of human gut bacterial species to their niche. INTRO +106 116 microbiota taxonomy_domain More importantly, this makes diet a tractable way to manipulate the abundance and metabolic output of the microbiota toward improved human health. INTRO +133 138 human species More importantly, this makes diet a tractable way to manipulate the abundance and metabolic output of the microbiota toward improved human health. INTRO +68 88 complex carbohydrate chemical However, there is a paucity of data regarding how the vast array of complex carbohydrate structures are selectively recognized and imported by members of the microbiota, a critical process that enables these organisms to thrive in the competitive gut environment. INTRO +158 168 microbiota taxonomy_domain However, there is a paucity of data regarding how the vast array of complex carbohydrate structures are selectively recognized and imported by members of the microbiota, a critical process that enables these organisms to thrive in the competitive gut environment. INTRO +4 9 human species The human gut bacteria Bacteroidetes share a profound capacity for dietary glycan degradation, with many species containing >250 predicted carbohydrate-active enzymes (CAZymes), compared to 50 to 100 within many Firmicutes and only 17 in the human genome devoted toward carbohydrate utilization. INTRO +14 22 bacteria taxonomy_domain The human gut bacteria Bacteroidetes share a profound capacity for dietary glycan degradation, with many species containing >250 predicted carbohydrate-active enzymes (CAZymes), compared to 50 to 100 within many Firmicutes and only 17 in the human genome devoted toward carbohydrate utilization. INTRO +23 36 Bacteroidetes taxonomy_domain The human gut bacteria Bacteroidetes share a profound capacity for dietary glycan degradation, with many species containing >250 predicted carbohydrate-active enzymes (CAZymes), compared to 50 to 100 within many Firmicutes and only 17 in the human genome devoted toward carbohydrate utilization. INTRO +75 81 glycan chemical The human gut bacteria Bacteroidetes share a profound capacity for dietary glycan degradation, with many species containing >250 predicted carbohydrate-active enzymes (CAZymes), compared to 50 to 100 within many Firmicutes and only 17 in the human genome devoted toward carbohydrate utilization. INTRO +212 222 Firmicutes taxonomy_domain The human gut bacteria Bacteroidetes share a profound capacity for dietary glycan degradation, with many species containing >250 predicted carbohydrate-active enzymes (CAZymes), compared to 50 to 100 within many Firmicutes and only 17 in the human genome devoted toward carbohydrate utilization. INTRO +242 247 human species The human gut bacteria Bacteroidetes share a profound capacity for dietary glycan degradation, with many species containing >250 predicted carbohydrate-active enzymes (CAZymes), compared to 50 to 100 within many Firmicutes and only 17 in the human genome devoted toward carbohydrate utilization. INTRO +28 41 Bacteroidetes taxonomy_domain A remarkable feature of the Bacteroidetes is the packaging of genes for carbohydrate catabolism into discrete polysaccharide utilization loci (PUL), which are transcriptionally regulated by specific substrate signatures. INTRO +110 141 polysaccharide utilization loci gene A remarkable feature of the Bacteroidetes is the packaging of genes for carbohydrate catabolism into discrete polysaccharide utilization loci (PUL), which are transcriptionally regulated by specific substrate signatures. INTRO +143 146 PUL gene A remarkable feature of the Bacteroidetes is the packaging of genes for carbohydrate catabolism into discrete polysaccharide utilization loci (PUL), which are transcriptionally regulated by specific substrate signatures. INTRO +15 18 PUL gene The archetypal PUL-encoded system is the starch utilization system (Sus) (Fig. 1B) of Bacteroides thetaiotaomicron. INTRO +41 66 starch utilization system complex_assembly The archetypal PUL-encoded system is the starch utilization system (Sus) (Fig. 1B) of Bacteroides thetaiotaomicron. INTRO +68 71 Sus complex_assembly The archetypal PUL-encoded system is the starch utilization system (Sus) (Fig. 1B) of Bacteroides thetaiotaomicron. INTRO +86 114 Bacteroides thetaiotaomicron species The archetypal PUL-encoded system is the starch utilization system (Sus) (Fig. 1B) of Bacteroides thetaiotaomicron. INTRO +4 7 Sus complex_assembly The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +19 33 lipid-anchored protein_state The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +50 62 endo-amylase protein_type The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +64 68 SusG protein The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +72 98 TonB-dependent transporter protein_type The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +100 104 TBDT protein_type The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +107 111 SusC protein The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +127 143 oligosaccharides chemical The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +175 197 starch-binding protein protein_type The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +199 203 SusD protein The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +220 253 carbohydrate-binding lipoproteins protein_type The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +255 259 SusE protein The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +264 268 SusF protein The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +290 306 exo-glucosidases protein_type The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +308 312 SusA protein The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +317 321 SusB protein The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +338 345 glucose chemical The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm. INTRO +18 21 PUL gene The importance of PUL as a successful evolutionary strategy is underscored by the observation that Bacteroidetes such as B. thetaiotaomicron and Bacteroides ovatus devote ~18% of their genomes to these systems. INTRO +99 112 Bacteroidetes taxonomy_domain The importance of PUL as a successful evolutionary strategy is underscored by the observation that Bacteroidetes such as B. thetaiotaomicron and Bacteroides ovatus devote ~18% of their genomes to these systems. INTRO +121 140 B. thetaiotaomicron species The importance of PUL as a successful evolutionary strategy is underscored by the observation that Bacteroidetes such as B. thetaiotaomicron and Bacteroides ovatus devote ~18% of their genomes to these systems. INTRO +145 163 Bacteroides ovatus species The importance of PUL as a successful evolutionary strategy is underscored by the observation that Bacteroidetes such as B. thetaiotaomicron and Bacteroides ovatus devote ~18% of their genomes to these systems. INTRO +88 91 PUL gene Moving beyond seminal genomic and transcriptomic analyses, the current state-of-the-art PUL characterization involves combined reverse-genetic, biochemical, and structural studies to illuminate the molecular details of PUL function. INTRO +127 179 reverse-genetic, biochemical, and structural studies experimental_method Moving beyond seminal genomic and transcriptomic analyses, the current state-of-the-art PUL characterization involves combined reverse-genetic, biochemical, and structural studies to illuminate the molecular details of PUL function. INTRO +219 222 PUL gene Moving beyond seminal genomic and transcriptomic analyses, the current state-of-the-art PUL characterization involves combined reverse-genetic, biochemical, and structural studies to illuminate the molecular details of PUL function. INTRO +0 10 Xyloglucan chemical Xyloglucan and the Bacteroides ovatus xyloglucan utilization locus (XyGUL). (A) Representative structures of common xyloglucans using the Consortium for Functional Glycomics Symbol Nomenclature (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml). FIG +19 37 Bacteroides ovatus species Xyloglucan and the Bacteroides ovatus xyloglucan utilization locus (XyGUL). (A) Representative structures of common xyloglucans using the Consortium for Functional Glycomics Symbol Nomenclature (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml). FIG +38 66 xyloglucan utilization locus gene Xyloglucan and the Bacteroides ovatus xyloglucan utilization locus (XyGUL). (A) Representative structures of common xyloglucans using the Consortium for Functional Glycomics Symbol Nomenclature (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml). FIG +68 73 XyGUL gene Xyloglucan and the Bacteroides ovatus xyloglucan utilization locus (XyGUL). (A) Representative structures of common xyloglucans using the Consortium for Functional Glycomics Symbol Nomenclature (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml). FIG +95 105 structures evidence Xyloglucan and the Bacteroides ovatus xyloglucan utilization locus (XyGUL). (A) Representative structures of common xyloglucans using the Consortium for Functional Glycomics Symbol Nomenclature (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml). FIG +116 127 xyloglucans chemical Xyloglucan and the Bacteroides ovatus xyloglucan utilization locus (XyGUL). (A) Representative structures of common xyloglucans using the Consortium for Functional Glycomics Symbol Nomenclature (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml). FIG +19 26 BoXyGUL gene Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. FIG +27 39 glycosidases protein_type Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. FIG +41 44 GHs protein_type Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. FIG +64 75 solanaceous taxonomy_domain Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. FIG +76 86 xyloglucan chemical Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. FIG +92 97 BtSus gene Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. FIG +102 109 BoXyGUL gene Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. FIG +131 138 BoXyGUL gene Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. FIG +230 241 xyloglucans chemical Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides. FIG +16 22 SGBP-A protein The location of SGBP-A/B is presented in this work; the location of GH5 has been empirically determined, and the enzymes have been placed based upon their predicted cellular location. FIG +23 24 B protein The location of SGBP-A/B is presented in this work; the location of GH5 has been empirically determined, and the enzymes have been placed based upon their predicted cellular location. FIG +68 71 GH5 protein The location of SGBP-A/B is presented in this work; the location of GH5 has been empirically determined, and the enzymes have been placed based upon their predicted cellular location. FIG +66 69 PUL gene We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B. ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG). INTRO +102 107 human species We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B. ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG). INTRO +122 141 B. ovatus ATCC 8483 species We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B. ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG). INTRO +175 180 plant taxonomy_domain We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B. ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG). INTRO +191 198 glycans chemical We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B. ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG). INTRO +204 215 xyloglucans chemical We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B. ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG). INTRO +217 220 XyG chemical We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B. ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG). INTRO +0 3 XyG chemical XyG variants (Fig. 1A) constitute up to 25% of the dry weight of common vegetables. INTRO +72 82 vegetables taxonomy_domain XyG variants (Fig. 1A) constitute up to 25% of the dry weight of common vegetables. INTRO +17 26 Sus locus gene Analogous to the Sus locus, the xyloglucan utilization locus (XyGUL) encodes a cohort of carbohydrate-binding, -hydrolyzing, and -importing proteins (Fig. 1B and C). INTRO +32 60 xyloglucan utilization locus gene Analogous to the Sus locus, the xyloglucan utilization locus (XyGUL) encodes a cohort of carbohydrate-binding, -hydrolyzing, and -importing proteins (Fig. 1B and C). INTRO +62 67 XyGUL gene Analogous to the Sus locus, the xyloglucan utilization locus (XyGUL) encodes a cohort of carbohydrate-binding, -hydrolyzing, and -importing proteins (Fig. 1B and C). INTRO +89 148 carbohydrate-binding, -hydrolyzing, and -importing proteins protein_type Analogous to the Sus locus, the xyloglucan utilization locus (XyGUL) encodes a cohort of carbohydrate-binding, -hydrolyzing, and -importing proteins (Fig. 1B and C). INTRO +14 34 glycoside hydrolases protein_type The number of glycoside hydrolases (GHs) encoded by the XyGUL is, however, more expansive than that by the Sus locus (Fig. 1B), which reflects the greater complexity of glycosidic linkages found in XyG vis-à-vis starch. INTRO +36 39 GHs protein_type The number of glycoside hydrolases (GHs) encoded by the XyGUL is, however, more expansive than that by the Sus locus (Fig. 1B), which reflects the greater complexity of glycosidic linkages found in XyG vis-à-vis starch. INTRO +56 61 XyGUL gene The number of glycoside hydrolases (GHs) encoded by the XyGUL is, however, more expansive than that by the Sus locus (Fig. 1B), which reflects the greater complexity of glycosidic linkages found in XyG vis-à-vis starch. INTRO +107 116 Sus locus gene The number of glycoside hydrolases (GHs) encoded by the XyGUL is, however, more expansive than that by the Sus locus (Fig. 1B), which reflects the greater complexity of glycosidic linkages found in XyG vis-à-vis starch. INTRO +198 201 XyG chemical The number of glycoside hydrolases (GHs) encoded by the XyGUL is, however, more expansive than that by the Sus locus (Fig. 1B), which reflects the greater complexity of glycosidic linkages found in XyG vis-à-vis starch. INTRO +212 218 starch chemical The number of glycoside hydrolases (GHs) encoded by the XyGUL is, however, more expansive than that by the Sus locus (Fig. 1B), which reflects the greater complexity of glycosidic linkages found in XyG vis-à-vis starch. INTRO +95 98 GHs protein_type Whereas our previous study focused on the characterization of the linkage specificity of these GHs, a key outstanding question regarding this locus is how XyG recognition is mediated at the cell surface. INTRO +155 158 XyG chemical Whereas our previous study focused on the characterization of the linkage specificity of these GHs, a key outstanding question regarding this locus is how XyG recognition is mediated at the cell surface. INTRO +18 43 starch utilization system complex_assembly In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +47 66 B. thetaiotaomicron species In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +133 153 starch-binding sites site In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +177 208 surface glycan-binding proteins protein_type In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +210 215 SGBPs protein_type In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +233 240 amylase protein_type In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +241 245 SusG protein In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +258 262 SusD protein In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +275 279 SusE protein In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +298 302 SusF protein In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +370 374 SusD protein In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +402 408 starch chemical In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +436 440 SusE protein In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +442 446 SusF protein In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +452 456 SusG protein In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +457 470 binding sites site In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +508 522 polysaccharide chemical In the archetypal starch utilization system of B. thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide. INTRO +0 13 Bacteroidetes taxonomy_domain Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as “putative lipoproteins”. INTRO +14 17 PUL gene Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as “putative lipoproteins”. INTRO +50 54 SusC protein Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as “putative lipoproteins”. INTRO +59 63 SusD protein Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as “putative lipoproteins”. INTRO +127 131 susD gene Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as “putative lipoproteins”. INTRO +141 147 susE/F gene Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as “putative lipoproteins”. INTRO +187 195 putative protein_state Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as “putative lipoproteins”. INTRO +196 208 lipoproteins protein_type Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as “putative lipoproteins”. INTRO +63 69 susE/F gene The genes coding for these proteins, sometimes referred to as “susE/F positioned,” display products with a wide variation in amino acid sequence and which have little or no homology to other PUL-encoded proteins or known carbohydrate-binding proteins. INTRO +191 194 PUL gene The genes coding for these proteins, sometimes referred to as “susE/F positioned,” display products with a wide variation in amino acid sequence and which have little or no homology to other PUL-encoded proteins or known carbohydrate-binding proteins. INTRO +221 250 carbohydrate-binding proteins protein_type The genes coding for these proteins, sometimes referred to as “susE/F positioned,” display products with a wide variation in amino acid sequence and which have little or no homology to other PUL-encoded proteins or known carbohydrate-binding proteins. INTRO +7 10 Sus complex_assembly As the Sus SGBPs remain the only structurally characterized cohort to date, we therefore wondered whether such glycan binding and function are extended to other PUL that target more complex and heterogeneous polysaccharides, such as XyG. INTRO +11 16 SGBPs protein_type As the Sus SGBPs remain the only structurally characterized cohort to date, we therefore wondered whether such glycan binding and function are extended to other PUL that target more complex and heterogeneous polysaccharides, such as XyG. INTRO +111 117 glycan chemical As the Sus SGBPs remain the only structurally characterized cohort to date, we therefore wondered whether such glycan binding and function are extended to other PUL that target more complex and heterogeneous polysaccharides, such as XyG. INTRO +161 164 PUL gene As the Sus SGBPs remain the only structurally characterized cohort to date, we therefore wondered whether such glycan binding and function are extended to other PUL that target more complex and heterogeneous polysaccharides, such as XyG. INTRO +208 223 polysaccharides chemical As the Sus SGBPs remain the only structurally characterized cohort to date, we therefore wondered whether such glycan binding and function are extended to other PUL that target more complex and heterogeneous polysaccharides, such as XyG. INTRO +233 236 XyG chemical As the Sus SGBPs remain the only structurally characterized cohort to date, we therefore wondered whether such glycan binding and function are extended to other PUL that target more complex and heterogeneous polysaccharides, such as XyG. INTRO +30 72 functional and structural characterization experimental_method We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo. INTRO +80 92 noncatalytic protein_state We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo. INTRO +93 98 SGBPs protein_type We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo. INTRO +110 122 Bacova_02651 gene We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo. INTRO +127 139 Bacova_02650 gene We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo. INTRO +147 152 XyGUL gene We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo. INTRO +174 180 SGBP-A protein We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo. INTRO +185 191 SGBP-B protein We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo. INTRO +9 64 biochemical, structural, and reverse-genetic approaches experimental_method Combined biochemical, structural, and reverse-genetic approaches clearly illuminate the distinct, yet complementary, functions that these two proteins play in XyG recognition as it impacts the physiology of B. ovatus. INTRO +159 162 XyG chemical Combined biochemical, structural, and reverse-genetic approaches clearly illuminate the distinct, yet complementary, functions that these two proteins play in XyG recognition as it impacts the physiology of B. ovatus. INTRO +207 216 B. ovatus species Combined biochemical, structural, and reverse-genetic approaches clearly illuminate the distinct, yet complementary, functions that these two proteins play in XyG recognition as it impacts the physiology of B. ovatus. INTRO +60 66 glycan chemical These data extend our current understanding of the Sus-like glycan uptake paradigm within the Bacteroidetes and reveals how the complex dietary polysaccharide xyloglucan is recognized at the cell surface. INTRO +94 107 Bacteroidetes taxonomy_domain These data extend our current understanding of the Sus-like glycan uptake paradigm within the Bacteroidetes and reveals how the complex dietary polysaccharide xyloglucan is recognized at the cell surface. INTRO +144 158 polysaccharide chemical These data extend our current understanding of the Sus-like glycan uptake paradigm within the Bacteroidetes and reveals how the complex dietary polysaccharide xyloglucan is recognized at the cell surface. INTRO +159 169 xyloglucan chemical These data extend our current understanding of the Sus-like glycan uptake paradigm within the Bacteroidetes and reveals how the complex dietary polysaccharide xyloglucan is recognized at the cell surface. INTRO +0 6 SGBP-A protein SGBP-A and SGBP-B are cell-surface-localized, xyloglucan-specific binding proteins. RESULTS +11 17 SGBP-B protein SGBP-A and SGBP-B are cell-surface-localized, xyloglucan-specific binding proteins. RESULTS +22 82 cell-surface-localized, xyloglucan-specific binding proteins protein_type SGBP-A and SGBP-B are cell-surface-localized, xyloglucan-specific binding proteins. RESULTS +0 6 SGBP-A protein SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig. 1B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B. thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work. RESULTS +23 28 XyGUL gene SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig. 1B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B. thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work. RESULTS +39 51 Bacova_02651 gene SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig. 1B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B. thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work. RESULTS +138 157 B. thetaiotaomicron species SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig. 1B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B. thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work. RESULTS +158 162 SusD protein SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig. 1B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B. thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work. RESULTS +194 212 SusD-like proteins protein_type SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig. 1B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B. thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work. RESULTS +237 242 XyGUL gene SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig. 1B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B. thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work. RESULTS +13 19 SGBP-B protein In contrast, SGBP-B, encoded by locus tag Bacova_02650, displays little sequence similarity to the products of similarly positioned genes in syntenic XyGUL nor to any other gene product among the diversity of Bacteroidetes PUL. RESULTS +42 54 Bacova_02650 gene In contrast, SGBP-B, encoded by locus tag Bacova_02650, displays little sequence similarity to the products of similarly positioned genes in syntenic XyGUL nor to any other gene product among the diversity of Bacteroidetes PUL. RESULTS +150 155 XyGUL gene In contrast, SGBP-B, encoded by locus tag Bacova_02650, displays little sequence similarity to the products of similarly positioned genes in syntenic XyGUL nor to any other gene product among the diversity of Bacteroidetes PUL. RESULTS +209 222 Bacteroidetes taxonomy_domain In contrast, SGBP-B, encoded by locus tag Bacova_02650, displays little sequence similarity to the products of similarly positioned genes in syntenic XyGUL nor to any other gene product among the diversity of Bacteroidetes PUL. RESULTS +223 226 PUL gene In contrast, SGBP-B, encoded by locus tag Bacova_02650, displays little sequence similarity to the products of similarly positioned genes in syntenic XyGUL nor to any other gene product among the diversity of Bacteroidetes PUL. RESULTS +34 38 SusC protein Whereas sequence similarity among SusC/SusD homolog pairs often serves as a hallmark for PUL identification, the sequence similarities of downstream genes encoding SGBPs are generally too low to allow reliable bioinformatic classification of their products into protein families, let alone prediction of function. RESULTS +39 43 SusD protein Whereas sequence similarity among SusC/SusD homolog pairs often serves as a hallmark for PUL identification, the sequence similarities of downstream genes encoding SGBPs are generally too low to allow reliable bioinformatic classification of their products into protein families, let alone prediction of function. RESULTS +89 92 PUL gene Whereas sequence similarity among SusC/SusD homolog pairs often serves as a hallmark for PUL identification, the sequence similarities of downstream genes encoding SGBPs are generally too low to allow reliable bioinformatic classification of their products into protein families, let alone prediction of function. RESULTS +164 169 SGBPs protein_type Whereas sequence similarity among SusC/SusD homolog pairs often serves as a hallmark for PUL identification, the sequence similarities of downstream genes encoding SGBPs are generally too low to allow reliable bioinformatic classification of their products into protein families, let alone prediction of function. RESULTS +103 106 PUL gene Hence, there is a critical need for the elucidation of detailed structure-function relationships among PUL SGBPs, in light of the manifold glycan structures in nature. RESULTS +107 112 SGBPs protein_type Hence, there is a critical need for the elucidation of detailed structure-function relationships among PUL SGBPs, in light of the manifold glycan structures in nature. RESULTS +139 145 glycan chemical Hence, there is a critical need for the elucidation of detailed structure-function relationships among PUL SGBPs, in light of the manifold glycan structures in nature. RESULTS +0 18 Immunofluorescence experimental_method Immunofluorescence of formaldehyde-fixed, nonpermeabilized cells grown in minimal medium with XyG as the sole carbon source to induce XyGUL expression, reveals that both SGBP-A and SGBP-B are presented on the cell surface by N-terminal lipidation, as predicted by signal peptide analysis with SignalP (Fig. 2). RESULTS +94 97 XyG chemical Immunofluorescence of formaldehyde-fixed, nonpermeabilized cells grown in minimal medium with XyG as the sole carbon source to induce XyGUL expression, reveals that both SGBP-A and SGBP-B are presented on the cell surface by N-terminal lipidation, as predicted by signal peptide analysis with SignalP (Fig. 2). RESULTS +134 139 XyGUL gene Immunofluorescence of formaldehyde-fixed, nonpermeabilized cells grown in minimal medium with XyG as the sole carbon source to induce XyGUL expression, reveals that both SGBP-A and SGBP-B are presented on the cell surface by N-terminal lipidation, as predicted by signal peptide analysis with SignalP (Fig. 2). RESULTS +170 176 SGBP-A protein Immunofluorescence of formaldehyde-fixed, nonpermeabilized cells grown in minimal medium with XyG as the sole carbon source to induce XyGUL expression, reveals that both SGBP-A and SGBP-B are presented on the cell surface by N-terminal lipidation, as predicted by signal peptide analysis with SignalP (Fig. 2). RESULTS +181 187 SGBP-B protein Immunofluorescence of formaldehyde-fixed, nonpermeabilized cells grown in minimal medium with XyG as the sole carbon source to induce XyGUL expression, reveals that both SGBP-A and SGBP-B are presented on the cell surface by N-terminal lipidation, as predicted by signal peptide analysis with SignalP (Fig. 2). RESULTS +236 246 lipidation ptm Immunofluorescence of formaldehyde-fixed, nonpermeabilized cells grown in minimal medium with XyG as the sole carbon source to induce XyGUL expression, reveals that both SGBP-A and SGBP-B are presented on the cell surface by N-terminal lipidation, as predicted by signal peptide analysis with SignalP (Fig. 2). RESULTS +10 15 SGBPs protein_type Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B. ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig. 1B and C). RESULTS +53 94 cell-surface-localized endo-xyloglucanase protein_type Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B. ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig. 1B and C). RESULTS +95 104 B. ovatus species Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B. ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig. 1B and C). RESULTS +105 108 GH5 protein Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B. ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig. 1B and C). RESULTS +110 115 BoGH5 protein Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B. ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig. 1B and C). RESULTS +139 142 XyG chemical Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B. ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig. 1B and C). RESULTS +185 199 SusC-like TBDT protein_type Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B. ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig. 1B and C). RESULTS +207 212 XyGUL gene Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B. ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig. 1B and C). RESULTS +0 6 SGBP-A protein SGBP-A and SGBP-B visualized by immunofluorescence. FIG +11 17 SGBP-B protein SGBP-A and SGBP-B visualized by immunofluorescence. FIG +32 50 immunofluorescence experimental_method SGBP-A and SGBP-B visualized by immunofluorescence. FIG +33 42 B. ovatus species Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +83 86 XyG chemical Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +128 134 SGBP-A protein Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +138 144 SGBP-B protein Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +210 217 Overlay experimental_method Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +221 249 bright-field and FITC images evidence Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +253 262 B. ovatus species Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +299 306 Overlay experimental_method Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +310 338 bright-field and FITC images evidence Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +342 351 B. ovatus species Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +388 406 Bright-field image evidence Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +410 417 ΔSGBP-B mutant Formalin-fixed, nonpermeabilized B. ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B. ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +4 15 FITC images evidence (D) FITC images of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +19 26 ΔSGBP-B mutant (D) FITC images of ΔSGBP-B cells labeled with anti-SGBP-B antibodies. FIG +6 13 lacking protein_state Cells lacking SGBP-A (ΔSGBP-A) do not grow on XyG and therefore could not be tested in parallel. FIG +14 20 SGBP-A protein Cells lacking SGBP-A (ΔSGBP-A) do not grow on XyG and therefore could not be tested in parallel. FIG +22 29 ΔSGBP-A mutant Cells lacking SGBP-A (ΔSGBP-A) do not grow on XyG and therefore could not be tested in parallel. FIG +46 49 XyG chemical Cells lacking SGBP-A (ΔSGBP-A) do not grow on XyG and therefore could not be tested in parallel. FIG +71 91 glycoside hydrolases protein_type In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). RESULTS +99 104 XyGUL gene In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). RESULTS +130 143 affinity PAGE experimental_method In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). RESULTS +148 180 isothermal titration calorimetry experimental_method In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). RESULTS +182 185 ITC experimental_method In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). RESULTS +213 219 SGBP-A protein In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). RESULTS +224 230 SGBP-B protein In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). RESULTS +245 272 xyloglucan-binding proteins protein_type In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). RESULTS +274 291 affinity constant evidence In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). RESULTS +293 295 Ka evidence In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 × 105 M−1 and 4.98 × 104 M−1, respectively [23]). RESULTS +11 24 affinity PAGE experimental_method Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. RESULTS +61 67 SGBP-A protein Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. RESULTS +143 165 hydroxyethyl cellulose chemical Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. RESULTS +167 170 HEC chemical Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. RESULTS +174 189 β(1 → 4)-glucan chemical Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. RESULTS +216 250 mixed-linkage β(1→3)/β(1→4)-glucan chemical Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. RESULTS +252 255 MLG chemical Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. RESULTS +261 272 glucomannan chemical Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. RESULTS +274 276 GM chemical Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. RESULTS +284 292 glucosyl chemical Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. RESULTS +297 305 mannosyl chemical Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. RESULTS +360 374 polysaccharide chemical Additional affinity PAGE analysis (Fig. 3) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a β(1 → 4)-glucan] and limited affinity for mixed-linkage β(1→3)/β(1→4)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition. RESULTS +13 19 SGBP-B protein In contrast, SGBP-B bound to HEC more weakly than SGBP-A and did not bind to MLG or GM. RESULTS +29 32 HEC chemical In contrast, SGBP-B bound to HEC more weakly than SGBP-A and did not bind to MLG or GM. RESULTS +50 56 SGBP-A protein In contrast, SGBP-B bound to HEC more weakly than SGBP-A and did not bind to MLG or GM. RESULTS +77 80 MLG chemical In contrast, SGBP-B bound to HEC more weakly than SGBP-A and did not bind to MLG or GM. RESULTS +84 86 GM chemical In contrast, SGBP-B bound to HEC more weakly than SGBP-A and did not bind to MLG or GM. RESULTS +8 12 SGBP protein_type Neither SGBP recognized galactomannan (GGM), starch, carboxymethylcellulose, or mucin (see Fig. S1 in the supplemental material). RESULTS +24 37 galactomannan chemical Neither SGBP recognized galactomannan (GGM), starch, carboxymethylcellulose, or mucin (see Fig. S1 in the supplemental material). RESULTS +39 42 GGM chemical Neither SGBP recognized galactomannan (GGM), starch, carboxymethylcellulose, or mucin (see Fig. S1 in the supplemental material). RESULTS +45 51 starch chemical Neither SGBP recognized galactomannan (GGM), starch, carboxymethylcellulose, or mucin (see Fig. S1 in the supplemental material). RESULTS +53 75 carboxymethylcellulose chemical Neither SGBP recognized galactomannan (GGM), starch, carboxymethylcellulose, or mucin (see Fig. S1 in the supplemental material). RESULTS +80 85 mucin chemical Neither SGBP recognized galactomannan (GGM), starch, carboxymethylcellulose, or mucin (see Fig. S1 in the supplemental material). RESULTS +60 66 SGBP-A protein Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). RESULTS +71 77 SGBP-B protein Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). RESULTS +82 85 XyG chemical Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). RESULTS +135 151 XyG-specific GHs protein_type Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). RESULTS +159 164 XyGUL gene Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). RESULTS +217 226 B. ovatus species Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). RESULTS +240 243 PUL gene Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). RESULTS +248 251 MLG chemical Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). RESULTS +253 255 GM chemical Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). RESULTS +261 264 GGM chemical Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B. ovatus has discrete PUL for MLG, GM, and GGM (11). RESULTS +24 52 carbohydrate-binding modules site Notably, the absence of carbohydrate-binding modules in the GHs encoded by the XyGUL implies that noncatalytic recognition of xyloglucan is mediated entirely by SGBP-A and -B. RESULTS +60 63 GHs protein_type Notably, the absence of carbohydrate-binding modules in the GHs encoded by the XyGUL implies that noncatalytic recognition of xyloglucan is mediated entirely by SGBP-A and -B. RESULTS +79 84 XyGUL gene Notably, the absence of carbohydrate-binding modules in the GHs encoded by the XyGUL implies that noncatalytic recognition of xyloglucan is mediated entirely by SGBP-A and -B. RESULTS +126 136 xyloglucan chemical Notably, the absence of carbohydrate-binding modules in the GHs encoded by the XyGUL implies that noncatalytic recognition of xyloglucan is mediated entirely by SGBP-A and -B. RESULTS +161 167 SGBP-A protein Notably, the absence of carbohydrate-binding modules in the GHs encoded by the XyGUL implies that noncatalytic recognition of xyloglucan is mediated entirely by SGBP-A and -B. RESULTS +172 174 -B protein Notably, the absence of carbohydrate-binding modules in the GHs encoded by the XyGUL implies that noncatalytic recognition of xyloglucan is mediated entirely by SGBP-A and -B. RESULTS +0 6 SGBP-A protein SGBP-A and SGBP-B preferentially bind xyloglucan. FIG +11 17 SGBP-B protein SGBP-A and SGBP-B preferentially bind xyloglucan. FIG +38 48 xyloglucan chemical SGBP-A and SGBP-B preferentially bind xyloglucan. FIG +0 24 Affinity electrophoresis experimental_method Affinity electrophoresis (10% acrylamide) of SGBP-A and SGBP-B with BSA as a control protein. FIG +45 51 SGBP-A protein Affinity electrophoresis (10% acrylamide) of SGBP-A and SGBP-B with BSA as a control protein. FIG +56 62 SGBP-B protein Affinity electrophoresis (10% acrylamide) of SGBP-A and SGBP-B with BSA as a control protein. FIG +68 71 BSA protein Affinity electrophoresis (10% acrylamide) of SGBP-A and SGBP-B with BSA as a control protein. FIG +52 55 BSA protein All samples were loaded on the same gel next to the BSA controls; thin black lines indicate where intervening lanes were removed from the final image for both space and clarity. FIG +18 32 polysaccharide chemical The percentage of polysaccharide incorporated into each native gel is displayed. FIG +13 31 endo-xyloglucanase protein_type The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +39 44 XyGUL gene The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +46 51 BoGH5 protein The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +80 94 polysaccharide chemical The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +109 117 glucosyl chemical The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +139 165 xylogluco-oligosaccharides chemical The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +167 172 XyGOs chemical The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +187 200 Glc4 backbone structure_element The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +206 241 variable side-chain galactosylation structure_element The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +243 248 XyGO1 chemical The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +312 350 controlled digestion and fractionation experimental_method The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +354 383 size exclusion chromatography experimental_method The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +421 437 oligosaccharides chemical The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +445 450 XyGO2 chemical The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig. 1A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig. 1A; n = 2). RESULTS +0 3 ITC experimental_method ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). RESULTS +22 28 SGBP-A protein ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). RESULTS +38 41 XyG chemical ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). RESULTS +42 56 polysaccharide chemical ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). RESULTS +61 66 XyGO2 chemical ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). RESULTS +79 92 Glc8 backbone structure_element ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). RESULTS +117 127 affinities evidence ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). RESULTS +149 154 XyGO1 chemical ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). RESULTS +156 169 Glc4 backbone structure_element ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table 1; see Fig. S2 and S3 in the supplemental material). RESULTS +11 17 SGBP-B protein Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +23 31 bound to protein_state Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +32 35 XyG chemical Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +40 45 XyGO2 chemical Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +71 81 affinities evidence Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +107 109 Ka evidence Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +158 164 SGBP-A protein Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +186 192 SGBP-A protein Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +194 200 SGBP-B protein Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +206 214 bound to protein_state Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +215 220 XyGO1 chemical Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +230 238 affinity evidence Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +248 270 minimal repeating unit structure_element Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +288 290 Ka evidence Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +340 343 XyG chemical Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +348 353 XyGO2 chemical Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2. RESULTS +42 56 polysaccharide chemical Together, these data clearly suggest that polysaccharide binding of both SGBPs is fulfilled by a dimer of the minimal repeat, corresponding to XyGO2 (cf. RESULTS +73 78 SGBPs protein_type Together, these data clearly suggest that polysaccharide binding of both SGBPs is fulfilled by a dimer of the minimal repeat, corresponding to XyGO2 (cf. RESULTS +97 102 dimer oligomeric_state Together, these data clearly suggest that polysaccharide binding of both SGBPs is fulfilled by a dimer of the minimal repeat, corresponding to XyGO2 (cf. RESULTS +110 124 minimal repeat structure_element Together, these data clearly suggest that polysaccharide binding of both SGBPs is fulfilled by a dimer of the minimal repeat, corresponding to XyGO2 (cf. RESULTS +143 148 XyGO2 chemical Together, these data clearly suggest that polysaccharide binding of both SGBPs is fulfilled by a dimer of the minimal repeat, corresponding to XyGO2 (cf. RESULTS +19 32 affinity PAGE experimental_method The observation by affinity PAGE that these proteins specifically recognize XyG is further substantiated by their lack of binding for the undecorated oligosaccharide cellotetraose (Table 1; see Fig. S3). RESULTS +76 79 XyG chemical The observation by affinity PAGE that these proteins specifically recognize XyG is further substantiated by their lack of binding for the undecorated oligosaccharide cellotetraose (Table 1; see Fig. S3). RESULTS +150 165 oligosaccharide chemical The observation by affinity PAGE that these proteins specifically recognize XyG is further substantiated by their lack of binding for the undecorated oligosaccharide cellotetraose (Table 1; see Fig. S3). RESULTS +166 179 cellotetraose chemical The observation by affinity PAGE that these proteins specifically recognize XyG is further substantiated by their lack of binding for the undecorated oligosaccharide cellotetraose (Table 1; see Fig. S3). RESULTS +13 19 SGBP-A protein Furthermore, SGBP-A binds cellohexaose with ~770-fold weaker affinity than XyG, while SGBP-B displays no detectable binding to this linear hexasaccharide. RESULTS +26 38 cellohexaose chemical Furthermore, SGBP-A binds cellohexaose with ~770-fold weaker affinity than XyG, while SGBP-B displays no detectable binding to this linear hexasaccharide. RESULTS +61 69 affinity evidence Furthermore, SGBP-A binds cellohexaose with ~770-fold weaker affinity than XyG, while SGBP-B displays no detectable binding to this linear hexasaccharide. RESULTS +75 78 XyG chemical Furthermore, SGBP-A binds cellohexaose with ~770-fold weaker affinity than XyG, while SGBP-B displays no detectable binding to this linear hexasaccharide. RESULTS +86 92 SGBP-B protein Furthermore, SGBP-A binds cellohexaose with ~770-fold weaker affinity than XyG, while SGBP-B displays no detectable binding to this linear hexasaccharide. RESULTS +139 153 hexasaccharide chemical Furthermore, SGBP-A binds cellohexaose with ~770-fold weaker affinity than XyG, while SGBP-B displays no detectable binding to this linear hexasaccharide. RESULTS +48 53 XyGUL gene To provide molecular-level insight into how the XyGUL SGBPs equip B. ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms. RESULTS +54 59 SGBPs protein_type To provide molecular-level insight into how the XyGUL SGBPs equip B. ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms. RESULTS +66 75 B. ovatus species To provide molecular-level insight into how the XyGUL SGBPs equip B. ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms. RESULTS +100 103 XyG chemical To provide molecular-level insight into how the XyGUL SGBPs equip B. ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms. RESULTS +143 164 X-ray crystallography experimental_method To provide molecular-level insight into how the XyGUL SGBPs equip B. ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms. RESULTS +182 188 SGBP-A protein To provide molecular-level insight into how the XyGUL SGBPs equip B. ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms. RESULTS +193 199 SGPB-B protein To provide molecular-level insight into how the XyGUL SGBPs equip B. ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms. RESULTS +203 232 oligosaccharide-complex forms complex_assembly To provide molecular-level insight into how the XyGUL SGBPs equip B. ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms. RESULTS +40 49 wild-type protein_state Summary of thermodynamic parameters for wild-type SGBP-A and SGBP-B obtained by isothermal titration calorimetry at 25°Ca TABLE +50 56 SGBP-A protein Summary of thermodynamic parameters for wild-type SGBP-A and SGBP-B obtained by isothermal titration calorimetry at 25°Ca TABLE +61 67 SGBP-B protein Summary of thermodynamic parameters for wild-type SGBP-A and SGBP-B obtained by isothermal titration calorimetry at 25°Ca TABLE +80 112 isothermal titration calorimetry experimental_method Summary of thermodynamic parameters for wild-type SGBP-A and SGBP-B obtained by isothermal titration calorimetry at 25°Ca TABLE +22 24 ΔG evidence "Carbohydrate Ka (M−1) ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) SGBP-A SGBP-B SGBP-A SGBP-B SGBP-A SGBP-B SGBP-A SGBP-B XyGb (4.4 ± 0.1) × 105 (5.7 ± 0.2) × 104 −7.7 −6.5 −14 ± 3 −14 ± 2 −6.5 −7.6 XyGO2c 3.0 × 105 2.0 × 104 −7.5 −5.9 −17.2 −17.6 −9.7 −11.7 XyGO1 NBd (2.4 ± 0.1) × 103 NB −4.6 NB −4.4 ± 0.2 NB 0.2 Cellohexaose 568.0 ± 291.0 NB −3.8 NB −16 ± 8 NB −12.7 NB Cellotetraose NB NB NB NB NB NB NB NB " TABLE +0 6 SGBP-A protein SGBP-A is a SusD homolog with an extensive glycan-binding platform. RESULTS +12 16 SusD protein SGBP-A is a SusD homolog with an extensive glycan-binding platform. RESULTS +43 66 glycan-binding platform site SGBP-A is a SusD homolog with an extensive glycan-binding platform. RESULTS +68 77 structure evidence As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). RESULTS +81 84 apo protein_state As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). RESULTS +85 91 SGBP-A protein As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). RESULTS +101 106 Rwork evidence As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). RESULTS +116 121 Rfree evidence As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). RESULTS +140 149 28 to 546 residue_range As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). RESULTS +184 208 “SusD-like” protein fold structure_element As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). RESULTS +227 252 tetratrico-peptide repeat structure_element As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). RESULTS +254 257 TPR structure_element As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). RESULTS +294 303 structure evidence As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36 Å, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table 2) displays the canonical “SusD-like” protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig. 4A). RESULTS +14 20 SGBP-A protein Specifically, SGBP-A overlays B. thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2 Å for 363 Cα pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig. 4C). RESULTS +21 29 overlays experimental_method Specifically, SGBP-A overlays B. thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2 Å for 363 Cα pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig. 4C). RESULTS +30 49 B. thetaiotaomicron species Specifically, SGBP-A overlays B. thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2 Å for 363 Cα pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig. 4C). RESULTS +50 54 SusD protein Specifically, SGBP-A overlays B. thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2 Å for 363 Cα pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig. 4C). RESULTS +56 62 BtSusD protein Specifically, SGBP-A overlays B. thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2 Å for 363 Cα pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig. 4C). RESULTS +71 97 root mean square deviation evidence Specifically, SGBP-A overlays B. thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2 Å for 363 Cα pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig. 4C). RESULTS +99 103 RMSD evidence Specifically, SGBP-A overlays B. thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2 Å for 363 Cα pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig. 4C). RESULTS +0 17 Cocrystallization experimental_method Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. RESULTS +21 27 SGBP-A protein Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. RESULTS +33 38 XyGO2 chemical Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. RESULTS +51 68 substrate complex complex_assembly Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. RESULTS +69 78 structure evidence Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. RESULTS +87 92 Rwork evidence Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. RESULTS +102 107 Rfree evidence Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. RESULTS +126 135 36 to 546 residue_range Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. RESULTS +189 201 binding-site site Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. RESULTS +222 241 XyG binding protein protein_type Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3 Å, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig. 4A and B; Table 2) that revealed the distinct binding-site architecture of the XyG binding protein. RESULTS +4 16 SGBP-A:XyGO2 complex_assembly The SGBP-A:XyGO2 complex superimposes closely with the apo structure (RMSD of 0.6 Å) and demonstrates that no major conformational change occurs upon substrate binding; small deviations in the orientation of several surface loops are likely the result of differential crystal packing. RESULTS +25 37 superimposes experimental_method The SGBP-A:XyGO2 complex superimposes closely with the apo structure (RMSD of 0.6 Å) and demonstrates that no major conformational change occurs upon substrate binding; small deviations in the orientation of several surface loops are likely the result of differential crystal packing. RESULTS +55 58 apo protein_state The SGBP-A:XyGO2 complex superimposes closely with the apo structure (RMSD of 0.6 Å) and demonstrates that no major conformational change occurs upon substrate binding; small deviations in the orientation of several surface loops are likely the result of differential crystal packing. RESULTS +59 68 structure evidence The SGBP-A:XyGO2 complex superimposes closely with the apo structure (RMSD of 0.6 Å) and demonstrates that no major conformational change occurs upon substrate binding; small deviations in the orientation of several surface loops are likely the result of differential crystal packing. RESULTS +70 74 RMSD evidence The SGBP-A:XyGO2 complex superimposes closely with the apo structure (RMSD of 0.6 Å) and demonstrates that no major conformational change occurs upon substrate binding; small deviations in the orientation of several surface loops are likely the result of differential crystal packing. RESULTS +61 80 ligand-binding site site It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). RESULTS +84 93 conserved protein_state It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). RESULTS +102 108 SGBP-A protein It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). RESULTS +113 117 SusD protein It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). RESULTS +127 133 SGBP-A protein It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). RESULTS +157 174 aromatic platform site It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). RESULTS +211 214 XyG chemical It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). RESULTS +252 255 XyG chemical It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). RESULTS +310 314 site site It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). RESULTS +322 326 SusD protein It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). RESULTS +372 379 amylose chemical It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-Å-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-Å-long, site within SusD that complements the helical conformation of amylose (Fig. 4C and D). RESULTS +10 19 structure evidence Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures. FIG +23 29 SGBP-A protein Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures. FIG +31 43 Bacova_02651 gene Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures. FIG +50 57 Overlay experimental_method Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures. FIG +61 67 SGBP-A protein Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures. FIG +77 80 apo protein_state Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures. FIG +95 100 XyGO2 chemical Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures. FIG +108 118 structures evidence Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures. FIG +4 7 apo protein_state The apo structure is color ramped from blue to red. FIG +8 17 structure evidence The apo structure is color ramped from blue to red. FIG +3 11 omit map evidence An omit map (2σ) for XyGO2 (orange and red sticks) is displayed. FIG +21 26 XyGO2 chemical An omit map (2σ) for XyGO2 (orange and red sticks) is displayed. FIG +25 33 omit map evidence (B) Close-up view of the omit map as in panel A, rotated 90° clockwise. FIG +4 11 Overlay experimental_method (C) Overlay of the Cα backbones of SGBP-A (black) with XyGO2 (orange and red spheres) and BtSusD (blue) with maltoheptaose (pink and red spheres), highlighting the conservation of the glycan-binding site location. FIG +35 41 SGBP-A protein (C) Overlay of the Cα backbones of SGBP-A (black) with XyGO2 (orange and red spheres) and BtSusD (blue) with maltoheptaose (pink and red spheres), highlighting the conservation of the glycan-binding site location. FIG +55 60 XyGO2 chemical (C) Overlay of the Cα backbones of SGBP-A (black) with XyGO2 (orange and red spheres) and BtSusD (blue) with maltoheptaose (pink and red spheres), highlighting the conservation of the glycan-binding site location. FIG +90 96 BtSusD protein (C) Overlay of the Cα backbones of SGBP-A (black) with XyGO2 (orange and red spheres) and BtSusD (blue) with maltoheptaose (pink and red spheres), highlighting the conservation of the glycan-binding site location. FIG +109 122 maltoheptaose chemical (C) Overlay of the Cα backbones of SGBP-A (black) with XyGO2 (orange and red spheres) and BtSusD (blue) with maltoheptaose (pink and red spheres), highlighting the conservation of the glycan-binding site location. FIG +184 203 glycan-binding site site (C) Overlay of the Cα backbones of SGBP-A (black) with XyGO2 (orange and red spheres) and BtSusD (blue) with maltoheptaose (pink and red spheres), highlighting the conservation of the glycan-binding site location. FIG +20 26 SGBP-A protein (D) Close-up of the SGBP-A (black and orange) and SusD (blue and pink) glycan-binding sites. FIG +50 54 SusD protein (D) Close-up of the SGBP-A (black and orange) and SusD (blue and pink) glycan-binding sites. FIG +71 91 glycan-binding sites site (D) Close-up of the SGBP-A (black and orange) and SusD (blue and pink) glycan-binding sites. FIG +31 50 glycan-binding site site The approximate length of each glycan-binding site is displayed, colored to match the protein structures. (E) Stereo view of the xyloglucan-binding site of SGBP-A, displaying all residues within 4 Å of the ligand. FIG +86 104 protein structures evidence The approximate length of each glycan-binding site is displayed, colored to match the protein structures. (E) Stereo view of the xyloglucan-binding site of SGBP-A, displaying all residues within 4 Å of the ligand. FIG +129 152 xyloglucan-binding site site The approximate length of each glycan-binding site is displayed, colored to match the protein structures. (E) Stereo view of the xyloglucan-binding site of SGBP-A, displaying all residues within 4 Å of the ligand. FIG +156 162 SGBP-A protein The approximate length of each glycan-binding site is displayed, colored to match the protein structures. (E) Stereo view of the xyloglucan-binding site of SGBP-A, displaying all residues within 4 Å of the ligand. FIG +13 20 glucose chemical The backbone glucose residues are numbered from the nonreducing end; xylose residues are labeled X1 and X2. FIG +69 75 xylose chemical The backbone glucose residues are numbered from the nonreducing end; xylose residues are labeled X1 and X2. FIG +97 99 X1 residue_name_number The backbone glucose residues are numbered from the nonreducing end; xylose residues are labeled X1 and X2. FIG +104 106 X2 residue_name_number The backbone glucose residues are numbered from the nonreducing end; xylose residues are labeled X1 and X2. FIG +10 39 hydrogen-bonding interactions bond_interaction Potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. FIG +28 36 glucosyl chemical Seven of the eight backbone glucosyl residues of XyGO2 could be convincingly modeled in the ligand electron density, and only two α(1→6)-linked xylosyl residues were observed (Fig. 4B; cf. RESULTS +49 54 XyGO2 chemical Seven of the eight backbone glucosyl residues of XyGO2 could be convincingly modeled in the ligand electron density, and only two α(1→6)-linked xylosyl residues were observed (Fig. 4B; cf. RESULTS +92 115 ligand electron density evidence Seven of the eight backbone glucosyl residues of XyGO2 could be convincingly modeled in the ligand electron density, and only two α(1→6)-linked xylosyl residues were observed (Fig. 4B; cf. RESULTS +130 151 α(1→6)-linked xylosyl chemical Seven of the eight backbone glucosyl residues of XyGO2 could be convincingly modeled in the ligand electron density, and only two α(1→6)-linked xylosyl residues were observed (Fig. 4B; cf. RESULTS +12 28 electron density evidence Indeed, the electron density for the ligand suggests some disorder, which may arise from multiple oligosaccharide orientations along the binding site. RESULTS +98 113 oligosaccharide chemical Indeed, the electron density for the ligand suggests some disorder, which may arise from multiple oligosaccharide orientations along the binding site. RESULTS +137 149 binding site site Indeed, the electron density for the ligand suggests some disorder, which may arise from multiple oligosaccharide orientations along the binding site. RESULTS +24 27 W82 residue_name_number Three aromatic residues—W82, W283, W306—comprise the flat platform that stacks along the naturally twisted β-glucan backbone (Fig. 4E). RESULTS +29 33 W283 residue_name_number Three aromatic residues—W82, W283, W306—comprise the flat platform that stacks along the naturally twisted β-glucan backbone (Fig. 4E). RESULTS +35 39 W306 residue_name_number Three aromatic residues—W82, W283, W306—comprise the flat platform that stacks along the naturally twisted β-glucan backbone (Fig. 4E). RESULTS +53 66 flat platform site Three aromatic residues—W82, W283, W306—comprise the flat platform that stacks along the naturally twisted β-glucan backbone (Fig. 4E). RESULTS +72 78 stacks bond_interaction Three aromatic residues—W82, W283, W306—comprise the flat platform that stacks along the naturally twisted β-glucan backbone (Fig. 4E). RESULTS +107 115 β-glucan chemical Three aromatic residues—W82, W283, W306—comprise the flat platform that stacks along the naturally twisted β-glucan backbone (Fig. 4E). RESULTS +34 42 platform site The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table 3; see Fig. S4 in the supplemental material). RESULTS +86 90 W82A mutant The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table 3; see Fig. S4 in the supplemental material). RESULTS +91 96 W283A mutant The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table 3; see Fig. S4 in the supplemental material). RESULTS +97 102 W306A mutant The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table 3; see Fig. S4 in the supplemental material). RESULTS +103 109 mutant protein_state The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table 3; see Fig. S4 in the supplemental material). RESULTS +113 119 SGBP-A protein The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table 3; see Fig. S4 in the supplemental material). RESULTS +132 139 SGBP-A* mutant The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table 3; see Fig. S4 in the supplemental material). RESULTS +144 177 completely devoid of XyG affinity protein_state The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table 3; see Fig. S4 in the supplemental material). RESULTS +77 81 W82A mutant Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding. RESULTS +82 88 mutant protein_state Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding. RESULTS +137 139 Ka evidence Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding. RESULTS +150 153 XyG chemical Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding. RESULTS +165 170 W306A mutant Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding. RESULTS +171 183 substitution experimental_method Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding. RESULTS +195 216 abolishes XyG binding protein_state Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding. RESULTS +47 71 hydrophobic interactions bond_interaction Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3. RESULTS +98 127 hydrogen-bonding interactions bond_interaction Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3. RESULTS +137 143 ligand chemical Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3. RESULTS +167 170 R65 residue_name_number Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3. RESULTS +172 175 N83 residue_name_number Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3. RESULTS +181 185 S308 residue_name_number Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3. RESULTS +209 213 Glc5 residue_name_number Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3. RESULTS +218 222 Glc3 residue_name_number Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3. RESULTS +32 55 saccharide-binding data evidence Most surprising in light of the saccharide-binding data, however, was a lack of extensive recognition of the XyG side chains; only Y84 appeared to provide a hydrophobic interface for a xylosyl residue (Xyl1). RESULTS +109 112 XyG chemical Most surprising in light of the saccharide-binding data, however, was a lack of extensive recognition of the XyG side chains; only Y84 appeared to provide a hydrophobic interface for a xylosyl residue (Xyl1). RESULTS +131 134 Y84 residue_name_number Most surprising in light of the saccharide-binding data, however, was a lack of extensive recognition of the XyG side chains; only Y84 appeared to provide a hydrophobic interface for a xylosyl residue (Xyl1). RESULTS +157 178 hydrophobic interface site Most surprising in light of the saccharide-binding data, however, was a lack of extensive recognition of the XyG side chains; only Y84 appeared to provide a hydrophobic interface for a xylosyl residue (Xyl1). RESULTS +185 192 xylosyl chemical Most surprising in light of the saccharide-binding data, however, was a lack of extensive recognition of the XyG side chains; only Y84 appeared to provide a hydrophobic interface for a xylosyl residue (Xyl1). RESULTS +202 206 Xyl1 residue_name_number Most surprising in light of the saccharide-binding data, however, was a lack of extensive recognition of the XyG side chains; only Y84 appeared to provide a hydrophobic interface for a xylosyl residue (Xyl1). RESULTS +65 71 SGBP-A protein Summary of thermodynamic parameters for site-directed mutants of SGBP-A and SGBP-B obtained by ITC with XyG at 25°Ca TABLE +76 82 SGBP-B protein Summary of thermodynamic parameters for site-directed mutants of SGBP-A and SGBP-B obtained by ITC with XyG at 25°Ca TABLE +95 98 ITC experimental_method Summary of thermodynamic parameters for site-directed mutants of SGBP-A and SGBP-B obtained by ITC with XyG at 25°Ca TABLE +104 107 XyG chemical Summary of thermodynamic parameters for site-directed mutants of SGBP-A and SGBP-B obtained by ITC with XyG at 25°Ca TABLE +13 15 Ka evidence "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +16 18 ΔG evidence "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +34 36 ΔH evidence "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +52 55 TΔS evidence "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +92 98 SGBP-A protein "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +99 103 W82A mutant "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +104 109 W283A mutant "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +110 115 W306A mutant "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +134 140 SGBP-A protein "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +141 145 W82A mutant "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +178 184 SGBP-A protein "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +185 189 W306 residue_name_number "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +208 214 SGBP-B protein "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +215 222 230–489 residue_range "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +271 277 SGBP-B protein "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +278 283 Y363A mutant "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +334 340 SGBP-B protein "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +341 346 W364A mutant "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +373 379 SGBP-B protein "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +380 385 F414A mutant "Protein name Ka ΔG (kcal ⋅ mol−1) ΔH (kcal ⋅ mol−1) TΔS (kcal ⋅ mol−1) Fold changeb M−1 SGBP-A(W82A W283A W306A) ND NB NB NB NB SGBP-A(W82A)c 4.9 9.1 × 104 −6.8 −6.3 0.5 SGBP-A(W306) ND NB NB NB NB SGBP-B(230–489) 0.7 (8.6 ± 0.20) × 104 −6.7 −14.9 ± 0.1 −8.2 SGBP-B(Y363A) 19.7 (2.9 ± 0.10) × 103 −4.7 −18.1 ± 0.1 −13.3 SGBP-B(W364A) ND Weak Weak Weak Weak SGBP-B(F414A) 3.2 (1.80 ± 0.03) × 104 −5.8 −11.4 ± 0.1 −5.6 " TABLE +75 80 XyGO2 chemical Binding thermodynamics are based on the concentration of the binding unit, XyGO2. TABLE +26 28 Ka evidence Weak binding represents a Ka of <500 M−1. TABLE +0 2 Ka evidence Ka fold change = Ka of wild-type protein/Ka of mutant protein for xyloglucan binding. TABLE +17 19 Ka evidence Ka fold change = Ka of wild-type protein/Ka of mutant protein for xyloglucan binding. TABLE +23 32 wild-type protein_state Ka fold change = Ka of wild-type protein/Ka of mutant protein for xyloglucan binding. TABLE +41 43 Ka evidence Ka fold change = Ka of wild-type protein/Ka of mutant protein for xyloglucan binding. TABLE +66 76 xyloglucan chemical Ka fold change = Ka of wild-type protein/Ka of mutant protein for xyloglucan binding. TABLE +0 6 SGBP-B protein SGBP-B has a multimodular structure with a single, C-terminal glycan-binding domain. RESULTS +62 83 glycan-binding domain structure_element SGBP-B has a multimodular structure with a single, C-terminal glycan-binding domain. RESULTS +4 21 crystal structure evidence The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +25 36 full-length protein_state The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +37 43 SGBP-B protein The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +44 59 in complex with protein_state The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +60 65 XyGO2 chemical The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +75 80 Rwork evidence The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +90 95 Rfree evidence The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +114 123 34 to 489 residue_range The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +156 165 structure evidence The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +184 223 tandem immunoglobulin (Ig)-like domains structure_element The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +233 234 A structure_element The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +236 237 B structure_element The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +243 244 C structure_element The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +284 309 xyloglucan-binding domain structure_element The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +318 319 D structure_element The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37 Å, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table 2) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig. 5A). RESULTS +8 9 A structure_element Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). RESULTS +11 12 B structure_element Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). RESULTS +18 19 C structure_element Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). RESULTS +36 52 β-sandwich folds structure_element Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). RESULTS +62 63 B structure_element Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). RESULTS +74 84 134 to 230 residue_range Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). RESULTS +90 91 C structure_element Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). RESULTS +102 112 231 to 313 residue_range Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). RESULTS +121 133 superimposed experimental_method Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). RESULTS +146 147 A structure_element Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). RESULTS +158 167 34 to 133 residue_range Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). RESULTS +174 179 RMSDs evidence Domains A, B, and C display similar β-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2 Å, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively). RESULTS +0 13 These domains structure_element These domains also display similarity to the C-terminal β-sandwich domains of many GH13 enzymes, including the cyclodextrin glucanotransferase of Geobacillus stearothermophilus (Fig. 5B). RESULTS +56 74 β-sandwich domains structure_element These domains also display similarity to the C-terminal β-sandwich domains of many GH13 enzymes, including the cyclodextrin glucanotransferase of Geobacillus stearothermophilus (Fig. 5B). RESULTS +83 95 GH13 enzymes protein_type These domains also display similarity to the C-terminal β-sandwich domains of many GH13 enzymes, including the cyclodextrin glucanotransferase of Geobacillus stearothermophilus (Fig. 5B). RESULTS +111 142 cyclodextrin glucanotransferase protein_type These domains also display similarity to the C-terminal β-sandwich domains of many GH13 enzymes, including the cyclodextrin glucanotransferase of Geobacillus stearothermophilus (Fig. 5B). RESULTS +146 176 Geobacillus stearothermophilus species These domains also display similarity to the C-terminal β-sandwich domains of many GH13 enzymes, including the cyclodextrin glucanotransferase of Geobacillus stearothermophilus (Fig. 5B). RESULTS +0 12 Such domains structure_element Such domains are not typically involved in carbohydrate binding. RESULTS +43 55 carbohydrate chemical Such domains are not typically involved in carbohydrate binding. RESULTS +8 25 visual inspection experimental_method Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig. S5 in the supplemental material), indicates that these domains do not contribute to XyG capture. RESULTS +33 39 SGBP-B protein Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig. S5 in the supplemental material), indicates that these domains do not contribute to XyG capture. RESULTS +40 49 structure evidence Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig. S5 in the supplemental material), indicates that these domains do not contribute to XyG capture. RESULTS +91 92 A structure_element Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig. S5 in the supplemental material), indicates that these domains do not contribute to XyG capture. RESULTS +97 98 B structure_element Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig. S5 in the supplemental material), indicates that these domains do not contribute to XyG capture. RESULTS +111 124 affinity PAGE experimental_method Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig. S5 in the supplemental material), indicates that these domains do not contribute to XyG capture. RESULTS +228 231 XyG chemical Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig. S5 in the supplemental material), indicates that these domains do not contribute to XyG capture. RESULTS +19 29 production experimental_method On the other hand, production of the fused domains C and D in tandem (SGBP-B residues 230 to 489) retains complete binding of xyloglucan in vitro, with the observed slight increase in affinity likely arising from a reduced potential for steric hindrance of the smaller protein construct during polysaccharide interactions (Table 3). RESULTS +37 58 fused domains C and D mutant On the other hand, production of the fused domains C and D in tandem (SGBP-B residues 230 to 489) retains complete binding of xyloglucan in vitro, with the observed slight increase in affinity likely arising from a reduced potential for steric hindrance of the smaller protein construct during polysaccharide interactions (Table 3). RESULTS +70 76 SGBP-B protein On the other hand, production of the fused domains C and D in tandem (SGBP-B residues 230 to 489) retains complete binding of xyloglucan in vitro, with the observed slight increase in affinity likely arising from a reduced potential for steric hindrance of the smaller protein construct during polysaccharide interactions (Table 3). RESULTS +86 96 230 to 489 residue_range On the other hand, production of the fused domains C and D in tandem (SGBP-B residues 230 to 489) retains complete binding of xyloglucan in vitro, with the observed slight increase in affinity likely arising from a reduced potential for steric hindrance of the smaller protein construct during polysaccharide interactions (Table 3). RESULTS +126 136 xyloglucan chemical On the other hand, production of the fused domains C and D in tandem (SGBP-B residues 230 to 489) retains complete binding of xyloglucan in vitro, with the observed slight increase in affinity likely arising from a reduced potential for steric hindrance of the smaller protein construct during polysaccharide interactions (Table 3). RESULTS +294 308 polysaccharide chemical On the other hand, production of the fused domains C and D in tandem (SGBP-B residues 230 to 489) retains complete binding of xyloglucan in vitro, with the observed slight increase in affinity likely arising from a reduced potential for steric hindrance of the smaller protein construct during polysaccharide interactions (Table 3). RESULTS +18 29 full-length protein_state While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). RESULTS +49 50 D structure_element While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). RESULTS +89 109 XyG-binding proteins protein_type While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). RESULTS +127 133 SGBP-B protein While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). RESULTS +148 169 xylan-binding protein protein_type While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). RESULTS +170 182 Bacova_04391 protein While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). RESULTS +211 216 xylan chemical While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). RESULTS +227 230 PUL gene While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). RESULTS +234 243 B. ovatus species While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B. ovatus (Fig. 5C). RESULTS +4 29 structure-based alignment experimental_method The structure-based alignment of these proteins reveals 17% sequence identity, with a core RMSD of 3.6 Å for 253 aligned residues. RESULTS +91 95 RMSD evidence The structure-based alignment of these proteins reveals 17% sequence identity, with a core RMSD of 3.6 Å for 253 aligned residues. RESULTS +51 63 Bacova_04391 protein While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +79 91 binding site site While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +116 120 W241 residue_name_number While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +125 129 Y404 residue_name_number While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +157 174 XyGO binding site site While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +178 184 SGBP-B protein While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +199 231 opposing, clamp-like arrangement protein_state While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +235 249 these residues structure_element While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +253 265 Bacova_04391 protein While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +295 321 planar surface arrangement site While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +329 337 residues structure_element While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +357 360 XyG chemical While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +364 370 SGBP-B protein While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below). RESULTS +26 32 SGBP-B protein Multimodular structure of SGBP-B (Bacova_02650). (A) Full-length structure of SGBP-B, color coded by domain as indicated. FIG +34 46 Bacova_02650 gene Multimodular structure of SGBP-B (Bacova_02650). (A) Full-length structure of SGBP-B, color coded by domain as indicated. FIG +53 64 Full-length protein_state Multimodular structure of SGBP-B (Bacova_02650). (A) Full-length structure of SGBP-B, color coded by domain as indicated. FIG +65 74 structure evidence Multimodular structure of SGBP-B (Bacova_02650). (A) Full-length structure of SGBP-B, color coded by domain as indicated. FIG +78 84 SGBP-B protein Multimodular structure of SGBP-B (Bacova_02650). (A) Full-length structure of SGBP-B, color coded by domain as indicated. FIG +0 8 Prolines residue_name Prolines between domains are indicated as spheres. FIG +3 11 omit map evidence An omit map (2σ) for XyGO2 is displayed to highlight the location of the glycan-binding site. FIG +21 26 XyGO2 chemical An omit map (2σ) for XyGO2 is displayed to highlight the location of the glycan-binding site. FIG +73 92 glycan-binding site site An omit map (2σ) for XyGO2 is displayed to highlight the location of the glycan-binding site. FIG +15 21 SGBP-B protein (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). FIG +30 31 A structure_element (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). FIG +33 34 B structure_element (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). FIG +40 41 C structure_element (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). FIG +85 99 Ig-like domain structure_element (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). FIG +107 128 G. stearothermophilus species (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). FIG +129 160 cyclodextrin glucanotransferase protein_type (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). FIG +181 191 375 to 493 residue_range (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). FIG +211 218 overlay experimental_method (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). FIG +222 228 SGBP-B protein (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). FIG +240 252 Bacova_04391 protein (B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) Cα overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink). FIG +13 21 omit map evidence (D) Close-up omit map for the XyGO2 ligand, contoured at 2σ. (E) Stereo view of the xyloglucan-binding site of SGBP-B, displaying all residues within 4 Å of the ligand. FIG +30 35 XyGO2 chemical (D) Close-up omit map for the XyGO2 ligand, contoured at 2σ. (E) Stereo view of the xyloglucan-binding site of SGBP-B, displaying all residues within 4 Å of the ligand. FIG +84 107 xyloglucan-binding site site (D) Close-up omit map for the XyGO2 ligand, contoured at 2σ. (E) Stereo view of the xyloglucan-binding site of SGBP-B, displaying all residues within 4 Å of the ligand. FIG +111 117 SGBP-B protein (D) Close-up omit map for the XyGO2 ligand, contoured at 2σ. (E) Stereo view of the xyloglucan-binding site of SGBP-B, displaying all residues within 4 Å of the ligand. FIG +13 20 glucose chemical The backbone glucose residues are numbered from the nonreducing end, xylose residues are shown as X1, X2, and X3, potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. FIG +69 75 xylose chemical The backbone glucose residues are numbered from the nonreducing end, xylose residues are shown as X1, X2, and X3, potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. FIG +98 100 X1 residue_name_number The backbone glucose residues are numbered from the nonreducing end, xylose residues are shown as X1, X2, and X3, potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. FIG +102 104 X2 residue_name_number The backbone glucose residues are numbered from the nonreducing end, xylose residues are shown as X1, X2, and X3, potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. FIG +110 112 X3 residue_name_number The backbone glucose residues are numbered from the nonreducing end, xylose residues are shown as X1, X2, and X3, potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. FIG +124 153 hydrogen-bonding interactions bond_interaction The backbone glucose residues are numbered from the nonreducing end, xylose residues are shown as X1, X2, and X3, potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms. FIG +27 36 structure evidence Inspection of the tertiary structure indicates that domains C and D are effectively inseparable, with a contact interface of 396 Å2. RESULTS +60 61 C structure_element Inspection of the tertiary structure indicates that domains C and D are effectively inseparable, with a contact interface of 396 Å2. RESULTS +66 67 D structure_element Inspection of the tertiary structure indicates that domains C and D are effectively inseparable, with a contact interface of 396 Å2. RESULTS +8 9 A structure_element Domains A, B, and C do not pack against each other. RESULTS +11 12 B structure_element Domains A, B, and C do not pack against each other. RESULTS +18 19 C structure_element Domains A, B, and C do not pack against each other. RESULTS +14 34 five-residue linkers structure_element Moreover, the five-residue linkers between these first three domains all feature a proline as the middle residue, suggesting significant conformational rigidity (Fig. 5A). RESULTS +83 90 proline residue_name Moreover, the five-residue linkers between these first three domains all feature a proline as the middle residue, suggesting significant conformational rigidity (Fig. 5A). RESULTS +98 112 middle residue structure_element Moreover, the five-residue linkers between these first three domains all feature a proline as the middle residue, suggesting significant conformational rigidity (Fig. 5A). RESULTS +81 88 proline residue_name Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface. RESULTS +99 114 Ig-like domains structure_element Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface. RESULTS +136 148 Bacova_04391 protein Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface. RESULTS +157 180 starch-binding proteins protein_type Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface. RESULTS +181 185 SusE protein Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface. RESULTS +190 194 SusF protein Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface. RESULTS +285 304 glycan binding site site Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface. RESULTS +16 22 SGBP-B protein Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. RESULTS +123 143 eight-residue linker structure_element Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. RESULTS +169 178 lipidated protein_state Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. RESULTS +179 182 Cys residue_name Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. RESULTS +184 187 C28 residue_name_number Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. RESULTS +197 211 first β-strand structure_element Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. RESULTS +222 223 A structure_element Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. RESULTS +231 254 outer membrane proteins protein_type Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. RESULTS +268 284 Sus-like systems complex_assembly Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. RESULTS +303 339 10- to 20-amino-acid flexible linker structure_element Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. RESULTS +352 361 lipidated protein_state Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. RESULTS +362 365 Cys residue_name Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first β-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element. RESULTS +17 40 outer membrane-anchored protein_state Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-β-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface. RESULTS +41 59 endo-xyloglucanase protein_type Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-β-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface. RESULTS +60 65 BoGH5 protein Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-β-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface. RESULTS +73 78 XyGUL gene Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-β-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface. RESULTS +90 118 100-amino-acid, all-β-strand structure_element Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-β-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface. RESULTS +120 137 N-terminal module structure_element Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-β-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface. RESULTS +142 157 flexible linker structure_element Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-β-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface. RESULTS +216 232 catalytic module structure_element Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-β-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface. RESULTS +0 3 XyG chemical XyG binds to domain D of SGBP-B at the concave interface of the top β-sheet, with binding mediated by loops connecting the β-strands. RESULTS +4 12 binds to protein_state XyG binds to domain D of SGBP-B at the concave interface of the top β-sheet, with binding mediated by loops connecting the β-strands. RESULTS +20 21 D structure_element XyG binds to domain D of SGBP-B at the concave interface of the top β-sheet, with binding mediated by loops connecting the β-strands. RESULTS +25 31 SGBP-B protein XyG binds to domain D of SGBP-B at the concave interface of the top β-sheet, with binding mediated by loops connecting the β-strands. RESULTS +39 56 concave interface site XyG binds to domain D of SGBP-B at the concave interface of the top β-sheet, with binding mediated by loops connecting the β-strands. RESULTS +68 75 β-sheet structure_element XyG binds to domain D of SGBP-B at the concave interface of the top β-sheet, with binding mediated by loops connecting the β-strands. RESULTS +102 107 loops structure_element XyG binds to domain D of SGBP-B at the concave interface of the top β-sheet, with binding mediated by loops connecting the β-strands. RESULTS +123 132 β-strands structure_element XyG binds to domain D of SGBP-B at the concave interface of the top β-sheet, with binding mediated by loops connecting the β-strands. RESULTS +4 12 glucosyl chemical Six glucosyl residues, comprising the main chain, and three branching xylosyl residues of XyGO2 can be modeled in the density (Fig. 5D; cf. RESULTS +70 77 xylosyl chemical Six glucosyl residues, comprising the main chain, and three branching xylosyl residues of XyGO2 can be modeled in the density (Fig. 5D; cf. RESULTS +90 95 XyGO2 chemical Six glucosyl residues, comprising the main chain, and three branching xylosyl residues of XyGO2 can be modeled in the density (Fig. 5D; cf. RESULTS +118 125 density evidence Six glucosyl residues, comprising the main chain, and three branching xylosyl residues of XyGO2 can be modeled in the density (Fig. 5D; cf. RESULTS +82 119 cello- and xylogluco-oligosaccharides chemical The backbone is flat, with less of the “twisted-ribbon” geometry observed in some cello- and xylogluco-oligosaccharides. RESULTS +4 21 aromatic platform site The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). RESULTS +33 37 W330 residue_name_number The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). RESULTS +39 43 W364 residue_name_number The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). RESULTS +49 53 Y363 residue_name_number The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). RESULTS +65 73 glucosyl chemical The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). RESULTS +100 106 longer protein_state The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). RESULTS +107 115 platform site The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). RESULTS +119 125 SGBP-A protein The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). RESULTS +146 154 glucosyl chemical The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig. 5E). RESULTS +4 9 Y363A mutant The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table 3; see Fig. S6 in the supplemental material). RESULTS +10 30 site-directed mutant experimental_method The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table 3; see Fig. S6 in the supplemental material). RESULTS +34 40 SGBP-B protein The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table 3; see Fig. S6 in the supplemental material). RESULTS +76 78 Ka evidence The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table 3; see Fig. S6 in the supplemental material). RESULTS +83 86 XyG chemical The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table 3; see Fig. S6 in the supplemental material). RESULTS +98 103 W364A mutant The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table 3; see Fig. S6 in the supplemental material). RESULTS +104 110 mutant protein_state The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table 3; see Fig. S6 in the supplemental material). RESULTS +111 128 lacks XyG binding protein_state The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table 3; see Fig. S6 in the supplemental material). RESULTS +61 69 β-glucan chemical There are no additional contacts between the protein and the β-glucan backbone and surprisingly few interactions with the side-chain xylosyl residues, despite that fact that ITC data demonstrate that SGBP-B does not measurably bind the cellohexaose (Table 1). RESULTS +133 140 xylosyl chemical There are no additional contacts between the protein and the β-glucan backbone and surprisingly few interactions with the side-chain xylosyl residues, despite that fact that ITC data demonstrate that SGBP-B does not measurably bind the cellohexaose (Table 1). RESULTS +174 177 ITC experimental_method There are no additional contacts between the protein and the β-glucan backbone and surprisingly few interactions with the side-chain xylosyl residues, despite that fact that ITC data demonstrate that SGBP-B does not measurably bind the cellohexaose (Table 1). RESULTS +200 206 SGBP-B protein There are no additional contacts between the protein and the β-glucan backbone and surprisingly few interactions with the side-chain xylosyl residues, despite that fact that ITC data demonstrate that SGBP-B does not measurably bind the cellohexaose (Table 1). RESULTS +236 248 cellohexaose chemical There are no additional contacts between the protein and the β-glucan backbone and surprisingly few interactions with the side-chain xylosyl residues, despite that fact that ITC data demonstrate that SGBP-B does not measurably bind the cellohexaose (Table 1). RESULTS +0 4 F414 residue_name_number F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1. RESULTS +5 11 stacks bond_interaction F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1. RESULTS +21 28 xylosyl chemical F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1. RESULTS +40 44 Glc3 residue_name_number F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1. RESULTS +52 56 Q407 residue_name_number F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1. RESULTS +75 91 hydrogen bonding bond_interaction F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1. RESULTS +107 114 xylosyl chemical F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1. RESULTS +123 127 Xyl1 residue_name_number F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1. RESULTS +17 22 F414A mutant Surprisingly, an F414A mutant of SGBP-B displays only a mild 3-fold decrease in the Ka value for XyG, again suggesting that glycan recognition is primarily mediated via contact with the β-glucan backbone (Table 3; see Fig. S6). RESULTS +23 29 mutant protein_state Surprisingly, an F414A mutant of SGBP-B displays only a mild 3-fold decrease in the Ka value for XyG, again suggesting that glycan recognition is primarily mediated via contact with the β-glucan backbone (Table 3; see Fig. S6). RESULTS +33 39 SGBP-B protein Surprisingly, an F414A mutant of SGBP-B displays only a mild 3-fold decrease in the Ka value for XyG, again suggesting that glycan recognition is primarily mediated via contact with the β-glucan backbone (Table 3; see Fig. S6). RESULTS +84 86 Ka evidence Surprisingly, an F414A mutant of SGBP-B displays only a mild 3-fold decrease in the Ka value for XyG, again suggesting that glycan recognition is primarily mediated via contact with the β-glucan backbone (Table 3; see Fig. S6). RESULTS +97 100 XyG chemical Surprisingly, an F414A mutant of SGBP-B displays only a mild 3-fold decrease in the Ka value for XyG, again suggesting that glycan recognition is primarily mediated via contact with the β-glucan backbone (Table 3; see Fig. S6). RESULTS +124 130 glycan chemical Surprisingly, an F414A mutant of SGBP-B displays only a mild 3-fold decrease in the Ka value for XyG, again suggesting that glycan recognition is primarily mediated via contact with the β-glucan backbone (Table 3; see Fig. S6). RESULTS +11 19 residues structure_element Additional residues surrounding the binding site, including Y369 and E412, may contribute to the recognition of more highly decorated XyG, but precisely how this is mediated is presently unclear. RESULTS +36 48 binding site site Additional residues surrounding the binding site, including Y369 and E412, may contribute to the recognition of more highly decorated XyG, but precisely how this is mediated is presently unclear. RESULTS +60 64 Y369 residue_name_number Additional residues surrounding the binding site, including Y369 and E412, may contribute to the recognition of more highly decorated XyG, but precisely how this is mediated is presently unclear. RESULTS +69 73 E412 residue_name_number Additional residues surrounding the binding site, including Y369 and E412, may contribute to the recognition of more highly decorated XyG, but precisely how this is mediated is presently unclear. RESULTS +134 137 XyG chemical Additional residues surrounding the binding site, including Y369 and E412, may contribute to the recognition of more highly decorated XyG, but precisely how this is mediated is presently unclear. RESULTS +50 56 SGBP-B protein Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). RESULTS +57 67 xyloglucan chemical Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). RESULTS +84 90 solved experimental_method Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). RESULTS +95 112 crystal structure evidence Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). RESULTS +120 136 fused CD domains mutant Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). RESULTS +137 152 in complex with protein_state Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). RESULTS +153 158 XyGO2 chemical Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). RESULTS +168 173 Rwork evidence Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). RESULTS +183 188 Rfree evidence Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). RESULTS +207 217 230 to 489 residue_range Hoping to achieve a higher-resolution view of the SGBP-B–xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57 Å, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table 2). RESULTS +4 14 CD domains structure_element The CD domains of the truncated and full-length proteins superimpose with a 0.4-Å RMSD of the Cα backbone, with no differences in the position of any of the glycan-binding residues (see Fig. S7A in the supplemental material). RESULTS +22 31 truncated protein_state The CD domains of the truncated and full-length proteins superimpose with a 0.4-Å RMSD of the Cα backbone, with no differences in the position of any of the glycan-binding residues (see Fig. S7A in the supplemental material). RESULTS +36 47 full-length protein_state The CD domains of the truncated and full-length proteins superimpose with a 0.4-Å RMSD of the Cα backbone, with no differences in the position of any of the glycan-binding residues (see Fig. S7A in the supplemental material). RESULTS +57 68 superimpose experimental_method The CD domains of the truncated and full-length proteins superimpose with a 0.4-Å RMSD of the Cα backbone, with no differences in the position of any of the glycan-binding residues (see Fig. S7A in the supplemental material). RESULTS +82 86 RMSD evidence The CD domains of the truncated and full-length proteins superimpose with a 0.4-Å RMSD of the Cα backbone, with no differences in the position of any of the glycan-binding residues (see Fig. S7A in the supplemental material). RESULTS +157 180 glycan-binding residues site The CD domains of the truncated and full-length proteins superimpose with a 0.4-Å RMSD of the Cα backbone, with no differences in the position of any of the glycan-binding residues (see Fig. S7A in the supplemental material). RESULTS +6 13 density evidence While density is observed for XyGO2, the ligand could not be unambiguously modeled into this density to achieve a reasonable fit between the X-ray data and the known stereochemistry of the sugar (see Fig. S7B and C). RESULTS +30 35 XyGO2 chemical While density is observed for XyGO2, the ligand could not be unambiguously modeled into this density to achieve a reasonable fit between the X-ray data and the known stereochemistry of the sugar (see Fig. S7B and C). RESULTS +93 100 density evidence While density is observed for XyGO2, the ligand could not be unambiguously modeled into this density to achieve a reasonable fit between the X-ray data and the known stereochemistry of the sugar (see Fig. S7B and C). RESULTS +141 151 X-ray data evidence While density is observed for XyGO2, the ligand could not be unambiguously modeled into this density to achieve a reasonable fit between the X-ray data and the known stereochemistry of the sugar (see Fig. S7B and C). RESULTS +48 66 crystal structures evidence While this may occur for a number of reasons in crystal structures, it is likely that the poor ligand density even at higher resolution is due to movement or multiple orientations of the sugar averaged throughout the lattice. RESULTS +187 192 sugar chemical While this may occur for a number of reasons in crystal structures, it is likely that the poor ligand density even at higher resolution is due to movement or multiple orientations of the sugar averaged throughout the lattice. RESULTS +0 6 SGBP-A protein SGBP-A and SGBP-B have distinct, coordinated functions in vivo. RESULTS +11 17 SGBP-B protein SGBP-A and SGBP-B have distinct, coordinated functions in vivo. RESULTS +22 28 glycan chemical The similarity of the glycan specificity of SGBP-A and SGBP-B presents an intriguing conundrum regarding their individual roles in XyG utilization by B. ovatus. RESULTS +44 50 SGBP-A protein The similarity of the glycan specificity of SGBP-A and SGBP-B presents an intriguing conundrum regarding their individual roles in XyG utilization by B. ovatus. RESULTS +55 61 SGBP-B protein The similarity of the glycan specificity of SGBP-A and SGBP-B presents an intriguing conundrum regarding their individual roles in XyG utilization by B. ovatus. RESULTS +131 134 XyG chemical The similarity of the glycan specificity of SGBP-A and SGBP-B presents an intriguing conundrum regarding their individual roles in XyG utilization by B. ovatus. RESULTS +150 159 B. ovatus species The similarity of the glycan specificity of SGBP-A and SGBP-B presents an intriguing conundrum regarding their individual roles in XyG utilization by B. ovatus. RESULTS +32 38 SGBP-A protein To disentangle the functions of SGBP-A and SGBP-B in XyG recognition and uptake, we created individual in-frame deletion and complementation mutant strains of B. ovatus. RESULTS +43 49 SGBP-B protein To disentangle the functions of SGBP-A and SGBP-B in XyG recognition and uptake, we created individual in-frame deletion and complementation mutant strains of B. ovatus. RESULTS +53 56 XyG chemical To disentangle the functions of SGBP-A and SGBP-B in XyG recognition and uptake, we created individual in-frame deletion and complementation mutant strains of B. ovatus. RESULTS +103 147 in-frame deletion and complementation mutant experimental_method To disentangle the functions of SGBP-A and SGBP-B in XyG recognition and uptake, we created individual in-frame deletion and complementation mutant strains of B. ovatus. RESULTS +159 168 B. ovatus species To disentangle the functions of SGBP-A and SGBP-B in XyG recognition and uptake, we created individual in-frame deletion and complementation mutant strains of B. ovatus. RESULTS +9 27 growth experiments experimental_method In these growth experiments, overnight cultures of strains grown on minimal medium plus glucose were back-diluted 1:100-fold into minimal medium containing 5 mg/ml of the reported carbohydrate. RESULTS +88 95 glucose chemical In these growth experiments, overnight cultures of strains grown on minimal medium plus glucose were back-diluted 1:100-fold into minimal medium containing 5 mg/ml of the reported carbohydrate. RESULTS +180 192 carbohydrate chemical In these growth experiments, overnight cultures of strains grown on minimal medium plus glucose were back-diluted 1:100-fold into minimal medium containing 5 mg/ml of the reported carbohydrate. RESULTS +10 17 glucose chemical Growth on glucose displayed the shortest lag time for each strain, and so lag times were normalized for each carbohydrate by subtracting the lag time of that strain in glucose (Fig. 6; see Fig. S8 in the supplemental material). RESULTS +41 49 lag time evidence Growth on glucose displayed the shortest lag time for each strain, and so lag times were normalized for each carbohydrate by subtracting the lag time of that strain in glucose (Fig. 6; see Fig. S8 in the supplemental material). RESULTS +74 83 lag times evidence Growth on glucose displayed the shortest lag time for each strain, and so lag times were normalized for each carbohydrate by subtracting the lag time of that strain in glucose (Fig. 6; see Fig. S8 in the supplemental material). RESULTS +109 121 carbohydrate chemical Growth on glucose displayed the shortest lag time for each strain, and so lag times were normalized for each carbohydrate by subtracting the lag time of that strain in glucose (Fig. 6; see Fig. S8 in the supplemental material). RESULTS +141 149 lag time evidence Growth on glucose displayed the shortest lag time for each strain, and so lag times were normalized for each carbohydrate by subtracting the lag time of that strain in glucose (Fig. 6; see Fig. S8 in the supplemental material). RESULTS +168 175 glucose chemical Growth on glucose displayed the shortest lag time for each strain, and so lag times were normalized for each carbohydrate by subtracting the lag time of that strain in glucose (Fig. 6; see Fig. S8 in the supplemental material). RESULTS +29 34 XyGUL gene A strain in which the entire XyGUL is deleted displays a lag of 24.5 h during growth on glucose compared to the isogenic parental wild-type (WT) Δtdk strain, for which exponential growth lags for 19.8 h (see Fig. S8D). RESULTS +38 45 deleted experimental_method A strain in which the entire XyGUL is deleted displays a lag of 24.5 h during growth on glucose compared to the isogenic parental wild-type (WT) Δtdk strain, for which exponential growth lags for 19.8 h (see Fig. S8D). RESULTS +57 60 lag evidence A strain in which the entire XyGUL is deleted displays a lag of 24.5 h during growth on glucose compared to the isogenic parental wild-type (WT) Δtdk strain, for which exponential growth lags for 19.8 h (see Fig. S8D). RESULTS +88 95 glucose chemical A strain in which the entire XyGUL is deleted displays a lag of 24.5 h during growth on glucose compared to the isogenic parental wild-type (WT) Δtdk strain, for which exponential growth lags for 19.8 h (see Fig. S8D). RESULTS +130 139 wild-type protein_state A strain in which the entire XyGUL is deleted displays a lag of 24.5 h during growth on glucose compared to the isogenic parental wild-type (WT) Δtdk strain, for which exponential growth lags for 19.8 h (see Fig. S8D). RESULTS +141 143 WT protein_state A strain in which the entire XyGUL is deleted displays a lag of 24.5 h during growth on glucose compared to the isogenic parental wild-type (WT) Δtdk strain, for which exponential growth lags for 19.8 h (see Fig. S8D). RESULTS +145 149 Δtdk mutant A strain in which the entire XyGUL is deleted displays a lag of 24.5 h during growth on glucose compared to the isogenic parental wild-type (WT) Δtdk strain, for which exponential growth lags for 19.8 h (see Fig. S8D). RESULTS +187 191 lags evidence A strain in which the entire XyGUL is deleted displays a lag of 24.5 h during growth on glucose compared to the isogenic parental wild-type (WT) Δtdk strain, for which exponential growth lags for 19.8 h (see Fig. S8D). RESULTS +151 158 glucose chemical It is unknown whether this is because cultures were not normalized by the starting optical density (OD) or viable cells or reflects a minor defect for glucose utilization. RESULTS +91 98 glucose chemical The former seems more likely as the growth rates are nearly identical for these strains on glucose and xylose. RESULTS +103 109 xylose chemical The former seems more likely as the growth rates are nearly identical for these strains on glucose and xylose. RESULTS +4 10 ΔXyGUL mutant The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. RESULTS +15 17 WT protein_state The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. RESULTS +18 22 Δtdk mutant The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. RESULTS +50 59 lag times evidence The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. RESULTS +63 69 xylose chemical The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. RESULTS +183 191 lag time evidence The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. RESULTS +195 201 xylose chemical The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. RESULTS +211 213 WT protein_state The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. RESULTS +214 218 Δtdk mutant The ΔXyGUL and WT Δtdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT Δtdk strain. RESULTS +0 15 Complementation experimental_method Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). RESULTS +23 30 ΔSGBP-A mutant Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). RESULTS +39 46 ΔSGBP-A mutant Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). RESULTS +48 54 SGBP-A protein Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). RESULTS +75 84 wild-type protein_state Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). RESULTS +94 104 xyloglucan chemical Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). RESULTS +109 114 XyGO1 chemical Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). RESULTS +187 189 WT protein_state Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). RESULTS +190 194 Δtdk mutant Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). RESULTS +205 210 XyGO2 chemical Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). RESULTS +250 256 SGBP-B protein Complementation of the ΔSGBP-A strain (ΔSGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT Δtdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig. S8C and F). RESULTS +35 40 XyGO2 chemical The reason for this observation on XyGO2 is unclear, as the ΔSGBP-B mutant does not have a significantly different growth rate from the WT on XyGO2. RESULTS +60 67 ΔSGBP-B mutant The reason for this observation on XyGO2 is unclear, as the ΔSGBP-B mutant does not have a significantly different growth rate from the WT on XyGO2. RESULTS +68 74 mutant protein_state The reason for this observation on XyGO2 is unclear, as the ΔSGBP-B mutant does not have a significantly different growth rate from the WT on XyGO2. RESULTS +136 138 WT protein_state The reason for this observation on XyGO2 is unclear, as the ΔSGBP-B mutant does not have a significantly different growth rate from the WT on XyGO2. RESULTS +142 147 XyGO2 chemical The reason for this observation on XyGO2 is unclear, as the ΔSGBP-B mutant does not have a significantly different growth rate from the WT on XyGO2. RESULTS +17 22 XyGUL gene Growth of select XyGUL mutants on xyloglucan and oligosaccharides. FIG +34 44 xyloglucan chemical Growth of select XyGUL mutants on xyloglucan and oligosaccharides. FIG +49 65 oligosaccharides chemical Growth of select XyGUL mutants on xyloglucan and oligosaccharides. FIG +0 9 B. ovatus species B. ovatus mutants were created in a thymidine kinase deletion (Δtdk) mutant as described previously. FIG +36 61 thymidine kinase deletion mutant B. ovatus mutants were created in a thymidine kinase deletion (Δtdk) mutant as described previously. FIG +63 67 Δtdk mutant B. ovatus mutants were created in a thymidine kinase deletion (Δtdk) mutant as described previously. FIG +0 7 SGBP-A* mutant SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649. FIG +20 32 Bacova_02651 gene SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649. FIG +34 38 W82A mutant SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649. FIG +39 44 W283A mutant SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649. FIG +45 50 W306A mutant SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649. FIG +68 71 GH9 protein SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649. FIG +80 92 Bacova_02649 gene SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649. FIG +63 66 XyG chemical Growth was measured over time in minimal medium containing (A) XyG, (B) XyGO2, (C) XyGO1, (D) glucose, and (E) xylose. FIG +72 77 XyGO2 chemical Growth was measured over time in minimal medium containing (A) XyG, (B) XyGO2, (C) XyGO1, (D) glucose, and (E) xylose. FIG +83 88 XyGO1 chemical Growth was measured over time in minimal medium containing (A) XyG, (B) XyGO2, (C) XyGO1, (D) glucose, and (E) xylose. FIG +94 101 glucose chemical Growth was measured over time in minimal medium containing (A) XyG, (B) XyGO2, (C) XyGO1, (D) glucose, and (E) xylose. FIG +111 117 xylose chemical Growth was measured over time in minimal medium containing (A) XyG, (B) XyGO2, (C) XyGO1, (D) glucose, and (E) xylose. FIG +115 123 lag time evidence In panel F, the growth rate of each strain on the five carbon sources is displayed, and in panel G, the normalized lag time of each culture, relative to its growth on glucose, is displayed. FIG +167 174 glucose chemical In panel F, the growth rate of each strain on the five carbon sources is displayed, and in panel G, the normalized lag time of each culture, relative to its growth on glucose, is displayed. FIG +79 81 WT protein_state Solid bars indicate conditions that are not statistically significant from the WT Δtdk cultures grown on the indicated carbohydrate, while open bars indicate a P value of <0.005 compared to the WT Δtdk strain. FIG +82 86 Δtdk mutant Solid bars indicate conditions that are not statistically significant from the WT Δtdk cultures grown on the indicated carbohydrate, while open bars indicate a P value of <0.005 compared to the WT Δtdk strain. FIG +119 131 carbohydrate chemical Solid bars indicate conditions that are not statistically significant from the WT Δtdk cultures grown on the indicated carbohydrate, while open bars indicate a P value of <0.005 compared to the WT Δtdk strain. FIG +194 196 WT protein_state Solid bars indicate conditions that are not statistically significant from the WT Δtdk cultures grown on the indicated carbohydrate, while open bars indicate a P value of <0.005 compared to the WT Δtdk strain. FIG +197 201 Δtdk mutant Solid bars indicate conditions that are not statistically significant from the WT Δtdk cultures grown on the indicated carbohydrate, while open bars indicate a P value of <0.005 compared to the WT Δtdk strain. FIG +184 191 ΔSGBP-A mutant Conditions denoted by the same letter (b, c, or d) are not statistically significant from each other but are significantly different from the condition labeled “a.” Complementation of ΔSGBP-A and ΔSBGP-B was performed by allelic exchange of the wild-type genes back into the genome for expression via the native promoter: these growth curves, quantified rates and lag times are displayed in Fig. S8 in the supplemental material. FIG +196 203 ΔSBGP-B mutant Conditions denoted by the same letter (b, c, or d) are not statistically significant from each other but are significantly different from the condition labeled “a.” Complementation of ΔSGBP-A and ΔSBGP-B was performed by allelic exchange of the wild-type genes back into the genome for expression via the native promoter: these growth curves, quantified rates and lag times are displayed in Fig. S8 in the supplemental material. FIG +245 254 wild-type protein_state Conditions denoted by the same letter (b, c, or d) are not statistically significant from each other but are significantly different from the condition labeled “a.” Complementation of ΔSGBP-A and ΔSBGP-B was performed by allelic exchange of the wild-type genes back into the genome for expression via the native promoter: these growth curves, quantified rates and lag times are displayed in Fig. S8 in the supplemental material. FIG +364 373 lag times evidence Conditions denoted by the same letter (b, c, or d) are not statistically significant from each other but are significantly different from the condition labeled “a.” Complementation of ΔSGBP-A and ΔSBGP-B was performed by allelic exchange of the wild-type genes back into the genome for expression via the native promoter: these growth curves, quantified rates and lag times are displayed in Fig. S8 in the supplemental material. FIG +4 11 ΔSGBP-A mutant The ΔSGBP-A (ΔBacova_02651) strain (cf. RESULTS +13 26 ΔBacova_02651 mutant The ΔSGBP-A (ΔBacova_02651) strain (cf. RESULTS +47 50 XyG chemical Fig. 1B) was completely incapable of growth on XyG, XyGO1, and XyGO2, indicating that SGBP-A is essential for XyG utilization (Fig. 6). RESULTS +52 57 XyGO1 chemical Fig. 1B) was completely incapable of growth on XyG, XyGO1, and XyGO2, indicating that SGBP-A is essential for XyG utilization (Fig. 6). RESULTS +63 68 XyGO2 chemical Fig. 1B) was completely incapable of growth on XyG, XyGO1, and XyGO2, indicating that SGBP-A is essential for XyG utilization (Fig. 6). RESULTS +86 92 SGBP-A protein Fig. 1B) was completely incapable of growth on XyG, XyGO1, and XyGO2, indicating that SGBP-A is essential for XyG utilization (Fig. 6). RESULTS +110 113 XyG chemical Fig. 1B) was completely incapable of growth on XyG, XyGO1, and XyGO2, indicating that SGBP-A is essential for XyG utilization (Fig. 6). RESULTS +56 59 Sus complex_assembly This result mirrors our previous data for the canonical Sus of B. thetaiotaomicron, which revealed that a homologous ΔsusD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins. RESULTS +63 82 B. thetaiotaomicron species This result mirrors our previous data for the canonical Sus of B. thetaiotaomicron, which revealed that a homologous ΔsusD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins. RESULTS +117 122 ΔsusD mutant This result mirrors our previous data for the canonical Sus of B. thetaiotaomicron, which revealed that a homologous ΔsusD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins. RESULTS +123 129 mutant protein_state This result mirrors our previous data for the canonical Sus of B. thetaiotaomicron, which revealed that a homologous ΔsusD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins. RESULTS +151 157 starch chemical This result mirrors our previous data for the canonical Sus of B. thetaiotaomicron, which revealed that a homologous ΔsusD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins. RESULTS +161 183 malto-oligosaccharides chemical This result mirrors our previous data for the canonical Sus of B. thetaiotaomicron, which revealed that a homologous ΔsusD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins. RESULTS +237 240 PUL gene This result mirrors our previous data for the canonical Sus of B. thetaiotaomicron, which revealed that a homologous ΔsusD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins. RESULTS +98 102 SusD protein More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +104 119 complementation experimental_method More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +123 128 ΔsusD mutant More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +134 139 SusD* mutant More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +143 170 triple site-directed mutant protein_state More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +172 176 W96A mutant More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +177 182 W320A mutant More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +183 188 Y296A mutant More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +195 217 ablates glycan binding protein_state More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +228 247 B. thetaiotaomicron species More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +258 280 malto-oligosaccharides chemical More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +285 291 starch chemical More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +297 300 sus gene More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +329 336 maltose chemical More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of ΔsusD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B. thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition. RESULTS +27 33 SGBP-A protein Similarly, the function of SGBP-A extends beyond glycan binding. RESULTS +49 55 glycan chemical Similarly, the function of SGBP-A extends beyond glycan binding. RESULTS +0 15 Complementation experimental_method Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +19 26 ΔSGBP-A mutant Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +36 43 SGBP-A* mutant Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +45 49 W82A mutant Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +50 55 W283A mutant Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +56 61 W306A mutant Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +83 91 not bind protein_state Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +92 95 XyG chemical Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +116 119 XyG chemical Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +124 129 XyGOs chemical Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +139 146 ΔSGBP-A mutant Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +148 155 SGBP-A* mutant Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +202 204 WT protein_state Complementation of ΔSGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig. 6; ΔSGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT. RESULTS +38 50 carbohydrate chemical In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5 g/liter maltose was required to restore growth of the ΔsusD::SusD* strain on starch, which nonetheless occurred following an extended lag phase. RESULTS +62 66 SusD protein In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5 g/liter maltose was required to restore growth of the ΔsusD::SusD* strain on starch, which nonetheless occurred following an extended lag phase. RESULTS +228 235 maltose chemical In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5 g/liter maltose was required to restore growth of the ΔsusD::SusD* strain on starch, which nonetheless occurred following an extended lag phase. RESULTS +274 279 ΔsusD mutant In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5 g/liter maltose was required to restore growth of the ΔsusD::SusD* strain on starch, which nonetheless occurred following an extended lag phase. RESULTS +281 286 SusD* mutant In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5 g/liter maltose was required to restore growth of the ΔsusD::SusD* strain on starch, which nonetheless occurred following an extended lag phase. RESULTS +297 303 starch chemical In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5 g/liter maltose was required to restore growth of the ΔsusD::SusD* strain on starch, which nonetheless occurred following an extended lag phase. RESULTS +354 363 lag phase evidence In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5 g/liter maltose was required to restore growth of the ΔsusD::SusD* strain on starch, which nonetheless occurred following an extended lag phase. RESULTS +17 24 ΔSGBP-A mutant In contrast, the ΔSGBP-A::SGBP-A* strain does not display an extended lag time on any of the xyloglucan substrates compared to the WT (Fig. 6). RESULTS +26 33 SGBP-A* mutant In contrast, the ΔSGBP-A::SGBP-A* strain does not display an extended lag time on any of the xyloglucan substrates compared to the WT (Fig. 6). RESULTS +70 78 lag time evidence In contrast, the ΔSGBP-A::SGBP-A* strain does not display an extended lag time on any of the xyloglucan substrates compared to the WT (Fig. 6). RESULTS +93 103 xyloglucan chemical In contrast, the ΔSGBP-A::SGBP-A* strain does not display an extended lag time on any of the xyloglucan substrates compared to the WT (Fig. 6). RESULTS +131 133 WT protein_state In contrast, the ΔSGBP-A::SGBP-A* strain does not display an extended lag time on any of the xyloglucan substrates compared to the WT (Fig. 6). RESULTS +13 19 glycan chemical The specific glycan signal that upregulates BoXyGUL is currently unknown. RESULTS +44 51 BoXyGUL gene The specific glycan signal that upregulates BoXyGUL is currently unknown. RESULTS +68 74 glycan chemical From our present data, we cannot eliminate the possibility that the glycan binding by SGBP-A enhances transcriptional activation of the XyGUL. RESULTS +86 92 SGBP-A protein From our present data, we cannot eliminate the possibility that the glycan binding by SGBP-A enhances transcriptional activation of the XyGUL. RESULTS +136 141 XyGUL gene From our present data, we cannot eliminate the possibility that the glycan binding by SGBP-A enhances transcriptional activation of the XyGUL. RESULTS +49 55 SGBP-A protein However, the modest rate defect displayed by the SGBP-A::SGBP-A* strain suggests that recognition of XyG and product import is somewhat less efficient in these cells. RESULTS +57 64 SGBP-A* mutant However, the modest rate defect displayed by the SGBP-A::SGBP-A* strain suggests that recognition of XyG and product import is somewhat less efficient in these cells. RESULTS +101 104 XyG chemical However, the modest rate defect displayed by the SGBP-A::SGBP-A* strain suggests that recognition of XyG and product import is somewhat less efficient in these cells. RESULTS +18 25 ΔSGBP-B mutant Intriguingly, the ΔSGBP-B strain (ΔBacova_02650) (cf. RESULTS +34 47 ΔBacova_02650 mutant Intriguingly, the ΔSGBP-B strain (ΔBacova_02650) (cf. RESULTS +49 52 XyG chemical Fig. 1B) exhibited a minor growth defect on both XyG and XyGO2, with rates 84.6% and 93.9% that of the WT Δtdk strain. RESULTS +57 62 XyGO2 chemical Fig. 1B) exhibited a minor growth defect on both XyG and XyGO2, with rates 84.6% and 93.9% that of the WT Δtdk strain. RESULTS +103 105 WT protein_state Fig. 1B) exhibited a minor growth defect on both XyG and XyGO2, with rates 84.6% and 93.9% that of the WT Δtdk strain. RESULTS +106 110 Δtdk mutant Fig. 1B) exhibited a minor growth defect on both XyG and XyGO2, with rates 84.6% and 93.9% that of the WT Δtdk strain. RESULTS +23 30 ΔSGBP-B mutant However, growth of the ΔSGBP-B strain on XyGO1 was 54.2% the rate of the parental strain, despite the fact that SGBP-B binds this substrate ca. RESULTS +41 46 XyGO1 chemical However, growth of the ΔSGBP-B strain on XyGO1 was 54.2% the rate of the parental strain, despite the fact that SGBP-B binds this substrate ca. RESULTS +112 118 SGBP-B protein However, growth of the ΔSGBP-B strain on XyGO1 was 54.2% the rate of the parental strain, despite the fact that SGBP-B binds this substrate ca. RESULTS +25 30 XyGO2 chemical 10-fold more weakly than XyGO2 and XyG (Fig. 6; Table 1). RESULTS +35 38 XyG chemical 10-fold more weakly than XyGO2 and XyG (Fig. 6; Table 1). RESULTS +31 37 SGBP-A protein As such, the data suggest that SGBP-A can compensate for the loss of function of SGBP-B on longer oligo- and polysaccharides, while SGBP-B may adapt the cell to recognize smaller oligosaccharides efficiently. RESULTS +81 87 SGBP-B protein As such, the data suggest that SGBP-A can compensate for the loss of function of SGBP-B on longer oligo- and polysaccharides, while SGBP-B may adapt the cell to recognize smaller oligosaccharides efficiently. RESULTS +98 124 oligo- and polysaccharides chemical As such, the data suggest that SGBP-A can compensate for the loss of function of SGBP-B on longer oligo- and polysaccharides, while SGBP-B may adapt the cell to recognize smaller oligosaccharides efficiently. RESULTS +132 138 SGBP-B protein As such, the data suggest that SGBP-A can compensate for the loss of function of SGBP-B on longer oligo- and polysaccharides, while SGBP-B may adapt the cell to recognize smaller oligosaccharides efficiently. RESULTS +179 195 oligosaccharides chemical As such, the data suggest that SGBP-A can compensate for the loss of function of SGBP-B on longer oligo- and polysaccharides, while SGBP-B may adapt the cell to recognize smaller oligosaccharides efficiently. RESULTS +10 23 double mutant protein_state Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. RESULTS +41 49 crippled protein_state Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. RESULTS +50 56 SGBP-A protein Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. RESULTS +63 74 deletion of experimental_method Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. RESULTS +75 81 SGBP-B protein Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. RESULTS +83 90 ΔSGBP-A mutant Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. RESULTS +92 99 SGBP-A* mutant Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. RESULTS +100 107 ΔSGBP-B mutant Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. RESULTS +131 139 lag time evidence Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. RESULTS +148 151 XyG chemical Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. RESULTS +156 161 XyGO2 chemical Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. RESULTS +174 179 XyGO1 chemical Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (ΔSGBP-A::SGBP-A*/ΔSGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1. RESULTS +39 45 SGBP-A protein Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B. ovatus to scavenge smaller XyGOs liberated by other gut commensals. RESULTS +50 56 SGBP-B protein Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B. ovatus to scavenge smaller XyGOs liberated by other gut commensals. RESULTS +110 113 XyG chemical Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B. ovatus to scavenge smaller XyGOs liberated by other gut commensals. RESULTS +114 128 polysaccharide chemical Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B. ovatus to scavenge smaller XyGOs liberated by other gut commensals. RESULTS +136 142 SGBP-B protein Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B. ovatus to scavenge smaller XyGOs liberated by other gut commensals. RESULTS +153 162 B. ovatus species Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B. ovatus to scavenge smaller XyGOs liberated by other gut commensals. RESULTS +183 188 XyGOs chemical Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B. ovatus to scavenge smaller XyGOs liberated by other gut commensals. RESULTS +24 30 SGBP-B protein This additional role of SGBP-B is especially notable in the context of studies on BtSusE and BtSusF (positioned similarly in the archetypal Sus locus) (Fig. 1B), for which growth defects on starch or malto-oligosaccharides have never been observed. RESULTS +82 88 BtSusE protein This additional role of SGBP-B is especially notable in the context of studies on BtSusE and BtSusF (positioned similarly in the archetypal Sus locus) (Fig. 1B), for which growth defects on starch or malto-oligosaccharides have never been observed. RESULTS +93 99 BtSusF protein This additional role of SGBP-B is especially notable in the context of studies on BtSusE and BtSusF (positioned similarly in the archetypal Sus locus) (Fig. 1B), for which growth defects on starch or malto-oligosaccharides have never been observed. RESULTS +140 149 Sus locus gene This additional role of SGBP-B is especially notable in the context of studies on BtSusE and BtSusF (positioned similarly in the archetypal Sus locus) (Fig. 1B), for which growth defects on starch or malto-oligosaccharides have never been observed. RESULTS +190 196 starch chemical This additional role of SGBP-B is especially notable in the context of studies on BtSusE and BtSusF (positioned similarly in the archetypal Sus locus) (Fig. 1B), for which growth defects on starch or malto-oligosaccharides have never been observed. RESULTS +200 222 malto-oligosaccharides chemical This additional role of SGBP-B is especially notable in the context of studies on BtSusE and BtSusF (positioned similarly in the archetypal Sus locus) (Fig. 1B), for which growth defects on starch or malto-oligosaccharides have never been observed. RESULTS +7 13 SGBP-A protein Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface. RESULTS +18 24 SGBP-B protein Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface. RESULTS +49 69 catalytically feeble protein_state Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface. RESULTS +70 88 endo-xyloglucanase protein_type Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface. RESULTS +89 92 GH9 protein Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface. RESULTS +144 147 GH5 protein Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface. RESULTS +175 181 glycan chemical Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface. RESULTS +9 48 combined deletion of the genes encoding experimental_method However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig. 6; ΔSGBP-B/ΔGH9). RESULTS +49 52 GH9 protein However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig. 6; ΔSGBP-B/ΔGH9). RESULTS +65 77 Bacova_02649 gene However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig. 6; ΔSGBP-B/ΔGH9). RESULTS +83 89 SGBP-B protein However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig. 6; ΔSGBP-B/ΔGH9). RESULTS +131 136 XyGO1 chemical However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig. 6; ΔSGBP-B/ΔGH9). RESULTS +146 153 ΔSGBP-B mutant However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig. 6; ΔSGBP-B/ΔGH9). RESULTS +154 158 ΔGH9 mutant However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig. 6; ΔSGBP-B/ΔGH9). RESULTS +17 23 SGBP-B protein The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. RESULTS +43 50 SGBP-A* mutant The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. RESULTS +66 73 ΔSGBP-A mutant The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. RESULTS +75 82 SGBP-A* mutant The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. RESULTS +84 91 ΔSGBP-B mutant The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. RESULTS +92 98 mutant protein_state The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. RESULTS +120 123 lag evidence The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. RESULTS +141 144 XyG chemical The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. RESULTS +149 175 xylogluco-oligosaccharides chemical The necessity of SGBP-B is elevated in the SGBP-A* strain, as the ΔSGBP-A::SGBP-A*/ ΔSGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle. RESULTS +28 31 lag evidence The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B. thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs. RESULTS +91 95 SusD protein The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B. thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs. RESULTS +99 120 malto-oligosaccharide chemical The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B. thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs. RESULTS +132 151 B. thetaiotaomicron species The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B. thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs. RESULTS +167 170 lag evidence The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B. thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs. RESULTS +224 234 xyloglucan chemical The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B. thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs. RESULTS +272 278 glycan chemical The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B. thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs. RESULTS +300 305 SGBPs protein_type The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B. thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs. RESULTS +36 45 B. ovatus species Our previous work demonstrates that B. ovatus cells grown in minimal medium plus glucose express low levels of the XyGUL transcript. RESULTS +81 88 glucose chemical Our previous work demonstrates that B. ovatus cells grown in minimal medium plus glucose express low levels of the XyGUL transcript. RESULTS +115 120 XyGUL gene Our previous work demonstrates that B. ovatus cells grown in minimal medium plus glucose express low levels of the XyGUL transcript. RESULTS +74 81 glucose chemical Thus, in our experiments, we presume that each strain, initially grown in glucose, expresses low levels of the XyGUL transcript and thus low levels of the XyGUL-encoded surface proteins, including the vanguard GH5. RESULTS +111 116 XyGUL gene Thus, in our experiments, we presume that each strain, initially grown in glucose, expresses low levels of the XyGUL transcript and thus low levels of the XyGUL-encoded surface proteins, including the vanguard GH5. RESULTS +155 160 XyGUL gene Thus, in our experiments, we presume that each strain, initially grown in glucose, expresses low levels of the XyGUL transcript and thus low levels of the XyGUL-encoded surface proteins, including the vanguard GH5. RESULTS +210 213 GH5 protein Thus, in our experiments, we presume that each strain, initially grown in glucose, expresses low levels of the XyGUL transcript and thus low levels of the XyGUL-encoded surface proteins, including the vanguard GH5. RESULTS +19 25 glycan chemical Presumably without glycan binding by the SGBPs, the GH5 protein cannot efficiently process xyloglucan, and/or the lack of SGBP function prevents efficient capture and import of the processed oligosaccharides. RESULTS +41 46 SGBPs protein_type Presumably without glycan binding by the SGBPs, the GH5 protein cannot efficiently process xyloglucan, and/or the lack of SGBP function prevents efficient capture and import of the processed oligosaccharides. RESULTS +52 55 GH5 protein Presumably without glycan binding by the SGBPs, the GH5 protein cannot efficiently process xyloglucan, and/or the lack of SGBP function prevents efficient capture and import of the processed oligosaccharides. RESULTS +91 101 xyloglucan chemical Presumably without glycan binding by the SGBPs, the GH5 protein cannot efficiently process xyloglucan, and/or the lack of SGBP function prevents efficient capture and import of the processed oligosaccharides. RESULTS +122 126 SGBP protein_type Presumably without glycan binding by the SGBPs, the GH5 protein cannot efficiently process xyloglucan, and/or the lack of SGBP function prevents efficient capture and import of the processed oligosaccharides. RESULTS +191 207 oligosaccharides chemical Presumably without glycan binding by the SGBPs, the GH5 protein cannot efficiently process xyloglucan, and/or the lack of SGBP function prevents efficient capture and import of the processed oligosaccharides. RESULTS +54 60 glycan chemical It may then be that only after a sufficient amount of glycan is processed and imported by the cell is XyGUL upregulated and exponential growth on the glycan can begin. RESULTS +102 107 XyGUL gene It may then be that only after a sufficient amount of glycan is processed and imported by the cell is XyGUL upregulated and exponential growth on the glycan can begin. RESULTS +150 156 glycan chemical It may then be that only after a sufficient amount of glycan is processed and imported by the cell is XyGUL upregulated and exponential growth on the glycan can begin. RESULTS +68 74 SGBP-A protein We hypothesize that during exponential growth the essential role of SGBP-A extends beyond glycan recognition, perhaps due to a critical interaction with the TBDT. RESULTS +90 96 glycan chemical We hypothesize that during exponential growth the essential role of SGBP-A extends beyond glycan recognition, perhaps due to a critical interaction with the TBDT. RESULTS +157 161 TBDT protein_type We hypothesize that during exponential growth the essential role of SGBP-A extends beyond glycan recognition, perhaps due to a critical interaction with the TBDT. RESULTS +7 12 BtSus gene In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. RESULTS +14 18 SusD protein In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. RESULTS +27 31 TBDT protein_type In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. RESULTS +32 36 SusC protein In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. RESULTS +103 109 glycan chemical In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. RESULTS +150 155 ΔsusD mutant In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. RESULTS +156 162 mutant protein_state In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. RESULTS +178 184 starch chemical In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. RESULTS +192 197 ΔsusD mutant In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. RESULTS +199 204 SusD* mutant In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. RESULTS +238 263 transcriptional activator protein_type In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. RESULTS +271 281 sus operon gene In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a ΔsusD mutant cannot grow on starch, but a ΔsusD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied. RESULTS +77 83 SGBP-A protein Likewise, such cognate interactions between homologous protein pairs such as SGBP-A and its TBDT may underlie our observation that a ΔSGBP-A mutant cannot grow on xyloglucan. RESULTS +92 96 TBDT protein_type Likewise, such cognate interactions between homologous protein pairs such as SGBP-A and its TBDT may underlie our observation that a ΔSGBP-A mutant cannot grow on xyloglucan. RESULTS +133 140 ΔSGBP-A mutant Likewise, such cognate interactions between homologous protein pairs such as SGBP-A and its TBDT may underlie our observation that a ΔSGBP-A mutant cannot grow on xyloglucan. RESULTS +141 147 mutant protein_state Likewise, such cognate interactions between homologous protein pairs such as SGBP-A and its TBDT may underlie our observation that a ΔSGBP-A mutant cannot grow on xyloglucan. RESULTS +163 173 xyloglucan chemical Likewise, such cognate interactions between homologous protein pairs such as SGBP-A and its TBDT may underlie our observation that a ΔSGBP-A mutant cannot grow on xyloglucan. RESULTS +20 23 Sus complex_assembly However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides. RESULTS +34 48 elimination of experimental_method However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides. RESULTS +49 53 SusE protein However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides. RESULTS +58 62 SusF protein However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides. RESULTS +89 95 starch chemical However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides. RESULTS +97 103 SGBP-B protein However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides. RESULTS +156 182 xylogluco-oligosaccharides chemical However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides. RESULTS +27 41 microorganisms taxonomy_domain The ability of gut-adapted microorganisms to thrive in the gastrointestinal tract is critically dependent upon their ability to efficiently recognize, cleave, and import glycans. RESULTS +170 177 glycans chemical The ability of gut-adapted microorganisms to thrive in the gastrointestinal tract is critically dependent upon their ability to efficiently recognize, cleave, and import glycans. RESULTS +4 9 human species The human gut, in particular, is a densely packed ecosystem with hundreds of species, in which there is potential for both competition and synergy in the utilization of different substrates. RESULTS +32 45 Bacteroidetes taxonomy_domain Recent work has elucidated that Bacteroidetes cross-feed during growth on many glycans; the glycoside hydrolases expressed by one species liberate oligosaccharides for consumption by other members of the community. RESULTS +79 86 glycans chemical Recent work has elucidated that Bacteroidetes cross-feed during growth on many glycans; the glycoside hydrolases expressed by one species liberate oligosaccharides for consumption by other members of the community. RESULTS +92 112 glycoside hydrolases protein_type Recent work has elucidated that Bacteroidetes cross-feed during growth on many glycans; the glycoside hydrolases expressed by one species liberate oligosaccharides for consumption by other members of the community. RESULTS +147 163 oligosaccharides chemical Recent work has elucidated that Bacteroidetes cross-feed during growth on many glycans; the glycoside hydrolases expressed by one species liberate oligosaccharides for consumption by other members of the community. RESULTS +20 26 glycan chemical Thus, understanding glycan capture at the cell surface is fundamental to explaining, and eventually predicting, how the carbohydrate content of the diet shapes the gut community structure as well as its causative health effects. RESULTS +30 61 surface glycan binding proteins protein_type Here, we demonstrate that the surface glycan binding proteins encoded within the BoXyGUL play unique and essential roles in the acquisition of the ubiquitous and abundant vegetable polysaccharide xyloglucan. RESULTS +81 88 BoXyGUL gene Here, we demonstrate that the surface glycan binding proteins encoded within the BoXyGUL play unique and essential roles in the acquisition of the ubiquitous and abundant vegetable polysaccharide xyloglucan. RESULTS +171 180 vegetable taxonomy_domain Here, we demonstrate that the surface glycan binding proteins encoded within the BoXyGUL play unique and essential roles in the acquisition of the ubiquitous and abundant vegetable polysaccharide xyloglucan. RESULTS +181 195 polysaccharide chemical Here, we demonstrate that the surface glycan binding proteins encoded within the BoXyGUL play unique and essential roles in the acquisition of the ubiquitous and abundant vegetable polysaccharide xyloglucan. RESULTS +196 206 xyloglucan chemical Here, we demonstrate that the surface glycan binding proteins encoded within the BoXyGUL play unique and essential roles in the acquisition of the ubiquitous and abundant vegetable polysaccharide xyloglucan. RESULTS +71 76 SGBPs protein_type Yet, a number of questions remain regarding the molecular interplay of SGBPs with their cotranscribed cohort of glycoside hydrolases and TonB-dependent transporters. RESULTS +112 132 glycoside hydrolases protein_type Yet, a number of questions remain regarding the molecular interplay of SGBPs with their cotranscribed cohort of glycoside hydrolases and TonB-dependent transporters. RESULTS +137 164 TonB-dependent transporters protein_type Yet, a number of questions remain regarding the molecular interplay of SGBPs with their cotranscribed cohort of glycoside hydrolases and TonB-dependent transporters. RESULTS +38 44 glycan chemical A particularly understudied aspect of glycan utilization is the mechanism of import via TBDTs (SusC homologs) (Fig. 1), which are ubiquitous and defining components of all PUL. RESULTS +88 93 TBDTs protein_type A particularly understudied aspect of glycan utilization is the mechanism of import via TBDTs (SusC homologs) (Fig. 1), which are ubiquitous and defining components of all PUL. RESULTS +95 99 SusC protein A particularly understudied aspect of glycan utilization is the mechanism of import via TBDTs (SusC homologs) (Fig. 1), which are ubiquitous and defining components of all PUL. RESULTS +172 175 PUL gene A particularly understudied aspect of glycan utilization is the mechanism of import via TBDTs (SusC homologs) (Fig. 1), which are ubiquitous and defining components of all PUL. RESULTS +0 3 PUL gene PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP. RESULTS +12 17 TBDTs protein_type PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP. RESULTS +21 34 Bacteroidetes taxonomy_domain PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP. RESULTS +74 94 iron-targeting TBDTs protein_type PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP. RESULTS +105 119 Proteobacteria taxonomy_domain PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP. RESULTS +168 190 glycan-importing TBDTs protein_type PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP. RESULTS +211 215 SGBP protein_type PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP. RESULTS +33 39 BtSusC protein A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus. RESULTS +40 44 TBDT protein_type A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus. RESULTS +53 57 SusD protein A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus. RESULTS +58 62 SGBP protein_type A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus. RESULTS +169 175 glycan chemical A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus. RESULTS +187 190 PUL gene A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus. RESULTS +194 219 Capnocytophaga canimorsus species A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus. RESULTS +55 59 SusD protein Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig. 1C). RESULTS +68 74 SGBP-A protein Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig. 1C). RESULTS +91 94 XyG chemical Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig. 1C). RESULTS +149 152 XyG chemical Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig. 1C). RESULTS +193 199 glycan chemical Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig. 1C). RESULTS +219 234 SusC-like TBDTs protein_type Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig. 1C). RESULTS +243 258 SusD-like SGBPs protein_type Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig. 1C). RESULTS +300 306 glycan chemical Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig. 1C). RESULTS +120 124 TBDT protein_type It is yet presently unclear whether this interaction is static or dynamic and to what extent the association of cognate TBDT/SGBPs is dependent upon the structure of the carbohydrate to be imported. RESULTS +125 130 SGBPs protein_type It is yet presently unclear whether this interaction is static or dynamic and to what extent the association of cognate TBDT/SGBPs is dependent upon the structure of the carbohydrate to be imported. RESULTS +170 182 carbohydrate chemical It is yet presently unclear whether this interaction is static or dynamic and to what extent the association of cognate TBDT/SGBPs is dependent upon the structure of the carbohydrate to be imported. RESULTS +59 64 TBDTs protein_type On the other hand, there is clear evidence for independent TBDTs in Bacteroidetes that do not require SGBP association for activity. RESULTS +68 81 Bacteroidetes taxonomy_domain On the other hand, there is clear evidence for independent TBDTs in Bacteroidetes that do not require SGBP association for activity. RESULTS +102 106 SGBP protein_type On the other hand, there is clear evidence for independent TBDTs in Bacteroidetes that do not require SGBP association for activity. RESULTS +61 65 nanO gene For example, it was recently demonstrated that expression of nanO, which encodes a SusC-like TBDT as part of a sialic-acid-targeting PUL from B. fragilis, restored growth on this monosaccharide in a mutant strain of E. coli. RESULTS +83 97 SusC-like TBDT protein_type For example, it was recently demonstrated that expression of nanO, which encodes a SusC-like TBDT as part of a sialic-acid-targeting PUL from B. fragilis, restored growth on this monosaccharide in a mutant strain of E. coli. RESULTS +133 136 PUL gene For example, it was recently demonstrated that expression of nanO, which encodes a SusC-like TBDT as part of a sialic-acid-targeting PUL from B. fragilis, restored growth on this monosaccharide in a mutant strain of E. coli. RESULTS +142 153 B. fragilis species For example, it was recently demonstrated that expression of nanO, which encodes a SusC-like TBDT as part of a sialic-acid-targeting PUL from B. fragilis, restored growth on this monosaccharide in a mutant strain of E. coli. RESULTS +179 193 monosaccharide chemical For example, it was recently demonstrated that expression of nanO, which encodes a SusC-like TBDT as part of a sialic-acid-targeting PUL from B. fragilis, restored growth on this monosaccharide in a mutant strain of E. coli. RESULTS +216 223 E. coli species For example, it was recently demonstrated that expression of nanO, which encodes a SusC-like TBDT as part of a sialic-acid-targeting PUL from B. fragilis, restored growth on this monosaccharide in a mutant strain of E. coli. RESULTS +38 42 susD gene In this instance, coexpression of the susD-like gene nanU was not required, nor did the expression of the nanU gene enhance growth kinetics. RESULTS +53 57 nanU gene In this instance, coexpression of the susD-like gene nanU was not required, nor did the expression of the nanU gene enhance growth kinetics. RESULTS +106 110 nanU gene In this instance, coexpression of the susD-like gene nanU was not required, nor did the expression of the nanU gene enhance growth kinetics. RESULTS +27 33 BT1762 gene Similarly, the deletion of BT1762 encoding a fructan-targeting SusD-like protein in B. thetaiotaomicron did not result in a dramatic loss of growth on fructans. RESULTS +45 80 fructan-targeting SusD-like protein protein_type Similarly, the deletion of BT1762 encoding a fructan-targeting SusD-like protein in B. thetaiotaomicron did not result in a dramatic loss of growth on fructans. RESULTS +84 103 B. thetaiotaomicron species Similarly, the deletion of BT1762 encoding a fructan-targeting SusD-like protein in B. thetaiotaomicron did not result in a dramatic loss of growth on fructans. RESULTS +151 159 fructans chemical Similarly, the deletion of BT1762 encoding a fructan-targeting SusD-like protein in B. thetaiotaomicron did not result in a dramatic loss of growth on fructans. RESULTS +33 47 SusD-like SGBP protein_type Thus, the strict dependence on a SusD-like SGBP for glycan uptake in the Bacteroidetes may be variable and substrate dependent. RESULTS +52 58 glycan chemical Thus, the strict dependence on a SusD-like SGBP for glycan uptake in the Bacteroidetes may be variable and substrate dependent. RESULTS +73 86 Bacteroidetes taxonomy_domain Thus, the strict dependence on a SusD-like SGBP for glycan uptake in the Bacteroidetes may be variable and substrate dependent. RESULTS +53 58 TBDTs protein_type Furthermore, considering the broader distribution of TBDTs in PUL lacking SGBPs (sometimes known as carbohydrate utilization containing TBDT [CUT] loci; see reference and reviewed in reference) across bacterial phyla, it appears that the intimate biophysical association of these substrate-transport and -binding proteins is the result of specific evolution within the Bacteroidetes. RESULTS +62 65 PUL gene Furthermore, considering the broader distribution of TBDTs in PUL lacking SGBPs (sometimes known as carbohydrate utilization containing TBDT [CUT] loci; see reference and reviewed in reference) across bacterial phyla, it appears that the intimate biophysical association of these substrate-transport and -binding proteins is the result of specific evolution within the Bacteroidetes. RESULTS +74 79 SGBPs protein_type Furthermore, considering the broader distribution of TBDTs in PUL lacking SGBPs (sometimes known as carbohydrate utilization containing TBDT [CUT] loci; see reference and reviewed in reference) across bacterial phyla, it appears that the intimate biophysical association of these substrate-transport and -binding proteins is the result of specific evolution within the Bacteroidetes. RESULTS +100 151 carbohydrate utilization containing TBDT [CUT] loci gene Furthermore, considering the broader distribution of TBDTs in PUL lacking SGBPs (sometimes known as carbohydrate utilization containing TBDT [CUT] loci; see reference and reviewed in reference) across bacterial phyla, it appears that the intimate biophysical association of these substrate-transport and -binding proteins is the result of specific evolution within the Bacteroidetes. RESULTS +201 210 bacterial taxonomy_domain Furthermore, considering the broader distribution of TBDTs in PUL lacking SGBPs (sometimes known as carbohydrate utilization containing TBDT [CUT] loci; see reference and reviewed in reference) across bacterial phyla, it appears that the intimate biophysical association of these substrate-transport and -binding proteins is the result of specific evolution within the Bacteroidetes. RESULTS +369 382 Bacteroidetes taxonomy_domain Furthermore, considering the broader distribution of TBDTs in PUL lacking SGBPs (sometimes known as carbohydrate utilization containing TBDT [CUT] loci; see reference and reviewed in reference) across bacterial phyla, it appears that the intimate biophysical association of these substrate-transport and -binding proteins is the result of specific evolution within the Bacteroidetes. RESULTS +49 67 SusD-like proteins protein_type Equally intriguing is the observation that while SusD-like proteins such as SGBP-A share moderate primary and high tertiary structural conservation, the genes for the SGBPs encoded immediately downstream (Fig. 1B [sometimes referred to as “susE positioned”]) encode glycan-binding lipoproteins with little or no sequence or structural conservation, even among syntenic PUL that target the same polysaccharide. RESULTS +76 82 SGBP-A protein Equally intriguing is the observation that while SusD-like proteins such as SGBP-A share moderate primary and high tertiary structural conservation, the genes for the SGBPs encoded immediately downstream (Fig. 1B [sometimes referred to as “susE positioned”]) encode glycan-binding lipoproteins with little or no sequence or structural conservation, even among syntenic PUL that target the same polysaccharide. RESULTS +167 172 SGBPs protein_type Equally intriguing is the observation that while SusD-like proteins such as SGBP-A share moderate primary and high tertiary structural conservation, the genes for the SGBPs encoded immediately downstream (Fig. 1B [sometimes referred to as “susE positioned”]) encode glycan-binding lipoproteins with little or no sequence or structural conservation, even among syntenic PUL that target the same polysaccharide. RESULTS +266 293 glycan-binding lipoproteins protein_type Equally intriguing is the observation that while SusD-like proteins such as SGBP-A share moderate primary and high tertiary structural conservation, the genes for the SGBPs encoded immediately downstream (Fig. 1B [sometimes referred to as “susE positioned”]) encode glycan-binding lipoproteins with little or no sequence or structural conservation, even among syntenic PUL that target the same polysaccharide. RESULTS +369 372 PUL gene Equally intriguing is the observation that while SusD-like proteins such as SGBP-A share moderate primary and high tertiary structural conservation, the genes for the SGBPs encoded immediately downstream (Fig. 1B [sometimes referred to as “susE positioned”]) encode glycan-binding lipoproteins with little or no sequence or structural conservation, even among syntenic PUL that target the same polysaccharide. RESULTS +394 408 polysaccharide chemical Equally intriguing is the observation that while SusD-like proteins such as SGBP-A share moderate primary and high tertiary structural conservation, the genes for the SGBPs encoded immediately downstream (Fig. 1B [sometimes referred to as “susE positioned”]) encode glycan-binding lipoproteins with little or no sequence or structural conservation, even among syntenic PUL that target the same polysaccharide. RESULTS +21 26 XyGUL gene Such is the case for XyGUL from related Bacteroides species, which may encode either one or two of these predicted SGBPs, and these proteins vary considerably in length. RESULTS +40 51 Bacteroides taxonomy_domain Such is the case for XyGUL from related Bacteroides species, which may encode either one or two of these predicted SGBPs, and these proteins vary considerably in length. RESULTS +115 120 SGBPs protein_type Such is the case for XyGUL from related Bacteroides species, which may encode either one or two of these predicted SGBPs, and these proteins vary considerably in length. RESULTS +38 43 SGBPs protein_type The extremely low similarity of these SGBPs is striking in light of the moderate sequence conservation observed among homologous GHs in syntenic PUL. RESULTS +129 132 GHs protein_type The extremely low similarity of these SGBPs is striking in light of the moderate sequence conservation observed among homologous GHs in syntenic PUL. RESULTS +145 148 PUL gene The extremely low similarity of these SGBPs is striking in light of the moderate sequence conservation observed among homologous GHs in syntenic PUL. RESULTS +47 52 SGBPs protein_type This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface. RESULTS +72 78 BtSusE protein This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface. RESULTS +83 89 BtSusF protein This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface. RESULTS +98 103 XyGUL gene This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface. RESULTS +104 110 SGBP-B protein This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface. RESULTS +152 166 polysaccharide chemical This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface. RESULTS +236 242 glycan chemical This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface. RESULTS +58 66 bacteria taxonomy_domain Because the intestinal ecosystem is a dense consortium of bacteria that must compete for their nutrients, these multimodular SGBPs may reflect ongoing evolutionary experiments to enhance glycan uptake efficiency. RESULTS +125 130 SGBPs protein_type Because the intestinal ecosystem is a dense consortium of bacteria that must compete for their nutrients, these multimodular SGBPs may reflect ongoing evolutionary experiments to enhance glycan uptake efficiency. RESULTS +187 193 glycan chemical Because the intestinal ecosystem is a dense consortium of bacteria that must compete for their nutrients, these multimodular SGBPs may reflect ongoing evolutionary experiments to enhance glycan uptake efficiency. RESULTS +38 43 SGBPs protein_type Whether organisms that express longer SGBPs, extending further above the cell surface toward the extracellular environment, are better equipped to compete for available carbohydrates is presently unknown. RESULTS +169 182 carbohydrates chemical Whether organisms that express longer SGBPs, extending further above the cell surface toward the extracellular environment, are better equipped to compete for available carbohydrates is presently unknown. RESULTS +102 129 carbohydrate-binding motifs structure_element However, the natural diversity of these proteins represents a rich source for the discovery of unique carbohydrate-binding motifs to both inform gut microbiology and generate new, specific carbohydrate analytical reagents. RESULTS +189 201 carbohydrate chemical However, the natural diversity of these proteins represents a rich source for the discovery of unique carbohydrate-binding motifs to both inform gut microbiology and generate new, specific carbohydrate analytical reagents. RESULTS +77 108 surface-glycan binding proteins protein_type In conclusion, the present study further illuminates the essential role that surface-glycan binding proteins play in facilitating the catabolism of complex dietary carbohydrates by Bacteroidetes. RESULTS +164 177 carbohydrates chemical In conclusion, the present study further illuminates the essential role that surface-glycan binding proteins play in facilitating the catabolism of complex dietary carbohydrates by Bacteroidetes. RESULTS +181 194 Bacteroidetes taxonomy_domain In conclusion, the present study further illuminates the essential role that surface-glycan binding proteins play in facilitating the catabolism of complex dietary carbohydrates by Bacteroidetes. RESULTS +32 40 bacteria taxonomy_domain The ability of our resident gut bacteria to recognize polysaccharides is the first committed step of glycan consumption by these organisms, a critical process that influences the community structure and thus the metabolic output (i.e., short-chain fatty acid and metabolite profile) of these organisms. RESULTS +54 69 polysaccharides chemical The ability of our resident gut bacteria to recognize polysaccharides is the first committed step of glycan consumption by these organisms, a critical process that influences the community structure and thus the metabolic output (i.e., short-chain fatty acid and metabolite profile) of these organisms. RESULTS +101 107 glycan chemical The ability of our resident gut bacteria to recognize polysaccharides is the first committed step of glycan consumption by these organisms, a critical process that influences the community structure and thus the metabolic output (i.e., short-chain fatty acid and metabolite profile) of these organisms. RESULTS +29 35 glycan chemical A molecular understanding of glycan uptake by human gut bacteria is therefore central to the development of strategies to improve human health through manipulation of the microbiota. RESULTS +46 51 human species A molecular understanding of glycan uptake by human gut bacteria is therefore central to the development of strategies to improve human health through manipulation of the microbiota. RESULTS +56 64 bacteria taxonomy_domain A molecular understanding of glycan uptake by human gut bacteria is therefore central to the development of strategies to improve human health through manipulation of the microbiota. RESULTS +130 135 human species A molecular understanding of glycan uptake by human gut bacteria is therefore central to the development of strategies to improve human health through manipulation of the microbiota. RESULTS +171 181 microbiota taxonomy_domain A molecular understanding of glycan uptake by human gut bacteria is therefore central to the development of strategies to improve human health through manipulation of the microbiota. RESULTS diff --git a/annotation_CSV/PMC4850288.csv b/annotation_CSV/PMC4850288.csv new file mode 100644 index 0000000000000000000000000000000000000000..c14f26b0f807c85480745b05085555ff7cded9d2 --- /dev/null +++ b/annotation_CSV/PMC4850288.csv @@ -0,0 +1,710 @@ +anno_start anno_end anno_text entity_type sentence section +0 17 Crystal Structure evidence Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome* TITLE +22 38 Activity Studies experimental_method Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome* TITLE +46 49 C11 protein_type Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome* TITLE +50 68 Cysteine Peptidase protein_type Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome* TITLE +74 96 Parabacteroides merdae species Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome* TITLE +104 109 Human species Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome* TITLE +0 27 Clan CD cysteine peptidases protein_type Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. ABSTRACT +61 71 peptidases protein_type Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. ABSTRACT +85 94 mammalian taxonomy_domain Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. ABSTRACT +95 103 caspases protein_type Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. ABSTRACT +17 35 clostripain family protein_type However, for the clostripain family (denoted C11), little is currently known. ABSTRACT +45 48 C11 protein_type However, for the clostripain family (denoted C11), little is currently known. ABSTRACT +28 45 crystal structure evidence Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. ABSTRACT +51 54 C11 protein_type Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. ABSTRACT +72 77 human species Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. ABSTRACT +82 91 bacterium taxonomy_domain Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. ABSTRACT +93 115 Parabacteroides merdae species Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. ABSTRACT +117 122 PmC11 protein Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. ABSTRACT +0 5 PmC11 protein PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. ABSTRACT +11 20 monomeric oligomeric_state PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. ABSTRACT +21 39 cysteine peptidase protein_type PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. ABSTRACT +58 94 extended caspase-like α/β/α sandwich structure_element PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. ABSTRACT +110 127 C-terminal domain structure_element PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. ABSTRACT +40 67 clan CD cysteine peptidases protein_type It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. ABSTRACT +77 83 Lys147 residue_name_number These studies also revealed a well ordered break in the polypeptide chain at Lys147, resulting in a large conformational rearrangement close to the active site. ABSTRACT +148 159 active site site These studies also revealed a well ordered break in the polypeptide chain at Lys147, resulting in a large conformational rearrangement close to the active site. ABSTRACT +0 32 Biochemical and kinetic analysis experimental_method Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. ABSTRACT +42 48 Lys147 residue_name_number Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. ABSTRACT +58 88 intramolecular processing site site Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. ABSTRACT +98 106 cleavage ptm Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. ABSTRACT +123 138 full activation protein_state Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. ABSTRACT +146 152 enzyme protein Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. ABSTRACT +0 5 PmC11 protein PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity. ABSTRACT +13 34 acidic binding pocket site PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity. ABSTRACT +102 105 Arg residue_name PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity. ABSTRACT +110 113 Lys residue_name PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity. ABSTRACT +117 119 P1 residue_number PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity. ABSTRACT +141 145 Ca2+ chemical PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity. ABSTRACT +77 82 PmC11 protein Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms. ABSTRACT +123 137 C11 peptidases protein_type Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms. ABSTRACT +0 19 Cysteine peptidases protein_type Cysteine peptidases play crucial roles in the virulence of bacterial and other eukaryotic pathogens. INTRO +59 68 bacterial taxonomy_domain Cysteine peptidases play crucial roles in the virulence of bacterial and other eukaryotic pathogens. INTRO +79 89 eukaryotic taxonomy_domain Cysteine peptidases play crucial roles in the virulence of bacterial and other eukaryotic pathogens. INTRO +34 41 clan CD protein_type In the MEROPS peptidase database, clan CD contains groups (or families) of cysteine peptidases that share some highly conserved structural elements. INTRO +75 94 cysteine peptidases protein_type In the MEROPS peptidase database, clan CD contains groups (or families) of cysteine peptidases that share some highly conserved structural elements. INTRO +111 127 highly conserved protein_state In the MEROPS peptidase database, clan CD contains groups (or families) of cysteine peptidases that share some highly conserved structural elements. INTRO +0 16 Clan CD families protein_type Clan CD families are typically described using the name of their archetypal, or founding, member and also given an identification number preceded by a “C,” to denote cysteine peptidase. INTRO +166 184 cysteine peptidase protein_type Clan CD families are typically described using the name of their archetypal, or founding, member and also given an identification number preceded by a “C,” to denote cysteine peptidase. INTRO +110 128 crystal structures evidence Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +166 174 legumain protein Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +176 179 C13 protein_type Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +182 189 caspase protein Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +191 195 C14a protein_type Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +198 209 paracaspase protein Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +211 217 C14b(P protein_type Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +220 231 metacaspase protein Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +233 239 C14b(M protein_type Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +242 251 gingipain protein Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +253 256 C25 protein_type Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +267 292 cysteine peptidase domain structure_element Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +294 297 CPD structure_element Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +318 321 C80 protein_type Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80). INTRO +43 54 clostripain protein No structural information is available for clostripain (C11), separase (C50), or PrtH-peptidase (C85). INTRO +56 59 C11 protein_type No structural information is available for clostripain (C11), separase (C50), or PrtH-peptidase (C85). INTRO +62 70 separase protein No structural information is available for clostripain (C11), separase (C50), or PrtH-peptidase (C85). INTRO +72 75 C50 protein_type No structural information is available for clostripain (C11), separase (C50), or PrtH-peptidase (C85). INTRO +81 95 PrtH-peptidase protein No structural information is available for clostripain (C11), separase (C50), or PrtH-peptidase (C85). INTRO +97 100 C85 protein_type No structural information is available for clostripain (C11), separase (C50), or PrtH-peptidase (C85). INTRO +0 15 Clan CD enzymes protein_type Clan CD enzymes have a highly conserved His/Cys catalytic dyad and exhibit strict specificity for the P1 residue of their substrates. INTRO +23 39 highly conserved protein_state Clan CD enzymes have a highly conserved His/Cys catalytic dyad and exhibit strict specificity for the P1 residue of their substrates. INTRO +40 62 His/Cys catalytic dyad site Clan CD enzymes have a highly conserved His/Cys catalytic dyad and exhibit strict specificity for the P1 residue of their substrates. INTRO +102 104 P1 residue_number Clan CD enzymes have a highly conserved His/Cys catalytic dyad and exhibit strict specificity for the P1 residue of their substrates. INTRO +37 44 clan CD protein_type However, despite these similarities, clan CD forms a functionally diverse group of enzymes: the overall structural diversity between (and at times within) the various families provides these peptidases with a wide variety of substrate specificities and activation mechanisms. INTRO +191 201 peptidases protein_type However, despite these similarities, clan CD forms a functionally diverse group of enzymes: the overall structural diversity between (and at times within) the various families provides these peptidases with a wide variety of substrate specificities and activation mechanisms. INTRO +75 84 mammalian taxonomy_domain The archetypal and arguably most notable family in the clan is that of the mammalian caspases (C14a), although clan CD members are distributed throughout the entire phylogenetic kingdom and are often required in fundamental biological processes. INTRO +85 93 caspases protein_type The archetypal and arguably most notable family in the clan is that of the mammalian caspases (C14a), although clan CD members are distributed throughout the entire phylogenetic kingdom and are often required in fundamental biological processes. INTRO +95 99 C14a protein_type The archetypal and arguably most notable family in the clan is that of the mammalian caspases (C14a), although clan CD members are distributed throughout the entire phylogenetic kingdom and are often required in fundamental biological processes. INTRO +111 118 clan CD protein_type The archetypal and arguably most notable family in the clan is that of the mammalian caspases (C14a), although clan CD members are distributed throughout the entire phylogenetic kingdom and are often required in fundamental biological processes. INTRO +70 73 C11 protein_type Interestingly, little is known about the structure or function of the C11 proteins, despite their widespread distribution and its archetypal member, clostripain from Clostridium histolyticum, first reported in the literature in 1938. INTRO +149 160 clostripain protein Interestingly, little is known about the structure or function of the C11 proteins, despite their widespread distribution and its archetypal member, clostripain from Clostridium histolyticum, first reported in the literature in 1938. INTRO +166 190 Clostridium histolyticum species Interestingly, little is known about the structure or function of the C11 proteins, despite their widespread distribution and its archetypal member, clostripain from Clostridium histolyticum, first reported in the literature in 1938. INTRO +0 11 Clostripain protein Clostripain has been described as an arginine-specific peptidase with a requirement for Ca2+ and loss of an internal nonapeptide for full activation; lack of structural information on the family appears to have prohibited further investigation. INTRO +37 64 arginine-specific peptidase protein_type Clostripain has been described as an arginine-specific peptidase with a requirement for Ca2+ and loss of an internal nonapeptide for full activation; lack of structural information on the family appears to have prohibited further investigation. INTRO +88 92 Ca2+ chemical Clostripain has been described as an arginine-specific peptidase with a requirement for Ca2+ and loss of an internal nonapeptide for full activation; lack of structural information on the family appears to have prohibited further investigation. INTRO +108 128 internal nonapeptide structure_element Clostripain has been described as an arginine-specific peptidase with a requirement for Ca2+ and loss of an internal nonapeptide for full activation; lack of structural information on the family appears to have prohibited further investigation. INTRO +133 148 full activation protein_state Clostripain has been described as an arginine-specific peptidase with a requirement for Ca2+ and loss of an internal nonapeptide for full activation; lack of structural information on the family appears to have prohibited further investigation. INTRO +56 64 bacteria taxonomy_domain As part of an ongoing project to characterize commensal bacteria in the microbiome that inhabit the human gut, the structure of C11 peptidase, PmC11, from Parabacteroides merdae was determined using the Joint Center for Structural Genomics (JCSG)4 HTP structural biology pipeline. INTRO +100 105 human species As part of an ongoing project to characterize commensal bacteria in the microbiome that inhabit the human gut, the structure of C11 peptidase, PmC11, from Parabacteroides merdae was determined using the Joint Center for Structural Genomics (JCSG)4 HTP structural biology pipeline. INTRO +115 124 structure evidence As part of an ongoing project to characterize commensal bacteria in the microbiome that inhabit the human gut, the structure of C11 peptidase, PmC11, from Parabacteroides merdae was determined using the Joint Center for Structural Genomics (JCSG)4 HTP structural biology pipeline. INTRO +128 141 C11 peptidase protein_type As part of an ongoing project to characterize commensal bacteria in the microbiome that inhabit the human gut, the structure of C11 peptidase, PmC11, from Parabacteroides merdae was determined using the Joint Center for Structural Genomics (JCSG)4 HTP structural biology pipeline. INTRO +143 148 PmC11 protein As part of an ongoing project to characterize commensal bacteria in the microbiome that inhabit the human gut, the structure of C11 peptidase, PmC11, from Parabacteroides merdae was determined using the Joint Center for Structural Genomics (JCSG)4 HTP structural biology pipeline. INTRO +155 177 Parabacteroides merdae species As part of an ongoing project to characterize commensal bacteria in the microbiome that inhabit the human gut, the structure of C11 peptidase, PmC11, from Parabacteroides merdae was determined using the Joint Center for Structural Genomics (JCSG)4 HTP structural biology pipeline. INTRO +4 26 structure was analyzed experimental_method The structure was analyzed, and the enzyme was biochemically characterized to provide the first structure/function correlation for a C11 peptidase. INTRO +47 74 biochemically characterized experimental_method The structure was analyzed, and the enzyme was biochemically characterized to provide the first structure/function correlation for a C11 peptidase. INTRO +133 146 C11 peptidase protein_type The structure was analyzed, and the enzyme was biochemically characterized to provide the first structure/function correlation for a C11 peptidase. INTRO +0 9 Structure evidence Structure of PmC11 RESULTS +13 18 PmC11 protein Structure of PmC11 RESULTS +4 21 crystal structure evidence The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like α/β/α sandwich architecture comprised of a central nine-stranded β-sheet, with an unusual C-terminal domain (CTD), starting at Lys250. RESULTS +29 49 catalytically active protein_state The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like α/β/α sandwich architecture comprised of a central nine-stranded β-sheet, with an unusual C-terminal domain (CTD), starting at Lys250. RESULTS +58 63 PmC11 protein The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like α/β/α sandwich architecture comprised of a central nine-stranded β-sheet, with an unusual C-terminal domain (CTD), starting at Lys250. RESULTS +76 112 extended caspase-like α/β/α sandwich structure_element The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like α/β/α sandwich architecture comprised of a central nine-stranded β-sheet, with an unusual C-terminal domain (CTD), starting at Lys250. RESULTS +149 170 nine-stranded β-sheet structure_element The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like α/β/α sandwich architecture comprised of a central nine-stranded β-sheet, with an unusual C-terminal domain (CTD), starting at Lys250. RESULTS +188 205 C-terminal domain structure_element The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like α/β/α sandwich architecture comprised of a central nine-stranded β-sheet, with an unusual C-terminal domain (CTD), starting at Lys250. RESULTS +207 210 CTD structure_element The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like α/β/α sandwich architecture comprised of a central nine-stranded β-sheet, with an unusual C-terminal domain (CTD), starting at Lys250. RESULTS +225 231 Lys250 residue_name_number The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like α/β/α sandwich architecture comprised of a central nine-stranded β-sheet, with an unusual C-terminal domain (CTD), starting at Lys250. RESULTS +2 17 single cleavage ptm A single cleavage was observed in the polypeptide chain at Lys147 (Fig. 1, A and B), where both ends of the cleavage site are fully visible and well ordered in the electron density. RESULTS +59 65 Lys147 residue_name_number A single cleavage was observed in the polypeptide chain at Lys147 (Fig. 1, A and B), where both ends of the cleavage site are fully visible and well ordered in the electron density. RESULTS +108 121 cleavage site site A single cleavage was observed in the polypeptide chain at Lys147 (Fig. 1, A and B), where both ends of the cleavage site are fully visible and well ordered in the electron density. RESULTS +164 180 electron density evidence A single cleavage was observed in the polypeptide chain at Lys147 (Fig. 1, A and B), where both ends of the cleavage site are fully visible and well ordered in the electron density. RESULTS +12 33 nine-stranded β-sheet structure_element The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. RESULTS +35 40 β1–β9 structure_element The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. RESULTS +45 50 PmC11 protein The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. RESULTS +67 75 parallel structure_element The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. RESULTS +86 109 anti-parallel β-strands structure_element The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. RESULTS +169 178 structure evidence The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. RESULTS +191 200 α-helices structure_element The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. RESULTS +211 216 α1–α2 structure_element The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. RESULTS +221 226 α4–α7 structure_element The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. RESULTS +252 259 β-sheet structure_element The central nine-stranded β-sheet (β1–β9) of PmC11 consists of six parallel and three anti-parallel β-strands with 4↑3↓2↑1↑5↑6↑7↓8↓9↑ topology (Fig. 1A) and the overall structure includes 14 α-helices with six (α1–α2 and α4–α7) closely surrounding the β-sheet in an approximately parallel orientation. RESULTS +0 7 Helices structure_element Helices α1, α7, and α6 are located on one side of the β-sheet with α2, α4, and α5 on the opposite side (Fig. 1A). RESULTS +8 10 α1 structure_element Helices α1, α7, and α6 are located on one side of the β-sheet with α2, α4, and α5 on the opposite side (Fig. 1A). RESULTS +12 14 α7 structure_element Helices α1, α7, and α6 are located on one side of the β-sheet with α2, α4, and α5 on the opposite side (Fig. 1A). RESULTS +20 22 α6 structure_element Helices α1, α7, and α6 are located on one side of the β-sheet with α2, α4, and α5 on the opposite side (Fig. 1A). RESULTS +54 61 β-sheet structure_element Helices α1, α7, and α6 are located on one side of the β-sheet with α2, α4, and α5 on the opposite side (Fig. 1A). RESULTS +67 69 α2 structure_element Helices α1, α7, and α6 are located on one side of the β-sheet with α2, α4, and α5 on the opposite side (Fig. 1A). RESULTS +71 73 α4 structure_element Helices α1, α7, and α6 are located on one side of the β-sheet with α2, α4, and α5 on the opposite side (Fig. 1A). RESULTS +79 81 α5 structure_element Helices α1, α7, and α6 are located on one side of the β-sheet with α2, α4, and α5 on the opposite side (Fig. 1A). RESULTS +0 5 Helix structure_element Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. RESULTS +6 8 α3 structure_element Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. RESULTS +32 36 loop structure_element Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. RESULTS +47 49 β5 structure_element Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. RESULTS +51 53 L5 structure_element Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. RESULTS +75 81 Lys147 residue_name_number Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. RESULTS +82 95 cleavage site site Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. RESULTS +107 109 L5 structure_element Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. RESULTS +114 116 α3 structure_element Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. RESULTS +148 155 β-sheet structure_element Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. RESULTS +171 174 CTD structure_element Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. RESULTS +194 196 α8 structure_element Helix α3 sits at the end of the loop following β5 (L5), just preceding the Lys147 cleavage site, with both L5 and α3 pointing away from the central β-sheet and toward the CTD, which starts with α8. RESULTS +4 13 structure evidence The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +38 48 β-hairpins structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +50 55 βA–βB structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +60 65 βD–βE structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +73 86 small β-sheet structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +88 93 βC–βF structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +155 157 βC structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +167 170 α11 structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +172 175 α12 structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +180 182 β9 structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +192 194 βF structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +207 212 βD-βE structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +213 220 hairpin structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +243 246 CTD structure_element The structure also includes two short β-hairpins (βA–βB and βD–βE) and a small β-sheet (βC–βF), which is formed from two distinct regions of the sequence (βC precedes α11, α12 and β9, whereas βF follows the βD-βE hairpin) in the middle of the CTD (Fig. 1B). RESULTS +0 17 Crystal structure evidence Crystal structure of a C11 peptidase from P. merdae. FIG +23 36 C11 peptidase protein_type Crystal structure of a C11 peptidase from P. merdae. FIG +42 51 P. merdae species Crystal structure of a C11 peptidase from P. merdae. FIG +4 30 primary sequence alignment experimental_method A, primary sequence alignment of PmC11 (Uniprot ID A7A9N3) and clostripain (Uniprot ID P09870) from C. histolyticum with identical residues highlighted in gray shading. FIG +34 39 PmC11 protein A, primary sequence alignment of PmC11 (Uniprot ID A7A9N3) and clostripain (Uniprot ID P09870) from C. histolyticum with identical residues highlighted in gray shading. FIG +64 75 clostripain protein A, primary sequence alignment of PmC11 (Uniprot ID A7A9N3) and clostripain (Uniprot ID P09870) from C. histolyticum with identical residues highlighted in gray shading. FIG +101 116 C. histolyticum species A, primary sequence alignment of PmC11 (Uniprot ID A7A9N3) and clostripain (Uniprot ID P09870) from C. histolyticum with identical residues highlighted in gray shading. FIG +27 32 PmC11 protein The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. FIG +42 59 crystal structure evidence The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. FIG +113 118 PmC11 protein The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. FIG +119 133 catalytic dyad site The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. FIG +135 162 autocatalytic cleavage site site The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. FIG +164 170 Lys147 residue_name_number The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. FIG +177 194 S1 binding pocket site The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. FIG +195 198 Asp residue_name The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. FIG +200 206 Asp177 residue_name_number The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively. FIG +11 16 loops structure_element Connecting loops are colored gray, the main β-sheet is in orange, with other strands in olive, α-helices are in blue, and the nonapeptide linker of clostripain that is excised upon autocleavage is underlined in red. FIG +44 51 β-sheet structure_element Connecting loops are colored gray, the main β-sheet is in orange, with other strands in olive, α-helices are in blue, and the nonapeptide linker of clostripain that is excised upon autocleavage is underlined in red. FIG +95 104 α-helices structure_element Connecting loops are colored gray, the main β-sheet is in orange, with other strands in olive, α-helices are in blue, and the nonapeptide linker of clostripain that is excised upon autocleavage is underlined in red. FIG +126 144 nonapeptide linker structure_element Connecting loops are colored gray, the main β-sheet is in orange, with other strands in olive, α-helices are in blue, and the nonapeptide linker of clostripain that is excised upon autocleavage is underlined in red. FIG +148 159 clostripain protein Connecting loops are colored gray, the main β-sheet is in orange, with other strands in olive, α-helices are in blue, and the nonapeptide linker of clostripain that is excised upon autocleavage is underlined in red. FIG +181 193 autocleavage ptm Connecting loops are colored gray, the main β-sheet is in orange, with other strands in olive, α-helices are in blue, and the nonapeptide linker of clostripain that is excised upon autocleavage is underlined in red. FIG +21 35 catalytic site site Sequences around the catalytic site of clostripain and PmC11 align well. FIG +39 50 clostripain protein Sequences around the catalytic site of clostripain and PmC11 align well. FIG +55 60 PmC11 protein Sequences around the catalytic site of clostripain and PmC11 align well. FIG +23 28 PmC11 protein B, topology diagram of PmC11 colored as in A except that additional (non-core) β-strands are in yellow. FIG +79 88 β-strands structure_element B, topology diagram of PmC11 colored as in A except that additional (non-core) β-strands are in yellow. FIG +44 51 β-sheet structure_element Helices found on either side of the central β-sheet are shown above and below the sheet, respectively. FIG +82 87 sheet structure_element Helices found on either side of the central β-sheet are shown above and below the sheet, respectively. FIG +20 34 catalytic dyad site The position of the catalytic dyad (H, C) and the processing site (Lys147) are highlighted. FIG +36 37 H residue_name The position of the catalytic dyad (H, C) and the processing site (Lys147) are highlighted. FIG +39 40 C residue_name The position of the catalytic dyad (H, C) and the processing site (Lys147) are highlighted. FIG +50 65 processing site site The position of the catalytic dyad (H, C) and the processing site (Lys147) are highlighted. FIG +67 73 Lys147 residue_name_number The position of the catalytic dyad (H, C) and the processing site (Lys147) are highlighted. FIG +19 28 β-strands structure_element Helices (1–14) and β-strands (1–9 and A-F) are numbered from the N terminus. FIG +4 21 core caspase-fold structure_element The core caspase-fold is highlighted in a box. FIG +25 30 PmC11 protein C, tertiary structure of PmC11. FIG +33 38 PmC11 protein The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. FIG +62 69 β-sheet structure_element The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. FIG +77 82 helix structure_element The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. FIG +83 85 α5 structure_element The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. FIG +91 98 helices structure_element The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. FIG +99 101 α8 structure_element The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. FIG +103 106 α11 structure_element The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. FIG +112 115 α13 structure_element The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. FIG +125 142 C-terminal domain structure_element The N and C termini (N and C) of PmC11 along with the central β-sheet (1–9), helix α5, and helices α8, α11, and α13 from the C-terminal domain, are all labeled. FIG +33 40 β-sheet structure_element Loops are colored gray, the main β-sheet is in orange, with other β-strands in yellow, and α-helices are in blue. FIG +66 75 β-strands structure_element Loops are colored gray, the main β-sheet is in orange, with other β-strands in yellow, and α-helices are in blue. FIG +91 100 α-helices structure_element Loops are colored gray, the main β-sheet is in orange, with other β-strands in yellow, and α-helices are in blue. FIG +4 7 CTD structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +11 16 PmC11 protein The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +34 54 tight helical bundle structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +67 74 helices structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +75 81 α8–α14 structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +95 102 strands structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +103 105 βC structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +110 112 βF structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +118 127 β-hairpin structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +128 133 βD–βE structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +139 142 CTD structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +205 207 α3 structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +209 211 α5 structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +213 215 β9 structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +225 230 loops structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +243 245 β8 structure_element The CTD of PmC11 is composed of a tight helical bundle formed from helices α8–α14 and includes strands βC and βF, and β-hairpin βD–βE. The CTD sits entirely on one side of the enzyme interacting only with α3, α5, β9, and the loops surrounding β8. RESULTS +49 51 α5 structure_element Of the interacting secondary structure elements, α5 is perhaps the most interesting. RESULTS +0 10 This helix structure_element This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C). RESULTS +34 48 hydrogen bonds bond_interaction This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C). RESULTS +58 61 CTD structure_element This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C). RESULTS +77 88 salt bridge bond_interaction This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C). RESULTS +90 96 Arg191 residue_name_number This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C). RESULTS +97 103 Asp255 residue_name_number This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C). RESULTS +130 133 CTD structure_element This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C). RESULTS +154 163 main core structure_element This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C). RESULTS +0 5 PmC11 protein PmC11 is, as expected, most structurally similar to other members of clan CD with the top hits in a search of known structures being caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively) (Table 2). RESULTS +69 76 clan CD protein_type PmC11 is, as expected, most structurally similar to other members of clan CD with the top hits in a search of known structures being caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively) (Table 2). RESULTS +116 126 structures evidence PmC11 is, as expected, most structurally similar to other members of clan CD with the top hits in a search of known structures being caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively) (Table 2). RESULTS +133 142 caspase-7 protein PmC11 is, as expected, most structurally similar to other members of clan CD with the top hits in a search of known structures being caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively) (Table 2). RESULTS +144 155 gingipain-K protein PmC11 is, as expected, most structurally similar to other members of clan CD with the top hits in a search of known structures being caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively) (Table 2). RESULTS +161 169 legumain protein PmC11 is, as expected, most structurally similar to other members of clan CD with the top hits in a search of known structures being caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively) (Table 2). RESULTS +4 21 C-terminal domain structure_element The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold. RESULTS +35 40 PmC11 protein The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold. RESULTS +48 55 clan CD protein_type The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold. RESULTS +60 81 structure comparisons experimental_method The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold. RESULTS +86 103 this domain alone structure_element The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold. RESULTS +142 150 DaliLite experimental_method The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold. RESULTS +152 160 PDBeFold experimental_method The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold. RESULTS +59 66 clan CD protein_type As the archetypal and arguably most well studied member of clan CD, the caspases were used as the basis to investigate the structure/function relationships in PmC11, with caspase-7 as the representative member. RESULTS +72 80 caspases protein_type As the archetypal and arguably most well studied member of clan CD, the caspases were used as the basis to investigate the structure/function relationships in PmC11, with caspase-7 as the representative member. RESULTS +159 164 PmC11 protein As the archetypal and arguably most well studied member of clan CD, the caspases were used as the basis to investigate the structure/function relationships in PmC11, with caspase-7 as the representative member. RESULTS +171 180 caspase-7 protein As the archetypal and arguably most well studied member of clan CD, the caspases were used as the basis to investigate the structure/function relationships in PmC11, with caspase-7 as the representative member. RESULTS +19 28 β-strands structure_element Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). RESULTS +32 37 PmC11 protein Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). RESULTS +39 44 β1–β2 structure_element Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). RESULTS +49 54 β5–β8 structure_element Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). RESULTS +87 107 six-stranded β-sheet structure_element Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). RESULTS +117 125 caspases protein_type Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). RESULTS +132 139 strands structure_element Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). RESULTS +140 142 β3 structure_element Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). RESULTS +144 146 β4 structure_element Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). RESULTS +152 154 β9 structure_element Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). RESULTS +186 200 core structure structure_element Six of the central β-strands in PmC11 (β1–β2 and β5–β8) share the same topology as the six-stranded β-sheet found in caspases, with strands β3, β4, and β9 located on the outside of this core structure (Fig. 1B, box). RESULTS +0 6 His133 residue_name_number His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). RESULTS +11 17 Cys179 residue_name_number His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). RESULTS +73 80 caspase protein_type His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). RESULTS +81 95 catalytic dyad site His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). RESULTS +107 114 clan CD protein_type His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). RESULTS +115 125 structures evidence His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). RESULTS +147 154 strands structure_element His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). RESULTS +155 157 β5 structure_element His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). RESULTS +162 164 β6 structure_element His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands β5 and β6, respectively (Figs. 1, A and B, and 2A). RESULTS +2 29 multiple sequence alignment experimental_method A multiple sequence alignment of C11 proteins revealed that these residues are highly conserved (data not shown). RESULTS +33 36 C11 protein_type A multiple sequence alignment of C11 proteins revealed that these residues are highly conserved (data not shown). RESULTS +79 95 highly conserved protein_state A multiple sequence alignment of C11 proteins revealed that these residues are highly conserved (data not shown). RESULTS +11 33 PDBeFOLD superposition experimental_method Summary of PDBeFOLD superposition of structures found to be most similar to PmC11 in the PBD based on DaliLite TABLE +76 81 PmC11 protein Summary of PDBeFOLD superposition of structures found to be most similar to PmC11 in the PBD based on DaliLite TABLE +102 110 DaliLite experimental_method Summary of PDBeFOLD superposition of structures found to be most similar to PmC11 in the PBD based on DaliLite TABLE +0 43 Biochemical and structural characterization experimental_method Biochemical and structural characterization of PmC11. FIG +47 52 PmC11 protein Biochemical and structural characterization of PmC11. FIG +54 59 PmC11 protein A, ribbon representation of the overall structure of PmC11 illustrating the catalytic site, cleavage site displacement, and potential S1 binding site. FIG +77 91 catalytic site site A, ribbon representation of the overall structure of PmC11 illustrating the catalytic site, cleavage site displacement, and potential S1 binding site. FIG +135 150 S1 binding site site A, ribbon representation of the overall structure of PmC11 illustrating the catalytic site, cleavage site displacement, and potential S1 binding site. FIG +12 21 structure evidence The overall structure of PmC11 is shown in gray, looking down into the catalytic site with the catalytic dyad in red. FIG +25 30 PmC11 protein The overall structure of PmC11 is shown in gray, looking down into the catalytic site with the catalytic dyad in red. FIG +71 85 catalytic site site The overall structure of PmC11 is shown in gray, looking down into the catalytic site with the catalytic dyad in red. FIG +95 109 catalytic dyad site The overall structure of PmC11 is shown in gray, looking down into the catalytic site with the catalytic dyad in red. FIG +20 43 autolytic cleavage site site The two ends of the autolytic cleavage site (Lys147 and Ala148, green) are displaced by 19.5 Å (thin black line) from one another and residues in the potential substrate binding pocket are highlighted in blue. FIG +45 51 Lys147 residue_name_number The two ends of the autolytic cleavage site (Lys147 and Ala148, green) are displaced by 19.5 Å (thin black line) from one another and residues in the potential substrate binding pocket are highlighted in blue. FIG +56 62 Ala148 residue_name_number The two ends of the autolytic cleavage site (Lys147 and Ala148, green) are displaced by 19.5 Å (thin black line) from one another and residues in the potential substrate binding pocket are highlighted in blue. FIG +160 184 substrate binding pocket site The two ends of the autolytic cleavage site (Lys147 and Ala148, green) are displaced by 19.5 Å (thin black line) from one another and residues in the potential substrate binding pocket are highlighted in blue. FIG +3 32 size exclusion chromatography experimental_method B, size exclusion chromatography of PmC11. FIG +36 41 PmC11 protein B, size exclusion chromatography of PmC11. FIG +20 27 monomer oligomeric_state PmC11 migrates as a monomer with a molecular mass around 41 kDa calculated from protein standards of known molecular weights. FIG +63 71 SDS-PAGE experimental_method Elution fractions across the major peak (1–6) were analyzed by SDS-PAGE on a 4–12% gel in MES buffer. FIG +7 13 active protein_state C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. FIG +22 27 PmC11 protein C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. FIG +45 55 PmC11C179A mutant C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. FIG +64 74 PmC11K147A mutant C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. FIG +97 105 SDS-PAGE experimental_method C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. FIG +119 131 Western blot experimental_method C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. FIG +187 192 PmC11 protein C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. FIG +193 206 autoprocesses ptm C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. FIG +225 235 PmC11C179A mutant C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. FIG +240 250 PmC11K147A mutant C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. FIG +264 278 autoprocessing ptm C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro. FIG +34 39 PmC11 protein D, cysteine peptidase activity of PmC11. FIG +7 11 Vmax evidence Km and Vmax of PmC11 and K147A mutant were determined by monitoring change in the fluorescence corresponding to AMC release from Bz-R-AMC. FIG +15 20 PmC11 protein Km and Vmax of PmC11 and K147A mutant were determined by monitoring change in the fluorescence corresponding to AMC release from Bz-R-AMC. FIG +25 30 K147A mutant Km and Vmax of PmC11 and K147A mutant were determined by monitoring change in the fluorescence corresponding to AMC release from Bz-R-AMC. FIG +129 137 Bz-R-AMC chemical Km and Vmax of PmC11 and K147A mutant were determined by monitoring change in the fluorescence corresponding to AMC release from Bz-R-AMC. FIG +3 28 intermolecular processing ptm E, intermolecular processing of PmC11C179A by PmC11. FIG +32 42 PmC11C179A mutant E, intermolecular processing of PmC11C179A by PmC11. FIG +46 51 PmC11 protein E, intermolecular processing of PmC11C179A by PmC11. FIG +89 94 PmC11 protein PmC11C179A (20 μg) was incubated overnight at 37 °C with increasing amounts of processed PmC11 and analyzed on a 10% SDS-PAGE gel. FIG +117 125 SDS-PAGE experimental_method PmC11C179A (20 μg) was incubated overnight at 37 °C with increasing amounts of processed PmC11 and analyzed on a 10% SDS-PAGE gel. FIG +9 19 PmC11C179A mutant Inactive PmC11C179A was not processed to a major extent by active PmC11 until around a ratio of 1:4 (5 μg of active PmC11). FIG +59 65 active protein_state Inactive PmC11C179A was not processed to a major extent by active PmC11 until around a ratio of 1:4 (5 μg of active PmC11). FIG +66 71 PmC11 protein Inactive PmC11C179A was not processed to a major extent by active PmC11 until around a ratio of 1:4 (5 μg of active PmC11). FIG +109 115 active protein_state Inactive PmC11C179A was not processed to a major extent by active PmC11 until around a ratio of 1:4 (5 μg of active PmC11). FIG +116 121 PmC11 protein Inactive PmC11C179A was not processed to a major extent by active PmC11 until around a ratio of 1:4 (5 μg of active PmC11). FIG +26 32 active protein_state A single lane of 20 μg of active PmC11 (labeled 20) is shown for comparison. FIG +33 38 PmC11 protein A single lane of 20 μg of active PmC11 (labeled 20) is shown for comparison. FIG +3 11 activity evidence F, activity of PmC11 against basic substrates. FIG +15 20 PmC11 protein F, activity of PmC11 against basic substrates. FIG +38 43 PmC11 protein G, electrostatic surface potential of PmC11 shown in a similar orientation, where blue and red denote positively and negatively charged surface potential, respectively, contoured at ±5 kT/e. FIG +20 34 catalytic dyad site The position of the catalytic dyad, one potential key substrate binding residue Asp177, and the ends of the cleavage site Lys147 and Ala148 are indicated. FIG +50 79 key substrate binding residue site The position of the catalytic dyad, one potential key substrate binding residue Asp177, and the ends of the cleavage site Lys147 and Ala148 are indicated. FIG +80 86 Asp177 residue_name_number The position of the catalytic dyad, one potential key substrate binding residue Asp177, and the ends of the cleavage site Lys147 and Ala148 are indicated. FIG +108 121 cleavage site site The position of the catalytic dyad, one potential key substrate binding residue Asp177, and the ends of the cleavage site Lys147 and Ala148 are indicated. FIG +122 128 Lys147 residue_name_number The position of the catalytic dyad, one potential key substrate binding residue Asp177, and the ends of the cleavage site Lys147 and Ala148 are indicated. FIG +133 139 Ala148 residue_name_number The position of the catalytic dyad, one potential key substrate binding residue Asp177, and the ends of the cleavage site Lys147 and Ala148 are indicated. FIG +12 21 α-helices structure_element Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +38 45 β-sheet structure_element Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +49 54 PmC11 protein Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +56 58 α1 structure_element Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +60 62 α2 structure_element Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +64 66 α4 structure_element Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +68 70 α6 structure_element Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +76 78 α7 structure_element Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +123 145 structurally conserved protein_state Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +146 153 helices structure_element Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +157 165 caspases protein_type Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +187 194 clan CD protein_type Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +214 217 C80 protein_type Five of the α-helices surrounding the β-sheet of PmC11 (α1, α2, α4, α6, and α7) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80. RESULTS +20 36 extended β-sheet structure_element Other than its more extended β-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven α-helices and four β-strands after β8. RESULTS +38 43 PmC11 protein Other than its more extended β-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven α-helices and four β-strands after β8. RESULTS +82 89 clan CD protein_type Other than its more extended β-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven α-helices and four β-strands after β8. RESULTS +127 130 CTD structure_element Other than its more extended β-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven α-helices and four β-strands after β8. RESULTS +156 165 α-helices structure_element Other than its more extended β-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven α-helices and four β-strands after β8. RESULTS +175 184 β-strands structure_element Other than its more extended β-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven α-helices and four β-strands after β8. RESULTS +191 193 β8 structure_element Other than its more extended β-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven α-helices and four β-strands after β8. RESULTS +0 14 Autoprocessing ptm Autoprocessing of PmC11 RESULTS +18 23 PmC11 protein Autoprocessing of PmC11 RESULTS +0 12 Purification experimental_method Purification of recombinant PmC11 (molecular mass = 42.6 kDa) revealed partial processing into two cleavage products of 26.4 and 16.2 kDa, related to the observed cleavage at Lys147 in the crystal structure (Fig. 2A). RESULTS +28 33 PmC11 protein Purification of recombinant PmC11 (molecular mass = 42.6 kDa) revealed partial processing into two cleavage products of 26.4 and 16.2 kDa, related to the observed cleavage at Lys147 in the crystal structure (Fig. 2A). RESULTS +163 171 cleavage ptm Purification of recombinant PmC11 (molecular mass = 42.6 kDa) revealed partial processing into two cleavage products of 26.4 and 16.2 kDa, related to the observed cleavage at Lys147 in the crystal structure (Fig. 2A). RESULTS +175 181 Lys147 residue_name_number Purification of recombinant PmC11 (molecular mass = 42.6 kDa) revealed partial processing into two cleavage products of 26.4 and 16.2 kDa, related to the observed cleavage at Lys147 in the crystal structure (Fig. 2A). RESULTS +189 206 crystal structure evidence Purification of recombinant PmC11 (molecular mass = 42.6 kDa) revealed partial processing into two cleavage products of 26.4 and 16.2 kDa, related to the observed cleavage at Lys147 in the crystal structure (Fig. 2A). RESULTS +0 10 Incubation experimental_method Incubation of PmC11 at 37 °C for 16 h, resulted in a fully processed enzyme that remained as an intact monomer when applied to a size-exclusion column (Fig. 2B). RESULTS +14 19 PmC11 protein Incubation of PmC11 at 37 °C for 16 h, resulted in a fully processed enzyme that remained as an intact monomer when applied to a size-exclusion column (Fig. 2B). RESULTS +53 68 fully processed protein_state Incubation of PmC11 at 37 °C for 16 h, resulted in a fully processed enzyme that remained as an intact monomer when applied to a size-exclusion column (Fig. 2B). RESULTS +96 102 intact protein_state Incubation of PmC11 at 37 °C for 16 h, resulted in a fully processed enzyme that remained as an intact monomer when applied to a size-exclusion column (Fig. 2B). RESULTS +103 110 monomer oligomeric_state Incubation of PmC11 at 37 °C for 16 h, resulted in a fully processed enzyme that remained as an intact monomer when applied to a size-exclusion column (Fig. 2B). RESULTS +11 24 cleavage site site The single cleavage site of PmC11 at Lys147 is found immediately after α3, in loop L5 within the central β-sheet (Figs. 1, A and B, and 2A). RESULTS +28 33 PmC11 protein The single cleavage site of PmC11 at Lys147 is found immediately after α3, in loop L5 within the central β-sheet (Figs. 1, A and B, and 2A). RESULTS +37 43 Lys147 residue_name_number The single cleavage site of PmC11 at Lys147 is found immediately after α3, in loop L5 within the central β-sheet (Figs. 1, A and B, and 2A). RESULTS +71 73 α3 structure_element The single cleavage site of PmC11 at Lys147 is found immediately after α3, in loop L5 within the central β-sheet (Figs. 1, A and B, and 2A). RESULTS +78 82 loop structure_element The single cleavage site of PmC11 at Lys147 is found immediately after α3, in loop L5 within the central β-sheet (Figs. 1, A and B, and 2A). RESULTS +83 85 L5 structure_element The single cleavage site of PmC11 at Lys147 is found immediately after α3, in loop L5 within the central β-sheet (Figs. 1, A and B, and 2A). RESULTS +105 112 β-sheet structure_element The single cleavage site of PmC11 at Lys147 is found immediately after α3, in loop L5 within the central β-sheet (Figs. 1, A and B, and 2A). RESULTS +20 33 cleavage site site The two ends of the cleavage site are remarkably well ordered in the crystal structure and displaced from one another by 19.5 Å (Fig. 2A). RESULTS +69 86 crystal structure evidence The two ends of the cleavage site are remarkably well ordered in the crystal structure and displaced from one another by 19.5 Å (Fig. 2A). RESULTS +37 50 cleavage site site Moreover, the C-terminal side of the cleavage site resides near the catalytic dyad with Ala148 being 4.5 and 5.7 Å from His133 and Cys179, respectively. RESULTS +68 82 catalytic dyad site Moreover, the C-terminal side of the cleavage site resides near the catalytic dyad with Ala148 being 4.5 and 5.7 Å from His133 and Cys179, respectively. RESULTS +88 94 Ala148 residue_name_number Moreover, the C-terminal side of the cleavage site resides near the catalytic dyad with Ala148 being 4.5 and 5.7 Å from His133 and Cys179, respectively. RESULTS +120 126 His133 residue_name_number Moreover, the C-terminal side of the cleavage site resides near the catalytic dyad with Ala148 being 4.5 and 5.7 Å from His133 and Cys179, respectively. RESULTS +131 137 Cys179 residue_name_number Moreover, the C-terminal side of the cleavage site resides near the catalytic dyad with Ala148 being 4.5 and 5.7 Å from His133 and Cys179, respectively. RESULTS +43 48 helix structure_element Consequently, it appears feasible that the helix attached to Lys147 (α3) could be responsible for steric autoinhibition of PmC11 when Lys147 is covalently bonded to Ala148. RESULTS +61 67 Lys147 residue_name_number Consequently, it appears feasible that the helix attached to Lys147 (α3) could be responsible for steric autoinhibition of PmC11 when Lys147 is covalently bonded to Ala148. RESULTS +69 71 α3 structure_element Consequently, it appears feasible that the helix attached to Lys147 (α3) could be responsible for steric autoinhibition of PmC11 when Lys147 is covalently bonded to Ala148. RESULTS +123 128 PmC11 protein Consequently, it appears feasible that the helix attached to Lys147 (α3) could be responsible for steric autoinhibition of PmC11 when Lys147 is covalently bonded to Ala148. RESULTS +134 140 Lys147 residue_name_number Consequently, it appears feasible that the helix attached to Lys147 (α3) could be responsible for steric autoinhibition of PmC11 when Lys147 is covalently bonded to Ala148. RESULTS +165 171 Ala148 residue_name_number Consequently, it appears feasible that the helix attached to Lys147 (α3) could be responsible for steric autoinhibition of PmC11 when Lys147 is covalently bonded to Ala148. RESULTS +10 18 cleavage ptm Thus, the cleavage would be required for full activation of PmC11. RESULTS +41 56 full activation protein_state Thus, the cleavage would be required for full activation of PmC11. RESULTS +60 65 PmC11 protein Thus, the cleavage would be required for full activation of PmC11. RESULTS +78 88 PmC11C179A mutant To investigate this possibility, two mutant forms of the enzyme were created: PmC11C179A (a catalytically inactive mutant) and PmC11K147A (a cleavage-site mutant). RESULTS +92 121 catalytically inactive mutant protein_state To investigate this possibility, two mutant forms of the enzyme were created: PmC11C179A (a catalytically inactive mutant) and PmC11K147A (a cleavage-site mutant). RESULTS +127 137 PmC11K147A mutant To investigate this possibility, two mutant forms of the enzyme were created: PmC11C179A (a catalytically inactive mutant) and PmC11K147A (a cleavage-site mutant). RESULTS +141 161 cleavage-site mutant protein_state To investigate this possibility, two mutant forms of the enzyme were created: PmC11C179A (a catalytically inactive mutant) and PmC11K147A (a cleavage-site mutant). RESULTS +8 16 SDS-PAGE experimental_method Initial SDS-PAGE and Western blot analysis of both mutants revealed no discernible processing occurred as compared with active PmC11 (Fig. 2C). RESULTS +21 33 Western blot experimental_method Initial SDS-PAGE and Western blot analysis of both mutants revealed no discernible processing occurred as compared with active PmC11 (Fig. 2C). RESULTS +120 126 active protein_state Initial SDS-PAGE and Western blot analysis of both mutants revealed no discernible processing occurred as compared with active PmC11 (Fig. 2C). RESULTS +127 132 PmC11 protein Initial SDS-PAGE and Western blot analysis of both mutants revealed no discernible processing occurred as compared with active PmC11 (Fig. 2C). RESULTS +4 14 PmC11K147A mutant The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D). RESULTS +15 21 mutant protein_state The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D). RESULTS +54 67 reaction rate evidence The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D). RESULTS +69 73 Vmax evidence The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D). RESULTS +89 91 WT protein_state The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D). RESULTS +103 120 reaction velocity evidence The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D). RESULTS +124 129 PmC11 protein The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D). RESULTS +164 174 PmC11K147A mutant The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D). RESULTS +39 44 PmC11 protein Taken together, these data reveal that PmC11 requires processing at Lys147 for optimum activity. RESULTS +68 74 Lys147 residue_name_number Taken together, these data reveal that PmC11 requires processing at Lys147 for optimum activity. RESULTS +88 98 PmC11C179A mutant To investigate whether processing is a result of intra- or intermolecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of processed and activated PmC11. RESULTS +99 105 mutant protein_state To investigate whether processing is a result of intra- or intermolecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of processed and activated PmC11. RESULTS +110 150 incubated with increasing concentrations experimental_method To investigate whether processing is a result of intra- or intermolecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of processed and activated PmC11. RESULTS +154 163 processed protein_state To investigate whether processing is a result of intra- or intermolecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of processed and activated PmC11. RESULTS +168 177 activated protein_state To investigate whether processing is a result of intra- or intermolecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of processed and activated PmC11. RESULTS +178 183 PmC11 protein To investigate whether processing is a result of intra- or intermolecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of processed and activated PmC11. RESULTS +62 72 PmC11C179A mutant These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). RESULTS +80 86 active protein_state These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). RESULTS +94 115 at low concentrations experimental_method These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). RESULTS +119 124 PmC11 protein These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). RESULTS +187 193 active protein_state These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). RESULTS +202 207 PmC11 protein These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). RESULTS +208 218 PmC11C179A mutant These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). RESULTS +224 250 increased to ∼1:10 and 1:4 experimental_method These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). RESULTS +289 301 ratio of 1:1 experimental_method These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to ∼1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E). RESULTS +19 27 cleavage ptm This suggests that cleavage of PmC11C179A was most likely an effect of the increasing concentration of PmC11 and intermolecular cleavage. RESULTS +31 41 PmC11C179A mutant This suggests that cleavage of PmC11C179A was most likely an effect of the increasing concentration of PmC11 and intermolecular cleavage. RESULTS +103 108 PmC11 protein This suggests that cleavage of PmC11C179A was most likely an effect of the increasing concentration of PmC11 and intermolecular cleavage. RESULTS +42 50 pro-form protein_state Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147. RESULTS +54 59 PmC11 protein Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147. RESULTS +63 76 autoinhibited protein_state Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147. RESULTS +93 95 L5 structure_element Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147. RESULTS +119 130 active site site Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147. RESULTS +141 164 intramolecular cleavage ptm Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147. RESULTS +168 174 Lys147 residue_name_number Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147. RESULTS +5 13 cleavage ptm This cleavage subsequently allows movement of the region containing Lys147 and the active site to open up for substrate access. RESULTS +68 74 Lys147 residue_name_number This cleavage subsequently allows movement of the region containing Lys147 and the active site to open up for substrate access. RESULTS +83 94 active site site This cleavage subsequently allows movement of the region containing Lys147 and the active site to open up for substrate access. RESULTS +98 102 open protein_state This cleavage subsequently allows movement of the region containing Lys147 and the active site to open up for substrate access. RESULTS +25 30 PmC11 protein Substrate Specificity of PmC11 RESULTS +4 26 autocatalytic cleavage ptm The autocatalytic cleavage of PmC11 at Lys147 (sequence KLK∧A) demonstrates that the enzyme accepts substrates with Lys in the P1 position. RESULTS +30 35 PmC11 protein The autocatalytic cleavage of PmC11 at Lys147 (sequence KLK∧A) demonstrates that the enzyme accepts substrates with Lys in the P1 position. RESULTS +39 45 Lys147 residue_name_number The autocatalytic cleavage of PmC11 at Lys147 (sequence KLK∧A) demonstrates that the enzyme accepts substrates with Lys in the P1 position. RESULTS +116 119 Lys residue_name The autocatalytic cleavage of PmC11 at Lys147 (sequence KLK∧A) demonstrates that the enzyme accepts substrates with Lys in the P1 position. RESULTS +127 129 P1 residue_number The autocatalytic cleavage of PmC11 at Lys147 (sequence KLK∧A) demonstrates that the enzyme accepts substrates with Lys in the P1 position. RESULTS +13 18 PmC11 protein As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. RESULTS +62 65 Pro residue_name As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. RESULTS +69 72 Asp residue_name As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. RESULTS +76 78 P1 residue_number As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. RESULTS +87 93 active protein_state As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. RESULTS +136 138 P1 residue_number As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. RESULTS +147 155 Bz-R-AMC chemical As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. RESULTS +157 166 Z-GGR-AMC chemical As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. RESULTS +172 183 BOC-VLK-AMC chemical As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC. RESULTS +59 62 Arg residue_name The rate of cleavage was ∼3-fold greater toward the single Arg substrate Bz-R-AMC than for the other two (Fig. 2F) and, unexpectedly, PmC11 showed no activity toward BOC-K-AMC. RESULTS +73 81 Bz-R-AMC chemical The rate of cleavage was ∼3-fold greater toward the single Arg substrate Bz-R-AMC than for the other two (Fig. 2F) and, unexpectedly, PmC11 showed no activity toward BOC-K-AMC. RESULTS +134 139 PmC11 protein The rate of cleavage was ∼3-fold greater toward the single Arg substrate Bz-R-AMC than for the other two (Fig. 2F) and, unexpectedly, PmC11 showed no activity toward BOC-K-AMC. RESULTS +166 175 BOC-K-AMC chemical The rate of cleavage was ∼3-fold greater toward the single Arg substrate Bz-R-AMC than for the other two (Fig. 2F) and, unexpectedly, PmC11 showed no activity toward BOC-K-AMC. RESULTS +27 32 PmC11 protein These results confirm that PmC11 accepts substrates containing Arg or Lys in P1 with a possible preference for Arg. RESULTS +63 66 Arg residue_name These results confirm that PmC11 accepts substrates containing Arg or Lys in P1 with a possible preference for Arg. RESULTS +70 73 Lys residue_name These results confirm that PmC11 accepts substrates containing Arg or Lys in P1 with a possible preference for Arg. RESULTS +77 79 P1 residue_number These results confirm that PmC11 accepts substrates containing Arg or Lys in P1 with a possible preference for Arg. RESULTS +111 114 Arg residue_name These results confirm that PmC11 accepts substrates containing Arg or Lys in P1 with a possible preference for Arg. RESULTS +4 18 catalytic dyad site The catalytic dyad of PmC11 sits near the bottom of an open pocket on the surface of the enzyme at a conserved location in the clan CD family. RESULTS +22 27 PmC11 protein The catalytic dyad of PmC11 sits near the bottom of an open pocket on the surface of the enzyme at a conserved location in the clan CD family. RESULTS +55 59 open protein_state The catalytic dyad of PmC11 sits near the bottom of an open pocket on the surface of the enzyme at a conserved location in the clan CD family. RESULTS +60 66 pocket site The catalytic dyad of PmC11 sits near the bottom of an open pocket on the surface of the enzyme at a conserved location in the clan CD family. RESULTS +101 119 conserved location protein_state The catalytic dyad of PmC11 sits near the bottom of an open pocket on the surface of the enzyme at a conserved location in the clan CD family. RESULTS +132 141 CD family protein_type The catalytic dyad of PmC11 sits near the bottom of an open pocket on the surface of the enzyme at a conserved location in the clan CD family. RESULTS +4 9 PmC11 protein The PmC11 structure reveals that the catalytic dyad forms part of a large acidic pocket (Fig. 2G), consistent with a binding site for a basic substrate. RESULTS +10 19 structure evidence The PmC11 structure reveals that the catalytic dyad forms part of a large acidic pocket (Fig. 2G), consistent with a binding site for a basic substrate. RESULTS +37 51 catalytic dyad site The PmC11 structure reveals that the catalytic dyad forms part of a large acidic pocket (Fig. 2G), consistent with a binding site for a basic substrate. RESULTS +74 87 acidic pocket site The PmC11 structure reveals that the catalytic dyad forms part of a large acidic pocket (Fig. 2G), consistent with a binding site for a basic substrate. RESULTS +117 129 binding site site The PmC11 structure reveals that the catalytic dyad forms part of a large acidic pocket (Fig. 2G), consistent with a binding site for a basic substrate. RESULTS +5 11 pocket site This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A). RESULTS +66 71 Asn50 residue_name_number This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A). RESULTS +73 79 Asp177 residue_name_number This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A). RESULTS +85 91 Thr204 residue_name_number This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A). RESULTS +97 103 Gly134 residue_name_number This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A). RESULTS +105 111 Asp207 residue_name_number This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A). RESULTS +117 123 Met205 residue_name_number This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A). RESULTS +149 155 pocket site This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A). RESULTS +54 74 structurally similar protein_state Interestingly, these residues are in regions that are structurally similar to those involved in the S1 binding pockets of other clan CD members (shown in Ref.). RESULTS +100 118 S1 binding pockets site Interestingly, these residues are in regions that are structurally similar to those involved in the S1 binding pockets of other clan CD members (shown in Ref.). RESULTS +128 143 clan CD members protein_type Interestingly, these residues are in regions that are structurally similar to those involved in the S1 binding pockets of other clan CD members (shown in Ref.). RESULTS +8 13 PmC11 protein Because PmC11 recognizes basic substrates, the tetrapeptide inhibitor Z-VRPR-FMK was tested as an enzyme inhibitor and was found to inhibit both the autoprocessing and activity of PmC11 (Fig. 3A). RESULTS +70 80 Z-VRPR-FMK chemical Because PmC11 recognizes basic substrates, the tetrapeptide inhibitor Z-VRPR-FMK was tested as an enzyme inhibitor and was found to inhibit both the autoprocessing and activity of PmC11 (Fig. 3A). RESULTS +132 139 inhibit protein_state Because PmC11 recognizes basic substrates, the tetrapeptide inhibitor Z-VRPR-FMK was tested as an enzyme inhibitor and was found to inhibit both the autoprocessing and activity of PmC11 (Fig. 3A). RESULTS +149 163 autoprocessing ptm Because PmC11 recognizes basic substrates, the tetrapeptide inhibitor Z-VRPR-FMK was tested as an enzyme inhibitor and was found to inhibit both the autoprocessing and activity of PmC11 (Fig. 3A). RESULTS +180 185 PmC11 protein Because PmC11 recognizes basic substrates, the tetrapeptide inhibitor Z-VRPR-FMK was tested as an enzyme inhibitor and was found to inhibit both the autoprocessing and activity of PmC11 (Fig. 3A). RESULTS +0 10 Z-VRPR-FMK chemical Z-VRPR-FMK was also shown to bind to the enzyme: a size-shift was observed, by SDS-PAGE analysis, in the larger processed product of PmC11 suggesting that the inhibitor bound to the active site (Fig. 3B). RESULTS +51 61 size-shift evidence Z-VRPR-FMK was also shown to bind to the enzyme: a size-shift was observed, by SDS-PAGE analysis, in the larger processed product of PmC11 suggesting that the inhibitor bound to the active site (Fig. 3B). RESULTS +79 87 SDS-PAGE experimental_method Z-VRPR-FMK was also shown to bind to the enzyme: a size-shift was observed, by SDS-PAGE analysis, in the larger processed product of PmC11 suggesting that the inhibitor bound to the active site (Fig. 3B). RESULTS +133 138 PmC11 protein Z-VRPR-FMK was also shown to bind to the enzyme: a size-shift was observed, by SDS-PAGE analysis, in the larger processed product of PmC11 suggesting that the inhibitor bound to the active site (Fig. 3B). RESULTS +159 174 inhibitor bound protein_state Z-VRPR-FMK was also shown to bind to the enzyme: a size-shift was observed, by SDS-PAGE analysis, in the larger processed product of PmC11 suggesting that the inhibitor bound to the active site (Fig. 3B). RESULTS +182 193 active site site Z-VRPR-FMK was also shown to bind to the enzyme: a size-shift was observed, by SDS-PAGE analysis, in the larger processed product of PmC11 suggesting that the inhibitor bound to the active site (Fig. 3B). RESULTS +2 19 structure overlay experimental_method A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +23 28 PmC11 protein A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +38 57 MALT1-paracacaspase protein A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +59 66 MALT1-P protein A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +72 79 complex protein_state A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +85 95 Z-VRPR-FMK chemical A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +115 120 PmC11 protein A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +121 125 dyad site A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +169 175 active protein_state A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +176 183 MALT1-P protein A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +193 198 Asn50 residue_name_number A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +200 206 Asp177 residue_name_number A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +212 218 Asp207 residue_name_number A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +255 262 MALT1-P protein A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +263 289 inhibitor binding residues site A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +291 297 Asp365 residue_name_number A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +299 305 Asp462 residue_name_number A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +311 317 Glu500 residue_name_number A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +333 341 VRPR-FMK chemical A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +347 354 MALT1-P protein A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +378 383 PmC11 protein A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +402 420 structural overlay experimental_method A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.). RESULTS +0 6 Asp177 residue_name_number Asp177 is located near the catalytic cysteine and is conserved throughout the C11 family, suggesting it is the primary S1 binding site residue. RESULTS +27 36 catalytic protein_state Asp177 is located near the catalytic cysteine and is conserved throughout the C11 family, suggesting it is the primary S1 binding site residue. RESULTS +37 45 cysteine residue_name Asp177 is located near the catalytic cysteine and is conserved throughout the C11 family, suggesting it is the primary S1 binding site residue. RESULTS +53 73 conserved throughout protein_state Asp177 is located near the catalytic cysteine and is conserved throughout the C11 family, suggesting it is the primary S1 binding site residue. RESULTS +78 88 C11 family protein_type Asp177 is located near the catalytic cysteine and is conserved throughout the C11 family, suggesting it is the primary S1 binding site residue. RESULTS +119 142 S1 binding site residue site Asp177 is located near the catalytic cysteine and is conserved throughout the C11 family, suggesting it is the primary S1 binding site residue. RESULTS +7 16 structure evidence In the structure of PmC11, Asp207 resides on a flexible loop pointing away from the S1 binding pocket (Fig. 3C). RESULTS +20 25 PmC11 protein In the structure of PmC11, Asp207 resides on a flexible loop pointing away from the S1 binding pocket (Fig. 3C). RESULTS +27 33 Asp207 residue_name_number In the structure of PmC11, Asp207 resides on a flexible loop pointing away from the S1 binding pocket (Fig. 3C). RESULTS +56 60 loop structure_element In the structure of PmC11, Asp207 resides on a flexible loop pointing away from the S1 binding pocket (Fig. 3C). RESULTS +84 101 S1 binding pocket site In the structure of PmC11, Asp207 resides on a flexible loop pointing away from the S1 binding pocket (Fig. 3C). RESULTS +14 18 loop structure_element However, this loop has been shown to be important for substrate binding in clan CD and this residue could easily rotate and be involved in substrate binding in PmC11. RESULTS +75 82 clan CD protein_type However, this loop has been shown to be important for substrate binding in clan CD and this residue could easily rotate and be involved in substrate binding in PmC11. RESULTS +160 165 PmC11 protein However, this loop has been shown to be important for substrate binding in clan CD and this residue could easily rotate and be involved in substrate binding in PmC11. RESULTS +6 11 Asn50 residue_name_number Thus, Asn50, Asp177, and Asp207 are most likely responsible for the substrate specificity of PmC11. RESULTS +13 19 Asp177 residue_name_number Thus, Asn50, Asp177, and Asp207 are most likely responsible for the substrate specificity of PmC11. RESULTS +25 31 Asp207 residue_name_number Thus, Asn50, Asp177, and Asp207 are most likely responsible for the substrate specificity of PmC11. RESULTS +93 98 PmC11 protein Thus, Asn50, Asp177, and Asp207 are most likely responsible for the substrate specificity of PmC11. RESULTS +0 6 Asp177 residue_name_number Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C). RESULTS +10 26 highly conserved protein_state Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C). RESULTS +42 64 clan CD C11 peptidases protein_type Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C). RESULTS +141 156 clan CD enzymes protein_type Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C). RESULTS +241 251 Z-VRPR-FMK chemical Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C). RESULTS +257 262 PmC11 protein Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C). RESULTS +266 274 overlaid experimental_method Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C). RESULTS +282 289 MALT1-P protein Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C). RESULTS +290 299 structure evidence Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C). RESULTS +0 5 PmC11 protein PmC11 binds and is inhibited by Z-VRPR-FMK and does not require Ca2+ for activity. FIG +32 42 Z-VRPR-FMK chemical PmC11 binds and is inhibited by Z-VRPR-FMK and does not require Ca2+ for activity. FIG +64 68 Ca2+ chemical PmC11 binds and is inhibited by Z-VRPR-FMK and does not require Ca2+ for activity. FIG +35 45 Z-VRPR-FMK chemical A, PmC11 activity is inhibited by Z-VRPR-FMK. FIG +12 20 Bz-R-AMC chemical Cleavage of Bz-R-AMC by PmC11 was measured in a fluorometric activity assay with (+, purple) and without (−, red) Z-VRPR-FMK. FIG +24 29 PmC11 protein Cleavage of Bz-R-AMC by PmC11 was measured in a fluorometric activity assay with (+, purple) and without (−, red) Z-VRPR-FMK. FIG +48 75 fluorometric activity assay experimental_method Cleavage of Bz-R-AMC by PmC11 was measured in a fluorometric activity assay with (+, purple) and without (−, red) Z-VRPR-FMK. FIG +114 124 Z-VRPR-FMK chemical Cleavage of Bz-R-AMC by PmC11 was measured in a fluorometric activity assay with (+, purple) and without (−, red) Z-VRPR-FMK. FIG +3 18 gel-shift assay experimental_method B, gel-shift assay reveals that Z-VRPR-FMK binds to PmC11. FIG +32 42 Z-VRPR-FMK chemical B, gel-shift assay reveals that Z-VRPR-FMK binds to PmC11. FIG +52 57 PmC11 protein B, gel-shift assay reveals that Z-VRPR-FMK binds to PmC11. FIG +10 19 incubated experimental_method PmC11 was incubated with (+) or without (−) Z-VRPR-FMK and the samples analyzed on a 10% SDS-PAGE gel. FIG +44 54 Z-VRPR-FMK chemical PmC11 was incubated with (+) or without (−) Z-VRPR-FMK and the samples analyzed on a 10% SDS-PAGE gel. FIG +89 97 SDS-PAGE experimental_method PmC11 was incubated with (+) or without (−) Z-VRPR-FMK and the samples analyzed on a 10% SDS-PAGE gel. FIG +2 12 size shift evidence A size shift can be observed in the larger processed product of PmC11 (26.1 kDa). FIG +64 69 PmC11 protein A size shift can be observed in the larger processed product of PmC11 (26.1 kDa). FIG +3 8 PmC11 protein C, PmC11 with the Z-VRPR-FMK from the MALT1-paracacaspase (MALT1-P) superimposed. FIG +18 28 Z-VRPR-FMK chemical C, PmC11 with the Z-VRPR-FMK from the MALT1-paracacaspase (MALT1-P) superimposed. FIG +38 57 MALT1-paracacaspase protein C, PmC11 with the Z-VRPR-FMK from the MALT1-paracacaspase (MALT1-P) superimposed. FIG +59 66 MALT1-P protein C, PmC11 with the Z-VRPR-FMK from the MALT1-paracacaspase (MALT1-P) superimposed. FIG +68 80 superimposed experimental_method C, PmC11 with the Z-VRPR-FMK from the MALT1-paracacaspase (MALT1-P) superimposed. FIG +2 38 three-dimensional structural overlay experimental_method A three-dimensional structural overlay of Z-VRPR-FMK from the MALT1-P complex onto PmC11. FIG +42 52 Z-VRPR-FMK chemical A three-dimensional structural overlay of Z-VRPR-FMK from the MALT1-P complex onto PmC11. FIG +62 69 MALT1-P protein A three-dimensional structural overlay of Z-VRPR-FMK from the MALT1-P complex onto PmC11. FIG +83 88 PmC11 protein A three-dimensional structural overlay of Z-VRPR-FMK from the MALT1-P complex onto PmC11. FIG +32 42 Z-VRPR-FMK chemical The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11. FIG +58 71 superposition experimental_method The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11. FIG +79 84 PmC11 protein The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11. FIG +89 96 MALTI_P protein The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11. FIG +97 107 structures evidence The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11. FIG +135 146 active site site The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11. FIG +150 155 PmC11 protein The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11. FIG +83 104 binding site residues site Residues surrounding the inhibitor are labeled and represent potentially important binding site residues, labeled in black and shown in an atomic representation. FIG +52 57 PmC11 protein C, divalent cations do not increase the activity of PmC11. FIG +16 24 Bz-R-AMC chemical The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). FIG +28 33 PmC11 protein The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). FIG +78 82 Ca2+ chemical The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). FIG +84 88 Mn2+ chemical The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). FIG +90 94 Zn2+ chemical The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). FIG +96 100 Co2+ chemical The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). FIG +102 106 Cu2+ chemical The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). FIG +108 112 Mg2+ chemical The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). FIG +118 122 Fe3+ chemical The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). FIG +128 132 EGTA chemical The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). FIG +160 203 relative fluorescence measured against time experimental_method The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min). FIG +4 23 addition of cations experimental_method The addition of cations produced no improvement in activity of PmC11 when compared in the presence of EGTA, suggesting that PmC11 does not require metal ions for proteolytic activity. FIG +63 68 PmC11 protein The addition of cations produced no improvement in activity of PmC11 when compared in the presence of EGTA, suggesting that PmC11 does not require metal ions for proteolytic activity. FIG +102 106 EGTA chemical The addition of cations produced no improvement in activity of PmC11 when compared in the presence of EGTA, suggesting that PmC11 does not require metal ions for proteolytic activity. FIG +124 129 PmC11 protein The addition of cations produced no improvement in activity of PmC11 when compared in the presence of EGTA, suggesting that PmC11 does not require metal ions for proteolytic activity. FIG +13 17 Cu2+ chemical Furthermore, Cu2+, Fe2+, and Zn2+ appear to inhibit PmC11. FIG +19 23 Fe2+ chemical Furthermore, Cu2+, Fe2+, and Zn2+ appear to inhibit PmC11. FIG +29 33 Zn2+ chemical Furthermore, Cu2+, Fe2+, and Zn2+ appear to inhibit PmC11. FIG +44 51 inhibit protein_state Furthermore, Cu2+, Fe2+, and Zn2+ appear to inhibit PmC11. FIG +52 57 PmC11 protein Furthermore, Cu2+, Fe2+, and Zn2+ appear to inhibit PmC11. FIG +16 27 Clostripain protein Comparison with Clostripain RESULTS +0 11 Clostripain protein Clostripain from C. histolyticum is the founding member of the C11 family of peptidases and contains an additional 149 residues compared with PmC11. RESULTS +17 32 C. histolyticum species Clostripain from C. histolyticum is the founding member of the C11 family of peptidases and contains an additional 149 residues compared with PmC11. RESULTS +63 73 C11 family protein_type Clostripain from C. histolyticum is the founding member of the C11 family of peptidases and contains an additional 149 residues compared with PmC11. RESULTS +77 87 peptidases protein_type Clostripain from C. histolyticum is the founding member of the C11 family of peptidases and contains an additional 149 residues compared with PmC11. RESULTS +115 127 149 residues residue_range Clostripain from C. histolyticum is the founding member of the C11 family of peptidases and contains an additional 149 residues compared with PmC11. RESULTS +142 147 PmC11 protein Clostripain from C. histolyticum is the founding member of the C11 family of peptidases and contains an additional 149 residues compared with PmC11. RESULTS +2 29 multiple sequence alignment experimental_method A multiple sequence alignment revealed that most of the secondary structural elements are conserved between the two enzymes, although they are only ∼23% identical (Fig. 1A). RESULTS +56 85 secondary structural elements structure_element A multiple sequence alignment revealed that most of the secondary structural elements are conserved between the two enzymes, although they are only ∼23% identical (Fig. 1A). RESULTS +90 99 conserved protein_state A multiple sequence alignment revealed that most of the secondary structural elements are conserved between the two enzymes, although they are only ∼23% identical (Fig. 1A). RESULTS +14 19 PmC11 protein Nevertheless, PmC11 may be a good model for the core structure of clostripain. RESULTS +66 77 clostripain protein Nevertheless, PmC11 may be a good model for the core structure of clostripain. RESULTS +4 32 primary structural alignment experimental_method The primary structural alignment also shows that the catalytic dyad in PmC11 is structurally conserved in clostripain (Fig. 1A). RESULTS +53 67 catalytic dyad site The primary structural alignment also shows that the catalytic dyad in PmC11 is structurally conserved in clostripain (Fig. 1A). RESULTS +71 76 PmC11 protein The primary structural alignment also shows that the catalytic dyad in PmC11 is structurally conserved in clostripain (Fig. 1A). RESULTS +80 102 structurally conserved protein_state The primary structural alignment also shows that the catalytic dyad in PmC11 is structurally conserved in clostripain (Fig. 1A). RESULTS +106 117 clostripain protein The primary structural alignment also shows that the catalytic dyad in PmC11 is structurally conserved in clostripain (Fig. 1A). RESULTS +7 12 PmC11 protein Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A). RESULTS +14 25 clostripain protein Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A). RESULTS +34 48 cleavage sites site Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A). RESULTS +50 56 Arg181 residue_name_number Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A). RESULTS +61 67 Arg190 residue_name_number Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A). RESULTS +104 115 nonapeptide structure_element Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A). RESULTS +137 152 full activation protein_state Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A). RESULTS +15 21 Arg190 residue_name_number Interestingly, Arg190 was found to align with Lys147 in PmC11. RESULTS +46 52 Lys147 residue_name_number Interestingly, Arg190 was found to align with Lys147 in PmC11. RESULTS +56 61 PmC11 protein Interestingly, Arg190 was found to align with Lys147 in PmC11. RESULTS +35 53 S1-binding residue site In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A). RESULTS +57 62 PmC11 protein In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A). RESULTS +63 69 Asp177 residue_name_number In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A). RESULTS +75 83 overlays experimental_method In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A). RESULTS +121 155 P1 specificity determining residue site In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A). RESULTS +159 170 clostripain protein In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A). RESULTS +172 178 Asp229 residue_name_number In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A). RESULTS +14 25 clostripain protein As studies on clostripain revealed addition of Ca2+ ions are required for full activation, the Ca2+ dependence of PmC11 was examined. RESULTS +47 51 Ca2+ chemical As studies on clostripain revealed addition of Ca2+ ions are required for full activation, the Ca2+ dependence of PmC11 was examined. RESULTS +74 89 full activation protein_state As studies on clostripain revealed addition of Ca2+ ions are required for full activation, the Ca2+ dependence of PmC11 was examined. RESULTS +95 99 Ca2+ chemical As studies on clostripain revealed addition of Ca2+ ions are required for full activation, the Ca2+ dependence of PmC11 was examined. RESULTS +114 119 PmC11 protein As studies on clostripain revealed addition of Ca2+ ions are required for full activation, the Ca2+ dependence of PmC11 was examined. RESULTS +14 18 Ca2+ chemical Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). RESULTS +35 40 PmC11 protein Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). RESULTS +92 96 Mg2+ chemical Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). RESULTS +98 102 Mn2+ chemical Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). RESULTS +104 108 Co2+ chemical Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). RESULTS +110 114 Fe2+ chemical Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). RESULTS +116 120 Zn2+ chemical Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). RESULTS +126 130 Cu2+ chemical Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). RESULTS +155 160 PmC11 protein Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D). RESULTS +30 34 EGTA chemical In support of these findings, EGTA did not inhibit PmC11 suggesting that, unlike clostripain, PmC11 does not require Ca2+ or other divalent cations, for activity. RESULTS +51 56 PmC11 protein In support of these findings, EGTA did not inhibit PmC11 suggesting that, unlike clostripain, PmC11 does not require Ca2+ or other divalent cations, for activity. RESULTS +81 92 clostripain protein In support of these findings, EGTA did not inhibit PmC11 suggesting that, unlike clostripain, PmC11 does not require Ca2+ or other divalent cations, for activity. RESULTS +94 99 PmC11 protein In support of these findings, EGTA did not inhibit PmC11 suggesting that, unlike clostripain, PmC11 does not require Ca2+ or other divalent cations, for activity. RESULTS +117 121 Ca2+ chemical In support of these findings, EGTA did not inhibit PmC11 suggesting that, unlike clostripain, PmC11 does not require Ca2+ or other divalent cations, for activity. RESULTS +4 21 crystal structure evidence The crystal structure of PmC11 now provides three-dimensional information for a member of the clostripain C11 family of cysteine peptidases. DISCUSS +25 30 PmC11 protein The crystal structure of PmC11 now provides three-dimensional information for a member of the clostripain C11 family of cysteine peptidases. DISCUSS +94 105 clostripain protein The crystal structure of PmC11 now provides three-dimensional information for a member of the clostripain C11 family of cysteine peptidases. DISCUSS +106 116 C11 family protein_type The crystal structure of PmC11 now provides three-dimensional information for a member of the clostripain C11 family of cysteine peptidases. DISCUSS +120 139 cysteine peptidases protein_type The crystal structure of PmC11 now provides three-dimensional information for a member of the clostripain C11 family of cysteine peptidases. DISCUSS +58 73 clan CD members protein_type The enzyme exhibits all of the key structural elements of clan CD members, but is unusual in that it has a nine-stranded central β-sheet with a novel C-terminal domain. DISCUSS +129 136 β-sheet structure_element The enzyme exhibits all of the key structural elements of clan CD members, but is unusual in that it has a nine-stranded central β-sheet with a novel C-terminal domain. DISCUSS +150 167 C-terminal domain structure_element The enzyme exhibits all of the key structural elements of clan CD members, but is unusual in that it has a nine-stranded central β-sheet with a novel C-terminal domain. DISCUSS +29 34 PmC11 protein The structural similarity of PmC11 with its nearest structural neighbors in the PDB is decidedly low, overlaying better with six-stranded caspase-7 than any of the other larger members of the clan (Table 2). DISCUSS +138 147 caspase-7 protein The structural similarity of PmC11 with its nearest structural neighbors in the PDB is decidedly low, overlaying better with six-stranded caspase-7 than any of the other larger members of the clan (Table 2). DISCUSS +29 34 PmC11 protein The substrate specificity of PmC11 is Arg/Lys and the crystal structure revealed an acidic pocket for specific binding of such basic substrates. DISCUSS +38 41 Arg residue_name The substrate specificity of PmC11 is Arg/Lys and the crystal structure revealed an acidic pocket for specific binding of such basic substrates. DISCUSS +42 45 Lys residue_name The substrate specificity of PmC11 is Arg/Lys and the crystal structure revealed an acidic pocket for specific binding of such basic substrates. DISCUSS +54 71 crystal structure evidence The substrate specificity of PmC11 is Arg/Lys and the crystal structure revealed an acidic pocket for specific binding of such basic substrates. DISCUSS +84 97 acidic pocket site The substrate specificity of PmC11 is Arg/Lys and the crystal structure revealed an acidic pocket for specific binding of such basic substrates. DISCUSS +17 26 structure evidence In addition, the structure suggested a mechanism of self-inhibition in both PmC11 and clostripain and an activation mechanism that requires autoprocessing. DISCUSS +76 81 PmC11 protein In addition, the structure suggested a mechanism of self-inhibition in both PmC11 and clostripain and an activation mechanism that requires autoprocessing. DISCUSS +86 97 clostripain protein In addition, the structure suggested a mechanism of self-inhibition in both PmC11 and clostripain and an activation mechanism that requires autoprocessing. DISCUSS +140 154 autoprocessing ptm In addition, the structure suggested a mechanism of self-inhibition in both PmC11 and clostripain and an activation mechanism that requires autoprocessing. DISCUSS +0 5 PmC11 protein PmC11 differs from clostripain in that is does not appear to require divalent cations for activation. DISCUSS +19 30 clostripain protein PmC11 differs from clostripain in that is does not appear to require divalent cations for activation. DISCUSS +25 32 clan CD protein_type Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7. DISCUSS +41 51 processing ptm Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7. DISCUSS +56 71 full activation protein_state Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7. DISCUSS +82 90 legumain protein Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7. DISCUSS +92 103 gingipain-R protein Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7. DISCUSS +105 114 MARTX-CPD protein Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7. DISCUSS +124 141 effector caspases protein_type Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7. DISCUSS +148 157 caspase-7 protein Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7. DISCUSS +13 30 effector caspases protein_type To date, the effector caspases are the only group of enzymes that require cleavage of a loop within the central β-sheet. DISCUSS +74 82 cleavage ptm To date, the effector caspases are the only group of enzymes that require cleavage of a loop within the central β-sheet. DISCUSS +88 92 loop structure_element To date, the effector caspases are the only group of enzymes that require cleavage of a loop within the central β-sheet. DISCUSS +112 119 β-sheet structure_element To date, the effector caspases are the only group of enzymes that require cleavage of a loop within the central β-sheet. DISCUSS +25 30 PmC11 protein This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases. DISCUSS +45 53 cleavage ptm This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases. DISCUSS +54 58 loop structure_element This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases. DISCUSS +106 114 caspases protein_type This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases. DISCUSS +131 140 catalytic protein_state This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases. DISCUSS +141 144 His residue_name This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases. DISCUSS +174 177 Cys residue_name This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases. DISCUSS +185 193 caspases protein_type This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases. DISCUSS +10 25 clan CD members protein_type All other clan CD members requiring cleavage for full activation do so at sites external to their central sheets. DISCUSS +36 44 cleavage ptm All other clan CD members requiring cleavage for full activation do so at sites external to their central sheets. DISCUSS +49 64 full activation protein_state All other clan CD members requiring cleavage for full activation do so at sites external to their central sheets. DISCUSS +74 79 sites site All other clan CD members requiring cleavage for full activation do so at sites external to their central sheets. DISCUSS +106 112 sheets structure_element All other clan CD members requiring cleavage for full activation do so at sites external to their central sheets. DISCUSS +4 12 caspases protein_type The caspases and gingipain-R both undergo intermolecular (trans) cleavage and legumain and MARTX-CPD are reported to perform intramolecular (cis) cleavage. DISCUSS +17 28 gingipain-R protein The caspases and gingipain-R both undergo intermolecular (trans) cleavage and legumain and MARTX-CPD are reported to perform intramolecular (cis) cleavage. DISCUSS +42 73 intermolecular (trans) cleavage ptm The caspases and gingipain-R both undergo intermolecular (trans) cleavage and legumain and MARTX-CPD are reported to perform intramolecular (cis) cleavage. DISCUSS +78 86 legumain protein The caspases and gingipain-R both undergo intermolecular (trans) cleavage and legumain and MARTX-CPD are reported to perform intramolecular (cis) cleavage. DISCUSS +91 100 MARTX-CPD protein The caspases and gingipain-R both undergo intermolecular (trans) cleavage and legumain and MARTX-CPD are reported to perform intramolecular (cis) cleavage. DISCUSS +125 154 intramolecular (cis) cleavage ptm The caspases and gingipain-R both undergo intermolecular (trans) cleavage and legumain and MARTX-CPD are reported to perform intramolecular (cis) cleavage. DISCUSS +32 39 clan CD protein_type In addition, several members of clan CD exhibit self-inhibition, whereby regions of the enzyme block access to the active site. DISCUSS +73 80 regions structure_element In addition, several members of clan CD exhibit self-inhibition, whereby regions of the enzyme block access to the active site. DISCUSS +115 126 active site site In addition, several members of clan CD exhibit self-inhibition, whereby regions of the enzyme block access to the active site. DISCUSS +5 10 PmC11 protein Like PmC11, these structures show preformed catalytic machinery and, for a substrate to gain access, movement and/or cleavage of the blocking region is required. DISCUSS +117 125 cleavage ptm Like PmC11, these structures show preformed catalytic machinery and, for a substrate to gain access, movement and/or cleavage of the blocking region is required. DISCUSS +133 148 blocking region structure_element Like PmC11, these structures show preformed catalytic machinery and, for a substrate to gain access, movement and/or cleavage of the blocking region is required. DISCUSS +4 13 structure evidence The structure of PmC11 gives the first insight into this class of relatively unexplored family of proteins and should allow important catalytic and substrate binding residues to be identified in a variety of orthologues. DISCUSS +17 22 PmC11 protein The structure of PmC11 gives the first insight into this class of relatively unexplored family of proteins and should allow important catalytic and substrate binding residues to be identified in a variety of orthologues. DISCUSS +48 53 PmC11 protein Indeed, insights gained from an analysis of the PmC11 structure revealed the identity of the Trypanosoma brucei PNT1 protein as a C11 cysteine peptidase with an essential role in organelle replication. DISCUSS +54 63 structure evidence Indeed, insights gained from an analysis of the PmC11 structure revealed the identity of the Trypanosoma brucei PNT1 protein as a C11 cysteine peptidase with an essential role in organelle replication. DISCUSS +93 111 Trypanosoma brucei species Indeed, insights gained from an analysis of the PmC11 structure revealed the identity of the Trypanosoma brucei PNT1 protein as a C11 cysteine peptidase with an essential role in organelle replication. DISCUSS +112 116 PNT1 protein Indeed, insights gained from an analysis of the PmC11 structure revealed the identity of the Trypanosoma brucei PNT1 protein as a C11 cysteine peptidase with an essential role in organelle replication. DISCUSS +130 152 C11 cysteine peptidase protein_type Indeed, insights gained from an analysis of the PmC11 structure revealed the identity of the Trypanosoma brucei PNT1 protein as a C11 cysteine peptidase with an essential role in organelle replication. DISCUSS +4 9 PmC11 protein The PmC11 structure should provide a good basis for structural modeling and, given the importance of other clan CD enzymes, this work should also advance the exploration of these peptidases and potentially identify new biologically important substrates. DISCUSS +10 19 structure evidence The PmC11 structure should provide a good basis for structural modeling and, given the importance of other clan CD enzymes, this work should also advance the exploration of these peptidases and potentially identify new biologically important substrates. DISCUSS +52 71 structural modeling experimental_method The PmC11 structure should provide a good basis for structural modeling and, given the importance of other clan CD enzymes, this work should also advance the exploration of these peptidases and potentially identify new biologically important substrates. DISCUSS +107 122 clan CD enzymes protein_type The PmC11 structure should provide a good basis for structural modeling and, given the importance of other clan CD enzymes, this work should also advance the exploration of these peptidases and potentially identify new biologically important substrates. DISCUSS +179 189 peptidases protein_type The PmC11 structure should provide a good basis for structural modeling and, given the importance of other clan CD enzymes, this work should also advance the exploration of these peptidases and potentially identify new biologically important substrates. DISCUSS diff --git a/annotation_CSV/PMC4852598.csv b/annotation_CSV/PMC4852598.csv new file mode 100644 index 0000000000000000000000000000000000000000..61b4d137274296de1da176432c884f331ca43d8d --- /dev/null +++ b/annotation_CSV/PMC4852598.csv @@ -0,0 +1,1229 @@ +anno_start anno_end anno_text entity_type sentence section +21 25 Mep2 protein_type Structural basis for Mep2 ammonium transceptor activation by phosphorylation TITLE +26 46 ammonium transceptor protein_type Structural basis for Mep2 ammonium transceptor activation by phosphorylation TITLE +61 76 phosphorylation ptm Structural basis for Mep2 ammonium transceptor activation by phosphorylation TITLE +0 13 Mep2 proteins protein_type Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. ABSTRACT +18 24 fungal taxonomy_domain Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. ABSTRACT +25 37 transceptors protein_type Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. ABSTRACT +69 77 ammonium chemical Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. ABSTRACT +89 95 fungal taxonomy_domain Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. ABSTRACT +0 4 Mep2 protein_type Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. ABSTRACT +38 53 phosphorylation ptm Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. ABSTRACT +15 39 X-ray crystal structures evidence Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. ABSTRACT +47 51 Mep2 protein_type Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. ABSTRACT +69 93 Saccharomyces cerevisiae species Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. ABSTRACT +98 114 Candida albicans species Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. ABSTRACT +170 182 transporters protein_type Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. ABSTRACT +187 205 not phosphorylated protein_state Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. ABSTRACT +221 227 closed protein_state Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. ABSTRACT +229 237 inactive protein_state Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. ABSTRACT +16 20 open protein_state Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. ABSTRACT +21 30 bacterial taxonomy_domain Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. ABSTRACT +31 52 ammonium transporters protein_type Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. ABSTRACT +54 72 non-phosphorylated protein_state Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. ABSTRACT +73 77 Mep2 protein_type Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. ABSTRACT +97 114 cytoplasmic loops structure_element Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. ABSTRACT +123 140 C-terminal region structure_element Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. ABSTRACT +142 145 CTR structure_element Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. ABSTRACT +174 178 exit site Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. ABSTRACT +186 193 channel site Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. ABSTRACT +215 219 His2 residue_name_number Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. ABSTRACT +227 241 twin-His motif structure_element Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. ABSTRACT +4 24 phosphorylation site site The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. ABSTRACT +32 35 CTR structure_element The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. ABSTRACT +39 57 solvent accessible protein_state The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. ABSTRACT +75 100 negatively charged pocket site The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. ABSTRACT +121 133 channel exit site The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. ABSTRACT +4 21 crystal structure evidence The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. ABSTRACT +25 50 phosphorylation-mimicking protein_state The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. ABSTRACT +51 64 Mep2 variants mutant The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. ABSTRACT +70 81 C. albicans species The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. ABSTRACT +121 130 conserved protein_state The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. ABSTRACT +172 175 CTR structure_element The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. ABSTRACT +58 68 eukaryotic taxonomy_domain The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation. ABSTRACT +69 77 ammonium chemical The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation. ABSTRACT +91 106 phosphorylation ptm The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation. ABSTRACT +1 14 Mep2 proteins protein_type Mep2 proteins are tightly regulated fungal ammonium transporters. ABSTRACT +37 43 fungal taxonomy_domain Mep2 proteins are tightly regulated fungal ammonium transporters. ABSTRACT +44 65 ammonium transporters protein_type Mep2 proteins are tightly regulated fungal ammonium transporters. ABSTRACT +29 47 crystal structures evidence Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria. ABSTRACT +51 57 closed protein_state Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria. ABSTRACT +68 81 Mep2 proteins protein_type Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria. ABSTRACT +123 145 by comparing them with experimental_method Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria. ABSTRACT +150 154 open protein_state Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria. ABSTRACT +155 176 ammonium transporters protein_type Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria. ABSTRACT +180 188 bacteria taxonomy_domain Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria. ABSTRACT +0 12 Transceptors protein_type Transceptors are membrane proteins that function not only as transporters but also as receptors/sensors during nutrient sensing to activate downstream signalling pathways. INTRO +17 34 membrane proteins protein_type Transceptors are membrane proteins that function not only as transporters but also as receptors/sensors during nutrient sensing to activate downstream signalling pathways. INTRO +20 32 transceptors protein_type A common feature of transceptors is that they are induced when cells are starved for their substrate. INTRO +39 63 Saccharomyces cerevisiae species While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +64 76 transceptors protein_type While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +81 90 phosphate chemical While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +92 97 Pho84 protein While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +100 111 amino acids chemical While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +113 117 Gap1 protein While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +123 131 ammonium chemical While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +133 137 Mep2 protein While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +140 152 transceptors protein_type While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +166 183 higher eukaryotes taxonomy_domain While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +210 219 mammalian taxonomy_domain While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +220 225 SNAT2 protein While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +226 248 amino-acid transporter protein_type While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +257 262 GLUT2 protein While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +263 282 glucose transporter protein_type While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). INTRO +71 83 transceptors protein_type One of the most important unresolved questions in the field is how the transceptors couple to downstream signalling pathways. INTRO +92 103 transporter protein_type One hypothesis is that downstream signalling is dependent on a specific conformation of the transporter. INTRO +0 4 Mep2 protein_type Mep2 (methylammonium (MA) permease) proteins are ammonium transceptors that are ubiquitous in fungi. INTRO +5 44 (methylammonium (MA) permease) proteins protein_type Mep2 (methylammonium (MA) permease) proteins are ammonium transceptors that are ubiquitous in fungi. INTRO +49 70 ammonium transceptors protein_type Mep2 (methylammonium (MA) permease) proteins are ammonium transceptors that are ubiquitous in fungi. INTRO +94 99 fungi taxonomy_domain Mep2 (methylammonium (MA) permease) proteins are ammonium transceptors that are ubiquitous in fungi. INTRO +19 52 Amt/Mep/Rh family of transporters protein_type They belong to the Amt/Mep/Rh family of transporters that are present in all kingdoms of life and they take up ammonium from the extracellular environment. INTRO +73 93 all kingdoms of life taxonomy_domain They belong to the Amt/Mep/Rh family of transporters that are present in all kingdoms of life and they take up ammonium from the extracellular environment. INTRO +111 119 ammonium chemical They belong to the Amt/Mep/Rh family of transporters that are present in all kingdoms of life and they take up ammonium from the extracellular environment. INTRO +0 5 Fungi taxonomy_domain Fungi typically have more than one Mep paralogue, for example, Mep1-3 in S. cerevisiae. INTRO +35 38 Mep protein_type Fungi typically have more than one Mep paralogue, for example, Mep1-3 in S. cerevisiae. INTRO +63 69 Mep1-3 protein Fungi typically have more than one Mep paralogue, for example, Mep1-3 in S. cerevisiae. INTRO +73 86 S. cerevisiae species Fungi typically have more than one Mep paralogue, for example, Mep1-3 in S. cerevisiae. INTRO +15 28 Mep2 proteins protein_type Of these, only Mep2 proteins function as ammonium receptors/sensors in fungal development. INTRO +41 49 ammonium chemical Of these, only Mep2 proteins function as ammonium receptors/sensors in fungal development. INTRO +71 77 fungal taxonomy_domain Of these, only Mep2 proteins function as ammonium receptors/sensors in fungal development. INTRO +41 45 Mep2 protein Under conditions of nitrogen limitation, Mep2 initiates a signalling cascade that results in a switch from the yeast form to filamentous (pseudohyphal) growth that may be required for fungal pathogenicity. INTRO +184 190 fungal taxonomy_domain Under conditions of nitrogen limitation, Mep2 initiates a signalling cascade that results in a switch from the yeast form to filamentous (pseudohyphal) growth that may be required for fungal pathogenicity. INTRO +25 37 transceptors protein_type As is the case for other transceptors, it is not clear how Mep2 interacts with downstream signalling partners, but the protein kinase A and mitogen-activated protein kinase pathways have been proposed as downstream effectors of Mep2 (refs). INTRO +59 63 Mep2 protein As is the case for other transceptors, it is not clear how Mep2 interacts with downstream signalling partners, but the protein kinase A and mitogen-activated protein kinase pathways have been proposed as downstream effectors of Mep2 (refs). INTRO +228 232 Mep2 protein As is the case for other transceptors, it is not clear how Mep2 interacts with downstream signalling partners, but the protein kinase A and mitogen-activated protein kinase pathways have been proposed as downstream effectors of Mep2 (refs). INTRO +14 18 Mep1 protein Compared with Mep1 and Mep3, Mep2 is highly expressed and functions as a low-capacity, high-affinity transporter in the uptake of MA. INTRO +23 27 Mep3 protein Compared with Mep1 and Mep3, Mep2 is highly expressed and functions as a low-capacity, high-affinity transporter in the uptake of MA. INTRO +29 33 Mep2 protein Compared with Mep1 and Mep3, Mep2 is highly expressed and functions as a low-capacity, high-affinity transporter in the uptake of MA. INTRO +37 53 highly expressed protein_state Compared with Mep1 and Mep3, Mep2 is highly expressed and functions as a low-capacity, high-affinity transporter in the uptake of MA. INTRO +130 132 MA chemical Compared with Mep1 and Mep3, Mep2 is highly expressed and functions as a low-capacity, high-affinity transporter in the uptake of MA. INTRO +13 17 Mep2 protein In addition, Mep2 is also important for uptake of ammonium produced by growth on other nitrogen sources. INTRO +50 58 ammonium chemical In addition, Mep2 is also important for uptake of ammonium produced by growth on other nitrogen sources. INTRO +87 95 nitrogen chemical In addition, Mep2 is also important for uptake of ammonium produced by growth on other nitrogen sources. INTRO +26 31 human species With the exception of the human RhCG structure, no structural information is available for eukaryotic ammonium transporters. INTRO +32 36 RhCG protein With the exception of the human RhCG structure, no structural information is available for eukaryotic ammonium transporters. INTRO +37 46 structure evidence With the exception of the human RhCG structure, no structural information is available for eukaryotic ammonium transporters. INTRO +91 101 eukaryotic taxonomy_domain With the exception of the human RhCG structure, no structural information is available for eukaryotic ammonium transporters. INTRO +102 123 ammonium transporters protein_type With the exception of the human RhCG structure, no structural information is available for eukaryotic ammonium transporters. INTRO +21 30 bacterial taxonomy_domain By contrast, several bacterial Amt orthologues have been characterized in detail via high-resolution crystal structures and a number of molecular dynamics (MD) studies. INTRO +31 34 Amt protein_type By contrast, several bacterial Amt orthologues have been characterized in detail via high-resolution crystal structures and a number of molecular dynamics (MD) studies. INTRO +101 119 crystal structures evidence By contrast, several bacterial Amt orthologues have been characterized in detail via high-resolution crystal structures and a number of molecular dynamics (MD) studies. INTRO +136 154 molecular dynamics experimental_method By contrast, several bacterial Amt orthologues have been characterized in detail via high-resolution crystal structures and a number of molecular dynamics (MD) studies. INTRO +156 158 MD experimental_method By contrast, several bacterial Amt orthologues have been characterized in detail via high-resolution crystal structures and a number of molecular dynamics (MD) studies. INTRO +15 25 structures evidence All the solved structures including that of RhCG are very similar, establishing the basic architecture of ammonium transporters. INTRO +44 48 RhCG protein All the solved structures including that of RhCG are very similar, establishing the basic architecture of ammonium transporters. INTRO +106 127 ammonium transporters protein_type All the solved structures including that of RhCG are very similar, establishing the basic architecture of ammonium transporters. INTRO +18 24 stable protein_state The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. INTRO +25 32 trimers oligomeric_state The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. INTRO +44 51 monomer oligomeric_state The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. INTRO +62 75 transmembrane structure_element The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. INTRO +77 79 TM structure_element The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. INTRO +81 88 helices structure_element The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. INTRO +95 110 central channel site The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. INTRO +132 140 ammonium chemical The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. INTRO +4 14 structures evidence All structures show the transporters in open conformations. INTRO +24 36 transporters protein_type All structures show the transporters in open conformations. INTRO +40 44 open protein_state All structures show the transporters in open conformations. INTRO +48 55 ammonia chemical Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport). INTRO +117 133 Amt/Mep proteins protein_type Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport). INTRO +146 152 active protein_state Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport). INTRO +154 179 electrogenic transporters protein_type Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport). INTRO +190 194 NH4+ chemical Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport). INTRO +208 211 NH3 chemical Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport). INTRO +212 214 H+ chemical Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport). INTRO +2 18 highly conserved protein_state A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport. INTRO +27 34 channel site A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport. INTRO +42 51 histidine residue_name A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport. INTRO +72 86 twin-His motif structure_element A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport. INTRO +128 131 NH3 chemical A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport. INTRO +150 157 channel site A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport. INTRO +165 168 NH3 chemical A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport. INTRO +169 171 H+ chemical A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport. INTRO +0 8 Ammonium chemical Ammonium transport is tightly regulated. INTRO +3 10 animals taxonomy_domain In animals, this is due to toxicity of elevated intracellular ammonium levels, whereas for microorganisms ammonium is a preferred nitrogen source. INTRO +62 70 ammonium chemical In animals, this is due to toxicity of elevated intracellular ammonium levels, whereas for microorganisms ammonium is a preferred nitrogen source. INTRO +91 105 microorganisms taxonomy_domain In animals, this is due to toxicity of elevated intracellular ammonium levels, whereas for microorganisms ammonium is a preferred nitrogen source. INTRO +106 114 ammonium chemical In animals, this is due to toxicity of elevated intracellular ammonium levels, whereas for microorganisms ammonium is a preferred nitrogen source. INTRO +3 11 bacteria taxonomy_domain In bacteria, amt genes are present in an operon with glnK, encoding a PII-like signal transduction class protein. INTRO +13 16 amt gene In bacteria, amt genes are present in an operon with glnK, encoding a PII-like signal transduction class protein. INTRO +53 57 glnK gene In bacteria, amt genes are present in an operon with glnK, encoding a PII-like signal transduction class protein. INTRO +70 112 PII-like signal transduction class protein protein_type In bacteria, amt genes are present in an operon with glnK, encoding a PII-like signal transduction class protein. INTRO +22 34 Amt proteins protein_type By binding tightly to Amt proteins without inducing a conformational change in the transporter, GlnK sterically blocks ammonium conductance when nitrogen levels are sufficient. INTRO +83 94 transporter protein_type By binding tightly to Amt proteins without inducing a conformational change in the transporter, GlnK sterically blocks ammonium conductance when nitrogen levels are sufficient. INTRO +96 100 GlnK protein_type By binding tightly to Amt proteins without inducing a conformational change in the transporter, GlnK sterically blocks ammonium conductance when nitrogen levels are sufficient. INTRO +119 127 ammonium chemical By binding tightly to Amt proteins without inducing a conformational change in the transporter, GlnK sterically blocks ammonium conductance when nitrogen levels are sufficient. INTRO +20 28 nitrogen chemical Under conditions of nitrogen limitation, GlnK becomes uridylated, blocking its ability to bind and inhibit Amt proteins. INTRO +41 45 GlnK protein_type Under conditions of nitrogen limitation, GlnK becomes uridylated, blocking its ability to bind and inhibit Amt proteins. INTRO +54 64 uridylated protein_state Under conditions of nitrogen limitation, GlnK becomes uridylated, blocking its ability to bind and inhibit Amt proteins. INTRO +107 119 Amt proteins protein_type Under conditions of nitrogen limitation, GlnK becomes uridylated, blocking its ability to bind and inhibit Amt proteins. INTRO +13 23 eukaryotes taxonomy_domain Importantly, eukaryotes do not have GlnK orthologues and have a different mechanism for regulation of ammonium transport activity. INTRO +36 40 GlnK protein_type Importantly, eukaryotes do not have GlnK orthologues and have a different mechanism for regulation of ammonium transport activity. INTRO +102 110 ammonium chemical Importantly, eukaryotes do not have GlnK orthologues and have a different mechanism for regulation of ammonium transport activity. INTRO +3 9 plants taxonomy_domain In plants, transporter phosphorylation and dephosphorylation are known to regulate activity. INTRO +11 22 transporter protein_type In plants, transporter phosphorylation and dephosphorylation are known to regulate activity. INTRO +23 38 phosphorylation ptm In plants, transporter phosphorylation and dephosphorylation are known to regulate activity. INTRO +43 60 dephosphorylation ptm In plants, transporter phosphorylation and dephosphorylation are known to regulate activity. INTRO +3 16 S. cerevisiae species In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change. INTRO +18 33 phosphorylation ptm In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change. INTRO +37 43 Ser457 residue_name_number In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change. INTRO +55 72 C-terminal region structure_element In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change. INTRO +74 77 CTR structure_element In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change. INTRO +127 131 Mep2 protein_type In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change. INTRO +30 34 Mep2 protein_type To elucidate the mechanism of Mep2 transport regulation, we present here X-ray crystal structures of the Mep2 transceptors from S. cerevisiae and C. albicans. INTRO +73 97 X-ray crystal structures evidence To elucidate the mechanism of Mep2 transport regulation, we present here X-ray crystal structures of the Mep2 transceptors from S. cerevisiae and C. albicans. INTRO +105 122 Mep2 transceptors protein_type To elucidate the mechanism of Mep2 transport regulation, we present here X-ray crystal structures of the Mep2 transceptors from S. cerevisiae and C. albicans. INTRO +128 141 S. cerevisiae species To elucidate the mechanism of Mep2 transport regulation, we present here X-ray crystal structures of the Mep2 transceptors from S. cerevisiae and C. albicans. INTRO +146 157 C. albicans species To elucidate the mechanism of Mep2 transport regulation, we present here X-ray crystal structures of the Mep2 transceptors from S. cerevisiae and C. albicans. INTRO +4 14 structures evidence The structures are similar to each other but show considerable differences to all other ammonium transporter structures. INTRO +88 108 ammonium transporter protein_type The structures are similar to each other but show considerable differences to all other ammonium transporter structures. INTRO +109 119 structures evidence The structures are similar to each other but show considerable differences to all other ammonium transporter structures. INTRO +50 63 Mep2 proteins protein_type The most striking difference is the fact that the Mep2 proteins have closed conformations. INTRO +69 75 closed protein_state The most striking difference is the fact that the Mep2 proteins have closed conformations. INTRO +13 33 phosphorylation site site The putative phosphorylation site is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. INTRO +37 55 solvent accessible protein_state The putative phosphorylation site is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. INTRO +73 98 negatively charged pocket site The putative phosphorylation site is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. INTRO +119 131 channel exit site The putative phosphorylation site is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. INTRO +4 12 channels site The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR. INTRO +16 49 phosphorylation-mimicking mutants protein_state The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR. INTRO +53 64 C. albicans species The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR. INTRO +65 69 Mep2 protein The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR. INTRO +80 86 closed protein_state The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR. INTRO +134 143 conserved protein_state The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR. INTRO +156 159 CTR structure_element The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR. INTRO +16 25 structure evidence Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport. INTRO +42 54 Mep2 variant mutant Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport. INTRO +55 62 lacking protein_state Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport. INTRO +67 74 segment structure_element Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport. INTRO +90 110 phosphorylation site site Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport. INTRO +203 213 eukaryotic taxonomy_domain Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport. INTRO +214 222 ammonium chemical Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport. INTRO +24 28 Mep2 protein_type General architecture of Mep2 ammonium transceptors RESULTS +29 50 ammonium transceptors protein_type General architecture of Mep2 ammonium transceptors RESULTS +4 8 Mep2 protein The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). RESULTS +20 33 S. cerevisiae species The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). RESULTS +35 41 ScMep2 protein The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). RESULTS +47 60 overexpressed experimental_method The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). RESULTS +64 77 S. cerevisiae species The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). RESULTS +103 126 structure determination experimental_method The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). RESULTS +130 151 X-ray crystallography experimental_method The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). RESULTS +186 207 molecular replacement experimental_method The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). RESULTS +209 211 MR experimental_method The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). RESULTS +222 237 archaebacterial taxonomy_domain The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). RESULTS +238 243 Amt-1 protein The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). RESULTS +244 253 structure evidence The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). RESULTS +40 49 structure evidence Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure–function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals. RESULTS +89 95 ScMep2 protein Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure–function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals. RESULTS +120 146 structure–function studies experimental_method Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure–function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals. RESULTS +162 168 fungal taxonomy_domain Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure–function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals. RESULTS +169 173 Mep2 protein_type Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure–function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals. RESULTS +204 234 overexpressed and screened for experimental_method Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure–function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals. RESULTS +255 263 crystals evidence Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure–function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals. RESULTS +10 14 Mep2 protein Of these, Mep2 from C. albicans (CaMep2) showed superior stability in relatively harsh detergents such as nonyl-glucoside, allowing structure determination in two different crystal forms to high resolution (up to 1.5 Å). RESULTS +20 31 C. albicans species Of these, Mep2 from C. albicans (CaMep2) showed superior stability in relatively harsh detergents such as nonyl-glucoside, allowing structure determination in two different crystal forms to high resolution (up to 1.5 Å). RESULTS +33 39 CaMep2 protein Of these, Mep2 from C. albicans (CaMep2) showed superior stability in relatively harsh detergents such as nonyl-glucoside, allowing structure determination in two different crystal forms to high resolution (up to 1.5 Å). RESULTS +132 155 structure determination experimental_method Of these, Mep2 from C. albicans (CaMep2) showed superior stability in relatively harsh detergents such as nonyl-glucoside, allowing structure determination in two different crystal forms to high resolution (up to 1.5 Å). RESULTS +173 186 crystal forms evidence Of these, Mep2 from C. albicans (CaMep2) showed superior stability in relatively harsh detergents such as nonyl-glucoside, allowing structure determination in two different crystal forms to high resolution (up to 1.5 Å). RESULTS +67 73 CaMep2 protein Despite different crystal packing (Supplementary Table 1), the two CaMep2 structures are identical to each other and very similar to ScMep2 (Cα r.m.s.d. RESULTS +74 84 structures evidence Despite different crystal packing (Supplementary Table 1), the two CaMep2 structures are identical to each other and very similar to ScMep2 (Cα r.m.s.d. RESULTS +133 139 ScMep2 protein Despite different crystal packing (Supplementary Table 1), the two CaMep2 structures are identical to each other and very similar to ScMep2 (Cα r.m.s.d. RESULTS +144 152 r.m.s.d. evidence Despite different crystal packing (Supplementary Table 1), the two CaMep2 structures are identical to each other and very similar to ScMep2 (Cα r.m.s.d. RESULTS +1 27 root mean square deviation evidence (root mean square deviation)=0.7 Å for 434 residues), with the main differences confined to the N terminus and the CTR (Fig. 1). RESULTS +115 118 CTR structure_element (root mean square deviation)=0.7 Å for 434 residues), with the main differences confined to the N terminus and the CTR (Fig. 1). RESULTS +0 16 Electron density evidence Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered. RESULTS +100 102 43 residue_range Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered. RESULTS +104 110 ScMep2 protein Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered. RESULTS +116 118 25 residue_range Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered. RESULTS +129 135 CaMep2 protein Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered. RESULTS +148 164 poorly conserved protein_state Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered. RESULTS +180 190 disordered protein_state Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered. RESULTS +5 18 Mep2 proteins protein_type Both Mep2 proteins show the archetypal trimeric assemblies in which each monomer consists of 11 TM helices surrounding a central pore. RESULTS +39 47 trimeric oligomeric_state Both Mep2 proteins show the archetypal trimeric assemblies in which each monomer consists of 11 TM helices surrounding a central pore. RESULTS +73 80 monomer oligomeric_state Both Mep2 proteins show the archetypal trimeric assemblies in which each monomer consists of 11 TM helices surrounding a central pore. RESULTS +96 106 TM helices structure_element Both Mep2 proteins show the archetypal trimeric assemblies in which each monomer consists of 11 TM helices surrounding a central pore. RESULTS +121 133 central pore structure_element Both Mep2 proteins show the archetypal trimeric assemblies in which each monomer consists of 11 TM helices surrounding a central pore. RESULTS +56 77 ammonium binding site site Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG. RESULTS +83 91 Phe gate site Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG. RESULTS +100 114 twin-His motif structure_element Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG. RESULTS +126 145 hydrophobic channel site Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG. RESULTS +191 200 bacterial taxonomy_domain Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG. RESULTS +201 213 transporters protein_type Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG. RESULTS +218 222 RhCG protein Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG. RESULTS +65 71 CaMep2 protein In the remainder of the manuscript, we will specifically discuss CaMep2 due to the superior resolution of the structure. RESULTS +110 119 structure evidence In the remainder of the manuscript, we will specifically discuss CaMep2 due to the superior resolution of the structure. RESULTS +64 70 ScMep2 protein Unless specifically stated, the drawn conclusions also apply to ScMep2. RESULTS +34 38 Mep2 protein While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. RESULTS +65 76 prokaryotic taxonomy_domain While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. RESULTS +77 89 transporters protein_type While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. RESULTS +94 102 r.m.s.d. evidence While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. RESULTS +108 113 Amt-1 protein While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. RESULTS +190 209 intracellular loops structure_element While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. RESULTS +211 215 ICLs structure_element While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. RESULTS +217 221 ICL1 structure_element While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. RESULTS +226 230 ICL3 structure_element While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. RESULTS +240 243 CTR structure_element While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. RESULTS +21 34 Mep2 proteins protein_type The N termini of the Mep2 proteins are ∼20–25 residues longer compared with their bacterial counterparts (Figs 1 and 2), substantially increasing the size of the extracellular domain. RESULTS +40 45 20–25 residue_range The N termini of the Mep2 proteins are ∼20–25 residues longer compared with their bacterial counterparts (Figs 1 and 2), substantially increasing the size of the extracellular domain. RESULTS +82 91 bacterial taxonomy_domain The N termini of the Mep2 proteins are ∼20–25 residues longer compared with their bacterial counterparts (Figs 1 and 2), substantially increasing the size of the extracellular domain. RESULTS +162 182 extracellular domain structure_element The N termini of the Mep2 proteins are ∼20–25 residues longer compared with their bacterial counterparts (Figs 1 and 2), substantially increasing the size of the extracellular domain. RESULTS +32 39 monomer oligomeric_state Moreover, the N terminus of one monomer interacts with the extended extracellular loop ECL5 of a neighbouring monomer. RESULTS +68 86 extracellular loop structure_element Moreover, the N terminus of one monomer interacts with the extended extracellular loop ECL5 of a neighbouring monomer. RESULTS +87 91 ECL5 structure_element Moreover, the N terminus of one monomer interacts with the extended extracellular loop ECL5 of a neighbouring monomer. RESULTS +110 117 monomer oligomeric_state Moreover, the N terminus of one monomer interacts with the extended extracellular loop ECL5 of a neighbouring monomer. RESULTS +55 74 extracellular loops structure_element Together with additional, smaller differences in other extracellular loops, these changes generate a distinct vestibule leading to the ammonium binding site that is much more pronounced than in the bacterial proteins. RESULTS +110 119 vestibule structure_element Together with additional, smaller differences in other extracellular loops, these changes generate a distinct vestibule leading to the ammonium binding site that is much more pronounced than in the bacterial proteins. RESULTS +135 156 ammonium binding site site Together with additional, smaller differences in other extracellular loops, these changes generate a distinct vestibule leading to the ammonium binding site that is much more pronounced than in the bacterial proteins. RESULTS +198 207 bacterial taxonomy_domain Together with additional, smaller differences in other extracellular loops, these changes generate a distinct vestibule leading to the ammonium binding site that is much more pronounced than in the bacterial proteins. RESULTS +15 24 vestibule structure_element The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins. RESULTS +49 56 monomer oligomeric_state The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins. RESULTS +107 111 Mep2 protein The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins. RESULTS +112 118 trimer oligomeric_state The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins. RESULTS +143 148 plant taxonomy_domain The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins. RESULTS +149 161 AMT proteins protein_type The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins. RESULTS +34 49 deletion mutant protein_state However, given that an N-terminal deletion mutant (2-27Δ) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear. RESULTS +51 56 2-27Δ mutant However, given that an N-terminal deletion mutant (2-27Δ) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear. RESULTS +75 84 wild-type protein_state However, given that an N-terminal deletion mutant (2-27Δ) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear. RESULTS +86 88 WT protein_state However, given that an N-terminal deletion mutant (2-27Δ) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear. RESULTS +90 94 Mep2 protein However, given that an N-terminal deletion mutant (2-27Δ) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear. RESULTS +106 114 ammonium chemical However, given that an N-terminal deletion mutant (2-27Δ) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear. RESULTS +194 198 Mep2 protein However, given that an N-terminal deletion mutant (2-27Δ) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear. RESULTS +0 4 Mep2 protein Mep2 channels are closed by a two-tier channel block RESULTS +5 13 channels site Mep2 channels are closed by a two-tier channel block RESULTS +18 24 closed protein_state Mep2 channels are closed by a two-tier channel block RESULTS +39 52 channel block structure_element Mep2 channels are closed by a two-tier channel block RESULTS +36 40 Mep2 protein The largest differences between the Mep2 structures and the other known ammonium transporter structures are located on the intracellular side of the membrane. RESULTS +41 51 structures evidence The largest differences between the Mep2 structures and the other known ammonium transporter structures are located on the intracellular side of the membrane. RESULTS +72 92 ammonium transporter protein_type The largest differences between the Mep2 structures and the other known ammonium transporter structures are located on the intracellular side of the membrane. RESULTS +93 103 structures evidence The largest differences between the Mep2 structures and the other known ammonium transporter structures are located on the intracellular side of the membrane. RESULTS +23 27 Mep2 protein In the vicinity of the Mep2 channel exit, the cytoplasmic end of TM2 has unwound, generating a longer ICL1 even though there are no insertions in this region compared to the bacterial proteins (Figs 2 and 4). RESULTS +28 40 channel exit site In the vicinity of the Mep2 channel exit, the cytoplasmic end of TM2 has unwound, generating a longer ICL1 even though there are no insertions in this region compared to the bacterial proteins (Figs 2 and 4). RESULTS +65 68 TM2 structure_element In the vicinity of the Mep2 channel exit, the cytoplasmic end of TM2 has unwound, generating a longer ICL1 even though there are no insertions in this region compared to the bacterial proteins (Figs 2 and 4). RESULTS +102 106 ICL1 structure_element In the vicinity of the Mep2 channel exit, the cytoplasmic end of TM2 has unwound, generating a longer ICL1 even though there are no insertions in this region compared to the bacterial proteins (Figs 2 and 4). RESULTS +174 183 bacterial taxonomy_domain In the vicinity of the Mep2 channel exit, the cytoplasmic end of TM2 has unwound, generating a longer ICL1 even though there are no insertions in this region compared to the bacterial proteins (Figs 2 and 4). RESULTS +0 4 ICL1 structure_element ICL1 has also moved inwards relative to its position in the bacterial Amts. RESULTS +60 69 bacterial taxonomy_domain ICL1 has also moved inwards relative to its position in the bacterial Amts. RESULTS +70 74 Amts protein_type ICL1 has also moved inwards relative to its position in the bacterial Amts. RESULTS +61 65 ICL1 structure_element The largest backbone movements of equivalent residues within ICL1 are ∼10 Å, markedly affecting the conserved basic RxK motif (Fig. 4). RESULTS +100 109 conserved protein_state The largest backbone movements of equivalent residues within ICL1 are ∼10 Å, markedly affecting the conserved basic RxK motif (Fig. 4). RESULTS +110 115 basic protein_state The largest backbone movements of equivalent residues within ICL1 are ∼10 Å, markedly affecting the conserved basic RxK motif (Fig. 4). RESULTS +116 125 RxK motif structure_element The largest backbone movements of equivalent residues within ICL1 are ∼10 Å, markedly affecting the conserved basic RxK motif (Fig. 4). RESULTS +18 23 Arg54 residue_name_number The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). RESULTS +60 65 Amt-1 protein The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). RESULTS +119 124 Lys55 residue_name_number The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). RESULTS +167 172 Lys56 residue_name_number The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). RESULTS +180 185 basic protein_state The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). RESULTS +186 191 motif structure_element The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). RESULTS +231 235 Mep2 protein The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). RESULTS +236 246 structures evidence The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). RESULTS +283 288 Amt-1 protein The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). RESULTS +28 37 RxK motif structure_element In addition to changing the RxK motif, the movement of ICL1 has another, crucial functional consequence. RESULTS +55 59 ICL1 structure_element In addition to changing the RxK motif, the movement of ICL1 has another, crucial functional consequence. RESULTS +25 28 TM1 structure_element At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). RESULTS +67 87 relatively conserved protein_state At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). RESULTS +88 93 Tyr49 residue_name_number At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). RESULTS +95 100 Tyr53 residue_name_number At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). RESULTS +104 110 ScMep2 protein At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). RESULTS +127 140 hydrogen bond bond_interaction At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). RESULTS +174 194 absolutely conserved protein_state At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). RESULTS +195 201 His342 residue_name_number At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). RESULTS +209 223 twin-His motif structure_element At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). RESULTS +225 231 His348 residue_name_number At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). RESULTS +235 241 ScMep2 protein At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). RESULTS +256 263 channel site At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). RESULTS +3 12 bacterial taxonomy_domain In bacterial Amt proteins, this Tyr side chain is rotated ∼4 Å away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2). RESULTS +13 25 Amt proteins protein_type In bacterial Amt proteins, this Tyr side chain is rotated ∼4 Å away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2). RESULTS +32 35 Tyr residue_name In bacterial Amt proteins, this Tyr side chain is rotated ∼4 Å away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2). RESULTS +113 116 TM1 structure_element In bacterial Amt proteins, this Tyr side chain is rotated ∼4 Å away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2). RESULTS +130 137 channel site In bacterial Amt proteins, this Tyr side chain is rotated ∼4 Å away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2). RESULTS +138 142 open protein_state In bacterial Amt proteins, this Tyr side chain is rotated ∼4 Å away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2). RESULTS +151 160 histidine residue_name In bacterial Amt proteins, this Tyr side chain is rotated ∼4 Å away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2). RESULTS +14 18 ICL1 structure_element Compared with ICL1, the backbone conformational changes observed for the neighbouring ICL2 are smaller, but large shifts are nevertheless observed for the conserved residues Glu140 and Arg141 (Fig. 4). RESULTS +86 90 ICL2 structure_element Compared with ICL1, the backbone conformational changes observed for the neighbouring ICL2 are smaller, but large shifts are nevertheless observed for the conserved residues Glu140 and Arg141 (Fig. 4). RESULTS +155 164 conserved protein_state Compared with ICL1, the backbone conformational changes observed for the neighbouring ICL2 are smaller, but large shifts are nevertheless observed for the conserved residues Glu140 and Arg141 (Fig. 4). RESULTS +174 180 Glu140 residue_name_number Compared with ICL1, the backbone conformational changes observed for the neighbouring ICL2 are smaller, but large shifts are nevertheless observed for the conserved residues Glu140 and Arg141 (Fig. 4). RESULTS +185 191 Arg141 residue_name_number Compared with ICL1, the backbone conformational changes observed for the neighbouring ICL2 are smaller, but large shifts are nevertheless observed for the conserved residues Glu140 and Arg141 (Fig. 4). RESULTS +23 27 ICL3 structure_element Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to ∼10 Å and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5). RESULTS +40 65 pseudo-symmetrical halves structure_element Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to ∼10 Å and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5). RESULTS +67 72 TM1-5 structure_element Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to ∼10 Å and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5). RESULTS +77 83 TM6-10 structure_element Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to ∼10 Å and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5). RESULTS +92 103 transporter protein_type Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to ∼10 Å and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5). RESULTS +180 187 channel site Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to ∼10 Å and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5). RESULTS +14 27 channel block structure_element This two-tier channel block likely ensures that very little ammonium transport will take place under nitrogen-sufficient conditions. RESULTS +60 68 ammonium chemical This two-tier channel block likely ensures that very little ammonium transport will take place under nitrogen-sufficient conditions. RESULTS +101 109 nitrogen chemical This two-tier channel block likely ensures that very little ammonium transport will take place under nitrogen-sufficient conditions. RESULTS +4 10 closed protein_state The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. RESULTS +24 31 channel site The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. RESULTS +55 65 no density evidence The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. RESULTS +93 101 ammonium chemical The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. RESULTS +106 111 water chemical The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. RESULTS +157 161 Mep2 protein The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. RESULTS +162 169 channel site The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. RESULTS +183 197 twin-His motif structure_element The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. RESULTS +37 43 ScMep2 protein Significantly, this is also true for ScMep2, which was crystallized in the presence of 0.2 M ammonium ions (see Methods section). RESULTS +55 67 crystallized experimental_method Significantly, this is also true for ScMep2, which was crystallized in the presence of 0.2 M ammonium ions (see Methods section). RESULTS +93 101 ammonium chemical Significantly, this is also true for ScMep2, which was crystallized in the presence of 0.2 M ammonium ions (see Methods section). RESULTS +20 24 Mep2 protein The final region in Mep2 that shows large differences compared with the bacterial transporters is the CTR. RESULTS +72 81 bacterial taxonomy_domain The final region in Mep2 that shows large differences compared with the bacterial transporters is the CTR. RESULTS +82 94 transporters protein_type The final region in Mep2 that shows large differences compared with the bacterial transporters is the CTR. RESULTS +102 105 CTR structure_element The final region in Mep2 that shows large differences compared with the bacterial transporters is the CTR. RESULTS +3 7 Mep2 protein In Mep2, the CTR has moved away and makes relatively few contacts with the main body of the transporter, generating a more elongated protein (Figs 1 and 4). RESULTS +13 16 CTR structure_element In Mep2, the CTR has moved away and makes relatively few contacts with the main body of the transporter, generating a more elongated protein (Figs 1 and 4). RESULTS +75 84 main body structure_element In Mep2, the CTR has moved away and makes relatively few contacts with the main body of the transporter, generating a more elongated protein (Figs 1 and 4). RESULTS +92 103 transporter protein_type In Mep2, the CTR has moved away and makes relatively few contacts with the main body of the transporter, generating a more elongated protein (Figs 1 and 4). RESULTS +123 132 elongated protein_state In Mep2, the CTR has moved away and makes relatively few contacts with the main body of the transporter, generating a more elongated protein (Figs 1 and 4). RESULTS +20 30 structures evidence By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. RESULTS +34 43 bacterial taxonomy_domain By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. RESULTS +58 61 CTR structure_element By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. RESULTS +89 104 N-terminal half structure_element By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. RESULTS +112 124 transporters protein_type By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. RESULTS +143 148 TM1-5 structure_element By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. RESULTS +171 178 compact protein_state By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. RESULTS +179 188 structure evidence By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. RESULTS +49 70 universally conserved protein_state This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +91 94 CTR structure_element This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +105 111 Arg415 residue_name_number This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +112 115 370 residue_number This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +118 124 Glu421 residue_name_number This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +125 128 376 residue_number This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +131 137 Gly424 residue_name_number This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +138 141 379 residue_number This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +144 150 Asp426 residue_name_number This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +151 154 381 residue_number This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +160 167 Tyr 435 residue_name_number This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +168 171 390 residue_number This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +176 182 CaMep2 protein This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +183 188 Amt-1 protein This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). RESULTS +36 50 ‘ExxGxD' motif structure_element These residues include those of the ‘ExxGxD' motif, which when mutated generate inactive transporters. RESULTS +63 70 mutated experimental_method These residues include those of the ‘ExxGxD' motif, which when mutated generate inactive transporters. RESULTS +80 88 inactive protein_state These residues include those of the ‘ExxGxD' motif, which when mutated generate inactive transporters. RESULTS +89 101 transporters protein_type These residues include those of the ‘ExxGxD' motif, which when mutated generate inactive transporters. RESULTS +3 8 Amt-1 protein In Amt-1 and other bacterial ammonium transporters, these CTR residues interact with residues within the N-terminal half of the protein. RESULTS +19 28 bacterial taxonomy_domain In Amt-1 and other bacterial ammonium transporters, these CTR residues interact with residues within the N-terminal half of the protein. RESULTS +29 50 ammonium transporters protein_type In Amt-1 and other bacterial ammonium transporters, these CTR residues interact with residues within the N-terminal half of the protein. RESULTS +58 61 CTR structure_element In Amt-1 and other bacterial ammonium transporters, these CTR residues interact with residues within the N-terminal half of the protein. RESULTS +105 120 N-terminal half structure_element In Amt-1 and other bacterial ammonium transporters, these CTR residues interact with residues within the N-terminal half of the protein. RESULTS +17 23 Tyr390 residue_name_number On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3. RESULTS +36 41 Amt-1 protein On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3. RESULTS +45 60 hydrogen bonded bond_interaction On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3. RESULTS +88 97 conserved protein_state On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3. RESULTS +98 104 His185 residue_name_number On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3. RESULTS +130 134 loop structure_element On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3. RESULTS +135 139 ICL3 structure_element On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3. RESULTS +20 24 ICL3 structure_element At the other end of ICL3, the backbone carbonyl groups of Gly172 and Lys173 are hydrogen bonded to the side chain of Arg370. RESULTS +58 64 Gly172 residue_name_number At the other end of ICL3, the backbone carbonyl groups of Gly172 and Lys173 are hydrogen bonded to the side chain of Arg370. RESULTS +69 75 Lys173 residue_name_number At the other end of ICL3, the backbone carbonyl groups of Gly172 and Lys173 are hydrogen bonded to the side chain of Arg370. RESULTS +80 95 hydrogen bonded bond_interaction At the other end of ICL3, the backbone carbonyl groups of Gly172 and Lys173 are hydrogen bonded to the side chain of Arg370. RESULTS +117 123 Arg370 residue_name_number At the other end of ICL3, the backbone carbonyl groups of Gly172 and Lys173 are hydrogen bonded to the side chain of Arg370. RESULTS +31 39 modelled experimental_method Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). RESULTS +47 53 active protein_state Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). RESULTS +55 73 non-phosphorylated protein_state Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). RESULTS +74 79 plant taxonomy_domain Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). RESULTS +80 89 AtAmt-1;1 protein Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). RESULTS +90 99 structure evidence Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). RESULTS +114 118 Y467 residue_name_number Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). RESULTS +119 123 H239 residue_name_number Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). RESULTS +128 132 D458 residue_name_number Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). RESULTS +133 136 K71 residue_name_number Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). RESULTS +45 48 CTR structure_element The result of these interactions is that the CTR ‘hugs' the N-terminal half of the transporters (Fig. 4). RESULTS +60 75 N-terminal half structure_element The result of these interactions is that the CTR ‘hugs' the N-terminal half of the transporters (Fig. 4). RESULTS +83 95 transporters protein_type The result of these interactions is that the CTR ‘hugs' the N-terminal half of the transporters (Fig. 4). RESULTS +19 25 Asp381 residue_name_number Also noteworthy is Asp381, the side chain of which interacts strongly with the positive dipole on the N-terminal end of TM2. RESULTS +120 123 TM2 structure_element Also noteworthy is Asp381, the side chain of which interacts strongly with the positive dipole on the N-terminal end of TM2. RESULTS +93 97 open protein_state This interaction in the centre of the protein may be particularly important to stabilize the open conformations of ammonium transporters. RESULTS +115 136 ammonium transporters protein_type This interaction in the centre of the protein may be particularly important to stabilize the open conformations of ammonium transporters. RESULTS +7 11 Mep2 protein In the Mep2 structures, none of the interactions mentioned above are present. RESULTS +12 22 structures evidence In the Mep2 structures, none of the interactions mentioned above are present. RESULTS +0 27 Phosphorylation target site site Phosphorylation target site is at the periphery of Mep2 RESULTS +51 55 Mep2 protein Phosphorylation target site is at the periphery of Mep2 RESULTS +51 57 Ser457 residue_name_number Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. RESULTS +61 67 ScMep2 protein Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. RESULTS +86 92 Ser453 residue_name_number Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. RESULTS +96 102 CaMep2 protein Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. RESULTS +107 121 phosphorylated protein_state Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. RESULTS +129 150 TORC1 effector kinase protein_type Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. RESULTS +151 155 Npr1 protein Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. RESULTS +162 170 nitrogen chemical Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. RESULTS +7 17 absence of protein_state In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). RESULTS +18 22 Npr1 protein In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). RESULTS +24 39 plasmid-encoded experimental_method In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). RESULTS +40 42 WT protein_state In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). RESULTS +43 47 Mep2 protein In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). RESULTS +53 66 S. cerevisiae species In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). RESULTS +67 74 mep1-3Δ mutant In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). RESULTS +83 94 triple mepΔ mutant In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). RESULTS +143 151 ammonium chemical In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). RESULTS +173 184 transporter protein_type In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). RESULTS +188 196 inactive protein_state In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). RESULTS +16 41 phosphorylation-mimicking protein_state Conversely, the phosphorylation-mimicking S457D variant is active both in the triple mepΔ background and in a triple mepΔ npr1Δ strain (Fig. 3). RESULTS +42 47 S457D mutant Conversely, the phosphorylation-mimicking S457D variant is active both in the triple mepΔ background and in a triple mepΔ npr1Δ strain (Fig. 3). RESULTS +59 65 active protein_state Conversely, the phosphorylation-mimicking S457D variant is active both in the triple mepΔ background and in a triple mepΔ npr1Δ strain (Fig. 3). RESULTS +78 89 triple mepΔ mutant Conversely, the phosphorylation-mimicking S457D variant is active both in the triple mepΔ background and in a triple mepΔ npr1Δ strain (Fig. 3). RESULTS +110 127 triple mepΔ npr1Δ mutant Conversely, the phosphorylation-mimicking S457D variant is active both in the triple mepΔ background and in a triple mepΔ npr1Δ strain (Fig. 3). RESULTS +0 8 Mutation experimental_method Mutation of other potential phosphorylation sites in the CTR did not support growth in the npr1Δ background. RESULTS +28 49 phosphorylation sites site Mutation of other potential phosphorylation sites in the CTR did not support growth in the npr1Δ background. RESULTS +57 60 CTR structure_element Mutation of other potential phosphorylation sites in the CTR did not support growth in the npr1Δ background. RESULTS +91 96 npr1Δ mutant Mutation of other potential phosphorylation sites in the CTR did not support growth in the npr1Δ background. RESULTS +38 53 phosphorylation ptm Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake. RESULTS +57 63 Ser457 residue_name_number Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake. RESULTS +57 63 Ser457 residue_name_number Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake. RESULTS +74 78 Mep2 protein Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake. RESULTS +79 86 channel site Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake. RESULTS +96 104 ammonium chemical Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake. RESULTS +0 6 Ser457 residue_name_number Ser457 is located in a part of the CTR that is conserved in a subgroup of Mep2 proteins, but which is not present in bacterial proteins (Fig. 2). RESULTS +35 38 CTR structure_element Ser457 is located in a part of the CTR that is conserved in a subgroup of Mep2 proteins, but which is not present in bacterial proteins (Fig. 2). RESULTS +47 56 conserved protein_state Ser457 is located in a part of the CTR that is conserved in a subgroup of Mep2 proteins, but which is not present in bacterial proteins (Fig. 2). RESULTS +74 87 Mep2 proteins protein_type Ser457 is located in a part of the CTR that is conserved in a subgroup of Mep2 proteins, but which is not present in bacterial proteins (Fig. 2). RESULTS +117 126 bacterial taxonomy_domain Ser457 is located in a part of the CTR that is conserved in a subgroup of Mep2 proteins, but which is not present in bacterial proteins (Fig. 2). RESULTS +5 12 segment structure_element This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). RESULTS +23 30 450–457 residue_range This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). RESULTS +34 40 ScMep2 protein This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). RESULTS +45 52 446–453 residue_range This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). RESULTS +56 62 CaMep2 protein This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). RESULTS +78 104 autoinhibitory (AI) region structure_element This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). RESULTS +132 139 removal experimental_method This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). RESULTS +153 159 active protein_state This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). RESULTS +160 171 transporter protein_type This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). RESULTS +179 189 absence of protein_state This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). RESULTS +190 194 Npr1 protein This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). RESULTS +13 22 AI region structure_element Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). RESULTS +31 35 Npr1 protein Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). RESULTS +36 56 phosphorylation site site Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). RESULTS +70 80 structures evidence Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). RESULTS +111 120 AI region structure_element Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). RESULTS +145 148 CTR structure_element Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). RESULTS +191 197 trimer oligomeric_state Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). RESULTS +219 228 bacterial taxonomy_domain Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). RESULTS +229 239 structures evidence Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). RESULTS +4 13 AI region structure_element The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). RESULTS +52 55 TM2 structure_element The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). RESULTS +60 63 TM4 structure_element The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). RESULTS +88 97 main body structure_element The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). RESULTS +105 116 transporter protein_type The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). RESULTS +126 129 CTR structure_element The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). RESULTS +189 195 Val447 residue_name_number The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). RESULTS +197 203 Asp449 residue_name_number The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). RESULTS +205 211 Pro450 residue_name_number The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). RESULTS +216 222 Arg452 residue_name_number The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). RESULTS +4 14 AI regions structure_element The AI regions have very similar conformations in CaMep2 and ScMep2, despite considerable differences in the rest of the CTR (Fig. 6). RESULTS +50 56 CaMep2 protein The AI regions have very similar conformations in CaMep2 and ScMep2, despite considerable differences in the rest of the CTR (Fig. 6). RESULTS +61 67 ScMep2 protein The AI regions have very similar conformations in CaMep2 and ScMep2, despite considerable differences in the rest of the CTR (Fig. 6). RESULTS +121 124 CTR structure_element The AI regions have very similar conformations in CaMep2 and ScMep2, despite considerable differences in the rest of the CTR (Fig. 6). RESULTS +16 34 Npr1 target serine site Strikingly, the Npr1 target serine residue is located at the periphery of the trimer, far away (∼30 Å) from any channel exit (Fig. 6). RESULTS +78 84 trimer oligomeric_state Strikingly, the Npr1 target serine residue is located at the periphery of the trimer, far away (∼30 Å) from any channel exit (Fig. 6). RESULTS +112 124 channel exit site Strikingly, the Npr1 target serine residue is located at the periphery of the trimer, far away (∼30 Å) from any channel exit (Fig. 6). RESULTS +45 51 trimer oligomeric_state Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6). RESULTS +57 73 electron density evidence Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6). RESULTS +82 88 serine residue_name Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6). RESULTS +113 117 Mep2 protein Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6). RESULTS +118 128 structures evidence Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6). RESULTS +152 170 non-phosphorylated protein_state Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6). RESULTS +132 150 non-phosphorylated protein_state This makes sense since the proteins were expressed in rich medium and confirms the recent suggestion by Boeckstaens et al. that the non-phosphorylated form of Mep2 corresponds to the inactive state. RESULTS +159 163 Mep2 protein This makes sense since the proteins were expressed in rich medium and confirms the recent suggestion by Boeckstaens et al. that the non-phosphorylated form of Mep2 corresponds to the inactive state. RESULTS +183 191 inactive protein_state This makes sense since the proteins were expressed in rich medium and confirms the recent suggestion by Boeckstaens et al. that the non-phosphorylated form of Mep2 corresponds to the inactive state. RESULTS +4 10 ScMep2 protein For ScMep2, Ser457 is the most C-terminal residue for which electron density is visible, indicating that the region beyond Ser457 is disordered. RESULTS +12 18 Ser457 residue_name_number For ScMep2, Ser457 is the most C-terminal residue for which electron density is visible, indicating that the region beyond Ser457 is disordered. RESULTS +60 76 electron density evidence For ScMep2, Ser457 is the most C-terminal residue for which electron density is visible, indicating that the region beyond Ser457 is disordered. RESULTS +123 129 Ser457 residue_name_number For ScMep2, Ser457 is the most C-terminal residue for which electron density is visible, indicating that the region beyond Ser457 is disordered. RESULTS +133 143 disordered protein_state For ScMep2, Ser457 is the most C-terminal residue for which electron density is visible, indicating that the region beyond Ser457 is disordered. RESULTS +3 9 CaMep2 protein In CaMep2, the visible part of the sequence extends for two residues beyond Ser453 (Fig. 6). RESULTS +76 82 Ser453 residue_name_number In CaMep2, the visible part of the sequence extends for two residues beyond Ser453 (Fig. 6). RESULTS +28 36 disorder protein_state The peripheral location and disorder of the CTR beyond the kinase target site should facilitate the phosphorylation by Npr1. RESULTS +44 47 CTR structure_element The peripheral location and disorder of the CTR beyond the kinase target site should facilitate the phosphorylation by Npr1. RESULTS +59 77 kinase target site site The peripheral location and disorder of the CTR beyond the kinase target site should facilitate the phosphorylation by Npr1. RESULTS +100 115 phosphorylation ptm The peripheral location and disorder of the CTR beyond the kinase target site should facilitate the phosphorylation by Npr1. RESULTS +119 123 Npr1 protein The peripheral location and disorder of the CTR beyond the kinase target site should facilitate the phosphorylation by Npr1. RESULTS +4 14 disordered protein_state The disordered part of the CTR is not conserved in ammonium transporters (Fig. 2), suggesting that it is not important for transport. RESULTS +27 30 CTR structure_element The disordered part of the CTR is not conserved in ammonium transporters (Fig. 2), suggesting that it is not important for transport. RESULTS +34 47 not conserved protein_state The disordered part of the CTR is not conserved in ammonium transporters (Fig. 2), suggesting that it is not important for transport. RESULTS +51 72 ammonium transporters protein_type The disordered part of the CTR is not conserved in ammonium transporters (Fig. 2), suggesting that it is not important for transport. RESULTS +17 23 ScMep2 protein Interestingly, a ScMep2 457Δ truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1. RESULTS +24 28 457Δ mutant Interestingly, a ScMep2 457Δ truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1. RESULTS +29 46 truncation mutant protein_state Interestingly, a ScMep2 457Δ truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1. RESULTS +83 89 Ser457 residue_name_number Interestingly, a ScMep2 457Δ truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1. RESULTS +118 130 low activity protein_state Interestingly, a ScMep2 457Δ truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1. RESULTS +211 226 phosphorylation ptm Interestingly, a ScMep2 457Δ truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1. RESULTS +230 234 Npr1 protein Interestingly, a ScMep2 457Δ truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1. RESULTS +9 15 mutant mutant The same mutant lacking the His-tag has WT properties (Supplementary Fig. 1b), confirming that the region following the phosphorylation site is dispensable for function. RESULTS +16 35 lacking the His-tag protein_state The same mutant lacking the His-tag has WT properties (Supplementary Fig. 1b), confirming that the region following the phosphorylation site is dispensable for function. RESULTS +40 42 WT protein_state The same mutant lacking the His-tag has WT properties (Supplementary Fig. 1b), confirming that the region following the phosphorylation site is dispensable for function. RESULTS +120 140 phosphorylation site site The same mutant lacking the His-tag has WT properties (Supplementary Fig. 1b), confirming that the region following the phosphorylation site is dispensable for function. RESULTS +0 4 Mep2 protein Mep2 lacking the AI region is conformationally heterogeneous RESULTS +5 12 lacking protein_state Mep2 lacking the AI region is conformationally heterogeneous RESULTS +17 26 AI region structure_element Mep2 lacking the AI region is conformationally heterogeneous RESULTS +30 60 conformationally heterogeneous protein_state Mep2 lacking the AI region is conformationally heterogeneous RESULTS +11 17 Ser457 residue_name_number Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. RESULTS +18 21 453 residue_number Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. RESULTS +38 50 channel exit site Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. RESULTS +89 104 phosphorylation ptm Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. RESULTS +115 119 Mep2 protein Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. RESULTS +120 127 channel site Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. RESULTS +143 149 active protein_state Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. RESULTS +150 161 transporter protein_type Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. RESULTS +33 48 phosphorylation ptm Boeckstaens et al. proposed that phosphorylation does not affect channel activity directly, but instead relieves inhibition by the AI region. RESULTS +131 140 AI region structure_element Boeckstaens et al. proposed that phosphorylation does not affect channel activity directly, but instead relieves inhibition by the AI region. RESULTS +58 64 ScMep2 protein The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. RESULTS +65 73 449-485Δ mutant The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. RESULTS +74 89 deletion mutant protein_state The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. RESULTS +90 97 lacking protein_state The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. RESULTS +102 111 AI region structure_element The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. RESULTS +115 128 highly active protein_state The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. RESULTS +132 134 MA chemical The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. RESULTS +154 165 triple mepΔ mutant The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. RESULTS +170 187 triple mepΔ npr1Δ mutant The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. RESULTS +220 232 Mep2 variant mutant The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. RESULTS +239 258 constitutively open protein_state The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. RESULTS +259 266 channel site The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. RESULTS +56 60 446Δ mutant We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. RESULTS +61 67 mutant protein_state We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. RESULTS +125 149 constructed and purified experimental_method We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. RESULTS +164 170 CaMep2 protein We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. RESULTS +171 175 442Δ mutant We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. RESULTS +176 193 truncation mutant protein_state We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. RESULTS +198 208 determined experimental_method We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. RESULTS +213 230 crystal structure evidence We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. RESULTS +4 13 structure evidence The structure shows that removal of the AI region markedly increases the dynamics of the cytoplasmic parts of the transporter. RESULTS +25 35 removal of experimental_method The structure shows that removal of the AI region markedly increases the dynamics of the cytoplasmic parts of the transporter. RESULTS +40 49 AI region structure_element The structure shows that removal of the AI region markedly increases the dynamics of the cytoplasmic parts of the transporter. RESULTS +89 106 cytoplasmic parts structure_element The structure shows that removal of the AI region markedly increases the dynamics of the cytoplasmic parts of the transporter. RESULTS +114 125 transporter protein_type The structure shows that removal of the AI region markedly increases the dynamics of the cytoplasmic parts of the transporter. RESULTS +47 56 AI region structure_element This is not unexpected given the fact that the AI region bridges the CTR and the main body of Mep2 (Fig. 6). RESULTS +69 72 CTR structure_element This is not unexpected given the fact that the AI region bridges the CTR and the main body of Mep2 (Fig. 6). RESULTS +81 90 main body structure_element This is not unexpected given the fact that the AI region bridges the CTR and the main body of Mep2 (Fig. 6). RESULTS +94 98 Mep2 protein This is not unexpected given the fact that the AI region bridges the CTR and the main body of Mep2 (Fig. 6). RESULTS +0 7 Density evidence Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). RESULTS +12 16 ICL3 structure_element Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). RESULTS +25 28 CTR structure_element Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). RESULTS +44 50 Arg415 residue_name_number Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). RESULTS +69 73 442Δ mutant Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). RESULTS +74 80 mutant protein_state Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). RESULTS +90 97 density evidence Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). RESULTS +112 116 ICLs structure_element Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). RESULTS +127 131 ICL1 structure_element Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). RESULTS +176 185 structure evidence Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). RESULTS +28 33 Tyr49 residue_name_number Interestingly, however, the Tyr49-His342 hydrogen bond that closes the channel in the WT protein is still present (Fig. 7 and Supplementary Fig. 2). RESULTS +34 40 His342 residue_name_number Interestingly, however, the Tyr49-His342 hydrogen bond that closes the channel in the WT protein is still present (Fig. 7 and Supplementary Fig. 2). RESULTS +41 54 hydrogen bond bond_interaction Interestingly, however, the Tyr49-His342 hydrogen bond that closes the channel in the WT protein is still present (Fig. 7 and Supplementary Fig. 2). RESULTS +86 88 WT protein_state Interestingly, however, the Tyr49-His342 hydrogen bond that closes the channel in the WT protein is still present (Fig. 7 and Supplementary Fig. 2). RESULTS +54 60 active protein_state Why then does this mutant appear to be constitutively active? We propose two possibilities. RESULTS +26 30 open protein_state The first one is that the open state is disfavoured by crystallization because of lower stability or due to crystal packing constraints. RESULTS +55 70 crystallization experimental_method The first one is that the open state is disfavoured by crystallization because of lower stability or due to crystal packing constraints. RESULTS +35 56 Tyr–His hydrogen bond site The second possibility is that the Tyr–His hydrogen bond has to be disrupted by the incoming substrate to open the channel. RESULTS +106 110 open protein_state The second possibility is that the Tyr–His hydrogen bond has to be disrupted by the incoming substrate to open the channel. RESULTS +41 44 NH3 chemical The latter model would fit well with the NH3/H+ symport model in which the proton is relayed by the twin-His motif. RESULTS +45 47 H+ chemical The latter model would fit well with the NH3/H+ symport model in which the proton is relayed by the twin-His motif. RESULTS +100 114 twin-His motif structure_element The latter model would fit well with the NH3/H+ symport model in which the proton is relayed by the twin-His motif. RESULTS +22 43 Tyr–His hydrogen bond site The importance of the Tyr–His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3). RESULTS +80 87 removal experimental_method The importance of the Tyr–His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3). RESULTS +95 101 ScMep2 protein The importance of the Tyr–His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3). RESULTS +102 106 Y53A mutant The importance of the Tyr–His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3). RESULTS +107 113 mutant protein_state The importance of the Tyr–His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3). RESULTS +127 148 constitutively active protein_state The importance of the Tyr–His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3). RESULTS +149 160 transporter protein_type The importance of the Tyr–His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3). RESULTS +0 15 Phosphorylation ptm Phosphorylation causes a conformational change in the CTR RESULTS +54 57 CTR structure_element Phosphorylation causes a conformational change in the CTR RESULTS +7 11 Mep2 protein Do the Mep2 structures provide any clues regarding the potential effect of phosphorylation? RESULTS +12 22 structures evidence Do the Mep2 structures provide any clues regarding the potential effect of phosphorylation? RESULTS +75 90 phosphorylation ptm Do the Mep2 structures provide any clues regarding the potential effect of phosphorylation? RESULTS +27 33 Ser457 residue_name_number The side-chain hydroxyl of Ser457/453 is located in a well-defined electronegative pocket that is solvent accessible (Fig. 6). RESULTS +34 37 453 residue_number The side-chain hydroxyl of Ser457/453 is located in a well-defined electronegative pocket that is solvent accessible (Fig. 6). RESULTS +67 89 electronegative pocket site The side-chain hydroxyl of Ser457/453 is located in a well-defined electronegative pocket that is solvent accessible (Fig. 6). RESULTS +98 116 solvent accessible protein_state The side-chain hydroxyl of Ser457/453 is located in a well-defined electronegative pocket that is solvent accessible (Fig. 6). RESULTS +25 31 serine residue_name The closest atoms to the serine hydroxyl group are the backbone carbonyl atoms of Asp419, Glu420 and Glu421, which are 3–4 Å away. RESULTS +82 88 Asp419 residue_name_number The closest atoms to the serine hydroxyl group are the backbone carbonyl atoms of Asp419, Glu420 and Glu421, which are 3–4 Å away. RESULTS +90 96 Glu420 residue_name_number The closest atoms to the serine hydroxyl group are the backbone carbonyl atoms of Asp419, Glu420 and Glu421, which are 3–4 Å away. RESULTS +101 107 Glu421 residue_name_number The closest atoms to the serine hydroxyl group are the backbone carbonyl atoms of Asp419, Glu420 and Glu421, which are 3–4 Å away. RESULTS +26 41 phosphorylation ptm We therefore predict that phosphorylation of Ser453 will result in steric clashes as well as electrostatic repulsion, which in turn might cause substantial conformational changes within the CTR. RESULTS +45 51 Ser453 residue_name_number We therefore predict that phosphorylation of Ser453 will result in steric clashes as well as electrostatic repulsion, which in turn might cause substantial conformational changes within the CTR. RESULTS +190 193 CTR structure_element We therefore predict that phosphorylation of Ser453 will result in steric clashes as well as electrostatic repulsion, which in turn might cause substantial conformational changes within the CTR. RESULTS +28 38 determined experimental_method To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). RESULTS +43 52 structure evidence To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). RESULTS +60 85 phosphorylation-mimicking protein_state To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). RESULTS +86 97 R452D/S453D mutant To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). RESULTS +125 134 DD mutant mutant To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). RESULTS +179 201 additional mutation of experimental_method To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). RESULTS +206 214 arginine residue_name To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). RESULTS +229 249 phosphorylation site site To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). RESULTS +342 351 phosphate chemical To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). RESULTS +423 432 AI region structure_element To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). RESULTS +442 451 main body structure_element To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). RESULTS +459 470 transporter protein_type To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). RESULTS +4 12 ammonium chemical The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). RESULTS +36 49 S. cerevisiae species The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). RESULTS +65 74 DD mutant mutant The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). RESULTS +98 100 WT protein_state The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). RESULTS +101 105 Mep2 protein The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). RESULTS +114 119 S453D mutant The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). RESULTS +120 126 mutant protein_state The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). RESULTS +205 216 triple mepΔ mutant The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). RESULTS +18 28 AI segment structure_element Unexpectedly, the AI segment containing the mutated residues has only undergone a slight shift compared with the WT protein (Fig. 8 and Supplementary Fig. 3). RESULTS +113 115 WT protein_state Unexpectedly, the AI segment containing the mutated residues has only undergone a slight shift compared with the WT protein (Fig. 8 and Supplementary Fig. 3). RESULTS +17 26 conserved protein_state By contrast, the conserved part of the CTR has undergone a large conformational change involving formation of a 12-residue-long α-helix from Leu427 to Asp438. RESULTS +39 42 CTR structure_element By contrast, the conserved part of the CTR has undergone a large conformational change involving formation of a 12-residue-long α-helix from Leu427 to Asp438. RESULTS +112 135 12-residue-long α-helix structure_element By contrast, the conserved part of the CTR has undergone a large conformational change involving formation of a 12-residue-long α-helix from Leu427 to Asp438. RESULTS +141 157 Leu427 to Asp438 residue_range By contrast, the conserved part of the CTR has undergone a large conformational change involving formation of a 12-residue-long α-helix from Leu427 to Asp438. RESULTS +22 35 Glu420-Leu423 residue_range In addition, residues Glu420-Leu423 including Glu421 of the ExxGxD motif are now disordered (Fig. 8 and Supplementary Fig. 3). RESULTS +46 52 Glu421 residue_name_number In addition, residues Glu420-Leu423 including Glu421 of the ExxGxD motif are now disordered (Fig. 8 and Supplementary Fig. 3). RESULTS +60 72 ExxGxD motif structure_element In addition, residues Glu420-Leu423 including Glu421 of the ExxGxD motif are now disordered (Fig. 8 and Supplementary Fig. 3). RESULTS +81 91 disordered protein_state In addition, residues Glu420-Leu423 including Glu421 of the ExxGxD motif are now disordered (Fig. 8 and Supplementary Fig. 3). RESULTS +77 97 ammonium transporter protein_type This is the first time a large conformational change has been observed in an ammonium transporter as a result of a mutation, and confirms previous hypotheses that phosphorylation causes structural changes in the CTR. RESULTS +115 123 mutation experimental_method This is the first time a large conformational change has been observed in an ammonium transporter as a result of a mutation, and confirms previous hypotheses that phosphorylation causes structural changes in the CTR. RESULTS +163 178 phosphorylation ptm This is the first time a large conformational change has been observed in an ammonium transporter as a result of a mutation, and confirms previous hypotheses that phosphorylation causes structural changes in the CTR. RESULTS +212 215 CTR structure_element This is the first time a large conformational change has been observed in an ammonium transporter as a result of a mutation, and confirms previous hypotheses that phosphorylation causes structural changes in the CTR. RESULTS +47 52 R452D mutant To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the ‘single D' S453D mutant. RESULTS +111 121 determined experimental_method To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the ‘single D' S453D mutant. RESULTS +126 135 structure evidence To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the ‘single D' S453D mutant. RESULTS +144 152 single D mutant To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the ‘single D' S453D mutant. RESULTS +154 159 S453D mutant To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the ‘single D' S453D mutant. RESULTS +160 166 mutant protein_state To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the ‘single D' S453D mutant. RESULTS +57 65 single D mutant As shown in Supplementary Fig. 4, the consequence of the single D mutation is very similar to that of the DD substitution, with conformational changes and increased dynamics confined to the conserved part of the CTR (Supplementary Fig. 4). RESULTS +66 74 mutation experimental_method As shown in Supplementary Fig. 4, the consequence of the single D mutation is very similar to that of the DD substitution, with conformational changes and increased dynamics confined to the conserved part of the CTR (Supplementary Fig. 4). RESULTS +106 121 DD substitution mutant As shown in Supplementary Fig. 4, the consequence of the single D mutation is very similar to that of the DD substitution, with conformational changes and increased dynamics confined to the conserved part of the CTR (Supplementary Fig. 4). RESULTS +190 199 conserved protein_state As shown in Supplementary Fig. 4, the consequence of the single D mutation is very similar to that of the DD substitution, with conformational changes and increased dynamics confined to the conserved part of the CTR (Supplementary Fig. 4). RESULTS +212 215 CTR structure_element As shown in Supplementary Fig. 4, the consequence of the single D mutation is very similar to that of the DD substitution, with conformational changes and increased dynamics confined to the conserved part of the CTR (Supplementary Fig. 4). RESULTS +18 36 crystal structures evidence To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). RESULTS +56 65 modelling experimental_method To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). RESULTS +70 72 MD experimental_method To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). RESULTS +84 86 WT protein_state To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). RESULTS +87 93 CaMep2 protein To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). RESULTS +99 108 DD mutant mutant To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). RESULTS +113 127 phosphorylated protein_state To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). RESULTS +137 142 S453J mutant To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). RESULTS +7 9 WT protein_state In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453. RESULTS +10 19 structure evidence In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453. RESULTS +41 47 Asp419 residue_name_number In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453. RESULTS +49 55 Glu420 residue_name_number In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453. RESULTS +60 66 Glu421 residue_name_number In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453. RESULTS +78 94 hydrogen bonding bond_interaction In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453. RESULTS +107 113 Ser453 residue_name_number In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453. RESULTS +16 18 MD experimental_method After 200 ns of MD simulation, the interactions between these residues and Ser453 remain intact. RESULTS +19 29 simulation experimental_method After 200 ns of MD simulation, the interactions between these residues and Ser453 remain intact. RESULTS +75 81 Ser453 residue_name_number After 200 ns of MD simulation, the interactions between these residues and Ser453 remain intact. RESULTS +36 44 r.m.s.d. evidence The protein backbone has an average r.m.s.d. of only ∼3 Å during the 200-ns simulation, indicating that the protein is stable. RESULTS +76 86 simulation experimental_method The protein backbone has an average r.m.s.d. of only ∼3 Å during the 200-ns simulation, indicating that the protein is stable. RESULTS +119 125 stable protein_state The protein backbone has an average r.m.s.d. of only ∼3 Å during the 200-ns simulation, indicating that the protein is stable. RESULTS +93 99 stable protein_state There is flexibility in the side chains of the acidic residues so that they are able to form stable hydrogen bonds with Ser453. RESULTS +100 114 hydrogen bonds bond_interaction There is flexibility in the side chains of the acidic residues so that they are able to form stable hydrogen bonds with Ser453. RESULTS +120 126 Ser453 residue_name_number There is flexibility in the side chains of the acidic residues so that they are able to form stable hydrogen bonds with Ser453. RESULTS +26 40 hydrogen bonds bond_interaction In particular, persistent hydrogen bonds are observed between the Ser453 hydroxyl group and the acidic group of Glu420, and also between the amine group of Ser453 and the backbone carbonyl of Glu420 (Supplementary Fig. 5). RESULTS +66 72 Ser453 residue_name_number In particular, persistent hydrogen bonds are observed between the Ser453 hydroxyl group and the acidic group of Glu420, and also between the amine group of Ser453 and the backbone carbonyl of Glu420 (Supplementary Fig. 5). RESULTS +112 118 Glu420 residue_name_number In particular, persistent hydrogen bonds are observed between the Ser453 hydroxyl group and the acidic group of Glu420, and also between the amine group of Ser453 and the backbone carbonyl of Glu420 (Supplementary Fig. 5). RESULTS +156 162 Ser453 residue_name_number In particular, persistent hydrogen bonds are observed between the Ser453 hydroxyl group and the acidic group of Glu420, and also between the amine group of Ser453 and the backbone carbonyl of Glu420 (Supplementary Fig. 5). RESULTS +192 198 Glu420 residue_name_number In particular, persistent hydrogen bonds are observed between the Ser453 hydroxyl group and the acidic group of Glu420, and also between the amine group of Ser453 and the backbone carbonyl of Glu420 (Supplementary Fig. 5). RESULTS +4 13 DD mutant mutant The DD mutant is also stable during the simulations, but the average backbone r.m.s.d of ∼3.6 Å suggests slightly more conformational flexibility than WT. RESULTS +22 28 stable protein_state The DD mutant is also stable during the simulations, but the average backbone r.m.s.d of ∼3.6 Å suggests slightly more conformational flexibility than WT. RESULTS +40 51 simulations experimental_method The DD mutant is also stable during the simulations, but the average backbone r.m.s.d of ∼3.6 Å suggests slightly more conformational flexibility than WT. RESULTS +78 85 r.m.s.d evidence The DD mutant is also stable during the simulations, but the average backbone r.m.s.d of ∼3.6 Å suggests slightly more conformational flexibility than WT. RESULTS +151 153 WT protein_state The DD mutant is also stable during the simulations, but the average backbone r.m.s.d of ∼3.6 Å suggests slightly more conformational flexibility than WT. RESULTS +7 17 simulation experimental_method As the simulation proceeds, the side chains of the acidic residues move away from Asp452 and Asp453, presumably to avoid electrostatic repulsion. RESULTS +82 88 Asp452 residue_name_number As the simulation proceeds, the side chains of the acidic residues move away from Asp452 and Asp453, presumably to avoid electrostatic repulsion. RESULTS +93 99 Asp453 residue_name_number As the simulation proceeds, the side chains of the acidic residues move away from Asp452 and Asp453, presumably to avoid electrostatic repulsion. RESULTS +17 25 distance evidence For example, the distance between the Asp453 acidic oxygens and the Glu420 acidic oxygens increases from ∼7 to >22 Å after 200 ns simulations, and thus these residues are not interacting. RESULTS +38 44 Asp453 residue_name_number For example, the distance between the Asp453 acidic oxygens and the Glu420 acidic oxygens increases from ∼7 to >22 Å after 200 ns simulations, and thus these residues are not interacting. RESULTS +68 74 Glu420 residue_name_number For example, the distance between the Asp453 acidic oxygens and the Glu420 acidic oxygens increases from ∼7 to >22 Å after 200 ns simulations, and thus these residues are not interacting. RESULTS +130 141 simulations experimental_method For example, the distance between the Asp453 acidic oxygens and the Glu420 acidic oxygens increases from ∼7 to >22 Å after 200 ns simulations, and thus these residues are not interacting. RESULTS +15 34 structurally stable protein_state The protein is structurally stable throughout the simulation with little deviation in the other parts of the protein. RESULTS +50 60 simulation experimental_method The protein is structurally stable throughout the simulation with little deviation in the other parts of the protein. RESULTS +13 18 S453J mutant Finally, the S453J mutant is also stable throughout the 200-ns simulation and has an average backbone deviation of ∼3.8 Å, which is similar to the DD mutant. RESULTS +19 25 mutant protein_state Finally, the S453J mutant is also stable throughout the 200-ns simulation and has an average backbone deviation of ∼3.8 Å, which is similar to the DD mutant. RESULTS +34 40 stable protein_state Finally, the S453J mutant is also stable throughout the 200-ns simulation and has an average backbone deviation of ∼3.8 Å, which is similar to the DD mutant. RESULTS +63 73 simulation experimental_method Finally, the S453J mutant is also stable throughout the 200-ns simulation and has an average backbone deviation of ∼3.8 Å, which is similar to the DD mutant. RESULTS +147 156 DD mutant mutant Finally, the S453J mutant is also stable throughout the 200-ns simulation and has an average backbone deviation of ∼3.8 Å, which is similar to the DD mutant. RESULTS +46 52 Arg452 residue_name_number The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant. RESULTS +57 63 Sep453 residue_name_number The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant. RESULTS +91 101 simulation experimental_method The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant. RESULTS +144 150 Asp452 residue_name_number The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant. RESULTS +155 161 Asp453 residue_name_number The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant. RESULTS +169 178 DD mutant mutant The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant. RESULTS +4 12 distance evidence The distance between the phosphate of Sep453 and the acidic oxygen atoms of Glu420 is initially ∼11 Å, but increases to >30 Å after 200 ns. RESULTS +25 34 phosphate chemical The distance between the phosphate of Sep453 and the acidic oxygen atoms of Glu420 is initially ∼11 Å, but increases to >30 Å after 200 ns. RESULTS +38 44 Sep453 residue_name_number The distance between the phosphate of Sep453 and the acidic oxygen atoms of Glu420 is initially ∼11 Å, but increases to >30 Å after 200 ns. RESULTS +76 82 Glu420 residue_name_number The distance between the phosphate of Sep453 and the acidic oxygen atoms of Glu420 is initially ∼11 Å, but increases to >30 Å after 200 ns. RESULTS +4 15 short helix structure_element The short helix formed by residues Leu427 to Asp438 unravels during the simulations to a disordered state. RESULTS +35 51 Leu427 to Asp438 residue_range The short helix formed by residues Leu427 to Asp438 unravels during the simulations to a disordered state. RESULTS +72 83 simulations experimental_method The short helix formed by residues Leu427 to Asp438 unravels during the simulations to a disordered state. RESULTS +89 99 disordered protein_state The short helix formed by residues Leu427 to Asp438 unravels during the simulations to a disordered state. RESULTS +10 12 MD experimental_method Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR. RESULTS +13 24 simulations experimental_method Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR. RESULTS +53 71 crystal structures evidence Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR. RESULTS +77 92 phosphorylation ptm Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR. RESULTS +133 142 conserved protein_state Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR. RESULTS +155 158 CTR structure_element Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR. RESULTS +44 66 phosphomimetic mutants mutant However, the conformational changes for the phosphomimetic mutants in the crystals are confined to the CTR (Fig. 8), and the channels are still closed (Supplementary Fig. 2). RESULTS +74 82 crystals evidence However, the conformational changes for the phosphomimetic mutants in the crystals are confined to the CTR (Fig. 8), and the channels are still closed (Supplementary Fig. 2). RESULTS +103 106 CTR structure_element However, the conformational changes for the phosphomimetic mutants in the crystals are confined to the CTR (Fig. 8), and the channels are still closed (Supplementary Fig. 2). RESULTS +125 133 channels site However, the conformational changes for the phosphomimetic mutants in the crystals are confined to the CTR (Fig. 8), and the channels are still closed (Supplementary Fig. 2). RESULTS +144 150 closed protein_state However, the conformational changes for the phosphomimetic mutants in the crystals are confined to the CTR (Fig. 8), and the channels are still closed (Supplementary Fig. 2). RESULTS +37 44 mutants mutant One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). RESULTS +71 84 phosphoserine residue_name One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). RESULTS +115 120 S453D mutant One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). RESULTS +125 135 DD mutants mutant One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). RESULTS +140 152 fully active protein_state One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). RESULTS +160 170 absence of protein_state One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). RESULTS +171 175 Npr1 protein One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). RESULTS +194 203 mutations experimental_method One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). RESULTS +227 242 phosphorylation ptm One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). RESULTS +18 23 S453D mutant The fact that the S453D structure was obtained in the presence of 10 mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels. RESULTS +24 33 structure evidence The fact that the S453D structure was obtained in the presence of 10 mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels. RESULTS +72 80 ammonium chemical The fact that the S453D structure was obtained in the presence of 10 mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels. RESULTS +104 119 crystallization experimental_method The fact that the S453D structure was obtained in the presence of 10 mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels. RESULTS +136 142 closed protein_state The fact that the S453D structure was obtained in the presence of 10 mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels. RESULTS +157 161 Mep2 protein The fact that the S453D structure was obtained in the presence of 10 mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels. RESULTS +162 170 channels site The fact that the S453D structure was obtained in the presence of 10 mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels. RESULTS +16 36 ammonium transporter protein_type Knowledge about ammonium transporter structure has been obtained from experimental and theoretical studies on bacterial family members. DISCUSS +37 46 structure evidence Knowledge about ammonium transporter structure has been obtained from experimental and theoretical studies on bacterial family members. DISCUSS +110 119 bacterial taxonomy_domain Knowledge about ammonium transporter structure has been obtained from experimental and theoretical studies on bacterial family members. DISCUSS +25 56 biochemical and genetic studies experimental_method In addition, a number of biochemical and genetic studies are available for bacterial, fungal and plant proteins. DISCUSS +75 84 bacterial taxonomy_domain In addition, a number of biochemical and genetic studies are available for bacterial, fungal and plant proteins. DISCUSS +86 92 fungal taxonomy_domain In addition, a number of biochemical and genetic studies are available for bacterial, fungal and plant proteins. DISCUSS +97 102 plant taxonomy_domain In addition, a number of biochemical and genetic studies are available for bacterial, fungal and plant proteins. DISCUSS +159 167 ammonium chemical These efforts have advanced our knowledge considerably but have not yet yielded atomic-level answers to several important mechanistic questions, including how ammonium transport is regulated in eukaryotes and the mechanism of ammonium signalling. DISCUSS +194 204 eukaryotes taxonomy_domain These efforts have advanced our knowledge considerably but have not yet yielded atomic-level answers to several important mechanistic questions, including how ammonium transport is regulated in eukaryotes and the mechanism of ammonium signalling. DISCUSS +226 234 ammonium chemical These efforts have advanced our knowledge considerably but have not yet yielded atomic-level answers to several important mechanistic questions, including how ammonium transport is regulated in eukaryotes and the mechanism of ammonium signalling. DISCUSS +3 23 Arabidopsis thaliana species In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. DISCUSS +24 31 Amt-1;1 protein In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. DISCUSS +33 48 phosphorylation ptm In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. DISCUSS +56 59 CTR structure_element In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. DISCUSS +68 72 T460 residue_name_number In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. DISCUSS +98 106 ammonium chemical In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. DISCUSS +158 176 non-phosphorylated protein_state In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. DISCUSS +191 196 plant taxonomy_domain In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. DISCUSS +197 208 transporter protein_type In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. DISCUSS +212 216 open protein_state In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. DISCUSS +15 39 phosphomimetic mutations mutant Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation. DISCUSS +60 67 monomer oligomeric_state Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation. DISCUSS +90 96 trimer oligomeric_state Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation. DISCUSS +158 161 CTR structure_element Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation. DISCUSS +196 204 ammonium chemical Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation. DISCUSS +228 243 phosphorylation ptm Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation. DISCUSS +48 53 plant taxonomy_domain Owing to the lack of structural information for plant AMTs, the details of channel closure and inter-monomer crosstalk are not yet clear. DISCUSS +54 58 AMTs protein_type Owing to the lack of structural information for plant AMTs, the details of channel closure and inter-monomer crosstalk are not yet clear. DISCUSS +75 82 channel site Owing to the lack of structural information for plant AMTs, the details of channel closure and inter-monomer crosstalk are not yet clear. DISCUSS +21 26 plant taxonomy_domain Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. DISCUSS +27 39 transporters protein_type Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. DISCUSS +45 53 inactive protein_state Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. DISCUSS +64 77 Mep2 proteins protein_type Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. DISCUSS +103 111 ammonium chemical Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. DISCUSS +116 134 non-phosphorylated protein_state Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. DISCUSS +141 149 channels site Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. DISCUSS +159 165 closed protein_state Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. DISCUSS +23 35 transporters protein_type The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. DISCUSS +44 55 A. thaliana species The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. DISCUSS +56 63 Amt-1;1 protein The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. DISCUSS +68 72 Mep2 protein The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. DISCUSS +107 122 phosphorylation ptm The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. DISCUSS +124 136 inactivation protein_state The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. DISCUSS +140 146 plants taxonomy_domain The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. DISCUSS +151 161 activation protein_state The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. DISCUSS +165 170 fungi taxonomy_domain The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. DISCUSS +3 8 fungi taxonomy_domain In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion. DISCUSS +21 29 ammonium chemical In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion. DISCUSS +59 80 ammonium transporters protein_type In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion. DISCUSS +111 119 ammonium chemical In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion. DISCUSS +145 153 ammonium chemical In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion. DISCUSS +168 171 Ato protein_type In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion. DISCUSS +172 184 transporters protein_type In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion. DISCUSS +25 35 structures evidence By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. DISCUSS +39 45 closed protein_state By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. DISCUSS +46 67 ammonium transporters protein_type By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. DISCUSS +72 81 comparing experimental_method By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. DISCUSS +88 98 structures evidence By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. DISCUSS +108 124 permanently open protein_state By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. DISCUSS +125 134 bacterial taxonomy_domain By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. DISCUSS +165 169 Mep2 protein_type By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. DISCUSS +170 177 channel site By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. DISCUSS +220 223 CTR structure_element By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. DISCUSS +228 232 ICL1 structure_element By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. DISCUSS +237 241 ICL3 structure_element By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. DISCUSS +54 57 CTR structure_element More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a). DISCUSS +62 66 ICL1 structure_element More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a). DISCUSS +67 71 ICL3 structure_element More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a). DISCUSS +83 87 open protein_state More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a). DISCUSS +88 100 transporters protein_type More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a). DISCUSS +124 128 ICL3 structure_element More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a). DISCUSS +160 167 channel site More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a). DISCUSS +13 17 ICL1 structure_element In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group. DISCUSS +59 66 channel site In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group. DISCUSS +87 91 His2 residue_name_number In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group. DISCUSS +101 115 twin-His motif structure_element In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group. DISCUSS +120 136 hydrogen bonding bond_interaction In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group. DISCUSS +144 160 highly conserved protein_state In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group. DISCUSS +161 169 tyrosine residue_name In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group. DISCUSS +5 20 phosphorylation ptm Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9). DISCUSS +28 32 Npr1 protein Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9). DISCUSS +33 39 kinase protein_type Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9). DISCUSS +55 63 nitrogen chemical Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9). DISCUSS +98 107 conserved protein_state Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9). DISCUSS +108 120 ExxGxD motif structure_element Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9). DISCUSS +170 177 channel site Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9). DISCUSS +17 40 structural similarities evidence Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel. DISCUSS +48 56 TM parts structure_element Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel. DISCUSS +60 64 Mep2 protein Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel. DISCUSS +69 76 AfAmt-1 protein Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel. DISCUSS +100 107 channel site Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel. DISCUSS +188 195 channel site Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel. DISCUSS +16 23 channel site How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3. DISCUSS +79 83 open protein_state How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3. DISCUSS +133 136 CTR structure_element How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3. DISCUSS +171 175 ICL3 structure_element How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3. DISCUSS +196 203 monomer oligomeric_state How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3. DISCUSS +265 272 channel site How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3. DISCUSS +300 304 ICL3 structure_element How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3. DISCUSS +31 39 monomers oligomeric_state Owing to the crosstalk between monomers, a single phosphorylation event might lead to opening of the entire trimer, although this has not yet been tested (Fig. 9b). DISCUSS +50 65 phosphorylation ptm Owing to the crosstalk between monomers, a single phosphorylation event might lead to opening of the entire trimer, although this has not yet been tested (Fig. 9b). DISCUSS +108 114 trimer oligomeric_state Owing to the crosstalk between monomers, a single phosphorylation event might lead to opening of the entire trimer, although this has not yet been tested (Fig. 9b). DISCUSS +15 19 Mep2 protein_type Whether or not Mep2 channel opening requires, in addition to phosphorylation, disruption of the Tyr–His2 interaction by the ammonium substrate is not yet clear. DISCUSS +20 27 channel site Whether or not Mep2 channel opening requires, in addition to phosphorylation, disruption of the Tyr–His2 interaction by the ammonium substrate is not yet clear. DISCUSS +61 76 phosphorylation ptm Whether or not Mep2 channel opening requires, in addition to phosphorylation, disruption of the Tyr–His2 interaction by the ammonium substrate is not yet clear. DISCUSS +96 116 Tyr–His2 interaction site Whether or not Mep2 channel opening requires, in addition to phosphorylation, disruption of the Tyr–His2 interaction by the ammonium substrate is not yet clear. DISCUSS +124 132 ammonium chemical Whether or not Mep2 channel opening requires, in addition to phosphorylation, disruption of the Tyr–His2 interaction by the ammonium substrate is not yet clear. DISCUSS +40 44 Mep2 protein Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. DISCUSS +45 53 channels site Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. DISCUSS +70 80 eukaryotic taxonomy_domain Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. DISCUSS +81 102 ammonium transporters protein_type Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. DISCUSS +108 123 structural data evidence Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. DISCUSS +185 188 CTR structure_element Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. DISCUSS +193 210 cytoplasmic loops structure_element Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. DISCUSS +237 243 closed protein_state Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. DISCUSS +248 252 open protein_state Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. DISCUSS +43 56 Mep2 proteins protein_type However, even the otherwise highly similar Mep2 proteins of S. cerevisiae and C. albicans have different structures for their CTRs (Fig. 1 and Supplementary Fig. 6). DISCUSS +60 73 S. cerevisiae species However, even the otherwise highly similar Mep2 proteins of S. cerevisiae and C. albicans have different structures for their CTRs (Fig. 1 and Supplementary Fig. 6). DISCUSS +78 89 C. albicans species However, even the otherwise highly similar Mep2 proteins of S. cerevisiae and C. albicans have different structures for their CTRs (Fig. 1 and Supplementary Fig. 6). DISCUSS +105 115 structures evidence However, even the otherwise highly similar Mep2 proteins of S. cerevisiae and C. albicans have different structures for their CTRs (Fig. 1 and Supplementary Fig. 6). DISCUSS +126 130 CTRs structure_element However, even the otherwise highly similar Mep2 proteins of S. cerevisiae and C. albicans have different structures for their CTRs (Fig. 1 and Supplementary Fig. 6). DISCUSS +17 26 AI region structure_element In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary. DISCUSS +34 37 CTR structure_element In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary. DISCUSS +53 69 Npr1 kinase site site In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary. DISCUSS +73 82 conserved protein_state In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary. DISCUSS +103 109 fungal taxonomy_domain In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary. DISCUSS +110 122 transporters protein_type In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary. DISCUSS +40 60 absolutely conserved protein_state Nevertheless, given the central role of absolutely conserved residues within the ICL1-ICL3-CTR interaction network (Fig. 4), we propose that the structural basics of fungal ammonium transporter activation are conserved. DISCUSS +81 114 ICL1-ICL3-CTR interaction network site Nevertheless, given the central role of absolutely conserved residues within the ICL1-ICL3-CTR interaction network (Fig. 4), we propose that the structural basics of fungal ammonium transporter activation are conserved. DISCUSS +166 172 fungal taxonomy_domain Nevertheless, given the central role of absolutely conserved residues within the ICL1-ICL3-CTR interaction network (Fig. 4), we propose that the structural basics of fungal ammonium transporter activation are conserved. DISCUSS +173 181 ammonium chemical Nevertheless, given the central role of absolutely conserved residues within the ICL1-ICL3-CTR interaction network (Fig. 4), we propose that the structural basics of fungal ammonium transporter activation are conserved. DISCUSS +209 218 conserved protein_state Nevertheless, given the central role of absolutely conserved residues within the ICL1-ICL3-CTR interaction network (Fig. 4), we propose that the structural basics of fungal ammonium transporter activation are conserved. DISCUSS +14 18 Mep2 protein_type The fact that Mep2 orthologues of distantly related fungi are fully functional in ammonium transport and signalling in S. cerevisiae supports this notion. DISCUSS +52 57 fungi taxonomy_domain The fact that Mep2 orthologues of distantly related fungi are fully functional in ammonium transport and signalling in S. cerevisiae supports this notion. DISCUSS +82 90 ammonium chemical The fact that Mep2 orthologues of distantly related fungi are fully functional in ammonium transport and signalling in S. cerevisiae supports this notion. DISCUSS +119 132 S. cerevisiae species The fact that Mep2 orthologues of distantly related fungi are fully functional in ammonium transport and signalling in S. cerevisiae supports this notion. DISCUSS +33 41 tyrosine residue_name It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins. DISCUSS +67 71 His2 residue_name_number It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins. DISCUSS +75 91 highly conserved protein_state It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins. DISCUSS +95 101 fungal taxonomy_domain It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins. DISCUSS +102 106 Mep2 protein_type It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins. DISCUSS +140 162 Tyr–His2 hydrogen bond site It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins. DISCUSS +189 194 close protein_state It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins. DISCUSS +195 208 Mep2 proteins protein_type It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins. DISCUSS +16 21 plant taxonomy_domain With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus. DISCUSS +22 26 AMTs protein_type With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus. DISCUSS +54 69 phosphorylation ptm With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus. DISCUSS +73 77 T460 residue_name_number With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus. DISCUSS +145 149 pore site With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus. DISCUSS +158 168 C terminus structure_element With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus. DISCUSS +38 52 homology model experimental_method This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. DISCUSS +57 64 Amt-1;1 protein This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. DISCUSS +79 83 open protein_state This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. DISCUSS +85 100 archaebacterial taxonomy_domain This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. DISCUSS +101 108 AfAmt-1 protein This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. DISCUSS +109 118 structure evidence This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. DISCUSS +145 155 C terminus structure_element This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. DISCUSS +159 166 Amt-1;1 protein This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. DISCUSS +208 215 monomer oligomeric_state This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. DISCUSS +4 8 Mep2 protein Our Mep2 structures show that this assumption may not be correct (Fig. 4 and Supplementary Fig. 6). DISCUSS +9 19 structures evidence Our Mep2 structures show that this assumption may not be correct (Fig. 4 and Supplementary Fig. 6). DISCUSS +72 75 CTR structure_element In addition, the considerable differences between structurally resolved CTR domains means that the exact environment of T460 in Amt-1;1 is also not known (Supplementary Fig. 6). DISCUSS +120 124 T460 residue_name_number In addition, the considerable differences between structurally resolved CTR domains means that the exact environment of T460 in Amt-1;1 is also not known (Supplementary Fig. 6). DISCUSS +128 135 Amt-1;1 protein In addition, the considerable differences between structurally resolved CTR domains means that the exact environment of T460 in Amt-1;1 is also not known (Supplementary Fig. 6). DISCUSS +23 45 structural information evidence Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). DISCUSS +120 127 Amt-1;1 protein Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). DISCUSS +195 198 CTR structure_element Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). DISCUSS +203 207 ICL1 structure_element Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). DISCUSS +208 212 ICL3 structure_element Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). DISCUSS +227 231 Y467 residue_name_number Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). DISCUSS +232 236 H239 residue_name_number Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). DISCUSS +241 245 D458 residue_name_number Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). DISCUSS +246 249 K71 residue_name_number Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). DISCUSS +37 43 CaMep2 protein There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters. DISCUSS +44 49 Tyr49 residue_name_number There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters. DISCUSS +53 58 plant taxonomy_domain There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters. DISCUSS +59 63 AMTs protein_type There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters. DISCUSS +83 105 Tyr–His2 hydrogen bond site There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters. DISCUSS +121 125 Mep2 protein There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters. DISCUSS +152 158 closed protein_state There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters. DISCUSS +168 173 plant taxonomy_domain There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters. DISCUSS +174 186 transporters protein_type There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters. DISCUSS +16 58 intra-monomeric CTR-ICL1/ICL3 interactions site We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. DISCUSS +98 104 fungal taxonomy_domain We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. DISCUSS +109 114 plant taxonomy_domain We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. DISCUSS +115 136 ammonium transporters protein_type We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. DISCUSS +166 170 open protein_state We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. DISCUSS +171 179 channels site We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. DISCUSS +193 200 lack of protein_state We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. DISCUSS +232 240 inactive protein_state We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. DISCUSS +64 85 phosphorylation sites site The need to regulate in opposite ways may be the reason why the phosphorylation sites are in different parts of the CTR, that is, centrally located close to the ExxGxD motif in AMTs and peripherally in Mep2. DISCUSS +116 119 CTR structure_element The need to regulate in opposite ways may be the reason why the phosphorylation sites are in different parts of the CTR, that is, centrally located close to the ExxGxD motif in AMTs and peripherally in Mep2. DISCUSS +161 173 ExxGxD motif structure_element The need to regulate in opposite ways may be the reason why the phosphorylation sites are in different parts of the CTR, that is, centrally located close to the ExxGxD motif in AMTs and peripherally in Mep2. DISCUSS +177 181 AMTs protein_type The need to regulate in opposite ways may be the reason why the phosphorylation sites are in different parts of the CTR, that is, centrally located close to the ExxGxD motif in AMTs and peripherally in Mep2. DISCUSS +202 206 Mep2 protein The need to regulate in opposite ways may be the reason why the phosphorylation sites are in different parts of the CTR, that is, centrally located close to the ExxGxD motif in AMTs and peripherally in Mep2. DISCUSS +13 28 phosphorylation ptm In this way, phosphorylation can either lead to channel closing (in the case of AMTs) or channel opening in the case of Mep2. DISCUSS +48 55 channel site In this way, phosphorylation can either lead to channel closing (in the case of AMTs) or channel opening in the case of Mep2. DISCUSS +80 84 AMTs protein_type In this way, phosphorylation can either lead to channel closing (in the case of AMTs) or channel opening in the case of Mep2. DISCUSS +89 96 channel site In this way, phosphorylation can either lead to channel closing (in the case of AMTs) or channel opening in the case of Mep2. DISCUSS +120 124 Mep2 protein In this way, phosphorylation can either lead to channel closing (in the case of AMTs) or channel opening in the case of Mep2. DISCUSS +64 81 certain mutations mutant Our model also provides an explanation for the observation that certain mutations within the CTR completely abolish transport activity. DISCUSS +93 96 CTR structure_element Our model also provides an explanation for the observation that certain mutations within the CTR completely abolish transport activity. DISCUSS +45 52 glycine residue_name An example of an inactivating residue is the glycine of the ExxGxD motif of the CTR. DISCUSS +60 72 ExxGxD motif structure_element An example of an inactivating residue is the glycine of the ExxGxD motif of the CTR. DISCUSS +80 83 CTR structure_element An example of an inactivating residue is the glycine of the ExxGxD motif of the CTR. DISCUSS +0 8 Mutation experimental_method Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). DISCUSS +26 30 G393 residue_name_number Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). DISCUSS +34 40 EcAmtB protein Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). DISCUSS +42 46 G456 residue_name_number Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). DISCUSS +50 59 AtAmt-1;1 protein Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). DISCUSS +73 85 transporters protein_type Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). DISCUSS +100 116 Escherichia coli species Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). DISCUSS +117 121 AmtB protein Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). DISCUSS +126 137 A. thaliana species Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). DISCUSS +138 145 Amt-1;1 protein Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). DISCUSS +54 57 CTR structure_element Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2. DISCUSS +98 101 CTR structure_element Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2. DISCUSS +106 110 ICL1 structure_element Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2. DISCUSS +111 115 ICL3 structure_element Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2. DISCUSS +139 145 closed protein_state Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2. DISCUSS +192 196 Mep2 protein Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2. DISCUSS +51 66 phosphorylation ptm Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters. DISCUSS +102 112 aquaporins protein_type Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters. DISCUSS +117 134 urea transporters protein_type Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters. DISCUSS +175 185 eukaryotic taxonomy_domain Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters. DISCUSS +186 194 channels protein_type Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters. DISCUSS +199 211 transporters protein_type Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters. DISCUSS +10 25 phosphorylation ptm Recently, phosphorylation was also shown to modulate substrate affinity in nitrate transporters. DISCUSS +75 95 nitrate transporters protein_type Recently, phosphorylation was also shown to modulate substrate affinity in nitrate transporters. DISCUSS +16 24 ammonium chemical With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs). DISCUSS +36 51 phosphorylation ptm With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs). DISCUSS +85 96 A. thaliana species With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs). DISCUSS +97 101 AMTs protein_type With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs). DISCUSS +110 123 S. cerevisiae species With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs). DISCUSS +124 128 Mep2 protein With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs). DISCUSS +13 23 absence of protein_state However, the absence of GlnK proteins in eukaryotes suggests that phosphorylation-based regulation of ammonium transport may be widespread. DISCUSS +24 37 GlnK proteins protein_type However, the absence of GlnK proteins in eukaryotes suggests that phosphorylation-based regulation of ammonium transport may be widespread. DISCUSS +41 51 eukaryotes taxonomy_domain However, the absence of GlnK proteins in eukaryotes suggests that phosphorylation-based regulation of ammonium transport may be widespread. DISCUSS +66 81 phosphorylation ptm However, the absence of GlnK proteins in eukaryotes suggests that phosphorylation-based regulation of ammonium transport may be widespread. DISCUSS +102 110 ammonium chemical However, the absence of GlnK proteins in eukaryotes suggests that phosphorylation-based regulation of ammonium transport may be widespread. DISCUSS +16 20 Mep2 protein_type With respect to Mep2-mediated signalling to induce pseudohyphal growth, two models have been put forward as to how this occurs and why it is specific to Mep2 proteins. DISCUSS +153 166 Mep2 proteins protein_type With respect to Mep2-mediated signalling to induce pseudohyphal growth, two models have been put forward as to how this occurs and why it is specific to Mep2 proteins. DISCUSS +142 163 ammonium transporters protein_type In one model, signalling is proposed to depend on the nature of the transported substrate, which might be different in certain subfamilies of ammonium transporters (for example, Mep1/Mep3 versus Mep2). DISCUSS +178 182 Mep1 protein In one model, signalling is proposed to depend on the nature of the transported substrate, which might be different in certain subfamilies of ammonium transporters (for example, Mep1/Mep3 versus Mep2). DISCUSS +183 187 Mep3 protein In one model, signalling is proposed to depend on the nature of the transported substrate, which might be different in certain subfamilies of ammonium transporters (for example, Mep1/Mep3 versus Mep2). DISCUSS +195 199 Mep2 protein In one model, signalling is proposed to depend on the nature of the transported substrate, which might be different in certain subfamilies of ammonium transporters (for example, Mep1/Mep3 versus Mep2). DISCUSS +13 16 NH3 chemical For example, NH3 uniport or symport of NH3/H+ might result in changes in local pH, but NH4+ uniport might not, and this difference might determine signalling. DISCUSS +39 42 NH3 chemical For example, NH3 uniport or symport of NH3/H+ might result in changes in local pH, but NH4+ uniport might not, and this difference might determine signalling. DISCUSS +43 45 H+ chemical For example, NH3 uniport or symport of NH3/H+ might result in changes in local pH, but NH4+ uniport might not, and this difference might determine signalling. DISCUSS +87 91 NH4+ chemical For example, NH3 uniport or symport of NH3/H+ might result in changes in local pH, but NH4+ uniport might not, and this difference might determine signalling. DISCUSS +84 88 Mep2 protein In the other model, signalling is thought to require a distinct conformation of the Mep2 transporter occurring during the transport cycle. DISCUSS +89 100 transporter protein_type In the other model, signalling is thought to require a distinct conformation of the Mep2 transporter occurring during the transport cycle. DISCUSS +118 128 structures evidence While the current study does not specifically address the mechanism of signalling underlying pseudohyphal growth, our structures do show that Mep2 proteins can assume different conformations. DISCUSS +142 155 Mep2 proteins protein_type While the current study does not specifically address the mechanism of signalling underlying pseudohyphal growth, our structures do show that Mep2 proteins can assume different conformations. DISCUSS +17 25 ammonium chemical It is clear that ammonium transport across biomembranes remains a fascinating and challenging field in large part due to the unique properties of the substrate. DISCUSS +4 8 Mep2 protein Our Mep2 structural work now provides a foundation for future studies to uncover the details of the structural changes that occur during eukaryotic ammonium transport and signaling, and to assess the possibility to utilize small molecules to shut down ammonium sensing and downstream signalling pathways in pathogenic fungi. DISCUSS +137 147 eukaryotic taxonomy_domain Our Mep2 structural work now provides a foundation for future studies to uncover the details of the structural changes that occur during eukaryotic ammonium transport and signaling, and to assess the possibility to utilize small molecules to shut down ammonium sensing and downstream signalling pathways in pathogenic fungi. DISCUSS +148 156 ammonium chemical Our Mep2 structural work now provides a foundation for future studies to uncover the details of the structural changes that occur during eukaryotic ammonium transport and signaling, and to assess the possibility to utilize small molecules to shut down ammonium sensing and downstream signalling pathways in pathogenic fungi. DISCUSS +252 260 ammonium chemical Our Mep2 structural work now provides a foundation for future studies to uncover the details of the structural changes that occur during eukaryotic ammonium transport and signaling, and to assess the possibility to utilize small molecules to shut down ammonium sensing and downstream signalling pathways in pathogenic fungi. DISCUSS +318 323 fungi taxonomy_domain Our Mep2 structural work now provides a foundation for future studies to uncover the details of the structural changes that occur during eukaryotic ammonium transport and signaling, and to assess the possibility to utilize small molecules to shut down ammonium sensing and downstream signalling pathways in pathogenic fungi. DISCUSS +0 24 X-ray crystal structures evidence X-ray crystal structures of Mep2 transceptors. FIG +28 32 Mep2 protein X-ray crystal structures of Mep2 transceptors. FIG +33 45 transceptors protein_type X-ray crystal structures of Mep2 transceptors. FIG +4 11 Monomer oligomeric_state (a) Monomer cartoon models viewed from the side for (left) A. fulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and C. albicans Mep2 (right). FIG +71 76 Amt-1 protein (a) Monomer cartoon models viewed from the side for (left) A. fulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and C. albicans Mep2 (right). FIG +92 105 S. cerevisiae species (a) Monomer cartoon models viewed from the side for (left) A. fulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and C. albicans Mep2 (right). FIG +106 110 Mep2 protein (a) Monomer cartoon models viewed from the side for (left) A. fulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and C. albicans Mep2 (right). FIG +124 135 C. albicans species (a) Monomer cartoon models viewed from the side for (left) A. fulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and C. albicans Mep2 (right). FIG +136 140 Mep2 protein (a) Monomer cartoon models viewed from the side for (left) A. fulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and C. albicans Mep2 (right). FIG +19 23 ICL1 structure_element The region showing ICL1 (blue), ICL3 (green) and the CTR (red) is boxed for comparison. FIG +32 36 ICL3 structure_element The region showing ICL1 (blue), ICL3 (green) and the CTR (red) is boxed for comparison. FIG +53 56 CTR structure_element The region showing ICL1 (blue), ICL3 (green) and the CTR (red) is boxed for comparison. FIG +4 10 CaMep2 protein (b) CaMep2 trimer viewed from the intracellular side (right). FIG +11 17 trimer oligomeric_state (b) CaMep2 trimer viewed from the intracellular side (right). FIG +4 11 monomer oligomeric_state One monomer is coloured as in a and one monomer is coloured by B-factor (blue, low; red; high). FIG +40 47 monomer oligomeric_state One monomer is coloured as in a and one monomer is coloured by B-factor (blue, low; red; high). FIG +4 7 CTR structure_element The CTR is boxed. FIG +5 12 Overlay experimental_method (c) Overlay of ScMep2 (grey) and CaMep2 (rainbow), illustrating the differences in the CTRs. FIG +16 22 ScMep2 protein (c) Overlay of ScMep2 (grey) and CaMep2 (rainbow), illustrating the differences in the CTRs. FIG +34 40 CaMep2 protein (c) Overlay of ScMep2 (grey) and CaMep2 (rainbow), illustrating the differences in the CTRs. FIG +88 92 CTRs structure_element (c) Overlay of ScMep2 (grey) and CaMep2 (rainbow), illustrating the differences in the CTRs. FIG +0 21 Sequence conservation evidence Sequence conservation in ammonium transporters. FIG +25 46 ammonium transporters protein_type Sequence conservation in ammonium transporters. FIG +0 18 ClustalW alignment experimental_method ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E. coli AmtB and A. thaliana Amt-1;1. FIG +22 28 CaMep2 protein ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E. coli AmtB and A. thaliana Amt-1;1. FIG +30 36 ScMep2 protein ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E. coli AmtB and A. thaliana Amt-1;1. FIG +38 49 A. fulgidus species ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E. coli AmtB and A. thaliana Amt-1;1. FIG +50 55 Amt-1 protein ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E. coli AmtB and A. thaliana Amt-1;1. FIG +65 69 AmtB protein ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E. coli AmtB and A. thaliana Amt-1;1. FIG +74 85 A. thaliana species ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E. coli AmtB and A. thaliana Amt-1;1. FIG +86 93 Amt-1;1 protein ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E. coli AmtB and A. thaliana Amt-1;1. FIG +46 52 CaMep2 protein The secondary structure elements observed for CaMep2 are indicated, with the numbers corresponding to the centre of the TM segment. FIG +120 130 TM segment structure_element The secondary structure elements observed for CaMep2 are indicated, with the numbers corresponding to the centre of the TM segment. FIG +4 13 conserved protein_state The conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, the conserved ExxGxD motif of the CTR in red and the AI region in yellow. FIG +14 23 RxK motif structure_element The conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, the conserved ExxGxD motif of the CTR in red and the AI region in yellow. FIG +27 31 ICL1 structure_element The conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, the conserved ExxGxD motif of the CTR in red and the AI region in yellow. FIG +54 62 ER motif structure_element The conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, the conserved ExxGxD motif of the CTR in red and the AI region in yellow. FIG +66 70 ICL2 structure_element The conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, the conserved ExxGxD motif of the CTR in red and the AI region in yellow. FIG +84 93 conserved protein_state The conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, the conserved ExxGxD motif of the CTR in red and the AI region in yellow. FIG +94 106 ExxGxD motif structure_element The conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, the conserved ExxGxD motif of the CTR in red and the AI region in yellow. FIG +114 117 CTR structure_element The conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, the conserved ExxGxD motif of the CTR in red and the AI region in yellow. FIG +133 142 AI region structure_element The conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, the conserved ExxGxD motif of the CTR in red and the AI region in yellow. FIG +77 85 Phe gate site Coloured residues are functionally important and correspond to those of the Phe gate (blue), the binding site Trp residue (magenta) and the twin-His motif (red). FIG +98 110 binding site site Coloured residues are functionally important and correspond to those of the Phe gate (blue), the binding site Trp residue (magenta) and the twin-His motif (red). FIG +111 114 Trp residue_name Coloured residues are functionally important and correspond to those of the Phe gate (blue), the binding site Trp residue (magenta) and the twin-His motif (red). FIG +4 20 Npr1 kinase site site The Npr1 kinase site in the AI region is highlighted pink. FIG +28 37 AI region structure_element The Npr1 kinase site in the AI region is highlighted pink. FIG +39 45 CaMep2 protein The grey sequences at the C termini of CaMep2 and ScMep2 are not visible in the structures and are likely disordered. FIG +50 56 ScMep2 protein The grey sequences at the C termini of CaMep2 and ScMep2 are not visible in the structures and are likely disordered. FIG +80 90 structures evidence The grey sequences at the C termini of CaMep2 and ScMep2 are not visible in the structures and are likely disordered. FIG +99 116 likely disordered protein_state The grey sequences at the C termini of CaMep2 and ScMep2 are not visible in the structures and are likely disordered. FIG +0 6 Growth experimental_method Growth of ScMep2 variants on low ammonium medium. FIG +10 25 ScMep2 variants mutant Growth of ScMep2 variants on low ammonium medium. FIG +8 19 triple mepΔ mutant (a) The triple mepΔ strain (black) and triple mepΔ npr1Δ strain (grey) containing plasmids expressing WT and variant ScMep2 were grown on minimal medium containing 1 mM ammonium sulphate. FIG +102 104 WT protein_state (a) The triple mepΔ strain (black) and triple mepΔ npr1Δ strain (grey) containing plasmids expressing WT and variant ScMep2 were grown on minimal medium containing 1 mM ammonium sulphate. FIG +109 123 variant ScMep2 mutant (a) The triple mepΔ strain (black) and triple mepΔ npr1Δ strain (grey) containing plasmids expressing WT and variant ScMep2 were grown on minimal medium containing 1 mM ammonium sulphate. FIG +129 152 grown on minimal medium experimental_method (a) The triple mepΔ strain (black) and triple mepΔ npr1Δ strain (grey) containing plasmids expressing WT and variant ScMep2 were grown on minimal medium containing 1 mM ammonium sulphate. FIG +169 186 ammonium sulphate chemical (a) The triple mepΔ strain (black) and triple mepΔ npr1Δ strain (grey) containing plasmids expressing WT and variant ScMep2 were grown on minimal medium containing 1 mM ammonium sulphate. FIG +15 27 cell density evidence The quantified cell density reflects logarithmic growth after 24 h. Error bars are the s.d. for three replicates of each strain (b) The strains used in a were also serially diluted and spotted onto minimal agar plates containing glutamate (0.1%) or ammonium sulphate (1 mM), and grown for 3 days at 30 °C. FIG +229 238 glutamate chemical The quantified cell density reflects logarithmic growth after 24 h. Error bars are the s.d. for three replicates of each strain (b) The strains used in a were also serially diluted and spotted onto minimal agar plates containing glutamate (0.1%) or ammonium sulphate (1 mM), and grown for 3 days at 30 °C. FIG +249 266 ammonium sulphate chemical The quantified cell density reflects logarithmic growth after 24 h. Error bars are the s.d. for three replicates of each strain (b) The strains used in a were also serially diluted and spotted onto minimal agar plates containing glutamate (0.1%) or ammonium sulphate (1 mM), and grown for 3 days at 30 °C. FIG +31 35 Mep2 protein Structural differences between Mep2 and bacterial ammonium transporters. FIG +40 49 bacterial taxonomy_domain Structural differences between Mep2 and bacterial ammonium transporters. FIG +4 8 ICL1 structure_element (a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing unwinding and inward movement in the fungal protein. (b) Stereo diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in AfAmt-1 (light colours) and CaMep2 (dark colours). FIG +12 19 AfAmt-1 protein (a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing unwinding and inward movement in the fungal protein. (b) Stereo diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in AfAmt-1 (light colours) and CaMep2 (dark colours). FIG +37 43 CaMep2 protein (a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing unwinding and inward movement in the fungal protein. (b) Stereo diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in AfAmt-1 (light colours) and CaMep2 (dark colours). FIG +102 108 fungal taxonomy_domain (a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing unwinding and inward movement in the fungal protein. (b) Stereo diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in AfAmt-1 (light colours) and CaMep2 (dark colours). FIG +164 168 ICL1 structure_element (a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing unwinding and inward movement in the fungal protein. (b) Stereo diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in AfAmt-1 (light colours) and CaMep2 (dark colours). FIG +170 174 ICL3 structure_element (a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing unwinding and inward movement in the fungal protein. (b) Stereo diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in AfAmt-1 (light colours) and CaMep2 (dark colours). FIG +191 194 CTR structure_element (a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing unwinding and inward movement in the fungal protein. (b) Stereo diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in AfAmt-1 (light colours) and CaMep2 (dark colours). FIG +204 211 AfAmt-1 protein (a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing unwinding and inward movement in the fungal protein. (b) Stereo diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in AfAmt-1 (light colours) and CaMep2 (dark colours). FIG +232 238 CaMep2 protein (a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing unwinding and inward movement in the fungal protein. (b) Stereo diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in AfAmt-1 (light colours) and CaMep2 (dark colours). FIG +35 44 RxK motif structure_element The side chains of residues in the RxK motif as well as those of Tyr49 and His342 are labelled. FIG +65 70 Tyr49 residue_name_number The side chains of residues in the RxK motif as well as those of Tyr49 and His342 are labelled. FIG +75 81 His342 residue_name_number The side chains of residues in the RxK motif as well as those of Tyr49 and His342 are labelled. FIG +22 28 CaMep2 protein The numbering is for CaMep2. FIG +4 13 Conserved protein_state (c) Conserved residues in ICL1-3 and the CTR. FIG +26 32 ICL1-3 structure_element (c) Conserved residues in ICL1-3 and the CTR. FIG +41 44 CTR structure_element (c) Conserved residues in ICL1-3 and the CTR. FIG +27 33 CaMep2 protein Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the large differences in conformation of the conserved residues in ICL1 (RxK motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). FIG +45 52 AfAmt-1 protein Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the large differences in conformation of the conserved residues in ICL1 (RxK motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). FIG +112 121 conserved protein_state Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the large differences in conformation of the conserved residues in ICL1 (RxK motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). FIG +134 138 ICL1 structure_element Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the large differences in conformation of the conserved residues in ICL1 (RxK motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). FIG +158 162 ICL2 structure_element Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the large differences in conformation of the conserved residues in ICL1 (RxK motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). FIG +164 172 ER motif structure_element Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the large differences in conformation of the conserved residues in ICL1 (RxK motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). FIG +181 185 ICL3 structure_element Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the large differences in conformation of the conserved residues in ICL1 (RxK motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). FIG +202 205 CTR structure_element Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the large differences in conformation of the conserved residues in ICL1 (RxK motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). FIG +48 58 structures evidence The labelled residues are analogous within both structures. FIG +30 36 trimer oligomeric_state In b and c, the centre of the trimer is at top. FIG +20 24 Mep2 protein Channel closures in Mep2. FIG +11 24 superposition experimental_method (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +28 35 AfAmt-1 protein (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +40 46 CaMep2 protein (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +75 83 Phe gate site (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +85 89 His2 residue_name_number (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +97 111 twin-His motif structure_element (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +120 128 tyrosine residue_name (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +137 140 Y49 residue_name_number (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +144 147 TM1 structure_element (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +161 174 hydrogen bond bond_interaction (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +180 184 His2 residue_name_number (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +188 194 CaMep2 protein (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +271 284 channel block structure_element (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +314 320 CaMep2 protein (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. FIG +4 8 Npr1 protein The Npr1 kinase target Ser453 is dephosphorylated and located in an electronegative pocket. FIG +9 15 kinase protein_type The Npr1 kinase target Ser453 is dephosphorylated and located in an electronegative pocket. FIG +23 29 Ser453 residue_name_number The Npr1 kinase target Ser453 is dephosphorylated and located in an electronegative pocket. FIG +33 49 dephosphorylated protein_state The Npr1 kinase target Ser453 is dephosphorylated and located in an electronegative pocket. FIG +68 90 electronegative pocket site The Npr1 kinase target Ser453 is dephosphorylated and located in an electronegative pocket. FIG +19 25 CaMep2 protein (a) Stereoviews of CaMep2 showing 2Fo–Fc electron density (contoured at 1.0 σ) for CTR residues Asp419-Met422 and for Tyr446-Thr455 of the AI region. FIG +83 86 CTR structure_element (a) Stereoviews of CaMep2 showing 2Fo–Fc electron density (contoured at 1.0 σ) for CTR residues Asp419-Met422 and for Tyr446-Thr455 of the AI region. FIG +96 109 Asp419-Met422 residue_range (a) Stereoviews of CaMep2 showing 2Fo–Fc electron density (contoured at 1.0 σ) for CTR residues Asp419-Met422 and for Tyr446-Thr455 of the AI region. FIG +118 131 Tyr446-Thr455 residue_range (a) Stereoviews of CaMep2 showing 2Fo–Fc electron density (contoured at 1.0 σ) for CTR residues Asp419-Met422 and for Tyr446-Thr455 of the AI region. FIG +139 148 AI region structure_element (a) Stereoviews of CaMep2 showing 2Fo–Fc electron density (contoured at 1.0 σ) for CTR residues Asp419-Met422 and for Tyr446-Thr455 of the AI region. FIG +4 19 phosphorylation ptm The phosphorylation target residue Ser453 is labelled in bold. FIG +35 41 Ser453 residue_name_number The phosphorylation target residue Ser453 is labelled in bold. FIG +4 11 Overlay experimental_method (b) Overlay of the CTRs of ScMep2 (grey) and CaMep2 (green), showing the similar electronegative environment surrounding the phosphorylation site (P). FIG +19 23 CTRs structure_element (b) Overlay of the CTRs of ScMep2 (grey) and CaMep2 (green), showing the similar electronegative environment surrounding the phosphorylation site (P). FIG +27 33 ScMep2 protein (b) Overlay of the CTRs of ScMep2 (grey) and CaMep2 (green), showing the similar electronegative environment surrounding the phosphorylation site (P). FIG +45 51 CaMep2 protein (b) Overlay of the CTRs of ScMep2 (grey) and CaMep2 (green), showing the similar electronegative environment surrounding the phosphorylation site (P). FIG +125 145 phosphorylation site site (b) Overlay of the CTRs of ScMep2 (grey) and CaMep2 (green), showing the similar electronegative environment surrounding the phosphorylation site (P). FIG +4 14 AI regions structure_element The AI regions are coloured magenta. FIG +28 32 Mep2 protein (c) Cytoplasmic view of the Mep2 trimer indicating the large distance between Ser453 and the channel exits (circles; Ile52 lining the channel exit is shown). FIG +33 39 trimer oligomeric_state (c) Cytoplasmic view of the Mep2 trimer indicating the large distance between Ser453 and the channel exits (circles; Ile52 lining the channel exit is shown). FIG +78 84 Ser453 residue_name_number (c) Cytoplasmic view of the Mep2 trimer indicating the large distance between Ser453 and the channel exits (circles; Ile52 lining the channel exit is shown). FIG +93 106 channel exits site (c) Cytoplasmic view of the Mep2 trimer indicating the large distance between Ser453 and the channel exits (circles; Ile52 lining the channel exit is shown). FIG +117 122 Ile52 residue_name_number (c) Cytoplasmic view of the Mep2 trimer indicating the large distance between Ser453 and the channel exits (circles; Ile52 lining the channel exit is shown). FIG +134 146 channel exit site (c) Cytoplasmic view of the Mep2 trimer indicating the large distance between Ser453 and the channel exits (circles; Ile52 lining the channel exit is shown). FIG +10 17 removal experimental_method Effect of removal of the AI region on Mep2 structure. FIG +25 34 AI region structure_element Effect of removal of the AI region on Mep2 structure. FIG +38 42 Mep2 protein Effect of removal of the AI region on Mep2 structure. FIG +43 52 structure evidence Effect of removal of the AI region on Mep2 structure. FIG +19 21 WT protein_state (a) Side views for WT CaMep2 (left) and the truncation mutant 442Δ (right). FIG +22 28 CaMep2 protein (a) Side views for WT CaMep2 (left) and the truncation mutant 442Δ (right). FIG +44 61 truncation mutant protein_state (a) Side views for WT CaMep2 (left) and the truncation mutant 442Δ (right). FIG +62 66 442Δ mutant (a) Side views for WT CaMep2 (left) and the truncation mutant 442Δ (right). FIG +78 86 disorder protein_state The latter is shown as a putty model according to B-factors to illustrate the disorder in the protein on the cytoplasmic side. FIG +42 56 superpositions experimental_method Missing regions are labelled. (b) Stereo superpositions of WT CaMep2 and the truncation mutant. FIG +60 62 WT protein_state Missing regions are labelled. (b) Stereo superpositions of WT CaMep2 and the truncation mutant. FIG +63 69 CaMep2 protein Missing regions are labelled. (b) Stereo superpositions of WT CaMep2 and the truncation mutant. FIG +78 95 truncation mutant protein_state Missing regions are labelled. (b) Stereo superpositions of WT CaMep2 and the truncation mutant. FIG +58 63 Tyr49 residue_name_number 2Fo–Fc electron density (contoured at 1.0 σ) for residues Tyr49 and His342 is shown for the truncation mutant. FIG +68 74 His342 residue_name_number 2Fo–Fc electron density (contoured at 1.0 σ) for residues Tyr49 and His342 is shown for the truncation mutant. FIG +92 109 truncation mutant protein_state 2Fo–Fc electron density (contoured at 1.0 σ) for residues Tyr49 and His342 is shown for the truncation mutant. FIG +0 15 Phosphorylation ptm Phosphorylation causes conformational changes in the CTR. FIG +53 56 CTR structure_element Phosphorylation causes conformational changes in the CTR. FIG +28 37 DD mutant mutant (a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2 superposed in grey for one of the monomers. FIG +38 44 trimer oligomeric_state (a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2 superposed in grey for one of the monomers. FIG +51 53 WT protein_state (a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2 superposed in grey for one of the monomers. FIG +54 60 CaMep2 protein (a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2 superposed in grey for one of the monomers. FIG +61 71 superposed experimental_method (a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2 superposed in grey for one of the monomers. FIG +95 103 monomers oligomeric_state (a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2 superposed in grey for one of the monomers. FIG +24 44 phosphorylation site site The arrow indicates the phosphorylation site. FIG +4 13 AI region structure_element The AI region is coloured magenta. FIG +4 11 Monomer oligomeric_state (b) Monomer side-view superposition of WT CaMep2 and the DD mutant, showing the conformational change and disorder around the ExxGxD motif. FIG +22 35 superposition experimental_method (b) Monomer side-view superposition of WT CaMep2 and the DD mutant, showing the conformational change and disorder around the ExxGxD motif. FIG +39 41 WT protein_state (b) Monomer side-view superposition of WT CaMep2 and the DD mutant, showing the conformational change and disorder around the ExxGxD motif. FIG +42 48 CaMep2 protein (b) Monomer side-view superposition of WT CaMep2 and the DD mutant, showing the conformational change and disorder around the ExxGxD motif. FIG +57 66 DD mutant mutant (b) Monomer side-view superposition of WT CaMep2 and the DD mutant, showing the conformational change and disorder around the ExxGxD motif. FIG +126 138 ExxGxD motif structure_element (b) Monomer side-view superposition of WT CaMep2 and the DD mutant, showing the conformational change and disorder around the ExxGxD motif. FIG +25 28 452 residue_number Side chains for residues 452 and 453 are shown as stick models. FIG +33 36 453 residue_number Side chains for residues 452 and 453 are shown as stick models. FIG +56 60 Mep2 protein Schematic model for phosphorylation-based regulation of Mep2 ammonium transporters. FIG +11 17 closed protein_state (a) In the closed, non-phosphorylated state (i), the CTR (magenta) and ICL3 (green) are far apart with the latter blocking the intracellular channel exit (indicated with a hatched circle). FIG +19 37 non-phosphorylated protein_state (a) In the closed, non-phosphorylated state (i), the CTR (magenta) and ICL3 (green) are far apart with the latter blocking the intracellular channel exit (indicated with a hatched circle). FIG +53 56 CTR structure_element (a) In the closed, non-phosphorylated state (i), the CTR (magenta) and ICL3 (green) are far apart with the latter blocking the intracellular channel exit (indicated with a hatched circle). FIG +71 75 ICL3 structure_element (a) In the closed, non-phosphorylated state (i), the CTR (magenta) and ICL3 (green) are far apart with the latter blocking the intracellular channel exit (indicated with a hatched circle). FIG +141 153 channel exit site (a) In the closed, non-phosphorylated state (i), the CTR (magenta) and ICL3 (green) are far apart with the latter blocking the intracellular channel exit (indicated with a hatched circle). FIG +5 20 phosphorylation ptm Upon phosphorylation and mimicked by the CaMep2 S453D and DD mutants (ii), the region around the ExxGxD motif undergoes a conformational change that results in the CTR interacting with the inward-moving ICL3, opening the channel (full circle) (iii). FIG +25 33 mimicked protein_state Upon phosphorylation and mimicked by the CaMep2 S453D and DD mutants (ii), the region around the ExxGxD motif undergoes a conformational change that results in the CTR interacting with the inward-moving ICL3, opening the channel (full circle) (iii). FIG +41 47 CaMep2 protein Upon phosphorylation and mimicked by the CaMep2 S453D and DD mutants (ii), the region around the ExxGxD motif undergoes a conformational change that results in the CTR interacting with the inward-moving ICL3, opening the channel (full circle) (iii). FIG +48 53 S453D mutant Upon phosphorylation and mimicked by the CaMep2 S453D and DD mutants (ii), the region around the ExxGxD motif undergoes a conformational change that results in the CTR interacting with the inward-moving ICL3, opening the channel (full circle) (iii). FIG +58 68 DD mutants mutant Upon phosphorylation and mimicked by the CaMep2 S453D and DD mutants (ii), the region around the ExxGxD motif undergoes a conformational change that results in the CTR interacting with the inward-moving ICL3, opening the channel (full circle) (iii). FIG +97 109 ExxGxD motif structure_element Upon phosphorylation and mimicked by the CaMep2 S453D and DD mutants (ii), the region around the ExxGxD motif undergoes a conformational change that results in the CTR interacting with the inward-moving ICL3, opening the channel (full circle) (iii). FIG +164 167 CTR structure_element Upon phosphorylation and mimicked by the CaMep2 S453D and DD mutants (ii), the region around the ExxGxD motif undergoes a conformational change that results in the CTR interacting with the inward-moving ICL3, opening the channel (full circle) (iii). FIG +203 207 ICL3 structure_element Upon phosphorylation and mimicked by the CaMep2 S453D and DD mutants (ii), the region around the ExxGxD motif undergoes a conformational change that results in the CTR interacting with the inward-moving ICL3, opening the channel (full circle) (iii). FIG +221 228 channel site Upon phosphorylation and mimicked by the CaMep2 S453D and DD mutants (ii), the region around the ExxGxD motif undergoes a conformational change that results in the CTR interacting with the inward-moving ICL3, opening the channel (full circle) (iii). FIG +4 8 open protein_state The open-channel Mep2 structure is represented by archaebacterial Amt-1 and shown in lighter colours consistent with Fig. 4. FIG +9 16 channel site The open-channel Mep2 structure is represented by archaebacterial Amt-1 and shown in lighter colours consistent with Fig. 4. FIG +17 21 Mep2 protein The open-channel Mep2 structure is represented by archaebacterial Amt-1 and shown in lighter colours consistent with Fig. 4. FIG +22 31 structure evidence The open-channel Mep2 structure is represented by archaebacterial Amt-1 and shown in lighter colours consistent with Fig. 4. FIG +50 65 archaebacterial taxonomy_domain The open-channel Mep2 structure is represented by archaebacterial Amt-1 and shown in lighter colours consistent with Fig. 4. FIG +66 71 Amt-1 protein The open-channel Mep2 structure is represented by archaebacterial Amt-1 and shown in lighter colours consistent with Fig. 4. FIG +71 76 plant taxonomy_domain As discussed in the text, similar structural arrangements may occur in plant AMTs. FIG +77 81 AMTs protein_type As discussed in the text, similar structural arrangements may occur in plant AMTs. FIG +26 30 open protein_state In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +31 38 channel site In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +58 76 non-phosphorylated protein_state In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +84 99 phosphorylation ptm In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +144 151 channel site In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +216 231 phosphorylation ptm In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +237 241 Mep2 protein In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +242 249 monomer oligomeric_state In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +271 275 open protein_state In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +276 284 channels site In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +299 305 trimer oligomeric_state In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +353 356 CTR structure_element In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +361 365 ICL3 structure_element In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG +384 391 monomer oligomeric_state In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). FIG diff --git a/annotation_CSV/PMC4854314.csv b/annotation_CSV/PMC4854314.csv new file mode 100644 index 0000000000000000000000000000000000000000..871c27f889a0b0e406e9a7e739553d3d01e779ad --- /dev/null +++ b/annotation_CSV/PMC4854314.csv @@ -0,0 +1,489 @@ +anno_start anno_end anno_text entity_type sentence section +0 3 RNA chemical RNA protects a nucleoprotein complex against radiation damage TITLE +15 28 nucleoprotein complex_assembly RNA protects a nucleoprotein complex against radiation damage TITLE +49 60 protein–RNA complex_assembly Systematic analysis of radiation damage within a protein–RNA complex over a large dose range (1.3–25 MGy) reveals significant differential susceptibility of RNA and protein. ABSTRACT +157 160 RNA chemical Systematic analysis of radiation damage within a protein–RNA complex over a large dose range (1.3–25 MGy) reveals significant differential susceptibility of RNA and protein. ABSTRACT +16 58 difference electron-density quantification experimental_method A new method of difference electron-density quantification is presented. ABSTRACT +24 77 macromolecular X-ray crystallographic data collection experimental_method Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. ABSTRACT +116 155 macromolecular structure determinations experimental_method Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. ABSTRACT +57 65 crystals evidence Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes. ABSTRACT +47 71 per-atom density changes evidence Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3–25.0 MGy) range and at higher resolution (1.98 Å) than the previous systematic specific damage study on a protein–DNA complex. ABSTRACT +244 247 DNA chemical Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3–25.0 MGy) range and at higher resolution (1.98 Å) than the previous systematic specific damage study on a protein–DNA complex. ABSTRACT +64 99 trp RNA-binding attenuation protein protein_type Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. ABSTRACT +101 105 TRAP complex_assembly Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. ABSTRACT +107 115 bound to protein_state Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. ABSTRACT +134 137 RNA chemical Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. ABSTRACT +29 32 RNA chemical Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein. ABSTRACT +24 28 TRAP complex_assembly The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. ABSTRACT +91 96 bound protein_state The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. ABSTRACT +97 100 RNA chemical The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. ABSTRACT +160 163 RNA chemical The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. ABSTRACT +33 37 TRAP complex_assembly The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. ABSTRACT +38 42 ring structure_element The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. ABSTRACT +95 98 Glu residue_name The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. ABSTRACT +103 106 Asp residue_name The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. ABSTRACT +129 132 RNA chemical The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. ABSTRACT +236 255 RNA-binding pockets site The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. ABSTRACT +88 91 Lys residue_name Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density. ABSTRACT +96 99 Phe residue_name Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density. ABSTRACT +117 120 RNA chemical Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density. ABSTRACT +147 163 electron density evidence Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density. ABSTRACT +150 173 structure determination experimental_method With the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100 K). INTRO +180 210 macromolecular crystallography experimental_method With the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100 K). INTRO +212 214 MX experimental_method With the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100 K). INTRO +133 141 crystals evidence Significant progress has been made in recent years in understanding the inevitable manifestations of X-ray-induced RD within protein crystals, and there is now a body of literature on possible strategies to mitigate the effects of RD (e.g. Zeldin, Brockhauser et al., 2013; Bourenkov & Popov, 2010). INTRO +91 93 MX experimental_method However, there is still no general consensus within the field on how to minimize RD during MX data collection, and debates on the dependence of RD progression on incident X-ray energy (Shimizu et al., 2007; Liebschner et al., 2015) and the efficacy of radical scavengers (Allan et al., 2013) have yet to be resolved. INTRO +140 159 diffraction pattern evidence Global radiation damage is observed within reciprocal space as the overall decay of the summed intensity of reflections detected within the diffraction pattern as dose increases (Garman, 2010; Murray & Garman, 2002). INTRO +174 176 MX experimental_method Dose is defined as the absorbed energy per unit mass of crystal in grays (Gy; 1 Gy = 1 J kg−1), and is the metric against which damage progression should be monitored during MX data collection, as opposed to time. INTRO +158 165 crystal evidence At 100 K, an experimental dose limit of 30 MGy has been recommended as an upper limit beyond which the biological information derived from any macromolecular crystal may be compromised (Owen et al., 2006). INTRO +1 26 Specific radiation damage experimental_method Specific radiation damage (SRD) is observed in the real-space electron density, and has been detected at much lower doses than any observable decay in the intensity of reflections. INTRO +28 31 SRD experimental_method Specific radiation damage (SRD) is observed in the real-space electron density, and has been detected at much lower doses than any observable decay in the intensity of reflections. INTRO +52 79 real-space electron density evidence Specific radiation damage (SRD) is observed in the real-space electron density, and has been detected at much lower doses than any observable decay in the intensity of reflections. INTRO +14 16 Se chemical Indeed, the C—Se bond in selenomethionine, the stability of which is key for the success of experimental phasing methods, can be cleaved at a dose as low as 2 MGy for a crystal maintained at 100 K (Holton, 2007). INTRO +25 41 selenomethionine chemical Indeed, the C—Se bond in selenomethionine, the stability of which is key for the success of experimental phasing methods, can be cleaved at a dose as low as 2 MGy for a crystal maintained at 100 K (Holton, 2007). INTRO +169 176 crystal evidence Indeed, the C—Se bond in selenomethionine, the stability of which is key for the success of experimental phasing methods, can be cleaved at a dose as low as 2 MGy for a crystal maintained at 100 K (Holton, 2007). INTRO +132 146 disulfide-bond ptm SRD has been well characterized in a large range of proteins, and is seen to follow a reproducible order: metallo-centre reduction, disulfide-bond cleavage, acidic residue decarboxylation and methionine methylthio cleavage (Ravelli & McSweeney, 2000; Burmeister, 2000; Weik et al., 2000; Yano et al., 2005). INTRO +112 125 MX-determined experimental_method There are a number of cases where SRD manifestations have compromised the biological information extracted from MX-determined structures at much lower doses than the recommended 30 MGy limit, leading to false structural interpretations of protein mechanisms. INTRO +126 136 structures evidence There are a number of cases where SRD manifestations have compromised the biological information extracted from MX-determined structures at much lower doses than the recommended 30 MGy limit, leading to false structural interpretations of protein mechanisms. INTRO +0 20 Active-site residues site Active-site residues appear to be particularly susceptible, particularly for photosensitive proteins and in instances where chemical strain is an intrinsic feature of the reaction mechanism. INTRO +14 37 structure determination experimental_method For instance, structure determination of the purple membrane protein bacterio­rhodopsin required careful corrections for radiation-induced structural changes before the correct photosensitive intermediate states could be isolated (Matsui et al., 2002). INTRO +69 87 bacterio­rhodopsin protein_type For instance, structure determination of the purple membrane protein bacterio­rhodopsin required careful corrections for radiation-induced structural changes before the correct photosensitive intermediate states could be isolated (Matsui et al., 2002). INTRO +66 77 active site site The significant chemical strain required for catalysis within the active site of phosphoserine aminotransferase has been observed to diminish during X-ray exposure (Dubnovitsky et al., 2005). INTRO +81 111 phosphoserine aminotransferase protein_type The significant chemical strain required for catalysis within the active site of phosphoserine aminotransferase has been observed to diminish during X-ray exposure (Dubnovitsky et al., 2005). INTRO +22 33 SRD studies experimental_method Since the majority of SRD studies to date have focused on proteins, much less is known about the effects of X-ray irradiation on the wider class of crystalline nucleoprotein complexes or how to correct for such radiation-induced structural changes. INTRO +160 173 nucleoprotein complex_assembly Since the majority of SRD studies to date have focused on proteins, much less is known about the effects of X-ray irradiation on the wider class of crystalline nucleoprotein complexes or how to correct for such radiation-induced structural changes. INTRO +53 56 DNA chemical Understanding RD to such complexes is crucial, since DNA is rarely naked within a cell, instead dynamically interacting with proteins, facilitating replication, transcription, modification and DNA repair. INTRO +193 196 DNA chemical Understanding RD to such complexes is crucial, since DNA is rarely naked within a cell, instead dynamically interacting with proteins, facilitating replication, transcription, modification and DNA repair. INTRO +24 37 nucleoprotein complex_assembly As of early 2016, >5400 nucleoprotein complex structures have been deposited within the PDB, with 91% solved by MX. INTRO +46 56 structures evidence As of early 2016, >5400 nucleoprotein complex structures have been deposited within the PDB, with 91% solved by MX. INTRO +112 114 MX experimental_method As of early 2016, >5400 nucleoprotein complex structures have been deposited within the PDB, with 91% solved by MX. INTRO +76 86 structures evidence It is essential to understand how these increasingly complex macromolecular structures are affected by the radiation used to solve them. INTRO +0 14 Nucleoproteins complex_assembly Nucleoproteins also represent one of the main targets of radiotherapy, and an insight into the damage mechanisms induced by X-ray irradiation could inform innovative treatments. INTRO +198 201 DNA chemical Investigations on sub-ionization-level LEEs (0–15 eV) interacting with both dried and aqueous oligonucleotides (Alizadeh & Sanche, 2014; Simons, 2006) concluded that resonant electron attachment to DNA bases and the sugar-phosphate backbone could lead to the preferential cleavage of strong (∼4 eV, 385 kJ mol−1) sugar-phosphate C—O covalent bonds within the DNA backbone and then base-sugar N1—C bonds, eventually leading to single-strand breakages (SSBs; Ptasińska & Sanche, 2007). INTRO +359 362 DNA chemical Investigations on sub-ionization-level LEEs (0–15 eV) interacting with both dried and aqueous oligonucleotides (Alizadeh & Sanche, 2014; Simons, 2006) concluded that resonant electron attachment to DNA bases and the sugar-phosphate backbone could lead to the preferential cleavage of strong (∼4 eV, 385 kJ mol−1) sugar-phosphate C—O covalent bonds within the DNA backbone and then base-sugar N1—C bonds, eventually leading to single-strand breakages (SSBs; Ptasińska & Sanche, 2007). INTRO +50 86 electron spin resonance spectroscopy experimental_method Electrons have been shown to be mobile at 77 K by electron spin resonance spectroscopy studies (Symons, 1997; Jones et al., 1987), with rapid electron quantum tunnelling and positive hole migration along the protein backbone and through stacked DNA bases indicated as a dominant mechanism by which oxidative and reductive damage localizes at distances from initial ionization sites at 100 K (O’Neill et al., 2002). INTRO +245 248 DNA chemical Electrons have been shown to be mobile at 77 K by electron spin resonance spectroscopy studies (Symons, 1997; Jones et al., 1987), with rapid electron quantum tunnelling and positive hole migration along the protein backbone and through stacked DNA bases indicated as a dominant mechanism by which oxidative and reductive damage localizes at distances from initial ionization sites at 100 K (O’Neill et al., 2002). INTRO +365 381 ionization sites site Electrons have been shown to be mobile at 77 K by electron spin resonance spectroscopy studies (Symons, 1997; Jones et al., 1987), with rapid electron quantum tunnelling and positive hole migration along the protein backbone and through stacked DNA bases indicated as a dominant mechanism by which oxidative and reductive damage localizes at distances from initial ionization sites at 100 K (O’Neill et al., 2002). INTRO +187 199 crystallized experimental_method The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein–DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8 Å; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1–44.6 MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015). INTRO +247 256 bacterial taxonomy_domain The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein–DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8 Å; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1–44.6 MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015). INTRO +265 268 DNA chemical The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein–DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8 Å; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1–44.6 MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015). INTRO +278 288 C.Esp1396I complex_assembly The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein–DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8 Å; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1–44.6 MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015). INTRO +460 463 DNA chemical The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein–DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8 Å; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1–44.6 MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015). INTRO +475 482 crystal evidence The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein–DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8 Å; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1–44.6 MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015). INTRO +91 106 AT-rich regions structure_element Only at doses above 20 MGy were precursors of phosphodiester-bond cleavage observed within AT-rich regions of the 35-mer DNA. INTRO +121 124 DNA chemical Only at doses above 20 MGy were precursors of phosphodiester-bond cleavage observed within AT-rich regions of the 35-mer DNA. INTRO +34 44 C.Esp1396I complex_assembly For crystalline complexes such as C.Esp1396I, whether the protein is intrinsically more susceptible to X-ray-induced damage or whether the protein scavenges electrons to protect the DNA remains unclear in the absence of a non-nucleic acid-bound protein control obtained under exactly the same crystallization and data-collection conditions. INTRO +182 185 DNA chemical For crystalline complexes such as C.Esp1396I, whether the protein is intrinsically more susceptible to X-ray-induced damage or whether the protein scavenges electrons to protect the DNA remains unclear in the absence of a non-nucleic acid-bound protein control obtained under exactly the same crystallization and data-collection conditions. INTRO +83 90 crystal evidence To monitor the effects of nucleic acid binding on protein damage susceptibility, a crystal containing two protein molecules per asymmetric unit, only one of which was bound to RNA, is reported here (Fig. 1 ▸). INTRO +167 175 bound to protein_state To monitor the effects of nucleic acid binding on protein damage susceptibility, a crystal containing two protein molecules per asymmetric unit, only one of which was bound to RNA, is reported here (Fig. 1 ▸). INTRO +176 179 RNA chemical To monitor the effects of nucleic acid binding on protein damage susceptibility, a crystal containing two protein molecules per asymmetric unit, only one of which was bound to RNA, is reported here (Fig. 1 ▸). INTRO +48 62 controlled SRD experimental_method Using newly developed methodology, we present a controlled SRD investigation at 1.98 Å resolution using a large (∼91 kDa) crystalline protein–RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53 bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002). INTRO +134 145 protein–RNA complex_assembly Using newly developed methodology, we present a controlled SRD investigation at 1.98 Å resolution using a large (∼91 kDa) crystalline protein–RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53 bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002). INTRO +155 190 trp RNA-binding attenuation protein protein_type Using newly developed methodology, we present a controlled SRD investigation at 1.98 Å resolution using a large (∼91 kDa) crystalline protein–RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53 bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002). INTRO +192 196 TRAP complex_assembly Using newly developed methodology, we present a controlled SRD investigation at 1.98 Å resolution using a large (∼91 kDa) crystalline protein–RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53 bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002). INTRO +198 206 bound to protein_state Using newly developed methodology, we present a controlled SRD investigation at 1.98 Å resolution using a large (∼91 kDa) crystalline protein–RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53 bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002). INTRO +215 218 RNA chemical Using newly developed methodology, we present a controlled SRD investigation at 1.98 Å resolution using a large (∼91 kDa) crystalline protein–RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53 bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002). INTRO +228 240 (GAGUU)10GAG chemical Using newly developed methodology, we present a controlled SRD investigation at 1.98 Å resolution using a large (∼91 kDa) crystalline protein–RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53 bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002). INTRO +0 4 TRAP complex_assembly TRAP consists of 11 identical subunits assembled into a ring with 11-fold rotational symmetry. INTRO +30 38 subunits structure_element TRAP consists of 11 identical subunits assembled into a ring with 11-fold rotational symmetry. INTRO +56 60 ring structure_element TRAP consists of 11 identical subunits assembled into a ring with 11-fold rotational symmetry. INTRO +29 32 K d evidence It binds with high affinity (K d ≃ 1.0 nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999). INTRO +46 49 RNA chemical It binds with high affinity (K d ≃ 1.0 nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999). INTRO +73 89 GAG/UAG triplets structure_element It binds with high affinity (K d ≃ 1.0 nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999). INTRO +116 134 spacer nucleotides structure_element It binds with high affinity (K d ≃ 1.0 nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999). INTRO +191 201 tryptophan chemical It binds with high affinity (K d ≃ 1.0 nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999). INTRO +224 241 Bacillus subtilis species It binds with high affinity (K d ≃ 1.0 nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999). INTRO +8 17 structure evidence In this structure, the bases of the G1-A2-G3 nucleotides form direct hydrogen bonds to the protein, unlike the U4-U5 nucleotides, which appear to be more flexible. INTRO +36 44 G1-A2-G3 chemical In this structure, the bases of the G1-A2-G3 nucleotides form direct hydrogen bonds to the protein, unlike the U4-U5 nucleotides, which appear to be more flexible. INTRO +69 83 hydrogen bonds bond_interaction In this structure, the bases of the G1-A2-G3 nucleotides form direct hydrogen bonds to the protein, unlike the U4-U5 nucleotides, which appear to be more flexible. INTRO +111 116 U4-U5 chemical In this structure, the bases of the G1-A2-G3 nucleotides form direct hydrogen bonds to the protein, unlike the U4-U5 nucleotides, which appear to be more flexible. INTRO +33 35 MX experimental_method Ten successive 1.98 Å resolution MX data sets were collected from the same TRAP–RNA crystal to analyse X-ray-induced structural changes over a large dose range (d 1 = 1.3 MGy to d 10 = 25.0 MGy). INTRO +75 83 TRAP–RNA complex_assembly Ten successive 1.98 Å resolution MX data sets were collected from the same TRAP–RNA crystal to analyse X-ray-induced structural changes over a large dose range (d 1 = 1.3 MGy to d 10 = 25.0 MGy). INTRO +84 91 crystal evidence Ten successive 1.98 Å resolution MX data sets were collected from the same TRAP–RNA crystal to analyse X-ray-induced structural changes over a large dose range (d 1 = 1.3 MGy to d 10 = 25.0 MGy). INTRO +57 78 electron-density maps evidence To avoid the previous necessity for visual inspection of electron-density maps to detect SRD sites, a computational approach was designed to quantify the electron-density change for each refined atom with increasing dose, thus providing a rapid systematic method for SRD study on such large multimeric complexes. INTRO +89 98 SRD sites site To avoid the previous necessity for visual inspection of electron-density maps to detect SRD sites, a computational approach was designed to quantify the electron-density change for each refined atom with increasing dose, thus providing a rapid systematic method for SRD study on such large multimeric complexes. INTRO +154 177 electron-density change evidence To avoid the previous necessity for visual inspection of electron-density maps to detect SRD sites, a computational approach was designed to quantify the electron-density change for each refined atom with increasing dose, thus providing a rapid systematic method for SRD study on such large multimeric complexes. INTRO +62 66 TRAP complex_assembly By employing the high 11-fold structural symmetry within each TRAP macromolecule, this approach permitted a thorough statistical quantification of the RD effects of RNA binding to TRAP. INTRO +165 168 RNA chemical By employing the high 11-fold structural symmetry within each TRAP macromolecule, this approach permitted a thorough statistical quantification of the RD effects of RNA binding to TRAP. INTRO +180 184 TRAP complex_assembly By employing the high 11-fold structural symmetry within each TRAP macromolecule, this approach permitted a thorough statistical quantification of the RD effects of RNA binding to TRAP. INTRO +0 43 Per-atom quantification of electron density experimental_method Per-atom quantification of electron density   RESULTS +175 198 electron-density change evidence To quantify the exact effects of nucleic acid binding to a protein on SRD susceptibility, a high-throughput and automated pipeline was created to systematically calculate the electron-density change for every refined atom within the TRAP–RNA structure as a function of dose. RESULTS +233 241 TRAP–RNA complex_assembly To quantify the exact effects of nucleic acid binding to a protein on SRD susceptibility, a high-throughput and automated pipeline was created to systematically calculate the electron-density change for every refined atom within the TRAP–RNA structure as a function of dose. RESULTS +242 251 structure evidence To quantify the exact effects of nucleic acid binding to a protein on SRD susceptibility, a high-throughput and automated pipeline was created to systematically calculate the electron-density change for every refined atom within the TRAP–RNA structure as a function of dose. RESULTS +49 70 density–dose dynamics evidence This provides an atom-specific quantification of density–dose dynamics, which was previously lacking within the field. RESULTS +36 45 SRD sites site Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) − F obs(d 1) Fourier difference map peaks in terms of the sigma (σ) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible. RESULTS +73 125 F obs(d n) − F obs(d 1) Fourier difference map peaks evidence Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) − F obs(d 1) Fourier difference map peaks in terms of the sigma (σ) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible. RESULTS +142 147 sigma evidence Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) − F obs(d 1) Fourier difference map peaks in terms of the sigma (σ) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible. RESULTS +149 150 σ evidence Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) − F obs(d 1) Fourier difference map peaks in terms of the sigma (σ) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible. RESULTS +181 200 standard deviations evidence Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) − F obs(d 1) Fourier difference map peaks in terms of the sigma (σ) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible. RESULTS +210 241 mean map electron-density value evidence Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) − F obs(d 1) Fourier difference map peaks in terms of the sigma (σ) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible. RESULTS +15 16 σ evidence However, these σ levels depend on the standard deviation values of the map, which can deviate between data sets, and are thus unsuitable for quantitative comparison of density between different dose data sets. RESULTS +38 56 standard deviation evidence However, these σ levels depend on the standard deviation values of the map, which can deviate between data sets, and are thus unsuitable for quantitative comparison of density between different dose data sets. RESULTS +71 74 map evidence However, these σ levels depend on the standard deviation values of the map, which can deviate between data sets, and are thus unsuitable for quantitative comparison of density between different dose data sets. RESULTS +168 175 density evidence However, these σ levels depend on the standard deviation values of the map, which can deviate between data sets, and are thus unsuitable for quantitative comparison of density between different dose data sets. RESULTS +23 50 maximum density-loss metric evidence Instead, we use here a maximum density-loss metric (D loss), which is the per-atom equivalent of the magnitude of these negative Fourier difference map peaks in units of e Å−3. RESULTS +52 58 D loss evidence Instead, we use here a maximum density-loss metric (D loss), which is the per-atom equivalent of the magnitude of these negative Fourier difference map peaks in units of e Å−3. RESULTS +120 157 negative Fourier difference map peaks evidence Instead, we use here a maximum density-loss metric (D loss), which is the per-atom equivalent of the magnitude of these negative Fourier difference map peaks in units of e Å−3. RESULTS +15 21 D loss evidence Large positive D loss values indicate radiation-induced atomic disordering reproducibly throughout the unit cells with respect to the initial low-dose data set. RESULTS +9 17 TRAP–RNA complex_assembly For each TRAP–RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 ▸ a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9 MGy; Fig. 3 ▸ a). RESULTS +32 45 D loss metric evidence For each TRAP–RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 ▸ a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9 MGy; Fig. 3 ▸ a). RESULTS +102 105 SRD experimental_method For each TRAP–RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 ▸ a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9 MGy; Fig. 3 ▸ a). RESULTS +131 134 Glu residue_name For each TRAP–RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 ▸ a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9 MGy; Fig. 3 ▸ a). RESULTS +139 142 Asp residue_name For each TRAP–RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 ▸ a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9 MGy; Fig. 3 ▸ a). RESULTS +188 202 difference map evidence For each TRAP–RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 ▸ a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9 MGy; Fig. 3 ▸ a). RESULTS +21 25 TRAP complex_assembly The main sequence of TRAP does not contain any Trp and Cys residues (and thus contains no disulfide bonds). RESULTS +47 50 Trp residue_name The main sequence of TRAP does not contain any Trp and Cys residues (and thus contains no disulfide bonds). RESULTS +55 58 Cys residue_name The main sequence of TRAP does not contain any Trp and Cys residues (and thus contains no disulfide bonds). RESULTS +14 17 Trp chemical The substrate Trp amino-acid ligands also exhibited disordering of the free terminal carboxyl groups at higher doses (Fig. 2 ▸ a); however, no clear Fourier difference peaks could be observed visually. RESULTS +149 173 Fourier difference peaks evidence The substrate Trp amino-acid ligands also exhibited disordering of the free terminal carboxyl groups at higher doses (Fig. 2 ▸ a); however, no clear Fourier difference peaks could be observed visually. RESULTS +46 49 Gly residue_name Even for radiation-insensitive residues (e.g. Gly) the average D loss increases with dose: this is the effect of global radiation damage, since as dose increases the electron density associated with each refined atom becomes weaker as the atomic occupancy decreases (Fig. 2 ▸ b). RESULTS +63 69 D loss evidence Even for radiation-insensitive residues (e.g. Gly) the average D loss increases with dose: this is the effect of global radiation damage, since as dose increases the electron density associated with each refined atom becomes weaker as the atomic occupancy decreases (Fig. 2 ▸ b). RESULTS +166 182 electron density evidence Even for radiation-insensitive residues (e.g. Gly) the average D loss increases with dose: this is the effect of global radiation damage, since as dose increases the electron density associated with each refined atom becomes weaker as the atomic occupancy decreases (Fig. 2 ▸ b). RESULTS +5 8 Glu residue_name Only Glu and Asp residues exhibit a rate of D loss increase that consistently exceeds the average decay (Fig. 2 ▸ b, dashed line) at each dose. RESULTS +13 16 Asp residue_name Only Glu and Asp residues exhibit a rate of D loss increase that consistently exceeds the average decay (Fig. 2 ▸ b, dashed line) at each dose. RESULTS +44 50 D loss evidence Only Glu and Asp residues exhibit a rate of D loss increase that consistently exceeds the average decay (Fig. 2 ▸ b, dashed line) at each dose. RESULTS +12 18 D loss evidence The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts. RESULTS +90 93 Glu residue_name The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts. RESULTS +108 111 Asp residue_name The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts. RESULTS +321 337 hydrogen-bonding bond_interaction The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts. RESULTS +352 355 CO2 chemical The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts. RESULTS +406 414 oxidized protein_state The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts. RESULTS +415 418 Glu residue_name The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts. RESULTS +444 447 Asp residue_name The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts. RESULTS +0 3 RNA chemical RNA is less susceptible to electron-density loss than protein within the TRAP–RNA complex   RESULTS +27 43 electron-density evidence RNA is less susceptible to electron-density loss than protein within the TRAP–RNA complex   RESULTS +73 81 TRAP–RNA complex_assembly RNA is less susceptible to electron-density loss than protein within the TRAP–RNA complex   RESULTS +0 20 Visual inspection of experimental_method Visual inspection of Fourier difference maps illustrated the clear lack of RNA electron-density degradation with increasing dose compared with the obvious protein damage manifestations (Figs. 3 ▸ b and 3 ▸ c). RESULTS +21 44 Fourier difference maps evidence Visual inspection of Fourier difference maps illustrated the clear lack of RNA electron-density degradation with increasing dose compared with the obvious protein damage manifestations (Figs. 3 ▸ b and 3 ▸ c). RESULTS +75 78 RNA chemical Visual inspection of Fourier difference maps illustrated the clear lack of RNA electron-density degradation with increasing dose compared with the obvious protein damage manifestations (Figs. 3 ▸ b and 3 ▸ c). RESULTS +79 107 electron-density degradation evidence Visual inspection of Fourier difference maps illustrated the clear lack of RNA electron-density degradation with increasing dose compared with the obvious protein damage manifestations (Figs. 3 ▸ b and 3 ▸ c). RESULTS +82 85 RNA chemical Only at the highest doses investigated (>20 MGy) was density loss observed at the RNA phosphate and C—O bonds of the phosphodiester backbone. RESULTS +20 26 D loss evidence However, the median D loss was lower by a factor of >2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0 MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly Cα atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3). RESULTS +59 62 RNA chemical However, the median D loss was lower by a factor of >2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0 MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly Cα atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3). RESULTS +80 83 Glu residue_name However, the median D loss was lower by a factor of >2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0 MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly Cα atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3). RESULTS +88 91 Asp residue_name However, the median D loss was lower by a factor of >2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0 MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly Cα atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3). RESULTS +207 210 Gly residue_name However, the median D loss was lower by a factor of >2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0 MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly Cα atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3). RESULTS +227 231 TRAP complex_assembly However, the median D loss was lower by a factor of >2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0 MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly Cα atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3). RESULTS +0 3 RNA chemical RNA binding protects radiation-sensitive residues   RESULTS +44 48 TRAP complex_assembly For the large number of acidic residues per TRAP ring (four Asp and six Glu residues per protein monomer), a strong dependence of decarboxylation susceptibility on local environment was observed (Fig. 4 ▸). RESULTS +49 53 ring structure_element For the large number of acidic residues per TRAP ring (four Asp and six Glu residues per protein monomer), a strong dependence of decarboxylation susceptibility on local environment was observed (Fig. 4 ▸). RESULTS +60 63 Asp residue_name For the large number of acidic residues per TRAP ring (four Asp and six Glu residues per protein monomer), a strong dependence of decarboxylation susceptibility on local environment was observed (Fig. 4 ▸). RESULTS +72 75 Glu residue_name For the large number of acidic residues per TRAP ring (four Asp and six Glu residues per protein monomer), a strong dependence of decarboxylation susceptibility on local environment was observed (Fig. 4 ▸). RESULTS +97 104 monomer oligomeric_state For the large number of acidic residues per TRAP ring (four Asp and six Glu residues per protein monomer), a strong dependence of decarboxylation susceptibility on local environment was observed (Fig. 4 ▸). RESULTS +9 12 Glu residue_name For each Glu Cδ or Asp Cγ atom, D loss provided a direct measure of the rate of side-chain carboxyl-group disordering and subsequent decarboxylation. RESULTS +19 22 Asp residue_name For each Glu Cδ or Asp Cγ atom, D loss provided a direct measure of the rate of side-chain carboxyl-group disordering and subsequent decarboxylation. RESULTS +32 38 D loss evidence For each Glu Cδ or Asp Cγ atom, D loss provided a direct measure of the rate of side-chain carboxyl-group disordering and subsequent decarboxylation. RESULTS +59 67 nonbound protein_state For acidic residues with no differing interactions between nonbound and bound TRAP (Fig. 4 ▸ a), similar damage was apparent between the two rings within the asymmetric unit, as expected. RESULTS +72 77 bound protein_state For acidic residues with no differing interactions between nonbound and bound TRAP (Fig. 4 ▸ a), similar damage was apparent between the two rings within the asymmetric unit, as expected. RESULTS +78 82 TRAP complex_assembly For acidic residues with no differing interactions between nonbound and bound TRAP (Fig. 4 ▸ a), similar damage was apparent between the two rings within the asymmetric unit, as expected. RESULTS +9 13 TRAP complex_assembly However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 ▸ b), and for residues at the ring–ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 ▸ c). RESULTS +39 61 RNA-binding interfaces site However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 ▸ b), and for residues at the ring–ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 ▸ c). RESULTS +103 111 nonbound protein_state However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 ▸ b), and for residues at the ring–ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 ▸ c). RESULTS +112 116 TRAP complex_assembly However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 ▸ b), and for residues at the ring–ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 ▸ c). RESULTS +155 175 ring–ring interfaces site However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 ▸ b), and for residues at the ring–ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 ▸ c). RESULTS +215 220 bound protein_state However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 ▸ b), and for residues at the ring–ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 ▸ c). RESULTS +221 225 TRAP complex_assembly However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 ▸ b), and for residues at the ring–ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 ▸ c). RESULTS +23 28 Glu36 residue_name_number Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. RESULTS +30 35 Asp39 residue_name_number Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. RESULTS +40 45 Glu42 residue_name_number Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. RESULTS +63 66 RNA chemical Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. RESULTS +102 106 TRAP complex_assembly Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. RESULTS +107 111 ring structure_element Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. RESULTS +112 120 subunits structure_element Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. RESULTS +147 162 density changes evidence Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. RESULTS +0 26 Hotelling’s T-squared test experimental_method Hotelling’s T-squared test (the multivariate counterpart of Student’s t-test) was used to reject the null hypothesis that the means of the D loss metric were equal for the bound and nonbound groups in Fig. 5 ▸. RESULTS +60 76 Student’s t-test experimental_method Hotelling’s T-squared test (the multivariate counterpart of Student’s t-test) was used to reject the null hypothesis that the means of the D loss metric were equal for the bound and nonbound groups in Fig. 5 ▸. RESULTS +139 152 D loss metric evidence Hotelling’s T-squared test (the multivariate counterpart of Student’s t-test) was used to reject the null hypothesis that the means of the D loss metric were equal for the bound and nonbound groups in Fig. 5 ▸. RESULTS +172 177 bound protein_state Hotelling’s T-squared test (the multivariate counterpart of Student’s t-test) was used to reject the null hypothesis that the means of the D loss metric were equal for the bound and nonbound groups in Fig. 5 ▸. RESULTS +182 190 nonbound protein_state Hotelling’s T-squared test (the multivariate counterpart of Student’s t-test) was used to reject the null hypothesis that the means of the D loss metric were equal for the bound and nonbound groups in Fig. 5 ▸. RESULTS +27 33 D loss evidence A significant reduction in D loss is seen for Glu36 in RNA-bound compared with nonbound TRAP, indicative of a lower rate of side-chain decarboxylation (Fig. 5 ▸ a; p = 6.06 × 10−5). RESULTS +46 51 Glu36 residue_name_number A significant reduction in D loss is seen for Glu36 in RNA-bound compared with nonbound TRAP, indicative of a lower rate of side-chain decarboxylation (Fig. 5 ▸ a; p = 6.06 × 10−5). RESULTS +55 64 RNA-bound protein_state A significant reduction in D loss is seen for Glu36 in RNA-bound compared with nonbound TRAP, indicative of a lower rate of side-chain decarboxylation (Fig. 5 ▸ a; p = 6.06 × 10−5). RESULTS +79 87 nonbound protein_state A significant reduction in D loss is seen for Glu36 in RNA-bound compared with nonbound TRAP, indicative of a lower rate of side-chain decarboxylation (Fig. 5 ▸ a; p = 6.06 × 10−5). RESULTS +88 92 TRAP complex_assembly A significant reduction in D loss is seen for Glu36 in RNA-bound compared with nonbound TRAP, indicative of a lower rate of side-chain decarboxylation (Fig. 5 ▸ a; p = 6.06 × 10−5). RESULTS +9 13 TRAP complex_assembly For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base. RESULTS +14 18 ring structure_element For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base. RESULTS +19 26 subunit structure_element For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base. RESULTS +32 37 Glu36 residue_name_number For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base. RESULTS +82 96 hydrogen bonds bond_interaction For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base. RESULTS +125 127 G3 residue_name_number For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base. RESULTS +128 131 RNA chemical For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base. RESULTS +17 22 Asp39 residue_name_number In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RESULTS +30 47 TRAP–(GAGUU)10GAG complex_assembly In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RESULTS +48 57 structure evidence In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RESULTS +90 104 hydrogen bonds bond_interaction In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RESULTS +112 114 G1 residue_name_number In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RESULTS +142 161 TRAP–RNA interfaces site In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RESULTS +171 176 Glu36 residue_name_number In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RESULTS +180 182 G3 residue_name_number In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RESULTS +210 217 density evidence In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RESULTS +235 238 RNA chemical In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RESULTS +275 280 Asp39 residue_name_number In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RESULTS +290 295 Glu36 residue_name_number In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925). RESULTS +0 3 RNA chemical RNA binding reduces radiation-induced disorder on the atomic scale   RESULTS +20 25 Glu42 residue_name_number One oxygen (O∊1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O∊2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999). RESULTS +44 57 hydrogen bond bond_interaction One oxygen (O∊1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O∊2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999). RESULTS +70 75 water chemical One oxygen (O∊1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O∊2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999). RESULTS +88 111 TRAP RNA-binding pocket site One oxygen (O∊1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O∊2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999). RESULTS +154 165 salt-bridge bond_interaction One oxygen (O∊1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O∊2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999). RESULTS +183 188 Arg58 residue_name_number One oxygen (O∊1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O∊2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999). RESULTS +0 11 Salt-bridge bond_interaction Salt-bridge interactions have previously been suggested to reduce the glutamate decarboxylation rate within the large (∼62.4 kDa) myrosinase protein structure (Burmeister, 2000). RESULTS +70 79 glutamate residue_name Salt-bridge interactions have previously been suggested to reduce the glutamate decarboxylation rate within the large (∼62.4 kDa) myrosinase protein structure (Burmeister, 2000). RESULTS +130 140 myrosinase protein_type Salt-bridge interactions have previously been suggested to reduce the glutamate decarboxylation rate within the large (∼62.4 kDa) myrosinase protein structure (Burmeister, 2000). RESULTS +149 158 structure evidence Salt-bridge interactions have previously been suggested to reduce the glutamate decarboxylation rate within the large (∼62.4 kDa) myrosinase protein structure (Burmeister, 2000). RESULTS +50 65 D loss dynamics evidence A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. RESULTS +74 82 nonbound protein_state A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. RESULTS +83 88 bound protein_state A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. RESULTS +89 94 Glu42 residue_name_number A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. RESULTS +145 150 Glu42 residue_name_number A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. RESULTS +235 246 salt-bridge bond_interaction A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. RESULTS +278 281 RNA chemical A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. RESULTS +303 308 water chemical A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. RESULTS +318 331 hydrogen bond bond_interaction A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. RESULTS +399 409 absence of protein_state A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. RESULTS +410 413 RNA chemical A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. RESULTS +4 27 density-change dynamics evidence The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate. RESULTS +73 78 bound protein_state The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate. RESULTS +83 91 nonbound protein_state The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate. RESULTS +92 96 TRAP complex_assembly The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate. RESULTS +106 111 Glu42 residue_name_number The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate. RESULTS +169 172 RNA chemical The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate. RESULTS +195 206 salt-bridge bond_interaction The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate. RESULTS +251 256 Glu42 residue_name_number The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate. RESULTS +4 7 RNA chemical The RNA-stabilizing effect was not restricted to radiation-sensitive acidic residues. RESULTS +18 23 Phe32 residue_name_number The side chain of Phe32 stacks against the G3 base within the 11 TRAP RNA-binding interfaces (Antson et al., 1999). RESULTS +43 45 G3 residue_name_number The side chain of Phe32 stacks against the G3 base within the 11 TRAP RNA-binding interfaces (Antson et al., 1999). RESULTS +65 92 TRAP RNA-binding interfaces site The side chain of Phe32 stacks against the G3 base within the 11 TRAP RNA-binding interfaces (Antson et al., 1999). RESULTS +26 32 D loss evidence With increasing dose, the D loss associated with the Phe32 side chain was significantly reduced upon RNA binding (Fig. 5 ▸ e; Phe32 Cζ; p = 0.0014), an indication that radiation-induced conformation disordering of Phe32 had been reduced. RESULTS +53 58 Phe32 residue_name_number With increasing dose, the D loss associated with the Phe32 side chain was significantly reduced upon RNA binding (Fig. 5 ▸ e; Phe32 Cζ; p = 0.0014), an indication that radiation-induced conformation disordering of Phe32 had been reduced. RESULTS +101 104 RNA chemical With increasing dose, the D loss associated with the Phe32 side chain was significantly reduced upon RNA binding (Fig. 5 ▸ e; Phe32 Cζ; p = 0.0014), an indication that radiation-induced conformation disordering of Phe32 had been reduced. RESULTS +126 131 Phe32 residue_name_number With increasing dose, the D loss associated with the Phe32 side chain was significantly reduced upon RNA binding (Fig. 5 ▸ e; Phe32 Cζ; p = 0.0014), an indication that radiation-induced conformation disordering of Phe32 had been reduced. RESULTS +214 219 Phe32 residue_name_number With increasing dose, the D loss associated with the Phe32 side chain was significantly reduced upon RNA binding (Fig. 5 ▸ e; Phe32 Cζ; p = 0.0014), an indication that radiation-induced conformation disordering of Phe32 had been reduced. RESULTS +23 28 Lys37 residue_name_number The extended aliphatic Lys37 side chain stacks against the nearby G1 base, making a series of nonpolar contacts within each RNA-binding interface. RESULTS +66 68 G1 residue_name_number The extended aliphatic Lys37 side chain stacks against the nearby G1 base, making a series of nonpolar contacts within each RNA-binding interface. RESULTS +94 111 nonpolar contacts bond_interaction The extended aliphatic Lys37 side chain stacks against the nearby G1 base, making a series of nonpolar contacts within each RNA-binding interface. RESULTS +124 145 RNA-binding interface site The extended aliphatic Lys37 side chain stacks against the nearby G1 base, making a series of nonpolar contacts within each RNA-binding interface. RESULTS +4 10 D loss evidence The D loss for Lys37 side-chain atoms was also reduced when stacked against the G1 base (Fig. 5 ▸ f; p = 0.0243 for Lys37 C∊ atoms). RESULTS +15 20 Lys37 residue_name_number The D loss for Lys37 side-chain atoms was also reduced when stacked against the G1 base (Fig. 5 ▸ f; p = 0.0243 for Lys37 C∊ atoms). RESULTS +60 67 stacked bond_interaction The D loss for Lys37 side-chain atoms was also reduced when stacked against the G1 base (Fig. 5 ▸ f; p = 0.0243 for Lys37 C∊ atoms). RESULTS +80 82 G1 residue_name_number The D loss for Lys37 side-chain atoms was also reduced when stacked against the G1 base (Fig. 5 ▸ f; p = 0.0243 for Lys37 C∊ atoms). RESULTS +116 121 Lys37 residue_name_number The D loss for Lys37 side-chain atoms was also reduced when stacked against the G1 base (Fig. 5 ▸ f; p = 0.0243 for Lys37 C∊ atoms). RESULTS +15 20 Phe32 residue_name_number Representative Phe32 and Lys37 atoms were selected to illustrate these trends. RESULTS +25 30 Lys37 residue_name_number Representative Phe32 and Lys37 atoms were selected to illustrate these trends. RESULTS +6 8 MX experimental_method Here, MX radiation-induced specific structural changes within the large TRAP–RNA assembly over a large dose range (1.3–25.0 MGy) have been analysed using a high-throughput quantitative approach, providing a measure of the electron-density distribution for each refined atom with increasing dose, D loss. DISCUSS +72 80 TRAP–RNA complex_assembly Here, MX radiation-induced specific structural changes within the large TRAP–RNA assembly over a large dose range (1.3–25.0 MGy) have been analysed using a high-throughput quantitative approach, providing a measure of the electron-density distribution for each refined atom with increasing dose, D loss. DISCUSS +222 251 electron-density distribution evidence Here, MX radiation-induced specific structural changes within the large TRAP–RNA assembly over a large dose range (1.3–25.0 MGy) have been analysed using a high-throughput quantitative approach, providing a measure of the electron-density distribution for each refined atom with increasing dose, D loss. DISCUSS +296 302 D loss evidence Here, MX radiation-induced specific structural changes within the large TRAP–RNA assembly over a large dose range (1.3–25.0 MGy) have been analysed using a high-throughput quantitative approach, providing a measure of the electron-density distribution for each refined atom with increasing dose, D loss. DISCUSS +118 120 MX experimental_method Compared with previous studies, the results provide a further step in the detailed characterization of SRD effects in MX. DISCUSS +173 225 F obs(d n) − F obs(d 1) Fourier difference map peaks evidence Our method­ology, which eliminated tedious and error-prone visual inspection, permitted the determination on a per-atom basis of the most damaged sites, as characterized by F obs(d n) − F obs(d 1) Fourier difference map peaks between successive data sets collected from the same crystal. DISCUSS +279 286 crystal evidence Our method­ology, which eliminated tedious and error-prone visual inspection, permitted the determination on a per-atom basis of the most damaged sites, as characterized by F obs(d n) − F obs(d 1) Fourier difference map peaks between successive data sets collected from the same crystal. DISCUSS +65 68 RNA chemical Here, it provided the precision required to quantify the role of RNA in the damage susceptibilities of equivalent atoms between RNA-bound and nonbound TRAP, but it is applicable to any MX SRD study. DISCUSS +128 137 RNA-bound protein_state Here, it provided the precision required to quantify the role of RNA in the damage susceptibilities of equivalent atoms between RNA-bound and nonbound TRAP, but it is applicable to any MX SRD study. DISCUSS +142 150 nonbound protein_state Here, it provided the precision required to quantify the role of RNA in the damage susceptibilities of equivalent atoms between RNA-bound and nonbound TRAP, but it is applicable to any MX SRD study. DISCUSS +151 155 TRAP complex_assembly Here, it provided the precision required to quantify the role of RNA in the damage susceptibilities of equivalent atoms between RNA-bound and nonbound TRAP, but it is applicable to any MX SRD study. DISCUSS +185 187 MX experimental_method Here, it provided the precision required to quantify the role of RNA in the damage susceptibilities of equivalent atoms between RNA-bound and nonbound TRAP, but it is applicable to any MX SRD study. DISCUSS +4 7 RNA chemical The RNA was found to be substantially more radiation-resistant than the protein, even at the highest doses investigated (∼25.0 MGy), which is in strong concurrence with our previous SRD investigation of the C.Esp1396I protein–DNA complex (Bury et al., 2015). DISCUSS +43 62 radiation-resistant protein_state The RNA was found to be substantially more radiation-resistant than the protein, even at the highest doses investigated (∼25.0 MGy), which is in strong concurrence with our previous SRD investigation of the C.Esp1396I protein–DNA complex (Bury et al., 2015). DISCUSS +182 199 SRD investigation experimental_method The RNA was found to be substantially more radiation-resistant than the protein, even at the highest doses investigated (∼25.0 MGy), which is in strong concurrence with our previous SRD investigation of the C.Esp1396I protein–DNA complex (Bury et al., 2015). DISCUSS +207 217 C.Esp1396I complex_assembly The RNA was found to be substantially more radiation-resistant than the protein, even at the highest doses investigated (∼25.0 MGy), which is in strong concurrence with our previous SRD investigation of the C.Esp1396I protein–DNA complex (Bury et al., 2015). DISCUSS +226 229 DNA chemical The RNA was found to be substantially more radiation-resistant than the protein, even at the highest doses investigated (∼25.0 MGy), which is in strong concurrence with our previous SRD investigation of the C.Esp1396I protein–DNA complex (Bury et al., 2015). DISCUSS +60 95 F obs(d n) − F obs(d 1) map density evidence Consistent with that study, at high doses of above ∼20 MGy, F obs(d n) − F obs(d 1) map density was detected around P, O3′ and O5′ atoms of the RNA backbone, with no significant difference density localized to RNA ribose and basic subunits. DISCUSS +144 147 RNA chemical Consistent with that study, at high doses of above ∼20 MGy, F obs(d n) − F obs(d 1) map density was detected around P, O3′ and O5′ atoms of the RNA backbone, with no significant difference density localized to RNA ribose and basic subunits. DISCUSS +178 196 difference density evidence Consistent with that study, at high doses of above ∼20 MGy, F obs(d n) − F obs(d 1) map density was detected around P, O3′ and O5′ atoms of the RNA backbone, with no significant difference density localized to RNA ribose and basic subunits. DISCUSS +210 213 RNA chemical Consistent with that study, at high doses of above ∼20 MGy, F obs(d n) − F obs(d 1) map density was detected around P, O3′ and O5′ atoms of the RNA backbone, with no significant difference density localized to RNA ribose and basic subunits. DISCUSS +231 239 subunits structure_element Consistent with that study, at high doses of above ∼20 MGy, F obs(d n) − F obs(d 1) map density was detected around P, O3′ and O5′ atoms of the RNA backbone, with no significant difference density localized to RNA ribose and basic subunits. DISCUSS +0 3 RNA chemical RNA backbone disordering thus appears to be the main radiation-induced effect in RNA, with the protein–base interactions maintained even at high doses (>20 MGy). DISCUSS +81 84 RNA chemical RNA backbone disordering thus appears to be the main radiation-induced effect in RNA, with the protein–base interactions maintained even at high doses (>20 MGy). DISCUSS +4 6 U4 residue_name_number The U4 phosphate exhibited marginally larger D loss values above 20 MGy than G1, A2 and G3 (Supplementary Fig. S4). DISCUSS +7 16 phosphate chemical The U4 phosphate exhibited marginally larger D loss values above 20 MGy than G1, A2 and G3 (Supplementary Fig. S4). DISCUSS +45 51 D loss evidence The U4 phosphate exhibited marginally larger D loss values above 20 MGy than G1, A2 and G3 (Supplementary Fig. S4). DISCUSS +77 79 G1 residue_name_number The U4 phosphate exhibited marginally larger D loss values above 20 MGy than G1, A2 and G3 (Supplementary Fig. S4). DISCUSS +81 83 A2 residue_name_number The U4 phosphate exhibited marginally larger D loss values above 20 MGy than G1, A2 and G3 (Supplementary Fig. S4). DISCUSS +88 90 G3 residue_name_number The U4 phosphate exhibited marginally larger D loss values above 20 MGy than G1, A2 and G3 (Supplementary Fig. S4). DISCUSS +6 8 U4 residue_name_number Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. DISCUSS +100 104 TRAP complex_assembly Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. DISCUSS +113 118 water chemical Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. DISCUSS +128 141 hydrogen bond bond_interaction Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. DISCUSS +175 183 subunits structure_element Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. DISCUSS +197 202 Arg58 residue_name_number Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. DISCUSS +203 216 hydrogen bond bond_interaction Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. DISCUSS +245 253 subunits structure_element Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. DISCUSS +271 273 U4 residue_name_number Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. DISCUSS +274 280 D loss evidence Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. DISCUSS +34 37 RNA chemical At 25.0 MGy, the magnitude of the RNA backbone D loss was of the same order as for the radiation-insensitive Gly Cα atoms and on average less than half that of the acidic residues of the protein (Supplementary Fig. S3). DISCUSS +47 53 D loss evidence At 25.0 MGy, the magnitude of the RNA backbone D loss was of the same order as for the radiation-insensitive Gly Cα atoms and on average less than half that of the acidic residues of the protein (Supplementary Fig. S3). DISCUSS +109 112 Gly residue_name At 25.0 MGy, the magnitude of the RNA backbone D loss was of the same order as for the radiation-insensitive Gly Cα atoms and on average less than half that of the acidic residues of the protein (Supplementary Fig. S3). DISCUSS +72 75 RNA chemical Consequently, no clear single-strand breaks could be located, and since RNA-binding within the current TRAP–(GAGUU)10GAG complex is mediated predominantly through base–protein interactions, the biological integrity of the RNA complex was dictated by the rate at which protein decarboxylation occurred. DISCUSS +103 120 TRAP–(GAGUU)10GAG complex_assembly Consequently, no clear single-strand breaks could be located, and since RNA-binding within the current TRAP–(GAGUU)10GAG complex is mediated predominantly through base–protein interactions, the biological integrity of the RNA complex was dictated by the rate at which protein decarboxylation occurred. DISCUSS +222 225 RNA chemical Consequently, no clear single-strand breaks could be located, and since RNA-binding within the current TRAP–(GAGUU)10GAG complex is mediated predominantly through base–protein interactions, the biological integrity of the RNA complex was dictated by the rate at which protein decarboxylation occurred. DISCUSS +0 3 RNA chemical RNA interacting with TRAP was shown to offer significant protection against radiation-induced structural changes. DISCUSS +21 25 TRAP complex_assembly RNA interacting with TRAP was shown to offer significant protection against radiation-induced structural changes. DISCUSS +5 10 Glu36 residue_name_number Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively). DISCUSS +15 20 Asp39 residue_name_number Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively). DISCUSS +38 41 RNA chemical Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively). DISCUSS +60 74 hydrogen bonds bond_interaction Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively). DISCUSS +78 85 guanine chemical Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively). DISCUSS +93 95 G3 residue_name_number Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively). DISCUSS +100 102 G1 residue_name_number Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively). DISCUSS +23 28 Asp39 residue_name_number However, compared with Asp39, Glu36 is strikingly less decarboxylated when bound to RNA (Fig. 4 ▸). DISCUSS +30 35 Glu36 residue_name_number However, compared with Asp39, Glu36 is strikingly less decarboxylated when bound to RNA (Fig. 4 ▸). DISCUSS +75 83 bound to protein_state However, compared with Asp39, Glu36 is strikingly less decarboxylated when bound to RNA (Fig. 4 ▸). DISCUSS +84 87 RNA chemical However, compared with Asp39, Glu36 is strikingly less decarboxylated when bound to RNA (Fig. 4 ▸). DISCUSS +40 83 mutagenesis and nucleoside analogue studies experimental_method This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). DISCUSS +133 135 G1 residue_name_number This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). DISCUSS +164 168 TRAP complex_assembly This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). DISCUSS +187 189 A2 residue_name_number This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). DISCUSS +194 196 G3 residue_name_number This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). DISCUSS +232 252 RNA-binding affinity evidence This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). DISCUSS +256 260 TRAP complex_assembly This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). DISCUSS +262 265 K d evidence This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). DISCUSS +4 9 Glu36 residue_name_number For Glu36 and Asp39, no direct quantitative correlation could be established between hydrogen-bond length and D loss (linear R 2 of <0.23 for all doses; Supplementary Fig. S5). DISCUSS +14 19 Asp39 residue_name_number For Glu36 and Asp39, no direct quantitative correlation could be established between hydrogen-bond length and D loss (linear R 2 of <0.23 for all doses; Supplementary Fig. S5). DISCUSS +85 98 hydrogen-bond bond_interaction For Glu36 and Asp39, no direct quantitative correlation could be established between hydrogen-bond length and D loss (linear R 2 of <0.23 for all doses; Supplementary Fig. S5). DISCUSS +110 116 D loss evidence For Glu36 and Asp39, no direct quantitative correlation could be established between hydrogen-bond length and D loss (linear R 2 of <0.23 for all doses; Supplementary Fig. S5). DISCUSS +118 128 linear R 2 evidence For Glu36 and Asp39, no direct quantitative correlation could be established between hydrogen-bond length and D loss (linear R 2 of <0.23 for all doses; Supplementary Fig. S5). DISCUSS +69 74 Glu36 residue_name_number Thus, another factor must be responsible for this clear reduction in Glu36 CO2 decarboxyl­ation in RNA-bound TRAP. DISCUSS +99 108 RNA-bound protein_state Thus, another factor must be responsible for this clear reduction in Glu36 CO2 decarboxyl­ation in RNA-bound TRAP. DISCUSS +109 113 TRAP complex_assembly Thus, another factor must be responsible for this clear reduction in Glu36 CO2 decarboxyl­ation in RNA-bound TRAP. DISCUSS +4 9 Glu36 residue_name_number The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. DISCUSS +53 67 hydrogen bonds bond_interaction The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. DISCUSS +71 76 His34 residue_name_number The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. DISCUSS +81 86 Lys56 residue_name_number The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. DISCUSS +121 130 conserved protein_state The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. DISCUSS +147 149 G3 residue_name_number The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. DISCUSS +231 236 Glu36 residue_name_number The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. DISCUSS +240 249 RNA-bound protein_state The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. DISCUSS +250 254 TRAP complex_assembly The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. DISCUSS +5 13 bound to protein_state When bound to RNA, the average solvent-accessible area of the Glu36 side-chain O atoms is reduced from ∼15 to 0 Å2. DISCUSS +14 17 RNA chemical When bound to RNA, the average solvent-accessible area of the Glu36 side-chain O atoms is reduced from ∼15 to 0 Å2. DISCUSS +62 67 Glu36 residue_name_number When bound to RNA, the average solvent-accessible area of the Glu36 side-chain O atoms is reduced from ∼15 to 0 Å2. DISCUSS +46 51 Glu36 residue_name_number We propose that with no solvent accessibility Glu36 decarboxylation is inhibited, since the CO2-formation rate K 2 is greatly reduced, and suggest that steric hindrance prevents each radicalized Glu36 CO2 group from achieving the planar conformation required for complete dissociation from TRAP. DISCUSS +92 114 CO2-formation rate K 2 evidence We propose that with no solvent accessibility Glu36 decarboxylation is inhibited, since the CO2-formation rate K 2 is greatly reduced, and suggest that steric hindrance prevents each radicalized Glu36 CO2 group from achieving the planar conformation required for complete dissociation from TRAP. DISCUSS +195 200 Glu36 residue_name_number We propose that with no solvent accessibility Glu36 decarboxylation is inhibited, since the CO2-formation rate K 2 is greatly reduced, and suggest that steric hindrance prevents each radicalized Glu36 CO2 group from achieving the planar conformation required for complete dissociation from TRAP. DISCUSS +290 294 TRAP complex_assembly We propose that with no solvent accessibility Glu36 decarboxylation is inhibited, since the CO2-formation rate K 2 is greatly reduced, and suggest that steric hindrance prevents each radicalized Glu36 CO2 group from achieving the planar conformation required for complete dissociation from TRAP. DISCUSS +4 36 electron-recombination rate K −1 evidence The electron-recombination rate K −1 remains high, however, owing to rapid electron migration through the protein–RNA complex to refill the Glu36 positive hole (the precursor for Glu decarboxylation). DISCUSS +106 117 protein–RNA complex_assembly The electron-recombination rate K −1 remains high, however, owing to rapid electron migration through the protein–RNA complex to refill the Glu36 positive hole (the precursor for Glu decarboxylation). DISCUSS +140 145 Glu36 residue_name_number The electron-recombination rate K −1 remains high, however, owing to rapid electron migration through the protein–RNA complex to refill the Glu36 positive hole (the precursor for Glu decarboxylation). DISCUSS +146 159 positive hole site The electron-recombination rate K −1 remains high, however, owing to rapid electron migration through the protein–RNA complex to refill the Glu36 positive hole (the precursor for Glu decarboxylation). DISCUSS +179 182 Glu residue_name The electron-recombination rate K −1 remains high, however, owing to rapid electron migration through the protein–RNA complex to refill the Glu36 positive hole (the precursor for Glu decarboxylation). DISCUSS +5 8 RNA chemical Upon RNA binding, the Asp39 side-chain carboxyl group solvent-accessible area changes from ∼75 to 35 Å2, still allowing a high CO2-formation rate K 2. DISCUSS +22 27 Asp39 residue_name_number Upon RNA binding, the Asp39 side-chain carboxyl group solvent-accessible area changes from ∼75 to 35 Å2, still allowing a high CO2-formation rate K 2. DISCUSS +127 130 CO2 chemical Upon RNA binding, the Asp39 side-chain carboxyl group solvent-accessible area changes from ∼75 to 35 Å2, still allowing a high CO2-formation rate K 2. DISCUSS +141 149 rate K 2 evidence Upon RNA binding, the Asp39 side-chain carboxyl group solvent-accessible area changes from ∼75 to 35 Å2, still allowing a high CO2-formation rate K 2. DISCUSS +86 93 crystal evidence The prevalence of radical attack from solvent channels surrounding the protein in the crystal is a questionable cause, considering previous observations indicating that the strongly oxidizing hydroxyl radical is immobile at 100 K (Allan et al., 2013; Owen et al., 2012). DISCUSS +40 44 with protein_state By comparing equivalent acidic residues with and without RNA, we have now deconvoluted the role of solvent accessibility from other local protein environment factors, and thus propose a suitable mechanism by which exceptionally low solvent accessibility can reduce the rate of decarboxylation. DISCUSS +49 56 without protein_state By comparing equivalent acidic residues with and without RNA, we have now deconvoluted the role of solvent accessibility from other local protein environment factors, and thus propose a suitable mechanism by which exceptionally low solvent accessibility can reduce the rate of decarboxylation. DISCUSS +57 60 RNA chemical By comparing equivalent acidic residues with and without RNA, we have now deconvoluted the role of solvent accessibility from other local protein environment factors, and thus propose a suitable mechanism by which exceptionally low solvent accessibility can reduce the rate of decarboxylation. DISCUSS +17 39 RNA-binding interfaces site Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 ▸ c). DISCUSS +41 44 RNA chemical Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 ▸ c). DISCUSS +102 107 Glu50 residue_name_number Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 ▸ c). DISCUSS +109 114 Glu71 residue_name_number Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 ▸ c). DISCUSS +119 124 Glu73 residue_name_number Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 ▸ c). DISCUSS +180 184 TRAP complex_assembly Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 ▸ c). DISCUSS +185 190 rings structure_element Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 ▸ c). DISCUSS +89 94 bound protein_state However, for each of these residues the exact crystal contacts are not preserved between bound and nonbound TRAP or even between monomers within one TRAP ring. DISCUSS +99 107 nonbound protein_state However, for each of these residues the exact crystal contacts are not preserved between bound and nonbound TRAP or even between monomers within one TRAP ring. DISCUSS +108 112 TRAP complex_assembly However, for each of these residues the exact crystal contacts are not preserved between bound and nonbound TRAP or even between monomers within one TRAP ring. DISCUSS +149 153 TRAP complex_assembly However, for each of these residues the exact crystal contacts are not preserved between bound and nonbound TRAP or even between monomers within one TRAP ring. DISCUSS +154 158 ring structure_element However, for each of these residues the exact crystal contacts are not preserved between bound and nonbound TRAP or even between monomers within one TRAP ring. DISCUSS +16 21 bound protein_state For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. DISCUSS +22 26 TRAP complex_assembly For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. DISCUSS +28 33 Glu73 residue_name_number For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. DISCUSS +61 67 lysine residue_name For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. DISCUSS +86 94 subunits structure_element For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. DISCUSS +107 115 nonbound protein_state For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. DISCUSS +116 120 TRAP complex_assembly For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. DISCUSS +152 157 Glu73 residue_name_number For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. DISCUSS +202 208 waters chemical For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. DISCUSS +217 224 subunit structure_element For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. DISCUSS +95 113 SRD investigations experimental_method Radiation-induced side-chain conformational changes have been poorly characterized in previous SRD investigations owing to their strong dependence on packing density and geometric strain. DISCUSS +205 207 MX experimental_method Such structural changes are known to have significant roles within enzymatic pathways, and experimenters must be aware of these possible confounding factors when assigning true functional mechanisms using MX. DISCUSS +22 25 RNA chemical Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively. DISCUSS +37 41 TRAP complex_assembly Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively. DISCUSS +95 99 TRAP complex_assembly Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively. DISCUSS +128 133 Lys37 residue_name_number Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively. DISCUSS +138 143 Phe32 residue_name_number Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively. DISCUSS +169 171 G1 residue_name_number Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively. DISCUSS +176 178 G3 residue_name_number Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively. DISCUSS +46 49 Tyr residue_name It has been suggested (Burmeister, 2000) that Tyr residues can lose their aromatic –OH group owing to radiation-induced effects; however, no energetically favourable pathway for –OH cleavage exists and this has not been detected in aqueous radiation-chemistry studies. DISCUSS +3 7 TRAP complex_assembly In TRAP, D loss increased at a similar rate for both the Tyr O atoms and aromatic ring atoms, suggesting that full ring conformational disordering is more likely. DISCUSS +9 15 D loss evidence In TRAP, D loss increased at a similar rate for both the Tyr O atoms and aromatic ring atoms, suggesting that full ring conformational disordering is more likely. DISCUSS +57 60 Tyr residue_name In TRAP, D loss increased at a similar rate for both the Tyr O atoms and aromatic ring atoms, suggesting that full ring conformational disordering is more likely. DISCUSS +82 86 ring structure_element In TRAP, D loss increased at a similar rate for both the Tyr O atoms and aromatic ring atoms, suggesting that full ring conformational disordering is more likely. DISCUSS +115 119 ring structure_element In TRAP, D loss increased at a similar rate for both the Tyr O atoms and aromatic ring atoms, suggesting that full ring conformational disordering is more likely. DISCUSS +35 59 Fourier difference peaks evidence Indeed, no convincing reproducible Fourier difference peaks above the background map noise were observed around any Tyr terminal –OH groups. DISCUSS +81 84 map evidence Indeed, no convincing reproducible Fourier difference peaks above the background map noise were observed around any Tyr terminal –OH groups. DISCUSS +116 119 Tyr residue_name Indeed, no convincing reproducible Fourier difference peaks above the background map noise were observed around any Tyr terminal –OH groups. DISCUSS +4 7 RNA chemical The RNA-stabilization effects on protein are observed at short ranges and are restricted to within the RNA-binding interfaces around the TRAP ring. DISCUSS +103 125 RNA-binding interfaces site The RNA-stabilization effects on protein are observed at short ranges and are restricted to within the RNA-binding interfaces around the TRAP ring. DISCUSS +137 141 TRAP complex_assembly The RNA-stabilization effects on protein are observed at short ranges and are restricted to within the RNA-binding interfaces around the TRAP ring. DISCUSS +142 146 ring structure_element The RNA-stabilization effects on protein are observed at short ranges and are restricted to within the RNA-binding interfaces around the TRAP ring. DISCUSS +13 18 Asp17 residue_name_number For example, Asp17 is located ∼6.8 Å from the G1 base, outside the RNA-binding interfaces, and has indistinguishable Cγ atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p > 0.9). DISCUSS +46 48 G1 residue_name_number For example, Asp17 is located ∼6.8 Å from the G1 base, outside the RNA-binding interfaces, and has indistinguishable Cγ atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p > 0.9). DISCUSS +67 89 RNA-binding interfaces site For example, Asp17 is located ∼6.8 Å from the G1 base, outside the RNA-binding interfaces, and has indistinguishable Cγ atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p > 0.9). DISCUSS +127 145 loss dose-dynamics evidence For example, Asp17 is located ∼6.8 Å from the G1 base, outside the RNA-binding interfaces, and has indistinguishable Cγ atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p > 0.9). DISCUSS +154 163 RNA-bound protein_state For example, Asp17 is located ∼6.8 Å from the G1 base, outside the RNA-binding interfaces, and has indistinguishable Cγ atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p > 0.9). DISCUSS +168 176 nonbound protein_state For example, Asp17 is located ∼6.8 Å from the G1 base, outside the RNA-binding interfaces, and has indistinguishable Cγ atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p > 0.9). DISCUSS +177 181 TRAP complex_assembly For example, Asp17 is located ∼6.8 Å from the G1 base, outside the RNA-binding interfaces, and has indistinguishable Cγ atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p > 0.9). DISCUSS +214 225 DNA–protein complex_assembly An increase in the dose at which functionally important residues remain intact has biological ramifications for understanding the mechanisms at which ionizing radiation damage is mitigated within naturally forming DNA–protein and RNA–protein complexes. DISCUSS +230 241 RNA–protein complex_assembly An increase in the dose at which functionally important residues remain intact has biological ramifications for understanding the mechanisms at which ionizing radiation damage is mitigated within naturally forming DNA–protein and RNA–protein complexes. DISCUSS +55 64 DNA-bound protein_state Observations of lower protein radiation-sensitivity in DNA-bound forms have been recorded in solution at RT at much lower doses (∼1 kGy) than those used for typical MX experiments [e.g. an oestrogen response element–receptor complex (Stísová et al., 2006) and a DNA glycosylase and its abasic DNA target site (Gillard et al., 2004)]. DISCUSS +165 167 MX experimental_method Observations of lower protein radiation-sensitivity in DNA-bound forms have been recorded in solution at RT at much lower doses (∼1 kGy) than those used for typical MX experiments [e.g. an oestrogen response element–receptor complex (Stísová et al., 2006) and a DNA glycosylase and its abasic DNA target site (Gillard et al., 2004)]. DISCUSS +262 277 DNA glycosylase protein_type Observations of lower protein radiation-sensitivity in DNA-bound forms have been recorded in solution at RT at much lower doses (∼1 kGy) than those used for typical MX experiments [e.g. an oestrogen response element–receptor complex (Stísová et al., 2006) and a DNA glycosylase and its abasic DNA target site (Gillard et al., 2004)]. DISCUSS +286 308 abasic DNA target site site Observations of lower protein radiation-sensitivity in DNA-bound forms have been recorded in solution at RT at much lower doses (∼1 kGy) than those used for typical MX experiments [e.g. an oestrogen response element–receptor complex (Stísová et al., 2006) and a DNA glycosylase and its abasic DNA target site (Gillard et al., 2004)]. DISCUSS +191 194 DNA chemical In these studies, the main damaging species is predicted to be the oxidizing hydroxyl radical produced through solvent irradiation, which is known to add to double covalent bonds within both DNA and RNA bases to induce strand breaks and base modification (Spotheim-Maurizot & Davídková, 2011; Chance et al., 1997). DISCUSS +199 202 RNA chemical In these studies, the main damaging species is predicted to be the oxidizing hydroxyl radical produced through solvent irradiation, which is known to add to double covalent bonds within both DNA and RNA bases to induce strand breaks and base modification (Spotheim-Maurizot & Davídková, 2011; Chance et al., 1997). DISCUSS +44 47 DNA chemical It was suggested that physical screening of DNA by protein shielded the DNA–protein interaction sites from radical damage, yielding an extended life-dose for the nucleoprotein complex compared with separate protein and DNA constituents at RT. DISCUSS +72 101 DNA–protein interaction sites site It was suggested that physical screening of DNA by protein shielded the DNA–protein interaction sites from radical damage, yielding an extended life-dose for the nucleoprotein complex compared with separate protein and DNA constituents at RT. DISCUSS +219 222 DNA chemical It was suggested that physical screening of DNA by protein shielded the DNA–protein interaction sites from radical damage, yielding an extended life-dose for the nucleoprotein complex compared with separate protein and DNA constituents at RT. DISCUSS +24 26 MX experimental_method However, in the current MX study at 100 K, the main damaging species are believed to be migrating LEEs and holes produced directly within the protein–RNA components or in closely associated solvent. DISCUSS +142 153 protein–RNA complex_assembly However, in the current MX study at 100 K, the main damaging species are believed to be migrating LEEs and holes produced directly within the protein–RNA components or in closely associated solvent. DISCUSS +62 75 nucleoprotein complex_assembly The results presented here suggest that biologically relevant nucleoprotein complexes also exhibit prolonged life-doses under the effect of LEE-induced structural changes, involving direct physical protection of key RNA-binding residues. DISCUSS +216 236 RNA-binding residues site The results presented here suggest that biologically relevant nucleoprotein complexes also exhibit prolonged life-doses under the effect of LEE-induced structural changes, involving direct physical protection of key RNA-binding residues. DISCUSS +93 98 bound protein_state Such reduced radiation-sensitivity in this case ensures that the interacting protein remains bound long enough to the RNA to complete its function, even whilst exposed to ionizing radiation. DISCUSS +118 121 RNA chemical Such reduced radiation-sensitivity in this case ensures that the interacting protein remains bound long enough to the RNA to complete its function, even whilst exposed to ionizing radiation. DISCUSS +11 19 nonbound protein_state Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 ▸). DISCUSS +20 24 TRAP complex_assembly Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 ▸). DISCUSS +82 104 RNA-binding interfaces site Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 ▸). DISCUSS +106 111 Asp39 residue_name_number Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 ▸). DISCUSS +113 118 Glu36 residue_name_number Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 ▸). DISCUSS +120 125 Glu42 residue_name_number Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 ▸). DISCUSS +128 132 TRAP complex_assembly When exposed to X-rays, these residues will be preferentially damaged by X-rays and subsequently reduce the affinity with which TRAP binds to RNA. DISCUSS +142 145 RNA chemical When exposed to X-rays, these residues will be preferentially damaged by X-rays and subsequently reduce the affinity with which TRAP binds to RNA. DISCUSS +116 119 RNA chemical Within the cellular environment, this mechanism could reduce the risk that radiation-damaged proteins might bind to RNA, thus avoiding the detrimental introduction of incorrect DNA-repair, transcriptional and base-modification pathways. DISCUSS +177 180 DNA chemical Within the cellular environment, this mechanism could reduce the risk that radiation-damaged proteins might bind to RNA, thus avoiding the detrimental introduction of incorrect DNA-repair, transcriptional and base-modification pathways. DISCUSS +4 21 TRAP–(GAGUU)10GAG complex_assembly The TRAP–(GAGUU)10GAG complex asymmetric unit (PDB entry 1gtf; Hopcroft et al., 2002). FIG +0 5 Bound protein_state Bound tryptophan ligands are represented as coloured spheres. FIG +6 16 tryptophan chemical Bound tryptophan ligands are represented as coloured spheres. FIG +0 3 RNA chemical RNA is shown is yellow. FIG +62 70 TRAP–RNA complex_assembly (a) Electron-density loss sites as indicated by D loss in the TRAP–RNA complex crystal by residue/nucleotide type for five doses [sites determined above the 4× average D loss threshold, calculated over the TRAP–RNA structure for the first difference map: F obs(d 2) − F obs(d 1)]. FIG +79 86 crystal evidence (a) Electron-density loss sites as indicated by D loss in the TRAP–RNA complex crystal by residue/nucleotide type for five doses [sites determined above the 4× average D loss threshold, calculated over the TRAP–RNA structure for the first difference map: F obs(d 2) − F obs(d 1)]. FIG +206 214 TRAP–RNA complex_assembly (a) Electron-density loss sites as indicated by D loss in the TRAP–RNA complex crystal by residue/nucleotide type for five doses [sites determined above the 4× average D loss threshold, calculated over the TRAP–RNA structure for the first difference map: F obs(d 2) − F obs(d 1)]. FIG +215 224 structure evidence (a) Electron-density loss sites as indicated by D loss in the TRAP–RNA complex crystal by residue/nucleotide type for five doses [sites determined above the 4× average D loss threshold, calculated over the TRAP–RNA structure for the first difference map: F obs(d 2) − F obs(d 1)]. FIG +239 253 difference map evidence (a) Electron-density loss sites as indicated by D loss in the TRAP–RNA complex crystal by residue/nucleotide type for five doses [sites determined above the 4× average D loss threshold, calculated over the TRAP–RNA structure for the first difference map: F obs(d 2) − F obs(d 1)]. FIG +72 75 DWD evidence (b) Average D loss for each residue/nucleotide type with respect to the DWD (diffraction-weighted dose; Zeldin, Brock­hauser et al., 2013). FIG +77 102 diffraction-weighted dose evidence (b) Average D loss for each residue/nucleotide type with respect to the DWD (diffraction-weighted dose; Zeldin, Brock­hauser et al., 2013). FIG +21 25 TRAP complex_assembly Only a subset of key TRAP residue types are included. FIG +46 50 TRAP complex_assembly The average D loss (calculated over the whole TRAP asymmetric unit) is shown at each dose (dashed line). FIG +13 31 difference density evidence In (a) clear difference density is observed around the Glu42 carboxyl side chain in chain H, within the lowest dose difference map at d 2 = 3.9 MGy. FIG +55 60 Glu42 residue_name_number In (a) clear difference density is observed around the Glu42 carboxyl side chain in chain H, within the lowest dose difference map at d 2 = 3.9 MGy. FIG +104 130 lowest dose difference map evidence In (a) clear difference density is observed around the Glu42 carboxyl side chain in chain H, within the lowest dose difference map at d 2 = 3.9 MGy. FIG +130 133 RNA chemical Radiation-induced protein disordering is evident across the large dose range (b, c); in comparison, no clear deterioration of the RNA density was observed. FIG +134 141 density evidence Radiation-induced protein disordering is evident across the large dose range (b, c); in comparison, no clear deterioration of the RNA density was observed. FIG +53 56 Glu residue_name D loss calculated for all side-chain carboxyl group Glu Cδ and Asp Cγ atoms within the TRAP–RNA complex for a dose of 19.3 MGy (d 8). FIG +64 67 Asp residue_name D loss calculated for all side-chain carboxyl group Glu Cδ and Asp Cγ atoms within the TRAP–RNA complex for a dose of 19.3 MGy (d 8). FIG +88 96 TRAP–RNA complex_assembly D loss calculated for all side-chain carboxyl group Glu Cδ and Asp Cγ atoms within the TRAP–RNA complex for a dose of 19.3 MGy (d 8). FIG +64 69 bound protein_state Residues have been grouped by amino-acid number, and split into bound and nonbound groupings, with each bar representing the mean calculated over 11 equivalent atoms around a TRAP ring. FIG +74 82 nonbound protein_state Residues have been grouped by amino-acid number, and split into bound and nonbound groupings, with each bar representing the mean calculated over 11 equivalent atoms around a TRAP ring. FIG +175 179 TRAP complex_assembly Residues have been grouped by amino-acid number, and split into bound and nonbound groupings, with each bar representing the mean calculated over 11 equivalent atoms around a TRAP ring. FIG +180 184 ring structure_element Residues have been grouped by amino-acid number, and split into bound and nonbound groupings, with each bar representing the mean calculated over 11 equivalent atoms around a TRAP ring. FIG +29 34 Glu36 residue_name_number D loss against dose for (a) Glu36 Cδ, (b) Asp39 Cγ, (c) Glu42 O∊1, (d) Glu42 O∊2, (e) Phe32 Cζ and (f) Lys37 C∊ atoms. FIG +43 48 Asp39 residue_name_number D loss against dose for (a) Glu36 Cδ, (b) Asp39 Cγ, (c) Glu42 O∊1, (d) Glu42 O∊2, (e) Phe32 Cζ and (f) Lys37 C∊ atoms. FIG +57 62 Glu42 residue_name_number D loss against dose for (a) Glu36 Cδ, (b) Asp39 Cγ, (c) Glu42 O∊1, (d) Glu42 O∊2, (e) Phe32 Cζ and (f) Lys37 C∊ atoms. FIG +72 77 Glu42 residue_name_number D loss against dose for (a) Glu36 Cδ, (b) Asp39 Cγ, (c) Glu42 O∊1, (d) Glu42 O∊2, (e) Phe32 Cζ and (f) Lys37 C∊ atoms. FIG +87 92 Phe32 residue_name_number D loss against dose for (a) Glu36 Cδ, (b) Asp39 Cγ, (c) Glu42 O∊1, (d) Glu42 O∊2, (e) Phe32 Cζ and (f) Lys37 C∊ atoms. FIG +104 109 Lys37 residue_name_number D loss against dose for (a) Glu36 Cδ, (b) Asp39 Cγ, (c) Glu42 O∊1, (d) Glu42 O∊2, (e) Phe32 Cζ and (f) Lys37 C∊ atoms. FIG +67 72 bound protein_state 95% CI are included for each set of 11 equivalent atoms grouped as bound/nonbound. FIG +73 81 nonbound protein_state 95% CI are included for each set of 11 equivalent atoms grouped as bound/nonbound. FIG +0 21 RNA-binding interface site RNA-binding interface interactions are shown for TRAP chain N, with the F obs(d 7) − F obs(d 1) Fourier difference map (dose 16.7 MGy) overlaid and contoured at a ±4σ level. FIG +49 53 TRAP complex_assembly RNA-binding interface interactions are shown for TRAP chain N, with the F obs(d 7) − F obs(d 1) Fourier difference map (dose 16.7 MGy) overlaid and contoured at a ±4σ level. FIG diff --git a/annotation_CSV/PMC4855620.csv b/annotation_CSV/PMC4855620.csv new file mode 100644 index 0000000000000000000000000000000000000000..a130e757af000cd201bc151d458946e4e4511bbd --- /dev/null +++ b/annotation_CSV/PMC4855620.csv @@ -0,0 +1,564 @@ +anno_start anno_end anno_text entity_type sentence section +39 60 peptide binding‐motif structure_element Structure‐activity relationship of the peptide binding‐motif mediating the BRCA2:RAD51 protein–protein interaction TITLE +75 86 BRCA2:RAD51 complex_assembly Structure‐activity relationship of the peptide binding‐motif mediating the BRCA2:RAD51 protein–protein interaction TITLE +1 6 RAD51 protein RAD51 is a recombinase involved in the homologous recombination of double‐strand breaks in DNA. ABSTRACT +12 23 recombinase protein_type RAD51 is a recombinase involved in the homologous recombination of double‐strand breaks in DNA. ABSTRACT +0 5 RAD51 protein RAD51 forms oligomers by binding to another molecule of RAD51 via an ‘FxxA’ motif, and the same recognition sequence is similarly utilised to bind BRCA2. ABSTRACT +12 21 oligomers oligomeric_state RAD51 forms oligomers by binding to another molecule of RAD51 via an ‘FxxA’ motif, and the same recognition sequence is similarly utilised to bind BRCA2. ABSTRACT +56 61 RAD51 protein RAD51 forms oligomers by binding to another molecule of RAD51 via an ‘FxxA’ motif, and the same recognition sequence is similarly utilised to bind BRCA2. ABSTRACT +70 74 FxxA structure_element RAD51 forms oligomers by binding to another molecule of RAD51 via an ‘FxxA’ motif, and the same recognition sequence is similarly utilised to bind BRCA2. ABSTRACT +147 152 BRCA2 protein RAD51 forms oligomers by binding to another molecule of RAD51 via an ‘FxxA’ motif, and the same recognition sequence is similarly utilised to bind BRCA2. ABSTRACT +33 41 mutation experimental_method We have tabulated the effects of mutation of this sequence, across a variety of experimental methods and from relevant mutations observed in the clinic. ABSTRACT +7 14 mutants protein_state We use mutants of a tetrapeptide sequence to probe the binding interaction, using both isothermal titration calorimetry and X‐ray crystallography. ABSTRACT +20 32 tetrapeptide chemical We use mutants of a tetrapeptide sequence to probe the binding interaction, using both isothermal titration calorimetry and X‐ray crystallography. ABSTRACT +87 119 isothermal titration calorimetry experimental_method We use mutants of a tetrapeptide sequence to probe the binding interaction, using both isothermal titration calorimetry and X‐ray crystallography. ABSTRACT +124 145 X‐ray crystallography experimental_method We use mutants of a tetrapeptide sequence to probe the binding interaction, using both isothermal titration calorimetry and X‐ray crystallography. ABSTRACT +39 68 tetrapeptide mutational study experimental_method Where possible, comparison between our tetrapeptide mutational study and the previously reported mutations is made, discrepancies are discussed and the importance of secondary structure in interpreting alanine scanning and mutational data of this nature is considered. ABSTRACT +202 218 alanine scanning experimental_method Where possible, comparison between our tetrapeptide mutational study and the previously reported mutations is made, discrepancies are discussed and the importance of secondary structure in interpreting alanine scanning and mutational data of this nature is considered. ABSTRACT +0 10 Eukaryotic taxonomy_domain Eukaryotic RAD51, archeal RadA and prokaryotic RecA are a family of ATP‐dependent recombinases involved in homologous recombination (HR) of double‐strand breaks in DNA 1. ABBR +11 16 RAD51 protein Eukaryotic RAD51, archeal RadA and prokaryotic RecA are a family of ATP‐dependent recombinases involved in homologous recombination (HR) of double‐strand breaks in DNA 1. ABBR +18 25 archeal taxonomy_domain Eukaryotic RAD51, archeal RadA and prokaryotic RecA are a family of ATP‐dependent recombinases involved in homologous recombination (HR) of double‐strand breaks in DNA 1. ABBR +26 30 RadA protein Eukaryotic RAD51, archeal RadA and prokaryotic RecA are a family of ATP‐dependent recombinases involved in homologous recombination (HR) of double‐strand breaks in DNA 1. ABBR +35 46 prokaryotic taxonomy_domain Eukaryotic RAD51, archeal RadA and prokaryotic RecA are a family of ATP‐dependent recombinases involved in homologous recombination (HR) of double‐strand breaks in DNA 1. ABBR +47 51 RecA protein Eukaryotic RAD51, archeal RadA and prokaryotic RecA are a family of ATP‐dependent recombinases involved in homologous recombination (HR) of double‐strand breaks in DNA 1. ABBR +68 94 ATP‐dependent recombinases protein_type Eukaryotic RAD51, archeal RadA and prokaryotic RecA are a family of ATP‐dependent recombinases involved in homologous recombination (HR) of double‐strand breaks in DNA 1. ABBR +0 5 RAD51 protein RAD51 interacts with BRCA2, and is thought to localise RAD51 to sites of DNA damage 2, 3. ABBR +21 26 BRCA2 protein RAD51 interacts with BRCA2, and is thought to localise RAD51 to sites of DNA damage 2, 3. ABBR +55 60 RAD51 protein RAD51 interacts with BRCA2, and is thought to localise RAD51 to sites of DNA damage 2, 3. ABBR +5 10 BRCA2 protein Both BRCA2 and RAD51 together are vital for helping to repair and maintain a high fidelity in DNA replication. ABBR +15 20 RAD51 protein Both BRCA2 and RAD51 together are vital for helping to repair and maintain a high fidelity in DNA replication. ABBR +0 5 BRCA2 protein BRCA2 especially has garnered much attention in a clinical context, as many mutations have been identified that drive an increased risk of cancer in individuals 4, 5. ABBR +33 44 BRCA2:RAD51 complex_assembly Although the inactivation of the BRCA2:RAD51 DNA repair pathway can cause genomic instability and eventual tumour development, an inability to repair breaks in DNA may also engender a sensitivity to ionising radiation 6, 7. ABBR +71 82 BRCA2:RAD51 complex_assembly For this reason it is hypothesised that in tumour cells with an intact BRCA2:RAD51 repair pathway, small molecules which prevent the interaction between RAD51 and BRCA2 may confer radiosensitivity by disabling the HR pathway 8. ABBR +153 158 RAD51 protein For this reason it is hypothesised that in tumour cells with an intact BRCA2:RAD51 repair pathway, small molecules which prevent the interaction between RAD51 and BRCA2 may confer radiosensitivity by disabling the HR pathway 8. ABBR +163 168 BRCA2 protein For this reason it is hypothesised that in tumour cells with an intact BRCA2:RAD51 repair pathway, small molecules which prevent the interaction between RAD51 and BRCA2 may confer radiosensitivity by disabling the HR pathway 8. ABBR +62 73 BRC repeats structure_element The interaction between the two proteins is mediated by eight BRC repeats, which are characterised by a conserved ‘FxxA’ motif 9. ABBR +104 113 conserved protein_state The interaction between the two proteins is mediated by eight BRC repeats, which are characterised by a conserved ‘FxxA’ motif 9. ABBR +114 128 ‘FxxA’ motif 9 structure_element The interaction between the two proteins is mediated by eight BRC repeats, which are characterised by a conserved ‘FxxA’ motif 9. ABBR +0 5 RAD51 protein RAD51 and RadA proteins also contain an ‘FxxA’ sequence (FTTA for human RAD51) through which it can bind to other RAD51 and RadA molecules, and oligomerise to form higher order filament structures on DNA. ABBR +10 14 RadA protein RAD51 and RadA proteins also contain an ‘FxxA’ sequence (FTTA for human RAD51) through which it can bind to other RAD51 and RadA molecules, and oligomerise to form higher order filament structures on DNA. ABBR +41 45 FxxA structure_element RAD51 and RadA proteins also contain an ‘FxxA’ sequence (FTTA for human RAD51) through which it can bind to other RAD51 and RadA molecules, and oligomerise to form higher order filament structures on DNA. ABBR +57 61 FTTA structure_element RAD51 and RadA proteins also contain an ‘FxxA’ sequence (FTTA for human RAD51) through which it can bind to other RAD51 and RadA molecules, and oligomerise to form higher order filament structures on DNA. ABBR +66 71 human species RAD51 and RadA proteins also contain an ‘FxxA’ sequence (FTTA for human RAD51) through which it can bind to other RAD51 and RadA molecules, and oligomerise to form higher order filament structures on DNA. ABBR +72 77 RAD51 protein RAD51 and RadA proteins also contain an ‘FxxA’ sequence (FTTA for human RAD51) through which it can bind to other RAD51 and RadA molecules, and oligomerise to form higher order filament structures on DNA. ABBR +114 119 RAD51 protein RAD51 and RadA proteins also contain an ‘FxxA’ sequence (FTTA for human RAD51) through which it can bind to other RAD51 and RadA molecules, and oligomerise to form higher order filament structures on DNA. ABBR +124 128 RadA protein RAD51 and RadA proteins also contain an ‘FxxA’ sequence (FTTA for human RAD51) through which it can bind to other RAD51 and RadA molecules, and oligomerise to form higher order filament structures on DNA. ABBR +11 15 FxxA structure_element The common FxxA motifs of both the BRC repeats and RAD51 oligomerisation sequence are recognised by the same FxxA‐binding site of RAD51. ABBR +35 46 BRC repeats structure_element The common FxxA motifs of both the BRC repeats and RAD51 oligomerisation sequence are recognised by the same FxxA‐binding site of RAD51. ABBR +51 56 RAD51 protein The common FxxA motifs of both the BRC repeats and RAD51 oligomerisation sequence are recognised by the same FxxA‐binding site of RAD51. ABBR +57 81 oligomerisation sequence structure_element The common FxxA motifs of both the BRC repeats and RAD51 oligomerisation sequence are recognised by the same FxxA‐binding site of RAD51. ABBR +109 126 FxxA‐binding site site The common FxxA motifs of both the BRC repeats and RAD51 oligomerisation sequence are recognised by the same FxxA‐binding site of RAD51. ABBR +130 135 RAD51 protein The common FxxA motifs of both the BRC repeats and RAD51 oligomerisation sequence are recognised by the same FxxA‐binding site of RAD51. ABBR +73 87 binding energy evidence In general, the dominant contribution of certain residues to the overall binding energy of a protein–protein interaction are known as ‘hot‐spot’ residues. ABBR +135 143 hot‐spot site In general, the dominant contribution of certain residues to the overall binding energy of a protein–protein interaction are known as ‘hot‐spot’ residues. ABBR +84 91 pockets site Interestingly, small molecule inhibitors of PPIs are often found to occupy the same pockets which are otherwise occupied by hot‐spot residues in the native complex. ABBR +124 132 hot‐spot site Interestingly, small molecule inhibitors of PPIs are often found to occupy the same pockets which are otherwise occupied by hot‐spot residues in the native complex. ABBR +149 155 native protein_state Interestingly, small molecule inhibitors of PPIs are often found to occupy the same pockets which are otherwise occupied by hot‐spot residues in the native complex. ABBR +46 55 hot‐spots site It is therefore of great interest to identify hot‐spots in an effort to guide drug discovery efforts against a PPI. ABBR +49 67 strongly conserved protein_state Further, a correlation between residues that are strongly conserved and hot‐spot residues has been reported 10. ABBR +72 80 hot‐spot site Further, a correlation between residues that are strongly conserved and hot‐spot residues has been reported 10. ABBR +84 97 phenylalanine residue_name Purely based on the amino acid consensus sequence reported by Pellegrini et al., 11 phenylalanine and alanine would both be expected to be hot‐spots and to a lesser extent, threonine. ABBR +102 109 alanine residue_name Purely based on the amino acid consensus sequence reported by Pellegrini et al., 11 phenylalanine and alanine would both be expected to be hot‐spots and to a lesser extent, threonine. ABBR +139 148 hot‐spots site Purely based on the amino acid consensus sequence reported by Pellegrini et al., 11 phenylalanine and alanine would both be expected to be hot‐spots and to a lesser extent, threonine. ABBR +173 182 threonine residue_name Purely based on the amino acid consensus sequence reported by Pellegrini et al., 11 phenylalanine and alanine would both be expected to be hot‐spots and to a lesser extent, threonine. ABBR +38 54 highly conserved protein_state However, whilst the identification of highly conserved residues may be a good starting point for identifying hot‐spots, experimental validation by mutation of these sequences is vital. ABBR +109 118 hot‐spots site However, whilst the identification of highly conserved residues may be a good starting point for identifying hot‐spots, experimental validation by mutation of these sequences is vital. ABBR +147 155 mutation experimental_method However, whilst the identification of highly conserved residues may be a good starting point for identifying hot‐spots, experimental validation by mutation of these sequences is vital. ABBR +34 38 FxxA structure_element The importance of residues in the FxxA motif has been probed by a variety of techniques, collated in Table 1. ABBR +9 17 mutating experimental_method Briefly, mutating phenylalanine to glutamic acid inactivated the BRC4 peptide and prevented RAD51 oligomerisation 11, 12. ABBR +18 31 phenylalanine residue_name Briefly, mutating phenylalanine to glutamic acid inactivated the BRC4 peptide and prevented RAD51 oligomerisation 11, 12. ABBR +35 48 glutamic acid residue_name Briefly, mutating phenylalanine to glutamic acid inactivated the BRC4 peptide and prevented RAD51 oligomerisation 11, 12. ABBR +49 60 inactivated protein_state Briefly, mutating phenylalanine to glutamic acid inactivated the BRC4 peptide and prevented RAD51 oligomerisation 11, 12. ABBR +65 69 BRC4 chemical Briefly, mutating phenylalanine to glutamic acid inactivated the BRC4 peptide and prevented RAD51 oligomerisation 11, 12. ABBR +92 97 RAD51 protein Briefly, mutating phenylalanine to glutamic acid inactivated the BRC4 peptide and prevented RAD51 oligomerisation 11, 12. ABBR +2 25 phenylalanine‐truncated protein_state A phenylalanine‐truncated BRC4 is also found to be inactive 13, however, introducing a tryptophan for phenylalanine was found to have no significant effect on BRC4 affinity 12. ABBR +26 30 BRC4 chemical A phenylalanine‐truncated BRC4 is also found to be inactive 13, however, introducing a tryptophan for phenylalanine was found to have no significant effect on BRC4 affinity 12. ABBR +51 59 inactive protein_state A phenylalanine‐truncated BRC4 is also found to be inactive 13, however, introducing a tryptophan for phenylalanine was found to have no significant effect on BRC4 affinity 12. ABBR +73 84 introducing experimental_method A phenylalanine‐truncated BRC4 is also found to be inactive 13, however, introducing a tryptophan for phenylalanine was found to have no significant effect on BRC4 affinity 12. ABBR +87 97 tryptophan residue_name A phenylalanine‐truncated BRC4 is also found to be inactive 13, however, introducing a tryptophan for phenylalanine was found to have no significant effect on BRC4 affinity 12. ABBR +102 115 phenylalanine residue_name A phenylalanine‐truncated BRC4 is also found to be inactive 13, however, introducing a tryptophan for phenylalanine was found to have no significant effect on BRC4 affinity 12. ABBR +159 163 BRC4 chemical A phenylalanine‐truncated BRC4 is also found to be inactive 13, however, introducing a tryptophan for phenylalanine was found to have no significant effect on BRC4 affinity 12. ABBR +164 172 affinity evidence A phenylalanine‐truncated BRC4 is also found to be inactive 13, however, introducing a tryptophan for phenylalanine was found to have no significant effect on BRC4 affinity 12. ABBR +2 11 glutamine residue_name A glutamine replacing the histidine in BRC4 maintains BRC4 activity 13. ABBR +12 21 replacing experimental_method A glutamine replacing the histidine in BRC4 maintains BRC4 activity 13. ABBR +26 35 histidine residue_name A glutamine replacing the histidine in BRC4 maintains BRC4 activity 13. ABBR +39 43 BRC4 chemical A glutamine replacing the histidine in BRC4 maintains BRC4 activity 13. ABBR +54 58 BRC4 chemical A glutamine replacing the histidine in BRC4 maintains BRC4 activity 13. ABBR +15 19 BRC3 chemical The ability of BRC3 to interact with RAD51 nucleoprotein filaments is disrupted when threonine is mutated to an alanine 3. ABBR +37 42 RAD51 protein The ability of BRC3 to interact with RAD51 nucleoprotein filaments is disrupted when threonine is mutated to an alanine 3. ABBR +85 94 threonine residue_name The ability of BRC3 to interact with RAD51 nucleoprotein filaments is disrupted when threonine is mutated to an alanine 3. ABBR +98 105 mutated experimental_method The ability of BRC3 to interact with RAD51 nucleoprotein filaments is disrupted when threonine is mutated to an alanine 3. ABBR +112 119 alanine residue_name The ability of BRC3 to interact with RAD51 nucleoprotein filaments is disrupted when threonine is mutated to an alanine 3. ABBR +11 19 mutating experimental_method Similarly, mutating alanine to glutamic acid in the RAD51 oligomerisation sequence 11 or to serine in BRC4 13 leads to loss of interaction in both cases. ABBR +20 27 alanine residue_name Similarly, mutating alanine to glutamic acid in the RAD51 oligomerisation sequence 11 or to serine in BRC4 13 leads to loss of interaction in both cases. ABBR +31 44 glutamic acid residue_name Similarly, mutating alanine to glutamic acid in the RAD51 oligomerisation sequence 11 or to serine in BRC4 13 leads to loss of interaction in both cases. ABBR +52 57 RAD51 protein Similarly, mutating alanine to glutamic acid in the RAD51 oligomerisation sequence 11 or to serine in BRC4 13 leads to loss of interaction in both cases. ABBR +58 82 oligomerisation sequence structure_element Similarly, mutating alanine to glutamic acid in the RAD51 oligomerisation sequence 11 or to serine in BRC4 13 leads to loss of interaction in both cases. ABBR +92 98 serine residue_name Similarly, mutating alanine to glutamic acid in the RAD51 oligomerisation sequence 11 or to serine in BRC4 13 leads to loss of interaction in both cases. ABBR +102 106 BRC4 chemical Similarly, mutating alanine to glutamic acid in the RAD51 oligomerisation sequence 11 or to serine in BRC4 13 leads to loss of interaction in both cases. ABBR +4 8 BRC5 chemical The BRC5 repeat in humans has serine in the place of alanine, and is thought to be a nonbinding repeat 12. ABBR +19 25 humans species The BRC5 repeat in humans has serine in the place of alanine, and is thought to be a nonbinding repeat 12. ABBR +30 36 serine residue_name The BRC5 repeat in humans has serine in the place of alanine, and is thought to be a nonbinding repeat 12. ABBR +53 60 alanine residue_name The BRC5 repeat in humans has serine in the place of alanine, and is thought to be a nonbinding repeat 12. ABBR +85 102 nonbinding repeat structure_element The BRC5 repeat in humans has serine in the place of alanine, and is thought to be a nonbinding repeat 12. ABBR +43 47 FxxA structure_element Mutations identified in the clinic, in the FxxA region of the BRC repeats of BRCA2 are collated in Table 1 14. ABBR +62 73 BRC repeats structure_element Mutations identified in the clinic, in the FxxA region of the BRC repeats of BRCA2 are collated in Table 1 14. ABBR +77 82 BRCA2 protein Mutations identified in the clinic, in the FxxA region of the BRC repeats of BRCA2 are collated in Table 1 14. ABBR +163 168 RAD51 protein For completeness, we present them here with this caveat, and to make the comment that these clinical mutations are consistent with abrogating the interaction with RAD51. ABBR +11 15 FxxA structure_element Summary of FxxA‐relevant mutations previously reported and degree of characterisation. TABLE +50 54 FxxA structure_element The mutation, relevant peptide context, resulting FxxA motif sequence and experimental technique for each entry is given. TABLE +27 31 FxxA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +62 67 RAD51 protein "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +69 73 FTTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +75 79 F86E mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +80 84 ETTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +85 104 Immunoprecipitation experimental_method "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +121 125 BRC4 chemical "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +127 131 FHTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +133 139 F1524E mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +140 144 EHTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +145 162 Competitive ELISA experimental_method "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +174 182 inactive protein_state "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +185 189 BRC4 chemical "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +191 195 FHTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +197 203 F1524W mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +204 208 WHTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +209 226 Competitive ELISA experimental_method "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +253 255 WT protein_state "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +258 262 BRC4 chemical "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +264 268 FHTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +270 276 F1524V mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +277 281 VHTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +282 287 BRCA2 protein "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +314 318 BRC4 chemical "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +320 324 FHTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +326 332 ΔF1524 mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +334 337 HTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +354 363 RAD51‐DNA complex_assembly "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +383 391 inactive protein_state "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +394 398 BRC4 chemical "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +400 404 FHTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +406 412 H1525Q mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +413 417 FQTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +434 443 RAD51‐DNA complex_assembly "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +477 481 BRC7 chemical "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +483 487 FSTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +489 495 S1979R mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +496 500 FRTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +501 506 BRCA2 protein "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +533 537 BRC3 chemical "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +539 543 FQTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +545 551 T1430A mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +552 556 FQAA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +557 566 RAD51:DNA complex_assembly "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +567 582 bandshift assay experimental_method "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +593 601 inactive protein_state "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +604 608 BRC3 chemical "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +610 614 FQTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +616 622 T1430A mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +623 627 FQAA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +628 648 Electron microscopic experimental_method "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +700 708 inactive protein_state "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +711 715 BRC1 chemical "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +717 721 FRTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +723 729 T1011R mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +730 734 FRRA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +735 740 BRCA2 protein "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +767 771 BRC2 chemical "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +773 777 FYSA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +779 785 S1221P mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +786 790 FYPA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +791 796 BRCA2 protein "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +823 827 BRC2 chemical "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +829 833 FYSA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +835 841 S1221Y mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +842 846 FYYA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +847 852 BRCA2 protein "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +879 884 RAD51 protein "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +886 890 FTTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +892 896 A89E mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +897 901 FTTE structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +902 921 Immunoprecipitation experimental_method "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +938 942 BRC4 chemical "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +944 948 FHTA structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +950 956 A1527S mutant "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +957 961 FHTS structure_element "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +978 987 RAD51‐DNA complex_assembly "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +1007 1015 inactive protein_state "Mutation contexta Mutation FxxA motif Technique used Effect RAD51 (FTTA) F86E ETTA Immunoprecipitation 11 No binding BRC4 (FHTA) F1524E EHTA Competitive ELISA 12 Peptide inactive BRC4 (FHTA) F1524W WHTA Competitive ELISA 12 Comparable activity to WT BRC4 (FHTA) F1524V VHTA BRCA2 mutations database 14 – BRC4 (FHTA) ΔF1524 ‐HTA Dissociation of RAD51‐DNA complex 13 Peptide inactive BRC4 (FHTA) H1525Q FQTA Dissociation of RAD51‐DNA complex 13 Comparable activity BRC7 (FSTA) S1979R FRTA BRCA2 mutations database 14 – BRC3 (FQTA) T1430A FQAA RAD51:DNA bandshift assay 3 Peptide inactive BRC3 (FQTA) T1430A FQAA Electron microscopic visualisation of nucleoprotein filaments 3 Peptide inactive BRC1 (FRTA) T1011R FRRA BRCA2 mutations database 14 – BRC2 (FYSA) S1221P FYPA BRCA2 mutations database 14 – BRC2 (FYSA) S1221Y FYYA BRCA2 mutations database 14 – RAD51 (FTTA) A89E FTTE Immunoprecipitation 11 No binding BRC4 (FHTA) A1527S FHTS Dissociation of RAD51‐DNA complex 13 Peptide inactive " TABLE +4 13 wild‐type protein_state The wild‐type FxxA sequence is indicated in parenthesis. TABLE +14 18 FxxA structure_element The wild‐type FxxA sequence is indicated in parenthesis. TABLE +51 71 systematic mutations experimental_method In this work, we report the most detailed study of systematic mutations of peptides to probe the FxxA‐binding motif to date. ABBR +97 115 FxxA‐binding motif structure_element In this work, we report the most detailed study of systematic mutations of peptides to probe the FxxA‐binding motif to date. ABBR +27 40 tetrapeptides chemical We have chosen to focus on tetrapeptides, which allows us to examine the effect of mutation on the fundamental unit of binding, FxxA, rather than in the context of either the BRC repeat or self‐oligomerisation sequence. ABBR +83 91 mutation experimental_method We have chosen to focus on tetrapeptides, which allows us to examine the effect of mutation on the fundamental unit of binding, FxxA, rather than in the context of either the BRC repeat or self‐oligomerisation sequence. ABBR +128 132 FxxA structure_element We have chosen to focus on tetrapeptides, which allows us to examine the effect of mutation on the fundamental unit of binding, FxxA, rather than in the context of either the BRC repeat or self‐oligomerisation sequence. ABBR +175 185 BRC repeat structure_element We have chosen to focus on tetrapeptides, which allows us to examine the effect of mutation on the fundamental unit of binding, FxxA, rather than in the context of either the BRC repeat or self‐oligomerisation sequence. ABBR +189 218 self‐oligomerisation sequence structure_element We have chosen to focus on tetrapeptides, which allows us to examine the effect of mutation on the fundamental unit of binding, FxxA, rather than in the context of either the BRC repeat or self‐oligomerisation sequence. ABBR +0 10 Affinities evidence Affinities of peptides were measured directly using Isothermal Titration Calorimetry (ITC) and the structures of many of the peptides bound to humanised RadA were determined by X‐ray crystallography. ABBR +52 84 Isothermal Titration Calorimetry experimental_method Affinities of peptides were measured directly using Isothermal Titration Calorimetry (ITC) and the structures of many of the peptides bound to humanised RadA were determined by X‐ray crystallography. ABBR +86 89 ITC experimental_method Affinities of peptides were measured directly using Isothermal Titration Calorimetry (ITC) and the structures of many of the peptides bound to humanised RadA were determined by X‐ray crystallography. ABBR +99 109 structures evidence Affinities of peptides were measured directly using Isothermal Titration Calorimetry (ITC) and the structures of many of the peptides bound to humanised RadA were determined by X‐ray crystallography. ABBR +134 142 bound to protein_state Affinities of peptides were measured directly using Isothermal Titration Calorimetry (ITC) and the structures of many of the peptides bound to humanised RadA were determined by X‐ray crystallography. ABBR +143 152 humanised protein_state Affinities of peptides were measured directly using Isothermal Titration Calorimetry (ITC) and the structures of many of the peptides bound to humanised RadA were determined by X‐ray crystallography. ABBR +153 157 RadA protein Affinities of peptides were measured directly using Isothermal Titration Calorimetry (ITC) and the structures of many of the peptides bound to humanised RadA were determined by X‐ray crystallography. ABBR +177 198 X‐ray crystallography experimental_method Affinities of peptides were measured directly using Isothermal Titration Calorimetry (ITC) and the structures of many of the peptides bound to humanised RadA were determined by X‐ray crystallography. ABBR +11 14 ITC experimental_method The use of ITC is generally perceived as a gold‐standard in protein–ligand characterisation, rather than a competitive assay which may be prone to aggregation artefacts. ABBR +107 124 competitive assay experimental_method The use of ITC is generally perceived as a gold‐standard in protein–ligand characterisation, rather than a competitive assay which may be prone to aggregation artefacts. ABBR +0 9 Wild‐type protein_state Wild‐type human RAD51, however, is a heterogeneous mixture of oligomers and when monomerised by mutation, is highly unstable. ABBR +10 15 human species Wild‐type human RAD51, however, is a heterogeneous mixture of oligomers and when monomerised by mutation, is highly unstable. ABBR +16 21 RAD51 protein Wild‐type human RAD51, however, is a heterogeneous mixture of oligomers and when monomerised by mutation, is highly unstable. ABBR +62 71 oligomers oligomeric_state Wild‐type human RAD51, however, is a heterogeneous mixture of oligomers and when monomerised by mutation, is highly unstable. ABBR +81 92 monomerised oligomeric_state Wild‐type human RAD51, however, is a heterogeneous mixture of oligomers and when monomerised by mutation, is highly unstable. ABBR +96 104 mutation experimental_method Wild‐type human RAD51, however, is a heterogeneous mixture of oligomers and when monomerised by mutation, is highly unstable. ABBR +109 124 highly unstable protein_state Wild‐type human RAD51, however, is a heterogeneous mixture of oligomers and when monomerised by mutation, is highly unstable. ABBR +56 62 stable protein_state In this context, we have previously reported the use of stable monomeric forms of RAD51, humanised from Pyrococcus furiosus homologue RadA, for ITC experiments and X‐ray crystallography 8, 15. ABBR +63 72 monomeric oligomeric_state In this context, we have previously reported the use of stable monomeric forms of RAD51, humanised from Pyrococcus furiosus homologue RadA, for ITC experiments and X‐ray crystallography 8, 15. ABBR +82 87 RAD51 protein In this context, we have previously reported the use of stable monomeric forms of RAD51, humanised from Pyrococcus furiosus homologue RadA, for ITC experiments and X‐ray crystallography 8, 15. ABBR +89 98 humanised protein_state In this context, we have previously reported the use of stable monomeric forms of RAD51, humanised from Pyrococcus furiosus homologue RadA, for ITC experiments and X‐ray crystallography 8, 15. ABBR +104 123 Pyrococcus furiosus species In this context, we have previously reported the use of stable monomeric forms of RAD51, humanised from Pyrococcus furiosus homologue RadA, for ITC experiments and X‐ray crystallography 8, 15. ABBR +134 138 RadA protein In this context, we have previously reported the use of stable monomeric forms of RAD51, humanised from Pyrococcus furiosus homologue RadA, for ITC experiments and X‐ray crystallography 8, 15. ABBR +144 147 ITC experimental_method In this context, we have previously reported the use of stable monomeric forms of RAD51, humanised from Pyrococcus furiosus homologue RadA, for ITC experiments and X‐ray crystallography 8, 15. ABBR +164 185 X‐ray crystallography experimental_method In this context, we have previously reported the use of stable monomeric forms of RAD51, humanised from Pyrococcus furiosus homologue RadA, for ITC experiments and X‐ray crystallography 8, 15. ABBR +16 20 FxxA structure_element Conservation of FxxA motif (A) BRC4 peptide (green cartoon) bound to truncated human RAD51 (grey surface) (PDB: 1n0w, 11). FIG +31 35 BRC4 chemical Conservation of FxxA motif (A) BRC4 peptide (green cartoon) bound to truncated human RAD51 (grey surface) (PDB: 1n0w, 11). FIG +60 68 bound to protein_state Conservation of FxxA motif (A) BRC4 peptide (green cartoon) bound to truncated human RAD51 (grey surface) (PDB: 1n0w, 11). FIG +69 78 truncated protein_state Conservation of FxxA motif (A) BRC4 peptide (green cartoon) bound to truncated human RAD51 (grey surface) (PDB: 1n0w, 11). FIG +79 84 human species Conservation of FxxA motif (A) BRC4 peptide (green cartoon) bound to truncated human RAD51 (grey surface) (PDB: 1n0w, 11). FIG +85 90 RAD51 protein Conservation of FxxA motif (A) BRC4 peptide (green cartoon) bound to truncated human RAD51 (grey surface) (PDB: 1n0w, 11). FIG +35 58 FxxA interaction pocket site The blue dashed box highlights the FxxA interaction pocket. FIG +41 46 RAD51 protein (B) Two interacting protein molecules of RAD51 from Saccharomyces cerevisiae are shown. FIG +52 76 Saccharomyces cerevisiae species (B) Two interacting protein molecules of RAD51 from Saccharomyces cerevisiae are shown. FIG +4 9 RAD51 protein One RAD51 (green cartoon) interacts with another molecule of RAD51 (grey and pink surface) via the FxxA pocket indicated by the dashed blue box. FIG +61 66 RAD51 protein One RAD51 (green cartoon) interacts with another molecule of RAD51 (grey and pink surface) via the FxxA pocket indicated by the dashed blue box. FIG +99 110 FxxA pocket site One RAD51 (green cartoon) interacts with another molecule of RAD51 (grey and pink surface) via the FxxA pocket indicated by the dashed blue box. FIG +29 34 RAD51 protein The N‐terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation. FIG +35 43 protomer oligomeric_state The N‐terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation. FIG +130 138 protomer oligomeric_state The N‐terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation. FIG +141 170 FxxA oligomerisation sequence structure_element The N‐terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation. FIG +208 212 FxxA structure_element The N‐terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation. FIG +230 235 human species The N‐terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation. FIG +236 247 BRC repeats structure_element The N‐terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation. FIG +266 276 eukaryotic taxonomy_domain The N‐terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation. FIG +277 283 RAD51s protein_type The N‐terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation. FIG +291 296 RadAs protein_type The N‐terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation. FIG +8 29 mutated and truncated experimental_method We have mutated and truncated the tetrapeptide epitope FHTA, and examined the effects both structurally and on the binding affinity with humanised RadA. As a comparative reference, we are using the FHTA sequence derived from the most tightly binding BRC repeat, BRC4 22. RESULTS +34 46 tetrapeptide chemical We have mutated and truncated the tetrapeptide epitope FHTA, and examined the effects both structurally and on the binding affinity with humanised RadA. As a comparative reference, we are using the FHTA sequence derived from the most tightly binding BRC repeat, BRC4 22. RESULTS +55 59 FHTA structure_element We have mutated and truncated the tetrapeptide epitope FHTA, and examined the effects both structurally and on the binding affinity with humanised RadA. As a comparative reference, we are using the FHTA sequence derived from the most tightly binding BRC repeat, BRC4 22. RESULTS +115 131 binding affinity evidence We have mutated and truncated the tetrapeptide epitope FHTA, and examined the effects both structurally and on the binding affinity with humanised RadA. As a comparative reference, we are using the FHTA sequence derived from the most tightly binding BRC repeat, BRC4 22. RESULTS +137 146 humanised protein_state We have mutated and truncated the tetrapeptide epitope FHTA, and examined the effects both structurally and on the binding affinity with humanised RadA. As a comparative reference, we are using the FHTA sequence derived from the most tightly binding BRC repeat, BRC4 22. RESULTS +147 151 RadA protein We have mutated and truncated the tetrapeptide epitope FHTA, and examined the effects both structurally and on the binding affinity with humanised RadA. As a comparative reference, we are using the FHTA sequence derived from the most tightly binding BRC repeat, BRC4 22. RESULTS +198 202 FHTA structure_element We have mutated and truncated the tetrapeptide epitope FHTA, and examined the effects both structurally and on the binding affinity with humanised RadA. As a comparative reference, we are using the FHTA sequence derived from the most tightly binding BRC repeat, BRC4 22. RESULTS +250 260 BRC repeat structure_element We have mutated and truncated the tetrapeptide epitope FHTA, and examined the effects both structurally and on the binding affinity with humanised RadA. As a comparative reference, we are using the FHTA sequence derived from the most tightly binding BRC repeat, BRC4 22. RESULTS +262 266 BRC4 chemical We have mutated and truncated the tetrapeptide epitope FHTA, and examined the effects both structurally and on the binding affinity with humanised RadA. As a comparative reference, we are using the FHTA sequence derived from the most tightly binding BRC repeat, BRC4 22. RESULTS +22 34 N‐acetylated protein_state The peptides used are N‐acetylated and C‐amide terminated in order to provide the most relevant peptide in the context of a longer peptide chain. RESULTS +49 52 K D evidence A summary of the peptide sequence, PDB codes and K D data measured by ITC with the corresponding ΔH and TΔS values are collated in Table 2. RESULTS +70 73 ITC experimental_method A summary of the peptide sequence, PDB codes and K D data measured by ITC with the corresponding ΔH and TΔS values are collated in Table 2. RESULTS +97 99 ΔH evidence A summary of the peptide sequence, PDB codes and K D data measured by ITC with the corresponding ΔH and TΔS values are collated in Table 2. RESULTS +104 107 TΔS evidence A summary of the peptide sequence, PDB codes and K D data measured by ITC with the corresponding ΔH and TΔS values are collated in Table 2. RESULTS +0 7 Phe1524 residue_name_number Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210. RESULTS +11 15 BRC4 chemical Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210. RESULTS +33 47 surface pocket site Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210. RESULTS +51 56 human species Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210. RESULTS +57 62 RAD51 protein Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210. RESULTS +115 121 Met158 residue_name_number Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210. RESULTS +123 129 Ile160 residue_name_number Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210. RESULTS +131 137 Ala192 residue_name_number Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210. RESULTS +139 145 Leu203 residue_name_number Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210. RESULTS +150 156 Met210 residue_name_number Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210. RESULTS +15 31 highly conserved protein_state The residue is highly conserved across BRC repeats and oligomerisation sequences. RESULTS +39 50 BRC repeats structure_element The residue is highly conserved across BRC repeats and oligomerisation sequences. RESULTS +55 80 oligomerisation sequences structure_element The residue is highly conserved across BRC repeats and oligomerisation sequences. RESULTS +26 35 truncated protein_state Consistent with this, the truncated HTA tripeptide could not be detected to bind to humanised, monomeric RadA, HumRadA2 (Table 2, entry 13). RESULTS +36 39 HTA structure_element Consistent with this, the truncated HTA tripeptide could not be detected to bind to humanised, monomeric RadA, HumRadA2 (Table 2, entry 13). RESULTS +40 50 tripeptide chemical Consistent with this, the truncated HTA tripeptide could not be detected to bind to humanised, monomeric RadA, HumRadA2 (Table 2, entry 13). RESULTS +84 93 humanised protein_state Consistent with this, the truncated HTA tripeptide could not be detected to bind to humanised, monomeric RadA, HumRadA2 (Table 2, entry 13). RESULTS +95 104 monomeric oligomeric_state Consistent with this, the truncated HTA tripeptide could not be detected to bind to humanised, monomeric RadA, HumRadA2 (Table 2, entry 13). RESULTS +105 109 RadA protein Consistent with this, the truncated HTA tripeptide could not be detected to bind to humanised, monomeric RadA, HumRadA2 (Table 2, entry 13). RESULTS +111 119 HumRadA2 mutant Consistent with this, the truncated HTA tripeptide could not be detected to bind to humanised, monomeric RadA, HumRadA2 (Table 2, entry 13). RESULTS +53 65 substituting experimental_method As previously discussed, there is some evidence that substituting a tryptophan for the phenylalanine at this position was tolerated in the context of BRC4 12. RESULTS +68 78 tryptophan residue_name As previously discussed, there is some evidence that substituting a tryptophan for the phenylalanine at this position was tolerated in the context of BRC4 12. RESULTS +87 100 phenylalanine residue_name As previously discussed, there is some evidence that substituting a tryptophan for the phenylalanine at this position was tolerated in the context of BRC4 12. RESULTS +150 154 BRC4 chemical As previously discussed, there is some evidence that substituting a tryptophan for the phenylalanine at this position was tolerated in the context of BRC4 12. RESULTS +15 19 WHTA structure_element Therefore, the WHTA peptide was tested and found to not only be tolerated, but to increase the binding affinity of the peptide approximately threefold. RESULTS +95 111 binding affinity evidence Therefore, the WHTA peptide was tested and found to not only be tolerated, but to increase the binding affinity of the peptide approximately threefold. RESULTS +27 39 tetrapeptide chemical The second position of the tetrapeptide was found to be largely invariant to changes in the side chains that were investigated. RESULTS +43 48 RAD51 protein The residue makes no interactions with the RAD51 protein, but may make an internal hydrogen bond with Thr1520 in the context of BRC4, Fig. 3A. RESULTS +83 96 hydrogen bond bond_interaction The residue makes no interactions with the RAD51 protein, but may make an internal hydrogen bond with Thr1520 in the context of BRC4, Fig. 3A. RESULTS +102 109 Thr1520 residue_name_number The residue makes no interactions with the RAD51 protein, but may make an internal hydrogen bond with Thr1520 in the context of BRC4, Fig. 3A. RESULTS +128 132 BRC4 chemical The residue makes no interactions with the RAD51 protein, but may make an internal hydrogen bond with Thr1520 in the context of BRC4, Fig. 3A. RESULTS +0 9 Replacing experimental_method Replacing the histidine with an asparagine, chosen to potentially mimic the hydrogen bond donor–acceptor nature of histidine, resulted in a moderate, twofold decrease in potency (Table 2, entry 4). RESULTS +14 23 histidine residue_name Replacing the histidine with an asparagine, chosen to potentially mimic the hydrogen bond donor–acceptor nature of histidine, resulted in a moderate, twofold decrease in potency (Table 2, entry 4). RESULTS +32 42 asparagine residue_name Replacing the histidine with an asparagine, chosen to potentially mimic the hydrogen bond donor–acceptor nature of histidine, resulted in a moderate, twofold decrease in potency (Table 2, entry 4). RESULTS +76 89 hydrogen bond bond_interaction Replacing the histidine with an asparagine, chosen to potentially mimic the hydrogen bond donor–acceptor nature of histidine, resulted in a moderate, twofold decrease in potency (Table 2, entry 4). RESULTS +115 124 histidine residue_name Replacing the histidine with an asparagine, chosen to potentially mimic the hydrogen bond donor–acceptor nature of histidine, resulted in a moderate, twofold decrease in potency (Table 2, entry 4). RESULTS +0 8 Mutating experimental_method Mutating to an alanine, recapitulated the potency of FHTA, implying that the interactions made by histidine do not contribute overall to binding affinity (Table 2, entry 3). RESULTS +15 22 alanine residue_name Mutating to an alanine, recapitulated the potency of FHTA, implying that the interactions made by histidine do not contribute overall to binding affinity (Table 2, entry 3). RESULTS +53 57 FHTA structure_element Mutating to an alanine, recapitulated the potency of FHTA, implying that the interactions made by histidine do not contribute overall to binding affinity (Table 2, entry 3). RESULTS +98 107 histidine residue_name Mutating to an alanine, recapitulated the potency of FHTA, implying that the interactions made by histidine do not contribute overall to binding affinity (Table 2, entry 3). RESULTS +137 153 binding affinity evidence Mutating to an alanine, recapitulated the potency of FHTA, implying that the interactions made by histidine do not contribute overall to binding affinity (Table 2, entry 3). RESULTS +0 4 FPTA structure_element FPTA was also tested, but was found to have no affinity for the protein (Table 2, entry 5). RESULTS +47 55 affinity evidence FPTA was also tested, but was found to have no affinity for the protein (Table 2, entry 5). RESULTS +26 33 proline residue_name Modelling suggests that a proline in the second position would be expected to clash sterically with the surface of the protein, and provides a rationale for the lack of binding observed. RESULTS +0 9 Threonine residue_name Threonine was mutated to an alanine, resulting in only a moderately weaker K D (twofold, Table 2, entry 7). RESULTS +14 21 mutated experimental_method Threonine was mutated to an alanine, resulting in only a moderately weaker K D (twofold, Table 2, entry 7). RESULTS +28 35 alanine residue_name Threonine was mutated to an alanine, resulting in only a moderately weaker K D (twofold, Table 2, entry 7). RESULTS +75 78 K D evidence Threonine was mutated to an alanine, resulting in only a moderately weaker K D (twofold, Table 2, entry 7). RESULTS +20 32 tetrapeptide chemical In the context of a tetrapeptide at least, this result implies a lack of importance of a threonine at this position. RESULTS +89 98 threonine residue_name In the context of a tetrapeptide at least, this result implies a lack of importance of a threonine at this position. RESULTS +35 42 proline residue_name Interestingly, it was found that a proline at this position improved the affinity almost threefold, to 113 μm (Table 2, entry 6). RESULTS +73 81 affinity evidence Interestingly, it was found that a proline at this position improved the affinity almost threefold, to 113 μm (Table 2, entry 6). RESULTS +16 24 mutation experimental_method This beneficial mutation was incorporated with another previously identified variant to produce the peptide WHPA. RESULTS +108 112 WHPA structure_element This beneficial mutation was incorporated with another previously identified variant to produce the peptide WHPA. RESULTS +28 41 phenylalanine residue_name While the importance of the phenylalanine may be possible to predict from examination of the crystal structure, the alanine appears to be of much less importance in this regard. RESULTS +93 110 crystal structure evidence While the importance of the phenylalanine may be possible to predict from examination of the crystal structure, the alanine appears to be of much less importance in this regard. RESULTS +116 123 alanine residue_name While the importance of the phenylalanine may be possible to predict from examination of the crystal structure, the alanine appears to be of much less importance in this regard. RESULTS +18 34 highly conserved protein_state It is, however, a highly conserved residue and clearly of interest for systematic mutation. RESULTS +0 8 Removing experimental_method Removing the alanine residue entirely produced the truncated tripeptide FHT, which did not bind (Table 2, entry 12). RESULTS +13 20 alanine residue_name Removing the alanine residue entirely produced the truncated tripeptide FHT, which did not bind (Table 2, entry 12). RESULTS +51 60 truncated protein_state Removing the alanine residue entirely produced the truncated tripeptide FHT, which did not bind (Table 2, entry 12). RESULTS +61 71 tripeptide chemical Removing the alanine residue entirely produced the truncated tripeptide FHT, which did not bind (Table 2, entry 12). RESULTS +72 75 FHT structure_element Removing the alanine residue entirely produced the truncated tripeptide FHT, which did not bind (Table 2, entry 12). RESULTS +26 46 α‐amino butyric acid chemical The unnatural amino acid, α‐amino butyric acid (U), was introduced at the fourth position, positioning an ethyl group into the alanine pocket (Table 2, entry 9). RESULTS +48 49 U chemical The unnatural amino acid, α‐amino butyric acid (U), was introduced at the fourth position, positioning an ethyl group into the alanine pocket (Table 2, entry 9). RESULTS +127 141 alanine pocket site The unnatural amino acid, α‐amino butyric acid (U), was introduced at the fourth position, positioning an ethyl group into the alanine pocket (Table 2, entry 9). RESULTS +50 58 affinity evidence Perhaps surprisingly, it was accommodated and the affinity dropped only by twofold as compared to FHTA. RESULTS +98 102 FHTA structure_element Perhaps surprisingly, it was accommodated and the affinity dropped only by twofold as compared to FHTA. RESULTS +21 29 removing experimental_method The effect of simply removing the β‐carbon of alanine, by mutation to glycine (FHTG), produced an approximately sixfold drop in binding affinity (Table 2, entry 8). RESULTS +46 53 alanine residue_name The effect of simply removing the β‐carbon of alanine, by mutation to glycine (FHTG), produced an approximately sixfold drop in binding affinity (Table 2, entry 8). RESULTS +58 69 mutation to experimental_method The effect of simply removing the β‐carbon of alanine, by mutation to glycine (FHTG), produced an approximately sixfold drop in binding affinity (Table 2, entry 8). RESULTS +70 77 glycine residue_name The effect of simply removing the β‐carbon of alanine, by mutation to glycine (FHTG), produced an approximately sixfold drop in binding affinity (Table 2, entry 8). RESULTS +79 83 FHTG structure_element The effect of simply removing the β‐carbon of alanine, by mutation to glycine (FHTG), produced an approximately sixfold drop in binding affinity (Table 2, entry 8). RESULTS +128 144 binding affinity evidence The effect of simply removing the β‐carbon of alanine, by mutation to glycine (FHTG), produced an approximately sixfold drop in binding affinity (Table 2, entry 8). RESULTS +42 49 alanine residue_name This is in line with the observation that alanine is not 100% conserved and some archeal RadA proteins contain a glycine in the place of alanine 23. RESULTS +53 71 not 100% conserved protein_state This is in line with the observation that alanine is not 100% conserved and some archeal RadA proteins contain a glycine in the place of alanine 23. RESULTS +81 88 archeal taxonomy_domain This is in line with the observation that alanine is not 100% conserved and some archeal RadA proteins contain a glycine in the place of alanine 23. RESULTS +89 102 RadA proteins protein_type This is in line with the observation that alanine is not 100% conserved and some archeal RadA proteins contain a glycine in the place of alanine 23. RESULTS +113 120 glycine residue_name This is in line with the observation that alanine is not 100% conserved and some archeal RadA proteins contain a glycine in the place of alanine 23. RESULTS +137 144 alanine residue_name This is in line with the observation that alanine is not 100% conserved and some archeal RadA proteins contain a glycine in the place of alanine 23. RESULTS +0 27 Structural characterisation experimental_method Structural characterisation of peptide complexes RESULTS +0 10 Structures evidence Structures of the key tetrapeptides were solved by soaking into crystals of a humanised form of RAD51, HumRadA1, which we have previously reported as a convenient surrogate system for RAD51 crystallography 15. RESULTS +22 35 tetrapeptides chemical Structures of the key tetrapeptides were solved by soaking into crystals of a humanised form of RAD51, HumRadA1, which we have previously reported as a convenient surrogate system for RAD51 crystallography 15. RESULTS +51 63 soaking into experimental_method Structures of the key tetrapeptides were solved by soaking into crystals of a humanised form of RAD51, HumRadA1, which we have previously reported as a convenient surrogate system for RAD51 crystallography 15. RESULTS +64 72 crystals evidence Structures of the key tetrapeptides were solved by soaking into crystals of a humanised form of RAD51, HumRadA1, which we have previously reported as a convenient surrogate system for RAD51 crystallography 15. RESULTS +78 87 humanised protein_state Structures of the key tetrapeptides were solved by soaking into crystals of a humanised form of RAD51, HumRadA1, which we have previously reported as a convenient surrogate system for RAD51 crystallography 15. RESULTS +96 101 RAD51 protein Structures of the key tetrapeptides were solved by soaking into crystals of a humanised form of RAD51, HumRadA1, which we have previously reported as a convenient surrogate system for RAD51 crystallography 15. RESULTS +103 111 HumRadA1 mutant Structures of the key tetrapeptides were solved by soaking into crystals of a humanised form of RAD51, HumRadA1, which we have previously reported as a convenient surrogate system for RAD51 crystallography 15. RESULTS +184 189 RAD51 protein Structures of the key tetrapeptides were solved by soaking into crystals of a humanised form of RAD51, HumRadA1, which we have previously reported as a convenient surrogate system for RAD51 crystallography 15. RESULTS +190 205 crystallography experimental_method Structures of the key tetrapeptides were solved by soaking into crystals of a humanised form of RAD51, HumRadA1, which we have previously reported as a convenient surrogate system for RAD51 crystallography 15. RESULTS +57 78 crystallographic data evidence The corresponding PDB codes are indicated in Table 2 and crystallographic data are found in the Supporting Information. RESULTS +4 14 structures evidence All structures are of high resolution (1.2–1.7 Å) and the electron density for the peptide was clearly visible after the first refinement using unliganded RadA coordinates (Fig. S1). RESULTS +58 74 electron density evidence All structures are of high resolution (1.2–1.7 Å) and the electron density for the peptide was clearly visible after the first refinement using unliganded RadA coordinates (Fig. S1). RESULTS +144 154 unliganded protein_state All structures are of high resolution (1.2–1.7 Å) and the electron density for the peptide was clearly visible after the first refinement using unliganded RadA coordinates (Fig. S1). RESULTS +155 159 RadA protein All structures are of high resolution (1.2–1.7 Å) and the electron density for the peptide was clearly visible after the first refinement using unliganded RadA coordinates (Fig. S1). RESULTS +32 48 binding analysis experimental_method Some of the SAR observed in the binding analysis can be interpreted in terms of these X‐ray crystal structures. RESULTS +86 91 X‐ray experimental_method Some of the SAR observed in the binding analysis can be interpreted in terms of these X‐ray crystal structures. RESULTS +92 110 crystal structures evidence Some of the SAR observed in the binding analysis can be interpreted in terms of these X‐ray crystal structures. RESULTS +16 23 overlay experimental_method For example, an overlay of the bound poses of the ligands FHTA and FHPA (Fig. 2B) reveals a high similarity in the binding modes, indicating that the conformational rigidity conferred by the proline is compatible with the FHTA‐binding mode, and a reduction in an entropic penalty of binding may be the source of the improvement in affinity. RESULTS +58 62 FHTA structure_element For example, an overlay of the bound poses of the ligands FHTA and FHPA (Fig. 2B) reveals a high similarity in the binding modes, indicating that the conformational rigidity conferred by the proline is compatible with the FHTA‐binding mode, and a reduction in an entropic penalty of binding may be the source of the improvement in affinity. RESULTS +67 71 FHPA structure_element For example, an overlay of the bound poses of the ligands FHTA and FHPA (Fig. 2B) reveals a high similarity in the binding modes, indicating that the conformational rigidity conferred by the proline is compatible with the FHTA‐binding mode, and a reduction in an entropic penalty of binding may be the source of the improvement in affinity. RESULTS +191 198 proline residue_name For example, an overlay of the bound poses of the ligands FHTA and FHPA (Fig. 2B) reveals a high similarity in the binding modes, indicating that the conformational rigidity conferred by the proline is compatible with the FHTA‐binding mode, and a reduction in an entropic penalty of binding may be the source of the improvement in affinity. RESULTS +222 226 FHTA structure_element For example, an overlay of the bound poses of the ligands FHTA and FHPA (Fig. 2B) reveals a high similarity in the binding modes, indicating that the conformational rigidity conferred by the proline is compatible with the FHTA‐binding mode, and a reduction in an entropic penalty of binding may be the source of the improvement in affinity. RESULTS +263 279 entropic penalty evidence For example, an overlay of the bound poses of the ligands FHTA and FHPA (Fig. 2B) reveals a high similarity in the binding modes, indicating that the conformational rigidity conferred by the proline is compatible with the FHTA‐binding mode, and a reduction in an entropic penalty of binding may be the source of the improvement in affinity. RESULTS +331 339 affinity evidence For example, an overlay of the bound poses of the ligands FHTA and FHPA (Fig. 2B) reveals a high similarity in the binding modes, indicating that the conformational rigidity conferred by the proline is compatible with the FHTA‐binding mode, and a reduction in an entropic penalty of binding may be the source of the improvement in affinity. RESULTS +0 4 WHTA structure_element WHTA peptide shows a relative dislocation when compared to FHTA (Fig 2A), with the entire ligand backbone of WHTA shifted to accommodate the change in the position of the main chain carbon of the first residue, as the larger indole side chain fills the Phe pocket. RESULTS +59 63 FHTA structure_element WHTA peptide shows a relative dislocation when compared to FHTA (Fig 2A), with the entire ligand backbone of WHTA shifted to accommodate the change in the position of the main chain carbon of the first residue, as the larger indole side chain fills the Phe pocket. RESULTS +109 113 WHTA structure_element WHTA peptide shows a relative dislocation when compared to FHTA (Fig 2A), with the entire ligand backbone of WHTA shifted to accommodate the change in the position of the main chain carbon of the first residue, as the larger indole side chain fills the Phe pocket. RESULTS +253 263 Phe pocket site WHTA peptide shows a relative dislocation when compared to FHTA (Fig 2A), with the entire ligand backbone of WHTA shifted to accommodate the change in the position of the main chain carbon of the first residue, as the larger indole side chain fills the Phe pocket. RESULTS +44 51 alanine residue_name This shift is translated all the way to the alanine side chain. RESULTS +25 33 mutation experimental_method It is possible that this mutation is beneficial in the tetrapeptide context and neutral in the full‐length BRC4 context because the smaller peptide is less constrained and allowed to explore more conformations. RESULTS +55 67 tetrapeptide chemical It is possible that this mutation is beneficial in the tetrapeptide context and neutral in the full‐length BRC4 context because the smaller peptide is less constrained and allowed to explore more conformations. RESULTS +95 106 full‐length protein_state It is possible that this mutation is beneficial in the tetrapeptide context and neutral in the full‐length BRC4 context because the smaller peptide is less constrained and allowed to explore more conformations. RESULTS +107 111 BRC4 chemical It is possible that this mutation is beneficial in the tetrapeptide context and neutral in the full‐length BRC4 context because the smaller peptide is less constrained and allowed to explore more conformations. RESULTS +31 41 tryptophan residue_name An attempt to combine both the tryptophan and proline mutations, however, led to no improvement for WHPA peptide compared to FHTA. RESULTS +46 53 proline residue_name An attempt to combine both the tryptophan and proline mutations, however, led to no improvement for WHPA peptide compared to FHTA. RESULTS +54 63 mutations experimental_method An attempt to combine both the tryptophan and proline mutations, however, led to no improvement for WHPA peptide compared to FHTA. RESULTS +100 104 WHPA structure_element An attempt to combine both the tryptophan and proline mutations, however, led to no improvement for WHPA peptide compared to FHTA. RESULTS +125 129 FHTA structure_element An attempt to combine both the tryptophan and proline mutations, however, led to no improvement for WHPA peptide compared to FHTA. RESULTS +72 76 WHTA structure_element One possible explanation is that the ‘shifted’ binding mode observed in WHTA was not compatible with the conformational restriction that the proline of WHPA introduced. RESULTS +141 148 proline residue_name One possible explanation is that the ‘shifted’ binding mode observed in WHTA was not compatible with the conformational restriction that the proline of WHPA introduced. RESULTS +152 156 WHPA structure_element One possible explanation is that the ‘shifted’ binding mode observed in WHTA was not compatible with the conformational restriction that the proline of WHPA introduced. RESULTS +46 53 Overlay experimental_method Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan). FIG +59 63 FHTA structure_element Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan). FIG +75 79 WHTA structure_element Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan). FIG +156 169 Superposition experimental_method Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan). FIG +173 177 FHTA structure_element Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan). FIG +189 193 FHPA structure_element Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan). FIG +253 260 Overlay experimental_method Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan). FIG +264 268 FHTU structure_element Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan). FIG +278 282 FHTA structure_element Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan). FIG +294 298 FHTG structure_element Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan). FIG +4 22 thermodynamic data evidence The thermodynamic data of peptide binding are also shown in Table 2. Although we have both thermodynamic data and high‐quality X‐ray structural information for some of the mutant peptides, we do not attempt to interpret differences in thermodynamic profiles between ligands, that is, to analyse ΔΔH and ΔΔS. RESULTS +91 109 thermodynamic data evidence The thermodynamic data of peptide binding are also shown in Table 2. Although we have both thermodynamic data and high‐quality X‐ray structural information for some of the mutant peptides, we do not attempt to interpret differences in thermodynamic profiles between ligands, that is, to analyse ΔΔH and ΔΔS. RESULTS +127 132 X‐ray experimental_method The thermodynamic data of peptide binding are also shown in Table 2. Although we have both thermodynamic data and high‐quality X‐ray structural information for some of the mutant peptides, we do not attempt to interpret differences in thermodynamic profiles between ligands, that is, to analyse ΔΔH and ΔΔS. RESULTS +133 155 structural information evidence The thermodynamic data of peptide binding are also shown in Table 2. Although we have both thermodynamic data and high‐quality X‐ray structural information for some of the mutant peptides, we do not attempt to interpret differences in thermodynamic profiles between ligands, that is, to analyse ΔΔH and ΔΔS. RESULTS +172 178 mutant protein_state The thermodynamic data of peptide binding are also shown in Table 2. Although we have both thermodynamic data and high‐quality X‐ray structural information for some of the mutant peptides, we do not attempt to interpret differences in thermodynamic profiles between ligands, that is, to analyse ΔΔH and ΔΔS. RESULTS +179 187 peptides chemical The thermodynamic data of peptide binding are also shown in Table 2. Although we have both thermodynamic data and high‐quality X‐ray structural information for some of the mutant peptides, we do not attempt to interpret differences in thermodynamic profiles between ligands, that is, to analyse ΔΔH and ΔΔS. RESULTS +235 257 thermodynamic profiles evidence The thermodynamic data of peptide binding are also shown in Table 2. Although we have both thermodynamic data and high‐quality X‐ray structural information for some of the mutant peptides, we do not attempt to interpret differences in thermodynamic profiles between ligands, that is, to analyse ΔΔH and ΔΔS. RESULTS +295 298 ΔΔH evidence The thermodynamic data of peptide binding are also shown in Table 2. Although we have both thermodynamic data and high‐quality X‐ray structural information for some of the mutant peptides, we do not attempt to interpret differences in thermodynamic profiles between ligands, that is, to analyse ΔΔH and ΔΔS. RESULTS +303 306 ΔΔS evidence The thermodynamic data of peptide binding are also shown in Table 2. Although we have both thermodynamic data and high‐quality X‐ray structural information for some of the mutant peptides, we do not attempt to interpret differences in thermodynamic profiles between ligands, that is, to analyse ΔΔH and ΔΔS. RESULTS +9 11 ΔH evidence Although ΔH and ΔS are tabulated, the K Ds measured are relatively weak and necessarily performed under low c‐value conditions. RESULTS +16 18 ΔS evidence Although ΔH and ΔS are tabulated, the K Ds measured are relatively weak and necessarily performed under low c‐value conditions. RESULTS +38 42 K Ds evidence Although ΔH and ΔS are tabulated, the K Ds measured are relatively weak and necessarily performed under low c‐value conditions. RESULTS +87 89 ΔH evidence In this experimental regime, nonsigmoidal curves are generated and therefore errors in ΔH are expected to be much higher than the errors from model fitting given in Table 2 16. RESULTS +3 5 ΔS evidence As ΔS is derived from ΔG by subtracting ΔH, errors in ΔH will be correlated with errors in ΔS, giving rise to a ‘phantom’ enthalpy–entropy compensation. RESULTS +22 24 ΔG evidence As ΔS is derived from ΔG by subtracting ΔH, errors in ΔH will be correlated with errors in ΔS, giving rise to a ‘phantom’ enthalpy–entropy compensation. RESULTS +40 42 ΔH evidence As ΔS is derived from ΔG by subtracting ΔH, errors in ΔH will be correlated with errors in ΔS, giving rise to a ‘phantom’ enthalpy–entropy compensation. RESULTS +54 56 ΔH evidence As ΔS is derived from ΔG by subtracting ΔH, errors in ΔH will be correlated with errors in ΔS, giving rise to a ‘phantom’ enthalpy–entropy compensation. RESULTS +91 93 ΔS evidence As ΔS is derived from ΔG by subtracting ΔH, errors in ΔH will be correlated with errors in ΔS, giving rise to a ‘phantom’ enthalpy–entropy compensation. RESULTS +125 128 ΔΔH evidence Such effects have been discussed by Klebe 24 and Chodera and Mobley 25 and will frustrate attempts to interpret the measured ΔΔH and ΔΔS. RESULTS +133 136 ΔΔS evidence Such effects have been discussed by Klebe 24 and Chodera and Mobley 25 and will frustrate attempts to interpret the measured ΔΔH and ΔΔS. RESULTS +4 13 conserved protein_state The conserved phenylalanine and alanine residues of the FHTA sequence were both found to be essential for binding by ITC. RESULTS +14 27 phenylalanine residue_name The conserved phenylalanine and alanine residues of the FHTA sequence were both found to be essential for binding by ITC. RESULTS +32 39 alanine residue_name The conserved phenylalanine and alanine residues of the FHTA sequence were both found to be essential for binding by ITC. RESULTS +56 60 FHTA structure_element The conserved phenylalanine and alanine residues of the FHTA sequence were both found to be essential for binding by ITC. RESULTS +117 120 ITC experimental_method The conserved phenylalanine and alanine residues of the FHTA sequence were both found to be essential for binding by ITC. RESULTS +31 40 histidine residue_name Conversely the second position histidine residue, corresponding to the unconserved His1525 in the BRC4 sequence, could be mutated without significant effect on the peptide affinity. RESULTS +71 82 unconserved protein_state Conversely the second position histidine residue, corresponding to the unconserved His1525 in the BRC4 sequence, could be mutated without significant effect on the peptide affinity. RESULTS +83 90 His1525 residue_name_number Conversely the second position histidine residue, corresponding to the unconserved His1525 in the BRC4 sequence, could be mutated without significant effect on the peptide affinity. RESULTS +98 102 BRC4 chemical Conversely the second position histidine residue, corresponding to the unconserved His1525 in the BRC4 sequence, could be mutated without significant effect on the peptide affinity. RESULTS +122 129 mutated experimental_method Conversely the second position histidine residue, corresponding to the unconserved His1525 in the BRC4 sequence, could be mutated without significant effect on the peptide affinity. RESULTS +164 180 peptide affinity evidence Conversely the second position histidine residue, corresponding to the unconserved His1525 in the BRC4 sequence, could be mutated without significant effect on the peptide affinity. RESULTS +37 45 hot‐spot site The more general correlation between hot‐spot residues in protein–protein interactions and the high conservation of such residues has been previously reported 10, 26. RESULTS +95 112 high conservation protein_state The more general correlation between hot‐spot residues in protein–protein interactions and the high conservation of such residues has been previously reported 10, 26. RESULTS +28 44 highly conserved protein_state Interestingly, however, the highly conserved threonine residue could be mutated without affecting the peptide affinity. RESULTS +45 54 threonine residue_name Interestingly, however, the highly conserved threonine residue could be mutated without affecting the peptide affinity. RESULTS +72 79 mutated experimental_method Interestingly, however, the highly conserved threonine residue could be mutated without affecting the peptide affinity. RESULTS +102 118 peptide affinity evidence Interestingly, however, the highly conserved threonine residue could be mutated without affecting the peptide affinity. RESULTS +49 66 high conservation protein_state This unexpected result, in the light of its very high conservation in the BRC and oligomerisation sequences, begs the question of what the role of Thr1526 is and highlights a potential pitfall and need for caution in the experimental design of alanine mutation studies. RESULTS +74 77 BRC structure_element This unexpected result, in the light of its very high conservation in the BRC and oligomerisation sequences, begs the question of what the role of Thr1526 is and highlights a potential pitfall and need for caution in the experimental design of alanine mutation studies. RESULTS +82 107 oligomerisation sequences structure_element This unexpected result, in the light of its very high conservation in the BRC and oligomerisation sequences, begs the question of what the role of Thr1526 is and highlights a potential pitfall and need for caution in the experimental design of alanine mutation studies. RESULTS +147 154 Thr1526 residue_name_number This unexpected result, in the light of its very high conservation in the BRC and oligomerisation sequences, begs the question of what the role of Thr1526 is and highlights a potential pitfall and need for caution in the experimental design of alanine mutation studies. RESULTS +244 268 alanine mutation studies experimental_method This unexpected result, in the light of its very high conservation in the BRC and oligomerisation sequences, begs the question of what the role of Thr1526 is and highlights a potential pitfall and need for caution in the experimental design of alanine mutation studies. RESULTS +7 11 FHTA structure_element As the FHTA peptide is potentially a surrogate peptide for both the BRC repeat peptides and the RAD51 self‐oligomerisation peptide, it is useful to examine the role of Thr1526 (BRC4) and the analogous Thr87 (RAD51) in both binding contexts in more detail. RESULTS +12 19 peptide chemical As the FHTA peptide is potentially a surrogate peptide for both the BRC repeat peptides and the RAD51 self‐oligomerisation peptide, it is useful to examine the role of Thr1526 (BRC4) and the analogous Thr87 (RAD51) in both binding contexts in more detail. RESULTS +68 78 BRC repeat structure_element As the FHTA peptide is potentially a surrogate peptide for both the BRC repeat peptides and the RAD51 self‐oligomerisation peptide, it is useful to examine the role of Thr1526 (BRC4) and the analogous Thr87 (RAD51) in both binding contexts in more detail. RESULTS +96 101 RAD51 protein As the FHTA peptide is potentially a surrogate peptide for both the BRC repeat peptides and the RAD51 self‐oligomerisation peptide, it is useful to examine the role of Thr1526 (BRC4) and the analogous Thr87 (RAD51) in both binding contexts in more detail. RESULTS +102 130 self‐oligomerisation peptide structure_element As the FHTA peptide is potentially a surrogate peptide for both the BRC repeat peptides and the RAD51 self‐oligomerisation peptide, it is useful to examine the role of Thr1526 (BRC4) and the analogous Thr87 (RAD51) in both binding contexts in more detail. RESULTS +168 175 Thr1526 residue_name_number As the FHTA peptide is potentially a surrogate peptide for both the BRC repeat peptides and the RAD51 self‐oligomerisation peptide, it is useful to examine the role of Thr1526 (BRC4) and the analogous Thr87 (RAD51) in both binding contexts in more detail. RESULTS +177 181 BRC4 chemical As the FHTA peptide is potentially a surrogate peptide for both the BRC repeat peptides and the RAD51 self‐oligomerisation peptide, it is useful to examine the role of Thr1526 (BRC4) and the analogous Thr87 (RAD51) in both binding contexts in more detail. RESULTS +201 206 Thr87 residue_name_number As the FHTA peptide is potentially a surrogate peptide for both the BRC repeat peptides and the RAD51 self‐oligomerisation peptide, it is useful to examine the role of Thr1526 (BRC4) and the analogous Thr87 (RAD51) in both binding contexts in more detail. RESULTS +208 213 RAD51 protein As the FHTA peptide is potentially a surrogate peptide for both the BRC repeat peptides and the RAD51 self‐oligomerisation peptide, it is useful to examine the role of Thr1526 (BRC4) and the analogous Thr87 (RAD51) in both binding contexts in more detail. RESULTS +9 18 structure evidence Only one structure of BRC4 is published in complex with human RAD51 (PDB: 1n0w). RESULTS +22 26 BRC4 chemical Only one structure of BRC4 is published in complex with human RAD51 (PDB: 1n0w). RESULTS +40 55 in complex with protein_state Only one structure of BRC4 is published in complex with human RAD51 (PDB: 1n0w). RESULTS +56 61 human species Only one structure of BRC4 is published in complex with human RAD51 (PDB: 1n0w). RESULTS +62 67 RAD51 protein Only one structure of BRC4 is published in complex with human RAD51 (PDB: 1n0w). RESULTS +36 40 BRC4 chemical Figure 3A shows the binding pose of BRC4 when bound to RAD51 and the intrapeptide hydrogen bonds that are made by BRC4. RESULTS +46 54 bound to protein_state Figure 3A shows the binding pose of BRC4 when bound to RAD51 and the intrapeptide hydrogen bonds that are made by BRC4. RESULTS +55 60 RAD51 protein Figure 3A shows the binding pose of BRC4 when bound to RAD51 and the intrapeptide hydrogen bonds that are made by BRC4. RESULTS +82 96 hydrogen bonds bond_interaction Figure 3A shows the binding pose of BRC4 when bound to RAD51 and the intrapeptide hydrogen bonds that are made by BRC4. RESULTS +114 118 BRC4 chemical Figure 3A shows the binding pose of BRC4 when bound to RAD51 and the intrapeptide hydrogen bonds that are made by BRC4. RESULTS +6 13 Phe1524 residue_name_number While Phe1524 and Ala1527 are buried in hydrophobic pockets on the surface, His1525 is close enough to form a hydrogen bond with the carbonyl of Thr1520, but the rotamer of His1525, supported by clearly positioned water molecules, is not compatible with hydrogen bonding. RESULTS +18 25 Ala1527 residue_name_number While Phe1524 and Ala1527 are buried in hydrophobic pockets on the surface, His1525 is close enough to form a hydrogen bond with the carbonyl of Thr1520, but the rotamer of His1525, supported by clearly positioned water molecules, is not compatible with hydrogen bonding. RESULTS +40 59 hydrophobic pockets site While Phe1524 and Ala1527 are buried in hydrophobic pockets on the surface, His1525 is close enough to form a hydrogen bond with the carbonyl of Thr1520, but the rotamer of His1525, supported by clearly positioned water molecules, is not compatible with hydrogen bonding. RESULTS +76 83 His1525 residue_name_number While Phe1524 and Ala1527 are buried in hydrophobic pockets on the surface, His1525 is close enough to form a hydrogen bond with the carbonyl of Thr1520, but the rotamer of His1525, supported by clearly positioned water molecules, is not compatible with hydrogen bonding. RESULTS +110 123 hydrogen bond bond_interaction While Phe1524 and Ala1527 are buried in hydrophobic pockets on the surface, His1525 is close enough to form a hydrogen bond with the carbonyl of Thr1520, but the rotamer of His1525, supported by clearly positioned water molecules, is not compatible with hydrogen bonding. RESULTS +145 152 Thr1520 residue_name_number While Phe1524 and Ala1527 are buried in hydrophobic pockets on the surface, His1525 is close enough to form a hydrogen bond with the carbonyl of Thr1520, but the rotamer of His1525, supported by clearly positioned water molecules, is not compatible with hydrogen bonding. RESULTS +173 180 His1525 residue_name_number While Phe1524 and Ala1527 are buried in hydrophobic pockets on the surface, His1525 is close enough to form a hydrogen bond with the carbonyl of Thr1520, but the rotamer of His1525, supported by clearly positioned water molecules, is not compatible with hydrogen bonding. RESULTS +214 219 water chemical While Phe1524 and Ala1527 are buried in hydrophobic pockets on the surface, His1525 is close enough to form a hydrogen bond with the carbonyl of Thr1520, but the rotamer of His1525, supported by clearly positioned water molecules, is not compatible with hydrogen bonding. RESULTS +254 270 hydrogen bonding bond_interaction While Phe1524 and Ala1527 are buried in hydrophobic pockets on the surface, His1525 is close enough to form a hydrogen bond with the carbonyl of Thr1520, but the rotamer of His1525, supported by clearly positioned water molecules, is not compatible with hydrogen bonding. RESULTS +6 13 Thr1520 residue_name_number Also, Thr1520 is constrained by crystal contacts in this structure. RESULTS +57 66 structure evidence Also, Thr1520 is constrained by crystal contacts in this structure. RESULTS +0 20 Lack of conservation protein_state Lack of conservation of this residue supports the idea that this interaction is not crucial for RAD51:BRC repeat binding. RESULTS +96 112 RAD51:BRC repeat complex_assembly Lack of conservation of this residue supports the idea that this interaction is not crucial for RAD51:BRC repeat binding. RESULTS +23 27 BRC4 chemical (A) Highlight of intra‐BRC4 interactions when bound to RAD51 (omitted for clarity) (PDB: 1n0w), with key residues shown in colour. (B) Intrapeptide interactions from oligomerisation epitope of S. cerevisiae RAD51 when bound to next RAD51 in the filament (PDB: 1szp). FIG +46 54 bound to protein_state (A) Highlight of intra‐BRC4 interactions when bound to RAD51 (omitted for clarity) (PDB: 1n0w), with key residues shown in colour. (B) Intrapeptide interactions from oligomerisation epitope of S. cerevisiae RAD51 when bound to next RAD51 in the filament (PDB: 1szp). FIG +55 60 RAD51 protein (A) Highlight of intra‐BRC4 interactions when bound to RAD51 (omitted for clarity) (PDB: 1n0w), with key residues shown in colour. (B) Intrapeptide interactions from oligomerisation epitope of S. cerevisiae RAD51 when bound to next RAD51 in the filament (PDB: 1szp). FIG +166 189 oligomerisation epitope structure_element (A) Highlight of intra‐BRC4 interactions when bound to RAD51 (omitted for clarity) (PDB: 1n0w), with key residues shown in colour. (B) Intrapeptide interactions from oligomerisation epitope of S. cerevisiae RAD51 when bound to next RAD51 in the filament (PDB: 1szp). FIG +193 206 S. cerevisiae species (A) Highlight of intra‐BRC4 interactions when bound to RAD51 (omitted for clarity) (PDB: 1n0w), with key residues shown in colour. (B) Intrapeptide interactions from oligomerisation epitope of S. cerevisiae RAD51 when bound to next RAD51 in the filament (PDB: 1szp). FIG +208 213 RAD51 protein (A) Highlight of intra‐BRC4 interactions when bound to RAD51 (omitted for clarity) (PDB: 1n0w), with key residues shown in colour. (B) Intrapeptide interactions from oligomerisation epitope of S. cerevisiae RAD51 when bound to next RAD51 in the filament (PDB: 1szp). FIG +219 227 bound to protein_state (A) Highlight of intra‐BRC4 interactions when bound to RAD51 (omitted for clarity) (PDB: 1n0w), with key residues shown in colour. (B) Intrapeptide interactions from oligomerisation epitope of S. cerevisiae RAD51 when bound to next RAD51 in the filament (PDB: 1szp). FIG +233 238 RAD51 protein (A) Highlight of intra‐BRC4 interactions when bound to RAD51 (omitted for clarity) (PDB: 1n0w), with key residues shown in colour. (B) Intrapeptide interactions from oligomerisation epitope of S. cerevisiae RAD51 when bound to next RAD51 in the filament (PDB: 1szp). FIG +33 46 S. cerevisiae species Residue numbering relates to the S. cerevisiae RAD51 protein, the corresponding human residues are in parentheses. FIG +48 53 RAD51 protein Residue numbering relates to the S. cerevisiae RAD51 protein, the corresponding human residues are in parentheses. FIG +81 86 human species Residue numbering relates to the S. cerevisiae RAD51 protein, the corresponding human residues are in parentheses. FIG +9 18 threonine residue_name Either a threonine or serine is most commonly found in the third position of the FxxA motif. RESULTS +22 28 serine residue_name Either a threonine or serine is most commonly found in the third position of the FxxA motif. RESULTS +81 85 FxxA structure_element Either a threonine or serine is most commonly found in the third position of the FxxA motif. RESULTS +0 7 Thr1526 residue_name_number Thr1526 makes no direct interactions with the RAD51 protein, but instead forms a hydrogen bond network with the highly conserved S1528 and K1530 (Fig. 1C). RESULTS +46 51 RAD51 protein Thr1526 makes no direct interactions with the RAD51 protein, but instead forms a hydrogen bond network with the highly conserved S1528 and K1530 (Fig. 1C). RESULTS +81 102 hydrogen bond network bond_interaction Thr1526 makes no direct interactions with the RAD51 protein, but instead forms a hydrogen bond network with the highly conserved S1528 and K1530 (Fig. 1C). RESULTS +112 128 highly conserved protein_state Thr1526 makes no direct interactions with the RAD51 protein, but instead forms a hydrogen bond network with the highly conserved S1528 and K1530 (Fig. 1C). RESULTS +129 134 S1528 residue_name_number Thr1526 makes no direct interactions with the RAD51 protein, but instead forms a hydrogen bond network with the highly conserved S1528 and K1530 (Fig. 1C). RESULTS +139 144 K1530 residue_name_number Thr1526 makes no direct interactions with the RAD51 protein, but instead forms a hydrogen bond network with the highly conserved S1528 and K1530 (Fig. 1C). RESULTS +4 31 high degree of conservation protein_state The high degree of conservation of these three residues suggests an important possible role in facilitating a turn and stabilising the conformation of the peptide as it continues its way to a second interaction site on the side of RAD51. RESULTS +199 215 interaction site site The high degree of conservation of these three residues suggests an important possible role in facilitating a turn and stabilising the conformation of the peptide as it continues its way to a second interaction site on the side of RAD51. RESULTS +231 236 RAD51 protein The high degree of conservation of these three residues suggests an important possible role in facilitating a turn and stabilising the conformation of the peptide as it continues its way to a second interaction site on the side of RAD51. RESULTS +34 45 RAD51:RAD51 complex_assembly With respect to understanding the RAD51:RAD51 interaction, no human crystal structure has been published, however, several oligomeric structures of archaeal RadA as well that of Saccharomyces cerevisiae RAD51 have been reported 27, 28, 29. RESULTS +62 67 human species With respect to understanding the RAD51:RAD51 interaction, no human crystal structure has been published, however, several oligomeric structures of archaeal RadA as well that of Saccharomyces cerevisiae RAD51 have been reported 27, 28, 29. RESULTS +68 85 crystal structure evidence With respect to understanding the RAD51:RAD51 interaction, no human crystal structure has been published, however, several oligomeric structures of archaeal RadA as well that of Saccharomyces cerevisiae RAD51 have been reported 27, 28, 29. RESULTS +134 144 structures evidence With respect to understanding the RAD51:RAD51 interaction, no human crystal structure has been published, however, several oligomeric structures of archaeal RadA as well that of Saccharomyces cerevisiae RAD51 have been reported 27, 28, 29. RESULTS +148 156 archaeal taxonomy_domain With respect to understanding the RAD51:RAD51 interaction, no human crystal structure has been published, however, several oligomeric structures of archaeal RadA as well that of Saccharomyces cerevisiae RAD51 have been reported 27, 28, 29. RESULTS +157 161 RadA protein With respect to understanding the RAD51:RAD51 interaction, no human crystal structure has been published, however, several oligomeric structures of archaeal RadA as well that of Saccharomyces cerevisiae RAD51 have been reported 27, 28, 29. RESULTS +178 202 Saccharomyces cerevisiae species With respect to understanding the RAD51:RAD51 interaction, no human crystal structure has been published, however, several oligomeric structures of archaeal RadA as well that of Saccharomyces cerevisiae RAD51 have been reported 27, 28, 29. RESULTS +203 208 RAD51 protein With respect to understanding the RAD51:RAD51 interaction, no human crystal structure has been published, however, several oligomeric structures of archaeal RadA as well that of Saccharomyces cerevisiae RAD51 have been reported 27, 28, 29. RESULTS +35 39 FxxA structure_element Figure 3B shows a highlight of the FxxA portion of oligomerisation peptide from the S. cerevisiae RAD51 structure, with residues in parentheses corresponding to the human RAD51 protein. RESULTS +51 74 oligomerisation peptide structure_element Figure 3B shows a highlight of the FxxA portion of oligomerisation peptide from the S. cerevisiae RAD51 structure, with residues in parentheses corresponding to the human RAD51 protein. RESULTS +84 97 S. cerevisiae species Figure 3B shows a highlight of the FxxA portion of oligomerisation peptide from the S. cerevisiae RAD51 structure, with residues in parentheses corresponding to the human RAD51 protein. RESULTS +98 103 RAD51 protein Figure 3B shows a highlight of the FxxA portion of oligomerisation peptide from the S. cerevisiae RAD51 structure, with residues in parentheses corresponding to the human RAD51 protein. RESULTS +104 113 structure evidence Figure 3B shows a highlight of the FxxA portion of oligomerisation peptide from the S. cerevisiae RAD51 structure, with residues in parentheses corresponding to the human RAD51 protein. RESULTS +165 170 human species Figure 3B shows a highlight of the FxxA portion of oligomerisation peptide from the S. cerevisiae RAD51 structure, with residues in parentheses corresponding to the human RAD51 protein. RESULTS +171 176 RAD51 protein Figure 3B shows a highlight of the FxxA portion of oligomerisation peptide from the S. cerevisiae RAD51 structure, with residues in parentheses corresponding to the human RAD51 protein. RESULTS +4 13 conserved protein_state The conserved threonine residue at the third position forms a hydrogen bond with the peptide backbone amide, which forms the base of an α‐helix. RESULTS +14 23 threonine residue_name The conserved threonine residue at the third position forms a hydrogen bond with the peptide backbone amide, which forms the base of an α‐helix. RESULTS +62 75 hydrogen bond bond_interaction The conserved threonine residue at the third position forms a hydrogen bond with the peptide backbone amide, which forms the base of an α‐helix. RESULTS +136 143 α‐helix structure_element The conserved threonine residue at the third position forms a hydrogen bond with the peptide backbone amide, which forms the base of an α‐helix. RESULTS +60 69 threonine residue_name In both structural contexts, the role of the third position threonine in FxxA seems to be in stabilising secondary structure; a β‐turn in the case of BRC binding and an α‐helix in the case of RAD51 oligomerisation. RESULTS +73 77 FxxA structure_element In both structural contexts, the role of the third position threonine in FxxA seems to be in stabilising secondary structure; a β‐turn in the case of BRC binding and an α‐helix in the case of RAD51 oligomerisation. RESULTS +128 134 β‐turn structure_element In both structural contexts, the role of the third position threonine in FxxA seems to be in stabilising secondary structure; a β‐turn in the case of BRC binding and an α‐helix in the case of RAD51 oligomerisation. RESULTS +150 153 BRC structure_element In both structural contexts, the role of the third position threonine in FxxA seems to be in stabilising secondary structure; a β‐turn in the case of BRC binding and an α‐helix in the case of RAD51 oligomerisation. RESULTS +169 176 α‐helix structure_element In both structural contexts, the role of the third position threonine in FxxA seems to be in stabilising secondary structure; a β‐turn in the case of BRC binding and an α‐helix in the case of RAD51 oligomerisation. RESULTS +192 197 RAD51 protein In both structural contexts, the role of the third position threonine in FxxA seems to be in stabilising secondary structure; a β‐turn in the case of BRC binding and an α‐helix in the case of RAD51 oligomerisation. RESULTS +7 19 tetrapeptide chemical In the tetrapeptide context these secondary interactions are not present and mutation of threonine to alanine would be expected to have little effect on affinity. RESULTS +77 85 mutation experimental_method In the tetrapeptide context these secondary interactions are not present and mutation of threonine to alanine would be expected to have little effect on affinity. RESULTS +89 98 threonine residue_name In the tetrapeptide context these secondary interactions are not present and mutation of threonine to alanine would be expected to have little effect on affinity. RESULTS +102 109 alanine residue_name In the tetrapeptide context these secondary interactions are not present and mutation of threonine to alanine would be expected to have little effect on affinity. RESULTS +153 161 affinity evidence In the tetrapeptide context these secondary interactions are not present and mutation of threonine to alanine would be expected to have little effect on affinity. RESULTS +69 85 peptide affinity evidence In line with this, although we observe a slight twofold weakening of peptide affinity, the effect is far from being as drastic or inactivating as reported in longer peptide backgrounds 3. It would be interesting to investigate the importance of this residue in the context of the BRC4 peptide, and the oligomerisation peptide. RESULTS +280 284 BRC4 chemical In line with this, although we observe a slight twofold weakening of peptide affinity, the effect is far from being as drastic or inactivating as reported in longer peptide backgrounds 3. It would be interesting to investigate the importance of this residue in the context of the BRC4 peptide, and the oligomerisation peptide. RESULTS +302 325 oligomerisation peptide structure_element In line with this, although we observe a slight twofold weakening of peptide affinity, the effect is far from being as drastic or inactivating as reported in longer peptide backgrounds 3. It would be interesting to investigate the importance of this residue in the context of the BRC4 peptide, and the oligomerisation peptide. RESULTS +28 35 alanine residue_name Rather than indifference to alanine mutation, a significant effect, via lack of secondary structure stabilisation, would be predicted, as indeed has been reported for BRC3 3. RESULTS +36 44 mutation experimental_method Rather than indifference to alanine mutation, a significant effect, via lack of secondary structure stabilisation, would be predicted, as indeed has been reported for BRC3 3. RESULTS +167 171 BRC3 chemical Rather than indifference to alanine mutation, a significant effect, via lack of secondary structure stabilisation, would be predicted, as indeed has been reported for BRC3 3. RESULTS +20 24 FxxA structure_element Two residues in the FxxA motif, phenylalanine and alanine, are highly conserved (Fig 4a). CONCL +32 45 phenylalanine residue_name Two residues in the FxxA motif, phenylalanine and alanine, are highly conserved (Fig 4a). CONCL +50 57 alanine residue_name Two residues in the FxxA motif, phenylalanine and alanine, are highly conserved (Fig 4a). CONCL +63 79 highly conserved protein_state Two residues in the FxxA motif, phenylalanine and alanine, are highly conserved (Fig 4a). CONCL +0 13 Phenylalanine residue_name Phenylalanine mutated to tryptophan, in the context of the tetrapeptide improved potency, contrary to the reported result of comparable activity in the context of BRC4 12. CONCL +14 24 mutated to experimental_method Phenylalanine mutated to tryptophan, in the context of the tetrapeptide improved potency, contrary to the reported result of comparable activity in the context of BRC4 12. CONCL +25 35 tryptophan residue_name Phenylalanine mutated to tryptophan, in the context of the tetrapeptide improved potency, contrary to the reported result of comparable activity in the context of BRC4 12. CONCL +59 71 tetrapeptide chemical Phenylalanine mutated to tryptophan, in the context of the tetrapeptide improved potency, contrary to the reported result of comparable activity in the context of BRC4 12. CONCL +163 167 BRC4 chemical Phenylalanine mutated to tryptophan, in the context of the tetrapeptide improved potency, contrary to the reported result of comparable activity in the context of BRC4 12. CONCL +0 7 Proline residue_name Proline at the third position similarly improved potency. CONCL +21 29 mutating experimental_method Activity was lost by mutating the terminal alanine to glycine, but recovered somewhat with the novel α‐amino butyric acid (U). CONCL +43 50 alanine residue_name Activity was lost by mutating the terminal alanine to glycine, but recovered somewhat with the novel α‐amino butyric acid (U). CONCL +54 61 glycine residue_name Activity was lost by mutating the terminal alanine to glycine, but recovered somewhat with the novel α‐amino butyric acid (U). CONCL +101 121 α‐amino butyric acid chemical Activity was lost by mutating the terminal alanine to glycine, but recovered somewhat with the novel α‐amino butyric acid (U). CONCL +123 124 U chemical Activity was lost by mutating the terminal alanine to glycine, but recovered somewhat with the novel α‐amino butyric acid (U). CONCL +0 9 Threonine residue_name Threonine was found to be relatively unimportant in the tetrapeptides but has been previously reported to be crucial in the context of BRC3. CONCL +56 69 tetrapeptides chemical Threonine was found to be relatively unimportant in the tetrapeptides but has been previously reported to be crucial in the context of BRC3. CONCL +135 139 BRC3 chemical Threonine was found to be relatively unimportant in the tetrapeptides but has been previously reported to be crucial in the context of BRC3. CONCL +58 67 threonine residue_name The reason for this disconnection is suggested to be that threonine plays a role in stabilising the β‐turn in the BRC repeats, which is absent in the tetrapeptides studied. CONCL +100 106 β‐turn structure_element The reason for this disconnection is suggested to be that threonine plays a role in stabilising the β‐turn in the BRC repeats, which is absent in the tetrapeptides studied. CONCL +114 125 BRC repeats structure_element The reason for this disconnection is suggested to be that threonine plays a role in stabilising the β‐turn in the BRC repeats, which is absent in the tetrapeptides studied. CONCL +150 163 tetrapeptides chemical The reason for this disconnection is suggested to be that threonine plays a role in stabilising the β‐turn in the BRC repeats, which is absent in the tetrapeptides studied. CONCL +46 54 hot‐spot site This may lead to a more general caution, that hot‐spot data should be interpreted by considering the bound interaction with the protein, as well as the potential role in stabilising the bound peptide secondary structure. CONCL +78 106 alanine‐scanning experiments experimental_method In either case, the requirement for structural data in correctly interpreting alanine‐scanning experiments is reinforced. CONCL +32 36 FxxA structure_element Summary of key observations (A) FxxA motif sequence conservation of Rad51 oligomerisation sequences and BRC repeats. (B) Highlight of SAR identified for the tetrapeptide. FIG +68 73 Rad51 protein Summary of key observations (A) FxxA motif sequence conservation of Rad51 oligomerisation sequences and BRC repeats. (B) Highlight of SAR identified for the tetrapeptide. FIG +104 115 BRC repeats structure_element Summary of key observations (A) FxxA motif sequence conservation of Rad51 oligomerisation sequences and BRC repeats. (B) Highlight of SAR identified for the tetrapeptide. FIG +157 169 tetrapeptide chemical Summary of key observations (A) FxxA motif sequence conservation of Rad51 oligomerisation sequences and BRC repeats. (B) Highlight of SAR identified for the tetrapeptide. FIG +19 21 ΔG evidence The differences in ΔG for different peptide variants relative to FHTA are shown in the bar chart with colouring matching with the structural overlay below. (C) Overlay of tetrapeptide structures, with wild‐type FHTA peptide across the figure for reference and truncated segments of mutated residues shown in each panel. FIG +65 69 FHTA structure_element The differences in ΔG for different peptide variants relative to FHTA are shown in the bar chart with colouring matching with the structural overlay below. (C) Overlay of tetrapeptide structures, with wild‐type FHTA peptide across the figure for reference and truncated segments of mutated residues shown in each panel. FIG +130 148 structural overlay experimental_method The differences in ΔG for different peptide variants relative to FHTA are shown in the bar chart with colouring matching with the structural overlay below. (C) Overlay of tetrapeptide structures, with wild‐type FHTA peptide across the figure for reference and truncated segments of mutated residues shown in each panel. FIG +160 167 Overlay experimental_method The differences in ΔG for different peptide variants relative to FHTA are shown in the bar chart with colouring matching with the structural overlay below. (C) Overlay of tetrapeptide structures, with wild‐type FHTA peptide across the figure for reference and truncated segments of mutated residues shown in each panel. FIG +171 183 tetrapeptide chemical The differences in ΔG for different peptide variants relative to FHTA are shown in the bar chart with colouring matching with the structural overlay below. (C) Overlay of tetrapeptide structures, with wild‐type FHTA peptide across the figure for reference and truncated segments of mutated residues shown in each panel. FIG +184 194 structures evidence The differences in ΔG for different peptide variants relative to FHTA are shown in the bar chart with colouring matching with the structural overlay below. (C) Overlay of tetrapeptide structures, with wild‐type FHTA peptide across the figure for reference and truncated segments of mutated residues shown in each panel. FIG +201 210 wild‐type protein_state The differences in ΔG for different peptide variants relative to FHTA are shown in the bar chart with colouring matching with the structural overlay below. (C) Overlay of tetrapeptide structures, with wild‐type FHTA peptide across the figure for reference and truncated segments of mutated residues shown in each panel. FIG +211 215 FHTA structure_element The differences in ΔG for different peptide variants relative to FHTA are shown in the bar chart with colouring matching with the structural overlay below. (C) Overlay of tetrapeptide structures, with wild‐type FHTA peptide across the figure for reference and truncated segments of mutated residues shown in each panel. FIG +17 21 WHTA structure_element Purple carbon is WHTA, light blue is FATA, yellow is FHPA, cyan is FHTG and grey carbon is FHTA. FIG +37 41 FATA structure_element Purple carbon is WHTA, light blue is FATA, yellow is FHPA, cyan is FHTG and grey carbon is FHTA. FIG +53 57 FHPA structure_element Purple carbon is WHTA, light blue is FATA, yellow is FHPA, cyan is FHTG and grey carbon is FHTA. FIG +67 71 FHTG structure_element Purple carbon is WHTA, light blue is FATA, yellow is FHPA, cyan is FHTG and grey carbon is FHTA. FIG +91 95 FHTA structure_element Purple carbon is WHTA, light blue is FATA, yellow is FHPA, cyan is FHTG and grey carbon is FHTA. FIG +46 50 FHTG structure_element Note the C‐terminal amide changes position in FHTG without the anchoring methyl group. FIG diff --git a/annotation_CSV/PMC4857006.csv b/annotation_CSV/PMC4857006.csv new file mode 100644 index 0000000000000000000000000000000000000000..56e2c7341baa9bda9c8a27bbcd0717133de0c84b --- /dev/null +++ b/annotation_CSV/PMC4857006.csv @@ -0,0 +1,1359 @@ +anno_start anno_end anno_text entity_type sentence section +12 15 DNA chemical Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2 TITLE +31 46 Topoisomerase 2 protein_type Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2 TITLE +47 50 DNA chemical Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2 TITLE +73 77 Tdp2 protein Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2 TITLE +0 9 Mammalian taxonomy_domain Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT +10 41 Tyrosyl-DNA phosphodiesterase 2 protein Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT +43 47 Tdp2 protein Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT +58 73 Topoisomerase 2 protein_type Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT +75 79 Top2 protein_type Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT +81 84 DNA chemical Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT +117 121 Top2 protein_type Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT +136 139 DNA chemical Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT +0 4 Tdp2 protein Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. ABSTRACT +81 85 Top2 protein_type Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. ABSTRACT +18 42 X-ray crystal structures evidence Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT +46 57 ligand-free protein_state Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT +58 62 Tdp2 protein Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT +67 75 Tdp2-DNA complex_assembly Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT +112 115 DNA chemical Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT +130 137 dynamic protein_state Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT +138 142 Tdp2 protein Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT +143 158 active site lid structure_element Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT +168 192 substrate binding trench site Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT +230 233 DNA chemical Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT +262 266 Top2 protein_type Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT +23 27 Tdp2 protein Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. ABSTRACT +63 74 mutagenesis experimental_method Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. ABSTRACT +79 98 biochemical studies experimental_method Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. ABSTRACT +116 120 Mg2+ chemical Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. ABSTRACT +149 191 phosphotyrosyl-arginine cation-π interface site Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. ABSTRACT +22 26 Tdp2 protein We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT +27 38 active site site We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT +48 55 ablates protein_state We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT +56 60 Tdp2 protein We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT +61 65 Mg2+ chemical We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT +106 110 Tdp2 protein We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT +128 136 tyrosine residue_name We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT +206 215 etoposide chemical We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT +61 65 Tdp2 protein Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. ABSTRACT +94 97 DNA chemical Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. ABSTRACT +117 121 Top2 protein_type Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. ABSTRACT +165 169 Tdp2 protein Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. ABSTRACT +287 291 Top2 protein_type Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. ABSTRACT +8 11 DNA chemical Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. INTRO +41 44 DNA chemical Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. INTRO +49 64 RNA polymerases protein_type Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. INTRO +94 97 DNA chemical Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. INTRO +138 141 DNA chemical Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. INTRO +205 208 DNA chemical Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. INTRO +0 14 Topoisomerases protein_type Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division. INTRO +35 38 DNA chemical Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division. INTRO +94 97 DNA chemical Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division. INTRO +121 124 DNA chemical Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division. INTRO +4 13 mammalian taxonomy_domain The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions. INTRO +14 36 type II topoisomerases protein_type The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions. INTRO +37 42 Top2α protein The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions. INTRO +47 52 Top2β protein The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions. INTRO +92 95 DNA chemical The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions. INTRO +17 21 Top2 protein_type Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO +22 25 DNA chemical Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO +92 115 phosphotyrosyl linkages ptm Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO +128 140 5′-phosphate chemical Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO +175 186 active site site Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO +187 191 Top2 protein_type Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO +192 200 tyrosine residue_name Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO +215 219 Top2 protein_type Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO +240 246 Top2cc complex_assembly Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO +4 10 Top2cc complex_assembly The Top2cc protein–DNA adduct is a unique threat to genomic integrity which must be resolved to prevent catastrophic Top2cc collisions with the cellular replication and transcription machineries. INTRO +19 22 DNA chemical The Top2cc protein–DNA adduct is a unique threat to genomic integrity which must be resolved to prevent catastrophic Top2cc collisions with the cellular replication and transcription machineries. INTRO +117 123 Top2cc complex_assembly The Top2cc protein–DNA adduct is a unique threat to genomic integrity which must be resolved to prevent catastrophic Top2cc collisions with the cellular replication and transcription machineries. INTRO +30 34 Top2 protein_type To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin. INTRO +111 120 etoposide chemical To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin. INTRO +122 132 teniposide chemical To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin. INTRO +137 148 doxorubicin chemical To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin. INTRO +13 17 Top2 protein_type Importantly, Top2 is also poisoned when it engages abundant endogenous DNA damage not limited to but including ribonucleotides, abasic sites and alkylation damage such as exocyclic DNA adducts arising from bioactivation of the vinyl chloride carcinogen (Figure 1A). INTRO +71 74 DNA chemical Importantly, Top2 is also poisoned when it engages abundant endogenous DNA damage not limited to but including ribonucleotides, abasic sites and alkylation damage such as exocyclic DNA adducts arising from bioactivation of the vinyl chloride carcinogen (Figure 1A). INTRO +181 184 DNA chemical Importantly, Top2 is also poisoned when it engages abundant endogenous DNA damage not limited to but including ribonucleotides, abasic sites and alkylation damage such as exocyclic DNA adducts arising from bioactivation of the vinyl chloride carcinogen (Figure 1A). INTRO +15 18 DNA chemical In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. INTRO +36 42 Top2cc complex_assembly In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. INTRO +53 56 DNA chemical In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. INTRO +117 120 DNA chemical In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. INTRO +160 164 Top2 protein_type In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. INTRO +165 168 DNA chemical In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. INTRO +27 30 DNA chemical The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc. INTRO +46 52 Top2cc complex_assembly The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc. INTRO +71 74 DNA chemical The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc. INTRO +145 148 DNA chemical The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc. INTRO +221 227 Top2cc complex_assembly The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc. INTRO +27 30 DNA chemical Precisely how the cellular DNA repair machinery navigates these complex lesions is an important aspect of Top2cc repair that has not yet been explored. INTRO +106 112 Top2cc complex_assembly Precisely how the cellular DNA repair machinery navigates these complex lesions is an important aspect of Top2cc repair that has not yet been explored. INTRO +0 4 Tdp2 protein Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts. FIG +15 38 phosphotyrosyl linkages ptm Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts. FIG +50 53 DNA chemical Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts. FIG +15 18 DNA chemical (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG +65 68 DNA chemical (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG +121 125 Top2 protein_type (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG +135 139 Top2 protein_type (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG +158 164 Top2cc complex_assembly (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG +196 200 Top2 protein_type (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG +228 243 phosphotyrosine residue_name (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG +0 4 Tdp2 protein Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. FIG +23 38 phosphotyrosine residue_name Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. FIG +68 72 Top2 protein_type Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. FIG +81 84 DNA chemical Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. FIG +97 109 5′-phosphate chemical Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. FIG +129 132 DNA chemical Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. FIG +4 7 DNA chemical (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG +85 103 Tdp2 enzyme assays experimental_method (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG +112 124 deoxyadenine chemical (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG +126 128 dA chemical (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG +131 144 Ethenoadenine chemical (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG +146 148 ϵA chemical (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG +156 167 abasic site site (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG +169 172 THF chemical (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG +0 14 Phosphotyrosyl ptm Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG +44 52 mTdp2cat structure_element Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG +62 75 p-nitrophenol chemical Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG +134 142 mTdp2cat structure_element Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG +143 157 reaction rates evidence Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG +161 174 p–nitrophenol chemical Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG +184 187 DNA chemical Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG +252 255 PNP chemical Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG +272 280 mTdp2cat structure_element Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG +0 8 P-values evidence P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG +37 43 t-test experimental_method P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG +111 120 Structure evidence P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG +124 132 mTdp2cat structure_element P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG +133 141 bound to protein_state P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG +142 158 5′-phosphate DNA chemical P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG +188 190 ϵA chemical P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG +0 22 DNA binding β2Hβ–grasp site DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. FIG +128 131 DNA chemical DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. FIG +0 22 DNA binding β2Hβ–grasp site DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. FIG +128 131 DNA chemical DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. FIG +51 60 Structure evidence PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow). FIG +64 72 mTdp2cat structure_element PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow). FIG +73 81 bound to protein_state PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow). FIG +82 98 5′-phosphate DNA chemical PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow). FIG +128 131 THF chemical PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow). FIG +51 60 Structure evidence PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG +64 72 mTdp2cat structure_element PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG +80 90 absence of protein_state PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG +91 94 DNA chemical PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG +107 115 extended protein_state PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG +116 128 3-helix loop structure_element PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG +135 139 open protein_state PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG +160 177 DNA-binding grasp site PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG +189 196 monomer oligomeric_state PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG +197 198 E structure_element PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG +206 209 apo protein_state PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG +210 219 structure evidence PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG +0 31 Tyrosyl DNA phosphodiesterase 2 protein Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons. INTRO +33 37 Tdp2 protein Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons. INTRO +59 76 5′-phosphotyrosyl ptm Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons. INTRO +78 82 5′-Y ptm Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons. INTRO +84 92 linkages ptm Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons. INTRO +160 164 Top2 protein_type Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons. INTRO +0 4 Tdp2 protein Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair. INTRO +5 14 knockdown experimental_method Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair. INTRO +52 61 etoposide chemical Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair. INTRO +158 162 Tdp2 protein Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair. INTRO +175 181 Top2cc complex_assembly Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair. INTRO +0 4 Tdp2 protein Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression. INTRO +76 82 mutant protein_state Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression. INTRO +83 86 p53 protein Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression. INTRO +106 112 mutant protein_state Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression. INTRO +113 116 p53 protein Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression. INTRO +18 22 Tdp2 protein The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO +36 49 topoisomerase protein_type The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO +98 103 human species The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO +104 108 TDP2 protein The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO +237 244 loss of protein_state The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO +245 249 Tdp2 protein The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO +259 264 Top2β protein The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO +20 24 TDP2 protein It is possible that TDP2 single nucleotide polymorphisms (SNPs) encode mutations that impact Tdp2 function, but the molecular underpinnings for such Tdp2 deficiencies are not understood. INTRO +93 97 Tdp2 protein It is possible that TDP2 single nucleotide polymorphisms (SNPs) encode mutations that impact Tdp2 function, but the molecular underpinnings for such Tdp2 deficiencies are not understood. INTRO +149 153 Tdp2 protein It is possible that TDP2 single nucleotide polymorphisms (SNPs) encode mutations that impact Tdp2 function, but the molecular underpinnings for such Tdp2 deficiencies are not understood. INTRO +39 44 X-ray experimental_method Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO +45 63 crystal structures evidence Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO +71 99 minimal catalytically active protein_state Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO +100 136 endonuclease/exonuclease/phosphatase structure_element Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO +138 141 EEP structure_element Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO +153 158 mouse taxonomy_domain Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO +159 163 Tdp2 protein Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO +165 173 mTdp2cat structure_element Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO +175 183 bound to protein_state Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO +186 189 DNA chemical Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO +213 230 5′-phosphorylated protein_state Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO +56 60 Tdp2 protein However, important questions regarding the mechanism of Tdp2 engagement and processing of DNA damage remain. INTRO +90 93 DNA chemical However, important questions regarding the mechanism of Tdp2 engagement and processing of DNA damage remain. INTRO +24 28 Tdp2 protein First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. INTRO +39 62 phosphotyrosyl linkages ptm First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. INTRO +81 84 DNA chemical First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. INTRO +106 112 Top2cc complex_assembly First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. INTRO +169 172 DNA chemical First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. INTRO +191 202 active site site First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. INTRO +9 20 metal-bound protein_state Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization. INTRO +21 25 Tdp2 protein Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization. INTRO +26 36 structures evidence Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization. INTRO +64 68 Mg2+ chemical Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization. INTRO +32 56 structure-function study experimental_method Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO +64 68 Tdp2 protein Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO +120 125 X-ray experimental_method Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO +126 136 structures evidence Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO +140 151 ligand-free protein_state Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO +152 156 Tdp2 protein Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO +162 166 Tdp2 protein Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO +167 175 bound to protein_state Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO +198 217 1-N6-etheno-adenine chemical Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO +219 222 DNA chemical Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO +28 47 structural analysis experimental_method Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO +49 60 mutagenesis experimental_method Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO +62 79 functional assays experimental_method Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO +84 121 quanyum mechanics/molecular mechanics experimental_method Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO +123 128 QM/MM experimental_method Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO +130 138 modeling experimental_method Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO +146 150 Tdp2 protein Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO +194 198 Tdp2 protein Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO +227 256 tyrosyl DNA phosphodiesterase protein_type Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO +295 298 DNA chemical Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO +26 29 DNA chemical We further establish that DNA damage binding in the Tdp2 active site is linked to conformational change and binding of metal cofactor. INTRO +52 56 Tdp2 protein We further establish that DNA damage binding in the Tdp2 active site is linked to conformational change and binding of metal cofactor. INTRO +57 68 active site site We further establish that DNA damage binding in the Tdp2 active site is linked to conformational change and binding of metal cofactor. INTRO +27 31 Tdp2 protein Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO +41 48 ablates protein_state Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO +53 57 Tdp2 protein Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO +58 83 single metal binding site site Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO +88 92 Tdp2 protein Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO +147 151 Top2 protein_type Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO +172 181 mammalian taxonomy_domain Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO +0 4 Tdp2 protein Tdp2 processing of compound DNA damage RESULTS +28 31 DNA chemical Tdp2 processing of compound DNA damage RESULTS +11 15 Top2 protein_type Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A). RESULTS +48 51 DNA chemical Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A). RESULTS +69 72 DNA chemical Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A). RESULTS +92 110 1-N6-ethenoadenine chemical Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A). RESULTS +112 114 ϵA chemical Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A). RESULTS +8 12 Tdp2 protein Whether Tdp2 processes phosphotyrosyl linkages within these diverse structural contexts is not known. RESULTS +23 46 phosphotyrosyl linkages ptm Whether Tdp2 processes phosphotyrosyl linkages within these diverse structural contexts is not known. RESULTS +28 47 EDC coupling method experimental_method To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods). RESULTS +72 85 p-nitrophenol chemical To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods). RESULTS +87 90 PNP chemical To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods). RESULTS +137 140 DNA chemical To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods). RESULTS +56 61 mouse taxonomy_domain We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS +62 66 Tdp2 protein We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS +67 83 catalytic domain structure_element We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS +85 93 mTdp2cat structure_element We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS +106 109 PNP chemical We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS +135 148 topoisomerase protein_type We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS +149 157 tyrosine residue_name We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS +200 203 DNA chemical We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS +223 241 colorimetric assay experimental_method We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS +18 22 Tdp2 protein We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). RESULTS +44 47 PNP chemical We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). RESULTS +100 106 dA-PNP chemical We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). RESULTS +108 114 ϵA-PNP chemical We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). RESULTS +141 163 tetrahydrofuran spacer chemical We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). RESULTS +165 168 THF chemical We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). RESULTS +6 10 Tdp2 protein Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage. RESULTS +31 54 phosphotyrosyl linkages ptm Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage. RESULTS +123 126 DNA chemical Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage. RESULTS +139 142 DNA chemical Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage. RESULTS +38 42 Tdp2 protein To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS +57 63 Top2cc complex_assembly To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS +82 85 DNA chemical To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS +97 124 crystallized and determined experimental_method To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS +125 130 X-ray experimental_method To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS +131 149 crystal structures evidence To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS +153 161 mTdp2cat structure_element To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS +162 170 bound to protein_state To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS +171 187 5′-phosphate DNA chemical To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS +213 218 5′-ϵA chemical To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS +272 275 DNA chemical To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS +289 295 5′-THF chemical To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS +9 17 Tdp2-DNA complex_assembly In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS +26 36 structures evidence In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS +38 46 mTdp2cat structure_element In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS +56 70 mixed α-β fold structure_element In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS +93 129 12-stranded anti-parallel β-sandwich structure_element In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS +182 186 Tdp2 protein In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS +187 198 active site site In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS +70 87 DNA-binding cleft site One half of the molecule contributes to formation of the walls of the DNA-binding cleft that embraces the terminal position of the damaged DNA substrate. RESULTS +139 142 DNA chemical One half of the molecule contributes to formation of the walls of the DNA-binding cleft that embraces the terminal position of the damaged DNA substrate. RESULTS +7 23 DNA lesion-bound protein_state In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS +39 42 DNA chemical In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS +65 76 β-2-helix-β structure_element In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS +78 82 β2Hβ structure_element In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS +85 90 grasp structure_element In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS +98 109 helical cap structure_element In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS +120 144 substrate binding trench site In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS +160 165 ssDNA chemical In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS +202 213 active site site In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS +34 43 structure evidence A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS +47 55 DNA-free protein_state A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS +56 60 Tdp2 protein A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS +62 65 apo protein_state A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS +100 104 loop structure_element A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS +108 131 conformationally mobile protein_state A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS +159 162 DNA chemical A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS +57 59 ϵA chemical The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage. RESULTS +93 97 Tdp2 protein The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage. RESULTS +106 120 5′-tyrosylated protein_state The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage. RESULTS +162 165 DNA chemical The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage. RESULTS +4 9 5′-ϵA chemical The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). RESULTS +50 54 Tdp2 protein The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). RESULTS +55 88 van Der Waals interaction surface site The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). RESULTS +115 131 hydrophobic wall site The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). RESULTS +183 189 Leu315 residue_name_number The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). RESULTS +194 200 Ile317 residue_name_number The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). RESULTS +0 10 Structures evidence Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning. FIG +14 22 mTdp2cat structure_element Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning. FIG +23 31 bound to protein_state Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning. FIG +32 35 DNA chemical Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning. FIG +57 61 Top2 protein_type Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning. FIG +4 13 Structure evidence (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +17 25 mTdp2cat structure_element (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +26 34 bound to protein_state (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +35 51 5′-phosphate DNA chemical (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +81 83 ϵA chemical (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +94 98 Mg2+ chemical (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +130 136 waters chemical (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +0 8 mTdp2cat structure_element mTdp2cat is colored by electrostatic surface potential (red = negative, blue = positive, gray = neutral/hydrophobic). FIG +4 44 σ-A weighted 2Fo-Fc electron density map evidence (B) σ-A weighted 2Fo-Fc electron density map (at 1.43 Å resolution, contoured at 2.0 σ) for the ϵA DNA complex. FIG +96 102 ϵA DNA chemical (B) σ-A weighted 2Fo-Fc electron density map (at 1.43 Å resolution, contoured at 2.0 σ) for the ϵA DNA complex. FIG +4 6 ϵA chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +43 56 hydrogen bond bond_interaction The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +66 68 ϵA chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +89 94 water chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +124 133 Structure evidence The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +137 145 mTdp2cat structure_element The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +146 154 bound to protein_state The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +155 171 5′-phosphate DNA chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +201 204 THF chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +215 219 Mg2+ chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +251 257 waters chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG +0 8 mTdp2cat structure_element mTdp2cat is colored with red (electronegative), blue (electropositive) and gray (hydrophobic) electrostatic surface potential displayed. FIG +33 73 σ-A weighted 2Fo-Fc electron density map evidence PDB entry 5INK is displayed. (D) σ-A weighted 2Fo-Fc electron density map (at 2.15 Å resolution, contoured at 2.0 σ) for THF-DNA complex. FIG +121 128 THF-DNA complex_assembly PDB entry 5INK is displayed. (D) σ-A weighted 2Fo-Fc electron density map (at 2.15 Å resolution, contoured at 2.0 σ) for THF-DNA complex. FIG +4 7 THF chemical The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes. FIG +33 46 hydrogen bond bond_interaction The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes. FIG +56 59 THF chemical The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes. FIG +80 85 water chemical The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes. FIG +24 34 determined experimental_method For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). RESULTS +37 46 structure evidence For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). RESULTS +63 73 5′-adenine chemical For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). RESULTS +75 80 5′-dA chemical For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). RESULTS +82 90 bound to protein_state For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). RESULTS +91 95 Tdp2 protein For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). RESULTS +2 20 structural overlay experimental_method A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). RESULTS +127 132 bound protein_state A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). RESULTS +147 150 DNA chemical A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). RESULTS +167 169 ϵA chemical A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). RESULTS +171 173 dA chemical A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). RESULTS +178 180 dC chemical A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). RESULTS +67 69 ϵG chemical Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS +71 73 ϵT chemical Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS +111 115 Tdp2 protein Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS +116 127 active site site Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS +132 152 planar base stacking bond_interaction Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS +162 173 active site site Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS +181 197 hydrophobic wall site Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS +205 209 β2Hβ structure_element Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS +14 32 abasic deoxyribose chemical Likewise, the abasic deoxyribose analog THF substrate binds similar to the alkylated and non-alkylated substrates, but with a slight alteration in the approach of the 5′-terminus (Figure 2C). RESULTS +40 43 THF chemical Likewise, the abasic deoxyribose analog THF substrate binds similar to the alkylated and non-alkylated substrates, but with a slight alteration in the approach of the 5′-terminus (Figure 2C). RESULTS +22 32 absence of protein_state Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D). RESULTS +58 61 THF chemical Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D). RESULTS +104 109 water chemical Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D). RESULTS +173 177 Mg2+ chemical Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D). RESULTS +178 200 ion coordination shell bond_interaction Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D). RESULTS +108 111 THF chemical These collective differences may explain the slight, but statistically significant elevated activity on the THF substrate (Figure 1C). RESULTS +29 33 Tdp2 protein Structural plasticity in the Tdp2 DNA binding trench RESULTS +34 52 DNA binding trench site Structural plasticity in the Tdp2 DNA binding trench RESULTS +29 45 DNA-damage bound protein_state An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). RESULTS +66 70 Tdp2 protein An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). RESULTS +71 82 active site site An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). RESULTS +119 124 water chemical An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). RESULTS +170 184 catalytic core site An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). RESULTS +193 215 DNA binding β2Hβ-grasp site An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). RESULTS +68 78 β2Hβ-grasp site In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358. RESULTS +92 106 hydrogen bonds bond_interaction In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358. RESULTS +138 142 Mg2+ chemical In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358. RESULTS +150 156 Asp358 residue_name_number In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358. RESULTS +15 39 hydrophobic interactions bond_interaction The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement. RESULTS +56 60 β2Hβ structure_element The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement. RESULTS +61 70 DNA-bound protein_state The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement. RESULTS +131 135 β2Hβ structure_element The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement. RESULTS +158 161 DNA chemical The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement. RESULTS +28 40 crystallized experimental_method To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS +41 45 Tdp2 protein To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS +53 63 absence of protein_state To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS +64 67 DNA chemical To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS +85 93 DNA free protein_state To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS +94 98 Tdp2 protein To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS +99 108 structure evidence To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS +33 37 Tdp2 protein Conformational plasticity in the Tdp2 active site. FIG +38 49 active site site Conformational plasticity in the Tdp2 active site. FIG +8 12 open protein_state (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG +14 21 3-helix structure_element (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG +44 52 flexible protein_state (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG +53 69 active-site loop structure_element (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG +82 89 monomer oligomeric_state (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG +90 91 E structure_element (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG +99 107 DNA-free protein_state (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG +108 116 mTdp2cat structure_element (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG +117 126 structure evidence (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG +160 164 T309 residue_name_number (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG +198 201 EEP structure_element (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG +4 23 β2Hβ docking pocket site The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. FIG +61 65 N312 residue_name_number The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. FIG +67 71 N314 residue_name_number The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. FIG +76 80 L315 residue_name_number The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. FIG +94 109 solvent-exposed protein_state The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. FIG +44 50 closed protein_state Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG +51 55 β2Hβ structure_element Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG +76 88 mTdp2cat–DNA complex_assembly Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG +97 106 structure evidence Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG +118 123 5′-ϵA chemical Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG +150 154 T309 residue_name_number Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG +190 212 β2Hβ DNA-binding grasp site Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG +223 237 hydrogen bonds bond_interaction Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG +257 261 Y321 residue_name_number Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG +269 273 N314 residue_name_number Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG +296 315 β2Hβ docking pocket site Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG +53 69 active site loop structure_element Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +99 108 promoters oligomeric_state Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +116 124 DNA-free protein_state Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +125 133 mTdp2cat structure_element Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +208 226 sequence alignment experimental_method Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +264 280 electron density evidence Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +301 328 Limited trypsin proteolysis experimental_method Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +369 377 flexible protein_state Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +378 394 active-site loop structure_element Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +0 8 mTdp2cat structure_element mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG +9 11 WT protein_state mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG +28 36 mTdp2cat structure_element mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG +37 42 D358N mutant mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG +79 87 presence protein_state mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG +91 101 absence of protein_state mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG +102 106 Mg2+ chemical mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG +156 159 DNA chemical mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG +28 36 SDS-PAGE experimental_method Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +98 130 Limited chymotrypsin proteolysis experimental_method Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +171 179 flexible protein_state Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +180 196 active-site loop structure_element Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG +40 48 mTdp2cat structure_element Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. FIG +49 51 WT protein_state Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. FIG +69 77 mTdp2cat structure_element Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. FIG +78 83 D358N mutant Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. FIG +108 120 chymotrypsin experimental_method Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. FIG +132 139 trypsin experimental_method Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. FIG +29 33 Tdp2 protein This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers. RESULTS +95 106 active site site This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers. RESULTS +107 111 Mg2+ chemical This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers. RESULTS +126 149 substrate binding loops structure_element This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers. RESULTS +178 187 protomers oligomeric_state This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers. RESULTS +33 48 DNA ligand-free protein_state The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B). RESULTS +67 89 active site β2Hβ-grasp site The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B). RESULTS +150 159 DNA-bound protein_state The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B). RESULTS +161 167 closed protein_state The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B). RESULTS +168 190 β2Hβ DNA binding grasp site The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B). RESULTS +7 14 monomer oligomeric_state In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS +16 25 chain ‘E’ structure_element In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS +32 37 grasp structure_element In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS +49 53 open protein_state In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS +55 67 3-helix loop structure_element In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS +109 112 EEP structure_element In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS +113 127 catalytic core site In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS +4 12 monomers oligomeric_state Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density. RESULTS +27 37 disordered protein_state Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density. RESULTS +67 83 DNA binding loop structure_element Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density. RESULTS +106 122 electron density evidence Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density. RESULTS +35 43 DNA-free protein_state The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). RESULTS +44 56 crystal form evidence The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). RESULTS +61 67 closed protein_state The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). RESULTS +68 72 β2Hβ structure_element The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). RESULTS +99 108 DNA bound protein_state The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). RESULTS +109 119 structures evidence The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). RESULTS +20 24 Tdp2 protein Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding. RESULTS +25 28 DNA chemical Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding. RESULTS +66 72 closed protein_state Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding. RESULTS +85 95 β2Hβ grasp site Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding. RESULTS +27 35 extended protein_state A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes. RESULTS +36 43 3-helix structure_element A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes. RESULTS +72 94 substrate-binding loop structure_element A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes. RESULTS +8 12 open protein_state In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS +28 41 Asn312-Leu315 residue_range In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS +62 73 active site site In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS +78 93 solvent-exposed protein_state In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS +128 134 Thr309 residue_name_number In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS +183 189 pocket site In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS +197 200 EEP structure_element In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS +220 224 loop structure_element In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS +10 16 Thr309 residue_name_number Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. RESULTS +53 69 cis–peptide bond bond_interaction Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. RESULTS +78 84 Asp308 residue_name_number Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. RESULTS +85 91 Thr309 residue_name_number Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. RESULTS +115 145 short antiparallel beta-strand structure_element Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. RESULTS +153 157 β2Hβ structure_element Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. RESULTS +19 25 closed protein_state By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). RESULTS +26 36 β2Hβ grasp site By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). RESULTS +64 70 Asn312 residue_name_number By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). RESULTS +75 81 Asn314 residue_name_number By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). RESULTS +99 119 β2Hβ docking pockets site By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). RESULTS +125 131 Leu315 residue_name_number By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). RESULTS +23 29 closed protein_state To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS +30 34 β2Hβ structure_element To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS +49 55 Thr309 residue_name_number To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS +76 79 EEP structure_element To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS +87 93 pocket site To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS +168 172 β2Hβ structure_element To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS +173 193 antiparallel β-sheet structure_element To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS +21 27 closed protein_state Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS +28 38 β2Hβ-grasp site Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS +69 80 active site site Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS +91 104 hydrogen bond bond_interaction Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS +113 119 Trp307 residue_name_number Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS +128 153 Mg2+ coordinating residue site Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS +154 160 Asp358 residue_name_number Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS +20 28 DNA free protein_state Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS +29 38 structure evidence Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS +71 77 closed protein_state Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS +78 86 monomers oligomeric_state Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS +103 107 Mg2+ chemical Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS +121 133 active sites site Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS +145 153 monomers oligomeric_state Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS +159 163 open protein_state Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS +210 228 metal binding site site Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS +71 74 DNA chemical Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site. RESULTS +147 169 β2Hβ DNA binding grasp site Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site. RESULTS +217 221 Mg2+ chemical Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site. RESULTS +237 241 Tdp2 protein Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site. RESULTS +242 253 active site site Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site. RESULTS +12 16 Mg2+ chemical To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E). RESULTS +21 24 DNA chemical To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E). RESULTS +35 39 Tdp2 protein To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E). RESULTS +81 89 mTdp2cat structure_element To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E). RESULTS +110 154 limited trypsin and chymotrypsin proteolysis experimental_method To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E). RESULTS +0 17 In the absence of protein_state In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS +18 21 DNA chemical In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS +25 30 Mg2+, chemical In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS +31 39 mTdp2cat structure_element In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS +82 99 DNA binding grasp site In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS +115 122 trypsin experimental_method In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS +124 130 Arg316 residue_name_number In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS +156 168 chymotrypsin experimental_method In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS +170 176 Trp307 residue_name_number In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS +181 187 Leu315 residue_name_number In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS +15 20 Mg2+, chemical By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage. RESULTS +45 49 Mg2+ chemical By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage. RESULTS +50 53 DNA chemical By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage. RESULTS +109 117 mTdp2cat structure_element By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage. RESULTS +27 31 Mg2+ chemical Interestingly, addition of Mg2+ alone protects against proteolysis as well. RESULTS +24 28 Mg2+ chemical This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS +45 51 closed protein_state This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS +72 82 β2Hβ-grasp site This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS +103 127 hydrogen-bonding network bond_interaction This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS +133 139 Asp358 residue_name_number This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS +167 177 β2Hβ-grasp site This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS +186 192 Trp307 residue_name_number This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS +219 223 Tdp2 protein This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS +224 235 active site site This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS +37 41 Tdp2 protein To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS +73 78 human species To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS +83 88 mouse taxonomy_domain To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS +89 93 Tdp2 protein To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS +103 113 determined experimental_method To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS +133 142 structure evidence To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS +150 155 human species To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS +156 163 Tdp2cat structure_element To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS +171 179 bound to protein_state To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS +182 192 DNA 5′-PO4 chemical To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS +19 24 human species Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). RESULTS +25 37 hTdp2cat-DNA complex_assembly Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). RESULTS +46 55 structure evidence Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). RESULTS +63 71 mTdp2cat structure_element Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). RESULTS +72 81 DNA bound protein_state Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). RESULTS +129 138 DNA-bound protein_state Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). RESULTS +21 29 mTdp2cat structure_element Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B). RESULTS +61 69 hTdp2cat structure_element Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B). RESULTS +70 92 substrate binding loop structure_element Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B). RESULTS +117 121 Mg2+ chemical Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B). RESULTS +126 129 DNA chemical Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B). RESULTS +6 11 X-ray experimental_method Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS +12 22 structures evidence Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS +27 55 limited proteolysis analysis experimental_method Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS +70 73 DNA chemical Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS +122 131 conserved protein_state Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS +147 157 vertebrate taxonomy_domain Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS +158 162 Tdp2 protein Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS +0 4 Tdp2 protein Tdp2 metal ion dependence RESULTS +32 57 X-ray structural analyses experimental_method Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site. RESULTS +90 94 Mg2+ chemical Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site. RESULTS +101 109 bound in protein_state Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site. RESULTS +114 118 Tdp2 protein Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site. RESULTS +119 130 active site site Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site. RESULTS +18 26 DNA-free protein_state This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS +40 56 DNA damage bound protein_state This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS +73 95 reaction product-bound protein_state This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS +96 109 crystal forms evidence This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS +113 118 mouse taxonomy_domain This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS +138 146 D. rerio species This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS +168 178 C. elegans species This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS +179 183 Tdp2 protein This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS +18 38 biochemical analysis experimental_method However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction. RESULTS +100 104 Tdp2 protein However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction. RESULTS +105 137 phosphotyrosyl phosphodiesterase protein_type However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction. RESULTS +34 38 Mg2+ chemical In these experiments, at limiting Mg2+ concentrations, Ca2+ addition to Tdp2 reactions stimulated activity. RESULTS +55 59 Ca2+ chemical In these experiments, at limiting Mg2+ concentrations, Ca2+ addition to Tdp2 reactions stimulated activity. RESULTS +72 76 Tdp2 protein In these experiments, at limiting Mg2+ concentrations, Ca2+ addition to Tdp2 reactions stimulated activity. RESULTS +64 78 phosphotyrosyl ptm While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site. RESULTS +96 100 Tdp2 protein While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site. RESULTS +164 187 metal ion binding sites site While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site. RESULTS +203 214 active site site While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site. RESULTS +9 24 divalent metals chemical In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies. RESULTS +51 55 Tdp2 protein In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies. RESULTS +64 67 DNA chemical In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies. RESULTS +109 122 active center site In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies. RESULTS +49 53 Tdp2 protein To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS +54 71 phosphodiesterase protein_type To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS +95 119 metal ion binding assays experimental_method To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS +132 150 crystal structures evidence To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS +158 169 presence of protein_state To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS +177 192 divalent metals chemical To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS +194 198 Mn2+ chemical To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS +203 208 Ca2+) chemical To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS +251 255 Tdp2 protein To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS +256 288 phosphotyrosyl phosphodiesterase protein_type To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS +33 37 Tdp2 protein Metal cofactor interactions with Tdp2. (A) Intrinsic tryptophan fluorescence of mTdp2cat was used to monitor a conformational response to divalent metal ion binding. FIG +43 76 Intrinsic tryptophan fluorescence evidence Metal cofactor interactions with Tdp2. (A) Intrinsic tryptophan fluorescence of mTdp2cat was used to monitor a conformational response to divalent metal ion binding. FIG +80 88 mTdp2cat structure_element Metal cofactor interactions with Tdp2. (A) Intrinsic tryptophan fluorescence of mTdp2cat was used to monitor a conformational response to divalent metal ion binding. FIG +7 11 Mg2+ chemical Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG +15 19 Ca2+ chemical Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG +25 33 titrated experimental_method Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG +41 49 presence protein_state Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG +53 63 absence of protein_state Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG +64 72 5′-P DNA chemical Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG +82 105 tryptophan fluorescence evidence Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG +5 9 Mg2+ chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +14 18 Ca2+ chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +79 102 tryptophan fluorescence evidence Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +106 114 mTdp2cat structure_element Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +122 130 presence protein_state Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +135 145 absence of protein_state Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +146 149 DNA chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +157 162 D358N mutant Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +163 174 active site site Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +175 181 mutant protein_state Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +185 193 mTdp2cat structure_element Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +197 209 unresponsive protein_state Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +213 217 Mg2+ chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +223 231 mTdp2cat structure_element Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +254 259 T5PNP chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +287 291 Mg2+ chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +296 300 Ca2+ chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG +0 3 PNP chemical PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG +65 69 Mg2+ chemical PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG +95 102 absence protein_state PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG +106 117 presence of protein_state PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG +129 133 Ca2+ chemical PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG +172 212 σ-A weighted 2Fo-Fc electron density map evidence PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG +224 265 model-phased anomalous difference Fourier evidence PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG +276 280 maps evidence PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG +289 306 mTdp2cat–DNA–Mn2+ complex_assembly PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG +346 350 Mn2+ chemical PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG +18 50 anomalous difference Fourier map evidence A 53σ peak in the anomalous difference Fourier map (data collected at λ = 1.5418 Å) supports Mn2+ as the identity of this atom. FIG +93 97 Mn2+ chemical A 53σ peak in the anomalous difference Fourier map (data collected at λ = 1.5418 Å) supports Mn2+ as the identity of this atom. FIG +18 22 Ca2+ chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG +30 34 Ca2+ chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG +47 50 DNA chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG +74 78 Mg2+ chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG +88 92 Mg2+ chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG +105 108 DNA chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG +127 139 mTdp2cat–DNA complex_assembly (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG +140 150 structures evidence (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG +162 166 Ca2+ chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG +180 205 5′-phosphate binding mode site (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG +4 15 proteolysis experimental_method Our proteolysis results indicate a Mg2+-dependent Tdp2 conformational response to metal binding. RESULTS +35 39 Mg2+ chemical Our proteolysis results indicate a Mg2+-dependent Tdp2 conformational response to metal binding. RESULTS +50 54 Tdp2 protein Our proteolysis results indicate a Mg2+-dependent Tdp2 conformational response to metal binding. RESULTS +4 8 Tdp2 protein The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. RESULTS +9 20 active site site The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. RESULTS +31 41 tryptophan residue_name The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. RESULTS +70 90 metal binding center site The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. RESULTS +106 139 intrinsic tryptophan fluorescence evidence The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. RESULTS +190 198 mTdp2cat structure_element The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. RESULTS +76 84 presence protein_state These data were an excellent fit to a single-site binding model both in the presence and absence of DNA (Figure 4A). RESULTS +89 99 absence of protein_state These data were an excellent fit to a single-site binding model both in the presence and absence of DNA (Figure 4A). RESULTS +100 103 DNA chemical These data were an excellent fit to a single-site binding model both in the presence and absence of DNA (Figure 4A). RESULTS +23 27 Mg2+ chemical This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS +28 30 Kd evidence This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS +70 87 Hill coefficients evidence This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS +124 142 metal binding site site This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS +155 163 presence protein_state This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS +168 178 absence of protein_state This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS +179 182 DNA chemical This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS +56 60 Tdp2 protein We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5′-phosphate by mTdp2cat. RESULTS +73 109 p-nitrophenyl-thymidine-5′-phosphate chemical We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5′-phosphate by mTdp2cat. RESULTS +113 121 mTdp2cat structure_element We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5′-phosphate by mTdp2cat. RESULTS +72 75 DNA chemical This small molecule substrate is not expected to be influenced by metal–DNA coordination outside of the active site. RESULTS +104 115 active site site This small molecule substrate is not expected to be influenced by metal–DNA coordination outside of the active site. RESULTS +23 27 Ca2+ chemical Inclusion of ultrapure Ca2+ (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting Mg2+ (Figure 4B). RESULTS +103 107 Tdp2 protein Inclusion of ultrapure Ca2+ (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting Mg2+ (Figure 4B). RESULTS +149 153 Mg2+ chemical Inclusion of ultrapure Ca2+ (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting Mg2+ (Figure 4B). RESULTS +22 32 titrations experimental_method We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. RESULTS +38 43 human species We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. RESULTS +44 51 hTdp2FL protein We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. RESULTS +56 64 hTdp2cat structure_element We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. RESULTS +136 140 Mg2+ chemical We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. RESULTS +159 163 Ca2+ chemical We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. RESULTS +15 37 metal binding analyses experimental_method Overall, these metal binding analyses are consistent with a single metal ion mediated reaction. RESULTS +72 76 Tdp2 protein To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS +77 88 active site site To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS +104 122 crystal structures evidence To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS +126 142 soaking crystals experimental_method To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS +176 183 support protein_state To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS +185 189 Mn2+ chemical To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS +194 201 inhibit protein_state To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS +203 207 Ca2+ chemical To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS +224 228 Tdp2 protein To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS +0 33 Anomalous difference Fourier maps evidence Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS +41 54 Tdp2–DNA–Mn2+ complex_assembly Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS +77 89 binding site site Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS +94 98 Mn2+ chemical Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS +107 111 Tdp2 protein Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS +112 123 active site site Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS +142 165 octahedral coordination bond_interaction Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS +195 199 Mn2+ chemical Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS +4 8 Mn2+ chemical The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. RESULTS +34 38 Tdp2 protein The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. RESULTS +39 50 active site site The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. RESULTS +66 76 Mg2+-bound protein_state The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. RESULTS +138 142 Mn2+ chemical The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. RESULTS +161 165 Tdp2 protein The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. RESULTS +19 40 co-complex structures evidence In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site. RESULTS +46 50 Ca2+ chemical In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site. RESULTS +81 85 Ca2+ chemical In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site. RESULTS +148 157 Mg2+ site site In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site. RESULTS +9 13 Ca2+ chemical Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site. RESULTS +22 46 octahedrally coordinated bond_interaction Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site. RESULTS +76 80 Ca2+ chemical Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site. RESULTS +124 128 Ca2+ chemical Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site. RESULTS +149 162 Mg2+ ion site site Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site. RESULTS +15 53 bi-dentate inner sphere metal contacts bond_interaction Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS +63 67 Ca2+ chemical Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS +75 81 Glu162 residue_name_number Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS +94 128 active site phosphate-binding mode site Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS +147 153 5′-PO4 chemical Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS +165 169 Tdp2 protein Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS +170 181 active site site Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS +71 75 Ca2+ chemical Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS +112 116 Tdp2 protein Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS +131 151 divalent metal bound protein_state Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS +152 156 Tdp2 protein Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS +157 167 structures evidence Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS +192 196 Ca2+ chemical Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS +224 228 Tdp2 protein Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS +13 17 Tdp2 protein Modeling the Tdp2 reaction coordinate RESULTS +56 60 Mg2+ chemical Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS +75 84 simulated experimental_method Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS +89 93 Tdp2 protein Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS +119 140 hybrid QM/MM modeling experimental_method Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS +147 151 Tdp2 protein Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS +152 169 substrate analog- protein_state Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS +174 187 product-bound protein_state Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS +188 198 structures evidence Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS +9 28 structural analyses experimental_method Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. RESULTS +45 58 superposition experimental_method Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. RESULTS +64 67 DNA chemical Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. RESULTS +85 100 5′-aminohexanol chemical Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. RESULTS +115 121 5′-PO4 chemical Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. RESULTS +155 159 Tdp2 protein Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. RESULTS +53 58 water chemical In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct. RESULTS +76 91 hydrogen bonded bond_interaction In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct. RESULTS +95 101 Asp272 residue_name_number In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct. RESULTS +106 112 Asn274 residue_name_number In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct. RESULTS +35 39 Tdp2 protein Structure-function analysis of the Tdp2 reaction mechanism. FIG +41 56 phosphotyrosine residue_name (A) Proposed mechanism for hydrolysis of phosphotyrosine bond by Tdp2. FIG +65 69 Tdp2 protein (A) Proposed mechanism for hydrolysis of phosphotyrosine bond by Tdp2. FIG +27 39 binding-site site Residues in green form the binding-site for the 5′-tyrosine (red) and phosphate, yellow bind the 5′ nucleotide and blue bind nucleotides 2–3. FIG +48 59 5′-tyrosine residue_name Residues in green form the binding-site for the 5′-tyrosine (red) and phosphate, yellow bind the 5′ nucleotide and blue bind nucleotides 2–3. FIG +70 79 phosphate chemical Residues in green form the binding-site for the 5′-tyrosine (red) and phosphate, yellow bind the 5′ nucleotide and blue bind nucleotides 2–3. FIG +34 39 mTdp2 protein Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom. FIG +53 64 Free energy evidence Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom. FIG +76 92 QM/MM simulation experimental_method Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom. FIG +144 149 water chemical Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom. FIG +57 69 mTdp2cat-DNA complex_assembly Reaction proceeds from right to left. (C) Models for the mTdp2cat-DNA complex during the QM/MM reaction path simulation showing the substrate (left, tan), transition state intermediate (center, cyan) and product (right, pink) states. FIG +89 119 QM/MM reaction path simulation experimental_method Reaction proceeds from right to left. (C) Models for the mTdp2cat-DNA complex during the QM/MM reaction path simulation showing the substrate (left, tan), transition state intermediate (center, cyan) and product (right, pink) states. FIG +34 39 mTdp2 protein Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. FIG +53 84 Electrostatic surface potential evidence Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. FIG +100 118 5′-phosphotyrosine residue_name Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. FIG +157 168 presence of protein_state Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. FIG +171 191 cation–π interaction bond_interaction Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. FIG +222 228 Arg216 residue_name_number Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. FIG +0 23 Electrostatic potential evidence Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG +161 170 wild-type protein_state Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG +175 181 mutant protein_state Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG +182 187 human species Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG +188 191 MBP experimental_method Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG +192 200 hTdp2cat structure_element Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG +201 216 fusion proteins experimental_method Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG +11 14 PNP chemical Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm. FIG +20 23 PNP chemical Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm. FIG +24 33 phosphate chemical Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm. FIG +38 43 T5PNP chemical Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm. FIG +0 14 Reaction rates evidence Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d. FIG +68 76 wildtype protein_state Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d. FIG +77 80 MBP experimental_method Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d. FIG +81 89 hTdp2cat structure_element Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d. FIG +11 16 hTdp2 protein Mutants of hTdp2 (black) and the equivalent residue in mTdp2 (tan) are indicated. FIG +55 60 mTdp2 protein Mutants of hTdp2 (black) and the equivalent residue in mTdp2 (tan) are indicated. FIG +65 70 water chemical We examined the energy profile of the nucleophilic attack of the water molecule by using the distance between the water oxygen and the P atom on the phosphate moiety as the sole reaction coordinate in the present calculation (Figure 5B and C). RESULTS +114 119 water chemical We examined the energy profile of the nucleophilic attack of the water molecule by using the distance between the water oxygen and the P atom on the phosphate moiety as the sole reaction coordinate in the present calculation (Figure 5B and C). RESULTS +62 70 mTdp2cat structure_element A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS +71 86 5′–aminohexanol chemical A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS +104 113 structure evidence A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS +132 140 tyrosine residue_name A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS +155 170 5′-aminohexanol chemical A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS +187 191 Mg2+ chemical A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS +209 215 waters chemical A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS +225 234 mTdp2-DNA complex_assembly A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS +243 252 structure evidence A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS +298 327 molecular dynamics simulation experimental_method A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS +6 24 QM/MM optimization experimental_method After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å). RESULTS +160 168 mTdp2cat structure_element After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å). RESULTS +169 184 5′-aminohexanol chemical After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å). RESULTS +202 211 structure evidence After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å). RESULTS +10 15 water chemical Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length. RESULTS +48 54 Asp272 residue_name_number Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length. RESULTS +80 93 hydrogen bond bond_interaction Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length. RESULTS +123 137 phosphotyrosyl ptm Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length. RESULTS +35 45 simulation experimental_method In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. RESULTS +54 59 water chemical In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. RESULTS +108 121 hydrogen bond bond_interaction In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. RESULTS +147 152 water chemical In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. RESULTS +157 163 Asp272 residue_name_number In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. RESULTS +190 195 water chemical In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. RESULTS +25 30 water chemical The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction. RESULTS +62 75 hydrogen bond bond_interaction The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction. RESULTS +81 87 Asn274 residue_name_number The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction. RESULTS +155 160 water chemical The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction. RESULTS +27 41 phosphotyrosyl ptm Concomitant with this, the phosphotyrosyl O–P bond weakens (d = 1.89 Å), and the formation of the penta-covalent transition state (Figure 5C ‘ii-transition state’) is observed. RESULTS +57 66 phosphate chemical The final steps show inversion of stereochemistry at the phosphate, along with lengthening and breaking of the phosphotyrosyl O–P bond. RESULTS +111 125 phosphotyrosyl ptm The final steps show inversion of stereochemistry at the phosphate, along with lengthening and breaking of the phosphotyrosyl O–P bond. RESULTS +78 83 water chemical Product formation is coupled to a transfer of a proton from the nucleophillic water to Asp272, consistent with the proposed function for this residue as the catalytic base. RESULTS +87 93 Asp272 residue_name_number Product formation is coupled to a transfer of a proton from the nucleophillic water to Asp272, consistent with the proposed function for this residue as the catalytic base. RESULTS +55 62 His 359 residue_name_number Of note, both nitrogens of the imidazole side chain of His 359 require protonation for stability of the simulation. RESULTS +104 114 simulation experimental_method Of note, both nitrogens of the imidazole side chain of His 359 require protonation for stability of the simulation. RESULTS +0 7 Asp 326 residue_name_number Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +16 29 hydrogen bond bond_interaction Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +40 46 His359 residue_name_number Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +69 80 salt bridge bond_interaction Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +101 111 protonated protein_state Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +120 126 His359 residue_name_number Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +170 173 Asp residue_name Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +174 177 His residue_name Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +190 193 EEP structure_element Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +204 208 APE1 protein Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +229 232 pKa evidence Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +241 244 His residue_name Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +302 315 hydrogen bond bond_interaction Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +328 345 doubly protonated protein_state Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +346 352 His359 residue_name_number Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +361 370 phosphate chemical Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +426 430 Mg2+ chemical Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +449 455 His359 residue_name_number Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +485 491 H-bond bond_interaction Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +501 507 Asp326 residue_name_number Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS +23 32 structure evidence In the final optimized structure, the observed product state (Figure 5C, ‘iii-product’) is found in a conformation that is 7.4 kcal mol−1 more stable than the initial reactive state (Figure 5B). RESULTS +4 12 tyrosine residue_name The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS +34 48 coordinated to bond_interaction The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS +53 57 Mg2+ chemical The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS +118 122 Mg2+ chemical The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS +148 153 water chemical The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS +196 205 phosphate chemical The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS +220 226 Glu162 residue_name_number The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS +259 263 Mg2+ chemical The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS +48 62 QM/MM modeling experimental_method An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group. RESULTS +99 103 Top2 protein_type An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group. RESULTS +104 112 tyrosine residue_name An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group. RESULTS +10 19 conserved protein_state A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS +30 37 Tyr 188 residue_name_number A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS +39 46 Arg 216 residue_name_number A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS +51 58 Ser 239 residue_name_number A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS +81 90 conserved protein_state A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS +91 95 Top2 protein_type A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS +96 119 tyrosine binding pocket site A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS +16 36 cation–π interaction bond_interaction We propose this cation–π interaction further contributes to tuned stabilization of the negatively charged phenolate reaction product. RESULTS +34 57 electrostatic potential evidence Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). RESULTS +65 79 phosphotyrosyl ptm Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). RESULTS +116 124 presence protein_state Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). RESULTS +129 139 absence of protein_state Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). RESULTS +144 150 Arg216 residue_name_number Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). RESULTS +171 177 Arg216 residue_name_number Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). RESULTS +45 65 cation–π interaction bond_interaction We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A). RESULTS +127 133 Arg216 residue_name_number We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A). RESULTS +143 145 QM experimental_method We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A). RESULTS +160 162 MM experimental_method We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A). RESULTS +223 239 energy penalties evidence We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A). RESULTS +9 15 Arg216 residue_name_number Removing Arg216 from the quantum subsystem incurs an ∼2 kcal mol−1 penalty in the transition state and product complex. RESULTS +30 36 Arg216 residue_name_number Removing the +1 charge on the Arg216 has a minimal impact on the transition state, but incurs an additional ∼2 kcal mol−1 penalty in the product complex. RESULTS +12 26 QM/MM modeling experimental_method Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis. RESULTS +62 66 Tdp2 protein Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis. RESULTS +114 118 Mg2+ chemical Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis. RESULTS +170 174 Tdp2 protein Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis. RESULTS +185 203 5′-phosphotyrosine residue_name Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis. RESULTS +0 4 Tdp2 protein Tdp2 mutational analysis RESULTS +5 24 mutational analysis experimental_method Tdp2 mutational analysis RESULTS +27 31 Tdp2 protein To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +95 100 mouse taxonomy_domain To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +101 105 Tdp2 protein To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +106 124 crystal structures evidence To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +186 191 mouse taxonomy_domain To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +205 228 engineered and purified experimental_method To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +238 243 human species To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +244 247 MBP experimental_method To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +248 256 hTdp2cat structure_element To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +257 263 mutant protein_state To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +325 330 human species To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +367 376 mutations experimental_method To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +380 384 Tdp2 protein To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +457 468 tyrosylated protein_state To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +469 472 DNA chemical To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +484 488 5′-Y ptm To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +491 514 p-nitrophenyl phosphate chemical To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +516 520 PNPP chemical To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +526 572 thymidine 5′-monophosphate p-nitrophenyl ester chemical To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +574 579 T5PNP chemical To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS +132 136 Tdp2 protein By analyzing activities on this nested set of chemically related substrates we aimed to dissect structure-activity relationships of Tdp2 catalysis. RESULTS +13 22 mutations experimental_method For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS +33 37 Tdp2 protein For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS +38 49 active site site For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS +64 78 phosphotyrosyl ptm For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS +174 177 DNA chemical For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS +224 228 5′-Y ptm For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS +233 238 T5PNP chemical For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS +247 251 PNPP chemical For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS +0 18 Structural results evidence Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage. RESULTS +23 37 QM/MM modeling experimental_method Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage. RESULTS +47 54 mAsp272 residue_name_number Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage. RESULTS +67 72 water chemical Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage. RESULTS +130 152 phosphotyrosyl linkage ptm Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage. RESULTS +74 81 mutated experimental_method To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. RESULTS +85 87 to experimental_method To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. RESULTS +90 93 His residue_name To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. RESULTS +183 186 Leu residue_name To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. RESULTS +191 194 Met residue_name To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. RESULTS +228 246 water-binding site site To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. RESULTS +38 44 hD262N mutant Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis. RESULTS +65 78 substitutions experimental_method Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis. RESULTS +127 134 hAsp262 residue_name_number Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis. RESULTS +136 143 mAsp272 residue_name_number Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis. RESULTS +9 16 mutated experimental_method Next, we mutated key elements of the mobile loop (β2Hβ hydrophobic wall, Figure 2A and C). RESULTS +50 71 β2Hβ hydrophobic wall site Next, we mutated key elements of the mobile loop (β2Hβ hydrophobic wall, Figure 2A and C). RESULTS +0 9 Mutations experimental_method Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity). RESULTS +10 16 hI307A mutant Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity). RESULTS +18 24 hL305A mutant Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity). RESULTS +26 32 hL305F mutant Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity). RESULTS +37 43 hL305W mutant Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity). RESULTS +4 10 hL305W mutant The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS +97 118 β2Hβ hydrophobic wall site The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS +167 170 DNA chemical The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS +181 185 5′-Y ptm The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS +234 238 β2Hβ structure_element The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS +318 331 substitutions experimental_method The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS +354 358 PNPP chemical The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS +45 80 enzyme substrate cation–π interface site Third, we altered properties of the proposed enzyme substrate cation–π interface. RESULTS +31 37 mutant protein_state No activity was detected for a mutant that removes the positive charge at this position (hR206A). RESULTS +89 95 hR206A mutant No activity was detected for a mutant that removes the positive charge at this position (hR206A). RESULTS +29 35 pocket site The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS +70 81 replacement experimental_method The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS +85 92 hArg206 residue_name_number The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS +94 101 mArg216 residue_name_number The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS +110 116 lysine residue_name The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS +183 187 5′-Y ptm The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS +215 220 T5PNP chemical The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS +224 228 PNPP chemical The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS +11 19 mutation experimental_method Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. RESULTS +23 30 hTyr178 residue_name_number Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. RESULTS +63 70 hArg206 residue_name_number Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. RESULTS +72 79 mArg216 residue_name_number Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. RESULTS +135 140 Y178F mutant Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. RESULTS +145 150 Y178W mutant Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. RESULTS +35 42 hHis351 residue_name_number Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair. RESULTS +43 50 hAsp316 residue_name_number Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair. RESULTS +52 59 mAsp326 residue_name_number Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair. RESULTS +60 67 mHis359 residue_name_number Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair. RESULTS +14 23 mutations experimental_method We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS +29 36 removed experimental_method We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS +76 89 hydrogen bond bond_interaction We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS +91 97 hH351Q mutant We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS +131 134 pKa evidence We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS +142 151 Histidine residue_name We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS +153 159 hD316N mutant We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS +63 74 active site site Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS +86 95 aspartate residue_name Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS +97 103 mobile protein_state Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS +104 130 substrate engagement loops structure_element Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS +149 170 cation–π interactions bond_interaction Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS +176 187 active site site Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS +217 235 charge interaction bond_interaction Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS +250 254 Tdp2 protein Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS +2 6 Tdp2 protein A Tdp2 active site single nucleotide polymorphism impairs Tdp2 function RESULTS +7 18 active site site A Tdp2 active site single nucleotide polymorphism impairs Tdp2 function RESULTS +58 62 Tdp2 protein A Tdp2 active site single nucleotide polymorphism impairs Tdp2 function RESULTS +44 48 TDP2 protein Recently, it was found that inactivation of TDP2 by a splice-site mutation is associated with neurological disease and confers hypersensitivity to Top2 poisons. RESULTS +147 151 Top2 protein_type Recently, it was found that inactivation of TDP2 by a splice-site mutation is associated with neurological disease and confers hypersensitivity to Top2 poisons. RESULTS +22 27 human species We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini. RESULTS +78 82 Tdp2 protein We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini. RESULTS +83 86 DNA chemical We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini. RESULTS +150 154 Tdp2 protein We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini. RESULTS +180 183 DNA chemical We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini. RESULTS +26 31 human species We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS +32 36 TDP2 protein We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS +115 141 DNA processing active site site We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS +143 154 rs199602263 gene We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS +206 213 hAsp350 residue_name_number We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS +218 221 Asn residue_name We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS +227 237 rs77273535 gene We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS +287 294 hIle307 residue_name_number We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS +299 302 Val residue_name We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS +12 18 hD350N mutant We show the hD350N substitution severely impairs activity on all substrates tested in vitro, whereas hI307V only has a mild impact on catalysis (Figure 6B–D). RESULTS +19 31 substitution experimental_method We show the hD350N substitution severely impairs activity on all substrates tested in vitro, whereas hI307V only has a mild impact on catalysis (Figure 6B–D). RESULTS +101 107 hI307V mutant We show the hD350N substitution severely impairs activity on all substrates tested in vitro, whereas hI307V only has a mild impact on catalysis (Figure 6B–D). RESULTS +39 44 D350N mutant To better understand the basis for the D350N catalytic defect, we analyzed the structural environment of this substitution based on the high-resolution structures of mTdp2cat (Figure 6A). RESULTS +152 162 structures evidence To better understand the basis for the D350N catalytic defect, we analyzed the structural environment of this substitution based on the high-resolution structures of mTdp2cat (Figure 6A). RESULTS +166 174 mTdp2cat structure_element To better understand the basis for the D350N catalytic defect, we analyzed the structural environment of this substitution based on the high-resolution structures of mTdp2cat (Figure 6A). RESULTS +19 23 Tdp2 protein Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS +31 35 Mg2+ chemical Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS +40 69 octahedral coordination shell bond_interaction Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS +96 120 hydrogen-bonding network bond_interaction Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS +133 140 hAsp350 residue_name_number Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS +142 149 mAsp358 residue_name_number Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS +171 180 DNA-bound protein_state Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS +201 228 β2Hβ substrate-binding loop structure_element Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS +237 253 hydrogen bonding bond_interaction Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS +257 264 mTrp307 residue_name_number Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS +6 13 hAsp350 residue_name_number Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations. RESULTS +15 22 mAsp358 residue_name_number Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations. RESULTS +61 72 active site site Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations. RESULTS +90 112 substrate binding loop structure_element Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations. RESULTS +0 4 Tdp2 protein Tdp2 SNPs impair function. (A) Active site residues mutated by TDP2 SNPs. FIG +31 42 Active site site Tdp2 SNPs impair function. (A) Active site residues mutated by TDP2 SNPs. FIG +63 67 TDP2 protein Tdp2 SNPs impair function. (A) Active site residues mutated by TDP2 SNPs. FIG +0 5 D350N mutant D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG +7 12 mTdp2 protein D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG +13 18 D358N mutant D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG +24 29 I307V mutant D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG +31 36 mTdp2 protein D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG +37 42 I317V mutant D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG +44 57 substitutions experimental_method D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG +78 82 Tdp2 protein D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG +83 94 active site site D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG +118 126 mTdp2cat structure_element D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG +127 136 structure evidence D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG +27 35 SDS-PAGE experimental_method (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG +52 54 WT protein_state (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG +59 65 mutant protein_state (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG +66 69 MBP experimental_method (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG +70 78 hTdp2cat structure_element (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG +139 141 WT protein_state (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG +146 152 mutant protein_state (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG +153 156 MBP experimental_method (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG +157 165 hTdp2cat structure_element (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG +180 218 5′–phosphotyrosyl–DNA oligonucleotides chemical (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG +227 238 fluorescein chemical (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG +135 148 TBE-urea PAGE experimental_method Samples were withdrawn from reactions, neutralized with TBE-urea loading dye at the indicated timepoints, and electrophoresed on a 20% TBE-urea PAGE. FIG +25 27 WT protein_state (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. FIG +42 48 mutant protein_state (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. FIG +49 54 human species (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. FIG +55 58 MBP experimental_method (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. FIG +59 67 hTdp2cat structure_element (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. FIG +99 103 Tdp2 protein (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. FIG +26 29 MBP experimental_method Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. FIG +30 38 hTdp2cat structure_element Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. FIG +60 62 WT protein_state Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. FIG +79 103 5′-Y DNA oligonucleotide chemical Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. FIG +127 132 T5PNP chemical Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. FIG +148 152 PNPP chemical Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. FIG +11 14 PNP chemical Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C. FIG +20 33 PNP phosphate chemical Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C. FIG +35 39 PNPP chemical Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C. FIG +110 114 5′-Y ptm Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C. FIG +160 175 gel based assay experimental_method Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C. FIG +38 44 hD350N mutant To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). RESULTS +46 52 mD358N mutant To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). RESULTS +65 92 crystallized and determined experimental_method To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). RESULTS +97 106 structure evidence To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). RESULTS +114 122 DNA-free protein_state To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). RESULTS +135 141 mD358N mutant To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). RESULTS +5 14 structure evidence This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS +25 30 D358N mutant This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS +31 39 mutation experimental_method This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS +53 66 hydrogen bond bond_interaction This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS +75 81 Asp358 residue_name_number This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS +86 92 Trp307 residue_name_number This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS +117 123 Asn358 residue_name_number This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS +141 147 Trp307 residue_name_number This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS +19 35 electron density evidence Consequently, poor electron density is visible for the β2Hβ loop which is mostly disordered (Supplementary Figure S6). RESULTS +55 64 β2Hβ loop structure_element Consequently, poor electron density is visible for the β2Hβ loop which is mostly disordered (Supplementary Figure S6). RESULTS +81 91 disordered protein_state Consequently, poor electron density is visible for the β2Hβ loop which is mostly disordered (Supplementary Figure S6). RESULTS +9 13 Mg2+ chemical Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS +58 60 WT protein_state Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS +61 68 mTdpcat protein Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS +69 77 crystals evidence Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS +99 109 metal site site Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS +113 123 unoccupied protein_state Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS +131 137 mD358N mutant Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS +138 146 crystals evidence Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS +50 61 active site site Therefore, metal-regulated opening/closure of the active site may modulate Tdp2 activity, and D350N is sufficient to block both metal binding and conformational change. RESULTS +75 79 Tdp2 protein Therefore, metal-regulated opening/closure of the active site may modulate Tdp2 activity, and D350N is sufficient to block both metal binding and conformational change. RESULTS +94 99 D350N mutant Therefore, metal-regulated opening/closure of the active site may modulate Tdp2 activity, and D350N is sufficient to block both metal binding and conformational change. RESULTS +38 44 hD350N mutant In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS +46 52 mD358N mutant In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS +62 66 Mg2+ chemical In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS +90 123 intrinsic tryptophan fluorescence evidence In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS +151 155 Mg2+ chemical In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS +167 178 active site site In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS +258 262 Tdp2 protein In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS +275 279 loop structure_element In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS +0 4 Tdp2 protein Tdp2 facilitates NHEJ repair of 5′-phosphotyrosine adducted DSBs RESULTS +32 50 5′-phosphotyrosine residue_name Tdp2 facilitates NHEJ repair of 5′-phosphotyrosine adducted DSBs RESULTS +13 17 Tdp2 protein Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. RESULTS +18 44 structure/activity studies experimental_method Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. RESULTS +61 78 5′-detyrosylation ptm Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. RESULTS +79 82 DNA chemical Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. RESULTS +141 145 Tdp2 protein Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. RESULTS +169 173 Top2 protein_type Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. RESULTS +38 52 5′-tyrosylated protein_state Accordingly, we demonstrate here that 5′-tyrosylated ends are sufficient to severely impair an in vitro reconstituted mammalian NHEJ reaction (Figure 7A, lanes 3 and 6), unless supplemented with catalytic quantities of hTdp2FL (Figure 7A, lane 8). RESULTS +118 127 mammalian taxonomy_domain Accordingly, we demonstrate here that 5′-tyrosylated ends are sufficient to severely impair an in vitro reconstituted mammalian NHEJ reaction (Figure 7A, lanes 3 and 6), unless supplemented with catalytic quantities of hTdp2FL (Figure 7A, lane 8). RESULTS +219 226 hTdp2FL protein Accordingly, we demonstrate here that 5′-tyrosylated ends are sufficient to severely impair an in vitro reconstituted mammalian NHEJ reaction (Figure 7A, lanes 3 and 6), unless supplemented with catalytic quantities of hTdp2FL (Figure 7A, lane 8). RESULTS +15 23 hTdp2cat structure_element Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS +56 63 hTdp2FL protein Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS +108 131 catalytically deficient protein_state Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS +132 137 E152Q mutant Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS +138 144 mutant protein_state Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS +149 157 inactive protein_state Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS +200 204 Tdp2 protein Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS +255 269 phosphotyrosyl ptm Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS +48 56 tyrosine residue_name We confirmed that efficient joining of the same tyrosine-adducted substrate in cells (Figure 7B) was dependent on both NHEJ (reduced over 10-fold in ligase IV deficient HCT 116 cells; Supplementary Figure S7B), and Tdp2 (reduced 5-fold in Tdp2 deficient MEFs; Figure 7C). RESULTS +215 219 Tdp2 protein We confirmed that efficient joining of the same tyrosine-adducted substrate in cells (Figure 7B) was dependent on both NHEJ (reduced over 10-fold in ligase IV deficient HCT 116 cells; Supplementary Figure S7B), and Tdp2 (reduced 5-fold in Tdp2 deficient MEFs; Figure 7C). RESULTS +239 243 Tdp2 protein We confirmed that efficient joining of the same tyrosine-adducted substrate in cells (Figure 7B) was dependent on both NHEJ (reduced over 10-fold in ligase IV deficient HCT 116 cells; Supplementary Figure S7B), and Tdp2 (reduced 5-fold in Tdp2 deficient MEFs; Figure 7C). RESULTS +144 148 Tdp2 protein Moreover, products with error (i.e. junctions have missing sequence flanking the adducted terminus) are twice as frequent in cells deficient in Tdp2 (Figure 7D). RESULTS +52 60 tyrosine residue_name Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex). RESULTS +81 85 Tdp2 protein Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex). RESULTS +95 109 detyrosylation ptm Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex). RESULTS +213 220 Artemis protein Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex). RESULTS +228 244 Mre11/Rad50/Nbs1 complex_assembly Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex). RESULTS +11 15 Tdp2 protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG +16 27 active site site Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG +60 64 Tdp2 protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG +80 83 Cy5 chemical Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG +108 120 5′-phosphate chemical Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG +144 158 5′-tyrosylated protein_state Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG +199 201 Ku protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG +207 218 NHEJ ligase protein_type Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG +220 225 XRCC4 protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG +227 236 ligase IV protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG +241 244 XLF protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG +262 269 hTdp2FL protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG +49 60 native PAGE experimental_method Concatemer ligation products were detected by 5% native PAGE. FIG +24 51 cellular end joining assays experimental_method (B) Workflow diagram of cellular end joining assays. FIG +0 3 DNA chemical DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells. FIG +20 38 5′-phosphotyrosine residue_name DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells. FIG +111 120 mammalian taxonomy_domain DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells. FIG +11 14 DNA chemical After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG +65 69 qPCR experimental_method After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG +73 83 sequencing experimental_method After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG +102 106 qPCR experimental_method After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG +160 171 tyrosylated protein_state After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG +205 213 wildtype protein_state After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG +227 231 Tdp2 protein After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG +245 249 Tdp2 protein After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG +277 285 wildtype protein_state After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG +299 306 hTDP2FL protein After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG +390 392 WT protein_state After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG +53 80 cellular end-joining assays experimental_method Error bars, s.d, n = 3. (D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate. FIG +127 137 sequencing experimental_method Error bars, s.d, n = 3. (D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate. FIG +152 174 end-joining error rate evidence Error bars, s.d, n = 3. (D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate. FIG +28 53 Clonogenic survival assay experimental_method Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3. FIG +57 59 WT protein_state Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3. FIG +61 65 Tdp2 protein Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3. FIG +151 160 etoposide chemical Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3. FIG +32 41 wild-type protein_state We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C). RESULTS +46 52 mutant protein_state We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C). RESULTS +53 60 hTdp2FL protein We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C). RESULTS +84 88 Tdp2 protein We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C). RESULTS +99 104 mouse taxonomy_domain We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C). RESULTS +28 31 DNA chemical Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +37 52 phosphotyrosine residue_name Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +177 186 wild-type protein_state Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +187 192 mouse taxonomy_domain Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +206 210 Tdp2 protein Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +214 219 mouse taxonomy_domain Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +235 244 wild-type protein_state Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +245 250 human species Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +251 255 Tdp2 protein Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +261 265 Tdp2 protein Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +294 299 I307V mutant Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +300 307 variant protein_state Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +308 313 human species Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +314 318 Tdp2 protein Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS +78 82 Tdp2 protein In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N. RESULTS +133 137 Tdp2 protein In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N. RESULTS +161 165 Tdp2 protein In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N. RESULTS +166 171 D350N mutant In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N. RESULTS +71 75 Tdp2 protein Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS +96 100 Tdp2 protein Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS +132 137 D350N mutant Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS +168 177 wild type protein_state Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS +178 182 Tdp2 protein Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS +186 191 hTdp2 protein Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS +192 197 I307V mutant Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS +14 23 wild type protein_state Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS +27 32 I307V mutant Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS +33 38 human species Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS +39 43 Tdp2 protein Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS +47 51 Tdp2 protein Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS +114 123 etoposide chemical Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS +150 159 wild-type protein_state Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS +176 190 overexpression experimental_method Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS +194 199 human species Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS +200 205 D350N mutant Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS +206 210 Tdp2 protein Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS +9 14 D350N mutant The rare D350N variant is thus inactive by all metrics analyzed. RESULTS +15 22 variant protein_state The rare D350N variant is thus inactive by all metrics analyzed. RESULTS +31 39 inactive protein_state The rare D350N variant is thus inactive by all metrics analyzed. RESULTS +32 37 I307V mutant By comparison the more frequent I307V has only mild effects on in vitro activity, and no detectable impact on cellular assays. RESULTS +0 4 Top2 protein_type Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable. DISCUSS +99 102 DNA chemical Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable. DISCUSS +115 119 Top2 protein_type Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable. DISCUSS +120 123 DNA chemical Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable. DISCUSS +42 45 DNA chemical Understanding how cells cope with complex DNA breaks bearing topoisomerase–DNA protein crosslinks is key to deciphering individual responses to chemotherapeutic outcomes and genotoxic agents that poison Top2. DISCUSS +75 78 DNA chemical Understanding how cells cope with complex DNA breaks bearing topoisomerase–DNA protein crosslinks is key to deciphering individual responses to chemotherapeutic outcomes and genotoxic agents that poison Top2. DISCUSS +203 207 Top2 protein_type Understanding how cells cope with complex DNA breaks bearing topoisomerase–DNA protein crosslinks is key to deciphering individual responses to chemotherapeutic outcomes and genotoxic agents that poison Top2. DISCUSS +14 25 mutagenesis experimental_method Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +30 47 functional assays experimental_method Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +57 61 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +62 72 structures evidence Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +73 90 in the absence of protein_state Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +91 98 ligands chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +103 118 in complex with protein_state Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +119 122 DNA chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +158 162 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +163 166 DNA chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +205 209 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +210 221 active site site Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +268 271 DNA chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +320 323 DNA chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +345 349 Top2 protein_type Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +382 401 structural analysis experimental_method Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +415 433 mutational studies experimental_method Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +438 462 QM/MM molecular modeling experimental_method Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +470 474 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +557 560 EEP structure_element Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +578 598 phosphoryl hydrolase protein_type Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +620 624 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +625 636 active site site Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +640 664 conformationally plastic protein_state Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +710 713 DNA chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +718 722 Mg2+ chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +769 773 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +793 797 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +798 809 active site site Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS +31 35 Tdp2 protein This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing. DISCUSS +62 65 DNA chemical This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing. DISCUSS +149 153 Tdp2 protein This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing. DISCUSS +154 157 DNA chemical This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing. DISCUSS +0 4 Tdp2 protein Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS +74 78 Top1 protein_type Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS +83 87 Top2 protein_type Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS +188 192 Tdp2 protein Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS +215 218 DNA chemical Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS +238 242 Top2 protein_type Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS +257 261 Top2 protein_type Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS +262 265 DNA chemical Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS +26 29 DNA chemical The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. DISCUSS +52 56 Top2 protein_type The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. DISCUSS +57 60 DNA chemical The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. DISCUSS +97 101 Tdp2 protein The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. DISCUSS +156 169 topoisomerase protein_type The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. DISCUSS +185 196 active site site The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. DISCUSS +4 8 Tdp2 protein The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways. DISCUSS +9 37 substrate interaction groove site The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways. DISCUSS +50 53 DNA chemical The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways. DISCUSS +11 38 nucleic acid binding trench site First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS +57 64 dynamic protein_state First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS +65 93 β2Hβ DNA damage-binding loop structure_element First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS +148 171 phosphotyrosyl linkages ptm First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS +217 219 ϵA chemical First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS +292 305 base-stacking bond_interaction First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS +306 322 hydrophobic wall site First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS +330 339 β2Hβ-loop structure_element First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS +14 19 QM/MM experimental_method Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal. DISCUSS +68 88 cation–π interaction bond_interaction Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal. DISCUSS +125 129 Tdp2 protein Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal. DISCUSS +138 141 DNA chemical Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal. DISCUSS +4 22 strictly conserved protein_state The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group. DISCUSS +23 34 active site site The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group. DISCUSS +35 41 Arg216 residue_name_number The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group. DISCUSS +137 151 phosphotyrosyl ptm The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group. DISCUSS +58 86 substrate cation–π interface site To our knowledge, this is the first proposed example of a substrate cation–π interface exploited to promote a phosphoryl-transfer reaction. DISCUSS +85 89 Tdp2 protein This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS +166 172 Top2cc complex_assembly This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS +174 186 picornaviral taxonomy_domain This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS +195 198 RNA chemical This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS +203 220 Hepatitis B Virus taxonomy_domain This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS +222 225 HBV taxonomy_domain This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS +235 238 DNA chemical This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS +21 24 EEP structure_element By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. DISCUSS +25 34 nucleases protein_type By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. DISCUSS +43 47 Ape1 protein By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. DISCUSS +52 56 Ape2 protein By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. DISCUSS +77 80 DNA chemical By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. DISCUSS +159 163 Tdp2 protein By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. DISCUSS +26 30 Tdp2 protein The dynamic nature of the Tdp2 active site presents opportunities for enzyme regulation. DISCUSS +31 42 active site site The dynamic nature of the Tdp2 active site presents opportunities for enzyme regulation. DISCUSS +56 60 Tdp2 protein However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 β2Hβ-loop is unknown. DISCUSS +102 106 Tdp2 protein However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 β2Hβ-loop is unknown. DISCUSS +107 116 β2Hβ-loop structure_element However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 β2Hβ-loop is unknown. DISCUSS +35 39 Top2 protein_type We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS +63 66 DNA chemical We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS +132 136 Tdp2 protein We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS +137 148 active site site We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS +178 182 Tdp2 protein We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS +222 226 Top2 protein_type We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS +306 310 Top2 protein_type We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS +29 39 structures evidence Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS +43 48 mouse taxonomy_domain Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS +71 81 C. elegans species Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS +82 86 Tdp2 protein Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS +129 133 Tdp2 protein Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS +134 145 active site site Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS +151 156 worms taxonomy_domain Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS +160 163 man taxonomy_domain Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS +83 116 intrinsic tryptophan fluorescence experimental_method Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS +118 143 crystallographic analysis experimental_method Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS +180 191 mutagenesis experimental_method Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS +193 216 Ca2+ inhibition studies experimental_method Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS +221 235 QM/MM analysis experimental_method Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS +271 275 Mg2+ chemical Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS +285 289 Tdp2 protein Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS +0 9 Etoposide chemical Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS +20 24 Top2 protein_type Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS +74 78 Tdp2 protein Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS +107 112 human species Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS +151 155 Top2 protein_type Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS +174 183 etoposide chemical Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS +188 199 doxyrubicin chemical Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS +6 10 Tdp2 protein Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection. DISCUSS +28 33 human species Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection. DISCUSS +117 121 TDP2 protein Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection. DISCUSS +259 263 Top2 protein_type Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection. DISCUSS +277 282 viral taxonomy_domain Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection. DISCUSS +8 12 Tdp2 protein Lastly, Tdp2 inhibitors may synergize or potentiate cytotoxic effects of current anticancer treatments that target Tdp2. DISCUSS +115 119 Tdp2 protein Lastly, Tdp2 inhibitors may synergize or potentiate cytotoxic effects of current anticancer treatments that target Tdp2. DISCUSS +98 102 Tdp2 protein Thus, we anticipate this atomic-level and mechanistic definition of the molecular determinants of Tdp2 catalysis and conformational changes driven by DNA–protein and protein–protein interactions will foster unique strategies for the development of Tdp2 targeted small molecule interventions. DISCUSS +150 153 DNA chemical Thus, we anticipate this atomic-level and mechanistic definition of the molecular determinants of Tdp2 catalysis and conformational changes driven by DNA–protein and protein–protein interactions will foster unique strategies for the development of Tdp2 targeted small molecule interventions. DISCUSS +248 252 Tdp2 protein Thus, we anticipate this atomic-level and mechanistic definition of the molecular determinants of Tdp2 catalysis and conformational changes driven by DNA–protein and protein–protein interactions will foster unique strategies for the development of Tdp2 targeted small molecule interventions. DISCUSS diff --git a/annotation_CSV/PMC4869123.csv b/annotation_CSV/PMC4869123.csv new file mode 100644 index 0000000000000000000000000000000000000000..11a5a3ddbf11ec980a07a4400fa0332a1fa35dfd --- /dev/null +++ b/annotation_CSV/PMC4869123.csv @@ -0,0 +1,923 @@ +anno_start anno_end anno_text entity_type sentence section +19 25 IL-17A protein Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide TITLE +30 37 IL-17RA protein Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide TITLE +65 72 peptide chemical Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide TITLE +0 6 IL-17A protein IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. ABSTRACT +29 37 cytokine protein_type IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. ABSTRACT +11 21 antibodies protein_type Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. ABSTRACT +33 39 IL-17A protein Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. ABSTRACT +2 41 high affinity IL-17A peptide antagonist chemical A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. ABSTRACT +43 46 HAP chemical A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. ABSTRACT +51 62 15 residues residue_range A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. ABSTRACT +86 109 phage-display screening experimental_method A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. ABSTRACT +122 157 saturation mutagenesis optimization experimental_method A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. ABSTRACT +162 186 amino acid substitutions experimental_method A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. ABSTRACT +0 3 HAP chemical HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. ABSTRACT +26 32 IL-17A protein HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. ABSTRACT +69 77 cytokine protein_type HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. ABSTRACT +87 95 receptor protein_type HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. ABSTRACT +97 104 IL-17RA protein HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. ABSTRACT +18 23 human species Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. ABSTRACT +31 34 HAP chemical Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. ABSTRACT +83 92 cytokines protein_type Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. ABSTRACT +0 25 Crystal structure studies experimental_method Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. ABSTRACT +44 47 HAP chemical Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. ABSTRACT +70 76 IL-17A protein Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. ABSTRACT +77 82 dimer oligomeric_state Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. ABSTRACT +27 30 HAP chemical The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. ABSTRACT +38 46 β-strand structure_element The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. ABSTRACT +72 78 IL-17A protein The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. ABSTRACT +79 87 monomers oligomeric_state The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. ABSTRACT +126 133 α helix structure_element The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. ABSTRACT +155 162 IL-17RA protein The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. ABSTRACT +198 204 IL-17A protein The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. ABSTRACT +14 29 IL-17 cytokines protein_type The family of IL-17 cytokines and receptors consists of six polypeptides, IL-17A-F, and five receptors, IL-17RA-E. IL-17A is secreted from activated Th17 cells, and several innate immune T cell types including macrophages, neutrophils, natural killer cells, and dendritic cells. INTRO +74 82 IL-17A-F protein The family of IL-17 cytokines and receptors consists of six polypeptides, IL-17A-F, and five receptors, IL-17RA-E. IL-17A is secreted from activated Th17 cells, and several innate immune T cell types including macrophages, neutrophils, natural killer cells, and dendritic cells. INTRO +104 113 IL-17RA-E protein The family of IL-17 cytokines and receptors consists of six polypeptides, IL-17A-F, and five receptors, IL-17RA-E. IL-17A is secreted from activated Th17 cells, and several innate immune T cell types including macrophages, neutrophils, natural killer cells, and dendritic cells. INTRO +115 121 IL-17A protein The family of IL-17 cytokines and receptors consists of six polypeptides, IL-17A-F, and five receptors, IL-17RA-E. IL-17A is secreted from activated Th17 cells, and several innate immune T cell types including macrophages, neutrophils, natural killer cells, and dendritic cells. INTRO +0 6 IL-17A protein IL-17A signals through a specific cell surface receptor complex which consists of IL-17RA and IL-17RC. INTRO +47 55 receptor protein_type IL-17A signals through a specific cell surface receptor complex which consists of IL-17RA and IL-17RC. INTRO +82 89 IL-17RA protein IL-17A signals through a specific cell surface receptor complex which consists of IL-17RA and IL-17RC. INTRO +94 101 IL-17RC protein IL-17A signals through a specific cell surface receptor complex which consists of IL-17RA and IL-17RC. INTRO +0 6 IL-17A protein IL-17A’s downstream signaling leads to increased production of inflammatory cytokines such as IL-6, IL-8, CCL-20 and CXCL1 by various mechanisms including stimulation of transcription and stabilization of mRNA. INTRO +76 85 cytokines protein_type IL-17A’s downstream signaling leads to increased production of inflammatory cytokines such as IL-6, IL-8, CCL-20 and CXCL1 by various mechanisms including stimulation of transcription and stabilization of mRNA. INTRO +94 98 IL-6 protein_type IL-17A’s downstream signaling leads to increased production of inflammatory cytokines such as IL-6, IL-8, CCL-20 and CXCL1 by various mechanisms including stimulation of transcription and stabilization of mRNA. INTRO +100 104 IL-8 protein_type IL-17A’s downstream signaling leads to increased production of inflammatory cytokines such as IL-6, IL-8, CCL-20 and CXCL1 by various mechanisms including stimulation of transcription and stabilization of mRNA. INTRO +106 112 CCL-20 protein_type IL-17A’s downstream signaling leads to increased production of inflammatory cytokines such as IL-6, IL-8, CCL-20 and CXCL1 by various mechanisms including stimulation of transcription and stabilization of mRNA. INTRO +117 122 CXCL1 protein_type IL-17A’s downstream signaling leads to increased production of inflammatory cytokines such as IL-6, IL-8, CCL-20 and CXCL1 by various mechanisms including stimulation of transcription and stabilization of mRNA. INTRO +205 209 mRNA chemical IL-17A’s downstream signaling leads to increased production of inflammatory cytokines such as IL-6, IL-8, CCL-20 and CXCL1 by various mechanisms including stimulation of transcription and stabilization of mRNA. INTRO +58 65 IL-17RA protein Although various cell types have been reported to express IL-17RA, the highest responses to IL-17A come from epithelial cells, endothelial cells, keratinocytes and fibroblasts. INTRO +92 98 IL-17A protein Although various cell types have been reported to express IL-17RA, the highest responses to IL-17A come from epithelial cells, endothelial cells, keratinocytes and fibroblasts. INTRO +0 6 IL-17A protein IL-17A and its signaling is important in host defense against certain fungal and bacterial infections as demonstrated by patients with autoantibodies against IL-17A and IL-17F, or with inborn errors of IL-17 immunity. INTRO +158 164 IL-17A protein IL-17A and its signaling is important in host defense against certain fungal and bacterial infections as demonstrated by patients with autoantibodies against IL-17A and IL-17F, or with inborn errors of IL-17 immunity. INTRO +169 175 IL-17F protein IL-17A and its signaling is important in host defense against certain fungal and bacterial infections as demonstrated by patients with autoantibodies against IL-17A and IL-17F, or with inborn errors of IL-17 immunity. INTRO +202 207 IL-17 protein_type IL-17A and its signaling is important in host defense against certain fungal and bacterial infections as demonstrated by patients with autoantibodies against IL-17A and IL-17F, or with inborn errors of IL-17 immunity. INTRO +39 45 IL-17A protein In addition to its physiological role, IL-17A is a key pathogenic factor in inflammatory and autoimmune diseases. INTRO +61 71 antibodies protein_type In phase II and III clinical trials, neutralizing monoclonal antibodies against IL-17A (secukinumab and ixekizumab) or its receptor IL-17RA (brodalumab) are highly efficacious in treating moderate to severe plaque psoriasis and psoriatic arthritis. INTRO +80 86 IL-17A protein In phase II and III clinical trials, neutralizing monoclonal antibodies against IL-17A (secukinumab and ixekizumab) or its receptor IL-17RA (brodalumab) are highly efficacious in treating moderate to severe plaque psoriasis and psoriatic arthritis. INTRO +88 99 secukinumab chemical In phase II and III clinical trials, neutralizing monoclonal antibodies against IL-17A (secukinumab and ixekizumab) or its receptor IL-17RA (brodalumab) are highly efficacious in treating moderate to severe plaque psoriasis and psoriatic arthritis. INTRO +104 114 ixekizumab chemical In phase II and III clinical trials, neutralizing monoclonal antibodies against IL-17A (secukinumab and ixekizumab) or its receptor IL-17RA (brodalumab) are highly efficacious in treating moderate to severe plaque psoriasis and psoriatic arthritis. INTRO +123 131 receptor protein_type In phase II and III clinical trials, neutralizing monoclonal antibodies against IL-17A (secukinumab and ixekizumab) or its receptor IL-17RA (brodalumab) are highly efficacious in treating moderate to severe plaque psoriasis and psoriatic arthritis. INTRO +132 139 IL-17RA protein In phase II and III clinical trials, neutralizing monoclonal antibodies against IL-17A (secukinumab and ixekizumab) or its receptor IL-17RA (brodalumab) are highly efficacious in treating moderate to severe plaque psoriasis and psoriatic arthritis. INTRO +141 151 brodalumab chemical In phase II and III clinical trials, neutralizing monoclonal antibodies against IL-17A (secukinumab and ixekizumab) or its receptor IL-17RA (brodalumab) are highly efficacious in treating moderate to severe plaque psoriasis and psoriatic arthritis. INTRO +0 11 Secukinumab chemical Secukinumab has been approved recently as a new psoriasis drug by the US Food and Drug Administration (Cosentyx™). INTRO +103 112 Cosentyx™ chemical Secukinumab has been approved recently as a new psoriasis drug by the US Food and Drug Administration (Cosentyx™). INTRO +50 56 IL-17A protein In addition to psoriasis and psoriatic arthritis, IL-17A blockade has also shown preclinical and clinical efficacies in ankylosing spondylitis and rheumatoid arthritis. INTRO +6 21 IL-17 cytokines protein_type Among IL-17 cytokines, IL-17A and IL-17F share the highest homology. INTRO +23 29 IL-17A protein Among IL-17 cytokines, IL-17A and IL-17F share the highest homology. INTRO +34 40 IL-17F protein Among IL-17 cytokines, IL-17A and IL-17F share the highest homology. INTRO +24 32 covalent protein_state These polypeptides form covalent homodimers, and IL-17A and IL-17F also form an IL-17A/IL-17F hetereodimer. INTRO +33 43 homodimers oligomeric_state These polypeptides form covalent homodimers, and IL-17A and IL-17F also form an IL-17A/IL-17F hetereodimer. INTRO +49 55 IL-17A protein These polypeptides form covalent homodimers, and IL-17A and IL-17F also form an IL-17A/IL-17F hetereodimer. INTRO +60 66 IL-17F protein These polypeptides form covalent homodimers, and IL-17A and IL-17F also form an IL-17A/IL-17F hetereodimer. INTRO +80 93 IL-17A/IL-17F complex_assembly These polypeptides form covalent homodimers, and IL-17A and IL-17F also form an IL-17A/IL-17F hetereodimer. INTRO +94 106 hetereodimer oligomeric_state These polypeptides form covalent homodimers, and IL-17A and IL-17F also form an IL-17A/IL-17F hetereodimer. INTRO +0 10 Structures evidence Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. INTRO +25 28 apo protein_state Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. INTRO +29 35 IL-17F protein Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. INTRO +44 56 complex with protein_state Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. INTRO +57 64 IL-17RA protein Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. INTRO +70 73 apo protein_state Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. INTRO +74 80 IL-17A protein Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. INTRO +86 98 complex with protein_state Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. INTRO +102 110 antibody protein_type Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. INTRO +111 114 Fab structure_element Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. INTRO +124 136 complex with protein_state Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. INTRO +137 144 IL-17RA protein Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. INTRO +9 19 structures evidence In these structures, both IL-17A and IL-17F adopt a cysteine-knot fold with two intramolecular disulfides and two interchain disulfide bonds that covalently link two monomers. INTRO +26 32 IL-17A protein In these structures, both IL-17A and IL-17F adopt a cysteine-knot fold with two intramolecular disulfides and two interchain disulfide bonds that covalently link two monomers. INTRO +37 43 IL-17F protein In these structures, both IL-17A and IL-17F adopt a cysteine-knot fold with two intramolecular disulfides and two interchain disulfide bonds that covalently link two monomers. INTRO +52 65 cysteine-knot structure_element In these structures, both IL-17A and IL-17F adopt a cysteine-knot fold with two intramolecular disulfides and two interchain disulfide bonds that covalently link two monomers. INTRO +95 105 disulfides ptm In these structures, both IL-17A and IL-17F adopt a cysteine-knot fold with two intramolecular disulfides and two interchain disulfide bonds that covalently link two monomers. INTRO +125 140 disulfide bonds ptm In these structures, both IL-17A and IL-17F adopt a cysteine-knot fold with two intramolecular disulfides and two interchain disulfide bonds that covalently link two monomers. INTRO +166 174 monomers oligomeric_state In these structures, both IL-17A and IL-17F adopt a cysteine-knot fold with two intramolecular disulfides and two interchain disulfide bonds that covalently link two monomers. INTRO +116 122 IL-17A protein There has been active research in identifying orally available chemical entities that would functionally antagonize IL-17A-mediated signaling. INTRO +141 166 IL-17A/IL-17RA interfaces site Developing small molecules targeting protein-protein interactions is difficult with particular challenges associated with the large, shallow IL-17A/IL-17RA interfaces. INTRO +6 13 IL-17RA protein Since IL-17RA is a shared receptor for at least IL-17A, IL-17F, IL-17A/IL-17F and IL-17E, we chose to seek IL-17A-specific inhibitors that may have more defined pharmacological responses than IL-17RA inhibitors. INTRO +26 34 receptor protein_type Since IL-17RA is a shared receptor for at least IL-17A, IL-17F, IL-17A/IL-17F and IL-17E, we chose to seek IL-17A-specific inhibitors that may have more defined pharmacological responses than IL-17RA inhibitors. INTRO +48 54 IL-17A protein Since IL-17RA is a shared receptor for at least IL-17A, IL-17F, IL-17A/IL-17F and IL-17E, we chose to seek IL-17A-specific inhibitors that may have more defined pharmacological responses than IL-17RA inhibitors. INTRO +56 62 IL-17F protein Since IL-17RA is a shared receptor for at least IL-17A, IL-17F, IL-17A/IL-17F and IL-17E, we chose to seek IL-17A-specific inhibitors that may have more defined pharmacological responses than IL-17RA inhibitors. INTRO +64 77 IL-17A/IL-17F complex_assembly Since IL-17RA is a shared receptor for at least IL-17A, IL-17F, IL-17A/IL-17F and IL-17E, we chose to seek IL-17A-specific inhibitors that may have more defined pharmacological responses than IL-17RA inhibitors. INTRO +82 88 IL-17E protein Since IL-17RA is a shared receptor for at least IL-17A, IL-17F, IL-17A/IL-17F and IL-17E, we chose to seek IL-17A-specific inhibitors that may have more defined pharmacological responses than IL-17RA inhibitors. INTRO +107 113 IL-17A protein Since IL-17RA is a shared receptor for at least IL-17A, IL-17F, IL-17A/IL-17F and IL-17E, we chose to seek IL-17A-specific inhibitors that may have more defined pharmacological responses than IL-17RA inhibitors. INTRO +192 199 IL-17RA protein Since IL-17RA is a shared receptor for at least IL-17A, IL-17F, IL-17A/IL-17F and IL-17E, we chose to seek IL-17A-specific inhibitors that may have more defined pharmacological responses than IL-17RA inhibitors. INTRO +39 78 high affinity IL-17A peptide antagonist chemical Our efforts resulted in discovery of a high affinity IL-17A peptide antagonist (HAP), which we attempted to increase the functional production and pharmacokinetics after fusing HAP to antibodies for evaluation as a bispecific therapeutic in animal studies. INTRO +80 83 HAP chemical Our efforts resulted in discovery of a high affinity IL-17A peptide antagonist (HAP), which we attempted to increase the functional production and pharmacokinetics after fusing HAP to antibodies for evaluation as a bispecific therapeutic in animal studies. INTRO +170 176 fusing experimental_method Our efforts resulted in discovery of a high affinity IL-17A peptide antagonist (HAP), which we attempted to increase the functional production and pharmacokinetics after fusing HAP to antibodies for evaluation as a bispecific therapeutic in animal studies. INTRO +177 180 HAP chemical Our efforts resulted in discovery of a high affinity IL-17A peptide antagonist (HAP), which we attempted to increase the functional production and pharmacokinetics after fusing HAP to antibodies for evaluation as a bispecific therapeutic in animal studies. INTRO +184 194 antibodies protein_type Our efforts resulted in discovery of a high affinity IL-17A peptide antagonist (HAP), which we attempted to increase the functional production and pharmacokinetics after fusing HAP to antibodies for evaluation as a bispecific therapeutic in animal studies. INTRO +63 71 uncapped protein_state Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR). INTRO +72 75 HAP chemical Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR). INTRO +167 170 HAP chemical Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR). INTRO +186 203 complex structure evidence Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR). INTRO +207 213 IL-17A protein Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR). INTRO +219 222 HAP chemical Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR). INTRO +274 294 peptide optimization experimental_method Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR). INTRO +299 330 structure activity relationship experimental_method Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR). INTRO +332 335 SAR experimental_method Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR). INTRO +18 24 IL-17A protein Identification of IL-17A peptide inhibitors RESULTS +33 38 human species Peptides specifically binding to human IL-17A were identified from phage panning using cyclic and linear peptide libraries (Supplementary Figure S1). RESULTS +39 45 IL-17A protein Peptides specifically binding to human IL-17A were identified from phage panning using cyclic and linear peptide libraries (Supplementary Figure S1). RESULTS +67 80 phage panning experimental_method Peptides specifically binding to human IL-17A were identified from phage panning using cyclic and linear peptide libraries (Supplementary Figure S1). RESULTS +87 122 cyclic and linear peptide libraries experimental_method Peptides specifically binding to human IL-17A were identified from phage panning using cyclic and linear peptide libraries (Supplementary Figure S1). RESULTS +0 20 Positive phage pools experimental_method Positive phage pools were then sub-cloned into a maltose-binding protein (MBP) fusion system. RESULTS +31 41 sub-cloned experimental_method Positive phage pools were then sub-cloned into a maltose-binding protein (MBP) fusion system. RESULTS +49 92 maltose-binding protein (MBP) fusion system experimental_method Positive phage pools were then sub-cloned into a maltose-binding protein (MBP) fusion system. RESULTS +104 137 enzyme-linked immunosorbent assay experimental_method Single clones were isolated and sub-cultured in growth medium, and culture supernatants were used in an enzyme-linked immunosorbent assay (ELISA) to identify specific IL-17A-binding clones. RESULTS +139 144 ELISA experimental_method Single clones were isolated and sub-cultured in growth medium, and culture supernatants were used in an enzyme-linked immunosorbent assay (ELISA) to identify specific IL-17A-binding clones. RESULTS +167 173 IL-17A protein Single clones were isolated and sub-cultured in growth medium, and culture supernatants were used in an enzyme-linked immunosorbent assay (ELISA) to identify specific IL-17A-binding clones. RESULTS +71 83 biotinylated protein_state The positive binding supernatants were tested for the ability to block biotinylated IL-17A signaling through IL-17RA in an IL-17A/IL-17RA competition ELISA assay where unlabeled IL-17A was used as positive control to inhibit biotinylated IL-17A binding. RESULTS +84 90 IL-17A protein The positive binding supernatants were tested for the ability to block biotinylated IL-17A signaling through IL-17RA in an IL-17A/IL-17RA competition ELISA assay where unlabeled IL-17A was used as positive control to inhibit biotinylated IL-17A binding. RESULTS +109 116 IL-17RA protein The positive binding supernatants were tested for the ability to block biotinylated IL-17A signaling through IL-17RA in an IL-17A/IL-17RA competition ELISA assay where unlabeled IL-17A was used as positive control to inhibit biotinylated IL-17A binding. RESULTS +123 137 IL-17A/IL-17RA complex_assembly The positive binding supernatants were tested for the ability to block biotinylated IL-17A signaling through IL-17RA in an IL-17A/IL-17RA competition ELISA assay where unlabeled IL-17A was used as positive control to inhibit biotinylated IL-17A binding. RESULTS +138 161 competition ELISA assay experimental_method The positive binding supernatants were tested for the ability to block biotinylated IL-17A signaling through IL-17RA in an IL-17A/IL-17RA competition ELISA assay where unlabeled IL-17A was used as positive control to inhibit biotinylated IL-17A binding. RESULTS +178 184 IL-17A protein The positive binding supernatants were tested for the ability to block biotinylated IL-17A signaling through IL-17RA in an IL-17A/IL-17RA competition ELISA assay where unlabeled IL-17A was used as positive control to inhibit biotinylated IL-17A binding. RESULTS +225 237 biotinylated protein_state The positive binding supernatants were tested for the ability to block biotinylated IL-17A signaling through IL-17RA in an IL-17A/IL-17RA competition ELISA assay where unlabeled IL-17A was used as positive control to inhibit biotinylated IL-17A binding. RESULTS +238 244 IL-17A protein The positive binding supernatants were tested for the ability to block biotinylated IL-17A signaling through IL-17RA in an IL-17A/IL-17RA competition ELISA assay where unlabeled IL-17A was used as positive control to inhibit biotinylated IL-17A binding. RESULTS +59 65 IL-17A protein Approximately 10% of the clones that specifically bound to IL-17A also prevented the cytokine from binding to IL-17RA. RESULTS +85 93 cytokine protein_type Approximately 10% of the clones that specifically bound to IL-17A also prevented the cytokine from binding to IL-17RA. RESULTS +110 117 IL-17RA protein Approximately 10% of the clones that specifically bound to IL-17A also prevented the cytokine from binding to IL-17RA. RESULTS +26 38 phage clones experimental_method Sequences identified from phage clones were chemically synthesized (Supplementary Table 1) and tested for inhibition of IL-17A binding to IL-17RA (Table 1). RESULTS +44 66 chemically synthesized experimental_method Sequences identified from phage clones were chemically synthesized (Supplementary Table 1) and tested for inhibition of IL-17A binding to IL-17RA (Table 1). RESULTS +120 126 IL-17A protein Sequences identified from phage clones were chemically synthesized (Supplementary Table 1) and tested for inhibition of IL-17A binding to IL-17RA (Table 1). RESULTS +138 145 IL-17RA protein Sequences identified from phage clones were chemically synthesized (Supplementary Table 1) and tested for inhibition of IL-17A binding to IL-17RA (Table 1). RESULTS +16 25 peptide 1 chemical A 15-mer linear peptide 1 was shown to block IL-17A/IL-17RA binding with an IC50 of 80 nM in the competition ELISA assay (Table 1). RESULTS +45 59 IL-17A/IL-17RA complex_assembly A 15-mer linear peptide 1 was shown to block IL-17A/IL-17RA binding with an IC50 of 80 nM in the competition ELISA assay (Table 1). RESULTS +76 80 IC50 evidence A 15-mer linear peptide 1 was shown to block IL-17A/IL-17RA binding with an IC50 of 80 nM in the competition ELISA assay (Table 1). RESULTS +97 120 competition ELISA assay experimental_method A 15-mer linear peptide 1 was shown to block IL-17A/IL-17RA binding with an IC50 of 80 nM in the competition ELISA assay (Table 1). RESULTS +34 61 cell-based functional assay experimental_method This peptide was then tested in a cell-based functional assay wherein production of GRO-α in BJ human fibroblast cells was measured as a function of IL-17A stimulation using 1 ng/ml IL-17A. RESULTS +84 89 GRO-α protein This peptide was then tested in a cell-based functional assay wherein production of GRO-α in BJ human fibroblast cells was measured as a function of IL-17A stimulation using 1 ng/ml IL-17A. RESULTS +96 101 human species This peptide was then tested in a cell-based functional assay wherein production of GRO-α in BJ human fibroblast cells was measured as a function of IL-17A stimulation using 1 ng/ml IL-17A. RESULTS +149 155 IL-17A protein This peptide was then tested in a cell-based functional assay wherein production of GRO-α in BJ human fibroblast cells was measured as a function of IL-17A stimulation using 1 ng/ml IL-17A. RESULTS +182 188 IL-17A protein This peptide was then tested in a cell-based functional assay wherein production of GRO-α in BJ human fibroblast cells was measured as a function of IL-17A stimulation using 1 ng/ml IL-17A. RESULTS +0 9 Peptide 1 chemical Peptide 1 was found to be active in this functional assay with an IC50 of 370 nM. RESULTS +41 57 functional assay experimental_method Peptide 1 was found to be active in this functional assay with an IC50 of 370 nM. RESULTS +66 70 IC50 evidence Peptide 1 was found to be active in this functional assay with an IC50 of 370 nM. RESULTS +16 22 IL-17A protein Optimization of IL-17A peptide inhibitors RESULTS +2 5 SAR experimental_method A SAR campaign was undertaken to improve the potency of peptide 1. RESULTS +56 65 peptide 1 chemical A SAR campaign was undertaken to improve the potency of peptide 1. RESULTS +3 15 alanine scan experimental_method An alanine scan of peptide 2, an analogue of 1 with a lysine to arginine substitution at position 14, was initiated. RESULTS +19 28 peptide 2 chemical An alanine scan of peptide 2, an analogue of 1 with a lysine to arginine substitution at position 14, was initiated. RESULTS +45 46 1 chemical An alanine scan of peptide 2, an analogue of 1 with a lysine to arginine substitution at position 14, was initiated. RESULTS +54 60 lysine residue_name An alanine scan of peptide 2, an analogue of 1 with a lysine to arginine substitution at position 14, was initiated. RESULTS +64 72 arginine residue_name An alanine scan of peptide 2, an analogue of 1 with a lysine to arginine substitution at position 14, was initiated. RESULTS +73 85 substitution experimental_method An alanine scan of peptide 2, an analogue of 1 with a lysine to arginine substitution at position 14, was initiated. RESULTS +98 100 14 residue_number An alanine scan of peptide 2, an analogue of 1 with a lysine to arginine substitution at position 14, was initiated. RESULTS +5 12 alanine residue_name When alanine was already present (positions 7 and 15), substitution was made with lysine (Table 1, peptides 3–17). RESULTS +44 45 7 residue_number When alanine was already present (positions 7 and 15), substitution was made with lysine (Table 1, peptides 3–17). RESULTS +50 52 15 residue_number When alanine was already present (positions 7 and 15), substitution was made with lysine (Table 1, peptides 3–17). RESULTS +55 67 substitution experimental_method When alanine was already present (positions 7 and 15), substitution was made with lysine (Table 1, peptides 3–17). RESULTS +82 88 lysine residue_name When alanine was already present (positions 7 and 15), substitution was made with lysine (Table 1, peptides 3–17). RESULTS +99 112 peptides 3–17 chemical When alanine was already present (positions 7 and 15), substitution was made with lysine (Table 1, peptides 3–17). RESULTS +10 11 1 residue_number Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA. RESULTS +13 14 2 residue_number Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA. RESULTS +16 17 4 residue_number Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA. RESULTS +19 20 5 residue_number Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA. RESULTS +22 23 7 residue_number Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA. RESULTS +25 27 14 residue_number Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA. RESULTS +32 34 15 residue_number Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA. RESULTS +124 140 binding affinity evidence Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA. RESULTS +160 174 IL-17A/IL-17RA complex_assembly Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA. RESULTS +175 192 competition ELISA experimental_method Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA. RESULTS +27 28 5 residue_number In particular, at position 5 (13), substitution of methionine with alanine resulted in a seven fold improvement in potency (80 nM versus 11 nM respectively). RESULTS +30 32 13 chemical In particular, at position 5 (13), substitution of methionine with alanine resulted in a seven fold improvement in potency (80 nM versus 11 nM respectively). RESULTS +35 47 substitution experimental_method In particular, at position 5 (13), substitution of methionine with alanine resulted in a seven fold improvement in potency (80 nM versus 11 nM respectively). RESULTS +51 61 methionine residue_name In particular, at position 5 (13), substitution of methionine with alanine resulted in a seven fold improvement in potency (80 nM versus 11 nM respectively). RESULTS +67 74 alanine residue_name In particular, at position 5 (13), substitution of methionine with alanine resulted in a seven fold improvement in potency (80 nM versus 11 nM respectively). RESULTS +44 56 substitution experimental_method In order to rapidly evaluate the effects of substitution of natural amino acids at tolerant positions identified by the alanine scan, the lead sequence was subjected to site-specific saturation mutagenesis using MBP. RESULTS +120 132 alanine scan experimental_method In order to rapidly evaluate the effects of substitution of natural amino acids at tolerant positions identified by the alanine scan, the lead sequence was subjected to site-specific saturation mutagenesis using MBP. RESULTS +169 205 site-specific saturation mutagenesis experimental_method In order to rapidly evaluate the effects of substitution of natural amino acids at tolerant positions identified by the alanine scan, the lead sequence was subjected to site-specific saturation mutagenesis using MBP. RESULTS +212 215 MBP experimental_method In order to rapidly evaluate the effects of substitution of natural amino acids at tolerant positions identified by the alanine scan, the lead sequence was subjected to site-specific saturation mutagenesis using MBP. RESULTS +46 58 alanine scan experimental_method Each of the seven positions identified by the alanine scan was individually modified while keeping the rest of the sequence constant. RESULTS +27 28 2 residue_number Modifications at positions 2 and 14 were shown to display improvement in binding affinity (data not shown). RESULTS +33 35 14 residue_number Modifications at positions 2 and 14 were shown to display improvement in binding affinity (data not shown). RESULTS +73 89 binding affinity evidence Modifications at positions 2 and 14 were shown to display improvement in binding affinity (data not shown). RESULTS +25 40 point mutations experimental_method Peptides with beneficial point mutations at positions 2, 5, and 14 were synthesized and evaluated in the competition ELISA (Table 1). RESULTS +54 55 2 residue_number Peptides with beneficial point mutations at positions 2, 5, and 14 were synthesized and evaluated in the competition ELISA (Table 1). RESULTS +57 58 5 residue_number Peptides with beneficial point mutations at positions 2, 5, and 14 were synthesized and evaluated in the competition ELISA (Table 1). RESULTS +64 66 14 residue_number Peptides with beneficial point mutations at positions 2, 5, and 14 were synthesized and evaluated in the competition ELISA (Table 1). RESULTS +72 83 synthesized experimental_method Peptides with beneficial point mutations at positions 2, 5, and 14 were synthesized and evaluated in the competition ELISA (Table 1). RESULTS +105 122 competition ELISA experimental_method Peptides with beneficial point mutations at positions 2, 5, and 14 were synthesized and evaluated in the competition ELISA (Table 1). RESULTS +20 23 V2H mutant Two of the changes, V2H (18) or V2T (21) displayed improved binding in the competition ELISA. RESULTS +25 27 18 chemical Two of the changes, V2H (18) or V2T (21) displayed improved binding in the competition ELISA. RESULTS +32 35 V2T mutant Two of the changes, V2H (18) or V2T (21) displayed improved binding in the competition ELISA. RESULTS +37 39 21 chemical Two of the changes, V2H (18) or V2T (21) displayed improved binding in the competition ELISA. RESULTS +75 92 competition ELISA experimental_method Two of the changes, V2H (18) or V2T (21) displayed improved binding in the competition ELISA. RESULTS +10 21 replacement experimental_method Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +25 35 methionine residue_name Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +48 49 5 residue_number Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +55 62 alanine residue_name Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +118 128 isoleucine residue_name Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +130 132 24 chemical Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +135 142 leucine residue_name Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +144 146 25 chemical Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +152 158 valine residue_name Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +160 162 26 chemical Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +256 262 valine residue_name Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +267 277 isoleucine residue_name Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +278 290 replacements experimental_method Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +310 317 alanine residue_name Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. RESULTS +0 12 Introduction experimental_method Introduction of a methionine (27) or a carboxamide (28 and 29) at position 14 was shown to improve the binding affinity of the lead peptide. RESULTS +18 28 methionine residue_name Introduction of a methionine (27) or a carboxamide (28 and 29) at position 14 was shown to improve the binding affinity of the lead peptide. RESULTS +30 32 27 chemical Introduction of a methionine (27) or a carboxamide (28 and 29) at position 14 was shown to improve the binding affinity of the lead peptide. RESULTS +39 50 carboxamide chemical Introduction of a methionine (27) or a carboxamide (28 and 29) at position 14 was shown to improve the binding affinity of the lead peptide. RESULTS +52 54 28 chemical Introduction of a methionine (27) or a carboxamide (28 and 29) at position 14 was shown to improve the binding affinity of the lead peptide. RESULTS +59 61 29 chemical Introduction of a methionine (27) or a carboxamide (28 and 29) at position 14 was shown to improve the binding affinity of the lead peptide. RESULTS +75 77 14 residue_number Introduction of a methionine (27) or a carboxamide (28 and 29) at position 14 was shown to improve the binding affinity of the lead peptide. RESULTS +103 119 binding affinity evidence Introduction of a methionine (27) or a carboxamide (28 and 29) at position 14 was shown to improve the binding affinity of the lead peptide. RESULTS +60 78 binding affinities evidence In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion. RESULTS +117 127 MBP fusion experimental_method In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion. RESULTS +153 165 substitution experimental_method In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion. RESULTS +169 175 valine residue_name In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion. RESULTS +188 189 2 residue_number In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion. RESULTS +195 205 tryptophan residue_name In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion. RESULTS +207 209 22 chemical In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion. RESULTS +249 257 affinity evidence In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion. RESULTS +303 313 MBP fusion experimental_method In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion. RESULTS +52 55 SAR experimental_method Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17 nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2). RESULTS +99 109 peptide 30 chemical Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17 nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2). RESULTS +117 138 high affinity peptide chemical Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17 nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2). RESULTS +140 143 HAP chemical Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17 nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2). RESULTS +172 178 IL-17A protein Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17 nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2). RESULTS +197 202 human species Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17 nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2). RESULTS +233 237 IC50 evidence Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17 nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2). RESULTS +289 294 phage experimental_method Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17 nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2). RESULTS +295 304 peptide 1 chemical Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17 nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2). RESULTS +78 81 HAP chemical We also examined the effect of removing the acetyl group at the N-terminus of HAP (which is present in all the peptides made, see Supplementary Material). RESULTS +4 13 un-capped protein_state The un-capped peptide (31) had an IC50 of 420 nM in the cell-based assay. RESULTS +14 26 peptide (31) chemical The un-capped peptide (31) had an IC50 of 420 nM in the cell-based assay. RESULTS +34 38 IC50 evidence The un-capped peptide (31) had an IC50 of 420 nM in the cell-based assay. RESULTS +56 72 cell-based assay experimental_method The un-capped peptide (31) had an IC50 of 420 nM in the cell-based assay. RESULTS +33 35 31 chemical The loss of cellular activity of 31 was most likely due to the degradation of the N-terminus of 31, since peptide 31 was shown to be able to bind to IL-17A with similar affinity as HAP itself. RESULTS +96 98 31 chemical The loss of cellular activity of 31 was most likely due to the degradation of the N-terminus of 31, since peptide 31 was shown to be able to bind to IL-17A with similar affinity as HAP itself. RESULTS +114 116 31 chemical The loss of cellular activity of 31 was most likely due to the degradation of the N-terminus of 31, since peptide 31 was shown to be able to bind to IL-17A with similar affinity as HAP itself. RESULTS +149 155 IL-17A protein The loss of cellular activity of 31 was most likely due to the degradation of the N-terminus of 31, since peptide 31 was shown to be able to bind to IL-17A with similar affinity as HAP itself. RESULTS +181 184 HAP chemical The loss of cellular activity of 31 was most likely due to the degradation of the N-terminus of 31, since peptide 31 was shown to be able to bind to IL-17A with similar affinity as HAP itself. RESULTS +52 68 antibody fusions experimental_method Furthermore, our previous work had reported that in antibody fusions the uncapped peptide was degraded under cell assay conditions with removal of the first 1-3 residues to inactive products with the same N-terminal sequences as peptides 32–34. RESULTS +73 81 uncapped protein_state Furthermore, our previous work had reported that in antibody fusions the uncapped peptide was degraded under cell assay conditions with removal of the first 1-3 residues to inactive products with the same N-terminal sequences as peptides 32–34. RESULTS +82 89 peptide chemical Furthermore, our previous work had reported that in antibody fusions the uncapped peptide was degraded under cell assay conditions with removal of the first 1-3 residues to inactive products with the same N-terminal sequences as peptides 32–34. RESULTS +136 146 removal of experimental_method Furthermore, our previous work had reported that in antibody fusions the uncapped peptide was degraded under cell assay conditions with removal of the first 1-3 residues to inactive products with the same N-terminal sequences as peptides 32–34. RESULTS +151 169 first 1-3 residues residue_range Furthermore, our previous work had reported that in antibody fusions the uncapped peptide was degraded under cell assay conditions with removal of the first 1-3 residues to inactive products with the same N-terminal sequences as peptides 32–34. RESULTS +229 243 peptides 32–34 chemical Furthermore, our previous work had reported that in antibody fusions the uncapped peptide was degraded under cell assay conditions with removal of the first 1-3 residues to inactive products with the same N-terminal sequences as peptides 32–34. RESULTS +14 19 32–34 chemical In this work, 32–34 are capped by protective acetyl group and reflect the same inactivity as reported. RESULTS +24 30 capped protein_state In this work, 32–34 are capped by protective acetyl group and reflect the same inactivity as reported. RESULTS +11 22 truncations experimental_method C-terminal truncations showed a more gradual reduction in activity (35–37; Table 2). RESULTS +68 73 35–37 chemical C-terminal truncations showed a more gradual reduction in activity (35–37; Table 2). RESULTS +6 17 deletion of experimental_method After deletion of three amino acids from the C-terminal end (37), the peptide is no longer active. RESULTS +18 35 three amino acids residue_range After deletion of three amino acids from the C-terminal end (37), the peptide is no longer active. RESULTS +61 63 37 chemical After deletion of three amino acids from the C-terminal end (37), the peptide is no longer active. RESULTS +16 19 HAP chemical Dimerization of HAP can further increase its potency RESULTS +27 33 IL-17A protein We reasoned that since the IL-17A protein is almost exclusively present in a dimeric form, dimerizing the IL-17A binding peptides could result in an improvement in binding affinity and inhibitory activity. RESULTS +77 84 dimeric oligomeric_state We reasoned that since the IL-17A protein is almost exclusively present in a dimeric form, dimerizing the IL-17A binding peptides could result in an improvement in binding affinity and inhibitory activity. RESULTS +91 101 dimerizing oligomeric_state We reasoned that since the IL-17A protein is almost exclusively present in a dimeric form, dimerizing the IL-17A binding peptides could result in an improvement in binding affinity and inhibitory activity. RESULTS +106 112 IL-17A protein We reasoned that since the IL-17A protein is almost exclusively present in a dimeric form, dimerizing the IL-17A binding peptides could result in an improvement in binding affinity and inhibitory activity. RESULTS +164 180 binding affinity evidence We reasoned that since the IL-17A protein is almost exclusively present in a dimeric form, dimerizing the IL-17A binding peptides could result in an improvement in binding affinity and inhibitory activity. RESULTS +0 10 Homodimers oligomeric_state Homodimers of HAP were made through attachment of polyethylene glycol (PEG) spacers of different lengths at amino acids 4, 7 and 14, as these positions were identified in the alanine scan analysis as not contributing significantly to the activity, and at each N-terminus (Supplementary Table S2). RESULTS +14 17 HAP chemical Homodimers of HAP were made through attachment of polyethylene glycol (PEG) spacers of different lengths at amino acids 4, 7 and 14, as these positions were identified in the alanine scan analysis as not contributing significantly to the activity, and at each N-terminus (Supplementary Table S2). RESULTS +50 69 polyethylene glycol chemical Homodimers of HAP were made through attachment of polyethylene glycol (PEG) spacers of different lengths at amino acids 4, 7 and 14, as these positions were identified in the alanine scan analysis as not contributing significantly to the activity, and at each N-terminus (Supplementary Table S2). RESULTS +71 74 PEG chemical Homodimers of HAP were made through attachment of polyethylene glycol (PEG) spacers of different lengths at amino acids 4, 7 and 14, as these positions were identified in the alanine scan analysis as not contributing significantly to the activity, and at each N-terminus (Supplementary Table S2). RESULTS +120 121 4 residue_number Homodimers of HAP were made through attachment of polyethylene glycol (PEG) spacers of different lengths at amino acids 4, 7 and 14, as these positions were identified in the alanine scan analysis as not contributing significantly to the activity, and at each N-terminus (Supplementary Table S2). RESULTS +123 124 7 residue_number Homodimers of HAP were made through attachment of polyethylene glycol (PEG) spacers of different lengths at amino acids 4, 7 and 14, as these positions were identified in the alanine scan analysis as not contributing significantly to the activity, and at each N-terminus (Supplementary Table S2). RESULTS +129 131 14 residue_number Homodimers of HAP were made through attachment of polyethylene glycol (PEG) spacers of different lengths at amino acids 4, 7 and 14, as these positions were identified in the alanine scan analysis as not contributing significantly to the activity, and at each N-terminus (Supplementary Table S2). RESULTS +175 196 alanine scan analysis experimental_method Homodimers of HAP were made through attachment of polyethylene glycol (PEG) spacers of different lengths at amino acids 4, 7 and 14, as these positions were identified in the alanine scan analysis as not contributing significantly to the activity, and at each N-terminus (Supplementary Table S2). RESULTS +34 50 pentafluoroester chemical Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. RESULTS +52 55 PFP chemical Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. RESULTS +99 102 PEG chemical Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. RESULTS +108 117 histidine residue_name Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. RESULTS +130 131 2 residue_number Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. RESULTS +140 146 lysine residue_name Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. RESULTS +159 161 15 residue_number Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. RESULTS +181 190 threonine residue_name Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. RESULTS +195 209 dimethyllysine residue_name Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. RESULTS +280 290 peptide 38 chemical Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. RESULTS +328 331 HAP chemical Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. RESULTS +36 43 dimeric oligomeric_state This exercise revealed that several dimeric peptides with the longer PEG21 spacer were significantly more potent than the monomer peptide in the cell-based assay (Supplementary Table S2). RESULTS +44 52 peptides chemical This exercise revealed that several dimeric peptides with the longer PEG21 spacer were significantly more potent than the monomer peptide in the cell-based assay (Supplementary Table S2). RESULTS +69 74 PEG21 chemical This exercise revealed that several dimeric peptides with the longer PEG21 spacer were significantly more potent than the monomer peptide in the cell-based assay (Supplementary Table S2). RESULTS +122 129 monomer oligomeric_state This exercise revealed that several dimeric peptides with the longer PEG21 spacer were significantly more potent than the monomer peptide in the cell-based assay (Supplementary Table S2). RESULTS +145 161 cell-based assay experimental_method This exercise revealed that several dimeric peptides with the longer PEG21 spacer were significantly more potent than the monomer peptide in the cell-based assay (Supplementary Table S2). RESULTS +0 10 Peptide 45 chemical Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1 nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21 nM (Supplementary Scheme S2). RESULTS +12 21 dimerized oligomeric_state Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1 nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21 nM (Supplementary Scheme S2). RESULTS +42 47 PEG21 chemical Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1 nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21 nM (Supplementary Scheme S2). RESULTS +67 69 14 residue_number Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1 nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21 nM (Supplementary Scheme S2). RESULTS +145 149 IC50 evidence Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1 nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21 nM (Supplementary Scheme S2). RESULTS +232 239 monomer oligomeric_state Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1 nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21 nM (Supplementary Scheme S2). RESULTS +262 267 PEG21 chemical Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1 nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21 nM (Supplementary Scheme S2). RESULTS +286 288 14 residue_number Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1 nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21 nM (Supplementary Scheme S2). RESULTS +292 302 peptide 48 chemical Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1 nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21 nM (Supplementary Scheme S2). RESULTS +310 314 IC50 evidence Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1 nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21 nM (Supplementary Scheme S2). RESULTS +36 43 dimeric oligomeric_state The species cross-reactivity of the dimeric peptide 45 and HAP were assessed in a murine functional cell assay using 15 ng/ml murine IL-17A. RESULTS +44 54 peptide 45 chemical The species cross-reactivity of the dimeric peptide 45 and HAP were assessed in a murine functional cell assay using 15 ng/ml murine IL-17A. RESULTS +59 62 HAP chemical The species cross-reactivity of the dimeric peptide 45 and HAP were assessed in a murine functional cell assay using 15 ng/ml murine IL-17A. RESULTS +82 110 murine functional cell assay experimental_method The species cross-reactivity of the dimeric peptide 45 and HAP were assessed in a murine functional cell assay using 15 ng/ml murine IL-17A. RESULTS +126 132 murine taxonomy_domain The species cross-reactivity of the dimeric peptide 45 and HAP were assessed in a murine functional cell assay using 15 ng/ml murine IL-17A. RESULTS +133 139 IL-17A protein The species cross-reactivity of the dimeric peptide 45 and HAP were assessed in a murine functional cell assay using 15 ng/ml murine IL-17A. RESULTS +0 10 Peptide 45 chemical Peptide 45 blocked the receptor binding of murine IL-17A although with potency two orders of magnitude weaker than that observed against human IL-17A (IC50 = 41 nM vs IC50 = 0.1 nM, respectively). RESULTS +23 31 receptor protein_type Peptide 45 blocked the receptor binding of murine IL-17A although with potency two orders of magnitude weaker than that observed against human IL-17A (IC50 = 41 nM vs IC50 = 0.1 nM, respectively). RESULTS +43 49 murine taxonomy_domain Peptide 45 blocked the receptor binding of murine IL-17A although with potency two orders of magnitude weaker than that observed against human IL-17A (IC50 = 41 nM vs IC50 = 0.1 nM, respectively). RESULTS +50 56 IL-17A protein Peptide 45 blocked the receptor binding of murine IL-17A although with potency two orders of magnitude weaker than that observed against human IL-17A (IC50 = 41 nM vs IC50 = 0.1 nM, respectively). RESULTS +137 142 human species Peptide 45 blocked the receptor binding of murine IL-17A although with potency two orders of magnitude weaker than that observed against human IL-17A (IC50 = 41 nM vs IC50 = 0.1 nM, respectively). RESULTS +143 149 IL-17A protein Peptide 45 blocked the receptor binding of murine IL-17A although with potency two orders of magnitude weaker than that observed against human IL-17A (IC50 = 41 nM vs IC50 = 0.1 nM, respectively). RESULTS +151 155 IC50 evidence Peptide 45 blocked the receptor binding of murine IL-17A although with potency two orders of magnitude weaker than that observed against human IL-17A (IC50 = 41 nM vs IC50 = 0.1 nM, respectively). RESULTS +167 171 IC50 evidence Peptide 45 blocked the receptor binding of murine IL-17A although with potency two orders of magnitude weaker than that observed against human IL-17A (IC50 = 41 nM vs IC50 = 0.1 nM, respectively). RESULTS +4 11 monomer oligomeric_state The monomer HAP was much weaker (IC50 >1 μM) in inhibiting murine IL-17A signaling (Supplementary Figure S4). RESULTS +12 15 HAP chemical The monomer HAP was much weaker (IC50 >1 μM) in inhibiting murine IL-17A signaling (Supplementary Figure S4). RESULTS +33 37 IC50 evidence The monomer HAP was much weaker (IC50 >1 μM) in inhibiting murine IL-17A signaling (Supplementary Figure S4). RESULTS +59 65 murine taxonomy_domain The monomer HAP was much weaker (IC50 >1 μM) in inhibiting murine IL-17A signaling (Supplementary Figure S4). RESULTS +66 72 IL-17A protein The monomer HAP was much weaker (IC50 >1 μM) in inhibiting murine IL-17A signaling (Supplementary Figure S4). RESULTS +13 20 dimeric oligomeric_state Although the dimeric peptide 45 is much more potent than HAP in the cell-based assay, in subsequent studies we decided to focus our efforts solely on characterizations of the monomeric peptide HAP in hopes to identify smaller peptide inhibitors containing the best minimal functional group. RESULTS +21 31 peptide 45 chemical Although the dimeric peptide 45 is much more potent than HAP in the cell-based assay, in subsequent studies we decided to focus our efforts solely on characterizations of the monomeric peptide HAP in hopes to identify smaller peptide inhibitors containing the best minimal functional group. RESULTS +57 60 HAP chemical Although the dimeric peptide 45 is much more potent than HAP in the cell-based assay, in subsequent studies we decided to focus our efforts solely on characterizations of the monomeric peptide HAP in hopes to identify smaller peptide inhibitors containing the best minimal functional group. RESULTS +68 84 cell-based assay experimental_method Although the dimeric peptide 45 is much more potent than HAP in the cell-based assay, in subsequent studies we decided to focus our efforts solely on characterizations of the monomeric peptide HAP in hopes to identify smaller peptide inhibitors containing the best minimal functional group. RESULTS +175 184 monomeric oligomeric_state Although the dimeric peptide 45 is much more potent than HAP in the cell-based assay, in subsequent studies we decided to focus our efforts solely on characterizations of the monomeric peptide HAP in hopes to identify smaller peptide inhibitors containing the best minimal functional group. RESULTS +193 196 HAP chemical Although the dimeric peptide 45 is much more potent than HAP in the cell-based assay, in subsequent studies we decided to focus our efforts solely on characterizations of the monomeric peptide HAP in hopes to identify smaller peptide inhibitors containing the best minimal functional group. RESULTS +29 32 HAP chemical Orthogonal assays to confirm HAP antagonism RESULTS +43 46 HAP chemical To further characterize the interaction of HAP with IL-17A, we set out to determine its in vitro binding affinity, specificity and kinetic profile using Surface Plasmon Resonance (SPR) methods (Fig. 1A). RESULTS +52 58 IL-17A protein To further characterize the interaction of HAP with IL-17A, we set out to determine its in vitro binding affinity, specificity and kinetic profile using Surface Plasmon Resonance (SPR) methods (Fig. 1A). RESULTS +97 113 binding affinity evidence To further characterize the interaction of HAP with IL-17A, we set out to determine its in vitro binding affinity, specificity and kinetic profile using Surface Plasmon Resonance (SPR) methods (Fig. 1A). RESULTS +131 146 kinetic profile evidence To further characterize the interaction of HAP with IL-17A, we set out to determine its in vitro binding affinity, specificity and kinetic profile using Surface Plasmon Resonance (SPR) methods (Fig. 1A). RESULTS +153 178 Surface Plasmon Resonance experimental_method To further characterize the interaction of HAP with IL-17A, we set out to determine its in vitro binding affinity, specificity and kinetic profile using Surface Plasmon Resonance (SPR) methods (Fig. 1A). RESULTS +180 183 SPR experimental_method To further characterize the interaction of HAP with IL-17A, we set out to determine its in vitro binding affinity, specificity and kinetic profile using Surface Plasmon Resonance (SPR) methods (Fig. 1A). RESULTS +0 3 HAP chemical HAP binds to immobilized human IL-17A homodimer tightly (Table 3). RESULTS +25 30 human species HAP binds to immobilized human IL-17A homodimer tightly (Table 3). RESULTS +31 37 IL-17A protein HAP binds to immobilized human IL-17A homodimer tightly (Table 3). RESULTS +38 47 homodimer oligomeric_state HAP binds to immobilized human IL-17A homodimer tightly (Table 3). RESULTS +23 31 affinity evidence It has slightly weaker affinity for human IL-17A/F heterodimer and >10 fold weaker affinity for mouse IL-17A (Table 3). RESULTS +36 41 human species It has slightly weaker affinity for human IL-17A/F heterodimer and >10 fold weaker affinity for mouse IL-17A (Table 3). RESULTS +42 50 IL-17A/F complex_assembly It has slightly weaker affinity for human IL-17A/F heterodimer and >10 fold weaker affinity for mouse IL-17A (Table 3). RESULTS +51 62 heterodimer oligomeric_state It has slightly weaker affinity for human IL-17A/F heterodimer and >10 fold weaker affinity for mouse IL-17A (Table 3). RESULTS +83 91 affinity evidence It has slightly weaker affinity for human IL-17A/F heterodimer and >10 fold weaker affinity for mouse IL-17A (Table 3). RESULTS +96 101 mouse taxonomy_domain It has slightly weaker affinity for human IL-17A/F heterodimer and >10 fold weaker affinity for mouse IL-17A (Table 3). RESULTS +102 108 IL-17A protein It has slightly weaker affinity for human IL-17A/F heterodimer and >10 fold weaker affinity for mouse IL-17A (Table 3). RESULTS +0 3 HAP chemical HAP does not show significant binding to immobilized human IL-17F homodimer or IL-17RA at concentrations up to 100 nM. RESULTS +53 58 human species HAP does not show significant binding to immobilized human IL-17F homodimer or IL-17RA at concentrations up to 100 nM. RESULTS +59 65 IL-17F protein HAP does not show significant binding to immobilized human IL-17F homodimer or IL-17RA at concentrations up to 100 nM. RESULTS +66 75 homodimer oligomeric_state HAP does not show significant binding to immobilized human IL-17F homodimer or IL-17RA at concentrations up to 100 nM. RESULTS +79 86 IL-17RA protein HAP does not show significant binding to immobilized human IL-17F homodimer or IL-17RA at concentrations up to 100 nM. RESULTS +52 57 human species Additionally, we investigated the antagonism of the human IL-17A/IL-17RA interaction by HAP using orthogonal methods including SPR and Förster resonance energy transfer (FRET) competition assays (Fig. 1B,C). RESULTS +58 72 IL-17A/IL-17RA complex_assembly Additionally, we investigated the antagonism of the human IL-17A/IL-17RA interaction by HAP using orthogonal methods including SPR and Förster resonance energy transfer (FRET) competition assays (Fig. 1B,C). RESULTS +88 91 HAP chemical Additionally, we investigated the antagonism of the human IL-17A/IL-17RA interaction by HAP using orthogonal methods including SPR and Förster resonance energy transfer (FRET) competition assays (Fig. 1B,C). RESULTS +127 130 SPR experimental_method Additionally, we investigated the antagonism of the human IL-17A/IL-17RA interaction by HAP using orthogonal methods including SPR and Förster resonance energy transfer (FRET) competition assays (Fig. 1B,C). RESULTS +135 194 Förster resonance energy transfer (FRET) competition assays experimental_method Additionally, we investigated the antagonism of the human IL-17A/IL-17RA interaction by HAP using orthogonal methods including SPR and Förster resonance energy transfer (FRET) competition assays (Fig. 1B,C). RESULTS +30 36 IL-17A protein In both assays, incubation of IL-17A with HAP effectively blocks the binding of IL-17A to immobilized IL-17RA with similar sub-nM IC50 (Table 3). RESULTS +42 45 HAP chemical In both assays, incubation of IL-17A with HAP effectively blocks the binding of IL-17A to immobilized IL-17RA with similar sub-nM IC50 (Table 3). RESULTS +80 86 IL-17A protein In both assays, incubation of IL-17A with HAP effectively blocks the binding of IL-17A to immobilized IL-17RA with similar sub-nM IC50 (Table 3). RESULTS +90 101 immobilized protein_state In both assays, incubation of IL-17A with HAP effectively blocks the binding of IL-17A to immobilized IL-17RA with similar sub-nM IC50 (Table 3). RESULTS +102 109 IL-17RA protein In both assays, incubation of IL-17A with HAP effectively blocks the binding of IL-17A to immobilized IL-17RA with similar sub-nM IC50 (Table 3). RESULTS +130 134 IC50 evidence In both assays, incubation of IL-17A with HAP effectively blocks the binding of IL-17A to immobilized IL-17RA with similar sub-nM IC50 (Table 3). RESULTS +0 3 HAP chemical HAP blocks IL-17A signaling in a human primary cell assay RESULTS +11 17 IL-17A protein HAP blocks IL-17A signaling in a human primary cell assay RESULTS +33 38 human species HAP blocks IL-17A signaling in a human primary cell assay RESULTS +13 19 IL-17A protein While either IL-17A or TNF-α alone can stimulate the release of multiple inflammatory cytokines, when acting together they can synergistically enhance each other’s effects (Supplementary Figure S5). RESULTS +23 28 TNF-α protein While either IL-17A or TNF-α alone can stimulate the release of multiple inflammatory cytokines, when acting together they can synergistically enhance each other’s effects (Supplementary Figure S5). RESULTS +86 95 cytokines protein_type While either IL-17A or TNF-α alone can stimulate the release of multiple inflammatory cytokines, when acting together they can synergistically enhance each other’s effects (Supplementary Figure S5). RESULTS +31 37 IL-17A protein These integrative responses to IL-17A and TNF-α in human keratinocytes have been reported to account for key inflammatory pathogenic circuits in psoriasis. RESULTS +42 47 TNF-α protein These integrative responses to IL-17A and TNF-α in human keratinocytes have been reported to account for key inflammatory pathogenic circuits in psoriasis. RESULTS +51 56 human species These integrative responses to IL-17A and TNF-α in human keratinocytes have been reported to account for key inflammatory pathogenic circuits in psoriasis. RESULTS +24 27 HAP chemical Thus, we chose to study HAP’s efficacy in blocking the production of IL-8, IL-6 and CCL-20 by primary human keratinocytes stimulated by IL-17A in the presence of TNF-α, an assay which may be more disease-relevant. RESULTS +69 73 IL-8 protein_type Thus, we chose to study HAP’s efficacy in blocking the production of IL-8, IL-6 and CCL-20 by primary human keratinocytes stimulated by IL-17A in the presence of TNF-α, an assay which may be more disease-relevant. RESULTS +75 79 IL-6 protein_type Thus, we chose to study HAP’s efficacy in blocking the production of IL-8, IL-6 and CCL-20 by primary human keratinocytes stimulated by IL-17A in the presence of TNF-α, an assay which may be more disease-relevant. RESULTS +84 90 CCL-20 protein_type Thus, we chose to study HAP’s efficacy in blocking the production of IL-8, IL-6 and CCL-20 by primary human keratinocytes stimulated by IL-17A in the presence of TNF-α, an assay which may be more disease-relevant. RESULTS +102 107 human species Thus, we chose to study HAP’s efficacy in blocking the production of IL-8, IL-6 and CCL-20 by primary human keratinocytes stimulated by IL-17A in the presence of TNF-α, an assay which may be more disease-relevant. RESULTS +136 142 IL-17A protein Thus, we chose to study HAP’s efficacy in blocking the production of IL-8, IL-6 and CCL-20 by primary human keratinocytes stimulated by IL-17A in the presence of TNF-α, an assay which may be more disease-relevant. RESULTS +162 167 TNF-α protein Thus, we chose to study HAP’s efficacy in blocking the production of IL-8, IL-6 and CCL-20 by primary human keratinocytes stimulated by IL-17A in the presence of TNF-α, an assay which may be more disease-relevant. RESULTS +0 3 HAP chemical HAP inhibits the production of all three cytokines in a dose-dependent fashion (Fig. 1D). RESULTS +41 50 cytokines protein_type HAP inhibits the production of all three cytokines in a dose-dependent fashion (Fig. 1D). RESULTS +38 42 IL-8 protein_type Significantly, the baseline levels of IL-8, IL-6 and CCL-20 stimulated by TNF-α alone are not inhibited by HAP, further indicating the selectivity of HAP (Fig. 1D). RESULTS +44 48 IL-6 protein_type Significantly, the baseline levels of IL-8, IL-6 and CCL-20 stimulated by TNF-α alone are not inhibited by HAP, further indicating the selectivity of HAP (Fig. 1D). RESULTS +53 59 CCL-20 protein_type Significantly, the baseline levels of IL-8, IL-6 and CCL-20 stimulated by TNF-α alone are not inhibited by HAP, further indicating the selectivity of HAP (Fig. 1D). RESULTS +74 79 TNF-α protein Significantly, the baseline levels of IL-8, IL-6 and CCL-20 stimulated by TNF-α alone are not inhibited by HAP, further indicating the selectivity of HAP (Fig. 1D). RESULTS +107 110 HAP chemical Significantly, the baseline levels of IL-8, IL-6 and CCL-20 stimulated by TNF-α alone are not inhibited by HAP, further indicating the selectivity of HAP (Fig. 1D). RESULTS +150 153 HAP chemical Significantly, the baseline levels of IL-8, IL-6 and CCL-20 stimulated by TNF-α alone are not inhibited by HAP, further indicating the selectivity of HAP (Fig. 1D). RESULTS +172 177 TNF-α protein Such pharmacological selectivity may be important to suppress inflammatory pathogenic circuits in psoriasis, while sparing the anti-infectious immune responses produced by TNF-α. RESULTS +20 24 IC50 evidence The relatively high IC50 values in this assay (Table 3) are probably due to the high IL-17A concentration (100 ng/ml) needed for detection of IL-6. RESULTS +85 91 IL-17A protein The relatively high IC50 values in this assay (Table 3) are probably due to the high IL-17A concentration (100 ng/ml) needed for detection of IL-6. RESULTS +142 146 IL-6 protein_type The relatively high IC50 values in this assay (Table 3) are probably due to the high IL-17A concentration (100 ng/ml) needed for detection of IL-6. RESULTS +34 40 IL-17A protein As a reference, a commercial anti-IL-17A antibody (R&D Systems) inhibits the production of IL-8 with an IC50 of 13(±6) nM (N = 3). RESULTS +41 49 antibody protein_type As a reference, a commercial anti-IL-17A antibody (R&D Systems) inhibits the production of IL-8 with an IC50 of 13(±6) nM (N = 3). RESULTS +91 95 IL-8 protein_type As a reference, a commercial anti-IL-17A antibody (R&D Systems) inhibits the production of IL-8 with an IC50 of 13(±6) nM (N = 3). RESULTS +104 108 IC50 evidence As a reference, a commercial anti-IL-17A antibody (R&D Systems) inhibits the production of IL-8 with an IC50 of 13(±6) nM (N = 3). RESULTS +12 16 IC50 evidence Indeed, the IC50 was 14(±9) nM (N = 12) for HAP inhibition of IL-8 production when only 5 ng/ml IL-17A was used in this assay. RESULTS +44 47 HAP chemical Indeed, the IC50 was 14(±9) nM (N = 12) for HAP inhibition of IL-8 production when only 5 ng/ml IL-17A was used in this assay. RESULTS +62 66 IL-8 protein_type Indeed, the IC50 was 14(±9) nM (N = 12) for HAP inhibition of IL-8 production when only 5 ng/ml IL-17A was used in this assay. RESULTS +96 102 IL-17A protein Indeed, the IC50 was 14(±9) nM (N = 12) for HAP inhibition of IL-8 production when only 5 ng/ml IL-17A was used in this assay. RESULTS +34 40 IL-17A protein In patients, the concentration of IL-17A in psoriatic lesions is reported to be 0.01 ng/ml, well below the EC50 (5–10ng/ml) of IL-17A induced IL-8 production in vitro. RESULTS +127 133 IL-17A protein In patients, the concentration of IL-17A in psoriatic lesions is reported to be 0.01 ng/ml, well below the EC50 (5–10ng/ml) of IL-17A induced IL-8 production in vitro. RESULTS +142 146 IL-8 protein_type In patients, the concentration of IL-17A in psoriatic lesions is reported to be 0.01 ng/ml, well below the EC50 (5–10ng/ml) of IL-17A induced IL-8 production in vitro. RESULTS +11 30 keratinocytes assay experimental_method Similar to keratinocytes assay results, while HAP inhibits IL-17A stimulated IL-6 production by BJ human fibroblast potently (IC50 of 17 nM), it does not inhibit TNF-α stimulated IL-6 production at concentrations up to 10 μM (Supplementary Figure S2). RESULTS +46 49 HAP chemical Similar to keratinocytes assay results, while HAP inhibits IL-17A stimulated IL-6 production by BJ human fibroblast potently (IC50 of 17 nM), it does not inhibit TNF-α stimulated IL-6 production at concentrations up to 10 μM (Supplementary Figure S2). RESULTS +59 65 IL-17A protein Similar to keratinocytes assay results, while HAP inhibits IL-17A stimulated IL-6 production by BJ human fibroblast potently (IC50 of 17 nM), it does not inhibit TNF-α stimulated IL-6 production at concentrations up to 10 μM (Supplementary Figure S2). RESULTS +77 81 IL-6 protein_type Similar to keratinocytes assay results, while HAP inhibits IL-17A stimulated IL-6 production by BJ human fibroblast potently (IC50 of 17 nM), it does not inhibit TNF-α stimulated IL-6 production at concentrations up to 10 μM (Supplementary Figure S2). RESULTS +99 104 human species Similar to keratinocytes assay results, while HAP inhibits IL-17A stimulated IL-6 production by BJ human fibroblast potently (IC50 of 17 nM), it does not inhibit TNF-α stimulated IL-6 production at concentrations up to 10 μM (Supplementary Figure S2). RESULTS +126 130 IC50 evidence Similar to keratinocytes assay results, while HAP inhibits IL-17A stimulated IL-6 production by BJ human fibroblast potently (IC50 of 17 nM), it does not inhibit TNF-α stimulated IL-6 production at concentrations up to 10 μM (Supplementary Figure S2). RESULTS +162 167 TNF-α protein Similar to keratinocytes assay results, while HAP inhibits IL-17A stimulated IL-6 production by BJ human fibroblast potently (IC50 of 17 nM), it does not inhibit TNF-α stimulated IL-6 production at concentrations up to 10 μM (Supplementary Figure S2). RESULTS +179 183 IL-6 protein_type Similar to keratinocytes assay results, while HAP inhibits IL-17A stimulated IL-6 production by BJ human fibroblast potently (IC50 of 17 nM), it does not inhibit TNF-α stimulated IL-6 production at concentrations up to 10 μM (Supplementary Figure S2). RESULTS +0 43 Crystallization and structure determination experimental_method Crystallization and structure determination RESULTS +10 32 crystallization trials experimental_method Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals. RESULTS +44 62 co-crystallization experimental_method Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals. RESULTS +69 76 soaking experimental_method Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals. RESULTS +77 80 HAP chemical Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals. RESULTS +96 99 apo protein_state Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals. RESULTS +100 106 IL-17A protein Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals. RESULTS +107 115 crystals evidence Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals. RESULTS +138 148 IL-17A/HAP complex_assembly Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals. RESULTS +157 165 crystals evidence Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals. RESULTS +18 21 HAP chemical We theorized that HAP binding induced large conformational changes in IL-17A that led to the difficulty of getting an IL-17A/HAP binary complex crystal. RESULTS +70 76 IL-17A protein We theorized that HAP binding induced large conformational changes in IL-17A that led to the difficulty of getting an IL-17A/HAP binary complex crystal. RESULTS +118 128 IL-17A/HAP complex_assembly We theorized that HAP binding induced large conformational changes in IL-17A that led to the difficulty of getting an IL-17A/HAP binary complex crystal. RESULTS +144 151 crystal evidence We theorized that HAP binding induced large conformational changes in IL-17A that led to the difficulty of getting an IL-17A/HAP binary complex crystal. RESULTS +20 28 antibody protein_type It is known that an antibody antigen-binding fragment (Fab) can be used as crystallization chaperones in crystallizing difficult targets. RESULTS +29 53 antigen-binding fragment structure_element It is known that an antibody antigen-binding fragment (Fab) can be used as crystallization chaperones in crystallizing difficult targets. RESULTS +55 58 Fab structure_element It is known that an antibody antigen-binding fragment (Fab) can be used as crystallization chaperones in crystallizing difficult targets. RESULTS +21 24 HAP chemical We hypothesized that HAP may target the N-terminal of IL-17A which is known to be more flexible than its C-terminal and conformational changes needed for HAP binding may be more likely there. RESULTS +54 60 IL-17A protein We hypothesized that HAP may target the N-terminal of IL-17A which is known to be more flexible than its C-terminal and conformational changes needed for HAP binding may be more likely there. RESULTS +154 157 HAP chemical We hypothesized that HAP may target the N-terminal of IL-17A which is known to be more flexible than its C-terminal and conformational changes needed for HAP binding may be more likely there. RESULTS +15 23 antibody protein_type We designed an antibody Fab known to target the C-terminal half of IL-17A based on a published IL-17A/Fab complex crystal structure, and produced it in HEK293 cells. RESULTS +24 27 Fab structure_element We designed an antibody Fab known to target the C-terminal half of IL-17A based on a published IL-17A/Fab complex crystal structure, and produced it in HEK293 cells. RESULTS +48 63 C-terminal half structure_element We designed an antibody Fab known to target the C-terminal half of IL-17A based on a published IL-17A/Fab complex crystal structure, and produced it in HEK293 cells. RESULTS +67 73 IL-17A protein We designed an antibody Fab known to target the C-terminal half of IL-17A based on a published IL-17A/Fab complex crystal structure, and produced it in HEK293 cells. RESULTS +95 105 IL-17A/Fab complex_assembly We designed an antibody Fab known to target the C-terminal half of IL-17A based on a published IL-17A/Fab complex crystal structure, and produced it in HEK293 cells. RESULTS +114 131 crystal structure evidence We designed an antibody Fab known to target the C-terminal half of IL-17A based on a published IL-17A/Fab complex crystal structure, and produced it in HEK293 cells. RESULTS +6 15 SPR assay experimental_method In an SPR assay HAP and this Fab were able to co-bind IL-17A without large changes in their binding affinities and kinetics, confirming our hypothesis (Supplementary Figure S6). RESULTS +16 19 HAP chemical In an SPR assay HAP and this Fab were able to co-bind IL-17A without large changes in their binding affinities and kinetics, confirming our hypothesis (Supplementary Figure S6). RESULTS +29 32 Fab structure_element In an SPR assay HAP and this Fab were able to co-bind IL-17A without large changes in their binding affinities and kinetics, confirming our hypothesis (Supplementary Figure S6). RESULTS +54 60 IL-17A protein In an SPR assay HAP and this Fab were able to co-bind IL-17A without large changes in their binding affinities and kinetics, confirming our hypothesis (Supplementary Figure S6). RESULTS +92 110 binding affinities evidence In an SPR assay HAP and this Fab were able to co-bind IL-17A without large changes in their binding affinities and kinetics, confirming our hypothesis (Supplementary Figure S6). RESULTS +115 123 kinetics evidence In an SPR assay HAP and this Fab were able to co-bind IL-17A without large changes in their binding affinities and kinetics, confirming our hypothesis (Supplementary Figure S6). RESULTS +61 64 HAP chemical Furthermore, since it binds to an area far away from that of HAP (see below), this Fab should have minimum effects on HAP binding conformation. RESULTS +83 86 Fab structure_element Furthermore, since it binds to an area far away from that of HAP (see below), this Fab should have minimum effects on HAP binding conformation. RESULTS +118 121 HAP chemical Furthermore, since it binds to an area far away from that of HAP (see below), this Fab should have minimum effects on HAP binding conformation. RESULTS +0 8 Crystals evidence Crystals of Fab/IL-17A/HAP ternary complex were obtained readily in crystallization screens. RESULTS +12 26 Fab/IL-17A/HAP complex_assembly Crystals of Fab/IL-17A/HAP ternary complex were obtained readily in crystallization screens. RESULTS +68 91 crystallization screens experimental_method Crystals of Fab/IL-17A/HAP ternary complex were obtained readily in crystallization screens. RESULTS +0 15 Crystallization experimental_method Crystallization of IL-17A and its binding partners was accomplished using two forms of IL-17A. RESULTS +19 25 IL-17A protein Crystallization of IL-17A and its binding partners was accomplished using two forms of IL-17A. RESULTS +87 93 IL-17A protein Crystallization of IL-17A and its binding partners was accomplished using two forms of IL-17A. RESULTS +64 70 IL-17A protein These were, respectively, a presumably more homogeneous form of IL-17A that lacked the disordered N-terminal peptide and a full-length form of the cytokine with a full complement of disulfide bonds. RESULTS +76 82 lacked protein_state These were, respectively, a presumably more homogeneous form of IL-17A that lacked the disordered N-terminal peptide and a full-length form of the cytokine with a full complement of disulfide bonds. RESULTS +87 97 disordered protein_state These were, respectively, a presumably more homogeneous form of IL-17A that lacked the disordered N-terminal peptide and a full-length form of the cytokine with a full complement of disulfide bonds. RESULTS +98 116 N-terminal peptide structure_element These were, respectively, a presumably more homogeneous form of IL-17A that lacked the disordered N-terminal peptide and a full-length form of the cytokine with a full complement of disulfide bonds. RESULTS +123 134 full-length protein_state These were, respectively, a presumably more homogeneous form of IL-17A that lacked the disordered N-terminal peptide and a full-length form of the cytokine with a full complement of disulfide bonds. RESULTS +147 155 cytokine protein_type These were, respectively, a presumably more homogeneous form of IL-17A that lacked the disordered N-terminal peptide and a full-length form of the cytokine with a full complement of disulfide bonds. RESULTS +182 197 disulfide bonds ptm These were, respectively, a presumably more homogeneous form of IL-17A that lacked the disordered N-terminal peptide and a full-length form of the cytokine with a full complement of disulfide bonds. RESULTS +0 8 Crystals evidence Crystals of the Fab/truncated IL-17A/HAP complex diffracted to 2.2 Å, and the Fab/full length IL-17A/HAP complex diffracted to 3.0 Å (Supplementary Table S3). RESULTS +16 40 Fab/truncated IL-17A/HAP complex_assembly Crystals of the Fab/truncated IL-17A/HAP complex diffracted to 2.2 Å, and the Fab/full length IL-17A/HAP complex diffracted to 3.0 Å (Supplementary Table S3). RESULTS +78 104 Fab/full length IL-17A/HAP complex_assembly Crystals of the Fab/truncated IL-17A/HAP complex diffracted to 2.2 Å, and the Fab/full length IL-17A/HAP complex diffracted to 3.0 Å (Supplementary Table S3). RESULTS +5 15 structures evidence Both structures were solved by molecular replacement. RESULTS +31 52 molecular replacement experimental_method Both structures were solved by molecular replacement. RESULTS +15 27 crystallized experimental_method Both complexes crystallized in the space group of P321, with half the complex (1 Fab/1 IL-17A monomer/1 HAP) in the asymmetric unit. RESULTS +81 84 Fab structure_element Both complexes crystallized in the space group of P321, with half the complex (1 Fab/1 IL-17A monomer/1 HAP) in the asymmetric unit. RESULTS +87 93 IL-17A protein Both complexes crystallized in the space group of P321, with half the complex (1 Fab/1 IL-17A monomer/1 HAP) in the asymmetric unit. RESULTS +94 101 monomer oligomeric_state Both complexes crystallized in the space group of P321, with half the complex (1 Fab/1 IL-17A monomer/1 HAP) in the asymmetric unit. RESULTS +104 107 HAP chemical Both complexes crystallized in the space group of P321, with half the complex (1 Fab/1 IL-17A monomer/1 HAP) in the asymmetric unit. RESULTS +4 10 intact protein_state The intact complex can be generated by applying crystallographic 2-fold symmetry. RESULTS +0 18 Electron densities evidence Electron densities for HAP residues Ile1-Asn14 were readily interpretable with the exception of Lys15, which is disordered. RESULTS +23 26 HAP chemical Electron densities for HAP residues Ile1-Asn14 were readily interpretable with the exception of Lys15, which is disordered. RESULTS +36 46 Ile1-Asn14 residue_range Electron densities for HAP residues Ile1-Asn14 were readily interpretable with the exception of Lys15, which is disordered. RESULTS +96 101 Lys15 residue_name_number Electron densities for HAP residues Ile1-Asn14 were readily interpretable with the exception of Lys15, which is disordered. RESULTS +112 122 disordered protein_state Electron densities for HAP residues Ile1-Asn14 were readily interpretable with the exception of Lys15, which is disordered. RESULTS +34 51 complex structure evidence When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered. RESULTS +67 78 full length protein_state When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered. RESULTS +79 85 IL-17A protein When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered. RESULTS +114 123 truncated protein_state When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered. RESULTS +124 130 IL-17A protein When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered. RESULTS +154 160 Cys106 residue_name_number When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered. RESULTS +162 168 Ser106 residue_name_number When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered. RESULTS +176 185 truncated protein_state When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered. RESULTS +186 192 IL-17A protein When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered. RESULTS +204 214 disordered protein_state When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered. RESULTS +0 6 Cys106 residue_name_number Cys106 is covalently linked to Cys10 that resides in the disordered N-terminal peptide in the full length IL-17A. RESULTS +31 36 Cys10 residue_name_number Cys106 is covalently linked to Cys10 that resides in the disordered N-terminal peptide in the full length IL-17A. RESULTS +57 67 disordered protein_state Cys106 is covalently linked to Cys10 that resides in the disordered N-terminal peptide in the full length IL-17A. RESULTS +68 86 N-terminal peptide structure_element Cys106 is covalently linked to Cys10 that resides in the disordered N-terminal peptide in the full length IL-17A. RESULTS +94 105 full length protein_state Cys106 is covalently linked to Cys10 that resides in the disordered N-terminal peptide in the full length IL-17A. RESULTS +106 112 IL-17A protein Cys106 is covalently linked to Cys10 that resides in the disordered N-terminal peptide in the full length IL-17A. RESULTS +8 17 structure evidence Overall structure of Fab/IL-17A/HAP complex RESULTS +21 35 Fab/IL-17A/HAP complex_assembly Overall structure of Fab/IL-17A/HAP complex RESULTS +37 46 structure evidence In a similar manner to the published structure of Fab/IL-17A complex, two Fab molecules bind symmetrically to the C-terminal of the cytokine dimer, interacting with epitopes from both monomers (Fig. 2A). RESULTS +50 60 Fab/IL-17A complex_assembly In a similar manner to the published structure of Fab/IL-17A complex, two Fab molecules bind symmetrically to the C-terminal of the cytokine dimer, interacting with epitopes from both monomers (Fig. 2A). RESULTS +74 77 Fab structure_element In a similar manner to the published structure of Fab/IL-17A complex, two Fab molecules bind symmetrically to the C-terminal of the cytokine dimer, interacting with epitopes from both monomers (Fig. 2A). RESULTS +132 140 cytokine protein_type In a similar manner to the published structure of Fab/IL-17A complex, two Fab molecules bind symmetrically to the C-terminal of the cytokine dimer, interacting with epitopes from both monomers (Fig. 2A). RESULTS +141 146 dimer oligomeric_state In a similar manner to the published structure of Fab/IL-17A complex, two Fab molecules bind symmetrically to the C-terminal of the cytokine dimer, interacting with epitopes from both monomers (Fig. 2A). RESULTS +184 192 monomers oligomeric_state In a similar manner to the published structure of Fab/IL-17A complex, two Fab molecules bind symmetrically to the C-terminal of the cytokine dimer, interacting with epitopes from both monomers (Fig. 2A). RESULTS +14 17 HAP chemical Two copies of HAP bind to the N-terminal of the cytokine dimer, also symmetrically, and each HAP molecule also interacts with both IL-17A monomers (Fig. 2). RESULTS +48 56 cytokine protein_type Two copies of HAP bind to the N-terminal of the cytokine dimer, also symmetrically, and each HAP molecule also interacts with both IL-17A monomers (Fig. 2). RESULTS +57 62 dimer oligomeric_state Two copies of HAP bind to the N-terminal of the cytokine dimer, also symmetrically, and each HAP molecule also interacts with both IL-17A monomers (Fig. 2). RESULTS +93 96 HAP chemical Two copies of HAP bind to the N-terminal of the cytokine dimer, also symmetrically, and each HAP molecule also interacts with both IL-17A monomers (Fig. 2). RESULTS +131 137 IL-17A protein Two copies of HAP bind to the N-terminal of the cytokine dimer, also symmetrically, and each HAP molecule also interacts with both IL-17A monomers (Fig. 2). RESULTS +138 146 monomers oligomeric_state Two copies of HAP bind to the N-terminal of the cytokine dimer, also symmetrically, and each HAP molecule also interacts with both IL-17A monomers (Fig. 2). RESULTS +31 42 Secukinumab chemical Based on disclosed epitopes of Secukinumab and Ixekizumab, HAP binds to IL-17A at an area that is also different from those of those two antibodies. RESULTS +47 57 Ixekizumab chemical Based on disclosed epitopes of Secukinumab and Ixekizumab, HAP binds to IL-17A at an area that is also different from those of those two antibodies. RESULTS +59 62 HAP chemical Based on disclosed epitopes of Secukinumab and Ixekizumab, HAP binds to IL-17A at an area that is also different from those of those two antibodies. RESULTS +72 78 IL-17A protein Based on disclosed epitopes of Secukinumab and Ixekizumab, HAP binds to IL-17A at an area that is also different from those of those two antibodies. RESULTS +137 147 antibodies protein_type Based on disclosed epitopes of Secukinumab and Ixekizumab, HAP binds to IL-17A at an area that is also different from those of those two antibodies. RESULTS +15 25 5 residues residue_range The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. RESULTS +29 32 HAP chemical The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. RESULTS +34 40 1IHVTI chemical The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. RESULTS +50 61 amphipathic protein_state The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. RESULTS +62 70 β-strand structure_element The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. RESULTS +92 102 β-strand 4 structure_element The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. RESULTS +110 116 IL-17A protein The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. RESULTS +117 124 monomer oligomeric_state The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. RESULTS +129 139 β-strand 0 structure_element The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. RESULTS +170 176 IL-17A protein The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. RESULTS +192 199 monomer oligomeric_state The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. RESULTS +5 13 β-strand structure_element This β-strand is parallel to both strands 0 and 4 (Fig. 3B). RESULTS +34 49 strands 0 and 4 structure_element This β-strand is parallel to both strands 0 and 4 (Fig. 3B). RESULTS +0 9 Strands 0 structure_element Strands 0 of two IL-17A monomer are antiparallel, as appeared in other IL-17A structures. RESULTS +17 23 IL-17A protein Strands 0 of two IL-17A monomer are antiparallel, as appeared in other IL-17A structures. RESULTS +24 31 monomer oligomeric_state Strands 0 of two IL-17A monomer are antiparallel, as appeared in other IL-17A structures. RESULTS +71 77 IL-17A protein Strands 0 of two IL-17A monomer are antiparallel, as appeared in other IL-17A structures. RESULTS +78 88 structures evidence Strands 0 of two IL-17A monomer are antiparallel, as appeared in other IL-17A structures. RESULTS +15 25 8 residues residue_range The C-terminal 8 residues of the HAP that are ordered in the structure, 7ADLWDWIN, form an amphipathic α-helix interacting with the second IL-17A monomer. RESULTS +33 36 HAP chemical The C-terminal 8 residues of the HAP that are ordered in the structure, 7ADLWDWIN, form an amphipathic α-helix interacting with the second IL-17A monomer. RESULTS +61 70 structure evidence The C-terminal 8 residues of the HAP that are ordered in the structure, 7ADLWDWIN, form an amphipathic α-helix interacting with the second IL-17A monomer. RESULTS +72 81 7ADLWDWIN chemical The C-terminal 8 residues of the HAP that are ordered in the structure, 7ADLWDWIN, form an amphipathic α-helix interacting with the second IL-17A monomer. RESULTS +91 102 amphipathic protein_state The C-terminal 8 residues of the HAP that are ordered in the structure, 7ADLWDWIN, form an amphipathic α-helix interacting with the second IL-17A monomer. RESULTS +103 110 α-helix structure_element The C-terminal 8 residues of the HAP that are ordered in the structure, 7ADLWDWIN, form an amphipathic α-helix interacting with the second IL-17A monomer. RESULTS +139 145 IL-17A protein The C-terminal 8 residues of the HAP that are ordered in the structure, 7ADLWDWIN, form an amphipathic α-helix interacting with the second IL-17A monomer. RESULTS +146 153 monomer oligomeric_state The C-terminal 8 residues of the HAP that are ordered in the structure, 7ADLWDWIN, form an amphipathic α-helix interacting with the second IL-17A monomer. RESULTS +0 4 Pro6 residue_name_number Pro6 of HAP makes a transition between the N-terminal β-strand and the C-terminal α-helix of HAP. RESULTS +8 11 HAP chemical Pro6 of HAP makes a transition between the N-terminal β-strand and the C-terminal α-helix of HAP. RESULTS +54 62 β-strand structure_element Pro6 of HAP makes a transition between the N-terminal β-strand and the C-terminal α-helix of HAP. RESULTS +82 89 α-helix structure_element Pro6 of HAP makes a transition between the N-terminal β-strand and the C-terminal α-helix of HAP. RESULTS +93 96 HAP chemical Pro6 of HAP makes a transition between the N-terminal β-strand and the C-terminal α-helix of HAP. RESULTS +20 34 IL-17A/IL-17RA complex_assembly As a comparison, an IL-17A/IL-17RA complex structure (PDB code 4HSA) is also shown with IL-17A in the same orientation (Fig. 2C). RESULTS +35 52 complex structure evidence As a comparison, an IL-17A/IL-17RA complex structure (PDB code 4HSA) is also shown with IL-17A in the same orientation (Fig. 2C). RESULTS +88 94 IL-17A protein As a comparison, an IL-17A/IL-17RA complex structure (PDB code 4HSA) is also shown with IL-17A in the same orientation (Fig. 2C). RESULTS +24 30 IL-17A protein Inhibition mechanism of IL-17A signaling by HAP RESULTS +44 47 HAP chemical Inhibition mechanism of IL-17A signaling by HAP RESULTS +0 7 IL-17RA protein IL-17RA binds IL-17A at three regions on the IL-17A homodimer. RESULTS +14 20 IL-17A protein IL-17RA binds IL-17A at three regions on the IL-17A homodimer. RESULTS +45 51 IL-17A protein IL-17RA binds IL-17A at three regions on the IL-17A homodimer. RESULTS +52 61 homodimer oligomeric_state IL-17RA binds IL-17A at three regions on the IL-17A homodimer. RESULTS +0 3 HAP chemical HAP binds IL-17A at region I. Region I is formed by residues at the ends of β strands 0 and 4, and from loops 1–2 and 3–4 of IL-17A (Fig. 2). RESULTS +10 16 IL-17A protein HAP binds IL-17A at region I. Region I is formed by residues at the ends of β strands 0 and 4, and from loops 1–2 and 3–4 of IL-17A (Fig. 2). RESULTS +20 28 region I structure_element HAP binds IL-17A at region I. Region I is formed by residues at the ends of β strands 0 and 4, and from loops 1–2 and 3–4 of IL-17A (Fig. 2). RESULTS +30 38 Region I structure_element HAP binds IL-17A at region I. Region I is formed by residues at the ends of β strands 0 and 4, and from loops 1–2 and 3–4 of IL-17A (Fig. 2). RESULTS +76 93 β strands 0 and 4 structure_element HAP binds IL-17A at region I. Region I is formed by residues at the ends of β strands 0 and 4, and from loops 1–2 and 3–4 of IL-17A (Fig. 2). RESULTS +104 113 loops 1–2 structure_element HAP binds IL-17A at region I. Region I is formed by residues at the ends of β strands 0 and 4, and from loops 1–2 and 3–4 of IL-17A (Fig. 2). RESULTS +118 121 3–4 structure_element HAP binds IL-17A at region I. Region I is formed by residues at the ends of β strands 0 and 4, and from loops 1–2 and 3–4 of IL-17A (Fig. 2). RESULTS +125 131 IL-17A protein HAP binds IL-17A at region I. Region I is formed by residues at the ends of β strands 0 and 4, and from loops 1–2 and 3–4 of IL-17A (Fig. 2). RESULTS +26 34 region I structure_element Conformational changes in region I induced by HAP binding alone may allosterically affect IL-17RA binding, but more importantly, the α-helix of HAP directly competes with IL-17RA for binding to IL-17A (Fig. 3). RESULTS +46 49 HAP chemical Conformational changes in region I induced by HAP binding alone may allosterically affect IL-17RA binding, but more importantly, the α-helix of HAP directly competes with IL-17RA for binding to IL-17A (Fig. 3). RESULTS +90 97 IL-17RA protein Conformational changes in region I induced by HAP binding alone may allosterically affect IL-17RA binding, but more importantly, the α-helix of HAP directly competes with IL-17RA for binding to IL-17A (Fig. 3). RESULTS +133 140 α-helix structure_element Conformational changes in region I induced by HAP binding alone may allosterically affect IL-17RA binding, but more importantly, the α-helix of HAP directly competes with IL-17RA for binding to IL-17A (Fig. 3). RESULTS +144 147 HAP chemical Conformational changes in region I induced by HAP binding alone may allosterically affect IL-17RA binding, but more importantly, the α-helix of HAP directly competes with IL-17RA for binding to IL-17A (Fig. 3). RESULTS +171 178 IL-17RA protein Conformational changes in region I induced by HAP binding alone may allosterically affect IL-17RA binding, but more importantly, the α-helix of HAP directly competes with IL-17RA for binding to IL-17A (Fig. 3). RESULTS +194 200 IL-17A protein Conformational changes in region I induced by HAP binding alone may allosterically affect IL-17RA binding, but more importantly, the α-helix of HAP directly competes with IL-17RA for binding to IL-17A (Fig. 3). RESULTS +46 53 α helix structure_element The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). RESULTS +57 60 HAP chemical The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). RESULTS +65 71 IL-17A protein The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). RESULTS +80 85 Trp12 residue_name_number The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). RESULTS +89 92 HAP chemical The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). RESULTS +111 129 hydrophobic pocket site The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). RESULTS +133 139 IL-17A protein The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). RESULTS +169 175 Phe110 residue_name_number The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). RESULTS +177 182 Tyr62 residue_name_number The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). RESULTS +184 189 Pro59 residue_name_number The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). RESULTS +225 231 Arg101 residue_name_number The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). RESULTS +4 9 Trp12 residue_name_number The Trp12 side chain of HAP donates a hydrogen bond to the main chain oxygen of Pro69 of IL-17A. RESULTS +24 27 HAP chemical The Trp12 side chain of HAP donates a hydrogen bond to the main chain oxygen of Pro69 of IL-17A. RESULTS +38 51 hydrogen bond bond_interaction The Trp12 side chain of HAP donates a hydrogen bond to the main chain oxygen of Pro69 of IL-17A. RESULTS +80 85 Pro69 residue_name_number The Trp12 side chain of HAP donates a hydrogen bond to the main chain oxygen of Pro69 of IL-17A. RESULTS +89 95 IL-17A protein The Trp12 side chain of HAP donates a hydrogen bond to the main chain oxygen of Pro69 of IL-17A. RESULTS +23 29 Arg101 residue_name_number The positively charged Arg101 side chain of the IL-17A engages in a charge-helix dipole interaction with the main chain oxygen of Trp12. RESULTS +48 54 IL-17A protein The positively charged Arg101 side chain of the IL-17A engages in a charge-helix dipole interaction with the main chain oxygen of Trp12. RESULTS +68 99 charge-helix dipole interaction bond_interaction The positively charged Arg101 side chain of the IL-17A engages in a charge-helix dipole interaction with the main chain oxygen of Trp12. RESULTS +130 135 Trp12 residue_name_number The positively charged Arg101 side chain of the IL-17A engages in a charge-helix dipole interaction with the main chain oxygen of Trp12. RESULTS +14 18 Leu9 residue_name_number Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. RESULTS +23 28 Ile13 residue_name_number Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. RESULTS +36 39 HAP chemical Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. RESULTS +45 69 hydrophobic interactions bond_interaction Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. RESULTS +75 81 IL-17A protein Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. RESULTS +91 95 Asp8 residue_name_number Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. RESULTS +111 124 hydrogen bond bond_interaction Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. RESULTS +129 150 ion pair interactions bond_interaction Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. RESULTS +156 161 Tyr62 residue_name_number Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. RESULTS +166 172 Lys114 residue_name_number Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. RESULTS +176 182 IL-17A protein Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. RESULTS +3 11 region I structure_element In region I, an IL-17RA peptide interacts with IL-17A in a very similar fashion to the α-helix of HAP. RESULTS +16 23 IL-17RA protein In region I, an IL-17RA peptide interacts with IL-17A in a very similar fashion to the α-helix of HAP. RESULTS +47 53 IL-17A protein In region I, an IL-17RA peptide interacts with IL-17A in a very similar fashion to the α-helix of HAP. RESULTS +87 94 α-helix structure_element In region I, an IL-17RA peptide interacts with IL-17A in a very similar fashion to the α-helix of HAP. RESULTS +98 101 HAP chemical In region I, an IL-17RA peptide interacts with IL-17A in a very similar fashion to the α-helix of HAP. RESULTS +4 11 IL-17RA protein The IL-17RA peptide has sequences of 27LDDSWI, and part of the peptide is also α-helical (Fig. 3B). RESULTS +37 45 27LDDSWI chemical The IL-17RA peptide has sequences of 27LDDSWI, and part of the peptide is also α-helical (Fig. 3B). RESULTS +79 88 α-helical structure_element The IL-17RA peptide has sequences of 27LDDSWI, and part of the peptide is also α-helical (Fig. 3B). RESULTS +0 4 Leu7 residue_name_number Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B). RESULTS +6 11 Trp31 residue_name_number Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B). RESULTS +16 21 Ile32 residue_name_number Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B). RESULTS +25 32 IL-17RA protein Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B). RESULTS +83 89 IL-17A protein Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B). RESULTS +93 97 Leu9 residue_name_number Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B). RESULTS +99 104 Trp12 residue_name_number Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B). RESULTS +109 114 Ile13 residue_name_number Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B). RESULTS +118 121 HAP chemical Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B). RESULTS +19 26 α-helix structure_element In this sense, the α-helix of HAP with a sequence of 9LWDWI is a good mimetic of the 27LDDSWI peptide of IL-17RA. RESULTS +30 33 HAP chemical In this sense, the α-helix of HAP with a sequence of 9LWDWI is a good mimetic of the 27LDDSWI peptide of IL-17RA. RESULTS +53 59 9LWDWI chemical In this sense, the α-helix of HAP with a sequence of 9LWDWI is a good mimetic of the 27LDDSWI peptide of IL-17RA. RESULTS +85 93 27LDDSWI chemical In this sense, the α-helix of HAP with a sequence of 9LWDWI is a good mimetic of the 27LDDSWI peptide of IL-17RA. RESULTS +105 112 IL-17RA protein In this sense, the α-helix of HAP with a sequence of 9LWDWI is a good mimetic of the 27LDDSWI peptide of IL-17RA. RESULTS +4 12 β-strand structure_element The β-strand of HAP has no equivalent in IL-17RA. RESULTS +16 19 HAP chemical The β-strand of HAP has no equivalent in IL-17RA. RESULTS +41 48 IL-17RA protein The β-strand of HAP has no equivalent in IL-17RA. RESULTS +23 33 β-strand 0 structure_element However, it mimics the β-strand 0 of IL-17A. RESULTS +37 43 IL-17A protein However, it mimics the β-strand 0 of IL-17A. RESULTS +4 15 amphipathic protein_state The amphipathic β-strand of HAP orients the hydrophilic side chains of His2 and Thr4 outwards, and the hydrophobic side chains of Ile1, Val3 and Ile5 inward (Fig. 3A). RESULTS +16 24 β-strand structure_element The amphipathic β-strand of HAP orients the hydrophilic side chains of His2 and Thr4 outwards, and the hydrophobic side chains of Ile1, Val3 and Ile5 inward (Fig. 3A). RESULTS +28 31 HAP chemical The amphipathic β-strand of HAP orients the hydrophilic side chains of His2 and Thr4 outwards, and the hydrophobic side chains of Ile1, Val3 and Ile5 inward (Fig. 3A). RESULTS +71 75 His2 residue_name_number The amphipathic β-strand of HAP orients the hydrophilic side chains of His2 and Thr4 outwards, and the hydrophobic side chains of Ile1, Val3 and Ile5 inward (Fig. 3A). RESULTS +80 84 Thr4 residue_name_number The amphipathic β-strand of HAP orients the hydrophilic side chains of His2 and Thr4 outwards, and the hydrophobic side chains of Ile1, Val3 and Ile5 inward (Fig. 3A). RESULTS +130 134 Ile1 residue_name_number The amphipathic β-strand of HAP orients the hydrophilic side chains of His2 and Thr4 outwards, and the hydrophobic side chains of Ile1, Val3 and Ile5 inward (Fig. 3A). RESULTS +136 140 Val3 residue_name_number The amphipathic β-strand of HAP orients the hydrophilic side chains of His2 and Thr4 outwards, and the hydrophobic side chains of Ile1, Val3 and Ile5 inward (Fig. 3A). RESULTS +145 149 Ile5 residue_name_number The amphipathic β-strand of HAP orients the hydrophilic side chains of His2 and Thr4 outwards, and the hydrophobic side chains of Ile1, Val3 and Ile5 inward (Fig. 3A). RESULTS +0 10 β-strand 0 structure_element β-strand 0 in IL-17A is also amphipathic with the sequence of 21TVMVNLNI. RESULTS +14 20 IL-17A protein β-strand 0 in IL-17A is also amphipathic with the sequence of 21TVMVNLNI. RESULTS +29 40 amphipathic protein_state β-strand 0 in IL-17A is also amphipathic with the sequence of 21TVMVNLNI. RESULTS +62 72 21TVMVNLNI chemical β-strand 0 in IL-17A is also amphipathic with the sequence of 21TVMVNLNI. RESULTS +7 13 IL-17A protein In all IL-17A structures obtained to date, β-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward. RESULTS +14 24 structures evidence In all IL-17A structures obtained to date, β-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward. RESULTS +43 53 β-strand 0 structure_element In all IL-17A structures obtained to date, β-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward. RESULTS +93 98 Thr21 residue_name_number In all IL-17A structures obtained to date, β-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward. RESULTS +100 105 Asn25 residue_name_number In all IL-17A structures obtained to date, β-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward. RESULTS +110 115 Asn27 residue_name_number In all IL-17A structures obtained to date, β-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward. RESULTS +160 165 Val22 residue_name_number In all IL-17A structures obtained to date, β-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward. RESULTS +167 172 Val24 residue_name_number In all IL-17A structures obtained to date, β-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward. RESULTS +174 179 Leu26 residue_name_number In all IL-17A structures obtained to date, β-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward. RESULTS +184 189 Ile28 residue_name_number In all IL-17A structures obtained to date, β-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward. RESULTS +4 18 binding pocket site The binding pocket occupied by either Trp12 of HAP or Trp31 of IL-17RA is not formed in the apo IL-17A structure (Fig. 3C). RESULTS +38 43 Trp12 residue_name_number The binding pocket occupied by either Trp12 of HAP or Trp31 of IL-17RA is not formed in the apo IL-17A structure (Fig. 3C). RESULTS +47 50 HAP chemical The binding pocket occupied by either Trp12 of HAP or Trp31 of IL-17RA is not formed in the apo IL-17A structure (Fig. 3C). RESULTS +54 59 Trp31 residue_name_number The binding pocket occupied by either Trp12 of HAP or Trp31 of IL-17RA is not formed in the apo IL-17A structure (Fig. 3C). RESULTS +63 70 IL-17RA protein The binding pocket occupied by either Trp12 of HAP or Trp31 of IL-17RA is not formed in the apo IL-17A structure (Fig. 3C). RESULTS +92 95 apo protein_state The binding pocket occupied by either Trp12 of HAP or Trp31 of IL-17RA is not formed in the apo IL-17A structure (Fig. 3C). RESULTS +96 102 IL-17A protein The binding pocket occupied by either Trp12 of HAP or Trp31 of IL-17RA is not formed in the apo IL-17A structure (Fig. 3C). RESULTS +103 112 structure evidence The binding pocket occupied by either Trp12 of HAP or Trp31 of IL-17RA is not formed in the apo IL-17A structure (Fig. 3C). RESULTS +26 32 IL-17A protein Conformational changes of IL-17A are needed for both HAP and IL-17RA to bind to that region. RESULTS +53 56 HAP chemical Conformational changes of IL-17A are needed for both HAP and IL-17RA to bind to that region. RESULTS +61 68 IL-17RA protein Conformational changes of IL-17A are needed for both HAP and IL-17RA to bind to that region. RESULTS +17 20 HAP chemical Particularly for HAP, β-strands 0 have to shift out of the hydrophobic cleft formed by the main body of the IL-17A by as much as 10 Å between Cα atoms (Fig. 3C). RESULTS +22 33 β-strands 0 structure_element Particularly for HAP, β-strands 0 have to shift out of the hydrophobic cleft formed by the main body of the IL-17A by as much as 10 Å between Cα atoms (Fig. 3C). RESULTS +59 76 hydrophobic cleft site Particularly for HAP, β-strands 0 have to shift out of the hydrophobic cleft formed by the main body of the IL-17A by as much as 10 Å between Cα atoms (Fig. 3C). RESULTS +91 100 main body structure_element Particularly for HAP, β-strands 0 have to shift out of the hydrophobic cleft formed by the main body of the IL-17A by as much as 10 Å between Cα atoms (Fig. 3C). RESULTS +108 114 IL-17A protein Particularly for HAP, β-strands 0 have to shift out of the hydrophobic cleft formed by the main body of the IL-17A by as much as 10 Å between Cα atoms (Fig. 3C). RESULTS +19 22 apo protein_state Disruptions of the apo IL-17A structure by HAP binding are apparently compensated for by formation of the new interactions that involve almost the entire HAP molecule (Fig. 3B). RESULTS +23 29 IL-17A protein Disruptions of the apo IL-17A structure by HAP binding are apparently compensated for by formation of the new interactions that involve almost the entire HAP molecule (Fig. 3B). RESULTS +30 39 structure evidence Disruptions of the apo IL-17A structure by HAP binding are apparently compensated for by formation of the new interactions that involve almost the entire HAP molecule (Fig. 3B). RESULTS +43 46 HAP chemical Disruptions of the apo IL-17A structure by HAP binding are apparently compensated for by formation of the new interactions that involve almost the entire HAP molecule (Fig. 3B). RESULTS +154 157 HAP chemical Disruptions of the apo IL-17A structure by HAP binding are apparently compensated for by formation of the new interactions that involve almost the entire HAP molecule (Fig. 3B). RESULTS +33 36 SAR experimental_method Structure basis for the observed SAR of peptides RESULTS +4 14 IL-17A/HAP complex_assembly The IL-17A/HAP complex structure obtained is very consistent with the observed SAR of our identified peptide inhibitors, explaining well how the evolution of the initial phage peptide 1 to HAP and 45 improved its potency (Supplementary Figure S7). RESULTS +15 32 complex structure evidence The IL-17A/HAP complex structure obtained is very consistent with the observed SAR of our identified peptide inhibitors, explaining well how the evolution of the initial phage peptide 1 to HAP and 45 improved its potency (Supplementary Figure S7). RESULTS +79 82 SAR experimental_method The IL-17A/HAP complex structure obtained is very consistent with the observed SAR of our identified peptide inhibitors, explaining well how the evolution of the initial phage peptide 1 to HAP and 45 improved its potency (Supplementary Figure S7). RESULTS +170 175 phage experimental_method The IL-17A/HAP complex structure obtained is very consistent with the observed SAR of our identified peptide inhibitors, explaining well how the evolution of the initial phage peptide 1 to HAP and 45 improved its potency (Supplementary Figure S7). RESULTS +176 185 peptide 1 chemical The IL-17A/HAP complex structure obtained is very consistent with the observed SAR of our identified peptide inhibitors, explaining well how the evolution of the initial phage peptide 1 to HAP and 45 improved its potency (Supplementary Figure S7). RESULTS +189 192 HAP chemical The IL-17A/HAP complex structure obtained is very consistent with the observed SAR of our identified peptide inhibitors, explaining well how the evolution of the initial phage peptide 1 to HAP and 45 improved its potency (Supplementary Figure S7). RESULTS +197 199 45 chemical The IL-17A/HAP complex structure obtained is very consistent with the observed SAR of our identified peptide inhibitors, explaining well how the evolution of the initial phage peptide 1 to HAP and 45 improved its potency (Supplementary Figure S7). RESULTS +37 42 Trp12 residue_name_number The important interactions involving Trp12 of HAP explain the >90 times drop in potency of the W12A variant (6 vs 1, Table 1). RESULTS +46 49 HAP chemical The important interactions involving Trp12 of HAP explain the >90 times drop in potency of the W12A variant (6 vs 1, Table 1). RESULTS +95 99 W12A mutant The important interactions involving Trp12 of HAP explain the >90 times drop in potency of the W12A variant (6 vs 1, Table 1). RESULTS +4 15 amphipathic protein_state The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). RESULTS +30 33 HAP chemical The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). RESULTS +34 42 β-strand structure_element The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). RESULTS +102 103 2 residue_number The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). RESULTS +108 109 4 residue_number The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). RESULTS +133 135 14 chemical The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). RESULTS +137 139 18 chemical The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). RESULTS +141 143 19 chemical The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). RESULTS +145 147 21 chemical The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). RESULTS +152 154 23 chemical The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). RESULTS +158 159 1 chemical The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). RESULTS +164 166 22 chemical The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). RESULTS +27 30 HAP chemical All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). RESULTS +47 54 β-sheet structure_element All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). RESULTS +60 76 β-stands 0 and 4 structure_element All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). RESULTS +80 86 IL-17A protein All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). RESULTS +107 117 removal of experimental_method All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). RESULTS +122 140 first 1–3 residues residue_range All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). RESULTS +177 180 HAP chemical All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). RESULTS +190 196 IL-17A protein All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). RESULTS +213 215 31 chemical All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). RESULTS +216 218 32 chemical All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). RESULTS +223 225 33 chemical All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). RESULTS +15 20 Asn14 residue_name_number The C-terminal Asn14 and Lys15 of HAP are not directly involved in interactions with IL-17A, and this is reflected in the gradual reduction in activity caused by C-terminal truncations (35 and 36, Table 2). RESULTS +25 30 Lys15 residue_name_number The C-terminal Asn14 and Lys15 of HAP are not directly involved in interactions with IL-17A, and this is reflected in the gradual reduction in activity caused by C-terminal truncations (35 and 36, Table 2). RESULTS +34 37 HAP chemical The C-terminal Asn14 and Lys15 of HAP are not directly involved in interactions with IL-17A, and this is reflected in the gradual reduction in activity caused by C-terminal truncations (35 and 36, Table 2). RESULTS +85 91 IL-17A protein The C-terminal Asn14 and Lys15 of HAP are not directly involved in interactions with IL-17A, and this is reflected in the gradual reduction in activity caused by C-terminal truncations (35 and 36, Table 2). RESULTS +173 184 truncations experimental_method The C-terminal Asn14 and Lys15 of HAP are not directly involved in interactions with IL-17A, and this is reflected in the gradual reduction in activity caused by C-terminal truncations (35 and 36, Table 2). RESULTS +186 188 35 chemical The C-terminal Asn14 and Lys15 of HAP are not directly involved in interactions with IL-17A, and this is reflected in the gradual reduction in activity caused by C-terminal truncations (35 and 36, Table 2). RESULTS +193 195 36 chemical The C-terminal Asn14 and Lys15 of HAP are not directly involved in interactions with IL-17A, and this is reflected in the gradual reduction in activity caused by C-terminal truncations (35 and 36, Table 2). RESULTS +13 20 monomer oligomeric_state Each peptide monomer in 45 may not necessarily be more potent than HAP, but two monomer peptides within the same molecule that can simultaneously bind to IL-17A can greatly improve its potency due to avidity effects. RESULTS +24 26 45 chemical Each peptide monomer in 45 may not necessarily be more potent than HAP, but two monomer peptides within the same molecule that can simultaneously bind to IL-17A can greatly improve its potency due to avidity effects. RESULTS +67 70 HAP chemical Each peptide monomer in 45 may not necessarily be more potent than HAP, but two monomer peptides within the same molecule that can simultaneously bind to IL-17A can greatly improve its potency due to avidity effects. RESULTS +80 87 monomer oligomeric_state Each peptide monomer in 45 may not necessarily be more potent than HAP, but two monomer peptides within the same molecule that can simultaneously bind to IL-17A can greatly improve its potency due to avidity effects. RESULTS +154 160 IL-17A protein Each peptide monomer in 45 may not necessarily be more potent than HAP, but two monomer peptides within the same molecule that can simultaneously bind to IL-17A can greatly improve its potency due to avidity effects. RESULTS +0 3 HAP chemical HAP targets region I of IL-17A, an area that has the least sequence conservation in IL-17 cytokines. RESULTS +12 20 region I structure_element HAP targets region I of IL-17A, an area that has the least sequence conservation in IL-17 cytokines. RESULTS +24 30 IL-17A protein HAP targets region I of IL-17A, an area that has the least sequence conservation in IL-17 cytokines. RESULTS +84 99 IL-17 cytokines protein_type HAP targets region I of IL-17A, an area that has the least sequence conservation in IL-17 cytokines. RESULTS +42 58 HAP binding site site This lack of sequence conservation in the HAP binding site explains the observed specificity of HAP binding to human IL-17A. RESULTS +96 99 HAP chemical This lack of sequence conservation in the HAP binding site explains the observed specificity of HAP binding to human IL-17A. RESULTS +111 116 human species This lack of sequence conservation in the HAP binding site explains the observed specificity of HAP binding to human IL-17A. RESULTS +117 123 IL-17A protein This lack of sequence conservation in the HAP binding site explains the observed specificity of HAP binding to human IL-17A. RESULTS +41 47 IL-17F protein For example, inspection of the published IL-17F crystal structure (PDB code 1JPY) revealed a pocket of IL-17F similar to that of IL-17A for W12 of HAP binding, but it is occupied by a Phe-Phe motif at the N-terminal peptide of IL-17F. RESULTS +48 65 crystal structure evidence For example, inspection of the published IL-17F crystal structure (PDB code 1JPY) revealed a pocket of IL-17F similar to that of IL-17A for W12 of HAP binding, but it is occupied by a Phe-Phe motif at the N-terminal peptide of IL-17F. RESULTS +93 99 pocket site For example, inspection of the published IL-17F crystal structure (PDB code 1JPY) revealed a pocket of IL-17F similar to that of IL-17A for W12 of HAP binding, but it is occupied by a Phe-Phe motif at the N-terminal peptide of IL-17F. RESULTS +103 109 IL-17F protein For example, inspection of the published IL-17F crystal structure (PDB code 1JPY) revealed a pocket of IL-17F similar to that of IL-17A for W12 of HAP binding, but it is occupied by a Phe-Phe motif at the N-terminal peptide of IL-17F. RESULTS +129 135 IL-17A protein For example, inspection of the published IL-17F crystal structure (PDB code 1JPY) revealed a pocket of IL-17F similar to that of IL-17A for W12 of HAP binding, but it is occupied by a Phe-Phe motif at the N-terminal peptide of IL-17F. RESULTS +140 143 W12 residue_name_number For example, inspection of the published IL-17F crystal structure (PDB code 1JPY) revealed a pocket of IL-17F similar to that of IL-17A for W12 of HAP binding, but it is occupied by a Phe-Phe motif at the N-terminal peptide of IL-17F. RESULTS +147 150 HAP chemical For example, inspection of the published IL-17F crystal structure (PDB code 1JPY) revealed a pocket of IL-17F similar to that of IL-17A for W12 of HAP binding, but it is occupied by a Phe-Phe motif at the N-terminal peptide of IL-17F. RESULTS +184 197 Phe-Phe motif structure_element For example, inspection of the published IL-17F crystal structure (PDB code 1JPY) revealed a pocket of IL-17F similar to that of IL-17A for W12 of HAP binding, but it is occupied by a Phe-Phe motif at the N-terminal peptide of IL-17F. RESULTS +227 233 IL-17F protein For example, inspection of the published IL-17F crystal structure (PDB code 1JPY) revealed a pocket of IL-17F similar to that of IL-17A for W12 of HAP binding, but it is occupied by a Phe-Phe motif at the N-terminal peptide of IL-17F. RESULTS +5 18 Phe-Phe motif structure_element This Phe-Phe motif is missing in IL-17A. RESULTS +22 29 missing protein_state This Phe-Phe motif is missing in IL-17A. RESULTS +33 39 IL-17A protein This Phe-Phe motif is missing in IL-17A. RESULTS +0 19 Sequence alignments experimental_method Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal β-strand of HAP. RESULTS +28 33 human species Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal β-strand of HAP. RESULTS +38 43 mouse taxonomy_domain Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal β-strand of HAP. RESULTS +44 50 IL-17A protein Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal β-strand of HAP. RESULTS +72 78 IL-17A protein Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal β-strand of HAP. RESULTS +110 113 HAP chemical Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal β-strand of HAP. RESULTS +145 153 strand 0 structure_element Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal β-strand of HAP. RESULTS +157 163 IL-17A protein Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal β-strand of HAP. RESULTS +200 208 β-strand structure_element Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal β-strand of HAP. RESULTS +212 215 HAP chemical Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal β-strand of HAP. RESULTS +3 8 human species In human IL-17A the sequences are 21TVMVNLNI, and in mouse they are 21NVKVNLKV. RESULTS +9 15 IL-17A protein In human IL-17A the sequences are 21TVMVNLNI, and in mouse they are 21NVKVNLKV. RESULTS +34 44 21TVMVNLNI chemical In human IL-17A the sequences are 21TVMVNLNI, and in mouse they are 21NVKVNLKV. RESULTS +53 58 mouse taxonomy_domain In human IL-17A the sequences are 21TVMVNLNI, and in mouse they are 21NVKVNLKV. RESULTS +68 78 21NVKVNLKV chemical In human IL-17A the sequences are 21TVMVNLNI, and in mouse they are 21NVKVNLKV. RESULTS +23 36 phage display experimental_method Using a combination of phage display and SAR we have discovered novel peptides that are IL-17A antagonists. DISCUSS +41 44 SAR experimental_method Using a combination of phage display and SAR we have discovered novel peptides that are IL-17A antagonists. DISCUSS +88 94 IL-17A protein Using a combination of phage display and SAR we have discovered novel peptides that are IL-17A antagonists. DISCUSS +23 26 HAP chemical One of those peptides, HAP, also shows activity in inhibiting the production of multiple inflammatory cytokines by primary human keratinocytes stimulated by IL-17A and TNF-α, a disease relevant-model. DISCUSS +102 111 cytokines protein_type One of those peptides, HAP, also shows activity in inhibiting the production of multiple inflammatory cytokines by primary human keratinocytes stimulated by IL-17A and TNF-α, a disease relevant-model. DISCUSS +123 128 human species One of those peptides, HAP, also shows activity in inhibiting the production of multiple inflammatory cytokines by primary human keratinocytes stimulated by IL-17A and TNF-α, a disease relevant-model. DISCUSS +157 163 IL-17A protein One of those peptides, HAP, also shows activity in inhibiting the production of multiple inflammatory cytokines by primary human keratinocytes stimulated by IL-17A and TNF-α, a disease relevant-model. DISCUSS +168 173 TNF-α protein One of those peptides, HAP, also shows activity in inhibiting the production of multiple inflammatory cytokines by primary human keratinocytes stimulated by IL-17A and TNF-α, a disease relevant-model. DISCUSS +13 23 determined experimental_method We have also determined the complex structure of IL-17A/HAP, which provides the structural basis for HAP’s antagonism to IL-17A signaling. DISCUSS +28 45 complex structure evidence We have also determined the complex structure of IL-17A/HAP, which provides the structural basis for HAP’s antagonism to IL-17A signaling. DISCUSS +49 59 IL-17A/HAP complex_assembly We have also determined the complex structure of IL-17A/HAP, which provides the structural basis for HAP’s antagonism to IL-17A signaling. DISCUSS +101 104 HAP chemical We have also determined the complex structure of IL-17A/HAP, which provides the structural basis for HAP’s antagonism to IL-17A signaling. DISCUSS +121 127 IL-17A protein We have also determined the complex structure of IL-17A/HAP, which provides the structural basis for HAP’s antagonism to IL-17A signaling. DISCUSS +7 13 IL-17A protein During IL-17A signaling, IL-17A binds to one copy of IL-17RA and one copy of IL-17RC. DISCUSS +25 31 IL-17A protein During IL-17A signaling, IL-17A binds to one copy of IL-17RA and one copy of IL-17RC. DISCUSS +53 60 IL-17RA protein During IL-17A signaling, IL-17A binds to one copy of IL-17RA and one copy of IL-17RC. DISCUSS +77 84 IL-17RC protein During IL-17A signaling, IL-17A binds to one copy of IL-17RA and one copy of IL-17RC. DISCUSS +6 9 apo protein_state Since apo IL-17A is a homodimer with 2 fold symmetry, IL-17RA potentially can bind to either face of the IL-17A dimer. DISCUSS +10 16 IL-17A protein Since apo IL-17A is a homodimer with 2 fold symmetry, IL-17RA potentially can bind to either face of the IL-17A dimer. DISCUSS +22 31 homodimer oligomeric_state Since apo IL-17A is a homodimer with 2 fold symmetry, IL-17RA potentially can bind to either face of the IL-17A dimer. DISCUSS +54 61 IL-17RA protein Since apo IL-17A is a homodimer with 2 fold symmetry, IL-17RA potentially can bind to either face of the IL-17A dimer. DISCUSS +105 111 IL-17A protein Since apo IL-17A is a homodimer with 2 fold symmetry, IL-17RA potentially can bind to either face of the IL-17A dimer. DISCUSS +112 117 dimer oligomeric_state Since apo IL-17A is a homodimer with 2 fold symmetry, IL-17RA potentially can bind to either face of the IL-17A dimer. DISCUSS +9 12 HAP chemical With two HAP molecules covering both faces of the IL-17A dimer, HAP can block IL-17RA approaching from either face. DISCUSS +50 56 IL-17A protein With two HAP molecules covering both faces of the IL-17A dimer, HAP can block IL-17RA approaching from either face. DISCUSS +57 62 dimer oligomeric_state With two HAP molecules covering both faces of the IL-17A dimer, HAP can block IL-17RA approaching from either face. DISCUSS +64 67 HAP chemical With two HAP molecules covering both faces of the IL-17A dimer, HAP can block IL-17RA approaching from either face. DISCUSS +78 85 IL-17RA protein With two HAP molecules covering both faces of the IL-17A dimer, HAP can block IL-17RA approaching from either face. DISCUSS +36 53 crystal structure evidence To form the 1:2 complex observed in crystal structure, it is important that there is no strong negative cooperativity in the binding of two HAP molecules. DISCUSS +140 143 HAP chemical To form the 1:2 complex observed in crystal structure, it is important that there is no strong negative cooperativity in the binding of two HAP molecules. DISCUSS +12 60 native electrospray ionization mass spectrometry experimental_method In fact, in native electrospray ionization mass spectrometry analysis only 1:2 IL-17A/HAP complex was observed even when IL-17A was in excess (Supplementary Figure S8), indicating a positive binding cooperativity that favors inhibition of IL-17RA binding by HAP. DISCUSS +79 89 IL-17A/HAP complex_assembly In fact, in native electrospray ionization mass spectrometry analysis only 1:2 IL-17A/HAP complex was observed even when IL-17A was in excess (Supplementary Figure S8), indicating a positive binding cooperativity that favors inhibition of IL-17RA binding by HAP. DISCUSS +121 127 IL-17A protein In fact, in native electrospray ionization mass spectrometry analysis only 1:2 IL-17A/HAP complex was observed even when IL-17A was in excess (Supplementary Figure S8), indicating a positive binding cooperativity that favors inhibition of IL-17RA binding by HAP. DISCUSS +239 246 IL-17RA protein In fact, in native electrospray ionization mass spectrometry analysis only 1:2 IL-17A/HAP complex was observed even when IL-17A was in excess (Supplementary Figure S8), indicating a positive binding cooperativity that favors inhibition of IL-17RA binding by HAP. DISCUSS +258 261 HAP chemical In fact, in native electrospray ionization mass spectrometry analysis only 1:2 IL-17A/HAP complex was observed even when IL-17A was in excess (Supplementary Figure S8), indicating a positive binding cooperativity that favors inhibition of IL-17RA binding by HAP. DISCUSS +0 3 HAP chemical HAP, with only 15 residues, can achieve almost the same binding affinity as the much larger IL-17RA molecule, indicating a more efficient way of binding to IL-17A. DISCUSS +15 26 15 residues residue_range HAP, with only 15 residues, can achieve almost the same binding affinity as the much larger IL-17RA molecule, indicating a more efficient way of binding to IL-17A. DISCUSS +56 72 binding affinity evidence HAP, with only 15 residues, can achieve almost the same binding affinity as the much larger IL-17RA molecule, indicating a more efficient way of binding to IL-17A. DISCUSS +92 99 IL-17RA protein HAP, with only 15 residues, can achieve almost the same binding affinity as the much larger IL-17RA molecule, indicating a more efficient way of binding to IL-17A. DISCUSS +156 162 IL-17A protein HAP, with only 15 residues, can achieve almost the same binding affinity as the much larger IL-17RA molecule, indicating a more efficient way of binding to IL-17A. DISCUSS +19 25 IL-17A protein The interaction of IL-17A with IL-17RA has an extensive interface, covering ~2,200 Å2 surface area of IL-17A. DISCUSS +31 38 IL-17RA protein The interaction of IL-17A with IL-17RA has an extensive interface, covering ~2,200 Å2 surface area of IL-17A. DISCUSS +56 65 interface site The interaction of IL-17A with IL-17RA has an extensive interface, covering ~2,200 Å2 surface area of IL-17A. DISCUSS +102 108 IL-17A protein The interaction of IL-17A with IL-17RA has an extensive interface, covering ~2,200 Å2 surface area of IL-17A. DISCUSS +39 71 IL-17A/IL-17RA binding interface site Due to the discontinuous nature of the IL-17A/IL-17RA binding interface, it is classified as having tertiary structural epitopes on both binding partners, and is therefore hard to target using small molecules. DISCUSS +15 18 HAP chemical Our studies of HAP demonstrated an uncommon mode of action for a peptide in inhibiting such a difficult protein-protein interaction target, and suggest further possible improvements in its binding potency. DISCUSS +29 32 HAP chemical One way of further improving HAP’s potency is by dimerization. DISCUSS +21 24 HAP chemical Homo-dimerization of HAP (45) achieved sub-nanomolar potency against human IL-17A in cell assay. DISCUSS +26 28 45 chemical Homo-dimerization of HAP (45) achieved sub-nanomolar potency against human IL-17A in cell assay. DISCUSS +69 74 human species Homo-dimerization of HAP (45) achieved sub-nanomolar potency against human IL-17A in cell assay. DISCUSS +75 81 IL-17A protein Homo-dimerization of HAP (45) achieved sub-nanomolar potency against human IL-17A in cell assay. DISCUSS +7 24 crystal structure evidence In the crystal structure, the distance between the carbonyl of Asn14 of one HAP molecule and the N-terminus of the second is only 15.7 Å, suggesting the potential for more potent dimeric peptides to be designed by using linkers of different lengths at different positions. DISCUSS +63 68 Asn14 residue_name_number In the crystal structure, the distance between the carbonyl of Asn14 of one HAP molecule and the N-terminus of the second is only 15.7 Å, suggesting the potential for more potent dimeric peptides to be designed by using linkers of different lengths at different positions. DISCUSS +76 79 HAP chemical In the crystal structure, the distance between the carbonyl of Asn14 of one HAP molecule and the N-terminus of the second is only 15.7 Å, suggesting the potential for more potent dimeric peptides to be designed by using linkers of different lengths at different positions. DISCUSS +179 186 dimeric oligomeric_state In the crystal structure, the distance between the carbonyl of Asn14 of one HAP molecule and the N-terminus of the second is only 15.7 Å, suggesting the potential for more potent dimeric peptides to be designed by using linkers of different lengths at different positions. DISCUSS +187 195 peptides chemical In the crystal structure, the distance between the carbonyl of Asn14 of one HAP molecule and the N-terminus of the second is only 15.7 Å, suggesting the potential for more potent dimeric peptides to be designed by using linkers of different lengths at different positions. DISCUSS +31 34 HAP chemical Another direction of improving HAP is by reducing its size. DISCUSS +23 40 crystal structure evidence As demonstrated by the crystal structure, binding of the α-helix of HAP should be sufficient for preventing IL-17RA binding to IL-17A. DISCUSS +57 64 α-helix structure_element As demonstrated by the crystal structure, binding of the α-helix of HAP should be sufficient for preventing IL-17RA binding to IL-17A. DISCUSS +68 71 HAP chemical As demonstrated by the crystal structure, binding of the α-helix of HAP should be sufficient for preventing IL-17RA binding to IL-17A. DISCUSS +108 115 IL-17RA protein As demonstrated by the crystal structure, binding of the α-helix of HAP should be sufficient for preventing IL-17RA binding to IL-17A. DISCUSS +127 133 IL-17A protein As demonstrated by the crystal structure, binding of the α-helix of HAP should be sufficient for preventing IL-17RA binding to IL-17A. DISCUSS +94 101 α-helix structure_element Theoretically, it is possible to design chemicals such as stapled α-helical peptides to block α-helix-mediated IL-17A/IL-17RA interactions. DISCUSS +111 125 IL-17A/IL-17RA complex_assembly Theoretically, it is possible to design chemicals such as stapled α-helical peptides to block α-helix-mediated IL-17A/IL-17RA interactions. DISCUSS +37 43 IL-17A protein In summary, these peptide-based anti-IL-17A modalities could be further developed as alternative therapeutic options to the reported monoclonal antibodies. DISCUSS +144 154 antibodies protein_type In summary, these peptide-based anti-IL-17A modalities could be further developed as alternative therapeutic options to the reported monoclonal antibodies. DISCUSS +67 73 IL-17A protein We are also very interested in finding non-peptidic small molecule IL-17A antagonists, and HAP can be used as an excellent tool peptide. DISCUSS +91 94 HAP chemical We are also very interested in finding non-peptidic small molecule IL-17A antagonists, and HAP can be used as an excellent tool peptide. DISCUSS +48 58 structures evidence The strategy utilized in generating the complex structures of HAP may also be useful for enabling structure based design of some known small molecule IL-17A antagonists. DISCUSS +62 65 HAP chemical The strategy utilized in generating the complex structures of HAP may also be useful for enabling structure based design of some known small molecule IL-17A antagonists. DISCUSS +11 14 HAP chemical Binding of HAP to IL-17A and inhibition of IL-17A/IL-17RA are measured by SPR, FRET and cell-based assays. FIG +18 24 IL-17A protein Binding of HAP to IL-17A and inhibition of IL-17A/IL-17RA are measured by SPR, FRET and cell-based assays. FIG +43 57 IL-17A/IL-17RA complex_assembly Binding of HAP to IL-17A and inhibition of IL-17A/IL-17RA are measured by SPR, FRET and cell-based assays. FIG +74 77 SPR experimental_method Binding of HAP to IL-17A and inhibition of IL-17A/IL-17RA are measured by SPR, FRET and cell-based assays. FIG +79 83 FRET experimental_method Binding of HAP to IL-17A and inhibition of IL-17A/IL-17RA are measured by SPR, FRET and cell-based assays. FIG +88 105 cell-based assays experimental_method Binding of HAP to IL-17A and inhibition of IL-17A/IL-17RA are measured by SPR, FRET and cell-based assays. FIG +12 15 SPR experimental_method (A) Typical SPR sensorgrams (black) of HAP at indicated concentrations binding to biotinylated human IL-17A immobilized on a streptavidin chip surface, fitted with single site binding model curves (red). FIG +16 27 sensorgrams evidence (A) Typical SPR sensorgrams (black) of HAP at indicated concentrations binding to biotinylated human IL-17A immobilized on a streptavidin chip surface, fitted with single site binding model curves (red). FIG +39 42 HAP chemical (A) Typical SPR sensorgrams (black) of HAP at indicated concentrations binding to biotinylated human IL-17A immobilized on a streptavidin chip surface, fitted with single site binding model curves (red). FIG +82 94 biotinylated protein_state (A) Typical SPR sensorgrams (black) of HAP at indicated concentrations binding to biotinylated human IL-17A immobilized on a streptavidin chip surface, fitted with single site binding model curves (red). FIG +95 100 human species (A) Typical SPR sensorgrams (black) of HAP at indicated concentrations binding to biotinylated human IL-17A immobilized on a streptavidin chip surface, fitted with single site binding model curves (red). FIG +101 107 IL-17A protein (A) Typical SPR sensorgrams (black) of HAP at indicated concentrations binding to biotinylated human IL-17A immobilized on a streptavidin chip surface, fitted with single site binding model curves (red). FIG +164 196 single site binding model curves evidence (A) Typical SPR sensorgrams (black) of HAP at indicated concentrations binding to biotinylated human IL-17A immobilized on a streptavidin chip surface, fitted with single site binding model curves (red). FIG +20 22 ka evidence Kinetic parameters (ka, kd) were obtained by a global fit using three concentrations in triplicate. FIG +24 26 kd evidence Kinetic parameters (ka, kd) were obtained by a global fit using three concentrations in triplicate. FIG +0 2 KD evidence KD determined by the standard equation, KD = kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA. FIG +40 42 KD evidence KD determined by the standard equation, KD = kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA. FIG +45 47 kd evidence KD determined by the standard equation, KD = kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA. FIG +48 50 ka evidence KD determined by the standard equation, KD = kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA. FIG +56 59 HAP chemical KD determined by the standard equation, KD = kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA. FIG +69 72 SPR experimental_method KD determined by the standard equation, KD = kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA. FIG +86 92 IL-17A protein KD determined by the standard equation, KD = kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA. FIG +104 115 immobilized protein_state KD determined by the standard equation, KD = kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA. FIG +116 123 IL-17RA protein KD determined by the standard equation, KD = kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA. FIG +96 102 IL-17A protein Data are mean and error bars of +/− standard deviation of three measurements. (C) Inhibition of IL-17A and IL-17RA binding by HAP measured by FRET assay. FIG +107 114 IL-17RA protein Data are mean and error bars of +/− standard deviation of three measurements. (C) Inhibition of IL-17A and IL-17RA binding by HAP measured by FRET assay. FIG +126 129 HAP chemical Data are mean and error bars of +/− standard deviation of three measurements. (C) Inhibition of IL-17A and IL-17RA binding by HAP measured by FRET assay. FIG +142 152 FRET assay experimental_method Data are mean and error bars of +/− standard deviation of three measurements. (C) Inhibition of IL-17A and IL-17RA binding by HAP measured by FRET assay. FIG +121 124 HAP chemical Data are mean and error bars of +/− standard deviation from 299 experiments, each performed in duplicate. (D) Example of HAP selective inhibition of the production of IL-8 (triangles), IL-6 (squares) and CCL-20 (circles) by primary human keratinocyte cells synergistically stimulated by 100 ng/ml IL-17A and 10 ng/ml TNF-α. FIG +167 171 IL-8 protein_type Data are mean and error bars of +/− standard deviation from 299 experiments, each performed in duplicate. (D) Example of HAP selective inhibition of the production of IL-8 (triangles), IL-6 (squares) and CCL-20 (circles) by primary human keratinocyte cells synergistically stimulated by 100 ng/ml IL-17A and 10 ng/ml TNF-α. FIG +185 189 IL-6 protein_type Data are mean and error bars of +/− standard deviation from 299 experiments, each performed in duplicate. (D) Example of HAP selective inhibition of the production of IL-8 (triangles), IL-6 (squares) and CCL-20 (circles) by primary human keratinocyte cells synergistically stimulated by 100 ng/ml IL-17A and 10 ng/ml TNF-α. FIG +204 210 CCL-20 protein_type Data are mean and error bars of +/− standard deviation from 299 experiments, each performed in duplicate. (D) Example of HAP selective inhibition of the production of IL-8 (triangles), IL-6 (squares) and CCL-20 (circles) by primary human keratinocyte cells synergistically stimulated by 100 ng/ml IL-17A and 10 ng/ml TNF-α. FIG +232 237 human species Data are mean and error bars of +/− standard deviation from 299 experiments, each performed in duplicate. (D) Example of HAP selective inhibition of the production of IL-8 (triangles), IL-6 (squares) and CCL-20 (circles) by primary human keratinocyte cells synergistically stimulated by 100 ng/ml IL-17A and 10 ng/ml TNF-α. FIG +297 303 IL-17A protein Data are mean and error bars of +/− standard deviation from 299 experiments, each performed in duplicate. (D) Example of HAP selective inhibition of the production of IL-8 (triangles), IL-6 (squares) and CCL-20 (circles) by primary human keratinocyte cells synergistically stimulated by 100 ng/ml IL-17A and 10 ng/ml TNF-α. FIG +317 322 TNF-α protein Data are mean and error bars of +/− standard deviation from 299 experiments, each performed in duplicate. (D) Example of HAP selective inhibition of the production of IL-8 (triangles), IL-6 (squares) and CCL-20 (circles) by primary human keratinocyte cells synergistically stimulated by 100 ng/ml IL-17A and 10 ng/ml TNF-α. FIG +0 3 HAP chemical HAP does not inhibit the baseline production of IL-6, IL-8 and CCL-20 stimulated by 10 ng/ml TNF-α alone (gray lines and symbols). FIG +48 52 IL-6 protein_type HAP does not inhibit the baseline production of IL-6, IL-8 and CCL-20 stimulated by 10 ng/ml TNF-α alone (gray lines and symbols). FIG +54 58 IL-8 protein_type HAP does not inhibit the baseline production of IL-6, IL-8 and CCL-20 stimulated by 10 ng/ml TNF-α alone (gray lines and symbols). FIG +63 69 CCL-20 protein_type HAP does not inhibit the baseline production of IL-6, IL-8 and CCL-20 stimulated by 10 ng/ml TNF-α alone (gray lines and symbols). FIG +93 98 TNF-α protein HAP does not inhibit the baseline production of IL-6, IL-8 and CCL-20 stimulated by 10 ng/ml TNF-α alone (gray lines and symbols). FIG +8 17 structure evidence Overall structure of the Fab/IL-17A/HAP complex in ribbon presentation. FIG +25 39 Fab/IL-17A/HAP complex_assembly Overall structure of the Fab/IL-17A/HAP complex in ribbon presentation. FIG +4 7 HAP chemical Two HAP molecules are colored blue and red, and IL-17A monomers are colored ice blue and pink, respectively. FIG +48 54 IL-17A protein Two HAP molecules are colored blue and red, and IL-17A monomers are colored ice blue and pink, respectively. FIG +55 63 monomers oligomeric_state Two HAP molecules are colored blue and red, and IL-17A monomers are colored ice blue and pink, respectively. FIG +29 42 binding sites site (A) Overview of the distinct binding sites of Fab and HAP to IL-17A. FIG +46 49 Fab structure_element (A) Overview of the distinct binding sites of Fab and HAP to IL-17A. FIG +54 57 HAP chemical (A) Overview of the distinct binding sites of Fab and HAP to IL-17A. FIG +61 67 IL-17A protein (A) Overview of the distinct binding sites of Fab and HAP to IL-17A. FIG +25 35 IL-17A/HAP complex_assembly (B) Close-in view of the IL-17A/HAP structure. FIG +36 45 structure evidence (B) Close-in view of the IL-17A/HAP structure. FIG +0 6 IL-17A protein IL-17A β-strands are labelled. FIG +7 16 β-strands structure_element IL-17A β-strands are labelled. FIG +16 21 bound protein_state Each of the two bound HAP interacts with both monomers of the IL-17A dimer. FIG +22 25 HAP chemical Each of the two bound HAP interacts with both monomers of the IL-17A dimer. FIG +46 54 monomers oligomeric_state Each of the two bound HAP interacts with both monomers of the IL-17A dimer. FIG +62 68 IL-17A protein Each of the two bound HAP interacts with both monomers of the IL-17A dimer. FIG +69 74 dimer oligomeric_state Each of the two bound HAP interacts with both monomers of the IL-17A dimer. FIG +25 39 IL-17A/IL-17RA complex_assembly (C) As a comparison, the IL-17A/IL-17RA complex was shown with IL-17A in the same orientation. FIG +63 69 IL-17A protein (C) As a comparison, the IL-17A/IL-17RA complex was shown with IL-17A in the same orientation. FIG +21 45 IL-17A/IL-17RA interface site Three distinct areas IL-17A/IL-17RA interface are labeled. FIG +35 49 IL-17A/IL-17RA complex_assembly Mechanism of the inhibition of the IL-17A/IL-17RA interaction by HAP. FIG +65 68 HAP chemical Mechanism of the inhibition of the IL-17A/IL-17RA interaction by HAP. FIG +4 7 HAP chemical (A) HAP binds at region I of IL-17A. FIG +17 25 region I structure_element (A) HAP binds at region I of IL-17A. FIG +29 35 IL-17A protein (A) HAP binds at region I of IL-17A. FIG +0 6 IL-17A protein IL-17A dimer is in surface presentation (β-strands 0 shown as ribbons for clarity). FIG +7 12 dimer oligomeric_state IL-17A dimer is in surface presentation (β-strands 0 shown as ribbons for clarity). FIG +41 52 β-strands 0 structure_element IL-17A dimer is in surface presentation (β-strands 0 shown as ribbons for clarity). FIG +0 18 Polar interactions bond_interaction Polar interactions are shown in dashes. FIG +0 3 HAP chemical HAP residues as well as key IL-17A residues are labeled. FIG +28 34 IL-17A protein HAP residues as well as key IL-17A residues are labeled. FIG +19 22 HAP chemical For clarity, a few HAP residues are also shown in stick model with carbon atoms colored green, oxygen in red and nitrogen in blue. FIG +4 10 I-17RA protein (B) I-17RA (ribbon in gold) peptide Leu27-Ile32 binds to the same area as the HAP α-helix. FIG +36 47 Leu27-Ile32 residue_range (B) I-17RA (ribbon in gold) peptide Leu27-Ile32 binds to the same area as the HAP α-helix. FIG +78 81 HAP chemical (B) I-17RA (ribbon in gold) peptide Leu27-Ile32 binds to the same area as the HAP α-helix. FIG +82 89 α-helix structure_element (B) I-17RA (ribbon in gold) peptide Leu27-Ile32 binds to the same area as the HAP α-helix. FIG +0 5 Trp31 residue_name_number Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +9 16 IL-17RA protein Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +35 41 pocket site Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +45 51 IL-17A protein Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +55 60 Trp12 residue_name_number Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +64 67 HAP chemical Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +91 98 overlay experimental_method Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +108 111 HAP chemical Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +125 136 β-strands 0 structure_element Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +151 161 IL-17A/HAP complex_assembly Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +177 180 apo protein_state Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +181 187 IL-17A protein Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +188 197 structure evidence Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +225 233 region I structure_element Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +237 243 IL-17A protein Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +279 286 β-stand structure_element Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +291 298 α-helix structure_element Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +306 309 HAP chemical Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. FIG +16 34 Trp binding pocket site Notice that the Trp binding pocket for W12 of HAP or W31 of IL-17RA is missing in the apo structure. FIG +39 42 W12 residue_name_number Notice that the Trp binding pocket for W12 of HAP or W31 of IL-17RA is missing in the apo structure. FIG +46 49 HAP chemical Notice that the Trp binding pocket for W12 of HAP or W31 of IL-17RA is missing in the apo structure. FIG +53 56 W31 residue_name_number Notice that the Trp binding pocket for W12 of HAP or W31 of IL-17RA is missing in the apo structure. FIG +60 67 IL-17RA protein Notice that the Trp binding pocket for W12 of HAP or W31 of IL-17RA is missing in the apo structure. FIG +86 89 apo protein_state Notice that the Trp binding pocket for W12 of HAP or W31 of IL-17RA is missing in the apo structure. FIG +90 99 structure evidence Notice that the Trp binding pocket for W12 of HAP or W31 of IL-17RA is missing in the apo structure. FIG +0 26 ELISA competition activity experimental_method ELISA competition activity of peptide analogues of 1. TABLE diff --git a/annotation_CSV/PMC4871749.csv b/annotation_CSV/PMC4871749.csv new file mode 100644 index 0000000000000000000000000000000000000000..678142307f42fb7a8a42aecfd8ce0725c025a381 --- /dev/null +++ b/annotation_CSV/PMC4871749.csv @@ -0,0 +1,412 @@ +anno_start anno_end anno_text entity_type sentence section +4 9 Taf14 protein The Taf14 YEATS domain is a reader of histone crotonylation TITLE +10 22 YEATS domain structure_element The Taf14 YEATS domain is a reader of histone crotonylation TITLE +38 45 histone protein_type The Taf14 YEATS domain is a reader of histone crotonylation TITLE +46 59 crotonylation ptm The Taf14 YEATS domain is a reader of histone crotonylation TITLE +21 28 histone protein_type The discovery of new histone modifications is unfolding at startling rates, however, the identification of effectors capable of interpreting these modifications has lagged behind. ABSTRACT +19 31 YEATS domain structure_element Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. ABSTRACT +58 65 histone protein_type Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. ABSTRACT +66 72 lysine residue_name Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. ABSTRACT +73 86 crotonylation ptm Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. ABSTRACT +17 22 Taf14 protein We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity. ABSTRACT +23 35 YEATS domain structure_element We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity. ABSTRACT +44 58 crotonyllysine residue_name We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity. ABSTRACT +72 86 π-π-π-stacking bond_interaction We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity. ABSTRACT +112 125 YEATS domains structure_element We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity. ABSTRACT +131 145 crotonyllysine residue_name We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity. ABSTRACT +0 13 Crotonylation ptm Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin. INTRO +17 23 lysine residue_name Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin. INTRO +34 48 crotonyllysine residue_name Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin. INTRO +50 53 Kcr residue_name Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin. INTRO +93 100 histone protein_type Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin. INTRO +150 159 mammalian taxonomy_domain Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin. INTRO +4 18 crotonyllysine residue_name The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. INTRO +27 34 histone protein_type The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. INTRO +35 37 H3 protein_type The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. INTRO +37 40 K18 residue_name_number The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. INTRO +56 60 p300 protein The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. INTRO +64 89 histone acetyltransferase protein_type The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. INTRO +111 122 acetylation ptm The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. INTRO +61 75 crotonyllysine residue_name Owing to some differences in their genomic distribution, the crotonyllysine and acetyllysine (Kac) modifications have been linked to distinct functional outcomes. INTRO +80 92 acetyllysine residue_name Owing to some differences in their genomic distribution, the crotonyllysine and acetyllysine (Kac) modifications have been linked to distinct functional outcomes. INTRO +94 97 Kac residue_name Owing to some differences in their genomic distribution, the crotonyllysine and acetyllysine (Kac) modifications have been linked to distinct functional outcomes. INTRO +0 4 p300 protein p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation. INTRO +15 22 histone protein_type p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation. INTRO +23 36 crotonylation ptm p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation. INTRO +129 133 p300 protein p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation. INTRO +144 155 acetylation ptm p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation. INTRO +53 67 crotonyllysine residue_name The discovery of individual biological roles for the crotonyllysine and acetyllysine marks suggests that these PTMs can be read by distinct readers. INTRO +72 84 acetyllysine residue_name The discovery of individual biological roles for the crotonyllysine and acetyllysine marks suggests that these PTMs can be read by distinct readers. INTRO +18 30 acetyllysine residue_name While a number of acetyllysine readers have been identified and characterized, a specific reader of the crotonyllysine mark remains unknown (reviewed in). INTRO +104 118 crotonyllysine residue_name While a number of acetyllysine readers have been identified and characterized, a specific reader of the crotonyllysine mark remains unknown (reviewed in). INTRO +19 31 bromodomains structure_element A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity. INTRO +33 36 BDs structure_element A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity. INTRO +65 67 BD structure_element A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity. INTRO +98 110 crotonylated protein_state A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity. INTRO +153 163 acetylated protein_state A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity. INTRO +189 201 bromodomains structure_element A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity. INTRO +242 256 crotonyllysine residue_name A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity. INTRO +14 26 acetyllysine residue_name The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +81 86 YEATS structure_element The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +88 92 Yaf9 protein The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +94 97 ENL protein The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +99 102 AF9 protein The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +104 109 Taf14 protein The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +111 115 Sas5 protein The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +128 133 human species The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +134 137 AF9 protein The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +142 147 yeast taxonomy_domain The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +148 153 Taf14 protein The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +185 192 histone protein_type The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +198 200 H3 protein_type The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +200 204 K9ac ptm The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. INTRO +4 16 acetyllysine residue_name The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. INTRO +41 44 AF9 protein The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. INTRO +45 57 YEATS domain structure_element The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. INTRO +98 123 histone methyltransferase protein_type The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. INTRO +124 129 DOT1L protein The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. INTRO +133 135 H3 protein_type The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. INTRO +135 139 K9ac ptm The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. INTRO +169 174 DOT1L protein The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. INTRO +184 186 H3 protein_type The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. INTRO +186 189 K79 residue_name_number The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. INTRO +190 201 methylation ptm The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. INTRO +68 73 yeast taxonomy_domain Similarly, activation of a subset of genes and DNA damage repair in yeast require the acetyllysine binding activity of the Taf14 YEATS domain. INTRO +86 98 acetyllysine residue_name Similarly, activation of a subset of genes and DNA damage repair in yeast require the acetyllysine binding activity of the Taf14 YEATS domain. INTRO +123 128 Taf14 protein Similarly, activation of a subset of genes and DNA damage repair in yeast require the acetyllysine binding activity of the Taf14 YEATS domain. INTRO +129 141 YEATS domain structure_element Similarly, activation of a subset of genes and DNA damage repair in yeast require the acetyllysine binding activity of the Taf14 YEATS domain. INTRO +45 50 Taf14 protein Consistent with its role in gene regulation, Taf14 was identified as a core component of the transcription factor complexes TFIID and TFIIF. INTRO +124 129 TFIID complex_assembly Consistent with its role in gene regulation, Taf14 was identified as a core component of the transcription factor complexes TFIID and TFIIF. INTRO +134 139 TFIIF complex_assembly Consistent with its role in gene regulation, Taf14 was identified as a core component of the transcription factor complexes TFIID and TFIIF. INTRO +9 14 Taf14 protein However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. INTRO +82 87 INO80 complex_assembly However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. INTRO +89 96 SWI/SNF complex_assembly However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. INTRO +101 104 RSC complex_assembly However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. INTRO +114 139 histone acetyltransferase protein_type However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. INTRO +148 152 NuA3 complex_assembly However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. INTRO +188 193 Taf14 protein However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. INTRO +33 38 Taf14 protein In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. INTRO +39 51 YEATS domain structure_element In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. INTRO +67 81 crotonyllysine residue_name In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. INTRO +96 103 histone protein_type In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. INTRO +104 106 H3 protein_type In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. INTRO +107 119 crotonylated protein_state In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. INTRO +123 131 lysine 9 residue_name_number In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. INTRO +133 135 H3 protein_type In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. INTRO +135 139 K9cr ptm In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. INTRO +14 16 H3 protein_type We found that H3K9cr is present in yeast and is dynamically regulated. INTRO +16 20 K9cr ptm We found that H3K9cr is present in yeast and is dynamically regulated. INTRO +35 40 yeast taxonomy_domain We found that H3K9cr is present in yeast and is dynamically regulated. INTRO +56 58 H3 protein_type To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). INTRO +58 62 K9cr ptm To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). INTRO +83 100 crystal structure evidence To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). INTRO +108 113 Taf14 protein To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). INTRO +114 126 YEATS domain structure_element To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). INTRO +127 142 in complex with protein_state To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). INTRO +143 153 H3K9cr5-13 chemical To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). INTRO +164 168 5–13 residue_range To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). INTRO +172 174 H3 protein_type To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). INTRO +4 9 Taf14 protein The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). INTRO +10 22 YEATS domain structure_element The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). INTRO +33 66 immunoglobin-like β sandwich fold structure_element The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). INTRO +84 107 anti-parallel β strands structure_element The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). INTRO +124 129 loops structure_element The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). INTRO +142 154 binding site site The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). INTRO +159 161 H3 protein_type The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). INTRO +161 165 K9cr ptm The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). INTRO +4 6 H3 protein_type The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c). INTRO +6 10 K9cr ptm The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c). INTRO +30 51 extended conformation protein_state The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c). INTRO +88 97 β strands structure_element The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c). INTRO +159 164 water chemical The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c). INTRO +174 188 hydrogen bonds bond_interaction The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c). INTRO +195 206 salt bridge bond_interaction The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c). INTRO +33 47 crotonyllysine residue_name The most striking feature of the crotonyllysine recognition mechanism is the unique coordination of crotonylated lysine residue. INTRO +100 112 crotonylated protein_state The most striking feature of the crotonyllysine recognition mechanism is the unique coordination of crotonylated lysine residue. INTRO +113 119 lysine residue_name The most striking feature of the crotonyllysine recognition mechanism is the unique coordination of crotonylated lysine residue. INTRO +33 37 K9cr ptm The fully extended side chain of K9cr transverses the narrow tunnel, crossing the β sandwich at right angle in a corkscrew-like manner (Fig. 1b and Supplementary Figure 1b). INTRO +82 92 β sandwich structure_element The fully extended side chain of K9cr transverses the narrow tunnel, crossing the β sandwich at right angle in a corkscrew-like manner (Fig. 1b and Supplementary Figure 1b). INTRO +11 19 crotonyl chemical The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic π-π-π-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c). INTRO +46 51 Trp81 residue_name_number The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic π-π-π-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c). INTRO +56 61 Phe62 residue_name_number The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic π-π-π-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c). INTRO +184 192 crotonyl chemical The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic π-π-π-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c). INTRO +251 265 π-π-π-stacking bond_interaction The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic π-π-π-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c). INTRO +18 23 Trp81 residue_name_number The side chain of Trp81 appears to adopt two conformations, one of which provides maximum π-stacking with the alkene functional group while the other rotamer affords maximum π-stacking with the amide π electrons (Supplementary Fig. 1c). INTRO +90 100 π-stacking bond_interaction The side chain of Trp81 appears to adopt two conformations, one of which provides maximum π-stacking with the alkene functional group while the other rotamer affords maximum π-stacking with the amide π electrons (Supplementary Fig. 1c). INTRO +174 184 π-stacking bond_interaction The side chain of Trp81 appears to adopt two conformations, one of which provides maximum π-stacking with the alkene functional group while the other rotamer affords maximum π-stacking with the amide π electrons (Supplementary Fig. 1c). INTRO +25 30 Trp81 residue_name_number The dual conformation of Trp81 is likely due to the conjugated nature of the C=C and C=O π-orbitals within the crotonyl functional group. INTRO +111 119 crotonyl chemical The dual conformation of Trp81 is likely due to the conjugated nature of the C=C and C=O π-orbitals within the crotonyl functional group. INTRO +15 29 π-π-π stacking bond_interaction In addition to π-π-π stacking, the crotonyl group is stabilized by a set of hydrogen bonds and electrostatic interactions. INTRO +35 43 crotonyl chemical In addition to π-π-π stacking, the crotonyl group is stabilized by a set of hydrogen bonds and electrostatic interactions. INTRO +76 90 hydrogen bonds bond_interaction In addition to π-π-π stacking, the crotonyl group is stabilized by a set of hydrogen bonds and electrostatic interactions. INTRO +95 121 electrostatic interactions bond_interaction In addition to π-π-π stacking, the crotonyl group is stabilized by a set of hydrogen bonds and electrostatic interactions. INTRO +4 10 π bond bond_interaction The π bond conjugation of the crotonyl group gives rise to a dipole moment of the alkene moiety, resulting in a partial positive charge on the β-carbon (Cβ) and a partial negative charge on the α-carbon (Cα). INTRO +30 38 crotonyl chemical The π bond conjugation of the crotonyl group gives rise to a dipole moment of the alkene moiety, resulting in a partial positive charge on the β-carbon (Cβ) and a partial negative charge on the α-carbon (Cα). INTRO +59 81 electrostatic contacts bond_interaction This provides the capability for the alkene moiety to form electrostatic contacts, as Cα and Cβ lay within electrostatic interaction distances of the carbonyl oxygen of Gln79 and of the hydroxyl group of Thr61, respectively. INTRO +107 132 electrostatic interaction bond_interaction This provides the capability for the alkene moiety to form electrostatic contacts, as Cα and Cβ lay within electrostatic interaction distances of the carbonyl oxygen of Gln79 and of the hydroxyl group of Thr61, respectively. INTRO +169 174 Gln79 residue_name_number This provides the capability for the alkene moiety to form electrostatic contacts, as Cα and Cβ lay within electrostatic interaction distances of the carbonyl oxygen of Gln79 and of the hydroxyl group of Thr61, respectively. INTRO +204 209 Thr61 residue_name_number This provides the capability for the alkene moiety to form electrostatic contacts, as Cα and Cβ lay within electrostatic interaction distances of the carbonyl oxygen of Gln79 and of the hydroxyl group of Thr61, respectively. INTRO +22 27 Thr61 residue_name_number The hydroxyl group of Thr61 also participates in a hydrogen bond with the amide nitrogen of the K9cr side chain (Fig. 1d). INTRO +51 64 hydrogen bond bond_interaction The hydroxyl group of Thr61 also participates in a hydrogen bond with the amide nitrogen of the K9cr side chain (Fig. 1d). INTRO +96 100 K9cr ptm The hydroxyl group of Thr61 also participates in a hydrogen bond with the amide nitrogen of the K9cr side chain (Fig. 1d). INTRO +26 31 Thr61 residue_name_number The fixed position of the Thr61 hydroxyl group, which facilitates interactions with both the amide and Cα of K9cr, is achieved through a hydrogen bond with imidazole ring of His59. INTRO +109 113 K9cr ptm The fixed position of the Thr61 hydroxyl group, which facilitates interactions with both the amide and Cα of K9cr, is achieved through a hydrogen bond with imidazole ring of His59. INTRO +137 150 hydrogen bond bond_interaction The fixed position of the Thr61 hydroxyl group, which facilitates interactions with both the amide and Cα of K9cr, is achieved through a hydrogen bond with imidazole ring of His59. INTRO +174 179 His59 residue_name_number The fixed position of the Thr61 hydroxyl group, which facilitates interactions with both the amide and Cα of K9cr, is achieved through a hydrogen bond with imidazole ring of His59. INTRO +23 27 K9cr ptm Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d). INTRO +45 58 hydrogen bond bond_interaction Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d). INTRO +123 128 Trp81 residue_name_number Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d). INTRO +143 148 water chemical Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d). INTRO +158 171 hydrogen bond bond_interaction Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d). INTRO +208 213 Gly82 residue_name_number Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d). INTRO +64 69 Taf14 protein This distinctive mechanism was corroborated through mapping the Taf14 YEATS-H3K9cr binding interface in solution using NMR chemical shift perturbation analysis (Supplementary Fig. 2a, b). INTRO +70 100 YEATS-H3K9cr binding interface site This distinctive mechanism was corroborated through mapping the Taf14 YEATS-H3K9cr binding interface in solution using NMR chemical shift perturbation analysis (Supplementary Fig. 2a, b). INTRO +119 159 NMR chemical shift perturbation analysis experimental_method This distinctive mechanism was corroborated through mapping the Taf14 YEATS-H3K9cr binding interface in solution using NMR chemical shift perturbation analysis (Supplementary Fig. 2a, b). INTRO +15 20 Taf14 protein Binding of the Taf14 YEATS domain to H3K9cr is robust. INTRO +21 33 YEATS domain structure_element Binding of the Taf14 YEATS domain to H3K9cr is robust. INTRO +37 39 H3 protein_type Binding of the Taf14 YEATS domain to H3K9cr is robust. INTRO +39 43 K9cr ptm Binding of the Taf14 YEATS domain to H3K9cr is robust. INTRO +4 25 dissociation constant evidence The dissociation constant (Kd) for the Taf14 YEATS-H3K9cr5-13 complex was found to be 9.5 μM, as measured by fluorescence spectroscopy (Supplementary Fig. 2c). INTRO +27 29 Kd evidence The dissociation constant (Kd) for the Taf14 YEATS-H3K9cr5-13 complex was found to be 9.5 μM, as measured by fluorescence spectroscopy (Supplementary Fig. 2c). INTRO +39 61 Taf14 YEATS-H3K9cr5-13 complex_assembly The dissociation constant (Kd) for the Taf14 YEATS-H3K9cr5-13 complex was found to be 9.5 μM, as measured by fluorescence spectroscopy (Supplementary Fig. 2c). INTRO +109 134 fluorescence spectroscopy experimental_method The dissociation constant (Kd) for the Taf14 YEATS-H3K9cr5-13 complex was found to be 9.5 μM, as measured by fluorescence spectroscopy (Supplementary Fig. 2c). INTRO +30 48 binding affinities evidence This value is in the range of binding affinities exhibited by the majority of histone readers, thus attesting to the physiological relevance of the H3K9cr recognition by Taf14. INTRO +148 150 H3 protein_type This value is in the range of binding affinities exhibited by the majority of histone readers, thus attesting to the physiological relevance of the H3K9cr recognition by Taf14. INTRO +150 154 K9cr ptm This value is in the range of binding affinities exhibited by the majority of histone readers, thus attesting to the physiological relevance of the H3K9cr recognition by Taf14. INTRO +170 175 Taf14 protein This value is in the range of binding affinities exhibited by the majority of histone readers, thus attesting to the physiological relevance of the H3K9cr recognition by Taf14. INTRO +21 23 H3 protein_type To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). INTRO +23 27 K9cr ptm To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). INTRO +42 47 yeast taxonomy_domain To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). INTRO +62 81 whole cell extracts experimental_method To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). INTRO +111 116 yeast taxonomy_domain To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). INTRO +145 166 Western blot analysis experimental_method To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). INTRO +201 203 H3 protein_type To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). INTRO +203 207 K9cr ptm To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). INTRO +209 211 H3 protein_type To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). INTRO +211 215 K9ac ptm To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). INTRO +220 222 H3 protein_type To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). INTRO +5 7 H3 protein_type Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. INTRO +7 11 K9cr ptm Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. INTRO +16 18 H3 protein_type Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. INTRO +18 22 K9ac ptm Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. INTRO +40 45 yeast taxonomy_domain Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. INTRO +46 54 histones protein_type Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. INTRO +102 104 H3 protein_type Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. INTRO +104 108 K9cr ptm Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. INTRO +122 127 yeast taxonomy_domain Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. INTRO +17 19 H3 protein_type We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). INTRO +19 23 K9cr ptm We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). INTRO +55 81 histone acetyltransferases protein_type We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). INTRO +83 87 HATs protein_type We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). INTRO +93 113 histone deacetylases protein_type We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). INTRO +115 120 HDACs protein_type We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). INTRO +50 55 yeast taxonomy_domain Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. INTRO +77 82 yeast taxonomy_domain Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. INTRO +83 87 HATs protein_type Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. INTRO +89 93 HAT1 protein Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. INTRO +95 99 Gcn5 protein Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. INTRO +105 111 Rtt109 protein Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. INTRO +116 121 HDACs protein_type Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. INTRO +123 127 Rpd3 protein Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. INTRO +129 133 Hos1 protein Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. INTRO +139 143 Hos2 protein Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. INTRO +150 157 deleted experimental_method Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. INTRO +52 54 H3 protein_type As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains. INTRO +54 58 K9cr ptm As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains. INTRO +112 115 HAT protein_type As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains. INTRO +116 124 deletion experimental_method As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains. INTRO +182 186 HDAC protein_type As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains. INTRO +187 195 deletion experimental_method As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains. INTRO +33 35 H3 protein_type Furthermore, fluctuations in the H3K9cr levels were more substantial than fluctuations in the corresponding H3K9ac levels. INTRO +35 39 K9cr ptm Furthermore, fluctuations in the H3K9cr levels were more substantial than fluctuations in the corresponding H3K9ac levels. INTRO +108 110 H3 protein_type Furthermore, fluctuations in the H3K9cr levels were more substantial than fluctuations in the corresponding H3K9ac levels. INTRO +110 114 K9ac ptm Furthermore, fluctuations in the H3K9cr levels were more substantial than fluctuations in the corresponding H3K9ac levels. INTRO +36 38 H3 protein_type Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs. INTRO +38 42 K9cr ptm Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs. INTRO +77 82 yeast taxonomy_domain Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs. INTRO +174 178 HATs protein_type Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs. INTRO +183 188 HDACs protein_type Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs. INTRO +36 46 acetylated protein_state We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. INTRO +47 54 histone protein_type We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. INTRO +66 71 Taf14 protein We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. INTRO +72 84 YEATS domain structure_element We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. INTRO +93 103 acetylated protein_state We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. INTRO +104 106 H3 protein_type We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. INTRO +106 108 K9 residue_name_number We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. INTRO +163 165 H3 protein_type We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. INTRO +165 169 K9cr ptm We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. INTRO +19 24 Taf14 protein The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). INTRO +33 47 crotonyllysine residue_name The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). INTRO +69 80 1H,15N HSQC experimental_method The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). INTRO +110 120 H3K9cr5-13 chemical The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). INTRO +124 134 H3K9ac5-13 chemical The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). INTRO +147 155 titrated experimental_method The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). INTRO +165 176 15N-labeled protein_state The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). INTRO +177 182 Taf14 protein The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). INTRO +183 195 YEATS domain structure_element The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). INTRO +11 13 H3 protein_type Binding of H3K9cr induced resonance changes in slow exchange regime on the NMR time scale, indicative of strong interaction. INTRO +13 17 K9cr ptm Binding of H3K9cr induced resonance changes in slow exchange regime on the NMR time scale, indicative of strong interaction. INTRO +26 43 resonance changes evidence Binding of H3K9cr induced resonance changes in slow exchange regime on the NMR time scale, indicative of strong interaction. INTRO +75 78 NMR experimental_method Binding of H3K9cr induced resonance changes in slow exchange regime on the NMR time scale, indicative of strong interaction. INTRO +24 26 H3 protein_type In contrast, binding of H3K9ac resulted in an intermediate exchange, which is characteristic of a weaker association. INTRO +26 30 K9ac ptm In contrast, binding of H3K9ac resulted in an intermediate exchange, which is characteristic of a weaker association. INTRO +13 23 crosspeaks evidence Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). INTRO +27 32 Gly80 residue_name_number Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). INTRO +37 42 Trp81 residue_name_number Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). INTRO +50 62 YEATS domain structure_element Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). INTRO +90 92 H3 protein_type Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). INTRO +92 96 K9cr ptm Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). INTRO +101 103 H3 protein_type Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). INTRO +103 107 K9ac ptm Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). INTRO +171 218 crotonyllysine and acetyllysine binding pockets site Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). INTRO +41 46 Trp81 residue_name_number These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c). INTRO +104 106 H3 protein_type These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c). INTRO +106 110 K9cr ptm These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c). INTRO +131 133 H3 protein_type These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c). INTRO +133 137 K9ac ptm These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c). INTRO +136 148 YEATS-H3K9cr complex_assembly One of the conformations, characterized by the π stacking involving two aromatic residues and the alkene group, is observed only in the YEATS-H3K9cr complex. INTRO +25 30 Taf14 protein To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). INTRO +31 43 YEATS domain structure_element To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). INTRO +91 101 acyllysine residue_name To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). INTRO +122 147 solution pull-down assays experimental_method To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). INTRO +154 156 H3 protein_type To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). INTRO +166 176 acetylated protein_state To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). INTRO +178 191 propionylated protein_state To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). INTRO +193 204 butyrylated protein_state To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). INTRO +210 222 crotonylated protein_state To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). INTRO +226 234 lysine 9 residue_name_number To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). INTRO +245 249 1–20 residue_range To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). INTRO +253 255 H3 protein_type To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). INTRO +53 58 Taf14 protein As shown in Figure 2d and Supplementary Fig. 5a, the Taf14 YEATS domain binds more strongly to H3K9cr1-20, as compared to other acylated histone peptides. INTRO +59 71 YEATS domain structure_element As shown in Figure 2d and Supplementary Fig. 5a, the Taf14 YEATS domain binds more strongly to H3K9cr1-20, as compared to other acylated histone peptides. INTRO +95 105 H3K9cr1-20 chemical As shown in Figure 2d and Supplementary Fig. 5a, the Taf14 YEATS domain binds more strongly to H3K9cr1-20, as compared to other acylated histone peptides. INTRO +128 136 acylated protein_state As shown in Figure 2d and Supplementary Fig. 5a, the Taf14 YEATS domain binds more strongly to H3K9cr1-20, as compared to other acylated histone peptides. INTRO +19 21 H3 protein_type The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. INTRO +21 25 K9cr ptm The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. INTRO +31 33 H3 protein_type The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. INTRO +33 37 K9ac ptm The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. INTRO +39 41 H3 protein_type The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. INTRO +41 45 K9pr ptm The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. INTRO +50 52 H3 protein_type The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. INTRO +52 56 K9bu ptm The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. INTRO +74 107 1H,15N HSQC titration experiments experimental_method The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. INTRO +12 22 H3K9ac1-20 chemical Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). INTRO +24 34 H3K9pr1-20 chemical Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). INTRO +40 50 H3K9bu1-20 chemical Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). INTRO +67 95 chemical shift perturbations evidence Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). INTRO +103 108 Taf14 protein Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). INTRO +109 121 YEATS domain structure_element Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). INTRO +236 246 H3K9cr1-20 chemical Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). INTRO +18 20 H3 protein_type We concluded that H3K9cr is the preferred target of this domain. INTRO +20 24 K9cr ptm We concluded that H3K9cr is the preferred target of this domain. INTRO +5 36 comparative structural analysis experimental_method From comparative structural analysis of the YEATS complexes, Gly80 emerged as candidate residue potentially responsible for the preference for crotonyllysine. INTRO +61 66 Gly80 residue_name_number From comparative structural analysis of the YEATS complexes, Gly80 emerged as candidate residue potentially responsible for the preference for crotonyllysine. INTRO +143 157 crotonyllysine residue_name From comparative structural analysis of the YEATS complexes, Gly80 emerged as candidate residue potentially responsible for the preference for crotonyllysine. INTRO +123 131 crotonyl chemical In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. INTRO +143 150 mutated protein_state In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. INTRO +151 156 Gly80 residue_name_number In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. INTRO +193 203 mutants of protein_state In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. INTRO +204 209 Gly80 residue_name_number In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. INTRO +255 263 acylated protein_state In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. INTRO +289 294 Gly80 residue_name_number In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. INTRO +13 21 mutation experimental_method In contrast, mutation of Val24, a residue located on another side of Trp81, had no effect on binding (Fig. 2d and Supplementary Fig. 5a, c). INTRO +25 30 Val24 residue_name_number In contrast, mutation of Val24, a residue located on another side of Trp81, had no effect on binding (Fig. 2d and Supplementary Fig. 5a, c). INTRO +69 74 Trp81 residue_name_number In contrast, mutation of Val24, a residue located on another side of Trp81, had no effect on binding (Fig. 2d and Supplementary Fig. 5a, c). INTRO +31 45 crotonyllysine residue_name To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). INTRO +49 58 conserved protein_state To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). INTRO +70 75 human species To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). INTRO +76 89 YEATS domains structure_element To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). INTRO +93 114 pull-down experiments experimental_method To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). INTRO +141 151 acetylated protein_state To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). INTRO +153 166 propionylated protein_state To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). INTRO +168 179 butyrylated protein_state To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). INTRO +185 197 crotonylated protein_state To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). INTRO +198 205 histone protein_type To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). INTRO +18 31 YEATS domains structure_element We found that all YEATS domains tested are capable of binding to crotonyllysine peptides, though they display variable preferences for the acyl moieties. INTRO +65 79 crotonyllysine residue_name We found that all YEATS domains tested are capable of binding to crotonyllysine peptides, though they display variable preferences for the acyl moieties. INTRO +6 12 YEATS2 protein While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well. INTRO +17 20 ENL protein While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well. INTRO +48 60 crotonylated protein_state While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well. INTRO +71 76 GAS41 protein While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well. INTRO +81 84 AF9 protein While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well. INTRO +91 99 acylated protein_state While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well. INTRO +11 23 YEATS domain structure_element Unlike the YEATS domain, a known acetyllysine reader, bromodomain, does not recognize crotonyllysine. INTRO +33 52 acetyllysine reader protein_type Unlike the YEATS domain, a known acetyllysine reader, bromodomain, does not recognize crotonyllysine. INTRO +54 65 bromodomain structure_element Unlike the YEATS domain, a known acetyllysine reader, bromodomain, does not recognize crotonyllysine. INTRO +86 100 crotonyllysine residue_name Unlike the YEATS domain, a known acetyllysine reader, bromodomain, does not recognize crotonyllysine. INTRO +26 29 BDs structure_element We assayed a large set of BDs in pull-down experiments and found that this module is highly specific for acetyllysine and propionyllysine containing peptides (Supplementary Fig. 7). INTRO +33 54 pull-down experiments experimental_method We assayed a large set of BDs in pull-down experiments and found that this module is highly specific for acetyllysine and propionyllysine containing peptides (Supplementary Fig. 7). INTRO +105 117 acetyllysine residue_name We assayed a large set of BDs in pull-down experiments and found that this module is highly specific for acetyllysine and propionyllysine containing peptides (Supplementary Fig. 7). INTRO +122 137 propionyllysine residue_name We assayed a large set of BDs in pull-down experiments and found that this module is highly specific for acetyllysine and propionyllysine containing peptides (Supplementary Fig. 7). INTRO +9 21 bromodomains structure_element However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports. INTRO +109 123 crotonyllysine residue_name However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports. INTRO +143 146 BDs structure_element However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports. INTRO +150 154 TAF1 protein However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports. INTRO +159 163 BRD2 protein However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports. INTRO +35 47 YEATS domain structure_element These results demonstrate that the YEATS domain is currently the sole reader of crotonyllysine. INTRO +80 94 crotonyllysine residue_name These results demonstrate that the YEATS domain is currently the sole reader of crotonyllysine. INTRO +38 50 YEATS domain structure_element In conclusion, we have identified the YEATS domain of Taf14 as the first reader of histone crotonylation. INTRO +54 59 Taf14 protein In conclusion, we have identified the YEATS domain of Taf14 as the first reader of histone crotonylation. INTRO +83 90 histone protein_type In conclusion, we have identified the YEATS domain of Taf14 as the first reader of histone crotonylation. INTRO +91 104 crotonylation ptm In conclusion, we have identified the YEATS domain of Taf14 as the first reader of histone crotonylation. INTRO +71 85 π-π-π-stacking bond_interaction The unique and previously unobserved aromatic-amide/aliphatic-aromatic π-π-π-stacking mechanism facilitates the specific recognition of the crotonyl moiety. INTRO +140 148 crotonyl chemical The unique and previously unobserved aromatic-amide/aliphatic-aromatic π-π-π-stacking mechanism facilitates the specific recognition of the crotonyl moiety. INTRO +28 30 H3 protein_type We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs. INTRO +30 34 K9cr ptm We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs. INTRO +45 50 yeast taxonomy_domain We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs. INTRO +83 87 HATs protein_type We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs. INTRO +92 97 HDACs protein_type We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs. INTRO +42 52 acyllysine residue_name As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. INTRO +68 73 Taf14 protein As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. INTRO +74 86 YEATS domain structure_element As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. INTRO +210 224 crotonyllysine residue_name As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. INTRO +276 281 Taf14 protein As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. INTRO +309 323 crotonyllysine residue_name As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. INTRO +328 340 acetyllysine residue_name As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. INTRO +44 58 crotonyllysine residue_name Furthermore, the functional significance of crotonyllysine recognition by other YEATS proteins will be of great importance to elucidate and compare. INTRO +80 85 YEATS protein_type Furthermore, the functional significance of crotonyllysine recognition by other YEATS proteins will be of great importance to elucidate and compare. INTRO +48 50 H3 protein_type The structural mechanism for the recognition of H3K9cr FIG +50 54 K9cr ptm The structural mechanism for the recognition of H3K9cr FIG +26 40 crotonyllysine residue_name (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. FIG +50 67 crystal structure evidence (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. FIG +75 80 Taf14 protein (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. FIG +81 93 YEATS domain structure_element (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. FIG +102 117 in complex with protein_state (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. FIG +122 132 H3K9cr5-13 chemical (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. FIG +154 156 H3 protein_type (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. FIG +156 160 K9cr ptm (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. FIG +218 254 electrostatic and polar interactions bond_interaction (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. FIG +264 269 Taf14 protein (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. FIG +270 282 YEATS domain structure_element (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. FIG +8 22 π-π-π stacking bond_interaction (d) The π-π-π stacking mechanism involving the alkene moiety of crotonyllysine. FIG +64 78 crotonyllysine residue_name (d) The π-π-π stacking mechanism involving the alkene moiety of crotonyllysine. FIG +0 2 H3 protein_type H3K9cr is a selective target of the Taf14 YEATS domain FIG +2 6 K9cr ptm H3K9cr is a selective target of the Taf14 YEATS domain FIG +36 41 Taf14 protein H3K9cr is a selective target of the Taf14 YEATS domain FIG +42 54 YEATS domain structure_element H3K9cr is a selective target of the Taf14 YEATS domain FIG +7 19 Western blot experimental_method (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. FIG +53 55 H3 protein_type (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. FIG +55 59 K9cr ptm (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. FIG +64 66 H3 protein_type (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. FIG +66 70 K9ac ptm (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. FIG +74 83 wild type protein_state (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. FIG +85 87 WT protein_state (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. FIG +90 93 HAT protein_type (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. FIG +107 111 HDAC protein_type (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. FIG +112 120 deletion experimental_method (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. FIG +121 126 yeast taxonomy_domain (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. FIG +6 8 H3 protein_type Total H3 was used as a loading control. FIG +17 28 1H,15N HSQC experimental_method (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. FIG +29 36 spectra evidence (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. FIG +40 45 Taf14 protein (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. FIG +46 51 YEATS structure_element (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. FIG +64 74 H3K9cr5-13 chemical (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. FIG +79 89 H3K9ac5-13 chemical (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. FIG +104 112 titrated experimental_method (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. FIG +0 7 Spectra evidence Spectra are color coded according to the protein:peptide molar ratio. FIG +4 16 Western blot experimental_method (d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides. FIG +29 53 peptide pull-down assays experimental_method (d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides. FIG +60 69 wild-type protein_state (d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides. FIG +74 81 mutated protein_state (d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides. FIG +82 87 Taf14 protein (d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides. FIG +88 101 YEATS domains structure_element (d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides. FIG diff --git a/annotation_CSV/PMC4872110.csv b/annotation_CSV/PMC4872110.csv new file mode 100644 index 0000000000000000000000000000000000000000..fe2dc1b3435079cd52c63822942e9970af2a23a0 --- /dev/null +++ b/annotation_CSV/PMC4872110.csv @@ -0,0 +1,1284 @@ +anno_start anno_end anno_text entity_type sentence section +0 26 Ribosome biogenesis factor protein_type Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans TITLE +27 31 Tsr3 protein Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans TITLE +39 69 aminocarboxypropyl transferase protein_type Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans TITLE +86 94 18S rRNA chemical Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans TITLE +116 121 yeast taxonomy_domain Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans TITLE +126 132 humans species Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans TITLE +44 54 eukaryotic taxonomy_domain The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. ABSTRACT +55 59 rRNA chemical The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. ABSTRACT +67 76 conserved protein_state The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. ABSTRACT +77 90 hypermodified protein_state The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. ABSTRACT +91 101 nucleotide chemical The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. ABSTRACT +102 147 N1-methyl-N3-aminocarboxypropyl-pseudouridine chemical The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. ABSTRACT +149 156 m1acp3Ψ chemical The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. ABSTRACT +178 184 P-site site The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. ABSTRACT +185 189 tRNA chemical The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. ABSTRACT +211 219 18S rRNA chemical The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. ABSTRACT +6 26 S-adenosylmethionine chemical While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. ABSTRACT +63 81 aminocarboxypropyl chemical While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. ABSTRACT +83 86 acp chemical While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. ABSTRACT +143 146 acp chemical While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. ABSTRACT +61 65 Tsr3 protein Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. ABSTRACT +95 100 yeast taxonomy_domain Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. ABSTRACT +105 110 human species Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. ABSTRACT +25 29 Tsr3 protein In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. ABSTRACT +30 37 mutants protein_state In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. ABSTRACT +58 61 acp chemical In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. ABSTRACT +110 122 20S pre-rRNA chemical In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. ABSTRACT +4 21 crystal structure evidence The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. ABSTRACT +25 33 archaeal taxonomy_domain The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. ABSTRACT +34 38 Tsr3 protein The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. ABSTRACT +77 111 SPOUT-class RNA-methyltransferases protein_type The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. ABSTRACT +127 143 SAM binding mode site The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. ABSTRACT +12 28 SAM binding mode site This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. ABSTRACT +42 46 Tsr3 protein This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. ABSTRACT +61 64 acp chemical This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. ABSTRACT +93 96 SAM chemical This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. ABSTRACT +14 18 Tsr3 protein Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes. ABSTRACT +57 72 acp transferase protein_type Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes. ABSTRACT +0 10 Eukaryotic taxonomy_domain Eukaryotic ribosome biogenesis is highly complex and requires a large number of non-ribosomal proteins and small non-coding RNAs in addition to ribosomal RNAs (rRNAs) and proteins. INTRO +107 128 small non-coding RNAs chemical Eukaryotic ribosome biogenesis is highly complex and requires a large number of non-ribosomal proteins and small non-coding RNAs in addition to ribosomal RNAs (rRNAs) and proteins. INTRO +144 158 ribosomal RNAs chemical Eukaryotic ribosome biogenesis is highly complex and requires a large number of non-ribosomal proteins and small non-coding RNAs in addition to ribosomal RNAs (rRNAs) and proteins. INTRO +160 165 rRNAs chemical Eukaryotic ribosome biogenesis is highly complex and requires a large number of non-ribosomal proteins and small non-coding RNAs in addition to ribosomal RNAs (rRNAs) and proteins. INTRO +7 17 eukaryotic taxonomy_domain During eukaryotic ribosome biogenesis several dozens of rRNA nucleotides become chemically modified. INTRO +56 60 rRNA chemical During eukaryotic ribosome biogenesis several dozens of rRNA nucleotides become chemically modified. INTRO +61 72 nucleotides chemical During eukaryotic ribosome biogenesis several dozens of rRNA nucleotides become chemically modified. INTRO +18 22 rRNA chemical The most abundant rRNA modifications are methylations at the 2′-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs). INTRO +41 53 methylations ptm The most abundant rRNA modifications are methylations at the 2′-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs). INTRO +67 73 ribose chemical The most abundant rRNA modifications are methylations at the 2′-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs). INTRO +105 112 uridine chemical The most abundant rRNA modifications are methylations at the 2′-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs). INTRO +125 138 pseudouridine chemical The most abundant rRNA modifications are methylations at the 2′-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs). INTRO +153 196 small nucleolar ribonucleoprotein particles complex_assembly The most abundant rRNA modifications are methylations at the 2′-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs). INTRO +198 205 snoRNPs complex_assembly The most abundant rRNA modifications are methylations at the 2′-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs). INTRO +13 16 18S chemical In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes. INTRO +21 24 25S chemical In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes. INTRO +26 31 yeast taxonomy_domain In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes. INTRO +34 37 28S chemical In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes. INTRO +39 45 humans species In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes. INTRO +47 52 rRNAs chemical In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes. INTRO +119 125 snoRNA chemical In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes. INTRO +3 27 Saccharomyces cerevisiae species In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA. INTRO +28 36 18S rRNA chemical In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA. INTRO +56 68 methylations ptm In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA. INTRO +74 86 acetylations ptm In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA. INTRO +100 123 3-amino-3-carboxypropyl chemical In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA. INTRO +125 128 acp chemical In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA. INTRO +161 173 methylations ptm In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA. INTRO +193 201 25S rRNA chemical In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA. INTRO +9 15 humans species While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. INTRO +20 28 18S rRNA chemical While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. INTRO +52 68 highly conserved protein_state While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. INTRO +88 93 yeast taxonomy_domain While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. INTRO +126 132 ScRrp8 protein While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. INTRO +133 138 HsNML protein While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. INTRO +140 146 ScRcm1 protein While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. INTRO +147 154 HsNSUN5 protein While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. INTRO +159 165 ScNop2 protein While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. INTRO +166 173 HsNSUN1 protein While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. INTRO +209 214 human species While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. INTRO +215 223 28S rRNA chemical While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA. INTRO +0 13 Ribosomal RNA chemical Ribosomal RNA modifications have been suggested to optimize ribosome function, although in most cases this remains to be clearly established. INTRO +35 38 RNA chemical They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations). INTRO +73 87 hydrogen bonds bond_interaction They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations). INTRO +89 103 pseudouridines chemical They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations). INTRO +115 128 base stacking bond_interaction They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations). INTRO +130 144 pseudouridines chemical They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations). INTRO +149 166 base methylations ptm They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations). INTRO +215 234 ribose methylations ptm They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations). INTRO +14 18 rRNA chemical Most modified rRNA nucleotides cluster in the vicinity of the decoding or the peptidyl transferase center, suggesting an influence on ribosome functionality and stability. INTRO +19 30 nucleotides chemical Most modified rRNA nucleotides cluster in the vicinity of the decoding or the peptidyl transferase center, suggesting an influence on ribosome functionality and stability. INTRO +62 70 decoding site Most modified rRNA nucleotides cluster in the vicinity of the decoding or the peptidyl transferase center, suggesting an influence on ribosome functionality and stability. INTRO +78 105 peptidyl transferase center site Most modified rRNA nucleotides cluster in the vicinity of the decoding or the peptidyl transferase center, suggesting an influence on ribosome functionality and stability. INTRO +11 15 rRNA chemical Defects of rRNA modification enzymes often lead to disturbed ribosome biogenesis or functionally impaired ribosomes, although the lack of individual rRNA modifications often has no or only a slight influence on the cell. INTRO +149 153 rRNA chemical Defects of rRNA modification enzymes often lead to disturbed ribosome biogenesis or functionally impaired ribosomes, although the lack of individual rRNA modifications often has no or only a slight influence on the cell. INTRO +59 80 loop capping helix 31 structure_element The chemically most complex modification is located in the loop capping helix 31 of 18S rRNA (Supplementary Figure S1B). INTRO +84 92 18S rRNA chemical The chemically most complex modification is located in the loop capping helix 31 of 18S rRNA (Supplementary Figure S1B). INTRO +8 15 uridine residue_name There a uridine (U1191 in yeast) is modified to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ, Figure 1A). INTRO +17 22 U1191 residue_name_number There a uridine (U1191 in yeast) is modified to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ, Figure 1A). INTRO +26 31 yeast taxonomy_domain There a uridine (U1191 in yeast) is modified to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ, Figure 1A). INTRO +48 98 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine chemical There a uridine (U1191 in yeast) is modified to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ, Figure 1A). INTRO +100 107 m1acp3Ψ chemical There a uridine (U1191 in yeast) is modified to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ, Figure 1A). INTRO +55 62 hamster taxonomy_domain This base modification was first described in 1968 for hamster cells and is conserved in eukaryotes. INTRO +76 88 conserved in protein_state This base modification was first described in 1968 for hamster cells and is conserved in eukaryotes. INTRO +89 99 eukaryotes taxonomy_domain This base modification was first described in 1968 for hamster cells and is conserved in eukaryotes. INTRO +5 18 hypermodified protein_state This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. INTRO +19 29 nucleotide chemical This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. INTRO +55 61 P-site site This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. INTRO +62 66 tRNA chemical This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. INTRO +117 122 snR35 chemical This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. INTRO +123 128 H/ACA structure_element This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. INTRO +129 135 snoRNP complex_assembly This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. INTRO +157 164 uridine chemical This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. INTRO +170 183 pseudouridine chemical This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine. INTRO +32 61 SPOUT-class methyltransferase protein_type In a second step, the essential SPOUT-class methyltransferase Nep1/Emg1 modifies the pseudouridine to N1-methylpseudouridine. INTRO +62 66 Nep1 protein In a second step, the essential SPOUT-class methyltransferase Nep1/Emg1 modifies the pseudouridine to N1-methylpseudouridine. INTRO +67 71 Emg1 protein In a second step, the essential SPOUT-class methyltransferase Nep1/Emg1 modifies the pseudouridine to N1-methylpseudouridine. INTRO +85 98 pseudouridine chemical In a second step, the essential SPOUT-class methyltransferase Nep1/Emg1 modifies the pseudouridine to N1-methylpseudouridine. INTRO +102 124 N1-methylpseudouridine chemical In a second step, the essential SPOUT-class methyltransferase Nep1/Emg1 modifies the pseudouridine to N1-methylpseudouridine. INTRO +0 11 Methylation ptm Methylation can only occur once pseudouridylation has taken place, as the latter reaction generates the substrate for the former. INTRO +32 49 pseudouridylation ptm Methylation can only occur once pseudouridylation has taken place, as the latter reaction generates the substrate for the former. INTRO +10 13 acp chemical The final acp modification leading to N1-methyl-N3-aminocarboxypropyl-pseudouridine occurs late during 40S biogenesis in the cytoplasm, while the two former reactions are taking place in the nucleolus and nucleus, and is independent from pseudouridylation or methylation. INTRO +38 83 N1-methyl-N3-aminocarboxypropyl-pseudouridine chemical The final acp modification leading to N1-methyl-N3-aminocarboxypropyl-pseudouridine occurs late during 40S biogenesis in the cytoplasm, while the two former reactions are taking place in the nucleolus and nucleus, and is independent from pseudouridylation or methylation. INTRO +103 106 40S complex_assembly The final acp modification leading to N1-methyl-N3-aminocarboxypropyl-pseudouridine occurs late during 40S biogenesis in the cytoplasm, while the two former reactions are taking place in the nucleolus and nucleus, and is independent from pseudouridylation or methylation. INTRO +238 255 pseudouridylation ptm The final acp modification leading to N1-methyl-N3-aminocarboxypropyl-pseudouridine occurs late during 40S biogenesis in the cytoplasm, while the two former reactions are taking place in the nucleolus and nucleus, and is independent from pseudouridylation or methylation. INTRO +51 71 S-adenosylmethionine chemical Both the methyl and the acp group are derived from S-adenosylmethionine (SAM), but the enzyme responsible for acp modification remained elusive for more than 40 years. INTRO +73 76 SAM chemical Both the methyl and the acp group are derived from S-adenosylmethionine (SAM), but the enzyme responsible for acp modification remained elusive for more than 40 years. INTRO +110 113 acp chemical Both the methyl and the acp group are derived from S-adenosylmethionine (SAM), but the enzyme responsible for acp modification remained elusive for more than 40 years. INTRO +0 4 Tsr3 protein Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +22 25 acp chemical Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +42 50 18S rRNA chemical Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +54 59 yeast taxonomy_domain Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +64 69 human species Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +75 88 Hypermodified protein_state Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +89 99 nucleotide chemical Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +100 107 m1acp3Ψ chemical Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +139 156 pseudouridylation ptm Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +170 178 snoRNP35 complex_assembly Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +180 194 N1-methylation ptm Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +208 225 methyltransferase protein_type Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +226 230 Nep1 protein Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +238 241 acp chemical Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +268 272 Tsr3 protein Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3Ψ is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3. FIG +50 73 14C-incorporation assay experimental_method The asterisk indicates the C1-atom labeled in the 14C-incorporation assay. FIG +4 11 RP-HPLC experimental_method (B) RP-HPLC elution profile of yeast 18S rRNA nucleosides. FIG +12 27 elution profile evidence (B) RP-HPLC elution profile of yeast 18S rRNA nucleosides. FIG +31 36 yeast taxonomy_domain (B) RP-HPLC elution profile of yeast 18S rRNA nucleosides. FIG +37 45 18S rRNA chemical (B) RP-HPLC elution profile of yeast 18S rRNA nucleosides. FIG +46 57 nucleosides chemical (B) RP-HPLC elution profile of yeast 18S rRNA nucleosides. FIG +0 13 Hypermodified protein_state Hypermodified m1acp3Ψ elutes at 7.4 min (wild type, left profile) and is missing in Δtsr3 (middle profile) and Δnep1 Δnop6 mutants (right profile). FIG +14 21 m1acp3Ψ chemical Hypermodified m1acp3Ψ elutes at 7.4 min (wild type, left profile) and is missing in Δtsr3 (middle profile) and Δnep1 Δnop6 mutants (right profile). FIG +41 50 wild type protein_state Hypermodified m1acp3Ψ elutes at 7.4 min (wild type, left profile) and is missing in Δtsr3 (middle profile) and Δnep1 Δnop6 mutants (right profile). FIG +84 89 Δtsr3 mutant Hypermodified m1acp3Ψ elutes at 7.4 min (wild type, left profile) and is missing in Δtsr3 (middle profile) and Δnep1 Δnop6 mutants (right profile). FIG +111 122 Δnep1 Δnop6 mutant Hypermodified m1acp3Ψ elutes at 7.4 min (wild type, left profile) and is missing in Δtsr3 (middle profile) and Δnep1 Δnop6 mutants (right profile). FIG +4 11 14C-acp chemical (C) 14C-acp labeling of 18S rRNAs. FIG +24 33 18S rRNAs chemical (C) 14C-acp labeling of 18S rRNAs. FIG +0 9 Wild type protein_state Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). FIG +11 13 WT protein_state Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). FIG +35 43 18S rRNA chemical Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). FIG +45 51 U1191U mutant Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). FIG +62 69 14C-acp chemical Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). FIG +90 97 14C-acp chemical Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). FIG +123 129 U1191A mutant Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). FIG +130 136 mutant protein_state Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). FIG +153 161 18S rRNA chemical Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). FIG +163 169 U1191A mutant Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). FIG +175 180 Δtsr3 mutant Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). FIG +190 195 Δtsr3 mutant Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the U1191A mutant plasmid encoded 18S rRNA (U1191A) and Δtsr3 mutants (Δtsr3). FIG +21 37 ethidium bromide chemical Upper lanes show the ethidium bromide staining of the 18S rRNAs for quantification. FIG +54 63 18S rRNAs chemical Upper lanes show the ethidium bromide staining of the 18S rRNAs for quantification. FIG +82 107 Primer extension analysis experimental_method All samples were loaded on the gel with two different amounts of 5 and 10 μl. (D) Primer extension analysis of acp modification in yeast 18S rRNA (right gel) including a sequencing ladder (left gel). FIG +111 114 acp chemical All samples were loaded on the gel with two different amounts of 5 and 10 μl. (D) Primer extension analysis of acp modification in yeast 18S rRNA (right gel) including a sequencing ladder (left gel). FIG +131 136 yeast taxonomy_domain All samples were loaded on the gel with two different amounts of 5 and 10 μl. (D) Primer extension analysis of acp modification in yeast 18S rRNA (right gel) including a sequencing ladder (left gel). FIG +137 145 18S rRNA chemical All samples were loaded on the gel with two different amounts of 5 and 10 μl. (D) Primer extension analysis of acp modification in yeast 18S rRNA (right gel) including a sequencing ladder (left gel). FIG +40 44 1191 residue_number The primer extension stop at nucleotide 1191 is missing exclusively in Δtsr3 mutants and Δtsr3 Δsnr35 recombinants. FIG +71 76 Δtsr3 mutant The primer extension stop at nucleotide 1191 is missing exclusively in Δtsr3 mutants and Δtsr3 Δsnr35 recombinants. FIG +89 101 Δtsr3 Δsnr35 mutant The primer extension stop at nucleotide 1191 is missing exclusively in Δtsr3 mutants and Δtsr3 Δsnr35 recombinants. FIG +4 29 Primer extension analysis experimental_method (E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel). FIG +33 38 human species (E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel). FIG +39 47 18S rRNA chemical (E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel). FIG +54 69 siRNA knockdown experimental_method (E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel). FIG +73 79 HsNEP1 protein (E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel). FIG +80 84 EMG1 protein (E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel). FIG +108 114 HsTSR3 protein (E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel). FIG +72 78 siRNAs chemical The primer extension arrest is reduced in HTC116 cells transfected with siRNAs 544 and 545. FIG +18 23 siRNA chemical The efficiency of siRNA mediated HsTSR3 repression correlates with the primer extension signals (see Supplementary Figure S2A). FIG +33 39 HsTSR3 protein The efficiency of siRNA mediated HsTSR3 repression correlates with the primer extension signals (see Supplementary Figure S2A). FIG +71 95 primer extension signals evidence The efficiency of siRNA mediated HsTSR3 repression correlates with the primer extension signals (see Supplementary Figure S2A). FIG +11 14 acp chemical Only a few acp transferring enzymes have been characterized until now. INTRO +27 37 wybutosine chemical During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. INTRO +51 61 nucleoside chemical During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. INTRO +73 83 eukaryotic taxonomy_domain During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. INTRO +88 96 archaeal taxonomy_domain During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. INTRO +97 110 phenylalanine chemical During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. INTRO +111 115 tRNA chemical During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. INTRO +117 121 Tyw2 protein During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. INTRO +123 128 Trm12 protein During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. INTRO +132 137 yeast taxonomy_domain During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. INTRO +152 155 acp chemical During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. INTRO +167 170 SAM chemical During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom. INTRO +0 8 Archaeal taxonomy_domain Archaeal Tyw2 has a structure very similar to Rossmann-fold (class I) RNA-methyltransferases, but its distinctive SAM-binding mode enables the transfer of the acp group instead of the methyl group of the cofactor. INTRO +9 13 Tyw2 protein Archaeal Tyw2 has a structure very similar to Rossmann-fold (class I) RNA-methyltransferases, but its distinctive SAM-binding mode enables the transfer of the acp group instead of the methyl group of the cofactor. INTRO +20 29 structure evidence Archaeal Tyw2 has a structure very similar to Rossmann-fold (class I) RNA-methyltransferases, but its distinctive SAM-binding mode enables the transfer of the acp group instead of the methyl group of the cofactor. INTRO +46 92 Rossmann-fold (class I) RNA-methyltransferases protein_type Archaeal Tyw2 has a structure very similar to Rossmann-fold (class I) RNA-methyltransferases, but its distinctive SAM-binding mode enables the transfer of the acp group instead of the methyl group of the cofactor. INTRO +114 130 SAM-binding mode site Archaeal Tyw2 has a structure very similar to Rossmann-fold (class I) RNA-methyltransferases, but its distinctive SAM-binding mode enables the transfer of the acp group instead of the methyl group of the cofactor. INTRO +159 162 acp chemical Archaeal Tyw2 has a structure very similar to Rossmann-fold (class I) RNA-methyltransferases, but its distinctive SAM-binding mode enables the transfer of the acp group instead of the methyl group of the cofactor. INTRO +8 11 acp chemical Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism. INTRO +51 61 diphtamide chemical Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism. INTRO +93 96 acp chemical Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism. INTRO +123 126 SAM chemical Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism. INTRO +151 160 histidine residue_name Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism. INTRO +172 182 eukaryotic taxonomy_domain Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism. INTRO +183 214 translation elongation factor 2 protein_type Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism. INTRO +53 58 yeast taxonomy_domain In a recent bioinformatic study, the uncharacterized yeast gene YOR006c was predicted to be involved in ribosome biogenesis. INTRO +64 71 YOR006c gene In a recent bioinformatic study, the uncharacterized yeast gene YOR006c was predicted to be involved in ribosome biogenesis. INTRO +6 22 highly conserved protein_state It is highly conserved among eukaryotes and archaea (Supplementary Figure S1A) and its deletion leads to an accumulation of the 20S pre-rRNA precursor of 18S rRNA, suggesting an influence on D-site cleavage during the maturation of the small ribosomal subunit. INTRO +29 39 eukaryotes taxonomy_domain It is highly conserved among eukaryotes and archaea (Supplementary Figure S1A) and its deletion leads to an accumulation of the 20S pre-rRNA precursor of 18S rRNA, suggesting an influence on D-site cleavage during the maturation of the small ribosomal subunit. INTRO +44 51 archaea taxonomy_domain It is highly conserved among eukaryotes and archaea (Supplementary Figure S1A) and its deletion leads to an accumulation of the 20S pre-rRNA precursor of 18S rRNA, suggesting an influence on D-site cleavage during the maturation of the small ribosomal subunit. INTRO +128 140 20S pre-rRNA chemical It is highly conserved among eukaryotes and archaea (Supplementary Figure S1A) and its deletion leads to an accumulation of the 20S pre-rRNA precursor of 18S rRNA, suggesting an influence on D-site cleavage during the maturation of the small ribosomal subunit. INTRO +154 162 18S rRNA chemical It is highly conserved among eukaryotes and archaea (Supplementary Figure S1A) and its deletion leads to an accumulation of the 20S pre-rRNA precursor of 18S rRNA, suggesting an influence on D-site cleavage during the maturation of the small ribosomal subunit. INTRO +191 197 D-site site It is highly conserved among eukaryotes and archaea (Supplementary Figure S1A) and its deletion leads to an accumulation of the 20S pre-rRNA precursor of 18S rRNA, suggesting an influence on D-site cleavage during the maturation of the small ribosomal subunit. INTRO +15 22 YOR006C gene On this basis, YOR006C was renamed ‘Twenty S rRNA accumulation 3′ (TSR3). INTRO +36 64 Twenty S rRNA accumulation 3 protein On this basis, YOR006C was renamed ‘Twenty S rRNA accumulation 3′ (TSR3). INTRO +67 71 TSR3 protein On this basis, YOR006C was renamed ‘Twenty S rRNA accumulation 3′ (TSR3). INTRO +93 101 18S rRNA chemical However, its function remained unclear although recently a putative nuclease function during 18S rRNA maturation was predicted. INTRO +18 22 Tsr3 protein Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3Ψ in yeast and human cells. INTRO +42 57 acp transferase protein_type Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3Ψ in yeast and human cells. INTRO +114 127 hypermodified protein_state Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3Ψ in yeast and human cells. INTRO +128 138 nucleotide chemical Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3Ψ in yeast and human cells. INTRO +139 146 m1acp3Ψ chemical Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3Ψ in yeast and human cells. INTRO +150 155 yeast taxonomy_domain Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3Ψ in yeast and human cells. INTRO +160 165 human species Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3Ψ in yeast and human cells. INTRO +18 41 catalytically defective protein_state Furthermore using catalytically defective mutants of yeast Tsr3 we demonstrated that the acp modification is required for 18S rRNA maturation. INTRO +53 58 yeast taxonomy_domain Furthermore using catalytically defective mutants of yeast Tsr3 we demonstrated that the acp modification is required for 18S rRNA maturation. INTRO +59 63 Tsr3 protein Furthermore using catalytically defective mutants of yeast Tsr3 we demonstrated that the acp modification is required for 18S rRNA maturation. INTRO +89 92 acp chemical Furthermore using catalytically defective mutants of yeast Tsr3 we demonstrated that the acp modification is required for 18S rRNA maturation. INTRO +122 130 18S rRNA chemical Furthermore using catalytically defective mutants of yeast Tsr3 we demonstrated that the acp modification is required for 18S rRNA maturation. INTRO +18 36 crystal structures evidence Surprisingly, the crystal structures of archaeal homologs revealed that Tsr3 is structurally similar to the SPOUT-class RNA methyltransferases. INTRO +40 48 archaeal taxonomy_domain Surprisingly, the crystal structures of archaeal homologs revealed that Tsr3 is structurally similar to the SPOUT-class RNA methyltransferases. INTRO +72 76 Tsr3 protein Surprisingly, the crystal structures of archaeal homologs revealed that Tsr3 is structurally similar to the SPOUT-class RNA methyltransferases. INTRO +108 142 SPOUT-class RNA methyltransferases protein_type Surprisingly, the crystal structures of archaeal homologs revealed that Tsr3 is structurally similar to the SPOUT-class RNA methyltransferases. INTRO +55 70 acp transferase protein_type In contrast, the only other structurally characterized acp transferase enzyme Tyw2 belongs to the Rossmann-fold class of methyltransferase proteins. INTRO +78 82 Tyw2 protein In contrast, the only other structurally characterized acp transferase enzyme Tyw2 belongs to the Rossmann-fold class of methyltransferase proteins. INTRO +98 147 Rossmann-fold class of methyltransferase proteins protein_type In contrast, the only other structurally characterized acp transferase enzyme Tyw2 belongs to the Rossmann-fold class of methyltransferase proteins. INTRO +97 100 SAM chemical Interestingly, the two structurally very different enzymes use similar strategies in binding the SAM-cofactor in order to ensure that in contrast to methyltransferases the acp and not the methyl group of SAM is transferred to the substrate. INTRO +149 167 methyltransferases protein_type Interestingly, the two structurally very different enzymes use similar strategies in binding the SAM-cofactor in order to ensure that in contrast to methyltransferases the acp and not the methyl group of SAM is transferred to the substrate. INTRO +172 175 acp chemical Interestingly, the two structurally very different enzymes use similar strategies in binding the SAM-cofactor in order to ensure that in contrast to methyltransferases the acp and not the methyl group of SAM is transferred to the substrate. INTRO +204 207 SAM chemical Interestingly, the two structurally very different enzymes use similar strategies in binding the SAM-cofactor in order to ensure that in contrast to methyltransferases the acp and not the methyl group of SAM is transferred to the substrate. INTRO +0 4 Tsr3 protein Tsr3 is the enzyme responsible for 18S rRNA acp modification in yeast and humans RESULTS +35 43 18S rRNA chemical Tsr3 is the enzyme responsible for 18S rRNA acp modification in yeast and humans RESULTS +44 47 acp chemical Tsr3 is the enzyme responsible for 18S rRNA acp modification in yeast and humans RESULTS +64 69 yeast taxonomy_domain Tsr3 is the enzyme responsible for 18S rRNA acp modification in yeast and humans RESULTS +74 80 humans species Tsr3 is the enzyme responsible for 18S rRNA acp modification in yeast and humans RESULTS +4 17 S. cerevisiae species The S. cerevisiae 18S rRNA acp transferase was identified in a systematic genetic screen where numerous deletion mutants from the EUROSCARF strain collection (www.euroscarf.de) were analyzed by HPLC for alterations in 18S rRNA base modifications. RESULTS +18 42 18S rRNA acp transferase protein_type The S. cerevisiae 18S rRNA acp transferase was identified in a systematic genetic screen where numerous deletion mutants from the EUROSCARF strain collection (www.euroscarf.de) were analyzed by HPLC for alterations in 18S rRNA base modifications. RESULTS +194 198 HPLC experimental_method The S. cerevisiae 18S rRNA acp transferase was identified in a systematic genetic screen where numerous deletion mutants from the EUROSCARF strain collection (www.euroscarf.de) were analyzed by HPLC for alterations in 18S rRNA base modifications. RESULTS +218 226 18S rRNA chemical The S. cerevisiae 18S rRNA acp transferase was identified in a systematic genetic screen where numerous deletion mutants from the EUROSCARF strain collection (www.euroscarf.de) were analyzed by HPLC for alterations in 18S rRNA base modifications. RESULTS +8 13 Δtsr3 mutant For the Δtsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant Δnep1, where a shoulder at ∼ 7.4 min elution time was missing in the elution profile. RESULTS +34 54 HPLC elution profile evidence For the Δtsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant Δnep1, where a shoulder at ∼ 7.4 min elution time was missing in the elution profile. RESULTS +58 66 18S rRNA chemical For the Δtsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant Δnep1, where a shoulder at ∼ 7.4 min elution time was missing in the elution profile. RESULTS +67 78 nucleosides chemical For the Δtsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant Δnep1, where a shoulder at ∼ 7.4 min elution time was missing in the elution profile. RESULTS +123 157 pseudouridine-N1 methyltransferase protein_type For the Δtsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant Δnep1, where a shoulder at ∼ 7.4 min elution time was missing in the elution profile. RESULTS +158 164 mutant protein_state For the Δtsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant Δnep1, where a shoulder at ∼ 7.4 min elution time was missing in the elution profile. RESULTS +165 170 Δnep1 mutant For the Δtsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant Δnep1, where a shoulder at ∼ 7.4 min elution time was missing in the elution profile. RESULTS +55 61 ESI-MS experimental_method As previously reported this shoulder was identified by ESI-MS as corresponding to m1acp3Ψ. RESULTS +82 89 m1acp3Ψ chemical As previously reported this shoulder was identified by ESI-MS as corresponding to m1acp3Ψ. RESULTS +49 52 acp chemical In order to directly analyze the presence of the acp modification of nucleotide 1191 we used an in vivo14C incorporation assay with 1-14C-methionine. RESULTS +69 79 nucleotide chemical In order to directly analyze the presence of the acp modification of nucleotide 1191 we used an in vivo14C incorporation assay with 1-14C-methionine. RESULTS +80 84 1191 residue_number In order to directly analyze the presence of the acp modification of nucleotide 1191 we used an in vivo14C incorporation assay with 1-14C-methionine. RESULTS +96 126 in vivo14C incorporation assay experimental_method In order to directly analyze the presence of the acp modification of nucleotide 1191 we used an in vivo14C incorporation assay with 1-14C-methionine. RESULTS +132 148 1-14C-methionine chemical In order to directly analyze the presence of the acp modification of nucleotide 1191 we used an in vivo14C incorporation assay with 1-14C-methionine. RESULTS +12 15 acp chemical Whereas the acp labeling of 18S rRNA was clearly present in the wild type strain no radioactive labeling could be observed in a Δtsr3 strain (Figure 1C). RESULTS +28 36 18S rRNA chemical Whereas the acp labeling of 18S rRNA was clearly present in the wild type strain no radioactive labeling could be observed in a Δtsr3 strain (Figure 1C). RESULTS +64 73 wild type protein_state Whereas the acp labeling of 18S rRNA was clearly present in the wild type strain no radioactive labeling could be observed in a Δtsr3 strain (Figure 1C). RESULTS +128 133 Δtsr3 mutant Whereas the acp labeling of 18S rRNA was clearly present in the wild type strain no radioactive labeling could be observed in a Δtsr3 strain (Figure 1C). RESULTS +44 54 18S U1191A mutant No radioactive labeling was detected in the 18S U1191A mutant which served as a control for the specificity of the 14C-aminocarboxypropyl incorporation. RESULTS +55 61 mutant protein_state No radioactive labeling was detected in the 18S U1191A mutant which served as a control for the specificity of the 14C-aminocarboxypropyl incorporation. RESULTS +115 137 14C-aminocarboxypropyl chemical No radioactive labeling was detected in the 18S U1191A mutant which served as a control for the specificity of the 14C-aminocarboxypropyl incorporation. RESULTS +30 33 acp chemical As previously shown, only the acp but none of the other modifications at U1191 of yeast 18S rRNA blocks reverse transcriptase activity. RESULTS +73 78 U1191 residue_name_number As previously shown, only the acp but none of the other modifications at U1191 of yeast 18S rRNA blocks reverse transcriptase activity. RESULTS +82 87 yeast taxonomy_domain As previously shown, only the acp but none of the other modifications at U1191 of yeast 18S rRNA blocks reverse transcriptase activity. RESULTS +88 96 18S rRNA chemical As previously shown, only the acp but none of the other modifications at U1191 of yeast 18S rRNA blocks reverse transcriptase activity. RESULTS +30 33 acp chemical Therefore the presence of the acp modification can be directly assessed by primer extension. RESULTS +75 91 primer extension experimental_method Therefore the presence of the acp modification can be directly assessed by primer extension. RESULTS +11 20 wild-type protein_state Indeed, in wild-type yeast a strong primer extension stop signal occurred at position 1192. RESULTS +21 26 yeast taxonomy_domain Indeed, in wild-type yeast a strong primer extension stop signal occurred at position 1192. RESULTS +36 64 primer extension stop signal evidence Indeed, in wild-type yeast a strong primer extension stop signal occurred at position 1192. RESULTS +86 90 1192 residue_number Indeed, in wild-type yeast a strong primer extension stop signal occurred at position 1192. RESULTS +18 23 Δtsr3 mutant In contrast, in a Δtsr3 mutant no primer extension stop signal was present at this position. RESULTS +24 30 mutant protein_state In contrast, in a Δtsr3 mutant no primer extension stop signal was present at this position. RESULTS +18 24 Δsnr35 mutant As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. RESULTS +25 33 deletion experimental_method As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. RESULTS +45 62 pseudouridylation ptm As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. RESULTS +67 81 N1-methylation ptm As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. RESULTS +96 101 acp3U chemical As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. RESULTS +119 124 Δnep1 mutant As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. RESULTS +147 160 pseudouridine chemical As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. RESULTS +164 178 not methylated protein_state As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. RESULTS +193 198 acp3Ψ chemical As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. RESULTS +202 230 primer extension stop signal evidence As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. RESULTS +262 271 wild type protein_state As expected, in a Δsnr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a Δnep1 deletion strain where pseudouridine is not methylated (resulting in acp3Ψ) a primer extension stop signal of similar intensity as in the wild type was observed. RESULTS +5 17 Δtsr3 Δsnr35 mutant In a Δtsr3 Δsnr35 double deletion strain the 18S rRNA contains an unmodified U and the primer extension stop signal was missing (Figure 1D). RESULTS +45 53 18S rRNA chemical In a Δtsr3 Δsnr35 double deletion strain the 18S rRNA contains an unmodified U and the primer extension stop signal was missing (Figure 1D). RESULTS +66 76 unmodified protein_state In a Δtsr3 Δsnr35 double deletion strain the 18S rRNA contains an unmodified U and the primer extension stop signal was missing (Figure 1D). RESULTS +77 78 U chemical In a Δtsr3 Δsnr35 double deletion strain the 18S rRNA contains an unmodified U and the primer extension stop signal was missing (Figure 1D). RESULTS +4 8 Tsr3 protein The Tsr3 protein is highly conserved in yeast and humans (50% identity). RESULTS +20 36 highly conserved protein_state The Tsr3 protein is highly conserved in yeast and humans (50% identity). RESULTS +40 45 yeast taxonomy_domain The Tsr3 protein is highly conserved in yeast and humans (50% identity). RESULTS +50 56 humans species The Tsr3 protein is highly conserved in yeast and humans (50% identity). RESULTS +0 5 Human species Human 18S rRNA has also been shown to contain m1acp3Ψ in the 18S rRNA at position 1248. RESULTS +6 14 18S rRNA chemical Human 18S rRNA has also been shown to contain m1acp3Ψ in the 18S rRNA at position 1248. RESULTS +46 53 m1acp3Ψ ptm Human 18S rRNA has also been shown to contain m1acp3Ψ in the 18S rRNA at position 1248. RESULTS +61 69 18S rRNA chemical Human 18S rRNA has also been shown to contain m1acp3Ψ in the 18S rRNA at position 1248. RESULTS +82 86 1248 residue_number Human 18S rRNA has also been shown to contain m1acp3Ψ in the 18S rRNA at position 1248. RESULTS +6 30 siRNA-mediated depletion experimental_method After siRNA-mediated depletion of Tsr3 in human colon carcinoma HCT116(+/+) cells the acp primer extension arrest was reduced in comparison to cells transfected with a non-targeting scramble siRNA control (Figure 1E, compare lanes 544 and scramble). RESULTS +34 38 Tsr3 protein After siRNA-mediated depletion of Tsr3 in human colon carcinoma HCT116(+/+) cells the acp primer extension arrest was reduced in comparison to cells transfected with a non-targeting scramble siRNA control (Figure 1E, compare lanes 544 and scramble). RESULTS +42 47 human species After siRNA-mediated depletion of Tsr3 in human colon carcinoma HCT116(+/+) cells the acp primer extension arrest was reduced in comparison to cells transfected with a non-targeting scramble siRNA control (Figure 1E, compare lanes 544 and scramble). RESULTS +86 113 acp primer extension arrest evidence After siRNA-mediated depletion of Tsr3 in human colon carcinoma HCT116(+/+) cells the acp primer extension arrest was reduced in comparison to cells transfected with a non-targeting scramble siRNA control (Figure 1E, compare lanes 544 and scramble). RESULTS +191 196 siRNA chemical After siRNA-mediated depletion of Tsr3 in human colon carcinoma HCT116(+/+) cells the acp primer extension arrest was reduced in comparison to cells transfected with a non-targeting scramble siRNA control (Figure 1E, compare lanes 544 and scramble). RESULTS +18 23 siRNA chemical The efficiency of siRNA-mediated depletion was established by RT-qPCR and found to be very high with siRNA 544 (Supplementary Figure S2A, remaining TSR3 mRNA level of 2%). RESULTS +62 69 RT-qPCR experimental_method The efficiency of siRNA-mediated depletion was established by RT-qPCR and found to be very high with siRNA 544 (Supplementary Figure S2A, remaining TSR3 mRNA level of 2%). RESULTS +101 106 siRNA chemical The efficiency of siRNA-mediated depletion was established by RT-qPCR and found to be very high with siRNA 544 (Supplementary Figure S2A, remaining TSR3 mRNA level of 2%). RESULTS +148 152 TSR3 protein The efficiency of siRNA-mediated depletion was established by RT-qPCR and found to be very high with siRNA 544 (Supplementary Figure S2A, remaining TSR3 mRNA level of 2%). RESULTS +35 40 siRNA chemical By comparison, treating cells with siRNA 545, which only reduced the TSR3 mRNA to 20%, did not markedly reduced the acp signal. RESULTS +69 73 TSR3 protein By comparison, treating cells with siRNA 545, which only reduced the TSR3 mRNA to 20%, did not markedly reduced the acp signal. RESULTS +116 119 acp chemical By comparison, treating cells with siRNA 545, which only reduced the TSR3 mRNA to 20%, did not markedly reduced the acp signal. RESULTS +42 48 HsTsr3 protein This suggests that low residual levels of HsTsr3 are sufficient to modify the RNA. RESULTS +78 81 RNA chemical This suggests that low residual levels of HsTsr3 are sufficient to modify the RNA. RESULTS +6 12 HsTsr3 protein Thus, HsTsr3 is also responsible for the acp modification of 18S rRNA nucleotide Ψ1248 in helix 31. RESULTS +41 44 acp chemical Thus, HsTsr3 is also responsible for the acp modification of 18S rRNA nucleotide Ψ1248 in helix 31. RESULTS +61 69 18S rRNA chemical Thus, HsTsr3 is also responsible for the acp modification of 18S rRNA nucleotide Ψ1248 in helix 31. RESULTS +70 80 nucleotide chemical Thus, HsTsr3 is also responsible for the acp modification of 18S rRNA nucleotide Ψ1248 in helix 31. RESULTS +81 86 Ψ1248 ptm Thus, HsTsr3 is also responsible for the acp modification of 18S rRNA nucleotide Ψ1248 in helix 31. RESULTS +90 98 helix 31 structure_element Thus, HsTsr3 is also responsible for the acp modification of 18S rRNA nucleotide Ψ1248 in helix 31. RESULTS +11 16 yeast taxonomy_domain Similar to yeast, siRNA-mediated depletion of the Ψ1248 N1-methyltransferase Nep1/Emg1 had no influence on the primer extension arrest (Figure 1E). RESULTS +18 42 siRNA-mediated depletion experimental_method Similar to yeast, siRNA-mediated depletion of the Ψ1248 N1-methyltransferase Nep1/Emg1 had no influence on the primer extension arrest (Figure 1E). RESULTS +50 76 Ψ1248 N1-methyltransferase protein_type Similar to yeast, siRNA-mediated depletion of the Ψ1248 N1-methyltransferase Nep1/Emg1 had no influence on the primer extension arrest (Figure 1E). RESULTS +77 81 Nep1 protein Similar to yeast, siRNA-mediated depletion of the Ψ1248 N1-methyltransferase Nep1/Emg1 had no influence on the primer extension arrest (Figure 1E). RESULTS +82 86 Emg1 protein Similar to yeast, siRNA-mediated depletion of the Ψ1248 N1-methyltransferase Nep1/Emg1 had no influence on the primer extension arrest (Figure 1E). RESULTS +111 134 primer extension arrest evidence Similar to yeast, siRNA-mediated depletion of the Ψ1248 N1-methyltransferase Nep1/Emg1 had no influence on the primer extension arrest (Figure 1E). RESULTS +31 36 Δtsr3 mutant Phenotypic characterization of Δtsr3 mutants RESULTS +13 16 acp chemical Although the acp modification of 18S rRNA is highly conserved in eukaryotes, yeast Δtsr3 mutants showed only a minor growth defect. RESULTS +33 41 18S rRNA chemical Although the acp modification of 18S rRNA is highly conserved in eukaryotes, yeast Δtsr3 mutants showed only a minor growth defect. RESULTS +45 61 highly conserved protein_state Although the acp modification of 18S rRNA is highly conserved in eukaryotes, yeast Δtsr3 mutants showed only a minor growth defect. RESULTS +65 75 eukaryotes taxonomy_domain Although the acp modification of 18S rRNA is highly conserved in eukaryotes, yeast Δtsr3 mutants showed only a minor growth defect. RESULTS +77 82 yeast taxonomy_domain Although the acp modification of 18S rRNA is highly conserved in eukaryotes, yeast Δtsr3 mutants showed only a minor growth defect. RESULTS +83 88 Δtsr3 mutant Although the acp modification of 18S rRNA is highly conserved in eukaryotes, yeast Δtsr3 mutants showed only a minor growth defect. RESULTS +13 18 Δtsr3 mutant However, the Δtsr3 deletion was synthetic sick with a Δsnr35 deletion preventing pseudouridylation and Nep1-catalyzed methylation of nucleotide 1191 (Figure 2A). RESULTS +54 60 Δsnr35 mutant However, the Δtsr3 deletion was synthetic sick with a Δsnr35 deletion preventing pseudouridylation and Nep1-catalyzed methylation of nucleotide 1191 (Figure 2A). RESULTS +81 98 pseudouridylation ptm However, the Δtsr3 deletion was synthetic sick with a Δsnr35 deletion preventing pseudouridylation and Nep1-catalyzed methylation of nucleotide 1191 (Figure 2A). RESULTS +103 107 Nep1 protein However, the Δtsr3 deletion was synthetic sick with a Δsnr35 deletion preventing pseudouridylation and Nep1-catalyzed methylation of nucleotide 1191 (Figure 2A). RESULTS +144 148 1191 residue_number However, the Δtsr3 deletion was synthetic sick with a Δsnr35 deletion preventing pseudouridylation and Nep1-catalyzed methylation of nucleotide 1191 (Figure 2A). RESULTS +64 75 Δtsr3 Δnep1 mutant Interestingly, no increased growth defect could be observed for Δtsr3 Δnep1 recombinants containing the nep1 suppressor mutation Δnop6 as well as for Δtsr3 Δsnr35 Δnep1 recombinants with unmodified U1191 (Supplementary Figure S2D and E). RESULTS +104 108 nep1 gene Interestingly, no increased growth defect could be observed for Δtsr3 Δnep1 recombinants containing the nep1 suppressor mutation Δnop6 as well as for Δtsr3 Δsnr35 Δnep1 recombinants with unmodified U1191 (Supplementary Figure S2D and E). RESULTS +129 134 Δnop6 mutant Interestingly, no increased growth defect could be observed for Δtsr3 Δnep1 recombinants containing the nep1 suppressor mutation Δnop6 as well as for Δtsr3 Δsnr35 Δnep1 recombinants with unmodified U1191 (Supplementary Figure S2D and E). RESULTS +150 168 Δtsr3 Δsnr35 Δnep1 mutant Interestingly, no increased growth defect could be observed for Δtsr3 Δnep1 recombinants containing the nep1 suppressor mutation Δnop6 as well as for Δtsr3 Δsnr35 Δnep1 recombinants with unmodified U1191 (Supplementary Figure S2D and E). RESULTS +187 197 unmodified protein_state Interestingly, no increased growth defect could be observed for Δtsr3 Δnep1 recombinants containing the nep1 suppressor mutation Δnop6 as well as for Δtsr3 Δsnr35 Δnep1 recombinants with unmodified U1191 (Supplementary Figure S2D and E). RESULTS +198 203 U1191 residue_name_number Interestingly, no increased growth defect could be observed for Δtsr3 Δnep1 recombinants containing the nep1 suppressor mutation Δnop6 as well as for Δtsr3 Δsnr35 Δnep1 recombinants with unmodified U1191 (Supplementary Figure S2D and E). RESULTS +31 36 yeast taxonomy_domain Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +37 41 TSR3 protein Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +52 57 Δtrs3 mutant Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +63 68 human species Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +69 73 TSR3 protein Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +85 91 siRNAs chemical Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +134 139 yeast taxonomy_domain Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +140 144 Tsr3 protein Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +160 165 yeast taxonomy_domain Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +166 175 wild type protein_state Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +177 182 Δtsr3 mutant Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +184 190 Δsnr35 mutant Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +195 207 Δtsr3 Δsnr35 mutant Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +258 263 Δtsr3 mutant Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +264 268 TSR3 protein Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +269 275 Δsnr35 mutant Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +276 281 SNR35 protein Phenotypic characterization of yeast TSR3 deletion (Δtrs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, Δtsr3, Δsnr35 and Δtsr3 Δsnr35 segregants after meiosis and tetrad dissection of Δtsr3/TSR3 Δsnr35/SNR35 heterozygous diploids. FIG +4 9 Δtsr3 mutant The Δtsr3 deletion is synthetic sick with a Δsnr35 deletion preventing U1191 pseudouridylation. FIG +44 50 Δsnr35 mutant The Δtsr3 deletion is synthetic sick with a Δsnr35 deletion preventing U1191 pseudouridylation. FIG +71 76 U1191 residue_name_number The Δtsr3 deletion is synthetic sick with a Δsnr35 deletion preventing U1191 pseudouridylation. FIG +7 28 agar diffusion assays experimental_method (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. FIG +33 38 yeast taxonomy_domain (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. FIG +39 44 Δtsr3 mutant (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. FIG +45 60 deletion mutant protein_state (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. FIG +94 105 paromomycin chemical (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. FIG +110 122 hygromycin B chemical (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. FIG +172 178 Δsnr35 mutant (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. FIG +184 206 Northern blot analysis experimental_method (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. FIG +246 261 siRNA depletion experimental_method (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. FIG +265 271 HsTSR3 protein (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. FIG +273 279 siRNAs chemical (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. FIG +309 314 siRNA chemical (B) In agar diffusion assays the yeast Δtsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with Δsnr35. (C) Northern blot analysis with an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control. FIG +20 24 18SE chemical The accumulation of 18SE and 47S and/or 45S pre-RNAs is enforced upon HsTSR3 depletion. FIG +29 32 47S chemical The accumulation of 18SE and 47S and/or 45S pre-RNAs is enforced upon HsTSR3 depletion. FIG +40 52 45S pre-RNAs chemical The accumulation of 18SE and 47S and/or 45S pre-RNAs is enforced upon HsTSR3 depletion. FIG +70 76 HsTSR3 protein The accumulation of 18SE and 47S and/or 45S pre-RNAs is enforced upon HsTSR3 depletion. FIG +45 48 18S chemical Right gel: Ethidium bromide staining showing 18S and 28S rRNAs. FIG +53 62 28S rRNAs chemical Right gel: Ethidium bromide staining showing 18S and 28S rRNAs. FIG +32 37 yeast taxonomy_domain (D) Cytoplasmic localization of yeast Tsr3 shown by fluorescence microscopy of GFP-fused Tsr3. FIG +38 42 Tsr3 protein (D) Cytoplasmic localization of yeast Tsr3 shown by fluorescence microscopy of GFP-fused Tsr3. FIG +52 75 fluorescence microscopy experimental_method (D) Cytoplasmic localization of yeast Tsr3 shown by fluorescence microscopy of GFP-fused Tsr3. FIG +79 93 GFP-fused Tsr3 mutant (D) Cytoplasmic localization of yeast Tsr3 shown by fluorescence microscopy of GFP-fused Tsr3. FIG +20 54 differential interference contrast experimental_method From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +56 59 DIC experimental_method From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +84 92 GFP-Tsr3 mutant From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +114 124 Nop56-mRFP mutant From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +159 167 GFP-Tsr3 mutant From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +168 178 Nop56-mRFP mutant From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +184 187 DIC experimental_method From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +193 208 Elution profile evidence From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +222 249 sucrose gradient separation experimental_method From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +253 258 yeast taxonomy_domain From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +259 277 ribosomal subunits complex_assembly From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +282 291 polysomes complex_assembly From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +309 321 western blot experimental_method From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +334 338 3xHA chemical From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +346 350 Tsr3 protein From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +352 361 Tsr3-3xHA mutant From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +369 377 SDS-PAGE experimental_method From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part). FIG +4 8 TSR3 protein The TSR3 gene was genetically modified at its native locus, resulting in a C-terminal fusion of Tsr3 with a 3xHA epitope expressed by the native promotor in yeast strain CEN.BM258-5B. FIG +86 92 fusion protein_state The TSR3 gene was genetically modified at its native locus, resulting in a C-terminal fusion of Tsr3 with a 3xHA epitope expressed by the native promotor in yeast strain CEN.BM258-5B. FIG +96 100 Tsr3 protein The TSR3 gene was genetically modified at its native locus, resulting in a C-terminal fusion of Tsr3 with a 3xHA epitope expressed by the native promotor in yeast strain CEN.BM258-5B. FIG +108 112 3xHA chemical The TSR3 gene was genetically modified at its native locus, resulting in a C-terminal fusion of Tsr3 with a 3xHA epitope expressed by the native promotor in yeast strain CEN.BM258-5B. FIG +157 162 yeast taxonomy_domain The TSR3 gene was genetically modified at its native locus, resulting in a C-terminal fusion of Tsr3 with a 3xHA epitope expressed by the native promotor in yeast strain CEN.BM258-5B. FIG +21 24 acp chemical The influence of the acp modification of nucleotide 1191 on ribosome function was analyzed by treating Δtsr3 mutants with protein synthesis inhibitors. RESULTS +41 51 nucleotide chemical The influence of the acp modification of nucleotide 1191 on ribosome function was analyzed by treating Δtsr3 mutants with protein synthesis inhibitors. RESULTS +52 56 1191 residue_number The influence of the acp modification of nucleotide 1191 on ribosome function was analyzed by treating Δtsr3 mutants with protein synthesis inhibitors. RESULTS +103 108 Δtsr3 mutant The influence of the acp modification of nucleotide 1191 on ribosome function was analyzed by treating Δtsr3 mutants with protein synthesis inhibitors. RESULTS +35 39 nep1 gene Similar to a temperature-sensitive nep1 mutant, the Δtsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown). RESULTS +40 46 mutant protein_state Similar to a temperature-sensitive nep1 mutant, the Δtsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown). RESULTS +52 57 Δtsr3 mutant Similar to a temperature-sensitive nep1 mutant, the Δtsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown). RESULTS +94 105 paromomycin chemical Similar to a temperature-sensitive nep1 mutant, the Δtsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown). RESULTS +134 146 hygromycin B chemical Similar to a temperature-sensitive nep1 mutant, the Δtsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown). RESULTS +171 175 G418 chemical Similar to a temperature-sensitive nep1 mutant, the Δtsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown). RESULTS +179 192 cycloheximide chemical Similar to a temperature-sensitive nep1 mutant, the Δtsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown). RESULTS +59 70 paromomycin chemical In accordance with the synthetic sick growth phenotype the paromomycin and hygromycin B hypersensitivity further increased in a Δtsr3 Δsnr35 recombination strain (Figure 2B). RESULTS +75 87 hygromycin B chemical In accordance with the synthetic sick growth phenotype the paromomycin and hygromycin B hypersensitivity further increased in a Δtsr3 Δsnr35 recombination strain (Figure 2B). RESULTS +128 140 Δtsr3 Δsnr35 mutant In accordance with the synthetic sick growth phenotype the paromomycin and hygromycin B hypersensitivity further increased in a Δtsr3 Δsnr35 recombination strain (Figure 2B). RESULTS +5 10 yeast taxonomy_domain In a yeast Δtsr3 strain as well as in the Δtsr3 Δsnr35 recombinant 20S pre-rRNA accumulated significantly and the level of mature 18S rRNA was reduced (Supplementary Figures S2C and S3D), as reported previously. RESULTS +11 16 Δtsr3 mutant In a yeast Δtsr3 strain as well as in the Δtsr3 Δsnr35 recombinant 20S pre-rRNA accumulated significantly and the level of mature 18S rRNA was reduced (Supplementary Figures S2C and S3D), as reported previously. RESULTS +42 54 Δtsr3 Δsnr35 mutant In a yeast Δtsr3 strain as well as in the Δtsr3 Δsnr35 recombinant 20S pre-rRNA accumulated significantly and the level of mature 18S rRNA was reduced (Supplementary Figures S2C and S3D), as reported previously. RESULTS +67 79 20S pre-rRNA chemical In a yeast Δtsr3 strain as well as in the Δtsr3 Δsnr35 recombinant 20S pre-rRNA accumulated significantly and the level of mature 18S rRNA was reduced (Supplementary Figures S2C and S3D), as reported previously. RESULTS +130 138 18S rRNA chemical In a yeast Δtsr3 strain as well as in the Δtsr3 Δsnr35 recombinant 20S pre-rRNA accumulated significantly and the level of mature 18S rRNA was reduced (Supplementary Figures S2C and S3D), as reported previously. RESULTS +18 26 20S rRNA chemical A minor effect on 20S rRNA accumulation was also observed for Δsnr35, but - probably due to different strain backgrounds – to a weaker extent than described earlier. RESULTS +62 68 Δsnr35 mutant A minor effect on 20S rRNA accumulation was also observed for Δsnr35, but - probably due to different strain backgrounds – to a weaker extent than described earlier. RESULTS +3 8 human species In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B). RESULTS +20 32 depletion of experimental_method In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B). RESULTS +33 39 HsTsr3 protein In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B). RESULTS +91 96 human species In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B). RESULTS +97 109 20S pre-rRNA chemical In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B). RESULTS +121 126 18S-E chemical In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B). RESULTS +172 176 Tsr3 protein In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B). RESULTS +198 206 18S rRNA chemical In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B). RESULTS +102 107 yeast taxonomy_domain Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast Δtsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B). RESULTS +108 113 Δtsr3 mutant Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast Δtsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B). RESULTS +141 144 35S complex_assembly Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast Δtsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B). RESULTS +178 182 Tsr3 protein Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast Δtsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B). RESULTS +192 197 human species Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast Δtsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B). RESULTS +209 212 47S complex_assembly Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast Δtsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B). RESULTS +213 216 45S complex_assembly Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast Δtsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B). RESULTS +247 260 Northern blot experimental_method Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast Δtsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B). RESULTS +33 41 18S rRNA chemical Consistent with its role in late 18S rRNA processing, TSR3 deletion leads to a ribosomal subunit imbalance with a reduced 40S to 60S ratio of 0.81 (σ = 0.024) which was further increased in a Δtsr3 Δsnr35 recombinant to 0.73 (σ = 0.023) (Supplementary Figure S2F). RESULTS +54 58 TSR3 protein Consistent with its role in late 18S rRNA processing, TSR3 deletion leads to a ribosomal subunit imbalance with a reduced 40S to 60S ratio of 0.81 (σ = 0.024) which was further increased in a Δtsr3 Δsnr35 recombinant to 0.73 (σ = 0.023) (Supplementary Figure S2F). RESULTS +122 125 40S complex_assembly Consistent with its role in late 18S rRNA processing, TSR3 deletion leads to a ribosomal subunit imbalance with a reduced 40S to 60S ratio of 0.81 (σ = 0.024) which was further increased in a Δtsr3 Δsnr35 recombinant to 0.73 (σ = 0.023) (Supplementary Figure S2F). RESULTS +129 132 60S complex_assembly Consistent with its role in late 18S rRNA processing, TSR3 deletion leads to a ribosomal subunit imbalance with a reduced 40S to 60S ratio of 0.81 (σ = 0.024) which was further increased in a Δtsr3 Δsnr35 recombinant to 0.73 (σ = 0.023) (Supplementary Figure S2F). RESULTS +192 204 Δtsr3 Δsnr35 mutant Consistent with its role in late 18S rRNA processing, TSR3 deletion leads to a ribosomal subunit imbalance with a reduced 40S to 60S ratio of 0.81 (σ = 0.024) which was further increased in a Δtsr3 Δsnr35 recombinant to 0.73 (σ = 0.023) (Supplementary Figure S2F). RESULTS +3 20 polysome profiles evidence In polysome profiles, a reduced level of 80S ribosomes and a strong signal for free 60S subunits was observed in line with the 40S subunit deficiency (Supplementary Figure S2G). RESULTS +41 54 80S ribosomes complex_assembly In polysome profiles, a reduced level of 80S ribosomes and a strong signal for free 60S subunits was observed in line with the 40S subunit deficiency (Supplementary Figure S2G). RESULTS +84 87 60S complex_assembly In polysome profiles, a reduced level of 80S ribosomes and a strong signal for free 60S subunits was observed in line with the 40S subunit deficiency (Supplementary Figure S2G). RESULTS +127 130 40S complex_assembly In polysome profiles, a reduced level of 80S ribosomes and a strong signal for free 60S subunits was observed in line with the 40S subunit deficiency (Supplementary Figure S2G). RESULTS +25 29 Tsr3 protein Cellular localization of Tsr3 in S. cerevisiae RESULTS +33 46 S. cerevisiae species Cellular localization of Tsr3 in S. cerevisiae RESULTS +0 23 Fluorescence microscopy experimental_method Fluorescence microscopy of GFP-tagged Tsr3 localized the fusion protein in the cytoplasm of yeast cells and no co-localization with the nucleolar marker protein Nop56 could be observed (Figure 2D). RESULTS +27 37 GFP-tagged protein_state Fluorescence microscopy of GFP-tagged Tsr3 localized the fusion protein in the cytoplasm of yeast cells and no co-localization with the nucleolar marker protein Nop56 could be observed (Figure 2D). RESULTS +38 42 Tsr3 protein Fluorescence microscopy of GFP-tagged Tsr3 localized the fusion protein in the cytoplasm of yeast cells and no co-localization with the nucleolar marker protein Nop56 could be observed (Figure 2D). RESULTS +92 97 yeast taxonomy_domain Fluorescence microscopy of GFP-tagged Tsr3 localized the fusion protein in the cytoplasm of yeast cells and no co-localization with the nucleolar marker protein Nop56 could be observed (Figure 2D). RESULTS +161 166 Nop56 protein Fluorescence microscopy of GFP-tagged Tsr3 localized the fusion protein in the cytoplasm of yeast cells and no co-localization with the nucleolar marker protein Nop56 could be observed (Figure 2D). RESULTS +63 66 acp chemical This agrees with previous biochemical data suggesting that the acp modification of 18S rRNA occurs late during 40S subunit biogenesis in the cytoplasm, and makes an additional nuclear localization as reported in a previous large-scale analysis unlikely. RESULTS +83 91 18S rRNA chemical This agrees with previous biochemical data suggesting that the acp modification of 18S rRNA occurs late during 40S subunit biogenesis in the cytoplasm, and makes an additional nuclear localization as reported in a previous large-scale analysis unlikely. RESULTS +111 114 40S complex_assembly This agrees with previous biochemical data suggesting that the acp modification of 18S rRNA occurs late during 40S subunit biogenesis in the cytoplasm, and makes an additional nuclear localization as reported in a previous large-scale analysis unlikely. RESULTS +6 34 polysome gradient separation experimental_method After polysome gradient separation C-terminally epitope-labeled Tsr3-3xHA was exclusively detectable in the low-density fraction (Figure 2E). RESULTS +64 73 Tsr3-3xHA mutant After polysome gradient separation C-terminally epitope-labeled Tsr3-3xHA was exclusively detectable in the low-density fraction (Figure 2E). RESULTS +5 39 distribution on a density gradient evidence Such distribution on a density gradient suggests that Tsr3 only interacts transiently with pre-40S subunits, which presumably explains why it was not characterized in pre-ribosome affinity purifications. RESULTS +54 58 Tsr3 protein Such distribution on a density gradient suggests that Tsr3 only interacts transiently with pre-40S subunits, which presumably explains why it was not characterized in pre-ribosome affinity purifications. RESULTS +91 107 pre-40S subunits complex_assembly Such distribution on a density gradient suggests that Tsr3 only interacts transiently with pre-40S subunits, which presumably explains why it was not characterized in pre-ribosome affinity purifications. RESULTS +167 202 pre-ribosome affinity purifications experimental_method Such distribution on a density gradient suggests that Tsr3 only interacts transiently with pre-40S subunits, which presumably explains why it was not characterized in pre-ribosome affinity purifications. RESULTS +0 9 Structure evidence Structure of Tsr3 RESULTS +13 17 Tsr3 protein Structure of Tsr3 RESULTS +34 47 S. cerevisiae species Searches for sequence homologs of S. cerevisiae Tsr3 (ScTsr3) by us and others revealed that the genomes of many archaea contain genes encoding Tsr3-like proteins. RESULTS +48 52 Tsr3 protein Searches for sequence homologs of S. cerevisiae Tsr3 (ScTsr3) by us and others revealed that the genomes of many archaea contain genes encoding Tsr3-like proteins. RESULTS +54 60 ScTsr3 protein Searches for sequence homologs of S. cerevisiae Tsr3 (ScTsr3) by us and others revealed that the genomes of many archaea contain genes encoding Tsr3-like proteins. RESULTS +113 120 archaea taxonomy_domain Searches for sequence homologs of S. cerevisiae Tsr3 (ScTsr3) by us and others revealed that the genomes of many archaea contain genes encoding Tsr3-like proteins. RESULTS +144 162 Tsr3-like proteins protein_type Searches for sequence homologs of S. cerevisiae Tsr3 (ScTsr3) by us and others revealed that the genomes of many archaea contain genes encoding Tsr3-like proteins. RESULTS +15 23 archaeal taxonomy_domain However, these archaeal homologs are significantly smaller than ScTsr3 (∼190 aa in archaea vs. 313 aa in yeast) due to shortened N- and C-termini (Supplementary Figure S1A). RESULTS +64 70 ScTsr3 protein However, these archaeal homologs are significantly smaller than ScTsr3 (∼190 aa in archaea vs. 313 aa in yeast) due to shortened N- and C-termini (Supplementary Figure S1A). RESULTS +83 90 archaea taxonomy_domain However, these archaeal homologs are significantly smaller than ScTsr3 (∼190 aa in archaea vs. 313 aa in yeast) due to shortened N- and C-termini (Supplementary Figure S1A). RESULTS +105 110 yeast taxonomy_domain However, these archaeal homologs are significantly smaller than ScTsr3 (∼190 aa in archaea vs. 313 aa in yeast) due to shortened N- and C-termini (Supplementary Figure S1A). RESULTS +41 45 Tsr3 protein To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a Δtsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C). RESULTS +56 62 ScTsr3 protein To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a Δtsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C). RESULTS +109 125 highly conserved protein_state To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a Δtsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C). RESULTS +144 153 expressed experimental_method To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a Δtsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C). RESULTS +159 164 Δtsr3 mutant To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a Δtsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C). RESULTS +165 171 mutant protein_state To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a Δtsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C). RESULTS +200 216 primer extension experimental_method To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a Δtsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C). RESULTS +233 250 Northern blotting experimental_method To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a Δtsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C). RESULTS +11 22 truncations experimental_method N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected. RESULTS +32 37 45 aa residue_range N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected. RESULTS +53 64 truncations experimental_method N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected. RESULTS +74 79 76 aa residue_range N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected. RESULTS +89 92 acp chemical N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected. RESULTS +128 139 full-length protein_state N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected. RESULTS +187 198 20S pre-RNA chemical N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected. RESULTS +7 11 Tsr3 protein Even a Tsr3 fragment with a 90 aa C-terminal truncation showed a residual primer extension stop, whereas N-terminal truncations exceeding 46 aa almost completely abolished the primer extension arrest (Figure 3B). RESULTS +28 33 90 aa residue_range Even a Tsr3 fragment with a 90 aa C-terminal truncation showed a residual primer extension stop, whereas N-terminal truncations exceeding 46 aa almost completely abolished the primer extension arrest (Figure 3B). RESULTS +138 143 46 aa residue_range Even a Tsr3 fragment with a 90 aa C-terminal truncation showed a residual primer extension stop, whereas N-terminal truncations exceeding 46 aa almost completely abolished the primer extension arrest (Figure 3B). RESULTS +27 32 yeast taxonomy_domain Domain characterization of yeast Tsr3 and correlation of acp modification with late 18S rRNA processing steps. (A) Scheme of the TSR3 gene with truncation positions in the open reading frame. FIG +33 37 Tsr3 protein Domain characterization of yeast Tsr3 and correlation of acp modification with late 18S rRNA processing steps. (A) Scheme of the TSR3 gene with truncation positions in the open reading frame. FIG +57 60 acp chemical Domain characterization of yeast Tsr3 and correlation of acp modification with late 18S rRNA processing steps. (A) Scheme of the TSR3 gene with truncation positions in the open reading frame. FIG +84 92 18S rRNA chemical Domain characterization of yeast Tsr3 and correlation of acp modification with late 18S rRNA processing steps. (A) Scheme of the TSR3 gene with truncation positions in the open reading frame. FIG +129 133 TSR3 protein Domain characterization of yeast Tsr3 and correlation of acp modification with late 18S rRNA processing steps. (A) Scheme of the TSR3 gene with truncation positions in the open reading frame. FIG +0 4 TSR3 protein TSR3 fragments of different length were expressed under the native promotor from multicopy plasmids in a Δtsr3 deletion strain. FIG +105 110 Δtsr3 mutant TSR3 fragments of different length were expressed under the native promotor from multicopy plasmids in a Δtsr3 deletion strain. FIG +4 29 Primer extension analysis experimental_method (B) Primer extension analysis of 18S rRNA acp modification in yeast cells expressing the indicated TSR3 fragments. FIG +33 41 18S rRNA chemical (B) Primer extension analysis of 18S rRNA acp modification in yeast cells expressing the indicated TSR3 fragments. FIG +42 45 acp chemical (B) Primer extension analysis of 18S rRNA acp modification in yeast cells expressing the indicated TSR3 fragments. FIG +62 67 yeast taxonomy_domain (B) Primer extension analysis of 18S rRNA acp modification in yeast cells expressing the indicated TSR3 fragments. FIG +99 103 TSR3 protein (B) Primer extension analysis of 18S rRNA acp modification in yeast cells expressing the indicated TSR3 fragments. FIG +11 20 deletions experimental_method N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type. FIG +24 26 36 residue_range N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type. FIG +30 32 45 residue_range N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type. FIG +60 69 deletions experimental_method N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type. FIG +73 75 43 residue_range N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type. FIG +79 81 76 residue_range N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type. FIG +98 119 primer extension stop evidence N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type. FIG +138 147 wild type protein_state N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type. FIG +0 4 Tsr3 protein Tsr3 fragments 37–223 or 46–223 cause a nearly complete loss of the arrest signal. FIG +15 21 37–223 residue_range Tsr3 fragments 37–223 or 46–223 cause a nearly complete loss of the arrest signal. FIG +25 31 46–223 residue_range Tsr3 fragments 37–223 or 46–223 cause a nearly complete loss of the arrest signal. FIG +32 36 Tsr3 protein The box highlights the shortest Tsr3 fragment (aa 46–270) with wild type activity (strong primer extension block). (C) Northern blot analysis of 20S pre-rRNA accumulation. FIG +50 56 46–270 residue_range The box highlights the shortest Tsr3 fragment (aa 46–270) with wild type activity (strong primer extension block). (C) Northern blot analysis of 20S pre-rRNA accumulation. FIG +63 72 wild type protein_state The box highlights the shortest Tsr3 fragment (aa 46–270) with wild type activity (strong primer extension block). (C) Northern blot analysis of 20S pre-rRNA accumulation. FIG +90 112 primer extension block evidence The box highlights the shortest Tsr3 fragment (aa 46–270) with wild type activity (strong primer extension block). (C) Northern blot analysis of 20S pre-rRNA accumulation. FIG +119 132 Northern blot experimental_method The box highlights the shortest Tsr3 fragment (aa 46–270) with wild type activity (strong primer extension block). (C) Northern blot analysis of 20S pre-rRNA accumulation. FIG +145 157 20S pre-rRNA chemical The box highlights the shortest Tsr3 fragment (aa 46–270) with wild type activity (strong primer extension block). (C) Northern blot analysis of 20S pre-rRNA accumulation. FIG +7 15 20S rRNA chemical A weak 20S rRNA signal, indicating normal processing, is observed for Tsr3 fragment 46–270 (highlighted in a box) showing its functionality. FIG +70 74 Tsr3 protein A weak 20S rRNA signal, indicating normal processing, is observed for Tsr3 fragment 46–270 (highlighted in a box) showing its functionality. FIG +84 90 46–270 residue_range A weak 20S rRNA signal, indicating normal processing, is observed for Tsr3 fragment 46–270 (highlighted in a box) showing its functionality. FIG +52 57 Δtsr3 mutant Strong 20S rRNA accumulation similar to that of the Δtsr3 deletion is observed for Tsr3 fragments 37–223 or 46–223. FIG +58 66 deletion experimental_method Strong 20S rRNA accumulation similar to that of the Δtsr3 deletion is observed for Tsr3 fragments 37–223 or 46–223. FIG +83 87 Tsr3 protein Strong 20S rRNA accumulation similar to that of the Δtsr3 deletion is observed for Tsr3 fragments 37–223 or 46–223. FIG +98 104 37–223 residue_range Strong 20S rRNA accumulation similar to that of the Δtsr3 deletion is observed for Tsr3 fragments 37–223 or 46–223. FIG +108 114 46–223 residue_range Strong 20S rRNA accumulation similar to that of the Δtsr3 deletion is observed for Tsr3 fragments 37–223 or 46–223. FIG +10 18 archaeal taxonomy_domain Thus, the archaeal homologs correspond to the functional core of Tsr3. RESULTS +65 69 Tsr3 protein Thus, the archaeal homologs correspond to the functional core of Tsr3. RESULTS +44 48 Tsr3 protein In order to define the structural basis for Tsr3 function, homologs from thermophilic archaea were screened for crystallization. RESULTS +73 93 thermophilic archaea taxonomy_domain In order to define the structural basis for Tsr3 function, homologs from thermophilic archaea were screened for crystallization. RESULTS +112 127 crystallization experimental_method In order to define the structural basis for Tsr3 function, homologs from thermophilic archaea were screened for crystallization. RESULTS +14 22 archaeal taxonomy_domain We focused on archaeal species containing a putative Nep1 homolog suggesting that these species are in principle capable of synthesizing N1-methyl-N3-acp-pseudouridine. RESULTS +53 57 Nep1 protein We focused on archaeal species containing a putative Nep1 homolog suggesting that these species are in principle capable of synthesizing N1-methyl-N3-acp-pseudouridine. RESULTS +137 167 N1-methyl-N3-acp-pseudouridine chemical We focused on archaeal species containing a putative Nep1 homolog suggesting that these species are in principle capable of synthesizing N1-methyl-N3-acp-pseudouridine. RESULTS +17 25 crystals evidence Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +44 48 Tsr3 protein Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +71 83 crenarchaeal taxonomy_domain Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +92 115 Vulcanisaeta distributa species Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +117 123 VdTsr3 protein Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +129 152 Sulfolobus solfataricus species Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +154 160 SsTsr3 protein Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +179 185 VdTsr3 protein Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +196 202 SsTsr3 protein Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +222 228 ScTsr3 protein Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +229 240 core region structure_element Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +242 248 ScTsr3 protein Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +252 258 46–223 residue_range Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46–223). RESULTS +10 25 S. solfataricus species While for S. solfataricus the existence of a modified nucleotide of unknown chemical composition in the loop capping helix 31 of its 16S rRNA has been demonstrated, no information regarding rRNA modifications is yet available for V. distributa. RESULTS +54 64 nucleotide chemical While for S. solfataricus the existence of a modified nucleotide of unknown chemical composition in the loop capping helix 31 of its 16S rRNA has been demonstrated, no information regarding rRNA modifications is yet available for V. distributa. RESULTS +104 125 loop capping helix 31 structure_element While for S. solfataricus the existence of a modified nucleotide of unknown chemical composition in the loop capping helix 31 of its 16S rRNA has been demonstrated, no information regarding rRNA modifications is yet available for V. distributa. RESULTS +133 141 16S rRNA chemical While for S. solfataricus the existence of a modified nucleotide of unknown chemical composition in the loop capping helix 31 of its 16S rRNA has been demonstrated, no information regarding rRNA modifications is yet available for V. distributa. RESULTS +230 243 V. distributa species While for S. solfataricus the existence of a modified nucleotide of unknown chemical composition in the loop capping helix 31 of its 16S rRNA has been demonstrated, no information regarding rRNA modifications is yet available for V. distributa. RESULTS +0 8 Crystals evidence Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +12 18 VdTsr3 protein Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +63 71 crystals evidence Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +75 81 SsTsr3 protein Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +121 127 VdTsr3 protein Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +145 157 crystallized experimental_method Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +158 173 in complex with protein_state Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +174 184 endogenous protein_state Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +186 193 E. coli species Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +195 198 SAM chemical Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +231 237 SsTsr3 protein Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +238 246 crystals evidence Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +276 279 apo protein_state Crystals of VdTsr3 diffracted to a resolution of 1.6 Å whereas crystals of SsTsr3 diffracted to 2.25 Å. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state. RESULTS +4 13 structure evidence The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM). RESULTS +17 23 VdTsr3 protein The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM). RESULTS +49 96 single-wavelength anomalous diffraction phasing experimental_method The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM). RESULTS +98 104 Se-SAD experimental_method The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM). RESULTS +111 113 Se chemical The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM). RESULTS +138 154 selenomethionine chemical The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM). RESULTS +159 181 seleno-substituted SAM chemical The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM). RESULTS +4 13 structure evidence The structure of SsTsr3 was solved by molecular replacement using VdTsr3 as a search model (see Supplementary Table S1 for data collection and refinement statistics). RESULTS +17 23 SsTsr3 protein The structure of SsTsr3 was solved by molecular replacement using VdTsr3 as a search model (see Supplementary Table S1 for data collection and refinement statistics). RESULTS +38 59 molecular replacement experimental_method The structure of SsTsr3 was solved by molecular replacement using VdTsr3 as a search model (see Supplementary Table S1 for data collection and refinement statistics). RESULTS +66 72 VdTsr3 protein The structure of SsTsr3 was solved by molecular replacement using VdTsr3 as a search model (see Supplementary Table S1 for data collection and refinement statistics). RESULTS +4 13 structure evidence The structure of VdTsr3 can be divided into two domains (Figure 4A). RESULTS +17 23 VdTsr3 protein The structure of VdTsr3 can be divided into two domains (Figure 4A). RESULTS +4 21 N-terminal domain structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +26 30 1–92 residue_range The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +44 57 α/β-structure structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +76 110 five-stranded all-parallel β-sheet structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +145 148 β5↑ structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +149 152 β3↑ structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +153 156 β4↑ structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +157 160 β1↑ structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +161 164 β2↑ structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +170 175 loops structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +187 189 β1 structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +194 196 β2 structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +198 200 β3 structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +205 207 β4 structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +212 214 β4 structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +219 221 β5 structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +230 239 α-helices structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +240 242 α1 structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +244 246 α2 structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +251 253 α3 structure_element The N-terminal domain (aa 1–92) has a mixed α/β-structure centered around a five-stranded all-parallel β-sheet (Figure 4B) with the strand order β5↑-β3↑-β4↑-β1↑-β2↑. The loops connecting β1 and β2, β3 and β4 and β4 and β5 include α-helices α1, α2 and α3, respectively. RESULTS +4 8 loop structure_element The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. RESULTS +20 22 β2 structure_element The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. RESULTS +27 29 β3 structure_element The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. RESULTS +58 67 310-helix structure_element The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. RESULTS +69 76 Helices structure_element The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. RESULTS +77 79 α1 structure_element The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. RESULTS +84 86 α2 structure_element The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. RESULTS +118 139 five-stranded β-sheet structure_element The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. RESULTS +146 148 α3 structure_element The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. RESULTS +176 183 β-sheet structure_element The loop connecting β2 and β3 contains a single turn of a 310-helix. Helices α1 and α2 are located on one side of the five-stranded β-sheet while α3 packs against the opposite β-sheet surface. RESULTS +4 21 C-terminal domain structure_element The C-terminal domain (aa 93–184) has a globular all α-helical structure comprising α-helices α4 to α9. RESULTS +26 32 93–184 residue_range The C-terminal domain (aa 93–184) has a globular all α-helical structure comprising α-helices α4 to α9. RESULTS +40 72 globular all α-helical structure structure_element The C-terminal domain (aa 93–184) has a globular all α-helical structure comprising α-helices α4 to α9. RESULTS +84 93 α-helices structure_element The C-terminal domain (aa 93–184) has a globular all α-helical structure comprising α-helices α4 to α9. RESULTS +94 102 α4 to α9 structure_element The C-terminal domain (aa 93–184) has a globular all α-helical structure comprising α-helices α4 to α9. RESULTS +23 40 C-terminal domain structure_element Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects β-strand β3 and α-helix α2 of the N-terminal domain. RESULTS +42 47 92 aa residue_range Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects β-strand β3 and α-helix α2 of the N-terminal domain. RESULTS +88 92 loop structure_element Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects β-strand β3 and α-helix α2 of the N-terminal domain. RESULTS +108 116 β-strand structure_element Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects β-strand β3 and α-helix α2 of the N-terminal domain. RESULTS +117 119 β3 structure_element Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects β-strand β3 and α-helix α2 of the N-terminal domain. RESULTS +124 131 α-helix structure_element Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects β-strand β3 and α-helix α2 of the N-terminal domain. RESULTS +132 134 α2 structure_element Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects β-strand β3 and α-helix α2 of the N-terminal domain. RESULTS +142 159 N-terminal domain structure_element Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects β-strand β3 and α-helix α2 of the N-terminal domain. RESULTS +10 16 VdTsr3 protein Thus, the VdTsr3 structure contains a deep trefoil knot. RESULTS +17 26 structure evidence Thus, the VdTsr3 structure contains a deep trefoil knot. RESULTS +38 55 deep trefoil knot structure_element Thus, the VdTsr3 structure contains a deep trefoil knot. RESULTS +4 13 structure evidence The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. RESULTS +17 23 SsTsr3 protein The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. RESULTS +31 34 apo protein_state The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. RESULTS +68 74 VdTsr3 protein The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. RESULTS +95 99 RMSD evidence The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. RESULTS +180 189 structure evidence The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. RESULTS +241 248 α-helix structure_element The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. RESULTS +249 251 α8 structure_element The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. RESULTS +260 270 absence of protein_state The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. RESULTS +271 278 α-helix structure_element The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. RESULTS +279 281 α9 structure_element The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. RESULTS +285 291 SsTsr3 protein The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent Cα atoms of 1.1 Å. The only significant difference in the global structure of the two proteins is the presence of an extended α-helix α8 and the absence of α-helix α9 in SsTsr3. RESULTS +0 4 Tsr3 protein Tsr3 has a fold similar to SPOUT-class RNA methyltransferases. (A) Cartoon representation of the X-ray structure of VdTsr3 in two orientations. FIG +27 61 SPOUT-class RNA methyltransferases protein_type Tsr3 has a fold similar to SPOUT-class RNA methyltransferases. (A) Cartoon representation of the X-ray structure of VdTsr3 in two orientations. FIG +97 112 X-ray structure evidence Tsr3 has a fold similar to SPOUT-class RNA methyltransferases. (A) Cartoon representation of the X-ray structure of VdTsr3 in two orientations. FIG +116 122 VdTsr3 protein Tsr3 has a fold similar to SPOUT-class RNA methyltransferases. (A) Cartoon representation of the X-ray structure of VdTsr3 in two orientations. FIG +0 9 β-strands structure_element β-strands are colored in crimson whereas α-helices in the N-terminal domain are colored light blue and α-helices in the C-terminal domain are colored dark blue. FIG +41 50 α-helices structure_element β-strands are colored in crimson whereas α-helices in the N-terminal domain are colored light blue and α-helices in the C-terminal domain are colored dark blue. FIG +58 75 N-terminal domain structure_element β-strands are colored in crimson whereas α-helices in the N-terminal domain are colored light blue and α-helices in the C-terminal domain are colored dark blue. FIG +103 112 α-helices structure_element β-strands are colored in crimson whereas α-helices in the N-terminal domain are colored light blue and α-helices in the C-terminal domain are colored dark blue. FIG +120 137 C-terminal domain structure_element β-strands are colored in crimson whereas α-helices in the N-terminal domain are colored light blue and α-helices in the C-terminal domain are colored dark blue. FIG +10 30 S-adenosylmethionine chemical The bound S-adenosylmethionine is shown in a stick representation and colored by atom type. FIG +38 54 topological knot structure_element A red arrow marks the location of the topological knot in the structure. (B) Secondary structure representation of the VdTsr3 structure. FIG +62 71 structure evidence A red arrow marks the location of the topological knot in the structure. (B) Secondary structure representation of the VdTsr3 structure. FIG +119 125 VdTsr3 protein A red arrow marks the location of the topological knot in the structure. (B) Secondary structure representation of the VdTsr3 structure. FIG +126 135 structure evidence A red arrow marks the location of the topological knot in the structure. (B) Secondary structure representation of the VdTsr3 structure. FIG +44 68 Structural superposition experimental_method The color coding is the same as in (A). (C) Structural superposition of the X-ray structures of VdTsr3 in the SAM-bound state (red) and SsTsr3 (blue) in the apo state. FIG +76 92 X-ray structures evidence The color coding is the same as in (A). (C) Structural superposition of the X-ray structures of VdTsr3 in the SAM-bound state (red) and SsTsr3 (blue) in the apo state. FIG +96 102 VdTsr3 protein The color coding is the same as in (A). (C) Structural superposition of the X-ray structures of VdTsr3 in the SAM-bound state (red) and SsTsr3 (blue) in the apo state. FIG +110 119 SAM-bound protein_state The color coding is the same as in (A). (C) Structural superposition of the X-ray structures of VdTsr3 in the SAM-bound state (red) and SsTsr3 (blue) in the apo state. FIG +136 142 SsTsr3 protein The color coding is the same as in (A). (C) Structural superposition of the X-ray structures of VdTsr3 in the SAM-bound state (red) and SsTsr3 (blue) in the apo state. FIG +157 160 apo protein_state The color coding is the same as in (A). (C) Structural superposition of the X-ray structures of VdTsr3 in the SAM-bound state (red) and SsTsr3 (blue) in the apo state. FIG +21 28 α-helix structure_element The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +29 31 α8 structure_element The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +51 57 SsTsr3 protein The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +65 72 α-helix structure_element The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +73 75 α9 structure_element The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +101 107 VdTsr3 protein The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +165 173 S. pombe species The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +174 179 Trm10 protein The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +194 227 SPOUT-class RNA methyltransferase protein_type The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +257 261 Tsr3 protein The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +266 279 superposition experimental_method The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +287 293 VdTsr3 protein The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +298 303 Trm10 protein The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +304 320 X-ray structures evidence The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +334 359 Analytical gel filtration experimental_method The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +360 368 profiles evidence The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +373 379 VdTsr3 protein The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +390 396 SsTsr3 protein The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +432 441 monomeric oligomeric_state The locations of the α-helix α8 which is longer in SsTsr3 and of α-helix α9 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)—the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution. FIG +0 2 Vd species Vd, Vulcanisaeta distributa; Ss, Sulfolobus solfataricus. FIG +4 27 Vulcanisaeta distributa species Vd, Vulcanisaeta distributa; Ss, Sulfolobus solfataricus. FIG +29 31 Ss species Vd, Vulcanisaeta distributa; Ss, Sulfolobus solfataricus. FIG +33 56 Sulfolobus solfataricus species Vd, Vulcanisaeta distributa; Ss, Sulfolobus solfataricus. FIG +0 21 Structure predictions experimental_method Structure predictions suggested that Tsr3 might contain a so-called RLI domain which contains a ‘bacterial like’ ferredoxin fold and binds two iron-sulfur clusters through eight conserved cysteine residues. RESULTS +37 41 Tsr3 protein Structure predictions suggested that Tsr3 might contain a so-called RLI domain which contains a ‘bacterial like’ ferredoxin fold and binds two iron-sulfur clusters through eight conserved cysteine residues. RESULTS +68 78 RLI domain structure_element Structure predictions suggested that Tsr3 might contain a so-called RLI domain which contains a ‘bacterial like’ ferredoxin fold and binds two iron-sulfur clusters through eight conserved cysteine residues. RESULTS +97 128 bacterial like’ ferredoxin fold structure_element Structure predictions suggested that Tsr3 might contain a so-called RLI domain which contains a ‘bacterial like’ ferredoxin fold and binds two iron-sulfur clusters through eight conserved cysteine residues. RESULTS +178 187 conserved protein_state Structure predictions suggested that Tsr3 might contain a so-called RLI domain which contains a ‘bacterial like’ ferredoxin fold and binds two iron-sulfur clusters through eight conserved cysteine residues. RESULTS +188 196 cysteine residue_name Structure predictions suggested that Tsr3 might contain a so-called RLI domain which contains a ‘bacterial like’ ferredoxin fold and binds two iron-sulfur clusters through eight conserved cysteine residues. RESULTS +40 50 RLI-domain structure_element However, no structural similarity to an RLI-domain was detectable. RESULTS +54 83 alanine replacement mutations experimental_method This is in accordance with the functional analysis of alanine replacement mutations of cysteine residues in ScTsr3 (Supplementary Figure S3). RESULTS +87 95 cysteine residue_name This is in accordance with the functional analysis of alanine replacement mutations of cysteine residues in ScTsr3 (Supplementary Figure S3). RESULTS +108 114 ScTsr3 protein This is in accordance with the functional analysis of alanine replacement mutations of cysteine residues in ScTsr3 (Supplementary Figure S3). RESULTS +4 21 β-strand topology structure_element The β-strand topology and the deep C-terminal trefoil knot of archaeal Tsr3 are the structural hallmarks of the SPOUT-class RNA-methyltransferase fold. RESULTS +46 58 trefoil knot structure_element The β-strand topology and the deep C-terminal trefoil knot of archaeal Tsr3 are the structural hallmarks of the SPOUT-class RNA-methyltransferase fold. RESULTS +62 70 archaeal taxonomy_domain The β-strand topology and the deep C-terminal trefoil knot of archaeal Tsr3 are the structural hallmarks of the SPOUT-class RNA-methyltransferase fold. RESULTS +71 75 Tsr3 protein The β-strand topology and the deep C-terminal trefoil knot of archaeal Tsr3 are the structural hallmarks of the SPOUT-class RNA-methyltransferase fold. RESULTS +112 145 SPOUT-class RNA-methyltransferase protein_type The β-strand topology and the deep C-terminal trefoil knot of archaeal Tsr3 are the structural hallmarks of the SPOUT-class RNA-methyltransferase fold. RESULTS +47 58 DALI search experimental_method The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. RESULTS +66 88 tRNA methyltransferase protein_type The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. RESULTS +89 94 Trm10 protein The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. RESULTS +96 108 DALI Z-score evidence The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. RESULTS +150 152 G9 residue_name_number The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. RESULTS +153 155 A9 residue_name_number The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. RESULTS +164 172 archaeal taxonomy_domain The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. RESULTS +177 187 eukaryotic taxonomy_domain The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. RESULTS +188 193 tRNAs chemical The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. RESULTS +203 206 SAM chemical The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor. RESULTS +17 21 Tsr3 protein In comparison to Tsr3 the central β-sheet element of Trm10 is extended by one additional β-strand pairing to β2. RESULTS +34 49 β-sheet element structure_element In comparison to Tsr3 the central β-sheet element of Trm10 is extended by one additional β-strand pairing to β2. RESULTS +53 58 Trm10 protein In comparison to Tsr3 the central β-sheet element of Trm10 is extended by one additional β-strand pairing to β2. RESULTS +89 97 β-strand structure_element In comparison to Tsr3 the central β-sheet element of Trm10 is extended by one additional β-strand pairing to β2. RESULTS +109 111 β2 structure_element In comparison to Tsr3 the central β-sheet element of Trm10 is extended by one additional β-strand pairing to β2. RESULTS +17 29 trefoil knot structure_element Furthermore, the trefoil knot of Trm10 is not as deep as that of Tsr3 (Figure 4D). RESULTS +33 38 Trm10 protein Furthermore, the trefoil knot of Trm10 is not as deep as that of Tsr3 (Figure 4D). RESULTS +65 69 Tsr3 protein Furthermore, the trefoil knot of Trm10 is not as deep as that of Tsr3 (Figure 4D). RESULTS +15 19 Nep1 protein Interestingly, Nep1—the enzyme preceding Tsr3 in the biosynthetic pathway for the synthesis of m1acp3Ψ—also belongs to the SPOUT-class of RNA methyltransferases. RESULTS +41 45 Tsr3 protein Interestingly, Nep1—the enzyme preceding Tsr3 in the biosynthetic pathway for the synthesis of m1acp3Ψ—also belongs to the SPOUT-class of RNA methyltransferases. RESULTS +95 102 m1acp3Ψ chemical Interestingly, Nep1—the enzyme preceding Tsr3 in the biosynthetic pathway for the synthesis of m1acp3Ψ—also belongs to the SPOUT-class of RNA methyltransferases. RESULTS +123 160 SPOUT-class of RNA methyltransferases protein_type Interestingly, Nep1—the enzyme preceding Tsr3 in the biosynthetic pathway for the synthesis of m1acp3Ψ—also belongs to the SPOUT-class of RNA methyltransferases. RESULTS +45 49 Nep1 protein However, the structural similarities between Nep1 and Tsr3 (DALI Z-score 4.4) are less pronounced than between Tsr3 and Trm10. RESULTS +54 58 Tsr3 protein However, the structural similarities between Nep1 and Tsr3 (DALI Z-score 4.4) are less pronounced than between Tsr3 and Trm10. RESULTS +60 72 DALI Z-score evidence However, the structural similarities between Nep1 and Tsr3 (DALI Z-score 4.4) are less pronounced than between Tsr3 and Trm10. RESULTS +111 115 Tsr3 protein However, the structural similarities between Nep1 and Tsr3 (DALI Z-score 4.4) are less pronounced than between Tsr3 and Trm10. RESULTS +120 125 Trm10 protein However, the structural similarities between Nep1 and Tsr3 (DALI Z-score 4.4) are less pronounced than between Tsr3 and Trm10. RESULTS +5 39 SPOUT-class RNA-methyltransferases protein_type Most SPOUT-class RNA-methyltransferases are homodimers. RESULTS +44 54 homodimers oligomeric_state Most SPOUT-class RNA-methyltransferases are homodimers. RESULTS +23 28 Trm10 protein A notable exception is Trm10. RESULTS +0 14 Gel filtration experimental_method Gel filtration experiments with both VdTsr3 and SsTsr3 (Figure 4E) showed that both proteins are monomeric in solution thereby extending the structural similarities to Trm10. RESULTS +37 43 VdTsr3 protein Gel filtration experiments with both VdTsr3 and SsTsr3 (Figure 4E) showed that both proteins are monomeric in solution thereby extending the structural similarities to Trm10. RESULTS +48 54 SsTsr3 protein Gel filtration experiments with both VdTsr3 and SsTsr3 (Figure 4E) showed that both proteins are monomeric in solution thereby extending the structural similarities to Trm10. RESULTS +97 106 monomeric oligomeric_state Gel filtration experiments with both VdTsr3 and SsTsr3 (Figure 4E) showed that both proteins are monomeric in solution thereby extending the structural similarities to Trm10. RESULTS +168 173 Trm10 protein Gel filtration experiments with both VdTsr3 and SsTsr3 (Figure 4E) showed that both proteins are monomeric in solution thereby extending the structural similarities to Trm10. RESULTS +89 92 acp chemical So far, structural information is only available for one other enzyme that transfers the acp group from SAM to an RNA nucleotide. RESULTS +104 107 SAM chemical So far, structural information is only available for one other enzyme that transfers the acp group from SAM to an RNA nucleotide. RESULTS +114 117 RNA chemical So far, structural information is only available for one other enzyme that transfers the acp group from SAM to an RNA nucleotide. RESULTS +118 128 nucleotide chemical So far, structural information is only available for one other enzyme that transfers the acp group from SAM to an RNA nucleotide. RESULTS +13 17 Tyw2 protein This enzyme, Tyw2, is part of the biosynthesis pathway of wybutosine nucleotides in tRNAs. RESULTS +58 80 wybutosine nucleotides chemical This enzyme, Tyw2, is part of the biosynthesis pathway of wybutosine nucleotides in tRNAs. RESULTS +84 89 tRNAs chemical This enzyme, Tyw2, is part of the biosynthesis pathway of wybutosine nucleotides in tRNAs. RESULTS +54 58 Tsr3 protein However, there are no structural similarities between Tsr3 and Tyw2, which contains an all-parallel β-sheet of a different topology and no knot structure. RESULTS +63 67 Tyw2 protein However, there are no structural similarities between Tsr3 and Tyw2, which contains an all-parallel β-sheet of a different topology and no knot structure. RESULTS +87 107 all-parallel β-sheet structure_element However, there are no structural similarities between Tsr3 and Tyw2, which contains an all-parallel β-sheet of a different topology and no knot structure. RESULTS +139 153 knot structure structure_element However, there are no structural similarities between Tsr3 and Tyw2, which contains an all-parallel β-sheet of a different topology and no knot structure. RESULTS +9 13 Tyw2 protein Instead, Tyw2 has a fold typical for the class-I-or Rossmann-fold class of methyltransferases (Supplementary Figure S5B). RESULTS +41 93 class-I-or Rossmann-fold class of methyltransferases protein_type Instead, Tyw2 has a fold typical for the class-I-or Rossmann-fold class of methyltransferases (Supplementary Figure S5B). RESULTS +20 24 Tsr3 protein Cofactor binding of Tsr3 RESULTS +4 20 SAM-binding site site The SAM-binding site of Tsr3 is located in a deep crevice between the N- and C-terminal domains in the vicinity of the trefoil knot as typical for SPOUT-class RNA-methyltransferases (Figure 4A). RESULTS +24 28 Tsr3 protein The SAM-binding site of Tsr3 is located in a deep crevice between the N- and C-terminal domains in the vicinity of the trefoil knot as typical for SPOUT-class RNA-methyltransferases (Figure 4A). RESULTS +70 95 N- and C-terminal domains structure_element The SAM-binding site of Tsr3 is located in a deep crevice between the N- and C-terminal domains in the vicinity of the trefoil knot as typical for SPOUT-class RNA-methyltransferases (Figure 4A). RESULTS +119 131 trefoil knot structure_element The SAM-binding site of Tsr3 is located in a deep crevice between the N- and C-terminal domains in the vicinity of the trefoil knot as typical for SPOUT-class RNA-methyltransferases (Figure 4A). RESULTS +147 181 SPOUT-class RNA-methyltransferases protein_type The SAM-binding site of Tsr3 is located in a deep crevice between the N- and C-terminal domains in the vicinity of the trefoil knot as typical for SPOUT-class RNA-methyltransferases (Figure 4A). RESULTS +4 11 adenine chemical The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). RESULTS +50 64 hydrogen bonds bond_interaction The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). RESULTS +115 118 L93 residue_name_number The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). RESULTS +138 140 β5 structure_element The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). RESULTS +214 218 Y108 residue_name_number The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). RESULTS +234 238 loop structure_element The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). RESULTS +250 252 β5 structure_element The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). RESULTS +260 270 N-terminal structure_element The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). RESULTS +275 277 α4 structure_element The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). RESULTS +285 302 C-terminal domain structure_element The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding β5 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting β5 in the N-terminal and α4 in the C-terminal domain (Figure 5A). RESULTS +17 24 adenine chemical Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +33 36 SAM chemical Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +52 84 hydrophobic packing interactions bond_interaction Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +109 112 L45 residue_name_number Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +114 116 β3 structure_element Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +119 122 P47 residue_name_number Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +127 130 W73 residue_name_number Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +132 134 α3 structure_element Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +143 160 N-terminal domain structure_element Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +177 180 L93 residue_name_number Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +182 186 L110 residue_name_number Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +200 204 loop structure_element Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +216 218 β5 structure_element Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +223 225 α4 structure_element Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +231 235 A115 residue_name_number Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +237 239 α5 structure_element Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +248 265 C-terminal domain structure_element Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (β3), P47 and W73 (α3) in the N-terminal domain as well as with L93, L110 (both in the loop connecting β5 and α4) and A115 (α5) in the C-terminal domain. RESULTS +4 10 ribose chemical The ribose 2′ and 3′ hydroxyl groups of SAM are hydrogen bonded to the backbone carbonyl group of I69. RESULTS +40 43 SAM chemical The ribose 2′ and 3′ hydroxyl groups of SAM are hydrogen bonded to the backbone carbonyl group of I69. RESULTS +48 63 hydrogen bonded bond_interaction The ribose 2′ and 3′ hydroxyl groups of SAM are hydrogen bonded to the backbone carbonyl group of I69. RESULTS +98 101 I69 residue_name_number The ribose 2′ and 3′ hydroxyl groups of SAM are hydrogen bonded to the backbone carbonyl group of I69. RESULTS +4 7 acp chemical The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in α1 as well as the backbone amide group of T112 in α4 (C-terminal domain). RESULTS +22 25 SAM chemical The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in α1 as well as the backbone amide group of T112 in α4 (C-terminal domain). RESULTS +50 66 hydrogen bonding bond_interaction The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in α1 as well as the backbone amide group of T112 in α4 (C-terminal domain). RESULTS +151 154 T19 residue_name_number The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in α1 as well as the backbone amide group of T112 in α4 (C-terminal domain). RESULTS +158 160 α1 structure_element The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in α1 as well as the backbone amide group of T112 in α4 (C-terminal domain). RESULTS +200 204 T112 residue_name_number The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in α1 as well as the backbone amide group of T112 in α4 (C-terminal domain). RESULTS +208 210 α4 structure_element The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in α1 as well as the backbone amide group of T112 in α4 (C-terminal domain). RESULTS +212 229 C-terminal domain structure_element The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in α1 as well as the backbone amide group of T112 in α4 (C-terminal domain). RESULTS +38 41 SAM chemical Most importantly, the methyl group of SAM is buried in a hydrophobic pocket formed by the sidechains of W73 and A76 both located in α3 (Figure 5A and B). RESULTS +57 75 hydrophobic pocket site Most importantly, the methyl group of SAM is buried in a hydrophobic pocket formed by the sidechains of W73 and A76 both located in α3 (Figure 5A and B). RESULTS +104 107 W73 residue_name_number Most importantly, the methyl group of SAM is buried in a hydrophobic pocket formed by the sidechains of W73 and A76 both located in α3 (Figure 5A and B). RESULTS +112 115 A76 residue_name_number Most importantly, the methyl group of SAM is buried in a hydrophobic pocket formed by the sidechains of W73 and A76 both located in α3 (Figure 5A and B). RESULTS +132 134 α3 structure_element Most importantly, the methyl group of SAM is buried in a hydrophobic pocket formed by the sidechains of W73 and A76 both located in α3 (Figure 5A and B). RESULTS +0 3 W73 residue_name_number W73 is highly conserved in all known Tsr3 proteins, whereas A76 can be replaced by other hydrophobic amino acids. RESULTS +7 23 highly conserved protein_state W73 is highly conserved in all known Tsr3 proteins, whereas A76 can be replaced by other hydrophobic amino acids. RESULTS +37 50 Tsr3 proteins protein_type W73 is highly conserved in all known Tsr3 proteins, whereas A76 can be replaced by other hydrophobic amino acids. RESULTS +60 63 A76 residue_name_number W73 is highly conserved in all known Tsr3 proteins, whereas A76 can be replaced by other hydrophobic amino acids. RESULTS +101 112 amino acids chemical W73 is highly conserved in all known Tsr3 proteins, whereas A76 can be replaced by other hydrophobic amino acids. RESULTS +118 140 RNA-methyltransferases protein_type Consequently, the accessibility of this methyl group for a nucleophilic attack is strongly reduced in comparison with RNA-methyltransferases such as Trm10 (Figure 5B, C). RESULTS +149 154 Trm10 protein Consequently, the accessibility of this methyl group for a nucleophilic attack is strongly reduced in comparison with RNA-methyltransferases such as Trm10 (Figure 5B, C). RESULTS +17 20 acp chemical In contrast, the acp side chain of SAM is accessible for reactions in the Tsr3-bound state (Figure 5B). RESULTS +35 38 SAM chemical In contrast, the acp side chain of SAM is accessible for reactions in the Tsr3-bound state (Figure 5B). RESULTS +74 84 Tsr3-bound protein_state In contrast, the acp side chain of SAM is accessible for reactions in the Tsr3-bound state (Figure 5B). RESULTS +0 3 SAM chemical SAM-binding by Tsr3. FIG +15 19 Tsr3 protein SAM-binding by Tsr3. FIG +25 43 SAM-binding pocket site (A) Close-up view of the SAM-binding pocket of VdTsr3. FIG +47 53 VdTsr3 protein (A) Close-up view of the SAM-binding pocket of VdTsr3. FIG +48 54 sulfur chemical Nitrogen atoms are dark blue, oxygen atoms red, sulfur atoms orange, carbon atoms of the protein light blue and carbon atoms of SAM yellow. FIG +128 131 SAM chemical Nitrogen atoms are dark blue, oxygen atoms red, sulfur atoms orange, carbon atoms of the protein light blue and carbon atoms of SAM yellow. FIG +0 14 Hydrogen bonds bond_interaction Hydrogen bonds are indicated by dashed lines. FIG +33 36 acp chemical (B) Solvent accessibility of the acp group of SAM bound to VdTsr3. FIG +46 49 SAM chemical (B) Solvent accessibility of the acp group of SAM bound to VdTsr3. FIG +50 58 bound to protein_state (B) Solvent accessibility of the acp group of SAM bound to VdTsr3. FIG +59 65 VdTsr3 protein (B) Solvent accessibility of the acp group of SAM bound to VdTsr3. FIG +87 90 SAM chemical The solvent accessible surface of the protein is shown in semitransparent gray whereas SAM is show in a stick representation. FIG +53 56 acp chemical A red arrow indicates the reactive CH2-moiety of the acp group. (C) Solvent accessibility of the SAM methyl group for SAM bound to the RNA methyltransferase Trm10. FIG +97 100 SAM chemical A red arrow indicates the reactive CH2-moiety of the acp group. (C) Solvent accessibility of the SAM methyl group for SAM bound to the RNA methyltransferase Trm10. FIG +118 121 SAM chemical A red arrow indicates the reactive CH2-moiety of the acp group. (C) Solvent accessibility of the SAM methyl group for SAM bound to the RNA methyltransferase Trm10. FIG +122 130 bound to protein_state A red arrow indicates the reactive CH2-moiety of the acp group. (C) Solvent accessibility of the SAM methyl group for SAM bound to the RNA methyltransferase Trm10. FIG +135 156 RNA methyltransferase protein_type A red arrow indicates the reactive CH2-moiety of the acp group. (C) Solvent accessibility of the SAM methyl group for SAM bound to the RNA methyltransferase Trm10. FIG +157 162 Trm10 protein A red arrow indicates the reactive CH2-moiety of the acp group. (C) Solvent accessibility of the SAM methyl group for SAM bound to the RNA methyltransferase Trm10. FIG +0 5 Bound protein_state Bound SAM was modelled based on the X-ray structure of the Trm10/SAH-complex (pdb4jwf). FIG +6 9 SAM chemical Bound SAM was modelled based on the X-ray structure of the Trm10/SAH-complex (pdb4jwf). FIG +36 51 X-ray structure evidence Bound SAM was modelled based on the X-ray structure of the Trm10/SAH-complex (pdb4jwf). FIG +59 68 Trm10/SAH complex_assembly Bound SAM was modelled based on the X-ray structure of the Trm10/SAH-complex (pdb4jwf). FIG +26 29 SAM chemical A red arrow indicates the SAM methyl group. (D) Binding of SAM analogs to SsTsr3. FIG +59 62 SAM chemical A red arrow indicates the SAM methyl group. (D) Binding of SAM analogs to SsTsr3. FIG +74 80 SsTsr3 protein A red arrow indicates the SAM methyl group. (D) Binding of SAM analogs to SsTsr3. FIG +0 40 Tryptophan fluorescence quenching curves evidence Tryptophan fluorescence quenching curves upon addition of SAM (blue), 5′-methyl-thioadenosine (red) and SAH (black). FIG +58 61 SAM chemical Tryptophan fluorescence quenching curves upon addition of SAM (blue), 5′-methyl-thioadenosine (red) and SAH (black). FIG +70 93 5′-methyl-thioadenosine chemical Tryptophan fluorescence quenching curves upon addition of SAM (blue), 5′-methyl-thioadenosine (red) and SAH (black). FIG +104 107 SAH chemical Tryptophan fluorescence quenching curves upon addition of SAM (blue), 5′-methyl-thioadenosine (red) and SAH (black). FIG +15 30 14C-labeled SAM chemical (E) Binding of 14C-labeled SAM to SsTsr3. FIG +34 40 SsTsr3 protein (E) Binding of 14C-labeled SAM to SsTsr3. FIG +22 25 SAM chemical Radioactively labeled SAM is retained on a filter in the presence of SsTsr3. FIG +57 68 presence of protein_state Radioactively labeled SAM is retained on a filter in the presence of SsTsr3. FIG +69 75 SsTsr3 protein Radioactively labeled SAM is retained on a filter in the presence of SsTsr3. FIG +22 25 SAM chemical Addition of unlabeled SAM competes with the binding of labeled SAM. FIG +63 66 SAM chemical Addition of unlabeled SAM competes with the binding of labeled SAM. FIG +2 6 W66A mutant A W66A-mutant of SsTsr3 (W73 in VdTsr3) does not bind SAM. FIG +7 13 mutant protein_state A W66A-mutant of SsTsr3 (W73 in VdTsr3) does not bind SAM. FIG +17 23 SsTsr3 protein A W66A-mutant of SsTsr3 (W73 in VdTsr3) does not bind SAM. FIG +25 28 W73 residue_name_number A W66A-mutant of SsTsr3 (W73 in VdTsr3) does not bind SAM. FIG +32 38 VdTsr3 protein A W66A-mutant of SsTsr3 (W73 in VdTsr3) does not bind SAM. FIG +54 57 SAM chemical A W66A-mutant of SsTsr3 (W73 in VdTsr3) does not bind SAM. FIG +4 20 Primer extension experimental_method (F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in Δtsr3 cells expressing Tsr3-S62D, -E111A or –W114A. FIG +59 62 acp chemical (F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in Δtsr3 cells expressing Tsr3-S62D, -E111A or –W114A. FIG +79 84 yeast taxonomy_domain (F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in Δtsr3 cells expressing Tsr3-S62D, -E111A or –W114A. FIG +85 93 18S rRNA chemical (F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in Δtsr3 cells expressing Tsr3-S62D, -E111A or –W114A. FIG +97 102 Δtsr3 mutant (F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in Δtsr3 cells expressing Tsr3-S62D, -E111A or –W114A. FIG +120 129 Tsr3-S62D mutant (F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in Δtsr3 cells expressing Tsr3-S62D, -E111A or –W114A. FIG +131 137 -E111A mutant (F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in Δtsr3 cells expressing Tsr3-S62D, -E111A or –W114A. FIG +141 147 –W114A mutant (F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in Δtsr3 cells expressing Tsr3-S62D, -E111A or –W114A. FIG +23 35 20S pre-rRNA chemical This correlates with a 20S pre-rRNA accumulation comparable to the Δtsr3 deletion (right: northern blot). FIG +67 72 Δtsr3 mutant This correlates with a 20S pre-rRNA accumulation comparable to the Δtsr3 deletion (right: northern blot). FIG +90 103 northern blot experimental_method This correlates with a 20S pre-rRNA accumulation comparable to the Δtsr3 deletion (right: northern blot). FIG +0 11 3xHA tagged protein_state 3xHA tagged Tsr3 mutants are expressed comparable to the wild type as shown by western blot (lower left). FIG +12 16 Tsr3 protein 3xHA tagged Tsr3 mutants are expressed comparable to the wild type as shown by western blot (lower left). FIG +17 24 mutants protein_state 3xHA tagged Tsr3 mutants are expressed comparable to the wild type as shown by western blot (lower left). FIG +57 66 wild type protein_state 3xHA tagged Tsr3 mutants are expressed comparable to the wild type as shown by western blot (lower left). FIG +79 91 western blot experimental_method 3xHA tagged Tsr3 mutants are expressed comparable to the wild type as shown by western blot (lower left). FIG +0 18 Binding affinities evidence Binding affinities for SAM and its analogs 5′-methylthioadenosin and SAH to SsTsr3 were measured using tryptophan fluorescence quenching. RESULTS +23 26 SAM chemical Binding affinities for SAM and its analogs 5′-methylthioadenosin and SAH to SsTsr3 were measured using tryptophan fluorescence quenching. RESULTS +43 64 5′-methylthioadenosin chemical Binding affinities for SAM and its analogs 5′-methylthioadenosin and SAH to SsTsr3 were measured using tryptophan fluorescence quenching. RESULTS +69 72 SAH chemical Binding affinities for SAM and its analogs 5′-methylthioadenosin and SAH to SsTsr3 were measured using tryptophan fluorescence quenching. RESULTS +76 82 SsTsr3 protein Binding affinities for SAM and its analogs 5′-methylthioadenosin and SAH to SsTsr3 were measured using tryptophan fluorescence quenching. RESULTS +103 136 tryptophan fluorescence quenching experimental_method Binding affinities for SAM and its analogs 5′-methylthioadenosin and SAH to SsTsr3 were measured using tryptophan fluorescence quenching. RESULTS +0 6 VdTsr3 protein VdTsr3 could not be used in these experiments since we could not purify it in a stable SAM-free form. RESULTS +80 86 stable protein_state VdTsr3 could not be used in these experiments since we could not purify it in a stable SAM-free form. RESULTS +87 95 SAM-free protein_state VdTsr3 could not be used in these experiments since we could not purify it in a stable SAM-free form. RESULTS +0 6 SsTsr3 protein SsTsr3 bound SAM with a KD of 6.5 μM, which is similar to SAM-KD's reported for several SPOUT-class methyltransferases. RESULTS +7 12 bound protein_state SsTsr3 bound SAM with a KD of 6.5 μM, which is similar to SAM-KD's reported for several SPOUT-class methyltransferases. RESULTS +13 16 SAM chemical SsTsr3 bound SAM with a KD of 6.5 μM, which is similar to SAM-KD's reported for several SPOUT-class methyltransferases. RESULTS +24 26 KD evidence SsTsr3 bound SAM with a KD of 6.5 μM, which is similar to SAM-KD's reported for several SPOUT-class methyltransferases. RESULTS +58 66 SAM-KD's evidence SsTsr3 bound SAM with a KD of 6.5 μM, which is similar to SAM-KD's reported for several SPOUT-class methyltransferases. RESULTS +88 118 SPOUT-class methyltransferases protein_type SsTsr3 bound SAM with a KD of 6.5 μM, which is similar to SAM-KD's reported for several SPOUT-class methyltransferases. RESULTS +0 21 5′-methylthioadenosin chemical 5′-methylthioadenosin—the reaction product after the acp-transfer—binds only ∼2.5-fold weaker (KD = 16.7 μM) compared to SAM. RESULTS +53 56 acp chemical 5′-methylthioadenosin—the reaction product after the acp-transfer—binds only ∼2.5-fold weaker (KD = 16.7 μM) compared to SAM. RESULTS +121 124 SAM chemical 5′-methylthioadenosin—the reaction product after the acp-transfer—binds only ∼2.5-fold weaker (KD = 16.7 μM) compared to SAM. RESULTS +0 22 S-adenosylhomocysteine chemical S-adenosylhomocysteine which lacks the methyl group of SAM binds with significantly lower affinity (KD = 55.5 μM) (Figure 5D). RESULTS +55 58 SAM chemical S-adenosylhomocysteine which lacks the methyl group of SAM binds with significantly lower affinity (KD = 55.5 μM) (Figure 5D). RESULTS +90 98 affinity evidence S-adenosylhomocysteine which lacks the methyl group of SAM binds with significantly lower affinity (KD = 55.5 μM) (Figure 5D). RESULTS +100 102 KD evidence S-adenosylhomocysteine which lacks the methyl group of SAM binds with significantly lower affinity (KD = 55.5 μM) (Figure 5D). RESULTS +23 46 hydrophobic interaction bond_interaction This suggests that the hydrophobic interaction between SAM's methyl group and the hydrophobic pocket of Tsr3 is thermodynamically important for the interaction. RESULTS +55 58 SAM chemical This suggests that the hydrophobic interaction between SAM's methyl group and the hydrophobic pocket of Tsr3 is thermodynamically important for the interaction. RESULTS +82 100 hydrophobic pocket site This suggests that the hydrophobic interaction between SAM's methyl group and the hydrophobic pocket of Tsr3 is thermodynamically important for the interaction. RESULTS +104 108 Tsr3 protein This suggests that the hydrophobic interaction between SAM's methyl group and the hydrophobic pocket of Tsr3 is thermodynamically important for the interaction. RESULTS +31 45 hydrogen bonds bond_interaction On the other hand, the loss of hydrogen bonds between the acp sidechain carboxylate group and the protein appears to be thermodynamically less important but these hydrogen bonds might play a crucial role for the proper orientation of the cofactor side chain in the substrate binding pocket. RESULTS +58 61 acp chemical On the other hand, the loss of hydrogen bonds between the acp sidechain carboxylate group and the protein appears to be thermodynamically less important but these hydrogen bonds might play a crucial role for the proper orientation of the cofactor side chain in the substrate binding pocket. RESULTS +163 177 hydrogen bonds bond_interaction On the other hand, the loss of hydrogen bonds between the acp sidechain carboxylate group and the protein appears to be thermodynamically less important but these hydrogen bonds might play a crucial role for the proper orientation of the cofactor side chain in the substrate binding pocket. RESULTS +265 289 substrate binding pocket site On the other hand, the loss of hydrogen bonds between the acp sidechain carboxylate group and the protein appears to be thermodynamically less important but these hydrogen bonds might play a crucial role for the proper orientation of the cofactor side chain in the substrate binding pocket. RESULTS +15 19 W66A mutant Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E). RESULTS +20 28 mutation experimental_method Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E). RESULTS +30 33 W73 residue_name_number Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E). RESULTS +37 43 VdTsr3 protein Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E). RESULTS +48 54 SsTsr3 protein Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E). RESULTS +80 91 SAM-binding evidence Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E). RESULTS +97 117 filter binding assay experimental_method Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E). RESULTS +134 143 wild type protein_state Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E). RESULTS +15 30 W to A mutation experimental_method Furthermore, a W to A mutation at the equivalent position W114 in ScTsr3 strongly reduced the in vivo acp transferase activity (Figure 5F). RESULTS +58 62 W114 residue_name_number Furthermore, a W to A mutation at the equivalent position W114 in ScTsr3 strongly reduced the in vivo acp transferase activity (Figure 5F). RESULTS +66 72 ScTsr3 protein Furthermore, a W to A mutation at the equivalent position W114 in ScTsr3 strongly reduced the in vivo acp transferase activity (Figure 5F). RESULTS +102 117 acp transferase protein_type Furthermore, a W to A mutation at the equivalent position W114 in ScTsr3 strongly reduced the in vivo acp transferase activity (Figure 5F). RESULTS +33 36 T19 residue_name_number The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). RESULTS +67 70 SAM chemical The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). RESULTS +85 94 mutations experimental_method The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). RESULTS +98 101 T17 residue_name_number The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). RESULTS +103 106 T19 residue_name_number The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). RESULTS +110 116 VdTsr3 protein The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). RESULTS +128 129 A residue_name The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). RESULTS +133 134 D residue_name The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). RESULTS +171 191 SAM-binding affinity evidence The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). RESULTS +195 201 SsTsr3 protein The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). RESULTS +203 205 KD evidence The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively). RESULTS +16 24 mutation experimental_method Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F). RESULTS +52 55 S62 residue_name_number Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F). RESULTS +59 65 ScTsr3 protein Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F). RESULTS +69 70 D residue_name Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F). RESULTS +83 84 A residue_name Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F). RESULTS +106 109 acp chemical Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F). RESULTS +144 169 primer extension analysis experimental_method Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F). RESULTS +4 7 acp chemical The acp-transfer reaction catalyzed by Tsr3 most likely requires the presence of a catalytic base in order to abstract a proton from the N3 imino group of the modified pseudouridine. RESULTS +39 43 Tsr3 protein The acp-transfer reaction catalyzed by Tsr3 most likely requires the presence of a catalytic base in order to abstract a proton from the N3 imino group of the modified pseudouridine. RESULTS +168 181 pseudouridine chemical The acp-transfer reaction catalyzed by Tsr3 most likely requires the presence of a catalytic base in order to abstract a proton from the N3 imino group of the modified pseudouridine. RESULTS +18 21 D70 residue_name_number The side chain of D70 (VdTsr3) located in β4 is ∼5 Å away from the SAM sulfur atom. RESULTS +23 29 VdTsr3 protein The side chain of D70 (VdTsr3) located in β4 is ∼5 Å away from the SAM sulfur atom. RESULTS +42 44 β4 structure_element The side chain of D70 (VdTsr3) located in β4 is ∼5 Å away from the SAM sulfur atom. RESULTS +67 70 SAM chemical The side chain of D70 (VdTsr3) located in β4 is ∼5 Å away from the SAM sulfur atom. RESULTS +16 28 conserved as protein_state This residue is conserved as D or E both in archaeal and eukaryotic Tsr3 homologs. RESULTS +29 30 D residue_name This residue is conserved as D or E both in archaeal and eukaryotic Tsr3 homologs. RESULTS +34 35 E residue_name This residue is conserved as D or E both in archaeal and eukaryotic Tsr3 homologs. RESULTS +44 52 archaeal taxonomy_domain This residue is conserved as D or E both in archaeal and eukaryotic Tsr3 homologs. RESULTS +57 67 eukaryotic taxonomy_domain This residue is conserved as D or E both in archaeal and eukaryotic Tsr3 homologs. RESULTS +68 72 Tsr3 protein This residue is conserved as D or E both in archaeal and eukaryotic Tsr3 homologs. RESULTS +0 9 Mutations experimental_method Mutations of the corresponding residue in SsTsr3 to A (D63) does not significantly alter the SAM-binding affinity of the protein (KD = 11.0 μM). RESULTS +42 48 SsTsr3 protein Mutations of the corresponding residue in SsTsr3 to A (D63) does not significantly alter the SAM-binding affinity of the protein (KD = 11.0 μM). RESULTS +52 53 A residue_name Mutations of the corresponding residue in SsTsr3 to A (D63) does not significantly alter the SAM-binding affinity of the protein (KD = 11.0 μM). RESULTS +55 58 D63 residue_name_number Mutations of the corresponding residue in SsTsr3 to A (D63) does not significantly alter the SAM-binding affinity of the protein (KD = 11.0 μM). RESULTS +93 113 SAM-binding affinity evidence Mutations of the corresponding residue in SsTsr3 to A (D63) does not significantly alter the SAM-binding affinity of the protein (KD = 11.0 μM). RESULTS +130 132 KD evidence Mutations of the corresponding residue in SsTsr3 to A (D63) does not significantly alter the SAM-binding affinity of the protein (KD = 11.0 μM). RESULTS +13 21 mutation experimental_method However, the mutation of the corresponding residue of ScTsr3 (E111A) leads to a significant decrease of the acp transferase activity in vivo (Figure 5F). RESULTS +54 60 ScTsr3 protein However, the mutation of the corresponding residue of ScTsr3 (E111A) leads to a significant decrease of the acp transferase activity in vivo (Figure 5F). RESULTS +62 67 E111A mutant However, the mutation of the corresponding residue of ScTsr3 (E111A) leads to a significant decrease of the acp transferase activity in vivo (Figure 5F). RESULTS +108 123 acp transferase protein_type However, the mutation of the corresponding residue of ScTsr3 (E111A) leads to a significant decrease of the acp transferase activity in vivo (Figure 5F). RESULTS +0 3 RNA chemical RNA-binding of Tsr3 RESULTS +15 19 Tsr3 protein RNA-binding of Tsr3 RESULTS +0 48 Analysis of the electrostatic surface properties experimental_method Analysis of the electrostatic surface properties of VdTsr3 clearly identified positively charged surface patches in the vicinity of the SAM-binding site suggesting a putative RNA-binding site (Figure 6A). RESULTS +52 58 VdTsr3 protein Analysis of the electrostatic surface properties of VdTsr3 clearly identified positively charged surface patches in the vicinity of the SAM-binding site suggesting a putative RNA-binding site (Figure 6A). RESULTS +78 112 positively charged surface patches site Analysis of the electrostatic surface properties of VdTsr3 clearly identified positively charged surface patches in the vicinity of the SAM-binding site suggesting a putative RNA-binding site (Figure 6A). RESULTS +136 152 SAM-binding site site Analysis of the electrostatic surface properties of VdTsr3 clearly identified positively charged surface patches in the vicinity of the SAM-binding site suggesting a putative RNA-binding site (Figure 6A). RESULTS +175 191 RNA-binding site site Analysis of the electrostatic surface properties of VdTsr3 clearly identified positively charged surface patches in the vicinity of the SAM-binding site suggesting a putative RNA-binding site (Figure 6A). RESULTS +34 37 MES chemical Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix α1. RESULTS +58 75 crystal structure evidence Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix α1. RESULTS +79 85 VdTsr3 protein Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix α1. RESULTS +86 98 complexed to protein_state Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix α1. RESULTS +117 120 K22 residue_name_number Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix α1. RESULTS +124 129 helix structure_element Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix α1. RESULTS +130 132 α1 structure_element Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix α1. RESULTS +23 30 sulfate chemical Its negatively charged sulfate group might mimic an RNA backbone phosphate. RESULTS +52 55 RNA chemical Its negatively charged sulfate group might mimic an RNA backbone phosphate. RESULTS +0 5 Helix structure_element Helix α1 contains two more positively charged amino acids K17 and R25 as does the loop preceding it (R9). RESULTS +6 8 α1 structure_element Helix α1 contains two more positively charged amino acids K17 and R25 as does the loop preceding it (R9). RESULTS +58 61 K17 residue_name_number Helix α1 contains two more positively charged amino acids K17 and R25 as does the loop preceding it (R9). RESULTS +66 69 R25 residue_name_number Helix α1 contains two more positively charged amino acids K17 and R25 as does the loop preceding it (R9). RESULTS +82 86 loop structure_element Helix α1 contains two more positively charged amino acids K17 and R25 as does the loop preceding it (R9). RESULTS +101 103 R9 residue_name_number Helix α1 contains two more positively charged amino acids K17 and R25 as does the loop preceding it (R9). RESULTS +68 73 helix structure_element A second cluster of positively charged residues is found in or near helix α3 (K74, R75, K82, R85 and K87). RESULTS +74 76 α3 structure_element A second cluster of positively charged residues is found in or near helix α3 (K74, R75, K82, R85 and K87). RESULTS +78 81 K74 residue_name_number A second cluster of positively charged residues is found in or near helix α3 (K74, R75, K82, R85 and K87). RESULTS +83 86 R75 residue_name_number A second cluster of positively charged residues is found in or near helix α3 (K74, R75, K82, R85 and K87). RESULTS +88 91 K82 residue_name_number A second cluster of positively charged residues is found in or near helix α3 (K74, R75, K82, R85 and K87). RESULTS +93 96 R85 residue_name_number A second cluster of positively charged residues is found in or near helix α3 (K74, R75, K82, R85 and K87). RESULTS +101 104 K87 residue_name_number A second cluster of positively charged residues is found in or near helix α3 (K74, R75, K82, R85 and K87). RESULTS +30 39 conserved protein_state Some of these amino acids are conserved between archaeal and eukaryotic Tsr3 (Supplementary Figure S1A). RESULTS +48 56 archaeal taxonomy_domain Some of these amino acids are conserved between archaeal and eukaryotic Tsr3 (Supplementary Figure S1A). RESULTS +61 71 eukaryotic taxonomy_domain Some of these amino acids are conserved between archaeal and eukaryotic Tsr3 (Supplementary Figure S1A). RESULTS +72 76 Tsr3 protein Some of these amino acids are conserved between archaeal and eukaryotic Tsr3 (Supplementary Figure S1A). RESULTS +7 24 C-terminal domain structure_element In the C-terminal domain, the surface exposed α-helices α5 and α7 carry a significant amount of positively charged amino acids. RESULTS +46 55 α-helices structure_element In the C-terminal domain, the surface exposed α-helices α5 and α7 carry a significant amount of positively charged amino acids. RESULTS +56 58 α5 structure_element In the C-terminal domain, the surface exposed α-helices α5 and α7 carry a significant amount of positively charged amino acids. RESULTS +63 65 α7 structure_element In the C-terminal domain, the surface exposed α-helices α5 and α7 carry a significant amount of positively charged amino acids. RESULTS +2 17 triple mutation experimental_method A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues. RESULTS +25 34 conserved protein_state A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues. RESULTS +63 66 R60 residue_name_number A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues. RESULTS +68 71 K65 residue_name_number A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues. RESULTS +76 80 R131 residue_name_number A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues. RESULTS +84 85 A residue_name A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues. RESULTS +89 95 ScTsr3 protein A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues. RESULTS +148 163 acp transferase protein_type A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues. RESULTS +15 19 Tsr3 protein RNA-binding of Tsr3. FIG +56 62 VdTsr3 protein (A) Electrostatic charge distribution on the surface of VdTsr3. FIG +0 3 SAM chemical SAM is shown in a stick representation. FIG +59 62 MES chemical Also shown in stick representation is a negatively charged MES ion. FIG +0 9 Conserved protein_state Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red. FIG +16 27 amino acids chemical Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red. FIG +87 95 helix 31 structure_element Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red. FIG +129 134 rRNAs chemical Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red. FIG +138 151 S. cerevisiae species Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red. FIG +156 171 S. solfataricus species Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red. FIG +197 210 hypermodified protein_state Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red. FIG +211 221 nucleotide chemical Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red. FIG +4 19 S. solfataricus species For S. solfataricus the chemical identity of the hypermodified nucleotide is not known but the existence of NEP1 and TSR3 homologs suggest that it is indeed N1-methyl-N3-acp-pseudouridine. FIG +49 62 hypermodified protein_state For S. solfataricus the chemical identity of the hypermodified nucleotide is not known but the existence of NEP1 and TSR3 homologs suggest that it is indeed N1-methyl-N3-acp-pseudouridine. FIG +63 73 nucleotide chemical For S. solfataricus the chemical identity of the hypermodified nucleotide is not known but the existence of NEP1 and TSR3 homologs suggest that it is indeed N1-methyl-N3-acp-pseudouridine. FIG +108 112 NEP1 protein For S. solfataricus the chemical identity of the hypermodified nucleotide is not known but the existence of NEP1 and TSR3 homologs suggest that it is indeed N1-methyl-N3-acp-pseudouridine. FIG +117 121 TSR3 protein For S. solfataricus the chemical identity of the hypermodified nucleotide is not known but the existence of NEP1 and TSR3 homologs suggest that it is indeed N1-methyl-N3-acp-pseudouridine. FIG +157 187 N1-methyl-N3-acp-pseudouridine chemical For S. solfataricus the chemical identity of the hypermodified nucleotide is not known but the existence of NEP1 and TSR3 homologs suggest that it is indeed N1-methyl-N3-acp-pseudouridine. FIG +15 21 SsTsr3 protein (C) Binding of SsTsr3 to RNA. FIG +25 28 RNA chemical (C) Binding of SsTsr3 to RNA. FIG +3 15 fluoresceine chemical 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +24 27 RNA chemical 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +73 79 native protein_state 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +81 86 20mer oligomeric_state 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +105 115 stabilized protein_state 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +117 125 20mer_GC oligomeric_state 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +135 143 helix 31 structure_element 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +175 179 rRNA chemical 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +185 200 S. solfataricus species 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +206 238 titrated with increasing amounts experimental_method 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +242 248 SsTsr3 protein 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +272 284 fluoresceine chemical 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +285 308 fluorescence anisotropy evidence 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +339 352 binding curve evidence 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +354 359 20mer oligomeric_state 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +367 375 20mer_GC oligomeric_state 5′-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer – see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer – red, 20mer_GC – blue). FIG +0 12 Oligo-U9-RNA chemical Oligo-U9-RNA was used for comparison (black). FIG +4 12 20mer_GC oligomeric_state The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +13 16 RNA chemical The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +26 34 titrated experimental_method The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +40 46 SsTsr3 protein The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +71 74 SAM chemical The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +89 96 Mutants protein_state The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +100 106 ScTsr3 protein The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +107 110 R60 residue_name_number The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +112 115 K65 residue_name_number The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +119 123 R131 residue_name_number The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +139 142 K17 residue_name_number The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +144 147 K22 residue_name_number The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +152 155 R91 residue_name_number The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +159 165 VdTsr3 protein The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +167 176 expressed experimental_method The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +180 185 Δtsr3 mutant The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +186 191 yeast taxonomy_domain The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +205 226 primer extension stop evidence The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +245 254 wild type protein_state The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in Δtsr3 yeast cells show a primer extension stop comparable to the wild type. FIG +0 40 Combination of the three point mutations experimental_method Combination of the three point mutations (R60A/K65A/R131A) leads to a strongly reduced acp modification of 18S rRNA. FIG +42 46 R60A mutant Combination of the three point mutations (R60A/K65A/R131A) leads to a strongly reduced acp modification of 18S rRNA. FIG +47 51 K65A mutant Combination of the three point mutations (R60A/K65A/R131A) leads to a strongly reduced acp modification of 18S rRNA. FIG +52 57 R131A mutant Combination of the three point mutations (R60A/K65A/R131A) leads to a strongly reduced acp modification of 18S rRNA. FIG +87 90 acp chemical Combination of the three point mutations (R60A/K65A/R131A) leads to a strongly reduced acp modification of 18S rRNA. FIG +107 115 18S rRNA chemical Combination of the three point mutations (R60A/K65A/R131A) leads to a strongly reduced acp modification of 18S rRNA. FIG +50 54 Tsr3 protein In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5′-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements. RESULTS +58 66 titrated experimental_method In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5′-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements. RESULTS +67 73 SsTsr3 protein In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5′-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements. RESULTS +86 96 RNase-free protein_state In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5′-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements. RESULTS +110 122 fluoresceine chemical In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5′-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements. RESULTS +131 134 RNA chemical In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5′-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements. RESULTS +154 162 affinity evidence In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5′-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements. RESULTS +166 202 fluorescence anisotropy measurements experimental_method In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5′-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements. RESULTS +0 6 SsTsr3 protein SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). RESULTS +14 17 apo protein_state SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). RESULTS +24 29 bound protein_state SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). RESULTS +32 37 20mer oligomeric_state SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). RESULTS +38 41 RNA chemical SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). RESULTS +59 67 helix 31 structure_element SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). RESULTS +71 86 S. solfataricus species SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). RESULTS +87 95 16S rRNA chemical SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). RESULTS +115 117 KD evidence SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). RESULTS +153 160 hairpin structure_element SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). RESULTS +201 209 20mer-GC oligomeric_state SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). RESULTS +218 220 KD evidence SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 μM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 μM (Figure 6C). RESULTS +18 28 oligoU-RNA chemical A single stranded oligoU-RNA bound with a 10-fold-reduced affinity (6.0 μM). RESULTS +29 34 bound protein_state A single stranded oligoU-RNA bound with a 10-fold-reduced affinity (6.0 μM). RESULTS +58 66 affinity evidence A single stranded oligoU-RNA bound with a 10-fold-reduced affinity (6.0 μM). RESULTS +38 41 SAM chemical The presence of saturating amounts of SAM (2 mM) did not have a significant influence on the RNA-affinity of SsTsr3 (KD of 1.7 μM for the 20mer-GC-RNA) suggesting no cooperativity in substrate binding. RESULTS +93 105 RNA-affinity evidence The presence of saturating amounts of SAM (2 mM) did not have a significant influence on the RNA-affinity of SsTsr3 (KD of 1.7 μM for the 20mer-GC-RNA) suggesting no cooperativity in substrate binding. RESULTS +109 115 SsTsr3 protein The presence of saturating amounts of SAM (2 mM) did not have a significant influence on the RNA-affinity of SsTsr3 (KD of 1.7 μM for the 20mer-GC-RNA) suggesting no cooperativity in substrate binding. RESULTS +117 119 KD evidence The presence of saturating amounts of SAM (2 mM) did not have a significant influence on the RNA-affinity of SsTsr3 (KD of 1.7 μM for the 20mer-GC-RNA) suggesting no cooperativity in substrate binding. RESULTS +138 146 20mer-GC oligomeric_state The presence of saturating amounts of SAM (2 mM) did not have a significant influence on the RNA-affinity of SsTsr3 (KD of 1.7 μM for the 20mer-GC-RNA) suggesting no cooperativity in substrate binding. RESULTS +147 150 RNA chemical The presence of saturating amounts of SAM (2 mM) did not have a significant influence on the RNA-affinity of SsTsr3 (KD of 1.7 μM for the 20mer-GC-RNA) suggesting no cooperativity in substrate binding. RESULTS +0 5 U1191 residue_name_number U1191 is the only hypermodified base in the yeast 18S rRNA and is strongly conserved in eukaryotes. DISCUSS +18 31 hypermodified protein_state U1191 is the only hypermodified base in the yeast 18S rRNA and is strongly conserved in eukaryotes. DISCUSS +44 49 yeast taxonomy_domain U1191 is the only hypermodified base in the yeast 18S rRNA and is strongly conserved in eukaryotes. DISCUSS +50 58 18S rRNA chemical U1191 is the only hypermodified base in the yeast 18S rRNA and is strongly conserved in eukaryotes. DISCUSS +66 84 strongly conserved protein_state U1191 is the only hypermodified base in the yeast 18S rRNA and is strongly conserved in eukaryotes. DISCUSS +88 98 eukaryotes taxonomy_domain U1191 is the only hypermodified base in the yeast 18S rRNA and is strongly conserved in eukaryotes. DISCUSS +17 67 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine chemical The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3). DISCUSS +69 76 m1acp3Ψ chemical The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3). DISCUSS +158 163 H/ACA structure_element The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3). DISCUSS +164 170 snoRNP complex_assembly The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3). DISCUSS +172 177 snR35 protein The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3). DISCUSS +204 208 Nep1 protein The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3). DISCUSS +209 213 Emg1 protein The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3). DISCUSS +218 222 Tsr3 protein The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3Ψ) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3). DISCUSS +24 34 eukaryotic taxonomy_domain This makes it unique in eukaryotic rRNA modification. DISCUSS +35 39 rRNA chemical This makes it unique in eukaryotic rRNA modification. DISCUSS +4 11 m1acp3Ψ chemical The m1acp3Ψ base is located at the tip of helix 31 on the 18S rRNA (Supplementary Figure S1B) which, together with helices 18, 24, 34 and 44, contribute to building the decoding center of the small ribosomal subunit. DISCUSS +42 50 helix 31 structure_element The m1acp3Ψ base is located at the tip of helix 31 on the 18S rRNA (Supplementary Figure S1B) which, together with helices 18, 24, 34 and 44, contribute to building the decoding center of the small ribosomal subunit. DISCUSS +58 66 18S rRNA chemical The m1acp3Ψ base is located at the tip of helix 31 on the 18S rRNA (Supplementary Figure S1B) which, together with helices 18, 24, 34 and 44, contribute to building the decoding center of the small ribosomal subunit. DISCUSS +115 140 helices 18, 24, 34 and 44 structure_element The m1acp3Ψ base is located at the tip of helix 31 on the 18S rRNA (Supplementary Figure S1B) which, together with helices 18, 24, 34 and 44, contribute to building the decoding center of the small ribosomal subunit. DISCUSS +24 29 acp3U chemical A similar modification (acp3U) was identified in Haloferax volcanii and corresponding modified nucleotides were also shown to occur in other archaea. DISCUSS +49 67 Haloferax volcanii species A similar modification (acp3U) was identified in Haloferax volcanii and corresponding modified nucleotides were also shown to occur in other archaea. DISCUSS +95 106 nucleotides chemical A similar modification (acp3U) was identified in Haloferax volcanii and corresponding modified nucleotides were also shown to occur in other archaea. DISCUSS +141 148 archaea taxonomy_domain A similar modification (acp3U) was identified in Haloferax volcanii and corresponding modified nucleotides were also shown to occur in other archaea. DISCUSS +14 18 TSR3 protein As shown here TSR3 encodes the transferase catalyzing the acp modification as the last step in the biosynthesis of m1acp3Ψ in yeast and human cells. DISCUSS +58 61 acp chemical As shown here TSR3 encodes the transferase catalyzing the acp modification as the last step in the biosynthesis of m1acp3Ψ in yeast and human cells. DISCUSS +115 122 m1acp3Ψ chemical As shown here TSR3 encodes the transferase catalyzing the acp modification as the last step in the biosynthesis of m1acp3Ψ in yeast and human cells. DISCUSS +126 131 yeast taxonomy_domain As shown here TSR3 encodes the transferase catalyzing the acp modification as the last step in the biosynthesis of m1acp3Ψ in yeast and human cells. DISCUSS +136 141 human species As shown here TSR3 encodes the transferase catalyzing the acp modification as the last step in the biosynthesis of m1acp3Ψ in yeast and human cells. DISCUSS +14 22 archaeal taxonomy_domain Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. DISCUSS +23 27 Tsr3 protein Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. DISCUSS +34 43 structure evidence Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. DISCUSS +55 89 SPOUT-class RNA methyltransferases protein_type Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. DISCUSS +163 166 acp chemical Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. DISCUSS +192 210 SAM-binding pocket site Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. DISCUSS +228 231 acp chemical Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. DISCUSS +263 266 SAM chemical Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. DISCUSS +274 277 RNA chemical Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate. DISCUSS +38 72 Rossmann-fold Tyw2 acp transferase protein_type Similar to the structurally unrelated Rossmann-fold Tyw2 acp transferase, the SAM methyl group of Tsr3 is bound in an inaccessible hydrophobic pocket whereas the acp side chain becomes accessible for a nucleophilic attack by the N3 of pseudouridine. DISCUSS +78 81 SAM chemical Similar to the structurally unrelated Rossmann-fold Tyw2 acp transferase, the SAM methyl group of Tsr3 is bound in an inaccessible hydrophobic pocket whereas the acp side chain becomes accessible for a nucleophilic attack by the N3 of pseudouridine. DISCUSS +98 102 Tsr3 protein Similar to the structurally unrelated Rossmann-fold Tyw2 acp transferase, the SAM methyl group of Tsr3 is bound in an inaccessible hydrophobic pocket whereas the acp side chain becomes accessible for a nucleophilic attack by the N3 of pseudouridine. DISCUSS +131 149 hydrophobic pocket site Similar to the structurally unrelated Rossmann-fold Tyw2 acp transferase, the SAM methyl group of Tsr3 is bound in an inaccessible hydrophobic pocket whereas the acp side chain becomes accessible for a nucleophilic attack by the N3 of pseudouridine. DISCUSS +162 165 acp chemical Similar to the structurally unrelated Rossmann-fold Tyw2 acp transferase, the SAM methyl group of Tsr3 is bound in an inaccessible hydrophobic pocket whereas the acp side chain becomes accessible for a nucleophilic attack by the N3 of pseudouridine. DISCUSS +235 248 pseudouridine chemical Similar to the structurally unrelated Rossmann-fold Tyw2 acp transferase, the SAM methyl group of Tsr3 is bound in an inaccessible hydrophobic pocket whereas the acp side chain becomes accessible for a nucleophilic attack by the N3 of pseudouridine. DISCUSS +49 70 RNA methyltransferase protein_type In contrast, in the structurally closely related RNA methyltransferase Trm10 the methyl group of the cofactor SAM is accessible whereas its acp side chain is buried inside the protein. DISCUSS +71 76 Trm10 protein In contrast, in the structurally closely related RNA methyltransferase Trm10 the methyl group of the cofactor SAM is accessible whereas its acp side chain is buried inside the protein. DISCUSS +110 113 SAM chemical In contrast, in the structurally closely related RNA methyltransferase Trm10 the methyl group of the cofactor SAM is accessible whereas its acp side chain is buried inside the protein. DISCUSS +140 143 acp chemical In contrast, in the structurally closely related RNA methyltransferase Trm10 the methyl group of the cofactor SAM is accessible whereas its acp side chain is buried inside the protein. DISCUSS +34 63 SAM-dependent acp transferase protein_type This suggests that enzymes with a SAM-dependent acp transferase activity might have evolved from SAM-dependent methyltransferases by slight modifications of the SAM-binding pocket. DISCUSS +97 129 SAM-dependent methyltransferases protein_type This suggests that enzymes with a SAM-dependent acp transferase activity might have evolved from SAM-dependent methyltransferases by slight modifications of the SAM-binding pocket. DISCUSS +161 179 SAM-binding pocket site This suggests that enzymes with a SAM-dependent acp transferase activity might have evolved from SAM-dependent methyltransferases by slight modifications of the SAM-binding pocket. DISCUSS +30 45 acp transferase protein_type Thus, additional examples for acp transferase enzymes might be found with similarities to other structural classes of methyltransferases. DISCUSS +118 136 methyltransferases protein_type Thus, additional examples for acp transferase enzymes might be found with similarities to other structural classes of methyltransferases. DISCUSS +15 19 Nep1 protein In contrast to Nep1, the enzyme preceding Tsr3 in the m1acp3Ψ biosynthesis pathway, Tsr3 binds rather weakly and with little specificity to its isolated substrate RNA. DISCUSS +42 46 Tsr3 protein In contrast to Nep1, the enzyme preceding Tsr3 in the m1acp3Ψ biosynthesis pathway, Tsr3 binds rather weakly and with little specificity to its isolated substrate RNA. DISCUSS +54 61 m1acp3Ψ chemical In contrast to Nep1, the enzyme preceding Tsr3 in the m1acp3Ψ biosynthesis pathway, Tsr3 binds rather weakly and with little specificity to its isolated substrate RNA. DISCUSS +84 88 Tsr3 protein In contrast to Nep1, the enzyme preceding Tsr3 in the m1acp3Ψ biosynthesis pathway, Tsr3 binds rather weakly and with little specificity to its isolated substrate RNA. DISCUSS +163 166 RNA chemical In contrast to Nep1, the enzyme preceding Tsr3 in the m1acp3Ψ biosynthesis pathway, Tsr3 binds rather weakly and with little specificity to its isolated substrate RNA. DISCUSS +19 23 Tsr3 protein This suggests that Tsr3 is not stably incorporated into pre-ribosomal particles and that its binding to the nascent ribosomal subunit possibly requires additional interactions with other pre-ribosomal components. DISCUSS +56 79 pre-ribosomal particles complex_assembly This suggests that Tsr3 is not stably incorporated into pre-ribosomal particles and that its binding to the nascent ribosomal subunit possibly requires additional interactions with other pre-ribosomal components. DISCUSS +116 133 ribosomal subunit complex_assembly This suggests that Tsr3 is not stably incorporated into pre-ribosomal particles and that its binding to the nascent ribosomal subunit possibly requires additional interactions with other pre-ribosomal components. DISCUSS +17 42 sucrose gradient analysis experimental_method Consistently, in sucrose gradient analysis, Tsr3 was found in low-molecular weight fractions rather than with pre-ribosome containing high-molecular weight fractions. DISCUSS +44 48 Tsr3 protein Consistently, in sucrose gradient analysis, Tsr3 was found in low-molecular weight fractions rather than with pre-ribosome containing high-molecular weight fractions. DISCUSS +110 122 pre-ribosome complex_assembly Consistently, in sucrose gradient analysis, Tsr3 was found in low-molecular weight fractions rather than with pre-ribosome containing high-molecular weight fractions. DISCUSS +76 81 rRNAs chemical In contrast to several enzymes that catalyze base specific modifications in rRNAs Tsr3 is not an essential protein. DISCUSS +82 86 Tsr3 protein In contrast to several enzymes that catalyze base specific modifications in rRNAs Tsr3 is not an essential protein. DISCUSS +17 54 small subunit rRNA methyltransferases protein_type Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7). DISCUSS +58 62 Dim1 protein Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7). DISCUSS +64 69 Bud23 protein Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7). DISCUSS +74 78 Nep1 protein Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7). DISCUSS +79 83 Emg1 protein Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7). DISCUSS +133 137 rRNA chemical Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7). DISCUSS +183 190 pre-RNA chemical Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7). DISCUSS +25 29 Tsr3 protein In contrast, for several Tsr3 mutants (SAM-binding and cysteine mutations) we found a systematic correlation between the loss of acp modification and the efficiency of 18S rRNA maturation. DISCUSS +39 50 SAM-binding protein_state In contrast, for several Tsr3 mutants (SAM-binding and cysteine mutations) we found a systematic correlation between the loss of acp modification and the efficiency of 18S rRNA maturation. DISCUSS +55 73 cysteine mutations protein_state In contrast, for several Tsr3 mutants (SAM-binding and cysteine mutations) we found a systematic correlation between the loss of acp modification and the efficiency of 18S rRNA maturation. DISCUSS +129 132 acp chemical In contrast, for several Tsr3 mutants (SAM-binding and cysteine mutations) we found a systematic correlation between the loss of acp modification and the efficiency of 18S rRNA maturation. DISCUSS +168 176 18S rRNA chemical In contrast, for several Tsr3 mutants (SAM-binding and cysteine mutations) we found a systematic correlation between the loss of acp modification and the efficiency of 18S rRNA maturation. DISCUSS +55 59 rRNA chemical This demonstrates that, unlike the other small subunit rRNA base modifications, the acp modification is required for efficient pre-rRNA processing. DISCUSS +84 87 acp chemical This demonstrates that, unlike the other small subunit rRNA base modifications, the acp modification is required for efficient pre-rRNA processing. DISCUSS +127 135 pre-rRNA chemical This demonstrates that, unlike the other small subunit rRNA base modifications, the acp modification is required for efficient pre-rRNA processing. DISCUSS +10 88 structural, functional, and CRAC (cross-linking and cDNA analysis) experiments experimental_method Recently, structural, functional, and CRAC (cross-linking and cDNA analysis) experiments of late assembly factors involved in cytoplasmic processing of 40S subunits, along with cryo-EM studies of the late pre-40S subunits have provided important insights into late pre-40S processing. DISCUSS +152 164 40S subunits complex_assembly Recently, structural, functional, and CRAC (cross-linking and cDNA analysis) experiments of late assembly factors involved in cytoplasmic processing of 40S subunits, along with cryo-EM studies of the late pre-40S subunits have provided important insights into late pre-40S processing. DISCUSS +177 184 cryo-EM experimental_method Recently, structural, functional, and CRAC (cross-linking and cDNA analysis) experiments of late assembly factors involved in cytoplasmic processing of 40S subunits, along with cryo-EM studies of the late pre-40S subunits have provided important insights into late pre-40S processing. DISCUSS +200 204 late protein_state Recently, structural, functional, and CRAC (cross-linking and cDNA analysis) experiments of late assembly factors involved in cytoplasmic processing of 40S subunits, along with cryo-EM studies of the late pre-40S subunits have provided important insights into late pre-40S processing. DISCUSS +205 221 pre-40S subunits complex_assembly Recently, structural, functional, and CRAC (cross-linking and cDNA analysis) experiments of late assembly factors involved in cytoplasmic processing of 40S subunits, along with cryo-EM studies of the late pre-40S subunits have provided important insights into late pre-40S processing. DISCUSS +265 272 pre-40S complex_assembly Recently, structural, functional, and CRAC (cross-linking and cDNA analysis) experiments of late assembly factors involved in cytoplasmic processing of 40S subunits, along with cryo-EM studies of the late pre-40S subunits have provided important insights into late pre-40S processing. DISCUSS +55 72 pre-40S particles complex_assembly Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. DISCUSS +81 89 20S rRNA chemical Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. DISCUSS +109 131 non-ribosomal proteins protein_type Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. DISCUSS +146 165 D-site endonuclease protein_type Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. DISCUSS +166 170 Nob1 protein Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. DISCUSS +182 186 Tsr1 protein Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. DISCUSS +199 205 GTPase protein_type Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. DISCUSS +210 214 Rio2 protein Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. DISCUSS +231 243 mRNA channel site Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. DISCUSS +252 279 initiator tRNA binding site site Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation. DISCUSS +45 48 GTP chemical After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. DISCUSS +105 118 decoding site site After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. DISCUSS +124 136 20S pre-rRNA chemical After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. DISCUSS +160 164 Nob1 protein After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. DISCUSS +177 183 site D site After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. DISCUSS +215 222 pre-40S complex_assembly After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. DISCUSS +227 239 60S subunits complex_assembly After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. DISCUSS +243 261 80S-like particles complex_assembly After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. DISCUSS +302 307 eIF5B protein After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B. DISCUSS +86 97 40S subunit complex_assembly The cleavage step most likely acts as a quality control check that ensures the proper 40S subunit assembly with only completely processed precursors. DISCUSS +9 27 termination factor protein_type Finally, termination factor Rli1, an ATPase, promotes the dissociation of assembly factors and the 80S-like complex dissociates and releases the mature 40S subunit. DISCUSS +28 32 Rli1 protein Finally, termination factor Rli1, an ATPase, promotes the dissociation of assembly factors and the 80S-like complex dissociates and releases the mature 40S subunit. DISCUSS +37 43 ATPase protein_type Finally, termination factor Rli1, an ATPase, promotes the dissociation of assembly factors and the 80S-like complex dissociates and releases the mature 40S subunit. DISCUSS +99 115 80S-like complex complex_assembly Finally, termination factor Rli1, an ATPase, promotes the dissociation of assembly factors and the 80S-like complex dissociates and releases the mature 40S subunit. DISCUSS +145 151 mature protein_state Finally, termination factor Rli1, an ATPase, promotes the dissociation of assembly factors and the 80S-like complex dissociates and releases the mature 40S subunit. DISCUSS +152 163 40S subunit complex_assembly Finally, termination factor Rli1, an ATPase, promotes the dissociation of assembly factors and the 80S-like complex dissociates and releases the mature 40S subunit. DISCUSS +43 46 acp chemical Interestingly, differences in the level of acp modification were demonstrated for different steps of the cytoplasmic pre-40S subunit maturation after analyzing purified 20S pre-rRNAs using different purification bait proteins. DISCUSS +117 132 pre-40S subunit complex_assembly Interestingly, differences in the level of acp modification were demonstrated for different steps of the cytoplasmic pre-40S subunit maturation after analyzing purified 20S pre-rRNAs using different purification bait proteins. DISCUSS +169 182 20S pre-rRNAs chemical Interestingly, differences in the level of acp modification were demonstrated for different steps of the cytoplasmic pre-40S subunit maturation after analyzing purified 20S pre-rRNAs using different purification bait proteins. DISCUSS +18 34 pre-40S subunits complex_assembly Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified. DISCUSS +56 81 ribosome assembly factors protein_type Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified. DISCUSS +82 86 Tsr1 protein Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified. DISCUSS +88 92 Ltv1 protein Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified. DISCUSS +94 98 Enp1 protein Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified. DISCUSS +103 107 Rio2 protein Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified. DISCUSS +135 147 acp modified protein_state Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified. DISCUSS +18 34 pre-40S subunits complex_assembly In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits. DISCUSS +46 50 Nob1 protein In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits. DISCUSS +55 59 Rio1 protein In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits. DISCUSS +87 99 60S subunits complex_assembly In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits. DISCUSS +103 121 80S-like particles complex_assembly In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits. DISCUSS +129 132 acp chemical In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits. DISCUSS +167 173 mature protein_state In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits. DISCUSS +174 186 40S subunits complex_assembly In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits. DISCUSS +10 13 acp chemical Thus, the acp transfer to m1Ψ1191 occurs during the step at which Rio2 leaves the pre-40S particle. DISCUSS +26 33 m1Ψ1191 residue_name_number Thus, the acp transfer to m1Ψ1191 occurs during the step at which Rio2 leaves the pre-40S particle. DISCUSS +66 70 Rio2 protein Thus, the acp transfer to m1Ψ1191 occurs during the step at which Rio2 leaves the pre-40S particle. DISCUSS +82 98 pre-40S particle complex_assembly Thus, the acp transfer to m1Ψ1191 occurs during the step at which Rio2 leaves the pre-40S particle. DISCUSS +42 45 acp chemical These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1. DISCUSS +67 79 pre-20S rRNA chemical These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1. DISCUSS +109 112 acp chemical These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1. DISCUSS +155 159 Rio2 protein These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1. DISCUSS +190 203 decoding site site These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1. DISCUSS +213 219 D-site site These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1. DISCUSS +232 236 Nob1 protein These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1. DISCUSS +26 29 acp chemical The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the Ψ1191 residue that receives the acp modification. DISCUSS +47 51 Rio2 protein The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the Ψ1191 residue that receives the acp modification. DISCUSS +81 94 CRAC analysis experimental_method The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the Ψ1191 residue that receives the acp modification. DISCUSS +108 112 Rio2 protein The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the Ψ1191 residue that receives the acp modification. DISCUSS +122 130 helix 31 structure_element The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the Ψ1191 residue that receives the acp modification. DISCUSS +143 148 Ψ1191 residue_name_number The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the Ψ1191 residue that receives the acp modification. DISCUSS +175 178 acp chemical The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the Ψ1191 residue that receives the acp modification. DISCUSS +11 15 Rio2 protein Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. DISCUSS +44 48 Tsr3 protein Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. DISCUSS +52 60 helix 31 structure_element Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. DISCUSS +66 69 acp chemical Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. DISCUSS +104 108 Rio2 protein Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. DISCUSS +129 132 acp chemical Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. DISCUSS +149 156 m1Ψ1191 residue_name_number Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. DISCUSS +207 215 20S rRNA chemical Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. DISCUSS +238 242 Rio2 protein Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. DISCUSS +246 254 helix 31 structure_element Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. DISCUSS +288 296 pre-rRNA chemical Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1Ψ1191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA. DISCUSS +27 31 Tsr3 protein In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. DISCUSS +78 81 acp chemical In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. DISCUSS +95 108 hypermodified protein_state In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. DISCUSS +109 116 m1acp3Ψ chemical In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. DISCUSS +117 127 nucleotide chemical In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. DISCUSS +140 144 1191 residue_number In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. DISCUSS +146 151 yeast taxonomy_domain In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. DISCUSS +154 158 1248 residue_number In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. DISCUSS +160 166 humans species In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. DISCUSS +171 179 18S rRNA chemical In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. DISCUSS +239 249 eukaryotic taxonomy_domain In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. DISCUSS +250 278 small ribosomal subunit rRNA chemical In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3Ψ nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications. DISCUSS +48 51 acp chemical The current data together with the finding that acp modification takes place at the very last step in pre-40S subunit maturation indicate that the acp modification probably supports the formation of the decoding site and efficient 20S pre-rRNA D-site cleavage. DISCUSS +102 117 pre-40S subunit complex_assembly The current data together with the finding that acp modification takes place at the very last step in pre-40S subunit maturation indicate that the acp modification probably supports the formation of the decoding site and efficient 20S pre-rRNA D-site cleavage. DISCUSS +147 150 acp chemical The current data together with the finding that acp modification takes place at the very last step in pre-40S subunit maturation indicate that the acp modification probably supports the formation of the decoding site and efficient 20S pre-rRNA D-site cleavage. DISCUSS +203 216 decoding site site The current data together with the finding that acp modification takes place at the very last step in pre-40S subunit maturation indicate that the acp modification probably supports the formation of the decoding site and efficient 20S pre-rRNA D-site cleavage. DISCUSS +231 243 20S pre-rRNA chemical The current data together with the finding that acp modification takes place at the very last step in pre-40S subunit maturation indicate that the acp modification probably supports the formation of the decoding site and efficient 20S pre-rRNA D-site cleavage. DISCUSS +244 250 D-site site The current data together with the finding that acp modification takes place at the very last step in pre-40S subunit maturation indicate that the acp modification probably supports the formation of the decoding site and efficient 20S pre-rRNA D-site cleavage. DISCUSS +17 32 structural data evidence Furthermore, our structural data unravelled how the regioselectivity of SAM-dependent group transfer reactions can be tuned by distinct small evolutionary adaptions of the ligand binding pocket of SAM-binding enzymes. DISCUSS +72 75 SAM chemical Furthermore, our structural data unravelled how the regioselectivity of SAM-dependent group transfer reactions can be tuned by distinct small evolutionary adaptions of the ligand binding pocket of SAM-binding enzymes. DISCUSS +172 193 ligand binding pocket site Furthermore, our structural data unravelled how the regioselectivity of SAM-dependent group transfer reactions can be tuned by distinct small evolutionary adaptions of the ligand binding pocket of SAM-binding enzymes. DISCUSS +197 216 SAM-binding enzymes protein_type Furthermore, our structural data unravelled how the regioselectivity of SAM-dependent group transfer reactions can be tuned by distinct small evolutionary adaptions of the ligand binding pocket of SAM-binding enzymes. DISCUSS diff --git a/annotation_CSV/PMC4880283.csv b/annotation_CSV/PMC4880283.csv new file mode 100644 index 0000000000000000000000000000000000000000..cce945abd87110167b8a526cec44c09b6f6f1150 --- /dev/null +++ b/annotation_CSV/PMC4880283.csv @@ -0,0 +1,784 @@ +anno_start anno_end anno_text entity_type sentence section +0 18 Crystal Structures evidence Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana TITLE +31 44 Sugar Kinases protein_type Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana TITLE +50 82 Synechococcus Elongatus PCC 7942 species Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana TITLE +87 107 Arabidopsis Thaliana species Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana TITLE +18 57 Synechococcus elongatus strain PCC 7942 species The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. ABSTRACT +77 89 sugar kinase protein_type The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. ABSTRACT +91 96 SePSK protein The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. ABSTRACT +145 162 xylulose kinase-1 protein The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. ABSTRACT +164 170 AtXK-1 protein The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. ABSTRACT +177 197 Arabidopsis thaliana species The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. ABSTRACT +0 18 Sequence alignment experimental_method Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. ABSTRACT +38 45 kinases protein_type Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. ABSTRACT +60 98 ribulokinase-like carbohydrate kinases protein_type Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. ABSTRACT +116 148 FGGY family carbohydrate kinases protein_type Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. ABSTRACT +8 14 solved experimental_method Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. ABSTRACT +19 29 structures evidence Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. ABSTRACT +33 38 SePSK protein Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. ABSTRACT +43 49 AtXK-1 protein Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. ABSTRACT +64 67 apo protein_state Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. ABSTRACT +78 93 in complex with protein_state Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. ABSTRACT +94 104 nucleotide chemical Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. ABSTRACT +73 80 kinases protein_type The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates. ABSTRACT +92 95 ATP chemical The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates. ABSTRACT +123 144 absence of substrates protein_state The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates. ABSTRACT +17 33 enzymatic assays experimental_method In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose. ABSTRACT +49 54 SePSK protein In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose. ABSTRACT +91 101 D-ribulose chemical In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose. ABSTRACT +50 55 SePSK protein In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. ABSTRACT +60 66 solved experimental_method In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. ABSTRACT +71 80 structure evidence In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. ABSTRACT +84 89 SePSK protein In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. ABSTRACT +90 105 in complex with protein_state In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. ABSTRACT +106 116 D-ribulose chemical In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. ABSTRACT +141 166 substrate binding pockets site In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. ABSTRACT +170 175 SePSK protein In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. ABSTRACT +6 36 mutation and activity analysis experimental_method Using mutation and activity analysis, we further verified the key residues important for its catalytic activity. ABSTRACT +14 35 structural comparison experimental_method Moreover, our structural comparison with other family members suggests that there are major conformational changes in SePSK upon substrate binding, facilitating the catalytic process. ABSTRACT +118 123 SePSK protein Moreover, our structural comparison with other family members suggests that there are major conformational changes in SePSK upon substrate binding, facilitating the catalytic process. ABSTRACT +169 174 SePSK protein Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1. ABSTRACT +211 216 plant taxonomy_domain Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1. ABSTRACT +227 233 AtXK-1 protein Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1. ABSTRACT +0 13 Carbohydrates chemical Carbohydrates are essential cellular compounds involved in the metabolic processes present in all organisms. INTRO +0 15 Phosphorylation ptm Phosphorylation is one of the various pivotal modifications of carbohydrates, and is catalyzed by specific sugar kinases. INTRO +63 76 carbohydrates chemical Phosphorylation is one of the various pivotal modifications of carbohydrates, and is catalyzed by specific sugar kinases. INTRO +107 120 sugar kinases protein_type Phosphorylation is one of the various pivotal modifications of carbohydrates, and is catalyzed by specific sugar kinases. INTRO +6 13 kinases protein_type These kinases exhibit considerable differences in their folding pattern and substrate specificity. INTRO +9 26 sequence analysis experimental_method Based on sequence analysis, they can be divided into four families, namely HSP 70_NBD family, FGGY family, Mer_B like family and Parm_like family. INTRO +75 92 HSP 70_NBD family protein_type Based on sequence analysis, they can be divided into four families, namely HSP 70_NBD family, FGGY family, Mer_B like family and Parm_like family. INTRO +94 105 FGGY family protein_type Based on sequence analysis, they can be divided into four families, namely HSP 70_NBD family, FGGY family, Mer_B like family and Parm_like family. INTRO +107 124 Mer_B like family protein_type Based on sequence analysis, they can be divided into four families, namely HSP 70_NBD family, FGGY family, Mer_B like family and Parm_like family. INTRO +129 145 Parm_like family protein_type Based on sequence analysis, they can be divided into four families, namely HSP 70_NBD family, FGGY family, Mer_B like family and Parm_like family. INTRO +4 36 FGGY family carbohydrate kinases protein_type The FGGY family carbohydrate kinases contain different types of sugar kinases, all of which possess different catalytic substrates with preferences for short-chained sugar substrates, ranging from triose to heptose. INTRO +64 77 sugar kinases protein_type The FGGY family carbohydrate kinases contain different types of sugar kinases, all of which possess different catalytic substrates with preferences for short-chained sugar substrates, ranging from triose to heptose. INTRO +166 171 sugar chemical The FGGY family carbohydrate kinases contain different types of sugar kinases, all of which possess different catalytic substrates with preferences for short-chained sugar substrates, ranging from triose to heptose. INTRO +197 203 triose chemical The FGGY family carbohydrate kinases contain different types of sugar kinases, all of which possess different catalytic substrates with preferences for short-chained sugar substrates, ranging from triose to heptose. INTRO +207 214 heptose chemical The FGGY family carbohydrate kinases contain different types of sugar kinases, all of which possess different catalytic substrates with preferences for short-chained sugar substrates, ranging from triose to heptose. INTRO +6 11 sugar chemical These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. INTRO +31 41 L-ribulose chemical These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. INTRO +43 53 erythritol chemical These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. INTRO +55 65 L-fuculose chemical These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. INTRO +67 77 D-glycerol chemical These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. INTRO +79 90 D-gluconate chemical These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. INTRO +92 102 L-xylulose chemical These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. INTRO +104 114 D-ribulose chemical These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. INTRO +116 128 L-rhamnulose chemical These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. INTRO +133 143 D-xylulose chemical These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. INTRO +0 10 Structures evidence Structures reported in the Protein Data Bank of the FGGY family carbohydrate kinases exhibit a similar overall architecture containing two protein domains, one of which is responsible for the binding of substrate, while the second is used for binding cofactor ATP. INTRO +52 84 FGGY family carbohydrate kinases protein_type Structures reported in the Protein Data Bank of the FGGY family carbohydrate kinases exhibit a similar overall architecture containing two protein domains, one of which is responsible for the binding of substrate, while the second is used for binding cofactor ATP. INTRO +260 263 ATP chemical Structures reported in the Protein Data Bank of the FGGY family carbohydrate kinases exhibit a similar overall architecture containing two protein domains, one of which is responsible for the binding of substrate, while the second is used for binding cofactor ATP. INTRO +10 25 binding pockets site While the binding pockets for substrates are at the same position, each FGGY family carbohydrate kinases uses different substrate-binding residues, resulting in high substrate specificity. INTRO +72 104 FGGY family carbohydrate kinases protein_type While the binding pockets for substrates are at the same position, each FGGY family carbohydrate kinases uses different substrate-binding residues, resulting in high substrate specificity. INTRO +120 146 substrate-binding residues site While the binding pockets for substrates are at the same position, each FGGY family carbohydrate kinases uses different substrate-binding residues, resulting in high substrate specificity. INTRO +0 15 Synpcc7942_2462 gene Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids. INTRO +25 38 cyanobacteria taxonomy_domain Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids. INTRO +39 71 Synechococcus elongatus PCC 7942 species Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids. INTRO +91 103 sugar kinase protein_type Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids. INTRO +105 110 SePSK protein Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids. INTRO +122 128 kinase protein_type Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids. INTRO +138 141 426 residue_range Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids. INTRO +4 13 At2g21370 gene The At2g21370 gene product from Arabidopsis thaliana, xylulose kinase-1 (AtXK-1), whose mature form contains 436 amino acids, is located in the chloroplast (ChloroP 1.1 Server). INTRO +32 52 Arabidopsis thaliana species The At2g21370 gene product from Arabidopsis thaliana, xylulose kinase-1 (AtXK-1), whose mature form contains 436 amino acids, is located in the chloroplast (ChloroP 1.1 Server). INTRO +54 71 xylulose kinase-1 protein The At2g21370 gene product from Arabidopsis thaliana, xylulose kinase-1 (AtXK-1), whose mature form contains 436 amino acids, is located in the chloroplast (ChloroP 1.1 Server). INTRO +73 79 AtXK-1 protein The At2g21370 gene product from Arabidopsis thaliana, xylulose kinase-1 (AtXK-1), whose mature form contains 436 amino acids, is located in the chloroplast (ChloroP 1.1 Server). INTRO +88 99 mature form protein_state The At2g21370 gene product from Arabidopsis thaliana, xylulose kinase-1 (AtXK-1), whose mature form contains 436 amino acids, is located in the chloroplast (ChloroP 1.1 Server). INTRO +109 112 436 residue_range The At2g21370 gene product from Arabidopsis thaliana, xylulose kinase-1 (AtXK-1), whose mature form contains 436 amino acids, is located in the chloroplast (ChloroP 1.1 Server). INTRO +0 5 SePSK protein SePSK and AtXK-1 display a sequence identity of 44.9%, and belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. INTRO +10 16 AtXK-1 protein SePSK and AtXK-1 display a sequence identity of 44.9%, and belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. INTRO +73 111 ribulokinase-like carbohydrate kinases protein_type SePSK and AtXK-1 display a sequence identity of 44.9%, and belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. INTRO +129 161 FGGY family carbohydrate kinases protein_type SePSK and AtXK-1 display a sequence identity of 44.9%, and belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. INTRO +51 66 phosphorylation ptm Members of this sub-family are responsible for the phosphorylation of sugars similar to L-ribulose and D-ribulose. INTRO +70 76 sugars chemical Members of this sub-family are responsible for the phosphorylation of sugars similar to L-ribulose and D-ribulose. INTRO +88 98 L-ribulose chemical Members of this sub-family are responsible for the phosphorylation of sugars similar to L-ribulose and D-ribulose. INTRO +103 113 D-ribulose chemical Members of this sub-family are responsible for the phosphorylation of sugars similar to L-ribulose and D-ribulose. INTRO +46 84 ribulokinase-like carbohydrate kinases protein_type The sequence and the substrate specificity of ribulokinase-like carbohydrate kinases are different, but they share the common folding feature with two domains. INTRO +0 8 Domain I structure_element Domain I exhibits a ribonuclease H-like folding pattern, and is responsible for the substrate binding, while domain II possesses an actin-like ATPase domain that binds cofactor ATP. INTRO +20 55 ribonuclease H-like folding pattern structure_element Domain I exhibits a ribonuclease H-like folding pattern, and is responsible for the substrate binding, while domain II possesses an actin-like ATPase domain that binds cofactor ATP. INTRO +109 118 domain II structure_element Domain I exhibits a ribonuclease H-like folding pattern, and is responsible for the substrate binding, while domain II possesses an actin-like ATPase domain that binds cofactor ATP. INTRO +132 156 actin-like ATPase domain structure_element Domain I exhibits a ribonuclease H-like folding pattern, and is responsible for the substrate binding, while domain II possesses an actin-like ATPase domain that binds cofactor ATP. INTRO +177 180 ATP chemical Domain I exhibits a ribonuclease H-like folding pattern, and is responsible for the substrate binding, while domain II possesses an actin-like ATPase domain that binds cofactor ATP. INTRO +13 29 xylulose kinases protein_type Two possible xylulose kinases (xylulose kinase-1: XK-1 and xylulose kinase-2: XK-2) from Arabidopsis thaliana were previously proposed. INTRO +31 48 xylulose kinase-1 protein Two possible xylulose kinases (xylulose kinase-1: XK-1 and xylulose kinase-2: XK-2) from Arabidopsis thaliana were previously proposed. INTRO +50 54 XK-1 protein Two possible xylulose kinases (xylulose kinase-1: XK-1 and xylulose kinase-2: XK-2) from Arabidopsis thaliana were previously proposed. INTRO +59 76 xylulose kinase-2 protein Two possible xylulose kinases (xylulose kinase-1: XK-1 and xylulose kinase-2: XK-2) from Arabidopsis thaliana were previously proposed. INTRO +78 82 XK-2 protein Two possible xylulose kinases (xylulose kinase-1: XK-1 and xylulose kinase-2: XK-2) from Arabidopsis thaliana were previously proposed. INTRO +89 109 Arabidopsis thaliana species Two possible xylulose kinases (xylulose kinase-1: XK-1 and xylulose kinase-2: XK-2) from Arabidopsis thaliana were previously proposed. INTRO +18 22 XK-2 protein It was shown that XK-2 (At5g49650) located in the cytosol is indeed xylulose kinase. INTRO +24 33 At5g49650 gene It was shown that XK-2 (At5g49650) located in the cytosol is indeed xylulose kinase. INTRO +68 83 xylulose kinase protein_type It was shown that XK-2 (At5g49650) located in the cytosol is indeed xylulose kinase. INTRO +25 29 XK-1 protein However, the function of XK-1 (At2g21370) inside the chloroplast stroma has remained unknown. INTRO +31 40 At2g21370 gene However, the function of XK-1 (At2g21370) inside the chloroplast stroma has remained unknown. INTRO +0 5 SePSK protein SePSK from Synechococcus elongatus strain PCC 7942 is the homolog of AtXK-1, though its physiological function and substrates remain unclear. INTRO +11 50 Synechococcus elongatus strain PCC 7942 species SePSK from Synechococcus elongatus strain PCC 7942 is the homolog of AtXK-1, though its physiological function and substrates remain unclear. INTRO +69 75 AtXK-1 protein SePSK from Synechococcus elongatus strain PCC 7942 is the homolog of AtXK-1, though its physiological function and substrates remain unclear. INTRO +104 122 crystal structures evidence In order to obtain functional and structural information about these two proteins, here we reported the crystal structures of SePSK and AtXK-1. INTRO +126 131 SePSK protein In order to obtain functional and structural information about these two proteins, here we reported the crystal structures of SePSK and AtXK-1. INTRO +136 142 AtXK-1 protein In order to obtain functional and structural information about these two proteins, here we reported the crystal structures of SePSK and AtXK-1. INTRO +63 68 SePSK protein Our findings provide new details of the catalytic mechanism of SePSK and lay the foundation for future studies into its homologs in eukaryotes. INTRO +132 142 eukaryotes taxonomy_domain Our findings provide new details of the catalytic mechanism of SePSK and lay the foundation for future studies into its homologs in eukaryotes. INTRO +8 18 structures evidence Overall structures of apo-SePSK and apo-AtXK-1 RESULTS +22 25 apo protein_state Overall structures of apo-SePSK and apo-AtXK-1 RESULTS +26 31 SePSK protein Overall structures of apo-SePSK and apo-AtXK-1 RESULTS +36 39 apo protein_state Overall structures of apo-SePSK and apo-AtXK-1 RESULTS +40 46 AtXK-1 protein Overall structures of apo-SePSK and apo-AtXK-1 RESULTS +25 30 SePSK protein The attempt to solve the SePSK structure by molecular replacement method failed with ribulokinase from Bacillus halodurans (PDB code: 3QDK, 15.7% sequence identity) as an initial model. RESULTS +31 40 structure evidence The attempt to solve the SePSK structure by molecular replacement method failed with ribulokinase from Bacillus halodurans (PDB code: 3QDK, 15.7% sequence identity) as an initial model. RESULTS +44 72 molecular replacement method experimental_method The attempt to solve the SePSK structure by molecular replacement method failed with ribulokinase from Bacillus halodurans (PDB code: 3QDK, 15.7% sequence identity) as an initial model. RESULTS +85 97 ribulokinase protein The attempt to solve the SePSK structure by molecular replacement method failed with ribulokinase from Bacillus halodurans (PDB code: 3QDK, 15.7% sequence identity) as an initial model. RESULTS +103 122 Bacillus halodurans species The attempt to solve the SePSK structure by molecular replacement method failed with ribulokinase from Bacillus halodurans (PDB code: 3QDK, 15.7% sequence identity) as an initial model. RESULTS +18 76 single isomorphous replacement anomalous scattering method experimental_method We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study. RESULTS +78 83 SIRAS experimental_method We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study. RESULTS +116 119 apo protein_state We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study. RESULTS +120 125 SePSK protein We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study. RESULTS +126 135 structure evidence We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study. RESULTS +180 183 apo protein_state We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study. RESULTS +184 189 SePSK protein We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study. RESULTS +190 199 structure evidence We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study. RESULTS +212 239 molecular replacement model experimental_method We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study. RESULTS +259 269 structures evidence We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study. RESULTS +4 23 structural analysis experimental_method Our structural analysis showed that apo-SePSK consists of one SePSK protein molecule in an asymmetric unit. RESULTS +36 39 apo protein_state Our structural analysis showed that apo-SePSK consists of one SePSK protein molecule in an asymmetric unit. RESULTS +40 45 SePSK protein Our structural analysis showed that apo-SePSK consists of one SePSK protein molecule in an asymmetric unit. RESULTS +62 67 SePSK protein Our structural analysis showed that apo-SePSK consists of one SePSK protein molecule in an asymmetric unit. RESULTS +41 45 Val2 residue_name_number The amino-acid residues were traced from Val2 to His419, except for the Met1 residue and the seven residues at the C-termini. RESULTS +49 55 His419 residue_name_number The amino-acid residues were traced from Val2 to His419, except for the Met1 residue and the seven residues at the C-termini. RESULTS +72 76 Met1 residue_name_number The amino-acid residues were traced from Val2 to His419, except for the Met1 residue and the seven residues at the C-termini. RESULTS +0 3 Apo protein_state Apo-SePSK contains two domains referred to further on as domain I and domain II (Fig 1A). RESULTS +4 9 SePSK protein Apo-SePSK contains two domains referred to further on as domain I and domain II (Fig 1A). RESULTS +57 65 domain I structure_element Apo-SePSK contains two domains referred to further on as domain I and domain II (Fig 1A). RESULTS +70 79 domain II structure_element Apo-SePSK contains two domains referred to further on as domain I and domain II (Fig 1A). RESULTS +0 8 Domain I structure_element Domain I consists of non-contiguous portions of the polypeptide chains (aa. RESULTS +0 5 2–228 residue_range 2–228 and aa. RESULTS +0 7 402–419 residue_range 402–419), exhibiting 11 α-helices and 11 β-sheets. RESULTS +24 33 α-helices structure_element 402–419), exhibiting 11 α-helices and 11 β-sheets. RESULTS +41 49 β-sheets structure_element 402–419), exhibiting 11 α-helices and 11 β-sheets. RESULTS +37 39 α4 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +40 42 α5 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +43 46 α11 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +47 50 α18 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +52 54 β3 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +55 57 β2 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +58 60 β1 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +61 63 β6 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +64 67 β19 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +68 71 β20 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +72 75 β17 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +80 83 α21 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +84 87 α32 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +123 125 A1 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +127 129 B1 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +134 136 A2 structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +153 164 core region structure_element Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. RESULTS +18 26 β-sheets structure_element In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. RESULTS +28 30 β7 structure_element In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. RESULTS +32 35 β10 structure_element In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. RESULTS +37 40 β12 structure_element In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. RESULTS +45 48 β16 structure_element In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. RESULTS +59 68 α-helices structure_element In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. RESULTS +70 72 α8 structure_element In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. RESULTS +74 76 α9 structure_element In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. RESULTS +78 81 α13 structure_element In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. RESULTS +83 86 α14 structure_element In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. RESULTS +91 94 α15 structure_element In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. RESULTS +123 134 core region structure_element In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. RESULTS +0 9 Domain II structure_element Domain II is comprised of aa. RESULTS +0 7 229–401 residue_range 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +28 30 B2 structure_element 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +32 35 β31 structure_element 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +36 39 β29 structure_element 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +40 43 β22 structure_element 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +44 47 β23 structure_element 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +48 51 β25 structure_element 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +52 55 β24 structure_element 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +61 63 A3 structure_element 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +65 68 α26 structure_element 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +69 72 α27 structure_element 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +73 76 α28 structure_element 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +77 80 α30 structure_element 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). RESULTS +7 12 SePSK protein In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +13 22 structure evidence In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +24 26 B1 structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +31 33 B2 structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +52 54 A1 structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +56 58 A2 structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +63 65 A3 structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +81 90 structure evidence In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +101 103 A1 structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +104 106 B1 structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +107 109 A2 structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +110 112 B2 structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +113 115 A3 structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +117 118 α structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +119 120 β structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +121 122 α structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +123 124 β structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +125 126 α structure_element In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +186 218 FGGY family carbohydrate kinases protein_type In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). RESULTS +23 28 SePSK protein The overall folding of SePSK resembles a clip, with A2 of domain I acting as a hinge region. RESULTS +52 54 A2 structure_element The overall folding of SePSK resembles a clip, with A2 of domain I acting as a hinge region. RESULTS +58 66 domain I structure_element The overall folding of SePSK resembles a clip, with A2 of domain I acting as a hinge region. RESULTS +79 91 hinge region structure_element The overall folding of SePSK resembles a clip, with A2 of domain I acting as a hinge region. RESULTS +8 18 structures evidence Overall structures of SePSK and AtXK-1. FIG +22 27 SePSK protein Overall structures of SePSK and AtXK-1. FIG +32 38 AtXK-1 protein Overall structures of SePSK and AtXK-1. FIG +22 31 structure evidence (A) Three-dimensional structure of apo-SePSK. FIG +35 38 apo protein_state (A) Three-dimensional structure of apo-SePSK. FIG +39 44 SePSK protein (A) Three-dimensional structure of apo-SePSK. FIG +49 56 α-helix structure_element The secondary structural elements are indicated (α-helix: cyan, β-sheet: yellow). FIG +64 71 β-sheet structure_element The secondary structural elements are indicated (α-helix: cyan, β-sheet: yellow). FIG +22 31 structure evidence (B) Three-dimensional structure of apo-AtXK-1. FIG +35 38 apo protein_state (B) Three-dimensional structure of apo-AtXK-1. FIG +39 45 AtXK-1 protein (B) Three-dimensional structure of apo-AtXK-1. FIG +49 56 α-helix structure_element The secondary structural elements are indicated (α-helix: green, β-sheet: wheat). FIG +65 72 β-sheet structure_element The secondary structural elements are indicated (α-helix: green, β-sheet: wheat). FIG +0 3 Apo protein_state Apo-AtXK-1 exhibits a folding pattern similar to that of SePSK in line with their high sequence identity (Fig 1B and S1 Fig). RESULTS +4 10 AtXK-1 protein Apo-AtXK-1 exhibits a folding pattern similar to that of SePSK in line with their high sequence identity (Fig 1B and S1 Fig). RESULTS +57 62 SePSK protein Apo-AtXK-1 exhibits a folding pattern similar to that of SePSK in line with their high sequence identity (Fig 1B and S1 Fig). RESULTS +9 22 superposition experimental_method However, superposition of structures of AtXK-1 and SePSK shows some differences, especially at the loop regions. RESULTS +26 36 structures evidence However, superposition of structures of AtXK-1 and SePSK shows some differences, especially at the loop regions. RESULTS +40 46 AtXK-1 protein However, superposition of structures of AtXK-1 and SePSK shows some differences, especially at the loop regions. RESULTS +51 56 SePSK protein However, superposition of structures of AtXK-1 and SePSK shows some differences, especially at the loop regions. RESULTS +99 111 loop regions structure_element However, superposition of structures of AtXK-1 and SePSK shows some differences, especially at the loop regions. RESULTS +42 47 loop3 structure_element A considerable difference is found in the loop3 linking β3 and α4, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part. RESULTS +56 58 β3 structure_element A considerable difference is found in the loop3 linking β3 and α4, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part. RESULTS +63 65 α4 structure_element A considerable difference is found in the loop3 linking β3 and α4, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part. RESULTS +97 103 AtXK-1 protein A considerable difference is found in the loop3 linking β3 and α4, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part. RESULTS +104 113 structure evidence A considerable difference is found in the loop3 linking β3 and α4, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part. RESULTS +128 133 SePSK protein A considerable difference is found in the loop3 linking β3 and α4, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part. RESULTS +134 143 structure evidence A considerable difference is found in the loop3 linking β3 and α4, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part. RESULTS +45 55 structures evidence The corresponding residues between these two structures (SePSK-Lys35 and AtXK-1-Lys48) have a distance of 15.4 Å (S3 Fig). RESULTS +57 62 SePSK protein The corresponding residues between these two structures (SePSK-Lys35 and AtXK-1-Lys48) have a distance of 15.4 Å (S3 Fig). RESULTS +63 68 Lys35 residue_name_number The corresponding residues between these two structures (SePSK-Lys35 and AtXK-1-Lys48) have a distance of 15.4 Å (S3 Fig). RESULTS +73 79 AtXK-1 protein The corresponding residues between these two structures (SePSK-Lys35 and AtXK-1-Lys48) have a distance of 15.4 Å (S3 Fig). RESULTS +80 85 Lys48 residue_name_number The corresponding residues between these two structures (SePSK-Lys35 and AtXK-1-Lys48) have a distance of 15.4 Å (S3 Fig). RESULTS +0 15 Activity assays experimental_method Activity assays of SePSK and AtXK-1 RESULTS +19 24 SePSK protein Activity assays of SePSK and AtXK-1 RESULTS +29 35 AtXK-1 protein Activity assays of SePSK and AtXK-1 RESULTS +71 92 structural comparison experimental_method In order to understand the function of these two kinases, we performed structural comparison using Dali server. RESULTS +99 110 Dali server experimental_method In order to understand the function of these two kinases, we performed structural comparison using Dali server. RESULTS +4 14 structures evidence The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases. RESULTS +39 44 SePSK protein The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases. RESULTS +49 64 xylulose kinase protein_type The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases. RESULTS +66 81 glycerol kinase protein_type The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases. RESULTS +86 101 ribulose kinase protein_type The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases. RESULTS +117 122 SePSK protein The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases. RESULTS +127 133 AtXK-1 protein The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases. RESULTS +168 175 kinases protein_type The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases. RESULTS +47 50 ATP chemical We first tested whether both enzymes possessed ATP hydrolysis activity in the absence of substrates. RESULTS +78 88 absence of protein_state We first tested whether both enzymes possessed ATP hydrolysis activity in the absence of substrates. RESULTS +25 30 SePSK protein As shown in Fig 2A, both SePSK and AtXK-1 exhibited ATP hydrolysis activity. RESULTS +35 41 AtXK-1 protein As shown in Fig 2A, both SePSK and AtXK-1 exhibited ATP hydrolysis activity. RESULTS +52 55 ATP chemical As shown in Fig 2A, both SePSK and AtXK-1 exhibited ATP hydrolysis activity. RESULTS +65 80 xylulose kinase protein_type This finding is in agreement with a previous result showing that xylulose kinase (PDB code: 2ITM) possessed ATP hydrolysis activity without adding substrate. RESULTS +108 111 ATP chemical This finding is in agreement with a previous result showing that xylulose kinase (PDB code: 2ITM) possessed ATP hydrolysis activity without adding substrate. RESULTS +44 49 SePSK protein To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays. RESULTS +54 60 AtXK-1 protein To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays. RESULTS +104 114 D-ribulose chemical To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays. RESULTS +116 126 L-ribulose chemical To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays. RESULTS +128 138 D-xylulose chemical To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays. RESULTS +140 150 L-xylulose chemical To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays. RESULTS +155 163 Glycerol chemical To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays. RESULTS +178 203 enzymatic activity assays experimental_method To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays. RESULTS +24 27 ATP chemical As shown in Fig 2B, the ATP hydrolysis activity of SePSK greatly increased upon adding D-ribulose than adding other potential substrates, suggesting that it has D-ribulose kinase activity. RESULTS +51 56 SePSK protein As shown in Fig 2B, the ATP hydrolysis activity of SePSK greatly increased upon adding D-ribulose than adding other potential substrates, suggesting that it has D-ribulose kinase activity. RESULTS +87 97 D-ribulose chemical As shown in Fig 2B, the ATP hydrolysis activity of SePSK greatly increased upon adding D-ribulose than adding other potential substrates, suggesting that it has D-ribulose kinase activity. RESULTS +161 178 D-ribulose kinase protein_type As shown in Fig 2B, the ATP hydrolysis activity of SePSK greatly increased upon adding D-ribulose than adding other potential substrates, suggesting that it has D-ribulose kinase activity. RESULTS +35 38 ATP chemical In contrary, limited increasing of ATP hydrolysis activity was detected for AtXK-1 upon addition of D-ribulose (Fig 2C), despite its structural similarity with SePSK. RESULTS +76 82 AtXK-1 protein In contrary, limited increasing of ATP hydrolysis activity was detected for AtXK-1 upon addition of D-ribulose (Fig 2C), despite its structural similarity with SePSK. RESULTS +100 110 D-ribulose chemical In contrary, limited increasing of ATP hydrolysis activity was detected for AtXK-1 upon addition of D-ribulose (Fig 2C), despite its structural similarity with SePSK. RESULTS +160 165 SePSK protein In contrary, limited increasing of ATP hydrolysis activity was detected for AtXK-1 upon addition of D-ribulose (Fig 2C), despite its structural similarity with SePSK. RESULTS +4 29 enzymatic activity assays experimental_method The enzymatic activity assays of SePSK and AtXK-1. FIG +33 38 SePSK protein The enzymatic activity assays of SePSK and AtXK-1. FIG +43 49 AtXK-1 protein The enzymatic activity assays of SePSK and AtXK-1. FIG +8 11 ATP chemical (A) The ATP hydrolysis activity of SePSK and AtXK-1. FIG +35 40 SePSK protein (A) The ATP hydrolysis activity of SePSK and AtXK-1. FIG +45 51 AtXK-1 protein (A) The ATP hydrolysis activity of SePSK and AtXK-1. FIG +5 10 SePSK protein Both SePSK and AtXK-1 showed ATP hydrolysis activity in the absence of substrate. FIG +15 21 AtXK-1 protein Both SePSK and AtXK-1 showed ATP hydrolysis activity in the absence of substrate. FIG +29 32 ATP chemical Both SePSK and AtXK-1 showed ATP hydrolysis activity in the absence of substrate. FIG +60 70 absence of protein_state Both SePSK and AtXK-1 showed ATP hydrolysis activity in the absence of substrate. FIG +10 13 ATP chemical While the ATP hydrolysis activity of SePSK greatly increases upon addition of D-ribulose (DR). FIG +37 42 SePSK protein While the ATP hydrolysis activity of SePSK greatly increases upon addition of D-ribulose (DR). FIG +78 88 D-ribulose chemical While the ATP hydrolysis activity of SePSK greatly increases upon addition of D-ribulose (DR). FIG +90 92 DR chemical While the ATP hydrolysis activity of SePSK greatly increases upon addition of D-ribulose (DR). FIG +8 11 ATP chemical (B) The ATP hydrolysis activity of SePSK with addition of five different substrates. FIG +35 40 SePSK protein (B) The ATP hydrolysis activity of SePSK with addition of five different substrates. FIG +19 21 DR chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +23 33 D-ribulose chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +36 38 LR chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +40 50 L-ribulose chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +53 55 DX chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +57 67 D-xylulose chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +70 72 LX chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +74 84 L-xylulose chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +90 93 GLY chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +95 103 Glycerol chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +114 117 ATP chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +141 146 SePSK protein The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +151 157 AtXK-1 protein The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +174 184 D-ribulose chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +194 197 ATP chemical The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +221 230 wild-type protein_state The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +232 234 WT protein_state The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +263 268 SePSK protein The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. FIG +29 34 SePSK protein Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK. FIG +39 42 D8A mutant Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK. FIG +43 48 SePSK protein Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK. FIG +50 54 T11A mutant Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK. FIG +55 60 SePSK protein Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK. FIG +65 70 D221A mutant Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK. FIG +71 76 SePSK protein Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK. FIG +4 7 ATP chemical The ATP hydrolysis activity measured via luminescent ADP-Glo assay (Promega). FIG +41 66 luminescent ADP-Glo assay experimental_method The ATP hydrolysis activity measured via luminescent ADP-Glo assay (Promega). FIG +41 46 SePSK protein To understand the catalytic mechanism of SePSK, we performed structural comparisons among xylulose kinase, glycerol kinase, ribulose kinase and SePSK. RESULTS +61 83 structural comparisons experimental_method To understand the catalytic mechanism of SePSK, we performed structural comparisons among xylulose kinase, glycerol kinase, ribulose kinase and SePSK. RESULTS +90 105 xylulose kinase protein_type To understand the catalytic mechanism of SePSK, we performed structural comparisons among xylulose kinase, glycerol kinase, ribulose kinase and SePSK. RESULTS +107 122 glycerol kinase protein_type To understand the catalytic mechanism of SePSK, we performed structural comparisons among xylulose kinase, glycerol kinase, ribulose kinase and SePSK. RESULTS +124 139 ribulose kinase protein_type To understand the catalytic mechanism of SePSK, we performed structural comparisons among xylulose kinase, glycerol kinase, ribulose kinase and SePSK. RESULTS +144 149 SePSK protein To understand the catalytic mechanism of SePSK, we performed structural comparisons among xylulose kinase, glycerol kinase, ribulose kinase and SePSK. RESULTS +53 55 D8 residue_name_number Our results suggested that three conserved residues (D8, T11 and D221 of SePSK) play an important role in SePSK function. RESULTS +57 60 T11 residue_name_number Our results suggested that three conserved residues (D8, T11 and D221 of SePSK) play an important role in SePSK function. RESULTS +65 69 D221 residue_name_number Our results suggested that three conserved residues (D8, T11 and D221 of SePSK) play an important role in SePSK function. RESULTS +73 78 SePSK protein Our results suggested that three conserved residues (D8, T11 and D221 of SePSK) play an important role in SePSK function. RESULTS +106 111 SePSK protein Our results suggested that three conserved residues (D8, T11 and D221 of SePSK) play an important role in SePSK function. RESULTS +0 9 Mutations experimental_method Mutations of the corresponding residue in xylulose kinase and glycerol kinase from Escherichia coli greatly reduced their activity. RESULTS +42 57 xylulose kinase protein_type Mutations of the corresponding residue in xylulose kinase and glycerol kinase from Escherichia coli greatly reduced their activity. RESULTS +62 77 glycerol kinase protein_type Mutations of the corresponding residue in xylulose kinase and glycerol kinase from Escherichia coli greatly reduced their activity. RESULTS +83 99 Escherichia coli species Mutations of the corresponding residue in xylulose kinase and glycerol kinase from Escherichia coli greatly reduced their activity. RESULTS +52 57 SePSK protein To identify the function of these three residues of SePSK, we constructed D8A, T11A and D221A mutants. RESULTS +74 77 D8A mutant To identify the function of these three residues of SePSK, we constructed D8A, T11A and D221A mutants. RESULTS +79 83 T11A mutant To identify the function of these three residues of SePSK, we constructed D8A, T11A and D221A mutants. RESULTS +88 93 D221A mutant To identify the function of these three residues of SePSK, we constructed D8A, T11A and D221A mutants. RESULTS +94 101 mutants protein_state To identify the function of these three residues of SePSK, we constructed D8A, T11A and D221A mutants. RESULTS +6 31 enzymatic activity assays experimental_method Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D). RESULTS +99 102 ATP chemical Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D). RESULTS +127 137 D-ribulose chemical Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D). RESULTS +151 160 wild type protein_state Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D). RESULTS +256 271 phosphorylation ptm Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D). RESULTS +272 282 D-ribulose chemical Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D). RESULTS +317 322 SePSK protein Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D). RESULTS +0 5 SePSK protein SePSK and AtXK-1 possess a similar ATP binding site RESULTS +10 16 AtXK-1 protein SePSK and AtXK-1 possess a similar ATP binding site RESULTS +35 51 ATP binding site site SePSK and AtXK-1 possess a similar ATP binding site RESULTS +39 44 SePSK protein To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +49 55 AtXK-1 protein To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +56 71 in complex with protein_state To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +72 75 ATP chemical To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +80 86 soaked experimental_method To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +91 94 apo protein_state To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +95 103 crystals evidence To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +137 140 ATP chemical To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +159 169 structures evidence To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +173 178 SePSK protein To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +183 189 AtXK-1 protein To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +190 200 bound with protein_state To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +201 204 ATP chemical To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. RESULTS +8 18 structures evidence In both structures, a strong electron density was found in the conserved ATP binding pocket, but can only be fitted with an ADP molecule (S4 Fig). RESULTS +29 45 electron density evidence In both structures, a strong electron density was found in the conserved ATP binding pocket, but can only be fitted with an ADP molecule (S4 Fig). RESULTS +63 72 conserved protein_state In both structures, a strong electron density was found in the conserved ATP binding pocket, but can only be fitted with an ADP molecule (S4 Fig). RESULTS +73 91 ATP binding pocket site In both structures, a strong electron density was found in the conserved ATP binding pocket, but can only be fitted with an ADP molecule (S4 Fig). RESULTS +124 127 ADP chemical In both structures, a strong electron density was found in the conserved ATP binding pocket, but can only be fitted with an ADP molecule (S4 Fig). RESULTS +13 23 structures evidence Thus the two structures were named ADP-SePSK and ADP-AtXK-1, respectively. RESULTS +35 44 ADP-SePSK complex_assembly Thus the two structures were named ADP-SePSK and ADP-AtXK-1, respectively. RESULTS +49 59 ADP-AtXK-1 complex_assembly Thus the two structures were named ADP-SePSK and ADP-AtXK-1, respectively. RESULTS +19 37 electron densities evidence The extremely weak electron densities of ATP γ-phosphate in both structures suggest that the γ-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1. RESULTS +47 56 phosphate chemical The extremely weak electron densities of ATP γ-phosphate in both structures suggest that the γ-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1. RESULTS +65 75 structures evidence The extremely weak electron densities of ATP γ-phosphate in both structures suggest that the γ-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1. RESULTS +95 104 phosphate chemical The extremely weak electron densities of ATP γ-phosphate in both structures suggest that the γ-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1. RESULTS +114 117 ATP chemical The extremely weak electron densities of ATP γ-phosphate in both structures suggest that the γ-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1. RESULTS +154 159 SePSK protein The extremely weak electron densities of ATP γ-phosphate in both structures suggest that the γ-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1. RESULTS +164 170 AtXK-1 protein The extremely weak electron densities of ATP γ-phosphate in both structures suggest that the γ-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1. RESULTS +36 61 enzymatic activity assays experimental_method This result was consistent with our enzymatic activity assays where SePSK and AtXK-1 showed ATP hydrolysis activity without adding any substrates (Fig 2A and 2C). RESULTS +68 73 SePSK protein This result was consistent with our enzymatic activity assays where SePSK and AtXK-1 showed ATP hydrolysis activity without adding any substrates (Fig 2A and 2C). RESULTS +78 84 AtXK-1 protein This result was consistent with our enzymatic activity assays where SePSK and AtXK-1 showed ATP hydrolysis activity without adding any substrates (Fig 2A and 2C). RESULTS +92 95 ATP chemical This result was consistent with our enzymatic activity assays where SePSK and AtXK-1 showed ATP hydrolysis activity without adding any substrates (Fig 2A and 2C). RESULTS +23 26 ATP chemical To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP. RESULTS +31 37 soaked experimental_method To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP. RESULTS +42 50 crystals evidence To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP. RESULTS +54 57 apo protein_state To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP. RESULTS +58 63 SePSK protein To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP. RESULTS +68 71 apo protein_state To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP. RESULTS +72 78 AtXK-1 protein To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP. RESULTS +105 112 AMP-PNP chemical To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP. RESULTS +27 45 electron densities evidence However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. RESULTS +51 60 phosphate chemical However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. RESULTS +70 77 AMP-PNP chemical However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. RESULTS +79 86 AMP-PNP chemical However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. RESULTS +89 98 phosphate chemical However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. RESULTS +122 135 AMP-PNP-SePSK complex_assembly However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. RESULTS +140 154 AMP-PNP-AtXK-1 complex_assembly However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. RESULTS +155 165 structures evidence However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. RESULTS +198 201 ATP chemical However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. RESULTS +204 213 phosphate chemical However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. RESULTS +6 15 phosphate chemical The γ-phosphate group of ATP is transferred to the sugar substrate during the reaction process, so this flexibility might be important for the ability of these kinases. RESULTS +25 28 ATP chemical The γ-phosphate group of ATP is transferred to the sugar substrate during the reaction process, so this flexibility might be important for the ability of these kinases. RESULTS +51 56 sugar chemical The γ-phosphate group of ATP is transferred to the sugar substrate during the reaction process, so this flexibility might be important for the ability of these kinases. RESULTS +160 167 kinases protein_type The γ-phosphate group of ATP is transferred to the sugar substrate during the reaction process, so this flexibility might be important for the ability of these kinases. RESULTS +12 22 structures evidence The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. RESULTS +60 63 ADP chemical The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. RESULTS +68 75 AMP-PNP chemical The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. RESULTS +83 97 AMP-PNP-AtXK-1 complex_assembly The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. RESULTS +99 109 ADP-AtXK-1 complex_assembly The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. RESULTS +111 120 ADP-SePSK complex_assembly The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. RESULTS +125 138 AMP-PNP-SePSK complex_assembly The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. RESULTS +139 149 structures evidence The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. RESULTS +195 204 structure evidence The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. RESULTS +208 221 AMP-PNP-SePSK complex_assembly The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. RESULTS +24 29 SePSK protein As shown in Fig 3A, one SePSK protein molecule is in an asymmetric unit with one AMP-PNP molecule. RESULTS +81 88 AMP-PNP chemical As shown in Fig 3A, one SePSK protein molecule is in an asymmetric unit with one AMP-PNP molecule. RESULTS +4 11 AMP-PNP chemical The AMP-PNP is bound at the domain II, where it fits well inside a positively charged groove. RESULTS +28 37 domain II structure_element The AMP-PNP is bound at the domain II, where it fits well inside a positively charged groove. RESULTS +67 92 positively charged groove site The AMP-PNP is bound at the domain II, where it fits well inside a positively charged groove. RESULTS +4 26 AMP-PNP binding pocket site The AMP-PNP binding pocket consists of four α-helices (α26, α28, α27 and α30) and forms a shape resembling a half-fist (Fig 3A and 3B). RESULTS +39 53 four α-helices structure_element The AMP-PNP binding pocket consists of four α-helices (α26, α28, α27 and α30) and forms a shape resembling a half-fist (Fig 3A and 3B). RESULTS +55 58 α26 structure_element The AMP-PNP binding pocket consists of four α-helices (α26, α28, α27 and α30) and forms a shape resembling a half-fist (Fig 3A and 3B). RESULTS +60 63 α28 structure_element The AMP-PNP binding pocket consists of four α-helices (α26, α28, α27 and α30) and forms a shape resembling a half-fist (Fig 3A and 3B). RESULTS +65 68 α27 structure_element The AMP-PNP binding pocket consists of four α-helices (α26, α28, α27 and α30) and forms a shape resembling a half-fist (Fig 3A and 3B). RESULTS +73 76 α30 structure_element The AMP-PNP binding pocket consists of four α-helices (α26, α28, α27 and α30) and forms a shape resembling a half-fist (Fig 3A and 3B). RESULTS +90 118 shape resembling a half-fist protein_state The AMP-PNP binding pocket consists of four α-helices (α26, α28, α27 and α30) and forms a shape resembling a half-fist (Fig 3A and 3B). RESULTS +22 29 AMP-PNP chemical The head group of the AMP-PNP is embedded in a pocket surrounded by Trp383, Asn380, Gly376 and Gly377. RESULTS +47 53 pocket site The head group of the AMP-PNP is embedded in a pocket surrounded by Trp383, Asn380, Gly376 and Gly377. RESULTS +68 74 Trp383 residue_name_number The head group of the AMP-PNP is embedded in a pocket surrounded by Trp383, Asn380, Gly376 and Gly377. RESULTS +76 82 Asn380 residue_name_number The head group of the AMP-PNP is embedded in a pocket surrounded by Trp383, Asn380, Gly376 and Gly377. RESULTS +84 90 Gly376 residue_name_number The head group of the AMP-PNP is embedded in a pocket surrounded by Trp383, Asn380, Gly376 and Gly377. RESULTS +95 101 Gly377 residue_name_number The head group of the AMP-PNP is embedded in a pocket surrounded by Trp383, Asn380, Gly376 and Gly377. RESULTS +19 26 AMP-PNP chemical The purine ring of AMP-PNP is positioned in parallel to the indole ring of Trp383. RESULTS +75 81 Trp383 residue_name_number The purine ring of AMP-PNP is positioned in parallel to the indole ring of Trp383. RESULTS +19 34 hydrogen-bonded bond_interaction In addition, it is hydrogen-bonded with the side chain amide of Asn380 (Fig 3B). RESULTS +64 70 Asn380 residue_name_number In addition, it is hydrogen-bonded with the side chain amide of Asn380 (Fig 3B). RESULTS +12 19 AMP-PNP chemical The tail of AMP-PNP points to the hinge region of SePSK, and its α-phosphate and β-phosphate groups are stabilized by Gly376 and Ser243, respectively. RESULTS +34 46 hinge region structure_element The tail of AMP-PNP points to the hinge region of SePSK, and its α-phosphate and β-phosphate groups are stabilized by Gly376 and Ser243, respectively. RESULTS +50 55 SePSK protein The tail of AMP-PNP points to the hinge region of SePSK, and its α-phosphate and β-phosphate groups are stabilized by Gly376 and Ser243, respectively. RESULTS +67 76 phosphate chemical The tail of AMP-PNP points to the hinge region of SePSK, and its α-phosphate and β-phosphate groups are stabilized by Gly376 and Ser243, respectively. RESULTS +83 92 phosphate chemical The tail of AMP-PNP points to the hinge region of SePSK, and its α-phosphate and β-phosphate groups are stabilized by Gly376 and Ser243, respectively. RESULTS +118 124 Gly376 residue_name_number The tail of AMP-PNP points to the hinge region of SePSK, and its α-phosphate and β-phosphate groups are stabilized by Gly376 and Ser243, respectively. RESULTS +129 135 Ser243 residue_name_number The tail of AMP-PNP points to the hinge region of SePSK, and its α-phosphate and β-phosphate groups are stabilized by Gly376 and Ser243, respectively. RESULTS +15 24 structure evidence Together, this structure clearly shows that the AMP-PNP-β-phosphate is sticking out of the ATP binding pocket, thus the γ-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein. RESULTS +48 55 AMP-PNP chemical Together, this structure clearly shows that the AMP-PNP-β-phosphate is sticking out of the ATP binding pocket, thus the γ-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein. RESULTS +58 67 phosphate chemical Together, this structure clearly shows that the AMP-PNP-β-phosphate is sticking out of the ATP binding pocket, thus the γ-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein. RESULTS +91 109 ATP binding pocket site Together, this structure clearly shows that the AMP-PNP-β-phosphate is sticking out of the ATP binding pocket, thus the γ-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein. RESULTS +122 131 phosphate chemical Together, this structure clearly shows that the AMP-PNP-β-phosphate is sticking out of the ATP binding pocket, thus the γ-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein. RESULTS +168 176 domain I structure_element Together, this structure clearly shows that the AMP-PNP-β-phosphate is sticking out of the ATP binding pocket, thus the γ-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein. RESULTS +181 190 domain II structure_element Together, this structure clearly shows that the AMP-PNP-β-phosphate is sticking out of the ATP binding pocket, thus the γ-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein. RESULTS +0 9 Structure evidence Structure of SePSK in complex with AMP-PNP. FIG +13 18 SePSK protein Structure of SePSK in complex with AMP-PNP. FIG +19 34 in complex with protein_state Structure of SePSK in complex with AMP-PNP. FIG +35 42 AMP-PNP chemical Structure of SePSK in complex with AMP-PNP. FIG +8 24 electron density evidence (A) The electron density of AMP-PNP. FIG +28 35 AMP-PNP chemical (A) The electron density of AMP-PNP. FIG +4 9 SePSK protein The SePSK structure is shown in the electrostatic potential surface mode. FIG +10 19 structure evidence The SePSK structure is shown in the electrostatic potential surface mode. FIG +4 11 AMP-PNP chemical The AMP-PNP is depicted as sticks with its ǀFoǀ-ǀFcǀ map contoured at 3 σ shown as cyan mesh. FIG +43 56 ǀFoǀ-ǀFcǀ map evidence The AMP-PNP is depicted as sticks with its ǀFoǀ-ǀFcǀ map contoured at 3 σ shown as cyan mesh. FIG +8 30 AMP-PNP binding pocket site (B) The AMP-PNP binding pocket. FIG +12 19 AMP-PNP chemical The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383). FIG +23 36 sandwiched by bond_interaction The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383). FIG +52 58 Leu293 residue_name_number The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383). FIG +60 66 Gly376 residue_name_number The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383). FIG +68 74 Gly377 residue_name_number The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383). FIG +79 85 Trp383 residue_name_number The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383). FIG +9 18 α-helices structure_element The four α-helices (α26, α28, α27 and α30) are labeled in red. FIG +20 23 α26 structure_element The four α-helices (α26, α28, α27 and α30) are labeled in red. FIG +25 28 α28 structure_element The four α-helices (α26, α28, α27 and α30) are labeled in red. FIG +30 33 α27 structure_element The four α-helices (α26, α28, α27 and α30) are labeled in red. FIG +38 41 α30 structure_element The four α-helices (α26, α28, α27 and α30) are labeled in red. FIG +4 11 AMP-PNP chemical The AMP-PNP and coordinated residues are shown as sticks. FIG +14 36 substrate binding site site The potential substrate binding site in SePSK RESULTS +40 45 SePSK protein The potential substrate binding site in SePSK RESULTS +21 36 activity assays experimental_method The results from our activity assays suggested that SePSK has D-ribulose kinase activity. RESULTS +52 57 SePSK protein The results from our activity assays suggested that SePSK has D-ribulose kinase activity. RESULTS +62 79 D-ribulose kinase protein_type The results from our activity assays suggested that SePSK has D-ribulose kinase activity. RESULTS +53 58 SePSK protein To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. RESULTS +63 73 D-ribulose chemical To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. RESULTS +79 82 apo protein_state To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. RESULTS +83 88 SePSK protein To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. RESULTS +89 114 crystals were soaked into experimental_method To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. RESULTS +119 128 reservoir experimental_method To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. RESULTS +140 150 D-ribulose chemical To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. RESULTS +152 155 RBL chemical To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. RESULTS +165 174 RBL-SePSK complex_assembly To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. RESULTS +175 184 structure evidence To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. RESULTS +189 195 solved experimental_method To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. RESULTS +33 51 electron densities evidence As shown in S6 Fig, two residual electron densities are visible in domain I, which can be interpreted as two D-ribulose molecules with reasonable fit. RESULTS +67 75 domain I structure_element As shown in S6 Fig, two residual electron densities are visible in domain I, which can be interpreted as two D-ribulose molecules with reasonable fit. RESULTS +109 119 D-ribulose chemical As shown in S6 Fig, two residual electron densities are visible in domain I, which can be interpreted as two D-ribulose molecules with reasonable fit. RESULTS +76 86 D-ribulose chemical As shown in Fig 4A, the nearest distance between the carbon skeleton of two D-ribulose molecules are approx. RESULTS +7 11 RBL1 residue_name_number 7.1 Å (RBL1-C4 and RBL2-C1). RESULTS +19 23 RBL2 residue_name_number 7.1 Å (RBL1-C4 and RBL2-C1). RESULTS +0 4 RBL1 residue_name_number RBL1 is located in the pocket consisting of α21 and the loop between β6 and β7. RESULTS +23 29 pocket site RBL1 is located in the pocket consisting of α21 and the loop between β6 and β7. RESULTS +44 47 α21 structure_element RBL1 is located in the pocket consisting of α21 and the loop between β6 and β7. RESULTS +56 60 loop structure_element RBL1 is located in the pocket consisting of α21 and the loop between β6 and β7. RESULTS +69 78 β6 and β7 structure_element RBL1 is located in the pocket consisting of α21 and the loop between β6 and β7. RESULTS +17 21 RBL1 residue_name_number The O4 and O5 of RBL1 are coordinated with the side chain carboxyl group of Asp221. RESULTS +26 42 coordinated with bond_interaction The O4 and O5 of RBL1 are coordinated with the side chain carboxyl group of Asp221. RESULTS +76 82 Asp221 residue_name_number The O4 and O5 of RBL1 are coordinated with the side chain carboxyl group of Asp221. RESULTS +23 27 RBL1 residue_name_number Furthermore, the O2 of RBL1 interacts with the main chain amide nitrogen of Ser72 (Fig 4B). RESULTS +28 42 interacts with bond_interaction Furthermore, the O2 of RBL1 interacts with the main chain amide nitrogen of Ser72 (Fig 4B). RESULTS +76 81 Ser72 residue_name_number Furthermore, the O2 of RBL1 interacts with the main chain amide nitrogen of Ser72 (Fig 4B). RESULTS +5 11 pocket site This pocket is at a similar position of substrate binding site of other sugar kinase, such as L-ribulokinase (PDB code: 3QDK) (S7 Fig). RESULTS +40 62 substrate binding site site This pocket is at a similar position of substrate binding site of other sugar kinase, such as L-ribulokinase (PDB code: 3QDK) (S7 Fig). RESULTS +72 84 sugar kinase protein_type This pocket is at a similar position of substrate binding site of other sugar kinase, such as L-ribulokinase (PDB code: 3QDK) (S7 Fig). RESULTS +94 108 L-ribulokinase protein This pocket is at a similar position of substrate binding site of other sugar kinase, such as L-ribulokinase (PDB code: 3QDK) (S7 Fig). RESULTS +9 30 structural comparison experimental_method However, structural comparison shows that the substrate ligating residues between the two structures are not strictly conserved. RESULTS +90 100 structures evidence However, structural comparison shows that the substrate ligating residues between the two structures are not strictly conserved. RESULTS +105 127 not strictly conserved protein_state However, structural comparison shows that the substrate ligating residues between the two structures are not strictly conserved. RESULTS +13 23 structures evidence Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +50 54 RBL1 residue_name_number Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +58 67 RBL-SePSK complex_assembly Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +68 77 structure evidence Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +82 87 Ser72 residue_name_number Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +89 95 Asp221 residue_name_number Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +100 106 Ser222 residue_name_number Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +140 150 L-ribulose chemical Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +156 170 L-ribulokinase protein Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +175 180 Ala96 residue_name_number Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +182 188 Lys208 residue_name_number Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +190 196 Asp274 residue_name_number Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +201 207 Glu329 residue_name_number Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). RESULTS +0 6 Glu329 residue_name_number Glu329 in 3QDK has no counterpart in RBL-SePSK structure. RESULTS +37 46 RBL-SePSK complex_assembly Glu329 in 3QDK has no counterpart in RBL-SePSK structure. RESULTS +47 56 structure evidence Glu329 in 3QDK has no counterpart in RBL-SePSK structure. RESULTS +22 28 Lys208 residue_name_number In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. RESULTS +32 46 L-ribulokinase protein In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. RESULTS +78 84 Lys163 residue_name_number In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. RESULTS +89 98 RBL-SePSK complex_assembly In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. RESULTS +99 108 structure evidence In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. RESULTS +114 127 hydrogen bond bond_interaction In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. RESULTS +131 137 Lys163 residue_name_number In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. RESULTS +192 201 α-helices structure_element In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. RESULTS +203 205 α9 structure_element In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. RESULTS +210 213 α13 structure_element In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. RESULTS +218 223 SePSK protein In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. RESULTS +15 25 D-ribulose chemical The binding of D-ribulose (RBL) with SePSK. FIG +27 30 RBL chemical The binding of D-ribulose (RBL) with SePSK. FIG +37 42 SePSK protein The binding of D-ribulose (RBL) with SePSK. FIG +8 43 electrostatic potential surface map evidence (A) The electrostatic potential surface map of RBL-SePSK and a zoom-in view of RBL binding site. FIG +47 56 RBL-SePSK complex_assembly (A) The electrostatic potential surface map of RBL-SePSK and a zoom-in view of RBL binding site. FIG +79 95 RBL binding site site (A) The electrostatic potential surface map of RBL-SePSK and a zoom-in view of RBL binding site. FIG +4 8 RBL1 residue_name_number The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK. FIG +13 17 RBL2 residue_name_number The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK. FIG +65 75 D-ribulose chemical The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK. FIG +87 91 RBL1 residue_name_number The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK. FIG +96 100 RBL2 residue_name_number The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK. FIG +107 112 SePSK protein The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK. FIG +4 7 RBL chemical The RBL molecules (carbon atoms colored yellow) and amino acid residues of SePSK (carbon atoms colored green) involved in RBL interaction are shown as sticks. FIG +75 80 SePSK protein The RBL molecules (carbon atoms colored yellow) and amino acid residues of SePSK (carbon atoms colored green) involved in RBL interaction are shown as sticks. FIG +122 125 RBL chemical The RBL molecules (carbon atoms colored yellow) and amino acid residues of SePSK (carbon atoms colored green) involved in RBL interaction are shown as sticks. FIG +4 18 hydrogen bonds bond_interaction The hydrogen bonds are indicated by the black dashed lines and the numbers near the dashed lines are the distances (Å). (C) The binding affinity assays of SePSK with D-ribulose. FIG +128 151 binding affinity assays experimental_method The hydrogen bonds are indicated by the black dashed lines and the numbers near the dashed lines are the distances (Å). (C) The binding affinity assays of SePSK with D-ribulose. FIG +155 160 SePSK protein The hydrogen bonds are indicated by the black dashed lines and the numbers near the dashed lines are the distances (Å). (C) The binding affinity assays of SePSK with D-ribulose. FIG +166 176 D-ribulose chemical The hydrogen bonds are indicated by the black dashed lines and the numbers near the dashed lines are the distances (Å). (C) The binding affinity assays of SePSK with D-ribulose. FIG +0 25 Single-cycle kinetic data experimental_method Single-cycle kinetic data are reflecting the interaction of SePSK and D8A-SePSK with D-ribulose. FIG +60 65 SePSK protein Single-cycle kinetic data are reflecting the interaction of SePSK and D8A-SePSK with D-ribulose. FIG +70 73 D8A mutant Single-cycle kinetic data are reflecting the interaction of SePSK and D8A-SePSK with D-ribulose. FIG +74 79 SePSK protein Single-cycle kinetic data are reflecting the interaction of SePSK and D8A-SePSK with D-ribulose. FIG +85 95 D-ribulose chemical Single-cycle kinetic data are reflecting the interaction of SePSK and D8A-SePSK with D-ribulose. FIG +26 37 sensorgrams evidence It shows two experimental sensorgrams after minus the empty sensorgrams. FIG +60 71 sensorgrams evidence It shows two experimental sensorgrams after minus the empty sensorgrams. FIG +92 101 wild type protein_state The original data is shown as black curve, and the fitted data is shown as different color (wild type SePSK: red curve, D8A-SePSK: green curve). FIG +102 107 SePSK protein The original data is shown as black curve, and the fitted data is shown as different color (wild type SePSK: red curve, D8A-SePSK: green curve). FIG +120 123 D8A mutant The original data is shown as black curve, and the fitted data is shown as different color (wild type SePSK: red curve, D8A-SePSK: green curve). FIG +124 129 SePSK protein The original data is shown as black curve, and the fitted data is shown as different color (wild type SePSK: red curve, D8A-SePSK: green curve). FIG +0 26 Dissociation rate constant evidence Dissociation rate constant of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. FIG +30 39 wild type protein_state Dissociation rate constant of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. FIG +44 47 D8A mutant Dissociation rate constant of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. FIG +48 53 SePSK protein Dissociation rate constant of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. FIG +4 18 binding pocket site The binding pocket of RBL2 with relatively weak electron density is near the N-terminal region of SePSK and is negatively charged. RESULTS +22 26 RBL2 residue_name_number The binding pocket of RBL2 with relatively weak electron density is near the N-terminal region of SePSK and is negatively charged. RESULTS +48 64 electron density evidence The binding pocket of RBL2 with relatively weak electron density is near the N-terminal region of SePSK and is negatively charged. RESULTS +98 103 SePSK protein The binding pocket of RBL2 with relatively weak electron density is near the N-terminal region of SePSK and is negatively charged. RESULTS +18 22 Asp8 residue_name_number The side chain of Asp8 interacts strongly with O3 and O4 of RBL2. RESULTS +23 46 interacts strongly with bond_interaction The side chain of Asp8 interacts strongly with O3 and O4 of RBL2. RESULTS +60 64 RBL2 residue_name_number The side chain of Asp8 interacts strongly with O3 and O4 of RBL2. RESULTS +22 27 Ser12 residue_name_number The hydroxyl group of Ser12 coordinates with O2 of RBL2. RESULTS +28 44 coordinates with bond_interaction The hydroxyl group of Ser12 coordinates with O2 of RBL2. RESULTS +51 55 RBL2 residue_name_number The hydroxyl group of Ser12 coordinates with O2 of RBL2. RESULTS +32 37 Gly13 residue_name_number The backbone amide nitrogens of Gly13 and Arg15 also keep hydrogen bonds with RBL2 (Fig 4B). RESULTS +42 47 Arg15 residue_name_number The backbone amide nitrogens of Gly13 and Arg15 also keep hydrogen bonds with RBL2 (Fig 4B). RESULTS +58 72 hydrogen bonds bond_interaction The backbone amide nitrogens of Gly13 and Arg15 also keep hydrogen bonds with RBL2 (Fig 4B). RESULTS +78 82 RBL2 residue_name_number The backbone amide nitrogens of Gly13 and Arg15 also keep hydrogen bonds with RBL2 (Fig 4B). RESULTS +0 21 Structural comparison experimental_method Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. RESULTS +25 30 SePSK protein Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. RESULTS +35 41 AtXK-1 protein Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. RESULTS +64 83 RBL1 binding pocket site Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. RESULTS +87 96 conserved protein_state Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. RESULTS +102 113 RBL2 pocket site Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. RESULTS +130 136 AtXK-1 protein Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. RESULTS +137 146 structure evidence Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. RESULTS +200 204 RBL2 residue_name_number Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. RESULTS +209 225 highly conserved protein_state Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. RESULTS +7 16 RBL-SePSK complex_assembly In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. RESULTS +17 26 structure evidence In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. RESULTS +36 49 hydrogen bond bond_interaction In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. RESULTS +69 73 RBL2 residue_name_number In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. RESULTS +78 83 Ser12 residue_name_number In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. RESULTS +107 113 AtXK-1 protein In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. RESULTS +114 123 structure evidence In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. RESULTS +129 142 hydrogen bond bond_interaction In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. RESULTS +175 180 Ser22 residue_name_number In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. RESULTS +71 79 β-sheets structure_element This break is probably induced by the conformational change of the two β-sheets (β1 and β2), with the result that the linking loop (loop 1) is located further away from the RBL2 binding site. RESULTS +81 83 β1 structure_element This break is probably induced by the conformational change of the two β-sheets (β1 and β2), with the result that the linking loop (loop 1) is located further away from the RBL2 binding site. RESULTS +88 90 β2 structure_element This break is probably induced by the conformational change of the two β-sheets (β1 and β2), with the result that the linking loop (loop 1) is located further away from the RBL2 binding site. RESULTS +118 130 linking loop structure_element This break is probably induced by the conformational change of the two β-sheets (β1 and β2), with the result that the linking loop (loop 1) is located further away from the RBL2 binding site. RESULTS +132 138 loop 1 structure_element This break is probably induced by the conformational change of the two β-sheets (β1 and β2), with the result that the linking loop (loop 1) is located further away from the RBL2 binding site. RESULTS +173 190 RBL2 binding site site This break is probably induced by the conformational change of the two β-sheets (β1 and β2), with the result that the linking loop (loop 1) is located further away from the RBL2 binding site. RESULTS +37 43 AtXK-1 protein This change might be the reason that AtXK-1 only shows limited increasing in its ATP hydrolysis ability upon adding D-ribulose as a substrate after comparing with SePSK (Fig 2C). RESULTS +81 84 ATP chemical This change might be the reason that AtXK-1 only shows limited increasing in its ATP hydrolysis ability upon adding D-ribulose as a substrate after comparing with SePSK (Fig 2C). RESULTS +116 126 D-ribulose chemical This change might be the reason that AtXK-1 only shows limited increasing in its ATP hydrolysis ability upon adding D-ribulose as a substrate after comparing with SePSK (Fig 2C). RESULTS +163 168 SePSK protein This change might be the reason that AtXK-1 only shows limited increasing in its ATP hydrolysis ability upon adding D-ribulose as a substrate after comparing with SePSK (Fig 2C). RESULTS +4 9 SePSK protein Our SePSK structure shows that the Asp8 residue forms strong hydrogen bond with RBL2 (Fig 4B). RESULTS +10 19 structure evidence Our SePSK structure shows that the Asp8 residue forms strong hydrogen bond with RBL2 (Fig 4B). RESULTS +35 39 Asp8 residue_name_number Our SePSK structure shows that the Asp8 residue forms strong hydrogen bond with RBL2 (Fig 4B). RESULTS +61 74 hydrogen bond bond_interaction Our SePSK structure shows that the Asp8 residue forms strong hydrogen bond with RBL2 (Fig 4B). RESULTS +80 84 RBL2 residue_name_number Our SePSK structure shows that the Asp8 residue forms strong hydrogen bond with RBL2 (Fig 4B). RESULTS +17 33 enzymatic assays experimental_method In addition, our enzymatic assays indicated that Asp8 is important for the activity of SePSK (Fig 2D). RESULTS +49 53 Asp8 residue_name_number In addition, our enzymatic assays indicated that Asp8 is important for the activity of SePSK (Fig 2D). RESULTS +87 92 SePSK protein In addition, our enzymatic assays indicated that Asp8 is important for the activity of SePSK (Fig 2D). RESULTS +49 65 binding affinity evidence To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method. RESULTS +70 80 D-ribulose chemical To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method. RESULTS +89 98 wild type protein_state To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method. RESULTS +100 102 WT protein_state To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method. RESULTS +108 111 D8A mutant To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method. RESULTS +112 118 mutant protein_state To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method. RESULTS +122 127 SePSK protein To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method. RESULTS +136 168 surface plasmon resonance method experimental_method To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method. RESULTS +28 36 affinity evidence The results showed that the affinity of D8A-SePSK with D-ribulose is weaker than that of WT with a reduction of approx. RESULTS +40 43 D8A mutant The results showed that the affinity of D8A-SePSK with D-ribulose is weaker than that of WT with a reduction of approx. RESULTS +44 49 SePSK protein The results showed that the affinity of D8A-SePSK with D-ribulose is weaker than that of WT with a reduction of approx. RESULTS +55 65 D-ribulose chemical The results showed that the affinity of D8A-SePSK with D-ribulose is weaker than that of WT with a reduction of approx. RESULTS +89 91 WT protein_state The results showed that the affinity of D8A-SePSK with D-ribulose is weaker than that of WT with a reduction of approx. RESULTS +0 26 Dissociation rate constant evidence Dissociation rate constant (Kd) of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. RESULTS +28 30 Kd evidence Dissociation rate constant (Kd) of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. RESULTS +35 44 wild type protein_state Dissociation rate constant (Kd) of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. RESULTS +49 52 D8A mutant Dissociation rate constant (Kd) of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. RESULTS +53 58 SePSK protein Dissociation rate constant (Kd) of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. RESULTS +29 52 second RBL binding site site The results implied that the second RBL binding site plays a role in the D-ribulose kinase function of SePSK. RESULTS +73 90 D-ribulose kinase protein_type The results implied that the second RBL binding site plays a role in the D-ribulose kinase function of SePSK. RESULTS +103 108 SePSK protein The results implied that the second RBL binding site plays a role in the D-ribulose kinase function of SePSK. RESULTS +47 57 D-ribulose chemical However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact. RESULTS +67 82 crystal soaking experimental_method However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact. RESULTS +115 131 electron density evidence However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact. RESULTS +135 139 RBL2 residue_name_number However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact. RESULTS +170 189 second binding site site However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact. RESULTS +193 203 D-ribulose chemical However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact. RESULTS +207 212 SePSK protein However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact. RESULTS +35 40 SePSK protein Simulated conformational change of SePSK during the catalytic process RESULTS +60 68 domain I structure_element It was reported earlier that the crossing angle between the domain I and domain II in FGGY family carbohydrate kinases is different. RESULTS +73 82 domain II structure_element It was reported earlier that the crossing angle between the domain I and domain II in FGGY family carbohydrate kinases is different. RESULTS +86 118 FGGY family carbohydrate kinases protein_type It was reported earlier that the crossing angle between the domain I and domain II in FGGY family carbohydrate kinases is different. RESULTS +79 82 ATP chemical In addition, this difference may be caused by the binding of substrates and/or ATP. RESULTS +39 51 sugar kinase protein_type As reported previously, members of the sugar kinase family undergo a conformational change to narrow the crossing angle between two domains and reduce the distance between substrate and ATP in order to facilitate the catalytic reaction of phosphorylation of sugar substrates. RESULTS +186 189 ATP chemical As reported previously, members of the sugar kinase family undergo a conformational change to narrow the crossing angle between two domains and reduce the distance between substrate and ATP in order to facilitate the catalytic reaction of phosphorylation of sugar substrates. RESULTS +239 254 phosphorylation ptm As reported previously, members of the sugar kinase family undergo a conformational change to narrow the crossing angle between two domains and reduce the distance between substrate and ATP in order to facilitate the catalytic reaction of phosphorylation of sugar substrates. RESULTS +16 26 structures evidence After comparing structures of apo-SePSK, RBL-SePSK and AMP-PNP-SePSK, we noticed that these structures presented here are similar. RESULTS +30 33 apo protein_state After comparing structures of apo-SePSK, RBL-SePSK and AMP-PNP-SePSK, we noticed that these structures presented here are similar. RESULTS +34 39 SePSK protein After comparing structures of apo-SePSK, RBL-SePSK and AMP-PNP-SePSK, we noticed that these structures presented here are similar. RESULTS +41 50 RBL-SePSK complex_assembly After comparing structures of apo-SePSK, RBL-SePSK and AMP-PNP-SePSK, we noticed that these structures presented here are similar. RESULTS +55 68 AMP-PNP-SePSK complex_assembly After comparing structures of apo-SePSK, RBL-SePSK and AMP-PNP-SePSK, we noticed that these structures presented here are similar. RESULTS +92 102 structures evidence After comparing structures of apo-SePSK, RBL-SePSK and AMP-PNP-SePSK, we noticed that these structures presented here are similar. RESULTS +0 11 Superposing experimental_method Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). RESULTS +16 26 structures evidence Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). RESULTS +30 39 RBL-SePSK complex_assembly Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). RESULTS +44 57 AMP-PNP-SePSK complex_assembly Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). RESULTS +110 117 AMP-PNP chemical Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). RESULTS +120 129 phosphate chemical Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). RESULTS +134 138 RBL1 residue_name_number Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). RESULTS +139 143 RBL2 residue_name_number Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). RESULTS +154 158 RBL1 residue_name_number Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). RESULTS +170 174 RBL2 residue_name_number Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). RESULTS +44 53 phosphate chemical This distance is too long to transfer the γ-phosphate group from ATP to the substrate. RESULTS +65 68 ATP chemical This distance is too long to transfer the γ-phosphate group from ATP to the substrate. RESULTS +25 30 SePSK protein Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates. RESULTS +60 69 structure evidence Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates. RESULTS +95 105 structures evidence Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates. RESULTS +109 114 SePSK protein Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates. RESULTS +129 133 open protein_state Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates. RESULTS +208 214 closed protein_state Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates. RESULTS +269 284 phosphorylation ptm Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates. RESULTS +53 63 simulation experimental_method For studying such potential conformational change, a simulation on the Hingeprot Server was performed to predict the movement of different SePSK domains. RESULTS +71 87 Hingeprot Server experimental_method For studying such potential conformational change, a simulation on the Hingeprot Server was performed to predict the movement of different SePSK domains. RESULTS +139 144 SePSK protein For studying such potential conformational change, a simulation on the Hingeprot Server was performed to predict the movement of different SePSK domains. RESULTS +24 32 domain I structure_element The results showed that domain I and domain II are closer to each other with Ala228 and Thr401 in A2 as Hinge-residues. RESULTS +37 46 domain II structure_element The results showed that domain I and domain II are closer to each other with Ala228 and Thr401 in A2 as Hinge-residues. RESULTS +77 83 Ala228 residue_name_number The results showed that domain I and domain II are closer to each other with Ala228 and Thr401 in A2 as Hinge-residues. RESULTS +88 94 Thr401 residue_name_number The results showed that domain I and domain II are closer to each other with Ala228 and Thr401 in A2 as Hinge-residues. RESULTS +98 100 A2 structure_element The results showed that domain I and domain II are closer to each other with Ala228 and Thr401 in A2 as Hinge-residues. RESULTS +104 118 Hinge-residues structure_element The results showed that domain I and domain II are closer to each other with Ala228 and Thr401 in A2 as Hinge-residues. RESULTS +28 33 SePSK protein Based on the above results, SePSK is divided into two rigid parts. RESULTS +4 12 domain I structure_element The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +16 25 RBL-SePSK complex_assembly The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +31 36 1–228 residue_range The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +42 49 402–421 residue_range The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +59 68 domain II structure_element The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +72 85 AMP-PNP-SePSK complex_assembly The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +91 98 229–401 residue_range The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +105 115 superposed experimental_method The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +121 131 structures evidence The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +143 146 apo protein_state The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +147 153 AtXK-1 protein The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +155 158 apo protein_state The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +159 164 SePSK protein The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +166 181 xylulose kinase protein_type The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +187 212 Lactobacillus acidophilus species The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +238 242 S58W mutant The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +243 249 mutant protein_state The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +258 273 glycerol kinase protein_type The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +279 295 Escherichia coli species The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). RESULTS +15 28 superposition experimental_method The results of superposition displayed different crossing angle between these two domains. RESULTS +6 19 superposition experimental_method After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). RESULTS +38 45 AMP-PNP chemical After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). RESULTS +48 57 phosphate chemical After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). RESULTS +90 94 RBL1 residue_name_number After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). RESULTS +106 116 superposed experimental_method After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). RESULTS +122 128 AtXK-1 protein After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). RESULTS +138 148 superposed experimental_method After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). RESULTS +154 159 SePSK protein After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). RESULTS +169 179 superposed experimental_method After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). RESULTS +202 212 superposed experimental_method After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). RESULTS +28 35 AMP-PNP chemical Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). RESULTS +38 47 phosphate chemical Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). RESULTS +80 84 RBL2 residue_name_number Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). RESULTS +96 106 superposed experimental_method Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). RESULTS +112 118 AtXK-1 protein Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). RESULTS +128 138 superposed experimental_method Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). RESULTS +144 149 SePSK protein Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). RESULTS +159 169 superposed experimental_method Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). RESULTS +188 195 AMP-PNP chemical Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). RESULTS +198 207 phosphate chemical Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). RESULTS +223 227 RBL2 residue_name_number Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). RESULTS +234 247 superposition experimental_method Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). RESULTS +22 26 RBL2 residue_name_number This distance between RBL2 and AMP-PNP-γ-phosphate is close enough to facilitate phosphate transferring. RESULTS +31 38 AMP-PNP chemical This distance between RBL2 and AMP-PNP-γ-phosphate is close enough to facilitate phosphate transferring. RESULTS +41 50 phosphate chemical This distance between RBL2 and AMP-PNP-γ-phosphate is close enough to facilitate phosphate transferring. RESULTS +81 90 phosphate chemical This distance between RBL2 and AMP-PNP-γ-phosphate is close enough to facilitate phosphate transferring. RESULTS +14 27 superposition experimental_method Together, our superposition results provided snapshots of the conformational changes at different catalytic stages of SePSK and potentially revealed the closed form of SePSK. RESULTS +118 123 SePSK protein Together, our superposition results provided snapshots of the conformational changes at different catalytic stages of SePSK and potentially revealed the closed form of SePSK. RESULTS +153 159 closed protein_state Together, our superposition results provided snapshots of the conformational changes at different catalytic stages of SePSK and potentially revealed the closed form of SePSK. RESULTS +168 173 SePSK protein Together, our superposition results provided snapshots of the conformational changes at different catalytic stages of SePSK and potentially revealed the closed form of SePSK. RESULTS +35 40 SePSK protein Simulated conformational change of SePSK during the catalytic process. FIG +4 14 structures evidence The structures are shown as cartoon and the ligands are shown as sticks. FIG +0 8 Domain I structure_element Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. FIG +14 30 D-ribulose-SePSK complex_assembly Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. FIG +43 52 Domain II structure_element Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. FIG +58 71 AMP-PNP-SePSK complex_assembly Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. FIG +83 93 superposed experimental_method Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. FIG +99 102 apo protein_state Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. FIG +103 109 AtXK-1 protein Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. FIG +117 120 apo protein_state Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. FIG +121 126 SePSK protein Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. FIG +92 95 RBL chemical The numbers near the black dashed lines show the distances (Å) between two nearest atoms of RBL and AMP-PNP. FIG +100 107 AMP-PNP chemical The numbers near the black dashed lines show the distances (Å) between two nearest atoms of RBL and AMP-PNP. FIG +16 49 structural and enzymatic analyses experimental_method In summary, our structural and enzymatic analyses provide evidence that SePSK shows D-ribulose kinase activity, and exhibits the conserved features of FGGY family carbohydrate kinases. RESULTS +72 77 SePSK protein In summary, our structural and enzymatic analyses provide evidence that SePSK shows D-ribulose kinase activity, and exhibits the conserved features of FGGY family carbohydrate kinases. RESULTS +84 101 D-ribulose kinase protein_type In summary, our structural and enzymatic analyses provide evidence that SePSK shows D-ribulose kinase activity, and exhibits the conserved features of FGGY family carbohydrate kinases. RESULTS +151 183 FGGY family carbohydrate kinases protein_type In summary, our structural and enzymatic analyses provide evidence that SePSK shows D-ribulose kinase activity, and exhibits the conserved features of FGGY family carbohydrate kinases. RESULTS +6 15 conserved site Three conserved residues in SePSK were identified to be essential for this function. RESULTS +28 33 SePSK protein Three conserved residues in SePSK were identified to be essential for this function. RESULTS +70 75 SePSK protein Our results provide the detailed information about the interaction of SePSK with ATP and substrates. RESULTS +81 84 ATP chemical Our results provide the detailed information about the interaction of SePSK with ATP and substrates. RESULTS +10 34 structural superposition experimental_method Moreover, structural superposition results enable us to visualize the conformational change of SePSK during the catalytic process. RESULTS +95 100 SePSK protein Moreover, structural superposition results enable us to visualize the conformational change of SePSK during the catalytic process. RESULTS +112 117 SePSK protein In conclusion, our results provide important information for a more detailed understanding of the mechanisms of SePSK and other members of FGGY family carbohydrate kinases. RESULTS +139 171 FGGY family carbohydrate kinases protein_type In conclusion, our results provide important information for a more detailed understanding of the mechanisms of SePSK and other members of FGGY family carbohydrate kinases. RESULTS diff --git a/annotation_CSV/PMC4885502.csv b/annotation_CSV/PMC4885502.csv new file mode 100644 index 0000000000000000000000000000000000000000..aea59af098753a297f2c6b3851009b8f979cc218 --- /dev/null +++ b/annotation_CSV/PMC4885502.csv @@ -0,0 +1,578 @@ +anno_start anno_end anno_text entity_type sentence section +6 13 Cryo-EM experimental_method Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase TITLE +78 101 Glutamate Dehydrogenase protein_type Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase TITLE +0 24 Cryo-electron microscopy experimental_method Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. ABSTRACT +26 33 cryo-EM experimental_method Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. ABSTRACT +75 85 structures evidence Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. ABSTRACT +30 53 glutamate dehydrogenase protein_type We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. ABSTRACT +55 58 GDH protein_type We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. ABSTRACT +132 141 glutamate chemical We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. ABSTRACT +17 20 GDH protein_type Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. ABSTRACT +39 46 cryo-EM experimental_method Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. ABSTRACT +47 57 structures evidence Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. ABSTRACT +106 109 GDH protein_type Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. ABSTRACT +151 169 crystal structures evidence Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. ABSTRACT +41 45 NADH chemical We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an “open” or “closed” state. ABSTRACT +71 74 GTP chemical We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an “open” or “closed” state. ABSTRACT +140 144 open protein_state We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an “open” or “closed” state. ABSTRACT +150 156 closed protein_state We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an “open” or “closed” state. ABSTRACT +12 21 structure evidence Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. ABSTRACT +25 29 NADH chemical Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. ABSTRACT +37 48 active site site Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. ABSTRACT +72 76 open protein_state Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. ABSTRACT +81 87 closed protein_state Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. ABSTRACT +132 147 regulatory site site Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. ABSTRACT +117 138 X-ray crystallography experimental_method Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes. ABSTRACT +140 147 cryo-EM experimental_method Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes. ABSTRACT +19 43 cryo-electron microscopy experimental_method Recent advances in cryo-electron microscopy (cryo-EM) allow determination of structures of small protein complexes and membrane proteins at near-atomic resolution, marking a critical shift in the structural biology field. INTRO +45 52 cryo-EM experimental_method Recent advances in cryo-electron microscopy (cryo-EM) allow determination of structures of small protein complexes and membrane proteins at near-atomic resolution, marking a critical shift in the structural biology field. INTRO +77 87 structures evidence Recent advances in cryo-electron microscopy (cryo-EM) allow determination of structures of small protein complexes and membrane proteins at near-atomic resolution, marking a critical shift in the structural biology field. INTRO +150 165 crystallization experimental_method One specific area of broad general interest in drug discovery is the localization of bound ligands and cofactors under conditions in which efforts at crystallization have not been successful because of structural heterogeneity. INTRO +7 14 cryo-EM experimental_method Recent cryo-EM analyses have already demonstrated that it is now possible to use single-particle cryo-EM methods to localize small bound ligands or inhibitors on target proteins. INTRO +81 104 single-particle cryo-EM experimental_method Recent cryo-EM analyses have already demonstrated that it is now possible to use single-particle cryo-EM methods to localize small bound ligands or inhibitors on target proteins. INTRO +37 46 mammalian taxonomy_domain Here, we address this question using mammalian glutamate dehydrogenase as an example. INTRO +47 70 glutamate dehydrogenase protein_type Here, we address this question using mammalian glutamate dehydrogenase as an example. INTRO +0 23 Glutamate dehydrogenase protein_type Glutamate dehydrogenase (GDH) is a highly conserved enzyme expressed in most organisms. INTRO +25 28 GDH protein_type Glutamate dehydrogenase (GDH) is a highly conserved enzyme expressed in most organisms. INTRO +35 51 highly conserved protein_state Glutamate dehydrogenase (GDH) is a highly conserved enzyme expressed in most organisms. INTRO +0 3 GDH protein_type GDH plays a central role in glutamate metabolism by catalyzing the reversible oxidative deamination of glutamate to generate α-ketoglutarate and ammonia, with the concomitant transfer of a pair of electrons to either NAD+ or NADP+. INTRO +28 37 glutamate chemical GDH plays a central role in glutamate metabolism by catalyzing the reversible oxidative deamination of glutamate to generate α-ketoglutarate and ammonia, with the concomitant transfer of a pair of electrons to either NAD+ or NADP+. INTRO +103 112 glutamate chemical GDH plays a central role in glutamate metabolism by catalyzing the reversible oxidative deamination of glutamate to generate α-ketoglutarate and ammonia, with the concomitant transfer of a pair of electrons to either NAD+ or NADP+. INTRO +125 140 α-ketoglutarate chemical GDH plays a central role in glutamate metabolism by catalyzing the reversible oxidative deamination of glutamate to generate α-ketoglutarate and ammonia, with the concomitant transfer of a pair of electrons to either NAD+ or NADP+. INTRO +145 152 ammonia chemical GDH plays a central role in glutamate metabolism by catalyzing the reversible oxidative deamination of glutamate to generate α-ketoglutarate and ammonia, with the concomitant transfer of a pair of electrons to either NAD+ or NADP+. INTRO +217 221 NAD+ chemical GDH plays a central role in glutamate metabolism by catalyzing the reversible oxidative deamination of glutamate to generate α-ketoglutarate and ammonia, with the concomitant transfer of a pair of electrons to either NAD+ or NADP+. INTRO +225 230 NADP+ chemical GDH plays a central role in glutamate metabolism by catalyzing the reversible oxidative deamination of glutamate to generate α-ketoglutarate and ammonia, with the concomitant transfer of a pair of electrons to either NAD+ or NADP+. INTRO +14 17 GDH protein_type Regulation of GDH is tightly controlled through multiple allosteric mechanisms. INTRO +10 50 biochemical and crystallographic studies experimental_method Extensive biochemical and crystallographic studies have characterized the enzymatic activity of GDH and its modulation by a chemically diverse group of compounds such as nucleotides, amino acids, steroid hormones, antipsychotic drugs, and natural products. INTRO +96 99 GDH protein_type Extensive biochemical and crystallographic studies have characterized the enzymatic activity of GDH and its modulation by a chemically diverse group of compounds such as nucleotides, amino acids, steroid hormones, antipsychotic drugs, and natural products. INTRO +0 30 X-ray crystallographic studies experimental_method X-ray crystallographic studies have shown that the functional unit of GDH is a homohexamer composed of a trimer of dimers, with a 3-fold axis and an equatorial plane that define its D3 symmetry (Fig. 1A). INTRO +70 73 GDH protein_type X-ray crystallographic studies have shown that the functional unit of GDH is a homohexamer composed of a trimer of dimers, with a 3-fold axis and an equatorial plane that define its D3 symmetry (Fig. 1A). INTRO +79 90 homohexamer oligomeric_state X-ray crystallographic studies have shown that the functional unit of GDH is a homohexamer composed of a trimer of dimers, with a 3-fold axis and an equatorial plane that define its D3 symmetry (Fig. 1A). INTRO +105 111 trimer oligomeric_state X-ray crystallographic studies have shown that the functional unit of GDH is a homohexamer composed of a trimer of dimers, with a 3-fold axis and an equatorial plane that define its D3 symmetry (Fig. 1A). INTRO +115 121 dimers oligomeric_state X-ray crystallographic studies have shown that the functional unit of GDH is a homohexamer composed of a trimer of dimers, with a 3-fold axis and an equatorial plane that define its D3 symmetry (Fig. 1A). INTRO +12 20 protomer oligomeric_state Each 56-kDa protomer consists of three domains. INTRO +30 45 dimer interface site The first is located near the dimer interface and forms the core of the hexamer. INTRO +72 79 hexamer oligomeric_state The first is located near the dimer interface and forms the core of the hexamer. INTRO +14 39 nucleotide-binding domain structure_element The second, a nucleotide-binding domain (NBD) with a Rossmann fold, defines one face of the catalytic cleft bounded by the core domain. INTRO +41 44 NBD structure_element The second, a nucleotide-binding domain (NBD) with a Rossmann fold, defines one face of the catalytic cleft bounded by the core domain. INTRO +53 66 Rossmann fold structure_element The second, a nucleotide-binding domain (NBD) with a Rossmann fold, defines one face of the catalytic cleft bounded by the core domain. INTRO +92 107 catalytic cleft site The second, a nucleotide-binding domain (NBD) with a Rossmann fold, defines one face of the catalytic cleft bounded by the core domain. INTRO +32 35 NBD structure_element During the catalytic cycle, the NBD executes a large movement, hinged around a “pivot” helix, that closes the catalytic cleft, and drives a large conformational change in the hexamer from open to closed states (Fig. 1B). INTRO +79 92 “pivot” helix structure_element During the catalytic cycle, the NBD executes a large movement, hinged around a “pivot” helix, that closes the catalytic cleft, and drives a large conformational change in the hexamer from open to closed states (Fig. 1B). INTRO +110 125 catalytic cleft site During the catalytic cycle, the NBD executes a large movement, hinged around a “pivot” helix, that closes the catalytic cleft, and drives a large conformational change in the hexamer from open to closed states (Fig. 1B). INTRO +175 182 hexamer oligomeric_state During the catalytic cycle, the NBD executes a large movement, hinged around a “pivot” helix, that closes the catalytic cleft, and drives a large conformational change in the hexamer from open to closed states (Fig. 1B). INTRO +188 192 open protein_state During the catalytic cycle, the NBD executes a large movement, hinged around a “pivot” helix, that closes the catalytic cleft, and drives a large conformational change in the hexamer from open to closed states (Fig. 1B). INTRO +196 202 closed protein_state During the catalytic cycle, the NBD executes a large movement, hinged around a “pivot” helix, that closes the catalytic cleft, and drives a large conformational change in the hexamer from open to closed states (Fig. 1B). INTRO +30 37 antenna structure_element The third domain, dubbed the “antenna,” is an evolutionary acquisition in protista and animals. INTRO +74 82 protista taxonomy_domain The third domain, dubbed the “antenna,” is an evolutionary acquisition in protista and animals. INTRO +87 94 animals taxonomy_domain The third domain, dubbed the “antenna,” is an evolutionary acquisition in protista and animals. INTRO +0 8 Antennae structure_element Antennae of adjacent protomers in each trimer intercalate to form a bundle, perpendicular to the pivot helices, that protrudes along the distal extremes of the 3-fold axis. INTRO +21 30 protomers oligomeric_state Antennae of adjacent protomers in each trimer intercalate to form a bundle, perpendicular to the pivot helices, that protrudes along the distal extremes of the 3-fold axis. INTRO +39 45 trimer oligomeric_state Antennae of adjacent protomers in each trimer intercalate to form a bundle, perpendicular to the pivot helices, that protrudes along the distal extremes of the 3-fold axis. INTRO +97 110 pivot helices structure_element Antennae of adjacent protomers in each trimer intercalate to form a bundle, perpendicular to the pivot helices, that protrudes along the distal extremes of the 3-fold axis. INTRO +7 15 protomer oligomeric_state When a protomer undergoes a conformational change, the rotation of its pivot helix is transferred through the antenna to the adjacent subunit. INTRO +71 82 pivot helix structure_element When a protomer undergoes a conformational change, the rotation of its pivot helix is transferred through the antenna to the adjacent subunit. INTRO +110 117 antenna structure_element When a protomer undergoes a conformational change, the rotation of its pivot helix is transferred through the antenna to the adjacent subunit. INTRO +134 141 subunit structure_element When a protomer undergoes a conformational change, the rotation of its pivot helix is transferred through the antenna to the adjacent subunit. INTRO +21 28 antenna structure_element The influence of the antenna, present only in protozoan and metazoan enzymes, has been proposed to explain its cooperative behavior, which is absent in bacterial homologs. INTRO +46 55 protozoan taxonomy_domain The influence of the antenna, present only in protozoan and metazoan enzymes, has been proposed to explain its cooperative behavior, which is absent in bacterial homologs. INTRO +60 68 metazoan taxonomy_domain The influence of the antenna, present only in protozoan and metazoan enzymes, has been proposed to explain its cooperative behavior, which is absent in bacterial homologs. INTRO +152 161 bacterial taxonomy_domain The influence of the antenna, present only in protozoan and metazoan enzymes, has been proposed to explain its cooperative behavior, which is absent in bacterial homologs. INTRO +51 54 GDH protein_type Structure and quaternary conformational changes in GDH. (A) Views of open (PDB ID 1NR7) and closed (PDB 3MW9) states of the GDH hexamer, shown in ribbon representation perpendicular to the 2-fold symmetry axis (side view, top) and 3-fold symmetry axis (top view, bottom). FIG +69 73 open protein_state Structure and quaternary conformational changes in GDH. (A) Views of open (PDB ID 1NR7) and closed (PDB 3MW9) states of the GDH hexamer, shown in ribbon representation perpendicular to the 2-fold symmetry axis (side view, top) and 3-fold symmetry axis (top view, bottom). FIG +92 98 closed protein_state Structure and quaternary conformational changes in GDH. (A) Views of open (PDB ID 1NR7) and closed (PDB 3MW9) states of the GDH hexamer, shown in ribbon representation perpendicular to the 2-fold symmetry axis (side view, top) and 3-fold symmetry axis (top view, bottom). FIG +124 127 GDH protein_type Structure and quaternary conformational changes in GDH. (A) Views of open (PDB ID 1NR7) and closed (PDB 3MW9) states of the GDH hexamer, shown in ribbon representation perpendicular to the 2-fold symmetry axis (side view, top) and 3-fold symmetry axis (top view, bottom). FIG +128 135 hexamer oligomeric_state Structure and quaternary conformational changes in GDH. (A) Views of open (PDB ID 1NR7) and closed (PDB 3MW9) states of the GDH hexamer, shown in ribbon representation perpendicular to the 2-fold symmetry axis (side view, top) and 3-fold symmetry axis (top view, bottom). FIG +11 20 protomers oligomeric_state Only three protomers are shown in the top view for purposes of visual clarity. FIG +137 141 open protein_state The dashed lines and arrows, respectively, highlight the slight extension in length, and twist in shape that occurs with transition from open to the closed state. FIG +149 155 closed protein_state The dashed lines and arrows, respectively, highlight the slight extension in length, and twist in shape that occurs with transition from open to the closed state. FIG +4 8 open protein_state The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. FIG +28 38 unliganded protein_state The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. FIG +39 42 GDH protein_type The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. FIG +56 62 closed protein_state The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. FIG +73 77 NADH chemical The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. FIG +79 82 GTP chemical The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. FIG +88 97 glutamate chemical The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. FIG +98 103 bound protein_state The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. FIG +109 122 Superposition experimental_method The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. FIG +126 136 structures evidence The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. FIG +141 147 closed protein_state The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. FIG +152 156 open protein_state The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. FIG +24 30 closed protein_state The transition between “closed” and “open” states of GDH is modulated by two allosteric sites in each protomer (Fig. 1A), which are differentially bound by GTP (an inhibitor) and ADP (an activator). INTRO +37 41 open protein_state The transition between “closed” and “open” states of GDH is modulated by two allosteric sites in each protomer (Fig. 1A), which are differentially bound by GTP (an inhibitor) and ADP (an activator). INTRO +53 56 GDH protein_type The transition between “closed” and “open” states of GDH is modulated by two allosteric sites in each protomer (Fig. 1A), which are differentially bound by GTP (an inhibitor) and ADP (an activator). INTRO +77 93 allosteric sites site The transition between “closed” and “open” states of GDH is modulated by two allosteric sites in each protomer (Fig. 1A), which are differentially bound by GTP (an inhibitor) and ADP (an activator). INTRO +102 110 protomer oligomeric_state The transition between “closed” and “open” states of GDH is modulated by two allosteric sites in each protomer (Fig. 1A), which are differentially bound by GTP (an inhibitor) and ADP (an activator). INTRO +147 155 bound by protein_state The transition between “closed” and “open” states of GDH is modulated by two allosteric sites in each protomer (Fig. 1A), which are differentially bound by GTP (an inhibitor) and ADP (an activator). INTRO +156 159 GTP chemical The transition between “closed” and “open” states of GDH is modulated by two allosteric sites in each protomer (Fig. 1A), which are differentially bound by GTP (an inhibitor) and ADP (an activator). INTRO +179 182 ADP chemical The transition between “closed” and “open” states of GDH is modulated by two allosteric sites in each protomer (Fig. 1A), which are differentially bound by GTP (an inhibitor) and ADP (an activator). INTRO +44 47 GDH protein_type These allosteric modulators tightly control GDH function in vivo. INTRO +42 53 pivot helix structure_element In the first site, which sits next to the pivot helix at the base of the antenna (the “GTP binding site”), GTP binding is known to act as an inhibitor, preventing release of the reaction product from the catalytic site by stabilizing the closed conformation of the catalytic cleft. INTRO +73 80 antenna structure_element In the first site, which sits next to the pivot helix at the base of the antenna (the “GTP binding site”), GTP binding is known to act as an inhibitor, preventing release of the reaction product from the catalytic site by stabilizing the closed conformation of the catalytic cleft. INTRO +87 103 GTP binding site site In the first site, which sits next to the pivot helix at the base of the antenna (the “GTP binding site”), GTP binding is known to act as an inhibitor, preventing release of the reaction product from the catalytic site by stabilizing the closed conformation of the catalytic cleft. INTRO +107 110 GTP chemical In the first site, which sits next to the pivot helix at the base of the antenna (the “GTP binding site”), GTP binding is known to act as an inhibitor, preventing release of the reaction product from the catalytic site by stabilizing the closed conformation of the catalytic cleft. INTRO +204 218 catalytic site site In the first site, which sits next to the pivot helix at the base of the antenna (the “GTP binding site”), GTP binding is known to act as an inhibitor, preventing release of the reaction product from the catalytic site by stabilizing the closed conformation of the catalytic cleft. INTRO +238 244 closed protein_state In the first site, which sits next to the pivot helix at the base of the antenna (the “GTP binding site”), GTP binding is known to act as an inhibitor, preventing release of the reaction product from the catalytic site by stabilizing the closed conformation of the catalytic cleft. INTRO +265 280 catalytic cleft site In the first site, which sits next to the pivot helix at the base of the antenna (the “GTP binding site”), GTP binding is known to act as an inhibitor, preventing release of the reaction product from the catalytic site by stabilizing the closed conformation of the catalytic cleft. INTRO +15 30 regulatory site site In the second “regulatory site”, which is situated near the pivot helix between adjacent protomers, ADP acts as an activator of enzymatic activity, presumably by hastening the opening of the catalytic cleft that leads to the release of the reaction product. INTRO +60 71 pivot helix structure_element In the second “regulatory site”, which is situated near the pivot helix between adjacent protomers, ADP acts as an activator of enzymatic activity, presumably by hastening the opening of the catalytic cleft that leads to the release of the reaction product. INTRO +89 98 protomers oligomeric_state In the second “regulatory site”, which is situated near the pivot helix between adjacent protomers, ADP acts as an activator of enzymatic activity, presumably by hastening the opening of the catalytic cleft that leads to the release of the reaction product. INTRO +100 103 ADP chemical In the second “regulatory site”, which is situated near the pivot helix between adjacent protomers, ADP acts as an activator of enzymatic activity, presumably by hastening the opening of the catalytic cleft that leads to the release of the reaction product. INTRO +191 206 catalytic cleft site In the second “regulatory site”, which is situated near the pivot helix between adjacent protomers, ADP acts as an activator of enzymatic activity, presumably by hastening the opening of the catalytic cleft that leads to the release of the reaction product. INTRO +56 60 NADH chemical Interestingly, it has also been shown that the coenzyme NADH can bind to the regulatory site (also bound by the activator ADP), exerting a converse, inhibitory effect on GDH product release, although the role this may play in vivo is not entirely clear. INTRO +77 92 regulatory site site Interestingly, it has also been shown that the coenzyme NADH can bind to the regulatory site (also bound by the activator ADP), exerting a converse, inhibitory effect on GDH product release, although the role this may play in vivo is not entirely clear. INTRO +99 107 bound by protein_state Interestingly, it has also been shown that the coenzyme NADH can bind to the regulatory site (also bound by the activator ADP), exerting a converse, inhibitory effect on GDH product release, although the role this may play in vivo is not entirely clear. INTRO +122 125 ADP chemical Interestingly, it has also been shown that the coenzyme NADH can bind to the regulatory site (also bound by the activator ADP), exerting a converse, inhibitory effect on GDH product release, although the role this may play in vivo is not entirely clear. INTRO +170 173 GDH protein_type Interestingly, it has also been shown that the coenzyme NADH can bind to the regulatory site (also bound by the activator ADP), exerting a converse, inhibitory effect on GDH product release, although the role this may play in vivo is not entirely clear. INTRO +28 46 crystal structures evidence Although there are numerous crystal structures available for GDH in complex with cofactors and nucleotides, they are limited to the combinations that have been amenable to crystallization. INTRO +61 64 GDH protein_type Although there are numerous crystal structures available for GDH in complex with cofactors and nucleotides, they are limited to the combinations that have been amenable to crystallization. INTRO +65 80 in complex with protein_state Although there are numerous crystal structures available for GDH in complex with cofactors and nucleotides, they are limited to the combinations that have been amenable to crystallization. INTRO +172 187 crystallization experimental_method Although there are numerous crystal structures available for GDH in complex with cofactors and nucleotides, they are limited to the combinations that have been amenable to crystallization. INTRO +11 16 X-ray experimental_method Nearly all X-ray structures of mammalian GDH are in the closed conformation, and the few structures that are in the open conformation are at lower resolution (Table 1). INTRO +17 27 structures evidence Nearly all X-ray structures of mammalian GDH are in the closed conformation, and the few structures that are in the open conformation are at lower resolution (Table 1). INTRO +31 40 mammalian taxonomy_domain Nearly all X-ray structures of mammalian GDH are in the closed conformation, and the few structures that are in the open conformation are at lower resolution (Table 1). INTRO +41 44 GDH protein_type Nearly all X-ray structures of mammalian GDH are in the closed conformation, and the few structures that are in the open conformation are at lower resolution (Table 1). INTRO +56 62 closed protein_state Nearly all X-ray structures of mammalian GDH are in the closed conformation, and the few structures that are in the open conformation are at lower resolution (Table 1). INTRO +89 99 structures evidence Nearly all X-ray structures of mammalian GDH are in the closed conformation, and the few structures that are in the open conformation are at lower resolution (Table 1). INTRO +116 120 open protein_state Nearly all X-ray structures of mammalian GDH are in the closed conformation, and the few structures that are in the open conformation are at lower resolution (Table 1). INTRO +9 19 structures evidence Of those structures in the closed conformation, most include NAD[P]H, GTP, and glutamate (or, alternately, NAD+, GTP, and α-ketoglutarate). INTRO +27 33 closed protein_state Of those structures in the closed conformation, most include NAD[P]H, GTP, and glutamate (or, alternately, NAD+, GTP, and α-ketoglutarate). INTRO +61 68 NAD[P]H chemical Of those structures in the closed conformation, most include NAD[P]H, GTP, and glutamate (or, alternately, NAD+, GTP, and α-ketoglutarate). INTRO +70 73 GTP chemical Of those structures in the closed conformation, most include NAD[P]H, GTP, and glutamate (or, alternately, NAD+, GTP, and α-ketoglutarate). INTRO +79 88 glutamate chemical Of those structures in the closed conformation, most include NAD[P]H, GTP, and glutamate (or, alternately, NAD+, GTP, and α-ketoglutarate). INTRO +107 111 NAD+ chemical Of those structures in the closed conformation, most include NAD[P]H, GTP, and glutamate (or, alternately, NAD+, GTP, and α-ketoglutarate). INTRO +113 116 GTP chemical Of those structures in the closed conformation, most include NAD[P]H, GTP, and glutamate (or, alternately, NAD+, GTP, and α-ketoglutarate). INTRO +122 137 α-ketoglutarate chemical Of those structures in the closed conformation, most include NAD[P]H, GTP, and glutamate (or, alternately, NAD+, GTP, and α-ketoglutarate). INTRO +37 40 GTP chemical However, the effects of coenzyme and GTP, bound alone or in concert in the absence of glutamate, have not been analyzed by crystallographic methods. INTRO +42 53 bound alone protein_state However, the effects of coenzyme and GTP, bound alone or in concert in the absence of glutamate, have not been analyzed by crystallographic methods. INTRO +75 85 absence of protein_state However, the effects of coenzyme and GTP, bound alone or in concert in the absence of glutamate, have not been analyzed by crystallographic methods. INTRO +86 95 glutamate chemical However, the effects of coenzyme and GTP, bound alone or in concert in the absence of glutamate, have not been analyzed by crystallographic methods. INTRO +16 56 single-particle cryo-electron microscopy experimental_method Here, we report single-particle cryo-electron microscopy (cryo-EM) studies that show that under these conditions enzyme complexes coexist in both closed and open conformations. INTRO +58 65 cryo-EM experimental_method Here, we report single-particle cryo-electron microscopy (cryo-EM) studies that show that under these conditions enzyme complexes coexist in both closed and open conformations. INTRO +146 152 closed protein_state Here, we report single-particle cryo-electron microscopy (cryo-EM) studies that show that under these conditions enzyme complexes coexist in both closed and open conformations. INTRO +157 161 open protein_state Here, we report single-particle cryo-electron microscopy (cryo-EM) studies that show that under these conditions enzyme complexes coexist in both closed and open conformations. INTRO +17 27 structures evidence We show that the structures in both states can be resolved at near-atomic resolution, suggesting a molecular mechanism for synergistic inhibition of GDH by NADH and GTP (see Table 2 for detailed information on all cryo-EM-derived structures that we report in this work). INTRO +149 152 GDH protein_type We show that the structures in both states can be resolved at near-atomic resolution, suggesting a molecular mechanism for synergistic inhibition of GDH by NADH and GTP (see Table 2 for detailed information on all cryo-EM-derived structures that we report in this work). INTRO +156 160 NADH chemical We show that the structures in both states can be resolved at near-atomic resolution, suggesting a molecular mechanism for synergistic inhibition of GDH by NADH and GTP (see Table 2 for detailed information on all cryo-EM-derived structures that we report in this work). INTRO +165 168 GTP chemical We show that the structures in both states can be resolved at near-atomic resolution, suggesting a molecular mechanism for synergistic inhibition of GDH by NADH and GTP (see Table 2 for detailed information on all cryo-EM-derived structures that we report in this work). INTRO +214 221 cryo-EM experimental_method We show that the structures in both states can be resolved at near-atomic resolution, suggesting a molecular mechanism for synergistic inhibition of GDH by NADH and GTP (see Table 2 for detailed information on all cryo-EM-derived structures that we report in this work). INTRO +230 240 structures evidence We show that the structures in both states can be resolved at near-atomic resolution, suggesting a molecular mechanism for synergistic inhibition of GDH by NADH and GTP (see Table 2 for detailed information on all cryo-EM-derived structures that we report in this work). INTRO +0 5 X-ray experimental_method X-ray structures of mammalian GDH reported in both the open and closed conformations TABLE +6 16 structures evidence X-ray structures of mammalian GDH reported in both the open and closed conformations TABLE +20 29 mammalian taxonomy_domain X-ray structures of mammalian GDH reported in both the open and closed conformations TABLE +30 33 GDH protein_type X-ray structures of mammalian GDH reported in both the open and closed conformations TABLE +55 59 open protein_state X-ray structures of mammalian GDH reported in both the open and closed conformations TABLE +64 70 closed protein_state X-ray structures of mammalian GDH reported in both the open and closed conformations TABLE +0 3 GDH protein "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +45 47 WT protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +48 52 NADH chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +55 58 GLU chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +61 64 GTP chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +70 76 Closed protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +83 85 WT protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +86 89 Glu chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +91 94 GTP chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +96 101 NADPH chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +122 128 Closed protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +135 137 WT protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +138 141 Glu chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +143 148 NADPH chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +150 153 GTP chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +168 174 Closed protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +181 183 WT protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +184 187 apo protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +193 197 Open protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +204 206 WT protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +207 212 NADPH chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +214 223 glutamate chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +229 232 GTP chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +238 244 Closed protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +251 253 WT protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +254 259 NADPH chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +262 265 GLU chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +268 271 GTP chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +274 278 Zinc chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +284 290 Closed protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +298 300 WT protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +301 306 NADPH chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +308 311 Glu chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +313 316 GTP chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +339 345 Closed protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +350 352 WT protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +353 356 NAD chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +358 361 PO4 chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +367 381 2-oxoglutarate chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +387 393 Closed protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +400 402 WT protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +403 408 NADPH chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +411 414 GLU chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +425 431 Closed protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +445 451 mutant protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +452 455 apo protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +461 465 Open protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +472 474 WT protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +475 478 apo protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +484 488 Open protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +495 497 WT protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +498 501 ADP chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +507 511 Open protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +518 520 WT protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +521 526 NADPH chemical "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +564 568 Open protein_state "GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 " TABLE +0 7 Cryo-EM experimental_method Cryo-EM structures of mammalian GDH determined for this study TABLE +8 18 structures evidence Cryo-EM structures of mammalian GDH determined for this study TABLE +22 31 mammalian taxonomy_domain Cryo-EM structures of mammalian GDH determined for this study TABLE +32 35 GDH protein_type Cryo-EM structures of mammalian GDH determined for this study TABLE +0 3 GDH protein_type "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +63 65 WT protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +66 69 apo protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +84 88 Open protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +102 104 WT protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +105 108 GTP chemical "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +123 127 Open protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +141 143 WT protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +144 148 NADH chemical "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +163 167 Open protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +181 183 WT protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +184 188 NADH chemical "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +203 209 Closed protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +223 225 WT protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +226 230 NADH chemical "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +233 236 GTP chemical "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +251 255 Open protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +269 271 WT protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +272 276 NADH chemical "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +279 282 GTP chemical "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +297 303 Closed protein_state "GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 " TABLE +43 46 apo protein_state To explore the conformational landscape of apo-GDH, we first determined its structure in the absence of any added ligands (Supplemental Fig. 1, Fig. 2, A–C). RESULTS +47 50 GDH protein To explore the conformational landscape of apo-GDH, we first determined its structure in the absence of any added ligands (Supplemental Fig. 1, Fig. 2, A–C). RESULTS +76 85 structure evidence To explore the conformational landscape of apo-GDH, we first determined its structure in the absence of any added ligands (Supplemental Fig. 1, Fig. 2, A–C). RESULTS +93 103 absence of protein_state To explore the conformational landscape of apo-GDH, we first determined its structure in the absence of any added ligands (Supplemental Fig. 1, Fig. 2, A–C). RESULTS +4 15 density map evidence The density map, refined to an average resolution of ∼3.0 Å (Supplemental Fig. 2), is in the open conformation and closely matches the model of unliganded GDH derived by X-ray crystallography at 3.3 Å resolution (PDB ID 1NR7). RESULTS +93 97 open protein_state The density map, refined to an average resolution of ∼3.0 Å (Supplemental Fig. 2), is in the open conformation and closely matches the model of unliganded GDH derived by X-ray crystallography at 3.3 Å resolution (PDB ID 1NR7). RESULTS +144 154 unliganded protein_state The density map, refined to an average resolution of ∼3.0 Å (Supplemental Fig. 2), is in the open conformation and closely matches the model of unliganded GDH derived by X-ray crystallography at 3.3 Å resolution (PDB ID 1NR7). RESULTS +155 158 GDH protein The density map, refined to an average resolution of ∼3.0 Å (Supplemental Fig. 2), is in the open conformation and closely matches the model of unliganded GDH derived by X-ray crystallography at 3.3 Å resolution (PDB ID 1NR7). RESULTS +170 191 X-ray crystallography experimental_method The density map, refined to an average resolution of ∼3.0 Å (Supplemental Fig. 2), is in the open conformation and closely matches the model of unliganded GDH derived by X-ray crystallography at 3.3 Å resolution (PDB ID 1NR7). RESULTS +81 87 ResMap experimental_method The variation in local resolution from the core to the periphery, as reported by ResMap (Supplemental Fig. 3D), is consistent with the B-factor gradient observed in the crystal structure (Supplemental Fig. 3A). RESULTS +135 152 B-factor gradient evidence The variation in local resolution from the core to the periphery, as reported by ResMap (Supplemental Fig. 3D), is consistent with the B-factor gradient observed in the crystal structure (Supplemental Fig. 3A). RESULTS +169 186 crystal structure evidence The variation in local resolution from the core to the periphery, as reported by ResMap (Supplemental Fig. 3D), is consistent with the B-factor gradient observed in the crystal structure (Supplemental Fig. 3A). RESULTS +64 68 open protein_state Extensive classification without imposing symmetry yielded only open structures and failed to detect any closed catalytic cleft in the unliganded enzyme, suggesting that all six protomers are in the open conformation. RESULTS +69 79 structures evidence Extensive classification without imposing symmetry yielded only open structures and failed to detect any closed catalytic cleft in the unliganded enzyme, suggesting that all six protomers are in the open conformation. RESULTS +105 111 closed protein_state Extensive classification without imposing symmetry yielded only open structures and failed to detect any closed catalytic cleft in the unliganded enzyme, suggesting that all six protomers are in the open conformation. RESULTS +112 127 catalytic cleft site Extensive classification without imposing symmetry yielded only open structures and failed to detect any closed catalytic cleft in the unliganded enzyme, suggesting that all six protomers are in the open conformation. RESULTS +135 145 unliganded protein_state Extensive classification without imposing symmetry yielded only open structures and failed to detect any closed catalytic cleft in the unliganded enzyme, suggesting that all six protomers are in the open conformation. RESULTS +178 187 protomers oligomeric_state Extensive classification without imposing symmetry yielded only open structures and failed to detect any closed catalytic cleft in the unliganded enzyme, suggesting that all six protomers are in the open conformation. RESULTS +199 203 open protein_state Extensive classification without imposing symmetry yielded only open structures and failed to detect any closed catalytic cleft in the unliganded enzyme, suggesting that all six protomers are in the open conformation. RESULTS +37 42 loops structure_element Consistent with this conclusion, the loops connecting the β-strands of the Rossmann fold are well-defined (Fig. 2B), implying that there is little movement at the NBD, as the transition between closed and open states is associated with NBD movement (Fig. 1B). RESULTS +58 67 β-strands structure_element Consistent with this conclusion, the loops connecting the β-strands of the Rossmann fold are well-defined (Fig. 2B), implying that there is little movement at the NBD, as the transition between closed and open states is associated with NBD movement (Fig. 1B). RESULTS +75 88 Rossmann fold structure_element Consistent with this conclusion, the loops connecting the β-strands of the Rossmann fold are well-defined (Fig. 2B), implying that there is little movement at the NBD, as the transition between closed and open states is associated with NBD movement (Fig. 1B). RESULTS +163 166 NBD structure_element Consistent with this conclusion, the loops connecting the β-strands of the Rossmann fold are well-defined (Fig. 2B), implying that there is little movement at the NBD, as the transition between closed and open states is associated with NBD movement (Fig. 1B). RESULTS +194 200 closed protein_state Consistent with this conclusion, the loops connecting the β-strands of the Rossmann fold are well-defined (Fig. 2B), implying that there is little movement at the NBD, as the transition between closed and open states is associated with NBD movement (Fig. 1B). RESULTS +205 209 open protein_state Consistent with this conclusion, the loops connecting the β-strands of the Rossmann fold are well-defined (Fig. 2B), implying that there is little movement at the NBD, as the transition between closed and open states is associated with NBD movement (Fig. 1B). RESULTS +236 239 NBD structure_element Consistent with this conclusion, the loops connecting the β-strands of the Rossmann fold are well-defined (Fig. 2B), implying that there is little movement at the NBD, as the transition between closed and open states is associated with NBD movement (Fig. 1B). RESULTS +0 7 Cryo-EM experimental_method Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at ∼3 Å resolution. FIG +8 18 structures evidence Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at ∼3 Å resolution. FIG +22 25 GDH protein Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at ∼3 Å resolution. FIG +29 39 unliganded protein_state Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at ∼3 Å resolution. FIG +44 54 NADH-bound protein_state Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at ∼3 Å resolution. FIG +75 82 cryo-EM experimental_method Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at ∼3 Å resolution. FIG +83 86 map evidence Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at ∼3 Å resolution. FIG +90 100 unliganded protein_state Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at ∼3 Å resolution. FIG +101 104 GDH protein Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at ∼3 Å resolution. FIG +23 34 density map evidence (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. FIG +67 99 Rossmann nucleotide binding fold structure_element (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. FIG +105 130 pivot and antenna helices structure_element (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. FIG +142 152 unliganded protein_state (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. FIG +153 156 GDH protein (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. FIG +157 160 map evidence (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. FIG +166 173 Cryo-EM experimental_method (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. FIG +182 194 density maps evidence (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. FIG +250 253 GDH protein (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. FIG +257 265 bound to protein_state (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. FIG +279 283 NADH chemical (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. FIG +5 13 protomer oligomeric_state Each protomer is shown in a different color and densities for NADH bound in both regulatory (red) and catalytic (purple) sites on one protomer are indicated. FIG +48 57 densities evidence Each protomer is shown in a different color and densities for NADH bound in both regulatory (red) and catalytic (purple) sites on one protomer are indicated. FIG +62 66 NADH chemical Each protomer is shown in a different color and densities for NADH bound in both regulatory (red) and catalytic (purple) sites on one protomer are indicated. FIG +67 75 bound in protein_state Each protomer is shown in a different color and densities for NADH bound in both regulatory (red) and catalytic (purple) sites on one protomer are indicated. FIG +81 91 regulatory site Each protomer is shown in a different color and densities for NADH bound in both regulatory (red) and catalytic (purple) sites on one protomer are indicated. FIG +102 111 catalytic site Each protomer is shown in a different color and densities for NADH bound in both regulatory (red) and catalytic (purple) sites on one protomer are indicated. FIG +121 126 sites site Each protomer is shown in a different color and densities for NADH bound in both regulatory (red) and catalytic (purple) sites on one protomer are indicated. FIG +134 142 protomer oligomeric_state Each protomer is shown in a different color and densities for NADH bound in both regulatory (red) and catalytic (purple) sites on one protomer are indicated. FIG +99 103 open protein_state The overall quaternary structures of the two conformations are essentially the same as that of the open and closed states observed by X-ray crystallography. FIG +108 114 closed protein_state The overall quaternary structures of the two conformations are essentially the same as that of the open and closed states observed by X-ray crystallography. FIG +134 155 X-ray crystallography experimental_method The overall quaternary structures of the two conformations are essentially the same as that of the open and closed states observed by X-ray crystallography. FIG +5 8 GDH protein When GDH is bound to NADH, GTP, and glutamate, the enzyme adopts a closed conformation; this “abortive complex” has been determined to 2.4-Å resolution by X-ray crystallography (PDB 3MW9). RESULTS +12 20 bound to protein_state When GDH is bound to NADH, GTP, and glutamate, the enzyme adopts a closed conformation; this “abortive complex” has been determined to 2.4-Å resolution by X-ray crystallography (PDB 3MW9). RESULTS +21 25 NADH chemical When GDH is bound to NADH, GTP, and glutamate, the enzyme adopts a closed conformation; this “abortive complex” has been determined to 2.4-Å resolution by X-ray crystallography (PDB 3MW9). RESULTS +27 30 GTP chemical When GDH is bound to NADH, GTP, and glutamate, the enzyme adopts a closed conformation; this “abortive complex” has been determined to 2.4-Å resolution by X-ray crystallography (PDB 3MW9). RESULTS +36 45 glutamate chemical When GDH is bound to NADH, GTP, and glutamate, the enzyme adopts a closed conformation; this “abortive complex” has been determined to 2.4-Å resolution by X-ray crystallography (PDB 3MW9). RESULTS +67 73 closed protein_state When GDH is bound to NADH, GTP, and glutamate, the enzyme adopts a closed conformation; this “abortive complex” has been determined to 2.4-Å resolution by X-ray crystallography (PDB 3MW9). RESULTS +155 176 X-ray crystallography experimental_method When GDH is bound to NADH, GTP, and glutamate, the enzyme adopts a closed conformation; this “abortive complex” has been determined to 2.4-Å resolution by X-ray crystallography (PDB 3MW9). RESULTS +9 27 crystal structures evidence However, crystal structures of GDH bound only to NADH or to GTP have not yet been reported. RESULTS +31 34 GDH protein However, crystal structures of GDH bound only to NADH or to GTP have not yet been reported. RESULTS +35 48 bound only to protein_state However, crystal structures of GDH bound only to NADH or to GTP have not yet been reported. RESULTS +49 53 NADH chemical However, crystal structures of GDH bound only to NADH or to GTP have not yet been reported. RESULTS +57 59 to protein_state However, crystal structures of GDH bound only to NADH or to GTP have not yet been reported. RESULTS +60 63 GTP chemical However, crystal structures of GDH bound only to NADH or to GTP have not yet been reported. RESULTS +22 26 NADH chemical To test the effect of NADH binding on GDH conformation in solution, we determined the structure of this binary complex using cryo-EM methods combined with three-dimensional classification. RESULTS +38 41 GDH protein To test the effect of NADH binding on GDH conformation in solution, we determined the structure of this binary complex using cryo-EM methods combined with three-dimensional classification. RESULTS +86 95 structure evidence To test the effect of NADH binding on GDH conformation in solution, we determined the structure of this binary complex using cryo-EM methods combined with three-dimensional classification. RESULTS +125 132 cryo-EM experimental_method To test the effect of NADH binding on GDH conformation in solution, we determined the structure of this binary complex using cryo-EM methods combined with three-dimensional classification. RESULTS +155 187 three-dimensional classification experimental_method To test the effect of NADH binding on GDH conformation in solution, we determined the structure of this binary complex using cryo-EM methods combined with three-dimensional classification. RESULTS +46 50 open protein_state Two dominant conformational states, in an all open or all closed conformation were detected, segregated (Fig. 2D), and further refined to near-atomic resolution (∼3.3 Å; Supplemental Fig. 2). RESULTS +58 64 closed protein_state Two dominant conformational states, in an all open or all closed conformation were detected, segregated (Fig. 2D), and further refined to near-atomic resolution (∼3.3 Å; Supplemental Fig. 2). RESULTS +0 9 Densities evidence Densities for 12 molecules of bound NADH were identified in maps of both open and closed states (Supplemental Fig. 4). RESULTS +30 35 bound protein_state Densities for 12 molecules of bound NADH were identified in maps of both open and closed states (Supplemental Fig. 4). RESULTS +36 40 NADH chemical Densities for 12 molecules of bound NADH were identified in maps of both open and closed states (Supplemental Fig. 4). RESULTS +60 64 maps evidence Densities for 12 molecules of bound NADH were identified in maps of both open and closed states (Supplemental Fig. 4). RESULTS +73 77 open protein_state Densities for 12 molecules of bound NADH were identified in maps of both open and closed states (Supplemental Fig. 4). RESULTS +82 88 closed protein_state Densities for 12 molecules of bound NADH were identified in maps of both open and closed states (Supplemental Fig. 4). RESULTS +4 14 NADH-bound protein_state The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map. RESULTS +15 21 closed protein_state The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map. RESULTS +47 56 structure evidence The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map. RESULTS +95 116 X-ray crystallography experimental_method The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map. RESULTS +142 149 density evidence The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map. RESULTS +167 170 GTP chemical The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map. RESULTS +175 184 glutamate chemical The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map. RESULTS +203 210 cryo-EM experimental_method The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map. RESULTS +219 222 map evidence The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map. RESULTS +18 28 NADH-bound protein_state Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by ∼7°, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D). RESULTS +29 35 closed protein_state Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by ∼7°, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D). RESULTS +56 66 NADH-bound protein_state Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by ∼7°, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D). RESULTS +67 71 open protein_state Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by ∼7°, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D). RESULTS +114 129 catalytic cleft site Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by ∼7°, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D). RESULTS +133 139 closed protein_state Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by ∼7°, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D). RESULTS +148 152 NBDs structure_element Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by ∼7°, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D). RESULTS +229 240 pivot helix structure_element Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by ∼7°, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D). RESULTS +303 311 antennae structure_element Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by ∼7°, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D). RESULTS +21 31 NADH-bound protein_state A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. RESULTS +32 36 open protein_state A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. RESULTS +41 47 closed protein_state A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. RESULTS +94 101 helix 5 structure_element A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. RESULTS +112 119 171–186 residue_range A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. RESULTS +152 160 β-sheets structure_element A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. RESULTS +218 223 57–97 residue_range A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. RESULTS +225 232 122–130 residue_range A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. RESULTS +238 247 α-helix 2 structure_element A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. RESULTS +258 263 36–54 residue_range A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. RESULTS +298 303 helix structure_element A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. RESULTS +21 36 catalytic cleft site Thus, closure of the catalytic cleft is accompanied by a quaternary structural change that can be described as a global bending of the structure about an axis that runs parallel to the pivot helix, accompanied by an expansion of the core (Figs. 1A and 2D). RESULTS +135 144 structure evidence Thus, closure of the catalytic cleft is accompanied by a quaternary structural change that can be described as a global bending of the structure about an axis that runs parallel to the pivot helix, accompanied by an expansion of the core (Figs. 1A and 2D). RESULTS +185 196 pivot helix structure_element Thus, closure of the catalytic cleft is accompanied by a quaternary structural change that can be described as a global bending of the structure about an axis that runs parallel to the pivot helix, accompanied by an expansion of the core (Figs. 1A and 2D). RESULTS +25 33 GDH/NADH complex_assembly Detailed analysis of the GDH/NADH structures shows that both the adenosine and nicotinamide moieties of NADH bind to the catalytic site within the NBD in nearly the same orientation in both the open and the closed states, and display closely comparable interactions with the Rossmann fold (Fig. 3, A and B). RESULTS +34 44 structures evidence Detailed analysis of the GDH/NADH structures shows that both the adenosine and nicotinamide moieties of NADH bind to the catalytic site within the NBD in nearly the same orientation in both the open and the closed states, and display closely comparable interactions with the Rossmann fold (Fig. 3, A and B). RESULTS +104 108 NADH chemical Detailed analysis of the GDH/NADH structures shows that both the adenosine and nicotinamide moieties of NADH bind to the catalytic site within the NBD in nearly the same orientation in both the open and the closed states, and display closely comparable interactions with the Rossmann fold (Fig. 3, A and B). RESULTS +121 135 catalytic site site Detailed analysis of the GDH/NADH structures shows that both the adenosine and nicotinamide moieties of NADH bind to the catalytic site within the NBD in nearly the same orientation in both the open and the closed states, and display closely comparable interactions with the Rossmann fold (Fig. 3, A and B). RESULTS +147 150 NBD structure_element Detailed analysis of the GDH/NADH structures shows that both the adenosine and nicotinamide moieties of NADH bind to the catalytic site within the NBD in nearly the same orientation in both the open and the closed states, and display closely comparable interactions with the Rossmann fold (Fig. 3, A and B). RESULTS +194 198 open protein_state Detailed analysis of the GDH/NADH structures shows that both the adenosine and nicotinamide moieties of NADH bind to the catalytic site within the NBD in nearly the same orientation in both the open and the closed states, and display closely comparable interactions with the Rossmann fold (Fig. 3, A and B). RESULTS +207 213 closed protein_state Detailed analysis of the GDH/NADH structures shows that both the adenosine and nicotinamide moieties of NADH bind to the catalytic site within the NBD in nearly the same orientation in both the open and the closed states, and display closely comparable interactions with the Rossmann fold (Fig. 3, A and B). RESULTS +275 288 Rossmann fold structure_element Detailed analysis of the GDH/NADH structures shows that both the adenosine and nicotinamide moieties of NADH bind to the catalytic site within the NBD in nearly the same orientation in both the open and the closed states, and display closely comparable interactions with the Rossmann fold (Fig. 3, A and B). RESULTS +7 22 regulatory site site At the regulatory site, where either ADP can bind as an activator or NADH can bind as an inhibitor, the binding of the adenine moiety of NADH is nearly identical between the two conformers. RESULTS +37 40 ADP chemical At the regulatory site, where either ADP can bind as an activator or NADH can bind as an inhibitor, the binding of the adenine moiety of NADH is nearly identical between the two conformers. RESULTS +69 73 NADH chemical At the regulatory site, where either ADP can bind as an activator or NADH can bind as an inhibitor, the binding of the adenine moiety of NADH is nearly identical between the two conformers. RESULTS +137 141 NADH chemical At the regulatory site, where either ADP can bind as an activator or NADH can bind as an inhibitor, the binding of the adenine moiety of NADH is nearly identical between the two conformers. RESULTS +7 13 closed protein_state In the closed state, the nicotinamide group is oriented toward the center of the hexamer, inserted into a narrow cavity between two adjacent subunits in the trimer. RESULTS +81 88 hexamer oligomeric_state In the closed state, the nicotinamide group is oriented toward the center of the hexamer, inserted into a narrow cavity between two adjacent subunits in the trimer. RESULTS +113 119 cavity site In the closed state, the nicotinamide group is oriented toward the center of the hexamer, inserted into a narrow cavity between two adjacent subunits in the trimer. RESULTS +141 149 subunits structure_element In the closed state, the nicotinamide group is oriented toward the center of the hexamer, inserted into a narrow cavity between two adjacent subunits in the trimer. RESULTS +157 163 trimer oligomeric_state In the closed state, the nicotinamide group is oriented toward the center of the hexamer, inserted into a narrow cavity between two adjacent subunits in the trimer. RESULTS +41 45 NADH chemical There are extensive interactions between NADH and the residues lining this cavity, which may explain the well-defined density of this portion of NADH in the closed state. RESULTS +75 81 cavity site There are extensive interactions between NADH and the residues lining this cavity, which may explain the well-defined density of this portion of NADH in the closed state. RESULTS +118 125 density evidence There are extensive interactions between NADH and the residues lining this cavity, which may explain the well-defined density of this portion of NADH in the closed state. RESULTS +145 149 NADH chemical There are extensive interactions between NADH and the residues lining this cavity, which may explain the well-defined density of this portion of NADH in the closed state. RESULTS +157 163 closed protein_state There are extensive interactions between NADH and the residues lining this cavity, which may explain the well-defined density of this portion of NADH in the closed state. RESULTS +20 24 open protein_state In contrast, in the open conformation, the cavity present in the closed state becomes too narrow for the nicotinamide group; instead, the group is oriented in the opposite direction, parallel to the pivot helix with the amido group extending toward the C-terminal end of the helix. RESULTS +43 49 cavity site In contrast, in the open conformation, the cavity present in the closed state becomes too narrow for the nicotinamide group; instead, the group is oriented in the opposite direction, parallel to the pivot helix with the amido group extending toward the C-terminal end of the helix. RESULTS +65 71 closed protein_state In contrast, in the open conformation, the cavity present in the closed state becomes too narrow for the nicotinamide group; instead, the group is oriented in the opposite direction, parallel to the pivot helix with the amido group extending toward the C-terminal end of the helix. RESULTS +199 210 pivot helix structure_element In contrast, in the open conformation, the cavity present in the closed state becomes too narrow for the nicotinamide group; instead, the group is oriented in the opposite direction, parallel to the pivot helix with the amido group extending toward the C-terminal end of the helix. RESULTS +275 280 helix structure_element In contrast, in the open conformation, the cavity present in the closed state becomes too narrow for the nicotinamide group; instead, the group is oriented in the opposite direction, parallel to the pivot helix with the amido group extending toward the C-terminal end of the helix. RESULTS +17 21 NADH chemical Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. FIG +38 68 catalytic and regulatory sites site Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. FIG +77 81 NADH chemical Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. FIG +82 89 density evidence Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. FIG +123 138 catalytic sites site Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. FIG +142 148 closed protein_state Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. FIG +157 161 open protein_state Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. FIG +181 185 NADH chemical Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. FIG +186 193 density evidence Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. FIG +224 240 regulatory sites site Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. FIG +244 250 closed protein_state Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. FIG +259 263 open protein_state Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. FIG +85 91 closed protein_state Although there is a difference in orientation of the nicotinamide moiety between the closed and open states in the regulatory site, in both structures the adenine portion of NADH has a similar binding pocket and is located in almost exactly the same position as ADP, a potent activator of GDH function (Supplemental Fig. 5). RESULTS +96 100 open protein_state Although there is a difference in orientation of the nicotinamide moiety between the closed and open states in the regulatory site, in both structures the adenine portion of NADH has a similar binding pocket and is located in almost exactly the same position as ADP, a potent activator of GDH function (Supplemental Fig. 5). RESULTS +115 130 regulatory site site Although there is a difference in orientation of the nicotinamide moiety between the closed and open states in the regulatory site, in both structures the adenine portion of NADH has a similar binding pocket and is located in almost exactly the same position as ADP, a potent activator of GDH function (Supplemental Fig. 5). RESULTS +140 150 structures evidence Although there is a difference in orientation of the nicotinamide moiety between the closed and open states in the regulatory site, in both structures the adenine portion of NADH has a similar binding pocket and is located in almost exactly the same position as ADP, a potent activator of GDH function (Supplemental Fig. 5). RESULTS +174 178 NADH chemical Although there is a difference in orientation of the nicotinamide moiety between the closed and open states in the regulatory site, in both structures the adenine portion of NADH has a similar binding pocket and is located in almost exactly the same position as ADP, a potent activator of GDH function (Supplemental Fig. 5). RESULTS +193 207 binding pocket site Although there is a difference in orientation of the nicotinamide moiety between the closed and open states in the regulatory site, in both structures the adenine portion of NADH has a similar binding pocket and is located in almost exactly the same position as ADP, a potent activator of GDH function (Supplemental Fig. 5). RESULTS +262 265 ADP chemical Although there is a difference in orientation of the nicotinamide moiety between the closed and open states in the regulatory site, in both structures the adenine portion of NADH has a similar binding pocket and is located in almost exactly the same position as ADP, a potent activator of GDH function (Supplemental Fig. 5). RESULTS +289 292 GDH protein Although there is a difference in orientation of the nicotinamide moiety between the closed and open states in the regulatory site, in both structures the adenine portion of NADH has a similar binding pocket and is located in almost exactly the same position as ADP, a potent activator of GDH function (Supplemental Fig. 5). RESULTS +7 11 open protein_state In the open state, the binding of ADP or NADH is further stabilized by His209, a residue that undergoes a large movement during the transition from open to closed conformation (Fig. 3, C and D). RESULTS +34 37 ADP chemical In the open state, the binding of ADP or NADH is further stabilized by His209, a residue that undergoes a large movement during the transition from open to closed conformation (Fig. 3, C and D). RESULTS +41 45 NADH chemical In the open state, the binding of ADP or NADH is further stabilized by His209, a residue that undergoes a large movement during the transition from open to closed conformation (Fig. 3, C and D). RESULTS +71 77 His209 residue_name_number In the open state, the binding of ADP or NADH is further stabilized by His209, a residue that undergoes a large movement during the transition from open to closed conformation (Fig. 3, C and D). RESULTS +148 152 open protein_state In the open state, the binding of ADP or NADH is further stabilized by His209, a residue that undergoes a large movement during the transition from open to closed conformation (Fig. 3, C and D). RESULTS +156 162 closed protein_state In the open state, the binding of ADP or NADH is further stabilized by His209, a residue that undergoes a large movement during the transition from open to closed conformation (Fig. 3, C and D). RESULTS +7 11 open protein_state In the open conformation, the distance between His209 and the α-phosphate of NADH is ∼4.4 Å, which is comparable with the corresponding distance in the ADP-bound conformation. RESULTS +47 53 His209 residue_name_number In the open conformation, the distance between His209 and the α-phosphate of NADH is ∼4.4 Å, which is comparable with the corresponding distance in the ADP-bound conformation. RESULTS +77 81 NADH chemical In the open conformation, the distance between His209 and the α-phosphate of NADH is ∼4.4 Å, which is comparable with the corresponding distance in the ADP-bound conformation. RESULTS +152 161 ADP-bound protein_state In the open conformation, the distance between His209 and the α-phosphate of NADH is ∼4.4 Å, which is comparable with the corresponding distance in the ADP-bound conformation. RESULTS +7 13 closed protein_state In the closed conformation, however, this key histidine residue is >10.5 Å away from the nearest phosphate group on NADH, altering a critical stabilization point within the regulatory site. RESULTS +46 55 histidine residue_name In the closed conformation, however, this key histidine residue is >10.5 Å away from the nearest phosphate group on NADH, altering a critical stabilization point within the regulatory site. RESULTS +116 120 NADH chemical In the closed conformation, however, this key histidine residue is >10.5 Å away from the nearest phosphate group on NADH, altering a critical stabilization point within the regulatory site. RESULTS +173 188 regulatory site site In the closed conformation, however, this key histidine residue is >10.5 Å away from the nearest phosphate group on NADH, altering a critical stabilization point within the regulatory site. RESULTS +48 52 NADH chemical This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity. RESULTS +60 64 open protein_state This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity. RESULTS +71 86 regulatory site site This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity. RESULTS +122 125 ADP chemical This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity. RESULTS +147 151 NADH chemical This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity. RESULTS +159 165 closed protein_state This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity. RESULTS +172 187 regulatory site site This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity. RESULTS +276 280 NADH chemical This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity. RESULTS +285 288 ADP chemical This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity. RESULTS +292 295 GDH protein This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity. RESULTS +7 17 absence of protein_state In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +18 22 NADH chemical In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +24 27 GTP chemical In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +44 47 GDH protein In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +55 76 dissociation constant evidence In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +88 95 Cryo-EM experimental_method In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +108 111 GDH protein In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +112 126 incubated with protein_state In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +127 130 GTP chemical In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +145 154 structure evidence In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +215 219 open protein_state In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +265 269 NBDs structure_element In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +277 281 open protein_state In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. RESULTS +4 11 density evidence The density for GTP is not very well defined, suggesting considerable wobble in the binding site. RESULTS +16 19 GTP chemical The density for GTP is not very well defined, suggesting considerable wobble in the binding site. RESULTS +84 96 binding site site The density for GTP is not very well defined, suggesting considerable wobble in the binding site. RESULTS +0 11 Subtraction experimental_method Subtraction of the GTP-bound map with that of the apo state shows that GTP binding can nevertheless be visualized specifically in the GTP binding site (Supplemental Fig. 6). RESULTS +19 28 GTP-bound protein_state Subtraction of the GTP-bound map with that of the apo state shows that GTP binding can nevertheless be visualized specifically in the GTP binding site (Supplemental Fig. 6). RESULTS +29 32 map evidence Subtraction of the GTP-bound map with that of the apo state shows that GTP binding can nevertheless be visualized specifically in the GTP binding site (Supplemental Fig. 6). RESULTS +50 53 apo protein_state Subtraction of the GTP-bound map with that of the apo state shows that GTP binding can nevertheless be visualized specifically in the GTP binding site (Supplemental Fig. 6). RESULTS +71 74 GTP chemical Subtraction of the GTP-bound map with that of the apo state shows that GTP binding can nevertheless be visualized specifically in the GTP binding site (Supplemental Fig. 6). RESULTS +134 150 GTP binding site site Subtraction of the GTP-bound map with that of the apo state shows that GTP binding can nevertheless be visualized specifically in the GTP binding site (Supplemental Fig. 6). RESULTS +28 31 GTP chemical Importantly, the binding of GTP alone does not appear to drive the transition from the open to the closed state of GDH. RESULTS +87 91 open protein_state Importantly, the binding of GTP alone does not appear to drive the transition from the open to the closed state of GDH. RESULTS +99 105 closed protein_state Importantly, the binding of GTP alone does not appear to drive the transition from the open to the closed state of GDH. RESULTS +115 118 GDH protein Importantly, the binding of GTP alone does not appear to drive the transition from the open to the closed state of GDH. RESULTS +32 36 NADH chemical To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. RESULTS +41 44 GTP chemical To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. RESULTS +72 76 open protein_state To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. RESULTS +80 86 closed protein_state To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. RESULTS +110 120 determined experimental_method To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. RESULTS +121 131 structures evidence To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. RESULTS +135 138 GDH protein To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. RESULTS +139 154 in complex with protein_state To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. RESULTS +160 164 NADH chemical To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. RESULTS +169 172 GTP chemical To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. RESULTS +178 185 without protein_state To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. RESULTS +186 195 glutamate chemical To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. RESULTS +5 9 NADH chemical When NADH and GTP are both present, classification reveals the presence of both closed and open GDH conformations, similar to the condition when only NADH is present (Fig. 4, A and B). RESULTS +14 17 GTP chemical When NADH and GTP are both present, classification reveals the presence of both closed and open GDH conformations, similar to the condition when only NADH is present (Fig. 4, A and B). RESULTS +36 50 classification experimental_method When NADH and GTP are both present, classification reveals the presence of both closed and open GDH conformations, similar to the condition when only NADH is present (Fig. 4, A and B). RESULTS +63 74 presence of protein_state When NADH and GTP are both present, classification reveals the presence of both closed and open GDH conformations, similar to the condition when only NADH is present (Fig. 4, A and B). RESULTS +80 86 closed protein_state When NADH and GTP are both present, classification reveals the presence of both closed and open GDH conformations, similar to the condition when only NADH is present (Fig. 4, A and B). RESULTS +91 95 open protein_state When NADH and GTP are both present, classification reveals the presence of both closed and open GDH conformations, similar to the condition when only NADH is present (Fig. 4, A and B). RESULTS +96 99 GDH protein When NADH and GTP are both present, classification reveals the presence of both closed and open GDH conformations, similar to the condition when only NADH is present (Fig. 4, A and B). RESULTS +150 154 NADH chemical When NADH and GTP are both present, classification reveals the presence of both closed and open GDH conformations, similar to the condition when only NADH is present (Fig. 4, A and B). RESULTS +0 37 Reconstruction without classification experimental_method Reconstruction without classification, however, yields a structure clearly in the closed conformation, suggesting that, in coordination with NADH, GTP may further stabilize the closed conformation. RESULTS +57 66 structure evidence Reconstruction without classification, however, yields a structure clearly in the closed conformation, suggesting that, in coordination with NADH, GTP may further stabilize the closed conformation. RESULTS +82 88 closed protein_state Reconstruction without classification, however, yields a structure clearly in the closed conformation, suggesting that, in coordination with NADH, GTP may further stabilize the closed conformation. RESULTS +141 145 NADH chemical Reconstruction without classification, however, yields a structure clearly in the closed conformation, suggesting that, in coordination with NADH, GTP may further stabilize the closed conformation. RESULTS +147 150 GTP chemical Reconstruction without classification, however, yields a structure clearly in the closed conformation, suggesting that, in coordination with NADH, GTP may further stabilize the closed conformation. RESULTS +177 183 closed protein_state Reconstruction without classification, however, yields a structure clearly in the closed conformation, suggesting that, in coordination with NADH, GTP may further stabilize the closed conformation. RESULTS +16 19 GTP chemical The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate. RESULTS +27 31 open protein_state The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate. RESULTS +36 42 closed protein_state The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate. RESULTS +57 69 GDH/NADH/GTP complex_assembly The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate. RESULTS +104 121 crystal structure evidence The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate. RESULTS +138 149 presence of protein_state The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate. RESULTS +150 154 NADH chemical The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate. RESULTS +156 159 GTP chemical The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate. RESULTS +165 174 glutamate chemical The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate. RESULTS +26 30 NADH chemical Likewise, the position of NADH in the open and closed states closely resembles the position of NADH in the GDH/NADH open and closed structures. RESULTS +38 42 open protein_state Likewise, the position of NADH in the open and closed states closely resembles the position of NADH in the GDH/NADH open and closed structures. RESULTS +47 53 closed protein_state Likewise, the position of NADH in the open and closed states closely resembles the position of NADH in the GDH/NADH open and closed structures. RESULTS +95 99 NADH chemical Likewise, the position of NADH in the open and closed states closely resembles the position of NADH in the GDH/NADH open and closed structures. RESULTS +107 115 GDH/NADH complex_assembly Likewise, the position of NADH in the open and closed states closely resembles the position of NADH in the GDH/NADH open and closed structures. RESULTS +116 120 open protein_state Likewise, the position of NADH in the open and closed states closely resembles the position of NADH in the GDH/NADH open and closed structures. RESULTS +125 131 closed protein_state Likewise, the position of NADH in the open and closed states closely resembles the position of NADH in the GDH/NADH open and closed structures. RESULTS +132 142 structures evidence Likewise, the position of NADH in the open and closed states closely resembles the position of NADH in the GDH/NADH open and closed structures. RESULTS +31 35 open protein_state One key difference between the open and closed states of these structures is the position of the His209 residue: As mentioned above, His209 swings away from the adenine moiety of NADH in the closed state. RESULTS +40 46 closed protein_state One key difference between the open and closed states of these structures is the position of the His209 residue: As mentioned above, His209 swings away from the adenine moiety of NADH in the closed state. RESULTS +63 73 structures evidence One key difference between the open and closed states of these structures is the position of the His209 residue: As mentioned above, His209 swings away from the adenine moiety of NADH in the closed state. RESULTS +97 103 His209 residue_name_number One key difference between the open and closed states of these structures is the position of the His209 residue: As mentioned above, His209 swings away from the adenine moiety of NADH in the closed state. RESULTS +133 139 His209 residue_name_number One key difference between the open and closed states of these structures is the position of the His209 residue: As mentioned above, His209 swings away from the adenine moiety of NADH in the closed state. RESULTS +179 183 NADH chemical One key difference between the open and closed states of these structures is the position of the His209 residue: As mentioned above, His209 swings away from the adenine moiety of NADH in the closed state. RESULTS +191 197 closed protein_state One key difference between the open and closed states of these structures is the position of the His209 residue: As mentioned above, His209 swings away from the adenine moiety of NADH in the closed state. RESULTS +5 8 GTP chemical When GTP is present in the GTP binding site, His209 instead interacts with GTP, probably stabilizing the closed conformation (Fig. 4, C and D). RESULTS +27 43 GTP binding site site When GTP is present in the GTP binding site, His209 instead interacts with GTP, probably stabilizing the closed conformation (Fig. 4, C and D). RESULTS +45 51 His209 residue_name_number When GTP is present in the GTP binding site, His209 instead interacts with GTP, probably stabilizing the closed conformation (Fig. 4, C and D). RESULTS +75 78 GTP chemical When GTP is present in the GTP binding site, His209 instead interacts with GTP, probably stabilizing the closed conformation (Fig. 4, C and D). RESULTS +105 111 closed protein_state When GTP is present in the GTP binding site, His209 instead interacts with GTP, probably stabilizing the closed conformation (Fig. 4, C and D). RESULTS +6 9 GTP chemical Thus, GTP binding to GDH appears synergistic with NADH and displaces the conformational landscape toward the closed state. RESULTS +21 24 GDH protein Thus, GTP binding to GDH appears synergistic with NADH and displaces the conformational landscape toward the closed state. RESULTS +50 54 NADH chemical Thus, GTP binding to GDH appears synergistic with NADH and displaces the conformational landscape toward the closed state. RESULTS +109 115 closed protein_state Thus, GTP binding to GDH appears synergistic with NADH and displaces the conformational landscape toward the closed state. RESULTS +0 7 Cryo-EM experimental_method Cryo-EM structure of GDH bound to both NADH and GTP. FIG +8 17 structure evidence Cryo-EM structure of GDH bound to both NADH and GTP. FIG +21 24 GDH protein Cryo-EM structure of GDH bound to both NADH and GTP. FIG +25 33 bound to protein_state Cryo-EM structure of GDH bound to both NADH and GTP. FIG +39 43 NADH chemical Cryo-EM structure of GDH bound to both NADH and GTP. FIG +48 51 GTP chemical Cryo-EM structure of GDH bound to both NADH and GTP. FIG +34 38 open protein_state (A, B) Observation of co-existing open (A) and closed (B) conformations in the GDH-NADH-GTP ternary complex. FIG +47 53 closed protein_state (A, B) Observation of co-existing open (A) and closed (B) conformations in the GDH-NADH-GTP ternary complex. FIG +79 91 GDH-NADH-GTP complex_assembly (A, B) Observation of co-existing open (A) and closed (B) conformations in the GDH-NADH-GTP ternary complex. FIG +0 9 Densities evidence Densities for GTP (yellow) as well as NADH bound to both catalytic (purple) and regulatory (red) sites in each protomer are shown. FIG +14 17 GTP chemical Densities for GTP (yellow) as well as NADH bound to both catalytic (purple) and regulatory (red) sites in each protomer are shown. FIG +38 42 NADH chemical Densities for GTP (yellow) as well as NADH bound to both catalytic (purple) and regulatory (red) sites in each protomer are shown. FIG +43 51 bound to protein_state Densities for GTP (yellow) as well as NADH bound to both catalytic (purple) and regulatory (red) sites in each protomer are shown. FIG +57 66 catalytic site Densities for GTP (yellow) as well as NADH bound to both catalytic (purple) and regulatory (red) sites in each protomer are shown. FIG +80 90 regulatory site Densities for GTP (yellow) as well as NADH bound to both catalytic (purple) and regulatory (red) sites in each protomer are shown. FIG +97 102 sites site Densities for GTP (yellow) as well as NADH bound to both catalytic (purple) and regulatory (red) sites in each protomer are shown. FIG +111 119 protomer oligomeric_state Densities for GTP (yellow) as well as NADH bound to both catalytic (purple) and regulatory (red) sites in each protomer are shown. FIG +56 71 regulatory site site (C, D) Detailed inspection of the interactions near the regulatory site show that the orientation of His209 switches between the two states, which may allow interactions with bound GTP in the closed (D), but not open (C) conformation. FIG +101 107 His209 residue_name_number (C, D) Detailed inspection of the interactions near the regulatory site show that the orientation of His209 switches between the two states, which may allow interactions with bound GTP in the closed (D), but not open (C) conformation. FIG +175 180 bound protein_state (C, D) Detailed inspection of the interactions near the regulatory site show that the orientation of His209 switches between the two states, which may allow interactions with bound GTP in the closed (D), but not open (C) conformation. FIG +181 184 GTP chemical (C, D) Detailed inspection of the interactions near the regulatory site show that the orientation of His209 switches between the two states, which may allow interactions with bound GTP in the closed (D), but not open (C) conformation. FIG +192 198 closed protein_state (C, D) Detailed inspection of the interactions near the regulatory site show that the orientation of His209 switches between the two states, which may allow interactions with bound GTP in the closed (D), but not open (C) conformation. FIG +212 216 open protein_state (C, D) Detailed inspection of the interactions near the regulatory site show that the orientation of His209 switches between the two states, which may allow interactions with bound GTP in the closed (D), but not open (C) conformation. FIG +4 22 structural studies experimental_method Our structural studies thus establish that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states. RESULTS +58 61 GTP chemical Our structural studies thus establish that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states. RESULTS +65 70 bound protein_state Our structural studies thus establish that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states. RESULTS +72 76 NADH chemical Our structural studies thus establish that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states. RESULTS +102 132 catalytic and regulatory sites site Our structural studies thus establish that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states. RESULTS +146 150 open protein_state Our structural studies thus establish that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states. RESULTS +155 161 closed protein_state Our structural studies thus establish that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states. RESULTS +33 37 NADH chemical Whereas the orientation in which NADH binds at the catalytic site is similar for both conformations, the orientation of the nicotinamide portion of NADH in the regulatory site is different between the open and closed conformations (Figs. 3 and 4). RESULTS +51 65 catalytic site site Whereas the orientation in which NADH binds at the catalytic site is similar for both conformations, the orientation of the nicotinamide portion of NADH in the regulatory site is different between the open and closed conformations (Figs. 3 and 4). RESULTS +148 152 NADH chemical Whereas the orientation in which NADH binds at the catalytic site is similar for both conformations, the orientation of the nicotinamide portion of NADH in the regulatory site is different between the open and closed conformations (Figs. 3 and 4). RESULTS +160 175 regulatory site site Whereas the orientation in which NADH binds at the catalytic site is similar for both conformations, the orientation of the nicotinamide portion of NADH in the regulatory site is different between the open and closed conformations (Figs. 3 and 4). RESULTS +201 205 open protein_state Whereas the orientation in which NADH binds at the catalytic site is similar for both conformations, the orientation of the nicotinamide portion of NADH in the regulatory site is different between the open and closed conformations (Figs. 3 and 4). RESULTS +210 216 closed protein_state Whereas the orientation in which NADH binds at the catalytic site is similar for both conformations, the orientation of the nicotinamide portion of NADH in the regulatory site is different between the open and closed conformations (Figs. 3 and 4). RESULTS +7 13 closed protein_state In the closed state, the nicotinamide moiety is inserted into a well-defined cavity at the interface between two adjacent protomers in the trimer. RESULTS +77 83 cavity site In the closed state, the nicotinamide moiety is inserted into a well-defined cavity at the interface between two adjacent protomers in the trimer. RESULTS +91 100 interface site In the closed state, the nicotinamide moiety is inserted into a well-defined cavity at the interface between two adjacent protomers in the trimer. RESULTS +122 131 protomers oligomeric_state In the closed state, the nicotinamide moiety is inserted into a well-defined cavity at the interface between two adjacent protomers in the trimer. RESULTS +139 145 trimer oligomeric_state In the closed state, the nicotinamide moiety is inserted into a well-defined cavity at the interface between two adjacent protomers in the trimer. RESULTS +25 31 cavity site As mentioned above, this cavity is much narrower in the open state, suggesting that this cavity may be unavailable to the NADH nicotinamide moiety when the enzyme is in the open conformation. RESULTS +56 60 open protein_state As mentioned above, this cavity is much narrower in the open state, suggesting that this cavity may be unavailable to the NADH nicotinamide moiety when the enzyme is in the open conformation. RESULTS +89 95 cavity site As mentioned above, this cavity is much narrower in the open state, suggesting that this cavity may be unavailable to the NADH nicotinamide moiety when the enzyme is in the open conformation. RESULTS +122 126 NADH chemical As mentioned above, this cavity is much narrower in the open state, suggesting that this cavity may be unavailable to the NADH nicotinamide moiety when the enzyme is in the open conformation. RESULTS +173 177 open protein_state As mentioned above, this cavity is much narrower in the open state, suggesting that this cavity may be unavailable to the NADH nicotinamide moiety when the enzyme is in the open conformation. RESULTS +72 79 density evidence These structural features provide a potential explanation of the weaker density for the nicotinamide moiety of NADH in the open state, and may account for the higher reported affinity of NADH for the closed state. RESULTS +111 115 NADH chemical These structural features provide a potential explanation of the weaker density for the nicotinamide moiety of NADH in the open state, and may account for the higher reported affinity of NADH for the closed state. RESULTS +123 127 open protein_state These structural features provide a potential explanation of the weaker density for the nicotinamide moiety of NADH in the open state, and may account for the higher reported affinity of NADH for the closed state. RESULTS +187 191 NADH chemical These structural features provide a potential explanation of the weaker density for the nicotinamide moiety of NADH in the open state, and may account for the higher reported affinity of NADH for the closed state. RESULTS +200 206 closed protein_state These structural features provide a potential explanation of the weaker density for the nicotinamide moiety of NADH in the open state, and may account for the higher reported affinity of NADH for the closed state. RESULTS +93 97 open protein_state The role of the nicotinamide moiety in acting as a wedge that prevents the transition to the open conformation also suggests a structural explanation of the mechanism by which NADH binding inhibits the activity of the enzyme by stabilizing the closed conformation state. RESULTS +176 180 NADH chemical The role of the nicotinamide moiety in acting as a wedge that prevents the transition to the open conformation also suggests a structural explanation of the mechanism by which NADH binding inhibits the activity of the enzyme by stabilizing the closed conformation state. RESULTS +244 250 closed protein_state The role of the nicotinamide moiety in acting as a wedge that prevents the transition to the open conformation also suggests a structural explanation of the mechanism by which NADH binding inhibits the activity of the enzyme by stabilizing the closed conformation state. RESULTS +23 30 cryo-EM experimental_method The rapid emergence of cryo-EM as a tool for near-atomic resolution structure determination provides new opportunities for complementing atomic resolution information from X-ray crystallography, as illustrated here with GDH. RESULTS +68 91 structure determination experimental_method The rapid emergence of cryo-EM as a tool for near-atomic resolution structure determination provides new opportunities for complementing atomic resolution information from X-ray crystallography, as illustrated here with GDH. RESULTS +172 193 X-ray crystallography experimental_method The rapid emergence of cryo-EM as a tool for near-atomic resolution structure determination provides new opportunities for complementing atomic resolution information from X-ray crystallography, as illustrated here with GDH. RESULTS +220 223 GDH protein The rapid emergence of cryo-EM as a tool for near-atomic resolution structure determination provides new opportunities for complementing atomic resolution information from X-ray crystallography, as illustrated here with GDH. RESULTS +130 139 structure evidence Perhaps the most important contribution of these methods is the prospect that when there are discrete subpopulations present, the structure of each state can be determined at near-atomic resolution. What we demonstrate here with GDH is that by employing three-dimensional image classification approaches, we not only can isolate distinct, coexisting conformations, but we can also localize small molecule ligands in each of these conformations. RESULTS +229 232 GDH protein Perhaps the most important contribution of these methods is the prospect that when there are discrete subpopulations present, the structure of each state can be determined at near-atomic resolution. What we demonstrate here with GDH is that by employing three-dimensional image classification approaches, we not only can isolate distinct, coexisting conformations, but we can also localize small molecule ligands in each of these conformations. RESULTS +254 303 three-dimensional image classification approaches experimental_method Perhaps the most important contribution of these methods is the prospect that when there are discrete subpopulations present, the structure of each state can be determined at near-atomic resolution. What we demonstrate here with GDH is that by employing three-dimensional image classification approaches, we not only can isolate distinct, coexisting conformations, but we can also localize small molecule ligands in each of these conformations. RESULTS diff --git a/annotation_CSV/PMC4887163.csv b/annotation_CSV/PMC4887163.csv new file mode 100644 index 0000000000000000000000000000000000000000..25de794518cafcc99b68a4b6ed982427bbd7159d --- /dev/null +++ b/annotation_CSV/PMC4887163.csv @@ -0,0 +1,857 @@ +anno_start anno_end anno_text entity_type sentence section +19 34 T cell receptor protein_type Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity TITLE +66 73 insulin chemical Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity TITLE +46 69 T cell antigen receptor complex_assembly However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. ABSTRACT +71 74 TCR complex_assembly However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. ABSTRACT +108 150 peptide–major histocompatibility complexes complex_assembly However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. ABSTRACT +152 156 pMHC complex_assembly However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. ABSTRACT +49 54 human species Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. ABSTRACT +56 69 preproinsulin protein Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. ABSTRACT +80 83 MHC complex_assembly Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. ABSTRACT +29 39 structures evidence We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. ABSTRACT +47 54 1E6 TCR complex_assembly We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. ABSTRACT +55 63 bound to protein_state We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. ABSTRACT +66 89 altered peptide ligands chemical We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. ABSTRACT +20 30 structures evidence Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. ABSTRACT +149 156 1E6 TCR complex_assembly Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. ABSTRACT +30 37 1E6 TCR complex_assembly Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. ABSTRACT +60 94 antipathogen-like binding affinity evidence Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. ABSTRACT +150 174 major histocompatibility complex_assembly T cells perform an essential role in adaptive immunity by interrogating the host proteome for anomalies, classically by recognizing peptides bound in major histocompatibility (MHC) molecules at the cell surface. INTRO +176 179 MHC complex_assembly T cells perform an essential role in adaptive immunity by interrogating the host proteome for anomalies, classically by recognizing peptides bound in major histocompatibility (MHC) molecules at the cell surface. INTRO +64 79 highly variable protein_state Recent data supports the notion that, to perform this role, the highly variable αβ T cell antigen receptor (TCR) must be able to recognize thousands, if not millions, of different peptide ligands. INTRO +80 106 αβ T cell antigen receptor complex_assembly Recent data supports the notion that, to perform this role, the highly variable αβ T cell antigen receptor (TCR) must be able to recognize thousands, if not millions, of different peptide ligands. INTRO +108 111 TCR complex_assembly Recent data supports the notion that, to perform this role, the highly variable αβ T cell antigen receptor (TCR) must be able to recognize thousands, if not millions, of different peptide ligands. INTRO +69 73 TCRs complex_assembly This ability is required to enable the estimated 25 million distinct TCRs expressed in humans to provide effective immune coverage against all possible foreign peptide antigens. INTRO +87 93 humans species This ability is required to enable the estimated 25 million distinct TCRs expressed in humans to provide effective immune coverage against all possible foreign peptide antigens. INTRO +29 33 TCRs complex_assembly Several mechanisms, by which TCRs could bind to a large number of different peptide-MHC (pMHC), have been proposed. INTRO +76 87 peptide-MHC complex_assembly Several mechanisms, by which TCRs could bind to a large number of different peptide-MHC (pMHC), have been proposed. INTRO +89 93 pMHC complex_assembly Several mechanisms, by which TCRs could bind to a large number of different peptide-MHC (pMHC), have been proposed. INTRO +0 10 Structures evidence Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations. INTRO +14 23 unligated protein_state Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations. INTRO +28 35 ligated protein_state Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations. INTRO +36 40 TCRs complex_assembly Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations. INTRO +61 64 TCR complex_assembly Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations. INTRO +65 99 complementarity determining region structure_element Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations. INTRO +101 104 CDR structure_element Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations. INTRO +106 111 loops structure_element Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations. INTRO +178 182 loop structure_element Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations. INTRO +5 8 MHC complex_assembly Both MHC and peptide have also been shown to undergo structural changes upon TCR binding, mediating an induced fit between the TCR and pMHC. INTRO +13 20 peptide chemical Both MHC and peptide have also been shown to undergo structural changes upon TCR binding, mediating an induced fit between the TCR and pMHC. INTRO +77 80 TCR complex_assembly Both MHC and peptide have also been shown to undergo structural changes upon TCR binding, mediating an induced fit between the TCR and pMHC. INTRO +127 130 TCR complex_assembly Both MHC and peptide have also been shown to undergo structural changes upon TCR binding, mediating an induced fit between the TCR and pMHC. INTRO +135 139 pMHC complex_assembly Both MHC and peptide have also been shown to undergo structural changes upon TCR binding, mediating an induced fit between the TCR and pMHC. INTRO +29 35 murine taxonomy_domain Other studies, mainly in the murine system, have demonstrated that the same TCR can interact with different pMHCs using a common or divergent modality. INTRO +76 79 TCR complex_assembly Other studies, mainly in the murine system, have demonstrated that the same TCR can interact with different pMHCs using a common or divergent modality. INTRO +108 113 pMHCs complex_assembly Other studies, mainly in the murine system, have demonstrated that the same TCR can interact with different pMHCs using a common or divergent modality. INTRO +24 30 murine taxonomy_domain Recent studies in model murine systems demonstrate that TCR cross-reactivity can be governed by recognition of a conserved region in the peptide that allows tolerance of peptide sequence variation outside of this hotspot. INTRO +56 59 TCR complex_assembly Recent studies in model murine systems demonstrate that TCR cross-reactivity can be governed by recognition of a conserved region in the peptide that allows tolerance of peptide sequence variation outside of this hotspot. INTRO +34 39 human species We recently reported that the 1E6 human CD8+ T cell clone — which mediates the destruction of β cells through the recognition of a major, HLA-A*0201–restricted, preproinsulin signal peptide (ALWGPDPAAA15–24) — can recognize upwards of 1 million different peptides. INTRO +138 148 HLA-A*0201 protein We recently reported that the 1E6 human CD8+ T cell clone — which mediates the destruction of β cells through the recognition of a major, HLA-A*0201–restricted, preproinsulin signal peptide (ALWGPDPAAA15–24) — can recognize upwards of 1 million different peptides. INTRO +161 174 preproinsulin protein We recently reported that the 1E6 human CD8+ T cell clone — which mediates the destruction of β cells through the recognition of a major, HLA-A*0201–restricted, preproinsulin signal peptide (ALWGPDPAAA15–24) — can recognize upwards of 1 million different peptides. INTRO +175 189 signal peptide structure_element We recently reported that the 1E6 human CD8+ T cell clone — which mediates the destruction of β cells through the recognition of a major, HLA-A*0201–restricted, preproinsulin signal peptide (ALWGPDPAAA15–24) — can recognize upwards of 1 million different peptides. INTRO +191 206 ALWGPDPAAA15–24 chemical We recently reported that the 1E6 human CD8+ T cell clone — which mediates the destruction of β cells through the recognition of a major, HLA-A*0201–restricted, preproinsulin signal peptide (ALWGPDPAAA15–24) — can recognize upwards of 1 million different peptides. INTRO +28 49 HLA-A*0201–ALWGPDPAAA complex_assembly CD8+ T cells that recognize HLA-A*0201–ALWGPDPAAA have been shown to populate insulitic lesions in patients with type 1 diabetes (T1D). INTRO +25 28 TCR complex_assembly We demonstrated that the TCR from the 1E6 T cell clone bound to HLA-A*0201–ALWGPDPAAA using a limited footprint and very weak binding affinity. INTRO +55 63 bound to protein_state We demonstrated that the TCR from the 1E6 T cell clone bound to HLA-A*0201–ALWGPDPAAA using a limited footprint and very weak binding affinity. INTRO +64 85 HLA-A*0201–ALWGPDPAAA complex_assembly We demonstrated that the TCR from the 1E6 T cell clone bound to HLA-A*0201–ALWGPDPAAA using a limited footprint and very weak binding affinity. INTRO +126 142 binding affinity evidence We demonstrated that the TCR from the 1E6 T cell clone bound to HLA-A*0201–ALWGPDPAAA using a limited footprint and very weak binding affinity. INTRO +86 91 human species This first experimental evidence of a high level of CD8+ T cell cross-reactivity in a human autoimmune disease system hinted toward molecular mimicry by a more potent pathogenic peptide as a potential mechanism leading to β cell destruction. INTRO +9 15 solved experimental_method Here, we solved the structure of the 1E6 TCR with 7 altered peptide ligands (APLs) determined by our previously published combinatorial peptide library (CPL) screening, 2 of which mapped within human pathogens. INTRO +20 29 structure evidence Here, we solved the structure of the 1E6 TCR with 7 altered peptide ligands (APLs) determined by our previously published combinatorial peptide library (CPL) screening, 2 of which mapped within human pathogens. INTRO +37 44 1E6 TCR complex_assembly Here, we solved the structure of the 1E6 TCR with 7 altered peptide ligands (APLs) determined by our previously published combinatorial peptide library (CPL) screening, 2 of which mapped within human pathogens. INTRO +52 75 altered peptide ligands chemical Here, we solved the structure of the 1E6 TCR with 7 altered peptide ligands (APLs) determined by our previously published combinatorial peptide library (CPL) screening, 2 of which mapped within human pathogens. INTRO +77 81 APLs chemical Here, we solved the structure of the 1E6 TCR with 7 altered peptide ligands (APLs) determined by our previously published combinatorial peptide library (CPL) screening, 2 of which mapped within human pathogens. INTRO +122 167 combinatorial peptide library (CPL) screening experimental_method Here, we solved the structure of the 1E6 TCR with 7 altered peptide ligands (APLs) determined by our previously published combinatorial peptide library (CPL) screening, 2 of which mapped within human pathogens. INTRO +194 199 human species Here, we solved the structure of the 1E6 TCR with 7 altered peptide ligands (APLs) determined by our previously published combinatorial peptide library (CPL) screening, 2 of which mapped within human pathogens. INTRO +6 10 APLs chemical These APLs differed from the natural preproinsulin peptide by up to 7 of 10 residues. INTRO +37 50 preproinsulin protein These APLs differed from the natural preproinsulin peptide by up to 7 of 10 residues. INTRO +8 14 solved experimental_method We also solved the structure of each unligated APL to investigate whether structural changes occurred before or after binding — which, combined with an in-depth cellular and biophysical analysis of the 1E6 interaction with each APL, demonstrated the molecular mechanism mediating the high level of cross-reactivity exhibited by this preproinsulin-reactive human CD8+ T cell clone. INTRO +19 28 structure evidence We also solved the structure of each unligated APL to investigate whether structural changes occurred before or after binding — which, combined with an in-depth cellular and biophysical analysis of the 1E6 interaction with each APL, demonstrated the molecular mechanism mediating the high level of cross-reactivity exhibited by this preproinsulin-reactive human CD8+ T cell clone. INTRO +37 46 unligated protein_state We also solved the structure of each unligated APL to investigate whether structural changes occurred before or after binding — which, combined with an in-depth cellular and biophysical analysis of the 1E6 interaction with each APL, demonstrated the molecular mechanism mediating the high level of cross-reactivity exhibited by this preproinsulin-reactive human CD8+ T cell clone. INTRO +47 50 APL chemical We also solved the structure of each unligated APL to investigate whether structural changes occurred before or after binding — which, combined with an in-depth cellular and biophysical analysis of the 1E6 interaction with each APL, demonstrated the molecular mechanism mediating the high level of cross-reactivity exhibited by this preproinsulin-reactive human CD8+ T cell clone. INTRO +161 194 cellular and biophysical analysis experimental_method We also solved the structure of each unligated APL to investigate whether structural changes occurred before or after binding — which, combined with an in-depth cellular and biophysical analysis of the 1E6 interaction with each APL, demonstrated the molecular mechanism mediating the high level of cross-reactivity exhibited by this preproinsulin-reactive human CD8+ T cell clone. INTRO +228 231 APL chemical We also solved the structure of each unligated APL to investigate whether structural changes occurred before or after binding — which, combined with an in-depth cellular and biophysical analysis of the 1E6 interaction with each APL, demonstrated the molecular mechanism mediating the high level of cross-reactivity exhibited by this preproinsulin-reactive human CD8+ T cell clone. INTRO +333 346 preproinsulin protein We also solved the structure of each unligated APL to investigate whether structural changes occurred before or after binding — which, combined with an in-depth cellular and biophysical analysis of the 1E6 interaction with each APL, demonstrated the molecular mechanism mediating the high level of cross-reactivity exhibited by this preproinsulin-reactive human CD8+ T cell clone. INTRO +356 361 human species We also solved the structure of each unligated APL to investigate whether structural changes occurred before or after binding — which, combined with an in-depth cellular and biophysical analysis of the 1E6 interaction with each APL, demonstrated the molecular mechanism mediating the high level of cross-reactivity exhibited by this preproinsulin-reactive human CD8+ T cell clone. INTRO +32 36 APLs chemical The 1E6 T cell clone recognizes APLs across a large dynamic range. RESULTS +166 179 preproinsulin protein We have previously demonstrated that the 1E6 T cell clone can recognize over 1 million different peptides with a potency comparable with, or better than, the cognate preproinsulin peptide ALWGPDPAAA. RESULTS +188 198 ALWGPDPAAA chemical We have previously demonstrated that the 1E6 T cell clone can recognize over 1 million different peptides with a potency comparable with, or better than, the cognate preproinsulin peptide ALWGPDPAAA. RESULTS +57 61 APLs chemical From this large functional scan, we selected 7 different APLs that activated the 1E6 T cell clone across a wide (4-log) functional range (Table 1). RESULTS +23 33 MVWGPDPLYV chemical Two of these peptides, MVWGPDPLYV and RQFGPDWIVA (bold text signifies amino acids that are different from the index preproinsulin–derived sequence), are contained within the proteomes of the human pathogens Bacteroides fragilis/thetaiotaomicron and Clostridium asparagiforme, respectively. RESULTS +38 48 RQFGPDWIVA chemical Two of these peptides, MVWGPDPLYV and RQFGPDWIVA (bold text signifies amino acids that are different from the index preproinsulin–derived sequence), are contained within the proteomes of the human pathogens Bacteroides fragilis/thetaiotaomicron and Clostridium asparagiforme, respectively. RESULTS +116 129 preproinsulin protein Two of these peptides, MVWGPDPLYV and RQFGPDWIVA (bold text signifies amino acids that are different from the index preproinsulin–derived sequence), are contained within the proteomes of the human pathogens Bacteroides fragilis/thetaiotaomicron and Clostridium asparagiforme, respectively. RESULTS +191 196 human species Two of these peptides, MVWGPDPLYV and RQFGPDWIVA (bold text signifies amino acids that are different from the index preproinsulin–derived sequence), are contained within the proteomes of the human pathogens Bacteroides fragilis/thetaiotaomicron and Clostridium asparagiforme, respectively. RESULTS +207 244 Bacteroides fragilis/thetaiotaomicron species Two of these peptides, MVWGPDPLYV and RQFGPDWIVA (bold text signifies amino acids that are different from the index preproinsulin–derived sequence), are contained within the proteomes of the human pathogens Bacteroides fragilis/thetaiotaomicron and Clostridium asparagiforme, respectively. RESULTS +249 274 Clostridium asparagiforme species Two of these peptides, MVWGPDPLYV and RQFGPDWIVA (bold text signifies amino acids that are different from the index preproinsulin–derived sequence), are contained within the proteomes of the human pathogens Bacteroides fragilis/thetaiotaomicron and Clostridium asparagiforme, respectively. RESULTS +0 30 Competitive functional testing experimental_method Competitive functional testing revealed that the preproinsulin-derived sequence ALWGPDPAAA was one of the least potent targets for 1E6, with only the MVWGPDPLYV and YLGGPDFPTI demonstrating a similar low-activity profile in MIP-1β secretion and target killing assays (Figure 1, A and B). RESULTS +49 62 preproinsulin protein Competitive functional testing revealed that the preproinsulin-derived sequence ALWGPDPAAA was one of the least potent targets for 1E6, with only the MVWGPDPLYV and YLGGPDFPTI demonstrating a similar low-activity profile in MIP-1β secretion and target killing assays (Figure 1, A and B). RESULTS +80 90 ALWGPDPAAA chemical Competitive functional testing revealed that the preproinsulin-derived sequence ALWGPDPAAA was one of the least potent targets for 1E6, with only the MVWGPDPLYV and YLGGPDFPTI demonstrating a similar low-activity profile in MIP-1β secretion and target killing assays (Figure 1, A and B). RESULTS +150 160 MVWGPDPLYV chemical Competitive functional testing revealed that the preproinsulin-derived sequence ALWGPDPAAA was one of the least potent targets for 1E6, with only the MVWGPDPLYV and YLGGPDFPTI demonstrating a similar low-activity profile in MIP-1β secretion and target killing assays (Figure 1, A and B). RESULTS +165 175 YLGGPDFPTI chemical Competitive functional testing revealed that the preproinsulin-derived sequence ALWGPDPAAA was one of the least potent targets for 1E6, with only the MVWGPDPLYV and YLGGPDFPTI demonstrating a similar low-activity profile in MIP-1β secretion and target killing assays (Figure 1, A and B). RESULTS +224 230 MIP-1β protein Competitive functional testing revealed that the preproinsulin-derived sequence ALWGPDPAAA was one of the least potent targets for 1E6, with only the MVWGPDPLYV and YLGGPDFPTI demonstrating a similar low-activity profile in MIP-1β secretion and target killing assays (Figure 1, A and B). RESULTS +4 14 RQFGPDWIVA chemical The RQFGPDWIVA sequence (present in C. asparagiforme) activated the 1E6 T cell with around 1 log–greater potency compared with ALWGPDPAAA. RESULTS +36 52 C. asparagiforme species The RQFGPDWIVA sequence (present in C. asparagiforme) activated the 1E6 T cell with around 1 log–greater potency compared with ALWGPDPAAA. RESULTS +127 137 ALWGPDPAAA chemical The RQFGPDWIVA sequence (present in C. asparagiforme) activated the 1E6 T cell with around 1 log–greater potency compared with ALWGPDPAAA. RESULTS +38 48 RQFGPDFPTI chemical At the other end of the spectrum, the RQFGPDFPTI peptide stimulated MIP-1β release and killing by 1E6 at an exogenous peptide concentration 2–3 logs lower compared with ALWGPDPAAA. RESULTS +68 74 MIP-1β protein At the other end of the spectrum, the RQFGPDFPTI peptide stimulated MIP-1β release and killing by 1E6 at an exogenous peptide concentration 2–3 logs lower compared with ALWGPDPAAA. RESULTS +169 179 ALWGPDPAAA chemical At the other end of the spectrum, the RQFGPDFPTI peptide stimulated MIP-1β release and killing by 1E6 at an exogenous peptide concentration 2–3 logs lower compared with ALWGPDPAAA. RESULTS +55 59 pMHC complex_assembly The pattern of peptide potency was closely mirrored by pMHC tetramer staining experiments (Figure 1C and plots shown in Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI85679DS1). RESULTS +60 77 tetramer staining experimental_method The pattern of peptide potency was closely mirrored by pMHC tetramer staining experiments (Figure 1C and plots shown in Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI85679DS1). RESULTS +10 23 A2-RQFGPDFPTI chemical Here, the A2-RQFGPDFPTI tetramer stained 1E6 with the greatest MFI, gradually decreasing to the weakest tetramers: A2-MVWGPDPLYV and -YLGGPDFPTI. RESULTS +24 32 tetramer oligomeric_state Here, the A2-RQFGPDFPTI tetramer stained 1E6 with the greatest MFI, gradually decreasing to the weakest tetramers: A2-MVWGPDPLYV and -YLGGPDFPTI. RESULTS +104 113 tetramers oligomeric_state Here, the A2-RQFGPDFPTI tetramer stained 1E6 with the greatest MFI, gradually decreasing to the weakest tetramers: A2-MVWGPDPLYV and -YLGGPDFPTI. RESULTS +115 128 A2-MVWGPDPLYV chemical Here, the A2-RQFGPDFPTI tetramer stained 1E6 with the greatest MFI, gradually decreasing to the weakest tetramers: A2-MVWGPDPLYV and -YLGGPDFPTI. RESULTS +134 144 YLGGPDFPTI chemical Here, the A2-RQFGPDFPTI tetramer stained 1E6 with the greatest MFI, gradually decreasing to the weakest tetramers: A2-MVWGPDPLYV and -YLGGPDFPTI. RESULTS +55 67 thermal melt experimental_method To parallel the functional analysis, we also performed thermal melt (Tm) experiments using synchrotron radiation circular dichroism (SRCD) to investigate the stability of each APL (Figure 1D). RESULTS +69 71 Tm evidence To parallel the functional analysis, we also performed thermal melt (Tm) experiments using synchrotron radiation circular dichroism (SRCD) to investigate the stability of each APL (Figure 1D). RESULTS +91 131 synchrotron radiation circular dichroism experimental_method To parallel the functional analysis, we also performed thermal melt (Tm) experiments using synchrotron radiation circular dichroism (SRCD) to investigate the stability of each APL (Figure 1D). RESULTS +133 137 SRCD experimental_method To parallel the functional analysis, we also performed thermal melt (Tm) experiments using synchrotron radiation circular dichroism (SRCD) to investigate the stability of each APL (Figure 1D). RESULTS +176 179 APL chemical To parallel the functional analysis, we also performed thermal melt (Tm) experiments using synchrotron radiation circular dichroism (SRCD) to investigate the stability of each APL (Figure 1D). RESULTS +13 15 Tm evidence The range of Tm was between 49.4°C (RQFGPDWIVA) and 60.3°C (YQFGPDFPIA), with an average approximately 55°C, similar to our previous findings. RESULTS +36 46 RQFGPDWIVA chemical The range of Tm was between 49.4°C (RQFGPDWIVA) and 60.3°C (YQFGPDFPIA), with an average approximately 55°C, similar to our previous findings. RESULTS +60 70 YQFGPDFPIA chemical The range of Tm was between 49.4°C (RQFGPDWIVA) and 60.3°C (YQFGPDFPIA), with an average approximately 55°C, similar to our previous findings. RESULTS +74 91 tetramer staining experimental_method This pattern of stability did not correlate with the T cell activation or tetramer staining experiments, indicating that peptide binding to the MHC do not explain ligand potency. RESULTS +144 147 MHC complex_assembly This pattern of stability did not correlate with the T cell activation or tetramer staining experiments, indicating that peptide binding to the MHC do not explain ligand potency. RESULTS +4 11 1E6 TCR complex_assembly The 1E6 TCR can bind peptides with strong antipathogen-like affinities. RESULTS +65 69 TCRs complex_assembly We, and others, have previously demonstrated that antipathogenic TCRs tend to bind with stronger affinity compared with self-reactive TCRs, likely a consequence of the deletion of T cells with high-affinity self-reactive TCR during thymic selection. RESULTS +97 105 affinity evidence We, and others, have previously demonstrated that antipathogenic TCRs tend to bind with stronger affinity compared with self-reactive TCRs, likely a consequence of the deletion of T cells with high-affinity self-reactive TCR during thymic selection. RESULTS +134 138 TCRs complex_assembly We, and others, have previously demonstrated that antipathogenic TCRs tend to bind with stronger affinity compared with self-reactive TCRs, likely a consequence of the deletion of T cells with high-affinity self-reactive TCR during thymic selection. RESULTS +198 206 affinity evidence We, and others, have previously demonstrated that antipathogenic TCRs tend to bind with stronger affinity compared with self-reactive TCRs, likely a consequence of the deletion of T cells with high-affinity self-reactive TCR during thymic selection. RESULTS +221 224 TCR complex_assembly We, and others, have previously demonstrated that antipathogenic TCRs tend to bind with stronger affinity compared with self-reactive TCRs, likely a consequence of the deletion of T cells with high-affinity self-reactive TCR during thymic selection. RESULTS +35 42 1E6 TCR complex_assembly In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell–derived TCR with a biologically relevant ligand (KD > 200 μM; KD, equilibrium binding constant). RESULTS +43 48 bound protein_state In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell–derived TCR with a biologically relevant ligand (KD > 200 μM; KD, equilibrium binding constant). RESULTS +61 74 preproinsulin protein In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell–derived TCR with a biologically relevant ligand (KD > 200 μM; KD, equilibrium binding constant). RESULTS +84 94 ALWGPDPAAA chemical In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell–derived TCR with a biologically relevant ligand (KD > 200 μM; KD, equilibrium binding constant). RESULTS +113 121 affinity evidence In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell–derived TCR with a biologically relevant ligand (KD > 200 μM; KD, equilibrium binding constant). RESULTS +148 153 human species In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell–derived TCR with a biologically relevant ligand (KD > 200 μM; KD, equilibrium binding constant). RESULTS +174 177 TCR complex_assembly In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell–derived TCR with a biologically relevant ligand (KD > 200 μM; KD, equilibrium binding constant). RESULTS +215 217 KD evidence In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell–derived TCR with a biologically relevant ligand (KD > 200 μM; KD, equilibrium binding constant). RESULTS +228 230 KD evidence In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell–derived TCR with a biologically relevant ligand (KD > 200 μM; KD, equilibrium binding constant). RESULTS +232 260 equilibrium binding constant evidence In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell–derived TCR with a biologically relevant ligand (KD > 200 μM; KD, equilibrium binding constant). RESULTS +0 25 Surface plasmon resonance experimental_method Surface plasmon resonance (SPR) analysis of the 1E6 TCR–pMHC interaction for all 7 APLs (Figure 2, A–H) demonstrated that stronger binding affinity (represented as ΔG°, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson’s correlation analysis value of 0.8 (P = 0.01) (Figure 2I). RESULTS +27 30 SPR experimental_method Surface plasmon resonance (SPR) analysis of the 1E6 TCR–pMHC interaction for all 7 APLs (Figure 2, A–H) demonstrated that stronger binding affinity (represented as ΔG°, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson’s correlation analysis value of 0.8 (P = 0.01) (Figure 2I). RESULTS +48 55 1E6 TCR complex_assembly Surface plasmon resonance (SPR) analysis of the 1E6 TCR–pMHC interaction for all 7 APLs (Figure 2, A–H) demonstrated that stronger binding affinity (represented as ΔG°, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson’s correlation analysis value of 0.8 (P = 0.01) (Figure 2I). RESULTS +56 60 pMHC complex_assembly Surface plasmon resonance (SPR) analysis of the 1E6 TCR–pMHC interaction for all 7 APLs (Figure 2, A–H) demonstrated that stronger binding affinity (represented as ΔG°, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson’s correlation analysis value of 0.8 (P = 0.01) (Figure 2I). RESULTS +83 87 APLs chemical Surface plasmon resonance (SPR) analysis of the 1E6 TCR–pMHC interaction for all 7 APLs (Figure 2, A–H) demonstrated that stronger binding affinity (represented as ΔG°, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson’s correlation analysis value of 0.8 (P = 0.01) (Figure 2I). RESULTS +131 147 binding affinity evidence Surface plasmon resonance (SPR) analysis of the 1E6 TCR–pMHC interaction for all 7 APLs (Figure 2, A–H) demonstrated that stronger binding affinity (represented as ΔG°, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson’s correlation analysis value of 0.8 (P = 0.01) (Figure 2I). RESULTS +164 167 ΔG° evidence Surface plasmon resonance (SPR) analysis of the 1E6 TCR–pMHC interaction for all 7 APLs (Figure 2, A–H) demonstrated that stronger binding affinity (represented as ΔG°, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson’s correlation analysis value of 0.8 (P = 0.01) (Figure 2I). RESULTS +204 208 EC50 evidence Surface plasmon resonance (SPR) analysis of the 1E6 TCR–pMHC interaction for all 7 APLs (Figure 2, A–H) demonstrated that stronger binding affinity (represented as ΔG°, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson’s correlation analysis value of 0.8 (P = 0.01) (Figure 2I). RESULTS +325 355 Pearson’s correlation analysis experimental_method Surface plasmon resonance (SPR) analysis of the 1E6 TCR–pMHC interaction for all 7 APLs (Figure 2, A–H) demonstrated that stronger binding affinity (represented as ΔG°, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson’s correlation analysis value of 0.8 (P = 0.01) (Figure 2I). RESULTS +121 131 affinities evidence It should be noted that this correlation, although consistent with the T cell killing experiments, uses only approximate affinities calculated for the 2 weakest ligands. RESULTS +75 95 TCR binding affinity evidence First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 μM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 μM, equivalent to the binding affinity of the very strongest antipathogen TCRs. RESULTS +126 150 1E6 TCR binding affinity evidence First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 μM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 μM, equivalent to the binding affinity of the very strongest antipathogen TCRs. RESULTS +159 172 A2-MVWGPDPLYV chemical First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 μM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 μM, equivalent to the binding affinity of the very strongest antipathogen TCRs. RESULTS +185 187 KD evidence First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 μM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 μM, equivalent to the binding affinity of the very strongest antipathogen TCRs. RESULTS +211 218 1E6 TCR complex_assembly First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 μM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 μM, equivalent to the binding affinity of the very strongest antipathogen TCRs. RESULTS +219 227 bound to protein_state First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 μM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 μM, equivalent to the binding affinity of the very strongest antipathogen TCRs. RESULTS +228 241 A2-RQFGPDFPTI chemical First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 μM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 μM, equivalent to the binding affinity of the very strongest antipathogen TCRs. RESULTS +247 249 KD evidence First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 μM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 μM, equivalent to the binding affinity of the very strongest antipathogen TCRs. RESULTS +278 294 binding affinity evidence First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 μM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 μM, equivalent to the binding affinity of the very strongest antipathogen TCRs. RESULTS +330 334 TCRs complex_assembly First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 μM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 μM, equivalent to the binding affinity of the very strongest antipathogen TCRs. RESULTS +11 18 1E6 TCR complex_assembly Third, the 1E6 TCR bound to A2-RQFGPDWIVA peptide, within the C. asparagiforme proteome, with approximately 4-fold stronger affinity than A2-ALWGPDPAAA, demonstrating the potential for a pathogen-derived antigen to initiate a response to the self-derived sequence. RESULTS +19 27 bound to protein_state Third, the 1E6 TCR bound to A2-RQFGPDWIVA peptide, within the C. asparagiforme proteome, with approximately 4-fold stronger affinity than A2-ALWGPDPAAA, demonstrating the potential for a pathogen-derived antigen to initiate a response to the self-derived sequence. RESULTS +28 41 A2-RQFGPDWIVA chemical Third, the 1E6 TCR bound to A2-RQFGPDWIVA peptide, within the C. asparagiforme proteome, with approximately 4-fold stronger affinity than A2-ALWGPDPAAA, demonstrating the potential for a pathogen-derived antigen to initiate a response to the self-derived sequence. RESULTS +62 78 C. asparagiforme species Third, the 1E6 TCR bound to A2-RQFGPDWIVA peptide, within the C. asparagiforme proteome, with approximately 4-fold stronger affinity than A2-ALWGPDPAAA, demonstrating the potential for a pathogen-derived antigen to initiate a response to the self-derived sequence. RESULTS +124 132 affinity evidence Third, the 1E6 TCR bound to A2-RQFGPDWIVA peptide, within the C. asparagiforme proteome, with approximately 4-fold stronger affinity than A2-ALWGPDPAAA, demonstrating the potential for a pathogen-derived antigen to initiate a response to the self-derived sequence. RESULTS +138 151 A2-ALWGPDPAAA chemical Third, the 1E6 TCR bound to A2-RQFGPDWIVA peptide, within the C. asparagiforme proteome, with approximately 4-fold stronger affinity than A2-ALWGPDPAAA, demonstrating the potential for a pathogen-derived antigen to initiate a response to the self-derived sequence. RESULTS +53 71 binding affinities evidence Finally, these data demonstrate the largest range of binding affinities reported for a natural, endogenous human TCR of more than 3 logs of magnitude (A2-MVWGPDPLYV vs. A2-RQFGPDFPTI). RESULTS +96 106 endogenous protein_state Finally, these data demonstrate the largest range of binding affinities reported for a natural, endogenous human TCR of more than 3 logs of magnitude (A2-MVWGPDPLYV vs. A2-RQFGPDFPTI). RESULTS +107 112 human species Finally, these data demonstrate the largest range of binding affinities reported for a natural, endogenous human TCR of more than 3 logs of magnitude (A2-MVWGPDPLYV vs. A2-RQFGPDFPTI). RESULTS +113 116 TCR complex_assembly Finally, these data demonstrate the largest range of binding affinities reported for a natural, endogenous human TCR of more than 3 logs of magnitude (A2-MVWGPDPLYV vs. A2-RQFGPDFPTI). RESULTS +151 164 A2-MVWGPDPLYV chemical Finally, these data demonstrate the largest range of binding affinities reported for a natural, endogenous human TCR of more than 3 logs of magnitude (A2-MVWGPDPLYV vs. A2-RQFGPDFPTI). RESULTS +169 182 A2-RQFGPDFPTI chemical Finally, these data demonstrate the largest range of binding affinities reported for a natural, endogenous human TCR of more than 3 logs of magnitude (A2-MVWGPDPLYV vs. A2-RQFGPDFPTI). RESULTS +15 23 affinity evidence To confirm the affinity spread detected by SPR, and to evaluate whether experiments performed using soluble molecules were biologically relevant to events at the T cell surface, we determined the effective 2D affinity of each APL using an adhesion frequency assay in which a human rbc coated in pMHC acted as an adhesion sensor. RESULTS +43 46 SPR experimental_method To confirm the affinity spread detected by SPR, and to evaluate whether experiments performed using soluble molecules were biologically relevant to events at the T cell surface, we determined the effective 2D affinity of each APL using an adhesion frequency assay in which a human rbc coated in pMHC acted as an adhesion sensor. RESULTS +206 217 2D affinity evidence To confirm the affinity spread detected by SPR, and to evaluate whether experiments performed using soluble molecules were biologically relevant to events at the T cell surface, we determined the effective 2D affinity of each APL using an adhesion frequency assay in which a human rbc coated in pMHC acted as an adhesion sensor. RESULTS +226 229 APL chemical To confirm the affinity spread detected by SPR, and to evaluate whether experiments performed using soluble molecules were biologically relevant to events at the T cell surface, we determined the effective 2D affinity of each APL using an adhesion frequency assay in which a human rbc coated in pMHC acted as an adhesion sensor. RESULTS +239 263 adhesion frequency assay experimental_method To confirm the affinity spread detected by SPR, and to evaluate whether experiments performed using soluble molecules were biologically relevant to events at the T cell surface, we determined the effective 2D affinity of each APL using an adhesion frequency assay in which a human rbc coated in pMHC acted as an adhesion sensor. RESULTS +275 280 human species To confirm the affinity spread detected by SPR, and to evaluate whether experiments performed using soluble molecules were biologically relevant to events at the T cell surface, we determined the effective 2D affinity of each APL using an adhesion frequency assay in which a human rbc coated in pMHC acted as an adhesion sensor. RESULTS +295 299 pMHC complex_assembly To confirm the affinity spread detected by SPR, and to evaluate whether experiments performed using soluble molecules were biologically relevant to events at the T cell surface, we determined the effective 2D affinity of each APL using an adhesion frequency assay in which a human rbc coated in pMHC acted as an adhesion sensor. RESULTS +18 21 SPR experimental_method In agreement with SPR experiments, the range of 2D affinities we detected differed by around 3 logs, with the A2-MVWGPDPLYV generating the weakest 2D affinity (2.6 × 10–5 AcKa μm4) and A2-RQFGPDFPTI the strongest (4.5 × 10–2 AcKa μm4) (Figure 2J). RESULTS +48 61 2D affinities evidence In agreement with SPR experiments, the range of 2D affinities we detected differed by around 3 logs, with the A2-MVWGPDPLYV generating the weakest 2D affinity (2.6 × 10–5 AcKa μm4) and A2-RQFGPDFPTI the strongest (4.5 × 10–2 AcKa μm4) (Figure 2J). RESULTS +110 123 A2-MVWGPDPLYV chemical In agreement with SPR experiments, the range of 2D affinities we detected differed by around 3 logs, with the A2-MVWGPDPLYV generating the weakest 2D affinity (2.6 × 10–5 AcKa μm4) and A2-RQFGPDFPTI the strongest (4.5 × 10–2 AcKa μm4) (Figure 2J). RESULTS +147 158 2D affinity evidence In agreement with SPR experiments, the range of 2D affinities we detected differed by around 3 logs, with the A2-MVWGPDPLYV generating the weakest 2D affinity (2.6 × 10–5 AcKa μm4) and A2-RQFGPDFPTI the strongest (4.5 × 10–2 AcKa μm4) (Figure 2J). RESULTS +171 175 AcKa evidence In agreement with SPR experiments, the range of 2D affinities we detected differed by around 3 logs, with the A2-MVWGPDPLYV generating the weakest 2D affinity (2.6 × 10–5 AcKa μm4) and A2-RQFGPDFPTI the strongest (4.5 × 10–2 AcKa μm4) (Figure 2J). RESULTS +185 198 A2-RQFGPDFPTI chemical In agreement with SPR experiments, the range of 2D affinities we detected differed by around 3 logs, with the A2-MVWGPDPLYV generating the weakest 2D affinity (2.6 × 10–5 AcKa μm4) and A2-RQFGPDFPTI the strongest (4.5 × 10–2 AcKa μm4) (Figure 2J). RESULTS +225 229 AcKa evidence In agreement with SPR experiments, the range of 2D affinities we detected differed by around 3 logs, with the A2-MVWGPDPLYV generating the weakest 2D affinity (2.6 × 10–5 AcKa μm4) and A2-RQFGPDFPTI the strongest (4.5 × 10–2 AcKa μm4) (Figure 2J). RESULTS +12 23 3D affinity evidence As with the 3D affinity measurements, the 2D affinity measurements correlated well with the EC50 values for each ligand (Figure 2K) demonstrating a strong correlation (Pearson’s correlation = 0.8, P = 0.01) between T cell antigen sensitivity and TCR binding affinity. RESULTS +42 53 2D affinity evidence As with the 3D affinity measurements, the 2D affinity measurements correlated well with the EC50 values for each ligand (Figure 2K) demonstrating a strong correlation (Pearson’s correlation = 0.8, P = 0.01) between T cell antigen sensitivity and TCR binding affinity. RESULTS +92 96 EC50 evidence As with the 3D affinity measurements, the 2D affinity measurements correlated well with the EC50 values for each ligand (Figure 2K) demonstrating a strong correlation (Pearson’s correlation = 0.8, P = 0.01) between T cell antigen sensitivity and TCR binding affinity. RESULTS +168 189 Pearson’s correlation evidence As with the 3D affinity measurements, the 2D affinity measurements correlated well with the EC50 values for each ligand (Figure 2K) demonstrating a strong correlation (Pearson’s correlation = 0.8, P = 0.01) between T cell antigen sensitivity and TCR binding affinity. RESULTS +197 198 P evidence As with the 3D affinity measurements, the 2D affinity measurements correlated well with the EC50 values for each ligand (Figure 2K) demonstrating a strong correlation (Pearson’s correlation = 0.8, P = 0.01) between T cell antigen sensitivity and TCR binding affinity. RESULTS +246 266 TCR binding affinity evidence As with the 3D affinity measurements, the 2D affinity measurements correlated well with the EC50 values for each ligand (Figure 2K) demonstrating a strong correlation (Pearson’s correlation = 0.8, P = 0.01) between T cell antigen sensitivity and TCR binding affinity. RESULTS +62 73 3D affinity evidence Of note, these data demonstrate a close agreement between the 3D affinity values generated using SPR and 2D affinity values generated using adhesion frequency assays. RESULTS +97 100 SPR experimental_method Of note, these data demonstrate a close agreement between the 3D affinity values generated using SPR and 2D affinity values generated using adhesion frequency assays. RESULTS +105 116 2D affinity evidence Of note, these data demonstrate a close agreement between the 3D affinity values generated using SPR and 2D affinity values generated using adhesion frequency assays. RESULTS +4 11 1E6 TCR complex_assembly The 1E6 TCR uses a consensus binding mode to engage multiple APLs. RESULTS +61 65 APLs chemical The 1E6 TCR uses a consensus binding mode to engage multiple APLs. RESULTS +13 22 structure evidence Our previous structure of the 1E6-A2-ALWGPDPAAA complex demonstrated a limited binding footprint between the TCR and pMHC. RESULTS +30 47 1E6-A2-ALWGPDPAAA complex_assembly Our previous structure of the 1E6-A2-ALWGPDPAAA complex demonstrated a limited binding footprint between the TCR and pMHC. RESULTS +79 96 binding footprint site Our previous structure of the 1E6-A2-ALWGPDPAAA complex demonstrated a limited binding footprint between the TCR and pMHC. RESULTS +109 112 TCR complex_assembly Our previous structure of the 1E6-A2-ALWGPDPAAA complex demonstrated a limited binding footprint between the TCR and pMHC. RESULTS +117 121 pMHC complex_assembly Our previous structure of the 1E6-A2-ALWGPDPAAA complex demonstrated a limited binding footprint between the TCR and pMHC. RESULTS +87 103 binding affinity evidence The low number of contacts between the 2 molecules most likely contributed to the weak binding affinity of the interaction. RESULTS +47 54 1E6 TCR complex_assembly In order to examine the mechanism by which the 1E6 TCR engaged a wide range of peptides with divergent binding affinities, we solved the structure of the 1E6 TCR in complex with all 7 APLs used in Figure 2. RESULTS +103 121 binding affinities evidence In order to examine the mechanism by which the 1E6 TCR engaged a wide range of peptides with divergent binding affinities, we solved the structure of the 1E6 TCR in complex with all 7 APLs used in Figure 2. RESULTS +126 132 solved experimental_method In order to examine the mechanism by which the 1E6 TCR engaged a wide range of peptides with divergent binding affinities, we solved the structure of the 1E6 TCR in complex with all 7 APLs used in Figure 2. RESULTS +137 146 structure evidence In order to examine the mechanism by which the 1E6 TCR engaged a wide range of peptides with divergent binding affinities, we solved the structure of the 1E6 TCR in complex with all 7 APLs used in Figure 2. RESULTS +154 161 1E6 TCR complex_assembly In order to examine the mechanism by which the 1E6 TCR engaged a wide range of peptides with divergent binding affinities, we solved the structure of the 1E6 TCR in complex with all 7 APLs used in Figure 2. RESULTS +162 177 in complex with protein_state In order to examine the mechanism by which the 1E6 TCR engaged a wide range of peptides with divergent binding affinities, we solved the structure of the 1E6 TCR in complex with all 7 APLs used in Figure 2. RESULTS +184 188 APLs chemical In order to examine the mechanism by which the 1E6 TCR engaged a wide range of peptides with divergent binding affinities, we solved the structure of the 1E6 TCR in complex with all 7 APLs used in Figure 2. RESULTS +4 14 structures evidence All structures were solved in space group P1 to 2–3 Å resolution with crystallographic Rwork/Rfree ratios within accepted limits as shown in the theoretically expected distribution (ref. and Supplemental Table 1). RESULTS +20 26 solved experimental_method All structures were solved in space group P1 to 2–3 Å resolution with crystallographic Rwork/Rfree ratios within accepted limits as shown in the theoretically expected distribution (ref. and Supplemental Table 1). RESULTS +87 105 Rwork/Rfree ratios evidence All structures were solved in space group P1 to 2–3 Å resolution with crystallographic Rwork/Rfree ratios within accepted limits as shown in the theoretically expected distribution (ref. and Supplemental Table 1). RESULTS +4 11 1E6 TCR complex_assembly The 1E6 TCR used a very similar overall binding modality to engage all of the APLs, with root mean square deviation ranging between 0.81 and 1.12 Å2 (compared with 1E6-A2-ALWGPDPAAA). RESULTS +78 82 APLs chemical The 1E6 TCR used a very similar overall binding modality to engage all of the APLs, with root mean square deviation ranging between 0.81 and 1.12 Å2 (compared with 1E6-A2-ALWGPDPAAA). RESULTS +89 115 root mean square deviation evidence The 1E6 TCR used a very similar overall binding modality to engage all of the APLs, with root mean square deviation ranging between 0.81 and 1.12 Å2 (compared with 1E6-A2-ALWGPDPAAA). RESULTS +164 181 1E6-A2-ALWGPDPAAA complex_assembly The 1E6 TCR used a very similar overall binding modality to engage all of the APLs, with root mean square deviation ranging between 0.81 and 1.12 Å2 (compared with 1E6-A2-ALWGPDPAAA). RESULTS +96 116 TCR binding affinity evidence The relatively broad range of buried surface areas (1,670–1,920 Å2) did not correlate well with TCR binding affinity (Pearson’s correlation = 0.45, P = 0.2). RESULTS +118 139 Pearson’s correlation evidence The relatively broad range of buried surface areas (1,670–1,920 Å2) did not correlate well with TCR binding affinity (Pearson’s correlation = 0.45, P = 0.2). RESULTS +148 149 P evidence The relatively broad range of buried surface areas (1,670–1,920 Å2) did not correlate well with TCR binding affinity (Pearson’s correlation = 0.45, P = 0.2). RESULTS +4 34 surface complementarity values evidence The surface complementarity values (0.52–0.7) correlated slightly with affinity (Pearson’s correlation = 0.7, P = 0.05) but could not explain all differences in binding (Figure 3A and Table 2). RESULTS +71 79 affinity evidence The surface complementarity values (0.52–0.7) correlated slightly with affinity (Pearson’s correlation = 0.7, P = 0.05) but could not explain all differences in binding (Figure 3A and Table 2). RESULTS +81 102 Pearson’s correlation evidence The surface complementarity values (0.52–0.7) correlated slightly with affinity (Pearson’s correlation = 0.7, P = 0.05) but could not explain all differences in binding (Figure 3A and Table 2). RESULTS +110 111 P evidence The surface complementarity values (0.52–0.7) correlated slightly with affinity (Pearson’s correlation = 0.7, P = 0.05) but could not explain all differences in binding (Figure 3A and Table 2). RESULTS +4 7 TCR complex_assembly The TCR CDR loops were in a very similar position in all complexes, apart from some slight deviations in the TCR β-chain (Figure 3B); the peptides were all presented in a similar conformation (Figure 3C); and there was minimal variation in crossing angles of the TCR (42.3°–45.6°) (Figure 3D). RESULTS +8 17 CDR loops structure_element The TCR CDR loops were in a very similar position in all complexes, apart from some slight deviations in the TCR β-chain (Figure 3B); the peptides were all presented in a similar conformation (Figure 3C); and there was minimal variation in crossing angles of the TCR (42.3°–45.6°) (Figure 3D). RESULTS +109 112 TCR complex_assembly The TCR CDR loops were in a very similar position in all complexes, apart from some slight deviations in the TCR β-chain (Figure 3B); the peptides were all presented in a similar conformation (Figure 3C); and there was minimal variation in crossing angles of the TCR (42.3°–45.6°) (Figure 3D). RESULTS +113 120 β-chain structure_element The TCR CDR loops were in a very similar position in all complexes, apart from some slight deviations in the TCR β-chain (Figure 3B); the peptides were all presented in a similar conformation (Figure 3C); and there was minimal variation in crossing angles of the TCR (42.3°–45.6°) (Figure 3D). RESULTS +263 266 TCR complex_assembly The TCR CDR loops were in a very similar position in all complexes, apart from some slight deviations in the TCR β-chain (Figure 3B); the peptides were all presented in a similar conformation (Figure 3C); and there was minimal variation in crossing angles of the TCR (42.3°–45.6°) (Figure 3D). RESULTS +13 20 1E6 TCR complex_assembly Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. RESULTS +66 69 APL chemical Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. RESULTS +79 82 TCR complex_assembly Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. RESULTS +83 90 α-chain structure_element Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. RESULTS +111 122 MHC class I complex_assembly Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. RESULTS +124 128 MHCI complex_assembly Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. RESULTS +130 138 α2-helix structure_element Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. RESULTS +147 150 TCR complex_assembly Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. RESULTS +151 158 β-chain structure_element Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. RESULTS +168 172 MHCI complex_assembly Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. RESULTS +173 182 α-1 helix structure_element Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. RESULTS +46 56 interfaces site However, subtle differences in the respective interfaces were apparent (discussed below) and resulted in altered binding affinities of the respective complexes. RESULTS +113 131 binding affinities evidence However, subtle differences in the respective interfaces were apparent (discussed below) and resulted in altered binding affinities of the respective complexes. RESULTS +25 32 1E6 TCR complex_assembly Interactions between the 1E6 TCR and different APLs are focused around a conserved GPD peptide motif. RESULTS +47 51 APLs chemical Interactions between the 1E6 TCR and different APLs are focused around a conserved GPD peptide motif. RESULTS +73 82 conserved protein_state Interactions between the 1E6 TCR and different APLs are focused around a conserved GPD peptide motif. RESULTS +83 100 GPD peptide motif structure_element Interactions between the 1E6 TCR and different APLs are focused around a conserved GPD peptide motif. RESULTS +30 45 atomic analysis experimental_method We next performed an in-depth atomic analysis of the contacts between the 1E6 TCR and each APL to determine the structural basis for the altered T cell peptide sensitivities and TCR binding affinities (Table 2). RESULTS +74 81 1E6 TCR complex_assembly We next performed an in-depth atomic analysis of the contacts between the 1E6 TCR and each APL to determine the structural basis for the altered T cell peptide sensitivities and TCR binding affinities (Table 2). RESULTS +91 94 APL chemical We next performed an in-depth atomic analysis of the contacts between the 1E6 TCR and each APL to determine the structural basis for the altered T cell peptide sensitivities and TCR binding affinities (Table 2). RESULTS +178 200 TCR binding affinities evidence We next performed an in-depth atomic analysis of the contacts between the 1E6 TCR and each APL to determine the structural basis for the altered T cell peptide sensitivities and TCR binding affinities (Table 2). RESULTS +40 47 1E6 TCR complex_assembly Concomitant with our global analysis of 1E6 TCR binding to the APLs, we observed a common interaction element, consistent with our previous findings, that utilized TCR residues Tyr97α and Trp97β, forming an aromatic cap over a central GPD motif that was present in all of the APLs (Figure 4). RESULTS +63 67 APLs chemical Concomitant with our global analysis of 1E6 TCR binding to the APLs, we observed a common interaction element, consistent with our previous findings, that utilized TCR residues Tyr97α and Trp97β, forming an aromatic cap over a central GPD motif that was present in all of the APLs (Figure 4). RESULTS +164 167 TCR complex_assembly Concomitant with our global analysis of 1E6 TCR binding to the APLs, we observed a common interaction element, consistent with our previous findings, that utilized TCR residues Tyr97α and Trp97β, forming an aromatic cap over a central GPD motif that was present in all of the APLs (Figure 4). RESULTS +177 183 Tyr97α residue_name_number Concomitant with our global analysis of 1E6 TCR binding to the APLs, we observed a common interaction element, consistent with our previous findings, that utilized TCR residues Tyr97α and Trp97β, forming an aromatic cap over a central GPD motif that was present in all of the APLs (Figure 4). RESULTS +188 194 Trp97β residue_name_number Concomitant with our global analysis of 1E6 TCR binding to the APLs, we observed a common interaction element, consistent with our previous findings, that utilized TCR residues Tyr97α and Trp97β, forming an aromatic cap over a central GPD motif that was present in all of the APLs (Figure 4). RESULTS +207 219 aromatic cap structure_element Concomitant with our global analysis of 1E6 TCR binding to the APLs, we observed a common interaction element, consistent with our previous findings, that utilized TCR residues Tyr97α and Trp97β, forming an aromatic cap over a central GPD motif that was present in all of the APLs (Figure 4). RESULTS +235 244 GPD motif structure_element Concomitant with our global analysis of 1E6 TCR binding to the APLs, we observed a common interaction element, consistent with our previous findings, that utilized TCR residues Tyr97α and Trp97β, forming an aromatic cap over a central GPD motif that was present in all of the APLs (Figure 4). RESULTS +276 280 APLs chemical Concomitant with our global analysis of 1E6 TCR binding to the APLs, we observed a common interaction element, consistent with our previous findings, that utilized TCR residues Tyr97α and Trp97β, forming an aromatic cap over a central GPD motif that was present in all of the APLs (Figure 4). RESULTS +29 32 TCR complex_assembly Interactions between these 2 TCR and 3 peptide residues accounted for 41%–50% of the total contacts across all complexes (Table 2), demonstrating the conserved peptide centric binding mode utilized by the 1E6 TCR. RESULTS +150 159 conserved protein_state Interactions between these 2 TCR and 3 peptide residues accounted for 41%–50% of the total contacts across all complexes (Table 2), demonstrating the conserved peptide centric binding mode utilized by the 1E6 TCR. RESULTS +205 212 1E6 TCR complex_assembly Interactions between these 2 TCR and 3 peptide residues accounted for 41%–50% of the total contacts across all complexes (Table 2), demonstrating the conserved peptide centric binding mode utilized by the 1E6 TCR. RESULTS +84 92 TCR-pMHC complex_assembly This fixed anchoring between the 2 molecules was important for stabilization of the TCR-pMHC complex, as — although other peptides without the ‘GDP’ motif were tested and shown to activate the 1E6 T cell clone — we were unable to measure robust affinities using SPR (data not shown). RESULTS +143 154 ‘GDP’ motif structure_element This fixed anchoring between the 2 molecules was important for stabilization of the TCR-pMHC complex, as — although other peptides without the ‘GDP’ motif were tested and shown to activate the 1E6 T cell clone — we were unable to measure robust affinities using SPR (data not shown). RESULTS +245 255 affinities evidence This fixed anchoring between the 2 molecules was important for stabilization of the TCR-pMHC complex, as — although other peptides without the ‘GDP’ motif were tested and shown to activate the 1E6 T cell clone — we were unable to measure robust affinities using SPR (data not shown). RESULTS +262 265 SPR experimental_method This fixed anchoring between the 2 molecules was important for stabilization of the TCR-pMHC complex, as — although other peptides without the ‘GDP’ motif were tested and shown to activate the 1E6 T cell clone — we were unable to measure robust affinities using SPR (data not shown). RESULTS +41 50 conserved protein_state These data support the requirement for a conserved interaction between the 1E6 TCR and the GPD motif, as we observed in our previously published 1E6-A2-ALWGPDPAAA structure. RESULTS +75 82 1E6 TCR complex_assembly These data support the requirement for a conserved interaction between the 1E6 TCR and the GPD motif, as we observed in our previously published 1E6-A2-ALWGPDPAAA structure. RESULTS +91 100 GPD motif structure_element These data support the requirement for a conserved interaction between the 1E6 TCR and the GPD motif, as we observed in our previously published 1E6-A2-ALWGPDPAAA structure. RESULTS +145 162 1E6-A2-ALWGPDPAAA complex_assembly These data support the requirement for a conserved interaction between the 1E6 TCR and the GPD motif, as we observed in our previously published 1E6-A2-ALWGPDPAAA structure. RESULTS +163 172 structure evidence These data support the requirement for a conserved interaction between the 1E6 TCR and the GPD motif, as we observed in our previously published 1E6-A2-ALWGPDPAAA structure. RESULTS +33 42 conserved protein_state Focused hotspot binding around a conserved GPD motif enables the 1E6 TCR to tolerate peptide degeneracy. RESULTS +43 52 GPD motif structure_element Focused hotspot binding around a conserved GPD motif enables the 1E6 TCR to tolerate peptide degeneracy. RESULTS +65 72 1E6 TCR complex_assembly Focused hotspot binding around a conserved GPD motif enables the 1E6 TCR to tolerate peptide degeneracy. RESULTS +13 20 1E6 TCR complex_assembly Although the 1E6 TCR formed a similar overall interaction with each APL, the stabilization between the TCR and the GPD motif enabled fine differences in the contact network with both the peptide and MHC surface that allowed discrimination between each ligand (Figure 5). RESULTS +68 71 APL chemical Although the 1E6 TCR formed a similar overall interaction with each APL, the stabilization between the TCR and the GPD motif enabled fine differences in the contact network with both the peptide and MHC surface that allowed discrimination between each ligand (Figure 5). RESULTS +103 106 TCR complex_assembly Although the 1E6 TCR formed a similar overall interaction with each APL, the stabilization between the TCR and the GPD motif enabled fine differences in the contact network with both the peptide and MHC surface that allowed discrimination between each ligand (Figure 5). RESULTS +115 124 GPD motif structure_element Although the 1E6 TCR formed a similar overall interaction with each APL, the stabilization between the TCR and the GPD motif enabled fine differences in the contact network with both the peptide and MHC surface that allowed discrimination between each ligand (Figure 5). RESULTS +157 172 contact network site Although the 1E6 TCR formed a similar overall interaction with each APL, the stabilization between the TCR and the GPD motif enabled fine differences in the contact network with both the peptide and MHC surface that allowed discrimination between each ligand (Figure 5). RESULTS +187 194 peptide chemical Although the 1E6 TCR formed a similar overall interaction with each APL, the stabilization between the TCR and the GPD motif enabled fine differences in the contact network with both the peptide and MHC surface that allowed discrimination between each ligand (Figure 5). RESULTS +199 210 MHC surface site Although the 1E6 TCR formed a similar overall interaction with each APL, the stabilization between the TCR and the GPD motif enabled fine differences in the contact network with both the peptide and MHC surface that allowed discrimination between each ligand (Figure 5). RESULTS +17 24 1E6 TCR complex_assembly For example, the 1E6 TCR made only 47 peptide contacts with A2-MVWGPDPLYV (KD = ~600 μM) compared with 63 and 57 contacts with A2-YQFGPDFPIA (KD = 7.4 μM) and A2-RQFGPDFPTI (KD = 0.5 μM), respectively. RESULTS +60 73 A2-MVWGPDPLYV chemical For example, the 1E6 TCR made only 47 peptide contacts with A2-MVWGPDPLYV (KD = ~600 μM) compared with 63 and 57 contacts with A2-YQFGPDFPIA (KD = 7.4 μM) and A2-RQFGPDFPTI (KD = 0.5 μM), respectively. RESULTS +75 77 KD evidence For example, the 1E6 TCR made only 47 peptide contacts with A2-MVWGPDPLYV (KD = ~600 μM) compared with 63 and 57 contacts with A2-YQFGPDFPIA (KD = 7.4 μM) and A2-RQFGPDFPTI (KD = 0.5 μM), respectively. RESULTS +127 140 A2-YQFGPDFPIA chemical For example, the 1E6 TCR made only 47 peptide contacts with A2-MVWGPDPLYV (KD = ~600 μM) compared with 63 and 57 contacts with A2-YQFGPDFPIA (KD = 7.4 μM) and A2-RQFGPDFPTI (KD = 0.5 μM), respectively. RESULTS +142 144 KD evidence For example, the 1E6 TCR made only 47 peptide contacts with A2-MVWGPDPLYV (KD = ~600 μM) compared with 63 and 57 contacts with A2-YQFGPDFPIA (KD = 7.4 μM) and A2-RQFGPDFPTI (KD = 0.5 μM), respectively. RESULTS +159 172 A2-RQFGPDFPTI chemical For example, the 1E6 TCR made only 47 peptide contacts with A2-MVWGPDPLYV (KD = ~600 μM) compared with 63 and 57 contacts with A2-YQFGPDFPIA (KD = 7.4 μM) and A2-RQFGPDFPTI (KD = 0.5 μM), respectively. RESULTS +174 176 KD evidence For example, the 1E6 TCR made only 47 peptide contacts with A2-MVWGPDPLYV (KD = ~600 μM) compared with 63 and 57 contacts with A2-YQFGPDFPIA (KD = 7.4 μM) and A2-RQFGPDFPTI (KD = 0.5 μM), respectively. RESULTS +64 84 TCR binding affinity evidence Although the number of peptide contacts was a good predictor of TCR binding affinity for some of the APLs, for others, the correlation was poor (Pearson’s correlation = 0.045, P = 0.92), possibly because of different resolutions for each complex structure. RESULTS +101 105 APLs chemical Although the number of peptide contacts was a good predictor of TCR binding affinity for some of the APLs, for others, the correlation was poor (Pearson’s correlation = 0.045, P = 0.92), possibly because of different resolutions for each complex structure. RESULTS +145 166 Pearson’s correlation evidence Although the number of peptide contacts was a good predictor of TCR binding affinity for some of the APLs, for others, the correlation was poor (Pearson’s correlation = 0.045, P = 0.92), possibly because of different resolutions for each complex structure. RESULTS +246 255 structure evidence Although the number of peptide contacts was a good predictor of TCR binding affinity for some of the APLs, for others, the correlation was poor (Pearson’s correlation = 0.045, P = 0.92), possibly because of different resolutions for each complex structure. RESULTS +17 24 1E6 TCR complex_assembly For example, the 1E6 TCR made 64 peptide contacts with A2-YLGGPDFPTI (KD = ~400 μM) compared with 43 contacts with A2-RQWGPDPAAV (KD = 7.8 μM). RESULTS +55 68 A2-YLGGPDFPTI chemical For example, the 1E6 TCR made 64 peptide contacts with A2-YLGGPDFPTI (KD = ~400 μM) compared with 43 contacts with A2-RQWGPDPAAV (KD = 7.8 μM). RESULTS +70 72 KD evidence For example, the 1E6 TCR made 64 peptide contacts with A2-YLGGPDFPTI (KD = ~400 μM) compared with 43 contacts with A2-RQWGPDPAAV (KD = 7.8 μM). RESULTS +115 128 A2-RQWGPDPAAV chemical For example, the 1E6 TCR made 64 peptide contacts with A2-YLGGPDFPTI (KD = ~400 μM) compared with 43 contacts with A2-RQWGPDPAAV (KD = 7.8 μM). RESULTS +130 132 KD evidence For example, the 1E6 TCR made 64 peptide contacts with A2-YLGGPDFPTI (KD = ~400 μM) compared with 43 contacts with A2-RQWGPDPAAV (KD = 7.8 μM). RESULTS +97 98 1 residue_number The most important peptide modification in terms of generating new contacts was peptide position 1. RESULTS +53 56 Arg residue_name The stronger ligands all encoded larger side chains (Arg or Tyr) at peptide position 1 (Figure 5, E–H), enabling interactions with 1E6 that were not present in the weaker APLs that lacked large side chains in this position (Figure 5, A, C, and D). RESULTS +60 63 Tyr residue_name The stronger ligands all encoded larger side chains (Arg or Tyr) at peptide position 1 (Figure 5, E–H), enabling interactions with 1E6 that were not present in the weaker APLs that lacked large side chains in this position (Figure 5, A, C, and D). RESULTS +85 86 1 residue_number The stronger ligands all encoded larger side chains (Arg or Tyr) at peptide position 1 (Figure 5, E–H), enabling interactions with 1E6 that were not present in the weaker APLs that lacked large side chains in this position (Figure 5, A, C, and D). RESULTS +171 175 APLs chemical The stronger ligands all encoded larger side chains (Arg or Tyr) at peptide position 1 (Figure 5, E–H), enabling interactions with 1E6 that were not present in the weaker APLs that lacked large side chains in this position (Figure 5, A, C, and D). RESULTS +34 41 1E6 TCR complex_assembly We have previously shown that the 1E6 TCR uses a rigid lock-and-key mechanism during binding to A2-ALWGPDPAAA. RESULTS +96 109 A2-ALWGPDPAAA chemical We have previously shown that the 1E6 TCR uses a rigid lock-and-key mechanism during binding to A2-ALWGPDPAAA. RESULTS +33 42 unligated protein_state These data demonstrated that the unligated structure of the 1E6 TCR was virtually identical to its ligated counterparts. RESULTS +43 52 structure evidence These data demonstrated that the unligated structure of the 1E6 TCR was virtually identical to its ligated counterparts. RESULTS +60 67 1E6 TCR complex_assembly These data demonstrated that the unligated structure of the 1E6 TCR was virtually identical to its ligated counterparts. RESULTS +99 106 ligated protein_state These data demonstrated that the unligated structure of the 1E6 TCR was virtually identical to its ligated counterparts. RESULTS +41 45 APLs chemical In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (ΔG) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2). RESULTS +132 151 free binding energy evidence In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (ΔG) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2). RESULTS +153 155 ΔG evidence In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (ΔG) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2). RESULTS +192 198 solved experimental_method In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (ΔG) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2). RESULTS +203 212 unligated protein_state In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (ΔG) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2). RESULTS +213 223 structures evidence In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (ΔG) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2). RESULTS +233 237 APLs chemical In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (ΔG) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2). RESULTS +243 256 A2-ALWGPDPAAA chemical In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (ΔG) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2). RESULTS +257 266 structure evidence In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (ΔG) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2). RESULTS +4 13 unligated protein_state The unligated A2-MVWGPDPLYV (KD = ~600 μM) structure revealed that the side chain Tyr9 swung around 8 Å in the complex structure, subsequently making contacts with TCR residues Asp30β and Asn51β (Figure 6A and Figure 5A, respectively). RESULTS +14 27 A2-MVWGPDPLYV chemical The unligated A2-MVWGPDPLYV (KD = ~600 μM) structure revealed that the side chain Tyr9 swung around 8 Å in the complex structure, subsequently making contacts with TCR residues Asp30β and Asn51β (Figure 6A and Figure 5A, respectively). RESULTS +29 31 KD evidence The unligated A2-MVWGPDPLYV (KD = ~600 μM) structure revealed that the side chain Tyr9 swung around 8 Å in the complex structure, subsequently making contacts with TCR residues Asp30β and Asn51β (Figure 6A and Figure 5A, respectively). RESULTS +43 52 structure evidence The unligated A2-MVWGPDPLYV (KD = ~600 μM) structure revealed that the side chain Tyr9 swung around 8 Å in the complex structure, subsequently making contacts with TCR residues Asp30β and Asn51β (Figure 6A and Figure 5A, respectively). RESULTS +82 86 Tyr9 residue_name_number The unligated A2-MVWGPDPLYV (KD = ~600 μM) structure revealed that the side chain Tyr9 swung around 8 Å in the complex structure, subsequently making contacts with TCR residues Asp30β and Asn51β (Figure 6A and Figure 5A, respectively). RESULTS +119 128 structure evidence The unligated A2-MVWGPDPLYV (KD = ~600 μM) structure revealed that the side chain Tyr9 swung around 8 Å in the complex structure, subsequently making contacts with TCR residues Asp30β and Asn51β (Figure 6A and Figure 5A, respectively). RESULTS +164 167 TCR complex_assembly The unligated A2-MVWGPDPLYV (KD = ~600 μM) structure revealed that the side chain Tyr9 swung around 8 Å in the complex structure, subsequently making contacts with TCR residues Asp30β and Asn51β (Figure 6A and Figure 5A, respectively). RESULTS +177 183 Asp30β residue_name_number The unligated A2-MVWGPDPLYV (KD = ~600 μM) structure revealed that the side chain Tyr9 swung around 8 Å in the complex structure, subsequently making contacts with TCR residues Asp30β and Asn51β (Figure 6A and Figure 5A, respectively). RESULTS +188 194 Asn51β residue_name_number The unligated A2-MVWGPDPLYV (KD = ~600 μM) structure revealed that the side chain Tyr9 swung around 8 Å in the complex structure, subsequently making contacts with TCR residues Asp30β and Asn51β (Figure 6A and Figure 5A, respectively). RESULTS +75 95 TCR binding affinity evidence This movement could result in an entropic penalty contributing to the weak TCR binding affinity we observed for this ligand. RESULTS +84 88 Asp6 residue_name_number Additional small movements in the Cα backbone of the peptide around peptide residue Asp6 were apparent in the A2-YLGGPDFPTI (KD = ~400 μM), A2-ALWGPDPAAA (KD = ~208 μM), and A2-RQFGPDWIVA (KD = 44.4 μM) structures (Figure 6, B, C, and E). RESULTS +110 123 A2-YLGGPDFPTI chemical Additional small movements in the Cα backbone of the peptide around peptide residue Asp6 were apparent in the A2-YLGGPDFPTI (KD = ~400 μM), A2-ALWGPDPAAA (KD = ~208 μM), and A2-RQFGPDWIVA (KD = 44.4 μM) structures (Figure 6, B, C, and E). RESULTS +125 127 KD evidence Additional small movements in the Cα backbone of the peptide around peptide residue Asp6 were apparent in the A2-YLGGPDFPTI (KD = ~400 μM), A2-ALWGPDPAAA (KD = ~208 μM), and A2-RQFGPDWIVA (KD = 44.4 μM) structures (Figure 6, B, C, and E). RESULTS +140 153 A2-ALWGPDPAAA chemical Additional small movements in the Cα backbone of the peptide around peptide residue Asp6 were apparent in the A2-YLGGPDFPTI (KD = ~400 μM), A2-ALWGPDPAAA (KD = ~208 μM), and A2-RQFGPDWIVA (KD = 44.4 μM) structures (Figure 6, B, C, and E). RESULTS +155 157 KD evidence Additional small movements in the Cα backbone of the peptide around peptide residue Asp6 were apparent in the A2-YLGGPDFPTI (KD = ~400 μM), A2-ALWGPDPAAA (KD = ~208 μM), and A2-RQFGPDWIVA (KD = 44.4 μM) structures (Figure 6, B, C, and E). RESULTS +174 187 A2-RQFGPDWIVA chemical Additional small movements in the Cα backbone of the peptide around peptide residue Asp6 were apparent in the A2-YLGGPDFPTI (KD = ~400 μM), A2-ALWGPDPAAA (KD = ~208 μM), and A2-RQFGPDWIVA (KD = 44.4 μM) structures (Figure 6, B, C, and E). RESULTS +189 191 KD evidence Additional small movements in the Cα backbone of the peptide around peptide residue Asp6 were apparent in the A2-YLGGPDFPTI (KD = ~400 μM), A2-ALWGPDPAAA (KD = ~208 μM), and A2-RQFGPDWIVA (KD = 44.4 μM) structures (Figure 6, B, C, and E). RESULTS +203 213 structures evidence Additional small movements in the Cα backbone of the peptide around peptide residue Asp6 were apparent in the A2-YLGGPDFPTI (KD = ~400 μM), A2-ALWGPDPAAA (KD = ~208 μM), and A2-RQFGPDWIVA (KD = 44.4 μM) structures (Figure 6, B, C, and E). RESULTS +4 13 unligated protein_state The unligated structures of A2-AQWGPDAAA, A2-RQWGPDPAAV, A2-YQFGPDFPIA, and A2-RQFGPDFPTI were virtually identical when in complex with 1E6 (Figure 6, D and F–H). RESULTS +14 24 structures evidence The unligated structures of A2-AQWGPDAAA, A2-RQWGPDPAAV, A2-YQFGPDFPIA, and A2-RQFGPDFPTI were virtually identical when in complex with 1E6 (Figure 6, D and F–H). RESULTS +28 40 A2-AQWGPDAAA chemical The unligated structures of A2-AQWGPDAAA, A2-RQWGPDPAAV, A2-YQFGPDFPIA, and A2-RQFGPDFPTI were virtually identical when in complex with 1E6 (Figure 6, D and F–H). RESULTS +42 55 A2-RQWGPDPAAV chemical The unligated structures of A2-AQWGPDAAA, A2-RQWGPDPAAV, A2-YQFGPDFPIA, and A2-RQFGPDFPTI were virtually identical when in complex with 1E6 (Figure 6, D and F–H). RESULTS +57 70 A2-YQFGPDFPIA chemical The unligated structures of A2-AQWGPDAAA, A2-RQWGPDPAAV, A2-YQFGPDFPIA, and A2-RQFGPDFPTI were virtually identical when in complex with 1E6 (Figure 6, D and F–H). RESULTS +76 89 A2-RQFGPDFPTI chemical The unligated structures of A2-AQWGPDAAA, A2-RQWGPDPAAV, A2-YQFGPDFPIA, and A2-RQFGPDFPTI were virtually identical when in complex with 1E6 (Figure 6, D and F–H). RESULTS +120 135 in complex with protein_state The unligated structures of A2-AQWGPDAAA, A2-RQWGPDPAAV, A2-YQFGPDFPIA, and A2-RQFGPDFPTI were virtually identical when in complex with 1E6 (Figure 6, D and F–H). RESULTS +23 35 A2-AQWGPDAAA chemical Apart from the case of A2-AQWGPDAAA (KD = 61.9 μM), these observations support the conclusion that the higher-affinity ligands required less conformational melding during binding, which could be energetically beneficial (lower entopic cost) during ligation with the 1E6 TCR. RESULTS +37 39 KD evidence Apart from the case of A2-AQWGPDAAA (KD = 61.9 μM), these observations support the conclusion that the higher-affinity ligands required less conformational melding during binding, which could be energetically beneficial (lower entopic cost) during ligation with the 1E6 TCR. RESULTS +110 118 affinity evidence Apart from the case of A2-AQWGPDAAA (KD = 61.9 μM), these observations support the conclusion that the higher-affinity ligands required less conformational melding during binding, which could be energetically beneficial (lower entopic cost) during ligation with the 1E6 TCR. RESULTS +266 273 1E6 TCR complex_assembly Apart from the case of A2-AQWGPDAAA (KD = 61.9 μM), these observations support the conclusion that the higher-affinity ligands required less conformational melding during binding, which could be energetically beneficial (lower entopic cost) during ligation with the 1E6 TCR. RESULTS +56 63 1E6 TCR complex_assembly Peptide modifications alter the interaction between the 1E6 TCR and the MHC surface. RESULTS +72 83 MHC surface site Peptide modifications alter the interaction between the 1E6 TCR and the MHC surface. RESULTS +35 38 TCR complex_assembly In addition to changes between the TCR and peptide component, we also observed that different APLs had different knock-on effects between the TCR and MHC. RESULTS +94 98 APLs chemical In addition to changes between the TCR and peptide component, we also observed that different APLs had different knock-on effects between the TCR and MHC. RESULTS +142 145 TCR complex_assembly In addition to changes between the TCR and peptide component, we also observed that different APLs had different knock-on effects between the TCR and MHC. RESULTS +150 153 MHC complex_assembly In addition to changes between the TCR and peptide component, we also observed that different APLs had different knock-on effects between the TCR and MHC. RESULTS +0 3 MHC complex_assembly MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). RESULTS +12 17 Arg65 residue_name_number MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). RESULTS +41 62 MHC restriction triad site MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). RESULTS +64 69 Arg65 residue_name_number MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). RESULTS +71 76 Ala69 residue_name_number MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). RESULTS +82 88 Gln155 residue_name_number MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). RESULTS +115 118 TCR complex_assembly MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). RESULTS +119 122 MHC complex_assembly MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). RESULTS +138 144 Gln155 residue_name_number MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). RESULTS +179 184 Ala69 residue_name_number MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). RESULTS +219 228 interface site MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). RESULTS +22 30 affinity evidence Generally, the weaker-affinity APLs made fewer contacts with the MHC surface (27–29 interactions) compared with the stronger-affinity APLs (29–35 contacts), consistent with a better Pearson’s correlation value (0.55) compared with TCR-peptide interactions versus affinity (0.045). RESULTS +31 35 APLs chemical Generally, the weaker-affinity APLs made fewer contacts with the MHC surface (27–29 interactions) compared with the stronger-affinity APLs (29–35 contacts), consistent with a better Pearson’s correlation value (0.55) compared with TCR-peptide interactions versus affinity (0.045). RESULTS +65 68 MHC complex_assembly Generally, the weaker-affinity APLs made fewer contacts with the MHC surface (27–29 interactions) compared with the stronger-affinity APLs (29–35 contacts), consistent with a better Pearson’s correlation value (0.55) compared with TCR-peptide interactions versus affinity (0.045). RESULTS +125 133 affinity evidence Generally, the weaker-affinity APLs made fewer contacts with the MHC surface (27–29 interactions) compared with the stronger-affinity APLs (29–35 contacts), consistent with a better Pearson’s correlation value (0.55) compared with TCR-peptide interactions versus affinity (0.045). RESULTS +134 138 APLs chemical Generally, the weaker-affinity APLs made fewer contacts with the MHC surface (27–29 interactions) compared with the stronger-affinity APLs (29–35 contacts), consistent with a better Pearson’s correlation value (0.55) compared with TCR-peptide interactions versus affinity (0.045). RESULTS +182 209 Pearson’s correlation value evidence Generally, the weaker-affinity APLs made fewer contacts with the MHC surface (27–29 interactions) compared with the stronger-affinity APLs (29–35 contacts), consistent with a better Pearson’s correlation value (0.55) compared with TCR-peptide interactions versus affinity (0.045). RESULTS +231 234 TCR complex_assembly Generally, the weaker-affinity APLs made fewer contacts with the MHC surface (27–29 interactions) compared with the stronger-affinity APLs (29–35 contacts), consistent with a better Pearson’s correlation value (0.55) compared with TCR-peptide interactions versus affinity (0.045). RESULTS +263 271 affinity evidence Generally, the weaker-affinity APLs made fewer contacts with the MHC surface (27–29 interactions) compared with the stronger-affinity APLs (29–35 contacts), consistent with a better Pearson’s correlation value (0.55) compared with TCR-peptide interactions versus affinity (0.045). RESULTS +41 44 TCR complex_assembly For instance, contacts were made between TCR residue Val53β and MHC residue Gln72 in all APLs except for in the weakest affinity ligand pair, 1E6-A2-MVWGPDPLYV, in which a subtle change in TCR conformation — probably mediated by different peptide contacts — abrogated this interaction (Figure 7A). RESULTS +53 59 Val53β residue_name_number For instance, contacts were made between TCR residue Val53β and MHC residue Gln72 in all APLs except for in the weakest affinity ligand pair, 1E6-A2-MVWGPDPLYV, in which a subtle change in TCR conformation — probably mediated by different peptide contacts — abrogated this interaction (Figure 7A). RESULTS +64 67 MHC complex_assembly For instance, contacts were made between TCR residue Val53β and MHC residue Gln72 in all APLs except for in the weakest affinity ligand pair, 1E6-A2-MVWGPDPLYV, in which a subtle change in TCR conformation — probably mediated by different peptide contacts — abrogated this interaction (Figure 7A). RESULTS +76 81 Gln72 residue_name_number For instance, contacts were made between TCR residue Val53β and MHC residue Gln72 in all APLs except for in the weakest affinity ligand pair, 1E6-A2-MVWGPDPLYV, in which a subtle change in TCR conformation — probably mediated by different peptide contacts — abrogated this interaction (Figure 7A). RESULTS +89 93 APLs chemical For instance, contacts were made between TCR residue Val53β and MHC residue Gln72 in all APLs except for in the weakest affinity ligand pair, 1E6-A2-MVWGPDPLYV, in which a subtle change in TCR conformation — probably mediated by different peptide contacts — abrogated this interaction (Figure 7A). RESULTS +120 128 affinity evidence For instance, contacts were made between TCR residue Val53β and MHC residue Gln72 in all APLs except for in the weakest affinity ligand pair, 1E6-A2-MVWGPDPLYV, in which a subtle change in TCR conformation — probably mediated by different peptide contacts — abrogated this interaction (Figure 7A). RESULTS +142 159 1E6-A2-MVWGPDPLYV complex_assembly For instance, contacts were made between TCR residue Val53β and MHC residue Gln72 in all APLs except for in the weakest affinity ligand pair, 1E6-A2-MVWGPDPLYV, in which a subtle change in TCR conformation — probably mediated by different peptide contacts — abrogated this interaction (Figure 7A). RESULTS +189 192 TCR complex_assembly For instance, contacts were made between TCR residue Val53β and MHC residue Gln72 in all APLs except for in the weakest affinity ligand pair, 1E6-A2-MVWGPDPLYV, in which a subtle change in TCR conformation — probably mediated by different peptide contacts — abrogated this interaction (Figure 7A). RESULTS +50 57 entropy evidence An energetic switch from unfavorable to favorable entropy (order-to-disorder) correlates with antigen potency. RESULTS +20 35 contact network site Our analysis of the contact network provided some clues that could explain the different antigen potencies and binding affinities between the 1E6 TCR and the different APLs. RESULTS +111 129 binding affinities evidence Our analysis of the contact network provided some clues that could explain the different antigen potencies and binding affinities between the 1E6 TCR and the different APLs. RESULTS +142 149 1E6 TCR complex_assembly Our analysis of the contact network provided some clues that could explain the different antigen potencies and binding affinities between the 1E6 TCR and the different APLs. RESULTS +168 172 APLs chemical Our analysis of the contact network provided some clues that could explain the different antigen potencies and binding affinities between the 1E6 TCR and the different APLs. RESULTS +17 24 1E6 TCR complex_assembly For example, the 1E6 TCR bound to A2-RQWGPDPAAV with the third strongest affinity (KD = 7.8 μM) but made fewer contacts than with A2-ALWGPDPAAA (KD = ~208 μM) (Table 2). RESULTS +25 33 bound to protein_state For example, the 1E6 TCR bound to A2-RQWGPDPAAV with the third strongest affinity (KD = 7.8 μM) but made fewer contacts than with A2-ALWGPDPAAA (KD = ~208 μM) (Table 2). RESULTS +34 47 A2-RQWGPDPAAV chemical For example, the 1E6 TCR bound to A2-RQWGPDPAAV with the third strongest affinity (KD = 7.8 μM) but made fewer contacts than with A2-ALWGPDPAAA (KD = ~208 μM) (Table 2). RESULTS +73 81 affinity evidence For example, the 1E6 TCR bound to A2-RQWGPDPAAV with the third strongest affinity (KD = 7.8 μM) but made fewer contacts than with A2-ALWGPDPAAA (KD = ~208 μM) (Table 2). RESULTS +83 85 KD evidence For example, the 1E6 TCR bound to A2-RQWGPDPAAV with the third strongest affinity (KD = 7.8 μM) but made fewer contacts than with A2-ALWGPDPAAA (KD = ~208 μM) (Table 2). RESULTS +130 143 A2-ALWGPDPAAA chemical For example, the 1E6 TCR bound to A2-RQWGPDPAAV with the third strongest affinity (KD = 7.8 μM) but made fewer contacts than with A2-ALWGPDPAAA (KD = ~208 μM) (Table 2). RESULTS +145 147 KD evidence For example, the 1E6 TCR bound to A2-RQWGPDPAAV with the third strongest affinity (KD = 7.8 μM) but made fewer contacts than with A2-ALWGPDPAAA (KD = ~208 μM) (Table 2). RESULTS +31 53 thermodynamic analysis experimental_method Thus, we performed an in-depth thermodynamic analysis of 6 of the ligands under investigation (Figure 8 and Supplemental Table 3). RESULTS +9 25 binding affinity evidence The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (ΔH°) and entropic (TΔS°) changes that contributed to the different binding affinities/potencies for each APL. RESULTS +42 55 A2-MVWGPDPLYV chemical The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (ΔH°) and entropic (TΔS°) changes that contributed to the different binding affinities/potencies for each APL. RESULTS +60 73 A2-YLGGPDFPTI chemical The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (ΔH°) and entropic (TΔS°) changes that contributed to the different binding affinities/potencies for each APL. RESULTS +156 165 enthalpic evidence The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (ΔH°) and entropic (TΔS°) changes that contributed to the different binding affinities/potencies for each APL. RESULTS +167 170 ΔH° evidence The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (ΔH°) and entropic (TΔS°) changes that contributed to the different binding affinities/potencies for each APL. RESULTS +176 184 entropic evidence The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (ΔH°) and entropic (TΔS°) changes that contributed to the different binding affinities/potencies for each APL. RESULTS +186 190 TΔS° evidence The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (ΔH°) and entropic (TΔS°) changes that contributed to the different binding affinities/potencies for each APL. RESULTS +234 252 binding affinities evidence The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (ΔH°) and entropic (TΔS°) changes that contributed to the different binding affinities/potencies for each APL. RESULTS +272 275 APL chemical The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (ΔH°) and entropic (TΔS°) changes that contributed to the different binding affinities/potencies for each APL. RESULTS +12 33 free binding energies evidence The overall free binding energies (ΔG°) were between –4.4 and –8.6 kcal/mol, reflecting the wide range of TCR binding affinities we observed for the different APLs. RESULTS +35 38 ΔG° evidence The overall free binding energies (ΔG°) were between –4.4 and –8.6 kcal/mol, reflecting the wide range of TCR binding affinities we observed for the different APLs. RESULTS +106 128 TCR binding affinities evidence The overall free binding energies (ΔG°) were between –4.4 and –8.6 kcal/mol, reflecting the wide range of TCR binding affinities we observed for the different APLs. RESULTS +159 163 APLs chemical The overall free binding energies (ΔG°) were between –4.4 and –8.6 kcal/mol, reflecting the wide range of TCR binding affinities we observed for the different APLs. RESULTS +77 85 affinity evidence The enthalpic contribution in each complex did not follow a clear trend with affinity, with all but the 1E6-A2-RQFGPDFPTI interaction (ΔH° = 6.3 kcal/mol) generating an energetically favorable enthalpy value (ΔH° = –3.7 to –11.4 kcal/mol); this indicated a net gain in electrostatic interactions during complex formation. RESULTS +104 121 1E6-A2-RQFGPDFPTI complex_assembly The enthalpic contribution in each complex did not follow a clear trend with affinity, with all but the 1E6-A2-RQFGPDFPTI interaction (ΔH° = 6.3 kcal/mol) generating an energetically favorable enthalpy value (ΔH° = –3.7 to –11.4 kcal/mol); this indicated a net gain in electrostatic interactions during complex formation. RESULTS +135 138 ΔH° evidence The enthalpic contribution in each complex did not follow a clear trend with affinity, with all but the 1E6-A2-RQFGPDFPTI interaction (ΔH° = 6.3 kcal/mol) generating an energetically favorable enthalpy value (ΔH° = –3.7 to –11.4 kcal/mol); this indicated a net gain in electrostatic interactions during complex formation. RESULTS +193 201 enthalpy evidence The enthalpic contribution in each complex did not follow a clear trend with affinity, with all but the 1E6-A2-RQFGPDFPTI interaction (ΔH° = 6.3 kcal/mol) generating an energetically favorable enthalpy value (ΔH° = –3.7 to –11.4 kcal/mol); this indicated a net gain in electrostatic interactions during complex formation. RESULTS +209 212 ΔH° evidence The enthalpic contribution in each complex did not follow a clear trend with affinity, with all but the 1E6-A2-RQFGPDFPTI interaction (ΔH° = 6.3 kcal/mol) generating an energetically favorable enthalpy value (ΔH° = –3.7 to –11.4 kcal/mol); this indicated a net gain in electrostatic interactions during complex formation. RESULTS +37 44 entropy evidence However, there was a clear switch in entropy between the weaker-affinity and stronger-affinity ligands, indicated by a strong Pearson’s correlation value between entropy and affinity (Pearson’s correlation value 0.93, P =0.007). RESULTS +64 72 affinity evidence However, there was a clear switch in entropy between the weaker-affinity and stronger-affinity ligands, indicated by a strong Pearson’s correlation value between entropy and affinity (Pearson’s correlation value 0.93, P =0.007). RESULTS +86 94 affinity evidence However, there was a clear switch in entropy between the weaker-affinity and stronger-affinity ligands, indicated by a strong Pearson’s correlation value between entropy and affinity (Pearson’s correlation value 0.93, P =0.007). RESULTS +126 153 Pearson’s correlation value evidence However, there was a clear switch in entropy between the weaker-affinity and stronger-affinity ligands, indicated by a strong Pearson’s correlation value between entropy and affinity (Pearson’s correlation value 0.93, P =0.007). RESULTS +162 169 entropy evidence However, there was a clear switch in entropy between the weaker-affinity and stronger-affinity ligands, indicated by a strong Pearson’s correlation value between entropy and affinity (Pearson’s correlation value 0.93, P =0.007). RESULTS +174 182 affinity evidence However, there was a clear switch in entropy between the weaker-affinity and stronger-affinity ligands, indicated by a strong Pearson’s correlation value between entropy and affinity (Pearson’s correlation value 0.93, P =0.007). RESULTS +184 211 Pearson’s correlation value evidence However, there was a clear switch in entropy between the weaker-affinity and stronger-affinity ligands, indicated by a strong Pearson’s correlation value between entropy and affinity (Pearson’s correlation value 0.93, P =0.007). RESULTS +218 219 P evidence However, there was a clear switch in entropy between the weaker-affinity and stronger-affinity ligands, indicated by a strong Pearson’s correlation value between entropy and affinity (Pearson’s correlation value 0.93, P =0.007). RESULTS +18 31 A2-ALWGPDPAAA chemical For instance, the A2-ALWGPDPAAA, A2-AQWGPDAAA, and A2-RQFGPDWIVA (KD = ~208 μM, KD = 61.9 μM, and KD = 44.4 μM, respectively) were all entropically unfavorable (TΔS° = –2.9 to –5.6 kcal/mol), indicating a net change from disorder to order. RESULTS +33 45 A2-AQWGPDAAA chemical For instance, the A2-ALWGPDPAAA, A2-AQWGPDAAA, and A2-RQFGPDWIVA (KD = ~208 μM, KD = 61.9 μM, and KD = 44.4 μM, respectively) were all entropically unfavorable (TΔS° = –2.9 to –5.6 kcal/mol), indicating a net change from disorder to order. RESULTS +51 64 A2-RQFGPDWIVA chemical For instance, the A2-ALWGPDPAAA, A2-AQWGPDAAA, and A2-RQFGPDWIVA (KD = ~208 μM, KD = 61.9 μM, and KD = 44.4 μM, respectively) were all entropically unfavorable (TΔS° = –2.9 to –5.6 kcal/mol), indicating a net change from disorder to order. RESULTS +66 68 KD evidence For instance, the A2-ALWGPDPAAA, A2-AQWGPDAAA, and A2-RQFGPDWIVA (KD = ~208 μM, KD = 61.9 μM, and KD = 44.4 μM, respectively) were all entropically unfavorable (TΔS° = –2.9 to –5.6 kcal/mol), indicating a net change from disorder to order. RESULTS +80 82 KD evidence For instance, the A2-ALWGPDPAAA, A2-AQWGPDAAA, and A2-RQFGPDWIVA (KD = ~208 μM, KD = 61.9 μM, and KD = 44.4 μM, respectively) were all entropically unfavorable (TΔS° = –2.9 to –5.6 kcal/mol), indicating a net change from disorder to order. RESULTS +98 100 KD evidence For instance, the A2-ALWGPDPAAA, A2-AQWGPDAAA, and A2-RQFGPDWIVA (KD = ~208 μM, KD = 61.9 μM, and KD = 44.4 μM, respectively) were all entropically unfavorable (TΔS° = –2.9 to –5.6 kcal/mol), indicating a net change from disorder to order. RESULTS +161 165 TΔS° evidence For instance, the A2-ALWGPDPAAA, A2-AQWGPDAAA, and A2-RQFGPDWIVA (KD = ~208 μM, KD = 61.9 μM, and KD = 44.4 μM, respectively) were all entropically unfavorable (TΔS° = –2.9 to –5.6 kcal/mol), indicating a net change from disorder to order. RESULTS +25 33 affinity evidence Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 μM), A2-YQFGPDFPIA (KD = 7.4 μM), and A2-RQFGPDFPTI (KD = 0.5 μM) exhibited favorable entropy (TΔS° = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules. RESULTS +42 55 A2-RQWGPDPAAV chemical Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 μM), A2-YQFGPDFPIA (KD = 7.4 μM), and A2-RQFGPDFPTI (KD = 0.5 μM) exhibited favorable entropy (TΔS° = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules. RESULTS +57 59 KD evidence Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 μM), A2-YQFGPDFPIA (KD = 7.4 μM), and A2-RQFGPDFPTI (KD = 0.5 μM) exhibited favorable entropy (TΔS° = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules. RESULTS +71 84 A2-YQFGPDFPIA chemical Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 μM), A2-YQFGPDFPIA (KD = 7.4 μM), and A2-RQFGPDFPTI (KD = 0.5 μM) exhibited favorable entropy (TΔS° = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules. RESULTS +86 88 KD evidence Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 μM), A2-YQFGPDFPIA (KD = 7.4 μM), and A2-RQFGPDFPTI (KD = 0.5 μM) exhibited favorable entropy (TΔS° = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules. RESULTS +104 117 A2-RQFGPDFPTI chemical Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 μM), A2-YQFGPDFPIA (KD = 7.4 μM), and A2-RQFGPDFPTI (KD = 0.5 μM) exhibited favorable entropy (TΔS° = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules. RESULTS +119 121 KD evidence Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 μM), A2-YQFGPDFPIA (KD = 7.4 μM), and A2-RQFGPDFPTI (KD = 0.5 μM) exhibited favorable entropy (TΔS° = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules. RESULTS +152 159 entropy evidence Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 μM), A2-YQFGPDFPIA (KD = 7.4 μM), and A2-RQFGPDFPTI (KD = 0.5 μM) exhibited favorable entropy (TΔS° = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules. RESULTS +161 165 TΔS° evidence Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 μM), A2-YQFGPDFPIA (KD = 7.4 μM), and A2-RQFGPDFPTI (KD = 0.5 μM) exhibited favorable entropy (TΔS° = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules. RESULTS +35 44 unligated protein_state Furthermore, the structures of the unligated pMHCs demonstrated that, for these stronger-affinity ligands, there was less conformational difference between the TCR ligated pMHCs compared with the weaker-affinity ligands (Figure 6). RESULTS +45 50 pMHCs complex_assembly Furthermore, the structures of the unligated pMHCs demonstrated that, for these stronger-affinity ligands, there was less conformational difference between the TCR ligated pMHCs compared with the weaker-affinity ligands (Figure 6). RESULTS +89 97 affinity evidence Furthermore, the structures of the unligated pMHCs demonstrated that, for these stronger-affinity ligands, there was less conformational difference between the TCR ligated pMHCs compared with the weaker-affinity ligands (Figure 6). RESULTS +160 163 TCR complex_assembly Furthermore, the structures of the unligated pMHCs demonstrated that, for these stronger-affinity ligands, there was less conformational difference between the TCR ligated pMHCs compared with the weaker-affinity ligands (Figure 6). RESULTS +164 171 ligated protein_state Furthermore, the structures of the unligated pMHCs demonstrated that, for these stronger-affinity ligands, there was less conformational difference between the TCR ligated pMHCs compared with the weaker-affinity ligands (Figure 6). RESULTS +172 177 pMHCs complex_assembly Furthermore, the structures of the unligated pMHCs demonstrated that, for these stronger-affinity ligands, there was less conformational difference between the TCR ligated pMHCs compared with the weaker-affinity ligands (Figure 6). RESULTS +203 211 affinity evidence Furthermore, the structures of the unligated pMHCs demonstrated that, for these stronger-affinity ligands, there was less conformational difference between the TCR ligated pMHCs compared with the weaker-affinity ligands (Figure 6). RESULTS +92 100 affinity evidence The potential requirement for a larger degree of induced fit during binding to these weaker-affinity ligands is consistent with the larger entropic penalties observed for these interactions. RESULTS +23 30 1E6 TCR complex_assembly Potential epitopes for 1E6 TCR occur commonly in the viral proteome. RESULTS +53 58 viral taxonomy_domain Potential epitopes for 1E6 TCR occur commonly in the viral proteome. RESULTS +56 64 peptides chemical We searched a database of over 1,924,572 unique decamer peptides from the proteome of viral pathogens that are known, or strongly suspected, to infect humans. RESULTS +86 91 viral taxonomy_domain We searched a database of over 1,924,572 unique decamer peptides from the proteome of viral pathogens that are known, or strongly suspected, to infect humans. RESULTS +151 157 humans species We searched a database of over 1,924,572 unique decamer peptides from the proteome of viral pathogens that are known, or strongly suspected, to infect humans. RESULTS +65 75 xxxGPDxxxx structure_element Three hundred forty-two of these decamers conformed to the motif xxxGPDxxxx. RESULTS +42 52 xOxGPDxxxO structure_element Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4). RESULTS +108 109 A residue_name Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4). RESULTS +110 111 V residue_name Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4). RESULTS +113 114 I residue_name Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4). RESULTS +116 117 L residue_name Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4). RESULTS +119 120 M residue_name Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4). RESULTS +122 123 Y residue_name Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4). RESULTS +125 126 F residue_name Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4). RESULTS +132 133 W residue_name Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4). RESULTS +162 172 HLA-A*0201 protein Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4). RESULTS +104 114 MVWGPDPLYV chemical Thus, there are many pathogen-encoded peptides that could act as agonists for the 1E6 T cell beyond the MVWGPDPLYV and RQFGPDWIVA sequences studied here. RESULTS +119 129 RQFGPDWIVA chemical Thus, there are many pathogen-encoded peptides that could act as agonists for the 1E6 T cell beyond the MVWGPDPLYV and RQFGPDWIVA sequences studied here. RESULTS +61 70 bacterial taxonomy_domain Extension of these analyses to include the larger genomes of bacterial pathogens would be expected to considerably increase these numbers. RESULTS +4 20 binding affinity evidence The binding affinity of the 1E6 TCR interaction with A2-RQFGPDWIVA is considerably higher than with the disease-implicated A2-ALWGPDPAAA sequence (KD = 44.4 μM and KD > 200 μM, respectively), highlighting how a pathogen-derived sequence might be capable of priming a 1E6-like T cell. RESULTS +28 35 1E6 TCR complex_assembly The binding affinity of the 1E6 TCR interaction with A2-RQFGPDWIVA is considerably higher than with the disease-implicated A2-ALWGPDPAAA sequence (KD = 44.4 μM and KD > 200 μM, respectively), highlighting how a pathogen-derived sequence might be capable of priming a 1E6-like T cell. RESULTS +53 66 A2-RQFGPDWIVA chemical The binding affinity of the 1E6 TCR interaction with A2-RQFGPDWIVA is considerably higher than with the disease-implicated A2-ALWGPDPAAA sequence (KD = 44.4 μM and KD > 200 μM, respectively), highlighting how a pathogen-derived sequence might be capable of priming a 1E6-like T cell. RESULTS +123 136 A2-ALWGPDPAAA chemical The binding affinity of the 1E6 TCR interaction with A2-RQFGPDWIVA is considerably higher than with the disease-implicated A2-ALWGPDPAAA sequence (KD = 44.4 μM and KD > 200 μM, respectively), highlighting how a pathogen-derived sequence might be capable of priming a 1E6-like T cell. RESULTS +147 149 KD evidence The binding affinity of the 1E6 TCR interaction with A2-RQFGPDWIVA is considerably higher than with the disease-implicated A2-ALWGPDPAAA sequence (KD = 44.4 μM and KD > 200 μM, respectively), highlighting how a pathogen-derived sequence might be capable of priming a 1E6-like T cell. RESULTS +164 166 KD evidence The binding affinity of the 1E6 TCR interaction with A2-RQFGPDWIVA is considerably higher than with the disease-implicated A2-ALWGPDPAAA sequence (KD = 44.4 μM and KD > 200 μM, respectively), highlighting how a pathogen-derived sequence might be capable of priming a 1E6-like T cell. RESULTS +91 94 TCR complex_assembly T cell antigen discrimination is governed by an interaction between the clonally expressed TCR and pMHC, mediated by the chemical characteristics of the interacting molecules. DISCUSS +99 103 pMHC complex_assembly T cell antigen discrimination is governed by an interaction between the clonally expressed TCR and pMHC, mediated by the chemical characteristics of the interacting molecules. DISCUSS +34 37 TCR complex_assembly It has recently become clear that TCR cross-reactivity with large numbers of different pMHC ligands is essential to plug holes in T cell immune coverage that pathogens could exploit. DISCUSS +87 91 pMHC complex_assembly It has recently become clear that TCR cross-reactivity with large numbers of different pMHC ligands is essential to plug holes in T cell immune coverage that pathogens could exploit. DISCUSS +19 28 interface site Flexibility at the interface between the TCR and pMHC, demonstrated in various studies, has been suggested as a mechanism mediating T cell cross-reactivity with multiple distinct epitopes. DISCUSS +41 44 TCR complex_assembly Flexibility at the interface between the TCR and pMHC, demonstrated in various studies, has been suggested as a mechanism mediating T cell cross-reactivity with multiple distinct epitopes. DISCUSS +49 53 pMHC complex_assembly Flexibility at the interface between the TCR and pMHC, demonstrated in various studies, has been suggested as a mechanism mediating T cell cross-reactivity with multiple distinct epitopes. DISCUSS +38 47 CDR loops structure_element This notion is attractive because the CDR loops, which form the TCR antigen-binding site, are usually the most flexible part of the TCR and have the ability to mold around differently shaped ligands. DISCUSS +64 88 TCR antigen-binding site site This notion is attractive because the CDR loops, which form the TCR antigen-binding site, are usually the most flexible part of the TCR and have the ability to mold around differently shaped ligands. DISCUSS +132 135 TCR complex_assembly This notion is attractive because the CDR loops, which form the TCR antigen-binding site, are usually the most flexible part of the TCR and have the ability to mold around differently shaped ligands. DISCUSS +109 112 TCR complex_assembly Focused binding around a minimal peptide motif has also been implicated as an alternative mechanism enabling TCR cross-reactivity. DISCUSS +69 81 alloreactive protein_state Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues. DISCUSS +82 88 murine taxonomy_domain Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues. DISCUSS +89 92 TCR complex_assembly Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues. DISCUSS +93 96 MHC complex_assembly Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues. DISCUSS +109 113 42F3 protein Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues. DISCUSS +114 117 TCR complex_assembly Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues. DISCUSS +122 127 H2-Ld protein Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues. DISCUSS +199 208 conserved protein_state Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues. DISCUSS +209 216 hotspot site Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues. DISCUSS +52 57 MHCII protein_type Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85–99 bound to HLA-DQ1). DISCUSS +69 76 Hy.1B11 protein Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85–99 bound to HLA-DQ1). DISCUSS +77 80 TCR complex_assembly Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85–99 bound to HLA-DQ1). DISCUSS +161 172 deep pocket site Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85–99 bound to HLA-DQ1). DISCUSS +202 203 2 residue_number Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85–99 bound to HLA-DQ1). DISCUSS +205 206 3 residue_number Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85–99 bound to HLA-DQ1). DISCUSS +212 213 5 residue_number Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85–99 bound to HLA-DQ1). DISCUSS +220 228 MBP85–99 protein Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85–99 bound to HLA-DQ1). DISCUSS +229 237 bound to protein_state Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85–99 bound to HLA-DQ1). DISCUSS +238 245 HLA-DQ1 protein Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85–99 bound to HLA-DQ1). DISCUSS +15 24 conserved protein_state This motif was conserved in at least 2 potential foreign peptides, originating from Herpes simplex virus and Pseudomonas aeruginosa, enabling TCR recognition of foreign epitopes. DISCUSS +84 104 Herpes simplex virus species This motif was conserved in at least 2 potential foreign peptides, originating from Herpes simplex virus and Pseudomonas aeruginosa, enabling TCR recognition of foreign epitopes. DISCUSS +109 131 Pseudomonas aeruginosa species This motif was conserved in at least 2 potential foreign peptides, originating from Herpes simplex virus and Pseudomonas aeruginosa, enabling TCR recognition of foreign epitopes. DISCUSS +142 145 TCR complex_assembly This motif was conserved in at least 2 potential foreign peptides, originating from Herpes simplex virus and Pseudomonas aeruginosa, enabling TCR recognition of foreign epitopes. DISCUSS +43 48 human species First, we currently know nothing about how human MHCI–restricted TCRs mediate cross-reactivity in the context of a clinically relevant model of autoimmunity, thought to be a major pathway of disease initiation in several autoimmune diseases. DISCUSS +49 53 MHCI complex_assembly First, we currently know nothing about how human MHCI–restricted TCRs mediate cross-reactivity in the context of a clinically relevant model of autoimmunity, thought to be a major pathway of disease initiation in several autoimmune diseases. DISCUSS +65 69 TCRs complex_assembly First, we currently know nothing about how human MHCI–restricted TCRs mediate cross-reactivity in the context of a clinically relevant model of autoimmunity, thought to be a major pathway of disease initiation in several autoimmune diseases. DISCUSS +84 87 TCR complex_assembly Second, molecular studies have not yet revealed a broad set of rules that determine TCR cross-reactivity because, with the exception of the allo–TCR-MHC pair of the 42F3 TCR and H2-Ld that did not encounter each other during T cell development, studies have been limited to structures of a TCR with only 2 or 3 different ligands. DISCUSS +140 144 allo protein_state Second, molecular studies have not yet revealed a broad set of rules that determine TCR cross-reactivity because, with the exception of the allo–TCR-MHC pair of the 42F3 TCR and H2-Ld that did not encounter each other during T cell development, studies have been limited to structures of a TCR with only 2 or 3 different ligands. DISCUSS +145 152 TCR-MHC complex_assembly Second, molecular studies have not yet revealed a broad set of rules that determine TCR cross-reactivity because, with the exception of the allo–TCR-MHC pair of the 42F3 TCR and H2-Ld that did not encounter each other during T cell development, studies have been limited to structures of a TCR with only 2 or 3 different ligands. DISCUSS +165 169 42F3 protein Second, molecular studies have not yet revealed a broad set of rules that determine TCR cross-reactivity because, with the exception of the allo–TCR-MHC pair of the 42F3 TCR and H2-Ld that did not encounter each other during T cell development, studies have been limited to structures of a TCR with only 2 or 3 different ligands. DISCUSS +170 173 TCR complex_assembly Second, molecular studies have not yet revealed a broad set of rules that determine TCR cross-reactivity because, with the exception of the allo–TCR-MHC pair of the 42F3 TCR and H2-Ld that did not encounter each other during T cell development, studies have been limited to structures of a TCR with only 2 or 3 different ligands. DISCUSS +178 183 H2-Ld protein Second, molecular studies have not yet revealed a broad set of rules that determine TCR cross-reactivity because, with the exception of the allo–TCR-MHC pair of the 42F3 TCR and H2-Ld that did not encounter each other during T cell development, studies have been limited to structures of a TCR with only 2 or 3 different ligands. DISCUSS +274 284 structures evidence Second, molecular studies have not yet revealed a broad set of rules that determine TCR cross-reactivity because, with the exception of the allo–TCR-MHC pair of the 42F3 TCR and H2-Ld that did not encounter each other during T cell development, studies have been limited to structures of a TCR with only 2 or 3 different ligands. DISCUSS +290 293 TCR complex_assembly Second, molecular studies have not yet revealed a broad set of rules that determine TCR cross-reactivity because, with the exception of the allo–TCR-MHC pair of the 42F3 TCR and H2-Ld that did not encounter each other during T cell development, studies have been limited to structures of a TCR with only 2 or 3 different ligands. DISCUSS +46 50 MHCI complex_assembly Here, we investigated a highly cross-reactive MHCI-restricted TCR isolated from a patient with T1D that recognizes an HLA-A*0201–restricted preproinsulin signal peptide (ALWGPDPAAA15–24). DISCUSS +62 65 TCR complex_assembly Here, we investigated a highly cross-reactive MHCI-restricted TCR isolated from a patient with T1D that recognizes an HLA-A*0201–restricted preproinsulin signal peptide (ALWGPDPAAA15–24). DISCUSS +118 128 HLA-A*0201 protein Here, we investigated a highly cross-reactive MHCI-restricted TCR isolated from a patient with T1D that recognizes an HLA-A*0201–restricted preproinsulin signal peptide (ALWGPDPAAA15–24). DISCUSS +140 153 preproinsulin protein Here, we investigated a highly cross-reactive MHCI-restricted TCR isolated from a patient with T1D that recognizes an HLA-A*0201–restricted preproinsulin signal peptide (ALWGPDPAAA15–24). DISCUSS +154 168 signal peptide structure_element Here, we investigated a highly cross-reactive MHCI-restricted TCR isolated from a patient with T1D that recognizes an HLA-A*0201–restricted preproinsulin signal peptide (ALWGPDPAAA15–24). DISCUSS +170 185 ALWGPDPAAA15–24 chemical Here, we investigated a highly cross-reactive MHCI-restricted TCR isolated from a patient with T1D that recognizes an HLA-A*0201–restricted preproinsulin signal peptide (ALWGPDPAAA15–24). DISCUSS +0 5 Human species Human CD8+ T cell clones expressing TCRs with this specificity mediate the destruction of β cells, have been found in islets early in infection, and are proposed to be a major driver of disease. DISCUSS +36 40 TCRs complex_assembly Human CD8+ T cell clones expressing TCRs with this specificity mediate the destruction of β cells, have been found in islets early in infection, and are proposed to be a major driver of disease. DISCUSS +3 9 solved experimental_method We solved the structure of the 1E6 TCR with 7 APLs to enable a comprehensive analysis of the molecular basis of TCR degeneracy. DISCUSS +14 23 structure evidence We solved the structure of the 1E6 TCR with 7 APLs to enable a comprehensive analysis of the molecular basis of TCR degeneracy. DISCUSS +31 38 1E6 TCR complex_assembly We solved the structure of the 1E6 TCR with 7 APLs to enable a comprehensive analysis of the molecular basis of TCR degeneracy. DISCUSS +46 50 APLs chemical We solved the structure of the 1E6 TCR with 7 APLs to enable a comprehensive analysis of the molecular basis of TCR degeneracy. DISCUSS +112 115 TCR complex_assembly We solved the structure of the 1E6 TCR with 7 APLs to enable a comprehensive analysis of the molecular basis of TCR degeneracy. DISCUSS +68 82 binding energy evidence Overall, the difference in antigen potency correlated well with the binding energy (ΔG° kcal/mol) of the 1E6 TCR for the different epitopes, which ranged from values of ΔG° = ~–4.4 to –8.6 kcal/mol (calculated from 3D affinity data) or 2D affinity values of AcKa = 2.5 × 10–5 to 4.4 × 10–2 μm4. DISCUSS +84 87 ΔG° evidence Overall, the difference in antigen potency correlated well with the binding energy (ΔG° kcal/mol) of the 1E6 TCR for the different epitopes, which ranged from values of ΔG° = ~–4.4 to –8.6 kcal/mol (calculated from 3D affinity data) or 2D affinity values of AcKa = 2.5 × 10–5 to 4.4 × 10–2 μm4. DISCUSS +105 112 1E6 TCR complex_assembly Overall, the difference in antigen potency correlated well with the binding energy (ΔG° kcal/mol) of the 1E6 TCR for the different epitopes, which ranged from values of ΔG° = ~–4.4 to –8.6 kcal/mol (calculated from 3D affinity data) or 2D affinity values of AcKa = 2.5 × 10–5 to 4.4 × 10–2 μm4. DISCUSS +169 172 ΔG° evidence Overall, the difference in antigen potency correlated well with the binding energy (ΔG° kcal/mol) of the 1E6 TCR for the different epitopes, which ranged from values of ΔG° = ~–4.4 to –8.6 kcal/mol (calculated from 3D affinity data) or 2D affinity values of AcKa = 2.5 × 10–5 to 4.4 × 10–2 μm4. DISCUSS +215 226 3D affinity evidence Overall, the difference in antigen potency correlated well with the binding energy (ΔG° kcal/mol) of the 1E6 TCR for the different epitopes, which ranged from values of ΔG° = ~–4.4 to –8.6 kcal/mol (calculated from 3D affinity data) or 2D affinity values of AcKa = 2.5 × 10–5 to 4.4 × 10–2 μm4. DISCUSS +236 247 2D affinity evidence Overall, the difference in antigen potency correlated well with the binding energy (ΔG° kcal/mol) of the 1E6 TCR for the different epitopes, which ranged from values of ΔG° = ~–4.4 to –8.6 kcal/mol (calculated from 3D affinity data) or 2D affinity values of AcKa = 2.5 × 10–5 to 4.4 × 10–2 μm4. DISCUSS +258 262 AcKa evidence Overall, the difference in antigen potency correlated well with the binding energy (ΔG° kcal/mol) of the 1E6 TCR for the different epitopes, which ranged from values of ΔG° = ~–4.4 to –8.6 kcal/mol (calculated from 3D affinity data) or 2D affinity values of AcKa = 2.5 × 10–5 to 4.4 × 10–2 μm4. DISCUSS +126 149 TCR 3D binding affinity evidence The weaker end of this spectrum extends our understanding of the limits in which T cells can functionally operate in terms of TCR 3D binding affinity and is in line with the types of very low affinity, yet fully functional self-reactive CD8+ T cells we have observed in tumor-infiltrating lymphocytes. DISCUSS +192 200 affinity evidence The weaker end of this spectrum extends our understanding of the limits in which T cells can functionally operate in terms of TCR 3D binding affinity and is in line with the types of very low affinity, yet fully functional self-reactive CD8+ T cells we have observed in tumor-infiltrating lymphocytes. DISCUSS +33 37 TCRs complex_assembly Previous studies of autoreactive TCRs have shown that their binding mode is generally atypical, either due to an unusual binding manner, weak TCR binding affinity, an unstable pMHC, or a combination of these factors. DISCUSS +142 162 TCR binding affinity evidence Previous studies of autoreactive TCRs have shown that their binding mode is generally atypical, either due to an unusual binding manner, weak TCR binding affinity, an unstable pMHC, or a combination of these factors. DISCUSS +167 175 unstable protein_state Previous studies of autoreactive TCRs have shown that their binding mode is generally atypical, either due to an unusual binding manner, weak TCR binding affinity, an unstable pMHC, or a combination of these factors. DISCUSS +176 180 pMHC complex_assembly Previous studies of autoreactive TCRs have shown that their binding mode is generally atypical, either due to an unusual binding manner, weak TCR binding affinity, an unstable pMHC, or a combination of these factors. DISCUSS +55 58 TCR complex_assembly Our data demonstrate the potential for an autoreactive TCR to bind with a conventional binding mode to a stable pMHC with antipathogen-like affinity (KD = 0.5 μM) depending on the peptide sequence. DISCUSS +105 111 stable protein_state Our data demonstrate the potential for an autoreactive TCR to bind with a conventional binding mode to a stable pMHC with antipathogen-like affinity (KD = 0.5 μM) depending on the peptide sequence. DISCUSS +112 116 pMHC complex_assembly Our data demonstrate the potential for an autoreactive TCR to bind with a conventional binding mode to a stable pMHC with antipathogen-like affinity (KD = 0.5 μM) depending on the peptide sequence. DISCUSS +140 148 affinity evidence Our data demonstrate the potential for an autoreactive TCR to bind with a conventional binding mode to a stable pMHC with antipathogen-like affinity (KD = 0.5 μM) depending on the peptide sequence. DISCUSS +150 152 KD evidence Our data demonstrate the potential for an autoreactive TCR to bind with a conventional binding mode to a stable pMHC with antipathogen-like affinity (KD = 0.5 μM) depending on the peptide sequence. DISCUSS +4 23 structural analysis experimental_method Our structural analysis revealed that the 1E6 TCR bound with a conserved conformation across all APLs investigated. DISCUSS +42 49 1E6 TCR complex_assembly Our structural analysis revealed that the 1E6 TCR bound with a conserved conformation across all APLs investigated. DISCUSS +50 55 bound protein_state Our structural analysis revealed that the 1E6 TCR bound with a conserved conformation across all APLs investigated. DISCUSS +63 85 conserved conformation protein_state Our structural analysis revealed that the 1E6 TCR bound with a conserved conformation across all APLs investigated. DISCUSS +97 101 APLs chemical Our structural analysis revealed that the 1E6 TCR bound with a conserved conformation across all APLs investigated. DISCUSS +73 76 TCR complex_assembly This binding orientation was mediated through a focused interaction with TCR residues Tyr97α and Trp97β that formed an aromatic cap over a central ‘GDP’ motif that was common to all APLs. DISCUSS +86 92 Tyr97α residue_name_number This binding orientation was mediated through a focused interaction with TCR residues Tyr97α and Trp97β that formed an aromatic cap over a central ‘GDP’ motif that was common to all APLs. DISCUSS +97 103 Trp97β residue_name_number This binding orientation was mediated through a focused interaction with TCR residues Tyr97α and Trp97β that formed an aromatic cap over a central ‘GDP’ motif that was common to all APLs. DISCUSS +119 131 aromatic cap structure_element This binding orientation was mediated through a focused interaction with TCR residues Tyr97α and Trp97β that formed an aromatic cap over a central ‘GDP’ motif that was common to all APLs. DISCUSS +147 158 ‘GDP’ motif structure_element This binding orientation was mediated through a focused interaction with TCR residues Tyr97α and Trp97β that formed an aromatic cap over a central ‘GDP’ motif that was common to all APLs. DISCUSS +182 186 APLs chemical This binding orientation was mediated through a focused interaction with TCR residues Tyr97α and Trp97β that formed an aromatic cap over a central ‘GDP’ motif that was common to all APLs. DISCUSS +54 63 GPD motif structure_element We have previously demonstrated the importance of the GPD motif using a peptide library scan, as well as a CPL scan approach. DISCUSS +72 92 peptide library scan experimental_method We have previously demonstrated the importance of the GPD motif using a peptide library scan, as well as a CPL scan approach. DISCUSS +107 115 CPL scan experimental_method We have previously demonstrated the importance of the GPD motif using a peptide library scan, as well as a CPL scan approach. DISCUSS +71 77 lacked protein_state Although the 1E6 T cell was able to activate weakly with peptides that lacked this motif, we were unable to robustly measure binding affinities or generate complex structures with these ligands, highlighting the central role of this interaction during 1E6 T cell antigen recognition. DISCUSS +125 143 binding affinities evidence Although the 1E6 T cell was able to activate weakly with peptides that lacked this motif, we were unable to robustly measure binding affinities or generate complex structures with these ligands, highlighting the central role of this interaction during 1E6 T cell antigen recognition. DISCUSS +164 174 structures evidence Although the 1E6 T cell was able to activate weakly with peptides that lacked this motif, we were unable to robustly measure binding affinities or generate complex structures with these ligands, highlighting the central role of this interaction during 1E6 T cell antigen recognition. DISCUSS +178 181 TCR complex_assembly This hotspot binding, defined as a localized cluster of interactions that dominate binding energy during protein-protein interactions, has been previously shown to contribute to TCR recognition of MHC as a mechanism that tunes T cell cross-reactivity by providing fixed anchor points that enable TCRs to tolerate a variable peptide cargo. DISCUSS +197 200 MHC complex_assembly This hotspot binding, defined as a localized cluster of interactions that dominate binding energy during protein-protein interactions, has been previously shown to contribute to TCR recognition of MHC as a mechanism that tunes T cell cross-reactivity by providing fixed anchor points that enable TCRs to tolerate a variable peptide cargo. DISCUSS +296 300 TCRs complex_assembly This hotspot binding, defined as a localized cluster of interactions that dominate binding energy during protein-protein interactions, has been previously shown to contribute to TCR recognition of MHC as a mechanism that tunes T cell cross-reactivity by providing fixed anchor points that enable TCRs to tolerate a variable peptide cargo. DISCUSS +40 43 TCR complex_assembly Alternatively, interactions between the TCR and peptide have been shown to dominate the energetic landscape during ligand engagement, ensuring that T cells retain peptide specificity. DISCUSS +38 45 1E6 TCR complex_assembly The binding mechanism utilized by the 1E6 TCR during pMHC recognition is consistent with both of these models. DISCUSS +53 57 pMHC complex_assembly The binding mechanism utilized by the 1E6 TCR during pMHC recognition is consistent with both of these models. DISCUSS +103 112 GPD motif structure_element Ligand engagement is dominated by peptide interactions, but hotspot-like interactions with the central GPD motif enable the 1E6 TCR to tolerate peptide residues that vary outside of this region, explaining how T cells expressing this TCR may cross-react with a large number of different peptides. DISCUSS +124 131 1E6 TCR complex_assembly Ligand engagement is dominated by peptide interactions, but hotspot-like interactions with the central GPD motif enable the 1E6 TCR to tolerate peptide residues that vary outside of this region, explaining how T cells expressing this TCR may cross-react with a large number of different peptides. DISCUSS +234 237 TCR complex_assembly Ligand engagement is dominated by peptide interactions, but hotspot-like interactions with the central GPD motif enable the 1E6 TCR to tolerate peptide residues that vary outside of this region, explaining how T cells expressing this TCR may cross-react with a large number of different peptides. DISCUSS +70 77 Hy.1B11 protein These findings are also analogous to the observed binding mode of the Hy.1B11 TCR, in which one aromatic residue of the TCR CDR3α loop anchored into a pocket created by a conserved peptide motif. DISCUSS +78 81 TCR complex_assembly These findings are also analogous to the observed binding mode of the Hy.1B11 TCR, in which one aromatic residue of the TCR CDR3α loop anchored into a pocket created by a conserved peptide motif. DISCUSS +120 123 TCR complex_assembly These findings are also analogous to the observed binding mode of the Hy.1B11 TCR, in which one aromatic residue of the TCR CDR3α loop anchored into a pocket created by a conserved peptide motif. DISCUSS +124 134 CDR3α loop structure_element These findings are also analogous to the observed binding mode of the Hy.1B11 TCR, in which one aromatic residue of the TCR CDR3α loop anchored into a pocket created by a conserved peptide motif. DISCUSS +59 62 TCR complex_assembly In both of these examples, self-recognition is mediated by TCR residues with aromatic side chains. DISCUSS +84 94 CDR2 loops structure_element Combined with evidence demonstrating that aromatic side chains are conserved in the CDR2 loops of TCRs from many species, we speculate that these aromatic residues could impart a level of “stickiness” to TCRs, which might be enriched in an autoimmune setting when the TCR often binds in a nonoptimal fashion. DISCUSS +98 102 TCRs complex_assembly Combined with evidence demonstrating that aromatic side chains are conserved in the CDR2 loops of TCRs from many species, we speculate that these aromatic residues could impart a level of “stickiness” to TCRs, which might be enriched in an autoimmune setting when the TCR often binds in a nonoptimal fashion. DISCUSS +204 208 TCRs complex_assembly Combined with evidence demonstrating that aromatic side chains are conserved in the CDR2 loops of TCRs from many species, we speculate that these aromatic residues could impart a level of “stickiness” to TCRs, which might be enriched in an autoimmune setting when the TCR often binds in a nonoptimal fashion. DISCUSS +268 271 TCR complex_assembly Combined with evidence demonstrating that aromatic side chains are conserved in the CDR2 loops of TCRs from many species, we speculate that these aromatic residues could impart a level of “stickiness” to TCRs, which might be enriched in an autoimmune setting when the TCR often binds in a nonoptimal fashion. DISCUSS +54 77 surface complementarity evidence Despite some weak statistical correlation between the surface complementarity (SC) and affinity, closer inspection of the interface revealed no obvious structural signature that could definitively explain the differences in antigen potency and TCR binding strength between the different ligands. DISCUSS +79 81 SC evidence Despite some weak statistical correlation between the surface complementarity (SC) and affinity, closer inspection of the interface revealed no obvious structural signature that could definitively explain the differences in antigen potency and TCR binding strength between the different ligands. DISCUSS +87 95 affinity evidence Despite some weak statistical correlation between the surface complementarity (SC) and affinity, closer inspection of the interface revealed no obvious structural signature that could definitively explain the differences in antigen potency and TCR binding strength between the different ligands. DISCUSS +122 131 interface site Despite some weak statistical correlation between the surface complementarity (SC) and affinity, closer inspection of the interface revealed no obvious structural signature that could definitively explain the differences in antigen potency and TCR binding strength between the different ligands. DISCUSS +224 239 antigen potency evidence Despite some weak statistical correlation between the surface complementarity (SC) and affinity, closer inspection of the interface revealed no obvious structural signature that could definitively explain the differences in antigen potency and TCR binding strength between the different ligands. DISCUSS +244 264 TCR binding strength evidence Despite some weak statistical correlation between the surface complementarity (SC) and affinity, closer inspection of the interface revealed no obvious structural signature that could definitively explain the differences in antigen potency and TCR binding strength between the different ligands. DISCUSS +102 123 central peptide bulge structure_element However, similar to our findings in other systems, modifications to residues outside of the canonical central peptide bulge were important for generating new interactions. DISCUSS +69 72 Arg residue_name For example, all of the stronger ligands encoded larger side chains (Arg or Tyr) at peptide position 1 that enabled new interactions with 1E6 not present with the Ala at this position in the natural preproinsulin peptide. DISCUSS +76 79 Tyr residue_name For example, all of the stronger ligands encoded larger side chains (Arg or Tyr) at peptide position 1 that enabled new interactions with 1E6 not present with the Ala at this position in the natural preproinsulin peptide. DISCUSS +101 102 1 residue_number For example, all of the stronger ligands encoded larger side chains (Arg or Tyr) at peptide position 1 that enabled new interactions with 1E6 not present with the Ala at this position in the natural preproinsulin peptide. DISCUSS +163 166 Ala residue_name For example, all of the stronger ligands encoded larger side chains (Arg or Tyr) at peptide position 1 that enabled new interactions with 1E6 not present with the Ala at this position in the natural preproinsulin peptide. DISCUSS +199 212 preproinsulin protein For example, all of the stronger ligands encoded larger side chains (Arg or Tyr) at peptide position 1 that enabled new interactions with 1E6 not present with the Ala at this position in the natural preproinsulin peptide. DISCUSS +213 220 peptide chemical For example, all of the stronger ligands encoded larger side chains (Arg or Tyr) at peptide position 1 that enabled new interactions with 1E6 not present with the Ala at this position in the natural preproinsulin peptide. DISCUSS +69 83 anchor residue structure_element These data also explain our previous findings that alteration of the anchor residue at peptide position 2 (Leu-Gln) has a direct effect on 1E6 TCR binding affinity because our structural analysis demonstrated that 1E6 made 3 additional bonds with A2-AQWGPDPAAA compared with A2-ALWGPDPAAA, consistent with the >3-fold stronger binding affinity. DISCUSS +104 105 2 residue_number These data also explain our previous findings that alteration of the anchor residue at peptide position 2 (Leu-Gln) has a direct effect on 1E6 TCR binding affinity because our structural analysis demonstrated that 1E6 made 3 additional bonds with A2-AQWGPDPAAA compared with A2-ALWGPDPAAA, consistent with the >3-fold stronger binding affinity. DISCUSS +107 114 Leu-Gln mutant These data also explain our previous findings that alteration of the anchor residue at peptide position 2 (Leu-Gln) has a direct effect on 1E6 TCR binding affinity because our structural analysis demonstrated that 1E6 made 3 additional bonds with A2-AQWGPDPAAA compared with A2-ALWGPDPAAA, consistent with the >3-fold stronger binding affinity. DISCUSS +139 163 1E6 TCR binding affinity evidence These data also explain our previous findings that alteration of the anchor residue at peptide position 2 (Leu-Gln) has a direct effect on 1E6 TCR binding affinity because our structural analysis demonstrated that 1E6 made 3 additional bonds with A2-AQWGPDPAAA compared with A2-ALWGPDPAAA, consistent with the >3-fold stronger binding affinity. DISCUSS +176 195 structural analysis experimental_method These data also explain our previous findings that alteration of the anchor residue at peptide position 2 (Leu-Gln) has a direct effect on 1E6 TCR binding affinity because our structural analysis demonstrated that 1E6 made 3 additional bonds with A2-AQWGPDPAAA compared with A2-ALWGPDPAAA, consistent with the >3-fold stronger binding affinity. DISCUSS +247 260 A2-AQWGPDPAAA chemical These data also explain our previous findings that alteration of the anchor residue at peptide position 2 (Leu-Gln) has a direct effect on 1E6 TCR binding affinity because our structural analysis demonstrated that 1E6 made 3 additional bonds with A2-AQWGPDPAAA compared with A2-ALWGPDPAAA, consistent with the >3-fold stronger binding affinity. DISCUSS +275 288 A2-ALWGPDPAAA chemical These data also explain our previous findings that alteration of the anchor residue at peptide position 2 (Leu-Gln) has a direct effect on 1E6 TCR binding affinity because our structural analysis demonstrated that 1E6 made 3 additional bonds with A2-AQWGPDPAAA compared with A2-ALWGPDPAAA, consistent with the >3-fold stronger binding affinity. DISCUSS +327 343 binding affinity evidence These data also explain our previous findings that alteration of the anchor residue at peptide position 2 (Leu-Gln) has a direct effect on 1E6 TCR binding affinity because our structural analysis demonstrated that 1E6 made 3 additional bonds with A2-AQWGPDPAAA compared with A2-ALWGPDPAAA, consistent with the >3-fold stronger binding affinity. DISCUSS +56 57 2 residue_number We have recently demonstrated how a suboptimal position 2 anchor in a melanoma-derived antigen can improve TCR binding through a similar mechanism. DISCUSS +58 64 anchor structure_element We have recently demonstrated how a suboptimal position 2 anchor in a melanoma-derived antigen can improve TCR binding through a similar mechanism. DISCUSS +107 110 TCR complex_assembly We have recently demonstrated how a suboptimal position 2 anchor in a melanoma-derived antigen can improve TCR binding through a similar mechanism. DISCUSS +94 98 pMHC complex_assembly These results challenge the notion that the most potent peptide antigens exhibit the greatest pMHC stability and have implications for the design of anchor residue–modified heteroclitic peptides for vaccination. DISCUSS +6 28 thermodynamic analysis experimental_method Early thermodynamic analysis of TCR-pMHC interactions suggested a common energetic signature, driven by favorable enthalpy (generally mediated through an increase in electrostatic interactions) and unfavorable entropy (changes from disorder to order). DISCUSS +32 40 TCR-pMHC complex_assembly Early thermodynamic analysis of TCR-pMHC interactions suggested a common energetic signature, driven by favorable enthalpy (generally mediated through an increase in electrostatic interactions) and unfavorable entropy (changes from disorder to order). DISCUSS +114 122 enthalpy evidence Early thermodynamic analysis of TCR-pMHC interactions suggested a common energetic signature, driven by favorable enthalpy (generally mediated through an increase in electrostatic interactions) and unfavorable entropy (changes from disorder to order). DISCUSS +210 217 entropy evidence Early thermodynamic analysis of TCR-pMHC interactions suggested a common energetic signature, driven by favorable enthalpy (generally mediated through an increase in electrostatic interactions) and unfavorable entropy (changes from disorder to order). DISCUSS +35 50 structural data evidence These parameters aligned well with structural data, demonstrating that TCRs engaged pMHC using an induced fit binding mode. DISCUSS +71 75 TCRs complex_assembly These parameters aligned well with structural data, demonstrating that TCRs engaged pMHC using an induced fit binding mode. DISCUSS +84 88 pMHC complex_assembly These parameters aligned well with structural data, demonstrating that TCRs engaged pMHC using an induced fit binding mode. DISCUSS +42 46 TCRs complex_assembly However, more recent data have shown that TCRs can utilize a range of energetic strategies during pMHC binding, currently with no obvious pattern in terms of TCR affinity, binding mechanism, or specificity (pathogen, cancer, or self-ligands). DISCUSS +98 102 pMHC complex_assembly However, more recent data have shown that TCRs can utilize a range of energetic strategies during pMHC binding, currently with no obvious pattern in terms of TCR affinity, binding mechanism, or specificity (pathogen, cancer, or self-ligands). DISCUSS +158 170 TCR affinity evidence However, more recent data have shown that TCRs can utilize a range of energetic strategies during pMHC binding, currently with no obvious pattern in terms of TCR affinity, binding mechanism, or specificity (pathogen, cancer, or self-ligands). DISCUSS +63 67 TCRs complex_assembly Although no energetic signature appears to exist for different TCRs, we used thermodynamic analysis here to explore whether changes in energetics could help explain ligand discrimination by a single TCR. DISCUSS +77 99 thermodynamic analysis experimental_method Although no energetic signature appears to exist for different TCRs, we used thermodynamic analysis here to explore whether changes in energetics could help explain ligand discrimination by a single TCR. DISCUSS +199 202 TCR complex_assembly Although no energetic signature appears to exist for different TCRs, we used thermodynamic analysis here to explore whether changes in energetics could help explain ligand discrimination by a single TCR. DISCUSS +67 97 Pearson’s correlation analysis experimental_method This analysis demonstrated a strong relationship (according to the Pearson’s correlation analysis) between the energetic signature used by the 1E6 TCR and the sensitivity of the 1E6 T cell clone to different APLs. DISCUSS +143 150 1E6 TCR complex_assembly This analysis demonstrated a strong relationship (according to the Pearson’s correlation analysis) between the energetic signature used by the 1E6 TCR and the sensitivity of the 1E6 T cell clone to different APLs. DISCUSS +208 212 APLs chemical This analysis demonstrated a strong relationship (according to the Pearson’s correlation analysis) between the energetic signature used by the 1E6 TCR and the sensitivity of the 1E6 T cell clone to different APLs. DISCUSS +11 14 APL chemical The weaker APL ligands were characterized by favorable enthalpy and unfavorable entropy, whereas the stronger ligands progressively shifted to favorable entropy. DISCUSS +55 63 enthalpy evidence The weaker APL ligands were characterized by favorable enthalpy and unfavorable entropy, whereas the stronger ligands progressively shifted to favorable entropy. DISCUSS +80 87 entropy evidence The weaker APL ligands were characterized by favorable enthalpy and unfavorable entropy, whereas the stronger ligands progressively shifted to favorable entropy. DISCUSS +153 160 entropy evidence The weaker APL ligands were characterized by favorable enthalpy and unfavorable entropy, whereas the stronger ligands progressively shifted to favorable entropy. DISCUSS +80 89 unligated protein_state These differences were consistent with a greater degree of movement between the unligated and ligated pMHCs for the weaker ligands, suggesting a greater requirement for disorder-to-order changes during TCR binding. DISCUSS +94 101 ligated protein_state These differences were consistent with a greater degree of movement between the unligated and ligated pMHCs for the weaker ligands, suggesting a greater requirement for disorder-to-order changes during TCR binding. DISCUSS +102 107 pMHCs complex_assembly These differences were consistent with a greater degree of movement between the unligated and ligated pMHCs for the weaker ligands, suggesting a greater requirement for disorder-to-order changes during TCR binding. DISCUSS +202 205 TCR complex_assembly These differences were consistent with a greater degree of movement between the unligated and ligated pMHCs for the weaker ligands, suggesting a greater requirement for disorder-to-order changes during TCR binding. DISCUSS +272 280 ΔG value evidence Thus, the enhanced antigen potency was probably mediated through a shift from an induced fit to a lock-and-key interaction between the stronger ligands (less requirement for energetically unfavorable disorder-to-order changes), resulting in a more energetically favorable ΔG value. DISCUSS +17 30 preproinsulin protein Importantly, the preproinsulin-derived epitope was one of the least potent peptides, demonstrating that the 1E6 T cell clone had the ability to respond to different peptide sequences with far greater potency. DISCUSS +4 14 RQFGPDWIVA chemical The RQFGPDWIVA peptide, which was substantially more potent than the preproinsulin peptide, is within the proteome of a common human pathogen (C. asparagiforme), demonstrating the potential for an encounter between a naive 1E6-like T cell and a foreign peptide with a more potent ligand that might then break self-tolerance. DISCUSS +69 82 preproinsulin protein The RQFGPDWIVA peptide, which was substantially more potent than the preproinsulin peptide, is within the proteome of a common human pathogen (C. asparagiforme), demonstrating the potential for an encounter between a naive 1E6-like T cell and a foreign peptide with a more potent ligand that might then break self-tolerance. DISCUSS +127 132 human species The RQFGPDWIVA peptide, which was substantially more potent than the preproinsulin peptide, is within the proteome of a common human pathogen (C. asparagiforme), demonstrating the potential for an encounter between a naive 1E6-like T cell and a foreign peptide with a more potent ligand that might then break self-tolerance. DISCUSS +143 159 C. asparagiforme species The RQFGPDWIVA peptide, which was substantially more potent than the preproinsulin peptide, is within the proteome of a common human pathogen (C. asparagiforme), demonstrating the potential for an encounter between a naive 1E6-like T cell and a foreign peptide with a more potent ligand that might then break self-tolerance. DISCUSS +81 86 human species Indeed, we found over 50 decamer peptides from the proteome of likely, or known, human viral pathogens alone that contained both the conserved central GPD motif and anchor residues at positions 2 and 10 that would enable binding to HLA-A*02:01. DISCUSS +87 92 viral taxonomy_domain Indeed, we found over 50 decamer peptides from the proteome of likely, or known, human viral pathogens alone that contained both the conserved central GPD motif and anchor residues at positions 2 and 10 that would enable binding to HLA-A*02:01. DISCUSS +133 142 conserved protein_state Indeed, we found over 50 decamer peptides from the proteome of likely, or known, human viral pathogens alone that contained both the conserved central GPD motif and anchor residues at positions 2 and 10 that would enable binding to HLA-A*02:01. DISCUSS +151 160 GPD motif structure_element Indeed, we found over 50 decamer peptides from the proteome of likely, or known, human viral pathogens alone that contained both the conserved central GPD motif and anchor residues at positions 2 and 10 that would enable binding to HLA-A*02:01. DISCUSS +165 180 anchor residues structure_element Indeed, we found over 50 decamer peptides from the proteome of likely, or known, human viral pathogens alone that contained both the conserved central GPD motif and anchor residues at positions 2 and 10 that would enable binding to HLA-A*02:01. DISCUSS +194 195 2 residue_number Indeed, we found over 50 decamer peptides from the proteome of likely, or known, human viral pathogens alone that contained both the conserved central GPD motif and anchor residues at positions 2 and 10 that would enable binding to HLA-A*02:01. DISCUSS +200 202 10 residue_number Indeed, we found over 50 decamer peptides from the proteome of likely, or known, human viral pathogens alone that contained both the conserved central GPD motif and anchor residues at positions 2 and 10 that would enable binding to HLA-A*02:01. DISCUSS +232 243 HLA-A*02:01 protein Indeed, we found over 50 decamer peptides from the proteome of likely, or known, human viral pathogens alone that contained both the conserved central GPD motif and anchor residues at positions 2 and 10 that would enable binding to HLA-A*02:01. DISCUSS +83 88 human species Further experiments will be required to determine whether any naturally presented, human pathogen–derived peptides act as active ligands for 1E6, but our work presented here demonstrates that it is at least feasible for an autoimmune TCR to bind to a different peptide sequence that could be present in a pathogen proteome with substantially higher affinity and potency than the interaction it might use to attack self-tissue. DISCUSS +234 237 TCR complex_assembly Further experiments will be required to determine whether any naturally presented, human pathogen–derived peptides act as active ligands for 1E6, but our work presented here demonstrates that it is at least feasible for an autoimmune TCR to bind to a different peptide sequence that could be present in a pathogen proteome with substantially higher affinity and potency than the interaction it might use to attack self-tissue. DISCUSS +349 357 affinity evidence Further experiments will be required to determine whether any naturally presented, human pathogen–derived peptides act as active ligands for 1E6, but our work presented here demonstrates that it is at least feasible for an autoimmune TCR to bind to a different peptide sequence that could be present in a pathogen proteome with substantially higher affinity and potency than the interaction it might use to attack self-tissue. DISCUSS +150 155 human species In summary, this investigation into the molecular basis of T cell cross-reactivity using a clinically relevant cytotoxic CD8+ T cell clone that kills human pancreatic β cells provides answers to a number of previously outstanding questions. DISCUSS +36 39 TCR complex_assembly First, our data shows that a single TCR has the potential to functionally (assessed through T cell activation) bind to different ligands with affinities ranging across 3 orders of magnitude. DISCUSS +142 152 affinities evidence First, our data shows that a single TCR has the potential to functionally (assessed through T cell activation) bind to different ligands with affinities ranging across 3 orders of magnitude. DISCUSS +96 101 human species Second, this is the first example in which ligands have been identified and characterized for a human autoreactive TCR that are substantially more potent than the natural self-ligand, demonstrating the potential for a pathogenic ligand to break self-tolerance and prime self-reactive T cells. DISCUSS +115 118 TCR complex_assembly Second, this is the first example in which ligands have been identified and characterized for a human autoreactive TCR that are substantially more potent than the natural self-ligand, demonstrating the potential for a pathogenic ligand to break self-tolerance and prime self-reactive T cells. DISCUSS +18 37 structural analysis experimental_method Third, this first structural analysis of a cross-reactive human MHCI–restricted autoimmune TCR showed that degeneracy was mediated through TCR-pMHC anchoring by a conserved minimal binding peptide motif. DISCUSS +58 63 human species Third, this first structural analysis of a cross-reactive human MHCI–restricted autoimmune TCR showed that degeneracy was mediated through TCR-pMHC anchoring by a conserved minimal binding peptide motif. DISCUSS +64 68 MHCI complex_assembly Third, this first structural analysis of a cross-reactive human MHCI–restricted autoimmune TCR showed that degeneracy was mediated through TCR-pMHC anchoring by a conserved minimal binding peptide motif. DISCUSS +91 94 TCR complex_assembly Third, this first structural analysis of a cross-reactive human MHCI–restricted autoimmune TCR showed that degeneracy was mediated through TCR-pMHC anchoring by a conserved minimal binding peptide motif. DISCUSS +139 147 TCR-pMHC complex_assembly Third, this first structural analysis of a cross-reactive human MHCI–restricted autoimmune TCR showed that degeneracy was mediated through TCR-pMHC anchoring by a conserved minimal binding peptide motif. DISCUSS +163 172 conserved protein_state Third, this first structural analysis of a cross-reactive human MHCI–restricted autoimmune TCR showed that degeneracy was mediated through TCR-pMHC anchoring by a conserved minimal binding peptide motif. DISCUSS +173 202 minimal binding peptide motif structure_element Third, this first structural analysis of a cross-reactive human MHCI–restricted autoimmune TCR showed that degeneracy was mediated through TCR-pMHC anchoring by a conserved minimal binding peptide motif. DISCUSS +9 12 TCR complex_assembly Finally, TCR ligand discrimination was characterized by an energetic shift from an enthalpically to entropically driven interaction. DISCUSS +80 93 preproinsulin protein Our demonstration of the molecular mechanism governing cross-reactivity by this preproinsulin reactive human CD8+ T cell clone supports the notion first put forward by Wucherpfennig and Strominger that molecular mimicry could mediate autoimmunity and has far-reaching implications for the complex nature of T cell antigen discrimination. DISCUSS +103 108 human species Our demonstration of the molecular mechanism governing cross-reactivity by this preproinsulin reactive human CD8+ T cell clone supports the notion first put forward by Wucherpfennig and Strominger that molecular mimicry could mediate autoimmunity and has far-reaching implications for the complex nature of T cell antigen discrimination. DISCUSS +62 66 APLs chemical The 1E6 T cell clone reacts with a broad sensitivity range to APLs. FIG +47 69 peptide dilution assay experimental_method (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). FIG +91 101 MVWGPDPLYV chemical (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). FIG +110 120 YLGGPDFPTI chemical (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). FIG +128 138 ALWGPDPAAA chemical (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). FIG +147 157 AQWGPDPAAA chemical (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). FIG +167 177 RQFGPDWIVA chemical (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). FIG +191 201 RQWGPDPAAV chemical (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). FIG +212 222 YQFGPDFPTA chemical (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). FIG +237 247 RQFGPDFPTI chemical (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). FIG +277 287 HLA-A*0201 protein (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). FIG +324 330 MIP-1β protein (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). FIG +57 66 tetramers oligomeric_state (C) The 1E6 T cell clone was stained, in duplicate, with tetramers composed of each APL (colored as above) presented by HLA-A*0201. (D) The stability of each APL (colored as above) was tested, in duplicate, using CD by recording the peak at 218 nm absorbance from 5°C–90°C. FIG +84 87 APL chemical (C) The 1E6 T cell clone was stained, in duplicate, with tetramers composed of each APL (colored as above) presented by HLA-A*0201. (D) The stability of each APL (colored as above) was tested, in duplicate, using CD by recording the peak at 218 nm absorbance from 5°C–90°C. FIG +120 130 HLA-A*0201 protein (C) The 1E6 T cell clone was stained, in duplicate, with tetramers composed of each APL (colored as above) presented by HLA-A*0201. (D) The stability of each APL (colored as above) was tested, in duplicate, using CD by recording the peak at 218 nm absorbance from 5°C–90°C. FIG +158 161 APL chemical (C) The 1E6 T cell clone was stained, in duplicate, with tetramers composed of each APL (colored as above) presented by HLA-A*0201. (D) The stability of each APL (colored as above) was tested, in duplicate, using CD by recording the peak at 218 nm absorbance from 5°C–90°C. FIG +213 215 CD experimental_method (C) The 1E6 T cell clone was stained, in duplicate, with tetramers composed of each APL (colored as above) presented by HLA-A*0201. (D) The stability of each APL (colored as above) was tested, in duplicate, using CD by recording the peak at 218 nm absorbance from 5°C–90°C. FIG +0 2 Tm evidence Tm values were calculated using a Boltzmann fit to each set of data. FIG +34 67 Boltzmann fit to each set of data experimental_method Tm values were calculated using a Boltzmann fit to each set of data. FIG +0 26 3D and 2D binding analysis experimental_method 3D and 2D binding analysis of the 1E6 TCR with A2-ALW and the APLs. FIG +34 41 1E6 TCR complex_assembly 3D and 2D binding analysis of the 1E6 TCR with A2-ALW and the APLs. FIG +47 53 A2-ALW chemical 3D and 2D binding analysis of the 1E6 TCR with A2-ALW and the APLs. FIG +62 66 APLs chemical 3D and 2D binding analysis of the 1E6 TCR with A2-ALW and the APLs. FIG +6 22 Binding affinity evidence (A–H) Binding affinity of the 1E6 TCR interaction at 25°C using SPR. FIG +30 37 1E6 TCR complex_assembly (A–H) Binding affinity of the 1E6 TCR interaction at 25°C using SPR. FIG +64 67 SPR experimental_method (A–H) Binding affinity of the 1E6 TCR interaction at 25°C using SPR. FIG +30 37 1E6 TCR complex_assembly Eight serial dilutions of the 1E6 TCR were measured (shown in the inset); representative data from 3 independent experiments are plotted. FIG +4 32 equilibrium binding constant evidence The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y= [P1x]/[P2 + X]). FIG +34 36 KD evidence The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y= [P1x]/[P2 + X]). FIG +69 88 nonlinear curve fit experimental_method The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y= [P1x]/[P2 + X]). FIG +41 48 1E6 TCR complex_assembly In order to calculate each response, the 1E6 TCR was also injected over a control sample (HLA-A*0201–ILAKFLHWL) that was deducted from the experimental data. FIG +90 110 HLA-A*0201–ILAKFLHWL complex_assembly In order to calculate each response, the 1E6 TCR was also injected over a control sample (HLA-A*0201–ILAKFLHWL) that was deducted from the experimental data. FIG +4 21 1E6-A2-MVWGPDPLYV complex_assembly (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +47 64 1E6-A2-YLGGPDFPTI complex_assembly (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +90 107 1E6-A2-ALWGPDPAAA complex_assembly (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +113 130 1E6-A2-AQWGPDPAAA complex_assembly (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +136 153 1E6-A2-RQFGPDWIVA complex_assembly (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +159 176 1E6-A2-RQWGPDPAAV complex_assembly (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +182 199 1E6-A2-YQFGPDFPTA complex_assembly (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +209 226 1E6-A2-RQFGPDFPTI complex_assembly (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +232 241 ΔG values evidence (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +259 262 SPR experimental_method (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +294 298 EC50 evidence (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +396 426 Pearson’s coefficient analysis experimental_method (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +428 429 r evidence (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +435 436 P evidence (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). FIG +4 25 Effective 2D affinity evidence (J) Effective 2D affinity (AcKa) calculated using adhesion frequency assays, using at least 5 cell pairs, and calculated as an average of 100 cell cell contacts. FIG +27 31 AcKa evidence (J) Effective 2D affinity (AcKa) calculated using adhesion frequency assays, using at least 5 cell pairs, and calculated as an average of 100 cell cell contacts. FIG +50 75 adhesion frequency assays experimental_method (J) Effective 2D affinity (AcKa) calculated using adhesion frequency assays, using at least 5 cell pairs, and calculated as an average of 100 cell cell contacts. FIG +4 25 Effective 2D affinity evidence (K) Effective 2D affinity plotted against 1/EC50 showing Pearson’s coefficient analysis (r) and P value. FIG +44 48 EC50 evidence (K) Effective 2D affinity plotted against 1/EC50 showing Pearson’s coefficient analysis (r) and P value. FIG +57 87 Pearson’s coefficient analysis experimental_method (K) Effective 2D affinity plotted against 1/EC50 showing Pearson’s coefficient analysis (r) and P value. FIG +89 90 r evidence (K) Effective 2D affinity plotted against 1/EC50 showing Pearson’s coefficient analysis (r) and P value. FIG +96 103 P value evidence (K) Effective 2D affinity plotted against 1/EC50 showing Pearson’s coefficient analysis (r) and P value. FIG +4 11 1E6 TCR complex_assembly The 1E6 TCR uses a conserved binding mode to engage A2-ALWGPDPAAA and the APLs. FIG +52 65 A2-ALWGPDPAAA chemical The 1E6 TCR uses a conserved binding mode to engage A2-ALWGPDPAAA and the APLs. FIG +74 78 APLs chemical The 1E6 TCR uses a conserved binding mode to engage A2-ALWGPDPAAA and the APLs. FIG +4 17 Superposition experimental_method (A) Superposition of the 1E6 TCR (multicolored illustration) in complex with all 7 APLs (multicolored sticks) and the A2-ALWGPDPAAA ligand using the HLA-A*0201 (gray illustration) molecule to align all of the structures. FIG +25 32 1E6 TCR complex_assembly (A) Superposition of the 1E6 TCR (multicolored illustration) in complex with all 7 APLs (multicolored sticks) and the A2-ALWGPDPAAA ligand using the HLA-A*0201 (gray illustration) molecule to align all of the structures. FIG +61 76 in complex with protein_state (A) Superposition of the 1E6 TCR (multicolored illustration) in complex with all 7 APLs (multicolored sticks) and the A2-ALWGPDPAAA ligand using the HLA-A*0201 (gray illustration) molecule to align all of the structures. FIG +83 87 APLs chemical (A) Superposition of the 1E6 TCR (multicolored illustration) in complex with all 7 APLs (multicolored sticks) and the A2-ALWGPDPAAA ligand using the HLA-A*0201 (gray illustration) molecule to align all of the structures. FIG +118 131 A2-ALWGPDPAAA chemical (A) Superposition of the 1E6 TCR (multicolored illustration) in complex with all 7 APLs (multicolored sticks) and the A2-ALWGPDPAAA ligand using the HLA-A*0201 (gray illustration) molecule to align all of the structures. FIG +149 159 HLA-A*0201 protein (A) Superposition of the 1E6 TCR (multicolored illustration) in complex with all 7 APLs (multicolored sticks) and the A2-ALWGPDPAAA ligand using the HLA-A*0201 (gray illustration) molecule to align all of the structures. FIG +192 197 align experimental_method (A) Superposition of the 1E6 TCR (multicolored illustration) in complex with all 7 APLs (multicolored sticks) and the A2-ALWGPDPAAA ligand using the HLA-A*0201 (gray illustration) molecule to align all of the structures. FIG +209 219 structures evidence (A) Superposition of the 1E6 TCR (multicolored illustration) in complex with all 7 APLs (multicolored sticks) and the A2-ALWGPDPAAA ligand using the HLA-A*0201 (gray illustration) molecule to align all of the structures. FIG +4 11 1E6 TCR complex_assembly The 1E6 TCR and each peptide are colored according to the APL used in the complex as in Figure 1. (B) Position of the 1E6 TCR CDR loops (multicolored lines) in each complex. FIG +58 61 APL chemical The 1E6 TCR and each peptide are colored according to the APL used in the complex as in Figure 1. (B) Position of the 1E6 TCR CDR loops (multicolored lines) in each complex. FIG +118 125 1E6 TCR complex_assembly The 1E6 TCR and each peptide are colored according to the APL used in the complex as in Figure 1. (B) Position of the 1E6 TCR CDR loops (multicolored lines) in each complex. FIG +126 135 CDR loops structure_element The 1E6 TCR and each peptide are colored according to the APL used in the complex as in Figure 1. (B) Position of the 1E6 TCR CDR loops (multicolored lines) in each complex. FIG +4 14 ALWGPDPAAA chemical The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The Cα backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 α1 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface). FIG +54 79 HLA-A*0201 binding groove site The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The Cα backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 α1 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface). FIG +137 140 APL chemical The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The Cα backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 α1 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface). FIG +191 201 HLA-A*0201 protein The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The Cα backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 α1 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface). FIG +202 212 α1 helices structure_element The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The Cα backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 α1 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface). FIG +238 252 Crossing angle evidence The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The Cα backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 α1 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface). FIG +260 267 1E6 TCR complex_assembly The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The Cα backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 α1 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface). FIG +360 370 ALWGPDPAAA chemical The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The Cα backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 α1 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface). FIG +394 402 bound in protein_state The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The Cα backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 α1 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface). FIG +407 432 HLA-A*0201 binding groove site The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The Cα backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 α1 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface). FIG +31 40 GPD motif structure_element A conserved interaction with a GPD motif underpins the 1E6 TCR interaction with the APLs. FIG +55 62 1E6 TCR complex_assembly A conserved interaction with a GPD motif underpins the 1E6 TCR interaction with the APLs. FIG +84 88 APLs chemical A conserved interaction with a GPD motif underpins the 1E6 TCR interaction with the APLs. FIG +20 27 1E6 TCR complex_assembly Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). FIG +57 63 Tyr97α residue_name_number Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). FIG +68 74 Tyr97β residue_name_number Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). FIG +117 120 TCR complex_assembly Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). FIG +121 136 in complex with protein_state Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). FIG +143 147 APLs chemical Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). FIG +177 190 A2-ALWGPDPAAA chemical Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). FIG +247 264 GPD peptide motif structure_element Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). FIG +309 313 APLs chemical Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). FIG +318 331 A2-ALWGPDPAAA chemical Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). FIG +332 347 in complex with protein_state Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). FIG +352 359 1E6 TCR complex_assembly Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). FIG +33 36 MHC complex_assembly The rest of the peptide, and the MHCα1 helix, are shown as a gray illustration. FIG +36 44 α1 helix structure_element The rest of the peptide, and the MHCα1 helix, are shown as a gray illustration. FIG +4 11 1E6 TCR complex_assembly The 1E6 TCR makes distinct peptide contacts with peripheral APL residues. FIG +60 63 APL chemical The 1E6 TCR makes distinct peptide contacts with peripheral APL residues. FIG +25 32 1E6 TCR complex_assembly Interactions between the 1E6 TCR and peptide residues outside of the conserved GPD motif. FIG +69 78 conserved protein_state Interactions between the 1E6 TCR and peptide residues outside of the conserved GPD motif. FIG +79 88 GPD motif structure_element Interactions between the 1E6 TCR and peptide residues outside of the conserved GPD motif. FIG +4 7 MHC complex_assembly The MHCα1 helix is shown in gray illustrations. FIG +7 15 α1 helix structure_element The MHCα1 helix is shown in gray illustrations. FIG +0 14 Hydrogen bonds bond_interaction Hydrogen bonds are shown as red dotted lines; van der Waals (vdW) contacts are shown as black dotted lines. FIG +46 74 van der Waals (vdW) contacts bond_interaction Hydrogen bonds are shown as red dotted lines; van der Waals (vdW) contacts are shown as black dotted lines. FIG +38 45 1E6 TCR complex_assembly Boxes show total contacts between the 1E6 TCR and each peptide ligand. FIG +28 35 1E6 TCR complex_assembly (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. FIG +72 85 A2-MVWGPDPLYV chemical (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. FIG +147 154 1E6 TCR complex_assembly (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. FIG +189 202 A2-YLGGPDFPTI chemical (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. FIG +262 269 1E6 TCR complex_assembly (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. FIG +305 318 A2-ALWGPDPAAA chemical (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. FIG +28 35 1E6 TCR complex_assembly (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. FIG +72 85 A2-MVWGPDPLYV chemical (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. FIG +147 154 1E6 TCR complex_assembly (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. FIG +189 202 A2-YLGGPDFPTI chemical (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. FIG +262 269 1E6 TCR complex_assembly (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. FIG +305 318 A2-ALWGPDPAAA chemical (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. FIG +28 35 1E6 TCR complex_assembly (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +72 85 A2-AQWGPDPAAA chemical (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +147 154 1E6 TCR complex_assembly (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +195 208 A2-RQFGPDWIVA chemical (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +274 281 1E6 TCR complex_assembly (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +319 332 A2-RQWGPDPAAV chemical (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +395 402 1E6 TCR complex_assembly (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +440 453 A2-YQFGPDFPTA chemical (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +516 523 1E6 TCR complex_assembly (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +559 572 A2-RQFGPDFPTI chemical (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +28 35 1E6 TCR complex_assembly (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +72 85 A2-AQWGPDPAAA chemical (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +147 154 1E6 TCR complex_assembly (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +195 208 A2-RQFGPDWIVA chemical (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +274 281 1E6 TCR complex_assembly (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +319 332 A2-RQWGPDPAAV chemical (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +395 402 1E6 TCR complex_assembly (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +440 453 A2-YQFGPDFPTA chemical (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +516 523 1E6 TCR complex_assembly (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +559 572 A2-RQFGPDFPTI chemical (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). FIG +14 21 ligated protein_state Comparison of ligated and unligated APLs. FIG +26 35 unligated protein_state Comparison of ligated and unligated APLs. FIG +36 40 APLs chemical Comparison of ligated and unligated APLs. FIG +0 13 Superposition experimental_method Superposition of each APL in unligated form and ligated to the 1E6 TCR. FIG +22 25 APL chemical Superposition of each APL in unligated form and ligated to the 1E6 TCR. FIG +29 38 unligated protein_state Superposition of each APL in unligated form and ligated to the 1E6 TCR. FIG +48 55 ligated protein_state Superposition of each APL in unligated form and ligated to the 1E6 TCR. FIG +63 70 1E6 TCR complex_assembly Superposition of each APL in unligated form and ligated to the 1E6 TCR. FIG +4 13 unligated protein_state All unligated pMHCs are shown as light green illustrations. FIG +14 19 pMHCs complex_assembly All unligated pMHCs are shown as light green illustrations. FIG +44 53 structure evidence Peptide sequences are shown underneath each structure aligned with the peptide structure. FIG +79 88 structure evidence Peptide sequences are shown underneath each structure aligned with the peptide structure. FIG +4 17 A2-MVWGPDPLYV chemical (A) A2-MVWGPDPLYV (black sticks). FIG +46 50 Tyr8 residue_name_number A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks). FIG +58 65 ligated protein_state A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks). FIG +73 82 unligated protein_state A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks). FIG +110 123 A2-YLGGPDFPTI chemical A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks). FIG +142 155 A2-ALWGPDPAAA chemical A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks). FIG +215 228 A2-AQWGPDPAAA chemical A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks). FIG +249 262 A2-RQFGPDWIVA chemical A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks). FIG +287 300 A2-RQWGPDPAAV chemical A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks). FIG +322 335 A2-YQFGPDFPTA chemical A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks). FIG +357 370 A2-RQFGPDFPTI chemical A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks). FIG +4 11 1E6 TCR complex_assembly The 1E6 TCR makes distinct peptide contacts with the MHC surface depending on the peptide cargo. FIG +53 64 MHC surface site The 1E6 TCR makes distinct peptide contacts with the MHC surface depending on the peptide cargo. FIG +25 32 1E6 TCR complex_assembly Interactions between the 1E6 TCR and the MHC α1 helix residues Arg65, Lys66, and Gln72. FIG +41 44 MHC complex_assembly Interactions between the 1E6 TCR and the MHC α1 helix residues Arg65, Lys66, and Gln72. FIG +45 53 α1 helix structure_element Interactions between the 1E6 TCR and the MHC α1 helix residues Arg65, Lys66, and Gln72. FIG +63 68 Arg65 residue_name_number Interactions between the 1E6 TCR and the MHC α1 helix residues Arg65, Lys66, and Gln72. FIG +70 75 Lys66 residue_name_number Interactions between the 1E6 TCR and the MHC α1 helix residues Arg65, Lys66, and Gln72. FIG +81 86 Gln72 residue_name_number Interactions between the 1E6 TCR and the MHC α1 helix residues Arg65, Lys66, and Gln72. FIG +0 14 Hydrogen bonds bond_interaction Hydrogen bonds are shown as red dotted lines; vdW contacts are shown as black dotted lines. FIG +46 49 vdW bond_interaction Hydrogen bonds are shown as red dotted lines; vdW contacts are shown as black dotted lines. FIG +0 3 MHC complex_assembly MHCα1 helix are shown in gray illustrations. FIG +3 11 α1 helix structure_element MHCα1 helix are shown in gray illustrations. FIG +38 45 1E6 TCR complex_assembly Boxes show total contacts between the 1E6 TCR and these key residues (green boxes are MHC residues; white boxes are TCR residues). FIG +86 89 MHC complex_assembly Boxes show total contacts between the 1E6 TCR and these key residues (green boxes are MHC residues; white boxes are TCR residues). FIG +116 119 TCR complex_assembly Boxes show total contacts between the 1E6 TCR and these key residues (green boxes are MHC residues; white boxes are TCR residues). FIG +0 22 Thermodynamic analysis experimental_method Thermodynamic analysis of the 1E6 TCR with A2-ALWGPDPAAA and the APLs. FIG +30 37 1E6 TCR complex_assembly Thermodynamic analysis of the 1E6 TCR with A2-ALWGPDPAAA and the APLs. FIG +43 56 A2-ALWGPDPAAA chemical Thermodynamic analysis of the 1E6 TCR with A2-ALWGPDPAAA and the APLs. FIG +65 69 APLs chemical Thermodynamic analysis of the 1E6 TCR with A2-ALWGPDPAAA and the APLs. FIG +30 37 1E6 TCR complex_assembly Eight serial dilutions of the 1E6 TCR were injected, in duplicate, over each immobilized APL and A2-ALW at 5°C, 13°C, 18°C, 25°C, 30°C, and 37°C. FIG +89 92 APL chemical Eight serial dilutions of the 1E6 TCR were injected, in duplicate, over each immobilized APL and A2-ALW at 5°C, 13°C, 18°C, 25°C, 30°C, and 37°C. FIG +97 103 A2-ALW chemical Eight serial dilutions of the 1E6 TCR were injected, in duplicate, over each immobilized APL and A2-ALW at 5°C, 13°C, 18°C, 25°C, 30°C, and 37°C. FIG +4 32 equilibrium binding constant evidence The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = [P1x]/[P2 + X]), and thermodynamic parameters were calculated according to the Gibbs-Helmholtz equation (ΔG° = ΔH − TΔS°). FIG +34 36 KD evidence The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = [P1x]/[P2 + X]), and thermodynamic parameters were calculated according to the Gibbs-Helmholtz equation (ΔG° = ΔH − TΔS°). FIG +69 88 nonlinear curve fit experimental_method The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = [P1x]/[P2 + X]), and thermodynamic parameters were calculated according to the Gibbs-Helmholtz equation (ΔG° = ΔH − TΔS°). FIG +173 197 Gibbs-Helmholtz equation experimental_method The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = [P1x]/[P2 + X]), and thermodynamic parameters were calculated according to the Gibbs-Helmholtz equation (ΔG° = ΔH − TΔS°). FIG +199 202 ΔG° evidence The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = [P1x]/[P2 + X]), and thermodynamic parameters were calculated according to the Gibbs-Helmholtz equation (ΔG° = ΔH − TΔS°). FIG +205 207 ΔH evidence The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = [P1x]/[P2 + X]), and thermodynamic parameters were calculated according to the Gibbs-Helmholtz equation (ΔG° = ΔH − TΔS°). FIG +210 214 TΔS° evidence The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = [P1x]/[P2 + X]), and thermodynamic parameters were calculated according to the Gibbs-Helmholtz equation (ΔG° = ΔH − TΔS°). FIG +4 25 binding free energies evidence The binding free energies, ΔG° (ΔG° = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van’t Hoff equation (RT ln KD = ΔH° – TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0] with T0 = 298 K). FIG +27 30 ΔG° evidence The binding free energies, ΔG° (ΔG° = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van’t Hoff equation (RT ln KD = ΔH° – TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0] with T0 = 298 K). FIG +32 35 ΔG° evidence The binding free energies, ΔG° (ΔG° = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van’t Hoff equation (RT ln KD = ΔH° – TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0] with T0 = 298 K). FIG +90 110 nonlinear regression experimental_method The binding free energies, ΔG° (ΔG° = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van’t Hoff equation (RT ln KD = ΔH° – TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0] with T0 = 298 K). FIG +135 154 van’t Hoff equation experimental_method The binding free energies, ΔG° (ΔG° = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van’t Hoff equation (RT ln KD = ΔH° – TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0] with T0 = 298 K). FIG +156 164 RT ln KD evidence The binding free energies, ΔG° (ΔG° = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van’t Hoff equation (RT ln KD = ΔH° – TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0] with T0 = 298 K). FIG +167 170 ΔH° evidence The binding free energies, ΔG° (ΔG° = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van’t Hoff equation (RT ln KD = ΔH° – TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0] with T0 = 298 K). FIG +173 177 TΔS° evidence The binding free energies, ΔG° (ΔG° = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van’t Hoff equation (RT ln KD = ΔH° – TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0] with T0 = 298 K). FIG +180 184 ΔCp° evidence The binding free energies, ΔG° (ΔG° = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van’t Hoff equation (RT ln KD = ΔH° – TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0] with T0 = 298 K). FIG +193 198 TΔCp° evidence The binding free energies, ΔG° (ΔG° = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van’t Hoff equation (RT ln KD = ΔH° – TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0] with T0 = 298 K). FIG +4 21 1E6-A2-ALWGPDPAAA complex_assembly (A) 1E6-A2-ALWGPDPAAA; (B) 1E6-A2-AQWGPDPAAA; (C) 1E6-A2-RQFGPDWIVA; (D) 1E6-A2-RQWGPDPAAV, (E) 1E6-A2-YQFGPDFPTA; and (F) 1E6-A2-RQFGPDFPTI. FIG +27 44 1E6-A2-AQWGPDPAAA complex_assembly (A) 1E6-A2-ALWGPDPAAA; (B) 1E6-A2-AQWGPDPAAA; (C) 1E6-A2-RQFGPDWIVA; (D) 1E6-A2-RQWGPDPAAV, (E) 1E6-A2-YQFGPDFPTA; and (F) 1E6-A2-RQFGPDFPTI. FIG +50 67 1E6-A2-RQFGPDWIVA complex_assembly (A) 1E6-A2-ALWGPDPAAA; (B) 1E6-A2-AQWGPDPAAA; (C) 1E6-A2-RQFGPDWIVA; (D) 1E6-A2-RQWGPDPAAV, (E) 1E6-A2-YQFGPDFPTA; and (F) 1E6-A2-RQFGPDFPTI. FIG +73 90 1E6-A2-RQWGPDPAAV complex_assembly (A) 1E6-A2-ALWGPDPAAA; (B) 1E6-A2-AQWGPDPAAA; (C) 1E6-A2-RQFGPDWIVA; (D) 1E6-A2-RQWGPDPAAV, (E) 1E6-A2-YQFGPDFPTA; and (F) 1E6-A2-RQFGPDFPTI. FIG +96 113 1E6-A2-YQFGPDFPTA complex_assembly (A) 1E6-A2-ALWGPDPAAA; (B) 1E6-A2-AQWGPDPAAA; (C) 1E6-A2-RQFGPDWIVA; (D) 1E6-A2-RQWGPDPAAV, (E) 1E6-A2-YQFGPDFPTA; and (F) 1E6-A2-RQFGPDFPTI. FIG +123 140 1E6-A2-RQFGPDFPTI complex_assembly (A) 1E6-A2-ALWGPDPAAA; (B) 1E6-A2-AQWGPDPAAA; (C) 1E6-A2-RQFGPDWIVA; (D) 1E6-A2-RQWGPDPAAV, (E) 1E6-A2-YQFGPDFPTA; and (F) 1E6-A2-RQFGPDFPTI. FIG +0 12 1E6 TCR-pMHC complex_assembly 1E6 TCR-pMHC contacts, affinity measurements and thermodynamics TABLE +23 44 affinity measurements experimental_method 1E6 TCR-pMHC contacts, affinity measurements and thermodynamics TABLE +49 63 thermodynamics experimental_method 1E6 TCR-pMHC contacts, affinity measurements and thermodynamics TABLE diff --git a/annotation_CSV/PMC4887326.csv b/annotation_CSV/PMC4887326.csv new file mode 100644 index 0000000000000000000000000000000000000000..f5ea2f95f6bd114fe44134038e802b55c107e13b --- /dev/null +++ b/annotation_CSV/PMC4887326.csv @@ -0,0 +1,943 @@ +anno_start anno_end anno_text entity_type sentence section +57 79 Pseudomonas aeruginosa species Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system TITLE +80 86 YfiBNR complex_assembly Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system TITLE +0 6 YfiBNR complex_assembly YfiBNR is a recently identified bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. ABSTRACT +32 62 bis-(3’-5’)-cyclic dimeric GMP chemical YfiBNR is a recently identified bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. ABSTRACT +64 72 c-di-GMP chemical YfiBNR is a recently identified bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. ABSTRACT +28 32 YfiB protein In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. ABSTRACT +93 97 YfiR protein In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. ABSTRACT +127 131 YfiN protein In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. ABSTRACT +209 213 YfiN protein In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. ABSTRACT +224 232 c-di-GMP chemical In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. ABSTRACT +20 38 crystal structures evidence Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. ABSTRACT +42 46 YfiB protein Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. ABSTRACT +47 52 alone protein_state Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. ABSTRACT +63 69 active protein_state Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. ABSTRACT +70 76 mutant protein_state Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. ABSTRACT +77 85 YfiBL43P mutant Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. ABSTRACT +86 100 complexed with protein_state Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. ABSTRACT +101 105 YfiR protein Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. ABSTRACT +0 19 Structural analyses experimental_method Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +53 73 compact conformation protein_state Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +81 88 dimeric oligomeric_state Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +89 93 YfiB protein Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +94 99 alone protein_state Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +101 109 YfiBL43P mutant Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +119 141 stretched conformation protein_state Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +151 160 activated protein_state Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +161 165 YfiB protein Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +183 196 peptidoglycan chemical Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +198 200 PG chemical Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +219 223 YfiR protein Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +225 233 YfiBL43P mutant Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +255 272 PG-binding pocket site Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +289 308 PG binding affinity evidence Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +314 323 wild-type protein_state Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +324 328 YfiB protein Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +384 388 YfiB protein Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. ABSTRACT +17 42 crystallographic analyses experimental_method In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. ABSTRACT +57 61 YfiR protein In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. ABSTRACT +68 78 Vitamin B6 chemical In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. ABSTRACT +80 83 VB6 chemical In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. ABSTRACT +88 93 L-Trp chemical In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. ABSTRACT +99 116 YfiB-binding site site In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. ABSTRACT +131 134 VB6 chemical In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. ABSTRACT +139 144 L-Trp chemical In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. ABSTRACT +164 172 YfiBL43P mutant In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. ABSTRACT +13 44 structural and biochemical data evidence Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system. ABSTRACT +92 98 YfiBNR complex_assembly Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system. ABSTRACT +0 30 Bis-(3’-5’)-cyclic dimeric GMP chemical Bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) is a ubiquitous second messenger that bacteria use to facilitate behavioral adaptations to their ever-changing environment. INTRO +32 40 c-di-GMP chemical Bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) is a ubiquitous second messenger that bacteria use to facilitate behavioral adaptations to their ever-changing environment. INTRO +80 88 bacteria taxonomy_domain Bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) is a ubiquitous second messenger that bacteria use to facilitate behavioral adaptations to their ever-changing environment. INTRO +15 23 c-di-GMP chemical An increase in c-di-GMP promotes biofilm formation, and a decrease results in biofilm degradation (Boehm et al.,; Duerig et al.,; Hickman et al.,; Jenal,; Romling et al.,). INTRO +4 12 c-di-GMP chemical The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +74 94 diguanylate cyclases protein_type The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +96 100 DGCs protein_type The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +118 126 c-di-GMP chemical The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +131 149 phosphodiesterases protein_type The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +151 155 PDEs protein_type The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +172 180 c-di-GMP chemical The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +304 321 N-terminal domain structure_element The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +400 420 relatively conserved protein_state The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +421 432 GGDEF motif structure_element The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +436 440 DGCs protein_type The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +444 462 EAL/HD-GYP domains structure_element The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +466 470 PDEs protein_type The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,). INTRO +69 78 bacterium taxonomy_domain Intriguingly, studies in diverse species have revealed that a single bacterium can have dozens of DGCs and PDEs (Hickman et al.,; Kirillina et al.,; Kulasakara et al.,; Tamayo et al.,). INTRO +98 102 DGCs protein_type Intriguingly, studies in diverse species have revealed that a single bacterium can have dozens of DGCs and PDEs (Hickman et al.,; Kirillina et al.,; Kulasakara et al.,; Tamayo et al.,). INTRO +107 111 PDEs protein_type Intriguingly, studies in diverse species have revealed that a single bacterium can have dozens of DGCs and PDEs (Hickman et al.,; Kirillina et al.,; Kulasakara et al.,; Tamayo et al.,). INTRO +3 25 Pseudomonas aeruginosa species In Pseudomonas aeruginosa in particular, 42 genes containing putative DGCs and/or PDEs were identified (Kulasakara et al.,). INTRO +70 74 DGCs protein_type In Pseudomonas aeruginosa in particular, 42 genes containing putative DGCs and/or PDEs were identified (Kulasakara et al.,). INTRO +82 86 PDEs protein_type In Pseudomonas aeruginosa in particular, 42 genes containing putative DGCs and/or PDEs were identified (Kulasakara et al.,). INTRO +59 67 c-di-GMP chemical The functional role of a number of downstream effectors of c-di-GMP has been characterized as affecting exopolysaccharide (EPS) production, transcription, motility, and surface attachment (Caly et al.,; Camilli and Bassler,; Ha and O’Toole,; Pesavento and Hengge,). INTRO +104 121 exopolysaccharide chemical The functional role of a number of downstream effectors of c-di-GMP has been characterized as affecting exopolysaccharide (EPS) production, transcription, motility, and surface attachment (Caly et al.,; Camilli and Bassler,; Ha and O’Toole,; Pesavento and Hengge,). INTRO +123 126 EPS chemical The functional role of a number of downstream effectors of c-di-GMP has been characterized as affecting exopolysaccharide (EPS) production, transcription, motility, and surface attachment (Caly et al.,; Camilli and Bassler,; Ha and O’Toole,; Pesavento and Hengge,). INTRO +33 41 c-di-GMP chemical However, due to the intricacy of c-di-GMP signaling networks and the diversity of experimental cues, the detailed mechanisms by which these signaling pathways specifically sense and integrate different inputs remain largely elusive. INTRO +38 46 bacteria taxonomy_domain Biofilm formation protects pathogenic bacteria from antibiotic treatment, and c-di-GMP-regulated biofilm formation has been extensively studied in P. aeruginosa (Evans,; Kirisits et al.,; Malone,; Reinhardt et al.,). INTRO +147 160 P. aeruginosa species Biofilm formation protects pathogenic bacteria from antibiotic treatment, and c-di-GMP-regulated biofilm formation has been extensively studied in P. aeruginosa (Evans,; Kirisits et al.,; Malone,; Reinhardt et al.,). INTRO +139 152 P. aeruginosa species In the lungs of cystic fibrosis (CF) patients, adherent biofilm formation and the appearance of small colony variant (SCV) morphologies of P. aeruginosa correlate with prolonged persistence of infection and poor lung function (Govan and Deretic,; Haussler et al.,; Haussler et al.,; Parsek and Singh,; Smith et al.,). INTRO +46 56 tripartite protein_state Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,). INTRO +57 65 c-di-GMP chemical Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,). INTRO +90 96 YfiBNR complex_assembly Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,). INTRO +112 118 AwsXRO complex_assembly Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,). INTRO +158 161 Tbp complex_assembly Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,). INTRO +183 200 genetic screening experimental_method Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,). INTRO +246 264 P. aeruginosa PAO1 species Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,). INTRO +4 10 YfiBNR complex_assembly The YfiBNR system contains three protein members and modulates intracellular c-di-GMP levels in response to signals received in the periplasm (Malone et al.,). INTRO +77 85 c-di-GMP chemical The YfiBNR system contains three protein members and modulates intracellular c-di-GMP levels in response to signals received in the periplasm (Malone et al.,). INTRO +54 76 Gram-negative bacteria taxonomy_domain More recently, this system was also reported in other Gram-negative bacteria, such as Escherichia coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), Klebsiella pneumonia (Huertas et al.,) and Yersinia pestis (Ren et al.,). INTRO +86 102 Escherichia coli species More recently, this system was also reported in other Gram-negative bacteria, such as Escherichia coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), Klebsiella pneumonia (Huertas et al.,) and Yersinia pestis (Ren et al.,). INTRO +165 185 Klebsiella pneumonia species More recently, this system was also reported in other Gram-negative bacteria, such as Escherichia coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), Klebsiella pneumonia (Huertas et al.,) and Yersinia pestis (Ren et al.,). INTRO +208 223 Yersinia pestis species More recently, this system was also reported in other Gram-negative bacteria, such as Escherichia coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), Klebsiella pneumonia (Huertas et al.,) and Yersinia pestis (Ren et al.,). INTRO +0 4 YfiN protein YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). INTRO +62 83 transmembrane helices structure_element YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). INTRO +99 111 Per-Arnt-Sim structure_element YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). INTRO +113 116 PAS structure_element YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). INTRO +161 172 HAMP domain structure_element YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). INTRO +208 225 histidine kinases protein_type YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). INTRO +227 244 adenylyl cyclases protein_type YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). INTRO +246 282 methyl-accepting chemotaxis proteins protein_type YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). INTRO +288 300 phosphatases protein_type YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). INTRO +319 331 GGDEF domain structure_element YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). INTRO +345 348 DGC protein_type YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC’s function (Giardina et al.,; Malone et al.,). INTRO +0 4 YfiN protein YfiN is repressed by specific interaction between its periplasmic PAS domain and the periplasmic protein YfiR (Malone et al.,). INTRO +8 20 repressed by protein_state YfiN is repressed by specific interaction between its periplasmic PAS domain and the periplasmic protein YfiR (Malone et al.,). INTRO +66 76 PAS domain structure_element YfiN is repressed by specific interaction between its periplasmic PAS domain and the periplasmic protein YfiR (Malone et al.,). INTRO +105 109 YfiR protein YfiN is repressed by specific interaction between its periplasmic PAS domain and the periplasmic protein YfiR (Malone et al.,). INTRO +0 4 YfiB protein YfiB is an OmpA/Pal-like outer-membrane lipoprotein (Parsons et al.,) that can activate YfiN by sequestering YfiR (Malone et al.,) in an unknown manner. INTRO +11 24 OmpA/Pal-like protein_type YfiB is an OmpA/Pal-like outer-membrane lipoprotein (Parsons et al.,) that can activate YfiN by sequestering YfiR (Malone et al.,) in an unknown manner. INTRO +40 51 lipoprotein protein_type YfiB is an OmpA/Pal-like outer-membrane lipoprotein (Parsons et al.,) that can activate YfiN by sequestering YfiR (Malone et al.,) in an unknown manner. INTRO +88 92 YfiN protein YfiB is an OmpA/Pal-like outer-membrane lipoprotein (Parsons et al.,) that can activate YfiN by sequestering YfiR (Malone et al.,) in an unknown manner. INTRO +109 113 YfiR protein YfiB is an OmpA/Pal-like outer-membrane lipoprotein (Parsons et al.,) that can activate YfiN by sequestering YfiR (Malone et al.,) in an unknown manner. INTRO +8 12 YfiB protein Whether YfiB directly recruits YfiR or recruits YfiR via a third partner is an open question. INTRO +31 35 YfiR protein Whether YfiB directly recruits YfiR or recruits YfiR via a third partner is an open question. INTRO +48 52 YfiR protein Whether YfiB directly recruits YfiR or recruits YfiR via a third partner is an open question. INTRO +27 31 YfiR protein After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,). INTRO +35 39 YfiB protein After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,). INTRO +45 53 c-di-GMP chemical After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,). INTRO +66 75 activated protein_state After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,). INTRO +76 80 YfiN protein After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,). INTRO +115 118 Pel chemical After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,). INTRO +123 126 Psl chemical After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,). INTRO +127 131 EPSs chemical After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,). INTRO +44 48 YfiN protein It has been reported that the activation of YfiN may be induced by redox-driven misfolding of YfiR (Giardina et al.,; Malone et al.,; Malone et al.,). INTRO +94 98 YfiR protein It has been reported that the activation of YfiN may be induced by redox-driven misfolding of YfiR (Giardina et al.,; Malone et al.,; Malone et al.,). INTRO +46 50 YfiR protein It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). INTRO +54 58 YfiB protein It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). INTRO +85 89 YfiB protein It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). INTRO +121 140 mutagenesis studies experimental_method It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). INTRO +162 181 activation residues structure_element It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). INTRO +185 189 YfiB protein It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). INTRO +234 243 predicted protein_state It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). INTRO +244 255 first helix structure_element It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). INTRO +263 281 periplasmic domain structure_element It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,). INTRO +61 71 PAS domain structure_element In addition, quorum sensing-related dephosphorylation of the PAS domain of YfiN may also be involved in the regulation (Ueda and Wood,; Xu et al.,). INTRO +75 79 YfiN protein In addition, quorum sensing-related dephosphorylation of the PAS domain of YfiN may also be involved in the regulation (Ueda and Wood,; Xu et al.,). INTRO +24 41 crystal structure evidence Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). INTRO +45 49 YfiR protein Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). INTRO +62 74 non-oxidized protein_state Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). INTRO +83 91 oxidized protein_state Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). INTRO +136 150 disulfide bond ptm Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). INTRO +152 157 Cys71 residue_name_number Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). INTRO +158 164 Cys110 residue_name_number Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). INTRO +220 226 Cys145 residue_name_number Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). INTRO +227 233 Cys152 residue_name_number Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). INTRO +252 258 Cys145 residue_name_number Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). INTRO +259 265 Cys152 residue_name_number Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). INTRO +328 332 YfiR protein Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,). INTRO +36 54 crystal structures evidence In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. INTRO +72 81 truncated protein_state In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. INTRO +90 94 YfiB protein In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. INTRO +96 102 34–168 residue_range In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. INTRO +108 112 YfiR protein In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. INTRO +113 128 in complex with protein_state In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. INTRO +132 138 active protein_state In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. INTRO +139 145 mutant protein_state In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. INTRO +146 154 YfiBL43P mutant In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34–168) and YfiR in complex with an active mutant YfiBL43P. INTRO +45 63 crystal structures evidence Most recently, Li and coworkers reported the crystal structures of YfiB (27–168) alone and YfiRC71S in complex with YfiB (59–168) (Li et al.,). INTRO +67 71 YfiB protein Most recently, Li and coworkers reported the crystal structures of YfiB (27–168) alone and YfiRC71S in complex with YfiB (59–168) (Li et al.,). INTRO +73 79 27–168 residue_range Most recently, Li and coworkers reported the crystal structures of YfiB (27–168) alone and YfiRC71S in complex with YfiB (59–168) (Li et al.,). INTRO +81 86 alone protein_state Most recently, Li and coworkers reported the crystal structures of YfiB (27–168) alone and YfiRC71S in complex with YfiB (59–168) (Li et al.,). INTRO +91 99 YfiRC71S mutant Most recently, Li and coworkers reported the crystal structures of YfiB (27–168) alone and YfiRC71S in complex with YfiB (59–168) (Li et al.,). INTRO +100 115 in complex with protein_state Most recently, Li and coworkers reported the crystal structures of YfiB (27–168) alone and YfiRC71S in complex with YfiB (59–168) (Li et al.,). INTRO +116 120 YfiB protein Most recently, Li and coworkers reported the crystal structures of YfiB (27–168) alone and YfiRC71S in complex with YfiB (59–168) (Li et al.,). INTRO +122 128 59–168 residue_range Most recently, Li and coworkers reported the crystal structures of YfiB (27–168) alone and YfiRC71S in complex with YfiB (59–168) (Li et al.,). INTRO +46 54 YfiBL43P mutant Compared with the reported complex structure, YfiBL43P in our YfiB-YfiR complex structure has additional visible N-terminal residues 44–58 that are shown to play essential roles in YfiB activation and biofilm formation. INTRO +62 71 YfiB-YfiR complex_assembly Compared with the reported complex structure, YfiBL43P in our YfiB-YfiR complex structure has additional visible N-terminal residues 44–58 that are shown to play essential roles in YfiB activation and biofilm formation. INTRO +80 89 structure evidence Compared with the reported complex structure, YfiBL43P in our YfiB-YfiR complex structure has additional visible N-terminal residues 44–58 that are shown to play essential roles in YfiB activation and biofilm formation. INTRO +133 138 44–58 residue_range Compared with the reported complex structure, YfiBL43P in our YfiB-YfiR complex structure has additional visible N-terminal residues 44–58 that are shown to play essential roles in YfiB activation and biofilm formation. INTRO +181 185 YfiB protein Compared with the reported complex structure, YfiBL43P in our YfiB-YfiR complex structure has additional visible N-terminal residues 44–58 that are shown to play essential roles in YfiB activation and biofilm formation. INTRO +103 107 YfiB protein Therefore, we are able to visualize the detailed allosteric arrangement of the N-terminal structure of YfiB and its important role in YfiB-YfiR interaction. INTRO +134 143 YfiB-YfiR complex_assembly Therefore, we are able to visualize the detailed allosteric arrangement of the N-terminal structure of YfiB and its important role in YfiB-YfiR interaction. INTRO +31 39 YfiBL43P mutant In addition, we found that the YfiBL43P shows a much higher PG-binding affinity than wild-type YfiB, most likely due to its more compact PG-binding pocket. INTRO +60 79 PG-binding affinity evidence In addition, we found that the YfiBL43P shows a much higher PG-binding affinity than wild-type YfiB, most likely due to its more compact PG-binding pocket. INTRO +85 94 wild-type protein_state In addition, we found that the YfiBL43P shows a much higher PG-binding affinity than wild-type YfiB, most likely due to its more compact PG-binding pocket. INTRO +95 99 YfiB protein In addition, we found that the YfiBL43P shows a much higher PG-binding affinity than wild-type YfiB, most likely due to its more compact PG-binding pocket. INTRO +137 154 PG-binding pocket site In addition, we found that the YfiBL43P shows a much higher PG-binding affinity than wild-type YfiB, most likely due to its more compact PG-binding pocket. INTRO +24 34 Vitamin B6 chemical Moreover, we found that Vitamin B6 (VB6) or L-Trp can bind YfiR with an affinity in the ten millimolar range. INTRO +36 39 VB6 chemical Moreover, we found that Vitamin B6 (VB6) or L-Trp can bind YfiR with an affinity in the ten millimolar range. INTRO +44 49 L-Trp chemical Moreover, we found that Vitamin B6 (VB6) or L-Trp can bind YfiR with an affinity in the ten millimolar range. INTRO +59 63 YfiR protein Moreover, we found that Vitamin B6 (VB6) or L-Trp can bind YfiR with an affinity in the ten millimolar range. INTRO +72 80 affinity evidence Moreover, we found that Vitamin B6 (VB6) or L-Trp can bind YfiR with an affinity in the ten millimolar range. INTRO +87 96 activated protein_state Together with functional data, these results provide new mechanistic insights into how activated YfiB sequesters YfiR and releases the suppression of YfiN. These findings may facilitate the development and optimization of anti-biofilm drugs for the treatment of chronic infections. INTRO +97 101 YfiB protein Together with functional data, these results provide new mechanistic insights into how activated YfiB sequesters YfiR and releases the suppression of YfiN. These findings may facilitate the development and optimization of anti-biofilm drugs for the treatment of chronic infections. INTRO +113 117 YfiR protein Together with functional data, these results provide new mechanistic insights into how activated YfiB sequesters YfiR and releases the suppression of YfiN. These findings may facilitate the development and optimization of anti-biofilm drugs for the treatment of chronic infections. INTRO +150 154 YfiN protein Together with functional data, these results provide new mechanistic insights into how activated YfiB sequesters YfiR and releases the suppression of YfiN. These findings may facilitate the development and optimization of anti-biofilm drugs for the treatment of chronic infections. INTRO +8 17 structure evidence Overall structure of YfiB RESULTS +21 25 YfiB protein Overall structure of YfiB RESULTS +16 29 crystal forms evidence We obtained two crystal forms of YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33), crystal forms I and II, belonging to space groups P21 and P41, respectively. RESULTS +33 37 YfiB protein We obtained two crystal forms of YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33), crystal forms I and II, belonging to space groups P21 and P41, respectively. RESULTS +48 54 34–168 residue_range We obtained two crystal forms of YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33), crystal forms I and II, belonging to space groups P21 and P41, respectively. RESULTS +56 63 lacking protein_state We obtained two crystal forms of YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33), crystal forms I and II, belonging to space groups P21 and P41, respectively. RESULTS +68 82 signal peptide structure_element We obtained two crystal forms of YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33), crystal forms I and II, belonging to space groups P21 and P41, respectively. RESULTS +97 101 1–26 residue_range We obtained two crystal forms of YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33), crystal forms I and II, belonging to space groups P21 and P41, respectively. RESULTS +127 132 27–33 residue_range We obtained two crystal forms of YfiB (residues 34–168, lacking the signal peptide from residues 1–26 and periplasmic residues 27–33), crystal forms I and II, belonging to space groups P21 and P41, respectively. RESULTS +8 17 structure evidence Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P FIG +21 25 YfiB protein Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P FIG +43 52 structure evidence Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P FIG +60 64 YfiB protein Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P FIG +65 72 monomer oligomeric_state Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P FIG +104 108 YfiB protein Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P FIG +109 116 monomer oligomeric_state Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P FIG +132 162 analytical ultracentrifugation experimental_method Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P FIG +190 199 wild-type protein_state Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P FIG +200 204 YfiB protein Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P FIG +209 217 YfiBL43P mutant Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P FIG +4 11 dimeric oligomeric_state Two dimeric types of YfiB dimer. (A–C) The “head to head” dimer. FIG +21 25 YfiB protein Two dimeric types of YfiB dimer. (A–C) The “head to head” dimer. FIG +26 31 dimer oligomeric_state Two dimeric types of YfiB dimer. (A–C) The “head to head” dimer. FIG +44 56 head to head protein_state Two dimeric types of YfiB dimer. (A–C) The “head to head” dimer. FIG +58 63 dimer oligomeric_state Two dimeric types of YfiB dimer. (A–C) The “head to head” dimer. FIG +5 17 back to back protein_state The “back to back” dimer. FIG +19 24 dimer oligomeric_state The “back to back” dimer. FIG +48 54 dimers oligomeric_state (A) and (E) indicate the front views of the two dimers, (B) and (F) indicate the top views of the two dimers, and (C) and (D) indicate the details of the two dimeric interfaces FIG +102 108 dimers oligomeric_state (A) and (E) indicate the front views of the two dimers, (B) and (F) indicate the top views of the two dimers, and (C) and (D) indicate the details of the two dimeric interfaces FIG +158 176 dimeric interfaces site (A) and (E) indicate the front views of the two dimers, (B) and (F) indicate the top views of the two dimers, and (C) and (D) indicate the details of the two dimeric interfaces FIG +4 21 crystal structure evidence The crystal structure of YfiB monomer consists of a five-stranded β-sheet (β1-2-5-3-4) flanked by five α-helices (α1–5) on one side. RESULTS +25 29 YfiB protein The crystal structure of YfiB monomer consists of a five-stranded β-sheet (β1-2-5-3-4) flanked by five α-helices (α1–5) on one side. RESULTS +30 37 monomer oligomeric_state The crystal structure of YfiB monomer consists of a five-stranded β-sheet (β1-2-5-3-4) flanked by five α-helices (α1–5) on one side. RESULTS +52 73 five-stranded β-sheet structure_element The crystal structure of YfiB monomer consists of a five-stranded β-sheet (β1-2-5-3-4) flanked by five α-helices (α1–5) on one side. RESULTS +75 85 β1-2-5-3-4 structure_element The crystal structure of YfiB monomer consists of a five-stranded β-sheet (β1-2-5-3-4) flanked by five α-helices (α1–5) on one side. RESULTS +98 112 five α-helices structure_element The crystal structure of YfiB monomer consists of a five-stranded β-sheet (β1-2-5-3-4) flanked by five α-helices (α1–5) on one side. RESULTS +114 118 α1–5 structure_element The crystal structure of YfiB monomer consists of a five-stranded β-sheet (β1-2-5-3-4) flanked by five α-helices (α1–5) on one side. RESULTS +30 40 helix turn structure_element In addition, there is a short helix turn connecting the β4 strand and α4 helix (Fig. 1A and 1B). RESULTS +56 65 β4 strand structure_element In addition, there is a short helix turn connecting the β4 strand and α4 helix (Fig. 1A and 1B). RESULTS +70 78 α4 helix structure_element In addition, there is a short helix turn connecting the β4 strand and α4 helix (Fig. 1A and 1B). RESULTS +43 50 dimeric oligomeric_state Each crystal form contains three different dimeric types of YfiB, two of which are present in both, suggesting that the rest of the dimeric types may result from crystal packing. RESULTS +60 64 YfiB protein Each crystal form contains three different dimeric types of YfiB, two of which are present in both, suggesting that the rest of the dimeric types may result from crystal packing. RESULTS +132 139 dimeric oligomeric_state Each crystal form contains three different dimeric types of YfiB, two of which are present in both, suggesting that the rest of the dimeric types may result from crystal packing. RESULTS +26 33 dimeric oligomeric_state Here, we refer to the two dimeric types as “head to head” and “back to back” according to the interacting mode (Fig. 2A and 2E), with the total buried surface areas being 316.8 Å2 and 554.3 Å2, respectively. RESULTS +44 56 head to head protein_state Here, we refer to the two dimeric types as “head to head” and “back to back” according to the interacting mode (Fig. 2A and 2E), with the total buried surface areas being 316.8 Å2 and 554.3 Å2, respectively. RESULTS +63 75 back to back protein_state Here, we refer to the two dimeric types as “head to head” and “back to back” according to the interacting mode (Fig. 2A and 2E), with the total buried surface areas being 316.8 Å2 and 554.3 Å2, respectively. RESULTS +5 17 head to head protein_state The “head to head” dimer exhibits a clamp shape. RESULTS +19 24 dimer oligomeric_state The “head to head” dimer exhibits a clamp shape. RESULTS +36 47 clamp shape protein_state The “head to head” dimer exhibits a clamp shape. RESULTS +35 59 hydrophobic interactions bond_interaction The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C). RESULTS +70 73 A37 residue_name_number The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C). RESULTS +78 81 I40 residue_name_number The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C). RESULTS +89 99 α1 helices structure_element The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C). RESULTS +101 104 L50 residue_name_number The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C). RESULTS +112 122 β1 strands structure_element The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C). RESULTS +128 131 W55 residue_name_number The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C). RESULTS +139 149 β2 strands structure_element The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C). RESULTS +178 206 hydrophobic interacting core site The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the α1 helices, L50 on the β1 strands, and W55 on the β2 strands of both molecules, making a hydrophobic interacting core (Fig. 2A–C). RESULTS +5 17 back to back protein_state The “back to back” dimer presents a Y shape. RESULTS +19 24 dimer oligomeric_state The “back to back” dimer presents a Y shape. RESULTS +36 43 Y shape protein_state The “back to back” dimer presents a Y shape. RESULTS +4 11 dimeric oligomeric_state The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). RESULTS +12 45 interaction is mainly hydrophilic bond_interaction The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). RESULTS +102 105 N68 residue_name_number The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). RESULTS +107 110 L69 residue_name_number The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). RESULTS +112 115 D70 residue_name_number The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). RESULTS +120 123 R71 residue_name_number The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). RESULTS +131 142 α2-α3 loops structure_element The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). RESULTS +147 151 R116 residue_name_number The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). RESULTS +156 160 S120 residue_name_number The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). RESULTS +168 178 α4 helices structure_element The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). RESULTS +221 242 hydrogen bond network site The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the α2-α3 loops and R116 and S120 on the α4 helices of both molecules, resulting in a complex hydrogen bond network (Fig. 2D–F). RESULTS +4 13 YfiB-YfiR complex_assembly The YfiB-YfiR interaction RESULTS +8 17 structure evidence Overall structure of the YfiB-YfiR complex and the conserved surface in YfiR. (A) The overall structure of the YfiB-YfiR complex. FIG +25 34 YfiB-YfiR complex_assembly Overall structure of the YfiB-YfiR complex and the conserved surface in YfiR. (A) The overall structure of the YfiB-YfiR complex. FIG +51 68 conserved surface site Overall structure of the YfiB-YfiR complex and the conserved surface in YfiR. (A) The overall structure of the YfiB-YfiR complex. FIG +72 76 YfiR protein Overall structure of the YfiB-YfiR complex and the conserved surface in YfiR. (A) The overall structure of the YfiB-YfiR complex. FIG +94 103 structure evidence Overall structure of the YfiB-YfiR complex and the conserved surface in YfiR. (A) The overall structure of the YfiB-YfiR complex. FIG +111 120 YfiB-YfiR complex_assembly Overall structure of the YfiB-YfiR complex and the conserved surface in YfiR. (A) The overall structure of the YfiB-YfiR complex. FIG +4 12 YfiBL43P mutant The YfiBL43P molecules are shown in cyan and yellow. FIG +4 8 YfiR protein The YfiR molecules are shown in green and magenta. FIG +63 87 Structural superposition experimental_method Two interacting regions are highlighted by red rectangles. (B) Structural superposition of apo YfiB and YfiR-bound YfiBL43P. FIG +91 94 apo protein_state Two interacting regions are highlighted by red rectangles. (B) Structural superposition of apo YfiB and YfiR-bound YfiBL43P. FIG +95 99 YfiB protein Two interacting regions are highlighted by red rectangles. (B) Structural superposition of apo YfiB and YfiR-bound YfiBL43P. FIG +104 114 YfiR-bound protein_state Two interacting regions are highlighted by red rectangles. (B) Structural superposition of apo YfiB and YfiR-bound YfiBL43P. FIG +115 123 YfiBL43P mutant Two interacting regions are highlighted by red rectangles. (B) Structural superposition of apo YfiB and YfiR-bound YfiBL43P. FIG +38 41 apo protein_state To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +42 46 YfiB protein To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +51 61 YfiR-bound protein_state To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +62 70 YfiBL43P mutant To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +76 79 apo protein_state To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +80 84 YfiB protein To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +119 124 34–70 residue_range To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +155 165 YfiR-bound protein_state To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +166 174 YfiBL43P mutant To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +209 214 44–70 residue_range To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +279 282 apo protein_state To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +283 287 YfiB protein To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +292 302 YfiR-bound protein_state To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +303 311 YfiBL43P mutant To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34–70 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44–70 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P. FIG +39 43 YfiB protein The residues proposed to contribute to YfiB activation are illustrated in sticks. FIG +20 23 apo protein_state The key residues in apo YfiB are shown in red and those in YfiBL43P are shown in blue. (D) Close-up views showing interactions in regions I and II. FIG +24 28 YfiB protein The key residues in apo YfiB are shown in red and those in YfiBL43P are shown in blue. (D) Close-up views showing interactions in regions I and II. FIG +59 67 YfiBL43P mutant The key residues in apo YfiB are shown in red and those in YfiBL43P are shown in blue. (D) Close-up views showing interactions in regions I and II. FIG +130 146 regions I and II structure_element The key residues in apo YfiB are shown in red and those in YfiBL43P are shown in blue. (D) Close-up views showing interactions in regions I and II. FIG +0 8 YfiBL43P mutant YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. FIG +13 17 YfiR protein YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. FIG +75 92 conserved surface site YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. FIG +96 100 YfiR protein YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. FIG +137 141 YfiB protein YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. FIG +151 159 residues structure_element YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. FIG +163 167 YfiR protein YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. FIG +201 205 YfiB protein YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. FIG +250 275 YfiN-interacting residues site YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks. FIG +36 61 YfiB-interacting residues site The red sticks, which represent the YfiB-interacting residues, are also responsible for the proposed interactions with YfiN FIG +119 123 YfiN protein The red sticks, which represent the YfiB-interacting residues, are also responsible for the proposed interactions with YfiN FIG +37 46 YfiB-YfiR complex_assembly To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). RESULTS +63 75 co-expressed experimental_method To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). RESULTS +76 80 YfiB protein To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). RESULTS +91 97 34–168 residue_range To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). RESULTS +103 107 YfiR protein To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). RESULTS +118 124 35–190 residue_range To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). RESULTS +126 133 lacking protein_state To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). RESULTS +138 152 signal peptide structure_element To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). RESULTS +235 244 no stable protein_state To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). RESULTS +256 265 YfiB-YfiR complex_assembly To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34–168) and YfiR (residues 35–190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,). RESULTS +26 43 single mutants of experimental_method It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,). RESULTS +44 47 Q39 residue_name_number It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,). RESULTS +49 52 L43 residue_name_number It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,). RESULTS +54 57 F48 residue_name_number It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,). RESULTS +62 65 W55 residue_name_number It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,). RESULTS +80 84 YfiB protein It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,). RESULTS +145 163 P. aeruginosa PAO1 species It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,). RESULTS +82 86 YfiB protein It is likely that these residues may be involved in the conformational changes of YfiB that are related to YfiR sequestration (Fig. 3C). RESULTS +107 111 YfiR protein It is likely that these residues may be involved in the conformational changes of YfiB that are related to YfiR sequestration (Fig. 3C). RESULTS +14 49 constructed two such single mutants experimental_method Therefore, we constructed two such single mutants of YfiB (YfiBL43P and YfiBF48S). RESULTS +53 57 YfiB protein Therefore, we constructed two such single mutants of YfiB (YfiBL43P and YfiBF48S). RESULTS +59 67 YfiBL43P mutant Therefore, we constructed two such single mutants of YfiB (YfiBL43P and YfiBF48S). RESULTS +72 80 YfiBF48S mutant Therefore, we constructed two such single mutants of YfiB (YfiBL43P and YfiBF48S). RESULTS +33 39 stable protein_state As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. RESULTS +40 52 complex with protein_state As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. RESULTS +53 57 YfiR protein As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. RESULTS +71 82 crystalized experimental_method As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. RESULTS +83 87 YfiR protein As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. RESULTS +88 103 in complex with protein_state As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. RESULTS +108 116 YfiBL43P mutant As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. RESULTS +117 123 mutant protein_state As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. RESULTS +139 148 structure evidence As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. RESULTS +173 194 molecular replacement experimental_method As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. RESULTS +201 205 YfiR protein As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. RESULTS +210 214 YfiB protein As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 Å resolution by molecular replacement using YfiR and YfiB as models. RESULTS +4 13 YfiB-YfiR complex_assembly The YfiB-YfiR complex is a 2:2 heterotetramer (Fig. 3A) in which the YfiR dimer is clamped by two separated YfiBL43P molecules with a total buried surface area of 3161.2 Å2. RESULTS +31 45 heterotetramer oligomeric_state The YfiB-YfiR complex is a 2:2 heterotetramer (Fig. 3A) in which the YfiR dimer is clamped by two separated YfiBL43P molecules with a total buried surface area of 3161.2 Å2. RESULTS +69 73 YfiR protein The YfiB-YfiR complex is a 2:2 heterotetramer (Fig. 3A) in which the YfiR dimer is clamped by two separated YfiBL43P molecules with a total buried surface area of 3161.2 Å2. RESULTS +74 79 dimer oligomeric_state The YfiB-YfiR complex is a 2:2 heterotetramer (Fig. 3A) in which the YfiR dimer is clamped by two separated YfiBL43P molecules with a total buried surface area of 3161.2 Å2. RESULTS +108 116 YfiBL43P mutant The YfiB-YfiR complex is a 2:2 heterotetramer (Fig. 3A) in which the YfiR dimer is clamped by two separated YfiBL43P molecules with a total buried surface area of 3161.2 Å2. RESULTS +4 8 YfiR protein The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +9 14 dimer oligomeric_state The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +50 62 non-oxidized protein_state The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +63 67 YfiR protein The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +68 73 dimer oligomeric_state The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +74 79 alone protein_state The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +106 112 Cys145 residue_name_number The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +113 119 Cys152 residue_name_number The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +131 146 disulfide bonds ptm The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +171 176 Cys71 residue_name_number The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +177 183 Cys110 residue_name_number The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +184 198 disulfide bond ptm The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +238 247 YfiB-YfiR complex_assembly The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex. RESULTS +42 50 YfiBL43P mutant The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +92 95 D70 residue_name_number The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +148 151 apo protein_state The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +152 156 YfiB protein The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +178 186 α1 helix structure_element The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +197 202 34–43 residue_range The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +224 244 electron density map evidence The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +254 262 α2 helix structure_element The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +267 269 β1 structure_element The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +274 284 β2 strands structure_element The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +315 319 loop structure_element The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +341 354 α-helix turns structure_element The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +392 396 YfiR protein The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +397 402 dimer oligomeric_state The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the α1 helix (residues 34–43) is invisible on the electron density map, and the α2 helix and β1 and β2 strands are rearranged to form a long loop containing two short α-helix turns (Fig. 3B and 3C), thus embracing the YfiR dimer. RESULTS +40 44 YfiB protein The observed changes in conformation of YfiB and the results of mutagenesis suggest a mechanism by which YfiB sequesters YfiR. RESULTS +64 75 mutagenesis experimental_method The observed changes in conformation of YfiB and the results of mutagenesis suggest a mechanism by which YfiB sequesters YfiR. RESULTS +105 109 YfiB protein The observed changes in conformation of YfiB and the results of mutagenesis suggest a mechanism by which YfiB sequesters YfiR. RESULTS +121 125 YfiR protein The observed changes in conformation of YfiB and the results of mutagenesis suggest a mechanism by which YfiB sequesters YfiR. RESULTS +4 23 YfiB-YfiR interface site The YfiB-YfiR interface can be divided into two regions (Fig. 3A and 3D). RESULTS +0 8 Region I structure_element Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). RESULTS +57 81 hydrophilic interactions bond_interaction Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). RESULTS +99 102 E45 residue_name_number Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). RESULTS +104 107 G47 residue_name_number Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). RESULTS +112 115 E53 residue_name_number Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). RESULTS +145 149 loop structure_element Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). RESULTS +153 157 YfiB protein Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). RESULTS +171 174 S57 residue_name_number Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). RESULTS +176 179 R60 residue_name_number Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). RESULTS +181 184 A89 residue_name_number Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). RESULTS +189 193 H177 residue_name_number Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). RESULTS +199 203 YfiR protein Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig. 3D-I(i)). RESULTS +20 47 hydrophobic anchoring sites site Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +57 65 region I structure_element Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +80 83 F48 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +88 91 W55 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +95 99 YfiB protein Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +122 139 hydrophobic cores site Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +212 215 S57 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +216 219 Q88 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +220 223 A89 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +224 227 N90 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +232 235 R60 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +236 240 R175 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +241 245 H177 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +249 253 YfiR protein Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +273 276 F57 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +280 284 YfiB protein Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +306 324 hydrophobic pocket site Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +335 339 L166 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +340 344 I169 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +345 349 V176 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +350 354 P178 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +355 359 L181 residue_name_number Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +363 367 YfiR protein Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig. 3D-I(ii)). RESULTS +3 12 region II structure_element In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. RESULTS +33 36 R96 residue_name_number In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. RESULTS +38 41 E98 residue_name_number In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. RESULTS +46 50 E157 residue_name_number In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. RESULTS +56 60 YfiB protein In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. RESULTS +94 98 E163 residue_name_number In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. RESULTS +100 104 S146 residue_name_number In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. RESULTS +109 113 R171 residue_name_number In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. RESULTS +119 123 YfiR protein In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively. RESULTS +33 37 I163 residue_name_number Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). RESULTS +42 46 V165 residue_name_number Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). RESULTS +52 56 YfiB protein Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). RESULTS +62 76 hydrogen bonds bond_interaction Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). RESULTS +101 105 L166 residue_name_number Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). RESULTS +110 114 A164 residue_name_number Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). RESULTS +120 124 YfiR protein Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). RESULTS +162 166 P166 residue_name_number Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). RESULTS +172 176 YfiB protein Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). RESULTS +210 214 R185 residue_name_number Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). RESULTS +220 224 YfiR protein Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig. 3D-II). RESULTS +38 62 hydrogen-bonding network site These two regions contribute a robust hydrogen-bonding network to the YfiB-YfiR interface, resulting in a tightly bound complex. RESULTS +70 89 YfiB-YfiR interface site These two regions contribute a robust hydrogen-bonding network to the YfiB-YfiR interface, resulting in a tightly bound complex. RESULTS +45 53 YfiBL43P mutant Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P. RESULTS +83 92 structure evidence Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P. RESULTS +98 102 YfiR protein Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P. RESULTS +103 108 dimer oligomeric_state Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P. RESULTS +126 156 analytical ultracentrifugation experimental_method Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P. RESULTS +202 211 wild-type protein_state Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P. RESULTS +212 216 YfiB protein Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P. RESULTS +221 229 YfiBL43P mutant Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P. RESULTS +24 33 wild-type protein_state The results showed that wild-type YfiB exists in both monomeric and dimeric states in solution, while YfiBL43P primarily adopts the monomer state in solution (Fig. 1C–D). RESULTS +34 38 YfiB protein The results showed that wild-type YfiB exists in both monomeric and dimeric states in solution, while YfiBL43P primarily adopts the monomer state in solution (Fig. 1C–D). RESULTS +54 63 monomeric oligomeric_state The results showed that wild-type YfiB exists in both monomeric and dimeric states in solution, while YfiBL43P primarily adopts the monomer state in solution (Fig. 1C–D). RESULTS +68 75 dimeric oligomeric_state The results showed that wild-type YfiB exists in both monomeric and dimeric states in solution, while YfiBL43P primarily adopts the monomer state in solution (Fig. 1C–D). RESULTS +102 110 YfiBL43P mutant The results showed that wild-type YfiB exists in both monomeric and dimeric states in solution, while YfiBL43P primarily adopts the monomer state in solution (Fig. 1C–D). RESULTS +132 139 monomer oligomeric_state The results showed that wild-type YfiB exists in both monomeric and dimeric states in solution, while YfiBL43P primarily adopts the monomer state in solution (Fig. 1C–D). RESULTS +37 41 YfiB protein This suggests that the N-terminus of YfiB plays an important role in forming the dimeric YfiB in solution and that the conformational change of residue L43 is associated with the stretch of the N-terminus and opening of the dimer. RESULTS +81 88 dimeric oligomeric_state This suggests that the N-terminus of YfiB plays an important role in forming the dimeric YfiB in solution and that the conformational change of residue L43 is associated with the stretch of the N-terminus and opening of the dimer. RESULTS +89 93 YfiB protein This suggests that the N-terminus of YfiB plays an important role in forming the dimeric YfiB in solution and that the conformational change of residue L43 is associated with the stretch of the N-terminus and opening of the dimer. RESULTS +152 155 L43 residue_name_number This suggests that the N-terminus of YfiB plays an important role in forming the dimeric YfiB in solution and that the conformational change of residue L43 is associated with the stretch of the N-terminus and opening of the dimer. RESULTS +224 229 dimer oligomeric_state This suggests that the N-terminus of YfiB plays an important role in forming the dimeric YfiB in solution and that the conformational change of residue L43 is associated with the stretch of the N-terminus and opening of the dimer. RESULTS +36 43 dimeric oligomeric_state Therefore, it is possible that both dimeric types might exist in solution. RESULTS +37 49 head to head protein_state For simplicity, we only discuss the “head to head” dimer in the following text. RESULTS +51 56 dimer oligomeric_state For simplicity, we only discuss the “head to head” dimer in the following text. RESULTS +4 19 PG-binding site site The PG-binding site of YfiB RESULTS +23 27 YfiB protein The PG-binding site of YfiB RESULTS +4 19 PG-binding site site The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. FIG +23 27 YfiB protein The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. FIG +33 57 Structural superposition experimental_method The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. FIG +65 81 PG-binding sites site The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. FIG +89 102 H. influenzae species The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. FIG +103 111 Pal/PG-P complex_assembly The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. FIG +124 134 YfiR-bound protein_state The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. FIG +135 143 YfiBL43P mutant The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. FIG +144 158 complexed with protein_state The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. FIG +159 166 sulfate chemical The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions. FIG +46 49 Pal protein_type (B) Close-up view showing the key residues of Pal interacting with the m-Dap5 ε-carboxylate group of PG-P. Pal is shown in wheat and PG-P is in magenta. FIG +71 91 m-Dap5 ε-carboxylate chemical (B) Close-up view showing the key residues of Pal interacting with the m-Dap5 ε-carboxylate group of PG-P. Pal is shown in wheat and PG-P is in magenta. FIG +101 105 PG-P chemical (B) Close-up view showing the key residues of Pal interacting with the m-Dap5 ε-carboxylate group of PG-P. Pal is shown in wheat and PG-P is in magenta. FIG +107 110 Pal protein_type (B) Close-up view showing the key residues of Pal interacting with the m-Dap5 ε-carboxylate group of PG-P. Pal is shown in wheat and PG-P is in magenta. FIG +133 137 PG-P chemical (B) Close-up view showing the key residues of Pal interacting with the m-Dap5 ε-carboxylate group of PG-P. Pal is shown in wheat and PG-P is in magenta. FIG +46 56 YfiR-bound protein_state (C) Close-up view showing the key residues of YfiR-bound YfiBL43P interacting with a sulfate ion. FIG +57 65 YfiBL43P mutant (C) Close-up view showing the key residues of YfiR-bound YfiBL43P interacting with a sulfate ion. FIG +85 92 sulfate chemical (C) Close-up view showing the key residues of YfiR-bound YfiBL43P interacting with a sulfate ion. FIG +0 10 YfiR-bound protein_state YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. FIG +11 19 YfiBL43P mutant YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. FIG +42 49 sulfate chemical YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. FIG +73 78 water chemical YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. FIG +104 128 Structural superposition experimental_method YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. FIG +136 152 PG-binding sites site YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. FIG +156 159 apo protein_state YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. FIG +160 164 YfiB protein YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. FIG +169 179 YfiR-bound protein_state YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. FIG +180 188 YfiBL43P mutant YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick. FIG +0 3 Apo protein_state Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +4 8 YfiB protein Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +32 42 YfiR-bound protein_state Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +43 51 YfiBL43P mutant Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +71 74 MST experimental_method Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +97 115 binding affinities evidence Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +123 127 YfiB protein Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +128 137 wild-type protein_state Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +146 154 YfiBL43P mutant Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +160 162 PG chemical Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +172 190 sequence alignment experimental_method Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +194 207 P. aeruginosa species Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +212 219 E. coli species Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +231 235 YfiB protein Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +237 240 Pal protein_type Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +249 267 periplasmic domain structure_element Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +271 275 OmpA protein_type Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA FIG +0 25 PG-associated lipoprotein protein_type PG-associated lipoprotein (Pal) is highly conserved in Gram-negative bacteria and anchors to the outer membrane through an N-terminal lipid attachment and to PG layer through its periplasmic domain, which is implicated in maintaining outer membrane integrity. RESULTS +27 30 Pal protein_type PG-associated lipoprotein (Pal) is highly conserved in Gram-negative bacteria and anchors to the outer membrane through an N-terminal lipid attachment and to PG layer through its periplasmic domain, which is implicated in maintaining outer membrane integrity. RESULTS +35 51 highly conserved protein_state PG-associated lipoprotein (Pal) is highly conserved in Gram-negative bacteria and anchors to the outer membrane through an N-terminal lipid attachment and to PG layer through its periplasmic domain, which is implicated in maintaining outer membrane integrity. RESULTS +55 77 Gram-negative bacteria taxonomy_domain PG-associated lipoprotein (Pal) is highly conserved in Gram-negative bacteria and anchors to the outer membrane through an N-terminal lipid attachment and to PG layer through its periplasmic domain, which is implicated in maintaining outer membrane integrity. RESULTS +158 160 PG chemical PG-associated lipoprotein (Pal) is highly conserved in Gram-negative bacteria and anchors to the outer membrane through an N-terminal lipid attachment and to PG layer through its periplasmic domain, which is implicated in maintaining outer membrane integrity. RESULTS +179 197 periplasmic domain structure_element PG-associated lipoprotein (Pal) is highly conserved in Gram-negative bacteria and anchors to the outer membrane through an N-terminal lipid attachment and to PG layer through its periplasmic domain, which is implicated in maintaining outer membrane integrity. RESULTS +9 26 homology modeling experimental_method Previous homology modeling studies suggested that YfiB contains a Pal-like PG-binding site (Parsons et al.,), and the mutation of two residues at this site, D102 and G105, reduces the ability for biofilm formation and surface attachment (Malone et al.,). RESULTS +50 54 YfiB protein Previous homology modeling studies suggested that YfiB contains a Pal-like PG-binding site (Parsons et al.,), and the mutation of two residues at this site, D102 and G105, reduces the ability for biofilm formation and surface attachment (Malone et al.,). RESULTS +66 90 Pal-like PG-binding site site Previous homology modeling studies suggested that YfiB contains a Pal-like PG-binding site (Parsons et al.,), and the mutation of two residues at this site, D102 and G105, reduces the ability for biofilm formation and surface attachment (Malone et al.,). RESULTS +118 142 mutation of two residues experimental_method Previous homology modeling studies suggested that YfiB contains a Pal-like PG-binding site (Parsons et al.,), and the mutation of two residues at this site, D102 and G105, reduces the ability for biofilm formation and surface attachment (Malone et al.,). RESULTS +157 161 D102 residue_name_number Previous homology modeling studies suggested that YfiB contains a Pal-like PG-binding site (Parsons et al.,), and the mutation of two residues at this site, D102 and G105, reduces the ability for biofilm formation and surface attachment (Malone et al.,). RESULTS +166 170 G105 residue_name_number Previous homology modeling studies suggested that YfiB contains a Pal-like PG-binding site (Parsons et al.,), and the mutation of two residues at this site, D102 and G105, reduces the ability for biofilm formation and surface attachment (Malone et al.,). RESULTS +7 16 YfiB-YfiR complex_assembly In the YfiB-YfiR complex, one sulfate ion is found at the bottom of each YfiBL43P molecule (Fig. 3A) and forms a strong hydrogen bond with D102 of YfiBL43P (Fig. 4A and 4C). RESULTS +30 37 sulfate chemical In the YfiB-YfiR complex, one sulfate ion is found at the bottom of each YfiBL43P molecule (Fig. 3A) and forms a strong hydrogen bond with D102 of YfiBL43P (Fig. 4A and 4C). RESULTS +73 81 YfiBL43P mutant In the YfiB-YfiR complex, one sulfate ion is found at the bottom of each YfiBL43P molecule (Fig. 3A) and forms a strong hydrogen bond with D102 of YfiBL43P (Fig. 4A and 4C). RESULTS +120 133 hydrogen bond bond_interaction In the YfiB-YfiR complex, one sulfate ion is found at the bottom of each YfiBL43P molecule (Fig. 3A) and forms a strong hydrogen bond with D102 of YfiBL43P (Fig. 4A and 4C). RESULTS +139 143 D102 residue_name_number In the YfiB-YfiR complex, one sulfate ion is found at the bottom of each YfiBL43P molecule (Fig. 3A) and forms a strong hydrogen bond with D102 of YfiBL43P (Fig. 4A and 4C). RESULTS +147 155 YfiBL43P mutant In the YfiB-YfiR complex, one sulfate ion is found at the bottom of each YfiBL43P molecule (Fig. 3A) and forms a strong hydrogen bond with D102 of YfiBL43P (Fig. 4A and 4C). RESULTS +0 24 Structural superposition experimental_method Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +33 41 YfiBL43P mutant Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +46 68 Haemophilus influenzae species Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +69 72 Pal protein_type Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +73 87 complexed with protein_state Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +101 124 peptidoglycan precursor chemical Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +126 130 PG-P chemical Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +133 184 UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala chemical Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +186 191 m-Dap chemical Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +195 215 meso-diaminopimelate chemical Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +271 278 sulfate chemical Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +317 337 m-Dap5 ϵ-carboxylate chemical Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +351 359 Pal/PG-P complex_assembly Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 ϵ-carboxylate group in the Pal/PG-P complex (Fig. 4A). RESULTS +7 15 Pal/PG-P complex_assembly In the Pal/PG-P complex structure, the m-Dap5 ϵ-carboxylate group interacts with the side-chain atoms of D71 and the main-chain amide of D37 (Fig. 4B). RESULTS +24 33 structure evidence In the Pal/PG-P complex structure, the m-Dap5 ϵ-carboxylate group interacts with the side-chain atoms of D71 and the main-chain amide of D37 (Fig. 4B). RESULTS +39 59 m-Dap5 ϵ-carboxylate chemical In the Pal/PG-P complex structure, the m-Dap5 ϵ-carboxylate group interacts with the side-chain atoms of D71 and the main-chain amide of D37 (Fig. 4B). RESULTS +105 108 D71 residue_name_number In the Pal/PG-P complex structure, the m-Dap5 ϵ-carboxylate group interacts with the side-chain atoms of D71 and the main-chain amide of D37 (Fig. 4B). RESULTS +137 140 D37 residue_name_number In the Pal/PG-P complex structure, the m-Dap5 ϵ-carboxylate group interacts with the side-chain atoms of D71 and the main-chain amide of D37 (Fig. 4B). RESULTS +18 28 YfiR-bound protein_state Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +29 37 YfiBL43P mutant Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +38 47 structure evidence Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +53 60 sulfate chemical Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +104 108 D102 residue_name_number Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +127 130 D71 residue_name_number Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +134 137 Pal protein_type Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +143 147 R117 residue_name_number Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +166 169 R86 residue_name_number Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +173 176 Pal protein_type Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +206 209 N68 residue_name_number Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +228 231 D37 residue_name_number Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +235 238 Pal protein_type Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal). RESULTS +12 17 water chemical Moreover, a water molecule was found to bridge the sulfate ion and the side chains of N67 and D102, strengthening the hydrogen bond network (Fig. 4C). RESULTS +51 58 sulfate chemical Moreover, a water molecule was found to bridge the sulfate ion and the side chains of N67 and D102, strengthening the hydrogen bond network (Fig. 4C). RESULTS +86 89 N67 residue_name_number Moreover, a water molecule was found to bridge the sulfate ion and the side chains of N67 and D102, strengthening the hydrogen bond network (Fig. 4C). RESULTS +94 98 D102 residue_name_number Moreover, a water molecule was found to bridge the sulfate ion and the side chains of N67 and D102, strengthening the hydrogen bond network (Fig. 4C). RESULTS +118 139 hydrogen bond network site Moreover, a water molecule was found to bridge the sulfate ion and the side chains of N67 and D102, strengthening the hydrogen bond network (Fig. 4C). RESULTS +13 31 sequence alignment experimental_method In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. RESULTS +35 39 YfiB protein In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. RESULTS +45 48 Pal protein_type In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. RESULTS +57 75 periplasmic domain structure_element In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. RESULTS +79 83 OmpA protein_type In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. RESULTS +105 120 PG-binding site site In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. RESULTS +134 137 N68 residue_name_number In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. RESULTS +142 146 D102 residue_name_number In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. RESULTS +151 167 highly conserved protein_state In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. RESULTS +262 266 YfiB protein In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig. 4G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB. RESULTS +15 28 superposition experimental_method Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. RESULTS +32 35 apo protein_state Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. RESULTS +36 40 YfiB protein Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. RESULTS +46 56 YfiR-bound protein_state Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. RESULTS +57 65 YfiBL43P mutant Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. RESULTS +84 101 PG-binding region site Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. RESULTS +135 157 different conformation protein_state Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. RESULTS +165 168 N68 residue_name_number Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. RESULTS +180 184 loop structure_element Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop. RESULTS +12 20 YfiBL43P mutant Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). RESULTS +26 29 N68 residue_name_number Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). RESULTS +41 45 loop structure_element Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). RESULTS +53 56 apo protein_state Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). RESULTS +57 61 YfiB protein Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). RESULTS +88 92 D102 residue_name_number Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). RESULTS +97 101 R117 residue_name_number Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). RESULTS +136 153 PG-binding pocket site Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). RESULTS +174 181 sulfate chemical Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). RESULTS +189 194 water chemical Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 Å, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig. 4D). RESULTS +32 34 PG chemical Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. RESULTS +54 62 inactive protein_state Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. RESULTS +63 67 YfiB protein Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. RESULTS +97 103 active protein_state Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. RESULTS +104 108 YfiB protein Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. RESULTS +143 168 microscale thermophoresis experimental_method Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. RESULTS +170 173 MST experimental_method Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. RESULTS +196 214 binding affinities evidence Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. RESULTS +218 220 PG chemical Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. RESULTS +224 233 wild-type protein_state Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. RESULTS +234 238 YfiB protein Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. RESULTS +243 251 YfiBL43P mutant Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively. RESULTS +31 50 PG-binding affinity evidence The results indicated that the PG-binding affinity of YfiBL43P is 65.5 μmol/L, which is about 16-fold stronger than that of wild-type YfiB (Kd = 1.1 mmol/L) (Fig. 4E–F). RESULTS +54 62 YfiBL43P mutant The results indicated that the PG-binding affinity of YfiBL43P is 65.5 μmol/L, which is about 16-fold stronger than that of wild-type YfiB (Kd = 1.1 mmol/L) (Fig. 4E–F). RESULTS +124 133 wild-type protein_state The results indicated that the PG-binding affinity of YfiBL43P is 65.5 μmol/L, which is about 16-fold stronger than that of wild-type YfiB (Kd = 1.1 mmol/L) (Fig. 4E–F). RESULTS +134 138 YfiB protein The results indicated that the PG-binding affinity of YfiBL43P is 65.5 μmol/L, which is about 16-fold stronger than that of wild-type YfiB (Kd = 1.1 mmol/L) (Fig. 4E–F). RESULTS +140 142 Kd evidence The results indicated that the PG-binding affinity of YfiBL43P is 65.5 μmol/L, which is about 16-fold stronger than that of wild-type YfiB (Kd = 1.1 mmol/L) (Fig. 4E–F). RESULTS +31 48 in the absence of protein_state As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation. RESULTS +49 53 YfiR protein As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation. RESULTS +91 110 PG-binding affinity evidence As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation. RESULTS +114 118 YfiB protein As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation. RESULTS +138 147 YfiB-YfiR complex_assembly As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation. RESULTS +203 207 YfiB protein As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation. RESULTS +227 260 stretched N-terminal conformation protein_state As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation. RESULTS +4 21 conserved surface site The conserved surface in YfiR is functional for binding YfiB and YfiN RESULTS +25 29 YfiR protein The conserved surface in YfiR is functional for binding YfiB and YfiN RESULTS +56 60 YfiB protein The conserved surface in YfiR is functional for binding YfiB and YfiN RESULTS +65 69 YfiN protein The conserved surface in YfiR is functional for binding YfiB and YfiN RESULTS +22 36 ConSurf Server experimental_method Calculation using the ConSurf Server (http://consurf.tau.ac.il/), which estimates the evolutionary conservation of amino acid positions and visualizes information on the structure surface, revealed a conserved surface on YfiR that contributes to the interaction with YfiB (Fig. 3E and 3F). RESULTS +86 111 evolutionary conservation evidence Calculation using the ConSurf Server (http://consurf.tau.ac.il/), which estimates the evolutionary conservation of amino acid positions and visualizes information on the structure surface, revealed a conserved surface on YfiR that contributes to the interaction with YfiB (Fig. 3E and 3F). RESULTS +170 187 structure surface site Calculation using the ConSurf Server (http://consurf.tau.ac.il/), which estimates the evolutionary conservation of amino acid positions and visualizes information on the structure surface, revealed a conserved surface on YfiR that contributes to the interaction with YfiB (Fig. 3E and 3F). RESULTS +200 217 conserved surface site Calculation using the ConSurf Server (http://consurf.tau.ac.il/), which estimates the evolutionary conservation of amino acid positions and visualizes information on the structure surface, revealed a conserved surface on YfiR that contributes to the interaction with YfiB (Fig. 3E and 3F). RESULTS +221 225 YfiR protein Calculation using the ConSurf Server (http://consurf.tau.ac.il/), which estimates the evolutionary conservation of amino acid positions and visualizes information on the structure surface, revealed a conserved surface on YfiR that contributes to the interaction with YfiB (Fig. 3E and 3F). RESULTS +267 271 YfiB protein Calculation using the ConSurf Server (http://consurf.tau.ac.il/), which estimates the evolutionary conservation of amino acid positions and visualizes information on the structure surface, revealed a conserved surface on YfiR that contributes to the interaction with YfiB (Fig. 3E and 3F). RESULTS +36 53 conserved surface site Interestingly, the majority of this conserved surface contributes to the interaction with YfiB (Fig. 3E and 3F). RESULTS +90 94 YfiB protein Interestingly, the majority of this conserved surface contributes to the interaction with YfiB (Fig. 3E and 3F). RESULTS +36 40 F151 residue_name_number Malone JG et al. have reported that F151, E163, I169 and Q187, located near the C-terminus of YfiR, comprise a putative YfiN binding site (Malone et al.,). RESULTS +42 46 E163 residue_name_number Malone JG et al. have reported that F151, E163, I169 and Q187, located near the C-terminus of YfiR, comprise a putative YfiN binding site (Malone et al.,). RESULTS +48 52 I169 residue_name_number Malone JG et al. have reported that F151, E163, I169 and Q187, located near the C-terminus of YfiR, comprise a putative YfiN binding site (Malone et al.,). RESULTS +57 61 Q187 residue_name_number Malone JG et al. have reported that F151, E163, I169 and Q187, located near the C-terminus of YfiR, comprise a putative YfiN binding site (Malone et al.,). RESULTS +94 98 YfiR protein Malone JG et al. have reported that F151, E163, I169 and Q187, located near the C-terminus of YfiR, comprise a putative YfiN binding site (Malone et al.,). RESULTS +120 137 YfiN binding site site Malone JG et al. have reported that F151, E163, I169 and Q187, located near the C-terminus of YfiR, comprise a putative YfiN binding site (Malone et al.,). RESULTS +46 63 conserved surface site Interestingly, these residues are part of the conserved surface of YfiR (Fig. 3G). RESULTS +67 71 YfiR protein Interestingly, these residues are part of the conserved surface of YfiR (Fig. 3G). RESULTS +0 4 F151 residue_name_number F151, E163 and I169 form a hydrophobic core while, Q187 is located at the end of the α6 helix. RESULTS +6 10 E163 residue_name_number F151, E163 and I169 form a hydrophobic core while, Q187 is located at the end of the α6 helix. RESULTS +15 19 I169 residue_name_number F151, E163 and I169 form a hydrophobic core while, Q187 is located at the end of the α6 helix. RESULTS +27 43 hydrophobic core site F151, E163 and I169 form a hydrophobic core while, Q187 is located at the end of the α6 helix. RESULTS +51 55 Q187 residue_name_number F151, E163 and I169 form a hydrophobic core while, Q187 is located at the end of the α6 helix. RESULTS +85 93 α6 helix structure_element F151, E163 and I169 form a hydrophobic core while, Q187 is located at the end of the α6 helix. RESULTS +0 4 E163 residue_name_number E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +9 13 I169 residue_name_number E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +18 43 YfiB-interacting residues site E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +47 51 YfiR protein E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +62 66 E163 residue_name_number E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +75 88 hydrogen bond bond_interaction E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +94 97 R96 residue_name_number E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +101 105 YfiB protein E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +123 127 I169 residue_name_number E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +155 159 L166 residue_name_number E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +160 164 I169 residue_name_number E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +165 169 V176 residue_name_number E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +170 174 P178 residue_name_number E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +175 179 L181 residue_name_number E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +180 196 hydrophobic core site E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +211 214 F57 residue_name_number E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +218 222 YfiB protein E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig. 3D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig. 3D-I(ii)). RESULTS +28 47 YfiB-YfiR interface site Collectively, a part of the YfiB-YfiR interface overlaps with the proposed YfiR-YfiN interface, suggesting alteration in the association-disassociation equilibrium of YfiR-YfiN and hence the ability of YfiB to sequester YfiR. RESULTS +75 94 YfiR-YfiN interface site Collectively, a part of the YfiB-YfiR interface overlaps with the proposed YfiR-YfiN interface, suggesting alteration in the association-disassociation equilibrium of YfiR-YfiN and hence the ability of YfiB to sequester YfiR. RESULTS +167 171 YfiR protein Collectively, a part of the YfiB-YfiR interface overlaps with the proposed YfiR-YfiN interface, suggesting alteration in the association-disassociation equilibrium of YfiR-YfiN and hence the ability of YfiB to sequester YfiR. RESULTS +172 176 YfiN protein Collectively, a part of the YfiB-YfiR interface overlaps with the proposed YfiR-YfiN interface, suggesting alteration in the association-disassociation equilibrium of YfiR-YfiN and hence the ability of YfiB to sequester YfiR. RESULTS +202 206 YfiB protein Collectively, a part of the YfiB-YfiR interface overlaps with the proposed YfiR-YfiN interface, suggesting alteration in the association-disassociation equilibrium of YfiR-YfiN and hence the ability of YfiB to sequester YfiR. RESULTS +220 224 YfiR protein Collectively, a part of the YfiB-YfiR interface overlaps with the proposed YfiR-YfiN interface, suggesting alteration in the association-disassociation equilibrium of YfiR-YfiN and hence the ability of YfiB to sequester YfiR. RESULTS +0 4 YfiR protein YfiR binds small molecules RESULTS +32 36 YfiR protein Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). RESULTS +51 55 YfiB protein Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). RESULTS +101 105 YfiN protein Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). RESULTS +177 181 YfiR protein Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). RESULTS +182 192 structures evidence Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). RESULTS +205 217 non-oxidized protein_state Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). RESULTS +226 234 oxidized protein_state Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). RESULTS +270 276 Cys145 residue_name_number Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). RESULTS +277 283 Cys152 residue_name_number Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). RESULTS +284 298 disulfide bond ptm Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). RESULTS +361 365 YfiR protein Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,). RESULTS +17 21 YfiR protein However, whether YfiR is involved in other regulatory mechanisms is still an open question. RESULTS +8 18 Structures evidence Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. FIG +22 31 VB6-bound protein_state Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. FIG +36 45 Trp-bound protein_state Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. FIG +46 50 YfiR protein Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. FIG +56 69 Superposition experimental_method Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. FIG +85 95 structures evidence Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. FIG +99 108 VB6-bound protein_state Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. FIG +113 122 Trp-bound protein_state Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. FIG +123 127 YfiR protein Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. FIG +176 180 YfiR protein Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. FIG +191 194 VB6 chemical Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. FIG +199 204 L-Trp chemical Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp. FIG +4 22 electron densities evidence The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB FIG +26 29 VB6 chemical The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB FIG +34 37 Trp chemical The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB FIG +87 101 |Fo|-|Fc| maps evidence The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB FIG +107 120 Superposition experimental_method The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB FIG +128 146 hydrophobic pocket site The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB FIG +150 154 YfiR protein The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB FIG +160 163 VB6 chemical The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB FIG +165 170 L-Trp chemical The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB FIG +175 178 F57 residue_name_number The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB FIG +182 186 YfiB protein The electron densities of VB6 and Trp are countered at 3.0σ and 2.3σ, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB FIG +16 27 Dali search experimental_method Intriguingly, a Dali search (Holm and Rosenstrom,) indicated that the closest homologs of YfiR shared the characteristic of being able to bind several structurally similar small molecules, such as L-Trp, L-Phe, B-group vitamins and their analogs, encouraging us to test whether YfiR can recognize these molecules. RESULTS +90 94 YfiR protein Intriguingly, a Dali search (Holm and Rosenstrom,) indicated that the closest homologs of YfiR shared the characteristic of being able to bind several structurally similar small molecules, such as L-Trp, L-Phe, B-group vitamins and their analogs, encouraging us to test whether YfiR can recognize these molecules. RESULTS +197 202 L-Trp chemical Intriguingly, a Dali search (Holm and Rosenstrom,) indicated that the closest homologs of YfiR shared the characteristic of being able to bind several structurally similar small molecules, such as L-Trp, L-Phe, B-group vitamins and their analogs, encouraging us to test whether YfiR can recognize these molecules. RESULTS +204 209 L-Phe chemical Intriguingly, a Dali search (Holm and Rosenstrom,) indicated that the closest homologs of YfiR shared the characteristic of being able to bind several structurally similar small molecules, such as L-Trp, L-Phe, B-group vitamins and their analogs, encouraging us to test whether YfiR can recognize these molecules. RESULTS +278 282 YfiR protein Intriguingly, a Dali search (Holm and Rosenstrom,) indicated that the closest homologs of YfiR shared the characteristic of being able to bind several structurally similar small molecules, such as L-Trp, L-Phe, B-group vitamins and their analogs, encouraging us to test whether YfiR can recognize these molecules. RESULTS +21 36 co-crystallized experimental_method For this purpose, we co-crystallized YfiR or soaked YfiR crystals with different small molecules, including L-Trp and B-group vitamins. RESULTS +37 41 YfiR protein For this purpose, we co-crystallized YfiR or soaked YfiR crystals with different small molecules, including L-Trp and B-group vitamins. RESULTS +45 51 soaked experimental_method For this purpose, we co-crystallized YfiR or soaked YfiR crystals with different small molecules, including L-Trp and B-group vitamins. RESULTS +52 56 YfiR protein For this purpose, we co-crystallized YfiR or soaked YfiR crystals with different small molecules, including L-Trp and B-group vitamins. RESULTS +57 65 crystals evidence For this purpose, we co-crystallized YfiR or soaked YfiR crystals with different small molecules, including L-Trp and B-group vitamins. RESULTS +108 113 L-Trp chemical For this purpose, we co-crystallized YfiR or soaked YfiR crystals with different small molecules, including L-Trp and B-group vitamins. RESULTS +30 52 small-molecule density evidence Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). RESULTS +60 69 VB6-bound protein_state Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). RESULTS +74 83 Trp-bound protein_state Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). RESULTS +84 88 YfiR protein Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). RESULTS +89 107 crystal structures evidence Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). RESULTS +138 148 structures evidence Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). RESULTS +154 158 YfiR protein Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). RESULTS +159 165 dimers oligomeric_state Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). RESULTS +179 187 oxidized protein_state Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). RESULTS +188 192 YfiR protein Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). RESULTS +193 202 structure evidence Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). RESULTS +221 236 disulfide bonds ptm Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig. 5A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,). RESULTS +23 26 VB6 chemical Functional analysis of VB6 and L-Trp. (A and B) The effect of increasing concentrations of VB6 or L-Trp on YfiBL43P-induced attachment (bars). FIG +31 36 L-Trp chemical Functional analysis of VB6 and L-Trp. (A and B) The effect of increasing concentrations of VB6 or L-Trp on YfiBL43P-induced attachment (bars). FIG +52 87 effect of increasing concentrations experimental_method Functional analysis of VB6 and L-Trp. (A and B) The effect of increasing concentrations of VB6 or L-Trp on YfiBL43P-induced attachment (bars). FIG +91 94 VB6 chemical Functional analysis of VB6 and L-Trp. (A and B) The effect of increasing concentrations of VB6 or L-Trp on YfiBL43P-induced attachment (bars). FIG +98 103 L-Trp chemical Functional analysis of VB6 and L-Trp. (A and B) The effect of increasing concentrations of VB6 or L-Trp on YfiBL43P-induced attachment (bars). FIG +107 115 YfiBL43P mutant Functional analysis of VB6 and L-Trp. (A and B) The effect of increasing concentrations of VB6 or L-Trp on YfiBL43P-induced attachment (bars). FIG +4 28 relative optical density evidence The relative optical density is represented as curves. FIG +0 9 Wild-type protein_state Wild-type YfiB is used as negative control. FIG +10 14 YfiB protein Wild-type YfiB is used as negative control. FIG +10 17 BIAcore experimental_method (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR FIG +40 58 binding affinities evidence (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR FIG +66 69 VB6 chemical (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR FIG +78 83 L-Trp chemical (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR FIG +89 93 YfiR protein (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR FIG +101 104 ITC experimental_method (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR FIG +127 136 titration experimental_method (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR FIG +144 148 YfiB protein (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR FIG +149 158 wild-type protein_state (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR FIG +182 190 YfiBL43P mutant (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR FIG +191 195 F57A mutant (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR FIG +201 205 YfiR protein (C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E–G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR FIG +0 19 Structural analyses experimental_method Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface. RESULTS +38 41 VB6 chemical Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface. RESULTS +46 51 L-Trp chemical Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface. RESULTS +66 74 bound at protein_state Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface. RESULTS +96 100 YfiR protein Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface. RESULTS +101 106 dimer oligomeric_state Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface. RESULTS +123 138 dimer interface site Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface. RESULTS +15 18 VB6 chemical Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +23 28 L-Trp chemical Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +59 77 hydrophobic pocket site Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +89 93 L166 residue_name_number Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +94 98 I169 residue_name_number Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +99 103 V176 residue_name_number Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +104 108 P178 residue_name_number Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +109 113 L181 residue_name_number Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +117 121 YfiR protein Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +139 153 binding pocket site Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +158 161 F57 residue_name_number Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +165 169 YfiB protein Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +190 199 YfiB-YfiR complex_assembly Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig. 5C). RESULTS +30 33 F57 residue_name_number To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). RESULTS +37 50 YfiBL43P-YfiR complex_assembly To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). RESULTS +68 86 binding affinities evidence To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). RESULTS +90 98 YfiBL43P mutant To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). RESULTS +103 111 YfiBL43P mutant To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). RESULTS +112 116 F57A mutant To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). RESULTS +121 125 YfiR protein To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). RESULTS +143 175 isothermal titration calorimetry experimental_method To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). RESULTS +177 180 ITC experimental_method To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC). RESULTS +19 21 Kd evidence The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). RESULTS +74 82 YfiBL43P mutant The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). RESULTS +87 95 YfiBL43P mutant The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). RESULTS +96 100 F57A mutant The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). RESULTS +135 143 YfiBL43P mutant The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). RESULTS +144 148 F57A mutant The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). RESULTS +149 155 mutant protein_state The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). RESULTS +191 207 binding affinity evidence The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). RESULTS +226 234 YfiBL43P mutant The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). RESULTS +235 241 mutant protein_state The results showed Kd values of 1.4 × 10−7 mol/L and 5.3 × 10−7 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig. 6F and 6G). RESULTS +66 69 VB6 chemical In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +74 79 L-Trp chemical In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +81 85 yfiB gene In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +90 97 deleted experimental_method In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +103 107 PAO1 species In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +108 117 wild-type protein_state In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +132 152 construct expressing experimental_method In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +157 165 YfiBL43P mutant In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +166 172 mutant protein_state In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +177 193 transformed into experimental_method In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +198 202 PAO1 species In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +203 208 ΔyfiB mutant In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +227 235 YfiBL43P mutant In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 ΔyfiB strain to trigger YfiBL43P-induced biofilm formation. RESULTS +0 36 Growth and surface attachment assays experimental_method Growth and surface attachment assays were carried out for the yfiB-L43P strain in the presence of increasing concentrations of VB6 or L-Trp. RESULTS +62 71 yfiB-L43P mutant Growth and surface attachment assays were carried out for the yfiB-L43P strain in the presence of increasing concentrations of VB6 or L-Trp. RESULTS +98 123 increasing concentrations experimental_method Growth and surface attachment assays were carried out for the yfiB-L43P strain in the presence of increasing concentrations of VB6 or L-Trp. RESULTS +127 130 VB6 chemical Growth and surface attachment assays were carried out for the yfiB-L43P strain in the presence of increasing concentrations of VB6 or L-Trp. RESULTS +134 139 L-Trp chemical Growth and surface attachment assays were carried out for the yfiB-L43P strain in the presence of increasing concentrations of VB6 or L-Trp. RESULTS +32 47 over-expression experimental_method As shown in Fig. 6A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4–6 mmol/L for VB6 and 6–10 mmol/L for L-Trp) could reduce the surface attachment. RESULTS +51 59 YfiBL43P mutant As shown in Fig. 6A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4–6 mmol/L for VB6 and 6–10 mmol/L for L-Trp) could reduce the surface attachment. RESULTS +124 133 yfiB-L43P mutant As shown in Fig. 6A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4–6 mmol/L for VB6 and 6–10 mmol/L for L-Trp) could reduce the surface attachment. RESULTS +179 182 VB6 chemical As shown in Fig. 6A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4–6 mmol/L for VB6 and 6–10 mmol/L for L-Trp) could reduce the surface attachment. RESULTS +186 191 L-Trp chemical As shown in Fig. 6A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4–6 mmol/L for VB6 and 6–10 mmol/L for L-Trp) could reduce the surface attachment. RESULTS +208 211 VB6 chemical As shown in Fig. 6A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4–6 mmol/L for VB6 and 6–10 mmol/L for L-Trp) could reduce the surface attachment. RESULTS +232 237 L-Trp chemical As shown in Fig. 6A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4–6 mmol/L for VB6 and 6–10 mmol/L for L-Trp) could reduce the surface attachment. RESULTS +56 59 VB6 chemical Interestingly, at a concentration higher than 8 mmol/L, VB6 lost its ability to inhibit biofilm formation, implying that the VB6-involving regulatory mechanism is highly complicated and remains to be further investigated. RESULTS +125 128 VB6 chemical Interestingly, at a concentration higher than 8 mmol/L, VB6 lost its ability to inhibit biofilm formation, implying that the VB6-involving regulatory mechanism is highly complicated and remains to be further investigated. RESULTS +14 17 VB6 chemical Of note, both VB6 and L-Trp have been reported to correlate with biofilm formation in certain Gram-negative bacteria (Grubman et al.,; Shimazaki et al.,). RESULTS +22 27 L-Trp chemical Of note, both VB6 and L-Trp have been reported to correlate with biofilm formation in certain Gram-negative bacteria (Grubman et al.,; Shimazaki et al.,). RESULTS +94 116 Gram-negative bacteria taxonomy_domain Of note, both VB6 and L-Trp have been reported to correlate with biofilm formation in certain Gram-negative bacteria (Grubman et al.,; Shimazaki et al.,). RESULTS +3 22 Helicobacter pylori species In Helicobacter pylori in particular, VB6 biosynthetic enzymes act as novel virulence factors, and VB6 is required for full motility and virulence (Grubman et al.,). RESULTS +38 41 VB6 chemical In Helicobacter pylori in particular, VB6 biosynthetic enzymes act as novel virulence factors, and VB6 is required for full motility and virulence (Grubman et al.,). RESULTS +99 102 VB6 chemical In Helicobacter pylori in particular, VB6 biosynthetic enzymes act as novel virulence factors, and VB6 is required for full motility and virulence (Grubman et al.,). RESULTS +4 11 E. coli species  In E. coli, mutants with decreased tryptophan synthesis show greater biofilm formation, and matured biofilm is degraded by L-tryptophan addition (Shimazaki et al.,). RESULTS +36 46 tryptophan chemical  In E. coli, mutants with decreased tryptophan synthesis show greater biofilm formation, and matured biofilm is degraded by L-tryptophan addition (Shimazaki et al.,). RESULTS +124 136 L-tryptophan chemical  In E. coli, mutants with decreased tryptophan synthesis show greater biofilm formation, and matured biofilm is degraded by L-tryptophan addition (Shimazaki et al.,). RESULTS +46 49 VB6 chemical To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. RESULTS +53 58 L-Trp chemical To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. RESULTS +67 71 YfiB protein To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. RESULTS +72 90 F57-binding pocket site To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. RESULTS +94 98 YfiR protein To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. RESULTS +172 190 binding affinities evidence To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. RESULTS +194 197 VB6 chemical To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. RESULTS +202 207 L-Trp chemical To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. RESULTS +212 216 YfiR protein To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. RESULTS +221 228 BIAcore experimental_method To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments. RESULTS +35 37 Kd evidence The results showed relatively weak Kd values of 35.2 mmol/L and 76.9 mmol/L for VB6 and L-Trp, respectively (Fig. 6C and 6D). RESULTS +80 83 VB6 chemical The results showed relatively weak Kd values of 35.2 mmol/L and 76.9 mmol/L for VB6 and L-Trp, respectively (Fig. 6C and 6D). RESULTS +88 93 L-Trp chemical The results showed relatively weak Kd values of 35.2 mmol/L and 76.9 mmol/L for VB6 and L-Trp, respectively (Fig. 6C and 6D). RESULTS +40 43 VB6 chemical Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation. RESULTS +47 52 L-Trp chemical Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation. RESULTS +65 69 YfiR protein Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation. RESULTS +80 83 VB6 chemical Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation. RESULTS +87 92 L-Trp chemical Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation. RESULTS +93 98 alone protein_state Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation. RESULTS +143 152 YfiB-YfiR complex_assembly Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation. RESULTS +189 192 VB6 chemical Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation. RESULTS +196 201 L-Trp chemical Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation. RESULTS +60 64 YfiB protein Previous studies suggested that in response to cell stress, YfiB in the outer membrane sequesters the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus inducing the diguanylate cyclase activity of YfiN to allow c-di-GMP production (Giardina et al.,; Malone et al.,; Malone et al.,). DISCUSS +122 126 YfiR protein Previous studies suggested that in response to cell stress, YfiB in the outer membrane sequesters the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus inducing the diguanylate cyclase activity of YfiN to allow c-di-GMP production (Giardina et al.,; Malone et al.,; Malone et al.,). DISCUSS +156 160 YfiN protein Previous studies suggested that in response to cell stress, YfiB in the outer membrane sequesters the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus inducing the diguanylate cyclase activity of YfiN to allow c-di-GMP production (Giardina et al.,; Malone et al.,; Malone et al.,). DISCUSS +237 241 YfiN protein Previous studies suggested that in response to cell stress, YfiB in the outer membrane sequesters the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus inducing the diguanylate cyclase activity of YfiN to allow c-di-GMP production (Giardina et al.,; Malone et al.,; Malone et al.,). DISCUSS +251 259 c-di-GMP chemical Previous studies suggested that in response to cell stress, YfiB in the outer membrane sequesters the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus inducing the diguanylate cyclase activity of YfiN to allow c-di-GMP production (Giardina et al.,; Malone et al.,; Malone et al.,). DISCUSS +20 38 crystal structures evidence Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. DISCUSS +42 46 YfiB protein Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. DISCUSS +47 52 alone protein_state Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. DISCUSS +60 66 active protein_state Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. DISCUSS +67 73 mutant protein_state Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. DISCUSS +74 82 YfiBL43P mutant Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. DISCUSS +83 98 in complex with protein_state Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. DISCUSS +99 103 YfiR protein Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. DISCUSS +121 125 YfiR protein Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. DISCUSS +138 150 complex with protein_state Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. DISCUSS +151 155 YfiB protein Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues. DISCUSS +4 28 structural data analysis experimental_method Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +44 53 activated protein_state Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +54 58 YfiB protein Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +66 84 N-terminal portion structure_element Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +121 143 stretched conformation protein_state Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +162 182 compact conformation protein_state Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +190 193 apo protein_state Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +194 198 YfiB protein Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +204 207 apo protein_state Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +208 212 YfiB protein Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +213 222 structure evidence Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +256 258 34 residue_number Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +265 285 compact conformation protein_state Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 Å in length. DISCUSS +19 33 preceding 8 aa residue_range In addition to the preceding 8 aa loop (from the lipid acceptor Cys26 to Gly34), the full length of the periplasmic portion of apo YfiB can reach approximately 60 Å. It was reported that the distance between the outer membrane and the cell wall is approximately 50 Å and that the thickness of the PG layer is approximately 70 Å (Matias et al.,). DISCUSS +34 38 loop structure_element In addition to the preceding 8 aa loop (from the lipid acceptor Cys26 to Gly34), the full length of the periplasmic portion of apo YfiB can reach approximately 60 Å. It was reported that the distance between the outer membrane and the cell wall is approximately 50 Å and that the thickness of the PG layer is approximately 70 Å (Matias et al.,). DISCUSS +64 78 Cys26 to Gly34 residue_range In addition to the preceding 8 aa loop (from the lipid acceptor Cys26 to Gly34), the full length of the periplasmic portion of apo YfiB can reach approximately 60 Å. It was reported that the distance between the outer membrane and the cell wall is approximately 50 Å and that the thickness of the PG layer is approximately 70 Å (Matias et al.,). DISCUSS +85 96 full length protein_state In addition to the preceding 8 aa loop (from the lipid acceptor Cys26 to Gly34), the full length of the periplasmic portion of apo YfiB can reach approximately 60 Å. It was reported that the distance between the outer membrane and the cell wall is approximately 50 Å and that the thickness of the PG layer is approximately 70 Å (Matias et al.,). DISCUSS +127 130 apo protein_state In addition to the preceding 8 aa loop (from the lipid acceptor Cys26 to Gly34), the full length of the periplasmic portion of apo YfiB can reach approximately 60 Å. It was reported that the distance between the outer membrane and the cell wall is approximately 50 Å and that the thickness of the PG layer is approximately 70 Å (Matias et al.,). DISCUSS +131 135 YfiB protein In addition to the preceding 8 aa loop (from the lipid acceptor Cys26 to Gly34), the full length of the periplasmic portion of apo YfiB can reach approximately 60 Å. It was reported that the distance between the outer membrane and the cell wall is approximately 50 Å and that the thickness of the PG layer is approximately 70 Å (Matias et al.,). DISCUSS +6 10 YfiB protein Thus, YfiB alone represents an inactive form that may only partially insert into the PG matrix. DISCUSS +11 16 alone protein_state Thus, YfiB alone represents an inactive form that may only partially insert into the PG matrix. DISCUSS +31 39 inactive protein_state Thus, YfiB alone represents an inactive form that may only partially insert into the PG matrix. DISCUSS +13 23 YfiR-bound protein_state By contrast, YfiR-bound YfiBL43P (residues 44–168) has a stretched conformation of approximately 55 Å in length. DISCUSS +24 32 YfiBL43P mutant By contrast, YfiR-bound YfiBL43P (residues 44–168) has a stretched conformation of approximately 55 Å in length. DISCUSS +43 49 44–168 residue_range By contrast, YfiR-bound YfiBL43P (residues 44–168) has a stretched conformation of approximately 55 Å in length. DISCUSS +57 79 stretched conformation protein_state By contrast, YfiR-bound YfiBL43P (residues 44–168) has a stretched conformation of approximately 55 Å in length. DISCUSS +19 54 17 preceding intracellular residues residue_range In addition to the 17 preceding intracellular residues (from the lipid acceptor Cys26 to Leu43), the length of the intracellular portion of active YfiB may extend over 100 Å, assuming a fully stretched conformation. DISCUSS +80 94 Cys26 to Leu43 residue_range In addition to the 17 preceding intracellular residues (from the lipid acceptor Cys26 to Leu43), the length of the intracellular portion of active YfiB may extend over 100 Å, assuming a fully stretched conformation. DISCUSS +140 146 active protein_state In addition to the 17 preceding intracellular residues (from the lipid acceptor Cys26 to Leu43), the length of the intracellular portion of active YfiB may extend over 100 Å, assuming a fully stretched conformation. DISCUSS +147 151 YfiB protein In addition to the 17 preceding intracellular residues (from the lipid acceptor Cys26 to Leu43), the length of the intracellular portion of active YfiB may extend over 100 Å, assuming a fully stretched conformation. DISCUSS +186 214 fully stretched conformation protein_state In addition to the 17 preceding intracellular residues (from the lipid acceptor Cys26 to Leu43), the length of the intracellular portion of active YfiB may extend over 100 Å, assuming a fully stretched conformation. DISCUSS +53 57 YfiB protein Provided that the diameter of the widest part of the YfiB dimer is approximately 64 Å, which is slightly smaller than the smallest diameter of the PG pore (70 Å) (Meroueh et al.,), the YfiB dimer should be able to penetrate the PG layer. DISCUSS +58 63 dimer oligomeric_state Provided that the diameter of the widest part of the YfiB dimer is approximately 64 Å, which is slightly smaller than the smallest diameter of the PG pore (70 Å) (Meroueh et al.,), the YfiB dimer should be able to penetrate the PG layer. DISCUSS +185 189 YfiB protein Provided that the diameter of the widest part of the YfiB dimer is approximately 64 Å, which is slightly smaller than the smallest diameter of the PG pore (70 Å) (Meroueh et al.,), the YfiB dimer should be able to penetrate the PG layer. DISCUSS +190 195 dimer oligomeric_state Provided that the diameter of the widest part of the YfiB dimer is approximately 64 Å, which is slightly smaller than the smallest diameter of the PG pore (70 Å) (Meroueh et al.,), the YfiB dimer should be able to penetrate the PG layer. DISCUSS +24 30 YfiBNR complex_assembly Regulatory model of the YfiBNR tripartite system. FIG +31 41 tripartite protein_state Regulatory model of the YfiBNR tripartite system. FIG +4 22 periplasmic domain structure_element The periplasmic domain of YfiB and the YfiB-YfiR complex are depicted according to the crystal structures. FIG +26 30 YfiB protein The periplasmic domain of YfiB and the YfiB-YfiR complex are depicted according to the crystal structures. FIG +39 48 YfiB-YfiR complex_assembly The periplasmic domain of YfiB and the YfiB-YfiR complex are depicted according to the crystal structures. FIG +87 105 crystal structures evidence The periplasmic domain of YfiB and the YfiB-YfiR complex are depicted according to the crystal structures. FIG +19 24 Cys26 residue_name_number The lipid acceptor Cys26 is indicated as blue ball. FIG +4 8 loop structure_element The loop connecting Cys26 and Gly34 of YfiB is modeled. FIG +20 25 Cys26 residue_name_number The loop connecting Cys26 and Gly34 of YfiB is modeled. FIG +30 35 Gly34 residue_name_number The loop connecting Cys26 and Gly34 of YfiB is modeled. FIG +39 43 YfiB protein The loop connecting Cys26 and Gly34 of YfiB is modeled. FIG +4 14 PAS domain structure_element The PAS domain of YfiN is shown as pink oval. FIG +18 22 YfiN protein The PAS domain of YfiN is shown as pink oval. FIG +5 14 activated protein_state Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN FIG +43 50 dimeric oligomeric_state Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN FIG +51 55 YfiB protein Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN FIG +74 94 compact conformation protein_state Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN FIG +100 122 stretched conformation protein_state Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN FIG +137 155 periplasmic domain structure_element Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN FIG +163 180 membrane-anchored protein_state Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN FIG +181 185 YfiB protein Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN FIG +231 235 YfiR protein Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN FIG +236 241 dimer oligomeric_state Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN FIG +276 280 YfiN protein Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN FIG +50 59 activated protein_state These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig. 7). DISCUSS +60 64 YfiB protein These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig. 7). DISCUSS +83 109 cell wall binding affinity evidence These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig. 7). DISCUSS +163 165 PG chemical These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig. 7). DISCUSS +214 218 YfiB protein These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig. 7). DISCUSS +290 294 loop structure_element These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig. 7). DISCUSS +332 334 PG chemical These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig. 7). DISCUSS +404 410 YfiBNR complex_assembly These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig. 7). DISCUSS +89 96 dimeric oligomeric_state In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity. DISCUSS +97 101 YfiB protein In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity. DISCUSS +105 114 activated protein_state In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity. DISCUSS +122 129 compact protein_state In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity. DISCUSS +131 139 inactive protein_state In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity. DISCUSS +140 152 conformation protein_state In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity. DISCUSS +158 180 stretched conformation protein_state In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity. DISCUSS +208 210 PG chemical In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity. DISCUSS +16 34 C-terminal portion structure_element This allows the C-terminal portion of the membrane-anchored YfiB to reach, bind and penetrate the cell wall and sequester the YfiR dimer. DISCUSS +42 59 membrane-anchored protein_state This allows the C-terminal portion of the membrane-anchored YfiB to reach, bind and penetrate the cell wall and sequester the YfiR dimer. DISCUSS +60 64 YfiB protein This allows the C-terminal portion of the membrane-anchored YfiB to reach, bind and penetrate the cell wall and sequester the YfiR dimer. DISCUSS +126 130 YfiR protein This allows the C-terminal portion of the membrane-anchored YfiB to reach, bind and penetrate the cell wall and sequester the YfiR dimer. DISCUSS +131 136 dimer oligomeric_state This allows the C-terminal portion of the membrane-anchored YfiB to reach, bind and penetrate the cell wall and sequester the YfiR dimer. DISCUSS +4 10 YfiBNR complex_assembly The YfiBNR system provides a good example of a delicate homeostatic system that integrates multiple signals to regulate the c-di-GMP level. DISCUSS +124 132 c-di-GMP chemical The YfiBNR system provides a good example of a delicate homeostatic system that integrates multiple signals to regulate the c-di-GMP level. DISCUSS +16 22 YfiBNR complex_assembly Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation. DISCUSS +34 56 functionally conserved protein_state Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation. DISCUSS +60 73 P. aeruginosa species Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation. DISCUSS +108 115 E. coli species Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation. DISCUSS +178 190 K. pneumonia species Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation. DISCUSS +213 222 Y. pestis species Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation. DISCUSS +256 264 c-di-GMP chemical Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation. DISCUSS +23 32 activated protein_state The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance. DISCUSS +33 37 YfiB protein The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance. DISCUSS +65 69 YfiN protein The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance. DISCUSS +95 101 YfiBNR complex_assembly The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance. DISCUSS +118 126 bacteria taxonomy_domain The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance. DISCUSS +169 177 c-di-GMP chemical The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance. DISCUSS diff --git a/annotation_CSV/PMC4888278.csv b/annotation_CSV/PMC4888278.csv new file mode 100644 index 0000000000000000000000000000000000000000..fe8f19f7658a115062bc83eec0c5f742cc0aab56 --- /dev/null +++ b/annotation_CSV/PMC4888278.csv @@ -0,0 +1,581 @@ +anno_start anno_end anno_text entity_type sentence section +36 44 RORgamma protein Structural determinant for inducing RORgamma specific inverse agonism triggered by a synthetic benzoxazinone ligand TITLE +95 108 benzoxazinone chemical Structural determinant for inducing RORgamma specific inverse agonism triggered by a synthetic benzoxazinone ligand TITLE +4 28 nuclear hormone receptor protein_type The nuclear hormone receptor RORγ regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. ABSTRACT +29 33 RORγ protein The nuclear hormone receptor RORγ regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. ABSTRACT +117 128 interleukin protein_type The nuclear hormone receptor RORγ regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. ABSTRACT +129 134 IL-17 protein_type The nuclear hormone receptor RORγ regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. ABSTRACT +33 37 RORγ protein This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. ABSTRACT +103 136 activation function 2 (AF2) helix structure_element This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. ABSTRACT +144 165 ligand binding domain structure_element This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. ABSTRACT +169 173 RORγ protein This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. ABSTRACT +180 189 conserved protein_state This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. ABSTRACT +190 207 LXXLL helix motif structure_element This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. ABSTRACT +26 30 RORγ protein Our goal was to develop a RORγ specific inverse agonist that would help down regulate pro-inflammatory gene transcription by disrupting the protein protein interaction with coactivator proteins as a therapeutic agent. ABSTRACT +40 55 inverse agonist protein_state Our goal was to develop a RORγ specific inverse agonist that would help down regulate pro-inflammatory gene transcription by disrupting the protein protein interaction with coactivator proteins as a therapeutic agent. ABSTRACT +42 55 benzoxazinone chemical We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. ABSTRACT +74 81 agonist protein_state We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. ABSTRACT +83 89 BIO592 chemical We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. ABSTRACT +95 110 inverse agonist protein_state We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. ABSTRACT +112 118 BIO399 chemical We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. ABSTRACT +140 156 FRET based assay experimental_method We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. ABSTRACT +17 26 AF2 helix structure_element We show that the AF2 helix of RORγ is proteolytically sensitive when inverse agonist BIO399 binds. ABSTRACT +30 34 RORγ protein We show that the AF2 helix of RORγ is proteolytically sensitive when inverse agonist BIO399 binds. ABSTRACT +38 63 proteolytically sensitive protein_state We show that the AF2 helix of RORγ is proteolytically sensitive when inverse agonist BIO399 binds. ABSTRACT +69 84 inverse agonist protein_state We show that the AF2 helix of RORγ is proteolytically sensitive when inverse agonist BIO399 binds. ABSTRACT +85 91 BIO399 chemical We show that the AF2 helix of RORγ is proteolytically sensitive when inverse agonist BIO399 binds. ABSTRACT +6 27 x-ray crystallography experimental_method Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of RORγ. ABSTRACT +67 80 benzoxazinone chemical Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of RORγ. ABSTRACT +81 88 agonist protein_state Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of RORγ. ABSTRACT +89 95 BIO592 chemical Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of RORγ. ABSTRACT +123 127 RORγ protein Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of RORγ. ABSTRACT +9 31 in vivo reporter assay experimental_method Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and β. ABSTRACT +50 65 inverse agonist protein_state Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and β. ABSTRACT +66 72 BIO399 chemical Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and β. ABSTRACT +99 103 RORγ protein Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and β. ABSTRACT +109 112 ROR protein_type Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and β. ABSTRACT +132 133 α protein Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and β. ABSTRACT +138 139 β protein Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and β. ABSTRACT +14 27 benzoxazinone chemical The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. ABSTRACT +54 64 FRET assay experimental_method The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. ABSTRACT +73 80 agonist protein_state The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. ABSTRACT +82 88 BIO592 chemical The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. ABSTRACT +93 108 inverse agonist protein_state The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. ABSTRACT +110 116 BIO399 chemical The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. ABSTRACT +161 168 agonist protein_state The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. ABSTRACT +185 189 RORγ protein The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. ABSTRACT +35 44 AF2 helix structure_element The proteolytic sensitivity of the AF2 helix of RORγ demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site. ABSTRACT +48 52 RORγ protein The proteolytic sensitivity of the AF2 helix of RORγ demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site. ABSTRACT +92 98 BIO399 chemical The proteolytic sensitivity of the AF2 helix of RORγ demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site. ABSTRACT +99 114 inverse agonist protein_state The proteolytic sensitivity of the AF2 helix of RORγ demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site. ABSTRACT +138 170 coactivator protein binding site site The proteolytic sensitivity of the AF2 helix of RORγ demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site. ABSTRACT +4 28 structural investigation experimental_method Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism. ABSTRACT +36 42 BIO592 chemical Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism. ABSTRACT +43 50 agonist protein_state Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism. ABSTRACT +55 61 BIO399 chemical Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism. ABSTRACT +62 77 inverse agonist protein_state Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism. ABSTRACT +78 88 structures evidence Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism. ABSTRACT +108 114 Met358 residue_name_number Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism. ABSTRACT +118 122 RORγ protein Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism. ABSTRACT +142 146 RORγ protein Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism. ABSTRACT +0 38 Retinoid-related orphan receptor gamma protein Retinoid-related orphan receptor gamma (RORγ) is a transcription factor belonging to a sub-family of nuclear receptors that includes two closely related members RORα and RORβ. INTRO +40 44 RORγ protein Retinoid-related orphan receptor gamma (RORγ) is a transcription factor belonging to a sub-family of nuclear receptors that includes two closely related members RORα and RORβ. INTRO +51 71 transcription factor protein_type Retinoid-related orphan receptor gamma (RORγ) is a transcription factor belonging to a sub-family of nuclear receptors that includes two closely related members RORα and RORβ. INTRO +101 118 nuclear receptors protein_type Retinoid-related orphan receptor gamma (RORγ) is a transcription factor belonging to a sub-family of nuclear receptors that includes two closely related members RORα and RORβ. INTRO +161 165 RORα protein Retinoid-related orphan receptor gamma (RORγ) is a transcription factor belonging to a sub-family of nuclear receptors that includes two closely related members RORα and RORβ. INTRO +170 174 RORβ protein Retinoid-related orphan receptor gamma (RORγ) is a transcription factor belonging to a sub-family of nuclear receptors that includes two closely related members RORα and RORβ. INTRO +68 72 RORs protein_type Even though a high degree of sequence similarity exists between the RORs, their functional roles in regulation for physiological processes involved in development and immunity are distinct. INTRO +20 24 RORγ protein During development, RORγ regulates the transcriptional genes involved in the functioning of multiple pro-inflammatory lymphocyte lineages including T helper cells (TH17cells) which are necessary for IL-17 production. INTRO +199 204 IL-17 protein_type During development, RORγ regulates the transcriptional genes involved in the functioning of multiple pro-inflammatory lymphocyte lineages including T helper cells (TH17cells) which are necessary for IL-17 production. INTRO +0 5 IL-17 protein_type IL-17 is a pro-inflammatory interleukin linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease; making its transcriptional regulation through RORγ an attractive therapeutic target. INTRO +28 39 interleukin protein_type IL-17 is a pro-inflammatory interleukin linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease; making its transcriptional regulation through RORγ an attractive therapeutic target. INTRO +197 201 RORγ protein IL-17 is a pro-inflammatory interleukin linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease; making its transcriptional regulation through RORγ an attractive therapeutic target. INTRO +0 4 RORγ protein RORγ consists of an N-terminal DNA binding domain (DBD) connected to a C-terminal ligand binding domain (LBD) via a flexible hinge region. INTRO +31 49 DNA binding domain structure_element RORγ consists of an N-terminal DNA binding domain (DBD) connected to a C-terminal ligand binding domain (LBD) via a flexible hinge region. INTRO +51 54 DBD structure_element RORγ consists of an N-terminal DNA binding domain (DBD) connected to a C-terminal ligand binding domain (LBD) via a flexible hinge region. INTRO +82 103 ligand binding domain structure_element RORγ consists of an N-terminal DNA binding domain (DBD) connected to a C-terminal ligand binding domain (LBD) via a flexible hinge region. INTRO +105 108 LBD structure_element RORγ consists of an N-terminal DNA binding domain (DBD) connected to a C-terminal ligand binding domain (LBD) via a flexible hinge region. INTRO +125 137 hinge region structure_element RORγ consists of an N-terminal DNA binding domain (DBD) connected to a C-terminal ligand binding domain (LBD) via a flexible hinge region. INTRO +4 7 DBD structure_element The DBD is composed of two zinc fingers that allow it to interact with specifically encoded regions on the DNA called the nuclear receptor response elements. INTRO +27 39 zinc fingers structure_element The DBD is composed of two zinc fingers that allow it to interact with specifically encoded regions on the DNA called the nuclear receptor response elements. INTRO +122 156 nuclear receptor response elements structure_element The DBD is composed of two zinc fingers that allow it to interact with specifically encoded regions on the DNA called the nuclear receptor response elements. INTRO +4 7 LBD structure_element The LBD consists of a coactivator protein binding pocket and a hydrophobic ligand binding site (LBS) which are responsible for regulating transcription. INTRO +22 56 coactivator protein binding pocket site The LBD consists of a coactivator protein binding pocket and a hydrophobic ligand binding site (LBS) which are responsible for regulating transcription. INTRO +63 94 hydrophobic ligand binding site site The LBD consists of a coactivator protein binding pocket and a hydrophobic ligand binding site (LBS) which are responsible for regulating transcription. INTRO +96 99 LBS site The LBD consists of a coactivator protein binding pocket and a hydrophobic ligand binding site (LBS) which are responsible for regulating transcription. INTRO +4 30 coactivator binding pocket site The coactivator binding pocket of RORγ recognizes a conserved helix motif LXXLL (where X can be any amino acid) on transcriptional coactivator complexes and recruits it to activate transcription. INTRO +34 38 RORγ protein The coactivator binding pocket of RORγ recognizes a conserved helix motif LXXLL (where X can be any amino acid) on transcriptional coactivator complexes and recruits it to activate transcription. INTRO +52 61 conserved protein_state The coactivator binding pocket of RORγ recognizes a conserved helix motif LXXLL (where X can be any amino acid) on transcriptional coactivator complexes and recruits it to activate transcription. INTRO +62 79 helix motif LXXLL structure_element The coactivator binding pocket of RORγ recognizes a conserved helix motif LXXLL (where X can be any amino acid) on transcriptional coactivator complexes and recruits it to activate transcription. INTRO +11 36 nuclear hormone receptors protein_type Like other nuclear hormone receptors, RORγ’s helix12 which makes up the C-termini of the LBD is an essential part of the coactivator binding pocket and is commonly referred to as the activation function helix 2 (AF2). INTRO +38 42 RORγ protein Like other nuclear hormone receptors, RORγ’s helix12 which makes up the C-termini of the LBD is an essential part of the coactivator binding pocket and is commonly referred to as the activation function helix 2 (AF2). INTRO +45 52 helix12 structure_element Like other nuclear hormone receptors, RORγ’s helix12 which makes up the C-termini of the LBD is an essential part of the coactivator binding pocket and is commonly referred to as the activation function helix 2 (AF2). INTRO +89 92 LBD structure_element Like other nuclear hormone receptors, RORγ’s helix12 which makes up the C-termini of the LBD is an essential part of the coactivator binding pocket and is commonly referred to as the activation function helix 2 (AF2). INTRO +121 147 coactivator binding pocket site Like other nuclear hormone receptors, RORγ’s helix12 which makes up the C-termini of the LBD is an essential part of the coactivator binding pocket and is commonly referred to as the activation function helix 2 (AF2). INTRO +183 210 activation function helix 2 structure_element Like other nuclear hormone receptors, RORγ’s helix12 which makes up the C-termini of the LBD is an essential part of the coactivator binding pocket and is commonly referred to as the activation function helix 2 (AF2). INTRO +212 215 AF2 structure_element Like other nuclear hormone receptors, RORγ’s helix12 which makes up the C-termini of the LBD is an essential part of the coactivator binding pocket and is commonly referred to as the activation function helix 2 (AF2). INTRO +3 7 RORγ protein In RORγ, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to π- π interactions between Tyr502 and Phe506. INTRO +33 42 AF2 helix structure_element In RORγ, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to π- π interactions between Tyr502 and Phe506. INTRO +64 90 coactivator binding pocket site In RORγ, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to π- π interactions between Tyr502 and Phe506. INTRO +108 119 salt bridge bond_interaction In RORγ, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to π- π interactions between Tyr502 and Phe506. INTRO +128 134 His479 residue_name_number In RORγ, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to π- π interactions between Tyr502 and Phe506. INTRO +139 145 Tyr502 residue_name_number In RORγ, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to π- π interactions between Tyr502 and Phe506. INTRO +161 178 π- π interactions bond_interaction In RORγ, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to π- π interactions between Tyr502 and Phe506. INTRO +187 193 Tyr502 residue_name_number In RORγ, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to π- π interactions between Tyr502 and Phe506. INTRO +198 204 Phe506 residue_name_number In RORγ, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to π- π interactions between Tyr502 and Phe506. INTRO +24 33 AF2 helix structure_element The conformation of the AF2 helix can be modulated through targeted ligands which bind the LBS and increase the binding of the coactivator protein (agonists) or disrupt binding (inverse agonists) thereby enhancing or inhibiting transcription. INTRO +91 94 LBS site The conformation of the AF2 helix can be modulated through targeted ligands which bind the LBS and increase the binding of the coactivator protein (agonists) or disrupt binding (inverse agonists) thereby enhancing or inhibiting transcription. INTRO +6 10 RORγ protein Since RORγ has been demonstrated to play an important role in pro-inflammatory gene expression patterns implicated in several major autoimmune diseases, our aim was to develop RORγ inverse agonists that would help down regulate pro-inflammatory gene transcription. INTRO +176 180 RORγ protein Since RORγ has been demonstrated to play an important role in pro-inflammatory gene expression patterns implicated in several major autoimmune diseases, our aim was to develop RORγ inverse agonists that would help down regulate pro-inflammatory gene transcription. INTRO +0 12 FRET results evidence FRET results for agonist BIO592 (a) and Inverse Agonist BIO399 (b) FIG +17 24 agonist protein_state FRET results for agonist BIO592 (a) and Inverse Agonist BIO399 (b) FIG +25 31 BIO592 chemical FRET results for agonist BIO592 (a) and Inverse Agonist BIO399 (b) FIG +40 55 Inverse Agonist protein_state FRET results for agonist BIO592 (a) and Inverse Agonist BIO399 (b) FIG +56 62 BIO399 chemical FRET results for agonist BIO592 (a) and Inverse Agonist BIO399 (b) FIG +52 65 benzoxazinone chemical Here we present the identification of two synthetic benzoxazinone RORγ ligands, a weak agonist BIO592 (Fig. 1a) and an inverse agonist BIO399 (Fig. 1b) which were identified using a Fluorescence Resonance Energy transfer (FRET) based assay that monitored coactivator peptide recruitment. INTRO +66 70 RORγ protein Here we present the identification of two synthetic benzoxazinone RORγ ligands, a weak agonist BIO592 (Fig. 1a) and an inverse agonist BIO399 (Fig. 1b) which were identified using a Fluorescence Resonance Energy transfer (FRET) based assay that monitored coactivator peptide recruitment. INTRO +87 94 agonist protein_state Here we present the identification of two synthetic benzoxazinone RORγ ligands, a weak agonist BIO592 (Fig. 1a) and an inverse agonist BIO399 (Fig. 1b) which were identified using a Fluorescence Resonance Energy transfer (FRET) based assay that monitored coactivator peptide recruitment. INTRO +95 101 BIO592 chemical Here we present the identification of two synthetic benzoxazinone RORγ ligands, a weak agonist BIO592 (Fig. 1a) and an inverse agonist BIO399 (Fig. 1b) which were identified using a Fluorescence Resonance Energy transfer (FRET) based assay that monitored coactivator peptide recruitment. INTRO +119 134 inverse agonist protein_state Here we present the identification of two synthetic benzoxazinone RORγ ligands, a weak agonist BIO592 (Fig. 1a) and an inverse agonist BIO399 (Fig. 1b) which were identified using a Fluorescence Resonance Energy transfer (FRET) based assay that monitored coactivator peptide recruitment. INTRO +135 141 BIO399 chemical Here we present the identification of two synthetic benzoxazinone RORγ ligands, a weak agonist BIO592 (Fig. 1a) and an inverse agonist BIO399 (Fig. 1b) which were identified using a Fluorescence Resonance Energy transfer (FRET) based assay that monitored coactivator peptide recruitment. INTRO +182 239 Fluorescence Resonance Energy transfer (FRET) based assay experimental_method Here we present the identification of two synthetic benzoxazinone RORγ ligands, a weak agonist BIO592 (Fig. 1a) and an inverse agonist BIO399 (Fig. 1b) which were identified using a Fluorescence Resonance Energy transfer (FRET) based assay that monitored coactivator peptide recruitment. INTRO +6 25 partial proteolysis experimental_method Using partial proteolysis in combination with mass spectrometry analysis we demonstrate that the AF2 helix of RORγ destabilizes upon BIO399 (inverse agonist) binding. INTRO +46 63 mass spectrometry experimental_method Using partial proteolysis in combination with mass spectrometry analysis we demonstrate that the AF2 helix of RORγ destabilizes upon BIO399 (inverse agonist) binding. INTRO +97 106 AF2 helix structure_element Using partial proteolysis in combination with mass spectrometry analysis we demonstrate that the AF2 helix of RORγ destabilizes upon BIO399 (inverse agonist) binding. INTRO +110 114 RORγ protein Using partial proteolysis in combination with mass spectrometry analysis we demonstrate that the AF2 helix of RORγ destabilizes upon BIO399 (inverse agonist) binding. INTRO +133 139 BIO399 chemical Using partial proteolysis in combination with mass spectrometry analysis we demonstrate that the AF2 helix of RORγ destabilizes upon BIO399 (inverse agonist) binding. INTRO +141 156 inverse agonist protein_state Using partial proteolysis in combination with mass spectrometry analysis we demonstrate that the AF2 helix of RORγ destabilizes upon BIO399 (inverse agonist) binding. INTRO +19 32 binding modes evidence Finally, comparing binding modes of our benzoxazinone RORγ crystal structures to other ROR structures, we hypothesize a new mode of action for achieving inverse agonism and selectivity. INTRO +40 53 benzoxazinone chemical Finally, comparing binding modes of our benzoxazinone RORγ crystal structures to other ROR structures, we hypothesize a new mode of action for achieving inverse agonism and selectivity. INTRO +54 58 RORγ protein Finally, comparing binding modes of our benzoxazinone RORγ crystal structures to other ROR structures, we hypothesize a new mode of action for achieving inverse agonism and selectivity. INTRO +59 77 crystal structures evidence Finally, comparing binding modes of our benzoxazinone RORγ crystal structures to other ROR structures, we hypothesize a new mode of action for achieving inverse agonism and selectivity. INTRO +87 90 ROR protein_type Finally, comparing binding modes of our benzoxazinone RORγ crystal structures to other ROR structures, we hypothesize a new mode of action for achieving inverse agonism and selectivity. INTRO +91 101 structures evidence Finally, comparing binding modes of our benzoxazinone RORγ crystal structures to other ROR structures, we hypothesize a new mode of action for achieving inverse agonism and selectivity. INTRO +8 24 FRET based assay experimental_method Using a FRET based assay we discovered agonist BIO592 (Fig. 1a) which increased the coactivator peptide TRAP220 recruitment to RORγ (EC50 0f 58nM and Emax of 130 %) and a potent inverse agonist BIO399 (Fig. 1b) which inhibited coactivator recruitment (IC50: 4.7nM). RESULTS +39 46 agonist protein_state Using a FRET based assay we discovered agonist BIO592 (Fig. 1a) which increased the coactivator peptide TRAP220 recruitment to RORγ (EC50 0f 58nM and Emax of 130 %) and a potent inverse agonist BIO399 (Fig. 1b) which inhibited coactivator recruitment (IC50: 4.7nM). RESULTS +47 53 BIO592 chemical Using a FRET based assay we discovered agonist BIO592 (Fig. 1a) which increased the coactivator peptide TRAP220 recruitment to RORγ (EC50 0f 58nM and Emax of 130 %) and a potent inverse agonist BIO399 (Fig. 1b) which inhibited coactivator recruitment (IC50: 4.7nM). RESULTS +104 111 TRAP220 chemical Using a FRET based assay we discovered agonist BIO592 (Fig. 1a) which increased the coactivator peptide TRAP220 recruitment to RORγ (EC50 0f 58nM and Emax of 130 %) and a potent inverse agonist BIO399 (Fig. 1b) which inhibited coactivator recruitment (IC50: 4.7nM). RESULTS +127 131 RORγ protein Using a FRET based assay we discovered agonist BIO592 (Fig. 1a) which increased the coactivator peptide TRAP220 recruitment to RORγ (EC50 0f 58nM and Emax of 130 %) and a potent inverse agonist BIO399 (Fig. 1b) which inhibited coactivator recruitment (IC50: 4.7nM). RESULTS +133 137 EC50 evidence Using a FRET based assay we discovered agonist BIO592 (Fig. 1a) which increased the coactivator peptide TRAP220 recruitment to RORγ (EC50 0f 58nM and Emax of 130 %) and a potent inverse agonist BIO399 (Fig. 1b) which inhibited coactivator recruitment (IC50: 4.7nM). RESULTS +150 154 Emax evidence Using a FRET based assay we discovered agonist BIO592 (Fig. 1a) which increased the coactivator peptide TRAP220 recruitment to RORγ (EC50 0f 58nM and Emax of 130 %) and a potent inverse agonist BIO399 (Fig. 1b) which inhibited coactivator recruitment (IC50: 4.7nM). RESULTS +178 193 inverse agonist protein_state Using a FRET based assay we discovered agonist BIO592 (Fig. 1a) which increased the coactivator peptide TRAP220 recruitment to RORγ (EC50 0f 58nM and Emax of 130 %) and a potent inverse agonist BIO399 (Fig. 1b) which inhibited coactivator recruitment (IC50: 4.7nM). RESULTS +194 200 BIO399 chemical Using a FRET based assay we discovered agonist BIO592 (Fig. 1a) which increased the coactivator peptide TRAP220 recruitment to RORγ (EC50 0f 58nM and Emax of 130 %) and a potent inverse agonist BIO399 (Fig. 1b) which inhibited coactivator recruitment (IC50: 4.7nM). RESULTS +252 256 IC50 evidence Using a FRET based assay we discovered agonist BIO592 (Fig. 1a) which increased the coactivator peptide TRAP220 recruitment to RORγ (EC50 0f 58nM and Emax of 130 %) and a potent inverse agonist BIO399 (Fig. 1b) which inhibited coactivator recruitment (IC50: 4.7nM). RESULTS +53 60 agonist protein_state Interestingly, the structural difference between the agonist BIO592 and inverse agonist BIO399 was minor; with the 2,3-dihydrobenzo[1,4]oxazepin-4-one ring system of BIO399 being 3 atoms larger than the benzo[1,4]oxazine-3-one ring system of BIO592. RESULTS +61 67 BIO592 chemical Interestingly, the structural difference between the agonist BIO592 and inverse agonist BIO399 was minor; with the 2,3-dihydrobenzo[1,4]oxazepin-4-one ring system of BIO399 being 3 atoms larger than the benzo[1,4]oxazine-3-one ring system of BIO592. RESULTS +72 87 inverse agonist protein_state Interestingly, the structural difference between the agonist BIO592 and inverse agonist BIO399 was minor; with the 2,3-dihydrobenzo[1,4]oxazepin-4-one ring system of BIO399 being 3 atoms larger than the benzo[1,4]oxazine-3-one ring system of BIO592. RESULTS +88 94 BIO399 chemical Interestingly, the structural difference between the agonist BIO592 and inverse agonist BIO399 was minor; with the 2,3-dihydrobenzo[1,4]oxazepin-4-one ring system of BIO399 being 3 atoms larger than the benzo[1,4]oxazine-3-one ring system of BIO592. RESULTS +115 150 2,3-dihydrobenzo[1,4]oxazepin-4-one chemical Interestingly, the structural difference between the agonist BIO592 and inverse agonist BIO399 was minor; with the 2,3-dihydrobenzo[1,4]oxazepin-4-one ring system of BIO399 being 3 atoms larger than the benzo[1,4]oxazine-3-one ring system of BIO592. RESULTS +166 172 BIO399 chemical Interestingly, the structural difference between the agonist BIO592 and inverse agonist BIO399 was minor; with the 2,3-dihydrobenzo[1,4]oxazepin-4-one ring system of BIO399 being 3 atoms larger than the benzo[1,4]oxazine-3-one ring system of BIO592. RESULTS +203 226 benzo[1,4]oxazine-3-one chemical Interestingly, the structural difference between the agonist BIO592 and inverse agonist BIO399 was minor; with the 2,3-dihydrobenzo[1,4]oxazepin-4-one ring system of BIO399 being 3 atoms larger than the benzo[1,4]oxazine-3-one ring system of BIO592. RESULTS +242 248 BIO592 chemical Interestingly, the structural difference between the agonist BIO592 and inverse agonist BIO399 was minor; with the 2,3-dihydrobenzo[1,4]oxazepin-4-one ring system of BIO399 being 3 atoms larger than the benzo[1,4]oxazine-3-one ring system of BIO592. RESULTS +152 158 BIO592 chemical In order to understand how small changes in the core ring system leads to inverse agonism, we wanted to structurally determine the binding mode of both BIO592 and BIO399 in the LBS of RORγ using x-ray crystallography. RESULTS +163 169 BIO399 chemical In order to understand how small changes in the core ring system leads to inverse agonism, we wanted to structurally determine the binding mode of both BIO592 and BIO399 in the LBS of RORγ using x-ray crystallography. RESULTS +177 180 LBS site In order to understand how small changes in the core ring system leads to inverse agonism, we wanted to structurally determine the binding mode of both BIO592 and BIO399 in the LBS of RORγ using x-ray crystallography. RESULTS +184 188 RORγ protein In order to understand how small changes in the core ring system leads to inverse agonism, we wanted to structurally determine the binding mode of both BIO592 and BIO399 in the LBS of RORγ using x-ray crystallography. RESULTS +195 216 x-ray crystallography experimental_method In order to understand how small changes in the core ring system leads to inverse agonism, we wanted to structurally determine the binding mode of both BIO592 and BIO399 in the LBS of RORγ using x-ray crystallography. RESULTS +0 9 Structure evidence Structure of the RORγ518-BIO592-EBI96 ternary complex is in a transcriptionally active conformation RESULTS +17 37 RORγ518-BIO592-EBI96 complex_assembly Structure of the RORγ518-BIO592-EBI96 ternary complex is in a transcriptionally active conformation RESULTS +80 86 active protein_state Structure of the RORγ518-BIO592-EBI96 ternary complex is in a transcriptionally active conformation RESULTS +7 24 ternary structure evidence a The ternary structure of RORγ518 BIO592 and EBI96. FIG +28 35 RORγ518 protein a The ternary structure of RORγ518 BIO592 and EBI96. FIG +36 42 BIO592 chemical a The ternary structure of RORγ518 BIO592 and EBI96. FIG +47 52 EBI96 chemical a The ternary structure of RORγ518 BIO592 and EBI96. FIG +2 6 RORγ protein b RORγ AF2 helix in the agonist conformation. FIG +7 16 AF2 helix structure_element b RORγ AF2 helix in the agonist conformation. FIG +24 31 agonist protein_state b RORγ AF2 helix in the agonist conformation. FIG +2 7 EBI96 chemical c EBI96 coactivator peptide bound in the coactivator pocket of RORγ FIG +28 36 bound in protein_state c EBI96 coactivator peptide bound in the coactivator pocket of RORγ FIG +41 59 coactivator pocket site c EBI96 coactivator peptide bound in the coactivator pocket of RORγ FIG +63 67 RORγ protein c EBI96 coactivator peptide bound in the coactivator pocket of RORγ FIG +0 7 RORγ518 protein RORγ518 bound to agonist BIO592 was crystallized with a truncated form of the coactivator peptide EBI96 to a resolution of 2.6 Å (Fig. 2a). RESULTS +8 16 bound to protein_state RORγ518 bound to agonist BIO592 was crystallized with a truncated form of the coactivator peptide EBI96 to a resolution of 2.6 Å (Fig. 2a). RESULTS +17 24 agonist protein_state RORγ518 bound to agonist BIO592 was crystallized with a truncated form of the coactivator peptide EBI96 to a resolution of 2.6 Å (Fig. 2a). RESULTS +25 31 BIO592 chemical RORγ518 bound to agonist BIO592 was crystallized with a truncated form of the coactivator peptide EBI96 to a resolution of 2.6 Å (Fig. 2a). RESULTS +36 48 crystallized experimental_method RORγ518 bound to agonist BIO592 was crystallized with a truncated form of the coactivator peptide EBI96 to a resolution of 2.6 Å (Fig. 2a). RESULTS +56 65 truncated protein_state RORγ518 bound to agonist BIO592 was crystallized with a truncated form of the coactivator peptide EBI96 to a resolution of 2.6 Å (Fig. 2a). RESULTS +98 103 EBI96 chemical RORγ518 bound to agonist BIO592 was crystallized with a truncated form of the coactivator peptide EBI96 to a resolution of 2.6 Å (Fig. 2a). RESULTS +4 13 structure evidence The structure of the ternary complex had features similar to other ROR agonist coactivator structures in a transcriptionally active canonical three layer helix fold with the AF2 helix in the agonist conformation. RESULTS +67 70 ROR protein_type The structure of the ternary complex had features similar to other ROR agonist coactivator structures in a transcriptionally active canonical three layer helix fold with the AF2 helix in the agonist conformation. RESULTS +71 78 agonist protein_state The structure of the ternary complex had features similar to other ROR agonist coactivator structures in a transcriptionally active canonical three layer helix fold with the AF2 helix in the agonist conformation. RESULTS +91 101 structures evidence The structure of the ternary complex had features similar to other ROR agonist coactivator structures in a transcriptionally active canonical three layer helix fold with the AF2 helix in the agonist conformation. RESULTS +107 131 transcriptionally active protein_state The structure of the ternary complex had features similar to other ROR agonist coactivator structures in a transcriptionally active canonical three layer helix fold with the AF2 helix in the agonist conformation. RESULTS +132 164 canonical three layer helix fold protein_state The structure of the ternary complex had features similar to other ROR agonist coactivator structures in a transcriptionally active canonical three layer helix fold with the AF2 helix in the agonist conformation. RESULTS +174 183 AF2 helix structure_element The structure of the ternary complex had features similar to other ROR agonist coactivator structures in a transcriptionally active canonical three layer helix fold with the AF2 helix in the agonist conformation. RESULTS +191 198 agonist protein_state The structure of the ternary complex had features similar to other ROR agonist coactivator structures in a transcriptionally active canonical three layer helix fold with the AF2 helix in the agonist conformation. RESULTS +4 11 agonist protein_state The agonist conformation is stabilized by a hydrogen bond between His479 and Tyr502, in addition to π-π interactions between His479, Tyr502 and Phe506 (Fig. 2b). RESULTS +44 57 hydrogen bond bond_interaction The agonist conformation is stabilized by a hydrogen bond between His479 and Tyr502, in addition to π-π interactions between His479, Tyr502 and Phe506 (Fig. 2b). RESULTS +66 72 His479 residue_name_number The agonist conformation is stabilized by a hydrogen bond between His479 and Tyr502, in addition to π-π interactions between His479, Tyr502 and Phe506 (Fig. 2b). RESULTS +77 83 Tyr502 residue_name_number The agonist conformation is stabilized by a hydrogen bond between His479 and Tyr502, in addition to π-π interactions between His479, Tyr502 and Phe506 (Fig. 2b). RESULTS +100 116 π-π interactions bond_interaction The agonist conformation is stabilized by a hydrogen bond between His479 and Tyr502, in addition to π-π interactions between His479, Tyr502 and Phe506 (Fig. 2b). RESULTS +125 131 His479 residue_name_number The agonist conformation is stabilized by a hydrogen bond between His479 and Tyr502, in addition to π-π interactions between His479, Tyr502 and Phe506 (Fig. 2b). RESULTS +133 139 Tyr502 residue_name_number The agonist conformation is stabilized by a hydrogen bond between His479 and Tyr502, in addition to π-π interactions between His479, Tyr502 and Phe506 (Fig. 2b). RESULTS +144 150 Phe506 residue_name_number The agonist conformation is stabilized by a hydrogen bond between His479 and Tyr502, in addition to π-π interactions between His479, Tyr502 and Phe506 (Fig. 2b). RESULTS +4 17 hydrogen bond bond_interaction The hydrogen bond between His479 and Tyr502 has been reported to be critical for RORγ agonist activity. RESULTS +26 32 His479 residue_name_number The hydrogen bond between His479 and Tyr502 has been reported to be critical for RORγ agonist activity. RESULTS +37 43 Tyr502 residue_name_number The hydrogen bond between His479 and Tyr502 has been reported to be critical for RORγ agonist activity. RESULTS +81 85 RORγ protein The hydrogen bond between His479 and Tyr502 has been reported to be critical for RORγ agonist activity. RESULTS +86 93 agonist protein_state The hydrogen bond between His479 and Tyr502 has been reported to be critical for RORγ agonist activity. RESULTS +36 47 mutagenesis experimental_method Disrupting this interaction through mutagenesis reduced transcriptional activity of RORγ. RESULTS +84 88 RORγ protein Disrupting this interaction through mutagenesis reduced transcriptional activity of RORγ. RESULTS +82 91 AF2 helix structure_element This reduced transcriptional activity has been attributed to the inability of the AF2 helix to complete the formation of the coactivator binding pocket necessary for coactivator proteins to bind. RESULTS +125 151 coactivator binding pocket site This reduced transcriptional activity has been attributed to the inability of the AF2 helix to complete the formation of the coactivator binding pocket necessary for coactivator proteins to bind. RESULTS +0 16 Electron density evidence Electron density for the coactivator peptide EBI96 was observed for residues EFPYLLSLLG which formed a α-helix stabilized through hydrophobic interactions with the coactivator binding pocket on RORγ (Fig. 2c). RESULTS +45 50 EBI96 chemical Electron density for the coactivator peptide EBI96 was observed for residues EFPYLLSLLG which formed a α-helix stabilized through hydrophobic interactions with the coactivator binding pocket on RORγ (Fig. 2c). RESULTS +77 87 EFPYLLSLLG structure_element Electron density for the coactivator peptide EBI96 was observed for residues EFPYLLSLLG which formed a α-helix stabilized through hydrophobic interactions with the coactivator binding pocket on RORγ (Fig. 2c). RESULTS +103 110 α-helix structure_element Electron density for the coactivator peptide EBI96 was observed for residues EFPYLLSLLG which formed a α-helix stabilized through hydrophobic interactions with the coactivator binding pocket on RORγ (Fig. 2c). RESULTS +130 154 hydrophobic interactions bond_interaction Electron density for the coactivator peptide EBI96 was observed for residues EFPYLLSLLG which formed a α-helix stabilized through hydrophobic interactions with the coactivator binding pocket on RORγ (Fig. 2c). RESULTS +164 190 coactivator binding pocket site Electron density for the coactivator peptide EBI96 was observed for residues EFPYLLSLLG which formed a α-helix stabilized through hydrophobic interactions with the coactivator binding pocket on RORγ (Fig. 2c). RESULTS +194 198 RORγ protein Electron density for the coactivator peptide EBI96 was observed for residues EFPYLLSLLG which formed a α-helix stabilized through hydrophobic interactions with the coactivator binding pocket on RORγ (Fig. 2c). RESULTS +49 58 conserved protein_state This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. RESULTS +59 72 charged clamp structure_element This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. RESULTS +103 107 Tyr7 residue_name_number This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. RESULTS +124 129 Leu11 residue_name_number This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. RESULTS +133 138 EBI96 chemical This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. RESULTS +144 158 hydrogen bonds bond_interaction This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. RESULTS +164 170 Glu504 residue_name_number This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. RESULTS +172 179 helix12 structure_element This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. RESULTS +185 191 Lys336 residue_name_number This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. RESULTS +193 199 helix3 structure_element This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. RESULTS +204 208 RORγ protein This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. RESULTS +18 31 charged clamp structure_element Formation of this charged clamp is essential for RORγ’s function for playing a role in transcriptional activation and this has been corroborated through mutagenic studies in this region. RESULTS +49 53 RORγ protein Formation of this charged clamp is essential for RORγ’s function for playing a role in transcriptional activation and this has been corroborated through mutagenic studies in this region. RESULTS +153 170 mutagenic studies experimental_method Formation of this charged clamp is essential for RORγ’s function for playing a role in transcriptional activation and this has been corroborated through mutagenic studies in this region. RESULTS +0 6 BIO592 chemical BIO592 binds in a collapsed conformation stabilizing the agonist conformation of RORγ RESULTS +18 27 collapsed protein_state BIO592 binds in a collapsed conformation stabilizing the agonist conformation of RORγ RESULTS +57 64 agonist protein_state BIO592 binds in a collapsed conformation stabilizing the agonist conformation of RORγ RESULTS +81 85 RORγ protein BIO592 binds in a collapsed conformation stabilizing the agonist conformation of RORγ RESULTS +29 36 agonist protein_state a Collapsed binding mode of agonist BIO592 in the hydrophobic LBS of RORγ. FIG +37 43 BIO592 chemical a Collapsed binding mode of agonist BIO592 in the hydrophobic LBS of RORγ. FIG +63 66 LBS site a Collapsed binding mode of agonist BIO592 in the hydrophobic LBS of RORγ. FIG +70 74 RORγ protein a Collapsed binding mode of agonist BIO592 in the hydrophobic LBS of RORγ. FIG +2 15 Benzoxazinone chemical b Benzoxazinone ring system of agonist BIO592 packing against His479 of RORγ stabilizing agonist conformation of the AF2 helix FIG +31 38 agonist protein_state b Benzoxazinone ring system of agonist BIO592 packing against His479 of RORγ stabilizing agonist conformation of the AF2 helix FIG +39 45 BIO592 chemical b Benzoxazinone ring system of agonist BIO592 packing against His479 of RORγ stabilizing agonist conformation of the AF2 helix FIG +62 68 His479 residue_name_number b Benzoxazinone ring system of agonist BIO592 packing against His479 of RORγ stabilizing agonist conformation of the AF2 helix FIG +72 76 RORγ protein b Benzoxazinone ring system of agonist BIO592 packing against His479 of RORγ stabilizing agonist conformation of the AF2 helix FIG +89 96 agonist protein_state b Benzoxazinone ring system of agonist BIO592 packing against His479 of RORγ stabilizing agonist conformation of the AF2 helix FIG +117 126 AF2 helix structure_element b Benzoxazinone ring system of agonist BIO592 packing against His479 of RORγ stabilizing agonist conformation of the AF2 helix FIG +0 6 BIO592 chemical BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +7 15 bound in protein_state BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +18 27 collapsed protein_state BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +56 59 LBS site BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +63 67 RORγ protein BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +77 83 xylene chemical BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +121 127 pocket site BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +135 159 hydrophobic interactions bond_interaction BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +165 171 Val376 residue_name_number BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +173 179 Phe378 residue_name_number BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +181 187 Phe388 residue_name_number BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +192 198 Phe401 residue_name_number BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +209 228 ethyl-benzoxazinone chemical BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +249 273 hydrophobic interactions bond_interaction BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +279 285 Trp317 residue_name_number BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +287 293 Leu324 residue_name_number BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +295 301 Met358 residue_name_number BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +303 309 Leu391 residue_name_number BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +311 318 Ile 400 residue_name_number BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +323 329 His479 residue_name_number BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). RESULTS +4 12 sulfonyl chemical The sulfonyl group faces the entrance of the pocket, while the CF3 makes a hydrophobic contact with Ala327. RESULTS +45 51 pocket site The sulfonyl group faces the entrance of the pocket, while the CF3 makes a hydrophobic contact with Ala327. RESULTS +75 94 hydrophobic contact bond_interaction The sulfonyl group faces the entrance of the pocket, while the CF3 makes a hydrophobic contact with Ala327. RESULTS +100 106 Ala327 residue_name_number The sulfonyl group faces the entrance of the pocket, while the CF3 makes a hydrophobic contact with Ala327. RESULTS +0 23 Hydrophobic interaction bond_interaction Hydrophobic interaction between the ethyl group of the benzoxazinone and His479 reinforce the His479 sidechain position for making the hydrogen bond with Tyr502 thereby stabilizing the agonist conformation (Fig. 3b). RESULTS +55 68 benzoxazinone chemical Hydrophobic interaction between the ethyl group of the benzoxazinone and His479 reinforce the His479 sidechain position for making the hydrogen bond with Tyr502 thereby stabilizing the agonist conformation (Fig. 3b). RESULTS +73 79 His479 residue_name_number Hydrophobic interaction between the ethyl group of the benzoxazinone and His479 reinforce the His479 sidechain position for making the hydrogen bond with Tyr502 thereby stabilizing the agonist conformation (Fig. 3b). RESULTS +94 100 His479 residue_name_number Hydrophobic interaction between the ethyl group of the benzoxazinone and His479 reinforce the His479 sidechain position for making the hydrogen bond with Tyr502 thereby stabilizing the agonist conformation (Fig. 3b). RESULTS +135 148 hydrogen bond bond_interaction Hydrophobic interaction between the ethyl group of the benzoxazinone and His479 reinforce the His479 sidechain position for making the hydrogen bond with Tyr502 thereby stabilizing the agonist conformation (Fig. 3b). RESULTS +154 160 Tyr502 residue_name_number Hydrophobic interaction between the ethyl group of the benzoxazinone and His479 reinforce the His479 sidechain position for making the hydrogen bond with Tyr502 thereby stabilizing the agonist conformation (Fig. 3b). RESULTS +185 192 agonist protein_state Hydrophobic interaction between the ethyl group of the benzoxazinone and His479 reinforce the His479 sidechain position for making the hydrogen bond with Tyr502 thereby stabilizing the agonist conformation (Fig. 3b). RESULTS +0 4 RORγ protein RORγ AF2 helix is sensitive to proteolysis in the presence of Inverse Agonist BIO399 RESULTS +5 14 AF2 helix structure_element RORγ AF2 helix is sensitive to proteolysis in the presence of Inverse Agonist BIO399 RESULTS +50 61 presence of protein_state RORγ AF2 helix is sensitive to proteolysis in the presence of Inverse Agonist BIO399 RESULTS +62 77 Inverse Agonist protein_state RORγ AF2 helix is sensitive to proteolysis in the presence of Inverse Agonist BIO399 RESULTS +78 84 BIO399 chemical RORγ AF2 helix is sensitive to proteolysis in the presence of Inverse Agonist BIO399 RESULTS +19 37 co-crystallization experimental_method Next, we attempted co-crystallization with the inverse agonist BIO399. RESULTS +47 62 inverse agonist protein_state Next, we attempted co-crystallization with the inverse agonist BIO399. RESULTS +63 69 BIO399 chemical Next, we attempted co-crystallization with the inverse agonist BIO399. RESULTS +19 34 crystallization experimental_method However, extensive crystallization efforts with BIO399 and RORγ518 or other AF2 intact constructs did not produce crystals. RESULTS +48 54 BIO399 chemical However, extensive crystallization efforts with BIO399 and RORγ518 or other AF2 intact constructs did not produce crystals. RESULTS +59 66 RORγ518 protein However, extensive crystallization efforts with BIO399 and RORγ518 or other AF2 intact constructs did not produce crystals. RESULTS +76 79 AF2 structure_element However, extensive crystallization efforts with BIO399 and RORγ518 or other AF2 intact constructs did not produce crystals. RESULTS +80 86 intact protein_state However, extensive crystallization efforts with BIO399 and RORγ518 or other AF2 intact constructs did not produce crystals. RESULTS +114 122 crystals evidence However, extensive crystallization efforts with BIO399 and RORγ518 or other AF2 intact constructs did not produce crystals. RESULTS +25 32 RORγ518 protein We hypothesized that the RORγ518 coactivator peptide interaction in the FRET assay was disrupted upon BIO399 binding and that a conformational rearrangement of the AF2 helix could have occurred, hindering crystallization. RESULTS +72 82 FRET assay experimental_method We hypothesized that the RORγ518 coactivator peptide interaction in the FRET assay was disrupted upon BIO399 binding and that a conformational rearrangement of the AF2 helix could have occurred, hindering crystallization. RESULTS +102 108 BIO399 chemical We hypothesized that the RORγ518 coactivator peptide interaction in the FRET assay was disrupted upon BIO399 binding and that a conformational rearrangement of the AF2 helix could have occurred, hindering crystallization. RESULTS +164 173 AF2 helix structure_element We hypothesized that the RORγ518 coactivator peptide interaction in the FRET assay was disrupted upon BIO399 binding and that a conformational rearrangement of the AF2 helix could have occurred, hindering crystallization. RESULTS +205 220 crystallization experimental_method We hypothesized that the RORγ518 coactivator peptide interaction in the FRET assay was disrupted upon BIO399 binding and that a conformational rearrangement of the AF2 helix could have occurred, hindering crystallization. RESULTS +34 41 RORγ518 protein Specific proteolytic positions on RORγ518 when treated with Actinase E alone (Green) or in the presence of BIO399 (Red) and shared proteolytic sites (Yellow) FIG +47 59 treated with experimental_method Specific proteolytic positions on RORγ518 when treated with Actinase E alone (Green) or in the presence of BIO399 (Red) and shared proteolytic sites (Yellow) FIG +60 70 Actinase E protein Specific proteolytic positions on RORγ518 when treated with Actinase E alone (Green) or in the presence of BIO399 (Red) and shared proteolytic sites (Yellow) FIG +95 106 presence of protein_state Specific proteolytic positions on RORγ518 when treated with Actinase E alone (Green) or in the presence of BIO399 (Red) and shared proteolytic sites (Yellow) FIG +107 113 BIO399 chemical Specific proteolytic positions on RORγ518 when treated with Actinase E alone (Green) or in the presence of BIO399 (Red) and shared proteolytic sites (Yellow) FIG +131 148 proteolytic sites site Specific proteolytic positions on RORγ518 when treated with Actinase E alone (Green) or in the presence of BIO399 (Red) and shared proteolytic sites (Yellow) FIG +21 30 AF2 helix structure_element The unfolding of the AF2 helix has been observed for other nuclear hormone receptors when bound to an inverse agonist or antagonist. RESULTS +59 84 nuclear hormone receptors protein_type The unfolding of the AF2 helix has been observed for other nuclear hormone receptors when bound to an inverse agonist or antagonist. RESULTS +90 98 bound to protein_state The unfolding of the AF2 helix has been observed for other nuclear hormone receptors when bound to an inverse agonist or antagonist. RESULTS +102 117 inverse agonist protein_state The unfolding of the AF2 helix has been observed for other nuclear hormone receptors when bound to an inverse agonist or antagonist. RESULTS +8 27 partial proteolysis experimental_method We used partial proteolysis in combination with mass spectrometry to determine if BIO399 was causing the AF2 helix to unfold. RESULTS +48 65 mass spectrometry experimental_method We used partial proteolysis in combination with mass spectrometry to determine if BIO399 was causing the AF2 helix to unfold. RESULTS +82 88 BIO399 chemical We used partial proteolysis in combination with mass spectrometry to determine if BIO399 was causing the AF2 helix to unfold. RESULTS +105 114 AF2 helix structure_element We used partial proteolysis in combination with mass spectrometry to determine if BIO399 was causing the AF2 helix to unfold. RESULTS +15 37 Actinase E proteolysis experimental_method Results of the Actinase E proteolysis experiments on RORγ518, the ternary complex of RORγ518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis. RESULTS +53 60 RORγ518 protein Results of the Actinase E proteolysis experiments on RORγ518, the ternary complex of RORγ518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis. RESULTS +85 92 RORγ518 protein Results of the Actinase E proteolysis experiments on RORγ518, the ternary complex of RORγ518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis. RESULTS +98 105 agonist protein_state Results of the Actinase E proteolysis experiments on RORγ518, the ternary complex of RORγ518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis. RESULTS +106 112 BIO592 chemical Results of the Actinase E proteolysis experiments on RORγ518, the ternary complex of RORγ518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis. RESULTS +129 134 EBI96 chemical Results of the Actinase E proteolysis experiments on RORγ518, the ternary complex of RORγ518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis. RESULTS +146 157 presence of protein_state Results of the Actinase E proteolysis experiments on RORγ518, the ternary complex of RORγ518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis. RESULTS +158 173 inverse agonist protein_state Results of the Actinase E proteolysis experiments on RORγ518, the ternary complex of RORγ518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis. RESULTS +174 180 BIO399 chemical Results of the Actinase E proteolysis experiments on RORγ518, the ternary complex of RORγ518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis. RESULTS +16 37 fragmentation pattern evidence Analysis of the fragmentation pattern showed minimal proteolytic removal of the AF2 helix by Actinase E on RORγ518 alone (ending at 504 to 506) and the ternary complex remained primarily intact (ending at 515/518) (Additional file 4). RESULTS +80 89 AF2 helix structure_element Analysis of the fragmentation pattern showed minimal proteolytic removal of the AF2 helix by Actinase E on RORγ518 alone (ending at 504 to 506) and the ternary complex remained primarily intact (ending at 515/518) (Additional file 4). RESULTS +93 103 Actinase E protein Analysis of the fragmentation pattern showed minimal proteolytic removal of the AF2 helix by Actinase E on RORγ518 alone (ending at 504 to 506) and the ternary complex remained primarily intact (ending at 515/518) (Additional file 4). RESULTS +107 114 RORγ518 protein Analysis of the fragmentation pattern showed minimal proteolytic removal of the AF2 helix by Actinase E on RORγ518 alone (ending at 504 to 506) and the ternary complex remained primarily intact (ending at 515/518) (Additional file 4). RESULTS +132 142 504 to 506 residue_range Analysis of the fragmentation pattern showed minimal proteolytic removal of the AF2 helix by Actinase E on RORγ518 alone (ending at 504 to 506) and the ternary complex remained primarily intact (ending at 515/518) (Additional file 4). RESULTS +152 167 ternary complex protein_state Analysis of the fragmentation pattern showed minimal proteolytic removal of the AF2 helix by Actinase E on RORγ518 alone (ending at 504 to 506) and the ternary complex remained primarily intact (ending at 515/518) (Additional file 4). RESULTS +205 208 515 residue_number Analysis of the fragmentation pattern showed minimal proteolytic removal of the AF2 helix by Actinase E on RORγ518 alone (ending at 504 to 506) and the ternary complex remained primarily intact (ending at 515/518) (Additional file 4). RESULTS +209 212 518 residue_number Analysis of the fragmentation pattern showed minimal proteolytic removal of the AF2 helix by Actinase E on RORγ518 alone (ending at 504 to 506) and the ternary complex remained primarily intact (ending at 515/518) (Additional file 4). RESULTS +16 27 presence of protein_state However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig. 4, Additional file 5). RESULTS +28 43 inverse agonist protein_state However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig. 4, Additional file 5). RESULTS +44 50 BIO399 chemical However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig. 4, Additional file 5). RESULTS +56 75 proteolytic pattern evidence However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig. 4, Additional file 5). RESULTS +178 181 494 residue_number However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig. 4, Additional file 5). RESULTS +182 185 495 residue_number However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig. 4, Additional file 5). RESULTS +226 235 AF2 helix structure_element However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig. 4, Additional file 5). RESULTS +259 262 APO protein_state However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig. 4, Additional file 5). RESULTS +266 289 ternary agonist complex protein_state However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig. 4, Additional file 5). RESULTS +18 35 cocrystallization experimental_method Several rounds of cocrystallization attempts with RORγ518 or other RORγ AF2 helix containing constructs complexed with BIO399 had not produced crystals. RESULTS +50 57 RORγ518 protein Several rounds of cocrystallization attempts with RORγ518 or other RORγ AF2 helix containing constructs complexed with BIO399 had not produced crystals. RESULTS +67 71 RORγ protein Several rounds of cocrystallization attempts with RORγ518 or other RORγ AF2 helix containing constructs complexed with BIO399 had not produced crystals. RESULTS +72 81 AF2 helix structure_element Several rounds of cocrystallization attempts with RORγ518 or other RORγ AF2 helix containing constructs complexed with BIO399 had not produced crystals. RESULTS +104 118 complexed with protein_state Several rounds of cocrystallization attempts with RORγ518 or other RORγ AF2 helix containing constructs complexed with BIO399 had not produced crystals. RESULTS +119 125 BIO399 chemical Several rounds of cocrystallization attempts with RORγ518 or other RORγ AF2 helix containing constructs complexed with BIO399 had not produced crystals. RESULTS +143 151 crystals evidence Several rounds of cocrystallization attempts with RORγ518 or other RORγ AF2 helix containing constructs complexed with BIO399 had not produced crystals. RESULTS +36 44 crystals evidence We attributed the inability to form crystals to the unfolding of the AF2 helix induced by BIO399. RESULTS +69 78 AF2 helix structure_element We attributed the inability to form crystals to the unfolding of the AF2 helix induced by BIO399. RESULTS +90 96 BIO399 chemical We attributed the inability to form crystals to the unfolding of the AF2 helix induced by BIO399. RESULTS +40 48 unfolded protein_state We reasoned that if we could remove the unfolded AF2 helix using proteolysis we could produce a binary complex more amenable to crystallization. RESULTS +49 58 AF2 helix structure_element We reasoned that if we could remove the unfolded AF2 helix using proteolysis we could produce a binary complex more amenable to crystallization. RESULTS +65 76 proteolysis experimental_method We reasoned that if we could remove the unfolded AF2 helix using proteolysis we could produce a binary complex more amenable to crystallization. RESULTS +128 143 crystallization experimental_method We reasoned that if we could remove the unfolded AF2 helix using proteolysis we could produce a binary complex more amenable to crystallization. RESULTS +0 13 AF2 truncated protein_state AF2 truncated RORγ BIO399 complex is more amenable to crystallization RESULTS +14 25 RORγ BIO399 complex_assembly AF2 truncated RORγ BIO399 complex is more amenable to crystallization RESULTS +54 69 crystallization experimental_method AF2 truncated RORγ BIO399 complex is more amenable to crystallization RESULTS +14 23 structure evidence a The binary structure of AF2-truncated RORγ and BIO399. FIG +27 40 AF2-truncated protein_state a The binary structure of AF2-truncated RORγ and BIO399. FIG +41 45 RORγ protein a The binary structure of AF2-truncated RORγ and BIO399. FIG +50 56 BIO399 chemical a The binary structure of AF2-truncated RORγ and BIO399. FIG +6 19 superposition experimental_method b The superposition of inverse agonist BIO399 (Cyan) and agonist BIO592 (Green). FIG +23 38 inverse agonist protein_state b The superposition of inverse agonist BIO399 (Cyan) and agonist BIO592 (Green). FIG +39 45 BIO399 chemical b The superposition of inverse agonist BIO399 (Cyan) and agonist BIO592 (Green). FIG +57 64 agonist protein_state b The superposition of inverse agonist BIO399 (Cyan) and agonist BIO592 (Green). FIG +65 71 BIO592 chemical b The superposition of inverse agonist BIO399 (Cyan) and agonist BIO592 (Green). FIG +14 20 Met358 residue_name_number c Movement of Met358 and His479 in the BIO399 (Cyan) and BIO592 (Green) structures FIG +25 31 His479 residue_name_number c Movement of Met358 and His479 in the BIO399 (Cyan) and BIO592 (Green) structures FIG +39 45 BIO399 chemical c Movement of Met358 and His479 in the BIO399 (Cyan) and BIO592 (Green) structures FIG +57 63 BIO592 chemical c Movement of Met358 and His479 in the BIO399 (Cyan) and BIO592 (Green) structures FIG +72 82 structures evidence c Movement of Met358 and His479 in the BIO399 (Cyan) and BIO592 (Green) structures FIG +4 14 Actinase E protein The Actinase E treated RORγ518 BIO399 ternary complex (aeRORγ493/4) co-crystallized readily in several PEG based conditions. RESULTS +23 37 RORγ518 BIO399 complex_assembly The Actinase E treated RORγ518 BIO399 ternary complex (aeRORγ493/4) co-crystallized readily in several PEG based conditions. RESULTS +55 66 aeRORγ493/4 complex_assembly The Actinase E treated RORγ518 BIO399 ternary complex (aeRORγ493/4) co-crystallized readily in several PEG based conditions. RESULTS +68 83 co-crystallized experimental_method The Actinase E treated RORγ518 BIO399 ternary complex (aeRORγ493/4) co-crystallized readily in several PEG based conditions. RESULTS +4 13 structure evidence The structure of aeRORγ493/4 BIO399 complex was solved to 2.3 Å and adopted a similar core fold to the BIO592 agonist crystal structure (Fig. 5a, Additional file 3). RESULTS +17 35 aeRORγ493/4 BIO399 complex_assembly The structure of aeRORγ493/4 BIO399 complex was solved to 2.3 Å and adopted a similar core fold to the BIO592 agonist crystal structure (Fig. 5a, Additional file 3). RESULTS +48 54 solved experimental_method The structure of aeRORγ493/4 BIO399 complex was solved to 2.3 Å and adopted a similar core fold to the BIO592 agonist crystal structure (Fig. 5a, Additional file 3). RESULTS +103 109 BIO592 chemical The structure of aeRORγ493/4 BIO399 complex was solved to 2.3 Å and adopted a similar core fold to the BIO592 agonist crystal structure (Fig. 5a, Additional file 3). RESULTS +110 117 agonist protein_state The structure of aeRORγ493/4 BIO399 complex was solved to 2.3 Å and adopted a similar core fold to the BIO592 agonist crystal structure (Fig. 5a, Additional file 3). RESULTS +118 135 crystal structure evidence The structure of aeRORγ493/4 BIO399 complex was solved to 2.3 Å and adopted a similar core fold to the BIO592 agonist crystal structure (Fig. 5a, Additional file 3). RESULTS +4 22 aeRORγ493/4 BIO399 complex_assembly The aeRORγ493/4 BIO399 structure diverged at the c-terminal end of Helix 11 from the RORγ518 BIO592 EBI96 structure, where helix 11 unwinds into a random coil after residue L475. RESULTS +23 32 structure evidence The aeRORγ493/4 BIO399 structure diverged at the c-terminal end of Helix 11 from the RORγ518 BIO592 EBI96 structure, where helix 11 unwinds into a random coil after residue L475. RESULTS +67 75 Helix 11 structure_element The aeRORγ493/4 BIO399 structure diverged at the c-terminal end of Helix 11 from the RORγ518 BIO592 EBI96 structure, where helix 11 unwinds into a random coil after residue L475. RESULTS +85 105 RORγ518 BIO592 EBI96 complex_assembly The aeRORγ493/4 BIO399 structure diverged at the c-terminal end of Helix 11 from the RORγ518 BIO592 EBI96 structure, where helix 11 unwinds into a random coil after residue L475. RESULTS +106 115 structure evidence The aeRORγ493/4 BIO399 structure diverged at the c-terminal end of Helix 11 from the RORγ518 BIO592 EBI96 structure, where helix 11 unwinds into a random coil after residue L475. RESULTS +123 131 helix 11 structure_element The aeRORγ493/4 BIO399 structure diverged at the c-terminal end of Helix 11 from the RORγ518 BIO592 EBI96 structure, where helix 11 unwinds into a random coil after residue L475. RESULTS +173 177 L475 residue_name_number The aeRORγ493/4 BIO399 structure diverged at the c-terminal end of Helix 11 from the RORγ518 BIO592 EBI96 structure, where helix 11 unwinds into a random coil after residue L475. RESULTS +0 15 Inverse agonist protein_state Inverse agonist BIO399 uses Met358 as a trigger for inverse agonism RESULTS +16 22 BIO399 chemical Inverse agonist BIO399 uses Met358 as a trigger for inverse agonism RESULTS +28 34 Met358 residue_name_number Inverse agonist BIO399 uses Met358 as a trigger for inverse agonism RESULTS +0 6 BIO399 chemical BIO399 binds to the ligand binding site of RORγ adopting a collapsed conformation as seen with BIO592 where the two compounds superimpose with an RMSD of 0.72 Å (Fig. 5b). RESULTS +20 39 ligand binding site site BIO399 binds to the ligand binding site of RORγ adopting a collapsed conformation as seen with BIO592 where the two compounds superimpose with an RMSD of 0.72 Å (Fig. 5b). RESULTS +43 47 RORγ protein BIO399 binds to the ligand binding site of RORγ adopting a collapsed conformation as seen with BIO592 where the two compounds superimpose with an RMSD of 0.72 Å (Fig. 5b). RESULTS +59 68 collapsed protein_state BIO399 binds to the ligand binding site of RORγ adopting a collapsed conformation as seen with BIO592 where the two compounds superimpose with an RMSD of 0.72 Å (Fig. 5b). RESULTS +95 101 BIO592 chemical BIO399 binds to the ligand binding site of RORγ adopting a collapsed conformation as seen with BIO592 where the two compounds superimpose with an RMSD of 0.72 Å (Fig. 5b). RESULTS +126 137 superimpose experimental_method BIO399 binds to the ligand binding site of RORγ adopting a collapsed conformation as seen with BIO592 where the two compounds superimpose with an RMSD of 0.72 Å (Fig. 5b). RESULTS +146 150 RMSD evidence BIO399 binds to the ligand binding site of RORγ adopting a collapsed conformation as seen with BIO592 where the two compounds superimpose with an RMSD of 0.72 Å (Fig. 5b). RESULTS +46 52 BIO399 chemical The majority of the side chains within 4 Å of BIO399 and BIO592 adopt similar rotomer conformations with the exceptions of Met358 and His479 (Fig. 5c). RESULTS +57 63 BIO592 chemical The majority of the side chains within 4 Å of BIO399 and BIO592 adopt similar rotomer conformations with the exceptions of Met358 and His479 (Fig. 5c). RESULTS +123 129 Met358 residue_name_number The majority of the side chains within 4 Å of BIO399 and BIO592 adopt similar rotomer conformations with the exceptions of Met358 and His479 (Fig. 5c). RESULTS +134 140 His479 residue_name_number The majority of the side chains within 4 Å of BIO399 and BIO592 adopt similar rotomer conformations with the exceptions of Met358 and His479 (Fig. 5c). RESULTS +4 26 difference density map evidence The difference density map showed clear positive density for Met358 in an alternate rotomer conformation compared to the one observed in the molecular replacement model or the other agonist containing models (Additional file 6). RESULTS +40 56 positive density evidence The difference density map showed clear positive density for Met358 in an alternate rotomer conformation compared to the one observed in the molecular replacement model or the other agonist containing models (Additional file 6). RESULTS +61 67 Met358 residue_name_number The difference density map showed clear positive density for Met358 in an alternate rotomer conformation compared to the one observed in the molecular replacement model or the other agonist containing models (Additional file 6). RESULTS +141 168 molecular replacement model experimental_method The difference density map showed clear positive density for Met358 in an alternate rotomer conformation compared to the one observed in the molecular replacement model or the other agonist containing models (Additional file 6). RESULTS +182 189 agonist protein_state The difference density map showed clear positive density for Met358 in an alternate rotomer conformation compared to the one observed in the molecular replacement model or the other agonist containing models (Additional file 6). RESULTS +19 25 Met358 residue_name_number We tried to refine Met358 in the same conformation as the molecular replacement model or the other agonist containing models, but the results clearly indicated that this was not possible, thus confirming the new rotamer conformation for the Met358 sidechain in the inverse agonist bound structure. RESULTS +58 85 molecular replacement model experimental_method We tried to refine Met358 in the same conformation as the molecular replacement model or the other agonist containing models, but the results clearly indicated that this was not possible, thus confirming the new rotamer conformation for the Met358 sidechain in the inverse agonist bound structure. RESULTS +99 106 agonist protein_state We tried to refine Met358 in the same conformation as the molecular replacement model or the other agonist containing models, but the results clearly indicated that this was not possible, thus confirming the new rotamer conformation for the Met358 sidechain in the inverse agonist bound structure. RESULTS +241 247 Met358 residue_name_number We tried to refine Met358 in the same conformation as the molecular replacement model or the other agonist containing models, but the results clearly indicated that this was not possible, thus confirming the new rotamer conformation for the Met358 sidechain in the inverse agonist bound structure. RESULTS +265 286 inverse agonist bound protein_state We tried to refine Met358 in the same conformation as the molecular replacement model or the other agonist containing models, but the results clearly indicated that this was not possible, thus confirming the new rotamer conformation for the Met358 sidechain in the inverse agonist bound structure. RESULTS +287 296 structure evidence We tried to refine Met358 in the same conformation as the molecular replacement model or the other agonist containing models, but the results clearly indicated that this was not possible, thus confirming the new rotamer conformation for the Met358 sidechain in the inverse agonist bound structure. RESULTS +38 44 Met358 residue_name_number The change in rotomer conformation of Met358 between the agonist and inverse agonist structures is attributed to the gem-dimethyl group on the larger 7 membered benzoxazinone ring system of BIO399. RESULTS +57 64 agonist protein_state The change in rotomer conformation of Met358 between the agonist and inverse agonist structures is attributed to the gem-dimethyl group on the larger 7 membered benzoxazinone ring system of BIO399. RESULTS +69 84 inverse agonist protein_state The change in rotomer conformation of Met358 between the agonist and inverse agonist structures is attributed to the gem-dimethyl group on the larger 7 membered benzoxazinone ring system of BIO399. RESULTS +85 95 structures evidence The change in rotomer conformation of Met358 between the agonist and inverse agonist structures is attributed to the gem-dimethyl group on the larger 7 membered benzoxazinone ring system of BIO399. RESULTS +161 174 benzoxazinone chemical The change in rotomer conformation of Met358 between the agonist and inverse agonist structures is attributed to the gem-dimethyl group on the larger 7 membered benzoxazinone ring system of BIO399. RESULTS +190 196 BIO399 chemical The change in rotomer conformation of Met358 between the agonist and inverse agonist structures is attributed to the gem-dimethyl group on the larger 7 membered benzoxazinone ring system of BIO399. RESULTS +4 14 comparison experimental_method The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c). RESULTS +26 36 structures evidence The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c). RESULTS +52 59 agonist protein_state The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c). RESULTS +89 95 BIO592 chemical The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c). RESULTS +96 105 structure evidence The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c). RESULTS +128 134 BIO399 chemical The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c). RESULTS +143 149 Met358 residue_name_number The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c). RESULTS +155 161 Phe506 residue_name_number The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c). RESULTS +169 178 AF2 helix structure_element The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c). RESULTS +195 201 Met358 residue_name_number The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c). RESULTS +247 251 RORγ protein The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c). RESULTS +0 6 BIO399 chemical BIO399 and Inverse agonist T0901317 bind in a collapsed conformation distinct from other RORγ Inverse Agonists Cocrystal structures RESULTS +11 26 Inverse agonist protein_state BIO399 and Inverse agonist T0901317 bind in a collapsed conformation distinct from other RORγ Inverse Agonists Cocrystal structures RESULTS +27 35 T0901317 chemical BIO399 and Inverse agonist T0901317 bind in a collapsed conformation distinct from other RORγ Inverse Agonists Cocrystal structures RESULTS +46 55 collapsed protein_state BIO399 and Inverse agonist T0901317 bind in a collapsed conformation distinct from other RORγ Inverse Agonists Cocrystal structures RESULTS +89 93 RORγ protein BIO399 and Inverse agonist T0901317 bind in a collapsed conformation distinct from other RORγ Inverse Agonists Cocrystal structures RESULTS +111 131 Cocrystal structures evidence BIO399 and Inverse agonist T0901317 bind in a collapsed conformation distinct from other RORγ Inverse Agonists Cocrystal structures RESULTS +3 10 Overlay experimental_method a Overlay of RORγ structures bound to BIO596 (Green), BIO399 (Cyan) and T0901317 (Pink). FIG +14 18 RORγ protein a Overlay of RORγ structures bound to BIO596 (Green), BIO399 (Cyan) and T0901317 (Pink). FIG +19 29 structures evidence a Overlay of RORγ structures bound to BIO596 (Green), BIO399 (Cyan) and T0901317 (Pink). FIG +30 38 bound to protein_state a Overlay of RORγ structures bound to BIO596 (Green), BIO399 (Cyan) and T0901317 (Pink). FIG +39 45 BIO596 chemical a Overlay of RORγ structures bound to BIO596 (Green), BIO399 (Cyan) and T0901317 (Pink). FIG +55 61 BIO399 chemical a Overlay of RORγ structures bound to BIO596 (Green), BIO399 (Cyan) and T0901317 (Pink). FIG +73 81 T0901317 chemical a Overlay of RORγ structures bound to BIO596 (Green), BIO399 (Cyan) and T0901317 (Pink). FIG +2 9 Overlay experimental_method b Overlay of M358 in RORγ structure BIO596 (Green), BIO399 (Cyan), Digoxin (Yellow), Compound 2 (Grey), Compound 48 (Salmon) and Compound 4j (Orange) FIG +13 17 M358 residue_name_number b Overlay of M358 in RORγ structure BIO596 (Green), BIO399 (Cyan), Digoxin (Yellow), Compound 2 (Grey), Compound 48 (Salmon) and Compound 4j (Orange) FIG +21 25 RORγ protein b Overlay of M358 in RORγ structure BIO596 (Green), BIO399 (Cyan), Digoxin (Yellow), Compound 2 (Grey), Compound 48 (Salmon) and Compound 4j (Orange) FIG +26 35 structure evidence b Overlay of M358 in RORγ structure BIO596 (Green), BIO399 (Cyan), Digoxin (Yellow), Compound 2 (Grey), Compound 48 (Salmon) and Compound 4j (Orange) FIG +36 42 BIO596 chemical b Overlay of M358 in RORγ structure BIO596 (Green), BIO399 (Cyan), Digoxin (Yellow), Compound 2 (Grey), Compound 48 (Salmon) and Compound 4j (Orange) FIG +52 58 BIO399 chemical b Overlay of M358 in RORγ structure BIO596 (Green), BIO399 (Cyan), Digoxin (Yellow), Compound 2 (Grey), Compound 48 (Salmon) and Compound 4j (Orange) FIG +67 74 Digoxin chemical b Overlay of M358 in RORγ structure BIO596 (Green), BIO399 (Cyan), Digoxin (Yellow), Compound 2 (Grey), Compound 48 (Salmon) and Compound 4j (Orange) FIG +4 24 co-crystal structure evidence The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. RESULTS +28 32 RORγ protein The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. RESULTS +38 46 T0901317 chemical The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. RESULTS +68 83 inverse agonist protein_state The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. RESULTS +87 91 RORγ protein The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. RESULTS +93 97 IC50 evidence The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. RESULTS +112 140 SRC1 displacement FRET assay experimental_method The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. RESULTS +148 152 IC50 evidence The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. RESULTS +168 178 FRET assay experimental_method The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. RESULTS +223 232 collapsed protein_state The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. RESULTS +261 270 structure evidence The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. RESULTS +274 280 BIO399 chemical The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. RESULTS +18 29 superimpose experimental_method The two compounds superimpose with an RMSD of 0.81 Å (Fig. 6a). RESULTS +38 42 RMSD evidence The two compounds superimpose with an RMSD of 0.81 Å (Fig. 6a). RESULTS +21 39 hexafluoropropanol chemical The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the RORγ BIO592 agonist structure. RESULTS +49 57 T0901317 chemical The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the RORγ BIO592 agonist structure. RESULTS +82 98 electron density evidence The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the RORγ BIO592 agonist structure. RESULTS +140 146 Met358 residue_name_number The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the RORγ BIO592 agonist structure. RESULTS +168 174 Phe506 residue_name_number The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the RORγ BIO592 agonist structure. RESULTS +182 186 RORγ protein The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the RORγ BIO592 agonist structure. RESULTS +187 193 BIO592 chemical The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the RORγ BIO592 agonist structure. RESULTS +194 201 agonist protein_state The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the RORγ BIO592 agonist structure. RESULTS +202 211 structure evidence The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the RORγ BIO592 agonist structure. RESULTS +30 36 Met358 residue_name_number We hypothesize that since the Met358 sidechain conformation in the T0901317 RORγ structure is not in the BIO399 conformation, this difference could account for the 10-fold reduction in the inverse agonism for T0901317 compared to BIO399 in the FRET assay. RESULTS +67 75 T0901317 chemical We hypothesize that since the Met358 sidechain conformation in the T0901317 RORγ structure is not in the BIO399 conformation, this difference could account for the 10-fold reduction in the inverse agonism for T0901317 compared to BIO399 in the FRET assay. RESULTS +76 80 RORγ protein We hypothesize that since the Met358 sidechain conformation in the T0901317 RORγ structure is not in the BIO399 conformation, this difference could account for the 10-fold reduction in the inverse agonism for T0901317 compared to BIO399 in the FRET assay. RESULTS +81 90 structure evidence We hypothesize that since the Met358 sidechain conformation in the T0901317 RORγ structure is not in the BIO399 conformation, this difference could account for the 10-fold reduction in the inverse agonism for T0901317 compared to BIO399 in the FRET assay. RESULTS +105 111 BIO399 chemical We hypothesize that since the Met358 sidechain conformation in the T0901317 RORγ structure is not in the BIO399 conformation, this difference could account for the 10-fold reduction in the inverse agonism for T0901317 compared to BIO399 in the FRET assay. RESULTS +209 217 T0901317 chemical We hypothesize that since the Met358 sidechain conformation in the T0901317 RORγ structure is not in the BIO399 conformation, this difference could account for the 10-fold reduction in the inverse agonism for T0901317 compared to BIO399 in the FRET assay. RESULTS +230 236 BIO399 chemical We hypothesize that since the Met358 sidechain conformation in the T0901317 RORγ structure is not in the BIO399 conformation, this difference could account for the 10-fold reduction in the inverse agonism for T0901317 compared to BIO399 in the FRET assay. RESULTS +244 254 FRET assay experimental_method We hypothesize that since the Met358 sidechain conformation in the T0901317 RORγ structure is not in the BIO399 conformation, this difference could account for the 10-fold reduction in the inverse agonism for T0901317 compared to BIO399 in the FRET assay. RESULTS +0 21 Co-crystal structures evidence Co-crystal structures of RORγ have been generated with several potent inverse agonists adopting a linear conformation distinct from the collapsed conformations seen for BIO399 and T090131718. RESULTS +25 29 RORγ protein Co-crystal structures of RORγ have been generated with several potent inverse agonists adopting a linear conformation distinct from the collapsed conformations seen for BIO399 and T090131718. RESULTS +98 104 linear protein_state Co-crystal structures of RORγ have been generated with several potent inverse agonists adopting a linear conformation distinct from the collapsed conformations seen for BIO399 and T090131718. RESULTS +136 145 collapsed protein_state Co-crystal structures of RORγ have been generated with several potent inverse agonists adopting a linear conformation distinct from the collapsed conformations seen for BIO399 and T090131718. RESULTS +169 175 BIO399 chemical Co-crystal structures of RORγ have been generated with several potent inverse agonists adopting a linear conformation distinct from the collapsed conformations seen for BIO399 and T090131718. RESULTS +180 190 T090131718 chemical Co-crystal structures of RORγ have been generated with several potent inverse agonists adopting a linear conformation distinct from the collapsed conformations seen for BIO399 and T090131718. RESULTS +4 19 inverse agonist protein_state The inverse agonist activity for these compounds has been attributed to orientating Trp317 to clash with Tyr502 or a direct inverse agonist hydrogen bonding event with His479, both of which would perturb the agonist conformation of RORγ. RESULTS +84 90 Trp317 residue_name_number The inverse agonist activity for these compounds has been attributed to orientating Trp317 to clash with Tyr502 or a direct inverse agonist hydrogen bonding event with His479, both of which would perturb the agonist conformation of RORγ. RESULTS +105 111 Tyr502 residue_name_number The inverse agonist activity for these compounds has been attributed to orientating Trp317 to clash with Tyr502 or a direct inverse agonist hydrogen bonding event with His479, both of which would perturb the agonist conformation of RORγ. RESULTS +124 139 inverse agonist protein_state The inverse agonist activity for these compounds has been attributed to orientating Trp317 to clash with Tyr502 or a direct inverse agonist hydrogen bonding event with His479, both of which would perturb the agonist conformation of RORγ. RESULTS +140 156 hydrogen bonding bond_interaction The inverse agonist activity for these compounds has been attributed to orientating Trp317 to clash with Tyr502 or a direct inverse agonist hydrogen bonding event with His479, both of which would perturb the agonist conformation of RORγ. RESULTS +168 174 His479 residue_name_number The inverse agonist activity for these compounds has been attributed to orientating Trp317 to clash with Tyr502 or a direct inverse agonist hydrogen bonding event with His479, both of which would perturb the agonist conformation of RORγ. RESULTS +208 215 agonist protein_state The inverse agonist activity for these compounds has been attributed to orientating Trp317 to clash with Tyr502 or a direct inverse agonist hydrogen bonding event with His479, both of which would perturb the agonist conformation of RORγ. RESULTS +232 236 RORγ protein The inverse agonist activity for these compounds has been attributed to orientating Trp317 to clash with Tyr502 or a direct inverse agonist hydrogen bonding event with His479, both of which would perturb the agonist conformation of RORγ. RESULTS +0 6 BIO399 chemical BIO399 neither orients the sidechain of Trp317 toward Tyr502 nor forms a hydrogen bond with His479 suggesting its mode of action is distinct from linear inverse agonists (Additional file 8). RESULTS +40 46 Trp317 residue_name_number BIO399 neither orients the sidechain of Trp317 toward Tyr502 nor forms a hydrogen bond with His479 suggesting its mode of action is distinct from linear inverse agonists (Additional file 8). RESULTS +54 60 Tyr502 residue_name_number BIO399 neither orients the sidechain of Trp317 toward Tyr502 nor forms a hydrogen bond with His479 suggesting its mode of action is distinct from linear inverse agonists (Additional file 8). RESULTS +73 86 hydrogen bond bond_interaction BIO399 neither orients the sidechain of Trp317 toward Tyr502 nor forms a hydrogen bond with His479 suggesting its mode of action is distinct from linear inverse agonists (Additional file 8). RESULTS +92 98 His479 residue_name_number BIO399 neither orients the sidechain of Trp317 toward Tyr502 nor forms a hydrogen bond with His479 suggesting its mode of action is distinct from linear inverse agonists (Additional file 8). RESULTS +14 29 inverse agonist protein_state In the linear inverse agonist crystal structures the side chain of Met358 resides in a similar position as the rotomer observed in RORγ agonist structures with BIO592 described here or as observed in the hydroxycholesterol derivatives and therefore would not trigger inverse agonism with these ligands (Fig. 6b). RESULTS +30 48 crystal structures evidence In the linear inverse agonist crystal structures the side chain of Met358 resides in a similar position as the rotomer observed in RORγ agonist structures with BIO592 described here or as observed in the hydroxycholesterol derivatives and therefore would not trigger inverse agonism with these ligands (Fig. 6b). RESULTS +67 73 Met358 residue_name_number In the linear inverse agonist crystal structures the side chain of Met358 resides in a similar position as the rotomer observed in RORγ agonist structures with BIO592 described here or as observed in the hydroxycholesterol derivatives and therefore would not trigger inverse agonism with these ligands (Fig. 6b). RESULTS +131 135 RORγ protein In the linear inverse agonist crystal structures the side chain of Met358 resides in a similar position as the rotomer observed in RORγ agonist structures with BIO592 described here or as observed in the hydroxycholesterol derivatives and therefore would not trigger inverse agonism with these ligands (Fig. 6b). RESULTS +136 143 agonist protein_state In the linear inverse agonist crystal structures the side chain of Met358 resides in a similar position as the rotomer observed in RORγ agonist structures with BIO592 described here or as observed in the hydroxycholesterol derivatives and therefore would not trigger inverse agonism with these ligands (Fig. 6b). RESULTS +144 154 structures evidence In the linear inverse agonist crystal structures the side chain of Met358 resides in a similar position as the rotomer observed in RORγ agonist structures with BIO592 described here or as observed in the hydroxycholesterol derivatives and therefore would not trigger inverse agonism with these ligands (Fig. 6b). RESULTS +160 166 BIO592 chemical In the linear inverse agonist crystal structures the side chain of Met358 resides in a similar position as the rotomer observed in RORγ agonist structures with BIO592 described here or as observed in the hydroxycholesterol derivatives and therefore would not trigger inverse agonism with these ligands (Fig. 6b). RESULTS +204 222 hydroxycholesterol chemical In the linear inverse agonist crystal structures the side chain of Met358 resides in a similar position as the rotomer observed in RORγ agonist structures with BIO592 described here or as observed in the hydroxycholesterol derivatives and therefore would not trigger inverse agonism with these ligands (Fig. 6b). RESULTS +0 6 BIO399 chemical BIO399 shows selectivity for RORγ over RORα and RORβ in a GAL4 Cellular Reporter Assay RESULTS +29 33 RORγ protein BIO399 shows selectivity for RORγ over RORα and RORβ in a GAL4 Cellular Reporter Assay RESULTS +39 43 RORα protein BIO399 shows selectivity for RORγ over RORα and RORβ in a GAL4 Cellular Reporter Assay RESULTS +48 52 RORβ protein BIO399 shows selectivity for RORγ over RORα and RORβ in a GAL4 Cellular Reporter Assay RESULTS +58 86 GAL4 Cellular Reporter Assay experimental_method BIO399 shows selectivity for RORγ over RORα and RORβ in a GAL4 Cellular Reporter Assay RESULTS +0 15 GAL4 cell assay experimental_method GAL4 cell assay selectivity profile for BIO399 toward RORα and RORβ in GAL4 TABLE +40 46 BIO399 chemical GAL4 cell assay selectivity profile for BIO399 toward RORα and RORβ in GAL4 TABLE +54 58 RORα protein GAL4 cell assay selectivity profile for BIO399 toward RORα and RORβ in GAL4 TABLE +63 67 RORβ protein GAL4 cell assay selectivity profile for BIO399 toward RORα and RORβ in GAL4 TABLE +71 75 GAL4 protein GAL4 cell assay selectivity profile for BIO399 toward RORα and RORβ in GAL4 TABLE +3 10 Overlay experimental_method a Overlay of RORα (yellow), β (pink) and γ (cyan) showing side chain differences at Met358 inverse agonism trigger position and (b) around the benzoxazinone ring system of BIO399 FIG +14 18 RORα protein a Overlay of RORα (yellow), β (pink) and γ (cyan) showing side chain differences at Met358 inverse agonism trigger position and (b) around the benzoxazinone ring system of BIO399 FIG +29 30 β protein a Overlay of RORα (yellow), β (pink) and γ (cyan) showing side chain differences at Met358 inverse agonism trigger position and (b) around the benzoxazinone ring system of BIO399 FIG +42 43 γ protein a Overlay of RORα (yellow), β (pink) and γ (cyan) showing side chain differences at Met358 inverse agonism trigger position and (b) around the benzoxazinone ring system of BIO399 FIG +85 91 Met358 residue_name_number a Overlay of RORα (yellow), β (pink) and γ (cyan) showing side chain differences at Met358 inverse agonism trigger position and (b) around the benzoxazinone ring system of BIO399 FIG +144 157 benzoxazinone chemical a Overlay of RORα (yellow), β (pink) and γ (cyan) showing side chain differences at Met358 inverse agonism trigger position and (b) around the benzoxazinone ring system of BIO399 FIG +173 179 BIO399 chemical a Overlay of RORα (yellow), β (pink) and γ (cyan) showing side chain differences at Met358 inverse agonism trigger position and (b) around the benzoxazinone ring system of BIO399 FIG +54 60 BIO399 chemical In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. RESULTS +63 86 cellular reporter assay experimental_method In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. RESULTS +113 135 ligand binding domains structure_element In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. RESULTS +139 142 ROR protein_type In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. RESULTS +143 144 α protein In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. RESULTS +146 147 β protein In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. RESULTS +152 153 γ protein In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. RESULTS +159 167 fused to experimental_method In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. RESULTS +172 190 DNA binding domain structure_element In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. RESULTS +198 220 transcriptional factor protein_type In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. RESULTS +221 225 GAL4 protein In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. RESULTS +4 7 ROR protein_type The ROR-GAL4 fusion proteins were expressed in cells with the luciferase reporter gene under the control of a GAL4 promoter. RESULTS +8 12 GAL4 protein The ROR-GAL4 fusion proteins were expressed in cells with the luciferase reporter gene under the control of a GAL4 promoter. RESULTS +110 114 GAL4 protein The ROR-GAL4 fusion proteins were expressed in cells with the luciferase reporter gene under the control of a GAL4 promoter. RESULTS +0 6 BIO399 chemical BIO399 inhibited the luciferase activity when added to the cells expressing the RORγ-GAL4 fusion with an in vivo IC50 of 42.5nM while showing >235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR α or β, respectively (Table 1). RESULTS +80 84 RORγ protein BIO399 inhibited the luciferase activity when added to the cells expressing the RORγ-GAL4 fusion with an in vivo IC50 of 42.5nM while showing >235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR α or β, respectively (Table 1). RESULTS +85 89 GAL4 protein BIO399 inhibited the luciferase activity when added to the cells expressing the RORγ-GAL4 fusion with an in vivo IC50 of 42.5nM while showing >235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR α or β, respectively (Table 1). RESULTS +113 117 IC50 evidence BIO399 inhibited the luciferase activity when added to the cells expressing the RORγ-GAL4 fusion with an in vivo IC50 of 42.5nM while showing >235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR α or β, respectively (Table 1). RESULTS +193 197 GAL4 protein BIO399 inhibited the luciferase activity when added to the cells expressing the RORγ-GAL4 fusion with an in vivo IC50 of 42.5nM while showing >235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR α or β, respectively (Table 1). RESULTS +211 214 LBD structure_element BIO399 inhibited the luciferase activity when added to the cells expressing the RORγ-GAL4 fusion with an in vivo IC50 of 42.5nM while showing >235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR α or β, respectively (Table 1). RESULTS +218 221 ROR protein_type BIO399 inhibited the luciferase activity when added to the cells expressing the RORγ-GAL4 fusion with an in vivo IC50 of 42.5nM while showing >235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR α or β, respectively (Table 1). RESULTS +222 223 α protein BIO399 inhibited the luciferase activity when added to the cells expressing the RORγ-GAL4 fusion with an in vivo IC50 of 42.5nM while showing >235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR α or β, respectively (Table 1). RESULTS +227 228 β protein BIO399 inhibited the luciferase activity when added to the cells expressing the RORγ-GAL4 fusion with an in vivo IC50 of 42.5nM while showing >235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR α or β, respectively (Table 1). RESULTS +4 7 LBS site The LBS of RORs share a high degree of similarity. RESULTS +11 15 RORs protein_type The LBS of RORs share a high degree of similarity. RESULTS +40 46 BIO399 chemical However, the inverse agonism trigger of BIO399, residue Met358, is a leucine in both RORα and β. RESULTS +56 62 Met358 residue_name_number However, the inverse agonism trigger of BIO399, residue Met358, is a leucine in both RORα and β. RESULTS +69 76 leucine residue_name However, the inverse agonism trigger of BIO399, residue Met358, is a leucine in both RORα and β. RESULTS +85 89 RORα protein However, the inverse agonism trigger of BIO399, residue Met358, is a leucine in both RORα and β. RESULTS +94 95 β protein However, the inverse agonism trigger of BIO399, residue Met358, is a leucine in both RORα and β. RESULTS +29 35 BIO399 chemical This selectivity profile for BIO399 is attributed to the shorter leucine side chain in RORα and β which would not reach the phenylalanine on the AF2 helix further underscoring the role of Met358 as a trigger for RORγ specific inverse agonism (Fig. 7a). RESULTS +65 72 leucine residue_name This selectivity profile for BIO399 is attributed to the shorter leucine side chain in RORα and β which would not reach the phenylalanine on the AF2 helix further underscoring the role of Met358 as a trigger for RORγ specific inverse agonism (Fig. 7a). RESULTS +87 91 RORα protein This selectivity profile for BIO399 is attributed to the shorter leucine side chain in RORα and β which would not reach the phenylalanine on the AF2 helix further underscoring the role of Met358 as a trigger for RORγ specific inverse agonism (Fig. 7a). RESULTS +96 97 β protein This selectivity profile for BIO399 is attributed to the shorter leucine side chain in RORα and β which would not reach the phenylalanine on the AF2 helix further underscoring the role of Met358 as a trigger for RORγ specific inverse agonism (Fig. 7a). RESULTS +124 137 phenylalanine residue_name This selectivity profile for BIO399 is attributed to the shorter leucine side chain in RORα and β which would not reach the phenylalanine on the AF2 helix further underscoring the role of Met358 as a trigger for RORγ specific inverse agonism (Fig. 7a). RESULTS +145 154 AF2 helix structure_element This selectivity profile for BIO399 is attributed to the shorter leucine side chain in RORα and β which would not reach the phenylalanine on the AF2 helix further underscoring the role of Met358 as a trigger for RORγ specific inverse agonism (Fig. 7a). RESULTS +188 194 Met358 residue_name_number This selectivity profile for BIO399 is attributed to the shorter leucine side chain in RORα and β which would not reach the phenylalanine on the AF2 helix further underscoring the role of Met358 as a trigger for RORγ specific inverse agonism (Fig. 7a). RESULTS +212 216 RORγ protein This selectivity profile for BIO399 is attributed to the shorter leucine side chain in RORα and β which would not reach the phenylalanine on the AF2 helix further underscoring the role of Met358 as a trigger for RORγ specific inverse agonism (Fig. 7a). RESULTS +13 17 RORα protein Furthermore, RORα contains two phenylalanine residues in its LBS whereas RORβ and γ have a leucine in the same position (Fig. 6b). RESULTS +31 44 phenylalanine residue_name Furthermore, RORα contains two phenylalanine residues in its LBS whereas RORβ and γ have a leucine in the same position (Fig. 6b). RESULTS +61 64 LBS site Furthermore, RORα contains two phenylalanine residues in its LBS whereas RORβ and γ have a leucine in the same position (Fig. 6b). RESULTS +73 77 RORβ protein Furthermore, RORα contains two phenylalanine residues in its LBS whereas RORβ and γ have a leucine in the same position (Fig. 6b). RESULTS +82 83 γ protein Furthermore, RORα contains two phenylalanine residues in its LBS whereas RORβ and γ have a leucine in the same position (Fig. 6b). RESULTS +91 98 leucine residue_name Furthermore, RORα contains two phenylalanine residues in its LBS whereas RORβ and γ have a leucine in the same position (Fig. 6b). RESULTS +28 41 phenylalanine residue_name We hypothesize that the two phenylalanine residues in the LBS of RORα occlude the dihydrobenzoxazepinone ring system of BIO399 from binding it and responsible for the increase in selectivity for RORα over β. RESULTS +58 61 LBS site We hypothesize that the two phenylalanine residues in the LBS of RORα occlude the dihydrobenzoxazepinone ring system of BIO399 from binding it and responsible for the increase in selectivity for RORα over β. RESULTS +65 69 RORα protein We hypothesize that the two phenylalanine residues in the LBS of RORα occlude the dihydrobenzoxazepinone ring system of BIO399 from binding it and responsible for the increase in selectivity for RORα over β. RESULTS +82 104 dihydrobenzoxazepinone chemical We hypothesize that the two phenylalanine residues in the LBS of RORα occlude the dihydrobenzoxazepinone ring system of BIO399 from binding it and responsible for the increase in selectivity for RORα over β. RESULTS +120 126 BIO399 chemical We hypothesize that the two phenylalanine residues in the LBS of RORα occlude the dihydrobenzoxazepinone ring system of BIO399 from binding it and responsible for the increase in selectivity for RORα over β. RESULTS +195 199 RORα protein We hypothesize that the two phenylalanine residues in the LBS of RORα occlude the dihydrobenzoxazepinone ring system of BIO399 from binding it and responsible for the increase in selectivity for RORα over β. RESULTS +205 206 β protein We hypothesize that the two phenylalanine residues in the LBS of RORα occlude the dihydrobenzoxazepinone ring system of BIO399 from binding it and responsible for the increase in selectivity for RORα over β. RESULTS +47 60 benzoxazinone chemical We have identified a novel series of synthetic benzoxazinone ligands which modulate the transcriptional activity of RORγ in a FRET based assay. CONCL +116 120 RORγ protein We have identified a novel series of synthetic benzoxazinone ligands which modulate the transcriptional activity of RORγ in a FRET based assay. CONCL +126 142 FRET based assay experimental_method We have identified a novel series of synthetic benzoxazinone ligands which modulate the transcriptional activity of RORγ in a FRET based assay. CONCL +6 25 partial proteolysis experimental_method Using partial proteolysis we show a conformational change which destabilizes the AF2 helix of RORγ when the inverse agonist BIO399 binds. CONCL +81 90 AF2 helix structure_element Using partial proteolysis we show a conformational change which destabilizes the AF2 helix of RORγ when the inverse agonist BIO399 binds. CONCL +94 98 RORγ protein Using partial proteolysis we show a conformational change which destabilizes the AF2 helix of RORγ when the inverse agonist BIO399 binds. CONCL +108 123 inverse agonist protein_state Using partial proteolysis we show a conformational change which destabilizes the AF2 helix of RORγ when the inverse agonist BIO399 binds. CONCL +124 130 BIO399 chemical Using partial proteolysis we show a conformational change which destabilizes the AF2 helix of RORγ when the inverse agonist BIO399 binds. CONCL +8 12 RORγ protein The two RORγ co-crystal structures reported here show how a small change to the core ring system can modulate the mode of action from agonist (BIO592) to inverse agonism (BIO399). CONCL +13 34 co-crystal structures evidence The two RORγ co-crystal structures reported here show how a small change to the core ring system can modulate the mode of action from agonist (BIO592) to inverse agonism (BIO399). CONCL +134 141 agonist protein_state The two RORγ co-crystal structures reported here show how a small change to the core ring system can modulate the mode of action from agonist (BIO592) to inverse agonism (BIO399). CONCL +143 149 BIO592 chemical The two RORγ co-crystal structures reported here show how a small change to the core ring system can modulate the mode of action from agonist (BIO592) to inverse agonism (BIO399). CONCL +171 177 BIO399 chemical The two RORγ co-crystal structures reported here show how a small change to the core ring system can modulate the mode of action from agonist (BIO592) to inverse agonism (BIO399). CONCL +67 71 RORγ protein Finally, we are reporting a newly identified trigger for achieving RORγ specific inverse agonism in an in vivo setting through Met358 which perturbs the agonist conformation of the AF2 helix and prevents coactivator protein binding. CONCL +127 133 Met358 residue_name_number Finally, we are reporting a newly identified trigger for achieving RORγ specific inverse agonism in an in vivo setting through Met358 which perturbs the agonist conformation of the AF2 helix and prevents coactivator protein binding. CONCL +153 160 agonist protein_state Finally, we are reporting a newly identified trigger for achieving RORγ specific inverse agonism in an in vivo setting through Met358 which perturbs the agonist conformation of the AF2 helix and prevents coactivator protein binding. CONCL +181 190 AF2 helix structure_element Finally, we are reporting a newly identified trigger for achieving RORγ specific inverse agonism in an in vivo setting through Met358 which perturbs the agonist conformation of the AF2 helix and prevents coactivator protein binding. CONCL diff --git a/annotation_CSV/PMC4896748.csv b/annotation_CSV/PMC4896748.csv new file mode 100644 index 0000000000000000000000000000000000000000..4cfb0481374cc722f931ff3a85eac838b2a782a2 --- /dev/null +++ b/annotation_CSV/PMC4896748.csv @@ -0,0 +1,2639 @@ +anno_start anno_end anno_text entity_type sentence section +9 16 cryo-EM experimental_method Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome TITLE +26 34 inchworm protein_state Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome TITLE +59 64 viral taxonomy_domain Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome TITLE +65 69 IRES site Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome TITLE +82 90 ribosome complex_assembly Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome TITLE +0 29 Internal ribosome entry sites site Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. ABSTRACT +31 36 IRESs site Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. ABSTRACT +77 82 viral taxonomy_domain Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. ABSTRACT +83 88 mRNAs chemical Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. ABSTRACT +6 30 electron cryo-microscopy experimental_method Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. ABSTRACT +69 77 ribosome complex_assembly Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. ABSTRACT +78 88 structures evidence Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. ABSTRACT +105 125 Taura syndrome virus species Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. ABSTRACT +126 130 IRES site Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. ABSTRACT +135 146 translocase protein_type Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. ABSTRACT +147 155 eEF2•GTP complex_assembly Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. ABSTRACT +156 166 bound with protein_state Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. ABSTRACT +167 175 sordarin chemical Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. ABSTRACT +4 14 structures evidence The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. ABSTRACT +39 43 IRES site The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. ABSTRACT +84 94 initiation protein_state The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. ABSTRACT +133 137 eEF2 protein The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. ABSTRACT +4 8 IRES site The IRES rearranges from extended to bent to extended conformations. ABSTRACT +25 33 extended protein_state The IRES rearranges from extended to bent to extended conformations. ABSTRACT +37 41 bent protein_state The IRES rearranges from extended to bent to extended conformations. ABSTRACT +45 53 extended protein_state The IRES rearranges from extended to bent to extended conformations. ABSTRACT +5 13 inchworm protein_state This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. ABSTRACT +81 84 40S complex_assembly This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. ABSTRACT +85 89 head structure_element This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. ABSTRACT +0 4 eEF2 protein eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. ABSTRACT +22 25 60S complex_assembly eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. ABSTRACT +26 33 subunit structure_element eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. ABSTRACT +61 64 40S complex_assembly eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. ABSTRACT +65 72 subunit structure_element eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. ABSTRACT +65 72 subunit structure_element eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. ABSTRACT +86 92 A site site eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. ABSTRACT +4 15 diphthamide ptm Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. ABSTRACT +38 40 IV structure_element Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. ABSTRACT +55 82 tRNA-mRNA-like pseudoknot I structure_element Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. ABSTRACT +84 87 PKI structure_element Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. ABSTRACT +96 100 IRES site Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. ABSTRACT +110 125 decoding center site Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. ABSTRACT +13 16 40S complex_assembly This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. ABSTRACT +39 43 head structure_element This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. ABSTRACT +63 67 IRES site This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. ABSTRACT +122 125 PKI structure_element This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. ABSTRACT +147 160 A and P sites site This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. ABSTRACT +4 14 structures evidence The structures suggest missing links in our understanding of tRNA translocation. ABSTRACT +61 65 tRNA chemical The structures suggest missing links in our understanding of tRNA translocation. ABSTRACT +0 5 Virus taxonomy_domain Virus propagation relies on the host translational apparatus. INTRO +33 38 mRNAs chemical To efficiently compete with host mRNAs and engage in translation under stress, some viral mRNAs undergo cap-independent translation. INTRO +84 89 viral taxonomy_domain To efficiently compete with host mRNAs and engage in translation under stress, some viral mRNAs undergo cap-independent translation. INTRO +90 95 mRNAs chemical To efficiently compete with host mRNAs and engage in translation under stress, some viral mRNAs undergo cap-independent translation. INTRO +13 41 internal ribosome entry site site To this end, internal ribosome entry site (IRES) RNAs are employed (reviewed in. INTRO +43 47 IRES site To this end, internal ribosome entry site (IRES) RNAs are employed (reviewed in. INTRO +49 53 RNAs chemical To this end, internal ribosome entry site (IRES) RNAs are employed (reviewed in. INTRO +3 7 IRES site An IRES is located at the 5’ untranslated region of the viral mRNA, preceding an open reading frame (ORF). INTRO +26 48 5’ untranslated region structure_element An IRES is located at the 5’ untranslated region of the viral mRNA, preceding an open reading frame (ORF). INTRO +56 61 viral taxonomy_domain An IRES is located at the 5’ untranslated region of the viral mRNA, preceding an open reading frame (ORF). INTRO +62 66 mRNA chemical An IRES is located at the 5’ untranslated region of the viral mRNA, preceding an open reading frame (ORF). INTRO +81 99 open reading frame structure_element An IRES is located at the 5’ untranslated region of the viral mRNA, preceding an open reading frame (ORF). INTRO +101 104 ORF structure_element An IRES is located at the 5’ untranslated region of the viral mRNA, preceding an open reading frame (ORF). INTRO +27 37 structured protein_state To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit. INTRO +38 42 IRES site To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit. INTRO +43 46 RNA chemical To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit. INTRO +66 69 40S complex_assembly To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit. INTRO +70 77 subunit structure_element To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit. INTRO +85 97 80S ribosome complex_assembly To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit. INTRO +169 174 small protein_state To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit. INTRO +175 178 40S complex_assembly To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit. INTRO +179 186 subunit structure_element To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit. INTRO +44 48 IRES site The canonical scenario of cap-dependent and IRES-dependent initiation involves positioning of the AUG start codon and the initiator tRNAMet in the ribosomal peptidyl-tRNA (P) site, facilitated by interaction with initiation factors. INTRO +132 139 tRNAMet chemical The canonical scenario of cap-dependent and IRES-dependent initiation involves positioning of the AUG start codon and the initiator tRNAMet in the ribosomal peptidyl-tRNA (P) site, facilitated by interaction with initiation factors. INTRO +157 179 peptidyl-tRNA (P) site site The canonical scenario of cap-dependent and IRES-dependent initiation involves positioning of the AUG start codon and the initiator tRNAMet in the ribosomal peptidyl-tRNA (P) site, facilitated by interaction with initiation factors. INTRO +213 231 initiation factors protein_type The canonical scenario of cap-dependent and IRES-dependent initiation involves positioning of the AUG start codon and the initiator tRNAMet in the ribosomal peptidyl-tRNA (P) site, facilitated by interaction with initiation factors. INTRO +35 49 aminoacyl-tRNA chemical Subsequent binding of an elongator aminoacyl-tRNA to the ribosomal A site transitions the initiation complex into the elongation cycle of translation. INTRO +67 73 A site site Subsequent binding of an elongator aminoacyl-tRNA to the ribosomal A site transitions the initiation complex into the elongation cycle of translation. INTRO +90 108 initiation complex complex_assembly Subsequent binding of an elongator aminoacyl-tRNA to the ribosomal A site transitions the initiation complex into the elongation cycle of translation. INTRO +37 42 tRNAs chemical Upon peptide bond formation, the two tRNAs and their respective mRNA codons translocate from the A and P to P and E (exit) sites, freeing the A site for the next elongator tRNA. INTRO +64 68 mRNA chemical Upon peptide bond formation, the two tRNAs and their respective mRNA codons translocate from the A and P to P and E (exit) sites, freeing the A site for the next elongator tRNA. INTRO +97 104 A and P site Upon peptide bond formation, the two tRNAs and their respective mRNA codons translocate from the A and P to P and E (exit) sites, freeing the A site for the next elongator tRNA. INTRO +108 128 P and E (exit) sites site Upon peptide bond formation, the two tRNAs and their respective mRNA codons translocate from the A and P to P and E (exit) sites, freeing the A site for the next elongator tRNA. INTRO +142 148 A site site Upon peptide bond formation, the two tRNAs and their respective mRNA codons translocate from the A and P to P and E (exit) sites, freeing the A site for the next elongator tRNA. INTRO +172 176 tRNA chemical Upon peptide bond formation, the two tRNAs and their respective mRNA codons translocate from the A and P to P and E (exit) sites, freeing the A site for the next elongator tRNA. INTRO +23 33 initiation protein_state An unusual strategy of initiation is used by intergenic-region (IGR) IRESs found in Dicistroviridae arthropod-infecting viruses. INTRO +45 62 intergenic-region structure_element An unusual strategy of initiation is used by intergenic-region (IGR) IRESs found in Dicistroviridae arthropod-infecting viruses. INTRO +64 67 IGR structure_element An unusual strategy of initiation is used by intergenic-region (IGR) IRESs found in Dicistroviridae arthropod-infecting viruses. INTRO +69 74 IRESs site An unusual strategy of initiation is used by intergenic-region (IGR) IRESs found in Dicistroviridae arthropod-infecting viruses. INTRO +84 109 Dicistroviridae arthropod species An unusual strategy of initiation is used by intergenic-region (IGR) IRESs found in Dicistroviridae arthropod-infecting viruses. INTRO +120 127 viruses taxonomy_domain An unusual strategy of initiation is used by intergenic-region (IGR) IRESs found in Dicistroviridae arthropod-infecting viruses. INTRO +14 20 shrimp taxonomy_domain These include shrimp-infecting Taura syndrome virus (TSV), and insect viruses Plautia stali intestine virus (PSIV) and Cricket paralysis virus (CrPV). INTRO +31 51 Taura syndrome virus species These include shrimp-infecting Taura syndrome virus (TSV), and insect viruses Plautia stali intestine virus (PSIV) and Cricket paralysis virus (CrPV). INTRO +53 56 TSV species These include shrimp-infecting Taura syndrome virus (TSV), and insect viruses Plautia stali intestine virus (PSIV) and Cricket paralysis virus (CrPV). INTRO +63 69 insect taxonomy_domain These include shrimp-infecting Taura syndrome virus (TSV), and insect viruses Plautia stali intestine virus (PSIV) and Cricket paralysis virus (CrPV). INTRO +78 107 Plautia stali intestine virus species These include shrimp-infecting Taura syndrome virus (TSV), and insect viruses Plautia stali intestine virus (PSIV) and Cricket paralysis virus (CrPV). INTRO +109 113 PSIV species These include shrimp-infecting Taura syndrome virus (TSV), and insect viruses Plautia stali intestine virus (PSIV) and Cricket paralysis virus (CrPV). INTRO +119 142 Cricket paralysis virus species These include shrimp-infecting Taura syndrome virus (TSV), and insect viruses Plautia stali intestine virus (PSIV) and Cricket paralysis virus (CrPV). INTRO +144 148 CrPV species These include shrimp-infecting Taura syndrome virus (TSV), and insect viruses Plautia stali intestine virus (PSIV) and Cricket paralysis virus (CrPV). INTRO +4 7 IGR structure_element The IGR IRES mRNAs do not contain an AUG start codon. INTRO +8 12 IRES site The IGR IRES mRNAs do not contain an AUG start codon. INTRO +13 18 mRNAs chemical The IGR IRES mRNAs do not contain an AUG start codon. INTRO +4 7 IGR structure_element The IGR-IRES-driven initiation does not involve initiator tRNAMet and initiation factors. INTRO +8 12 IRES site The IGR-IRES-driven initiation does not involve initiator tRNAMet and initiation factors. INTRO +20 30 initiation protein_state The IGR-IRES-driven initiation does not involve initiator tRNAMet and initiation factors. INTRO +58 65 tRNAMet chemical The IGR-IRES-driven initiation does not involve initiator tRNAMet and initiation factors. INTRO +70 80 initiation protein_state The IGR-IRES-driven initiation does not involve initiator tRNAMet and initiation factors. INTRO +23 28 IRESs site As such, this group of IRESs represents the most streamlined mechanism of eukaryotic translation initiation. INTRO +74 84 eukaryotic taxonomy_domain As such, this group of IRESs represents the most streamlined mechanism of eukaryotic translation initiation. INTRO +97 107 initiation protein_state As such, this group of IRESs represents the most streamlined mechanism of eukaryotic translation initiation. INTRO +26 35 bacterial taxonomy_domain A recent demonstration of bacterial translation initiation by an IGR IRES indicates that the IRESs take advantage of conserved structural and dynamic properties of the ribosome. INTRO +48 58 initiation protein_state A recent demonstration of bacterial translation initiation by an IGR IRES indicates that the IRESs take advantage of conserved structural and dynamic properties of the ribosome. INTRO +65 68 IGR structure_element A recent demonstration of bacterial translation initiation by an IGR IRES indicates that the IRESs take advantage of conserved structural and dynamic properties of the ribosome. INTRO +69 73 IRES site A recent demonstration of bacterial translation initiation by an IGR IRES indicates that the IRESs take advantage of conserved structural and dynamic properties of the ribosome. INTRO +93 98 IRESs site A recent demonstration of bacterial translation initiation by an IGR IRES indicates that the IRESs take advantage of conserved structural and dynamic properties of the ribosome. INTRO +168 176 ribosome complex_assembly A recent demonstration of bacterial translation initiation by an IGR IRES indicates that the IRESs take advantage of conserved structural and dynamic properties of the ribosome. INTRO +6 30 electron cryo-microscopy experimental_method Early electron cryo-microscopy (cryo-EM) studies have found that the CrPV IRES packs in the ribosome intersubunit space. INTRO +32 39 cryo-EM experimental_method Early electron cryo-microscopy (cryo-EM) studies have found that the CrPV IRES packs in the ribosome intersubunit space. INTRO +69 73 CrPV species Early electron cryo-microscopy (cryo-EM) studies have found that the CrPV IRES packs in the ribosome intersubunit space. INTRO +74 78 IRES site Early electron cryo-microscopy (cryo-EM) studies have found that the CrPV IRES packs in the ribosome intersubunit space. INTRO +92 100 ribosome complex_assembly Early electron cryo-microscopy (cryo-EM) studies have found that the CrPV IRES packs in the ribosome intersubunit space. INTRO +101 119 intersubunit space site Early electron cryo-microscopy (cryo-EM) studies have found that the CrPV IRES packs in the ribosome intersubunit space. INTRO +7 14 cryo-EM experimental_method Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +15 25 structures evidence Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +29 43 ribosome-bound protein_state Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +44 47 TSV species Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +48 52 IRES site Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +57 61 CrPV species Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +62 66 IRES site Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +81 84 IGR structure_element Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +85 90 IRESs site Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +104 107 ORF structure_element Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +135 143 ribosome complex_assembly Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +144 154 bound with protein_state Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +155 159 tRNA chemical Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +164 168 mRNA chemical Recent cryo-EM structures of ribosome-bound TSV IRES and CrPV IRES revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. INTRO +12 16 IRES site The ~200-nt IRES RNAs span from the A site beyond the E site. INTRO +17 21 RNAs chemical The ~200-nt IRES RNAs span from the A site beyond the E site. INTRO +36 42 A site site The ~200-nt IRES RNAs span from the A site beyond the E site. INTRO +54 60 E site site The ~200-nt IRES RNAs span from the A site beyond the E site. INTRO +2 11 conserved protein_state A conserved tRNA-mRNA–like structural element of pseudoknot I (PKI) interacts with the decoding center in the A site of the 40S subunit. INTRO +12 45 tRNA-mRNA–like structural element structure_element A conserved tRNA-mRNA–like structural element of pseudoknot I (PKI) interacts with the decoding center in the A site of the 40S subunit. INTRO +49 61 pseudoknot I structure_element A conserved tRNA-mRNA–like structural element of pseudoknot I (PKI) interacts with the decoding center in the A site of the 40S subunit. INTRO +63 66 PKI structure_element A conserved tRNA-mRNA–like structural element of pseudoknot I (PKI) interacts with the decoding center in the A site of the 40S subunit. INTRO +87 102 decoding center site A conserved tRNA-mRNA–like structural element of pseudoknot I (PKI) interacts with the decoding center in the A site of the 40S subunit. INTRO +110 116 A site site A conserved tRNA-mRNA–like structural element of pseudoknot I (PKI) interacts with the decoding center in the A site of the 40S subunit. INTRO +124 127 40S complex_assembly A conserved tRNA-mRNA–like structural element of pseudoknot I (PKI) interacts with the decoding center in the A site of the 40S subunit. INTRO +128 135 subunit structure_element A conserved tRNA-mRNA–like structural element of pseudoknot I (PKI) interacts with the decoding center in the A site of the 40S subunit. INTRO +4 30 codon-anticodon-like helix structure_element The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +34 37 PKI structure_element The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +77 98 universally conserved protein_state The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +99 114 decoding-center site The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +127 131 G577 residue_name_number The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +133 138 A1755 residue_name_number The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +143 148 A1756 residue_name_number The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +150 154 G530 residue_name_number The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +156 161 A1492 residue_name_number The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +166 171 A1493 residue_name_number The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +175 182 E. coli species The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +197 200 RNA chemical The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +205 209 rRNA chemical The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). INTRO +43 50 alanine residue_name The downstream initiation codon—coding for alanine—is placed in the mRNA tunnel, preceding the decoding center. INTRO +68 79 mRNA tunnel site The downstream initiation codon—coding for alanine—is placed in the mRNA tunnel, preceding the decoding center. INTRO +95 110 decoding center site The downstream initiation codon—coding for alanine—is placed in the mRNA tunnel, preceding the decoding center. INTRO +0 3 PKI structure_element PKI of IGR IRESs therefore mimics an A-site elongator tRNA interacting with an mRNA sense codon, but not a P-site initiator tRNAMet and the AUG start codon. INTRO +7 10 IGR structure_element PKI of IGR IRESs therefore mimics an A-site elongator tRNA interacting with an mRNA sense codon, but not a P-site initiator tRNAMet and the AUG start codon. INTRO +11 16 IRESs site PKI of IGR IRESs therefore mimics an A-site elongator tRNA interacting with an mRNA sense codon, but not a P-site initiator tRNAMet and the AUG start codon. INTRO +37 43 A-site site PKI of IGR IRESs therefore mimics an A-site elongator tRNA interacting with an mRNA sense codon, but not a P-site initiator tRNAMet and the AUG start codon. INTRO +54 58 tRNA chemical PKI of IGR IRESs therefore mimics an A-site elongator tRNA interacting with an mRNA sense codon, but not a P-site initiator tRNAMet and the AUG start codon. INTRO +79 83 mRNA chemical PKI of IGR IRESs therefore mimics an A-site elongator tRNA interacting with an mRNA sense codon, but not a P-site initiator tRNAMet and the AUG start codon. INTRO +107 113 P-site site PKI of IGR IRESs therefore mimics an A-site elongator tRNA interacting with an mRNA sense codon, but not a P-site initiator tRNAMet and the AUG start codon. INTRO +124 131 tRNAMet chemical PKI of IGR IRESs therefore mimics an A-site elongator tRNA interacting with an mRNA sense codon, but not a P-site initiator tRNAMet and the AUG start codon. INTRO +23 33 initiation protein_state How this non-canonical initiation complex transitions to the elongation step is not fully understood. INTRO +14 28 aminoacyl-tRNA chemical For a cognate aminoacyl-tRNA to bind the first viral mRNA codon, PKI has to be translocated from the A site, so that the first codon can be presented in the A site. INTRO +47 52 viral taxonomy_domain For a cognate aminoacyl-tRNA to bind the first viral mRNA codon, PKI has to be translocated from the A site, so that the first codon can be presented in the A site. INTRO +53 57 mRNA chemical For a cognate aminoacyl-tRNA to bind the first viral mRNA codon, PKI has to be translocated from the A site, so that the first codon can be presented in the A site. INTRO +65 68 PKI structure_element For a cognate aminoacyl-tRNA to bind the first viral mRNA codon, PKI has to be translocated from the A site, so that the first codon can be presented in the A site. INTRO +101 107 A site site For a cognate aminoacyl-tRNA to bind the first viral mRNA codon, PKI has to be translocated from the A site, so that the first codon can be presented in the A site. INTRO +157 163 A site site For a cognate aminoacyl-tRNA to bind the first viral mRNA codon, PKI has to be translocated from the A site, so that the first codon can be presented in the A site. INTRO +2 9 cryo-EM experimental_method A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state. INTRO +10 19 structure evidence A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state. INTRO +27 35 ribosome complex_assembly A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state. INTRO +36 46 bound with protein_state A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state. INTRO +49 53 CrPV species A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state. INTRO +54 58 IRES site A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state. INTRO +63 77 release factor protein_type A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state. INTRO +78 82 eRF1 protein A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state. INTRO +97 103 A site site A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state. INTRO +130 148 post-translocation protein_state A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state. INTRO +8 17 structure evidence In this structure, PKI is positioned in the P site and the first mRNA codon is located in the A site. INTRO +19 22 PKI structure_element In this structure, PKI is positioned in the P site and the first mRNA codon is located in the A site. INTRO +44 50 P site site In this structure, PKI is positioned in the P site and the first mRNA codon is located in the A site. INTRO +65 69 mRNA chemical In this structure, PKI is positioned in the P site and the first mRNA codon is located in the A site. INTRO +94 100 A site site In this structure, PKI is positioned in the P site and the first mRNA codon is located in the A site. INTRO +8 13 large protein_state How the large IRES RNA translocates within the ribosome, allowing PKI translocation from the A to P site is not known. INTRO +14 18 IRES site How the large IRES RNA translocates within the ribosome, allowing PKI translocation from the A to P site is not known. INTRO +19 22 RNA chemical How the large IRES RNA translocates within the ribosome, allowing PKI translocation from the A to P site is not known. INTRO +47 55 ribosome complex_assembly How the large IRES RNA translocates within the ribosome, allowing PKI translocation from the A to P site is not known. INTRO +66 69 PKI structure_element How the large IRES RNA translocates within the ribosome, allowing PKI translocation from the A to P site is not known. INTRO +93 104 A to P site site How the large IRES RNA translocates within the ribosome, allowing PKI translocation from the A to P site is not known. INTRO +29 32 PKI structure_element The structural similarity of PKI and the tRNA anticodon stem loop (ASL) bound to a codon suggests that their mechanisms of translocation are similar to some extent. INTRO +41 45 tRNA chemical The structural similarity of PKI and the tRNA anticodon stem loop (ASL) bound to a codon suggests that their mechanisms of translocation are similar to some extent. INTRO +46 65 anticodon stem loop structure_element The structural similarity of PKI and the tRNA anticodon stem loop (ASL) bound to a codon suggests that their mechanisms of translocation are similar to some extent. INTRO +67 70 ASL structure_element The structural similarity of PKI and the tRNA anticodon stem loop (ASL) bound to a codon suggests that their mechanisms of translocation are similar to some extent. INTRO +72 80 bound to protein_state The structural similarity of PKI and the tRNA anticodon stem loop (ASL) bound to a codon suggests that their mechanisms of translocation are similar to some extent. INTRO +21 25 IRES site Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +29 38 tRNA-mRNA complex_assembly Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +48 58 eukaryotic taxonomy_domain Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +59 78 elongation factor 2 protein Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +80 84 eEF2 protein Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +143 152 bacterial taxonomy_domain Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +153 157 EF-G protein Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +159 176 Pre-translocation protein_state Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +177 187 tRNA-bound protein_state Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +188 197 ribosomes complex_assembly Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +208 233 peptidyl- and deacyl-tRNA chemical Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +255 259 mRNA chemical Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +274 287 A and P sites site Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +296 306 2tRNA•mRNA complex_assembly Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2), a structural and functional homolog of the well-studied bacterial EF-G. Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). INTRO +17 27 2tRNA•mRNA complex_assembly Translocation of 2tRNA•mRNA involves two major large-scale ribosome rearrangements (Figure 1—figure supplement 1) (reviewed in). INTRO +59 67 ribosome complex_assembly Translocation of 2tRNA•mRNA involves two major large-scale ribosome rearrangements (Figure 1—figure supplement 1) (reviewed in). INTRO +18 27 bacterial taxonomy_domain First, studies of bacterial ribosomes showed that a ~10° rotation of the small subunit relative to the large subunit, known as intersubunit rotation, or ratcheting, is required for translocation. INTRO +28 37 ribosomes complex_assembly First, studies of bacterial ribosomes showed that a ~10° rotation of the small subunit relative to the large subunit, known as intersubunit rotation, or ratcheting, is required for translocation. INTRO +73 86 small subunit structure_element First, studies of bacterial ribosomes showed that a ~10° rotation of the small subunit relative to the large subunit, known as intersubunit rotation, or ratcheting, is required for translocation. INTRO +103 116 large subunit structure_element First, studies of bacterial ribosomes showed that a ~10° rotation of the small subunit relative to the large subunit, known as intersubunit rotation, or ratcheting, is required for translocation. INTRO +100 106 hybrid protein_state Intersubunit rotation occurs spontaneously upon peptidyl transfer, and is coupled with formation of hybrid tRNA states. INTRO +107 111 tRNA chemical Intersubunit rotation occurs spontaneously upon peptidyl transfer, and is coupled with formation of hybrid tRNA states. INTRO +7 14 rotated protein_state In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). INTRO +15 32 pre-translocation protein_state In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). INTRO +33 41 ribosome complex_assembly In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). INTRO +47 60 peptidyl-tRNA chemical In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). INTRO +71 77 A site site In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). INTRO +85 98 small subunit structure_element In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). INTRO +108 111 ASL structure_element In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). INTRO +120 126 P site site In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). INTRO +134 147 large subunit structure_element In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). INTRO +169 179 A/P hybrid protein_state In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). INTRO +18 29 deacyl-tRNA chemical Concurrently, the deacyl-tRNA interacts with the P site of the small subunit and the E site of the large subunit (P/E hybrid state). INTRO +49 55 P site site Concurrently, the deacyl-tRNA interacts with the P site of the small subunit and the E site of the large subunit (P/E hybrid state). INTRO +63 76 small subunit structure_element Concurrently, the deacyl-tRNA interacts with the P site of the small subunit and the E site of the large subunit (P/E hybrid state). INTRO +85 91 E site site Concurrently, the deacyl-tRNA interacts with the P site of the small subunit and the E site of the large subunit (P/E hybrid state). INTRO +99 112 large subunit structure_element Concurrently, the deacyl-tRNA interacts with the P site of the small subunit and the E site of the large subunit (P/E hybrid state). INTRO +114 124 P/E hybrid protein_state Concurrently, the deacyl-tRNA interacts with the P site of the small subunit and the E site of the large subunit (P/E hybrid state). INTRO +4 12 ribosome complex_assembly The ribosome can undergo spontaneous, thermally-driven forward-reverse rotation that shifts the two tRNAs between the hybrid and 'classical' states while the anticodon stem loops remain non-translocated. INTRO +100 105 tRNAs chemical The ribosome can undergo spontaneous, thermally-driven forward-reverse rotation that shifts the two tRNAs between the hybrid and 'classical' states while the anticodon stem loops remain non-translocated. INTRO +118 124 hybrid protein_state The ribosome can undergo spontaneous, thermally-driven forward-reverse rotation that shifts the two tRNAs between the hybrid and 'classical' states while the anticodon stem loops remain non-translocated. INTRO +130 139 classical protein_state The ribosome can undergo spontaneous, thermally-driven forward-reverse rotation that shifts the two tRNAs between the hybrid and 'classical' states while the anticodon stem loops remain non-translocated. INTRO +158 178 anticodon stem loops structure_element The ribosome can undergo spontaneous, thermally-driven forward-reverse rotation that shifts the two tRNAs between the hybrid and 'classical' states while the anticodon stem loops remain non-translocated. INTRO +186 202 non-translocated protein_state The ribosome can undergo spontaneous, thermally-driven forward-reverse rotation that shifts the two tRNAs between the hybrid and 'classical' states while the anticodon stem loops remain non-translocated. INTRO +11 15 EF-G protein Binding of EF-G next to the A site and reverse rotation of the small subunit results in translocation of both ASLs on the small subunit. INTRO +28 34 A site site Binding of EF-G next to the A site and reverse rotation of the small subunit results in translocation of both ASLs on the small subunit. INTRO +63 76 small subunit structure_element Binding of EF-G next to the A site and reverse rotation of the small subunit results in translocation of both ASLs on the small subunit. INTRO +110 114 ASLs structure_element Binding of EF-G next to the A site and reverse rotation of the small subunit results in translocation of both ASLs on the small subunit. INTRO +122 135 small subunit structure_element Binding of EF-G next to the A site and reverse rotation of the small subunit results in translocation of both ASLs on the small subunit. INTRO +0 4 EF-G protein EF-G is thought to 'unlock' the pre-translocation ribosome, allowing movement of the 2tRNA•mRNA complex, however the structural details of this unlocking are not known. INTRO +32 49 pre-translocation protein_state EF-G is thought to 'unlock' the pre-translocation ribosome, allowing movement of the 2tRNA•mRNA complex, however the structural details of this unlocking are not known. INTRO +50 58 ribosome complex_assembly EF-G is thought to 'unlock' the pre-translocation ribosome, allowing movement of the 2tRNA•mRNA complex, however the structural details of this unlocking are not known. INTRO +85 95 2tRNA•mRNA complex_assembly EF-G is thought to 'unlock' the pre-translocation ribosome, allowing movement of the 2tRNA•mRNA complex, however the structural details of this unlocking are not known. INTRO +77 81 head structure_element The second large-scale rearrangement involves rotation, or swiveling, of the head of the small subunit relative to the body. INTRO +89 102 small subunit structure_element The second large-scale rearrangement involves rotation, or swiveling, of the head of the small subunit relative to the body. INTRO +119 123 body structure_element The second large-scale rearrangement involves rotation, or swiveling, of the head of the small subunit relative to the body. INTRO +4 8 head structure_element The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +109 119 absence of protein_state The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +120 124 tRNA chemical The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +135 146 presence of protein_state The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +156 157 P site The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +158 159 E site The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +160 164 tRNA chemical The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +169 173 eEF2 protein The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +177 181 EF-G protein The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +183 216 Förster resonance energy transfer experimental_method The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +218 222 FRET experimental_method The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +242 246 head structure_element The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +261 268 rotated protein_state The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +269 282 small subunit structure_element The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +295 299 EF-G protein The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +321 331 2tRNA•mRNA complex_assembly The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA. INTRO +0 10 Structures evidence Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +18 26 70S•EF-G complex_assembly Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +35 45 bound with protein_state Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +50 69 nearly translocated protein_state Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +70 75 tRNAs chemical Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +104 108 head structure_element Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +121 132 mid-rotated protein_state Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +133 140 subunit structure_element Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +153 157 head structure_element Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +184 197 fully rotated protein_state Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +198 215 pre-translocation protein_state Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +226 237 non-rotated protein_state Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +238 256 post-translocation protein_state Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +257 271 70S•2tRNA•EF-G complex_assembly Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +272 282 structures evidence Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures. INTRO +23 27 head structure_element The structural role of head swivel is not fully understood. INTRO +4 8 head structure_element The head swivel was proposed to facilitate transition of the tRNA from the P to E site by widening a constriction between these sites on the 30S subunit. INTRO +61 65 tRNA chemical The head swivel was proposed to facilitate transition of the tRNA from the P to E site by widening a constriction between these sites on the 30S subunit. INTRO +75 86 P to E site site The head swivel was proposed to facilitate transition of the tRNA from the P to E site by widening a constriction between these sites on the 30S subunit. INTRO +101 113 constriction site The head swivel was proposed to facilitate transition of the tRNA from the P to E site by widening a constriction between these sites on the 30S subunit. INTRO +141 144 30S complex_assembly The head swivel was proposed to facilitate transition of the tRNA from the P to E site by widening a constriction between these sites on the 30S subunit. INTRO +145 152 subunit structure_element The head swivel was proposed to facilitate transition of the tRNA from the P to E site by widening a constriction between these sites on the 30S subunit. INTRO +145 152 subunit structure_element The head swivel was proposed to facilitate transition of the tRNA from the P to E site by widening a constriction between these sites on the 30S subunit. INTRO +25 28 ASL structure_element This widening allows the ASL to sample positions between the P and E sites. INTRO +61 74 P and E sites site This widening allows the ASL to sample positions between the P and E sites. INTRO +20 24 head structure_element Whether and how the head swivel mediates tRNA transition from the A to P site remains unknown. INTRO +41 45 tRNA chemical Whether and how the head swivel mediates tRNA transition from the A to P site remains unknown. INTRO +66 77 A to P site site Whether and how the head swivel mediates tRNA transition from the A to P site remains unknown. INTRO +14 28 70S•2tRNA•mRNA complex_assembly Comparison of 70S•2tRNA•mRNA and 80S•IRES translocation complexes. FIG +33 41 80S•IRES complex_assembly Comparison of 70S•2tRNA•mRNA and 80S•IRES translocation complexes. FIG +4 14 Structures evidence (a) Structures of bacterial 70S•2tRNA•mRNA translocation complexes, ordered according to the position of the translocating A->P tRNA (orange). FIG +18 27 bacterial taxonomy_domain (a) Structures of bacterial 70S•2tRNA•mRNA translocation complexes, ordered according to the position of the translocating A->P tRNA (orange). FIG +28 42 70S•2tRNA•mRNA complex_assembly (a) Structures of bacterial 70S•2tRNA•mRNA translocation complexes, ordered according to the position of the translocating A->P tRNA (orange). FIG +123 127 A->P site (a) Structures of bacterial 70S•2tRNA•mRNA translocation complexes, ordered according to the position of the translocating A->P tRNA (orange). FIG +128 132 tRNA chemical (a) Structures of bacterial 70S•2tRNA•mRNA translocation complexes, ordered according to the position of the translocating A->P tRNA (orange). FIG +20 27 subunit structure_element The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body), elongation factor G (EF-G) is shown in green. FIG +50 63 small subunit structure_element The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body), elongation factor G (EF-G) is shown in green. FIG +81 85 head structure_element The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body), elongation factor G (EF-G) is shown in green. FIG +105 109 body structure_element The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body), elongation factor G (EF-G) is shown in green. FIG +112 131 elongation factor G protein The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body), elongation factor G (EF-G) is shown in green. FIG +133 137 EF-G protein The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body), elongation factor G (EF-G) is shown in green. FIG +12 17 C1054 residue_name_number Nucleotides C1054, G966 and G693 of 16S rRNA are shown in black to denote the A, P and E sites, respectively. FIG +19 23 G966 residue_name_number Nucleotides C1054, G966 and G693 of 16S rRNA are shown in black to denote the A, P and E sites, respectively. FIG +28 32 G693 residue_name_number Nucleotides C1054, G966 and G693 of 16S rRNA are shown in black to denote the A, P and E sites, respectively. FIG +36 44 16S rRNA chemical Nucleotides C1054, G966 and G693 of 16S rRNA are shown in black to denote the A, P and E sites, respectively. FIG +78 94 A, P and E sites site Nucleotides C1054, G966 and G693 of 16S rRNA are shown in black to denote the A, P and E sites, respectively. FIG +19 22 30S complex_assembly The extents of the 30S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows. FIG +23 30 subunit structure_element The extents of the 30S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows. FIG +44 48 head structure_element The extents of the 30S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows. FIG +91 109 post-translocation protein_state The extents of the 30S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows. FIG +110 119 structure evidence The extents of the 30S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows. FIG +32 42 structures evidence References and PDB codes of the structures are shown. FIG +4 14 Structures evidence (b) Structures of the 80S•IRES complexes in the absence and presence of eEF2 (this work). FIG +22 30 80S•IRES complex_assembly (b) Structures of the 80S•IRES complexes in the absence and presence of eEF2 (this work). FIG +48 55 absence protein_state (b) Structures of the 80S•IRES complexes in the absence and presence of eEF2 (this work). FIG +60 71 presence of protein_state (b) Structures of the 80S•IRES complexes in the absence and presence of eEF2 (this work). FIG +72 76 eEF2 protein (b) Structures of the 80S•IRES complexes in the absence and presence of eEF2 (this work). FIG +20 27 subunit structure_element The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +50 63 small subunit structure_element The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +81 85 head structure_element The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +105 109 body structure_element The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +116 119 TSV species The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +120 124 IRES site The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +133 137 eEF2 protein The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +12 17 C1274 residue_name_number Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +19 24 U1191 residue_name_number Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +32 35 40S complex_assembly Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +36 40 head structure_element Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +45 49 G904 residue_name_number Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +57 65 platform structure_element Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +84 89 C1054 residue_name_number Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +91 95 G966 residue_name_number Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +100 104 G693 residue_name_number Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +108 115 E. coli species Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +116 124 16S rRNA chemical Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +159 175 A, P and E sites site Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +26 30 IRES site Unresolved regions of the IRES in densities for Structures III and V are shown in gray. FIG +34 43 densities evidence Unresolved regions of the IRES in densities for Structures III and V are shown in gray. FIG +48 68 Structures III and V evidence Unresolved regions of the IRES in densities for Structures III and V are shown in gray. FIG +26 30 IRES site Unresolved regions of the IRES in densities for Structures III and V are shown in gray. FIG +34 43 densities evidence Unresolved regions of the IRES in densities for Structures III and V are shown in gray. FIG +48 68 Structures III and V evidence Unresolved regions of the IRES in densities for Structures III and V are shown in gray. FIG +19 22 40S complex_assembly The extents of the 40S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows. FIG +23 30 subunit structure_element The extents of the 40S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows. FIG +44 48 head structure_element The extents of the 40S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows. FIG +91 109 post-translocation protein_state The extents of the 40S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows. FIG +110 119 structure evidence The extents of the 40S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows. FIG +13 20 cryo-EM experimental_method Schematic of cryo-EM refinement and classification procedures. FIG +4 13 particles experimental_method All particles were initially aligned to a single model. FIG +0 17 3D classification experimental_method 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. FIG +26 33 3D mask evidence 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. FIG +45 48 40S complex_assembly 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. FIG +49 53 head structure_element 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. FIG +55 58 TSV species 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. FIG +59 63 IRES site 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. FIG +68 72 eEF2 protein 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. FIG +91 96 stack bond_interaction 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. FIG +118 127 particles experimental_method 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. FIG +148 152 IRES site 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. FIG +157 161 eEF2 protein 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. FIG +11 28 3D classification experimental_method Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). FIG +37 44 2D mask evidence Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). FIG +56 59 PKI structure_element Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). FIG +71 73 IV structure_element Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). FIG +77 81 eEF2 protein Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). FIG +124 146 Structures I through V evidence Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). FIG +148 166 Sub-classification experimental_method Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). FIG +234 241 density evidence Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). FIG +249 252 PKI structure_element Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). FIG +313 322 particles experimental_method Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). FIG +330 344 sub-classified experimental_method Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). FIG +345 359 reconstruction evidence Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses). FIG +0 7 Cryo-EM experimental_method Cryo-EM density of Structures I-V. FIG +8 15 density evidence Cryo-EM density of Structures I-V. FIG +19 33 Structures I-V evidence Cryo-EM density of Structures I-V. FIG +21 25 maps evidence In panels (a-e), the maps are segmented and colored as in Figure 1. FIG +4 8 maps evidence The maps in all panels were B-softened by applying a B-factor of 30 Å2. FIG +6 13 Cryo-EM experimental_method (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +14 17 map evidence (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +21 52 Structures I, II, III, IV and V evidence (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +104 111 cryo-EM experimental_method (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +112 127 reconstructions evidence (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +144 151 Blocres experimental_method (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +180 211 Structures I, II, III, IV and V evidence (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +219 226 Cryo-EM experimental_method (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +227 234 density evidence (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +243 246 TSV species (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +247 251 IRES site (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +268 272 eEF2 protein (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +290 321 Structures I, II, III, IV and V evidence (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +327 352 Fourier shell correlation evidence (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +354 357 FSC evidence (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +359 365 curves evidence (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +370 384 Structures I-V evidence (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +508 511 FSC evidence (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +570 578 FREALIGN experimental_method (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +587 590 FSC evidence (a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency Å-1 and with Å. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC'). FIG +0 7 Cryo-EM experimental_method Cryo-EM structures of the 80S•TSV IRES bound with eEF2•GDP•sordarin. FIG +8 18 structures evidence Cryo-EM structures of the 80S•TSV IRES bound with eEF2•GDP•sordarin. FIG +26 38 80S•TSV IRES complex_assembly Cryo-EM structures of the 80S•TSV IRES bound with eEF2•GDP•sordarin. FIG +39 49 bound with protein_state Cryo-EM structures of the 80S•TSV IRES bound with eEF2•GDP•sordarin. FIG +50 67 eEF2•GDP•sordarin complex_assembly Cryo-EM structures of the 80S•TSV IRES bound with eEF2•GDP•sordarin. FIG +4 26 Structures I through V evidence (a) Structures I through V. In all panels, the large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +47 70 large ribosomal subunit structure_element (a) Structures I through V. In all panels, the large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +93 106 small subunit structure_element (a) Structures I through V. In all panels, the large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +124 128 head structure_element (a) Structures I through V. In all panels, the large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +148 152 body structure_element (a) Structures I through V. In all panels, the large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +159 162 TSV species (a) Structures I through V. In all panels, the large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +163 167 IRES site (a) Structures I through V. In all panels, the large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +176 180 eEF2 protein (a) Structures I through V. In all panels, the large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. FIG +12 17 C1274 residue_name_number Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +19 24 U1191 residue_name_number Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +32 35 40S complex_assembly Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +36 40 head structure_element Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +45 49 G904 residue_name_number Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +57 65 platform site Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +67 72 C1054 residue_name_number Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +74 78 G966 residue_name_number Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +83 87 G693 residue_name_number Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +91 98 E. coli species Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +99 107 16S rRNA chemical Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +142 158 A, P and E sites site Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. FIG +36 46 structures evidence (b) Schematic representation of the structures shown in panel a, denoting the conformations of the small subunit relative to the large subunit. FIG +99 112 small subunit structure_element (b) Schematic representation of the structures shown in panel a, denoting the conformations of the small subunit relative to the large subunit. FIG +129 142 large subunit structure_element (b) Schematic representation of the structures shown in panel a, denoting the conformations of the small subunit relative to the large subunit. FIG +0 16 A, P and E sites site A, P and E sites are shown as rectangles. FIG +37 48 non-rotated protein_state All measurements are relative to the non-rotated 80S•2tRNA•mRNA structure. FIG +49 63 80S•2tRNA•mRNA complex_assembly All measurements are relative to the non-rotated 80S•2tRNA•mRNA structure. FIG +64 73 structure evidence All measurements are relative to the non-rotated 80S•2tRNA•mRNA structure. FIG +37 48 non-rotated protein_state All measurements are relative to the non-rotated 80S•2tRNA•mRNA structure. FIG +49 63 80S•2tRNA•mRNA complex_assembly All measurements are relative to the non-rotated 80S•2tRNA•mRNA structure. FIG +64 73 structure evidence All measurements are relative to the non-rotated 80S•2tRNA•mRNA structure. FIG +48 72 structural visualization experimental_method We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +76 89 80S•IRES•eEF2 complex_assembly We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +136 140 IRES site We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +141 144 RNA chemical We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +202 205 PKI structure_element We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +215 226 A to P site site We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +234 247 small subunit structure_element We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +262 266 eEF2 protein We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +275 279 IRES site We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +304 308 IRES site We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +359 367 ribosome complex_assembly We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +380 384 tRNA chemical We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +445 448 40S complex_assembly We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +449 453 head structure_element We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +466 470 IRES site We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? INTRO +8 15 cryo-EM experimental_method We used cryo-EM to visualize 80S•TSV IRES complexes formed in the presence of eEF2•GTP and the translation inhibitor sordarin, which stabilizes eEF2 on the ribosome. INTRO +29 41 80S•TSV IRES complex_assembly We used cryo-EM to visualize 80S•TSV IRES complexes formed in the presence of eEF2•GTP and the translation inhibitor sordarin, which stabilizes eEF2 on the ribosome. INTRO +66 77 presence of protein_state We used cryo-EM to visualize 80S•TSV IRES complexes formed in the presence of eEF2•GTP and the translation inhibitor sordarin, which stabilizes eEF2 on the ribosome. INTRO +78 86 eEF2•GTP complex_assembly We used cryo-EM to visualize 80S•TSV IRES complexes formed in the presence of eEF2•GTP and the translation inhibitor sordarin, which stabilizes eEF2 on the ribosome. INTRO +117 125 sordarin chemical We used cryo-EM to visualize 80S•TSV IRES complexes formed in the presence of eEF2•GTP and the translation inhibitor sordarin, which stabilizes eEF2 on the ribosome. INTRO +144 148 eEF2 protein We used cryo-EM to visualize 80S•TSV IRES complexes formed in the presence of eEF2•GTP and the translation inhibitor sordarin, which stabilizes eEF2 on the ribosome. INTRO +156 164 ribosome complex_assembly We used cryo-EM to visualize 80S•TSV IRES complexes formed in the presence of eEF2•GTP and the translation inhibitor sordarin, which stabilizes eEF2 on the ribosome. INTRO +26 34 sordarin chemical Although the mechanism of sordarin action is not fully understood, the inhibitor does not affect the conformation of eEF2•GDPNP on the ribosome, rendering it an excellent tool in translocation studies. INTRO +117 127 eEF2•GDPNP complex_assembly Although the mechanism of sordarin action is not fully understood, the inhibitor does not affect the conformation of eEF2•GDPNP on the ribosome, rendering it an excellent tool in translocation studies. INTRO +135 143 ribosome complex_assembly Although the mechanism of sordarin action is not fully understood, the inhibitor does not affect the conformation of eEF2•GDPNP on the ribosome, rendering it an excellent tool in translocation studies. INTRO +0 33 Maximum-likelihood classification experimental_method Maximum-likelihood classification using FREALIGN identified five IRES-eEF2-bound ribosome structures within a single sample (Figures 1 and 2). INTRO +40 48 FREALIGN experimental_method Maximum-likelihood classification using FREALIGN identified five IRES-eEF2-bound ribosome structures within a single sample (Figures 1 and 2). INTRO +65 80 IRES-eEF2-bound protein_state Maximum-likelihood classification using FREALIGN identified five IRES-eEF2-bound ribosome structures within a single sample (Figures 1 and 2). INTRO +81 89 ribosome complex_assembly Maximum-likelihood classification using FREALIGN identified five IRES-eEF2-bound ribosome structures within a single sample (Figures 1 and 2). INTRO +90 100 structures evidence Maximum-likelihood classification using FREALIGN identified five IRES-eEF2-bound ribosome structures within a single sample (Figures 1 and 2). INTRO +4 14 structures evidence The structures differ in the positions and conformations of ribosomal subunits (Figures 1b and 2), IRES RNA (Figures 3 and 4) and eEF2 (Figures 5 and 6). INTRO +99 103 IRES site The structures differ in the positions and conformations of ribosomal subunits (Figures 1b and 2), IRES RNA (Figures 3 and 4) and eEF2 (Figures 5 and 6). INTRO +104 107 RNA chemical The structures differ in the positions and conformations of ribosomal subunits (Figures 1b and 2), IRES RNA (Figures 3 and 4) and eEF2 (Figures 5 and 6). INTRO +130 134 eEF2 protein The structures differ in the positions and conformations of ribosomal subunits (Figures 1b and 2), IRES RNA (Figures 3 and 4) and eEF2 (Figures 5 and 6). INTRO +17 27 structures evidence This ensemble of structures allowed us to reconstruct a sequence of steps in IRES translocation induced by eEF2. INTRO +77 81 IRES site This ensemble of structures allowed us to reconstruct a sequence of steps in IRES translocation induced by eEF2. INTRO +107 111 eEF2 protein This ensemble of structures allowed us to reconstruct a sequence of steps in IRES translocation induced by eEF2. INTRO +8 31 single-particle cryo-EM experimental_method We used single-particle cryo-EM and maximum-likelihood image classification in FREALIGN to obtain three-dimensional density maps from a single specimen. RESULTS +36 75 maximum-likelihood image classification experimental_method We used single-particle cryo-EM and maximum-likelihood image classification in FREALIGN to obtain three-dimensional density maps from a single specimen. RESULTS +79 87 FREALIGN experimental_method We used single-particle cryo-EM and maximum-likelihood image classification in FREALIGN to obtain three-dimensional density maps from a single specimen. RESULTS +116 128 density maps evidence We used single-particle cryo-EM and maximum-likelihood image classification in FREALIGN to obtain three-dimensional density maps from a single specimen. RESULTS +43 56 S. cerevisiae species The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. RESULTS +57 70 80S ribosomes complex_assembly The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. RESULTS +72 92 Taura syndrome virus species The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. RESULTS +93 97 IRES site The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. RESULTS +103 116 S. cerevisiae species The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. RESULTS +117 121 eEF2 protein The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. RESULTS +129 140 presence of protein_state The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. RESULTS +141 144 GTP chemical The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. RESULTS +153 157 eEF2 protein The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. RESULTS +188 196 sordarin chemical The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. RESULTS +0 40 Unsupervised cryo-EM data classification experimental_method Unsupervised cryo-EM data classification was combined with the use of three-dimensional and two-dimensional masking around the ribosomal A site (Figure 1—figure supplement 2). RESULTS +70 115 three-dimensional and two-dimensional masking experimental_method Unsupervised cryo-EM data classification was combined with the use of three-dimensional and two-dimensional masking around the ribosomal A site (Figure 1—figure supplement 2). RESULTS +137 143 A site site Unsupervised cryo-EM data classification was combined with the use of three-dimensional and two-dimensional masking around the ribosomal A site (Figure 1—figure supplement 2). RESULTS +28 45 80S•IRES•eEF2•GDP complex_assembly This approach revealed five 80S•IRES•eEF2•GDP structures at average resolutions of 3.5 to 4.2 Å, sufficient to locate IRES domains and to resolve individual residues in the core regions of the ribosome and eEF2 (Figures 3c,d, and 5f,h; see also Figure 1—figure supplement 2 and Figure 5—figure supplement 2), including the post-translational modification diphthamide 699 (Figure 3c). RESULTS +46 56 structures evidence This approach revealed five 80S•IRES•eEF2•GDP structures at average resolutions of 3.5 to 4.2 Å, sufficient to locate IRES domains and to resolve individual residues in the core regions of the ribosome and eEF2 (Figures 3c,d, and 5f,h; see also Figure 1—figure supplement 2 and Figure 5—figure supplement 2), including the post-translational modification diphthamide 699 (Figure 3c). RESULTS +118 122 IRES site This approach revealed five 80S•IRES•eEF2•GDP structures at average resolutions of 3.5 to 4.2 Å, sufficient to locate IRES domains and to resolve individual residues in the core regions of the ribosome and eEF2 (Figures 3c,d, and 5f,h; see also Figure 1—figure supplement 2 and Figure 5—figure supplement 2), including the post-translational modification diphthamide 699 (Figure 3c). RESULTS +193 201 ribosome complex_assembly This approach revealed five 80S•IRES•eEF2•GDP structures at average resolutions of 3.5 to 4.2 Å, sufficient to locate IRES domains and to resolve individual residues in the core regions of the ribosome and eEF2 (Figures 3c,d, and 5f,h; see also Figure 1—figure supplement 2 and Figure 5—figure supplement 2), including the post-translational modification diphthamide 699 (Figure 3c). RESULTS +206 210 eEF2 protein This approach revealed five 80S•IRES•eEF2•GDP structures at average resolutions of 3.5 to 4.2 Å, sufficient to locate IRES domains and to resolve individual residues in the core regions of the ribosome and eEF2 (Figures 3c,d, and 5f,h; see also Figure 1—figure supplement 2 and Figure 5—figure supplement 2), including the post-translational modification diphthamide 699 (Figure 3c). RESULTS +355 370 diphthamide 699 ptm This approach revealed five 80S•IRES•eEF2•GDP structures at average resolutions of 3.5 to 4.2 Å, sufficient to locate IRES domains and to resolve individual residues in the core regions of the ribosome and eEF2 (Figures 3c,d, and 5f,h; see also Figure 1—figure supplement 2 and Figure 5—figure supplement 2), including the post-translational modification diphthamide 699 (Figure 3c). RESULTS +30 52 Structures I through V evidence Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site. FIG +83 86 PKI structure_element Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site. FIG +96 107 A to P site site Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site. FIG +112 116 eEF2 protein Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site. FIG +132 138 A site site Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site. FIG +30 52 Structures I through V evidence Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site. FIG +83 86 PKI structure_element Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site. FIG +96 107 A to P site site Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site. FIG +112 116 eEF2 protein Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site. FIG +132 138 A site site Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site. FIG +29 32 40S complex_assembly (a) Rotational states of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +33 40 subunit structure_element (a) Rotational states of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +48 56 80S•IRES complex_assembly (a) Rotational states of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +57 66 structure evidence (a) Rotational states of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +68 72 INIT complex_assembly (a) Rotational states of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +91 104 80S•IRES•eEF2 complex_assembly (a) Rotational states of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +105 136 Structures I, II, III, IV and V evidence (a) Rotational states of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +9 18 structure evidence For each structure, the triangle outlines the contours of the 40S body; the lower angle illustrates the extent of intersubunit (body) rotation. FIG +62 65 40S complex_assembly For each structure, the triangle outlines the contours of the 40S body; the lower angle illustrates the extent of intersubunit (body) rotation. FIG +66 70 body structure_element For each structure, the triangle outlines the contours of the 40S body; the lower angle illustrates the extent of intersubunit (body) rotation. FIG +128 132 body structure_element For each structure, the triangle outlines the contours of the 40S body; the lower angle illustrates the extent of intersubunit (body) rotation. FIG +56 60 head structure_element The sizes of the arrows correspond to the extent of the head swivel (yellow) and subunit rotation (black). FIG +81 88 subunit structure_element The sizes of the arrows correspond to the extent of the head swivel (yellow) and subunit rotation (black). FIG +27 47 structural alignment experimental_method The views were obtained by structural alignment of the 25S rRNAs; the sarcin-ricin loop (SRL) of 25S rRNA is shown in gray for reference. FIG +55 64 25S rRNAs chemical The views were obtained by structural alignment of the 25S rRNAs; the sarcin-ricin loop (SRL) of 25S rRNA is shown in gray for reference. FIG +70 87 sarcin-ricin loop structure_element The views were obtained by structural alignment of the 25S rRNAs; the sarcin-ricin loop (SRL) of 25S rRNA is shown in gray for reference. FIG +89 92 SRL structure_element The views were obtained by structural alignment of the 25S rRNAs; the sarcin-ricin loop (SRL) of 25S rRNA is shown in gray for reference. FIG +97 105 25S rRNA chemical The views were obtained by structural alignment of the 25S rRNAs; the sarcin-ricin loop (SRL) of 25S rRNA is shown in gray for reference. FIG +58 61 40S complex_assembly (b) Solvent view (opposite from that shown in (a)) of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +62 69 subunit structure_element (b) Solvent view (opposite from that shown in (a)) of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +77 85 80S•IRES complex_assembly (b) Solvent view (opposite from that shown in (a)) of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +86 95 structure evidence (b) Solvent view (opposite from that shown in (a)) of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +97 101 INIT complex_assembly (b) Solvent view (opposite from that shown in (a)) of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +120 133 80S•IRES•eEF2 complex_assembly (b) Solvent view (opposite from that shown in (a)) of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +134 165 Structures I, II, III, IV and V evidence (b) Solvent view (opposite from that shown in (a)) of the 40S subunit in the 80S•IRES structure (INIT; PDB 3J6Y) and in 80S•IRES•eEF2 Structures I, II, III, IV and V (this work). FIG +4 14 structures evidence The structures are colored as in Figure 1. FIG +22 25 40S complex_assembly (a) Comparison of the 40S-subunit rotational states in Structures I through V, sampling a ~10° range between Structure I (fully rotated) and Structure V (non-rotated). FIG +26 33 subunit structure_element (a) Comparison of the 40S-subunit rotational states in Structures I through V, sampling a ~10° range between Structure I (fully rotated) and Structure V (non-rotated). FIG +55 77 Structures I through V evidence (a) Comparison of the 40S-subunit rotational states in Structures I through V, sampling a ~10° range between Structure I (fully rotated) and Structure V (non-rotated). FIG +109 120 Structure I evidence (a) Comparison of the 40S-subunit rotational states in Structures I through V, sampling a ~10° range between Structure I (fully rotated) and Structure V (non-rotated). FIG +122 135 fully rotated protein_state (a) Comparison of the 40S-subunit rotational states in Structures I through V, sampling a ~10° range between Structure I (fully rotated) and Structure V (non-rotated). FIG +141 152 Structure V evidence (a) Comparison of the 40S-subunit rotational states in Structures I through V, sampling a ~10° range between Structure I (fully rotated) and Structure V (non-rotated). FIG +154 165 non-rotated protein_state (a) Comparison of the 40S-subunit rotational states in Structures I through V, sampling a ~10° range between Structure I (fully rotated) and Structure V (non-rotated). FIG +0 17 18S ribosomal RNA chemical 18S ribosomal RNA is shown and ribosomal proteins are omitted for clarity. FIG +4 18 superpositions experimental_method The superpositions of Structures I-V were performed by structural alignments of the 25S ribosomal RNAs. FIG +22 36 Structures I-V evidence The superpositions of Structures I-V were performed by structural alignments of the 25S ribosomal RNAs. FIG +55 76 structural alignments experimental_method The superpositions of Structures I-V were performed by structural alignments of the 25S ribosomal RNAs. FIG +84 102 25S ribosomal RNAs chemical The superpositions of Structures I-V were performed by structural alignments of the 25S ribosomal RNAs. FIG +47 50 40S complex_assembly (b) Bar graph of the angles characterizing the 40S rotational and 40S head swiveling states in Structures I through V. Measurements for the two 80S•IRES (INIT) structures are included for comparison. FIG +66 69 40S complex_assembly (b) Bar graph of the angles characterizing the 40S rotational and 40S head swiveling states in Structures I through V. Measurements for the two 80S•IRES (INIT) structures are included for comparison. FIG +70 74 head structure_element (b) Bar graph of the angles characterizing the 40S rotational and 40S head swiveling states in Structures I through V. Measurements for the two 80S•IRES (INIT) structures are included for comparison. FIG +95 117 Structures I through V evidence (b) Bar graph of the angles characterizing the 40S rotational and 40S head swiveling states in Structures I through V. Measurements for the two 80S•IRES (INIT) structures are included for comparison. FIG +144 152 80S•IRES complex_assembly (b) Bar graph of the angles characterizing the 40S rotational and 40S head swiveling states in Structures I through V. Measurements for the two 80S•IRES (INIT) structures are included for comparison. FIG +154 158 INIT complex_assembly (b) Bar graph of the angles characterizing the 40S rotational and 40S head swiveling states in Structures I through V. Measurements for the two 80S•IRES (INIT) structures are included for comparison. FIG +160 170 structures evidence (b) Bar graph of the angles characterizing the 40S rotational and 40S head swiveling states in Structures I through V. Measurements for the two 80S•IRES (INIT) structures are included for comparison. FIG +22 25 40S complex_assembly (c) Comparison of the 40S conformations in Structures I through V shows distinct positions of the head relative to the body of the 40S subunit (head swivel). FIG +43 65 Structures I through V evidence (c) Comparison of the 40S conformations in Structures I through V shows distinct positions of the head relative to the body of the 40S subunit (head swivel). FIG +98 102 head structure_element (c) Comparison of the 40S conformations in Structures I through V shows distinct positions of the head relative to the body of the 40S subunit (head swivel). FIG +119 123 body structure_element (c) Comparison of the 40S conformations in Structures I through V shows distinct positions of the head relative to the body of the 40S subunit (head swivel). FIG +131 134 40S complex_assembly (c) Comparison of the 40S conformations in Structures I through V shows distinct positions of the head relative to the body of the 40S subunit (head swivel). FIG +135 142 subunit structure_element (c) Comparison of the 40S conformations in Structures I through V shows distinct positions of the head relative to the body of the 40S subunit (head swivel). FIG +144 148 head structure_element (c) Comparison of the 40S conformations in Structures I through V shows distinct positions of the head relative to the body of the 40S subunit (head swivel). FIG +20 32 non-swiveled protein_state Conformation of the non-swiveled 40S subunit in the S. cerevisiae 80S ribosome bound with two tRNAs is shown for reference (blue). FIG +33 36 40S complex_assembly Conformation of the non-swiveled 40S subunit in the S. cerevisiae 80S ribosome bound with two tRNAs is shown for reference (blue). FIG +37 44 subunit structure_element Conformation of the non-swiveled 40S subunit in the S. cerevisiae 80S ribosome bound with two tRNAs is shown for reference (blue). FIG +52 65 S. cerevisiae species Conformation of the non-swiveled 40S subunit in the S. cerevisiae 80S ribosome bound with two tRNAs is shown for reference (blue). FIG +66 78 80S ribosome complex_assembly Conformation of the non-swiveled 40S subunit in the S. cerevisiae 80S ribosome bound with two tRNAs is shown for reference (blue). FIG +79 89 bound with protein_state Conformation of the non-swiveled 40S subunit in the S. cerevisiae 80S ribosome bound with two tRNAs is shown for reference (blue). FIG +94 99 tRNAs chemical Conformation of the non-swiveled 40S subunit in the S. cerevisiae 80S ribosome bound with two tRNAs is shown for reference (blue). FIG +39 41 L1 structure_element (d) Comparison of conformations of the L1 and P stalks of the large subunit in Structures I through V with those in the 80S•IRES and tRNA-bound 80S structures. FIG +46 54 P stalks structure_element (d) Comparison of conformations of the L1 and P stalks of the large subunit in Structures I through V with those in the 80S•IRES and tRNA-bound 80S structures. FIG +62 75 large subunit structure_element (d) Comparison of conformations of the L1 and P stalks of the large subunit in Structures I through V with those in the 80S•IRES and tRNA-bound 80S structures. FIG +79 101 Structures I through V evidence (d) Comparison of conformations of the L1 and P stalks of the large subunit in Structures I through V with those in the 80S•IRES and tRNA-bound 80S structures. FIG +120 128 80S•IRES complex_assembly (d) Comparison of conformations of the L1 and P stalks of the large subunit in Structures I through V with those in the 80S•IRES and tRNA-bound 80S structures. FIG +133 143 tRNA-bound protein_state (d) Comparison of conformations of the L1 and P stalks of the large subunit in Structures I through V with those in the 80S•IRES and tRNA-bound 80S structures. FIG +144 147 80S complex_assembly (d) Comparison of conformations of the L1 and P stalks of the large subunit in Structures I through V with those in the 80S•IRES and tRNA-bound 80S structures. FIG +148 158 structures evidence (d) Comparison of conformations of the L1 and P stalks of the large subunit in Structures I through V with those in the 80S•IRES and tRNA-bound 80S structures. FIG +0 14 Superpositions experimental_method Superpositions were performed by structural alignments of 25S ribosomal RNAs. FIG +33 54 structural alignments experimental_method Superpositions were performed by structural alignments of 25S ribosomal RNAs. FIG +58 76 25S ribosomal RNAs chemical Superpositions were performed by structural alignments of 25S ribosomal RNAs. FIG +4 24 central protuberance structure_element The central protuberance (CP) is labeled. FIG +26 28 CP structure_element The central protuberance (CP) is labeled. FIG +35 38 PKI structure_element  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +50 52 IV structure_element  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +56 60 eEF2 protein  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +77 83 P site site  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +100 104 head structure_element  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +106 111 U1191 residue_name_number  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +117 121 body structure_element  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +123 128 C1637 residue_name_number  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +133 155 Structures I through V evidence  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +206 219 A and P sites site  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +229 239 initiation protein_state  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +247 251 INIT complex_assembly  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +273 291 post-translocation protein_state  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +292 303 Structure V evidence  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +398 401 40S complex_assembly  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +402 406 body structure_element  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +428 431 40S complex_assembly  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +432 436 head structure_element  (e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g) Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow. FIG +4 18 superpositions experimental_method The superpositions of structures were performed by structural alignments of the 18S ribosomal RNAs excluding the head region (nt 1150–1620). FIG +22 32 structures evidence The superpositions of structures were performed by structural alignments of the 18S ribosomal RNAs excluding the head region (nt 1150–1620). FIG +51 72 structural alignments experimental_method The superpositions of structures were performed by structural alignments of the 18S ribosomal RNAs excluding the head region (nt 1150–1620). FIG +80 98 18S ribosomal RNAs chemical The superpositions of structures were performed by structural alignments of the 18S ribosomal RNAs excluding the head region (nt 1150–1620). FIG +113 117 head structure_element The superpositions of structures were performed by structural alignments of the 18S ribosomal RNAs excluding the head region (nt 1150–1620). FIG +129 138 1150–1620 residue_range The superpositions of structures were performed by structural alignments of the 18S ribosomal RNAs excluding the head region (nt 1150–1620). FIG +4 14 structures evidence Our structures represent hitherto uncharacterized translocation complexes of the TSV IRES captured within globally distinct 80S conformations (Figures 1b and 2). RESULTS +81 84 TSV species Our structures represent hitherto uncharacterized translocation complexes of the TSV IRES captured within globally distinct 80S conformations (Figures 1b and 2). RESULTS +85 89 IRES site Our structures represent hitherto uncharacterized translocation complexes of the TSV IRES captured within globally distinct 80S conformations (Figures 1b and 2). RESULTS +124 127 80S complex_assembly Our structures represent hitherto uncharacterized translocation complexes of the TSV IRES captured within globally distinct 80S conformations (Figures 1b and 2). RESULTS +16 38 structures from I to V evidence We numbered the structures from I to V, according to the position of the tRNA-mRNA-like PKI on the 40S subunit (Figure 2—source data 1). RESULTS +73 82 tRNA-mRNA complex_assembly We numbered the structures from I to V, according to the position of the tRNA-mRNA-like PKI on the 40S subunit (Figure 2—source data 1). RESULTS +88 91 PKI structure_element We numbered the structures from I to V, according to the position of the tRNA-mRNA-like PKI on the 40S subunit (Figure 2—source data 1). RESULTS +99 102 40S complex_assembly We numbered the structures from I to V, according to the position of the tRNA-mRNA-like PKI on the 40S subunit (Figure 2—source data 1). RESULTS +103 110 subunit structure_element We numbered the structures from I to V, according to the position of the tRNA-mRNA-like PKI on the 40S subunit (Figure 2—source data 1). RESULTS +14 17 PKI structure_element Specifically, PKI is partially withdrawn from the A site in Structure I, and fully translocated to the P site in Structure V (Figure 4; see also Figure 3—figure supplement 1). RESULTS +50 56 A site site Specifically, PKI is partially withdrawn from the A site in Structure I, and fully translocated to the P site in Structure V (Figure 4; see also Figure 3—figure supplement 1). RESULTS +60 71 Structure I evidence Specifically, PKI is partially withdrawn from the A site in Structure I, and fully translocated to the P site in Structure V (Figure 4; see also Figure 3—figure supplement 1). RESULTS +77 95 fully translocated protein_state Specifically, PKI is partially withdrawn from the A site in Structure I, and fully translocated to the P site in Structure V (Figure 4; see also Figure 3—figure supplement 1). RESULTS +103 109 P site site Specifically, PKI is partially withdrawn from the A site in Structure I, and fully translocated to the P site in Structure V (Figure 4; see also Figure 3—figure supplement 1). RESULTS +113 124 Structure V evidence Specifically, PKI is partially withdrawn from the A site in Structure I, and fully translocated to the P site in Structure V (Figure 4; see also Figure 3—figure supplement 1). RESULTS +5 23 Structures I to IV evidence Thus Structures I to IV represent different positions of PKI between the A and P sites (Figure 2—source data 1), suggesting that these structures describe intermediate states of translocation. RESULTS +57 60 PKI structure_element Thus Structures I to IV represent different positions of PKI between the A and P sites (Figure 2—source data 1), suggesting that these structures describe intermediate states of translocation. RESULTS +73 86 A and P sites site Thus Structures I to IV represent different positions of PKI between the A and P sites (Figure 2—source data 1), suggesting that these structures describe intermediate states of translocation. RESULTS +135 145 structures evidence Thus Structures I to IV represent different positions of PKI between the A and P sites (Figure 2—source data 1), suggesting that these structures describe intermediate states of translocation. RESULTS +0 11 Structure V evidence Structure V corresponds to the post-translocation state. RESULTS +31 49 post-translocation protein_state Structure V corresponds to the post-translocation state. RESULTS +11 19 ribosome complex_assembly Changes in ribosome conformation and eEF2 positions are coupled with IRES movement through the ribosome RESULTS +37 41 eEF2 protein Changes in ribosome conformation and eEF2 positions are coupled with IRES movement through the ribosome RESULTS +69 73 IRES site Changes in ribosome conformation and eEF2 positions are coupled with IRES movement through the ribosome RESULTS +95 103 ribosome complex_assembly Changes in ribosome conformation and eEF2 positions are coupled with IRES movement through the ribosome RESULTS +10 28 post-translocation protein_state Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). RESULTS +29 42 S. cerevisiae species Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). RESULTS +43 55 80S ribosome complex_assembly Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). RESULTS +56 66 bound with protein_state Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). RESULTS +71 83 P and E site site Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). RESULTS +84 89 tRNAs chemical Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). RESULTS +106 120 80S•2tRNA•mRNA complex_assembly Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). RESULTS +141 148 subunit structure_element Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). RESULTS +166 170 head structure_element Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). RESULTS +171 175 body structure_element Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). RESULTS +209 217 ribosome complex_assembly Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). RESULTS +265 287 Structures I through V evidence Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). RESULTS +0 11 Structure I evidence Structure I comprises the most rotated ribosome conformation (~10°), characteristic of pre-translocation hybrid-tRNA states. RESULTS +26 38 most rotated protein_state Structure I comprises the most rotated ribosome conformation (~10°), characteristic of pre-translocation hybrid-tRNA states. RESULTS +39 47 ribosome complex_assembly Structure I comprises the most rotated ribosome conformation (~10°), characteristic of pre-translocation hybrid-tRNA states. RESULTS +87 104 pre-translocation protein_state Structure I comprises the most rotated ribosome conformation (~10°), characteristic of pre-translocation hybrid-tRNA states. RESULTS +105 116 hybrid-tRNA protein_state Structure I comprises the most rotated ribosome conformation (~10°), characteristic of pre-translocation hybrid-tRNA states. RESULTS +5 21 Structure I to V evidence From Structure I to V, the body of the small subunit undergoes backward (reverse) rotation (Figure 2b; see also Figure 1—figure supplement 2 and Figure 2—figure supplement 1). RESULTS +27 31 body structure_element From Structure I to V, the body of the small subunit undergoes backward (reverse) rotation (Figure 2b; see also Figure 1—figure supplement 2 and Figure 2—figure supplement 1). RESULTS +39 52 small subunit structure_element From Structure I to V, the body of the small subunit undergoes backward (reverse) rotation (Figure 2b; see also Figure 1—figure supplement 2 and Figure 2—figure supplement 1). RESULTS +0 21 Structures II and III evidence Structures II and III are in mid-rotation conformations (~5°). RESULTS +29 41 mid-rotation protein_state Structures II and III are in mid-rotation conformations (~5°). RESULTS +0 12 Structure IV evidence Structure IV adopts a slightly rotated conformation (~1°). RESULTS +22 38 slightly rotated protein_state Structure IV adopts a slightly rotated conformation (~1°). RESULTS +0 11 Structure V evidence Structure V is in a nearly non-rotated conformation (0.5°), very similar to that of post-translocation ribosome-tRNA complexes. RESULTS +27 38 non-rotated protein_state Structure V is in a nearly non-rotated conformation (0.5°), very similar to that of post-translocation ribosome-tRNA complexes. RESULTS +84 102 post-translocation protein_state Structure V is in a nearly non-rotated conformation (0.5°), very similar to that of post-translocation ribosome-tRNA complexes. RESULTS +103 116 ribosome-tRNA complex_assembly Structure V is in a nearly non-rotated conformation (0.5°), very similar to that of post-translocation ribosome-tRNA complexes. RESULTS +40 56 Structure I to V evidence Thus, intersubunit rotation of ~9° from Structure I to V covers a nearly complete range of relative subunit positions, similar to what was reported for tRNA-bound yeast, bacterial and mammalian ribosomes. RESULTS +100 107 subunit structure_element Thus, intersubunit rotation of ~9° from Structure I to V covers a nearly complete range of relative subunit positions, similar to what was reported for tRNA-bound yeast, bacterial and mammalian ribosomes. RESULTS +152 162 tRNA-bound protein_state Thus, intersubunit rotation of ~9° from Structure I to V covers a nearly complete range of relative subunit positions, similar to what was reported for tRNA-bound yeast, bacterial and mammalian ribosomes. RESULTS +163 168 yeast taxonomy_domain Thus, intersubunit rotation of ~9° from Structure I to V covers a nearly complete range of relative subunit positions, similar to what was reported for tRNA-bound yeast, bacterial and mammalian ribosomes. RESULTS +170 179 bacterial taxonomy_domain Thus, intersubunit rotation of ~9° from Structure I to V covers a nearly complete range of relative subunit positions, similar to what was reported for tRNA-bound yeast, bacterial and mammalian ribosomes. RESULTS +184 193 mammalian taxonomy_domain Thus, intersubunit rotation of ~9° from Structure I to V covers a nearly complete range of relative subunit positions, similar to what was reported for tRNA-bound yeast, bacterial and mammalian ribosomes. RESULTS +194 203 ribosomes complex_assembly Thus, intersubunit rotation of ~9° from Structure I to V covers a nearly complete range of relative subunit positions, similar to what was reported for tRNA-bound yeast, bacterial and mammalian ribosomes. RESULTS +0 3 40S complex_assembly 40S head swivel RESULTS +4 8 head structure_element 40S head swivel RESULTS +15 18 40S complex_assembly The pattern of 40S head swivel between the structures is different from that of intersubunit rotation (Figures 2c and d; see also Figure 2—source data 1). RESULTS +19 23 head structure_element The pattern of 40S head swivel between the structures is different from that of intersubunit rotation (Figures 2c and d; see also Figure 2—source data 1). RESULTS +43 53 structures evidence The pattern of 40S head swivel between the structures is different from that of intersubunit rotation (Figures 2c and d; see also Figure 2—source data 1). RESULTS +45 49 head structure_element As with the intersubunit rotation, the small head swivel (~1°) in the non-rotated Structure V is closest to that in the 80S•2tRNA•mRNA post-translocation ribosome. RESULTS +70 81 non-rotated protein_state As with the intersubunit rotation, the small head swivel (~1°) in the non-rotated Structure V is closest to that in the 80S•2tRNA•mRNA post-translocation ribosome. RESULTS +82 93 Structure V evidence As with the intersubunit rotation, the small head swivel (~1°) in the non-rotated Structure V is closest to that in the 80S•2tRNA•mRNA post-translocation ribosome. RESULTS +120 134 80S•2tRNA•mRNA complex_assembly As with the intersubunit rotation, the small head swivel (~1°) in the non-rotated Structure V is closest to that in the 80S•2tRNA•mRNA post-translocation ribosome. RESULTS +135 153 post-translocation protein_state As with the intersubunit rotation, the small head swivel (~1°) in the non-rotated Structure V is closest to that in the 80S•2tRNA•mRNA post-translocation ribosome. RESULTS +154 162 ribosome complex_assembly As with the intersubunit rotation, the small head swivel (~1°) in the non-rotated Structure V is closest to that in the 80S•2tRNA•mRNA post-translocation ribosome. RESULTS +15 32 pre-translocation protein_state However in the pre-translocation intermediates (from Structure I to IV), the beak of the head domain first turns toward the large subunit and then backs off (Figure 2—figure supplement 1). RESULTS +53 70 Structure I to IV evidence However in the pre-translocation intermediates (from Structure I to IV), the beak of the head domain first turns toward the large subunit and then backs off (Figure 2—figure supplement 1). RESULTS +89 93 head structure_element However in the pre-translocation intermediates (from Structure I to IV), the beak of the head domain first turns toward the large subunit and then backs off (Figure 2—figure supplement 1). RESULTS +124 137 large subunit structure_element However in the pre-translocation intermediates (from Structure I to IV), the beak of the head domain first turns toward the large subunit and then backs off (Figure 2—figure supplement 1). RESULTS +4 8 head structure_element The head samples a mid-swiveled position in Structure I (12°), then a highly-swiveled position in Structures II and III (17°) and a less swiveled position in Structure IV (14°). RESULTS +19 31 mid-swiveled protein_state The head samples a mid-swiveled position in Structure I (12°), then a highly-swiveled position in Structures II and III (17°) and a less swiveled position in Structure IV (14°). RESULTS +44 55 Structure I evidence The head samples a mid-swiveled position in Structure I (12°), then a highly-swiveled position in Structures II and III (17°) and a less swiveled position in Structure IV (14°). RESULTS +70 85 highly-swiveled protein_state The head samples a mid-swiveled position in Structure I (12°), then a highly-swiveled position in Structures II and III (17°) and a less swiveled position in Structure IV (14°). RESULTS +98 119 Structures II and III evidence The head samples a mid-swiveled position in Structure I (12°), then a highly-swiveled position in Structures II and III (17°) and a less swiveled position in Structure IV (14°). RESULTS +132 145 less swiveled protein_state The head samples a mid-swiveled position in Structure I (12°), then a highly-swiveled position in Structures II and III (17°) and a less swiveled position in Structure IV (14°). RESULTS +158 170 Structure IV evidence The head samples a mid-swiveled position in Structure I (12°), then a highly-swiveled position in Structures II and III (17°) and a less swiveled position in Structure IV (14°). RESULTS +12 16 head structure_element The maximum head swivel is observed in the mid-rotated complexes II and III, in which PKI transitions from the A to P site, while eEF2 occupies the A site partially. RESULTS +43 54 mid-rotated protein_state The maximum head swivel is observed in the mid-rotated complexes II and III, in which PKI transitions from the A to P site, while eEF2 occupies the A site partially. RESULTS +65 75 II and III evidence The maximum head swivel is observed in the mid-rotated complexes II and III, in which PKI transitions from the A to P site, while eEF2 occupies the A site partially. RESULTS +86 89 PKI structure_element The maximum head swivel is observed in the mid-rotated complexes II and III, in which PKI transitions from the A to P site, while eEF2 occupies the A site partially. RESULTS +111 122 A to P site site The maximum head swivel is observed in the mid-rotated complexes II and III, in which PKI transitions from the A to P site, while eEF2 occupies the A site partially. RESULTS +130 134 eEF2 protein The maximum head swivel is observed in the mid-rotated complexes II and III, in which PKI transitions from the A to P site, while eEF2 occupies the A site partially. RESULTS +148 154 A site site The maximum head swivel is observed in the mid-rotated complexes II and III, in which PKI transitions from the A to P site, while eEF2 occupies the A site partially. RESULTS +29 40 mid-rotated protein_state By comparison, the similarly mid-rotated (4°) 80S•TSV IRES initiation complex, in the absence of eEF2, adopts a mid-swiveled position (~10°) (Figure 2c). RESULTS +46 58 80S•TSV IRES complex_assembly By comparison, the similarly mid-rotated (4°) 80S•TSV IRES initiation complex, in the absence of eEF2, adopts a mid-swiveled position (~10°) (Figure 2c). RESULTS +59 69 initiation protein_state By comparison, the similarly mid-rotated (4°) 80S•TSV IRES initiation complex, in the absence of eEF2, adopts a mid-swiveled position (~10°) (Figure 2c). RESULTS +86 96 absence of protein_state By comparison, the similarly mid-rotated (4°) 80S•TSV IRES initiation complex, in the absence of eEF2, adopts a mid-swiveled position (~10°) (Figure 2c). RESULTS +97 101 eEF2 protein By comparison, the similarly mid-rotated (4°) 80S•TSV IRES initiation complex, in the absence of eEF2, adopts a mid-swiveled position (~10°) (Figure 2c). RESULTS +112 124 mid-swiveled protein_state By comparison, the similarly mid-rotated (4°) 80S•TSV IRES initiation complex, in the absence of eEF2, adopts a mid-swiveled position (~10°) (Figure 2c). RESULTS +32 36 eEF2 protein These observations suggest that eEF2 is necessary for inducing or stabilizing the large head swivel of the 40S subunit characteristic for IRES translocation intermediates. RESULTS +88 92 head structure_element These observations suggest that eEF2 is necessary for inducing or stabilizing the large head swivel of the 40S subunit characteristic for IRES translocation intermediates. RESULTS +107 110 40S complex_assembly These observations suggest that eEF2 is necessary for inducing or stabilizing the large head swivel of the 40S subunit characteristic for IRES translocation intermediates. RESULTS +111 118 subunit structure_element These observations suggest that eEF2 is necessary for inducing or stabilizing the large head swivel of the 40S subunit characteristic for IRES translocation intermediates. RESULTS +138 142 IRES site These observations suggest that eEF2 is necessary for inducing or stabilizing the large head swivel of the 40S subunit characteristic for IRES translocation intermediates. RESULTS +0 4 IRES site IRES rearrangements RESULTS +18 21 TSV species Comparison of the TSV IRES and eEF2 positions in Structures I through V. FIG +22 26 IRES site Comparison of the TSV IRES and eEF2 positions in Structures I through V. FIG +31 35 eEF2 protein Comparison of the TSV IRES and eEF2 positions in Structures I through V. FIG +49 71 Structures I through V evidence Comparison of the TSV IRES and eEF2 positions in Structures I through V. FIG +21 25 IRES site (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +30 34 eEF2 protein (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +42 52 initiation protein_state (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +54 71 pre-translocation protein_state (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +73 74 I evidence (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +80 98 post-translocation protein_state (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +100 101 V evidence (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +127 131 body structure_element (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +139 142 40S complex_assembly (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +143 150 subunit structure_element (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +143 150 subunit structure_element (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +184 188 IRES site (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +193 197 eEF2 protein (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +205 215 initiation protein_state (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +223 227 INIT complex_assembly (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +270 284 II, III and IV evidence (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +303 307 body structure_element (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +315 318 40S complex_assembly (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +319 326 subunit structure_element (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +319 326 subunit structure_element (a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown). FIG +33 54 structural alignments experimental_method  Superpositions were obtained by structural alignments of the 18S rRNAs excluding the head domains (nt 1150–1620). FIG +62 71 18S rRNAs chemical  Superpositions were obtained by structural alignments of the 18S rRNAs excluding the head domains (nt 1150–1620). FIG +86 90 head structure_element  Superpositions were obtained by structural alignments of the 18S rRNAs excluding the head domains (nt 1150–1620). FIG +103 112 1150–1620 residue_range  Superpositions were obtained by structural alignments of the 18S rRNAs excluding the head domains (nt 1150–1620). FIG +17 21 IRES site Positions of the IRES relative to proteins uS7, uS11 and eS25. FIG +43 46 uS7 protein Positions of the IRES relative to proteins uS7, uS11 and eS25. FIG +48 52 uS11 protein Positions of the IRES relative to proteins uS7, uS11 and eS25. FIG +57 61 eS25 protein Positions of the IRES relative to proteins uS7, uS11 and eS25. FIG +10 14 IRES site (a) Intra-IRES rearrangements from the 80S*IRES initiation structure (INIT; PDB 3J6Y,) to Structures I through V. For each structure (shown in red), the conformation from a preceding structure is shown in light red for comparison. FIG +39 47 80S*IRES complex_assembly (a) Intra-IRES rearrangements from the 80S*IRES initiation structure (INIT; PDB 3J6Y,) to Structures I through V. For each structure (shown in red), the conformation from a preceding structure is shown in light red for comparison. FIG +48 58 initiation protein_state (a) Intra-IRES rearrangements from the 80S*IRES initiation structure (INIT; PDB 3J6Y,) to Structures I through V. For each structure (shown in red), the conformation from a preceding structure is shown in light red for comparison. FIG +59 68 structure evidence (a) Intra-IRES rearrangements from the 80S*IRES initiation structure (INIT; PDB 3J6Y,) to Structures I through V. For each structure (shown in red), the conformation from a preceding structure is shown in light red for comparison. FIG +70 74 INIT complex_assembly (a) Intra-IRES rearrangements from the 80S*IRES initiation structure (INIT; PDB 3J6Y,) to Structures I through V. For each structure (shown in red), the conformation from a preceding structure is shown in light red for comparison. FIG +90 112 Structures I through V evidence (a) Intra-IRES rearrangements from the 80S*IRES initiation structure (INIT; PDB 3J6Y,) to Structures I through V. For each structure (shown in red), the conformation from a preceding structure is shown in light red for comparison. FIG +123 132 structure evidence (a) Intra-IRES rearrangements from the 80S*IRES initiation structure (INIT; PDB 3J6Y,) to Structures I through V. For each structure (shown in red), the conformation from a preceding structure is shown in light red for comparison. FIG +183 192 structure evidence (a) Intra-IRES rearrangements from the 80S*IRES initiation structure (INIT; PDB 3J6Y,) to Structures I through V. For each structure (shown in red), the conformation from a preceding structure is shown in light red for comparison. FIG +0 14 Superpositions experimental_method Superpositions were obtained by structural alignments of 18S rRNA. FIG +32 53 structural alignments experimental_method Superpositions were obtained by structural alignments of 18S rRNA. FIG +57 65 18S rRNA chemical Superpositions were obtained by structural alignments of 18S rRNA. FIG +21 25 IRES site (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +30 34 eEF2 protein (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +66 79 P- and E-site site (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +80 85 tRNAs chemical (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +93 101 80S•tRNA complex_assembly (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +132 136 IRES site (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +158 162 uS11 protein (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +164 176 40S platform site (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +182 185 uS7 protein (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +190 194 eS25 protein (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +196 199 40S complex_assembly (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +200 204 head structure_element (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +231 240 5′ domain structure_element (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +248 252 IRES site (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +260 270 initiation protein_state (b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S•tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5′ domain of the IRES in the initiation state (left panel). FIG +15 29 superpositions experimental_method In all panels, superpositions were obtained by structural alignments of the 18S rRNAs. FIG +47 68 structural alignments experimental_method In all panels, superpositions were obtained by structural alignments of the 18S rRNAs. FIG +76 85 18S rRNAs chemical In all panels, superpositions were obtained by structural alignments of the 18S rRNAs. FIG +26 36 initiation protein_state Ribosomal proteins of the initiation state are shown in gray for comparison. FIG +17 24 L1stalk structure_element Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S•tRNA structures and 80S•IRES structures I and V (this work). FIG +26 30 tRNA chemical Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S•tRNA structures and 80S•IRES structures I and V (this work). FIG +35 38 TSV species Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S•tRNA structures and 80S•IRES structures I and V (this work). FIG +39 43 IRES site Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S•tRNA structures and 80S•IRES structures I and V (this work). FIG +65 68 uS7 protein Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S•tRNA structures and 80S•IRES structures I and V (this work). FIG +73 77 eS25 protein Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S•tRNA structures and 80S•IRES structures I and V (this work). FIG +82 90 80S•tRNA complex_assembly Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S•tRNA structures and 80S•IRES structures I and V (this work). FIG +91 101 structures evidence Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S•tRNA structures and 80S•IRES structures I and V (this work). FIG +106 114 80S•IRES complex_assembly Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S•tRNA structures and 80S•IRES structures I and V (this work). FIG +115 133 structures I and V evidence Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S•tRNA structures and 80S•IRES structures I and V (this work). FIG +45 51 E site site The view shows the vicinity of the ribosomal E site. FIG +0 8 Loop 1.1 structure_element Loop 1.1 and stem loops 4 and 5 of the IRES are labeled. FIG +13 31 stem loops 4 and 5 structure_element Loop 1.1 and stem loops 4 and 5 of the IRES are labeled. FIG +39 43 IRES site Loop 1.1 and stem loops 4 and 5 of the IRES are labeled. FIG +20 38 stem loops 4 and 5 structure_element Interactions of the stem loops 4 and 5 of the TSV with proteins uS7 and eS25. FIG +46 49 TSV species Interactions of the stem loops 4 and 5 of the TSV with proteins uS7 and eS25. FIG +64 67 uS7 protein Interactions of the stem loops 4 and 5 of the TSV with proteins uS7 and eS25. FIG +72 76 eS25 protein Interactions of the stem loops 4 and 5 of the TSV with proteins uS7 and eS25. FIG +29 35 loop 3 structure_element Position and interactions of loop 3 (variable loop region) of the PKI domain in Structure V (this work) resembles those of the anticodon stem loop of the E-site tRNA (blue) in the 80S•2tRNA•mRNA complex. FIG +37 57 variable loop region structure_element Position and interactions of loop 3 (variable loop region) of the PKI domain in Structure V (this work) resembles those of the anticodon stem loop of the E-site tRNA (blue) in the 80S•2tRNA•mRNA complex. FIG +66 69 PKI structure_element Position and interactions of loop 3 (variable loop region) of the PKI domain in Structure V (this work) resembles those of the anticodon stem loop of the E-site tRNA (blue) in the 80S•2tRNA•mRNA complex. FIG +80 91 Structure V evidence Position and interactions of loop 3 (variable loop region) of the PKI domain in Structure V (this work) resembles those of the anticodon stem loop of the E-site tRNA (blue) in the 80S•2tRNA•mRNA complex. FIG +127 146 anticodon stem loop structure_element Position and interactions of loop 3 (variable loop region) of the PKI domain in Structure V (this work) resembles those of the anticodon stem loop of the E-site tRNA (blue) in the 80S•2tRNA•mRNA complex. FIG +154 160 E-site site Position and interactions of loop 3 (variable loop region) of the PKI domain in Structure V (this work) resembles those of the anticodon stem loop of the E-site tRNA (blue) in the 80S•2tRNA•mRNA complex. FIG +161 165 tRNA chemical Position and interactions of loop 3 (variable loop region) of the PKI domain in Structure V (this work) resembles those of the anticodon stem loop of the E-site tRNA (blue) in the 80S•2tRNA•mRNA complex. FIG +180 194 80S•2tRNA•mRNA complex_assembly Position and interactions of loop 3 (variable loop region) of the PKI domain in Structure V (this work) resembles those of the anticodon stem loop of the E-site tRNA (blue) in the 80S•2tRNA•mRNA complex. FIG +13 18 tRNAs chemical Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008–1043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt. FIG +27 30 TSV species Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008–1043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt. FIG +31 35 IRES site Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008–1043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt. FIG +52 65 A-site finger structure_element Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008–1043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt. FIG +76 85 1008–1043 residue_range Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008–1043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt. FIG +89 97 25S rRNA chemical Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008–1043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt. FIG +107 113 P site site Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008–1043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt. FIG +121 134 large subunit structure_element Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008–1043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt. FIG +147 155 helix 84 structure_element Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008–1043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt. FIG +159 167 25S rRNA chemical Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008–1043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt. FIG +0 9 2668–2687 residue_range 2668–2687) and protein uL5 (collectively labeled as central protuberance, CP, in the upper-row first figure, and individually labeled in the lower-row first figure). FIG +23 26 uL5 protein 2668–2687) and protein uL5 (collectively labeled as central protuberance, CP, in the upper-row first figure, and individually labeled in the lower-row first figure). FIG +52 72 central protuberance structure_element 2668–2687) and protein uL5 (collectively labeled as central protuberance, CP, in the upper-row first figure, and individually labeled in the lower-row first figure). FIG +74 76 CP structure_element 2668–2687) and protein uL5 (collectively labeled as central protuberance, CP, in the upper-row first figure, and individually labeled in the lower-row first figure). FIG +0 10 Structures evidence Structures of translocation complexes of the bacterial 70S ribosome bound with two tRNAs and yeast 80S complexes with tRNAs are shown in the upper row and labeled. FIG +45 54 bacterial taxonomy_domain Structures of translocation complexes of the bacterial 70S ribosome bound with two tRNAs and yeast 80S complexes with tRNAs are shown in the upper row and labeled. FIG +55 67 70S ribosome complex_assembly Structures of translocation complexes of the bacterial 70S ribosome bound with two tRNAs and yeast 80S complexes with tRNAs are shown in the upper row and labeled. FIG +68 78 bound with protein_state Structures of translocation complexes of the bacterial 70S ribosome bound with two tRNAs and yeast 80S complexes with tRNAs are shown in the upper row and labeled. FIG +83 88 tRNAs chemical Structures of translocation complexes of the bacterial 70S ribosome bound with two tRNAs and yeast 80S complexes with tRNAs are shown in the upper row and labeled. FIG +93 98 yeast taxonomy_domain Structures of translocation complexes of the bacterial 70S ribosome bound with two tRNAs and yeast 80S complexes with tRNAs are shown in the upper row and labeled. FIG +99 102 80S complex_assembly Structures of translocation complexes of the bacterial 70S ribosome bound with two tRNAs and yeast 80S complexes with tRNAs are shown in the upper row and labeled. FIG +103 117 complexes with protein_state Structures of translocation complexes of the bacterial 70S ribosome bound with two tRNAs and yeast 80S complexes with tRNAs are shown in the upper row and labeled. FIG +118 123 tRNAs chemical Structures of translocation complexes of the bacterial 70S ribosome bound with two tRNAs and yeast 80S complexes with tRNAs are shown in the upper row and labeled. FIG +0 10 Structures evidence Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the lower row and labeled. FIG +14 22 80S•IRES complex_assembly Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the lower row and labeled. FIG +40 50 absence of protein_state Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the lower row and labeled. FIG +51 55 eEF2 protein Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the lower row and labeled. FIG +57 61 INIT complex_assembly Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the lower row and labeled. FIG +85 96 presence of protein_state Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the lower row and labeled. FIG +97 101 eEF2 protein Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the lower row and labeled. FIG +20 23 TSV species Interactions of the TSV IRES with uL5 and eL42. FIG +24 28 IRES site Interactions of the TSV IRES with uL5 and eL42. FIG +34 37 uL5 protein Interactions of the TSV IRES with uL5 and eL42. FIG +42 46 eL42 protein Interactions of the TSV IRES with uL5 and eL42. FIG +0 10 Structures evidence Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the upper row and labeled. FIG +14 22 80S•IRES complex_assembly Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the upper row and labeled. FIG +40 50 absence of protein_state Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the upper row and labeled. FIG +51 55 eEF2 protein Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the upper row and labeled. FIG +57 61 INIT complex_assembly Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the upper row and labeled. FIG +85 96 presence of protein_state Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the upper row and labeled. FIG +97 101 eEF2 protein Structures of 80S•IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the upper row and labeled. FIG +0 10 Structures evidence Structures of the 80S complexes with tRNAs are shown in the lower row in a view similar to that for the 80S•IRES complex. FIG +18 21 80S complex_assembly Structures of the 80S complexes with tRNAs are shown in the lower row in a view similar to that for the 80S•IRES complex. FIG +22 36 complexes with protein_state Structures of the 80S complexes with tRNAs are shown in the lower row in a view similar to that for the 80S•IRES complex. FIG +37 42 tRNAs chemical Structures of the 80S complexes with tRNAs are shown in the lower row in a view similar to that for the 80S•IRES complex. FIG +104 112 80S•IRES complex_assembly Structures of the 80S complexes with tRNAs are shown in the lower row in a view similar to that for the 80S•IRES complex. FIG +17 21 IRES site Positions of the IRES relative to eEF2 and elements of the ribosome in Structures I through V. FIG +34 38 eEF2 protein Positions of the IRES relative to eEF2 and elements of the ribosome in Structures I through V. FIG +59 67 ribosome complex_assembly Positions of the IRES relative to eEF2 and elements of the ribosome in Structures I through V. FIG +71 93 Structures I through V evidence Positions of the IRES relative to eEF2 and elements of the ribosome in Structures I through V. FIG +14 23 structure evidence (a) Secondary structure of the TSV IRES. FIG +31 34 TSV species (a) Secondary structure of the TSV IRES. FIG +35 39 IRES site (a) Secondary structure of the TSV IRES. FIG +5 8 TSV species  The TSV IRES comprises two domains: the 5' domain (blue) and the PKI domain (red). FIG +9 13 IRES site  The TSV IRES comprises two domains: the 5' domain (blue) and the PKI domain (red). FIG +41 50 5' domain structure_element  The TSV IRES comprises two domains: the 5' domain (blue) and the PKI domain (red). FIG +66 69 PKI structure_element  The TSV IRES comprises two domains: the 5' domain (blue) and the PKI domain (red). FIG +4 22 open reading frame structure_element The open reading frame (gray) is immediately following pseudoknot I (PKI). FIG +55 67 pseudoknot I structure_element The open reading frame (gray) is immediately following pseudoknot I (PKI). FIG +69 72 PKI structure_element The open reading frame (gray) is immediately following pseudoknot I (PKI). FIG +22 31 structure evidence (b) Three-dimensional structure of the TSV IRES (Structure II). FIG +39 42 TSV species (b) Three-dimensional structure of the TSV IRES (Structure II). FIG +43 47 IRES site (b) Three-dimensional structure of the TSV IRES (Structure II). FIG +49 61 Structure II evidence (b) Three-dimensional structure of the TSV IRES (Structure II). FIG +16 26 stem loops structure_element Pseudoknots and stem loops are labeled and colored as in (a). FIG +21 25 IRES site (c) Positions of the IRES and eEF2 on the small subunit in Structures I to V. The initiation-state IRES is shown in gray. FIG +30 34 eEF2 protein (c) Positions of the IRES and eEF2 on the small subunit in Structures I to V. The initiation-state IRES is shown in gray. FIG +42 55 small subunit structure_element (c) Positions of the IRES and eEF2 on the small subunit in Structures I to V. The initiation-state IRES is shown in gray. FIG +59 76 Structures I to V evidence (c) Positions of the IRES and eEF2 on the small subunit in Structures I to V. The initiation-state IRES is shown in gray. FIG +82 92 initiation protein_state (c) Positions of the IRES and eEF2 on the small subunit in Structures I to V. The initiation-state IRES is shown in gray. FIG +99 103 IRES site (c) Positions of the IRES and eEF2 on the small subunit in Structures I to V. The initiation-state IRES is shown in gray. FIG +61 65 eEF2 protein  The insert shows density for interaction of diphthamide 699 (eEF2; green) with the codon-anticodon-like helix (PKI; red) in Structure V. (d and e) Density of the P site in Structure V shows that interactions of PKI with the 18S rRNA nucleotides (c) are nearly identical to those in the P site of the 2tRNA•mRNA-bound 70S ribosome (d). FIG +111 114 PKI structure_element  The insert shows density for interaction of diphthamide 699 (eEF2; green) with the codon-anticodon-like helix (PKI; red) in Structure V. (d and e) Density of the P site in Structure V shows that interactions of PKI with the 18S rRNA nucleotides (c) are nearly identical to those in the P site of the 2tRNA•mRNA-bound 70S ribosome (d). FIG +8 17 structure evidence In each structure, the TSV IRES adopts a distinct conformation in the intersubunit space of the ribosome (Figures 3 and 4). RESULTS +23 26 TSV species In each structure, the TSV IRES adopts a distinct conformation in the intersubunit space of the ribosome (Figures 3 and 4). RESULTS +27 31 IRES site In each structure, the TSV IRES adopts a distinct conformation in the intersubunit space of the ribosome (Figures 3 and 4). RESULTS +96 104 ribosome complex_assembly In each structure, the TSV IRES adopts a distinct conformation in the intersubunit space of the ribosome (Figures 3 and 4). RESULTS +4 8 IRES site The IRES (nt 6758–6952) consists of two globular parts (Figure 3a): the 5’-region (domains I and II, nt 6758–6888) and the PKI domain (domain III, nt 6889–6952). RESULTS +13 22 6758–6952 residue_range The IRES (nt 6758–6952) consists of two globular parts (Figure 3a): the 5’-region (domains I and II, nt 6758–6888) and the PKI domain (domain III, nt 6889–6952). RESULTS +72 81 5’-region structure_element The IRES (nt 6758–6952) consists of two globular parts (Figure 3a): the 5’-region (domains I and II, nt 6758–6888) and the PKI domain (domain III, nt 6889–6952). RESULTS +91 92 I structure_element The IRES (nt 6758–6952) consists of two globular parts (Figure 3a): the 5’-region (domains I and II, nt 6758–6888) and the PKI domain (domain III, nt 6889–6952). RESULTS +97 99 II structure_element The IRES (nt 6758–6952) consists of two globular parts (Figure 3a): the 5’-region (domains I and II, nt 6758–6888) and the PKI domain (domain III, nt 6889–6952). RESULTS +104 113 6758–6888 residue_range The IRES (nt 6758–6952) consists of two globular parts (Figure 3a): the 5’-region (domains I and II, nt 6758–6888) and the PKI domain (domain III, nt 6889–6952). RESULTS +123 126 PKI structure_element The IRES (nt 6758–6952) consists of two globular parts (Figure 3a): the 5’-region (domains I and II, nt 6758–6888) and the PKI domain (domain III, nt 6889–6952). RESULTS +142 145 III structure_element The IRES (nt 6758–6952) consists of two globular parts (Figure 3a): the 5’-region (domains I and II, nt 6758–6888) and the PKI domain (domain III, nt 6889–6952). RESULTS +150 159 6889–6952 residue_range The IRES (nt 6758–6952) consists of two globular parts (Figure 3a): the 5’-region (domains I and II, nt 6758–6888) and the PKI domain (domain III, nt 6889–6952). RESULTS +29 30 I structure_element We collectively term domains I and II the 5’ domain. RESULTS +35 37 II structure_element We collectively term domains I and II the 5’ domain. RESULTS +42 51 5’ domain structure_element We collectively term domains I and II the 5’ domain. RESULTS +4 7 PKI structure_element The PKI domain comprises PKI and stem loop 3 (SL3), which stacks on top of the stem of PKI. RESULTS +25 28 PKI structure_element The PKI domain comprises PKI and stem loop 3 (SL3), which stacks on top of the stem of PKI. RESULTS +33 44 stem loop 3 structure_element The PKI domain comprises PKI and stem loop 3 (SL3), which stacks on top of the stem of PKI. RESULTS +46 49 SL3 structure_element The PKI domain comprises PKI and stem loop 3 (SL3), which stacks on top of the stem of PKI. RESULTS +87 90 PKI structure_element The PKI domain comprises PKI and stem loop 3 (SL3), which stacks on top of the stem of PKI. RESULTS +46 49 PKI structure_element The 6953GCU triplet immediately following the PKI domain is the first codon of the open reading frame. RESULTS +83 101 open reading frame structure_element The 6953GCU triplet immediately following the PKI domain is the first codon of the open reading frame. RESULTS +7 16 eEF2-free protein_state In the eEF2-free 80S•IRES initiation complex (INIT), the bulk of the 5’-domain (nt. RESULTS +17 25 80S•IRES complex_assembly In the eEF2-free 80S•IRES initiation complex (INIT), the bulk of the 5’-domain (nt. RESULTS +26 36 initiation protein_state In the eEF2-free 80S•IRES initiation complex (INIT), the bulk of the 5’-domain (nt. RESULTS +46 50 INIT complex_assembly In the eEF2-free 80S•IRES initiation complex (INIT), the bulk of the 5’-domain (nt. RESULTS +69 78 5’-domain structure_element In the eEF2-free 80S•IRES initiation complex (INIT), the bulk of the 5’-domain (nt. RESULTS +0 9 6758–6888 residue_range 6758–6888) binds near the E site, contacting the ribosome mostly by means of three protruding structural elements: the L1.1 region and stem loops 4 and 5 (SL4 and SL5). RESULTS +26 32 E site site 6758–6888) binds near the E site, contacting the ribosome mostly by means of three protruding structural elements: the L1.1 region and stem loops 4 and 5 (SL4 and SL5). RESULTS +49 57 ribosome complex_assembly 6758–6888) binds near the E site, contacting the ribosome mostly by means of three protruding structural elements: the L1.1 region and stem loops 4 and 5 (SL4 and SL5). RESULTS +119 130 L1.1 region structure_element 6758–6888) binds near the E site, contacting the ribosome mostly by means of three protruding structural elements: the L1.1 region and stem loops 4 and 5 (SL4 and SL5). RESULTS +135 153 stem loops 4 and 5 structure_element 6758–6888) binds near the E site, contacting the ribosome mostly by means of three protruding structural elements: the L1.1 region and stem loops 4 and 5 (SL4 and SL5). RESULTS +155 158 SL4 structure_element 6758–6888) binds near the E site, contacting the ribosome mostly by means of three protruding structural elements: the L1.1 region and stem loops 4 and 5 (SL4 and SL5). RESULTS +163 166 SL5 structure_element 6758–6888) binds near the E site, contacting the ribosome mostly by means of three protruding structural elements: the L1.1 region and stem loops 4 and 5 (SL4 and SL5). RESULTS +3 21 Structures I to IV evidence In Structures I to IV, these contacts remain as in the initiation complex (Figure 1a). RESULTS +55 73 initiation complex complex_assembly In Structures I to IV, these contacts remain as in the initiation complex (Figure 1a). RESULTS +18 29 L1.1 region structure_element Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to). RESULTS +49 57 L1 stalk structure_element Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to). RESULTS +65 78 large subunit structure_element Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to). RESULTS +86 89 SL4 structure_element Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to). RESULTS +94 97 SL5 structure_element Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to). RESULTS +122 125 40S complex_assembly Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to). RESULTS +126 130 head structure_element Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to). RESULTS +158 161 uS7 protein Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to). RESULTS +163 167 uS11 protein Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to). RESULTS +172 176 eS25 protein Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to). RESULTS +3 18 Structures I-IV evidence In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). RESULTS +24 36 minor groove site In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). RESULTS +40 43 SL4 structure_element In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). RESULTS +51 60 6840–6846 residue_range In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). RESULTS +79 86 α-helix structure_element In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). RESULTS +90 93 uS7 protein In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). RESULTS +141 145 K212 residue_name_number In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). RESULTS +147 151 K213 residue_name_number In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). RESULTS +153 157 R219 residue_name_number In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). RESULTS +162 166 K222 residue_name_number In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). RESULTS +11 14 SL4 structure_element The tip of SL4 binds in the vicinity of R157 in the β-hairpin of uS7 and of Y58 in uS11. RESULTS +40 44 R157 residue_name_number The tip of SL4 binds in the vicinity of R157 in the β-hairpin of uS7 and of Y58 in uS11. RESULTS +52 61 β-hairpin structure_element The tip of SL4 binds in the vicinity of R157 in the β-hairpin of uS7 and of Y58 in uS11. RESULTS +65 68 uS7 protein The tip of SL4 binds in the vicinity of R157 in the β-hairpin of uS7 and of Y58 in uS11. RESULTS +76 79 Y58 residue_name_number The tip of SL4 binds in the vicinity of R157 in the β-hairpin of uS7 and of Y58 in uS11. RESULTS +83 87 uS11 protein The tip of SL4 binds in the vicinity of R157 in the β-hairpin of uS7 and of Y58 in uS11. RESULTS +4 16 minor groove site The minor groove of SL5 (at nt 6862–6868) contacts the positively charged region of eS25 (R49, R58 and R68) (Figure 3—figure supplement 4). RESULTS +20 23 SL5 structure_element The minor groove of SL5 (at nt 6862–6868) contacts the positively charged region of eS25 (R49, R58 and R68) (Figure 3—figure supplement 4). RESULTS +31 40 6862–6868 residue_range The minor groove of SL5 (at nt 6862–6868) contacts the positively charged region of eS25 (R49, R58 and R68) (Figure 3—figure supplement 4). RESULTS +84 88 eS25 protein The minor groove of SL5 (at nt 6862–6868) contacts the positively charged region of eS25 (R49, R58 and R68) (Figure 3—figure supplement 4). RESULTS +90 93 R49 residue_name_number The minor groove of SL5 (at nt 6862–6868) contacts the positively charged region of eS25 (R49, R58 and R68) (Figure 3—figure supplement 4). RESULTS +95 98 R58 residue_name_number The minor groove of SL5 (at nt 6862–6868) contacts the positively charged region of eS25 (R49, R58 and R68) (Figure 3—figure supplement 4). RESULTS +103 106 R68 residue_name_number The minor groove of SL5 (at nt 6862–6868) contacts the positively charged region of eS25 (R49, R58 and R68) (Figure 3—figure supplement 4). RESULTS +3 14 Structure V evidence In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20 Å away from its initial position (Figure 3—figure supplement 2c). RESULTS +29 36 density evidence In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20 Å away from its initial position (Figure 3—figure supplement 2c). RESULTS +41 44 SL5 structure_element In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20 Å away from its initial position (Figure 3—figure supplement 2c). RESULTS +72 75 SL5 structure_element In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20 Å away from its initial position (Figure 3—figure supplement 2c). RESULTS +79 85 mobile protein_state In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20 Å away from its initial position (Figure 3—figure supplement 2c). RESULTS +98 101 SL4 structure_element In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20 Å away from its initial position (Figure 3—figure supplement 2c). RESULTS +102 109 density evidence In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20 Å away from its initial position (Figure 3—figure supplement 2c). RESULTS +124 127 SL4 structure_element In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20 Å away from its initial position (Figure 3—figure supplement 2c). RESULTS +160 163 uS7 protein In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20 Å away from its initial position (Figure 3—figure supplement 2c). RESULTS +4 15 L1.1 region structure_element The L1.1 region remains in contact with the L1 stalk (Figure 3—figure supplement 3). RESULTS +44 52 L1 stalk structure_element The L1.1 region remains in contact with the L1 stalk (Figure 3—figure supplement 3). RESULTS +0 8 Inchworm protein_state Inchworm-like translocation of the TSV IRES. FIG +35 38 TSV species Inchworm-like translocation of the TSV IRES. FIG +39 43 IRES site Inchworm-like translocation of the TSV IRES. FIG +35 39 IRES site Conformations and positions of the IRES in the initiation state and in Structures I-V are shown relative to those of the A-, P- and E-site tRNAs. FIG +47 57 initiation protein_state Conformations and positions of the IRES in the initiation state and in Structures I-V are shown relative to those of the A-, P- and E-site tRNAs. FIG +71 85 Structures I-V evidence Conformations and positions of the IRES in the initiation state and in Structures I-V are shown relative to those of the A-, P- and E-site tRNAs. FIG +121 138 A-, P- and E-site site Conformations and positions of the IRES in the initiation state and in Structures I-V are shown relative to those of the A-, P- and E-site tRNAs. FIG +139 144 tRNAs chemical Conformations and positions of the IRES in the initiation state and in Structures I-V are shown relative to those of the A-, P- and E-site tRNAs. FIG +25 45 structural alignment experimental_method The view was obtained by structural alignment of the body domains of 18S rRNAs of the corresponding 80S structures. FIG +53 57 body structure_element The view was obtained by structural alignment of the body domains of 18S rRNAs of the corresponding 80S structures. FIG +69 78 18S rRNAs chemical The view was obtained by structural alignment of the body domains of 18S rRNAs of the corresponding 80S structures. FIG +100 103 80S complex_assembly The view was obtained by structural alignment of the body domains of 18S rRNAs of the corresponding 80S structures. FIG +104 114 structures evidence The view was obtained by structural alignment of the body domains of 18S rRNAs of the corresponding 80S structures. FIG +30 34 6848 residue_number Distances between nucleotides 6848 and 6913 in SL4 and PKI, respectively, are shown (see also Figure 2—source data 1). FIG +39 43 6913 residue_number Distances between nucleotides 6848 and 6913 in SL4 and PKI, respectively, are shown (see also Figure 2—source data 1). FIG +47 50 SL4 structure_element Distances between nucleotides 6848 and 6913 in SL4 and PKI, respectively, are shown (see also Figure 2—source data 1). FIG +55 58 PKI structure_element Distances between nucleotides 6848 and 6913 in SL4 and PKI, respectively, are shown (see also Figure 2—source data 1). FIG +17 21 IRES site The shape of the IRES changes considerably from the initiation state to Structures I through V, from an extended to compact to extended conformation (Figure 4; see also Figure 3—figure supplement 2a). RESULTS +52 62 initiation protein_state The shape of the IRES changes considerably from the initiation state to Structures I through V, from an extended to compact to extended conformation (Figure 4; see also Figure 3—figure supplement 2a). RESULTS +72 94 Structures I through V evidence The shape of the IRES changes considerably from the initiation state to Structures I through V, from an extended to compact to extended conformation (Figure 4; see also Figure 3—figure supplement 2a). RESULTS +104 112 extended protein_state The shape of the IRES changes considerably from the initiation state to Structures I through V, from an extended to compact to extended conformation (Figure 4; see also Figure 3—figure supplement 2a). RESULTS +116 123 compact protein_state The shape of the IRES changes considerably from the initiation state to Structures I through V, from an extended to compact to extended conformation (Figure 4; see also Figure 3—figure supplement 2a). RESULTS +127 135 extended protein_state The shape of the IRES changes considerably from the initiation state to Structures I through V, from an extended to compact to extended conformation (Figure 4; see also Figure 3—figure supplement 2a). RESULTS +11 29 Structures I to IV evidence Because in Structures I to IV the PKI domain shifts toward the P site, while the 5’ remains unchanged near the E site, the distance between the domains shortens (Figure 4). RESULTS +34 37 PKI structure_element Because in Structures I to IV the PKI domain shifts toward the P site, while the 5’ remains unchanged near the E site, the distance between the domains shortens (Figure 4). RESULTS +63 69 P site site Because in Structures I to IV the PKI domain shifts toward the P site, while the 5’ remains unchanged near the E site, the distance between the domains shortens (Figure 4). RESULTS +111 117 E site site Because in Structures I to IV the PKI domain shifts toward the P site, while the 5’ remains unchanged near the E site, the distance between the domains shortens (Figure 4). RESULTS +7 15 80S•IRES complex_assembly In the 80S•IRES initiation state, the A-site-bound PKI is separated from SL4 by almost 50 Å (Figure 4). RESULTS +16 26 initiation protein_state In the 80S•IRES initiation state, the A-site-bound PKI is separated from SL4 by almost 50 Å (Figure 4). RESULTS +38 50 A-site-bound protein_state In the 80S•IRES initiation state, the A-site-bound PKI is separated from SL4 by almost 50 Å (Figure 4). RESULTS +51 54 PKI structure_element In the 80S•IRES initiation state, the A-site-bound PKI is separated from SL4 by almost 50 Å (Figure 4). RESULTS +73 76 SL4 structure_element In the 80S•IRES initiation state, the A-site-bound PKI is separated from SL4 by almost 50 Å (Figure 4). RESULTS +3 22 Structures I and II evidence In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35 Å. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25 Å of SL4. RESULTS +28 31 PKI structure_element In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35 Å. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25 Å of SL4. RESULTS +64 70 A site site In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35 Å. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25 Å of SL4. RESULTS +93 96 SL4 structure_element In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35 Å. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25 Å of SL4. RESULTS +119 122 PKI structure_element In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35 Å. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25 Å of SL4. RESULTS +140 146 P site site In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35 Å. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25 Å of SL4. RESULTS +150 171 Structures III and IV evidence In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35 Å. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25 Å of SL4. RESULTS +177 180 PKI structure_element In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35 Å. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25 Å of SL4. RESULTS +218 221 SL4 structure_element In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35 Å. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25 Å of SL4. RESULTS +12 21 5’-domain structure_element Because the 5’-domain in the following structure (V) moves by ~20 Å along the 40S head, the IRES returns to an extended conformation (~45 Å) that is similar to that in the 80S•IRES initiation complex. RESULTS +39 52 structure (V) evidence Because the 5’-domain in the following structure (V) moves by ~20 Å along the 40S head, the IRES returns to an extended conformation (~45 Å) that is similar to that in the 80S•IRES initiation complex. RESULTS +78 81 40S complex_assembly Because the 5’-domain in the following structure (V) moves by ~20 Å along the 40S head, the IRES returns to an extended conformation (~45 Å) that is similar to that in the 80S•IRES initiation complex. RESULTS +82 86 head structure_element Because the 5’-domain in the following structure (V) moves by ~20 Å along the 40S head, the IRES returns to an extended conformation (~45 Å) that is similar to that in the 80S•IRES initiation complex. RESULTS +92 96 IRES site Because the 5’-domain in the following structure (V) moves by ~20 Å along the 40S head, the IRES returns to an extended conformation (~45 Å) that is similar to that in the 80S•IRES initiation complex. RESULTS +111 119 extended protein_state Because the 5’-domain in the following structure (V) moves by ~20 Å along the 40S head, the IRES returns to an extended conformation (~45 Å) that is similar to that in the 80S•IRES initiation complex. RESULTS +172 180 80S•IRES complex_assembly Because the 5’-domain in the following structure (V) moves by ~20 Å along the 40S head, the IRES returns to an extended conformation (~45 Å) that is similar to that in the 80S•IRES initiation complex. RESULTS +181 191 initiation protein_state Because the 5’-domain in the following structure (V) moves by ~20 Å along the 40S head, the IRES returns to an extended conformation (~45 Å) that is similar to that in the 80S•IRES initiation complex. RESULTS +22 26 IRES site Rearrangements of the IRES involve restructuring of several interactions with the ribosome. RESULTS +82 90 ribosome complex_assembly Rearrangements of the IRES involve restructuring of several interactions with the ribosome. RESULTS +3 14 Structure I evidence In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. RESULTS +16 19 SL3 structure_element In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. RESULTS +27 30 PKI structure_element In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. RESULTS +64 77 A-site finger structure_element In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. RESULTS +82 91 1008–1043 residue_range In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. RESULTS +95 103 25S rRNA chemical In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. RESULTS +113 119 P site site In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. RESULTS +127 130 60S complex_assembly In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. RESULTS +131 138 subunit structure_element In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. RESULTS +151 159 helix 84 structure_element In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. RESULTS +163 171 25S rRNA chemical In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. RESULTS +0 9 2668–2687 residue_range 2668–2687) and protein uL5 (Figure 3—figure supplement 6). RESULTS +23 26 uL5 protein 2668–2687) and protein uL5 (Figure 3—figure supplement 6). RESULTS +17 20 SL3 structure_element This position of SL3 is ~25 Å away from that in the 80S•IRES initiation state, in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively. RESULTS +52 60 80S•IRES complex_assembly This position of SL3 is ~25 Å away from that in the 80S•IRES initiation state, in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively. RESULTS +61 71 initiation protein_state This position of SL3 is ~25 Å away from that in the 80S•IRES initiation state, in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively. RESULTS +88 91 PKI structure_element This position of SL3 is ~25 Å away from that in the 80S•IRES initiation state, in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively. RESULTS +96 99 SL3 structure_element This position of SL3 is ~25 Å away from that in the 80S•IRES initiation state, in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively. RESULTS +118 121 ASL structure_element This position of SL3 is ~25 Å away from that in the 80S•IRES initiation state, in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively. RESULTS +126 131 elbow structure_element This position of SL3 is ~25 Å away from that in the 80S•IRES initiation state, in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively. RESULTS +139 145 A-site site This position of SL3 is ~25 Å away from that in the 80S•IRES initiation state, in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively. RESULTS +146 150 tRNA chemical This position of SL3 is ~25 Å away from that in the 80S•IRES initiation state, in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively. RESULTS +33 43 initiation protein_state As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA. RESULTS +53 64 Structure I evidence As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA. RESULTS +91 94 SL3 structure_element As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA. RESULTS +106 119 A-site finger structure_element As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA. RESULTS +159 176 pre-translocation protein_state As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA. RESULTS +177 180 A/P site As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA. RESULTS +185 189 A/P* site As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA. RESULTS +190 194 tRNA chemical As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA. RESULTS +71 84 P site region site The second set of major structural changes involves interaction of the P site region of the large subunit with the hinge point of the IRES bending between the 5´ domain and the PKI domain (nt. 6886–6890). RESULTS +92 105 large subunit structure_element The second set of major structural changes involves interaction of the P site region of the large subunit with the hinge point of the IRES bending between the 5´ domain and the PKI domain (nt. 6886–6890). RESULTS +115 126 hinge point structure_element The second set of major structural changes involves interaction of the P site region of the large subunit with the hinge point of the IRES bending between the 5´ domain and the PKI domain (nt. 6886–6890). RESULTS +134 138 IRES site The second set of major structural changes involves interaction of the P site region of the large subunit with the hinge point of the IRES bending between the 5´ domain and the PKI domain (nt. 6886–6890). RESULTS +159 168 5´ domain structure_element The second set of major structural changes involves interaction of the P site region of the large subunit with the hinge point of the IRES bending between the 5´ domain and the PKI domain (nt. 6886–6890). RESULTS +177 180 PKI structure_element The second set of major structural changes involves interaction of the P site region of the large subunit with the hinge point of the IRES bending between the 5´ domain and the PKI domain (nt. 6886–6890). RESULTS +193 202 6886–6890 residue_range The second set of major structural changes involves interaction of the P site region of the large subunit with the hinge point of the IRES bending between the 5´ domain and the PKI domain (nt. 6886–6890). RESULTS +7 18 highly bent protein_state In the highly bent Structures III and IV, the hinge region interacts with the universally conserved uL5 and the C-terminal tail of eL42 (Figure 3—figure supplement 7). RESULTS +19 40 Structures III and IV evidence In the highly bent Structures III and IV, the hinge region interacts with the universally conserved uL5 and the C-terminal tail of eL42 (Figure 3—figure supplement 7). RESULTS +46 58 hinge region structure_element In the highly bent Structures III and IV, the hinge region interacts with the universally conserved uL5 and the C-terminal tail of eL42 (Figure 3—figure supplement 7). RESULTS +78 99 universally conserved protein_state In the highly bent Structures III and IV, the hinge region interacts with the universally conserved uL5 and the C-terminal tail of eL42 (Figure 3—figure supplement 7). RESULTS +100 103 uL5 protein In the highly bent Structures III and IV, the hinge region interacts with the universally conserved uL5 and the C-terminal tail of eL42 (Figure 3—figure supplement 7). RESULTS +112 127 C-terminal tail structure_element In the highly bent Structures III and IV, the hinge region interacts with the universally conserved uL5 and the C-terminal tail of eL42 (Figure 3—figure supplement 7). RESULTS +131 135 eL42 protein In the highly bent Structures III and IV, the hinge region interacts with the universally conserved uL5 and the C-terminal tail of eL42 (Figure 3—figure supplement 7). RESULTS +16 24 extended protein_state However, in the extended conformations, these parts of the IRES and the 60S subunit are separated by more than 10 Å, suggesting that an interaction between them stabilizes the bent conformations but not the extended ones. RESULTS +59 63 IRES site However, in the extended conformations, these parts of the IRES and the 60S subunit are separated by more than 10 Å, suggesting that an interaction between them stabilizes the bent conformations but not the extended ones. RESULTS +72 75 60S complex_assembly However, in the extended conformations, these parts of the IRES and the 60S subunit are separated by more than 10 Å, suggesting that an interaction between them stabilizes the bent conformations but not the extended ones. RESULTS +76 83 subunit structure_element However, in the extended conformations, these parts of the IRES and the 60S subunit are separated by more than 10 Å, suggesting that an interaction between them stabilizes the bent conformations but not the extended ones. RESULTS +176 180 bent protein_state However, in the extended conformations, these parts of the IRES and the 60S subunit are separated by more than 10 Å, suggesting that an interaction between them stabilizes the bent conformations but not the extended ones. RESULTS +207 215 extended protein_state However, in the extended conformations, these parts of the IRES and the 60S subunit are separated by more than 10 Å, suggesting that an interaction between them stabilizes the bent conformations but not the extended ones. RESULTS +37 43 loop 3 structure_element Another local rearrangement concerns loop 3, also known as the variable loop region , which connects the ASL- and mRNA-like parts of PKI. RESULTS +63 83 variable loop region structure_element Another local rearrangement concerns loop 3, also known as the variable loop region , which connects the ASL- and mRNA-like parts of PKI. RESULTS +105 129 ASL- and mRNA-like parts structure_element Another local rearrangement concerns loop 3, also known as the variable loop region , which connects the ASL- and mRNA-like parts of PKI. RESULTS +133 136 PKI structure_element Another local rearrangement concerns loop 3, also known as the variable loop region , which connects the ASL- and mRNA-like parts of PKI. RESULTS +5 9 loop structure_element This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI and biochemical studies of unbound IRESs. RESULTS +32 55 Structures I through IV evidence This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI and biochemical studies of unbound IRESs. RESULTS +113 131 structural studies experimental_method This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI and biochemical studies of unbound IRESs. RESULTS +139 147 isolated protein_state This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI and biochemical studies of unbound IRESs. RESULTS +148 151 PKI structure_element This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI and biochemical studies of unbound IRESs. RESULTS +156 175 biochemical studies experimental_method This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI and biochemical studies of unbound IRESs. RESULTS +179 186 unbound protein_state This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI and biochemical studies of unbound IRESs. RESULTS +187 192 IRESs site This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI and biochemical studies of unbound IRESs. RESULTS +3 14 Structure V evidence In Structure V, loop 3 is bound in the 40S E site and the backbone of loop 3 near the codon-like part of PKI (at nt. RESULTS +16 22 loop 3 structure_element In Structure V, loop 3 is bound in the 40S E site and the backbone of loop 3 near the codon-like part of PKI (at nt. RESULTS +26 34 bound in protein_state In Structure V, loop 3 is bound in the 40S E site and the backbone of loop 3 near the codon-like part of PKI (at nt. RESULTS +39 42 40S complex_assembly In Structure V, loop 3 is bound in the 40S E site and the backbone of loop 3 near the codon-like part of PKI (at nt. RESULTS +43 49 E site site In Structure V, loop 3 is bound in the 40S E site and the backbone of loop 3 near the codon-like part of PKI (at nt. RESULTS +70 76 loop 3 structure_element In Structure V, loop 3 is bound in the 40S E site and the backbone of loop 3 near the codon-like part of PKI (at nt. RESULTS +86 101 codon-like part structure_element In Structure V, loop 3 is bound in the 40S E site and the backbone of loop 3 near the codon-like part of PKI (at nt. RESULTS +105 108 PKI structure_element In Structure V, loop 3 is bound in the 40S E site and the backbone of loop 3 near the codon-like part of PKI (at nt. RESULTS +0 9 6945–6946 residue_range 6945–6946) interacts with R148 and R157 in β-hairpin of uS7. RESULTS +26 30 R148 residue_name_number 6945–6946) interacts with R148 and R157 in β-hairpin of uS7. RESULTS +35 39 R157 residue_name_number 6945–6946) interacts with R148 and R157 in β-hairpin of uS7. RESULTS +43 52 β-hairpin structure_element 6945–6946) interacts with R148 and R157 in β-hairpin of uS7. RESULTS +56 59 uS7 protein 6945–6946) interacts with R148 and R157 in β-hairpin of uS7. RESULTS +19 25 loop 3 structure_element The interaction of loop 3 backbone with uS7 resembles that of the anticodon-stem loop of E-site tRNA in the post-translocation 80S•2tRNA•mRNA structure (Figure 3—figure supplement 5). RESULTS +40 43 uS7 protein The interaction of loop 3 backbone with uS7 resembles that of the anticodon-stem loop of E-site tRNA in the post-translocation 80S•2tRNA•mRNA structure (Figure 3—figure supplement 5). RESULTS +66 85 anticodon-stem loop structure_element The interaction of loop 3 backbone with uS7 resembles that of the anticodon-stem loop of E-site tRNA in the post-translocation 80S•2tRNA•mRNA structure (Figure 3—figure supplement 5). RESULTS +89 95 E-site site The interaction of loop 3 backbone with uS7 resembles that of the anticodon-stem loop of E-site tRNA in the post-translocation 80S•2tRNA•mRNA structure (Figure 3—figure supplement 5). RESULTS +96 100 tRNA chemical The interaction of loop 3 backbone with uS7 resembles that of the anticodon-stem loop of E-site tRNA in the post-translocation 80S•2tRNA•mRNA structure (Figure 3—figure supplement 5). RESULTS +108 126 post-translocation protein_state The interaction of loop 3 backbone with uS7 resembles that of the anticodon-stem loop of E-site tRNA in the post-translocation 80S•2tRNA•mRNA structure (Figure 3—figure supplement 5). RESULTS +127 141 80S•2tRNA•mRNA complex_assembly The interaction of loop 3 backbone with uS7 resembles that of the anticodon-stem loop of E-site tRNA in the post-translocation 80S•2tRNA•mRNA structure (Figure 3—figure supplement 5). RESULTS +142 151 structure evidence The interaction of loop 3 backbone with uS7 resembles that of the anticodon-stem loop of E-site tRNA in the post-translocation 80S•2tRNA•mRNA structure (Figure 3—figure supplement 5). RESULTS +12 18 loop 3 structure_element Ordering of loop 3 suggests that this flexible region contributes to the stabilization of the PKI domain in the post-translocation state. RESULTS +94 97 PKI structure_element Ordering of loop 3 suggests that this flexible region contributes to the stabilization of the PKI domain in the post-translocation state. RESULTS +112 130 post-translocation protein_state Ordering of loop 3 suggests that this flexible region contributes to the stabilization of the PKI domain in the post-translocation state. RESULTS +82 88 loop 3 structure_element This interpretation is consistent with the recent observation that alterations in loop 3 of the CrPV IRES result in decreased efficiency of translocation. RESULTS +96 100 CrPV species This interpretation is consistent with the recent observation that alterations in loop 3 of the CrPV IRES result in decreased efficiency of translocation. RESULTS +101 105 IRES site This interpretation is consistent with the recent observation that alterations in loop 3 of the CrPV IRES result in decreased efficiency of translocation. RESULTS +0 4 eEF2 protein eEF2 structures RESULTS +5 15 structures evidence eEF2 structures RESULTS +16 28 80S ribosome complex_assembly Elements of the 80S ribosome that contact eEF2 in Structures I through V. FIG +42 46 eEF2 protein Elements of the 80S ribosome that contact eEF2 in Structures I through V. FIG +50 72 Structures I through V evidence Elements of the 80S ribosome that contact eEF2 in Structures I through V. FIG +41 45 eEF2 protein The view and colors are as in Figure 5b: eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +65 69 IRES site The view and colors are as in Figure 5b: eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +70 73 RNA chemical The view and colors are as in Figure 5b: eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +82 85 40S complex_assembly The view and colors are as in Figure 5b: eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +86 93 subunit structure_element The view and colors are as in Figure 5b: eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +86 93 subunit structure_element The view and colors are as in Figure 5b: eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +114 117 60S complex_assembly The view and colors are as in Figure 5b: eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +0 7 Cryo-EM experimental_method Cryo-EM density of the GTPase region in Structures I and II. FIG +8 15 density evidence Cryo-EM density of the GTPase region in Structures I and II. FIG +23 36 GTPase region structure_element Cryo-EM density of the GTPase region in Structures I and II. FIG +40 59 Structures I and II evidence Cryo-EM density of the GTPase region in Structures I and II. FIG +4 17 switch loop I structure_element The switch loop I in Structure I is shown in blue. FIG +21 32 Structure I evidence The switch loop I in Structure I is shown in blue. FIG +29 42 switch loop I structure_element The putative position of the switch loop I, unresolved in the density of Structure II, is shown with a dashed line. FIG +62 69 density evidence The putative position of the switch loop I, unresolved in the density of Structure II, is shown with a dashed line. FIG +73 85 Structure II evidence The putative position of the switch loop I, unresolved in the density of Structure II, is shown with a dashed line. FIG +15 23 ribosome complex_assembly Colors for the ribosome and eEF2 are as in Figure 1. FIG +28 32 eEF2 protein Colors for the ribosome and eEF2 are as in Figure 1. FIG +34 38 eEF2 protein Conformations and interactions of eEF2. FIG +21 25 eEF2 protein (a) Conformations of eEF2 in Structures I-V and domain organization of eEF2 are shown. FIG +29 43 Structures I-V evidence (a) Conformations of eEF2 in Structures I-V and domain organization of eEF2 are shown. FIG +71 75 eEF2 protein (a) Conformations of eEF2 in Structures I-V and domain organization of eEF2 are shown. FIG +22 26 eEF2 protein Roman numerals denote eEF2 domains. FIG +0 13 Superposition experimental_method Superposition was obtained by structural alignment of domains I and II. FIG +30 50 structural alignment experimental_method Superposition was obtained by structural alignment of domains I and II. FIG +62 63 I structure_element Superposition was obtained by structural alignment of domains I and II. FIG +68 70 II structure_element Superposition was obtained by structural alignment of domains I and II. FIG +20 32 80S ribosome complex_assembly (b) Elements of the 80S ribosome in Structures I and V that contact eEF2. FIG +36 54 Structures I and V evidence (b) Elements of the 80S ribosome in Structures I and V that contact eEF2. FIG +68 72 eEF2 protein (b) Elements of the 80S ribosome in Structures I and V that contact eEF2. FIG +0 4 eEF2 protein eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +24 28 IRES site eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +29 32 RNA chemical eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +41 44 40S complex_assembly eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +45 52 subunit structure_element eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +73 76 60S complex_assembly eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal. FIG +35 48 eEF2•sordarin complex_assembly (c) Comparison of conformations of eEF2•sordarin in Structure I (light green) with those of free apo-eEF2 (magenta) and eEF2•sordarin (teal). FIG +52 63 Structure I evidence (c) Comparison of conformations of eEF2•sordarin in Structure I (light green) with those of free apo-eEF2 (magenta) and eEF2•sordarin (teal). FIG +92 96 free protein_state (c) Comparison of conformations of eEF2•sordarin in Structure I (light green) with those of free apo-eEF2 (magenta) and eEF2•sordarin (teal). FIG +97 100 apo protein_state (c) Comparison of conformations of eEF2•sordarin in Structure I (light green) with those of free apo-eEF2 (magenta) and eEF2•sordarin (teal). FIG +101 105 eEF2 protein (c) Comparison of conformations of eEF2•sordarin in Structure I (light green) with those of free apo-eEF2 (magenta) and eEF2•sordarin (teal). FIG +120 133 eEF2•sordarin complex_assembly (c) Comparison of conformations of eEF2•sordarin in Structure I (light green) with those of free apo-eEF2 (magenta) and eEF2•sordarin (teal). FIG +24 38 GTPase domains structure_element (d) Interactions of the GTPase domains with the 40S and 60S subunits in Structure I (colored in green/blue, eEF2; orange, 40S; cyan/teal, 60S) and in Structure II (gray). FIG +48 51 40S complex_assembly (d) Interactions of the GTPase domains with the 40S and 60S subunits in Structure I (colored in green/blue, eEF2; orange, 40S; cyan/teal, 60S) and in Structure II (gray). FIG +56 59 60S complex_assembly (d) Interactions of the GTPase domains with the 40S and 60S subunits in Structure I (colored in green/blue, eEF2; orange, 40S; cyan/teal, 60S) and in Structure II (gray). FIG +60 68 subunits structure_element (d) Interactions of the GTPase domains with the 40S and 60S subunits in Structure I (colored in green/blue, eEF2; orange, 40S; cyan/teal, 60S) and in Structure II (gray). FIG +72 83 Structure I evidence (d) Interactions of the GTPase domains with the 40S and 60S subunits in Structure I (colored in green/blue, eEF2; orange, 40S; cyan/teal, 60S) and in Structure II (gray). FIG +108 112 eEF2 protein (d) Interactions of the GTPase domains with the 40S and 60S subunits in Structure I (colored in green/blue, eEF2; orange, 40S; cyan/teal, 60S) and in Structure II (gray). FIG +122 125 40S complex_assembly (d) Interactions of the GTPase domains with the 40S and 60S subunits in Structure I (colored in green/blue, eEF2; orange, 40S; cyan/teal, 60S) and in Structure II (gray). FIG +138 141 60S complex_assembly (d) Interactions of the GTPase domains with the 40S and 60S subunits in Structure I (colored in green/blue, eEF2; orange, 40S; cyan/teal, 60S) and in Structure II (gray). FIG +150 162 Structure II evidence (d) Interactions of the GTPase domains with the 40S and 60S subunits in Structure I (colored in green/blue, eEF2; orange, 40S; cyan/teal, 60S) and in Structure II (gray). FIG +0 13 Switch loop I structure_element Switch loop I (SWI) in Structure I is in blue; dashed line shows the putative location of unresolved switch loop I in Structure II. FIG +15 18 SWI structure_element Switch loop I (SWI) in Structure I is in blue; dashed line shows the putative location of unresolved switch loop I in Structure II. FIG +23 34 Structure I evidence Switch loop I (SWI) in Structure I is in blue; dashed line shows the putative location of unresolved switch loop I in Structure II. FIG +101 114 switch loop I structure_element Switch loop I (SWI) in Structure I is in blue; dashed line shows the putative location of unresolved switch loop I in Structure II. FIG +118 130 Structure II evidence Switch loop I (SWI) in Structure I is in blue; dashed line shows the putative location of unresolved switch loop I in Structure II. FIG +0 13 Superposition experimental_method Superposition was obtained by structural alignment of the 25S rRNAs. FIG +30 50 structural alignment experimental_method Superposition was obtained by structural alignment of the 25S rRNAs. FIG +58 67 25S rRNAs chemical Superposition was obtained by structural alignment of the 25S rRNAs. FIG +22 30 GTP-like protein_state (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +47 55 eEF2•GDP complex_assembly (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +59 70 Structure I evidence (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +99 108 70S-bound protein_state (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +109 127 elongation factors protein_type (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +128 139 EF-Tu•GDPCP complex_assembly (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +151 172 EF-G•GDP•fusidic acid complex_assembly (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +212 219 Cryo-EM experimental_method (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +220 227 density evidence (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +236 257 guanosine diphosphate chemical (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +258 266 bound in protein_state (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +271 284 GTPase center site (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +305 322 sarcin-ricin loop structure_element (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +326 334 25S rRNA chemical (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +345 357 Structure II evidence (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +381 403 sordarin-binding sites site (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +411 425 ribosome-bound protein_state (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +440 452 Structure II evidence (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +467 471 eEF2 protein (e) Comparison of the GTP-like conformation of eEF2•GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu•GDPCP (teal) and EF-G•GDP•fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal). FIG +4 11 Cryo-EM experimental_method (h) Cryo-EM density showing the sordarin-binding pocket of eEF2 (Structure II). FIG +12 19 density evidence (h) Cryo-EM density showing the sordarin-binding pocket of eEF2 (Structure II). FIG +32 55 sordarin-binding pocket site (h) Cryo-EM density showing the sordarin-binding pocket of eEF2 (Structure II). FIG +59 63 eEF2 protein (h) Cryo-EM density showing the sordarin-binding pocket of eEF2 (Structure II). FIG +65 77 Structure II evidence (h) Cryo-EM density showing the sordarin-binding pocket of eEF2 (Structure II). FIG +0 8 Sordarin chemical Sordarin is shown in pink with oxygen atoms in red. FIG +0 17 Elongation factor protein_type Elongation factor eEF2 in all five structures is bound with GDP and sordarin (Figure 5). RESULTS +18 22 eEF2 protein Elongation factor eEF2 in all five structures is bound with GDP and sordarin (Figure 5). RESULTS +35 45 structures evidence Elongation factor eEF2 in all five structures is bound with GDP and sordarin (Figure 5). RESULTS +49 59 bound with protein_state Elongation factor eEF2 in all five structures is bound with GDP and sordarin (Figure 5). RESULTS +60 63 GDP chemical Elongation factor eEF2 in all five structures is bound with GDP and sordarin (Figure 5). RESULTS +68 76 sordarin chemical Elongation factor eEF2 in all five structures is bound with GDP and sordarin (Figure 5). RESULTS +4 21 elongation factor protein_type The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). RESULTS +48 60 superdomains structure_element The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). RESULTS +85 96 superdomain structure_element The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). RESULTS +111 128 G (GTPase) domain structure_element The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). RESULTS +137 138 I structure_element The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). RESULTS +151 153 II structure_element The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). RESULTS +157 174 linker domain III structure_element The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). RESULTS +193 204 superdomain structure_element The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). RESULTS +224 226 IV structure_element The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). RESULTS +231 232 V structure_element The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). RESULTS +7 9 IV structure_element Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation. RESULTS +32 36 body structure_element Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation. RESULTS +84 88 eEF2 protein Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation. RESULTS +92 96 EF-G protein Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation. RESULTS +98 114 ADP-ribosylation ptm Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation. RESULTS +118 122 eEF2 protein Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation. RESULTS +144 146 IV structure_element Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation. RESULTS +150 158 deletion experimental_method Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation. RESULTS +169 171 IV structure_element Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation. RESULTS +177 181 EF-G protein Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation. RESULTS +3 21 post-translocation protein_state In post-translocation-like 80S•tRNA•eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site. RESULTS +27 40 80S•tRNA•eEF2 complex_assembly In post-translocation-like 80S•tRNA•eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site. RESULTS +59 61 IV structure_element In post-translocation-like 80S•tRNA•eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site. RESULTS +75 78 40S complex_assembly In post-translocation-like 80S•tRNA•eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site. RESULTS +79 85 A site site In post-translocation-like 80S•tRNA•eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site. RESULTS +127 129 IV structure_element In post-translocation-like 80S•tRNA•eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site. RESULTS +150 154 tRNA chemical In post-translocation-like 80S•tRNA•eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site. RESULTS +164 175 A to P site site In post-translocation-like 80S•tRNA•eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site. RESULTS +0 3 GDP chemical GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex. RESULTS +11 21 structures evidence GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex. RESULTS +25 33 bound in protein_state GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex. RESULTS +38 51 GTPase center site GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex. RESULTS +78 86 sordarin chemical GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex. RESULTS +113 124 β-platforms structure_element GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex. RESULTS +136 139 III structure_element GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex. RESULTS +144 145 V structure_element GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex. RESULTS +176 185 structure evidence GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex. RESULTS +189 193 free protein_state GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex. RESULTS +194 207 eEF2•sordarin complex_assembly GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex. RESULTS +28 32 eEF2 protein The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD ≤ 2 Å), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2—source data 1). RESULTS +66 76 structures evidence The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD ≤ 2 Å), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2—source data 1). RESULTS +87 91 RMSD evidence The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD ≤ 2 Å), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2—source data 1). RESULTS +121 125 eEF2 protein The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD ≤ 2 Å), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2—source data 1). RESULTS +142 145 40S complex_assembly The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD ≤ 2 Å), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2—source data 1). RESULTS +146 153 subunit structure_element The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD ≤ 2 Å), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2—source data 1). RESULTS +190 193 40S complex_assembly The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD ≤ 2 Å), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2—source data 1). RESULTS +194 201 subunit structure_element The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD ≤ 2 Å), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2—source data 1). RESULTS +5 21 Structure I to V evidence From Structure I to V, eEF2 is rigidly attached to the GTPase-associated center of the 60S subunit. RESULTS +23 27 eEF2 protein From Structure I to V, eEF2 is rigidly attached to the GTPase-associated center of the 60S subunit. RESULTS +55 79 GTPase-associated center site From Structure I to V, eEF2 is rigidly attached to the GTPase-associated center of the 60S subunit. RESULTS +87 90 60S complex_assembly From Structure I to V, eEF2 is rigidly attached to the GTPase-associated center of the 60S subunit. RESULTS +91 98 subunit structure_element From Structure I to V, eEF2 is rigidly attached to the GTPase-associated center of the 60S subunit. RESULTS +4 28 GTPase-associated center site The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). RESULTS +43 50 P stalk structure_element The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). RESULTS +52 55 L11 structure_element The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). RESULTS +60 62 L7 structure_element The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). RESULTS +63 66 L12 structure_element The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). RESULTS +67 72 stalk structure_element The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). RESULTS +76 84 bacteria taxonomy_domain The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). RESULTS +94 111 sarcin-ricin loop structure_element The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). RESULTS +113 116 SRL structure_element The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). RESULTS +121 130 3012–3042 residue_range The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). RESULTS +12 20 25S rRNA chemical The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +21 38 helices 43 and 44 structure_element The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +46 53 P stalk structure_element The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +67 72 G1242 residue_name_number The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +77 82 A1270 residue_name_number The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +98 103 stack bond_interaction The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +107 111 V754 residue_name_number The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +116 120 Y744 residue_name_number The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +131 132 V structure_element The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +137 146 αββ motif structure_element The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +154 163 eukaryote taxonomy_domain The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +181 183 P0 protein The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +188 195 126–154 residue_range The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +230 244 long α-helix D structure_element The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +249 256 172–188 residue_range The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +265 278 GTPase domain structure_element The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +287 301 β-sheet region structure_element The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +306 313 246–263 residue_range The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +322 342 GTPase domain insert structure_element The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +347 356 G’ insert structure_element The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +361 365 eEF2 protein The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +405 409 eEF2 protein The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +414 418 EF-G protein The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same). RESULTS +13 24 P/L11 stalk structure_element Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). RESULTS +85 101 Structure I to V evidence Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). RESULTS +112 140 root-mean-square differences evidence Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). RESULTS +149 157 25S rRNA chemical Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). RESULTS +165 172 P stalk structure_element Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). RESULTS +177 186 1223–1286 residue_range Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). RESULTS +251 259 80S•IRES complex_assembly Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). RESULTS +275 285 absence of protein_state Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). RESULTS +286 290 eEF2 protein Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). RESULTS +302 316 80S•2tRNA•mRNA complex_assembly Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). RESULTS +330 337 P stalk structure_element Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). RESULTS +369 375 A site site Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). RESULTS +4 21 sarcin-ricin loop structure_element The sarcin-ricin loop interacts with the GTP-binding site of eEF2 (Figures 5d and f). RESULTS +41 57 GTP-binding site site The sarcin-ricin loop interacts with the GTP-binding site of eEF2 (Figures 5d and f). RESULTS +61 65 eEF2 protein The sarcin-ricin loop interacts with the GTP-binding site of eEF2 (Figures 5d and f). RESULTS +70 78 70S•EF-G complex_assembly While the overall mode of this interaction is similar to that seen in 70S•EF-G crystal structures, there is an important local difference between Structure I and Structures II-V in switch loop I, as discussed below. RESULTS +79 97 crystal structures evidence While the overall mode of this interaction is similar to that seen in 70S•EF-G crystal structures, there is an important local difference between Structure I and Structures II-V in switch loop I, as discussed below. RESULTS +146 157 Structure I evidence While the overall mode of this interaction is similar to that seen in 70S•EF-G crystal structures, there is an important local difference between Structure I and Structures II-V in switch loop I, as discussed below. RESULTS +162 177 Structures II-V evidence While the overall mode of this interaction is similar to that seen in 70S•EF-G crystal structures, there is an important local difference between Structure I and Structures II-V in switch loop I, as discussed below. RESULTS +181 194 switch loop I structure_element While the overall mode of this interaction is similar to that seen in 70S•EF-G crystal structures, there is an important local difference between Structure I and Structures II-V in switch loop I, as discussed below. RESULTS +31 57 positively-charged cluster site Repositioning (sliding) of the positively-charged cluster of domain IV of eEF2 over the phosphate backbone (red) of the 18S helices 33 and 34. FIG +68 70 IV structure_element Repositioning (sliding) of the positively-charged cluster of domain IV of eEF2 over the phosphate backbone (red) of the 18S helices 33 and 34. FIG +74 78 eEF2 protein Repositioning (sliding) of the positively-charged cluster of domain IV of eEF2 over the phosphate backbone (red) of the 18S helices 33 and 34. FIG +120 141 18S helices 33 and 34 structure_element Repositioning (sliding) of the positively-charged cluster of domain IV of eEF2 over the phosphate backbone (red) of the 18S helices 33 and 34. FIG +0 22 Structures I through V evidence Structures I through V are shown. FIG +25 29 eEF2 protein Electrostatic surface of eEF2 is shown; negatively and positively charged regions are shown in red and blue, respectively. FIG +25 45 structural alignment experimental_method The view was obtained by structural alignment of the 18S rRNAs. FIG +53 62 18S rRNAs chemical The view was obtained by structural alignment of the 18S rRNAs. FIG +16 20 eEF2 protein Interactions of eEF2 with the 40S subunit. FIG +30 33 40S complex_assembly Interactions of eEF2 with the 40S subunit. FIG +34 41 subunit structure_element Interactions of eEF2 with the 40S subunit. FIG +4 8 eEF2 protein (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +41 45 body structure_element (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +49 60 Structure I evidence (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +62 66 eEF2 protein (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +136 140 head structure_element (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +145 149 body structure_element (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +153 176 Structures II through V evidence (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +220 223 40S complex_assembly (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +257 261 eEF2 protein (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +315 319 eEF2 protein (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +329 332 40S complex_assembly (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +333 339 A site site (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +346 367 Structure I through V evidence (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +386 392 A-site site (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +406 410 eEF2 protein (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +412 423 Structure V evidence (a) eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b) Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown. FIG +25 39 superpositions experimental_method The view was obtained by superpositions of the body domains of 18S rRNAs. FIG +47 51 body structure_element The view was obtained by superpositions of the body domains of 18S rRNAs. FIG +63 72 18S rRNAs chemical The view was obtained by superpositions of the body domains of 18S rRNAs. FIG +25 46 Structure I through V evidence (c) Rearrangements, from Structure I through V, of a positively charged cluster of eEF2 (K613, R617 and R631) positioned over the phosphate backbone of 18S helices 33 and 34, suggesting a role of electrostatic interactions in eEF2 diffusion over the 40S surface. FIG +83 87 eEF2 protein (c) Rearrangements, from Structure I through V, of a positively charged cluster of eEF2 (K613, R617 and R631) positioned over the phosphate backbone of 18S helices 33 and 34, suggesting a role of electrostatic interactions in eEF2 diffusion over the 40S surface. FIG +89 93 K613 residue_name_number (c) Rearrangements, from Structure I through V, of a positively charged cluster of eEF2 (K613, R617 and R631) positioned over the phosphate backbone of 18S helices 33 and 34, suggesting a role of electrostatic interactions in eEF2 diffusion over the 40S surface. FIG +95 99 R617 residue_name_number (c) Rearrangements, from Structure I through V, of a positively charged cluster of eEF2 (K613, R617 and R631) positioned over the phosphate backbone of 18S helices 33 and 34, suggesting a role of electrostatic interactions in eEF2 diffusion over the 40S surface. FIG +104 108 R631 residue_name_number (c) Rearrangements, from Structure I through V, of a positively charged cluster of eEF2 (K613, R617 and R631) positioned over the phosphate backbone of 18S helices 33 and 34, suggesting a role of electrostatic interactions in eEF2 diffusion over the 40S surface. FIG +152 173 18S helices 33 and 34 structure_element (c) Rearrangements, from Structure I through V, of a positively charged cluster of eEF2 (K613, R617 and R631) positioned over the phosphate backbone of 18S helices 33 and 34, suggesting a role of electrostatic interactions in eEF2 diffusion over the 40S surface. FIG +196 222 electrostatic interactions bond_interaction (c) Rearrangements, from Structure I through V, of a positively charged cluster of eEF2 (K613, R617 and R631) positioned over the phosphate backbone of 18S helices 33 and 34, suggesting a role of electrostatic interactions in eEF2 diffusion over the 40S surface. FIG +226 230 eEF2 protein (c) Rearrangements, from Structure I through V, of a positively charged cluster of eEF2 (K613, R617 and R631) positioned over the phosphate backbone of 18S helices 33 and 34, suggesting a role of electrostatic interactions in eEF2 diffusion over the 40S surface. FIG +250 253 40S complex_assembly (c) Rearrangements, from Structure I through V, of a positively charged cluster of eEF2 (K613, R617 and R631) positioned over the phosphate backbone of 18S helices 33 and 34, suggesting a role of electrostatic interactions in eEF2 diffusion over the 40S surface. FIG +31 34 III structure_element (d) Shift of the tip of domain III of eEF2, interacting with uS12 upon reverse subunit rotation from Structure I to Structure V. Structure I colored as in Figure 1, except uS12, which is in purple; Structure V is in gray. FIG +38 42 eEF2 protein (d) Shift of the tip of domain III of eEF2, interacting with uS12 upon reverse subunit rotation from Structure I to Structure V. Structure I colored as in Figure 1, except uS12, which is in purple; Structure V is in gray. FIG +61 65 uS12 protein (d) Shift of the tip of domain III of eEF2, interacting with uS12 upon reverse subunit rotation from Structure I to Structure V. Structure I colored as in Figure 1, except uS12, which is in purple; Structure V is in gray. FIG +79 86 subunit structure_element (d) Shift of the tip of domain III of eEF2, interacting with uS12 upon reverse subunit rotation from Structure I to Structure V. Structure I colored as in Figure 1, except uS12, which is in purple; Structure V is in gray. FIG +101 127 Structure I to Structure V evidence (d) Shift of the tip of domain III of eEF2, interacting with uS12 upon reverse subunit rotation from Structure I to Structure V. Structure I colored as in Figure 1, except uS12, which is in purple; Structure V is in gray. FIG +129 140 Structure I evidence (d) Shift of the tip of domain III of eEF2, interacting with uS12 upon reverse subunit rotation from Structure I to Structure V. Structure I colored as in Figure 1, except uS12, which is in purple; Structure V is in gray. FIG +172 176 uS12 protein (d) Shift of the tip of domain III of eEF2, interacting with uS12 upon reverse subunit rotation from Structure I to Structure V. Structure I colored as in Figure 1, except uS12, which is in purple; Structure V is in gray. FIG +198 209 Structure V evidence (d) Shift of the tip of domain III of eEF2, interacting with uS12 upon reverse subunit rotation from Structure I to Structure V. Structure I colored as in Figure 1, except uS12, which is in purple; Structure V is in gray. FIG +66 84 Structures I and V evidence There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50 Å of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c), eEF2 undergoes moderate repositioning of domain IV (~3 Å; Figure 5a) and domain III (~5 Å; Figure 6d). RESULTS +96 100 free protein_state There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50 Å of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c), eEF2 undergoes moderate repositioning of domain IV (~3 Å; Figure 5a) and domain III (~5 Å; Figure 6d). RESULTS +101 105 eEF2 protein There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50 Å of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c), eEF2 undergoes moderate repositioning of domain IV (~3 Å; Figure 5a) and domain III (~5 Å; Figure 6d). RESULTS +175 186 superdomain structure_element There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50 Å of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c), eEF2 undergoes moderate repositioning of domain IV (~3 Å; Figure 5a) and domain III (~5 Å; Figure 6d). RESULTS +214 225 superdomain structure_element There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50 Å of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c), eEF2 undergoes moderate repositioning of domain IV (~3 Å; Figure 5a) and domain III (~5 Å; Figure 6d). RESULTS +239 243 eEF2 protein There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50 Å of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c), eEF2 undergoes moderate repositioning of domain IV (~3 Å; Figure 5a) and domain III (~5 Å; Figure 6d). RESULTS +287 289 IV structure_element There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50 Å of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c), eEF2 undergoes moderate repositioning of domain IV (~3 Å; Figure 5a) and domain III (~5 Å; Figure 6d). RESULTS +319 322 III structure_element There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50 Å of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c), eEF2 undergoes moderate repositioning of domain IV (~3 Å; Figure 5a) and domain III (~5 Å; Figure 6d). RESULTS +32 46 ribosome-bound protein_state This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit. RESULTS +47 51 eEF2 protein This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit. RESULTS +101 105 eEF2 protein This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit. RESULTS +106 118 superdomains structure_element This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit. RESULTS +132 133 I structure_element This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit. RESULTS +138 139 V structure_element This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit. RESULTS +148 172 GTPase-associated center site This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit. RESULTS +180 193 large subunit structure_element This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit. RESULTS +7 9 IV structure_element Domain IV of eEF2 binds at the 40S A site in Structures I to V but the mode of interaction differs in each complex (Figure 6). RESULTS +13 17 eEF2 protein Domain IV of eEF2 binds at the 40S A site in Structures I to V but the mode of interaction differs in each complex (Figure 6). RESULTS +31 34 40S complex_assembly Domain IV of eEF2 binds at the 40S A site in Structures I to V but the mode of interaction differs in each complex (Figure 6). RESULTS +35 41 A site site Domain IV of eEF2 binds at the 40S A site in Structures I to V but the mode of interaction differs in each complex (Figure 6). RESULTS +45 62 Structures I to V evidence Domain IV of eEF2 binds at the 40S A site in Structures I to V but the mode of interaction differs in each complex (Figure 6). RESULTS +8 12 eEF2 protein Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. RESULTS +40 43 60S complex_assembly Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. RESULTS +44 51 subunit structure_element Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. RESULTS +85 92 subunit structure_element Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. RESULTS +133 135 IV structure_element Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. RESULTS +145 151 A site site Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. RESULTS +160 178 Structures I and V evidence Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. RESULTS +189 192 40S complex_assembly Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. RESULTS +193 200 subunit structure_element Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. RESULTS +214 218 head structure_element Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. RESULTS +0 4 eEF2 protein eEF2 settles into the A site from Structure I to V, as the tip of domain IV shifts by ~10 Å relative to the body and by ~20 Å relative to the swiveling head. RESULTS +22 28 A site site eEF2 settles into the A site from Structure I to V, as the tip of domain IV shifts by ~10 Å relative to the body and by ~20 Å relative to the swiveling head. RESULTS +34 50 Structure I to V evidence eEF2 settles into the A site from Structure I to V, as the tip of domain IV shifts by ~10 Å relative to the body and by ~20 Å relative to the swiveling head. RESULTS +73 75 IV structure_element eEF2 settles into the A site from Structure I to V, as the tip of domain IV shifts by ~10 Å relative to the body and by ~20 Å relative to the swiveling head. RESULTS +108 112 body structure_element eEF2 settles into the A site from Structure I to V, as the tip of domain IV shifts by ~10 Å relative to the body and by ~20 Å relative to the swiveling head. RESULTS +152 156 head structure_element eEF2 settles into the A site from Structure I to V, as the tip of domain IV shifts by ~10 Å relative to the body and by ~20 Å relative to the swiveling head. RESULTS +13 17 eEF2 protein Modest intra-eEF2 shifts of domain IV between Structures I to V outline a stochastic trajectory (Figure 5a), consistent with local adjustments of the domain in the A site. RESULTS +35 37 IV structure_element Modest intra-eEF2 shifts of domain IV between Structures I to V outline a stochastic trajectory (Figure 5a), consistent with local adjustments of the domain in the A site. RESULTS +46 63 Structures I to V evidence Modest intra-eEF2 shifts of domain IV between Structures I to V outline a stochastic trajectory (Figure 5a), consistent with local adjustments of the domain in the A site. RESULTS +164 170 A site site Modest intra-eEF2 shifts of domain IV between Structures I to V outline a stochastic trajectory (Figure 5a), consistent with local adjustments of the domain in the A site. RESULTS +25 29 eEF2 protein At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). RESULTS +39 41 II structure_element At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). RESULTS +46 49 III structure_element At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). RESULTS +62 65 40S complex_assembly At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). RESULTS +66 70 body structure_element At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). RESULTS +94 99 48–52 residue_range At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). RESULTS +104 111 429–432 residue_range At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). RESULTS +115 123 18S rRNA chemical At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). RESULTS +124 131 helix 5 structure_element At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). RESULTS +136 140 uS12 protein At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). RESULTS +5 21 Structure I to V evidence From Structure I to V, these central domains migrate by ~10 Å along the 40S surface (Figure 6c). RESULTS +72 75 40S complex_assembly From Structure I to V, these central domains migrate by ~10 Å along the 40S surface (Figure 6c). RESULTS +14 18 eEF2 protein Comparison of eEF2 conformations reveals that in Structure V, domain III is displaced as a result of interaction with uS12, as discussed below. RESULTS +49 60 Structure V evidence Comparison of eEF2 conformations reveals that in Structure V, domain III is displaced as a result of interaction with uS12, as discussed below. RESULTS +69 72 III structure_element Comparison of eEF2 conformations reveals that in Structure V, domain III is displaced as a result of interaction with uS12, as discussed below. RESULTS +118 122 uS12 protein Comparison of eEF2 conformations reveals that in Structure V, domain III is displaced as a result of interaction with uS12, as discussed below. RESULTS +20 38 Structures I and V evidence In summary, between Structures I and V, a step-wise translocation of PKI by ~15 Å from the A to P site - within the 40S subunit – occurs simultaneously with the ~11 Å side-way entry of domain IV into the A site coupled with ~3 to 5 Å inter-domain rearrangements in eEF2. RESULTS +69 72 PKI structure_element In summary, between Structures I and V, a step-wise translocation of PKI by ~15 Å from the A to P site - within the 40S subunit – occurs simultaneously with the ~11 Å side-way entry of domain IV into the A site coupled with ~3 to 5 Å inter-domain rearrangements in eEF2. RESULTS +91 102 A to P site site In summary, between Structures I and V, a step-wise translocation of PKI by ~15 Å from the A to P site - within the 40S subunit – occurs simultaneously with the ~11 Å side-way entry of domain IV into the A site coupled with ~3 to 5 Å inter-domain rearrangements in eEF2. RESULTS +116 119 40S complex_assembly In summary, between Structures I and V, a step-wise translocation of PKI by ~15 Å from the A to P site - within the 40S subunit – occurs simultaneously with the ~11 Å side-way entry of domain IV into the A site coupled with ~3 to 5 Å inter-domain rearrangements in eEF2. RESULTS +120 127 subunit structure_element In summary, between Structures I and V, a step-wise translocation of PKI by ~15 Å from the A to P site - within the 40S subunit – occurs simultaneously with the ~11 Å side-way entry of domain IV into the A site coupled with ~3 to 5 Å inter-domain rearrangements in eEF2. RESULTS +192 194 IV structure_element In summary, between Structures I and V, a step-wise translocation of PKI by ~15 Å from the A to P site - within the 40S subunit – occurs simultaneously with the ~11 Å side-way entry of domain IV into the A site coupled with ~3 to 5 Å inter-domain rearrangements in eEF2. RESULTS +204 210 A site site In summary, between Structures I and V, a step-wise translocation of PKI by ~15 Å from the A to P site - within the 40S subunit – occurs simultaneously with the ~11 Å side-way entry of domain IV into the A site coupled with ~3 to 5 Å inter-domain rearrangements in eEF2. RESULTS +265 269 eEF2 protein In summary, between Structures I and V, a step-wise translocation of PKI by ~15 Å from the A to P site - within the 40S subunit – occurs simultaneously with the ~11 Å side-way entry of domain IV into the A site coupled with ~3 to 5 Å inter-domain rearrangements in eEF2. RESULTS +54 57 40S complex_assembly These shifts occur during the reverse rotation of the 40S body coupled with the forward-then-reverse head swivel. RESULTS +58 62 body structure_element These shifts occur during the reverse rotation of the 40S body coupled with the forward-then-reverse head swivel. RESULTS +101 105 head structure_element These shifts occur during the reverse rotation of the 40S body coupled with the forward-then-reverse head swivel. RESULTS +50 54 IRES site To elucidate the detailed structural mechanism of IRES translocation and the roles of eEF2 and ribosome rearrangements, we describe in the following sections the interactions of PKI and eEF2 with the ribosomal A and P sites in Structures I through V (Figure 2g; see also Figure 1—figure supplement 1). RESULTS +86 90 eEF2 protein To elucidate the detailed structural mechanism of IRES translocation and the roles of eEF2 and ribosome rearrangements, we describe in the following sections the interactions of PKI and eEF2 with the ribosomal A and P sites in Structures I through V (Figure 2g; see also Figure 1—figure supplement 1). RESULTS +95 103 ribosome complex_assembly To elucidate the detailed structural mechanism of IRES translocation and the roles of eEF2 and ribosome rearrangements, we describe in the following sections the interactions of PKI and eEF2 with the ribosomal A and P sites in Structures I through V (Figure 2g; see also Figure 1—figure supplement 1). RESULTS +178 181 PKI structure_element To elucidate the detailed structural mechanism of IRES translocation and the roles of eEF2 and ribosome rearrangements, we describe in the following sections the interactions of PKI and eEF2 with the ribosomal A and P sites in Structures I through V (Figure 2g; see also Figure 1—figure supplement 1). RESULTS +186 190 eEF2 protein To elucidate the detailed structural mechanism of IRES translocation and the roles of eEF2 and ribosome rearrangements, we describe in the following sections the interactions of PKI and eEF2 with the ribosomal A and P sites in Structures I through V (Figure 2g; see also Figure 1—figure supplement 1). RESULTS +210 223 A and P sites site To elucidate the detailed structural mechanism of IRES translocation and the roles of eEF2 and ribosome rearrangements, we describe in the following sections the interactions of PKI and eEF2 with the ribosomal A and P sites in Structures I through V (Figure 2g; see also Figure 1—figure supplement 1). RESULTS +227 249 Structures I through V evidence To elucidate the detailed structural mechanism of IRES translocation and the roles of eEF2 and ribosome rearrangements, we describe in the following sections the interactions of PKI and eEF2 with the ribosomal A and P sites in Structures I through V (Figure 2g; see also Figure 1—figure supplement 1). RESULTS +0 11 Structure I evidence Structure I represents a pre-translocation IRES and initial entry of eEF2 in a GTP-like state RESULTS +25 42 pre-translocation protein_state Structure I represents a pre-translocation IRES and initial entry of eEF2 in a GTP-like state RESULTS +43 47 IRES site Structure I represents a pre-translocation IRES and initial entry of eEF2 in a GTP-like state RESULTS +69 73 eEF2 protein Structure I represents a pre-translocation IRES and initial entry of eEF2 in a GTP-like state RESULTS +79 82 GTP chemical Structure I represents a pre-translocation IRES and initial entry of eEF2 in a GTP-like state RESULTS +7 20 fully rotated protein_state In the fully rotated Structure I, PKI is shifted toward the P site by ~3 Å relative to its position in the initiation complex but maintains interactions with the partially swiveled head. RESULTS +21 32 Structure I evidence In the fully rotated Structure I, PKI is shifted toward the P site by ~3 Å relative to its position in the initiation complex but maintains interactions with the partially swiveled head. RESULTS +34 37 PKI structure_element In the fully rotated Structure I, PKI is shifted toward the P site by ~3 Å relative to its position in the initiation complex but maintains interactions with the partially swiveled head. RESULTS +60 66 P site site In the fully rotated Structure I, PKI is shifted toward the P site by ~3 Å relative to its position in the initiation complex but maintains interactions with the partially swiveled head. RESULTS +107 125 initiation complex complex_assembly In the fully rotated Structure I, PKI is shifted toward the P site by ~3 Å relative to its position in the initiation complex but maintains interactions with the partially swiveled head. RESULTS +162 180 partially swiveled protein_state In the fully rotated Structure I, PKI is shifted toward the P site by ~3 Å relative to its position in the initiation complex but maintains interactions with the partially swiveled head. RESULTS +181 185 head structure_element In the fully rotated Structure I, PKI is shifted toward the P site by ~3 Å relative to its position in the initiation complex but maintains interactions with the partially swiveled head. RESULTS +7 11 head structure_element At the head, C1274 of the 18S rRNA (C1054 in E. coli) base pairs with the first nucleotide of the ORF immediately downstream of PKI. RESULTS +13 18 C1274 residue_name_number At the head, C1274 of the 18S rRNA (C1054 in E. coli) base pairs with the first nucleotide of the ORF immediately downstream of PKI. RESULTS +26 34 18S rRNA chemical At the head, C1274 of the 18S rRNA (C1054 in E. coli) base pairs with the first nucleotide of the ORF immediately downstream of PKI. RESULTS +36 41 C1054 residue_name_number At the head, C1274 of the 18S rRNA (C1054 in E. coli) base pairs with the first nucleotide of the ORF immediately downstream of PKI. RESULTS +45 52 E. coli species At the head, C1274 of the 18S rRNA (C1054 in E. coli) base pairs with the first nucleotide of the ORF immediately downstream of PKI. RESULTS +98 101 ORF structure_element At the head, C1274 of the 18S rRNA (C1054 in E. coli) base pairs with the first nucleotide of the ORF immediately downstream of PKI. RESULTS +128 131 PKI structure_element At the head, C1274 of the 18S rRNA (C1054 in E. coli) base pairs with the first nucleotide of the ORF immediately downstream of PKI. RESULTS +4 9 C1274 residue_name_number The C1274:G6953 base pair provides a stacking platform for the codon-anticodon–like helix of PKI. RESULTS +10 15 G6953 residue_name_number The C1274:G6953 base pair provides a stacking platform for the codon-anticodon–like helix of PKI. RESULTS +37 54 stacking platform site The C1274:G6953 base pair provides a stacking platform for the codon-anticodon–like helix of PKI. RESULTS +63 89 codon-anticodon–like helix structure_element The C1274:G6953 base pair provides a stacking platform for the codon-anticodon–like helix of PKI. RESULTS +93 96 PKI structure_element The C1274:G6953 base pair provides a stacking platform for the codon-anticodon–like helix of PKI. RESULTS +20 25 C1274 residue_name_number We therefore define C1274 as the foundation of the 'head A site'. RESULTS +52 56 head structure_element We therefore define C1274 as the foundation of the 'head A site'. RESULTS +57 63 A site site We therefore define C1274 as the foundation of the 'head A site'. RESULTS +20 25 U1191 residue_name_number Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +27 31 G966 residue_name_number Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +35 42 E. coli species Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +48 53 C1637 residue_name_number Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +55 60 C1400 residue_name_number Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +64 71 E. coli species Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +105 109 head structure_element Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +110 116 P site site Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +123 127 body structure_element Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +128 134 P site site Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +224 242 fully translocated protein_state Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +243 258 mRNA-tRNA helix structure_element Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +262 272 tRNA-bound protein_state Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +273 283 structures evidence Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +295 313 post-translocation protein_state Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +314 325 Structure V evidence Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below. RESULTS +36 40 eEF2 protein Interactions of the residues at the eEF2 tip with the decoding center of the IRES-bound ribosome. FIG +54 69 decoding center site Interactions of the residues at the eEF2 tip with the decoding center of the IRES-bound ribosome. FIG +77 87 IRES-bound protein_state Interactions of the residues at the eEF2 tip with the decoding center of the IRES-bound ribosome. FIG +88 96 ribosome complex_assembly Interactions of the residues at the eEF2 tip with the decoding center of the IRES-bound ribosome. FIG +20 35 decoding center site Key elements of the decoding center of the 'locked' initiation structure, 'unlocked' Structure I, and post-translocation Structure V (this work) are shown. FIG +44 50 locked protein_state Key elements of the decoding center of the 'locked' initiation structure, 'unlocked' Structure I, and post-translocation Structure V (this work) are shown. FIG +52 62 initiation protein_state Key elements of the decoding center of the 'locked' initiation structure, 'unlocked' Structure I, and post-translocation Structure V (this work) are shown. FIG +63 72 structure evidence Key elements of the decoding center of the 'locked' initiation structure, 'unlocked' Structure I, and post-translocation Structure V (this work) are shown. FIG +75 83 unlocked protein_state Key elements of the decoding center of the 'locked' initiation structure, 'unlocked' Structure I, and post-translocation Structure V (this work) are shown. FIG +85 96 Structure I evidence Key elements of the decoding center of the 'locked' initiation structure, 'unlocked' Structure I, and post-translocation Structure V (this work) are shown. FIG +102 120 post-translocation protein_state Key elements of the decoding center of the 'locked' initiation structure, 'unlocked' Structure I, and post-translocation Structure V (this work) are shown. FIG +121 132 Structure V evidence Key elements of the decoding center of the 'locked' initiation structure, 'unlocked' Structure I, and post-translocation Structure V (this work) are shown. FIG +4 29 histidine-diphthamide tip site The histidine-diphthamide tip of eEF2 is shown in green. FIG +33 37 eEF2 protein The histidine-diphthamide tip of eEF2 is shown in green. FIG +4 30 codon-anticodon-like helix structure_element The codon-anticodon-like helix of PKI is shown in red, the downstream first codon of the ORF in magenta. FIG +34 37 PKI structure_element The codon-anticodon-like helix of PKI is shown in red, the downstream first codon of the ORF in magenta. FIG +89 92 ORF structure_element The codon-anticodon-like helix of PKI is shown in red, the downstream first codon of the ORF in magenta. FIG +19 27 18S rRNA chemical Nucleotides of the 18S rRNA body are in orange and head in yellow; 25S rRNA nucleotide A2256 is blue. FIG +28 32 body structure_element Nucleotides of the 18S rRNA body are in orange and head in yellow; 25S rRNA nucleotide A2256 is blue. FIG +51 55 head structure_element Nucleotides of the 18S rRNA body are in orange and head in yellow; 25S rRNA nucleotide A2256 is blue. FIG +67 75 25S rRNA chemical Nucleotides of the 18S rRNA body are in orange and head in yellow; 25S rRNA nucleotide A2256 is blue. FIG +87 92 A2256 residue_name_number Nucleotides of the 18S rRNA body are in orange and head in yellow; 25S rRNA nucleotide A2256 is blue. FIG +0 13 A and P sites site A and P sites are schematically demarcated by dotted lines. FIG +19 22 PKI structure_element The interaction of PKI with the 40S body is substantially rearranged relative to that in the initiation state. RESULTS +32 35 40S complex_assembly The interaction of PKI with the 40S body is substantially rearranged relative to that in the initiation state. RESULTS +36 40 body structure_element The interaction of PKI with the 40S body is substantially rearranged relative to that in the initiation state. RESULTS +93 103 initiation protein_state The interaction of PKI with the 40S body is substantially rearranged relative to that in the initiation state. RESULTS +15 18 PKI structure_element In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes. RESULTS +58 79 universally conserved protein_state In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes. RESULTS +80 95 decoding-center site In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes. RESULTS +108 112 G577 residue_name_number In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes. RESULTS +114 119 A1755 residue_name_number In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes. RESULTS +124 129 A1756 residue_name_number In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes. RESULTS +132 136 body structure_element In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes. RESULTS +137 143 A site site In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes. RESULTS +157 163 A-site site In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes. RESULTS +164 174 tRNA bound protein_state In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes. RESULTS +3 14 Structure I evidence In Structure I, PKI does not contact these nucleotides (Figures 2g and 7). RESULTS +16 19 PKI structure_element In Structure I, PKI does not contact these nucleotides (Figures 2g and 7). RESULTS +16 20 eEF2 protein The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). RESULTS +28 31 40S complex_assembly The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). RESULTS +32 39 subunit structure_element The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). RESULTS +32 39 subunit structure_element The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). RESULTS +43 54 Structure I evidence The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). RESULTS +90 108 Structures II to V evidence The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). RESULTS +114 125 translocase protein_type The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). RESULTS +145 148 40S complex_assembly The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). RESULTS +149 153 body structure_element The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). RESULTS +179 183 head structure_element The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). RESULTS +7 9 IV structure_element Domain IV is partially engaged with the body A site. RESULTS +40 44 body structure_element Domain IV is partially engaged with the body A site. RESULTS +45 51 A site site Domain IV is partially engaged with the body A site. RESULTS +18 20 IV structure_element The tip of domain IV is wedged between PKI and decoding-center nucleotides A1755 and A1756, which are bulged out of h44. RESULTS +39 42 PKI structure_element The tip of domain IV is wedged between PKI and decoding-center nucleotides A1755 and A1756, which are bulged out of h44. RESULTS +47 62 decoding-center site The tip of domain IV is wedged between PKI and decoding-center nucleotides A1755 and A1756, which are bulged out of h44. RESULTS +75 80 A1755 residue_name_number The tip of domain IV is wedged between PKI and decoding-center nucleotides A1755 and A1756, which are bulged out of h44. RESULTS +85 90 A1756 residue_name_number The tip of domain IV is wedged between PKI and decoding-center nucleotides A1755 and A1756, which are bulged out of h44. RESULTS +22 49 histidine-diphthamide triad site This tip contains the histidine-diphthamide triad (H583, H694 and Diph699), which interacts with the codon-anticodon-like helix of PKI and A1756 (Figure 7). RESULTS +51 55 H583 residue_name_number This tip contains the histidine-diphthamide triad (H583, H694 and Diph699), which interacts with the codon-anticodon-like helix of PKI and A1756 (Figure 7). RESULTS +57 61 H694 residue_name_number This tip contains the histidine-diphthamide triad (H583, H694 and Diph699), which interacts with the codon-anticodon-like helix of PKI and A1756 (Figure 7). RESULTS +66 73 Diph699 ptm This tip contains the histidine-diphthamide triad (H583, H694 and Diph699), which interacts with the codon-anticodon-like helix of PKI and A1756 (Figure 7). RESULTS +101 127 codon-anticodon-like helix structure_element This tip contains the histidine-diphthamide triad (H583, H694 and Diph699), which interacts with the codon-anticodon-like helix of PKI and A1756 (Figure 7). RESULTS +131 134 PKI structure_element This tip contains the histidine-diphthamide triad (H583, H694 and Diph699), which interacts with the codon-anticodon-like helix of PKI and A1756 (Figure 7). RESULTS +139 144 A1756 residue_name_number This tip contains the histidine-diphthamide triad (H583, H694 and Diph699), which interacts with the codon-anticodon-like helix of PKI and A1756 (Figure 7). RESULTS +0 22 Histidines 583 and 694 residue_name_number Histidines 583 and 694 interact with the phosphate backbone of the anticodon-like strand (at G6907 and C6908). RESULTS +67 88 anticodon-like strand structure_element Histidines 583 and 694 interact with the phosphate backbone of the anticodon-like strand (at G6907 and C6908). RESULTS +93 98 G6907 residue_name_number Histidines 583 and 694 interact with the phosphate backbone of the anticodon-like strand (at G6907 and C6908). RESULTS +103 108 C6908 residue_name_number Histidines 583 and 694 interact with the phosphate backbone of the anticodon-like strand (at G6907 and C6908). RESULTS +0 11 Diphthamide ptm Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-Å long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1. RESULTS +55 64 conserved protein_state Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-Å long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1. RESULTS +68 76 archaeal taxonomy_domain Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-Å long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1. RESULTS +81 91 eukaryotic taxonomy_domain Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-Å long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1. RESULTS +92 95 EF2 protein Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-Å long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1. RESULTS +108 111 699 residue_number Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-Å long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1. RESULTS +115 128 S. cerevisiae species Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-Å long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1. RESULTS +222 231 histidine residue_name Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-Å long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1. RESULTS +26 33 Diph699 ptm The trimethylamino end of Diph699 packs over A1756 (Figure 7). RESULTS +45 50 A1756 residue_name_number The trimethylamino end of Diph699 packs over A1756 (Figure 7). RESULTS +56 68 minor-groove site The opposite surface of the tail is oriented toward the minor-groove side of the second base pair of the codon-anticodon helix (G6906:C6951). RESULTS +105 126 codon-anticodon helix structure_element The opposite surface of the tail is oriented toward the minor-groove side of the second base pair of the codon-anticodon helix (G6906:C6951). RESULTS +128 133 G6906 residue_name_number The opposite surface of the tail is oriented toward the minor-groove side of the second base pair of the codon-anticodon helix (G6906:C6951). RESULTS +134 139 C6951 residue_name_number The opposite surface of the tail is oriented toward the minor-groove side of the second base pair of the codon-anticodon helix (G6906:C6951). RESULTS +29 39 initiation protein_state Thus, in comparison with the initiation state, the histidine-diphthamide tip of eEF2 replaces the codon-anticodon–like helix of PKI. RESULTS +51 76 histidine-diphthamide tip site Thus, in comparison with the initiation state, the histidine-diphthamide tip of eEF2 replaces the codon-anticodon–like helix of PKI. RESULTS +80 84 eEF2 protein Thus, in comparison with the initiation state, the histidine-diphthamide tip of eEF2 replaces the codon-anticodon–like helix of PKI. RESULTS +98 124 codon-anticodon–like helix structure_element Thus, in comparison with the initiation state, the histidine-diphthamide tip of eEF2 replaces the codon-anticodon–like helix of PKI. RESULTS +128 131 PKI structure_element Thus, in comparison with the initiation state, the histidine-diphthamide tip of eEF2 replaces the codon-anticodon–like helix of PKI. RESULTS +36 41 A1755 residue_name_number The splitting of the interaction of A1755-A1756 and PKI is achieved by providing the histidine-diphthamine tip as a binding partner for both A1756 and the minor groove of the codon-anticodon helix (Figure 7). RESULTS +42 47 A1756 residue_name_number The splitting of the interaction of A1755-A1756 and PKI is achieved by providing the histidine-diphthamine tip as a binding partner for both A1756 and the minor groove of the codon-anticodon helix (Figure 7). RESULTS +52 55 PKI structure_element The splitting of the interaction of A1755-A1756 and PKI is achieved by providing the histidine-diphthamine tip as a binding partner for both A1756 and the minor groove of the codon-anticodon helix (Figure 7). RESULTS +85 110 histidine-diphthamine tip site The splitting of the interaction of A1755-A1756 and PKI is achieved by providing the histidine-diphthamine tip as a binding partner for both A1756 and the minor groove of the codon-anticodon helix (Figure 7). RESULTS +141 146 A1756 residue_name_number The splitting of the interaction of A1755-A1756 and PKI is achieved by providing the histidine-diphthamine tip as a binding partner for both A1756 and the minor groove of the codon-anticodon helix (Figure 7). RESULTS +155 167 minor groove site The splitting of the interaction of A1755-A1756 and PKI is achieved by providing the histidine-diphthamine tip as a binding partner for both A1756 and the minor groove of the codon-anticodon helix (Figure 7). RESULTS +175 196 codon-anticodon helix structure_element The splitting of the interaction of A1755-A1756 and PKI is achieved by providing the histidine-diphthamine tip as a binding partner for both A1756 and the minor groove of the codon-anticodon helix (Figure 7). RESULTS +10 28 Structures II to V evidence Unlike in Structures II to V, the conformation of the eEF2 GTPase center in Structure I resembles that of a GTP-bound translocase (Figure 5e). RESULTS +54 58 eEF2 protein Unlike in Structures II to V, the conformation of the eEF2 GTPase center in Structure I resembles that of a GTP-bound translocase (Figure 5e). RESULTS +59 72 GTPase center site Unlike in Structures II to V, the conformation of the eEF2 GTPase center in Structure I resembles that of a GTP-bound translocase (Figure 5e). RESULTS +76 87 Structure I evidence Unlike in Structures II to V, the conformation of the eEF2 GTPase center in Structure I resembles that of a GTP-bound translocase (Figure 5e). RESULTS +108 117 GTP-bound protein_state Unlike in Structures II to V, the conformation of the eEF2 GTPase center in Structure I resembles that of a GTP-bound translocase (Figure 5e). RESULTS +118 129 translocase protein_type Unlike in Structures II to V, the conformation of the eEF2 GTPase center in Structure I resembles that of a GTP-bound translocase (Figure 5e). RESULTS +3 24 translational GTPases protein_type In translational GTPases, switch loops I and II are involved in the GTPase activity (reviewed in). RESULTS +26 47 switch loops I and II structure_element In translational GTPases, switch loops I and II are involved in the GTPase activity (reviewed in). RESULTS +68 74 GTPase protein_type In translational GTPases, switch loops I and II are involved in the GTPase activity (reviewed in). RESULTS +0 14 Switch loop II structure_element Switch loop II (aa 105–110), which carries the catalytic H108 (H92 in E. coli EF-G; is well resolved in all five structures. RESULTS +19 26 105–110 residue_range Switch loop II (aa 105–110), which carries the catalytic H108 (H92 in E. coli EF-G; is well resolved in all five structures. RESULTS +47 56 catalytic protein_state Switch loop II (aa 105–110), which carries the catalytic H108 (H92 in E. coli EF-G; is well resolved in all five structures. RESULTS +57 61 H108 residue_name_number Switch loop II (aa 105–110), which carries the catalytic H108 (H92 in E. coli EF-G; is well resolved in all five structures. RESULTS +63 66 H92 residue_name_number Switch loop II (aa 105–110), which carries the catalytic H108 (H92 in E. coli EF-G; is well resolved in all five structures. RESULTS +70 77 E. coli species Switch loop II (aa 105–110), which carries the catalytic H108 (H92 in E. coli EF-G; is well resolved in all five structures. RESULTS +78 82 EF-G protein Switch loop II (aa 105–110), which carries the catalytic H108 (H92 in E. coli EF-G; is well resolved in all five structures. RESULTS +113 123 structures evidence Switch loop II (aa 105–110), which carries the catalytic H108 (H92 in E. coli EF-G; is well resolved in all five structures. RESULTS +4 13 histidine residue_name The histidine resides next to the backbone of G3028 of the sarcin-ricin loop and near the diphosphate of GDP (Figure 5e). RESULTS +46 51 G3028 residue_name_number The histidine resides next to the backbone of G3028 of the sarcin-ricin loop and near the diphosphate of GDP (Figure 5e). RESULTS +59 76 sarcin-ricin loop structure_element The histidine resides next to the backbone of G3028 of the sarcin-ricin loop and near the diphosphate of GDP (Figure 5e). RESULTS +105 108 GDP chemical The histidine resides next to the backbone of G3028 of the sarcin-ricin loop and near the diphosphate of GDP (Figure 5e). RESULTS +13 26 switch loop I structure_element By contrast, switch loop I (aa 50–70 in S. cerevisiae eEF2) is resolved only in Structure I (Figure 5—figure supplement 2). RESULTS +31 36 50–70 residue_range By contrast, switch loop I (aa 50–70 in S. cerevisiae eEF2) is resolved only in Structure I (Figure 5—figure supplement 2). RESULTS +40 53 S. cerevisiae species By contrast, switch loop I (aa 50–70 in S. cerevisiae eEF2) is resolved only in Structure I (Figure 5—figure supplement 2). RESULTS +54 58 eEF2 protein By contrast, switch loop I (aa 50–70 in S. cerevisiae eEF2) is resolved only in Structure I (Figure 5—figure supplement 2). RESULTS +80 91 Structure I evidence By contrast, switch loop I (aa 50–70 in S. cerevisiae eEF2) is resolved only in Structure I (Figure 5—figure supplement 2). RESULTS +27 31 loop structure_element The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). RESULTS +36 41 50–60 residue_range The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). RESULTS +76 84 helix 14 structure_element The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). RESULTS +86 96 415CAAA418 structure_element The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). RESULTS +105 113 18S rRNA chemical The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). RESULTS +121 124 40S complex_assembly The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). RESULTS +125 132 subunit structure_element The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). RESULTS +125 132 subunit structure_element The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). RESULTS +137 144 helix A structure_element The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). RESULTS +149 154 32–42 residue_range The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). RESULTS +159 163 eEF2 protein The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). RESULTS +0 6 Bulged protein_state Bulged A416 interacts with the switch loop in the vicinity of D53. RESULTS +7 11 A416 residue_name_number Bulged A416 interacts with the switch loop in the vicinity of D53. RESULTS +31 42 switch loop structure_element Bulged A416 interacts with the switch loop in the vicinity of D53. RESULTS +62 65 D53 residue_name_number Bulged A416 interacts with the switch loop in the vicinity of D53. RESULTS +8 11 GDP chemical Next to GDP, the C-terminal part of the switch loop (aa 61–67) adopts a helical fold. RESULTS +40 51 switch loop structure_element Next to GDP, the C-terminal part of the switch loop (aa 61–67) adopts a helical fold. RESULTS +56 61 61–67 residue_range Next to GDP, the C-terminal part of the switch loop (aa 61–67) adopts a helical fold. RESULTS +72 84 helical fold protein_state Next to GDP, the C-terminal part of the switch loop (aa 61–67) adopts a helical fold. RESULTS +30 33 SWI structure_element As such, the conformations of SWI and the GTPase center in general are similar to those observed in ribosome-bound EF-Tu and EF-G in the presence of GTP analogs. RESULTS +42 55 GTPase center site As such, the conformations of SWI and the GTPase center in general are similar to those observed in ribosome-bound EF-Tu and EF-G in the presence of GTP analogs. RESULTS +100 114 ribosome-bound protein_state As such, the conformations of SWI and the GTPase center in general are similar to those observed in ribosome-bound EF-Tu and EF-G in the presence of GTP analogs. RESULTS +115 120 EF-Tu protein As such, the conformations of SWI and the GTPase center in general are similar to those observed in ribosome-bound EF-Tu and EF-G in the presence of GTP analogs. RESULTS +125 129 EF-G protein As such, the conformations of SWI and the GTPase center in general are similar to those observed in ribosome-bound EF-Tu and EF-G in the presence of GTP analogs. RESULTS +137 148 presence of protein_state As such, the conformations of SWI and the GTPase center in general are similar to those observed in ribosome-bound EF-Tu and EF-G in the presence of GTP analogs. RESULTS +149 152 GTP chemical As such, the conformations of SWI and the GTPase center in general are similar to those observed in ribosome-bound EF-Tu and EF-G in the presence of GTP analogs. RESULTS +0 12 Structure II evidence Structure II reveals PKI between the body A and P sites and eEF2 partially advanced into the A site RESULTS +21 24 PKI structure_element Structure II reveals PKI between the body A and P sites and eEF2 partially advanced into the A site RESULTS +37 41 body structure_element Structure II reveals PKI between the body A and P sites and eEF2 partially advanced into the A site RESULTS +42 55 A and P sites site Structure II reveals PKI between the body A and P sites and eEF2 partially advanced into the A site RESULTS +60 64 eEF2 protein Structure II reveals PKI between the body A and P sites and eEF2 partially advanced into the A site RESULTS +93 99 A site site Structure II reveals PKI between the body A and P sites and eEF2 partially advanced into the A site RESULTS +3 15 Structure II evidence In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4 Å toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site. RESULTS +29 40 Structure I evidence In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4 Å toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site. RESULTS +42 45 PKI structure_element In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4 Å toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site. RESULTS +75 78 40S complex_assembly In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4 Å toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site. RESULTS +79 83 body structure_element In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4 Å toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site. RESULTS +112 118 P site site In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4 Å toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site. RESULTS +149 157 stacking bond_interaction In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4 Å toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site. RESULTS +161 166 C1274 residue_name_number In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4 Å toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site. RESULTS +174 178 head structure_element In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4 Å toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site. RESULTS +179 185 A site site In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4 Å toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site. RESULTS +35 38 PKI structure_element Thus, the intermediate position of PKI is possible due to a large swivel of the head relative to the body, which brings the head A site close to the body P site. RESULTS +80 84 head structure_element Thus, the intermediate position of PKI is possible due to a large swivel of the head relative to the body, which brings the head A site close to the body P site. RESULTS +101 105 body structure_element Thus, the intermediate position of PKI is possible due to a large swivel of the head relative to the body, which brings the head A site close to the body P site. RESULTS +124 128 head structure_element Thus, the intermediate position of PKI is possible due to a large swivel of the head relative to the body, which brings the head A site close to the body P site. RESULTS +129 135 A site site Thus, the intermediate position of PKI is possible due to a large swivel of the head relative to the body, which brings the head A site close to the body P site. RESULTS +149 153 body structure_element Thus, the intermediate position of PKI is possible due to a large swivel of the head relative to the body, which brings the head A site close to the body P site. RESULTS +154 160 P site site Thus, the intermediate position of PKI is possible due to a large swivel of the head relative to the body, which brings the head A site close to the body P site. RESULTS +7 9 IV structure_element Domain IV of eEF2 is further entrenched in the A site by ~3 Å relative to the body and ~8 Å relative to the head, preserving its interactions with PKI. RESULTS +13 17 eEF2 protein Domain IV of eEF2 is further entrenched in the A site by ~3 Å relative to the body and ~8 Å relative to the head, preserving its interactions with PKI. RESULTS +47 53 A site site Domain IV of eEF2 is further entrenched in the A site by ~3 Å relative to the body and ~8 Å relative to the head, preserving its interactions with PKI. RESULTS +78 82 body structure_element Domain IV of eEF2 is further entrenched in the A site by ~3 Å relative to the body and ~8 Å relative to the head, preserving its interactions with PKI. RESULTS +108 112 head structure_element Domain IV of eEF2 is further entrenched in the A site by ~3 Å relative to the body and ~8 Å relative to the head, preserving its interactions with PKI. RESULTS +147 150 PKI structure_element Domain IV of eEF2 is further entrenched in the A site by ~3 Å relative to the body and ~8 Å relative to the head, preserving its interactions with PKI. RESULTS +4 19 decoding center site The decoding center residues A1755 and A1756 are rearranged to pack inside helix 44, making room for eEF2. RESULTS +29 34 A1755 residue_name_number The decoding center residues A1755 and A1756 are rearranged to pack inside helix 44, making room for eEF2. RESULTS +39 44 A1756 residue_name_number The decoding center residues A1755 and A1756 are rearranged to pack inside helix 44, making room for eEF2. RESULTS +75 83 helix 44 structure_element The decoding center residues A1755 and A1756 are rearranged to pack inside helix 44, making room for eEF2. RESULTS +101 105 eEF2 protein The decoding center residues A1755 and A1756 are rearranged to pack inside helix 44, making room for eEF2. RESULTS +21 36 decoding center site This conformation of decoding center residues is also observed in the absence of A-site ligands. RESULTS +70 80 absence of protein_state This conformation of decoding center residues is also observed in the absence of A-site ligands. RESULTS +81 87 A-site site This conformation of decoding center residues is also observed in the absence of A-site ligands. RESULTS +4 18 head interface site The head interface of domain IV interacts with the 40S head (Figure 6a). RESULTS +29 31 IV structure_element The head interface of domain IV interacts with the 40S head (Figure 6a). RESULTS +51 54 40S complex_assembly The head interface of domain IV interacts with the 40S head (Figure 6a). RESULTS +55 59 head structure_element The head interface of domain IV interacts with the 40S head (Figure 6a). RESULTS +8 34 positively charged surface site Here, a positively charged surface of eEF2, formed by K613, R617 and R631 contacts the phosphate backbone of helix 33 (Figures 6c; see also Figure 6—figure supplement 1). RESULTS +38 42 eEF2 protein Here, a positively charged surface of eEF2, formed by K613, R617 and R631 contacts the phosphate backbone of helix 33 (Figures 6c; see also Figure 6—figure supplement 1). RESULTS +54 58 K613 residue_name_number Here, a positively charged surface of eEF2, formed by K613, R617 and R631 contacts the phosphate backbone of helix 33 (Figures 6c; see also Figure 6—figure supplement 1). RESULTS +60 64 R617 residue_name_number Here, a positively charged surface of eEF2, formed by K613, R617 and R631 contacts the phosphate backbone of helix 33 (Figures 6c; see also Figure 6—figure supplement 1). RESULTS +69 73 R631 residue_name_number Here, a positively charged surface of eEF2, formed by K613, R617 and R631 contacts the phosphate backbone of helix 33 (Figures 6c; see also Figure 6—figure supplement 1). RESULTS +109 117 helix 33 structure_element Here, a positively charged surface of eEF2, formed by K613, R617 and R631 contacts the phosphate backbone of helix 33 (Figures 6c; see also Figure 6—figure supplement 1). RESULTS +0 13 Structure III evidence Structure III represents a highly bent IRES with PKI captured between the head A and P sites RESULTS +27 38 highly bent protein_state Structure III represents a highly bent IRES with PKI captured between the head A and P sites RESULTS +39 43 IRES site Structure III represents a highly bent IRES with PKI captured between the head A and P sites RESULTS +49 52 PKI structure_element Structure III represents a highly bent IRES with PKI captured between the head A and P sites RESULTS +74 78 head structure_element Structure III represents a highly bent IRES with PKI captured between the head A and P sites RESULTS +79 92 A and P sites site Structure III represents a highly bent IRES with PKI captured between the head A and P sites RESULTS +28 32 head structure_element Consistent with the similar head swivels in Structure III and Structure II, relative positions of the 40S head A site and body P site remain as in Structure II. RESULTS +44 57 Structure III evidence Consistent with the similar head swivels in Structure III and Structure II, relative positions of the 40S head A site and body P site remain as in Structure II. RESULTS +62 74 Structure II evidence Consistent with the similar head swivels in Structure III and Structure II, relative positions of the 40S head A site and body P site remain as in Structure II. RESULTS +102 105 40S complex_assembly Consistent with the similar head swivels in Structure III and Structure II, relative positions of the 40S head A site and body P site remain as in Structure II. RESULTS +106 110 head structure_element Consistent with the similar head swivels in Structure III and Structure II, relative positions of the 40S head A site and body P site remain as in Structure II. RESULTS +111 117 A site site Consistent with the similar head swivels in Structure III and Structure II, relative positions of the 40S head A site and body P site remain as in Structure II. RESULTS +122 126 body structure_element Consistent with the similar head swivels in Structure III and Structure II, relative positions of the 40S head A site and body P site remain as in Structure II. RESULTS +127 133 P site site Consistent with the similar head swivels in Structure III and Structure II, relative positions of the 40S head A site and body P site remain as in Structure II. RESULTS +147 159 Structure II evidence Consistent with the similar head swivels in Structure III and Structure II, relative positions of the 40S head A site and body P site remain as in Structure II. RESULTS +15 25 structures evidence Among the five structures, the PKI domain is least ordered in Structure III and lacks density for SL3. RESULTS +31 34 PKI structure_element Among the five structures, the PKI domain is least ordered in Structure III and lacks density for SL3. RESULTS +62 75 Structure III evidence Among the five structures, the PKI domain is least ordered in Structure III and lacks density for SL3. RESULTS +86 93 density evidence Among the five structures, the PKI domain is least ordered in Structure III and lacks density for SL3. RESULTS +98 101 SL3 structure_element Among the five structures, the PKI domain is least ordered in Structure III and lacks density for SL3. RESULTS +4 7 map evidence The map allows placement of PKI at the body P site (Figure 1—figure supplement 3). RESULTS +28 31 PKI structure_element The map allows placement of PKI at the body P site (Figure 1—figure supplement 3). RESULTS +39 43 body structure_element The map allows placement of PKI at the body P site (Figure 1—figure supplement 3). RESULTS +44 50 P site site The map allows placement of PKI at the body P site (Figure 1—figure supplement 3). RESULTS +9 22 Structure III evidence Thus, in Structure III, PKI has translocated along the 40S body, but the head remains fully swiveled so that PKI is between the head A and P sites. RESULTS +24 27 PKI structure_element Thus, in Structure III, PKI has translocated along the 40S body, but the head remains fully swiveled so that PKI is between the head A and P sites. RESULTS +55 58 40S complex_assembly Thus, in Structure III, PKI has translocated along the 40S body, but the head remains fully swiveled so that PKI is between the head A and P sites. RESULTS +59 63 body structure_element Thus, in Structure III, PKI has translocated along the 40S body, but the head remains fully swiveled so that PKI is between the head A and P sites. RESULTS +73 77 head structure_element Thus, in Structure III, PKI has translocated along the 40S body, but the head remains fully swiveled so that PKI is between the head A and P sites. RESULTS +86 100 fully swiveled protein_state Thus, in Structure III, PKI has translocated along the 40S body, but the head remains fully swiveled so that PKI is between the head A and P sites. RESULTS +109 112 PKI structure_element Thus, in Structure III, PKI has translocated along the 40S body, but the head remains fully swiveled so that PKI is between the head A and P sites. RESULTS +128 132 head structure_element Thus, in Structure III, PKI has translocated along the 40S body, but the head remains fully swiveled so that PKI is between the head A and P sites. RESULTS +133 146 A and P sites site Thus, in Structure III, PKI has translocated along the 40S body, but the head remains fully swiveled so that PKI is between the head A and P sites. RESULTS +24 27 map evidence Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191). RESULTS +57 60 PKI structure_element Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191). RESULTS +109 113 body structure_element Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191). RESULTS +114 120 P site site Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191). RESULTS +128 138 absence of protein_state Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191). RESULTS +139 147 stacking bond_interaction Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191). RESULTS +176 180 head structure_element Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191). RESULTS +181 187 A site site Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191). RESULTS +189 194 C1274 residue_name_number Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191). RESULTS +199 205 P site site Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191). RESULTS +207 212 U1191 residue_name_number Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191). RESULTS +16 20 eEF2 protein The position of eEF2 is similar to that in Structure II. RESULTS +43 55 Structure II evidence The position of eEF2 is similar to that in Structure II. RESULTS +0 12 Structure IV evidence Structure IV represents a highly bent IRES with PKI partially accommodated in the P site RESULTS +26 37 highly bent protein_state Structure IV represents a highly bent IRES with PKI partially accommodated in the P site RESULTS +38 42 IRES site Structure IV represents a highly bent IRES with PKI partially accommodated in the P site RESULTS +48 51 PKI structure_element Structure IV represents a highly bent IRES with PKI partially accommodated in the P site RESULTS +82 88 P site site Structure IV represents a highly bent IRES with PKI partially accommodated in the P site RESULTS +3 15 Structure IV evidence In Structure IV, the 40S subunit is almost non-rotated relative to the 60S subunit, and the 40S head is mid-swiveled. RESULTS +21 24 40S complex_assembly In Structure IV, the 40S subunit is almost non-rotated relative to the 60S subunit, and the 40S head is mid-swiveled. RESULTS +25 32 subunit structure_element In Structure IV, the 40S subunit is almost non-rotated relative to the 60S subunit, and the 40S head is mid-swiveled. RESULTS +25 32 subunit structure_element In Structure IV, the 40S subunit is almost non-rotated relative to the 60S subunit, and the 40S head is mid-swiveled. RESULTS +43 54 non-rotated protein_state In Structure IV, the 40S subunit is almost non-rotated relative to the 60S subunit, and the 40S head is mid-swiveled. RESULTS +71 74 60S complex_assembly In Structure IV, the 40S subunit is almost non-rotated relative to the 60S subunit, and the 40S head is mid-swiveled. RESULTS +75 82 subunit structure_element In Structure IV, the 40S subunit is almost non-rotated relative to the 60S subunit, and the 40S head is mid-swiveled. RESULTS +92 95 40S complex_assembly In Structure IV, the 40S subunit is almost non-rotated relative to the 60S subunit, and the 40S head is mid-swiveled. RESULTS +96 100 head structure_element In Structure IV, the 40S subunit is almost non-rotated relative to the 60S subunit, and the 40S head is mid-swiveled. RESULTS +104 116 mid-swiveled protein_state In Structure IV, the 40S subunit is almost non-rotated relative to the 60S subunit, and the 40S head is mid-swiveled. RESULTS +17 21 head structure_element Unwinding of the head moves the head P-site residue U1191 and body P-site residue C1637 closer together, resulting in a partially restored 40S P site. RESULTS +32 36 head structure_element Unwinding of the head moves the head P-site residue U1191 and body P-site residue C1637 closer together, resulting in a partially restored 40S P site. RESULTS +37 43 P-site site Unwinding of the head moves the head P-site residue U1191 and body P-site residue C1637 closer together, resulting in a partially restored 40S P site. RESULTS +52 57 U1191 residue_name_number Unwinding of the head moves the head P-site residue U1191 and body P-site residue C1637 closer together, resulting in a partially restored 40S P site. RESULTS +62 66 body structure_element Unwinding of the head moves the head P-site residue U1191 and body P-site residue C1637 closer together, resulting in a partially restored 40S P site. RESULTS +67 73 P-site site Unwinding of the head moves the head P-site residue U1191 and body P-site residue C1637 closer together, resulting in a partially restored 40S P site. RESULTS +82 87 C1637 residue_name_number Unwinding of the head moves the head P-site residue U1191 and body P-site residue C1637 closer together, resulting in a partially restored 40S P site. RESULTS +139 142 40S complex_assembly Unwinding of the head moves the head P-site residue U1191 and body P-site residue C1637 closer together, resulting in a partially restored 40S P site. RESULTS +143 149 P site site Unwinding of the head moves the head P-site residue U1191 and body P-site residue C1637 closer together, resulting in a partially restored 40S P site. RESULTS +8 13 C1637 residue_name_number Whereas C1637 forms a stacking platform for the last base pair of PKI, U1191 does not yet stack on PKI because the head remains partially swiveled. RESULTS +22 39 stacking platform site Whereas C1637 forms a stacking platform for the last base pair of PKI, U1191 does not yet stack on PKI because the head remains partially swiveled. RESULTS +66 69 PKI structure_element Whereas C1637 forms a stacking platform for the last base pair of PKI, U1191 does not yet stack on PKI because the head remains partially swiveled. RESULTS +71 76 U1191 residue_name_number Whereas C1637 forms a stacking platform for the last base pair of PKI, U1191 does not yet stack on PKI because the head remains partially swiveled. RESULTS +90 95 stack bond_interaction Whereas C1637 forms a stacking platform for the last base pair of PKI, U1191 does not yet stack on PKI because the head remains partially swiveled. RESULTS +99 102 PKI structure_element Whereas C1637 forms a stacking platform for the last base pair of PKI, U1191 does not yet stack on PKI because the head remains partially swiveled. RESULTS +115 119 head structure_element Whereas C1637 forms a stacking platform for the last base pair of PKI, U1191 does not yet stack on PKI because the head remains partially swiveled. RESULTS +13 16 PKI structure_element This renders PKI partially accommodated in the P site (Figure 2g). RESULTS +47 53 P site site This renders PKI partially accommodated in the P site (Figure 2g). RESULTS +17 20 40S complex_assembly Unwinding of the 40S head also positions the head A site closer to the body A site. RESULTS +21 25 head structure_element Unwinding of the 40S head also positions the head A site closer to the body A site. RESULTS +45 49 head structure_element Unwinding of the 40S head also positions the head A site closer to the body A site. RESULTS +50 56 A site site Unwinding of the 40S head also positions the head A site closer to the body A site. RESULTS +71 75 body structure_element Unwinding of the 40S head also positions the head A site closer to the body A site. RESULTS +76 82 A site site Unwinding of the 40S head also positions the head A site closer to the body A site. RESULTS +34 38 eEF2 protein This results in rearrangements of eEF2 interactions with the head, allowing eEF2 to advance further into the A site. RESULTS +61 65 head structure_element This results in rearrangements of eEF2 interactions with the head, allowing eEF2 to advance further into the A site. RESULTS +76 80 eEF2 protein This results in rearrangements of eEF2 interactions with the head, allowing eEF2 to advance further into the A site. RESULTS +109 115 A site site This results in rearrangements of eEF2 interactions with the head, allowing eEF2 to advance further into the A site. RESULTS +17 43 head-interacting interface site To this end, the head-interacting interface of domain IV slides along the surface of the head by 5 Å. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6—figure supplement 1). RESULTS +54 56 IV structure_element To this end, the head-interacting interface of domain IV slides along the surface of the head by 5 Å. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6—figure supplement 1). RESULTS +89 93 head structure_element To this end, the head-interacting interface of domain IV slides along the surface of the head by 5 Å. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6—figure supplement 1). RESULTS +102 109 Helix A structure_element To this end, the head-interacting interface of domain IV slides along the surface of the head by 5 Å. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6—figure supplement 1). RESULTS +120 122 IV structure_element To this end, the head-interacting interface of domain IV slides along the surface of the head by 5 Å. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6—figure supplement 1). RESULTS +161 164 h34 structure_element To this end, the head-interacting interface of domain IV slides along the surface of the head by 5 Å. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6—figure supplement 1). RESULTS +199 203 K613 residue_name_number To this end, the head-interacting interface of domain IV slides along the surface of the head by 5 Å. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6—figure supplement 1). RESULTS +205 209 R617 residue_name_number To this end, the head-interacting interface of domain IV slides along the surface of the head by 5 Å. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6—figure supplement 1). RESULTS +214 218 R631 residue_name_number To this end, the head-interacting interface of domain IV slides along the surface of the head by 5 Å. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6—figure supplement 1). RESULTS +251 254 h33 structure_element To this end, the head-interacting interface of domain IV slides along the surface of the head by 5 Å. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6—figure supplement 1). RESULTS +0 11 Structure V evidence Structure V represents an extended IRES with PKI fully accommodated in the P site and domain IV of eEF2 in the A site RESULTS +26 34 extended protein_state Structure V represents an extended IRES with PKI fully accommodated in the P site and domain IV of eEF2 in the A site RESULTS +35 39 IRES site Structure V represents an extended IRES with PKI fully accommodated in the P site and domain IV of eEF2 in the A site RESULTS +45 48 PKI structure_element Structure V represents an extended IRES with PKI fully accommodated in the P site and domain IV of eEF2 in the A site RESULTS +75 81 P site site Structure V represents an extended IRES with PKI fully accommodated in the P site and domain IV of eEF2 in the A site RESULTS +93 95 IV structure_element Structure V represents an extended IRES with PKI fully accommodated in the P site and domain IV of eEF2 in the A site RESULTS +99 103 eEF2 protein Structure V represents an extended IRES with PKI fully accommodated in the P site and domain IV of eEF2 in the A site RESULTS +111 117 A site site Structure V represents an extended IRES with PKI fully accommodated in the P site and domain IV of eEF2 in the A site RESULTS +7 25 nearly non-rotated protein_state In the nearly non-rotated and non-swiveled ribosome conformation in Structure V closely resembling that of the post-translocation 80S•2tRNA•mRNA complex, PKI is fully accommodated in the P site. RESULTS +30 42 non-swiveled protein_state In the nearly non-rotated and non-swiveled ribosome conformation in Structure V closely resembling that of the post-translocation 80S•2tRNA•mRNA complex, PKI is fully accommodated in the P site. RESULTS +43 51 ribosome complex_assembly In the nearly non-rotated and non-swiveled ribosome conformation in Structure V closely resembling that of the post-translocation 80S•2tRNA•mRNA complex, PKI is fully accommodated in the P site. RESULTS +68 79 Structure V evidence In the nearly non-rotated and non-swiveled ribosome conformation in Structure V closely resembling that of the post-translocation 80S•2tRNA•mRNA complex, PKI is fully accommodated in the P site. RESULTS +111 129 post-translocation protein_state In the nearly non-rotated and non-swiveled ribosome conformation in Structure V closely resembling that of the post-translocation 80S•2tRNA•mRNA complex, PKI is fully accommodated in the P site. RESULTS +130 144 80S•2tRNA•mRNA complex_assembly In the nearly non-rotated and non-swiveled ribosome conformation in Structure V closely resembling that of the post-translocation 80S•2tRNA•mRNA complex, PKI is fully accommodated in the P site. RESULTS +154 157 PKI structure_element In the nearly non-rotated and non-swiveled ribosome conformation in Structure V closely resembling that of the post-translocation 80S•2tRNA•mRNA complex, PKI is fully accommodated in the P site. RESULTS +187 193 P site site In the nearly non-rotated and non-swiveled ribosome conformation in Structure V closely resembling that of the post-translocation 80S•2tRNA•mRNA complex, PKI is fully accommodated in the P site. RESULTS +4 30 codon-anticodon–like helix structure_element The codon-anticodon–like helix is stacked on P-site residues U1191 and C1637 (Figure 3d), analogous to stacking of the tRNA-mRNA helix (Figure 3e). RESULTS +45 51 P-site site The codon-anticodon–like helix is stacked on P-site residues U1191 and C1637 (Figure 3d), analogous to stacking of the tRNA-mRNA helix (Figure 3e). RESULTS +61 66 U1191 residue_name_number The codon-anticodon–like helix is stacked on P-site residues U1191 and C1637 (Figure 3d), analogous to stacking of the tRNA-mRNA helix (Figure 3e). RESULTS +71 76 C1637 residue_name_number The codon-anticodon–like helix is stacked on P-site residues U1191 and C1637 (Figure 3d), analogous to stacking of the tRNA-mRNA helix (Figure 3e). RESULTS +103 111 stacking bond_interaction The codon-anticodon–like helix is stacked on P-site residues U1191 and C1637 (Figure 3d), analogous to stacking of the tRNA-mRNA helix (Figure 3e). RESULTS +119 128 tRNA-mRNA complex_assembly The codon-anticodon–like helix is stacked on P-site residues U1191 and C1637 (Figure 3d), analogous to stacking of the tRNA-mRNA helix (Figure 3e). RESULTS +129 134 helix structure_element The codon-anticodon–like helix is stacked on P-site residues U1191 and C1637 (Figure 3d), analogous to stacking of the tRNA-mRNA helix (Figure 3e). RESULTS +35 39 eEF2 protein A notable conformational change in eEF2 from that in the preceding Structures is visible in the position of domain III, which contacts uS12 (Figure 6d). RESULTS +67 77 Structures evidence A notable conformational change in eEF2 from that in the preceding Structures is visible in the position of domain III, which contacts uS12 (Figure 6d). RESULTS +115 118 III structure_element A notable conformational change in eEF2 from that in the preceding Structures is visible in the position of domain III, which contacts uS12 (Figure 6d). RESULTS +135 139 uS12 protein A notable conformational change in eEF2 from that in the preceding Structures is visible in the position of domain III, which contacts uS12 (Figure 6d). RESULTS +3 14 Structure V evidence In Structure V, protein uS12 is shifted along with the 40S body as a result of intersubunit rotation. RESULTS +24 28 uS12 protein In Structure V, protein uS12 is shifted along with the 40S body as a result of intersubunit rotation. RESULTS +55 58 40S complex_assembly In Structure V, protein uS12 is shifted along with the 40S body as a result of intersubunit rotation. RESULTS +59 63 body structure_element In Structure V, protein uS12 is shifted along with the 40S body as a result of intersubunit rotation. RESULTS +18 22 uS12 protein In this position, uS12 forms extensive interactions with eEF2 domains II and III. RESULTS +57 61 eEF2 protein In this position, uS12 forms extensive interactions with eEF2 domains II and III. RESULTS +70 72 II structure_element In this position, uS12 forms extensive interactions with eEF2 domains II and III. RESULTS +77 80 III structure_element In this position, uS12 forms extensive interactions with eEF2 domains II and III. RESULTS +18 33 C-terminal tail structure_element Specifically, the C-terminal tail of uS12 packs against the β-barrel of domain II, while the β-barrel of uS12 packs against helix A of domain III. RESULTS +37 41 uS12 protein Specifically, the C-terminal tail of uS12 packs against the β-barrel of domain II, while the β-barrel of uS12 packs against helix A of domain III. RESULTS +60 68 β-barrel structure_element Specifically, the C-terminal tail of uS12 packs against the β-barrel of domain II, while the β-barrel of uS12 packs against helix A of domain III. RESULTS +79 81 II structure_element Specifically, the C-terminal tail of uS12 packs against the β-barrel of domain II, while the β-barrel of uS12 packs against helix A of domain III. RESULTS +93 101 β-barrel structure_element Specifically, the C-terminal tail of uS12 packs against the β-barrel of domain II, while the β-barrel of uS12 packs against helix A of domain III. RESULTS +105 109 uS12 protein Specifically, the C-terminal tail of uS12 packs against the β-barrel of domain II, while the β-barrel of uS12 packs against helix A of domain III. RESULTS +124 131 helix A structure_element Specifically, the C-terminal tail of uS12 packs against the β-barrel of domain II, while the β-barrel of uS12 packs against helix A of domain III. RESULTS +142 145 III structure_element Specifically, the C-terminal tail of uS12 packs against the β-barrel of domain II, while the β-barrel of uS12 packs against helix A of domain III. RESULTS +23 30 helix A structure_element This shifts the tip of helix A of domain III (at aa 500) by ~5 Å (relative to that in Structure I) toward domain I. Although domain III remains in contact with domain V, the shift occurs in the direction that could eventually disconnect the β-platforms of these domains. RESULTS +41 44 III structure_element This shifts the tip of helix A of domain III (at aa 500) by ~5 Å (relative to that in Structure I) toward domain I. Although domain III remains in contact with domain V, the shift occurs in the direction that could eventually disconnect the β-platforms of these domains. RESULTS +52 55 500 residue_number This shifts the tip of helix A of domain III (at aa 500) by ~5 Å (relative to that in Structure I) toward domain I. Although domain III remains in contact with domain V, the shift occurs in the direction that could eventually disconnect the β-platforms of these domains. RESULTS +86 97 Structure I evidence This shifts the tip of helix A of domain III (at aa 500) by ~5 Å (relative to that in Structure I) toward domain I. Although domain III remains in contact with domain V, the shift occurs in the direction that could eventually disconnect the β-platforms of these domains. RESULTS +113 114 I structure_element This shifts the tip of helix A of domain III (at aa 500) by ~5 Å (relative to that in Structure I) toward domain I. Although domain III remains in contact with domain V, the shift occurs in the direction that could eventually disconnect the β-platforms of these domains. RESULTS +132 135 III structure_element This shifts the tip of helix A of domain III (at aa 500) by ~5 Å (relative to that in Structure I) toward domain I. Although domain III remains in contact with domain V, the shift occurs in the direction that could eventually disconnect the β-platforms of these domains. RESULTS +167 168 V structure_element This shifts the tip of helix A of domain III (at aa 500) by ~5 Å (relative to that in Structure I) toward domain I. Although domain III remains in contact with domain V, the shift occurs in the direction that could eventually disconnect the β-platforms of these domains. RESULTS +241 252 β-platforms structure_element This shifts the tip of helix A of domain III (at aa 500) by ~5 Å (relative to that in Structure I) toward domain I. Although domain III remains in contact with domain V, the shift occurs in the direction that could eventually disconnect the β-platforms of these domains. RESULTS +7 9 IV structure_element Domain IV of eEF2 is fully accommodated in the A site. RESULTS +13 17 eEF2 protein Domain IV of eEF2 is fully accommodated in the A site. RESULTS +47 53 A site site Domain IV of eEF2 is fully accommodated in the A site. RESULTS +23 41 open reading frame structure_element The first codon of the open reading frame is also positioned in the A site, with bases exposed toward eEF2 (Figure 7), resembling the conformations of the A-site codons in EF-G-bound 70S complexes. RESULTS +68 74 A site site The first codon of the open reading frame is also positioned in the A site, with bases exposed toward eEF2 (Figure 7), resembling the conformations of the A-site codons in EF-G-bound 70S complexes. RESULTS +102 106 eEF2 protein The first codon of the open reading frame is also positioned in the A site, with bases exposed toward eEF2 (Figure 7), resembling the conformations of the A-site codons in EF-G-bound 70S complexes. RESULTS +155 161 A-site site The first codon of the open reading frame is also positioned in the A site, with bases exposed toward eEF2 (Figure 7), resembling the conformations of the A-site codons in EF-G-bound 70S complexes. RESULTS +172 182 EF-G-bound protein_state The first codon of the open reading frame is also positioned in the A site, with bases exposed toward eEF2 (Figure 7), resembling the conformations of the A-site codons in EF-G-bound 70S complexes. RESULTS +183 186 70S complex_assembly The first codon of the open reading frame is also positioned in the A site, with bases exposed toward eEF2 (Figure 7), resembling the conformations of the A-site codons in EF-G-bound 70S complexes. RESULTS +20 30 Structures evidence As in the preceding Structures, the histidine-diphthamide tip is bound in the minor groove of the P-site codon-anticodon helix. RESULTS +36 61 histidine-diphthamide tip site As in the preceding Structures, the histidine-diphthamide tip is bound in the minor groove of the P-site codon-anticodon helix. RESULTS +65 73 bound in protein_state As in the preceding Structures, the histidine-diphthamide tip is bound in the minor groove of the P-site codon-anticodon helix. RESULTS +78 90 minor groove site As in the preceding Structures, the histidine-diphthamide tip is bound in the minor groove of the P-site codon-anticodon helix. RESULTS +98 104 P-site site As in the preceding Structures, the histidine-diphthamide tip is bound in the minor groove of the P-site codon-anticodon helix. RESULTS +105 126 codon-anticodon helix structure_element As in the preceding Structures, the histidine-diphthamide tip is bound in the minor groove of the P-site codon-anticodon helix. RESULTS +0 7 Diph699 ptm Diph699 slightly rearranges, relative to that in Structure I (Figure 7), and interacts with four out of six codon-anticodon nucleotides. RESULTS +49 60 Structure I evidence Diph699 slightly rearranges, relative to that in Structure I (Figure 7), and interacts with four out of six codon-anticodon nucleotides. RESULTS +31 36 G6907 residue_name_number The imidazole moiety stacks on G6907 (corresponding to nt 36 in the tRNA anticodon) and hydrogen bonds with O2’ of G6906 (nt 35 of tRNA). RESULTS +68 72 tRNA chemical The imidazole moiety stacks on G6907 (corresponding to nt 36 in the tRNA anticodon) and hydrogen bonds with O2’ of G6906 (nt 35 of tRNA). RESULTS +88 102 hydrogen bonds bond_interaction The imidazole moiety stacks on G6907 (corresponding to nt 36 in the tRNA anticodon) and hydrogen bonds with O2’ of G6906 (nt 35 of tRNA). RESULTS +115 120 G6906 residue_name_number The imidazole moiety stacks on G6907 (corresponding to nt 36 in the tRNA anticodon) and hydrogen bonds with O2’ of G6906 (nt 35 of tRNA). RESULTS +131 135 tRNA chemical The imidazole moiety stacks on G6907 (corresponding to nt 36 in the tRNA anticodon) and hydrogen bonds with O2’ of G6906 (nt 35 of tRNA). RESULTS +17 28 diphthamide ptm The amide at the diphthamide end interacts with N2 of G6906 and O2 and O2’ of C6951 (corresponding to nt 2 of the codon). RESULTS +54 59 G6906 residue_name_number The amide at the diphthamide end interacts with N2 of G6906 and O2 and O2’ of C6951 (corresponding to nt 2 of the codon). RESULTS +78 83 C6951 residue_name_number The amide at the diphthamide end interacts with N2 of G6906 and O2 and O2’ of C6951 (corresponding to nt 2 of the codon). RESULTS +58 63 C6952 residue_name_number The trimethylamino-group is positioned over the ribose of C6952 (codon nt 3). RESULTS +0 4 IRES site IRES translocation mechanism DISCUSS +42 52 initiation protein_state Animation showing the transition from the initiation 80S•TSV IRES structures (Koh et al., 2014) to eEF2-bound Structures I through V (this work). FIG +53 65 80S•TSV IRES complex_assembly Animation showing the transition from the initiation 80S•TSV IRES structures (Koh et al., 2014) to eEF2-bound Structures I through V (this work). FIG +66 76 structures evidence Animation showing the transition from the initiation 80S•TSV IRES structures (Koh et al., 2014) to eEF2-bound Structures I through V (this work). FIG +99 109 eEF2-bound protein_state Animation showing the transition from the initiation 80S•TSV IRES structures (Koh et al., 2014) to eEF2-bound Structures I through V (this work). FIG +110 132 Structures I through V evidence Animation showing the transition from the initiation 80S•TSV IRES structures (Koh et al., 2014) to eEF2-bound Structures I through V (this work). FIG +80 84 head structure_element Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. FIG +92 95 40S complex_assembly Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. FIG +96 103 subunit structure_element Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. FIG +185 188 40S complex_assembly Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. FIG +189 196 subunit structure_element Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. FIG +207 210 40S complex_assembly Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. FIG +211 215 head structure_element Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. FIG +318 321 40S complex_assembly Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. FIG +322 329 subunit structure_element Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. FIG +358 373 decoding center site Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. FIG +375 381 A site site Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. FIG +391 397 P site site Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2—figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice. FIG +34 39 C1274 residue_name_number In scenes 1, 2 and 3, nucleotides C1274, U1191 of the 40S head and G904 of the 40S platform are shown in black to denote the A, P and E sites, respectively. FIG +41 46 U1191 residue_name_number In scenes 1, 2 and 3, nucleotides C1274, U1191 of the 40S head and G904 of the 40S platform are shown in black to denote the A, P and E sites, respectively. FIG +54 57 40S complex_assembly In scenes 1, 2 and 3, nucleotides C1274, U1191 of the 40S head and G904 of the 40S platform are shown in black to denote the A, P and E sites, respectively. FIG +58 62 head structure_element In scenes 1, 2 and 3, nucleotides C1274, U1191 of the 40S head and G904 of the 40S platform are shown in black to denote the A, P and E sites, respectively. FIG +67 71 G904 residue_name_number In scenes 1, 2 and 3, nucleotides C1274, U1191 of the 40S head and G904 of the 40S platform are shown in black to denote the A, P and E sites, respectively. FIG +79 91 40S platform site In scenes 1, 2 and 3, nucleotides C1274, U1191 of the 40S head and G904 of the 40S platform are shown in black to denote the A, P and E sites, respectively. FIG +125 141 A, P and E sites site In scenes 1, 2 and 3, nucleotides C1274, U1191 of the 40S head and G904 of the 40S platform are shown in black to denote the A, P and E sites, respectively. FIG +12 17 C1274 residue_name_number In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange. FIG +22 27 U1191 residue_name_number In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange. FIG +61 65 G577 residue_name_number In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange. FIG +67 72 A1755 residue_name_number In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange. FIG +77 82 A1756 residue_name_number In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange. FIG +90 93 40S complex_assembly In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange. FIG +94 98 body structure_element In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange. FIG +99 105 A site site In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange. FIG +110 115 C1637 residue_name_number In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange. FIG +123 127 body structure_element In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange. FIG +128 134 P site site In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange. FIG +34 44 structures evidence In this work we have captured the structures of the TSV IRES, whose PKI samples positions between the A and P sites (Structures I–IV), as well as in the P site (Structure V). DISCUSS +52 55 TSV species In this work we have captured the structures of the TSV IRES, whose PKI samples positions between the A and P sites (Structures I–IV), as well as in the P site (Structure V). DISCUSS +56 60 IRES site In this work we have captured the structures of the TSV IRES, whose PKI samples positions between the A and P sites (Structures I–IV), as well as in the P site (Structure V). DISCUSS +68 71 PKI structure_element In this work we have captured the structures of the TSV IRES, whose PKI samples positions between the A and P sites (Structures I–IV), as well as in the P site (Structure V). DISCUSS +102 115 A and P sites site In this work we have captured the structures of the TSV IRES, whose PKI samples positions between the A and P sites (Structures I–IV), as well as in the P site (Structure V). DISCUSS +117 132 Structures I–IV evidence In this work we have captured the structures of the TSV IRES, whose PKI samples positions between the A and P sites (Structures I–IV), as well as in the P site (Structure V). DISCUSS +153 159 P site site In this work we have captured the structures of the TSV IRES, whose PKI samples positions between the A and P sites (Structures I–IV), as well as in the P site (Structure V). DISCUSS +161 172 Structure V evidence In this work we have captured the structures of the TSV IRES, whose PKI samples positions between the A and P sites (Structures I–IV), as well as in the P site (Structure V). DISCUSS +54 64 initiation protein_state We propose that together with the previously reported initiation state, these structures represent the trajectory of eEF2-induced IRES translocation (shown as an animation in http://labs.umassmed.edu/korostelevlab/msc/iresmovie.gif and Video 1). DISCUSS +78 88 structures evidence We propose that together with the previously reported initiation state, these structures represent the trajectory of eEF2-induced IRES translocation (shown as an animation in http://labs.umassmed.edu/korostelevlab/msc/iresmovie.gif and Video 1). DISCUSS +117 121 eEF2 protein We propose that together with the previously reported initiation state, these structures represent the trajectory of eEF2-induced IRES translocation (shown as an animation in http://labs.umassmed.edu/korostelevlab/msc/iresmovie.gif and Video 1). DISCUSS +130 134 IRES site We propose that together with the previously reported initiation state, these structures represent the trajectory of eEF2-induced IRES translocation (shown as an animation in http://labs.umassmed.edu/korostelevlab/msc/iresmovie.gif and Video 1). DISCUSS +4 14 structures evidence Our structures reveal previously unseen intermediate states of eEF2 or EF-G engagement with the A site, providing the structural basis for the mechanism of translocase action. DISCUSS +63 67 eEF2 protein Our structures reveal previously unseen intermediate states of eEF2 or EF-G engagement with the A site, providing the structural basis for the mechanism of translocase action. DISCUSS +71 75 EF-G protein Our structures reveal previously unseen intermediate states of eEF2 or EF-G engagement with the A site, providing the structural basis for the mechanism of translocase action. DISCUSS +96 102 A site site Our structures reveal previously unseen intermediate states of eEF2 or EF-G engagement with the A site, providing the structural basis for the mechanism of translocase action. DISCUSS +156 167 translocase protein_type Our structures reveal previously unseen intermediate states of eEF2 or EF-G engagement with the A site, providing the structural basis for the mechanism of translocase action. DISCUSS +56 64 eEF2•GTP complex_assembly Furthermore, they provide insight into the mechanism of eEF2•GTP association with the pre-translocation ribosome and eEF2•GDP dissociation from the post-translocation ribosome, also delineating the mechanism of translation inhibition by the antifungal drug sordarin. DISCUSS +86 103 pre-translocation protein_state Furthermore, they provide insight into the mechanism of eEF2•GTP association with the pre-translocation ribosome and eEF2•GDP dissociation from the post-translocation ribosome, also delineating the mechanism of translation inhibition by the antifungal drug sordarin. DISCUSS +104 112 ribosome complex_assembly Furthermore, they provide insight into the mechanism of eEF2•GTP association with the pre-translocation ribosome and eEF2•GDP dissociation from the post-translocation ribosome, also delineating the mechanism of translation inhibition by the antifungal drug sordarin. DISCUSS +117 125 eEF2•GDP complex_assembly Furthermore, they provide insight into the mechanism of eEF2•GTP association with the pre-translocation ribosome and eEF2•GDP dissociation from the post-translocation ribosome, also delineating the mechanism of translation inhibition by the antifungal drug sordarin. DISCUSS +148 166 post-translocation protein_state Furthermore, they provide insight into the mechanism of eEF2•GTP association with the pre-translocation ribosome and eEF2•GDP dissociation from the post-translocation ribosome, also delineating the mechanism of translation inhibition by the antifungal drug sordarin. DISCUSS +167 175 ribosome complex_assembly Furthermore, they provide insight into the mechanism of eEF2•GTP association with the pre-translocation ribosome and eEF2•GDP dissociation from the post-translocation ribosome, also delineating the mechanism of translation inhibition by the antifungal drug sordarin. DISCUSS +257 265 sordarin chemical Furthermore, they provide insight into the mechanism of eEF2•GTP association with the pre-translocation ribosome and eEF2•GDP dissociation from the post-translocation ribosome, also delineating the mechanism of translation inhibition by the antifungal drug sordarin. DISCUSS +37 47 structures evidence In summary, the reported ensemble of structures substantially enhances our understanding of the translocation mechanism, including that of tRNAs as discussed below. DISCUSS +139 144 tRNAs chemical In summary, the reported ensemble of structures substantially enhances our understanding of the translocation mechanism, including that of tRNAs as discussed below. DISCUSS +21 24 TSV species Translocation of the TSV IRES on the 40S subunit globally resembles a step of an inchworm (Figure 4; see also Figure 3—figure supplement 2). DISCUSS +25 29 IRES site Translocation of the TSV IRES on the 40S subunit globally resembles a step of an inchworm (Figure 4; see also Figure 3—figure supplement 2). DISCUSS +37 40 40S complex_assembly Translocation of the TSV IRES on the 40S subunit globally resembles a step of an inchworm (Figure 4; see also Figure 3—figure supplement 2). DISCUSS +41 48 subunit structure_element Translocation of the TSV IRES on the 40S subunit globally resembles a step of an inchworm (Figure 4; see also Figure 3—figure supplement 2). DISCUSS +81 89 inchworm protein_state Translocation of the TSV IRES on the 40S subunit globally resembles a step of an inchworm (Figure 4; see also Figure 3—figure supplement 2). DISCUSS +14 24 initiation protein_state At the start (initiation state), the IRES adopts an extended conformation (extended inchworm). DISCUSS +37 41 IRES site At the start (initiation state), the IRES adopts an extended conformation (extended inchworm). DISCUSS +52 60 extended protein_state At the start (initiation state), the IRES adopts an extended conformation (extended inchworm). DISCUSS +75 92 extended inchworm protein_state At the start (initiation state), the IRES adopts an extended conformation (extended inchworm). DISCUSS +4 15 front 'legs structure_element The front 'legs' (SL4 and SL5) of the 5’-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2). DISCUSS +18 21 SL4 structure_element The front 'legs' (SL4 and SL5) of the 5’-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2). DISCUSS +26 29 SL5 structure_element The front 'legs' (SL4 and SL5) of the 5’-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2). DISCUSS +38 47 5’-domain structure_element The front 'legs' (SL4 and SL5) of the 5’-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2). DISCUSS +49 58 front end structure_element The front 'legs' (SL4 and SL5) of the 5’-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2). DISCUSS +80 83 40S complex_assembly The front 'legs' (SL4 and SL5) of the 5’-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2). DISCUSS +84 88 head structure_element The front 'legs' (SL4 and SL5) of the 5’-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2). DISCUSS +98 101 uS7 protein The front 'legs' (SL4 and SL5) of the 5’-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2). DISCUSS +103 107 uS11 protein The front 'legs' (SL4 and SL5) of the 5’-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2). DISCUSS +112 116 eS25 protein The front 'legs' (SL4 and SL5) of the 5’-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2). DISCUSS +0 3 PKI structure_element PKI, representing the hind end, is bound in the A site. DISCUSS +22 30 hind end structure_element PKI, representing the hind end, is bound in the A site. DISCUSS +35 43 bound in protein_state PKI, representing the hind end, is bound in the A site. DISCUSS +48 54 A site site PKI, representing the hind end, is bound in the A site. DISCUSS +23 41 Structures I to IV evidence In the first sub-step (Structures I to IV), the hind end advances from the A to the P site and approaches the front end, which remains attached to the 40S surface. DISCUSS +48 56 hind end structure_element In the first sub-step (Structures I to IV), the hind end advances from the A to the P site and approaches the front end, which remains attached to the 40S surface. DISCUSS +75 90 A to the P site site In the first sub-step (Structures I to IV), the hind end advances from the A to the P site and approaches the front end, which remains attached to the 40S surface. DISCUSS +110 119 front end structure_element In the first sub-step (Structures I to IV), the hind end advances from the A to the P site and approaches the front end, which remains attached to the 40S surface. DISCUSS +151 154 40S complex_assembly In the first sub-step (Structures I to IV), the hind end advances from the A to the P site and approaches the front end, which remains attached to the 40S surface. DISCUSS +35 38 PKI structure_element This shortens the distance between PKI and SL4 by up to 20 Å relative to the initiating IRES structure, resulting in a bent IRES conformation (bent inchworm). DISCUSS +43 46 SL4 structure_element This shortens the distance between PKI and SL4 by up to 20 Å relative to the initiating IRES structure, resulting in a bent IRES conformation (bent inchworm). DISCUSS +88 92 IRES site This shortens the distance between PKI and SL4 by up to 20 Å relative to the initiating IRES structure, resulting in a bent IRES conformation (bent inchworm). DISCUSS +93 102 structure evidence This shortens the distance between PKI and SL4 by up to 20 Å relative to the initiating IRES structure, resulting in a bent IRES conformation (bent inchworm). DISCUSS +119 123 bent protein_state This shortens the distance between PKI and SL4 by up to 20 Å relative to the initiating IRES structure, resulting in a bent IRES conformation (bent inchworm). DISCUSS +124 128 IRES site This shortens the distance between PKI and SL4 by up to 20 Å relative to the initiating IRES structure, resulting in a bent IRES conformation (bent inchworm). DISCUSS +143 156 bent inchworm protein_state This shortens the distance between PKI and SL4 by up to 20 Å relative to the initiating IRES structure, resulting in a bent IRES conformation (bent inchworm). DISCUSS +9 27 Structures IV to V evidence Finally (Structures IV to V), as the hind end is accommodated in the P site, the front 'legs' advance by departing from their initial binding sites. DISCUSS +37 45 hind end structure_element Finally (Structures IV to V), as the hind end is accommodated in the P site, the front 'legs' advance by departing from their initial binding sites. DISCUSS +69 75 P site site Finally (Structures IV to V), as the hind end is accommodated in the P site, the front 'legs' advance by departing from their initial binding sites. DISCUSS +81 93 front 'legs' structure_element Finally (Structures IV to V), as the hind end is accommodated in the P site, the front 'legs' advance by departing from their initial binding sites. DISCUSS +126 147 initial binding sites site Finally (Structures IV to V), as the hind end is accommodated in the P site, the front 'legs' advance by departing from their initial binding sites. DISCUSS +18 22 IRES site This converts the IRES into an extended conformation, rendering the inchworm prepared for the next translocation step. DISCUSS +31 39 extended protein_state This converts the IRES into an extended conformation, rendering the inchworm prepared for the next translocation step. DISCUSS +68 76 inchworm protein_state This converts the IRES into an extended conformation, rendering the inchworm prepared for the next translocation step. DISCUSS +27 31 head structure_element Notably, at all steps, the head of the IRES inchworm (L1.1 region) is supported by the mobile L1 stalk. DISCUSS +39 43 IRES site Notably, at all steps, the head of the IRES inchworm (L1.1 region) is supported by the mobile L1 stalk. DISCUSS +44 52 inchworm protein_state Notably, at all steps, the head of the IRES inchworm (L1.1 region) is supported by the mobile L1 stalk. DISCUSS +54 65 L1.1 region structure_element Notably, at all steps, the head of the IRES inchworm (L1.1 region) is supported by the mobile L1 stalk. DISCUSS +87 93 mobile protein_state Notably, at all steps, the head of the IRES inchworm (L1.1 region) is supported by the mobile L1 stalk. DISCUSS +94 102 L1 stalk structure_element Notably, at all steps, the head of the IRES inchworm (L1.1 region) is supported by the mobile L1 stalk. DISCUSS +7 25 post-translocation protein_state In the post-translocation CrPV IRES structure, the 5’-domain similarly protrudes between the subunits and interacts with the L1 stalk, as in the initiation state for this IRES. DISCUSS +26 30 CrPV species In the post-translocation CrPV IRES structure, the 5’-domain similarly protrudes between the subunits and interacts with the L1 stalk, as in the initiation state for this IRES. DISCUSS +31 35 IRES site In the post-translocation CrPV IRES structure, the 5’-domain similarly protrudes between the subunits and interacts with the L1 stalk, as in the initiation state for this IRES. DISCUSS +36 45 structure evidence In the post-translocation CrPV IRES structure, the 5’-domain similarly protrudes between the subunits and interacts with the L1 stalk, as in the initiation state for this IRES. DISCUSS +51 60 5’-domain structure_element In the post-translocation CrPV IRES structure, the 5’-domain similarly protrudes between the subunits and interacts with the L1 stalk, as in the initiation state for this IRES. DISCUSS +125 133 L1 stalk structure_element In the post-translocation CrPV IRES structure, the 5’-domain similarly protrudes between the subunits and interacts with the L1 stalk, as in the initiation state for this IRES. DISCUSS +145 155 initiation protein_state In the post-translocation CrPV IRES structure, the 5’-domain similarly protrudes between the subunits and interacts with the L1 stalk, as in the initiation state for this IRES. DISCUSS +171 175 IRES site In the post-translocation CrPV IRES structure, the 5’-domain similarly protrudes between the subunits and interacts with the L1 stalk, as in the initiation state for this IRES. DISCUSS +46 49 TSV species This underlines structural similarity for the TSV and CrPV IRES translocation mechanisms. DISCUSS +54 58 CrPV species This underlines structural similarity for the TSV and CrPV IRES translocation mechanisms. DISCUSS +59 63 IRES site This underlines structural similarity for the TSV and CrPV IRES translocation mechanisms. DISCUSS +61 67 A site site Upon translocation, the GCU start codon is positioned in the A site (Structure V), ready for interaction with Ala-tRNAAla upon eEF2 departure. DISCUSS +69 80 Structure V evidence Upon translocation, the GCU start codon is positioned in the A site (Structure V), ready for interaction with Ala-tRNAAla upon eEF2 departure. DISCUSS +110 121 Ala-tRNAAla chemical Upon translocation, the GCU start codon is positioned in the A site (Structure V), ready for interaction with Ala-tRNAAla upon eEF2 departure. DISCUSS +127 131 eEF2 protein Upon translocation, the GCU start codon is positioned in the A site (Structure V), ready for interaction with Ala-tRNAAla upon eEF2 departure. DISCUSS +59 62 IGR structure_element Recent studies have shown that in some cases a fraction of IGR IRES-driven translation results from an alternative reading frame, which is shifted by one nucleotide relative to the normal ORF. DISCUSS +63 67 IRES site Recent studies have shown that in some cases a fraction of IGR IRES-driven translation results from an alternative reading frame, which is shifted by one nucleotide relative to the normal ORF. DISCUSS +188 191 ORF structure_element Recent studies have shown that in some cases a fraction of IGR IRES-driven translation results from an alternative reading frame, which is shifted by one nucleotide relative to the normal ORF. DISCUSS +78 92 aminoacyl-tRNA chemical One of the mechanistic scenarios (discussed in) involves binding of the first aminoacyl-tRNA to the post-translocated IRES mRNA frame shifted by one nucleotide (predominantly a +1 frame shift). DISCUSS +100 117 post-translocated protein_state One of the mechanistic scenarios (discussed in) involves binding of the first aminoacyl-tRNA to the post-translocated IRES mRNA frame shifted by one nucleotide (predominantly a +1 frame shift). DISCUSS +118 122 IRES site One of the mechanistic scenarios (discussed in) involves binding of the first aminoacyl-tRNA to the post-translocated IRES mRNA frame shifted by one nucleotide (predominantly a +1 frame shift). DISCUSS +123 127 mRNA chemical One of the mechanistic scenarios (discussed in) involves binding of the first aminoacyl-tRNA to the post-translocated IRES mRNA frame shifted by one nucleotide (predominantly a +1 frame shift). DISCUSS +7 17 structures evidence In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs. DISCUSS +23 27 IRES site In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs. DISCUSS +44 59 decoding center site In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs. DISCUSS +62 78 pre-translocated protein_state In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs. DISCUSS +82 100 fully translocated protein_state In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs. DISCUSS +101 104 ORF structure_element In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs. DISCUSS +143 146 ORF structure_element In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs. DISCUSS +164 168 eEF2 protein In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs. DISCUSS +227 231 IRES site In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs. DISCUSS +232 237 mRNAs chemical In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs. DISCUSS +61 65 eEF2 protein It is likely that alternative frame setting occurs following eEF2 release and that this depends on transient displacement of the start codon in the decoding center, allowing binding of the corresponding amino acyl-tRNA to an off-frame codon. DISCUSS +148 163 decoding center site It is likely that alternative frame setting occurs following eEF2 release and that this depends on transient displacement of the start codon in the decoding center, allowing binding of the corresponding amino acyl-tRNA to an off-frame codon. DISCUSS +203 218 amino acyl-tRNA chemical It is likely that alternative frame setting occurs following eEF2 release and that this depends on transient displacement of the start codon in the decoding center, allowing binding of the corresponding amino acyl-tRNA to an off-frame codon. DISCUSS +8 26 structural studies experimental_method Further structural studies involving 80S•IRES•tRNA complexes are necessary to understand the mechanisms underlying alternative reading frame selection. DISCUSS +37 50 80S•IRES•tRNA complex_assembly Further structural studies involving 80S•IRES•tRNA complexes are necessary to understand the mechanisms underlying alternative reading frame selection. DISCUSS +4 15 presence of protein_state The presence of several translocation complexes in a single sample suggests that the structures represent equilibrium states of forward and reverse translocation of the IRES, which interconvert among each other. DISCUSS +85 95 structures evidence The presence of several translocation complexes in a single sample suggests that the structures represent equilibrium states of forward and reverse translocation of the IRES, which interconvert among each other. DISCUSS +169 173 IRES site The presence of several translocation complexes in a single sample suggests that the structures represent equilibrium states of forward and reverse translocation of the IRES, which interconvert among each other. DISCUSS +61 66 IRESs site This is consistent with the observations that the intergenic IRESs are prone to reverse translocation. DISCUSS +14 46 biochemical toe-printing studies experimental_method Specifically, biochemical toe-printing studies in the presence of eEF2•GTP identified IRES in a non-translocated position unless eEF1a•aa-tRNA is also present. DISCUSS +54 65 presence of protein_state Specifically, biochemical toe-printing studies in the presence of eEF2•GTP identified IRES in a non-translocated position unless eEF1a•aa-tRNA is also present. DISCUSS +66 74 eEF2•GTP complex_assembly Specifically, biochemical toe-printing studies in the presence of eEF2•GTP identified IRES in a non-translocated position unless eEF1a•aa-tRNA is also present. DISCUSS +86 90 IRES site Specifically, biochemical toe-printing studies in the presence of eEF2•GTP identified IRES in a non-translocated position unless eEF1a•aa-tRNA is also present. DISCUSS +96 112 non-translocated protein_state Specifically, biochemical toe-printing studies in the presence of eEF2•GTP identified IRES in a non-translocated position unless eEF1a•aa-tRNA is also present. DISCUSS +129 142 eEF1a•aa-tRNA complex_assembly Specifically, biochemical toe-printing studies in the presence of eEF2•GTP identified IRES in a non-translocated position unless eEF1a•aa-tRNA is also present. DISCUSS +29 33 IRES site These findings indicate that IRES translocation by eEF2 is futile: the IRES returns to the A site upon releasing eEF2•GDP unless an amino-acyl tRNA enters the A site and blocks IRES back-translocation. DISCUSS +51 55 eEF2 protein These findings indicate that IRES translocation by eEF2 is futile: the IRES returns to the A site upon releasing eEF2•GDP unless an amino-acyl tRNA enters the A site and blocks IRES back-translocation. DISCUSS +71 75 IRES site These findings indicate that IRES translocation by eEF2 is futile: the IRES returns to the A site upon releasing eEF2•GDP unless an amino-acyl tRNA enters the A site and blocks IRES back-translocation. DISCUSS +91 97 A site site These findings indicate that IRES translocation by eEF2 is futile: the IRES returns to the A site upon releasing eEF2•GDP unless an amino-acyl tRNA enters the A site and blocks IRES back-translocation. DISCUSS +113 121 eEF2•GDP complex_assembly These findings indicate that IRES translocation by eEF2 is futile: the IRES returns to the A site upon releasing eEF2•GDP unless an amino-acyl tRNA enters the A site and blocks IRES back-translocation. DISCUSS +132 147 amino-acyl tRNA chemical These findings indicate that IRES translocation by eEF2 is futile: the IRES returns to the A site upon releasing eEF2•GDP unless an amino-acyl tRNA enters the A site and blocks IRES back-translocation. DISCUSS +159 165 A site site These findings indicate that IRES translocation by eEF2 is futile: the IRES returns to the A site upon releasing eEF2•GDP unless an amino-acyl tRNA enters the A site and blocks IRES back-translocation. DISCUSS +177 181 IRES site These findings indicate that IRES translocation by eEF2 is futile: the IRES returns to the A site upon releasing eEF2•GDP unless an amino-acyl tRNA enters the A site and blocks IRES back-translocation. DISCUSS +24 41 post-translocated protein_state This contrasts with the post-translocated 2tRNA•mRNA complex, in which the classical P and E-site tRNAs are stabilized in the non-rotated ribosome after translocase release. DISCUSS +42 52 2tRNA•mRNA complex_assembly This contrasts with the post-translocated 2tRNA•mRNA complex, in which the classical P and E-site tRNAs are stabilized in the non-rotated ribosome after translocase release. DISCUSS +85 97 P and E-site site This contrasts with the post-translocated 2tRNA•mRNA complex, in which the classical P and E-site tRNAs are stabilized in the non-rotated ribosome after translocase release. DISCUSS +98 103 tRNAs chemical This contrasts with the post-translocated 2tRNA•mRNA complex, in which the classical P and E-site tRNAs are stabilized in the non-rotated ribosome after translocase release. DISCUSS +126 137 non-rotated protein_state This contrasts with the post-translocated 2tRNA•mRNA complex, in which the classical P and E-site tRNAs are stabilized in the non-rotated ribosome after translocase release. DISCUSS +138 146 ribosome complex_assembly This contrasts with the post-translocated 2tRNA•mRNA complex, in which the classical P and E-site tRNAs are stabilized in the non-rotated ribosome after translocase release. DISCUSS +153 164 translocase protein_type This contrasts with the post-translocated 2tRNA•mRNA complex, in which the classical P and E-site tRNAs are stabilized in the non-rotated ribosome after translocase release. DISCUSS +32 50 post-translocation protein_state Thus, the meta-stability of the post-translocation IRES is likely due to the absence of stabilizing structural features present in the 2tRNA•mRNA complex. DISCUSS +51 55 IRES site Thus, the meta-stability of the post-translocation IRES is likely due to the absence of stabilizing structural features present in the 2tRNA•mRNA complex. DISCUSS +77 87 absence of protein_state Thus, the meta-stability of the post-translocation IRES is likely due to the absence of stabilizing structural features present in the 2tRNA•mRNA complex. DISCUSS +135 145 2tRNA•mRNA complex_assembly Thus, the meta-stability of the post-translocation IRES is likely due to the absence of stabilizing structural features present in the 2tRNA•mRNA complex. DISCUSS +7 17 initiation protein_state In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +29 33 IRES site In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +46 63 pre-translocation protein_state In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +64 74 2tRNA•mRNA complex_assembly In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +98 101 A/P site In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +102 106 tRNA chemical In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +107 126 anticodon-stem loop structure_element In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +131 136 elbow structure_element In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +144 150 A site site In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +159 162 P/E site In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +163 167 tRNA chemical In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +168 173 elbow structure_element In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +189 197 L1 stalk structure_element In the initiation state, the IRES resembles a pre-translocation 2tRNA•mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk. DISCUSS +12 31 anticodon-stem loop structure_element Because the anticodon-stem loop of the A-tRNA is sufficient for translocation completion, we ascribe the meta-stability of the post-translocation IRES to the absence of the P/E-tRNA elements, either the ASL or the acceptor arm, or both. DISCUSS +39 40 A site Because the anticodon-stem loop of the A-tRNA is sufficient for translocation completion, we ascribe the meta-stability of the post-translocation IRES to the absence of the P/E-tRNA elements, either the ASL or the acceptor arm, or both. DISCUSS +41 45 tRNA chemical Because the anticodon-stem loop of the A-tRNA is sufficient for translocation completion, we ascribe the meta-stability of the post-translocation IRES to the absence of the P/E-tRNA elements, either the ASL or the acceptor arm, or both. DISCUSS +127 145 post-translocation protein_state Because the anticodon-stem loop of the A-tRNA is sufficient for translocation completion, we ascribe the meta-stability of the post-translocation IRES to the absence of the P/E-tRNA elements, either the ASL or the acceptor arm, or both. DISCUSS +146 150 IRES site Because the anticodon-stem loop of the A-tRNA is sufficient for translocation completion, we ascribe the meta-stability of the post-translocation IRES to the absence of the P/E-tRNA elements, either the ASL or the acceptor arm, or both. DISCUSS +158 168 absence of protein_state Because the anticodon-stem loop of the A-tRNA is sufficient for translocation completion, we ascribe the meta-stability of the post-translocation IRES to the absence of the P/E-tRNA elements, either the ASL or the acceptor arm, or both. DISCUSS +173 176 P/E site Because the anticodon-stem loop of the A-tRNA is sufficient for translocation completion, we ascribe the meta-stability of the post-translocation IRES to the absence of the P/E-tRNA elements, either the ASL or the acceptor arm, or both. DISCUSS +177 181 tRNA chemical Because the anticodon-stem loop of the A-tRNA is sufficient for translocation completion, we ascribe the meta-stability of the post-translocation IRES to the absence of the P/E-tRNA elements, either the ASL or the acceptor arm, or both. DISCUSS +203 206 ASL structure_element Because the anticodon-stem loop of the A-tRNA is sufficient for translocation completion, we ascribe the meta-stability of the post-translocation IRES to the absence of the P/E-tRNA elements, either the ASL or the acceptor arm, or both. DISCUSS +29 32 SL4 structure_element Furthermore, interactions of SL4 and SL5 with the 40S subunit likely contribute to stabilization of pre-translocation structures. DISCUSS +37 40 SL5 structure_element Furthermore, interactions of SL4 and SL5 with the 40S subunit likely contribute to stabilization of pre-translocation structures. DISCUSS +50 53 40S complex_assembly Furthermore, interactions of SL4 and SL5 with the 40S subunit likely contribute to stabilization of pre-translocation structures. DISCUSS +54 61 subunit structure_element Furthermore, interactions of SL4 and SL5 with the 40S subunit likely contribute to stabilization of pre-translocation structures. DISCUSS +100 117 pre-translocation protein_state Furthermore, interactions of SL4 and SL5 with the 40S subunit likely contribute to stabilization of pre-translocation structures. DISCUSS +118 128 structures evidence Furthermore, interactions of SL4 and SL5 with the 40S subunit likely contribute to stabilization of pre-translocation structures. DISCUSS +21 24 40S complex_assembly Partitioned roles of 40S subunit rearrangements DISCUSS +25 32 subunit structure_element Partitioned roles of 40S subunit rearrangements DISCUSS +4 14 structures evidence Our structures delineate the mechanistic functions for intersubunit rotation and head swivel in translocation. DISCUSS +81 85 head structure_element Our structures delineate the mechanistic functions for intersubunit rotation and head swivel in translocation. DISCUSS +43 47 eEF2 protein Specifically, intersubunit rotation allows eEF2 entry into the A site, while the head swivel mediates PKI translocation. DISCUSS +63 69 A site site Specifically, intersubunit rotation allows eEF2 entry into the A site, while the head swivel mediates PKI translocation. DISCUSS +81 85 head structure_element Specifically, intersubunit rotation allows eEF2 entry into the A site, while the head swivel mediates PKI translocation. DISCUSS +102 105 PKI structure_element Specifically, intersubunit rotation allows eEF2 entry into the A site, while the head swivel mediates PKI translocation. DISCUSS +63 78 cryo-EM studies experimental_method Various degrees of intersubunit rotation have been observed in cryo-EM studies of the 80S•IRES initiation complexes. DISCUSS +86 94 80S•IRES complex_assembly Various degrees of intersubunit rotation have been observed in cryo-EM studies of the 80S•IRES initiation complexes. DISCUSS +95 105 initiation protein_state Various degrees of intersubunit rotation have been observed in cryo-EM studies of the 80S•IRES initiation complexes. DISCUSS +23 31 subunits structure_element This suggests that the subunits are capable of spontaneous rotation, as is the case for tRNA-bound pre-translocation complexes. DISCUSS +88 98 tRNA-bound protein_state This suggests that the subunits are capable of spontaneous rotation, as is the case for tRNA-bound pre-translocation complexes. DISCUSS +99 116 pre-translocation protein_state This suggests that the subunits are capable of spontaneous rotation, as is the case for tRNA-bound pre-translocation complexes. DISCUSS +4 21 pre-translocation protein_state The pre-translocation Structure I with eEF2 least advanced into the A site adopts a fully rotated conformation. DISCUSS +22 33 Structure I evidence The pre-translocation Structure I with eEF2 least advanced into the A site adopts a fully rotated conformation. DISCUSS +39 43 eEF2 protein The pre-translocation Structure I with eEF2 least advanced into the A site adopts a fully rotated conformation. DISCUSS +68 74 A site site The pre-translocation Structure I with eEF2 least advanced into the A site adopts a fully rotated conformation. DISCUSS +84 110 fully rotated conformation protein_state The pre-translocation Structure I with eEF2 least advanced into the A site adopts a fully rotated conformation. DISCUSS +35 51 Structure I to V evidence Reverse intersubunit rotation from Structure I to V shifts the translocation tunnel (the tunnel between the A, P and E sites) toward eEF2, which is rigidly attached to the 60S subunit. DISCUSS +63 83 translocation tunnel site Reverse intersubunit rotation from Structure I to V shifts the translocation tunnel (the tunnel between the A, P and E sites) toward eEF2, which is rigidly attached to the 60S subunit. DISCUSS +89 95 tunnel site Reverse intersubunit rotation from Structure I to V shifts the translocation tunnel (the tunnel between the A, P and E sites) toward eEF2, which is rigidly attached to the 60S subunit. DISCUSS +108 124 A, P and E sites site Reverse intersubunit rotation from Structure I to V shifts the translocation tunnel (the tunnel between the A, P and E sites) toward eEF2, which is rigidly attached to the 60S subunit. DISCUSS +133 137 eEF2 protein Reverse intersubunit rotation from Structure I to V shifts the translocation tunnel (the tunnel between the A, P and E sites) toward eEF2, which is rigidly attached to the 60S subunit. DISCUSS +172 175 60S complex_assembly Reverse intersubunit rotation from Structure I to V shifts the translocation tunnel (the tunnel between the A, P and E sites) toward eEF2, which is rigidly attached to the 60S subunit. DISCUSS +176 183 subunit structure_element Reverse intersubunit rotation from Structure I to V shifts the translocation tunnel (the tunnel between the A, P and E sites) toward eEF2, which is rigidly attached to the 60S subunit. DISCUSS +12 16 eEF2 protein This allows eEF2 to move into the A site. DISCUSS +34 40 A site site This allows eEF2 to move into the A site. DISCUSS +67 71 eEF2 protein As such, reverse intersubunit rotation facilitates full docking of eEF2 in the A site. DISCUSS +79 85 A site site As such, reverse intersubunit rotation facilitates full docking of eEF2 in the A site. DISCUSS +12 37 histidine-diphthamide tip site Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site. DISCUSS +41 45 eEF2 protein Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site. DISCUSS +47 51 H583 residue_name_number Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site. DISCUSS +53 57 H694 residue_name_number Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site. DISCUSS +62 69 Diph699 ptm Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site. DISCUSS +87 113 codon-anticodon-like helix structure_element Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site. DISCUSS +117 120 PKI structure_element Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site. DISCUSS +122 126 eEF2 protein Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site. DISCUSS +153 156 PKI structure_element Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site. DISCUSS +168 174 A site site Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site. DISCUSS +4 8 head structure_element The head swivel allows gradual translocation of PKI to the P site, first with respect to the body and then to the head. DISCUSS +48 51 PKI structure_element The head swivel allows gradual translocation of PKI to the P site, first with respect to the body and then to the head. DISCUSS +59 65 P site site The head swivel allows gradual translocation of PKI to the P site, first with respect to the body and then to the head. DISCUSS +93 97 body structure_element The head swivel allows gradual translocation of PKI to the P site, first with respect to the body and then to the head. DISCUSS +114 118 head structure_element The head swivel allows gradual translocation of PKI to the P site, first with respect to the body and then to the head. DISCUSS +4 18 fully swiveled protein_state The fully swiveled conformations of Structures II and III represent the mid-point of translocation, in which PKI relocates between the head A site and body P site. DISCUSS +36 57 Structures II and III evidence The fully swiveled conformations of Structures II and III represent the mid-point of translocation, in which PKI relocates between the head A site and body P site. DISCUSS +109 112 PKI structure_element The fully swiveled conformations of Structures II and III represent the mid-point of translocation, in which PKI relocates between the head A site and body P site. DISCUSS +135 139 head structure_element The fully swiveled conformations of Structures II and III represent the mid-point of translocation, in which PKI relocates between the head A site and body P site. DISCUSS +140 146 A site site The fully swiveled conformations of Structures II and III represent the mid-point of translocation, in which PKI relocates between the head A site and body P site. DISCUSS +151 155 body structure_element The fully swiveled conformations of Structures II and III represent the mid-point of translocation, in which PKI relocates between the head A site and body P site. DISCUSS +156 162 P site site The fully swiveled conformations of Structures II and III represent the mid-point of translocation, in which PKI relocates between the head A site and body P site. DISCUSS +56 66 2tRNA•mRNA complex_assembly We note that such mid-states have not been observed for 2tRNA•mRNA, but their formation can explain the formation of subsequent pe/E hybrid and ap/P chimeric structures (Figure 1—figure supplement 1). DISCUSS +128 139 pe/E hybrid protein_state We note that such mid-states have not been observed for 2tRNA•mRNA, but their formation can explain the formation of subsequent pe/E hybrid and ap/P chimeric structures (Figure 1—figure supplement 1). DISCUSS +144 157 ap/P chimeric protein_state We note that such mid-states have not been observed for 2tRNA•mRNA, but their formation can explain the formation of subsequent pe/E hybrid and ap/P chimeric structures (Figure 1—figure supplement 1). DISCUSS +158 168 structures evidence We note that such mid-states have not been observed for 2tRNA•mRNA, but their formation can explain the formation of subsequent pe/E hybrid and ap/P chimeric structures (Figure 1—figure supplement 1). DISCUSS +20 38 Structure III to V evidence Reverse swivel from Structure III to V brings the head to the non-swiveled position, restoring the A and P sites on the small subunit. DISCUSS +50 54 head structure_element Reverse swivel from Structure III to V brings the head to the non-swiveled position, restoring the A and P sites on the small subunit. DISCUSS +62 74 non-swiveled protein_state Reverse swivel from Structure III to V brings the head to the non-swiveled position, restoring the A and P sites on the small subunit. DISCUSS +99 112 A and P sites site Reverse swivel from Structure III to V brings the head to the non-swiveled position, restoring the A and P sites on the small subunit. DISCUSS +120 133 small subunit structure_element Reverse swivel from Structure III to V brings the head to the non-swiveled position, restoring the A and P sites on the small subunit. DISCUSS +17 21 eEF2 protein The functions of eEF2 in translocation DISCUSS +88 109 ribosomal translocase protein_type To our knowledge, our work provides the first high-resolution view of the dynamics of a ribosomal translocase that is inferred from an ensemble of structures sampled under uniform conditions. DISCUSS +147 157 structures evidence To our knowledge, our work provides the first high-resolution view of the dynamics of a ribosomal translocase that is inferred from an ensemble of structures sampled under uniform conditions. DISCUSS +4 14 structures evidence The structures, therefore, offer a unique opportunity to address the role of the elongation factors during translocation. DISCUSS +81 99 elongation factors protein_type The structures, therefore, offer a unique opportunity to address the role of the elongation factors during translocation. DISCUSS +0 12 Translocases protein_type Translocases are efficient enzymes. DISCUSS +10 18 ribosome complex_assembly While the ribosome itself has the capacity to translocate in the absence of the translocase, spontaneous translocation is slow. DISCUSS +65 75 absence of protein_state While the ribosome itself has the capacity to translocate in the absence of the translocase, spontaneous translocation is slow. DISCUSS +80 91 translocase protein_type While the ribosome itself has the capacity to translocate in the absence of the translocase, spontaneous translocation is slow. DISCUSS +0 4 EF-G protein EF-G enhances the translocation rate by several orders of magnitude, aided by an additional 2- to 50-fold boost from GTP hydrolysis. DISCUSS +117 120 GTP chemical EF-G enhances the translocation rate by several orders of magnitude, aided by an additional 2- to 50-fold boost from GTP hydrolysis. DISCUSS +19 29 structures evidence Due to the lack of structures of translocation intermediates, the mechanistic role of eEF2/EF-G is not fully understood. DISCUSS +86 90 eEF2 protein Due to the lack of structures of translocation intermediates, the mechanistic role of eEF2/EF-G is not fully understood. DISCUSS +91 95 EF-G protein Due to the lack of structures of translocation intermediates, the mechanistic role of eEF2/EF-G is not fully understood. DISCUSS +4 17 80S•IRES•eEF2 complex_assembly The 80S•IRES•eEF2 structures reported here suggest two main roles for eEF2 in translocation. DISCUSS +18 28 structures evidence The 80S•IRES•eEF2 structures reported here suggest two main roles for eEF2 in translocation. DISCUSS +70 74 eEF2 protein The 80S•IRES•eEF2 structures reported here suggest two main roles for eEF2 in translocation. DISCUSS +56 59 PKI structure_element As discussed above, the first role is to directly shift PKI out of the A site upon spontaneous reverse intersubunit rotation. DISCUSS +71 77 A site site As discussed above, the first role is to directly shift PKI out of the A site upon spontaneous reverse intersubunit rotation. DISCUSS +7 17 structures evidence In our structures, the tip of domain IV docks next to PKI, with diphthamide 699 fit into the minor groove of the codon-anticodon-like helix of PKI (Figure 7). DISCUSS +37 39 IV structure_element In our structures, the tip of domain IV docks next to PKI, with diphthamide 699 fit into the minor groove of the codon-anticodon-like helix of PKI (Figure 7). DISCUSS +54 57 PKI structure_element In our structures, the tip of domain IV docks next to PKI, with diphthamide 699 fit into the minor groove of the codon-anticodon-like helix of PKI (Figure 7). DISCUSS +64 79 diphthamide 699 ptm In our structures, the tip of domain IV docks next to PKI, with diphthamide 699 fit into the minor groove of the codon-anticodon-like helix of PKI (Figure 7). DISCUSS +93 105 minor groove site In our structures, the tip of domain IV docks next to PKI, with diphthamide 699 fit into the minor groove of the codon-anticodon-like helix of PKI (Figure 7). DISCUSS +113 139 codon-anticodon-like helix structure_element In our structures, the tip of domain IV docks next to PKI, with diphthamide 699 fit into the minor groove of the codon-anticodon-like helix of PKI (Figure 7). DISCUSS +143 146 PKI structure_element In our structures, the tip of domain IV docks next to PKI, with diphthamide 699 fit into the minor groove of the codon-anticodon-like helix of PKI (Figure 7). DISCUSS +46 50 eEF2 protein This arrangement rationalizes inactivation of eEF2 by diphtheria toxin, which catalyzes ADP-ribosylation of the diphthamide (reviewed in). DISCUSS +54 70 diphtheria toxin protein_type This arrangement rationalizes inactivation of eEF2 by diphtheria toxin, which catalyzes ADP-ribosylation of the diphthamide (reviewed in). DISCUSS +88 104 ADP-ribosylation ptm This arrangement rationalizes inactivation of eEF2 by diphtheria toxin, which catalyzes ADP-ribosylation of the diphthamide (reviewed in). DISCUSS +112 123 diphthamide ptm This arrangement rationalizes inactivation of eEF2 by diphtheria toxin, which catalyzes ADP-ribosylation of the diphthamide (reviewed in). DISCUSS +11 26 ADP-ribosylates ptm The enzyme ADP-ribosylates the NE2 atom of the imidazole ring, which in our structures interacts with the first two residues of the anticodon-like strand of PKI. DISCUSS +76 86 structures evidence The enzyme ADP-ribosylates the NE2 atom of the imidazole ring, which in our structures interacts with the first two residues of the anticodon-like strand of PKI. DISCUSS +132 153 anticodon-like strand structure_element The enzyme ADP-ribosylates the NE2 atom of the imidazole ring, which in our structures interacts with the first two residues of the anticodon-like strand of PKI. DISCUSS +157 160 PKI structure_element The enzyme ADP-ribosylates the NE2 atom of the imidazole ring, which in our structures interacts with the first two residues of the anticodon-like strand of PKI. DISCUSS +10 13 ADP chemical The bulky ADP-ribosyl moiety at this position would disrupt the interaction, rendering eEF2 unable to bind to the A site and/or stalled on ribosomes in a non-productive conformation. DISCUSS +87 91 eEF2 protein The bulky ADP-ribosyl moiety at this position would disrupt the interaction, rendering eEF2 unable to bind to the A site and/or stalled on ribosomes in a non-productive conformation. DISCUSS +114 120 A site site The bulky ADP-ribosyl moiety at this position would disrupt the interaction, rendering eEF2 unable to bind to the A site and/or stalled on ribosomes in a non-productive conformation. DISCUSS +139 148 ribosomes complex_assembly The bulky ADP-ribosyl moiety at this position would disrupt the interaction, rendering eEF2 unable to bind to the A site and/or stalled on ribosomes in a non-productive conformation. DISCUSS +3 7 eEF2 protein As eEF2 shifts PKI toward the P site in the course of reverse intersubunit rotation, the 60S-attached translocase migrates along the surface of the 40S subunit, guided by electrostatic interactions. DISCUSS +15 18 PKI structure_element As eEF2 shifts PKI toward the P site in the course of reverse intersubunit rotation, the 60S-attached translocase migrates along the surface of the 40S subunit, guided by electrostatic interactions. DISCUSS +30 36 P site site As eEF2 shifts PKI toward the P site in the course of reverse intersubunit rotation, the 60S-attached translocase migrates along the surface of the 40S subunit, guided by electrostatic interactions. DISCUSS +89 101 60S-attached protein_state As eEF2 shifts PKI toward the P site in the course of reverse intersubunit rotation, the 60S-attached translocase migrates along the surface of the 40S subunit, guided by electrostatic interactions. DISCUSS +102 113 translocase protein_type As eEF2 shifts PKI toward the P site in the course of reverse intersubunit rotation, the 60S-attached translocase migrates along the surface of the 40S subunit, guided by electrostatic interactions. DISCUSS +148 151 40S complex_assembly As eEF2 shifts PKI toward the P site in the course of reverse intersubunit rotation, the 60S-attached translocase migrates along the surface of the 40S subunit, guided by electrostatic interactions. DISCUSS +152 159 subunit structure_element As eEF2 shifts PKI toward the P site in the course of reverse intersubunit rotation, the 60S-attached translocase migrates along the surface of the 40S subunit, guided by electrostatic interactions. DISCUSS +171 197 electrostatic interactions bond_interaction As eEF2 shifts PKI toward the P site in the course of reverse intersubunit rotation, the 60S-attached translocase migrates along the surface of the 40S subunit, guided by electrostatic interactions. DISCUSS +0 26 Positively-charged patches site Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +38 40 II structure_element Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +45 48 III structure_element Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +50 54 R391 residue_name_number Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +56 60 K394 residue_name_number Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +62 66 R433 residue_name_number Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +68 72 R510 residue_name_number Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +78 80 IV structure_element Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +82 86 K613 residue_name_number Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +88 92 R617 residue_name_number Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +94 98 R609 residue_name_number Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +100 104 R631 residue_name_number Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +106 110 K651 residue_name_number Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +123 127 rRNA chemical Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +135 138 40S complex_assembly Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +139 143 body structure_element Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +145 147 h5 structure_element Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +153 157 head structure_element Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +159 162 h18 structure_element Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +167 170 h33 structure_element Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +171 174 h34 structure_element Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively. DISCUSS +4 14 Structures evidence The Structures reveal hopping of the positive clusters over rRNA helices. DISCUSS +60 64 rRNA chemical The Structures reveal hopping of the positive clusters over rRNA helices. DISCUSS +65 72 helices structure_element The Structures reveal hopping of the positive clusters over rRNA helices. DISCUSS +21 40 Structures II and V evidence For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19 Å (for Cα of R617) from the phosphate backbone of h33 (at nt 1261–1264) to that of the neighboring h34 (at nt 1442–1445). DISCUSS +46 50 K613 residue_name_number For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19 Å (for Cα of R617) from the phosphate backbone of h33 (at nt 1261–1264) to that of the neighboring h34 (at nt 1442–1445). DISCUSS +51 55 R617 residue_name_number For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19 Å (for Cα of R617) from the phosphate backbone of h33 (at nt 1261–1264) to that of the neighboring h34 (at nt 1442–1445). DISCUSS +56 60 R631 residue_name_number For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19 Å (for Cα of R617) from the phosphate backbone of h33 (at nt 1261–1264) to that of the neighboring h34 (at nt 1442–1445). DISCUSS +79 81 IV structure_element For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19 Å (for Cα of R617) from the phosphate backbone of h33 (at nt 1261–1264) to that of the neighboring h34 (at nt 1442–1445). DISCUSS +107 111 R617 residue_name_number For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19 Å (for Cα of R617) from the phosphate backbone of h33 (at nt 1261–1264) to that of the neighboring h34 (at nt 1442–1445). DISCUSS +144 147 h33 structure_element For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19 Å (for Cα of R617) from the phosphate backbone of h33 (at nt 1261–1264) to that of the neighboring h34 (at nt 1442–1445). DISCUSS +155 164 1261–1264 residue_range For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19 Å (for Cα of R617) from the phosphate backbone of h33 (at nt 1261–1264) to that of the neighboring h34 (at nt 1442–1445). DISCUSS +193 196 h34 structure_element For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19 Å (for Cα of R617) from the phosphate backbone of h33 (at nt 1261–1264) to that of the neighboring h34 (at nt 1442–1445). DISCUSS +204 213 1442–1445 residue_range For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19 Å (for Cα of R617) from the phosphate backbone of h33 (at nt 1261–1264) to that of the neighboring h34 (at nt 1442–1445). DISCUSS +17 21 eEF2 protein Thus, sliding of eEF2 involves reorganization of electrostatic, perhaps isoenergetic interactions, echoing those implied in extraordinarily fast ribosome inactivation rates by the small-protein ribotoxins and in fast protein association and diffusion along DNA. DISCUSS +49 97 electrostatic, perhaps isoenergetic interactions bond_interaction Thus, sliding of eEF2 involves reorganization of electrostatic, perhaps isoenergetic interactions, echoing those implied in extraordinarily fast ribosome inactivation rates by the small-protein ribotoxins and in fast protein association and diffusion along DNA. DISCUSS +145 153 ribosome complex_assembly Thus, sliding of eEF2 involves reorganization of electrostatic, perhaps isoenergetic interactions, echoing those implied in extraordinarily fast ribosome inactivation rates by the small-protein ribotoxins and in fast protein association and diffusion along DNA. DISCUSS +0 10 Comparison experimental_method Comparison of our structures with the 80S•IRES initiation structure reveals the structural basis for the second key function of the translocase: 'unlocking' of intrasubunit rearrangements that are required for step-wise translocation of PKI on the small subunit. DISCUSS +18 28 structures evidence Comparison of our structures with the 80S•IRES initiation structure reveals the structural basis for the second key function of the translocase: 'unlocking' of intrasubunit rearrangements that are required for step-wise translocation of PKI on the small subunit. DISCUSS +38 46 80S•IRES complex_assembly Comparison of our structures with the 80S•IRES initiation structure reveals the structural basis for the second key function of the translocase: 'unlocking' of intrasubunit rearrangements that are required for step-wise translocation of PKI on the small subunit. DISCUSS +47 57 initiation protein_state Comparison of our structures with the 80S•IRES initiation structure reveals the structural basis for the second key function of the translocase: 'unlocking' of intrasubunit rearrangements that are required for step-wise translocation of PKI on the small subunit. DISCUSS +58 67 structure evidence Comparison of our structures with the 80S•IRES initiation structure reveals the structural basis for the second key function of the translocase: 'unlocking' of intrasubunit rearrangements that are required for step-wise translocation of PKI on the small subunit. DISCUSS +132 143 translocase protein_type Comparison of our structures with the 80S•IRES initiation structure reveals the structural basis for the second key function of the translocase: 'unlocking' of intrasubunit rearrangements that are required for step-wise translocation of PKI on the small subunit. DISCUSS +237 240 PKI structure_element Comparison of our structures with the 80S•IRES initiation structure reveals the structural basis for the second key function of the translocase: 'unlocking' of intrasubunit rearrangements that are required for step-wise translocation of PKI on the small subunit. DISCUSS +248 261 small subunit structure_element Comparison of our structures with the 80S•IRES initiation structure reveals the structural basis for the second key function of the translocase: 'unlocking' of intrasubunit rearrangements that are required for step-wise translocation of PKI on the small subunit. DISCUSS +27 46 ribosome•2tRNA•mRNA complex_assembly The unlocking model of the ribosome•2tRNA•mRNA pre-translocation complex has been proposed decades ago and functional requirement of the translocase in this process has been implicated. DISCUSS +47 64 pre-translocation protein_state The unlocking model of the ribosome•2tRNA•mRNA pre-translocation complex has been proposed decades ago and functional requirement of the translocase in this process has been implicated. DISCUSS +137 148 translocase protein_type The unlocking model of the ribosome•2tRNA•mRNA pre-translocation complex has been proposed decades ago and functional requirement of the translocase in this process has been implicated. DISCUSS +59 65 locked protein_state However, the structural and mechanistic definitions of the locked and unlocked states have remained unclear, ranging from the globally distinct ribosome conformations to unknown local rearrangements, e.g. those in the decoding center. DISCUSS +70 78 unlocked protein_state However, the structural and mechanistic definitions of the locked and unlocked states have remained unclear, ranging from the globally distinct ribosome conformations to unknown local rearrangements, e.g. those in the decoding center. DISCUSS +144 152 ribosome complex_assembly However, the structural and mechanistic definitions of the locked and unlocked states have remained unclear, ranging from the globally distinct ribosome conformations to unknown local rearrangements, e.g. those in the decoding center. DISCUSS +218 233 decoding center site However, the structural and mechanistic definitions of the locked and unlocked states have remained unclear, ranging from the globally distinct ribosome conformations to unknown local rearrangements, e.g. those in the decoding center. DISCUSS +0 9 FRET data evidence FRET data indicate that translocation of 2tRNA•mRNA on the 70S ribosome requires a forward-and-reverse head swivel, which may be related to the unlocking phenomenon. DISCUSS +41 51 2tRNA•mRNA complex_assembly FRET data indicate that translocation of 2tRNA•mRNA on the 70S ribosome requires a forward-and-reverse head swivel, which may be related to the unlocking phenomenon. DISCUSS +59 71 70S ribosome complex_assembly FRET data indicate that translocation of 2tRNA•mRNA on the 70S ribosome requires a forward-and-reverse head swivel, which may be related to the unlocking phenomenon. DISCUSS +103 107 head structure_element FRET data indicate that translocation of 2tRNA•mRNA on the 70S ribosome requires a forward-and-reverse head swivel, which may be related to the unlocking phenomenon. DISCUSS +37 54 pre-translocation protein_state Whereas intersubunit rotation of the pre-translocation complex occurs spontaneously, the head swivel is induced by the eEF2/EF-G translocase, consistent with requirement of eEF2 for unlocking. DISCUSS +89 93 head structure_element Whereas intersubunit rotation of the pre-translocation complex occurs spontaneously, the head swivel is induced by the eEF2/EF-G translocase, consistent with requirement of eEF2 for unlocking. DISCUSS +119 123 eEF2 protein Whereas intersubunit rotation of the pre-translocation complex occurs spontaneously, the head swivel is induced by the eEF2/EF-G translocase, consistent with requirement of eEF2 for unlocking. DISCUSS +124 128 EF-G protein Whereas intersubunit rotation of the pre-translocation complex occurs spontaneously, the head swivel is induced by the eEF2/EF-G translocase, consistent with requirement of eEF2 for unlocking. DISCUSS +129 140 translocase protein_type Whereas intersubunit rotation of the pre-translocation complex occurs spontaneously, the head swivel is induced by the eEF2/EF-G translocase, consistent with requirement of eEF2 for unlocking. DISCUSS +173 177 eEF2 protein Whereas intersubunit rotation of the pre-translocation complex occurs spontaneously, the head swivel is induced by the eEF2/EF-G translocase, consistent with requirement of eEF2 for unlocking. DISCUSS +0 18 Structural studies experimental_method Structural studies revealed large head swivels in various 70S•tRNA•EF-G and 80S•tRNA•eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase. DISCUSS +34 38 head structure_element Structural studies revealed large head swivels in various 70S•tRNA•EF-G and 80S•tRNA•eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase. DISCUSS +58 71 70S•tRNA•EF-G complex_assembly Structural studies revealed large head swivels in various 70S•tRNA•EF-G and 80S•tRNA•eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase. DISCUSS +76 89 80S•tRNA•eEF2 complex_assembly Structural studies revealed large head swivels in various 70S•tRNA•EF-G and 80S•tRNA•eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase. DISCUSS +113 119 locked protein_state Structural studies revealed large head swivels in various 70S•tRNA•EF-G and 80S•tRNA•eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase. DISCUSS +121 135 complexes with protein_state Structural studies revealed large head swivels in various 70S•tRNA•EF-G and 80S•tRNA•eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase. DISCUSS +140 146 A site site Structural studies revealed large head swivels in various 70S•tRNA•EF-G and 80S•tRNA•eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase. DISCUSS +163 167 tRNA chemical Structural studies revealed large head swivels in various 70S•tRNA•EF-G and 80S•tRNA•eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase. DISCUSS +175 185 absence of protein_state Structural studies revealed large head swivels in various 70S•tRNA•EF-G and 80S•tRNA•eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase. DISCUSS +190 201 translocase protein_type Structural studies revealed large head swivels in various 70S•tRNA•EF-G and 80S•tRNA•eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase. DISCUSS +4 14 structures evidence Our structures suggest that eEF2 induces head swivel by 'unlocking' the head-body interactions (Figure 7). DISCUSS +28 32 eEF2 protein Our structures suggest that eEF2 induces head swivel by 'unlocking' the head-body interactions (Figure 7). DISCUSS +41 45 head structure_element Our structures suggest that eEF2 induces head swivel by 'unlocking' the head-body interactions (Figure 7). DISCUSS +72 76 head structure_element Our structures suggest that eEF2 induces head swivel by 'unlocking' the head-body interactions (Figure 7). DISCUSS +77 81 body structure_element Our structures suggest that eEF2 induces head swivel by 'unlocking' the head-body interactions (Figure 7). DISCUSS +15 18 ASL structure_element Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains. DISCUSS +26 32 A site site Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains. DISCUSS +47 65 structural studies experimental_method Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains. DISCUSS +69 78 bacterial taxonomy_domain Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains. DISCUSS +79 88 ribosomes complex_assembly Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains. DISCUSS +103 117 domain closure protein_state Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains. DISCUSS +126 139 small subunit structure_element Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains. DISCUSS +172 176 head structure_element Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains. DISCUSS +178 186 shoulder structure_element Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains. DISCUSS +191 195 body structure_element Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains. DISCUSS +35 39 tRNA chemical The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +47 53 A site site The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +58 66 stacking bond_interaction The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +74 78 head structure_element The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +79 85 A site site The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +87 92 C1274 residue_name_number The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +96 109 S. cerevisiae species The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +113 118 C1054 residue_name_number The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +122 129 E. coli species The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +157 161 body structure_element The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +162 168 A-site site The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +181 186 A1755 residue_name_number The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +191 196 A1756 residue_name_number The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +198 203 A1492 residue_name_number The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +208 213 A1493 residue_name_number The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +217 224 E. coli species The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli). DISCUSS +6 12 locked protein_state This 'locked' state is identical to that observed for PKI in the 80S•IRES initiation structures in the absence of eEF2. DISCUSS +54 57 PKI structure_element This 'locked' state is identical to that observed for PKI in the 80S•IRES initiation structures in the absence of eEF2. DISCUSS +65 73 80S•IRES complex_assembly This 'locked' state is identical to that observed for PKI in the 80S•IRES initiation structures in the absence of eEF2. DISCUSS +74 84 initiation protein_state This 'locked' state is identical to that observed for PKI in the 80S•IRES initiation structures in the absence of eEF2. DISCUSS +85 95 structures evidence This 'locked' state is identical to that observed for PKI in the 80S•IRES initiation structures in the absence of eEF2. DISCUSS +103 113 absence of protein_state This 'locked' state is identical to that observed for PKI in the 80S•IRES initiation structures in the absence of eEF2. DISCUSS +114 118 eEF2 protein This 'locked' state is identical to that observed for PKI in the 80S•IRES initiation structures in the absence of eEF2. DISCUSS +0 11 Structure I evidence Structure I demonstrates that at an early pre-translocation step, the histidine-diphthamide tip of eEF2 is wedged between A1755 and A1756 and PKI. DISCUSS +42 59 pre-translocation protein_state Structure I demonstrates that at an early pre-translocation step, the histidine-diphthamide tip of eEF2 is wedged between A1755 and A1756 and PKI. DISCUSS +70 95 histidine-diphthamide tip site Structure I demonstrates that at an early pre-translocation step, the histidine-diphthamide tip of eEF2 is wedged between A1755 and A1756 and PKI. DISCUSS +99 103 eEF2 protein Structure I demonstrates that at an early pre-translocation step, the histidine-diphthamide tip of eEF2 is wedged between A1755 and A1756 and PKI. DISCUSS +122 127 A1755 residue_name_number Structure I demonstrates that at an early pre-translocation step, the histidine-diphthamide tip of eEF2 is wedged between A1755 and A1756 and PKI. DISCUSS +132 137 A1756 residue_name_number Structure I demonstrates that at an early pre-translocation step, the histidine-diphthamide tip of eEF2 is wedged between A1755 and A1756 and PKI. DISCUSS +142 145 PKI structure_element Structure I demonstrates that at an early pre-translocation step, the histidine-diphthamide tip of eEF2 is wedged between A1755 and A1756 and PKI. DISCUSS +28 31 PKI structure_element This destabilization allows PKI to detach from the body A site upon spontaneous reverse 40S body rotation, while maintaining interactions with the head A site. DISCUSS +51 55 body structure_element This destabilization allows PKI to detach from the body A site upon spontaneous reverse 40S body rotation, while maintaining interactions with the head A site. DISCUSS +56 62 A site site This destabilization allows PKI to detach from the body A site upon spontaneous reverse 40S body rotation, while maintaining interactions with the head A site. DISCUSS +88 91 40S complex_assembly This destabilization allows PKI to detach from the body A site upon spontaneous reverse 40S body rotation, while maintaining interactions with the head A site. DISCUSS +92 96 body structure_element This destabilization allows PKI to detach from the body A site upon spontaneous reverse 40S body rotation, while maintaining interactions with the head A site. DISCUSS +147 151 head structure_element This destabilization allows PKI to detach from the body A site upon spontaneous reverse 40S body rotation, while maintaining interactions with the head A site. DISCUSS +152 158 A site site This destabilization allows PKI to detach from the body A site upon spontaneous reverse 40S body rotation, while maintaining interactions with the head A site. DISCUSS +23 33 head-bound protein_state Destabilization of the head-bound PKI at the body A site thus allows mobility of the head relative to the body. DISCUSS +34 37 PKI structure_element Destabilization of the head-bound PKI at the body A site thus allows mobility of the head relative to the body. DISCUSS +45 49 body structure_element Destabilization of the head-bound PKI at the body A site thus allows mobility of the head relative to the body. DISCUSS +50 56 A site site Destabilization of the head-bound PKI at the body A site thus allows mobility of the head relative to the body. DISCUSS +85 89 head structure_element Destabilization of the head-bound PKI at the body A site thus allows mobility of the head relative to the body. DISCUSS +106 110 body structure_element Destabilization of the head-bound PKI at the body A site thus allows mobility of the head relative to the body. DISCUSS +4 25 histidine-diphthamide ptm The histidine-diphthamide-induced disengagement of PKI from A1755 and A1756 therefore provides the structural definition for the 'unlocking' mode of eEF2 action. DISCUSS +51 54 PKI structure_element The histidine-diphthamide-induced disengagement of PKI from A1755 and A1756 therefore provides the structural definition for the 'unlocking' mode of eEF2 action. DISCUSS +60 65 A1755 residue_name_number The histidine-diphthamide-induced disengagement of PKI from A1755 and A1756 therefore provides the structural definition for the 'unlocking' mode of eEF2 action. DISCUSS +70 75 A1756 residue_name_number The histidine-diphthamide-induced disengagement of PKI from A1755 and A1756 therefore provides the structural definition for the 'unlocking' mode of eEF2 action. DISCUSS +149 153 eEF2 protein The histidine-diphthamide-induced disengagement of PKI from A1755 and A1756 therefore provides the structural definition for the 'unlocking' mode of eEF2 action. DISCUSS +16 26 structures evidence In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2. DISCUSS +58 62 eEF2 protein In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2. DISCUSS +99 102 PKI structure_element In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2. DISCUSS +107 111 eEF2 protein In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2. DISCUSS +139 151 P and A site site In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2. DISCUSS +186 189 40S complex_assembly In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2. DISCUSS +190 194 body structure_element In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2. DISCUSS +208 212 head structure_element In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2. DISCUSS +268 274 A site site In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2. DISCUSS +278 282 eEF2 protein In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2. DISCUSS +25 28 PKI structure_element Observation of different PKI conformations sampling a range of positions between the A and P sites in the presence of eEF2•GDP implies that thermal fluctuations of the 40S head domain are sufficient for translocation along the energetically flat trajectory. DISCUSS +85 98 A and P sites site Observation of different PKI conformations sampling a range of positions between the A and P sites in the presence of eEF2•GDP implies that thermal fluctuations of the 40S head domain are sufficient for translocation along the energetically flat trajectory. DISCUSS +106 117 presence of protein_state Observation of different PKI conformations sampling a range of positions between the A and P sites in the presence of eEF2•GDP implies that thermal fluctuations of the 40S head domain are sufficient for translocation along the energetically flat trajectory. DISCUSS +118 126 eEF2•GDP complex_assembly Observation of different PKI conformations sampling a range of positions between the A and P sites in the presence of eEF2•GDP implies that thermal fluctuations of the 40S head domain are sufficient for translocation along the energetically flat trajectory. DISCUSS +168 171 40S complex_assembly Observation of different PKI conformations sampling a range of positions between the A and P sites in the presence of eEF2•GDP implies that thermal fluctuations of the 40S head domain are sufficient for translocation along the energetically flat trajectory. DISCUSS +172 176 head structure_element Observation of different PKI conformations sampling a range of positions between the A and P sites in the presence of eEF2•GDP implies that thermal fluctuations of the 40S head domain are sufficient for translocation along the energetically flat trajectory. DISCUSS +14 18 eEF2 protein Insights into eEF2 association with and dissociation from the ribosome DISCUSS +62 70 ribosome complex_assembly Insights into eEF2 association with and dissociation from the ribosome DISCUSS +37 41 eEF2 protein The conformational rearrangements in eEF2 from Structure I through Structure V provide insights into the mechanisms of eEF2 association with the pre-translocation ribosome and dissociation from the post-translocation ribosome. DISCUSS +47 58 Structure I evidence The conformational rearrangements in eEF2 from Structure I through Structure V provide insights into the mechanisms of eEF2 association with the pre-translocation ribosome and dissociation from the post-translocation ribosome. DISCUSS +67 78 Structure V evidence The conformational rearrangements in eEF2 from Structure I through Structure V provide insights into the mechanisms of eEF2 association with the pre-translocation ribosome and dissociation from the post-translocation ribosome. DISCUSS +119 123 eEF2 protein The conformational rearrangements in eEF2 from Structure I through Structure V provide insights into the mechanisms of eEF2 association with the pre-translocation ribosome and dissociation from the post-translocation ribosome. DISCUSS +145 162 pre-translocation protein_state The conformational rearrangements in eEF2 from Structure I through Structure V provide insights into the mechanisms of eEF2 association with the pre-translocation ribosome and dissociation from the post-translocation ribosome. DISCUSS +163 171 ribosome complex_assembly The conformational rearrangements in eEF2 from Structure I through Structure V provide insights into the mechanisms of eEF2 association with the pre-translocation ribosome and dissociation from the post-translocation ribosome. DISCUSS +198 216 post-translocation protein_state The conformational rearrangements in eEF2 from Structure I through Structure V provide insights into the mechanisms of eEF2 association with the pre-translocation ribosome and dissociation from the post-translocation ribosome. DISCUSS +217 225 ribosome complex_assembly The conformational rearrangements in eEF2 from Structure I through Structure V provide insights into the mechanisms of eEF2 association with the pre-translocation ribosome and dissociation from the post-translocation ribosome. DISCUSS +12 22 structures evidence In all five structures, the GTPase domain is attached to the P stalk and the sarcin-ricin loop. DISCUSS +28 41 GTPase domain structure_element In all five structures, the GTPase domain is attached to the P stalk and the sarcin-ricin loop. DISCUSS +61 68 P stalk structure_element In all five structures, the GTPase domain is attached to the P stalk and the sarcin-ricin loop. DISCUSS +77 94 sarcin-ricin loop structure_element In all five structures, the GTPase domain is attached to the P stalk and the sarcin-ricin loop. DISCUSS +7 20 fully-rotated protein_state In the fully-rotated pre-translocation-like Structure I, an additional interaction exists. DISCUSS +21 38 pre-translocation protein_state In the fully-rotated pre-translocation-like Structure I, an additional interaction exists. DISCUSS +44 55 Structure I evidence In the fully-rotated pre-translocation-like Structure I, an additional interaction exists. DISCUSS +6 19 switch loop I structure_element Here, switch loop I interacts with helix 14 (415CAAA418) of the 18S rRNA. DISCUSS +35 43 helix 14 structure_element Here, switch loop I interacts with helix 14 (415CAAA418) of the 18S rRNA. DISCUSS +45 55 415CAAA418 structure_element Here, switch loop I interacts with helix 14 (415CAAA418) of the 18S rRNA. DISCUSS +64 72 18S rRNA chemical Here, switch loop I interacts with helix 14 (415CAAA418) of the 18S rRNA. DISCUSS +31 44 GTPase center site This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +56 65 GTP-bound protein_state This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +115 136 translational GTPases protein_type This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +144 155 presence of protein_state This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +156 159 GTP chemical This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +179 187 80S•eEF2 complex_assembly This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +196 206 bound with protein_state This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +232 241 GDP•AlF4– complex_assembly This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +247 258 switch loop structure_element This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +280 284 A416 residue_name_number This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +286 296 invariable protein_state This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +297 301 A344 residue_name_number This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +305 312 E. coli species This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +317 321 A463 residue_name_number This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +325 335 H. sapiens species This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S•eEF2 complex bound with a transition-state mimic GDP•AlF4–. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens). DISCUSS +0 9 Mutations experimental_method Mutations of residues flanking A344 in E. coli 16S rRNA modestly inhibit translation but do not specifically affect EF-G-mediated translocation. DISCUSS +31 35 A344 residue_name_number Mutations of residues flanking A344 in E. coli 16S rRNA modestly inhibit translation but do not specifically affect EF-G-mediated translocation. DISCUSS +39 46 E. coli species Mutations of residues flanking A344 in E. coli 16S rRNA modestly inhibit translation but do not specifically affect EF-G-mediated translocation. DISCUSS +47 55 16S rRNA chemical Mutations of residues flanking A344 in E. coli 16S rRNA modestly inhibit translation but do not specifically affect EF-G-mediated translocation. DISCUSS +116 120 EF-G protein Mutations of residues flanking A344 in E. coli 16S rRNA modestly inhibit translation but do not specifically affect EF-G-mediated translocation. DISCUSS +23 27 A344 residue_name_number However, the effect of A344 mutation on translation was not addressed in that study, leaving the question open whether this residue is critical for eEF2/EF-G function. DISCUSS +28 36 mutation experimental_method However, the effect of A344 mutation on translation was not addressed in that study, leaving the question open whether this residue is critical for eEF2/EF-G function. DISCUSS +148 152 eEF2 protein However, the effect of A344 mutation on translation was not addressed in that study, leaving the question open whether this residue is critical for eEF2/EF-G function. DISCUSS +153 157 EF-G protein However, the effect of A344 mutation on translation was not addressed in that study, leaving the question open whether this residue is critical for eEF2/EF-G function. DISCUSS +24 27 h14 structure_element The interaction between h14 and switch loop I is not resolved in Structures II to V, in all of which the small subunit is partially rotated or non-rotated, so that helix 14 is placed at least 6 Å farther from eEF2 (Figure 5d). DISCUSS +32 45 switch loop I structure_element The interaction between h14 and switch loop I is not resolved in Structures II to V, in all of which the small subunit is partially rotated or non-rotated, so that helix 14 is placed at least 6 Å farther from eEF2 (Figure 5d). DISCUSS +65 83 Structures II to V evidence The interaction between h14 and switch loop I is not resolved in Structures II to V, in all of which the small subunit is partially rotated or non-rotated, so that helix 14 is placed at least 6 Å farther from eEF2 (Figure 5d). DISCUSS +105 118 small subunit structure_element The interaction between h14 and switch loop I is not resolved in Structures II to V, in all of which the small subunit is partially rotated or non-rotated, so that helix 14 is placed at least 6 Å farther from eEF2 (Figure 5d). DISCUSS +122 139 partially rotated protein_state The interaction between h14 and switch loop I is not resolved in Structures II to V, in all of which the small subunit is partially rotated or non-rotated, so that helix 14 is placed at least 6 Å farther from eEF2 (Figure 5d). DISCUSS +143 154 non-rotated protein_state The interaction between h14 and switch loop I is not resolved in Structures II to V, in all of which the small subunit is partially rotated or non-rotated, so that helix 14 is placed at least 6 Å farther from eEF2 (Figure 5d). DISCUSS +164 172 helix 14 structure_element The interaction between h14 and switch loop I is not resolved in Structures II to V, in all of which the small subunit is partially rotated or non-rotated, so that helix 14 is placed at least 6 Å farther from eEF2 (Figure 5d). DISCUSS +209 213 eEF2 protein The interaction between h14 and switch loop I is not resolved in Structures II to V, in all of which the small subunit is partially rotated or non-rotated, so that helix 14 is placed at least 6 Å farther from eEF2 (Figure 5d). DISCUSS +51 59 ribosome complex_assembly We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase. DISCUSS +65 78 fully rotated protein_state We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase. DISCUSS +79 82 40S complex_assembly We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase. DISCUSS +83 90 subunit structure_element We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase. DISCUSS +98 115 pre-translocation protein_state We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase. DISCUSS +116 124 ribosome complex_assembly We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase. DISCUSS +137 156 interaction surface site We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase. DISCUSS +176 183 P stalk structure_element We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase. DISCUSS +188 191 SRL structure_element We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase. DISCUSS +212 221 GTP-bound protein_state We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase. DISCUSS +222 233 translocase protein_type We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase. DISCUSS +85 92 rotated protein_state This structural basis rationalizes the observation of transient stabilization of the rotated 70S ribosome upon EF-G•GTP binding and prior to translocation. DISCUSS +93 105 70S ribosome complex_assembly This structural basis rationalizes the observation of transient stabilization of the rotated 70S ribosome upon EF-G•GTP binding and prior to translocation. DISCUSS +111 119 EF-G•GTP complex_assembly This structural basis rationalizes the observation of transient stabilization of the rotated 70S ribosome upon EF-G•GTP binding and prior to translocation. DISCUSS +4 17 least rotated protein_state The least rotated conformation of the post-translocation Structure V suggests conformational changes that may trigger eEF2 release from the ribosome at the end of translocation. DISCUSS +38 56 post-translocation protein_state The least rotated conformation of the post-translocation Structure V suggests conformational changes that may trigger eEF2 release from the ribosome at the end of translocation. DISCUSS +57 68 Structure V evidence The least rotated conformation of the post-translocation Structure V suggests conformational changes that may trigger eEF2 release from the ribosome at the end of translocation. DISCUSS +118 122 eEF2 protein The least rotated conformation of the post-translocation Structure V suggests conformational changes that may trigger eEF2 release from the ribosome at the end of translocation. DISCUSS +140 148 ribosome complex_assembly The least rotated conformation of the post-translocation Structure V suggests conformational changes that may trigger eEF2 release from the ribosome at the end of translocation. DISCUSS +50 54 eEF2 protein The most pronounced inter-domain rearrangement in eEF2 involves movement of domain III. DISCUSS +83 86 III structure_element The most pronounced inter-domain rearrangement in eEF2 involves movement of domain III. DISCUSS +7 14 rotated protein_state In the rotated or mid-rotated Structures I through III, this domain remains rigidly associated with domain V and the N-terminal superdomain and does not undergo noticeable rearrangements. DISCUSS +18 29 mid-rotated protein_state In the rotated or mid-rotated Structures I through III, this domain remains rigidly associated with domain V and the N-terminal superdomain and does not undergo noticeable rearrangements. DISCUSS +30 54 Structures I through III evidence In the rotated or mid-rotated Structures I through III, this domain remains rigidly associated with domain V and the N-terminal superdomain and does not undergo noticeable rearrangements. DISCUSS +107 108 V structure_element In the rotated or mid-rotated Structures I through III, this domain remains rigidly associated with domain V and the N-terminal superdomain and does not undergo noticeable rearrangements. DISCUSS +128 139 superdomain structure_element In the rotated or mid-rotated Structures I through III, this domain remains rigidly associated with domain V and the N-terminal superdomain and does not undergo noticeable rearrangements. DISCUSS +3 14 Structure V evidence In Structure V, however, the tip of helix A of domain III is displaced toward domain I by ~5 Å relative to that in mid-rotated or fully rotated structures. DISCUSS +36 43 helix A structure_element In Structure V, however, the tip of helix A of domain III is displaced toward domain I by ~5 Å relative to that in mid-rotated or fully rotated structures. DISCUSS +54 57 III structure_element In Structure V, however, the tip of helix A of domain III is displaced toward domain I by ~5 Å relative to that in mid-rotated or fully rotated structures. DISCUSS +85 86 I structure_element In Structure V, however, the tip of helix A of domain III is displaced toward domain I by ~5 Å relative to that in mid-rotated or fully rotated structures. DISCUSS +115 126 mid-rotated protein_state In Structure V, however, the tip of helix A of domain III is displaced toward domain I by ~5 Å relative to that in mid-rotated or fully rotated structures. DISCUSS +130 143 fully rotated protein_state In Structure V, however, the tip of helix A of domain III is displaced toward domain I by ~5 Å relative to that in mid-rotated or fully rotated structures. DISCUSS +144 154 structures evidence In Structure V, however, the tip of helix A of domain III is displaced toward domain I by ~5 Å relative to that in mid-rotated or fully rotated structures. DISCUSS +55 58 40S complex_assembly This displacement is caused by the 8 Å movement of the 40S body protein uS12 upon reverse intersubunit rotation from Structure I to V (Figure 6d). DISCUSS +59 63 body structure_element This displacement is caused by the 8 Å movement of the 40S body protein uS12 upon reverse intersubunit rotation from Structure I to V (Figure 6d). DISCUSS +72 76 uS12 protein This displacement is caused by the 8 Å movement of the 40S body protein uS12 upon reverse intersubunit rotation from Structure I to V (Figure 6d). DISCUSS +117 133 Structure I to V evidence This displacement is caused by the 8 Å movement of the 40S body protein uS12 upon reverse intersubunit rotation from Structure I to V (Figure 6d). DISCUSS +36 39 III structure_element We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the β-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the β-platforms are nearly perpendicular. DISCUSS +43 47 uS12 protein We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the β-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the β-platforms are nearly perpendicular. DISCUSS +89 93 eEF2 protein We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the β-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the β-platforms are nearly perpendicular. DISCUSS +113 123 β-platform structure_element We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the β-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the β-platforms are nearly perpendicular. DISCUSS +134 137 III structure_element We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the β-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the β-platforms are nearly perpendicular. DISCUSS +158 159 V structure_element We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the β-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the β-platforms are nearly perpendicular. DISCUSS +215 219 free protein_state We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the β-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the β-platforms are nearly perpendicular. DISCUSS +220 224 eEF2 protein We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the β-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the β-platforms are nearly perpendicular. DISCUSS +229 233 EF-G protein We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the β-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the β-platforms are nearly perpendicular. DISCUSS +247 258 β-platforms structure_element We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the β-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the β-platforms are nearly perpendicular. DISCUSS +21 32 Structure V evidence As we discuss below, Structure V captures a 'pre-unstacking' state due to stabilization of the interface between domains III and V by sordarin. DISCUSS +45 59 pre-unstacking protein_state As we discuss below, Structure V captures a 'pre-unstacking' state due to stabilization of the interface between domains III and V by sordarin. DISCUSS +95 104 interface site As we discuss below, Structure V captures a 'pre-unstacking' state due to stabilization of the interface between domains III and V by sordarin. DISCUSS +121 124 III structure_element As we discuss below, Structure V captures a 'pre-unstacking' state due to stabilization of the interface between domains III and V by sordarin. DISCUSS +129 130 V structure_element As we discuss below, Structure V captures a 'pre-unstacking' state due to stabilization of the interface between domains III and V by sordarin. DISCUSS +134 142 sordarin chemical As we discuss below, Structure V captures a 'pre-unstacking' state due to stabilization of the interface between domains III and V by sordarin. DISCUSS +0 8 Sordarin chemical Sordarin stabilizes GDP-bound eEF2 on the ribosome DISCUSS +20 29 GDP-bound protein_state Sordarin stabilizes GDP-bound eEF2 on the ribosome DISCUSS +30 34 eEF2 protein Sordarin stabilizes GDP-bound eEF2 on the ribosome DISCUSS +42 50 ribosome complex_assembly Sordarin stabilizes GDP-bound eEF2 on the ribosome DISCUSS +0 8 Sordarin chemical Sordarin is a potent antifungal antibiotic that inhibits translation. DISCUSS +9 32 biochemical experiments experimental_method Based on biochemical experiments, two alternative mechanisms of action were proposed: sordarin either prevents eEF2 departure by inhibiting GTP hydrolysis or acts after GTP hydrolysis. DISCUSS +86 94 sordarin chemical Based on biochemical experiments, two alternative mechanisms of action were proposed: sordarin either prevents eEF2 departure by inhibiting GTP hydrolysis or acts after GTP hydrolysis. DISCUSS +111 115 eEF2 protein Based on biochemical experiments, two alternative mechanisms of action were proposed: sordarin either prevents eEF2 departure by inhibiting GTP hydrolysis or acts after GTP hydrolysis. DISCUSS +140 143 GTP chemical Based on biochemical experiments, two alternative mechanisms of action were proposed: sordarin either prevents eEF2 departure by inhibiting GTP hydrolysis or acts after GTP hydrolysis. DISCUSS +169 172 GTP chemical Based on biochemical experiments, two alternative mechanisms of action were proposed: sordarin either prevents eEF2 departure by inhibiting GTP hydrolysis or acts after GTP hydrolysis. DISCUSS +41 49 eEF2•GTP complex_assembly Although our complex was assembled using eEF2•GTP, density maps clearly show GDP and Mg2+ in each structure (Figure 5g). DISCUSS +51 63 density maps evidence Although our complex was assembled using eEF2•GTP, density maps clearly show GDP and Mg2+ in each structure (Figure 5g). DISCUSS +77 80 GDP chemical Although our complex was assembled using eEF2•GTP, density maps clearly show GDP and Mg2+ in each structure (Figure 5g). DISCUSS +85 89 Mg2+ chemical Although our complex was assembled using eEF2•GTP, density maps clearly show GDP and Mg2+ in each structure (Figure 5g). DISCUSS +98 107 structure evidence Although our complex was assembled using eEF2•GTP, density maps clearly show GDP and Mg2+ in each structure (Figure 5g). DISCUSS +4 14 structures evidence Our structures therefore indicate that sordarin stalls eEF2 on the ribosome in the GDP-bound form, i.e. following GTP hydrolysis and phosphate release. DISCUSS +39 47 sordarin chemical Our structures therefore indicate that sordarin stalls eEF2 on the ribosome in the GDP-bound form, i.e. following GTP hydrolysis and phosphate release. DISCUSS +55 59 eEF2 protein Our structures therefore indicate that sordarin stalls eEF2 on the ribosome in the GDP-bound form, i.e. following GTP hydrolysis and phosphate release. DISCUSS +67 75 ribosome complex_assembly Our structures therefore indicate that sordarin stalls eEF2 on the ribosome in the GDP-bound form, i.e. following GTP hydrolysis and phosphate release. DISCUSS +83 92 GDP-bound protein_state Our structures therefore indicate that sordarin stalls eEF2 on the ribosome in the GDP-bound form, i.e. following GTP hydrolysis and phosphate release. DISCUSS +114 117 GTP chemical Our structures therefore indicate that sordarin stalls eEF2 on the ribosome in the GDP-bound form, i.e. following GTP hydrolysis and phosphate release. DISCUSS +56 73 pre-translocation protein_state The mechanism of stalling is suggested by comparison of pre-translocation and post-translocation structures in our ensemble. DISCUSS +78 96 post-translocation protein_state The mechanism of stalling is suggested by comparison of pre-translocation and post-translocation structures in our ensemble. DISCUSS +97 107 structures evidence The mechanism of stalling is suggested by comparison of pre-translocation and post-translocation structures in our ensemble. DISCUSS +12 22 structures evidence In all five structures, sordarin is bound between domains III and V of eEF2, stabilized by hydrophobic interactions identical to those in the isolated eEF2•sordarin complex (Figures 5g and h). DISCUSS +24 32 sordarin chemical In all five structures, sordarin is bound between domains III and V of eEF2, stabilized by hydrophobic interactions identical to those in the isolated eEF2•sordarin complex (Figures 5g and h). DISCUSS +36 41 bound protein_state In all five structures, sordarin is bound between domains III and V of eEF2, stabilized by hydrophobic interactions identical to those in the isolated eEF2•sordarin complex (Figures 5g and h). DISCUSS +58 61 III structure_element In all five structures, sordarin is bound between domains III and V of eEF2, stabilized by hydrophobic interactions identical to those in the isolated eEF2•sordarin complex (Figures 5g and h). DISCUSS +66 67 V structure_element In all five structures, sordarin is bound between domains III and V of eEF2, stabilized by hydrophobic interactions identical to those in the isolated eEF2•sordarin complex (Figures 5g and h). DISCUSS +71 75 eEF2 protein In all five structures, sordarin is bound between domains III and V of eEF2, stabilized by hydrophobic interactions identical to those in the isolated eEF2•sordarin complex (Figures 5g and h). DISCUSS +91 115 hydrophobic interactions bond_interaction In all five structures, sordarin is bound between domains III and V of eEF2, stabilized by hydrophobic interactions identical to those in the isolated eEF2•sordarin complex (Figures 5g and h). DISCUSS +142 150 isolated protein_state In all five structures, sordarin is bound between domains III and V of eEF2, stabilized by hydrophobic interactions identical to those in the isolated eEF2•sordarin complex (Figures 5g and h). DISCUSS +151 164 eEF2•sordarin complex_assembly In all five structures, sordarin is bound between domains III and V of eEF2, stabilized by hydrophobic interactions identical to those in the isolated eEF2•sordarin complex (Figures 5g and h). DISCUSS +7 25 nearly non-rotated protein_state In the nearly non-rotated post-translocation Structure V, the tip of domain III is shifted, however the interface between domains III and V remains unchanged, suggesting strong stabilization of this interface by sordarin. DISCUSS +26 44 post-translocation protein_state In the nearly non-rotated post-translocation Structure V, the tip of domain III is shifted, however the interface between domains III and V remains unchanged, suggesting strong stabilization of this interface by sordarin. DISCUSS +45 56 Structure V evidence In the nearly non-rotated post-translocation Structure V, the tip of domain III is shifted, however the interface between domains III and V remains unchanged, suggesting strong stabilization of this interface by sordarin. DISCUSS +76 79 III structure_element In the nearly non-rotated post-translocation Structure V, the tip of domain III is shifted, however the interface between domains III and V remains unchanged, suggesting strong stabilization of this interface by sordarin. DISCUSS +104 113 interface site In the nearly non-rotated post-translocation Structure V, the tip of domain III is shifted, however the interface between domains III and V remains unchanged, suggesting strong stabilization of this interface by sordarin. DISCUSS +130 133 III structure_element In the nearly non-rotated post-translocation Structure V, the tip of domain III is shifted, however the interface between domains III and V remains unchanged, suggesting strong stabilization of this interface by sordarin. DISCUSS +138 139 V structure_element In the nearly non-rotated post-translocation Structure V, the tip of domain III is shifted, however the interface between domains III and V remains unchanged, suggesting strong stabilization of this interface by sordarin. DISCUSS +199 208 interface site In the nearly non-rotated post-translocation Structure V, the tip of domain III is shifted, however the interface between domains III and V remains unchanged, suggesting strong stabilization of this interface by sordarin. DISCUSS +212 220 sordarin chemical In the nearly non-rotated post-translocation Structure V, the tip of domain III is shifted, however the interface between domains III and V remains unchanged, suggesting strong stabilization of this interface by sordarin. DISCUSS +13 24 Structure V evidence We note that Structure V is slightly more rotated than the 80S•2tRNA•mRNA complex in the absence of eEF2•sordarin, implying that sordarin interferes with the final stages of reverse rotation of the post-translocation ribosome. DISCUSS +59 73 80S•2tRNA•mRNA complex_assembly We note that Structure V is slightly more rotated than the 80S•2tRNA•mRNA complex in the absence of eEF2•sordarin, implying that sordarin interferes with the final stages of reverse rotation of the post-translocation ribosome. DISCUSS +89 99 absence of protein_state We note that Structure V is slightly more rotated than the 80S•2tRNA•mRNA complex in the absence of eEF2•sordarin, implying that sordarin interferes with the final stages of reverse rotation of the post-translocation ribosome. DISCUSS +100 113 eEF2•sordarin complex_assembly We note that Structure V is slightly more rotated than the 80S•2tRNA•mRNA complex in the absence of eEF2•sordarin, implying that sordarin interferes with the final stages of reverse rotation of the post-translocation ribosome. DISCUSS +129 137 sordarin chemical We note that Structure V is slightly more rotated than the 80S•2tRNA•mRNA complex in the absence of eEF2•sordarin, implying that sordarin interferes with the final stages of reverse rotation of the post-translocation ribosome. DISCUSS +198 216 post-translocation protein_state We note that Structure V is slightly more rotated than the 80S•2tRNA•mRNA complex in the absence of eEF2•sordarin, implying that sordarin interferes with the final stages of reverse rotation of the post-translocation ribosome. DISCUSS +217 225 ribosome complex_assembly We note that Structure V is slightly more rotated than the 80S•2tRNA•mRNA complex in the absence of eEF2•sordarin, implying that sordarin interferes with the final stages of reverse rotation of the post-translocation ribosome. DISCUSS +16 24 sordarin chemical We propose that sordarin acts to prevent full reverse rotation and release of eEF2•GDP by stabilizing the interdomain interface and thus blocking uS12-induced disengagement of domain III from domain V. DISCUSS +78 86 eEF2•GDP complex_assembly We propose that sordarin acts to prevent full reverse rotation and release of eEF2•GDP by stabilizing the interdomain interface and thus blocking uS12-induced disengagement of domain III from domain V. DISCUSS +106 127 interdomain interface site We propose that sordarin acts to prevent full reverse rotation and release of eEF2•GDP by stabilizing the interdomain interface and thus blocking uS12-induced disengagement of domain III from domain V. DISCUSS +146 150 uS12 protein We propose that sordarin acts to prevent full reverse rotation and release of eEF2•GDP by stabilizing the interdomain interface and thus blocking uS12-induced disengagement of domain III from domain V. DISCUSS +183 186 III structure_element We propose that sordarin acts to prevent full reverse rotation and release of eEF2•GDP by stabilizing the interdomain interface and thus blocking uS12-induced disengagement of domain III from domain V. DISCUSS +199 200 V structure_element We propose that sordarin acts to prevent full reverse rotation and release of eEF2•GDP by stabilizing the interdomain interface and thus blocking uS12-induced disengagement of domain III from domain V. DISCUSS +17 21 tRNA chemical Implications for tRNA and mRNA translocation during translation DISCUSS +26 30 mRNA chemical Implications for tRNA and mRNA translocation during translation DISCUSS +25 29 tRNA chemical Because translocation of tRNA must involve large-scale dynamics, this step has long been regarded as the most puzzling step of translation. DISCUSS +32 36 tRNA chemical Intersubunit rearrangements and tRNA hybrid states have been proposed to play key roles half a century ago. DISCUSS +37 43 hybrid protein_state Intersubunit rearrangements and tRNA hybrid states have been proposed to play key roles half a century ago. DISCUSS +22 26 body structure_element Despite an impressive body of biochemical, fluorescence and structural data accumulated since then, translocation remains the least understood step of elongation. DISCUSS +30 75 biochemical, fluorescence and structural data evidence Despite an impressive body of biochemical, fluorescence and structural data accumulated since then, translocation remains the least understood step of elongation. DISCUSS +32 40 ribosome complex_assembly The structural understanding of ribosome and tRNA dynamics has been greatly aided by a wealth of X-ray and cryo-EM structures (reviewed in). DISCUSS +45 49 tRNA chemical The structural understanding of ribosome and tRNA dynamics has been greatly aided by a wealth of X-ray and cryo-EM structures (reviewed in). DISCUSS +97 102 X-ray experimental_method The structural understanding of ribosome and tRNA dynamics has been greatly aided by a wealth of X-ray and cryo-EM structures (reviewed in). DISCUSS +107 114 cryo-EM experimental_method The structural understanding of ribosome and tRNA dynamics has been greatly aided by a wealth of X-ray and cryo-EM structures (reviewed in). DISCUSS +115 125 structures evidence The structural understanding of ribosome and tRNA dynamics has been greatly aided by a wealth of X-ray and cryo-EM structures (reviewed in). DISCUSS +30 34 eEF2 protein However, visualization of the eEF2/EF-G-induced translocation is confined to very early pre-EF-G-entry states and late (almost translocated or fully translocated) states, leaving most of the path from the A to the P site uncharacterized (Figure 1—figure supplement 1). DISCUSS +35 39 EF-G protein However, visualization of the eEF2/EF-G-induced translocation is confined to very early pre-EF-G-entry states and late (almost translocated or fully translocated) states, leaving most of the path from the A to the P site uncharacterized (Figure 1—figure supplement 1). DISCUSS +88 102 pre-EF-G-entry protein_state However, visualization of the eEF2/EF-G-induced translocation is confined to very early pre-EF-G-entry states and late (almost translocated or fully translocated) states, leaving most of the path from the A to the P site uncharacterized (Figure 1—figure supplement 1). DISCUSS +120 139 almost translocated protein_state However, visualization of the eEF2/EF-G-induced translocation is confined to very early pre-EF-G-entry states and late (almost translocated or fully translocated) states, leaving most of the path from the A to the P site uncharacterized (Figure 1—figure supplement 1). DISCUSS +143 161 fully translocated protein_state However, visualization of the eEF2/EF-G-induced translocation is confined to very early pre-EF-G-entry states and late (almost translocated or fully translocated) states, leaving most of the path from the A to the P site uncharacterized (Figure 1—figure supplement 1). DISCUSS +205 220 A to the P site site However, visualization of the eEF2/EF-G-induced translocation is confined to very early pre-EF-G-entry states and late (almost translocated or fully translocated) states, leaving most of the path from the A to the P site uncharacterized (Figure 1—figure supplement 1). DISCUSS +69 73 tRNA chemical Our study provides new insights into the structural understanding of tRNA translocation. DISCUSS +23 27 tRNA chemical First, we propose that tRNA and IRES translocations occur via the same general trajectory. DISCUSS +32 36 IRES site First, we propose that tRNA and IRES translocations occur via the same general trajectory. DISCUSS +35 43 ribosome complex_assembly This is evident from the fact that ribosome rearrangements in translocation are inherent to the ribosome and likely occur in similar ways in both cases. DISCUSS +96 104 ribosome complex_assembly This is evident from the fact that ribosome rearrangements in translocation are inherent to the ribosome and likely occur in similar ways in both cases. DISCUSS +39 47 ribosome complex_assembly Furthermore, the step-wise coupling of ribosome dynamics with IRES translocation is overall consistent with that observed for 2tRNA•mRNA translocation in solution. DISCUSS +62 66 IRES site Furthermore, the step-wise coupling of ribosome dynamics with IRES translocation is overall consistent with that observed for 2tRNA•mRNA translocation in solution. DISCUSS +126 136 2tRNA•mRNA complex_assembly Furthermore, the step-wise coupling of ribosome dynamics with IRES translocation is overall consistent with that observed for 2tRNA•mRNA translocation in solution. DISCUSS +13 49 fluorescence and biochemical studies experimental_method For example, fluorescence and biochemical studies revealed that the early pre-translocation EF-G-bound ribosomes are fully rotated and translocation of the tRNA-mRNA complex occurs during reverse rotation of the small subunit, coupled with head swivel. DISCUSS +74 91 pre-translocation protein_state For example, fluorescence and biochemical studies revealed that the early pre-translocation EF-G-bound ribosomes are fully rotated and translocation of the tRNA-mRNA complex occurs during reverse rotation of the small subunit, coupled with head swivel. DISCUSS +92 102 EF-G-bound protein_state For example, fluorescence and biochemical studies revealed that the early pre-translocation EF-G-bound ribosomes are fully rotated and translocation of the tRNA-mRNA complex occurs during reverse rotation of the small subunit, coupled with head swivel. DISCUSS +103 112 ribosomes complex_assembly For example, fluorescence and biochemical studies revealed that the early pre-translocation EF-G-bound ribosomes are fully rotated and translocation of the tRNA-mRNA complex occurs during reverse rotation of the small subunit, coupled with head swivel. DISCUSS +117 130 fully rotated protein_state For example, fluorescence and biochemical studies revealed that the early pre-translocation EF-G-bound ribosomes are fully rotated and translocation of the tRNA-mRNA complex occurs during reverse rotation of the small subunit, coupled with head swivel. DISCUSS +156 165 tRNA-mRNA complex_assembly For example, fluorescence and biochemical studies revealed that the early pre-translocation EF-G-bound ribosomes are fully rotated and translocation of the tRNA-mRNA complex occurs during reverse rotation of the small subunit, coupled with head swivel. DISCUSS +212 225 small subunit structure_element For example, fluorescence and biochemical studies revealed that the early pre-translocation EF-G-bound ribosomes are fully rotated and translocation of the tRNA-mRNA complex occurs during reverse rotation of the small subunit, coupled with head swivel. DISCUSS +240 244 head structure_element For example, fluorescence and biochemical studies revealed that the early pre-translocation EF-G-bound ribosomes are fully rotated and translocation of the tRNA-mRNA complex occurs during reverse rotation of the small subunit, coupled with head swivel. DISCUSS +16 24 ribosome complex_assembly The sequence of ribosome rearrangements during IRES translocation also agrees with that inferred from 70S•EF-G structures, including those in which the A-to-P-site translocating tRNA was not present. DISCUSS +47 51 IRES site The sequence of ribosome rearrangements during IRES translocation also agrees with that inferred from 70S•EF-G structures, including those in which the A-to-P-site translocating tRNA was not present. DISCUSS +102 110 70S•EF-G complex_assembly The sequence of ribosome rearrangements during IRES translocation also agrees with that inferred from 70S•EF-G structures, including those in which the A-to-P-site translocating tRNA was not present. DISCUSS +111 121 structures evidence The sequence of ribosome rearrangements during IRES translocation also agrees with that inferred from 70S•EF-G structures, including those in which the A-to-P-site translocating tRNA was not present. DISCUSS +152 163 A-to-P-site site The sequence of ribosome rearrangements during IRES translocation also agrees with that inferred from 70S•EF-G structures, including those in which the A-to-P-site translocating tRNA was not present. DISCUSS +178 182 tRNA chemical The sequence of ribosome rearrangements during IRES translocation also agrees with that inferred from 70S•EF-G structures, including those in which the A-to-P-site translocating tRNA was not present. DISCUSS +52 60 ribosome complex_assembly Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7–9°) body and a partly rotated head (5–7.5°), in agreement with the conformation of our Structure I. The most swiveled head (18–21°) was observed in a mid-rotated ribosome (3–5°) of a later translocation intermediate TIpost, similar to the conformation of our Structure III. DISCUSS +93 100 rotated protein_state Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7–9°) body and a partly rotated head (5–7.5°), in agreement with the conformation of our Structure I. The most swiveled head (18–21°) was observed in a mid-rotated ribosome (3–5°) of a later translocation intermediate TIpost, similar to the conformation of our Structure III. DISCUSS +108 112 body structure_element Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7–9°) body and a partly rotated head (5–7.5°), in agreement with the conformation of our Structure I. The most swiveled head (18–21°) was observed in a mid-rotated ribosome (3–5°) of a later translocation intermediate TIpost, similar to the conformation of our Structure III. DISCUSS +119 133 partly rotated protein_state Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7–9°) body and a partly rotated head (5–7.5°), in agreement with the conformation of our Structure I. The most swiveled head (18–21°) was observed in a mid-rotated ribosome (3–5°) of a later translocation intermediate TIpost, similar to the conformation of our Structure III. DISCUSS +134 138 head structure_element Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7–9°) body and a partly rotated head (5–7.5°), in agreement with the conformation of our Structure I. The most swiveled head (18–21°) was observed in a mid-rotated ribosome (3–5°) of a later translocation intermediate TIpost, similar to the conformation of our Structure III. DISCUSS +191 202 Structure I evidence Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7–9°) body and a partly rotated head (5–7.5°), in agreement with the conformation of our Structure I. The most swiveled head (18–21°) was observed in a mid-rotated ribosome (3–5°) of a later translocation intermediate TIpost, similar to the conformation of our Structure III. DISCUSS +208 221 most swiveled protein_state Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7–9°) body and a partly rotated head (5–7.5°), in agreement with the conformation of our Structure I. The most swiveled head (18–21°) was observed in a mid-rotated ribosome (3–5°) of a later translocation intermediate TIpost, similar to the conformation of our Structure III. DISCUSS +222 226 head structure_element Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7–9°) body and a partly rotated head (5–7.5°), in agreement with the conformation of our Structure I. The most swiveled head (18–21°) was observed in a mid-rotated ribosome (3–5°) of a later translocation intermediate TIpost, similar to the conformation of our Structure III. DISCUSS +254 265 mid-rotated protein_state Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7–9°) body and a partly rotated head (5–7.5°), in agreement with the conformation of our Structure I. The most swiveled head (18–21°) was observed in a mid-rotated ribosome (3–5°) of a later translocation intermediate TIpost, similar to the conformation of our Structure III. DISCUSS +266 274 ribosome complex_assembly Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7–9°) body and a partly rotated head (5–7.5°), in agreement with the conformation of our Structure I. The most swiveled head (18–21°) was observed in a mid-rotated ribosome (3–5°) of a later translocation intermediate TIpost, similar to the conformation of our Structure III. DISCUSS +363 376 Structure III evidence Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7–9°) body and a partly rotated head (5–7.5°), in agreement with the conformation of our Structure I. The most swiveled head (18–21°) was observed in a mid-rotated ribosome (3–5°) of a later translocation intermediate TIpost, similar to the conformation of our Structure III. DISCUSS +83 94 A-to-P-site site Overall, these correlations suggest that the intermediate locations of the elusive A-to-P-site translocating tRNA are similar to those of PKI in our structures. DISCUSS +109 113 tRNA chemical Overall, these correlations suggest that the intermediate locations of the elusive A-to-P-site translocating tRNA are similar to those of PKI in our structures. DISCUSS +138 141 PKI structure_element Overall, these correlations suggest that the intermediate locations of the elusive A-to-P-site translocating tRNA are similar to those of PKI in our structures. DISCUSS +149 159 structures evidence Overall, these correlations suggest that the intermediate locations of the elusive A-to-P-site translocating tRNA are similar to those of PKI in our structures. DISCUSS +12 22 structures evidence Second, the structures clarify the structural basis of the often-used but structurally undefined terms 'locking' and 'unlocking' with respect to the pre-translocation complex (Figure 6f). DISCUSS +149 166 pre-translocation protein_state Second, the structures clarify the structural basis of the often-used but structurally undefined terms 'locking' and 'unlocking' with respect to the pre-translocation complex (Figure 6f). DISCUSS +12 29 pre-translocation protein_state We deem the pre-translocation complex locked, because the A-site bound ASL-mRNA is stabilized by interactions with the decoding center. DISCUSS +38 44 locked protein_state We deem the pre-translocation complex locked, because the A-site bound ASL-mRNA is stabilized by interactions with the decoding center. DISCUSS +58 70 A-site bound protein_state We deem the pre-translocation complex locked, because the A-site bound ASL-mRNA is stabilized by interactions with the decoding center. DISCUSS +75 79 mRNA chemical We deem the pre-translocation complex locked, because the A-site bound ASL-mRNA is stabilized by interactions with the decoding center. DISCUSS +119 134 decoding center site We deem the pre-translocation complex locked, because the A-site bound ASL-mRNA is stabilized by interactions with the decoding center. DISCUSS +42 51 classical protein_state These interactions are maintained for the classical- and hybrid-state tRNAs in the spontaneously sampled non-rotated and rotated ribosomes, respectively. DISCUSS +57 63 hybrid protein_state These interactions are maintained for the classical- and hybrid-state tRNAs in the spontaneously sampled non-rotated and rotated ribosomes, respectively. DISCUSS +70 75 tRNAs chemical These interactions are maintained for the classical- and hybrid-state tRNAs in the spontaneously sampled non-rotated and rotated ribosomes, respectively. DISCUSS +105 116 non-rotated protein_state These interactions are maintained for the classical- and hybrid-state tRNAs in the spontaneously sampled non-rotated and rotated ribosomes, respectively. DISCUSS +121 128 rotated protein_state These interactions are maintained for the classical- and hybrid-state tRNAs in the spontaneously sampled non-rotated and rotated ribosomes, respectively. DISCUSS +129 138 ribosomes complex_assembly These interactions are maintained for the classical- and hybrid-state tRNAs in the spontaneously sampled non-rotated and rotated ribosomes, respectively. DISCUSS +37 58 codon-anticodon helix structure_element Unlocking involves separation of the codon-anticodon helix from the decoding center residues by the protruding tip of eEF2/EF-G (Figure 7), occurring in the fully rotated ribosome at an early pre-translocation step. DISCUSS +68 83 decoding center site Unlocking involves separation of the codon-anticodon helix from the decoding center residues by the protruding tip of eEF2/EF-G (Figure 7), occurring in the fully rotated ribosome at an early pre-translocation step. DISCUSS +118 122 eEF2 protein Unlocking involves separation of the codon-anticodon helix from the decoding center residues by the protruding tip of eEF2/EF-G (Figure 7), occurring in the fully rotated ribosome at an early pre-translocation step. DISCUSS +123 127 EF-G protein Unlocking involves separation of the codon-anticodon helix from the decoding center residues by the protruding tip of eEF2/EF-G (Figure 7), occurring in the fully rotated ribosome at an early pre-translocation step. DISCUSS +157 170 fully rotated protein_state Unlocking involves separation of the codon-anticodon helix from the decoding center residues by the protruding tip of eEF2/EF-G (Figure 7), occurring in the fully rotated ribosome at an early pre-translocation step. DISCUSS +171 179 ribosome complex_assembly Unlocking involves separation of the codon-anticodon helix from the decoding center residues by the protruding tip of eEF2/EF-G (Figure 7), occurring in the fully rotated ribosome at an early pre-translocation step. DISCUSS +192 209 pre-translocation protein_state Unlocking involves separation of the codon-anticodon helix from the decoding center residues by the protruding tip of eEF2/EF-G (Figure 7), occurring in the fully rotated ribosome at an early pre-translocation step. DISCUSS +19 23 head structure_element This unlatches the head, allowing creation of hitherto elusive intermediate tRNA positions during spontaneous reverse body rotation. DISCUSS +76 80 tRNA chemical This unlatches the head, allowing creation of hitherto elusive intermediate tRNA positions during spontaneous reverse body rotation. DISCUSS +118 122 body structure_element This unlatches the head, allowing creation of hitherto elusive intermediate tRNA positions during spontaneous reverse body rotation. DISCUSS +46 50 head structure_element Third, our findings uncover a new role of the head swivel. DISCUSS +54 66 constriction site Previous studies showed that this movement widens the constriction ('gate') between the P and E sites, thus allowing the P-tRNA passage to the E site. DISCUSS +69 73 gate site Previous studies showed that this movement widens the constriction ('gate') between the P and E sites, thus allowing the P-tRNA passage to the E site. DISCUSS +88 101 P and E sites site Previous studies showed that this movement widens the constriction ('gate') between the P and E sites, thus allowing the P-tRNA passage to the E site. DISCUSS +121 122 P site Previous studies showed that this movement widens the constriction ('gate') between the P and E sites, thus allowing the P-tRNA passage to the E site. DISCUSS +123 127 tRNA chemical Previous studies showed that this movement widens the constriction ('gate') between the P and E sites, thus allowing the P-tRNA passage to the E site. DISCUSS +143 149 E site site Previous studies showed that this movement widens the constriction ('gate') between the P and E sites, thus allowing the P-tRNA passage to the E site. DISCUSS +20 24 gate site In addition to the 'gate-opening' role, we now show that the head swivel brings the head A site to the body P site, allowing a step-wise conveying of the codon-anticodon helix between the A and P sites. DISCUSS +61 65 head structure_element In addition to the 'gate-opening' role, we now show that the head swivel brings the head A site to the body P site, allowing a step-wise conveying of the codon-anticodon helix between the A and P sites. DISCUSS +84 88 head structure_element In addition to the 'gate-opening' role, we now show that the head swivel brings the head A site to the body P site, allowing a step-wise conveying of the codon-anticodon helix between the A and P sites. DISCUSS +89 95 A site site In addition to the 'gate-opening' role, we now show that the head swivel brings the head A site to the body P site, allowing a step-wise conveying of the codon-anticodon helix between the A and P sites. DISCUSS +103 107 body structure_element In addition to the 'gate-opening' role, we now show that the head swivel brings the head A site to the body P site, allowing a step-wise conveying of the codon-anticodon helix between the A and P sites. DISCUSS +108 114 P site site In addition to the 'gate-opening' role, we now show that the head swivel brings the head A site to the body P site, allowing a step-wise conveying of the codon-anticodon helix between the A and P sites. DISCUSS +154 175 codon-anticodon helix structure_element In addition to the 'gate-opening' role, we now show that the head swivel brings the head A site to the body P site, allowing a step-wise conveying of the codon-anticodon helix between the A and P sites. DISCUSS +188 201 A and P sites site In addition to the 'gate-opening' role, we now show that the head swivel brings the head A site to the body P site, allowing a step-wise conveying of the codon-anticodon helix between the A and P sites. DISCUSS +36 45 particles experimental_method Finally, the similar populations of particles (within a 2X range) in our 80S•IRES•eEF2 reconstructions (Figure 1—figure supplement 2) suggest that the intermediate translocation states sample several energetically similar and interconverting conformations. DISCUSS +73 86 80S•IRES•eEF2 complex_assembly Finally, the similar populations of particles (within a 2X range) in our 80S•IRES•eEF2 reconstructions (Figure 1—figure supplement 2) suggest that the intermediate translocation states sample several energetically similar and interconverting conformations. DISCUSS +87 102 reconstructions evidence Finally, the similar populations of particles (within a 2X range) in our 80S•IRES•eEF2 reconstructions (Figure 1—figure supplement 2) suggest that the intermediate translocation states sample several energetically similar and interconverting conformations. DISCUSS +156 164 ribosome complex_assembly This is consistent with the idea of a rather flat energy landscape of translocation, suggested by recent work that measured mechanical work produced by the ribosome during translocation. DISCUSS +139 160 codon-anticodon helix structure_element Our findings implicate, however, that the energy landscape is not completely flat and contains local minima for transient positions of the codon-anticodon helix between the A and P sites. DISCUSS +173 186 A and P sites site Our findings implicate, however, that the energy landscape is not completely flat and contains local minima for transient positions of the codon-anticodon helix between the A and P sites. DISCUSS +17 20 PKI structure_element The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4 Å each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2—source data 1). DISCUSS +41 45 body structure_element The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4 Å each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2—source data 1). DISCUSS +68 72 head structure_element The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4 Å each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2—source data 1). DISCUSS +117 135 initiation complex complex_assembly The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4 Å each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2—source data 1). DISCUSS +139 140 I evidence The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4 Å each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2—source data 1). DISCUSS +146 147 I evidence The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4 Å each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2—source data 1). DISCUSS +151 153 II evidence The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4 Å each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2—source data 1). DISCUSS +168 171 PKI structure_element The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4 Å each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2—source data 1). DISCUSS +212 216 body structure_element The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4 Å each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2—source data 1). DISCUSS +217 223 P site site The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4 Å each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2—source data 1). DISCUSS +227 251 Structures III, IV and V evidence The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4 Å each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2—source data 1). DISCUSS +12 15 PKI structure_element Movement of PKI relative to the head occurs during the subsequent reverse swivel in three 3–7 Å sub-steps (II to III to IV to V). DISCUSS +32 36 head structure_element Movement of PKI relative to the head occurs during the subsequent reverse swivel in three 3–7 Å sub-steps (II to III to IV to V). DISCUSS +107 127 II to III to IV to V evidence Movement of PKI relative to the head occurs during the subsequent reverse swivel in three 3–7 Å sub-steps (II to III to IV to V). DISCUSS +48 52 maps evidence We note that four of our near-atomic resolution maps comprised ~30,000 particles each, the minimum number required for a near-atomic-resolution reconstruction of the ribosome. DISCUSS +71 80 particles experimental_method We note that four of our near-atomic resolution maps comprised ~30,000 particles each, the minimum number required for a near-atomic-resolution reconstruction of the ribosome. DISCUSS +121 158 near-atomic-resolution reconstruction evidence We note that four of our near-atomic resolution maps comprised ~30,000 particles each, the minimum number required for a near-atomic-resolution reconstruction of the ribosome. DISCUSS +166 174 ribosome complex_assembly We note that four of our near-atomic resolution maps comprised ~30,000 particles each, the minimum number required for a near-atomic-resolution reconstruction of the ribosome. DISCUSS +15 20 viral taxonomy_domain Translation of viral mRNA DISCUSS +21 25 mRNA chemical Translation of viral mRNA DISCUSS +80 83 IGR structure_element Our work sheds light on the dynamic mechanism of cap-independent translation by IGR IRESs, tightly coupled with the universally conserved dynamic properties of the ribosome. DISCUSS +84 89 IRESs site Our work sheds light on the dynamic mechanism of cap-independent translation by IGR IRESs, tightly coupled with the universally conserved dynamic properties of the ribosome. DISCUSS +116 137 universally conserved protein_state Our work sheds light on the dynamic mechanism of cap-independent translation by IGR IRESs, tightly coupled with the universally conserved dynamic properties of the ribosome. DISCUSS +164 172 ribosome complex_assembly Our work sheds light on the dynamic mechanism of cap-independent translation by IGR IRESs, tightly coupled with the universally conserved dynamic properties of the ribosome. DISCUSS +4 11 cryo-EM experimental_method The cryo-EM structures demonstrate that the TSV IRES structurally and dynamically represents a chimera of the 2tRNA•mRNA translocating complex (A/P-tRNA • P/E-tRNA • mRNA). DISCUSS +12 22 structures evidence The cryo-EM structures demonstrate that the TSV IRES structurally and dynamically represents a chimera of the 2tRNA•mRNA translocating complex (A/P-tRNA • P/E-tRNA • mRNA). DISCUSS +44 47 TSV species The cryo-EM structures demonstrate that the TSV IRES structurally and dynamically represents a chimera of the 2tRNA•mRNA translocating complex (A/P-tRNA • P/E-tRNA • mRNA). DISCUSS +48 52 IRES site The cryo-EM structures demonstrate that the TSV IRES structurally and dynamically represents a chimera of the 2tRNA•mRNA translocating complex (A/P-tRNA • P/E-tRNA • mRNA). DISCUSS +110 120 2tRNA•mRNA complex_assembly The cryo-EM structures demonstrate that the TSV IRES structurally and dynamically represents a chimera of the 2tRNA•mRNA translocating complex (A/P-tRNA • P/E-tRNA • mRNA). DISCUSS +144 170 A/P-tRNA • P/E-tRNA • mRNA complex_assembly The cryo-EM structures demonstrate that the TSV IRES structurally and dynamically represents a chimera of the 2tRNA•mRNA translocating complex (A/P-tRNA • P/E-tRNA • mRNA). DISCUSS +12 22 2tRNA•mRNA complex_assembly Like in the 2tRNA•mRNA translocating complex in which the two tRNAs move independently of each other, the PKI domain moves relative to the 5´-domain, causing the IRES to undergo an inchworm-walk translocation. DISCUSS +62 67 tRNAs chemical Like in the 2tRNA•mRNA translocating complex in which the two tRNAs move independently of each other, the PKI domain moves relative to the 5´-domain, causing the IRES to undergo an inchworm-walk translocation. DISCUSS +106 109 PKI structure_element Like in the 2tRNA•mRNA translocating complex in which the two tRNAs move independently of each other, the PKI domain moves relative to the 5´-domain, causing the IRES to undergo an inchworm-walk translocation. DISCUSS +139 148 5´-domain structure_element Like in the 2tRNA•mRNA translocating complex in which the two tRNAs move independently of each other, the PKI domain moves relative to the 5´-domain, causing the IRES to undergo an inchworm-walk translocation. DISCUSS +162 166 IRES site Like in the 2tRNA•mRNA translocating complex in which the two tRNAs move independently of each other, the PKI domain moves relative to the 5´-domain, causing the IRES to undergo an inchworm-walk translocation. DISCUSS +181 189 inchworm protein_state Like in the 2tRNA•mRNA translocating complex in which the two tRNAs move independently of each other, the PKI domain moves relative to the 5´-domain, causing the IRES to undergo an inchworm-walk translocation. DISCUSS +42 46 IRES site A large structural difference between the IRES and the 2tRNA•mRNA complex exists, however, in that the IRES lacks three out of six tRNA-like domains involved in tRNA translocation. DISCUSS +55 65 2tRNA•mRNA complex_assembly A large structural difference between the IRES and the 2tRNA•mRNA complex exists, however, in that the IRES lacks three out of six tRNA-like domains involved in tRNA translocation. DISCUSS +103 107 IRES site A large structural difference between the IRES and the 2tRNA•mRNA complex exists, however, in that the IRES lacks three out of six tRNA-like domains involved in tRNA translocation. DISCUSS +108 113 lacks protein_state A large structural difference between the IRES and the 2tRNA•mRNA complex exists, however, in that the IRES lacks three out of six tRNA-like domains involved in tRNA translocation. DISCUSS +131 148 tRNA-like domains structure_element A large structural difference between the IRES and the 2tRNA•mRNA complex exists, however, in that the IRES lacks three out of six tRNA-like domains involved in tRNA translocation. DISCUSS +161 165 tRNA chemical A large structural difference between the IRES and the 2tRNA•mRNA complex exists, however, in that the IRES lacks three out of six tRNA-like domains involved in tRNA translocation. DISCUSS +73 77 IRES site This difference likely accounts for the inefficient translocation of the IRES, which is difficult to stabilize in the post-translocation state and therefore is prone to reverse translocation. DISCUSS +118 136 post-translocation protein_state This difference likely accounts for the inefficient translocation of the IRES, which is difficult to stabilize in the post-translocation state and therefore is prone to reverse translocation. DISCUSS +39 42 TSV species Although structurally handicapped, the TSV IRES manages to translocate by employing ribosome dynamics that are remarkably similar to that in 2tRNA•mRNA translocation. DISCUSS +43 47 IRES site Although structurally handicapped, the TSV IRES manages to translocate by employing ribosome dynamics that are remarkably similar to that in 2tRNA•mRNA translocation. DISCUSS +84 92 ribosome complex_assembly Although structurally handicapped, the TSV IRES manages to translocate by employing ribosome dynamics that are remarkably similar to that in 2tRNA•mRNA translocation. DISCUSS +141 151 2tRNA•mRNA complex_assembly Although structurally handicapped, the TSV IRES manages to translocate by employing ribosome dynamics that are remarkably similar to that in 2tRNA•mRNA translocation. DISCUSS +18 26 ribosome complex_assembly The uniformity of ribosome dynamics underscores the idea that translocation is an inherent and structurally-optimized property of the ribosome, supported also by translocation activity in the absence of the elongation factor. DISCUSS +134 142 ribosome complex_assembly The uniformity of ribosome dynamics underscores the idea that translocation is an inherent and structurally-optimized property of the ribosome, supported also by translocation activity in the absence of the elongation factor. DISCUSS +192 202 absence of protein_state The uniformity of ribosome dynamics underscores the idea that translocation is an inherent and structurally-optimized property of the ribosome, supported also by translocation activity in the absence of the elongation factor. DISCUSS +207 224 elongation factor protein_type The uniformity of ribosome dynamics underscores the idea that translocation is an inherent and structurally-optimized property of the ribosome, supported also by translocation activity in the absence of the elongation factor. DISCUSS +91 94 60S complex_assembly This property is rendered by the relative mobility of the three major building blocks, the 60S subunit and the 40S head and body, assisted by ligand-interacting extensions including the L1 stalk and the P stalk. DISCUSS +95 102 subunit structure_element This property is rendered by the relative mobility of the three major building blocks, the 60S subunit and the 40S head and body, assisted by ligand-interacting extensions including the L1 stalk and the P stalk. DISCUSS +111 114 40S complex_assembly This property is rendered by the relative mobility of the three major building blocks, the 60S subunit and the 40S head and body, assisted by ligand-interacting extensions including the L1 stalk and the P stalk. DISCUSS +115 119 head structure_element This property is rendered by the relative mobility of the three major building blocks, the 60S subunit and the 40S head and body, assisted by ligand-interacting extensions including the L1 stalk and the P stalk. DISCUSS +124 128 body structure_element This property is rendered by the relative mobility of the three major building blocks, the 60S subunit and the 40S head and body, assisted by ligand-interacting extensions including the L1 stalk and the P stalk. DISCUSS +142 171 ligand-interacting extensions structure_element This property is rendered by the relative mobility of the three major building blocks, the 60S subunit and the 40S head and body, assisted by ligand-interacting extensions including the L1 stalk and the P stalk. DISCUSS +186 194 L1 stalk structure_element This property is rendered by the relative mobility of the three major building blocks, the 60S subunit and the 40S head and body, assisted by ligand-interacting extensions including the L1 stalk and the P stalk. DISCUSS +203 210 P stalk structure_element This property is rendered by the relative mobility of the three major building blocks, the 60S subunit and the 40S head and body, assisted by ligand-interacting extensions including the L1 stalk and the P stalk. DISCUSS +11 16 IRESs site Intergenic IRESs, in turn, represent a striking example of convergent molecular evolution. DISCUSS +0 5 Viral taxonomy_domain Viral mRNAs have evolved to adopt an atypical structure to employ the inherent ribosome dynamics, to be able to hijack the host translational machinery in a simple fashion. DISCUSS +6 11 mRNAs chemical Viral mRNAs have evolved to adopt an atypical structure to employ the inherent ribosome dynamics, to be able to hijack the host translational machinery in a simple fashion. DISCUSS +46 55 structure evidence Viral mRNAs have evolved to adopt an atypical structure to employ the inherent ribosome dynamics, to be able to hijack the host translational machinery in a simple fashion. DISCUSS +79 87 ribosome complex_assembly Viral mRNAs have evolved to adopt an atypical structure to employ the inherent ribosome dynamics, to be able to hijack the host translational machinery in a simple fashion. DISCUSS +9 16 cryo-EM experimental_method Ensemble cryo-EM DISCUSS +66 74 ribosome complex_assembly Our current understanding of macromolecular machines, such as the ribosome, is often limited by a gap between biophysical/biochemical studies and structural studies. DISCUSS +110 141 biophysical/biochemical studies experimental_method Our current understanding of macromolecular machines, such as the ribosome, is often limited by a gap between biophysical/biochemical studies and structural studies. DISCUSS +146 164 structural studies experimental_method Our current understanding of macromolecular machines, such as the ribosome, is often limited by a gap between biophysical/biochemical studies and structural studies. DISCUSS +13 46 Förster resonance energy transfer experimental_method For example, Förster resonance energy transfer can provide insight into the macromolecular dynamics of an assembly at the single-molecule level but is limited to specifically labeled locations within the assembly. DISCUSS +16 34 crystal structures evidence High-resolution crystal structures, on the other hand, can provide static images of an assembly, and the structural dynamics can only be inferred by comparing structures that are usually obtained in different experiments and under different, often non-native, conditions. DISCUSS +159 169 structures evidence High-resolution crystal structures, on the other hand, can provide static images of an assembly, and the structural dynamics can only be inferred by comparing structures that are usually obtained in different experiments and under different, often non-native, conditions. DISCUSS +0 7 Cryo-EM experimental_method Cryo-EM offers the possibility of obtaining integrated information of both structure and dynamics as demonstrated in lower-resolution studies of bacterial ribosome complexes. DISCUSS +75 84 structure evidence Cryo-EM offers the possibility of obtaining integrated information of both structure and dynamics as demonstrated in lower-resolution studies of bacterial ribosome complexes. DISCUSS +145 154 bacterial taxonomy_domain Cryo-EM offers the possibility of obtaining integrated information of both structure and dynamics as demonstrated in lower-resolution studies of bacterial ribosome complexes. DISCUSS +155 163 ribosome complex_assembly Cryo-EM offers the possibility of obtaining integrated information of both structure and dynamics as demonstrated in lower-resolution studies of bacterial ribosome complexes. DISCUSS +65 73 ribosome complex_assembly This is presumably one of the reasons why most recent studies of ribosome complexes have focused on a single high-resolution structure despite the non-uniform local resolution of the maps that likely reflects structural heterogeneity. DISCUSS +125 134 structure evidence This is presumably one of the reasons why most recent studies of ribosome complexes have focused on a single high-resolution structure despite the non-uniform local resolution of the maps that likely reflects structural heterogeneity. DISCUSS +183 187 maps evidence This is presumably one of the reasons why most recent studies of ribosome complexes have focused on a single high-resolution structure despite the non-uniform local resolution of the maps that likely reflects structural heterogeneity. DISCUSS +32 40 FREALIGN experimental_method The computational efficiency of FREALIGN has allowed us to classify a relatively large dataset (1.1 million particles) into 15 classes (Figure 1—figure supplement 2) and obtain eight near-atomic-resolution structures from it. DISCUSS +108 117 particles experimental_method The computational efficiency of FREALIGN has allowed us to classify a relatively large dataset (1.1 million particles) into 15 classes (Figure 1—figure supplement 2) and obtain eight near-atomic-resolution structures from it. DISCUSS +206 216 structures evidence The computational efficiency of FREALIGN has allowed us to classify a relatively large dataset (1.1 million particles) into 15 classes (Figure 1—figure supplement 2) and obtain eight near-atomic-resolution structures from it. DISCUSS +63 72 particles experimental_method The classification, which followed an initial alignment of all particles to a single reference, required about 130,000 CPU hours or about five to six full days on a 1000-CPU cluster. DISCUSS +11 18 cryo-EM experimental_method Therefore, cryo-EM has the potential to become a standard tool for uncovering detailed dynamic pathways of complex macromolecular machines. DISCUSS diff --git a/annotation_CSV/PMC4918759.csv b/annotation_CSV/PMC4918759.csv new file mode 100644 index 0000000000000000000000000000000000000000..b4ba4d0b5b565ae9770b624917df2873c81ba401 --- /dev/null +++ b/annotation_CSV/PMC4918759.csv @@ -0,0 +1,804 @@ +anno_start anno_end anno_text entity_type sentence section +0 10 Structures evidence Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity TITLE +14 19 human species Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity TITLE +20 25 ADAR2 protein Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity TITLE +26 34 bound to protein_state Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity TITLE +35 40 dsRNA chemical Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity TITLE +0 5 ADARs protein_type ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function. ABSTRACT +7 41 adenosine deaminases acting on RNA protein_type ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function. ABSTRACT +47 62 editing enzymes protein_type ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function. ABSTRACT +76 85 adenosine residue_name ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function. ABSTRACT +87 88 A residue_name ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function. ABSTRACT +93 100 inosine residue_name ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function. ABSTRACT +102 103 I residue_name ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function. ABSTRACT +108 118 duplex RNA structure_element ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function. ABSTRACT +178 181 RNA chemical ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function. ABSTRACT +25 29 ADAR protein_type Our understanding of the ADAR reaction mechanism, origin of editing site selectivity and effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. ABSTRACT +60 72 editing site site Our understanding of the ADAR reaction mechanism, origin of editing site selectivity and effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. ABSTRACT +151 166 structural data evidence Our understanding of the ADAR reaction mechanism, origin of editing site selectivity and effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. ABSTRACT +184 189 ADARs protein_type Our understanding of the ADAR reaction mechanism, origin of editing site selectivity and effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. ABSTRACT +190 198 bound to protein_state Our understanding of the ADAR reaction mechanism, origin of editing site selectivity and effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. ABSTRACT +209 213 RNAs chemical Our understanding of the ADAR reaction mechanism, origin of editing site selectivity and effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. ABSTRACT +22 40 crystal structures evidence Here we describe four crystal structures of the deaminase domain of human ADAR2 bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. ABSTRACT +48 64 deaminase domain structure_element Here we describe four crystal structures of the deaminase domain of human ADAR2 bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. ABSTRACT +68 73 human species Here we describe four crystal structures of the deaminase domain of human ADAR2 bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. ABSTRACT +74 79 ADAR2 protein Here we describe four crystal structures of the deaminase domain of human ADAR2 bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. ABSTRACT +80 88 bound to protein_state Here we describe four crystal structures of the deaminase domain of human ADAR2 bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. ABSTRACT +89 101 RNA duplexes structure_element Here we describe four crystal structures of the deaminase domain of human ADAR2 bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. ABSTRACT +6 16 structures evidence These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis for ADAR deaminase domain’s dsRNA specificity, its base-flipping mechanism, and nearest neighbor preferences. ABSTRACT +32 60 structure-guided mutagenesis experimental_method These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis for ADAR deaminase domain’s dsRNA specificity, its base-flipping mechanism, and nearest neighbor preferences. ABSTRACT +65 93 RNA-modification experiments experimental_method These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis for ADAR deaminase domain’s dsRNA specificity, its base-flipping mechanism, and nearest neighbor preferences. ABSTRACT +117 121 ADAR protein_type These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis for ADAR deaminase domain’s dsRNA specificity, its base-flipping mechanism, and nearest neighbor preferences. ABSTRACT +122 138 deaminase domain structure_element These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis for ADAR deaminase domain’s dsRNA specificity, its base-flipping mechanism, and nearest neighbor preferences. ABSTRACT +141 146 dsRNA chemical These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis for ADAR deaminase domain’s dsRNA specificity, its base-flipping mechanism, and nearest neighbor preferences. ABSTRACT +16 21 ADAR2 protein In addition, an ADAR2-specific RNA-binding loop was identified near the enzyme active site rationalizing differences in selectivity observed between different ADARs. ABSTRACT +31 47 RNA-binding loop structure_element In addition, an ADAR2-specific RNA-binding loop was identified near the enzyme active site rationalizing differences in selectivity observed between different ADARs. ABSTRACT +79 90 active site site In addition, an ADAR2-specific RNA-binding loop was identified near the enzyme active site rationalizing differences in selectivity observed between different ADARs. ABSTRACT +159 164 ADARs protein_type In addition, an ADAR2-specific RNA-binding loop was identified near the enzyme active site rationalizing differences in selectivity observed between different ADARs. ABSTRACT +85 89 ADAR protein_type Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease. ABSTRACT +116 121 human species Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease. ABSTRACT +0 3 RNA chemical RNA editing reactions alter a transcript’s genomically encoded sequence by inserting, deleting or modifying nucleotides. INTRO +15 24 adenosine residue_name Deamination of adenosine (A), the most common form of RNA editing in humans, generates inosine (I) at the corresponding nucleotide position. INTRO +26 27 A residue_name Deamination of adenosine (A), the most common form of RNA editing in humans, generates inosine (I) at the corresponding nucleotide position. INTRO +54 57 RNA chemical Deamination of adenosine (A), the most common form of RNA editing in humans, generates inosine (I) at the corresponding nucleotide position. INTRO +69 75 humans species Deamination of adenosine (A), the most common form of RNA editing in humans, generates inosine (I) at the corresponding nucleotide position. INTRO +87 94 inosine residue_name Deamination of adenosine (A), the most common form of RNA editing in humans, generates inosine (I) at the corresponding nucleotide position. INTRO +96 97 I residue_name Deamination of adenosine (A), the most common form of RNA editing in humans, generates inosine (I) at the corresponding nucleotide position. INTRO +6 7 I residue_name Since I base pairs with cytidine (C), it functions like guanosine (G) in cellular processes such as splicing, translation and reverse transcription. INTRO +24 32 cytidine residue_name Since I base pairs with cytidine (C), it functions like guanosine (G) in cellular processes such as splicing, translation and reverse transcription. INTRO +34 35 C residue_name Since I base pairs with cytidine (C), it functions like guanosine (G) in cellular processes such as splicing, translation and reverse transcription. INTRO +56 65 guanosine residue_name Since I base pairs with cytidine (C), it functions like guanosine (G) in cellular processes such as splicing, translation and reverse transcription. INTRO +67 68 G residue_name Since I base pairs with cytidine (C), it functions like guanosine (G) in cellular processes such as splicing, translation and reverse transcription. INTRO +48 51 RNA chemical A to I editing has wide-ranging consequences on RNA function including altering miRNA recognition sites, redirecting splicing and changing the meaning of specific codons. INTRO +80 103 miRNA recognition sites site A to I editing has wide-ranging consequences on RNA function including altering miRNA recognition sites, redirecting splicing and changing the meaning of specific codons. INTRO +50 56 humans species Two different enzymes carry out A to I editing in humans; ADAR1 and ADAR2. INTRO +58 63 ADAR1 protein Two different enzymes carry out A to I editing in humans; ADAR1 and ADAR2. INTRO +68 73 ADAR2 protein Two different enzymes carry out A to I editing in humans; ADAR1 and ADAR2. INTRO +0 4 ADAR protein_type ADAR activity is required for nervous system function and altered editing has been linked to neurological disorders such as epilepsy and Prader Willi Syndrome. INTRO +30 35 ADAR1 protein In addition, mutations in the ADAR1 gene are known to cause the autoimmune disease Aicardi-Goutieres Syndrome (AGS) and the skin disorder Dyschromatosis Symmetrica Hereditaria (DSH). INTRO +81 85 mRNA chemical Hyper editing has been observed at certain sites in cancer cells, such as in the mRNA for AZIN1 (antizyme inhibitor 1). INTRO +90 95 AZIN1 protein Hyper editing has been observed at certain sites in cancer cells, such as in the mRNA for AZIN1 (antizyme inhibitor 1). INTRO +97 117 antizyme inhibitor 1 protein Hyper editing has been observed at certain sites in cancer cells, such as in the mRNA for AZIN1 (antizyme inhibitor 1). INTRO +122 150 glioma-associated oncogene 1 protein However, hypo editing also occurs in cancer-derived cell lines exemplified by reduced editing observed in the message for glioma-associated oncogene 1 (Gli1). INTRO +152 156 Gli1 protein However, hypo editing also occurs in cancer-derived cell lines exemplified by reduced editing observed in the message for glioma-associated oncogene 1 (Gli1). INTRO +4 8 ADAR protein_type The ADAR proteins have a modular structure with double stranded RNA binding domains (dsRBDs) and a C-terminal deaminase domain (see Fig. 1a for hADAR2 domains). INTRO +48 83 double stranded RNA binding domains structure_element The ADAR proteins have a modular structure with double stranded RNA binding domains (dsRBDs) and a C-terminal deaminase domain (see Fig. 1a for hADAR2 domains). INTRO +85 91 dsRBDs structure_element The ADAR proteins have a modular structure with double stranded RNA binding domains (dsRBDs) and a C-terminal deaminase domain (see Fig. 1a for hADAR2 domains). INTRO +110 126 deaminase domain structure_element The ADAR proteins have a modular structure with double stranded RNA binding domains (dsRBDs) and a C-terminal deaminase domain (see Fig. 1a for hADAR2 domains). INTRO +144 150 hADAR2 protein The ADAR proteins have a modular structure with double stranded RNA binding domains (dsRBDs) and a C-terminal deaminase domain (see Fig. 1a for hADAR2 domains). INTRO +0 5 ADARs protein_type ADARs efficiently deaminate specific adenosines in duplex RNA while leaving most adenosines unmodified. INTRO +37 47 adenosines residue_name ADARs efficiently deaminate specific adenosines in duplex RNA while leaving most adenosines unmodified. INTRO +51 61 duplex RNA structure_element ADARs efficiently deaminate specific adenosines in duplex RNA while leaving most adenosines unmodified. INTRO +81 91 adenosines residue_name ADARs efficiently deaminate specific adenosines in duplex RNA while leaving most adenosines unmodified. INTRO +17 26 adenosine residue_name The mechanism of adenosine deamination requires ADAR to flip the reactive base out of an RNA double helix to access its active site. INTRO +48 52 ADAR protein_type The mechanism of adenosine deamination requires ADAR to flip the reactive base out of an RNA double helix to access its active site. INTRO +89 105 RNA double helix chemical The mechanism of adenosine deamination requires ADAR to flip the reactive base out of an RNA double helix to access its active site. INTRO +120 131 active site site The mechanism of adenosine deamination requires ADAR to flip the reactive base out of an RNA double helix to access its active site. INTRO +48 58 duplex RNA structure_element How an enzyme could accomplish this task with a duplex RNA substrate is not known. INTRO +20 24 ADAR protein_type Furthermore, how an ADAR deaminase domain contributes to editing site selectivity is also unknown, since no structures of ADAR deaminase domain-RNA complexes have been reported. INTRO +25 41 deaminase domain structure_element Furthermore, how an ADAR deaminase domain contributes to editing site selectivity is also unknown, since no structures of ADAR deaminase domain-RNA complexes have been reported. INTRO +57 69 editing site site Furthermore, how an ADAR deaminase domain contributes to editing site selectivity is also unknown, since no structures of ADAR deaminase domain-RNA complexes have been reported. INTRO +108 118 structures evidence Furthermore, how an ADAR deaminase domain contributes to editing site selectivity is also unknown, since no structures of ADAR deaminase domain-RNA complexes have been reported. INTRO +122 147 ADAR deaminase domain-RNA complex_assembly Furthermore, how an ADAR deaminase domain contributes to editing site selectivity is also unknown, since no structures of ADAR deaminase domain-RNA complexes have been reported. INTRO +56 61 human species To address these knowledge gaps, we set out to trap the human ADAR2 deaminase domain (aa299–701, hADAR2d) bound to different duplex RNAs and solve structures for the resulting complexes using x-ray crystallography. INTRO +62 67 ADAR2 protein To address these knowledge gaps, we set out to trap the human ADAR2 deaminase domain (aa299–701, hADAR2d) bound to different duplex RNAs and solve structures for the resulting complexes using x-ray crystallography. INTRO +68 84 deaminase domain structure_element To address these knowledge gaps, we set out to trap the human ADAR2 deaminase domain (aa299–701, hADAR2d) bound to different duplex RNAs and solve structures for the resulting complexes using x-ray crystallography. INTRO +88 95 299–701 residue_range To address these knowledge gaps, we set out to trap the human ADAR2 deaminase domain (aa299–701, hADAR2d) bound to different duplex RNAs and solve structures for the resulting complexes using x-ray crystallography. INTRO +97 104 hADAR2d mutant To address these knowledge gaps, we set out to trap the human ADAR2 deaminase domain (aa299–701, hADAR2d) bound to different duplex RNAs and solve structures for the resulting complexes using x-ray crystallography. INTRO +106 114 bound to protein_state To address these knowledge gaps, we set out to trap the human ADAR2 deaminase domain (aa299–701, hADAR2d) bound to different duplex RNAs and solve structures for the resulting complexes using x-ray crystallography. INTRO +125 136 duplex RNAs structure_element To address these knowledge gaps, we set out to trap the human ADAR2 deaminase domain (aa299–701, hADAR2d) bound to different duplex RNAs and solve structures for the resulting complexes using x-ray crystallography. INTRO +147 157 structures evidence To address these knowledge gaps, we set out to trap the human ADAR2 deaminase domain (aa299–701, hADAR2d) bound to different duplex RNAs and solve structures for the resulting complexes using x-ray crystallography. INTRO +192 213 x-ray crystallography experimental_method To address these knowledge gaps, we set out to trap the human ADAR2 deaminase domain (aa299–701, hADAR2d) bound to different duplex RNAs and solve structures for the resulting complexes using x-ray crystallography. INTRO +44 47 RNA chemical We then evaluated the importance of protein-RNA contacts using structure-guided mutagenesis and RNA-modification experiments coupled with adenosine deamination kinetics. INTRO +63 91 structure-guided mutagenesis experimental_method We then evaluated the importance of protein-RNA contacts using structure-guided mutagenesis and RNA-modification experiments coupled with adenosine deamination kinetics. INTRO +96 124 RNA-modification experiments experimental_method We then evaluated the importance of protein-RNA contacts using structure-guided mutagenesis and RNA-modification experiments coupled with adenosine deamination kinetics. INTRO +138 168 adenosine deamination kinetics experimental_method We then evaluated the importance of protein-RNA contacts using structure-guided mutagenesis and RNA-modification experiments coupled with adenosine deamination kinetics. INTRO +13 20 flipped protein_state Trapping the flipped conformation RESULTS +4 8 ADAR protein_type The ADAR reaction involves the formation of a hydrated intermediate that loses ammonia to generate the inosine-containing product RNA (for reaction scheme see Fig. 1b). RESULTS +103 110 inosine residue_name The ADAR reaction involves the formation of a hydrated intermediate that loses ammonia to generate the inosine-containing product RNA (for reaction scheme see Fig. 1b). RESULTS +130 133 RNA chemical The ADAR reaction involves the formation of a hydrated intermediate that loses ammonia to generate the inosine-containing product RNA (for reaction scheme see Fig. 1b). RESULTS +46 61 8-azanebularine chemical The covalent hydrate of the nucleoside analog 8-azanebularine (N) mimics the proposed high-energy intermediate (for reaction scheme see Fig. 1b). RESULTS +63 64 N chemical The covalent hydrate of the nucleoside analog 8-azanebularine (N) mimics the proposed high-energy intermediate (for reaction scheme see Fig. 1b). RESULTS +13 20 hADAR2d mutant For trapping hADAR2d bound to RNA for crystallography, we incorporated 8-azanebularine into duplex RNAs shown recently to be excellent substrates for deamination by hADAR2d (for duplex sequence see Fig. 1c) (for characterization of protein–RNA complex see Supplementary Fig. 1). RESULTS +21 29 bound to protein_state For trapping hADAR2d bound to RNA for crystallography, we incorporated 8-azanebularine into duplex RNAs shown recently to be excellent substrates for deamination by hADAR2d (for duplex sequence see Fig. 1c) (for characterization of protein–RNA complex see Supplementary Fig. 1). RESULTS +30 33 RNA chemical For trapping hADAR2d bound to RNA for crystallography, we incorporated 8-azanebularine into duplex RNAs shown recently to be excellent substrates for deamination by hADAR2d (for duplex sequence see Fig. 1c) (for characterization of protein–RNA complex see Supplementary Fig. 1). RESULTS +38 53 crystallography experimental_method For trapping hADAR2d bound to RNA for crystallography, we incorporated 8-azanebularine into duplex RNAs shown recently to be excellent substrates for deamination by hADAR2d (for duplex sequence see Fig. 1c) (for characterization of protein–RNA complex see Supplementary Fig. 1). RESULTS +71 86 8-azanebularine chemical For trapping hADAR2d bound to RNA for crystallography, we incorporated 8-azanebularine into duplex RNAs shown recently to be excellent substrates for deamination by hADAR2d (for duplex sequence see Fig. 1c) (for characterization of protein–RNA complex see Supplementary Fig. 1). RESULTS +92 103 duplex RNAs structure_element For trapping hADAR2d bound to RNA for crystallography, we incorporated 8-azanebularine into duplex RNAs shown recently to be excellent substrates for deamination by hADAR2d (for duplex sequence see Fig. 1c) (for characterization of protein–RNA complex see Supplementary Fig. 1). RESULTS +165 172 hADAR2d mutant For trapping hADAR2d bound to RNA for crystallography, we incorporated 8-azanebularine into duplex RNAs shown recently to be excellent substrates for deamination by hADAR2d (for duplex sequence see Fig. 1c) (for characterization of protein–RNA complex see Supplementary Fig. 1). RESULTS +240 243 RNA chemical For trapping hADAR2d bound to RNA for crystallography, we incorporated 8-azanebularine into duplex RNAs shown recently to be excellent substrates for deamination by hADAR2d (for duplex sequence see Fig. 1c) (for characterization of protein–RNA complex see Supplementary Fig. 1). RESULTS +40 44 Bdf2 chemical In addition, for one of these duplexes (Bdf2), we positioned the 8-azanebularine opposite either uridine or cytidine to mimic an A-U pair or A-C mismatch at the editing site creating a total of three different RNA substrates for structural studies (Fig. 1c). RESULTS +65 80 8-azanebularine chemical In addition, for one of these duplexes (Bdf2), we positioned the 8-azanebularine opposite either uridine or cytidine to mimic an A-U pair or A-C mismatch at the editing site creating a total of three different RNA substrates for structural studies (Fig. 1c). RESULTS +97 104 uridine residue_name In addition, for one of these duplexes (Bdf2), we positioned the 8-azanebularine opposite either uridine or cytidine to mimic an A-U pair or A-C mismatch at the editing site creating a total of three different RNA substrates for structural studies (Fig. 1c). RESULTS +108 116 cytidine residue_name In addition, for one of these duplexes (Bdf2), we positioned the 8-azanebularine opposite either uridine or cytidine to mimic an A-U pair or A-C mismatch at the editing site creating a total of three different RNA substrates for structural studies (Fig. 1c). RESULTS +129 130 A residue_name In addition, for one of these duplexes (Bdf2), we positioned the 8-azanebularine opposite either uridine or cytidine to mimic an A-U pair or A-C mismatch at the editing site creating a total of three different RNA substrates for structural studies (Fig. 1c). RESULTS +131 132 U residue_name In addition, for one of these duplexes (Bdf2), we positioned the 8-azanebularine opposite either uridine or cytidine to mimic an A-U pair or A-C mismatch at the editing site creating a total of three different RNA substrates for structural studies (Fig. 1c). RESULTS +141 142 A residue_name In addition, for one of these duplexes (Bdf2), we positioned the 8-azanebularine opposite either uridine or cytidine to mimic an A-U pair or A-C mismatch at the editing site creating a total of three different RNA substrates for structural studies (Fig. 1c). RESULTS +143 144 C residue_name In addition, for one of these duplexes (Bdf2), we positioned the 8-azanebularine opposite either uridine or cytidine to mimic an A-U pair or A-C mismatch at the editing site creating a total of three different RNA substrates for structural studies (Fig. 1c). RESULTS +161 173 editing site site In addition, for one of these duplexes (Bdf2), we positioned the 8-azanebularine opposite either uridine or cytidine to mimic an A-U pair or A-C mismatch at the editing site creating a total of three different RNA substrates for structural studies (Fig. 1c). RESULTS +210 213 RNA chemical In addition, for one of these duplexes (Bdf2), we positioned the 8-azanebularine opposite either uridine or cytidine to mimic an A-U pair or A-C mismatch at the editing site creating a total of three different RNA substrates for structural studies (Fig. 1c). RESULTS +4 11 hADAR2d mutant The hADAR2d protein (without RNA bound) has been previously crystallized and structurally characterized revealing features of the active site including the presence of zinc. RESULTS +21 38 without RNA bound protein_state The hADAR2d protein (without RNA bound) has been previously crystallized and structurally characterized revealing features of the active site including the presence of zinc. RESULTS +60 72 crystallized experimental_method The hADAR2d protein (without RNA bound) has been previously crystallized and structurally characterized revealing features of the active site including the presence of zinc. RESULTS +130 141 active site site The hADAR2d protein (without RNA bound) has been previously crystallized and structurally characterized revealing features of the active site including the presence of zinc. RESULTS +168 172 zinc chemical The hADAR2d protein (without RNA bound) has been previously crystallized and structurally characterized revealing features of the active site including the presence of zinc. RESULTS +16 41 inositol hexakisphosphate chemical In addition, an inositol hexakisphosphate (IHP) molecule was found buried in the core of the protein hydrogen bonded to numerous conserved polar residues. RESULTS +43 46 IHP chemical In addition, an inositol hexakisphosphate (IHP) molecule was found buried in the core of the protein hydrogen bonded to numerous conserved polar residues. RESULTS +101 116 hydrogen bonded bond_interaction In addition, an inositol hexakisphosphate (IHP) molecule was found buried in the core of the protein hydrogen bonded to numerous conserved polar residues. RESULTS +4 19 crystallization experimental_method For crystallization of hADAR2d-RNA complexes, we used both the wild type (WT) deaminase domain and a mutant (E488Q) that has enhanced catalytic activity. RESULTS +23 34 hADAR2d-RNA complex_assembly For crystallization of hADAR2d-RNA complexes, we used both the wild type (WT) deaminase domain and a mutant (E488Q) that has enhanced catalytic activity. RESULTS +63 72 wild type protein_state For crystallization of hADAR2d-RNA complexes, we used both the wild type (WT) deaminase domain and a mutant (E488Q) that has enhanced catalytic activity. RESULTS +74 76 WT protein_state For crystallization of hADAR2d-RNA complexes, we used both the wild type (WT) deaminase domain and a mutant (E488Q) that has enhanced catalytic activity. RESULTS +78 94 deaminase domain structure_element For crystallization of hADAR2d-RNA complexes, we used both the wild type (WT) deaminase domain and a mutant (E488Q) that has enhanced catalytic activity. RESULTS +101 107 mutant protein_state For crystallization of hADAR2d-RNA complexes, we used both the wild type (WT) deaminase domain and a mutant (E488Q) that has enhanced catalytic activity. RESULTS +109 114 E488Q mutant For crystallization of hADAR2d-RNA complexes, we used both the wild type (WT) deaminase domain and a mutant (E488Q) that has enhanced catalytic activity. RESULTS +49 95 X-ray diffraction data collection and solution experimental_method A description of the crystallization conditions, X-ray diffraction data collection and solution of the structures can be found in Online Methods. RESULTS +103 113 structures evidence A description of the crystallization conditions, X-ray diffraction data collection and solution of the structures can be found in Online Methods. RESULTS +13 16 RNA chemical Four protein-RNA combinations generated diffracting crystals that resulted in high-resolution structures (hADAR2d WT–Bdf2-U, hADAR2d WT–Bdf2-C, hADAR2d E488Q–Bdf2-C, hADAR2d E488Q–Gli1) (Table 1). RESULTS +52 60 crystals evidence Four protein-RNA combinations generated diffracting crystals that resulted in high-resolution structures (hADAR2d WT–Bdf2-U, hADAR2d WT–Bdf2-C, hADAR2d E488Q–Bdf2-C, hADAR2d E488Q–Gli1) (Table 1). RESULTS +94 104 structures evidence Four protein-RNA combinations generated diffracting crystals that resulted in high-resolution structures (hADAR2d WT–Bdf2-U, hADAR2d WT–Bdf2-C, hADAR2d E488Q–Bdf2-C, hADAR2d E488Q–Gli1) (Table 1). RESULTS +106 123 hADAR2d WT–Bdf2-U complex_assembly Four protein-RNA combinations generated diffracting crystals that resulted in high-resolution structures (hADAR2d WT–Bdf2-U, hADAR2d WT–Bdf2-C, hADAR2d E488Q–Bdf2-C, hADAR2d E488Q–Gli1) (Table 1). RESULTS +125 142 hADAR2d WT–Bdf2-C complex_assembly Four protein-RNA combinations generated diffracting crystals that resulted in high-resolution structures (hADAR2d WT–Bdf2-U, hADAR2d WT–Bdf2-C, hADAR2d E488Q–Bdf2-C, hADAR2d E488Q–Gli1) (Table 1). RESULTS +144 164 hADAR2d E488Q–Bdf2-C complex_assembly Four protein-RNA combinations generated diffracting crystals that resulted in high-resolution structures (hADAR2d WT–Bdf2-U, hADAR2d WT–Bdf2-C, hADAR2d E488Q–Bdf2-C, hADAR2d E488Q–Gli1) (Table 1). RESULTS +166 184 hADAR2d E488Q–Gli1 complex_assembly Four protein-RNA combinations generated diffracting crystals that resulted in high-resolution structures (hADAR2d WT–Bdf2-U, hADAR2d WT–Bdf2-C, hADAR2d E488Q–Bdf2-C, hADAR2d E488Q–Gli1) (Table 1). RESULTS +50 53 RNA chemical In each of these complexes, the protein binds the RNA on one face of the duplex over ~ 20 bp using a positively charged surface near the zinc-containing active site (Fig. 2, Supplementary Fig. 2a). RESULTS +137 164 zinc-containing active site site In each of these complexes, the protein binds the RNA on one face of the duplex over ~ 20 bp using a positively charged surface near the zinc-containing active site (Fig. 2, Supplementary Fig. 2a). RESULTS +10 22 binding site site The large binding site (1493 Å2 RNA surface area and 1277 Å2 protein surface area buried) observed for hADAR2d is consistent with recent footprinting studies. RESULTS +103 110 hADAR2d mutant The large binding site (1493 Å2 RNA surface area and 1277 Å2 protein surface area buried) observed for hADAR2d is consistent with recent footprinting studies. RESULTS +137 157 footprinting studies experimental_method The large binding site (1493 Å2 RNA surface area and 1277 Å2 protein surface area buried) observed for hADAR2d is consistent with recent footprinting studies. RESULTS +20 23 RNA chemical Both strands of the RNA contact the protein with the majority of these interactions mediated through the phosphodiester-ribose backbone near the editing site (Fig. 2c, Supplementary Fig. 2 b–d). RESULTS +145 157 editing site site Both strands of the RNA contact the protein with the majority of these interactions mediated through the phosphodiester-ribose backbone near the editing site (Fig. 2c, Supplementary Fig. 2 b–d). RESULTS +4 14 structures evidence The structures show a large deviation from A-form RNA conformation at the editing site (Fig. 2, Fig. 3, Supplementary Video 1). RESULTS +43 49 A-form structure_element The structures show a large deviation from A-form RNA conformation at the editing site (Fig. 2, Fig. 3, Supplementary Video 1). RESULTS +50 53 RNA chemical The structures show a large deviation from A-form RNA conformation at the editing site (Fig. 2, Fig. 3, Supplementary Video 1). RESULTS +74 86 editing site site The structures show a large deviation from A-form RNA conformation at the editing site (Fig. 2, Fig. 3, Supplementary Video 1). RESULTS +4 19 8-azanebularine chemical The 8-azanebularine is flipped out of the helix and bound into the active site as its covalent hydrate where it interacts with several amino acids including V351, T375, K376, E396 and R455 (Fig. 3a, Supplementary Fig. 3a). RESULTS +23 34 flipped out protein_state The 8-azanebularine is flipped out of the helix and bound into the active site as its covalent hydrate where it interacts with several amino acids including V351, T375, K376, E396 and R455 (Fig. 3a, Supplementary Fig. 3a). RESULTS +42 47 helix structure_element The 8-azanebularine is flipped out of the helix and bound into the active site as its covalent hydrate where it interacts with several amino acids including V351, T375, K376, E396 and R455 (Fig. 3a, Supplementary Fig. 3a). RESULTS +52 62 bound into protein_state The 8-azanebularine is flipped out of the helix and bound into the active site as its covalent hydrate where it interacts with several amino acids including V351, T375, K376, E396 and R455 (Fig. 3a, Supplementary Fig. 3a). RESULTS +67 78 active site site The 8-azanebularine is flipped out of the helix and bound into the active site as its covalent hydrate where it interacts with several amino acids including V351, T375, K376, E396 and R455 (Fig. 3a, Supplementary Fig. 3a). RESULTS +157 161 V351 residue_name_number The 8-azanebularine is flipped out of the helix and bound into the active site as its covalent hydrate where it interacts with several amino acids including V351, T375, K376, E396 and R455 (Fig. 3a, Supplementary Fig. 3a). RESULTS +163 167 T375 residue_name_number The 8-azanebularine is flipped out of the helix and bound into the active site as its covalent hydrate where it interacts with several amino acids including V351, T375, K376, E396 and R455 (Fig. 3a, Supplementary Fig. 3a). RESULTS +169 173 K376 residue_name_number The 8-azanebularine is flipped out of the helix and bound into the active site as its covalent hydrate where it interacts with several amino acids including V351, T375, K376, E396 and R455 (Fig. 3a, Supplementary Fig. 3a). RESULTS +175 179 E396 residue_name_number The 8-azanebularine is flipped out of the helix and bound into the active site as its covalent hydrate where it interacts with several amino acids including V351, T375, K376, E396 and R455 (Fig. 3a, Supplementary Fig. 3a). RESULTS +184 188 R455 residue_name_number The 8-azanebularine is flipped out of the helix and bound into the active site as its covalent hydrate where it interacts with several amino acids including V351, T375, K376, E396 and R455 (Fig. 3a, Supplementary Fig. 3a). RESULTS +18 22 E396 residue_name_number The side chain of E396 H-bonds to purine N1 and O6. RESULTS +23 30 H-bonds bond_interaction The side chain of E396 H-bonds to purine N1 and O6. RESULTS +34 40 purine chemical The side chain of E396 H-bonds to purine N1 and O6. RESULTS +57 61 E396 residue_name_number This interaction was expected given the proposed role of E396 in mediating proton transfers to and from N1 of the substrate adenosine. RESULTS +124 133 adenosine residue_name This interaction was expected given the proposed role of E396 in mediating proton transfers to and from N1 of the substrate adenosine. RESULTS +19 34 8-azanebularine chemical The 2’-hydroxyl of 8-azanebularine H-bonds to the backbone carbonyl of T375 while the T375 side chain contacts its 3’-phosphodiester. RESULTS +35 42 H-bonds bond_interaction The 2’-hydroxyl of 8-azanebularine H-bonds to the backbone carbonyl of T375 while the T375 side chain contacts its 3’-phosphodiester. RESULTS +71 75 T375 residue_name_number The 2’-hydroxyl of 8-azanebularine H-bonds to the backbone carbonyl of T375 while the T375 side chain contacts its 3’-phosphodiester. RESULTS +86 90 T375 residue_name_number The 2’-hydroxyl of 8-azanebularine H-bonds to the backbone carbonyl of T375 while the T375 side chain contacts its 3’-phosphodiester. RESULTS +0 4 R455 residue_name_number R455 and K376 help position the flipped nucleotide in the active site by fastening the phosphate backbone flanking the editing site. RESULTS +9 13 K376 residue_name_number R455 and K376 help position the flipped nucleotide in the active site by fastening the phosphate backbone flanking the editing site. RESULTS +32 39 flipped protein_state R455 and K376 help position the flipped nucleotide in the active site by fastening the phosphate backbone flanking the editing site. RESULTS +40 50 nucleotide chemical R455 and K376 help position the flipped nucleotide in the active site by fastening the phosphate backbone flanking the editing site. RESULTS +58 69 active site site R455 and K376 help position the flipped nucleotide in the active site by fastening the phosphate backbone flanking the editing site. RESULTS +119 131 editing site site R455 and K376 help position the flipped nucleotide in the active site by fastening the phosphate backbone flanking the editing site. RESULTS +4 8 R455 residue_name_number The R455 side chain ion pairs with the 5’-phosphodiester of 8-azanebularine while the K376 side chain contacts its 3’-phosphodiester. RESULTS +20 29 ion pairs bond_interaction The R455 side chain ion pairs with the 5’-phosphodiester of 8-azanebularine while the K376 side chain contacts its 3’-phosphodiester. RESULTS +60 75 8-azanebularine chemical The R455 side chain ion pairs with the 5’-phosphodiester of 8-azanebularine while the K376 side chain contacts its 3’-phosphodiester. RESULTS +86 90 K376 residue_name_number The R455 side chain ion pairs with the 5’-phosphodiester of 8-azanebularine while the K376 side chain contacts its 3’-phosphodiester. RESULTS +26 30 V351 residue_name_number Lastly, the side chain of V351 provides a hydrophobic surface for interaction with the nucleobase of the edited nucleotide. RESULTS +42 61 hydrophobic surface site Lastly, the side chain of V351 provides a hydrophobic surface for interaction with the nucleobase of the edited nucleotide. RESULTS +105 111 edited protein_state Lastly, the side chain of V351 provides a hydrophobic surface for interaction with the nucleobase of the edited nucleotide. RESULTS +112 122 nucleotide chemical Lastly, the side chain of V351 provides a hydrophobic surface for interaction with the nucleobase of the edited nucleotide. RESULTS +0 3 RNA chemical RNA binding does not alter IHP binding or the H-bonding network linking IHP to the active site. RESULTS +27 30 IHP chemical RNA binding does not alter IHP binding or the H-bonding network linking IHP to the active site. RESULTS +46 63 H-bonding network site RNA binding does not alter IHP binding or the H-bonding network linking IHP to the active site. RESULTS +72 75 IHP chemical RNA binding does not alter IHP binding or the H-bonding network linking IHP to the active site. RESULTS +83 94 active site site RNA binding does not alter IHP binding or the H-bonding network linking IHP to the active site. RESULTS +0 5 ADARs protein_type ADARs use a unique mechanism to modify duplex RNA RESULTS +39 49 duplex RNA structure_element ADARs use a unique mechanism to modify duplex RNA RESULTS +4 9 ADAR2 protein The ADAR2 base-flipping loop, bearing residue 488, approaches the RNA duplex from the minor groove side at the editing site. RESULTS +10 28 base-flipping loop structure_element The ADAR2 base-flipping loop, bearing residue 488, approaches the RNA duplex from the minor groove side at the editing site. RESULTS +46 49 488 residue_number The ADAR2 base-flipping loop, bearing residue 488, approaches the RNA duplex from the minor groove side at the editing site. RESULTS +66 76 RNA duplex structure_element The ADAR2 base-flipping loop, bearing residue 488, approaches the RNA duplex from the minor groove side at the editing site. RESULTS +86 98 minor groove site The ADAR2 base-flipping loop, bearing residue 488, approaches the RNA duplex from the minor groove side at the editing site. RESULTS +111 123 editing site site The ADAR2 base-flipping loop, bearing residue 488, approaches the RNA duplex from the minor groove side at the editing site. RESULTS +98 109 flipped out protein_state The side chain of this amino acid penetrates the helix where it occupies the space vacated by the flipped out base and H-bonds to the complementary strand orphaned base and to the 2’ hydroxyl of the nucleotide immediately 5’ to the editing site (Figs. 3b, 3c). RESULTS +110 114 base chemical The side chain of this amino acid penetrates the helix where it occupies the space vacated by the flipped out base and H-bonds to the complementary strand orphaned base and to the 2’ hydroxyl of the nucleotide immediately 5’ to the editing site (Figs. 3b, 3c). RESULTS +119 126 H-bonds bond_interaction The side chain of this amino acid penetrates the helix where it occupies the space vacated by the flipped out base and H-bonds to the complementary strand orphaned base and to the 2’ hydroxyl of the nucleotide immediately 5’ to the editing site (Figs. 3b, 3c). RESULTS +155 163 orphaned protein_state The side chain of this amino acid penetrates the helix where it occupies the space vacated by the flipped out base and H-bonds to the complementary strand orphaned base and to the 2’ hydroxyl of the nucleotide immediately 5’ to the editing site (Figs. 3b, 3c). RESULTS +164 168 base chemical The side chain of this amino acid penetrates the helix where it occupies the space vacated by the flipped out base and H-bonds to the complementary strand orphaned base and to the 2’ hydroxyl of the nucleotide immediately 5’ to the editing site (Figs. 3b, 3c). RESULTS +232 244 editing site site The side chain of this amino acid penetrates the helix where it occupies the space vacated by the flipped out base and H-bonds to the complementary strand orphaned base and to the 2’ hydroxyl of the nucleotide immediately 5’ to the editing site (Figs. 3b, 3c). RESULTS +12 22 structures evidence In the four structures reported here, three different combinations of helix-penetrating residue and orphan base are observed (i.e. E488 + U, E488 + C and Q488 + C) and all three combinations show the same side chain and base positions (Figs. 3b, 3c, Supplementary Fig. 4a for overlay of all three). RESULTS +100 106 orphan protein_state In the four structures reported here, three different combinations of helix-penetrating residue and orphan base are observed (i.e. E488 + U, E488 + C and Q488 + C) and all three combinations show the same side chain and base positions (Figs. 3b, 3c, Supplementary Fig. 4a for overlay of all three). RESULTS +107 111 base chemical In the four structures reported here, three different combinations of helix-penetrating residue and orphan base are observed (i.e. E488 + U, E488 + C and Q488 + C) and all three combinations show the same side chain and base positions (Figs. 3b, 3c, Supplementary Fig. 4a for overlay of all three). RESULTS +131 135 E488 residue_name_number In the four structures reported here, three different combinations of helix-penetrating residue and orphan base are observed (i.e. E488 + U, E488 + C and Q488 + C) and all three combinations show the same side chain and base positions (Figs. 3b, 3c, Supplementary Fig. 4a for overlay of all three). RESULTS +138 139 U residue_name In the four structures reported here, three different combinations of helix-penetrating residue and orphan base are observed (i.e. E488 + U, E488 + C and Q488 + C) and all three combinations show the same side chain and base positions (Figs. 3b, 3c, Supplementary Fig. 4a for overlay of all three). RESULTS +141 145 E488 residue_name_number In the four structures reported here, three different combinations of helix-penetrating residue and orphan base are observed (i.e. E488 + U, E488 + C and Q488 + C) and all three combinations show the same side chain and base positions (Figs. 3b, 3c, Supplementary Fig. 4a for overlay of all three). RESULTS +148 149 C residue_name In the four structures reported here, three different combinations of helix-penetrating residue and orphan base are observed (i.e. E488 + U, E488 + C and Q488 + C) and all three combinations show the same side chain and base positions (Figs. 3b, 3c, Supplementary Fig. 4a for overlay of all three). RESULTS +154 158 Q488 residue_name_number In the four structures reported here, three different combinations of helix-penetrating residue and orphan base are observed (i.e. E488 + U, E488 + C and Q488 + C) and all three combinations show the same side chain and base positions (Figs. 3b, 3c, Supplementary Fig. 4a for overlay of all three). RESULTS +161 162 C residue_name In the four structures reported here, three different combinations of helix-penetrating residue and orphan base are observed (i.e. E488 + U, E488 + C and Q488 + C) and all three combinations show the same side chain and base positions (Figs. 3b, 3c, Supplementary Fig. 4a for overlay of all three). RESULTS +276 283 overlay experimental_method In the four structures reported here, three different combinations of helix-penetrating residue and orphan base are observed (i.e. E488 + U, E488 + C and Q488 + C) and all three combinations show the same side chain and base positions (Figs. 3b, 3c, Supplementary Fig. 4a for overlay of all three). RESULTS +21 33 complex with protein_state For instance, in the complex with hADAR2d E488Q and the Bdf2-C duplex, the protein recognizes an orphaned C by donating H-bonds from Nε2 to cytosine N3 and from its backbone NH to cytosine O2 (Fig. 3b). RESULTS +34 41 hADAR2d mutant For instance, in the complex with hADAR2d E488Q and the Bdf2-C duplex, the protein recognizes an orphaned C by donating H-bonds from Nε2 to cytosine N3 and from its backbone NH to cytosine O2 (Fig. 3b). RESULTS +42 47 E488Q mutant For instance, in the complex with hADAR2d E488Q and the Bdf2-C duplex, the protein recognizes an orphaned C by donating H-bonds from Nε2 to cytosine N3 and from its backbone NH to cytosine O2 (Fig. 3b). RESULTS +56 69 Bdf2-C duplex chemical For instance, in the complex with hADAR2d E488Q and the Bdf2-C duplex, the protein recognizes an orphaned C by donating H-bonds from Nε2 to cytosine N3 and from its backbone NH to cytosine O2 (Fig. 3b). RESULTS +97 105 orphaned protein_state For instance, in the complex with hADAR2d E488Q and the Bdf2-C duplex, the protein recognizes an orphaned C by donating H-bonds from Nε2 to cytosine N3 and from its backbone NH to cytosine O2 (Fig. 3b). RESULTS +106 107 C residue_name For instance, in the complex with hADAR2d E488Q and the Bdf2-C duplex, the protein recognizes an orphaned C by donating H-bonds from Nε2 to cytosine N3 and from its backbone NH to cytosine O2 (Fig. 3b). RESULTS +120 127 H-bonds bond_interaction For instance, in the complex with hADAR2d E488Q and the Bdf2-C duplex, the protein recognizes an orphaned C by donating H-bonds from Nε2 to cytosine N3 and from its backbone NH to cytosine O2 (Fig. 3b). RESULTS +140 148 cytosine residue_name For instance, in the complex with hADAR2d E488Q and the Bdf2-C duplex, the protein recognizes an orphaned C by donating H-bonds from Nε2 to cytosine N3 and from its backbone NH to cytosine O2 (Fig. 3b). RESULTS +180 188 cytosine residue_name For instance, in the complex with hADAR2d E488Q and the Bdf2-C duplex, the protein recognizes an orphaned C by donating H-bonds from Nε2 to cytosine N3 and from its backbone NH to cytosine O2 (Fig. 3b). RESULTS +7 19 complex with protein_state In the complex with hADAR2d WT and the Bdf2-U duplex, a similar interaction is observed with the E488 backbone NH hydrogen bonded to the uracil O2 and the E488 side chain H-bonded to the uracil N3H (Fig. 3c). RESULTS +20 27 hADAR2d mutant In the complex with hADAR2d WT and the Bdf2-U duplex, a similar interaction is observed with the E488 backbone NH hydrogen bonded to the uracil O2 and the E488 side chain H-bonded to the uracil N3H (Fig. 3c). RESULTS +28 30 WT protein_state In the complex with hADAR2d WT and the Bdf2-U duplex, a similar interaction is observed with the E488 backbone NH hydrogen bonded to the uracil O2 and the E488 side chain H-bonded to the uracil N3H (Fig. 3c). RESULTS +39 52 Bdf2-U duplex chemical In the complex with hADAR2d WT and the Bdf2-U duplex, a similar interaction is observed with the E488 backbone NH hydrogen bonded to the uracil O2 and the E488 side chain H-bonded to the uracil N3H (Fig. 3c). RESULTS +97 101 E488 residue_name_number In the complex with hADAR2d WT and the Bdf2-U duplex, a similar interaction is observed with the E488 backbone NH hydrogen bonded to the uracil O2 and the E488 side chain H-bonded to the uracil N3H (Fig. 3c). RESULTS +114 129 hydrogen bonded bond_interaction In the complex with hADAR2d WT and the Bdf2-U duplex, a similar interaction is observed with the E488 backbone NH hydrogen bonded to the uracil O2 and the E488 side chain H-bonded to the uracil N3H (Fig. 3c). RESULTS +137 143 uracil residue_name In the complex with hADAR2d WT and the Bdf2-U duplex, a similar interaction is observed with the E488 backbone NH hydrogen bonded to the uracil O2 and the E488 side chain H-bonded to the uracil N3H (Fig. 3c). RESULTS +155 159 E488 residue_name_number In the complex with hADAR2d WT and the Bdf2-U duplex, a similar interaction is observed with the E488 backbone NH hydrogen bonded to the uracil O2 and the E488 side chain H-bonded to the uracil N3H (Fig. 3c). RESULTS +171 179 H-bonded bond_interaction In the complex with hADAR2d WT and the Bdf2-U duplex, a similar interaction is observed with the E488 backbone NH hydrogen bonded to the uracil O2 and the E488 side chain H-bonded to the uracil N3H (Fig. 3c). RESULTS +187 193 uracil residue_name In the complex with hADAR2d WT and the Bdf2-U duplex, a similar interaction is observed with the E488 backbone NH hydrogen bonded to the uracil O2 and the E488 side chain H-bonded to the uracil N3H (Fig. 3c). RESULTS +19 24 E488Q mutant Interestingly, the E488Q mutant was discovered in a screen for highly active ADAR2 mutants and this residue was suggested to be involved in base flipping given its effect on editing substrates with a fluorescent nucleobase at the editing site. RESULTS +25 31 mutant protein_state Interestingly, the E488Q mutant was discovered in a screen for highly active ADAR2 mutants and this residue was suggested to be involved in base flipping given its effect on editing substrates with a fluorescent nucleobase at the editing site. RESULTS +63 76 highly active protein_state Interestingly, the E488Q mutant was discovered in a screen for highly active ADAR2 mutants and this residue was suggested to be involved in base flipping given its effect on editing substrates with a fluorescent nucleobase at the editing site. RESULTS +77 82 ADAR2 protein Interestingly, the E488Q mutant was discovered in a screen for highly active ADAR2 mutants and this residue was suggested to be involved in base flipping given its effect on editing substrates with a fluorescent nucleobase at the editing site. RESULTS +83 90 mutants protein_state Interestingly, the E488Q mutant was discovered in a screen for highly active ADAR2 mutants and this residue was suggested to be involved in base flipping given its effect on editing substrates with a fluorescent nucleobase at the editing site. RESULTS +230 242 editing site site Interestingly, the E488Q mutant was discovered in a screen for highly active ADAR2 mutants and this residue was suggested to be involved in base flipping given its effect on editing substrates with a fluorescent nucleobase at the editing site. RESULTS +0 5 ADARs protein_type ADARs react preferentially with adenosines in A•C mismatches and A-U pairs over A•A and A•G mismatches. RESULTS +32 42 adenosines residue_name ADARs react preferentially with adenosines in A•C mismatches and A-U pairs over A•A and A•G mismatches. RESULTS +46 49 A•C structure_element ADARs react preferentially with adenosines in A•C mismatches and A-U pairs over A•A and A•G mismatches. RESULTS +65 74 A-U pairs structure_element ADARs react preferentially with adenosines in A•C mismatches and A-U pairs over A•A and A•G mismatches. RESULTS +80 83 A•A structure_element ADARs react preferentially with adenosines in A•C mismatches and A-U pairs over A•A and A•G mismatches. RESULTS +88 91 A•G structure_element ADARs react preferentially with adenosines in A•C mismatches and A-U pairs over A•A and A•G mismatches. RESULTS +2 8 purine chemical A purine at the orphan base position (in its anti conformation) would clash with the 488 residue explaining the preference for pyrimidines here. RESULTS +16 22 orphan protein_state A purine at the orphan base position (in its anti conformation) would clash with the 488 residue explaining the preference for pyrimidines here. RESULTS +23 27 base chemical A purine at the orphan base position (in its anti conformation) would clash with the 488 residue explaining the preference for pyrimidines here. RESULTS +85 88 488 residue_number A purine at the orphan base position (in its anti conformation) would clash with the 488 residue explaining the preference for pyrimidines here. RESULTS +127 138 pyrimidines chemical A purine at the orphan base position (in its anti conformation) would clash with the 488 residue explaining the preference for pyrimidines here. RESULTS +23 26 488 residue_number The interaction of the 488 residue with the orphaned base is reminiscent of an interaction observed for Hha I DNA methyltransfersase (MTase), a duplex DNA modifying enzyme that also uses a base flipping mechanism to access 2’-deoxycytidine (dC) for methylation. RESULTS +44 52 orphaned protein_state The interaction of the 488 residue with the orphaned base is reminiscent of an interaction observed for Hha I DNA methyltransfersase (MTase), a duplex DNA modifying enzyme that also uses a base flipping mechanism to access 2’-deoxycytidine (dC) for methylation. RESULTS +53 57 base chemical The interaction of the 488 residue with the orphaned base is reminiscent of an interaction observed for Hha I DNA methyltransfersase (MTase), a duplex DNA modifying enzyme that also uses a base flipping mechanism to access 2’-deoxycytidine (dC) for methylation. RESULTS +104 132 Hha I DNA methyltransfersase protein_type The interaction of the 488 residue with the orphaned base is reminiscent of an interaction observed for Hha I DNA methyltransfersase (MTase), a duplex DNA modifying enzyme that also uses a base flipping mechanism to access 2’-deoxycytidine (dC) for methylation. RESULTS +134 139 MTase protein_type The interaction of the 488 residue with the orphaned base is reminiscent of an interaction observed for Hha I DNA methyltransfersase (MTase), a duplex DNA modifying enzyme that also uses a base flipping mechanism to access 2’-deoxycytidine (dC) for methylation. RESULTS +144 154 duplex DNA structure_element The interaction of the 488 residue with the orphaned base is reminiscent of an interaction observed for Hha I DNA methyltransfersase (MTase), a duplex DNA modifying enzyme that also uses a base flipping mechanism to access 2’-deoxycytidine (dC) for methylation. RESULTS +223 239 2’-deoxycytidine residue_name The interaction of the 488 residue with the orphaned base is reminiscent of an interaction observed for Hha I DNA methyltransfersase (MTase), a duplex DNA modifying enzyme that also uses a base flipping mechanism to access 2’-deoxycytidine (dC) for methylation. RESULTS +241 243 dC residue_name The interaction of the 488 residue with the orphaned base is reminiscent of an interaction observed for Hha I DNA methyltransfersase (MTase), a duplex DNA modifying enzyme that also uses a base flipping mechanism to access 2’-deoxycytidine (dC) for methylation. RESULTS +17 21 Q237 residue_name_number For that enzyme, Q237 H-bonds to an orphaned dG while it fills the void left by the flipped out dC (Supplementary Fig. 4b). RESULTS +22 29 H-bonds bond_interaction For that enzyme, Q237 H-bonds to an orphaned dG while it fills the void left by the flipped out dC (Supplementary Fig. 4b). RESULTS +36 44 orphaned protein_state For that enzyme, Q237 H-bonds to an orphaned dG while it fills the void left by the flipped out dC (Supplementary Fig. 4b). RESULTS +45 47 dG residue_name For that enzyme, Q237 H-bonds to an orphaned dG while it fills the void left by the flipped out dC (Supplementary Fig. 4b). RESULTS +84 95 flipped out protein_state For that enzyme, Q237 H-bonds to an orphaned dG while it fills the void left by the flipped out dC (Supplementary Fig. 4b). RESULTS +96 98 dC residue_name For that enzyme, Q237 H-bonds to an orphaned dG while it fills the void left by the flipped out dC (Supplementary Fig. 4b). RESULTS +17 24 glycine residue_name In addition, two glycine residues flank Q237 allowing the loop to adopt the conformation necessary for penetration into the helix. RESULTS +40 44 Q237 residue_name_number In addition, two glycine residues flank Q237 allowing the loop to adopt the conformation necessary for penetration into the helix. RESULTS +58 62 loop structure_element In addition, two glycine residues flank Q237 allowing the loop to adopt the conformation necessary for penetration into the helix. RESULTS +124 129 helix structure_element In addition, two glycine residues flank Q237 allowing the loop to adopt the conformation necessary for penetration into the helix. RESULTS +4 17 flipping loop structure_element The flipping loop in ADAR2 (i.e. aa487–489) also has the helix-penetrating residue flanked by glycines. RESULTS +21 26 ADAR2 protein The flipping loop in ADAR2 (i.e. aa487–489) also has the helix-penetrating residue flanked by glycines. RESULTS +35 42 487–489 residue_range The flipping loop in ADAR2 (i.e. aa487–489) also has the helix-penetrating residue flanked by glycines. RESULTS +94 102 glycines residue_name The flipping loop in ADAR2 (i.e. aa487–489) also has the helix-penetrating residue flanked by glycines. RESULTS +32 41 DNA MTase protein_type However, unlike the case of the DNA MTase that approaches the DNA from the major groove, the ADAR2 loop approaches the duplex from the minor groove side. RESULTS +62 65 DNA chemical However, unlike the case of the DNA MTase that approaches the DNA from the major groove, the ADAR2 loop approaches the duplex from the minor groove side. RESULTS +75 87 major groove site However, unlike the case of the DNA MTase that approaches the DNA from the major groove, the ADAR2 loop approaches the duplex from the minor groove side. RESULTS +93 98 ADAR2 protein However, unlike the case of the DNA MTase that approaches the DNA from the major groove, the ADAR2 loop approaches the duplex from the minor groove side. RESULTS +99 103 loop structure_element However, unlike the case of the DNA MTase that approaches the DNA from the major groove, the ADAR2 loop approaches the duplex from the minor groove side. RESULTS +119 125 duplex structure_element However, unlike the case of the DNA MTase that approaches the DNA from the major groove, the ADAR2 loop approaches the duplex from the minor groove side. RESULTS +135 147 minor groove site However, unlike the case of the DNA MTase that approaches the DNA from the major groove, the ADAR2 loop approaches the duplex from the minor groove side. RESULTS +52 73 intercalating residue site Such an approach requires deeper penetration of the intercalating residue to access the H-bonding sites on the orphaned base, necessitating an additional conformational change in the RNA duplex. RESULTS +88 103 H-bonding sites site Such an approach requires deeper penetration of the intercalating residue to access the H-bonding sites on the orphaned base, necessitating an additional conformational change in the RNA duplex. RESULTS +111 119 orphaned protein_state Such an approach requires deeper penetration of the intercalating residue to access the H-bonding sites on the orphaned base, necessitating an additional conformational change in the RNA duplex. RESULTS +120 124 base chemical Such an approach requires deeper penetration of the intercalating residue to access the H-bonding sites on the orphaned base, necessitating an additional conformational change in the RNA duplex. RESULTS +183 193 RNA duplex structure_element Such an approach requires deeper penetration of the intercalating residue to access the H-bonding sites on the orphaned base, necessitating an additional conformational change in the RNA duplex. RESULTS +70 82 editing site site This change includes shifting of the base pairs immediately 5’ to the editing site toward the helical axis and a widening of the major groove opposite the editing site (Figs. 4a, 4b, Supplementary Video 1). RESULTS +129 141 major groove site This change includes shifting of the base pairs immediately 5’ to the editing site toward the helical axis and a widening of the major groove opposite the editing site (Figs. 4a, 4b, Supplementary Video 1). RESULTS +155 167 editing site site This change includes shifting of the base pairs immediately 5’ to the editing site toward the helical axis and a widening of the major groove opposite the editing site (Figs. 4a, 4b, Supplementary Video 1). RESULTS +19 36 hADAR2d WT–Bdf2-U complex_assembly "In the case of the hADAR2d WT–Bdf2-U RNA, this shift is accompanied by a shearing of the U11-A13' base pair with U11 shifted further in the direction of the major groove creating an unusual U-A ""wobble"" interaction with adenine N6 and N1 within H-bonding distance to uracil N3H and O2, respectively (Fig. 4c, Supplementary Fig. 3b)." RESULTS +37 40 RNA chemical "In the case of the hADAR2d WT–Bdf2-U RNA, this shift is accompanied by a shearing of the U11-A13' base pair with U11 shifted further in the direction of the major groove creating an unusual U-A ""wobble"" interaction with adenine N6 and N1 within H-bonding distance to uracil N3H and O2, respectively (Fig. 4c, Supplementary Fig. 3b)." RESULTS +89 92 U11 residue_name_number "In the case of the hADAR2d WT–Bdf2-U RNA, this shift is accompanied by a shearing of the U11-A13' base pair with U11 shifted further in the direction of the major groove creating an unusual U-A ""wobble"" interaction with adenine N6 and N1 within H-bonding distance to uracil N3H and O2, respectively (Fig. 4c, Supplementary Fig. 3b)." RESULTS +93 96 A13 residue_name_number "In the case of the hADAR2d WT–Bdf2-U RNA, this shift is accompanied by a shearing of the U11-A13' base pair with U11 shifted further in the direction of the major groove creating an unusual U-A ""wobble"" interaction with adenine N6 and N1 within H-bonding distance to uracil N3H and O2, respectively (Fig. 4c, Supplementary Fig. 3b)." RESULTS +113 116 U11 residue_name_number "In the case of the hADAR2d WT–Bdf2-U RNA, this shift is accompanied by a shearing of the U11-A13' base pair with U11 shifted further in the direction of the major groove creating an unusual U-A ""wobble"" interaction with adenine N6 and N1 within H-bonding distance to uracil N3H and O2, respectively (Fig. 4c, Supplementary Fig. 3b)." RESULTS +157 169 major groove site "In the case of the hADAR2d WT–Bdf2-U RNA, this shift is accompanied by a shearing of the U11-A13' base pair with U11 shifted further in the direction of the major groove creating an unusual U-A ""wobble"" interaction with adenine N6 and N1 within H-bonding distance to uracil N3H and O2, respectively (Fig. 4c, Supplementary Fig. 3b)." RESULTS +190 202 "U-A ""wobble""" structure_element "In the case of the hADAR2d WT–Bdf2-U RNA, this shift is accompanied by a shearing of the U11-A13' base pair with U11 shifted further in the direction of the major groove creating an unusual U-A ""wobble"" interaction with adenine N6 and N1 within H-bonding distance to uracil N3H and O2, respectively (Fig. 4c, Supplementary Fig. 3b)." RESULTS +220 227 adenine residue_name "In the case of the hADAR2d WT–Bdf2-U RNA, this shift is accompanied by a shearing of the U11-A13' base pair with U11 shifted further in the direction of the major groove creating an unusual U-A ""wobble"" interaction with adenine N6 and N1 within H-bonding distance to uracil N3H and O2, respectively (Fig. 4c, Supplementary Fig. 3b)." RESULTS +245 254 H-bonding bond_interaction "In the case of the hADAR2d WT–Bdf2-U RNA, this shift is accompanied by a shearing of the U11-A13' base pair with U11 shifted further in the direction of the major groove creating an unusual U-A ""wobble"" interaction with adenine N6 and N1 within H-bonding distance to uracil N3H and O2, respectively (Fig. 4c, Supplementary Fig. 3b)." RESULTS +267 273 uracil residue_name "In the case of the hADAR2d WT–Bdf2-U RNA, this shift is accompanied by a shearing of the U11-A13' base pair with U11 shifted further in the direction of the major groove creating an unusual U-A ""wobble"" interaction with adenine N6 and N1 within H-bonding distance to uracil N3H and O2, respectively (Fig. 4c, Supplementary Fig. 3b)." RESULTS +92 99 adenine residue_name This type of wobble pair has been observed before and requires either the imino tautomer of adenine or the enol tautomer of uracil. RESULTS +124 130 uracil residue_name This type of wobble pair has been observed before and requires either the imino tautomer of adenine or the enol tautomer of uracil. RESULTS +4 8 ADAR protein_type The ADAR-induced distortion in RNA conformation results in a kink in the RNA strand opposite the editing site (Fig. 4b). RESULTS +31 34 RNA chemical The ADAR-induced distortion in RNA conformation results in a kink in the RNA strand opposite the editing site (Fig. 4b). RESULTS +61 65 kink structure_element The ADAR-induced distortion in RNA conformation results in a kink in the RNA strand opposite the editing site (Fig. 4b). RESULTS +73 76 RNA chemical The ADAR-induced distortion in RNA conformation results in a kink in the RNA strand opposite the editing site (Fig. 4b). RESULTS +97 109 editing site site The ADAR-induced distortion in RNA conformation results in a kink in the RNA strand opposite the editing site (Fig. 4b). RESULTS +5 9 kink structure_element This kink is stabilized by interactions of the side chains of R510 and S495 with phosphodiesters in the RNA backbone of the unedited strand (Fig. 4a). RESULTS +62 66 R510 residue_name_number This kink is stabilized by interactions of the side chains of R510 and S495 with phosphodiesters in the RNA backbone of the unedited strand (Fig. 4a). RESULTS +71 75 S495 residue_name_number This kink is stabilized by interactions of the side chains of R510 and S495 with phosphodiesters in the RNA backbone of the unedited strand (Fig. 4a). RESULTS +104 107 RNA chemical This kink is stabilized by interactions of the side chains of R510 and S495 with phosphodiesters in the RNA backbone of the unedited strand (Fig. 4a). RESULTS +15 20 ADAR2 protein Interestingly, ADAR2’s flipping loop approach from the minor groove side is like that seen with certain DNA repair glycosylases (e.g. UDG, HOGG1, and AAG) that also project intercalating residues from loops bound in the minor groove (Supplementary Fig. 5a). RESULTS +23 36 flipping loop structure_element Interestingly, ADAR2’s flipping loop approach from the minor groove side is like that seen with certain DNA repair glycosylases (e.g. UDG, HOGG1, and AAG) that also project intercalating residues from loops bound in the minor groove (Supplementary Fig. 5a). RESULTS +55 67 minor groove site Interestingly, ADAR2’s flipping loop approach from the minor groove side is like that seen with certain DNA repair glycosylases (e.g. UDG, HOGG1, and AAG) that also project intercalating residues from loops bound in the minor groove (Supplementary Fig. 5a). RESULTS +104 127 DNA repair glycosylases protein_type Interestingly, ADAR2’s flipping loop approach from the minor groove side is like that seen with certain DNA repair glycosylases (e.g. UDG, HOGG1, and AAG) that also project intercalating residues from loops bound in the minor groove (Supplementary Fig. 5a). RESULTS +134 137 UDG protein Interestingly, ADAR2’s flipping loop approach from the minor groove side is like that seen with certain DNA repair glycosylases (e.g. UDG, HOGG1, and AAG) that also project intercalating residues from loops bound in the minor groove (Supplementary Fig. 5a). RESULTS +139 144 HOGG1 protein Interestingly, ADAR2’s flipping loop approach from the minor groove side is like that seen with certain DNA repair glycosylases (e.g. UDG, HOGG1, and AAG) that also project intercalating residues from loops bound in the minor groove (Supplementary Fig. 5a). RESULTS +150 153 AAG protein Interestingly, ADAR2’s flipping loop approach from the minor groove side is like that seen with certain DNA repair glycosylases (e.g. UDG, HOGG1, and AAG) that also project intercalating residues from loops bound in the minor groove (Supplementary Fig. 5a). RESULTS +201 206 loops structure_element Interestingly, ADAR2’s flipping loop approach from the minor groove side is like that seen with certain DNA repair glycosylases (e.g. UDG, HOGG1, and AAG) that also project intercalating residues from loops bound in the minor groove (Supplementary Fig. 5a). RESULTS +207 215 bound in protein_state Interestingly, ADAR2’s flipping loop approach from the minor groove side is like that seen with certain DNA repair glycosylases (e.g. UDG, HOGG1, and AAG) that also project intercalating residues from loops bound in the minor groove (Supplementary Fig. 5a). RESULTS +220 232 minor groove site Interestingly, ADAR2’s flipping loop approach from the minor groove side is like that seen with certain DNA repair glycosylases (e.g. UDG, HOGG1, and AAG) that also project intercalating residues from loops bound in the minor groove (Supplementary Fig. 5a). RESULTS +42 52 DNA duplex chemical However, these enzymes typically bend the DNA duplex at the site of modification to allow for penetration of intercalating residues and damage recognition. RESULTS +6 13 hADAR2d mutant While hADAR2d clearly alters the duplex conformation to gain access to the modification site from the minor groove, it does not bend the RNA duplex (Figs. 2a, 2b, 4b). RESULTS +102 114 minor groove site While hADAR2d clearly alters the duplex conformation to gain access to the modification site from the minor groove, it does not bend the RNA duplex (Figs. 2a, 2b, 4b). RESULTS +137 147 RNA duplex structure_element While hADAR2d clearly alters the duplex conformation to gain access to the modification site from the minor groove, it does not bend the RNA duplex (Figs. 2a, 2b, 4b). RESULTS +13 18 ADARs protein_type Furthermore, ADARs do not modify duplex DNA. RESULTS +33 43 duplex DNA structure_element Furthermore, ADARs do not modify duplex DNA. RESULTS +4 7 DNA chemical The DNA B-form helix has groove widths and depths that would prevent productive interactions with ADAR. RESULTS +8 20 B-form helix structure_element The DNA B-form helix has groove widths and depths that would prevent productive interactions with ADAR. RESULTS +98 102 ADAR protein_type The DNA B-form helix has groove widths and depths that would prevent productive interactions with ADAR. RESULTS +14 18 ADAR protein_type For instance, ADAR can readily penetrate an A-form helix from the minor groove side and place the helix-penetrating residue in the space occupied by the editing site base (Supplementary Fig. 6). RESULTS +44 56 A-form helix structure_element For instance, ADAR can readily penetrate an A-form helix from the minor groove side and place the helix-penetrating residue in the space occupied by the editing site base (Supplementary Fig. 6). RESULTS +66 78 minor groove site For instance, ADAR can readily penetrate an A-form helix from the minor groove side and place the helix-penetrating residue in the space occupied by the editing site base (Supplementary Fig. 6). RESULTS +153 165 editing site site For instance, ADAR can readily penetrate an A-form helix from the minor groove side and place the helix-penetrating residue in the space occupied by the editing site base (Supplementary Fig. 6). RESULTS +43 55 minor groove site However, this residue cannot penetrate the minor groove enough to occupy the base position in a B-form helix (Supplementary Fig. 6). RESULTS +96 108 B-form helix structure_element However, this residue cannot penetrate the minor groove enough to occupy the base position in a B-form helix (Supplementary Fig. 6). RESULTS +13 16 DNA chemical Furthermore, DNA lacks the 2’ hydroxyls that are used by ADAR for substrate recognition (Fig. 2c). RESULTS +57 61 ADAR protein_type Furthermore, DNA lacks the 2’ hydroxyls that are used by ADAR for substrate recognition (Fig. 2c). RESULTS +6 13 hADAR2d mutant Thus, hADAR2d uses a substrate recognition and base flipping mechanism with similarities to other known nucleic acid-modifying enzymes but uniquely suited for reaction with adenosine in the context of duplex RNA. RESULTS +104 134 nucleic acid-modifying enzymes protein_type Thus, hADAR2d uses a substrate recognition and base flipping mechanism with similarities to other known nucleic acid-modifying enzymes but uniquely suited for reaction with adenosine in the context of duplex RNA. RESULTS +173 182 adenosine residue_name Thus, hADAR2d uses a substrate recognition and base flipping mechanism with similarities to other known nucleic acid-modifying enzymes but uniquely suited for reaction with adenosine in the context of duplex RNA. RESULTS +201 211 duplex RNA structure_element Thus, hADAR2d uses a substrate recognition and base flipping mechanism with similarities to other known nucleic acid-modifying enzymes but uniquely suited for reaction with adenosine in the context of duplex RNA. RESULTS +0 10 Structures evidence Structures explain nearest neighbor preferences RESULTS +0 5 ADARs protein_type ADARs have a preference for editing adenosines with 5’ nearest neighbor U (or A) and 3’ nearest neighbor G. The ADAR2 flipping loop occupies the minor groove spanning the three base pairs that include the nearest neighbor nucleotides flanking the edited base (Figs. 3b, 3c). RESULTS +36 46 adenosines residue_name ADARs have a preference for editing adenosines with 5’ nearest neighbor U (or A) and 3’ nearest neighbor G. The ADAR2 flipping loop occupies the minor groove spanning the three base pairs that include the nearest neighbor nucleotides flanking the edited base (Figs. 3b, 3c). RESULTS +72 73 U residue_name ADARs have a preference for editing adenosines with 5’ nearest neighbor U (or A) and 3’ nearest neighbor G. The ADAR2 flipping loop occupies the minor groove spanning the three base pairs that include the nearest neighbor nucleotides flanking the edited base (Figs. 3b, 3c). RESULTS +78 79 A residue_name ADARs have a preference for editing adenosines with 5’ nearest neighbor U (or A) and 3’ nearest neighbor G. The ADAR2 flipping loop occupies the minor groove spanning the three base pairs that include the nearest neighbor nucleotides flanking the edited base (Figs. 3b, 3c). RESULTS +105 106 G residue_name ADARs have a preference for editing adenosines with 5’ nearest neighbor U (or A) and 3’ nearest neighbor G. The ADAR2 flipping loop occupies the minor groove spanning the three base pairs that include the nearest neighbor nucleotides flanking the edited base (Figs. 3b, 3c). RESULTS +112 117 ADAR2 protein ADARs have a preference for editing adenosines with 5’ nearest neighbor U (or A) and 3’ nearest neighbor G. The ADAR2 flipping loop occupies the minor groove spanning the three base pairs that include the nearest neighbor nucleotides flanking the edited base (Figs. 3b, 3c). RESULTS +118 131 flipping loop structure_element ADARs have a preference for editing adenosines with 5’ nearest neighbor U (or A) and 3’ nearest neighbor G. The ADAR2 flipping loop occupies the minor groove spanning the three base pairs that include the nearest neighbor nucleotides flanking the edited base (Figs. 3b, 3c). RESULTS +145 157 minor groove site ADARs have a preference for editing adenosines with 5’ nearest neighbor U (or A) and 3’ nearest neighbor G. The ADAR2 flipping loop occupies the minor groove spanning the three base pairs that include the nearest neighbor nucleotides flanking the edited base (Figs. 3b, 3c). RESULTS +68 69 U residue_name As described above, the base pair including the 5’ nearest neighbor U (U11-A13’ in the Bdf2 duplex) is shifted from the position it would occupy in a typical A-form helix to accommodate the loop (Fig. 4a). RESULTS +71 74 U11 residue_name_number As described above, the base pair including the 5’ nearest neighbor U (U11-A13’ in the Bdf2 duplex) is shifted from the position it would occupy in a typical A-form helix to accommodate the loop (Fig. 4a). RESULTS +75 78 A13 residue_name_number As described above, the base pair including the 5’ nearest neighbor U (U11-A13’ in the Bdf2 duplex) is shifted from the position it would occupy in a typical A-form helix to accommodate the loop (Fig. 4a). RESULTS +87 91 Bdf2 chemical As described above, the base pair including the 5’ nearest neighbor U (U11-A13’ in the Bdf2 duplex) is shifted from the position it would occupy in a typical A-form helix to accommodate the loop (Fig. 4a). RESULTS +158 170 A-form helix structure_element As described above, the base pair including the 5’ nearest neighbor U (U11-A13’ in the Bdf2 duplex) is shifted from the position it would occupy in a typical A-form helix to accommodate the loop (Fig. 4a). RESULTS +190 194 loop structure_element As described above, the base pair including the 5’ nearest neighbor U (U11-A13’ in the Bdf2 duplex) is shifted from the position it would occupy in a typical A-form helix to accommodate the loop (Fig. 4a). RESULTS +10 22 minor groove site Also, the minor groove edge of this pair is juxtaposed to the protein backbone at G489. RESULTS +82 86 G489 residue_name_number Also, the minor groove edge of this pair is juxtaposed to the protein backbone at G489. RESULTS +11 26 G-C or C-G pair structure_element Modeling a G-C or C-G pair at this position (i.e. 5’ G or 5’ C) suggests a 2-amino group in the minor groove would clash with the protein at G489 (Fig. 5a, Supplementary Fig. 7c). RESULTS +53 54 G residue_name Modeling a G-C or C-G pair at this position (i.e. 5’ G or 5’ C) suggests a 2-amino group in the minor groove would clash with the protein at G489 (Fig. 5a, Supplementary Fig. 7c). RESULTS +61 62 C residue_name Modeling a G-C or C-G pair at this position (i.e. 5’ G or 5’ C) suggests a 2-amino group in the minor groove would clash with the protein at G489 (Fig. 5a, Supplementary Fig. 7c). RESULTS +96 108 minor groove site Modeling a G-C or C-G pair at this position (i.e. 5’ G or 5’ C) suggests a 2-amino group in the minor groove would clash with the protein at G489 (Fig. 5a, Supplementary Fig. 7c). RESULTS +141 145 G489 residue_name_number Modeling a G-C or C-G pair at this position (i.e. 5’ G or 5’ C) suggests a 2-amino group in the minor groove would clash with the protein at G489 (Fig. 5a, Supplementary Fig. 7c). RESULTS +22 30 U-A pair structure_element Indeed, replacing the U-A pair adjacent to the editing site with a C-G pair in the Gli1 duplex substrate resulted in an 80% reduction in the rate of hADAR2d-catalyzed deamination (Figs. 5b, 5c). RESULTS +47 59 editing site site Indeed, replacing the U-A pair adjacent to the editing site with a C-G pair in the Gli1 duplex substrate resulted in an 80% reduction in the rate of hADAR2d-catalyzed deamination (Figs. 5b, 5c). RESULTS +67 75 C-G pair structure_element Indeed, replacing the U-A pair adjacent to the editing site with a C-G pair in the Gli1 duplex substrate resulted in an 80% reduction in the rate of hADAR2d-catalyzed deamination (Figs. 5b, 5c). RESULTS +83 87 Gli1 protein Indeed, replacing the U-A pair adjacent to the editing site with a C-G pair in the Gli1 duplex substrate resulted in an 80% reduction in the rate of hADAR2d-catalyzed deamination (Figs. 5b, 5c). RESULTS +149 156 hADAR2d mutant Indeed, replacing the U-A pair adjacent to the editing site with a C-G pair in the Gli1 duplex substrate resulted in an 80% reduction in the rate of hADAR2d-catalyzed deamination (Figs. 5b, 5c). RESULTS +176 184 U-A pair structure_element To determine whether this effect arises from an increase in local duplex stability from the C-G for U-A substitution or from the presence of the 2-amino group, we replaced the U-A pair with a U-2-aminopurine (2AP) pair. RESULTS +192 218 U-2-aminopurine (2AP) pair structure_element To determine whether this effect arises from an increase in local duplex stability from the C-G for U-A substitution or from the presence of the 2-amino group, we replaced the U-A pair with a U-2-aminopurine (2AP) pair. RESULTS +0 3 2AP structure_element 2AP is an adenosine analog that forms a base pair with uridine of similar stability to a U-A pair, but places an amino group in the minor groove (Fig. 5b). RESULTS +10 19 adenosine residue_name 2AP is an adenosine analog that forms a base pair with uridine of similar stability to a U-A pair, but places an amino group in the minor groove (Fig. 5b). RESULTS +55 62 uridine residue_name 2AP is an adenosine analog that forms a base pair with uridine of similar stability to a U-A pair, but places an amino group in the minor groove (Fig. 5b). RESULTS +89 97 U-A pair structure_element 2AP is an adenosine analog that forms a base pair with uridine of similar stability to a U-A pair, but places an amino group in the minor groove (Fig. 5b). RESULTS +132 144 minor groove site 2AP is an adenosine analog that forms a base pair with uridine of similar stability to a U-A pair, but places an amino group in the minor groove (Fig. 5b). RESULTS +136 148 minor groove site Importantly, this substitution also resulted in an 80% reduction in rate, illustrating the detrimental effect of the amino group in the minor groove at this location. RESULTS +32 38 hADAR2 protein These observations suggest that hADAR2’s 5’ nearest neighbor preference for U (or A) is due to a destabilizing clash with the protein backbone at G489 that results from the presence of an amino group in the minor groove at this location for sequences with 5’ nearest neighbor G or C. However, the observed clash is not severe and the enzyme would be able to accommodate G or C 5’ nearest neighbors by slight structural perturbations, explaining why this sequence preference is not an absolute requirement. RESULTS +76 77 U residue_name These observations suggest that hADAR2’s 5’ nearest neighbor preference for U (or A) is due to a destabilizing clash with the protein backbone at G489 that results from the presence of an amino group in the minor groove at this location for sequences with 5’ nearest neighbor G or C. However, the observed clash is not severe and the enzyme would be able to accommodate G or C 5’ nearest neighbors by slight structural perturbations, explaining why this sequence preference is not an absolute requirement. RESULTS +82 83 A residue_name These observations suggest that hADAR2’s 5’ nearest neighbor preference for U (or A) is due to a destabilizing clash with the protein backbone at G489 that results from the presence of an amino group in the minor groove at this location for sequences with 5’ nearest neighbor G or C. However, the observed clash is not severe and the enzyme would be able to accommodate G or C 5’ nearest neighbors by slight structural perturbations, explaining why this sequence preference is not an absolute requirement. RESULTS +146 150 G489 residue_name_number These observations suggest that hADAR2’s 5’ nearest neighbor preference for U (or A) is due to a destabilizing clash with the protein backbone at G489 that results from the presence of an amino group in the minor groove at this location for sequences with 5’ nearest neighbor G or C. However, the observed clash is not severe and the enzyme would be able to accommodate G or C 5’ nearest neighbors by slight structural perturbations, explaining why this sequence preference is not an absolute requirement. RESULTS +207 219 minor groove site These observations suggest that hADAR2’s 5’ nearest neighbor preference for U (or A) is due to a destabilizing clash with the protein backbone at G489 that results from the presence of an amino group in the minor groove at this location for sequences with 5’ nearest neighbor G or C. However, the observed clash is not severe and the enzyme would be able to accommodate G or C 5’ nearest neighbors by slight structural perturbations, explaining why this sequence preference is not an absolute requirement. RESULTS +276 277 G residue_name These observations suggest that hADAR2’s 5’ nearest neighbor preference for U (or A) is due to a destabilizing clash with the protein backbone at G489 that results from the presence of an amino group in the minor groove at this location for sequences with 5’ nearest neighbor G or C. However, the observed clash is not severe and the enzyme would be able to accommodate G or C 5’ nearest neighbors by slight structural perturbations, explaining why this sequence preference is not an absolute requirement. RESULTS +281 282 C residue_name These observations suggest that hADAR2’s 5’ nearest neighbor preference for U (or A) is due to a destabilizing clash with the protein backbone at G489 that results from the presence of an amino group in the minor groove at this location for sequences with 5’ nearest neighbor G or C. However, the observed clash is not severe and the enzyme would be able to accommodate G or C 5’ nearest neighbors by slight structural perturbations, explaining why this sequence preference is not an absolute requirement. RESULTS +370 371 G residue_name These observations suggest that hADAR2’s 5’ nearest neighbor preference for U (or A) is due to a destabilizing clash with the protein backbone at G489 that results from the presence of an amino group in the minor groove at this location for sequences with 5’ nearest neighbor G or C. However, the observed clash is not severe and the enzyme would be able to accommodate G or C 5’ nearest neighbors by slight structural perturbations, explaining why this sequence preference is not an absolute requirement. RESULTS +375 376 C residue_name These observations suggest that hADAR2’s 5’ nearest neighbor preference for U (or A) is due to a destabilizing clash with the protein backbone at G489 that results from the presence of an amino group in the minor groove at this location for sequences with 5’ nearest neighbor G or C. However, the observed clash is not severe and the enzyme would be able to accommodate G or C 5’ nearest neighbors by slight structural perturbations, explaining why this sequence preference is not an absolute requirement. RESULTS +15 26 hADAR2d-RNA complex_assembly In each of the hADAR2d-RNA structures reported here, the backbone carbonyl oxygen at S486 accepts an H-bond from the 2-amino group of the G on the 3’ side of the edited nucleotide (Fig. 5d). RESULTS +27 37 structures evidence In each of the hADAR2d-RNA structures reported here, the backbone carbonyl oxygen at S486 accepts an H-bond from the 2-amino group of the G on the 3’ side of the edited nucleotide (Fig. 5d). RESULTS +85 89 S486 residue_name_number In each of the hADAR2d-RNA structures reported here, the backbone carbonyl oxygen at S486 accepts an H-bond from the 2-amino group of the G on the 3’ side of the edited nucleotide (Fig. 5d). RESULTS +101 107 H-bond bond_interaction In each of the hADAR2d-RNA structures reported here, the backbone carbonyl oxygen at S486 accepts an H-bond from the 2-amino group of the G on the 3’ side of the edited nucleotide (Fig. 5d). RESULTS +138 139 G residue_name In each of the hADAR2d-RNA structures reported here, the backbone carbonyl oxygen at S486 accepts an H-bond from the 2-amino group of the G on the 3’ side of the edited nucleotide (Fig. 5d). RESULTS +0 7 Guanine residue_name Guanine is the only common nucleobase that presents an H-bond donor in the RNA minor groove suggesting that other nucleotides in this position would reduce editing efficiency. RESULTS +55 61 H-bond bond_interaction Guanine is the only common nucleobase that presents an H-bond donor in the RNA minor groove suggesting that other nucleotides in this position would reduce editing efficiency. RESULTS +75 91 RNA minor groove site Guanine is the only common nucleobase that presents an H-bond donor in the RNA minor groove suggesting that other nucleotides in this position would reduce editing efficiency. RESULTS +8 16 mutating experimental_method Indeed, mutating this base to A, C or U, while maintaining base pairing at this position, reduced the rate of deamination by hADAR2d in Gli1 mRNA model substrates (Supplementary Fig. 7 a–b). RESULTS +30 31 A residue_name Indeed, mutating this base to A, C or U, while maintaining base pairing at this position, reduced the rate of deamination by hADAR2d in Gli1 mRNA model substrates (Supplementary Fig. 7 a–b). RESULTS +33 34 C residue_name Indeed, mutating this base to A, C or U, while maintaining base pairing at this position, reduced the rate of deamination by hADAR2d in Gli1 mRNA model substrates (Supplementary Fig. 7 a–b). RESULTS +38 39 U residue_name Indeed, mutating this base to A, C or U, while maintaining base pairing at this position, reduced the rate of deamination by hADAR2d in Gli1 mRNA model substrates (Supplementary Fig. 7 a–b). RESULTS +125 132 hADAR2d mutant Indeed, mutating this base to A, C or U, while maintaining base pairing at this position, reduced the rate of deamination by hADAR2d in Gli1 mRNA model substrates (Supplementary Fig. 7 a–b). RESULTS +136 140 Gli1 protein Indeed, mutating this base to A, C or U, while maintaining base pairing at this position, reduced the rate of deamination by hADAR2d in Gli1 mRNA model substrates (Supplementary Fig. 7 a–b). RESULTS +141 145 mRNA chemical Indeed, mutating this base to A, C or U, while maintaining base pairing at this position, reduced the rate of deamination by hADAR2d in Gli1 mRNA model substrates (Supplementary Fig. 7 a–b). RESULTS +52 53 G residue_name To test the importance of the amino group on the 3’ G in the hADAR2d reaction, we prepared RNA duplex substrates with purine analogs on the 3’ side of the edited A (Fig. 5e). RESULTS +61 68 hADAR2d mutant To test the importance of the amino group on the 3’ G in the hADAR2d reaction, we prepared RNA duplex substrates with purine analogs on the 3’ side of the edited A (Fig. 5e). RESULTS +91 101 RNA duplex structure_element To test the importance of the amino group on the 3’ G in the hADAR2d reaction, we prepared RNA duplex substrates with purine analogs on the 3’ side of the edited A (Fig. 5e). RESULTS +155 161 edited protein_state To test the importance of the amino group on the 3’ G in the hADAR2d reaction, we prepared RNA duplex substrates with purine analogs on the 3’ side of the edited A (Fig. 5e). RESULTS +162 163 A residue_name To test the importance of the amino group on the 3’ G in the hADAR2d reaction, we prepared RNA duplex substrates with purine analogs on the 3’ side of the edited A (Fig. 5e). RESULTS +12 13 G residue_name We tested a G analog that lacks the 2-amino group (inosine, I) and one that blocks access to this amino group (N2-methylguanosine (N2MeG). RESULTS +51 58 inosine residue_name We tested a G analog that lacks the 2-amino group (inosine, I) and one that blocks access to this amino group (N2-methylguanosine (N2MeG). RESULTS +60 61 I residue_name We tested a G analog that lacks the 2-amino group (inosine, I) and one that blocks access to this amino group (N2-methylguanosine (N2MeG). RESULTS +30 31 A residue_name In addition, we compared a 3’ A to a 3’ 2AP since 2AP could form the H-bonding interaction observed with S486. RESULTS +40 43 2AP structure_element In addition, we compared a 3’ A to a 3’ 2AP since 2AP could form the H-bonding interaction observed with S486. RESULTS +50 53 2AP structure_element In addition, we compared a 3’ A to a 3’ 2AP since 2AP could form the H-bonding interaction observed with S486. RESULTS +69 90 H-bonding interaction bond_interaction In addition, we compared a 3’ A to a 3’ 2AP since 2AP could form the H-bonding interaction observed with S486. RESULTS +105 109 S486 residue_name_number In addition, we compared a 3’ A to a 3’ 2AP since 2AP could form the H-bonding interaction observed with S486. RESULTS +59 66 hADAR2d mutant We found the substrate with a 3’ N2MeG to be unreactive to hADAR2d-catalyzed deamination confirming the importance of the observed close approach by the protein to the 3’ G 2-amino group (Fig. 5f). RESULTS +171 172 G residue_name We found the substrate with a 3’ N2MeG to be unreactive to hADAR2d-catalyzed deamination confirming the importance of the observed close approach by the protein to the 3’ G 2-amino group (Fig. 5f). RESULTS +37 38 I residue_name In addition, the substrate with a 3’ I displayed a reduced deamination rate compared to the substrate with a 3’ G suggesting the observed H-bond to the 2-amino group contributes to the 3’ nearest neighbor selectivity (Fig. 5f). RESULTS +51 75 reduced deamination rate evidence In addition, the substrate with a 3’ I displayed a reduced deamination rate compared to the substrate with a 3’ G suggesting the observed H-bond to the 2-amino group contributes to the 3’ nearest neighbor selectivity (Fig. 5f). RESULTS +112 113 G residue_name In addition, the substrate with a 3’ I displayed a reduced deamination rate compared to the substrate with a 3’ G suggesting the observed H-bond to the 2-amino group contributes to the 3’ nearest neighbor selectivity (Fig. 5f). RESULTS +138 144 H-bond bond_interaction In addition, the substrate with a 3’ I displayed a reduced deamination rate compared to the substrate with a 3’ G suggesting the observed H-bond to the 2-amino group contributes to the 3’ nearest neighbor selectivity (Fig. 5f). RESULTS +100 103 2AP structure_element This conclusion is further supported by the observation that deamination in the substrate with a 3’ 2AP is faster than in the substrate with a 3’ A (Fig. 5f). RESULTS +146 147 A residue_name This conclusion is further supported by the observation that deamination in the substrate with a 3’ 2AP is faster than in the substrate with a 3’ A (Fig. 5f). RESULTS +0 17 RNA-binding loops structure_element RNA-binding loops of the ADAR catalytic domain RESULTS +25 29 ADAR protein_type RNA-binding loops of the ADAR catalytic domain RESULTS +30 46 catalytic domain structure_element RNA-binding loops of the ADAR catalytic domain RESULTS +4 14 structures evidence The structures reported here identify RNA-binding loops of the ADAR catalytic domain and suggest roles for several amino acids not previously known to be important for editing, either substrate binding or catalysis (Fig. 6). RESULTS +38 55 RNA-binding loops structure_element The structures reported here identify RNA-binding loops of the ADAR catalytic domain and suggest roles for several amino acids not previously known to be important for editing, either substrate binding or catalysis (Fig. 6). RESULTS +63 67 ADAR protein_type The structures reported here identify RNA-binding loops of the ADAR catalytic domain and suggest roles for several amino acids not previously known to be important for editing, either substrate binding or catalysis (Fig. 6). RESULTS +68 84 catalytic domain structure_element The structures reported here identify RNA-binding loops of the ADAR catalytic domain and suggest roles for several amino acids not previously known to be important for editing, either substrate binding or catalysis (Fig. 6). RESULTS +19 23 R510 residue_name_number The side chain for R510 ion-pairs with the 3’ phosphodiester of the orphaned nucleotide (Figs. 3a, 3c). RESULTS +24 33 ion-pairs bond_interaction The side chain for R510 ion-pairs with the 3’ phosphodiester of the orphaned nucleotide (Figs. 3a, 3c). RESULTS +68 76 orphaned protein_state The side chain for R510 ion-pairs with the 3’ phosphodiester of the orphaned nucleotide (Figs. 3a, 3c). RESULTS +77 87 nucleotide chemical The side chain for R510 ion-pairs with the 3’ phosphodiester of the orphaned nucleotide (Figs. 3a, 3c). RESULTS +16 25 conserved protein_state This residue is conserved in ADAR2s and ADAR1s, but is glutamine in the editing-inactive ADAR3s (Supplementary Table 1). RESULTS +29 35 ADAR2s protein_type This residue is conserved in ADAR2s and ADAR1s, but is glutamine in the editing-inactive ADAR3s (Supplementary Table 1). RESULTS +40 46 ADAR1s protein_type This residue is conserved in ADAR2s and ADAR1s, but is glutamine in the editing-inactive ADAR3s (Supplementary Table 1). RESULTS +55 64 glutamine residue_name This residue is conserved in ADAR2s and ADAR1s, but is glutamine in the editing-inactive ADAR3s (Supplementary Table 1). RESULTS +72 88 editing-inactive protein_state This residue is conserved in ADAR2s and ADAR1s, but is glutamine in the editing-inactive ADAR3s (Supplementary Table 1). RESULTS +89 95 ADAR3s protein_type This residue is conserved in ADAR2s and ADAR1s, but is glutamine in the editing-inactive ADAR3s (Supplementary Table 1). RESULTS +0 8 Mutation experimental_method Mutation of hADAR2d at this site to either glutamine (R510Q) or to alanine (R510A) reduced the measured deamination rate constant by approximately an order of magnitude (Fig. 6c). RESULTS +12 19 hADAR2d mutant Mutation of hADAR2d at this site to either glutamine (R510Q) or to alanine (R510A) reduced the measured deamination rate constant by approximately an order of magnitude (Fig. 6c). RESULTS +43 52 glutamine residue_name Mutation of hADAR2d at this site to either glutamine (R510Q) or to alanine (R510A) reduced the measured deamination rate constant by approximately an order of magnitude (Fig. 6c). RESULTS +54 59 R510Q mutant Mutation of hADAR2d at this site to either glutamine (R510Q) or to alanine (R510A) reduced the measured deamination rate constant by approximately an order of magnitude (Fig. 6c). RESULTS +67 74 alanine residue_name Mutation of hADAR2d at this site to either glutamine (R510Q) or to alanine (R510A) reduced the measured deamination rate constant by approximately an order of magnitude (Fig. 6c). RESULTS +76 81 R510A mutant Mutation of hADAR2d at this site to either glutamine (R510Q) or to alanine (R510A) reduced the measured deamination rate constant by approximately an order of magnitude (Fig. 6c). RESULTS +104 129 deamination rate constant evidence Mutation of hADAR2d at this site to either glutamine (R510Q) or to alanine (R510A) reduced the measured deamination rate constant by approximately an order of magnitude (Fig. 6c). RESULTS +79 83 G593 residue_name_number In addition, the contact point near the 5’ end of the unedited strand involves G593, K594 and R348, residues completely conserved in the family of ADAR2s (Fig. 2c, Supplementary Table 1). RESULTS +85 89 K594 residue_name_number In addition, the contact point near the 5’ end of the unedited strand involves G593, K594 and R348, residues completely conserved in the family of ADAR2s (Fig. 2c, Supplementary Table 1). RESULTS +94 98 R348 residue_name_number In addition, the contact point near the 5’ end of the unedited strand involves G593, K594 and R348, residues completely conserved in the family of ADAR2s (Fig. 2c, Supplementary Table 1). RESULTS +109 129 completely conserved protein_state In addition, the contact point near the 5’ end of the unedited strand involves G593, K594 and R348, residues completely conserved in the family of ADAR2s (Fig. 2c, Supplementary Table 1). RESULTS +147 153 ADAR2s protein_type In addition, the contact point near the 5’ end of the unedited strand involves G593, K594 and R348, residues completely conserved in the family of ADAR2s (Fig. 2c, Supplementary Table 1). RESULTS +0 8 Mutation experimental_method Mutation of any of these residues to alanine (G593A, K594A, R348A) substantially reduces editing activity (Fig. 6c). RESULTS +37 44 alanine residue_name Mutation of any of these residues to alanine (G593A, K594A, R348A) substantially reduces editing activity (Fig. 6c). RESULTS +46 51 G593A mutant Mutation of any of these residues to alanine (G593A, K594A, R348A) substantially reduces editing activity (Fig. 6c). RESULTS +53 58 K594A mutant Mutation of any of these residues to alanine (G593A, K594A, R348A) substantially reduces editing activity (Fig. 6c). RESULTS +60 65 R348A mutant Mutation of any of these residues to alanine (G593A, K594A, R348A) substantially reduces editing activity (Fig. 6c). RESULTS +13 21 mutation experimental_method In addition, mutation of G593 to glutamic acid (G593E) resulted in a nearly two orders of magnitude reduction in rate, consistent with proximity of this residue to the negatively charged phosphodiester backbone of the RNA (Fig. 6c). RESULTS +25 29 G593 residue_name_number In addition, mutation of G593 to glutamic acid (G593E) resulted in a nearly two orders of magnitude reduction in rate, consistent with proximity of this residue to the negatively charged phosphodiester backbone of the RNA (Fig. 6c). RESULTS +33 46 glutamic acid residue_name In addition, mutation of G593 to glutamic acid (G593E) resulted in a nearly two orders of magnitude reduction in rate, consistent with proximity of this residue to the negatively charged phosphodiester backbone of the RNA (Fig. 6c). RESULTS +48 53 G593E mutant In addition, mutation of G593 to glutamic acid (G593E) resulted in a nearly two orders of magnitude reduction in rate, consistent with proximity of this residue to the negatively charged phosphodiester backbone of the RNA (Fig. 6c). RESULTS +218 221 RNA chemical In addition, mutation of G593 to glutamic acid (G593E) resulted in a nearly two orders of magnitude reduction in rate, consistent with proximity of this residue to the negatively charged phosphodiester backbone of the RNA (Fig. 6c). RESULTS +0 3 RNA chemical RNA binding leads to an ordering of the 454–477 loop, which was disordered in the RNA-free hADAR2d structure (Fig. 1d, green) (Supplementary Video 2). RESULTS +40 47 454–477 residue_range RNA binding leads to an ordering of the 454–477 loop, which was disordered in the RNA-free hADAR2d structure (Fig. 1d, green) (Supplementary Video 2). RESULTS +48 52 loop structure_element RNA binding leads to an ordering of the 454–477 loop, which was disordered in the RNA-free hADAR2d structure (Fig. 1d, green) (Supplementary Video 2). RESULTS +64 74 disordered protein_state RNA binding leads to an ordering of the 454–477 loop, which was disordered in the RNA-free hADAR2d structure (Fig. 1d, green) (Supplementary Video 2). RESULTS +82 90 RNA-free protein_state RNA binding leads to an ordering of the 454–477 loop, which was disordered in the RNA-free hADAR2d structure (Fig. 1d, green) (Supplementary Video 2). RESULTS +91 98 hADAR2d mutant RNA binding leads to an ordering of the 454–477 loop, which was disordered in the RNA-free hADAR2d structure (Fig. 1d, green) (Supplementary Video 2). RESULTS +99 108 structure evidence RNA binding leads to an ordering of the 454–477 loop, which was disordered in the RNA-free hADAR2d structure (Fig. 1d, green) (Supplementary Video 2). RESULTS +5 9 loop structure_element This loop binds the RNA duplex contacting the minor groove near the editing site and inserting into the adjacent major groove (Fig. 6e). RESULTS +20 30 RNA duplex structure_element This loop binds the RNA duplex contacting the minor groove near the editing site and inserting into the adjacent major groove (Fig. 6e). RESULTS +46 58 minor groove site This loop binds the RNA duplex contacting the minor groove near the editing site and inserting into the adjacent major groove (Fig. 6e). RESULTS +68 80 editing site site This loop binds the RNA duplex contacting the minor groove near the editing site and inserting into the adjacent major groove (Fig. 6e). RESULTS +113 125 major groove site This loop binds the RNA duplex contacting the minor groove near the editing site and inserting into the adjacent major groove (Fig. 6e). RESULTS +5 9 loop structure_element This loop sequence is conserved in ADAR2s but different in the family of ADAR1s (Fig. 6d). RESULTS +22 31 conserved protein_state This loop sequence is conserved in ADAR2s but different in the family of ADAR1s (Fig. 6d). RESULTS +35 41 ADAR2s protein_type This loop sequence is conserved in ADAR2s but different in the family of ADAR1s (Fig. 6d). RESULTS +73 79 ADAR1s protein_type This loop sequence is conserved in ADAR2s but different in the family of ADAR1s (Fig. 6d). RESULTS +51 56 ADARs protein_type The substantial difference in sequence between the ADARs in this RNA-binding loop suggests differences in editing site selectivity between the two ADARs arise, at least in part, from differences in how this loop binds RNA substrates. RESULTS +65 81 RNA-binding loop structure_element The substantial difference in sequence between the ADARs in this RNA-binding loop suggests differences in editing site selectivity between the two ADARs arise, at least in part, from differences in how this loop binds RNA substrates. RESULTS +106 118 editing site site The substantial difference in sequence between the ADARs in this RNA-binding loop suggests differences in editing site selectivity between the two ADARs arise, at least in part, from differences in how this loop binds RNA substrates. RESULTS +147 152 ADARs protein_type The substantial difference in sequence between the ADARs in this RNA-binding loop suggests differences in editing site selectivity between the two ADARs arise, at least in part, from differences in how this loop binds RNA substrates. RESULTS +207 211 loop structure_element The substantial difference in sequence between the ADARs in this RNA-binding loop suggests differences in editing site selectivity between the two ADARs arise, at least in part, from differences in how this loop binds RNA substrates. RESULTS +218 221 RNA chemical The substantial difference in sequence between the ADARs in this RNA-binding loop suggests differences in editing site selectivity between the two ADARs arise, at least in part, from differences in how this loop binds RNA substrates. RESULTS +57 87 nucleic acid modifying enzymes protein_type Base flipping is a well-characterized mechanism by which nucleic acid modifying enzymes gain access to sites of reaction that are otherwise buried in base-paired structures. DISCUSS +162 172 structures evidence Base flipping is a well-characterized mechanism by which nucleic acid modifying enzymes gain access to sites of reaction that are otherwise buried in base-paired structures. DISCUSS +0 14 DNA methylases protein_type DNA methylases, DNA repair glycosylases and RNA loop modifying enzymes are known that flip a nucleotide out of a base pair. DISCUSS +16 39 DNA repair glycosylases protein_type DNA methylases, DNA repair glycosylases and RNA loop modifying enzymes are known that flip a nucleotide out of a base pair. DISCUSS +44 70 RNA loop modifying enzymes protein_type DNA methylases, DNA repair glycosylases and RNA loop modifying enzymes are known that flip a nucleotide out of a base pair. DISCUSS +93 103 nucleotide chemical DNA methylases, DNA repair glycosylases and RNA loop modifying enzymes are known that flip a nucleotide out of a base pair. DISCUSS +48 69 base-flipping enzymes protein_type However, none of the structurally characterized base-flipping enzymes access their reactive sites from within a normal base-paired RNA duplex. DISCUSS +83 97 reactive sites site However, none of the structurally characterized base-flipping enzymes access their reactive sites from within a normal base-paired RNA duplex. DISCUSS +112 130 normal base-paired protein_state However, none of the structurally characterized base-flipping enzymes access their reactive sites from within a normal base-paired RNA duplex. DISCUSS +131 141 RNA duplex structure_element However, none of the structurally characterized base-flipping enzymes access their reactive sites from within a normal base-paired RNA duplex. DISCUSS +70 80 RNA duplex structure_element We are aware of one other protein-induced nucleotide flipping from an RNA duplex region. DISCUSS +0 9 Bacterial taxonomy_domain Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b). DISCUSS +10 29 initiation factor 1 protein Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b). DISCUSS +31 34 IF1 protein Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b). DISCUSS +49 70 30S ribosomal subunit complex_assembly Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b). DISCUSS +74 82 helix 44 structure_element Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b). DISCUSS +86 93 16S RNA chemical Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b). DISCUSS +99 104 A1492 residue_name_number Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b). DISCUSS +109 114 A1493 residue_name_number Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b). DISCUSS +115 126 flipped out protein_state Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b). DISCUSS +144 154 bound into protein_state Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b). DISCUSS +155 170 protein pockets site Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b). DISCUSS +44 60 highly distorted protein_state However, these nucleotides are located in a highly distorted and dynamic duplex region containing several mismatches and are predisposed to undergo this conformational change. DISCUSS +65 72 dynamic protein_state However, these nucleotides are located in a highly distorted and dynamic duplex region containing several mismatches and are predisposed to undergo this conformational change. DISCUSS +73 86 duplex region structure_element However, these nucleotides are located in a highly distorted and dynamic duplex region containing several mismatches and are predisposed to undergo this conformational change. DISCUSS +62 68 normal protein_state Thus, this system is not illustrative of base flipping from a normal duplex and does not involve an enzyme that must carryout a chemical reaction on the flipped out nucleotide. DISCUSS +153 164 flipped out protein_state Thus, this system is not illustrative of base flipping from a normal duplex and does not involve an enzyme that must carryout a chemical reaction on the flipped out nucleotide. DISCUSS +165 175 nucleotide chemical Thus, this system is not illustrative of base flipping from a normal duplex and does not involve an enzyme that must carryout a chemical reaction on the flipped out nucleotide. DISCUSS +6 30 RNA modification enzymes protein_type Other RNA modification enzymes are known that flip nucleotides out of loops, even from base pairs in loop regions (pseudoU synthetase, tRNA transglycosylase, and restrictocin bound to sarcin/ricin loop of 28S rRNA) (Supplementary Fig. 5b). DISCUSS +115 133 pseudoU synthetase protein_type Other RNA modification enzymes are known that flip nucleotides out of loops, even from base pairs in loop regions (pseudoU synthetase, tRNA transglycosylase, and restrictocin bound to sarcin/ricin loop of 28S rRNA) (Supplementary Fig. 5b). DISCUSS +135 156 tRNA transglycosylase protein_type Other RNA modification enzymes are known that flip nucleotides out of loops, even from base pairs in loop regions (pseudoU synthetase, tRNA transglycosylase, and restrictocin bound to sarcin/ricin loop of 28S rRNA) (Supplementary Fig. 5b). DISCUSS +162 174 restrictocin protein Other RNA modification enzymes are known that flip nucleotides out of loops, even from base pairs in loop regions (pseudoU synthetase, tRNA transglycosylase, and restrictocin bound to sarcin/ricin loop of 28S rRNA) (Supplementary Fig. 5b). DISCUSS +175 183 bound to protein_state Other RNA modification enzymes are known that flip nucleotides out of loops, even from base pairs in loop regions (pseudoU synthetase, tRNA transglycosylase, and restrictocin bound to sarcin/ricin loop of 28S rRNA) (Supplementary Fig. 5b). DISCUSS +184 201 sarcin/ricin loop structure_element Other RNA modification enzymes are known that flip nucleotides out of loops, even from base pairs in loop regions (pseudoU synthetase, tRNA transglycosylase, and restrictocin bound to sarcin/ricin loop of 28S rRNA) (Supplementary Fig. 5b). DISCUSS +205 213 28S rRNA chemical Other RNA modification enzymes are known that flip nucleotides out of loops, even from base pairs in loop regions (pseudoU synthetase, tRNA transglycosylase, and restrictocin bound to sarcin/ricin loop of 28S rRNA) (Supplementary Fig. 5b). DISCUSS +12 30 modification sites site Because the modification sites are not flanked on both sides by normal duplex, these enzymes do not experience the same limits in approach to the substrate that ADARs do. DISCUSS +64 70 normal protein_state Because the modification sites are not flanked on both sides by normal duplex, these enzymes do not experience the same limits in approach to the substrate that ADARs do. DISCUSS +71 77 duplex structure_element Because the modification sites are not flanked on both sides by normal duplex, these enzymes do not experience the same limits in approach to the substrate that ADARs do. DISCUSS +161 166 ADARs protein_type Because the modification sites are not flanked on both sides by normal duplex, these enzymes do not experience the same limits in approach to the substrate that ADARs do. DISCUSS +14 19 ADARs protein_type The fact that ADARs must induce flipping from a normal duplex has implications on its preference for adenosines flanked by certain base pairs, a phenomenon that was not well understood prior to this work. DISCUSS +48 54 normal protein_state The fact that ADARs must induce flipping from a normal duplex has implications on its preference for adenosines flanked by certain base pairs, a phenomenon that was not well understood prior to this work. DISCUSS +55 61 duplex structure_element The fact that ADARs must induce flipping from a normal duplex has implications on its preference for adenosines flanked by certain base pairs, a phenomenon that was not well understood prior to this work. DISCUSS +101 111 adenosines residue_name The fact that ADARs must induce flipping from a normal duplex has implications on its preference for adenosines flanked by certain base pairs, a phenomenon that was not well understood prior to this work. DISCUSS +7 17 structures evidence In our structures, the flipped out 8-azanebularine is hydrated, mimicking the tetrahedral intermediate predicted for deamination of adenosine (Figs. 1b, 3a, Supplementary Fig. 3 a–b). DISCUSS +23 34 flipped out protein_state In our structures, the flipped out 8-azanebularine is hydrated, mimicking the tetrahedral intermediate predicted for deamination of adenosine (Figs. 1b, 3a, Supplementary Fig. 3 a–b). DISCUSS +35 50 8-azanebularine chemical In our structures, the flipped out 8-azanebularine is hydrated, mimicking the tetrahedral intermediate predicted for deamination of adenosine (Figs. 1b, 3a, Supplementary Fig. 3 a–b). DISCUSS +132 141 adenosine residue_name In our structures, the flipped out 8-azanebularine is hydrated, mimicking the tetrahedral intermediate predicted for deamination of adenosine (Figs. 1b, 3a, Supplementary Fig. 3 a–b). DISCUSS +11 26 8-azanebularine chemical Our use of 8-azanebularine, with its high propensity to form a covalent hydrate, allowed us to capture a true mimic of the tetrahedral intermediate and reveal the interactions between the deaminase active site and the reactive nucleotide. DISCUSS +188 197 deaminase protein_type Our use of 8-azanebularine, with its high propensity to form a covalent hydrate, allowed us to capture a true mimic of the tetrahedral intermediate and reveal the interactions between the deaminase active site and the reactive nucleotide. DISCUSS +198 209 active site site Our use of 8-azanebularine, with its high propensity to form a covalent hydrate, allowed us to capture a true mimic of the tetrahedral intermediate and reveal the interactions between the deaminase active site and the reactive nucleotide. DISCUSS +13 28 8-azanebularine chemical In addition, 8-azanebularine was found to adopt a 2’-endo sugar pucker with its 2’-hydroxyl H-bonded to the protein backbone carbonyl at T375. DISCUSS +92 100 H-bonded bond_interaction In addition, 8-azanebularine was found to adopt a 2’-endo sugar pucker with its 2’-hydroxyl H-bonded to the protein backbone carbonyl at T375. DISCUSS +137 141 T375 residue_name_number In addition, 8-azanebularine was found to adopt a 2’-endo sugar pucker with its 2’-hydroxyl H-bonded to the protein backbone carbonyl at T375. DISCUSS +79 83 zinc chemical The 2’ endo conformation appears to facilitate access of the nucleobase to the zinc-bound water for nucleophilic attack at C6. DISCUSS +90 95 water chemical The 2’ endo conformation appears to facilitate access of the nucleobase to the zinc-bound water for nucleophilic attack at C6. DISCUSS +14 35 base-flipping enzymes protein_type Several other base-flipping enzymes stabilize the altered nucleic acid conformation by intercalation of an amino acid side chain into the space vacated by the flipped out base. DISCUSS +159 170 flipped out protein_state Several other base-flipping enzymes stabilize the altered nucleic acid conformation by intercalation of an amino acid side chain into the space vacated by the flipped out base. DISCUSS +171 175 base chemical Several other base-flipping enzymes stabilize the altered nucleic acid conformation by intercalation of an amino acid side chain into the space vacated by the flipped out base. DISCUSS +4 10 hADAR2 protein For hADAR2, E488 serves this role. DISCUSS +12 16 E488 residue_name_number For hADAR2, E488 serves this role. DISCUSS +11 21 structures evidence In the two structures with wild type hADAR2, the E488 residue and orphan base are in nearly identical positions (see Supplementary Fig. 4a for overlay). DISCUSS +27 36 wild type protein_state In the two structures with wild type hADAR2, the E488 residue and orphan base are in nearly identical positions (see Supplementary Fig. 4a for overlay). DISCUSS +37 43 hADAR2 protein In the two structures with wild type hADAR2, the E488 residue and orphan base are in nearly identical positions (see Supplementary Fig. 4a for overlay). DISCUSS +49 53 E488 residue_name_number In the two structures with wild type hADAR2, the E488 residue and orphan base are in nearly identical positions (see Supplementary Fig. 4a for overlay). DISCUSS +66 72 orphan protein_state In the two structures with wild type hADAR2, the E488 residue and orphan base are in nearly identical positions (see Supplementary Fig. 4a for overlay). DISCUSS +73 77 base chemical In the two structures with wild type hADAR2, the E488 residue and orphan base are in nearly identical positions (see Supplementary Fig. 4a for overlay). DISCUSS +143 150 overlay experimental_method In the two structures with wild type hADAR2, the E488 residue and orphan base are in nearly identical positions (see Supplementary Fig. 4a for overlay). DISCUSS +10 14 E488 residue_name_number Thus, the E488 side chain directly contacts each orphan base, likely by accepting an H-bond from uracil N3H or by donating an H-bond to cytidine N3. DISCUSS +49 55 orphan protein_state Thus, the E488 side chain directly contacts each orphan base, likely by accepting an H-bond from uracil N3H or by donating an H-bond to cytidine N3. DISCUSS +56 60 base chemical Thus, the E488 side chain directly contacts each orphan base, likely by accepting an H-bond from uracil N3H or by donating an H-bond to cytidine N3. DISCUSS +85 91 H-bond bond_interaction Thus, the E488 side chain directly contacts each orphan base, likely by accepting an H-bond from uracil N3H or by donating an H-bond to cytidine N3. DISCUSS +97 103 uracil residue_name Thus, the E488 side chain directly contacts each orphan base, likely by accepting an H-bond from uracil N3H or by donating an H-bond to cytidine N3. DISCUSS +126 132 H-bond bond_interaction Thus, the E488 side chain directly contacts each orphan base, likely by accepting an H-bond from uracil N3H or by donating an H-bond to cytidine N3. DISCUSS +136 144 cytidine residue_name Thus, the E488 side chain directly contacts each orphan base, likely by accepting an H-bond from uracil N3H or by donating an H-bond to cytidine N3. DISCUSS +32 36 E488 residue_name_number The latter interaction requires E488 to be protonated. DISCUSS +43 53 protonated protein_state The latter interaction requires E488 to be protonated. DISCUSS +4 7 pKa evidence The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation. DISCUSS +11 15 E488 residue_name_number The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation. DISCUSS +23 31 ADAR-RNA complex_assembly The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation. DISCUSS +80 86 H-bond bond_interaction The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation. DISCUSS +106 114 cytidine residue_name The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation. DISCUSS +247 257 protonated protein_state The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation. DISCUSS +306 315 glutamine residue_name The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation. DISCUSS +330 346 fully protonated protein_state The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation. DISCUSS +417 422 E488Q mutant The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation. DISCUSS +423 429 mutant protein_state The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation. DISCUSS +473 477 E488 residue_name_number The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation. DISCUSS +20 27 hADAR2d mutant The interactions of hADAR2d with base pairs adjacent to the editing site adenosine explain the known 5’ and 3’ nearest neighbor preferences (Fig. 5). DISCUSS +60 72 editing site site The interactions of hADAR2d with base pairs adjacent to the editing site adenosine explain the known 5’ and 3’ nearest neighbor preferences (Fig. 5). DISCUSS +73 82 adenosine residue_name The interactions of hADAR2d with base pairs adjacent to the editing site adenosine explain the known 5’ and 3’ nearest neighbor preferences (Fig. 5). DISCUSS +33 38 ADAR2 protein While these studies indicate the ADAR2 catalytic domain makes an important contact to the 3’ nearest neighbor G, Stefl et al. suggested the 3’ G preference arises from dsRBD binding selectivity for ADAR2. DISCUSS +39 55 catalytic domain structure_element While these studies indicate the ADAR2 catalytic domain makes an important contact to the 3’ nearest neighbor G, Stefl et al. suggested the 3’ G preference arises from dsRBD binding selectivity for ADAR2. DISCUSS +110 111 G residue_name While these studies indicate the ADAR2 catalytic domain makes an important contact to the 3’ nearest neighbor G, Stefl et al. suggested the 3’ G preference arises from dsRBD binding selectivity for ADAR2. DISCUSS +143 144 G residue_name While these studies indicate the ADAR2 catalytic domain makes an important contact to the 3’ nearest neighbor G, Stefl et al. suggested the 3’ G preference arises from dsRBD binding selectivity for ADAR2. DISCUSS +168 173 dsRBD structure_element While these studies indicate the ADAR2 catalytic domain makes an important contact to the 3’ nearest neighbor G, Stefl et al. suggested the 3’ G preference arises from dsRBD binding selectivity for ADAR2. DISCUSS +198 203 ADAR2 protein While these studies indicate the ADAR2 catalytic domain makes an important contact to the 3’ nearest neighbor G, Stefl et al. suggested the 3’ G preference arises from dsRBD binding selectivity for ADAR2. DISCUSS +35 40 ADAR2 protein These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. DISCUSS +43 49 dsRBDs structure_element These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. DISCUSS +50 58 bound to protein_state These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. DISCUSS +89 92 NMR experimental_method These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. DISCUSS +107 115 isolated protein_state These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. DISCUSS +116 122 dsRBDs structure_element These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. DISCUSS +124 131 lacking protein_state These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. DISCUSS +136 152 deaminase domain structure_element These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. DISCUSS +164 167 RNA chemical These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. DISCUSS +195 201 GluR-B protein These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. DISCUSS +202 210 R/G site site These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. DISCUSS +211 214 RNA chemical These authors reported a model for ADAR2’s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA. DISCUSS +44 45 G residue_name They describe an interaction wherein the 3’ G 2-amino group H-bonds to the backbone carbonyl of S258 found in the β1-β2 loop of ADAR2’s dsRBDII. DISCUSS +60 67 H-bonds bond_interaction They describe an interaction wherein the 3’ G 2-amino group H-bonds to the backbone carbonyl of S258 found in the β1-β2 loop of ADAR2’s dsRBDII. DISCUSS +96 100 S258 residue_name_number They describe an interaction wherein the 3’ G 2-amino group H-bonds to the backbone carbonyl of S258 found in the β1-β2 loop of ADAR2’s dsRBDII. DISCUSS +114 124 β1-β2 loop structure_element They describe an interaction wherein the 3’ G 2-amino group H-bonds to the backbone carbonyl of S258 found in the β1-β2 loop of ADAR2’s dsRBDII. DISCUSS +128 133 ADAR2 protein They describe an interaction wherein the 3’ G 2-amino group H-bonds to the backbone carbonyl of S258 found in the β1-β2 loop of ADAR2’s dsRBDII. DISCUSS +136 143 dsRBDII structure_element They describe an interaction wherein the 3’ G 2-amino group H-bonds to the backbone carbonyl of S258 found in the β1-β2 loop of ADAR2’s dsRBDII. DISCUSS +27 31 S486 residue_name_number It is not possible for the S486-3’G interaction we describe here and the S258-3’G interaction reported by Stefl et al. to exist in the same complex since both involve protein loops bound in the RNA minor groove at the same location. DISCUSS +34 35 G residue_name It is not possible for the S486-3’G interaction we describe here and the S258-3’G interaction reported by Stefl et al. to exist in the same complex since both involve protein loops bound in the RNA minor groove at the same location. DISCUSS +73 77 S258 residue_name_number It is not possible for the S486-3’G interaction we describe here and the S258-3’G interaction reported by Stefl et al. to exist in the same complex since both involve protein loops bound in the RNA minor groove at the same location. DISCUSS +80 81 G residue_name It is not possible for the S486-3’G interaction we describe here and the S258-3’G interaction reported by Stefl et al. to exist in the same complex since both involve protein loops bound in the RNA minor groove at the same location. DISCUSS +181 189 bound in protein_state It is not possible for the S486-3’G interaction we describe here and the S258-3’G interaction reported by Stefl et al. to exist in the same complex since both involve protein loops bound in the RNA minor groove at the same location. DISCUSS +194 197 RNA chemical It is not possible for the S486-3’G interaction we describe here and the S258-3’G interaction reported by Stefl et al. to exist in the same complex since both involve protein loops bound in the RNA minor groove at the same location. DISCUSS +198 210 minor groove site It is not possible for the S486-3’G interaction we describe here and the S258-3’G interaction reported by Stefl et al. to exist in the same complex since both involve protein loops bound in the RNA minor groove at the same location. DISCUSS +12 22 structures evidence Because our structures have captured the edited nucleotide in the conformation required to access the active site, the interactions observed here are highly likely to occur during the deamination reaction at the editing site. DISCUSS +41 47 edited protein_state Because our structures have captured the edited nucleotide in the conformation required to access the active site, the interactions observed here are highly likely to occur during the deamination reaction at the editing site. DISCUSS +48 58 nucleotide chemical Because our structures have captured the edited nucleotide in the conformation required to access the active site, the interactions observed here are highly likely to occur during the deamination reaction at the editing site. DISCUSS +102 113 active site site Because our structures have captured the edited nucleotide in the conformation required to access the active site, the interactions observed here are highly likely to occur during the deamination reaction at the editing site. DISCUSS +212 224 editing site site Because our structures have captured the edited nucleotide in the conformation required to access the active site, the interactions observed here are highly likely to occur during the deamination reaction at the editing site. DISCUSS +15 21 dsRBDs structure_element However, since dsRBDs are known to bind promiscuously with duplex RNA, it is possible that the S258-3’G interaction found in a complex lacking the deaminase domain is not relevant to catalysis at the editing site. DISCUSS +59 69 duplex RNA structure_element However, since dsRBDs are known to bind promiscuously with duplex RNA, it is possible that the S258-3’G interaction found in a complex lacking the deaminase domain is not relevant to catalysis at the editing site. DISCUSS +95 99 S258 residue_name_number However, since dsRBDs are known to bind promiscuously with duplex RNA, it is possible that the S258-3’G interaction found in a complex lacking the deaminase domain is not relevant to catalysis at the editing site. DISCUSS +102 103 G residue_name However, since dsRBDs are known to bind promiscuously with duplex RNA, it is possible that the S258-3’G interaction found in a complex lacking the deaminase domain is not relevant to catalysis at the editing site. DISCUSS +135 146 lacking the protein_state However, since dsRBDs are known to bind promiscuously with duplex RNA, it is possible that the S258-3’G interaction found in a complex lacking the deaminase domain is not relevant to catalysis at the editing site. DISCUSS +147 163 deaminase domain structure_element However, since dsRBDs are known to bind promiscuously with duplex RNA, it is possible that the S258-3’G interaction found in a complex lacking the deaminase domain is not relevant to catalysis at the editing site. DISCUSS +200 212 editing site site However, since dsRBDs are known to bind promiscuously with duplex RNA, it is possible that the S258-3’G interaction found in a complex lacking the deaminase domain is not relevant to catalysis at the editing site. DISCUSS +25 29 ADAR protein_type It is also possible that ADAR dsRBD and catalytic domain binding are sequential, with release of the dsRBD from the RNA taking place prior to catalytic domain engagement and base flipping. DISCUSS +30 35 dsRBD structure_element It is also possible that ADAR dsRBD and catalytic domain binding are sequential, with release of the dsRBD from the RNA taking place prior to catalytic domain engagement and base flipping. DISCUSS +40 56 catalytic domain structure_element It is also possible that ADAR dsRBD and catalytic domain binding are sequential, with release of the dsRBD from the RNA taking place prior to catalytic domain engagement and base flipping. DISCUSS +101 106 dsRBD structure_element It is also possible that ADAR dsRBD and catalytic domain binding are sequential, with release of the dsRBD from the RNA taking place prior to catalytic domain engagement and base flipping. DISCUSS +116 119 RNA chemical It is also possible that ADAR dsRBD and catalytic domain binding are sequential, with release of the dsRBD from the RNA taking place prior to catalytic domain engagement and base flipping. DISCUSS +142 158 catalytic domain structure_element It is also possible that ADAR dsRBD and catalytic domain binding are sequential, with release of the dsRBD from the RNA taking place prior to catalytic domain engagement and base flipping. DISCUSS +85 90 human species Aicardi-Goutieres Syndrome (AGS) and Dyschromatosis Symmetrica Hereditaria (DSH) are human diseases caused by mutations in the human ADAR1 gene and several of the disease-associated mutations are found in the deaminase domain. DISCUSS +127 132 human species Aicardi-Goutieres Syndrome (AGS) and Dyschromatosis Symmetrica Hereditaria (DSH) are human diseases caused by mutations in the human ADAR1 gene and several of the disease-associated mutations are found in the deaminase domain. DISCUSS +133 138 ADAR1 protein Aicardi-Goutieres Syndrome (AGS) and Dyschromatosis Symmetrica Hereditaria (DSH) are human diseases caused by mutations in the human ADAR1 gene and several of the disease-associated mutations are found in the deaminase domain. DISCUSS +209 225 deaminase domain structure_element Aicardi-Goutieres Syndrome (AGS) and Dyschromatosis Symmetrica Hereditaria (DSH) are human diseases caused by mutations in the human ADAR1 gene and several of the disease-associated mutations are found in the deaminase domain. DISCUSS +26 45 RNA binding surface site Given the conservation in RNA binding surface and active site residues, we expect the hADAR1 catalytic domain to bind RNA with a similar orientation of the helix found in our hADAR2d-RNA structures. DISCUSS +50 61 active site site Given the conservation in RNA binding surface and active site residues, we expect the hADAR1 catalytic domain to bind RNA with a similar orientation of the helix found in our hADAR2d-RNA structures. DISCUSS +86 92 hADAR1 protein Given the conservation in RNA binding surface and active site residues, we expect the hADAR1 catalytic domain to bind RNA with a similar orientation of the helix found in our hADAR2d-RNA structures. DISCUSS +93 109 catalytic domain structure_element Given the conservation in RNA binding surface and active site residues, we expect the hADAR1 catalytic domain to bind RNA with a similar orientation of the helix found in our hADAR2d-RNA structures. DISCUSS +118 121 RNA chemical Given the conservation in RNA binding surface and active site residues, we expect the hADAR1 catalytic domain to bind RNA with a similar orientation of the helix found in our hADAR2d-RNA structures. DISCUSS +175 186 hADAR2d-RNA complex_assembly Given the conservation in RNA binding surface and active site residues, we expect the hADAR1 catalytic domain to bind RNA with a similar orientation of the helix found in our hADAR2d-RNA structures. DISCUSS +187 197 structures evidence Given the conservation in RNA binding surface and active site residues, we expect the hADAR1 catalytic domain to bind RNA with a similar orientation of the helix found in our hADAR2d-RNA structures. DISCUSS +69 80 hADAR2d-RNA complex_assembly When one maps the locations of the AGS-associated mutations onto the hADAR2d-RNA complex, two mutations involve residues in close proximity to the RNA (< 4 Å) (Supplementary Fig. 8a). DISCUSS +147 150 RNA chemical When one maps the locations of the AGS-associated mutations onto the hADAR2d-RNA complex, two mutations involve residues in close proximity to the RNA (< 4 Å) (Supplementary Fig. 8a). DISCUSS +0 4 G487 residue_name_number G487 of hADAR2 is found on the flipping loop near the RNA (Fig. 3b). DISCUSS +8 14 hADAR2 protein G487 of hADAR2 is found on the flipping loop near the RNA (Fig. 3b). DISCUSS +31 44 flipping loop structure_element G487 of hADAR2 is found on the flipping loop near the RNA (Fig. 3b). DISCUSS +54 57 RNA chemical G487 of hADAR2 is found on the flipping loop near the RNA (Fig. 3b). DISCUSS +17 21 loop structure_element Sequence in this loop is highly conserved among ADARs and corresponds to G1007 in hADAR1 (Supplementary Table 2). DISCUSS +25 41 highly conserved protein_state Sequence in this loop is highly conserved among ADARs and corresponds to G1007 in hADAR1 (Supplementary Table 2). DISCUSS +48 53 ADARs protein_type Sequence in this loop is highly conserved among ADARs and corresponds to G1007 in hADAR1 (Supplementary Table 2). DISCUSS +73 78 G1007 residue_name_number Sequence in this loop is highly conserved among ADARs and corresponds to G1007 in hADAR1 (Supplementary Table 2). DISCUSS +82 88 hADAR1 protein Sequence in this loop is highly conserved among ADARs and corresponds to G1007 in hADAR1 (Supplementary Table 2). DISCUSS +3 11 arginine residue_name An arginine at this position would preclude close approach of the flipping loop to the RNA, preventing E1008 insertion and base flipping into the active site (Supplementary Fig. 8b). DISCUSS +66 79 flipping loop structure_element An arginine at this position would preclude close approach of the flipping loop to the RNA, preventing E1008 insertion and base flipping into the active site (Supplementary Fig. 8b). DISCUSS +87 90 RNA chemical An arginine at this position would preclude close approach of the flipping loop to the RNA, preventing E1008 insertion and base flipping into the active site (Supplementary Fig. 8b). DISCUSS +103 108 E1008 residue_name_number An arginine at this position would preclude close approach of the flipping loop to the RNA, preventing E1008 insertion and base flipping into the active site (Supplementary Fig. 8b). DISCUSS +146 157 active site site An arginine at this position would preclude close approach of the flipping loop to the RNA, preventing E1008 insertion and base flipping into the active site (Supplementary Fig. 8b). DISCUSS +49 55 G1007R mutant This is consistent with the observation that the G1007R mutation in hADAR1 inhibits RNA editing activity. DISCUSS +68 74 hADAR1 protein This is consistent with the observation that the G1007R mutation in hADAR1 inhibits RNA editing activity. DISCUSS +84 87 RNA chemical This is consistent with the observation that the G1007R mutation in hADAR1 inhibits RNA editing activity. DISCUSS +6 10 K376 residue_name_number Also, K376 forms salt bridges with both the 5’ and 3’ phosphodiesters of the guanosine on the 3’ side of the editing site (Fig. 2). DISCUSS +17 29 salt bridges bond_interaction Also, K376 forms salt bridges with both the 5’ and 3’ phosphodiesters of the guanosine on the 3’ side of the editing site (Fig. 2). DISCUSS +77 86 guanosine residue_name Also, K376 forms salt bridges with both the 5’ and 3’ phosphodiesters of the guanosine on the 3’ side of the editing site (Fig. 2). DISCUSS +109 121 editing site site Also, K376 forms salt bridges with both the 5’ and 3’ phosphodiesters of the guanosine on the 3’ side of the editing site (Fig. 2). DISCUSS +29 35 hADAR1 protein The corresponding residue in hADAR1 (R892) could form similar contacts and the R892H mutation would likely alter this interaction. DISCUSS +37 41 R892 residue_name_number The corresponding residue in hADAR1 (R892) could form similar contacts and the R892H mutation would likely alter this interaction. DISCUSS +79 84 R892H mutant The corresponding residue in hADAR1 (R892) could form similar contacts and the R892H mutation would likely alter this interaction. DISCUSS +16 26 structures evidence In summary, the structures described here establish human ADAR2 as a base-flipping enzyme that uses a unique mechanism well suited for modifying duplex RNA. DISCUSS +52 57 human species In summary, the structures described here establish human ADAR2 as a base-flipping enzyme that uses a unique mechanism well suited for modifying duplex RNA. DISCUSS +58 63 ADAR2 protein In summary, the structures described here establish human ADAR2 as a base-flipping enzyme that uses a unique mechanism well suited for modifying duplex RNA. DISCUSS +145 155 duplex RNA structure_element In summary, the structures described here establish human ADAR2 as a base-flipping enzyme that uses a unique mechanism well suited for modifying duplex RNA. DISCUSS +74 78 ADAR protein_type In addition, this work provides a basis for understanding the role of the ADAR catalytic domain in determining editing site selectivity and additional structural context to evaluate the impact of ADAR mutations associated with human disease. DISCUSS +79 95 catalytic domain structure_element In addition, this work provides a basis for understanding the role of the ADAR catalytic domain in determining editing site selectivity and additional structural context to evaluate the impact of ADAR mutations associated with human disease. DISCUSS +111 123 editing site site In addition, this work provides a basis for understanding the role of the ADAR catalytic domain in determining editing site selectivity and additional structural context to evaluate the impact of ADAR mutations associated with human disease. DISCUSS +196 200 ADAR protein_type In addition, this work provides a basis for understanding the role of the ADAR catalytic domain in determining editing site selectivity and additional structural context to evaluate the impact of ADAR mutations associated with human disease. DISCUSS +227 232 human species In addition, this work provides a basis for understanding the role of the ADAR catalytic domain in determining editing site selectivity and additional structural context to evaluate the impact of ADAR mutations associated with human disease. DISCUSS +0 5 Human species Human ADAR2 and modified RNAs for crystallography FIG +6 11 ADAR2 protein Human ADAR2 and modified RNAs for crystallography FIG +25 29 RNAs chemical Human ADAR2 and modified RNAs for crystallography FIG +34 49 crystallography experimental_method Human ADAR2 and modified RNAs for crystallography FIG +18 23 human species a, Domain map for human ADAR2 b, ADAR reaction showing intermediate and 8-azanebularine (N) hydrate that mimics this structure c, Duplex RNAs used for crystallization. FIG +24 29 ADAR2 protein a, Domain map for human ADAR2 b, ADAR reaction showing intermediate and 8-azanebularine (N) hydrate that mimics this structure c, Duplex RNAs used for crystallization. FIG +33 37 ADAR protein_type a, Domain map for human ADAR2 b, ADAR reaction showing intermediate and 8-azanebularine (N) hydrate that mimics this structure c, Duplex RNAs used for crystallization. FIG +72 99 8-azanebularine (N) hydrate chemical a, Domain map for human ADAR2 b, ADAR reaction showing intermediate and 8-azanebularine (N) hydrate that mimics this structure c, Duplex RNAs used for crystallization. FIG +117 126 structure evidence a, Domain map for human ADAR2 b, ADAR reaction showing intermediate and 8-azanebularine (N) hydrate that mimics this structure c, Duplex RNAs used for crystallization. FIG +130 141 Duplex RNAs structure_element a, Domain map for human ADAR2 b, ADAR reaction showing intermediate and 8-azanebularine (N) hydrate that mimics this structure c, Duplex RNAs used for crystallization. FIG +151 166 crystallization experimental_method a, Domain map for human ADAR2 b, ADAR reaction showing intermediate and 8-azanebularine (N) hydrate that mimics this structure c, Duplex RNAs used for crystallization. FIG +0 11 Bdf2 duplex chemical Bdf2 duplex sequence is derived from an editing site found in S. cerevisiae Bdf2 mRNA and Gli1 duplex has sequence surrounding the human Gli1 mRNA editing site. FIG +40 52 editing site site Bdf2 duplex sequence is derived from an editing site found in S. cerevisiae Bdf2 mRNA and Gli1 duplex has sequence surrounding the human Gli1 mRNA editing site. FIG +62 75 S. cerevisiae species Bdf2 duplex sequence is derived from an editing site found in S. cerevisiae Bdf2 mRNA and Gli1 duplex has sequence surrounding the human Gli1 mRNA editing site. FIG +76 85 Bdf2 mRNA chemical Bdf2 duplex sequence is derived from an editing site found in S. cerevisiae Bdf2 mRNA and Gli1 duplex has sequence surrounding the human Gli1 mRNA editing site. FIG +90 94 Gli1 protein Bdf2 duplex sequence is derived from an editing site found in S. cerevisiae Bdf2 mRNA and Gli1 duplex has sequence surrounding the human Gli1 mRNA editing site. FIG +131 136 human species Bdf2 duplex sequence is derived from an editing site found in S. cerevisiae Bdf2 mRNA and Gli1 duplex has sequence surrounding the human Gli1 mRNA editing site. FIG +137 141 Gli1 protein Bdf2 duplex sequence is derived from an editing site found in S. cerevisiae Bdf2 mRNA and Gli1 duplex has sequence surrounding the human Gli1 mRNA editing site. FIG +142 146 mRNA chemical Bdf2 duplex sequence is derived from an editing site found in S. cerevisiae Bdf2 mRNA and Gli1 duplex has sequence surrounding the human Gli1 mRNA editing site. FIG +147 159 editing site site Bdf2 duplex sequence is derived from an editing site found in S. cerevisiae Bdf2 mRNA and Gli1 duplex has sequence surrounding the human Gli1 mRNA editing site. FIG +0 9 Structure evidence Structure of hADAR2d E488Q bound to the Bdf2-C RNA duplex at 2.75 Å resolution FIG +13 20 hADAR2d mutant Structure of hADAR2d E488Q bound to the Bdf2-C RNA duplex at 2.75 Å resolution FIG +21 26 E488Q mutant Structure of hADAR2d E488Q bound to the Bdf2-C RNA duplex at 2.75 Å resolution FIG +27 35 bound to protein_state Structure of hADAR2d E488Q bound to the Bdf2-C RNA duplex at 2.75 Å resolution FIG +40 57 Bdf2-C RNA duplex chemical Structure of hADAR2d E488Q bound to the Bdf2-C RNA duplex at 2.75 Å resolution FIG +42 47 dsRNA chemical a, View of structure perpendicular to the dsRNA helical axis. FIG +47 58 flipped out protein_state Colors correspond to those in Figs. 1a and 1c; flipped out base N is highlighted red, zinc in grey space-filling sphere, Q488 in yellow, previously disordered aa454–477 loop in green and inositol hexakisphosphate (IHP) in space filling. FIG +86 90 zinc chemical Colors correspond to those in Figs. 1a and 1c; flipped out base N is highlighted red, zinc in grey space-filling sphere, Q488 in yellow, previously disordered aa454–477 loop in green and inositol hexakisphosphate (IHP) in space filling. FIG +121 125 Q488 residue_name_number Colors correspond to those in Figs. 1a and 1c; flipped out base N is highlighted red, zinc in grey space-filling sphere, Q488 in yellow, previously disordered aa454–477 loop in green and inositol hexakisphosphate (IHP) in space filling. FIG +148 158 disordered protein_state Colors correspond to those in Figs. 1a and 1c; flipped out base N is highlighted red, zinc in grey space-filling sphere, Q488 in yellow, previously disordered aa454–477 loop in green and inositol hexakisphosphate (IHP) in space filling. FIG +161 168 454–477 residue_range Colors correspond to those in Figs. 1a and 1c; flipped out base N is highlighted red, zinc in grey space-filling sphere, Q488 in yellow, previously disordered aa454–477 loop in green and inositol hexakisphosphate (IHP) in space filling. FIG +169 173 loop structure_element Colors correspond to those in Figs. 1a and 1c; flipped out base N is highlighted red, zinc in grey space-filling sphere, Q488 in yellow, previously disordered aa454–477 loop in green and inositol hexakisphosphate (IHP) in space filling. FIG +187 212 inositol hexakisphosphate chemical Colors correspond to those in Figs. 1a and 1c; flipped out base N is highlighted red, zinc in grey space-filling sphere, Q488 in yellow, previously disordered aa454–477 loop in green and inositol hexakisphosphate (IHP) in space filling. FIG +214 217 IHP chemical Colors correspond to those in Figs. 1a and 1c; flipped out base N is highlighted red, zinc in grey space-filling sphere, Q488 in yellow, previously disordered aa454–477 loop in green and inositol hexakisphosphate (IHP) in space filling. FIG +39 46 hADAR2d mutant A transparent surface is shown for the hADAR2d protein. FIG +31 36 dsRNA chemical b, View of structure along the dsRNA helical axis. FIG +35 42 hADAR2d mutant c, Summary of the contacts between hADAR2d E488Q and the Bdf2-C RNA duplex. FIG +43 48 E488Q mutant c, Summary of the contacts between hADAR2d E488Q and the Bdf2-C RNA duplex. FIG +57 74 Bdf2-C RNA duplex chemical c, Summary of the contacts between hADAR2d E488Q and the Bdf2-C RNA duplex. FIG +0 4 ADAR protein_type ADAR recognition of the flipped out and orphaned nucleotides FIG +24 35 flipped out protein_state ADAR recognition of the flipped out and orphaned nucleotides FIG +40 48 orphaned protein_state ADAR recognition of the flipped out and orphaned nucleotides FIG +49 60 nucleotides chemical ADAR recognition of the flipped out and orphaned nucleotides FIG +19 31 editing site site a, Contacts to the editing site nucleotide (N) in the active site. FIG +32 42 nucleotide chemical a, Contacts to the editing site nucleotide (N) in the active site. FIG +54 65 active site site a, Contacts to the editing site nucleotide (N) in the active site. FIG +3 9 Orphan protein_state b, Orphan nucleotide recognition in the hADAR2d E488Q–Bdf2-C complex. FIG +10 20 nucleotide chemical b, Orphan nucleotide recognition in the hADAR2d E488Q–Bdf2-C complex. FIG +40 60 hADAR2d E488Q–Bdf2-C complex_assembly b, Orphan nucleotide recognition in the hADAR2d E488Q–Bdf2-C complex. FIG +3 9 Orphan protein_state c, Orphan nucleotide recognition in the hADAR2d WT–Bdf2-U complex. FIG +10 20 nucleotide chemical c, Orphan nucleotide recognition in the hADAR2d WT–Bdf2-U complex. FIG +40 57 hADAR2d WT–Bdf2-U complex_assembly c, Orphan nucleotide recognition in the hADAR2d WT–Bdf2-U complex. FIG +6 10 ADAR protein_type Other ADAR-induced changes in RNA conformation FIG +30 33 RNA chemical Other ADAR-induced changes in RNA conformation FIG +3 10 hADAR2d mutant a, hADAR2d shifts the position of U11-A13’ base pair from ideal A-form RNA helix (yellow). FIG +34 37 U11 residue_name_number a, hADAR2d shifts the position of U11-A13’ base pair from ideal A-form RNA helix (yellow). FIG +38 41 A13 residue_name_number a, hADAR2d shifts the position of U11-A13’ base pair from ideal A-form RNA helix (yellow). FIG +64 80 A-form RNA helix structure_element a, hADAR2d shifts the position of U11-A13’ base pair from ideal A-form RNA helix (yellow). FIG +3 10 Overlay experimental_method b, Overlay of Bdf2 duplex RNA and idealized A form duplex of same sequence (yellow) illustrating kink in strand and widening of major groove opposite editing site induced by hADAR2d. FIG +14 29 Bdf2 duplex RNA chemical b, Overlay of Bdf2 duplex RNA and idealized A form duplex of same sequence (yellow) illustrating kink in strand and widening of major groove opposite editing site induced by hADAR2d. FIG +44 57 A form duplex structure_element b, Overlay of Bdf2 duplex RNA and idealized A form duplex of same sequence (yellow) illustrating kink in strand and widening of major groove opposite editing site induced by hADAR2d. FIG +128 140 major groove site b, Overlay of Bdf2 duplex RNA and idealized A form duplex of same sequence (yellow) illustrating kink in strand and widening of major groove opposite editing site induced by hADAR2d. FIG +150 162 editing site site b, Overlay of Bdf2 duplex RNA and idealized A form duplex of same sequence (yellow) illustrating kink in strand and widening of major groove opposite editing site induced by hADAR2d. FIG +174 181 hADAR2d mutant b, Overlay of Bdf2 duplex RNA and idealized A form duplex of same sequence (yellow) illustrating kink in strand and widening of major groove opposite editing site induced by hADAR2d. FIG +20 23 A13 residue_name_number c, Unusual “wobble” A13’-U11 interaction in the hADAR2d WT–Bdf2-U complex shown in stick with H-bond indicated with yellow dashes and distances shown in Å. The position of this base pair in the hADAR2d E488Q–Bdf2-C duplex is shown in wire with H-bonds shown with gray dashes. FIG +25 28 U11 residue_name_number c, Unusual “wobble” A13’-U11 interaction in the hADAR2d WT–Bdf2-U complex shown in stick with H-bond indicated with yellow dashes and distances shown in Å. The position of this base pair in the hADAR2d E488Q–Bdf2-C duplex is shown in wire with H-bonds shown with gray dashes. FIG +48 65 hADAR2d WT–Bdf2-U complex_assembly c, Unusual “wobble” A13’-U11 interaction in the hADAR2d WT–Bdf2-U complex shown in stick with H-bond indicated with yellow dashes and distances shown in Å. The position of this base pair in the hADAR2d E488Q–Bdf2-C duplex is shown in wire with H-bonds shown with gray dashes. FIG +94 100 H-bond bond_interaction c, Unusual “wobble” A13’-U11 interaction in the hADAR2d WT–Bdf2-U complex shown in stick with H-bond indicated with yellow dashes and distances shown in Å. The position of this base pair in the hADAR2d E488Q–Bdf2-C duplex is shown in wire with H-bonds shown with gray dashes. FIG +194 214 hADAR2d E488Q–Bdf2-C complex_assembly c, Unusual “wobble” A13’-U11 interaction in the hADAR2d WT–Bdf2-U complex shown in stick with H-bond indicated with yellow dashes and distances shown in Å. The position of this base pair in the hADAR2d E488Q–Bdf2-C duplex is shown in wire with H-bonds shown with gray dashes. FIG +244 251 H-bonds bond_interaction c, Unusual “wobble” A13’-U11 interaction in the hADAR2d WT–Bdf2-U complex shown in stick with H-bond indicated with yellow dashes and distances shown in Å. The position of this base pair in the hADAR2d E488Q–Bdf2-C duplex is shown in wire with H-bonds shown with gray dashes. FIG +18 30 editing site site Interactions with editing site nearest neighbor nucleotides FIG +48 59 nucleotides chemical Interactions with editing site nearest neighbor nucleotides FIG +7 19 minor groove site a, The minor groove edge of the U11-A13’ base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5’ nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine). FIG +32 35 U11 residue_name_number a, The minor groove edge of the U11-A13’ base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5’ nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine). FIG +36 39 A13 residue_name_number a, The minor groove edge of the U11-A13’ base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5’ nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine). FIG +60 71 Bdf2 duplex chemical a, The minor groove edge of the U11-A13’ base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5’ nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine). FIG +83 87 G489 residue_name_number a, The minor groove edge of the U11-A13’ base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5’ nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine). FIG +102 110 C-G pair structure_element a, The minor groove edge of the U11-A13’ base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5’ nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine). FIG +154 155 G residue_name a, The minor groove edge of the U11-A13’ base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5’ nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine). FIG +173 183 RNA duplex structure_element a, The minor groove edge of the U11-A13’ base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5’ nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine). FIG +263 275 editing site site a, The minor groove edge of the U11-A13’ base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5’ nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine). FIG +294 297 2AP structure_element a, The minor groove edge of the U11-A13’ base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5’ nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine). FIG +300 313 2-aminopurine structure_element a, The minor groove edge of the U11-A13’ base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5’ nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine). FIG +17 43 deamination rate constants evidence c, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 5’ nearest neighbors; krel = kobs/(kobs for unmodified RNA). FIG +47 54 hADAR2d mutant c, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 5’ nearest neighbors; krel = kobs/(kobs for unmodified RNA). FIG +62 74 editing site site c, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 5’ nearest neighbors; krel = kobs/(kobs for unmodified RNA). FIG +75 84 adenosine residue_name c, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 5’ nearest neighbors; krel = kobs/(kobs for unmodified RNA). FIG +144 148 krel evidence c, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 5’ nearest neighbors; krel = kobs/(kobs for unmodified RNA). FIG +151 155 kobs evidence c, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 5’ nearest neighbors; krel = kobs/(kobs for unmodified RNA). FIG +157 161 kobs evidence c, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 5’ nearest neighbors; krel = kobs/(kobs for unmodified RNA). FIG +166 176 unmodified protein_state c, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 5’ nearest neighbors; krel = kobs/(kobs for unmodified RNA). FIG +177 180 RNA chemical c, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 5’ nearest neighbors; krel = kobs/(kobs for unmodified RNA). FIG +3 9 hADAR2 protein d, hADAR2 S486 backbone H-bond with 3’ G 2-amino group; e, RNA duplex substrates prepared with different 3’ nearest neighbor nucleotides adjacent to editing site indicated in red (I = inosine, N2MeG = N2-methylguanosine, 2AP = 2-aminopurine). FIG +10 14 S486 residue_name_number d, hADAR2 S486 backbone H-bond with 3’ G 2-amino group; e, RNA duplex substrates prepared with different 3’ nearest neighbor nucleotides adjacent to editing site indicated in red (I = inosine, N2MeG = N2-methylguanosine, 2AP = 2-aminopurine). FIG +24 30 H-bond bond_interaction d, hADAR2 S486 backbone H-bond with 3’ G 2-amino group; e, RNA duplex substrates prepared with different 3’ nearest neighbor nucleotides adjacent to editing site indicated in red (I = inosine, N2MeG = N2-methylguanosine, 2AP = 2-aminopurine). FIG +39 40 G residue_name d, hADAR2 S486 backbone H-bond with 3’ G 2-amino group; e, RNA duplex substrates prepared with different 3’ nearest neighbor nucleotides adjacent to editing site indicated in red (I = inosine, N2MeG = N2-methylguanosine, 2AP = 2-aminopurine). FIG +59 69 RNA duplex structure_element d, hADAR2 S486 backbone H-bond with 3’ G 2-amino group; e, RNA duplex substrates prepared with different 3’ nearest neighbor nucleotides adjacent to editing site indicated in red (I = inosine, N2MeG = N2-methylguanosine, 2AP = 2-aminopurine). FIG +149 161 editing site site d, hADAR2 S486 backbone H-bond with 3’ G 2-amino group; e, RNA duplex substrates prepared with different 3’ nearest neighbor nucleotides adjacent to editing site indicated in red (I = inosine, N2MeG = N2-methylguanosine, 2AP = 2-aminopurine). FIG +180 181 I residue_name d, hADAR2 S486 backbone H-bond with 3’ G 2-amino group; e, RNA duplex substrates prepared with different 3’ nearest neighbor nucleotides adjacent to editing site indicated in red (I = inosine, N2MeG = N2-methylguanosine, 2AP = 2-aminopurine). FIG +184 191 inosine residue_name d, hADAR2 S486 backbone H-bond with 3’ G 2-amino group; e, RNA duplex substrates prepared with different 3’ nearest neighbor nucleotides adjacent to editing site indicated in red (I = inosine, N2MeG = N2-methylguanosine, 2AP = 2-aminopurine). FIG +221 224 2AP structure_element d, hADAR2 S486 backbone H-bond with 3’ G 2-amino group; e, RNA duplex substrates prepared with different 3’ nearest neighbor nucleotides adjacent to editing site indicated in red (I = inosine, N2MeG = N2-methylguanosine, 2AP = 2-aminopurine). FIG +227 240 2-aminopurine structure_element d, hADAR2 S486 backbone H-bond with 3’ G 2-amino group; e, RNA duplex substrates prepared with different 3’ nearest neighbor nucleotides adjacent to editing site indicated in red (I = inosine, N2MeG = N2-methylguanosine, 2AP = 2-aminopurine). FIG +17 43 deamination rate constants evidence f, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 3’ nearest neighbors. FIG +47 54 hADAR2d mutant f, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 3’ nearest neighbors. FIG +62 74 editing site site f, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 3’ nearest neighbors. FIG +75 84 adenosine residue_name f, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 3’ nearest neighbors. FIG +0 4 krel evidence krel = kobs/(kobs for unmodified RNA). FIG +7 11 kobs evidence krel = kobs/(kobs for unmodified RNA). FIG +13 17 kobs evidence krel = kobs/(kobs for unmodified RNA). FIG +22 32 unmodified protein_state krel = kobs/(kobs for unmodified RNA). FIG +33 36 RNA chemical krel = kobs/(kobs for unmodified RNA). FIG +0 17 RNA-binding loops structure_element RNA-binding loops in the ADAR catalytic domain FIG +25 29 ADAR protein_type RNA-binding loops in the ADAR catalytic domain FIG +30 46 catalytic domain structure_element RNA-binding loops in the ADAR catalytic domain FIG +3 9 hADAR2 protein a, hADAR2 residues that contact phosphodiester backbone near 5’ end of unedited strand. FIG +39 60 protein-RNA interface site b, Location of mutations introduced at protein-RNA interface. FIG +17 43 deamination rate constants evidence c, Comparison of deamination rate constants of the different hADAR2d mutants (Log scale). FIG +61 68 hADAR2d mutant c, Comparison of deamination rate constants of the different hADAR2d mutants (Log scale). FIG +0 4 krel evidence krel = kobs for mutant/kobs for WT. FIG +7 11 kobs evidence krel = kobs for mutant/kobs for WT. FIG +16 22 mutant protein_state krel = kobs for mutant/kobs for WT. FIG +23 27 kobs evidence krel = kobs for mutant/kobs for WT. FIG +32 34 WT protein_state krel = kobs for mutant/kobs for WT. FIG +3 21 Sequence alignment experimental_method d, Sequence alignment of ADAR2s (A2) and ADAR1s (A1) from different organisms with different levels of conservation colored (Yellow: conserved in all ADAR1s and ADAR2s, red: conserved in ADAR2s, blue: conserved in ADAR1s. FIG +25 31 ADAR2s protein_type d, Sequence alignment of ADAR2s (A2) and ADAR1s (A1) from different organisms with different levels of conservation colored (Yellow: conserved in all ADAR1s and ADAR2s, red: conserved in ADAR2s, blue: conserved in ADAR1s. FIG +41 47 ADAR1s protein_type d, Sequence alignment of ADAR2s (A2) and ADAR1s (A1) from different organisms with different levels of conservation colored (Yellow: conserved in all ADAR1s and ADAR2s, red: conserved in ADAR2s, blue: conserved in ADAR1s. FIG +133 142 conserved protein_state d, Sequence alignment of ADAR2s (A2) and ADAR1s (A1) from different organisms with different levels of conservation colored (Yellow: conserved in all ADAR1s and ADAR2s, red: conserved in ADAR2s, blue: conserved in ADAR1s. FIG +150 156 ADAR1s protein_type d, Sequence alignment of ADAR2s (A2) and ADAR1s (A1) from different organisms with different levels of conservation colored (Yellow: conserved in all ADAR1s and ADAR2s, red: conserved in ADAR2s, blue: conserved in ADAR1s. FIG +161 167 ADAR2s protein_type d, Sequence alignment of ADAR2s (A2) and ADAR1s (A1) from different organisms with different levels of conservation colored (Yellow: conserved in all ADAR1s and ADAR2s, red: conserved in ADAR2s, blue: conserved in ADAR1s. FIG +174 183 conserved protein_state d, Sequence alignment of ADAR2s (A2) and ADAR1s (A1) from different organisms with different levels of conservation colored (Yellow: conserved in all ADAR1s and ADAR2s, red: conserved in ADAR2s, blue: conserved in ADAR1s. FIG +187 193 ADAR2s protein_type d, Sequence alignment of ADAR2s (A2) and ADAR1s (A1) from different organisms with different levels of conservation colored (Yellow: conserved in all ADAR1s and ADAR2s, red: conserved in ADAR2s, blue: conserved in ADAR1s. FIG +201 210 conserved protein_state d, Sequence alignment of ADAR2s (A2) and ADAR1s (A1) from different organisms with different levels of conservation colored (Yellow: conserved in all ADAR1s and ADAR2s, red: conserved in ADAR2s, blue: conserved in ADAR1s. FIG +214 220 ADAR1s protein_type d, Sequence alignment of ADAR2s (A2) and ADAR1s (A1) from different organisms with different levels of conservation colored (Yellow: conserved in all ADAR1s and ADAR2s, red: conserved in ADAR2s, blue: conserved in ADAR1s. FIG +22 52 ADAR-specific RNA-binding loop structure_element e, Interaction of the ADAR-specific RNA-binding loop near the 5’ end of the edited strand. FIG +23 36 not conserved protein_state Colors as in d, white: not conserved, flipped out base is shown in pink. FIG +38 49 flipped out protein_state Colors as in d, white: not conserved, flipped out base is shown in pink. FIG +50 54 base chemical Colors as in d, white: not conserved, flipped out base is shown in pink. FIG diff --git a/annotation_CSV/PMC4918766.csv b/annotation_CSV/PMC4918766.csv new file mode 100644 index 0000000000000000000000000000000000000000..cd33afb871ade2fc9ce50d29b390b9aeaecd09ee --- /dev/null +++ b/annotation_CSV/PMC4918766.csv @@ -0,0 +1,1028 @@ +anno_start anno_end anno_text entity_type sentence section +44 56 binding-site site Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger TITLE +74 98 sodium-calcium exchanger protein_type Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger TITLE +0 19 Na+/Ca2+ exchangers protein_type Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. ABSTRACT +32 35 Na+ chemical Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. ABSTRACT +113 117 Ca2+ chemical Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. ABSTRACT +146 150 Ca2+ chemical Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. ABSTRACT +92 111 structural analysis experimental_method Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT +119 128 exchanger protein_type Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT +134 158 Methanococcus jannaschii species Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT +160 166 NCX_Mj protein Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT +168 176 bound to protein_state Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT +177 180 Na+ chemical Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT +182 186 Ca2+ chemical Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT +190 194 Sr2+ chemical Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT +228 231 apo protein_state Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT +130 133 Na+ chemical This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. ABSTRACT +135 139 Ca2+ chemical This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. ABSTRACT +44 80 conformational free-energy landscape evidence An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. ABSTRACT +84 90 NCX_Mj protein An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. ABSTRACT +104 117 ion-occupancy protein_state An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. ABSTRACT +135 183 enhanced-sampling molecular-dynamics simulations experimental_method An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. ABSTRACT +207 225 crystal structures evidence An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. ABSTRACT +6 18 calculations experimental_method These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. ABSTRACT +58 65 outward protein_state These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. ABSTRACT +69 75 inward protein_state These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. ABSTRACT +182 185 Na+ chemical These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. ABSTRACT +186 190 Ca2+ chemical These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. ABSTRACT +0 19 Na+/Ca2+ exchangers protein_type Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis. INTRO +21 24 NCX protein_type Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis. INTRO +66 70 Ca2+ chemical Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis. INTRO +0 3 NCX protein_type NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry. INTRO +52 56 Ca2+ chemical NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry. INTRO +138 141 Na+ chemical NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry. INTRO +166 169 Na+ chemical NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry. INTRO +175 179 Ca2+ chemical NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry. INTRO +17 20 NCX protein_type The mechanism of NCX proteins is therefore highly likely to be consistent with the alternating-access model of secondary-active transport. INTRO +47 50 NCX protein_type The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves. INTRO +67 93 membrane-spanning segments structure_element The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves. INTRO +121 127 halves structure_element The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves. INTRO +14 20 halves structure_element Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO +32 48 highly conserved protein_state Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO +72 80 α-repeat structure_element Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO +142 152 eukaryotic taxonomy_domain Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO +153 156 NCX protein_type Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO +166 172 halves structure_element Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO +198 229 intracellular regulatory domain structure_element Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO +240 246 absent protein_state Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO +250 259 microbial taxonomy_domain Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO +260 263 NCX protein_type Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO +91 94 NCX protein_type Despite a long history of physiological and functional studies, the molecular mechanism of NCX has been elusive, owing to the lack of structural information. INTRO +29 38 structure evidence Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein. INTRO +42 48 NCX_Mj protein Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein. INTRO +54 78 Methanococcus jannaschii species Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein. INTRO +138 141 NCX protein_type Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein. INTRO +5 14 structure evidence This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +25 34 exchanger protein_type This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +41 55 outward-facing protein_state This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +95 112 ion-binding sites site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +126 134 internal site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +136 140 Sint site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +143 151 external site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +153 157 Sext site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +160 172 Ca2+-binding site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +174 177 SCa site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +183 189 middle site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +191 195 Smid site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +241 254 occluded from protein_state This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO +53 63 eukaryotic taxonomy_domain With similar ion exchange properties to those of its eukaryotic counterparts, NCX_Mj provides a compelling model system to investigate the structural basis for the specificity, stoichiometry and mechanism of the ion-exchange reaction catalyzed by NCX. INTRO +78 84 NCX_Mj protein With similar ion exchange properties to those of its eukaryotic counterparts, NCX_Mj provides a compelling model system to investigate the structural basis for the specificity, stoichiometry and mechanism of the ion-exchange reaction catalyzed by NCX. INTRO +247 250 NCX protein_type With similar ion exchange properties to those of its eukaryotic counterparts, NCX_Mj provides a compelling model system to investigate the structural basis for the specificity, stoichiometry and mechanism of the ion-exchange reaction catalyzed by NCX. INTRO +43 53 structures evidence In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO +57 71 outward-facing protein_state In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO +72 81 wild-type protein_state In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO +82 88 NCX_Mj protein In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO +89 104 in complex with protein_state In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO +105 108 Na+ chemical In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO +110 114 Ca2+ chemical In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO +119 123 Sr2+ chemical In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO +6 16 structures evidence These structures reveal the mode of recognition of these ions, their relative affinities, and the mechanism of extracellular ion exchange, for a well-defined, functional conformation in a membrane-like environment. INTRO +33 63 molecular-dynamics simulations experimental_method An independent analysis based on molecular-dynamics simulations demonstrates that the structures capture mechanistically relevant states. INTRO +86 96 structures evidence An independent analysis based on molecular-dynamics simulations demonstrates that the structures capture mechanistically relevant states. INTRO +6 18 calculations experimental_method These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism. INTRO +66 80 outward-facing protein_state These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism. INTRO +81 90 exchanger protein_type These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism. INTRO +143 156 inward-facing protein_state These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism. INTRO +14 17 Na+ chemical Extracellular Na+ binding RESULTS +27 48 central binding sites site The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. RESULTS +87 93 NCX_Mj protein The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. RESULTS +94 103 structure evidence The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. RESULTS +141 144 Na+ chemical The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. RESULTS +149 153 Ca2+ chemical The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. RESULTS +169 177 crystals evidence The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. RESULTS +73 76 Na+ chemical To conclusively clarify this assignment, we first set out to examine the Na+ occupancy of these sites without Ca2+. RESULTS +110 114 Ca2+ chemical To conclusively clarify this assignment, we first set out to examine the Na+ occupancy of these sites without Ca2+. RESULTS +0 8 Crystals evidence Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique. RESULTS +30 34 NaCl chemical Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique. RESULTS +45 64 lipidic cubic phase experimental_method Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique. RESULTS +66 69 LCP experimental_method Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique. RESULTS +41 44 LCP experimental_method The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods). RESULTS +132 136 NaCl chemical The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods). RESULTS +141 145 EGTA chemical The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods). RESULTS +0 17 X-ray diffraction experimental_method X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS +34 42 crystals evidence X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS +54 57 Na+ chemical X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS +85 114 electron-density distribution evidence X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS +124 128 Sext site X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS +130 133 SCa site X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS +138 142 Sint site X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS +157 160 Na+ chemical X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS +0 20 Occupancy refinement experimental_method Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). RESULTS +35 38 Na+ chemical Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). RESULTS +52 56 Sint site Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). RESULTS +61 64 SCa site Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). RESULTS +72 75 Na+ chemical Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). RESULTS +131 135 Sint site Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). RESULTS +19 22 Na+ chemical Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). RESULTS +26 30 Sext site Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). RESULTS +70 77 density evidence Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). RESULTS +106 109 Na+ chemical Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). RESULTS +130 134 Sext site Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). RESULTS +174 177 Na+ chemical Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). RESULTS +4 7 Na+ chemical The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated. RESULTS +22 25 SCa site The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated. RESULTS +57 60 Na+ chemical The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated. RESULTS +62 66 Ca2+ chemical The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated. RESULTS +121 125 Smid site The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated. RESULTS +41 46 water chemical Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study. RESULTS +70 73 Na+ chemical Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study. RESULTS +87 91 Smid site Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study. RESULTS +122 132 simulation experimental_method Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study. RESULTS +11 27 electron density evidence First, the electron density at Smid does not depend significantly on the Na+ concentration. RESULTS +31 35 Smid site First, the electron density at Smid does not depend significantly on the Na+ concentration. RESULTS +73 76 Na+ chemical First, the electron density at Smid does not depend significantly on the Na+ concentration. RESULTS +45 49 Smid site Second, the protein coordination geometry at Smid is clearly suboptimal for Na+ (Supplementary Fig. 1d). RESULTS +76 79 Na+ chemical Second, the protein coordination geometry at Smid is clearly suboptimal for Na+ (Supplementary Fig. 1d). RESULTS +4 9 water chemical The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS +22 26 Smid site The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS +33 47 hydrogen-bonds bond_interaction The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS +57 73 highly conserved protein_state The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS +74 79 Glu54 residue_name_number The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS +84 90 Glu213 residue_name_number The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS +158 168 coordinate bond_interaction The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS +178 181 Na+ chemical The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS +190 194 Sext site The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS +196 199 SCa site The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS +204 208 Sint site The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS +45 50 Glu54 residue_name_number It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS +55 61 Glu213 residue_name_number It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS +81 87 Asp240 residue_name_number It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS +102 106 Smid site It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS +127 130 Asn residue_name It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS +134 144 eukaryotic taxonomy_domain It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS +145 148 NCX protein_type It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS +159 169 protonated protein_state It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS +206 216 simulation experimental_method It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS +0 3 Na+ chemical Na+-dependent conformational change RESULTS +4 10 NCX_Mj protein The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2). RESULTS +11 21 structures evidence The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2). RESULTS +33 36 Na+ chemical The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2). RESULTS +69 72 Na+ chemical The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2). RESULTS +84 88 Sext site The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2). RESULTS +5 8 Na+ chemical When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS +18 22 Sext site When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS +26 30 high protein_state When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS +51 66 N-terminal half structure_element When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS +70 73 TM7 structure_element When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS +91 104 short helices structure_element When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS +106 110 TM7a structure_element When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS +115 119 TM7b structure_element When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS +0 4 TM7b structure_element TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d). RESULTS +23 44 central binding sites site TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d). RESULTS +103 109 Ala206 residue_name_number TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d). RESULTS +110 122 coordinating bond_interaction TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d). RESULTS +127 130 Na+ chemical TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d). RESULTS +14 18 Sext site However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS +27 32 empty protein_state However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS +36 39 low protein_state However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS +40 43 Na+ chemical However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS +60 64 TM7a structure_element However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS +69 73 TM7b structure_element However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS +103 108 helix structure_element However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS +146 152 Ala206 residue_name_number However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS +0 4 TM7a structure_element TM7a also forms hydrophobic contacts with the C-terminal half of TM6. RESULTS +16 36 hydrophobic contacts bond_interaction TM7a also forms hydrophobic contacts with the C-terminal half of TM6. RESULTS +46 61 C-terminal half structure_element TM7a also forms hydrophobic contacts with the C-terminal half of TM6. RESULTS +65 68 TM6 structure_element TM7a also forms hydrophobic contacts with the C-terminal half of TM6. RESULTS +33 42 structure evidence These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b). RESULTS +48 51 Na+ chemical These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b). RESULTS +55 59 Sext site These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b). RESULTS +107 114 helices structure_element These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b). RESULTS +38 41 TM6 structure_element This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation. RESULTS +46 49 TM1 structure_element This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation. RESULTS +138 149 transporter protein_type This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation. RESULTS +166 179 inward-facing protein_state This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation. RESULTS +21 26 TM7ab structure_element The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). RESULTS +84 88 Sext site The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). RESULTS +93 97 Smid site The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). RESULTS +105 108 SCa site The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). RESULTS +113 117 Sint site The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). RESULTS +125 133 occluded protein_state The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). RESULTS +10 20 structures evidence Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS +24 28 high protein_state Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS +33 36 low protein_state Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS +37 40 Na+ chemical Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS +70 84 outward-facing protein_state Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS +85 93 occluded protein_state Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS +98 112 partially open protein_state Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS +47 50 Na+ chemical This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa. RESULTS +64 68 Sext site This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa. RESULTS +85 88 Na+ chemical This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa. RESULTS +106 110 Sint site This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa. RESULTS +115 118 SCa site This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa. RESULTS +4 41 crystallographic titration experiment experimental_method Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS +61 65 K1/2 evidence Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS +74 77 Na+ chemical Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS +145 149 Sext site Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS +153 171 partially occupied protein_state Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS +180 186 NCX_Mj protein Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS +187 194 crystal evidence Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS +220 228 occluded protein_state Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS +233 247 partially open protein_state Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS +26 38 Na+ affinity evidence This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes. RESULTS +69 72 Na+ chemical This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes. RESULTS +100 103 NCX protein_type This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes. RESULTS +118 128 eukaryotes taxonomy_domain This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes. RESULTS +21 24 Na+ chemical The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. RESULTS +105 116 transporter protein_type The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. RESULTS +150 166 Hill coefficient evidence The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. RESULTS +174 177 Na+ chemical The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. RESULTS +210 220 eukaryotic taxonomy_domain The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. RESULTS +221 224 NCX protein_type The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. RESULTS +14 18 Ca2+ chemical Extracellular Ca2+ and Sr2+ binding and their competition with Na+ RESULTS +23 27 Sr2+ chemical Extracellular Ca2+ and Sr2+ binding and their competition with Na+ RESULTS +63 66 Na+ chemical Extracellular Ca2+ and Sr2+ binding and their competition with Na+ RESULTS +17 21 Ca2+ chemical To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods). RESULTS +31 37 NCX_Mj protein To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods). RESULTS +56 59 Na+ chemical To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods). RESULTS +70 91 titrated the crystals experimental_method To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods). RESULTS +97 101 Sr2+ chemical To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods). RESULTS +0 4 Sr2+ chemical Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity. RESULTS +23 26 NCX protein_type Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity. RESULTS +40 44 Ca2+ chemical Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity. RESULTS +75 78 Na+ chemical Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity. RESULTS +94 120 electron-density intensity evidence Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity. RESULTS +0 23 Protein crystals soaked experimental_method Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). RESULTS +35 39 Sr2+ chemical Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). RESULTS +51 54 Na+ chemical Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). RESULTS +73 94 electron-density peak evidence Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). RESULTS +103 106 SCa site Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). RESULTS +139 143 Sr2+ chemical Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). RESULTS +4 15 Sr2+-loaded protein_state The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations. RESULTS +16 22 NCX_Mj protein The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations. RESULTS +23 32 structure evidence The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations. RESULTS +44 58 partially open protein_state The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations. RESULTS +88 91 Na+ chemical The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations. RESULTS +11 15 Sr2+ chemical Binding of Sr2+, however, excludes Na+ entirely. RESULTS +35 38 Na+ chemical Binding of Sr2+, however, excludes Na+ entirely. RESULTS +0 18 Crystal titrations experimental_method Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS +24 34 decreasing experimental_method Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS +35 39 Sr2+ chemical Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS +43 53 increasing experimental_method Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS +54 57 Na+ chemical Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS +76 80 Sr2+ chemical Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS +94 108 outward-facing protein_state Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS +109 115 NCX_Mj protein Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS +170 173 Na+ chemical Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS +16 19 Na+ chemical Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS +30 34 Sr2+ chemical Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS +36 39 Na+ chemical Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS +60 64 Sr2+ chemical Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS +88 94 NCX_Mj protein Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS +102 112 Na+-loaded protein_state Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS +114 128 fully occluded protein_state Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS +8 29 titration experiments experimental_method Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +42 46 Ca2+ chemical Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +51 55 Sr2+ chemical Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +67 73 NCX_Mj protein Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +100 129 electron density distribution evidence Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +135 153 crystals soaked in experimental_method Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +154 158 high protein_state Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +159 163 Ca2+ chemical Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +168 171 low protein_state Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +172 175 Na+ chemical Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +192 196 Ca2+ chemical Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +209 213 Smid site Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +225 228 SCa site Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +252 255 SCa site Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS +11 15 Ca2+ chemical Binding of Ca2+ to both sites simultaneously is highly improbable due to their close proximity, and at least one water molecule can be discerned coordinating the ion (Fig. 3b). RESULTS +113 118 water chemical Binding of Ca2+ to both sites simultaneously is highly improbable due to their close proximity, and at least one water molecule can be discerned coordinating the ion (Fig. 3b). RESULTS +145 157 coordinating bond_interaction Binding of Ca2+ to both sites simultaneously is highly improbable due to their close proximity, and at least one water molecule can be discerned coordinating the ion (Fig. 3b). RESULTS +4 11 partial protein_state The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS +12 16 Ca2+ chemical The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS +17 26 occupancy protein_state The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS +30 34 Smid site The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS +55 61 Asp240 residue_name_number The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS +107 117 coordinate bond_interaction The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS +118 122 Ca2+ chemical The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS +9 45 functional and computational studies experimental_method Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport. RESULTS +65 71 Asp240 residue_name_number Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport. RESULTS +80 90 protonated protein_state Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport. RESULTS +16 19 NCX protein_type Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. RESULTS +29 35 Asp240 residue_name_number Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. RESULTS +39 50 substituted experimental_method Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. RESULTS +54 57 Asn residue_name Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. RESULTS +97 101 Ca2+ chemical Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. RESULTS +113 117 Smid site Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. RESULTS +0 3 SCa site SCa is therefore the functional Ca2+ site. RESULTS +32 41 Ca2+ site site SCa is therefore the functional Ca2+ site. RESULTS +13 17 Sr2+ chemical Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). RESULTS +19 23 Ca2+ chemical Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). RESULTS +39 47 affinity evidence Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). RESULTS +51 65 outward-facing protein_state Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). RESULTS +66 72 NCX_Mj protein Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). RESULTS +105 108 Na+ chemical Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). RESULTS +32 66 physiological and biochemical data evidence This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side. RESULTS +76 86 eukaryotic taxonomy_domain This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side. RESULTS +87 90 NCX protein_type This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side. RESULTS +95 101 NCX_Mj protein This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side. RESULTS +131 144 Ca2+ affinity evidence This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side. RESULTS +18 50 crystallographic titration assay experimental_method Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS +61 65 Ca2+ chemical Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS +92 100 affinity evidence Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS +147 162 Ca2+ affinities evidence Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS +196 199 NCX protein_type Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS +201 203 Km evidence Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS +219 225 NCX_Mj protein Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS +227 229 Km evidence Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS +22 51 crystal titration experiments experimental_method Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +78 91 binding sites site Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +95 109 outward-facing protein_state Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +110 116 NCX_Mj protein Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +148 152 Sint site Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +157 161 Sext site Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +166 169 Na+ chemical Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +187 190 SCa site Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +222 226 Ca2+ chemical Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +251 254 Na+ chemical Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +279 289 simulation experimental_method Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +308 312 Sr2+ chemical Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +314 318 Smid site Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +352 356 Ca2+ chemical Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +378 382 Smid site Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +410 415 water chemical Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS +4 21 ion-binding sites site The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS +25 31 NCX_Mj protein The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS +70 73 Na+ chemical The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS +122 125 Na+ chemical The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS +130 134 Ca2+ chemical The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS +139 143 Sr2+ chemical The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS +188 198 eukaryotic taxonomy_domain The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS +199 209 exchangers protein_type The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS +2 11 structure evidence A structure of NCX_Mj without Na+ or Ca2+ bound RESULTS +15 21 NCX_Mj protein A structure of NCX_Mj without Na+ or Ca2+ bound RESULTS +22 29 without protein_state A structure of NCX_Mj without Na+ or Ca2+ bound RESULTS +30 33 Na+ chemical A structure of NCX_Mj without Na+ or Ca2+ bound RESULTS +37 41 Ca2+ chemical A structure of NCX_Mj without Na+ or Ca2+ bound RESULTS +42 47 bound protein_state A structure of NCX_Mj without Na+ or Ca2+ bound RESULTS +3 6 apo protein_state An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM). RESULTS +16 30 outward-facing protein_state An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM). RESULTS +31 37 NCX_Mj protein An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM). RESULTS +140 143 Na+ chemical An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM). RESULTS +158 162 Ca2+ chemical An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM). RESULTS +29 32 apo protein_state We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS +39 48 structure evidence We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS +52 58 NCX_Mj protein We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS +63 76 crystallizing experimental_method We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS +92 100 lower pH protein_state We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS +112 122 absence of protein_state We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS +123 126 Na+ chemical We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS +5 14 structure evidence This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. RESULTS +33 47 partially open protein_state This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. RESULTS +48 57 structure evidence This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. RESULTS +67 70 Na+ chemical This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. RESULTS +85 89 Ca2+ chemical This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. RESULTS +97 101 Sr2+ chemical This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. RESULTS +7 12 TM7ab structure_element First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites. RESULTS +28 46 extracellular half structure_element First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites. RESULTS +54 57 TM6 structure_element First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites. RESULTS +62 65 TM1 structure_element First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites. RESULTS +168 181 binding sites site First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites. RESULTS +8 13 Glu54 residue_name_number Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS +18 24 Glu213 residue_name_number Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS +58 71 binding sites site Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS +91 105 hydrogen-bonds bond_interaction Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS +132 148 ion coordination bond_interaction Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS +156 172 fully Na+-loaded protein_state Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS +173 182 structure evidence Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS +13 26 binding sites site Although the binding sites are thus fully accessible to the external solution (Fig. 3e), the lack of electron density therein indicates no ions or ordered solvent molecules. RESULTS +36 52 fully accessible protein_state Although the binding sites are thus fully accessible to the external solution (Fig. 3e), the lack of electron density therein indicates no ions or ordered solvent molecules. RESULTS +101 117 electron density evidence Although the binding sites are thus fully accessible to the external solution (Fig. 3e), the lack of electron density therein indicates no ions or ordered solvent molecules. RESULTS +5 8 apo protein_state This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. RESULTS +9 18 structure evidence This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. RESULTS +49 57 unloaded protein_state This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. RESULTS +59 63 open protein_state This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. RESULTS +73 87 outward-facing protein_state This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. RESULTS +88 94 NCX_Mj protein This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. RESULTS +20 29 structure evidence Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind. RESULTS +46 62 fully protonated protein_state Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind. RESULTS +76 87 transporter protein_type Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind. RESULTS +98 101 Na+ chemical Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind. RESULTS +106 110 Ca2+ chemical Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind. RESULTS +49 69 computer simulations experimental_method Such interpretation would be consistent with the computer simulations reported below. RESULTS +8 24 transport assays experimental_method Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS +28 34 NCX_Mj protein Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS +63 74 presence of protein_state Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS +75 78 Na+ chemical Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS +82 86 Ca2+ chemical Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS +88 94 low pH protein_state Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS +95 106 inactivates protein_state Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS +54 60 NCX_Mj protein Ion occupancy determines the free-energy landscape of NCX_Mj RESULTS +5 34 secondary-active transporters protein_type That secondary-active transporters are able to harness an electrochemical gradient of one substrate to power the uphill transport of another relies on a seemingly simple principle: they must not transition between outward- and inward-open conformations unless in two precise substrate occupancy states. RESULTS +214 222 outward- protein_state That secondary-active transporters are able to harness an electrochemical gradient of one substrate to power the uphill transport of another relies on a seemingly simple principle: they must not transition between outward- and inward-open conformations unless in two precise substrate occupancy states. RESULTS +227 238 inward-open protein_state That secondary-active transporters are able to harness an electrochemical gradient of one substrate to power the uphill transport of another relies on a seemingly simple principle: they must not transition between outward- and inward-open conformations unless in two precise substrate occupancy states. RESULTS +0 3 NCX protein_type NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS +33 36 Na+ chemical NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS +42 46 Ca2+ chemical NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS +78 88 antiporter protein_type NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS +90 100 symporters protein_type NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS +217 222 bound protein_state NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS +234 237 apo protein_state NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS +64 100 conformational free-energy landscape evidence To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations. RESULTS +104 110 NCX_Mj protein To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations. RESULTS +175 193 molecular dynamics experimental_method To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations. RESULTS +195 197 MD experimental_method To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations. RESULTS +199 210 simulations experimental_method To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations. RESULTS +62 71 structure evidence This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above. RESULTS +75 81 NCX_Mj protein This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above. RESULTS +104 128 crystallographic studies experimental_method This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above. RESULTS +44 54 structures evidence As it happens, the results confirm that the structures now available are representing interconverting states of the functional cycle of NCX_Mj, while revealing how the alternating-access mechanism is controlled by the ion-occupancy state. RESULTS +136 142 NCX_Mj protein As it happens, the results confirm that the structures now available are representing interconverting states of the functional cycle of NCX_Mj, while revealing how the alternating-access mechanism is controlled by the ion-occupancy state. RESULTS +24 38 MD simulations experimental_method A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy. RESULTS +97 103 NCX_Mj protein A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy. RESULTS +104 113 structure evidence A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy. RESULTS +134 151 ion-binding sites site A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy. RESULTS +23 32 simulated experimental_method Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b). RESULTS +37 53 outward-occluded protein_state Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b). RESULTS +131 139 high-Na+ protein_state Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b). RESULTS +140 157 crystal structure evidence Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b). RESULTS +9 12 Na+ chemical That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS +25 29 Sext site That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS +31 34 SCa site That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS +40 44 Sint site That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS +52 56 D240 residue_name_number That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS +60 70 protonated protein_state That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS +77 82 water chemical That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS +101 105 Smid site That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS +4 7 Na+ chemical The Na+ ion at Sext was then relocated from the site to the bulk solution (Methods), and this system was then allowed to evolve freely in time. RESULTS +15 19 Sext site The Na+ ion at Sext was then relocated from the site to the bulk solution (Methods), and this system was then allowed to evolve freely in time. RESULTS +4 7 Na+ chemical The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out. RESULTS +16 19 SCa site The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out. RESULTS +24 28 Sint site The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out. RESULTS +75 85 simulation experimental_method The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out. RESULTS +14 25 simulations experimental_method These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures. RESULTS +61 72 transporter protein_type These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures. RESULTS +116 134 crystal structures evidence These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures. RESULTS +45 48 Na+ chemical The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a). RESULTS +54 58 Sext site The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a). RESULTS +84 89 TM7ab structure_element The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a). RESULTS +7 10 Na+ chemical When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS +20 25 bound protein_state When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS +27 32 TM7ab structure_element When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS +80 99 α-helical fragments structure_element When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS +150 164 hydrogen-bonds bond_interaction When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS +173 177 F202 residue_name_number When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS +182 186 A206 residue_name_number When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS +192 196 T203 residue_name_number When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS +201 205 F207 residue_name_number When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS +25 29 Sext site This distortion occludes Sext from the exterior (Fig. 4d, 4h-i) and appears to be induced by the Na+ ion itself, which pulls the carbonyl group of A206 into its coordination sphere (Fig. 4g). RESULTS +97 100 Na+ chemical This distortion occludes Sext from the exterior (Fig. 4d, 4h-i) and appears to be induced by the Na+ ion itself, which pulls the carbonyl group of A206 into its coordination sphere (Fig. 4g). RESULTS +147 151 A206 residue_name_number This distortion occludes Sext from the exterior (Fig. 4d, 4h-i) and appears to be induced by the Na+ ion itself, which pulls the carbonyl group of A206 into its coordination sphere (Fig. 4g). RESULTS +5 9 Sext site With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS +10 15 empty protein_state With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS +26 31 TM7ab structure_element With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS +50 57 α-helix structure_element With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS +111 114 TM3 structure_element With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS +119 122 TM7 structure_element With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS +145 150 water chemical With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS +202 206 Sext site With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS +233 244 transporter protein_type With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS +248 266 no longer occluded protein_state With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS +16 19 Na+ chemical Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c). RESULTS +25 28 SCa site Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c). RESULTS +33 37 Sint site Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c). RESULTS +55 58 TM7 structure_element The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e). RESULTS +63 66 TM2 structure_element The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e). RESULTS +151 154 Na+ chemical The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e). RESULTS +162 165 SCa site The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e). RESULTS +0 3 TM1 structure_element TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c). RESULTS +8 11 TM6 structure_element TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c). RESULTS +76 92 outward-occluded protein_state TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c). RESULTS +38 53 aqueous channel site Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i). RESULTS +76 79 SCa site Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i). RESULTS +84 88 Sint site Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i). RESULTS +4 15 transporter protein_type The transporter thus becomes fully outward-open. RESULTS +29 47 fully outward-open protein_state The transporter thus becomes fully outward-open. RESULTS +111 120 exchanger protein_type To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods). RESULTS +167 182 MD calculations experimental_method To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods). RESULTS +242 258 outward-occluded protein_state To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods). RESULTS +263 281 fully outward-open protein_state To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods). RESULTS +312 333 free-energy landscape evidence To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods). RESULTS +68 71 Na+ chemical As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS +75 79 Sext site As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS +81 84 SCa site As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS +89 93 Sint site As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS +100 103 Na+ chemical As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS +112 115 SCa site As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS +120 124 Sint site As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS +130 137 without protein_state As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS +138 141 Na+ chemical As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS +6 18 calculations experimental_method These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape. RESULTS +40 43 Na+ chemical These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape. RESULTS +67 78 transporter protein_type These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape. RESULTS +108 144 conformational free-energy landscape evidence These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape. RESULTS +9 18 Na+ sites site When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). RESULTS +44 63 free-energy minimum evidence When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). RESULTS +168 173 TM7ab structure_element When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). RESULTS +205 208 TM2 structure_element When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). RESULTS +213 216 TM3 structure_element When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). RESULTS +229 242 binding sites site When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). RESULTS +40 51 transporter protein_type At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). RESULTS +64 74 metastable protein_state At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). RESULTS +76 85 half-open protein_state At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). RESULTS +109 114 TM7ab structure_element At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). RESULTS +142 146 Sext site At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). RESULTS +150 154 open protein_state At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). RESULTS +4 7 Na+ chemical The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c). RESULTS +15 19 Sext site The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c). RESULTS +28 45 fully coordinated protein_state The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c). RESULTS +62 67 water chemical The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c). RESULTS +111 115 A206 residue_name_number The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c). RESULTS +147 151 F202 residue_name_number The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c). RESULTS +154 158 A206 residue_name_number The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c). RESULTS +161 174 hydrogen-bond bond_interaction The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c). RESULTS +5 14 semi-open protein_state This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS +91 94 Na+ chemical This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS +109 112 SCa site This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS +117 121 Sint site This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS +127 130 Na+ chemical This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS +185 188 Na+ chemical This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS +192 196 Sext site This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS +212 222 metastable protein_state This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS +92 107 aqueous channel site Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS +141 144 SCa site Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS +153 156 TM7 structure_element Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS +161 164 TM2 structure_element Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS +178 192 gating helices structure_element Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS +193 196 TM1 structure_element Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS +201 204 TM6 structure_element Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS +219 223 open protein_state Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS +23 44 free-energy landscape evidence Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a). RESULTS +54 72 partially occupied protein_state Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a). RESULTS +101 109 occluded protein_state Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a). RESULTS +34 37 Na+ chemical Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS +48 51 SCa site Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS +56 60 Sint site Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS +82 103 free-energy landscape evidence Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS +111 122 transporter protein_type Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS +170 180 fully open protein_state Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS +205 221 aqueous channels site Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS +22 30 occluded protein_state The transition to the occluded state in this apo state is again energetically unfeasible. RESULTS +45 48 apo protein_state The transition to the occluded state in this apo state is again energetically unfeasible. RESULTS +67 71 open protein_state From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS +76 85 semi-open protein_state From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS +132 135 Na+ chemical From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS +175 178 Na+ chemical From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS +287 308 free-energy landscape evidence From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS +316 327 transporter protein_type From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS +346 354 occluded protein_state From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS +392 417 Na+ occupancy is complete protein_state From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS +41 44 Na+ chemical This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS +93 103 Ca2+-bound protein_state This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS +144 155 transporter protein_type This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS +223 235 outward-open protein_state This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS +276 280 Ca2+ chemical This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS +295 303 occluded protein_state This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS +331 340 semi-open protein_state This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS +385 396 inward-open protein_state This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS +26 28 H+ chemical By contrast, occupancy by H+, which as mentioned are not transported, might be compatible with a semi-open state as well as with the fully open conformation, but should not be conducive to occlusion. RESULTS +97 106 semi-open protein_state By contrast, occupancy by H+, which as mentioned are not transported, might be compatible with a semi-open state as well as with the fully open conformation, but should not be conducive to occlusion. RESULTS +133 143 fully open protein_state By contrast, occupancy by H+, which as mentioned are not transported, might be compatible with a semi-open state as well as with the fully open conformation, but should not be conducive to occlusion. RESULTS +42 71 enhanced-sampling simulations experimental_method To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS +80 84 Ca2+ protein_state To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS +89 97 H+-bound protein_state To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS +108 122 outward-facing protein_state To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS +123 129 NCX_Mj protein To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS +169 172 Na+ chemical To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS +249 259 structures evidence To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS +267 277 Ca2+-bound protein_state To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS +4 14 calculated experimental_method The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +15 36 free-energy landscape evidence The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +41 51 Ca2+-bound protein_state The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +52 58 NCX_Mj protein The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +103 107 Ca2+ chemical The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +149 155 NCX_Mj protein The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +176 180 Ca2+ chemical The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +186 194 occluded protein_state The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +196 206 dehydrated protein_state The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +274 283 exchanger protein_type The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +303 312 semi-open protein_state The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +317 321 open protein_state The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +356 360 Ca2+ chemical The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +373 376 Na+ chemical The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +402 425 aqueous access channels site The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +439 443 Sext site The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +448 451 SCa site The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS +13 24 protonation protein_state By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +28 33 Glu54 residue_name_number By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +38 44 Glu213 residue_name_number By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +55 63 occluded protein_state By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +133 139 NCX_Mj protein By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +159 166 protons chemical By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +176 184 H+-bound protein_state By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +204 213 exchanger protein_type By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +228 237 semi-open protein_state By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +267 273 low pH protein_state By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +275 278 apo protein_state By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +279 296 crystal structure evidence By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +302 304 H+ chemical By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS +21 54 systematic computational analysis experimental_method Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. RESULTS +58 72 outward-facing protein_state Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. RESULTS +73 79 NCX_Mj protein Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. RESULTS +168 186 Na+/Ca2+ exchanger protein_type Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. RESULTS +253 274 free-energy landscape evidence Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. RESULTS +335 343 occluded protein_state Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. RESULTS +5 13 occluded protein_state This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. RESULTS +74 81 outward protein_state This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. RESULTS +86 97 inward-open protein_state This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. RESULTS +137 148 dehydration protein_state This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. RESULTS +189 207 complete occupancy protein_state This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. RESULTS +215 228 binding sites site This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. RESULTS +78 85 outward protein_state The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized. DISCUSS +91 102 inward-open protein_state The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized. DISCUSS +128 144 secondary-active protein_state The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized. DISCUSS +145 156 transporter protein_type The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized. DISCUSS +32 43 antiporters protein_type It is however also non-trivial: antiporters, for example, do not undergo the alternating-access transition without a cargo, but this is precisely how membrane symporters reset their transport cycles. DISCUSS +150 169 membrane symporters protein_type It is however also non-trivial: antiporters, for example, do not undergo the alternating-access transition without a cargo, but this is precisely how membrane symporters reset their transport cycles. DISCUSS +35 45 antiporter protein_type Similarly puzzling is that a given antiporter will undergo this transition upon recognition of substrates of different charge, size and number. DISCUSS +67 77 antiporter protein_type Yet, when multiple species are to be co-translocated, by either an antiporter or a symporter, partial occupancies must not be conducive to the alternating-access switch. DISCUSS +83 92 symporter protein_type Yet, when multiple species are to be co-translocated, by either an antiporter or a symporter, partial occupancies must not be conducive to the alternating-access switch. DISCUSS +143 168 alternating-access switch site Yet, when multiple species are to be co-translocated, by either an antiporter or a symporter, partial occupancies must not be conducive to the alternating-access switch. DISCUSS +103 121 structural studies experimental_method Here, we have provided novel insights into this intriguing mechanism of conformational control through structural studies and quantitative molecular simulations of a Na+/Ca2+ exchanger. DISCUSS +126 160 quantitative molecular simulations experimental_method Here, we have provided novel insights into this intriguing mechanism of conformational control through structural studies and quantitative molecular simulations of a Na+/Ca2+ exchanger. DISCUSS +166 184 Na+/Ca2+ exchanger protein_type Here, we have provided novel insights into this intriguing mechanism of conformational control through structural studies and quantitative molecular simulations of a Na+/Ca2+ exchanger. DISCUSS +29 35 NCX_Mj protein Specifically, our studies of NCX_Mj reveal the mechanism of forward ion exchange (Fig. 7). DISCUSS +25 39 outward-facing protein_state The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS +40 46 NCX_Mj protein The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS +55 68 inward-facing protein_state The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS +69 87 crystal structures evidence The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS +99 117 Ca2+/H+ exchangers protein_type The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS +168 171 NCX protein_type The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS +209 212 TM1 structure_element The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS +217 220 TM6 structure_element The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS +249 260 transporter protein_type The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS +56 76 extracellular region structure_element Here, we demonstrate that conformational changes in the extracellular region of the TM2-TM3 and TM7-TM8 bundle precede and are necessary for the transition, and are associated with ion recognition and/or release. DISCUSS +84 91 TM2-TM3 structure_element Here, we demonstrate that conformational changes in the extracellular region of the TM2-TM3 and TM7-TM8 bundle precede and are necessary for the transition, and are associated with ion recognition and/or release. DISCUSS +96 110 TM7-TM8 bundle structure_element Here, we demonstrate that conformational changes in the extracellular region of the TM2-TM3 and TM7-TM8 bundle precede and are necessary for the transition, and are associated with ion recognition and/or release. DISCUSS +48 63 N-terminal half structure_element The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS +67 70 TM7 structure_element The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS +72 77 TM7ab structure_element The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS +123 126 TM2 structure_element The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS +131 134 TM3 structure_element The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS +151 156 TM7ab structure_element The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS +213 229 aqueous channels site The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS +247 264 ion-binding sites site The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS +30 33 TM7 structure_element Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. DISCUSS +71 88 ion-binding sites site Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. DISCUSS +123 126 TM6 structure_element Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. DISCUSS +145 148 TM6 structure_element Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. DISCUSS +153 156 TM1 structure_element Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. DISCUSS +180 193 inward-facing protein_state Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. DISCUSS +4 22 crystal structures evidence The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS +26 32 NCX_Mj protein The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS +60 64 Na+, chemical The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS +65 70 Ca2+, chemical The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS +71 75 Sr2+ chemical The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS +79 81 H+ chemical The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS +82 87 bound protein_state The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS +101 110 exchanger protein_type The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS +115 126 transporter protein_type These states can only represent a subset among all possible, but they ought to reflect inherent preferences of the transporter, modulated by the experimental conditions. DISCUSS +20 27 crystal evidence For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS +31 37 NCX_Mj protein For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS +41 44 LCP experimental_method For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS +50 68 extracellular half structure_element For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS +76 90 gating helices structure_element For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS +92 95 TM6 structure_element For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS +100 103 TM1 structure_element For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS +190 207 ion-binding sites site For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS +235 238 apo protein_state For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS +240 246 low pH protein_state For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS +247 256 structure evidence For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS +17 50 calculated free-energy landscapes evidence Nonetheless, the calculated free-energy landscapes, derived without knowledge of the experimental data, reassuringly confirm that the crystallized structures correspond to mechanistically relevant, interconverting states. DISCUSS +134 157 crystallized structures evidence Nonetheless, the calculated free-energy landscapes, derived without knowledge of the experimental data, reassuringly confirm that the crystallized structures correspond to mechanistically relevant, interconverting states. DISCUSS +4 15 simulations experimental_method The simulations also demonstrate how this landscape is drastically re-shaped upon each ion-binding event. DISCUSS +58 66 occluded protein_state Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry. DISCUSS +130 136 NCX_Mj protein Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry. DISCUSS +146 149 Na+ chemical Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry. DISCUSS +151 155 Ca2+ chemical Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry. DISCUSS +89 101 transporters protein_type We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms. DISCUSS +140 150 symporters protein_type We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms. DISCUSS +155 166 antiporters protein_type We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms. DISCUSS +197 205 occluded protein_state We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms. DISCUSS +228 272 protein conformational free-energy landscape evidence We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms. DISCUSS +122 128 NCX_Mj protein In multiple ways, our findings provide an explanation for, existing functional, biochemical and biophysical data for both NCX_Mj and its eukaryotic homologues. DISCUSS +137 147 eukaryotic taxonomy_domain In multiple ways, our findings provide an explanation for, existing functional, biochemical and biophysical data for both NCX_Mj and its eukaryotic homologues. DISCUSS +48 70 ion-binding affinities evidence The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above. DISCUSS +89 116 crystallographic titrations experimental_method The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above. DISCUSS +125 127 Km evidence The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above. DISCUSS +132 143 K1/2 values evidence The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above. DISCUSS +168 185 functional assays experimental_method The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above. DISCUSS +113 119 NCX_Mj protein Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d). DISCUSS +132 135 NCX protein_type Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d). DISCUSS +193 199 NCX_Mj protein Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d). DISCUSS +200 210 structures evidence Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d). DISCUSS +4 25 crystallographic data evidence The crystallographic data also provides the long-sought structural basis for the ‘two-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger. DISCUSS +149 159 eukaryotic taxonomy_domain The crystallographic data also provides the long-sought structural basis for the ‘two-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger. DISCUSS +160 163 NCX protein_type The crystallographic data also provides the long-sought structural basis for the ‘two-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger. DISCUSS +212 218 NCX_Mj protein The crystallographic data also provides the long-sought structural basis for the ‘two-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger. DISCUSS +237 255 Na+/Ca2+ exchanger protein_type The crystallographic data also provides the long-sought structural basis for the ‘two-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger. DISCUSS +18 36 crystal titrations experimental_method Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +66 69 Na+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +70 74 Ca2+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +91 95 Sint site Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +100 103 SCa site Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +111 115 Ca2+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +120 123 Na+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +162 165 Na+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +211 214 Na+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +239 242 Na+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +261 266 water chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +280 284 Smid site Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +313 317 Ca2+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +334 350 Hill coefficient evidence Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +361 364 Na+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +389 393 Ca2+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS +4 8 Sext site The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. DISCUSS +51 66 activation site site The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. DISCUSS +78 81 Na+ chemical The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. DISCUSS +127 130 Na+ chemical The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. DISCUSS +149 152 Na+ chemical The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. DISCUSS +199 207 occluded protein_state The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. DISCUSS +26 30 Ca2+ chemical Interestingly, binding of Ca2+ to Smid appears to be also possible, but available evidence indicates that this event transiently blocks the exchange cycle. DISCUSS +34 38 Smid site Interestingly, binding of Ca2+ to Smid appears to be also possible, but available evidence indicates that this event transiently blocks the exchange cycle. DISCUSS +8 18 structures evidence Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS +22 28 NCX_Mj protein Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS +29 37 bound to protein_state Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS +38 42 Cd2+ chemical Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS +46 50 Mn2+ chemical Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS +104 108 Smid site Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS +123 127 Sr2+ chemical Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS +142 145 SCa site Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS +182 185 NCX protein_type Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS +199 206 calcium chemical Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS +37 43 NCX_Mj protein Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS +72 76 Ca2+ chemical Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS +80 83 Na+ chemical Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS +147 174 hydrogen-deuterium exchange experimental_method Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS +176 179 HDX experimental_method Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS +184 190 NCX_Mj protein Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS +199 207 presence protein_state Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS +211 221 absence of protein_state Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS +259 273 outward-facing protein_state Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS +36 40 Ca2+ chemical Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices. DISCUSS +44 47 Na+ chemical Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices. DISCUSS +89 97 HDX rate evidence Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices. DISCUSS +132 148 α-repeat helices structure_element Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices. DISCUSS +231 248 ion-binding sites site We interpret these observations as reflecting that the solvent accessibility of the protein interior is diminished upon ion recognition, consistent with our finding that opening and closing of extracellular aqueous pathways to the ion-binding sites depend on ion occupancy state. DISCUSS +80 88 occluded protein_state In addition, the increased compactness of the protein tertiary structure in the occluded state would also slow down the dynamics of the secondary-structure elements, and thus further reduce the HDX rate. DISCUSS +194 202 HDX rate evidence In addition, the increased compactness of the protein tertiary structure in the occluded state would also slow down the dynamics of the secondary-structure elements, and thus further reduce the HDX rate. DISCUSS +70 78 HDX rate evidence Our data would also explain the observation that the reduction in the HDX rate is comparable for Na+ and Ca2+, as well as the finding that the degree of deuterium incorporation remains non-negligible even under saturating ion concentrations. DISCUSS +97 100 Na+ chemical Our data would also explain the observation that the reduction in the HDX rate is comparable for Na+ and Ca2+, as well as the finding that the degree of deuterium incorporation remains non-negligible even under saturating ion concentrations. DISCUSS +105 109 Ca2+ chemical Our data would also explain the observation that the reduction in the HDX rate is comparable for Na+ and Ca2+, as well as the finding that the degree of deuterium incorporation remains non-negligible even under saturating ion concentrations. DISCUSS +7 40 calculated free-energy landscapes evidence As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. DISCUSS +47 50 Na+ chemical As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. DISCUSS +55 59 Ca2+ chemical As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. DISCUSS +88 99 transporter protein_type As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. DISCUSS +136 145 ion-bound protein_state As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. DISCUSS +219 229 fully open protein_state As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. DISCUSS +0 3 Na+ chemical Na+ binding to outward-facing NCX_Mj. FIG +15 29 outward-facing protein_state Na+ binding to outward-facing NCX_Mj. FIG +30 36 NCX_Mj protein Na+ binding to outward-facing NCX_Mj. FIG +12 21 structure evidence (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. FIG +25 31 native protein_state (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. FIG +32 46 outward-facing protein_state (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. FIG +47 53 NCX_Mj protein (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. FIG +59 73 crystals grown experimental_method (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. FIG +84 87 Na+ chemical (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. FIG +36 39 Na+ chemical Colored spheres represent the bound Na+ (green) and water (red). FIG +52 57 water chemical Colored spheres represent the bound Na+ (green) and water (red). FIG +50 71 central binding sites site (b) Structural details and definition of the four central binding sites. FIG +4 20 electron density evidence The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG +39 57 Fo-Fc ion omit map evidence The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG +78 82 Smid site The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG +98 103 water chemical The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG +130 134 Sext site The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG +136 139 SCa site The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG +144 148 Sint site The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG +152 155 Na+ chemical The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG +89 92 Na+ chemical Further details are shown in Supplementary Fig. 1. (c) Concentration-dependent change in Na+ occupancy (see also Table 1). FIG +4 25 Fo – Fc ion-omit maps evidence All Fo – Fc ion-omit maps are calculated to 2.4 Å and contoured at 3σ for comparison. FIG +20 24 A206 residue_name_number The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively). FIG +39 42 Na+ chemical The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively). FIG +85 99 partially open protein_state The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively). FIG +107 115 occluded protein_state The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively). FIG +148 151 Na+ chemical The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively). FIG +9 12 Na+ chemical At 20 mM Na+, both conformations co-exist. FIG +75 80 water chemical No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites. FIG +101 104 SCa site No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites. FIG +106 110 Sint site No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites. FIG +115 119 Smid site No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites. FIG +0 3 Na+ chemical Na+-occupancy dependent conformational change in NCX_Mj. FIG +49 55 NCX_Mj protein Na+-occupancy dependent conformational change in NCX_Mj. FIG +4 19 Superimposition experimental_method (a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations. FIG +27 33 NCX_Mj protein (a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations. FIG +34 52 crystal structures evidence (a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations. FIG +128 131 Na+ chemical (a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations. FIG +25 34 interface site (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). FIG +43 46 TM6 structure_element (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). FIG +51 56 TM7ab structure_element (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). FIG +64 70 NCX_Mj protein (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). FIG +71 81 structures evidence (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). FIG +107 110 Na+ chemical (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). FIG +47 56 structure evidence Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail. FIG +60 63 low protein_state Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail. FIG +64 67 Na+ chemical Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail. FIG +25 42 Na+-binding sites site (c) Close-up view of the Na+-binding sites. FIG +11 15 Sext site The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere. FIG +28 37 structure evidence The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere. FIG +41 44 low protein_state The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere. FIG +45 48 Na+ chemical The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere. FIG +56 60 A206 residue_name_number Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen. FIG +78 89 coordinates bond_interaction Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen. FIG +90 93 Na+ chemical Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen. FIG +97 101 Sext site Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen. FIG +47 64 ion binding sites site (d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+]. FIG +72 82 structures evidence (d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+]. FIG +86 90 high protein_state (d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+]. FIG +95 98 low protein_state (d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+]. FIG +100 103 Na+ chemical (d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+]. FIG +9 25 solvent channels site Putative solvent channels are represented as light-purple surfaces. FIG +28 31 apo protein_state Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +32 41 structure evidence Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +45 51 NCX_Mj protein Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +66 70 Sr2+ chemical Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +99 102 SCa site Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +106 123 crystals titrated experimental_method Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +135 139 Sr2+ chemical Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +151 154 Na+ chemical Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +21 25 Sr2+ chemical Residues involved in Sr2+ coordination are labeled. FIG +98 107 Na+-bound protein_state There are no significant changes in the side-chains involved in ion coordination, relative to the Na+-bound state. FIG +0 3 T50 residue_name_number T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms. FIG +8 12 T209 residue_name_number T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms. FIG +30 40 coordinate bond_interaction T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms. FIG +41 45 Sr2+ chemical T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms. FIG +5 8 Na+ chemical High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel). FIG +53 57 Sr2+ chemical High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel). FIG +70 76 NCX_Mj protein High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel). FIG +84 92 occluded protein_state High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel). FIG +25 41 Fo – Fc omit map evidence The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. FIG +49 58 structure evidence The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. FIG +67 70 Na+ chemical The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. FIG +128 135 density evidence The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. FIG +141 144 Na+ chemical The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. FIG +154 157 H2O chemical The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. FIG +4 8 Ca2+ chemical (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +42 45 SCa site (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +49 53 Smid site (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +57 74 crystals titrated experimental_method (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +86 90 Ca2+ chemical (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +102 105 Na+ chemical (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG +60 75 Superimposition experimental_method The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG +79 85 NCX_Mj protein The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG +86 96 structures evidence The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG +113 116 Na+ chemical The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG +169 179 absence of protein_state The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG +180 183 Na+ chemical The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG +188 192 pH 4 protein_state The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG +223 226 apo protein_state The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG +259 276 ion-binding sites site The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG +284 287 apo protein_state The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG +292 299 high H+ protein_state The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG +19 22 E54 residue_name_number The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison. FIG +27 31 E213 residue_name_number The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison. FIG +41 48 low Na+ protein_state The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison. FIG +49 58 structure evidence The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison. FIG +36 40 Sint site White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. FIG +42 46 Smid site White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. FIG +47 50 SCa site White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. FIG +99 116 ion-binding sites site White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. FIG +120 123 apo protein_state White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. FIG +124 130 NCX_Mj protein White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. FIG +27 36 structure evidence Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+. FIG +40 56 outward-occluded protein_state Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+. FIG +58 76 fully Na+-occupied protein_state Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+. FIG +77 83 NCX_Mj protein Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+. FIG +132 135 Na+ chemical Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+. FIG +19 29 simulation experimental_method (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +43 49 NCX_Mj protein (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +65 68 Na+ chemical (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +69 77 bound at protein_state (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +78 82 Sext site (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +84 87 SCa site (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +92 96 Sint site (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +139 142 Na+ chemical (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +143 156 bound only at protein_state (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +157 160 SCa site (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +165 169 Sint site (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +240 255 N-terminal half structure_element (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +259 262 TM7 structure_element (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +264 269 TM7ab structure_element (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +284 287 Na+ chemical (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG +19 39 simulation snapshots evidence (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG +61 64 Na+ chemical (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG +65 73 bound at protein_state (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG +74 77 SCa site (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG +82 86 Sint site (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG +125 132 without protein_state (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG +137 140 Na+ chemical (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG +141 146 bound protein_state (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG +20 38 ion-binding region site (d) Close-up of the ion-binding region in the fully Na+-occupied state. FIG +46 64 fully Na+-occupied protein_state (d) Close-up of the ion-binding region in the fully Na+-occupied state. FIG +30 33 TM2 structure_element Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state. FIG +35 38 TM3 structure_element Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state. FIG +43 46 TM7 structure_element Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state. FIG +87 105 ion-binding region site Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state. FIG +113 135 partially Na+-occupied protein_state Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state. FIG +20 38 ion-binding region site (f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f). FIG +46 54 Na+-free protein_state (f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f). FIG +142 153 simulations experimental_method (f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f). FIG +162 175 Na+-occupancy protein_state (f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f). FIG +63 101 Bias-Exchange Metadynamics simulations experimental_method These descriptors were employed as collective variables in the Bias-Exchange Metadynamics simulations (Methods). FIG +4 29 Probability distributions evidence (g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1). FIG +74 90 hydrogen-bonding bond_interaction (g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1). FIG +102 107 TM7ab structure_element (g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1). FIG +223 227 Sext site (g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1). FIG +348 351 SCa site (g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1). FIG +108 111 NCX protein_type Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. FIG +117 165 Calculated conformational free-energy landscapes evidence Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. FIG +170 184 outward-facing protein_state Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. FIG +185 191 NCX_Mj protein Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. FIG +211 214 Na+ chemical Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. FIG +254 267 no ions bound protein_state Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. FIG +4 15 free energy evidence The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods). FIG +106 122 aqueous channels site The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods). FIG +138 142 Sext site The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods). FIG +147 150 SCa site The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods). FIG +22 38 X-ray structures evidence Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. FIG +42 48 NCX_Mj protein Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. FIG +61 65 high protein_state Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. FIG +70 73 low protein_state Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. FIG +74 77 Na+ chemical Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. FIG +112 118 low pH protein_state Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. FIG +4 23 Density isosurfaces evidence (b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state. FIG +28 33 water chemical (b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state. FIG +63 81 ion-binding region site (b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state. FIG +120 153 conformational free-energy minima evidence (b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state. FIG +0 3 Na+ chemical Na+ ions are shown as green spheres. FIG +8 33 inverted-topology repeats structure_element The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine). FIG +41 52 transporter protein_type The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine). FIG +53 62 structure evidence The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine). FIG +111 116 TM1-5 structure_element The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine). FIG +126 132 TM6-10 structure_element The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine). FIG +26 44 ion-binding region site (c) Close-up views of the ion-binding region in the same conformational free-energy minima. FIG +57 90 conformational free-energy minima evidence (c) Close-up views of the ion-binding region in the same conformational free-energy minima. FIG +25 28 Na+ chemical Key residues involved in Na+ and water coordination (W) are highlighted (sticks, black lines). FIG +33 38 water chemical Key residues involved in Na+ and water coordination (W) are highlighted (sticks, black lines). FIG +4 22 water-density maps evidence The water-density maps in (b) is shown here as a grey mesh. FIG +5 9 D240 residue_name_number Note D240 is protonated, while E54 and E213 are ionized. FIG +31 34 E54 residue_name_number Note D240 is protonated, while E54 and E213 are ionized. FIG +39 43 E213 residue_name_number Note D240 is protonated, while E54 and E213 are ionized. FIG +108 111 NCX protein_type Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. FIG +117 150 Calculated free-energy landscapes evidence Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. FIG +155 169 outward-facing protein_state Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. FIG +170 176 NCX_Mj protein Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. FIG +186 190 Ca2+ chemical Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. FIG +199 215 fully protonated protein_state Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. FIG +4 15 free energy evidence The free energy is plotted as in Fig. 5. FIG +4 8 Ca2+ chemical For Ca2+, a map is shown in which a correction for the charge-transfer between the ion and the protein is introduced, alongside the uncorrected map (see Supplementary Notes 3-4 and Supplementary Fig. 5-6). FIG +12 15 map evidence For Ca2+, a map is shown in which a correction for the charge-transfer between the ion and the protein is introduced, alongside the uncorrected map (see Supplementary Notes 3-4 and Supplementary Fig. 5-6). FIG +144 147 map evidence For Ca2+, a map is shown in which a correction for the charge-transfer between the ion and the protein is introduced, alongside the uncorrected map (see Supplementary Notes 3-4 and Supplementary Fig. 5-6). FIG +16 19 map evidence The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). FIG +39 43 open protein_state The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). FIG +66 75 semi-open protein_state The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). FIG +80 88 occluded protein_state The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). FIG +166 174 bound to protein_state The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). FIG +218 221 Na+ chemical The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). FIG +22 40 crystal structures evidence Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study. FIG +58 62 Ca2+ chemical Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study. FIG +84 90 low pH protein_state Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study. FIG +95 102 high H+ protein_state Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study. FIG +4 29 Water-density isosurfaces evidence (b) Water-density isosurfaces analogous to those in Fig. 5 are shown for each of the major conformational free-energy minima in the free-energy maps. FIG +106 124 free-energy minima evidence (b) Water-density isosurfaces analogous to those in Fig. 5 are shown for each of the major conformational free-energy minima in the free-energy maps. FIG +132 148 free-energy maps evidence (b) Water-density isosurfaces analogous to those in Fig. 5 are shown for each of the major conformational free-energy minima in the free-energy maps. FIG +4 8 Ca2+ chemical The Ca2+ ion is shown as a red sphere; the protein is shown as in Fig. 5. (c) Close-up views of the ion-binding region in the same conformational free-energy minima. FIG +100 118 ion-binding region site The Ca2+ ion is shown as a red sphere; the protein is shown as in Fig. 5. (c) Close-up views of the ion-binding region in the same conformational free-energy minima. FIG +131 164 conformational free-energy minima evidence The Ca2+ ion is shown as a red sphere; the protein is shown as in Fig. 5. (c) Close-up views of the ion-binding region in the same conformational free-energy minima. FIG +25 29 Ca2+ chemical Key residues involved in Ca2+ and water coordination (W) are highlighted (sticks, black lines). FIG +34 39 water chemical Key residues involved in Ca2+ and water coordination (W) are highlighted (sticks, black lines). FIG +4 22 water-density maps evidence The water-density maps in (b) are shown here as a grey mesh. FIG +7 15 occluded protein_state In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG +27 31 Ca2+ chemical In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG +32 37 bound protein_state In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG +39 44 helix structure_element In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG +45 50 TM7ab structure_element In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG +83 97 fully occupied protein_state In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG +98 101 Na+ chemical In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG +128 134 Ala206 residue_name_number In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG +143 171 hydrogen-bonding interaction bond_interaction In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG +177 183 Ser210 residue_name_number In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG +62 65 NCX protein_type Structural mechanism of extracellular forward ion exchange in NCX. FIG +23 28 Ala47 residue_name_number The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG +33 37 TM2b structure_element The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG +43 49 Ala206 residue_name_number The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG +54 58 TM7b structure_element The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG +84 89 Glu54 residue_name_number The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG +94 98 TM2c structure_element The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG +104 110 Glu213 residue_name_number The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG +115 119 TM7c structure_element The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG +39 53 gating helices structure_element The green open cylinders represent the gating helices TM1 and TM6. FIG +54 57 TM1 structure_element The green open cylinders represent the gating helices TM1 and TM6. FIG +62 65 TM6 structure_element The green open cylinders represent the gating helices TM1 and TM6. FIG +32 50 crystal structures evidence Asterisks mark the states whose crystal structures have been determined in this study. FIG +65 98 calculated free-energy landscapes evidence These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. FIG +120 131 Ca2+-loaded protein_state These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. FIG +132 146 outward-facing protein_state These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. FIG +147 155 occluded protein_state These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. FIG +170 178 unloaded protein_state These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. FIG +180 190 fully open protein_state These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. FIG diff --git a/annotation_CSV/PMC4919469.csv b/annotation_CSV/PMC4919469.csv new file mode 100644 index 0000000000000000000000000000000000000000..32c87934160ca0b3c80348b6d22fe1f6c8200379 --- /dev/null +++ b/annotation_CSV/PMC4919469.csv @@ -0,0 +1,1625 @@ +anno_start anno_end anno_text entity_type sentence section +41 46 Cdc42 protein Investigation of the Interaction between Cdc42 and Its Effector TOCA1 TITLE +64 69 TOCA1 protein Investigation of the Interaction between Cdc42 and Its Effector TOCA1 TITLE +0 54 Transducer of Cdc42-dependent actin assembly protein 1 protein Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. ABSTRACT +56 61 TOCA1 protein Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. ABSTRACT +85 111 Rho family small G protein protein_type Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. ABSTRACT +112 117 Cdc42 protein Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. ABSTRACT +33 38 F-BAR structure_element It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. ABSTRACT +59 73 Src homology 3 structure_element It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. ABSTRACT +75 78 SH3 structure_element It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. ABSTRACT +93 128 G protein-binding homology region 1 structure_element It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. ABSTRACT +130 133 HR1 structure_element It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. ABSTRACT +0 5 TOCA1 protein TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. ABSTRACT +17 22 Cdc42 protein TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. ABSTRACT +32 37 actin protein_type TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. ABSTRACT +8 14 solved experimental_method We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. ABSTRACT +19 28 structure evidence We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. ABSTRACT +36 39 HR1 structure_element We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. ABSTRACT +50 55 TOCA1 protein We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. ABSTRACT +77 92 structural data evidence We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. ABSTRACT +23 28 TOCA1 protein We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. ABSTRACT +29 32 HR1 structure_element We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. ABSTRACT +59 63 CIP4 protein We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. ABSTRACT +64 67 HR1 structure_element We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. ABSTRACT +136 139 HR1 structure_element We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. ABSTRACT +45 49 TOCA protein We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. ABSTRACT +50 53 HR1 structure_element We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. ABSTRACT +64 69 Cdc42 protein We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. ABSTRACT +112 117 Cdc42 protein We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. ABSTRACT +126 151 G protein-binding regions site We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. ABSTRACT +155 160 TOCA1 protein We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. ABSTRACT +181 220 Wiskott-Aldrich syndrome protein family protein_type We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. ABSTRACT +222 228 N-WASP protein We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. ABSTRACT +0 5 TOCA1 protein TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. ABSTRACT +12 17 Cdc42 protein TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. ABSTRACT +89 95 N-WASP protein TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. ABSTRACT +96 120 G protein-binding region site TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. ABSTRACT +125 130 Cdc42 protein TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. ABSTRACT +0 3 NMR experimental_method NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. ABSTRACT +30 50 Cdc42-binding domain site NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. ABSTRACT +56 62 N-WASP protein NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. ABSTRACT +83 88 TOCA1 protein NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. ABSTRACT +89 92 HR1 structure_element NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. ABSTRACT +98 103 Cdc42 protein NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. ABSTRACT +117 123 N-WASP protein NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. ABSTRACT +143 148 TOCA1 protein NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. ABSTRACT +149 152 HR1 structure_element NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. ABSTRACT +19 24 TOCA1 protein This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. ABSTRACT +36 41 Cdc42 protein This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. ABSTRACT +66 71 Cdc42 protein This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. ABSTRACT +140 158 binding affinities evidence This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. ABSTRACT +203 208 TOCA1 protein This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. ABSTRACT +212 218 N-WASP protein This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. ABSTRACT +4 19 Ras superfamily protein_type The Ras superfamily of small GTPases comprises over 150 members that regulate a multitude of cellular processes in eukaryotes. INTRO +23 36 small GTPases protein_type The Ras superfamily of small GTPases comprises over 150 members that regulate a multitude of cellular processes in eukaryotes. INTRO +115 125 eukaryotes taxonomy_domain The Ras superfamily of small GTPases comprises over 150 members that regulate a multitude of cellular processes in eukaryotes. INTRO +99 102 Ras protein_type The superfamily can be divided into five families based on structural and functional similarities: Ras, Rho, Rab, Arf, and Ran. INTRO +104 107 Rho protein_type The superfamily can be divided into five families based on structural and functional similarities: Ras, Rho, Rab, Arf, and Ran. INTRO +109 112 Rab protein_type The superfamily can be divided into five families based on structural and functional similarities: Ras, Rho, Rab, Arf, and Ran. INTRO +114 117 Arf protein_type The superfamily can be divided into five families based on structural and functional similarities: Ras, Rho, Rab, Arf, and Ran. INTRO +123 126 Ran protein_type The superfamily can be divided into five families based on structural and functional similarities: Ras, Rho, Rab, Arf, and Ran. INTRO +72 80 G domain structure_element All members share a well defined core structure of ∼20 kDa known as the G domain, which is responsible for guanine nucleotide binding. INTRO +107 125 guanine nucleotide chemical All members share a well defined core structure of ∼20 kDa known as the G domain, which is responsible for guanine nucleotide binding. INTRO +39 45 active protein_state These molecular switches cycle between active, GTP-bound, and inactive, GDP-bound, states with the help of auxiliary proteins. INTRO +47 56 GTP-bound protein_state These molecular switches cycle between active, GTP-bound, and inactive, GDP-bound, states with the help of auxiliary proteins. INTRO +62 70 inactive protein_state These molecular switches cycle between active, GTP-bound, and inactive, GDP-bound, states with the help of auxiliary proteins. INTRO +72 81 GDP-bound protein_state These molecular switches cycle between active, GTP-bound, and inactive, GDP-bound, states with the help of auxiliary proteins. INTRO +4 39 guanine nucleotide exchange factors protein_type The guanine nucleotide exchange factors mediate formation of the active state by promoting the dissociation of GDP, allowing GTP to bind. INTRO +65 71 active protein_state The guanine nucleotide exchange factors mediate formation of the active state by promoting the dissociation of GDP, allowing GTP to bind. INTRO +111 114 GDP chemical The guanine nucleotide exchange factors mediate formation of the active state by promoting the dissociation of GDP, allowing GTP to bind. INTRO +125 128 GTP chemical The guanine nucleotide exchange factors mediate formation of the active state by promoting the dissociation of GDP, allowing GTP to bind. INTRO +4 30 GTPase-activating proteins protein_type The GTPase-activating proteins stimulate the rate of intrinsic GTP hydrolysis, mediating the return to the inactive state (reviewed in Ref.). INTRO +63 66 GTP chemical The GTPase-activating proteins stimulate the rate of intrinsic GTP hydrolysis, mediating the return to the inactive state (reviewed in Ref.). INTRO +107 115 inactive protein_state The GTPase-activating proteins stimulate the rate of intrinsic GTP hydrolysis, mediating the return to the inactive state (reviewed in Ref.). INTRO +28 44 small G proteins protein_type The overall conformation of small G proteins in the active and inactive states is similar, but they differ significantly in two main regions known as switch I and switch II. INTRO +52 58 active protein_state The overall conformation of small G proteins in the active and inactive states is similar, but they differ significantly in two main regions known as switch I and switch II. INTRO +63 71 inactive protein_state The overall conformation of small G proteins in the active and inactive states is similar, but they differ significantly in two main regions known as switch I and switch II. INTRO +150 158 switch I site The overall conformation of small G proteins in the active and inactive states is similar, but they differ significantly in two main regions known as switch I and switch II. INTRO +163 172 switch II site The overall conformation of small G proteins in the active and inactive states is similar, but they differ significantly in two main regions known as switch I and switch II. INTRO +75 84 GTP-bound protein_state These regions are responsible for “sensing” the nucleotide state, with the GTP-bound state showing greater rigidity and the GDP-bound state adopting a more relaxed conformation (reviewed in Ref.). INTRO +124 133 GDP-bound protein_state These regions are responsible for “sensing” the nucleotide state, with the GTP-bound state showing greater rigidity and the GDP-bound state adopting a more relaxed conformation (reviewed in Ref.). INTRO +7 13 active protein_state In the active state, G proteins bind to an array of downstream effectors, through which they exert their extensive roles within the cell. INTRO +21 31 G proteins protein_type In the active state, G proteins bind to an array of downstream effectors, through which they exert their extensive roles within the cell. INTRO +4 14 structures evidence The structures of more than 60 small G protein·effector complexes have been solved, and, not surprisingly, the switch regions have been implicated in a large proportion of the G protein-effector interactions (reviewed in Ref.). INTRO +37 46 G protein protein_type The structures of more than 60 small G protein·effector complexes have been solved, and, not surprisingly, the switch regions have been implicated in a large proportion of the G protein-effector interactions (reviewed in Ref.). INTRO +76 82 solved experimental_method The structures of more than 60 small G protein·effector complexes have been solved, and, not surprisingly, the switch regions have been implicated in a large proportion of the G protein-effector interactions (reviewed in Ref.). INTRO +111 125 switch regions site The structures of more than 60 small G protein·effector complexes have been solved, and, not surprisingly, the switch regions have been implicated in a large proportion of the G protein-effector interactions (reviewed in Ref.). INTRO +176 185 G protein protein_type The structures of more than 60 small G protein·effector complexes have been solved, and, not surprisingly, the switch regions have been implicated in a large proportion of the G protein-effector interactions (reviewed in Ref.). INTRO +134 143 G protein protein_type However, because each of the 150 members of the superfamily interacts with multiple effectors, there are still a huge number of known G protein-effector interactions that have not yet been studied structurally. INTRO +4 14 Rho family protein_type The Rho family comprises 20 members, of which three, RhoA, Rac1, and Cdc42, have been relatively well studied. INTRO +53 57 RhoA protein The Rho family comprises 20 members, of which three, RhoA, Rac1, and Cdc42, have been relatively well studied. INTRO +59 63 Rac1 protein The Rho family comprises 20 members, of which three, RhoA, Rac1, and Cdc42, have been relatively well studied. INTRO +69 74 Cdc42 protein The Rho family comprises 20 members, of which three, RhoA, Rac1, and Cdc42, have been relatively well studied. INTRO +0 4 RhoA protein RhoA acts to rearrange existing actin structures to form stress fibers, whereas Rac1 and Cdc42 promote de novo actin polymerization to form lamellipodia and filopodia, respectively. INTRO +80 84 Rac1 protein RhoA acts to rearrange existing actin structures to form stress fibers, whereas Rac1 and Cdc42 promote de novo actin polymerization to form lamellipodia and filopodia, respectively. INTRO +89 94 Cdc42 protein RhoA acts to rearrange existing actin structures to form stress fibers, whereas Rac1 and Cdc42 promote de novo actin polymerization to form lamellipodia and filopodia, respectively. INTRO +12 16 RhoA protein A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. INTRO +21 25 Rac1 protein A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. INTRO +86 117 protein kinase C-related kinase protein_type A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. INTRO +119 122 PRK protein_type A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. INTRO +123 124 6 protein_type A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. INTRO +144 149 Cdc42 protein A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. INTRO +175 199 Wiskott-Aldrich syndrome protein_type A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. INTRO +201 205 WASP protein_type A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. INTRO +236 266 Cdc42-dependent actin assembly protein_type A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. INTRO +268 272 TOCA protein_type A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics. INTRO +0 5 Cdc42 protein Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). INTRO +17 22 TOCA1 protein Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). INTRO +68 79 WASP family protein_type Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). INTRO +81 87 N-WASP protein Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). INTRO +166 171 Cdc42 protein Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). INTRO +176 213 phosphatidylinositol 4,5-bisphosphate chemical Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). INTRO +215 224 PI(4,5)P2 chemical Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). INTRO +0 6 N-WASP protein N-WASP exists in an autoinhibited conformation, which is released upon PI(4,5)P2 and Cdc42 binding or by other factors, such as phosphorylation. INTRO +20 46 autoinhibited conformation protein_state N-WASP exists in an autoinhibited conformation, which is released upon PI(4,5)P2 and Cdc42 binding or by other factors, such as phosphorylation. INTRO +71 80 PI(4,5)P2 chemical N-WASP exists in an autoinhibited conformation, which is released upon PI(4,5)P2 and Cdc42 binding or by other factors, such as phosphorylation. INTRO +85 90 Cdc42 protein N-WASP exists in an autoinhibited conformation, which is released upon PI(4,5)P2 and Cdc42 binding or by other factors, such as phosphorylation. INTRO +29 47 C-terminal regions structure_element Following their release, the C-terminal regions of N-WASP are free to interact with G-actin and a known nucleator of actin assembly, the Arp2/3 complex. INTRO +51 57 N-WASP protein Following their release, the C-terminal regions of N-WASP are free to interact with G-actin and a known nucleator of actin assembly, the Arp2/3 complex. INTRO +84 91 G-actin protein_type Following their release, the C-terminal regions of N-WASP are free to interact with G-actin and a known nucleator of actin assembly, the Arp2/3 complex. INTRO +137 143 Arp2/3 complex_assembly Following their release, the C-terminal regions of N-WASP are free to interact with G-actin and a known nucleator of actin assembly, the Arp2/3 complex. INTRO +18 23 TOCA1 protein The importance of TOCA1 in actin polymerization has been demonstrated in a range of in vitro and in vivo studies, but the exact role of TOCA1 in the many pathways involving actin assembly remains unclear. INTRO +136 141 TOCA1 protein The importance of TOCA1 in actin polymerization has been demonstrated in a range of in vitro and in vivo studies, but the exact role of TOCA1 in the many pathways involving actin assembly remains unclear. INTRO +32 37 TOCA1 protein The most widely studied role of TOCA1 is in membrane invagination and endocytosis, although it has also been implicated in filopodia formation, neurite elongation, transcriptional reprogramming via nuclear actin, and interaction with ZO-1 at tight junctions. INTRO +206 211 actin protein_type The most widely studied role of TOCA1 is in membrane invagination and endocytosis, although it has also been implicated in filopodia formation, neurite elongation, transcriptional reprogramming via nuclear actin, and interaction with ZO-1 at tight junctions. INTRO +234 238 ZO-1 protein The most widely studied role of TOCA1 is in membrane invagination and endocytosis, although it has also been implicated in filopodia formation, neurite elongation, transcriptional reprogramming via nuclear actin, and interaction with ZO-1 at tight junctions. INTRO +0 5 TOCA1 protein TOCA1 comprises an N-terminal F-BAR domain, a central homology region 1 (HR1) domain, and a C-terminal SH3 domain. INTRO +30 35 F-BAR structure_element TOCA1 comprises an N-terminal F-BAR domain, a central homology region 1 (HR1) domain, and a C-terminal SH3 domain. INTRO +46 71 central homology region 1 structure_element TOCA1 comprises an N-terminal F-BAR domain, a central homology region 1 (HR1) domain, and a C-terminal SH3 domain. INTRO +73 76 HR1 structure_element TOCA1 comprises an N-terminal F-BAR domain, a central homology region 1 (HR1) domain, and a C-terminal SH3 domain. INTRO +103 106 SH3 structure_element TOCA1 comprises an N-terminal F-BAR domain, a central homology region 1 (HR1) domain, and a C-terminal SH3 domain. INTRO +4 9 F-BAR structure_element The F-BAR domain is a known dimerization, membrane-binding, and membrane-deforming module found in a number of cell signaling proteins. INTRO +4 9 TOCA1 protein The TOCA1 SH3 domain has many known binding partners, including N-WASP and dynamin. INTRO +10 13 SH3 structure_element The TOCA1 SH3 domain has many known binding partners, including N-WASP and dynamin. INTRO +64 70 N-WASP protein The TOCA1 SH3 domain has many known binding partners, including N-WASP and dynamin. INTRO +75 82 dynamin protein The TOCA1 SH3 domain has many known binding partners, including N-WASP and dynamin. INTRO +4 7 HR1 structure_element The HR1 domain has been directly implicated in the interaction between TOCA1 and Cdc42, representing the first Cdc42-HR1 domain interaction to be identified. INTRO +71 76 TOCA1 protein The HR1 domain has been directly implicated in the interaction between TOCA1 and Cdc42, representing the first Cdc42-HR1 domain interaction to be identified. INTRO +81 86 Cdc42 protein The HR1 domain has been directly implicated in the interaction between TOCA1 and Cdc42, representing the first Cdc42-HR1 domain interaction to be identified. INTRO +111 116 Cdc42 protein The HR1 domain has been directly implicated in the interaction between TOCA1 and Cdc42, representing the first Cdc42-HR1 domain interaction to be identified. INTRO +117 120 HR1 structure_element The HR1 domain has been directly implicated in the interaction between TOCA1 and Cdc42, representing the first Cdc42-HR1 domain interaction to be identified. INTRO +6 9 HR1 structure_element Other HR1 domains studied so far, including those from the PRK family, have been found to bind their cognate Rho family G protein-binding partner with high specificity and affinities in the nanomolar range. INTRO +59 69 PRK family protein_type Other HR1 domains studied so far, including those from the PRK family, have been found to bind their cognate Rho family G protein-binding partner with high specificity and affinities in the nanomolar range. INTRO +120 129 G protein protein_type Other HR1 domains studied so far, including those from the PRK family, have been found to bind their cognate Rho family G protein-binding partner with high specificity and affinities in the nanomolar range. INTRO +172 182 affinities evidence Other HR1 domains studied so far, including those from the PRK family, have been found to bind their cognate Rho family G protein-binding partner with high specificity and affinities in the nanomolar range. INTRO +4 14 structures evidence The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. INTRO +22 26 PRK1 protein The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. INTRO +27 31 HR1a structure_element The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. INTRO +42 54 complex with protein_state The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. INTRO +55 59 RhoA protein The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. INTRO +68 72 HR1b structure_element The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. INTRO +83 95 complex with protein_state The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. INTRO +96 100 Rac1 protein The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. INTRO +115 118 HR1 structure_element The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. INTRO +139 164 anti-parallel coiled-coil structure_element The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. INTRO +189 198 G protein protein_type The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. INTRO +224 231 helices structure_element The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices. INTRO +12 36 G protein switch regions site Both of the G protein switch regions are involved in the interaction. INTRO +4 20 coiled-coil fold structure_element The coiled-coil fold is shared by the HR1 domain of the TOCA family protein, CIP4, and, based on sequence homology, by TOCA1 itself. INTRO +38 41 HR1 structure_element The coiled-coil fold is shared by the HR1 domain of the TOCA family protein, CIP4, and, based on sequence homology, by TOCA1 itself. INTRO +56 75 TOCA family protein protein_type The coiled-coil fold is shared by the HR1 domain of the TOCA family protein, CIP4, and, based on sequence homology, by TOCA1 itself. INTRO +77 81 CIP4 protein The coiled-coil fold is shared by the HR1 domain of the TOCA family protein, CIP4, and, based on sequence homology, by TOCA1 itself. INTRO +119 124 TOCA1 protein The coiled-coil fold is shared by the HR1 domain of the TOCA family protein, CIP4, and, based on sequence homology, by TOCA1 itself. INTRO +6 9 HR1 structure_element These HR1 domains, however, show specificity for Cdc42, rather than RhoA or Rac1. INTRO +49 54 Cdc42 protein These HR1 domains, however, show specificity for Cdc42, rather than RhoA or Rac1. INTRO +68 72 RhoA protein These HR1 domains, however, show specificity for Cdc42, rather than RhoA or Rac1. INTRO +76 80 Rac1 protein These HR1 domains, however, show specificity for Cdc42, rather than RhoA or Rac1. INTRO +14 17 HR1 structure_element How different HR1 domain proteins distinguish their specific G protein partners remains only partially understood, and structural characterization of a novel G protein-HR1 domain interaction would add to the growing body of information pertaining to these protein complexes. INTRO +61 70 G protein protein_type How different HR1 domain proteins distinguish their specific G protein partners remains only partially understood, and structural characterization of a novel G protein-HR1 domain interaction would add to the growing body of information pertaining to these protein complexes. INTRO +158 167 G protein protein_type How different HR1 domain proteins distinguish their specific G protein partners remains only partially understood, and structural characterization of a novel G protein-HR1 domain interaction would add to the growing body of information pertaining to these protein complexes. INTRO +168 171 HR1 structure_element How different HR1 domain proteins distinguish their specific G protein partners remains only partially understood, and structural characterization of a novel G protein-HR1 domain interaction would add to the growing body of information pertaining to these protein complexes. INTRO +64 69 TOCA1 protein Furthermore, the biological function of the interaction between TOCA1 and Cdc42 remains poorly understood, and so far there has been no biophysical or structural insight. INTRO +74 79 Cdc42 protein Furthermore, the biological function of the interaction between TOCA1 and Cdc42 remains poorly understood, and so far there has been no biophysical or structural insight. INTRO +20 25 TOCA1 protein The interactions of TOCA1 and N-WASP with Cdc42 as well as with each other have raised questions as to whether the two Cdc42 effectors can interact with a single molecule of Cdc42 simultaneously. INTRO +30 36 N-WASP protein The interactions of TOCA1 and N-WASP with Cdc42 as well as with each other have raised questions as to whether the two Cdc42 effectors can interact with a single molecule of Cdc42 simultaneously. INTRO +42 47 Cdc42 protein The interactions of TOCA1 and N-WASP with Cdc42 as well as with each other have raised questions as to whether the two Cdc42 effectors can interact with a single molecule of Cdc42 simultaneously. INTRO +119 124 Cdc42 protein The interactions of TOCA1 and N-WASP with Cdc42 as well as with each other have raised questions as to whether the two Cdc42 effectors can interact with a single molecule of Cdc42 simultaneously. INTRO +174 179 Cdc42 protein The interactions of TOCA1 and N-WASP with Cdc42 as well as with each other have raised questions as to whether the two Cdc42 effectors can interact with a single molecule of Cdc42 simultaneously. INTRO +53 58 Cdc42 protein There is some evidence for a ternary complex between Cdc42, N-WASP, and TOCA1, but there was no direct demonstration of simultaneous contacts between the two effectors and a single molecule of Cdc42. INTRO +60 66 N-WASP protein There is some evidence for a ternary complex between Cdc42, N-WASP, and TOCA1, but there was no direct demonstration of simultaneous contacts between the two effectors and a single molecule of Cdc42. INTRO +72 77 TOCA1 protein There is some evidence for a ternary complex between Cdc42, N-WASP, and TOCA1, but there was no direct demonstration of simultaneous contacts between the two effectors and a single molecule of Cdc42. INTRO +193 198 Cdc42 protein There is some evidence for a ternary complex between Cdc42, N-WASP, and TOCA1, but there was no direct demonstration of simultaneous contacts between the two effectors and a single molecule of Cdc42. INTRO +52 62 structures evidence Nonetheless, the substantial difference between the structures of the G protein-binding regions of the two effectors is intriguing and implies that they bind to Cdc42 quite differently, providing motivation for investigating the possibility that Cdc42 can bind both effectors concurrently. INTRO +70 95 G protein-binding regions site Nonetheless, the substantial difference between the structures of the G protein-binding regions of the two effectors is intriguing and implies that they bind to Cdc42 quite differently, providing motivation for investigating the possibility that Cdc42 can bind both effectors concurrently. INTRO +161 166 Cdc42 protein Nonetheless, the substantial difference between the structures of the G protein-binding regions of the two effectors is intriguing and implies that they bind to Cdc42 quite differently, providing motivation for investigating the possibility that Cdc42 can bind both effectors concurrently. INTRO +246 251 Cdc42 protein Nonetheless, the substantial difference between the structures of the G protein-binding regions of the two effectors is intriguing and implies that they bind to Cdc42 quite differently, providing motivation for investigating the possibility that Cdc42 can bind both effectors concurrently. INTRO +0 4 WASP protein_type WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. INTRO +20 25 Cdc42 protein WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. INTRO +32 41 conserved protein_state WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. INTRO +43 69 unstructured binding motif structure_element WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. INTRO +83 124 Cdc42- and Rac-interactive binding region structure_element WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. INTRO +126 130 CRIB structure_element WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. INTRO +148 170 intermolecular β-sheet structure_element WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. INTRO +200 217 β2 and β3 strands structure_element WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. INTRO +221 226 Cdc42 protein WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular β-sheet, expanding the anti-parallel β2 and β3 strands of Cdc42. INTRO +17 37 TOCA family proteins protein_type In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1. INTRO +70 73 HR1 structure_element In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1. INTRO +99 117 triple coiled-coil structure_element In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1. INTRO +123 132 switch II site In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1. INTRO +136 140 Rac1 protein In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1. INTRO +151 155 HR1b structure_element In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1. INTRO +166 170 PRK1 protein In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1. INTRO +21 33 solution NMR experimental_method Here, we present the solution NMR structure of the HR1 domain of TOCA1, providing the first structural data for this protein. INTRO +34 43 structure evidence Here, we present the solution NMR structure of the HR1 domain of TOCA1, providing the first structural data for this protein. INTRO +51 54 HR1 structure_element Here, we present the solution NMR structure of the HR1 domain of TOCA1, providing the first structural data for this protein. INTRO +65 70 TOCA1 protein Here, we present the solution NMR structure of the HR1 domain of TOCA1, providing the first structural data for this protein. INTRO +92 107 structural data evidence Here, we present the solution NMR structure of the HR1 domain of TOCA1, providing the first structural data for this protein. INTRO +50 54 TOCA protein_type We also present data pertaining to binding of the TOCA HR1 domain to Cdc42, which is the first biophysical description of an HR1 domain binding this particular Rho family small G protein. INTRO +55 58 HR1 structure_element We also present data pertaining to binding of the TOCA HR1 domain to Cdc42, which is the first biophysical description of an HR1 domain binding this particular Rho family small G protein. INTRO +69 74 Cdc42 protein We also present data pertaining to binding of the TOCA HR1 domain to Cdc42, which is the first biophysical description of an HR1 domain binding this particular Rho family small G protein. INTRO +125 128 HR1 structure_element We also present data pertaining to binding of the TOCA HR1 domain to Cdc42, which is the first biophysical description of an HR1 domain binding this particular Rho family small G protein. INTRO +160 186 Rho family small G protein protein_type We also present data pertaining to binding of the TOCA HR1 domain to Cdc42, which is the first biophysical description of an HR1 domain binding this particular Rho family small G protein. INTRO +62 67 Cdc42 protein Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics. INTRO +76 101 G protein-binding regions site Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics. INTRO +105 110 TOCA1 protein Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics. INTRO +115 121 N-WASP protein Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics. INTRO +160 169 G protein protein_type Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics. INTRO +216 221 Cdc42 protein Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics. INTRO +223 229 N-WASP protein Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics. INTRO +235 240 TOCA1 protein Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics. INTRO +269 274 actin protein_type Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics. INTRO +0 5 Cdc42 protein Cdc42-TOCA1 Binding RESULTS +6 11 TOCA1 protein Cdc42-TOCA1 Binding RESULTS +0 5 TOCA1 protein TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. RESULTS +24 31 Xenopus taxonomy_domain TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. RESULTS +68 73 Cdc42 protein TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. RESULTS +124 135 Cdc42·GTPγS complex_assembly TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. RESULTS +147 156 Cdc42·GDP complex_assembly TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. RESULTS +163 167 Rac1 protein TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. RESULTS +172 176 RhoA protein TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. RESULTS +206 232 Rho family binding modules site TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. RESULTS +256 259 HR1 structure_element TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. RESULTS +270 275 TOCA1 protein TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. RESULTS +298 303 Cdc42 protein TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42·GTPγS but not to Cdc42·GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42. RESULTS +4 14 C. elegans species The C. elegans TOCA1 orthologues also bind to Cdc42 via their consensus HR1 domain. RESULTS +15 20 TOCA1 protein The C. elegans TOCA1 orthologues also bind to Cdc42 via their consensus HR1 domain. RESULTS +46 51 Cdc42 protein The C. elegans TOCA1 orthologues also bind to Cdc42 via their consensus HR1 domain. RESULTS +72 75 HR1 structure_element The C. elegans TOCA1 orthologues also bind to Cdc42 via their consensus HR1 domain. RESULTS +4 7 HR1 structure_element The HR1 domains from the PRK family bind their G protein partners with a high affinity, exhibiting a range of submicromolar dissociation constants (Kd) as low as 26 nm. RESULTS +25 35 PRK family protein_type The HR1 domains from the PRK family bind their G protein partners with a high affinity, exhibiting a range of submicromolar dissociation constants (Kd) as low as 26 nm. RESULTS +47 56 G protein protein_type The HR1 domains from the PRK family bind their G protein partners with a high affinity, exhibiting a range of submicromolar dissociation constants (Kd) as low as 26 nm. RESULTS +124 146 dissociation constants evidence The HR1 domains from the PRK family bind their G protein partners with a high affinity, exhibiting a range of submicromolar dissociation constants (Kd) as low as 26 nm. RESULTS +148 150 Kd evidence The HR1 domains from the PRK family bind their G protein partners with a high affinity, exhibiting a range of submicromolar dissociation constants (Kd) as low as 26 nm. RESULTS +2 4 Kd evidence A Kd in the nanomolar range was therefore expected for the interaction of the TOCA1 HR1 domain with Cdc42. RESULTS +78 83 TOCA1 protein A Kd in the nanomolar range was therefore expected for the interaction of the TOCA1 HR1 domain with Cdc42. RESULTS +84 87 HR1 structure_element A Kd in the nanomolar range was therefore expected for the interaction of the TOCA1 HR1 domain with Cdc42. RESULTS +100 105 Cdc42 protein A Kd in the nanomolar range was therefore expected for the interaction of the TOCA1 HR1 domain with Cdc42. RESULTS +16 29 X. tropicalis species We generated an X. tropicalis TOCA1 HR1 domain construct encompassing residues 330–426. RESULTS +30 35 TOCA1 protein We generated an X. tropicalis TOCA1 HR1 domain construct encompassing residues 330–426. RESULTS +36 39 HR1 structure_element We generated an X. tropicalis TOCA1 HR1 domain construct encompassing residues 330–426. RESULTS +79 86 330–426 residue_range We generated an X. tropicalis TOCA1 HR1 domain construct encompassing residues 330–426. RESULTS +35 38 HR1 structure_element This region comprises the complete HR1 domain based on secondary structure predictions and sequence alignments with another TOCA family member, CIP4, whose structure has been determined. RESULTS +91 110 sequence alignments experimental_method This region comprises the complete HR1 domain based on secondary structure predictions and sequence alignments with another TOCA family member, CIP4, whose structure has been determined. RESULTS +124 135 TOCA family protein_type This region comprises the complete HR1 domain based on secondary structure predictions and sequence alignments with another TOCA family member, CIP4, whose structure has been determined. RESULTS +144 148 CIP4 protein This region comprises the complete HR1 domain based on secondary structure predictions and sequence alignments with another TOCA family member, CIP4, whose structure has been determined. RESULTS +156 165 structure evidence This region comprises the complete HR1 domain based on secondary structure predictions and sequence alignments with another TOCA family member, CIP4, whose structure has been determined. RESULTS +24 37 [3H]GTP·Cdc42 complex_assembly The interaction between [3H]GTP·Cdc42 and a C-terminally His-tagged TOCA1 HR1 domain construct was investigated using SPA. RESULTS +57 67 His-tagged protein_state The interaction between [3H]GTP·Cdc42 and a C-terminally His-tagged TOCA1 HR1 domain construct was investigated using SPA. RESULTS +68 73 TOCA1 protein The interaction between [3H]GTP·Cdc42 and a C-terminally His-tagged TOCA1 HR1 domain construct was investigated using SPA. RESULTS +74 77 HR1 structure_element The interaction between [3H]GTP·Cdc42 and a C-terminally His-tagged TOCA1 HR1 domain construct was investigated using SPA. RESULTS +118 121 SPA experimental_method The interaction between [3H]GTP·Cdc42 and a C-terminally His-tagged TOCA1 HR1 domain construct was investigated using SPA. RESULTS +4 20 binding isotherm evidence The binding isotherm for the interaction is shown in Fig. 1A, together with the Cdc42-PAK interaction as a positive control. RESULTS +80 85 Cdc42 protein The binding isotherm for the interaction is shown in Fig. 1A, together with the Cdc42-PAK interaction as a positive control. RESULTS +86 89 PAK protein The binding isotherm for the interaction is shown in Fig. 1A, together with the Cdc42-PAK interaction as a positive control. RESULTS +15 20 TOCA1 protein The binding of TOCA1 HR1 to Cdc42 was unexpectedly weak, with a Kd of >1 μm. RESULTS +21 24 HR1 structure_element The binding of TOCA1 HR1 to Cdc42 was unexpectedly weak, with a Kd of >1 μm. RESULTS +28 33 Cdc42 protein The binding of TOCA1 HR1 to Cdc42 was unexpectedly weak, with a Kd of >1 μm. RESULTS +64 66 Kd evidence The binding of TOCA1 HR1 to Cdc42 was unexpectedly weak, with a Kd of >1 μm. RESULTS +36 38 Kd evidence It was not possible to estimate the Kd more accurately using direct SPA experiments, because saturation could not be reached due to nonspecific signal at higher protein concentrations. RESULTS +68 71 SPA experimental_method It was not possible to estimate the Kd more accurately using direct SPA experiments, because saturation could not be reached due to nonspecific signal at higher protein concentrations. RESULTS +4 9 TOCA1 protein The TOCA1 HR1-Cdc42 interaction is low affinity. FIG +10 13 HR1 structure_element The TOCA1 HR1-Cdc42 interaction is low affinity. FIG +14 19 Cdc42 protein The TOCA1 HR1-Cdc42 interaction is low affinity. FIG +24 45 direct binding assays experimental_method A, curves derived from direct binding assays in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-PAK or HR1-His6 in SPAs. FIG +87 106 Cdc42Δ7Q61L·[3H]GTP complex_assembly A, curves derived from direct binding assays in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-PAK or HR1-His6 in SPAs. FIG +112 121 incubated experimental_method A, curves derived from direct binding assays in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-PAK or HR1-His6 in SPAs. FIG +133 140 GST-PAK mutant A, curves derived from direct binding assays in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-PAK or HR1-His6 in SPAs. FIG +144 152 HR1-His6 mutant A, curves derived from direct binding assays in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-PAK or HR1-His6 in SPAs. FIG +156 160 SPAs experimental_method A, curves derived from direct binding assays in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-PAK or HR1-His6 in SPAs. FIG +4 7 SPA experimental_method The SPA signal was corrected by subtraction of control data with no GST-PAK or HR1-His6. FIG +68 75 GST-PAK mutant The SPA signal was corrected by subtraction of control data with no GST-PAK or HR1-His6. FIG +79 87 HR1-His6 mutant The SPA signal was corrected by subtraction of control data with no GST-PAK or HR1-His6. FIG +26 42 binding isotherm evidence The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. FIG +63 65 Kd evidence The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. FIG +132 147 competition SPA experimental_method The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. FIG +214 217 ACK protein The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. FIG +218 221 GBD structure_element The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. FIG +229 232 HR1 structure_element The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. FIG +244 252 titrated experimental_method The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. FIG +264 271 GST-ACK mutant The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. FIG +289 308 Cdc42Δ7Q61L·[3H]GTP complex_assembly The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. FIG +312 323 full-length protein_state The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. FIG +324 341 Cdc42Q61L·[3H]GTP complex_assembly The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42Δ7Q61L·[3H]GTP or full-length Cdc42Q61L·[3H]GTP. FIG +4 6 Kd evidence The Kd values derived for the ACK GBD with Cdc42Δ7 and full-length Cdc42 were 0.032 ± 0.01 and 0.011 ± 0.01 μm, respectively. FIG +30 33 ACK protein The Kd values derived for the ACK GBD with Cdc42Δ7 and full-length Cdc42 were 0.032 ± 0.01 and 0.011 ± 0.01 μm, respectively. FIG +34 37 GBD structure_element The Kd values derived for the ACK GBD with Cdc42Δ7 and full-length Cdc42 were 0.032 ± 0.01 and 0.011 ± 0.01 μm, respectively. FIG +43 50 Cdc42Δ7 mutant The Kd values derived for the ACK GBD with Cdc42Δ7 and full-length Cdc42 were 0.032 ± 0.01 and 0.011 ± 0.01 μm, respectively. FIG +55 66 full-length protein_state The Kd values derived for the ACK GBD with Cdc42Δ7 and full-length Cdc42 were 0.032 ± 0.01 and 0.011 ± 0.01 μm, respectively. FIG +67 72 Cdc42 protein The Kd values derived for the ACK GBD with Cdc42Δ7 and full-length Cdc42 were 0.032 ± 0.01 and 0.011 ± 0.01 μm, respectively. FIG +4 6 Kd evidence The Kd values derived for the TOCA1 HR1 with Cdc42Δ7 and full-length Cdc42 were 6.05 ± 1.96 and 5.39 ± 1.69 μm, respectively. FIG +30 35 TOCA1 protein The Kd values derived for the TOCA1 HR1 with Cdc42Δ7 and full-length Cdc42 were 6.05 ± 1.96 and 5.39 ± 1.69 μm, respectively. FIG +36 39 HR1 structure_element The Kd values derived for the TOCA1 HR1 with Cdc42Δ7 and full-length Cdc42 were 6.05 ± 1.96 and 5.39 ± 1.69 μm, respectively. FIG +45 52 Cdc42Δ7 mutant The Kd values derived for the TOCA1 HR1 with Cdc42Δ7 and full-length Cdc42 were 6.05 ± 1.96 and 5.39 ± 1.69 μm, respectively. FIG +57 68 full-length protein_state The Kd values derived for the TOCA1 HR1 with Cdc42Δ7 and full-length Cdc42 were 6.05 ± 1.96 and 5.39 ± 1.69 μm, respectively. FIG +69 74 Cdc42 protein The Kd values derived for the TOCA1 HR1 with Cdc42Δ7 and full-length Cdc42 were 6.05 ± 1.96 and 5.39 ± 1.69 μm, respectively. FIG +100 103 HR1 structure_element It was possible that the low affinity observed was due to negative effects of immobilization of the HR1 domain, so other methods were employed, which utilized untagged proteins. RESULTS +159 167 untagged protein_state It was possible that the low affinity observed was due to negative effects of immobilization of the HR1 domain, so other methods were employed, which utilized untagged proteins. RESULTS +0 32 Isothermal titration calorimetry experimental_method Isothermal titration calorimetry was carried out, but no heat changes were observed at a range of concentrations and temperatures (data not shown), suggesting that the interaction is predominantly entropically driven. RESULTS +6 15 G protein protein_type Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK. RESULTS +16 19 HR1 structure_element Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK. RESULTS +93 116 Infrared interferometry experimental_method Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK. RESULTS +122 133 immobilized protein_state Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK. RESULTS +134 139 Cdc42 protein Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK. RESULTS +189 194 TOCA1 protein Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK. RESULTS +195 198 HR1 structure_element Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK. RESULTS +229 232 ACK protein Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK. RESULTS +4 12 affinity evidence The affinity was therefore determined using competition SPAs. RESULTS +44 60 competition SPAs experimental_method The affinity was therefore determined using competition SPAs. RESULTS +15 25 GST fusion experimental_method A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. RESULTS +33 36 GBD structure_element A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. RESULTS +40 43 ACK protein A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. RESULTS +81 86 Cdc42 protein A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. RESULTS +106 119 [3H]GTP·Cdc42 complex_assembly A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. RESULTS +153 178 increasing concentrations experimental_method A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. RESULTS +182 190 untagged protein_state A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. RESULTS +191 196 TOCA1 protein A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. RESULTS +197 200 HR1 structure_element A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. RESULTS +15 22 GST-ACK mutant Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. RESULTS +23 26 GBD structure_element Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. RESULTS +27 35 bound to protein_state Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. RESULTS +36 49 [3H]GTP·Cdc42 complex_assembly Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. RESULTS +53 57 free protein_state Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. RESULTS +58 61 ACK protein Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. RESULTS +62 65 GBD structure_element Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. RESULTS +141 146 Cdc42 protein Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. RESULTS +26 42 binding isotherm evidence The data were fitted to a binding isotherm describing competition. RESULTS +0 4 Free protein_state Free ACK competed with itself with an affinity of 32 nm, similar to the value obtained by direct binding of 23 nm. RESULTS +5 8 ACK protein Free ACK competed with itself with an affinity of 32 nm, similar to the value obtained by direct binding of 23 nm. RESULTS +38 46 affinity evidence Free ACK competed with itself with an affinity of 32 nm, similar to the value obtained by direct binding of 23 nm. RESULTS +90 104 direct binding experimental_method Free ACK competed with itself with an affinity of 32 nm, similar to the value obtained by direct binding of 23 nm. RESULTS +4 9 TOCA1 protein The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 μm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments. RESULTS +10 13 HR1 structure_element The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 μm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments. RESULTS +50 57 GST-ACK mutant The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 μm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments. RESULTS +62 67 bound protein_state The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 μm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments. RESULTS +76 84 affinity evidence The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 μm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments. RESULTS +138 146 affinity evidence The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 μm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments. RESULTS +163 189 direct binding experiments experimental_method The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 μm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments. RESULTS +4 9 Cdc42 protein The Cdc42 construct used in the binding assays has seven residues deleted from the C terminus to facilitate purification. RESULTS +32 46 binding assays experimental_method The Cdc42 construct used in the binding assays has seven residues deleted from the C terminus to facilitate purification. RESULTS +51 65 seven residues residue_range The Cdc42 construct used in the binding assays has seven residues deleted from the C terminus to facilitate purification. RESULTS +66 73 deleted experimental_method The Cdc42 construct used in the binding assays has seven residues deleted from the C terminus to facilitate purification. RESULTS +46 55 G protein protein_type These residues are not generally required for G protein-effector interactions, including the interaction between RhoA and the PRK1 HR1a domain. RESULTS +113 117 RhoA protein These residues are not generally required for G protein-effector interactions, including the interaction between RhoA and the PRK1 HR1a domain. RESULTS +126 130 PRK1 protein These residues are not generally required for G protein-effector interactions, including the interaction between RhoA and the PRK1 HR1a domain. RESULTS +131 135 HR1a structure_element These residues are not generally required for G protein-effector interactions, including the interaction between RhoA and the PRK1 HR1a domain. RESULTS +31 35 Rac1 protein In contrast, the C terminus of Rac1 contains a polybasic sequence, which is crucial for Rac1 binding to the HR1b domain from PRK1. RESULTS +88 92 Rac1 protein In contrast, the C terminus of Rac1 contains a polybasic sequence, which is crucial for Rac1 binding to the HR1b domain from PRK1. RESULTS +108 112 HR1b structure_element In contrast, the C terminus of Rac1 contains a polybasic sequence, which is crucial for Rac1 binding to the HR1b domain from PRK1. RESULTS +125 129 PRK1 protein In contrast, the C terminus of Rac1 contains a polybasic sequence, which is crucial for Rac1 binding to the HR1b domain from PRK1. RESULTS +16 24 affinity evidence As the observed affinity between TOCA1 HR1 and Cdc42 was much lower than expected, we reasoned that the C terminus of Cdc42 might be necessary for a high affinity interaction. RESULTS +33 38 TOCA1 protein As the observed affinity between TOCA1 HR1 and Cdc42 was much lower than expected, we reasoned that the C terminus of Cdc42 might be necessary for a high affinity interaction. RESULTS +39 42 HR1 structure_element As the observed affinity between TOCA1 HR1 and Cdc42 was much lower than expected, we reasoned that the C terminus of Cdc42 might be necessary for a high affinity interaction. RESULTS +47 52 Cdc42 protein As the observed affinity between TOCA1 HR1 and Cdc42 was much lower than expected, we reasoned that the C terminus of Cdc42 might be necessary for a high affinity interaction. RESULTS +118 123 Cdc42 protein As the observed affinity between TOCA1 HR1 and Cdc42 was much lower than expected, we reasoned that the C terminus of Cdc42 might be necessary for a high affinity interaction. RESULTS +154 162 affinity evidence As the observed affinity between TOCA1 HR1 and Cdc42 was much lower than expected, we reasoned that the C terminus of Cdc42 might be necessary for a high affinity interaction. RESULTS +4 23 binding experiments experimental_method The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). RESULTS +43 54 full-length protein_state The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). RESULTS +55 68 [3H]GTP·Cdc42 complex_assembly The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). RESULTS +78 86 affinity evidence The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). RESULTS +94 97 HR1 structure_element The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). RESULTS +109 120 full-length protein_state The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). RESULTS +121 126 Cdc42 protein The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). RESULTS +146 154 affinity evidence The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). RESULTS +159 168 truncated protein_state The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). RESULTS +169 174 Cdc42 protein The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). RESULTS +176 178 Kd evidence The binding experiments were repeated with full-length [3H]GTP·Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd ≈ 5 μm; Fig. 1C). RESULTS +10 27 C-terminal region structure_element Thus, the C-terminal region of Cdc42 is not required for maximal binding of TOCA1 HR1. RESULTS +31 36 Cdc42 protein Thus, the C-terminal region of Cdc42 is not required for maximal binding of TOCA1 HR1. RESULTS +76 81 TOCA1 protein Thus, the C-terminal region of Cdc42 is not required for maximal binding of TOCA1 HR1. RESULTS +82 85 HR1 structure_element Thus, the C-terminal region of Cdc42 is not required for maximal binding of TOCA1 HR1. RESULTS +41 51 affinities evidence Another possible explanation for the low affinities observed was that the HR1 domain alone is not sufficient for maximal binding of the TOCA proteins to Cdc42 and that the other domains are required. RESULTS +74 77 HR1 structure_element Another possible explanation for the low affinities observed was that the HR1 domain alone is not sufficient for maximal binding of the TOCA proteins to Cdc42 and that the other domains are required. RESULTS +85 90 alone protein_state Another possible explanation for the low affinities observed was that the HR1 domain alone is not sufficient for maximal binding of the TOCA proteins to Cdc42 and that the other domains are required. RESULTS +136 149 TOCA proteins protein_type Another possible explanation for the low affinities observed was that the HR1 domain alone is not sufficient for maximal binding of the TOCA proteins to Cdc42 and that the other domains are required. RESULTS +153 158 Cdc42 protein Another possible explanation for the low affinities observed was that the HR1 domain alone is not sufficient for maximal binding of the TOCA proteins to Cdc42 and that the other domains are required. RESULTS +8 22 GST pull-downs experimental_method Indeed, GST pull-downs performed with in vitro translated human TOCA1 fragments had suggested that residues N-terminal to the HR1 domain may be required to stabilize the HR1 domain structure. RESULTS +58 63 human species Indeed, GST pull-downs performed with in vitro translated human TOCA1 fragments had suggested that residues N-terminal to the HR1 domain may be required to stabilize the HR1 domain structure. RESULTS +64 69 TOCA1 protein Indeed, GST pull-downs performed with in vitro translated human TOCA1 fragments had suggested that residues N-terminal to the HR1 domain may be required to stabilize the HR1 domain structure. RESULTS +126 129 HR1 structure_element Indeed, GST pull-downs performed with in vitro translated human TOCA1 fragments had suggested that residues N-terminal to the HR1 domain may be required to stabilize the HR1 domain structure. RESULTS +170 173 HR1 structure_element Indeed, GST pull-downs performed with in vitro translated human TOCA1 fragments had suggested that residues N-terminal to the HR1 domain may be required to stabilize the HR1 domain structure. RESULTS +18 21 BAR structure_element Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. RESULTS +26 29 SH3 structure_element Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. RESULTS +86 96 G proteins protein_type Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. RESULTS +107 110 BAR structure_element Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. RESULTS +121 130 Arfaptin2 protein Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. RESULTS +140 144 Rac1 protein Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. RESULTS +149 153 Arl1 protein Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. RESULTS +165 168 SH3 structure_element Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. RESULTS +209 213 Rac1 protein Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. RESULTS +222 256 guanine nucleotide exchange factor protein Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. RESULTS +258 263 β-PIX protein Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, β-PIX. RESULTS +0 5 TOCA1 protein TOCA1 dimerizes via its F-BAR domain, which could also affect Cdc42 binding, for example by presenting two HR1 domains for Cdc42 interactions. RESULTS +6 11 dimer oligomeric_state TOCA1 dimerizes via its F-BAR domain, which could also affect Cdc42 binding, for example by presenting two HR1 domains for Cdc42 interactions. RESULTS +24 29 F-BAR structure_element TOCA1 dimerizes via its F-BAR domain, which could also affect Cdc42 binding, for example by presenting two HR1 domains for Cdc42 interactions. RESULTS +62 67 Cdc42 protein TOCA1 dimerizes via its F-BAR domain, which could also affect Cdc42 binding, for example by presenting two HR1 domains for Cdc42 interactions. RESULTS +107 110 HR1 structure_element TOCA1 dimerizes via its F-BAR domain, which could also affect Cdc42 binding, for example by presenting two HR1 domains for Cdc42 interactions. RESULTS +123 128 Cdc42 protein TOCA1 dimerizes via its F-BAR domain, which could also affect Cdc42 binding, for example by presenting two HR1 domains for Cdc42 interactions. RESULTS +8 13 TOCA1 protein Various TOCA1 fragments (Fig. 2A) were therefore assessed for binding to full-length Cdc42 by direct SPA. RESULTS +73 84 full-length protein_state Various TOCA1 fragments (Fig. 2A) were therefore assessed for binding to full-length Cdc42 by direct SPA. RESULTS +85 90 Cdc42 protein Various TOCA1 fragments (Fig. 2A) were therefore assessed for binding to full-length Cdc42 by direct SPA. RESULTS +101 104 SPA experimental_method Various TOCA1 fragments (Fig. 2A) were therefore assessed for binding to full-length Cdc42 by direct SPA. RESULTS +13 18 F-BAR structure_element The isolated F-BAR domain showed no binding to full-length Cdc42 (Fig. 2B). RESULTS +47 58 full-length protein_state The isolated F-BAR domain showed no binding to full-length Cdc42 (Fig. 2B). RESULTS +59 64 Cdc42 protein The isolated F-BAR domain showed no binding to full-length Cdc42 (Fig. 2B). RESULTS +0 11 Full-length protein_state Full-length TOCA1 and ΔSH3 TOCA1 bound with micromolar affinity (Fig. 2B), in a similar manner to the isolated HR1 domain (Fig. 1A). RESULTS +12 17 TOCA1 protein Full-length TOCA1 and ΔSH3 TOCA1 bound with micromolar affinity (Fig. 2B), in a similar manner to the isolated HR1 domain (Fig. 1A). RESULTS +22 26 ΔSH3 mutant Full-length TOCA1 and ΔSH3 TOCA1 bound with micromolar affinity (Fig. 2B), in a similar manner to the isolated HR1 domain (Fig. 1A). RESULTS +27 32 TOCA1 protein Full-length TOCA1 and ΔSH3 TOCA1 bound with micromolar affinity (Fig. 2B), in a similar manner to the isolated HR1 domain (Fig. 1A). RESULTS +33 38 bound protein_state Full-length TOCA1 and ΔSH3 TOCA1 bound with micromolar affinity (Fig. 2B), in a similar manner to the isolated HR1 domain (Fig. 1A). RESULTS +111 114 HR1 structure_element Full-length TOCA1 and ΔSH3 TOCA1 bound with micromolar affinity (Fig. 2B), in a similar manner to the isolated HR1 domain (Fig. 1A). RESULTS +4 11 HR1-SH3 mutant The HR1-SH3 protein could not be purified to homogeneity as a fusion protein, so it was assayed in competition assays after cleavage of the His tag. RESULTS +99 117 competition assays experimental_method The HR1-SH3 protein could not be purified to homogeneity as a fusion protein, so it was assayed in competition assays after cleavage of the His tag. RESULTS +29 36 GST-ACK mutant This construct competed with GST-ACK GBD to give a similar affinity to the HR1 domain alone (Kd = 4.6 ± 4 μm; Fig. 2C). RESULTS +37 40 GBD structure_element This construct competed with GST-ACK GBD to give a similar affinity to the HR1 domain alone (Kd = 4.6 ± 4 μm; Fig. 2C). RESULTS +75 78 HR1 structure_element This construct competed with GST-ACK GBD to give a similar affinity to the HR1 domain alone (Kd = 4.6 ± 4 μm; Fig. 2C). RESULTS +86 91 alone protein_state This construct competed with GST-ACK GBD to give a similar affinity to the HR1 domain alone (Kd = 4.6 ± 4 μm; Fig. 2C). RESULTS +93 95 Kd evidence This construct competed with GST-ACK GBD to give a similar affinity to the HR1 domain alone (Kd = 4.6 ± 4 μm; Fig. 2C). RESULTS +44 49 TOCA1 protein Taken together, these data suggest that the TOCA1 HR1 domain is sufficient for maximal binding and that this binding is of a relatively low affinity compared with many other Cdc42·effector complexes. RESULTS +50 53 HR1 structure_element Taken together, these data suggest that the TOCA1 HR1 domain is sufficient for maximal binding and that this binding is of a relatively low affinity compared with many other Cdc42·effector complexes. RESULTS +174 179 Cdc42 protein Taken together, these data suggest that the TOCA1 HR1 domain is sufficient for maximal binding and that this binding is of a relatively low affinity compared with many other Cdc42·effector complexes. RESULTS +4 13 Cdc42-HR1 complex_assembly The Cdc42-HR1 interaction is of low affinity in the context of full-length protein and in TOCA1 paralogues. FIG +63 74 full-length protein_state The Cdc42-HR1 interaction is of low affinity in the context of full-length protein and in TOCA1 paralogues. FIG +90 95 TOCA1 protein The Cdc42-HR1 interaction is of low affinity in the context of full-length protein and in TOCA1 paralogues. FIG +29 34 TOCA1 protein A, diagram illustrating the TOCA1 constructs assayed for Cdc42 binding. FIG +58 63 Cdc42 protein A, diagram illustrating the TOCA1 constructs assayed for Cdc42 binding. FIG +71 85 binding curves evidence Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs. FIG +99 120 direct binding assays experimental_method Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs. FIG +163 182 Cdc42Δ7Q61L·[3H]GTP complex_assembly Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs. FIG +188 197 incubated experimental_method Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs. FIG +209 216 GST-ACK mutant Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs. FIG +220 230 His-tagged protein_state Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs. FIG +231 236 TOCA1 protein Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs. FIG +266 270 SPAs experimental_method Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42Δ7Q61L·[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs. FIG +4 7 SPA experimental_method The SPA signal was corrected by subtraction of control data with no fusion protein. FIG +26 42 binding isotherm evidence The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal. FIG +63 65 Kd evidence The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal. FIG +32 47 competition SPA experimental_method C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. FIG +113 118 TOCA1 protein C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. FIG +119 126 HR1-SH3 mutant C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. FIG +137 145 titrated experimental_method C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. FIG +157 164 GST-ACK mutant C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. FIG +175 194 Cdc42Δ7Q61L·[3H]GTP complex_assembly C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. FIG +202 205 HR1 structure_element C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. FIG +217 220 HR1 structure_element C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. FIG +230 238 titrated experimental_method C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. FIG +250 257 GST-ACK mutant C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. FIG +268 287 Cdc42FLQ61L·[3H]GTP complex_assembly C–E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42Δ7Q61L·[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L·[3H]GTP. FIG +24 29 TOCA1 protein The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly. RESULTS +30 33 HR1 structure_element The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly. RESULTS +34 39 Cdc42 protein The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly. RESULTS +99 104 Cdc42 protein The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly. RESULTS +113 133 TOCA family proteins protein_type The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly. RESULTS +135 140 FBP17 protein The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly. RESULTS +145 149 CIP4 protein The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly. RESULTS +4 7 HR1 structure_element The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain. RESULTS +21 26 FBP17 protein The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain. RESULTS +31 35 CIP4 protein The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain. RESULTS +41 49 purified experimental_method The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain. RESULTS +66 71 Cdc42 protein The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain. RESULTS +83 99 competition SPAs experimental_method The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain. RESULTS +141 146 TOCA1 protein The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain. RESULTS +147 150 HR1 structure_element The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain. RESULTS +4 14 affinities evidence The affinities of both the FBP17 and CIP4 HR1 domains were also in the low micromolar range (10 and 5 μm, respectively) (Fig. 2, D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family. RESULTS +27 32 FBP17 protein The affinities of both the FBP17 and CIP4 HR1 domains were also in the low micromolar range (10 and 5 μm, respectively) (Fig. 2, D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family. RESULTS +37 41 CIP4 protein The affinities of both the FBP17 and CIP4 HR1 domains were also in the low micromolar range (10 and 5 μm, respectively) (Fig. 2, D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family. RESULTS +42 45 HR1 structure_element The affinities of both the FBP17 and CIP4 HR1 domains were also in the low micromolar range (10 and 5 μm, respectively) (Fig. 2, D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family. RESULTS +186 191 Cdc42 protein The affinities of both the FBP17 and CIP4 HR1 domains were also in the low micromolar range (10 and 5 μm, respectively) (Fig. 2, D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family. RESULTS +224 235 TOCA family protein_type The affinities of both the FBP17 and CIP4 HR1 domains were also in the low micromolar range (10 and 5 μm, respectively) (Fig. 2, D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family. RESULTS +0 9 Structure evidence Structure of the TOCA1 HR1 Domain RESULTS +17 22 TOCA1 protein Structure of the TOCA1 HR1 Domain RESULTS +23 26 HR1 structure_element Structure of the TOCA1 HR1 Domain RESULTS +12 17 TOCA1 protein Because the TOCA1 HR1 domain was sufficient for maximal Cdc42-binding, we used this construct for structural studies. RESULTS +18 21 HR1 structure_element Because the TOCA1 HR1 domain was sufficient for maximal Cdc42-binding, we used this construct for structural studies. RESULTS +56 61 Cdc42 protein Because the TOCA1 HR1 domain was sufficient for maximal Cdc42-binding, we used this construct for structural studies. RESULTS +40 45 TOCA1 protein Initial experiments were performed with TOCA1 residues 324–426, but we observed that the N terminus was cleaved during purification to yield a new N terminus at residue 330 (data not shown). RESULTS +55 62 324–426 residue_range Initial experiments were performed with TOCA1 residues 324–426, but we observed that the N terminus was cleaved during purification to yield a new N terminus at residue 330 (data not shown). RESULTS +169 172 330 residue_number Initial experiments were performed with TOCA1 residues 324–426, but we observed that the N terminus was cleaved during purification to yield a new N terminus at residue 330 (data not shown). RESULTS +56 63 330–426 residue_range We therefore engineered a construct comprising residues 330–426 to produce the minimal, stable HR1 domain. RESULTS +79 86 minimal protein_state We therefore engineered a construct comprising residues 330–426 to produce the minimal, stable HR1 domain. RESULTS +88 94 stable protein_state We therefore engineered a construct comprising residues 330–426 to produce the minimal, stable HR1 domain. RESULTS +95 98 HR1 structure_element We therefore engineered a construct comprising residues 330–426 to produce the minimal, stable HR1 domain. RESULTS +21 35 NOE restraints evidence 2,778 non-degenerate NOE restraints were used in initial structure calculations (1,791 unambiguous and 987 ambiguous), derived from three-dimensional 15N-separated NOESY and 13C-separated NOESY experiments. RESULTS +57 79 structure calculations experimental_method 2,778 non-degenerate NOE restraints were used in initial structure calculations (1,791 unambiguous and 987 ambiguous), derived from three-dimensional 15N-separated NOESY and 13C-separated NOESY experiments. RESULTS +150 169 15N-separated NOESY experimental_method 2,778 non-degenerate NOE restraints were used in initial structure calculations (1,791 unambiguous and 987 ambiguous), derived from three-dimensional 15N-separated NOESY and 13C-separated NOESY experiments. RESULTS +174 193 13C-separated NOESY experimental_method 2,778 non-degenerate NOE restraints were used in initial structure calculations (1,791 unambiguous and 987 ambiguous), derived from three-dimensional 15N-separated NOESY and 13C-separated NOESY experiments. RESULTS +29 33 NOEs evidence There were 1,845 unambiguous NOEs and 757 ambiguous NOEs after eight iterations. RESULTS +52 56 NOEs evidence There were 1,845 unambiguous NOEs and 757 ambiguous NOEs after eight iterations. RESULTS +4 14 structures evidence 100 structures were calculated in the final iteration; the 50 lowest energy structures were water-refined; and of these, the 35 lowest energy structures were analyzed. RESULTS +20 30 calculated experimental_method 100 structures were calculated in the final iteration; the 50 lowest energy structures were water-refined; and of these, the 35 lowest energy structures were analyzed. RESULTS +76 86 structures evidence 100 structures were calculated in the final iteration; the 50 lowest energy structures were water-refined; and of these, the 35 lowest energy structures were analyzed. RESULTS +142 152 structures evidence 100 structures were calculated in the final iteration; the 50 lowest energy structures were water-refined; and of these, the 35 lowest energy structures were analyzed. RESULTS +27 30 HR1 structure_element Table 1 indicates that the HR1 domain structure is well defined by the NMR data. RESULTS +38 47 structure evidence Table 1 indicates that the HR1 domain structure is well defined by the NMR data. RESULTS +71 74 NMR experimental_method Table 1 indicates that the HR1 domain structure is well defined by the NMR data. RESULTS +12 47 average root mean square deviations evidence a , the average root mean square deviations for the ensemble ± S.D. TABLE +24 33 structure evidence b c, values for the structure that is closest to the mean. TABLE +4 13 structure evidence The structure closest to the mean is shown in Fig. 3A. RESULTS +8 17 α-helices structure_element The two α-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (PDB code 2KE4) and PRK1 (PDB codes 1CXZ and 1URF). RESULTS +25 28 HR1 structure_element The two α-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (PDB code 2KE4) and PRK1 (PDB codes 1CXZ and 1URF). RESULTS +56 81 anti-parallel coiled-coil structure_element The two α-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (PDB code 2KE4) and PRK1 (PDB codes 1CXZ and 1URF). RESULTS +134 137 HR1 structure_element The two α-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (PDB code 2KE4) and PRK1 (PDB codes 1CXZ and 1URF). RESULTS +149 153 CIP4 protein The two α-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (PDB code 2KE4) and PRK1 (PDB codes 1CXZ and 1URF). RESULTS +174 178 PRK1 protein The two α-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (PDB code 2KE4) and PRK1 (PDB codes 1CXZ and 1URF). RESULTS +2 20 sequence alignment experimental_method A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B. RESULTS +74 79 TOCA1 protein A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B. RESULTS +84 88 CIP4 protein A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B. RESULTS +89 92 HR1 structure_element A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B. RESULTS +109 113 HR1a structure_element A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B. RESULTS +118 122 HR1b structure_element A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B. RESULTS +136 140 PRK1 protein A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B. RESULTS +4 13 structure evidence The structure of the TOCA1 HR1 domain. FIG +21 26 TOCA1 protein The structure of the TOCA1 HR1 domain. FIG +27 30 HR1 structure_element The structure of the TOCA1 HR1 domain. FIG +17 22 trace evidence A, the backbone trace of the 35 lowest energy structures of the HR1 domain overlaid with the structure closest to the mean is shown alongside a schematic representation of the structure closest to the mean. FIG +47 57 structures evidence A, the backbone trace of the 35 lowest energy structures of the HR1 domain overlaid with the structure closest to the mean is shown alongside a schematic representation of the structure closest to the mean. FIG +65 68 HR1 structure_element A, the backbone trace of the 35 lowest energy structures of the HR1 domain overlaid with the structure closest to the mean is shown alongside a schematic representation of the structure closest to the mean. FIG +94 103 structure evidence A, the backbone trace of the 35 lowest energy structures of the HR1 domain overlaid with the structure closest to the mean is shown alongside a schematic representation of the structure closest to the mean. FIG +177 186 structure evidence A, the backbone trace of the 35 lowest energy structures of the HR1 domain overlaid with the structure closest to the mean is shown alongside a schematic representation of the structure closest to the mean. FIG +50 57 330–333 residue_range Flexible regions at the N and C termini (residues 330–333 and 421–426) are omitted for clarity. FIG +62 69 421–426 residue_range Flexible regions at the N and C termini (residues 330–333 and 421–426) are omitted for clarity. FIG +5 23 sequence alignment experimental_method B, a sequence alignment of the HR1 domains from TOCA1, CIP4, and PRK1. FIG +31 34 HR1 structure_element B, a sequence alignment of the HR1 domains from TOCA1, CIP4, and PRK1. FIG +48 53 TOCA1 protein B, a sequence alignment of the HR1 domains from TOCA1, CIP4, and PRK1. FIG +55 59 CIP4 protein B, a sequence alignment of the HR1 domains from TOCA1, CIP4, and PRK1. FIG +65 69 PRK1 protein B, a sequence alignment of the HR1 domains from TOCA1, CIP4, and PRK1. FIG +42 48 Stride experimental_method The secondary structure was deduced using Stride, based on the Ramachandran angles, and is indicated as follows: gray, turn; yellow, α-helix; blue, 310 helix; white, coil. FIG +63 82 Ramachandran angles evidence The secondary structure was deduced using Stride, based on the Ramachandran angles, and is indicated as follows: gray, turn; yellow, α-helix; blue, 310 helix; white, coil. FIG +133 140 α-helix structure_element The secondary structure was deduced using Stride, based on the Ramachandran angles, and is indicated as follows: gray, turn; yellow, α-helix; blue, 310 helix; white, coil. FIG +148 157 310 helix structure_element The secondary structure was deduced using Stride, based on the Ramachandran angles, and is indicated as follows: gray, turn; yellow, α-helix; blue, 310 helix; white, coil. FIG +42 47 TOCA1 protein C, a close-up of the N-terminal region of TOCA1 HR1, indicating some of the NOEs defining its position with respect to the two α-helices. FIG +48 51 HR1 structure_element C, a close-up of the N-terminal region of TOCA1 HR1, indicating some of the NOEs defining its position with respect to the two α-helices. FIG +76 80 NOEs evidence C, a close-up of the N-terminal region of TOCA1 HR1, indicating some of the NOEs defining its position with respect to the two α-helices. FIG +127 136 α-helices structure_element C, a close-up of the N-terminal region of TOCA1 HR1, indicating some of the NOEs defining its position with respect to the two α-helices. FIG +14 28 NOE restraints evidence Dotted lines, NOE restraints. FIG +21 36 interhelix loop structure_element D, a close-up of the interhelix loop region showing some of the contacts between the loop and helix 1. FIG +85 89 loop structure_element D, a close-up of the interhelix loop region showing some of the contacts between the loop and helix 1. FIG +94 101 helix 1 structure_element D, a close-up of the interhelix loop region showing some of the contacts between the loop and helix 1. FIG +7 11 HR1a structure_element In the HR1a domain of PRK1, a region N-terminal to helix 1 forms a short α-helix, which packs against both helices of the HR1 domain. RESULTS +22 26 PRK1 protein In the HR1a domain of PRK1, a region N-terminal to helix 1 forms a short α-helix, which packs against both helices of the HR1 domain. RESULTS +51 58 helix 1 structure_element In the HR1a domain of PRK1, a region N-terminal to helix 1 forms a short α-helix, which packs against both helices of the HR1 domain. RESULTS +67 80 short α-helix structure_element In the HR1a domain of PRK1, a region N-terminal to helix 1 forms a short α-helix, which packs against both helices of the HR1 domain. RESULTS +122 125 HR1 structure_element In the HR1a domain of PRK1, a region N-terminal to helix 1 forms a short α-helix, which packs against both helices of the HR1 domain. RESULTS +15 20 TOCA1 protein This region of TOCA1 HR1 (residues 334–340) is well defined in the family of structures (Fig. 3A) but does not form an α-helix. RESULTS +21 24 HR1 structure_element This region of TOCA1 HR1 (residues 334–340) is well defined in the family of structures (Fig. 3A) but does not form an α-helix. RESULTS +35 42 334–340 residue_range This region of TOCA1 HR1 (residues 334–340) is well defined in the family of structures (Fig. 3A) but does not form an α-helix. RESULTS +77 87 structures evidence This region of TOCA1 HR1 (residues 334–340) is well defined in the family of structures (Fig. 3A) but does not form an α-helix. RESULTS +119 126 α-helix structure_element This region of TOCA1 HR1 (residues 334–340) is well defined in the family of structures (Fig. 3A) but does not form an α-helix. RESULTS +47 61 NOE restraints evidence It instead forms a series of turns, defined by NOE restraints observed between residues separated by one (residues 332–334, 333–335, etc.) or two (residues 337–340) residues in the sequence and the φ and ψ angles, assessed using Stride. RESULTS +115 122 332–334 residue_range It instead forms a series of turns, defined by NOE restraints observed between residues separated by one (residues 332–334, 333–335, etc.) or two (residues 337–340) residues in the sequence and the φ and ψ angles, assessed using Stride. RESULTS +124 131 333–335 residue_range It instead forms a series of turns, defined by NOE restraints observed between residues separated by one (residues 332–334, 333–335, etc.) or two (residues 337–340) residues in the sequence and the φ and ψ angles, assessed using Stride. RESULTS +156 163 337–340 residue_range It instead forms a series of turns, defined by NOE restraints observed between residues separated by one (residues 332–334, 333–335, etc.) or two (residues 337–340) residues in the sequence and the φ and ψ angles, assessed using Stride. RESULTS +198 212 φ and ψ angles evidence It instead forms a series of turns, defined by NOE restraints observed between residues separated by one (residues 332–334, 333–335, etc.) or two (residues 337–340) residues in the sequence and the φ and ψ angles, assessed using Stride. RESULTS +229 235 Stride experimental_method It instead forms a series of turns, defined by NOE restraints observed between residues separated by one (residues 332–334, 333–335, etc.) or two (residues 337–340) residues in the sequence and the φ and ψ angles, assessed using Stride. RESULTS +92 99 334–340 residue_range These turns cause the chain to reverse direction, allowing the N-terminal segment (residues 334–340) to contact both helices of the HR1 domain. RESULTS +132 135 HR1 structure_element These turns cause the chain to reverse direction, allowing the N-terminal segment (residues 334–340) to contact both helices of the HR1 domain. RESULTS +11 15 NOEs evidence Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +38 45 Leu-334 residue_name_number Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +47 54 Glu-335 residue_name_number Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +60 67 Asp-336 residue_name_number Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +73 80 Trp-413 residue_name_number Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +84 91 helix 2 structure_element Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +93 100 Leu-334 residue_name_number Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +106 113 Lys-409 residue_name_number Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +117 124 helix 2 structure_element Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +130 137 Phe-337 residue_name_number Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +142 149 Ser-338 residue_name_number Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +155 162 Arg-345 residue_name_number Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +164 171 Arg-348 residue_name_number Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +177 184 Leu-349 residue_name_number Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +188 195 helix 1 structure_element Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1. RESULTS +8 17 α-helices structure_element The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 310 helices (residues 381–383 and 386–389). RESULTS +21 26 TOCA1 protein The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 310 helices (residues 381–383 and 386–389). RESULTS +27 30 HR1 structure_element The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 310 helices (residues 381–383 and 386–389). RESULTS +55 59 loop structure_element The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 310 helices (residues 381–383 and 386–389). RESULTS +85 92 380–389 residue_range The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 310 helices (residues 381–383 and 386–389). RESULTS +112 129 short 310 helices structure_element The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 310 helices (residues 381–383 and 386–389). RESULTS +140 147 381–383 residue_range The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 310 helices (residues 381–383 and 386–389). RESULTS +152 159 386–389 residue_range The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 310 helices (residues 381–383 and 386–389). RESULTS +50 61 loop region structure_element Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D). RESULTS +80 87 helix 1 structure_element Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D). RESULTS +162 169 Asn-380 residue_name_number Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D). RESULTS +174 181 Met-383 residue_name_number Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D). RESULTS +189 200 loop region structure_element Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D). RESULTS +205 212 Tyr-377 residue_name_number Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D). RESULTS +217 224 Val-376 residue_name_number Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D). RESULTS +228 235 helix 1 structure_element Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D). RESULTS +34 41 Gly-384 residue_name_number The backbone NH and CHα groups of Gly-384 and Asp-385 also show NOEs with the side chain of Tyr-377. RESULTS +46 53 Asp-385 residue_name_number The backbone NH and CHα groups of Gly-384 and Asp-385 also show NOEs with the side chain of Tyr-377. RESULTS +92 99 Tyr-377 residue_name_number The backbone NH and CHα groups of Gly-384 and Asp-385 also show NOEs with the side chain of Tyr-377. RESULTS +12 17 TOCA1 protein Mapping the TOCA1 and Cdc42 Binding Interfaces RESULTS +22 46 Cdc42 Binding Interfaces site Mapping the TOCA1 and Cdc42 Binding Interfaces RESULTS +4 28 HR1TOCA1-Cdc42 interface site The HR1TOCA1-Cdc42 interface was investigated using NMR spectroscopy. RESULTS +52 68 NMR spectroscopy experimental_method The HR1TOCA1-Cdc42 interface was investigated using NMR spectroscopy. RESULTS +12 20 15N HSQC experimental_method A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. RESULTS +49 52 15N chemical A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. RESULTS +53 60 labeled protein_state A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. RESULTS +61 66 TOCA1 protein A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. RESULTS +67 70 HR1 structure_element A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. RESULTS +85 96 presence of protein_state A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. RESULTS +97 122 increasing concentrations experimental_method A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. RESULTS +126 135 unlabeled protein_state A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. RESULTS +136 154 Cdc42Δ7Q61L·GMPPNP complex_assembly A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. RESULTS +166 187 Cdc42-binding surface site A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. RESULTS +20 28 15N HSQC experimental_method A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A). RESULTS +29 36 spectra evidence A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A). RESULTS +40 44 free protein_state A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A). RESULTS +45 48 HR1 structure_element A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A). RESULTS +53 56 HR1 structure_element A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A). RESULTS +64 75 presence of protein_state A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A). RESULTS +83 88 Cdc42 protein A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A). RESULTS +93 98 Cdc42 protein This behavior cannot be explained by the increase in molecular mass (from 12 to 33 kDa) when Cdc42 binds and is more likely to be due to conformational exchange. RESULTS +8 36 chemical shift perturbations experimental_method Overall chemical shift perturbations (CSPs) were calculated for each residue, whereas those that had disappeared were assigned a shift change of 0.2 (Fig. 4B). RESULTS +38 42 CSPs experimental_method Overall chemical shift perturbations (CSPs) were calculated for each residue, whereas those that had disappeared were assigned a shift change of 0.2 (Fig. 4B). RESULTS +33 36 CSP experimental_method A peak that disappeared or had a CSP above the mean CSP for the spectrum was considered to be significantly affected. RESULTS +52 55 CSP experimental_method A peak that disappeared or had a CSP above the mean CSP for the spectrum was considered to be significantly affected. RESULTS +12 27 binding surface site Mapping the binding surface of Cdc42 onto the TOCA1 HR1 domain. FIG +31 36 Cdc42 protein Mapping the binding surface of Cdc42 onto the TOCA1 HR1 domain. FIG +46 51 TOCA1 protein Mapping the binding surface of Cdc42 onto the TOCA1 HR1 domain. FIG +52 55 HR1 structure_element Mapping the binding surface of Cdc42 onto the TOCA1 HR1 domain. FIG +8 16 15N HSQC experimental_method A, the 15N HSQC of 200 μm TOCA1 HR1 domain is shown in the free form (black) and in the presence of a 4-fold molar excess of Cdc42Δ7Q61L·GMPPNP (red). FIG +27 32 TOCA1 protein A, the 15N HSQC of 200 μm TOCA1 HR1 domain is shown in the free form (black) and in the presence of a 4-fold molar excess of Cdc42Δ7Q61L·GMPPNP (red). FIG +33 36 HR1 structure_element A, the 15N HSQC of 200 μm TOCA1 HR1 domain is shown in the free form (black) and in the presence of a 4-fold molar excess of Cdc42Δ7Q61L·GMPPNP (red). FIG +60 69 free form protein_state A, the 15N HSQC of 200 μm TOCA1 HR1 domain is shown in the free form (black) and in the presence of a 4-fold molar excess of Cdc42Δ7Q61L·GMPPNP (red). FIG +89 100 presence of protein_state A, the 15N HSQC of 200 μm TOCA1 HR1 domain is shown in the free form (black) and in the presence of a 4-fold molar excess of Cdc42Δ7Q61L·GMPPNP (red). FIG +126 144 Cdc42Δ7Q61L·GMPPNP complex_assembly A, the 15N HSQC of 200 μm TOCA1 HR1 domain is shown in the free form (black) and in the presence of a 4-fold molar excess of Cdc42Δ7Q61L·GMPPNP (red). FIG +3 7 CSPs experimental_method B, CSPs were calculated as described under “Experimental Procedures” and are shown for backbone and side chain NH groups. FIG +9 12 CSP experimental_method The mean CSP is marked with a red line. FIG +33 44 presence of protein_state Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.2 but were excluded when calculating the mean CSP and are indicated with open bars. FIG +45 50 Cdc42 protein Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.2 but were excluded when calculating the mean CSP and are indicated with open bars. FIG +67 70 CSP experimental_method Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.2 but were excluded when calculating the mean CSP and are indicated with open bars. FIG +122 125 CSP experimental_method Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.2 but were excluded when calculating the mean CSP and are indicated with open bars. FIG +70 73 CSP experimental_method Those that were not traceable due to spectral overlap were assigned a CSP of zero and are marked with an asterisk below the bar. FIG +34 38 CSPs experimental_method Residues with affected side chain CSPs derived from 13C HSQCs are marked with green asterisks above the bars. FIG +52 61 13C HSQCs experimental_method Residues with affected side chain CSPs derived from 13C HSQCs are marked with green asterisks above the bars. FIG +37 40 HR1 structure_element C, a schematic representation of the HR1 domain. FIG +81 92 Cdc42 bound protein_state Residues with significantly affected backbone or side chain chemical shifts when Cdc42 bound and that are buried are colored dark blue, whereas those that are solvent-accessible are colored yellow. FIG +159 177 solvent-accessible protein_state Residues with significantly affected backbone or side chain chemical shifts when Cdc42 bound and that are buried are colored dark blue, whereas those that are solvent-accessible are colored yellow. FIG +77 95 solvent-accessible protein_state Residues with significantly affected backbone and side chain groups that are solvent-accessible are colored red. FIG +18 32 binding region site A close-up of the binding region is shown, with affected side chain heavy atoms shown as sticks. FIG +7 31 G protein-binding region site D, the G protein-binding region is marked in red onto structures of the HR1 domains as indicated. FIG +54 64 structures evidence D, the G protein-binding region is marked in red onto structures of the HR1 domains as indicated. FIG +72 75 HR1 structure_element D, the G protein-binding region is marked in red onto structures of the HR1 domains as indicated. FIG +0 22 15N HSQC shift mapping experimental_method 15N HSQC shift mapping experiments report on changes to amide groups, which are mainly inaccessible because they are buried inside the helices and are involved in hydrogen bonds. RESULTS +135 142 helices structure_element 15N HSQC shift mapping experiments report on changes to amide groups, which are mainly inaccessible because they are buried inside the helices and are involved in hydrogen bonds. RESULTS +163 177 hydrogen bonds bond_interaction 15N HSQC shift mapping experiments report on changes to amide groups, which are mainly inaccessible because they are buried inside the helices and are involved in hydrogen bonds. RESULTS +11 19 13C HSQC experimental_method Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement. RESULTS +24 52 methyl-selective SOFAST-HMQC experimental_method Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement. RESULTS +87 90 15N chemical Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement. RESULTS +91 94 13C chemical Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement. RESULTS +95 102 labeled protein_state Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement. RESULTS +103 108 TOCA1 protein Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement. RESULTS +109 112 HR1 structure_element Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement. RESULTS +47 58 presence of protein_state Side chains whose CH groups disappeared in the presence of Cdc42 are marked on the graph in Fig. 4B with green asterisks. RESULTS +59 64 Cdc42 protein Side chains whose CH groups disappeared in the presence of Cdc42 are marked on the graph in Fig. 4B with green asterisks. RESULTS +0 5 TOCA1 protein TOCA1 residues whose signals were affected by Cdc42 binding were mapped onto the structure of TOCA1 HR1 (Fig. 4C). RESULTS +46 51 Cdc42 protein TOCA1 residues whose signals were affected by Cdc42 binding were mapped onto the structure of TOCA1 HR1 (Fig. 4C). RESULTS +81 90 structure evidence TOCA1 residues whose signals were affected by Cdc42 binding were mapped onto the structure of TOCA1 HR1 (Fig. 4C). RESULTS +94 99 TOCA1 protein TOCA1 residues whose signals were affected by Cdc42 binding were mapped onto the structure of TOCA1 HR1 (Fig. 4C). RESULTS +100 103 HR1 structure_element TOCA1 residues whose signals were affected by Cdc42 binding were mapped onto the structure of TOCA1 HR1 (Fig. 4C). RESULTS +45 56 coiled-coil structure_element The changes were localized to one end of the coiled-coil, and the binding site appeared to include residues from both α-helices and the loop region that joins them. RESULTS +66 78 binding site site The changes were localized to one end of the coiled-coil, and the binding site appeared to include residues from both α-helices and the loop region that joins them. RESULTS +118 127 α-helices structure_element The changes were localized to one end of the coiled-coil, and the binding site appeared to include residues from both α-helices and the loop region that joins them. RESULTS +136 147 loop region structure_element The changes were localized to one end of the coiled-coil, and the binding site appeared to include residues from both α-helices and the loop region that joins them. RESULTS +20 37 interhelical loop structure_element The residues in the interhelical loop and helix 1 that contact each other (Fig. 3D) show shift changes in their backbone NH and side chains in the presence of Cdc42. RESULTS +42 49 helix 1 structure_element The residues in the interhelical loop and helix 1 that contact each other (Fig. 3D) show shift changes in their backbone NH and side chains in the presence of Cdc42. RESULTS +147 158 presence of protein_state The residues in the interhelical loop and helix 1 that contact each other (Fig. 3D) show shift changes in their backbone NH and side chains in the presence of Cdc42. RESULTS +159 164 Cdc42 protein The residues in the interhelical loop and helix 1 that contact each other (Fig. 3D) show shift changes in their backbone NH and side chains in the presence of Cdc42. RESULTS +31 38 Asn-380 residue_name_number For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. RESULTS +60 67 Val-376 residue_name_number For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. RESULTS +72 79 Tyr-377 residue_name_number For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. RESULTS +134 138 free protein_state For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. RESULTS +139 144 TOCA1 protein For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. RESULTS +145 148 HR1 structure_element For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. RESULTS +149 158 structure evidence For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. RESULTS +212 216 loop structure_element For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. RESULTS +4 26 chemical shift mapping experimental_method The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D). RESULTS +50 74 G protein-binding region site The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D). RESULTS +82 87 TOCA1 protein The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D). RESULTS +88 91 HR1 structure_element The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D). RESULTS +133 137 CIP4 protein The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D). RESULTS +142 146 PRK1 protein The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D). RESULTS +147 150 HR1 structure_element The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D). RESULTS +18 21 15N experimental_method The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. RESULTS +26 33 13C NMR experimental_method The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. RESULTS +68 71 15N chemical The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. RESULTS +72 90 Cdc42Δ7Q61L·GMPPNP complex_assembly The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. RESULTS +94 97 15N chemical The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. RESULTS +98 101 13C chemical The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. RESULTS +103 121 Cdc42Δ7Q61L·GMPPNP complex_assembly The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. RESULTS +129 140 presence of protein_state The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. RESULTS +141 150 unlabeled protein_state The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. RESULTS +151 154 HR1 structure_element The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. RESULTS +12 15 CSP experimental_method The overall CSP was calculated for each residue. RESULTS +21 28 labeled protein_state As was the case when labeled HR1 was observed, several peaks were shifted in the complex, but many disappeared, indicating exchange on an unfavorable, millisecond time scale (Fig. 5A). RESULTS +29 32 HR1 structure_element As was the case when labeled HR1 was observed, several peaks were shifted in the complex, but many disappeared, indicating exchange on an unfavorable, millisecond time scale (Fig. 5A). RESULTS +93 115 constant time 13C HSQC experimental_method Detailed side chain data could not be obtained for all residues due to spectral overlap, but constant time 13C HSQC and methyl-selective SOFAST-HMQC experiments provided further information on certain well resolved side chains (marked with green asterisks in Fig. 5B). RESULTS +120 148 methyl-selective SOFAST-HMQC experimental_method Detailed side chain data could not be obtained for all residues due to spectral overlap, but constant time 13C HSQC and methyl-selective SOFAST-HMQC experiments provided further information on certain well resolved side chains (marked with green asterisks in Fig. 5B). RESULTS +12 27 binding surface site Mapping the binding surface of the HR1 domain onto Cdc42. FIG +35 38 HR1 structure_element Mapping the binding surface of the HR1 domain onto Cdc42. FIG +51 56 Cdc42 protein Mapping the binding surface of the HR1 domain onto Cdc42. FIG +8 16 15N HSQC experimental_method A, the 15N HSQC of Cdc42Δ7Q61L·GMPPNP is shown in its free form (black) and in the presence of excess TOCA1 HR1 domain (1:2.2, red). FIG +20 38 Cdc42Δ7Q61L·GMPPNP complex_assembly A, the 15N HSQC of Cdc42Δ7Q61L·GMPPNP is shown in its free form (black) and in the presence of excess TOCA1 HR1 domain (1:2.2, red). FIG +55 64 free form protein_state A, the 15N HSQC of Cdc42Δ7Q61L·GMPPNP is shown in its free form (black) and in the presence of excess TOCA1 HR1 domain (1:2.2, red). FIG +84 95 presence of protein_state A, the 15N HSQC of Cdc42Δ7Q61L·GMPPNP is shown in its free form (black) and in the presence of excess TOCA1 HR1 domain (1:2.2, red). FIG +103 108 TOCA1 protein A, the 15N HSQC of Cdc42Δ7Q61L·GMPPNP is shown in its free form (black) and in the presence of excess TOCA1 HR1 domain (1:2.2, red). FIG +109 112 HR1 structure_element A, the 15N HSQC of Cdc42Δ7Q61L·GMPPNP is shown in its free form (black) and in the presence of excess TOCA1 HR1 domain (1:2.2, red). FIG +3 7 CSPs experimental_method B, CSPs are shown for backbone NH groups. FIG +32 35 CSP experimental_method The red line indicates the mean CSP, plus one S.D. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.1 and are indicated with open bars. FIG +84 95 presence of protein_state The red line indicates the mean CSP, plus one S.D. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.1 and are indicated with open bars. FIG +96 101 Cdc42 protein The red line indicates the mean CSP, plus one S.D. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.1 and are indicated with open bars. FIG +118 121 CSP experimental_method The red line indicates the mean CSP, plus one S.D. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.1 and are indicated with open bars. FIG +35 43 13C HSQC experimental_method Residues with disappeared peaks in 13C HSQC experiments are marked on the chart with green asterisks. FIG +97 100 NMR experimental_method C, the residues with significantly affected backbone and side chain groups are highlighted on an NMR structure of free Cdc42Δ7Q61L·GMPPNP; those that are buried are colored dark blue, whereas those that are solvent-accessible are colored red. FIG +101 110 structure evidence C, the residues with significantly affected backbone and side chain groups are highlighted on an NMR structure of free Cdc42Δ7Q61L·GMPPNP; those that are buried are colored dark blue, whereas those that are solvent-accessible are colored red. FIG +114 118 free protein_state C, the residues with significantly affected backbone and side chain groups are highlighted on an NMR structure of free Cdc42Δ7Q61L·GMPPNP; those that are buried are colored dark blue, whereas those that are solvent-accessible are colored red. FIG +119 137 Cdc42Δ7Q61L·GMPPNP complex_assembly C, the residues with significantly affected backbone and side chain groups are highlighted on an NMR structure of free Cdc42Δ7Q61L·GMPPNP; those that are buried are colored dark blue, whereas those that are solvent-accessible are colored red. FIG +207 225 solvent-accessible protein_state C, the residues with significantly affected backbone and side chain groups are highlighted on an NMR structure of free Cdc42Δ7Q61L·GMPPNP; those that are buried are colored dark blue, whereas those that are solvent-accessible are colored red. FIG +101 119 solvent-accessible protein_state Residues with either side chain or backbone groups affected are colored blue if buried and yellow if solvent-accessible. FIG +34 47 shift mapping experimental_method Residues without information from shift mapping are colored gray. FIG +4 12 flexible protein_state The flexible switch regions are circled. FIG +13 27 switch regions site The flexible switch regions are circled. FIG +38 64 mean chemical shift change evidence As many of the peaks disappeared, the mean chemical shift change was relatively low, so a threshold of the mean plus one S.D. value was used to define a significant CSP. RESULTS +165 168 CSP experimental_method As many of the peaks disappeared, the mean chemical shift change was relatively low, so a threshold of the mean plus one S.D. value was used to define a significant CSP. RESULTS +13 27 switch regions site Parts of the switch regions (Fig. 5, B and C) are invisible in NMR spectra recorded on free Cdc42 due to conformational exchange. RESULTS +63 66 NMR experimental_method Parts of the switch regions (Fig. 5, B and C) are invisible in NMR spectra recorded on free Cdc42 due to conformational exchange. RESULTS +67 74 spectra evidence Parts of the switch regions (Fig. 5, B and C) are invisible in NMR spectra recorded on free Cdc42 due to conformational exchange. RESULTS +87 91 free protein_state Parts of the switch regions (Fig. 5, B and C) are invisible in NMR spectra recorded on free Cdc42 due to conformational exchange. RESULTS +92 97 Cdc42 protein Parts of the switch regions (Fig. 5, B and C) are invisible in NMR spectra recorded on free Cdc42 due to conformational exchange. RESULTS +6 20 switch regions site These switch regions become visible in Cdc42 and other small G protein·effector complexes due to a decrease in conformational freedom upon complex formation. RESULTS +39 44 Cdc42 protein These switch regions become visible in Cdc42 and other small G protein·effector complexes due to a decrease in conformational freedom upon complex formation. RESULTS +61 70 G protein protein_type These switch regions become visible in Cdc42 and other small G protein·effector complexes due to a decrease in conformational freedom upon complex formation. RESULTS +4 18 switch regions site The switch regions of Cdc42 did not, however, become visible in the presence of the TOCA1 HR1 domain. RESULTS +22 27 Cdc42 protein The switch regions of Cdc42 did not, however, become visible in the presence of the TOCA1 HR1 domain. RESULTS +68 79 presence of protein_state The switch regions of Cdc42 did not, however, become visible in the presence of the TOCA1 HR1 domain. RESULTS +84 89 TOCA1 protein The switch regions of Cdc42 did not, however, become visible in the presence of the TOCA1 HR1 domain. RESULTS +90 93 HR1 structure_element The switch regions of Cdc42 did not, however, become visible in the presence of the TOCA1 HR1 domain. RESULTS +8 14 Ser-30 residue_name_number Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. RESULTS +18 26 switch I site Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. RESULTS +31 37 Arg-66 residue_name_number Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. RESULTS +39 45 Arg-68 residue_name_number Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. RESULTS +47 53 Leu-70 residue_name_number Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. RESULTS +59 65 Ser-71 residue_name_number Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. RESULTS +69 78 switch II site Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. RESULTS +94 98 free protein_state Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. RESULTS +99 104 Cdc42 protein Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. RESULTS +126 137 presence of protein_state Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. RESULTS +142 145 HR1 structure_element Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain. RESULTS +23 37 switch regions site This suggests that the switch regions are not rigidified in the HR1 complex and are still in conformational exchange. RESULTS +64 67 HR1 structure_element This suggests that the switch regions are not rigidified in the HR1 complex and are still in conformational exchange. RESULTS +56 59 NMR experimental_method Nevertheless, mapping of the affected residues onto the NMR structure of free Cdc42Δ7Q61L·GMPPNP (Fig. 5C)8 shows that, although they are relatively widespread compared with changes in the HR1 domain, in general, they are on the face of the protein that includes the switches. RESULTS +60 69 structure evidence Nevertheless, mapping of the affected residues onto the NMR structure of free Cdc42Δ7Q61L·GMPPNP (Fig. 5C)8 shows that, although they are relatively widespread compared with changes in the HR1 domain, in general, they are on the face of the protein that includes the switches. RESULTS +73 77 free protein_state Nevertheless, mapping of the affected residues onto the NMR structure of free Cdc42Δ7Q61L·GMPPNP (Fig. 5C)8 shows that, although they are relatively widespread compared with changes in the HR1 domain, in general, they are on the face of the protein that includes the switches. RESULTS +78 96 Cdc42Δ7Q61L·GMPPNP complex_assembly Nevertheless, mapping of the affected residues onto the NMR structure of free Cdc42Δ7Q61L·GMPPNP (Fig. 5C)8 shows that, although they are relatively widespread compared with changes in the HR1 domain, in general, they are on the face of the protein that includes the switches. RESULTS +189 192 HR1 structure_element Nevertheless, mapping of the affected residues onto the NMR structure of free Cdc42Δ7Q61L·GMPPNP (Fig. 5C)8 shows that, although they are relatively widespread compared with changes in the HR1 domain, in general, they are on the face of the protein that includes the switches. RESULTS +267 275 switches site Nevertheless, mapping of the affected residues onto the NMR structure of free Cdc42Δ7Q61L·GMPPNP (Fig. 5C)8 shows that, although they are relatively widespread compared with changes in the HR1 domain, in general, they are on the face of the protein that includes the switches. RESULTS +13 30 binding interface site Although the binding interface may be overestimated, this suggests that the switch regions are involved in binding to TOCA1. RESULTS +76 90 switch regions site Although the binding interface may be overestimated, this suggests that the switch regions are involved in binding to TOCA1. RESULTS +118 123 TOCA1 protein Although the binding interface may be overestimated, this suggests that the switch regions are involved in binding to TOCA1. RESULTS +13 28 Cdc42·TOCA1 HR1 complex_assembly Modeling the Cdc42·TOCA1 HR1 Complex RESULTS +4 18 Cdc42·HR1TOCA1 complex_assembly The Cdc42·HR1TOCA1 complex was not amenable to full structural analysis due to the weak interaction and the extensive exchange broadening seen in the NMR experiments. RESULTS +150 153 NMR experimental_method The Cdc42·HR1TOCA1 complex was not amenable to full structural analysis due to the weak interaction and the extensive exchange broadening seen in the NMR experiments. RESULTS +0 7 HADDOCK experimental_method HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above. RESULTS +38 56 rigid body docking experimental_method HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above. RESULTS +70 80 structures evidence HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above. RESULTS +84 88 free protein_state HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above. RESULTS +89 92 HR1 structure_element HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above. RESULTS +104 109 Cdc42 protein HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above. RESULTS +164 185 titration experiments experimental_method HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above. RESULTS +23 26 HR1 structure_element The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42. RESULTS +50 55 Cdc42 protein The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42. RESULTS +147 154 HADDOCK experimental_method The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42. RESULTS +193 196 HR1 structure_element The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42. RESULTS +233 238 Cdc42 protein The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42. RESULTS +286 291 Cdc42 protein The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42. RESULTS +28 54 root mean square deviation evidence The cluster with the lowest root mean square deviation from the lowest energy structure is assumed to be the best model. RESULTS +78 87 structure evidence The cluster with the lowest root mean square deviation from the lowest energy structure is assumed to be the best model. RESULTS +42 45 HR1 structure_element By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. RESULTS +88 92 HR1a structure_element By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. RESULTS +103 107 PRK1 protein By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. RESULTS +108 116 bound to protein_state By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. RESULTS +117 121 RhoA protein By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. RESULTS +130 134 HR1b structure_element By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. RESULTS +142 150 bound to protein_state By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. RESULTS +151 155 Rac1 protein By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. RESULTS +75 84 Rac1-HR1b complex_assembly A representative model from this cluster is shown in Fig. 6A alongside the Rac1-HR1b structure (PDB code 2RMK) in Fig. 6B. RESULTS +85 94 structure evidence A representative model from this cluster is shown in Fig. 6A alongside the Rac1-HR1b structure (PDB code 2RMK) in Fig. 6B. RESULTS +9 18 Cdc42·HR1 complex_assembly Model of Cdc42·HR1 complex. FIG +34 43 Cdc42·HR1 complex_assembly A, a representative model of the Cdc42·HR1 complex from the cluster closest to the lowest energy model produced using HADDOCK. FIG +119 126 HADDOCK experimental_method A, a representative model of the Cdc42·HR1 complex from the cluster closest to the lowest energy model produced using HADDOCK. FIG +12 17 Cdc42 protein Residues of Cdc42 that are affected in the presence of the HR1 domain but are not in close proximity to it are colored in red and labeled. FIG +43 54 presence of protein_state Residues of Cdc42 that are affected in the presence of the HR1 domain but are not in close proximity to it are colored in red and labeled. FIG +59 62 HR1 structure_element Residues of Cdc42 that are affected in the presence of the HR1 domain but are not in close proximity to it are colored in red and labeled. FIG +3 12 structure evidence B, structure of Rac1 in complex with the HR1b domain of PRK1 (PDB code 2RMK). FIG +16 20 Rac1 protein B, structure of Rac1 in complex with the HR1b domain of PRK1 (PDB code 2RMK). FIG +21 36 in complex with protein_state B, structure of Rac1 in complex with the HR1b domain of PRK1 (PDB code 2RMK). FIG +41 45 HR1b structure_element B, structure of Rac1 in complex with the HR1b domain of PRK1 (PDB code 2RMK). FIG +56 60 PRK1 protein B, structure of Rac1 in complex with the HR1b domain of PRK1 (PDB code 2RMK). FIG +3 21 sequence alignment experimental_method C, sequence alignment of RhoA, Cdc42 and Rac1. FIG +25 29 RhoA protein C, sequence alignment of RhoA, Cdc42 and Rac1. FIG +31 36 Cdc42 protein C, sequence alignment of RhoA, Cdc42 and Rac1. FIG +41 45 Rac1 protein C, sequence alignment of RhoA, Cdc42 and Rac1. FIG +20 24 RhoA protein Contact residues of RhoA and Rac1 to PRK1 HR1a and HR1b, respectively, are colored cyan. FIG +29 33 Rac1 protein Contact residues of RhoA and Rac1 to PRK1 HR1a and HR1b, respectively, are colored cyan. FIG +37 41 PRK1 protein Contact residues of RhoA and Rac1 to PRK1 HR1a and HR1b, respectively, are colored cyan. FIG +42 46 HR1a structure_element Contact residues of RhoA and Rac1 to PRK1 HR1a and HR1b, respectively, are colored cyan. FIG +51 55 HR1b structure_element Contact residues of RhoA and Rac1 to PRK1 HR1a and HR1b, respectively, are colored cyan. FIG +12 17 Cdc42 protein Residues of Cdc42 that disappear or show chemical shift changes in the presence of TOCA1 are colored cyan if also identified as contacts in RhoA and Rac1 and yellow if they are not. FIG +71 82 presence of protein_state Residues of Cdc42 that disappear or show chemical shift changes in the presence of TOCA1 are colored cyan if also identified as contacts in RhoA and Rac1 and yellow if they are not. FIG +83 88 TOCA1 protein Residues of Cdc42 that disappear or show chemical shift changes in the presence of TOCA1 are colored cyan if also identified as contacts in RhoA and Rac1 and yellow if they are not. FIG +140 144 RhoA protein Residues of Cdc42 that disappear or show chemical shift changes in the presence of TOCA1 are colored cyan if also identified as contacts in RhoA and Rac1 and yellow if they are not. FIG +149 153 Rac1 protein Residues of Cdc42 that disappear or show chemical shift changes in the presence of TOCA1 are colored cyan if also identified as contacts in RhoA and Rac1 and yellow if they are not. FIG +23 27 Rac1 protein Residues equivalent to Rac1 and RhoA contact sites but that are invisible in free Cdc42 are gray. FIG +32 36 RhoA protein Residues equivalent to Rac1 and RhoA contact sites but that are invisible in free Cdc42 are gray. FIG +37 50 contact sites site Residues equivalent to Rac1 and RhoA contact sites but that are invisible in free Cdc42 are gray. FIG +77 81 free protein_state Residues equivalent to Rac1 and RhoA contact sites but that are invisible in free Cdc42 are gray. FIG +82 87 Cdc42 protein Residues equivalent to Rac1 and RhoA contact sites but that are invisible in free Cdc42 are gray. FIG +30 39 Cdc42·HR1 complex_assembly D, regions of interest of the Cdc42·HR1 domain model. FIG +23 33 structures evidence The four lowest energy structures in the chosen HADDOCK cluster are shown overlaid, with the residues of interest shown as sticks and labeled. FIG +48 55 HADDOCK experimental_method The four lowest energy structures in the chosen HADDOCK cluster are shown overlaid, with the residues of interest shown as sticks and labeled. FIG +28 33 TOCA1 protein Cdc42 is shown in cyan, and TOCA1 is shown in purple. FIG +2 20 sequence alignment experimental_method A sequence alignment of RhoA, Cdc42, and Rac1 is shown in Fig. 6C. RESULTS +24 28 RhoA protein A sequence alignment of RhoA, Cdc42, and Rac1 is shown in Fig. 6C. RESULTS +30 35 Cdc42 protein A sequence alignment of RhoA, Cdc42, and Rac1 is shown in Fig. 6C. RESULTS +41 45 Rac1 protein A sequence alignment of RhoA, Cdc42, and Rac1 is shown in Fig. 6C. RESULTS +4 8 RhoA protein The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins. RESULTS +13 17 Rac1 protein The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins. RESULTS +42 56 switch regions site The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins. RESULTS +78 85 spectra evidence The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins. RESULTS +89 94 Cdc42 protein The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins. RESULTS +119 128 conserved protein_state The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins. RESULTS +147 157 G proteins protein_type The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins. RESULTS +8 13 Cdc42 protein Several Cdc42 residues identified by chemical shift mapping are not in close contact in the Cdc42·TOCA1 model (Fig. 6A). RESULTS +37 59 chemical shift mapping experimental_method Several Cdc42 residues identified by chemical shift mapping are not in close contact in the Cdc42·TOCA1 model (Fig. 6A). RESULTS +92 103 Cdc42·TOCA1 complex_assembly Several Cdc42 residues identified by chemical shift mapping are not in close contact in the Cdc42·TOCA1 model (Fig. 6A). RESULTS +48 54 Thr-24 residue_name_number Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +54 59 Cdc42 protein Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +61 68 Leu-160 residue_name_number Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +68 73 Cdc42 protein Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +79 86 Lys-163 residue_name_number Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +86 91 Cdc42 protein Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +108 116 switch I site Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +184 190 switch site Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +198 204 Glu-95 residue_name_number Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +204 209 Cdc42 protein Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +214 220 Lys-96 residue_name_number Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +220 225 Cdc42 protein Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +237 242 helix structure_element Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +250 259 switch II site Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. RESULTS +40 51 Cdc42·TOCA1 complex_assembly Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. RESULTS +110 114 RhoA protein Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. RESULTS +118 122 Rac1 protein Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. RESULTS +141 146 Gln-2 residue_name_number Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. RESULTS +146 151 Cdc42 protein Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. RESULTS +153 159 Lys-16 residue_name_number Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. RESULTS +159 164 Cdc42 protein Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. RESULTS +166 172 Thr-52 residue_name_number Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. RESULTS +172 177 Cdc42 protein Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. RESULTS +183 189 Arg-68 residue_name_number Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. RESULTS +189 194 Cdc42 protein Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. RESULTS +0 6 Lys-16 residue_name_number Lys-16Cdc42 is unlikely to be a contact residue because it is involved in nucleotide binding, but the others may represent specific Cdc42-TOCA1 contacts. RESULTS +6 11 Cdc42 protein Lys-16Cdc42 is unlikely to be a contact residue because it is involved in nucleotide binding, but the others may represent specific Cdc42-TOCA1 contacts. RESULTS +132 143 Cdc42-TOCA1 complex_assembly Lys-16Cdc42 is unlikely to be a contact residue because it is involved in nucleotide binding, but the others may represent specific Cdc42-TOCA1 contacts. RESULTS +20 26 N-WASP protein Competition between N-WASP and TOCA1 RESULTS +31 36 TOCA1 protein Competition between N-WASP and TOCA1 RESULTS +106 111 TOCA1 protein From the known interactions and effects of the proteins in biological systems, it has been suggested that TOCA1 and N-WASP could bind Cdc42 simultaneously. RESULTS +116 122 N-WASP protein From the known interactions and effects of the proteins in biological systems, it has been suggested that TOCA1 and N-WASP could bind Cdc42 simultaneously. RESULTS +134 139 Cdc42 protein From the known interactions and effects of the proteins in biological systems, it has been suggested that TOCA1 and N-WASP could bind Cdc42 simultaneously. RESULTS +38 56 Cdc42·N-WASP·TOCA1 complex_assembly Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. RESULTS +81 85 FRET evidence Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. RESULTS +107 110 RFP chemical Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. RESULTS +111 116 TOCA1 protein Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. RESULTS +121 124 GFP chemical Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. RESULTS +125 131 N-WASP protein Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. RESULTS +174 180 N-WASP protein Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. RESULTS +181 187 mutant protein_state Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. RESULTS +218 223 Cdc42 protein Studies in CHO cells indicated that a Cdc42·N-WASP·TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42. RESULTS +3 10 overlay experimental_method An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). RESULTS +18 25 HADDOCK experimental_method An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). RESULTS +26 31 model evidence An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). RESULTS +39 53 Cdc42·HR1TOCA1 complex_assembly An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). RESULTS +70 79 structure evidence An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). RESULTS +83 88 Cdc42 protein An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). RESULTS +89 104 in complex with protein_state An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). RESULTS +109 112 GBD structure_element An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). RESULTS +120 126 N-WASP protein An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). RESULTS +138 142 WASP protein An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). RESULTS +175 178 HR1 structure_element An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). RESULTS +183 200 GBD binding sites site An overlay of the HADDOCK model of the Cdc42·HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A). RESULTS +19 30 presence of protein_state Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. RESULTS +35 40 TOCA1 protein Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. RESULTS +41 44 HR1 structure_element Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. RESULTS +72 76 CRIB structure_element Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. RESULTS +80 84 WASP protein Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. RESULTS +101 106 Cdc42 protein Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. RESULTS +147 151 CRIB structure_element Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. RESULTS +199 203 WASP protein Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. RESULTS +240 245 TOCA1 protein Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. RESULTS +246 249 HR1 structure_element Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP would interfere sterically with the TOCA1 HR1. RESULTS +18 22 WASP protein A basic region in WASP including three lysines (residues 230–232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A). RESULTS +39 46 lysines residue_name A basic region in WASP including three lysines (residues 230–232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A). RESULTS +57 64 230–232 residue_range A basic region in WASP including three lysines (residues 230–232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A). RESULTS +90 94 CRIB structure_element A basic region in WASP including three lysines (residues 230–232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A). RESULTS +204 215 presence of protein_state A basic region in WASP including three lysines (residues 230–232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A). RESULTS +216 221 TOCA1 protein A basic region in WASP including three lysines (residues 230–232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A). RESULTS +222 225 HR1 structure_element A basic region in WASP including three lysines (residues 230–232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A). RESULTS +4 10 N-WASP protein The N-WASP GBD displaces the TOCA1 HR1 domain. FIG +11 14 GBD structure_element The N-WASP GBD displaces the TOCA1 HR1 domain. FIG +29 34 TOCA1 protein The N-WASP GBD displaces the TOCA1 HR1 domain. FIG +35 38 HR1 structure_element The N-WASP GBD displaces the TOCA1 HR1 domain. FIG +21 32 Cdc42·TOCA1 complex_assembly A, the model of the Cdc42·TOCA1 HR1 domain complex overlaid with the Cdc42-WASP structure. FIG +33 36 HR1 structure_element A, the model of the Cdc42·TOCA1 HR1 domain complex overlaid with the Cdc42-WASP structure. FIG +70 80 Cdc42-WASP complex_assembly A, the model of the Cdc42·TOCA1 HR1 domain complex overlaid with the Cdc42-WASP structure. FIG +81 90 structure evidence A, the model of the Cdc42·TOCA1 HR1 domain complex overlaid with the Cdc42-WASP structure. FIG +29 34 TOCA1 protein Cdc42 is shown in green, and TOCA1 is shown in purple. FIG +9 13 CRIB structure_element The core CRIB region of WASP is shown in red, whereas its basic region is shown in orange and the C-terminal region required for maximal affinity is shown in cyan. FIG +24 28 WASP protein The core CRIB region of WASP is shown in red, whereas its basic region is shown in orange and the C-terminal region required for maximal affinity is shown in cyan. FIG +44 49 Cdc42 protein A semitransparent surface representation of Cdc42 and WASP is shown overlaid with the schematic. FIG +54 58 WASP protein A semitransparent surface representation of Cdc42 and WASP is shown overlaid with the schematic. FIG +3 18 competition SPA experimental_method B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42Δ7Q61L·[3H]GTP. FIG +80 86 N-WASP protein B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42Δ7Q61L·[3H]GTP. FIG +87 90 GBD structure_element B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42Δ7Q61L·[3H]GTP. FIG +101 109 titrated experimental_method B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42Δ7Q61L·[3H]GTP. FIG +121 128 GST-ACK mutant B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42Δ7Q61L·[3H]GTP. FIG +132 140 GST-WASP mutant B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42Δ7Q61L·[3H]GTP. FIG +141 144 GBD structure_element B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42Δ7Q61L·[3H]GTP. FIG +155 174 Cdc42Δ7Q61L·[3H]GTP complex_assembly B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42Δ7Q61L·[3H]GTP. FIG +27 35 15N HSQC experimental_method C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. FIG +46 64 Cdc42Δ7Q61L·GMPPNP complex_assembly C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. FIG +98 103 TOCA1 protein C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. FIG +104 107 HR1 structure_element C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. FIG +120 126 N-WASP protein C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. FIG +127 130 GBD structure_element C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. FIG +158 162 TOCA protein C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. FIG +163 166 HR1 structure_element C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. FIG +196 202 N-WASP protein C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. FIG +203 206 GBD structure_element C, Selected regions of the 15N HSQC of 145 μm Cdc42Δ7Q61L·GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD. FIG +27 35 15N HSQC experimental_method D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. FIG +46 51 TOCA1 protein D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. FIG +52 55 HR1 structure_element D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. FIG +63 78 in complex with protein_state D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. FIG +79 84 Cdc42 protein D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. FIG +92 99 absence protein_state D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. FIG +104 115 presence of protein_state D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. FIG +120 126 N-WASP protein D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. FIG +127 130 GBD structure_element D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. FIG +156 161 Cdc42 protein D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. FIG +171 174 HR1 structure_element D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. FIG +185 191 N-WASP protein D, selected regions of the 15N HSQC of 600 μm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP. FIG +3 9 N-WASP protein An N-WASP GBD construct was produced, and its affinity for Cdc42 was measured by competition SPA (Fig. 7B). RESULTS +10 13 GBD structure_element An N-WASP GBD construct was produced, and its affinity for Cdc42 was measured by competition SPA (Fig. 7B). RESULTS +46 54 affinity evidence An N-WASP GBD construct was produced, and its affinity for Cdc42 was measured by competition SPA (Fig. 7B). RESULTS +59 64 Cdc42 protein An N-WASP GBD construct was produced, and its affinity for Cdc42 was measured by competition SPA (Fig. 7B). RESULTS +81 96 competition SPA experimental_method An N-WASP GBD construct was produced, and its affinity for Cdc42 was measured by competition SPA (Fig. 7B). RESULTS +4 6 Kd evidence The Kd that was determined (37 nm) is consistent with the previously reported affinity. RESULTS +78 86 affinity evidence The Kd that was determined (37 nm) is consistent with the previously reported affinity. RESULTS +0 9 Unlabeled protein_state Unlabeled N-WASP GBD was titrated into 15N-Cdc42Δ7Q61L·GMPPNP, and the backbone NH groups were monitored using HSQCs (Fig. 7C). RESULTS +10 16 N-WASP protein Unlabeled N-WASP GBD was titrated into 15N-Cdc42Δ7Q61L·GMPPNP, and the backbone NH groups were monitored using HSQCs (Fig. 7C). RESULTS +17 20 GBD structure_element Unlabeled N-WASP GBD was titrated into 15N-Cdc42Δ7Q61L·GMPPNP, and the backbone NH groups were monitored using HSQCs (Fig. 7C). RESULTS +25 33 titrated experimental_method Unlabeled N-WASP GBD was titrated into 15N-Cdc42Δ7Q61L·GMPPNP, and the backbone NH groups were monitored using HSQCs (Fig. 7C). RESULTS +39 42 15N chemical Unlabeled N-WASP GBD was titrated into 15N-Cdc42Δ7Q61L·GMPPNP, and the backbone NH groups were monitored using HSQCs (Fig. 7C). RESULTS +43 61 Cdc42Δ7Q61L·GMPPNP complex_assembly Unlabeled N-WASP GBD was titrated into 15N-Cdc42Δ7Q61L·GMPPNP, and the backbone NH groups were monitored using HSQCs (Fig. 7C). RESULTS +111 116 HSQCs experimental_method Unlabeled N-WASP GBD was titrated into 15N-Cdc42Δ7Q61L·GMPPNP, and the backbone NH groups were monitored using HSQCs (Fig. 7C). RESULTS +0 9 Unlabeled protein_state Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. RESULTS +10 13 HR1 structure_element Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. RESULTS +13 18 TOCA1 protein Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. RESULTS +41 53 Cdc42·N-WASP complex_assembly Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. RESULTS +109 115 N-WASP protein Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. RESULTS +116 119 GBD structure_element Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. RESULTS +150 161 presence of protein_state Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. RESULTS +181 184 HR1 structure_element Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. RESULTS +184 189 TOCA1 protein Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. RESULTS +84 89 Cdc42 protein These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). RESULTS +98 104 N-WASP protein These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). RESULTS +105 108 GBD structure_element These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). RESULTS +117 122 TOCA1 protein These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). RESULTS +123 126 HR1 structure_element These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). RESULTS +162 164 Kd evidence These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). RESULTS +204 209 TOCA1 protein These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). RESULTS +210 212 Kd evidence These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). RESULTS +221 227 N-WASP protein These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). RESULTS +228 230 Kd evidence These experiments were recorded at sufficiently high protein concentrations (145 μm Cdc42, 145 μm N-WASP GBD, 725 μm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd ≈ 5 μm, N-WASP Kd = 37 nm). RESULTS +20 24 HSQC experimental_method A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +49 52 15N chemical A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +53 58 Cdc42 protein A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +59 64 alone protein_state A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +73 84 presence of protein_state A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +85 90 TOCA1 protein A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +91 94 HR1 structure_element A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +96 102 N-WASP protein A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +103 106 GBD structure_element A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +132 139 spectra evidence A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +147 158 presence of protein_state A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +159 165 N-WASP protein A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +177 188 presence of protein_state A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +194 200 N-WASP protein A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +205 210 TOCA1 protein A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +211 214 HR1 structure_element A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C). RESULTS +13 16 15N chemical Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. RESULTS +17 22 TOCA1 protein Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. RESULTS +23 26 HR1 structure_element Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. RESULTS +48 59 presence of protein_state Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. RESULTS +60 69 unlabeled protein_state Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. RESULTS +70 88 Cdc42Δ7Q61L·GMPPNP complex_assembly Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. RESULTS +147 156 unlabeled protein_state Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. RESULTS +157 163 N-WASP protein Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. RESULTS +164 167 GBD structure_element Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. RESULTS +4 12 spectrum evidence The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +18 24 N-WASP protein The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +29 34 TOCA1 protein The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +79 83 free protein_state The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +84 87 HR1 structure_element The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +108 116 spectrum evidence The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +124 135 presence of protein_state The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +147 153 N-WASP protein The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +183 188 TOCA1 protein The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +189 192 HR1 structure_element The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +193 197 free protein_state The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +202 209 complex protein_state The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +210 217 spectra evidence The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (Fig. 7D). RESULTS +27 30 NMR experimental_method When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain. RESULTS +87 91 free protein_state When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain. RESULTS +96 101 bound protein_state When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain. RESULTS +130 138 spectrum evidence When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain. RESULTS +192 196 free protein_state When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain. RESULTS +201 206 bound protein_state When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain. RESULTS +207 210 HR1 structure_element When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain. RESULTS +88 90 Kd evidence Again, the experiments were recorded on protein samples far in excess of the individual Kd values (600 μm each protein). RESULTS +29 32 HR1 structure_element These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. RESULTS +58 63 Cdc42 protein These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. RESULTS +67 73 N-WASP protein These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. RESULTS +112 117 TOCA1 protein These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. RESULTS +118 121 HR1 structure_element These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. RESULTS +123 129 N-WASP protein These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. RESULTS +130 133 GBD structure_element These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. RESULTS +139 144 Cdc42 protein These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. RESULTS +85 90 Cdc42 protein Taken together, the data in Fig. 7, C and D, indicate unidirectional competition for Cdc42 binding in which the N-WASP GBD displaces TOCA1 HR1 but not vice versa. RESULTS +112 118 N-WASP protein Taken together, the data in Fig. 7, C and D, indicate unidirectional competition for Cdc42 binding in which the N-WASP GBD displaces TOCA1 HR1 but not vice versa. RESULTS +119 122 GBD structure_element Taken together, the data in Fig. 7, C and D, indicate unidirectional competition for Cdc42 binding in which the N-WASP GBD displaces TOCA1 HR1 but not vice versa. RESULTS +133 138 TOCA1 protein Taken together, the data in Fig. 7, C and D, indicate unidirectional competition for Cdc42 binding in which the N-WASP GBD displaces TOCA1 HR1 but not vice versa. RESULTS +139 142 HR1 structure_element Taken together, the data in Fig. 7, C and D, indicate unidirectional competition for Cdc42 binding in which the N-WASP GBD displaces TOCA1 HR1 but not vice versa. RESULTS +78 83 TOCA1 protein To extend these studies to a more complex system and to assess the ability of TOCA1 HR1 to compete with full-length N-WASP, pyrene actin assays were employed. RESULTS +84 87 HR1 structure_element To extend these studies to a more complex system and to assess the ability of TOCA1 HR1 to compete with full-length N-WASP, pyrene actin assays were employed. RESULTS +104 115 full-length protein_state To extend these studies to a more complex system and to assess the ability of TOCA1 HR1 to compete with full-length N-WASP, pyrene actin assays were employed. RESULTS +116 122 N-WASP protein To extend these studies to a more complex system and to assess the ability of TOCA1 HR1 to compete with full-length N-WASP, pyrene actin assays were employed. RESULTS +124 143 pyrene actin assays experimental_method To extend these studies to a more complex system and to assess the ability of TOCA1 HR1 to compete with full-length N-WASP, pyrene actin assays were employed. RESULTS +68 80 pyrene actin chemical These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization. RESULTS +94 101 Xenopus taxonomy_domain These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization. RESULTS +132 137 TOCA1 protein These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization. RESULTS +138 141 HR1 structure_element These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization. RESULTS +152 158 N-WASP protein These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization. RESULTS +159 162 GBD structure_element These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization. RESULTS +201 206 actin protein_type These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization. RESULTS +0 5 Actin protein_type Actin polymerization in all cases was initiated by the addition of PI(4,5)P2-containing liposomes. RESULTS +67 76 PI(4,5)P2 chemical Actin polymerization in all cases was initiated by the addition of PI(4,5)P2-containing liposomes. RESULTS +0 5 Actin protein_type Actin polymerization triggered by the addition of PI(4,5)P2-containing liposomes has previously been shown to depend on TOCA1 and N-WASP. RESULTS +50 59 PI(4,5)P2 chemical Actin polymerization triggered by the addition of PI(4,5)P2-containing liposomes has previously been shown to depend on TOCA1 and N-WASP. RESULTS +120 125 TOCA1 protein Actin polymerization triggered by the addition of PI(4,5)P2-containing liposomes has previously been shown to depend on TOCA1 and N-WASP. RESULTS +130 136 N-WASP protein Actin polymerization triggered by the addition of PI(4,5)P2-containing liposomes has previously been shown to depend on TOCA1 and N-WASP. RESULTS +11 17 N-WASP protein Endogenous N-WASP is present at ∼100 nm in Xenopus extracts, whereas TOCA1 is present at a 10-fold lower concentration than N-WASP. RESULTS +43 50 Xenopus taxonomy_domain Endogenous N-WASP is present at ∼100 nm in Xenopus extracts, whereas TOCA1 is present at a 10-fold lower concentration than N-WASP. RESULTS +69 74 TOCA1 protein Endogenous N-WASP is present at ∼100 nm in Xenopus extracts, whereas TOCA1 is present at a 10-fold lower concentration than N-WASP. RESULTS +124 130 N-WASP protein Endogenous N-WASP is present at ∼100 nm in Xenopus extracts, whereas TOCA1 is present at a 10-fold lower concentration than N-WASP. RESULTS +4 12 addition experimental_method The addition of the isolated N-WASP GBD significantly inhibited the polymerization of actin at concentrations as low as 100 nm and completely abolished polymerization at higher concentrations (Fig. 8). RESULTS +29 35 N-WASP protein The addition of the isolated N-WASP GBD significantly inhibited the polymerization of actin at concentrations as low as 100 nm and completely abolished polymerization at higher concentrations (Fig. 8). RESULTS +36 39 GBD structure_element The addition of the isolated N-WASP GBD significantly inhibited the polymerization of actin at concentrations as low as 100 nm and completely abolished polymerization at higher concentrations (Fig. 8). RESULTS +86 91 actin protein_type The addition of the isolated N-WASP GBD significantly inhibited the polymerization of actin at concentrations as low as 100 nm and completely abolished polymerization at higher concentrations (Fig. 8). RESULTS +4 7 GBD structure_element The GBD presumably acts as a dominant negative, sequestering endogenous Cdc42 and preventing endogenous full-length N-WASP from binding and becoming activated. RESULTS +72 77 Cdc42 protein The GBD presumably acts as a dominant negative, sequestering endogenous Cdc42 and preventing endogenous full-length N-WASP from binding and becoming activated. RESULTS +93 103 endogenous protein_state The GBD presumably acts as a dominant negative, sequestering endogenous Cdc42 and preventing endogenous full-length N-WASP from binding and becoming activated. RESULTS +104 115 full-length protein_state The GBD presumably acts as a dominant negative, sequestering endogenous Cdc42 and preventing endogenous full-length N-WASP from binding and becoming activated. RESULTS +116 122 N-WASP protein The GBD presumably acts as a dominant negative, sequestering endogenous Cdc42 and preventing endogenous full-length N-WASP from binding and becoming activated. RESULTS +4 12 addition experimental_method The addition of the TOCA1 HR1 domain to 100 μm had no significant effect on the rate of actin polymerization or maximum fluorescence. RESULTS +20 25 TOCA1 protein The addition of the TOCA1 HR1 domain to 100 μm had no significant effect on the rate of actin polymerization or maximum fluorescence. RESULTS +26 29 HR1 structure_element The addition of the TOCA1 HR1 domain to 100 μm had no significant effect on the rate of actin polymerization or maximum fluorescence. RESULTS +88 93 actin protein_type The addition of the TOCA1 HR1 domain to 100 μm had no significant effect on the rate of actin polymerization or maximum fluorescence. RESULTS +112 132 maximum fluorescence evidence The addition of the TOCA1 HR1 domain to 100 μm had no significant effect on the rate of actin polymerization or maximum fluorescence. RESULTS +24 34 endogenous protein_state This is consistent with endogenous N-WASP, activated by other components of the assay, outcompeting the TOCA1 HR1 domain for Cdc42 binding. RESULTS +35 41 N-WASP protein This is consistent with endogenous N-WASP, activated by other components of the assay, outcompeting the TOCA1 HR1 domain for Cdc42 binding. RESULTS +104 109 TOCA1 protein This is consistent with endogenous N-WASP, activated by other components of the assay, outcompeting the TOCA1 HR1 domain for Cdc42 binding. RESULTS +110 113 HR1 structure_element This is consistent with endogenous N-WASP, activated by other components of the assay, outcompeting the TOCA1 HR1 domain for Cdc42 binding. RESULTS +125 130 Cdc42 protein This is consistent with endogenous N-WASP, activated by other components of the assay, outcompeting the TOCA1 HR1 domain for Cdc42 binding. RESULTS +35 53 Cdc42·N-WASP·TOCA1 complex_assembly Actin polymerization downstream of Cdc42·N-WASP·TOCA1 is inhibited by excess N-WASP GBD but not by the TOCA1 HR1 domain. FIG +57 66 inhibited protein_state Actin polymerization downstream of Cdc42·N-WASP·TOCA1 is inhibited by excess N-WASP GBD but not by the TOCA1 HR1 domain. FIG +77 83 N-WASP protein Actin polymerization downstream of Cdc42·N-WASP·TOCA1 is inhibited by excess N-WASP GBD but not by the TOCA1 HR1 domain. FIG +84 87 GBD structure_element Actin polymerization downstream of Cdc42·N-WASP·TOCA1 is inhibited by excess N-WASP GBD but not by the TOCA1 HR1 domain. FIG +103 108 TOCA1 protein Actin polymerization downstream of Cdc42·N-WASP·TOCA1 is inhibited by excess N-WASP GBD but not by the TOCA1 HR1 domain. FIG +109 112 HR1 structure_element Actin polymerization downstream of Cdc42·N-WASP·TOCA1 is inhibited by excess N-WASP GBD but not by the TOCA1 HR1 domain. FIG +0 19 Fluorescence curves evidence Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated. FIG +53 64 presence of protein_state Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated. FIG +65 90 increasing concentrations experimental_method Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated. FIG +94 100 N-WASP protein Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated. FIG +101 104 GBD structure_element Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated. FIG +108 113 TOCA1 protein Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated. FIG +114 117 HR1 structure_element Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated. FIG +4 9 Cdc42 protein The Cdc42-TOCA1 Interaction DISCUSS +10 15 TOCA1 protein The Cdc42-TOCA1 Interaction DISCUSS +4 9 TOCA1 protein The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). DISCUSS +10 13 HR1 structure_element The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). DISCUSS +21 26 alone protein_state The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). DISCUSS +45 50 Cdc42 protein The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). DISCUSS +77 85 affinity evidence The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). DISCUSS +93 98 TOCA1 protein The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). DISCUSS +99 102 HR1 structure_element The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). DISCUSS +114 119 Cdc42 protein The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). DISCUSS +139 141 Kd evidence The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd ≈ 5 μm). DISCUSS +46 52 N-WASP protein This is over 100 times lower than that of the N-WASP GBD (Kd = 37 nm) and considerably lower than other known G protein-HR1 domain interactions. DISCUSS +53 56 GBD structure_element This is over 100 times lower than that of the N-WASP GBD (Kd = 37 nm) and considerably lower than other known G protein-HR1 domain interactions. DISCUSS +58 60 Kd evidence This is over 100 times lower than that of the N-WASP GBD (Kd = 37 nm) and considerably lower than other known G protein-HR1 domain interactions. DISCUSS +110 119 G protein protein_type This is over 100 times lower than that of the N-WASP GBD (Kd = 37 nm) and considerably lower than other known G protein-HR1 domain interactions. DISCUSS +120 123 HR1 structure_element This is over 100 times lower than that of the N-WASP GBD (Kd = 37 nm) and considerably lower than other known G protein-HR1 domain interactions. DISCUSS +31 48 C-terminal region structure_element The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. DISCUSS +52 57 Cdc42 protein The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. DISCUSS +104 109 TOCA1 protein The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. DISCUSS +159 163 Rac1 protein The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. DISCUSS +172 176 HR1b structure_element The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. DISCUSS +187 191 PRK1 protein The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. DISCUSS +216 220 PRK1 protein The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. DISCUSS +221 225 HR1a structure_element The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. DISCUSS +226 230 RhoA protein The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. DISCUSS +9 26 binding interface site A single binding interface on both the HR1 domain and Cdc42 can be concluded from the data presented here. DISCUSS +39 42 HR1 structure_element A single binding interface on both the HR1 domain and Cdc42 can be concluded from the data presented here. DISCUSS +54 59 Cdc42 protein A single binding interface on both the HR1 domain and Cdc42 can be concluded from the data presented here. DISCUSS +17 27 interfaces site Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions (Fig. 4), and the lowest energy model produced in rigid body docking resembles previously studied G protein·HR1 complexes (Fig. 6). DISCUSS +63 72 G protein protein_type Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions (Fig. 4), and the lowest energy model produced in rigid body docking resembles previously studied G protein·HR1 complexes (Fig. 6). DISCUSS +73 76 HR1 structure_element Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions (Fig. 4), and the lowest energy model produced in rigid body docking resembles previously studied G protein·HR1 complexes (Fig. 6). DISCUSS +122 127 model evidence Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions (Fig. 4), and the lowest energy model produced in rigid body docking resembles previously studied G protein·HR1 complexes (Fig. 6). DISCUSS +140 158 rigid body docking experimental_method Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions (Fig. 4), and the lowest energy model produced in rigid body docking resembles previously studied G protein·HR1 complexes (Fig. 6). DISCUSS +188 201 G protein·HR1 complex_assembly Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions (Fig. 4), and the lowest energy model produced in rigid body docking resembles previously studied G protein·HR1 complexes (Fig. 6). DISCUSS +124 127 HR1 structure_element It seems, therefore, that the interaction, despite its relatively low affinity, is specific and sterically similar to other HR1 domain-G protein interactions. DISCUSS +135 144 G protein protein_type It seems, therefore, that the interaction, despite its relatively low affinity, is specific and sterically similar to other HR1 domain-G protein interactions. DISCUSS +4 9 TOCA1 protein The TOCA1 HR1 domain is a left-handed coiled-coil comparable with other known HR1 domains. DISCUSS +10 13 HR1 structure_element The TOCA1 HR1 domain is a left-handed coiled-coil comparable with other known HR1 domains. DISCUSS +38 49 coiled-coil structure_element The TOCA1 HR1 domain is a left-handed coiled-coil comparable with other known HR1 domains. DISCUSS +78 81 HR1 structure_element The TOCA1 HR1 domain is a left-handed coiled-coil comparable with other known HR1 domains. DISCUSS +33 44 coiled-coil structure_element A short region N-terminal to the coiled-coil exhibits a series of turns and contacts residues of both helices of the coiled-coil (Fig. 3). DISCUSS +117 128 coiled-coil structure_element A short region N-terminal to the coiled-coil exhibits a series of turns and contacts residues of both helices of the coiled-coil (Fig. 3). DISCUSS +30 34 CIP4 protein The corresponding sequence in CIP4 also includes a series of turns but is flexible, whereas in the HR1a domain of PRK1, the equivalent region adopts an α-helical structure that packs against the coiled-coil. DISCUSS +99 103 HR1a structure_element The corresponding sequence in CIP4 also includes a series of turns but is flexible, whereas in the HR1a domain of PRK1, the equivalent region adopts an α-helical structure that packs against the coiled-coil. DISCUSS +114 118 PRK1 protein The corresponding sequence in CIP4 also includes a series of turns but is flexible, whereas in the HR1a domain of PRK1, the equivalent region adopts an α-helical structure that packs against the coiled-coil. DISCUSS +152 171 α-helical structure structure_element The corresponding sequence in CIP4 also includes a series of turns but is flexible, whereas in the HR1a domain of PRK1, the equivalent region adopts an α-helical structure that packs against the coiled-coil. DISCUSS +195 206 coiled-coil structure_element The corresponding sequence in CIP4 also includes a series of turns but is flexible, whereas in the HR1a domain of PRK1, the equivalent region adopts an α-helical structure that packs against the coiled-coil. DISCUSS +4 12 contacts bond_interaction The contacts between the N-terminal region and the coiled-coil are predominantly hydrophobic in both cases, but sequence-specific contacts do not appear to be conserved. DISCUSS +51 62 coiled-coil structure_element The contacts between the N-terminal region and the coiled-coil are predominantly hydrophobic in both cases, but sequence-specific contacts do not appear to be conserved. DISCUSS +81 92 hydrophobic bond_interaction The contacts between the N-terminal region and the coiled-coil are predominantly hydrophobic in both cases, but sequence-specific contacts do not appear to be conserved. DISCUSS +32 59 G protein-binding interface site This region is distant from the G protein-binding interface of the HR1 domains, so the structural differences may relate to the structure and regulation of these domains rather than their G protein interactions. DISCUSS +67 70 HR1 structure_element This region is distant from the G protein-binding interface of the HR1 domains, so the structural differences may relate to the structure and regulation of these domains rather than their G protein interactions. DISCUSS +188 197 G protein protein_type This region is distant from the G protein-binding interface of the HR1 domains, so the structural differences may relate to the structure and regulation of these domains rather than their G protein interactions. DISCUSS +4 22 interhelical loops structure_element The interhelical loops of TOCA1 and CIP4 differ from the same region in the HR1 domains of PRK1 in that they are longer and contain two short stretches of 310-helix. DISCUSS +26 31 TOCA1 protein The interhelical loops of TOCA1 and CIP4 differ from the same region in the HR1 domains of PRK1 in that they are longer and contain two short stretches of 310-helix. DISCUSS +36 40 CIP4 protein The interhelical loops of TOCA1 and CIP4 differ from the same region in the HR1 domains of PRK1 in that they are longer and contain two short stretches of 310-helix. DISCUSS +76 79 HR1 structure_element The interhelical loops of TOCA1 and CIP4 differ from the same region in the HR1 domains of PRK1 in that they are longer and contain two short stretches of 310-helix. DISCUSS +91 95 PRK1 protein The interhelical loops of TOCA1 and CIP4 differ from the same region in the HR1 domains of PRK1 in that they are longer and contain two short stretches of 310-helix. DISCUSS +155 164 310-helix structure_element The interhelical loops of TOCA1 and CIP4 differ from the same region in the HR1 domains of PRK1 in that they are longer and contain two short stretches of 310-helix. DISCUSS +28 53 G protein-binding surface site This region lies within the G protein-binding surface of all of the HR1 domains (Fig. 4D). DISCUSS +68 71 HR1 structure_element This region lies within the G protein-binding surface of all of the HR1 domains (Fig. 4D). DISCUSS +0 5 TOCA1 protein TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. DISCUSS +10 14 CIP4 protein TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. DISCUSS +35 40 Cdc42 protein TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. DISCUSS +54 58 HR1a structure_element TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. DISCUSS +69 73 PRK1 protein TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. DISCUSS +91 95 RhoA protein TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. DISCUSS +100 104 Rac1 protein TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. DISCUSS +114 118 HR1b structure_element TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. DISCUSS +135 139 Rac1 protein TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. DISCUSS +34 39 TOCA1 protein The structural features shared by TOCA1 and CIP4 may therefore be related to Cdc42 binding specificity and the low affinities. DISCUSS +44 48 CIP4 protein The structural features shared by TOCA1 and CIP4 may therefore be related to Cdc42 binding specificity and the low affinities. DISCUSS +77 82 Cdc42 protein The structural features shared by TOCA1 and CIP4 may therefore be related to Cdc42 binding specificity and the low affinities. DISCUSS +3 7 free protein_state In free TOCA1, the side chains of the interhelical region make extensive contacts with residues in helix 1. DISCUSS +8 13 TOCA1 protein In free TOCA1, the side chains of the interhelical region make extensive contacts with residues in helix 1. DISCUSS +38 57 interhelical region structure_element In free TOCA1, the side chains of the interhelical region make extensive contacts with residues in helix 1. DISCUSS +99 106 helix 1 structure_element In free TOCA1, the side chains of the interhelical region make extensive contacts with residues in helix 1. DISCUSS +57 68 presence of protein_state Many of these residues are significantly affected in the presence of Cdc42, so it is likely that the conformation of this loop is altered in the Cdc42 complex. DISCUSS +69 74 Cdc42 protein Many of these residues are significantly affected in the presence of Cdc42, so it is likely that the conformation of this loop is altered in the Cdc42 complex. DISCUSS +122 126 loop structure_element Many of these residues are significantly affected in the presence of Cdc42, so it is likely that the conformation of this loop is altered in the Cdc42 complex. DISCUSS +145 150 Cdc42 protein Many of these residues are significantly affected in the presence of Cdc42, so it is likely that the conformation of this loop is altered in the Cdc42 complex. DISCUSS +67 75 mutation experimental_method These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42. DISCUSS +79 85 Met383 residue_name_number These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42. DISCUSS +86 92 Gly384 residue_name_number These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42. DISCUSS +93 99 Asp385 residue_name_number These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42. DISCUSS +103 109 Ile383 residue_name_number These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42. DISCUSS +110 116 Ser384 residue_name_number These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42. DISCUSS +117 123 Thr385 residue_name_number These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42. DISCUSS +134 139 TOCA1 protein These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42. DISCUSS +151 156 Cdc42 protein These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42. DISCUSS +18 23 model evidence The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. DISCUSS +36 43 HADDOCK experimental_method The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. DISCUSS +92 101 titration evidence The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. DISCUSS +121 124 NMR experimental_method The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. DISCUSS +125 135 structures evidence The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. DISCUSS +139 143 RhoA protein The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. DISCUSS +148 152 Rac1 protein The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. DISCUSS +153 168 in complex with protein_state The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. DISCUSS +175 178 HR1 structure_element The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners. DISCUSS +13 19 Phe-56 residue_name_number For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42·HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A). DISCUSS +19 24 Cdc42 protein For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42·HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A). DISCUSS +50 54 free protein_state For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42·HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A). DISCUSS +55 60 Cdc42 protein For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42·HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A). DISCUSS +64 78 Cdc42·HR1TOCA1 complex_assembly For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42·HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A). DISCUSS +96 101 TOCA1 protein For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42·HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A). DISCUSS +102 105 HR1 structure_element For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42·HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A). DISCUSS +0 6 Phe-56 residue_name_number Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation. DISCUSS +6 11 Cdc42 protein Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation. DISCUSS +24 27 Trp residue_name Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation. DISCUSS +36 40 Rac1 protein Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation. DISCUSS +45 49 RhoA protein Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation. DISCUSS +87 95 switch I site Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation. DISCUSS +101 106 Cdc42 protein Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation. DISCUSS +122 125 ACK protein Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation. DISCUSS +55 60 Cdc42 protein This residue has also been identified as important for Cdc42-WASP binding. DISCUSS +61 65 WASP protein This residue has also been identified as important for Cdc42-WASP binding. DISCUSS +0 6 Phe-56 residue_name_number Phe-56Cdc42 is therefore likely to be involved in the Cdc42-TOCA1 interaction, probably by stabilizing the position of switch I. DISCUSS +6 11 Cdc42 protein Phe-56Cdc42 is therefore likely to be involved in the Cdc42-TOCA1 interaction, probably by stabilizing the position of switch I. DISCUSS +54 59 Cdc42 protein Phe-56Cdc42 is therefore likely to be involved in the Cdc42-TOCA1 interaction, probably by stabilizing the position of switch I. DISCUSS +60 65 TOCA1 protein Phe-56Cdc42 is therefore likely to be involved in the Cdc42-TOCA1 interaction, probably by stabilizing the position of switch I. DISCUSS +119 127 switch I site Phe-56Cdc42 is therefore likely to be involved in the Cdc42-TOCA1 interaction, probably by stabilizing the position of switch I. DISCUSS +39 53 Cdc42·HR1TOCA1 complex_assembly Some residues that are affected in the Cdc42·HR1TOCA1 complex but do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) may contact HR1TOCA1 directly (Fig. 6D). DISCUSS +107 111 RhoA protein Some residues that are affected in the Cdc42·HR1TOCA1 complex but do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) may contact HR1TOCA1 directly (Fig. 6D). DISCUSS +115 119 Rac1 protein Some residues that are affected in the Cdc42·HR1TOCA1 complex but do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) may contact HR1TOCA1 directly (Fig. 6D). DISCUSS +142 145 HR1 structure_element Some residues that are affected in the Cdc42·HR1TOCA1 complex but do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) may contact HR1TOCA1 directly (Fig. 6D). DISCUSS +145 150 TOCA1 protein Some residues that are affected in the Cdc42·HR1TOCA1 complex but do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) may contact HR1TOCA1 directly (Fig. 6D). DISCUSS +0 5 Gln-2 residue_name_number Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. DISCUSS +5 10 Cdc42 protein Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. DISCUSS +71 80 Cdc42·ACK complex_assembly Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. DISCUSS +99 106 Val-376 residue_name_number Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. DISCUSS +106 111 TOCA1 protein Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. DISCUSS +116 123 Asn-380 residue_name_number Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. DISCUSS +123 128 TOCA1 protein Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. DISCUSS +180 197 interhelical loop structure_element Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. DISCUSS +206 217 first helix structure_element Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. DISCUSS +225 230 TOCA1 protein Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. DISCUSS +231 242 coiled-coil structure_element Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil. DISCUSS +0 6 Thr-52 residue_name_number Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. DISCUSS +6 11 Cdc42 protein Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. DISCUSS +74 77 ACK protein Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. DISCUSS +109 112 HR1 structure_element Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. DISCUSS +112 117 TOCA1 protein Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. DISCUSS +118 125 helix 1 structure_element Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. DISCUSS +140 147 Lys-372 residue_name_number Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. DISCUSS +147 152 TOCA1 protein Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. DISCUSS +189 193 Rac1 protein Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. DISCUSS +197 203 Asn-52 residue_name_number Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. DISCUSS +203 207 Rac1 protein Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1. DISCUSS +0 4 N52T mutant N52T is one of a combination of seven residues found to confer ACK binding on Rac1 and so may represent a specific Cdc42-effector contact residue. DISCUSS +63 66 ACK protein N52T is one of a combination of seven residues found to confer ACK binding on Rac1 and so may represent a specific Cdc42-effector contact residue. DISCUSS +78 82 Rac1 protein N52T is one of a combination of seven residues found to confer ACK binding on Rac1 and so may represent a specific Cdc42-effector contact residue. DISCUSS +115 120 Cdc42 protein N52T is one of a combination of seven residues found to confer ACK binding on Rac1 and so may represent a specific Cdc42-effector contact residue. DISCUSS +27 34 Lys-372 residue_name_number The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b. DISCUSS +34 39 TOCA1 protein The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b. DISCUSS +43 47 PRK1 protein The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b. DISCUSS +51 57 Glu-58 residue_name_number The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b. DISCUSS +57 61 HR1a structure_element The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b. DISCUSS +65 72 Gln-151 residue_name_number The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b. DISCUSS +72 76 HR1b structure_element The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b. DISCUSS +0 6 Thr-52 residue_name_number Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact. DISCUSS +6 11 Cdc42 protein Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact. DISCUSS +12 19 Lys-372 residue_name_number Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact. DISCUSS +19 24 TOCA1 protein Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact. DISCUSS +60 65 Cdc42 protein Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact. DISCUSS +66 69 HR1 structure_element Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact. DISCUSS +69 74 TOCA1 protein Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact. DISCUSS +0 6 Arg-68 residue_name_number Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. DISCUSS +6 11 Cdc42 protein Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. DISCUSS +15 24 switch II site Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. DISCUSS +48 55 Glu-395 residue_name_number Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. DISCUSS +55 60 TOCA1 protein Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. DISCUSS +122 131 switch II site Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. DISCUSS +135 140 Cdc42 protein Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. DISCUSS +145 152 helix 2 structure_element Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. DISCUSS +160 163 HR1 structure_element Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. DISCUSS +15 18 Arg residue_name The equivalent Arg in Rac1 and RhoA is pointing away from the HR1 domains of PRK1. DISCUSS +22 26 Rac1 protein The equivalent Arg in Rac1 and RhoA is pointing away from the HR1 domains of PRK1. DISCUSS +31 35 RhoA protein The equivalent Arg in Rac1 and RhoA is pointing away from the HR1 domains of PRK1. DISCUSS +62 65 HR1 structure_element The equivalent Arg in Rac1 and RhoA is pointing away from the HR1 domains of PRK1. DISCUSS +77 81 PRK1 protein The equivalent Arg in Rac1 and RhoA is pointing away from the HR1 domains of PRK1. DISCUSS +38 43 Cdc42 protein The importance of this residue in the Cdc42-TOCA1 interaction remains unclear, although its mutation reduces binding to RhoGAP, suggesting that it can be involved in Cdc42 interactions. DISCUSS +44 49 TOCA1 protein The importance of this residue in the Cdc42-TOCA1 interaction remains unclear, although its mutation reduces binding to RhoGAP, suggesting that it can be involved in Cdc42 interactions. DISCUSS +92 100 mutation experimental_method The importance of this residue in the Cdc42-TOCA1 interaction remains unclear, although its mutation reduces binding to RhoGAP, suggesting that it can be involved in Cdc42 interactions. DISCUSS +120 126 RhoGAP protein The importance of this residue in the Cdc42-TOCA1 interaction remains unclear, although its mutation reduces binding to RhoGAP, suggesting that it can be involved in Cdc42 interactions. DISCUSS +166 171 Cdc42 protein The importance of this residue in the Cdc42-TOCA1 interaction remains unclear, although its mutation reduces binding to RhoGAP, suggesting that it can be involved in Cdc42 interactions. DISCUSS +4 22 solution structure evidence The solution structure of the TOCA1 HR1 domain presented here, along with the model of the HR1TOCA1·Cdc42 complex is consistent with a conserved mode of binding across the known HR1 domain-Rho family interactions, despite their differing affinities. DISCUSS +30 35 TOCA1 protein The solution structure of the TOCA1 HR1 domain presented here, along with the model of the HR1TOCA1·Cdc42 complex is consistent with a conserved mode of binding across the known HR1 domain-Rho family interactions, despite their differing affinities. DISCUSS +36 39 HR1 structure_element The solution structure of the TOCA1 HR1 domain presented here, along with the model of the HR1TOCA1·Cdc42 complex is consistent with a conserved mode of binding across the known HR1 domain-Rho family interactions, despite their differing affinities. DISCUSS +91 105 HR1TOCA1·Cdc42 complex_assembly The solution structure of the TOCA1 HR1 domain presented here, along with the model of the HR1TOCA1·Cdc42 complex is consistent with a conserved mode of binding across the known HR1 domain-Rho family interactions, despite their differing affinities. DISCUSS +178 181 HR1 structure_element The solution structure of the TOCA1 HR1 domain presented here, along with the model of the HR1TOCA1·Cdc42 complex is consistent with a conserved mode of binding across the known HR1 domain-Rho family interactions, despite their differing affinities. DISCUSS +36 72 structural and thermodynamic studies experimental_method The weak binding prevented detailed structural and thermodynamic studies of the complex. DISCUSS +13 31 structural studies experimental_method Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. DISCUSS +39 44 TOCA1 protein Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. DISCUSS +45 48 HR1 structure_element Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. DISCUSS +71 93 chemical shift mapping experimental_method Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. DISCUSS +161 166 Cdc42 protein Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. DISCUSS +167 170 HR1 structure_element Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. DISCUSS +170 175 TOCA1 protein Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. DISCUSS +180 184 RhoA protein Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. DISCUSS +185 189 Rac1 protein Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. DISCUSS +190 193 HR1 structure_element Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. DISCUSS +193 197 PRK1 protein Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding. DISCUSS +63 66 HR1 structure_element We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures (Tm) and Rac binders being more rigid. DISCUSS +125 146 Rho family G proteins protein_type We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures (Tm) and Rac binders being more rigid. DISCUSS +165 168 HR1 structure_element We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures (Tm) and Rac binders being more rigid. DISCUSS +236 256 melting temperatures evidence We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures (Tm) and Rac binders being more rigid. DISCUSS +258 260 Tm evidence We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures (Tm) and Rac binders being more rigid. DISCUSS +266 269 Rac protein_type We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures (Tm) and Rac binders being more rigid. DISCUSS +4 6 Tm evidence The Tm of the TOCA1 HR1 domain is 61.9 °C (data not shown), which is the highest Tm that we have measured for an HR1 domain thus far. DISCUSS +14 19 TOCA1 protein The Tm of the TOCA1 HR1 domain is 61.9 °C (data not shown), which is the highest Tm that we have measured for an HR1 domain thus far. DISCUSS +20 23 HR1 structure_element The Tm of the TOCA1 HR1 domain is 61.9 °C (data not shown), which is the highest Tm that we have measured for an HR1 domain thus far. DISCUSS +81 83 Tm evidence The Tm of the TOCA1 HR1 domain is 61.9 °C (data not shown), which is the highest Tm that we have measured for an HR1 domain thus far. DISCUSS +113 116 HR1 structure_element The Tm of the TOCA1 HR1 domain is 61.9 °C (data not shown), which is the highest Tm that we have measured for an HR1 domain thus far. DISCUSS +28 33 TOCA1 protein As such, the ability of the TOCA1 HR1 domain to bind to Cdc42 (a close relative of Rac1 rather than RhoA) fits this trend. DISCUSS +34 37 HR1 structure_element As such, the ability of the TOCA1 HR1 domain to bind to Cdc42 (a close relative of Rac1 rather than RhoA) fits this trend. DISCUSS +56 61 Cdc42 protein As such, the ability of the TOCA1 HR1 domain to bind to Cdc42 (a close relative of Rac1 rather than RhoA) fits this trend. DISCUSS +83 87 Rac1 protein As such, the ability of the TOCA1 HR1 domain to bind to Cdc42 (a close relative of Rac1 rather than RhoA) fits this trend. DISCUSS +100 104 RhoA protein As such, the ability of the TOCA1 HR1 domain to bind to Cdc42 (a close relative of Rac1 rather than RhoA) fits this trend. DISCUSS +61 86 G protein-binding regions site An investigation into the local motions, particularly in the G protein-binding regions, may offer further insight into the differential specificities and affinities of G protein-HR1 domain interactions. DISCUSS +168 177 G protein protein_type An investigation into the local motions, particularly in the G protein-binding regions, may offer further insight into the differential specificities and affinities of G protein-HR1 domain interactions. DISCUSS +178 181 HR1 structure_element An investigation into the local motions, particularly in the G protein-binding regions, may offer further insight into the differential specificities and affinities of G protein-HR1 domain interactions. DISCUSS +24 29 Cdc42 protein The low affinity of the Cdc42-HR1TOCA1 interaction is consistent with a tightly spatially and temporally regulated pathway, requiring combinatorial signals leading to a series of coincident weak interactions that elicit full activation. DISCUSS +30 33 HR1 structure_element The low affinity of the Cdc42-HR1TOCA1 interaction is consistent with a tightly spatially and temporally regulated pathway, requiring combinatorial signals leading to a series of coincident weak interactions that elicit full activation. DISCUSS +33 38 TOCA1 protein The low affinity of the Cdc42-HR1TOCA1 interaction is consistent with a tightly spatially and temporally regulated pathway, requiring combinatorial signals leading to a series of coincident weak interactions that elicit full activation. DISCUSS +4 7 HR1 structure_element The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family. DISCUSS +27 46 TOCA family members protein_type The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family. DISCUSS +48 52 CIP4 protein The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family. DISCUSS +57 62 FBP17 protein The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family. DISCUSS +106 111 Cdc42 protein The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family. DISCUSS +192 211 HR1 domain proteins protein_type The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family. DISCUSS +232 242 PRK family protein_type The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family. DISCUSS +24 32 HR1TOCA1 structure_element The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. DISCUSS +33 38 Cdc42 protein The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. DISCUSS +104 109 TOCA1 protein The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. DISCUSS +113 120 Xenopus taxonomy_domain The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. DISCUSS +169 174 TOCA1 protein The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. DISCUSS +179 184 Cdc42 protein The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. DISCUSS +222 227 TOCA1 protein The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. DISCUSS +319 328 activated protein_state The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. DISCUSS +329 334 Cdc42 protein The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (∼10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42. DISCUSS +27 38 TOCA family protein_type Evidence suggests that the TOCA family of proteins are recruited to the membrane via an interaction between their F-BAR domain and specific signaling lipids. DISCUSS +114 119 F-BAR structure_element Evidence suggests that the TOCA family of proteins are recruited to the membrane via an interaction between their F-BAR domain and specific signaling lipids. DISCUSS +13 39 electrostatic interactions bond_interaction For example, electrostatic interactions between the F-BAR domain and the membrane are required for TOCA1 recruitment to membrane vesicles and tubules, and TOCA1-dependent actin polymerization is known to depend specifically on PI(4,5)P2. DISCUSS +52 57 F-BAR structure_element For example, electrostatic interactions between the F-BAR domain and the membrane are required for TOCA1 recruitment to membrane vesicles and tubules, and TOCA1-dependent actin polymerization is known to depend specifically on PI(4,5)P2. DISCUSS +99 104 TOCA1 protein For example, electrostatic interactions between the F-BAR domain and the membrane are required for TOCA1 recruitment to membrane vesicles and tubules, and TOCA1-dependent actin polymerization is known to depend specifically on PI(4,5)P2. DISCUSS +155 160 TOCA1 protein For example, electrostatic interactions between the F-BAR domain and the membrane are required for TOCA1 recruitment to membrane vesicles and tubules, and TOCA1-dependent actin polymerization is known to depend specifically on PI(4,5)P2. DISCUSS +227 236 PI(4,5)P2 chemical For example, electrostatic interactions between the F-BAR domain and the membrane are required for TOCA1 recruitment to membrane vesicles and tubules, and TOCA1-dependent actin polymerization is known to depend specifically on PI(4,5)P2. DISCUSS +17 25 isolated experimental_method Furthermore, the isolated F-BAR domain of FBP17 has been shown to induce membrane tubulation of brain liposomes and BAR domain proteins that promote tubulation cluster on membranes at high densities. DISCUSS +26 31 F-BAR structure_element Furthermore, the isolated F-BAR domain of FBP17 has been shown to induce membrane tubulation of brain liposomes and BAR domain proteins that promote tubulation cluster on membranes at high densities. DISCUSS +42 47 FBP17 protein Furthermore, the isolated F-BAR domain of FBP17 has been shown to induce membrane tubulation of brain liposomes and BAR domain proteins that promote tubulation cluster on membranes at high densities. DISCUSS +116 119 BAR structure_element Furthermore, the isolated F-BAR domain of FBP17 has been shown to induce membrane tubulation of brain liposomes and BAR domain proteins that promote tubulation cluster on membranes at high densities. DISCUSS +51 56 TOCA1 protein Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. DISCUSS +74 76 Kd evidence Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. DISCUSS +80 85 F-BAR structure_element Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. DISCUSS +86 91 dimer oligomeric_state Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. DISCUSS +141 146 FCHo2 protein Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. DISCUSS +147 152 F-BAR structure_element Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. DISCUSS +186 191 Cdc42 protein Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. DISCUSS +192 195 HR1 structure_element Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. DISCUSS +195 200 TOCA1 protein Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 μm)) and that of the Cdc42-HR1TOCA1 interaction. DISCUSS +0 5 Cdc42 protein Cdc42-HR1TOCA1 binding would then be favorable, as long as coincident activation of Cdc42 had occurred, leading to stabilization of TOCA1 at the membrane and downstream activation of N-WASP. DISCUSS +6 9 HR1 structure_element Cdc42-HR1TOCA1 binding would then be favorable, as long as coincident activation of Cdc42 had occurred, leading to stabilization of TOCA1 at the membrane and downstream activation of N-WASP. DISCUSS +9 14 TOCA1 protein Cdc42-HR1TOCA1 binding would then be favorable, as long as coincident activation of Cdc42 had occurred, leading to stabilization of TOCA1 at the membrane and downstream activation of N-WASP. DISCUSS +84 89 Cdc42 protein Cdc42-HR1TOCA1 binding would then be favorable, as long as coincident activation of Cdc42 had occurred, leading to stabilization of TOCA1 at the membrane and downstream activation of N-WASP. DISCUSS +132 137 TOCA1 protein Cdc42-HR1TOCA1 binding would then be favorable, as long as coincident activation of Cdc42 had occurred, leading to stabilization of TOCA1 at the membrane and downstream activation of N-WASP. DISCUSS +183 189 N-WASP protein Cdc42-HR1TOCA1 binding would then be favorable, as long as coincident activation of Cdc42 had occurred, leading to stabilization of TOCA1 at the membrane and downstream activation of N-WASP. DISCUSS +28 32 WASP protein_type It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. DISCUSS +37 43 N-WASP protein It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. DISCUSS +73 79 folded protein_state It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. DISCUSS +81 89 inactive protein_state It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. DISCUSS +95 103 unfolded protein_state It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. DISCUSS +105 111 active protein_state It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. DISCUSS +128 136 affinity evidence It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. DISCUSS +140 145 Cdc42 protein It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. DISCUSS +154 162 unfolded protein_state It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. DISCUSS +163 167 WASP protein_type It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced. DISCUSS +4 12 unfolded protein_state The unfolded, high affinity state of WASP is represented by a short peptide, the GBD, which binds with a low nanomolar affinity to Cdc42. DISCUSS +37 41 WASP protein_type The unfolded, high affinity state of WASP is represented by a short peptide, the GBD, which binds with a low nanomolar affinity to Cdc42. DISCUSS +68 75 peptide chemical The unfolded, high affinity state of WASP is represented by a short peptide, the GBD, which binds with a low nanomolar affinity to Cdc42. DISCUSS +81 84 GBD structure_element The unfolded, high affinity state of WASP is represented by a short peptide, the GBD, which binds with a low nanomolar affinity to Cdc42. DISCUSS +131 136 Cdc42 protein The unfolded, high affinity state of WASP is represented by a short peptide, the GBD, which binds with a low nanomolar affinity to Cdc42. DISCUSS +38 46 affinity evidence In contrast, the best estimate of the affinity of full-length WASP for Cdc42 is low micromolar. DISCUSS +50 61 full-length protein_state In contrast, the best estimate of the affinity of full-length WASP for Cdc42 is low micromolar. DISCUSS +62 66 WASP protein_type In contrast, the best estimate of the affinity of full-length WASP for Cdc42 is low micromolar. DISCUSS +71 76 Cdc42 protein In contrast, the best estimate of the affinity of full-length WASP for Cdc42 is low micromolar. DISCUSS +7 15 inactive protein_state In the inactive state of WASP, the actin- and Arp2/3-binding VCA domain contacts the GBD, competing for Cdc42 binding. DISCUSS +25 29 WASP protein_type In the inactive state of WASP, the actin- and Arp2/3-binding VCA domain contacts the GBD, competing for Cdc42 binding. DISCUSS +46 52 Arp2/3 complex_assembly In the inactive state of WASP, the actin- and Arp2/3-binding VCA domain contacts the GBD, competing for Cdc42 binding. DISCUSS +61 64 VCA structure_element In the inactive state of WASP, the actin- and Arp2/3-binding VCA domain contacts the GBD, competing for Cdc42 binding. DISCUSS +85 88 GBD structure_element In the inactive state of WASP, the actin- and Arp2/3-binding VCA domain contacts the GBD, competing for Cdc42 binding. DISCUSS +104 109 Cdc42 protein In the inactive state of WASP, the actin- and Arp2/3-binding VCA domain contacts the GBD, competing for Cdc42 binding. DISCUSS +21 26 Cdc42 protein The high affinity of Cdc42 for the unfolded, active form pushes the equilibrium in favor of (N-)WASP activation. DISCUSS +35 43 unfolded protein_state The high affinity of Cdc42 for the unfolded, active form pushes the equilibrium in favor of (N-)WASP activation. DISCUSS +45 51 active protein_state The high affinity of Cdc42 for the unfolded, active form pushes the equilibrium in favor of (N-)WASP activation. DISCUSS +92 100 (N-)WASP protein The high affinity of Cdc42 for the unfolded, active form pushes the equilibrium in favor of (N-)WASP activation. DISCUSS +11 20 PI(4,5)P2 chemical Binding of PI(4,5)P2 to the basic region just N-terminal to the GBD further favors the active conformation. DISCUSS +64 67 GBD structure_element Binding of PI(4,5)P2 to the basic region just N-terminal to the GBD further favors the active conformation. DISCUSS +87 93 active protein_state Binding of PI(4,5)P2 to the basic region just N-terminal to the GBD further favors the active conformation. DISCUSS +69 89 WASP/N-WASP proteins protein_type A substantial body of data has illuminated the complex regulation of WASP/N-WASP proteins, and current evidence suggests that these allosteric activation mechanisms and oligomerization combine to regulate WASP activity, allowing the synchronization and integration of multiple potential activation signals (reviewed in Ref.). DISCUSS +205 209 WASP protein_type A substantial body of data has illuminated the complex regulation of WASP/N-WASP proteins, and current evidence suggests that these allosteric activation mechanisms and oligomerization combine to regulate WASP activity, allowing the synchronization and integration of multiple potential activation signals (reviewed in Ref.). DISCUSS +17 22 TOCA1 protein We envisage that TOCA1 is first recruited to the appropriate membrane in response to PI(4,5)P2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1. DISCUSS +85 94 PI(4,5)P2 chemical We envisage that TOCA1 is first recruited to the appropriate membrane in response to PI(4,5)P2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1. DISCUSS +103 108 F-BAR structure_element We envisage that TOCA1 is first recruited to the appropriate membrane in response to PI(4,5)P2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1. DISCUSS +166 171 F-BAR structure_element We envisage that TOCA1 is first recruited to the appropriate membrane in response to PI(4,5)P2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1. DISCUSS +181 186 dimer oligomeric_state We envisage that TOCA1 is first recruited to the appropriate membrane in response to PI(4,5)P2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1. DISCUSS +197 202 TOCA1 protein We envisage that TOCA1 is first recruited to the appropriate membrane in response to PI(4,5)P2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1. DISCUSS +0 5 Cdc42 protein Cdc42 is activated in response to co-incident signals and can then bind to TOCA1, further stabilizing TOCA1 at the membrane. DISCUSS +75 80 TOCA1 protein Cdc42 is activated in response to co-incident signals and can then bind to TOCA1, further stabilizing TOCA1 at the membrane. DISCUSS +102 107 TOCA1 protein Cdc42 is activated in response to co-incident signals and can then bind to TOCA1, further stabilizing TOCA1 at the membrane. DISCUSS +0 5 TOCA1 protein TOCA1 can then recruit N-WASP via an interaction between its SH3 domain and the N-WASP proline-rich region. DISCUSS +23 29 N-WASP protein TOCA1 can then recruit N-WASP via an interaction between its SH3 domain and the N-WASP proline-rich region. DISCUSS +61 64 SH3 structure_element TOCA1 can then recruit N-WASP via an interaction between its SH3 domain and the N-WASP proline-rich region. DISCUSS +80 86 N-WASP protein TOCA1 can then recruit N-WASP via an interaction between its SH3 domain and the N-WASP proline-rich region. DISCUSS +87 106 proline-rich region structure_element TOCA1 can then recruit N-WASP via an interaction between its SH3 domain and the N-WASP proline-rich region. DISCUSS +19 25 N-WASP protein The recruitment of N-WASP alone and of the N-WASP·WIP complex by TOCA1 and FBP17 has been demonstrated. DISCUSS +26 31 alone protein_state The recruitment of N-WASP alone and of the N-WASP·WIP complex by TOCA1 and FBP17 has been demonstrated. DISCUSS +43 53 N-WASP·WIP complex_assembly The recruitment of N-WASP alone and of the N-WASP·WIP complex by TOCA1 and FBP17 has been demonstrated. DISCUSS +65 70 TOCA1 protein The recruitment of N-WASP alone and of the N-WASP·WIP complex by TOCA1 and FBP17 has been demonstrated. DISCUSS +75 80 FBP17 protein The recruitment of N-WASP alone and of the N-WASP·WIP complex by TOCA1 and FBP17 has been demonstrated. DISCUSS +0 3 WIP protein WIP inhibits the activation of N-WASP by Cdc42, an effect that is reversed by TOCA1. DISCUSS +31 37 N-WASP protein WIP inhibits the activation of N-WASP by Cdc42, an effect that is reversed by TOCA1. DISCUSS +41 46 Cdc42 protein WIP inhibits the activation of N-WASP by Cdc42, an effect that is reversed by TOCA1. DISCUSS +78 83 TOCA1 protein WIP inhibits the activation of N-WASP by Cdc42, an effect that is reversed by TOCA1. DISCUSS +35 38 WIP protein It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42. DISCUSS +43 48 TOCA1 protein It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42. DISCUSS +86 92 N-WASP protein It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42. DISCUSS +99 104 TOCA1 protein It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42. DISCUSS +118 126 unfolded protein_state It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42. DISCUSS +128 134 active protein_state It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42. DISCUSS +151 157 N-WASP protein It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42. DISCUSS +190 195 Cdc42 protein It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42. DISCUSS +0 5 TOCA1 protein TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state. DISCUSS +24 30 N-WASP protein TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state. DISCUSS +82 87 TOCA1 protein TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state. DISCUSS +143 152 PI(4,5)P2 chemical TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state. DISCUSS +157 162 Cdc42 protein TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state. DISCUSS +199 205 N-WASP protein TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state. DISCUSS +257 265 unfolded protein_state TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state. DISCUSS +267 273 active protein_state TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state. DISCUSS +23 34 full-length protein_state In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. DISCUSS +35 40 TOCA1 protein In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. DISCUSS +45 51 N-WASP protein In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. DISCUSS +79 89 affinities evidence In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. DISCUSS +94 100 active protein_state In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. DISCUSS +101 106 Cdc42 protein In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. DISCUSS +119 127 unfolded protein_state In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. DISCUSS +129 135 active protein_state In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. DISCUSS +154 162 affinity evidence In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. DISCUSS +166 172 N-WASP protein In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. DISCUSS +177 182 Cdc42 protein In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. DISCUSS +4 16 binding data evidence Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. DISCUSS +30 35 TOCA1 protein Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. DISCUSS +36 39 HR1 structure_element Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. DISCUSS +89 92 NMR experimental_method Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. DISCUSS +119 128 stability protein_state Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. DISCUSS +132 137 TOCA1 protein Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. DISCUSS +138 141 HR1 structure_element Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. DISCUSS +208 219 presence of protein_state Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. DISCUSS +220 225 Cdc42 protein Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. DISCUSS +3 14 full-length protein_state As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. DISCUSS +15 20 TOCA1 protein As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. DISCUSS +29 37 isolated protein_state As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. DISCUSS +38 41 HR1 structure_element As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. DISCUSS +54 59 Cdc42 protein As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. DISCUSS +89 95 N-WASP protein As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. DISCUSS +96 101 Cdc42 protein As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. DISCUSS +142 148 N-WASP protein As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. DISCUSS +149 152 GBD structure_element As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. DISCUSS +179 184 TOCA1 protein As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. DISCUSS +185 188 HR1 structure_element As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. DISCUSS +193 198 Cdc42 protein As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. DISCUSS +42 51 PI(4,5)P2 chemical A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization. DISCUSS +53 62 activated protein_state A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization. DISCUSS +63 68 Cdc42 protein A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization. DISCUSS +73 78 TOCA1 protein A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization. DISCUSS +121 126 TOCA1 protein A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization. DISCUSS +141 156 full activation protein_state A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization. DISCUSS +160 166 N-WASP protein A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization. DISCUSS +99 103 WASP protein In such an array of molecules localized to a discrete region of the membrane, it is plausible that WASP could bind to a second Cdc42 molecule rather than displacing TOCA1 from its cognate Cdc42. DISCUSS +127 132 Cdc42 protein In such an array of molecules localized to a discrete region of the membrane, it is plausible that WASP could bind to a second Cdc42 molecule rather than displacing TOCA1 from its cognate Cdc42. DISCUSS +165 170 TOCA1 protein In such an array of molecules localized to a discrete region of the membrane, it is plausible that WASP could bind to a second Cdc42 molecule rather than displacing TOCA1 from its cognate Cdc42. DISCUSS +188 193 Cdc42 protein In such an array of molecules localized to a discrete region of the membrane, it is plausible that WASP could bind to a second Cdc42 molecule rather than displacing TOCA1 from its cognate Cdc42. DISCUSS +4 7 NMR experimental_method Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD. DISCUSS +12 25 affinity data evidence Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD. DISCUSS +76 81 TOCA1 protein Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD. DISCUSS +82 85 HR1 structure_element Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD. DISCUSS +93 99 N-WASP protein Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD. DISCUSS +100 103 GBD structure_element Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD. DISCUSS +13 18 TOCA1 protein Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP·WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding. DISCUSS +35 40 Cdc42 protein Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP·WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding. DISCUSS +64 74 N-WASP·WIP complex_assembly Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP·WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding. DISCUSS +117 122 Cdc42 protein Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP·WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding. DISCUSS +144 150 N-WASP protein Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP·WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding. DISCUSS +160 165 TOCA1 protein Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP·WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding. DISCUSS +166 171 Cdc42 protein Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP·WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding. DISCUSS +18 27 MGD → IST mutant The commonly used MGD → IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP·WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP. DISCUSS +29 52 Cdc42-binding deficient protein_state The commonly used MGD → IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP·WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP. DISCUSS +64 69 TOCA1 protein The commonly used MGD → IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP·WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP. DISCUSS +108 118 N-WASP·WIP complex_assembly The commonly used MGD → IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP·WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP. DISCUSS +169 174 Cdc42 protein The commonly used MGD → IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP·WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP. DISCUSS +175 178 HR1 structure_element The commonly used MGD → IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP·WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP. DISCUSS +178 183 TOCA1 protein The commonly used MGD → IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP·WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP. DISCUSS +230 236 N-WASP protein The commonly used MGD → IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP·WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP. DISCUSS +65 70 TOCA1 protein In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. DISCUSS +86 91 Cdc42 protein In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. DISCUSS +101 107 N-WASP protein In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. DISCUSS +132 138 N-WASP protein In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. DISCUSS +149 154 TOCA1 protein In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. DISCUSS +164 169 bound protein_state In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. DISCUSS +170 175 Cdc42 protein In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. DISCUSS +191 206 fully activated protein_state In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. DISCUSS +236 241 Cdc42 protein In light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. DISCUSS +17 22 TOCA1 protein Potentially, the TOCA1-Cdc42 interaction functions to position N-WASP and Cdc42 such that they are poised to interact with high affinity. DISCUSS +23 28 Cdc42 protein Potentially, the TOCA1-Cdc42 interaction functions to position N-WASP and Cdc42 such that they are poised to interact with high affinity. DISCUSS +63 69 N-WASP protein Potentially, the TOCA1-Cdc42 interaction functions to position N-WASP and Cdc42 such that they are poised to interact with high affinity. DISCUSS +74 79 Cdc42 protein Potentially, the TOCA1-Cdc42 interaction functions to position N-WASP and Cdc42 such that they are poised to interact with high affinity. DISCUSS +27 32 TOCA1 protein The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP·WIP-induced actin polymerization. DISCUSS +38 43 Cdc42 protein The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP·WIP-induced actin polymerization. DISCUSS +56 64 bound to protein_state The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP·WIP-induced actin polymerization. DISCUSS +65 71 N-WASP protein The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP·WIP-induced actin polymerization. DISCUSS +107 112 TOCA1 protein The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP·WIP-induced actin polymerization. DISCUSS +133 143 N-WASP·WIP complex_assembly The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP·WIP-induced actin polymerization. DISCUSS +60 66 N-WASP protein There is an advantage to such an effector handover, in that N-WASP would only be robustly recruited when F-BAR domains are already present. DISCUSS +105 110 F-BAR structure_element There is an advantage to such an effector handover, in that N-WASP would only be robustly recruited when F-BAR domains are already present. DISCUSS +47 52 F-BAR structure_element Hence, actin polymerization cannot occur until F-BAR domains are poised for membrane distortion. DISCUSS +17 31 Cdc42·HR1TOCA1 complex_assembly Our model of the Cdc42·HR1TOCA1 complex indicates a mechanism by which such a handover could take place (Fig. 9) because it shows that the effector binding sites only partially overlap on Cdc42. DISCUSS +139 161 effector binding sites site Our model of the Cdc42·HR1TOCA1 complex indicates a mechanism by which such a handover could take place (Fig. 9) because it shows that the effector binding sites only partially overlap on Cdc42. DISCUSS +188 193 Cdc42 protein Our model of the Cdc42·HR1TOCA1 complex indicates a mechanism by which such a handover could take place (Fig. 9) because it shows that the effector binding sites only partially overlap on Cdc42. DISCUSS +4 10 lysine residue_name The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound. DISCUSS +85 89 WASP protein The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound. DISCUSS +90 95 Cdc42 protein The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound. DISCUSS +121 127 N-WASP protein The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound. DISCUSS +163 168 Cdc42 protein The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound. DISCUSS +183 188 TOCA1 protein The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound. DISCUSS +189 192 HR1 structure_element The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound. DISCUSS +211 216 bound protein_state The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound. DISCUSS +83 88 Cdc42 protein It has been postulated that the initial interactions between this basic region and Cdc42 could stabilize the active conformation of WASP, leading to high affinity binding between the core CRIB and Cdc42. DISCUSS +109 115 active protein_state It has been postulated that the initial interactions between this basic region and Cdc42 could stabilize the active conformation of WASP, leading to high affinity binding between the core CRIB and Cdc42. DISCUSS +132 136 WASP protein It has been postulated that the initial interactions between this basic region and Cdc42 could stabilize the active conformation of WASP, leading to high affinity binding between the core CRIB and Cdc42. DISCUSS +188 192 CRIB structure_element It has been postulated that the initial interactions between this basic region and Cdc42 could stabilize the active conformation of WASP, leading to high affinity binding between the core CRIB and Cdc42. DISCUSS +197 202 Cdc42 protein It has been postulated that the initial interactions between this basic region and Cdc42 could stabilize the active conformation of WASP, leading to high affinity binding between the core CRIB and Cdc42. DISCUSS +34 38 CRIB structure_element The region C-terminal to the core CRIB, required for maximal affinity binding, would then fully displace the TOCA1 HR1. DISCUSS +109 114 TOCA1 protein The region C-terminal to the core CRIB, required for maximal affinity binding, would then fully displace the TOCA1 HR1. DISCUSS +115 118 HR1 structure_element The region C-terminal to the core CRIB, required for maximal affinity binding, would then fully displace the TOCA1 HR1. DISCUSS +42 60 Cdc42·N-WASP·TOCA1 complex_assembly A simplified model of the early stages of Cdc42·N-WASP·TOCA1-dependent actin polymerization. FIG +9 14 TOCA1 protein Step 1, TOCA1 is recruited to the membrane via its F-BAR domain and/or Cdc42 interactions. FIG +52 57 F-BAR structure_element Step 1, TOCA1 is recruited to the membrane via its F-BAR domain and/or Cdc42 interactions. FIG +72 77 Cdc42 protein Step 1, TOCA1 is recruited to the membrane via its F-BAR domain and/or Cdc42 interactions. FIG +84 91 monomer oligomeric_state F-BAR oligomerization is expected to occur following membrane binding, but a single monomer is shown for clarity. FIG +8 14 N-WASP protein Step 2, N-WASP exists in an inactive, folded conformation. FIG +28 36 inactive protein_state Step 2, N-WASP exists in an inactive, folded conformation. FIG +38 44 folded protein_state Step 2, N-WASP exists in an inactive, folded conformation. FIG +4 9 TOCA1 protein The TOCA1 SH3 domain interacts with N-WASP, causing an activatory allosteric effect. FIG +10 13 SH3 structure_element The TOCA1 SH3 domain interacts with N-WASP, causing an activatory allosteric effect. FIG +36 42 N-WASP protein The TOCA1 SH3 domain interacts with N-WASP, causing an activatory allosteric effect. FIG +4 7 HR1 structure_element The HR1TOCA1-Cdc42 and SH3TOCA1-N-WASP interactions position Cdc42 and N-WASP for binding. FIG +23 26 SH3 structure_element The HR1TOCA1-Cdc42 and SH3TOCA1-N-WASP interactions position Cdc42 and N-WASP for binding. FIG +61 66 Cdc42 protein The HR1TOCA1-Cdc42 and SH3TOCA1-N-WASP interactions position Cdc42 and N-WASP for binding. FIG +71 77 N-WASP protein The HR1TOCA1-Cdc42 and SH3TOCA1-N-WASP interactions position Cdc42 and N-WASP for binding. FIG +8 34 electrostatic interactions bond_interaction Step 3, electrostatic interactions between Cdc42 and the basic region upstream of the CRIB initiate Cdc42·N-WASP binding. FIG +43 48 Cdc42 protein Step 3, electrostatic interactions between Cdc42 and the basic region upstream of the CRIB initiate Cdc42·N-WASP binding. FIG +86 90 CRIB structure_element Step 3, electrostatic interactions between Cdc42 and the basic region upstream of the CRIB initiate Cdc42·N-WASP binding. FIG +100 112 Cdc42·N-WASP complex_assembly Step 3, electrostatic interactions between Cdc42 and the basic region upstream of the CRIB initiate Cdc42·N-WASP binding. FIG +17 21 CRIB structure_element Step 4, the core CRIB binds with high affinity while the region C-terminal to the CRIB displaces the TOCA1 HR1 domain and increases the affinity of the N-WASP-Cdc42 interaction further. FIG +82 86 CRIB structure_element Step 4, the core CRIB binds with high affinity while the region C-terminal to the CRIB displaces the TOCA1 HR1 domain and increases the affinity of the N-WASP-Cdc42 interaction further. FIG +101 106 TOCA1 protein Step 4, the core CRIB binds with high affinity while the region C-terminal to the CRIB displaces the TOCA1 HR1 domain and increases the affinity of the N-WASP-Cdc42 interaction further. FIG +107 110 HR1 structure_element Step 4, the core CRIB binds with high affinity while the region C-terminal to the CRIB displaces the TOCA1 HR1 domain and increases the affinity of the N-WASP-Cdc42 interaction further. FIG +152 158 N-WASP protein Step 4, the core CRIB binds with high affinity while the region C-terminal to the CRIB displaces the TOCA1 HR1 domain and increases the affinity of the N-WASP-Cdc42 interaction further. FIG +4 7 VCA structure_element The VCA domain is released for downstream interactions, and actin polymerization proceeds. FIG +5 27 WASP homology 1 domain structure_element WH1, WASP homology 1 domain; PP, proline-rich region; VCA, verprolin homology, cofilin homology, acidic region. FIG +29 31 PP structure_element WH1, WASP homology 1 domain; PP, proline-rich region; VCA, verprolin homology, cofilin homology, acidic region. FIG +33 52 proline-rich region structure_element WH1, WASP homology 1 domain; PP, proline-rich region; VCA, verprolin homology, cofilin homology, acidic region. FIG +54 57 VCA structure_element WH1, WASP homology 1 domain; PP, proline-rich region; VCA, verprolin homology, cofilin homology, acidic region. FIG +59 110 verprolin homology, cofilin homology, acidic region structure_element WH1, WASP homology 1 domain; PP, proline-rich region; VCA, verprolin homology, cofilin homology, acidic region. FIG +53 58 TOCA1 protein In conclusion, the data presented here show that the TOCA1 HR1 domain is sufficient for Cdc42 binding in vitro and that the interaction is of micromolar affinity, lower than that of other G protein-HR1 domain interactions. DISCUSS +59 62 HR1 structure_element In conclusion, the data presented here show that the TOCA1 HR1 domain is sufficient for Cdc42 binding in vitro and that the interaction is of micromolar affinity, lower than that of other G protein-HR1 domain interactions. DISCUSS +88 93 Cdc42 protein In conclusion, the data presented here show that the TOCA1 HR1 domain is sufficient for Cdc42 binding in vitro and that the interaction is of micromolar affinity, lower than that of other G protein-HR1 domain interactions. DISCUSS +188 197 G protein protein_type In conclusion, the data presented here show that the TOCA1 HR1 domain is sufficient for Cdc42 binding in vitro and that the interaction is of micromolar affinity, lower than that of other G protein-HR1 domain interactions. DISCUSS +198 201 HR1 structure_element In conclusion, the data presented here show that the TOCA1 HR1 domain is sufficient for Cdc42 binding in vitro and that the interaction is of micromolar affinity, lower than that of other G protein-HR1 domain interactions. DISCUSS +14 17 HR1 structure_element The analogous HR1 domains from other TOCA1 family members, FBP17 and CIP4, also exhibit micromolar affinity for Cdc42. DISCUSS +37 49 TOCA1 family protein_type The analogous HR1 domains from other TOCA1 family members, FBP17 and CIP4, also exhibit micromolar affinity for Cdc42. DISCUSS +59 64 FBP17 protein The analogous HR1 domains from other TOCA1 family members, FBP17 and CIP4, also exhibit micromolar affinity for Cdc42. DISCUSS +69 73 CIP4 protein The analogous HR1 domains from other TOCA1 family members, FBP17 and CIP4, also exhibit micromolar affinity for Cdc42. DISCUSS +112 117 Cdc42 protein The analogous HR1 domains from other TOCA1 family members, FBP17 and CIP4, also exhibit micromolar affinity for Cdc42. DISCUSS +15 20 TOCA1 protein A role for the TOCA1-, FBP17-, and CIP4-Cdc42 interactions in the recruitment of these proteins to the membrane therefore appears unlikely. DISCUSS +23 28 FBP17 protein A role for the TOCA1-, FBP17-, and CIP4-Cdc42 interactions in the recruitment of these proteins to the membrane therefore appears unlikely. DISCUSS +35 39 CIP4 protein A role for the TOCA1-, FBP17-, and CIP4-Cdc42 interactions in the recruitment of these proteins to the membrane therefore appears unlikely. DISCUSS +40 45 Cdc42 protein A role for the TOCA1-, FBP17-, and CIP4-Cdc42 interactions in the recruitment of these proteins to the membrane therefore appears unlikely. DISCUSS +62 67 F-BAR structure_element Instead, our findings agree with earlier suggestions that the F-BAR domain is responsible for membrane recruitment. DISCUSS +16 21 Cdc42 protein The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. DISCUSS +22 27 TOCA1 protein The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. DISCUSS +95 104 activated protein_state The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. DISCUSS +105 110 Cdc42 protein The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. DISCUSS +115 121 N-WASP protein The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. DISCUSS +131 146 full activation protein_state The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. DISCUSS +150 156 N-WASP protein The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. DISCUSS +185 190 F-BAR structure_element The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. DISCUSS +226 232 N-WASP protein The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation. DISCUSS +54 58 free protein_state We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway. DISCUSS +63 68 bound protein_state We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway. DISCUSS +70 76 active protein_state We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway. DISCUSS +81 89 inactive protein_state We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway. DISCUSS +90 95 Cdc42 protein We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway. DISCUSS +97 108 TOCA family protein_type We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway. DISCUSS +114 118 WASP protein_type We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway. DISCUSS +204 208 WASP protein_type Our data are therefore easily reconciled with the dynamic instability models described in relation to the formation of endocytic vesicles and with the current data pertaining to the complex activation of WASP/N-WASP pathways by allosteric and oligomeric effects. DISCUSS +209 215 N-WASP protein Our data are therefore easily reconciled with the dynamic instability models described in relation to the formation of endocytic vesicles and with the current data pertaining to the complex activation of WASP/N-WASP pathways by allosteric and oligomeric effects. DISCUSS +46 51 TOCA1 protein It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding. DISCUSS +56 62 N-WASP protein It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding. DISCUSS +75 80 Cdc42 protein It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding. DISCUSS +105 111 N-WASP protein It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding. DISCUSS +136 141 TOCA1 protein It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding. DISCUSS +146 151 Cdc42 protein It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding. DISCUSS +92 96 WASP protein We therefore postulate an effector handover mechanism based on current evidence surrounding WASP/N-WASP activation and our model of the Cdc42·HR1TOCA1 complex. DISCUSS +97 103 N-WASP protein We therefore postulate an effector handover mechanism based on current evidence surrounding WASP/N-WASP activation and our model of the Cdc42·HR1TOCA1 complex. DISCUSS +136 150 Cdc42·HR1TOCA1 complex_assembly We therefore postulate an effector handover mechanism based on current evidence surrounding WASP/N-WASP activation and our model of the Cdc42·HR1TOCA1 complex. DISCUSS +24 29 TOCA1 protein The displacement of the TOCA1 HR1 domain from Cdc42 by N-WASP may represent a unidirectional step in the pathway of Cdc42·N-WASP·TOCA1-dependent actin assembly. DISCUSS +30 33 HR1 structure_element The displacement of the TOCA1 HR1 domain from Cdc42 by N-WASP may represent a unidirectional step in the pathway of Cdc42·N-WASP·TOCA1-dependent actin assembly. DISCUSS +46 51 Cdc42 protein The displacement of the TOCA1 HR1 domain from Cdc42 by N-WASP may represent a unidirectional step in the pathway of Cdc42·N-WASP·TOCA1-dependent actin assembly. DISCUSS +55 61 N-WASP protein The displacement of the TOCA1 HR1 domain from Cdc42 by N-WASP may represent a unidirectional step in the pathway of Cdc42·N-WASP·TOCA1-dependent actin assembly. DISCUSS +116 134 Cdc42·N-WASP·TOCA1 complex_assembly The displacement of the TOCA1 HR1 domain from Cdc42 by N-WASP may represent a unidirectional step in the pathway of Cdc42·N-WASP·TOCA1-dependent actin assembly. DISCUSS diff --git a/annotation_CSV/PMC4937829.csv b/annotation_CSV/PMC4937829.csv new file mode 100644 index 0000000000000000000000000000000000000000..16c7805b38f9d97cbb17d3facd577002c0e0095e --- /dev/null +++ b/annotation_CSV/PMC4937829.csv @@ -0,0 +1,634 @@ +anno_start anno_end anno_text entity_type sentence section +12 21 chaperone protein_type Visualizing chaperone-assisted protein folding TITLE +30 40 structures evidence Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. ABSTRACT +20 29 chaperone protein_type One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. ABSTRACT +96 105 chaperone protein_type One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. ABSTRACT +154 164 chaperones protein_type One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. ABSTRACT +81 102 X-ray crystallography experimental_method To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ). ABSTRACT +111 150 Residual Electron and Anomalous Density experimental_method To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ). ABSTRACT +152 156 READ experimental_method To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ). ABSTRACT +0 4 READ experimental_method READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. ABSTRACT +92 110 immunity protein 7 protein READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. ABSTRACT +112 115 Im7 protein READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. ABSTRACT +117 132 in complex with protein_state READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. ABSTRACT +137 144 E. coli species READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. ABSTRACT +145 154 chaperone protein_type READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. ABSTRACT +155 158 Spy protein READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. ABSTRACT +85 88 Im7 protein This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy. ABSTRACT +95 103 bound to protein_state This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy. ABSTRACT +104 107 Spy protein This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy. ABSTRACT +24 38 Spy-associated protein_state The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone. ABSTRACT +39 42 Im7 protein The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone. ABSTRACT +78 86 unfolded protein_state The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone. ABSTRACT +100 106 folded protein_state The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone. ABSTRACT +111 117 native protein_state The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone. ABSTRACT +199 207 bound to protein_state The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone. ABSTRACT +210 219 chaperone protein_type The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone. ABSTRACT +16 33 structural models evidence High-resolution structural models of protein-protein interactions are critical for obtaining mechanistic insights into biological processes. INTRO +47 61 highly dynamic protein_state However, many protein-protein interactions are highly dynamic, making it difficult to obtain high-resolution data. INTRO +45 86 intrinsically or conditionally disordered protein_state Particularly challenging are interactions of intrinsically or conditionally disordered sections of proteins with their partner proteins. INTRO +19 40 X-ray crystallography experimental_method Recent advances in X-ray crystallography and NMR spectroscopy continue to improve our ability to analyze biomolecules that exist in multiple conformations. INTRO +45 61 NMR spectroscopy experimental_method Recent advances in X-ray crystallography and NMR spectroscopy continue to improve our ability to analyze biomolecules that exist in multiple conformations. INTRO +0 21 X-ray crystallography experimental_method X-ray crystallography has historically provided valuable information on small-scale conformational changes, but observing large-amplitude heterogeneous conformational changes often falls beyond the reach of current crystallographic techniques. INTRO +0 3 NMR experimental_method NMR can theoretically be used to determine heterogeneous ensembles, but in practice, this proves to be very challenging. INTRO +27 37 chaperones protein_type It is clear that molecular chaperones aid in protein folding. INTRO +31 40 chaperone protein_type Structural characterization of chaperone-assisted protein folding likely would help bring clarity to this question. INTRO +0 17 Structural models evidence Structural models of chaperone-substrate complexes have recently begun to provide information as to how a chaperone can recognize its substrate. INTRO +21 30 chaperone protein_type Structural models of chaperone-substrate complexes have recently begun to provide information as to how a chaperone can recognize its substrate. INTRO +106 115 chaperone protein_type Structural models of chaperone-substrate complexes have recently begun to provide information as to how a chaperone can recognize its substrate. INTRO +25 35 chaperones protein_type However, the impact that chaperones have on their substrates, and how these interactions affect the folding process remain largely unknown. INTRO +9 19 chaperones protein_type For most chaperones, it is still unclear whether the chaperone actively participates in and affects the folding of the substrate proteins, or merely provides a suitable microenvironment enabling the substrate to fold on its own. INTRO +53 62 chaperone protein_type For most chaperones, it is still unclear whether the chaperone actively participates in and affects the folding of the substrate proteins, or merely provides a suitable microenvironment enabling the substrate to fold on its own. INTRO +44 53 chaperone protein_type This is a truly fundamental question in the chaperone field, and one that has eluded the community largely because of the highly dynamic nature of the chaperone-substrate complexes. INTRO +122 136 highly dynamic protein_state This is a truly fundamental question in the chaperone field, and one that has eluded the community largely because of the highly dynamic nature of the chaperone-substrate complexes. INTRO +151 160 chaperone protein_type This is a truly fundamental question in the chaperone field, and one that has eluded the community largely because of the highly dynamic nature of the chaperone-substrate complexes. INTRO +46 61 ATP-independent protein_state To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy. INTRO +62 78 Escherichia coli species To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy. INTRO +91 100 chaperone protein_type To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy. INTRO +101 104 Spy protein To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy. INTRO +0 3 Spy protein Spy prevents protein aggregation and aids in protein folding under various stress conditions, including treatment with tannin and butanol. INTRO +119 125 tannin chemical Spy prevents protein aggregation and aids in protein folding under various stress conditions, including treatment with tannin and butanol. INTRO +130 137 butanol chemical Spy prevents protein aggregation and aids in protein folding under various stress conditions, including treatment with tannin and butanol. INTRO +25 28 Spy protein We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy. INTRO +83 86 Im7 protein We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy. INTRO +126 129 Im7 protein We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy. INTRO +158 161 Spy protein We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy. INTRO +4 21 crystal structure evidence The crystal structure of Spy revealed that it forms a thin α-helical homodimeric cradle. INTRO +25 28 Spy protein The crystal structure of Spy revealed that it forms a thin α-helical homodimeric cradle. INTRO +69 80 homodimeric oligomeric_state The crystal structure of Spy revealed that it forms a thin α-helical homodimeric cradle. INTRO +81 87 cradle site The crystal structure of Spy revealed that it forms a thin α-helical homodimeric cradle. INTRO +0 36 Crosslinking and genetic experiments experimental_method Crosslinking and genetic experiments suggested that Spy interacts with substrates somewhere on its concave side. INTRO +52 55 Spy protein Crosslinking and genetic experiments suggested that Spy interacts with substrates somewhere on its concave side. INTRO +17 38 X-ray crystallography experimental_method By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex. INTRO +75 93 crystal structures evidence By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex. INTRO +138 146 ensemble evidence By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex. INTRO +154 161 dynamic protein_state By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex. INTRO +162 169 Spy:Im7 complex_assembly By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex. INTRO +38 47 chaperone protein_type This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones. INTRO +103 106 Im7 protein This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones. INTRO +136 144 bound to protein_state This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones. INTRO +151 161 chaperones protein_type This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones. INTRO +0 13 Crystallizing experimental_method Crystallizing the Spy:Im7 complex RESULTS +18 25 Spy:Im7 complex_assembly Crystallizing the Spy:Im7 complex RESULTS +27 35 crystals evidence We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence. RESULTS +57 60 Spy protein We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence. RESULTS +167 193 crystallization conditions experimental_method We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence. RESULTS +207 210 Spy protein We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence. RESULTS +211 219 crystals evidence We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence. RESULTS +227 238 presence of protein_state We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence. RESULTS +275 282 absence protein_state We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence. RESULTS +13 21 screened experimental_method We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +22 48 crystallization conditions experimental_method We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +53 56 Spy protein We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +123 131 unfolded protein_state We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +132 138 bovine taxonomy_domain We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +139 147 α-casein chemical We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +157 166 wild-type protein_state We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +168 170 WT protein_state We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +172 179 E. coli species We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +180 183 Im7 protein We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +188 196 unfolded protein_state We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +208 211 Im7 protein We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +213 217 L18A mutant We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +218 222 L19A mutant We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +223 227 L37A mutant We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +238 253 N-terminal half structure_element We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +257 260 Im7 protein We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +262 269 Im76-45 mutant We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +301 320 Spy-binding portion structure_element We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +324 327 Im7 protein We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine α-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7. RESULTS +49 64 co-crystallized experimental_method We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals. RESULTS +65 69 with protein_state We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals. RESULTS +70 73 Spy protein We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals. RESULTS +88 91 Spy protein We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals. RESULTS +92 97 alone protein_state We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals. RESULTS +112 120 crystals evidence We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals. RESULTS +11 42 crystal washing and dissolution experimental_method Subsequent crystal washing and dissolution experiments confirmed the presence of the substrates in the co-crystals (Supplementary Fig. 2). RESULTS +103 114 co-crystals experimental_method Subsequent crystal washing and dissolution experiments confirmed the presence of the substrates in the co-crystals (Supplementary Fig. 2). RESULTS +4 12 crystals evidence The crystals diffracted to ~1.8 Å resolution. RESULTS +8 19 Spy:Im76-45 complex_assembly We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes. RESULTS +20 36 selenomethionine chemical We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes. RESULTS +37 45 crystals evidence We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes. RESULTS +63 102 single-wavelength anomalous diffraction experimental_method We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes. RESULTS +104 107 SAD experimental_method We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes. RESULTS +171 174 Spy protein We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes. RESULTS +95 111 electron density evidence However, modeling of the substrate in the complex proved to be a substantial challenge, as the electron density of the substrate was discontinuous and fragmented. RESULTS +9 32 minimal binding portion structure_element Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a). RESULTS +36 39 Im7 protein Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a). RESULTS +41 48 Im76-45 mutant Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a). RESULTS +74 90 electron density evidence Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a). RESULTS +36 43 density evidence We hypothesized that the fragmented density was due to multiple, partially occupied conformations of the substrate bound within the crystal. RESULTS +132 139 crystal evidence We hypothesized that the fragmented density was due to multiple, partially occupied conformations of the substrate bound within the crystal. RESULTS +72 93 X-ray crystallography experimental_method Such residual density is typically not considered usable by traditional X-ray crystallography methods. RESULTS +51 66 chaperone-bound protein_state Thus, we developed a new approach to interpret the chaperone-bound substrate in multiple conformations. RESULTS +0 4 READ experimental_method READ: a strategy to visualize heterogeneous and dynamic biomolecules RESULTS +17 26 structure evidence To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density. RESULTS +61 64 Spy protein To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density. RESULTS +127 131 READ experimental_method To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density. RESULTS +137 176 Residual Electron and Anomalous Density experimental_method To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density. RESULTS +72 75 Spy protein We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate. RESULTS +86 96 co-crystal evidence We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate. RESULTS +122 131 structure evidence We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate. RESULTS +139 145 folded protein_state We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate. RESULTS +146 152 domain structure_element We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate. RESULTS +156 159 Spy protein We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate. RESULTS +186 211 residual electron density evidence We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate. RESULTS +223 230 dynamic protein_state We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate. RESULTS +47 55 flexible protein_state (2) We then labeled individual residues in the flexible regions of the substrate with the strong anomalous scatterer iodine, which serves to locate these residues in three-dimensional space using their anomalous density. RESULTS +117 123 iodine chemical (2) We then labeled individual residues in the flexible regions of the substrate with the strong anomalous scatterer iodine, which serves to locate these residues in three-dimensional space using their anomalous density. RESULTS +202 219 anomalous density evidence (2) We then labeled individual residues in the flexible regions of the substrate with the strong anomalous scatterer iodine, which serves to locate these residues in three-dimensional space using their anomalous density. RESULTS +17 35 molecular dynamics experimental_method (3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density. RESULTS +37 39 MD experimental_method (3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density. RESULTS +41 52 simulations experimental_method (3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density. RESULTS +121 128 dynamic protein_state (3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density. RESULTS +155 182 sample-and-select algorithm experimental_method (3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density. RESULTS +257 287 residual and anomalous density evidence (3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density. RESULTS +73 81 flexible protein_state Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues. RESULTS +112 118 iodine chemical Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues. RESULTS +124 149 residual electron density evidence Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues. RESULTS +203 211 flexible protein_state Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues. RESULTS +4 20 electron density evidence The electron density then allowed us to connect the labeled residues of the substrate by confining the protein chain within regions of detectable density. RESULTS +146 153 density evidence The electron density then allowed us to connect the labeled residues of the substrate by confining the protein chain within regions of detectable density. RESULTS +117 124 crystal evidence In this way, the two forms of data together were able to describe multiple conformations of the substrate within the crystal. RESULTS +47 51 READ experimental_method As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy. RESULTS +109 127 Spy-binding domain structure_element As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy. RESULTS +131 134 Im7 protein As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy. RESULTS +142 149 Im76-45 mutant As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy. RESULTS +164 172 bound to protein_state As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy. RESULTS +173 176 Spy protein As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy. RESULTS +25 29 READ experimental_method However, we believe that READ will prove generally applicable to visualizing heterogeneous and dynamic complexes that have previously escaped detailed structural analysis. RESULTS +11 15 READ experimental_method Collecting READ data for the Spy:Im76-45 complex RESULTS +29 40 Spy:Im76-45 complex_assembly Collecting READ data for the Spy:Im76-45 complex RESULTS +13 27 READ technique experimental_method To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3). RESULTS +69 78 chaperone protein_type To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3). RESULTS +79 82 Spy protein To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3). RESULTS +96 103 Im76-45 mutant To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3). RESULTS +138 141 NMR experimental_method To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3). RESULTS +162 169 Im76-45 mutant To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3). RESULTS +189 197 unfolded protein_state To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3). RESULTS +209 215 folded protein_state To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3). RESULTS +243 246 Im7 protein To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3). RESULTS +10 29 binding experiments experimental_method Moreover, binding experiments indicated that Im76-45 comprises the entire Spy-binding region. RESULTS +45 52 Im76-45 mutant Moreover, binding experiments indicated that Im76-45 comprises the entire Spy-binding region. RESULTS +74 92 Spy-binding region site Moreover, binding experiments indicated that Im76-45 comprises the entire Spy-binding region. RESULTS +37 43 iodine chemical To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe). RESULTS +48 56 replaced experimental_method To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe). RESULTS +63 70 Im76-45 mutant To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe). RESULTS +114 133 4-iodophenylalanine chemical To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe). RESULTS +135 141 pI-Phe chemical To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe). RESULTS +11 31 anomalous scattering evidence Its strong anomalous scattering allowed us to track the positions of these individual Im76-45 residues one at a time, potentially even if the residue was found in several locations in the same crystal. RESULTS +86 93 Im76-45 mutant Its strong anomalous scattering allowed us to track the positions of these individual Im76-45 residues one at a time, potentially even if the residue was found in several locations in the same crystal. RESULTS +193 200 crystal evidence Its strong anomalous scattering allowed us to track the positions of these individual Im76-45 residues one at a time, potentially even if the residue was found in several locations in the same crystal. RESULTS +8 23 co-crystallized experimental_method We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2). RESULTS +24 27 Spy protein We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2). RESULTS +42 49 Im76-45 mutant We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2). RESULTS +97 103 pI-Phe chemical We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2). RESULTS +104 116 substitution experimental_method We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2). RESULTS +147 156 collected experimental_method We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2). RESULTS +157 171 anomalous data evidence We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2). RESULTS +186 197 Spy:Im76-45 complex_assembly We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2). RESULTS +20 40 electron density map evidence Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site. RESULTS +72 89 anomalous signals evidence Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site. RESULTS +105 111 cradle site Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site. RESULTS +115 118 Spy protein Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site. RESULTS +153 156 Im7 protein Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site. RESULTS +157 179 substrate binding site site Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site. RESULTS +31 38 density evidence Consistent with the fragmented density, however, we observed multiple iodine positions for seven of the eight substituted residues. RESULTS +70 76 iodine chemical Consistent with the fragmented density, however, we observed multiple iodine positions for seven of the eight substituted residues. RESULTS +43 46 Im7 protein Together, these results indicated that the Im7 substrate binds Spy in multiple conformations. RESULTS +63 66 Spy protein Together, these results indicated that the Im7 substrate binds Spy in multiple conformations. RESULTS +0 4 READ experimental_method READ sample-and-select procedure RESULTS +5 22 sample-and-select experimental_method READ sample-and-select procedure RESULTS +42 49 Im76-45 mutant To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes. RESULTS +63 71 bound to protein_state To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes. RESULTS +72 75 Spy protein To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes. RESULTS +93 118 residual electron density evidence To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes. RESULTS +127 144 anomalous signals evidence To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes. RESULTS +154 160 pI-Phe chemical To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes. RESULTS +173 184 Spy:Im76-45 complex_assembly To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes. RESULTS +41 50 chaperone protein_type To generate an accurate depiction of the chaperone-substrate interactions, we devised a selection protocol based on a sample-and-select procedure employed in NMR spectroscopy. RESULTS +118 135 sample-and-select experimental_method To generate an accurate depiction of the chaperone-substrate interactions, we devised a selection protocol based on a sample-and-select procedure employed in NMR spectroscopy. RESULTS +158 174 NMR spectroscopy experimental_method To generate an accurate depiction of the chaperone-substrate interactions, we devised a selection protocol based on a sample-and-select procedure employed in NMR spectroscopy. RESULTS +38 55 genetic algorithm experimental_method During each round of the selection, a genetic algorithm alters the ensemble and its agreement to the experimental data is re-evaluated. RESULTS +104 129 residual electron density evidence If successful, the selection identifies the smallest group of specific conformations that best fits the residual electron density and anomalous signals. RESULTS +134 151 anomalous signals evidence If successful, the selection identifies the smallest group of specific conformations that best fits the residual electron density and anomalous signals. RESULTS +4 8 READ experimental_method The READ sample-and-select algorithm is diagrammed in Fig. 2. RESULTS +9 36 sample-and-select algorithm experimental_method The READ sample-and-select algorithm is diagrammed in Fig. 2. RESULTS +76 85 chaperone protein_type Prior to performing the selection, we generated a large and diverse pool of chaperone-substrate complexes using coarse-grained MD simulations in a pseudo-crystal environment (Fig. 2 and Supplementary Fig. 4). RESULTS +112 141 coarse-grained MD simulations experimental_method Prior to performing the selection, we generated a large and diverse pool of chaperone-substrate complexes using coarse-grained MD simulations in a pseudo-crystal environment (Fig. 2 and Supplementary Fig. 4). RESULTS +147 173 pseudo-crystal environment experimental_method Prior to performing the selection, we generated a large and diverse pool of chaperone-substrate complexes using coarse-grained MD simulations in a pseudo-crystal environment (Fig. 2 and Supplementary Fig. 4). RESULTS +4 30 coarse-grained simulations experimental_method The coarse-grained simulations are based on a single-residue resolution model for protein folding and were extended here to describe Spy-Im76-45 binding events (Online Methods). RESULTS +133 144 Spy-Im76-45 complex_assembly The coarse-grained simulations are based on a single-residue resolution model for protein folding and were extended here to describe Spy-Im76-45 binding events (Online Methods). RESULTS +30 49 binding simulations experimental_method The initial conditions of the binding simulations are not biased toward a particular conformation of the substrate or any specific chaperone-substrate interaction (Online Methods). RESULTS +131 140 chaperone protein_type The initial conditions of the binding simulations are not biased toward a particular conformation of the substrate or any specific chaperone-substrate interaction (Online Methods). RESULTS +0 7 Im76-45 mutant Im76-45 binds and unbinds to Spy throughout the simulations. RESULTS +29 32 Spy protein Im76-45 binds and unbinds to Spy throughout the simulations. RESULTS +48 59 simulations experimental_method Im76-45 binds and unbinds to Spy throughout the simulations. RESULTS +82 91 chaperone protein_type This strategy allows a wide range of substrate conformations to interact with the chaperone. RESULTS +9 11 MD experimental_method From the MD simulations, we extracted ~10,000 diverse Spy:Im76-45 complexes to be used by the ensuing selection. RESULTS +12 23 simulations experimental_method From the MD simulations, we extracted ~10,000 diverse Spy:Im76-45 complexes to be used by the ensuing selection. RESULTS +54 65 Spy:Im76-45 complex_assembly From the MD simulations, we extracted ~10,000 diverse Spy:Im76-45 complexes to be used by the ensuing selection. RESULTS +44 47 Spy protein Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate. RESULTS +48 53 dimer oligomeric_state Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate. RESULTS +54 62 bound to protein_state Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate. RESULTS +72 79 Im76-45 mutant Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate. RESULTS +113 166 residual electron and anomalous crystallographic data evidence This pool was then used by the selection algorithm to identify the minimal ensemble that best satisfies both the residual electron and anomalous crystallographic data. RESULTS +4 24 anomalous scattering evidence The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals. RESULTS +78 84 pI-Phe chemical The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals. RESULTS +99 105 iodine chemical The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals. RESULTS +263 269 iodine chemical The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals. RESULTS +270 287 anomalous signals evidence The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective Cα atom in each coarse-grained conformer by 6.5 Å. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals. RESULTS +28 53 residual electron density evidence Simultaneously, it uses the residual electron density to help choose ensembles. RESULTS +12 38 electron density selection experimental_method To make the electron density selection practical, we needed to develop a method to rapidly evaluate the agreement between the selected sub-ensembles and the experimental electron density on-the-fly during the selection procedure. RESULTS +170 186 electron density evidence To make the electron density selection practical, we needed to develop a method to rapidly evaluate the agreement between the selected sub-ensembles and the experimental electron density on-the-fly during the selection procedure. RESULTS +79 108 2mFo−DFc electron density map evidence To accomplish this task, we generated a compressed version of the experimental 2mFo−DFc electron density map for use in the selection. RESULTS +39 42 map evidence This process provided us with a target map that the ensuing selection tried to recapitulate. RESULTS +65 68 map evidence To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations. RESULTS +95 102 density evidence To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations. RESULTS +150 157 Im76-45 mutant To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations. RESULTS +165 176 Spy:Im76-45 complex_assembly To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations. RESULTS +177 179 MD experimental_method To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations. RESULTS +180 191 simulations experimental_method To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations. RESULTS +41 55 coarse-grained experimental_method For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods). RESULTS +56 58 MD experimental_method For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods). RESULTS +69 85 electron density evidence For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods). RESULTS +109 116 Im76-45 mutant For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods). RESULTS +156 176 electron density map evidence For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the Cα positions of Im76-45 was extracted and used to construct an electron density map (Online Methods). RESULTS +17 38 electron density maps evidence These individual electron density maps from the separate conformers could then be combined (Fig. 2) and compared to the averaged experimental electron density map as part of the selection algorithm. RESULTS +142 162 electron density map evidence These individual electron density maps from the separate conformers could then be combined (Fig. 2) and compared to the averaged experimental electron density map as part of the selection algorithm. RESULTS +56 62 iodine chemical This approach allowed us to simultaneously use both the iodine anomalous signals and the residual electron density in the selection procedure. RESULTS +63 80 anomalous signals evidence This approach allowed us to simultaneously use both the iodine anomalous signals and the residual electron density in the selection procedure. RESULTS +89 114 residual electron density evidence This approach allowed us to simultaneously use both the iodine anomalous signals and the residual electron density in the selection procedure. RESULTS +51 53 MD experimental_method The selection resulted in small ensembles from the MD pool that best fit the READ data (Fig. 1c,d). RESULTS +77 81 READ experimental_method The selection resulted in small ensembles from the MD pool that best fit the READ data (Fig. 1c,d). RESULTS +36 47 Spy:Im76-45 complex_assembly Before analyzing the details of the Spy:Im76-45 complex, we first engaged in a series of validation tests to verify the ensemble and selection procedure (Supplementary Note 1, Figures 1c,d, Supplemental Figures 5-7). RESULTS +105 110 RFree evidence Of note, the final six-membered ensemble was the largest ensemble that could simultaneously decrease the RFree and pass the 10-fold cross-validation test. RESULTS +124 153 10-fold cross-validation test experimental_method Of note, the final six-membered ensemble was the largest ensemble that could simultaneously decrease the RFree and pass the 10-fold cross-validation test. RESULTS +59 66 Im76-45 mutant This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1). RESULTS +80 88 bound to protein_state This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1). RESULTS +93 102 chaperone protein_type This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1). RESULTS +103 106 Spy protein This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1). RESULTS +28 31 Im7 protein Folding and interactions of Im7 while bound to Spy RESULTS +38 46 bound to protein_state Folding and interactions of Im7 while bound to Spy RESULTS +47 50 Spy protein Folding and interactions of Im7 while bound to Spy RESULTS +44 48 READ experimental_method Our results showed that by using this novel READ approach, we were able to obtain structural information about the dynamic interaction of a chaperone with its substrate protein. RESULTS +140 149 chaperone protein_type Our results showed that by using this novel READ approach, we were able to obtain structural information about the dynamic interaction of a chaperone with its substrate protein. RESULTS +95 104 chaperone protein_type We were particularly interested in finding answers to one of the most fundamental questions in chaperone biology—how does chaperone binding affect substrate structure and vice versa. RESULTS +122 131 chaperone protein_type We were particularly interested in finding answers to one of the most fundamental questions in chaperone biology—how does chaperone binding affect substrate structure and vice versa. RESULTS +28 38 structures evidence By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound. RESULTS +69 76 Im76-45 mutant By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound. RESULTS +77 85 bound to protein_state By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound. RESULTS +86 89 Spy protein By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound. RESULTS +108 115 Im76-45 mutant By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound. RESULTS +163 168 bound protein_state By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound. RESULTS +71 79 unfolded protein_state We found these conformations to be highly heterogeneous and to include unfolded, partially folded, and native-like states (Fig. 3). RESULTS +81 97 partially folded protein_state We found these conformations to be highly heterogeneous and to include unfolded, partially folded, and native-like states (Fig. 3). RESULTS +103 114 native-like protein_state We found these conformations to be highly heterogeneous and to include unfolded, partially folded, and native-like states (Fig. 3). RESULTS +35 42 Im76-45 mutant The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a). RESULTS +72 75 Spy protein The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a). RESULTS +76 82 cradle site The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a). RESULTS +205 211 cradle site The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a). RESULTS +17 28 contact map evidence We constructed a contact map of the complex, which shows the frequency of interactions for chaperone-substrate residue pairs (Fig. 4). RESULTS +91 100 chaperone protein_type We constructed a contact map of the complex, which shows the frequency of interactions for chaperone-substrate residue pairs (Fig. 4). RESULTS +26 43 interaction sites site We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). RESULTS +47 50 Spy protein We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). RESULTS +82 88 Arg122 residue_name_number We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). RESULTS +90 96 Thr124 residue_name_number We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). RESULTS +102 107 Phe29 residue_name_number We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). RESULTS +147 156 chaperone protein_type We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). RESULTS +158 163 Arg61 residue_name_number We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). RESULTS +165 170 Arg43 residue_name_number We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). RESULTS +172 177 Lys47 residue_name_number We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). RESULTS +179 184 His96 residue_name_number We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). RESULTS +190 195 Met46 residue_name_number We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46). RESULTS +4 27 Spy-contacting residues site The Spy-contacting residues comprise a mixture of charged, polar, and hydrophobic residues. RESULTS +45 52 Im76-45 mutant Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle. RESULTS +112 115 Spy protein Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle. RESULTS +116 122 cradle site Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle. RESULTS +182 188 cradle site Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle. RESULTS +101 108 Im76-45 mutant This mixture suggests the importance of both electrostatic and hydrophobic components in binding the Im76-45 ensemble. RESULTS +72 79 Im76-45 mutant With respect to the substrate, we observed that nearly every residue in Im76-45 is in contact with Spy (Fig. 4a). RESULTS +99 102 Spy protein With respect to the substrate, we observed that nearly every residue in Im76-45 is in contact with Spy (Fig. 4a). RESULTS +64 71 Im76-45 mutant However, we did notice that despite this uniformity, regions of Im76-45 preferentially interact with different regions in Spy (Fig. 4b). RESULTS +122 125 Spy protein However, we did notice that despite this uniformity, regions of Im76-45 preferentially interact with different regions in Spy (Fig. 4b). RESULTS +17 32 N-terminal half structure_element For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface. RESULTS +36 43 Im76-45 mutant For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface. RESULTS +75 78 Spy protein For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface. RESULTS +79 85 cradle site For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface. RESULTS +99 114 C-terminal half structure_element For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface. RESULTS +157 160 Spy protein For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface. RESULTS +163 178 concave surface site For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy’s concave surface. RESULTS +35 42 Im76-45 mutant Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly. RESULTS +63 71 unfolded protein_state Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly. RESULTS +79 85 native protein_state Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly. RESULTS +115 118 Spy protein Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly. RESULTS +12 24 least-folded protein_state Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3). RESULTS +25 32 Im76-45 mutant Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3). RESULTS +95 98 Spy protein Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3). RESULTS +117 128 most-folded protein_state Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3). RESULTS +64 71 Im76-45 mutant This shift in contacts is likely due to hydrophobic residues of Im76-45 preferentially forming intra-molecular contacts upon folding (i.e., hydrophobic collapse), effectively removing themselves from the interaction sites. RESULTS +204 221 interaction sites site This shift in contacts is likely due to hydrophobic residues of Im76-45 preferentially forming intra-molecular contacts upon folding (i.e., hydrophobic collapse), effectively removing themselves from the interaction sites. RESULTS +35 48 binding sites site The diversity of conformations and binding sites observed here emphasizes the dynamic and heterogeneous nature of the chaperone-substrate ensemble. RESULTS +118 127 chaperone protein_type The diversity of conformations and binding sites observed here emphasizes the dynamic and heterogeneous nature of the chaperone-substrate ensemble. RESULTS +79 86 Im76-45 mutant Although we do not yet have time resolution data of these various snapshots of Im76-45, this ensemble illustrates how a substrate samples its folding landscape while bound to a chaperone. RESULTS +166 174 bound to protein_state Although we do not yet have time resolution data of these various snapshots of Im76-45, this ensemble illustrates how a substrate samples its folding landscape while bound to a chaperone. RESULTS +177 186 chaperone protein_type Although we do not yet have time resolution data of these various snapshots of Im76-45, this ensemble illustrates how a substrate samples its folding landscape while bound to a chaperone. RESULTS +0 3 Spy protein Spy changes conformation upon substrate binding RESULTS +14 23 structure evidence Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2). RESULTS +27 30 Spy protein Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2). RESULTS +38 53 substrate-bound protein_state Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2). RESULTS +58 61 apo protein_state Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2). RESULTS +87 90 Spy protein Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2). RESULTS +91 96 dimer oligomeric_state Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2). RESULTS +28 31 Spy protein Upon substrate binding, the Spy dimer twists 9° about its center relative to its apo form. RESULTS +32 37 dimer oligomeric_state Upon substrate binding, the Spy dimer twists 9° about its center relative to its apo form. RESULTS +81 84 apo protein_state Upon substrate binding, the Spy dimer twists 9° about its center relative to its apo form. RESULTS +99 102 Spy protein This twist yields asymmetry and results in substantially different interaction patterns in the two Spy monomers (Fig. 4b). RESULTS +103 111 monomers oligomeric_state This twist yields asymmetry and results in substantially different interaction patterns in the two Spy monomers (Fig. 4b). RESULTS +67 70 Spy protein It is possible that this twist serves to increase heterogeneity in Spy by providing more binding poses. RESULTS +35 48 linker region structure_element Additionally, we observed that the linker region (residues 47–57) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate. RESULTS +59 64 47–57 residue_range Additionally, we observed that the linker region (residues 47–57) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate. RESULTS +69 72 Spy protein Additionally, we observed that the linker region (residues 47–57) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate. RESULTS +134 144 disordered protein_state Additionally, we observed that the linker region (residues 47–57) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate. RESULTS +42 45 Spy protein This increased disorder might explain how Spy is able to recognize and bind different substrates and/or differing conformations of the same substrate. RESULTS +56 59 Spy protein Importantly, we observed the same structural changes in Spy regardless of which of the four substrates was bound (Fig. 5b, Table 1). RESULTS +4 8 RMSD evidence The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site. RESULTS +21 32 well-folded protein_state The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site. RESULTS +45 48 Spy protein The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site. RESULTS +61 70 chaperone protein_type The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site. RESULTS +138 161 competition experiments experimental_method The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site. RESULTS +214 217 Spy protein The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site. RESULTS +328 344 Spy binding site site The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 Å. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site. RESULTS +21 31 chaperones protein_type To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate. DISCUSS +112 116 READ experimental_method To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate. DISCUSS +148 171 conformational ensemble evidence To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate. DISCUSS +179 188 chaperone protein_type To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate. DISCUSS +189 192 Spy protein To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate. DISCUSS +193 211 bound to substrate protein_state To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate. DISCUSS +24 31 Im76-45 mutant As a substrate, we used Im76-45, the chaperone-interacting portion of the protein-folding model protein Im7. DISCUSS +37 66 chaperone-interacting portion structure_element As a substrate, we used Im76-45, the chaperone-interacting portion of the protein-folding model protein Im7. DISCUSS +104 107 Im7 protein As a substrate, we used Im76-45, the chaperone-interacting portion of the protein-folding model protein Im7. DISCUSS +7 22 chaperone-bound protein_state In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states. DISCUSS +33 40 Im76-45 mutant In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states. DISCUSS +49 57 unfolded protein_state In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states. DISCUSS +69 75 folded protein_state In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states. DISCUSS +81 87 native protein_state In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states. DISCUSS +117 126 chaperone protein_type The ensemble provides an unprecedented description of the conformations that a substrate assumes while exploring its chaperone-associated folding landscape. DISCUSS +15 24 chaperone protein_type This substrate-chaperone ensemble helps accomplish the longstanding goal of obtaining a detailed view of how a chaperone aids protein folding. DISCUSS +111 120 chaperone protein_type This substrate-chaperone ensemble helps accomplish the longstanding goal of obtaining a detailed view of how a chaperone aids protein folding. DISCUSS +24 27 Im7 protein We recently showed that Im7 can fold while remaining continuously bound to Spy. DISCUSS +53 74 continuously bound to protein_state We recently showed that Im7 can fold while remaining continuously bound to Spy. DISCUSS +75 78 Spy protein We recently showed that Im7 can fold while remaining continuously bound to Spy. DISCUSS +20 28 ensemble evidence The high-resolution ensemble obtained here now provides insight into exactly how this occurs. DISCUSS +4 14 structures evidence The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy. DISCUSS +22 30 ensemble evidence The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy. DISCUSS +103 112 chaperone protein_type The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy. DISCUSS +159 174 concave surface site The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy. DISCUSS +178 181 Spy protein The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy. DISCUSS +4 12 ensemble evidence The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone. DISCUSS +39 42 Spy protein The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone. DISCUSS +55 74 amphipathic surface site The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone. DISCUSS +146 154 bound to protein_state The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone. DISCUSS +159 168 chaperone protein_type The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone. DISCUSS +88 98 chaperones protein_type This model is consistent with previous studies postulating that the flexible binding of chaperones allows for substrate protein folding. DISCUSS +16 31 concave surface site The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates. DISCUSS +35 38 Spy protein The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates. DISCUSS +113 116 Spy protein The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates. DISCUSS +139 149 chaperones protein_type The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates. DISCUSS +15 18 Spy protein In contrast to Spy’s binding hotspots, Im76-45 displays substantially less specificity in its binding sites. DISCUSS +21 37 binding hotspots site In contrast to Spy’s binding hotspots, Im76-45 displays substantially less specificity in its binding sites. DISCUSS +39 46 Im76-45 mutant In contrast to Spy’s binding hotspots, Im76-45 displays substantially less specificity in its binding sites. DISCUSS +94 107 binding sites site In contrast to Spy’s binding hotspots, Im76-45 displays substantially less specificity in its binding sites. DISCUSS +11 18 Im76-45 mutant Nearly all Im76-45 residues come in contact with Spy. DISCUSS +49 52 Spy protein Nearly all Im76-45 residues come in contact with Spy. DISCUSS +0 8 Unfolded protein_state Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic. DISCUSS +44 47 Spy protein Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic. DISCUSS +61 101 hydrophobic and hydrophilic interactions bond_interaction Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic. DISCUSS +126 137 native-like protein_state Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic. DISCUSS +54 69 ATP-independent protein_state This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate. DISCUSS +70 79 chaperone protein_type This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate. DISCUSS +88 96 unfolded protein_state This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate. DISCUSS +129 153 hydrophobic interactions bond_interaction This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate. DISCUSS +211 230 hydrophobic regions site This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate. DISCUSS +153 162 chaperone protein_type Once the substrate begins to fold within this protected environment, it progressively buries its own hydrophobic residues, and its interactions with the chaperone shift towards becoming more electrostatic. DISCUSS +44 47 Spy protein Notably, the most frequent contacts between Spy and Im76-45 are charge-charge interactions. DISCUSS +52 59 Im76-45 mutant Notably, the most frequent contacts between Spy and Im76-45 are charge-charge interactions. DISCUSS +64 90 charge-charge interactions bond_interaction Notably, the most frequent contacts between Spy and Im76-45 are charge-charge interactions. DISCUSS +23 26 Im7 protein The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states. DISCUSS +36 41 Glu21 residue_name_number The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states. DISCUSS +43 48 Asp32 residue_name_number The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states. DISCUSS +54 59 Asp35 residue_name_number The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states. DISCUSS +85 88 Im7 protein The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states. DISCUSS +116 119 Spy protein The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states. DISCUSS +141 147 cradle site The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states. DISCUSS +160 168 unfolded protein_state The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states. DISCUSS +173 184 native-like protein_state The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy’s positively charged cradle in both the unfolded and native-like states. DISCUSS +9 14 Asp32 residue_name_number Residues Asp32 and Asp35 are close to each other in the folded state of Im7. DISCUSS +19 24 Asp35 residue_name_number Residues Asp32 and Asp35 are close to each other in the folded state of Im7. DISCUSS +56 62 folded protein_state Residues Asp32 and Asp35 are close to each other in the folded state of Im7. DISCUSS +72 75 Im7 protein Residues Asp32 and Asp35 are close to each other in the folded state of Im7. DISCUSS +71 74 Im7 protein This proximity likely causes electrostatic repulsion that destabilizes Im7’s native state. DISCUSS +77 83 native protein_state This proximity likely causes electrostatic repulsion that destabilizes Im7’s native state. DISCUSS +17 20 Spy protein Interaction with Spy’s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation. DISCUSS +96 101 Asp32 residue_name_number Interaction with Spy’s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation. DISCUSS +106 111 Asp35 residue_name_number Interaction with Spy’s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation. DISCUSS +147 167 helical conformation protein_state Interaction with Spy’s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation. DISCUSS +19 43 hydrophobic interactions bond_interaction As inter-molecular hydrophobic interactions between Spy and the substrate become progressively replaced by intra-molecular interactions within the substrate, the affinity between chaperone and substrates could decrease, eventually leading to release of the folded client protein. DISCUSS +52 55 Spy protein As inter-molecular hydrophobic interactions between Spy and the substrate become progressively replaced by intra-molecular interactions within the substrate, the affinity between chaperone and substrates could decrease, eventually leading to release of the folded client protein. DISCUSS +179 188 chaperone protein_type As inter-molecular hydrophobic interactions between Spy and the substrate become progressively replaced by intra-molecular interactions within the substrate, the affinity between chaperone and substrates could decrease, eventually leading to release of the folded client protein. DISCUSS +257 263 folded protein_state As inter-molecular hydrophobic interactions between Spy and the substrate become progressively replaced by intra-molecular interactions within the substrate, the affinity between chaperone and substrates could decrease, eventually leading to release of the folded client protein. DISCUSS +24 48 genetic selection system experimental_method Recently, we employed a genetic selection system to improve the chaperone activity of Spy. DISCUSS +64 73 chaperone protein_type Recently, we employed a genetic selection system to improve the chaperone activity of Spy. DISCUSS +86 89 Spy protein Recently, we employed a genetic selection system to improve the chaperone activity of Spy. DISCUSS +34 37 Spy protein This selection resulted in “Super Spy” variants that were more effective at both preventing aggregation and promoting protein folding. DISCUSS +39 47 variants protein_state This selection resulted in “Super Spy” variants that were more effective at both preventing aggregation and promoting protein folding. DISCUSS +24 29 bound protein_state In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function. DISCUSS +30 37 Im76-45 mutant In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function. DISCUSS +38 46 ensemble evidence In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function. DISCUSS +125 134 chaperone protein_type In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function. DISCUSS +42 45 Spy protein Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both. DISCUSS +46 54 variants protein_state Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both. DISCUSS +62 67 bound protein_state Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both. DISCUSS +68 71 Im7 protein Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both. DISCUSS +85 87 WT protein_state Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both. DISCUSS +88 91 Spy protein Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both. DISCUSS +103 112 chaperone protein_type Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both. DISCUSS +141 153 H/D exchange experimental_method Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both. DISCUSS +4 12 ensemble evidence Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a). DISCUSS +44 47 Spy protein Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a). DISCUSS +48 57 mutations protein_state Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a). DISCUSS +59 63 H96L mutant Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a). DISCUSS +68 73 Q100L mutant Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a). DISCUSS +92 117 chaperone contact surface site Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a). DISCUSS +132 139 Im76-45 mutant Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a). DISCUSS +14 26 co-structure evidence Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker. DISCUSS +45 49 L32P mutant Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker. DISCUSS +80 83 Spy protein Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker. DISCUSS +130 146 N-terminal helix structure_element Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker. DISCUSS +189 199 disordered protein_state Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker. DISCUSS +200 206 linker structure_element Moreover, our co-structure suggests that the L32P substitution, which increases Spy’s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker. DISCUSS +37 40 Spy protein This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a). DISCUSS +51 61 structures evidence This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a). DISCUSS +76 89 linker region structure_element This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a). DISCUSS +128 131 apo protein_state This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a). DISCUSS +41 54 linker region structure_element By sampling multiple conformations, this linker region may allow diverse substrate conformations to be accommodated. DISCUSS +12 15 Spy protein Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding. DISCUSS +16 25 mutations protein_state Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding. DISCUSS +27 32 F115I mutant Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding. DISCUSS +37 42 F115L mutant Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding. DISCUSS +39 46 Im76-45 mutant This residue does not directly contact Im76-45 in our READ-derived ensemble. DISCUSS +54 58 READ experimental_method This residue does not directly contact Im76-45 in our READ-derived ensemble. DISCUSS +67 75 ensemble evidence This residue does not directly contact Im76-45 in our READ-derived ensemble. DISCUSS +14 17 Spy protein Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b). DISCUSS +21 29 bound to protein_state Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b). DISCUSS +41 45 F115 residue_name_number Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b). DISCUSS +68 82 hydrogen bonds bond_interaction Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b). DISCUSS +88 94 Tyr104 residue_name_number Instead, when Spy is bound to substrate, F115 engages in close CH⋯π hydrogen bonds with Tyr104 (Fig. 6b). DISCUSS +56 72 C-terminal helix structure_element This interaction presumably reduces the mobility of the C-terminal helix. DISCUSS +4 9 F115I mutant The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence. DISCUSS +10 11 L mutant The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence. DISCUSS +46 60 hydrogen bonds bond_interaction The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence. DISCUSS +66 90 hydrophobic interactions bond_interaction The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence. DISCUSS +51 59 flexible protein_state As a result, the C-terminus, and possibly also the flexible linker, is likely to become more flexible and thus more accommodating of different conformations of substrates. DISCUSS +60 66 linker structure_element As a result, the C-terminus, and possibly also the flexible linker, is likely to become more flexible and thus more accommodating of different conformations of substrates. DISCUSS +27 35 ensemble evidence Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions. DISCUSS +49 52 Spy protein Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions. DISCUSS +53 61 variants protein_state Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions. DISCUSS +152 161 chaperone protein_type Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions. DISCUSS +47 56 chaperone protein_type Despite extensive studies, exactly how complex chaperone machines help proteins fold remains controversial. DISCUSS +29 38 chaperone protein_type Our study indicates that the chaperone Spy employs a simple surface binding approach that allows the substrate to explore various conformations and form transiently favorable interactions while being protected from aggregation. DISCUSS +39 42 Spy protein Our study indicates that the chaperone Spy employs a simple surface binding approach that allows the substrate to explore various conformations and form transiently favorable interactions while being protected from aggregation. DISCUSS +29 39 chaperones protein_type We speculate that many other chaperones could utilize a similar strategy. DISCUSS +0 3 ATP chemical ATP and co-chaperone dependencies may have emerged later through evolution to better modulate and control chaperone action. DISCUSS +11 20 chaperone protein_type ATP and co-chaperone dependencies may have emerged later through evolution to better modulate and control chaperone action. DISCUSS +106 115 chaperone protein_type ATP and co-chaperone dependencies may have emerged later through evolution to better modulate and control chaperone action. DISCUSS +29 38 chaperone protein_type In addition to insights into chaperone function, this work presents a new method for determining heterogeneous structural ensembles via a hybrid methodology of X-ray crystallography and computational modeling. DISCUSS +160 181 X-ray crystallography experimental_method In addition to insights into chaperone function, this work presents a new method for determining heterogeneous structural ensembles via a hybrid methodology of X-ray crystallography and computational modeling. DISCUSS +186 208 computational modeling experimental_method In addition to insights into chaperone function, this work presents a new method for determining heterogeneous structural ensembles via a hybrid methodology of X-ray crystallography and computational modeling. DISCUSS +35 45 disordered protein_state Heterogeneous dynamic complexes or disordered regions of single proteins, once considered solely approachable by NMR spectroscopy, can now be visualized through X-ray crystallography. DISCUSS +113 129 NMR spectroscopy experimental_method Heterogeneous dynamic complexes or disordered regions of single proteins, once considered solely approachable by NMR spectroscopy, can now be visualized through X-ray crystallography. DISCUSS +161 182 X-ray crystallography experimental_method Heterogeneous dynamic complexes or disordered regions of single proteins, once considered solely approachable by NMR spectroscopy, can now be visualized through X-ray crystallography. DISCUSS +50 67 2mFo−DFc omit map evidence Crystallographic data and ensemble selection. (a) 2mFo−DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 σ. FIG +80 87 Im76-45 mutant Crystallographic data and ensemble selection. (a) 2mFo−DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 σ. FIG +92 107 flexible linker structure_element Crystallographic data and ensemble selection. (a) 2mFo−DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 σ. FIG +108 124 electron density evidence Crystallographic data and ensemble selection. (a) 2mFo−DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 σ. FIG +21 28 density evidence This is the residual density that is used in the READ selection. FIG +49 53 READ experimental_method This is the residual density that is used in the READ selection. FIG +18 24 iodine chemical (b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence. FIG +49 66 anomalous signals evidence (b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence. FIG +73 79 pI-Phe chemical (b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence. FIG +80 93 substitutions experimental_method (b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence. FIG +9 15 iodine chemical Multiple iodine positions were detected for most residues. FIG +26 33 Im76-45 mutant Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black). FIG +34 50 electron density evidence Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black). FIG +59 83 anomalous iodine signals evidence Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black). FIG +158 160 MD experimental_method Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black). FIG +4 17 cost function evidence The cost function, χ2, decreases as the agreement to the experimental data increases and is defined in the Online Methods. FIG +19 21 χ2 evidence The cost function, χ2, decreases as the agreement to the experimental data increases and is defined in the Online Methods. FIG +17 21 READ experimental_method Flowchart of the READ sample-and-select process. FIG +22 39 sample-and-select experimental_method Flowchart of the READ sample-and-select process. FIG +0 11 Spy:Im76-45 complex_assembly Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here. FIG +34 38 RMSD evidence Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here. FIG +42 48 native protein_state Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here. FIG +58 65 Im76-45 mutant Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here. FIG +111 115 READ experimental_method Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here. FIG +0 3 Spy protein Spy is depicted as a gray surface and the Im76-45 conformer is shown as orange balls. FIG +42 49 Im76-45 mutant Spy is depicted as a gray surface and the Im76-45 conformer is shown as orange balls. FIG +52 56 READ experimental_method Atoms that were either not directly selected in the READ procedure, or whose position could not be justified based on agreement with the residual electron density were removed, leading to non-contiguous sections. FIG +137 162 residual electron density evidence Atoms that were either not directly selected in the READ procedure, or whose position could not be justified based on agreement with the residual electron density were removed, leading to non-contiguous sections. FIG +52 59 Im76-45 mutant Dashed lines connect non-contiguous segments of the Im76-45 substrate. FIG +16 19 Spy protein Residues of the Spy flexible linker region that fit the residual electron density are shown as larger gray spheres. FIG +29 42 linker region structure_element Residues of the Spy flexible linker region that fit the residual electron density are shown as larger gray spheres. FIG +56 81 residual electron density evidence Residues of the Spy flexible linker region that fit the residual electron density are shown as larger gray spheres. FIG +40 44 RMSD evidence Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic. FIG +70 76 native protein_state Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic. FIG +86 93 Im76-45 mutant Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic. FIG +141 148 Im76-45 mutant Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic. FIG +153 156 Spy protein Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic. FIG +0 12 Contact maps evidence Contact maps of Spy:Im76-45 complex. FIG +16 27 Spy:Im76-45 complex_assembly Contact maps of Spy:Im76-45 complex. FIG +4 15 Spy:Im76-45 complex_assembly (a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures. FIG +16 27 contact map evidence (a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures. FIG +47 52 bound protein_state (a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures. FIG +53 56 Spy protein (a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures. FIG +57 62 dimer oligomeric_state (a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures. FIG +75 82 Im76-45 mutant (a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures. FIG +91 101 structures evidence (a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures. FIG +13 20 Im76-45 mutant For clarity, Im76-45 is represented with a single conformation. FIG +51 68 contact frequency evidence The frequency plotted is calculated as the average contact frequency from Spy to every residue of Im76-45 and vice-versa. FIG +74 77 Spy protein The frequency plotted is calculated as the average contact frequency from Spy to every residue of Im76-45 and vice-versa. FIG +98 105 Im76-45 mutant The frequency plotted is calculated as the average contact frequency from Spy to every residue of Im76-45 and vice-versa. FIG +68 75 Im76-45 mutant As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45. FIG +88 91 Spy protein As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45. FIG +97 108 contact map evidence As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45. FIG +137 149 contact maps evidence As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45. FIG +153 164 Spy:Im76-45 complex_assembly As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45. FIG +20 23 Spy protein Contacts to the two Spy monomers are depicted separately. FIG +24 32 monomers oligomeric_state Contacts to the two Spy monomers are depicted separately. FIG +14 22 flexible protein_state Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps. FIG +23 36 linker region structure_element Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps. FIG +40 43 Spy protein Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps. FIG +54 59 47–57 residue_range Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps. FIG +90 102 contact maps evidence Note that the flexible linker region of Spy (residues 47–57) is not represented in the 2D contact maps. FIG +0 3 Spy protein Spy conformation changes upon substrate binding. FIG +4 11 Overlay experimental_method (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +15 18 apo protein_state (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +19 22 Spy protein (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +48 53 bound protein_state (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +54 57 Spy protein (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +71 78 Overlay experimental_method (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +82 84 WT protein_state (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +85 88 Spy protein (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +89 97 bound to protein_state (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +98 105 Im76-45 mutant (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +115 119 H96L mutant (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +120 123 Spy protein (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +124 132 bound to protein_state (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +133 136 Im7 protein (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +137 141 L18A mutant (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +142 147 L19 A mutant (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +147 151 L13A mutant (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +160 164 H96L mutant (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +165 168 Spy protein (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +169 177 bound to protein_state (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +178 180 WT protein_state (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +181 184 Im7 protein (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +199 201 WT protein_state (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +202 205 Spy protein (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +206 214 bound to protein_state (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +215 221 casein chemical (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +236 253 Competition assay experimental_method (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +262 269 Im76-45 mutant (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +284 287 Im7 protein (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +288 292 L18A mutant (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +293 297 L19A mutant (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +298 302 L37A mutant (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +303 307 H40W mutant (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +321 333 binding site site (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +337 340 Spy protein (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +350 378 substrate competition assays experimental_method (a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8). FIG +15 18 Spy protein Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green). FIG +19 32 linker region structure_element Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green). FIG +53 56 Spy protein Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green). FIG +74 77 Spy protein Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green). FIG +78 91 linker region structure_element Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green). FIG +132 135 apo protein_state Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green). FIG +212 217 bound protein_state Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green). FIG +4 8 F115 residue_name_number (b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility. FIG +13 16 L32 residue_name_number (b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility. FIG +24 27 Spy protein (b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility. FIG +30 43 linker region structure_element (b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility. FIG +51 57 cradle site (b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility. FIG +70 73 Spy protein (b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility. FIG +95 108 linker region structure_element (b) F115 and L32 tether Spy’s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility. FIG +10 13 Spy protein The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region. FIG +22 27 F115L mutant The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region. FIG +29 34 F115I mutant The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region. FIG +40 44 L32P mutant The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region. FIG +121 134 linker region structure_element The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region. FIG +0 3 L32 residue_name_number L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes. FIG +5 9 F115 residue_name_number L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes. FIG +15 19 Y104 residue_name_number L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes. FIG +98 101 Spy protein L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes. FIG +102 111 mutations protein_state L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes. FIG +118 132 hydrogen bonds bond_interaction L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH⋯π hydrogen bonds are depicted by orange dashes. FIG diff --git a/annotation_CSV/PMC4968113.csv b/annotation_CSV/PMC4968113.csv new file mode 100644 index 0000000000000000000000000000000000000000..fe3d0183ba09e9564ed7e664f00853cb50b1c252 --- /dev/null +++ b/annotation_CSV/PMC4968113.csv @@ -0,0 +1,1246 @@ +anno_start anno_end anno_text entity_type sentence section +26 31 human species Structural diversity in a human antibody germline library TITLE +32 40 antibody protein_type Structural diversity in a human antibody germline library TITLE +11 19 antibody protein_type To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined. ABSTRACT +49 67 crystal structures evidence To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined. ABSTRACT +125 143 kappa light chains structure_element To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined. ABSTRACT +168 180 heavy chains structure_element To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined. ABSTRACT +9 21 heavy chains structure_element All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. ABSTRACT +29 54 antigen-binding fragments structure_element All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. ABSTRACT +56 60 Fabs structure_element All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. ABSTRACT +76 110 complementarity-determining region structure_element All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. ABSTRACT +112 115 CDR structure_element All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. ABSTRACT +117 119 H3 structure_element All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. ABSTRACT +152 155 Fab structure_element All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. ABSTRACT +156 165 structure evidence All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. ABSTRACT +4 22 structure analyses experimental_method The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. ABSTRACT +58 68 structures evidence The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. ABSTRACT +80 90 structures evidence The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. ABSTRACT +98 102 CDRs structure_element The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. ABSTRACT +111 116 VH:VL complex_assembly The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. ABSTRACT +117 137 packing interactions bond_interaction The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. ABSTRACT +4 7 CDR structure_element The CDR conformations for the most part are tightly clustered, especially for the ones with shorter lengths. ABSTRACT +4 10 longer protein_state The longer CDRs with tandem glycines or serines have more conformational diversity than the others. ABSTRACT +11 15 CDRs structure_element The longer CDRs with tandem glycines or serines have more conformational diversity than the others. ABSTRACT +28 36 glycines residue_name The longer CDRs with tandem glycines or serines have more conformational diversity than the others. ABSTRACT +40 47 serines residue_name The longer CDRs with tandem glycines or serines have more conformational diversity than the others. ABSTRACT +0 3 CDR structure_element CDR H3, despite having the same amino acid sequence, exhibits the largest conformational diversity. ABSTRACT +4 6 H3 structure_element CDR H3, despite having the same amino acid sequence, exhibits the largest conformational diversity. ABSTRACT +18 28 structures evidence About half of the structures have CDR H3 conformations similar to that of the parent; the others diverge significantly. ABSTRACT +34 37 CDR structure_element About half of the structures have CDR H3 conformations similar to that of the parent; the others diverge significantly. ABSTRACT +38 40 H3 structure_element About half of the structures have CDR H3 conformations similar to that of the parent; the others diverge significantly. ABSTRACT +27 30 CDR structure_element One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing. ABSTRACT +31 33 H3 structure_element One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing. ABSTRACT +148 153 heavy structure_element One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing. ABSTRACT +158 169 light chain structure_element One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing. ABSTRACT +4 16 stem regions structure_element The stem regions of 14 of the variant pairs are in the ‘kinked’ conformation, and only 2 are in the extended conformation. ABSTRACT +56 62 kinked protein_state The stem regions of 14 of the variant pairs are in the ‘kinked’ conformation, and only 2 are in the extended conformation. ABSTRACT +100 108 extended protein_state The stem regions of 14 of the variant pairs are in the ‘kinked’ conformation, and only 2 are in the extended conformation. ABSTRACT +19 21 VH structure_element The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees. ABSTRACT +26 28 VL structure_element The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees. ABSTRACT +73 81 antibody protein_type The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees. ABSTRACT +82 91 structure evidence The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees. ABSTRACT +101 112 tilt angles evidence The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees. ABSTRACT +10 20 structures evidence Two of 16 structures showed particularly large variations in the tilt angles when compared with the other pairings. ABSTRACT +65 76 tilt angles evidence Two of 16 structures showed particularly large variations in the tilt angles when compared with the other pairings. ABSTRACT +4 14 structures evidence The structures and their analyses provide a rich foundation for future antibody modeling and engineering efforts. ABSTRACT +71 79 antibody protein_type The structures and their analyses provide a rich foundation for future antibody modeling and engineering efforts. ABSTRACT +24 34 antibodies protein_type At present, therapeutic antibodies are the largest class of biotherapeutic proteins that are in clinical trials. INTRO +22 32 antibodies protein_type The use of monoclonal antibodies as therapeutics began in the early 1980s, and their composition has transitioned from murine antibodies to generally less immunogenic humanized and human antibodies. INTRO +119 125 murine taxonomy_domain The use of monoclonal antibodies as therapeutics began in the early 1980s, and their composition has transitioned from murine antibodies to generally less immunogenic humanized and human antibodies. INTRO +126 136 antibodies protein_type The use of monoclonal antibodies as therapeutics began in the early 1980s, and their composition has transitioned from murine antibodies to generally less immunogenic humanized and human antibodies. INTRO +181 186 human species The use of monoclonal antibodies as therapeutics began in the early 1980s, and their composition has transitioned from murine antibodies to generally less immunogenic humanized and human antibodies. INTRO +187 197 antibodies protein_type The use of monoclonal antibodies as therapeutics began in the early 1980s, and their composition has transitioned from murine antibodies to generally less immunogenic humanized and human antibodies. INTRO +42 47 human species The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies. INTRO +48 58 antibodies protein_type The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies. INTRO +78 82 mice taxonomy_domain The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies. INTRO +94 99 human species The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies. INTRO +100 108 antibody protein_type The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies. INTRO +144 149 human species The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies. INTRO +163 181 in vitro selection experimental_method The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies. INTRO +187 205 antibody libraries experimental_method The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies. INTRO +17 25 antibody protein_type Once a candidate antibody is identified, protein engineering is usually required to produce a molecule with the right biophysical and functional properties. INTRO +41 60 protein engineering experimental_method Once a candidate antibody is identified, protein engineering is usually required to produce a molecule with the right biophysical and functional properties. INTRO +63 80 atomic structures evidence All engineering efforts are guided by our understanding of the atomic structures of antibodies. INTRO +84 94 antibodies protein_type All engineering efforts are guided by our understanding of the atomic structures of antibodies. INTRO +21 38 crystal structure evidence In such efforts, the crystal structure of the specific antibody may not be available, but modeling can be used to guide the engineering efforts. INTRO +55 63 antibody protein_type In such efforts, the crystal structure of the specific antibody may not be available, but modeling can be used to guide the engineering efforts. INTRO +8 16 antibody protein_type Today's antibody modeling approaches, which normally focus on the variable region, are being developed by the application of structural principles and insights that are evolving as our knowledge of antibody structures continues to expand. INTRO +66 81 variable region structure_element Today's antibody modeling approaches, which normally focus on the variable region, are being developed by the application of structural principles and insights that are evolving as our knowledge of antibody structures continues to expand. INTRO +198 206 antibody protein_type Today's antibody modeling approaches, which normally focus on the variable region, are being developed by the application of structural principles and insights that are evolving as our knowledge of antibody structures continues to expand. INTRO +207 217 structures evidence Today's antibody modeling approaches, which normally focus on the variable region, are being developed by the application of structural principles and insights that are evolving as our knowledge of antibody structures continues to expand. INTRO +36 46 antibodies protein_type Our current structural knowledge of antibodies is based on a multitude of studies that used many techniques to gain insight into the functional and structural properties of this class of macromolecule. INTRO +15 23 antibody protein_type Five different antibody isotypes occur, IgG, IgD, IgE, IgA and IgM, and each isotype has a unique role in the adaptive immune system. INTRO +40 43 IgG protein Five different antibody isotypes occur, IgG, IgD, IgE, IgA and IgM, and each isotype has a unique role in the adaptive immune system. INTRO +45 48 IgD protein Five different antibody isotypes occur, IgG, IgD, IgE, IgA and IgM, and each isotype has a unique role in the adaptive immune system. INTRO +50 53 IgE protein Five different antibody isotypes occur, IgG, IgD, IgE, IgA and IgM, and each isotype has a unique role in the adaptive immune system. INTRO +55 58 IgA protein Five different antibody isotypes occur, IgG, IgD, IgE, IgA and IgM, and each isotype has a unique role in the adaptive immune system. INTRO +63 66 IgM protein Five different antibody isotypes occur, IgG, IgD, IgE, IgA and IgM, and each isotype has a unique role in the adaptive immune system. INTRO +0 3 IgG protein IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. INTRO +5 8 IgD protein IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. INTRO +13 16 IgE protein IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. INTRO +44 56 heavy chains structure_element IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. INTRO +58 61 HCs structure_element IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. INTRO +69 81 light chains structure_element IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. INTRO +83 86 LCs structure_element IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. INTRO +103 118 disulfide bonds ptm IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. INTRO +126 129 IgA protein IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. INTRO +134 137 IgM protein IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. INTRO +175 185 antibodies protein_type IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively. INTRO +9 12 IgG protein Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. INTRO +14 17 IgD protein Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. INTRO +22 25 IgA protein Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. INTRO +51 59 variable structure_element Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. INTRO +61 62 V structure_element Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. INTRO +70 78 constant structure_element Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. INTRO +80 81 C structure_element Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. INTRO +98 101 IgE protein Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. INTRO +106 109 IgM protein Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. INTRO +164 172 C domain structure_element Isotypes IgG, IgD and IgA each have 4 domains, one variable (V) and 3 constant (C) domains, while IgE and IgM each have the same 4 domains along with an additional C domain. INTRO +53 54 J structure_element These multimeric forms are linked with an additional J chain. INTRO +4 7 LCs structure_element The LCs that associate with the HCs are divided into 2 functionally indistinguishable classes, κ and λ. INTRO +32 35 HCs structure_element The LCs that associate with the HCs are divided into 2 functionally indistinguishable classes, κ and λ. INTRO +95 96 κ structure_element The LCs that associate with the HCs are divided into 2 functionally indistinguishable classes, κ and λ. INTRO +101 102 λ structure_element The LCs that associate with the HCs are divided into 2 functionally indistinguishable classes, κ and λ. INTRO +5 6 κ structure_element Both κ and λ polypeptide chains are composed of a single V domain and a single C domain. INTRO +11 12 λ structure_element Both κ and λ polypeptide chains are composed of a single V domain and a single C domain. INTRO +57 65 V domain structure_element Both κ and λ polypeptide chains are composed of a single V domain and a single C domain. INTRO +79 87 C domain structure_element Both κ and λ polypeptide chains are composed of a single V domain and a single C domain. INTRO +4 9 heavy structure_element The heavy and light chains are composed of structural domains that have ∼110 amino acid residues. INTRO +14 26 light chains structure_element The heavy and light chains are composed of structural domains that have ∼110 amino acid residues. INTRO +43 61 structural domains structure_element The heavy and light chains are composed of structural domains that have ∼110 amino acid residues. INTRO +72 96 ∼110 amino acid residues residue_range The heavy and light chains are composed of structural domains that have ∼110 amino acid residues. INTRO +70 89 immunoglobulin fold structure_element These domains have a common folding pattern often referred to as the “immunoglobulin fold,” formed by the packing together of 2 anti-parallel β-sheets. INTRO +128 150 anti-parallel β-sheets structure_element These domains have a common folding pattern often referred to as the “immunoglobulin fold,” formed by the packing together of 2 anti-parallel β-sheets. INTRO +4 25 immunoglobulin chains protein_type All immunoglobulin chains have an N-terminal V domain followed by 1 to 4 C domains, depending upon the chain type. INTRO +45 53 V domain structure_element All immunoglobulin chains have an N-terminal V domain followed by 1 to 4 C domains, depending upon the chain type. INTRO +73 82 C domains structure_element All immunoglobulin chains have an N-terminal V domain followed by 1 to 4 C domains, depending upon the chain type. INTRO +3 13 antibodies protein_type In antibodies, the heavy and light chain V domains pack together forming the antigen combining site. INTRO +19 40 heavy and light chain structure_element In antibodies, the heavy and light chain V domains pack together forming the antigen combining site. INTRO +41 50 V domains structure_element In antibodies, the heavy and light chain V domains pack together forming the antigen combining site. INTRO +77 99 antigen combining site site In antibodies, the heavy and light chain V domains pack together forming the antigen combining site. INTRO +81 89 antibody protein_type This site, which interacts with the antigen (or target), is the focus of current antibody modeling efforts. INTRO +5 21 interaction site site This interaction site is composed of 6 complementarity-determining regions (CDRs) that were identified in early antibody amino acid sequence analyses to be hypervariable in nature, and thus are responsible for the sequence and structural diversity of our antibody repertoire. INTRO +39 74 complementarity-determining regions structure_element This interaction site is composed of 6 complementarity-determining regions (CDRs) that were identified in early antibody amino acid sequence analyses to be hypervariable in nature, and thus are responsible for the sequence and structural diversity of our antibody repertoire. INTRO +76 80 CDRs structure_element This interaction site is composed of 6 complementarity-determining regions (CDRs) that were identified in early antibody amino acid sequence analyses to be hypervariable in nature, and thus are responsible for the sequence and structural diversity of our antibody repertoire. INTRO +112 149 antibody amino acid sequence analyses experimental_method This interaction site is composed of 6 complementarity-determining regions (CDRs) that were identified in early antibody amino acid sequence analyses to be hypervariable in nature, and thus are responsible for the sequence and structural diversity of our antibody repertoire. INTRO +156 169 hypervariable protein_state This interaction site is composed of 6 complementarity-determining regions (CDRs) that were identified in early antibody amino acid sequence analyses to be hypervariable in nature, and thus are responsible for the sequence and structural diversity of our antibody repertoire. INTRO +255 263 antibody protein_type This interaction site is composed of 6 complementarity-determining regions (CDRs) that were identified in early antibody amino acid sequence analyses to be hypervariable in nature, and thus are responsible for the sequence and structural diversity of our antibody repertoire. INTRO +30 41 CDR regions structure_element The sequence diversity of the CDR regions presents a substantial challenge to antibody modeling. INTRO +78 86 antibody protein_type The sequence diversity of the CDR regions presents a substantial challenge to antibody modeling. INTRO +20 39 structural analysis experimental_method However, an initial structural analysis of the combining sites of the small set of structures of immunoglobulin fragments available in the 1980s found that 5 of the 6 hypervariable loops or CDRs had canonical structures (a limited set of main-chain conformations). INTRO +47 62 combining sites site However, an initial structural analysis of the combining sites of the small set of structures of immunoglobulin fragments available in the 1980s found that 5 of the 6 hypervariable loops or CDRs had canonical structures (a limited set of main-chain conformations). INTRO +83 93 structures evidence However, an initial structural analysis of the combining sites of the small set of structures of immunoglobulin fragments available in the 1980s found that 5 of the 6 hypervariable loops or CDRs had canonical structures (a limited set of main-chain conformations). INTRO +167 186 hypervariable loops structure_element However, an initial structural analysis of the combining sites of the small set of structures of immunoglobulin fragments available in the 1980s found that 5 of the 6 hypervariable loops or CDRs had canonical structures (a limited set of main-chain conformations). INTRO +190 194 CDRs structure_element However, an initial structural analysis of the combining sites of the small set of structures of immunoglobulin fragments available in the 1980s found that 5 of the 6 hypervariable loops or CDRs had canonical structures (a limited set of main-chain conformations). INTRO +2 5 CDR structure_element A CDR canonical structure is defined by its length and conserved residues located in the hypervariable loop and framework residues (V-region residues that are not part of the CDRs). INTRO +89 107 hypervariable loop structure_element A CDR canonical structure is defined by its length and conserved residues located in the hypervariable loop and framework residues (V-region residues that are not part of the CDRs). INTRO +112 130 framework residues structure_element A CDR canonical structure is defined by its length and conserved residues located in the hypervariable loop and framework residues (V-region residues that are not part of the CDRs). INTRO +132 140 V-region structure_element A CDR canonical structure is defined by its length and conserved residues located in the hypervariable loop and framework residues (V-region residues that are not part of the CDRs). INTRO +175 179 CDRs structure_element A CDR canonical structure is defined by its length and conserved residues located in the hypervariable loop and framework residues (V-region residues that are not part of the CDRs). INTRO +24 32 antibody protein_type Furthermore, studies of antibody sequences revealed that the total number of canonical structures are limited for each CDR, indicating possibly that antigen recognition may be affected by structural restrictions at the antigen-binding site. INTRO +119 122 CDR structure_element Furthermore, studies of antibody sequences revealed that the total number of canonical structures are limited for each CDR, indicating possibly that antigen recognition may be affected by structural restrictions at the antigen-binding site. INTRO +219 239 antigen-binding site site Furthermore, studies of antibody sequences revealed that the total number of canonical structures are limited for each CDR, indicating possibly that antigen recognition may be affected by structural restrictions at the antigen-binding site. INTRO +29 37 CDR loop structure_element Later studies found that the CDR loop length is the primary determining factor of antigen-binding site topography because it is the primary factor for determining a canonical structure. INTRO +82 102 antigen-binding site site Later studies found that the CDR loop length is the primary determining factor of antigen-binding site topography because it is the primary factor for determining a canonical structure. INTRO +66 68 LC structure_element Additional efforts have led to our current understanding that the LC CDRs L1, L2, and L3 have preferred sets of canonical structures based on length and amino acid sequence composition. INTRO +69 73 CDRs structure_element Additional efforts have led to our current understanding that the LC CDRs L1, L2, and L3 have preferred sets of canonical structures based on length and amino acid sequence composition. INTRO +74 76 L1 structure_element Additional efforts have led to our current understanding that the LC CDRs L1, L2, and L3 have preferred sets of canonical structures based on length and amino acid sequence composition. INTRO +78 80 L2 structure_element Additional efforts have led to our current understanding that the LC CDRs L1, L2, and L3 have preferred sets of canonical structures based on length and amino acid sequence composition. INTRO +86 88 L3 structure_element Additional efforts have led to our current understanding that the LC CDRs L1, L2, and L3 have preferred sets of canonical structures based on length and amino acid sequence composition. INTRO +43 45 H1 structure_element This was also found to be the case for the H1 and H2 CDRs. INTRO +50 52 H2 structure_element This was also found to be the case for the H1 and H2 CDRs. INTRO +53 57 CDRs structure_element This was also found to be the case for the H1 and H2 CDRs. INTRO +63 67 CDRs structure_element Classification schemes for the canonical structures of these 5 CDRs have emerged and evolved as the number of depositions in the Protein Data Bank of Fab fragments of antibodies grow. INTRO +150 153 Fab structure_element Classification schemes for the canonical structures of these 5 CDRs have emerged and evolved as the number of depositions in the Protein Data Bank of Fab fragments of antibodies grow. INTRO +167 177 antibodies protein_type Classification schemes for the canonical structures of these 5 CDRs have emerged and evolved as the number of depositions in the Protein Data Bank of Fab fragments of antibodies grow. INTRO +26 29 CDR structure_element Recently, a comprehensive CDR classification scheme was reported identifying 72 clusters of conformations observed in antibody structures. INTRO +118 126 antibody protein_type Recently, a comprehensive CDR classification scheme was reported identifying 72 clusters of conformations observed in antibody structures. INTRO +127 137 structures evidence Recently, a comprehensive CDR classification scheme was reported identifying 72 clusters of conformations observed in antibody structures. INTRO +42 45 CDR structure_element The knowledge and predictability of these CDR canonical structures have greatly advanced antibody modeling efforts. INTRO +56 66 structures evidence The knowledge and predictability of these CDR canonical structures have greatly advanced antibody modeling efforts. INTRO +89 97 antibody protein_type The knowledge and predictability of these CDR canonical structures have greatly advanced antibody modeling efforts. INTRO +15 19 CDRs structure_element In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence. INTRO +20 22 L1 structure_element In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence. INTRO +24 26 L2 structure_element In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence. INTRO +28 30 L3 structure_element In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence. INTRO +32 34 H1 structure_element In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence. INTRO +39 41 H2 structure_element In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence. INTRO +56 66 structures evidence In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence. INTRO +90 93 CDR structure_element In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence. INTRO +94 96 H3 structure_element In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence. INTRO +96 101 loops structure_element Some clustering of conformations was observed for the shortest lengths; however, for the longer loops, only the portions nearest the framework (torso, stem or anchor region) were found to have defined conformations. INTRO +133 142 framework structure_element Some clustering of conformations was observed for the shortest lengths; however, for the longer loops, only the portions nearest the framework (torso, stem or anchor region) were found to have defined conformations. INTRO +144 149 torso structure_element Some clustering of conformations was observed for the shortest lengths; however, for the longer loops, only the portions nearest the framework (torso, stem or anchor region) were found to have defined conformations. INTRO +151 155 stem structure_element Some clustering of conformations was observed for the shortest lengths; however, for the longer loops, only the portions nearest the framework (torso, stem or anchor region) were found to have defined conformations. INTRO +159 172 anchor region structure_element Some clustering of conformations was observed for the shortest lengths; however, for the longer loops, only the portions nearest the framework (torso, stem or anchor region) were found to have defined conformations. INTRO +7 19 torso region structure_element In the torso region, 2 primary groups could be identified, which led to sequence-based rules that can predict with some degree of reliability the conformation of the stem region. INTRO +166 177 stem region structure_element In the torso region, 2 primary groups could be identified, which led to sequence-based rules that can predict with some degree of reliability the conformation of the stem region. INTRO +5 11 kinked protein_state The “kinked” or “bulged” conformation is the most prevalent, but an “extended” or “non-bulged” conformation is also, but less frequently, observed. INTRO +17 23 bulged protein_state The “kinked” or “bulged” conformation is the most prevalent, but an “extended” or “non-bulged” conformation is also, but less frequently, observed. INTRO +69 77 extended protein_state The “kinked” or “bulged” conformation is the most prevalent, but an “extended” or “non-bulged” conformation is also, but less frequently, observed. INTRO +83 93 non-bulged protein_state The “kinked” or “bulged” conformation is the most prevalent, but an “extended” or “non-bulged” conformation is also, but less frequently, observed. INTRO +83 96 anchor region structure_element The cataloging and development of the rules for predicting the conformation of the anchor region of CDR H3 continue to be refined, producing new insight into the CDR H3 conformations and new tools for antibody engineering. INTRO +100 103 CDR structure_element The cataloging and development of the rules for predicting the conformation of the anchor region of CDR H3 continue to be refined, producing new insight into the CDR H3 conformations and new tools for antibody engineering. INTRO +104 106 H3 structure_element The cataloging and development of the rules for predicting the conformation of the anchor region of CDR H3 continue to be refined, producing new insight into the CDR H3 conformations and new tools for antibody engineering. INTRO +162 165 CDR structure_element The cataloging and development of the rules for predicting the conformation of the anchor region of CDR H3 continue to be refined, producing new insight into the CDR H3 conformations and new tools for antibody engineering. INTRO +166 168 H3 structure_element The cataloging and development of the rules for predicting the conformation of the anchor region of CDR H3 continue to be refined, producing new insight into the CDR H3 conformations and new tools for antibody engineering. INTRO +201 209 antibody protein_type The cataloging and development of the rules for predicting the conformation of the anchor region of CDR H3 continue to be refined, producing new insight into the CDR H3 conformations and new tools for antibody engineering. INTRO +8 16 antibody protein_type Current antibody modeling approaches take advantage of the most recent advances in homology modeling, the evolving understanding of the CDR canonical structures, the emerging rules for CDR H3 modeling and the growing body of antibody structural data available from the PDB. INTRO +83 100 homology modeling experimental_method Current antibody modeling approaches take advantage of the most recent advances in homology modeling, the evolving understanding of the CDR canonical structures, the emerging rules for CDR H3 modeling and the growing body of antibody structural data available from the PDB. INTRO +136 139 CDR structure_element Current antibody modeling approaches take advantage of the most recent advances in homology modeling, the evolving understanding of the CDR canonical structures, the emerging rules for CDR H3 modeling and the growing body of antibody structural data available from the PDB. INTRO +150 160 structures evidence Current antibody modeling approaches take advantage of the most recent advances in homology modeling, the evolving understanding of the CDR canonical structures, the emerging rules for CDR H3 modeling and the growing body of antibody structural data available from the PDB. INTRO +185 188 CDR structure_element Current antibody modeling approaches take advantage of the most recent advances in homology modeling, the evolving understanding of the CDR canonical structures, the emerging rules for CDR H3 modeling and the growing body of antibody structural data available from the PDB. INTRO +189 191 H3 structure_element Current antibody modeling approaches take advantage of the most recent advances in homology modeling, the evolving understanding of the CDR canonical structures, the emerging rules for CDR H3 modeling and the growing body of antibody structural data available from the PDB. INTRO +225 233 antibody protein_type Current antibody modeling approaches take advantage of the most recent advances in homology modeling, the evolving understanding of the CDR canonical structures, the emerging rules for CDR H3 modeling and the growing body of antibody structural data available from the PDB. INTRO +7 36 antibody modeling assessments experimental_method Recent antibody modeling assessments show continued improvement in the quality of the models being generated by a variety of modeling methods. INTRO +9 17 antibody protein_type Although antibody modeling is improving, the latest assessment revealed a number of challenges that need to be overcome to provide accurate 3-dimensional models of antibody V regions, including accuracies in the modeling of CDR H3. INTRO +164 172 antibody protein_type Although antibody modeling is improving, the latest assessment revealed a number of challenges that need to be overcome to provide accurate 3-dimensional models of antibody V regions, including accuracies in the modeling of CDR H3. INTRO +173 182 V regions structure_element Although antibody modeling is improving, the latest assessment revealed a number of challenges that need to be overcome to provide accurate 3-dimensional models of antibody V regions, including accuracies in the modeling of CDR H3. INTRO +224 227 CDR structure_element Although antibody modeling is improving, the latest assessment revealed a number of challenges that need to be overcome to provide accurate 3-dimensional models of antibody V regions, including accuracies in the modeling of CDR H3. INTRO +228 230 H3 structure_element Although antibody modeling is improving, the latest assessment revealed a number of challenges that need to be overcome to provide accurate 3-dimensional models of antibody V regions, including accuracies in the modeling of CDR H3. INTRO +137 145 antibody protein_type The need for improvement in this area was also highlighted in a recent study reporting an approach and results that may influence future antibody modeling efforts. INTRO +29 58 antibody modeling assessments experimental_method One important finding of the antibody modeling assessments was that errors in the structural templates that are used as the basis for homology models can propagate into the final models, producing inaccuracies that may negatively influence the predictive nature of the V region model. INTRO +134 149 homology models experimental_method One important finding of the antibody modeling assessments was that errors in the structural templates that are used as the basis for homology models can propagate into the final models, producing inaccuracies that may negatively influence the predictive nature of the V region model. INTRO +269 277 V region structure_element One important finding of the antibody modeling assessments was that errors in the structural templates that are used as the basis for homology models can propagate into the final models, producing inaccuracies that may negatively influence the predictive nature of the V region model. INTRO +11 19 antibody protein_type To support antibody engineering and therapeutic development efforts, a phage library was designed and constructed based on a limited number of scaffolds built with frequently used human germ-line IGV and IGJ gene segments that encode antigen combining sites suitable for recognition of peptides and proteins. INTRO +71 84 phage library experimental_method To support antibody engineering and therapeutic development efforts, a phage library was designed and constructed based on a limited number of scaffolds built with frequently used human germ-line IGV and IGJ gene segments that encode antigen combining sites suitable for recognition of peptides and proteins. INTRO +180 185 human species To support antibody engineering and therapeutic development efforts, a phage library was designed and constructed based on a limited number of scaffolds built with frequently used human germ-line IGV and IGJ gene segments that encode antigen combining sites suitable for recognition of peptides and proteins. INTRO +196 199 IGV structure_element To support antibody engineering and therapeutic development efforts, a phage library was designed and constructed based on a limited number of scaffolds built with frequently used human germ-line IGV and IGJ gene segments that encode antigen combining sites suitable for recognition of peptides and proteins. INTRO +204 207 IGJ structure_element To support antibody engineering and therapeutic development efforts, a phage library was designed and constructed based on a limited number of scaffolds built with frequently used human germ-line IGV and IGJ gene segments that encode antigen combining sites suitable for recognition of peptides and proteins. INTRO +234 257 antigen combining sites site To support antibody engineering and therapeutic development efforts, a phage library was designed and constructed based on a limited number of scaffolds built with frequently used human germ-line IGV and IGJ gene segments that encode antigen combining sites suitable for recognition of peptides and proteins. INTRO +5 8 Fab structure_element This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +34 36 HC structure_element This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +48 56 IGHV1-69 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +58 63 H1-69 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +66 74 IGHV3-23 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +76 81 H3-23 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +87 95 IGHV5-51 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +96 101 H5-51 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +110 112 LC structure_element This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +128 129 κ structure_element This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +132 140 IGKV1-39 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +142 147 L1-39 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +150 158 IGKV3-11 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +160 165 L3-11 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +168 176 IGKV3-20 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +178 183 L3-20 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +189 196 IGKV4-1 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +198 202 L4-1 mutant This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all κ), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1). INTRO +98 108 structures evidence Selection of these genes was based on the high frequency of their use and their cognate canonical structures that were found binding to peptides and proteins, as well as their ability to be expressed in bacteria and displayed on filamentous phage. INTRO +190 211 expressed in bacteria experimental_method Selection of these genes was based on the high frequency of their use and their cognate canonical structures that were found binding to peptides and proteins, as well as their ability to be expressed in bacteria and displayed on filamentous phage. INTRO +216 246 displayed on filamentous phage experimental_method Selection of these genes was based on the high frequency of their use and their cognate canonical structures that were found binding to peptides and proteins, as well as their ability to be expressed in bacteria and displayed on filamentous phage. INTRO +70 75 human species The implementation of the library involves the diversification of the human germline genes to mimic that found in natural human libraries. INTRO +122 127 human species The implementation of the library involves the diversification of the human germline genes to mimic that found in natural human libraries. INTRO +4 36 crystal structure determinations experimental_method The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development. INTRO +41 60 structural analyses experimental_method The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development. INTRO +77 81 Fabs structure_element The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development. INTRO +128 138 structures evidence The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development. INTRO +151 153 HC structure_element The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development. INTRO +164 172 IGHV3-53 mutant The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development. INTRO +174 179 H3-53 mutant The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development. INTRO +200 203 LCs structure_element The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development. INTRO +252 260 antibody protein_type The crystal structure determinations and structural analyses of all germline Fabs in the library described above along with the structures of a fourth HC germline, IGHV3-53 (H3-53), paired with the 4 LCs of the library have been carried out to support antibody therapeutic development. INTRO +7 10 HCs structure_element All 16 HCs of the Fabs have the same CDR H3 that was reported in an earlier Fab structure. INTRO +18 22 Fabs structure_element All 16 HCs of the Fabs have the same CDR H3 that was reported in an earlier Fab structure. INTRO +37 40 CDR structure_element All 16 HCs of the Fabs have the same CDR H3 that was reported in an earlier Fab structure. INTRO +41 43 H3 structure_element All 16 HCs of the Fabs have the same CDR H3 that was reported in an earlier Fab structure. INTRO +76 79 Fab structure_element All 16 HCs of the Fabs have the same CDR H3 that was reported in an earlier Fab structure. INTRO +80 89 structure evidence All 16 HCs of the Fabs have the same CDR H3 that was reported in an earlier Fab structure. INTRO +47 49 VH structure_element This is the first systematic study of the same VH and VL structures in the context of different pairings. INTRO +54 56 VL structure_element This is the first systematic study of the same VH and VL structures in the context of different pairings. INTRO +57 67 structures evidence This is the first systematic study of the same VH and VL structures in the context of different pairings. INTRO +58 68 structures evidence The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +80 90 structures evidence The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +98 100 L1 structure_element The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +102 104 L2 structure_element The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +106 108 L3 structure_element The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +110 112 H1 structure_element The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +117 119 H2 structure_element The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +120 124 CDRs structure_element The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +130 140 structures evidence The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +148 151 CDR structure_element The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +152 155 H3s structure_element The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +165 170 VH:VL complex_assembly The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +171 191 packing interactions bond_interaction The structure analyses include comparisons of the overall structures, canonical structures of the L1, L2, L3, H1 and H2 CDRs, the structures of all CDR H3s, and the VH:VL packing interactions. INTRO +4 14 structures evidence The structures and their analyses provide a foundation for future antibody engineering and structure determination efforts. INTRO +66 74 antibody protein_type The structures and their analyses provide a foundation for future antibody engineering and structure determination efforts. INTRO +0 18 Crystal structures evidence Crystal structures RESULTS +0 12 Crystal data evidence Crystal data, X-ray data, and refinement statistics. TABLE +14 24 X-ray data evidence Crystal data, X-ray data, and refinement statistics. TABLE +30 51 refinement statistics evidence Crystal data, X-ray data, and refinement statistics. TABLE +12 24 Crystal data evidence (Continued) Crystal data, X-ray data, and refinement statistics. TABLE +26 36 X-ray data evidence (Continued) Crystal data, X-ray data, and refinement statistics. TABLE +42 63 refinement statistics evidence (Continued) Crystal data, X-ray data, and refinement statistics. TABLE +12 24 Crystal data evidence (Continued) Crystal data, X-ray data, and refinement statistics. TABLE +26 36 X-ray data evidence (Continued) Crystal data, X-ray data, and refinement statistics. TABLE +42 63 refinement statistics evidence (Continued) Crystal data, X-ray data, and refinement statistics. TABLE +12 24 Crystal data evidence (Continued) Crystal data, X-ray data, and refinement statistics. TABLE +26 36 X-ray data evidence (Continued) Crystal data, X-ray data, and refinement statistics. TABLE +42 63 refinement statistics evidence (Continued) Crystal data, X-ray data, and refinement statistics. TABLE +4 22 crystal structures evidence The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +28 44 germline library experimental_method The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +60 64 Fabs structure_element The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +90 93 HCs structure_element The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +95 100 H1-69 mutant The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +102 107 H3-23 mutant The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +109 114 H3-53 mutant The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +119 124 H5-51 mutant The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +132 135 LCs structure_element The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +137 142 L1-39 mutant The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +144 149 L3-11 mutant The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +151 156 L3-20 mutant The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +161 165 L4-1 mutant The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined. RESULTS +4 7 Fab structure_element The Fab heavy and light chain sequences for the variants numbered according to Chothia are shown in Fig. S1. RESULTS +18 29 light chain structure_element The Fab heavy and light chain sequences for the variants numbered according to Chothia are shown in Fig. S1. RESULTS +19 22 HCs structure_element The four different HCs all have the same CDR H3 sequence, ARYDGIYGELDF. RESULTS +41 44 CDR structure_element The four different HCs all have the same CDR H3 sequence, ARYDGIYGELDF. RESULTS +45 47 H3 structure_element The four different HCs all have the same CDR H3 sequence, ARYDGIYGELDF. RESULTS +58 70 ARYDGIYGELDF structure_element The four different HCs all have the same CDR H3 sequence, ARYDGIYGELDF. RESULTS +0 15 Crystallization experimental_method Crystallization of the 16 Fabs was previously reported. RESULTS +26 30 Fabs structure_element Crystallization of the 16 Fabs was previously reported. RESULTS +18 26 crystals evidence Three sets of the crystals were isomorphous with nearly identical unit cells (Table 1). RESULTS +18 29 H3-23:L3-11 complex_assembly These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121. RESULTS +34 44 H3-23:L4-1 complex_assembly These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121. RESULTS +61 72 H3-53:L1-39 complex_assembly These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121. RESULTS +74 85 H3-53:L3-11 complex_assembly These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121. RESULTS +90 101 H3-53:L3-20 complex_assembly These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121. RESULTS +120 131 H5-51:L1-39 complex_assembly These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121. RESULTS +133 144 H5-51:L3-11 complex_assembly These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121. RESULTS +149 160 H5-51:L3-20 complex_assembly These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121. RESULTS +72 80 PEG 3350 chemical Variations occur in the pH (buffer) and the additives, and, in group 3, PEG 3350 is the precipitant for one variants while ammonium sulfate is the precipitant for the other two. RESULTS +123 139 ammonium sulfate chemical Variations occur in the pH (buffer) and the additives, and, in group 3, PEG 3350 is the precipitant for one variants while ammonium sulfate is the precipitant for the other two. RESULTS +22 35 crystal forms evidence The similarity in the crystal forms is attributed in part to cross-seeding using the microseed matrix screening for groups 2 and 3. RESULTS +85 111 microseed matrix screening experimental_method The similarity in the crystal forms is attributed in part to cross-seeding using the microseed matrix screening for groups 2 and 3. RESULTS +4 22 crystal structures evidence The crystal structures of the 16 Fabs have been determined at resolutions ranging from 3.3 Å to 1.65 Å (Table 1). RESULTS +33 37 Fabs structure_element The crystal structures of the 16 Fabs have been determined at resolutions ranging from 3.3 Å to 1.65 Å (Table 1). RESULTS +14 17 Fab structure_element The number of Fab molecules in the crystallographic asymmetric unit varies from 1 (for 12 Fabs) to 2 (for 4 Fabs). RESULTS +90 94 Fabs structure_element The number of Fab molecules in the crystallographic asymmetric unit varies from 1 (for 12 Fabs) to 2 (for 4 Fabs). RESULTS +108 112 Fabs structure_element The number of Fab molecules in the crystallographic asymmetric unit varies from 1 (for 12 Fabs) to 2 (for 4 Fabs). RESULTS +12 22 structures evidence Overall the structures are fairly complete, and, as can be expected, the models for the higher resolution structures are more complete than those for the lower resolution structures (Table S1). RESULTS +106 116 structures evidence Overall the structures are fairly complete, and, as can be expected, the models for the higher resolution structures are more complete than those for the lower resolution structures (Table S1). RESULTS +171 181 structures evidence Overall the structures are fairly complete, and, as can be expected, the models for the higher resolution structures are more complete than those for the lower resolution structures (Table S1). RESULTS +16 19 HCs structure_element Invariably, the HCs have more disorder than the LCs. RESULTS +30 38 disorder protein_state Invariably, the HCs have more disorder than the LCs. RESULTS +48 51 LCs structure_element Invariably, the HCs have more disorder than the LCs. RESULTS +8 10 LC structure_element For the LC, the disorder is observed at 2 of the C-terminal residues with few exceptions. RESULTS +16 24 disorder protein_state For the LC, the disorder is observed at 2 of the C-terminal residues with few exceptions. RESULTS +58 60 LC structure_element Apart from the C-terminus, only a few surface residues in LC are disordered. RESULTS +65 75 disordered protein_state Apart from the C-terminus, only a few surface residues in LC are disordered. RESULTS +4 7 HCs structure_element The HCs feature the largest number of disordered residues, with the lower resolution structures having the most. RESULTS +38 48 disordered protein_state The HCs feature the largest number of disordered residues, with the lower resolution structures having the most. RESULTS +85 95 structures evidence The HCs feature the largest number of disordered residues, with the lower resolution structures having the most. RESULTS +53 63 disordered protein_state The C-terminal residues including the 6xHis tags are disordered in all 16 structures. RESULTS +74 84 structures evidence The C-terminal residues including the 6xHis tags are disordered in all 16 structures. RESULTS +93 103 structures evidence In addition to these, 2 primary disordered stretches of residues are observed in a number of structures (Table S1). RESULTS +17 21 loop structure_element One involves the loop connecting the first 2 β-strands of the constant domain (in all Fabs except H3-23:L1-39, H3-23:L3-11 and H3-53:L1-39). RESULTS +45 54 β-strands structure_element One involves the loop connecting the first 2 β-strands of the constant domain (in all Fabs except H3-23:L1-39, H3-23:L3-11 and H3-53:L1-39). RESULTS +62 77 constant domain structure_element One involves the loop connecting the first 2 β-strands of the constant domain (in all Fabs except H3-23:L1-39, H3-23:L3-11 and H3-53:L1-39). RESULTS +86 90 Fabs structure_element One involves the loop connecting the first 2 β-strands of the constant domain (in all Fabs except H3-23:L1-39, H3-23:L3-11 and H3-53:L1-39). RESULTS +98 109 H3-23:L1-39 complex_assembly One involves the loop connecting the first 2 β-strands of the constant domain (in all Fabs except H3-23:L1-39, H3-23:L3-11 and H3-53:L1-39). RESULTS +111 122 H3-23:L3-11 complex_assembly One involves the loop connecting the first 2 β-strands of the constant domain (in all Fabs except H3-23:L1-39, H3-23:L3-11 and H3-53:L1-39). RESULTS +127 138 H3-53:L1-39 complex_assembly One involves the loop connecting the first 2 β-strands of the constant domain (in all Fabs except H3-23:L1-39, H3-23:L3-11 and H3-53:L1-39). RESULTS +24 27 CDR structure_element The other is located in CDR H3 (in H5-51:L3-11, H5-51:L3-20 and in one of 2 copies of H3-23:L4-1). RESULTS +28 30 H3 structure_element The other is located in CDR H3 (in H5-51:L3-11, H5-51:L3-20 and in one of 2 copies of H3-23:L4-1). RESULTS +35 46 H5-51:L3-11 complex_assembly The other is located in CDR H3 (in H5-51:L3-11, H5-51:L3-20 and in one of 2 copies of H3-23:L4-1). RESULTS +48 59 H5-51:L3-20 complex_assembly The other is located in CDR H3 (in H5-51:L3-11, H5-51:L3-20 and in one of 2 copies of H3-23:L4-1). RESULTS +86 96 H3-23:L4-1 complex_assembly The other is located in CDR H3 (in H5-51:L3-11, H5-51:L3-20 and in one of 2 copies of H3-23:L4-1). RESULTS +0 3 CDR structure_element CDR H1 and CDR H2 also show some degree of disorder, but to a lesser extent. RESULTS +4 6 H1 structure_element CDR H1 and CDR H2 also show some degree of disorder, but to a lesser extent. RESULTS +11 14 CDR structure_element CDR H1 and CDR H2 also show some degree of disorder, but to a lesser extent. RESULTS +15 17 H2 structure_element CDR H1 and CDR H2 also show some degree of disorder, but to a lesser extent. RESULTS +43 51 disorder protein_state CDR H1 and CDR H2 also show some degree of disorder, but to a lesser extent. RESULTS +0 3 CDR structure_element CDR canonical structures RESULTS +14 24 structures evidence CDR canonical structures RESULTS +8 11 CDR structure_element Several CDR definitions have evolved over decades of antibody research. RESULTS +53 61 antibody protein_type Several CDR definitions have evolved over decades of antibody research. RESULTS +41 44 CDR structure_element Depending on the focus of the study, the CDR boundaries differ slightly between various definitions. RESULTS +25 28 CDR structure_element In this work, we use the CDR definition of North et al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr. RESULTS +126 130 CDRs structure_element In this work, we use the CDR definition of North et al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr. RESULTS +131 133 H1 structure_element In this work, we use the CDR definition of North et al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr. RESULTS +138 140 H3 structure_element In this work, we use the CDR definition of North et al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr. RESULTS +169 172 Cys residue_name In this work, we use the CDR definition of North et al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr. RESULTS +181 184 CDR structure_element In this work, we use the CDR definition of North et al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr. RESULTS +185 187 L2 structure_element In this work, we use the CDR definition of North et al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr. RESULTS +253 256 Tyr residue_name In this work, we use the CDR definition of North et al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr. RESULTS +0 3 CDR structure_element CDR H1 RESULTS +4 6 H1 structure_element CDR H1 RESULTS +4 17 superposition experimental_method The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +21 24 CDR structure_element The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +25 27 H1 structure_element The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +46 51 HC:LC complex_assembly The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +63 75 heavy chains structure_element The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +81 86 H1-69 mutant The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +92 97 H3-23 mutant The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +103 108 H3-53 mutant The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +117 122 H5-51 mutant The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +0 4 CDRs structure_element CDRs are defined using the Dunbrack convention [12]. TABLE +32 35 Fab structure_element Assignments for 2 copies of the Fab in the asymmetric unit are given for 5 structures. TABLE +75 85 structures evidence Assignments for 2 copies of the Fab in the asymmetric unit are given for 5 structures. TABLE +23 27 CDRs structure_element No assignment (NA) for CDRs with missing residues. TABLE +9 12 HCs structure_element The four HCs feature CDR H1 of the same length, and their sequences are highly similar (Table 2). RESULTS +21 24 CDR structure_element The four HCs feature CDR H1 of the same length, and their sequences are highly similar (Table 2). RESULTS +25 27 H1 structure_element The four HCs feature CDR H1 of the same length, and their sequences are highly similar (Table 2). RESULTS +4 7 CDR structure_element The CDR H1 backbone conformations for all variants for each of the HCs are shown in Fig. 1. RESULTS +8 10 H1 structure_element The CDR H1 backbone conformations for all variants for each of the HCs are shown in Fig. 1. RESULTS +67 70 HCs structure_element The CDR H1 backbone conformations for all variants for each of the HCs are shown in Fig. 1. RESULTS +13 16 HCs structure_element Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig. 1B-D). RESULTS +18 23 H3-23 mutant Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig. 1B-D). RESULTS +25 30 H3-53 mutant Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig. 1B-D). RESULTS +35 40 H5-51 mutant Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig. 1B-D). RESULTS +77 84 H1-13-1 mutant Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig. 1B-D). RESULTS +155 158 Fab structure_element Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig. 1B-D). RESULTS +159 169 structures evidence Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig. 1B-D). RESULTS +190 201 rmsd values evidence Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig. 1B-D). RESULTS +31 36 H3-53 mutant Some deviation is observed for H3-53, mostly due to H3-53:L4-1, which exhibits a significant degree of disorder in CDR H1. RESULTS +52 62 H3-53:L4-1 complex_assembly Some deviation is observed for H3-53, mostly due to H3-53:L4-1, which exhibits a significant degree of disorder in CDR H1. RESULTS +115 118 CDR structure_element Some deviation is observed for H3-53, mostly due to H3-53:L4-1, which exhibits a significant degree of disorder in CDR H1. RESULTS +119 121 H1 structure_element Some deviation is observed for H3-53, mostly due to H3-53:L4-1, which exhibits a significant degree of disorder in CDR H1. RESULTS +4 20 electron density evidence The electron density for the backbone is weak and discontinuous, and completely missing for several side chains. RESULTS +4 7 CDR structure_element The CDR H1 structures with H1-69 shown in Fig. 1A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20. RESULTS +8 10 H1 structure_element The CDR H1 structures with H1-69 shown in Fig. 1A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20. RESULTS +11 21 structures evidence The CDR H1 structures with H1-69 shown in Fig. 1A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20. RESULTS +27 32 H1-69 mutant The CDR H1 structures with H1-69 shown in Fig. 1A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20. RESULTS +83 93 structures evidence The CDR H1 structures with H1-69 shown in Fig. 1A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20. RESULTS +109 112 LCs structure_element The CDR H1 structures with H1-69 shown in Fig. 1A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20. RESULTS +144 147 Fab structure_element The CDR H1 structures with H1-69 shown in Fig. 1A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20. RESULTS +172 183 H1-69:L3-11 complex_assembly The CDR H1 structures with H1-69 shown in Fig. 1A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20. RESULTS +188 199 H1-69:L3-20 complex_assembly The CDR H1 structures with H1-69 shown in Fig. 1A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20. RESULTS +24 27 Fab structure_element In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10. RESULTS +28 38 structures evidence In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10. RESULTS +69 79 structures evidence In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10. RESULTS +88 95 H1-13-1 mutant In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10. RESULTS +97 104 H1-13-3 mutant In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10. RESULTS +106 113 H1-13-4 mutant In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10. RESULTS +115 122 H1-13-6 mutant In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10. RESULTS +127 135 H1-13-10 mutant In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10. RESULTS +22 27 H1-69 mutant A major difference of H1-69 from the other germlines in the experimental data set is the presence of Gly instead of Phe or Tyr at position 27 (residue 5 of 13 in CDR H1). RESULTS +101 104 Gly residue_name A major difference of H1-69 from the other germlines in the experimental data set is the presence of Gly instead of Phe or Tyr at position 27 (residue 5 of 13 in CDR H1). RESULTS +116 119 Phe residue_name A major difference of H1-69 from the other germlines in the experimental data set is the presence of Gly instead of Phe or Tyr at position 27 (residue 5 of 13 in CDR H1). RESULTS +123 126 Tyr residue_name A major difference of H1-69 from the other germlines in the experimental data set is the presence of Gly instead of Phe or Tyr at position 27 (residue 5 of 13 in CDR H1). RESULTS +139 141 27 residue_number A major difference of H1-69 from the other germlines in the experimental data set is the presence of Gly instead of Phe or Tyr at position 27 (residue 5 of 13 in CDR H1). RESULTS +162 165 CDR structure_element A major difference of H1-69 from the other germlines in the experimental data set is the presence of Gly instead of Phe or Tyr at position 27 (residue 5 of 13 in CDR H1). RESULTS +166 168 H1 structure_element A major difference of H1-69 from the other germlines in the experimental data set is the presence of Gly instead of Phe or Tyr at position 27 (residue 5 of 13 in CDR H1). RESULTS +0 7 Glycine residue_name Glycine introduces the possibility of a higher degree of conformational flexibility that undoubtedly translates to the differences observed, and contributes to the elevated thermal parameters for the atoms in the amino acid residues in this region. RESULTS +0 3 CDR structure_element CDR H2 RESULTS +4 6 H2 structure_element CDR H2 RESULTS +4 17 superposition experimental_method The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +21 24 CDR structure_element The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +25 27 H2 structure_element The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +46 51 HC:LC complex_assembly The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +63 75 heavy chains structure_element The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +81 86 H1-69 mutant The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +92 97 H3-23 mutant The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +103 108 H3-53 mutant The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +117 122 H5-51 mutant The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51. FIG +28 31 CDR structure_element The canonical structures of CDR H2 have fairly consistent conformations (Table 2, Fig. 2). RESULTS +32 34 H2 structure_element The canonical structures of CDR H2 have fairly consistent conformations (Table 2, Fig. 2). RESULTS +14 17 HCs structure_element Each of the 4 HCs adopts only one canonical structure regardless of the pairing LC. RESULTS +80 82 LC structure_element Each of the 4 HCs adopts only one canonical structure regardless of the pairing LC. RESULTS +10 15 H1-69 mutant Germlines H1-69 and H5-51 have the same canonical structure assignment H2-10-1, H3-23 has H2-10-2, and H3-53 has H2-9-3. RESULTS +20 25 H5-51 mutant Germlines H1-69 and H5-51 have the same canonical structure assignment H2-10-1, H3-23 has H2-10-2, and H3-53 has H2-9-3. RESULTS +71 78 H2-10-1 mutant Germlines H1-69 and H5-51 have the same canonical structure assignment H2-10-1, H3-23 has H2-10-2, and H3-53 has H2-9-3. RESULTS +80 85 H3-23 mutant Germlines H1-69 and H5-51 have the same canonical structure assignment H2-10-1, H3-23 has H2-10-2, and H3-53 has H2-9-3. RESULTS +90 97 H2-10-2 mutant Germlines H1-69 and H5-51 have the same canonical structure assignment H2-10-1, H3-23 has H2-10-2, and H3-53 has H2-9-3. RESULTS +103 108 H3-53 mutant Germlines H1-69 and H5-51 have the same canonical structure assignment H2-10-1, H3-23 has H2-10-2, and H3-53 has H2-9-3. RESULTS +113 119 H2-9-3 mutant Germlines H1-69 and H5-51 have the same canonical structure assignment H2-10-1, H3-23 has H2-10-2, and H3-53 has H2-9-3. RESULTS +35 38 CDR structure_element The conformations for all of these CDR H2s are tightly clustered (Fig. 2). RESULTS +39 42 H2s structure_element The conformations for all of these CDR H2s are tightly clustered (Fig. 2). RESULTS +27 30 Fab structure_element In one case, in the second Fab of H1-69:L3-20, CDR H2 is partially disordered (Δ55-60). RESULTS +34 45 H1-69:L3-20 complex_assembly In one case, in the second Fab of H1-69:L3-20, CDR H2 is partially disordered (Δ55-60). RESULTS +47 50 CDR structure_element In one case, in the second Fab of H1-69:L3-20, CDR H2 is partially disordered (Δ55-60). RESULTS +51 53 H2 structure_element In one case, in the second Fab of H1-69:L3-20, CDR H2 is partially disordered (Δ55-60). RESULTS +57 77 partially disordered protein_state In one case, in the second Fab of H1-69:L3-20, CDR H2 is partially disordered (Δ55-60). RESULTS +79 85 Δ55-60 mutant In one case, in the second Fab of H1-69:L3-20, CDR H2 is partially disordered (Δ55-60). RESULTS +37 40 CDR structure_element Although three of the germlines have CDR H2 of the same length, 10 residues, they adopt 2 distinctively different conformations depending mostly on the residue at position 71 from the so-called CDR H4. RESULTS +41 43 H2 structure_element Although three of the germlines have CDR H2 of the same length, 10 residues, they adopt 2 distinctively different conformations depending mostly on the residue at position 71 from the so-called CDR H4. RESULTS +64 75 10 residues residue_range Although three of the germlines have CDR H2 of the same length, 10 residues, they adopt 2 distinctively different conformations depending mostly on the residue at position 71 from the so-called CDR H4. RESULTS +172 174 71 residue_number Although three of the germlines have CDR H2 of the same length, 10 residues, they adopt 2 distinctively different conformations depending mostly on the residue at position 71 from the so-called CDR H4. RESULTS +194 197 CDR structure_element Although three of the germlines have CDR H2 of the same length, 10 residues, they adopt 2 distinctively different conformations depending mostly on the residue at position 71 from the so-called CDR H4. RESULTS +198 200 H4 structure_element Although three of the germlines have CDR H2 of the same length, 10 residues, they adopt 2 distinctively different conformations depending mostly on the residue at position 71 from the so-called CDR H4. RESULTS +0 5 Arg71 residue_name_number Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site. RESULTS +9 14 H3-23 mutant Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site. RESULTS +39 43 CDRs structure_element Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site. RESULTS +44 46 H2 structure_element Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site. RESULTS +51 53 H4 structure_element Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site. RESULTS +98 101 CDR structure_element Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site. RESULTS +102 104 H2 structure_element Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site. RESULTS +121 123 54 residue_number Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site. RESULTS +145 165 antigen binding site site Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site. RESULTS +10 15 H1-69 mutant Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. RESULTS +20 25 H5-51 mutant Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. RESULTS +44 49 human species Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. RESULTS +74 77 Ala residue_name Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. RESULTS +90 92 71 residue_number Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. RESULTS +122 123 H structure_element Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. RESULTS +124 130 Pro52a residue_name_number Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. RESULTS +154 157 CDR structure_element Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. RESULTS +158 160 H4 structure_element Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. RESULTS +192 194 53 residue_number Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. RESULTS +199 201 54 residue_number Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen. RESULTS +17 20 CDR structure_element Conformations of CDR H2 in H1-69 and H5-51, both of which have canonical structure H2-10-1, show little deviation within each set of 4 structures. RESULTS +21 23 H2 structure_element Conformations of CDR H2 in H1-69 and H5-51, both of which have canonical structure H2-10-1, show little deviation within each set of 4 structures. RESULTS +27 32 H1-69 mutant Conformations of CDR H2 in H1-69 and H5-51, both of which have canonical structure H2-10-1, show little deviation within each set of 4 structures. RESULTS +37 42 H5-51 mutant Conformations of CDR H2 in H1-69 and H5-51, both of which have canonical structure H2-10-1, show little deviation within each set of 4 structures. RESULTS +83 90 H2-10-1 mutant Conformations of CDR H2 in H1-69 and H5-51, both of which have canonical structure H2-10-1, show little deviation within each set of 4 structures. RESULTS +135 145 structures evidence Conformations of CDR H2 in H1-69 and H5-51, both of which have canonical structure H2-10-1, show little deviation within each set of 4 structures. RESULTS +45 48 CDR structure_element However, there is a significant shift of the CDR as a rigid body when the 2 sets are superimposed. RESULTS +85 97 superimposed experimental_method However, there is a significant shift of the CDR as a rigid body when the 2 sets are superimposed. RESULTS +49 52 CDR structure_element Most likely this is the result of interaction of CDR H2 with CDR H1, namely with the residue at position 33 (residue 11 of 13 in CDR H1). RESULTS +53 55 H2 structure_element Most likely this is the result of interaction of CDR H2 with CDR H1, namely with the residue at position 33 (residue 11 of 13 in CDR H1). RESULTS +61 64 CDR structure_element Most likely this is the result of interaction of CDR H2 with CDR H1, namely with the residue at position 33 (residue 11 of 13 in CDR H1). RESULTS +65 67 H1 structure_element Most likely this is the result of interaction of CDR H2 with CDR H1, namely with the residue at position 33 (residue 11 of 13 in CDR H1). RESULTS +105 107 33 residue_number Most likely this is the result of interaction of CDR H2 with CDR H1, namely with the residue at position 33 (residue 11 of 13 in CDR H1). RESULTS +129 132 CDR structure_element Most likely this is the result of interaction of CDR H2 with CDR H1, namely with the residue at position 33 (residue 11 of 13 in CDR H1). RESULTS +133 135 H1 structure_element Most likely this is the result of interaction of CDR H2 with CDR H1, namely with the residue at position 33 (residue 11 of 13 in CDR H1). RESULTS +9 14 H1-69 mutant Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center. RESULTS +19 22 Ala residue_name Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center. RESULTS +35 37 33 residue_number Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center. RESULTS +49 54 H5-51 mutant Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center. RESULTS +64 66 33 residue_number Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center. RESULTS +90 93 Trp residue_name Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center. RESULTS +116 117 H structure_element Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center. RESULTS +118 123 Tyr52 residue_name_number Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center. RESULTS +135 138 CDR structure_element Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center. RESULTS +139 141 H2 structure_element Germline H1-69 has Ala at position 33 whereas in H5-51 position 33 is occupied by a bulky Trp, which stacks against H-Tyr52 and drives CDR H2 away from the center. RESULTS +0 3 CDR structure_element CDR L1 RESULTS +4 6 L1 structure_element CDR L1 RESULTS +4 17 superposition experimental_method The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +21 24 CDR structure_element The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +25 27 L1 structure_element The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +46 51 HC:LC complex_assembly The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +63 75 light chains structure_element The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +81 86 L1-39 mutant The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +92 97 L3-11 mutant The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +103 108 L3-20 mutant The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +117 121 L4-1 mutant The superposition of CDR L1 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +9 11 LC structure_element The four LC CDRs L1 feature 3 different lengths (11, 12 and 17 residues) having a total of 4 different canonical structure assignments. RESULTS +12 16 CDRs structure_element The four LC CDRs L1 feature 3 different lengths (11, 12 and 17 residues) having a total of 4 different canonical structure assignments. RESULTS +17 19 L1 structure_element The four LC CDRs L1 feature 3 different lengths (11, 12 and 17 residues) having a total of 4 different canonical structure assignments. RESULTS +49 51 11 residue_range The four LC CDRs L1 feature 3 different lengths (11, 12 and 17 residues) having a total of 4 different canonical structure assignments. RESULTS +53 55 12 residue_range The four LC CDRs L1 feature 3 different lengths (11, 12 and 17 residues) having a total of 4 different canonical structure assignments. RESULTS +60 62 17 residue_range The four LC CDRs L1 feature 3 different lengths (11, 12 and 17 residues) having a total of 4 different canonical structure assignments. RESULTS +9 12 LCs structure_element Of these LCs, L1-39 and L3-11 have the same canonical structure, L1-11-1, and superimpose very well (Fig. 3A, B). RESULTS +14 19 L1-39 mutant Of these LCs, L1-39 and L3-11 have the same canonical structure, L1-11-1, and superimpose very well (Fig. 3A, B). RESULTS +24 29 L3-11 mutant Of these LCs, L1-39 and L3-11 have the same canonical structure, L1-11-1, and superimpose very well (Fig. 3A, B). RESULTS +65 72 L1-11-1 mutant Of these LCs, L1-39 and L3-11 have the same canonical structure, L1-11-1, and superimpose very well (Fig. 3A, B). RESULTS +78 89 superimpose experimental_method Of these LCs, L1-39 and L3-11 have the same canonical structure, L1-11-1, and superimpose very well (Fig. 3A, B). RESULTS +21 26 L3-20 mutant For the remaining 2, L3-20 has 2 different assignments, L1-12-1 and L1-12-2, while L4-1 has a single assignment, L1-17-1. RESULTS +56 63 L1-12-1 mutant For the remaining 2, L3-20 has 2 different assignments, L1-12-1 and L1-12-2, while L4-1 has a single assignment, L1-17-1. RESULTS +68 75 L1-12-2 mutant For the remaining 2, L3-20 has 2 different assignments, L1-12-1 and L1-12-2, while L4-1 has a single assignment, L1-17-1. RESULTS +83 87 L4-1 mutant For the remaining 2, L3-20 has 2 different assignments, L1-12-1 and L1-12-2, while L4-1 has a single assignment, L1-17-1. RESULTS +113 120 L1-17-1 mutant For the remaining 2, L3-20 has 2 different assignments, L1-12-1 and L1-12-2, while L4-1 has a single assignment, L1-17-1. RESULTS +0 4 L4-1 mutant L4-1 has the longest CDR L1, composed of 17 amino acid residues (Fig. 3D). RESULTS +21 24 CDR structure_element L4-1 has the longest CDR L1, composed of 17 amino acid residues (Fig. 3D). RESULTS +25 27 L1 structure_element L4-1 has the longest CDR L1, composed of 17 amino acid residues (Fig. 3D). RESULTS +41 63 17 amino acid residues residue_range L4-1 has the longest CDR L1, composed of 17 amino acid residues (Fig. 3D). RESULTS +55 59 rmsd evidence Despite this, the conformations are tightly clustered (rmsd is 0.20 Å). RESULTS +34 46 stem regions structure_element The backbone conformations of the stem regions superimpose well. RESULTS +52 55 30a residue_number Some changes in conformation occur between residues 30a and 30f (residues 8 and 13 of 17 in CDR L1). RESULTS +60 63 30f residue_number Some changes in conformation occur between residues 30a and 30f (residues 8 and 13 of 17 in CDR L1). RESULTS +74 75 8 residue_number Some changes in conformation occur between residues 30a and 30f (residues 8 and 13 of 17 in CDR L1). RESULTS +80 82 13 residue_number Some changes in conformation occur between residues 30a and 30f (residues 8 and 13 of 17 in CDR L1). RESULTS +86 88 17 residue_number Some changes in conformation occur between residues 30a and 30f (residues 8 and 13 of 17 in CDR L1). RESULTS +92 95 CDR structure_element Some changes in conformation occur between residues 30a and 30f (residues 8 and 13 of 17 in CDR L1). RESULTS +96 98 L1 structure_element Some changes in conformation occur between residues 30a and 30f (residues 8 and 13 of 17 in CDR L1). RESULTS +23 34 loop region structure_element This is the tip of the loop region, which appears to have similar conformations that fan out the structures because of the slight differences in torsion angles in the backbone near Tyr30a and Lys30f. RESULTS +97 107 structures evidence This is the tip of the loop region, which appears to have similar conformations that fan out the structures because of the slight differences in torsion angles in the backbone near Tyr30a and Lys30f. RESULTS +181 187 Tyr30a residue_name_number This is the tip of the loop region, which appears to have similar conformations that fan out the structures because of the slight differences in torsion angles in the backbone near Tyr30a and Lys30f. RESULTS +192 198 Lys30f residue_name_number This is the tip of the loop region, which appears to have similar conformations that fan out the structures because of the slight differences in torsion angles in the backbone near Tyr30a and Lys30f. RESULTS +0 5 L3-20 mutant L3-20 is the most variable in CDR L1 among the 4 germlines as indicated by an rmsd of 0.54 Å (Fig. 3C). RESULTS +30 33 CDR structure_element L3-20 is the most variable in CDR L1 among the 4 germlines as indicated by an rmsd of 0.54 Å (Fig. 3C). RESULTS +34 36 L1 structure_element L3-20 is the most variable in CDR L1 among the 4 germlines as indicated by an rmsd of 0.54 Å (Fig. 3C). RESULTS +78 82 rmsd evidence L3-20 is the most variable in CDR L1 among the 4 germlines as indicated by an rmsd of 0.54 Å (Fig. 3C). RESULTS +4 14 structures evidence Two structures, H3-53:L3-20 and H5-51:L3-20 are assigned to canonical structure L1-12-1 with virtually identical backbone conformations. RESULTS +16 27 H3-53:L3-20 complex_assembly Two structures, H3-53:L3-20 and H5-51:L3-20 are assigned to canonical structure L1-12-1 with virtually identical backbone conformations. RESULTS +32 43 H5-51:L3-20 complex_assembly Two structures, H3-53:L3-20 and H5-51:L3-20 are assigned to canonical structure L1-12-1 with virtually identical backbone conformations. RESULTS +80 87 L1-12-1 mutant Two structures, H3-53:L3-20 and H5-51:L3-20 are assigned to canonical structure L1-12-1 with virtually identical backbone conformations. RESULTS +21 32 H3-23:L3-20 complex_assembly The third structure, H3-23:L3-20, has CDR L1 as L1-12-2, which deviates from L1-12-1 at residues 29-32, i.e., at the site of insertion with respect to the 11-residue CDR. RESULTS +38 41 CDR structure_element The third structure, H3-23:L3-20, has CDR L1 as L1-12-2, which deviates from L1-12-1 at residues 29-32, i.e., at the site of insertion with respect to the 11-residue CDR. RESULTS +42 44 L1 structure_element The third structure, H3-23:L3-20, has CDR L1 as L1-12-2, which deviates from L1-12-1 at residues 29-32, i.e., at the site of insertion with respect to the 11-residue CDR. RESULTS +48 55 L1-12-2 mutant The third structure, H3-23:L3-20, has CDR L1 as L1-12-2, which deviates from L1-12-1 at residues 29-32, i.e., at the site of insertion with respect to the 11-residue CDR. RESULTS +77 84 L1-12-1 mutant The third structure, H3-23:L3-20, has CDR L1 as L1-12-2, which deviates from L1-12-1 at residues 29-32, i.e., at the site of insertion with respect to the 11-residue CDR. RESULTS +97 102 29-32 residue_range The third structure, H3-23:L3-20, has CDR L1 as L1-12-2, which deviates from L1-12-1 at residues 29-32, i.e., at the site of insertion with respect to the 11-residue CDR. RESULTS +155 165 11-residue residue_range The third structure, H3-23:L3-20, has CDR L1 as L1-12-2, which deviates from L1-12-1 at residues 29-32, i.e., at the site of insertion with respect to the 11-residue CDR. RESULTS +166 169 CDR structure_element The third structure, H3-23:L3-20, has CDR L1 as L1-12-2, which deviates from L1-12-1 at residues 29-32, i.e., at the site of insertion with respect to the 11-residue CDR. RESULTS +30 41 H1-69:L3-20 complex_assembly The fourth member of the set, H1-69:L3-20, was crystallized with 2 Fabs in the asymmetric unit. RESULTS +47 59 crystallized experimental_method The fourth member of the set, H1-69:L3-20, was crystallized with 2 Fabs in the asymmetric unit. RESULTS +67 71 Fabs structure_element The fourth member of the set, H1-69:L3-20, was crystallized with 2 Fabs in the asymmetric unit. RESULTS +20 23 CDR structure_element The conformation of CDR L1 in these 2 Fabs is slightly different, and both conformations fall somewhere between L1-12-1 and L1-12-2. RESULTS +24 26 L1 structure_element The conformation of CDR L1 in these 2 Fabs is slightly different, and both conformations fall somewhere between L1-12-1 and L1-12-2. RESULTS +38 42 Fabs structure_element The conformation of CDR L1 in these 2 Fabs is slightly different, and both conformations fall somewhere between L1-12-1 and L1-12-2. RESULTS +112 119 L1-12-1 mutant The conformation of CDR L1 in these 2 Fabs is slightly different, and both conformations fall somewhere between L1-12-1 and L1-12-2. RESULTS +124 131 L1-12-2 mutant The conformation of CDR L1 in these 2 Fabs is slightly different, and both conformations fall somewhere between L1-12-1 and L1-12-2. RESULTS +42 51 structure evidence This reflects the lack of accuracy in the structure due to low resolution of the X-ray data (3.3 Å). RESULTS +81 91 X-ray data evidence This reflects the lack of accuracy in the structure due to low resolution of the X-ray data (3.3 Å). RESULTS +0 3 CDR structure_element CDR L2 RESULTS +4 6 L2 structure_element CDR L2 RESULTS +4 17 superposition experimental_method The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +21 24 CDR structure_element The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +25 27 L2 structure_element The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +46 51 HC:LC complex_assembly The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +63 75 light chains structure_element The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +81 86 L1-39 mutant The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +92 97 L3-11 mutant The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +103 108 L3-20 mutant The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +117 121 L4-1 mutant The superposition of CDR L2 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +9 12 LCs structure_element All four LCs have CDR L2 of the same length and canonical structure, L2-8-1 (Table 2). RESULTS +18 21 CDR structure_element All four LCs have CDR L2 of the same length and canonical structure, L2-8-1 (Table 2). RESULTS +22 24 L2 structure_element All four LCs have CDR L2 of the same length and canonical structure, L2-8-1 (Table 2). RESULTS +69 75 L2-8-1 mutant All four LCs have CDR L2 of the same length and canonical structure, L2-8-1 (Table 2). RESULTS +4 7 CDR structure_element The CDR L2 conformations for each of the LCs paired with the 4 HCs are clustered more tightly than any of the other CDRs (rmsd values are in the range 0.09-0.16 Å), and all 4 sets have virtually the same conformation despite the sequence diversity of the loop. RESULTS +8 10 L2 structure_element The CDR L2 conformations for each of the LCs paired with the 4 HCs are clustered more tightly than any of the other CDRs (rmsd values are in the range 0.09-0.16 Å), and all 4 sets have virtually the same conformation despite the sequence diversity of the loop. RESULTS +41 44 LCs structure_element The CDR L2 conformations for each of the LCs paired with the 4 HCs are clustered more tightly than any of the other CDRs (rmsd values are in the range 0.09-0.16 Å), and all 4 sets have virtually the same conformation despite the sequence diversity of the loop. RESULTS +63 66 HCs structure_element The CDR L2 conformations for each of the LCs paired with the 4 HCs are clustered more tightly than any of the other CDRs (rmsd values are in the range 0.09-0.16 Å), and all 4 sets have virtually the same conformation despite the sequence diversity of the loop. RESULTS +116 120 CDRs structure_element The CDR L2 conformations for each of the LCs paired with the 4 HCs are clustered more tightly than any of the other CDRs (rmsd values are in the range 0.09-0.16 Å), and all 4 sets have virtually the same conformation despite the sequence diversity of the loop. RESULTS +122 126 rmsd evidence The CDR L2 conformations for each of the LCs paired with the 4 HCs are clustered more tightly than any of the other CDRs (rmsd values are in the range 0.09-0.16 Å), and all 4 sets have virtually the same conformation despite the sequence diversity of the loop. RESULTS +255 259 loop structure_element The CDR L2 conformations for each of the LCs paired with the 4 HCs are clustered more tightly than any of the other CDRs (rmsd values are in the range 0.09-0.16 Å), and all 4 sets have virtually the same conformation despite the sequence diversity of the loop. RESULTS +0 3 CDR structure_element CDR L3 RESULTS +4 6 L3 structure_element CDR L3 RESULTS +4 17 superposition experimental_method The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +21 24 CDR structure_element The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +25 27 L3 structure_element The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +46 51 HC:LC complex_assembly The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +63 75 light chains structure_element The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +81 86 L1-39 mutant The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +92 97 L3-11 mutant The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +103 108 L3-20 mutant The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +117 121 L4-1 mutant The superposition of CDR L3 backbones for all HC:LC pairs with light chains: (A) L1-39, (B) L3-11, (C) L3-20 and (D) L4-1. FIG +8 11 CDR structure_element As with CDR L2, all 4 LCs have CDR L3 of the same length and canonical structure, L3-9-cis7-1 (Table 2). RESULTS +12 14 L2 structure_element As with CDR L2, all 4 LCs have CDR L3 of the same length and canonical structure, L3-9-cis7-1 (Table 2). RESULTS +22 25 LCs structure_element As with CDR L2, all 4 LCs have CDR L3 of the same length and canonical structure, L3-9-cis7-1 (Table 2). RESULTS +31 34 CDR structure_element As with CDR L2, all 4 LCs have CDR L3 of the same length and canonical structure, L3-9-cis7-1 (Table 2). RESULTS +35 37 L3 structure_element As with CDR L2, all 4 LCs have CDR L3 of the same length and canonical structure, L3-9-cis7-1 (Table 2). RESULTS +71 80 structure evidence As with CDR L2, all 4 LCs have CDR L3 of the same length and canonical structure, L3-9-cis7-1 (Table 2). RESULTS +82 93 L3-9-cis7-1 mutant As with CDR L2, all 4 LCs have CDR L3 of the same length and canonical structure, L3-9-cis7-1 (Table 2). RESULTS +21 24 CDR structure_element The conformations of CDR L3 for L1-39, L3-11, and particularly for L320, are not as tightly clustered as those of L4-1 (Fig. 5). RESULTS +25 27 L3 structure_element The conformations of CDR L3 for L1-39, L3-11, and particularly for L320, are not as tightly clustered as those of L4-1 (Fig. 5). RESULTS +32 37 L1-39 mutant The conformations of CDR L3 for L1-39, L3-11, and particularly for L320, are not as tightly clustered as those of L4-1 (Fig. 5). RESULTS +39 44 L3-11 mutant The conformations of CDR L3 for L1-39, L3-11, and particularly for L320, are not as tightly clustered as those of L4-1 (Fig. 5). RESULTS +114 118 L4-1 mutant The conformations of CDR L3 for L1-39, L3-11, and particularly for L320, are not as tightly clustered as those of L4-1 (Fig. 5). RESULTS +82 87 90-92 residue_range The slight conformational variability occurs in the region of amino acid residues 90-92, which is in contact with CDR H3. RESULTS +114 117 CDR structure_element The slight conformational variability occurs in the region of amino acid residues 90-92, which is in contact with CDR H3. RESULTS +118 120 H3 structure_element The slight conformational variability occurs in the region of amino acid residues 90-92, which is in contact with CDR H3. RESULTS +0 3 CDR structure_element CDR H3 conformational diversity RESULTS +4 6 H3 structure_element CDR H3 conformational diversity RESULTS +29 33 Fabs structure_element As mentioned earlier, all 16 Fabs have the same CDR H3, for which the amino acid sequence is derived from the anti-CCL2 antibody CNTO 888. RESULTS +48 51 CDR structure_element As mentioned earlier, all 16 Fabs have the same CDR H3, for which the amino acid sequence is derived from the anti-CCL2 antibody CNTO 888. RESULTS +52 54 H3 structure_element As mentioned earlier, all 16 Fabs have the same CDR H3, for which the amino acid sequence is derived from the anti-CCL2 antibody CNTO 888. RESULTS +120 128 antibody protein_type As mentioned earlier, all 16 Fabs have the same CDR H3, for which the amino acid sequence is derived from the anti-CCL2 antibody CNTO 888. RESULTS +129 137 CNTO 888 chemical As mentioned earlier, all 16 Fabs have the same CDR H3, for which the amino acid sequence is derived from the anti-CCL2 antibody CNTO 888. RESULTS +4 8 loop structure_element The loop and the 2 β-strands of the CDR H3 in this ‘parent’ structure are stabilized by H-bonds between the carbonyl oxygen and peptide nitrogen atoms in the 2 strands. RESULTS +19 28 β-strands structure_element The loop and the 2 β-strands of the CDR H3 in this ‘parent’ structure are stabilized by H-bonds between the carbonyl oxygen and peptide nitrogen atoms in the 2 strands. RESULTS +36 39 CDR structure_element The loop and the 2 β-strands of the CDR H3 in this ‘parent’ structure are stabilized by H-bonds between the carbonyl oxygen and peptide nitrogen atoms in the 2 strands. RESULTS +40 42 H3 structure_element The loop and the 2 β-strands of the CDR H3 in this ‘parent’ structure are stabilized by H-bonds between the carbonyl oxygen and peptide nitrogen atoms in the 2 strands. RESULTS +60 69 structure evidence The loop and the 2 β-strands of the CDR H3 in this ‘parent’ structure are stabilized by H-bonds between the carbonyl oxygen and peptide nitrogen atoms in the 2 strands. RESULTS +88 95 H-bonds bond_interaction The loop and the 2 β-strands of the CDR H3 in this ‘parent’ structure are stabilized by H-bonds between the carbonyl oxygen and peptide nitrogen atoms in the 2 strands. RESULTS +32 35 CDR structure_element An interesting feature of these CDR H3 structures is the presence of a water molecule that interacts with the peptide nitrogens and carbonyl oxygens near the bridging loop connecting the 2 β-strands. RESULTS +36 38 H3 structure_element An interesting feature of these CDR H3 structures is the presence of a water molecule that interacts with the peptide nitrogens and carbonyl oxygens near the bridging loop connecting the 2 β-strands. RESULTS +39 49 structures evidence An interesting feature of these CDR H3 structures is the presence of a water molecule that interacts with the peptide nitrogens and carbonyl oxygens near the bridging loop connecting the 2 β-strands. RESULTS +71 76 water chemical An interesting feature of these CDR H3 structures is the presence of a water molecule that interacts with the peptide nitrogens and carbonyl oxygens near the bridging loop connecting the 2 β-strands. RESULTS +167 171 loop structure_element An interesting feature of these CDR H3 structures is the presence of a water molecule that interacts with the peptide nitrogens and carbonyl oxygens near the bridging loop connecting the 2 β-strands. RESULTS +189 198 β-strands structure_element An interesting feature of these CDR H3 structures is the presence of a water molecule that interacts with the peptide nitrogens and carbonyl oxygens near the bridging loop connecting the 2 β-strands. RESULTS +5 10 water chemical This water is present in both the bound (4DN4) and unbound (4DN3) forms of CNTO 888. RESULTS +34 39 bound protein_state This water is present in both the bound (4DN4) and unbound (4DN3) forms of CNTO 888. RESULTS +51 58 unbound protein_state This water is present in both the bound (4DN4) and unbound (4DN3) forms of CNTO 888. RESULTS +75 83 CNTO 888 chemical This water is present in both the bound (4DN4) and unbound (4DN3) forms of CNTO 888. RESULTS +4 15 stem region structure_element The stem region of CDR H3 in the parental Fab is in a ‘kinked’ conformation, in which the indole nitrogen of Trp103 forms a hydrogen bond with the carbonyl oxygen of Leu100b. RESULTS +19 22 CDR structure_element The stem region of CDR H3 in the parental Fab is in a ‘kinked’ conformation, in which the indole nitrogen of Trp103 forms a hydrogen bond with the carbonyl oxygen of Leu100b. RESULTS +23 25 H3 structure_element The stem region of CDR H3 in the parental Fab is in a ‘kinked’ conformation, in which the indole nitrogen of Trp103 forms a hydrogen bond with the carbonyl oxygen of Leu100b. RESULTS +42 45 Fab structure_element The stem region of CDR H3 in the parental Fab is in a ‘kinked’ conformation, in which the indole nitrogen of Trp103 forms a hydrogen bond with the carbonyl oxygen of Leu100b. RESULTS +55 61 kinked protein_state The stem region of CDR H3 in the parental Fab is in a ‘kinked’ conformation, in which the indole nitrogen of Trp103 forms a hydrogen bond with the carbonyl oxygen of Leu100b. RESULTS +109 115 Trp103 residue_name_number The stem region of CDR H3 in the parental Fab is in a ‘kinked’ conformation, in which the indole nitrogen of Trp103 forms a hydrogen bond with the carbonyl oxygen of Leu100b. RESULTS +124 137 hydrogen bond bond_interaction The stem region of CDR H3 in the parental Fab is in a ‘kinked’ conformation, in which the indole nitrogen of Trp103 forms a hydrogen bond with the carbonyl oxygen of Leu100b. RESULTS +166 173 Leu100b residue_name_number The stem region of CDR H3 in the parental Fab is in a ‘kinked’ conformation, in which the indole nitrogen of Trp103 forms a hydrogen bond with the carbonyl oxygen of Leu100b. RESULTS +22 28 Asp101 residue_name_number The carboxyl group of Asp101 forms a salt bridge with Arg94. RESULTS +37 48 salt bridge bond_interaction The carboxyl group of Asp101 forms a salt bridge with Arg94. RESULTS +54 59 Arg94 residue_name_number The carboxyl group of Asp101 forms a salt bridge with Arg94. RESULTS +34 47 superposition experimental_method Ribbon representations of (A) the superposition of all CDR H3s of the structures with complete backbone traces. (B) The CDR H3s rotated 90° about the y axis of the page. FIG +55 58 CDR structure_element Ribbon representations of (A) the superposition of all CDR H3s of the structures with complete backbone traces. (B) The CDR H3s rotated 90° about the y axis of the page. FIG +59 62 H3s structure_element Ribbon representations of (A) the superposition of all CDR H3s of the structures with complete backbone traces. (B) The CDR H3s rotated 90° about the y axis of the page. FIG +70 80 structures evidence Ribbon representations of (A) the superposition of all CDR H3s of the structures with complete backbone traces. (B) The CDR H3s rotated 90° about the y axis of the page. FIG +120 123 CDR structure_element Ribbon representations of (A) the superposition of all CDR H3s of the structures with complete backbone traces. (B) The CDR H3s rotated 90° about the y axis of the page. FIG +124 127 H3s structure_element Ribbon representations of (A) the superposition of all CDR H3s of the structures with complete backbone traces. (B) The CDR H3s rotated 90° about the y axis of the page. FIG +4 13 structure evidence The structure of each CDR H3 is represented with a different color. FIG +22 25 CDR structure_element The structure of each CDR H3 is represented with a different color. FIG +26 28 H3 structure_element The structure of each CDR H3 is represented with a different color. FIG +61 64 CDR structure_element Despite having the same amino acid sequence in all variants, CDR H3 has the highest degree of structural diversity and disorder of all of the CDRs in the experimental set. RESULTS +65 67 H3 structure_element Despite having the same amino acid sequence in all variants, CDR H3 has the highest degree of structural diversity and disorder of all of the CDRs in the experimental set. RESULTS +142 146 CDRs structure_element Despite having the same amino acid sequence in all variants, CDR H3 has the highest degree of structural diversity and disorder of all of the CDRs in the experimental set. RESULTS +16 19 Fab structure_element Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop. RESULTS +20 30 structures evidence Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop. RESULTS +83 94 H5-51:L3-11 complex_assembly Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop. RESULTS +96 106 H551:L3-20 complex_assembly Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop. RESULTS +111 121 H3-23:L4-1 complex_assembly Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop. RESULTS +136 140 Fabs structure_element Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop. RESULTS +148 155 missing protein_state Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop. RESULTS +157 167 disordered protein_state Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop. RESULTS +197 205 CDR loop structure_element Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop. RESULTS +20 24 Fabs structure_element Another four of the Fabs, H3-23:L1-39, H3-53:L1-39, H3-53:L3-11 and H3-53:L4-1 have missing side-chain atoms. RESULTS +26 37 H3-23:L1-39 complex_assembly Another four of the Fabs, H3-23:L1-39, H3-53:L1-39, H3-53:L3-11 and H3-53:L4-1 have missing side-chain atoms. RESULTS +39 50 H3-53:L1-39 complex_assembly Another four of the Fabs, H3-23:L1-39, H3-53:L1-39, H3-53:L3-11 and H3-53:L4-1 have missing side-chain atoms. RESULTS +52 63 H3-53:L3-11 complex_assembly Another four of the Fabs, H3-23:L1-39, H3-53:L1-39, H3-53:L3-11 and H3-53:L4-1 have missing side-chain atoms. RESULTS +68 78 H3-53:L4-1 complex_assembly Another four of the Fabs, H3-23:L1-39, H3-53:L1-39, H3-53:L3-11 and H3-53:L4-1 have missing side-chain atoms. RESULTS +18 21 CDR structure_element The variations in CDR H3 conformation are illustrated in Fig. 6 for the 18 Fab structures that have ordered backbone atoms. RESULTS +22 24 H3 structure_element The variations in CDR H3 conformation are illustrated in Fig. 6 for the 18 Fab structures that have ordered backbone atoms. RESULTS +75 78 Fab structure_element The variations in CDR H3 conformation are illustrated in Fig. 6 for the 18 Fab structures that have ordered backbone atoms. RESULTS +79 89 structures evidence The variations in CDR H3 conformation are illustrated in Fig. 6 for the 18 Fab structures that have ordered backbone atoms. RESULTS +40 46 kinked protein_state A comparison of representatives of the “kinked” and “extended” structures. FIG +53 61 extended protein_state A comparison of representatives of the “kinked” and “extended” structures. FIG +63 73 structures evidence A comparison of representatives of the “kinked” and “extended” structures. FIG +9 15 kinked protein_state (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. FIG +17 20 CDR structure_element (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. FIG +21 23 H3 structure_element (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. FIG +27 38 H1-69:L3-11 complex_assembly (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. FIG +120 127 Leu100b residue_name_number (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. FIG +134 140 Trp103 residue_name_number (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. FIG +146 151 Arg94 residue_name_number (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. FIG +159 165 Asp101 residue_name_number (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. FIG +175 180 Arg94 residue_name_number (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. FIG +189 195 Asp101 residue_name_number (A) The “kinked” CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2. FIG +9 17 extended protein_state (B) The “extended” CDR H3 of H1-69:L3-20 with green carbon atoms and yellow dashed lines connecting the H-bond pairs for Asp101 OD1 and OD2 and Trp103 NE1. FIG +19 22 CDR structure_element (B) The “extended” CDR H3 of H1-69:L3-20 with green carbon atoms and yellow dashed lines connecting the H-bond pairs for Asp101 OD1 and OD2 and Trp103 NE1. FIG +23 25 H3 structure_element (B) The “extended” CDR H3 of H1-69:L3-20 with green carbon atoms and yellow dashed lines connecting the H-bond pairs for Asp101 OD1 and OD2 and Trp103 NE1. FIG +29 40 H1-69:L3-20 complex_assembly (B) The “extended” CDR H3 of H1-69:L3-20 with green carbon atoms and yellow dashed lines connecting the H-bond pairs for Asp101 OD1 and OD2 and Trp103 NE1. FIG +121 127 Asp101 residue_name_number (B) The “extended” CDR H3 of H1-69:L3-20 with green carbon atoms and yellow dashed lines connecting the H-bond pairs for Asp101 OD1 and OD2 and Trp103 NE1. FIG +144 150 Trp103 residue_name_number (B) The “extended” CDR H3 of H1-69:L3-20 with green carbon atoms and yellow dashed lines connecting the H-bond pairs for Asp101 OD1 and OD2 and Trp103 NE1. FIG +16 19 Fab structure_element In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +20 30 structures evidence In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +32 43 H1-69:L1-39 complex_assembly In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +45 56 H1-69:L3-11 complex_assembly In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +60 64 Fabs structure_element In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +67 77 H1-69:L4-1 complex_assembly In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +79 90 H3-23:L3-11 complex_assembly In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +94 98 Fabs structure_element In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +101 112 H3-23:L3-20 complex_assembly In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +114 125 H3-53:L3-11 complex_assembly In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +127 138 H3-53:L3-20 complex_assembly In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +143 154 H5-51:L1-39 complex_assembly In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +160 164 CDRs structure_element In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3. RESULTS +19 29 structures evidence The bases of these structures have the ‘kinked’ conformation with the H-bond between Trp103 and Leu100b. RESULTS +40 46 kinked protein_state The bases of these structures have the ‘kinked’ conformation with the H-bond between Trp103 and Leu100b. RESULTS +85 91 Trp103 residue_name_number The bases of these structures have the ‘kinked’ conformation with the H-bond between Trp103 and Leu100b. RESULTS +96 103 Leu100b residue_name_number The bases of these structures have the ‘kinked’ conformation with the H-bond between Trp103 and Leu100b. RESULTS +17 20 CDR structure_element A representative CDR H3 structure for H1-69:L1-39 illustrating this is shown in Fig. 7A. RESULTS +21 23 H3 structure_element A representative CDR H3 structure for H1-69:L1-39 illustrating this is shown in Fig. 7A. RESULTS +24 33 structure evidence A representative CDR H3 structure for H1-69:L1-39 illustrating this is shown in Fig. 7A. RESULTS +38 49 H1-69:L1-39 complex_assembly A representative CDR H3 structure for H1-69:L1-39 illustrating this is shown in Fig. 7A. RESULTS +64 69 Tyr99 residue_name_number The largest backbone conformational deviation for the set is at Tyr99, where the C=O is rotated by 90° relative to that observed in 4DN3. RESULTS +48 58 structures evidence Also, it is worth noting that only one of these structures, H1-69:L4-1, has the conserved water molecule in CDR H3 observed in the 4DN3 and 4DN4 structures. RESULTS +60 70 H1-69:L4-1 complex_assembly Also, it is worth noting that only one of these structures, H1-69:L4-1, has the conserved water molecule in CDR H3 observed in the 4DN3 and 4DN4 structures. RESULTS +80 89 conserved protein_state Also, it is worth noting that only one of these structures, H1-69:L4-1, has the conserved water molecule in CDR H3 observed in the 4DN3 and 4DN4 structures. RESULTS +90 95 water chemical Also, it is worth noting that only one of these structures, H1-69:L4-1, has the conserved water molecule in CDR H3 observed in the 4DN3 and 4DN4 structures. RESULTS +108 111 CDR structure_element Also, it is worth noting that only one of these structures, H1-69:L4-1, has the conserved water molecule in CDR H3 observed in the 4DN3 and 4DN4 structures. RESULTS +112 114 H3 structure_element Also, it is worth noting that only one of these structures, H1-69:L4-1, has the conserved water molecule in CDR H3 observed in the 4DN3 and 4DN4 structures. RESULTS +145 155 structures evidence Also, it is worth noting that only one of these structures, H1-69:L4-1, has the conserved water molecule in CDR H3 observed in the 4DN3 and 4DN4 structures. RESULTS +24 27 Fab structure_element In fact, it is the only Fab in the set that has a water molecule present at this site. RESULTS +50 55 water chemical In fact, it is the only Fab in the set that has a water molecule present at this site. RESULTS +4 7 CDR structure_element The CDR H3 for this structure is shown in Fig. S3. RESULTS +8 10 H3 structure_element The CDR H3 for this structure is shown in Fig. S3. RESULTS +20 29 structure evidence The CDR H3 for this structure is shown in Fig. S3. RESULTS +16 20 Fabs structure_element The remaining 8 Fabs can be grouped into 5 different conformational classes. RESULTS +13 17 Fabs structure_element Three of the Fabs, H3-23:L1-39, H3-23:L4-1 and H3-53:L1-39, have distinctive conformations. RESULTS +19 30 H3-23:L1-39 complex_assembly Three of the Fabs, H3-23:L1-39, H3-23:L4-1 and H3-53:L1-39, have distinctive conformations. RESULTS +32 42 H3-23:L4-1 complex_assembly Three of the Fabs, H3-23:L1-39, H3-23:L4-1 and H3-53:L1-39, have distinctive conformations. RESULTS +47 58 H3-53:L1-39 complex_assembly Three of the Fabs, H3-23:L1-39, H3-23:L4-1 and H3-53:L1-39, have distinctive conformations. RESULTS +4 16 stem regions structure_element The stem regions in these 3 cases are in the ‘kinked’ conformation consistent with that observed for 4DN3. RESULTS +46 52 kinked protein_state The stem regions in these 3 cases are in the ‘kinked’ conformation consistent with that observed for 4DN3. RESULTS +19 23 Fabs structure_element The five remaining Fabs, H5-51:L4-1 (2 copies), H1-69:L3-20 (2 copies) and H3-53:L4-1, have 3 different CDR H3 conformations (Fig. S4). RESULTS +25 35 H5-51:L4-1 complex_assembly The five remaining Fabs, H5-51:L4-1 (2 copies), H1-69:L3-20 (2 copies) and H3-53:L4-1, have 3 different CDR H3 conformations (Fig. S4). RESULTS +48 59 H1-69:L3-20 complex_assembly The five remaining Fabs, H5-51:L4-1 (2 copies), H1-69:L3-20 (2 copies) and H3-53:L4-1, have 3 different CDR H3 conformations (Fig. S4). RESULTS +75 85 H3-53:L4-1 complex_assembly The five remaining Fabs, H5-51:L4-1 (2 copies), H1-69:L3-20 (2 copies) and H3-53:L4-1, have 3 different CDR H3 conformations (Fig. S4). RESULTS +104 107 CDR structure_element The five remaining Fabs, H5-51:L4-1 (2 copies), H1-69:L3-20 (2 copies) and H3-53:L4-1, have 3 different CDR H3 conformations (Fig. S4). RESULTS +108 110 H3 structure_element The five remaining Fabs, H5-51:L4-1 (2 copies), H1-69:L3-20 (2 copies) and H3-53:L4-1, have 3 different CDR H3 conformations (Fig. S4). RESULTS +4 16 stem regions structure_element The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the ‘kinked’ conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the ‘extended’ conformation (Fig. 7B). RESULTS +20 23 CDR structure_element The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the ‘kinked’ conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the ‘extended’ conformation (Fig. 7B). RESULTS +24 26 H3 structure_element The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the ‘kinked’ conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the ‘extended’ conformation (Fig. 7B). RESULTS +35 45 H5-51:L4-1 complex_assembly The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the ‘kinked’ conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the ‘extended’ conformation (Fig. 7B). RESULTS +46 50 Fabs structure_element The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the ‘kinked’ conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the ‘extended’ conformation (Fig. 7B). RESULTS +63 69 kinked protein_state The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the ‘kinked’ conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the ‘extended’ conformation (Fig. 7B). RESULTS +118 129 H1-69:L3-20 complex_assembly The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the ‘kinked’ conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the ‘extended’ conformation (Fig. 7B). RESULTS +139 149 H3-53:L4-1 complex_assembly The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the ‘kinked’ conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the ‘extended’ conformation (Fig. 7B). RESULTS +162 170 extended protein_state The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the ‘kinked’ conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the ‘extended’ conformation (Fig. 7B). RESULTS +0 5 VH:VL complex_assembly VH:VL domain packing RESULTS +4 6 VH structure_element The VH and VL domains have a β-sandwich structure (also often referred as a Greek key motif) and each is composed of a 4-stranded and a 5-stranded antiparallel β-sheets. RESULTS +11 13 VL structure_element The VH and VL domains have a β-sandwich structure (also often referred as a Greek key motif) and each is composed of a 4-stranded and a 5-stranded antiparallel β-sheets. RESULTS +29 49 β-sandwich structure structure_element The VH and VL domains have a β-sandwich structure (also often referred as a Greek key motif) and each is composed of a 4-stranded and a 5-stranded antiparallel β-sheets. RESULTS +76 91 Greek key motif structure_element The VH and VL domains have a β-sandwich structure (also often referred as a Greek key motif) and each is composed of a 4-stranded and a 5-stranded antiparallel β-sheets. RESULTS +119 168 4-stranded and a 5-stranded antiparallel β-sheets structure_element The VH and VL domains have a β-sandwich structure (also often referred as a Greek key motif) and each is composed of a 4-stranded and a 5-stranded antiparallel β-sheets. RESULTS +44 63 5-stranded β-sheets structure_element The two domains pack together such that the 5-stranded β-sheets, which have hydrophobic surfaces, interact with each other bringing the CDRs from both the VH and VL domains into close proximity. RESULTS +136 140 CDRs structure_element The two domains pack together such that the 5-stranded β-sheets, which have hydrophobic surfaces, interact with each other bringing the CDRs from both the VH and VL domains into close proximity. RESULTS +155 157 VH structure_element The two domains pack together such that the 5-stranded β-sheets, which have hydrophobic surfaces, interact with each other bringing the CDRs from both the VH and VL domains into close proximity. RESULTS +162 164 VL structure_element The two domains pack together such that the 5-stranded β-sheets, which have hydrophobic surfaces, interact with each other bringing the CDRs from both the VH and VL domains into close proximity. RESULTS +65 81 domain interface site The domain packing of the variants was assessed by computing the domain interface interactions, the VH:VL tilt angles, the buried surface area and surface complementarity. RESULTS +100 105 VH:VL complex_assembly The domain packing of the variants was assessed by computing the domain interface interactions, the VH:VL tilt angles, the buried surface area and surface complementarity. RESULTS +106 117 tilt angles evidence The domain packing of the variants was assessed by computing the domain interface interactions, the VH:VL tilt angles, the buried surface area and surface complementarity. RESULTS +0 15 VH:VL interface site VH:VL interface amino acid residue interactions RESULTS +4 13 conserved protein_state The conserved VH:VL interactions as viewed along the VH/VL axis. FIG +14 19 VH:VL complex_assembly The conserved VH:VL interactions as viewed along the VH/VL axis. FIG +53 55 VH structure_element The conserved VH:VL interactions as viewed along the VH/VL axis. FIG +56 58 VL structure_element The conserved VH:VL interactions as viewed along the VH/VL axis. FIG +4 6 VH structure_element The VH residues are in blue, the VL residues are in orange. FIG +33 35 VL structure_element The VH residues are in blue, the VL residues are in orange. FIG +4 19 VH:VL interface site The VH:VL interface is pseudosymmetric, and involves 2 stretches of the polypeptide chain from each domain, namely CDR3 and the framework region between CDRs 1 and 2. RESULTS +23 38 pseudosymmetric protein_state The VH:VL interface is pseudosymmetric, and involves 2 stretches of the polypeptide chain from each domain, namely CDR3 and the framework region between CDRs 1 and 2. RESULTS +115 119 CDR3 structure_element The VH:VL interface is pseudosymmetric, and involves 2 stretches of the polypeptide chain from each domain, namely CDR3 and the framework region between CDRs 1 and 2. RESULTS +128 144 framework region structure_element The VH:VL interface is pseudosymmetric, and involves 2 stretches of the polypeptide chain from each domain, namely CDR3 and the framework region between CDRs 1 and 2. RESULTS +153 165 CDRs 1 and 2 structure_element The VH:VL interface is pseudosymmetric, and involves 2 stretches of the polypeptide chain from each domain, namely CDR3 and the framework region between CDRs 1 and 2. RESULTS +21 44 antiparallel β-hairpins structure_element These stretches form antiparallel β-hairpins within the internal 5-stranded β-sheet. RESULTS +65 83 5-stranded β-sheet structure_element These stretches form antiparallel β-hairpins within the internal 5-stranded β-sheet. RESULTS +110 114 Fabs structure_element There are a few principal inter-domain interactions that are conserved not only in the experimental set of 16 Fabs, but in all human antibodies. RESULTS +127 132 human species There are a few principal inter-domain interactions that are conserved not only in the experimental set of 16 Fabs, but in all human antibodies. RESULTS +133 143 antibodies protein_type There are a few principal inter-domain interactions that are conserved not only in the experimental set of 16 Fabs, but in all human antibodies. RESULTS +29 42 hydrogen bond bond_interaction They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +51 52 L structure_element They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +53 58 Gln38 residue_name_number They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +63 64 H structure_element They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +65 70 Gln39 residue_name_number They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +75 76 H structure_element They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +77 82 Leu45 residue_name_number They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +88 106 hydrophobic pocket site They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +115 116 L structure_element They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +117 122 Phe98 residue_name_number They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +124 125 L structure_element They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +126 131 Tyr87 residue_name_number They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +136 137 L structure_element They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +138 143 Pro44 residue_name_number They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +148 149 L structure_element They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +150 155 Pro44 residue_name_number They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +172 173 H structure_element They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +174 180 Trp103 residue_name_number They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +189 190 L structure_element They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +191 196 Ala43 residue_name_number They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +218 219 H structure_element They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +220 225 Tyr91 residue_name_number They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig. 8). RESULTS +22 23 L structure_element With the exception of L-Ala43, all other residues are conserved in human germlines. RESULTS +24 29 Ala43 residue_name_number With the exception of L-Ala43, all other residues are conserved in human germlines. RESULTS +67 72 human species With the exception of L-Ala43, all other residues are conserved in human germlines. RESULTS +9 11 43 residue_number Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved. RESULTS +45 48 Ser residue_name Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved. RESULTS +50 53 Val residue_name Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved. RESULTS +57 60 Pro residue_name Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved. RESULTS +68 72 L4-1 mutant Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved. RESULTS +83 106 hydrophobic interaction bond_interaction Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved. RESULTS +112 113 H structure_element Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved. RESULTS +114 119 Tyr91 residue_name_number Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved. RESULTS +56 61 VH:VL complex_assembly These core interactions provide enough stability to the VH:VL dimer so that additional VH-VL contacts can tolerate amino acid sequence variations in CDRs H3 and L3 that form part of the VH:VL interface. RESULTS +62 67 dimer oligomeric_state These core interactions provide enough stability to the VH:VL dimer so that additional VH-VL contacts can tolerate amino acid sequence variations in CDRs H3 and L3 that form part of the VH:VL interface. RESULTS +87 101 VH-VL contacts site These core interactions provide enough stability to the VH:VL dimer so that additional VH-VL contacts can tolerate amino acid sequence variations in CDRs H3 and L3 that form part of the VH:VL interface. RESULTS +149 153 CDRs structure_element These core interactions provide enough stability to the VH:VL dimer so that additional VH-VL contacts can tolerate amino acid sequence variations in CDRs H3 and L3 that form part of the VH:VL interface. RESULTS +154 156 H3 structure_element These core interactions provide enough stability to the VH:VL dimer so that additional VH-VL contacts can tolerate amino acid sequence variations in CDRs H3 and L3 that form part of the VH:VL interface. RESULTS +161 163 L3 structure_element These core interactions provide enough stability to the VH:VL dimer so that additional VH-VL contacts can tolerate amino acid sequence variations in CDRs H3 and L3 that form part of the VH:VL interface. RESULTS +186 201 VH:VL interface site These core interactions provide enough stability to the VH:VL dimer so that additional VH-VL contacts can tolerate amino acid sequence variations in CDRs H3 and L3 that form part of the VH:VL interface. RESULTS +16 27 20 residues residue_range In total, about 20 residues are involved in the VH:VL interactions on each side (Fig. S5). RESULTS +48 53 VH:VL complex_assembly In total, about 20 residues are involved in the VH:VL interactions on each side (Fig. S5). RESULTS +24 41 framework regions structure_element Half of them are in the framework regions and those residues (except residue 61 in HC, which is actually in CDR2 in Kabat's definition) are conserved in the set of 16 Fabs. RESULTS +77 79 61 residue_number Half of them are in the framework regions and those residues (except residue 61 in HC, which is actually in CDR2 in Kabat's definition) are conserved in the set of 16 Fabs. RESULTS +83 85 HC structure_element Half of them are in the framework regions and those residues (except residue 61 in HC, which is actually in CDR2 in Kabat's definition) are conserved in the set of 16 Fabs. RESULTS +108 112 CDR2 structure_element Half of them are in the framework regions and those residues (except residue 61 in HC, which is actually in CDR2 in Kabat's definition) are conserved in the set of 16 Fabs. RESULTS +167 171 Fabs structure_element Half of them are in the framework regions and those residues (except residue 61 in HC, which is actually in CDR2 in Kabat's definition) are conserved in the set of 16 Fabs. RESULTS +25 26 H structure_element One notable exception is H-Trp47, which exhibits 2 conformations of the indole ring. RESULTS +27 32 Trp47 residue_name_number One notable exception is H-Trp47, which exhibits 2 conformations of the indole ring. RESULTS +15 25 structures evidence In most of the structures, it has the χ2 angle of ∼80°, while the ring is flipped over (χ2 = −100°) in H5-51:L3:11 and H5-51:L3-20. RESULTS +38 40 χ2 evidence In most of the structures, it has the χ2 angle of ∼80°, while the ring is flipped over (χ2 = −100°) in H5-51:L3:11 and H5-51:L3-20. RESULTS +88 90 χ2 evidence In most of the structures, it has the χ2 angle of ∼80°, while the ring is flipped over (χ2 = −100°) in H5-51:L3:11 and H5-51:L3-20. RESULTS +103 114 H5-51:L3:11 complex_assembly In most of the structures, it has the χ2 angle of ∼80°, while the ring is flipped over (χ2 = −100°) in H5-51:L3:11 and H5-51:L3-20. RESULTS +119 130 H5-51:L3-20 complex_assembly In most of the structures, it has the χ2 angle of ∼80°, while the ring is flipped over (χ2 = −100°) in H5-51:L3:11 and H5-51:L3-20. RESULTS +36 46 structures evidence Interestingly, these are the only 2 structures with residues missing in CDR H3 because of disorder, although both structures are determined at high resolution and the rest of the structure is well defined. RESULTS +61 68 missing protein_state Interestingly, these are the only 2 structures with residues missing in CDR H3 because of disorder, although both structures are determined at high resolution and the rest of the structure is well defined. RESULTS +72 75 CDR structure_element Interestingly, these are the only 2 structures with residues missing in CDR H3 because of disorder, although both structures are determined at high resolution and the rest of the structure is well defined. RESULTS +76 78 H3 structure_element Interestingly, these are the only 2 structures with residues missing in CDR H3 because of disorder, although both structures are determined at high resolution and the rest of the structure is well defined. RESULTS +114 124 structures evidence Interestingly, these are the only 2 structures with residues missing in CDR H3 because of disorder, although both structures are determined at high resolution and the rest of the structure is well defined. RESULTS +179 188 structure evidence Interestingly, these are the only 2 structures with residues missing in CDR H3 because of disorder, although both structures are determined at high resolution and the rest of the structure is well defined. RESULTS +30 33 CDR structure_element Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47. RESULTS +34 36 H3 structure_element Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47. RESULTS +46 51 VH:VL complex_assembly Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47. RESULTS +87 93 stable protein_state Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47. RESULTS +110 113 CDR structure_element Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47. RESULTS +114 116 H3 structure_element Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47. RESULTS +207 208 H structure_element Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47. RESULTS +209 214 Trp47 residue_name_number Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47. RESULTS +0 5 VH:VL complex_assembly VH:VL tilt angles RESULTS +6 17 tilt angles evidence VH:VL tilt angles RESULTS +28 30 VH structure_element The relative orientation of VH and VL has been measured in a number of different ways. RESULTS +35 37 VL structure_element The relative orientation of VH and VL has been measured in a number of different ways. RESULTS +24 32 ABangles experimental_method The first approach uses ABangles, the results of which are shown in Table S2. RESULTS +9 12 LCs structure_element The four LCs all are classified as Type A because they have a proline at position 44, and the results for each orientation parameter are within the range of values of this type reported by Dunbar and co-workers. RESULTS +62 69 proline residue_name The four LCs all are classified as Type A because they have a proline at position 44, and the results for each orientation parameter are within the range of values of this type reported by Dunbar and co-workers. RESULTS +82 84 44 residue_number The four LCs all are classified as Type A because they have a proline at position 44, and the results for each orientation parameter are within the range of values of this type reported by Dunbar and co-workers. RESULTS +111 132 orientation parameter evidence The four LCs all are classified as Type A because they have a proline at position 44, and the results for each orientation parameter are within the range of values of this type reported by Dunbar and co-workers. RESULTS +48 52 Fabs structure_element In fact, the parameter values for the set of 16 Fabs are in the middle of the distribution observed for 351 non-redundant antibody structures determined at 3.0 Å resolution or better. RESULTS +122 130 antibody protein_type In fact, the parameter values for the set of 16 Fabs are in the middle of the distribution observed for 351 non-redundant antibody structures determined at 3.0 Å resolution or better. RESULTS +131 141 structures evidence In fact, the parameter values for the set of 16 Fabs are in the middle of the distribution observed for 351 non-redundant antibody structures determined at 3.0 Å resolution or better. RESULTS +22 25 HC1 structure_element The only exception is HC1, which is shifted toward smaller angles with the mean value of 70.8° as compared to the distribution centered at 72° for the entire PDB. RESULTS +41 44 CDR structure_element This probably reflects the invariance of CDR H3 in the current set as opposed to the CDR H3 diversity in the PDB. RESULTS +45 47 H3 structure_element This probably reflects the invariance of CDR H3 in the current set as opposed to the CDR H3 diversity in the PDB. RESULTS +85 88 CDR structure_element This probably reflects the invariance of CDR H3 in the current set as opposed to the CDR H3 diversity in the PDB. RESULTS +89 91 H3 structure_element This probably reflects the invariance of CDR H3 in the current set as opposed to the CDR H3 diversity in the PDB. RESULTS +39 50 tilt angles evidence The second approach used for comparing tilt angles involved computing the difference in the tilt angles between all pairs of structures. RESULTS +74 84 difference evidence The second approach used for comparing tilt angles involved computing the difference in the tilt angles between all pairs of structures. RESULTS +92 103 tilt angles evidence The second approach used for comparing tilt angles involved computing the difference in the tilt angles between all pairs of structures. RESULTS +125 135 structures evidence The second approach used for comparing tilt angles involved computing the difference in the tilt angles between all pairs of structures. RESULTS +4 14 structures evidence For structures with 2 copies of the Fab in the asymmetric unit, only one structure was used. RESULTS +36 39 Fab structure_element For structures with 2 copies of the Fab in the asymmetric unit, only one structure was used. RESULTS +73 82 structure evidence For structures with 2 copies of the Fab in the asymmetric unit, only one structure was used. RESULTS +36 40 Fabs structure_element The differences between independent Fabs in the same structure are 4.9° for H1-69:L3-20, 1.6° for H1-69:L3-11, 1.4° for H3-23:L4-1, 3.3° for H3-23:L3-11, and 2.5° for H5-51:L4-1. RESULTS +53 62 structure evidence The differences between independent Fabs in the same structure are 4.9° for H1-69:L3-20, 1.6° for H1-69:L3-11, 1.4° for H3-23:L4-1, 3.3° for H3-23:L3-11, and 2.5° for H5-51:L4-1. RESULTS +76 87 H1-69:L3-20 complex_assembly The differences between independent Fabs in the same structure are 4.9° for H1-69:L3-20, 1.6° for H1-69:L3-11, 1.4° for H3-23:L4-1, 3.3° for H3-23:L3-11, and 2.5° for H5-51:L4-1. RESULTS +98 109 H1-69:L3-11 complex_assembly The differences between independent Fabs in the same structure are 4.9° for H1-69:L3-20, 1.6° for H1-69:L3-11, 1.4° for H3-23:L4-1, 3.3° for H3-23:L3-11, and 2.5° for H5-51:L4-1. RESULTS +120 130 H3-23:L4-1 complex_assembly The differences between independent Fabs in the same structure are 4.9° for H1-69:L3-20, 1.6° for H1-69:L3-11, 1.4° for H3-23:L4-1, 3.3° for H3-23:L3-11, and 2.5° for H5-51:L4-1. RESULTS +141 152 H3-23:L3-11 complex_assembly The differences between independent Fabs in the same structure are 4.9° for H1-69:L3-20, 1.6° for H1-69:L3-11, 1.4° for H3-23:L4-1, 3.3° for H3-23:L3-11, and 2.5° for H5-51:L4-1. RESULTS +167 177 H5-51:L4-1 complex_assembly The differences between independent Fabs in the same structure are 4.9° for H1-69:L3-20, 1.6° for H1-69:L3-11, 1.4° for H3-23:L4-1, 3.3° for H3-23:L3-11, and 2.5° for H5-51:L4-1. RESULTS +22 33 H1-69:L3-20 complex_assembly With the exception of H1-69:L3-20, the angles are within the range of 2-3° as are observed in the identical structures in the PDB. RESULTS +108 118 structures evidence With the exception of H1-69:L3-20, the angles are within the range of 2-3° as are observed in the identical structures in the PDB. RESULTS +3 14 H1-69:L3-20 complex_assembly In H1-69:L3-20, one of the Fabs is substantially disordered so that part of CDR H2 (the outer β-strand, residues 55-60) is completely missing. RESULTS +27 31 Fabs structure_element In H1-69:L3-20, one of the Fabs is substantially disordered so that part of CDR H2 (the outer β-strand, residues 55-60) is completely missing. RESULTS +49 59 disordered protein_state In H1-69:L3-20, one of the Fabs is substantially disordered so that part of CDR H2 (the outer β-strand, residues 55-60) is completely missing. RESULTS +76 79 CDR structure_element In H1-69:L3-20, one of the Fabs is substantially disordered so that part of CDR H2 (the outer β-strand, residues 55-60) is completely missing. RESULTS +80 82 H2 structure_element In H1-69:L3-20, one of the Fabs is substantially disordered so that part of CDR H2 (the outer β-strand, residues 55-60) is completely missing. RESULTS +94 102 β-strand structure_element In H1-69:L3-20, one of the Fabs is substantially disordered so that part of CDR H2 (the outer β-strand, residues 55-60) is completely missing. RESULTS +113 118 55-60 residue_range In H1-69:L3-20, one of the Fabs is substantially disordered so that part of CDR H2 (the outer β-strand, residues 55-60) is completely missing. RESULTS +58 60 VH structure_element This kind of disorder may compromise the integrity of the VH domain and its interaction with the VL. RESULTS +97 99 VL structure_element This kind of disorder may compromise the integrity of the VH domain and its interaction with the VL. RESULTS +13 16 Fab structure_element Indeed, this Fab has the largest twist angle HC2 within the experimental set that exceeds the mean value by 2.5 standard deviations (Table S2). RESULTS +33 44 twist angle evidence Indeed, this Fab has the largest twist angle HC2 within the experimental set that exceeds the mean value by 2.5 standard deviations (Table S2). RESULTS +45 48 HC2 structure_element Indeed, this Fab has the largest twist angle HC2 within the experimental set that exceeds the mean value by 2.5 standard deviations (Table S2). RESULTS +79 92 superposition experimental_method An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5°) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6°). FIG +100 102 VH structure_element An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5°) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6°). FIG +118 129 H1-69:L3-20 complex_assembly An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5°) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6°). FIG +141 152 H5-51:L1-39 complex_assembly An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5°) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6°). FIG +158 160 VL structure_element An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5°) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6°). FIG +219 229 H1-69:L4-1 complex_assembly An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5°) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6°). FIG +241 252 H5-51:L1-39 complex_assembly An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5°) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6°). FIG +258 260 VL structure_element An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5°) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6°). FIG +15 20 VH:VL complex_assembly Differences in VH:VL tilt angles. TABLE +21 32 tilt angles evidence Differences in VH:VL tilt angles. TABLE +4 15 differences evidence The differences in the tilt angle are shown for all pairs of V regions in Table 3. RESULTS +23 33 tilt angle evidence The differences in the tilt angle are shown for all pairs of V regions in Table 3. RESULTS +61 70 V regions structure_element The differences in the tilt angle are shown for all pairs of V regions in Table 3. RESULTS +32 42 tilt angle evidence The smallest differences in the tilt angle are between the Fabs in isomorphous crystal forms. RESULTS +59 63 Fabs structure_element The smallest differences in the tilt angle are between the Fabs in isomorphous crystal forms. RESULTS +79 92 crystal forms evidence The smallest differences in the tilt angle are between the Fabs in isomorphous crystal forms. RESULTS +30 40 tilt angle evidence The largest deviations in the tilt angle, up to 11.0°, are found for 2 structures, H1-69:L3-20 and H3-23:L3-20, that stand out from the other Fabs. RESULTS +71 81 structures evidence The largest deviations in the tilt angle, up to 11.0°, are found for 2 structures, H1-69:L3-20 and H3-23:L3-20, that stand out from the other Fabs. RESULTS +83 94 H1-69:L3-20 complex_assembly The largest deviations in the tilt angle, up to 11.0°, are found for 2 structures, H1-69:L3-20 and H3-23:L3-20, that stand out from the other Fabs. RESULTS +99 110 H3-23:L3-20 complex_assembly The largest deviations in the tilt angle, up to 11.0°, are found for 2 structures, H1-69:L3-20 and H3-23:L3-20, that stand out from the other Fabs. RESULTS +142 146 Fabs structure_element The largest deviations in the tilt angle, up to 11.0°, are found for 2 structures, H1-69:L3-20 and H3-23:L3-20, that stand out from the other Fabs. RESULTS +13 23 structures evidence One of the 2 structures, H1-69:L3-20, has its CDR H3 in the ‘extended’ conformation; the other structure has it in the ‘kinked’ conformation. RESULTS +25 36 H1-69:L3-20 complex_assembly One of the 2 structures, H1-69:L3-20, has its CDR H3 in the ‘extended’ conformation; the other structure has it in the ‘kinked’ conformation. RESULTS +46 49 CDR structure_element One of the 2 structures, H1-69:L3-20, has its CDR H3 in the ‘extended’ conformation; the other structure has it in the ‘kinked’ conformation. RESULTS +50 52 H3 structure_element One of the 2 structures, H1-69:L3-20, has its CDR H3 in the ‘extended’ conformation; the other structure has it in the ‘kinked’ conformation. RESULTS +61 69 extended protein_state One of the 2 structures, H1-69:L3-20, has its CDR H3 in the ‘extended’ conformation; the other structure has it in the ‘kinked’ conformation. RESULTS +120 126 kinked protein_state One of the 2 structures, H1-69:L3-20, has its CDR H3 in the ‘extended’ conformation; the other structure has it in the ‘kinked’ conformation. RESULTS +76 87 tilt angles evidence Two examples illustrating large (10.5°) and small (1.6°) differences in the tilt angles are shown in Fig. 9. RESULTS +0 5 VH:VL complex_assembly VH:VL buried surface area and complementarity RESULTS +0 5 VH:VL complex_assembly VH:VL surface areas and surface complementarity. TABLE +25 28 CDR structure_element Some side chain atoms in CDR H3 are missing. TABLE +29 31 H3 structure_element Some side chain atoms in CDR H3 are missing. TABLE +12 15 CDR structure_element Residues in CDR H3 are missing: YGE in H5-51:L3-11, GIY in H5-51:L3-20. TABLE +16 18 H3 structure_element Residues in CDR H3 are missing: YGE in H5-51:L3-11, GIY in H5-51:L3-20. TABLE +32 35 YGE structure_element Residues in CDR H3 are missing: YGE in H5-51:L3-11, GIY in H5-51:L3-20. TABLE +39 50 H5-51:L3-11 complex_assembly Residues in CDR H3 are missing: YGE in H5-51:L3-11, GIY in H5-51:L3-20. TABLE +52 55 GIY structure_element Residues in CDR H3 are missing: YGE in H5-51:L3-11, GIY in H5-51:L3-20. TABLE +59 70 H5-51:L3-20 complex_assembly Residues in CDR H3 are missing: YGE in H5-51:L3-11, GIY in H5-51:L3-20. TABLE +19 23 PISA experimental_method The results of the PISA contact surface calculation and surface complementarity calculation are shown in Table 4. RESULTS +24 51 contact surface calculation experimental_method The results of the PISA contact surface calculation and surface complementarity calculation are shown in Table 4. RESULTS +56 91 surface complementarity calculation experimental_method The results of the PISA contact surface calculation and surface complementarity calculation are shown in Table 4. RESULTS +4 13 interface site The interface areas are calculated as the average of the VH and VL contact surfaces. RESULTS +57 83 VH and VL contact surfaces site The interface areas are calculated as the average of the VH and VL contact surfaces. RESULTS +14 24 structures evidence Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness). RESULTS +30 33 CDR structure_element Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness). RESULTS +34 36 H3 structure_element Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness). RESULTS +70 77 missing protein_state Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness). RESULTS +99 109 interfaces site Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness). RESULTS +148 158 structures evidence Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness). RESULTS +164 172 complete protein_state Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness). RESULTS +173 177 CDRs structure_element Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness). RESULTS +212 216 Fabs structure_element Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness). RESULTS +10 18 complete protein_state Among the complete structures, the interface areas range from 684 to 836 Å2. RESULTS +19 29 structures evidence Among the complete structures, the interface areas range from 684 to 836 Å2. RESULTS +35 44 interface site Among the complete structures, the interface areas range from 684 to 836 Å2. RESULTS +21 31 structures evidence Interestingly, the 2 structures that have the largest tilt angle differences with the other variants, H3-23:L3-20 and H1-69:L3-20, have the smallest VH:VL interfaces, 684 and 725 Å2, respectively. RESULTS +54 76 tilt angle differences evidence Interestingly, the 2 structures that have the largest tilt angle differences with the other variants, H3-23:L3-20 and H1-69:L3-20, have the smallest VH:VL interfaces, 684 and 725 Å2, respectively. RESULTS +102 113 H3-23:L3-20 complex_assembly Interestingly, the 2 structures that have the largest tilt angle differences with the other variants, H3-23:L3-20 and H1-69:L3-20, have the smallest VH:VL interfaces, 684 and 725 Å2, respectively. RESULTS +118 129 H1-69:L3-20 complex_assembly Interestingly, the 2 structures that have the largest tilt angle differences with the other variants, H3-23:L3-20 and H1-69:L3-20, have the smallest VH:VL interfaces, 684 and 725 Å2, respectively. RESULTS +149 165 VH:VL interfaces site Interestingly, the 2 structures that have the largest tilt angle differences with the other variants, H3-23:L3-20 and H1-69:L3-20, have the smallest VH:VL interfaces, 684 and 725 Å2, respectively. RESULTS +0 11 H3-23:L3-20 complex_assembly H3-23:L3-20 is also unique in that it has the lowest value (0.676) of surface complementarity. RESULTS +70 93 surface complementarity evidence H3-23:L3-20 is also unique in that it has the lowest value (0.676) of surface complementarity. RESULTS +0 20 Melting temperatures evidence Melting temperatures for the 16 Fabs. TABLE +32 36 Fabs structure_element Melting temperatures for the 16 Fabs. TABLE +14 16 Tm evidence Colors: blue (Tm < 70°C), green (70°C < Tm < 73°C), yellow (73°C < Tm < 78°C), orange (Tm > 78°C). TABLE +40 42 Tm evidence Colors: blue (Tm < 70°C), green (70°C < Tm < 73°C), yellow (73°C < Tm < 78°C), orange (Tm > 78°C). TABLE +67 69 Tm evidence Colors: blue (Tm < 70°C), green (70°C < Tm < 73°C), yellow (73°C < Tm < 78°C), orange (Tm > 78°C). TABLE +87 89 Tm evidence Colors: blue (Tm < 70°C), green (70°C < Tm < 73°C), yellow (73°C < Tm < 78°C), orange (Tm > 78°C). TABLE +0 20 Melting temperatures evidence Melting temperatures (Tm) were measured for all Fabs using differential scanning calorimetry (Table 5). RESULTS +22 24 Tm evidence Melting temperatures (Tm) were measured for all Fabs using differential scanning calorimetry (Table 5). RESULTS +48 52 Fabs structure_element Melting temperatures (Tm) were measured for all Fabs using differential scanning calorimetry (Table 5). RESULTS +59 92 differential scanning calorimetry experimental_method Melting temperatures (Tm) were measured for all Fabs using differential scanning calorimetry (Table 5). RESULTS +31 33 LC structure_element It appears that for each given LC, the Fabs with germlines H1-69 and H3-23 are substantially more stable than those with germlines H3-53 and H5-51. RESULTS +39 43 Fabs structure_element It appears that for each given LC, the Fabs with germlines H1-69 and H3-23 are substantially more stable than those with germlines H3-53 and H5-51. RESULTS +59 64 H1-69 mutant It appears that for each given LC, the Fabs with germlines H1-69 and H3-23 are substantially more stable than those with germlines H3-53 and H5-51. RESULTS +69 74 H3-23 mutant It appears that for each given LC, the Fabs with germlines H1-69 and H3-23 are substantially more stable than those with germlines H3-53 and H5-51. RESULTS +98 104 stable protein_state It appears that for each given LC, the Fabs with germlines H1-69 and H3-23 are substantially more stable than those with germlines H3-53 and H5-51. RESULTS +131 136 H3-53 mutant It appears that for each given LC, the Fabs with germlines H1-69 and H3-23 are substantially more stable than those with germlines H3-53 and H5-51. RESULTS +141 146 H5-51 mutant It appears that for each given LC, the Fabs with germlines H1-69 and H3-23 are substantially more stable than those with germlines H3-53 and H5-51. RESULTS +13 18 L1-39 mutant In addition, L1-39 provides a much higher degree of stabilization than the other 3 LC germlines when combined with any of the HCs. RESULTS +83 85 LC structure_element In addition, L1-39 provides a much higher degree of stabilization than the other 3 LC germlines when combined with any of the HCs. RESULTS +126 129 HCs structure_element In addition, L1-39 provides a much higher degree of stabilization than the other 3 LC germlines when combined with any of the HCs. RESULTS +17 19 Tm evidence As a result, the Tm for pairs H1-69:L1-39 and H3-23:L1-39 is 12-13° higher than for pairs H3-53:L3-20, H3-53:L4-1, H5-51:L3-20 and H5-51:L4-1. RESULTS +30 41 H1-69:L1-39 complex_assembly As a result, the Tm for pairs H1-69:L1-39 and H3-23:L1-39 is 12-13° higher than for pairs H3-53:L3-20, H3-53:L4-1, H5-51:L3-20 and H5-51:L4-1. RESULTS +46 57 H3-23:L1-39 complex_assembly As a result, the Tm for pairs H1-69:L1-39 and H3-23:L1-39 is 12-13° higher than for pairs H3-53:L3-20, H3-53:L4-1, H5-51:L3-20 and H5-51:L4-1. RESULTS +90 101 H3-53:L3-20 complex_assembly As a result, the Tm for pairs H1-69:L1-39 and H3-23:L1-39 is 12-13° higher than for pairs H3-53:L3-20, H3-53:L4-1, H5-51:L3-20 and H5-51:L4-1. RESULTS +103 113 H3-53:L4-1 complex_assembly As a result, the Tm for pairs H1-69:L1-39 and H3-23:L1-39 is 12-13° higher than for pairs H3-53:L3-20, H3-53:L4-1, H5-51:L3-20 and H5-51:L4-1. RESULTS +115 126 H5-51:L3-20 complex_assembly As a result, the Tm for pairs H1-69:L1-39 and H3-23:L1-39 is 12-13° higher than for pairs H3-53:L3-20, H3-53:L4-1, H5-51:L3-20 and H5-51:L4-1. RESULTS +131 141 H5-51:L4-1 complex_assembly As a result, the Tm for pairs H1-69:L1-39 and H3-23:L1-39 is 12-13° higher than for pairs H3-53:L3-20, H3-53:L4-1, H5-51:L3-20 and H5-51:L4-1. RESULTS +89 107 crystal structures evidence These findings correlate well with the degree of conformational disorder observed in the crystal structures. RESULTS +9 12 CDR structure_element Parts of CDR H3 main chain are completely disordered, and were not modeled in Fabs H5-51:L3-20 and H5-51:L3-11 that have the lowest Tms in the set. RESULTS +13 15 H3 structure_element Parts of CDR H3 main chain are completely disordered, and were not modeled in Fabs H5-51:L3-20 and H5-51:L3-11 that have the lowest Tms in the set. RESULTS +42 52 disordered protein_state Parts of CDR H3 main chain are completely disordered, and were not modeled in Fabs H5-51:L3-20 and H5-51:L3-11 that have the lowest Tms in the set. RESULTS +78 82 Fabs structure_element Parts of CDR H3 main chain are completely disordered, and were not modeled in Fabs H5-51:L3-20 and H5-51:L3-11 that have the lowest Tms in the set. RESULTS +83 94 H5-51:L3-20 complex_assembly Parts of CDR H3 main chain are completely disordered, and were not modeled in Fabs H5-51:L3-20 and H5-51:L3-11 that have the lowest Tms in the set. RESULTS +99 110 H5-51:L3-11 complex_assembly Parts of CDR H3 main chain are completely disordered, and were not modeled in Fabs H5-51:L3-20 and H5-51:L3-11 that have the lowest Tms in the set. RESULTS +132 135 Tms evidence Parts of CDR H3 main chain are completely disordered, and were not modeled in Fabs H5-51:L3-20 and H5-51:L3-11 that have the lowest Tms in the set. RESULTS +3 19 electron density evidence No electron density is observed for a number of side chains in CDRs H3 and L3 in all Fabs with germline H3-53, which indicates loose packing of the variable domains. RESULTS +63 67 CDRs structure_element No electron density is observed for a number of side chains in CDRs H3 and L3 in all Fabs with germline H3-53, which indicates loose packing of the variable domains. RESULTS +68 70 H3 structure_element No electron density is observed for a number of side chains in CDRs H3 and L3 in all Fabs with germline H3-53, which indicates loose packing of the variable domains. RESULTS +75 77 L3 structure_element No electron density is observed for a number of side chains in CDRs H3 and L3 in all Fabs with germline H3-53, which indicates loose packing of the variable domains. RESULTS +85 89 Fabs structure_element No electron density is observed for a number of side chains in CDRs H3 and L3 in all Fabs with germline H3-53, which indicates loose packing of the variable domains. RESULTS +104 109 H3-53 mutant No electron density is observed for a number of side chains in CDRs H3 and L3 in all Fabs with germline H3-53, which indicates loose packing of the variable domains. RESULTS +148 164 variable domains structure_element No electron density is observed for a number of side chains in CDRs H3 and L3 in all Fabs with germline H3-53, which indicates loose packing of the variable domains. RESULTS +74 77 Tms evidence All those molecules are relatively unstable, as is reflected in their low Tms. RESULTS +30 65 systematic structural investigation experimental_method This is the first report of a systematic structural investigation of a phage germline library. DISCUSS +71 93 phage germline library experimental_method This is the first report of a systematic structural investigation of a phage germline library. DISCUSS +7 10 Fab structure_element The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +11 21 structures evidence The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +73 76 HCs structure_element The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +78 83 H1-69 mutant The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +85 90 H3-23 mutant The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +92 97 H3-53 mutant The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +103 108 H5-51 mutant The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +126 129 LCs structure_element The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +131 136 L1-39 mutant The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +138 143 L3-11 mutant The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +145 150 L3-20 mutant The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +155 159 L4-1 mutant The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +180 183 CDR structure_element The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +184 186 H3 structure_element The 16 Fab structures offer a unique look at all pairings of 4 different HCs (H1-69, H3-23, H3-53, and H5-51) and 4 different LCs (L1-39, L3-11, L3-20 and L4-1), all with the same CDR H3. DISCUSS +4 19 structural data evidence The structural data set taken as a whole provides insight into how the backbone conformations of the CDRs of a specific heavy or light chain vary when it is paired with 4 different light or heavy chains, respectively. DISCUSS +101 105 CDRs structure_element The structural data set taken as a whole provides insight into how the backbone conformations of the CDRs of a specific heavy or light chain vary when it is paired with 4 different light or heavy chains, respectively. DISCUSS +129 140 light chain structure_element The structural data set taken as a whole provides insight into how the backbone conformations of the CDRs of a specific heavy or light chain vary when it is paired with 4 different light or heavy chains, respectively. DISCUSS +190 202 heavy chains structure_element The structural data set taken as a whole provides insight into how the backbone conformations of the CDRs of a specific heavy or light chain vary when it is paired with 4 different light or heavy chains, respectively. DISCUSS +27 30 CDR structure_element A large variability in the CDR conformations for the sets of HCs and LCs is observed. DISCUSS +61 64 HCs structure_element A large variability in the CDR conformations for the sets of HCs and LCs is observed. DISCUSS +69 72 LCs structure_element A large variability in the CDR conformations for the sets of HCs and LCs is observed. DISCUSS +18 21 CDR structure_element In some cases the CDR conformations for all members of a set are virtually identical, for others subtle changes occur in a few members of a set, and in some cases larger deviations are observed within a set. DISCUSS +23 35 crystallized experimental_method The five variants that crystallized with 2 copies of the Fab in the asymmetric unit serve somewhat as controls for the influence of crystal packing on the conformations of the CDRs. DISCUSS +57 60 Fab structure_element The five variants that crystallized with 2 copies of the Fab in the asymmetric unit serve somewhat as controls for the influence of crystal packing on the conformations of the CDRs. DISCUSS +176 180 CDRs structure_element The five variants that crystallized with 2 copies of the Fab in the asymmetric unit serve somewhat as controls for the influence of crystal packing on the conformations of the CDRs. DISCUSS +17 27 structures evidence In four of the 5 structures the CDR conformations are consistent. DISCUSS +32 35 CDR structure_element In four of the 5 structures the CDR conformations are consistent. DISCUSS +26 37 H1-69:L3-20 complex_assembly In only one case, that of H1-69:L3-20 (the lowest resolution structure), do we see differences in the conformations of the 2 copies of CDRs H1 and L1. DISCUSS +61 70 structure evidence In only one case, that of H1-69:L3-20 (the lowest resolution structure), do we see differences in the conformations of the 2 copies of CDRs H1 and L1. DISCUSS +135 139 CDRs structure_element In only one case, that of H1-69:L3-20 (the lowest resolution structure), do we see differences in the conformations of the 2 copies of CDRs H1 and L1. DISCUSS +140 142 H1 structure_element In only one case, that of H1-69:L3-20 (the lowest resolution structure), do we see differences in the conformations of the 2 copies of CDRs H1 and L1. DISCUSS +147 149 L1 structure_element In only one case, that of H1-69:L3-20 (the lowest resolution structure), do we see differences in the conformations of the 2 copies of CDRs H1 and L1. DISCUSS +111 126 variable domain structure_element This variability is likely a result of 2 factors, crystal packing interactions and internal instability of the variable domain. DISCUSS +8 12 CDRs structure_element For the CDRs with canonical structures, the largest changes in conformation occur for CDR H1 of H1-69 and H3-53. DISCUSS +86 89 CDR structure_element For the CDRs with canonical structures, the largest changes in conformation occur for CDR H1 of H1-69 and H3-53. DISCUSS +90 92 H1 structure_element For the CDRs with canonical structures, the largest changes in conformation occur for CDR H1 of H1-69 and H3-53. DISCUSS +96 101 H1-69 mutant For the CDRs with canonical structures, the largest changes in conformation occur for CDR H1 of H1-69 and H3-53. DISCUSS +106 111 H3-53 mutant For the CDRs with canonical structures, the largest changes in conformation occur for CDR H1 of H1-69 and H3-53. DISCUSS +12 15 HCs structure_element The other 2 HCs, H3-23 and H5-51, have canonical structures that are remarkably well conserved (Fig. 1). DISCUSS +17 22 H3-23 mutant The other 2 HCs, H3-23 and H5-51, have canonical structures that are remarkably well conserved (Fig. 1). DISCUSS +27 32 H5-51 mutant The other 2 HCs, H3-23 and H5-51, have canonical structures that are remarkably well conserved (Fig. 1). DISCUSS +69 94 remarkably well conserved protein_state The other 2 HCs, H3-23 and H5-51, have canonical structures that are remarkably well conserved (Fig. 1). DISCUSS +9 12 HCs structure_element Of the 4 HCs, H1-69 has the greatest number of canonical structure assignments (Table 2). DISCUSS +14 19 H1-69 mutant Of the 4 HCs, H1-69 has the greatest number of canonical structure assignments (Table 2). DISCUSS +0 5 H1-69 mutant H1-69 is unique in having a pair of glycine residues at positions 26 and 27, which provide more conformational freedom in CDR H1. DISCUSS +36 43 glycine residue_name H1-69 is unique in having a pair of glycine residues at positions 26 and 27, which provide more conformational freedom in CDR H1. DISCUSS +66 68 26 residue_number H1-69 is unique in having a pair of glycine residues at positions 26 and 27, which provide more conformational freedom in CDR H1. DISCUSS +73 75 27 residue_number H1-69 is unique in having a pair of glycine residues at positions 26 and 27, which provide more conformational freedom in CDR H1. DISCUSS +96 118 conformational freedom protein_state H1-69 is unique in having a pair of glycine residues at positions 26 and 27, which provide more conformational freedom in CDR H1. DISCUSS +122 125 CDR structure_element H1-69 is unique in having a pair of glycine residues at positions 26 and 27, which provide more conformational freedom in CDR H1. DISCUSS +126 128 H1 structure_element H1-69 is unique in having a pair of glycine residues at positions 26 and 27, which provide more conformational freedom in CDR H1. DISCUSS +8 16 IGHV1-69 mutant Besides IGHV1-69, only the germlines of the VH4 family possess double glycines in CDR H1, and it will be interesting to see if they are also conformationally unstable. DISCUSS +44 47 VH4 structure_element Besides IGHV1-69, only the germlines of the VH4 family possess double glycines in CDR H1, and it will be interesting to see if they are also conformationally unstable. DISCUSS +70 78 glycines residue_name Besides IGHV1-69, only the germlines of the VH4 family possess double glycines in CDR H1, and it will be interesting to see if they are also conformationally unstable. DISCUSS +82 85 CDR structure_element Besides IGHV1-69, only the germlines of the VH4 family possess double glycines in CDR H1, and it will be interesting to see if they are also conformationally unstable. DISCUSS +86 88 H1 structure_element Besides IGHV1-69, only the germlines of the VH4 family possess double glycines in CDR H1, and it will be interesting to see if they are also conformationally unstable. DISCUSS +141 166 conformationally unstable protein_state Besides IGHV1-69, only the germlines of the VH4 family possess double glycines in CDR H1, and it will be interesting to see if they are also conformationally unstable. DISCUSS +14 19 VH:VL complex_assembly Having all 16 VH:VL pairs with the same CDR H3 provides some insights into why molecular modeling efforts of CDR H3 have proven so difficult. DISCUSS +40 43 CDR structure_element Having all 16 VH:VL pairs with the same CDR H3 provides some insights into why molecular modeling efforts of CDR H3 have proven so difficult. DISCUSS +44 46 H3 structure_element Having all 16 VH:VL pairs with the same CDR H3 provides some insights into why molecular modeling efforts of CDR H3 have proven so difficult. DISCUSS +109 112 CDR structure_element Having all 16 VH:VL pairs with the same CDR H3 provides some insights into why molecular modeling efforts of CDR H3 have proven so difficult. DISCUSS +113 115 H3 structure_element Having all 16 VH:VL pairs with the same CDR H3 provides some insights into why molecular modeling efforts of CDR H3 have proven so difficult. DISCUSS +69 73 Fabs structure_element As mentioned in the Results section, this data set is composed of 21 Fabs, since 5 of the 16 variants have 2 Fab copies in the asymmetric unit. DISCUSS +109 112 Fab structure_element As mentioned in the Results section, this data set is composed of 21 Fabs, since 5 of the 16 variants have 2 Fab copies in the asymmetric unit. DISCUSS +11 15 Fabs structure_element For the 18 Fabs with complete backbone atoms for CDR H3, 10 have conformations similar to that of the parent, while the others have significantly different conformations (Fig. 6). DISCUSS +49 52 CDR structure_element For the 18 Fabs with complete backbone atoms for CDR H3, 10 have conformations similar to that of the parent, while the others have significantly different conformations (Fig. 6). DISCUSS +53 55 H3 structure_element For the 18 Fabs with complete backbone atoms for CDR H3, 10 have conformations similar to that of the parent, while the others have significantly different conformations (Fig. 6). DISCUSS +28 31 CDR structure_element Thus, it is likely that the CDR H3 conformation is dependent upon 2 dominating factors: 1) amino acid sequence; and 2) VH and VL context. DISCUSS +32 34 H3 structure_element Thus, it is likely that the CDR H3 conformation is dependent upon 2 dominating factors: 1) amino acid sequence; and 2) VH and VL context. DISCUSS +119 121 VH structure_element Thus, it is likely that the CDR H3 conformation is dependent upon 2 dominating factors: 1) amino acid sequence; and 2) VH and VL context. DISCUSS +126 128 VL structure_element Thus, it is likely that the CDR H3 conformation is dependent upon 2 dominating factors: 1) amino acid sequence; and 2) VH and VL context. DISCUSS +103 108 VH:VL complex_assembly More than half of the variants retain the conformation of the parent despite having differences in the VH:VL pairing. DISCUSS +23 33 structures evidence This subset includes 2 structures with 2 copies of the Fab in the asymmetric unit, all of which are nearly identical in conformation. DISCUSS +55 58 Fab structure_element This subset includes 2 structures with 2 copies of the Fab in the asymmetric unit, all of which are nearly identical in conformation. DISCUSS +16 26 structures evidence The remaining 8 structures exhibit “non-parental” conformations, indicating that the VH and VL context can also be a dominating factor influencing CDR H3. DISCUSS +85 87 VH structure_element The remaining 8 structures exhibit “non-parental” conformations, indicating that the VH and VL context can also be a dominating factor influencing CDR H3. DISCUSS +92 94 VL structure_element The remaining 8 structures exhibit “non-parental” conformations, indicating that the VH and VL context can also be a dominating factor influencing CDR H3. DISCUSS +147 150 CDR structure_element The remaining 8 structures exhibit “non-parental” conformations, indicating that the VH and VL context can also be a dominating factor influencing CDR H3. DISCUSS +151 153 H3 structure_element The remaining 8 structures exhibit “non-parental” conformations, indicating that the VH and VL context can also be a dominating factor influencing CDR H3. DISCUSS +23 33 structures evidence This subset also has 2 structures with 2 Fab copies in the asymmetric unit. DISCUSS +41 44 Fab structure_element This subset also has 2 structures with 2 Fab copies in the asymmetric unit. DISCUSS +65 77 stem regions structure_element Interestingly, as described earlier, these 2 pairs differ in the stem regions with the H1-69:L3-20 pair in the ‘extended’ conformation and H5-51:L4-1 pair in the ‘kinked’ conformation. DISCUSS +87 98 H1-69:L3-20 complex_assembly Interestingly, as described earlier, these 2 pairs differ in the stem regions with the H1-69:L3-20 pair in the ‘extended’ conformation and H5-51:L4-1 pair in the ‘kinked’ conformation. DISCUSS +112 120 extended protein_state Interestingly, as described earlier, these 2 pairs differ in the stem regions with the H1-69:L3-20 pair in the ‘extended’ conformation and H5-51:L4-1 pair in the ‘kinked’ conformation. DISCUSS +139 149 H5-51:L4-1 complex_assembly Interestingly, as described earlier, these 2 pairs differ in the stem regions with the H1-69:L3-20 pair in the ‘extended’ conformation and H5-51:L4-1 pair in the ‘kinked’ conformation. DISCUSS +163 169 kinked protein_state Interestingly, as described earlier, these 2 pairs differ in the stem regions with the H1-69:L3-20 pair in the ‘extended’ conformation and H5-51:L4-1 pair in the ‘kinked’ conformation. DISCUSS +4 7 CDR structure_element The CDR H3 conformational analysis shows that, for each set of variants of one HC paired with the 4 different LCs, both “parental” and “non-parental” conformations are observed. DISCUSS +8 10 H3 structure_element The CDR H3 conformational analysis shows that, for each set of variants of one HC paired with the 4 different LCs, both “parental” and “non-parental” conformations are observed. DISCUSS +11 34 conformational analysis experimental_method The CDR H3 conformational analysis shows that, for each set of variants of one HC paired with the 4 different LCs, both “parental” and “non-parental” conformations are observed. DISCUSS +79 81 HC structure_element The CDR H3 conformational analysis shows that, for each set of variants of one HC paired with the 4 different LCs, both “parental” and “non-parental” conformations are observed. DISCUSS +110 113 LCs structure_element The CDR H3 conformational analysis shows that, for each set of variants of one HC paired with the 4 different LCs, both “parental” and “non-parental” conformations are observed. DISCUSS +74 76 LC structure_element The same variability is observed for the sets of variants composed of one LC paired with each of the 4 HCs. DISCUSS +103 106 HCs structure_element The same variability is observed for the sets of variants composed of one LC paired with each of the 4 HCs. DISCUSS +64 66 HC structure_element Thus, no patterns of conformational preference for a particular HC or LC emerge to shed any direct light on what drives the conformational differences. DISCUSS +70 72 LC structure_element Thus, no patterns of conformational preference for a particular HC or LC emerge to shed any direct light on what drives the conformational differences. DISCUSS +65 67 H3 structure_element This finding supports the hypothesis of Weitzner et al. that the H3 conformation is controlled both by its sequence and its environment. DISCUSS +49 59 tilt angle evidence In looking at a possible correlation between the tilt angle and the conformation of CDR H3, no clear trends are observed. DISCUSS +84 87 CDR structure_element In looking at a possible correlation between the tilt angle and the conformation of CDR H3, no clear trends are observed. DISCUSS +88 90 H3 structure_element In looking at a possible correlation between the tilt angle and the conformation of CDR H3, no clear trends are observed. DISCUSS +14 25 H1-69:L3-20 complex_assembly Two variants, H1-69:L3-20 and H3-23:L3-20, have the largest differences in the tilt angles compared to other variants as seen in Table 3. DISCUSS +30 41 H3-23:L3-20 complex_assembly Two variants, H1-69:L3-20 and H3-23:L3-20, have the largest differences in the tilt angles compared to other variants as seen in Table 3. DISCUSS +13 18 VH:VL complex_assembly The absolute VH:VL orientation parameters for the 2 Fabs (Table S2) show significant deviation in HL, LC1 and HC2 values (2-3 standard deviations from the mean). DISCUSS +19 41 orientation parameters evidence The absolute VH:VL orientation parameters for the 2 Fabs (Table S2) show significant deviation in HL, LC1 and HC2 values (2-3 standard deviations from the mean). DISCUSS +52 56 Fabs structure_element The absolute VH:VL orientation parameters for the 2 Fabs (Table S2) show significant deviation in HL, LC1 and HC2 values (2-3 standard deviations from the mean). DISCUSS +85 94 deviation evidence The absolute VH:VL orientation parameters for the 2 Fabs (Table S2) show significant deviation in HL, LC1 and HC2 values (2-3 standard deviations from the mean). DISCUSS +98 100 HL structure_element The absolute VH:VL orientation parameters for the 2 Fabs (Table S2) show significant deviation in HL, LC1 and HC2 values (2-3 standard deviations from the mean). DISCUSS +102 105 LC1 structure_element The absolute VH:VL orientation parameters for the 2 Fabs (Table S2) show significant deviation in HL, LC1 and HC2 values (2-3 standard deviations from the mean). DISCUSS +110 113 HC2 structure_element The absolute VH:VL orientation parameters for the 2 Fabs (Table S2) show significant deviation in HL, LC1 and HC2 values (2-3 standard deviations from the mean). DISCUSS +21 32 H3-23:L3-20 complex_assembly One of the variants, H3-23:L3-20, has the CDR H3 conformation similar to the parent, but the other, H1-69:L3-20, is different. DISCUSS +42 45 CDR structure_element One of the variants, H3-23:L3-20, has the CDR H3 conformation similar to the parent, but the other, H1-69:L3-20, is different. DISCUSS +46 48 H3 structure_element One of the variants, H3-23:L3-20, has the CDR H3 conformation similar to the parent, but the other, H1-69:L3-20, is different. DISCUSS +100 111 H1-69:L3-20 complex_assembly One of the variants, H3-23:L3-20, has the CDR H3 conformation similar to the parent, but the other, H1-69:L3-20, is different. DISCUSS +49 60 H1-69:L3-20 complex_assembly As noted in the Results section, the 2 variants, H1-69:L3-20 and H3-23:L3-20, are outliers in terms of the tilt angle; at the same time, both have the smallest VH:VL interface. DISCUSS +65 76 H3-23:L3-20 complex_assembly As noted in the Results section, the 2 variants, H1-69:L3-20 and H3-23:L3-20, are outliers in terms of the tilt angle; at the same time, both have the smallest VH:VL interface. DISCUSS +107 117 tilt angle evidence As noted in the Results section, the 2 variants, H1-69:L3-20 and H3-23:L3-20, are outliers in terms of the tilt angle; at the same time, both have the smallest VH:VL interface. DISCUSS +160 175 VH:VL interface site As noted in the Results section, the 2 variants, H1-69:L3-20 and H3-23:L3-20, are outliers in terms of the tilt angle; at the same time, both have the smallest VH:VL interface. DISCUSS +14 24 interfaces site These smaller interfaces may perhaps translate to a significant deviation in how VH is oriented relative to VL than the other variants. DISCUSS +81 83 VH structure_element These smaller interfaces may perhaps translate to a significant deviation in how VH is oriented relative to VL than the other variants. DISCUSS +108 110 VL structure_element These smaller interfaces may perhaps translate to a significant deviation in how VH is oriented relative to VL than the other variants. DISCUSS +76 81 VH:VL complex_assembly These deviations from the other variants can also be seen to some extent in VH:VL orientation parameters in Table S2, as well as in the smaller number of residues involved in the VH:VL interfaces of these 2 variants (Fig. S5). DISCUSS +179 195 VH:VL interfaces site These deviations from the other variants can also be seen to some extent in VH:VL orientation parameters in Table S2, as well as in the smaller number of residues involved in the VH:VL interfaces of these 2 variants (Fig. S5). DISCUSS +64 68 CDRs structure_element These differences undoubtedly influence the conformation of the CDRs, in particular CDR H1 (Fig. 1A) and CDR L1 (Fig. 3C), especially with the tandem glycines and multiple serines present, respectively. DISCUSS +84 87 CDR structure_element These differences undoubtedly influence the conformation of the CDRs, in particular CDR H1 (Fig. 1A) and CDR L1 (Fig. 3C), especially with the tandem glycines and multiple serines present, respectively. DISCUSS +88 90 H1 structure_element These differences undoubtedly influence the conformation of the CDRs, in particular CDR H1 (Fig. 1A) and CDR L1 (Fig. 3C), especially with the tandem glycines and multiple serines present, respectively. DISCUSS +105 108 CDR structure_element These differences undoubtedly influence the conformation of the CDRs, in particular CDR H1 (Fig. 1A) and CDR L1 (Fig. 3C), especially with the tandem glycines and multiple serines present, respectively. DISCUSS +109 111 L1 structure_element These differences undoubtedly influence the conformation of the CDRs, in particular CDR H1 (Fig. 1A) and CDR L1 (Fig. 3C), especially with the tandem glycines and multiple serines present, respectively. DISCUSS +150 158 glycines residue_name These differences undoubtedly influence the conformation of the CDRs, in particular CDR H1 (Fig. 1A) and CDR L1 (Fig. 3C), especially with the tandem glycines and multiple serines present, respectively. DISCUSS +172 179 serines residue_name These differences undoubtedly influence the conformation of the CDRs, in particular CDR H1 (Fig. 1A) and CDR L1 (Fig. 3C), especially with the tandem glycines and multiple serines present, respectively. DISCUSS +38 48 antibodies protein_type Pairing of different germlines yields antibodies with various degrees of stability. DISCUSS +20 40 melting temperatures evidence As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set. DISCUSS +52 57 H1-69 mutant As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set. DISCUSS +62 67 H3-23 mutant As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set. DISCUSS +72 74 HC structure_element As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set. DISCUSS +88 93 L1-39 mutant As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set. DISCUSS +98 100 LC structure_element As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set. DISCUSS +114 120 stable protein_state As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set. DISCUSS +121 125 Fabs structure_element As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set. DISCUSS +52 54 LC structure_element One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3. DISCUSS +64 69 L1-39 mutant One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3. DISCUSS +78 81 CDR structure_element One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3. DISCUSS +82 84 L3 structure_element One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3. DISCUSS +119 121 91 residue_number One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3. DISCUSS +126 128 94 residue_number One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3. DISCUSS +168 171 CDR structure_element One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3. DISCUSS +172 174 H3 structure_element One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3. DISCUSS +37 40 Tyr residue_name Other germlines have bulky residues, Tyr, Arg and Trp, at these positions, whereas L1-39 has Ser and Thr. DISCUSS +42 45 Arg residue_name Other germlines have bulky residues, Tyr, Arg and Trp, at these positions, whereas L1-39 has Ser and Thr. DISCUSS +50 53 Trp residue_name Other germlines have bulky residues, Tyr, Arg and Trp, at these positions, whereas L1-39 has Ser and Thr. DISCUSS +83 88 L1-39 mutant Other germlines have bulky residues, Tyr, Arg and Trp, at these positions, whereas L1-39 has Ser and Thr. DISCUSS +93 96 Ser residue_name Other germlines have bulky residues, Tyr, Arg and Trp, at these positions, whereas L1-39 has Ser and Thr. DISCUSS +101 104 Thr residue_name Other germlines have bulky residues, Tyr, Arg and Trp, at these positions, whereas L1-39 has Ser and Thr. DISCUSS +47 49 VL structure_element Various combinations of germline sequences for VL and VH impose certain constraints on CDR H3, which has to adapt to the environment. DISCUSS +54 56 VH structure_element Various combinations of germline sequences for VL and VH impose certain constraints on CDR H3, which has to adapt to the environment. DISCUSS +87 90 CDR structure_element Various combinations of germline sequences for VL and VH impose certain constraints on CDR H3, which has to adapt to the environment. DISCUSS +91 93 H3 structure_element Various combinations of germline sequences for VL and VH impose certain constraints on CDR H3, which has to adapt to the environment. DISCUSS +7 14 compact protein_state A more compact CDR L3 may be beneficial in this situation. DISCUSS +15 18 CDR structure_element A more compact CDR L3 may be beneficial in this situation. DISCUSS +19 21 L3 structure_element A more compact CDR L3 may be beneficial in this situation. DISCUSS +43 45 LC structure_element At the other end of the stability range is LC germline L3-20, which yields antibodies with the lowest Tms. DISCUSS +55 60 L3-20 mutant At the other end of the stability range is LC germline L3-20, which yields antibodies with the lowest Tms. DISCUSS +75 85 antibodies protein_type At the other end of the stability range is LC germline L3-20, which yields antibodies with the lowest Tms. DISCUSS +102 105 Tms evidence At the other end of the stability range is LC germline L3-20, which yields antibodies with the lowest Tms. DISCUSS +20 25 H3-53 mutant While pairings with H3-53 and H5-51 may be safely called a mismatch, those with H1-69 and H3-23 have Tms about 5-6° higher. DISCUSS +30 35 H5-51 mutant While pairings with H3-53 and H5-51 may be safely called a mismatch, those with H1-69 and H3-23 have Tms about 5-6° higher. DISCUSS +80 85 H1-69 mutant While pairings with H3-53 and H5-51 may be safely called a mismatch, those with H1-69 and H3-23 have Tms about 5-6° higher. DISCUSS +90 95 H3-23 mutant While pairings with H3-53 and H5-51 may be safely called a mismatch, those with H1-69 and H3-23 have Tms about 5-6° higher. DISCUSS +101 104 Tms evidence While pairings with H3-53 and H5-51 may be safely called a mismatch, those with H1-69 and H3-23 have Tms about 5-6° higher. DISCUSS +17 21 Fabs structure_element Curiously, the 2 Fabs, H1-69:L3-20 and H3-23:L3-20, deviate markedly in their tilt angles from the rest of the panel. DISCUSS +23 34 H1-69:L3-20 complex_assembly Curiously, the 2 Fabs, H1-69:L3-20 and H3-23:L3-20, deviate markedly in their tilt angles from the rest of the panel. DISCUSS +39 50 H3-23:L3-20 complex_assembly Curiously, the 2 Fabs, H1-69:L3-20 and H3-23:L3-20, deviate markedly in their tilt angles from the rest of the panel. DISCUSS +78 89 tilt angles evidence Curiously, the 2 Fabs, H1-69:L3-20 and H3-23:L3-20, deviate markedly in their tilt angles from the rest of the panel. DISCUSS +40 51 tilt angles evidence It is possible that by adopting extreme tilt angles the structure modulates CDR H3 and its environment, which apparently cannot be achieved solely by conformational rearrangement of the CDR. DISCUSS +56 65 structure evidence It is possible that by adopting extreme tilt angles the structure modulates CDR H3 and its environment, which apparently cannot be achieved solely by conformational rearrangement of the CDR. DISCUSS +76 79 CDR structure_element It is possible that by adopting extreme tilt angles the structure modulates CDR H3 and its environment, which apparently cannot be achieved solely by conformational rearrangement of the CDR. DISCUSS +80 82 H3 structure_element It is possible that by adopting extreme tilt angles the structure modulates CDR H3 and its environment, which apparently cannot be achieved solely by conformational rearrangement of the CDR. DISCUSS +186 189 CDR structure_element It is possible that by adopting extreme tilt angles the structure modulates CDR H3 and its environment, which apparently cannot be achieved solely by conformational rearrangement of the CDR. DISCUSS +22 37 VH:VL interface site Note that most of the VH:VL interface residues are invariant; therefore, significant change of the tilt angle must come with a penalty in free energy. DISCUSS +15 25 antibodies protein_type Yet, for the 2 antibodies, the total gain in stability merits the domain repacking. DISCUSS +30 33 Fab structure_element Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft. DISCUSS +50 52 Tm evidence Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft. DISCUSS +98 100 HC structure_element Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft. DISCUSS +105 107 LC structure_element Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft. DISCUSS +108 124 variable domains structure_element Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft. DISCUSS +143 146 CDR structure_element Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft. DISCUSS +147 149 H3 structure_element Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft. DISCUSS +157 168 VH:VL cleft site Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft. DISCUSS +167 176 structure evidence The final conformation represents an energetic minimum; however, in most cases it is very shallow, so that a single mutation can cause a dramatic rearrangement of the structure. DISCUSS +33 51 structural library experimental_method In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +100 103 HCs structure_element In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +109 112 LCs structure_element In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +132 135 CDR structure_element In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +136 138 H3 structure_element In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +173 181 antibody protein_type In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +182 191 structure evidence In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +276 278 L1 structure_element In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +280 282 L2 structure_element In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +284 286 L3 structure_element In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +288 290 H1 structure_element In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +295 297 H2 structure_element In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +298 302 CDRs structure_element In summary, the analysis of this structural library of germline variants composed of all pairs of 4 HCs and 4LCs, all with the same CDR H3, offers some unique insights into antibody structure and how pairing and sequence may influence, or not, the canonical structures of the L1, L2, L3, H1 and H2 CDRs. DISCUSS +18 21 CDR structure_element Comparison of the CDR H3s reveals a large set of variants with conformations similar to the parent, while a second set has significant conformational variability, indicating that both the sequence and the structural context define the CDR H3 conformation. DISCUSS +22 25 H3s structure_element Comparison of the CDR H3s reveals a large set of variants with conformations similar to the parent, while a second set has significant conformational variability, indicating that both the sequence and the structural context define the CDR H3 conformation. DISCUSS +235 238 CDR structure_element Comparison of the CDR H3s reveals a large set of variants with conformations similar to the parent, while a second set has significant conformational variability, indicating that both the sequence and the structural context define the CDR H3 conformation. DISCUSS +239 241 H3 structure_element Comparison of the CDR H3s reveals a large set of variants with conformations similar to the parent, while a second set has significant conformational variability, indicating that both the sequence and the structural context define the CDR H3 conformation. DISCUSS +39 50 H1-69:L3-20 complex_assembly Quite unexpectedly, 2 of the variants, H1-69:L3-20 and H3-53:L4-1, have the ‘extended’ stem region differing from the other 14 that have a ‘kinked’ stem region. DISCUSS +55 65 H3-53:L4-1 complex_assembly Quite unexpectedly, 2 of the variants, H1-69:L3-20 and H3-53:L4-1, have the ‘extended’ stem region differing from the other 14 that have a ‘kinked’ stem region. DISCUSS +77 85 extended protein_state Quite unexpectedly, 2 of the variants, H1-69:L3-20 and H3-53:L4-1, have the ‘extended’ stem region differing from the other 14 that have a ‘kinked’ stem region. DISCUSS +87 98 stem region structure_element Quite unexpectedly, 2 of the variants, H1-69:L3-20 and H3-53:L4-1, have the ‘extended’ stem region differing from the other 14 that have a ‘kinked’ stem region. DISCUSS +140 146 kinked protein_state Quite unexpectedly, 2 of the variants, H1-69:L3-20 and H3-53:L4-1, have the ‘extended’ stem region differing from the other 14 that have a ‘kinked’ stem region. DISCUSS +148 159 stem region structure_element Quite unexpectedly, 2 of the variants, H1-69:L3-20 and H3-53:L4-1, have the ‘extended’ stem region differing from the other 14 that have a ‘kinked’ stem region. DISCUSS +45 48 CDR structure_element These data reveal the difficulty of modeling CDR H3 accurately, as shown again in Antibody Modeling Assessment II. DISCUSS +49 51 H3 structure_element These data reveal the difficulty of modeling CDR H3 accurately, as shown again in Antibody Modeling Assessment II. DISCUSS +13 21 antibody protein_type Furthermore, antibody CDRs, H3 in particular, may go through conformational changes upon binding their targets, making structural prediction for docking purposes an even more difficult task. DISCUSS +22 26 CDRs structure_element Furthermore, antibody CDRs, H3 in particular, may go through conformational changes upon binding their targets, making structural prediction for docking purposes an even more difficult task. DISCUSS +28 30 H3 structure_element Furthermore, antibody CDRs, H3 in particular, may go through conformational changes upon binding their targets, making structural prediction for docking purposes an even more difficult task. DISCUSS +38 46 antibody protein_type Fortunately, for most applications of antibody modeling, such as engineering affinity and biophysical properties, an accurate CDR H3 structure is not always necessary. DISCUSS +126 129 CDR structure_element Fortunately, for most applications of antibody modeling, such as engineering affinity and biophysical properties, an accurate CDR H3 structure is not always necessary. DISCUSS +130 132 H3 structure_element Fortunately, for most applications of antibody modeling, such as engineering affinity and biophysical properties, an accurate CDR H3 structure is not always necessary. DISCUSS +133 142 structure evidence Fortunately, for most applications of antibody modeling, such as engineering affinity and biophysical properties, an accurate CDR H3 structure is not always necessary. DISCUSS +38 41 CDR structure_element For those applications where accurate CDR structures are essential, such as docking, the results in this work demonstrate the importance of experimental structures. DISCUSS +42 52 structures evidence For those applications where accurate CDR structures are essential, such as docking, the results in this work demonstrate the importance of experimental structures. DISCUSS +153 163 structures evidence For those applications where accurate CDR structures are essential, such as docking, the results in this work demonstrate the importance of experimental structures. DISCUSS +28 66 expression and crystallization methods experimental_method With the recent advances in expression and crystallization methods, Fab structures can be obtained rapidly. DISCUSS +68 71 Fab structure_element With the recent advances in expression and crystallization methods, Fab structures can be obtained rapidly. DISCUSS +72 82 structures evidence With the recent advances in expression and crystallization methods, Fab structures can be obtained rapidly. DISCUSS +23 26 Fab structure_element The set of 16 germline Fab structures offers a unique dataset to facilitate software development for antibody modeling. DISCUSS +27 37 structures evidence The set of 16 germline Fab structures offers a unique dataset to facilitate software development for antibody modeling. DISCUSS +101 109 antibody protein_type The set of 16 germline Fab structures offers a unique dataset to facilitate software development for antibody modeling. DISCUSS +65 75 structures evidence The results essentially support the underlying idea of canonical structures, indicating that most CDRs with germline sequences tend to adopt predefined conformations. DISCUSS +98 102 CDRs structure_element The results essentially support the underlying idea of canonical structures, indicating that most CDRs with germline sequences tend to adopt predefined conformations. DISCUSS +66 74 antibody protein_type From this point of view, a novel approach to design combinatorial antibody libraries would be to cover the range of CDR conformations that may not necessarily coincide with the germline usage in the human repertoire. DISCUSS +116 119 CDR structure_element From this point of view, a novel approach to design combinatorial antibody libraries would be to cover the range of CDR conformations that may not necessarily coincide with the germline usage in the human repertoire. DISCUSS +199 204 human species From this point of view, a novel approach to design combinatorial antibody libraries would be to cover the range of CDR conformations that may not necessarily coincide with the germline usage in the human repertoire. DISCUSS +80 90 antibodies protein_type This would insure more structural diversity, leading to a more diverse panel of antibodies that would bind to a broad spectrum of targets. DISCUSS diff --git a/annotation_IOB/all.tsv b/annotation_IOB/all.tsv new file mode 100644 index 0000000000000000000000000000000000000000..689b5ea79f2edf2c6ad34d894604c2cd2e84e5b1 --- /dev/null +++ b/annotation_IOB/all.tsv @@ -0,0 +1,264664 @@ +Molecular O +Dissection O +of O +Xyloglucan B-chemical +Recognition O +in O +a O +Prominent O +Human B-species +Gut O +Symbiont O + +Polysaccharide B-gene +utilization I-gene +loci I-gene +( O +PUL B-gene +) O +within O +the O +genomes O +of O +resident O +human B-species +gut O +Bacteroidetes B-taxonomy_domain +are O +central O +to O +the O +metabolism O +of O +the O +otherwise O +indigestible O +complex O +carbohydrates B-chemical +known O +as O +“ O +dietary O +fiber O +.” O +However O +, O +functional O +characterization O +of O +PUL B-gene +lags O +significantly O +behind O +sequencing O +efforts O +, O +which O +limits O +physiological O +understanding O +of O +the O +human B-species +- O +bacterial B-taxonomy_domain +symbiosis O +. 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O + +Xyloglucan B-chemical +and O +the O +Bacteroides B-species +ovatus I-species +xyloglucan B-gene +utilization I-gene +locus I-gene +( O +XyGUL B-gene +). O +( O +A O +) O +Representative O +structures B-evidence +of O +common O +xyloglucans B-chemical +using O +the O +Consortium O +for O +Functional O +Glycomics O +Symbol O +Nomenclature O +( O +http O +:// O +www O +. O +functionalglycomics O +. O +org O +/ O +static O +/ O +consortium O +/ O +Nomenclature O +. O +shtml O +). O + +Cleavage O +sites O +for O +BoXyGUL B-gene +glycosidases B-protein_type +( O +GHs B-protein_type +) O +are O +indicated O +for O +solanaceous B-taxonomy_domain +xyloglucan B-chemical +. O +( O +B O +) O +BtSus B-gene +and O +BoXyGUL B-gene +. O +( O +C O +) O +Localization O +of O +BoXyGUL B-gene +- O +encoded O +proteins O +in O +cellular O +membranes O +and O +concerted O +modes O +of O +action O +in O +the O +degradation O +of O +xyloglucans B-chemical +to O +monosaccharides O +. O + +The O +location O +of O +SGBP B-protein +- I-protein +A I-protein +/ O +B B-protein +is O +presented O +in O +this O +work O +; O +the O +location O +of O +GH5 B-protein +has O +been O +empirically O +determined O +, O +and O +the O +enzymes O +have O +been O +placed O +based O +upon O +their O +predicted O +cellular O +location O +. O + +We O +recently O +reported O +the O +detailed O +molecular O +characterization O +of O +a O +PUL B-gene +that O +confers O +the O +ability O +of O +the O +human B-species +gut O +commensal O +B B-species +. I-species +ovatus I-species +ATCC I-species +8483 I-species +to O +grow O +on O +a O +prominent O +family O +of O +plant B-taxonomy_domain +cell O +wall O +glycans B-chemical +, O +the O +xyloglucans B-chemical +( O +XyG B-chemical +). O + +XyG B-chemical +variants O +( O +Fig O +. O +1A O +) O +constitute O +up O +to O +25 O +% O +of O +the O +dry O +weight O +of O +common O +vegetables B-taxonomy_domain +. O + +Analogous O +to O +the O +Sus B-gene +locus I-gene +, O +the O +xyloglucan B-gene +utilization I-gene +locus I-gene +( O +XyGUL B-gene +) O +encodes O +a O +cohort O +of O +carbohydrate B-protein_type +- I-protein_type +binding I-protein_type +, I-protein_type +- I-protein_type +hydrolyzing I-protein_type +, I-protein_type +and I-protein_type +- I-protein_type +importing I-protein_type +proteins I-protein_type +( O +Fig O +. O +1B O +and O +C O +). O + +The O +number O +of O +glycoside B-protein_type +hydrolases I-protein_type +( O +GHs B-protein_type +) O +encoded O +by O +the O +XyGUL B-gene +is O +, O +however O +, O +more O +expansive O +than O +that O +by O +the O +Sus B-gene +locus I-gene +( O +Fig O +. O +1B O +), O +which O +reflects O +the O +greater O +complexity O +of O +glycosidic O +linkages O +found O +in O +XyG B-chemical +vis O +- O +à O +- O +vis O +starch B-chemical +. O + +Whereas O +our O +previous O +study O +focused O +on O +the O +characterization O +of O +the O +linkage O +specificity O +of O +these O +GHs B-protein_type +, O +a O +key O +outstanding O +question O +regarding O +this O +locus O +is O +how O +XyG B-chemical +recognition O +is O +mediated O +at O +the O +cell O +surface O +. O + +In O +the O +archetypal O +starch B-complex_assembly +utilization I-complex_assembly +system I-complex_assembly +of O +B B-species +. I-species +thetaiotaomicron I-species +, O +starch O +binding O +to O +the O +cell O +surface O +is O +mediated O +at O +eight O +distinct O +starch B-site +- I-site +binding I-site +sites I-site +distributed O +among O +four O +surface B-protein_type +glycan I-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +( O +SGBPs B-protein_type +): O +two O +within O +the O +amylase B-protein_type +SusG B-protein +, O +one O +within O +SusD B-protein +, O +two O +within O +SusE B-protein +, O +and O +three O +within O +SusF B-protein +. O +The O +functional O +redundancy O +of O +many O +of O +these O +sites O +is O +high O +: O +whereas O +SusD B-protein +is O +essential O +for O +growth O +on O +starch B-chemical +, O +combined O +mutations O +of O +the O +SusE B-protein +, O +SusF B-protein +, O +and O +SusG B-protein +binding B-site +sites I-site +are O +required O +to O +impair O +growth O +on O +the O +polysaccharide B-chemical +. O + +Bacteroidetes B-taxonomy_domain +PUL B-gene +ubiquitously O +encode O +homologs O +of O +SusC B-protein +and O +SusD B-protein +, O +as O +well O +as O +proteins O +whose O +genes O +are O +immediately O +downstream O +of O +susD B-gene +, O +akin O +to O +susE B-gene +/ I-gene +F I-gene +, O +and O +these O +are O +typically O +annotated O +as O +“ O +putative B-protein_state +lipoproteins B-protein_type +”. O + +The O +genes O +coding O +for O +these O +proteins O +, O +sometimes O +referred O +to O +as O +“ O +susE B-gene +/ I-gene +F I-gene +positioned O +,” O +display O +products O +with O +a O +wide O +variation O +in O +amino O +acid O +sequence O +and O +which O +have O +little O +or O +no O +homology O +to O +other O +PUL B-gene +- O +encoded O +proteins O +or O +known O +carbohydrate B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +. O + +As O +the O +Sus B-complex_assembly +SGBPs B-protein_type +remain O +the O +only O +structurally O +characterized O +cohort O +to O +date O +, O +we O +therefore O +wondered O +whether O +such O +glycan B-chemical +binding O +and O +function O +are O +extended O +to O +other O +PUL B-gene +that O +target O +more O +complex O +and O +heterogeneous O +polysaccharides B-chemical +, O +such O +as O +XyG B-chemical +. O + +We O +describe O +here O +the O +detailed O +functional B-experimental_method +and I-experimental_method +structural I-experimental_method +characterization I-experimental_method +of O +the O +noncatalytic B-protein_state +SGBPs B-protein_type +encoded O +by O +Bacova_02651 B-gene +and O +Bacova_02650 B-gene +of O +the O +XyGUL B-gene +, O +here O +referred O +to O +as O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +, O +to O +elucidate O +their O +molecular O +roles O +in O +carbohydrate O +acquisition O +in O +vivo O +. O + +Combined O +biochemical B-experimental_method +, I-experimental_method +structural I-experimental_method +, I-experimental_method +and I-experimental_method +reverse I-experimental_method +- I-experimental_method +genetic I-experimental_method +approaches I-experimental_method +clearly O +illuminate O +the O +distinct O +, O +yet O +complementary O +, O +functions O +that O +these O +two O +proteins O +play O +in O +XyG B-chemical +recognition O +as O +it O +impacts O +the O +physiology O +of O +B B-species +. I-species +ovatus I-species +. O + +These O +data O +extend O +our O +current O +understanding O +of O +the O +Sus O +- O +like O +glycan B-chemical +uptake O +paradigm O +within O +the O +Bacteroidetes B-taxonomy_domain +and O +reveals O +how O +the O +complex O +dietary O +polysaccharide B-chemical +xyloglucan B-chemical +is O +recognized O +at O +the O +cell O +surface O +. O + +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +are O +cell B-protein_type +- I-protein_type +surface I-protein_type +- I-protein_type +localized I-protein_type +, I-protein_type +xyloglucan I-protein_type +- I-protein_type +specific I-protein_type +binding I-protein_type +proteins I-protein_type +. O + +SGBP B-protein +- I-protein +A I-protein +, O +encoded O +by O +the O +XyGUL B-gene +locus O +tag O +Bacova_02651 B-gene +( O +Fig O +. O +1B O +), O +shares O +26 O +% O +amino O +acid O +sequence O +identity O +( O +40 O +% O +similarity O +) O +with O +its O +homolog O +, O +B B-species +. I-species +thetaiotaomicron I-species +SusD B-protein +, O +and O +similar O +homology O +with O +the O +SusD B-protein_type +- I-protein_type +like I-protein_type +proteins I-protein_type +encoded O +within O +syntenic O +XyGUL B-gene +identified O +in O +our O +earlier O +work O +. O + +In O +contrast O +, O +SGBP B-protein +- I-protein +B I-protein +, O +encoded O +by O +locus O +tag O +Bacova_02650 B-gene +, O +displays O +little O +sequence O +similarity O +to O +the O +products O +of O +similarly O +positioned O +genes O +in O +syntenic O +XyGUL B-gene +nor O +to O +any O +other O +gene O +product O +among O +the O +diversity O +of O +Bacteroidetes B-taxonomy_domain +PUL B-gene +. O + +Whereas O +sequence O +similarity O +among O +SusC B-protein +/ O +SusD B-protein +homolog O +pairs O +often O +serves O +as O +a O +hallmark O +for O +PUL B-gene +identification O +, O +the O +sequence O +similarities O +of O +downstream O +genes O +encoding O +SGBPs B-protein_type +are O +generally O +too O +low O +to O +allow O +reliable O +bioinformatic O +classification O +of O +their O +products O +into O +protein O +families O +, O +let O +alone O +prediction O +of O +function O +. O + +Hence O +, O +there O +is O +a O +critical O +need O +for O +the O +elucidation O +of O +detailed O +structure O +- O +function O +relationships O +among O +PUL B-gene +SGBPs B-protein_type +, O +in O +light O +of O +the O +manifold O +glycan B-chemical +structures O +in O +nature O +. O + +Immunofluorescence B-experimental_method +of O +formaldehyde O +- O +fixed O +, O +nonpermeabilized O +cells O +grown O +in O +minimal O +medium O +with O +XyG B-chemical +as O +the O +sole O +carbon O +source O +to O +induce O +XyGUL B-gene +expression O +, O +reveals O +that O +both O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +are O +presented O +on O +the O +cell O +surface O +by O +N O +- O +terminal O +lipidation B-ptm +, O +as O +predicted O +by O +signal O +peptide O +analysis O +with O +SignalP O +( O +Fig O +. O +2 O +). O + +Here O +, O +the O +SGBPs B-protein_type +very O +likely O +work O +in O +concert O +with O +the O +cell B-protein_type +- I-protein_type +surface I-protein_type +- I-protein_type +localized I-protein_type +endo I-protein_type +- I-protein_type +xyloglucanase I-protein_type +B B-species +. I-species +ovatus I-species +GH5 B-protein +( O +BoGH5 B-protein +) O +to O +recruit O +and O +cleave O +XyG B-chemical +for O +subsequent O +periplasmic O +import O +via O +the O +SusC B-protein_type +- I-protein_type +like I-protein_type +TBDT I-protein_type +of O +the O +XyGUL B-gene +( O +Fig O +. O +1B O +and O +C O +). O + +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +visualized O +by O +immunofluorescence B-experimental_method +. O + +Formalin O +- O +fixed O +, O +nonpermeabilized O +B B-species +. I-species +ovatus I-species +cells O +were O +grown O +in O +minimal O +medium O +plus O +XyG B-chemical +, O +probed O +with O +custom O +rabbit O +antibodies O +to O +SGBP B-protein +- I-protein +A I-protein +or O +SGBP B-protein +- I-protein +B I-protein +, O +and O +then O +stained O +with O +Alexa O +Fluor O +488 O +goat O +anti O +- O +rabbit O +IgG O +. O +( O +A O +) O +Overlay B-experimental_method +of O +bright B-evidence +- I-evidence +field I-evidence +and I-evidence +FITC I-evidence +images I-evidence +of O +B B-species +. I-species +ovatus I-species +cells O +labeled O +with O +anti O +- O +SGBP O +- O +A O +. O +( O +B O +) O +Overlay B-experimental_method +of O +bright B-evidence +- I-evidence +field I-evidence +and I-evidence +FITC I-evidence +images I-evidence +of O +B B-species +. I-species +ovatus I-species +cells O +labeled O +with O +anti O +- O +SGBP O +- O +B O +. O +( O +C O +) O +Bright B-evidence +- I-evidence +field I-evidence +image I-evidence +of O +ΔSGBP B-mutant +- I-mutant +B I-mutant +cells O +labeled O +with O +anti O +- O +SGBP O +- O +B O +antibodies O +. O + +( O +D O +) O +FITC B-evidence +images I-evidence +of O +ΔSGBP B-mutant +- I-mutant +B I-mutant +cells O +labeled O +with O +anti O +- O +SGBP O +- O +B O +antibodies O +. O + +Cells O +lacking B-protein_state +SGBP B-protein +- I-protein +A I-protein +( O +ΔSGBP B-mutant +- I-mutant +A I-mutant +) O +do O +not O +grow O +on O +XyG B-chemical +and O +therefore O +could O +not O +be O +tested O +in O +parallel O +. O + +In O +our O +initial O +study O +focused O +on O +the O +functional O +characterization O +of O +the O +glycoside B-protein_type +hydrolases I-protein_type +of O +the O +XyGUL B-gene +, O +we O +reported O +preliminary O +affinity B-experimental_method +PAGE I-experimental_method +and O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +( O +ITC B-experimental_method +) O +data O +indicating O +that O +both O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +are O +competent O +xyloglucan B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +( O +affinity B-evidence +constant I-evidence +[ O +Ka B-evidence +] O +values O +of O +3 O +. O +74 O +× O +105 O +M O +− O +1 O +and O +4 O +. O +98 O +× O +104 O +M O +− O +1 O +, O +respectively O +[ O +23 O +]). O + +Additional O +affinity B-experimental_method +PAGE I-experimental_method +analysis O +( O +Fig O +. O +3 O +) O +demonstrates O +that O +SGBP B-protein +- I-protein +A I-protein +also O +has O +moderate O +affinity O +for O +the O +artificial O +soluble O +cellulose O +derivative O +hydroxyethyl B-chemical +cellulose I-chemical +[ O +HEC B-chemical +; O +a O +β B-chemical +( I-chemical +1 I-chemical +→ I-chemical +4 I-chemical +)- I-chemical +glucan I-chemical +] O +and O +limited O +affinity O +for O +mixed B-chemical +- I-chemical +linkage I-chemical +β I-chemical +( I-chemical +1 I-chemical +→ I-chemical +3 I-chemical +)/ I-chemical +β I-chemical +( I-chemical +1 I-chemical +→ I-chemical +4 I-chemical +)- I-chemical +glucan I-chemical +( O +MLG B-chemical +) O +and O +glucomannan B-chemical +( O +GM B-chemical +; O +mixed O +glucosyl B-chemical +and O +mannosyl B-chemical +backbone O +), O +which O +together O +indicate O +general O +binding O +to O +polysaccharide B-chemical +backbone O +residues O +and O +major O +contributions O +from O +side O +- O +chain O +recognition O +. O + +In O +contrast O +, O +SGBP B-protein +- I-protein +B I-protein +bound O +to O +HEC B-chemical +more O +weakly O +than O +SGBP B-protein +- I-protein +A I-protein +and O +did O +not O +bind O +to O +MLG B-chemical +or O +GM B-chemical +. O + +Neither O +SGBP B-protein_type +recognized O +galactomannan B-chemical +( O +GGM B-chemical +), O +starch B-chemical +, O +carboxymethylcellulose B-chemical +, O +or O +mucin B-chemical +( O +see O +Fig O +. O +S1 O +in O +the O +supplemental O +material O +). O + +Together O +, O +these O +results O +highlight O +the O +high O +specificities O +of O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +for O +XyG B-chemical +, O +which O +is O +concordant O +with O +their O +association O +with O +XyG B-protein_type +- I-protein_type +specific I-protein_type +GHs I-protein_type +in O +the O +XyGUL B-gene +, O +as O +well O +as O +transcriptomic O +analysis O +indicating O +that O +B B-species +. I-species +ovatus I-species +has O +discrete O +PUL B-gene +for O +MLG B-chemical +, O +GM B-chemical +, O +and O +GGM B-chemical +( O +11 O +). O + +Notably O +, O +the O +absence O +of O +carbohydrate B-site +- I-site +binding I-site +modules I-site +in O +the O +GHs B-protein_type +encoded O +by O +the O +XyGUL B-gene +implies O +that O +noncatalytic O +recognition O +of O +xyloglucan B-chemical +is O +mediated O +entirely O +by O +SGBP B-protein +- I-protein +A I-protein +and O +- B-protein +B I-protein +. O + +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +preferentially O +bind O +xyloglucan B-chemical +. O + +Affinity B-experimental_method +electrophoresis I-experimental_method +( O +10 O +% O +acrylamide O +) O +of O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +with O +BSA B-protein +as O +a O +control O +protein O +. O + +All O +samples O +were O +loaded O +on O +the O +same O +gel O +next O +to O +the O +BSA B-protein +controls O +; O +thin O +black O +lines O +indicate O +where O +intervening O +lanes O +were O +removed O +from O +the O +final O +image O +for O +both O +space O +and O +clarity O +. O + +The O +percentage O +of O +polysaccharide B-chemical +incorporated O +into O +each O +native O +gel O +is O +displayed O +. O + +The O +vanguard O +endo B-protein_type +- I-protein_type +xyloglucanase I-protein_type +of O +the O +XyGUL B-gene +, O +BoGH5 B-protein +, O +preferentially O +cleaves O +the O +polysaccharide B-chemical +at O +unbranched O +glucosyl B-chemical +residues O +to O +generate O +xylogluco B-chemical +- I-chemical +oligosaccharides I-chemical +( O +XyGOs B-chemical +) O +comprising O +a O +Glc4 B-structure_element +backbone I-structure_element +with O +variable B-structure_element +side I-structure_element +- I-structure_element +chain I-structure_element +galactosylation I-structure_element +( O +XyGO1 B-chemical +) O +( O +Fig O +. O +1A O +; O +n O += O +1 O +) O +as O +the O +limit O +of O +digestion O +products O +in O +vitro O +; O +controlled B-experimental_method +digestion I-experimental_method +and I-experimental_method +fractionation I-experimental_method +by O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +allow O +the O +production O +of O +higher O +- O +order O +oligosaccharides B-chemical +( O +e O +. O +g O +., O +XyGO2 B-chemical +) O +( O +Fig O +. O +1A O +; O +n O += O +2 O +). O + +ITC B-experimental_method +demonstrates O +that O +SGBP B-protein +- I-protein +A I-protein +binds O +to O +XyG B-chemical +polysaccharide B-chemical +and O +XyGO2 B-chemical +( O +based O +on O +a O +Glc8 B-structure_element +backbone I-structure_element +) O +with O +essentially O +equal O +affinities B-evidence +, O +while O +no O +binding O +of O +XyGO1 B-chemical +( O +Glc4 B-structure_element +backbone I-structure_element +) O +was O +detectable O +( O +Table O +1 O +; O +see O +Fig O +. O +S2 O +and O +S3 O +in O +the O +supplemental O +material O +). O + +Similarly O +, O +SGBP B-protein +- I-protein +B I-protein +also O +bound B-protein_state +to I-protein_state +XyG B-chemical +and O +XyGO2 B-chemical +with O +approximately O +equal O +affinities B-evidence +, O +although O +in O +both O +cases O +, O +Ka B-evidence +values O +were O +nearly O +10 O +- O +fold O +lower O +than O +those O +for O +SGBP B-protein +- I-protein +A I-protein +. O +Also O +in O +contrast O +to O +SGBP B-protein +- I-protein +A I-protein +, O +SGBP B-protein +- I-protein +B I-protein +also O +bound B-protein_state +to I-protein_state +XyGO1 B-chemical +, O +yet O +the O +affinity B-evidence +for O +this O +minimal B-structure_element +repeating I-structure_element +unit I-structure_element +was O +poor O +, O +with O +a O +Ka B-evidence +value O +of O +ca O +. O +1 O +order O +of O +magnitude O +lower O +than O +for O +XyG B-chemical +and O +XyGO2 B-chemical +. O + +Together O +, O +these O +data O +clearly O +suggest O +that O +polysaccharide B-chemical +binding O +of O +both O +SGBPs B-protein_type +is O +fulfilled O +by O +a O +dimer B-oligomeric_state +of O +the O +minimal B-structure_element +repeat I-structure_element +, O +corresponding O +to O +XyGO2 B-chemical +( O +cf O +. O + +The O +observation O +by O +affinity B-experimental_method +PAGE I-experimental_method +that O +these O +proteins O +specifically O +recognize O +XyG B-chemical +is O +further O +substantiated O +by O +their O +lack O +of O +binding O +for O +the O +undecorated O +oligosaccharide B-chemical +cellotetraose B-chemical +( O +Table O +1 O +; O +see O +Fig O +. O +S3 O +). O + +Furthermore O +, O +SGBP B-protein +- I-protein +A I-protein +binds O +cellohexaose B-chemical +with O +~ O +770 O +- O +fold O +weaker O +affinity B-evidence +than O +XyG B-chemical +, O +while O +SGBP B-protein +- I-protein +B I-protein +displays O +no O +detectable O +binding O +to O +this O +linear O +hexasaccharide B-chemical +. O + +To O +provide O +molecular O +- O +level O +insight O +into O +how O +the O +XyGUL B-gene +SGBPs B-protein_type +equip O +B B-species +. I-species +ovatus I-species +to O +specifically O +harvest O +XyG B-chemical +from O +the O +gut O +environment O +, O +we O +performed O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +analysis O +of O +both O +SGBP B-protein +- I-protein +A I-protein +and O +SGPB B-protein +- I-protein +B I-protein +in O +oligosaccharide B-complex_assembly +- I-complex_assembly +complex I-complex_assembly +forms I-complex_assembly +. O + +Summary O +of O +thermodynamic O +parameters O +for O +wild B-protein_state +- I-protein_state +type I-protein_state +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +obtained O +by O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +at O +25 O +° O +Ca O + +Carbohydrate O +Ka O +( O +M O +− O +1 O +) O +ΔG B-evidence +( O +kcal O +⋅ O +mol O +− O +1 O +) O +ΔH O +( O +kcal O +⋅ O +mol O +− O +1 O +) O +TΔS O +( O +kcal O +⋅ O +mol O +− O +1 O +) O +SGBP O +- O +A O +SGBP O +- O +B O +SGBP O +- O +A O +SGBP O +- O +B O +SGBP O +- O +A O +SGBP O +- O +B O +SGBP O +- O +A O +SGBP O +- O +B O +XyGb O +( O +4 O +. O +4 O +± O +0 O +. O +1 O +) O +× O +105 O +( O +5 O +. O +7 O +± O +0 O +. O +2 O +) O +× O +104 O +− O +7 O +. O +7 O +− O +6 O +. O +5 O +− O +14 O +± O +3 O +− O +14 O +± O +2 O +− O +6 O +. O +5 O +− O +7 O +. O +6 O +XyGO2c O +3 O +. O +0 O +× O +105 O +2 O +. O +0 O +× O +104 O +− O +7 O +. O +5 O +− O +5 O +. O +9 O +− O +17 O +. O +2 O +− O +17 O +. O +6 O +− O +9 O +. O +7 O +− O +11 O +. O +7 O +XyGO1 O +NBd O +( O +2 O +. O +4 O +± O +0 O +. O +1 O +) O +× O +103 O +NB O +− O +4 O +. O +6 O +NB O +− O +4 O +. O +4 O +± O +0 O +. O +2 O +NB O +0 O +. O +2 O +Cellohexaose O +568 O +. O +0 O +± O +291 O +. O +0 O +NB O +− O +3 O +. O +8 O +NB O +− O +16 O +± O +8 O +NB O +− O +12 O +. O +7 O +NB O +Cellotetraose O +NB O +NB O +NB O +NB O +NB O +NB O +NB O +NB O + +SGBP B-protein +- I-protein +A I-protein +is O +a O +SusD B-protein +homolog O +with O +an O +extensive O +glycan B-site +- I-site +binding I-site +platform I-site +. O + +As O +anticipated O +by O +sequence O +similarity O +, O +the O +high O +- O +resolution O +tertiary O +structure B-evidence +of O +apo B-protein_state +- O +SGBP B-protein +- I-protein +A I-protein +( O +1 O +. O +36 O +Å O +, O +Rwork B-evidence += O +14 O +. O +7 O +%, O +Rfree B-evidence += O +17 O +. O +4 O +%, O +residues O +28 B-residue_range +to I-residue_range +546 I-residue_range +) O +( O +Table O +2 O +) O +displays O +the O +canonical O +“ B-structure_element +SusD I-structure_element +- I-structure_element +like I-structure_element +” I-structure_element +protein I-structure_element +fold I-structure_element +dominated O +by O +four O +tetratrico B-structure_element +- I-structure_element +peptide I-structure_element +repeat I-structure_element +( O +TPR B-structure_element +) O +motifs O +that O +cradle O +the O +rest O +of O +the O +structure B-evidence +( O +Fig O +. O +4A O +). O + +Specifically O +, O +SGBP B-protein +- I-protein +A I-protein +overlays B-experimental_method +B B-species +. I-species +thetaiotaomicron I-species +SusD B-protein +( O +BtSusD B-protein +) O +with O +a O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +RMSD B-evidence +) O +value O +of O +2 O +. O +2 O +Å O +for O +363 O +Cα O +pairs O +, O +which O +is O +notable O +given O +the O +26 O +% O +amino O +acid O +identity O +( O +40 O +% O +similarity O +) O +between O +these O +homologs O +( O +Fig O +. O +4C O +). O + +Cocrystallization B-experimental_method +of O +SGBP B-protein +- I-protein +A I-protein +with O +XyGO2 B-chemical +generated O +a O +substrate B-complex_assembly +complex I-complex_assembly +structure B-evidence +( O +2 O +. O +3 O +Å O +, O +Rwork B-evidence += O +21 O +. O +8 O +%, O +Rfree B-evidence += O +24 O +. O +8 O +%, O +residues O +36 B-residue_range +to I-residue_range +546 I-residue_range +) O +( O +Fig O +. O +4A O +and O +B O +; O +Table O +2 O +) O +that O +revealed O +the O +distinct O +binding B-site +- I-site +site I-site +architecture O +of O +the O +XyG B-protein_type +binding I-protein_type +protein I-protein_type +. O + +The O +SGBP B-complex_assembly +- I-complex_assembly +A I-complex_assembly +: I-complex_assembly +XyGO2 I-complex_assembly +complex O +superimposes B-experimental_method +closely O +with O +the O +apo B-protein_state +structure B-evidence +( O +RMSD B-evidence +of O +0 O +. O +6 O +Å O +) O +and O +demonstrates O +that O +no O +major O +conformational O +change O +occurs O +upon O +substrate O +binding O +; O +small O +deviations O +in O +the O +orientation O +of O +several O +surface O +loops O +are O +likely O +the O +result O +of O +differential O +crystal O +packing O +. O + +It O +is O +particularly O +notable O +that O +although O +the O +location O +of O +the O +ligand B-site +- I-site +binding I-site +site I-site +is O +conserved B-protein_state +between O +SGBP B-protein +- I-protein +A I-protein +and O +SusD B-protein +, O +that O +of O +SGBP B-protein +- I-protein +A I-protein +displays O +an O +~ O +29 O +- O +Å O +- O +long O +aromatic B-site +platform I-site +to O +accommodate O +the O +extended O +, O +linear O +XyG B-chemical +chain O +( O +see O +reference O +for O +a O +review O +of O +XyG B-chemical +secondary O +structure O +), O +versus O +the O +shorter O +, O +~ O +18 O +- O +Å O +- O +long O +, O +site B-site +within O +SusD B-protein +that O +complements O +the O +helical O +conformation O +of O +amylose B-chemical +( O +Fig O +. O +4C O +and O +D O +). O + +Molecular O +structure B-evidence +of O +SGBP B-protein +- I-protein +A I-protein +( O +Bacova_02651 B-gene +). O +( O +A O +) O +Overlay B-experimental_method +of O +SGBP B-protein +- I-protein +A I-protein +from O +the O +apo B-protein_state +( O +rainbow O +) O +and O +XyGO2 B-chemical +( O +gray O +) O +structures B-evidence +. O + +The O +apo B-protein_state +structure B-evidence +is O +color O +ramped O +from O +blue O +to O +red O +. O + +An O +omit B-evidence +map I-evidence +( O +2σ O +) O +for O +XyGO2 B-chemical +( O +orange O +and O +red O +sticks O +) O +is O +displayed O +. O + +( O +B O +) O +Close O +- O +up O +view O +of O +the O +omit B-evidence +map I-evidence +as O +in O +panel O +A O +, O +rotated O +90 O +° O +clockwise O +. O + +( O +C O +) O +Overlay B-experimental_method +of O +the O +Cα O +backbones O +of O +SGBP B-protein +- I-protein +A I-protein +( O +black O +) O +with O +XyGO2 B-chemical +( O +orange O +and O +red O +spheres O +) O +and O +BtSusD B-protein +( O +blue O +) O +with O +maltoheptaose B-chemical +( O +pink O +and O +red O +spheres O +), O +highlighting O +the O +conservation O +of O +the O +glycan B-site +- I-site +binding I-site +site I-site +location O +. O + +( O +D O +) O +Close O +- O +up O +of O +the O +SGBP B-protein +- I-protein +A I-protein +( O +black O +and O +orange O +) O +and O +SusD B-protein +( O +blue O +and O +pink O +) O +glycan B-site +- I-site +binding I-site +sites I-site +. O + +The O +approximate O +length O +of O +each O +glycan B-site +- I-site +binding I-site +site I-site +is O +displayed O +, O +colored O +to O +match O +the O +protein B-evidence +structures I-evidence +. O +( O +E O +) O +Stereo O +view O +of O +the O +xyloglucan B-site +- I-site +binding I-site +site I-site +of O +SGBP B-protein +- I-protein +A I-protein +, O +displaying O +all O +residues O +within O +4 O +Å O +of O +the O +ligand O +. O + +The O +backbone O +glucose B-chemical +residues O +are O +numbered O +from O +the O +nonreducing O +end O +; O +xylose B-chemical +residues O +are O +labeled O +X1 B-residue_name_number +and O +X2 B-residue_name_number +. O + +Potential O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +interactions I-bond_interaction +are O +shown O +as O +dashed O +lines O +, O +and O +the O +distance O +is O +shown O +in O +angstroms O +. O + +Seven O +of O +the O +eight O +backbone O +glucosyl B-chemical +residues O +of O +XyGO2 B-chemical +could O +be O +convincingly O +modeled O +in O +the O +ligand B-evidence +electron I-evidence +density I-evidence +, O +and O +only O +two O +α B-chemical +( I-chemical +1 I-chemical +→ I-chemical +6 I-chemical +)- I-chemical +linked I-chemical +xylosyl I-chemical +residues O +were O +observed O +( O +Fig O +. O +4B O +; O +cf O +. O + +Indeed O +, O +the O +electron B-evidence +density I-evidence +for O +the O +ligand O +suggests O +some O +disorder O +, O +which O +may O +arise O +from O +multiple O +oligosaccharide B-chemical +orientations O +along O +the O +binding B-site +site I-site +. O + +Three O +aromatic O +residues O +— O +W82 B-residue_name_number +, O +W283 B-residue_name_number +, O +W306 B-residue_name_number +— O +comprise O +the O +flat B-site +platform I-site +that O +stacks B-bond_interaction +along O +the O +naturally O +twisted O +β B-chemical +- I-chemical +glucan I-chemical +backbone O +( O +Fig O +. O +4E O +). O + +The O +functional O +importance O +of O +this O +platform B-site +is O +underscored O +by O +the O +observation O +that O +the O +W82A B-mutant +W283A B-mutant +W306A B-mutant +mutant B-protein_state +of O +SGBP B-protein +- I-protein +A I-protein +, O +designated O +SGBP B-mutant +- I-mutant +A I-mutant +*, I-mutant +is O +completely B-protein_state +devoid I-protein_state +of I-protein_state +XyG I-protein_state +affinity I-protein_state +( O +Table O +3 O +; O +see O +Fig O +. O +S4 O +in O +the O +supplemental O +material O +). O + +Dissection O +of O +the O +individual O +contribution O +of O +these O +residues O +reveals O +that O +the O +W82A B-mutant +mutant B-protein_state +displays O +a O +significant O +4 O +. O +9 O +- O +fold O +decrease O +in O +the O +Ka B-evidence +value O +for O +XyG B-chemical +, O +while O +the O +W306A B-mutant +substitution B-experimental_method +completely O +abolishes B-protein_state +XyG I-protein_state +binding I-protein_state +. O + +Contrasting O +with O +the O +clear O +importance O +of O +these O +hydrophobic B-bond_interaction +interactions I-bond_interaction +, O +there O +are O +remarkably O +few O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +interactions I-bond_interaction +with O +the O +ligand B-chemical +, O +which O +are O +provided O +by O +R65 B-residue_name_number +, O +N83 B-residue_name_number +, O +and O +S308 B-residue_name_number +, O +which O +are O +proximal O +to O +Glc5 B-residue_name_number +and O +Glc3 B-residue_name_number +. O + +Most O +surprising O +in O +light O +of O +the O +saccharide B-evidence +- I-evidence +binding I-evidence +data I-evidence +, O +however O +, O +was O +a O +lack O +of O +extensive O +recognition O +of O +the O +XyG B-chemical +side O +chains O +; O +only O +Y84 B-residue_name_number +appeared O +to O +provide O +a O +hydrophobic B-site +interface I-site +for O +a O +xylosyl B-chemical +residue O +( O +Xyl1 B-residue_name_number +). O + +Summary O +of O +thermodynamic O +parameters O +for O +site O +- O +directed O +mutants O +of O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +obtained O +by O +ITC B-experimental_method +with O +XyG B-chemical +at O +25 O +° O +Ca O + +Protein O +name O +Ka B-evidence +ΔG B-evidence +( O +kcal O +⋅ O +mol O +− O +1 O +) O +ΔH B-evidence +( O +kcal O +⋅ O +mol O +− O +1 O +) O +TΔS B-evidence +( O +kcal O +⋅ O +mol O +− O +1 O +) O +Fold O +changeb O +M O +− O +1 O +SGBP B-protein +- I-protein +A I-protein +( O +W82A B-mutant +W283A B-mutant +W306A B-mutant +) O +ND O +NB O +NB O +NB O +NB O +SGBP B-protein +- I-protein +A I-protein +( O +W82A B-mutant +) O +c O +4 O +. O +9 O +9 O +. O +1 O +× O +104 O +− O +6 O +. O +8 O +− O +6 O +. O +3 O +0 O +. O +5 O +SGBP B-protein +- I-protein +A I-protein +( O +W306 B-residue_name_number +) O +ND O +NB O +NB O +NB O +NB O +SGBP B-protein +- I-protein +B I-protein +( O +230 B-residue_range +– I-residue_range +489 I-residue_range +) O +0 O +. O +7 O +( O +8 O +. O +6 O +± O +0 O +. O +20 O +) O +× O +104 O +− O +6 O +. O +7 O +− O +14 O +. O +9 O +± O +0 O +. O +1 O +− O +8 O +. O +2 O +SGBP B-protein +- I-protein +B I-protein +( O +Y363A B-mutant +) O +19 O +. O +7 O +( O +2 O +. O +9 O +± O +0 O +. O +10 O +) O +× O +103 O +− O +4 O +. O +7 O +− O +18 O +. O +1 O +± O +0 O +. O +1 O +− O +13 O +. O +3 O +SGBP B-protein +- I-protein +B I-protein +( O +W364A B-mutant +) O +ND O +Weak O +Weak O +Weak O +Weak O +SGBP B-protein +- I-protein +B I-protein +( O +F414A B-mutant +) O +3 O +. O +2 O +( O +1 O +. O +80 O +± O +0 O +. O +03 O +) O +× O +104 O +− O +5 O +. O +8 O +− O +11 O +. O +4 O +± O +0 O +. O +1 O +− O +5 O +. O +6 O + +Binding O +thermodynamics O +are O +based O +on O +the O +concentration O +of O +the O +binding O +unit O +, O +XyGO2 B-chemical +. O + +Weak O +binding O +represents O +a O +Ka B-evidence +of O +< O +500 O +M O +− O +1 O +. O + +Ka B-evidence +fold O +change O += O +Ka B-evidence +of O +wild B-protein_state +- I-protein_state +type I-protein_state +protein O +/ O +Ka B-evidence +of O +mutant O +protein O +for O +xyloglucan B-chemical +binding O +. O + +SGBP B-protein +- I-protein +B I-protein +has O +a O +multimodular O +structure O +with O +a O +single O +, O +C O +- O +terminal O +glycan B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +. O + +The O +crystal B-evidence +structure I-evidence +of O +full B-protein_state +- I-protein_state +length I-protein_state +SGBP B-protein +- I-protein +B I-protein +in B-protein_state +complex I-protein_state +with I-protein_state +XyGO2 B-chemical +( O +2 O +. O +37 O +Å O +, O +Rwork B-evidence += O +19 O +. O +9 O +%, O +Rfree B-evidence += O +23 O +. O +9 O +%, O +residues O +34 B-residue_range +to I-residue_range +489 I-residue_range +) O +( O +Table O +2 O +) O +revealed O +an O +extended O +structure B-evidence +composed O +of O +three O +tandem B-structure_element +immunoglobulin I-structure_element +( I-structure_element +Ig I-structure_element +)- I-structure_element +like I-structure_element +domains I-structure_element +( O +domains O +A B-structure_element +, O +B B-structure_element +, O +and O +C B-structure_element +) O +followed O +at O +the O +C O +terminus O +by O +a O +novel O +xyloglucan B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +( O +domain O +D B-structure_element +) O +( O +Fig O +. O +5A O +). O + +Domains O +A B-structure_element +, O +B B-structure_element +, O +and O +C B-structure_element +display O +similar O +β B-structure_element +- I-structure_element +sandwich I-structure_element +folds I-structure_element +; O +domains O +B B-structure_element +( O +residues O +134 B-residue_range +to I-residue_range +230 I-residue_range +) O +and O +C B-structure_element +( O +residues O +231 B-residue_range +to I-residue_range +313 I-residue_range +) O +can O +be O +superimposed B-experimental_method +onto O +domain O +A B-structure_element +( O +residues O +34 B-residue_range +to I-residue_range +133 I-residue_range +) O +with O +RMSDs B-evidence +of O +1 O +. O +1 O +and O +1 O +. O +2 O +Å O +, O +respectively O +, O +for O +47 O +atom O +pairs O +( O +23 O +% O +and O +16 O +% O +sequence O +identity O +, O +respectively O +). O + +These B-structure_element +domains I-structure_element +also O +display O +similarity O +to O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sandwich I-structure_element +domains I-structure_element +of O +many O +GH13 B-protein_type +enzymes I-protein_type +, O +including O +the O +cyclodextrin B-protein_type +glucanotransferase I-protein_type +of O +Geobacillus B-species +stearothermophilus I-species +( O +Fig O +. O +5B O +). O + +Such B-structure_element +domains I-structure_element +are O +not O +typically O +involved O +in O +carbohydrate B-chemical +binding O +. O + +Indeed O +, O +visual B-experimental_method +inspection I-experimental_method +of O +the O +SGBP B-protein +- I-protein +B I-protein +structure B-evidence +, O +as O +well O +as O +individual O +production O +of O +the O +A B-structure_element +and O +B B-structure_element +domains O +and O +affinity B-experimental_method +PAGE I-experimental_method +analysis O +( O +see O +Fig O +. O +S5 O +in O +the O +supplemental O +material O +), O +indicates O +that O +these O +domains O +do O +not O +contribute O +to O +XyG B-chemical +capture O +. O + +On O +the O +other O +hand O +, O +production B-experimental_method +of O +the O +fused B-mutant +domains I-mutant +C I-mutant +and I-mutant +D I-mutant +in O +tandem O +( O +SGBP B-protein +- I-protein +B I-protein +residues O +230 B-residue_range +to I-residue_range +489 I-residue_range +) O +retains O +complete O +binding O +of O +xyloglucan B-chemical +in O +vitro O +, O +with O +the O +observed O +slight O +increase O +in O +affinity O +likely O +arising O +from O +a O +reduced O +potential O +for O +steric O +hindrance O +of O +the O +smaller O +protein O +construct O +during O +polysaccharide B-chemical +interactions O +( O +Table O +3 O +). O + +While O +neither O +the O +full B-protein_state +- I-protein_state +length I-protein_state +protein O +nor O +domain O +D B-structure_element +displays O +structural O +homology O +to O +known O +XyG B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +, O +the O +topology O +of O +SGBP B-protein +- I-protein +B I-protein +resembles O +the O +xylan B-protein_type +- I-protein_type +binding I-protein_type +protein I-protein_type +Bacova_04391 B-protein +( O +PDB O +3ORJ O +) O +encoded O +within O +a O +xylan B-chemical +- O +targeting O +PUL B-gene +of O +B B-species +. I-species +ovatus I-species +( O +Fig O +. O +5C O +). O + +The O +structure B-experimental_method +- I-experimental_method +based I-experimental_method +alignment I-experimental_method +of O +these O +proteins O +reveals O +17 O +% O +sequence O +identity O +, O +with O +a O +core O +RMSD B-evidence +of O +3 O +. O +6 O +Å O +for O +253 O +aligned O +residues O +. O + +While O +there O +is O +no O +substrate O +- O +complexed O +structure O +of O +Bacova_04391 B-protein +available O +, O +the O +binding B-site +site I-site +is O +predicted O +to O +include O +W241 B-residue_name_number +and O +Y404 B-residue_name_number +, O +which O +are O +proximal O +to O +the O +XyGO B-site +binding I-site +site I-site +in O +SGBP B-protein +- I-protein +B I-protein +. O +However O +, O +the O +opposing B-protein_state +, I-protein_state +clamp I-protein_state +- I-protein_state +like I-protein_state +arrangement I-protein_state +of O +these B-structure_element +residues I-structure_element +in O +Bacova_04391 B-protein +is O +clearly O +distinct O +from O +the O +planar B-site +surface I-site +arrangement I-site +of O +the O +residues B-structure_element +that O +interact O +with O +XyG B-chemical +in O +SGBP B-protein +- I-protein +B I-protein +( O +described O +below O +). O + +Multimodular O +structure O +of O +SGBP B-protein +- I-protein +B I-protein +( O +Bacova_02650 B-gene +). O +( O +A O +) O +Full B-protein_state +- I-protein_state +length I-protein_state +structure B-evidence +of O +SGBP B-protein +- I-protein +B I-protein +, O +color O +coded O +by O +domain O +as O +indicated O +. O + +Prolines B-residue_name +between O +domains O +are O +indicated O +as O +spheres O +. O + +An O +omit B-evidence +map I-evidence +( O +2σ O +) O +for O +XyGO2 B-chemical +is O +displayed O +to O +highlight O +the O +location O +of O +the O +glycan B-site +- I-site +binding I-site +site I-site +. O + +( O +B O +) O +Overlay O +of O +SGBP B-protein +- I-protein +B I-protein +domains O +A B-structure_element +, O +B B-structure_element +, O +and O +C B-structure_element +( O +colored O +as O +in O +panel O +A O +), O +with O +a O +C O +- O +terminal O +Ig B-structure_element +- I-structure_element +like I-structure_element +domain I-structure_element +of O +the O +G B-species +. I-species +stearothermophilus I-species +cyclodextrin B-protein_type +glucanotransferase I-protein_type +( O +PDB O +1CYG O +[ O +residues O +375 B-residue_range +to I-residue_range +493 I-residue_range +]) O +in O +green O +. O +( O +C O +) O +Cα O +overlay B-experimental_method +of O +SGBP B-protein +- I-protein +B I-protein +( O +gray O +) O +and O +Bacova_04391 B-protein +( O +PDB O +3ORJ O +) O +( O +pink O +). O + +( O +D O +) O +Close O +- O +up O +omit B-evidence +map I-evidence +for O +the O +XyGO2 B-chemical +ligand O +, O +contoured O +at O +2σ O +. O +( O +E O +) O +Stereo O +view O +of O +the O +xyloglucan B-site +- I-site +binding I-site +site I-site +of O +SGBP B-protein +- I-protein +B I-protein +, O +displaying O +all O +residues O +within O +4 O +Å O +of O +the O +ligand O +. O + +The O +backbone O +glucose B-chemical +residues O +are O +numbered O +from O +the O +nonreducing O +end O +, O +xylose B-chemical +residues O +are O +shown O +as O +X1 B-residue_name_number +, O +X2 B-residue_name_number +, O +and O +X3 B-residue_name_number +, O +potential O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +interactions I-bond_interaction +are O +shown O +as O +dashed O +lines O +, O +and O +the O +distance O +is O +shown O +in O +angstroms O +. O + +Inspection O +of O +the O +tertiary O +structure B-evidence +indicates O +that O +domains O +C B-structure_element +and O +D B-structure_element +are O +effectively O +inseparable O +, O +with O +a O +contact O +interface O +of O +396 O +Å2 O +. O + +Domains O +A B-structure_element +, O +B B-structure_element +, O +and O +C B-structure_element +do O +not O +pack O +against O +each O +other O +. O + +Moreover O +, O +the O +five B-structure_element +- I-structure_element +residue I-structure_element +linkers I-structure_element +between O +these O +first O +three O +domains O +all O +feature O +a O +proline B-residue_name +as O +the O +middle B-structure_element +residue I-structure_element +, O +suggesting O +significant O +conformational O +rigidity O +( O +Fig O +. O +5A O +). O + +Despite O +the O +lack O +of O +sequence O +and O +structural O +conservation O +, O +a O +similarly O +positioned O +proline B-residue_name +joins O +the O +Ig B-structure_element +- I-structure_element +like I-structure_element +domains I-structure_element +of O +the O +xylan O +- O +binding O +Bacova_04391 B-protein +and O +the O +starch B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +SusE B-protein +and O +SusF B-protein +. O +We O +speculate O +that O +this O +is O +a O +biologically O +important O +adaptation O +that O +serves O +to O +project O +the O +glycan B-site +binding I-site +site I-site +of O +these O +proteins O +far O +from O +the O +membrane O +surface O +. O + +Any O +mobility O +of O +SGBP B-protein +- I-protein +B I-protein +on O +the O +surface O +of O +the O +cell O +( O +beyond O +lateral O +diffusion O +within O +the O +membrane O +) O +is O +likely O +imparted O +by O +the O +eight B-structure_element +- I-structure_element +residue I-structure_element +linker I-structure_element +that O +spans O +the O +predicted O +lipidated B-protein_state +Cys B-residue_name +( O +C28 B-residue_name_number +) O +and O +the O +first B-structure_element +β I-structure_element +- I-structure_element +strand I-structure_element +of O +domain O +A B-structure_element +. O +Other O +outer B-protein_type +membrane I-protein_type +proteins I-protein_type +from O +various O +Sus B-complex_assembly +- I-complex_assembly +like I-complex_assembly +systems I-complex_assembly +possess O +a O +similar O +10 B-structure_element +- I-structure_element +to I-structure_element +20 I-structure_element +- I-structure_element +amino I-structure_element +- I-structure_element +acid I-structure_element +flexible I-structure_element +linker I-structure_element +between O +the O +lipidated B-protein_state +Cys B-residue_name +that O +tethers O +the O +protein O +to O +the O +outside O +the O +cell O +and O +the O +first O +secondary O +structure O +element O +. O + +Analogously O +, O +the O +outer B-protein_state +membrane I-protein_state +- I-protein_state +anchored I-protein_state +endo B-protein_type +- I-protein_type +xyloglucanase I-protein_type +BoGH5 B-protein +of O +the O +XyGUL B-gene +contains O +a O +100 B-structure_element +- I-structure_element +amino I-structure_element +- I-structure_element +acid I-structure_element +, I-structure_element +all I-structure_element +- I-structure_element +β I-structure_element +- I-structure_element +strand I-structure_element +, O +N B-structure_element +- I-structure_element +terminal I-structure_element +module I-structure_element +and O +flexible B-structure_element +linker I-structure_element +that O +imparts O +conformational O +flexibility O +and O +distances O +the O +catalytic B-structure_element +module I-structure_element +from O +the O +cell O +surface O +. O + +XyG B-chemical +binds B-protein_state +to I-protein_state +domain O +D B-structure_element +of O +SGBP B-protein +- I-protein +B I-protein +at O +the O +concave B-site +interface I-site +of O +the O +top O +β B-structure_element +- I-structure_element +sheet I-structure_element +, O +with O +binding O +mediated O +by O +loops B-structure_element +connecting O +the O +β B-structure_element +- I-structure_element +strands I-structure_element +. O + +Six O +glucosyl B-chemical +residues O +, O +comprising O +the O +main O +chain O +, O +and O +three O +branching O +xylosyl B-chemical +residues O +of O +XyGO2 B-chemical +can O +be O +modeled O +in O +the O +density B-evidence +( O +Fig O +. O +5D O +; O +cf O +. O + +The O +backbone O +is O +flat O +, O +with O +less O +of O +the O +“ O +twisted O +- O +ribbon O +” O +geometry O +observed O +in O +some O +cello B-chemical +- I-chemical +and I-chemical +xylogluco I-chemical +- I-chemical +oligosaccharides I-chemical +. O + +The O +aromatic B-site +platform I-site +created O +by O +W330 B-residue_name_number +, O +W364 B-residue_name_number +, O +and O +Y363 B-residue_name_number +spans O +four O +glucosyl B-chemical +residues O +, O +compared O +to O +the O +longer B-protein_state +platform B-site +of O +SGBP B-protein +- I-protein +A I-protein +, O +which O +supports O +six O +glucosyl B-chemical +residues O +( O +Fig O +. O +5E O +). O + +The O +Y363A B-mutant +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutant I-experimental_method +of O +SGBP B-protein +- I-protein +B I-protein +displays O +a O +20 O +- O +fold O +decrease O +in O +the O +Ka B-evidence +for O +XyG B-chemical +, O +while O +the O +W364A B-mutant +mutant B-protein_state +lacks B-protein_state +XyG I-protein_state +binding I-protein_state +( O +Table O +3 O +; O +see O +Fig O +. O +S6 O +in O +the O +supplemental O +material O +). O + +There O +are O +no O +additional O +contacts O +between O +the O +protein O +and O +the O +β B-chemical +- I-chemical +glucan I-chemical +backbone O +and O +surprisingly O +few O +interactions O +with O +the O +side O +- O +chain O +xylosyl B-chemical +residues O +, O +despite O +that O +fact O +that O +ITC B-experimental_method +data O +demonstrate O +that O +SGBP B-protein +- I-protein +B I-protein +does O +not O +measurably O +bind O +the O +cellohexaose B-chemical +( O +Table O +1 O +). O + +F414 B-residue_name_number +stacks B-bond_interaction +with O +the O +xylosyl B-chemical +residue O +of O +Glc3 B-residue_name_number +, O +while O +Q407 B-residue_name_number +is O +positioned O +for O +hydrogen B-bond_interaction +bonding I-bond_interaction +with O +the O +O4 O +of O +xylosyl B-chemical +residue O +Xyl1 B-residue_name_number +. O + +Surprisingly O +, O +an O +F414A B-mutant +mutant B-protein_state +of O +SGBP B-protein +- I-protein +B I-protein +displays O +only O +a O +mild O +3 O +- O +fold O +decrease O +in O +the O +Ka B-evidence +value O +for O +XyG B-chemical +, O +again O +suggesting O +that O +glycan B-chemical +recognition O +is O +primarily O +mediated O +via O +contact O +with O +the O +β O +- O +glucan O +backbone O +( O +Table O +3 O +; O +see O +Fig O +. O +S6 O +). O + +Additional O +residues B-structure_element +surrounding O +the O +binding B-site +site I-site +, O +including O +Y369 B-residue_name_number +and O +E412 B-residue_name_number +, O +may O +contribute O +to O +the O +recognition O +of O +more O +highly O +decorated O +XyG B-chemical +, O +but O +precisely O +how O +this O +is O +mediated O +is O +presently O +unclear O +. O + +Hoping O +to O +achieve O +a O +higher O +- O +resolution O +view O +of O +the O +SGBP B-protein +- I-protein +B I-protein +– O +xyloglucan B-chemical +interaction O +, O +we O +solved B-experimental_method +the O +crystal B-evidence +structure I-evidence +of O +the O +fused B-mutant +CD I-mutant +domains I-mutant +in B-protein_state +complex I-protein_state +with I-protein_state +XyGO2 B-chemical +( O +1 O +. O +57 O +Å O +, O +Rwork B-evidence += O +15 O +. O +6 O +%, O +Rfree B-evidence += O +17 O +. O +1 O +%, O +residues O +230 B-residue_range +to I-residue_range +489 I-residue_range +) O +( O +Table O +2 O +). O + +The O +CD B-structure_element +domains I-structure_element +of O +the O +truncated B-protein_state +and O +full B-protein_state +- I-protein_state +length I-protein_state +proteins O +superimpose B-experimental_method +with O +a O +0 O +. O +4 O +- O +Å O +RMSD B-evidence +of O +the O +Cα O +backbone O +, O +with O +no O +differences O +in O +the O +position O +of O +any O +of O +the O +glycan B-site +- I-site +binding I-site +residues I-site +( O +see O +Fig O +. O +S7A O +in O +the O +supplemental O +material O +). O + +While O +density B-evidence +is O +observed O +for O +XyGO2 B-chemical +, O +the O +ligand O +could O +not O +be O +unambiguously O +modeled O +into O +this O +density B-evidence +to O +achieve O +a O +reasonable O +fit O +between O +the O +X B-evidence +- I-evidence +ray I-evidence +data I-evidence +and O +the O +known O +stereochemistry O +of O +the O +sugar O +( O +see O +Fig O +. O +S7B O +and O +C O +). O + +While O +this O +may O +occur O +for O +a O +number O +of O +reasons O +in O +crystal B-evidence +structures I-evidence +, O +it O +is O +likely O +that O +the O +poor O +ligand O +density O +even O +at O +higher O +resolution O +is O +due O +to O +movement O +or O +multiple O +orientations O +of O +the O +sugar B-chemical +averaged O +throughout O +the O +lattice O +. O + +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +have O +distinct O +, O +coordinated O +functions O +in O +vivo O +. O + +The O +similarity O +of O +the O +glycan B-chemical +specificity O +of O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +presents O +an O +intriguing O +conundrum O +regarding O +their O +individual O +roles O +in O +XyG B-chemical +utilization O +by O +B B-species +. I-species +ovatus I-species +. O + +To O +disentangle O +the O +functions O +of O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +in O +XyG B-chemical +recognition O +and O +uptake O +, O +we O +created O +individual O +in B-experimental_method +- I-experimental_method +frame I-experimental_method +deletion I-experimental_method +and I-experimental_method +complementation I-experimental_method +mutant I-experimental_method +strains O +of O +B B-species +. I-species +ovatus I-species +. O + +In O +these O +growth B-experimental_method +experiments I-experimental_method +, O +overnight O +cultures O +of O +strains O +grown O +on O +minimal O +medium O +plus O +glucose B-chemical +were O +back O +- O +diluted O +1 O +: O +100 O +- O +fold O +into O +minimal O +medium O +containing O +5 O +mg O +/ O +ml O +of O +the O +reported O +carbohydrate B-chemical +. O + +Growth O +on O +glucose B-chemical +displayed O +the O +shortest O +lag B-evidence +time I-evidence +for O +each O +strain O +, O +and O +so O +lag B-evidence +times I-evidence +were O +normalized O +for O +each O +carbohydrate B-chemical +by O +subtracting O +the O +lag B-evidence +time I-evidence +of O +that O +strain O +in O +glucose B-chemical +( O +Fig O +. O +6 O +; O +see O +Fig O +. O +S8 O +in O +the O +supplemental O +material O +). O + +A O +strain O +in O +which O +the O +entire O +XyGUL B-gene +is O +deleted B-experimental_method +displays O +a O +lag B-evidence +of O +24 O +. O +5 O +h O +during O +growth O +on O +glucose B-chemical +compared O +to O +the O +isogenic O +parental O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +Δtdk B-mutant +strain O +, O +for O +which O +exponential O +growth O +lags B-evidence +for O +19 O +. O +8 O +h O +( O +see O +Fig O +. O +S8D O +). O + +It O +is O +unknown O +whether O +this O +is O +because O +cultures O +were O +not O +normalized O +by O +the O +starting O +optical O +density O +( O +OD O +) O +or O +viable O +cells O +or O +reflects O +a O +minor O +defect O +for O +glucose B-chemical +utilization O +. O + +The O +former O +seems O +more O +likely O +as O +the O +growth O +rates O +are O +nearly O +identical O +for O +these O +strains O +on O +glucose B-chemical +and O +xylose B-chemical +. O + +The O +ΔXyGUL B-mutant +and O +WT B-protein_state +Δtdk B-mutant +strains O +display O +normalized O +lag B-evidence +times I-evidence +on O +xylose B-chemical +within O +experimental O +error O +, O +and O +curiously O +some O +of O +the O +mutant O +and O +complemented O +strains O +display O +a O +nominally O +shorter O +lag B-evidence +time I-evidence +on O +xylose B-chemical +than O +the O +WT B-protein_state +Δtdk B-mutant +strain O +. O + +Complementation B-experimental_method +of O +the O +ΔSGBP B-mutant +- I-mutant +A I-mutant +strain O +( O +ΔSGBP B-mutant +- I-mutant +A I-mutant +:: O +SGBP B-protein +- I-protein +A I-protein +) O +restores O +growth O +to O +wild B-protein_state +- I-protein_state +type I-protein_state +rates O +on O +xyloglucan B-chemical +and O +XyGO1 B-chemical +, O +yet O +the O +calculated O +rate O +of O +the O +complemented O +strain O +is O +~ O +72 O +% O +that O +of O +the O +WT B-protein_state +Δtdk B-mutant +strain O +on O +XyGO2 B-chemical +; O +similar O +results O +were O +obtained O +for O +the O +SGBP B-protein +- I-protein +B I-protein +complemented O +strain O +despite O +the O +fact O +that O +the O +growth O +curves O +do O +not O +appear O +much O +different O +( O +see O +Fig O +. O +S8C O +and O +F O +). O + +The O +reason O +for O +this O +observation O +on O +XyGO2 B-chemical +is O +unclear O +, O +as O +the O +ΔSGBP B-mutant +- I-mutant +B I-mutant +mutant B-protein_state +does O +not O +have O +a O +significantly O +different O +growth O +rate O +from O +the O +WT B-protein_state +on O +XyGO2 B-chemical +. O + +Growth O +of O +select O +XyGUL B-gene +mutants O +on O +xyloglucan B-chemical +and O +oligosaccharides B-chemical +. O + +B B-species +. I-species +ovatus I-species +mutants O +were O +created O +in O +a O +thymidine B-mutant +kinase I-mutant +deletion I-mutant +( O +Δtdk B-mutant +) O +mutant O +as O +described O +previously O +. O + +SGBP B-mutant +- I-mutant +A I-mutant +* I-mutant +denotes O +the O +Bacova_02651 B-gene +( O +W82A B-mutant +W283A B-mutant +W306A B-mutant +) O +allele O +, O +and O +the O +GH9 B-protein +gene O +is O +Bacova_02649 B-gene +. O + +Growth O +was O +measured O +over O +time O +in O +minimal O +medium O +containing O +( O +A O +) O +XyG B-chemical +, O +( O +B O +) O +XyGO2 B-chemical +, O +( O +C O +) O +XyGO1 B-chemical +, O +( O +D O +) O +glucose B-chemical +, O +and O +( O +E O +) O +xylose B-chemical +. O + +In O +panel O +F O +, O +the O +growth O +rate O +of O +each O +strain O +on O +the O +five O +carbon O +sources O +is O +displayed O +, O +and O +in O +panel O +G O +, O +the O +normalized O +lag B-evidence +time I-evidence +of O +each O +culture O +, O +relative O +to O +its O +growth O +on O +glucose B-chemical +, O +is O +displayed O +. O + +Solid O +bars O +indicate O +conditions O +that O +are O +not O +statistically O +significant O +from O +the O +WT B-protein_state +Δtdk B-mutant +cultures O +grown O +on O +the O +indicated O +carbohydrate B-chemical +, O +while O +open O +bars O +indicate O +a O +P O +value O +of O +< O +0 O +. O +005 O +compared O +to O +the O +WT B-protein_state +Δtdk B-mutant +strain O +. O + +Conditions O +denoted O +by O +the O +same O +letter O +( O +b O +, O +c O +, O +or O +d O +) O +are O +not O +statistically O +significant O +from O +each O +other O +but O +are O +significantly O +different O +from O +the O +condition O +labeled O +“ O +a O +.” O +Complementation O +of O +ΔSGBP B-mutant +- I-mutant +A I-mutant +and O +ΔSBGP B-mutant +- I-mutant +B I-mutant +was O +performed O +by O +allelic O +exchange O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +genes O +back O +into O +the O +genome O +for O +expression O +via O +the O +native O +promoter O +: O +these O +growth O +curves O +, O +quantified O +rates O +and O +lag B-evidence +times I-evidence +are O +displayed O +in O +Fig O +. O +S8 O +in O +the O +supplemental O +material O +. O + +The O +ΔSGBP B-mutant +- I-mutant +A I-mutant +( O +ΔBacova_02651 B-mutant +) O +strain O +( O +cf O +. O + +Fig O +. O +1B O +) O +was O +completely O +incapable O +of O +growth O +on O +XyG B-chemical +, O +XyGO1 B-chemical +, O +and O +XyGO2 B-chemical +, O +indicating O +that O +SGBP B-protein +- I-protein +A I-protein +is O +essential O +for O +XyG B-chemical +utilization O +( O +Fig O +. O +6 O +). O + +This O +result O +mirrors O +our O +previous O +data O +for O +the O +canonical O +Sus B-complex_assembly +of O +B B-species +. I-species +thetaiotaomicron I-species +, O +which O +revealed O +that O +a O +homologous O +ΔsusD B-mutant +mutant B-protein_state +is O +unable O +to O +grow O +on O +starch B-chemical +or O +malto B-chemical +- I-chemical +oligosaccharides I-chemical +, O +despite O +normal O +cell O +surface O +expression O +of O +all O +other O +PUL B-gene +- O +encoded O +proteins O +. O + +More O +recently O +, O +we O +demonstrated O +that O +this O +phenotype O +is O +due O +to O +the O +loss O +of O +the O +physical O +presence O +of O +SusD B-protein +; O +complementation B-experimental_method +of O +ΔsusD B-mutant +with O +SusD B-mutant +*, I-mutant +a O +triple B-protein_state +site I-protein_state +- I-protein_state +directed I-protein_state +mutant I-protein_state +( O +W96A B-mutant +W320A B-mutant +Y296A B-mutant +) O +that O +ablates B-protein_state +glycan I-protein_state +binding I-protein_state +, O +restores O +B B-species +. I-species +thetaiotaomicron I-species +growth O +on O +malto B-chemical +- I-chemical +oligosaccharides I-chemical +and O +starch B-chemical +when O +sus B-gene +transcription O +is O +induced O +by O +maltose B-chemical +addition O +. O + +Similarly O +, O +the O +function O +of O +SGBP B-protein +- I-protein +A I-protein +extends O +beyond O +glycan B-chemical +binding O +. O + +Complementation B-experimental_method +of O +ΔSGBP B-mutant +- I-mutant +A I-mutant +with O +the O +SGBP B-mutant +- I-mutant +A I-mutant +* I-mutant +( O +W82A B-mutant +W283A B-mutant +W306A B-mutant +) O +variant O +, O +which O +does O +not B-protein_state +bind I-protein_state +XyG B-chemical +, O +supports O +growth O +on O +XyG B-chemical +and O +XyGOs B-chemical +( O +Fig O +. O +6 O +; O +ΔSGBP B-mutant +- I-mutant +A I-mutant +:: O +SGBP B-mutant +- I-mutant +A I-mutant +*), I-mutant +with O +growth O +rates O +that O +are O +~ O +70 O +% O +that O +of O +the O +WT B-protein_state +. O + +In O +previous O +studies O +, O +we O +observed O +that O +carbohydrate B-chemical +binding O +by O +SusD B-protein +enhanced O +the O +sensitivity O +of O +the O +cells O +to O +limiting O +concentrations O +of O +malto O +- O +oligosaccharides O +by O +several O +orders O +of O +magnitude O +, O +such O +that O +the O +addition O +of O +0 O +. O +5 O +g O +/ O +liter O +maltose B-chemical +was O +required O +to O +restore O +growth O +of O +the O +ΔsusD B-mutant +:: O +SusD B-mutant +* I-mutant +strain O +on O +starch B-chemical +, O +which O +nonetheless O +occurred O +following O +an O +extended O +lag B-evidence +phase I-evidence +. O + +In O +contrast O +, O +the O +ΔSGBP B-mutant +- I-mutant +A I-mutant +:: O +SGBP B-mutant +- I-mutant +A I-mutant +* I-mutant +strain O +does O +not O +display O +an O +extended O +lag B-evidence +time I-evidence +on O +any O +of O +the O +xyloglucan B-chemical +substrates O +compared O +to O +the O +WT B-protein_state +( O +Fig O +. O +6 O +). O + +The O +specific O +glycan B-chemical +signal O +that O +upregulates O +BoXyGUL B-gene +is O +currently O +unknown O +. O + +From O +our O +present O +data O +, O +we O +cannot O +eliminate O +the O +possibility O +that O +the O +glycan B-chemical +binding O +by O +SGBP B-protein +- I-protein +A I-protein +enhances O +transcriptional O +activation O +of O +the O +XyGUL B-gene +. O + +However O +, O +the O +modest O +rate O +defect O +displayed O +by O +the O +SGBP B-protein +- I-protein +A I-protein +:: O +SGBP B-mutant +- I-mutant +A I-mutant +* I-mutant +strain O +suggests O +that O +recognition O +of O +XyG B-chemical +and O +product O +import O +is O +somewhat O +less O +efficient O +in O +these O +cells O +. O + +Intriguingly O +, O +the O +ΔSGBP B-mutant +- I-mutant +B I-mutant +strain O +( O +ΔBacova_02650 B-mutant +) O +( O +cf O +. O + +Fig O +. O +1B O +) O +exhibited O +a O +minor O +growth O +defect O +on O +both O +XyG B-chemical +and O +XyGO2 B-chemical +, O +with O +rates O +84 O +. O +6 O +% O +and O +93 O +. O +9 O +% O +that O +of O +the O +WT B-protein_state +Δtdk B-mutant +strain O +. O + +However O +, O +growth O +of O +the O +ΔSGBP B-mutant +- I-mutant +B I-mutant +strain O +on O +XyGO1 B-chemical +was O +54 O +. O +2 O +% O +the O +rate O +of O +the O +parental O +strain O +, O +despite O +the O +fact O +that O +SGBP B-protein +- I-protein +B I-protein +binds O +this O +substrate O +ca O +. O + +10 O +- O +fold O +more O +weakly O +than O +XyGO2 B-chemical +and O +XyG B-chemical +( O +Fig O +. O +6 O +; O +Table O +1 O +). O + +As O +such O +, O +the O +data O +suggest O +that O +SGBP B-protein +- I-protein +A I-protein +can O +compensate O +for O +the O +loss O +of O +function O +of O +SGBP B-protein +- I-protein +B I-protein +on O +longer O +oligo B-chemical +- I-chemical +and I-chemical +polysaccharides I-chemical +, O +while O +SGBP B-protein +- I-protein +B I-protein +may O +adapt O +the O +cell O +to O +recognize O +smaller O +oligosaccharides B-chemical +efficiently O +. O + +Indeed O +, O +a O +double B-protein_state +mutant I-protein_state +, O +consisting O +of O +a O +crippled B-protein_state +SGBP B-protein +- I-protein +A I-protein +and O +a O +deletion B-experimental_method +of I-experimental_method +SGBP B-protein +- I-protein +B I-protein +( O +ΔSGBP B-mutant +- I-mutant +A I-mutant +:: O +SGBP B-mutant +- I-mutant +A I-mutant +*/ I-mutant +ΔSGBP B-mutant +- I-mutant +B I-mutant +), O +exhibits O +an O +extended O +lag B-evidence +time I-evidence +on O +both O +XyG B-chemical +and O +XyGO2 B-chemical +, O +as O +well O +as O +XyGO1 B-chemical +. O + +Taken O +together O +, O +the O +data O +indicate O +that O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +functionally O +complement O +each O +other O +in O +the O +capture O +of O +XyG B-chemical +polysaccharide B-chemical +, O +while O +SGBP B-protein +- I-protein +B I-protein +may O +allow O +B B-species +. I-species +ovatus I-species +to O +scavenge O +smaller O +XyGOs B-chemical +liberated O +by O +other O +gut O +commensals O +. O + +This O +additional O +role O +of O +SGBP B-protein +- I-protein +B I-protein +is O +especially O +notable O +in O +the O +context O +of O +studies O +on O +BtSusE B-protein +and O +BtSusF B-protein +( O +positioned O +similarly O +in O +the O +archetypal O +Sus B-gene +locus I-gene +) O +( O +Fig O +. O +1B O +), O +for O +which O +growth O +defects O +on O +starch B-chemical +or O +malto B-chemical +- I-chemical +oligosaccharides I-chemical +have O +never O +been O +observed O +. O + +Beyond O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +, O +we O +speculated O +that O +the O +catalytically B-protein_state +feeble I-protein_state +endo B-protein_type +- I-protein_type +xyloglucanase I-protein_type +GH9 B-protein +, O +which O +is O +expendable O +for O +growth O +in O +the O +presence O +of O +GH5 B-protein +, O +might O +also O +play O +a O +role O +in O +glycan B-chemical +binding O +to O +the O +cell O +surface O +. O + +However O +, O +combined B-experimental_method +deletion I-experimental_method +of I-experimental_method +the I-experimental_method +genes I-experimental_method +encoding I-experimental_method +GH9 B-protein +( O +encoded O +by O +Bacova_02649 B-gene +) O +and O +SGBP B-protein +- I-protein +B I-protein +does O +not O +exacerbate O +the O +growth O +defect O +on O +XyGO1 B-chemical +( O +Fig O +. O +6 O +; O +ΔSGBP B-mutant +- I-mutant +B I-mutant +/ O +ΔGH9 B-mutant +). O + +The O +necessity O +of O +SGBP B-protein +- I-protein +B I-protein +is O +elevated O +in O +the O +SGBP B-mutant +- I-mutant +A I-mutant +* I-mutant +strain O +, O +as O +the O +ΔSGBP B-mutant +- I-mutant +A I-mutant +:: O +SGBP B-mutant +- I-mutant +A I-mutant +*/ I-mutant +ΔSGBP B-mutant +- I-mutant +B I-mutant +mutant B-protein_state +displays O +an O +extended O +lag B-evidence +during O +growth O +on O +XyG B-chemical +and O +xylogluco B-chemical +- I-chemical +oligosaccharides I-chemical +, O +while O +growth O +rate O +differences O +are O +more O +subtle O +. O + +The O +precise O +reason O +for O +this O +lag B-evidence +is O +unclear O +, O +but O +recapitulating O +our O +findings O +on O +the O +role O +of O +SusD B-protein +in O +malto B-chemical +- I-chemical +oligosaccharide I-chemical +sensing O +in O +B B-species +. I-species +thetaiotaomicron I-species +, O +this O +extended O +lag B-evidence +may O +be O +due O +to O +inefficient O +import O +and O +thus O +sensing O +of O +xyloglucan B-chemical +in O +the O +environment O +in O +the O +absence O +of O +glycan B-chemical +binding O +by O +essential O +SGBPs B-protein_type +. O + +Our O +previous O +work O +demonstrates O +that O +B B-species +. I-species +ovatus I-species +cells O +grown O +in O +minimal O +medium O +plus O +glucose B-chemical +express O +low O +levels O +of O +the O +XyGUL B-gene +transcript O +. O + +Thus O +, O +in O +our O +experiments O +, O +we O +presume O +that O +each O +strain O +, O +initially O +grown O +in O +glucose B-chemical +, O +expresses O +low O +levels O +of O +the O +XyGUL B-gene +transcript O +and O +thus O +low O +levels O +of O +the O +XyGUL B-gene +- O +encoded O +surface O +proteins O +, O +including O +the O +vanguard O +GH5 B-protein +. O + +Presumably O +without O +glycan B-chemical +binding O +by O +the O +SGBPs B-protein_type +, O +the O +GH5 B-protein +protein O +cannot O +efficiently O +process O +xyloglucan B-chemical +, O +and O +/ O +or O +the O +lack O +of O +SGBP B-protein_type +function O +prevents O +efficient O +capture O +and O +import O +of O +the O +processed O +oligosaccharides B-chemical +. O + +It O +may O +then O +be O +that O +only O +after O +a O +sufficient O +amount O +of O +glycan B-chemical +is O +processed O +and O +imported O +by O +the O +cell O +is O +XyGUL B-gene +upregulated O +and O +exponential O +growth O +on O +the O +glycan B-chemical +can O +begin O +. O + +We O +hypothesize O +that O +during O +exponential O +growth O +the O +essential O +role O +of O +SGBP B-protein +- I-protein +A I-protein +extends O +beyond O +glycan B-chemical +recognition O +, O +perhaps O +due O +to O +a O +critical O +interaction O +with O +the O +TBDT B-protein_type +. O + +In O +the O +BtSus B-gene +, O +SusD B-protein +and O +the O +TBDT B-protein_type +SusC B-protein +interact O +, O +and O +we O +speculate O +that O +this O +interaction O +is O +necessary O +for O +glycan B-chemical +uptake O +, O +as O +suggested O +by O +the O +fact O +that O +a O +ΔsusD B-mutant +mutant B-protein_state +cannot O +grow O +on O +starch B-chemical +, O +but O +a O +ΔsusD B-mutant +:: O +SusD B-mutant +* I-mutant +strain O +regains O +this O +ability O +if O +a O +transcriptional B-protein_type +activator I-protein_type +of O +the O +sus B-gene +operon I-gene +is O +supplied O +. O + +Likewise O +, O +such O +cognate O +interactions O +between O +homologous O +protein O +pairs O +such O +as O +SGBP B-protein +- I-protein +A I-protein +and O +its O +TBDT B-protein_type +may O +underlie O +our O +observation O +that O +a O +ΔSGBP B-mutant +- I-mutant +A I-mutant +mutant B-protein_state +cannot O +grow O +on O +xyloglucan B-chemical +. O + +However O +, O +unlike O +the O +Sus B-complex_assembly +, O +in O +which O +elimination B-experimental_method +of I-experimental_method +SusE B-protein +and O +SusF B-protein +does O +not O +affect O +growth O +on O +starch B-chemical +, O +SGBP B-protein +- I-protein +B I-protein +appears O +to O +have O +a O +dedicated O +role O +in O +growth O +on O +small O +xylogluco B-chemical +- I-chemical +oligosaccharides I-chemical +. O + +The O +ability O +of O +gut O +- O +adapted O +microorganisms B-taxonomy_domain +to O +thrive O +in O +the O +gastrointestinal O +tract O +is O +critically O +dependent O +upon O +their O +ability O +to O +efficiently O +recognize O +, O +cleave O +, O +and O +import O +glycans B-chemical +. O + +The O +human B-species +gut O +, O +in O +particular O +, O +is O +a O +densely O +packed O +ecosystem O +with O +hundreds O +of O +species O +, O +in O +which O +there O +is O +potential O +for O +both O +competition O +and O +synergy O +in O +the O +utilization O +of O +different O +substrates O +. O + +Recent O +work O +has O +elucidated O +that O +Bacteroidetes B-taxonomy_domain +cross O +- O +feed O +during O +growth O +on O +many O +glycans B-chemical +; O +the O +glycoside B-protein_type +hydrolases I-protein_type +expressed O +by O +one O +species O +liberate O +oligosaccharides B-chemical +for O +consumption O +by O +other O +members O +of O +the O +community O +. O + +Thus O +, O +understanding O +glycan B-chemical +capture O +at O +the O +cell O +surface O +is O +fundamental O +to O +explaining O +, O +and O +eventually O +predicting O +, O +how O +the O +carbohydrate O +content O +of O +the O +diet O +shapes O +the O +gut O +community O +structure O +as O +well O +as O +its O +causative O +health O +effects O +. O + +Here O +, O +we O +demonstrate O +that O +the O +surface B-protein_type +glycan I-protein_type +binding I-protein_type +proteins I-protein_type +encoded O +within O +the O +BoXyGUL B-gene +play O +unique O +and O +essential O +roles O +in O +the O +acquisition O +of O +the O +ubiquitous O +and O +abundant O +vegetable B-taxonomy_domain +polysaccharide B-chemical +xyloglucan B-chemical +. O + +Yet O +, O +a O +number O +of O +questions O +remain O +regarding O +the O +molecular O +interplay O +of O +SGBPs B-protein_type +with O +their O +cotranscribed O +cohort O +of O +glycoside B-protein_type +hydrolases I-protein_type +and O +TonB B-protein_type +- I-protein_type +dependent I-protein_type +transporters I-protein_type +. O + +A O +particularly O +understudied O +aspect O +of O +glycan B-chemical +utilization O +is O +the O +mechanism O +of O +import O +via O +TBDTs B-protein_type +( O +SusC B-protein +homologs O +) O +( O +Fig O +. O +1 O +), O +which O +are O +ubiquitous O +and O +defining O +components O +of O +all O +PUL B-gene +. O + +PUL B-gene +- O +encoded O +TBDTs B-protein_type +in O +Bacteroidetes B-taxonomy_domain +are O +larger O +than O +the O +well O +- O +characterized O +iron B-protein_type +- I-protein_type +targeting I-protein_type +TBDTs I-protein_type +from O +many O +Proteobacteria B-taxonomy_domain +and O +are O +further O +distinguished O +as O +the O +only O +known O +glycan B-protein_type +- I-protein_type +importing I-protein_type +TBDTs I-protein_type +coexpressed O +with O +an O +SGBP B-protein_type +. O + +A O +direct O +interaction O +between O +the O +BtSusC B-protein +TBDT B-protein_type +and O +the O +SusD B-protein +SGBP B-protein_type +has O +been O +previously O +demonstrated O +, O +as O +has O +an O +interaction O +between O +the O +homologous O +components O +encoded O +by O +an O +N O +- O +glycan B-chemical +- O +scavenging O +PUL B-gene +of O +Capnocytophaga B-species +canimorsus I-species +. O + +Our O +observation O +here O +that O +the O +physical O +presence O +of O +the O +SusD B-protein +homolog O +SGBP B-protein +- I-protein +A I-protein +, O +independent O +of O +XyG B-chemical +- O +binding O +ability O +, O +is O +both O +necessary O +and O +sufficient O +for O +XyG B-chemical +utilization O +further O +supports O +a O +model O +of O +glycan B-chemical +import O +whereby O +the O +SusC B-protein_type +- I-protein_type +like I-protein_type +TBDTs I-protein_type +and O +the O +SusD B-protein_type +- I-protein_type +like I-protein_type +SGBPs I-protein_type +must O +be O +intimately O +associated O +to O +support O +glycan B-chemical +uptake O +( O +Fig O +. O +1C O +). O + +It O +is O +yet O +presently O +unclear O +whether O +this O +interaction O +is O +static O +or O +dynamic O +and O +to O +what O +extent O +the O +association O +of O +cognate O +TBDT B-protein_type +/ O +SGBPs B-protein_type +is O +dependent O +upon O +the O +structure O +of O +the O +carbohydrate B-chemical +to O +be O +imported O +. O + +On O +the O +other O +hand O +, O +there O +is O +clear O +evidence O +for O +independent O +TBDTs B-protein_type +in O +Bacteroidetes B-taxonomy_domain +that O +do O +not O +require O +SGBP B-protein_type +association O +for O +activity O +. O + +For O +example O +, O +it O +was O +recently O +demonstrated O +that O +expression O +of O +nanO B-gene +, O +which O +encodes O +a O +SusC B-protein_type +- I-protein_type +like I-protein_type +TBDT I-protein_type +as O +part O +of O +a O +sialic O +- O +acid O +- O +targeting O +PUL B-gene +from O +B B-species +. I-species +fragilis I-species +, O +restored O +growth O +on O +this O +monosaccharide B-chemical +in O +a O +mutant O +strain O +of O +E B-species +. I-species +coli I-species +. O + +In O +this O +instance O +, O +coexpression O +of O +the O +susD B-gene +- O +like O +gene O +nanU B-gene +was O +not O +required O +, O +nor O +did O +the O +expression O +of O +the O +nanU B-gene +gene O +enhance O +growth O +kinetics O +. O + +Similarly O +, O +the O +deletion O +of O +BT1762 B-gene +encoding O +a O +fructan B-protein_type +- I-protein_type +targeting I-protein_type +SusD I-protein_type +- I-protein_type +like I-protein_type +protein I-protein_type +in O +B B-species +. I-species +thetaiotaomicron I-species +did O +not O +result O +in O +a O +dramatic O +loss O +of O +growth O +on O +fructans B-chemical +. O + +Thus O +, O +the O +strict O +dependence O +on O +a O +SusD B-protein_type +- I-protein_type +like I-protein_type +SGBP I-protein_type +for O +glycan B-chemical +uptake O +in O +the O +Bacteroidetes B-taxonomy_domain +may O +be O +variable O +and O +substrate O +dependent O +. O + +Furthermore O +, O +considering O +the O +broader O +distribution O +of O +TBDTs B-protein_type +in O +PUL B-gene +lacking O +SGBPs B-protein_type +( O +sometimes O +known O +as O +carbohydrate B-gene +utilization I-gene +containing I-gene +TBDT I-gene +[ I-gene +CUT I-gene +] I-gene +loci I-gene +; O +see O +reference O +and O +reviewed O +in O +reference O +) O +across O +bacterial B-taxonomy_domain +phyla O +, O +it O +appears O +that O +the O +intimate O +biophysical O +association O +of O +these O +substrate O +- O +transport O +and O +- O +binding O +proteins O +is O +the O +result O +of O +specific O +evolution O +within O +the O +Bacteroidetes B-taxonomy_domain +. O + +Equally O +intriguing O +is O +the O +observation O +that O +while O +SusD B-protein_type +- I-protein_type +like I-protein_type +proteins I-protein_type +such O +as O +SGBP B-protein +- I-protein +A I-protein +share O +moderate O +primary O +and O +high O +tertiary O +structural O +conservation O +, O +the O +genes O +for O +the O +SGBPs B-protein_type +encoded O +immediately O +downstream O +( O +Fig O +. O +1B O +[ O +sometimes O +referred O +to O +as O +“ O +susE O +positioned O +”]) O +encode O +glycan B-protein_type +- I-protein_type +binding I-protein_type +lipoproteins I-protein_type +with O +little O +or O +no O +sequence O +or O +structural O +conservation O +, O +even O +among O +syntenic O +PUL B-gene +that O +target O +the O +same O +polysaccharide B-chemical +. O + +Such O +is O +the O +case O +for O +XyGUL B-gene +from O +related O +Bacteroides B-taxonomy_domain +species O +, O +which O +may O +encode O +either O +one O +or O +two O +of O +these O +predicted O +SGBPs B-protein_type +, O +and O +these O +proteins O +vary O +considerably O +in O +length O +. O + +The O +extremely O +low O +similarity O +of O +these O +SGBPs B-protein_type +is O +striking O +in O +light O +of O +the O +moderate O +sequence O +conservation O +observed O +among O +homologous O +GHs B-protein_type +in O +syntenic O +PUL B-gene +. O + +This O +, O +together O +with O +the O +observation O +that O +these O +SGBPs B-protein_type +, O +as O +exemplified O +by O +BtSusE B-protein +and O +BtSusF B-protein +and O +the O +XyGUL B-gene +SGBP B-protein +- I-protein +B I-protein +of O +the O +present O +study O +, O +are O +expendable O +for O +polysaccharide B-chemical +growth O +, O +implies O +a O +high O +degree O +of O +evolutionary O +flexibility O +to O +enhance O +glycan B-chemical +capture O +at O +the O +cell O +surface O +. O + +Because O +the O +intestinal O +ecosystem O +is O +a O +dense O +consortium O +of O +bacteria B-taxonomy_domain +that O +must O +compete O +for O +their O +nutrients O +, O +these O +multimodular O +SGBPs B-protein_type +may O +reflect O +ongoing O +evolutionary O +experiments O +to O +enhance O +glycan B-chemical +uptake O +efficiency O +. O + +Whether O +organisms O +that O +express O +longer O +SGBPs B-protein_type +, O +extending O +further O +above O +the O +cell O +surface O +toward O +the O +extracellular O +environment O +, O +are O +better O +equipped O +to O +compete O +for O +available O +carbohydrates B-chemical +is O +presently O +unknown O +. O + +However O +, O +the O +natural O +diversity O +of O +these O +proteins O +represents O +a O +rich O +source O +for O +the O +discovery O +of O +unique O +carbohydrate B-structure_element +- I-structure_element +binding I-structure_element +motifs I-structure_element +to O +both O +inform O +gut O +microbiology O +and O +generate O +new O +, O +specific O +carbohydrate B-chemical +analytical O +reagents O +. O + +In O +conclusion O +, O +the O +present O +study O +further O +illuminates O +the O +essential O +role O +that O +surface B-protein_type +- I-protein_type +glycan I-protein_type +binding I-protein_type +proteins I-protein_type +play O +in O +facilitating O +the O +catabolism O +of O +complex O +dietary O +carbohydrates B-chemical +by O +Bacteroidetes B-taxonomy_domain +. O + +The O +ability O +of O +our O +resident O +gut O +bacteria B-taxonomy_domain +to O +recognize O +polysaccharides B-chemical +is O +the O +first O +committed O +step O +of O +glycan B-chemical +consumption O +by O +these O +organisms O +, O +a O +critical O +process O +that O +influences O +the O +community O +structure O +and O +thus O +the O +metabolic O +output O +( O +i O +. O +e O +., O +short O +- O +chain O +fatty O +acid O +and O +metabolite O +profile O +) O +of O +these O +organisms O +. O + +A O +molecular O +understanding O +of O +glycan B-chemical +uptake O +by O +human B-species +gut O +bacteria B-taxonomy_domain +is O +therefore O +central O +to O +the O +development O +of O +strategies O +to O +improve O +human B-species +health O +through O +manipulation O +of O +the O +microbiota B-taxonomy_domain +. O + +Inhibiting O +complex O +IL B-protein +- I-protein +17A I-protein +and O +IL B-protein +- I-protein +17RA I-protein +interactions O +with O +a O +linear O +peptide B-chemical + +IL B-protein +- I-protein +17A I-protein +is O +a O +pro O +- O +inflammatory O +cytokine B-protein_type +that O +has O +been O +implicated O +in O +autoimmune O +and O +inflammatory O +diseases O +. O + +Monoclonal O +antibodies B-protein_type +inhibiting O +IL B-protein +- I-protein +17A I-protein +signaling O +have O +demonstrated O +remarkable O +efficacy O +, O +but O +an O +oral O +therapy O +is O +still O +lacking O +. O + +A O +high B-chemical +affinity I-chemical +IL I-chemical +- I-chemical +17A I-chemical +peptide I-chemical +antagonist I-chemical +( O +HAP B-chemical +) O +of O +15 B-residue_range +residues I-residue_range +was O +identified O +through O +phage B-experimental_method +- I-experimental_method +display I-experimental_method +screening I-experimental_method +followed O +by O +saturation B-experimental_method +mutagenesis I-experimental_method +optimization I-experimental_method +and O +amino B-experimental_method +acid I-experimental_method +substitutions I-experimental_method +. O + +HAP B-chemical +binds O +specifically O +to O +IL B-protein +- I-protein +17A I-protein +and O +inhibits O +the O +interaction O +of O +the O +cytokine B-protein_type +with O +its O +receptor B-protein_type +, O +IL B-protein +- I-protein +17RA I-protein +. O + +Tested O +in O +primary O +human B-species +cells O +, O +HAP B-chemical +blocked O +the O +production O +of O +multiple O +inflammatory O +cytokines B-protein_type +. O + +Crystal B-experimental_method +structure I-experimental_method +studies I-experimental_method +revealed O +that O +two O +HAP B-chemical +molecules O +bind O +to O +one O +IL B-protein +- I-protein +17A I-protein +dimer B-oligomeric_state +symmetrically O +. O + +The O +N O +- O +terminal O +portions O +of O +HAP B-chemical +form O +a O +β B-structure_element +- I-structure_element +strand I-structure_element +that O +inserts O +between O +two O +IL B-protein +- I-protein +17A I-protein +monomers B-oligomeric_state +while O +the O +C O +- O +terminal O +section O +forms O +an O +α B-structure_element +helix I-structure_element +that O +directly O +blocks O +IL B-protein +- I-protein +17RA I-protein +from O +binding O +to O +the O +same O +region O +of O +IL B-protein +- I-protein +17A I-protein +. O + +The O +family O +of O +IL B-protein_type +- I-protein_type +17 I-protein_type +cytokines I-protein_type +and O +receptors O +consists O +of O +six O +polypeptides O +, O +IL B-protein +- I-protein +17A I-protein +- I-protein +F I-protein +, O +and O +five O +receptors O +, O +IL B-protein +- I-protein +17RA I-protein +- I-protein +E I-protein +. O +IL B-protein +- I-protein +17A I-protein +is O +secreted O +from O +activated O +Th17 O +cells O +, O +and O +several O +innate O +immune O +T O +cell O +types O +including O +macrophages O +, O +neutrophils O +, O +natural O +killer O +cells O +, O +and O +dendritic O +cells O +. O + +IL B-protein +- I-protein +17A I-protein +signals O +through O +a O +specific O +cell O +surface O +receptor B-protein_type +complex O +which O +consists O +of O +IL B-protein +- I-protein +17RA I-protein +and O +IL B-protein +- I-protein +17RC I-protein +. O + +IL B-protein +- I-protein +17A I-protein +’ O +s O +downstream O +signaling O +leads O +to O +increased O +production O +of O +inflammatory O +cytokines B-protein_type +such O +as O +IL B-protein_type +- I-protein_type +6 I-protein_type +, O +IL B-protein_type +- I-protein_type +8 I-protein_type +, O +CCL B-protein_type +- I-protein_type +20 I-protein_type +and O +CXCL1 B-protein_type +by O +various O +mechanisms O +including O +stimulation O +of O +transcription O +and O +stabilization O +of O +mRNA B-chemical +. O + +Although O +various O +cell O +types O +have O +been O +reported O +to O +express O +IL B-protein +- I-protein +17RA I-protein +, O +the O +highest O +responses O +to O +IL B-protein +- I-protein +17A I-protein +come O +from O +epithelial O +cells O +, O +endothelial O +cells O +, O +keratinocytes O +and O +fibroblasts O +. O + +IL B-protein +- I-protein +17A I-protein +and O +its O +signaling O +is O +important O +in O +host O +defense O +against O +certain O +fungal O +and O +bacterial O +infections O +as O +demonstrated O +by O +patients O +with O +autoantibodies O +against O +IL B-protein +- I-protein +17A I-protein +and O +IL B-protein +- I-protein +17F I-protein +, O +or O +with O +inborn O +errors O +of O +IL B-protein_type +- I-protein_type +17 I-protein_type +immunity O +. O + +In O +addition O +to O +its O +physiological O +role O +, O +IL B-protein +- I-protein +17A I-protein +is O +a O +key O +pathogenic O +factor O +in O +inflammatory O +and O +autoimmune O +diseases O +. O + +In O +phase O +II O +and O +III O +clinical O +trials O +, O +neutralizing O +monoclonal O +antibodies B-protein_type +against O +IL B-protein +- I-protein +17A I-protein +( O +secukinumab B-chemical +and O +ixekizumab B-chemical +) O +or O +its O +receptor B-protein_type +IL B-protein +- I-protein +17RA I-protein +( O +brodalumab B-chemical +) O +are O +highly O +efficacious O +in O +treating O +moderate O +to O +severe O +plaque O +psoriasis O +and O +psoriatic O +arthritis O +. O + +Secukinumab B-chemical +has O +been O +approved O +recently O +as O +a O +new O +psoriasis O +drug O +by O +the O +US O +Food O +and O +Drug O +Administration O +( O +Cosentyx B-chemical +™). I-chemical + +In O +addition O +to O +psoriasis O +and O +psoriatic O +arthritis O +, O +IL B-protein +- I-protein +17A I-protein +blockade O +has O +also O +shown O +preclinical O +and O +clinical O +efficacies O +in O +ankylosing O +spondylitis O +and O +rheumatoid O +arthritis O +. O + +Among O +IL B-protein_type +- I-protein_type +17 I-protein_type +cytokines I-protein_type +, O +IL B-protein +- I-protein +17A I-protein +and O +IL B-protein +- I-protein +17F I-protein +share O +the O +highest O +homology O +. O + +These O +polypeptides O +form O +covalent B-protein_state +homodimers B-oligomeric_state +, O +and O +IL B-protein +- I-protein +17A I-protein +and O +IL B-protein +- I-protein +17F I-protein +also O +form O +an O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17F I-complex_assembly +hetereodimer B-oligomeric_state +. O + +Structures B-evidence +are O +known O +for O +apo B-protein_state +IL B-protein +- I-protein +17F I-protein +and O +its O +complex B-protein_state +with I-protein_state +IL B-protein +- I-protein +17RA I-protein +, O +for O +apo B-protein_state +IL B-protein +- I-protein +17A I-protein +, O +its O +complex B-protein_state +with I-protein_state +an O +antibody B-protein_type +Fab B-structure_element +, O +and O +its O +complex B-protein_state +with I-protein_state +IL B-protein +- I-protein +17RA I-protein +. O + +In O +these O +structures B-evidence +, O +both O +IL B-protein +- I-protein +17A I-protein +and O +IL B-protein +- I-protein +17F I-protein +adopt O +a O +cysteine B-structure_element +- I-structure_element +knot I-structure_element +fold O +with O +two O +intramolecular O +disulfides B-ptm +and O +two O +interchain O +disulfide B-ptm +bonds I-ptm +that O +covalently O +link O +two O +monomers B-oligomeric_state +. O + +There O +has O +been O +active O +research O +in O +identifying O +orally O +available O +chemical O +entities O +that O +would O +functionally O +antagonize O +IL B-protein +- I-protein +17A I-protein +- O +mediated O +signaling O +. O + +Developing O +small O +molecules O +targeting O +protein O +- O +protein O +interactions O +is O +difficult O +with O +particular O +challenges O +associated O +with O +the O +large O +, O +shallow O +IL B-site +- I-site +17A I-site +/ I-site +IL I-site +- I-site +17RA I-site +interfaces I-site +. O + +Since O +IL B-protein +- I-protein +17RA I-protein +is O +a O +shared O +receptor B-protein_type +for O +at O +least O +IL B-protein +- I-protein +17A I-protein +, O +IL B-protein +- I-protein +17F I-protein +, O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17F I-complex_assembly +and O +IL B-protein +- I-protein +17E I-protein +, O +we O +chose O +to O +seek O +IL B-protein +- I-protein +17A I-protein +- O +specific O +inhibitors O +that O +may O +have O +more O +defined O +pharmacological O +responses O +than O +IL B-protein +- I-protein +17RA I-protein +inhibitors O +. O + +Our O +efforts O +resulted O +in O +discovery O +of O +a O +high B-chemical +affinity I-chemical +IL I-chemical +- I-chemical +17A I-chemical +peptide I-chemical +antagonist I-chemical +( O +HAP B-chemical +), O +which O +we O +attempted O +to O +increase O +the O +functional O +production O +and O +pharmacokinetics O +after O +fusing B-experimental_method +HAP B-chemical +to O +antibodies B-protein_type +for O +evaluation O +as O +a O +bispecific O +therapeutic O +in O +animal O +studies O +. O + +Unfortunately O +, O +this O +past O +work O +revealed O +stability O +issues O +of O +the O +uncapped B-protein_state +HAP B-chemical +in O +cell O +culture O +Here O +, O +we O +provide O +the O +details O +of O +the O +discovery O +and O +optimization O +that O +led O +to O +HAP B-chemical +and O +report O +the O +complex B-evidence +structure I-evidence +of O +IL B-protein +- I-protein +17A I-protein +with O +HAP B-chemical +, O +which O +provides O +structure O +based O +rationalization O +of O +peptide B-experimental_method +optimization I-experimental_method +and O +structure B-experimental_method +activity I-experimental_method +relationship I-experimental_method +( O +SAR B-experimental_method +). O + +Identification O +of O +IL B-protein +- I-protein +17A I-protein +peptide O +inhibitors O + +Peptides O +specifically O +binding O +to O +human B-species +IL B-protein +- I-protein +17A I-protein +were O +identified O +from O +phage B-experimental_method +panning I-experimental_method +using O +cyclic B-experimental_method +and I-experimental_method +linear I-experimental_method +peptide I-experimental_method +libraries I-experimental_method +( O +Supplementary O +Figure O +S1 O +). O + +Positive B-experimental_method +phage I-experimental_method +pools I-experimental_method +were O +then O +sub B-experimental_method +- I-experimental_method +cloned I-experimental_method +into O +a O +maltose B-experimental_method +- I-experimental_method +binding I-experimental_method +protein I-experimental_method +( I-experimental_method +MBP I-experimental_method +) I-experimental_method +fusion I-experimental_method +system I-experimental_method +. O + +Single O +clones O +were O +isolated O +and O +sub O +- O +cultured O +in O +growth O +medium O +, O +and O +culture O +supernatants O +were O +used O +in O +an O +enzyme B-experimental_method +- I-experimental_method +linked I-experimental_method +immunosorbent I-experimental_method +assay I-experimental_method +( O +ELISA B-experimental_method +) O +to O +identify O +specific O +IL B-protein +- I-protein +17A I-protein +- O +binding O +clones O +. O + +The O +positive O +binding O +supernatants O +were O +tested O +for O +the O +ability O +to O +block O +biotinylated B-protein_state +IL B-protein +- I-protein +17A I-protein +signaling O +through O +IL B-protein +- I-protein +17RA I-protein +in O +an O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +competition B-experimental_method +ELISA I-experimental_method +assay I-experimental_method +where O +unlabeled O +IL B-protein +- I-protein +17A I-protein +was O +used O +as O +positive O +control O +to O +inhibit O +biotinylated B-protein_state +IL B-protein +- I-protein +17A I-protein +binding O +. O + +Approximately O +10 O +% O +of O +the O +clones O +that O +specifically O +bound O +to O +IL B-protein +- I-protein +17A I-protein +also O +prevented O +the O +cytokine B-protein_type +from O +binding O +to O +IL B-protein +- I-protein +17RA I-protein +. O + +Sequences O +identified O +from O +phage B-experimental_method +clones I-experimental_method +were O +chemically B-experimental_method +synthesized I-experimental_method +( O +Supplementary O +Table O +1 O +) O +and O +tested O +for O +inhibition O +of O +IL B-protein +- I-protein +17A I-protein +binding O +to O +IL B-protein +- I-protein +17RA I-protein +( O +Table O +1 O +). O + +A O +15 O +- O +mer O +linear O +peptide B-chemical +1 I-chemical +was O +shown O +to O +block O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +binding O +with O +an O +IC50 B-evidence +of O +80 O +nM O +in O +the O +competition B-experimental_method +ELISA I-experimental_method +assay I-experimental_method +( O +Table O +1 O +). O + +This O +peptide O +was O +then O +tested O +in O +a O +cell B-experimental_method +- I-experimental_method +based I-experimental_method +functional I-experimental_method +assay I-experimental_method +wherein O +production O +of O +GRO B-protein +- I-protein +α I-protein +in O +BJ O +human B-species +fibroblast O +cells O +was O +measured O +as O +a O +function O +of O +IL B-protein +- I-protein +17A I-protein +stimulation O +using O +1 O +ng O +/ O +ml O +IL B-protein +- I-protein +17A I-protein +. O + +Peptide B-chemical +1 I-chemical +was O +found O +to O +be O +active O +in O +this O +functional B-experimental_method +assay I-experimental_method +with O +an O +IC50 B-evidence +of O +370 O +nM O +. O + +Optimization O +of O +IL B-protein +- I-protein +17A I-protein +peptide O +inhibitors O + +A O +SAR B-experimental_method +campaign O +was O +undertaken O +to O +improve O +the O +potency O +of O +peptide B-chemical +1 I-chemical +. O + +An O +alanine B-experimental_method +scan I-experimental_method +of O +peptide B-chemical +2 I-chemical +, O +an O +analogue O +of O +1 B-chemical +with O +a O +lysine B-residue_name +to O +arginine B-residue_name +substitution B-experimental_method +at O +position O +14 B-residue_number +, O +was O +initiated O +. O + +When O +alanine B-residue_name +was O +already O +present O +( O +positions O +7 B-residue_number +and O +15 B-residue_number +), O +substitution B-experimental_method +was O +made O +with O +lysine B-residue_name +( O +Table O +1 O +, O +peptides B-chemical +3 I-chemical +– I-chemical +17 I-chemical +). O + +Positions O +1 B-residue_number +, O +2 B-residue_number +, O +4 B-residue_number +, O +5 B-residue_number +, O +7 B-residue_number +, O +14 B-residue_number +and O +15 B-residue_number +were O +shown O +to O +be O +amenable O +to O +substitution O +without O +significant O +loss O +( O +less O +than O +3 O +- O +fold O +) O +of O +binding B-evidence +affinity I-evidence +as O +measured O +by O +the O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +competition B-experimental_method +ELISA I-experimental_method +. O + +In O +particular O +, O +at O +position O +5 B-residue_number +( O +13 B-chemical +), O +substitution B-experimental_method +of O +methionine B-residue_name +with O +alanine B-residue_name +resulted O +in O +a O +seven O +fold O +improvement O +in O +potency O +( O +80 O +nM O +versus O +11 O +nM O +respectively O +). O + +In O +order O +to O +rapidly O +evaluate O +the O +effects O +of O +substitution B-experimental_method +of O +natural O +amino O +acids O +at O +tolerant O +positions O +identified O +by O +the O +alanine B-experimental_method +scan I-experimental_method +, O +the O +lead O +sequence O +was O +subjected O +to O +site B-experimental_method +- I-experimental_method +specific I-experimental_method +saturation I-experimental_method +mutagenesis I-experimental_method +using O +MBP B-experimental_method +. O + +Each O +of O +the O +seven O +positions O +identified O +by O +the O +alanine B-experimental_method +scan I-experimental_method +was O +individually O +modified O +while O +keeping O +the O +rest O +of O +the O +sequence O +constant O +. O + +Modifications O +at O +positions O +2 B-residue_number +and O +14 B-residue_number +were O +shown O +to O +display O +improvement O +in O +binding B-evidence +affinity I-evidence +( O +data O +not O +shown O +). O + +Peptides O +with O +beneficial O +point B-experimental_method +mutations I-experimental_method +at O +positions O +2 B-residue_number +, O +5 B-residue_number +, O +and O +14 B-residue_number +were O +synthesized B-experimental_method +and O +evaluated O +in O +the O +competition B-experimental_method +ELISA I-experimental_method +( O +Table O +1 O +). O + +Two O +of O +the O +changes O +, O +V2H B-mutant +( O +18 B-chemical +) O +or O +V2T B-mutant +( O +21 B-chemical +) O +displayed O +improved O +binding O +in O +the O +competition B-experimental_method +ELISA I-experimental_method +. O + +Since O +the O +replacement B-experimental_method +of O +methionine B-residue_name +at O +position O +5 B-residue_number +with O +alanine B-residue_name +was O +beneficial O +, O +the O +additional O +hydrophobic O +amino O +acids O +isoleucine B-residue_name +( O +24 B-chemical +), O +leucine B-residue_name +( O +25 B-chemical +) O +and O +valine B-residue_name +( O +26 B-chemical +) O +were O +evaluated O +and O +an O +additional O +two O +- O +three O +fold O +improvement O +in O +binding O +was O +observed O +for O +the O +valine B-residue_name +and O +isoleucine B-residue_name +replacements B-experimental_method +in O +comparison O +with O +alanine B-residue_name +. O + +Introduction B-experimental_method +of O +a O +methionine B-residue_name +( O +27 B-chemical +) O +or O +a O +carboxamide B-chemical +( O +28 B-chemical +and O +29 B-chemical +) O +at O +position O +14 B-residue_number +was O +shown O +to O +improve O +the O +binding B-evidence +affinity I-evidence +of O +the O +lead O +peptide O +. O + +In O +general O +, O +there O +was O +good O +agreement O +between O +the O +respective O +binding B-evidence +affinities I-evidence +of O +the O +synthesized O +peptides O +and O +their O +MBP B-experimental_method +fusion I-experimental_method +counterparts O +, O +except O +for O +substitution B-experimental_method +of O +valine B-residue_name +at O +position O +2 B-residue_number +to O +a O +tryptophan B-residue_name +( O +22 B-chemical +), O +which O +resulted O +in O +a O +fivefold O +loss O +of O +affinity B-evidence +, O +for O +the O +free O +peptide O +when O +compared O +with O +the O +MBP B-experimental_method +fusion I-experimental_method +. O + +Combining O +the O +key O +amino O +- O +acid O +residues O +identified O +by O +SAR B-experimental_method +into O +a O +single O +peptide O +sequence O +resulted O +in O +peptide B-chemical +30 I-chemical +, O +named O +high B-chemical +affinity I-chemical +peptide I-chemical +( O +HAP B-chemical +), O +that O +was O +found O +to O +inhibit O +IL B-protein +- I-protein +17A I-protein +signaling O +in O +a O +BJ O +human B-species +fibroblast O +cell O +assay O +with O +an O +IC50 B-evidence +of O +17 O +nM O +, O +a O +more O +than O +20 O +- O +fold O +improvement O +over O +the O +phage B-experimental_method +peptide B-chemical +1 I-chemical +( O +Table O +2 O +and O +Supplementary O +Figure O +S2 O +). O + +We O +also O +examined O +the O +effect O +of O +removing O +the O +acetyl O +group O +at O +the O +N O +- O +terminus O +of O +HAP B-chemical +( O +which O +is O +present O +in O +all O +the O +peptides O +made O +, O +see O +Supplementary O +Material O +). O + +The O +un B-protein_state +- I-protein_state +capped I-protein_state +peptide B-chemical +( I-chemical +31 I-chemical +) I-chemical +had O +an O +IC50 B-evidence +of O +420 O +nM O +in O +the O +cell B-experimental_method +- I-experimental_method +based I-experimental_method +assay I-experimental_method +. O + +The O +loss O +of O +cellular O +activity O +of O +31 B-chemical +was O +most O +likely O +due O +to O +the O +degradation O +of O +the O +N O +- O +terminus O +of O +31 B-chemical +, O +since O +peptide O +31 B-chemical +was O +shown O +to O +be O +able O +to O +bind O +to O +IL B-protein +- I-protein +17A I-protein +with O +similar O +affinity O +as O +HAP B-chemical +itself O +. O + +Furthermore O +, O +our O +previous O +work O +had O +reported O +that O +in O +antibody B-experimental_method +fusions I-experimental_method +the O +uncapped B-protein_state +peptide B-chemical +was O +degraded O +under O +cell O +assay O +conditions O +with O +removal B-experimental_method +of I-experimental_method +the O +first B-residue_range +1 I-residue_range +- I-residue_range +3 I-residue_range +residues I-residue_range +to O +inactive O +products O +with O +the O +same O +N O +- O +terminal O +sequences O +as O +peptides B-chemical +32 I-chemical +– I-chemical +34 I-chemical +. O + +In O +this O +work O +, O +32 B-chemical +– I-chemical +34 I-chemical +are O +capped B-protein_state +by O +protective O +acetyl O +group O +and O +reflect O +the O +same O +inactivity O +as O +reported O +. O + +C O +- O +terminal O +truncations B-experimental_method +showed O +a O +more O +gradual O +reduction O +in O +activity O +( O +35 B-chemical +– I-chemical +37 I-chemical +; O +Table O +2 O +). O + +After O +deletion B-experimental_method +of I-experimental_method +three B-residue_range +amino I-residue_range +acids I-residue_range +from O +the O +C O +- O +terminal O +end O +( O +37 B-chemical +), O +the O +peptide O +is O +no O +longer O +active O +. O + +Dimerization O +of O +HAP B-chemical +can O +further O +increase O +its O +potency O + +We O +reasoned O +that O +since O +the O +IL B-protein +- I-protein +17A I-protein +protein O +is O +almost O +exclusively O +present O +in O +a O +dimeric B-oligomeric_state +form O +, O +dimerizing B-oligomeric_state +the O +IL B-protein +- I-protein +17A I-protein +binding O +peptides O +could O +result O +in O +an O +improvement O +in O +binding B-evidence +affinity I-evidence +and O +inhibitory O +activity O +. O + +Homodimers B-oligomeric_state +of O +HAP B-chemical +were O +made O +through O +attachment O +of O +polyethylene B-chemical +glycol I-chemical +( O +PEG B-chemical +) O +spacers O +of O +different O +lengths O +at O +amino O +acids O +4 B-residue_number +, O +7 B-residue_number +and O +14 B-residue_number +, O +as O +these O +positions O +were O +identified O +in O +the O +alanine B-experimental_method +scan I-experimental_method +analysis I-experimental_method +as O +not O +contributing O +significantly O +to O +the O +activity O +, O +and O +at O +each O +N O +- O +terminus O +( O +Supplementary O +Table O +S2 O +). O + +Due O +to O +the O +high O +reactivity O +of O +the O +pentafluoroester B-chemical +( O +PFP B-chemical +) O +group O +used O +as O +the O +activating O +group O +in O +the O +PEG B-chemical +, O +the O +histidine B-residue_name +at O +position O +2 B-residue_number +and O +the O +lysine B-residue_name +at O +position O +15 B-residue_number +were O +replaced O +with O +threonine B-residue_name +and O +dimethyllysine B-residue_name +respectively O +to O +prevent O +formation O +of O +side O +products O +, O +which O +resulted O +in O +peptide B-chemical +38 I-chemical +that O +was O +comparable O +in O +activity O +with O +HAP B-chemical +. O + +This O +exercise O +revealed O +that O +several O +dimeric B-oligomeric_state +peptides B-chemical +with O +the O +longer O +PEG21 B-chemical +spacer O +were O +significantly O +more O +potent O +than O +the O +monomer B-oligomeric_state +peptide O +in O +the O +cell B-experimental_method +- I-experimental_method +based I-experimental_method +assay I-experimental_method +( O +Supplementary O +Table O +S2 O +). O + +Peptide B-chemical +45 I-chemical +, O +dimerized B-oligomeric_state +via O +attachment O +of O +a O +PEG21 B-chemical +spacer O +at O +position O +14 B-residue_number +( O +Supplementary O +Scheme O +S1 O +and O +Figure O +S3 O +), O +was O +the O +most O +potent O +with O +cellular O +IC50 B-evidence +of O +0 O +. O +1 O +nM O +. O +This O +significant O +improvement O +in O +antagonism O +was O +not O +seen O +in O +the O +peptide O +monomer B-oligomeric_state +functionalized O +with O +a O +PEG21 B-chemical +group O +at O +position O +14 B-residue_number +as O +peptide B-chemical +48 I-chemical +had O +an O +IC50 B-evidence +of O +21 O +nM O +( O +Supplementary O +Scheme O +S2 O +). O + +The O +species O +cross O +- O +reactivity O +of O +the O +dimeric B-oligomeric_state +peptide B-chemical +45 I-chemical +and O +HAP B-chemical +were O +assessed O +in O +a O +murine B-experimental_method +functional I-experimental_method +cell I-experimental_method +assay I-experimental_method +using O +15 O +ng O +/ O +ml O +murine B-taxonomy_domain +IL B-protein +- I-protein +17A I-protein +. O + +Peptide B-chemical +45 I-chemical +blocked O +the O +receptor B-protein_type +binding O +of O +murine B-taxonomy_domain +IL B-protein +- I-protein +17A I-protein +although O +with O +potency O +two O +orders O +of O +magnitude O +weaker O +than O +that O +observed O +against O +human B-species +IL B-protein +- I-protein +17A I-protein +( O +IC50 B-evidence += O +41 O +nM O +vs O +IC50 B-evidence += O +0 O +. O +1 O +nM O +, O +respectively O +). O + +The O +monomer B-oligomeric_state +HAP B-chemical +was O +much O +weaker O +( O +IC50 B-evidence +> O +1 O +μM O +) O +in O +inhibiting O +murine B-taxonomy_domain +IL B-protein +- I-protein +17A I-protein +signaling O +( O +Supplementary O +Figure O +S4 O +). O + +Although O +the O +dimeric B-oligomeric_state +peptide B-chemical +45 I-chemical +is O +much O +more O +potent O +than O +HAP B-chemical +in O +the O +cell B-experimental_method +- I-experimental_method +based I-experimental_method +assay I-experimental_method +, O +in O +subsequent O +studies O +we O +decided O +to O +focus O +our O +efforts O +solely O +on O +characterizations O +of O +the O +monomeric B-oligomeric_state +peptide O +HAP B-chemical +in O +hopes O +to O +identify O +smaller O +peptide O +inhibitors O +containing O +the O +best O +minimal O +functional O +group O +. O + +Orthogonal O +assays O +to O +confirm O +HAP B-chemical +antagonism O + +To O +further O +characterize O +the O +interaction O +of O +HAP B-chemical +with O +IL B-protein +- I-protein +17A I-protein +, O +we O +set O +out O +to O +determine O +its O +in O +vitro O +binding B-evidence +affinity I-evidence +, O +specificity O +and O +kinetic B-evidence +profile I-evidence +using O +Surface B-experimental_method +Plasmon I-experimental_method +Resonance I-experimental_method +( O +SPR B-experimental_method +) O +methods O +( O +Fig O +. O +1A O +). O + +HAP B-chemical +binds O +to O +immobilized O +human B-species +IL B-protein +- I-protein +17A I-protein +homodimer B-oligomeric_state +tightly O +( O +Table O +3 O +). O + +It O +has O +slightly O +weaker O +affinity B-evidence +for O +human B-species +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +F I-complex_assembly +heterodimer B-oligomeric_state +and O +> O +10 O +fold O +weaker O +affinity B-evidence +for O +mouse B-taxonomy_domain +IL B-protein +- I-protein +17A I-protein +( O +Table O +3 O +). O + +HAP B-chemical +does O +not O +show O +significant O +binding O +to O +immobilized O +human B-species +IL B-protein +- I-protein +17F I-protein +homodimer B-oligomeric_state +or O +IL B-protein +- I-protein +17RA I-protein +at O +concentrations O +up O +to O +100 O +nM O +. O + +Additionally O +, O +we O +investigated O +the O +antagonism O +of O +the O +human B-species +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +interaction O +by O +HAP B-chemical +using O +orthogonal O +methods O +including O +SPR B-experimental_method +and O +Förster B-experimental_method +resonance I-experimental_method +energy I-experimental_method +transfer I-experimental_method +( I-experimental_method +FRET I-experimental_method +) I-experimental_method +competition I-experimental_method +assays I-experimental_method +( O +Fig O +. O +1B O +, O +C O +). O + +In O +both O +assays O +, O +incubation O +of O +IL B-protein +- I-protein +17A I-protein +with O +HAP B-chemical +effectively O +blocks O +the O +binding O +of O +IL B-protein +- I-protein +17A I-protein +to O +immobilized B-protein_state +IL B-protein +- I-protein +17RA I-protein +with O +similar O +sub O +- O +nM O +IC50 B-evidence +( O +Table O +3 O +). O + +HAP B-chemical +blocks O +IL B-protein +- I-protein +17A I-protein +signaling O +in O +a O +human B-species +primary O +cell O +assay O + +While O +either O +IL B-protein +- I-protein +17A I-protein +or O +TNF B-protein +- I-protein +α I-protein +alone O +can O +stimulate O +the O +release O +of O +multiple O +inflammatory O +cytokines B-protein_type +, O +when O +acting O +together O +they O +can O +synergistically O +enhance O +each O +other O +’ O +s O +effects O +( O +Supplementary O +Figure O +S5 O +). O + +These O +integrative O +responses O +to O +IL B-protein +- I-protein +17A I-protein +and O +TNF B-protein +- I-protein +α I-protein +in O +human B-species +keratinocytes O +have O +been O +reported O +to O +account O +for O +key O +inflammatory O +pathogenic O +circuits O +in O +psoriasis O +. O + +Thus O +, O +we O +chose O +to O +study O +HAP B-chemical +’ O +s O +efficacy O +in O +blocking O +the O +production O +of O +IL B-protein_type +- I-protein_type +8 I-protein_type +, O +IL B-protein_type +- I-protein_type +6 I-protein_type +and O +CCL B-protein_type +- I-protein_type +20 I-protein_type +by O +primary O +human B-species +keratinocytes O +stimulated O +by O +IL B-protein +- I-protein +17A I-protein +in O +the O +presence O +of O +TNF B-protein +- I-protein +α I-protein +, O +an O +assay O +which O +may O +be O +more O +disease O +- O +relevant O +. O + +HAP B-chemical +inhibits O +the O +production O +of O +all O +three O +cytokines B-protein_type +in O +a O +dose O +- O +dependent O +fashion O +( O +Fig O +. O +1D O +). O + +Significantly O +, O +the O +baseline O +levels O +of O +IL B-protein_type +- I-protein_type +8 I-protein_type +, O +IL B-protein_type +- I-protein_type +6 I-protein_type +and O +CCL B-protein_type +- I-protein_type +20 I-protein_type +stimulated O +by O +TNF B-protein +- I-protein +α I-protein +alone O +are O +not O +inhibited O +by O +HAP B-chemical +, O +further O +indicating O +the O +selectivity O +of O +HAP B-chemical +( O +Fig O +. O +1D O +). O + +Such O +pharmacological O +selectivity O +may O +be O +important O +to O +suppress O +inflammatory O +pathogenic O +circuits O +in O +psoriasis O +, O +while O +sparing O +the O +anti O +- O +infectious O +immune O +responses O +produced O +by O +TNF B-protein +- I-protein +α I-protein +. O + +The O +relatively O +high O +IC50 B-evidence +values O +in O +this O +assay O +( O +Table O +3 O +) O +are O +probably O +due O +to O +the O +high O +IL B-protein +- I-protein +17A I-protein +concentration O +( O +100 O +ng O +/ O +ml O +) O +needed O +for O +detection O +of O +IL B-protein_type +- I-protein_type +6 I-protein_type +. O + +As O +a O +reference O +, O +a O +commercial O +anti O +- O +IL B-protein +- I-protein +17A I-protein +antibody B-protein_type +( O +R O +& O +D O +Systems O +) O +inhibits O +the O +production O +of O +IL B-protein_type +- I-protein_type +8 I-protein_type +with O +an O +IC50 B-evidence +of O +13 O +(± O +6 O +) O +nM O +( O +N O += O +3 O +). O + +Indeed O +, O +the O +IC50 B-evidence +was O +14 O +(± O +9 O +) O +nM O +( O +N O += O +12 O +) O +for O +HAP B-chemical +inhibition O +of O +IL B-protein_type +- I-protein_type +8 I-protein_type +production O +when O +only O +5 O +ng O +/ O +ml O +IL B-protein +- I-protein +17A I-protein +was O +used O +in O +this O +assay O +. O + +In O +patients O +, O +the O +concentration O +of O +IL B-protein +- I-protein +17A I-protein +in O +psoriatic O +lesions O +is O +reported O +to O +be O +0 O +. O +01 O +ng O +/ O +ml O +, O +well O +below O +the O +EC50 O +( O +5 O +– O +10ng O +/ O +ml O +) O +of O +IL B-protein +- I-protein +17A I-protein +induced O +IL B-protein_type +- I-protein_type +8 I-protein_type +production O +in O +vitro O +. O + +Similar O +to O +keratinocytes B-experimental_method +assay I-experimental_method +results O +, O +while O +HAP B-chemical +inhibits O +IL B-protein +- I-protein +17A I-protein +stimulated O +IL B-protein_type +- I-protein_type +6 I-protein_type +production O +by O +BJ O +human B-species +fibroblast O +potently O +( O +IC50 B-evidence +of O +17 O +nM O +), O +it O +does O +not O +inhibit O +TNF B-protein +- I-protein +α I-protein +stimulated O +IL B-protein_type +- I-protein_type +6 I-protein_type +production O +at O +concentrations O +up O +to O +10 O +μM O +( O +Supplementary O +Figure O +S2 O +). O + +Crystallization B-experimental_method +and I-experimental_method +structure I-experimental_method +determination I-experimental_method + +Extensive O +crystallization B-experimental_method +trials I-experimental_method +, O +either O +by O +co B-experimental_method +- I-experimental_method +crystallization I-experimental_method +or O +by O +soaking B-experimental_method +HAP B-chemical +into O +preformed O +apo B-protein_state +IL B-protein +- I-protein +17A I-protein +crystals B-evidence +, O +failed O +to O +lead O +to O +an O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O +crystals B-evidence +. O + +We O +theorized O +that O +HAP B-chemical +binding O +induced O +large O +conformational O +changes O +in O +IL B-protein +- I-protein +17A I-protein +that O +led O +to O +the O +difficulty O +of O +getting O +an O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +binary O +complex O +crystal B-evidence +. O + +It O +is O +known O +that O +an O +antibody B-protein_type +antigen B-structure_element +- I-structure_element +binding I-structure_element +fragment I-structure_element +( O +Fab B-structure_element +) O +can O +be O +used O +as O +crystallization O +chaperones O +in O +crystallizing O +difficult O +targets O +. O + +We O +hypothesized O +that O +HAP B-chemical +may O +target O +the O +N O +- O +terminal O +of O +IL B-protein +- I-protein +17A I-protein +which O +is O +known O +to O +be O +more O +flexible O +than O +its O +C O +- O +terminal O +and O +conformational O +changes O +needed O +for O +HAP B-chemical +binding O +may O +be O +more O +likely O +there O +. O + +We O +designed O +an O +antibody B-protein_type +Fab B-structure_element +known O +to O +target O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +based O +on O +a O +published O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +Fab I-complex_assembly +complex O +crystal B-evidence +structure I-evidence +, O +and O +produced O +it O +in O +HEK293 O +cells O +. O + +In O +an O +SPR B-experimental_method +assay I-experimental_method +HAP B-chemical +and O +this O +Fab B-structure_element +were O +able O +to O +co O +- O +bind O +IL B-protein +- I-protein +17A I-protein +without O +large O +changes O +in O +their O +binding B-evidence +affinities I-evidence +and O +kinetics B-evidence +, O +confirming O +our O +hypothesis O +( O +Supplementary O +Figure O +S6 O +). O + +Furthermore O +, O +since O +it O +binds O +to O +an O +area O +far O +away O +from O +that O +of O +HAP B-chemical +( O +see O +below O +), O +this O +Fab B-structure_element +should O +have O +minimum O +effects O +on O +HAP B-chemical +binding O +conformation O +. O + +Crystals B-evidence +of O +Fab B-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +ternary O +complex O +were O +obtained O +readily O +in O +crystallization B-experimental_method +screens I-experimental_method +. O + +Crystallization B-experimental_method +of O +IL B-protein +- I-protein +17A I-protein +and O +its O +binding O +partners O +was O +accomplished O +using O +two O +forms O +of O +IL B-protein +- I-protein +17A I-protein +. O + +These O +were O +, O +respectively O +, O +a O +presumably O +more O +homogeneous O +form O +of O +IL B-protein +- I-protein +17A I-protein +that O +lacked B-protein_state +the O +disordered B-protein_state +N B-structure_element +- I-structure_element +terminal I-structure_element +peptide I-structure_element +and O +a O +full B-protein_state +- I-protein_state +length I-protein_state +form O +of O +the O +cytokine B-protein_type +with O +a O +full O +complement O +of O +disulfide B-ptm +bonds I-ptm +. O + +Crystals B-evidence +of O +the O +Fab B-complex_assembly +/ I-complex_assembly +truncated I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O +diffracted O +to O +2 O +. O +2 O +Å O +, O +and O +the O +Fab B-complex_assembly +/ I-complex_assembly +full I-complex_assembly +length I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O +diffracted O +to O +3 O +. O +0 O +Å O +( O +Supplementary O +Table O +S3 O +). O + +Both O +structures B-evidence +were O +solved O +by O +molecular B-experimental_method +replacement I-experimental_method +. O + +Both O +complexes O +crystallized B-experimental_method +in O +the O +space O +group O +of O +P321 O +, O +with O +half O +the O +complex O +( O +1 O +Fab B-structure_element +/ O +1 O +IL B-protein +- I-protein +17A I-protein +monomer B-oligomeric_state +/ O +1 O +HAP B-chemical +) O +in O +the O +asymmetric O +unit O +. O + +The O +intact B-protein_state +complex O +can O +be O +generated O +by O +applying O +crystallographic O +2 O +- O +fold O +symmetry O +. O + +Electron B-evidence +densities I-evidence +for O +HAP B-chemical +residues O +Ile1 B-residue_range +- I-residue_range +Asn14 I-residue_range +were O +readily O +interpretable O +with O +the O +exception O +of O +Lys15 B-residue_name_number +, O +which O +is O +disordered B-protein_state +. O + +When O +considering O +the O +protein O +, O +the O +complex B-evidence +structure I-evidence +containing O +the O +full B-protein_state +length I-protein_state +IL B-protein +- I-protein +17A I-protein +is O +identical O +to O +that O +of O +the O +truncated B-protein_state +IL B-protein +- I-protein +17A I-protein +, O +with O +the O +exception O +of O +Cys106 B-residue_name_number +( O +Ser106 B-residue_name_number +in O +the O +truncated B-protein_state +IL B-protein +- I-protein +17A I-protein +), O +which O +is O +disordered B-protein_state +. O + +Cys106 B-residue_name_number +is O +covalently O +linked O +to O +Cys10 B-residue_name_number +that O +resides O +in O +the O +disordered B-protein_state +N B-structure_element +- I-structure_element +terminal I-structure_element +peptide I-structure_element +in O +the O +full B-protein_state +length I-protein_state +IL B-protein +- I-protein +17A I-protein +. O + +Overall O +structure B-evidence +of O +Fab B-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O + +In O +a O +similar O +manner O +to O +the O +published O +structure B-evidence +of O +Fab B-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17A I-complex_assembly +complex O +, O +two O +Fab B-structure_element +molecules O +bind O +symmetrically O +to O +the O +C O +- O +terminal O +of O +the O +cytokine B-protein_type +dimer B-oligomeric_state +, O +interacting O +with O +epitopes O +from O +both O +monomers B-oligomeric_state +( O +Fig O +. O +2A O +). O + +Two O +copies O +of O +HAP B-chemical +bind O +to O +the O +N O +- O +terminal O +of O +the O +cytokine B-protein_type +dimer B-oligomeric_state +, O +also O +symmetrically O +, O +and O +each O +HAP B-chemical +molecule O +also O +interacts O +with O +both O +IL B-protein +- I-protein +17A I-protein +monomers B-oligomeric_state +( O +Fig O +. O +2 O +). O + +Based O +on O +disclosed O +epitopes O +of O +Secukinumab B-chemical +and O +Ixekizumab B-chemical +, O +HAP B-chemical +binds O +to O +IL B-protein +- I-protein +17A I-protein +at O +an O +area O +that O +is O +also O +different O +from O +those O +of O +those O +two O +antibodies B-protein_type +. O + +The O +N O +- O +terminal O +5 B-residue_range +residues I-residue_range +of O +HAP B-chemical +, O +1IHVTI B-chemical +, O +form O +an O +amphipathic B-protein_state +β B-structure_element +- I-structure_element +strand I-structure_element +that O +inserts O +between O +β B-structure_element +- I-structure_element +strand I-structure_element +4 I-structure_element +of O +one O +IL B-protein +- I-protein +17A I-protein +monomer B-oligomeric_state +and O +β B-structure_element +- I-structure_element +strand I-structure_element +0 I-structure_element +( O +the O +first O +ordered O +peptide O +of O +IL B-protein +- I-protein +17A I-protein +) O +of O +the O +second O +monomer B-oligomeric_state +. O + +This O +β B-structure_element +- I-structure_element +strand I-structure_element +is O +parallel O +to O +both O +strands B-structure_element +0 I-structure_element +and I-structure_element +4 I-structure_element +( O +Fig O +. O +3B O +). O + +Strands B-structure_element +0 I-structure_element +of O +two O +IL B-protein +- I-protein +17A I-protein +monomer B-oligomeric_state +are O +antiparallel O +, O +as O +appeared O +in O +other O +IL B-protein +- I-protein +17A I-protein +structures B-evidence +. O + +The O +C O +- O +terminal O +8 B-residue_range +residues I-residue_range +of O +the O +HAP B-chemical +that O +are O +ordered O +in O +the O +structure B-evidence +, O +7ADLWDWIN B-chemical +, O +form O +an O +amphipathic B-protein_state +α B-structure_element +- I-structure_element +helix I-structure_element +interacting O +with O +the O +second O +IL B-protein +- I-protein +17A I-protein +monomer B-oligomeric_state +. O + +Pro6 B-residue_name_number +of O +HAP B-chemical +makes O +a O +transition O +between O +the O +N O +- O +terminal O +β B-structure_element +- I-structure_element +strand I-structure_element +and O +the O +C O +- O +terminal O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +HAP B-chemical +. O + +As O +a O +comparison O +, O +an O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +complex B-evidence +structure I-evidence +( O +PDB O +code O +4HSA O +) O +is O +also O +shown O +with O +IL B-protein +- I-protein +17A I-protein +in O +the O +same O +orientation O +( O +Fig O +. O +2C O +). O + +Inhibition O +mechanism O +of O +IL B-protein +- I-protein +17A I-protein +signaling O +by O +HAP B-chemical + +IL B-protein +- I-protein +17RA I-protein +binds O +IL B-protein +- I-protein +17A I-protein +at O +three O +regions O +on O +the O +IL B-protein +- I-protein +17A I-protein +homodimer B-oligomeric_state +. O + +HAP B-chemical +binds O +IL B-protein +- I-protein +17A I-protein +at O +region B-structure_element +I I-structure_element +. O +Region B-structure_element +I I-structure_element +is O +formed O +by O +residues O +at O +the O +ends O +of O +β B-structure_element +strands I-structure_element +0 I-structure_element +and I-structure_element +4 I-structure_element +, O +and O +from O +loops B-structure_element +1 I-structure_element +– I-structure_element +2 I-structure_element +and O +3 B-structure_element +– I-structure_element +4 I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +( O +Fig O +. O +2 O +). O + +Conformational O +changes O +in O +region B-structure_element +I I-structure_element +induced O +by O +HAP B-chemical +binding O +alone O +may O +allosterically O +affect O +IL B-protein +- I-protein +17RA I-protein +binding O +, O +but O +more O +importantly O +, O +the O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +HAP B-chemical +directly O +competes O +with O +IL B-protein +- I-protein +17RA I-protein +for O +binding O +to O +IL B-protein +- I-protein +17A I-protein +( O +Fig O +. O +3 O +). O + +The O +most O +significant O +interactions O +between O +the O +α B-structure_element +helix I-structure_element +of O +HAP B-chemical +and O +IL B-protein +- I-protein +17A I-protein +involve O +Trp12 B-residue_name_number +of O +HAP B-chemical +, O +which O +binds O +in O +a O +hydrophobic B-site +pocket I-site +in O +IL B-protein +- I-protein +17A I-protein +formed O +by O +the O +side O +chains O +of O +Phe110 B-residue_name_number +, O +Tyr62 B-residue_name_number +, O +Pro59 B-residue_name_number +and O +the O +hydrophobic O +portion O +of O +the O +Arg101 B-residue_name_number +side O +chain O +( O +Fig O +. O +3A O +). O + +The O +Trp12 B-residue_name_number +side O +chain O +of O +HAP B-chemical +donates O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +to O +the O +main O +chain O +oxygen O +of O +Pro69 B-residue_name_number +of O +IL B-protein +- I-protein +17A I-protein +. O + +The O +positively O +charged O +Arg101 B-residue_name_number +side O +chain O +of O +the O +IL B-protein +- I-protein +17A I-protein +engages O +in O +a O +charge B-bond_interaction +- I-bond_interaction +helix I-bond_interaction +dipole I-bond_interaction +interaction I-bond_interaction +with O +the O +main O +chain O +oxygen O +of O +Trp12 B-residue_name_number +. O + +Additionally O +, O +Leu9 B-residue_name_number +and O +Ile13 B-residue_name_number +of O +the O +HAP B-chemical +have O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +IL B-protein +- I-protein +17A I-protein +, O +and O +the O +Asp8 B-residue_name_number +side O +chain O +has O +hydrogen B-bond_interaction +bond I-bond_interaction +and O +ion B-bond_interaction +pair I-bond_interaction +interactions I-bond_interaction +with O +Tyr62 B-residue_name_number +and O +Lys114 B-residue_name_number +of O +IL B-protein +- I-protein +17A I-protein +, O +respectively O +. O + +In O +region B-structure_element +I I-structure_element +, O +an O +IL B-protein +- I-protein +17RA I-protein +peptide O +interacts O +with O +IL B-protein +- I-protein +17A I-protein +in O +a O +very O +similar O +fashion O +to O +the O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +HAP B-chemical +. O + +The O +IL B-protein +- I-protein +17RA I-protein +peptide O +has O +sequences O +of O +27LDDSWI B-chemical +, O +and O +part O +of O +the O +peptide O +is O +also O +α B-structure_element +- I-structure_element +helical I-structure_element +( O +Fig O +. O +3B O +). O + +Leu7 B-residue_name_number +, O +Trp31 B-residue_name_number +and O +Ile32 B-residue_name_number +of O +IL B-protein +- I-protein +17RA I-protein +interact O +very O +similarly O +with O +the O +same O +residues O +of O +IL B-protein +- I-protein +17A I-protein +as O +Leu9 B-residue_name_number +, O +Trp12 B-residue_name_number +and O +Ile13 B-residue_name_number +of O +HAP B-chemical +( O +Fig O +. O +3B O +). O + +In O +this O +sense O +, O +the O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +HAP B-chemical +with O +a O +sequence O +of O +9LWDWI B-chemical +is O +a O +good O +mimetic O +of O +the O +27LDDSWI B-chemical +peptide O +of O +IL B-protein +- I-protein +17RA I-protein +. O + +The O +β B-structure_element +- I-structure_element +strand I-structure_element +of O +HAP B-chemical +has O +no O +equivalent O +in O +IL B-protein +- I-protein +17RA I-protein +. O + +However O +, O +it O +mimics O +the O +β B-structure_element +- I-structure_element +strand I-structure_element +0 I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +. O + +The O +amphipathic B-protein_state +β B-structure_element +- I-structure_element +strand I-structure_element +of O +HAP B-chemical +orients O +the O +hydrophilic O +side O +chains O +of O +His2 B-residue_name_number +and O +Thr4 B-residue_name_number +outwards O +, O +and O +the O +hydrophobic O +side O +chains O +of O +Ile1 B-residue_name_number +, O +Val3 B-residue_name_number +and O +Ile5 B-residue_name_number +inward O +( O +Fig O +. O +3A O +). O + +β B-structure_element +- I-structure_element +strand I-structure_element +0 I-structure_element +in O +IL B-protein +- I-protein +17A I-protein +is O +also O +amphipathic B-protein_state +with O +the O +sequence O +of O +21TVMVNLNI B-chemical +. O + +In O +all O +IL B-protein +- I-protein +17A I-protein +structures B-evidence +obtained O +to O +date O +, O +β B-structure_element +- I-structure_element +strand I-structure_element +0 I-structure_element +orients O +the O +hydrophilic O +side O +chains O +of O +Thr21 B-residue_name_number +, O +Asn25 B-residue_name_number +and O +Asn27 B-residue_name_number +outward O +, O +and O +the O +hydrophobic O +side O +chains O +of O +Val22 B-residue_name_number +, O +Val24 B-residue_name_number +, O +Leu26 B-residue_name_number +and O +Ile28 B-residue_name_number +inward O +. O + +The O +binding B-site +pocket I-site +occupied O +by O +either O +Trp12 B-residue_name_number +of O +HAP B-chemical +or O +Trp31 B-residue_name_number +of O +IL B-protein +- I-protein +17RA I-protein +is O +not O +formed O +in O +the O +apo B-protein_state +IL B-protein +- I-protein +17A I-protein +structure B-evidence +( O +Fig O +. O +3C O +). O + +Conformational O +changes O +of O +IL B-protein +- I-protein +17A I-protein +are O +needed O +for O +both O +HAP B-chemical +and O +IL B-protein +- I-protein +17RA I-protein +to O +bind O +to O +that O +region O +. O + +Particularly O +for O +HAP B-chemical +, O +β B-structure_element +- I-structure_element +strands I-structure_element +0 I-structure_element +have O +to O +shift O +out O +of O +the O +hydrophobic B-site +cleft I-site +formed O +by O +the O +main B-structure_element +body I-structure_element +of O +the O +IL B-protein +- I-protein +17A I-protein +by O +as O +much O +as O +10 O +Å O +between O +Cα O +atoms O +( O +Fig O +. O +3C O +). O + +Disruptions O +of O +the O +apo B-protein_state +IL B-protein +- I-protein +17A I-protein +structure B-evidence +by O +HAP B-chemical +binding O +are O +apparently O +compensated O +for O +by O +formation O +of O +the O +new O +interactions O +that O +involve O +almost O +the O +entire O +HAP B-chemical +molecule O +( O +Fig O +. O +3B O +). O + +Structure O +basis O +for O +the O +observed O +SAR B-experimental_method +of O +peptides O + +The O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex B-evidence +structure I-evidence +obtained O +is O +very O +consistent O +with O +the O +observed O +SAR B-experimental_method +of O +our O +identified O +peptide O +inhibitors O +, O +explaining O +well O +how O +the O +evolution O +of O +the O +initial O +phage B-experimental_method +peptide B-chemical +1 I-chemical +to O +HAP B-chemical +and O +45 B-chemical +improved O +its O +potency O +( O +Supplementary O +Figure O +S7 O +). O + +The O +important O +interactions O +involving O +Trp12 B-residue_name_number +of O +HAP B-chemical +explain O +the O +> O +90 O +times O +drop O +in O +potency O +of O +the O +W12A B-mutant +variant O +( O +6 O +vs O +1 O +, O +Table O +1 O +). O + +The O +amphipathic B-protein_state +nature O +of O +the O +HAP B-chemical +β B-structure_element +- I-structure_element +strand I-structure_element +explains O +the O +preference O +of O +the O +hydrophilic O +residues O +at O +the O +2 B-residue_number +and O +4 B-residue_number +positions O +of O +peptides O +( O +14 B-chemical +, O +18 B-chemical +, O +19 B-chemical +, O +21 B-chemical +and O +23 B-chemical +vs O +1 B-chemical +and O +22 B-chemical +, O +Table O +1 O +). O + +All O +N O +- O +terminal O +residues O +of O +HAP B-chemical +are O +part O +of O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +with O +β B-structure_element +- I-structure_element +stands I-structure_element +0 I-structure_element +and I-structure_element +4 I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +, O +which O +explains O +why O +removal B-experimental_method +of I-experimental_method +the O +first B-residue_range +1 I-residue_range +– I-residue_range +3 I-residue_range +residues I-residue_range +completely O +abolishes O +the O +ability O +of O +HAP B-chemical +to O +block O +IL B-protein +- I-protein +17A I-protein +cell O +signaling O +( O +31 B-chemical +, O +32 B-chemical +and O +33 B-chemical +, O +Table O +2 O +). O + +The O +C O +- O +terminal O +Asn14 B-residue_name_number +and O +Lys15 B-residue_name_number +of O +HAP B-chemical +are O +not O +directly O +involved O +in O +interactions O +with O +IL B-protein +- I-protein +17A I-protein +, O +and O +this O +is O +reflected O +in O +the O +gradual O +reduction O +in O +activity O +caused O +by O +C O +- O +terminal O +truncations B-experimental_method +( O +35 B-chemical +and O +36 B-chemical +, O +Table O +2 O +). O + +Each O +peptide O +monomer B-oligomeric_state +in O +45 B-chemical +may O +not O +necessarily O +be O +more O +potent O +than O +HAP B-chemical +, O +but O +two O +monomer B-oligomeric_state +peptides O +within O +the O +same O +molecule O +that O +can O +simultaneously O +bind O +to O +IL B-protein +- I-protein +17A I-protein +can O +greatly O +improve O +its O +potency O +due O +to O +avidity O +effects O +. O + +HAP B-chemical +targets O +region B-structure_element +I I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +, O +an O +area O +that O +has O +the O +least O +sequence O +conservation O +in O +IL B-protein_type +- I-protein_type +17 I-protein_type +cytokines I-protein_type +. O + +This O +lack O +of O +sequence O +conservation O +in O +the O +HAP B-site +binding I-site +site I-site +explains O +the O +observed O +specificity O +of O +HAP B-chemical +binding O +to O +human B-species +IL B-protein +- I-protein +17A I-protein +. O + +For O +example O +, O +inspection O +of O +the O +published O +IL B-protein +- I-protein +17F I-protein +crystal B-evidence +structure I-evidence +( O +PDB O +code O +1JPY O +) O +revealed O +a O +pocket B-site +of O +IL B-protein +- I-protein +17F I-protein +similar O +to O +that O +of O +IL B-protein +- I-protein +17A I-protein +for O +W12 B-residue_name_number +of O +HAP B-chemical +binding O +, O +but O +it O +is O +occupied O +by O +a O +Phe B-structure_element +- I-structure_element +Phe I-structure_element +motif I-structure_element +at O +the O +N O +- O +terminal O +peptide O +of O +IL B-protein +- I-protein +17F I-protein +. O + +This O +Phe B-structure_element +- I-structure_element +Phe I-structure_element +motif I-structure_element +is O +missing B-protein_state +in O +IL B-protein +- I-protein +17A I-protein +. O + +Sequence B-experimental_method +alignments I-experimental_method +between O +human B-species +and O +mouse B-taxonomy_domain +IL B-protein +- I-protein +17A I-protein +indicated O +that O +among O +IL B-protein +- I-protein +17A I-protein +residues O +that O +interacting O +with O +HAP B-chemical +, O +majority O +differences O +occur O +in O +strand B-structure_element +0 I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +which O +interacts O +with O +the O +N O +- O +terminal O +β B-structure_element +- I-structure_element +strand I-structure_element +of O +HAP B-chemical +. O + +In O +human B-species +IL B-protein +- I-protein +17A I-protein +the O +sequences O +are O +21TVMVNLNI B-chemical +, O +and O +in O +mouse B-taxonomy_domain +they O +are O +21NVKVNLKV B-chemical +. O + +Using O +a O +combination O +of O +phage B-experimental_method +display I-experimental_method +and O +SAR B-experimental_method +we O +have O +discovered O +novel O +peptides O +that O +are O +IL B-protein +- I-protein +17A I-protein +antagonists O +. O + +One O +of O +those O +peptides O +, O +HAP B-chemical +, O +also O +shows O +activity O +in O +inhibiting O +the O +production O +of O +multiple O +inflammatory O +cytokines B-protein_type +by O +primary O +human B-species +keratinocytes O +stimulated O +by O +IL B-protein +- I-protein +17A I-protein +and O +TNF B-protein +- I-protein +α I-protein +, O +a O +disease O +relevant O +- O +model O +. O + +We O +have O +also O +determined B-experimental_method +the O +complex B-evidence +structure I-evidence +of O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +, O +which O +provides O +the O +structural O +basis O +for O +HAP B-chemical +’ O +s O +antagonism O +to O +IL B-protein +- I-protein +17A I-protein +signaling O +. O + +During O +IL B-protein +- I-protein +17A I-protein +signaling O +, O +IL B-protein +- I-protein +17A I-protein +binds O +to O +one O +copy O +of O +IL B-protein +- I-protein +17RA I-protein +and O +one O +copy O +of O +IL B-protein +- I-protein +17RC I-protein +. O + +Since O +apo B-protein_state +IL B-protein +- I-protein +17A I-protein +is O +a O +homodimer B-oligomeric_state +with O +2 O +fold O +symmetry O +, O +IL B-protein +- I-protein +17RA I-protein +potentially O +can O +bind O +to O +either O +face O +of O +the O +IL B-protein +- I-protein +17A I-protein +dimer B-oligomeric_state +. O + +With O +two O +HAP B-chemical +molecules O +covering O +both O +faces O +of O +the O +IL B-protein +- I-protein +17A I-protein +dimer B-oligomeric_state +, O +HAP B-chemical +can O +block O +IL B-protein +- I-protein +17RA I-protein +approaching O +from O +either O +face O +. O + +To O +form O +the O +1 O +: O +2 O +complex O +observed O +in O +crystal B-evidence +structure I-evidence +, O +it O +is O +important O +that O +there O +is O +no O +strong O +negative O +cooperativity O +in O +the O +binding O +of O +two O +HAP B-chemical +molecules O +. O + +In O +fact O +, O +in O +native B-experimental_method +electrospray I-experimental_method +ionization I-experimental_method +mass I-experimental_method +spectrometry I-experimental_method +analysis O +only O +1 O +: O +2 O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O +was O +observed O +even O +when O +IL B-protein +- I-protein +17A I-protein +was O +in O +excess O +( O +Supplementary O +Figure O +S8 O +), O +indicating O +a O +positive O +binding O +cooperativity O +that O +favors O +inhibition O +of O +IL B-protein +- I-protein +17RA I-protein +binding O +by O +HAP B-chemical +. O + +HAP B-chemical +, O +with O +only O +15 B-residue_range +residues I-residue_range +, O +can O +achieve O +almost O +the O +same O +binding B-evidence +affinity I-evidence +as O +the O +much O +larger O +IL B-protein +- I-protein +17RA I-protein +molecule O +, O +indicating O +a O +more O +efficient O +way O +of O +binding O +to O +IL B-protein +- I-protein +17A I-protein +. O + +The O +interaction O +of O +IL B-protein +- I-protein +17A I-protein +with O +IL B-protein +- I-protein +17RA I-protein +has O +an O +extensive O +interface B-site +, O +covering O +~ O +2 O +, O +200 O +Å2 O +surface O +area O +of O +IL B-protein +- I-protein +17A I-protein +. O + +Due O +to O +the O +discontinuous O +nature O +of O +the O +IL B-site +- I-site +17A I-site +/ I-site +IL I-site +- I-site +17RA I-site +binding I-site +interface I-site +, O +it O +is O +classified O +as O +having O +tertiary O +structural O +epitopes O +on O +both O +binding O +partners O +, O +and O +is O +therefore O +hard O +to O +target O +using O +small O +molecules O +. O + +Our O +studies O +of O +HAP B-chemical +demonstrated O +an O +uncommon O +mode O +of O +action O +for O +a O +peptide O +in O +inhibiting O +such O +a O +difficult O +protein O +- O +protein O +interaction O +target O +, O +and O +suggest O +further O +possible O +improvements O +in O +its O +binding O +potency O +. O + +One O +way O +of O +further O +improving O +HAP B-chemical +’ O +s O +potency O +is O +by O +dimerization O +. O + +Homo O +- O +dimerization O +of O +HAP B-chemical +( O +45 B-chemical +) O +achieved O +sub O +- O +nanomolar O +potency O +against O +human B-species +IL B-protein +- I-protein +17A I-protein +in O +cell O +assay O +. O + +In O +the O +crystal B-evidence +structure I-evidence +, O +the O +distance O +between O +the O +carbonyl O +of O +Asn14 B-residue_name_number +of O +one O +HAP B-chemical +molecule O +and O +the O +N O +- O +terminus O +of O +the O +second O +is O +only O +15 O +. O +7 O +Å O +, O +suggesting O +the O +potential O +for O +more O +potent O +dimeric B-oligomeric_state +peptides B-chemical +to O +be O +designed O +by O +using O +linkers O +of O +different O +lengths O +at O +different O +positions O +. O + +Another O +direction O +of O +improving O +HAP B-chemical +is O +by O +reducing O +its O +size O +. O + +As O +demonstrated O +by O +the O +crystal B-evidence +structure I-evidence +, O +binding O +of O +the O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +HAP B-chemical +should O +be O +sufficient O +for O +preventing O +IL B-protein +- I-protein +17RA I-protein +binding O +to O +IL B-protein +- I-protein +17A I-protein +. O + +Theoretically O +, O +it O +is O +possible O +to O +design O +chemicals O +such O +as O +stapled O +α O +- O +helical O +peptides O +to O +block O +α B-structure_element +- I-structure_element +helix I-structure_element +- O +mediated O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +interactions O +. O + +In O +summary O +, O +these O +peptide O +- O +based O +anti O +- O +IL B-protein +- I-protein +17A I-protein +modalities O +could O +be O +further O +developed O +as O +alternative O +therapeutic O +options O +to O +the O +reported O +monoclonal O +antibodies B-protein_type +. O + +We O +are O +also O +very O +interested O +in O +finding O +non O +- O +peptidic O +small O +molecule O +IL B-protein +- I-protein +17A I-protein +antagonists O +, O +and O +HAP B-chemical +can O +be O +used O +as O +an O +excellent O +tool O +peptide O +. O + +The O +strategy O +utilized O +in O +generating O +the O +complex O +structures B-evidence +of O +HAP B-chemical +may O +also O +be O +useful O +for O +enabling O +structure O +based O +design O +of O +some O +known O +small O +molecule O +IL O +- O +17A O +antagonists O +. 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O + +Kinetic O +parameters O +( O +ka B-evidence +, O +kd B-evidence +) O +were O +obtained O +by O +a O +global O +fit O +using O +three O +concentrations O +in O +triplicate O +. O + +KD B-evidence +determined O +by O +the O +standard O +equation O +, O +KD B-evidence += O +kd B-evidence +/ O +ka B-evidence +. O +( O +B O +) O +HAP B-chemical +inhibits O +SPR B-experimental_method +signaling O +of O +IL B-protein +- I-protein +17A I-protein +binding O +to O +immobilized B-protein_state +IL B-protein +- I-protein +17RA I-protein +. O + +Data O +are O +mean O +and O +error O +bars O +of O ++/− O +standard O +deviation O +of O +three O +measurements O +. O +( O +C O +) O +Inhibition O +of O +IL B-protein +- I-protein +17A I-protein +and O +IL B-protein +- I-protein +17RA I-protein +binding O +by O +HAP B-chemical +measured O +by O +FRET B-experimental_method +assay I-experimental_method +. O + +Data O +are O +mean O +and O +error O +bars O +of O ++/− O +standard O +deviation O +from O +299 O +experiments O +, O +each O +performed O +in O +duplicate O +. O +( O +D O +) O +Example O +of O +HAP B-chemical +selective O +inhibition O +of O +the O +production O +of O +IL B-protein_type +- I-protein_type +8 I-protein_type +( O +triangles O +), O +IL B-protein_type +- I-protein_type +6 I-protein_type +( O +squares O +) O +and O +CCL B-protein_type +- I-protein_type +20 I-protein_type +( O +circles O +) O +by O +primary O +human B-species +keratinocyte O +cells O +synergistically O +stimulated O +by O +100 O +ng O +/ O +ml O +IL B-protein +- I-protein +17A I-protein +and O +10 O +ng O +/ O +ml O +TNF B-protein +- I-protein +α I-protein +. O + +HAP B-chemical +does O +not O +inhibit O +the O +baseline O +production O +of O +IL B-protein_type +- I-protein_type +6 I-protein_type +, O +IL B-protein_type +- I-protein_type +8 I-protein_type +and O +CCL B-protein_type +- I-protein_type +20 I-protein_type +stimulated O +by O +10 O +ng O +/ O +ml O +TNF B-protein +- I-protein +α I-protein +alone O +( O +gray O +lines O +and O +symbols O +). O + +Overall O +structure B-evidence +of O +the O +Fab B-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O +in O +ribbon O +presentation O +. O + +Two O +HAP B-chemical +molecules O +are O +colored O +blue O +and O +red O +, O +and O +IL B-protein +- I-protein +17A I-protein +monomers B-oligomeric_state +are O +colored O +ice O +blue O +and O +pink O +, O +respectively O +. O + +( O +A O +) O +Overview O +of O +the O +distinct O +binding B-site +sites I-site +of O +Fab B-structure_element +and O +HAP B-chemical +to O +IL B-protein +- I-protein +17A I-protein +. O + +( O +B O +) O +Close O +- O +in O +view O +of O +the O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +structure B-evidence +. O + +IL B-protein +- I-protein +17A I-protein +β B-structure_element +- I-structure_element +strands I-structure_element +are O +labelled O +. O + +Each O +of O +the O +two O +bound B-protein_state +HAP B-chemical +interacts O +with O +both O +monomers B-oligomeric_state +of O +the O +IL B-protein +- I-protein +17A I-protein +dimer B-oligomeric_state +. O + +( O +C O +) O +As O +a O +comparison O +, O +the O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +complex O +was O +shown O +with O +IL B-protein +- I-protein +17A I-protein +in O +the O +same O +orientation O +. O + +Three O +distinct O +areas O +IL B-site +- I-site +17A I-site +/ I-site +IL I-site +- I-site +17RA I-site +interface I-site +are O +labeled O +. O + +Mechanism O +of O +the O +inhibition O +of O +the O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +interaction O +by O +HAP B-chemical +. O + +( O +A O +) O +HAP B-chemical +binds O +at O +region B-structure_element +I I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +. O + +IL B-protein +- I-protein +17A I-protein +dimer B-oligomeric_state +is O +in O +surface O +presentation O +( O +β B-structure_element +- I-structure_element +strands I-structure_element +0 I-structure_element +shown O +as O +ribbons O +for O +clarity O +). O + +Polar B-bond_interaction +interactions I-bond_interaction +are O +shown O +in O +dashes O +. O + +HAP B-chemical +residues O +as O +well O +as O +key O +IL B-protein +- I-protein +17A I-protein +residues O +are O +labeled O +. O + +For O +clarity O +, O +a O +few O +HAP B-chemical +residues O +are O +also O +shown O +in O +stick O +model O +with O +carbon O +atoms O +colored O +green O +, O +oxygen O +in O +red O +and O +nitrogen O +in O +blue O +. 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O + +Recently O +, O +we O +reported O +the O +crystal B-evidence +structure I-evidence +of O +NadA B-protein +, O +providing O +insights O +into O +its O +biological O +and O +immunological O +functions O +. O + +Recombinant O +NadA B-protein +elicits O +a O +strong O +bactericidal O +immune O +response O +and O +is O +therefore O +included O +in O +the O +Bexsero O +vaccine O +that O +protects O +against O +MenB B-species +and O +which O +was O +recently O +approved O +in O +over O +35 O +countries O +worldwide O +. O + +Previous O +studies O +revealed O +that O +nadA B-gene +expression O +levels O +are O +mainly O +regulated O +by O +the O +Neisseria B-protein +adhesin I-protein +A I-protein +Regulator I-protein +( O +NadR B-protein +). O + +Although O +additional O +factors O +influence O +nadA B-gene +expression O +, O +we O +focused O +on O +its O +regulation O +by O +NadR B-protein +, O +the O +major O +mediator O +of O +NadA B-protein +phase O +variable O +expression O +. O + +Studies O +of O +NadR B-protein +also O +have O +broader O +implications O +, O +since O +a O +genome O +- O +wide O +analysis O +of O +MenB B-species +wild B-protein_state +- I-protein_state +type I-protein_state +and O +nadR B-gene +knock B-protein_state +- I-protein_state +out I-protein_state +strains O +revealed O +that O +NadR B-protein +influences O +the O +regulation O +of O +> O +30 O +genes O +, O +including O +maf O +genes O +, O +from O +the O +multiple O +adhesin B-protein_type +family O +. O + +These O +genes O +encode O +a O +wide O +variety O +of O +proteins O +connected O +to O +many O +biological O +processes O +contributing O +to O +bacterial B-taxonomy_domain +survival O +, O +adaptation O +in O +the O +host O +niche O +, O +colonization O +and O +invasion O +. O + +NadR B-protein +belongs O +to O +the O +MarR B-protein_type +( O +Multiple B-protein_type +Antibiotic I-protein_type +Resistance I-protein_type +Regulator I-protein_type +) O +family O +, O +a O +group O +of O +ligand B-protein_type +- I-protein_type +responsive I-protein_type +transcriptional I-protein_type +regulators I-protein_type +ubiquitous O +in O +bacteria B-taxonomy_domain +and O +archaea B-taxonomy_domain +. O + +MarR B-protein_type +family O +proteins O +can O +promote O +bacterial B-taxonomy_domain +survival O +in O +the O +presence O +of O +antibiotics O +, O +toxic O +chemicals O +, O +organic O +solvents O +or O +reactive O +oxygen O +species O +and O +can O +regulate O +virulence O +factor O +expression O +. O + +MarR B-protein_type +homologues O +can O +act O +either O +as O +transcriptional O +repressors O +or O +as O +activators O +. O + +Although O +> O +50 O +MarR B-protein_type +family O +structures B-evidence +are O +known O +, O +a O +molecular O +understanding O +of O +their O +ligand O +- O +dependent O +regulatory O +mechanisms O +is O +still O +limited O +, O +often O +hampered O +by O +lack O +of O +identification O +of O +their O +ligands O +and O +/ O +or O +DNA O +targets O +. O + +A O +potentially O +interesting O +exception O +comes O +from O +the O +ligand B-protein_state +- I-protein_state +free I-protein_state +and O +salicylate B-protein_state +- I-protein_state +bound I-protein_state +forms O +of O +the O +Methanobacterium B-species +thermoautotrophicum I-species +protein O +MTH313 B-protein +which O +revealed O +that O +two O +salicylate B-chemical +molecules O +bind O +to O +one O +MTH313 B-protein +dimer B-oligomeric_state +and O +induce O +large O +conformational O +changes O +, O +apparently O +sufficient O +to O +prevent O +DNA O +binding O +. O + +However O +, O +the O +homologous O +archeal B-taxonomy_domain +Sulfolobus B-species +tokodaii I-species +protein O +ST1710 B-protein +presented O +essentially O +the O +same O +structure B-evidence +in O +ligand B-protein_state +- I-protein_state +free I-protein_state +and O +salicylate B-protein_state +- I-protein_state +bound I-protein_state +forms O +, O +apparently O +contrasting O +the O +mechanism O +proposed O +for O +MTH313 B-protein +. O + +Despite O +these O +apparent O +differences O +, O +MTH313 B-protein +and O +ST1710 B-protein +bind O +salicylate B-chemical +in O +approximately O +the O +same O +site O +, O +between O +their O +dimerization B-structure_element +and I-structure_element +DNA I-structure_element +- I-structure_element +binding I-structure_element +domains I-structure_element +. O + +However O +, O +it O +is O +unknown O +whether O +salicylate B-chemical +is O +a O +relevant O +in O +vivo O +ligand O +of O +either O +of O +these O +two O +proteins O +, O +which O +share O +~ O +20 O +% O +sequence O +identity O +with O +NadR B-protein +, O +rendering O +unclear O +the O +interpretation O +of O +these O +findings O +in O +relation O +to O +the O +regulatory O +mechanisms O +of O +NadR B-protein +or O +other O +MarR B-protein_type +family O +proteins O +. O + +NadR B-protein +binds O +nadA B-gene +on O +three O +different O +operators O +( O +OpI O +, O +OpII O +and O +OpIII O +). O + +The O +DNA O +- O +binding O +activity O +of O +NadR B-protein +is O +attenuated O +in O +vitro O +upon O +addition O +of O +various O +hydroxyphenylacetate B-chemical +( O +HPA B-chemical +) O +derivatives O +, O +including O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +4 B-chemical +- I-chemical +HPA I-chemical +is O +a O +small O +molecule O +derived O +from O +mammalian B-taxonomy_domain +aromatic O +amino O +acid O +catabolism O +and O +is O +released O +in O +human B-species +saliva O +, O +where O +it O +has O +been O +detected O +at O +micromolar O +concentration O +. O + +In O +the O +presence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +, O +NadR B-protein +is O +unable O +to O +bind O +the O +nadA B-gene +promoter O +and O +nadA B-gene +gene O +expression O +is O +induced O +. O + +In O +vivo O +, O +the O +presence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +in O +the O +host O +niche O +of O +N B-species +. I-species +meningitidis I-species +serves O +as O +an O +inducer O +of O +NadA B-protein +production O +, O +thereby O +promoting O +bacterial B-taxonomy_domain +adhesion O +to O +host O +cells O +. O + +Further O +, O +we O +recently O +reported O +that O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +, O +produced O +during O +inflammation O +, O +is O +another O +inducer O +of O +nadA B-gene +expression O +. O + +Extending O +our O +previous O +studies O +based O +on O +hydrogen B-experimental_method +- I-experimental_method +deuterium I-experimental_method +exchange I-experimental_method +mass I-experimental_method +spectrometry I-experimental_method +( O +HDX B-experimental_method +- I-experimental_method +MS I-experimental_method +), O +here O +we O +sought O +to O +reveal O +the O +molecular O +mechanisms O +and O +effects O +of O +NadR B-protein +/ O +HPA B-chemical +interactions O +via O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +, O +NMR B-experimental_method +spectroscopy I-experimental_method +and O +complementary O +biochemical B-experimental_method +and I-experimental_method +in I-experimental_method +vivo I-experimental_method +mutagenesis I-experimental_method +studies I-experimental_method +. O + +We O +obtained O +detailed O +new O +insights O +into O +ligand O +specificity O +, O +how O +the O +ligand O +allosterically O +influences O +the O +DNA O +- O +binding O +ability O +of O +NadR B-protein +, O +and O +the O +regulation O +of O +nadA B-gene +expression O +, O +thus O +also O +providing O +a O +deeper O +structural O +understanding O +of O +the O +ligand O +- O +responsive O +MarR B-protein_type +super O +- O +family O +. O + +Moreover O +, O +these O +findings O +are O +important O +because O +the O +activity O +of O +NadR B-protein +impacts O +the O +potential O +coverage O +provided O +by O +anti O +- O +NadA B-protein +antibodies O +elicited O +by O +the O +Bexsero O +vaccine O +and O +influences O +host O +- O +bacteria B-taxonomy_domain +interactions O +that O +contribute O +to O +meningococcal B-taxonomy_domain +pathogenesis O +. O + +NadR B-protein +is O +dimeric B-oligomeric_state +and O +is O +stabilized O +by O +specific O +hydroxyphenylacetate B-chemical +ligands O + +Recombinant O +NadR B-protein +was O +produced O +in O +E B-species +. I-species +coli I-species +using O +an O +expression B-experimental_method +construct I-experimental_method +prepared O +from O +N B-species +. I-species +meningitidis I-species +serogroup I-species +B I-species +strain I-species +MC58 I-species +. O + +Standard O +chromatographic O +techniques O +were O +used O +to O +obtain O +a O +highly O +purified O +sample O +of O +NadR B-protein +( O +see O +Materials O +and O +Methods O +). O + +In O +analytical B-experimental_method +size I-experimental_method +- I-experimental_method +exclusion I-experimental_method +high I-experimental_method +- I-experimental_method +performance I-experimental_method +liquid I-experimental_method +chromatography I-experimental_method +( O +SE B-experimental_method +- I-experimental_method +HPLC I-experimental_method +) O +experiments O +coupled O +with O +multi B-experimental_method +- I-experimental_method +angle I-experimental_method +laser I-experimental_method +light I-experimental_method +scattering I-experimental_method +( O +MALLS B-experimental_method +), O +NadR B-protein +presented O +a O +single O +species O +with O +an O +absolute O +molecular O +mass O +of O +35 O +kDa O +( O +S1 O +Fig O +). O + +These O +data O +showed O +that O +NadR B-protein +was O +dimeric B-oligomeric_state +in O +solution O +, O +since O +the O +theoretical O +molecular O +mass O +of O +the O +NadR B-protein +dimer B-oligomeric_state +is O +33 O +. O +73 O +kDa O +; O +and O +, O +there O +was O +no O +change O +in O +oligomeric O +state O +on O +addition O +of O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +The O +thermal O +stability O +of O +NadR B-protein +was O +examined O +using O +differential B-experimental_method +scanning I-experimental_method +calorimetry I-experimental_method +( O +DSC B-experimental_method +). O + +Since O +ligand O +- O +binding O +often O +increases O +protein O +stability O +, O +we O +also O +investigated O +the O +effect O +of O +various O +HPAs B-chemical +( O +Fig O +1A O +) O +on O +the O +melting B-evidence +temperature I-evidence +( O +Tm B-evidence +) O +of O +NadR B-protein +. O +As O +a O +control O +of O +specificity O +, O +we O +also O +tested O +salicylate B-chemical +, O +a O +known O +ligand O +of O +some O +MarR B-protein_type +proteins O +previously O +reported O +to O +increase O +the O +Tm B-evidence +of O +ST1710 B-protein +and O +MTH313 B-protein +. O + +The O +Tm B-evidence +of O +NadR B-protein +was O +67 O +. O +4 O +± O +0 O +. O +1 O +° O +C O +in O +the O +absence B-protein_state +of I-protein_state +ligand I-protein_state +, O +and O +was O +unaffected O +by O +salicylate B-chemical +. O + +However O +, O +an O +increased O +thermal O +stability O +was O +induced O +by O +4 B-chemical +- I-chemical +HPA I-chemical +and O +, O +to O +a O +lesser O +extent O +, O +by O +3 B-chemical +- I-chemical +HPA I-chemical +. O + +Interestingly O +, O +NadR B-protein +displayed O +the O +greatest O +Tm B-evidence +increase O +upon O +addition O +of O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +( O +Table O +1 O +and O +Fig O +1B O +). O + +Stability O +of O +NadR B-protein +is O +increased O +by O +small O +molecule O +ligands O +. O + +( O +A O +) O +Molecular O +structures O +of O +3 B-chemical +- I-chemical +HPA I-chemical +( O +MW O +152 O +. O +2 O +), O +4 B-chemical +- I-chemical +HPA I-chemical +( O +MW O +152 O +. O +2 O +), O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +( O +MW O +186 O +. O +6 O +) O +and O +salicylic B-chemical +acid I-chemical +( O +MW O +160 O +. O +1 O +). O +( O +B O +) O +DSC B-experimental_method +profiles B-evidence +, O +colored O +as O +follows O +: O +apo B-protein_state +- O +NadR B-protein +( O +violet O +), O +NadR B-complex_assembly ++ I-complex_assembly +salicylate I-complex_assembly +( O +red O +), O +NadR B-complex_assembly ++ I-complex_assembly +3 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +( O +green O +), O +NadR B-complex_assembly ++ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +( O +blue O +), O +NadR B-complex_assembly ++ I-complex_assembly +3Cl I-complex_assembly +, I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +( O +pink O +). O + +All O +DSC B-experimental_method +profiles B-evidence +are O +representative O +of O +triplicate O +experiments O +. O + +Melting B-evidence +- I-evidence +point I-evidence +( O +Tm B-evidence +) O +and O +its O +ligand O +- O +induced O +increase O +( O +ΔTm B-evidence +) O +derived O +from O +DSC B-experimental_method +thermostability B-experimental_method +experiments I-experimental_method +. O + +Dissociation B-evidence +constants I-evidence +( O +KD B-evidence +) O +of O +the O +NadR B-protein +/ O +ligand O +interactions O +from O +SPR B-experimental_method +steady I-experimental_method +- I-experimental_method +state I-experimental_method +binding I-experimental_method +experiments I-experimental_method +. O + +Ligand O +Tm B-evidence +(° O +C O +) O +ΔTm B-evidence +(° O +C O +) O +KD B-evidence +( O +mM O +) O +No O +ligand O +67 O +. O +4 O +± O +0 O +. O +1 O +n O +. O +a O +. O +n O +. O +a O +. O + +3 B-chemical +- I-chemical +HPA I-chemical +70 O +. O +0 O +± O +0 O +. O +1 O +2 O +. O +7 O +2 O +. O +7 O +± O +0 O +. O +1 O +4 B-chemical +- I-chemical +HPA I-chemical +70 O +. O +7 O +± O +0 O +. O +1 O +3 O +. O +3 O +1 O +. O +5 O +± O +0 O +. O +1 O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +71 O +. O +3 O +± O +0 O +. O +2 O +3 O +. O +9 O +1 O +. O +1 O +± O +0 O +. O +1 O + +NadR B-protein +displays O +distinct O +binding B-evidence +affinities I-evidence +for O +hydroxyphenylacetate B-chemical +ligands O + +To O +further O +investigate O +the O +binding O +of O +HPAs B-chemical +to O +NadR B-protein +, O +we O +used O +surface B-experimental_method +plasmon I-experimental_method +resonance I-experimental_method +( O +SPR B-experimental_method +). O + +The O +SPR B-experimental_method +sensorgrams B-evidence +revealed O +very O +fast O +association O +and O +dissociation O +events O +, O +typical O +of O +small O +molecule O +ligands O +, O +thus O +prohibiting O +a O +detailed O +study O +of O +binding O +kinetics O +. O + +However O +, O +steady B-experimental_method +- I-experimental_method +state I-experimental_method +SPR I-experimental_method +analyses O +of O +the O +NadR B-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +interactions O +allowed O +determination O +of O +the O +equilibrium B-evidence +dissociation I-evidence +constants I-evidence +( O +KD B-evidence +) O +( O +Table O +1 O +and O +S2 O +Fig O +). O + +The O +interactions O +of O +4 B-chemical +- I-chemical +HPA I-chemical +and O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +with O +NadR B-protein +exhibited O +KD B-evidence +values O +of O +1 O +. O +5 O +mM O +and O +1 O +. O +1 O +mM O +, O +respectively O +. O + +3 B-chemical +- I-chemical +HPA I-chemical +showed O +a O +weaker O +interaction O +, O +with O +a O +KD B-evidence +of O +2 O +. O +7 O +mM O +, O +while O +salicylate B-chemical +showed O +only O +a O +very O +weak O +response O +that O +did O +not O +reach O +saturation O +, O +indicating O +a O +non O +- O +specific O +interaction O +with O +NadR B-protein +. O +A O +ranking O +of O +these O +KD B-evidence +values O +showed O +that O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +was O +the O +tightest O +binder O +, O +and O +thus O +matched O +the O +ranking O +of O +ligand O +- O +induced O +Tm B-evidence +increases O +observed O +in O +the O +DSC B-experimental_method +experiments O +. O + +Although O +these O +KD B-evidence +values O +indicate O +rather O +weak O +interactions O +, O +they O +are O +similar O +to O +the O +values O +reported O +previously O +for O +the O +MarR B-protein_type +/ O +salicylate B-chemical +interaction O +( O +KD O +~ O +1 O +mM O +) O +and O +the O +MTH313 B-protein +/ O +salicylate B-chemical +interaction O +( O +KD O +2 O +– O +3 O +mM O +), O +and O +approximately O +20 O +- O +fold O +tighter O +than O +the O +ST1710 B-protein +/ O +salicylate B-chemical +interaction O +( O +KD O +~ O +20 O +mM O +). O + +Crystal B-evidence +structures I-evidence +of O +holo B-protein_state +- O +NadR B-protein +and O +apo B-protein_state +- O +NadR B-protein + +To O +fully O +characterize O +the O +NadR B-protein +/ O +HPA B-chemical +interactions O +, O +we O +sought O +to O +determine O +crystal B-evidence +structures I-evidence +of O +NadR B-protein +in O +ligand B-protein_state +- I-protein_state +bound I-protein_state +( O +holo B-protein_state +) O +and O +ligand B-protein_state +- I-protein_state +free I-protein_state +( O +apo B-protein_state +) O +forms O +. O + +First O +, O +we O +crystallized B-experimental_method +NadR B-protein +( O +a O +selenomethionine B-experimental_method +- I-experimental_method +labelled I-experimental_method +derivative I-experimental_method +) O +in O +the O +presence O +of O +a O +200 O +- O +fold O +molar O +excess O +of O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +The O +structure B-evidence +of O +the O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +complex O +was O +determined O +at O +2 O +. O +3 O +Å O +resolution O +using O +a O +combination O +of O +the O +single B-experimental_method +- I-experimental_method +wavelength I-experimental_method +anomalous I-experimental_method +dispersion I-experimental_method +( O +SAD B-experimental_method +) O +and O +molecular B-experimental_method +replacement I-experimental_method +( O +MR B-experimental_method +) O +methods O +, O +and O +was O +refined O +to O +R B-evidence +work I-evidence +/ I-evidence +R I-evidence +free I-evidence +values O +of O +20 O +. O +9 O +/ O +26 O +. O +0 O +% O +( O +Table O +2 O +). O + +Despite O +numerous O +attempts O +, O +we O +were O +unable O +to O +obtain O +high O +- O +quality O +crystals B-evidence +of O +NadR B-protein +complexed B-protein_state +with I-protein_state +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +, O +3 B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +, O +3 B-chemical +- I-chemical +HPA I-chemical +or O +DNA O +targets O +. O + +However O +, O +it O +was O +eventually O +possible O +to O +crystallize B-experimental_method +apo B-protein_state +- O +NadR B-protein +, O +and O +the O +structure B-evidence +was O +determined O +at O +2 O +. O +7 O +Å O +resolution O +by O +MR B-experimental_method +methods O +using O +the O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +complex O +as O +the O +search O +model O +. O + +The O +apo B-protein_state +- O +NadR B-protein +structure B-evidence +was O +refined O +to O +R B-evidence +work I-evidence +/ I-evidence +R I-evidence +free I-evidence +values O +of O +19 O +. O +1 O +/ O +26 O +. O +8 O +% O +( O +Table O +2 O +). O + +Data O +collection O +and O +refinement O +statistics O +for O +NadR B-protein +structures B-evidence +. O + +The O +asymmetric O +unit O +of O +the O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +crystals B-evidence +( O +holo B-protein_state +- O +NadR B-protein +) O +contained O +one O +NadR B-protein +homodimer B-oligomeric_state +, O +while O +the O +apo B-protein_state +- O +NadR B-protein +crystals B-evidence +contained O +two O +homodimers B-oligomeric_state +. O + +In O +the O +apo B-protein_state +- O +NadR B-protein +crystals B-evidence +, O +the O +two O +homodimers B-oligomeric_state +were O +related O +by O +a O +rotation O +of O +~ O +90 O +°; O +the O +observed O +association O +of O +the O +two O +dimers B-oligomeric_state +was O +presumably O +merely O +an O +effect O +of O +crystal O +packing O +, O +since O +the O +interface B-site +between O +the O +two O +homodimers B-oligomeric_state +is O +small O +(< O +550 O +Å2 O +of O +buried O +surface O +area O +), O +and O +is O +not O +predicted O +to O +be O +physiologically O +relevant O +by O +the O +PISA O +software O +. O + +Moreover O +, O +our O +SE B-experimental_method +- I-experimental_method +HPLC I-experimental_method +/ I-experimental_method +MALLS I-experimental_method +analyses O +( O +see O +above O +) O +revealed O +that O +in O +solution O +NadR B-protein +is O +dimeric B-oligomeric_state +, O +and O +previous O +studies O +using O +native B-experimental_method +mass I-experimental_method +spectrometry I-experimental_method +( O +MS B-experimental_method +) O +revealed O +dimers B-oligomeric_state +, O +not O +tetramers B-oligomeric_state +. O + +The O +NadR B-protein +homodimer B-oligomeric_state +bound B-protein_state +to I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +has O +a O +dimerization B-site +interface I-site +mostly O +involving O +the O +top O +of O +its O +‘ O +triangular B-protein_state +’ O +form O +, O +while O +the O +two O +DNA B-structure_element +- I-structure_element +binding I-structure_element +domains I-structure_element +are O +located O +at O +the O +base O +( O +Fig O +2A O +). O + +High O +- O +quality O +electron B-evidence +density I-evidence +maps I-evidence +allowed O +clear O +identification O +of O +the O +bound B-protein_state +ligand O +, O +4 B-chemical +- I-chemical +HPA I-chemical +( O +Fig O +2B O +). O + +The O +overall O +structure B-evidence +of O +NadR B-protein +shows O +dimensions O +of O +~ O +50 O +× O +65 O +× O +50 O +Å O +and O +a O +large O +homodimer B-site +interface I-site +that O +buries O +a O +total O +surface O +area O +of O +~ O +4800 O +Å2 O +. O + +Each O +NadR B-protein +monomer B-oligomeric_state +consists O +of O +six O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +two O +short B-structure_element +β I-structure_element +- I-structure_element +strands I-structure_element +, O +with O +helices B-structure_element +α1 B-structure_element +, O +α5 B-structure_element +, O +and O +α6 B-structure_element +forming O +the O +dimer B-site +interface I-site +. O + +Helices B-structure_element +α3 B-structure_element +and O +α4 B-structure_element +form O +a O +helix B-structure_element +- I-structure_element +turn I-structure_element +- I-structure_element +helix I-structure_element +motif I-structure_element +, O +followed O +by O +the O +“ O +wing B-structure_element +motif I-structure_element +” O +comprised O +of O +two O +short B-structure_element +antiparallel I-structure_element +β I-structure_element +- I-structure_element +strands I-structure_element +( O +β1 B-structure_element +- I-structure_element +β2 I-structure_element +) O +linked O +by O +a O +relatively O +long O +and O +flexible O +loop B-structure_element +. O + +Interestingly O +, O +in O +the O +α4 B-structure_element +- I-structure_element +β2 I-structure_element +region I-structure_element +, O +the O +stretch O +of O +residues O +from O +R64 B-residue_range +- I-residue_range +R91 I-residue_range +presents O +seven O +positively O +- O +charged O +side O +chains O +, O +all O +available O +for O +potential O +interactions O +with O +DNA B-chemical +. O + +Together O +, O +these O +structural O +elements O +constitute O +the O +winged B-structure_element +helix I-structure_element +- I-structure_element +turn I-structure_element +- I-structure_element +helix I-structure_element +( O +wHTH B-structure_element +) O +DNA B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +and O +, O +together O +with O +the O +dimeric B-oligomeric_state +organization O +, O +are O +the O +hallmarks O +of O +MarR B-protein_type +family O +structures B-evidence +. O + +The O +crystal B-evidence +structure I-evidence +of O +NadR B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +. O + +( O +A O +) O +The O +holo B-protein_state +- O +NadR B-protein +homodimer B-oligomeric_state +is O +depicted O +in O +green O +and O +blue O +for O +chains B-structure_element +A I-structure_element +and I-structure_element +B I-structure_element +respectively O +, O +while O +yellow O +sticks O +depict O +the O +4 B-chemical +- I-chemical +HPA I-chemical +ligand O +( O +labelled O +). O + +For O +simplicity O +, O +secondary O +structure O +elements O +are O +labelled O +for O +chain B-structure_element +B I-structure_element +only O +. O + +Red O +dashes O +show O +hypothetical O +positions O +of O +chain B-structure_element +B I-structure_element +residues O +88 B-residue_range +– I-residue_range +90 I-residue_range +that O +were O +not O +modeled O +due O +to O +lack O +of O +electron B-evidence +density I-evidence +. O + +( O +B O +) O +A O +zoom O +into O +the O +pocket B-site +occupied O +by O +4 B-chemical +- I-chemical +HPA I-chemical +shows O +that O +the O +ligand O +contacts O +both O +chains B-structure_element +A I-structure_element +and I-structure_element +B I-structure_element +; O +blue O +mesh O +shows O +electron B-evidence +density I-evidence +around O +4 B-chemical +- I-chemical +HPA I-chemical +calculated O +from O +a O +composite B-evidence +omit I-evidence +map I-evidence +( O +omitting O +4 B-chemical +- I-chemical +HPA I-chemical +), O +using O +phenix B-experimental_method +. O + +The O +map B-evidence +is O +contoured O +at O +1σ O +and O +the O +figure O +was O +prepared O +with O +a O +density B-evidence +mesh I-evidence +carve O +factor O +of O +1 O +. O +7 O +, O +using O +Pymol O +( O +www O +. O +pymol O +. O +org O +). O + +A O +single O +conserved B-protein_state +leucine B-residue_name +residue O +( O +L130 B-residue_name_number +) O +is O +crucial O +for O +dimerization O + +The O +NadR B-protein +dimer B-site +interface I-site +is O +formed O +by O +at O +least O +32 O +residues O +, O +which O +establish O +numerous O +inter O +- O +chain O +salt B-bond_interaction +bridges I-bond_interaction +or O +hydrogen B-bond_interaction +bonds I-bond_interaction +, O +and O +many O +hydrophobic B-bond_interaction +packing I-bond_interaction +interactions I-bond_interaction +( O +Fig O +3A O +and O +3B O +). O + +To O +determine O +which O +residues O +were O +most O +important O +for O +dimerization O +, O +we O +studied O +the O +interface B-site +in O +silico O +and O +identified O +several O +residues O +as O +potential O +mediators O +of O +key O +stabilizing O +interactions O +. O + +Using O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +, O +a O +panel O +of O +eight O +mutant B-protein_state +NadR B-protein +proteins O +was O +prepared O +( O +including O +mutations O +H7A B-mutant +, O +S9A B-mutant +, O +N11A B-mutant +, O +D112A B-mutant +, O +R114A B-mutant +, O +Y115A B-mutant +, O +K126A B-mutant +, O +L130K B-mutant +and O +L133K B-mutant +), O +sufficient O +to O +explore O +the O +entire O +dimer B-site +interface I-site +. O + +Each O +mutant B-protein_state +NadR B-protein +protein O +was O +purified O +, O +and O +then O +its O +oligomeric O +state O +was O +examined O +by O +analytical B-experimental_method +SE I-experimental_method +- I-experimental_method +HPLC I-experimental_method +. O + +Almost O +all O +the O +mutants O +showed O +the O +same O +elution O +profile O +as O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +NadR B-protein +protein O +. O + +Only O +the O +L130K B-mutant +mutation O +induced O +a O +notable O +change O +in O +the O +oligomeric O +state O +of O +NadR B-protein +( O +Fig O +3C O +). O + +Further O +, O +in O +SE B-experimental_method +- I-experimental_method +MALLS I-experimental_method +analyses O +, O +the O +L130K B-mutant +mutant B-protein_state +displayed O +two O +distinct O +species O +in O +solution O +, O +approximately O +80 O +% O +being O +monomeric B-oligomeric_state +( O +a O +19 O +kDa O +species O +), O +and O +only O +20 O +% O +retaining O +the O +typical O +native O +dimeric B-oligomeric_state +state O +( O +a O +35 O +kDa O +species O +) O +( O +Fig O +3D O +), O +demonstrating O +that O +Leu130 B-residue_name_number +is O +crucial O +for O +stable O +dimerization O +. O + +It O +is O +notable O +that O +L130 B-residue_name_number +is O +usually O +present O +as O +Leu B-residue_name +, O +or O +an O +alternative O +bulky O +hydrophobic O +amino O +acid O +( O +e O +. O +g O +. O +Phe B-residue_name +, O +Val B-residue_name +), O +in O +many O +MarR B-protein_type +family O +proteins O +, O +suggesting O +a O +conserved B-protein_state +role O +in O +stabilizing O +the O +dimer B-site +interface I-site +. O + +In O +contrast O +, O +most O +of O +the O +other O +residues O +identified O +in O +the O +NadR B-protein +dimer B-site +interface I-site +were O +poorly B-protein_state +conserved I-protein_state +in O +the O +MarR B-protein_type +family O +. O + +Analysis O +of O +the O +NadR B-protein +dimer B-site +interface I-site +. O + +( O +A O +) O +Both O +orientations O +show O +chain B-structure_element +A I-structure_element +, O +green O +backbone O +ribbon O +, O +colored O +red O +to O +highlight O +all O +locations O +involved O +in O +dimerization O +; O +namely O +, O +inter O +- O +chain O +salt B-bond_interaction +bridges I-bond_interaction +or O +hydrogen B-bond_interaction +bonds I-bond_interaction +involving O +Q4 B-residue_name_number +, O +S5 B-residue_name_number +, O +K6 B-residue_name_number +, O +H7 B-residue_name_number +, O +S9 B-residue_name_number +, O +I10 B-residue_name_number +, O +N11 B-residue_name_number +, O +I15 B-residue_name_number +, O +Q16 B-residue_name_number +, O +R18 B-residue_name_number +, O +D36 B-residue_name_number +, O +R43 B-residue_name_number +, O +A46 B-residue_name_number +, O +Q59 B-residue_name_number +, O +C61 B-residue_name_number +, O +Y104 B-residue_name_number +, O +D112 B-residue_name_number +, O +R114 B-residue_name_number +, O +Y115 B-residue_name_number +, O +D116 B-residue_name_number +, O +E119 B-residue_name_number +, O +K126 B-residue_name_number +, O +E136 B-residue_name_number +, O +E141 B-residue_name_number +, O +N145 B-residue_name_number +, O +and O +the O +hydrophobic B-bond_interaction +packing I-bond_interaction +interactions I-bond_interaction +involving O +I10 B-residue_name_number +, O +I12 B-residue_name_number +, O +L14 B-residue_name_number +, O +I15 B-residue_name_number +, O +R18 B-residue_name_number +, O +Y115 B-residue_name_number +, O +I118 B-residue_name_number +, O +L130 B-residue_name_number +, O +L133 B-residue_name_number +, O +L134 B-residue_name_number +and O +L137 B-residue_name_number +. O + +Chain B-structure_element +B I-structure_element +, O +grey O +surface O +, O +is O +marked O +blue O +to O +highlight O +residues O +probed O +by O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +( O +E136 B-residue_name_number +only O +makes O +a O +salt B-bond_interaction +bridge I-bond_interaction +with O +K126 B-residue_name_number +, O +therefore O +it O +was O +sufficient O +to O +make O +the O +K126A B-mutant +mutation O +to O +assess O +the O +importance O +of O +this O +ionic B-bond_interaction +interaction I-bond_interaction +; O +the O +H7 B-residue_name_number +position O +is O +labelled O +for O +monomer B-oligomeric_state +A B-structure_element +, O +since O +electron B-evidence +density I-evidence +was O +lacking O +for O +monomer B-oligomeric_state +B B-structure_element +). O +( O +B O +) O +A O +zoom O +into O +the O +environment O +of O +helix B-structure_element +α6 B-structure_element +to O +show O +how O +residue O +L130 B-residue_name_number +chain B-structure_element +B I-structure_element +( O +blue O +side O +chain O +) O +is O +a O +focus O +of O +hydrophobic B-bond_interaction +packing I-bond_interaction +interactions I-bond_interaction +with O +L130 B-residue_name_number +, O +L133 B-residue_name_number +, O +L134 B-residue_name_number +and O +L137 B-residue_name_number +of O +chain B-structure_element +A I-structure_element +( O +red O +side O +chains O +). O + +( O +C O +) O +SE B-experimental_method +- I-experimental_method +HPLC I-experimental_method +analyses O +of O +all O +mutant B-protein_state +forms O +of O +NadR B-protein +are O +compared O +with O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +protein O +. O + +The O +WT B-protein_state +and O +most O +of O +the O +mutants O +show O +a O +single O +elution O +peak O +with O +an O +absorbance O +maximum O +at O +17 O +. O +5 O +min O +. O + +Only O +the O +mutation O +L130K B-mutant +has O +a O +noteworthy O +effect O +on O +the O +oligomeric O +state O +, O +inducing O +a O +second O +peak O +with O +a O +longer O +retention O +time O +and O +a O +second O +peak O +maximum O +at O +18 O +. O +6 O +min O +. O + +To O +a O +much O +lesser O +extent O +, O +the O +L133K B-mutant +mutation O +also O +appears O +to O +induce O +a O +‘ O +shoulder O +’ O +to O +the O +main O +peak O +, O +suggesting O +very O +weak O +ability O +to O +disrupt O +the O +dimer B-oligomeric_state +. O +( O +D O +) O +SE B-experimental_method +- I-experimental_method +HPLC I-experimental_method +/ I-experimental_method +MALLS I-experimental_method +analyses O +of O +the O +L130K B-mutant +mutant B-protein_state +, O +shows O +20 O +% O +dimer B-oligomeric_state +and O +80 O +% O +monomer B-oligomeric_state +. O + +The O +holo B-protein_state +- O +NadR B-protein +structure B-evidence +presents O +only O +one O +occupied O +ligand B-site +- I-site +binding I-site +pocket I-site + +The O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +structure B-evidence +revealed O +the O +ligand B-site +- I-site +binding I-site +site I-site +nestled O +between O +the O +dimerization B-structure_element +and I-structure_element +DNA I-structure_element +- I-structure_element +binding I-structure_element +domains I-structure_element +( O +Fig O +2 O +). O + +The O +ligand O +showed O +a O +different O +position O +and O +orientation O +compared O +to O +salicylate B-chemical +complexed B-protein_state +with I-protein_state +MTH313 B-protein +and O +ST1710 B-protein +( O +see O +Discussion O +). O + +The O +binding B-site +pocket I-site +was O +almost O +entirely O +filled O +by O +4 B-chemical +- I-chemical +HPA I-chemical +and O +one O +water B-chemical +molecule O +, O +although O +there O +also O +remained O +a O +small O +tunnel B-site +2 O +- O +4Å O +in O +diameter O +and O +5 O +- O +6Å O +long O +leading O +from O +the O +pocket B-site +( O +proximal O +to O +the O +4 O +- O +hydroxyl O +position O +) O +to O +the O +protein O +surface O +. O + +The O +tunnel B-site +was O +lined O +with O +rather O +hydrophobic O +amino O +acids O +, O +and O +did O +not O +contain O +water B-chemical +molecules O +. O + +Unexpectedly O +, O +only O +one O +monomer B-oligomeric_state +of O +the O +holo B-protein_state +- O +NadR B-protein +homodimer B-oligomeric_state +contained O +4 B-chemical +- I-chemical +HPA I-chemical +in O +the O +binding B-site +pocket I-site +, O +whereas O +the O +corresponding O +pocket B-site +of O +the O +other O +monomer B-oligomeric_state +was O +unoccupied O +by O +ligand O +, O +despite O +the O +large O +excess O +of O +4 B-chemical +- I-chemical +HPA I-chemical +used O +in O +the O +crystallization O +conditions O +. O + +Inspection O +of O +the O +protein B-site +- I-site +ligand I-site +interaction I-site +network I-site +revealed O +no O +bonds O +from O +NadR B-protein +backbone O +groups O +to O +the O +ligand O +, O +but O +several O +key O +side O +chain O +mediated O +hydrogen B-bond_interaction +( I-bond_interaction +H I-bond_interaction +)- I-bond_interaction +bonds I-bond_interaction +and O +ionic B-bond_interaction +interactions I-bond_interaction +, O +most O +notably O +between O +the O +carboxylate O +group O +of O +4 B-chemical +- I-chemical +HPA I-chemical +and O +Ser9 B-residue_name_number +of O +chain B-structure_element +A I-structure_element +( O +SerA9 B-residue_name_number +), O +and O +chain B-structure_element +B I-structure_element +residues O +TrpB39 B-residue_name_number +, O +ArgB43 B-residue_name_number +and O +TyrB115 B-residue_name_number +( O +Fig O +4A O +). O + +At O +the O +other O +‘ O +end O +’ O +of O +the O +ligand O +, O +the O +4 O +- O +hydroxyl O +group O +was O +proximal O +to O +AspB36 B-residue_name_number +, O +with O +which O +it O +may O +establish O +an O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +( O +see O +bond O +distances O +in O +Table O +3 O +). O + +The O +water B-chemical +molecule O +observed O +in O +the O +pocket O +was O +bound O +by O +the O +carboxylate O +group O +and O +the O +side O +chains O +of O +SerA9 B-residue_name_number +and O +AsnA11 B-residue_name_number +. O + +Atomic O +details O +of O +NadR B-protein +/ O +HPA B-chemical +interactions O +. O + +A O +) O +A O +stereo O +- O +view O +zoom O +into O +the O +binding B-site +pocket I-site +showing O +side O +chain O +sticks O +for O +all O +interactions O +between O +NadR B-protein +and O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +Green O +and O +blue O +ribbons O +depict O +NadR B-protein +chains B-structure_element +A I-structure_element +and I-structure_element +B I-structure_element +, O +respectively O +. O + +4 B-chemical +- I-chemical +HPA I-chemical +is O +shown O +in O +yellow O +sticks O +, O +with O +oxygen O +atoms O +in O +red O +. O + +A O +water B-chemical +molecule O +is O +shown O +by O +the O +red O +sphere O +. O + +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +up O +to O +3 O +. O +6Å O +are O +shown O +as O +dashed O +lines O +. O + +The O +entire O +set O +of O +residues O +making O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +or O +non B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +contacts I-bond_interaction +with O +4 B-chemical +- I-chemical +HPA I-chemical +is O +as O +follows O +: O +SerA9 B-residue_name_number +, O +AsnA11 B-residue_name_number +, O +LeuB21 B-residue_name_number +, O +MetB22 B-residue_name_number +, O +PheB25 B-residue_name_number +, O +LeuB29 B-residue_name_number +, O +AspB36 B-residue_name_number +, O +TrpB39 B-residue_name_number +, O +ArgB43 B-residue_name_number +, O +ValB111 B-residue_name_number +and O +TyrB115 B-residue_name_number +( O +automated O +analysis O +performed O +using O +PDBsum B-experimental_method +and O +verified O +manually O +). O + +Residues O +AsnA11 B-residue_name_number +and O +ArgB18 B-residue_name_number +likely O +make O +indirect O +yet O +local O +contributions O +to O +ligand O +binding O +, O +mainly O +by O +stabilizing O +the O +position O +of O +AspB36 B-residue_name_number +. O + +Side O +chains O +mediating O +hydrophobic B-bond_interaction +interactions I-bond_interaction +are O +shown O +in O +orange O +. O +( O +B O +) O +A O +model O +was O +prepared O +to O +visualize O +putative O +interactions O +of O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +( O +pink O +) O +with O +NadR B-protein +, O +revealing O +the O +potential O +for O +additional O +contacts O +( O +dashed O +lines O +) O +of O +the O +chloro O +moiety O +( O +green O +stick O +) O +with O +LeuB29 B-residue_name_number +and O +AspB36 B-residue_name_number +. O + +List O +of O +4 B-chemical +- I-chemical +HPA I-chemical +atoms O +bound O +to O +NadR B-protein +via O +ionic B-bond_interaction +interactions I-bond_interaction +and O +/ O +or O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +. O + +4 B-chemical +- I-chemical +HPA I-chemical +atom O +NadR B-protein +residue O +/ O +atom O +Distance O +( O +Å O +) O +O2 O +TrpB39 B-residue_name_number +/ O +NE1 O +2 O +. O +83 O +O2 O +ArgB43 B-residue_name_number +/ O +NH1 O +2 O +. O +76 O +O1 O +ArgB43 B-residue_name_number +/ O +NH1 O +3 O +. O +84 O +O1 O +SerA9 B-residue_name_number +/ O +OG O +2 O +. O +75 O +O1 O +TyrB115 B-residue_name_number +/ O +OH O +2 O +. O +50 O +O2 O +Water B-chemical +(* O +Ser9 B-residue_name_number +/ O +Asn11 B-residue_name_number +) O +2 O +. O +88 O +OH O +AspB36 B-residue_name_number +/ O +OD1 O +/ O +OD2 O +3 O +. O +6 O +/ O +3 O +. O +7 O + +* O +Bond O +distance O +between O +the O +ligand O +carboxylate O +group O +and O +the O +water B-chemical +molecule O +, O +which O +in O +turn O +makes O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +to O +the O +SerA9 B-residue_name_number +and O +AsnA11 B-residue_name_number +side O +chains O +. O + +In O +addition O +to O +the O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +involving O +the O +carboxylate O +and O +hydroxyl O +groups O +of O +4 B-chemical +- I-chemical +HPA I-chemical +, O +binding O +of O +the O +phenyl O +moiety O +appeared O +to O +be O +stabilized O +by O +several O +van B-bond_interaction +der I-bond_interaction +Waals I-bond_interaction +’ I-bond_interaction +contacts I-bond_interaction +, O +particularly O +those O +involving O +the O +hydrophobic O +side O +chain O +atoms O +of O +LeuB21 B-residue_name_number +, O +MetB22 B-residue_name_number +, O +PheB25 B-residue_name_number +, O +LeuB29 B-residue_name_number +and O +ValB111 B-residue_name_number +( O +Fig O +4A O +). O + +Notably O +, O +the O +phenyl O +ring O +of O +PheB25 B-residue_name_number +was O +positioned O +parallel O +to O +the O +phenyl O +ring O +of O +4 B-chemical +- I-chemical +HPA I-chemical +, O +potentially O +forming O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +parallel I-bond_interaction +- I-bond_interaction +displaced I-bond_interaction +stacking I-bond_interaction +interactions I-bond_interaction +. O + +Consequently O +, O +residues O +in O +the O +4 B-site +- I-site +HPA I-site +binding I-site +pocket I-site +are O +mostly O +contributed O +by O +NadR B-protein +chain B-structure_element +B I-structure_element +, O +and O +effectively O +created O +a O +polar O +‘ O +floor O +’ O +and O +a O +hydrophobic O +‘ O +ceiling O +’, O +which O +house O +the O +ligand O +. O + +Collectively O +, O +this O +mixed O +network O +of O +polar B-bond_interaction +and I-bond_interaction +hydrophobic I-bond_interaction +interactions I-bond_interaction +endows O +NadR B-protein +with O +a O +strong O +recognition O +pattern O +for O +HPAs B-chemical +, O +with O +additional O +medium O +- O +range O +interactions O +potentially O +established O +with O +the O +hydroxyl O +group O +at O +the O +4 O +- O +position O +. O + +Structure O +- O +activity O +relationships O +: O +molecular O +basis O +of O +enhanced O +stabilization O +by O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical + +We O +modelled B-experimental_method +the O +binding O +of O +other O +HPAs B-chemical +by O +in B-experimental_method +silico I-experimental_method +superposition I-experimental_method +onto O +4 B-chemical +- I-chemical +HPA I-chemical +in O +the O +holo B-protein_state +- O +NadR B-protein +structure B-evidence +, O +and O +thereby O +obtained O +molecular O +explanations O +for O +the O +binding O +specificities O +of O +diverse O +ligands O +. O + +For O +example O +, O +similar O +to O +4 B-chemical +- I-chemical +HPA I-chemical +, O +the O +binding O +of O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +could O +involve O +multiple O +bonds O +towards O +the O +carboxylate O +group O +of O +the O +ligand O +and O +some O +to O +the O +4 O +- O +hydroxyl O +group O +. O + +Additionally O +, O +the O +side O +chains O +of O +LeuB29 B-residue_name_number +and O +AspB36 B-residue_name_number +would O +be O +only O +2 O +. O +6 O +– O +3 O +. O +5 O +Å O +from O +the O +chlorine O +atom O +, O +thus O +providing O +van B-bond_interaction +der I-bond_interaction +Waals I-bond_interaction +’ I-bond_interaction +interactions I-bond_interaction +or O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +generate O +the O +additional O +binding B-evidence +affinity I-evidence +observed O +for O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +( O +Fig O +4B O +). O + +The O +presence O +of O +a O +single O +hydroxyl O +group O +at O +position O +2 O +, O +as O +in O +2 B-chemical +- I-chemical +HPA I-chemical +, O +rather O +than O +at O +position O +4 O +, O +would O +eliminate O +the O +possibility O +of O +favorable O +interactions O +with O +AspB36 B-residue_name_number +, O +resulting O +in O +the O +lack O +of O +NadR B-protein +regulation O +by O +2 B-chemical +- I-chemical +HPA I-chemical +described O +previously O +. O + +Finally O +, O +salicylate B-chemical +is O +presumably O +unable O +to O +specifically O +bind O +NadR B-protein +due O +to O +the O +2 O +- O +hydroxyl O +substitution O +and O +the O +shorter O +aliphatic O +chain O +connecting O +its O +carboxylate O +group O +( O +Fig O +1A O +): O +the O +compound O +simply O +seems O +too O +small O +to O +simultaneously O +establish O +the O +network O +of O +beneficial O +bonds O +observed O +in O +the O +NadR B-protein +/ O +HPA B-chemical +interactions O +. O + +Analysis O +of O +the O +pockets B-site +reveals O +the O +molecular O +basis O +for O +asymmetric O +binding O +and O +stoichiometry O + +However O +, O +studies O +based O +on O +tryptophan B-experimental_method +fluorescence I-experimental_method +were O +confounded O +by O +the O +fluorescence O +of O +the O +HPA B-chemical +ligands O +, O +and O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +( O +ITC B-experimental_method +) O +was O +unfeasible O +due O +to O +the O +need O +for O +very O +high O +concentrations O +of O +NadR B-protein +in O +the O +ITC B-experimental_method +chamber O +( O +due O +to O +the O +relatively O +low O +affinity O +), O +which O +exceeded O +the O +solubility O +limits O +of O +the O +protein O +. O + +However O +, O +it O +was O +possible O +to O +calculate O +the O +binding B-evidence +stoichiometry I-evidence +of O +the O +NadR B-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +interactions O +using O +an O +SPR B-experimental_method +- O +based O +approach O +. O + +In O +SPR B-experimental_method +, O +the O +signal O +measured O +is O +proportional O +to O +the O +total O +molecular O +mass O +proximal O +to O +the O +sensor O +surface O +; O +consequently O +, O +if O +the O +molecular O +weights O +of O +the O +interactors O +are O +known O +, O +then O +the O +stoichiometry O +of O +the O +resulting O +complex O +can O +be O +determined O +. O + +This O +approach O +relies O +on O +the O +assumption O +that O +the O +captured O +protein O +(‘ O +the O +ligand O +’, O +according O +to O +SPR B-experimental_method +conventions O +) O +is O +100 O +% O +active O +and O +freely O +- O +accessible O +to O +potential O +interactors O +(‘ O +the O +analytes O +’). O + +Firstly O +, O +NadR B-protein +is O +expected O +to O +be O +covalently O +immobilized O +on O +the O +sensor O +chip O +as O +a O +dimer B-oligomeric_state +in O +random O +orientations O +, O +since O +it O +is O +a O +stable B-protein_state +dimer B-oligomeric_state +in O +solution O +and O +has O +sixteen O +lysines B-residue_name +well O +- O +distributed O +around O +its O +surface O +, O +all O +able O +to O +act O +as O +potential O +sites O +for O +amine O +coupling O +to O +the O +chip O +, O +and O +none O +of O +which O +are O +close O +to O +the O +ligand B-site +- I-site +binding I-site +pocket I-site +. O + +Secondly O +, O +the O +HPA B-chemical +analytes O +are O +all O +very O +small O +( O +MW O +150 O +– O +170 O +, O +Fig O +1A O +) O +and O +therefore O +are O +expected O +to O +be O +able O +to O +diffuse O +readily O +into O +all O +potential O +binding B-site +sites I-site +, O +irrespective O +of O +the O +random O +orientations O +of O +the O +immobilized O +NadR B-protein +dimers B-oligomeric_state +on O +the O +chip O +. O + +The O +stoichiometry O +of O +the O +NadR B-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +interactions O +was O +determined O +using O +Eq O +1 O +( O +see O +Materials O +and O +Methods O +), O +and O +revealed O +stoichiometries B-evidence +of O +1 O +. O +13 O +for O +4 B-chemical +- I-chemical +HPA I-chemical +, O +1 O +. O +02 O +for O +3 B-chemical +- I-chemical +HPA I-chemical +, O +and O +1 O +. O +21 O +for O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +, O +strongly O +suggesting O +that O +one O +NadR B-protein +dimer B-oligomeric_state +bound B-protein_state +to I-protein_state +1 O +HPA B-chemical +analyte O +molecule O +. O + +The O +crystallographic B-evidence +data I-evidence +, O +supported O +by O +the O +SPR B-experimental_method +studies O +of O +binding B-evidence +stoichiometry I-evidence +, O +revealed O +the O +lack O +of O +a O +second O +4 B-chemical +- I-chemical +HPA I-chemical +molecule O +in O +the O +homodimer B-oligomeric_state +, O +suggesting O +negative O +co O +- O +operativity O +, O +a O +phenomenon O +previously O +described O +for O +the O +MTH313 B-protein +/ O +salicylate B-chemical +interaction O +and O +for O +other O +MarR B-protein_type +family O +proteins O +. O + +To O +explore O +the O +molecular O +basis O +of O +asymmetry O +in O +holo B-protein_state +- O +NadR B-protein +, O +we O +superposed B-experimental_method +its O +ligand B-protein_state +- I-protein_state +free I-protein_state +monomer B-oligomeric_state +( O +chain B-structure_element +A I-structure_element +) O +onto O +the O +ligand B-protein_state +- I-protein_state +occupied I-protein_state +monomer B-oligomeric_state +( O +chain B-structure_element +B I-structure_element +). O + +Overall O +, O +the O +superposition B-experimental_method +revealed O +a O +high O +degree O +of O +structural O +similarity O +( O +Cα O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +rmsd B-evidence +) O +of O +1 O +. O +5Å O +), O +though O +on O +closer O +inspection O +a O +rotational O +difference O +of O +~ O +9 O +degrees O +along O +the O +long O +axis O +of O +helix B-structure_element +α6 B-structure_element +was O +observed O +, O +suggesting O +that O +4 B-chemical +- I-chemical +HPA I-chemical +induced O +a O +slight O +conformational O +change O +( O +Fig O +5A O +). O + +However O +, O +since O +residues O +of O +helix B-structure_element +α6 B-structure_element +were O +not O +directly O +involved O +in O +ligand O +binding O +, O +an O +explanation O +for O +the O +lack O +of O +4 B-chemical +- I-chemical +HPA I-chemical +in O +monomer B-oligomeric_state +A B-structure_element +did O +not O +emerge O +by O +analyzing O +only O +these O +backbone O +atom O +positions O +, O +suggesting O +that O +a O +more O +complex O +series O +of O +allosteric O +events O +may O +occur O +. O + +Indeed O +, O +we O +noted O +interesting O +differences O +in O +the O +side O +chains O +of O +Met22 B-residue_name_number +, O +Phe25 B-residue_name_number +and O +Arg43 B-residue_name_number +, O +which O +in O +monomer B-oligomeric_state +B B-structure_element +are O +used O +to O +contact O +the O +ligand O +while O +in O +monomer B-oligomeric_state +A B-structure_element +they O +partially O +occupied O +the O +pocket B-site +and O +collectively O +reduced O +its O +volume O +significantly O +. O + +Specifically O +, O +upon O +analysis O +with O +the O +CASTp B-experimental_method +software O +, O +the O +pocket B-site +in O +chain B-structure_element +B I-structure_element +containing O +the O +4 B-chemical +- I-chemical +HPA I-chemical +exhibited O +a O +total O +volume O +of O +approximately O +370 O +Å3 O +, O +while O +the O +pocket B-site +in O +chain B-structure_element +A I-structure_element +was O +occupied O +by O +these O +three O +side O +chains O +that O +adopted O +‘ O +inward B-protein_state +’ O +positions O +and O +thereby O +divided O +the O +space O +into O +a O +few O +much O +smaller O +pockets O +, O +each O +with O +volume O +< O +50 O +Å3 O +, O +evidently O +rendering O +chain B-structure_element +A I-structure_element +unfavorable O +for O +ligand O +binding O +. O + +Most O +notably O +, O +atomic O +clashes O +between O +the O +ligand O +and O +the O +side O +chains O +of O +MetA22 B-residue_name_number +, O +PheA25 B-residue_name_number +and O +ArgA43 B-residue_name_number +would O +occur O +if O +4 B-chemical +- I-chemical +HPA I-chemical +were O +present O +in O +the O +monomer B-oligomeric_state +A B-structure_element +pocket B-site +( O +Fig O +5B O +). O + +Subsequently O +, O +analyses O +of O +the O +pockets B-site +in O +apo B-protein_state +- O +NadR B-protein +revealed O +that O +in O +the O +absence B-protein_state +of I-protein_state +ligand I-protein_state +the O +long O +Arg43 B-residue_name_number +side O +chain O +was O +always O +in O +the O +open O +‘ O +outward B-protein_state +’ O +position O +compatible O +with O +binding O +to O +the O +4 B-chemical +- I-chemical +HPA I-chemical +carboxylate O +group O +. O + +In O +contrast O +, O +the O +apo B-protein_state +- O +form O +Met22 B-residue_name_number +and O +Phe25 B-residue_name_number +residues O +were O +still O +encroaching O +the O +spaces O +of O +the O +4 O +- O +hydroxyl O +group O +and O +the O +phenyl O +ring O +of O +the O +ligand O +, O +respectively O +( O +Fig O +5C O +). O + +The O +‘ O +outward B-protein_state +’ O +position O +of O +Arg43 B-residue_name_number +generated O +an O +open B-protein_state +apo B-protein_state +- O +form O +pocket B-site +with O +volume O +approximately O +380Å3 O +. O + +Taken O +together O +, O +these O +observations O +suggest O +that O +Arg43 B-residue_name_number +is O +a O +major O +determinant O +of O +ligand O +binding O +, O +and O +that O +its O +‘ O +inward B-protein_state +’ O +position O +inhibits O +the O +binding O +of O +4 B-chemical +- I-chemical +HPA I-chemical +to O +the O +empty O +pocket B-site +of O +holo B-protein_state +- O +NadR B-protein +. O + +Structural O +differences O +of O +NadR B-protein +in O +ligand B-protein_state +- I-protein_state +bound I-protein_state +or O +free B-protein_state +forms O +. O + +( O +A O +) O +Aligned B-experimental_method +monomers B-oligomeric_state +of O +holo B-protein_state +- O +NadR B-protein +( O +chain B-structure_element +A I-structure_element +: O +green O +; O +chain B-structure_element +B I-structure_element +: O +blue O +), O +reveal O +major O +overall O +differences O +by O +the O +shift O +of O +helix B-structure_element +α6 B-structure_element +. O +( O +B O +) O +Comparison B-experimental_method +of O +the O +two O +binding B-site +pockets I-site +in O +holo B-protein_state +- O +NadR B-protein +shows O +that O +in O +the O +ligand B-protein_state +- I-protein_state +free I-protein_state +monomer B-oligomeric_state +A B-structure_element +( O +green O +) O +residues O +Met22 B-residue_name_number +, O +Phe25 B-residue_name_number +and O +Arg43 B-residue_name_number +adopt O +‘ O +inward B-protein_state +’ O +positions O +( O +highlighted O +by O +arrows O +) O +compared O +to O +the O +ligand B-protein_state +- I-protein_state +occupied I-protein_state +pocket B-site +( O +blue O +residues O +); O +these O +‘ O +inward B-protein_state +’ O +conformations O +appear O +unfavorable O +for O +binding O +of O +4 B-chemical +- I-chemical +HPA I-chemical +due O +to O +clashes O +with O +the O +4 O +- O +hydroxyl O +group O +, O +the O +phenyl O +ring O +and O +the O +carboxylate O +group O +, O +respectively O +. O + +In O +these O +crystals B-evidence +, O +the O +ArgA43 B-residue_name_number +side O +chain O +showed O +two O +alternate O +conformations O +, O +modelled O +with O +50 O +% O +occupancy O +in O +each O +state O +, O +as O +indicated O +by O +the O +two O +‘ O +mirrored O +’ O +arrows O +. O + +The O +inner O +conformer O +is O +the O +one O +that O +would O +display O +major O +clashes O +if O +4 B-chemical +- I-chemical +HPA I-chemical +were O +present O +. O +( O +C O +) O +Comparison O +of O +the O +empty O +pocket B-site +from O +holo B-protein_state +- O +NadR B-protein +( O +green O +residues O +) O +with O +the O +four O +empty O +pockets B-site +of O +apo B-protein_state +- O +NadR B-protein +( O +grey O +residues O +), O +shows O +that O +in O +the O +absence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +the O +Arg43 B-residue_name_number +side O +chain O +is O +always O +observed O +in O +the O +‘ O +outward B-protein_state +’ O +conformation O +. O + +Finally O +, O +we O +applied O +15N B-experimental_method +heteronuclear I-experimental_method +solution I-experimental_method +NMR I-experimental_method +spectroscopy I-experimental_method +to O +examine O +the O +interaction O +of O +4 B-chemical +- I-chemical +HPA I-chemical +with O +apo B-protein_state +NadR B-protein +. O +We O +collected O +NMR B-experimental_method +spectra B-evidence +on O +NadR B-protein +in B-protein_state +the I-protein_state +presence I-protein_state +and O +absence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +( O +see O +Materials O +and O +Methods O +). O + +The O +1H B-experimental_method +- I-experimental_method +15N I-experimental_method +TROSY I-experimental_method +- I-experimental_method +HSQC I-experimental_method +spectrum B-evidence +of O +apo B-protein_state +- O +NadR B-protein +, O +acquired O +at O +25 O +° O +C O +, O +displayed O +approximately O +140 O +distinct O +peaks O +( O +Fig O +6A O +), O +most O +of O +which O +correspond O +to O +backbone O +amide O +N O +- O +H O +groups O +. O + +The O +broad O +spectral O +dispersion O +and O +the O +number O +of O +peaks O +observed O +, O +which O +is O +close O +to O +the O +number O +of O +expected O +backbone O +amide O +N O +- O +H O +groups O +for O +this O +polypeptide O +, O +confirmed O +that O +apo B-protein_state +- O +NadR B-protein +is O +well B-protein_state +- I-protein_state +folded I-protein_state +under O +these O +conditions O +and O +exhibits O +one O +conformation O +appreciable O +on O +the O +NMR B-experimental_method +timescale O +, O +i O +. O +e O +. O +in O +the O +NMR B-experimental_method +experiments O +at O +25 O +° O +C O +, O +two O +or O +more O +distinct O +conformations O +of O +apo B-protein_state +- O +NadR B-protein +monomers B-oligomeric_state +were O +not O +readily O +apparent O +. O + +Upon O +the O +addition O +of O +4 B-chemical +- I-chemical +HPA I-chemical +, O +over O +45 O +peaks O +showed O +chemical O +shift O +perturbations O +, O +i O +. O +e O +. O +changed O +position O +in O +the O +spectrum O +or O +disappeared O +, O +while O +the O +remaining O +peaks O +remained O +unchanged O +. O + +This O +observation O +showed O +that O +4 B-chemical +- I-chemical +HPA I-chemical +was O +able O +to O +bind O +NadR B-protein +and O +induce O +notable O +changes O +in O +specific O +regions O +of O +the O +protein O +. O + +NMR B-experimental_method +spectra B-evidence +of O +NadR B-protein +in B-protein_state +the I-protein_state +presence I-protein_state +and O +absence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +. O + +( O +A O +) O +Superposition B-experimental_method +of O +two O +1H B-experimental_method +- I-experimental_method +15N I-experimental_method +TROSY I-experimental_method +- I-experimental_method +HSQC I-experimental_method +spectra B-evidence +recorded O +at O +25 O +° O +C O +on O +apo B-protein_state +- O +NadR B-protein +( O +cyan O +) O +and O +on O +NadR B-protein +in O +the O +presence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +( O +red O +). O + +( O +B O +, O +C O +) O +Overlay B-experimental_method +of O +selected O +regions O +of O +the O +1H B-experimental_method +- I-experimental_method +15N I-experimental_method +TROSY I-experimental_method +- I-experimental_method +HSQC I-experimental_method +spectra B-evidence +acquired O +at O +25 O +° O +C O +of O +apo B-protein_state +- O +NadR B-protein +( O +cyan O +) O +and O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +( O +red O +) O +superimposed B-experimental_method +with O +the O +spectra B-evidence +acquired O +at O +10 O +° O +C O +of O +apo B-protein_state +- O +NadR B-protein +( O +blue O +) O +and O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +( O +green O +). O + +The O +spectra B-evidence +acquired O +at O +10 O +° O +C O +are O +excluded O +from O +panel O +A O +for O +simplicity O +. O + +However O +, O +in O +the O +presence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +, O +the O +1H B-experimental_method +- I-experimental_method +15N I-experimental_method +TROSY I-experimental_method +- I-experimental_method +HSQC I-experimental_method +spectrum B-evidence +of O +NadR B-protein +displayed O +approximately O +140 O +peaks O +, O +as O +for O +apo B-protein_state +- O +NadR B-protein +, O +i O +. O +e O +. O +two O +distinct O +stable O +conformations O +( O +that O +might O +have O +potentially O +revealed O +the O +molecular O +asymmetry O +observed O +crystallographically B-experimental_method +) O +were O +not O +notable O +. O + +Considering O +the O +small O +size O +, O +fast O +diffusion O +and O +relatively O +low O +binding B-evidence +affinity I-evidence +of O +4 B-chemical +- I-chemical +HPA I-chemical +, O +it O +would O +not O +be O +surprising O +if O +the O +ligand O +associates O +and O +dissociates O +rapidly O +on O +the O +NMR B-experimental_method +time O +scale O +, O +resulting O +in O +only O +one O +set O +of O +peaks O +whose O +chemical O +shifts O +represent O +the O +average O +environment O +of O +the O +bound B-protein_state +and O +unbound B-protein_state +states O +. O + +Interestingly O +, O +by O +cooling O +the O +samples O +to O +10 O +° O +C O +, O +we O +observed O +that O +a O +number O +of O +those O +peaks O +strongly O +affected O +by O +4 B-chemical +- I-chemical +HPA I-chemical +( O +and O +therefore O +likely O +to O +be O +in O +the O +ligand B-site +- I-site +binding I-site +site I-site +) O +demonstrated O +evidence O +of O +peak O +splitting O +, O +i O +. O +e O +. O +a O +tendency O +to O +become O +two O +distinct O +peaks O +rather O +than O +one O +single O +peak O +( O +Fig O +6B O +and O +6C O +). O + +These O +doubled O +peaks O +may O +therefore O +reveal O +that O +the O +cooler O +temperature O +partially O +trapped O +the O +existence O +in O +solution O +of O +two O +distinct O +states O +, O +in O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +, O +with O +minor O +conformational O +differences O +occurring O +at O +least O +in O +proximity O +to O +the O +binding B-site +pocket I-site +. O + +Although O +more O +comprehensive O +NMR B-experimental_method +experiments O +and O +full O +chemical O +shift O +assignment O +of O +the O +spectra B-evidence +would O +be O +required O +to O +precisely O +define O +this O +multi O +- O +state O +behavior O +, O +the O +NMR B-experimental_method +data O +clearly O +demonstrate O +that O +NadR B-protein +exhibits O +conformational O +flexibility O +which O +is O +modulated O +by O +4 B-chemical +- I-chemical +HPA I-chemical +in O +solution O +. O + +Apo B-protein_state +- O +NadR B-protein +structures B-evidence +reveal O +intrinsic O +conformational O +flexibility O + +The O +apo B-protein_state +- O +NadR B-protein +crystal B-evidence +structure I-evidence +contained O +two O +homodimers B-oligomeric_state +in O +the O +asymmetric O +unit O +( O +chains B-structure_element +A I-structure_element ++ I-structure_element +B I-structure_element +and O +chains B-structure_element +C I-structure_element ++ I-structure_element +D I-structure_element +). O + +Upon O +overall O +structural B-experimental_method +superposition I-experimental_method +, O +these O +dimers B-oligomeric_state +revealed O +a O +few O +minor O +differences O +in O +the O +α6 B-structure_element +helix I-structure_element +( O +a O +major O +component O +of O +the O +dimer B-site +interface I-site +) O +and O +the O +helices B-structure_element +α4 B-structure_element +- I-structure_element +α5 I-structure_element +( O +the O +DNA B-site +binding I-site +region I-site +), O +and O +an O +rmsd B-evidence +of O +1 O +. O +55Å O +( O +Fig O +7A O +). O + +Similarly O +, O +the O +entire O +holo B-protein_state +- O +homodimer B-oligomeric_state +could O +be O +closely B-experimental_method +superposed I-experimental_method +onto O +each O +of O +the O +apo B-protein_state +- O +homodimers B-oligomeric_state +, O +showing O +rmsd B-evidence +values O +of O +1 O +. O +29Å O +and O +1 O +. O +31Å O +, O +and O +with O +more O +notable O +differences O +in O +the O +α6 B-structure_element +helix I-structure_element +positions O +( O +Fig O +7B O +). O + +The O +slightly O +larger O +rmsd B-evidence +between O +the O +two O +apo B-protein_state +- O +homodimers B-oligomeric_state +, O +rather O +than O +between O +apo B-protein_state +- O +and O +holo B-protein_state +- O +homodimers B-oligomeric_state +, O +further O +indicate O +that O +apo B-protein_state +- O +NadR B-protein +possesses O +a O +notable O +degree O +of O +intrinsic O +conformational O +flexibility O +. O + +Overall O +apo B-protein_state +- O +and O +holo B-protein_state +- O +NadR B-protein +structures B-evidence +are O +similar O +. O + +( O +A O +) O +Pairwise B-experimental_method +alignment I-experimental_method +of O +the O +two O +distinct O +apo B-protein_state +- O +NadR B-protein +homodimers B-oligomeric_state +( O +AB B-structure_element +and O +CD B-structure_element +) O +present O +in O +the O +apo B-protein_state +- O +NadR B-protein +crystals B-evidence +. O +( O +B O +) O +Alignment B-experimental_method +of O +the O +holo B-protein_state +- O +NadR B-protein +homodimer B-oligomeric_state +( O +green O +and O +blue O +chains O +) O +onto O +the O +apo B-protein_state +- O +NadR B-protein +homodimers B-oligomeric_state +. O + +Here O +, O +larger O +differences O +are O +observed O +in O +the O +α6 B-structure_element +helices I-structure_element +( O +top O +). O + +4 B-chemical +- I-chemical +HPA I-chemical +stabilizes O +concerted O +conformational O +changes O +in O +NadR B-protein +that O +prevent O +DNA O +- O +binding O + +To O +further O +investigate O +the O +conformational O +rearrangements O +of O +NadR B-protein +, O +we O +performed O +local B-experimental_method +structural I-experimental_method +alignments I-experimental_method +using O +only O +a O +subset O +of O +residues O +in O +the O +DNA B-structure_element +- I-structure_element +binding I-structure_element +helix I-structure_element +( O +α4 B-structure_element +). O + +By O +selecting B-experimental_method +and O +aligning B-experimental_method +residues O +Arg64 B-residue_range +- I-residue_range +Ala77 I-residue_range +of O +one O +α4 B-structure_element +helix I-structure_element +per O +dimer B-oligomeric_state +, O +superposition B-experimental_method +of O +the O +holo B-protein_state +- O +homodimer B-oligomeric_state +onto O +the O +two O +apo B-protein_state +- O +homodimers B-oligomeric_state +revealed O +differences O +in O +the O +monomer B-oligomeric_state +conformations O +of O +each O +structure B-evidence +. O + +While O +one O +monomer B-oligomeric_state +from O +each O +structure B-evidence +was O +closely O +superimposable O +( O +Fig O +8A O +, O +left O +side O +), O +the O +second O +monomer B-oligomeric_state +displayed O +quite O +large O +differences O +( O +Fig O +8A O +, O +right O +side O +). O + +Most O +notably O +, O +the O +position O +of O +the O +DNA B-chemical +- O +binding O +helix B-structure_element +α4 B-structure_element +shifted O +by O +as O +much O +as O +6 O +Å O +( O +Fig O +8B O +). O + +Accordingly O +, O +helix B-structure_element +α4 B-structure_element +was O +also O +found O +to O +be O +one O +of O +the O +most O +dynamic O +regions O +in O +previous O +HDX B-experimental_method +- I-experimental_method +MS I-experimental_method +analyses O +of O +apo B-protein_state +- O +NadR B-protein +in O +solution O +. O + +Structural B-experimental_method +comparisons I-experimental_method +of O +NadR B-protein +and O +modelling O +of O +interactions O +with O +DNA B-chemical +. O + +( O +A O +) O +The O +holo B-protein_state +- O +homodimer B-oligomeric_state +structure B-evidence +is O +shown O +as O +green O +and O +blue O +cartoons O +, O +for O +chain B-structure_element +A I-structure_element +and I-structure_element +B I-structure_element +, O +respectively O +, O +while O +the O +two O +homodimers B-oligomeric_state +of O +apo B-protein_state +- O +NadR B-protein +are O +both O +cyan O +and O +pale O +blue O +for O +chains O +A B-structure_element +/ I-structure_element +C I-structure_element +and O +B B-structure_element +/ I-structure_element +D I-structure_element +, O +respectively O +. O + +The O +three O +homodimers B-oligomeric_state +( O +chains O +AB B-structure_element +holo B-protein_state +, O +AB B-structure_element +apo B-protein_state +, O +and O +CD B-structure_element +apo B-protein_state +) O +were O +overlaid B-experimental_method +by O +structural B-experimental_method +alignment I-experimental_method +exclusively O +of O +all O +heavy O +atoms O +in O +residues O +R64 B-residue_range +- I-residue_range +A77 I-residue_range +( O +shown O +in O +red O +, O +with O +side O +chain O +sticks O +) O +of O +chains O +A B-structure_element +holo B-protein_state +, O +A B-structure_element +apo B-protein_state +, O +and O +C B-structure_element +apo B-protein_state +, O +belonging O +to O +helix B-structure_element +α4 B-structure_element +( O +left O +). O + +The O +α4 B-structure_element +helices I-structure_element +aligned O +closely O +, O +Cα O +rmsd B-evidence +0 O +. O +2Å O +for O +14 O +residues O +. O + +( O +B O +) O +The O +relative O +positions O +of O +the O +α4 B-structure_element +helices I-structure_element +of O +the O +4 B-protein_state +- I-protein_state +HPA I-protein_state +- I-protein_state +bound I-protein_state +holo B-protein_state +homodimer B-oligomeric_state +chain B-structure_element +B I-structure_element +( O +blue O +), O +and O +of O +apo B-protein_state +homodimers B-oligomeric_state +AB B-structure_element +and O +CD B-structure_element +( O +showing O +chains B-structure_element +B I-structure_element +and I-structure_element +D I-structure_element +) O +in O +pale O +blue O +. O + +Dashes O +indicate O +the O +Ala77 B-residue_name_number +Cα O +atoms O +, O +in O +the O +most O +highly O +shifted O +region O +of O +the O +‘ O +non O +- O +fixed O +’ O +α4 B-structure_element +helix I-structure_element +. O + +( O +C O +) O +The O +double O +- O +stranded O +DNA B-chemical +molecule O +( O +grey O +cartoon O +) O +from O +the O +OhrR B-complex_assembly +- I-complex_assembly +ohrA I-complex_assembly +complex O +is O +shown O +after O +superposition B-experimental_method +with O +NadR B-protein +, O +to O +highlight O +the O +expected O +positions O +of O +the O +NadR B-protein +α4 B-structure_element +helices I-structure_element +in O +the O +DNA B-chemical +major O +grooves O +. O + +For O +clarity O +, O +only O +the O +α4 B-structure_element +helices I-structure_element +are O +shown O +in O +panels O +( O +B O +) O +and O +( O +C O +). O +( O +D O +) O +Upon O +comparison O +with O +the O +experimentally O +- O +determined O +OhrR B-complex_assembly +: I-complex_assembly +ohrA I-complex_assembly +structure B-evidence +( O +grey O +), O +the O +α4 B-structure_element +helix I-structure_element +of O +holo B-protein_state +- O +NadR B-protein +( O +blue O +) O +is O +shifted O +~ O +8Å O +out O +of O +the O +major O +groove O +. O + +However O +, O +structural B-experimental_method +comparisons I-experimental_method +revealed O +that O +the O +shift O +of O +holo B-protein_state +- O +NadR B-protein +helix B-structure_element +α4 B-structure_element +induced O +by O +the O +presence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +was O +also O +accompanied O +by O +several O +changes O +at O +the O +holo B-protein_state +dimer B-site +interface I-site +, O +while O +such O +extensive O +structural O +differences O +were O +not O +observed O +in O +the O +apo B-protein_state +dimer B-site +interfaces I-site +, O +particularly O +notable O +when O +comparing O +the O +α6 B-structure_element +helices I-structure_element +( O +S3 O +Fig O +). O + +In O +summary O +, O +compared O +to O +ligand B-protein_state +- I-protein_state +stabilized I-protein_state +holo B-protein_state +- O +NadR B-protein +, O +apo B-protein_state +- O +NadR B-protein +displayed O +an O +intrinsic O +flexibility O +focused O +in O +the O +DNA B-site +- I-site +binding I-site +region I-site +. O + +This O +was O +also O +evident O +in O +the O +greater O +disorder O +( O +i O +. O +e O +. O +less O +well O +- O +defined O +electron B-evidence +density I-evidence +) O +in O +the O +β1 B-structure_element +- I-structure_element +β2 I-structure_element +loops I-structure_element +of O +the O +apo B-protein_state +dimers B-oligomeric_state +( O +density B-evidence +for O +16 O +residues O +per O +dimer B-oligomeric_state +was O +missing O +) O +compared O +to O +the O +holo B-protein_state +dimer B-oligomeric_state +( O +density B-evidence +for O +only O +3 O +residues O +was O +missing O +). O + +In O +holo B-protein_state +- O +NadR B-protein +, O +the O +distance O +separating O +the O +two O +DNA O +- O +binding O +α4 B-structure_element +helices I-structure_element +was O +32 O +Å O +, O +while O +in O +apo B-protein_state +- O +NadR B-protein +it O +was O +29 O +Å O +for O +homodimer B-oligomeric_state +AB B-structure_element +, O +and O +34 O +Å O +for O +homodimer B-oligomeric_state +CD B-structure_element +( O +Fig O +8C O +). O + +Thus O +, O +the O +apo B-protein_state +- O +homodimer B-oligomeric_state +AB B-structure_element +presented O +the O +DNA B-structure_element +- I-structure_element +binding I-structure_element +helices I-structure_element +in O +a O +conformation O +similar O +to O +that O +observed O +in O +the O +protein O +: O +DNA O +complex O +of O +OhrR B-complex_assembly +: I-complex_assembly +ohrA I-complex_assembly +from O +Bacillus B-species +subtilis I-species +( O +Fig O +8C O +). O + +Interestingly O +, O +OhrR B-protein +contacts O +ohrA B-gene +across O +22 O +base O +pairs O +( O +bp O +), O +and O +similarly O +the O +main O +NadR B-protein +target B-site +sites I-site +identified O +in O +the O +nadA B-gene +promoter O +( O +the O +operators O +Op O +I O +and O +Op O +II O +) O +both O +span O +22 O +bp O +. O + +Pairwise B-experimental_method +superpositions I-experimental_method +showed O +that O +the O +NadR B-protein +apo B-protein_state +- O +homodimer B-oligomeric_state +AB B-structure_element +was O +the O +most O +similar O +to O +OhrR B-protein +( O +rmsd B-evidence +2 O +. O +6 O +Å O +), O +while O +the O +holo B-protein_state +- O +homodimer B-oligomeric_state +was O +the O +most O +divergent O +( O +rmsd B-evidence +3 O +. O +3 O +Å O +) O +( O +Fig O +8C O +). O + +Assuming O +the O +same O +DNA B-chemical +- O +binding O +mechanism O +is O +used O +by O +OhrR B-protein +and O +NadR B-protein +, O +the O +apo B-protein_state +- O +homodimer B-oligomeric_state +AB B-structure_element +seems O +ideally O +pre O +- O +configured O +for O +DNA B-chemical +binding O +, O +while O +4 B-chemical +- I-chemical +HPA I-chemical +appeared O +to O +stabilize O +holo B-protein_state +- O +NadR B-protein +in O +a O +conformation O +poorly O +suited O +for O +DNA B-chemical +binding O +. O + +Specifically O +, O +in O +addition O +to O +the O +different O +inter B-evidence +- I-evidence +helical I-evidence +translational I-evidence +distances I-evidence +, O +the O +α4 B-structure_element +helices I-structure_element +in O +the O +holo B-protein_state +- O +NadR B-protein +homodimer B-oligomeric_state +were O +also O +reoriented O +, O +resulting O +in O +movement O +of O +α4 B-structure_element +out O +of O +the O +major O +groove O +, O +by O +up O +to O +8Å O +, O +and O +presumably O +preventing O +efficient O +DNA B-chemical +binding O +in O +the O +presence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +( O +Fig O +8D O +). O + +When O +aligned B-experimental_method +with O +OhrR B-protein +, O +the O +apo B-protein_state +- O +homodimer B-oligomeric_state +CD B-structure_element +presented O +yet O +another O +different O +intermediate O +conformation O +( O +rmsd B-evidence +2 O +. O +9Å O +), O +apparently O +not O +ideally O +pre O +- O +configured O +for O +DNA B-chemical +binding O +, O +but O +which O +in O +solution O +can O +presumably O +readily O +adopt O +the O +AB B-structure_element +conformation O +due O +to O +the O +intrinsic O +flexibility O +described O +above O +. O + +NadR B-protein +residues O +His7 B-residue_name_number +, O +Ser9 B-residue_name_number +, O +Asn11 B-residue_name_number +and O +Phe25 B-residue_name_number +are O +essential O +for O +regulation O +of O +NadA B-protein +expression O +in O +vivo O + +While O +previous O +studies O +had O +correctly O +suggested O +the O +involvement O +of O +several O +NadR B-protein +residues O +in O +ligand O +binding O +, O +the O +crystal B-evidence +structures I-evidence +presented O +here O +revealed O +additional O +residues O +with O +previously O +unknown O +roles O +in O +dimerization O +and O +/ O +or O +binding O +to O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +To O +explore O +the O +functional O +involvement O +of O +these O +residues O +, O +we O +characterized O +the O +behavior O +of O +four O +new O +NadR B-protein +mutants O +( O +H7A B-mutant +, O +S9A B-mutant +, O +N11A B-mutant +and O +F25A B-mutant +) O +in O +an O +in O +vivo O +assay O +using O +the O +previously O +described O +MC58 B-mutant +- I-mutant +Δ1843 I-mutant +nadR B-gene +- O +null O +mutant B-protein_state +strain O +, O +which O +was O +complemented O +either O +by O +wild B-protein_state +- I-protein_state +type I-protein_state +nadR B-gene +or O +by O +the O +nadR B-gene +mutants B-protein_state +. O + +NadA B-protein +protein O +abundance O +levels O +were O +assessed O +by O +Western B-experimental_method +blotting I-experimental_method +to O +evaluate O +the O +ability O +of O +the O +NadR B-protein +mutants B-protein_state +to O +repress O +the O +nadA B-gene +promoter O +, O +in O +the O +presence O +or O +absence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +The O +nadR B-gene +H7A B-mutant +, O +S9A B-mutant +and O +F25A B-mutant +complemented O +strains O +showed O +hyper O +- O +repression O +of O +nadA B-gene +expression O +in O +vivo O +, O +i O +. O +e O +. O +these O +mutants O +repressed O +nadA B-gene +more O +efficiently O +than O +the O +NadR B-protein +WT B-protein_state +protein O +, O +either O +in O +the O +presence O +or O +absence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +, O +while O +complementation O +with O +wild B-protein_state +- I-protein_state +type I-protein_state +nadR B-gene +resulted O +in O +high O +production O +of O +NadA B-protein +only O +in O +the O +presence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +( O +Fig O +9 O +). O + +Interestingly O +, O +and O +on O +the O +contrary O +, O +the O +nadR B-gene +N11A B-mutant +complemented O +strain O +showed O +hypo O +- O +repression O +( O +i O +. O +e O +. O +exhibited O +high O +expression O +of O +nadA B-gene +both O +in O +absence O +and O +presence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +). O + +This O +mutagenesis B-experimental_method +data O +revealed O +that O +NadR B-protein +residues O +His7 B-residue_name_number +, O +Ser9 B-residue_name_number +, O +Asn11 B-residue_name_number +and O +Phe25 B-residue_name_number +play O +key O +roles O +in O +the O +ligand O +- O +mediated O +regulation O +of O +NadR B-protein +; O +they O +are O +each O +involved O +in O +the O +controlled O +de O +- O +repression O +of O +the O +nadA B-gene +promoter O +and O +synthesis O +of O +NadA B-protein +in O +response O +to O +4 B-chemical +- I-chemical +HPA I-chemical +in O +vivo O +. O + +Structure B-experimental_method +- I-experimental_method +based I-experimental_method +point I-experimental_method +mutations I-experimental_method +shed O +light O +on O +ligand O +- O +induced O +regulation O +of O +NadR B-protein +. O + +Western B-experimental_method +blot I-experimental_method +analyses O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +strain O +( O +lanes O +1 O +– O +2 O +) O +or O +isogenic O +nadR B-gene +knockout O +strains O +( O +ΔNadR B-mutant +) O +complemented O +to O +express O +the O +indicated O +NadR B-protein +WT B-protein_state +or O +mutant B-protein_state +proteins O +( O +lanes O +3 O +– O +12 O +) O +or O +not O +complemented O +( O +lanes O +13 O +– O +14 O +), O +grown O +in O +the O +presence O +( O +even O +lanes O +) O +or O +absence O +( O +odd O +lanes O +) O +of O +5mM O +4 B-chemical +- I-chemical +HPA I-chemical +, O +showing O +NadA B-protein +and O +NadR B-protein +expression O +. O + +Complementation O +of O +ΔNadR B-mutant +with O +WT B-protein_state +NadR B-protein +enables O +induction O +of O +nadA B-gene +expression O +by O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +The O +H7A B-mutant +, O +S9A B-mutant +and O +F25A B-mutant +mutants O +efficiently O +repress O +nadA B-gene +expression O +but O +are O +less O +ligand O +- O +responsive O +than O +WT B-protein_state +NadR B-protein +. O +The O +N11A B-mutant +mutant B-protein_state +does O +not O +efficiently O +repress O +nadA B-gene +expression O +either O +in O +presence O +or O +absence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +. O +( O +The O +protein O +abundance O +levels O +of O +the O +meningococcal B-taxonomy_domain +factor B-protein +H I-protein +binding I-protein +protein I-protein +( O +fHbp B-protein +) O +were O +used O +as O +a O +gel O +loading O +control O +). O + +NadA B-protein +is O +a O +surface O +- O +exposed O +meningococcal B-taxonomy_domain +protein O +contributing O +to O +pathogenesis O +, O +and O +is O +one O +of O +three O +main O +antigens O +present O +in O +the O +vaccine O +Bexsero O +. O + +A O +detailed O +understanding O +of O +the O +in O +vitro O +repression O +of O +nadA B-gene +expression O +by O +the O +transcriptional B-protein_type +regulator I-protein_type +NadR B-protein +is O +important O +, O +both O +because O +it O +is O +a O +relevant O +disease O +- O +related O +model O +of O +how O +small O +- O +molecule O +ligands O +can O +regulate O +MarR B-protein_type +family O +proteins O +and O +thereby O +impact O +bacterial B-taxonomy_domain +virulence O +, O +and O +because O +nadA B-gene +expression O +levels O +are O +linked O +to O +the O +prediction O +of O +vaccine O +coverage O +. O + +The O +repressive O +activity O +of O +NadR B-protein +can O +be O +relieved O +by O +hydroxyphenylacetate B-chemical +( O +HPA B-chemical +) O +ligands O +, O +and O +HDX B-experimental_method +- I-experimental_method +MS I-experimental_method +studies O +previously O +indicated O +that O +4 B-chemical +- I-chemical +HPA I-chemical +stabilizes O +dimeric B-oligomeric_state +NadR B-protein +in O +a O +configuration O +incompatible O +with O +DNA O +binding O +. O + +Despite O +these O +and O +other O +studies O +, O +the O +molecular O +mechanisms O +by O +which O +ligands O +regulate O +MarR B-protein_type +family O +proteins O +are O +relatively O +poorly O +understood O +and O +likely O +differ O +depending O +on O +the O +specific O +ligand O +. O + +Given O +the O +importance O +of O +NadR B-protein +- O +mediated O +regulation O +of O +NadA B-protein +levels O +in O +the O +contexts O +of O +meningococcal B-taxonomy_domain +pathogenesis O +, O +we O +sought O +to O +characterize O +NadR B-protein +, O +and O +its O +interaction O +with O +ligands O +, O +at O +atomic O +resolution O +. O + +Firstly O +, O +we O +confirmed O +that O +NadR B-protein +is O +dimeric B-oligomeric_state +in O +solution O +and O +demonstrated O +that O +it O +retains O +its O +dimeric B-oligomeric_state +state O +in O +the O +presence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +, O +indicating O +that O +induction O +of O +a O +monomeric B-oligomeric_state +status O +is O +not O +the O +manner O +by O +which O +4 B-chemical +- I-chemical +HPA I-chemical +regulates O +NadR B-protein +. O +These O +observations O +were O +in O +agreement O +with O +( O +i O +) O +a O +previous O +study O +of O +NadR B-protein +performed O +using O +SEC B-experimental_method +and O +mass B-experimental_method +spectrometry I-experimental_method +, O +and O +( O +ii O +) O +crystallographic B-experimental_method +studies I-experimental_method +showing O +that O +several O +MarR B-protein_type +homologues O +are O +dimeric B-oligomeric_state +. O + +We O +also O +used O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +site I-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +to O +identify O +an O +important O +conserved B-protein_state +residue O +, O +Leu130 B-residue_name_number +, O +which O +stabilizes O +the O +NadR B-protein +dimer B-site +interface I-site +, O +knowledge O +of O +which O +may O +also O +inform O +future O +studies O +to O +explore O +the O +regulatory O +mechanisms O +of O +other O +MarR B-protein_type +family O +proteins O +. O + +Secondly O +, O +we O +assessed B-experimental_method +the I-experimental_method +thermal I-experimental_method +stability I-experimental_method +and O +unfolding O +of O +NadR B-protein +in B-protein_state +the I-protein_state +presence I-protein_state +or O +absence B-protein_state +of I-protein_state +ligands O +. O + +All O +DSC B-experimental_method +profiles B-evidence +showed O +a O +single O +peak O +, O +suggesting O +that O +a O +single O +unfolding O +event O +simultaneously O +disrupted O +the O +dimer B-oligomeric_state +and O +the O +monomer B-oligomeric_state +. O + +HPA O +ligands O +specifically O +increased O +the O +stability O +of O +NadR B-protein +. O +The O +largest O +effects O +were O +induced O +by O +the O +naturally O +- O +occurring O +compounds O +4 B-chemical +- I-chemical +HPA I-chemical +and O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +, O +which O +, O +in O +SPR B-experimental_method +assays I-experimental_method +, O +were O +found O +to O +bind O +NadR B-protein +with O +KD B-evidence +values O +of O +1 O +. O +5 O +mM O +and O +1 O +. O +1 O +mM O +, O +respectively O +. O + +Although O +these O +NadR B-protein +/ O +HPA B-chemical +interactions O +appeared O +rather O +weak O +, O +their O +distinct O +affinities O +and O +specificities O +matched O +their O +in O +vitro O +effects O +and O +their O +biological O +relevance O +appears O +similar O +to O +previous O +proposals O +that O +certain O +small O +molecules O +, O +including O +some O +antibiotics O +, O +in O +the O +millimolar O +concentration O +range O +may O +be O +broad O +inhibitors O +of O +MarR B-protein_type +family O +proteins O +. O + +Indeed O +, O +4 B-chemical +- I-chemical +HPA I-chemical +is O +found O +in O +human B-species +saliva O +and O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +is O +produced O +during O +inflammatory O +processes O +, O +suggesting O +that O +these O +natural O +ligands O +are O +encountered O +by O +N B-species +. I-species +meningitidis I-species +in O +the O +mucosa O +of O +the O +oropharynx O +during O +infections O +. O + +It O +is O +also O +possible O +that O +NadR B-protein +responds O +to O +currently O +unidentified O +HPA B-chemical +analogues O +. O + +Indeed O +, O +in O +the O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +complex O +there O +was O +a O +water B-chemical +molecule O +close O +to O +the O +carboxylate O +group O +and O +also O +a O +small O +unfilled O +tunnel B-site +~ O +5Å O +long O +, O +both O +factors O +suggesting O +that O +alternative O +larger O +ligands O +could O +occupy O +the O +pocket O +. O + +The O +ability O +to O +respond O +to O +various O +ligands O +might O +enable O +NadR B-protein +in O +vivo O +to O +orchestrate O +multiple O +response O +mechanisms O +and O +modulate O +expression O +of O +genes O +other O +than O +nadA B-gene +. O +Ultimately O +, O +confirmation O +of O +the O +relevance O +of O +each O +ligand O +will O +require O +a O +deeper O +understanding O +of O +the O +available O +concentration O +in O +vivo O +in O +the O +host O +niche O +during O +bacterial B-taxonomy_domain +colonization O +and O +inflammation O +. O + +Here O +, O +we O +determined O +the O +first O +crystal B-evidence +structures I-evidence +of O +apo B-protein_state +- O +NadR B-protein +and O +holo B-protein_state +- O +NadR B-protein +. O +These O +experimentally O +- O +determined O +structures B-evidence +enabled O +a O +new O +detailed O +characterization O +of O +the O +ligand B-site +- I-site +binding I-site +pocket I-site +. O + +In O +holo B-protein_state +- O +NadR B-protein +, O +4 B-chemical +- I-chemical +HPA I-chemical +interacted O +directly O +with O +at O +least O +11 O +polar O +and O +hydrophobic O +residues O +. O + +Several O +, O +but O +not O +all O +, O +of O +these O +interactions O +were O +predicted O +previously O +by O +homology B-experimental_method +modelling I-experimental_method +combined O +with O +ligand B-experimental_method +docking I-experimental_method +in O +silico O +. O + +Subsequently O +, O +we O +established O +the O +functional O +importance O +of O +His7 B-residue_name_number +, O +Ser9 B-residue_name_number +, O +Asn11 B-residue_name_number +and O +Phe25 B-residue_name_number +in O +the O +in O +vitro O +response O +of O +meningococcus B-taxonomy_domain +to O +4 B-chemical +- I-chemical +HPA I-chemical +, O +via O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +. O + +More O +unexpectedly O +, O +the O +crystal B-evidence +structure I-evidence +revealed O +that O +only O +one O +molecule O +of O +4 B-chemical +- I-chemical +HPA I-chemical +was O +bound B-protein_state +per O +NadR B-protein +dimer B-oligomeric_state +. O + +We O +confirmed O +this O +stoichiometry O +in O +solution O +using O +SPR B-experimental_method +methods O +. O + +We O +also O +used O +heteronuclear B-experimental_method +NMR I-experimental_method +spectroscopy I-experimental_method +to O +detect O +substantial O +conformational O +changes O +of O +NadR B-protein +occurring O +in O +solution O +upon O +addition O +of O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +Moreover O +, O +NMR B-experimental_method +spectra B-evidence +at O +10 O +° O +C O +suggested O +the O +existence O +of O +two O +distinct O +conformations O +of O +NadR B-protein +in O +the O +vicinity O +of O +the O +ligand B-site +- I-site +binding I-site +pocket I-site +. O + +More O +powerfully O +, O +our O +unique O +crystallographic B-evidence +observation I-evidence +of O +this O +‘ O +occupied B-protein_state +vs O +unoccupied B-protein_state +site O +’ O +asymmetry O +in O +the O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +interaction O +is O +, O +to O +our O +knowledge O +, O +the O +first O +example O +reported O +for O +a O +MarR B-protein_type +family O +protein O +. O + +Structural B-experimental_method +analyses I-experimental_method +suggested O +that O +‘ O +inward B-protein_state +’ O +side O +chain O +positions O +of O +Met22 B-residue_name_number +, O +Phe25 B-residue_name_number +and O +especially O +Arg43 B-residue_name_number +precluded O +binding O +of O +a O +second O +ligand O +molecule O +. O + +Such O +a O +mechanism O +indicates O +negative O +cooperativity O +, O +which O +may O +enhance O +the O +ligand O +- O +responsiveness O +of O +NadR B-protein +. O + +Comparisons O +of O +the O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +complex O +with O +available O +MarR B-protein_type +family O +/ O +salicylate B-chemical +complexes O +revealed O +that O +4 B-chemical +- I-chemical +HPA I-chemical +has O +a O +previously O +unobserved O +binding O +mode O +. O + +Briefly O +, O +in O +the O +M B-species +. I-species +thermoautotrophicum I-species +MTH313 B-protein +dimer B-oligomeric_state +, O +one O +molecule O +of O +salicylate B-chemical +binds O +in O +the O +pocket B-site +of O +each O +monomer B-oligomeric_state +, O +though O +with O +two O +rather O +different O +positions O +and O +orientations O +, O +only O +one O +of O +which O +( O +site B-site +- I-site +1 I-site +) O +is O +thought O +to O +be O +biologically O +relevant O +( O +Fig O +10A O +). O + +In O +the O +S B-species +. I-species +tokodaii I-species +protein O +ST1710 B-protein +, O +salicylate B-chemical +binds O +to O +the O +same O +position O +in O +each O +monomer B-oligomeric_state +of O +the O +dimer B-oligomeric_state +, O +in O +a O +site O +equivalent O +to O +the O +putative O +biologically O +relevant O +site O +of O +MTH313 B-protein +( O +Fig O +10B O +). O + +Unlike O +other O +MarR B-protein_type +family O +proteins O +which O +revealed O +multiple O +ligand O +binding O +interactions O +, O +we O +observed O +only O +1 O +molecule O +of O +4 B-chemical +- I-chemical +HPA I-chemical +bound B-protein_state +to I-protein_state +NadR B-protein +, O +suggesting O +a O +more O +specific O +and O +less O +promiscuous O +interaction O +. O + +In O +NadR B-protein +, O +the O +single O +molecule O +of O +4 B-chemical +- I-chemical +HPA I-chemical +binds O +in O +a O +position O +distinctly O +different O +from O +the O +salicylate B-site +binding I-site +site I-site +: O +translated O +by O +> O +10 O +Å O +and O +with O +a O +180 O +° O +inverted O +orientation O +( O +Fig O +10C O +). O + +NadR B-protein +shows O +a O +ligand B-site +binding I-site +site I-site +distinct O +from O +other O +MarR B-protein_type +homologues O +. O + +( O +A O +) O +A O +structural B-experimental_method +alignment I-experimental_method +of O +MTH313 B-protein +chains B-structure_element +A I-structure_element +and I-structure_element +B I-structure_element +shows O +that O +salicylate B-chemical +is O +bound B-protein_state +in O +distinct O +locations O +in O +each O +monomer B-oligomeric_state +; O +site B-site +- I-site +1 I-site +( O +thought O +to O +be O +the O +biologically O +relevant O +site O +) O +and O +site B-site +- I-site +2 I-site +differ O +by O +~ O +7Å O +( O +indicated O +by O +black O +dotted O +line O +) O +and O +also O +by O +ligand O +orientation O +. O + +( O +B O +) O +A O +structural B-experimental_method +alignment I-experimental_method +of O +MTH313 B-protein +chain B-structure_element +A I-structure_element +and O +ST1710 B-protein +( O +pink O +) O +( O +Cα O +rmsd B-evidence +2 O +. O +3Å O +), O +shows O +that O +they O +bind O +salicylate B-chemical +in O +equivalent O +sites O +( O +differing O +by O +only O +~ O +3Å O +) O +and O +with O +the O +same O +orientation O +. O + +( O +C O +) O +Addition O +of O +holo B-protein_state +- O +NadR B-protein +( O +chain B-structure_element +B I-structure_element +, O +blue O +) O +to O +the O +alignment B-experimental_method +reveals O +that O +bound B-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +differs O +in O +position O +by O +> O +10 O +Å O +compared O +to O +salicylate B-chemical +, O +and O +adopts O +a O +novel O +orientation O +. O + +Interestingly O +, O +a O +crystal B-evidence +structure I-evidence +was O +previously O +reported O +for O +a O +functionally O +- O +uncharacterized O +meningococcal B-taxonomy_domain +homologue O +of O +NadR B-protein +, O +termed O +NMB1585 B-protein +, O +which O +shares O +16 O +% O +sequence O +identity O +with O +NadR B-protein +. O +The O +two O +structures B-evidence +can O +be O +closely O +aligned O +( O +rmsd B-evidence +2 O +. O +3 O +Å O +), O +but O +NMB1585 B-protein +appears O +unsuited O +for O +binding O +HPAs B-chemical +, O +since O +its O +corresponding O +‘ B-site +pocket I-site +’ O +region O +is O +occupied O +by O +several O +bulky O +hydrophobic O +side O +chains O +. O + +It O +can O +be O +speculated O +that O +MarR B-protein_type +family O +members O +have O +evolved O +separately O +to O +engage O +distinct O +signaling O +molecules O +, O +thus O +enabling O +bacteria B-taxonomy_domain +to O +use O +the O +overall O +conserved O +MarR B-protein_type +scaffold O +to O +adapt O +and O +respond O +to O +diverse O +changing O +environmental O +stimuli O +experienced O +in O +their O +natural O +niches O +. O + +Alternatively O +, O +it O +is O +possible O +that O +other O +MarR B-protein_type +homologues O +( O +e O +. O +g O +. O +NMB1585 B-protein +) O +may O +have O +no O +extant O +functional O +binding B-site +pocket I-site +and O +thus O +may O +have O +lost O +the O +ability O +to O +respond O +to O +a O +ligand O +, O +acting O +instead O +as O +constitutive O +DNA B-chemical +- O +binding O +regulatory O +proteins O +. O + +The O +apo B-protein_state +- O +NadR B-protein +crystal B-evidence +structures I-evidence +revealed O +two O +dimers B-oligomeric_state +with O +slightly O +different O +conformations O +, O +most O +divergent O +in O +the O +DNA B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +. O + +It O +is O +not O +unusual O +for O +a O +crystal B-evidence +structure I-evidence +to O +reveal O +multiple O +copies O +of O +the O +same O +protein O +in O +very O +slightly O +different O +conformations O +, O +which O +are O +likely O +representative O +of O +the O +lowest O +- O +energy O +conformations O +sampled O +by O +the O +dynamic O +ensemble O +of O +molecular O +states O +occurring O +in O +solution O +, O +and O +which O +likely O +have O +only O +small O +energetic O +differences O +, O +as O +described O +previously O +for O +MexR B-protein +( O +a O +MarR B-protein_type +protein O +) O +or O +more O +recently O +for O +the O +solute B-protein_type +- I-protein_type +binding I-protein_type +protein I-protein_type +FhuD2 B-protein +. O + +Further O +, O +the O +holo B-protein_state +- O +NadR B-protein +structure B-evidence +was O +overall O +more O +different O +from O +the O +two O +apo B-protein_state +- O +NadR B-protein +structures B-evidence +( O +rmsd B-evidence +values O +~ O +1 O +. O +3Å O +), O +suggesting O +that O +the O +ligand O +selected O +and O +stabilized O +yet O +another O +conformation O +of O +NadR B-protein +. O +These O +observations O +suggest O +that O +4 B-chemical +- I-chemical +HPA I-chemical +, O +and O +potentially O +other O +similar O +ligands O +, O +can O +shift O +the O +molecular O +equilibrium O +, O +changing O +the O +energy O +barriers O +that O +separate O +active B-protein_state +and O +inactive B-protein_state +states O +, O +and O +stabilizing O +the O +specific O +conformation O +of O +NadR B-protein +poorly O +suited O +to O +bind O +DNA B-chemical +. O + +Comparisons O +of O +the O +apo B-protein_state +- O +and O +holo B-protein_state +- O +NadR B-protein +structures B-evidence +revealed O +that O +the O +largest O +differences O +occurred O +in O +the O +DNA B-chemical +- O +binding O +helix B-structure_element +α4 B-structure_element +. O + +The O +shift O +of O +helix B-structure_element +α4 B-structure_element +in O +holo B-protein_state +- O +NadR B-protein +was O +also O +accompanied O +by O +rearrangements O +at O +the O +dimer B-site +interface I-site +, O +involving O +helices B-structure_element +α1 B-structure_element +, O +α5 B-structure_element +, O +and O +α6 B-structure_element +, O +and O +this O +holo B-protein_state +- O +form O +appeared O +poorly O +suited O +for O +DNA B-chemical +- O +binding O +when O +compared O +with O +the O +known O +OhrR B-complex_assembly +: I-complex_assembly +ohrA I-complex_assembly +complex O +. O + +While O +some O +flexibility O +of O +helix B-structure_element +α4 B-structure_element +was O +also O +observed O +in O +the O +two O +apo B-protein_state +- O +structures B-evidence +, O +concomitant O +changes O +in O +the O +dimer B-site +interfaces I-site +were O +not O +observed O +, O +possibly O +due O +to O +the O +absence B-protein_state +of I-protein_state +ligand I-protein_state +. O + +One O +of O +the O +two O +conformations O +of O +apo B-protein_state +- O +NadR B-protein +appeared O +ideally O +suited O +for O +DNA B-chemical +- O +binding O +. O + +Overall O +, O +these O +analyses O +suggest O +that O +the O +apo B-protein_state +- O +NadR B-protein +dimer B-oligomeric_state +has O +a O +pre O +- O +existing O +equilibrium O +that O +samples O +a O +variety O +of O +conformations O +, O +some O +of O +which O +are O +compatible O +with O +DNA B-chemical +binding O +. O + +The O +noted O +flexibility O +may O +also O +explain O +how O +NadR B-protein +can O +adapt O +to O +bind O +various O +DNA B-chemical +target O +sequences O +with O +slightly O +different O +structural O +features O +. O + +Subsequently O +, O +upon O +ligand O +binding O +, O +holo B-protein_state +- O +NadR B-protein +adopts O +a O +structure O +less O +suited O +for O +DNA B-chemical +- O +binding O +and O +this O +conformation O +is O +selected O +and O +stabilized O +by O +a O +network O +of O +protein O +- O +ligand O +interactions O +and O +concomitant O +rearrangements O +at O +the O +NadR B-protein +holo B-protein_state +dimer B-site +interface I-site +. O + +In O +an O +alternative O +and O +less O +extensive O +manner O +, O +the O +binding O +of O +two O +salicylate B-chemical +molecules O +to O +the O +M B-species +. I-species +thermoautotrophicum I-species +protein O +MTH313 B-protein +appeared O +to O +induce O +large O +changes O +in O +the O +wHTH B-structure_element +domain I-structure_element +, O +which O +was O +associated O +with O +reduced O +DNA O +- O +binding O +activity O +. O + +Here O +we O +have O +presented O +two O +new O +crystal B-evidence +structures I-evidence +of O +the O +transcription B-protein_type +factor I-protein_type +, O +NadR B-protein +, O +which O +regulates O +expression O +of O +the O +meningococcal B-taxonomy_domain +surface O +protein O +, O +virulence O +factor O +and O +vaccine O +antigen O +NadA B-protein +. O +Detailed O +structural B-experimental_method +analyses I-experimental_method +provided O +a O +molecular O +explanation O +for O +the O +ligand O +- O +responsive O +regulation O +by O +NadR B-protein +on O +the O +majority O +of O +the O +promoters O +of O +meningococcal B-taxonomy_domain +genes O +regulated O +by O +NadR B-protein +, O +including O +nadA B-gene +. O +Intriguingly O +, O +NadR B-protein +exhibits O +a O +reversed O +regulatory O +mechanism O +on O +a O +second O +class O +of O +promoters O +, O +including O +mafA B-gene +of O +the O +multiple O +adhesin O +family O +– O +i O +. O +e O +. O +NadR B-protein +represses O +these O +genes O +in O +the O +presence O +but O +not O +absence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +The O +latter O +may O +influence O +the O +surface O +abundance O +or O +secretion O +of O +maf O +proteins O +, O +an O +emerging O +class O +of O +highly B-protein_state +conserved I-protein_state +meningococcal B-taxonomy_domain +putative O +adhesins O +and O +toxins O +with O +many O +important O +roles O +. O + +Further O +work O +is O +required O +to O +investigate O +how O +the O +two O +different O +promoter O +types O +influence O +the O +ligand O +- O +responsiveness O +of O +NadR B-protein +during O +bacterial B-taxonomy_domain +infection O +and O +may O +provide O +insights O +into O +the O +regulatory O +mechanisms O +occurring O +during O +these O +host O +- O +pathogen O +interactions O +. O + +Ultimately O +, O +knowledge O +of O +the O +ligand O +- O +dependent O +activity O +of O +NadR B-protein +will O +continue O +to O +deepen O +our O +understanding O +of O +nadA B-gene +expression O +levels O +, O +which O +influence O +meningococcal B-taxonomy_domain +pathogenesis O +. O + +Structure O +of O +an O +OhrR O +- O +ohrA B-gene +operator O +complex O +reveals O +the O +DNA O +binding O +mechanism O +of O +the O +MarR O +family O + +The O +structure O +of O +NMB1585 B-protein +, O +a O +MarR O +- O +family O +regulator O +from O +Neisseria O +meningitidis O + +Structural O +determinant O +for O +inducing O +RORgamma B-protein +specific O +inverse O +agonism O +triggered O +by O +a O +synthetic O +benzoxazinone B-chemical +ligand O + +The O +nuclear B-protein_type +hormone I-protein_type +receptor I-protein_type +RORγ B-protein +regulates O +transcriptional O +genes O +involved O +in O +the O +production O +of O +the O +pro O +- O +inflammatory O +interleukin B-protein_type +IL B-protein_type +- I-protein_type +17 I-protein_type +which O +has O +been O +linked O +to O +autoimmune O +diseases O +such O +as O +rheumatoid O +arthritis O +, O +multiple O +sclerosis O +and O +inflammatory O +bowel O +disease O +. O + +This O +transcriptional O +activity O +of O +RORγ B-protein +is O +modulated O +through O +a O +protein O +- O +protein O +interaction O +involving O +the O +activation B-structure_element +function I-structure_element +2 I-structure_element +( I-structure_element +AF2 I-structure_element +) I-structure_element +helix I-structure_element +on O +the O +ligand B-structure_element +binding I-structure_element +domain I-structure_element +of O +RORγ B-protein +and O +a O +conserved B-protein_state +LXXLL B-structure_element +helix I-structure_element +motif I-structure_element +on O +coactivator O +proteins O +. O + +Our O +goal O +was O +to O +develop O +a O +RORγ B-protein +specific O +inverse B-protein_state +agonist I-protein_state +that O +would O +help O +down O +regulate O +pro O +- O +inflammatory O +gene O +transcription O +by O +disrupting O +the O +protein O +protein O +interaction O +with O +coactivator O +proteins O +as O +a O +therapeutic O +agent O +. O + +We O +identified O +a O +novel O +series O +of O +synthetic O +benzoxazinone B-chemical +ligands O +having O +an O +agonist B-protein_state +( O +BIO592 B-chemical +) O +and O +inverse B-protein_state +agonist I-protein_state +( O +BIO399 B-chemical +) O +mode O +of O +action O +in O +a O +FRET B-experimental_method +based I-experimental_method +assay I-experimental_method +. O + +We O +show O +that O +the O +AF2 B-structure_element +helix I-structure_element +of O +RORγ B-protein +is O +proteolytically B-protein_state +sensitive I-protein_state +when O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +binds O +. O + +Using O +x B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +we O +show O +how O +small O +modifications O +on O +the O +benzoxazinone B-chemical +agonist B-protein_state +BIO592 B-chemical +trigger O +inverse O +agonism O +of O +RORγ B-protein +. O + +Using O +an O +in B-experimental_method +vivo I-experimental_method +reporter I-experimental_method +assay I-experimental_method +, O +we O +show O +that O +the O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +displayed O +specificity O +for O +RORγ B-protein +over O +ROR B-protein_type +sub O +- O +family O +members O +α B-protein +and O +β B-protein +. O + +The O +synthetic O +benzoxazinone B-chemical +ligands O +identified O +in O +our O +FRET B-experimental_method +assay I-experimental_method +have O +an O +agonist B-protein_state +( O +BIO592 B-chemical +) O +or O +inverse B-protein_state +agonist I-protein_state +( O +BIO399 B-chemical +) O +effect O +by O +stabilizing O +or O +destabilizing O +the O +agonist B-protein_state +conformation O +of O +RORγ B-protein +. O + +The O +proteolytic O +sensitivity O +of O +the O +AF2 B-structure_element +helix I-structure_element +of O +RORγ B-protein +demonstrates O +that O +it O +destabilizes O +upon O +BIO399 B-chemical +inverse B-protein_state +agonist I-protein_state +binding O +perturbing O +the O +coactivator B-site +protein I-site +binding I-site +site I-site +. O + +Our O +structural B-experimental_method +investigation I-experimental_method +of O +the O +BIO592 B-chemical +agonist B-protein_state +and O +BIO399 B-chemical +inverse B-protein_state +agonist I-protein_state +structures B-evidence +identified O +residue O +Met358 B-residue_name_number +on O +RORγ B-protein +as O +the O +trigger O +for O +RORγ B-protein +specific O +inverse O +agonism O +. O + +Retinoid B-protein +- I-protein +related I-protein +orphan I-protein +receptor I-protein +gamma I-protein +( O +RORγ B-protein +) O +is O +a O +transcription B-protein_type +factor I-protein_type +belonging O +to O +a O +sub O +- O +family O +of O +nuclear B-protein_type +receptors I-protein_type +that O +includes O +two O +closely O +related O +members O +RORα B-protein +and O +RORβ B-protein +. O + +Even O +though O +a O +high O +degree O +of O +sequence O +similarity O +exists O +between O +the O +RORs B-protein_type +, O +their O +functional O +roles O +in O +regulation O +for O +physiological O +processes O +involved O +in O +development O +and O +immunity O +are O +distinct O +. O + +During O +development O +, O +RORγ B-protein +regulates O +the O +transcriptional O +genes O +involved O +in O +the O +functioning O +of O +multiple O +pro O +- O +inflammatory O +lymphocyte O +lineages O +including O +T O +helper O +cells O +( O +TH17cells O +) O +which O +are O +necessary O +for O +IL B-protein_type +- I-protein_type +17 I-protein_type +production O +. O + +IL B-protein_type +- I-protein_type +17 I-protein_type +is O +a O +pro O +- O +inflammatory O +interleukin B-protein_type +linked O +to O +autoimmune O +diseases O +such O +as O +rheumatoid O +arthritis O +, O +multiple O +sclerosis O +and O +inflammatory O +bowel O +disease O +; O +making O +its O +transcriptional O +regulation O +through O +RORγ B-protein +an O +attractive O +therapeutic O +target O +. O + +RORγ B-protein +consists O +of O +an O +N O +- O +terminal O +DNA B-structure_element +binding I-structure_element +domain I-structure_element +( O +DBD B-structure_element +) O +connected O +to O +a O +C O +- O +terminal O +ligand B-structure_element +binding I-structure_element +domain I-structure_element +( O +LBD B-structure_element +) O +via O +a O +flexible O +hinge B-structure_element +region I-structure_element +. O + +The O +DBD B-structure_element +is O +composed O +of O +two O +zinc B-structure_element +fingers I-structure_element +that O +allow O +it O +to O +interact O +with O +specifically O +encoded O +regions O +on O +the O +DNA O +called O +the O +nuclear B-structure_element +receptor I-structure_element +response I-structure_element +elements I-structure_element +. O + +The O +LBD B-structure_element +consists O +of O +a O +coactivator B-site +protein I-site +binding I-site +pocket I-site +and O +a O +hydrophobic B-site +ligand I-site +binding I-site +site I-site +( O +LBS B-site +) O +which O +are O +responsible O +for O +regulating O +transcription O +. O + +The O +coactivator B-site +binding I-site +pocket I-site +of O +RORγ B-protein +recognizes O +a O +conserved B-protein_state +helix B-structure_element +motif I-structure_element +LXXLL I-structure_element +( O +where O +X O +can O +be O +any O +amino O +acid O +) O +on O +transcriptional O +coactivator O +complexes O +and O +recruits O +it O +to O +activate O +transcription O +. O + +Like O +other O +nuclear B-protein_type +hormone I-protein_type +receptors I-protein_type +, O +RORγ B-protein +’ O +s O +helix12 B-structure_element +which O +makes O +up O +the O +C O +- O +termini O +of O +the O +LBD B-structure_element +is O +an O +essential O +part O +of O +the O +coactivator B-site +binding I-site +pocket I-site +and O +is O +commonly O +referred O +to O +as O +the O +activation B-structure_element +function I-structure_element +helix I-structure_element +2 I-structure_element +( O +AF2 B-structure_element +). O + +In O +RORγ B-protein +, O +the O +conformation O +of O +the O +AF2 B-structure_element +helix I-structure_element +required O +to O +form O +the O +coactivator B-site +binding I-site +pocket I-site +is O +mediated O +by O +a O +salt B-bond_interaction +bridge I-bond_interaction +between O +His479 B-residue_name_number +and O +Tyr502 B-residue_name_number +in O +addition O +to O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +interactions I-bond_interaction +between O +Tyr502 B-residue_name_number +and O +Phe506 B-residue_name_number +. O + +The O +conformation O +of O +the O +AF2 B-structure_element +helix I-structure_element +can O +be O +modulated O +through O +targeted O +ligands O +which O +bind O +the O +LBS B-site +and O +increase O +the O +binding O +of O +the O +coactivator O +protein O +( O +agonists O +) O +or O +disrupt O +binding O +( O +inverse O +agonists O +) O +thereby O +enhancing O +or O +inhibiting O +transcription O +. O + +Since O +RORγ B-protein +has O +been O +demonstrated O +to O +play O +an O +important O +role O +in O +pro O +- O +inflammatory O +gene O +expression O +patterns O +implicated O +in O +several O +major O +autoimmune O +diseases O +, O +our O +aim O +was O +to O +develop O +RORγ B-protein +inverse O +agonists O +that O +would O +help O +down O +regulate O +pro O +- O +inflammatory O +gene O +transcription O +. O + +FRET B-evidence +results I-evidence +for O +agonist B-protein_state +BIO592 B-chemical +( O +a O +) O +and O +Inverse B-protein_state +Agonist I-protein_state +BIO399 B-chemical +( O +b O +) O + +Here O +we O +present O +the O +identification O +of O +two O +synthetic O +benzoxazinone B-chemical +RORγ B-protein +ligands O +, O +a O +weak O +agonist B-protein_state +BIO592 B-chemical +( O +Fig O +. O +1a O +) O +and O +an O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +( O +Fig O +. O +1b O +) O +which O +were O +identified O +using O +a O +Fluorescence B-experimental_method +Resonance I-experimental_method +Energy I-experimental_method +transfer I-experimental_method +( I-experimental_method +FRET I-experimental_method +) I-experimental_method +based I-experimental_method +assay I-experimental_method +that O +monitored O +coactivator O +peptide O +recruitment O +. O + +Using O +partial B-experimental_method +proteolysis I-experimental_method +in O +combination O +with O +mass B-experimental_method +spectrometry I-experimental_method +analysis O +we O +demonstrate O +that O +the O +AF2 B-structure_element +helix I-structure_element +of O +RORγ B-protein +destabilizes O +upon O +BIO399 B-chemical +( O +inverse B-protein_state +agonist I-protein_state +) O +binding O +. O + +Finally O +, O +comparing O +binding B-evidence +modes I-evidence +of O +our O +benzoxazinone B-chemical +RORγ B-protein +crystal B-evidence +structures I-evidence +to O +other O +ROR B-protein_type +structures B-evidence +, O +we O +hypothesize O +a O +new O +mode O +of O +action O +for O +achieving O +inverse O +agonism O +and O +selectivity O +. O + +Using O +a O +FRET B-experimental_method +based I-experimental_method +assay I-experimental_method +we O +discovered O +agonist B-protein_state +BIO592 B-chemical +( O +Fig O +. O +1a O +) O +which O +increased O +the O +coactivator O +peptide O +TRAP220 B-chemical +recruitment O +to O +RORγ B-protein +( O +EC50 B-evidence +0f O +58nM O +and O +Emax B-evidence +of O +130 O +%) O +and O +a O +potent O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +( O +Fig O +. O +1b O +) O +which O +inhibited O +coactivator O +recruitment O +( O +IC50 B-evidence +: O +4 O +. O +7nM O +). O + +Interestingly O +, O +the O +structural O +difference O +between O +the O +agonist B-protein_state +BIO592 B-chemical +and O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +was O +minor O +; O +with O +the O +2 B-chemical +, I-chemical +3 I-chemical +- I-chemical +dihydrobenzo I-chemical +[ I-chemical +1 I-chemical +, I-chemical +4 I-chemical +] I-chemical +oxazepin I-chemical +- I-chemical +4 I-chemical +- I-chemical +one I-chemical +ring O +system O +of O +BIO399 B-chemical +being O +3 O +atoms O +larger O +than O +the O +benzo B-chemical +[ I-chemical +1 I-chemical +, I-chemical +4 I-chemical +] I-chemical +oxazine I-chemical +- I-chemical +3 I-chemical +- I-chemical +one I-chemical +ring O +system O +of O +BIO592 B-chemical +. O + +In O +order O +to O +understand O +how O +small O +changes O +in O +the O +core O +ring O +system O +leads O +to O +inverse O +agonism O +, O +we O +wanted O +to O +structurally O +determine O +the O +binding O +mode O +of O +both O +BIO592 B-chemical +and O +BIO399 B-chemical +in O +the O +LBS B-site +of O +RORγ B-protein +using O +x B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +Structure B-evidence +of O +the O +RORγ518 B-complex_assembly +- I-complex_assembly +BIO592 I-complex_assembly +- I-complex_assembly +EBI96 I-complex_assembly +ternary O +complex O +is O +in O +a O +transcriptionally O +active B-protein_state +conformation O + +a O +The O +ternary B-evidence +structure I-evidence +of O +RORγ518 B-protein +BIO592 B-chemical +and O +EBI96 B-chemical +. O + +b O +RORγ B-protein +AF2 B-structure_element +helix I-structure_element +in O +the O +agonist B-protein_state +conformation O +. O + +c O +EBI96 B-chemical +coactivator O +peptide O +bound B-protein_state +in I-protein_state +the O +coactivator B-site +pocket I-site +of O +RORγ B-protein + +RORγ518 B-protein +bound B-protein_state +to I-protein_state +agonist B-protein_state +BIO592 B-chemical +was O +crystallized B-experimental_method +with O +a O +truncated B-protein_state +form O +of O +the O +coactivator O +peptide O +EBI96 B-chemical +to O +a O +resolution O +of O +2 O +. O +6 O +Å O +( O +Fig O +. O +2a O +). O + +The O +structure B-evidence +of O +the O +ternary O +complex O +had O +features O +similar O +to O +other O +ROR B-protein_type +agonist B-protein_state +coactivator O +structures B-evidence +in O +a O +transcriptionally B-protein_state +active I-protein_state +canonical B-protein_state +three I-protein_state +layer I-protein_state +helix I-protein_state +fold I-protein_state +with O +the O +AF2 B-structure_element +helix I-structure_element +in O +the O +agonist B-protein_state +conformation O +. O + +The O +agonist B-protein_state +conformation O +is O +stabilized O +by O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +His479 B-residue_name_number +and O +Tyr502 B-residue_name_number +, O +in O +addition O +to O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +interactions I-bond_interaction +between O +His479 B-residue_name_number +, O +Tyr502 B-residue_name_number +and O +Phe506 B-residue_name_number +( O +Fig O +. O +2b O +). O + +The O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +His479 B-residue_name_number +and O +Tyr502 B-residue_name_number +has O +been O +reported O +to O +be O +critical O +for O +RORγ B-protein +agonist B-protein_state +activity O +. O + +Disrupting O +this O +interaction O +through O +mutagenesis B-experimental_method +reduced O +transcriptional O +activity O +of O +RORγ B-protein +. O + +This O +reduced O +transcriptional O +activity O +has O +been O +attributed O +to O +the O +inability O +of O +the O +AF2 B-structure_element +helix I-structure_element +to O +complete O +the O +formation O +of O +the O +coactivator B-site +binding I-site +pocket I-site +necessary O +for O +coactivator O +proteins O +to O +bind O +. O + +Electron B-evidence +density I-evidence +for O +the O +coactivator O +peptide O +EBI96 B-chemical +was O +observed O +for O +residues O +EFPYLLSLLG B-structure_element +which O +formed O +a O +α B-structure_element +- I-structure_element +helix I-structure_element +stabilized O +through O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +the O +coactivator B-site +binding I-site +pocket I-site +on O +RORγ B-protein +( O +Fig O +. O +2c O +). O + +This O +interaction O +is O +further O +stabilized O +through O +a O +conserved B-protein_state +charged B-structure_element +clamp I-structure_element +wherein O +the O +backbone O +amide O +of O +Tyr7 B-residue_name_number +and O +carbonyl O +of O +Leu11 B-residue_name_number +of O +EBI96 B-chemical +form O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +Glu504 B-residue_name_number +( O +helix12 B-structure_element +) O +and O +Lys336 B-residue_name_number +( O +helix3 B-structure_element +) O +of O +RORγ B-protein +. O + +Formation O +of O +this O +charged B-structure_element +clamp I-structure_element +is O +essential O +for O +RORγ B-protein +’ O +s O +function O +for O +playing O +a O +role O +in O +transcriptional O +activation O +and O +this O +has O +been O +corroborated O +through O +mutagenic B-experimental_method +studies I-experimental_method +in O +this O +region O +. O + +BIO592 B-chemical +binds O +in O +a O +collapsed B-protein_state +conformation O +stabilizing O +the O +agonist B-protein_state +conformation O +of O +RORγ B-protein + +a O +Collapsed O +binding O +mode O +of O +agonist B-protein_state +BIO592 B-chemical +in O +the O +hydrophobic O +LBS B-site +of O +RORγ B-protein +. O + +b O +Benzoxazinone B-chemical +ring O +system O +of O +agonist B-protein_state +BIO592 B-chemical +packing O +against O +His479 B-residue_name_number +of O +RORγ B-protein +stabilizing O +agonist B-protein_state +conformation O +of O +the O +AF2 B-structure_element +helix I-structure_element + +BIO592 B-chemical +bound B-protein_state +in I-protein_state +a O +collapsed B-protein_state +conformational O +state O +in O +the O +LBS B-site +of O +RORγ B-protein +with O +the O +xylene B-chemical +ring O +positioned O +at O +the O +bottom O +of O +the O +pocket B-site +making O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +Val376 B-residue_name_number +, O +Phe378 B-residue_name_number +, O +Phe388 B-residue_name_number +and O +Phe401 B-residue_name_number +, O +with O +the O +ethyl B-chemical +- I-chemical +benzoxazinone I-chemical +ring O +making O +several O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +Trp317 B-residue_name_number +, O +Leu324 B-residue_name_number +, O +Met358 B-residue_name_number +, O +Leu391 B-residue_name_number +, O +Ile B-residue_name_number +400 I-residue_name_number +and O +His479 B-residue_name_number +( O +Fig O +. O +3a O +, O +Additional O +file O +3 O +). O + +The O +sulfonyl B-chemical +group O +faces O +the O +entrance O +of O +the O +pocket B-site +, O +while O +the O +CF3 O +makes O +a O +hydrophobic B-bond_interaction +contact I-bond_interaction +with O +Ala327 B-residue_name_number +. O + +Hydrophobic B-bond_interaction +interaction I-bond_interaction +between O +the O +ethyl O +group O +of O +the O +benzoxazinone B-chemical +and O +His479 B-residue_name_number +reinforce O +the O +His479 B-residue_name_number +sidechain O +position O +for O +making O +the O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +Tyr502 B-residue_name_number +thereby O +stabilizing O +the O +agonist B-protein_state +conformation O +( O +Fig O +. O +3b O +). O + +RORγ B-protein +AF2 B-structure_element +helix I-structure_element +is O +sensitive O +to O +proteolysis O +in O +the O +presence B-protein_state +of I-protein_state +Inverse B-protein_state +Agonist I-protein_state +BIO399 B-chemical + +Next O +, O +we O +attempted O +co B-experimental_method +- I-experimental_method +crystallization I-experimental_method +with O +the O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +. O + +However O +, O +extensive O +crystallization B-experimental_method +efforts O +with O +BIO399 B-chemical +and O +RORγ518 B-protein +or O +other O +AF2 B-structure_element +intact B-protein_state +constructs O +did O +not O +produce O +crystals B-evidence +. O + +We O +hypothesized O +that O +the O +RORγ518 B-protein +coactivator O +peptide O +interaction O +in O +the O +FRET B-experimental_method +assay I-experimental_method +was O +disrupted O +upon O +BIO399 B-chemical +binding O +and O +that O +a O +conformational O +rearrangement O +of O +the O +AF2 B-structure_element +helix I-structure_element +could O +have O +occurred O +, O +hindering O +crystallization B-experimental_method +. O + +Specific O +proteolytic O +positions O +on O +RORγ518 B-protein +when O +treated B-experimental_method +with I-experimental_method +Actinase B-protein +E I-protein +alone O +( O +Green O +) O +or O +in O +the O +presence B-protein_state +of I-protein_state +BIO399 B-chemical +( O +Red O +) O +and O +shared O +proteolytic B-site +sites I-site +( O +Yellow O +) O + +The O +unfolding O +of O +the O +AF2 B-structure_element +helix I-structure_element +has O +been O +observed O +for O +other O +nuclear B-protein_type +hormone I-protein_type +receptors I-protein_type +when O +bound B-protein_state +to I-protein_state +an O +inverse B-protein_state +agonist I-protein_state +or O +antagonist O +. O + +We O +used O +partial B-experimental_method +proteolysis I-experimental_method +in O +combination O +with O +mass B-experimental_method +spectrometry I-experimental_method +to O +determine O +if O +BIO399 B-chemical +was O +causing O +the O +AF2 B-structure_element +helix I-structure_element +to O +unfold O +. O + +Results O +of O +the O +Actinase B-experimental_method +E I-experimental_method +proteolysis I-experimental_method +experiments O +on O +RORγ518 B-protein +, O +the O +ternary O +complex O +of O +RORγ518 B-protein +with O +agonist B-protein_state +BIO592 B-chemical +and O +coactivator O +EBI96 B-chemical +, O +or O +in O +the O +presence B-protein_state +of I-protein_state +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +supported O +our O +hypothesis O +. O + +Analysis O +of O +the O +fragmentation B-evidence +pattern I-evidence +showed O +minimal O +proteolytic O +removal O +of O +the O +AF2 B-structure_element +helix I-structure_element +by O +Actinase B-protein +E I-protein +on O +RORγ518 B-protein +alone O +( O +ending O +at O +504 B-residue_range +to I-residue_range +506 I-residue_range +) O +and O +the O +ternary B-protein_state +complex I-protein_state +remained O +primarily O +intact O +( O +ending O +at O +515 B-residue_number +/ O +518 B-residue_number +) O +( O +Additional O +file O +4 O +). O + +However O +, O +in O +the O +presence B-protein_state +of I-protein_state +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +, O +the O +proteolytic B-evidence +pattern I-evidence +showed O +significantly O +less O +protection O +, O +albeit O +the O +products O +were O +more O +heterogeneous O +( O +majority O +ending O +at O +494 B-residue_number +/ O +495 B-residue_number +), O +indicating O +the O +destabilization O +of O +the O +AF2 B-structure_element +helix I-structure_element +compared O +to O +either O +the O +APO B-protein_state +or O +ternary B-protein_state +agonist I-protein_state +complex I-protein_state +( O +Fig O +. O +4 O +, O +Additional O +file O +5 O +). O + +Several O +rounds O +of O +cocrystallization B-experimental_method +attempts O +with O +RORγ518 B-protein +or O +other O +RORγ B-protein +AF2 B-structure_element +helix I-structure_element +containing O +constructs O +complexed B-protein_state +with I-protein_state +BIO399 B-chemical +had O +not O +produced O +crystals B-evidence +. O + +We O +attributed O +the O +inability O +to O +form O +crystals B-evidence +to O +the O +unfolding O +of O +the O +AF2 B-structure_element +helix I-structure_element +induced O +by O +BIO399 B-chemical +. O + +We O +reasoned O +that O +if O +we O +could O +remove O +the O +unfolded B-protein_state +AF2 B-structure_element +helix I-structure_element +using O +proteolysis B-experimental_method +we O +could O +produce O +a O +binary O +complex O +more O +amenable O +to O +crystallization B-experimental_method +. O + +AF2 B-protein_state +truncated I-protein_state +RORγ B-complex_assembly +BIO399 I-complex_assembly +complex O +is O +more O +amenable O +to O +crystallization B-experimental_method + +a O +The O +binary O +structure B-evidence +of O +AF2 B-protein_state +- I-protein_state +truncated I-protein_state +RORγ B-protein +and O +BIO399 B-chemical +. O + +b O +The O +superposition B-experimental_method +of O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +( O +Cyan O +) O +and O +agonist B-protein_state +BIO592 B-chemical +( O +Green O +). O + +c O +Movement O +of O +Met358 B-residue_name_number +and O +His479 B-residue_name_number +in O +the O +BIO399 B-chemical +( O +Cyan O +) O +and O +BIO592 B-chemical +( O +Green O +) O +structures B-evidence + +The O +Actinase B-protein +E I-protein +treated O +RORγ518 B-complex_assembly +BIO399 I-complex_assembly +ternary O +complex O +( O +aeRORγ493 B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +) O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +readily O +in O +several O +PEG O +based O +conditions O +. O + +The O +structure B-evidence +of O +aeRORγ493 B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +BIO399 I-complex_assembly +complex O +was O +solved B-experimental_method +to O +2 O +. O +3 O +Å O +and O +adopted O +a O +similar O +core O +fold O +to O +the O +BIO592 B-chemical +agonist B-protein_state +crystal B-evidence +structure I-evidence +( O +Fig O +. O +5a O +, O +Additional O +file O +3 O +). O + +The O +aeRORγ493 B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +BIO399 I-complex_assembly +structure B-evidence +diverged O +at O +the O +c O +- O +terminal O +end O +of O +Helix B-structure_element +11 I-structure_element +from O +the O +RORγ518 B-complex_assembly +BIO592 I-complex_assembly +EBI96 I-complex_assembly +structure B-evidence +, O +where O +helix B-structure_element +11 I-structure_element +unwinds O +into O +a O +random O +coil O +after O +residue O +L475 B-residue_name_number +. O + +Inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +uses O +Met358 B-residue_name_number +as O +a O +trigger O +for O +inverse O +agonism O + +BIO399 B-chemical +binds O +to O +the O +ligand B-site +binding I-site +site I-site +of O +RORγ B-protein +adopting O +a O +collapsed B-protein_state +conformation O +as O +seen O +with O +BIO592 B-chemical +where O +the O +two O +compounds O +superimpose B-experimental_method +with O +an O +RMSD B-evidence +of O +0 O +. O +72 O +Å O +( O +Fig O +. O +5b O +). O + +The O +majority O +of O +the O +side O +chains O +within O +4 O +Å O +of O +BIO399 B-chemical +and O +BIO592 B-chemical +adopt O +similar O +rotomer O +conformations O +with O +the O +exceptions O +of O +Met358 B-residue_name_number +and O +His479 B-residue_name_number +( O +Fig O +. O +5c O +). O + +The O +difference B-evidence +density I-evidence +map I-evidence +showed O +clear O +positive B-evidence +density I-evidence +for O +Met358 B-residue_name_number +in O +an O +alternate O +rotomer O +conformation O +compared O +to O +the O +one O +observed O +in O +the O +molecular B-experimental_method +replacement I-experimental_method +model I-experimental_method +or O +the O +other O +agonist B-protein_state +containing O +models O +( O +Additional O +file O +6 O +). O + +We O +tried O +to O +refine O +Met358 B-residue_name_number +in O +the O +same O +conformation O +as O +the O +molecular B-experimental_method +replacement I-experimental_method +model I-experimental_method +or O +the O +other O +agonist B-protein_state +containing O +models O +, O +but O +the O +results O +clearly O +indicated O +that O +this O +was O +not O +possible O +, O +thus O +confirming O +the O +new O +rotamer O +conformation O +for O +the O +Met358 B-residue_name_number +sidechain O +in O +the O +inverse B-protein_state +agonist I-protein_state +bound I-protein_state +structure B-evidence +. O + +The O +change O +in O +rotomer O +conformation O +of O +Met358 B-residue_name_number +between O +the O +agonist B-protein_state +and O +inverse B-protein_state +agonist I-protein_state +structures B-evidence +is O +attributed O +to O +the O +gem O +- O +dimethyl O +group O +on O +the O +larger O +7 O +membered O +benzoxazinone B-chemical +ring O +system O +of O +BIO399 B-chemical +. O + +The O +comparison B-experimental_method +of O +the O +two O +structures B-evidence +shows O +that O +the O +agonist B-protein_state +conformation O +observed O +in O +the O +BIO592 B-chemical +structure B-evidence +would O +be O +perturbed O +by O +BIO399 B-chemical +pushing O +Met358 B-residue_name_number +into O +Phe506 B-residue_name_number +of O +the O +AF2 B-structure_element +helix I-structure_element +indicating O +that O +Met358 B-residue_name_number +is O +a O +trigger O +for O +inducing O +inverse O +agonism O +in O +RORγ B-protein +( O +Fig O +. O +5c O +). O + +BIO399 B-chemical +and O +Inverse B-protein_state +agonist I-protein_state +T0901317 B-chemical +bind O +in O +a O +collapsed B-protein_state +conformation O +distinct O +from O +other O +RORγ B-protein +Inverse O +Agonists O +Cocrystal B-evidence +structures I-evidence + +a O +Overlay B-experimental_method +of O +RORγ B-protein +structures B-evidence +bound B-protein_state +to I-protein_state +BIO596 B-chemical +( O +Green O +), O +BIO399 B-chemical +( O +Cyan O +) O +and O +T0901317 B-chemical +( O +Pink O +). O + +b O +Overlay B-experimental_method +of O +M358 B-residue_name_number +in O +RORγ B-protein +structure B-evidence +BIO596 B-chemical +( O +Green O +), O +BIO399 B-chemical +( O +Cyan O +), O +Digoxin B-chemical +( O +Yellow O +), O +Compound O +2 O +( O +Grey O +), O +Compound O +48 O +( O +Salmon O +) O +and O +Compound O +4j O +( O +Orange O +) O + +The O +co B-evidence +- I-evidence +crystal I-evidence +structure I-evidence +of O +RORγ B-protein +with O +T0901317 B-chemical +( O +PDB O +code O +: O +4NB6 O +), O +an O +inverse B-protein_state +agonist I-protein_state +of O +RORγ B-protein +( O +IC50 B-evidence +of O +54nM O +in O +an O +SRC1 B-experimental_method +displacement I-experimental_method +FRET I-experimental_method +assay I-experimental_method +and O +an O +IC50 B-evidence +of O +59nM O +in O +our O +FRET B-experimental_method +assay I-experimental_method +( O +Additional O +file O +7 O +)) O +shows O +that O +it O +adopts O +a O +collapsed B-protein_state +conformation O +similar O +to O +the O +structure B-evidence +of O +BIO399 B-chemical +described O +here O +. O + +The O +two O +compounds O +superimpose B-experimental_method +with O +an O +RMSD B-evidence +of O +0 O +. O +81 O +Å O +( O +Fig O +. O +6a O +). O + +The O +CF3 O +group O +on O +the O +hexafluoropropanol B-chemical +group O +of O +T0901317 B-chemical +was O +reported O +to O +fit O +the O +electron B-evidence +density I-evidence +in O +two O +conformations O +one O +of O +which O +pushes O +Met358 B-residue_name_number +into O +the O +vicinity O +of O +Phe506 B-residue_name_number +in O +the O +RORγ B-protein +BIO592 B-chemical +agonist B-protein_state +structure B-evidence +. O + +We O +hypothesize O +that O +since O +the O +Met358 B-residue_name_number +sidechain O +conformation O +in O +the O +T0901317 B-chemical +RORγ B-protein +structure B-evidence +is O +not O +in O +the O +BIO399 B-chemical +conformation O +, O +this O +difference O +could O +account O +for O +the O +10 O +- O +fold O +reduction O +in O +the O +inverse O +agonism O +for O +T0901317 B-chemical +compared O +to O +BIO399 B-chemical +in O +the O +FRET B-experimental_method +assay I-experimental_method +. O + +Co B-evidence +- I-evidence +crystal I-evidence +structures I-evidence +of O +RORγ B-protein +have O +been O +generated O +with O +several O +potent O +inverse O +agonists O +adopting O +a O +linear B-protein_state +conformation O +distinct O +from O +the O +collapsed B-protein_state +conformations O +seen O +for O +BIO399 B-chemical +and O +T090131718 B-chemical +. O + +The O +inverse B-protein_state +agonist I-protein_state +activity O +for O +these O +compounds O +has O +been O +attributed O +to O +orientating O +Trp317 B-residue_name_number +to O +clash O +with O +Tyr502 B-residue_name_number +or O +a O +direct O +inverse B-protein_state +agonist I-protein_state +hydrogen B-bond_interaction +bonding I-bond_interaction +event O +with O +His479 B-residue_name_number +, O +both O +of O +which O +would O +perturb O +the O +agonist B-protein_state +conformation O +of O +RORγ B-protein +. O + +BIO399 B-chemical +neither O +orients O +the O +sidechain O +of O +Trp317 B-residue_name_number +toward O +Tyr502 B-residue_name_number +nor O +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +His479 B-residue_name_number +suggesting O +its O +mode O +of O +action O +is O +distinct O +from O +linear O +inverse O +agonists O +( O +Additional O +file O +8 O +). O + +In O +the O +linear O +inverse B-protein_state +agonist I-protein_state +crystal B-evidence +structures I-evidence +the O +side O +chain O +of O +Met358 B-residue_name_number +resides O +in O +a O +similar O +position O +as O +the O +rotomer O +observed O +in O +RORγ B-protein +agonist B-protein_state +structures B-evidence +with O +BIO592 B-chemical +described O +here O +or O +as O +observed O +in O +the O +hydroxycholesterol B-chemical +derivatives O +and O +therefore O +would O +not O +trigger O +inverse O +agonism O +with O +these O +ligands O +( O +Fig O +. O +6b O +). O + +BIO399 B-chemical +shows O +selectivity O +for O +RORγ B-protein +over O +RORα B-protein +and O +RORβ B-protein +in O +a O +GAL4 B-experimental_method +Cellular I-experimental_method +Reporter I-experimental_method +Assay I-experimental_method + +GAL4 B-experimental_method +cell I-experimental_method +assay I-experimental_method +selectivity O +profile O +for O +BIO399 B-chemical +toward O +RORα B-protein +and O +RORβ B-protein +in O +GAL4 B-protein + +a O +Overlay B-experimental_method +of O +RORα B-protein +( O +yellow O +), O +β B-protein +( O +pink O +) O +and O +γ B-protein +( O +cyan O +) O +showing O +side O +chain O +differences O +at O +Met358 B-residue_name_number +inverse O +agonism O +trigger O +position O +and O +( O +b O +) O +around O +the O +benzoxazinone B-chemical +ring O +system O +of O +BIO399 B-chemical + +In O +order O +to O +assess O +the O +in O +vivo O +selectivity O +profile O +of O +BIO399 B-chemical +a O +cellular B-experimental_method +reporter I-experimental_method +assay I-experimental_method +was O +implemented O +where O +the O +ligand B-structure_element +binding I-structure_element +domains I-structure_element +of O +ROR B-protein_type +α B-protein +, O +β B-protein +and O +γ B-protein +were O +fused B-experimental_method +to I-experimental_method +the O +DNA B-structure_element +binding I-structure_element +domain I-structure_element +of O +the O +transcriptional B-protein_type +factor I-protein_type +GAL4 B-protein +. O + +The O +ROR B-protein_type +- O +GAL4 B-protein +fusion O +proteins O +were O +expressed O +in O +cells O +with O +the O +luciferase O +reporter O +gene O +under O +the O +control O +of O +a O +GAL4 B-protein +promoter O +. O + +BIO399 B-chemical +inhibited O +the O +luciferase O +activity O +when O +added O +to O +the O +cells O +expressing O +the O +RORγ B-protein +- O +GAL4 B-protein +fusion O +with O +an O +in O +vivo O +IC50 B-evidence +of O +42 O +. O +5nM O +while O +showing O +> O +235 O +and O +28 O +fold O +selectivity O +over O +cells O +expressing O +GAL4 B-protein +fused O +to O +the O +LBD B-structure_element +of O +ROR B-protein_type +α B-protein +or O +β B-protein +, O +respectively O +( O +Table O +1 O +). O + +The O +LBS B-site +of O +RORs B-protein_type +share O +a O +high O +degree O +of O +similarity O +. O + +However O +, O +the O +inverse O +agonism O +trigger O +of O +BIO399 B-chemical +, O +residue O +Met358 B-residue_name_number +, O +is O +a O +leucine B-residue_name +in O +both O +RORα B-protein +and O +β B-protein +. O + +This O +selectivity O +profile O +for O +BIO399 B-chemical +is O +attributed O +to O +the O +shorter O +leucine B-residue_name +side O +chain O +in O +RORα B-protein +and O +β B-protein +which O +would O +not O +reach O +the O +phenylalanine B-residue_name +on O +the O +AF2 B-structure_element +helix I-structure_element +further O +underscoring O +the O +role O +of O +Met358 B-residue_name_number +as O +a O +trigger O +for O +RORγ B-protein +specific O +inverse O +agonism O +( O +Fig O +. O +7a O +). O + +Furthermore O +, O +RORα B-protein +contains O +two O +phenylalanine B-residue_name +residues O +in O +its O +LBS B-site +whereas O +RORβ B-protein +and O +γ B-protein +have O +a O +leucine B-residue_name +in O +the O +same O +position O +( O +Fig O +. O +6b O +). O + +We O +hypothesize O +that O +the O +two O +phenylalanine B-residue_name +residues O +in O +the O +LBS B-site +of O +RORα B-protein +occlude O +the O +dihydrobenzoxazepinone B-chemical +ring O +system O +of O +BIO399 B-chemical +from O +binding O +it O +and O +responsible O +for O +the O +increase O +in O +selectivity O +for O +RORα B-protein +over O +β B-protein +. O + +We O +have O +identified O +a O +novel O +series O +of O +synthetic O +benzoxazinone B-chemical +ligands O +which O +modulate O +the O +transcriptional O +activity O +of O +RORγ B-protein +in O +a O +FRET B-experimental_method +based I-experimental_method +assay I-experimental_method +. O + +Using O +partial B-experimental_method +proteolysis I-experimental_method +we O +show O +a O +conformational O +change O +which O +destabilizes O +the O +AF2 B-structure_element +helix I-structure_element +of O +RORγ B-protein +when O +the O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +binds O +. O + +The O +two O +RORγ B-protein +co B-evidence +- I-evidence +crystal I-evidence +structures I-evidence +reported O +here O +show O +how O +a O +small O +change O +to O +the O +core O +ring O +system O +can O +modulate O +the O +mode O +of O +action O +from O +agonist B-protein_state +( O +BIO592 B-chemical +) O +to O +inverse O +agonism O +( O +BIO399 B-chemical +). O + +Finally O +, O +we O +are O +reporting O +a O +newly O +identified O +trigger O +for O +achieving O +RORγ B-protein +specific O +inverse O +agonism O +in O +an O +in O +vivo O +setting O +through O +Met358 B-residue_name_number +which O +perturbs O +the O +agonist B-protein_state +conformation O +of O +the O +AF2 B-structure_element +helix I-structure_element +and O +prevents O +coactivator O +protein O +binding O +. O + +The O +Structural O +Basis O +of O +Coenzyme B-chemical +A I-chemical +Recycling O +in O +a O +Bacterial B-taxonomy_domain +Organelle O + +Bacterial B-taxonomy_domain +Microcompartments B-complex_assembly +( O +BMCs B-complex_assembly +) O +are O +proteinaceous O +organelles O +that O +encapsulate O +critical O +segments O +of O +autotrophic O +and O +heterotrophic O +metabolic O +pathways O +; O +they O +are O +functionally O +diverse O +and O +are O +found O +across O +23 O +different O +phyla O +. O + +The O +majority O +of O +catabolic B-protein_state +BMCs B-complex_assembly +( O +metabolosomes B-complex_assembly +) O +compartmentalize O +a O +common O +core O +of O +enzymes O +to O +metabolize O +compounds O +via O +a O +toxic O +and O +/ O +or O +volatile O +aldehyde B-chemical +intermediate O +. O + +The O +core O +enzyme O +phosphotransacylase B-protein_type +( O +PTAC B-protein_type +) O +recycles O +Coenzyme B-chemical +A I-chemical +and O +generates O +an O +acyl B-chemical +phosphate I-chemical +that O +can O +serve O +as O +an O +energy O +source O +. O + +The O +PTAC B-protein_type +predominantly O +associated O +with O +metabolosomes B-complex_assembly +( O +PduL B-protein_type +) O +has O +no O +sequence O +homology O +to O +the O +PTAC B-protein_type +ubiquitous O +among O +fermentative B-taxonomy_domain +bacteria I-taxonomy_domain +( O +Pta B-protein_type +). O + +Here O +, O +we O +report O +two O +high O +- O +resolution O +PduL B-protein_type +crystal B-evidence +structures I-evidence +with B-protein_state +bound I-protein_state +substrates I-protein_state +. O + +The O +PduL B-protein_type +fold B-structure_element +is O +unrelated O +to O +that B-structure_element +of O +Pta B-protein_type +; O +it O +contains O +a O +dimetal B-site +active I-site +site I-site +involved O +in O +a O +catalytic O +mechanism O +distinct O +from O +that O +of O +the O +housekeeping B-protein_state +PTAC B-protein_type +. O + +Accordingly O +, O +PduL B-protein_type +and O +Pta B-protein_type +exemplify O +functional O +, O +but O +not O +structural O +, O +convergent O +evolution O +. O + +The O +PduL B-protein_type +structure B-evidence +, O +in O +the O +context O +of O +the O +catalytic O +core O +, O +completes O +our O +understanding O +of O +the O +structural O +basis O +of O +cofactor O +recycling O +in O +the O +metabolosome B-complex_assembly +lumen O +. O + +This O +study O +describes O +the O +structure B-evidence +of O +a O +novel O +phosphotransacylase B-protein_type +enzyme O +that O +facilitates O +the O +recycling O +of O +the O +essential O +cofactor O +acetyl B-chemical +- I-chemical +CoA I-chemical +within O +a O +bacterial B-taxonomy_domain +organelle O +and O +discusses O +the O +properties O +of O +the O +enzyme O +' O +s O +active B-site +site I-site +and O +how O +it O +is O +packaged O +into O +the O +organelle O +. O + +In O +metabolism O +, O +molecules O +with O +“ O +high O +- O +energy O +” O +bonds O +( O +e O +. O +g O +., O +ATP B-chemical +and O +Acetyl B-chemical +~ I-chemical +CoA I-chemical +) O +are O +critical O +for O +both O +catabolic O +and O +anabolic O +processes O +. O + +The O +phosphotransacylase B-protein_type +( O +Pta B-protein_type +) O +enzyme O +catalyzes O +the O +conversion O +between O +acyl B-chemical +- I-chemical +CoA I-chemical +and O +acyl B-chemical +- I-chemical +phosphate I-chemical +. O + +This O +reaction O +directly O +links O +an O +acyl B-chemical +- I-chemical +CoA I-chemical +with O +ATP B-chemical +generation O +via O +substrate O +- O +level O +phosphorylation O +, O +producing O +short B-chemical +- I-chemical +chain I-chemical +fatty I-chemical +acids I-chemical +( O +e O +. O +g O +., O +acetate B-chemical +), O +and O +also O +provides O +a O +path O +for O +short B-chemical +- I-chemical +chain I-chemical +fatty I-chemical +acids I-chemical +to O +enter O +central O +metabolism O +. O + +Due O +to O +this O +key O +function O +, O +Pta O +is O +conserved B-protein_state +across O +the O +bacterial B-taxonomy_domain +kingdom I-taxonomy_domain +. O + +Recently O +, O +a O +new O +type O +of O +phosphotransacylase B-protein_type +was O +described O +that O +shares O +no O +evolutionary O +relation O +to O +Pta B-protein_type +. O + +This O +enzyme O +, O +PduL B-protein_type +, O +is O +exclusively B-protein_state +associated O +with O +organelles O +called O +bacterial B-taxonomy_domain +microcompartments B-complex_assembly +, O +which O +are O +used O +to O +catabolize O +various O +compounds O +. O + +Not O +only O +does O +PduL B-protein_type +facilitate O +substrate O +level O +phosphorylation O +, O +but O +it O +also O +is O +critical O +for O +cofactor O +recycling O +within O +, O +and O +product O +efflux O +from O +, O +the O +organelle O +. O + +We O +solved B-experimental_method +the O +structure B-evidence +of O +this O +convergent B-protein_state +phosphotransacylase B-protein_type +and O +show O +that O +it O +is O +completely O +structurally O +different O +from O +Pta B-protein_type +, O +including O +its O +active B-site +site I-site +architecture O +. O + +Bacterial B-taxonomy_domain +Microcompartments B-complex_assembly +( O +BMCs B-complex_assembly +) O +are O +organelles O +that O +encapsulate O +enzymes O +for O +sequential O +biochemical O +reactions O +within O +a O +protein O +shell B-structure_element +. O + +The O +shell B-structure_element +is O +typically O +composed O +of O +three O +types O +of O +protein O +subunits O +, O +which O +form O +either O +hexagonal B-protein_state +( O +BMC B-complex_assembly +- I-complex_assembly +H I-complex_assembly +and O +BMC B-complex_assembly +- I-complex_assembly +T I-complex_assembly +) O +or O +pentagonal B-protein_state +( O +BMC B-complex_assembly +- I-complex_assembly +P I-complex_assembly +) O +tiles O +that O +assemble O +into O +a O +polyhedral B-protein_state +shell B-structure_element +. O + +The O +facets O +of O +the O +shell B-structure_element +are O +composed O +primarily O +of O +hexamers B-oligomeric_state +that O +are O +typically O +perforated O +by O +pores B-site +lined O +with O +highly B-protein_state +conserved I-protein_state +, O +polar B-protein_state +residues B-structure_element +that O +presumably O +function O +as O +the O +conduits O +for O +metabolites O +into O +and O +out O +of O +the O +shell B-structure_element +. O + +The O +vitamin B-complex_assembly +B12 I-complex_assembly +- I-complex_assembly +dependent I-complex_assembly +propanediol I-complex_assembly +- I-complex_assembly +utilizing I-complex_assembly +( I-complex_assembly +PDU I-complex_assembly +) I-complex_assembly +BMC I-complex_assembly +was O +one O +of O +the O +first O +functionally O +characterized O +catabolic B-protein_state +BMCs B-complex_assembly +; O +subsequently O +, O +other O +types O +have O +been O +implicated O +in O +the O +degradation O +of O +ethanolamine B-chemical +, O +choline B-chemical +, O +fucose B-chemical +, O +rhamnose B-chemical +, O +and O +ethanol B-chemical +, O +all O +of O +which O +produce O +different O +aldehyde B-chemical +intermediates O +( O +Table O +1 O +). O + +More O +recently O +, O +bioinformatic B-experimental_method +studies I-experimental_method +have O +demonstrated O +the O +widespread O +distribution O +of O +BMCs B-complex_assembly +among O +diverse O +bacterial B-taxonomy_domain +phyla I-taxonomy_domain +and O +grouped O +them O +into O +23 O +different O +functional O +types O +. O + +The O +reactions O +carried O +out O +in O +the O +majority O +of O +catabolic B-protein_state +BMCs B-complex_assembly +( O +also O +known O +as O +metabolosomes B-complex_assembly +) O +fit O +a O +generalized O +biochemical O +paradigm O +for O +the O +oxidation O +of O +aldehydes B-chemical +( O +Fig O +1 O +). O + +This O +involves O +a O +BMC B-complex_assembly +- O +encapsulated O +signature O +enzyme O +that O +generates O +a O +toxic O +and O +/ O +or O +volatile O +aldehyde B-chemical +that O +the O +BMC B-complex_assembly +shell B-structure_element +sequesters O +from O +the O +cytosol O +. O + +The O +aldehyde B-chemical +is O +subsequently O +converted O +into O +an O +acyl B-chemical +- I-chemical +CoA I-chemical +by O +aldehyde B-protein_type +dehydrogenase I-protein_type +, O +which O +uses O +NAD B-chemical ++ I-chemical +and O +CoA B-chemical +as O +cofactors O +. O + +These O +two O +cofactors O +are O +relatively O +large O +, O +and O +their O +diffusion O +across O +the O +protein B-structure_element +shell I-structure_element +is O +thought O +to O +be O +restricted O +, O +necessitating O +their O +regeneration O +within O +the O +BMC B-complex_assembly +lumen O +. O + +NAD B-chemical ++ I-chemical +is O +recycled O +via O +alcohol B-protein_type +dehydrogenase I-protein_type +, O +and O +CoA B-chemical +is O +recycled O +via O +phosphotransacetylase B-protein_type +( O +PTAC B-protein_type +) O +( O +Fig O +1 O +). O + +The O +final O +product O +of O +the O +BMC B-complex_assembly +, O +an O +acyl B-chemical +- I-chemical +phosphate I-chemical +, O +can O +then O +be O +used O +to O +generate O +ATP B-chemical +via O +acyl B-protein_type +kinase I-protein_type +, O +or O +revert O +back O +to O +acyl B-chemical +- I-chemical +CoA I-chemical +by O +Pta B-protein_type +for O +biosynthesis O +. O + +Collectively O +, O +the O +aldehyde B-protein_type +and I-protein_type +alcohol I-protein_type +dehydrogenases I-protein_type +, O +as O +well O +as O +the O +PTAC B-protein_type +, O +constitute O +the O +common O +metabolosome B-complex_assembly +core O +. O + +General O +biochemical O +model O +of O +aldehyde B-protein_state +- I-protein_state +degrading I-protein_state +BMCs B-complex_assembly +( O +metabolosomes B-complex_assembly +) O +illustrating O +the O +common O +metabolosome B-complex_assembly +core O +enzymes O +and O +reactions O +. O + +Substrates O +and O +cofactors O +involving O +the O +PTAC B-protein_type +reaction O +are O +shown O +in O +red O +; O +other O +substrates O +and O +enzymes O +are O +shown O +in O +black O +, O +and O +other O +cofactors O +are O +shown O +in O +gray O +. O + +Characterized O +and O +predicted O +catabolic B-protein_state +BMC B-complex_assembly +( O +metabolosome B-complex_assembly +) O +types O +that O +represent O +the O +aldehyde B-chemical +- O +degrading O +paradigm O +( O +for O +definition O +of O +types O +see O +Kerfeld O +and O +Erbilgin O +). O + +Name O +PTAC B-protein_type +Type O +Sequestered O +Aldehyde B-chemical +PDU B-complex_assembly +* O +PduL B-protein_type +propionaldehyde B-chemical +EUT1 B-complex_assembly +PTA_PTB B-protein_type +acetaldehyde B-chemical +EUT2 B-complex_assembly +PduL B-protein_type +acetaldehyde B-chemical +ETU B-complex_assembly +None O +acetaldehyde B-chemical +GRM1 B-complex_assembly +/ I-complex_assembly +CUT I-complex_assembly +PduL B-protein_type +acetaldehyde B-chemical +GRM2 B-complex_assembly +PduL B-protein_type +acetaldehyde B-chemical +GRM3 B-complex_assembly +*, I-complex_assembly +4 I-complex_assembly +PduL B-protein_type +propionaldehyde B-chemical +GRM5 B-complex_assembly +/ I-complex_assembly +GRP I-complex_assembly +PduL B-protein_type +propionaldehyde B-chemical +PVM B-complex_assembly +* O +PduL B-protein_type +lactaldehyde B-chemical +RMM1 B-complex_assembly +, I-complex_assembly +2 I-complex_assembly +None O +unknown O +SPU B-complex_assembly +PduL B-protein_type +unknown O + +* O +PduL B-protein_type +from O +these O +functional O +types O +of O +metabolosomes B-complex_assembly +were O +purified O +in O +this O +study O +. O + +The O +activities O +of O +core O +enzymes O +are O +not O +confined O +to O +BMC B-complex_assembly +- O +associated O +functions O +: O +aldehyde B-protein_type +and I-protein_type +alcohol I-protein_type +dehydrogenases I-protein_type +are O +utilized O +in O +diverse O +metabolic O +reactions O +, O +and O +PTAC B-protein_type +catalyzes O +a O +key O +biochemical O +reaction O +in O +the O +process O +of O +obtaining O +energy O +during O +fermentation O +. O + +The O +concerted O +functioning O +of O +a O +PTAC B-protein_type +and O +an O +acetate B-protein_type +kinase I-protein_type +( O +Ack B-protein_type +) O +is O +crucial O +for O +ATP B-chemical +generation O +in O +the O +fermentation O +of O +pyruvate B-chemical +to O +acetate B-chemical +( O +see O +Reactions O +1 O +and O +2 O +). O + +Both O +enzymes O +are O +, O +however O +, O +not O +restricted O +to O +fermentative B-taxonomy_domain +organisms I-taxonomy_domain +. O + +They O +can O +also O +work O +in O +the O +reverse O +direction O +to O +activate O +acetate B-chemical +to O +the O +CoA B-chemical +- I-chemical +thioester I-chemical +. O + +This O +occurs O +, O +for O +example O +, O +during O +acetoclastic O +methanogenesis O +in O +the O +archaeal B-taxonomy_domain +Methanosarcina B-taxonomy_domain +species I-taxonomy_domain +. O + +Reaction O +1 O +: O +acetyl B-chemical +- I-chemical +S I-chemical +- I-chemical +CoA I-chemical ++ O +Pi B-chemical +←→ O +acetyl B-chemical +phosphate I-chemical ++ O +CoA B-chemical +- I-chemical +SH I-chemical +( O +PTAC B-protein_type +) O + +Reaction O +2 O +: O +acetyl B-chemical +phosphate I-chemical ++ O +ADP B-chemical +←→ O +acetate B-chemical ++ O +ATP B-chemical +( O +Ack B-protein_type +) O + +The O +canonical O +PTAC B-protein_type +, O +Pta B-protein_type +, O +is O +an O +ancient O +enzyme O +found O +in O +some O +eukaryotes B-taxonomy_domain +and O +archaea B-taxonomy_domain +, O +and O +widespread O +among O +the O +bacteria B-taxonomy_domain +; O +90 O +% O +of O +the O +bacterial B-taxonomy_domain +genomes O +in O +the O +Integrated O +Microbial O +Genomes O +database O +contain O +a O +gene O +encoding O +the O +PTA_PTB B-protein_type +phosphotransacylase I-protein_type +( O +Pfam O +domain O +PF01515 B-structure_element +). O + +Pta B-protein_type +has O +been O +extensively O +characterized O +due O +to O +its O +key O +role O +in O +fermentation O +. O + +More O +recently O +, O +a O +second O +type O +of O +PTAC B-protein_type +without O +any O +sequence O +homology O +to O +Pta B-protein_type +was O +identified O +. O + +This O +protein O +, O +PduL B-protein_type +( O +Pfam O +domain O +PF06130 B-structure_element +), O +was O +shown O +to O +catalyze O +the O +conversion O +of O +propionyl B-chemical +- I-chemical +CoA I-chemical +to O +propionyl B-chemical +- I-chemical +phosphate I-chemical +and O +is O +associated O +with O +a O +BMC B-complex_assembly +involved O +in O +propanediol O +utilization O +, O +the O +PDU B-complex_assembly +BMC I-complex_assembly +. O + +Both O +pduL B-gene +and O +pta B-gene +genes O +can O +be O +found O +in O +genetic O +loci O +of O +functionally O +distinct O +BMCs B-complex_assembly +, O +although O +the O +PduL B-protein_type +type O +is O +much O +more O +prevalent O +, O +being O +found O +in O +all O +but O +one O +type O +of O +metabolosome B-gene +locus I-gene +: O +EUT1 B-gene +( O +Table O +1 O +). O + +Furthermore O +, O +in O +the O +Integrated O +Microbial O +Genomes O +Database O +, O +91 O +% O +of O +genomes O +that O +encode O +PF06130 B-structure_element +also O +encode O +genes O +for O +shell O +proteins O +. O + +As O +a O +member O +of O +the O +core O +biochemical O +machinery O +of O +functionally O +diverse O +aldehyde B-protein_state +- I-protein_state +oxidizing I-protein_state +metabolosomes B-complex_assembly +, O +PduL B-protein_type +must O +have O +a O +certain O +level O +of O +substrate O +plasticity O +( O +see O +Table O +1 O +) O +that O +is O +not O +required O +of O +Pta B-protein_type +, O +which O +has O +generally O +been O +observed O +to O +prefer O +acetyl B-chemical +- I-chemical +CoA I-chemical +. O +PduL B-protein_type +from O +the O +PDU B-complex_assembly +BMC I-complex_assembly +of O +Salmonella B-species +enterica I-species +favors O +propionyl B-chemical +- I-chemical +CoA I-chemical +over O +acetyl B-chemical +- I-chemical +CoA I-chemical +, O +and O +it O +is O +likely O +that O +PduL B-protein_type +orthologs O +in O +functionally O +diverse O +BMCs B-complex_assembly +would O +have O +substrate O +preferences O +for O +other O +CoA B-chemical +derivatives O +. O + +Another O +distinctive O +feature O +of O +BMC B-protein_state +- I-protein_state +associated I-protein_state +PduL B-protein_type +homologs O +is O +an O +N O +- O +terminal O +encapsulation B-structure_element +peptide I-structure_element +( O +EP B-structure_element +) O +that O +is O +thought O +to O +“ O +target O +” O +proteins O +for O +encapsulation O +by O +the O +BMC B-complex_assembly +shell B-structure_element +. O + +EPs B-structure_element +are O +frequently O +found O +on O +BMC B-protein_type +- I-protein_type +associated I-protein_type +proteins I-protein_type +and O +have O +been O +shown O +to O +interact O +with O +shell O +proteins O +. O + +EPs B-structure_element +have O +also O +been O +observed O +to O +cause O +proteins O +to O +aggregate O +, O +and O +this O +has O +recently O +been O +suggested O +to O +be O +functionally O +relevant O +as O +an O +initial O +step O +in O +metabolosome B-complex_assembly +assembly O +, O +in O +which O +a O +multifunctional O +protein O +core O +is O +formed O +, O +around O +which O +the O +shell B-structure_element +assembles O +. O + +Of O +the O +three O +common O +metabolosome B-complex_assembly +core O +enzymes O +, O +crystal B-evidence +structures I-evidence +are O +available O +for O +both O +the O +alcohol B-protein_type +and I-protein_type +aldehyde I-protein_type +dehydrogenases I-protein_type +. O + +In O +contrast O +, O +the O +structure B-evidence +of O +PduL B-protein_type +, O +the O +PTAC B-protein_type +found O +in O +the O +vast O +majority O +of O +catabolic B-protein_state +BMCs B-complex_assembly +, O +has O +not O +been O +determined O +. O + +This O +is O +a O +major O +gap O +in O +our O +understanding O +of O +metabolosome B-complex_assembly +- O +encapsulated O +biochemistry O +and O +cofactor O +recycling O +. O + +Moreover O +, O +it O +will O +be O +useful O +for O +guiding O +efforts O +to O +engineer O +novel O +BMC B-complex_assembly +cores O +for O +biotechnological O +applications O +. O + +The O +primary O +structure O +of O +PduL B-protein_type +homologs O +is O +subdivided O +into O +two O +PF06130 B-structure_element +domains O +, O +each O +roughly O +80 B-residue_range +residues I-residue_range +in I-residue_range +length I-residue_range +. O + +No O +available O +protein O +structures O +contain O +the O +PF06130 B-structure_element +domain O +, O +and O +homology B-experimental_method +searches I-experimental_method +using O +the O +primary O +structure O +of O +PduL B-protein_type +do O +not O +return O +any O +significant O +results O +that O +would O +allow O +prediction O +of O +the O +structure B-evidence +. O + +Moreover O +, O +the O +evident O +novelty O +of O +PduL B-protein_type +makes O +its O +structure B-evidence +interesting O +in O +the O +context O +of O +convergent O +evolution O +of O +PTAC B-protein_type +function O +; O +to O +- O +date O +, O +only O +the O +Pta B-protein_type +active B-site +site I-site +and O +catalytic O +mechanism O +is O +known O +. O + +Here O +we O +report O +high O +- O +resolution O +crystal B-evidence +structures I-evidence +of O +a O +PduL B-protein_type +- I-protein_type +type I-protein_type +PTAC I-protein_type +in O +both O +CoA B-protein_state +- I-protein_state +and O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +forms O +, O +completing O +our O +understanding O +of O +the O +structural O +basis O +of O +catalysis O +by O +the O +metabolosome B-complex_assembly +common O +core O +enzymes O +. O + +We O +propose O +a O +catalytic O +mechanism O +analogous O +but O +yet O +distinct O +from O +the O +ubiquitous O +Pta B-protein_type +enzyme O +, O +highlighting O +the O +functional O +convergence O +of O +two O +enzymes O +with O +completely O +different O +structures O +and O +metal O +requirements O +. O + +We O +also O +investigate O +the O +quaternary O +structures O +of O +three O +different O +PduL B-protein_type +homologs O +and O +situate O +our O +findings O +in O +the O +context O +of O +organelle O +biogenesis O +in O +functionally O +diverse O +BMCs B-complex_assembly +. O + +Structure B-experimental_method +Determination I-experimental_method +of O +PduL B-protein_type + +We O +cloned B-experimental_method +, I-experimental_method +expressed I-experimental_method +, I-experimental_method +and I-experimental_method +purified I-experimental_method +three O +different O +PduL B-protein_type +homologs O +from O +functionally O +distinct O +BMCs B-complex_assembly +( O +Table O +1 O +): O +from O +the O +well O +- O +studied O +pdu B-gene +locus I-gene +in O +S B-species +. I-species +enterica I-species +Typhimurium I-species +LT2 I-species +( O +sPduL B-protein +), O +from O +the O +recently O +characterized O +pvm B-gene +locus I-gene +in O +Planctomyces B-species +limnophilus I-species +( O +pPduL B-protein +), O +and O +from O +the O +grm3 B-gene +locus I-gene +in O +Rhodopseudomonas B-species +palustris I-species +BisB18 I-species +( O +rPduL B-protein +). O + +While O +purifying O +full B-protein_state +- I-protein_state +length I-protein_state +sPduL B-protein +, O +we O +observed O +a O +tendency O +to O +aggregation O +as O +described O +previously O +, O +with O +a O +large O +fraction O +of O +the O +expressed O +protein O +found O +in O +the O +insoluble O +fraction O +in O +a O +white O +, O +cake O +- O +like O +pellet O +. O + +Remarkably O +, O +after O +removing B-experimental_method +the O +N O +- O +terminal O +putative O +EP B-structure_element +( O +27 B-residue_range +amino I-residue_range +acids I-residue_range +), O +most O +of O +the O +sPduLΔEP B-mutant +protein O +was O +in O +the O +soluble O +fraction O +upon O +cell O +lysis O +. O + +Similar O +differences O +in O +solubility O +were O +observed O +for O +pPduL B-protein +and O +rPduL B-protein +when O +comparing O +EP B-protein_state +- I-protein_state +truncated I-protein_state +forms O +to O +the O +full B-protein_state +- I-protein_state +length I-protein_state +protein O +, O +but O +none O +were O +quite O +as O +dramatic O +as O +for O +sPduL B-protein +. O +We O +confirmed O +that O +all O +homologs O +were O +active B-protein_state +( O +S1a O +and O +S1b O +Fig O +). O + +Among O +these O +, O +we O +were O +only O +able O +to O +obtain O +diffraction B-evidence +- I-evidence +quality I-evidence +crystals I-evidence +of O +rPduL B-protein +after O +removing B-experimental_method +the O +N O +- O +terminal O +putative O +EP B-structure_element +( O +33 B-residue_range +amino I-residue_range +acids I-residue_range +, O +also O +see O +Fig O +2a O +) O +( O +rPduLΔEP B-mutant +). O + +Truncated B-protein_state +rPduLΔEP B-mutant +had O +comparable O +enzymatic O +activity O +to O +the O +full B-protein_state +- I-protein_state +length I-protein_state +enzyme O +( O +S1a O +Fig O +). O + +Structural O +overview O +of O +R B-species +. I-species +palustris I-species +PduL B-protein_type +from O +the O +grm3 B-gene +locus I-gene +. O + +( O +a O +) O +Primary O +and O +secondary O +structure O +of O +rPduL B-protein +( O +tubes O +represent O +α B-structure_element +- I-structure_element +helices I-structure_element +, O +arrows O +β B-structure_element +- I-structure_element +sheets I-structure_element +and O +dashed O +line O +residues O +disordered O +in O +the O +structure B-evidence +. O + +The O +first B-residue_range +33 I-residue_range +amino I-residue_range +acids I-residue_range +are O +present O +only O +in O +the O +wildtype O +construct O +and O +contains O +the O +predicted O +EP B-structure_element +alpha B-structure_element +helix I-structure_element +, O +α0 B-structure_element +); O +the O +truncated B-protein_state +rPduLΔEP B-mutant +that O +was O +crystallized B-experimental_method +begins O +with O +M B-residue_name +- O +G B-residue_name +- O +V B-residue_name +. O +Coloring O +is O +according O +to O +structural O +domains O +( O +domain B-structure_element +1 I-structure_element +D36 B-residue_range +- I-residue_range +N46 I-residue_range +/ O +Q155 B-residue_range +- I-residue_range +C224 I-residue_range +, O +blue O +; O +loop B-structure_element +insertion I-structure_element +G61 B-residue_range +- I-residue_range +E81 I-residue_range +, O +grey O +; O +domain B-structure_element +2 I-structure_element +R47 B-residue_range +- I-residue_range +F60 I-residue_range +/ O +E82 B-residue_range +- I-residue_range +A154 I-residue_range +, O +red O +). O + +Metal B-site +coordination I-site +residues I-site +are O +highlighted O +in O +light O +blue O +and O +CoA B-site +contacting I-site +residues I-site +in O +magenta O +, O +residues O +contacting O +the O +CoA B-chemical +of O +the O +other O +chain O +are O +also O +outlined O +. O + +( O +b O +) O +Cartoon O +representation O +of O +the O +structure B-evidence +colored O +by O +domains O +and O +including O +secondary O +structure B-evidence +numbering O +. O + +Coenzyme B-chemical +A I-chemical +is O +shown O +in O +magenta O +sticks O +and O +Zinc B-chemical +( O +grey O +) O +as O +spheres O +. O + +We O +collected B-experimental_method +a I-experimental_method +native I-experimental_method +dataset I-experimental_method +from O +rPduLΔEP B-mutant +crystals B-evidence +diffracting O +to O +a O +resolution O +of O +1 O +. O +54 O +Å O +( O +Table O +2 O +). O + +Using O +a O +mercury B-experimental_method +- I-experimental_method +derivative I-experimental_method +crystal I-experimental_method +form O +diffracting O +to O +1 O +. O +99 O +Å O +( O +Table O +2 O +), O +we O +obtained O +high O +quality O +electron B-evidence +density I-evidence +for O +model O +building O +and O +used O +the O +initial O +model O +to O +refine O +against O +the O +native O +data O +to O +Rwork B-evidence +/ O +Rfree B-evidence +values O +of O +18 O +. O +9 O +/ O +22 O +. O +1 O +%. O + +There O +are O +two O +PduL B-protein_type +molecules O +in O +the O +asymmetric O +unit O +of O +the O +P212121 O +unit O +cell O +. O + +We O +were O +able O +to O +fit O +all O +of O +the O +primary O +structure O +of O +PduLΔEP B-mutant +into O +the O +electron B-evidence +density I-evidence +with O +the O +exception O +of O +three O +amino O +acids O +at O +the O +N O +- O +terminus O +and O +two O +amino O +acids O +at O +the O +C O +- O +terminus O +( O +Fig O +2a O +); O +the O +model O +is O +of O +excellent O +quality O +( O +Table O +2 O +). O + +A O +CoA B-chemical +cofactor O +as O +well O +as O +two O +metal O +ions O +are O +clearly O +resolved O +in O +the O +density B-evidence +( O +for O +omit B-evidence +maps I-evidence +of O +CoA B-chemical +see O +S2 O +Fig O +). O + +Structurally O +, O +PduL B-protein_type +consists O +of O +two O +domains B-structure_element +( O +Fig O +2 O +, O +blue O +/ O +red O +), O +each O +a O +beta B-structure_element +- I-structure_element +barrel I-structure_element +that O +is O +capped O +on O +both O +ends O +by O +short O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +β B-structure_element +- I-structure_element +Barrel I-structure_element +1 I-structure_element +consists O +of O +the O +N O +- O +terminal O +β B-structure_element +strand I-structure_element +and O +β B-structure_element +strands I-structure_element +from O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +the O +polypeptide O +chain O +( O +β1 B-structure_element +, O +β10 B-structure_element +- I-structure_element +β14 I-structure_element +; O +residues O +37 B-residue_range +– I-residue_range +46 I-residue_range +and O +155 B-residue_range +– I-residue_range +224 I-residue_range +). O + +β B-structure_element +- I-structure_element +Barrel I-structure_element +2 I-structure_element +consists O +mainly O +of O +the O +central O +segment O +of O +primary O +structure O +( O +β2 B-structure_element +, O +β5 B-structure_element +– I-structure_element +β9 I-structure_element +; O +residues O +47 B-residue_range +– I-residue_range +60 I-residue_range +and O +82 B-residue_range +– I-residue_range +154 I-residue_range +) O +( O +Fig O +2 O +, O +red O +), O +but O +is O +interrupted O +by O +a O +short B-structure_element +two I-structure_element +- I-structure_element +strand I-structure_element +beta I-structure_element +sheet I-structure_element +( O +β3 B-structure_element +- I-structure_element +β4 I-structure_element +, O +residues O +61 B-residue_range +– I-residue_range +81 I-residue_range +). O + +This O +β B-structure_element +- I-structure_element +sheet I-structure_element +is O +involved O +in O +contacts O +between O +the O +two O +domains O +and O +forms O +a O +lid O +over O +the O +active B-site +site I-site +. O + +Residues O +in O +this O +region O +( O +Gln42 B-residue_name_number +, O +Pro43 B-residue_name_number +, O +Gly44 B-residue_name_number +), O +covering O +the O +active B-site +site I-site +, O +are O +strongly B-protein_state +conserved I-protein_state +( O +Fig O +3 O +). O + +This O +structural O +arrangement O +is O +completely O +different O +from O +the O +functionally O +related O +Pta B-protein_type +, O +which O +is O +composed O +of O +two O +domains B-structure_element +, O +each O +consisting O +of O +a O +central O +flat O +beta B-structure_element +sheet I-structure_element +with O +alpha B-structure_element +- I-structure_element +helices I-structure_element +on O +the O +top O +and O +bottom O +. O + +Primary O +structure O +conservation O +of O +the O +PduL B-protein_type +protein O +family O +. O + +Sequence O +logo O +calculated O +from O +the O +multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +PduL B-protein_type +homologs O +( O +see O +Materials O +and O +Methods O +), O +but O +not B-protein_state +including I-protein_state +putative O +EP B-structure_element +sequences O +. O + +Residues O +100 O +% O +conserved O +across O +all O +PduL B-protein_type +homologs O +in O +our O +dataset O +are O +noted O +with O +an O +asterisk O +, O +and O +residues O +conserved O +in O +over O +90 O +% O +of O +sequences O +are O +noted O +with O +a O +colon O +. O + +The O +sequences O +aligning O +to O +the O +PF06130 B-structure_element +domain O +( O +determined O +by O +BLAST O +) O +are O +highlighted O +in O +red O +and O +blue O +. O + +The O +position O +numbers O +shown O +correspond O +to O +the O +residue O +numbering O +of O +rPduL B-protein +; O +note O +that O +some O +positions O +in O +the O +logo O +represent O +gaps O +in O +the O +rPduL B-protein +sequence O +. O + +There O +are O +two O +PduL B-protein_type +molecules O +in O +the O +asymmetric O +unit O +forming O +a O +butterfly B-protein_state +- I-protein_state +shaped I-protein_state +dimer B-oligomeric_state +( O +Fig O +4c O +). O + +Consistent O +with O +this O +, O +results O +from O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +of O +rPduLΔEP B-mutant +suggest O +that O +it O +is O +a O +dimer B-oligomeric_state +in O +solution O +( O +Fig O +5e O +). O + +The O +interface B-site +between O +the O +two O +chains O +buries O +882 O +Å2 O +per O +monomer B-oligomeric_state +and O +is O +mainly O +formed O +by O +α B-structure_element +- I-structure_element +helices I-structure_element +2 I-structure_element +and I-structure_element +4 I-structure_element +and O +parts O +of O +β B-structure_element +- I-structure_element +sheets I-structure_element +12 I-structure_element +and I-structure_element +14 I-structure_element +, O +as O +well O +as O +a O +π O +– O +π O +stacking O +of O +the O +adenine B-chemical +moiety O +of O +CoA B-chemical +with O +Phe116 B-residue_name_number +of O +the O +adjacent O +chain O +( O +Fig O +4c O +). O + +The O +folds O +of O +the O +two O +chains O +in O +the O +asymmetric O +unit O +are O +very O +similar O +, O +superimposing B-experimental_method +with O +a O +rmsd B-evidence +of O +0 O +. O +16 O +Å O +over O +2 O +, O +306 O +aligned O +atom O +pairs O +. O + +The O +peripheral O +helices B-structure_element +and O +the O +short B-structure_element +antiparallel I-structure_element +β3 I-structure_element +– I-structure_element +4 I-structure_element +sheet I-structure_element +mediate O +most O +of O +the O +crystal O +contacts O +. O + +Details O +of O +active B-site +site I-site +, O +dimeric B-oligomeric_state +assembly O +, O +and O +sequence O +conservation O +of O +PduL B-protein_type +. O + +( O +a O +, O +b O +) O +Proposed O +active B-site +site I-site +of O +PduL B-protein_type +with O +relevant O +residues O +shown O +as O +sticks O +in O +atom O +coloring O +( O +nitrogen B-chemical +blue O +, O +oxygen B-chemical +red O +, O +sulfur B-chemical +yellow O +), O +zinc B-chemical +as O +grey O +colored O +spheres O +and O +coordinating O +ordered O +water B-chemical +molecules O +in O +red O +. O + +Distances O +between O +atom O +centers O +are O +indicated O +in O +Å O +. O +( O +a O +) O +Coenzyme B-chemical +A I-chemical +containing O +, O +( O +b O +) O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +structure B-evidence +. O + +( O +c O +) O +View O +of O +the O +dimer B-oligomeric_state +in O +the O +asymmetric O +unit O +from O +the O +side O +, O +domains B-structure_element +1 I-structure_element +and I-structure_element +2 I-structure_element +colored O +as O +in O +Fig O +2 O +and O +the O +two O +chains O +differentiated O +by O +blue O +/ O +red O +versus O +slate O +/ O +firebrick O +. O + +The O +asterisk O +and O +double O +arrow O +marks O +the O +location O +of O +the O +π B-bond_interaction +– I-bond_interaction +π I-bond_interaction +interaction I-bond_interaction +between O +F116 B-residue_name_number +and O +the O +CoA B-chemical +base O +of O +the O +other O +dimer B-oligomeric_state +chain O +. O + +( O +d O +) O +Surface O +representation O +of O +the O +structure B-evidence +with O +indicated O +conservation O +( O +red O +: O +high O +, O +white O +: O +intermediate O +, O +yellow O +: O +low O +). O + +Size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +of O +PduL B-protein_type +homologs O +. O + +( O +a O +)–( O +c O +): O +Chromatograms B-evidence +of O +sPduL B-protein +( O +a O +), O +rPduL B-protein +( O +b O +), O +and O +pPduL B-protein +( O +c O +) O +with O +( O +orange O +) O +or O +without O +( O +blue O +) O +the O +predicted O +EP B-structure_element +, O +post O +- O +nickel B-experimental_method +affinity I-experimental_method +purification I-experimental_method +, O +applied O +over O +a O +preparative O +size O +exclusion O +column O +( O +see O +Materials O +and O +Methods O +). O + +( O +d O +)–( O +f O +): O +Chromatograms B-evidence +of O +sPduL B-protein +( O +d O +), O +rPduL B-protein +( O +e O +), O +and O +pPduL B-protein +( O +f O +) O +post O +- O +preparative O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +with O +different O +size O +fractions O +separated O +, O +applied O +over O +an O +analytical O +size O +exclusion O +column O +( O +see O +Materials O +and O +Methods O +). O + +All O +chromatograms B-evidence +are O +cropped O +to O +show O +only O +the O +linear O +range O +of O +separation O +based O +on O +standard O +runs O +, O +shown O +in O +black O +squares O +with O +a O +dashed O +linear O +trend O +line O +. O + +Active B-site +Site I-site +Properties O + +CoA B-chemical +and O +the O +metal O +ions O +bind O +between O +the O +two O +domains O +, O +presumably O +in O +the O +active B-site +site I-site +( O +Figs O +2b O +and O +4a O +). O + +To O +identify O +the O +bound O +metals O +, O +we O +performed O +an O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +fluorescence I-experimental_method +scan I-experimental_method +on O +the O +crystals B-evidence +at O +various O +wavelengths O +( O +corresponding O +to O +the O +K O +- O +edges O +of O +Mn B-chemical +, O +Fe B-chemical +, O +Co B-chemical +, O +Ni B-chemical +, O +Cu B-chemical +, O +and O +Zn B-chemical +). O + +There O +was O +a O +large O +signal O +at O +the O +zinc O +edge O +, O +and O +we O +tested O +for O +the O +presence O +of O +zinc B-chemical +by O +collecting B-experimental_method +full I-experimental_method +data I-experimental_method +sets I-experimental_method +before I-experimental_method +and I-experimental_method +after I-experimental_method +the I-experimental_method +Zn I-experimental_method +K I-experimental_method +- I-experimental_method +edge I-experimental_method +( I-experimental_method +1 I-experimental_method +. I-experimental_method +2861 I-experimental_method +and I-experimental_method +1 I-experimental_method +. I-experimental_method +2822 I-experimental_method +Å I-experimental_method +, O +respectively O +). O + +The O +large O +differences O +between O +the O +anomalous O +signals O +confirm O +the O +presence O +of O +zinc B-chemical +at O +both O +metal O +sites O +( O +S3 O +Fig O +). O + +The O +first O +zinc B-chemical +ion O +( O +Zn1 B-chemical +) O +is O +in O +a O +tetrahedral O +coordination O +state O +with O +His48 B-residue_name_number +, O +His50 B-residue_name_number +, O +Glu109 B-residue_name_number +, O +and O +the O +CoA B-chemical +sulfur B-chemical +( O +Fig O +4a O +). O + +The O +second O +( O +Zn2 B-chemical +) O +is O +in O +octahedral O +coordination O +by O +three O +conserved B-protein_state +histidine B-residue_name +residues O +( O +His157 B-residue_name_number +, O +His159 B-residue_name_number +and O +His204 B-residue_name_number +) O +as O +well O +as O +three O +water B-chemical +molecules O +( O +Fig O +4a O +). O + +The O +nitrogen O +atom O +coordinating O +the O +zinc B-chemical +is O +the O +Nε O +in O +each O +histidine B-residue_name +residue O +, O +as O +is O +typical O +for O +this O +interaction O +. O + +When O +the O +crystals B-experimental_method +were I-experimental_method +soaked I-experimental_method +in O +a O +sodium B-chemical +phosphate I-chemical +solution O +for O +2 O +d O +prior O +to O +data O +collection O +, O +the O +CoA B-chemical +dissociates O +, O +and O +density B-evidence +for O +a O +phosphate B-chemical +molecule O +is O +visible O +at O +the O +active B-site +site I-site +( O +Table O +2 O +, O +Fig O +4b O +). O + +The O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +structure B-evidence +aligns B-experimental_method +well O +with O +the O +CoA B-protein_state +- I-protein_state +bound I-protein_state +structure B-evidence +( O +0 O +. O +43 O +Å O +rmsd B-evidence +over O +2 O +, O +361 O +atoms O +for O +the O +monomer B-oligomeric_state +, O +0 O +. O +83 O +Å O +over O +5 O +, O +259 O +aligned O +atoms O +for O +the O +dimer B-oligomeric_state +). O + +The O +phosphate B-chemical +contacts O +both O +zinc B-chemical +atoms O +( O +Fig O +4b O +) O +and O +replaces O +the O +coordination O +by O +CoA B-chemical +at O +Zn1 B-chemical +; O +the O +coordination O +for O +Zn2 B-chemical +changes O +from O +octahedral O +with O +three O +bound O +waters B-chemical +to O +tetrahedral O +with O +a O +phosphate B-chemical +ion O +as O +one O +of O +the O +ligands O +( O +Fig O +4b O +). O + +Conserved B-protein_state +Arg103 B-residue_name_number +seems O +to O +be O +involved O +in O +maintaining O +the O +phosphate B-chemical +in O +that O +position O +. O + +The O +two O +zinc B-chemical +atoms O +are O +slightly O +closer O +together O +in O +the O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +form O +( O +5 O +. O +8 O +Å O +vs O +6 O +. O +3 O +Å O +), O +possibly O +due O +to O +the O +bridging O +effect O +of O +the O +phosphate B-chemical +. O + +An O +additional O +phosphate B-chemical +molecule O +is O +bound O +at O +a O +crystal O +contact O +interface O +, O +perhaps O +accounting O +for O +the O +14 O +Å O +shorter O +c O +- O +axis O +in O +the O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +crystal O +form O +( O +Table O +2 O +). O + +Oligomeric O +States O +of O +PduL B-protein_type +Orthologs O +Are O +Influenced O +by O +the O +EP B-structure_element + +Interestingly O +, O +some O +of O +the O +residues O +important O +for O +dimerization O +of O +rPduL B-protein +, O +particularly O +Phe116 B-residue_name_number +, O +are O +poorly B-protein_state +conserved I-protein_state +across O +PduL B-protein_type +homologs O +associated O +with O +functionally O +diverse O +BMCs B-complex_assembly +( O +Figs O +4c O +and O +3 O +), O +suggesting O +that O +they O +may O +have O +alternative O +oligomeric O +states O +. O + +We O +tested O +this O +hypothesis O +by O +performing O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +on O +both O +full B-protein_state +- I-protein_state +length I-protein_state +and O +truncated O +variants O +( O +lacking B-protein_state +the O +EP B-structure_element +, O +ΔEP B-mutant +) O +of O +sPduL B-protein +, O +rPduL B-protein +, O +and O +pPduL B-protein +. O +These O +three O +homologs O +are O +found O +in O +functionally O +distinct O +BMCs B-complex_assembly +( O +Table O +1 O +). O + +It O +has O +been O +proposed O +that O +the O +catabolic B-protein_state +BMCs B-complex_assembly +may O +assemble O +in O +a O +core O +- O +first O +manner O +, O +with O +the O +luminal O +enzymes O +( O +signature O +enzyme O +, O +aldehyde B-protein_type +, I-protein_type +and I-protein_type +alcohol I-protein_type +dehydrogenases I-protein_type +and O +the O +BMC B-complex_assembly +PTAC B-protein_type +) O +forming O +an O +initial O +bolus O +, O +or O +prometabolosome O +, O +around O +which O +a O +shell B-structure_element +assembles O +. O + +Given O +the O +diversity O +of O +signature O +enzymes O +( O +Table O +1 O +), O +it O +is O +plausible O +that O +PduL B-protein_type +orthologs O +may O +adopt O +different O +oligomeric O +states O +that O +reflect O +the O +differences O +in O +the O +proteins O +being O +packaged O +with O +them O +in O +the O +organelle O +lumen O +. O + +We O +found O +that O +not O +only O +did O +the O +different O +orthologs O +appear O +to O +assemble O +into O +different O +oligomeric O +states O +, O +but O +that O +quaternary O +structure O +was O +dependent O +on O +whether O +or O +not O +the O +EP B-structure_element +was O +present O +. O + +Full B-protein_state +- I-protein_state +length I-protein_state +sPduL B-protein +was O +unstable O +in O +solution O +— O +precipitating O +over O +time O +— O +and O +eluted O +throughout O +the O +entire O +volume O +of O +a O +size O +exclusion O +column O +, O +indicating O +it O +was O +nonspecifically O +aggregating O +. O + +However O +, O +when O +the O +putative O +EP B-structure_element +( O +residues O +1 B-residue_range +– I-residue_range +27 I-residue_range +) O +was O +removed B-experimental_method +( O +sPduL B-mutant +ΔEP I-mutant +), O +the O +truncated B-protein_state +protein O +was O +stable O +and O +eluted O +as O +a O +single O +peak O +( O +Fig O +5a O +) O +consistent O +with O +the O +size O +of O +a O +monomer B-oligomeric_state +( O +Fig O +5d O +, O +blue O +curve O +). O + +In O +contrast O +, O +both O +full B-protein_state +- I-protein_state +length I-protein_state +rPduL B-protein +and O +pPduL B-protein +appeared O +to O +exist O +in O +two O +distinct O +oligomeric O +states O +( O +Fig O +5b O +and O +5c O +respectively O +, O +orange O +curves O +), O +one O +form O +of O +the O +approximate O +size O +of O +a O +dimer B-oligomeric_state +and O +the O +second O +, O +a O +higher O +molecular O +weight O +oligomer B-oligomeric_state +(~ O +150 O +kDa O +). O + +Upon O +deletion B-experimental_method +of O +the O +putative O +EP B-structure_element +( O +residues O +1 B-residue_range +– I-residue_range +47 I-residue_range +for O +rPduL B-protein +, O +and O +1 B-residue_range +– I-residue_range +20 I-residue_range +for O +pPduL B-protein +), O +there O +was O +a O +distinct O +change O +in O +the O +elution O +profiles O +( O +Fig O +5b O +and O +5c O +respectively O +, O +blue O +curves O +). O + +pPduLΔEP B-mutant +eluted O +as O +two O +smaller O +forms O +, O +possibly O +corresponding O +to O +a O +trimer B-oligomeric_state +and O +a O +monomer B-oligomeric_state +. O + +In O +contrast O +, O +rPduLΔEP B-mutant +eluted O +as O +one O +smaller O +oligomer O +, O +possibly O +a O +dimer B-oligomeric_state +. O + +We O +also O +analyzed O +purified O +rPduL B-protein +and O +rPduLΔEP B-mutant +by O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +coupled O +with O +multiangle B-experimental_method +light I-experimental_method +scattering I-experimental_method +( O +SEC B-experimental_method +- I-experimental_method +MALS I-experimental_method +) O +for O +a O +complementary O +approach O +to O +assessing O +oligomeric O +state O +. O + +SEC B-experimental_method +- I-experimental_method +MALS I-experimental_method +analysis O +of O +rPdulΔEP B-mutant +is O +consistent O +with O +a O +dimer B-oligomeric_state +( O +as O +observed O +in O +the O +crystal B-evidence +structure I-evidence +) O +with O +a O +weighted B-evidence +average I-evidence +( I-evidence +Mw I-evidence +) I-evidence +and I-evidence +number I-evidence +average I-evidence +( I-evidence +Mn I-evidence +) I-evidence +of I-evidence +the I-evidence +molar I-evidence +mass I-evidence +of O +58 O +. O +4 O +kDa O ++/− O +11 O +. O +2 O +% O +and O +58 O +. O +8 O +kDa O ++/− O +10 O +. O +9 O +%, O +respectively O +( O +S4a O +Fig O +). O + +rPduL B-protein +full B-protein_state +length I-protein_state +runs O +as O +Mw B-evidence += O +140 O +. O +3 O +kDa O ++/− O +1 O +. O +2 O +% O +and O +Mn B-evidence += O +140 O +. O +5 O +kDa O ++/− O +1 O +. O +2 O +%. O + +This O +corresponds O +to O +an O +oligomeric O +state O +of O +six B-oligomeric_state +subunits I-oligomeric_state +( O +calculated O +molecular B-evidence +weight I-evidence +of O +144 O +kDa O +). O + +Collectively O +, O +these O +data O +strongly O +suggest O +that O +the O +N O +- O +terminal O +EP B-structure_element +of O +PduL B-protein_type +plays O +a O +role O +in O +defining O +the O +quaternary O +structure O +of O +the O +protein O +. O + +The O +BMC B-complex_assembly +shell B-structure_element +not O +only O +sequesters O +specific O +enzymes O +but O +also O +their O +cofactors O +, O +thereby O +establishing O +a O +private O +cofactor O +pool O +dedicated O +to O +the O +encapsulated O +reactions O +. O + +In O +catabolic B-protein_state +BMCs B-complex_assembly +, O +CoA B-chemical +and O +NAD B-chemical ++ I-chemical +must O +be O +continually O +recycled O +within O +the O +organelle O +( O +Fig O +1 O +). O + +Homologs O +of O +the O +predominant O +cofactor O +utilizer O +( O +aldehyde B-protein_type +dehydrogenase I-protein_type +) O +and O +NAD B-chemical ++ I-chemical +regenerator O +( O +alcohol B-protein_type +dehydrogenase I-protein_type +) O +have O +been O +structurally O +characterized O +, O +but O +until O +now O +structural O +information O +was O +lacking O +for O +PduL B-protein_type +, O +which O +recycles O +CoA B-chemical +in O +the O +organelle O +lumen O +. O + +Curiously O +, O +while O +the O +housekeeping B-protein_state +Pta B-protein_type +could O +provide O +this O +function O +, O +and O +indeed O +does O +so O +in O +the O +case O +of O +one O +type O +of O +ethanolamine B-complex_assembly +- I-complex_assembly +utilizing I-complex_assembly +( I-complex_assembly +EUT I-complex_assembly +) I-complex_assembly +BMC I-complex_assembly +, O +the O +evolutionarily O +unrelated O +PduL B-protein_type +fulfills O +this O +function O +for O +the O +majority O +of O +metabolosomes B-complex_assembly +using O +a O +novel O +structure B-evidence +and O +active B-site +site I-site +for O +convergent O +evolution O +of O +function O +. O + +The O +Tertiary O +Structure O +of O +PduL B-protein_type +Is O +Formed O +by O +Discontinuous O +Segments O +of O +Primary O +Structure O + +The O +structure B-evidence +of O +PduL B-protein_type +consists O +of O +two B-structure_element +β I-structure_element +- I-structure_element +barrel I-structure_element +domains I-structure_element +capped O +by O +short B-structure_element +alpha I-structure_element +helical I-structure_element +segments I-structure_element +( O +Fig O +2b O +). O + +The O +two O +domains O +are O +structurally O +very O +similar O +( O +superimposing B-experimental_method +with O +a O +rmsd B-evidence +of O +1 O +. O +34 O +Å O +( O +over O +123 O +out O +of O +320 O +/ O +348 O +aligned O +backbone O +atoms O +, O +S5a O +Fig O +). O + +However O +, O +the O +amino O +acid O +sequences O +of O +the O +two O +domains O +are O +only O +16 O +% O +identical O +( O +mainly O +the O +RHxH B-structure_element +motif I-structure_element +, O +β2 B-structure_element +and O +β10 B-structure_element +), O +and O +34 O +% O +similar O +. O + +Our O +structure B-evidence +reveals O +that O +the O +two O +assigned O +PF06130 B-structure_element +domains O +( O +Fig O +3 O +) O +do O +not O +form O +structurally O +discrete O +units O +; O +this O +reduces O +the O +apparent O +sequence O +conservation O +at O +the O +level O +of O +primary O +structure O +. O + +One O +strand B-structure_element +of O +the O +domain B-structure_element +1 I-structure_element +beta B-structure_element +barrel I-structure_element +( O +shown O +in O +blue O +in O +Fig O +2 O +) O +is O +contributed O +by O +the O +N O +- O +terminus O +, O +while O +the O +rest O +of O +the O +domain O +is O +formed O +by O +the O +residues O +from O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +the O +protein B-protein_type +. O + +When O +aligned B-experimental_method +by O +structure B-evidence +, O +the O +β1 B-structure_element +strand I-structure_element +of O +the O +first B-structure_element +domain I-structure_element +( O +Fig O +2a O +and O +2b O +, O +blue O +) O +corresponds O +to O +the O +final B-structure_element +strand I-structure_element +of O +the O +second B-structure_element +domain I-structure_element +( O +β9 B-structure_element +), O +effectively O +making O +the O +domains O +continuous O +if O +the O +first O +strand O +was O +transplanted O +to O +the O +C O +- O +terminus O +. O + +Refined O +domain O +assignment O +based O +on O +our O +structure B-evidence +should O +be O +able O +to O +predict O +domains O +of O +PF06130 B-structure_element +homologs O +much O +more O +accurately O +. O + +The O +closest O +structural O +homolog O +of O +the O +PduL B-protein_type +barrel B-structure_element +domain I-structure_element +is O +a O +subdomain O +of O +a O +multienzyme O +complex O +, O +the O +alpha B-structure_element +subunit I-structure_element +of O +ethylbenzene B-protein_type +dehydrogenase I-protein_type +( O +S5b O +Fig O +, O +rmsd B-evidence +of O +2 O +. O +26 O +Å O +over O +226 O +aligned O +atoms O +consisting O +of O +one O +beta B-structure_element +barrel I-structure_element +and O +one O +capping B-structure_element +helix I-structure_element +). O + +In O +contrast O +to O +PduL B-protein_type +, O +there O +is O +only O +one O +barrel B-structure_element +present O +in O +ethylbenzene B-protein_type +dehydrogenase I-protein_type +, O +and O +there O +is O +no O +comparable O +active B-site +site I-site +arrangement O +. O + +The O +PduL B-protein_type +signature O +primary O +structure O +, O +two O +PF06130 B-structure_element +domains O +, O +occurs O +in O +some O +multidomain O +proteins O +, O +most O +of O +them O +annotated O +as O +Acks B-protein_type +, O +suggesting O +that O +PduL B-protein_type +may O +also O +replace O +Pta B-protein_type +in O +variants O +of O +the O +phosphotransacetylase B-protein_type +- O +Ack B-protein_type +pathway O +. O + +These O +PduL B-protein_type +homologs O +lack B-protein_state +EPs B-structure_element +, O +and O +their B-protein_type +fusion O +to O +Ack B-protein_type +may O +have O +evolved O +as O +a O +way O +to O +facilitate O +substrate O +channeling O +between O +the O +two O +enzymes O +. O + +Implications O +for O +Metabolosome B-complex_assembly +Core O +Assembly O + +For O +BMC B-complex_assembly +- O +encapsulated O +proteins O +to O +properly O +function O +together O +, O +they O +must O +be O +targeted O +to O +the O +lumen O +and O +assemble O +into O +an O +organization O +that O +facilitates O +substrate O +/ O +product O +channeling O +among O +the O +different O +catalytic B-site +sites I-site +of O +the O +signature O +and O +core O +enzymes O +. O + +The O +N B-structure_element +- I-structure_element +terminal I-structure_element +extension I-structure_element +on O +PduL B-protein_type +homologs O +may O +serve O +both O +of O +these O +functions O +. O + +The B-structure_element +extension I-structure_element +shares O +many O +features O +with O +previously O +characterized O +EPs B-structure_element +: O +it O +is O +present O +only O +in O +homologs O +associated O +with O +BMC B-gene +loci I-gene +, O +and O +it O +is O +predicted O +to O +form O +an O +amphipathic B-protein_state +α B-structure_element +- I-structure_element +helix I-structure_element +. O + +Moreover O +, O +its O +removal B-experimental_method +affects O +the O +oligomeric O +state O +of O +the O +protein O +. O + +EP B-structure_element +- O +mediated O +oligomerization O +has O +been O +observed O +for O +the O +signature O +and O +core O +BMC B-complex_assembly +enzymes O +; O +for O +example O +, O +full B-protein_state +- I-protein_state +length I-protein_state +propanediol B-protein_type +dehydratase I-protein_type +and O +ethanolamine B-protein_type +ammonia I-protein_type +- I-protein_type +lyase I-protein_type +( O +signature O +enzymes O +for O +PDU B-complex_assembly +and O +EUT B-complex_assembly +BMCs I-complex_assembly +) O +subunits O +are O +also O +insoluble O +, O +but O +become O +soluble O +upon O +removal O +of O +the O +predicted O +EP B-structure_element +. O + +sPduL B-protein +has O +also O +previously O +been O +reported O +to O +localize O +to O +inclusion O +bodies O +when O +overexpressed B-experimental_method +; O +we O +show O +here O +that O +this O +is O +dependent O +on O +the O +presence O +of O +the O +EP B-structure_element +. O + +This O +propensity O +of O +the O +EP B-structure_element +to O +cause O +proteins O +to O +form O +complexes O +( O +Fig O +5 O +) O +might O +not O +be O +a O +coincidence O +, O +but O +could O +be O +a O +necessary O +step O +in O +the O +assembly O +of O +BMCs B-complex_assembly +. O + +Structured O +aggregation O +of O +the O +core O +enzymes O +has O +been O +proposed O +to O +be O +the O +initial O +step O +in O +metabolosome B-complex_assembly +assembly O +and O +is O +known O +to O +be O +the O +first O +step O +of O +β O +- O +carboxysome O +biogenesis O +, O +where O +the O +core O +enzyme O +Ribulose B-protein_type +Bisphosphate I-protein_type +Carboxylase I-protein_type +/ I-protein_type +Oxygenase I-protein_type +( O +RuBisCO B-protein_type +) O +is O +aggregated O +by O +the O +CcmM B-protein_type +protein O +. O + +Likewise O +, O +CsoS2 B-protein_type +, O +a O +protein O +in O +the O +α B-complex_assembly +- I-complex_assembly +carboxysome I-complex_assembly +core O +, O +also O +aggregates O +when O +purified O +and O +is O +proposed O +to O +facilitate O +the O +nucleation O +and O +encapsulation O +of O +RuBisCO B-protein_type +molecules O +in O +the O +lumen O +of O +the O +organelle O +. O + +This O +role O +for O +EPs B-structure_element +in O +BMC B-complex_assembly +assembly O +is O +in O +addition O +to O +their O +interaction O +with O +shell O +proteins O +. O + +Moreover O +, O +the O +PduL B-protein_type +crystal B-evidence +structures I-evidence +offer O +a O +clue O +as O +to O +how O +required O +cofactors O +enter O +the O +BMC B-complex_assembly +lumen O +during O +assembly O +. O + +Free O +CoA B-chemical +and O +NAD B-chemical ++/ I-chemical +H B-chemical +could O +potentially O +be O +bound O +to O +the O +enzymes O +as O +the O +core O +assembles O +and O +is O +encapsulated O +. O + +Our O +PduL B-protein_type +crystals B-evidence +contained O +CoA B-chemical +that O +was O +captured O +from O +the O +Escherichia B-species +coli I-species +cytosol O +, O +indicating O +that O +the O +“ O +ground O +state O +” O +of O +PduL B-protein_type +is O +in O +the O +CoA B-protein_state +- I-protein_state +bound I-protein_state +form O +; O +this O +could O +provide O +an O +elegantly O +simple O +means O +of O +guaranteeing O +a O +1 O +: O +1 O +ratio O +of O +CoA B-complex_assembly +: I-complex_assembly +PduL I-complex_assembly +within O +the O +metabolosome B-complex_assembly +lumen O +. O + +Active B-site +Site I-site +Identification O +and O +Structural O +Insights O +into O +Catalysis O + +The O +active B-site +site I-site +of O +PduL B-protein_type +is O +formed O +at O +the O +interface B-site +of O +the O +two O +structural O +domains B-structure_element +( O +Fig O +2b O +). O + +As O +expected O +, O +the O +amino O +acid O +sequence O +conservation O +is O +highest O +in O +the O +region O +around O +the O +proposed O +active B-site +site I-site +( O +Fig O +4d O +); O +highly B-protein_state +conserved I-protein_state +residues O +are O +also O +involved O +in O +CoA B-chemical +binding O +( O +Figs O +2a O +and O +3 O +, O +residues O +Ser45 B-residue_name_number +, O +Lys70 B-residue_name_number +, O +Arg97 B-residue_name_number +, O +Leu99 B-residue_name_number +, O +His204 B-residue_name_number +, O +Asn211 B-residue_name_number +). O + +All O +of O +the O +metal B-site +- I-site +coordinating I-site +residues I-site +( O +Fig O +2a O +) O +are O +absolutely B-protein_state +conserved I-protein_state +, O +implicating O +them O +in O +catalysis O +or O +the O +correct O +spatial O +orientation O +of O +the O +substrates O +. O + +Arg103 B-residue_name_number +, O +which O +contacts O +the O +phosphate B-chemical +( O +Fig O +4b O +), O +is O +present O +in O +all O +PduL B-protein_type +homologs O +. O + +The O +close O +resemblance O +between O +the O +structures O +binding O +CoA B-chemical +and O +phosphate B-chemical +likely O +indicates O +that O +no O +large O +changes O +in O +protein O +conformation O +are O +involved O +in O +catalysis O +, O +and O +that O +our O +crystal B-evidence +structures I-evidence +are O +representative O +of O +the O +active B-protein_state +form O +. O + +The O +native O +substrate O +for O +the O +forward O +reaction O +of O +rPduL B-protein +and O +pPduL B-protein +, O +propionyl B-chemical +- I-chemical +CoA I-chemical +, O +most O +likely O +binds O +to O +the O +enzyme O +in O +the O +same O +way O +at O +the O +observed O +nucleotide B-chemical +and O +pantothenic B-chemical +acid I-chemical +moiety O +, O +but O +the O +propionyl O +group O +in O +the O +CoA B-chemical +- I-chemical +thioester I-chemical +might O +point O +in O +a O +different O +direction O +. O + +There O +is O +a O +pocket B-site +nearby O +the O +active B-site +site I-site +between O +the O +well B-protein_state +- I-protein_state +conserved I-protein_state +residues O +Ser45 B-residue_name_number +and O +Ala154 B-residue_name_number +, O +which O +could O +accommodate O +the O +propionyl O +group O +( O +S6 O +Fig O +). O + +A O +homology B-experimental_method +model I-experimental_method +of O +sPduL B-protein +indicates O +that O +the O +residues O +making O +up O +this O +pocket B-site +and O +the O +surrounding O +active B-site +site I-site +region O +are O +identical O +to O +that O +of O +rPduL B-protein +, O +which O +is O +not O +surprising O +, O +because O +these O +two O +homologs O +presumably O +have O +the O +same O +propionyl B-chemical +- I-chemical +CoA I-chemical +substrate O +. O + +The O +homology B-experimental_method +model I-experimental_method +of O +pPduL B-protein +also O +has O +identical O +residues O +making O +up O +the O +pocket B-site +, O +but O +with O +a O +key O +difference O +in O +the O +vicinity O +of O +the O +active B-site +site I-site +: O +Gln77 B-residue_name_number +of O +rPduL B-protein +is O +replaced O +by O +a O +tyrosine B-residue_name +( O +Tyr77 B-residue_name_number +) O +in O +pPduL B-protein +. O +The O +physiological O +substrate O +of O +pPduL B-protein +( O +Table O +1 O +) O +is O +thought O +to O +be O +lactyl B-chemical +- I-chemical +CoA I-chemical +, O +which O +contains O +an O +additional O +hydroxyl O +group O +relative O +to O +propionyl B-chemical +- I-chemical +CoA I-chemical +. O +The O +presence O +of O +an O +aromatic B-protein_state +residue B-structure_element +at O +this O +position O +may O +underlie O +the O +substrate O +preference O +of O +the O +PduL B-protein_type +enzyme O +from O +the O +pvm B-gene +locus I-gene +. O + +Indeed O +, O +in O +the O +majority O +of O +PduLs B-protein_type +encoded O +in O +pvm B-gene +loci I-gene +, O +Gln77 B-residue_name_number +is O +substituted O +by O +either O +a O +Tyr B-residue_name +or O +Phe B-residue_name +, O +whereas O +it O +is O +typically O +a O +Gln B-residue_name +or O +Glu B-residue_name +in O +PduLs B-protein_type +in O +all O +other O +BMC B-complex_assembly +types O +that O +degrade O +acetyl B-chemical +- I-chemical +or O +propionyl B-chemical +- I-chemical +CoA I-chemical +. O +A O +comparison B-experimental_method +of O +the O +PduL B-protein_type +active B-site +site I-site +to O +that O +of O +the O +functionally O +identical O +Pta B-protein_type +suggests O +that O +the O +two O +enzymes O +have O +distinctly O +different O +mechanisms O +. O + +The O +catalytic O +mechanism O +of O +Pta B-protein_type +involves O +the O +abstraction O +of O +a O +thiol O +hydrogen O +by O +an O +aspartate B-residue_name +residue O +, O +resulting O +in O +the O +nucleophilic O +attack O +of O +thiolate O +upon O +the O +carbonyl O +carbon O +of O +acetyl B-chemical +- I-chemical +phosphate I-chemical +, O +oriented O +by O +an O +arginine B-residue_name +and O +stabilized O +by O +a O +serine B-residue_name +— O +there O +are O +no O +metals O +involved O +. O + +In O +contrast O +, O +in O +the O +rPduL B-protein +structure B-evidence +, O +there O +are O +no O +conserved O +aspartate B-residue_name +residues O +in O +or O +around O +the O +active B-site +site I-site +, O +and O +the O +only O +well B-protein_state +- I-protein_state +conserved I-protein_state +glutamate B-residue_name +residue O +in O +the O +active B-site +site I-site +is O +involved O +in O +coordinating B-bond_interaction +one O +of O +the O +metal O +ions O +. O + +These O +observations O +strongly O +suggest O +that O +an O +acidic B-protein_state +residue B-structure_element +is O +not O +directly O +involved O +in O +catalysis O +by O +PduL B-protein_type +. O +Instead O +, O +the O +dimetal B-site +active I-site +site I-site +of O +PduL B-protein_type +may O +create O +a O +nucleophile O +from O +one O +of O +the O +hydroxyl O +groups O +on O +free O +phosphate B-chemical +to O +attack O +the O +carbonyl O +carbon O +of O +the O +thioester O +bond O +of O +an O +acyl B-chemical +- I-chemical +CoA I-chemical +. O +In O +the O +reverse O +direction O +, O +the O +metal O +ion O +( O +s O +) O +could O +stabilize O +the O +thiolate O +anion O +that O +would O +attack O +the O +carbonyl O +carbon O +of O +an O +acyl B-chemical +- I-chemical +phosphate I-chemical +; O +a O +similar O +mechanism O +has O +been O +described O +for O +phosphatases B-protein_type +where O +hydroxyl O +groups O +or O +hydroxide O +ions O +can O +act O +as O +a O +base O +when O +coordinated O +by O +a O +dimetal B-site +active I-site +site I-site +. O + +Our O +structures B-evidence +provide O +the O +foundation O +for O +studies O +to O +elucidate O +the O +details O +of O +the O +catalytic O +mechanism O +of O +PduL B-protein_type +. O +Conserved B-protein_state +residues O +in O +the O +active B-site +site I-site +that O +may O +contribute O +to O +substrate O +binding O +and O +/ O +or O +transition O +state O +stabilization O +include O +Ser127 B-residue_name_number +, O +Arg103 B-residue_name_number +, O +Arg194 B-residue_name_number +, O +Gln107 B-residue_name_number +, O +Gln74 B-residue_name_number +, O +and O +Gln B-residue_name_number +/ O +Glu77 B-residue_name_number +. O + +In O +the O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +crystal B-evidence +structure I-evidence +, O +Ser127 B-residue_name_number +and O +Arg103 B-residue_name_number +appear O +to O +position O +the O +phosphate B-chemical +( O +Fig O +4b O +). O + +Alternatively O +, O +Arg103 B-residue_name_number +might O +act O +as O +a O +base O +to O +render O +the O +phosphate B-chemical +more O +nucleophilic O +. O + +The O +functional O +groups O +of O +Gln74 B-residue_name_number +, O +Gln B-residue_name_number +/ O +Glu77 B-residue_name_number +, O +and O +Arg194 B-residue_name_number +are O +directed O +away O +from O +the O +active B-site +site I-site +in O +both O +CoA B-protein_state +and O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +crystal B-evidence +structures I-evidence +and O +do O +not O +appear O +to O +be O +involved O +in O +hydrogen B-bond_interaction +bonding I-bond_interaction +with O +these O +substrates O +, O +although O +they O +could O +be O +important O +for O +positioning O +an O +acyl B-chemical +- I-chemical +phosphate I-chemical +. O + +The O +free O +CoA B-protein_state +- I-protein_state +bound I-protein_state +form O +is O +presumably O +poised O +for O +attack O +upon O +an O +acyl B-chemical +- I-chemical +phosphate I-chemical +, O +indicating O +that O +the O +enzyme O +initially O +binds O +CoA B-chemical +as O +opposed O +to O +acyl B-chemical +- I-chemical +phosphate I-chemical +. O + +This O +hypothesis O +is O +strengthened O +by O +the O +fact O +that O +the O +CoA B-protein_state +- I-protein_state +bound I-protein_state +crystals B-evidence +were O +obtained O +without O +added O +CoA B-chemical +, O +indicating O +that O +the O +protein O +bound B-protein_state +CoA B-chemical +from O +the O +E B-species +. I-species +coli I-species +expression O +strain O +and O +retained O +it O +throughout O +purification O +and O +crystallization O +. O + +The O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +structure B-evidence +indicates O +that O +in O +the O +opposite O +reaction O +direction O +phosphate B-chemical +is O +bound O +first O +, O +and O +then O +an O +acyl B-chemical +- I-chemical +CoA I-chemical +enters O +. O + +The O +two O +high O +- O +resolution O +crystal B-evidence +structures I-evidence +presented O +here O +will O +serve O +as O +the O +foundation O +for O +mechanistic O +studies O +on O +this O +noncanonical O +PTAC B-protein_type +enzyme O +to O +determine O +how O +the O +dimetal B-site +active I-site +site I-site +functions O +to O +catalyze O +both O +forward O +and O +reverse O +reactions O +. O + +Functional O +, O +but O +Not O +Structural O +, O +Convergence O +of O +PduL B-protein_type +and O +Pta B-protein_type + +PduL B-protein_type +and O +Pta B-protein_type +are O +mechanistically O +and O +structurally O +distinct O +enzymes O +that O +catalyze O +the O +same O +reaction O +, O +a O +prime O +example O +of O +evolutionary O +convergence O +upon O +a O +function O +. O + +There O +are O +several O +examples O +of O +such O +functional O +convergence O +of O +enzymes O +, O +although O +typically O +the O +enzymes O +have O +independently O +evolved O +similar O +, O +or O +even O +identical O +active B-site +sites I-site +; O +for O +example O +, O +the O +carbonic B-protein_type +anhydrase I-protein_type +family O +. O + +However O +, O +apparently O +less O +frequent O +is O +functional O +convergence O +that O +is O +supported O +by O +distinctly O +different O +active B-site +sites I-site +and O +accordingly O +catalytic O +mechanism O +, O +as O +revealed O +by O +comparison O +of O +the O +structures O +of O +Pta B-protein_type +and O +PduL B-protein_type +. O +One O +well O +- O +studied O +example O +of O +this O +is O +the O +β B-protein_type +- I-protein_type +lactamase I-protein_type +family O +of O +enzymes O +, O +in O +which O +the O +active B-site +site I-site +of O +Class O +A O +and O +Class O +C O +enzymes O +involve O +serine O +- O +based O +catalysis O +, O +but O +Class O +B O +enzymes O +are O +metalloproteins B-protein_type +. O + +This O +is O +not O +surprising O +, O +as O +β B-protein_type +- I-protein_type +lactamases I-protein_type +are O +not O +so O +widespread O +among O +bacteria B-taxonomy_domain +and O +therefore O +would O +be O +expected O +to O +have O +evolved O +independently O +several O +times O +as O +a O +defense O +mechanism O +against O +β O +- O +lactam O +antibiotics O +. O + +However O +, O +nearly O +all O +bacteria B-taxonomy_domain +encode O +Pta B-protein_type +, O +and O +it O +is O +not O +immediately O +clear O +why O +the O +Pta B-protein_type +/ O +PduL B-protein_type +functional O +convergence O +should O +have O +evolved O +: O +it O +would O +seem O +to O +be O +evolutionarily O +more O +resourceful O +for O +the O +Pta B-gene +- I-gene +encoding I-gene +gene I-gene +to O +be O +duplicated O +and O +repurposed O +for O +BMCs B-complex_assembly +, O +as O +is O +apparently O +the O +case O +in O +one O +type O +of O +BMC B-complex_assembly +— I-complex_assembly +EUT1 I-complex_assembly +( O +Table O +1 O +). O + +There O +could O +be O +some O +intrinsic O +biochemical O +difference O +between O +the O +two O +enzymes O +that O +renders O +PduL B-protein_type +a O +more O +attractive O +candidate O +for O +encapsulation O +in O +a O +BMC B-complex_assembly +— O +for O +example O +, O +PduL B-protein_type +might O +be O +more O +amenable O +to O +tight O +packaging O +, O +or O +is O +better O +suited O +for O +the O +chemical O +microenvironment O +formed O +within O +the O +lumen O +of O +the O +BMC B-complex_assembly +, O +which O +can O +be O +quite O +different O +from O +the O +cytosol O +. O + +Further O +biochemical O +comparison O +between O +the O +two O +PTACs B-protein_type +will O +likely O +yield O +exciting O +results O +that O +could O +answer O +this O +evolutionary O +question O +. O + +BMCs B-complex_assembly +are O +now O +known O +to O +be O +widespread O +among O +the O +bacteria B-taxonomy_domain +and O +are O +involved O +in O +critical O +segments O +of O +both O +autotrophic O +and O +heterotrophic O +biochemical O +pathways O +that O +confer O +to O +the O +host O +organism O +a O +competitive O +( O +metabolic O +) O +advantage O +in O +select O +niches O +. O + +As O +one O +of O +the O +three O +common O +metabolosome B-complex_assembly +core O +enzymes O +, O +the O +structure B-evidence +of O +PduL B-protein_type +provides O +a O +key O +missing O +piece O +to O +our O +structural O +picture O +of O +the O +shared O +core O +biochemistry O +( O +Fig O +1 O +) O +of O +functionally O +diverse O +catabolic B-protein_state +BMCs B-complex_assembly +. O + +We O +have O +observed O +the O +oligomeric O +state O +differences O +of O +PduL B-protein_type +to O +correlate O +with O +the O +presence O +of O +an O +EP B-structure_element +, O +providing O +new O +insight O +into O +the O +function O +of O +this O +sequence O +extension O +in O +BMC B-complex_assembly +assembly O +. O + +Moreover O +, O +our O +results O +suggest O +a O +means O +for O +Coenzyme B-chemical +A I-chemical +incorporation O +during O +metabolosome B-complex_assembly +biogenesis O +. O + +A O +detailed O +understanding O +of O +the O +underlying O +principles O +governing O +the O +assembly O +and O +internal O +structural O +organization O +of O +BMCs B-complex_assembly +is O +a O +requisite O +for O +synthetic O +biologists O +to O +design O +custom O +nanoreactors O +that O +use O +BMC B-complex_assembly +architectures O +as O +a O +template O +. O + +Furthermore O +, O +given O +the O +growing O +number O +of O +metabolosomes B-complex_assembly +implicated O +in O +pathogenesis O +, O +the O +PduL B-protein_type +structure B-evidence +will O +be O +useful O +in O +the O +development O +of O +therapeutics O +. O + +The O +fact O +that O +PduL B-protein_type +is O +confined O +almost O +exclusively O +to O +metabolosomes B-complex_assembly +can O +be O +used O +to O +develop O +an O +inhibitor O +that O +blocks O +only O +PduL B-protein_type +and O +not O +Pta B-protein_type +as O +a O +way O +to O +selectively O +disrupt O +BMC B-complex_assembly +- O +based O +metabolism O +, O +while O +not O +affecting O +most O +commensal O +organisms O +that O +require O +PTAC B-protein_type +activity O +. O + +Biochemistry O +and O +Crystal B-evidence +Structure I-evidence +of O +Ectoine B-protein_type +Synthase I-protein_type +: O +A O +Metal B-protein_state +- I-protein_state +Containing I-protein_state +Member O +of O +the O +Cupin B-protein_type +Superfamily I-protein_type + +Ectoine B-chemical +is O +a O +compatible O +solute O +and O +chemical O +chaperone O +widely O +used O +by O +members O +of O +the O +Bacteria B-taxonomy_domain +and O +a O +few O +Archaea B-taxonomy_domain +to O +fend O +- O +off O +the O +detrimental O +effects O +of O +high O +external O +osmolarity O +on O +cellular O +physiology O +and O +growth O +. O + +Ectoine B-protein_type +synthase I-protein_type +( O +EctC B-protein_type +) O +catalyzes O +the O +last O +step O +in O +ectoine B-chemical +production O +and O +mediates O +the O +ring O +closure O +of O +the O +substrate O +N B-chemical +- I-chemical +gamma I-chemical +- I-chemical +acetyl I-chemical +- I-chemical +L I-chemical +- I-chemical +2 I-chemical +, I-chemical +4 I-chemical +- I-chemical +diaminobutyric I-chemical +acid I-chemical +through O +a O +water B-chemical +elimination O +reaction O +. O + +However O +, O +the O +crystal B-evidence +structure I-evidence +of O +ectoine B-protein_type +synthase I-protein_type +is O +not O +known O +and O +a O +clear O +understanding O +of O +how O +its O +fold O +contributes O +to O +enzyme O +activity O +is O +thus O +lacking O +. O + +Using O +the O +ectoine B-protein_type +synthase I-protein_type +from O +the O +cold O +- O +adapted O +marine B-taxonomy_domain +bacterium I-taxonomy_domain +Sphingopyxis B-species +alaskensis I-species +( O +Sa B-species +), O +we O +report O +here O +both O +a O +detailed O +biochemical O +characterization O +of O +the O +EctC B-protein +enzyme O +and O +the O +high O +- O +resolution O +crystal B-evidence +structure I-evidence +of O +its O +apo B-protein_state +- O +form O +. O + +Structural B-experimental_method +analysis I-experimental_method +classified O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +as O +a O +member O +of O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +EctC B-protein +forms O +a O +dimer B-oligomeric_state +with O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +arrangement O +, O +both O +in O +solution O +and O +in O +the O +crystal B-evidence +structure I-evidence +. O + +The O +interface B-site +of O +the O +dimer B-oligomeric_state +assembly O +is O +shaped O +through O +backbone O +- O +contacts O +and O +weak O +hydrophobic B-bond_interaction +interactions I-bond_interaction +mediated O +by O +two O +beta B-structure_element +- I-structure_element +sheets I-structure_element +within O +each O +monomer B-oligomeric_state +. O + +We O +show O +for O +the O +first O +time O +that O +ectoine B-protein_type +synthase I-protein_type +harbors O +a O +catalytically O +important O +metal B-chemical +co O +- O +factor O +; O +metal B-experimental_method +depletion I-experimental_method +and I-experimental_method +reconstitution I-experimental_method +experiments I-experimental_method +suggest O +that O +EctC B-protein +is O +probably O +an O +iron B-protein_state +- I-protein_state +dependent I-protein_state +enzyme O +. O + +We O +found O +that O +EctC B-protein +not O +only O +effectively O +converts O +its O +natural O +substrate O +N B-chemical +- I-chemical +gamma I-chemical +- I-chemical +acetyl I-chemical +- I-chemical +L I-chemical +- I-chemical +2 I-chemical +, I-chemical +4 I-chemical +- I-chemical +diaminobutyric I-chemical +acid I-chemical +into O +ectoine B-chemical +through O +a O +cyclocondensation O +reaction O +, O +but O +that O +it O +can O +also O +use O +the O +isomer O +N B-chemical +- I-chemical +alpha I-chemical +- I-chemical +acetyl I-chemical +- I-chemical +L I-chemical +- I-chemical +2 I-chemical +, I-chemical +4 I-chemical +- I-chemical +diaminobutyric I-chemical +acid I-chemical +as O +its O +substrate O +, O +albeit O +with O +substantially O +reduced O +catalytic B-evidence +efficiency I-evidence +. O + +Structure B-experimental_method +- I-experimental_method +guided I-experimental_method +site I-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +experiments O +targeting O +amino O +acid O +residues O +that O +are O +evolutionarily B-protein_state +highly I-protein_state +conserved I-protein_state +among O +the O +extended O +EctC B-protein_type +protein I-protein_type +family I-protein_type +, O +including O +those O +forming O +the O +presumptive O +iron B-site +- I-site +binding I-site +site I-site +, O +were O +conducted O +to O +functionally O +analyze O +the O +properties O +of O +the O +resulting O +EctC B-protein +variants O +. O + +An O +assessment O +of O +enzyme O +activity O +and O +iron B-chemical +content O +of O +these O +mutants O +give O +important O +clues O +for O +understanding O +the O +architecture O +of O +the O +active B-site +site I-site +positioned O +within O +the O +core O +of O +the O +EctC B-protein +cupin B-structure_element +barrel I-structure_element +. O + +Ectoine B-chemical +[( O +S B-chemical +)- I-chemical +2 I-chemical +- I-chemical +methyl I-chemical +- I-chemical +1 I-chemical +, I-chemical +4 I-chemical +, I-chemical +5 I-chemical +, I-chemical +6 I-chemical +- I-chemical +tetrahydropyrimidine I-chemical +- I-chemical +4 I-chemical +- I-chemical +carboxylic I-chemical +acid I-chemical +] O +and O +its O +derivative O +5 B-chemical +- I-chemical +hydroxyectoine I-chemical +[( O +4S B-chemical +, I-chemical +5S I-chemical +)- I-chemical +5 I-chemical +- I-chemical +hydroxy I-chemical +- I-chemical +2 I-chemical +- I-chemical +methyl I-chemical +- I-chemical +1 I-chemical +, I-chemical +4 I-chemical +, I-chemical +5 I-chemical +, I-chemical +6 I-chemical +- I-chemical +tetrahydropyrimidine I-chemical +- I-chemical +4 I-chemical +- I-chemical +carboxylic I-chemical +acid I-chemical +] O +are O +such O +compatible O +solutes O +. O + +Both O +marine B-taxonomy_domain +and I-taxonomy_domain +terrestrial I-taxonomy_domain +microorganisms I-taxonomy_domain +produce O +them O +widely O +in O +response O +to O +osmotic O +or O +temperature O +stress O +. O + +Synthesis O +of O +ectoine B-chemical +occurs O +from O +the O +intermediate O +metabolite O +L B-chemical +- I-chemical +aspartate I-chemical +- I-chemical +ß I-chemical +- I-chemical +semialdehyde I-chemical +and O +comprises O +the O +sequential O +activities O +of O +three O +enzymes O +: O +L B-protein_type +- I-protein_type +2 I-protein_type +, I-protein_type +4 I-protein_type +- I-protein_type +diaminobutyrate I-protein_type +transaminase I-protein_type +( O +EctB B-protein_type +; O +EC O +2 O +. O +6 O +. O +1 O +. O +76 O +), O +2 B-protein_type +, I-protein_type +4 I-protein_type +- I-protein_type +diaminobutyrate I-protein_type +acetyltransferase I-protein_type +( O +EctA B-protein_type +; O +EC O +2 O +. O +3 O +. O +1 O +. O +178 O +), O +and O +ectoine B-protein_type +synthase I-protein_type +( O +EctC B-protein_type +; O +EC O +4 O +. O +2 O +. O +1 O +. O +108 O +) O +( O +Fig O +1 O +). O + +The O +ectoine B-chemical +derivative O +5 B-chemical +- I-chemical +hydroxyectoine I-chemical +, O +a O +highly O +effective O +stress O +protectant O +in O +its O +own O +right O +, O +is O +synthesized O +by O +a O +substantial O +subgroup O +of O +the O +ectoine B-chemical +producers O +. O + +This O +stereospecific O +chemical O +modification O +of O +ectoine B-chemical +( O +Fig O +1 O +) O +is O +catalyzed O +by O +the O +ectoine B-protein_type +hydroxylase I-protein_type +( O +EctD B-protein_type +) O +( O +EC O +1 O +. O +14 O +. O +11 O +), O +a O +member O +of O +the O +non B-protein_type +- I-protein_type +heme I-protein_type +containing I-protein_type +iron I-protein_type +( I-protein_type +II I-protein_type +) I-protein_type +and I-protein_type +2 I-protein_type +- I-protein_type +oxoglutarate I-protein_type +- I-protein_type +dependent I-protein_type +dioxygenase I-protein_type +superfamily I-protein_type +. O + +The O +remarkable O +function O +preserving O +effects O +of O +ectoines B-chemical +for O +macromolecules O +and O +cells O +, O +frequently O +also O +addressed O +as O +chemical O +chaperones O +, O +led O +to O +a O +substantial O +interest O +in O +exploiting O +these O +compounds O +for O +biotechnological O +purposes O +and O +medical O +applications O +. O + +Biosynthetic O +routes O +for O +ectoine B-chemical +and O +5 B-chemical +- I-chemical +hydroxyectoine I-chemical +. O + +Scheme O +of O +the O +ectoine B-chemical +and O +5 B-chemical +- I-chemical +hydroxyectoine I-chemical +biosynthetic O +pathway O +. O + +Here O +we O +focus O +on O +ectoine B-protein_type +synthase I-protein_type +( O +EctC B-protein +), O +the O +key O +enzyme O +of O +the O +ectoine B-chemical +biosynthetic O +route O +( O +Fig O +1 O +). O + +Biochemical O +characterizations B-experimental_method +of O +ectoine B-protein_type +synthases I-protein_type +from O +the O +extremophiles B-taxonomy_domain +Halomonas B-species +elongata I-species +, O +Methylomicrobium B-species +alcaliphilum I-species +, O +and O +Acidiphilium B-species +cryptum I-species +, O +and O +from O +the O +nitrifying B-taxonomy_domain +archaeon I-taxonomy_domain +Nitrosopumilus B-species +maritimus I-species +have O +been O +carried O +out O +. O + +Each O +of O +these O +enzymes O +catalyzes O +as O +their O +main O +activity O +the O +cyclization O +of O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +acetyl I-chemical +- I-chemical +L I-chemical +- I-chemical +2 I-chemical +, I-chemical +4 I-chemical +- I-chemical +diaminobutyric I-chemical +acid I-chemical +( O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +), O +the O +reaction O +product O +of O +the O +2 B-protein_type +, I-protein_type +4 I-protein_type +- I-protein_type +diaminobutyrate I-protein_type +acetyltransferase I-protein_type +( O +EctA B-protein_type +), O +to O +ectoine B-chemical +with O +the O +concomitant O +release O +of O +a O +water B-chemical +molecule O +( O +Fig O +1 O +). O + +In O +side O +reactions O +, O +EctC B-protein +can O +promote O +the O +formation O +of O +the O +synthetic O +compatible O +solute O +5 B-chemical +- I-chemical +amino I-chemical +- I-chemical +3 I-chemical +, I-chemical +4 I-chemical +- I-chemical +dihydro I-chemical +- I-chemical +2H I-chemical +- I-chemical +pyrrole I-chemical +- I-chemical +2 I-chemical +- I-chemical +carboxylate I-chemical +( O +ADPC B-chemical +) O +through O +the O +cyclic O +condensation O +of O +two O +glutamine B-chemical +molecules O +and O +it O +also O +possesses O +a O +minor O +hydrolytic O +activity O +for O +ectoine B-chemical +and O +synthetic O +ectoine B-chemical +derivatives O +with O +either O +reduced O +or O +expanded O +ring O +sizes O +. O + +Although O +progress O +has O +been O +made O +with O +respect O +to O +the O +biochemical O +characterization O +of O +ectoine B-protein_type +synthase I-protein_type +, O +a O +clear O +understanding O +of O +how O +its O +structure B-evidence +contributes O +to O +its O +enzyme O +activity O +and O +reaction O +mechanism O +is O +still O +lacking O +. O +With O +this O +in O +mind O +, O +we O +have O +biochemically B-experimental_method +characterized I-experimental_method +the O +ectoine B-protein_type +synthase I-protein_type +from O +the O +cold O +- O +adapted O +marine B-taxonomy_domain +bacterium I-taxonomy_domain +Sphingopyxis B-species +alaskensis I-species +( O +Sa B-species +). O + +We O +demonstrate O +here O +for O +the O +first O +time O +that O +the O +ectoine B-protein_type +synthase I-protein_type +is O +a O +metal B-chemical +- O +dependent O +enzyme O +, O +with O +iron B-chemical +as O +the O +most O +likely O +physiologically O +relevant O +co O +- O +factor O +. O + +The O +EctC B-protein +protein O +forms O +a O +dimer B-oligomeric_state +in O +solution O +and O +our O +structural B-experimental_method +analysis I-experimental_method +identifies O +it O +as O +a O +member O +of O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +The O +two O +crystal B-evidence +structures I-evidence +that O +we O +report O +here O +for O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +with O +resolutions O +of O +1 O +. O +2 O +Å O +and O +2 O +. O +0 O +Å O +, O +respectively O +), O +and O +data O +derived O +from O +extensive O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +experiments O +targeting O +evolutionarily B-protein_state +highly I-protein_state +conserved I-protein_state +residues O +within O +the O +extended O +EctC B-protein_type +protein I-protein_type +family O +, O +provide O +a O +first O +view O +into O +the O +architecture O +of O +the O +catalytic B-site +core I-site +of O +the O +ectoine B-protein_type +synthase I-protein_type +. O + +Overproduction B-experimental_method +, O +purification B-experimental_method +and O +oligomeric O +state O +of O +the O +ectoine B-protein_type +synthase I-protein_type +in O +solution O + +We O +focused O +our O +biochemical B-experimental_method +and I-experimental_method +structural I-experimental_method +studies I-experimental_method +on O +the O +ectoine B-protein_type +synthase I-protein_type +from O +S B-species +. I-species +alaskensis I-species +[( O +Sa B-species +) O +EctC B-protein +], O +a O +cold O +- O +adapted O +marine B-taxonomy_domain +ultra I-taxonomy_domain +- I-taxonomy_domain +microbacterium I-taxonomy_domain +, O +from O +which O +we O +recently O +also O +determined O +the O +crystal B-evidence +structure I-evidence +of O +the O +ectoine B-protein_type +hydroxylase I-protein_type +( O +EctD B-protein_type +) O +in B-protein_state +complex I-protein_state +with I-protein_state +either O +its O +substrate O +or O +its O +reaction O +product O +. O + +We O +expressed O +a O +codon O +- O +optimized O +version O +of O +the O +S B-species +. I-species +alaskensis I-species +ectC B-gene +gene O +in O +E B-species +. I-species +coli I-species +to O +produce O +a O +recombinant O +protein O +with O +a O +carboxy O +- O +terminally O +attached O +Strep B-experimental_method +- I-experimental_method +tag I-experimental_method +II I-experimental_method +affinity I-experimental_method +peptide I-experimental_method +to O +allow O +purification O +of O +the O +( O +Sa B-species +) O +EctC B-protein +- O +Strep B-experimental_method +- I-experimental_method +Tag I-experimental_method +- I-experimental_method +II I-experimental_method +protein O +by O +affinity B-experimental_method +chromatography I-experimental_method +. O + +The O +( O +Sa B-species +) O +EctC B-protein +protein O +was O +overproduced O +and O +isolated O +with O +good O +yields O +( O +30 O +– O +40 O +mg O +L O +- O +1 O +of O +culture O +) O +and O +purity O +( O +S2a O +Fig O +). O + +Conventional O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +( O +SEC B-experimental_method +) O +has O +already O +shown O +that O +( O +Sa B-species +) O +EctC B-protein +preparations O +produced O +in O +this O +fashion O +are O +homogeneous O +and O +that O +the O +protein O +forms O +dimers B-oligomeric_state +in O +solution O +. O + +High B-experimental_method +performance I-experimental_method +liquid I-experimental_method +chromatography I-experimental_method +coupled O +with O +multi B-experimental_method +- I-experimental_method +angle I-experimental_method +light I-experimental_method +- I-experimental_method +scattering I-experimental_method +detection I-experimental_method +( O +HPLC B-experimental_method +- I-experimental_method +MALS I-experimental_method +) O +experiments O +carried O +out O +here O +confirmed O +that O +the O +purified O +( O +Sa B-species +) O +EctC B-protein +protein O +was O +mono O +- O +disperse O +and O +possessed O +a O +molecular O +mass O +of O +33 O +. O +0 O +± O +2 O +. O +3 O +kDa O +( O +S2b O +Fig O +). O + +This O +value O +corresponds O +very O +well O +with O +the O +theoretically O +calculated O +molecular O +mass O +of O +an O +( O +Sa B-species +) O +EctC B-protein +dimer B-oligomeric_state +( O +molecular O +mass O +of O +the O +monomer B-oligomeric_state +, O +including O +the O +Strep B-experimental_method +- I-experimental_method +tag I-experimental_method +II I-experimental_method +affinity I-experimental_method +peptide I-experimental_method +: O +16 O +. O +3 O +kDa O +). O + +Such O +a O +quaternary O +assembly O +as O +dimer B-oligomeric_state +has O +also O +been O +reported O +for O +the O +EctC B-protein_type +proteins I-protein_type +from O +H B-species +. I-species +elongata I-species +and O +N B-species +. I-species +maritimus I-species +. O + +Biochemical O +properties O +of O +the O +ectoine B-protein_type +synthase I-protein_type + +The O +EctA B-protein +- O +produced O +substrate O +of O +the O +ectoine B-protein_type +synthase I-protein_type +, O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +acetyl I-chemical +- I-chemical +L I-chemical +- I-chemical +2 I-chemical +, I-chemical +4 I-chemical +- I-chemical +diaminobutyric I-chemical +acid I-chemical +( O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +) O +( O +Fig O +1 O +), O +is O +commercially O +not O +available O +. O + +We O +used O +alkaline O +hydrolysis O +of O +ectoine B-chemical +and O +subsequent O +chromatography O +on O +silica O +gel O +columns O +to O +obtain O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +in O +chemically O +highly O +purified O +form O +( O +S1a O +Fig O +). O + +This O +procedure O +also O +yielded O +the O +isomer O +of O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +, O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +acetyl I-chemical +- I-chemical +L I-chemical +- I-chemical +2 I-chemical +, I-chemical +4 I-chemical +- I-chemical +diaminobutyric I-chemical +acid I-chemical +( O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +) O +( O +S1b O +Fig O +). O + +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +has O +so O +far O +not O +been O +considered O +as O +a O +substrate O +for O +EctC B-protein +, O +but O +microorganisms B-taxonomy_domain +that O +use O +ectoine B-chemical +as O +a O +nutrient O +produce O +it O +as O +an O +intermediate O +during O +catabolism O +. O + +Using O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +as O +the O +substrate O +, O +we O +initially O +evaluated O +a O +set O +of O +biochemical O +parameters O +of O +the O +recombinant O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +S B-species +. I-species +alaskensis I-species +, O +from O +which O +the O +studied O +ectoine B-protein_type +synthase I-protein_type +was O +originally O +derived O +, O +is O +a O +microorganism B-taxonomy_domain +that O +is O +well O +- O +adapted O +to O +a O +life O +in O +permanently O +cold O +ocean O +waters O +. O + +Consistent O +with O +the O +physicochemical O +attributes O +of O +this O +habitat O +, O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +was O +already O +enzymatically B-protein_state +active I-protein_state +at O +5 O +° O +C O +, O +had O +a O +temperature O +optimum O +of O +15 O +° O +C O +and O +was O +able O +to O +function O +over O +a O +broad O +range O +of O +temperatures O +( O +S3a O +Fig O +). O + +It O +possessed O +an O +alkaline B-protein_state +pH O +optimum O +of O +8 O +. O +5 O +( O +S3b O +Fig O +), O +a O +value O +similar O +to O +the O +ectoine B-protein_type +synthases I-protein_type +from O +the O +halo B-protein_state +- I-protein_state +tolerant I-protein_state +H B-species +. I-species +elongata I-species +( O +pH O +optimum O +of O +8 O +. O +5 O +to O +9 O +. O +0 O +), O +the O +alkaliphile B-taxonomy_domain +M B-species +. I-species +alcaliphilum I-species +( O +pH O +optimum O +of O +9 O +. O +0 O +), O +and O +the O +acidophile B-taxonomy_domain +Acidiphilium B-species +cryptum I-species +( O +pH O +optimum O +of O +8 O +. O +5 O +to O +9 O +. O +0 O +), O +whereas O +the O +EctC B-protein +protein O +from O +N B-species +. I-species +maritimus I-species +has O +a O +neutral B-protein_state +pH I-protein_state +optimum O +( O +pH O +7 O +. O +0 O +). O + +The O +salinity O +of O +the O +assay O +buffer O +had O +a O +significant O +influence O +on O +the O +maximal O +enzyme O +activity O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +An O +increase O +in O +either O +the O +NaCl B-chemical +or O +the O +KCl B-chemical +concentration O +led O +to O +an O +approximately O +5 O +- O +fold O +enhancement O +of O +the O +ectoine B-protein_type +synthase I-protein_type +activity O +. O + +The O +maximum O +enzyme O +activity O +of O +( O +Sa B-species +) O +EctC B-protein +occurred O +around O +250 O +mM O +NaCl B-chemical +or O +KCl B-chemical +, O +respectively O +. O + +( O +Sa B-species +) O +EctC B-protein +is O +a O +highly O +salt O +- O +tolerant O +enzyme O +since O +it O +exhibited O +substantial O +enzyme O +activity O +even O +at O +NaCl B-chemical +and O +KCl B-chemical +concentrations O +of O +1 O +M O +in O +the O +assay O +buffer O +( O +S3c O +and O +S3d O +Fig O +). O + +The O +stimulation O +of O +EctC B-protein +enzyme O +activity O +by O +salts O +has O +previously O +also O +been O +observed O +for O +other O +ectoine B-protein_type +synthases I-protein_type +. O + +The O +ectoine B-protein_type +synthase I-protein_type +is O +a O +metal B-protein_type +- I-protein_type +containing I-protein_type +protein I-protein_type + +Considerations O +based O +on O +bioinformatics O +suggests O +that O +EctC B-protein +belongs O +to O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +Most O +of O +these O +proteins O +contain O +catalytically O +important O +transition O +state O +metals O +such O +as O +iron B-chemical +, O +copper B-chemical +, O +zinc B-chemical +, O +manganese B-chemical +, O +cobalt B-chemical +, O +or O +nickel B-chemical +. O + +Cupins B-protein_type +contain O +two O +conserved B-protein_state +motifs O +: O +G B-structure_element +( I-structure_element +X I-structure_element +) I-structure_element +5HXH I-structure_element +( I-structure_element +X I-structure_element +) I-structure_element +3 I-structure_element +, I-structure_element +4E I-structure_element +( I-structure_element +X I-structure_element +) I-structure_element +6G I-structure_element +and O +G B-structure_element +( I-structure_element +X I-structure_element +) I-structure_element +5PXG I-structure_element +( I-structure_element +X I-structure_element +) I-structure_element +2H I-structure_element +( I-structure_element +X I-structure_element +) I-structure_element +3N I-structure_element +( O +the O +letters O +in O +bold O +represent O +those O +residues O +that O +often O +coordinate O +the O +metal B-chemical +). O + +Inspection O +of O +a O +previous O +alignment B-experimental_method +of I-experimental_method +the I-experimental_method +amino I-experimental_method +acid I-experimental_method +sequences I-experimental_method +of O +440 O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +revealed O +that O +the O +canonical O +metal B-structure_element +- I-structure_element +binding I-structure_element +motif I-structure_element +( O +s O +) O +of O +cupin B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +is O +not B-protein_state +conserved I-protein_state +among O +members O +of O +the O +extended O +ectoine B-protein_type +synthase I-protein_type +protein I-protein_type +family I-protein_type +. O + +An O +abbreviated O +alignment B-experimental_method +of I-experimental_method +the I-experimental_method +amino I-experimental_method +acid I-experimental_method +sequence I-experimental_method +of O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +is O +shown O +in O +Fig O +2 O +. O + +Abbreviated O +alignment B-experimental_method +of O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +. O + +The O +amino O +acid O +sequences O +of O +20 O +selected O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +are O +compared O +. O + +Strictly B-protein_state +conserved I-protein_state +amino O +acid O +residues O +are O +shown O +in O +yellow O +. O + +Dots O +shown O +above O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +sequence O +indicate O +residues O +likely O +to O +be O +involved O +in O +iron B-chemical +- O +binding O +( O +red O +), O +ligand O +- O +binding O +( O +green O +) O +and O +stabilization O +of O +the O +loop O +- O +architecture O +( O +blue O +). O + +The O +conserved B-protein_state +residue O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +with O +so O +- O +far O +undefined O +functions O +is O +indicated O +by O +a O +green O +dot O +circled O +in O +red O +. O + +Secondary O +structural O +elements O +( O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +β B-structure_element +- I-structure_element +sheets I-structure_element +) O +found O +in O +the O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structure I-evidence +are O +projected O +onto O +the O +amino O +acid O +sequences O +of O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +. O + +Since O +variations O +of O +the O +above O +- O +described O +metal B-structure_element +- I-structure_element +binding I-structure_element +motif I-structure_element +occur O +frequently O +, O +we O +experimentally O +investigated O +the O +presence O +and O +nature O +of O +the O +metal B-chemical +that O +might O +be O +contained O +in O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +by O +inductive B-experimental_method +- I-experimental_method +coupled I-experimental_method +plasma I-experimental_method +mass I-experimental_method +spectrometry I-experimental_method +( O +ICP B-experimental_method +- I-experimental_method +MS I-experimental_method +). O + +For O +this O +analysis O +we O +used O +recombinant O +( O +Sa B-species +) O +EctC B-protein +preparations O +from O +three O +independent O +protein O +overproduction O +and O +purification O +experiments O +. O + +The O +ICP B-experimental_method +- I-experimental_method +MS I-experimental_method +analyses O +yielded O +an O +iron B-chemical +content O +of O +0 O +. O +66 O +± O +0 O +. O +06 O +mol O +iron B-chemical +per O +mol O +of O +protein O +and O +the O +used O +( O +Sa B-species +) O +EctC B-protein +protein O +preparations O +also O +contained O +a O +minor O +amount O +of O +zinc B-chemical +( O +0 O +. O +08 O +mol O +zinc B-chemical +per O +mol O +of O +protein O +). O + +All O +other O +assayed O +metals O +( O +copper B-chemical +and O +nickel B-chemical +) O +were O +only O +present O +in O +trace O +amounts O +( O +0 O +. O +01 O +mol O +metal B-chemical +per O +mol O +of O +protein O +, O +respectively O +). O + +The O +presence O +of O +iron B-chemical +in O +these O +( O +Sa B-species +) O +EctC B-protein +protein O +preparations O +was O +further O +confirmed O +by O +a O +colorimetric B-experimental_method +method I-experimental_method +that O +is O +based O +on O +an O +iron B-chemical +- O +complexing O +reagent O +; O +this O +procedure O +yielded O +an O +iron B-chemical +- O +content O +of O +0 O +. O +84 O +± O +0 O +. O +05 O +mol O +per O +mol O +of O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +Hence O +, O +both O +ICP B-experimental_method +- I-experimental_method +MS I-experimental_method +and O +the O +colorimetric B-experimental_method +method I-experimental_method +clearly O +established O +that O +the O +recombinantly O +produced O +ectoine B-protein_type +synthase I-protein_type +from O +S B-species +. I-species +alaskensis I-species +is O +an O +iron B-chemical +- O +containing O +protein O +. O + +We O +note O +in O +this O +context O +, O +that O +the O +values O +obtained O +for O +the O +iron B-chemical +content O +of O +the O +( O +Sa B-species +) O +EctC B-protein +proteins O +varied O +by O +approximately O +10 O +to O +20 O +% O +between O +the O +two O +methods O +. O + +The O +reason O +for O +this O +difference O +is O +not O +known O +, O +but O +indicates O +that O +the O +well O +established O +colorimetric B-experimental_method +assay I-experimental_method +probably O +overestimates O +the O +iron B-chemical +content O +of O +( O +Sa B-species +) O +EctC B-protein +protein O +preparations O +to O +a O +certain O +degree O +. O + +A O +metal B-chemical +cofactor O +is O +important O +for O +the O +catalytic O +activity O +of O +EctC B-protein + +The O +iron B-chemical +detected O +in O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +preparations O +could O +serve O +a O +structural O +role O +, O +or O +most O +likely O +, O +could O +be O +critical O +for O +enzyme O +catalysis O +as O +is O +the O +case O +for O +many O +members O +of O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +To O +address O +these O +questions O +, O +we O +incubated B-experimental_method +the O +( O +Sa B-species +) O +EctC B-protein +enzyme O +with B-experimental_method +increasing I-experimental_method +concentrations I-experimental_method +of O +the O +metal B-chemical +chelator O +ethylene B-chemical +- I-chemical +diamine I-chemical +- I-chemical +tetraacetic I-chemical +- I-chemical +acid I-chemical +( O +EDTA B-chemical +) O +and O +subsequently O +assayed O +ectoine B-protein_type +synthase I-protein_type +activity O +. O + +The O +addition O +of O +very O +low O +concentrations O +of O +EDTA B-chemical +( O +0 O +. O +05 O +mM O +) O +to O +the O +EctC B-protein +enzyme O +already O +led O +to O +a O +noticeable O +inhibition O +of O +the O +ectoine B-protein_type +synthase I-protein_type +activity O +and O +the O +presence O +of O +1 O +mM O +EDTA B-chemical +completely O +inhibited O +the O +enzyme O +( O +Fig O +3a O +). O + +Dependency O +of O +the O +ectoine B-protein_type +synthase I-protein_type +activity O +on O +metals O +. O + +( O +a O +) O +Impact O +of O +the O +iron B-chemical +- O +chelator O +EDTA B-chemical +on O +the O +enzyme O +activity O +of O +the O +purified O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +Metal B-experimental_method +depletion I-experimental_method +and I-experimental_method +reconstitution I-experimental_method +experiments I-experimental_method +with O +( O +b O +) O +stoichiometric O +and O +( O +c O +) O +excess O +amounts O +of O +metals O +. O + +The O +( O +Sa B-species +) O +EctC B-protein +protein O +was O +present O +at O +a O +concentration O +of O +10 O +μM O +. O +The O +level O +of O +enzyme O +activity O +given O +in O +( O +b O +) O +is O +benchmarked O +relative O +to O +that O +of O +ectoine B-protein_type +synthase I-protein_type +enzyme B-experimental_method +assays I-experimental_method +in O +which O +1 O +mM O +FeCl2 B-chemical +was O +added O +. O + +We O +then O +took O +such O +an O +inactivated B-protein_state +enzyme O +preparation O +, O +removed O +the O +EDTA B-chemical +by O +dialysis B-experimental_method +, O +and O +added O +stoichiometric O +amounts O +( O +10 O +μM O +) O +of O +various O +metals O +to O +the O +( O +Sa B-species +) O +EctC B-protein +enzyme O +. O + +The O +addition O +of O +FeCl2 B-chemical +to O +the O +enzyme B-experimental_method +assay I-experimental_method +restored O +enzyme O +activity O +to O +about O +38 O +%, O +whereas O +the O +addition O +of O +ZnCl2 B-chemical +or O +CoCl2 B-chemical +rescued O +( O +Sa B-species +) O +EctC B-protein +enzyme O +activity O +only O +to O +5 O +% O +and O +3 O +%, O +respectively O +. O + +All O +other O +tested O +metals O +, O +including O +Fe3 B-chemical ++, I-chemical +were O +unable O +to O +restore O +activity O +( O +Fig O +3b O +). O + +When O +the O +concentration O +of O +the O +various O +metals O +in O +the O +enzyme B-experimental_method +assay I-experimental_method +was O +increased O +100 O +- O +fold O +, O +Fe2 B-chemical ++ I-chemical +exhibited O +again O +the O +strongest O +stimulating O +effect O +on O +enzyme O +activity O +, O +and O +rescued O +enzyme O +activity O +to O +a O +degree O +similar O +to O +that O +exhibited O +by O +( O +Sa B-species +) O +EctC B-protein +protein O +preparations O +that O +had O +not O +been O +inactivated O +through O +EDTA B-chemical +treatment O +( O +Fig O +3c O +). O + +However O +, O +a O +large O +molar O +excess O +of O +other O +transition O +- O +state O +metals O +( O +zinc B-chemical +, O +cobalt B-chemical +, O +nickel B-chemical +, O +copper B-chemical +, O +and O +manganese B-chemical +) O +typically O +found O +in O +members O +of O +the O +cupin B-protein_type +superfamily I-protein_type +allowed O +the O +partial O +rescue O +of O +ectoine B-protein_type +synthase I-protein_type +activity O +as O +well O +( O +Fig O +3c O +). O + +This O +is O +in O +line O +with O +literature O +data O +showing O +that O +cupin B-protein_type +- I-protein_type +type I-protein_type +enzymes I-protein_type +are O +often O +promiscuous O +with O +respect O +to O +the O +use O +of O +the O +catalytically O +important O +metal B-chemical +. O + +Kinetic O +parameters O +of O +EctC B-protein +for O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +and O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical + +Based O +on O +the O +data O +presented O +in O +S3 O +Fig O +, O +we O +formulated O +an O +optimized O +activity B-experimental_method +assay I-experimental_method +for O +the O +ectoine B-protein_type +synthase I-protein_type +of O +S B-species +. I-species +alaskensis I-species +and O +used O +it O +to O +determined O +the O +kinetic O +parameters O +for O +the O +( O +Sa B-species +) O +EctC B-protein +enzyme O +for O +both O +its O +natural O +substrate O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +and O +the O +isomer O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +. O + +The O +EctC B-protein +- O +catalyzed O +ring O +- O +closure O +of O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +to O +form O +ectoine B-chemical +exhibited O +Michaelis B-experimental_method +- I-experimental_method +Menten I-experimental_method +- I-experimental_method +kinetics I-experimental_method +with O +an O +apparent O +Km B-evidence +of O +4 O +. O +9 O +± O +0 O +. O +5 O +mM O +, O +a O +vmax B-evidence +of O +25 O +. O +0 O +± O +0 O +. O +8 O +U O +/ O +mg O +and O +a O +kcat B-evidence +of O +7 O +. O +2 O +s O +- O +1 O +( O +S4a O +Fig O +). O + +Given O +the O +chemical O +relatedness O +of O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +to O +the O +natural O +substrate O +( O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +) O +of O +the O +ectoine B-protein_type +synthase I-protein_type +( O +S1a O +and O +S1b O +Fig O +), O +we O +wondered O +whether O +( O +Sa B-species +) O +EctC B-protein +could O +also O +use O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +to O +produce O +ectoine B-chemical +. O + +( O +Sa B-species +) O +EctC B-protein +catalyzed O +this O +reaction O +with O +Michaelis B-experimental_method +- I-experimental_method +Menten I-experimental_method +- I-experimental_method +kinetics I-experimental_method +exhibiting O +an O +apparent O +Km B-evidence +of O +25 O +. O +4 O +± O +2 O +. O +9 O +mM O +, O +a O +vmax B-evidence +of O +24 O +. O +6 O +± O +1 O +. O +0 O +U O +/ O +mg O +and O +a O +kcat B-evidence +0 O +. O +6 O +s O +- O +1 O +( O +S4b O +Fig O +). O + +Hence O +, O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +is O +a O +newly O +recognized O +substrate O +for O +ectoine B-protein_type +synthase I-protein_type +. O + +However O +, O +both O +the O +affinity B-evidence +( O +Km B-evidence +) O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +and O +its O +catalytic B-evidence +efficiency I-evidence +( O +kcat B-evidence +/ I-evidence +Km I-evidence +) O +were O +strongly O +reduced O +in O +comparison O +with O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +. O + +The O +Km B-evidence +dropped O +fife O +- O +fold O +from O +4 O +. O +9 O +± O +0 O +. O +5 O +mM O +to O +25 O +. O +4 O +± O +2 O +. O +9 O +mM O +, O +and O +the O +catalytic B-evidence +efficiency I-evidence +was O +reduced O +from O +1 O +. O +47 O +mM O +- O +1 O +s O +- O +1 O +to O +0 O +. O +02 O +mM O +- O +1 O +s O +- O +1 O +, O +a O +73 O +- O +fold O +decrease O +. O + +Both O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +and O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +are O +concomitantly O +formed O +during O +the O +enzymatic O +hydrolysis O +of O +the O +ectoine B-chemical +ring O +during O +catabolism O +. O + +Our O +finding O +that O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +is O +a O +substrate O +for O +ectoine B-protein_type +synthase I-protein_type +has O +bearings O +for O +an O +understanding O +of O +the O +physiology O +of O +those O +microorganisms B-taxonomy_domain +that O +can O +both O +synthesize O +and O +catabolize O +ectoine B-chemical +. O + +However O +, O +these O +types O +of O +microorganisms B-taxonomy_domain +should O +still O +be O +able O +to O +largely O +avoid O +a O +futile O +cycle O +since O +the O +affinity B-evidence +of O +ectoine B-protein_type +synthase I-protein_type +for O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +and O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +, O +and O +its O +catalytic B-evidence +efficiency I-evidence +for O +the O +two O +compounds O +, O +differs O +substantially O +( O +S4a O +and O +S4b O +Fig O +). O + +Crystallization B-experimental_method +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O + +Since O +no O +crystal B-evidence +structure I-evidence +of O +ectoine B-protein_type +synthase I-protein_type +has O +been O +reported O +, O +we O +set O +out O +to O +crystallize B-experimental_method +the O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +Attempts O +to O +obtain O +crystals B-evidence +of O +( O +Sa B-species +) O +EctC B-protein +in B-protein_state +complex I-protein_state +either O +with O +its O +substrate O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +or O +its O +reaction O +product O +ectoine B-chemical +were O +not O +successful O +. O + +However O +, O +two O +crystal B-evidence +forms I-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +in O +the O +absence B-protein_state +of I-protein_state +the O +substrate O +were O +obtained O +. O + +Attempts O +to O +solve O +the O +crystal B-evidence +structure I-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +by O +molecular B-experimental_method +replacement I-experimental_method +has O +previously O +failed O +. O + +However O +, O +we O +were O +able O +to O +obtain O +crystals B-evidence +of O +form O +B O +that O +were O +derivatized O +with O +mercury B-chemical +and O +these O +diffracted O +up O +to O +2 O +. O +8 O +Å O +( O +S1 O +Table O +). O + +This O +dataset O +was O +used O +to O +derive O +an O +initial O +structural B-evidence +model I-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +, O +which O +in O +turn O +was O +employed O +as O +a O +template O +for O +molecular B-experimental_method +replacement I-experimental_method +to O +phase O +the O +native O +dataset O +( O +2 O +. O +0 O +Å O +) O +of O +crystal O +form O +B O +. O +After O +several O +rounds O +of O +manual O +model O +building O +and O +refinement O +, O +four O +monomers B-oligomeric_state +of O +( O +Sa B-species +) O +EctC B-protein +were O +identified O +and O +the O +crystal B-evidence +structure I-evidence +was O +refined O +to O +a O +final O +Rcryst B-evidence +of O +21 O +. O +1 O +% O +and O +an O +Rfree B-evidence +of O +24 O +. O +8 O +% O +( O +S1 O +Table O +). O + +Finally O +, O +a O +monomer B-oligomeric_state +of O +this O +structure B-evidence +was O +used O +as O +a O +template O +for O +molecular B-experimental_method +replacement I-experimental_method +to O +phase O +the O +high O +- O +resolution O +( O +1 O +. O +2 O +Å O +) O +dataset O +of O +crystal O +form O +A O +, O +which O +was O +subsequently O +refined O +to O +a O +final O +Rcryst B-evidence +of O +12 O +. O +4 O +% O +and O +an O +Rfree B-evidence +of O +14 O +. O +9 O +% O +( O +S1 O +Table O +). O + +Overall O +fold O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O + +The O +two O +EctC B-protein +structures B-evidence +that O +we O +determined O +revealed O +that O +the O +ectoine B-protein_type +synthase I-protein_type +belongs O +to O +the O +cupin B-protein_type +superfamily I-protein_type +with O +respect O +to O +its O +overall O +fold O +( O +Fig O +4a O +– O +4c O +). O + +However O +, O +they O +represent O +two O +different O +states O +of O +the O +137 B-residue_range +amino I-residue_range +acids I-residue_range +comprising O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +2 O +). O + +First O +, O +the O +1 O +. O +2 O +Å O +structure B-evidence +reveals O +the O +spatial O +configuration O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +ranging O +from O +amino O +acid O +Met B-residue_range +- I-residue_range +1 I-residue_range +to I-residue_range +Glu I-residue_range +- I-residue_range +115 I-residue_range +; O +hence O +, O +it O +lacks B-protein_state +22 B-residue_range +amino I-residue_range +acids I-residue_range +at O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +of O +the O +authentic O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +This O +structure B-evidence +adopts O +an O +open B-protein_state +conformation O +with O +respect O +to O +the O +typical O +fold O +of O +cupin B-structure_element +barrels I-structure_element +and O +is O +therefore O +termed O +in O +the O +following O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +( O +Fig O +4b O +). O + +In O +this O +structure B-evidence +no O +metal B-chemical +co O +- O +factor O +was O +identified O +. O + +The O +second O +crystal B-evidence +structure I-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +was O +solved B-experimental_method +at O +a O +resolution O +of O +2 O +. O +0 O +Å O +and O +contained O +four O +molecules O +of O +the O +protein O +in O +the O +asymmetric O +unit O +of O +which O +protomer B-oligomeric_state +A B-structure_element +comprised O +amino O +acid O +Met B-residue_range +- I-residue_range +1 I-residue_range +to I-residue_range +Gly I-residue_range +- I-residue_range +121 I-residue_range +and O +adopts O +a O +closed B-protein_state +conformation O +. O + +Hence O +, O +it O +still O +lacks B-protein_state +16 B-residue_range +amino I-residue_range +acid I-residue_range +residues O +of O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +of O +the O +authentic O +137 B-residue_range +amino I-residue_range +acids I-residue_range +comprising O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +2 O +). O + +We O +therefore O +cannot O +exclude O +that O +this O +crystal B-evidence +structure I-evidence +does O +not O +represent O +the O +fully B-protein_state +closed I-protein_state +state O +of O +the O +ectoine B-protein_type +synthase I-protein_type +; O +consequently O +, O +we O +tentatively O +termed O +it O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +. O + +Interestingly O +, O +the O +three O +other O +monomers B-oligomeric_state +present O +in O +the O +asymmetric O +unit O +all O +range O +from O +Met B-residue_range +- I-residue_range +1 I-residue_range +to I-residue_range +Glu I-residue_range +- I-residue_range +115 I-residue_range +and O +adopt O +a O +conformation O +similar O +to O +the O +“ O +open B-protein_state +” O +EctC B-protein +structure B-evidence +. O + +Overall O +structure B-evidence +of O +the O +“ O +open B-protein_state +” O +and O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +crystal B-evidence +structures I-evidence +of O +( O +Sa B-species +) O +EctC B-protein +. O + +( O +a O +) O +The O +overall O +structure B-evidence +of O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +resolved O +at O +2 O +. O +0 O +Å O +is O +depicted O +in O +green O +in O +a O +cartoon O +( O +upper O +panel O +) O +and O +surface O +( O +lower O +panel O +) O +representation O +. O + +The O +β B-structure_element +- I-structure_element +strands I-structure_element +are O +numbered O +β1 B-structure_element +- I-structure_element +β11 I-structure_element +and O +the O +helices B-structure_element +α B-structure_element +- I-structure_element +I I-structure_element +to I-structure_element +α I-structure_element +- I-structure_element +II I-structure_element +. O + +( O +b O +) O +The O +overall O +structure B-evidence +of O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +was O +resolved O +at O +1 O +. O +2 O +Å O +and O +is O +depicted O +in O +yellow O +in O +a O +cartoon O +( O +upper O +panel O +) O +and O +surface O +( O +lower O +panel O +) O +representation O +. O + +The O +entrance O +to O +the O +active B-site +site I-site +of O +the O +ectoine B-protein_type +synthase I-protein_type +is O +marked O +. O + +( O +c O +) O +Overlay B-experimental_method +of O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +and O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structures B-evidence +. O + +The O +overall O +structure B-evidence +of O +( O +Sa B-species +) O +EctC B-protein +is O +basically O +the O +same O +in O +both O +crystals B-evidence +except O +for O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +, O +which O +covers O +the O +entry O +of O +one O +side O +of O +the O +cupin B-structure_element +barrel I-structure_element +from O +the O +surroundings O +in O +monomer B-oligomeric_state +A B-structure_element +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +. O + +This O +is O +reflected O +by O +the O +calculated O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +RMSD B-evidence +) O +of O +the O +Cα O +atoms O +that O +was O +about O +0 O +. O +56 O +Å O +( O +over O +117 O +residues O +) O +when O +the O +four O +“ O +open B-protein_state +” O +monomers B-oligomeric_state +were O +compared O +with O +each O +other O +. O + +However O +, O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +monomer B-oligomeric_state +has O +a O +slightly O +higher O +RMSD B-evidence +of O +1 O +. O +4 O +Å O +( O +over O +117 O +residues O +) O +when O +compared O +with O +the O +“ O +open B-protein_state +” O +2 O +. O +0 O +Å O +structure B-evidence +. O + +Therefore O +, O +we O +describe O +in O +the O +following O +the O +overall O +structure B-evidence +for O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +form O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +and O +subsequently O +highlight O +the O +structural O +differences O +between O +the O +“ O +open B-protein_state +” O +and O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +forms O +in O +more O +detail O +. O + +The O +structure B-evidence +of O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +protein O +consists O +of O +11 O +β B-structure_element +- I-structure_element +strands I-structure_element +( O +β1 B-structure_element +- I-structure_element +β11 I-structure_element +) O +and O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +α B-structure_element +- I-structure_element +I I-structure_element +and O +α B-structure_element +- I-structure_element +II I-structure_element +) O +( O +Fig O +4a O +). O + +The O +β B-structure_element +- I-structure_element +strands I-structure_element +form O +two O +anti B-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +: O +β2 B-structure_element +β3 B-structure_element +, O +β4 B-structure_element +, O +β11 B-structure_element +, O +β6 B-structure_element +, O +and O +β9 B-structure_element +, O +and O +a O +smaller O +three B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +( O +β7 B-structure_element +, O +β8 B-structure_element +, O +and O +β10 B-structure_element +), O +respectively O +. O + +These O +two O +β B-structure_element +- I-structure_element +sheets I-structure_element +pack O +against O +each O +other O +, O +forming O +a O +cup B-structure_element +- I-structure_element +shaped I-structure_element +β I-structure_element +- I-structure_element +sandwich I-structure_element +with O +a O +topology O +characteristic O +for O +the O +cupin B-structure_element +- I-structure_element +fold I-structure_element +. O + +Hence O +, O +( O +Sa B-species +) O +EctC B-protein +adopts O +an O +overall O +bowl O +shape O +in O +which O +one O +side O +is O +opened O +towards O +the O +solvent O +( O +Fig O +4a O +to O +4c O +). O + +In O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +, O +a O +longer O +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +tail I-structure_element +is O +visible O +in O +the O +electron B-evidence +density I-evidence +, O +folding O +into O +a O +small B-structure_element +helix I-structure_element +( O +α B-structure_element +- I-structure_element +II I-structure_element +) O +that O +closes O +the O +active B-site +site I-site +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +4a O +). O + +The O +formation O +of O +this O +α B-structure_element +- I-structure_element +II I-structure_element +helix I-structure_element +induces O +a O +reorientation O +and O +shift O +of O +a O +long O +unstructured B-protein_state +loop B-structure_element +( O +as O +observed O +in O +the O +“ O +open B-protein_state +” O +structure B-evidence +) O +connecting O +β4 B-structure_element +and O +β6 B-structure_element +, O +resulting O +in O +the O +formation O +of O +the O +stable B-protein_state +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +as O +observed O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +state O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +4a O +). O + +Structural B-experimental_method +comparison I-experimental_method +analyses I-experimental_method +using O +the O +DALI B-experimental_method +server I-experimental_method +revealed O +that O +( O +Sa B-species +) O +EctC B-protein +adopts O +a O +fold O +similar O +to O +other O +members O +of O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +The O +highest O +structural O +similarities O +are O +observed O +for O +the O +Cupin B-protein +2 I-protein +conserved I-protein +barrel I-protein +domain I-protein +protein I-protein +( O +YP_751781 B-protein +. I-protein +1 I-protein +) O +from O +Shewanella B-species +frigidimarina I-species +( O +PDB O +accession O +code O +: O +2PFW O +) O +with O +a O +Z B-evidence +- I-evidence +score I-evidence +of O +13 O +. O +1 O +and O +an O +RMSD B-evidence +of O +2 O +. O +2 O +Å O +over O +104 O +Cα O +- O +atoms O +( O +structural O +data O +for O +this O +protein O +have O +been O +deposited O +in O +the O +PDB O +but O +no O +publication O +connected O +to O +this O +structure B-evidence +is O +currently O +available O +), O +a O +manganese B-protein +- I-protein +containing I-protein +cupin I-protein +( O +TM1459 B-protein +) O +from O +Thermotoga B-species +maritima I-species +( O +PDB O +accession O +code O +: O +1VJ2 O +) O +with O +a O +Z B-evidence +- I-evidence +score I-evidence +of O +12 O +. O +8 O +and O +an O +RMSD B-evidence +of O +2 O +. O +0 O +Å O +over O +103 O +Cα O +- O +atoms O +, O +the O +cyclase B-protein_type +RemF B-protein +from O +Streptomyces B-species +resistomycificus I-species +( O +PDB O +accession O +code O +: O +3HT1 O +with O +a O +Z B-evidence +- I-evidence +score I-evidence +of O +11 O +. O +9 O +and O +an O +RMSD B-evidence +of O +1 O +. O +9 O +Å O +over O +102 O +Cα O +- O +atoms O +), O +and O +an O +auxin B-protein +- I-protein +binding I-protein +protein I-protein +1 I-protein +from O +Zea B-species +mays I-species +( O +PDB O +accession O +code O +: O +1LR5 O +) O +with O +an O +Z B-evidence +- I-evidence +score I-evidence +of O +11 O +. O +8 O +and O +an O +RMSD B-evidence +of O +2 O +. O +8 O +Å O +over O +104 O +Cα O +- O +atoms O +). O + +Our O +data O +classify O +EctC B-protein +, O +in O +addition O +to O +the O +polyketide B-protein_type +cyclase I-protein_type +RemF B-protein +, O +as O +the O +second O +known O +cupin B-protein_type +- I-protein_type +related I-protein_type +enzyme O +that O +catalyze O +a O +cyclocondensation O +reaction O +. O + +Next O +to O +RemF B-protein +and O +the O +aldos B-protein_type +- I-protein_type +2 I-protein_type +- I-protein_type +ulose I-protein_type +dehydratase I-protein_type +/ O +isomerase B-protein_type +, O +the O +ectoine B-protein_type +synthase I-protein_type +is O +only O +the O +third O +characterized O +dehydratase B-protein_type +within O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +Analysis O +of O +the O +EctC B-protein +dimer B-site +interface I-site +as O +observed O +in O +the O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structure I-evidence + +Both O +the O +SEC B-experimental_method +analysis O +and O +the O +HPLC B-experimental_method +- I-experimental_method +MALS I-experimental_method +experiments O +( O +S2b O +Fig O +) O +have O +shown O +that O +the O +ectoine B-protein_type +synthase I-protein_type +from O +S B-species +. I-species +alaskensis I-species +is O +a O +dimer B-oligomeric_state +in O +solution O +. O + +The O +crystal B-evidence +structure I-evidence +of O +this O +protein O +reflects O +this O +quaternary O +arrangement O +. O + +In O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +crystal B-evidence +structure I-evidence +, O +( O +Sa B-species +) O +EctC B-protein +has O +crystallized B-experimental_method +as O +a O +dimer B-oligomeric_state +of O +dimers B-oligomeric_state +within O +the O +asymmetric O +unit O +. O + +This O +dimer B-oligomeric_state +( O +Fig O +5a O +and O +5b O +) O +is O +composed O +of O +two O +monomers B-oligomeric_state +arranged O +in O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +orientation O +and O +is O +stabilized O +via O +strong O +interactions O +mediated O +by O +two O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +strands I-structure_element +, O +β B-structure_element +- I-structure_element +strand I-structure_element +β1 B-structure_element +( O +sequence O +1MIVRN5 B-structure_element +) O +from O +monomer B-oligomeric_state +A B-structure_element +and O +β B-structure_element +- I-structure_element +strand I-structure_element +β8 B-structure_element +from O +monomer B-oligomeric_state +B B-structure_element +( O +sequence O +82GVMYAL87 B-structure_element +) O +( O +Fig O +5c O +). O + +The O +strong O +interactions O +between O +these O +β B-structure_element +- I-structure_element +strands I-structure_element +rely O +primarily O +on O +backbone O +contacts O +. O + +In O +addition O +to O +these O +interactions O +, O +some O +weaker O +hydrophobic B-bond_interaction +interactions I-bond_interaction +are O +also O +observed O +between O +the O +two O +monomers B-oligomeric_state +in O +some O +loops B-structure_element +connecting O +the O +β B-structure_element +- I-structure_element +strands I-structure_element +. O + +As O +calculated O +with O +PDBePISA B-experimental_method +, O +the O +surface O +area O +buried O +upon O +dimer B-oligomeric_state +formation O +is O +1462 O +Å2 O +, O +which O +is O +20 O +. O +5 O +% O +of O +the O +total O +accessible O +surface O +of O +a O +monomer B-oligomeric_state +of O +this O +protein O +. O + +Both O +values O +fall O +within O +the O +range O +for O +known O +functional O +dimers B-oligomeric_state +. O + +Crystal B-evidence +structure I-evidence +of O +( O +Sa B-species +) O +EctC B-protein +. O + +( O +a O +) O +Top O +- O +view O +of O +the O +dimer B-oligomeric_state +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +The O +position O +of O +the O +water B-chemical +molecule O +, O +described O +in O +detail O +in O +the O +text O +, O +is O +shown O +in O +one O +of O +the O +monomers B-oligomeric_state +as O +an O +orange O +sphere O +. O +( O +b O +) O +Side O +- O +view O +of O +a O +( O +Sa B-species +) O +EctC B-protein +dimer B-oligomeric_state +allowing O +an O +assessment O +of O +the O +dimer B-site +interface I-site +formed O +by O +two O +β B-structure_element +- I-structure_element +strands I-structure_element +of O +each O +monomer B-oligomeric_state +. O + +( O +c O +) O +Close O +- O +up O +representation O +of O +the O +dimer B-site +interface I-site +mediated O +by O +beta B-structure_element +- I-structure_element +strand I-structure_element +β1 B-structure_element +and O +β6 B-structure_element +. O + +In O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +, O +one O +monomer B-oligomeric_state +is O +present O +in O +the O +asymmetric O +unit O +. O + +We O +therefore O +inspected O +the O +crystal O +packing B-experimental_method +and O +analyzed O +the O +monomer B-oligomeric_state +- O +monomer B-oligomeric_state +interactions O +with O +symmetry O +related O +molecules O +to O +elucidate O +whether O +a O +physiologically O +relevant O +dimer B-oligomeric_state +could O +be O +deduced O +from O +this O +crystal B-evidence +form I-evidence +as O +well O +. O + +Indeed O +, O +a O +similar O +dimer B-oligomeric_state +configuration O +to O +the O +one O +described O +for O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +is O +observed O +with O +the O +same O +monomer B-oligomeric_state +- O +monomer B-oligomeric_state +interactions O +mediated O +by O +the O +two O +β B-structure_element +- I-structure_element +sheets I-structure_element +. O + +The O +crystallographic O +two O +- O +fold O +axis O +present O +within O +the O +crystal O +symmetry O +is O +located O +exactly O +in O +between O +the O +two O +monomers B-oligomeric_state +, O +resulting O +in O +a O +monomer B-oligomeric_state +within O +the O +asymmetric O +unit O +. O + +Hence O +, O +the O +same O +dimer B-oligomeric_state +observed O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +of O +( O +Sa B-species +) O +EctC B-protein +can O +also O +be O +observed O +in O +the O +“ O +open B-protein_state +” O +structure B-evidence +. O + +Interestingly O +, O +the O +proteins O +identified O +by O +the O +above O +- O +described O +DALI B-experimental_method +search I-experimental_method +not O +only O +have O +folds O +similar O +to O +EctC B-protein +, O +but O +are O +also O +functional O +dimers B-oligomeric_state +that O +adopt O +similar O +monomer B-oligomeric_state +- O +monomer B-oligomeric_state +interactions O +within O +the O +dimer B-oligomeric_state +assembly O +as O +deduced O +from O +the O +inspection O +of O +the O +corresponding O +PDB O +files O +( O +2PFW O +, O +3HT1 O +, O +1VJ2 O +, O +1LR5 O +). O + +Structural O +rearrangements O +of O +the O +flexible B-protein_state +( O +Sa B-species +) O +EctC B-protein +carboxy B-structure_element +- I-structure_element +terminus I-structure_element + +The O +cupin O +core O +represents O +the O +structural O +framework O +of O +ectoine B-protein_type +synthase I-protein_type +( O +Figs O +4 O +and O +5 O +). O + +The O +major O +difference O +in O +the O +two O +crystal B-evidence +structures I-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +reported O +here O +is O +the O +orientation O +of O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +. O + +Some O +amino O +acids O +located O +in O +the O +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +the O +137 B-residue_range +amino I-residue_range +acids I-residue_range +comprising O +( O +Sa B-species +) O +EctC B-protein +protein O +are O +highly B-protein_state +conserved I-protein_state +( O +Fig O +2 O +) O +within O +the O +extended B-protein_state +EctC B-protein_type +protein I-protein_type +family O +. O + +At O +the O +end O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β11 B-structure_element +, O +two O +consecutive O +conserved B-protein_state +proline B-residue_name +residues O +( O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +and O +Pro B-residue_name_number +- I-residue_name_number +110 I-residue_name_number +) O +are O +present O +that O +are O +responsible O +for O +a O +turn O +in O +the O +main O +chain O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +In O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +, O +the O +visible O +electron B-evidence +density I-evidence +of O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +is O +extended O +by O +7 B-residue_range +amino I-residue_range +acid I-residue_range +residues I-residue_range +and O +ends O +at O +position O +Gly B-residue_name_number +- I-residue_name_number +121 I-residue_name_number +. O + +These O +additional O +amino O +acids O +fold O +into O +a O +small B-structure_element +helix I-structure_element +, O +which O +seals O +the O +open B-protein_state +cavity B-site +of O +the O +cupin B-structure_element +- I-structure_element +fold I-structure_element +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +4a O +). O + +Furthermore O +, O +this O +helix B-structure_element +is O +stabilized O +via O +interactions O +with O +the O +loop B-structure_element +region I-structure_element +between O +β B-structure_element +- I-structure_element +strands I-structure_element +β4 B-structure_element +and O +β6 B-structure_element +, O +thereby O +inducing O +a O +structural O +rearrangement O +. O + +This O +induces O +the O +formation O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +, O +which O +is O +not O +present O +when O +the O +small B-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +helix I-structure_element +is O +absent B-protein_state +as O +observed O +in O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +. O + +As O +a O +result O +, O +the O +newly O +formed O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +is O +reoriented O +and O +moved O +by O +2 O +. O +4 O +Å O +within O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +( O +Fig O +4a O +to O +4c O +). O + +It O +is O +worth O +mentioning O +that O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +is O +located O +next O +to O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +, O +which O +in O +all O +likelihood O +involved O +in O +metal B-chemical +binding O +( O +see O +below O +). O + +The O +position O +of O +this O +His B-residue_name +residue O +is O +slightly O +shifted O +in O +both O +( O +Sa B-species +) O +EctC B-protein +structures B-evidence +, O +likely O +the O +result O +of O +the O +formation O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +. O + +Therefore O +the O +sealing O +of O +the O +cupin B-structure_element +fold I-structure_element +, O +as O +described O +above O +, O +seem O +to O +have O +an O +indirect O +influence O +on O +the O +architecture O +of O +the O +postulated O +iron B-site +- I-site +binding I-site +site I-site +. O + +The O +consecutive O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +and O +Pro B-residue_name_number +- I-residue_name_number +110 I-residue_name_number +residues O +found O +at O +the O +end O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β11are B-structure_element +highly B-protein_state +conserved I-protein_state +in O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +( O +Fig O +2 O +). O + +They O +are O +responsible O +for O +redirecting O +the O +main O +chain O +of O +the O +remaining O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +( O +27 B-residue_range +amino I-residue_range +acid I-residue_range +residues I-residue_range +) O +of O +( O +Sa B-species +) O +EctC B-protein +to O +close O +the O +cupin B-structure_element +fold I-structure_element +. O + +In O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +this O +results O +in O +a O +complete O +closure O +of O +the O +entry O +of O +the O +cupin B-structure_element +barrel I-structure_element +( O +Fig O +4a O +to O +4c O +). O + +In O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +, O +both O +proline B-residue_name +residues O +are O +visible O +in O +the O +electron B-evidence +density I-evidence +; O +however O +, O +almost O +directly O +after O +Pro B-residue_name_number +- I-residue_name_number +110 I-residue_name_number +, O +the O +electron B-evidence +density I-evidence +is O +drastically O +diminished O +caused O +by O +the O +flexibility O +of O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +. O + +A O +search O +for O +partners O +interacting O +with O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +revealed O +that O +it O +interacts O +via O +its O +backbone O +oxygen O +with O +the O +side O +chain O +of O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +as O +visible O +in O +both O +the O +“ O +open B-protein_state +” O +and O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structures B-evidence +. O + +The O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +/ O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +interaction O +ensures O +the O +stable B-protein_state +orientation O +of O +both O +proline B-residue_name +residues O +at O +the O +end O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β11 B-structure_element +. O + +Since O +these O +proline B-residue_name +residues O +are O +followed O +by O +the O +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +, O +the O +interaction O +of O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +with O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +will O +likely O +play O +a O +substantial O +role O +in O +spatially O +orienting O +this O +very O +flexible O +part O +of O +the O +protein O +. O + +In O +addition O +to O +the O +interactions O +between O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +and O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +, O +the O +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +( O +Sa B-species +) O +EctC B-protein +is O +held O +in O +position O +via O +an O +interaction O +of O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +with O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +, O +which O +stabilizes O +the O +conformation O +of O +the O +small B-structure_element +helix I-structure_element +in O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +further O +. O + +The O +interaction O +between O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +and O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +is O +only O +visible O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +where O +the O +partially B-protein_state +extended I-protein_state +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +is O +resolved O +in O +the O +electron B-evidence +density I-evidence +. O + +In O +the O +“ O +open B-protein_state +” O +structure B-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +, O +this O +interaction O +does O +not O +occur O +since O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +is O +rotated O +outwards O +( O +Fig O +6a O +and O +6b O +). O + +Hence O +, O +one O +might O +speculate O +that O +this O +missing O +interaction O +might O +be O +responsible O +for O +the O +flexibility O +of O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +in O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +and O +consequently O +results O +in O +less O +well O +defined O +electron B-evidence +density I-evidence +in O +this O +region O +. O + +Architecture O +of O +the O +presumed O +metal B-site +- I-site +binding I-site +site I-site +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +and O +its O +flexible B-protein_state +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +. O + +( O +a O +) O +The O +described O +water B-chemical +molecule O +( O +depicted O +as O +orange O +sphere O +) O +is O +bound O +via O +interactions O +with O +the O +side O +chains O +of O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +and O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +. O + +The O +position O +occupied O +by O +this O +water B-chemical +molecule O +represents O +probably O +the O +position O +of O +the O +Fe2 B-chemical ++ I-chemical +cofactor O +in O +the O +active B-site +side I-site +of O +the O +ectoine B-protein_type +synthase I-protein_type +. O + +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +interacts O +with O +the O +double B-structure_element +proline I-structure_element +motif I-structure_element +( O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +and O +Pro B-residue_name_number +- I-residue_name_number +110 I-residue_name_number +). O + +It O +is O +further O +stabilized O +via O +an O +interaction O +with O +the O +side O +chain O +of O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +which O +is O +localized O +in O +the O +flexible B-protein_state +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +( O +colored O +in O +orange O +) O +of O +( O +Sa B-species +) O +EctC B-protein +that O +is O +visible O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +. O + +( O +b O +) O +An O +overlay B-experimental_method +of O +the O +“ O +open B-protein_state +” O +( O +colored O +in O +light O +blue O +) O +and O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +colored O +in O +green O +) O +structure B-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +The O +putative O +iron B-site +binding I-site +site I-site +of O +( O +Sa B-species +) O +EctC B-protein + +In O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +of O +( O +Sa B-species +) O +EctC B-protein +, O +each O +of O +the O +four O +monomers B-oligomeric_state +in O +the O +asymmetric O +unit O +contains O +a O +relative O +strong O +electron B-evidence +density I-evidence +positioned O +within O +the O +cupin B-structure_element +barrel I-structure_element +. O + +Since O +( O +Sa B-species +) O +EctC B-protein +is O +a O +metal B-chemical +containing O +protein O +( O +Fig O +3 O +), O +we O +tried O +to O +fit O +either O +Fe2 B-chemical ++, I-chemical +or O +Zn2 B-chemical ++ I-chemical +ions O +into O +this O +density B-evidence +and O +also O +refined B-experimental_method +occupancy I-experimental_method +. O + +Only O +the O +refinement O +of O +Fe2 B-chemical ++ I-chemical +resulted O +in O +a O +visibly O +improved O +electron B-evidence +density I-evidence +, O +however O +with O +a O +low O +degree O +of O +occupancy O +. O + +This O +possible O +iron B-chemical +molecule O +is O +bound O +via O +interactions O +with O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +and O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +( O +Fig O +6a O +and O +6b O +). O + +The O +distance O +between O +the O +side O +chains O +of O +these O +residues O +and O +the O +( O +putative O +) O +iron B-chemical +co O +- O +factor O +is O +3 O +. O +1 O +Å O +for O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +2 O +. O +9 O +Å O +for O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +and O +2 O +. O +9 O +Å O +for O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +, O +respectively O +. O + +These O +distances O +are O +to O +long O +when O +compared O +to O +other O +iron B-site +binding I-site +sites I-site +, O +a O +fact O +that O +might O +be O +caused O +by O +the O +absence B-protein_state +of I-protein_state +the O +proper O +substrate O +in O +the O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structure I-evidence +. O + +Since O +both O +the O +refinement O +and O +the O +distance O +did O +not O +clearly O +identify O +an O +iron B-chemical +molecule O +, O +we O +decided O +to O +conservatively O +place O +a O +water B-chemical +molecule O +at O +this O +position O +. O + +The O +position O +of O +this O +water B-chemical +molecule O +is O +described O +in O +more O +detail O +below O +and O +is O +highlighted O +in O +Figs O +5a O +and O +5b O +and O +6a O +and O +6b O +as O +a O +sphere O +. O + +Interestingly O +, O +all O +three O +amino O +acids O +coordinating O +this O +water B-chemical +molecule O +are O +strictly B-protein_state +conserved I-protein_state +within O +an O +alignment B-experimental_method +of O +440 O +members O +of O +the O +EctC B-protein_type +protein I-protein_type +family O +( O +for O +an O +abbreviated O +alignment O +of O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +see O +Fig O +2 O +). O + +In O +the O +“ O +open B-protein_state +” O +structure B-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +, O +electron B-evidence +density I-evidence +is O +visible O +where O +the O +presumptive O +iron B-chemical +is O +positioned O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +. O + +However O +, O +this O +electron B-evidence +density I-evidence +fits O +perfectly O +to O +a O +water B-chemical +molecule O +and O +not O +to O +an O +iron B-chemical +, O +and O +the O +water B-chemical +molecule O +was O +clearly O +visible O +after O +the O +refinement O +at O +this O +high O +resolution O +( O +1 O +. O +2 O +Å O +) O +of O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +. O + +In O +a O +superimposition B-experimental_method +of O +both O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structures I-evidence +, O +the O +spatial O +arrangements O +of O +the O +side O +chains O +of O +the O +three O +amino O +acids O +( O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +and O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +) O +likely O +to O +contact O +the O +iron B-chemical +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +match O +nicely O +with O +those O +of O +the O +corresponding O +residues O +of O +the O +“ O +iron B-protein_state +- I-protein_state +free I-protein_state +” O +“ O +open B-protein_state +” O +structure B-evidence +( O +Fig O +6b O +). O + +Only O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +is O +slightly O +rotated O +inwards O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +, O +most O +likely O +due O +to O +formation O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +as O +described O +above O +. O + +Taken O +together O +, O +this O +observations O +indicate O +, O +that O +the O +architecture O +of O +the O +presumptive O +iron B-site +- I-site +binding I-site +site I-site +is O +pre O +- O +set O +for O +the O +binding O +of O +the O +catalytically O +important O +metal B-chemical +by O +the O +ectoine B-protein_type +synthase I-protein_type +. O + +Of O +note O +is O +the O +different O +spatial O +arrangement O +of O +the O +side O +- O +chain O +of O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +( O +located O +in O +a O +loop B-structure_element +after O +the O +end O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +) O +in O +the O +“ O +open B-protein_state +” O +and O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structures B-evidence +. O + +In O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +, O +the O +hydroxyl O +- O +group O +of O +the O +side O +- O +chain O +of O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +points O +towards O +the O +iron B-chemical +( O +Fig O +6a O +and O +6b O +), O +but O +the O +corresponding O +distance O +( O +3 O +. O +9 O +Å O +) O +makes O +it O +highly O +unlikely O +that O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +is O +directly O +involved O +in O +metal B-chemical +binding O +. O + +Nevertheless O +, O +its O +substitution B-experimental_method +by O +an O +Ala B-residue_name +residue O +causes O +a O +strong O +decrease O +in O +iron B-chemical +- O +content O +and O +enzyme O +activity O +of O +the O +mutant B-protein_state +protein O +( O +Table O +1 O +). O + +It O +becomes O +apparent O +from O +an O +overlay B-experimental_method +of O +the O +“ O +open B-protein_state +” O +and O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structures I-evidence +that O +the O +side O +- O +chain O +of O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +rotates O +away O +from O +the O +position O +of O +the O +presumptive O +iron B-chemical +, O +whereas O +the O +side O +- O +chains O +of O +those O +residues O +that O +probably O +contacting O +the O +metal B-chemical +directly O +[ O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +and O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +], O +remain O +in O +place O +( O +Fig O +6a O +and O +6b O +). O + +Since O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +is O +strictly B-protein_state +conserved I-protein_state +in O +an O +alignment B-experimental_method +of O +440 O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +( O +Fig O +2 O +), O +we O +speculate O +that O +it O +might O +be O +involved O +in O +contacting O +the O +substrate O +of O +the O +ectoine B-protein_type +synthase I-protein_type +and O +that O +the O +absence B-protein_state +of I-protein_state +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +in O +our O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structures I-evidence +might O +endow O +the O +side O +chain O +of O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +with O +extra O +spatial O +flexibility O +. O + +To O +further O +analyze O +the O +putative O +iron B-site +binding I-site +site I-site +( O +Fig O +6a O +), O +we O +performed O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +site I-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +and O +assessed O +the O +resulting O +( O +Sa B-species +) O +EctC B-protein +variants O +for O +their O +iron B-chemical +content O +and O +studied O +their O +enzyme O +activity O +. O + +When O +those O +three O +residues O +( O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +) O +that O +likely O +form O +the O +mono B-site +- I-site +nuclear I-site +iron I-site +center I-site +in O +the O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structure I-evidence +were O +individually O +replaced B-experimental_method +by O +an O +Ala B-residue_name +residue O +, O +both O +the O +catalytic O +activity O +and O +the O +iron B-chemical +content O +of O +the O +mutant B-protein_state +proteins O +was O +strongly O +reduced O +( O +Table O +1 O +). O + +For O +some O +of O +the O +presumptive O +iron B-site +- I-site +coordinating I-site +residues I-site +, O +additional O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +experiments O +were O +carried O +out O +. O + +To O +verify O +the O +importance O +of O +the O +negative O +charge O +in O +the O +position O +of O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +we O +created O +an O +Asp B-residue_name +variant B-protein_state +. O + +This O +mutant B-protein_state +protein O +rescued O +the O +enzyme O +activity O +and O +iron B-chemical +content O +of O +the O +Ala B-residue_name +substitution B-experimental_method +substantially O +( O +Table O +1 O +). O + +We O +also O +replaced B-experimental_method +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +with O +either O +a O +Phe B-residue_name +or O +a O +Trp B-residue_name +residue O +and O +both O +mutant B-protein_state +proteins O +largely O +lost O +their O +catalytic O +activity O +and O +iron B-chemical +content O +( O +Table O +1 O +) O +despite O +the O +fact O +that O +these O +substitutions O +were O +conservative O +. O + +Collectively O +, O +these O +data O +suggest O +that O +the O +hydroxyl O +group O +of O +the O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +side O +chain O +is O +needed O +for O +the O +binding O +of O +the O +iron B-chemical +( O +Fig O +6a O +). O + +We O +also O +replaced B-experimental_method +the O +presumptive O +iron B-site +- I-site +binding I-site +residue I-site +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +by O +an O +Asn B-residue_name +residue O +, O +yielding O +a O +( O +Sa B-species +) O +EctC B-protein +protein O +variant O +that O +possessed O +an O +enzyme O +activity O +of O +23 O +% O +and O +iron B-chemical +content O +of O +only O +14 O +% O +relative O +to O +that O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +protein O +( O +Table O +1 O +). O + +Collectively O +, O +the O +data O +addressing O +the O +functionality O +of O +the O +putative O +iron B-site +- I-site +coordinating I-site +residues I-site +( O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +) O +buttress O +our O +notion O +that O +the O +Fe2 B-chemical ++ I-chemical +present O +in O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +is O +of O +catalytic O +importance O +. O + +A O +chemically O +undefined O +ligand O +in O +the O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +provides O +clues O +for O +the O +binding O +of O +the O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +substrate O + +Despite O +considerable O +efforts O +, O +either O +by O +trying O +co B-experimental_method +- I-experimental_method +crystallization I-experimental_method +or O +soaking B-experimental_method +experiments I-experimental_method +, O +we O +were O +not O +able O +to O +obtain O +a O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structures I-evidence +that O +contained O +either O +the O +substrate O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +, O +or O +ectoine B-chemical +, O +the O +reaction O +product O +of O +ectoine B-protein_type +synthase I-protein_type +( O +Fig O +1 O +). O + +However O +, O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +where O +the O +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +loop I-structure_element +is O +largely O +resolved O +, O +a O +long O +stretched O +electron B-evidence +density I-evidence +feature O +was O +detected O +in O +the O +predicted O +active B-site +site I-site +of O +the O +enzyme O +; O +it O +remained O +visible O +after O +crystallographic B-experimental_method +refinement I-experimental_method +. O + +This O +is O +in O +contrast O +to O +the O +high O +- O +resolution O +“ O +open B-protein_state +” O +structure B-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +where O +no O +additional O +electron B-evidence +density I-evidence +was O +observed O +after O +refinement O +. O + +We O +tried O +to O +fit O +all O +compounds O +used O +in O +the O +buffers O +during O +purification B-experimental_method +and O +crystallization B-experimental_method +into O +the O +observed O +electron B-evidence +density I-evidence +, O +but O +none O +matched O +. O + +This O +observation O +indicates O +that O +the O +chemically O +undefined O +ligand O +was O +either O +trapped O +by O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +during O +its O +heterologous O +production O +in O +E B-species +. I-species +coli I-species +or O +during O +crystallization B-experimental_method +. O + +Since O +we O +used O +PEG B-chemical +molecules O +in O +the O +crystallization O +conditions O +, O +the O +observed O +density B-evidence +might O +stem O +from O +an O +ordered O +part O +of O +a O +PEG B-chemical +molecule O +, O +or O +low O +molecular O +weight O +PEG B-chemical +species O +that O +might O +have O +been O +present O +in O +the O +PEG B-chemical +preparation O +used O +in O +our O +experiments O +. O + +Estimating O +from O +the O +dimensions O +of O +the O +electron B-evidence +density I-evidence +feature I-evidence +, O +we O +modeled O +the O +chemically O +undefined O +compound O +trapped O +by O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +as O +a O +hexane B-chemical +- I-chemical +1 I-chemical +, I-chemical +6 I-chemical +- I-chemical +diol I-chemical +molecule O +( O +PDB O +identifier O +: O +HEZ O +) O +to O +best O +fit O +the O +observed O +electron B-evidence +density I-evidence +. O + +However O +, O +to O +the O +best O +of O +our O +knowledge O +, O +hexane B-chemical +- I-chemical +1 I-chemical +, I-chemical +6 I-chemical +- I-chemical +diol I-chemical +is O +not O +part O +of O +the O +E B-species +. I-species +coli I-species +metabolome O +. O + +Despite O +these O +notable O +limitations O +, O +we O +considered O +the O +serendipitously O +trapped O +compound O +as O +a O +mock O +ligand O +that O +might O +provide O +useful O +insights O +into O +the O +spatial O +positioning O +of O +the O +true O +EctC B-protein +substrate O +and O +those O +residues O +that O +coordinate O +it O +within O +the O +ectoine B-protein_type +synthase I-protein_type +active B-site +site I-site +. O + +We O +note O +that O +both O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +and O +hexane B-chemical +- I-chemical +1 I-chemical +, I-chemical +6 I-chemical +- I-chemical +diol I-chemical +are O +both O +C6 O +- O +compounds O +and O +display O +similar O +length O +( O +Fig O +7a O +). O + +A O +chemically O +undefined O +ligand O +is O +captured O +in O +the O +active B-site +site I-site +of O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structure I-evidence +. O + +( O +a O +) O +The O +observed O +electron B-evidence +density I-evidence +in O +the O +active B-site +site I-site +of O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +of O +( O +Sa B-species +) O +EctC B-protein +is O +modeled O +as O +a O +hexane B-chemical +- I-chemical +1 I-chemical +, I-chemical +6 I-chemical +- I-chemical +diol I-chemical +molecule O +and O +compared O +with O +the O +electron B-evidence +density I-evidence +of O +the O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +substrate O +of O +the O +ectoine B-protein_type +synthase I-protein_type +to O +emphasize O +the O +similarity O +in O +size O +of O +these O +compounds O +. O + +( O +b O +) O +The O +presumable O +binding B-site +site I-site +of O +the O +iron B-chemical +co O +- O +factor O +and O +of O +the O +modeled O +hexane B-chemical +- I-chemical +1 I-chemical +, I-chemical +6 I-chemical +- I-chemical +diol I-chemical +molecule O +is O +depicted O +. O + +The O +amino O +acid O +side O +chains O +involved O +in O +iron B-chemical +- O +ligand O +binding O +are O +colored O +in O +blue O +and O +those O +involved O +in O +the O +binding O +of O +the O +chemically O +undefined O +ligand O +are O +colored O +in O +green O +using O +a O +ball O +and O +stick O +representation O +. O + +The O +flexible B-protein_state +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +loop I-structure_element +of O +( O +Sa B-species +) O +EctC B-protein +is O +highlighted O +in O +orange O +. O + +The O +electron B-evidence +density I-evidence +was O +calculated O +as O +an O +omit B-evidence +map I-evidence +and O +contoured O +at O +1 O +. O +0 O +σ O +. O + +We O +refined B-experimental_method +the O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +with O +the O +trapped O +compound O +, O +and O +by O +doing O +so O +, O +the O +refinement O +parameters O +( O +especially O +R B-evidence +- I-evidence +and I-evidence +Rfree I-evidence +- I-evidence +factor I-evidence +) O +dropped O +by O +1 O +. O +5 O +%. O + +We O +also O +calculated O +an O +omit B-evidence +map I-evidence +and O +the O +electron B-evidence +density I-evidence +reappeared O +( O +Fig O +7b O +). O + +When O +analyzing O +the O +interactions O +of O +this O +compound O +within O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +, O +we O +found O +that O +it O +is O +bound B-protein_state +via O +interactions O +with O +Trp B-residue_name_number +- I-residue_name_number +21 I-residue_name_number +and O +Ser B-residue_name_number +- I-residue_name_number +23 I-residue_name_number +of O +β B-structure_element +- I-structure_element +sheet I-structure_element +β3 B-structure_element +, O +Thr B-residue_name_number +- I-residue_name_number +40 I-residue_name_number +located O +in O +β B-structure_element +- I-structure_element +sheet I-structure_element +β4 B-structure_element +, O +and O +Cys B-residue_name_number +- I-residue_name_number +105 I-residue_name_number +and O +Phe B-residue_name_number +- I-residue_name_number +107 I-residue_name_number +, O +which O +are O +both O +part O +of O +β B-structure_element +- I-structure_element +sheet I-structure_element +β11 B-structure_element +. O + +Remarkably O +, O +all O +of O +these O +residues O +are O +highly B-protein_state +conserved I-protein_state +throughout O +the O +extended O +EctC B-protein_type +protein I-protein_type +family O +( O +Fig O +2 O +). O + +Structure B-experimental_method +- I-experimental_method +guided I-experimental_method +site I-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +of O +the O +catalytic B-site +core I-site +of O +the O +ectoine B-protein_type +synthase I-protein_type + +In O +a O +previous O +alignment B-experimental_method +of I-experimental_method +the I-experimental_method +amino I-experimental_method +acid I-experimental_method +sequences I-experimental_method +of O +440 O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +, O +13 O +amino O +acids O +were O +identified O +as O +strictly B-protein_state +conserved I-protein_state +residues O +. O + +These O +correspond O +to O +amino O +acids O +Thr B-residue_name_number +- I-residue_name_number +40 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +, O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +, O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Gly B-residue_name_number +- I-residue_name_number +64 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +- O +Leu B-residue_name_number +- I-residue_name_number +87 I-residue_name_number +, O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +, O +Phe B-residue_name_number +- I-residue_name_number +107 I-residue_name_number +, O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +, O +Gly B-residue_name_number +- I-residue_name_number +113 I-residue_name_number +, O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +, O +and O +His B-residue_name_number +- I-residue_name_number +117 I-residue_name_number +in O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +2 O +). O + +Amino O +acid O +residues O +Gly B-residue_name_number +- I-residue_name_number +64 I-residue_name_number +, O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +, O +and O +Gly B-residue_name_number +- I-residue_name_number +113 I-residue_name_number +likely O +fulfill O +structural O +roles O +since O +they O +are O +positioned O +either O +at O +the O +end O +or O +at O +the O +beginning O +of O +β B-structure_element +- I-structure_element +strands I-structure_element +and O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +We O +considered O +the O +remaining O +ten O +residues O +as O +important O +either O +for O +ligand O +binding O +, O +for O +catalysis O +, O +or O +for O +the O +structurally O +correct O +orientation O +of O +the O +flexible B-protein_state +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +As O +described O +above O +, O +the O +side O +chains O +of O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +and O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +are O +probably O +involved O +in O +iron B-chemical +binding O +( O +Table O +1 O +and O +Fig O +6a O +). O + +In O +view O +of O +the O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +with O +the O +serendipitously O +trapped O +compound O +( O +Fig O +7b O +), O +we O +probed O +the O +functional O +importance O +of O +the O +seven O +residues O +that O +contact O +this O +ligand O +by O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +site I-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +( O +Table O +1 O +). O + +Each O +of O +these O +mutant B-protein_state +( O +Sa B-species +) O +EctC B-protein +proteins O +was O +overproduced O +in O +E B-species +. I-species +coli I-species +and O +purified O +by O +affinity B-experimental_method +chromatography I-experimental_method +; O +they O +all O +yielded O +pure O +and O +stable O +protein O +preparations O +. O + +We O +benchmarked O +the O +activity O +of O +the O +( O +Sa B-species +) O +EctC B-protein +variants O +in O +a O +single B-experimental_method +time I-experimental_method +- I-experimental_method +point I-experimental_method +enzyme I-experimental_method +assay I-experimental_method +under O +conditions O +where O +10 O +μM O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +Sa B-species +) O +EctC B-protein +protein O +converted O +almost O +completely O +the O +supplied O +10 O +mM O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +substrate O +to O +9 O +. O +33 O +mM O +ectoine B-chemical +within O +a O +time O +frame O +of O +20 O +min O +. O + +In O +addition O +, O +we O +determined O +the O +iron B-chemical +content O +of O +each O +of O +the O +mutant B-protein_state +( O +Sa B-species +) O +EctC B-protein +protein O +by O +a O +colorimetric B-experimental_method +assay I-experimental_method +( O +Table O +1 O +). O + +The O +side O +chains O +of O +the O +evolutionarily B-protein_state +conserved I-protein_state +Trp B-residue_name_number +- I-residue_name_number +21 I-residue_name_number +, O +Ser B-residue_name_number +- I-residue_name_number +23 I-residue_name_number +, O +Thr B-residue_name_number +- I-residue_name_number +40 I-residue_name_number +, O +Cys B-residue_name_number +- I-residue_name_number +105 I-residue_name_number +, O +and O +Phe B-residue_name_number +- I-residue_name_number +107 I-residue_name_number +residues O +( O +Fig O +2 O +) O +make O +contacts O +with O +the O +chemically O +undefined O +ligand O +that O +we O +observed O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +( O +Fig O +7b O +). O + +We O +replaced B-experimental_method +each O +of O +these O +residues O +with O +an O +Ala B-residue_name +residue O +and O +found O +that O +none O +of O +them O +had O +an O +influence O +on O +the O +iron B-chemical +content O +of O +the O +mutant B-protein_state +proteins O +. O + +Thr B-residue_name_number +- I-residue_name_number +40 I-residue_name_number +is O +positioned O +on O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +and O +its O +side O +chain O +protrudes O +into O +the O +lumen O +of O +the O +cupin B-structure_element +barrel I-structure_element +formed O +by O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +7b O +). O + +We O +also O +replaced B-experimental_method +Phe B-residue_name_number +- I-residue_name_number +107 I-residue_name_number +with O +either O +an O +Tyr B-residue_name +or O +an O +Trp B-residue_name +residue O +: O +the O +Phe B-mutant +- I-mutant +107 I-mutant +/ I-mutant +Tyr I-mutant +substitution B-experimental_method +possessed O +near O +wild B-protein_state +- I-protein_state +type I-protein_state +enzyme O +activity O +( O +about O +95 O +%) O +and O +the O +full O +iron B-chemical +content O +, O +but O +the O +Phe B-mutant +- I-mutant +107 I-mutant +/ I-mutant +Trp I-mutant +substitution B-experimental_method +possessed O +only O +12 O +% O +enzyme O +activity O +and O +72 O +% O +iron B-chemical +content O +compared O +to O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +protein O +. O + +The O +properties O +of O +these O +mutant B-protein_state +proteins O +indicate O +that O +the O +aromatic O +side O +chain O +at O +position O +107 B-residue_number +of O +( O +Sa B-species +) O +EctC B-protein +is O +of O +importance O +but O +that O +a O +substitution B-experimental_method +with O +a O +bulky O +aromatic O +side O +chain O +is O +strongly O +detrimental O +to O +enzyme O +activity O +and O +concomitantly O +moderately O +impairs O +iron B-chemical +binding O +. O + +Replacement B-experimental_method +of O +the O +only O +Cys B-residue_name +residue O +in O +( O +Sa B-species +) O +EctC B-protein +( O +Cys B-residue_name_number +- I-residue_name_number +105 I-residue_name_number +; O +Fig O +2 O +) O +by O +a O +Ser B-residue_name +residue O +, O +a O +configuration O +that O +is O +naturally O +found O +in O +two O +EctC B-protein_type +proteins I-protein_type +among O +440 O +inspected O +amino O +acid O +sequences O +, O +yielded O +a O +( O +Sa B-species +) O +EctC B-protein +variant B-protein_state +with O +84 O +% O +wild B-protein_state +- I-protein_state +type I-protein_state +activity O +and O +an O +iron B-chemical +content O +similar O +to O +that O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +protein O +. O + +However O +, O +the O +Cys B-mutant +- I-mutant +105 I-mutant +/ I-mutant +Ala I-mutant +variant B-protein_state +was O +practically O +catalytically B-protein_state +inactive I-protein_state +while O +largely O +maintaining O +its O +iron B-chemical +content O +( O +Table O +1 O +). O + +Since O +the O +side O +- O +chains O +of O +Cys B-residue_name +residues O +are O +chemically O +reactive O +and O +often O +participate O +in O +enzyme O +catalysis O +, O +Cys B-residue_name_number +- I-residue_name_number +105 I-residue_name_number +( O +or O +Ser B-residue_name_number +- I-residue_name_number +105 I-residue_name_number +) O +might O +serve O +such O +a O +role O +for O +ectoine B-protein_type +synthase I-protein_type +. O + +We O +observed O +two O +amino B-experimental_method +acid I-experimental_method +substitutions I-experimental_method +that O +simultaneously O +strongly O +affected O +enzyme O +activity O +and O +iron B-chemical +content O +; O +these O +were O +the O +Tyr B-mutant +- I-mutant +52 I-mutant +/ I-mutant +Ala I-mutant +and O +the O +His B-mutant +- I-mutant +55 I-mutant +/ I-mutant +Ala I-mutant +( O +Sa B-species +) O +EctC B-protein +protein O +variants O +( O +Table O +1 O +). O + +Based O +on O +the O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structures I-evidence +that O +we O +present O +here O +, O +we O +can O +currently O +not O +firmly O +understand O +why O +the O +replacement B-experimental_method +of O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +by O +Ala B-residue_name +impairs O +enzyme O +function O +and O +iron B-chemical +content O +so O +drastically O +( O +Table O +1 O +). O + +This O +is O +different O +for O +the O +His B-mutant +- I-mutant +55 I-mutant +/ I-mutant +Ala I-mutant +substitution O +. O + +The O +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +is O +held O +in O +its O +position O +via O +an O +interaction O +of O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +with O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +, O +where O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +in O +turn O +interacts O +with O +Pro B-residue_name_number +- I-residue_name_number +110 I-residue_name_number +( O +Fig O +6a O +and O +6b O +). O + +Each O +of O +these O +residues O +is O +evolutionarily B-protein_state +highly I-protein_state +conserved I-protein_state +. O + +The O +individual O +substitution B-experimental_method +of O +either O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +or O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +by O +an O +Ala B-residue_name +residue O +is O +predicted O +to O +disrupt O +this O +interactive B-site +network I-site +and O +therefore O +should O +affect O +enzyme O +activity O +. O + +Indeed O +, O +the O +Glu B-mutant +- I-mutant +115 I-mutant +/ I-mutant +Ala I-mutant +and O +the O +His B-mutant +- I-mutant +55 I-mutant +/ I-mutant +Ala I-mutant +substitutions O +possessed O +only O +21 O +% O +and O +16 O +% O +activity O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +protein O +, O +respectively O +( O +Table O +1 O +). O + +The O +Glu B-mutant +- I-mutant +115 I-mutant +/ I-mutant +Ala I-mutant +mutant B-protein_state +possessed O +wild B-protein_state +- I-protein_state +type I-protein_state +levels O +of O +iron B-chemical +, O +whereas O +the O +iron B-chemical +content O +of O +the O +His B-mutant +- I-mutant +55 I-mutant +/ I-mutant +Ala I-mutant +substitutions O +dropped O +to O +15 O +% O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +level O +( O +Table O +1 O +). O + +We O +also O +replaced B-experimental_method +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +with O +a O +negatively O +charged O +residue O +( O +Asp B-residue_name +); O +this O +( O +Sa B-species +) O +EctC B-protein +variant O +possessed O +wild B-protein_state +- I-protein_state +type I-protein_state +levels O +of O +iron B-chemical +and O +still O +exhibited O +77 O +% O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +enzyme O +activity O +. O + +Collectively O +, O +these O +data O +suggest O +that O +the O +correct O +positioning O +of O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +is O +of O +structural O +and O +functional O +importance O +for O +the O +activity O +of O +the O +ectoine B-protein_type +synthase I-protein_type +. O + +Residues O +Leu B-residue_name_number +- I-residue_name_number +87 I-residue_name_number +and O +Asp B-residue_name_number +- I-residue_name_number +91 I-residue_name_number +are O +highly B-protein_state +conserved I-protein_state +in O +the O +ectoine B-protein_type +synthase I-protein_type +protein O +family O +. O + +The O +replacement B-experimental_method +of O +Leu B-residue_name_number +- I-residue_name_number +87 I-residue_name_number +by O +Ala B-residue_name +led O +to O +a O +substantial O +drop O +in O +enzyme O +activity O +( O +Table O +1 O +). O + +Conversely O +, O +the O +replacement B-experimental_method +of O +Asp B-residue_name_number +- I-residue_name_number +91 I-residue_name_number +by O +Ala B-residue_name +and O +Glu B-residue_name +, O +resulted O +in O +( O +Sa B-species +) O +EctC B-protein +protein O +variants O +with O +80 O +% O +and O +98 O +% O +enzyme O +activity O +, O +respectively O +( O +Table O +1 O +). O + +We O +currently O +cannot O +comment O +on O +possible O +functional O +role O +Asp B-residue_name_number +- I-residue_name_number +91 I-residue_name_number +. O + +However O +, O +Leu B-residue_name_number +- I-residue_name_number +87 I-residue_name_number +is O +positioned O +at O +the O +end O +of O +one O +of O +the O +β B-structure_element +- I-structure_element +sheets I-structure_element +that O +form O +the O +dimer B-site +interface I-site +( O +Fig O +5c O +) O +and O +it O +might O +therefore O +possess O +a O +structural O +role O +. O + +It O +is O +also O +located O +near O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +one O +of O +the O +residues O +that O +probably O +coordinate O +the O +iron B-chemical +molecule O +with O +in O +the O +( O +Sa B-species +) O +EctC B-protein +active B-site +site I-site +( O +Fig O +6a O +) O +and O +therefore O +might O +exert O +indirect O +effects O +. O + +His B-residue_name_number +- I-residue_name_number +117 I-residue_name_number +is O +a O +strictly B-protein_state +conserved I-protein_state +residue O +and O +its O +substitution B-experimental_method +by O +an O +Ala B-residue_name +residue O +results O +in O +a O +drop O +of O +enzyme O +activity O +( O +down O +to O +44 O +%) O +and O +an O +iron B-chemical +content O +of O +83 O +% O +( O +Table O +1 O +). O + +We O +note O +that O +His B-residue_name_number +- I-residue_name_number +117 I-residue_name_number +is O +located O +close O +to O +the O +chemically O +undefined O +ligand O +in O +the O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +( O +Fig O +7b O +) O +and O +might O +thus O +play O +a O +role O +in O +contacting O +the O +natural O +substrate O +of O +the O +ectoine B-protein_type +synthase I-protein_type +. O + +As O +an O +internal O +control O +for O +our O +mutagenesis B-experimental_method +experiments I-experimental_method +, O +we O +also O +substituted B-experimental_method +Thr B-residue_name_number +- I-residue_name_number +41 I-residue_name_number +and O +His B-residue_name_number +- I-residue_name_number +51 I-residue_name_number +, O +two O +residues O +that O +are O +not B-protein_state +evolutionarily I-protein_state +conserved I-protein_state +in O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +with O +Ala B-residue_name +residues O +. O + +Both O +( O +Sa B-species +) O +EctC B-protein +protein O +variants O +exhibited O +wild B-protein_state +- I-protein_state +type I-protein_state +level O +enzyme O +activities O +and O +possessed O +a O +iron B-chemical +content O +matching O +that O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +Table O +1 O +). O + +This O +illustrates O +that O +not O +every O +amino O +acid O +substitution O +in O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +leads O +to O +an O +indiscriminate O +impairment O +of O +enzyme O +function O +and O +iron B-chemical +content O +. O + +The O +crystallographic B-evidence +data I-evidence +presented O +here O +firmly O +identify O +ectoine B-protein_type +synthase I-protein_type +( O +EctC B-protein +), O +an O +enzyme O +critical O +for O +the O +production O +of O +the O +microbial B-taxonomy_domain +cytoprotectant O +and O +chemical O +chaperone O +ectoine B-chemical +, O +as O +a O +new O +member O +of O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +The O +overall O +fold O +and O +bowl O +shape O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Figs O +4 O +and O +5 O +) O +with O +its O +11 O +β B-structure_element +- I-structure_element +strands I-structure_element +( O +β1 B-structure_element +- I-structure_element +β11 I-structure_element +) O +and O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +α B-structure_element +- I-structure_element +I I-structure_element +and O +α B-structure_element +- I-structure_element +II I-structure_element +) O +closely O +adheres O +to O +the O +design O +principles O +typically O +found O +in O +crystal B-evidence +structures I-evidence +of O +cupins B-protein_type +. O + +In O +addition O +to O +the O +ectoine B-protein_type +synthase I-protein_type +, O +the O +polyketide B-protein_type +cyclase I-protein_type +RemF B-protein +is O +the O +only O +other O +currently O +known O +cupin B-protein_type +- I-protein_type +related I-protein_type +enzyme O +that O +catalyze O +a O +cyclocondensation O +reaction O +although O +the O +substrates O +of O +EctC B-protein +and O +RemF B-protein +are O +rather O +different O +. O + +As O +a O +consequence O +of O +the O +structural O +relatedness O +of O +EctC B-protein +and O +RemF B-protein +and O +the O +type O +of O +chemical O +reaction O +these O +two O +enzymes O +catalyze O +, O +is O +now O +understandable O +why O +bona O +fide O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +are O +frequently O +( O +mis O +)- O +annotated O +in O +microbial B-taxonomy_domain +genome O +sequences O +as O +“ O +RemF B-protein_type +- I-protein_type +like I-protein_type +” O +proteins O +. O + +The O +pro B-taxonomy_domain +- I-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +members O +of O +the O +cupin B-protein_type +superfamily I-protein_type +perform O +a O +variety O +of O +both O +enzymatic O +and O +non O +- O +enzymatic O +functions O +that O +are O +built O +upon O +a O +common O +structural O +scaffold O +. O + +Most O +cupins B-protein_type +contain O +transition O +state O +metals O +that O +can O +promote O +different O +types O +of O +chemical O +reactions O +. O + +Except O +for O +some O +cupin B-protein_type +- I-protein_type +related I-protein_type +proteins I-protein_type +that O +seem O +to O +function O +as O +metallo B-protein_type +- I-protein_type +chaperones I-protein_type +, O +the O +bound B-protein_state +metal B-chemical +is O +typically O +an O +essential O +part O +of O +the O +active B-site +sites I-site +. O + +We O +report O +here O +for O +the O +first O +time O +that O +the O +ectoine B-protein_type +synthase I-protein_type +is O +a O +metal B-chemical +- O +dependent O +enzyme O +. O + +ICP B-experimental_method +- I-experimental_method +MS I-experimental_method +, O +metal B-experimental_method +- I-experimental_method +depletion I-experimental_method +and I-experimental_method +reconstitution I-experimental_method +experiments I-experimental_method +( O +Fig O +3 O +) O +consistently O +identify O +iron B-chemical +as O +the O +biologically O +most O +relevant O +metal B-chemical +for O +the O +EctC B-protein +- O +catalyzed O +cyclocondensation O +reaction O +. O + +However O +, O +as O +observed O +with O +other O +cupins B-protein_type +, O +EctC B-protein +is O +a O +somewhat O +promiscuous O +enzyme O +as O +far O +as O +the O +catalytically O +important O +metal B-chemical +is O +concerned O +when O +they O +are O +provided O +in O +large O +molar O +excess O +( O +Fig O +3c O +). O + +Although O +some O +uncertainty O +remains O +with O +respect O +to O +the O +precise O +identity O +of O +amino O +acid O +residues O +that O +participate O +in O +metal B-chemical +binding O +by O +( O +Sa B-species +) O +EctC B-protein +, O +our O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +site I-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +experiments O +targeting O +the O +presumptive O +iron B-site +- I-site +binding I-site +residues I-site +( O +Fig O +6a O +and O +6b O +) O +demonstrate O +that O +none O +of O +them O +can O +be O +spared O +( O +Table O +1 O +). O + +The O +architecture O +of O +the O +metal B-site +center I-site +of O +ectoine B-protein_type +synthase I-protein_type +seems O +to O +be O +subjected O +to O +considerable O +evolutionary O +constraints O +. O + +The O +three O +residues O +( O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +) O +that O +we O +deem O +to O +form O +it O +( O +Figs O +6 O +and O +7b O +) O +are O +strictly B-protein_state +conserved I-protein_state +in O +a O +large O +collection O +of O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +originating O +from O +16 O +bacterial B-taxonomy_domain +and O +three O +archaeal B-taxonomy_domain +phyla O +( O +Fig O +2 O +). O + +We O +also O +show O +here O +for O +the O +first O +time O +that O +, O +in O +addition O +to O +its O +natural O +substrate O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +, O +EctC B-protein +also O +converts O +the O +isomer O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +into O +ectoine B-chemical +, O +albeit O +with O +a O +73 O +- O +fold O +reduced O +catalytic B-evidence +efficiency I-evidence +( O +S3a O +and O +S3b O +Fig O +). O + +Hence O +, O +the O +active B-site +site I-site +of O +ectoine B-protein_type +synthase I-protein_type +must O +possess O +a O +certain O +degree O +of O +structural O +plasticity O +, O +a O +notion O +that O +is O +supported O +by O +the O +report O +on O +the O +EctC B-protein +- O +catalyzed O +formation O +of O +the O +synthetic O +compatible O +solute O +ADPC B-chemical +through O +the O +cyclic O +condensation O +of O +two O +glutamine B-chemical +molecules O +. O + +Our O +finding O +that O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +serves O +as O +a O +substrate O +for O +ectoine B-protein_type +synthase I-protein_type +has O +physiologically O +relevant O +ramifications O +for O +those O +microorganisms B-taxonomy_domain +that O +can O +both O +synthesize O +and O +catabolize O +ectoine B-chemical +, O +since O +they O +need O +to O +prevent O +a O +futile O +cycle O +of O +synthesis O +and O +degradation O +when O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +is O +produced O +as O +an O +intermediate O +in O +the O +catabolic O +route O +. O + +Although O +we O +cannot O +identify O +the O +true O +chemical O +nature O +of O +the O +C6 B-chemical +compound O +that O +was O +trapped O +in O +the O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +nor O +its O +precise O +origin O +, O +we O +treated O +this O +compound O +as O +a O +proxy O +for O +the O +natural O +substrate O +of O +ectoine B-protein_type +synthase I-protein_type +, O +which O +is O +a O +C6 O +compound O +as O +well O +( O +Fig O +7a O +). O + +We O +assumed O +that O +its O +location O +and O +mode O +of O +binding O +gives O +, O +in O +all O +likelihood O +, O +clues O +as O +to O +the O +position O +of O +the O +true O +substrate O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +within O +the O +EctC B-protein +active B-site +site I-site +. O + +Indeed O +, O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +of O +those O +five O +residues O +that O +contact O +the O +unknown O +C6 O +compound O +( O +Fig O +7b O +) O +yielded O +( O +Sa B-species +) O +EctC B-protein +variants O +with O +strongly O +impaired O +enzyme O +function O +but O +near O +wild B-protein_state +- I-protein_state +type I-protein_state +levels O +of O +iron B-chemical +( O +Table O +1 O +). O + +This O +set O +of O +data O +and O +the O +fact O +that O +the O +targeted O +residues O +are O +strongly B-protein_state +conserved I-protein_state +among O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +( O +Fig O +2 O +) O +is O +consistent O +with O +their O +potential O +role O +in O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +binding O +or O +enzyme O +catalysis O +. O + +We O +therefore O +surmise O +that O +our O +crystallographic B-evidence +data I-evidence +and O +the O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +study I-experimental_method +reported O +here O +provide O +a O +structural O +and O +functional O +view O +into O +the O +architecture O +of O +the O +EctC B-protein +active B-site +site I-site +( O +Fig O +7b O +). O + +The O +ectoine B-protein_type +synthase I-protein_type +from O +the O +cold O +- O +adapted O +marine B-taxonomy_domain +bacterium I-taxonomy_domain +S B-species +. I-species +alaskensis I-species +can O +be O +considered O +as O +a O +psychrophilic O +enzyme O +( O +S3a O +Fig O +), O +types O +of O +proteins O +with O +a O +considerable O +structural O +flexibility O +. O + +This O +probably O +worked O +to O +the O +detriment O +of O +our O +efforts O +in O +solving O +crystal B-evidence +structures I-evidence +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +( O +Sa B-species +) O +EctC B-protein +protein O +in B-protein_state +complex I-protein_state +with I-protein_state +either O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +or O +ectoine B-chemical +. O + +Because O +microbial B-taxonomy_domain +ectoine B-chemical +producers O +can O +colonize O +ecological O +niches O +with O +rather O +different O +physicochemical O +attributes O +, O +it O +seems O +promising O +to O +exploit O +this O +considerable O +biodiversity O +to O +identify O +EctC B-protein_type +proteins I-protein_type +with O +enhanced O +protein O +stability O +. O + +It O +is O +hoped O +that O +these O +can O +be O +further O +employed O +to O +obtain O +EctC B-protein +crystal B-evidence +structures I-evidence +with O +either O +the O +substrate O +or O +the O +reaction O +product O +. O + +Together O +with O +our O +finding O +that O +ectoine B-protein_type +synthase I-protein_type +is O +metal B-protein_state +dependent I-protein_state +, O +these O +crystal B-evidence +structures I-evidence +should O +allow O +a O +more O +detailed O +understanding O +of O +the O +chemistry O +underlying O +the O +EctC B-protein +- O +catalyzed O +cyclocondensation O +reaction O +. O + +Structures B-evidence +of O +human B-species +ADAR2 B-protein +bound B-protein_state +to I-protein_state +dsRNA B-chemical +reveal O +base O +- O +flipping O +mechanism O +and O +basis O +for O +site O +selectivity O + +ADARs B-protein_type +( O +adenosine B-protein_type +deaminases I-protein_type +acting I-protein_type +on I-protein_type +RNA I-protein_type +) O +are O +editing B-protein_type +enzymes I-protein_type +that O +convert O +adenosine B-residue_name +( O +A B-residue_name +) O +to O +inosine B-residue_name +( O +I B-residue_name +) O +in O +duplex B-structure_element +RNA I-structure_element +, O +a O +modification O +reaction O +with O +wide O +- O +ranging O +consequences O +on O +RNA B-chemical +function O +. O + +Our O +understanding O +of O +the O +ADAR B-protein_type +reaction O +mechanism O +, O +origin O +of O +editing B-site +site I-site +selectivity O +and O +effect O +of O +mutations O +is O +limited O +by O +the O +lack O +of O +high O +- O +resolution O +structural B-evidence +data I-evidence +for O +complexes O +of O +ADARs B-protein_type +bound B-protein_state +to I-protein_state +substrate O +RNAs B-chemical +. O + +Here O +we O +describe O +four O +crystal B-evidence +structures I-evidence +of O +the O +deaminase B-structure_element +domain I-structure_element +of O +human B-species +ADAR2 B-protein +bound B-protein_state +to I-protein_state +RNA B-structure_element +duplexes I-structure_element +bearing O +a O +mimic O +of O +the O +deamination O +reaction O +intermediate O +. O + +These O +structures B-evidence +, O +together O +with O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +mutagenesis I-experimental_method +and O +RNA B-experimental_method +- I-experimental_method +modification I-experimental_method +experiments I-experimental_method +, O +explain O +the O +basis O +for O +ADAR B-protein_type +deaminase B-structure_element +domain I-structure_element +’ O +s O +dsRNA B-chemical +specificity O +, O +its O +base O +- O +flipping O +mechanism O +, O +and O +nearest O +neighbor O +preferences O +. O + +In O +addition O +, O +an O +ADAR2 B-protein +- O +specific O +RNA B-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +was O +identified O +near O +the O +enzyme O +active B-site +site I-site +rationalizing O +differences O +in O +selectivity O +observed O +between O +different O +ADARs B-protein_type +. O + +Finally O +, O +our O +results O +provide O +a O +structural O +framework O +for O +understanding O +the O +effects O +of O +ADAR B-protein_type +mutations O +associated O +with O +human B-species +disease O +. O + +RNA B-chemical +editing O +reactions O +alter O +a O +transcript O +’ O +s O +genomically O +encoded O +sequence O +by O +inserting O +, O +deleting O +or O +modifying O +nucleotides O +. O + +Deamination O +of O +adenosine B-residue_name +( O +A B-residue_name +), O +the O +most O +common O +form O +of O +RNA B-chemical +editing O +in O +humans B-species +, O +generates O +inosine B-residue_name +( O +I B-residue_name +) O +at O +the O +corresponding O +nucleotide O +position O +. O + +Since O +I B-residue_name +base O +pairs O +with O +cytidine B-residue_name +( O +C B-residue_name +), O +it O +functions O +like O +guanosine B-residue_name +( O +G B-residue_name +) O +in O +cellular O +processes O +such O +as O +splicing O +, O +translation O +and O +reverse O +transcription O +. O + +A O +to O +I O +editing O +has O +wide O +- O +ranging O +consequences O +on O +RNA B-chemical +function O +including O +altering O +miRNA B-site +recognition I-site +sites I-site +, O +redirecting O +splicing O +and O +changing O +the O +meaning O +of O +specific O +codons O +. O + +Two O +different O +enzymes O +carry O +out O +A O +to O +I O +editing O +in O +humans B-species +; O +ADAR1 B-protein +and O +ADAR2 B-protein +. O + +ADAR B-protein_type +activity O +is O +required O +for O +nervous O +system O +function O +and O +altered O +editing O +has O +been O +linked O +to O +neurological O +disorders O +such O +as O +epilepsy O +and O +Prader O +Willi O +Syndrome O +. O + +In O +addition O +, O +mutations O +in O +the O +ADAR1 B-protein +gene O +are O +known O +to O +cause O +the O +autoimmune O +disease O +Aicardi O +- O +Goutieres O +Syndrome O +( O +AGS O +) O +and O +the O +skin O +disorder O +Dyschromatosis O +Symmetrica O +Hereditaria O +( O +DSH O +). O + +Hyper O +editing O +has O +been O +observed O +at O +certain O +sites O +in O +cancer O +cells O +, O +such O +as O +in O +the O +mRNA B-chemical +for O +AZIN1 B-protein +( O +antizyme B-protein +inhibitor I-protein +1 I-protein +). O + +However O +, O +hypo O +editing O +also O +occurs O +in O +cancer O +- O +derived O +cell O +lines O +exemplified O +by O +reduced O +editing O +observed O +in O +the O +message O +for O +glioma B-protein +- I-protein +associated I-protein +oncogene I-protein +1 I-protein +( O +Gli1 B-protein +). O + +The O +ADAR B-protein_type +proteins O +have O +a O +modular O +structure O +with O +double B-structure_element +stranded I-structure_element +RNA I-structure_element +binding I-structure_element +domains I-structure_element +( O +dsRBDs B-structure_element +) O +and O +a O +C O +- O +terminal O +deaminase B-structure_element +domain I-structure_element +( O +see O +Fig O +. O +1a O +for O +hADAR2 B-protein +domains O +). O + +ADARs B-protein_type +efficiently O +deaminate O +specific O +adenosines B-residue_name +in O +duplex B-structure_element +RNA I-structure_element +while O +leaving O +most O +adenosines B-residue_name +unmodified O +. O + +The O +mechanism O +of O +adenosine B-residue_name +deamination O +requires O +ADAR B-protein_type +to O +flip O +the O +reactive O +base O +out O +of O +an O +RNA B-chemical +double I-chemical +helix I-chemical +to O +access O +its O +active B-site +site I-site +. O + +How O +an O +enzyme O +could O +accomplish O +this O +task O +with O +a O +duplex B-structure_element +RNA I-structure_element +substrate O +is O +not O +known O +. O + +Furthermore O +, O +how O +an O +ADAR B-protein_type +deaminase B-structure_element +domain I-structure_element +contributes O +to O +editing B-site +site I-site +selectivity O +is O +also O +unknown O +, O +since O +no O +structures B-evidence +of O +ADAR B-complex_assembly +deaminase I-complex_assembly +domain I-complex_assembly +- I-complex_assembly +RNA I-complex_assembly +complexes O +have O +been O +reported O +. O + +To O +address O +these O +knowledge O +gaps O +, O +we O +set O +out O +to O +trap O +the O +human B-species +ADAR2 B-protein +deaminase B-structure_element +domain I-structure_element +( O +aa299 O +– B-residue_range +701 I-residue_range +, O +hADAR2d B-mutant +) O +bound B-protein_state +to I-protein_state +different O +duplex B-structure_element +RNAs I-structure_element +and O +solve O +structures B-evidence +for O +the O +resulting O +complexes O +using O +x B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +We O +then O +evaluated O +the O +importance O +of O +protein O +- O +RNA B-chemical +contacts O +using O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +mutagenesis I-experimental_method +and O +RNA B-experimental_method +- I-experimental_method +modification I-experimental_method +experiments I-experimental_method +coupled O +with O +adenosine B-experimental_method +deamination I-experimental_method +kinetics I-experimental_method +. O + +Trapping O +the O +flipped B-protein_state +conformation O + +The O +ADAR B-protein_type +reaction O +involves O +the O +formation O +of O +a O +hydrated O +intermediate O +that O +loses O +ammonia O +to O +generate O +the O +inosine B-residue_name +- O +containing O +product O +RNA B-chemical +( O +for O +reaction O +scheme O +see O +Fig O +. O +1b O +). O + +The O +covalent O +hydrate O +of O +the O +nucleoside O +analog O +8 B-chemical +- I-chemical +azanebularine I-chemical +( O +N B-chemical +) O +mimics O +the O +proposed O +high O +- O +energy O +intermediate O +( O +for O +reaction O +scheme O +see O +Fig O +. O +1b O +). O + +For O +trapping O +hADAR2d B-mutant +bound B-protein_state +to I-protein_state +RNA B-chemical +for O +crystallography B-experimental_method +, O +we O +incorporated O +8 B-chemical +- I-chemical +azanebularine I-chemical +into O +duplex B-structure_element +RNAs I-structure_element +shown O +recently O +to O +be O +excellent O +substrates O +for O +deamination O +by O +hADAR2d B-mutant +( O +for O +duplex O +sequence O +see O +Fig O +. O +1c O +) O +( O +for O +characterization O +of O +protein O +– O +RNA B-chemical +complex O +see O +Supplementary O +Fig O +. O +1 O +). O + +In O +addition O +, O +for O +one O +of O +these O +duplexes O +( O +Bdf2 B-chemical +), O +we O +positioned O +the O +8 B-chemical +- I-chemical +azanebularine I-chemical +opposite O +either O +uridine B-residue_name +or O +cytidine B-residue_name +to O +mimic O +an O +A B-residue_name +- O +U B-residue_name +pair O +or O +A B-residue_name +- O +C B-residue_name +mismatch O +at O +the O +editing B-site +site I-site +creating O +a O +total O +of O +three O +different O +RNA B-chemical +substrates O +for O +structural O +studies O +( O +Fig O +. O +1c O +). O + +The O +hADAR2d B-mutant +protein O +( O +without B-protein_state +RNA I-protein_state +bound I-protein_state +) O +has O +been O +previously O +crystallized B-experimental_method +and O +structurally O +characterized O +revealing O +features O +of O +the O +active B-site +site I-site +including O +the O +presence O +of O +zinc B-chemical +. O + +In O +addition O +, O +an O +inositol B-chemical +hexakisphosphate I-chemical +( O +IHP B-chemical +) O +molecule O +was O +found O +buried O +in O +the O +core O +of O +the O +protein O +hydrogen B-bond_interaction +bonded I-bond_interaction +to O +numerous O +conserved O +polar O +residues O +. O + +For O +crystallization B-experimental_method +of O +hADAR2d B-complex_assembly +- I-complex_assembly +RNA I-complex_assembly +complexes O +, O +we O +used O +both O +the O +wild B-protein_state +type I-protein_state +( O +WT B-protein_state +) O +deaminase B-structure_element +domain I-structure_element +and O +a O +mutant B-protein_state +( O +E488Q B-mutant +) O +that O +has O +enhanced O +catalytic O +activity O +. O + +A O +description O +of O +the O +crystallization O +conditions O +, O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +diffraction I-experimental_method +data I-experimental_method +collection I-experimental_method +and I-experimental_method +solution I-experimental_method +of O +the O +structures B-evidence +can O +be O +found O +in O +Online O +Methods O +. O + +Four O +protein O +- O +RNA B-chemical +combinations O +generated O +diffracting O +crystals B-evidence +that O +resulted O +in O +high O +- O +resolution O +structures B-evidence +( O +hADAR2d B-complex_assembly +WT I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +U I-complex_assembly +, O +hADAR2d B-complex_assembly +WT I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +C I-complex_assembly +, O +hADAR2d B-complex_assembly +E488Q I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +C I-complex_assembly +, O +hADAR2d B-complex_assembly +E488Q I-complex_assembly +– I-complex_assembly +Gli1 I-complex_assembly +) O +( O +Table O +1 O +). O + +In O +each O +of O +these O +complexes O +, O +the O +protein O +binds O +the O +RNA B-chemical +on O +one O +face O +of O +the O +duplex O +over O +~ O +20 O +bp O +using O +a O +positively O +charged O +surface O +near O +the O +zinc B-site +- I-site +containing I-site +active I-site +site I-site +( O +Fig O +. O +2 O +, O +Supplementary O +Fig O +. O +2a O +). O + +The O +large O +binding B-site +site I-site +( O +1493 O +Å2 O +RNA O +surface O +area O +and O +1277 O +Å2 O +protein O +surface O +area O +buried O +) O +observed O +for O +hADAR2d B-mutant +is O +consistent O +with O +recent O +footprinting B-experimental_method +studies I-experimental_method +. O + +Both O +strands O +of O +the O +RNA B-chemical +contact O +the O +protein O +with O +the O +majority O +of O +these O +interactions O +mediated O +through O +the O +phosphodiester O +- O +ribose O +backbone O +near O +the O +editing B-site +site I-site +( O +Fig O +. O +2c O +, O +Supplementary O +Fig O +. O +2 O +b O +– O +d O +). O + +The O +structures B-evidence +show O +a O +large O +deviation O +from O +A B-structure_element +- I-structure_element +form I-structure_element +RNA B-chemical +conformation O +at O +the O +editing B-site +site I-site +( O +Fig O +. O +2 O +, O +Fig O +. O +3 O +, O +Supplementary O +Video O +1 O +). O + +The O +8 B-chemical +- I-chemical +azanebularine I-chemical +is O +flipped B-protein_state +out I-protein_state +of O +the O +helix B-structure_element +and O +bound B-protein_state +into I-protein_state +the O +active B-site +site I-site +as O +its O +covalent O +hydrate O +where O +it O +interacts O +with O +several O +amino O +acids O +including O +V351 B-residue_name_number +, O +T375 B-residue_name_number +, O +K376 B-residue_name_number +, O +E396 B-residue_name_number +and O +R455 B-residue_name_number +( O +Fig O +. O +3a O +, O +Supplementary O +Fig O +. O +3a O +). O + +The O +side O +chain O +of O +E396 B-residue_name_number +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +purine B-chemical +N1 O +and O +O6 O +. O + +This O +interaction O +was O +expected O +given O +the O +proposed O +role O +of O +E396 B-residue_name_number +in O +mediating O +proton O +transfers O +to O +and O +from O +N1 O +of O +the O +substrate O +adenosine B-residue_name +. O + +The O +2 O +’- O +hydroxyl O +of O +8 B-chemical +- I-chemical +azanebularine I-chemical +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +the O +backbone O +carbonyl O +of O +T375 B-residue_name_number +while O +the O +T375 B-residue_name_number +side O +chain O +contacts O +its O +3 O +’- O +phosphodiester O +. O + +R455 B-residue_name_number +and O +K376 B-residue_name_number +help O +position O +the O +flipped B-protein_state +nucleotide B-chemical +in O +the O +active B-site +site I-site +by O +fastening O +the O +phosphate O +backbone O +flanking O +the O +editing B-site +site I-site +. O + +The O +R455 B-residue_name_number +side O +chain O +ion B-bond_interaction +pairs I-bond_interaction +with O +the O +5 O +’- O +phosphodiester O +of O +8 B-chemical +- I-chemical +azanebularine I-chemical +while O +the O +K376 B-residue_name_number +side O +chain O +contacts O +its O +3 O +’- O +phosphodiester O +. O + +Lastly O +, O +the O +side O +chain O +of O +V351 B-residue_name_number +provides O +a O +hydrophobic B-site +surface I-site +for O +interaction O +with O +the O +nucleobase O +of O +the O +edited B-protein_state +nucleotide B-chemical +. O + +RNA B-chemical +binding O +does O +not O +alter O +IHP B-chemical +binding O +or O +the O +H B-site +- I-site +bonding I-site +network I-site +linking O +IHP B-chemical +to O +the O +active B-site +site I-site +. O + +ADARs B-protein_type +use O +a O +unique O +mechanism O +to O +modify O +duplex B-structure_element +RNA I-structure_element + +The O +ADAR2 B-protein +base B-structure_element +- I-structure_element +flipping I-structure_element +loop I-structure_element +, O +bearing O +residue O +488 B-residue_number +, O +approaches O +the O +RNA B-structure_element +duplex I-structure_element +from O +the O +minor B-site +groove I-site +side O +at O +the O +editing B-site +site I-site +. O + +The O +side O +chain O +of O +this O +amino O +acid O +penetrates O +the O +helix O +where O +it O +occupies O +the O +space O +vacated O +by O +the O +flipped B-protein_state +out I-protein_state +base B-chemical +and O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +the O +complementary O +strand O +orphaned B-protein_state +base B-chemical +and O +to O +the O +2 O +’ O +hydroxyl O +of O +the O +nucleotide O +immediately O +5 O +’ O +to O +the O +editing B-site +site I-site +( O +Figs O +. O +3b O +, O +3c O +). O + +In O +the O +four O +structures B-evidence +reported O +here O +, O +three O +different O +combinations O +of O +helix O +- O +penetrating O +residue O +and O +orphan B-protein_state +base B-chemical +are O +observed O +( O +i O +. O +e O +. O +E488 B-residue_name_number ++ O +U B-residue_name +, O +E488 B-residue_name_number ++ O +C B-residue_name +and O +Q488 B-residue_name_number ++ O +C B-residue_name +) O +and O +all O +three O +combinations O +show O +the O +same O +side O +chain O +and O +base O +positions O +( O +Figs O +. O +3b O +, O +3c O +, O +Supplementary O +Fig O +. O +4a O +for O +overlay B-experimental_method +of O +all O +three O +). O + +For O +instance O +, O +in O +the O +complex B-protein_state +with I-protein_state +hADAR2d B-mutant +E488Q B-mutant +and O +the O +Bdf2 B-chemical +- I-chemical +C I-chemical +duplex I-chemical +, O +the O +protein O +recognizes O +an O +orphaned B-protein_state +C B-residue_name +by O +donating O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +from O +Nε2 O +to O +cytosine B-residue_name +N3 O +and O +from O +its O +backbone O +NH O +to O +cytosine B-residue_name +O2 O +( O +Fig O +. O +3b O +). O + +In O +the O +complex B-protein_state +with I-protein_state +hADAR2d B-mutant +WT B-protein_state +and O +the O +Bdf2 B-chemical +- I-chemical +U I-chemical +duplex I-chemical +, O +a O +similar O +interaction O +is O +observed O +with O +the O +E488 B-residue_name_number +backbone O +NH O +hydrogen B-bond_interaction +bonded I-bond_interaction +to O +the O +uracil B-residue_name +O2 O +and O +the O +E488 B-residue_name_number +side O +chain O +H B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +uracil B-residue_name +N3H O +( O +Fig O +. O +3c O +). O + +Interestingly O +, O +the O +E488Q B-mutant +mutant B-protein_state +was O +discovered O +in O +a O +screen O +for O +highly B-protein_state +active I-protein_state +ADAR2 B-protein +mutants B-protein_state +and O +this O +residue O +was O +suggested O +to O +be O +involved O +in O +base O +flipping O +given O +its O +effect O +on O +editing O +substrates O +with O +a O +fluorescent O +nucleobase O +at O +the O +editing B-site +site I-site +. O + +ADARs B-protein_type +react O +preferentially O +with O +adenosines B-residue_name +in O +A B-structure_element +• I-structure_element +C I-structure_element +mismatches O +and O +A B-structure_element +- I-structure_element +U I-structure_element +pairs I-structure_element +over O +A B-structure_element +• I-structure_element +A I-structure_element +and O +A B-structure_element +• I-structure_element +G I-structure_element +mismatches O +. O + +A O +purine B-chemical +at O +the O +orphan B-protein_state +base B-chemical +position O +( O +in O +its O +anti O +conformation O +) O +would O +clash O +with O +the O +488 B-residue_number +residue O +explaining O +the O +preference O +for O +pyrimidines B-chemical +here O +. O + +The O +interaction O +of O +the O +488 B-residue_number +residue O +with O +the O +orphaned B-protein_state +base B-chemical +is O +reminiscent O +of O +an O +interaction O +observed O +for O +Hha B-protein_type +I I-protein_type +DNA I-protein_type +methyltransfersase I-protein_type +( O +MTase B-protein_type +), O +a O +duplex B-structure_element +DNA I-structure_element +modifying O +enzyme O +that O +also O +uses O +a O +base O +flipping O +mechanism O +to O +access O +2 B-residue_name +’- I-residue_name +deoxycytidine I-residue_name +( O +dC B-residue_name +) O +for O +methylation O +. O + +For O +that O +enzyme O +, O +Q237 B-residue_name_number +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +an O +orphaned B-protein_state +dG B-residue_name +while O +it O +fills O +the O +void O +left O +by O +the O +flipped B-protein_state +out I-protein_state +dC B-residue_name +( O +Supplementary O +Fig O +. O +4b O +). O + +In O +addition O +, O +two O +glycine B-residue_name +residues O +flank O +Q237 B-residue_name_number +allowing O +the O +loop B-structure_element +to O +adopt O +the O +conformation O +necessary O +for O +penetration O +into O +the O +helix B-structure_element +. O + +The O +flipping B-structure_element +loop I-structure_element +in O +ADAR2 B-protein +( O +i O +. O +e O +. O +aa487 O +– B-residue_range +489 I-residue_range +) O +also O +has O +the O +helix O +- O +penetrating O +residue O +flanked O +by O +glycines B-residue_name +. O + +However O +, O +unlike O +the O +case O +of O +the O +DNA B-protein_type +MTase I-protein_type +that O +approaches O +the O +DNA B-chemical +from O +the O +major B-site +groove I-site +, O +the O +ADAR2 B-protein +loop B-structure_element +approaches O +the O +duplex B-structure_element +from O +the O +minor B-site +groove I-site +side O +. O + +Such O +an O +approach O +requires O +deeper O +penetration O +of O +the O +intercalating B-site +residue I-site +to O +access O +the O +H B-site +- I-site +bonding I-site +sites I-site +on O +the O +orphaned B-protein_state +base B-chemical +, O +necessitating O +an O +additional O +conformational O +change O +in O +the O +RNA B-structure_element +duplex I-structure_element +. O + +This O +change O +includes O +shifting O +of O +the O +base O +pairs O +immediately O +5 O +’ O +to O +the O +editing B-site +site I-site +toward O +the O +helical O +axis O +and O +a O +widening O +of O +the O +major B-site +groove I-site +opposite O +the O +editing B-site +site I-site +( O +Figs O +. O +4a O +, O +4b O +, O +Supplementary O +Video O +1 O +). O + +In O +the O +case O +of O +the O +hADAR2d B-complex_assembly +WT I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +U I-complex_assembly +RNA B-chemical +, O +this O +shift O +is O +accompanied O +by O +a O +shearing O +of O +the O +U11 B-residue_name_number +- O +A13 B-residue_name_number +' O +base O +pair O +with O +U11 B-residue_name_number +shifted O +further O +in O +the O +direction O +of O +the O +major B-site +groove I-site +creating O +an O +unusual O +U B-structure_element +- I-structure_element +A I-structure_element +"""" I-structure_element +wobble I-structure_element +"""" I-structure_element +interaction O +with O +adenine B-residue_name +N6 O +and O +N1 O +within O +H B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +distance O +to O +uracil B-residue_name +N3H O +and O +O2 O +, O +respectively O +( O +Fig O +. O +4c O +, O +Supplementary O +Fig O +. O +3b O +). O + +This O +type O +of O +wobble O +pair O +has O +been O +observed O +before O +and O +requires O +either O +the O +imino O +tautomer O +of O +adenine B-residue_name +or O +the O +enol O +tautomer O +of O +uracil B-residue_name +. O + +The O +ADAR B-protein_type +- O +induced O +distortion O +in O +RNA B-chemical +conformation O +results O +in O +a O +kink B-structure_element +in O +the O +RNA B-chemical +strand O +opposite O +the O +editing B-site +site I-site +( O +Fig O +. O +4b O +). O + +This O +kink B-structure_element +is O +stabilized O +by O +interactions O +of O +the O +side O +chains O +of O +R510 B-residue_name_number +and O +S495 B-residue_name_number +with O +phosphodiesters O +in O +the O +RNA B-chemical +backbone O +of O +the O +unedited O +strand O +( O +Fig O +. O +4a O +). O + +Interestingly O +, O +ADAR2 B-protein +’ O +s O +flipping B-structure_element +loop I-structure_element +approach O +from O +the O +minor B-site +groove I-site +side O +is O +like O +that O +seen O +with O +certain O +DNA B-protein_type +repair I-protein_type +glycosylases I-protein_type +( O +e O +. O +g O +. O +UDG B-protein +, O +HOGG1 B-protein +, O +and O +AAG B-protein +) O +that O +also O +project O +intercalating O +residues O +from O +loops B-structure_element +bound B-protein_state +in I-protein_state +the O +minor B-site +groove I-site +( O +Supplementary O +Fig O +. O +5a O +). O + +However O +, O +these O +enzymes O +typically O +bend O +the O +DNA B-chemical +duplex I-chemical +at O +the O +site O +of O +modification O +to O +allow O +for O +penetration O +of O +intercalating O +residues O +and O +damage O +recognition O +. O + +While O +hADAR2d B-mutant +clearly O +alters O +the O +duplex O +conformation O +to O +gain O +access O +to O +the O +modification O +site O +from O +the O +minor B-site +groove I-site +, O +it O +does O +not O +bend O +the O +RNA B-structure_element +duplex I-structure_element +( O +Figs O +. O +2a O +, O +2b O +, O +4b O +). O + +Furthermore O +, O +ADARs B-protein_type +do O +not O +modify O +duplex B-structure_element +DNA I-structure_element +. O + +The O +DNA B-chemical +B B-structure_element +- I-structure_element +form I-structure_element +helix I-structure_element +has O +groove O +widths O +and O +depths O +that O +would O +prevent O +productive O +interactions O +with O +ADAR B-protein_type +. O + +For O +instance O +, O +ADAR B-protein_type +can O +readily O +penetrate O +an O +A B-structure_element +- I-structure_element +form I-structure_element +helix I-structure_element +from O +the O +minor B-site +groove I-site +side O +and O +place O +the O +helix O +- O +penetrating O +residue O +in O +the O +space O +occupied O +by O +the O +editing B-site +site I-site +base O +( O +Supplementary O +Fig O +. O +6 O +). O + +However O +, O +this O +residue O +cannot O +penetrate O +the O +minor B-site +groove I-site +enough O +to O +occupy O +the O +base O +position O +in O +a O +B B-structure_element +- I-structure_element +form I-structure_element +helix I-structure_element +( O +Supplementary O +Fig O +. O +6 O +). O + +Furthermore O +, O +DNA B-chemical +lacks O +the O +2 O +’ O +hydroxyls O +that O +are O +used O +by O +ADAR B-protein_type +for O +substrate O +recognition O +( O +Fig O +. O +2c O +). O + +Thus O +, O +hADAR2d B-mutant +uses O +a O +substrate O +recognition O +and O +base O +flipping O +mechanism O +with O +similarities O +to O +other O +known O +nucleic B-protein_type +acid I-protein_type +- I-protein_type +modifying I-protein_type +enzymes I-protein_type +but O +uniquely O +suited O +for O +reaction O +with O +adenosine B-residue_name +in O +the O +context O +of O +duplex B-structure_element +RNA I-structure_element +. O + +Structures B-evidence +explain O +nearest O +neighbor O +preferences O + +ADARs B-protein_type +have O +a O +preference O +for O +editing O +adenosines B-residue_name +with O +5 O +’ O +nearest O +neighbor O +U B-residue_name +( O +or O +A B-residue_name +) O +and O +3 O +’ O +nearest O +neighbor O +G B-residue_name +. O +The O +ADAR2 B-protein +flipping B-structure_element +loop I-structure_element +occupies O +the O +minor B-site +groove I-site +spanning O +the O +three O +base O +pairs O +that O +include O +the O +nearest O +neighbor O +nucleotides O +flanking O +the O +edited O +base O +( O +Figs O +. O +3b O +, O +3c O +). O + +As O +described O +above O +, O +the O +base O +pair O +including O +the O +5 O +’ O +nearest O +neighbor O +U B-residue_name +( O +U11 B-residue_name_number +- O +A13 B-residue_name_number +’ O +in O +the O +Bdf2 B-chemical +duplex O +) O +is O +shifted O +from O +the O +position O +it O +would O +occupy O +in O +a O +typical O +A B-structure_element +- I-structure_element +form I-structure_element +helix I-structure_element +to O +accommodate O +the O +loop B-structure_element +( O +Fig O +. O +4a O +). O + +Also O +, O +the O +minor B-site +groove I-site +edge O +of O +this O +pair O +is O +juxtaposed O +to O +the O +protein O +backbone O +at O +G489 B-residue_name_number +. O + +Modeling O +a O +G B-structure_element +- I-structure_element +C I-structure_element +or I-structure_element +C I-structure_element +- I-structure_element +G I-structure_element +pair I-structure_element +at O +this O +position O +( O +i O +. O +e O +. O +5 O +’ O +G B-residue_name +or O +5 O +’ O +C B-residue_name +) O +suggests O +a O +2 O +- O +amino O +group O +in O +the O +minor B-site +groove I-site +would O +clash O +with O +the O +protein O +at O +G489 B-residue_name_number +( O +Fig O +. O +5a O +, O +Supplementary O +Fig O +. O +7c O +). O + +Indeed O +, O +replacing O +the O +U B-structure_element +- I-structure_element +A I-structure_element +pair I-structure_element +adjacent O +to O +the O +editing B-site +site I-site +with O +a O +C B-structure_element +- I-structure_element +G I-structure_element +pair I-structure_element +in O +the O +Gli1 B-protein +duplex O +substrate O +resulted O +in O +an O +80 O +% O +reduction O +in O +the O +rate O +of O +hADAR2d B-mutant +- O +catalyzed O +deamination O +( O +Figs O +. O +5b O +, O +5c O +). O + +To O +determine O +whether O +this O +effect O +arises O +from O +an O +increase O +in O +local O +duplex O +stability O +from O +the O +C O +- O +G O +for O +U O +- O +A O +substitution O +or O +from O +the O +presence O +of O +the O +2 O +- O +amino O +group O +, O +we O +replaced O +the O +U B-structure_element +- I-structure_element +A I-structure_element +pair I-structure_element +with O +a O +U B-structure_element +- I-structure_element +2 I-structure_element +- I-structure_element +aminopurine I-structure_element +( I-structure_element +2AP I-structure_element +) I-structure_element +pair I-structure_element +. O + +2AP B-structure_element +is O +an O +adenosine B-residue_name +analog O +that O +forms O +a O +base O +pair O +with O +uridine B-residue_name +of O +similar O +stability O +to O +a O +U B-structure_element +- I-structure_element +A I-structure_element +pair I-structure_element +, O +but O +places O +an O +amino O +group O +in O +the O +minor B-site +groove I-site +( O +Fig O +. O +5b O +). O + +Importantly O +, O +this O +substitution O +also O +resulted O +in O +an O +80 O +% O +reduction O +in O +rate O +, O +illustrating O +the O +detrimental O +effect O +of O +the O +amino O +group O +in O +the O +minor B-site +groove I-site +at O +this O +location O +. O + +These O +observations O +suggest O +that O +hADAR2 B-protein +’ O +s O +5 O +’ O +nearest O +neighbor O +preference O +for O +U B-residue_name +( O +or O +A B-residue_name +) O +is O +due O +to O +a O +destabilizing O +clash O +with O +the O +protein O +backbone O +at O +G489 B-residue_name_number +that O +results O +from O +the O +presence O +of O +an O +amino O +group O +in O +the O +minor B-site +groove I-site +at O +this O +location O +for O +sequences O +with O +5 O +’ O +nearest O +neighbor O +G B-residue_name +or O +C B-residue_name +. O +However O +, O +the O +observed O +clash O +is O +not O +severe O +and O +the O +enzyme O +would O +be O +able O +to O +accommodate O +G B-residue_name +or O +C B-residue_name +5 O +’ O +nearest O +neighbors O +by O +slight O +structural O +perturbations O +, O +explaining O +why O +this O +sequence O +preference O +is O +not O +an O +absolute O +requirement O +. O + +In O +each O +of O +the O +hADAR2d B-complex_assembly +- I-complex_assembly +RNA I-complex_assembly +structures B-evidence +reported O +here O +, O +the O +backbone O +carbonyl O +oxygen O +at O +S486 B-residue_name_number +accepts O +an O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +from O +the O +2 O +- O +amino O +group O +of O +the O +G B-residue_name +on O +the O +3 O +’ O +side O +of O +the O +edited O +nucleotide O +( O +Fig O +. O +5d O +). O + +Guanine B-residue_name +is O +the O +only O +common O +nucleobase O +that O +presents O +an O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +donor O +in O +the O +RNA B-site +minor I-site +groove I-site +suggesting O +that O +other O +nucleotides O +in O +this O +position O +would O +reduce O +editing O +efficiency O +. O + +Indeed O +, O +mutating B-experimental_method +this O +base O +to O +A B-residue_name +, O +C B-residue_name +or O +U B-residue_name +, O +while O +maintaining O +base O +pairing O +at O +this O +position O +, O +reduced O +the O +rate O +of O +deamination O +by O +hADAR2d B-mutant +in O +Gli1 B-protein +mRNA B-chemical +model O +substrates O +( O +Supplementary O +Fig O +. O +7 O +a O +– O +b O +). O + +To O +test O +the O +importance O +of O +the O +amino O +group O +on O +the O +3 O +’ O +G B-residue_name +in O +the O +hADAR2d B-mutant +reaction O +, O +we O +prepared O +RNA B-structure_element +duplex I-structure_element +substrates O +with O +purine O +analogs O +on O +the O +3 O +’ O +side O +of O +the O +edited B-protein_state +A B-residue_name +( O +Fig O +. O +5e O +). O + +We O +tested O +a O +G B-residue_name +analog O +that O +lacks O +the O +2 O +- O +amino O +group O +( O +inosine B-residue_name +, O +I B-residue_name +) O +and O +one O +that O +blocks O +access O +to O +this O +amino O +group O +( O +N2 O +- O +methylguanosine O +( O +N2MeG O +). O + +In O +addition O +, O +we O +compared O +a O +3 O +’ O +A B-residue_name +to O +a O +3 O +’ O +2AP B-structure_element +since O +2AP B-structure_element +could O +form O +the O +H B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +interaction I-bond_interaction +observed O +with O +S486 B-residue_name_number +. O + +We O +found O +the O +substrate O +with O +a O +3 O +’ O +N2MeG O +to O +be O +unreactive O +to O +hADAR2d B-mutant +- O +catalyzed O +deamination O +confirming O +the O +importance O +of O +the O +observed O +close O +approach O +by O +the O +protein O +to O +the O +3 O +’ O +G B-residue_name +2 O +- O +amino O +group O +( O +Fig O +. O +5f O +). O + +In O +addition O +, O +the O +substrate O +with O +a O +3 O +’ O +I B-residue_name +displayed O +a O +reduced B-evidence +deamination I-evidence +rate I-evidence +compared O +to O +the O +substrate O +with O +a O +3 O +’ O +G B-residue_name +suggesting O +the O +observed O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +to O +the O +2 O +- O +amino O +group O +contributes O +to O +the O +3 O +’ O +nearest O +neighbor O +selectivity O +( O +Fig O +. O +5f O +). O + +This O +conclusion O +is O +further O +supported O +by O +the O +observation O +that O +deamination O +in O +the O +substrate O +with O +a O +3 O +’ O +2AP B-structure_element +is O +faster O +than O +in O +the O +substrate O +with O +a O +3 O +’ O +A B-residue_name +( O +Fig O +. O +5f O +). O + +RNA B-structure_element +- I-structure_element +binding I-structure_element +loops I-structure_element +of O +the O +ADAR B-protein_type +catalytic B-structure_element +domain I-structure_element + +The O +structures B-evidence +reported O +here O +identify O +RNA B-structure_element +- I-structure_element +binding I-structure_element +loops I-structure_element +of O +the O +ADAR B-protein_type +catalytic B-structure_element +domain I-structure_element +and O +suggest O +roles O +for O +several O +amino O +acids O +not O +previously O +known O +to O +be O +important O +for O +editing O +, O +either O +substrate O +binding O +or O +catalysis O +( O +Fig O +. O +6 O +). O + +The O +side O +chain O +for O +R510 B-residue_name_number +ion B-bond_interaction +- I-bond_interaction +pairs I-bond_interaction +with O +the O +3 O +’ O +phosphodiester O +of O +the O +orphaned B-protein_state +nucleotide B-chemical +( O +Figs O +. O +3a O +, O +3c O +). O + +This O +residue O +is O +conserved B-protein_state +in O +ADAR2s B-protein_type +and O +ADAR1s B-protein_type +, O +but O +is O +glutamine B-residue_name +in O +the O +editing B-protein_state +- I-protein_state +inactive I-protein_state +ADAR3s B-protein_type +( O +Supplementary O +Table O +1 O +). O + +Mutation B-experimental_method +of O +hADAR2d B-mutant +at O +this O +site O +to O +either O +glutamine B-residue_name +( O +R510Q B-mutant +) O +or O +to O +alanine B-residue_name +( O +R510A B-mutant +) O +reduced O +the O +measured O +deamination B-evidence +rate I-evidence +constant I-evidence +by O +approximately O +an O +order O +of O +magnitude O +( O +Fig O +. O +6c O +). O + +In O +addition O +, O +the O +contact O +point O +near O +the O +5 O +’ O +end O +of O +the O +unedited O +strand O +involves O +G593 B-residue_name_number +, O +K594 B-residue_name_number +and O +R348 B-residue_name_number +, O +residues O +completely B-protein_state +conserved I-protein_state +in O +the O +family O +of O +ADAR2s B-protein_type +( O +Fig O +. O +2c O +, O +Supplementary O +Table O +1 O +). O + +Mutation B-experimental_method +of O +any O +of O +these O +residues O +to O +alanine B-residue_name +( O +G593A B-mutant +, O +K594A B-mutant +, O +R348A B-mutant +) O +substantially O +reduces O +editing O +activity O +( O +Fig O +. O +6c O +). O + +In O +addition O +, O +mutation B-experimental_method +of O +G593 B-residue_name_number +to O +glutamic B-residue_name +acid I-residue_name +( O +G593E B-mutant +) O +resulted O +in O +a O +nearly O +two O +orders O +of O +magnitude O +reduction O +in O +rate O +, O +consistent O +with O +proximity O +of O +this O +residue O +to O +the O +negatively O +charged O +phosphodiester O +backbone O +of O +the O +RNA B-chemical +( O +Fig O +. O +6c O +). O + +RNA B-chemical +binding O +leads O +to O +an O +ordering O +of O +the O +454 B-residue_range +– I-residue_range +477 I-residue_range +loop B-structure_element +, O +which O +was O +disordered B-protein_state +in O +the O +RNA B-protein_state +- I-protein_state +free I-protein_state +hADAR2d B-mutant +structure B-evidence +( O +Fig O +. O +1d O +, O +green O +) O +( O +Supplementary O +Video O +2 O +). O + +This O +loop B-structure_element +binds O +the O +RNA B-structure_element +duplex I-structure_element +contacting O +the O +minor B-site +groove I-site +near O +the O +editing B-site +site I-site +and O +inserting O +into O +the O +adjacent O +major B-site +groove I-site +( O +Fig O +. O +6e O +). O + +This O +loop B-structure_element +sequence O +is O +conserved B-protein_state +in O +ADAR2s B-protein_type +but O +different O +in O +the O +family O +of O +ADAR1s B-protein_type +( O +Fig O +. O +6d O +). O + +The O +substantial O +difference O +in O +sequence O +between O +the O +ADARs B-protein_type +in O +this O +RNA B-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +suggests O +differences O +in O +editing B-site +site I-site +selectivity O +between O +the O +two O +ADARs B-protein_type +arise O +, O +at O +least O +in O +part O +, O +from O +differences O +in O +how O +this O +loop B-structure_element +binds O +RNA B-chemical +substrates O +. O + +Base O +flipping O +is O +a O +well O +- O +characterized O +mechanism O +by O +which O +nucleic B-protein_type +acid I-protein_type +modifying I-protein_type +enzymes I-protein_type +gain O +access O +to O +sites O +of O +reaction O +that O +are O +otherwise O +buried O +in O +base O +- O +paired O +structures B-evidence +. O + +DNA B-protein_type +methylases I-protein_type +, O +DNA B-protein_type +repair I-protein_type +glycosylases I-protein_type +and O +RNA B-protein_type +loop I-protein_type +modifying I-protein_type +enzymes I-protein_type +are O +known O +that O +flip O +a O +nucleotide B-chemical +out O +of O +a O +base O +pair O +. O + +However O +, O +none O +of O +the O +structurally O +characterized O +base B-protein_type +- I-protein_type +flipping I-protein_type +enzymes I-protein_type +access O +their O +reactive B-site +sites I-site +from O +within O +a O +normal B-protein_state +base I-protein_state +- I-protein_state +paired I-protein_state +RNA B-structure_element +duplex I-structure_element +. O + +We O +are O +aware O +of O +one O +other O +protein O +- O +induced O +nucleotide O +flipping O +from O +an O +RNA B-structure_element +duplex I-structure_element +region O +. O + +Bacterial B-taxonomy_domain +initiation B-protein +factor I-protein +1 I-protein +( O +IF1 B-protein +) O +binds O +to O +the O +30S B-complex_assembly +ribosomal I-complex_assembly +subunit I-complex_assembly +at O +helix B-structure_element +44 I-structure_element +of O +16S B-chemical +RNA I-chemical +with O +A1492 B-residue_name_number +and O +A1493 B-residue_name_number +flipped B-protein_state +out I-protein_state +of O +the O +helix O +and O +bound B-protein_state +into I-protein_state +protein B-site +pockets I-site +( O +Supplementary O +Fig O +. O +5b O +). O + +However O +, O +these O +nucleotides O +are O +located O +in O +a O +highly B-protein_state +distorted I-protein_state +and O +dynamic B-protein_state +duplex B-structure_element +region I-structure_element +containing O +several O +mismatches O +and O +are O +predisposed O +to O +undergo O +this O +conformational O +change O +. O + +Thus O +, O +this O +system O +is O +not O +illustrative O +of O +base O +flipping O +from O +a O +normal B-protein_state +duplex O +and O +does O +not O +involve O +an O +enzyme O +that O +must O +carryout O +a O +chemical O +reaction O +on O +the O +flipped B-protein_state +out I-protein_state +nucleotide B-chemical +. O + +Other O +RNA B-protein_type +modification I-protein_type +enzymes I-protein_type +are O +known O +that O +flip O +nucleotides O +out O +of O +loops O +, O +even O +from O +base O +pairs O +in O +loop O +regions O +( O +pseudoU B-protein_type +synthetase I-protein_type +, O +tRNA B-protein_type +transglycosylase I-protein_type +, O +and O +restrictocin B-protein +bound B-protein_state +to I-protein_state +sarcin B-structure_element +/ I-structure_element +ricin I-structure_element +loop I-structure_element +of O +28S B-chemical +rRNA I-chemical +) O +( O +Supplementary O +Fig O +. O +5b O +). O + +Because O +the O +modification B-site +sites I-site +are O +not O +flanked O +on O +both O +sides O +by O +normal B-protein_state +duplex B-structure_element +, O +these O +enzymes O +do O +not O +experience O +the O +same O +limits O +in O +approach O +to O +the O +substrate O +that O +ADARs B-protein_type +do O +. O + +The O +fact O +that O +ADARs B-protein_type +must O +induce O +flipping O +from O +a O +normal B-protein_state +duplex B-structure_element +has O +implications O +on O +its O +preference O +for O +adenosines B-residue_name +flanked O +by O +certain O +base O +pairs O +, O +a O +phenomenon O +that O +was O +not O +well O +understood O +prior O +to O +this O +work O +. O + +In O +our O +structures B-evidence +, O +the O +flipped B-protein_state +out I-protein_state +8 B-chemical +- I-chemical +azanebularine I-chemical +is O +hydrated O +, O +mimicking O +the O +tetrahedral O +intermediate O +predicted O +for O +deamination O +of O +adenosine B-residue_name +( O +Figs O +. O +1b O +, O +3a O +, O +Supplementary O +Fig O +. O +3 O +a O +– O +b O +). O + +Our O +use O +of O +8 B-chemical +- I-chemical +azanebularine I-chemical +, O +with O +its O +high O +propensity O +to O +form O +a O +covalent O +hydrate O +, O +allowed O +us O +to O +capture O +a O +true O +mimic O +of O +the O +tetrahedral O +intermediate O +and O +reveal O +the O +interactions O +between O +the O +deaminase B-protein_type +active B-site +site I-site +and O +the O +reactive O +nucleotide O +. O + +In O +addition O +, O +8 B-chemical +- I-chemical +azanebularine I-chemical +was O +found O +to O +adopt O +a O +2 O +’- O +endo O +sugar O +pucker O +with O +its O +2 O +’- O +hydroxyl O +H B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +protein O +backbone O +carbonyl O +at O +T375 B-residue_name_number +. O + +The O +2 O +’ O +endo O +conformation O +appears O +to O +facilitate O +access O +of O +the O +nucleobase O +to O +the O +zinc B-chemical +- O +bound O +water B-chemical +for O +nucleophilic O +attack O +at O +C6 O +. O + +Several O +other O +base B-protein_type +- I-protein_type +flipping I-protein_type +enzymes I-protein_type +stabilize O +the O +altered O +nucleic O +acid O +conformation O +by O +intercalation O +of O +an O +amino O +acid O +side O +chain O +into O +the O +space O +vacated O +by O +the O +flipped B-protein_state +out I-protein_state +base B-chemical +. O + +For O +hADAR2 B-protein +, O +E488 B-residue_name_number +serves O +this O +role O +. O + +In O +the O +two O +structures B-evidence +with O +wild B-protein_state +type I-protein_state +hADAR2 B-protein +, O +the O +E488 B-residue_name_number +residue O +and O +orphan B-protein_state +base B-chemical +are O +in O +nearly O +identical O +positions O +( O +see O +Supplementary O +Fig O +. O +4a O +for O +overlay B-experimental_method +). O + +Thus O +, O +the O +E488 B-residue_name_number +side O +chain O +directly O +contacts O +each O +orphan B-protein_state +base B-chemical +, O +likely O +by O +accepting O +an O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +from O +uracil B-residue_name +N3H O +or O +by O +donating O +an O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +to O +cytidine B-residue_name +N3 O +. O + +The O +latter O +interaction O +requires O +E488 B-residue_name_number +to O +be O +protonated B-protein_state +. O + +The O +pKa B-evidence +of O +E488 B-residue_name_number +in O +the O +ADAR B-complex_assembly +- I-complex_assembly +RNA I-complex_assembly +complex O +has O +not O +been O +measured O +, O +but O +proximity O +to O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +acceptors O +, O +such O +as O +cytidine B-residue_name +N3 O +, O +and O +insertion O +between O +stacked O +nucleobases O +, O +would O +undoubtedly O +elevate O +this O +value O +and O +could O +lead O +to O +a O +substantial O +fraction O +in O +the O +protonated B-protein_state +state O +at O +physiologically O +relevant O +pH O +. O +Since O +the O +glutamine B-residue_name +side O +chain O +is O +fully B-protein_state +protonated I-protein_state +under O +physiologically O +relevant O +conditions O +, O +a O +rate O +enhancement O +for O +the O +E488Q B-mutant +mutant B-protein_state +would O +be O +expected O +if O +the O +reaction O +requires O +E488 B-residue_name_number +protonation O +. O + +The O +interactions O +of O +hADAR2d B-mutant +with O +base O +pairs O +adjacent O +to O +the O +editing B-site +site I-site +adenosine B-residue_name +explain O +the O +known O +5 O +’ O +and O +3 O +’ O +nearest O +neighbor O +preferences O +( O +Fig O +. O +5 O +). O + +While O +these O +studies O +indicate O +the O +ADAR2 B-protein +catalytic B-structure_element +domain I-structure_element +makes O +an O +important O +contact O +to O +the O +3 O +’ O +nearest O +neighbor O +G B-residue_name +, O +Stefl O +et O +al O +. O +suggested O +the O +3 O +’ O +G B-residue_name +preference O +arises O +from O +dsRBD B-structure_element +binding O +selectivity O +for O +ADAR2 B-protein +. O + +These O +authors O +reported O +a O +model O +for O +ADAR2 B-protein +’ O +s O +dsRBDs B-structure_element +bound B-protein_state +to I-protein_state +an O +editing O +substrate O +based O +on O +NMR B-experimental_method +data O +from O +the O +isolated B-protein_state +dsRBDs B-structure_element +( O +lacking B-protein_state +the O +deaminase B-structure_element +domain I-structure_element +) O +and O +short O +RNA B-chemical +fragments O +derived O +from O +the O +GluR B-protein +- I-protein +B I-protein +R B-site +/ I-site +G I-site +site I-site +RNA B-chemical +. O + +They O +describe O +an O +interaction O +wherein O +the O +3 O +’ O +G B-residue_name +2 O +- O +amino O +group O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +the O +backbone O +carbonyl O +of O +S258 B-residue_name_number +found O +in O +the O +β1 B-structure_element +- I-structure_element +β2 I-structure_element +loop I-structure_element +of O +ADAR2 B-protein +’ O +s O +dsRBDII B-structure_element +. O + +It O +is O +not O +possible O +for O +the O +S486 B-residue_name_number +- O +3 O +’ O +G B-residue_name +interaction O +we O +describe O +here O +and O +the O +S258 B-residue_name_number +- O +3 O +’ O +G B-residue_name +interaction O +reported O +by O +Stefl O +et O +al O +. O +to O +exist O +in O +the O +same O +complex O +since O +both O +involve O +protein O +loops O +bound B-protein_state +in I-protein_state +the O +RNA B-chemical +minor B-site +groove I-site +at O +the O +same O +location O +. O + +Because O +our O +structures B-evidence +have O +captured O +the O +edited B-protein_state +nucleotide B-chemical +in O +the O +conformation O +required O +to O +access O +the O +active B-site +site I-site +, O +the O +interactions O +observed O +here O +are O +highly O +likely O +to O +occur O +during O +the O +deamination O +reaction O +at O +the O +editing B-site +site I-site +. O + +However O +, O +since O +dsRBDs B-structure_element +are O +known O +to O +bind O +promiscuously O +with O +duplex B-structure_element +RNA I-structure_element +, O +it O +is O +possible O +that O +the O +S258 B-residue_name_number +- O +3 O +’ O +G B-residue_name +interaction O +found O +in O +a O +complex O +lacking B-protein_state +the I-protein_state +deaminase B-structure_element +domain I-structure_element +is O +not O +relevant O +to O +catalysis O +at O +the O +editing B-site +site I-site +. O + +It O +is O +also O +possible O +that O +ADAR B-protein_type +dsRBD B-structure_element +and O +catalytic B-structure_element +domain I-structure_element +binding O +are O +sequential O +, O +with O +release O +of O +the O +dsRBD B-structure_element +from O +the O +RNA B-chemical +taking O +place O +prior O +to O +catalytic B-structure_element +domain I-structure_element +engagement O +and O +base O +flipping O +. O + +Aicardi O +- O +Goutieres O +Syndrome O +( O +AGS O +) O +and O +Dyschromatosis O +Symmetrica O +Hereditaria O +( O +DSH O +) O +are O +human B-species +diseases O +caused O +by O +mutations O +in O +the O +human B-species +ADAR1 B-protein +gene O +and O +several O +of O +the O +disease O +- O +associated O +mutations O +are O +found O +in O +the O +deaminase B-structure_element +domain I-structure_element +. O + +Given O +the O +conservation O +in O +RNA B-site +binding I-site +surface I-site +and O +active B-site +site I-site +residues O +, O +we O +expect O +the O +hADAR1 B-protein +catalytic B-structure_element +domain I-structure_element +to O +bind O +RNA B-chemical +with O +a O +similar O +orientation O +of O +the O +helix O +found O +in O +our O +hADAR2d B-complex_assembly +- I-complex_assembly +RNA I-complex_assembly +structures B-evidence +. O + +When O +one O +maps O +the O +locations O +of O +the O +AGS O +- O +associated O +mutations O +onto O +the O +hADAR2d B-complex_assembly +- I-complex_assembly +RNA I-complex_assembly +complex O +, O +two O +mutations O +involve O +residues O +in O +close O +proximity O +to O +the O +RNA B-chemical +(< O +4 O +Å O +) O +( O +Supplementary O +Fig O +. O +8a O +). O + +G487 B-residue_name_number +of O +hADAR2 B-protein +is O +found O +on O +the O +flipping B-structure_element +loop I-structure_element +near O +the O +RNA B-chemical +( O +Fig O +. O +3b O +). O + +Sequence O +in O +this O +loop B-structure_element +is O +highly B-protein_state +conserved I-protein_state +among O +ADARs B-protein_type +and O +corresponds O +to O +G1007 B-residue_name_number +in O +hADAR1 B-protein +( O +Supplementary O +Table O +2 O +). O + +An O +arginine B-residue_name +at O +this O +position O +would O +preclude O +close O +approach O +of O +the O +flipping B-structure_element +loop I-structure_element +to O +the O +RNA B-chemical +, O +preventing O +E1008 B-residue_name_number +insertion O +and O +base O +flipping O +into O +the O +active B-site +site I-site +( O +Supplementary O +Fig O +. O +8b O +). O + +This O +is O +consistent O +with O +the O +observation O +that O +the O +G1007R B-mutant +mutation O +in O +hADAR1 B-protein +inhibits O +RNA B-chemical +editing O +activity O +. O + +Also O +, O +K376 B-residue_name_number +forms O +salt B-bond_interaction +bridges I-bond_interaction +with O +both O +the O +5 O +’ O +and O +3 O +’ O +phosphodiesters O +of O +the O +guanosine B-residue_name +on O +the O +3 O +’ O +side O +of O +the O +editing B-site +site I-site +( O +Fig O +. O +2 O +). O + +The O +corresponding O +residue O +in O +hADAR1 B-protein +( O +R892 B-residue_name_number +) O +could O +form O +similar O +contacts O +and O +the O +R892H B-mutant +mutation O +would O +likely O +alter O +this O +interaction O +. O + +In O +summary O +, O +the O +structures B-evidence +described O +here O +establish O +human B-species +ADAR2 B-protein +as O +a O +base O +- O +flipping O +enzyme O +that O +uses O +a O +unique O +mechanism O +well O +suited O +for O +modifying O +duplex B-structure_element +RNA I-structure_element +. O + +In O +addition O +, O +this O +work O +provides O +a O +basis O +for O +understanding O +the O +role O +of O +the O +ADAR B-protein_type +catalytic B-structure_element +domain I-structure_element +in O +determining O +editing B-site +site I-site +selectivity O +and O +additional O +structural O +context O +to O +evaluate O +the O +impact O +of O +ADAR B-protein_type +mutations O +associated O +with O +human B-species +disease O +. O + +Human B-species +ADAR2 B-protein +and O +modified O +RNAs B-chemical +for O +crystallography B-experimental_method + +a O +, O +Domain O +map O +for O +human B-species +ADAR2 B-protein +b O +, O +ADAR B-protein_type +reaction O +showing O +intermediate O +and O +8 B-chemical +- I-chemical +azanebularine I-chemical +( I-chemical +N I-chemical +) I-chemical +hydrate I-chemical +that O +mimics O +this O +structure B-evidence +c O +, O +Duplex B-structure_element +RNAs I-structure_element +used O +for O +crystallization B-experimental_method +. O + +Bdf2 B-chemical +duplex I-chemical +sequence O +is O +derived O +from O +an O +editing B-site +site I-site +found O +in O +S B-species +. I-species +cerevisiae I-species +Bdf2 B-chemical +mRNA I-chemical +and O +Gli1 B-protein +duplex O +has O +sequence O +surrounding O +the O +human B-species +Gli1 B-protein +mRNA B-chemical +editing B-site +site I-site +. O + +Structure B-evidence +of O +hADAR2d B-mutant +E488Q B-mutant +bound B-protein_state +to I-protein_state +the O +Bdf2 B-chemical +- I-chemical +C I-chemical +RNA I-chemical +duplex I-chemical +at O +2 O +. O +75 O +Å O +resolution O + +a O +, O +View O +of O +structure O +perpendicular O +to O +the O +dsRNA B-chemical +helical O +axis O +. O + +Colors O +correspond O +to O +those O +in O +Figs O +. O +1a O +and O +1c O +; O +flipped B-protein_state +out I-protein_state +base O +N O +is O +highlighted O +red O +, O +zinc B-chemical +in O +grey O +space O +- O +filling O +sphere O +, O +Q488 B-residue_name_number +in O +yellow O +, O +previously O +disordered B-protein_state +aa454 O +– B-residue_range +477 I-residue_range +loop B-structure_element +in O +green O +and O +inositol B-chemical +hexakisphosphate I-chemical +( O +IHP B-chemical +) O +in O +space O +filling O +. O + +A O +transparent O +surface O +is O +shown O +for O +the O +hADAR2d B-mutant +protein O +. O + +b O +, O +View O +of O +structure O +along O +the O +dsRNA B-chemical +helical O +axis O +. O + +c O +, O +Summary O +of O +the O +contacts O +between O +hADAR2d B-mutant +E488Q B-mutant +and O +the O +Bdf2 B-chemical +- I-chemical +C I-chemical +RNA I-chemical +duplex I-chemical +. O + +ADAR B-protein_type +recognition O +of O +the O +flipped B-protein_state +out I-protein_state +and O +orphaned B-protein_state +nucleotides B-chemical + +a O +, O +Contacts O +to O +the O +editing B-site +site I-site +nucleotide B-chemical +( O +N O +) O +in O +the O +active B-site +site I-site +. O + +b O +, O +Orphan B-protein_state +nucleotide B-chemical +recognition O +in O +the O +hADAR2d B-complex_assembly +E488Q I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +C I-complex_assembly +complex O +. O + +c O +, O +Orphan B-protein_state +nucleotide B-chemical +recognition O +in O +the O +hADAR2d B-complex_assembly +WT I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +U I-complex_assembly +complex O +. O + +Other O +ADAR B-protein_type +- O +induced O +changes O +in O +RNA B-chemical +conformation O + +a O +, O +hADAR2d B-mutant +shifts O +the O +position O +of O +U11 B-residue_name_number +- O +A13 B-residue_name_number +’ O +base O +pair O +from O +ideal O +A B-structure_element +- I-structure_element +form I-structure_element +RNA I-structure_element +helix I-structure_element +( O +yellow O +). O + +b O +, O +Overlay B-experimental_method +of O +Bdf2 B-chemical +duplex I-chemical +RNA I-chemical +and O +idealized O +A B-structure_element +form I-structure_element +duplex I-structure_element +of O +same O +sequence O +( O +yellow O +) O +illustrating O +kink O +in O +strand O +and O +widening O +of O +major B-site +groove I-site +opposite O +editing B-site +site I-site +induced O +by O +hADAR2d B-mutant +. O + +c O +, O +Unusual O +“ O +wobble O +” O +A13 B-residue_name_number +’- O +U11 B-residue_name_number +interaction O +in O +the O +hADAR2d B-complex_assembly +WT I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +U I-complex_assembly +complex O +shown O +in O +stick O +with O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +indicated O +with O +yellow O +dashes O +and O +distances O +shown O +in O +Å O +. O +The O +position O +of O +this O +base O +pair O +in O +the O +hADAR2d B-complex_assembly +E488Q I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +C I-complex_assembly +duplex O +is O +shown O +in O +wire O +with O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +shown O +with O +gray O +dashes O +. O + +Interactions O +with O +editing B-site +site I-site +nearest O +neighbor O +nucleotides B-chemical + +a O +, O +The O +minor B-site +groove I-site +edge O +of O +the O +U11 B-residue_name_number +- O +A13 B-residue_name_number +’ O +base O +pair O +from O +the O +Bdf2 B-chemical +duplex I-chemical +approaches O +G489 B-residue_name_number +; O +model O +with O +a O +C B-structure_element +- I-structure_element +G I-structure_element +pair I-structure_element +at O +this O +position O +suggests O +a O +clash O +with O +the O +G B-residue_name +2 O +- O +amino O +group O +b O +, O +RNA B-structure_element +duplex I-structure_element +substrates O +prepared O +with O +different O +5 O +’ O +nearest O +neighbor O +nucleotides O +adjacent O +to O +editing B-site +site I-site +indicated O +in O +red O +( O +2AP B-structure_element += O +2 B-structure_element +- I-structure_element +aminopurine I-structure_element +). O + +c O +, O +Comparison O +of O +deamination B-evidence +rate I-evidence +constants I-evidence +by O +hADAR2d B-mutant +at O +the O +editing B-site +site I-site +adenosine B-residue_name +( O +red O +) O +for O +duplexes O +bearing O +different O +5 O +’ O +nearest O +neighbors O +; O +krel B-evidence += O +kobs B-evidence +/( O +kobs B-evidence +for O +unmodified B-protein_state +RNA B-chemical +). O + +d O +, O +hADAR2 B-protein +S486 B-residue_name_number +backbone O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +with O +3 O +’ O +G B-residue_name +2 O +- O +amino O +group O +; O +e O +, O +RNA B-structure_element +duplex I-structure_element +substrates O +prepared O +with O +different O +3 O +’ O +nearest O +neighbor O +nucleotides O +adjacent O +to O +editing B-site +site I-site +indicated O +in O +red O +( O +I B-residue_name += O +inosine B-residue_name +, O +N2MeG O += O +N2 O +- O +methylguanosine O +, O +2AP B-structure_element += O +2 B-structure_element +- I-structure_element +aminopurine I-structure_element +). O + +f O +, O +Comparison O +of O +deamination B-evidence +rate I-evidence +constants I-evidence +by O +hADAR2d B-mutant +at O +the O +editing B-site +site I-site +adenosine B-residue_name +( O +red O +) O +for O +duplexes O +bearing O +different O +3 O +’ O +nearest O +neighbors O +. O + +krel B-evidence += O +kobs B-evidence +/( O +kobs B-evidence +for O +unmodified B-protein_state +RNA B-chemical +). O + +RNA B-structure_element +- I-structure_element +binding I-structure_element +loops I-structure_element +in O +the O +ADAR B-protein_type +catalytic B-structure_element +domain I-structure_element + +a O +, O +hADAR2 B-protein +residues O +that O +contact O +phosphodiester O +backbone O +near O +5 O +’ O +end O +of O +unedited O +strand O +. O + +b O +, O +Location O +of O +mutations O +introduced O +at O +protein B-site +- I-site +RNA I-site +interface I-site +. O + +c O +, O +Comparison O +of O +deamination B-evidence +rate I-evidence +constants I-evidence +of O +the O +different O +hADAR2d B-mutant +mutants O +( O +Log O +scale O +). O + +krel B-evidence += O +kobs B-evidence +for O +mutant B-protein_state +/ O +kobs B-evidence +for O +WT B-protein_state +. O + +d O +, O +Sequence B-experimental_method +alignment I-experimental_method +of O +ADAR2s B-protein_type +( O +A2 O +) O +and O +ADAR1s B-protein_type +( O +A1 O +) O +from O +different O +organisms O +with O +different O +levels O +of O +conservation O +colored O +( O +Yellow O +: O +conserved B-protein_state +in O +all O +ADAR1s B-protein_type +and O +ADAR2s B-protein_type +, O +red O +: O +conserved B-protein_state +in O +ADAR2s B-protein_type +, O +blue O +: O +conserved B-protein_state +in O +ADAR1s B-protein_type +. O + +e O +, O +Interaction O +of O +the O +ADAR B-structure_element +- I-structure_element +specific I-structure_element +RNA I-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +near O +the O +5 O +’ O +end O +of O +the O +edited O +strand O +. O + +Colors O +as O +in O +d O +, O +white O +: O +not B-protein_state +conserved I-protein_state +, O +flipped B-protein_state +out I-protein_state +base B-chemical +is O +shown O +in O +pink O +. O + +Structural O +basis O +for O +the O +regulation O +of O +enzymatic O +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +by O +domain O +- O +domain O +interactions O + +Regnase B-protein +- I-protein +1 I-protein +is O +an O +RNase B-protein_type +that O +directly O +cleaves O +mRNAs B-chemical +of O +inflammatory O +genes O +such O +as O +IL B-protein_type +- I-protein_type +6 I-protein_type +and O +IL B-protein_type +- I-protein_type +12p40 I-protein_type +, O +and O +negatively O +regulates O +cellular O +inflammatory O +responses O +. O + +Here O +, O +we O +report O +the O +structures B-evidence +of O +four O +domains O +of O +Regnase B-protein +- I-protein +1 I-protein +from O +Mus B-species +musculus I-species +— O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +NTD B-structure_element +), O +PilT B-structure_element +N I-structure_element +- I-structure_element +terminus I-structure_element +like I-structure_element +( O +PIN B-structure_element +) O +domain O +, O +zinc B-structure_element +finger I-structure_element +( O +ZF B-structure_element +) O +domain O +and O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +CTD B-structure_element +). O + +The O +PIN B-structure_element +domain O +harbors O +the O +RNase B-protein_type +catalytic B-site +center I-site +; O +however O +, O +it O +is O +insufficient O +for O +enzymatic O +activity O +. O + +We O +found O +that O +the O +NTD B-structure_element +associates O +with O +the O +PIN B-structure_element +domain O +and O +significantly O +enhances O +its O +RNase B-protein_type +activity O +. O + +The O +PIN B-structure_element +domain O +forms O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomer B-oligomeric_state +and O +the O +dimer B-site +interface I-site +overlaps O +with O +the O +NTD B-site +binding I-site +site I-site +. O + +Interestingly O +, O +mutations B-experimental_method +blocking O +PIN B-structure_element +oligomerization O +had O +no O +RNase B-protein_type +activity O +, O +indicating O +that O +both O +oligomerization O +and O +NTD B-structure_element +binding O +are O +crucial O +for O +RNase B-protein_type +activity O +in O +vitro O +. O + +These O +results O +suggest O +that O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O +is O +tightly O +controlled O +by O +both O +intramolecular O +( O +NTD B-structure_element +- O +PIN B-structure_element +) O +and O +intermolecular O +( O +PIN B-structure_element +- O +PIN B-structure_element +) O +interactions O +. O + +The O +initial O +sensing O +of O +infection O +is O +mediated O +by O +a O +set O +of O +pattern B-protein_type +- I-protein_type +recognition I-protein_type +receptors I-protein_type +( O +PRRs B-protein_type +) O +such O +Toll B-protein_type +- I-protein_type +like I-protein_type +receptors I-protein_type +( O +TLRs B-protein_type +) O +and O +the O +intracellular O +signaling O +cascades O +triggered O +by O +TLRs B-protein_type +evoke O +transcriptional O +expression O +of O +inflammatory O +mediators O +that O +coordinate O +the O +elimination O +of O +pathogens O +and O +infected O +cells O +. O + +Regnase B-protein +- I-protein +1 I-protein +( O +also O +known O +as O +Zc3h12a B-protein +and O +MCPIP1 B-protein +) O +is O +an O +RNase B-protein_type +whose O +expression O +level O +is O +stimulated O +by O +lipopolysaccharides B-chemical +and O +prevents O +autoimmune O +diseases O +by O +directly O +controlling O +the O +stability O +of O +mRNAs B-chemical +of O +inflammatory O +genes O +such O +as O +interleukin O +( B-protein_type +IL I-protein_type +)- I-protein_type +6 I-protein_type +, O +IL B-protein_type +- I-protein_type +1β I-protein_type +, O +IL B-protein_type +- I-protein_type +2 I-protein_type +, O +and O +IL B-protein_type +- I-protein_type +12p40 I-protein_type +. O + +Regnase B-protein +- I-protein +1 I-protein +accelerates O +target O +mRNA B-chemical +degradation O +via O +their O +3 B-structure_element +′- I-structure_element +terminal I-structure_element +untranslated I-structure_element +region I-structure_element +( O +3 B-structure_element +′ I-structure_element +UTR I-structure_element +), O +and O +also O +degrades O +its O +own O +mRNA B-chemical +. O + +Regnase B-protein +- I-protein +1 I-protein +is O +a O +member O +of O +Regnase B-protein_type +family I-protein_type +and O +is O +composed O +of O +a O +PilT B-structure_element +N I-structure_element +- I-structure_element +terminus I-structure_element +like I-structure_element +( O +PIN B-structure_element +) O +domain O +followed O +by O +a O +CCCH B-structure_element +- I-structure_element +type I-structure_element +zinc I-structure_element +– I-structure_element +finger I-structure_element +( O +ZF B-structure_element +) O +domain O +, O +which O +are O +conserved B-protein_state +among O +Regnase B-protein_type +family I-protein_type +members I-protein_type +. O + +Recently O +, O +the O +crystal B-evidence +structure I-evidence +of O +the O +Regnase B-protein +- I-protein +1 I-protein +PIN B-structure_element +domain O +derived O +from O +Homo B-species +sapiens I-species +was O +reported O +. O + +The O +structure B-evidence +combined O +with O +functional O +analyses O +revealed O +that O +four O +catalytically O +important O +Asp B-residue_name +residues O +form O +the O +catalytic B-site +center I-site +and O +stabilize O +Mg2 B-chemical ++ I-chemical +binding O +that O +is O +crucial O +for O +RNase B-protein_type +activity O +. O + +Several O +CCCH B-structure_element +- I-structure_element +type I-structure_element +ZF I-structure_element +motifs I-structure_element +in O +RNA B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +have O +been O +reported O +to O +directly O +bind O +RNA B-chemical +. O + +In O +addition O +, O +Regnase B-protein +- I-protein +1 I-protein +has O +been O +predicted O +to O +possess O +other O +domains O +in O +the O +N B-structure_element +- I-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +regions I-structure_element +. O + +However O +, O +the O +structure B-evidence +and O +function O +of O +the O +ZF B-structure_element +domain O +, O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +NTD B-structure_element +) O +and O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +CTD B-structure_element +) O +of O +Regnase B-protein +- I-protein +1 I-protein +have O +not O +been O +solved O +. O + +Here O +, O +we O +performed O +structural B-experimental_method +and I-experimental_method +functional I-experimental_method +analyses I-experimental_method +of O +individual O +domains O +of O +Regnase B-protein +- I-protein +1 I-protein +derived O +from O +Mus B-species +musculus I-species +in O +order O +to O +understand O +the O +catalytic O +activity O +in O +vitro O +. O + +Our O +data O +revealed O +that O +the O +catalytic O +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +is O +regulated O +through O +both O +intra O +and O +intermolecular O +domain O +interactions O +in O +vitro O +. O + +The O +NTD B-structure_element +plays O +a O +crucial O +role O +in O +efficient O +cleavage O +of O +target O +mRNA B-chemical +, O +through O +intramolecular O +NTD B-structure_element +- O +PIN B-structure_element +interactions O +. O + +Moreover O +, O +Regnase B-protein +- I-protein +1 I-protein +functions O +as O +a O +dimer B-oligomeric_state +through O +intermolecular O +PIN B-structure_element +- O +PIN B-structure_element +interactions O +during O +cleavage O +of O +target O +mRNA B-chemical +. O + +Our O +findings O +suggest O +that O +Regnase B-protein +- I-protein +1 I-protein +cleaves O +its O +target O +mRNA B-chemical +by O +an O +NTD B-protein_state +- I-protein_state +activated I-protein_state +functional B-protein_state +PIN B-structure_element +dimer B-oligomeric_state +, O +while O +the O +ZF B-structure_element +increases O +RNA B-chemical +affinity O +in O +the O +vicinity O +of O +the O +PIN B-structure_element +dimer B-oligomeric_state +. O + +Domain O +structures B-evidence +of O +Regnase B-protein +- I-protein +1 I-protein + +We O +analyzed O +Rengase B-protein +- I-protein +1 I-protein +derived O +from O +Mus B-species +musculus I-species +and O +solved B-experimental_method +the O +structures B-evidence +of O +the O +four O +domains O +; O +NTD B-structure_element +, O +PIN B-structure_element +, O +ZF B-structure_element +, O +and O +CTD B-structure_element +individually O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +or O +NMR B-experimental_method +( O +Fig O +. O +1a O +– O +e O +). O + +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +was O +attempted O +for O +the O +fragment O +containing O +both O +the O +PIN B-structure_element +and O +ZF B-structure_element +domains O +, O +however O +, O +electron B-evidence +density I-evidence +was O +observed O +only O +for O +the O +PIN B-structure_element +domain O +( O +Fig O +. O +1c O +), O +consistent O +with O +a O +previous O +report O +on O +Regnase B-protein +- I-protein +1 I-protein +derived O +from O +Homo B-species +sapiens I-species +. O + +This O +suggests O +that O +the O +PIN B-structure_element +and O +ZF B-structure_element +domains O +exist O +independently O +without O +interacting O +with O +each O +other O +. O + +The O +domain O +structures B-evidence +of O +NTD B-structure_element +, O +ZF B-structure_element +, O +and O +CTD B-structure_element +were O +determined O +by O +NMR B-experimental_method +( O +Fig O +. O +1b O +, O +d O +, O +e O +). O + +The O +NTD B-structure_element +and O +CTD B-structure_element +are O +both O +composed O +of O +three O +α B-structure_element +helices I-structure_element +, O +and O +structurally O +resemble O +ubiquitin B-protein +conjugating I-protein +enzyme I-protein +E2 I-protein +K I-protein +( O +PDB O +ID O +: O +3K9O O +) O +and O +ubiquitin B-protein +associated I-protein +protein I-protein +1 I-protein +( O +PDB O +ID O +: O +4AE4 O +), O +respectively O +, O +according O +to O +the O +Dali B-experimental_method +server I-experimental_method +. O + +Contribution O +of O +each O +domain O +of O +Regnase B-protein +- I-protein +1 I-protein +to O +the O +mRNA B-chemical +binding O +activity O + +Although O +the O +PIN B-structure_element +domain O +is O +responsible O +for O +the O +catalytic O +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +, O +the O +roles O +of O +the O +other O +domains O +are O +largely O +unknown O +. O + +First O +, O +we O +evaluated O +a O +role O +of O +the O +NTD B-structure_element +and O +ZF B-structure_element +domains O +for O +mRNA B-chemical +binding O +by O +an O +in B-experimental_method +vitro I-experimental_method +gel I-experimental_method +shift I-experimental_method +assay I-experimental_method +( O +Fig O +. O +1f O +). O + +Fluorescently B-protein_state +5 I-protein_state +′- I-protein_state +labeled I-protein_state +RNA B-chemical +corresponding O +to O +nucleotides O +82 O +– O +106 O +of O +the O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +3 B-structure_element +′ I-structure_element +UTR I-structure_element +and O +the O +catalytically O +inactive B-protein_state +mutant B-protein_state +( O +D226N B-mutant +and O +D244N B-mutant +) O +of O +Regnase B-protein +- I-protein +1 I-protein +— O +hereafter O +referred O +to O +as O +the O +DDNN B-mutant +mutant B-protein_state +— O +were O +utilized O +. O + +Upon O +addition O +of O +a O +larger O +amount O +of O +Regnase B-protein +- I-protein +1 I-protein +, O +the O +fluorescence B-evidence +of O +free B-protein_state +RNA B-chemical +decreased O +, O +indicating O +that O +Regnase B-protein +- I-protein +1 I-protein +bound B-protein_state +to I-protein_state +the O +RNA B-chemical +. O + +Based O +on O +the O +decrease O +in O +the O +free O +RNA B-chemical +fluorescence O +band O +, O +we O +evaluated O +the O +contribution O +of O +each O +domain O +of O +Regnase B-protein +- I-protein +1 I-protein +to O +RNA B-chemical +binding O +. O + +While O +the O +RNA B-chemical +binding O +ability O +was O +not O +significantly O +changed O +in O +the O +presence B-protein_state +of I-protein_state +NTD B-structure_element +, O +it O +increased O +in O +the O +presence B-protein_state +of I-protein_state +the O +ZF B-structure_element +domain O +( O +Fig O +. O +1f O +, O +g O +and O +Supplementary O +Fig O +. O +1 O +). O + +Direct O +binding O +of O +the O +ZF B-structure_element +domain O +and O +RNA B-chemical +were O +confirmed O +by O +NMR B-experimental_method +spectral B-evidence +changes I-evidence +. O + +The O +fitting O +of O +the O +titration B-evidence +curve I-evidence +of O +Y314 B-residue_name_number +resulted O +in O +an O +apparent O +dissociation B-evidence +constant I-evidence +( O +Kd B-evidence +) O +of O +10 O +± O +1 O +. O +1 O +μM O +( O +Supplementary O +Fig O +. O +2 O +). O + +These O +results O +indicate O +that O +not O +only O +the O +PIN B-structure_element +but O +also O +the O +ZF B-structure_element +domain O +contribute O +to O +RNA B-chemical +binding O +, O +while O +the O +NTD B-structure_element +is O +not O +likely O +to O +be O +involved O +in O +direct O +interaction O +with O +RNA B-chemical +. O + +Contribution O +of O +each O +domain O +of O +Regnase B-protein +- I-protein +1 I-protein +to O +RNase B-protein_type +activity O + +In O +order O +to O +characterize O +the O +role O +of O +each O +domain O +in O +the O +RNase B-protein_type +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +, O +we O +performed O +an O +in B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assay I-experimental_method +using O +fluorescently B-protein_state +5 I-protein_state +′- I-protein_state +labeled I-protein_state +RNA B-chemical +corresponding O +to O +nucleotides O +82 O +– O +106 O +of O +the O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +3 B-structure_element +′ I-structure_element +UTR I-structure_element +( O +Fig O +. O +1g O +). O + +Regnase B-protein +- I-protein +1 I-protein +constructs O +consisting O +of O +NTD B-mutant +- I-mutant +PIN I-mutant +- I-mutant +ZF I-mutant +completely O +cleaved O +the O +target O +mRNA B-chemical +and O +generated O +the O +cleaved O +products O +. O + +The O +apparent O +half O +- O +life O +( O +T1 O +/ O +2 O +) O +of O +the O +RNase B-protein_type +activity O +was O +about O +20 O +minutes O +. O + +Regnase B-protein +- I-protein +1 I-protein +lacking B-protein_state +the O +ZF B-structure_element +domain O +generated O +a O +smaller O +but O +appreciable O +amount O +of O +cleaved O +product O +( O +T1 O +/ O +2 O +~ O +70 O +minutes O +), O +while O +those O +lacking B-protein_state +the O +NTD B-structure_element +did O +not O +generate O +cleaved O +products O +( O +T1 O +/ O +2 O +> O +90 O +minutes O +). O + +It O +should O +be O +noted O +that O +NTD B-mutant +- I-mutant +PIN I-mutant +( I-mutant +DDNN I-mutant +)- I-mutant +ZF I-mutant +, O +which O +possesses O +the O +NTD B-structure_element +but O +lacks B-protein_state +the O +catalytic B-site +residues I-site +in O +PIN B-structure_element +, O +completely O +lost O +all O +RNase B-protein_type +activity O +( O +Fig O +. O +1g O +, O +right O +panel O +), O +as O +expected O +, O +confirming O +that O +the O +RNase B-protein_type +catalytic B-site +center I-site +is O +located O +in O +the O +PIN B-structure_element +domain O +. O + +Taken O +together O +with O +the O +results O +in O +the O +previous O +section O +, O +we O +conclude O +that O +the O +NTD B-structure_element +is O +crucial O +for O +the O +RNase B-protein_type +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +in O +vitro O +, O +although O +it O +does O +not O +contribute O +to O +the O +direct O +mRNA B-chemical +binding O +. O + +Dimer B-oligomeric_state +formation O +of O +the O +PIN B-structure_element +domains O + +During O +purification B-experimental_method +by O +gel B-experimental_method +filtration I-experimental_method +, O +the O +PIN B-structure_element +domain O +exhibited O +extremely O +asymmetric O +elution O +peaks O +in O +a O +concentration O +dependent O +manner O +( O +Fig O +. O +2a O +). O + +By O +comparison B-experimental_method +with I-experimental_method +the I-experimental_method +elution I-experimental_method +volume I-experimental_method +of I-experimental_method +standard I-experimental_method +marker I-experimental_method +proteins I-experimental_method +, O +the O +PIN B-structure_element +domain O +was O +assumed O +to O +be O +in O +equilibrium O +between O +a O +monomer B-oligomeric_state +and O +a O +dimer B-oligomeric_state +in O +solution O +at O +concentrations O +in O +the O +20 O +– O +200 O +μM O +range O +. O + +The O +crystal B-evidence +structure I-evidence +of O +the O +PIN B-structure_element +domain O +has O +been O +determined O +in O +three O +distinct O +crystal B-evidence +forms I-evidence +with O +a O +space O +group O +of O +P3121 O +( O +form O +I O +in O +this O +study O +and O +PDB O +ID O +3V33 O +), O +P3221 O +( O +form O +II O +in O +this O +study O +), O +and O +P41 O +( O +PDB O +ID O +3V32 O +and O +3V34 O +), O +respectively O +. O + +We O +found O +that O +the O +PIN B-structure_element +domain O +formed O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomer B-oligomeric_state +that O +was O +commonly O +observed O +in O +all O +three O +crystal B-evidence +forms I-evidence +in O +spite O +of O +the O +different O +crystallization O +conditions O +( O +Supplementary O +Fig O +. O +3 O +). O + +Mutation B-experimental_method +of O +Arg215 B-residue_name_number +, O +whose O +side O +chain O +faces O +to O +the O +opposite O +side O +of O +the O +oligomeric B-site +surface I-site +, O +to O +Glu B-residue_name +preserved O +the O +monomer B-oligomeric_state +/ O +dimer B-oligomeric_state +equilibrium O +, O +similar O +to O +the O +wild B-protein_state +type I-protein_state +. O + +On O +the O +other O +hand O +, O +single B-experimental_method +mutations I-experimental_method +of O +side O +chains O +involved O +in O +the O +PIN B-structure_element +– O +PIN B-structure_element +oligomeric O +interaction O +resulted O +in O +monomer B-oligomeric_state +formation O +, O +judging O +from O +gel B-experimental_method +filtration I-experimental_method +( O +Fig O +. O +2a O +, O +b O +). O + +Wild B-protein_state +type I-protein_state +and O +monomeric B-oligomeric_state +PIN B-structure_element +mutants B-protein_state +( O +P212A B-mutant +and O +D278R B-mutant +) O +were O +also O +analyzed O +by O +NMR B-experimental_method +. O + +The O +spectra B-evidence +indicate O +that O +the O +dimer B-site +interface I-site +of O +the O +wild B-protein_state +type I-protein_state +PIN B-structure_element +domain O +were O +significantly O +broadened O +compared O +to O +the O +monomeric B-oligomeric_state +mutants B-protein_state +( O +Supplementary O +Fig O +. O +4 O +). O + +These O +results O +indicate O +that O +the O +PIN B-structure_element +domain O +forms O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomer B-oligomeric_state +in O +solution O +similar O +to O +the O +crystal B-evidence +structure I-evidence +. O + +Interestingly O +, O +the O +monomeric B-oligomeric_state +PIN B-structure_element +mutants B-protein_state +P212A B-mutant +, O +R214A B-mutant +, O +and O +D278R B-mutant +had O +no O +significant O +RNase B-protein_type +activity O +for O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +in O +vitro O +( O +Fig O +. O +2c O +). O + +The O +side O +chains O +of O +these O +residues O +point O +away O +from O +the O +catalytic B-site +center I-site +on O +the O +same O +molecule O +( O +Fig O +. O +2b O +). O + +Therefore O +, O +we O +concluded O +that O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +PIN B-structure_element +dimerization O +, O +together O +with O +the O +NTD B-structure_element +, O +are O +required O +for O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O +in O +vitro O +. O + +Domain O +- O +domain O +interaction O +between O +the O +NTD B-structure_element +and O +the O +PIN B-structure_element +domain O + +While O +the O +NTD B-structure_element +does O +not O +contribute O +to O +RNA B-chemical +binding O +( O +Fig O +. O +1f O +, O +g O +, O +and O +Supplementary O +Fig O +. O +1 O +), O +it O +increases O +the O +RNase B-protein_type +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +( O +Fig O +. O +1h O +). O + +In O +order O +to O +gain O +insight O +into O +the O +molecular O +mechanism O +of O +the O +NTD B-structure_element +- O +mediated O +enhancement O +of O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O +, O +we O +further O +investigated O +the O +domain O +- O +domain O +interaction O +between O +the O +NTD B-structure_element +and O +the O +PIN B-structure_element +domain O +using O +NMR B-experimental_method +. O + +We O +used O +the O +catalytically B-protein_state +inactive I-protein_state +monomeric B-oligomeric_state +PIN B-structure_element +mutant B-protein_state +possessing O +both O +the O +DDNN B-mutant +and O +D278R B-mutant +mutations O +to O +avoid O +dimer B-oligomeric_state +formation O +of O +the O +PIN B-structure_element +domain O +. O + +The O +NMR B-experimental_method +signals O +from O +the O +PIN B-structure_element +domain O +( O +residues O +V177 B-residue_name_number +, O +F210 B-residue_range +- I-residue_range +T211 I-residue_range +, O +R214 B-residue_name_number +, O +F228 B-residue_range +- I-residue_range +L232 I-residue_range +, O +and O +F234 B-residue_range +- I-residue_range +S236 I-residue_range +) O +exhibited O +significant O +chemical O +shift O +changes O +upon O +addition B-experimental_method +of I-experimental_method +the O +NTD B-structure_element +( O +Fig O +. O +3a O +). O + +Likewise O +, O +upon O +addition B-experimental_method +of I-experimental_method +the O +PIN B-structure_element +domain O +, O +NMR B-experimental_method +signals O +derived O +from O +R56 B-residue_name_number +, O +L58 B-residue_range +- I-residue_range +G59 I-residue_range +, O +and O +V86 B-residue_range +- I-residue_range +H88 I-residue_range +in O +the O +NTD B-structure_element +exhibited O +large O +chemical O +shift O +changes O +and O +residues O +D53 B-residue_name_number +, O +F55 B-residue_name_number +, O +K57 B-residue_name_number +, O +Y60 B-residue_range +- I-residue_range +S61 I-residue_range +, O +V68 B-residue_name_number +, O +T80 B-residue_range +- I-residue_range +G83 I-residue_range +, O +L85 B-residue_name_number +, O +and O +G89 B-residue_name_number +of O +the O +NTD B-structure_element +as O +well O +as O +side O +chain O +amide O +signals O +of O +N79 B-residue_name_number +exhibited O +small O +but O +appreciable O +chemical O +shift O +changes O +( O +Fig O +. O +3b O +and O +Supplementary O +Fig O +. O +5 O +). O + +These O +results O +clearly O +indicate O +a O +direct O +interaction O +between O +the O +PIN B-structure_element +domain O +and O +the O +NTD B-structure_element +. O + +Based O +on O +the O +titration B-evidence +curve I-evidence +for O +the O +chemical B-evidence +shift I-evidence +changes I-evidence +of O +L58 B-residue_name_number +, O +the O +apparent O +Kd B-evidence +between O +the O +isolated O +NTD B-structure_element +and O +PIN B-structure_element +was O +estimated O +to O +be O +110 O +± O +5 O +. O +8 O +μM O +. O +Considering O +the O +fact O +that O +the O +NTD B-structure_element +and O +PIN B-structure_element +domains O +are O +attached O +by O +a O +linker B-structure_element +, O +the O +actual O +binding B-evidence +affinity I-evidence +is O +expected O +much O +higher O +in O +the O +native B-protein_state +protein O +. O + +Mapping O +the O +residues O +with O +chemical O +shift O +changes O +reveals O +the O +putative O +PIN B-site +/ I-site +NTD I-site +interface I-site +, O +which O +includes O +a O +helix B-structure_element +that O +harbors O +catalytic O +residues O +D225 B-residue_name_number +and O +D226 B-residue_name_number +on O +the O +PIN B-structure_element +domain O +( O +Fig O +. O +3a O +). O + +Interestingly O +, O +the O +putative O +binding B-site +site I-site +for O +the O +NTD B-structure_element +overlaps O +with O +the O +PIN B-site +- I-site +PIN I-site +dimer I-site +interface I-site +, O +implying O +that O +NTD B-structure_element +binding O +can O +“ O +terminate O +” O +PIN B-structure_element +- O +PIN B-structure_element +oligomerization O +( O +Fig O +. O +2b O +). O + +An O +in B-experimental_method +silico I-experimental_method +docking I-experimental_method +of O +the O +NTD B-structure_element +and O +PIN B-structure_element +domains O +using O +chemical B-evidence +shift I-evidence +restraints I-evidence +provided O +a O +model O +consistent O +with O +the O +NMR B-experimental_method +experiments O +( O +Fig O +. O +3c O +). O + +Residues O +critical O +for O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O + +To O +gain O +insight O +into O +the O +residues O +critical O +for O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O +, O +each O +basic O +or O +aromatic O +residue O +located O +around O +the O +catalytic B-site +site I-site +of O +the O +PIN B-structure_element +oligomer B-oligomeric_state +was O +mutated B-experimental_method +to I-experimental_method +alanine B-residue_name +, O +and O +the O +oligomerization O +and O +RNase B-protein_type +activity O +were O +investigated O +( O +Fig O +. O +4 O +). O + +From O +the O +gel B-experimental_method +filtration I-experimental_method +assays I-experimental_method +, O +all O +mutants B-protein_state +except O +R214A B-mutant +formed O +dimers B-oligomeric_state +, O +suggesting O +that O +any O +lack O +of O +RNase B-protein_type +activity O +in O +the O +mutants B-protein_state +, O +except O +R214A B-mutant +, O +was O +directly O +due O +to O +mutational O +effects O +of O +the O +specific O +residues O +and O +not O +to O +abrogation O +of O +dimer B-oligomeric_state +formation O +. O + +The O +W182A B-mutant +, O +R183A B-mutant +, O +and O +R214A B-mutant +mutants B-protein_state +markedly O +lost O +cleavage O +activity O +for O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +as O +well O +as O +for O +Regnase B-protein +- I-protein +1 I-protein +mRNA B-chemical +. O + +The O +K184A B-mutant +, O +R215A B-mutant +, O +and O +R220A B-mutant +mutants B-protein_state +moderately O +but O +significantly O +decreased O +the O +cleavage O +activity O +for O +both O +target O +mRNAs B-chemical +. O + +The O +importance O +of O +K219 B-residue_name_number +and O +R247 B-residue_name_number +was O +slightly O +different O +for O +IL B-protein_type +- I-protein_type +6 I-protein_type +and O +Regnase B-protein +- I-protein +1 I-protein +mRNA B-chemical +; O +both O +K219 B-residue_name_number +and O +R247 B-residue_name_number +were O +more O +important O +in O +the O +cleavage O +of O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +than O +for O +Regnase B-protein +- I-protein +1 I-protein +mRNA B-chemical +. O + +The O +other O +mutated O +residues O +— O +K152 B-residue_name_number +, O +R158 B-residue_name_number +, O +R188 B-residue_name_number +, O +R200 B-residue_name_number +, O +K204 B-residue_name_number +, O +K206 B-residue_name_number +, O +K257 B-residue_name_number +, O +and O +R258 B-residue_name_number +— O +were O +not O +critical O +for O +RNase B-protein_type +activity O +. O + +The O +importance O +of O +residues O +W182 B-residue_name_number +and O +R183 B-residue_name_number +can O +readily O +be O +understood O +in O +terms O +of O +the O +monomeric B-oligomeric_state +PIN B-structure_element +structure B-evidence +as O +they O +are O +located O +near O +to O +the O +RNase B-protein_type +catalytic B-site +site I-site +; O +however O +, O +the O +importance O +of O +residue O +K184 B-residue_name_number +, O +which O +points O +away O +from O +the O +active B-site +site I-site +is O +more O +easily O +rationalized O +in O +terms O +of O +the O +oligomeric O +structure B-evidence +, O +in O +which O +the O +“ O +secondary O +” O +chain O +’ O +s O +residue O +K184 B-residue_name_number +is O +positioned O +near O +the O +“ O +primary B-protein_state +” I-protein_state +chain O +’ O +s O +catalytic B-site +site I-site +( O +Fig O +. O +4 O +). O + +In O +contrast O +, O +R214 B-residue_name_number +is O +important O +for O +oligomerization O +of O +the O +PIN B-structure_element +domain O +and O +the O +“ O +secondary O +” O +chain O +’ O +s O +residue O +R214 B-residue_name_number +is O +also O +positioned O +near O +the O +“ O +primary B-protein_state +” O +chain O +’ O +s O +active B-site +site I-site +within O +the O +dimer B-site +interface I-site +. O + +It O +should O +be O +noted O +that O +the O +putative B-site +- I-site +RNA I-site +binding I-site +residues I-site +K184 B-residue_name_number +and O +R214 B-residue_name_number +are O +unique O +to O +Regnase B-protein +- I-protein +1 I-protein +among O +PIN B-structure_element +domains O +. O + +Molecular O +mechanism O +of O +target O +mRNA B-chemical +cleavage O +by O +the O +PIN B-structure_element +dimer B-oligomeric_state + +Our O +mutational B-experimental_method +experiments I-experimental_method +indicated O +that O +the O +observed O +dimer B-oligomeric_state +is O +functional O +and O +that O +the O +role O +of O +the O +secondary B-protein_state +PIN B-structure_element +domain O +is O +to O +position O +Regnase B-protein +- I-protein +1 I-protein +- O +unique O +RNA B-site +binding I-site +residues I-site +near O +the O +active B-site +site I-site +of O +the O +primary B-protein_state +PIN B-structure_element +domain O +. O + +If O +this O +model O +is O +correct O +, O +then O +we O +reasoned O +that O +a O +catalytically B-protein_state +inactive I-protein_state +PIN B-structure_element +and O +a O +PIN B-structure_element +lacking B-protein_state +the O +putative O +RNA B-site +- I-site +binding I-site +residues I-site +ought O +to O +be O +inactive B-protein_state +in O +isolation O +but O +become O +active B-protein_state +when O +mixed O +together O +. O + +In O +order O +to O +test O +this O +hypothesis O +, O +we O +performed O +in B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assays I-experimental_method +using O +combinations O +of O +Regnase B-protein +- I-protein +1 I-protein +mutants B-protein_state +that O +had O +no O +or O +decreased O +RNase B-protein_type +activities O +by O +themselves O +( O +Fig O +. O +5 O +). O + +One O +group O +consisted O +of O +catalytically B-protein_state +active I-protein_state +PIN B-structure_element +domains O +with O +mutation B-experimental_method +of I-experimental_method +basic O +residues O +found O +in O +the O +previous O +section O +to O +confer O +decreased O +RNase B-protein_type +activity O +( O +Fig O +. O +4 O +). O + +These O +were O +paired O +with O +a O +DDNN B-mutant +mutant B-protein_state +that O +had O +no O +RNase B-protein_type +activity O +by O +itself O +. O + +When O +any O +members O +of O +the O +two O +groups O +are O +mixed O +, O +two O +kinds O +of O +heterodimers B-oligomeric_state +can O +be O +formed O +: O +one O +is O +composed O +of O +a O +DDNN B-mutant +primary B-protein_state +PIN B-structure_element +and O +a O +basic O +residue O +mutant B-protein_state +secondary B-protein_state +PIN B-structure_element +and O +is O +expected O +to O +exhibit O +no O +RNase B-protein_type +activity O +; O +the O +other O +is O +composed O +of O +a O +basic O +residue O +mutant B-protein_state +primary B-protein_state +PIN B-structure_element +and O +a O +DDNN B-mutant +secondary B-protein_state +PIN B-structure_element +and O +is O +predicted O +to O +rescue O +RNase B-protein_type +activity O +( O +Fig O +. O +5a O +). O + +When O +we O +compared O +the O +fluorescence B-evidence +intensity I-evidence +of O +uncleaved B-protein_state +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +, O +basic O +residue O +mutants B-protein_state +W182A B-mutant +, O +K184A B-mutant +, O +R214A B-mutant +, O +and O +R220A B-mutant +were O +rescued O +upon O +addition O +of O +the O +DDNN B-mutant +mutant B-protein_state +( O +Fig O +. O +5b O +). O + +Consistently O +, O +when O +we O +compared O +the O +fluorescence B-evidence +intensity I-evidence +of O +the O +uncleaved B-protein_state +Regnase B-protein +- I-protein +1 I-protein +mRNA B-chemical +, O +basic O +residue O +mutants B-protein_state +K184A B-mutant +and O +R214A B-mutant +were O +rescued O +upon O +addition O +of O +the O +DDNN B-mutant +mutant B-protein_state +( O +Fig O +. O +5c O +). O + +Rescue O +of O +K184A B-mutant +and O +R214A B-mutant +by O +the O +DDNN B-mutant +mutant B-protein_state +was O +also O +confirmed O +by O +a O +significant O +increase O +in O +the O +cleaved O +products O +. O + +This O +is O +particularly O +significant O +because O +the O +side O +chains O +of O +K184 B-residue_name_number +and O +R214 B-residue_name_number +in O +the O +primary B-protein_state +PIN B-structure_element +are O +oriented O +away O +from O +their O +own O +catalytic B-site +center I-site +, O +while O +those O +in O +the O +secondary B-protein_state +PIN B-structure_element +face O +toward O +the O +catalytic B-site +center I-site +of O +the O +primary B-protein_state +PIN B-structure_element +. O + +R214 B-residue_name_number +is O +an O +important O +residue O +for O +dimer B-oligomeric_state +formation O +as O +shown O +in O +Fig O +. O +2 O +, O +therefore O +, O +R214A B-mutant +in O +the O +secondary B-protein_state +PIN B-structure_element +cannot O +dimerize O +. O + +According O +to O +the O +proposed O +model O +, O +an O +R214A B-mutant +PIN B-structure_element +domain O +can O +only O +form O +a O +dimer B-oligomeric_state +when O +the O +DDNN B-mutant +PIN B-structure_element +acts O +as O +the O +secondary B-protein_state +PIN B-structure_element +. O + +Taken O +together O +, O +the O +rescue O +experiments O +above O +support O +the O +proposed O +model O +in O +which O +the O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +dimer B-oligomeric_state +is O +functional O +in O +vitro O +. O + +We O +determined O +the O +individual O +domain O +structures B-evidence +of O +Regnase B-protein +- I-protein +1 I-protein +by O +NMR B-experimental_method +and O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +Although O +the O +function O +of O +the O +CTD B-structure_element +remains O +elusive O +, O +we O +revealed O +the O +functions O +of O +the O +NTD B-structure_element +, O +PIN B-structure_element +, O +and O +ZF B-structure_element +domains O +. O + +A O +Regnase B-protein +- I-protein +1 I-protein +construct O +consisting O +of O +PIN B-structure_element +and O +ZF B-structure_element +domains O +derived O +from O +Mus B-species +musculus I-species +was O +crystallized B-experimental_method +; O +however O +, O +the O +electron B-evidence +density I-evidence +of O +the O +ZF B-structure_element +domain O +was O +low O +, O +indicating O +that O +the O +ZF B-structure_element +domain O +is O +highly B-protein_state +mobile I-protein_state +in O +the O +absence B-protein_state +of I-protein_state +target O +mRNA B-chemical +or O +possibly O +other O +protein O +- O +protein O +interactions O +. O + +Our O +NMR B-experimental_method +experiments O +confirmed O +direct O +binding O +of O +the O +ZF B-structure_element +domain O +to O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +with O +a O +Kd B-evidence +of O +10 O +± O +1 O +. O +1 O +μM O +. O +Furthermore O +, O +an O +in B-experimental_method +vitro I-experimental_method +gel I-experimental_method +shift I-experimental_method +assay I-experimental_method +indicated O +that O +Regnase B-protein +- I-protein +1 I-protein +containing O +the O +ZF B-structure_element +domain O +enhanced O +target O +mRNA B-chemical +- O +binding O +, O +but O +the O +protein O +- O +RNA B-chemical +complex O +remained O +in O +the O +bottom O +of O +the O +well O +without O +entering O +into O +the O +polyacrylamide O +gel O +. O + +These O +results O +indicate O +that O +Regnase B-protein +- I-protein +1 I-protein +directly O +binds O +to O +RNA B-chemical +and O +precipitates O +under O +such O +experimental O +conditions O +. O + +Due O +to O +this O +limitation O +, O +it O +is O +difficult O +to O +perform O +further O +structural B-experimental_method +analyses I-experimental_method +of O +mRNA B-complex_assembly +- I-complex_assembly +Regnase I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +complexes O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +or O +NMR B-experimental_method +. O + +The O +previously O +reported O +crystal B-evidence +structure I-evidence +of O +the O +Regnase B-protein +- I-protein +1 I-protein +PIN B-structure_element +domain O +derived O +from O +Homo B-species +sapiens I-species +is O +nearly O +identical O +to O +the O +one O +derived O +from O +Mus B-species +musculus I-species +in O +this O +study O +, O +with O +a O +backbone O +RMSD B-evidence +of O +0 O +. O +2 O +Å O +. O +The O +amino O +acid O +sequences O +corresponding O +to O +PIN B-structure_element +( O +residues O +134 B-residue_range +– I-residue_range +295 I-residue_range +) O +are O +the O +two O +non O +- O +identical O +residues O +are O +substituted O +with O +similar O +amino O +acids O +. O + +Both O +the O +mouse B-taxonomy_domain +and O +human B-species +PIN B-structure_element +domains O +form O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomers B-oligomeric_state +in O +three O +distinct O +crystal B-evidence +forms I-evidence +. O + +Rao O +and O +co O +- O +workers O +previously O +argued O +that O +PIN B-structure_element +dimerization O +is O +likely O +to O +be O +a O +crystallographic O +artifact O +with O +no O +physiological O +significance O +, O +since O +monomers B-oligomeric_state +were O +dominant O +in O +their O +analytical B-experimental_method +ultra I-experimental_method +- I-experimental_method +centrifugation I-experimental_method +experiments O +. O + +In O +contrast O +, O +our O +gel B-experimental_method +filtration I-experimental_method +data O +, O +mutational B-experimental_method +analyses I-experimental_method +, O +and O +NMR B-experimental_method +spectra B-evidence +all O +indicate O +that O +the O +PIN B-structure_element +domain O +forms O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +dimer B-oligomeric_state +in O +solution O +in O +a O +manner O +similar O +to O +the O +crystal B-evidence +structure I-evidence +. O + +This O +inconsistency O +might O +be O +due O +to O +difference O +in O +the O +analytical O +methods O +and O +/ O +or O +protein O +concentrations O +used O +in O +each O +experiment O +, O +since O +the O +oligomer B-oligomeric_state +formation O +of O +PIN B-structure_element +was O +dependent O +on O +the O +protein O +concentration O +in O +our O +study O +. O + +Single B-experimental_method +mutations I-experimental_method +to O +residues O +involved O +in O +the O +putative O +oligomeric O +interaction O +of O +PIN B-structure_element +monomerized B-oligomeric_state +as O +expected O +and O +these O +mutants B-protein_state +lost O +their O +RNase B-protein_type +activity O +as O +well O +. O + +Since O +the O +NMR B-experimental_method +spectra B-evidence +of O +monomeric B-oligomeric_state +mutants B-protein_state +overlaps O +with O +those O +of O +the O +oligomeric O +forms O +, O +it O +is O +unlikely O +that O +the O +tertiary O +structure O +of O +the O +monomeric B-oligomeric_state +mutants B-protein_state +were O +affected O +by O +the O +mutations O +. O +( O +Supplementary O +Fig O +. O +4b O +, O +c O +). O + +Based O +on O +these O +observations O +, O +we O +concluded O +that O +PIN B-structure_element +- O +PIN B-structure_element +dimer B-oligomeric_state +formation O +is O +critical O +for O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O +in O +vitro O +. O + +Within O +the O +crystal B-evidence +structure I-evidence +of O +the O +PIN B-structure_element +dimer B-oligomeric_state +, O +the O +Regnase B-protein +- I-protein +1 I-protein +specific O +basic O +regions O +in O +both O +the O +“ O +primary B-protein_state +” O +and O +“ O +secondary B-protein_state +” O +PINs B-structure_element +are O +located O +around O +the O +catalytic B-site +site I-site +of O +the O +primary O +PIN B-structure_element +( O +Supplementary O +Fig O +. O +6 O +). O + +Moreover O +, O +our O +structure B-experimental_method +- I-experimental_method +based I-experimental_method +mutational I-experimental_method +analyses I-experimental_method +showed O +these O +two O +Regnase B-protein +- I-protein +1 I-protein +specific O +basic O +regions O +were O +essential O +for O +target O +mRNA B-chemical +cleavage O +in O +vitro O +. O + +The O +cleavage B-experimental_method +assay I-experimental_method +also O +showed O +that O +the O +NTD B-structure_element +is O +crucial O +for O +efficient O +mRNA B-chemical +cleavage O +. O + +Moreover O +, O +we O +found O +that O +the O +NTD B-structure_element +associates O +with O +the O +oligomeric B-site +surface I-site +of O +the O +primary B-protein_state +PIN B-structure_element +, O +docking O +to O +a O +helix B-structure_element +that O +harbors O +its O +catalytic B-site +residues I-site +( O +Figs O +2b O +and O +3a O +). O + +Taken O +together O +, O +this O +suggests O +that O +the O +NTD B-structure_element +and O +the O +PIN B-structure_element +domain O +compete O +for O +a O +common B-site +binding I-site +site I-site +. O + +The O +affinity B-evidence +of O +the O +domain O +- O +domain O +interaction O +between O +two O +PIN B-structure_element +domains O +( O +Kd B-evidence += O +~ O +10 O +− O +4 O +M O +) O +is O +similar O +to O +that O +of O +the O +NTD B-structure_element +- O +PIN B-structure_element +( O +Kd B-evidence += O +110 O +± O +5 O +. O +8 O +μM O +) O +interactions O +; O +however O +, O +the O +covalent O +connection O +corresponding O +to O +residues O +90 B-residue_range +– I-residue_range +133 I-residue_range +between O +the O +NTD B-structure_element +and O +the O +primary B-protein_state +PIN B-structure_element +will O +greatly O +enhance O +the O +intramolecular O +domain O +interaction O +in O +the O +case O +of O +full B-protein_state +- I-protein_state +length I-protein_state +Regnase B-protein +- I-protein +1 I-protein +. O + +While O +further O +analyses O +are O +necessary O +to O +prove O +this O +point O +, O +our O +preliminary O +docking B-experimental_method +and I-experimental_method +molecular I-experimental_method +dynamics I-experimental_method +simulations I-experimental_method +indicate O +that O +NTD B-structure_element +- O +binding O +rearranges O +the O +catalytic B-site +residues I-site +of O +the O +PIN B-structure_element +domain O +toward O +an O +active B-protein_state +conformation O +suitable O +for O +binding O +Mg2 B-chemical ++. I-chemical + +In O +this O +context O +, O +it O +is O +interesting O +that O +, O +in O +response O +to O +TCR O +stimulation O +, O +Malt1 B-protein +cleaves O +Regnase B-protein +- I-protein +1 I-protein +at O +R111 B-residue_name_number +to O +control O +immune O +responses O +in O +vivo O +. O + +This O +result O +is O +consistent O +with O +a O +model O +in O +which O +the O +NTD B-structure_element +acts O +as O +an O +enhancer O +, O +and O +cleavage O +of O +the O +linker B-structure_element +lowers O +enzymatic O +activity O +dramatically O +. O + +Based O +on O +these O +structural B-experimental_method +and I-experimental_method +functional I-experimental_method +analyses I-experimental_method +of O +Regnase B-protein +- I-protein +1 I-protein +domain O +- O +domain O +interactions O +, O +we O +performed O +docking B-experimental_method +simulations I-experimental_method +of O +the O +NTD B-structure_element +, O +PIN B-structure_element +dimer B-oligomeric_state +, O +and O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +. O + +We O +incorporated O +information O +from O +the O +cleavage B-site +site I-site +of O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +in O +vitro O +is O +indicated O +by O +denaturing O +polyacrylamide B-experimental_method +gel I-experimental_method +electrophoresis I-experimental_method +( O +Supplementary O +Fig O +. O +7a O +, O +b O +). O + +The O +docking B-experimental_method +result O +revealed O +multiple O +RNA B-chemical +binding O +modes O +that O +satisfied O +the O +experimental O +results O +in O +vitro O +( O +Supplementary O +Fig O +. O +7c O +, O +d O +), O +however O +, O +it O +should O +be O +noted O +that O +, O +in O +vivo O +, O +there O +would O +likely O +be O +many O +other O +RNA B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +that O +would O +protect O +loop B-structure_element +regions O +from O +cleavage O +by O +Regnase B-protein +- I-protein +1 I-protein +. O + +The O +overall O +model O +of O +regulation O +of O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O +through O +domain O +- O +domain O +interactions O +in O +vitro O +is O +summarized O +in O +Fig O +. O +6 O +. O + +In O +the O +absence B-protein_state +of I-protein_state +target O +mRNA B-chemical +, O +the O +PIN B-structure_element +domain O +forms O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomers B-oligomeric_state +at O +high O +concentration O +. O + +A O +fully B-protein_state +active I-protein_state +catalytic B-site +center I-site +can O +be O +formed O +only O +when O +the O +NTD B-structure_element +associates O +with O +the O +oligomer B-oligomeric_state +surface O +of O +the O +PIN B-structure_element +domain O +, O +which O +terminates O +the O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomer B-oligomeric_state +formation O +in O +one O +direction O +( O +primary B-protein_state +PIN B-structure_element +), O +and O +forms O +a O +functional B-protein_state +dimer B-oligomeric_state +together O +with O +the O +neighboring O +PIN B-structure_element +( O +secondary B-protein_state +PIN B-structure_element +). O + +While O +further O +investigations O +on O +the O +domain O +- O +domain O +interactions O +of O +Regnase B-protein +- I-protein +1 I-protein +in O +vivo O +are O +necessary O +, O +these O +intramolecular O +and O +intermolecular O +domain O +interactions O +of O +Regnase B-protein +- I-protein +1 I-protein +appear O +to O +structurally O +constrain O +Regnase B-protein +- I-protein +1activity I-protein +, O +which O +, O +in O +turn O +, O +enables O +tight O +regulation O +of O +immune O +responses O +. O + +Structural B-experimental_method +and I-experimental_method +functional I-experimental_method +analyses I-experimental_method +of O +Regnase B-protein +- I-protein +1 I-protein +. O + +( O +a O +) O +Domain O +architecture O +of O +Regnase B-protein +- I-protein +1 I-protein +. O +( O +b O +) O +Solution B-evidence +structure I-evidence +of O +the O +NTD B-structure_element +. O +( O +c O +) O +Crystal B-evidence +structure I-evidence +of O +the O +PIN B-structure_element +domain O +. O + +Catalytic B-protein_state +Asp B-residue_name +residues O +were O +shown O +in O +sticks O +. O + +( O +d O +) O +Solution B-evidence +structure I-evidence +of O +the O +ZF B-structure_element +domain O +. O + +Three O +Cys B-residue_name +residues O +and O +one O +His B-residue_name +residue O +responsible O +for O +Zn2 O ++- O +binding O +were O +shown O +in O +sticks O +. O + +( O +e O +) O +Solution B-evidence +structure I-evidence +of O +the O +CTD B-structure_element +. O + +All O +the O +structures B-evidence +were O +colored O +in O +rainbow O +from O +N O +- O +terminus O +( O +blue O +) O +to O +C O +- O +terminus O +( O +red O +). O + +( O +f O +) O +In B-experimental_method +vitro I-experimental_method +gel I-experimental_method +shift I-experimental_method +binding I-experimental_method +assay I-experimental_method +between O +Regnase B-protein +- I-protein +1 I-protein +and O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +. O + +Fluorescence B-evidence +intensity I-evidence +of O +the O +free B-protein_state +IL B-protein_type +- I-protein_type +6 I-protein_type +in O +each O +sample O +was O +indicated O +as O +the O +percentage O +against O +that O +in O +the O +absence B-protein_state +of I-protein_state +Regnase B-protein +- I-protein +1 I-protein +. O + +( O +g O +) O +Binding O +of O +Regnase B-protein +- I-protein +1 I-protein +and O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +was O +plotted O +. O + +The O +percentage O +of O +the O +bound O +IL B-protein_type +- I-protein_type +6 I-protein_type +was O +calculated O +based O +on O +the O +fluorescence B-evidence +intensities I-evidence +of O +the O +free O +IL B-protein_type +- I-protein_type +6 I-protein_type +quantified O +in O +( O +f O +). O + +( O +h O +) O +In B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assay I-experimental_method +of O +Regnase B-protein +- I-protein +1 I-protein +to O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +. O + +Fluorescence B-evidence +intensity I-evidence +of O +the O +uncleaved B-protein_state +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +was O +indicated O +as O +the O +percentage O +against O +that O +in O +the O +absence B-protein_state +of I-protein_state +Regnase B-protein +- I-protein +1 I-protein +. O + +Head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomer B-oligomeric_state +formation O +of O +the O +PIN B-structure_element +domain O +is O +crucial O +for O +the O +RNase B-protein_type +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +. O + +( O +a O +) O +Gel B-experimental_method +filtration I-experimental_method +analyses I-experimental_method +of O +the O +PIN B-structure_element +domain O +. O + +( O +b O +) O +Dimer B-oligomeric_state +structure B-evidence +of O +the O +PIN B-structure_element +domain O +. O + +Two O +PIN B-structure_element +molecules O +in O +the O +crystal B-evidence +were O +colored O +white O +and O +green O +, O +respectively O +. O + +Catalytic B-site +residues I-site +and O +mutated O +residues O +were O +shown O +in O +sticks O +. O + +Residues O +important O +for O +the O +oligomeric O +interaction O +were O +colored O +red O +, O +while O +R215 B-residue_name_number +that O +was O +dispensable O +for O +the O +oligomeric O +interaction O +was O +colored O +blue O +. O +( O +c O +) O +RNase B-protein_type +activity O +of O +monomeric B-oligomeric_state +mutants B-protein_state +for O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +was O +analyzed O +. O + +Domain O +- O +domain O +interaction O +between O +the O +NTD B-structure_element +and O +the O +PIN B-structure_element +domain O +. O + +( O +a O +) O +NMR B-experimental_method +analyses I-experimental_method +of O +the O +NTD B-structure_element +- O +binding O +to O +the O +PIN B-structure_element +domain O +. O + +The O +residues O +with O +significant O +chemical O +shift O +changes O +were O +labeled O +in O +the O +overlaid B-experimental_method +spectra B-evidence +( O +left O +) O +and O +colored O +red O +on O +the O +surface O +and O +ribbon O +structure O +of O +the O +PIN B-structure_element +domain O +( O +right O +). O + +Pro B-residue_name +and O +the O +residues O +without O +analysis O +were O +colored O +black O +and O +gray O +, O +respectively O +. O + +( O +b O +) O +NMR B-experimental_method +analyses I-experimental_method +of O +the O +PIN B-structure_element +- O +binding O +to O +the O +NTD B-structure_element +. O + +The O +residues O +with O +significant B-evidence +chemical I-evidence +shift I-evidence +changes I-evidence +were O +labeled O +in O +the O +overlaid B-experimental_method +spectra B-evidence +( O +left O +) O +and O +colored O +red O +, O +yellow O +, O +or O +green O +on O +the O +surface O +and O +ribbon O +structure O +of O +the O +NTD B-structure_element +. O + +S62 B-residue_name_number +was O +colored O +gray O +and O +excluded O +from O +the O +analysis O +, O +due O +to O +low O +signal O +intensity O +. O + +( O +c O +) O +Docking O +model O +of O +the O +NTD B-structure_element +and O +the O +PIN B-structure_element +domain O +. O + +The O +NTD B-structure_element +and O +the O +PIN B-structure_element +domain O +are O +shown O +in O +cyan O +and O +white O +, O +respectively O +. O + +Residues O +in O +close O +proximity O +(< O +5 O +Å O +) O +to O +each O +other O +in O +the O +docking B-evidence +structure I-evidence +were O +colored O +yellow O +. O + +Catalytic B-site +residues I-site +of O +the O +PIN B-structure_element +domain O +are O +shown O +in O +sticks O +, O +and O +the O +residues O +that O +exhibited O +significant B-evidence +chemical I-evidence +shift I-evidence +changes I-evidence +in O +( O +a O +, O +b O +) O +were O +labeled O +. O + +Critical O +residues O +in O +the O +PIN B-structure_element +domain O +for O +the O +RNase B-protein_type +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +. O + +( O +a O +) O +In B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assay I-experimental_method +of O +basic O +residue O +mutants B-protein_state +for O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +. O + +( O +b O +) O +In B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assay I-experimental_method +of O +basic O +residue O +mutants B-protein_state +for O +Regnase B-protein +- I-protein +1 I-protein +mRNA B-chemical +. O + +The O +fluorescence B-evidence +intensity I-evidence +of O +the O +uncleaved B-protein_state +mRNA B-chemical +was O +quantified O +and O +the O +results O +were O +mapped O +on O +the O +PIN B-structure_element +dimer B-oligomeric_state +structure B-evidence +. O + +Mutated O +basic O +residues O +were O +shown O +in O +sticks O +and O +those O +with O +significantly O +reduced O +RNase B-protein_type +activities O +were O +colored O +red O +or O +yellow O +. O + +Heterodimer O +formation O +by O +combination O +of O +the O +Regnase B-protein +- I-protein +1 I-protein +basic O +residue O +mutants B-protein_state +and O +the O +DDNN B-mutant +mutant B-protein_state +restored O +the O +RNase B-protein_type +activity O +. O + +( O +a O +) O +Cartoon O +representation O +of O +the O +concept O +of O +the O +experiment O +. O +( O +b O +) O +In B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assay I-experimental_method +of O +Regnase B-protein +- I-protein +1 I-protein +for O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +. O + +( O +c O +) O +In B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assay I-experimental_method +of O +Regnase B-protein +- I-protein +1 I-protein +for O +Regnase B-protein +- I-protein +1 I-protein +mRNA B-chemical +. O + +The O +fluorescence B-evidence +intensity I-evidence +of O +the O +uncleaved B-protein_state +mRNA B-chemical +was O +quantified O +and O +the O +results O +were O +mapped O +on O +the O +PIN B-structure_element +dimer B-oligomeric_state +. O + +The O +mutations O +whose O +RNase B-protein_type +activities O +were O +not O +increased O +in O +the O +presence B-protein_state +of I-protein_state +DDNN B-mutant +mutant B-protein_state +were O +colored O +in O +blue O +on O +the O +primary O +PIN B-structure_element +. O + +The O +mutations O +whose O +RNase B-protein_type +activities O +were O +restored O +in O +the O +presence B-protein_state +of I-protein_state +DDNN B-mutant +mutant B-protein_state +were O +colored O +in O +red O +or O +yellow O +on O +the O +primary O +PIN B-structure_element +. O + +Schematic O +representation O +of O +regulation O +of O +the O +Regnase B-protein +- I-protein +1 I-protein +catalytic O +activity O +through O +the O +domain O +- O +domain O +interactions O +. O + +Structure O +‐ O +activity O +relationship O +of O +the O +peptide B-structure_element +binding I-structure_element +‐ I-structure_element +motif I-structure_element +mediating O +the O +BRCA2 B-complex_assembly +: I-complex_assembly +RAD51 I-complex_assembly +protein O +– O +protein O +interaction O + +RAD51 B-protein +is O +a O +recombinase B-protein_type +involved O +in O +the O +homologous O +recombination O +of O +double O +‐ O +strand O +breaks O +in O +DNA O +. O + +RAD51 B-protein +forms O +oligomers B-oligomeric_state +by O +binding O +to O +another O +molecule O +of O +RAD51 B-protein +via O +an O +‘ O +FxxA B-structure_element +’ O +motif O +, O +and O +the O +same O +recognition O +sequence O +is O +similarly O +utilised O +to O +bind O +BRCA2 B-protein +. O + +We O +have O +tabulated O +the O +effects O +of O +mutation B-experimental_method +of O +this O +sequence O +, O +across O +a O +variety O +of O +experimental O +methods O +and O +from O +relevant O +mutations O +observed O +in O +the O +clinic O +. O + +We O +use O +mutants B-protein_state +of O +a O +tetrapeptide B-chemical +sequence O +to O +probe O +the O +binding O +interaction O +, O +using O +both O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +and O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +Where O +possible O +, O +comparison O +between O +our O +tetrapeptide B-experimental_method +mutational I-experimental_method +study I-experimental_method +and O +the O +previously O +reported O +mutations O +is O +made O +, O +discrepancies O +are O +discussed O +and O +the O +importance O +of O +secondary O +structure O +in O +interpreting O +alanine B-experimental_method +scanning I-experimental_method +and O +mutational O +data O +of O +this O +nature O +is O +considered O +. O + +Eukaryotic B-taxonomy_domain +RAD51 B-protein +, O +archeal B-taxonomy_domain +RadA B-protein +and O +prokaryotic B-taxonomy_domain +RecA B-protein +are O +a O +family O +of O +ATP B-protein_type +‐ I-protein_type +dependent I-protein_type +recombinases I-protein_type +involved O +in O +homologous O +recombination O +( O +HR O +) O +of O +double O +‐ O +strand O +breaks O +in O +DNA O +1 O +. O + +RAD51 B-protein +interacts O +with O +BRCA2 B-protein +, O +and O +is O +thought O +to O +localise O +RAD51 B-protein +to O +sites O +of O +DNA O +damage O +2 O +, O +3 O +. O + +Both O +BRCA2 B-protein +and O +RAD51 B-protein +together O +are O +vital O +for O +helping O +to O +repair O +and O +maintain O +a O +high O +fidelity O +in O +DNA O +replication O +. O + +BRCA2 B-protein +especially O +has O +garnered O +much O +attention O +in O +a O +clinical O +context O +, O +as O +many O +mutations O +have O +been O +identified O +that O +drive O +an O +increased O +risk O +of O +cancer O +in O +individuals O +4 O +, O +5 O +. O + +Although O +the O +inactivation O +of O +the O +BRCA2 B-complex_assembly +: I-complex_assembly +RAD51 I-complex_assembly +DNA O +repair O +pathway O +can O +cause O +genomic O +instability O +and O +eventual O +tumour O +development O +, O +an O +inability O +to O +repair O +breaks O +in O +DNA O +may O +also O +engender O +a O +sensitivity O +to O +ionising O +radiation O +6 O +, O +7 O +. O + +For O +this O +reason O +it O +is O +hypothesised O +that O +in O +tumour O +cells O +with O +an O +intact O +BRCA2 B-complex_assembly +: I-complex_assembly +RAD51 I-complex_assembly +repair O +pathway O +, O +small O +molecules O +which O +prevent O +the O +interaction O +between O +RAD51 B-protein +and O +BRCA2 B-protein +may O +confer O +radiosensitivity O +by O +disabling O +the O +HR O +pathway O +8 O +. O + +The O +interaction O +between O +the O +two O +proteins O +is O +mediated O +by O +eight O +BRC B-structure_element +repeats I-structure_element +, O +which O +are O +characterised O +by O +a O +conserved B-protein_state +‘ B-structure_element +FxxA I-structure_element +’ I-structure_element +motif I-structure_element +9 I-structure_element +. O + +RAD51 B-protein +and O +RadA B-protein +proteins O +also O +contain O +an O +‘ O +FxxA B-structure_element +’ O +sequence O +( O +FTTA B-structure_element +for O +human B-species +RAD51 B-protein +) O +through O +which O +it O +can O +bind O +to O +other O +RAD51 B-protein +and O +RadA B-protein +molecules O +, O +and O +oligomerise O +to O +form O +higher O +order O +filament O +structures O +on O +DNA O +. O + +The O +common O +FxxA B-structure_element +motifs O +of O +both O +the O +BRC B-structure_element +repeats I-structure_element +and O +RAD51 B-protein +oligomerisation B-structure_element +sequence I-structure_element +are O +recognised O +by O +the O +same O +FxxA B-site +‐ I-site +binding I-site +site I-site +of O +RAD51 B-protein +. O + +In O +general O +, O +the O +dominant O +contribution O +of O +certain O +residues O +to O +the O +overall O +binding B-evidence +energy I-evidence +of O +a O +protein O +– O +protein O +interaction O +are O +known O +as O +‘ O +hot B-site +‐ I-site +spot I-site +’ O +residues O +. O + +Interestingly O +, O +small O +molecule O +inhibitors O +of O +PPIs O +are O +often O +found O +to O +occupy O +the O +same O +pockets B-site +which O +are O +otherwise O +occupied O +by O +hot B-site +‐ I-site +spot I-site +residues O +in O +the O +native B-protein_state +complex O +. O + +It O +is O +therefore O +of O +great O +interest O +to O +identify O +hot B-site +‐ I-site +spots I-site +in O +an O +effort O +to O +guide O +drug O +discovery O +efforts O +against O +a O +PPI O +. O + +Further O +, O +a O +correlation O +between O +residues O +that O +are O +strongly B-protein_state +conserved I-protein_state +and O +hot B-site +‐ I-site +spot I-site +residues O +has O +been O +reported O +10 O +. O + +Purely O +based O +on O +the O +amino O +acid O +consensus O +sequence O +reported O +by O +Pellegrini O +et O +al O +., O +11 O +phenylalanine B-residue_name +and O +alanine B-residue_name +would O +both O +be O +expected O +to O +be O +hot B-site +‐ I-site +spots I-site +and O +to O +a O +lesser O +extent O +, O +threonine B-residue_name +. O + +However O +, O +whilst O +the O +identification O +of O +highly B-protein_state +conserved I-protein_state +residues O +may O +be O +a O +good O +starting O +point O +for O +identifying O +hot B-site +‐ I-site +spots I-site +, O +experimental O +validation O +by O +mutation B-experimental_method +of O +these O +sequences O +is O +vital O +. O + +The O +importance O +of O +residues O +in O +the O +FxxA B-structure_element +motif O +has O +been O +probed O +by O +a O +variety O +of O +techniques O +, O +collated O +in O +Table O +1 O +. O + +Briefly O +, O +mutating B-experimental_method +phenylalanine B-residue_name +to O +glutamic B-residue_name +acid I-residue_name +inactivated B-protein_state +the O +BRC4 B-chemical +peptide O +and O +prevented O +RAD51 B-protein +oligomerisation O +11 O +, O +12 O +. O + +A O +phenylalanine B-protein_state +‐ I-protein_state +truncated I-protein_state +BRC4 B-chemical +is O +also O +found O +to O +be O +inactive B-protein_state +13 O +, O +however O +, O +introducing B-experimental_method +a O +tryptophan B-residue_name +for O +phenylalanine B-residue_name +was O +found O +to O +have O +no O +significant O +effect O +on O +BRC4 B-chemical +affinity B-evidence +12 O +. O + +A O +glutamine B-residue_name +replacing B-experimental_method +the O +histidine B-residue_name +in O +BRC4 B-chemical +maintains O +BRC4 B-chemical +activity O +13 O +. O + +The O +ability O +of O +BRC3 B-chemical +to O +interact O +with O +RAD51 B-protein +nucleoprotein O +filaments O +is O +disrupted O +when O +threonine B-residue_name +is O +mutated B-experimental_method +to O +an O +alanine B-residue_name +3 O +. O + +Similarly O +, O +mutating B-experimental_method +alanine B-residue_name +to O +glutamic B-residue_name +acid I-residue_name +in O +the O +RAD51 B-protein +oligomerisation B-structure_element +sequence I-structure_element +11 O +or O +to O +serine B-residue_name +in O +BRC4 B-chemical +13 O +leads O +to O +loss O +of O +interaction O +in O +both O +cases O +. O + +The O +BRC5 B-chemical +repeat O +in O +humans B-species +has O +serine B-residue_name +in O +the O +place O +of O +alanine B-residue_name +, O +and O +is O +thought O +to O +be O +a O +nonbinding B-structure_element +repeat I-structure_element +12 O +. O + +Mutations O +identified O +in O +the O +clinic O +, O +in O +the O +FxxA B-structure_element +region O +of O +the O +BRC B-structure_element +repeats I-structure_element +of O +BRCA2 B-protein +are O +collated O +in O +Table O +1 O +14 O +. O + +For O +completeness O +, O +we O +present O +them O +here O +with O +this O +caveat O +, O +and O +to O +make O +the O +comment O +that O +these O +clinical O +mutations O +are O +consistent O +with O +abrogating O +the O +interaction O +with O +RAD51 B-protein +. O + +Summary O +of O +FxxA B-structure_element +‐ O +relevant O +mutations O +previously O +reported O +and O +degree O +of O +characterisation O +. O + +The O +mutation O +, O +relevant O +peptide O +context O +, O +resulting O +FxxA B-structure_element +motif O +sequence O +and O +experimental O +technique O +for O +each O +entry O +is O +given O +. O + +Mutation O +contexta O +Mutation O +FxxA B-structure_element +motif O +Technique O +used O +Effect O +RAD51 B-protein +( O +FTTA B-structure_element +) O +F86E B-mutant +ETTA B-structure_element +Immunoprecipitation B-experimental_method +11 O +No O +binding O +BRC4 B-chemical +( O +FHTA B-structure_element +) O +F1524E B-mutant +EHTA B-structure_element +Competitive B-experimental_method +ELISA I-experimental_method +12 O +Peptide O +inactive B-protein_state +BRC4 B-chemical +( O +FHTA B-structure_element +) O +F1524W B-mutant +WHTA B-structure_element +Competitive B-experimental_method +ELISA I-experimental_method +12 O +Comparable O +activity O +to O +WT B-protein_state +BRC4 B-chemical +( O +FHTA B-structure_element +) O +F1524V B-mutant +VHTA B-structure_element +BRCA2 B-protein +mutations O +database O +14 O +– O +BRC4 B-chemical +( O +FHTA B-structure_element +) O +ΔF1524 B-mutant +‐ O +HTA B-structure_element +Dissociation O +of O +RAD51 B-complex_assembly +‐ I-complex_assembly +DNA I-complex_assembly +complex O +13 O +Peptide O +inactive B-protein_state +BRC4 B-chemical +( O +FHTA B-structure_element +) O +H1525Q B-mutant +FQTA B-structure_element +Dissociation O +of O +RAD51 B-complex_assembly +‐ I-complex_assembly +DNA I-complex_assembly +complex O +13 O +Comparable O +activity O +BRC7 B-chemical +( O +FSTA B-structure_element +) O +S1979R B-mutant +FRTA B-structure_element +BRCA2 B-protein +mutations O +database O +14 O +– O +BRC3 B-chemical +( O +FQTA B-structure_element +) O +T1430A B-mutant +FQAA B-structure_element +RAD51 B-complex_assembly +: I-complex_assembly +DNA I-complex_assembly +bandshift B-experimental_method +assay I-experimental_method +3 O +Peptide O +inactive B-protein_state +BRC3 B-chemical +( O +FQTA B-structure_element +) O +T1430A B-mutant +FQAA B-structure_element +Electron B-experimental_method +microscopic I-experimental_method +visualisation O +of O +nucleoprotein O +filaments O +3 O +Peptide O +inactive B-protein_state +BRC1 B-chemical +( O +FRTA B-structure_element +) O +T1011R B-mutant +FRRA B-structure_element +BRCA2 B-protein +mutations O +database O +14 O +– O +BRC2 B-chemical +( O +FYSA B-structure_element +) O +S1221P B-mutant +FYPA B-structure_element +BRCA2 B-protein +mutations O +database O +14 O +– O +BRC2 B-chemical +( O +FYSA B-structure_element +) O +S1221Y B-mutant +FYYA B-structure_element +BRCA2 B-protein +mutations O +database O +14 O +– O +RAD51 B-protein +( O +FTTA B-structure_element +) O +A89E B-mutant +FTTE B-structure_element +Immunoprecipitation B-experimental_method +11 O +No O +binding O +BRC4 B-chemical +( O +FHTA B-structure_element +) O +A1527S B-mutant +FHTS B-structure_element +Dissociation O +of O +RAD51 B-complex_assembly +‐ I-complex_assembly +DNA I-complex_assembly +complex O +13 O +Peptide O +inactive B-protein_state + +The O +wild B-protein_state +‐ I-protein_state +type I-protein_state +FxxA B-structure_element +sequence O +is O +indicated O +in O +parenthesis O +. O + +In O +this O +work O +, O +we O +report O +the O +most O +detailed O +study O +of O +systematic B-experimental_method +mutations I-experimental_method +of O +peptides O +to O +probe O +the O +FxxA B-structure_element +‐ I-structure_element +binding I-structure_element +motif I-structure_element +to O +date O +. O + +We O +have O +chosen O +to O +focus O +on O +tetrapeptides B-chemical +, O +which O +allows O +us O +to O +examine O +the O +effect O +of O +mutation B-experimental_method +on O +the O +fundamental O +unit O +of O +binding O +, O +FxxA B-structure_element +, O +rather O +than O +in O +the O +context O +of O +either O +the O +BRC B-structure_element +repeat I-structure_element +or O +self B-structure_element +‐ I-structure_element +oligomerisation I-structure_element +sequence I-structure_element +. O + +Affinities B-evidence +of O +peptides O +were O +measured O +directly O +using O +Isothermal B-experimental_method +Titration I-experimental_method +Calorimetry I-experimental_method +( O +ITC B-experimental_method +) O +and O +the O +structures B-evidence +of O +many O +of O +the O +peptides O +bound B-protein_state +to I-protein_state +humanised B-protein_state +RadA B-protein +were O +determined O +by O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +The O +use O +of O +ITC B-experimental_method +is O +generally O +perceived O +as O +a O +gold O +‐ O +standard O +in O +protein O +– O +ligand O +characterisation O +, O +rather O +than O +a O +competitive B-experimental_method +assay I-experimental_method +which O +may O +be O +prone O +to O +aggregation O +artefacts O +. O + +Wild B-protein_state +‐ I-protein_state +type I-protein_state +human B-species +RAD51 B-protein +, O +however O +, O +is O +a O +heterogeneous O +mixture O +of O +oligomers B-oligomeric_state +and O +when O +monomerised B-oligomeric_state +by O +mutation B-experimental_method +, O +is O +highly B-protein_state +unstable I-protein_state +. O + +In O +this O +context O +, O +we O +have O +previously O +reported O +the O +use O +of O +stable B-protein_state +monomeric B-oligomeric_state +forms O +of O +RAD51 B-protein +, O +humanised B-protein_state +from O +Pyrococcus B-species +furiosus I-species +homologue O +RadA B-protein +, O +for O +ITC B-experimental_method +experiments O +and O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +8 O +, O +15 O +. O + +Conservation O +of O +FxxA B-structure_element +motif O +( O +A O +) O +BRC4 B-chemical +peptide O +( O +green O +cartoon O +) O +bound B-protein_state +to I-protein_state +truncated B-protein_state +human B-species +RAD51 B-protein +( O +grey O +surface O +) O +( O +PDB O +: O +1n0w O +, O +11 O +). O + +The O +blue O +dashed O +box O +highlights O +the O +FxxA B-site +interaction I-site +pocket I-site +. O + +( O +B O +) O +Two O +interacting O +protein O +molecules O +of O +RAD51 B-protein +from O +Saccharomyces B-species +cerevisiae I-species +are O +shown O +. O + +One O +RAD51 B-protein +( O +green O +cartoon O +) O +interacts O +with O +another O +molecule O +of O +RAD51 B-protein +( O +grey O +and O +pink O +surface O +) O +via O +the O +FxxA B-site +pocket I-site +indicated O +by O +the O +dashed O +blue O +box O +. O + +The O +N O +‐ O +terminal O +domain O +of O +one O +RAD51 B-protein +protomer B-oligomeric_state +is O +highlighted O +in O +pink O +for O +clarity O +and O +the O +green O +arrow O +indicates O +the O +location O +of O +this O +protomer B-oligomeric_state +' O +s O +FxxA B-structure_element +oligomerisation I-structure_element +sequence I-structure_element +( O +PDB O +: O +1szp O +, O +29 O +). O +( O +C O +) O +Conservation O +of O +FxxA B-structure_element +motif O +across O +the O +human B-species +BRC B-structure_element +repeats I-structure_element +and O +( O +D O +) O +across O +21 O +eukaryotic B-taxonomy_domain +RAD51s B-protein_type +and O +24 O +RadAs B-protein_type +, O +with O +the O +size O +of O +the O +letters O +proportional O +to O +the O +degree O +of O +conservation O +. O + +We O +have O +mutated B-experimental_method +and I-experimental_method +truncated I-experimental_method +the O +tetrapeptide B-chemical +epitope O +FHTA B-structure_element +, O +and O +examined O +the O +effects O +both O +structurally O +and O +on O +the O +binding B-evidence +affinity I-evidence +with O +humanised B-protein_state +RadA B-protein +. O +As O +a O +comparative O +reference O +, O +we O +are O +using O +the O +FHTA B-structure_element +sequence O +derived O +from O +the O +most O +tightly O +binding O +BRC B-structure_element +repeat I-structure_element +, O +BRC4 B-chemical +22 O +. O + +The O +peptides O +used O +are O +N B-protein_state +‐ I-protein_state +acetylated I-protein_state +and O +C O +‐ O +amide O +terminated O +in O +order O +to O +provide O +the O +most O +relevant O +peptide O +in O +the O +context O +of O +a O +longer O +peptide O +chain O +. O + +A O +summary O +of O +the O +peptide O +sequence O +, O +PDB O +codes O +and O +K B-evidence +D I-evidence +data O +measured O +by O +ITC B-experimental_method +with O +the O +corresponding O +ΔH B-evidence +and O +TΔS B-evidence +values O +are O +collated O +in O +Table O +2 O +. O + +Phe1524 B-residue_name_number +of O +BRC4 B-chemical +binds O +in O +a O +small O +surface B-site +pocket I-site +of O +human B-species +RAD51 B-protein +, O +defined O +by O +the O +hydrophobic O +side O +chains O +of O +residues O +Met158 B-residue_name_number +, O +Ile160 B-residue_name_number +, O +Ala192 B-residue_name_number +, O +Leu203 B-residue_name_number +and O +Met210 B-residue_name_number +. O + +The O +residue O +is O +highly B-protein_state +conserved I-protein_state +across O +BRC B-structure_element +repeats I-structure_element +and O +oligomerisation B-structure_element +sequences I-structure_element +. O + +Consistent O +with O +this O +, O +the O +truncated B-protein_state +HTA B-structure_element +tripeptide B-chemical +could O +not O +be O +detected O +to O +bind O +to O +humanised B-protein_state +, O +monomeric B-oligomeric_state +RadA B-protein +, O +HumRadA2 B-mutant +( O +Table O +2 O +, O +entry O +13 O +). O + +As O +previously O +discussed O +, O +there O +is O +some O +evidence O +that O +substituting B-experimental_method +a O +tryptophan B-residue_name +for O +the O +phenylalanine B-residue_name +at O +this O +position O +was O +tolerated O +in O +the O +context O +of O +BRC4 B-chemical +12 O +. O + +Therefore O +, O +the O +WHTA B-structure_element +peptide O +was O +tested O +and O +found O +to O +not O +only O +be O +tolerated O +, O +but O +to O +increase O +the O +binding B-evidence +affinity I-evidence +of O +the O +peptide O +approximately O +threefold O +. O + +The O +second O +position O +of O +the O +tetrapeptide B-chemical +was O +found O +to O +be O +largely O +invariant O +to O +changes O +in O +the O +side O +chains O +that O +were O +investigated O +. O + +The O +residue O +makes O +no O +interactions O +with O +the O +RAD51 B-protein +protein O +, O +but O +may O +make O +an O +internal O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +Thr1520 B-residue_name_number +in O +the O +context O +of O +BRC4 B-chemical +, O +Fig O +. O +3A O +. O + +Replacing B-experimental_method +the O +histidine B-residue_name +with O +an O +asparagine B-residue_name +, O +chosen O +to O +potentially O +mimic O +the O +hydrogen B-bond_interaction +bond I-bond_interaction +donor O +– O +acceptor O +nature O +of O +histidine B-residue_name +, O +resulted O +in O +a O +moderate O +, O +twofold O +decrease O +in O +potency O +( O +Table O +2 O +, O +entry O +4 O +). O + +Mutating B-experimental_method +to O +an O +alanine B-residue_name +, O +recapitulated O +the O +potency O +of O +FHTA B-structure_element +, O +implying O +that O +the O +interactions O +made O +by O +histidine B-residue_name +do O +not O +contribute O +overall O +to O +binding B-evidence +affinity I-evidence +( O +Table O +2 O +, O +entry O +3 O +). O + +FPTA B-structure_element +was O +also O +tested O +, O +but O +was O +found O +to O +have O +no O +affinity B-evidence +for O +the O +protein O +( O +Table O +2 O +, O +entry O +5 O +). O + +Modelling O +suggests O +that O +a O +proline B-residue_name +in O +the O +second O +position O +would O +be O +expected O +to O +clash O +sterically O +with O +the O +surface O +of O +the O +protein O +, O +and O +provides O +a O +rationale O +for O +the O +lack O +of O +binding O +observed O +. O + +Threonine B-residue_name +was O +mutated B-experimental_method +to O +an O +alanine B-residue_name +, O +resulting O +in O +only O +a O +moderately O +weaker O +K B-evidence +D I-evidence +( O +twofold O +, O +Table O +2 O +, O +entry O +7 O +). O + +In O +the O +context O +of O +a O +tetrapeptide B-chemical +at O +least O +, O +this O +result O +implies O +a O +lack O +of O +importance O +of O +a O +threonine B-residue_name +at O +this O +position O +. O + +Interestingly O +, O +it O +was O +found O +that O +a O +proline B-residue_name +at O +this O +position O +improved O +the O +affinity B-evidence +almost O +threefold O +, O +to O +113 O +μm O +( O +Table O +2 O +, O +entry O +6 O +). O + +This O +beneficial O +mutation B-experimental_method +was O +incorporated O +with O +another O +previously O +identified O +variant O +to O +produce O +the O +peptide O +WHPA B-structure_element +. O + +While O +the O +importance O +of O +the O +phenylalanine B-residue_name +may O +be O +possible O +to O +predict O +from O +examination O +of O +the O +crystal B-evidence +structure I-evidence +, O +the O +alanine B-residue_name +appears O +to O +be O +of O +much O +less O +importance O +in O +this O +regard O +. O + +It O +is O +, O +however O +, O +a O +highly B-protein_state +conserved I-protein_state +residue O +and O +clearly O +of O +interest O +for O +systematic O +mutation O +. O + +Removing B-experimental_method +the O +alanine B-residue_name +residue O +entirely O +produced O +the O +truncated B-protein_state +tripeptide B-chemical +FHT B-structure_element +, O +which O +did O +not O +bind O +( O +Table O +2 O +, O +entry O +12 O +). O + +The O +unnatural O +amino O +acid O +, O +α B-chemical +‐ I-chemical +amino I-chemical +butyric I-chemical +acid I-chemical +( O +U B-chemical +), O +was O +introduced O +at O +the O +fourth O +position O +, O +positioning O +an O +ethyl O +group O +into O +the O +alanine B-site +pocket I-site +( O +Table O +2 O +, O +entry O +9 O +). O + +Perhaps O +surprisingly O +, O +it O +was O +accommodated O +and O +the O +affinity B-evidence +dropped O +only O +by O +twofold O +as O +compared O +to O +FHTA B-structure_element +. O + +The O +effect O +of O +simply O +removing B-experimental_method +the O +β O +‐ O +carbon O +of O +alanine B-residue_name +, O +by O +mutation B-experimental_method +to I-experimental_method +glycine B-residue_name +( O +FHTG B-structure_element +), O +produced O +an O +approximately O +sixfold O +drop O +in O +binding B-evidence +affinity I-evidence +( O +Table O +2 O +, O +entry O +8 O +). O + +This O +is O +in O +line O +with O +the O +observation O +that O +alanine B-residue_name +is O +not B-protein_state +100 I-protein_state +% I-protein_state +conserved I-protein_state +and O +some O +archeal B-taxonomy_domain +RadA B-protein_type +proteins I-protein_type +contain O +a O +glycine B-residue_name +in O +the O +place O +of O +alanine B-residue_name +23 O +. O + +Structural B-experimental_method +characterisation I-experimental_method +of O +peptide O +complexes O + +Structures B-evidence +of O +the O +key O +tetrapeptides B-chemical +were O +solved O +by O +soaking B-experimental_method +into I-experimental_method +crystals B-evidence +of O +a O +humanised B-protein_state +form O +of O +RAD51 B-protein +, O +HumRadA1 B-mutant +, O +which O +we O +have O +previously O +reported O +as O +a O +convenient O +surrogate O +system O +for O +RAD51 B-protein +crystallography B-experimental_method +15 O +. O + +The O +corresponding O +PDB O +codes O +are O +indicated O +in O +Table O +2 O +and O +crystallographic B-evidence +data I-evidence +are O +found O +in O +the O +Supporting O +Information O +. O + +All O +structures B-evidence +are O +of O +high O +resolution O +( O +1 O +. O +2 O +– O +1 O +. O +7 O +Å O +) O +and O +the O +electron B-evidence +density I-evidence +for O +the O +peptide O +was O +clearly O +visible O +after O +the O +first O +refinement O +using O +unliganded B-protein_state +RadA B-protein +coordinates O +( O +Fig O +. O +S1 O +). O + +Some O +of O +the O +SAR O +observed O +in O +the O +binding B-experimental_method +analysis I-experimental_method +can O +be O +interpreted O +in O +terms O +of O +these O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystal B-evidence +structures I-evidence +. O + +For O +example O +, O +an O +overlay B-experimental_method +of O +the O +bound O +poses O +of O +the O +ligands O +FHTA B-structure_element +and O +FHPA B-structure_element +( O +Fig O +. O +2B O +) O +reveals O +a O +high O +similarity O +in O +the O +binding O +modes O +, O +indicating O +that O +the O +conformational O +rigidity O +conferred O +by O +the O +proline B-residue_name +is O +compatible O +with O +the O +FHTA B-structure_element +‐ O +binding O +mode O +, O +and O +a O +reduction O +in O +an O +entropic B-evidence +penalty I-evidence +of O +binding O +may O +be O +the O +source O +of O +the O +improvement O +in O +affinity B-evidence +. O + +WHTA B-structure_element +peptide O +shows O +a O +relative O +dislocation O +when O +compared O +to O +FHTA B-structure_element +( O +Fig O +2A O +), O +with O +the O +entire O +ligand O +backbone O +of O +WHTA B-structure_element +shifted O +to O +accommodate O +the O +change O +in O +the O +position O +of O +the O +main O +chain O +carbon O +of O +the O +first O +residue O +, O +as O +the O +larger O +indole O +side O +chain O +fills O +the O +Phe B-site +pocket I-site +. O + +This O +shift O +is O +translated O +all O +the O +way O +to O +the O +alanine B-residue_name +side O +chain O +. O + +It O +is O +possible O +that O +this O +mutation B-experimental_method +is O +beneficial O +in O +the O +tetrapeptide B-chemical +context O +and O +neutral O +in O +the O +full B-protein_state +‐ I-protein_state +length I-protein_state +BRC4 B-chemical +context O +because O +the O +smaller O +peptide O +is O +less O +constrained O +and O +allowed O +to O +explore O +more O +conformations O +. O + +An O +attempt O +to O +combine O +both O +the O +tryptophan B-residue_name +and O +proline B-residue_name +mutations B-experimental_method +, O +however O +, O +led O +to O +no O +improvement O +for O +WHPA B-structure_element +peptide O +compared O +to O +FHTA B-structure_element +. O + +One O +possible O +explanation O +is O +that O +the O +‘ O +shifted O +’ O +binding O +mode O +observed O +in O +WHTA B-structure_element +was O +not O +compatible O +with O +the O +conformational O +restriction O +that O +the O +proline B-residue_name +of O +WHPA B-structure_element +introduced O +. O + +Comparison O +of O +different O +peptide O +complexes O +( O +A O +) O +Overlay B-experimental_method +with O +FHTA B-structure_element +( O +grey O +) O +and O +WHTA B-structure_element +( O +purple O +) O +showing O +a O +small O +relative O +displacement O +of O +the O +peptide O +backbone O +. O +( O +B O +) O +Superposition B-experimental_method +of O +FHTA B-structure_element +( O +grey O +) O +and O +FHPA B-structure_element +( O +yellow O +), O +showing O +conservation O +of O +backbone O +orientation O +( O +C O +) O +Overlay B-experimental_method +of O +FHTU B-structure_element +( O +green O +), O +FHTA B-structure_element +( O +grey O +) O +and O +FHTG B-structure_element +( O +cyan O +). O + +The O +thermodynamic B-evidence +data I-evidence +of O +peptide O +binding O +are O +also O +shown O +in O +Table O +2 O +. O +Although O +we O +have O +both O +thermodynamic B-evidence +data I-evidence +and O +high O +‐ O +quality O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +structural B-evidence +information I-evidence +for O +some O +of O +the O +mutant B-protein_state +peptides B-chemical +, O +we O +do O +not O +attempt O +to O +interpret O +differences O +in O +thermodynamic B-evidence +profiles I-evidence +between O +ligands O +, O +that O +is O +, O +to O +analyse O +ΔΔH B-evidence +and O +ΔΔS B-evidence +. O + +Although O +ΔH B-evidence +and O +ΔS B-evidence +are O +tabulated O +, O +the O +K B-evidence +Ds I-evidence +measured O +are O +relatively O +weak O +and O +necessarily O +performed O +under O +low O +c O +‐ O +value O +conditions O +. O + +In O +this O +experimental O +regime O +, O +nonsigmoidal O +curves O +are O +generated O +and O +therefore O +errors O +in O +ΔH B-evidence +are O +expected O +to O +be O +much O +higher O +than O +the O +errors O +from O +model O +fitting O +given O +in O +Table O +2 O +16 O +. O + +As O +ΔS B-evidence +is O +derived O +from O +ΔG B-evidence +by O +subtracting O +ΔH B-evidence +, O +errors O +in O +ΔH B-evidence +will O +be O +correlated O +with O +errors O +in O +ΔS B-evidence +, O +giving O +rise O +to O +a O +‘ O +phantom O +’ O +enthalpy O +– O +entropy O +compensation O +. O + +Such O +effects O +have O +been O +discussed O +by O +Klebe O +24 O +and O +Chodera O +and O +Mobley O +25 O +and O +will O +frustrate O +attempts O +to O +interpret O +the O +measured O +ΔΔH B-evidence +and O +ΔΔS B-evidence +. O + +The O +conserved B-protein_state +phenylalanine B-residue_name +and O +alanine B-residue_name +residues O +of O +the O +FHTA B-structure_element +sequence O +were O +both O +found O +to O +be O +essential O +for O +binding O +by O +ITC B-experimental_method +. O + +Conversely O +the O +second O +position O +histidine B-residue_name +residue O +, O +corresponding O +to O +the O +unconserved B-protein_state +His1525 B-residue_name_number +in O +the O +BRC4 B-chemical +sequence O +, O +could O +be O +mutated B-experimental_method +without O +significant O +effect O +on O +the O +peptide B-evidence +affinity I-evidence +. O + +The O +more O +general O +correlation O +between O +hot B-site +‐ I-site +spot I-site +residues O +in O +protein O +– O +protein O +interactions O +and O +the O +high B-protein_state +conservation I-protein_state +of O +such O +residues O +has O +been O +previously O +reported O +10 O +, O +26 O +. O + +Interestingly O +, O +however O +, O +the O +highly B-protein_state +conserved I-protein_state +threonine B-residue_name +residue O +could O +be O +mutated B-experimental_method +without O +affecting O +the O +peptide B-evidence +affinity I-evidence +. O + +This O +unexpected O +result O +, O +in O +the O +light O +of O +its O +very O +high B-protein_state +conservation I-protein_state +in O +the O +BRC B-structure_element +and O +oligomerisation B-structure_element +sequences I-structure_element +, O +begs O +the O +question O +of O +what O +the O +role O +of O +Thr1526 B-residue_name_number +is O +and O +highlights O +a O +potential O +pitfall O +and O +need O +for O +caution O +in O +the O +experimental O +design O +of O +alanine B-experimental_method +mutation I-experimental_method +studies I-experimental_method +. O + +As O +the O +FHTA B-structure_element +peptide B-chemical +is O +potentially O +a O +surrogate O +peptide O +for O +both O +the O +BRC B-structure_element +repeat I-structure_element +peptides O +and O +the O +RAD51 B-protein +self B-structure_element +‐ I-structure_element +oligomerisation I-structure_element +peptide I-structure_element +, O +it O +is O +useful O +to O +examine O +the O +role O +of O +Thr1526 B-residue_name_number +( O +BRC4 B-chemical +) O +and O +the O +analogous O +Thr87 B-residue_name_number +( O +RAD51 B-protein +) O +in O +both O +binding O +contexts O +in O +more O +detail O +. O + +Only O +one O +structure B-evidence +of O +BRC4 B-chemical +is O +published O +in B-protein_state +complex I-protein_state +with I-protein_state +human B-species +RAD51 B-protein +( O +PDB O +: O +1n0w O +). O + +Figure O +3A O +shows O +the O +binding O +pose O +of O +BRC4 B-chemical +when O +bound B-protein_state +to I-protein_state +RAD51 B-protein +and O +the O +intrapeptide O +hydrogen B-bond_interaction +bonds I-bond_interaction +that O +are O +made O +by O +BRC4 B-chemical +. O + +While O +Phe1524 B-residue_name_number +and O +Ala1527 B-residue_name_number +are O +buried O +in O +hydrophobic B-site +pockets I-site +on O +the O +surface O +, O +His1525 B-residue_name_number +is O +close O +enough O +to O +form O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +carbonyl O +of O +Thr1520 B-residue_name_number +, O +but O +the O +rotamer O +of O +His1525 B-residue_name_number +, O +supported O +by O +clearly O +positioned O +water B-chemical +molecules O +, O +is O +not O +compatible O +with O +hydrogen B-bond_interaction +bonding I-bond_interaction +. O + +Also O +, O +Thr1520 B-residue_name_number +is O +constrained O +by O +crystal O +contacts O +in O +this O +structure B-evidence +. O + +Lack B-protein_state +of I-protein_state +conservation I-protein_state +of O +this O +residue O +supports O +the O +idea O +that O +this O +interaction O +is O +not O +crucial O +for O +RAD51 B-complex_assembly +: I-complex_assembly +BRC I-complex_assembly +repeat I-complex_assembly +binding O +. O + +( O +A O +) O +Highlight O +of O +intra O +‐ O +BRC4 B-chemical +interactions O +when O +bound B-protein_state +to I-protein_state +RAD51 B-protein +( O +omitted O +for O +clarity O +) O +( O +PDB O +: O +1n0w O +), O +with O +key O +residues O +shown O +in O +colour O +. O +( O +B O +) O +Intrapeptide O +interactions O +from O +oligomerisation B-structure_element +epitope I-structure_element +of O +S B-species +. I-species +cerevisiae I-species +RAD51 B-protein +when O +bound B-protein_state +to I-protein_state +next O +RAD51 B-protein +in O +the O +filament O +( O +PDB O +: O +1szp O +). O + +Residue O +numbering O +relates O +to O +the O +S B-species +. I-species +cerevisiae I-species +RAD51 B-protein +protein O +, O +the O +corresponding O +human B-species +residues O +are O +in O +parentheses O +. O + +Either O +a O +threonine B-residue_name +or O +serine B-residue_name +is O +most O +commonly O +found O +in O +the O +third O +position O +of O +the O +FxxA B-structure_element +motif O +. O + +Thr1526 B-residue_name_number +makes O +no O +direct O +interactions O +with O +the O +RAD51 B-protein +protein O +, O +but O +instead O +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +network I-bond_interaction +with O +the O +highly B-protein_state +conserved I-protein_state +S1528 B-residue_name_number +and O +K1530 B-residue_name_number +( O +Fig O +. O +1C O +). O + +The O +high B-protein_state +degree I-protein_state +of I-protein_state +conservation I-protein_state +of O +these O +three O +residues O +suggests O +an O +important O +possible O +role O +in O +facilitating O +a O +turn O +and O +stabilising O +the O +conformation O +of O +the O +peptide O +as O +it O +continues O +its O +way O +to O +a O +second O +interaction B-site +site I-site +on O +the O +side O +of O +RAD51 B-protein +. O + +With O +respect O +to O +understanding O +the O +RAD51 B-complex_assembly +: I-complex_assembly +RAD51 I-complex_assembly +interaction O +, O +no O +human B-species +crystal B-evidence +structure I-evidence +has O +been O +published O +, O +however O +, O +several O +oligomeric O +structures B-evidence +of O +archaeal B-taxonomy_domain +RadA B-protein +as O +well O +that O +of O +Saccharomyces B-species +cerevisiae I-species +RAD51 B-protein +have O +been O +reported O +27 O +, O +28 O +, O +29 O +. O + +Figure O +3B O +shows O +a O +highlight O +of O +the O +FxxA B-structure_element +portion O +of O +oligomerisation B-structure_element +peptide I-structure_element +from O +the O +S B-species +. I-species +cerevisiae I-species +RAD51 B-protein +structure B-evidence +, O +with O +residues O +in O +parentheses O +corresponding O +to O +the O +human B-species +RAD51 B-protein +protein O +. O + +The O +conserved B-protein_state +threonine B-residue_name +residue O +at O +the O +third O +position O +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +peptide O +backbone O +amide O +, O +which O +forms O +the O +base O +of O +an O +α B-structure_element +‐ I-structure_element +helix I-structure_element +. O + +In O +both O +structural O +contexts O +, O +the O +role O +of O +the O +third O +position O +threonine B-residue_name +in O +FxxA B-structure_element +seems O +to O +be O +in O +stabilising O +secondary O +structure O +; O +a O +β B-structure_element +‐ I-structure_element +turn I-structure_element +in O +the O +case O +of O +BRC B-structure_element +binding O +and O +an O +α B-structure_element +‐ I-structure_element +helix I-structure_element +in O +the O +case O +of O +RAD51 B-protein +oligomerisation O +. O + +In O +the O +tetrapeptide B-chemical +context O +these O +secondary O +interactions O +are O +not O +present O +and O +mutation B-experimental_method +of O +threonine B-residue_name +to O +alanine B-residue_name +would O +be O +expected O +to O +have O +little O +effect O +on O +affinity B-evidence +. O + +In O +line O +with O +this O +, O +although O +we O +observe O +a O +slight O +twofold O +weakening O +of O +peptide B-evidence +affinity I-evidence +, O +the O +effect O +is O +far O +from O +being O +as O +drastic O +or O +inactivating O +as O +reported O +in O +longer O +peptide O +backgrounds O +3 O +. O +It O +would O +be O +interesting O +to O +investigate O +the O +importance O +of O +this O +residue O +in O +the O +context O +of O +the O +BRC4 B-chemical +peptide O +, O +and O +the O +oligomerisation B-structure_element +peptide I-structure_element +. O + +Rather O +than O +indifference O +to O +alanine B-residue_name +mutation B-experimental_method +, O +a O +significant O +effect O +, O +via O +lack O +of O +secondary O +structure O +stabilisation O +, O +would O +be O +predicted O +, O +as O +indeed O +has O +been O +reported O +for O +BRC3 B-chemical +3 O +. O + +Two O +residues O +in O +the O +FxxA B-structure_element +motif O +, O +phenylalanine B-residue_name +and O +alanine B-residue_name +, O +are O +highly B-protein_state +conserved I-protein_state +( O +Fig O +4a O +). O + +Phenylalanine B-residue_name +mutated B-experimental_method +to I-experimental_method +tryptophan B-residue_name +, O +in O +the O +context O +of O +the O +tetrapeptide B-chemical +improved O +potency O +, O +contrary O +to O +the O +reported O +result O +of O +comparable O +activity O +in O +the O +context O +of O +BRC4 B-chemical +12 O +. O + +Proline B-residue_name +at O +the O +third O +position O +similarly O +improved O +potency O +. O + +Activity O +was O +lost O +by O +mutating B-experimental_method +the O +terminal O +alanine B-residue_name +to O +glycine B-residue_name +, O +but O +recovered O +somewhat O +with O +the O +novel O +α B-chemical +‐ I-chemical +amino I-chemical +butyric I-chemical +acid I-chemical +( O +U B-chemical +). O + +Threonine B-residue_name +was O +found O +to O +be O +relatively O +unimportant O +in O +the O +tetrapeptides B-chemical +but O +has O +been O +previously O +reported O +to O +be O +crucial O +in O +the O +context O +of O +BRC3 B-chemical +. O + +The O +reason O +for O +this O +disconnection O +is O +suggested O +to O +be O +that O +threonine B-residue_name +plays O +a O +role O +in O +stabilising O +the O +β B-structure_element +‐ I-structure_element +turn I-structure_element +in O +the O +BRC B-structure_element +repeats I-structure_element +, O +which O +is O +absent O +in O +the O +tetrapeptides B-chemical +studied O +. O + +This O +may O +lead O +to O +a O +more O +general O +caution O +, O +that O +hot B-site +‐ I-site +spot I-site +data O +should O +be O +interpreted O +by O +considering O +the O +bound O +interaction O +with O +the O +protein O +, O +as O +well O +as O +the O +potential O +role O +in O +stabilising O +the O +bound O +peptide O +secondary O +structure O +. O + +In O +either O +case O +, O +the O +requirement O +for O +structural O +data O +in O +correctly O +interpreting O +alanine B-experimental_method +‐ I-experimental_method +scanning I-experimental_method +experiments I-experimental_method +is O +reinforced O +. O + +Summary O +of O +key O +observations O +( O +A O +) O +FxxA B-structure_element +motif O +sequence O +conservation O +of O +Rad51 B-protein +oligomerisation O +sequences O +and O +BRC B-structure_element +repeats I-structure_element +. O +( O +B O +) O +Highlight O +of O +SAR O +identified O +for O +the O +tetrapeptide B-chemical +. O + +The O +differences O +in O +ΔG B-evidence +for O +different O +peptide O +variants O +relative O +to O +FHTA B-structure_element +are O +shown O +in O +the O +bar O +chart O +with O +colouring O +matching O +with O +the O +structural B-experimental_method +overlay I-experimental_method +below O +. O +( O +C O +) O +Overlay B-experimental_method +of O +tetrapeptide B-chemical +structures B-evidence +, O +with O +wild B-protein_state +‐ I-protein_state +type I-protein_state +FHTA B-structure_element +peptide O +across O +the O +figure O +for O +reference O +and O +truncated O +segments O +of O +mutated O +residues O +shown O +in O +each O +panel O +. O + +Purple O +carbon O +is O +WHTA B-structure_element +, O +light O +blue O +is O +FATA B-structure_element +, O +yellow O +is O +FHPA B-structure_element +, O +cyan O +is O +FHTG B-structure_element +and O +grey O +carbon O +is O +FHTA B-structure_element +. O + +Note O +the O +C O +‐ O +terminal O +amide O +changes O +position O +in O +FHTG B-structure_element +without O +the O +anchoring O +methyl O +group O +. O + +Crystal B-evidence +Structure I-evidence +and O +Activity B-experimental_method +Studies I-experimental_method +of O +the O +C11 B-protein_type +Cysteine B-protein_type +Peptidase I-protein_type +from O +Parabacteroides B-species +merdae I-species +in O +the O +Human B-species +Gut O +Microbiome O +* O + +Clan B-protein_type +CD I-protein_type +cysteine I-protein_type +peptidases I-protein_type +, O +a O +structurally O +related O +group O +of O +peptidases B-protein_type +that O +include O +mammalian B-taxonomy_domain +caspases B-protein_type +, O +exhibit O +a O +wide O +range O +of O +important O +functions O +, O +along O +with O +a O +variety O +of O +specificities O +and O +activation O +mechanisms O +. O + +However O +, O +for O +the O +clostripain B-protein_type +family I-protein_type +( O +denoted O +C11 B-protein_type +), O +little O +is O +currently O +known O +. O + +Here O +, O +we O +describe O +the O +first O +crystal B-evidence +structure I-evidence +of O +a O +C11 B-protein_type +protein O +from O +the O +human B-species +gut O +bacterium B-taxonomy_domain +, O +Parabacteroides B-species +merdae I-species +( O +PmC11 B-protein +), O +determined O +to O +1 O +. O +7 O +- O +Å O +resolution O +. O + +PmC11 B-protein +is O +a O +monomeric B-oligomeric_state +cysteine B-protein_type +peptidase I-protein_type +that O +comprises O +an O +extended B-structure_element +caspase I-structure_element +- I-structure_element +like I-structure_element +α I-structure_element +/ I-structure_element +β I-structure_element +/ I-structure_element +α I-structure_element +sandwich I-structure_element +and O +an O +unusual O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +. O + +It O +shares O +core O +structural O +elements O +with O +clan B-protein_type +CD I-protein_type +cysteine I-protein_type +peptidases I-protein_type +but O +otherwise O +structurally O +differs O +from O +the O +other O +families O +in O +the O +clan O +. O + +These O +studies O +also O +revealed O +a O +well O +ordered O +break O +in O +the O +polypeptide O +chain O +at O +Lys147 B-residue_name_number +, O +resulting O +in O +a O +large O +conformational O +rearrangement O +close O +to O +the O +active B-site +site I-site +. O + +Biochemical B-experimental_method +and I-experimental_method +kinetic I-experimental_method +analysis I-experimental_method +revealed O +Lys147 B-residue_name_number +to O +be O +an O +intramolecular B-site +processing I-site +site I-site +at O +which O +cleavage B-ptm +is O +required O +for O +full B-protein_state +activation I-protein_state +of O +the O +enzyme B-protein +, O +suggesting O +an O +autoinhibitory O +mechanism O +for O +self O +- O +preservation O +. O + +PmC11 B-protein +has O +an O +acidic B-site +binding I-site +pocket I-site +and O +a O +preference O +for O +basic O +substrates O +, O +and O +accepts O +substrates O +with O +Arg B-residue_name +and O +Lys B-residue_name +in O +P1 B-residue_number +and O +does O +not O +require O +Ca2 B-chemical ++ I-chemical +for O +activity O +. O + +Collectively O +, O +these O +data O +provide O +insights O +into O +the O +mechanism O +and O +activity O +of O +PmC11 B-protein +and O +a O +detailed O +framework O +for O +studies O +on O +C11 B-protein_type +peptidases I-protein_type +from O +other O +phylogenetic O +kingdoms O +. O + +Cysteine B-protein_type +peptidases I-protein_type +play O +crucial O +roles O +in O +the O +virulence O +of O +bacterial B-taxonomy_domain +and O +other O +eukaryotic B-taxonomy_domain +pathogens O +. O + +In O +the O +MEROPS O +peptidase O +database O +, O +clan B-protein_type +CD I-protein_type +contains O +groups O +( O +or O +families O +) O +of O +cysteine B-protein_type +peptidases I-protein_type +that O +share O +some O +highly B-protein_state +conserved I-protein_state +structural O +elements O +. O + +Clan B-protein_type +CD I-protein_type +families I-protein_type +are O +typically O +described O +using O +the O +name O +of O +their O +archetypal O +, O +or O +founding O +, O +member O +and O +also O +given O +an O +identification O +number O +preceded O +by O +a O +“ O +C O +,” O +to O +denote O +cysteine B-protein_type +peptidase I-protein_type +. O + +Although O +seven O +families O +( O +C14 O +is O +additionally O +split O +into O +three O +subfamilies O +) O +have O +been O +described O +for O +this O +clan O +, O +crystal B-evidence +structures I-evidence +have O +only O +been O +determined O +from O +four O +: O +legumain B-protein +( O +C13 B-protein_type +), O +caspase B-protein +( O +C14a B-protein_type +), O +paracaspase B-protein +( O +C14b B-protein_type +( I-protein_type +P I-protein_type +), O +metacaspase B-protein +( O +C14b B-protein_type +( I-protein_type +M I-protein_type +), O +gingipain B-protein +( O +C25 B-protein_type +), O +and O +the O +cysteine B-structure_element +peptidase I-structure_element +domain I-structure_element +( O +CPD B-structure_element +) O +of O +various O +toxins O +( O +C80 B-protein_type +). O + +No O +structural O +information O +is O +available O +for O +clostripain B-protein +( O +C11 B-protein_type +), O +separase B-protein +( O +C50 B-protein_type +), O +or O +PrtH B-protein +- I-protein +peptidase I-protein +( O +C85 B-protein_type +). O + +Clan B-protein_type +CD I-protein_type +enzymes I-protein_type +have O +a O +highly B-protein_state +conserved I-protein_state +His B-site +/ I-site +Cys I-site +catalytic I-site +dyad I-site +and O +exhibit O +strict O +specificity O +for O +the O +P1 B-residue_number +residue O +of O +their O +substrates O +. O + +However O +, O +despite O +these O +similarities O +, O +clan B-protein_type +CD I-protein_type +forms O +a O +functionally O +diverse O +group O +of O +enzymes O +: O +the O +overall O +structural O +diversity O +between O +( O +and O +at O +times O +within O +) O +the O +various O +families O +provides O +these O +peptidases B-protein_type +with O +a O +wide O +variety O +of O +substrate O +specificities O +and O +activation O +mechanisms O +. O + +The O +archetypal O +and O +arguably O +most O +notable O +family O +in O +the O +clan O +is O +that O +of O +the O +mammalian B-taxonomy_domain +caspases B-protein_type +( O +C14a B-protein_type +), O +although O +clan B-protein_type +CD I-protein_type +members O +are O +distributed O +throughout O +the O +entire O +phylogenetic O +kingdom O +and O +are O +often O +required O +in O +fundamental O +biological O +processes O +. O + +Interestingly O +, O +little O +is O +known O +about O +the O +structure O +or O +function O +of O +the O +C11 B-protein_type +proteins O +, O +despite O +their O +widespread O +distribution O +and O +its O +archetypal O +member O +, O +clostripain B-protein +from O +Clostridium B-species +histolyticum I-species +, O +first O +reported O +in O +the O +literature O +in O +1938 O +. O + +Clostripain B-protein +has O +been O +described O +as O +an O +arginine B-protein_type +- I-protein_type +specific I-protein_type +peptidase I-protein_type +with O +a O +requirement O +for O +Ca2 B-chemical ++ I-chemical +and O +loss O +of O +an O +internal B-structure_element +nonapeptide I-structure_element +for O +full B-protein_state +activation I-protein_state +; O +lack O +of O +structural O +information O +on O +the O +family O +appears O +to O +have O +prohibited O +further O +investigation O +. O + +As O +part O +of O +an O +ongoing O +project O +to O +characterize O +commensal O +bacteria B-taxonomy_domain +in O +the O +microbiome O +that O +inhabit O +the O +human B-species +gut O +, O +the O +structure B-evidence +of O +C11 B-protein_type +peptidase I-protein_type +, O +PmC11 B-protein +, O +from O +Parabacteroides B-species +merdae I-species +was O +determined O +using O +the O +Joint O +Center O +for O +Structural O +Genomics O +( O +JCSG O +) O +4 O +HTP O +structural O +biology O +pipeline O +. O + +The O +structure B-experimental_method +was I-experimental_method +analyzed I-experimental_method +, O +and O +the O +enzyme O +was O +biochemically B-experimental_method +characterized I-experimental_method +to O +provide O +the O +first O +structure O +/ O +function O +correlation O +for O +a O +C11 B-protein_type +peptidase I-protein_type +. O + +Structure B-evidence +of O +PmC11 B-protein + +The O +crystal B-evidence +structure I-evidence +of O +the O +catalytically B-protein_state +active I-protein_state +form O +of O +PmC11 B-protein +revealed O +an O +extended B-structure_element +caspase I-structure_element +- I-structure_element +like I-structure_element +α I-structure_element +/ I-structure_element +β I-structure_element +/ I-structure_element +α I-structure_element +sandwich I-structure_element +architecture O +comprised O +of O +a O +central O +nine B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +, O +with O +an O +unusual O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +CTD B-structure_element +), O +starting O +at O +Lys250 B-residue_name_number +. O + +A O +single B-ptm +cleavage I-ptm +was O +observed O +in O +the O +polypeptide O +chain O +at O +Lys147 B-residue_name_number +( O +Fig O +. O +1 O +, O +A O +and O +B O +), O +where O +both O +ends O +of O +the O +cleavage B-site +site I-site +are O +fully O +visible O +and O +well O +ordered O +in O +the O +electron B-evidence +density I-evidence +. O + +The O +central O +nine B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +( O +β1 B-structure_element +– I-structure_element +β9 I-structure_element +) O +of O +PmC11 B-protein +consists O +of O +six O +parallel B-structure_element +and O +three O +anti B-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +strands I-structure_element +with O +4 O +↑ O +3 O +↓ O +2 O +↑ O +1 O +↑ O +5 O +↑ O +6 O +↑ O +7 O +↓ O +8 O +↓ O +9 O +↑ O +topology O +( O +Fig O +. O +1A O +) O +and O +the O +overall O +structure B-evidence +includes O +14 O +α B-structure_element +- I-structure_element +helices I-structure_element +with O +six O +( O +α1 B-structure_element +– I-structure_element +α2 I-structure_element +and O +α4 B-structure_element +– I-structure_element +α7 I-structure_element +) O +closely O +surrounding O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +in O +an O +approximately O +parallel O +orientation O +. O + +Helices B-structure_element +α1 B-structure_element +, O +α7 B-structure_element +, O +and O +α6 B-structure_element +are O +located O +on O +one O +side O +of O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +with O +α2 B-structure_element +, O +α4 B-structure_element +, O +and O +α5 B-structure_element +on O +the O +opposite O +side O +( O +Fig O +. O +1A O +). O + +Helix B-structure_element +α3 B-structure_element +sits O +at O +the O +end O +of O +the O +loop B-structure_element +following O +β5 B-structure_element +( O +L5 B-structure_element +), O +just O +preceding O +the O +Lys147 B-residue_name_number +cleavage B-site +site I-site +, O +with O +both O +L5 B-structure_element +and O +α3 B-structure_element +pointing O +away O +from O +the O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +and O +toward O +the O +CTD B-structure_element +, O +which O +starts O +with O +α8 B-structure_element +. O + +The O +structure B-evidence +also O +includes O +two O +short O +β B-structure_element +- I-structure_element +hairpins I-structure_element +( O +βA B-structure_element +– I-structure_element +βB I-structure_element +and O +βD B-structure_element +– I-structure_element +βE I-structure_element +) O +and O +a O +small B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +( O +βC B-structure_element +– I-structure_element +βF I-structure_element +), O +which O +is O +formed O +from O +two O +distinct O +regions O +of O +the O +sequence O +( O +βC B-structure_element +precedes O +α11 B-structure_element +, O +α12 B-structure_element +and O +β9 B-structure_element +, O +whereas O +βF B-structure_element +follows O +the O +βD B-structure_element +- I-structure_element +βE I-structure_element +hairpin B-structure_element +) O +in O +the O +middle O +of O +the O +CTD B-structure_element +( O +Fig O +. O +1B O +). O + +Crystal B-evidence +structure I-evidence +of O +a O +C11 B-protein_type +peptidase I-protein_type +from O +P B-species +. I-species +merdae I-species +. O + +A O +, O +primary B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +PmC11 B-protein +( O +Uniprot O +ID O +A7A9N3 O +) O +and O +clostripain B-protein +( O +Uniprot O +ID O +P09870 O +) O +from O +C B-species +. I-species +histolyticum I-species +with O +identical O +residues O +highlighted O +in O +gray O +shading O +. O + +The O +secondary O +structure O +of O +PmC11 B-protein +from O +the O +crystal B-evidence +structure I-evidence +is O +mapped O +onto O +its O +sequence O +with O +the O +position O +of O +the O +PmC11 B-protein +catalytic B-site +dyad I-site +, O +autocatalytic B-site +cleavage I-site +site I-site +( O +Lys147 B-residue_name_number +), O +and O +S1 B-site +binding I-site +pocket I-site +Asp B-residue_name +( O +Asp177 B-residue_name_number +) O +highlighted O +by O +a O +red O +star O +, O +a O +red O +downturned O +triangle O +, O +and O +a O +red O +upturned O +triangle O +, O +respectively O +. O + +Connecting O +loops B-structure_element +are O +colored O +gray O +, O +the O +main O +β B-structure_element +- I-structure_element +sheet I-structure_element +is O +in O +orange O +, O +with O +other O +strands O +in O +olive O +, O +α B-structure_element +- I-structure_element +helices I-structure_element +are O +in O +blue O +, O +and O +the O +nonapeptide B-structure_element +linker I-structure_element +of O +clostripain B-protein +that O +is O +excised O +upon O +autocleavage B-ptm +is O +underlined O +in O +red O +. O + +Sequences O +around O +the O +catalytic B-site +site I-site +of O +clostripain B-protein +and O +PmC11 B-protein +align O +well O +. O + +B O +, O +topology O +diagram O +of O +PmC11 B-protein +colored O +as O +in O +A O +except O +that O +additional O +( O +non O +- O +core O +) O +β B-structure_element +- I-structure_element +strands I-structure_element +are O +in O +yellow O +. O + +Helices O +found O +on O +either O +side O +of O +the O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +are O +shown O +above O +and O +below O +the O +sheet B-structure_element +, O +respectively O +. O + +The O +position O +of O +the O +catalytic B-site +dyad I-site +( O +H B-residue_name +, O +C B-residue_name +) O +and O +the O +processing B-site +site I-site +( O +Lys147 B-residue_name_number +) O +are O +highlighted O +. O + +Helices O +( O +1 O +– O +14 O +) O +and O +β B-structure_element +- I-structure_element +strands I-structure_element +( O +1 O +– O +9 O +and O +A O +- O +F O +) O +are O +numbered O +from O +the O +N O +terminus O +. O + +The O +core B-structure_element +caspase I-structure_element +- I-structure_element +fold I-structure_element +is O +highlighted O +in O +a O +box O +. O + +C O +, O +tertiary O +structure O +of O +PmC11 B-protein +. O + +The O +N O +and O +C O +termini O +( O +N O +and O +C O +) O +of O +PmC11 B-protein +along O +with O +the O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +( O +1 O +– O +9 O +), O +helix B-structure_element +α5 B-structure_element +, O +and O +helices B-structure_element +α8 B-structure_element +, O +α11 B-structure_element +, O +and O +α13 B-structure_element +from O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +, O +are O +all O +labeled O +. O + +Loops O +are O +colored O +gray O +, O +the O +main O +β B-structure_element +- I-structure_element +sheet I-structure_element +is O +in O +orange O +, O +with O +other O +β B-structure_element +- I-structure_element +strands I-structure_element +in O +yellow O +, O +and O +α B-structure_element +- I-structure_element +helices I-structure_element +are O +in O +blue O +. O + +The O +CTD B-structure_element +of O +PmC11 B-protein +is O +composed O +of O +a O +tight B-structure_element +helical I-structure_element +bundle I-structure_element +formed O +from O +helices B-structure_element +α8 B-structure_element +– I-structure_element +α14 I-structure_element +and O +includes O +strands B-structure_element +βC B-structure_element +and O +βF B-structure_element +, O +and O +β B-structure_element +- I-structure_element +hairpin I-structure_element +βD B-structure_element +– I-structure_element +βE I-structure_element +. O +The O +CTD B-structure_element +sits O +entirely O +on O +one O +side O +of O +the O +enzyme O +interacting O +only O +with O +α3 B-structure_element +, O +α5 B-structure_element +, O +β9 B-structure_element +, O +and O +the O +loops B-structure_element +surrounding O +β8 B-structure_element +. O + +Of O +the O +interacting O +secondary O +structure O +elements O +, O +α5 B-structure_element +is O +perhaps O +the O +most O +interesting O +. O + +This B-structure_element +helix I-structure_element +makes O +a O +total O +of O +eight O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +CTD B-structure_element +, O +including O +one O +salt B-bond_interaction +bridge I-bond_interaction +( O +Arg191 B-residue_name_number +- O +Asp255 B-residue_name_number +) O +and O +is O +surrounded O +by O +the O +CTD B-structure_element +on O +one O +side O +and O +the O +main B-structure_element +core I-structure_element +of O +the O +enzyme O +on O +the O +other O +, O +acting O +like O +a O +linchpin O +holding O +both O +components O +together O +( O +Fig O +. O +1C O +). O + +PmC11 B-protein +is O +, O +as O +expected O +, O +most O +structurally O +similar O +to O +other O +members O +of O +clan B-protein_type +CD I-protein_type +with O +the O +top O +hits O +in O +a O +search O +of O +known O +structures B-evidence +being O +caspase B-protein +- I-protein +7 I-protein +, O +gingipain B-protein +- I-protein +K I-protein +, O +and O +legumain B-protein +( O +PBD O +codes O +4hq0 O +, O +4tkx O +, O +and O +4aw9 O +, O +respectively O +) O +( O +Table O +2 O +). O + +The O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +is O +unique O +to O +PmC11 B-protein +within O +clan B-protein_type +CD I-protein_type +and O +structure B-experimental_method +comparisons I-experimental_method +for O +this B-structure_element +domain I-structure_element +alone I-structure_element +does O +not O +produce O +any O +hits O +in O +the O +PDB O +( O +DaliLite B-experimental_method +, O +PDBeFold B-experimental_method +), O +suggesting O +a O +completely O +novel O +fold O +. O + +As O +the O +archetypal O +and O +arguably O +most O +well O +studied O +member O +of O +clan B-protein_type +CD I-protein_type +, O +the O +caspases B-protein_type +were O +used O +as O +the O +basis O +to O +investigate O +the O +structure O +/ O +function O +relationships O +in O +PmC11 B-protein +, O +with O +caspase B-protein +- I-protein +7 I-protein +as O +the O +representative O +member O +. O + +Six O +of O +the O +central O +β B-structure_element +- I-structure_element +strands I-structure_element +in O +PmC11 B-protein +( O +β1 B-structure_element +– I-structure_element +β2 I-structure_element +and O +β5 B-structure_element +– I-structure_element +β8 I-structure_element +) O +share O +the O +same O +topology O +as O +the O +six B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +found O +in O +caspases B-protein_type +, O +with O +strands B-structure_element +β3 B-structure_element +, O +β4 B-structure_element +, O +and O +β9 B-structure_element +located O +on O +the O +outside O +of O +this O +core B-structure_element +structure I-structure_element +( O +Fig O +. O +1B O +, O +box O +). O + +His133 B-residue_name_number +and O +Cys179 B-residue_name_number +were O +found O +at O +locations O +structurally O +homologous O +to O +the O +caspase B-protein_type +catalytic B-site +dyad I-site +, O +and O +other O +clan B-protein_type +CD I-protein_type +structures B-evidence +, O +at O +the O +C O +termini O +of O +strands B-structure_element +β5 B-structure_element +and O +β6 B-structure_element +, O +respectively O +( O +Figs O +. O +1 O +, O +A O +and O +B O +, O +and O +2A O +). O + +A O +multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +C11 B-protein_type +proteins O +revealed O +that O +these O +residues O +are O +highly B-protein_state +conserved I-protein_state +( O +data O +not O +shown O +). O + +Summary O +of O +PDBeFOLD B-experimental_method +superposition I-experimental_method +of O +structures O +found O +to O +be O +most O +similar O +to O +PmC11 B-protein +in O +the O +PBD O +based O +on O +DaliLite B-experimental_method + +Biochemical B-experimental_method +and I-experimental_method +structural I-experimental_method +characterization I-experimental_method +of O +PmC11 B-protein +. O + +A O +, O +ribbon O +representation O +of O +the O +overall O +structure O +of O +PmC11 B-protein +illustrating O +the O +catalytic B-site +site I-site +, O +cleavage O +site O +displacement O +, O +and O +potential O +S1 B-site +binding I-site +site I-site +. O + +The O +overall O +structure B-evidence +of O +PmC11 B-protein +is O +shown O +in O +gray O +, O +looking O +down O +into O +the O +catalytic B-site +site I-site +with O +the O +catalytic B-site +dyad I-site +in O +red O +. O + +The O +two O +ends O +of O +the O +autolytic B-site +cleavage I-site +site I-site +( O +Lys147 B-residue_name_number +and O +Ala148 B-residue_name_number +, O +green O +) O +are O +displaced O +by O +19 O +. O +5 O +Å O +( O +thin O +black O +line O +) O +from O +one O +another O +and O +residues O +in O +the O +potential O +substrate B-site +binding I-site +pocket I-site +are O +highlighted O +in O +blue O +. O + +B O +, O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +of O +PmC11 B-protein +. O + +PmC11 O +migrates O +as O +a O +monomer B-oligomeric_state +with O +a O +molecular O +mass O +around O +41 O +kDa O +calculated O +from O +protein O +standards O +of O +known O +molecular O +weights O +. O + +Elution O +fractions O +across O +the O +major O +peak O +( O +1 O +– O +6 O +) O +were O +analyzed O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +on O +a O +4 O +– O +12 O +% O +gel O +in O +MES O +buffer O +. O + +C O +, O +the O +active B-protein_state +form O +of O +PmC11 B-protein +and O +two O +mutants O +, O +PmC11C179A B-mutant +( O +C O +) O +and O +PmC11K147A B-mutant +( O +K O +), O +were O +examined O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +( O +lane O +1 O +) O +and O +Western B-experimental_method +blot I-experimental_method +analysis O +using O +an O +anti O +- O +His O +antibody O +( O +lane O +2 O +) O +show O +that O +PmC11 B-protein +autoprocesses B-ptm +, O +whereas O +mutants O +, O +PmC11C179A B-mutant +and O +PmC11K147A B-mutant +, O +do O +not O +show O +autoprocessing B-ptm +in O +vitro O +. O + +D O +, O +cysteine O +peptidase O +activity O +of O +PmC11 B-protein +. O + +Km O +and O +Vmax B-evidence +of O +PmC11 B-protein +and O +K147A B-mutant +mutant O +were O +determined O +by O +monitoring O +change O +in O +the O +fluorescence O +corresponding O +to O +AMC O +release O +from O +Bz B-chemical +- I-chemical +R I-chemical +- I-chemical +AMC I-chemical +. O + +E O +, O +intermolecular B-ptm +processing I-ptm +of O +PmC11C179A B-mutant +by O +PmC11 B-protein +. O + +PmC11C179A O +( O +20 O +μg O +) O +was O +incubated O +overnight O +at O +37 O +° O +C O +with O +increasing O +amounts O +of O +processed O +PmC11 B-protein +and O +analyzed O +on O +a O +10 O +% O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +gel O +. O + +Inactive O +PmC11C179A B-mutant +was O +not O +processed O +to O +a O +major O +extent O +by O +active B-protein_state +PmC11 B-protein +until O +around O +a O +ratio O +of O +1 O +: O +4 O +( O +5 O +μg O +of O +active B-protein_state +PmC11 B-protein +). O + +A O +single O +lane O +of O +20 O +μg O +of O +active B-protein_state +PmC11 B-protein +( O +labeled O +20 O +) O +is O +shown O +for O +comparison O +. O + +F O +, O +activity B-evidence +of O +PmC11 B-protein +against O +basic O +substrates O +. O + +G O +, O +electrostatic O +surface O +potential O +of O +PmC11 B-protein +shown O +in O +a O +similar O +orientation O +, O +where O +blue O +and O +red O +denote O +positively O +and O +negatively O +charged O +surface O +potential O +, O +respectively O +, O +contoured O +at O +± O +5 O +kT O +/ O +e O +. O + +The O +position O +of O +the O +catalytic B-site +dyad I-site +, O +one O +potential O +key B-site +substrate I-site +binding I-site +residue I-site +Asp177 B-residue_name_number +, O +and O +the O +ends O +of O +the O +cleavage B-site +site I-site +Lys147 B-residue_name_number +and O +Ala148 B-residue_name_number +are O +indicated O +. O + +Five O +of O +the O +α B-structure_element +- I-structure_element +helices I-structure_element +surrounding O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +PmC11 B-protein +( O +α1 B-structure_element +, O +α2 B-structure_element +, O +α4 B-structure_element +, O +α6 B-structure_element +, O +and O +α7 B-structure_element +) O +are O +found O +in O +similar O +positions O +to O +the O +five O +structurally B-protein_state +conserved I-protein_state +helices B-structure_element +in O +caspases B-protein_type +and O +other O +members O +of O +clan B-protein_type +CD I-protein_type +, O +apart O +from O +family O +C80 B-protein_type +. O + +Other O +than O +its O +more O +extended B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +, O +PmC11 B-protein +differs O +most O +significantly O +from O +other O +clan B-protein_type +CD I-protein_type +members O +at O +its O +C O +terminus O +, O +where O +the O +CTD B-structure_element +contains O +a O +further O +seven O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +four O +β B-structure_element +- I-structure_element +strands I-structure_element +after O +β8 B-structure_element +. O + +Autoprocessing B-ptm +of O +PmC11 B-protein + +Purification B-experimental_method +of O +recombinant O +PmC11 B-protein +( O +molecular O +mass O += O +42 O +. O +6 O +kDa O +) O +revealed O +partial O +processing O +into O +two O +cleavage O +products O +of O +26 O +. O +4 O +and O +16 O +. O +2 O +kDa O +, O +related O +to O +the O +observed O +cleavage B-ptm +at O +Lys147 B-residue_name_number +in O +the O +crystal B-evidence +structure I-evidence +( O +Fig O +. O +2A O +). O + +Incubation B-experimental_method +of O +PmC11 B-protein +at O +37 O +° O +C O +for O +16 O +h O +, O +resulted O +in O +a O +fully B-protein_state +processed I-protein_state +enzyme O +that O +remained O +as O +an O +intact B-protein_state +monomer B-oligomeric_state +when O +applied O +to O +a O +size O +- O +exclusion O +column O +( O +Fig O +. O +2B O +). O + +The O +single O +cleavage B-site +site I-site +of O +PmC11 B-protein +at O +Lys147 B-residue_name_number +is O +found O +immediately O +after O +α3 B-structure_element +, O +in O +loop B-structure_element +L5 B-structure_element +within O +the O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +( O +Figs O +. O +1 O +, O +A O +and O +B O +, O +and O +2A O +). O + +The O +two O +ends O +of O +the O +cleavage B-site +site I-site +are O +remarkably O +well O +ordered O +in O +the O +crystal B-evidence +structure I-evidence +and O +displaced O +from O +one O +another O +by O +19 O +. O +5 O +Å O +( O +Fig O +. O +2A O +). O + +Moreover O +, O +the O +C O +- O +terminal O +side O +of O +the O +cleavage B-site +site I-site +resides O +near O +the O +catalytic B-site +dyad I-site +with O +Ala148 B-residue_name_number +being O +4 O +. O +5 O +and O +5 O +. O +7 O +Å O +from O +His133 B-residue_name_number +and O +Cys179 B-residue_name_number +, O +respectively O +. O + +Consequently O +, O +it O +appears O +feasible O +that O +the O +helix B-structure_element +attached O +to O +Lys147 B-residue_name_number +( O +α3 B-structure_element +) O +could O +be O +responsible O +for O +steric O +autoinhibition O +of O +PmC11 B-protein +when O +Lys147 B-residue_name_number +is O +covalently O +bonded O +to O +Ala148 B-residue_name_number +. O + +Thus O +, O +the O +cleavage B-ptm +would O +be O +required O +for O +full B-protein_state +activation I-protein_state +of O +PmC11 B-protein +. O + +To O +investigate O +this O +possibility O +, O +two O +mutant O +forms O +of O +the O +enzyme O +were O +created O +: O +PmC11C179A B-mutant +( O +a O +catalytically B-protein_state +inactive I-protein_state +mutant I-protein_state +) O +and O +PmC11K147A B-mutant +( O +a O +cleavage B-protein_state +- I-protein_state +site I-protein_state +mutant I-protein_state +). O + +Initial O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +and O +Western B-experimental_method +blot I-experimental_method +analysis O +of O +both O +mutants O +revealed O +no O +discernible O +processing O +occurred O +as O +compared O +with O +active B-protein_state +PmC11 B-protein +( O +Fig O +. O +2C O +). O + +The O +PmC11K147A B-mutant +mutant B-protein_state +enzyme O +had O +a O +markedly O +different O +reaction B-evidence +rate I-evidence +( O +Vmax B-evidence +) O +compared O +with O +WT B-protein_state +, O +where O +the O +reaction B-evidence +velocity I-evidence +of O +PmC11 B-protein +was O +10 O +times O +greater O +than O +that O +of O +PmC11K147A B-mutant +( O +Fig O +. O +2D O +). O + +Taken O +together O +, O +these O +data O +reveal O +that O +PmC11 B-protein +requires O +processing O +at O +Lys147 B-residue_name_number +for O +optimum O +activity O +. O + +To O +investigate O +whether O +processing O +is O +a O +result O +of O +intra O +- O +or O +intermolecular O +cleavage O +, O +the O +PmC11C179A B-mutant +mutant B-protein_state +was O +incubated B-experimental_method +with I-experimental_method +increasing I-experimental_method +concentrations I-experimental_method +of O +processed B-protein_state +and O +activated B-protein_state +PmC11 B-protein +. O + +These O +studies O +revealed O +that O +there O +was O +no O +apparent O +cleavage O +of O +PmC11C179A B-mutant +by O +the O +active B-protein_state +enzyme O +at B-experimental_method +low I-experimental_method +concentrations I-experimental_method +of O +PmC11 B-protein +and O +that O +only O +limited O +cleavage O +was O +observed O +when O +the O +ratio O +of O +active B-protein_state +enzyme O +( O +PmC11 B-protein +: O +PmC11C179A B-mutant +) O +was O +increased B-experimental_method +to I-experimental_method +∼ I-experimental_method +1 I-experimental_method +: I-experimental_method +10 I-experimental_method +and I-experimental_method +1 I-experimental_method +: I-experimental_method +4 I-experimental_method +, O +with O +complete O +cleavage O +observed O +at O +a O +ratio B-experimental_method +of I-experimental_method +1 I-experimental_method +: I-experimental_method +1 I-experimental_method +( O +Fig O +. O +2E O +). O + +This O +suggests O +that O +cleavage B-ptm +of O +PmC11C179A B-mutant +was O +most O +likely O +an O +effect O +of O +the O +increasing O +concentration O +of O +PmC11 B-protein +and O +intermolecular O +cleavage O +. O + +Collectively O +, O +these O +data O +suggest O +that O +the O +pro B-protein_state +- I-protein_state +form I-protein_state +of O +PmC11 B-protein +is O +autoinhibited B-protein_state +by O +a O +section O +of O +L5 B-structure_element +blocking O +access O +to O +the O +active B-site +site I-site +, O +prior O +to O +intramolecular B-ptm +cleavage I-ptm +at O +Lys147 B-residue_name_number +. O + +This O +cleavage B-ptm +subsequently O +allows O +movement O +of O +the O +region O +containing O +Lys147 B-residue_name_number +and O +the O +active B-site +site I-site +to O +open B-protein_state +up O +for O +substrate O +access O +. O + +Substrate O +Specificity O +of O +PmC11 B-protein + +The O +autocatalytic B-ptm +cleavage I-ptm +of O +PmC11 B-protein +at O +Lys147 B-residue_name_number +( O +sequence O +KLK O +∧ O +A O +) O +demonstrates O +that O +the O +enzyme O +accepts O +substrates O +with O +Lys B-residue_name +in O +the O +P1 B-residue_number +position O +. O + +As O +expected O +, O +PmC11 B-protein +showed O +no O +activity O +against O +substrates O +with O +Pro B-residue_name +or O +Asp B-residue_name +in O +P1 B-residue_number +but O +was O +active B-protein_state +toward O +substrates O +with O +a O +basic O +residue O +in O +P1 B-residue_number +such O +as O +Bz B-chemical +- I-chemical +R I-chemical +- I-chemical +AMC I-chemical +, O +Z B-chemical +- I-chemical +GGR I-chemical +- I-chemical +AMC I-chemical +, O +and O +BOC B-chemical +- I-chemical +VLK I-chemical +- I-chemical +AMC I-chemical +. O + +The O +rate O +of O +cleavage O +was O +∼ O +3 O +- O +fold O +greater O +toward O +the O +single O +Arg B-residue_name +substrate O +Bz B-chemical +- I-chemical +R I-chemical +- I-chemical +AMC I-chemical +than O +for O +the O +other O +two O +( O +Fig O +. O +2F O +) O +and O +, O +unexpectedly O +, O +PmC11 B-protein +showed O +no O +activity O +toward O +BOC B-chemical +- I-chemical +K I-chemical +- I-chemical +AMC I-chemical +. O + +These O +results O +confirm O +that O +PmC11 B-protein +accepts O +substrates O +containing O +Arg B-residue_name +or O +Lys B-residue_name +in O +P1 B-residue_number +with O +a O +possible O +preference O +for O +Arg B-residue_name +. O + +The O +catalytic B-site +dyad I-site +of O +PmC11 B-protein +sits O +near O +the O +bottom O +of O +an O +open B-protein_state +pocket B-site +on O +the O +surface O +of O +the O +enzyme O +at O +a O +conserved B-protein_state +location I-protein_state +in O +the O +clan O +CD B-protein_type +family I-protein_type +. O + +The O +PmC11 B-protein +structure B-evidence +reveals O +that O +the O +catalytic B-site +dyad I-site +forms O +part O +of O +a O +large O +acidic B-site +pocket I-site +( O +Fig O +. O +2G O +), O +consistent O +with O +a O +binding B-site +site I-site +for O +a O +basic O +substrate O +. O + +This O +pocket B-site +is O +lined O +with O +the O +potential O +functional O +side O +chains O +of O +Asn50 B-residue_name_number +, O +Asp177 B-residue_name_number +, O +and O +Thr204 B-residue_name_number +with O +Gly134 B-residue_name_number +, O +Asp207 B-residue_name_number +, O +and O +Met205 B-residue_name_number +also O +contributing O +to O +the O +pocket B-site +( O +Fig O +. O +2A O +). O + +Interestingly O +, O +these O +residues O +are O +in O +regions O +that O +are O +structurally B-protein_state +similar I-protein_state +to O +those O +involved O +in O +the O +S1 B-site +binding I-site +pockets I-site +of O +other O +clan B-protein_type +CD I-protein_type +members I-protein_type +( O +shown O +in O +Ref O +.). O + +Because O +PmC11 B-protein +recognizes O +basic O +substrates O +, O +the O +tetrapeptide O +inhibitor O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +was O +tested O +as O +an O +enzyme O +inhibitor O +and O +was O +found O +to O +inhibit B-protein_state +both O +the O +autoprocessing B-ptm +and O +activity O +of O +PmC11 B-protein +( O +Fig O +. O +3A O +). O + +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +was O +also O +shown O +to O +bind O +to O +the O +enzyme O +: O +a O +size B-evidence +- I-evidence +shift I-evidence +was O +observed O +, O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +analysis O +, O +in O +the O +larger O +processed O +product O +of O +PmC11 B-protein +suggesting O +that O +the O +inhibitor B-protein_state +bound I-protein_state +to O +the O +active B-site +site I-site +( O +Fig O +. O +3B O +). O + +A O +structure B-experimental_method +overlay I-experimental_method +of O +PmC11 B-protein +with O +the O +MALT1 B-protein +- I-protein +paracacaspase I-protein +( O +MALT1 B-protein +- I-protein +P I-protein +), O +in O +complex B-protein_state +with O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +, O +revealed O +that O +the O +PmC11 B-protein +dyad B-site +sits O +in O +a O +very O +similar O +position O +to O +that O +of O +active B-protein_state +MALT1 B-protein +- I-protein +P I-protein +and O +that O +Asn50 B-residue_name_number +, O +Asp177 B-residue_name_number +, O +and O +Asp207 B-residue_name_number +superimpose O +well O +with O +the O +principal O +MALT1 B-protein +- I-protein +P I-protein +inhibitor B-site +binding I-site +residues I-site +( O +Asp365 B-residue_name_number +, O +Asp462 B-residue_name_number +, O +and O +Glu500 B-residue_name_number +, O +respectively O +( O +VRPR B-chemical +- I-chemical +FMK I-chemical +from O +MALT1 B-protein +- I-protein +P I-protein +with O +the O +corresponding O +PmC11 B-protein +residues O +from O +the O +structural B-experimental_method +overlay I-experimental_method +is O +shown O +in O +Fig O +. O +1D O +), O +as O +described O +in O +Ref O +.). O + +Asp177 B-residue_name_number +is O +located O +near O +the O +catalytic B-protein_state +cysteine B-residue_name +and O +is O +conserved B-protein_state +throughout I-protein_state +the O +C11 B-protein_type +family I-protein_type +, O +suggesting O +it O +is O +the O +primary O +S1 B-site +binding I-site +site I-site +residue I-site +. O + +In O +the O +structure B-evidence +of O +PmC11 B-protein +, O +Asp207 B-residue_name_number +resides O +on O +a O +flexible O +loop B-structure_element +pointing O +away O +from O +the O +S1 B-site +binding I-site +pocket I-site +( O +Fig O +. O +3C O +). O + +However O +, O +this O +loop B-structure_element +has O +been O +shown O +to O +be O +important O +for O +substrate O +binding O +in O +clan B-protein_type +CD I-protein_type +and O +this O +residue O +could O +easily O +rotate O +and O +be O +involved O +in O +substrate O +binding O +in O +PmC11 B-protein +. O + +Thus O +, O +Asn50 B-residue_name_number +, O +Asp177 B-residue_name_number +, O +and O +Asp207 B-residue_name_number +are O +most O +likely O +responsible O +for O +the O +substrate O +specificity O +of O +PmC11 B-protein +. O + +Asp177 B-residue_name_number +is O +highly B-protein_state +conserved I-protein_state +throughout O +the O +clan B-protein_type +CD I-protein_type +C11 I-protein_type +peptidases I-protein_type +and O +is O +thought O +to O +be O +primarily O +responsible O +for O +substrate O +specificity O +of O +the O +clan B-protein_type +CD I-protein_type +enzymes I-protein_type +, O +as O +also O +illustrated O +from O +the O +proximity O +of O +these O +residues O +relative O +to O +the O +inhibitor O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +when O +PmC11 B-protein +is O +overlaid B-experimental_method +on O +the O +MALT1 B-protein +- I-protein +P I-protein +structure B-evidence +( O +Fig O +. O +3C O +). O + +PmC11 B-protein +binds O +and O +is O +inhibited O +by O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +and O +does O +not O +require O +Ca2 B-chemical ++ I-chemical +for O +activity O +. O + +A O +, O +PmC11 O +activity O +is O +inhibited O +by O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +. O + +Cleavage O +of O +Bz B-chemical +- I-chemical +R I-chemical +- I-chemical +AMC I-chemical +by O +PmC11 B-protein +was O +measured O +in O +a O +fluorometric B-experimental_method +activity I-experimental_method +assay I-experimental_method +with O +(+, O +purple O +) O +and O +without O +(−, O +red O +) O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +. O + +B O +, O +gel B-experimental_method +- I-experimental_method +shift I-experimental_method +assay I-experimental_method +reveals O +that O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +binds O +to O +PmC11 B-protein +. O + +PmC11 O +was O +incubated B-experimental_method +with O +(+) O +or O +without O +(−) O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +and O +the O +samples O +analyzed O +on O +a O +10 O +% O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +gel O +. O + +A O +size B-evidence +shift I-evidence +can O +be O +observed O +in O +the O +larger O +processed O +product O +of O +PmC11 B-protein +( O +26 O +. O +1 O +kDa O +). O + +C O +, O +PmC11 B-protein +with O +the O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +from O +the O +MALT1 B-protein +- I-protein +paracacaspase I-protein +( O +MALT1 B-protein +- I-protein +P I-protein +) O +superimposed B-experimental_method +. O + +A O +three B-experimental_method +- I-experimental_method +dimensional I-experimental_method +structural I-experimental_method +overlay I-experimental_method +of O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +from O +the O +MALT1 B-protein +- I-protein +P I-protein +complex O +onto O +PmC11 B-protein +. O + +The O +position O +and O +orientation O +of O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +was O +taken O +from O +superposition B-experimental_method +of O +the O +PmC11 B-protein +and O +MALTI_P B-protein +structures B-evidence +and O +indicates O +the O +presumed O +active B-site +site I-site +of O +PmC11 B-protein +. O + +Residues O +surrounding O +the O +inhibitor O +are O +labeled O +and O +represent O +potentially O +important O +binding B-site +site I-site +residues I-site +, O +labeled O +in O +black O +and O +shown O +in O +an O +atomic O +representation O +. O + +C O +, O +divalent O +cations O +do O +not O +increase O +the O +activity O +of O +PmC11 B-protein +. O + +The O +cleavage O +of O +Bz B-chemical +- I-chemical +R I-chemical +- I-chemical +AMC I-chemical +by O +PmC11 B-protein +was O +measured O +in O +the O +presence O +of O +the O +cations O +Ca2 B-chemical ++, I-chemical +Mn2 B-chemical ++, I-chemical +Zn2 B-chemical ++, I-chemical +Co2 B-chemical ++, I-chemical +Cu2 B-chemical ++, I-chemical +Mg2 B-chemical ++, I-chemical +and O +Fe3 B-chemical ++ I-chemical +with O +EGTA B-chemical +as O +a O +negative O +control O +, O +and O +relative B-experimental_method +fluorescence I-experimental_method +measured I-experimental_method +against I-experimental_method +time I-experimental_method +( O +min O +). O + +The O +addition B-experimental_method +of I-experimental_method +cations I-experimental_method +produced O +no O +improvement O +in O +activity O +of O +PmC11 B-protein +when O +compared O +in O +the O +presence O +of O +EGTA B-chemical +, O +suggesting O +that O +PmC11 B-protein +does O +not O +require O +metal O +ions O +for O +proteolytic O +activity O +. O + +Furthermore O +, O +Cu2 B-chemical ++, I-chemical +Fe2 B-chemical ++, I-chemical +and O +Zn2 B-chemical ++ I-chemical +appear O +to O +inhibit B-protein_state +PmC11 B-protein +. O + +Comparison O +with O +Clostripain B-protein + +Clostripain B-protein +from O +C B-species +. I-species +histolyticum I-species +is O +the O +founding O +member O +of O +the O +C11 B-protein_type +family I-protein_type +of O +peptidases B-protein_type +and O +contains O +an O +additional O +149 B-residue_range +residues I-residue_range +compared O +with O +PmC11 B-protein +. O + +A O +multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +revealed O +that O +most O +of O +the O +secondary B-structure_element +structural I-structure_element +elements I-structure_element +are O +conserved B-protein_state +between O +the O +two O +enzymes O +, O +although O +they O +are O +only O +∼ O +23 O +% O +identical O +( O +Fig O +. O +1A O +). O + +Nevertheless O +, O +PmC11 B-protein +may O +be O +a O +good O +model O +for O +the O +core O +structure O +of O +clostripain B-protein +. O + +The O +primary B-experimental_method +structural I-experimental_method +alignment I-experimental_method +also O +shows O +that O +the O +catalytic B-site +dyad I-site +in O +PmC11 B-protein +is O +structurally B-protein_state +conserved I-protein_state +in O +clostripain B-protein +( O +Fig O +. O +1A O +). O + +Unlike O +PmC11 B-protein +, O +clostripain B-protein +has O +two O +cleavage B-site +sites I-site +( O +Arg181 B-residue_name_number +and O +Arg190 B-residue_name_number +), O +which O +results O +in O +the O +removal O +of O +a O +nonapeptide B-structure_element +, O +and O +is O +required O +for O +full B-protein_state +activation I-protein_state +of O +the O +enzyme O +( O +highlighted O +in O +Fig O +. O +1A O +). O + +Interestingly O +, O +Arg190 B-residue_name_number +was O +found O +to O +align O +with O +Lys147 B-residue_name_number +in O +PmC11 B-protein +. O + +In O +addition O +, O +the O +predicted O +primary O +S1 B-site +- I-site +binding I-site +residue I-site +in O +PmC11 B-protein +Asp177 B-residue_name_number +also O +overlays B-experimental_method +with O +the O +residue O +predicted O +to O +be O +the O +P1 B-site +specificity I-site +determining I-site +residue I-site +in O +clostripain B-protein +( O +Asp229 B-residue_name_number +, O +Fig O +. O +1A O +). O + +As O +studies O +on O +clostripain B-protein +revealed O +addition O +of O +Ca2 B-chemical ++ I-chemical +ions O +are O +required O +for O +full B-protein_state +activation I-protein_state +, O +the O +Ca2 B-chemical ++ I-chemical +dependence O +of O +PmC11 B-protein +was O +examined O +. O + +Surprisingly O +, O +Ca2 B-chemical ++ I-chemical +did O +not O +enhance O +PmC11 B-protein +activity O +and O +, O +furthermore O +, O +other O +divalent O +cations O +, O +Mg2 B-chemical ++, I-chemical +Mn2 B-chemical ++, I-chemical +Co2 B-chemical ++, I-chemical +Fe2 B-chemical ++, I-chemical +Zn2 B-chemical ++, I-chemical +and O +Cu2 B-chemical ++, I-chemical +were O +not O +necessary O +for O +PmC11 B-protein +activity O +( O +Fig O +. O +3D O +). O + +In O +support O +of O +these O +findings O +, O +EGTA B-chemical +did O +not O +inhibit O +PmC11 B-protein +suggesting O +that O +, O +unlike O +clostripain B-protein +, O +PmC11 B-protein +does O +not O +require O +Ca2 B-chemical ++ I-chemical +or O +other O +divalent O +cations O +, O +for O +activity O +. O + +The O +crystal B-evidence +structure I-evidence +of O +PmC11 B-protein +now O +provides O +three O +- O +dimensional O +information O +for O +a O +member O +of O +the O +clostripain B-protein +C11 B-protein_type +family I-protein_type +of O +cysteine B-protein_type +peptidases I-protein_type +. O + +The O +enzyme O +exhibits O +all O +of O +the O +key O +structural O +elements O +of O +clan B-protein_type +CD I-protein_type +members I-protein_type +, O +but O +is O +unusual O +in O +that O +it O +has O +a O +nine O +- O +stranded O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +with O +a O +novel O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +. O + +The O +structural O +similarity O +of O +PmC11 B-protein +with O +its O +nearest O +structural O +neighbors O +in O +the O +PDB O +is O +decidedly O +low O +, O +overlaying O +better O +with O +six O +- O +stranded O +caspase B-protein +- I-protein +7 I-protein +than O +any O +of O +the O +other O +larger O +members O +of O +the O +clan O +( O +Table O +2 O +). O + +The O +substrate O +specificity O +of O +PmC11 B-protein +is O +Arg B-residue_name +/ O +Lys B-residue_name +and O +the O +crystal B-evidence +structure I-evidence +revealed O +an O +acidic B-site +pocket I-site +for O +specific O +binding O +of O +such O +basic O +substrates O +. O + +In O +addition O +, O +the O +structure B-evidence +suggested O +a O +mechanism O +of O +self O +- O +inhibition O +in O +both O +PmC11 B-protein +and O +clostripain B-protein +and O +an O +activation O +mechanism O +that O +requires O +autoprocessing B-ptm +. O + +PmC11 B-protein +differs O +from O +clostripain B-protein +in O +that O +is O +does O +not O +appear O +to O +require O +divalent O +cations O +for O +activation O +. O + +Several O +other O +members O +of O +clan B-protein_type +CD I-protein_type +require O +processing B-ptm +for O +full B-protein_state +activation I-protein_state +including O +legumain B-protein +, O +gingipain B-protein +- I-protein +R I-protein +, O +MARTX B-protein +- I-protein +CPD I-protein +, O +and O +the O +effector B-protein_type +caspases I-protein_type +, O +e O +. O +g O +. O +caspase B-protein +- I-protein +7 I-protein +. O + +To O +date O +, O +the O +effector B-protein_type +caspases I-protein_type +are O +the O +only O +group O +of O +enzymes O +that O +require O +cleavage B-ptm +of O +a O +loop B-structure_element +within O +the O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +. O + +This O +is O +also O +the O +case O +in O +PmC11 B-protein +, O +although O +the O +cleavage B-ptm +loop B-structure_element +is O +structurally O +different O +to O +that O +found O +in O +the O +caspases B-protein_type +and O +follows O +the O +catalytic B-protein_state +His B-residue_name +( O +Fig O +. O +1A O +), O +as O +opposed O +to O +the O +Cys B-residue_name +in O +the O +caspases B-protein_type +. O + +All O +other O +clan B-protein_type +CD I-protein_type +members I-protein_type +requiring O +cleavage B-ptm +for O +full B-protein_state +activation I-protein_state +do O +so O +at O +sites B-site +external O +to O +their O +central O +sheets B-structure_element +. O + +The O +caspases B-protein_type +and O +gingipain B-protein +- I-protein +R I-protein +both O +undergo O +intermolecular B-ptm +( I-ptm +trans I-ptm +) I-ptm +cleavage I-ptm +and O +legumain B-protein +and O +MARTX B-protein +- I-protein +CPD I-protein +are O +reported O +to O +perform O +intramolecular B-ptm +( I-ptm +cis I-ptm +) I-ptm +cleavage I-ptm +. O + +In O +addition O +, O +several O +members O +of O +clan B-protein_type +CD I-protein_type +exhibit O +self O +- O +inhibition O +, O +whereby O +regions B-structure_element +of O +the O +enzyme O +block O +access O +to O +the O +active B-site +site I-site +. O + +Like O +PmC11 B-protein +, O +these O +structures O +show O +preformed O +catalytic O +machinery O +and O +, O +for O +a O +substrate O +to O +gain O +access O +, O +movement O +and O +/ O +or O +cleavage B-ptm +of O +the O +blocking B-structure_element +region I-structure_element +is O +required O +. O + +The O +structure B-evidence +of O +PmC11 B-protein +gives O +the O +first O +insight O +into O +this O +class O +of O +relatively O +unexplored O +family O +of O +proteins O +and O +should O +allow O +important O +catalytic O +and O +substrate O +binding O +residues O +to O +be O +identified O +in O +a O +variety O +of O +orthologues O +. O + +Indeed O +, O +insights O +gained O +from O +an O +analysis O +of O +the O +PmC11 B-protein +structure B-evidence +revealed O +the O +identity O +of O +the O +Trypanosoma B-species +brucei I-species +PNT1 B-protein +protein O +as O +a O +C11 B-protein_type +cysteine I-protein_type +peptidase I-protein_type +with O +an O +essential O +role O +in O +organelle O +replication O +. O + +The O +PmC11 B-protein +structure B-evidence +should O +provide O +a O +good O +basis O +for O +structural B-experimental_method +modeling I-experimental_method +and O +, O +given O +the O +importance O +of O +other O +clan B-protein_type +CD I-protein_type +enzymes I-protein_type +, O +this O +work O +should O +also O +advance O +the O +exploration O +of O +these O +peptidases B-protein_type +and O +potentially O +identify O +new O +biologically O +important O +substrates O +. O + +Ribosome B-protein_type +biogenesis I-protein_type +factor I-protein_type +Tsr3 B-protein +is O +the O +aminocarboxypropyl B-protein_type +transferase I-protein_type +responsible O +for O +18S B-chemical +rRNA I-chemical +hypermodification O +in O +yeast B-taxonomy_domain +and O +humans B-species + +The O +chemically O +most O +complex O +modification O +in O +eukaryotic B-taxonomy_domain +rRNA B-chemical +is O +the O +conserved B-protein_state +hypermodified B-protein_state +nucleotide B-chemical +N1 B-chemical +- I-chemical +methyl I-chemical +- I-chemical +N3 I-chemical +- I-chemical +aminocarboxypropyl I-chemical +- I-chemical +pseudouridine I-chemical +( O +m1acp3Ψ B-chemical +) O +located O +next O +to O +the O +P B-site +- I-site +site I-site +tRNA B-chemical +on O +the O +small O +subunit O +18S B-chemical +rRNA I-chemical +. O + +While O +S B-chemical +- I-chemical +adenosylmethionine I-chemical +was O +identified O +as O +the O +source O +of O +the O +aminocarboxypropyl B-chemical +( O +acp B-chemical +) O +group O +more O +than O +40 O +years O +ago O +the O +enzyme O +catalyzing O +the O +acp B-chemical +transfer O +remained O +elusive O +. O + +Here O +we O +identify O +the O +cytoplasmic O +ribosome O +biogenesis O +protein O +Tsr3 B-protein +as O +the O +responsible O +enzyme O +in O +yeast B-taxonomy_domain +and O +human B-species +cells O +. O + +In O +functionally O +impaired O +Tsr3 B-protein +- O +mutants B-protein_state +, O +a O +reduced O +level O +of O +acp B-chemical +modification O +directly O +correlates O +with O +increased O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +accumulation O +. O + +The O +crystal B-evidence +structure I-evidence +of O +archaeal B-taxonomy_domain +Tsr3 B-protein +homologs O +revealed O +the O +same O +fold O +as O +in O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +- I-protein_type +methyltransferases I-protein_type +but O +a O +distinct O +SAM B-site +binding I-site +mode I-site +. O + +This O +unique O +SAM B-site +binding I-site +mode I-site +explains O +why O +Tsr3 B-protein +transfers O +the O +acp B-chemical +and O +not O +the O +methyl O +group O +of O +SAM B-chemical +to O +its O +substrate O +. O + +Structurally O +, O +Tsr3 B-protein +therefore O +represents O +a O +novel O +class O +of O +acp B-protein_type +transferase I-protein_type +enzymes O +. O + +Eukaryotic B-taxonomy_domain +ribosome O +biogenesis O +is O +highly O +complex O +and O +requires O +a O +large O +number O +of O +non O +- O +ribosomal O +proteins O +and O +small B-chemical +non I-chemical +- I-chemical +coding I-chemical +RNAs I-chemical +in O +addition O +to O +ribosomal B-chemical +RNAs I-chemical +( O +rRNAs B-chemical +) O +and O +proteins O +. O + +During O +eukaryotic B-taxonomy_domain +ribosome O +biogenesis O +several O +dozens O +of O +rRNA B-chemical +nucleotides B-chemical +become O +chemically O +modified O +. O + +The O +most O +abundant O +rRNA B-chemical +modifications O +are O +methylations B-ptm +at O +the O +2 O +′- O +OH O +ribose B-chemical +moieties O +and O +isomerizations O +of O +uridine B-chemical +residues O +to O +pseudouridine B-chemical +, O +catalyzed O +by O +small B-complex_assembly +nucleolar I-complex_assembly +ribonucleoprotein I-complex_assembly +particles I-complex_assembly +( O +snoRNPs B-complex_assembly +). O + +In O +addition O +, O +18S B-chemical +and O +25S B-chemical +( O +yeast B-taxonomy_domain +)/ O +28S B-chemical +( O +humans B-species +) O +rRNAs B-chemical +contain O +several O +base O +modifications O +catalyzed O +by O +site O +- O +specific O +and O +snoRNA B-chemical +- O +independent O +enzymes O +. O + +In O +Saccharomyces B-species +cerevisiae I-species +18S B-chemical +rRNA I-chemical +contains O +four O +base O +methylations B-ptm +, O +two O +acetylations B-ptm +and O +a O +single O +3 B-chemical +- I-chemical +amino I-chemical +- I-chemical +3 I-chemical +- I-chemical +carboxypropyl I-chemical +( O +acp B-chemical +) O +modification O +, O +whereas O +six O +base O +methylations B-ptm +are O +present O +in O +the O +25S B-chemical +rRNA I-chemical +. O + +While O +in O +humans B-species +the O +18S B-chemical +rRNA I-chemical +base O +modifications O +are O +highly B-protein_state +conserved I-protein_state +, O +only O +three O +of O +the O +yeast B-taxonomy_domain +base O +modifications O +catalyzed O +by O +ScRrp8 B-protein +/ O +HsNML B-protein +, O +ScRcm1 B-protein +/ O +HsNSUN5 B-protein +and O +ScNop2 B-protein +/ O +HsNSUN1 B-protein +are O +preserved O +in O +the O +corresponding O +human B-species +28S B-chemical +rRNA I-chemical +. O + +Ribosomal B-chemical +RNA I-chemical +modifications O +have O +been O +suggested O +to O +optimize O +ribosome O +function O +, O +although O +in O +most O +cases O +this O +remains O +to O +be O +clearly O +established O +. O + +They O +might O +contribute O +to O +increased O +RNA B-chemical +stability O +by O +providing O +additional O +hydrogen B-bond_interaction +bonds I-bond_interaction +( O +pseudouridines B-chemical +), O +improved O +base B-bond_interaction +stacking I-bond_interaction +( O +pseudouridines B-chemical +and O +base B-ptm +methylations I-ptm +) O +or O +an O +increased O +resistance O +against O +hydrolysis O +( O +ribose B-ptm +methylations I-ptm +). O + +Most O +modified O +rRNA B-chemical +nucleotides B-chemical +cluster O +in O +the O +vicinity O +of O +the O +decoding B-site +or O +the O +peptidyl B-site +transferase I-site +center I-site +, O +suggesting O +an O +influence O +on O +ribosome O +functionality O +and O +stability O +. O + +Defects O +of O +rRNA B-chemical +modification O +enzymes O +often O +lead O +to O +disturbed O +ribosome O +biogenesis O +or O +functionally O +impaired O +ribosomes O +, O +although O +the O +lack O +of O +individual O +rRNA B-chemical +modifications O +often O +has O +no O +or O +only O +a O +slight O +influence O +on O +the O +cell O +. O + +The O +chemically O +most O +complex O +modification O +is O +located O +in O +the O +loop B-structure_element +capping I-structure_element +helix I-structure_element +31 I-structure_element +of O +18S B-chemical +rRNA I-chemical +( O +Supplementary O +Figure O +S1B O +). O + +There O +a O +uridine B-residue_name +( O +U1191 B-residue_name_number +in O +yeast B-taxonomy_domain +) O +is O +modified O +to O +1 B-chemical +- I-chemical +methyl I-chemical +- I-chemical +3 I-chemical +-( I-chemical +3 I-chemical +- I-chemical +amino I-chemical +- I-chemical +3 I-chemical +- I-chemical +carboxypropyl I-chemical +)- I-chemical +pseudouridine I-chemical +( O +m1acp3Ψ B-chemical +, O +Figure O +1A O +). O + +This O +base O +modification O +was O +first O +described O +in O +1968 O +for O +hamster B-taxonomy_domain +cells O +and O +is O +conserved B-protein_state +in I-protein_state +eukaryotes B-taxonomy_domain +. O + +This O +hypermodified B-protein_state +nucleotide B-chemical +, O +which O +is O +located O +at O +the O +P B-site +- I-site +site I-site +tRNA B-chemical +, O +is O +synthesized O +in O +three O +steps O +beginning O +with O +the O +snR35 B-chemical +H B-structure_element +/ I-structure_element +ACA I-structure_element +snoRNP B-complex_assembly +guided O +conversion O +of O +uridine B-chemical +into O +pseudouridine B-chemical +. O + +In O +a O +second O +step O +, O +the O +essential O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +methyltransferase I-protein_type +Nep1 B-protein +/ O +Emg1 B-protein +modifies O +the O +pseudouridine B-chemical +to O +N1 B-chemical +- I-chemical +methylpseudouridine I-chemical +. O + +Methylation B-ptm +can O +only O +occur O +once O +pseudouridylation B-ptm +has O +taken O +place O +, O +as O +the O +latter O +reaction O +generates O +the O +substrate O +for O +the O +former O +. O + +The O +final O +acp B-chemical +modification O +leading O +to O +N1 B-chemical +- I-chemical +methyl I-chemical +- I-chemical +N3 I-chemical +- I-chemical +aminocarboxypropyl I-chemical +- I-chemical +pseudouridine I-chemical +occurs O +late O +during O +40S B-complex_assembly +biogenesis O +in O +the O +cytoplasm O +, O +while O +the O +two O +former O +reactions O +are O +taking O +place O +in O +the O +nucleolus O +and O +nucleus O +, O +and O +is O +independent O +from O +pseudouridylation B-ptm +or O +methylation O +. O + +Both O +the O +methyl O +and O +the O +acp O +group O +are O +derived O +from O +S B-chemical +- I-chemical +adenosylmethionine I-chemical +( O +SAM B-chemical +), O +but O +the O +enzyme O +responsible O +for O +acp B-chemical +modification O +remained O +elusive O +for O +more O +than O +40 O +years O +. O + +Tsr3 B-protein +is O +necessary O +for O +acp B-chemical +modification O +of O +18S B-chemical +rRNA I-chemical +in O +yeast B-taxonomy_domain +and O +human B-species +. O +( O +A O +) O +Hypermodified B-protein_state +nucleotide B-chemical +m1acp3Ψ B-chemical +is O +synthesized O +in O +three O +steps O +: O +pseudouridylation B-ptm +catalyzed O +by O +snoRNP35 B-complex_assembly +, O +N1 B-ptm +- I-ptm +methylation I-ptm +catalyzed O +by O +methyltransferase B-protein_type +Nep1 B-protein +and O +N3 O +- O +acp B-chemical +modification O +catalyzed O +by O +Tsr3 B-protein +. O + +The O +asterisk O +indicates O +the O +C1 O +- O +atom O +labeled O +in O +the O +14C B-experimental_method +- I-experimental_method +incorporation I-experimental_method +assay I-experimental_method +. O + +( O +B O +) O +RP B-experimental_method +- I-experimental_method +HPLC I-experimental_method +elution B-evidence +profile I-evidence +of O +yeast B-taxonomy_domain +18S B-chemical +rRNA I-chemical +nucleosides B-chemical +. O + +Hypermodified B-protein_state +m1acp3Ψ B-chemical +elutes O +at O +7 O +. O +4 O +min O +( O +wild B-protein_state +type I-protein_state +, O +left O +profile O +) O +and O +is O +missing O +in O +Δtsr3 B-mutant +( O +middle O +profile O +) O +and O +Δnep1 B-mutant +Δnop6 I-mutant +mutants O +( O +right O +profile O +). O + +( O +C O +) O +14C B-chemical +- I-chemical +acp I-chemical +labeling O +of O +18S B-chemical +rRNAs I-chemical +. O + +Wild B-protein_state +type I-protein_state +( O +WT B-protein_state +) O +and O +plasmid O +encoded O +18S B-chemical +rRNA I-chemical +( O +U1191U B-mutant +) O +show O +the O +14C B-chemical +- I-chemical +acp I-chemical +signal O +, O +whereas O +the O +14C B-chemical +- I-chemical +acp I-chemical +signal O +is O +missing O +in O +the O +U1191A B-mutant +mutant B-protein_state +plasmid O +encoded O +18S B-chemical +rRNA I-chemical +( O +U1191A B-mutant +) O +and O +Δtsr3 B-mutant +mutants O +( O +Δtsr3 B-mutant +). O + +Upper O +lanes O +show O +the O +ethidium B-chemical +bromide I-chemical +staining O +of O +the O +18S B-chemical +rRNAs I-chemical +for O +quantification O +. O + +All O +samples O +were O +loaded O +on O +the O +gel O +with O +two O +different O +amounts O +of O +5 O +and O +10 O +μl O +. O +( O +D O +) O +Primer B-experimental_method +extension I-experimental_method +analysis I-experimental_method +of O +acp B-chemical +modification O +in O +yeast B-taxonomy_domain +18S B-chemical +rRNA I-chemical +( O +right O +gel O +) O +including O +a O +sequencing O +ladder O +( O +left O +gel O +). O + +The O +primer O +extension O +stop O +at O +nucleotide O +1191 B-residue_number +is O +missing O +exclusively O +in O +Δtsr3 B-mutant +mutants O +and O +Δtsr3 B-mutant +Δsnr35 I-mutant +recombinants O +. O + +( O +E O +) O +Primer B-experimental_method +extension I-experimental_method +analysis I-experimental_method +of O +human B-species +18S B-chemical +rRNA I-chemical +after O +siRNA B-experimental_method +knockdown I-experimental_method +of O +HsNEP1 B-protein +/ O +EMG1 B-protein +( O +541 O +, O +542 O +and O +543 O +) O +and O +HsTSR3 B-protein +( O +544 O +and O +545 O +) O +( O +right O +gel O +), O +including O +a O +sequencing O +ladder O +( O +left O +gel O +). O + +The O +primer O +extension O +arrest O +is O +reduced O +in O +HTC116 O +cells O +transfected O +with O +siRNAs B-chemical +544 O +and O +545 O +. O + +The O +efficiency O +of O +siRNA B-chemical +mediated O +HsTSR3 B-protein +repression O +correlates O +with O +the O +primer B-evidence +extension I-evidence +signals I-evidence +( O +see O +Supplementary O +Figure O +S2A O +). O + +Only O +a O +few O +acp B-chemical +transferring O +enzymes O +have O +been O +characterized O +until O +now O +. O + +During O +the O +biosynthesis O +of O +wybutosine B-chemical +, O +a O +tricyclic O +nucleoside B-chemical +present O +in O +eukaryotic B-taxonomy_domain +and O +archaeal B-taxonomy_domain +phenylalanine B-chemical +tRNA B-chemical +, O +Tyw2 B-protein +( O +Trm12 B-protein +in O +yeast B-taxonomy_domain +) O +transfers O +an O +acp B-chemical +group O +from O +SAM B-chemical +to O +an O +acidic O +carbon O +atom O +. O + +Archaeal B-taxonomy_domain +Tyw2 B-protein +has O +a O +structure B-evidence +very O +similar O +to O +Rossmann B-protein_type +- I-protein_type +fold I-protein_type +( I-protein_type +class I-protein_type +I I-protein_type +) I-protein_type +RNA I-protein_type +- I-protein_type +methyltransferases I-protein_type +, O +but O +its O +distinctive O +SAM B-site +- I-site +binding I-site +mode I-site +enables O +the O +transfer O +of O +the O +acp B-chemical +group O +instead O +of O +the O +methyl O +group O +of O +the O +cofactor O +. O + +Another O +acp B-chemical +modification O +has O +been O +described O +in O +the O +diphtamide B-chemical +biosynthesis O +pathway O +, O +where O +an O +acp B-chemical +group O +is O +transferred O +from O +SAM B-chemical +to O +the O +carbon O +atom O +of O +a O +histidine B-residue_name +residue O +of O +eukaryotic B-taxonomy_domain +translation B-protein_type +elongation I-protein_type +factor I-protein_type +2 I-protein_type +by O +use O +of O +a O +radical O +mechanism O +. O + +In O +a O +recent O +bioinformatic O +study O +, O +the O +uncharacterized O +yeast B-taxonomy_domain +gene O +YOR006c B-gene +was O +predicted O +to O +be O +involved O +in O +ribosome O +biogenesis O +. O + +It O +is O +highly B-protein_state +conserved I-protein_state +among O +eukaryotes B-taxonomy_domain +and O +archaea B-taxonomy_domain +( O +Supplementary O +Figure O +S1A O +) O +and O +its O +deletion O +leads O +to O +an O +accumulation O +of O +the O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +precursor O +of O +18S B-chemical +rRNA I-chemical +, O +suggesting O +an O +influence O +on O +D B-site +- I-site +site I-site +cleavage O +during O +the O +maturation O +of O +the O +small O +ribosomal O +subunit O +. O + +On O +this O +basis O +, O +YOR006C B-gene +was O +renamed O +‘ O +Twenty B-protein +S I-protein +rRNA I-protein +accumulation I-protein +3 I-protein +′ O +( O +TSR3 B-protein +). O + +However O +, O +its O +function O +remained O +unclear O +although O +recently O +a O +putative O +nuclease O +function O +during O +18S B-chemical +rRNA I-chemical +maturation O +was O +predicted O +. O + +Here O +, O +we O +identify O +Tsr3 B-protein +as O +the O +long O +- O +sought O +acp B-protein_type +transferase I-protein_type +that O +catalyzes O +the O +last O +step O +in O +the O +biosynthesis O +of O +the O +hypermodified B-protein_state +nucleotide B-chemical +m1acp3Ψ B-chemical +in O +yeast B-taxonomy_domain +and O +human B-species +cells O +. O + +Furthermore O +using O +catalytically B-protein_state +defective I-protein_state +mutants O +of O +yeast B-taxonomy_domain +Tsr3 B-protein +we O +demonstrated O +that O +the O +acp B-chemical +modification O +is O +required O +for O +18S B-chemical +rRNA I-chemical +maturation O +. O + +Surprisingly O +, O +the O +crystal B-evidence +structures I-evidence +of O +archaeal B-taxonomy_domain +homologs O +revealed O +that O +Tsr3 B-protein +is O +structurally O +similar O +to O +the O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +methyltransferases I-protein_type +. O + +In O +contrast O +, O +the O +only O +other O +structurally O +characterized O +acp B-protein_type +transferase I-protein_type +enzyme O +Tyw2 B-protein +belongs O +to O +the O +Rossmann B-protein_type +- I-protein_type +fold I-protein_type +class I-protein_type +of I-protein_type +methyltransferase I-protein_type +proteins I-protein_type +. O + +Interestingly O +, O +the O +two O +structurally O +very O +different O +enzymes O +use O +similar O +strategies O +in O +binding O +the O +SAM B-chemical +- O +cofactor O +in O +order O +to O +ensure O +that O +in O +contrast O +to O +methyltransferases B-protein_type +the O +acp B-chemical +and O +not O +the O +methyl O +group O +of O +SAM B-chemical +is O +transferred O +to O +the O +substrate O +. O + +Tsr3 B-protein +is O +the O +enzyme O +responsible O +for O +18S B-chemical +rRNA I-chemical +acp B-chemical +modification O +in O +yeast B-taxonomy_domain +and O +humans B-species + +The O +S B-species +. I-species +cerevisiae I-species +18S B-protein_type +rRNA I-protein_type +acp I-protein_type +transferase I-protein_type +was O +identified O +in O +a O +systematic O +genetic O +screen O +where O +numerous O +deletion O +mutants O +from O +the O +EUROSCARF O +strain O +collection O +( O +www O +. O +euroscarf O +. O +de O +) O +were O +analyzed O +by O +HPLC B-experimental_method +for O +alterations O +in O +18S B-chemical +rRNA I-chemical +base O +modifications O +. O + +For O +the O +Δtsr3 B-mutant +deletion O +strain O +the O +HPLC B-evidence +elution I-evidence +profile I-evidence +of O +18S B-chemical +rRNA I-chemical +nucleosides B-chemical +( O +Figure O +1B O +) O +was O +very O +similar O +to O +that O +of O +the O +pseudouridine B-protein_type +- I-protein_type +N1 I-protein_type +methyltransferase I-protein_type +mutant B-protein_state +Δnep1 B-mutant +, O +where O +a O +shoulder O +at O +∼ O +7 O +. O +4 O +min O +elution O +time O +was O +missing O +in O +the O +elution O +profile O +. O + +As O +previously O +reported O +this O +shoulder O +was O +identified O +by O +ESI B-experimental_method +- I-experimental_method +MS I-experimental_method +as O +corresponding O +to O +m1acp3Ψ B-chemical +. O + +In O +order O +to O +directly O +analyze O +the O +presence O +of O +the O +acp B-chemical +modification O +of O +nucleotide B-chemical +1191 B-residue_number +we O +used O +an O +in B-experimental_method +vivo14C I-experimental_method +incorporation I-experimental_method +assay I-experimental_method +with O +1 B-chemical +- I-chemical +14C I-chemical +- I-chemical +methionine I-chemical +. O + +Whereas O +the O +acp B-chemical +labeling O +of O +18S B-chemical +rRNA I-chemical +was O +clearly O +present O +in O +the O +wild B-protein_state +type I-protein_state +strain O +no O +radioactive O +labeling O +could O +be O +observed O +in O +a O +Δtsr3 B-mutant +strain O +( O +Figure O +1C O +). O + +No O +radioactive O +labeling O +was O +detected O +in O +the O +18S B-mutant +U1191A I-mutant +mutant B-protein_state +which O +served O +as O +a O +control O +for O +the O +specificity O +of O +the O +14C B-chemical +- I-chemical +aminocarboxypropyl I-chemical +incorporation O +. O + +As O +previously O +shown O +, O +only O +the O +acp B-chemical +but O +none O +of O +the O +other O +modifications O +at O +U1191 B-residue_name_number +of O +yeast B-taxonomy_domain +18S B-chemical +rRNA I-chemical +blocks O +reverse O +transcriptase O +activity O +. O + +Therefore O +the O +presence O +of O +the O +acp B-chemical +modification O +can O +be O +directly O +assessed O +by O +primer B-experimental_method +extension I-experimental_method +. O + +Indeed O +, O +in O +wild B-protein_state +- I-protein_state +type I-protein_state +yeast B-taxonomy_domain +a O +strong O +primer B-evidence +extension I-evidence +stop I-evidence +signal I-evidence +occurred O +at O +position O +1192 B-residue_number +. O + +In O +contrast O +, O +in O +a O +Δtsr3 B-mutant +mutant B-protein_state +no O +primer O +extension O +stop O +signal O +was O +present O +at O +this O +position O +. O + +As O +expected O +, O +in O +a O +Δsnr35 B-mutant +deletion B-experimental_method +preventing O +pseudouridylation B-ptm +and O +N1 B-ptm +- I-ptm +methylation I-ptm +( O +resulting O +in O +acp3U B-chemical +) O +as O +well O +as O +in O +a O +Δnep1 B-mutant +deletion O +strain O +where O +pseudouridine B-chemical +is O +not B-protein_state +methylated I-protein_state +( O +resulting O +in O +acp3Ψ B-chemical +) O +a O +primer B-evidence +extension I-evidence +stop I-evidence +signal I-evidence +of O +similar O +intensity O +as O +in O +the O +wild B-protein_state +type I-protein_state +was O +observed O +. O + +In O +a O +Δtsr3 B-mutant +Δsnr35 I-mutant +double O +deletion O +strain O +the O +18S B-chemical +rRNA I-chemical +contains O +an O +unmodified B-protein_state +U B-chemical +and O +the O +primer O +extension O +stop O +signal O +was O +missing O +( O +Figure O +1D O +). O + +The O +Tsr3 B-protein +protein O +is O +highly B-protein_state +conserved I-protein_state +in O +yeast B-taxonomy_domain +and O +humans B-species +( O +50 O +% O +identity O +). O + +Human B-species +18S B-chemical +rRNA I-chemical +has O +also O +been O +shown O +to O +contain O +m1acp3Ψ B-ptm +in O +the O +18S B-chemical +rRNA I-chemical +at O +position O +1248 B-residue_number +. O + +After O +siRNA B-experimental_method +- I-experimental_method +mediated I-experimental_method +depletion I-experimental_method +of O +Tsr3 B-protein +in O +human B-species +colon O +carcinoma O +HCT116 O +(+/+) O +cells O +the O +acp B-evidence +primer I-evidence +extension I-evidence +arrest I-evidence +was O +reduced O +in O +comparison O +to O +cells O +transfected O +with O +a O +non O +- O +targeting O +scramble O +siRNA B-chemical +control O +( O +Figure O +1E O +, O +compare O +lanes O +544 O +and O +scramble O +). O + +The O +efficiency O +of O +siRNA B-chemical +- O +mediated O +depletion O +was O +established O +by O +RT B-experimental_method +- I-experimental_method +qPCR I-experimental_method +and O +found O +to O +be O +very O +high O +with O +siRNA B-chemical +544 O +( O +Supplementary O +Figure O +S2A O +, O +remaining O +TSR3 B-protein +mRNA O +level O +of O +2 O +%). O + +By O +comparison O +, O +treating O +cells O +with O +siRNA B-chemical +545 O +, O +which O +only O +reduced O +the O +TSR3 B-protein +mRNA O +to O +20 O +%, O +did O +not O +markedly O +reduced O +the O +acp B-chemical +signal O +. O + +This O +suggests O +that O +low O +residual O +levels O +of O +HsTsr3 B-protein +are O +sufficient O +to O +modify O +the O +RNA B-chemical +. O + +Thus O +, O +HsTsr3 B-protein +is O +also O +responsible O +for O +the O +acp B-chemical +modification O +of O +18S B-chemical +rRNA I-chemical +nucleotide B-chemical +Ψ1248 B-ptm +in O +helix B-structure_element +31 I-structure_element +. O + +Similar O +to O +yeast B-taxonomy_domain +, O +siRNA B-experimental_method +- I-experimental_method +mediated I-experimental_method +depletion I-experimental_method +of O +the O +Ψ1248 B-protein_type +N1 I-protein_type +- I-protein_type +methyltransferase I-protein_type +Nep1 B-protein +/ O +Emg1 B-protein +had O +no O +influence O +on O +the O +primer B-evidence +extension I-evidence +arrest I-evidence +( O +Figure O +1E O +). O + +Phenotypic O +characterization O +of O +Δtsr3 B-mutant +mutants O + +Although O +the O +acp B-chemical +modification O +of O +18S B-chemical +rRNA I-chemical +is O +highly B-protein_state +conserved I-protein_state +in O +eukaryotes B-taxonomy_domain +, O +yeast B-taxonomy_domain +Δtsr3 B-mutant +mutants O +showed O +only O +a O +minor O +growth O +defect O +. O + +However O +, O +the O +Δtsr3 B-mutant +deletion O +was O +synthetic O +sick O +with O +a O +Δsnr35 B-mutant +deletion O +preventing O +pseudouridylation B-ptm +and O +Nep1 B-protein +- O +catalyzed O +methylation O +of O +nucleotide O +1191 B-residue_number +( O +Figure O +2A O +). O + +Interestingly O +, O +no O +increased O +growth O +defect O +could O +be O +observed O +for O +Δtsr3 B-mutant +Δnep1 I-mutant +recombinants O +containing O +the O +nep1 B-gene +suppressor O +mutation O +Δnop6 B-mutant +as O +well O +as O +for O +Δtsr3 B-mutant +Δsnr35 I-mutant +Δnep1 I-mutant +recombinants O +with O +unmodified B-protein_state +U1191 B-residue_name_number +( O +Supplementary O +Figure O +S2D O +and O +E O +). O + +Phenotypic O +characterization O +of O +yeast B-taxonomy_domain +TSR3 B-protein +deletion O +( O +Δtrs3 B-mutant +) O +and O +human B-species +TSR3 B-protein +depletion O +( O +siRNAs B-chemical +544 O +and O +545 O +) O +and O +cellular O +localization O +of O +yeast B-taxonomy_domain +Tsr3 B-protein +. O +( O +A O +) O +Growth O +of O +yeast B-taxonomy_domain +wild B-protein_state +type I-protein_state +, O +Δtsr3 B-mutant +, O +Δsnr35 B-mutant +and O +Δtsr3 B-mutant +Δsnr35 I-mutant +segregants O +after O +meiosis O +and O +tetrad O +dissection O +of O +Δtsr3 B-mutant +/ O +TSR3 B-protein +Δsnr35 B-mutant +/ O +SNR35 B-protein +heterozygous O +diploids O +. O + +The O +Δtsr3 B-mutant +deletion O +is O +synthetic O +sick O +with O +a O +Δsnr35 B-mutant +deletion O +preventing O +U1191 B-residue_name_number +pseudouridylation O +. O + +( O +B O +) O +In O +agar B-experimental_method +diffusion I-experimental_method +assays I-experimental_method +the O +yeast B-taxonomy_domain +Δtsr3 B-mutant +deletion B-protein_state +mutant I-protein_state +shows O +a O +hypersensitivity O +against O +paromomycin B-chemical +and O +hygromycin B-chemical +B I-chemical +which O +is O +further O +increased O +by O +recombination O +with O +Δsnr35 B-mutant +. O +( O +C O +) O +Northern B-experimental_method +blot I-experimental_method +analysis I-experimental_method +with O +an O +ITS1 O +hybridization O +probe O +after O +siRNA B-experimental_method +depletion I-experimental_method +of O +HsTSR3 B-protein +( O +siRNAs B-chemical +544 O +and O +545 O +) O +and O +a O +scrambled O +siRNA B-chemical +as O +control O +. O + +The O +accumulation O +of O +18SE B-chemical +and O +47S B-chemical +and O +/ O +or O +45S B-chemical +pre I-chemical +- I-chemical +RNAs I-chemical +is O +enforced O +upon O +HsTSR3 B-protein +depletion O +. O + +Right O +gel O +: O +Ethidium O +bromide O +staining O +showing O +18S B-chemical +and O +28S B-chemical +rRNAs I-chemical +. O + +( O +D O +) O +Cytoplasmic O +localization O +of O +yeast B-taxonomy_domain +Tsr3 B-protein +shown O +by O +fluorescence B-experimental_method +microscopy I-experimental_method +of O +GFP B-mutant +- I-mutant +fused I-mutant +Tsr3 I-mutant +. O + +From O +left O +to O +right O +: O +differential B-experimental_method +interference I-experimental_method +contrast I-experimental_method +( O +DIC B-experimental_method +), O +green O +fluorescence O +of O +GFP B-mutant +- I-mutant +Tsr3 I-mutant +, O +red O +fluorescence O +of O +Nop56 B-mutant +- I-mutant +mRFP I-mutant +as O +nucleolar O +marker O +, O +and O +merge O +of O +GFP B-mutant +- I-mutant +Tsr3 I-mutant +/ O +Nop56 B-mutant +- I-mutant +mRFP I-mutant +with O +DIC B-experimental_method +. O +( O +E O +) O +Elution B-evidence +profile I-evidence +( O +A254 O +) O +after O +sucrose B-experimental_method +gradient I-experimental_method +separation I-experimental_method +of O +yeast B-taxonomy_domain +ribosomal B-complex_assembly +subunits I-complex_assembly +and O +polysomes B-complex_assembly +( O +upper O +part O +) O +and O +western B-experimental_method +blot I-experimental_method +analysis O +of O +3xHA B-chemical +tagged O +Tsr3 B-protein +( O +Tsr3 B-mutant +- I-mutant +3xHA I-mutant +) O +after O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +separation O +of O +polysome O +profile O +fractions O +taken O +every O +20 O +s O +( O +lower O +part O +). O + +The O +TSR3 B-protein +gene O +was O +genetically O +modified O +at O +its O +native O +locus O +, O +resulting O +in O +a O +C O +- O +terminal O +fusion B-protein_state +of O +Tsr3 B-protein +with O +a O +3xHA B-chemical +epitope O +expressed O +by O +the O +native O +promotor O +in O +yeast B-taxonomy_domain +strain O +CEN O +. O +BM258 O +- O +5B O +. O + +The O +influence O +of O +the O +acp B-chemical +modification O +of O +nucleotide B-chemical +1191 B-residue_number +on O +ribosome O +function O +was O +analyzed O +by O +treating O +Δtsr3 B-mutant +mutants O +with O +protein O +synthesis O +inhibitors O +. O + +Similar O +to O +a O +temperature O +- O +sensitive O +nep1 B-gene +mutant B-protein_state +, O +the O +Δtsr3 B-mutant +deletion O +caused O +hypersensitivity O +to O +paromomycin B-chemical +and O +, O +to O +a O +lesser O +extent O +, O +to O +hygromycin B-chemical +B I-chemical +( O +Figure O +2B O +), O +but O +not O +to O +G418 B-chemical +or O +cycloheximide B-chemical +( O +data O +not O +shown O +). O + +In O +accordance O +with O +the O +synthetic O +sick O +growth O +phenotype O +the O +paromomycin B-chemical +and O +hygromycin B-chemical +B I-chemical +hypersensitivity O +further O +increased O +in O +a O +Δtsr3 B-mutant +Δsnr35 I-mutant +recombination O +strain O +( O +Figure O +2B O +). O + +In O +a O +yeast B-taxonomy_domain +Δtsr3 B-mutant +strain O +as O +well O +as O +in O +the O +Δtsr3 B-mutant +Δsnr35 I-mutant +recombinant O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +accumulated O +significantly O +and O +the O +level O +of O +mature O +18S B-chemical +rRNA I-chemical +was O +reduced O +( O +Supplementary O +Figures O +S2C O +and O +S3D O +), O +as O +reported O +previously O +. O + +A O +minor O +effect O +on O +20S B-chemical +rRNA I-chemical +accumulation O +was O +also O +observed O +for O +Δsnr35 B-mutant +, O +but O +- O +probably O +due O +to O +different O +strain O +backgrounds O +– O +to O +a O +weaker O +extent O +than O +described O +earlier O +. O + +In O +human B-species +cells O +, O +the O +depletion B-experimental_method +of I-experimental_method +HsTsr3 B-protein +in O +HCT116 O +(+/+) O +cells O +caused O +an O +accumulation O +of O +the O +human B-species +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +equivalent O +18S B-chemical +- I-chemical +E I-chemical +suggesting O +an O +evolutionary O +conserved O +role O +of O +Tsr3 B-protein +in O +the O +late O +steps O +of O +18S B-chemical +rRNA I-chemical +processing O +( O +Figure O +2C O +and O +Supplementary O +Figure O +S2B O +). O + +Surprisingly O +, O +early O +nucleolar O +processing O +reactions O +were O +also O +inhibited O +, O +and O +this O +was O +observed O +in O +both O +yeast B-taxonomy_domain +Δtsr3 B-mutant +cells O +( O +see O +accumulation O +of O +35S B-complex_assembly +in O +Supplementary O +Figure O +S2C O +) O +and O +Tsr3 B-protein +depleted O +human B-species +cells O +( O +see O +47S B-complex_assembly +/ O +45S B-complex_assembly +accumulation O +in O +Figure O +2C O +and O +Northern B-experimental_method +blot I-experimental_method +quantification O +in O +Supplementary O +Figure O +S2B O +). O + +Consistent O +with O +its O +role O +in O +late O +18S B-chemical +rRNA I-chemical +processing O +, O +TSR3 B-protein +deletion O +leads O +to O +a O +ribosomal O +subunit O +imbalance O +with O +a O +reduced O +40S B-complex_assembly +to O +60S B-complex_assembly +ratio O +of O +0 O +. O +81 O +( O +σ O += O +0 O +. O +024 O +) O +which O +was O +further O +increased O +in O +a O +Δtsr3 B-mutant +Δsnr35 I-mutant +recombinant O +to O +0 O +. O +73 O +( O +σ O += O +0 O +. O +023 O +) O +( O +Supplementary O +Figure O +S2F O +). O + +In O +polysome B-evidence +profiles I-evidence +, O +a O +reduced O +level O +of O +80S B-complex_assembly +ribosomes I-complex_assembly +and O +a O +strong O +signal O +for O +free O +60S B-complex_assembly +subunits O +was O +observed O +in O +line O +with O +the O +40S B-complex_assembly +subunit O +deficiency O +( O +Supplementary O +Figure O +S2G O +). O + +Cellular O +localization O +of O +Tsr3 B-protein +in O +S B-species +. I-species +cerevisiae I-species + +Fluorescence B-experimental_method +microscopy I-experimental_method +of O +GFP B-protein_state +- I-protein_state +tagged I-protein_state +Tsr3 B-protein +localized O +the O +fusion O +protein O +in O +the O +cytoplasm O +of O +yeast B-taxonomy_domain +cells O +and O +no O +co O +- O +localization O +with O +the O +nucleolar O +marker O +protein O +Nop56 B-protein +could O +be O +observed O +( O +Figure O +2D O +). O + +This O +agrees O +with O +previous O +biochemical O +data O +suggesting O +that O +the O +acp B-chemical +modification O +of O +18S B-chemical +rRNA I-chemical +occurs O +late O +during O +40S B-complex_assembly +subunit O +biogenesis O +in O +the O +cytoplasm O +, O +and O +makes O +an O +additional O +nuclear O +localization O +as O +reported O +in O +a O +previous O +large O +- O +scale O +analysis O +unlikely O +. O + +After O +polysome B-experimental_method +gradient I-experimental_method +separation I-experimental_method +C O +- O +terminally O +epitope O +- O +labeled O +Tsr3 B-mutant +- I-mutant +3xHA I-mutant +was O +exclusively O +detectable O +in O +the O +low O +- O +density O +fraction O +( O +Figure O +2E O +). O + +Such O +distribution B-evidence +on I-evidence +a I-evidence +density I-evidence +gradient I-evidence +suggests O +that O +Tsr3 B-protein +only O +interacts O +transiently O +with O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +subunits I-complex_assembly +, O +which O +presumably O +explains O +why O +it O +was O +not O +characterized O +in O +pre B-experimental_method +- I-experimental_method +ribosome I-experimental_method +affinity I-experimental_method +purifications I-experimental_method +. O + +Structure B-evidence +of O +Tsr3 B-protein + +Searches O +for O +sequence O +homologs O +of O +S B-species +. I-species +cerevisiae I-species +Tsr3 B-protein +( O +ScTsr3 B-protein +) O +by O +us O +and O +others O +revealed O +that O +the O +genomes O +of O +many O +archaea B-taxonomy_domain +contain O +genes O +encoding O +Tsr3 B-protein_type +- I-protein_type +like I-protein_type +proteins I-protein_type +. O + +However O +, O +these O +archaeal B-taxonomy_domain +homologs O +are O +significantly O +smaller O +than O +ScTsr3 B-protein +(∼ O +190 O +aa O +in O +archaea B-taxonomy_domain +vs O +. O +313 O +aa O +in O +yeast B-taxonomy_domain +) O +due O +to O +shortened O +N O +- O +and O +C O +- O +termini O +( O +Supplementary O +Figure O +S1A O +). O + +To O +locate O +the O +domains O +most O +important O +for O +Tsr3 B-protein +activity O +, O +ScTsr3 B-protein +fragments O +of O +different O +lengths O +containing O +the O +highly B-protein_state +conserved I-protein_state +central O +part O +were O +expressed B-experimental_method +in O +a O +Δtsr3 B-mutant +mutant B-protein_state +( O +Figure O +3A O +) O +and O +analyzed O +by O +primer B-experimental_method +extension I-experimental_method +( O +Figure O +3B O +) O +and O +Northern B-experimental_method +blotting I-experimental_method +( O +Figure O +3C O +). O + +N O +- O +terminal O +truncations B-experimental_method +of O +up O +to O +45 B-residue_range +aa I-residue_range +and O +C O +- O +terminal O +truncations B-experimental_method +of O +up O +to O +76 B-residue_range +aa I-residue_range +mediated O +acp B-chemical +modification O +as O +efficiently O +as O +the O +full B-protein_state +- I-protein_state +length I-protein_state +protein O +and O +no O +significant O +increased O +levels O +of O +20S B-chemical +pre I-chemical +- I-chemical +RNA I-chemical +were O +detected O +. O + +Even O +a O +Tsr3 B-protein +fragment O +with O +a O +90 B-residue_range +aa I-residue_range +C O +- O +terminal O +truncation O +showed O +a O +residual O +primer O +extension O +stop O +, O +whereas O +N O +- O +terminal O +truncations O +exceeding O +46 B-residue_range +aa I-residue_range +almost O +completely O +abolished O +the O +primer O +extension O +arrest O +( O +Figure O +3B O +). O + +Domain O +characterization O +of O +yeast B-taxonomy_domain +Tsr3 B-protein +and O +correlation O +of O +acp B-chemical +modification O +with O +late O +18S B-chemical +rRNA I-chemical +processing O +steps O +. O +( O +A O +) O +Scheme O +of O +the O +TSR3 B-protein +gene O +with O +truncation O +positions O +in O +the O +open O +reading O +frame O +. O + +TSR3 B-protein +fragments O +of O +different O +length O +were O +expressed O +under O +the O +native O +promotor O +from O +multicopy O +plasmids O +in O +a O +Δtsr3 B-mutant +deletion O +strain O +. O + +( O +B O +) O +Primer B-experimental_method +extension I-experimental_method +analysis I-experimental_method +of O +18S B-chemical +rRNA I-chemical +acp B-chemical +modification O +in O +yeast B-taxonomy_domain +cells O +expressing O +the O +indicated O +TSR3 B-protein +fragments O +. O + +N O +- O +terminal O +deletions B-experimental_method +of O +36 B-residue_range +or O +45 B-residue_range +amino O +acids O +and O +C O +- O +terminal O +deletions B-experimental_method +of O +43 B-residue_range +or O +76 B-residue_range +residues O +show O +a O +primer B-evidence +extension I-evidence +stop I-evidence +comparable O +to O +the O +wild B-protein_state +type I-protein_state +. O + +Tsr3 B-protein +fragments O +37 B-residue_range +– I-residue_range +223 I-residue_range +or O +46 B-residue_range +– I-residue_range +223 I-residue_range +cause O +a O +nearly O +complete O +loss O +of O +the O +arrest O +signal O +. O + +The O +box O +highlights O +the O +shortest O +Tsr3 B-protein +fragment O +( O +aa O +46 B-residue_range +– I-residue_range +270 I-residue_range +) O +with O +wild B-protein_state +type I-protein_state +activity O +( O +strong O +primer B-evidence +extension I-evidence +block I-evidence +). O +( O +C O +) O +Northern B-experimental_method +blot I-experimental_method +analysis O +of O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +accumulation O +. O + +A O +weak O +20S B-chemical +rRNA I-chemical +signal O +, O +indicating O +normal O +processing O +, O +is O +observed O +for O +Tsr3 B-protein +fragment O +46 B-residue_range +– I-residue_range +270 I-residue_range +( O +highlighted O +in O +a O +box O +) O +showing O +its O +functionality O +. O + +Strong O +20S O +rRNA O +accumulation O +similar O +to O +that O +of O +the O +Δtsr3 B-mutant +deletion B-experimental_method +is O +observed O +for O +Tsr3 B-protein +fragments O +37 B-residue_range +– I-residue_range +223 I-residue_range +or O +46 B-residue_range +– I-residue_range +223 I-residue_range +. O + +Thus O +, O +the O +archaeal B-taxonomy_domain +homologs O +correspond O +to O +the O +functional O +core O +of O +Tsr3 B-protein +. O + +In O +order O +to O +define O +the O +structural O +basis O +for O +Tsr3 B-protein +function O +, O +homologs O +from O +thermophilic B-taxonomy_domain +archaea I-taxonomy_domain +were O +screened O +for O +crystallization B-experimental_method +. O + +We O +focused O +on O +archaeal B-taxonomy_domain +species O +containing O +a O +putative O +Nep1 B-protein +homolog O +suggesting O +that O +these O +species O +are O +in O +principle O +capable O +of O +synthesizing O +N1 B-chemical +- I-chemical +methyl I-chemical +- I-chemical +N3 I-chemical +- I-chemical +acp I-chemical +- I-chemical +pseudouridine I-chemical +. O + +Well O +diffracting O +crystals B-evidence +were O +obtained O +for O +Tsr3 B-protein +homologs O +from O +the O +two O +crenarchaeal B-taxonomy_domain +species O +Vulcanisaeta B-species +distributa I-species +( O +VdTsr3 B-protein +) O +and O +Sulfolobus B-species +solfataricus I-species +( O +SsTsr3 B-protein +) O +which O +share O +36 O +% O +( O +VdTsr3 B-protein +) O +and O +38 O +% O +( O +SsTsr3 B-protein +) O +identity O +with O +the O +ScTsr3 B-protein +core B-structure_element +region I-structure_element +( O +ScTsr3 B-protein +aa O +46 B-residue_range +– I-residue_range +223 I-residue_range +). O + +While O +for O +S B-species +. I-species +solfataricus I-species +the O +existence O +of O +a O +modified O +nucleotide B-chemical +of O +unknown O +chemical O +composition O +in O +the O +loop B-structure_element +capping I-structure_element +helix I-structure_element +31 I-structure_element +of O +its O +16S B-chemical +rRNA I-chemical +has O +been O +demonstrated O +, O +no O +information O +regarding O +rRNA O +modifications O +is O +yet O +available O +for O +V B-species +. I-species +distributa I-species +. O + +Crystals B-evidence +of O +VdTsr3 B-protein +diffracted O +to O +a O +resolution O +of O +1 O +. O +6 O +Å O +whereas O +crystals B-evidence +of O +SsTsr3 B-protein +diffracted O +to O +2 O +. O +25 O +Å O +. O +Serendipitously O +, O +VdTsr3 B-protein +was O +purified O +and O +crystallized B-experimental_method +in B-protein_state +complex I-protein_state +with I-protein_state +endogenous B-protein_state +( O +E B-species +. I-species +coli I-species +) O +SAM B-chemical +( O +Supplementary O +Figure O +S4 O +) O +while O +SsTsr3 B-protein +crystals B-evidence +contained O +the O +protein O +in O +the O +apo B-protein_state +state O +. O + +The O +structure B-evidence +of O +VdTsr3 B-protein +was O +solved O +ab O +initio O +, O +by O +single B-experimental_method +- I-experimental_method +wavelength I-experimental_method +anomalous I-experimental_method +diffraction I-experimental_method +phasing I-experimental_method +( O +Se B-experimental_method +- I-experimental_method +SAD I-experimental_method +) O +with O +Se B-chemical +containing O +derivatives O +( O +selenomethionine B-chemical +and O +seleno B-chemical +- I-chemical +substituted I-chemical +SAM I-chemical +). O + +The O +structure B-evidence +of O +SsTsr3 B-protein +was O +solved O +by O +molecular B-experimental_method +replacement I-experimental_method +using O +VdTsr3 B-protein +as O +a O +search O +model O +( O +see O +Supplementary O +Table O +S1 O +for O +data O +collection O +and O +refinement O +statistics O +). O + +The O +structure B-evidence +of O +VdTsr3 B-protein +can O +be O +divided O +into O +two O +domains O +( O +Figure O +4A O +). O + +The O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +aa O +1 B-residue_range +– I-residue_range +92 I-residue_range +) O +has O +a O +mixed O +α B-structure_element +/ I-structure_element +β I-structure_element +- I-structure_element +structure I-structure_element +centered O +around O +a O +five B-structure_element +- I-structure_element +stranded I-structure_element +all I-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +( O +Figure O +4B O +) O +with O +the O +strand O +order O +β5 B-structure_element +↑- I-structure_element +β3 B-structure_element +↑- I-structure_element +β4 B-structure_element +↑- I-structure_element +β1 B-structure_element +↑- I-structure_element +β2 B-structure_element +↑. I-structure_element +The O +loops B-structure_element +connecting O +β1 B-structure_element +and O +β2 B-structure_element +, O +β3 B-structure_element +and O +β4 B-structure_element +and O +β4 B-structure_element +and O +β5 B-structure_element +include O +α B-structure_element +- I-structure_element +helices I-structure_element +α1 B-structure_element +, O +α2 B-structure_element +and O +α3 B-structure_element +, O +respectively O +. O + +The O +loop B-structure_element +connecting O +β2 B-structure_element +and O +β3 B-structure_element +contains O +a O +single O +turn O +of O +a O +310 B-structure_element +- I-structure_element +helix I-structure_element +. O +Helices B-structure_element +α1 B-structure_element +and O +α2 B-structure_element +are O +located O +on O +one O +side O +of O +the O +five B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +while O +α3 B-structure_element +packs O +against O +the O +opposite O +β B-structure_element +- I-structure_element +sheet I-structure_element +surface O +. O + +The O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +aa O +93 B-residue_range +– I-residue_range +184 I-residue_range +) O +has O +a O +globular B-structure_element +all I-structure_element +α I-structure_element +- I-structure_element +helical I-structure_element +structure I-structure_element +comprising O +α B-structure_element +- I-structure_element +helices I-structure_element +α4 B-structure_element +to I-structure_element +α9 I-structure_element +. O + +Remarkably O +, O +the O +entire O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +92 B-residue_range +aa I-residue_range +) O +of O +the O +protein O +is O +threaded O +through O +the O +loop B-structure_element +which O +connects O +β B-structure_element +- I-structure_element +strand I-structure_element +β3 B-structure_element +and O +α B-structure_element +- I-structure_element +helix I-structure_element +α2 B-structure_element +of O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +. O + +Thus O +, O +the O +VdTsr3 B-protein +structure B-evidence +contains O +a O +deep B-structure_element +trefoil I-structure_element +knot I-structure_element +. O + +The O +structure B-evidence +of O +SsTsr3 B-protein +in O +the O +apo B-protein_state +state O +is O +very O +similar O +to O +that O +of O +VdTsr3 B-protein +( O +Figure O +4C O +) O +with O +an O +RMSD B-evidence +for O +equivalent O +Cα O +atoms O +of O +1 O +. O +1 O +Å O +. O +The O +only O +significant O +difference O +in O +the O +global O +structure B-evidence +of O +the O +two O +proteins O +is O +the O +presence O +of O +an O +extended O +α B-structure_element +- I-structure_element +helix I-structure_element +α8 B-structure_element +and O +the O +absence B-protein_state +of I-protein_state +α B-structure_element +- I-structure_element +helix I-structure_element +α9 B-structure_element +in O +SsTsr3 B-protein +. O + +Tsr3 B-protein +has O +a O +fold O +similar O +to O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +methyltransferases I-protein_type +. O +( O +A O +) O +Cartoon O +representation O +of O +the O +X B-evidence +- I-evidence +ray I-evidence +structure I-evidence +of O +VdTsr3 B-protein +in O +two O +orientations O +. O + +β B-structure_element +- I-structure_element +strands I-structure_element +are O +colored O +in O +crimson O +whereas O +α B-structure_element +- I-structure_element +helices I-structure_element +in O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +are O +colored O +light O +blue O +and O +α B-structure_element +- I-structure_element +helices I-structure_element +in O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +are O +colored O +dark O +blue O +. O + +The O +bound O +S B-chemical +- I-chemical +adenosylmethionine I-chemical +is O +shown O +in O +a O +stick O +representation O +and O +colored O +by O +atom O +type O +. O + +A O +red O +arrow O +marks O +the O +location O +of O +the O +topological B-structure_element +knot I-structure_element +in O +the O +structure B-evidence +. O +( O +B O +) O +Secondary O +structure O +representation O +of O +the O +VdTsr3 B-protein +structure B-evidence +. O + +The O +color O +coding O +is O +the O +same O +as O +in O +( O +A O +). O +( O +C O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +X B-evidence +- I-evidence +ray I-evidence +structures I-evidence +of O +VdTsr3 B-protein +in O +the O +SAM B-protein_state +- I-protein_state +bound I-protein_state +state O +( O +red O +) O +and O +SsTsr3 B-protein +( O +blue O +) O +in O +the O +apo B-protein_state +state O +. O + +The O +locations O +of O +the O +α B-structure_element +- I-structure_element +helix I-structure_element +α8 B-structure_element +which O +is O +longer O +in O +SsTsr3 B-protein +and O +of O +α B-structure_element +- I-structure_element +helix I-structure_element +α9 B-structure_element +which O +is O +only O +present O +in O +VdTsr3 B-protein +are O +indicated O +. O +( O +D O +) O +Secondary O +structure O +cartoon O +( O +left O +) O +of O +S B-species +. I-species +pombe I-species +Trm10 B-protein +( O +pdb4jwf O +)— O +the O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +methyltransferase I-protein_type +structurally O +most O +similar O +to O +Tsr3 B-protein +and O +superposition B-experimental_method +of O +the O +VdTsr3 B-protein +and O +Trm10 B-protein +X B-evidence +- I-evidence +ray I-evidence +structures I-evidence +( O +right O +). O +( O +E O +) O +Analytical B-experimental_method +gel I-experimental_method +filtration I-experimental_method +profiles B-evidence +for O +VdTsr3 B-protein +( O +red O +) O +and O +SsTsr3 B-protein +( O +blue O +) O +show O +that O +both O +proteins O +are O +monomeric B-oligomeric_state +in O +solution O +. O + +Vd B-species +, O +Vulcanisaeta B-species +distributa I-species +; O +Ss B-species +, O +Sulfolobus B-species +solfataricus I-species +. O + +Structure B-experimental_method +predictions I-experimental_method +suggested O +that O +Tsr3 B-protein +might O +contain O +a O +so O +- O +called O +RLI B-structure_element +domain I-structure_element +which O +contains O +a O +‘ O +bacterial B-structure_element +like I-structure_element +’ I-structure_element +ferredoxin I-structure_element +fold I-structure_element +and O +binds O +two O +iron O +- O +sulfur O +clusters O +through O +eight O +conserved B-protein_state +cysteine B-residue_name +residues O +. O + +However O +, O +no O +structural O +similarity O +to O +an O +RLI B-structure_element +- I-structure_element +domain I-structure_element +was O +detectable O +. O + +This O +is O +in O +accordance O +with O +the O +functional O +analysis O +of O +alanine B-experimental_method +replacement I-experimental_method +mutations I-experimental_method +of O +cysteine B-residue_name +residues O +in O +ScTsr3 B-protein +( O +Supplementary O +Figure O +S3 O +). O + +The O +β B-structure_element +- I-structure_element +strand I-structure_element +topology I-structure_element +and O +the O +deep O +C O +- O +terminal O +trefoil B-structure_element +knot I-structure_element +of O +archaeal B-taxonomy_domain +Tsr3 B-protein +are O +the O +structural O +hallmarks O +of O +the O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +- I-protein_type +methyltransferase I-protein_type +fold O +. O + +The O +closest O +structural O +homolog O +identified O +in O +a O +DALI B-experimental_method +search I-experimental_method +is O +the O +tRNA B-protein_type +methyltransferase I-protein_type +Trm10 B-protein +( O +DALI B-evidence +Z I-evidence +- I-evidence +score I-evidence +6 O +. O +8 O +) O +which O +methylates O +the O +N1 O +nitrogen O +of O +G9 B-residue_name_number +/ O +A9 B-residue_name_number +in O +many O +archaeal B-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +tRNAs B-chemical +by O +using O +SAM B-chemical +as O +the O +methyl O +group O +donor O +. O + +In O +comparison O +to O +Tsr3 B-protein +the O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +element I-structure_element +of O +Trm10 B-protein +is O +extended O +by O +one O +additional O +β B-structure_element +- I-structure_element +strand I-structure_element +pairing O +to O +β2 B-structure_element +. O + +Furthermore O +, O +the O +trefoil B-structure_element +knot I-structure_element +of O +Trm10 B-protein +is O +not O +as O +deep O +as O +that O +of O +Tsr3 B-protein +( O +Figure O +4D O +). O + +Interestingly O +, O +Nep1 B-protein +— O +the O +enzyme O +preceding O +Tsr3 B-protein +in O +the O +biosynthetic O +pathway O +for O +the O +synthesis O +of O +m1acp3Ψ B-chemical +— O +also O +belongs O +to O +the O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +of I-protein_type +RNA I-protein_type +methyltransferases I-protein_type +. O + +However O +, O +the O +structural O +similarities O +between O +Nep1 B-protein +and O +Tsr3 B-protein +( O +DALI B-evidence +Z I-evidence +- I-evidence +score I-evidence +4 O +. O +4 O +) O +are O +less O +pronounced O +than O +between O +Tsr3 B-protein +and O +Trm10 B-protein +. O + +Most O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +- I-protein_type +methyltransferases I-protein_type +are O +homodimers B-oligomeric_state +. O + +A O +notable O +exception O +is O +Trm10 B-protein +. O + +Gel B-experimental_method +filtration I-experimental_method +experiments O +with O +both O +VdTsr3 B-protein +and O +SsTsr3 B-protein +( O +Figure O +4E O +) O +showed O +that O +both O +proteins O +are O +monomeric B-oligomeric_state +in O +solution O +thereby O +extending O +the O +structural O +similarities O +to O +Trm10 B-protein +. O + +So O +far O +, O +structural O +information O +is O +only O +available O +for O +one O +other O +enzyme O +that O +transfers O +the O +acp B-chemical +group O +from O +SAM B-chemical +to O +an O +RNA B-chemical +nucleotide B-chemical +. O + +This O +enzyme O +, O +Tyw2 B-protein +, O +is O +part O +of O +the O +biosynthesis O +pathway O +of O +wybutosine B-chemical +nucleotides I-chemical +in O +tRNAs B-chemical +. O + +However O +, O +there O +are O +no O +structural O +similarities O +between O +Tsr3 B-protein +and O +Tyw2 B-protein +, O +which O +contains O +an O +all B-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +of O +a O +different O +topology O +and O +no O +knot B-structure_element +structure I-structure_element +. O + +Instead O +, O +Tyw2 B-protein +has O +a O +fold O +typical O +for O +the O +class B-protein_type +- I-protein_type +I I-protein_type +- I-protein_type +or I-protein_type +Rossmann I-protein_type +- I-protein_type +fold I-protein_type +class I-protein_type +of I-protein_type +methyltransferases I-protein_type +( O +Supplementary O +Figure O +S5B O +). O + +Cofactor O +binding O +of O +Tsr3 B-protein + +The O +SAM B-site +- I-site +binding I-site +site I-site +of O +Tsr3 B-protein +is O +located O +in O +a O +deep O +crevice O +between O +the O +N B-structure_element +- I-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +domains I-structure_element +in O +the O +vicinity O +of O +the O +trefoil B-structure_element +knot I-structure_element +as O +typical O +for O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +- I-protein_type +methyltransferases I-protein_type +( O +Figure O +4A O +). O + +The O +adenine B-chemical +base O +of O +the O +cofactor O +is O +recognized O +by O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +its O +N1 O +nitrogen O +and O +the O +backbone O +amide O +of O +L93 B-residue_name_number +directly O +preceding O +β5 B-structure_element +as O +well O +as O +between O +its O +N6 O +- O +amino O +group O +and O +the O +backbone O +carbonyl O +group O +of O +Y108 B-residue_name_number +located O +in O +the O +loop B-structure_element +connecting O +β5 B-structure_element +in O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +and O +α4 B-structure_element +in O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +Figure O +5A O +). O + +Furthermore O +, O +the O +adenine B-chemical +base O +of O +SAM B-chemical +is O +involved O +in O +hydrophobic B-bond_interaction +packing I-bond_interaction +interactions I-bond_interaction +with O +the O +side O +chains O +of O +L45 B-residue_name_number +( O +β3 B-structure_element +), O +P47 B-residue_name_number +and O +W73 B-residue_name_number +( O +α3 B-structure_element +) O +in O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +as O +well O +as O +with O +L93 B-residue_name_number +, O +L110 B-residue_name_number +( O +both O +in O +the O +loop B-structure_element +connecting O +β5 B-structure_element +and O +α4 B-structure_element +) O +and O +A115 B-residue_name_number +( O +α5 B-structure_element +) O +in O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +. O + +The O +ribose B-chemical +2 O +′ O +and O +3 O +′ O +hydroxyl O +groups O +of O +SAM B-chemical +are O +hydrogen B-bond_interaction +bonded I-bond_interaction +to O +the O +backbone O +carbonyl O +group O +of O +I69 B-residue_name_number +. O + +The O +acp B-chemical +side O +chain O +of O +SAM B-chemical +is O +fixed O +in O +position O +by O +hydrogen B-bond_interaction +bonding I-bond_interaction +of O +its O +carboxylate O +group O +to O +the O +backbone O +amide O +and O +the O +side O +chain O +hydroxyl O +group O +of O +T19 B-residue_name_number +in O +α1 B-structure_element +as O +well O +as O +the O +backbone O +amide O +group O +of O +T112 B-residue_name_number +in O +α4 B-structure_element +( O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +). O + +Most O +importantly O +, O +the O +methyl O +group O +of O +SAM B-chemical +is O +buried O +in O +a O +hydrophobic B-site +pocket I-site +formed O +by O +the O +sidechains O +of O +W73 B-residue_name_number +and O +A76 B-residue_name_number +both O +located O +in O +α3 B-structure_element +( O +Figure O +5A O +and O +B O +). O + +W73 B-residue_name_number +is O +highly B-protein_state +conserved I-protein_state +in O +all O +known O +Tsr3 B-protein_type +proteins I-protein_type +, O +whereas O +A76 B-residue_name_number +can O +be O +replaced O +by O +other O +hydrophobic O +amino B-chemical +acids I-chemical +. O + +Consequently O +, O +the O +accessibility O +of O +this O +methyl O +group O +for O +a O +nucleophilic O +attack O +is O +strongly O +reduced O +in O +comparison O +with O +RNA B-protein_type +- I-protein_type +methyltransferases I-protein_type +such O +as O +Trm10 B-protein +( O +Figure O +5B O +, O +C O +). O + +In O +contrast O +, O +the O +acp B-chemical +side O +chain O +of O +SAM B-chemical +is O +accessible O +for O +reactions O +in O +the O +Tsr3 B-protein_state +- I-protein_state +bound I-protein_state +state O +( O +Figure O +5B O +). O + +SAM B-chemical +- O +binding O +by O +Tsr3 B-protein +. O + +( O +A O +) O +Close O +- O +up O +view O +of O +the O +SAM B-site +- I-site +binding I-site +pocket I-site +of O +VdTsr3 B-protein +. O + +Nitrogen O +atoms O +are O +dark O +blue O +, O +oxygen O +atoms O +red O +, O +sulfur B-chemical +atoms O +orange O +, O +carbon O +atoms O +of O +the O +protein O +light O +blue O +and O +carbon O +atoms O +of O +SAM B-chemical +yellow O +. O + +Hydrogen B-bond_interaction +bonds I-bond_interaction +are O +indicated O +by O +dashed O +lines O +. O + +( O +B O +) O +Solvent O +accessibility O +of O +the O +acp B-chemical +group O +of O +SAM B-chemical +bound B-protein_state +to I-protein_state +VdTsr3 B-protein +. O + +The O +solvent O +accessible O +surface O +of O +the O +protein O +is O +shown O +in O +semitransparent O +gray O +whereas O +SAM B-chemical +is O +show O +in O +a O +stick O +representation O +. O + +A O +red O +arrow O +indicates O +the O +reactive O +CH2 O +- O +moiety O +of O +the O +acp B-chemical +group O +. O +( O +C O +) O +Solvent O +accessibility O +of O +the O +SAM B-chemical +methyl O +group O +for O +SAM B-chemical +bound B-protein_state +to I-protein_state +the O +RNA B-protein_type +methyltransferase I-protein_type +Trm10 B-protein +. O + +Bound B-protein_state +SAM B-chemical +was O +modelled O +based O +on O +the O +X B-evidence +- I-evidence +ray I-evidence +structure I-evidence +of O +the O +Trm10 B-complex_assembly +/ I-complex_assembly +SAH I-complex_assembly +- O +complex O +( O +pdb4jwf O +). O + +A O +red O +arrow O +indicates O +the O +SAM B-chemical +methyl O +group O +. O +( O +D O +) O +Binding O +of O +SAM B-chemical +analogs O +to O +SsTsr3 B-protein +. O + +Tryptophan B-evidence +fluorescence I-evidence +quenching I-evidence +curves I-evidence +upon O +addition O +of O +SAM B-chemical +( O +blue O +), O +5 B-chemical +′- I-chemical +methyl I-chemical +- I-chemical +thioadenosine I-chemical +( O +red O +) O +and O +SAH B-chemical +( O +black O +). O + +( O +E O +) O +Binding O +of O +14C B-chemical +- I-chemical +labeled I-chemical +SAM I-chemical +to O +SsTsr3 B-protein +. O + +Radioactively O +labeled O +SAM B-chemical +is O +retained O +on O +a O +filter O +in O +the O +presence B-protein_state +of I-protein_state +SsTsr3 B-protein +. O + +Addition O +of O +unlabeled O +SAM B-chemical +competes O +with O +the O +binding O +of O +labeled O +SAM B-chemical +. O + +A O +W66A B-mutant +- O +mutant B-protein_state +of O +SsTsr3 B-protein +( O +W73 B-residue_name_number +in O +VdTsr3 B-protein +) O +does O +not O +bind O +SAM B-chemical +. O + +( O +F O +) O +Primer B-experimental_method +extension I-experimental_method +( O +upper O +left O +) O +shows O +a O +strongly O +reduced O +acp B-chemical +modification O +of O +yeast B-taxonomy_domain +18S B-chemical +rRNA I-chemical +in O +Δtsr3 B-mutant +cells O +expressing O +Tsr3 B-mutant +- I-mutant +S62D I-mutant +, O +- B-mutant +E111A I-mutant +or O +– B-mutant +W114A I-mutant +. O + +This O +correlates O +with O +a O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +accumulation O +comparable O +to O +the O +Δtsr3 B-mutant +deletion O +( O +right O +: O +northern B-experimental_method +blot I-experimental_method +). O + +3xHA B-protein_state +tagged I-protein_state +Tsr3 B-protein +mutants B-protein_state +are O +expressed O +comparable O +to O +the O +wild B-protein_state +type I-protein_state +as O +shown O +by O +western B-experimental_method +blot I-experimental_method +( O +lower O +left O +). O + +Binding B-evidence +affinities I-evidence +for O +SAM B-chemical +and O +its O +analogs O +5 B-chemical +′- I-chemical +methylthioadenosin I-chemical +and O +SAH B-chemical +to O +SsTsr3 B-protein +were O +measured O +using O +tryptophan B-experimental_method +fluorescence I-experimental_method +quenching I-experimental_method +. O + +VdTsr3 B-protein +could O +not O +be O +used O +in O +these O +experiments O +since O +we O +could O +not O +purify O +it O +in O +a O +stable B-protein_state +SAM B-protein_state +- I-protein_state +free I-protein_state +form O +. O + +SsTsr3 B-protein +bound B-protein_state +SAM B-chemical +with O +a O +KD B-evidence +of O +6 O +. O +5 O +μM O +, O +which O +is O +similar O +to O +SAM B-evidence +- I-evidence +KD I-evidence +' I-evidence +s I-evidence +reported O +for O +several O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +methyltransferases I-protein_type +. O + +5 B-chemical +′- I-chemical +methylthioadenosin I-chemical +— O +the O +reaction O +product O +after O +the O +acp B-chemical +- O +transfer O +— O +binds O +only O +∼ O +2 O +. O +5 O +- O +fold O +weaker O +( O +KD O += O +16 O +. O +7 O +μM O +) O +compared O +to O +SAM B-chemical +. O + +S B-chemical +- I-chemical +adenosylhomocysteine I-chemical +which O +lacks O +the O +methyl O +group O +of O +SAM B-chemical +binds O +with O +significantly O +lower O +affinity B-evidence +( O +KD B-evidence += O +55 O +. O +5 O +μM O +) O +( O +Figure O +5D O +). O + +This O +suggests O +that O +the O +hydrophobic B-bond_interaction +interaction I-bond_interaction +between O +SAM B-chemical +' O +s O +methyl O +group O +and O +the O +hydrophobic B-site +pocket I-site +of O +Tsr3 B-protein +is O +thermodynamically O +important O +for O +the O +interaction O +. O + +On O +the O +other O +hand O +, O +the O +loss O +of O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +the O +acp B-chemical +sidechain O +carboxylate O +group O +and O +the O +protein O +appears O +to O +be O +thermodynamically O +less O +important O +but O +these O +hydrogen B-bond_interaction +bonds I-bond_interaction +might O +play O +a O +crucial O +role O +for O +the O +proper O +orientation O +of O +the O +cofactor O +side O +chain O +in O +the O +substrate B-site +binding I-site +pocket I-site +. O + +Accordingly O +, O +a O +W66A B-mutant +- O +mutation B-experimental_method +( O +W73 B-residue_name_number +in O +VdTsr3 B-protein +) O +of O +SsTsr3 B-protein +significantly O +diminished O +SAM B-evidence +- I-evidence +binding I-evidence +in O +a O +filter B-experimental_method +binding I-experimental_method +assay I-experimental_method +compared O +to O +the O +wild B-protein_state +type I-protein_state +( O +Figure O +5E O +). O + +Furthermore O +, O +a O +W B-experimental_method +to I-experimental_method +A I-experimental_method +mutation I-experimental_method +at O +the O +equivalent O +position O +W114 B-residue_name_number +in O +ScTsr3 B-protein +strongly O +reduced O +the O +in O +vivo O +acp B-protein_type +transferase I-protein_type +activity O +( O +Figure O +5F O +). O + +The O +side O +chain O +hydroxyl O +group O +of O +T19 B-residue_name_number +seems O +of O +minor O +importance O +for O +SAM B-chemical +binding O +since O +mutations B-experimental_method +of O +T17 B-residue_name_number +( O +T19 B-residue_name_number +in O +VdTsr3 B-protein +) O +to O +either O +A B-residue_name +or O +D B-residue_name +did O +not O +significantly O +influence O +the O +SAM B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +SsTsr3 B-protein +( O +KD B-evidence +' O +s O += O +3 O +. O +9 O +or O +11 O +. O +2 O +mM O +, O +respectively O +). O + +Nevertheless O +, O +a O +mutation B-experimental_method +of O +the O +equivalent O +position O +S62 B-residue_name_number +of O +ScTsr3 B-protein +to O +D B-residue_name +, O +but O +not O +to O +A B-residue_name +, O +resulted O +in O +reduced O +acp B-chemical +modification O +in O +vivo O +, O +as O +shown O +by O +primer B-experimental_method +extension I-experimental_method +analysis I-experimental_method +( O +Figure O +5F O +). O + +The O +acp B-chemical +- O +transfer O +reaction O +catalyzed O +by O +Tsr3 B-protein +most O +likely O +requires O +the O +presence O +of O +a O +catalytic O +base O +in O +order O +to O +abstract O +a O +proton O +from O +the O +N3 O +imino O +group O +of O +the O +modified O +pseudouridine B-chemical +. O + +The O +side O +chain O +of O +D70 B-residue_name_number +( O +VdTsr3 B-protein +) O +located O +in O +β4 B-structure_element +is O +∼ O +5 O +Å O +away O +from O +the O +SAM B-chemical +sulfur O +atom O +. O + +This O +residue O +is O +conserved B-protein_state +as I-protein_state +D B-residue_name +or O +E B-residue_name +both O +in O +archaeal B-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +Tsr3 B-protein +homologs O +. O + +Mutations B-experimental_method +of O +the O +corresponding O +residue O +in O +SsTsr3 B-protein +to O +A B-residue_name +( O +D63 B-residue_name_number +) O +does O +not O +significantly O +alter O +the O +SAM B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +protein O +( O +KD B-evidence += O +11 O +. O +0 O +μM O +). O + +However O +, O +the O +mutation B-experimental_method +of O +the O +corresponding O +residue O +of O +ScTsr3 B-protein +( O +E111A B-mutant +) O +leads O +to O +a O +significant O +decrease O +of O +the O +acp B-protein_type +transferase I-protein_type +activity O +in O +vivo O +( O +Figure O +5F O +). O + +RNA B-chemical +- O +binding O +of O +Tsr3 B-protein + +Analysis B-experimental_method +of I-experimental_method +the I-experimental_method +electrostatic I-experimental_method +surface I-experimental_method +properties I-experimental_method +of O +VdTsr3 B-protein +clearly O +identified O +positively B-site +charged I-site +surface I-site +patches I-site +in O +the O +vicinity O +of O +the O +SAM B-site +- I-site +binding I-site +site I-site +suggesting O +a O +putative O +RNA B-site +- I-site +binding I-site +site I-site +( O +Figure O +6A O +). O + +Furthermore O +, O +a O +negatively O +charged O +MES B-chemical +- O +ion O +is O +found O +in O +the O +crystal B-evidence +structure I-evidence +of O +VdTsr3 B-protein +complexed B-protein_state +to I-protein_state +the O +side O +chain O +of O +K22 B-residue_name_number +in O +helix B-structure_element +α1 B-structure_element +. O + +Its O +negatively O +charged O +sulfate B-chemical +group O +might O +mimic O +an O +RNA B-chemical +backbone O +phosphate O +. O + +Helix B-structure_element +α1 B-structure_element +contains O +two O +more O +positively O +charged O +amino O +acids O +K17 B-residue_name_number +and O +R25 B-residue_name_number +as O +does O +the O +loop B-structure_element +preceding O +it O +( O +R9 B-residue_name_number +). O + +A O +second O +cluster O +of O +positively O +charged O +residues O +is O +found O +in O +or O +near O +helix B-structure_element +α3 B-structure_element +( O +K74 B-residue_name_number +, O +R75 B-residue_name_number +, O +K82 B-residue_name_number +, O +R85 B-residue_name_number +and O +K87 B-residue_name_number +). O + +Some O +of O +these O +amino O +acids O +are O +conserved B-protein_state +between O +archaeal B-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +Tsr3 B-protein +( O +Supplementary O +Figure O +S1A O +). O + +In O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +, O +the O +surface O +exposed O +α B-structure_element +- I-structure_element +helices I-structure_element +α5 B-structure_element +and O +α7 B-structure_element +carry O +a O +significant O +amount O +of O +positively O +charged O +amino O +acids O +. O + +A O +triple B-experimental_method +mutation I-experimental_method +of O +the O +conserved B-protein_state +positively O +charged O +residues O +R60 B-residue_name_number +, O +K65 B-residue_name_number +and O +R131 B-residue_name_number +to O +A B-residue_name +in O +ScTsr3 B-protein +resulted O +in O +a O +protein O +with O +a O +significantly O +impaired O +acp B-protein_type +transferase I-protein_type +activity O +in O +vivo O +( O +Figure O +6D O +) O +in O +line O +with O +an O +important O +functional O +role O +for O +these O +positively O +charged O +residues O +. O + +RNA O +- O +binding O +of O +Tsr3 B-protein +. O + +( O +A O +) O +Electrostatic O +charge O +distribution O +on O +the O +surface O +of O +VdTsr3 B-protein +. O + +SAM B-chemical +is O +shown O +in O +a O +stick O +representation O +. O + +Also O +shown O +in O +stick O +representation O +is O +a O +negatively O +charged O +MES B-chemical +ion O +. O + +Conserved B-protein_state +basic O +amino B-chemical +acids I-chemical +are O +labeled O +. O +( O +B O +) O +Comparison O +of O +the O +secondary O +structures O +of O +helix B-structure_element +31 I-structure_element +from O +the O +small O +ribosomal O +subunit O +rRNAs B-chemical +in O +S B-species +. I-species +cerevisiae I-species +and O +S B-species +. I-species +solfataricus I-species +with O +the O +location O +of O +the O +hypermodified B-protein_state +nucleotide B-chemical +indicated O +in O +red O +. O + +For O +S B-species +. I-species +solfataricus I-species +the O +chemical O +identity O +of O +the O +hypermodified B-protein_state +nucleotide B-chemical +is O +not O +known O +but O +the O +existence O +of O +NEP1 B-protein +and O +TSR3 B-protein +homologs O +suggest O +that O +it O +is O +indeed O +N1 B-chemical +- I-chemical +methyl I-chemical +- I-chemical +N3 I-chemical +- I-chemical +acp I-chemical +- I-chemical +pseudouridine I-chemical +. O + +( O +C O +) O +Binding O +of O +SsTsr3 B-protein +to O +RNA B-chemical +. O + +5 O +′- O +fluoresceine B-chemical +labeled O +RNA B-chemical +oligonucleotides O +corresponding O +either O +to O +the O +native B-protein_state +( O +20mer B-oligomeric_state +– O +see O +inset O +) O +or O +a O +stabilized B-protein_state +( O +20mer_GC B-oligomeric_state +- O +inset O +) O +helix B-structure_element +31 I-structure_element +of O +the O +small O +ribosomal O +subunit O +rRNA B-chemical +from O +S B-species +. I-species +solfataricus I-species +were O +titrated B-experimental_method +with I-experimental_method +increasing I-experimental_method +amounts I-experimental_method +of O +SsTsr3 B-protein +and O +the O +changes O +in O +the O +fluoresceine B-chemical +fluorescence B-evidence +anisotropy I-evidence +were O +measured O +and O +fitted O +to O +a O +binding B-evidence +curve I-evidence +( O +20mer B-oligomeric_state +– O +red O +, O +20mer_GC B-oligomeric_state +– O +blue O +). O + +Oligo B-chemical +- I-chemical +U9 I-chemical +- I-chemical +RNA I-chemical +was O +used O +for O +comparison O +( O +black O +). O + +The O +20mer_GC B-oligomeric_state +RNA B-chemical +was O +also O +titrated B-experimental_method +with O +SsTsr3 B-protein +in O +the O +presence O +of O +2 O +mM O +SAM B-chemical +( O +purple O +). O +( O +D O +) O +Mutants B-protein_state +of O +ScTsr3 B-protein +R60 B-residue_name_number +, O +K65 B-residue_name_number +or O +R131 B-residue_name_number +( O +equivalent O +to O +K17 B-residue_name_number +, O +K22 B-residue_name_number +and O +R91 B-residue_name_number +in O +VdTsr3 B-protein +) O +expressed B-experimental_method +in O +Δtsr3 B-mutant +yeast B-taxonomy_domain +cells O +show O +a O +primer B-evidence +extension I-evidence +stop I-evidence +comparable O +to O +the O +wild B-protein_state +type I-protein_state +. O + +Combination B-experimental_method +of I-experimental_method +the I-experimental_method +three I-experimental_method +point I-experimental_method +mutations I-experimental_method +( O +R60A B-mutant +/ O +K65A B-mutant +/ O +R131A B-mutant +) O +leads O +to O +a O +strongly O +reduced O +acp B-chemical +modification O +of O +18S B-chemical +rRNA I-chemical +. O + +In O +order O +to O +explore O +the O +RNA O +- O +ligand O +specificity O +of O +Tsr3 B-protein +we O +titrated B-experimental_method +SsTsr3 B-protein +prepared O +in O +RNase B-protein_state +- I-protein_state +free I-protein_state +form O +with O +5 O +′- O +fluoresceine B-chemical +- O +labeled O +RNA B-chemical +and O +determined O +the O +affinity B-evidence +by O +fluorescence B-experimental_method +anisotropy I-experimental_method +measurements I-experimental_method +. O + +SsTsr3 B-protein +in O +the O +apo B-protein_state +state O +bound B-protein_state +a O +20mer B-oligomeric_state +RNA B-chemical +corresponding O +to O +helix B-structure_element +31 I-structure_element +of O +S B-species +. I-species +solfataricus I-species +16S B-chemical +rRNA I-chemical +( O +Figure O +6B O +) O +with O +a O +KD B-evidence +of O +1 O +. O +9 O +μM O +and O +to O +a O +version O +of O +this O +hairpin B-structure_element +stabilized O +by O +additional O +GC O +base O +pairs O +( O +20mer B-oligomeric_state +- I-oligomeric_state +GC I-oligomeric_state +) O +with O +a O +KD B-evidence +of O +0 O +. O +6 O +μM O +( O +Figure O +6C O +). O + +A O +single O +stranded O +oligoU B-chemical +- I-chemical +RNA I-chemical +bound B-protein_state +with O +a O +10 O +- O +fold O +- O +reduced O +affinity B-evidence +( O +6 O +. O +0 O +μM O +). O + +The O +presence O +of O +saturating O +amounts O +of O +SAM B-chemical +( O +2 O +mM O +) O +did O +not O +have O +a O +significant O +influence O +on O +the O +RNA B-evidence +- I-evidence +affinity I-evidence +of O +SsTsr3 B-protein +( O +KD B-evidence +of O +1 O +. O +7 O +μM O +for O +the O +20mer B-oligomeric_state +- I-oligomeric_state +GC I-oligomeric_state +- O +RNA B-chemical +) O +suggesting O +no O +cooperativity O +in O +substrate O +binding O +. O + +U1191 B-residue_name_number +is O +the O +only O +hypermodified B-protein_state +base O +in O +the O +yeast B-taxonomy_domain +18S B-chemical +rRNA I-chemical +and O +is O +strongly B-protein_state +conserved I-protein_state +in O +eukaryotes B-taxonomy_domain +. O + +The O +formation O +of O +1 B-chemical +- I-chemical +methyl I-chemical +- I-chemical +3 I-chemical +-( I-chemical +3 I-chemical +- I-chemical +amino I-chemical +- I-chemical +3 I-chemical +- I-chemical +carboxypropyl I-chemical +)- I-chemical +pseudouridine I-chemical +( O +m1acp3Ψ B-chemical +) O +is O +very O +complex O +requiring O +three O +successive O +modification O +reactions O +involving O +one O +H B-structure_element +/ I-structure_element +ACA I-structure_element +snoRNP B-complex_assembly +( O +snR35 B-protein +) O +and O +two O +protein O +enzymes O +( O +Nep1 B-protein +/ O +Emg1 B-protein +and O +Tsr3 B-protein +). O + +This O +makes O +it O +unique O +in O +eukaryotic B-taxonomy_domain +rRNA B-chemical +modification O +. O + +The O +m1acp3Ψ B-chemical +base O +is O +located O +at O +the O +tip O +of O +helix B-structure_element +31 I-structure_element +on O +the O +18S B-chemical +rRNA I-chemical +( O +Supplementary O +Figure O +S1B O +) O +which O +, O +together O +with O +helices B-structure_element +18 I-structure_element +, I-structure_element +24 I-structure_element +, I-structure_element +34 I-structure_element +and I-structure_element +44 I-structure_element +, O +contribute O +to O +building O +the O +decoding O +center O +of O +the O +small O +ribosomal O +subunit O +. O + +A O +similar O +modification O +( O +acp3U B-chemical +) O +was O +identified O +in O +Haloferax B-species +volcanii I-species +and O +corresponding O +modified O +nucleotides B-chemical +were O +also O +shown O +to O +occur O +in O +other O +archaea B-taxonomy_domain +. O + +As O +shown O +here O +TSR3 B-protein +encodes O +the O +transferase O +catalyzing O +the O +acp B-chemical +modification O +as O +the O +last O +step O +in O +the O +biosynthesis O +of O +m1acp3Ψ B-chemical +in O +yeast B-taxonomy_domain +and O +human B-species +cells O +. O + +Unexpectedly O +, O +archaeal B-taxonomy_domain +Tsr3 B-protein +has O +a O +structure B-evidence +similar O +to O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +methyltransferases I-protein_type +, O +and O +it O +is O +the O +first O +example O +for O +an O +enzyme O +of O +this O +class O +transferring O +an O +acp B-chemical +group O +, O +due O +to O +a O +modified O +SAM B-site +- I-site +binding I-site +pocket I-site +that O +exposes O +the O +acp B-chemical +instead O +of O +the O +methyl O +group O +of O +SAM B-chemical +to O +its O +RNA B-chemical +substrate O +. O + +Similar O +to O +the O +structurally O +unrelated O +Rossmann B-protein_type +- I-protein_type +fold I-protein_type +Tyw2 I-protein_type +acp I-protein_type +transferase I-protein_type +, O +the O +SAM B-chemical +methyl O +group O +of O +Tsr3 B-protein +is O +bound O +in O +an O +inaccessible O +hydrophobic B-site +pocket I-site +whereas O +the O +acp B-chemical +side O +chain O +becomes O +accessible O +for O +a O +nucleophilic O +attack O +by O +the O +N3 O +of O +pseudouridine B-chemical +. O + +In O +contrast O +, O +in O +the O +structurally O +closely O +related O +RNA B-protein_type +methyltransferase I-protein_type +Trm10 B-protein +the O +methyl O +group O +of O +the O +cofactor O +SAM B-chemical +is O +accessible O +whereas O +its O +acp B-chemical +side O +chain O +is O +buried O +inside O +the O +protein O +. O + +This O +suggests O +that O +enzymes O +with O +a O +SAM B-protein_type +- I-protein_type +dependent I-protein_type +acp I-protein_type +transferase I-protein_type +activity O +might O +have O +evolved O +from O +SAM B-protein_type +- I-protein_type +dependent I-protein_type +methyltransferases I-protein_type +by O +slight O +modifications O +of O +the O +SAM B-site +- I-site +binding I-site +pocket I-site +. O + +Thus O +, O +additional O +examples O +for O +acp B-protein_type +transferase I-protein_type +enzymes O +might O +be O +found O +with O +similarities O +to O +other O +structural O +classes O +of O +methyltransferases B-protein_type +. O + +In O +contrast O +to O +Nep1 B-protein +, O +the O +enzyme O +preceding O +Tsr3 B-protein +in O +the O +m1acp3Ψ B-chemical +biosynthesis O +pathway O +, O +Tsr3 B-protein +binds O +rather O +weakly O +and O +with O +little O +specificity O +to O +its O +isolated O +substrate O +RNA B-chemical +. O + +This O +suggests O +that O +Tsr3 B-protein +is O +not O +stably O +incorporated O +into O +pre B-complex_assembly +- I-complex_assembly +ribosomal I-complex_assembly +particles I-complex_assembly +and O +that O +its O +binding O +to O +the O +nascent O +ribosomal B-complex_assembly +subunit I-complex_assembly +possibly O +requires O +additional O +interactions O +with O +other O +pre O +- O +ribosomal O +components O +. O + +Consistently O +, O +in O +sucrose B-experimental_method +gradient I-experimental_method +analysis I-experimental_method +, O +Tsr3 B-protein +was O +found O +in O +low O +- O +molecular O +weight O +fractions O +rather O +than O +with O +pre B-complex_assembly +- I-complex_assembly +ribosome I-complex_assembly +containing O +high O +- O +molecular O +weight O +fractions O +. O + +In O +contrast O +to O +several O +enzymes O +that O +catalyze O +base O +specific O +modifications O +in O +rRNAs B-chemical +Tsr3 B-protein +is O +not O +an O +essential O +protein O +. O + +Typically O +, O +other O +small B-protein_type +subunit I-protein_type +rRNA I-protein_type +methyltransferases I-protein_type +as O +Dim1 B-protein +, O +Bud23 B-protein +and O +Nep1 B-protein +/ O +Emg1 B-protein +carry O +dual O +functions O +, O +in O +ribosome O +biogenesis O +and O +rRNA B-chemical +modification O +, O +and O +it O +is O +their O +involvement O +in O +pre B-chemical +- I-chemical +RNA I-chemical +processing O +that O +is O +essential O +rather O +than O +their O +RNA O +- O +methylating O +activity O +(, O +discussed O +in O +7 O +). O + +In O +contrast O +, O +for O +several O +Tsr3 B-protein +mutants O +( O +SAM B-protein_state +- I-protein_state +binding I-protein_state +and O +cysteine B-protein_state +mutations I-protein_state +) O +we O +found O +a O +systematic O +correlation O +between O +the O +loss O +of O +acp B-chemical +modification O +and O +the O +efficiency O +of O +18S B-chemical +rRNA I-chemical +maturation O +. O + +This O +demonstrates O +that O +, O +unlike O +the O +other O +small O +subunit O +rRNA B-chemical +base O +modifications O +, O +the O +acp B-chemical +modification O +is O +required O +for O +efficient O +pre B-chemical +- I-chemical +rRNA I-chemical +processing O +. O + +Recently O +, O +structural B-experimental_method +, I-experimental_method +functional I-experimental_method +, I-experimental_method +and I-experimental_method +CRAC I-experimental_method +( I-experimental_method +cross I-experimental_method +- I-experimental_method +linking I-experimental_method +and I-experimental_method +cDNA I-experimental_method +analysis I-experimental_method +) I-experimental_method +experiments I-experimental_method +of O +late O +assembly O +factors O +involved O +in O +cytoplasmic O +processing O +of O +40S B-complex_assembly +subunits I-complex_assembly +, O +along O +with O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +studies O +of O +the O +late B-protein_state +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +subunits I-complex_assembly +have O +provided O +important O +insights O +into O +late O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +processing O +. O + +Apart O +from O +most O +of O +the O +ribosomal O +proteins O +, O +cytoplasmic O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +particles I-complex_assembly +contain O +20S B-chemical +rRNA I-chemical +and O +at O +least O +seven O +non B-protein_type +- I-protein_type +ribosomal I-protein_type +proteins I-protein_type +including O +the O +D B-protein_type +- I-protein_type +site I-protein_type +endonuclease I-protein_type +Nob1 B-protein +as O +well O +as O +Tsr1 B-protein +, O +a O +putative O +GTPase B-protein_type +and O +Rio2 B-protein +which O +block O +the O +mRNA B-site +channel I-site +and O +the O +initiator B-site +tRNA I-site +binding I-site +site I-site +, O +respectively O +, O +thus O +preventing O +translation O +initiation O +. O + +After O +structural O +changes O +, O +possibly O +driven O +by O +GTP B-chemical +hydrolysis O +, O +which O +go O +together O +with O +the O +formation O +of O +the O +decoding B-site +site I-site +, O +the O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +becomes O +accessible O +for O +Nob1 B-protein +cleavage O +at O +site B-site +D I-site +. O +This O +also O +involves O +joining O +of O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +and O +60S B-complex_assembly +subunits I-complex_assembly +to O +80S B-complex_assembly +- I-complex_assembly +like I-complex_assembly +particles I-complex_assembly +in O +a O +translation O +- O +like O +cycle O +promoted O +by O +eIF5B B-protein +. O + +The O +cleavage O +step O +most O +likely O +acts O +as O +a O +quality O +control O +check O +that O +ensures O +the O +proper O +40S B-complex_assembly +subunit I-complex_assembly +assembly O +with O +only O +completely O +processed O +precursors O +. O + +Finally O +, O +termination B-protein_type +factor I-protein_type +Rli1 B-protein +, O +an O +ATPase B-protein_type +, O +promotes O +the O +dissociation O +of O +assembly O +factors O +and O +the O +80S B-complex_assembly +- I-complex_assembly +like I-complex_assembly +complex I-complex_assembly +dissociates O +and O +releases O +the O +mature B-protein_state +40S B-complex_assembly +subunit I-complex_assembly +. O + +Interestingly O +, O +differences O +in O +the O +level O +of O +acp B-chemical +modification O +were O +demonstrated O +for O +different O +steps O +of O +the O +cytoplasmic O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +subunit I-complex_assembly +maturation O +after O +analyzing O +purified O +20S B-chemical +pre I-chemical +- I-chemical +rRNAs I-chemical +using O +different O +purification O +bait O +proteins O +. O + +Early O +cytoplasmic O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +subunits I-complex_assembly +still O +containing O +the O +ribosome B-protein_type +assembly I-protein_type +factors I-protein_type +Tsr1 B-protein +, O +Ltv1 B-protein +, O +Enp1 B-protein +and O +Rio2 B-protein +were O +not O +or O +only O +partially O +acp B-protein_state +modified I-protein_state +. O + +In O +contrast O +, O +late O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +subunits I-complex_assembly +containing O +Nob1 B-protein +and O +Rio1 B-protein +or O +already O +associated O +with O +60S B-complex_assembly +subunits I-complex_assembly +in O +80S B-complex_assembly +- I-complex_assembly +like I-complex_assembly +particles I-complex_assembly +showed O +acp B-chemical +modification O +levels O +comparable O +to O +mature B-protein_state +40S B-complex_assembly +subunits I-complex_assembly +. O + +Thus O +, O +the O +acp B-chemical +transfer O +to O +m1Ψ1191 B-residue_name_number +occurs O +during O +the O +step O +at O +which O +Rio2 B-protein +leaves O +the O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +particle I-complex_assembly +. O + +These O +data O +and O +the O +finding O +that O +a O +missing O +acp B-chemical +modification O +hinders O +pre B-chemical +- I-chemical +20S I-chemical +rRNA I-chemical +processing O +, O +suggest O +that O +the O +acp B-chemical +modification O +together O +with O +the O +release O +of O +Rio2 B-protein +promotes O +the O +formation O +of O +the O +decoding B-site +site I-site +and O +thus O +D B-site +- I-site +site I-site +cleavage O +by O +Nob1 B-protein +. O + +The O +interrelation O +between O +acp B-chemical +modification O +and O +Rio2 B-protein +release O +is O +also O +supported O +by O +CRAC B-experimental_method +analysis I-experimental_method +showing O +that O +Rio2 B-protein +binds O +to O +helix B-structure_element +31 I-structure_element +next O +to O +the O +Ψ1191 B-residue_name_number +residue O +that O +receives O +the O +acp B-chemical +modification O +. O + +Therefore O +, O +Rio2 B-protein +either O +blocks O +the O +access O +of O +Tsr3 B-protein +to O +helix B-structure_element +31 I-structure_element +, O +and O +acp B-chemical +modification O +can O +only O +occur O +after O +Rio2 B-protein +is O +released O +, O +or O +the O +acp B-chemical +modification O +of O +m1Ψ1191 B-residue_name_number +and O +putative O +subsequent O +conformational O +changes O +of O +20S B-chemical +rRNA I-chemical +weaken O +the O +binding O +of O +Rio2 B-protein +to O +helix B-structure_element +31 I-structure_element +and O +support O +its O +release O +from O +the O +pre B-chemical +- I-chemical +rRNA I-chemical +. O + +In O +summary O +, O +by O +identifying O +Tsr3 B-protein +as O +the O +enzyme O +responsible O +for O +introducing O +the O +acp B-chemical +group O +to O +the O +hypermodified B-protein_state +m1acp3Ψ B-chemical +nucleotide B-chemical +at O +position O +1191 B-residue_number +( O +yeast B-taxonomy_domain +)/ O +1248 B-residue_number +( O +humans B-species +) O +of O +18S B-chemical +rRNA I-chemical +we O +added O +one O +of O +the O +last O +remaining O +pieces O +to O +the O +puzzle O +of O +eukaryotic B-taxonomy_domain +small B-chemical +ribosomal I-chemical +subunit I-chemical +rRNA I-chemical +modifications O +. O + +The O +current O +data O +together O +with O +the O +finding O +that O +acp B-chemical +modification O +takes O +place O +at O +the O +very O +last O +step O +in O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +subunit I-complex_assembly +maturation O +indicate O +that O +the O +acp B-chemical +modification O +probably O +supports O +the O +formation O +of O +the O +decoding B-site +site I-site +and O +efficient O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +D B-site +- I-site +site I-site +cleavage O +. O + +Furthermore O +, O +our O +structural B-evidence +data I-evidence +unravelled O +how O +the O +regioselectivity O +of O +SAM B-chemical +- O +dependent O +group O +transfer O +reactions O +can O +be O +tuned O +by O +distinct O +small O +evolutionary O +adaptions O +of O +the O +ligand B-site +binding I-site +pocket I-site +of O +SAM B-protein_type +- I-protein_type +binding I-protein_type +enzymes I-protein_type +. O + +Structural O +insights O +into O +the O +regulatory O +mechanism O +of O +the O +Pseudomonas B-species +aeruginosa I-species +YfiBNR B-complex_assembly +system O + +YfiBNR B-complex_assembly +is O +a O +recently O +identified O +bis B-chemical +-( I-chemical +3 I-chemical +’- I-chemical +5 I-chemical +’)- I-chemical +cyclic I-chemical +dimeric I-chemical +GMP I-chemical +( O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +) O +signaling O +system O +in O +opportunistic O +pathogens O +. O + +In O +response O +to O +cell O +stress O +, O +YfiB B-protein +in O +the O +outer O +membrane O +can O +sequester O +the O +periplasmic O +protein O +YfiR B-protein +, O +releasing O +its O +inhibition O +of O +YfiN B-protein +on O +the O +inner O +membrane O +and O +thus O +provoking O +the O +diguanylate O +cyclase O +activity O +of O +YfiN B-protein +to O +induce O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +production O +. O + +Here O +, O +we O +report O +the O +crystal B-evidence +structures I-evidence +of O +YfiB B-protein +alone B-protein_state +and O +of O +an O +active B-protein_state +mutant B-protein_state +YfiBL43P B-mutant +complexed B-protein_state +with I-protein_state +YfiR B-protein +with O +2 O +: O +2 O +stoichiometry O +. O + +Structural B-experimental_method +analyses I-experimental_method +revealed O +that O +in O +contrast O +to O +the O +compact B-protein_state +conformation I-protein_state +of O +the O +dimeric B-oligomeric_state +YfiB B-protein +alone B-protein_state +, O +YfiBL43P B-mutant +adopts O +a O +stretched B-protein_state +conformation I-protein_state +allowing O +activated B-protein_state +YfiB B-protein +to O +penetrate O +the O +peptidoglycan B-chemical +( O +PG B-chemical +) O +layer O +and O +access O +YfiR B-protein +. O +YfiBL43P B-mutant +shows O +a O +more O +compact O +PG B-site +- I-site +binding I-site +pocket I-site +and O +much O +higher O +PG B-evidence +binding I-evidence +affinity I-evidence +than O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +, O +suggesting O +a O +tight O +correlation O +between O +PG O +binding O +and O +YfiB B-protein +activation O +. O + +In O +addition O +, O +our O +crystallographic B-experimental_method +analyses I-experimental_method +revealed O +that O +YfiR B-protein +binds O +Vitamin B-chemical +B6 I-chemical +( O +VB6 B-chemical +) O +or O +L B-chemical +- I-chemical +Trp I-chemical +at O +a O +YfiB B-site +- I-site +binding I-site +site I-site +and O +that O +both O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +are O +able O +to O +reduce O +YfiBL43P B-mutant +- O +induced O +biofilm O +formation O +. O + +Based O +on O +the O +structural B-evidence +and I-evidence +biochemical I-evidence +data I-evidence +, O +we O +propose O +an O +updated O +regulatory O +model O +of O +the O +YfiBNR B-complex_assembly +system O +. O + +Bis B-chemical +-( I-chemical +3 I-chemical +’- I-chemical +5 I-chemical +’)- I-chemical +cyclic I-chemical +dimeric I-chemical +GMP I-chemical +( O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +) O +is O +a O +ubiquitous O +second O +messenger O +that O +bacteria B-taxonomy_domain +use O +to O +facilitate O +behavioral O +adaptations O +to O +their O +ever O +- O +changing O +environment O +. O + +An O +increase O +in O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +promotes O +biofilm O +formation O +, O +and O +a O +decrease O +results O +in O +biofilm O +degradation O +( O +Boehm O +et O +al O +.,; O +Duerig O +et O +al O +.,; O +Hickman O +et O +al O +.,; O +Jenal O +,; O +Romling O +et O +al O +.,). O + +The O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +level O +is O +regulated O +by O +two O +reciprocal O +enzyme O +systems O +, O +namely O +, O +diguanylate B-protein_type +cyclases I-protein_type +( O +DGCs B-protein_type +) O +that O +synthesize O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +and O +phosphodiesterases B-protein_type +( O +PDEs B-protein_type +) O +that O +hydrolyze O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +( O +Kulasakara O +et O +al O +.,; O +Ross O +et O +al O +.,; O +Ross O +et O +al O +.,). O +Many O +of O +these O +enzymes O +are O +multiple O +- O +domain O +proteins O +containing O +a O +variable O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +that O +commonly O +acts O +as O +a O +signal O +sensor O +or O +transduction O +module O +, O +followed O +by O +the O +relatively B-protein_state +conserved I-protein_state +GGDEF B-structure_element +motif I-structure_element +in O +DGCs B-protein_type +or O +EAL B-structure_element +/ I-structure_element +HD I-structure_element +- I-structure_element +GYP I-structure_element +domains I-structure_element +in O +PDEs B-protein_type +( O +Hengge O +,; O +Navarro O +et O +al O +.,; O +Schirmer O +and O +Jenal O +,). O + +Intriguingly O +, O +studies O +in O +diverse O +species O +have O +revealed O +that O +a O +single O +bacterium B-taxonomy_domain +can O +have O +dozens O +of O +DGCs B-protein_type +and O +PDEs B-protein_type +( O +Hickman O +et O +al O +.,; O +Kirillina O +et O +al O +.,; O +Kulasakara O +et O +al O +.,; O +Tamayo O +et O +al O +.,). O + +In O +Pseudomonas B-species +aeruginosa I-species +in O +particular O +, O +42 O +genes O +containing O +putative O +DGCs B-protein_type +and O +/ O +or O +PDEs B-protein_type +were O +identified O +( O +Kulasakara O +et O +al O +.,). O + +The O +functional O +role O +of O +a O +number O +of O +downstream O +effectors O +of O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +has O +been O +characterized O +as O +affecting O +exopolysaccharide B-chemical +( O +EPS B-chemical +) O +production O +, O +transcription O +, O +motility O +, O +and O +surface O +attachment O +( O +Caly O +et O +al O +.,; O +Camilli O +and O +Bassler O +,; O +Ha O +and O +O O +’ O +Toole O +,; O +Pesavento O +and O +Hengge O +,). O + +However O +, O +due O +to O +the O +intricacy O +of O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +signaling O +networks O +and O +the O +diversity O +of O +experimental O +cues O +, O +the O +detailed O +mechanisms O +by O +which O +these O +signaling O +pathways O +specifically O +sense O +and O +integrate O +different O +inputs O +remain O +largely O +elusive O +. O + +Biofilm O +formation O +protects O +pathogenic O +bacteria B-taxonomy_domain +from O +antibiotic O +treatment O +, O +and O +c O +- O +di O +- O +GMP O +- O +regulated O +biofilm O +formation O +has O +been O +extensively O +studied O +in O +P B-species +. I-species +aeruginosa I-species +( O +Evans O +,; O +Kirisits O +et O +al O +.,; O +Malone O +,; O +Reinhardt O +et O +al O +.,). O + +In O +the O +lungs O +of O +cystic O +fibrosis O +( O +CF O +) O +patients O +, O +adherent O +biofilm O +formation O +and O +the O +appearance O +of O +small O +colony O +variant O +( O +SCV O +) O +morphologies O +of O +P B-species +. I-species +aeruginosa I-species +correlate O +with O +prolonged O +persistence O +of O +infection O +and O +poor O +lung O +function O +( O +Govan O +and O +Deretic O +,; O +Haussler O +et O +al O +.,; O +Haussler O +et O +al O +.,; O +Parsek O +and O +Singh O +,; O +Smith O +et O +al O +.,). O + +Recently O +, O +Malone O +and O +coworkers O +identified O +the O +tripartite B-protein_state +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +signaling O +module O +system O +YfiBNR B-complex_assembly +( O +also O +known O +as O +AwsXRO B-complex_assembly +( O +Beaumont O +et O +al O +.,; O +Giddens O +et O +al O +.,) O +or O +Tbp B-complex_assembly +( O +Ueda O +and O +Wood O +,)) O +by O +genetic B-experimental_method +screening I-experimental_method +for O +mutants O +that O +displayed O +SCV O +phenotypes O +in O +P B-species +. I-species +aeruginosa I-species +PAO1 I-species +( O +Malone O +et O +al O +.,; O +Malone O +et O +al O +.,). O + +The O +YfiBNR B-complex_assembly +system O +contains O +three O +protein O +members O +and O +modulates O +intracellular O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +levels O +in O +response O +to O +signals O +received O +in O +the O +periplasm O +( O +Malone O +et O +al O +.,). O + +More O +recently O +, O +this O +system O +was O +also O +reported O +in O +other O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +bacteria I-taxonomy_domain +, O +such O +as O +Escherichia B-species +coli I-species +( O +Hufnagel O +et O +al O +.,; O +Raterman O +et O +al O +.,; O +Sanchez O +- O +Torres O +et O +al O +.,), O +Klebsiella B-species +pneumonia I-species +( O +Huertas O +et O +al O +.,) O +and O +Yersinia B-species +pestis I-species +( O +Ren O +et O +al O +.,). O + +YfiN B-protein +is O +an O +integral O +inner O +- O +membrane O +protein O +with O +two O +potential O +transmembrane B-structure_element +helices I-structure_element +, O +a O +periplasmic O +Per B-structure_element +- I-structure_element +Arnt I-structure_element +- I-structure_element +Sim I-structure_element +( O +PAS B-structure_element +) O +domain O +, O +and O +cytosolic O +domains O +containing O +a O +HAMP B-structure_element +domain I-structure_element +( O +mediate O +input O +- O +output O +signaling O +in O +histidine B-protein_type +kinases I-protein_type +, O +adenylyl B-protein_type +cyclases I-protein_type +, O +methyl B-protein_type +- I-protein_type +accepting I-protein_type +chemotaxis I-protein_type +proteins I-protein_type +, O +and O +phosphatases B-protein_type +) O +and O +a O +C O +- O +terminal O +GGDEF B-structure_element +domain I-structure_element +indicating O +a O +DGC B-protein_type +’ O +s O +function O +( O +Giardina O +et O +al O +.,; O +Malone O +et O +al O +.,). O + +YfiN B-protein +is O +repressed B-protein_state +by I-protein_state +specific O +interaction O +between O +its O +periplasmic O +PAS B-structure_element +domain I-structure_element +and O +the O +periplasmic O +protein O +YfiR B-protein +( O +Malone O +et O +al O +.,). O + +YfiB B-protein +is O +an O +OmpA B-protein_type +/ I-protein_type +Pal I-protein_type +- I-protein_type +like I-protein_type +outer O +- O +membrane O +lipoprotein B-protein_type +( O +Parsons O +et O +al O +.,) O +that O +can O +activate O +YfiN B-protein +by O +sequestering O +YfiR B-protein +( O +Malone O +et O +al O +.,) O +in O +an O +unknown O +manner O +. O + +Whether O +YfiB B-protein +directly O +recruits O +YfiR B-protein +or O +recruits O +YfiR B-protein +via O +a O +third O +partner O +is O +an O +open O +question O +. O + +After O +the O +sequestration O +of O +YfiR B-protein +by O +YfiB B-protein +, O +the O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +produced O +by O +activated B-protein_state +YfiN B-protein +increases O +the O +biosynthesis O +of O +the O +Pel B-chemical +and O +Psl B-chemical +EPSs B-chemical +, O +resulting O +in O +the O +appearance O +of O +the O +SCV O +phenotype O +, O +which O +indicates O +enhanced O +biofilm O +formation O +( O +Malone O +et O +al O +.,). O + +It O +has O +been O +reported O +that O +the O +activation O +of O +YfiN B-protein +may O +be O +induced O +by O +redox O +- O +driven O +misfolding O +of O +YfiR B-protein +( O +Giardina O +et O +al O +.,; O +Malone O +et O +al O +.,; O +Malone O +et O +al O +.,). O + +It O +is O +also O +proposed O +that O +the O +sequestration O +of O +YfiR B-protein +by O +YfiB B-protein +can O +be O +induced O +by O +certain O +YfiB B-protein +- O +mediated O +cell O +wall O +stress O +, O +and O +mutagenesis B-experimental_method +studies I-experimental_method +revealed O +a O +number O +of O +activation B-structure_element +residues I-structure_element +of O +YfiB B-protein +that O +were O +located O +in O +close O +proximity O +to O +the O +predicted B-protein_state +first B-structure_element +helix I-structure_element +of O +the O +periplasmic B-structure_element +domain I-structure_element +( O +Malone O +et O +al O +.,). O + +In O +addition O +, O +quorum O +sensing O +- O +related O +dephosphorylation O +of O +the O +PAS B-structure_element +domain I-structure_element +of O +YfiN B-protein +may O +also O +be O +involved O +in O +the O +regulation O +( O +Ueda O +and O +Wood O +,; O +Xu O +et O +al O +.,). O + +Recently O +, O +we O +solved O +the O +crystal B-evidence +structure I-evidence +of O +YfiR B-protein +in O +both O +the O +non B-protein_state +- I-protein_state +oxidized I-protein_state +and O +the O +oxidized B-protein_state +states O +, O +revealing O +breakage O +/ O +formation O +of O +one O +disulfide B-ptm +bond I-ptm +( O +Cys71 B-residue_name_number +- O +Cys110 B-residue_name_number +) O +and O +local O +conformational O +change O +around O +the O +other O +one O +( O +Cys145 B-residue_name_number +- O +Cys152 B-residue_name_number +), O +indicating O +that O +Cys145 B-residue_name_number +- O +Cys152 B-residue_name_number +plays O +an O +important O +role O +in O +maintaining O +the O +correct O +folding O +of O +YfiR B-protein +( O +Yang O +et O +al O +.,). O + +In O +the O +present O +study O +, O +we O +solved O +the O +crystal B-evidence +structures I-evidence +of O +an O +N O +- O +terminal O +truncated B-protein_state +form O +of O +YfiB B-protein +( O +34 B-residue_range +– I-residue_range +168 I-residue_range +) O +and O +YfiR B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +an O +active B-protein_state +mutant B-protein_state +YfiBL43P B-mutant +. O + +Most O +recently O +, O +Li O +and O +coworkers O +reported O +the O +crystal B-evidence +structures I-evidence +of O +YfiB B-protein +( O +27 B-residue_range +– I-residue_range +168 I-residue_range +) O +alone B-protein_state +and O +YfiRC71S B-mutant +in B-protein_state +complex I-protein_state +with I-protein_state +YfiB B-protein +( O +59 B-residue_range +– I-residue_range +168 I-residue_range +) O +( O +Li O +et O +al O +.,). O + +Compared O +with O +the O +reported O +complex O +structure O +, O +YfiBL43P B-mutant +in O +our O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +structure B-evidence +has O +additional O +visible O +N O +- O +terminal O +residues O +44 B-residue_range +– I-residue_range +58 I-residue_range +that O +are O +shown O +to O +play O +essential O +roles O +in O +YfiB B-protein +activation O +and O +biofilm O +formation O +. O + +Therefore O +, O +we O +are O +able O +to O +visualize O +the O +detailed O +allosteric O +arrangement O +of O +the O +N O +- O +terminal O +structure O +of O +YfiB B-protein +and O +its O +important O +role O +in O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +interaction O +. O + +In O +addition O +, O +we O +found O +that O +the O +YfiBL43P B-mutant +shows O +a O +much O +higher O +PG B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +than O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +, O +most O +likely O +due O +to O +its O +more O +compact O +PG B-site +- I-site +binding I-site +pocket I-site +. O + +Moreover O +, O +we O +found O +that O +Vitamin B-chemical +B6 I-chemical +( O +VB6 B-chemical +) O +or O +L B-chemical +- I-chemical +Trp I-chemical +can O +bind O +YfiR B-protein +with O +an O +affinity B-evidence +in O +the O +ten O +millimolar O +range O +. O + +Together O +with O +functional O +data O +, O +these O +results O +provide O +new O +mechanistic O +insights O +into O +how O +activated B-protein_state +YfiB B-protein +sequesters O +YfiR B-protein +and O +releases O +the O +suppression O +of O +YfiN B-protein +. O +These O +findings O +may O +facilitate O +the O +development O +and O +optimization O +of O +anti O +- O +biofilm O +drugs O +for O +the O +treatment O +of O +chronic O +infections O +. O + +Overall O +structure B-evidence +of O +YfiB B-protein + +We O +obtained O +two O +crystal B-evidence +forms I-evidence +of O +YfiB B-protein +( O +residues O +34 B-residue_range +– I-residue_range +168 I-residue_range +, O +lacking B-protein_state +the O +signal B-structure_element +peptide I-structure_element +from O +residues O +1 B-residue_range +– I-residue_range +26 I-residue_range +and O +periplasmic O +residues O +27 B-residue_range +– I-residue_range +33 I-residue_range +), O +crystal O +forms O +I O +and O +II O +, O +belonging O +to O +space O +groups O +P21 O +and O +P41 O +, O +respectively O +. O + +Overall O +structure B-evidence +of O +YfiB B-protein +. O +( O +A O +) O +The O +overall O +structure B-evidence +of O +the O +YfiB B-protein +monomer B-oligomeric_state +. O +( O +B O +) O +A O +topology O +diagram O +of O +the O +YfiB B-protein +monomer B-oligomeric_state +. O +( O +C O +and O +D O +) O +The O +analytical B-experimental_method +ultracentrifugation I-experimental_method +experiment O +results O +for O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +and O +YfiBL43P B-mutant + +Two O +dimeric B-oligomeric_state +types O +of O +YfiB B-protein +dimer B-oligomeric_state +. O +( O +A O +– O +C O +) O +The O +“ O +head B-protein_state +to I-protein_state +head I-protein_state +” O +dimer B-oligomeric_state +. O + +The O +“ O +back B-protein_state +to I-protein_state +back I-protein_state +” O +dimer B-oligomeric_state +. O + +( O +A O +) O +and O +( O +E O +) O +indicate O +the O +front O +views O +of O +the O +two O +dimers B-oligomeric_state +, O +( O +B O +) O +and O +( O +F O +) O +indicate O +the O +top O +views O +of O +the O +two O +dimers B-oligomeric_state +, O +and O +( O +C O +) O +and O +( O +D O +) O +indicate O +the O +details O +of O +the O +two O +dimeric B-site +interfaces I-site + +The O +crystal B-evidence +structure I-evidence +of O +YfiB B-protein +monomer B-oligomeric_state +consists O +of O +a O +five B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +( O +β1 B-structure_element +- I-structure_element +2 I-structure_element +- I-structure_element +5 I-structure_element +- I-structure_element +3 I-structure_element +- I-structure_element +4 I-structure_element +) O +flanked O +by O +five B-structure_element +α I-structure_element +- I-structure_element +helices I-structure_element +( O +α1 B-structure_element +– I-structure_element +5 I-structure_element +) O +on O +one O +side O +. O + +In O +addition O +, O +there O +is O +a O +short O +helix B-structure_element +turn I-structure_element +connecting O +the O +β4 B-structure_element +strand I-structure_element +and O +α4 B-structure_element +helix I-structure_element +( O +Fig O +. O +1A O +and O +1B O +). O + +Each O +crystal O +form O +contains O +three O +different O +dimeric B-oligomeric_state +types O +of O +YfiB B-protein +, O +two O +of O +which O +are O +present O +in O +both O +, O +suggesting O +that O +the O +rest O +of O +the O +dimeric B-oligomeric_state +types O +may O +result O +from O +crystal O +packing O +. O + +Here O +, O +we O +refer O +to O +the O +two O +dimeric B-oligomeric_state +types O +as O +“ O +head B-protein_state +to I-protein_state +head I-protein_state +” O +and O +“ O +back B-protein_state +to I-protein_state +back I-protein_state +” O +according O +to O +the O +interacting O +mode O +( O +Fig O +. O +2A O +and O +2E O +), O +with O +the O +total O +buried O +surface O +areas O +being O +316 O +. O +8 O +Å2 O +and O +554 O +. O +3 O +Å2 O +, O +respectively O +. O + +The O +“ O +head B-protein_state +to I-protein_state +head I-protein_state +” O +dimer B-oligomeric_state +exhibits O +a O +clamp B-protein_state +shape I-protein_state +. O + +The O +dimerization O +occurs O +mainly O +via O +hydrophobic B-bond_interaction +interactions I-bond_interaction +formed O +by O +A37 B-residue_name_number +and O +I40 B-residue_name_number +on O +the O +α1 B-structure_element +helices I-structure_element +, O +L50 B-residue_name_number +on O +the O +β1 B-structure_element +strands I-structure_element +, O +and O +W55 B-residue_name_number +on O +the O +β2 B-structure_element +strands I-structure_element +of O +both O +molecules O +, O +making O +a O +hydrophobic B-site +interacting I-site +core I-site +( O +Fig O +. O +2A O +– O +C O +). O + +The O +“ O +back B-protein_state +to I-protein_state +back I-protein_state +” O +dimer B-oligomeric_state +presents O +a O +Y B-protein_state +shape I-protein_state +. O + +The O +dimeric B-oligomeric_state +interaction B-bond_interaction +is I-bond_interaction +mainly I-bond_interaction +hydrophilic I-bond_interaction +, O +occurring O +among O +the O +main O +- O +chain O +and O +side O +- O +chain O +atoms O +of O +N68 B-residue_name_number +, O +L69 B-residue_name_number +, O +D70 B-residue_name_number +and O +R71 B-residue_name_number +on O +the O +α2 B-structure_element +- I-structure_element +α3 I-structure_element +loops I-structure_element +and O +R116 B-residue_name_number +and O +S120 B-residue_name_number +on O +the O +α4 B-structure_element +helices I-structure_element +of O +both O +molecules O +, O +resulting O +in O +a O +complex O +hydrogen B-site +bond I-site +network I-site +( O +Fig O +. O +2D O +– O +F O +). O + +The O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +interaction O + +Overall O +structure B-evidence +of O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +and O +the O +conserved B-site +surface I-site +in O +YfiR B-protein +. O +( O +A O +) O +The O +overall O +structure B-evidence +of O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +. O + +The O +YfiBL43P B-mutant +molecules O +are O +shown O +in O +cyan O +and O +yellow O +. O + +The O +YfiR B-protein +molecules O +are O +shown O +in O +green O +and O +magenta O +. O + +Two O +interacting O +regions O +are O +highlighted O +by O +red O +rectangles O +. O +( O +B O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +apo B-protein_state +YfiB B-protein +and O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +. O + +To O +illustrate O +the O +differences O +between O +apo B-protein_state +YfiB B-protein +and O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +, O +the O +apo B-protein_state +YfiB B-protein +is O +shown O +in O +pink O +, O +except O +residues O +34 B-residue_range +– I-residue_range +70 I-residue_range +are O +shown O +in O +red O +, O +whereas O +the O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +is O +shown O +in O +cyan O +, O +except O +residues O +44 B-residue_range +– I-residue_range +70 I-residue_range +are O +shown O +in O +blue O +. O +( O +C O +) O +Close O +- O +up O +view O +of O +the O +differences O +between O +apo B-protein_state +YfiB B-protein +and O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +. O + +The O +residues O +proposed O +to O +contribute O +to O +YfiB B-protein +activation O +are O +illustrated O +in O +sticks O +. O + +The O +key O +residues O +in O +apo B-protein_state +YfiB B-protein +are O +shown O +in O +red O +and O +those O +in O +YfiBL43P B-mutant +are O +shown O +in O +blue O +. O +( O +D O +) O +Close O +- O +up O +views O +showing O +interactions O +in O +regions B-structure_element +I I-structure_element +and I-structure_element +II I-structure_element +. O + +YfiBL43P B-mutant +and O +YfiR B-protein +are O +shown O +in O +cyan O +and O +green O +, O +respectively O +. O +( O +E O +and O +F O +) O +The O +conserved B-site +surface I-site +in O +YfiR B-protein +contributes O +to O +the O +interaction O +with O +YfiB B-protein +. O +( O +G O +) O +The O +residues B-structure_element +of O +YfiR B-protein +responsible O +for O +interacting O +with O +YfiB B-protein +are O +shown O +in O +green O +sticks O +, O +and O +the O +proposed O +YfiN B-site +- I-site +interacting I-site +residues I-site +are O +shown O +in O +yellow O +sticks O +. O + +The O +red O +sticks O +, O +which O +represent O +the O +YfiB B-site +- I-site +interacting I-site +residues I-site +, O +are O +also O +responsible O +for O +the O +proposed O +interactions O +with O +YfiN B-protein + +To O +gain O +structural O +insights O +into O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +interaction O +, O +we O +co B-experimental_method +- I-experimental_method +expressed I-experimental_method +YfiB B-protein +( O +residues O +34 B-residue_range +– I-residue_range +168 I-residue_range +) O +and O +YfiR B-protein +( O +residues O +35 B-residue_range +– I-residue_range +190 I-residue_range +, O +lacking B-protein_state +the O +signal B-structure_element +peptide I-structure_element +), O +but O +failed O +to O +obtain O +the O +complex O +, O +in O +accordance O +with O +a O +previous O +report O +in O +which O +no B-protein_state +stable I-protein_state +complex O +of O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +was O +observed O +( O +Malone O +et O +al O +.,). O + +It O +has O +been O +reported O +that O +single B-experimental_method +mutants I-experimental_method +of I-experimental_method +Q39 B-residue_name_number +, O +L43 B-residue_name_number +, O +F48 B-residue_name_number +and O +W55 B-residue_name_number +contribute O +to O +YfiB B-protein +activation O +leading O +to O +the O +induction O +of O +the O +SCV O +phenotype O +in O +P B-species +. I-species +aeruginosa I-species +PAO1 I-species +( O +Malone O +et O +al O +.,). O + +It O +is O +likely O +that O +these O +residues O +may O +be O +involved O +in O +the O +conformational O +changes O +of O +YfiB B-protein +that O +are O +related O +to O +YfiR B-protein +sequestration O +( O +Fig O +. O +3C O +). O + +Therefore O +, O +we O +constructed B-experimental_method +two I-experimental_method +such I-experimental_method +single I-experimental_method +mutants I-experimental_method +of O +YfiB B-protein +( O +YfiBL43P B-mutant +and O +YfiBF48S B-mutant +). O + +As O +expected O +, O +both O +mutants O +form O +a O +stable B-protein_state +complex B-protein_state +with I-protein_state +YfiR B-protein +. O +Finally O +, O +we O +crystalized B-experimental_method +YfiR B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +YfiBL43P B-mutant +mutant B-protein_state +and O +solved O +the O +structure B-evidence +at O +1 O +. O +78 O +Å O +resolution O +by O +molecular B-experimental_method +replacement I-experimental_method +using O +YfiR B-protein +and O +YfiB B-protein +as O +models O +. O + +The O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +is O +a O +2 O +: O +2 O +heterotetramer B-oligomeric_state +( O +Fig O +. O +3A O +) O +in O +which O +the O +YfiR B-protein +dimer B-oligomeric_state +is O +clamped O +by O +two O +separated O +YfiBL43P B-mutant +molecules O +with O +a O +total O +buried O +surface O +area O +of O +3161 O +. O +2 O +Å2 O +. O + +The O +YfiR B-protein +dimer B-oligomeric_state +in O +the O +complex O +is O +identical O +to O +the O +non B-protein_state +- I-protein_state +oxidized I-protein_state +YfiR B-protein +dimer B-oligomeric_state +alone B-protein_state +( O +Yang O +et O +al O +.,), O +with O +only O +Cys145 B-residue_name_number +- O +Cys152 B-residue_name_number +of O +the O +two O +disulfide B-ptm +bonds I-ptm +well O +formed O +, O +suggesting O +Cys71 B-residue_name_number +- O +Cys110 B-residue_name_number +disulfide B-ptm +bond I-ptm +formation O +is O +not O +essential O +for O +forming O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +. O + +The O +N O +- O +terminal O +structural O +conformation O +of O +YfiBL43P B-mutant +, O +from O +the O +foremost O +N O +- O +terminus O +to O +residue O +D70 B-residue_name_number +, O +is O +significantly O +altered O +compared O +with O +that O +of O +the O +apo B-protein_state +YfiB B-protein +. O +The O +majority O +of O +the O +α1 B-structure_element +helix I-structure_element +( O +residues O +34 B-residue_range +– I-residue_range +43 I-residue_range +) O +is O +invisible O +on O +the O +electron B-evidence +density I-evidence +map I-evidence +, O +and O +the O +α2 B-structure_element +helix I-structure_element +and O +β1 B-structure_element +and O +β2 B-structure_element +strands I-structure_element +are O +rearranged O +to O +form O +a O +long O +loop B-structure_element +containing O +two O +short O +α B-structure_element +- I-structure_element +helix I-structure_element +turns I-structure_element +( O +Fig O +. O +3B O +and O +3C O +), O +thus O +embracing O +the O +YfiR B-protein +dimer B-oligomeric_state +. O + +The O +observed O +changes O +in O +conformation O +of O +YfiB B-protein +and O +the O +results O +of O +mutagenesis B-experimental_method +suggest O +a O +mechanism O +by O +which O +YfiB B-protein +sequesters O +YfiR B-protein +. O + +The O +YfiB B-site +- I-site +YfiR I-site +interface I-site +can O +be O +divided O +into O +two O +regions O +( O +Fig O +. O +3A O +and O +3D O +). O + +Region B-structure_element +I I-structure_element +is O +formed O +by O +numerous O +main O +- O +chain O +and O +side O +- O +chain O +hydrophilic B-bond_interaction +interactions I-bond_interaction +between O +residues O +E45 B-residue_name_number +, O +G47 B-residue_name_number +and O +E53 B-residue_name_number +from O +the O +N O +- O +terminal O +extended O +loop B-structure_element +of O +YfiB B-protein +and O +residues O +S57 B-residue_name_number +, O +R60 B-residue_name_number +, O +A89 B-residue_name_number +and O +H177 B-residue_name_number +from O +YfiR B-protein +( O +Fig O +. O +3D O +- O +I O +( O +i O +)). O + +Additionally O +, O +three O +hydrophobic B-site +anchoring I-site +sites I-site +exist O +in O +region B-structure_element +I I-structure_element +. O +The O +residues O +F48 B-residue_name_number +and O +W55 B-residue_name_number +of O +YfiB B-protein +are O +inserted O +into O +the O +hydrophobic B-site +cores I-site +mainly O +formed O +by O +the O +main O +chain O +and O +side O +chain O +carbon O +atoms O +of O +residues O +S57 B-residue_name_number +/ O +Q88 B-residue_name_number +/ O +A89 B-residue_name_number +/ O +N90 B-residue_name_number +and O +R60 B-residue_name_number +/ O +R175 B-residue_name_number +/ O +H177 B-residue_name_number +of O +YfiR B-protein +, O +respectively O +; O +and O +F57 B-residue_name_number +of O +YfiB B-protein +is O +inserted O +into O +the O +hydrophobic B-site +pocket I-site +formed O +by O +L166 B-residue_name_number +/ O +I169 B-residue_name_number +/ O +V176 B-residue_name_number +/ O +P178 B-residue_name_number +/ O +L181 B-residue_name_number +of O +YfiR B-protein +( O +Fig O +. O +3D O +- O +I O +( O +ii O +)). O + +In O +region B-structure_element +II I-structure_element +, O +the O +side O +chains O +of O +R96 B-residue_name_number +, O +E98 B-residue_name_number +and O +E157 B-residue_name_number +from O +YfiB B-protein +interact O +with O +the O +side O +chains O +of O +E163 B-residue_name_number +, O +S146 B-residue_name_number +and O +R171 B-residue_name_number +from O +YfiR B-protein +, O +respectively O +. O + +Additionally O +, O +the O +main O +chains O +of O +I163 B-residue_name_number +and O +V165 B-residue_name_number +from O +YfiB B-protein +form O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +main O +chains O +of O +L166 B-residue_name_number +and O +A164 B-residue_name_number +from O +YfiR B-protein +, O +respectively O +, O +and O +the O +main O +chain O +of O +P166 B-residue_name_number +from O +YfiB B-protein +interacts O +with O +the O +side O +chain O +of O +R185 B-residue_name_number +from O +YfiR B-protein +( O +Fig O +. O +3D O +- O +II O +). O + +These O +two O +regions O +contribute O +a O +robust O +hydrogen B-site +- I-site +bonding I-site +network I-site +to O +the O +YfiB B-site +- I-site +YfiR I-site +interface I-site +, O +resulting O +in O +a O +tightly O +bound O +complex O +. O + +Based O +on O +the O +observations O +that O +two O +separated O +YfiBL43P B-mutant +molecules O +form O +a O +2 O +: O +2 O +complex O +structure B-evidence +with O +YfiR B-protein +dimer B-oligomeric_state +, O +we O +performed O +an O +analytical B-experimental_method +ultracentrifugation I-experimental_method +experiment O +to O +check O +the O +oligomeric O +states O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +and O +YfiBL43P B-mutant +. O + +The O +results O +showed O +that O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +exists O +in O +both O +monomeric B-oligomeric_state +and O +dimeric B-oligomeric_state +states O +in O +solution O +, O +while O +YfiBL43P B-mutant +primarily O +adopts O +the O +monomer B-oligomeric_state +state O +in O +solution O +( O +Fig O +. O +1C O +– O +D O +). O + +This O +suggests O +that O +the O +N O +- O +terminus O +of O +YfiB B-protein +plays O +an O +important O +role O +in O +forming O +the O +dimeric B-oligomeric_state +YfiB B-protein +in O +solution O +and O +that O +the O +conformational O +change O +of O +residue O +L43 B-residue_name_number +is O +associated O +with O +the O +stretch O +of O +the O +N O +- O +terminus O +and O +opening O +of O +the O +dimer B-oligomeric_state +. O + +Therefore O +, O +it O +is O +possible O +that O +both O +dimeric B-oligomeric_state +types O +might O +exist O +in O +solution O +. O + +For O +simplicity O +, O +we O +only O +discuss O +the O +“ O +head B-protein_state +to I-protein_state +head I-protein_state +” O +dimer B-oligomeric_state +in O +the O +following O +text O +. O + +The O +PG B-site +- I-site +binding I-site +site I-site +of O +YfiB B-protein + +The O +PG B-site +- I-site +binding I-site +site I-site +in O +YfiB B-protein +. O +( O +A O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +PG B-site +- I-site +binding I-site +sites I-site +of O +the O +H B-species +. I-species +influenzae I-species +Pal B-complex_assembly +/ I-complex_assembly +PG I-complex_assembly +- I-complex_assembly +P I-complex_assembly +complex O +and O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +complexed B-protein_state +with I-protein_state +sulfate B-chemical +ions O +. O + +( O +B O +) O +Close O +- O +up O +view O +showing O +the O +key O +residues O +of O +Pal B-protein_type +interacting O +with O +the O +m B-chemical +- I-chemical +Dap5 I-chemical +ε I-chemical +- I-chemical +carboxylate I-chemical +group O +of O +PG B-chemical +- I-chemical +P I-chemical +. O +Pal B-protein_type +is O +shown O +in O +wheat O +and O +PG B-chemical +- I-chemical +P I-chemical +is O +in O +magenta O +. O + +( O +C O +) O +Close O +- O +up O +view O +showing O +the O +key O +residues O +of O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +interacting O +with O +a O +sulfate B-chemical +ion O +. O + +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +is O +shown O +in O +cyan O +; O +the O +sulfate B-chemical +ion O +, O +in O +green O +; O +and O +the O +water B-chemical +molecule O +, O +in O +yellow O +. O +( O +D O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +PG B-site +- I-site +binding I-site +sites I-site +of O +apo B-protein_state +YfiB B-protein +and O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +, O +the O +key O +residues O +are O +shown O +in O +stick O +. O + +Apo B-protein_state +YfiB B-protein +is O +shown O +in O +yellow O +and O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +in O +cyan O +. O +( O +E O +and O +F O +) O +MST B-experimental_method +data O +and O +analysis O +for O +binding B-evidence +affinities I-evidence +of O +( O +E O +) O +YfiB B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +and O +( O +F O +) O +YfiBL43P B-mutant +with O +PG B-chemical +. O +( O +G O +) O +The O +sequence B-experimental_method +alignment I-experimental_method +of O +P B-species +. I-species +aeruginosa I-species +and O +E B-species +. I-species +coli I-species +sources O +of O +YfiB B-protein +, O +Pal B-protein_type +and O +the O +periplasmic B-structure_element +domain I-structure_element +of O +OmpA B-protein_type + +PG B-protein_type +- I-protein_type +associated I-protein_type +lipoprotein I-protein_type +( O +Pal B-protein_type +) O +is O +highly B-protein_state +conserved I-protein_state +in O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +bacteria I-taxonomy_domain +and O +anchors O +to O +the O +outer O +membrane O +through O +an O +N O +- O +terminal O +lipid O +attachment O +and O +to O +PG B-chemical +layer O +through O +its O +periplasmic B-structure_element +domain I-structure_element +, O +which O +is O +implicated O +in O +maintaining O +outer O +membrane O +integrity O +. O + +Previous O +homology B-experimental_method +modeling I-experimental_method +studies O +suggested O +that O +YfiB B-protein +contains O +a O +Pal B-site +- I-site +like I-site +PG I-site +- I-site +binding I-site +site I-site +( O +Parsons O +et O +al O +.,), O +and O +the O +mutation B-experimental_method +of I-experimental_method +two I-experimental_method +residues I-experimental_method +at O +this O +site O +, O +D102 B-residue_name_number +and O +G105 B-residue_name_number +, O +reduces O +the O +ability O +for O +biofilm O +formation O +and O +surface O +attachment O +( O +Malone O +et O +al O +.,). O + +In O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +, O +one O +sulfate B-chemical +ion O +is O +found O +at O +the O +bottom O +of O +each O +YfiBL43P B-mutant +molecule O +( O +Fig O +. O +3A O +) O +and O +forms O +a O +strong O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +D102 B-residue_name_number +of O +YfiBL43P B-mutant +( O +Fig O +. O +4A O +and O +4C O +). O + +Structural B-experimental_method +superposition I-experimental_method +between O +YfiBL43P B-mutant +and O +Haemophilus B-species +influenzae I-species +Pal B-protein_type +complexed B-protein_state +with I-protein_state +biosynthetic O +peptidoglycan B-chemical +precursor I-chemical +( O +PG B-chemical +- I-chemical +P I-chemical +), O +UDP B-chemical +- I-chemical +N I-chemical +- I-chemical +acetylmuramyl I-chemical +- I-chemical +L I-chemical +- I-chemical +Ala I-chemical +- I-chemical +α I-chemical +- I-chemical +D I-chemical +- I-chemical +Glu I-chemical +- I-chemical +m I-chemical +- I-chemical +Dap I-chemical +- I-chemical +D I-chemical +- I-chemical +Ala I-chemical +- I-chemical +D I-chemical +- I-chemical +Ala I-chemical +( O +m B-chemical +- I-chemical +Dap I-chemical +is O +meso B-chemical +- I-chemical +diaminopimelate I-chemical +) O +( O +PDB O +code O +: O +2aiz O +) O +( O +Parsons O +et O +al O +.,), O +revealed O +that O +the O +sulfate B-chemical +ion O +is O +located O +at O +the O +position O +of O +the O +m B-chemical +- I-chemical +Dap5 I-chemical +ϵ I-chemical +- I-chemical +carboxylate I-chemical +group O +in O +the O +Pal B-complex_assembly +/ I-complex_assembly +PG I-complex_assembly +- I-complex_assembly +P I-complex_assembly +complex O +( O +Fig O +. O +4A O +). O + +In O +the O +Pal B-complex_assembly +/ I-complex_assembly +PG I-complex_assembly +- I-complex_assembly +P I-complex_assembly +complex O +structure B-evidence +, O +the O +m B-chemical +- I-chemical +Dap5 I-chemical +ϵ I-chemical +- I-chemical +carboxylate I-chemical +group O +interacts O +with O +the O +side O +- O +chain O +atoms O +of O +D71 B-residue_name_number +and O +the O +main O +- O +chain O +amide O +of O +D37 B-residue_name_number +( O +Fig O +. O +4B O +). O + +Similarly O +, O +in O +the O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +structure B-evidence +, O +the O +sulfate B-chemical +ion O +interacts O +with O +the O +side O +- O +chain O +atoms O +of O +D102 B-residue_name_number +( O +corresponding O +to O +D71 B-residue_name_number +in O +Pal B-protein_type +) O +and O +R117 B-residue_name_number +( O +corresponding O +to O +R86 B-residue_name_number +in O +Pal B-protein_type +) O +and O +the O +main O +- O +chain O +amide O +of O +N68 B-residue_name_number +( O +corresponding O +to O +D37 B-residue_name_number +in O +Pal B-protein_type +). O + +Moreover O +, O +a O +water B-chemical +molecule O +was O +found O +to O +bridge O +the O +sulfate B-chemical +ion O +and O +the O +side O +chains O +of O +N67 B-residue_name_number +and O +D102 B-residue_name_number +, O +strengthening O +the O +hydrogen B-site +bond I-site +network I-site +( O +Fig O +. O +4C O +). O + +In O +addition O +, O +sequence B-experimental_method +alignment I-experimental_method +of O +YfiB B-protein +with O +Pal B-protein_type +and O +the O +periplasmic B-structure_element +domain I-structure_element +of O +OmpA B-protein_type +( O +proteins O +containing O +PG B-site +- I-site +binding I-site +site I-site +) O +showed O +that O +N68 B-residue_name_number +and O +D102 B-residue_name_number +are O +highly B-protein_state +conserved I-protein_state +( O +Fig O +. O +4G O +, O +blue O +stars O +), O +suggesting O +that O +these O +residues O +contribute O +to O +the O +PG O +- O +binding O +ability O +of O +YfiB B-protein +. O + +Interestingly O +, O +superposition B-experimental_method +of O +apo B-protein_state +YfiB B-protein +with O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +revealed O +that O +the O +PG B-site +- I-site +binding I-site +region I-site +is O +largely O +altered O +mainly O +due O +to O +different B-protein_state +conformation I-protein_state +of O +the O +N68 B-residue_name_number +containing O +loop B-structure_element +. O + +Compared O +to O +YfiBL43P B-mutant +, O +the O +N68 B-residue_name_number +- O +containing O +loop B-structure_element +of O +the O +apo B-protein_state +YfiB B-protein +flips O +away O +about O +7 O +Å O +, O +and O +D102 B-residue_name_number +and O +R117 B-residue_name_number +swing O +slightly O +outward O +; O +thus O +, O +the O +PG B-site +- I-site +binding I-site +pocket I-site +is O +enlarged O +with O +no O +sulfate B-chemical +ion O +or O +water B-chemical +bound O +( O +Fig O +. O +4D O +). O + +Therefore O +, O +we O +proposed O +that O +the O +PG B-chemical +- O +binding O +ability O +of O +inactive B-protein_state +YfiB B-protein +might O +be O +weaker O +than O +that O +of O +active B-protein_state +YfiB B-protein +. O +To O +validate O +this O +, O +we O +performed O +a O +microscale B-experimental_method +thermophoresis I-experimental_method +( O +MST B-experimental_method +) O +assay O +to O +measure O +the O +binding B-evidence +affinities I-evidence +of O +PG B-chemical +to O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +and O +YfiBL43P B-mutant +, O +respectively O +. O + +The O +results O +indicated O +that O +the O +PG B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +YfiBL43P B-mutant +is O +65 O +. O +5 O +μmol O +/ O +L O +, O +which O +is O +about O +16 O +- O +fold O +stronger O +than O +that O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +( O +Kd B-evidence += O +1 O +. O +1 O +mmol O +/ O +L O +) O +( O +Fig O +. O +4E O +– O +F O +). O + +As O +the O +experiment O +is O +performed O +in B-protein_state +the I-protein_state +absence I-protein_state +of I-protein_state +YfiR B-protein +, O +it O +suggests O +that O +an O +increase O +in O +the O +PG B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +YfiB B-protein +is O +not O +a O +result O +of O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +interaction O +and O +is O +highly O +coupled O +to O +the O +activation O +of O +YfiB B-protein +characterized O +by O +a O +stretched B-protein_state +N I-protein_state +- I-protein_state +terminal I-protein_state +conformation I-protein_state +. O + +The O +conserved B-site +surface I-site +in O +YfiR B-protein +is O +functional O +for O +binding O +YfiB B-protein +and O +YfiN B-protein + +Calculation O +using O +the O +ConSurf B-experimental_method +Server I-experimental_method +( O +http O +:// O +consurf O +. O +tau O +. O +ac O +. O +il O +/), O +which O +estimates O +the O +evolutionary B-evidence +conservation I-evidence +of O +amino O +acid O +positions O +and O +visualizes O +information O +on O +the O +structure B-site +surface I-site +, O +revealed O +a O +conserved B-site +surface I-site +on O +YfiR B-protein +that O +contributes O +to O +the O +interaction O +with O +YfiB B-protein +( O +Fig O +. O +3E O +and O +3F O +). O + +Interestingly O +, O +the O +majority O +of O +this O +conserved B-site +surface I-site +contributes O +to O +the O +interaction O +with O +YfiB B-protein +( O +Fig O +. O +3E O +and O +3F O +). O + +Malone O +JG O +et O +al O +. O +have O +reported O +that O +F151 B-residue_name_number +, O +E163 B-residue_name_number +, O +I169 B-residue_name_number +and O +Q187 B-residue_name_number +, O +located O +near O +the O +C O +- O +terminus O +of O +YfiR B-protein +, O +comprise O +a O +putative O +YfiN B-site +binding I-site +site I-site +( O +Malone O +et O +al O +.,). O + +Interestingly O +, O +these O +residues O +are O +part O +of O +the O +conserved B-site +surface I-site +of O +YfiR B-protein +( O +Fig O +. O +3G O +). O + +F151 B-residue_name_number +, O +E163 B-residue_name_number +and O +I169 B-residue_name_number +form O +a O +hydrophobic B-site +core I-site +while O +, O +Q187 B-residue_name_number +is O +located O +at O +the O +end O +of O +the O +α6 B-structure_element +helix I-structure_element +. O + +E163 B-residue_name_number +and O +I169 B-residue_name_number +are O +YfiB B-site +- I-site +interacting I-site +residues I-site +of O +YfiR B-protein +, O +in O +which O +E163 B-residue_name_number +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +R96 B-residue_name_number +of O +YfiB B-protein +( O +Fig O +. O +3D O +- O +II O +) O +and O +I169 B-residue_name_number +is O +involved O +in O +forming O +the O +L166 B-residue_name_number +/ O +I169 B-residue_name_number +/ O +V176 B-residue_name_number +/ O +P178 B-residue_name_number +/ O +L181 B-residue_name_number +hydrophobic B-site +core I-site +for O +anchoring O +F57 B-residue_name_number +of O +YfiB B-protein +( O +Fig O +. O +3D O +- O +I O +( O +ii O +)). O + +Collectively O +, O +a O +part O +of O +the O +YfiB B-site +- I-site +YfiR I-site +interface I-site +overlaps O +with O +the O +proposed O +YfiR B-site +- I-site +YfiN I-site +interface I-site +, O +suggesting O +alteration O +in O +the O +association O +- O +disassociation O +equilibrium O +of O +YfiR B-protein +- O +YfiN B-protein +and O +hence O +the O +ability O +of O +YfiB B-protein +to O +sequester O +YfiR B-protein +. O + +YfiR B-protein +binds O +small O +molecules O + +Previous O +studies O +indicated O +that O +YfiR B-protein +constitutes O +a O +YfiB B-protein +- O +independent O +sensing O +device O +that O +can O +activate O +YfiN B-protein +in O +response O +to O +the O +redox O +status O +of O +the O +periplasm O +, O +and O +we O +have O +reported O +YfiR B-protein +structures B-evidence +in O +both O +the O +non B-protein_state +- I-protein_state +oxidized I-protein_state +and O +the O +oxidized B-protein_state +states O +earlier O +, O +revealing O +that O +the O +Cys145 B-residue_name_number +- O +Cys152 B-residue_name_number +disulfide B-ptm +bond I-ptm +plays O +an O +essential O +role O +in O +maintaining O +the O +correct O +folding O +of O +YfiR B-protein +( O +Yang O +et O +al O +.,). O + +However O +, O +whether O +YfiR B-protein +is O +involved O +in O +other O +regulatory O +mechanisms O +is O +still O +an O +open O +question O +. O + +Overall O +Structures B-evidence +of O +VB6 B-protein_state +- I-protein_state +bound I-protein_state +and O +Trp B-protein_state +- I-protein_state +bound I-protein_state +YfiR B-protein +. O +( O +A O +) O +Superposition B-experimental_method +of O +the O +overall O +structures B-evidence +of O +VB6 B-protein_state +- I-protein_state +bound I-protein_state +and O +Trp B-protein_state +- I-protein_state +bound I-protein_state +YfiR B-protein +. O +( O +B O +) O +Close O +- O +up O +views O +showing O +the O +key O +residues O +of O +YfiR B-protein +that O +bind O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +. O + +The O +electron B-evidence +densities I-evidence +of O +VB6 B-chemical +and O +Trp B-chemical +are O +countered O +at O +3 O +. O +0σ O +and O +2 O +. O +3σ O +, O +respectively O +, O +in O +| B-evidence +Fo I-evidence +|-| I-evidence +Fc I-evidence +| I-evidence +maps I-evidence +. O +( O +C O +) O +Superposition B-experimental_method +of O +the O +hydrophobic B-site +pocket I-site +of O +YfiR B-protein +with O +VB6 B-chemical +, O +L B-chemical +- I-chemical +Trp I-chemical +and O +F57 B-residue_name_number +of O +YfiB B-protein + +Intriguingly O +, O +a O +Dali B-experimental_method +search I-experimental_method +( O +Holm O +and O +Rosenstrom O +,) O +indicated O +that O +the O +closest O +homologs O +of O +YfiR B-protein +shared O +the O +characteristic O +of O +being O +able O +to O +bind O +several O +structurally O +similar O +small O +molecules O +, O +such O +as O +L B-chemical +- I-chemical +Trp I-chemical +, O +L B-chemical +- I-chemical +Phe I-chemical +, O +B O +- O +group O +vitamins O +and O +their O +analogs O +, O +encouraging O +us O +to O +test O +whether O +YfiR B-protein +can O +recognize O +these O +molecules O +. O + +For O +this O +purpose O +, O +we O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +YfiR B-protein +or O +soaked B-experimental_method +YfiR B-protein +crystals B-evidence +with O +different O +small O +molecules O +, O +including O +L B-chemical +- I-chemical +Trp I-chemical +and O +B O +- O +group O +vitamins O +. O + +Fortunately O +, O +we O +found O +obvious O +small B-evidence +- I-evidence +molecule I-evidence +density I-evidence +in O +the O +VB6 B-protein_state +- I-protein_state +bound I-protein_state +and O +Trp B-protein_state +- I-protein_state +bound I-protein_state +YfiR B-protein +crystal B-evidence +structures I-evidence +( O +Fig O +. O +5A O +and O +5B O +), O +and O +in O +both O +structures B-evidence +, O +the O +YfiR B-protein +dimers B-oligomeric_state +resemble O +the O +oxidized B-protein_state +YfiR B-protein +structure B-evidence +in O +which O +both O +two O +disulfide B-ptm +bonds I-ptm +are O +well O +formed O +( O +Yang O +et O +al O +.,). O + +Functional O +analysis O +of O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +. O +( O +A O +and O +B O +) O +The O +effect B-experimental_method +of I-experimental_method +increasing I-experimental_method +concentrations I-experimental_method +of O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +on O +YfiBL43P B-mutant +- O +induced O +attachment O +( O +bars O +). O + +The O +relative B-evidence +optical I-evidence +density I-evidence +is O +represented O +as O +curves O +. O + +Wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +is O +used O +as O +negative O +control O +. O + +( O +C O +and O +D O +) O +BIAcore B-experimental_method +data O +and O +analysis O +for O +binding B-evidence +affinities I-evidence +of O +( O +C O +) O +VB6 B-chemical +and O +( O +D O +) O +L B-chemical +- I-chemical +Trp I-chemical +with O +YfiR B-protein +. O +( O +E O +– O +G O +) O +ITC B-experimental_method +data O +and O +analysis O +for O +titration B-experimental_method +of O +( O +E O +) O +YfiB B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +, O +( O +F O +) O +YfiBL43P O +, O +and O +( O +G O +) O +YfiBL43P B-mutant +/ O +F57A B-mutant +into O +YfiR B-protein + +Structural B-experimental_method +analyses I-experimental_method +revealed O +that O +the O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +molecules O +are O +bound B-protein_state +at I-protein_state +the O +periphery O +of O +the O +YfiR B-protein +dimer B-oligomeric_state +, O +but O +not O +at O +the O +dimer B-site +interface I-site +. O + +Interestingly O +, O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +were O +found O +to O +occupy O +the O +same O +hydrophobic B-site +pocket I-site +, O +formed O +by O +L166 B-residue_name_number +/ O +I169 B-residue_name_number +/ O +V176 B-residue_name_number +/ O +P178 B-residue_name_number +/ O +L181 B-residue_name_number +of O +YfiR B-protein +, O +which O +is O +also O +a O +binding B-site +pocket I-site +for O +F57 B-residue_name_number +of O +YfiB B-protein +, O +as O +observed O +in O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +( O +Fig O +. O +5C O +). O + +To O +evaluate O +the O +importance O +of O +F57 B-residue_name_number +in O +YfiBL43P B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +interaction O +, O +the O +binding B-evidence +affinities I-evidence +of O +YfiBL43P B-mutant +and O +YfiBL43P B-mutant +/ O +F57A B-mutant +for O +YfiR B-protein +were O +measured O +by O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +( O +ITC B-experimental_method +). O + +The O +results O +showed O +Kd B-evidence +values O +of O +1 O +. O +4 O +× O +10 O +− O +7 O +mol O +/ O +L O +and O +5 O +. O +3 O +× O +10 O +− O +7 O +mol O +/ O +L O +for O +YfiBL43P B-mutant +and O +YfiBL43P B-mutant +/ O +F57A B-mutant +, O +respectively O +, O +revealing O +that O +the O +YfiBL43P B-mutant +/ O +F57A B-mutant +mutant B-protein_state +caused O +a O +3 O +. O +8 O +- O +fold O +reduction O +in O +the O +binding B-evidence +affinity I-evidence +compared O +with O +the O +YfiBL43P B-mutant +mutant B-protein_state +( O +Fig O +. O +6F O +and O +6G O +). O + +In O +parallel O +, O +to O +better O +understand O +the O +putative O +functional O +role O +of O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +, O +yfiB B-gene +was O +deleted B-experimental_method +in O +a O +PAO1 B-species +wild B-protein_state +- I-protein_state +type I-protein_state +strain O +, O +and O +a O +construct B-experimental_method +expressing I-experimental_method +the O +YfiBL43P B-mutant +mutant B-protein_state +was O +transformed B-experimental_method +into I-experimental_method +the O +PAO1 B-species +ΔyfiB B-mutant +strain O +to O +trigger O +YfiBL43P B-mutant +- O +induced O +biofilm O +formation O +. O + +Growth B-experimental_method +and I-experimental_method +surface I-experimental_method +attachment I-experimental_method +assays I-experimental_method +were O +carried O +out O +for O +the O +yfiB B-mutant +- I-mutant +L43P I-mutant +strain O +in O +the O +presence O +of O +increasing B-experimental_method +concentrations I-experimental_method +of O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +. O + +As O +shown O +in O +Fig O +. O +6A O +and O +6B O +, O +the O +over B-experimental_method +- I-experimental_method +expression I-experimental_method +of O +YfiBL43P B-mutant +induced O +strong O +surface O +attachment O +and O +much O +slower O +growth O +of O +the O +yfiB B-mutant +- I-mutant +L43P I-mutant +strain O +, O +and O +as O +expected O +, O +a O +certain O +amount O +of O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +( O +4 O +– O +6 O +mmol O +/ O +L O +for O +VB6 B-chemical +and O +6 O +– O +10 O +mmol O +/ O +L O +for O +L B-chemical +- I-chemical +Trp I-chemical +) O +could O +reduce O +the O +surface O +attachment O +. O + +Interestingly O +, O +at O +a O +concentration O +higher O +than O +8 O +mmol O +/ O +L O +, O +VB6 B-chemical +lost O +its O +ability O +to O +inhibit O +biofilm O +formation O +, O +implying O +that O +the O +VB6 B-chemical +- O +involving O +regulatory O +mechanism O +is O +highly O +complicated O +and O +remains O +to O +be O +further O +investigated O +. O + +Of O +note O +, O +both O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +have O +been O +reported O +to O +correlate O +with O +biofilm O +formation O +in O +certain O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +bacteria I-taxonomy_domain +( O +Grubman O +et O +al O +.,; O +Shimazaki O +et O +al O +.,). O + +In O +Helicobacter B-species +pylori I-species +in O +particular O +, O +VB6 B-chemical +biosynthetic O +enzymes O +act O +as O +novel O +virulence O +factors O +, O +and O +VB6 B-chemical +is O +required O +for O +full O +motility O +and O +virulence O +( O +Grubman O +et O +al O +.,). O + +In O +E B-species +. I-species +coli I-species +, O +mutants O +with O +decreased O +tryptophan B-chemical +synthesis O +show O +greater O +biofilm O +formation O +, O +and O +matured O +biofilm O +is O +degraded O +by O +L B-chemical +- I-chemical +tryptophan I-chemical +addition O +( O +Shimazaki O +et O +al O +.,). O + +To O +answer O +the O +question O +whether O +competition O +of O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +for O +the O +YfiB B-protein +F57 B-site +- I-site +binding I-site +pocket I-site +of O +YfiR B-protein +plays O +an O +essential O +role O +in O +inhibiting O +biofilm O +formation O +, O +we O +measured O +the O +binding B-evidence +affinities I-evidence +of O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +for O +YfiR B-protein +via O +BIAcore B-experimental_method +experiments O +. O + +The O +results O +showed O +relatively O +weak O +Kd B-evidence +values O +of O +35 O +. O +2 O +mmol O +/ O +L O +and O +76 O +. O +9 O +mmol O +/ O +L O +for O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +, O +respectively O +( O +Fig O +. O +6C O +and O +6D O +). O + +Based O +on O +our O +results O +, O +we O +concluded O +that O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +can O +bind O +to O +YfiR B-protein +, O +however O +, O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +alone B-protein_state +may O +have O +little O +effects O +in O +interrupting O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +interaction O +, O +the O +mechanism O +by O +which O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +inhibits O +biofilm O +formation O +remains O +unclear O +and O +requires O +further O +investigation O +. O + +Previous O +studies O +suggested O +that O +in O +response O +to O +cell O +stress O +, O +YfiB B-protein +in O +the O +outer O +membrane O +sequesters O +the O +periplasmic O +protein O +YfiR B-protein +, O +releasing O +its O +inhibition O +of O +YfiN B-protein +on O +the O +inner O +membrane O +and O +thus O +inducing O +the O +diguanylate O +cyclase O +activity O +of O +YfiN B-protein +to O +allow O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +production O +( O +Giardina O +et O +al O +.,; O +Malone O +et O +al O +.,; O +Malone O +et O +al O +.,). O + +Here O +, O +we O +report O +the O +crystal B-evidence +structures I-evidence +of O +YfiB B-protein +alone B-protein_state +and O +an O +active B-protein_state +mutant B-protein_state +YfiBL43P B-mutant +in B-protein_state +complex I-protein_state +with I-protein_state +YfiR B-protein +, O +indicating O +that O +YfiR B-protein +forms O +a O +2 O +: O +2 O +complex B-protein_state +with I-protein_state +YfiB B-protein +via O +a O +region O +composed O +of O +conserved O +residues O +. O + +Our O +structural B-experimental_method +data I-experimental_method +analysis I-experimental_method +shows O +that O +the O +activated B-protein_state +YfiB B-protein +has O +an O +N B-structure_element +- I-structure_element +terminal I-structure_element +portion I-structure_element +that O +is O +largely O +altered O +, O +adopting O +a O +stretched B-protein_state +conformation I-protein_state +compared O +with O +the O +compact B-protein_state +conformation I-protein_state +of O +the O +apo B-protein_state +YfiB B-protein +. O +The O +apo B-protein_state +YfiB B-protein +structure B-evidence +constructed O +beginning O +at O +residue O +34 B-residue_number +has O +a O +compact B-protein_state +conformation I-protein_state +of O +approximately O +45 O +Å O +in O +length O +. O + +In O +addition O +to O +the O +preceding B-residue_range +8 I-residue_range +aa I-residue_range +loop B-structure_element +( O +from O +the O +lipid O +acceptor O +Cys26 B-residue_range +to I-residue_range +Gly34 I-residue_range +), O +the O +full B-protein_state +length I-protein_state +of O +the O +periplasmic O +portion O +of O +apo B-protein_state +YfiB B-protein +can O +reach O +approximately O +60 O +Å O +. O +It O +was O +reported O +that O +the O +distance O +between O +the O +outer O +membrane O +and O +the O +cell O +wall O +is O +approximately O +50 O +Å O +and O +that O +the O +thickness O +of O +the O +PG O +layer O +is O +approximately O +70 O +Å O +( O +Matias O +et O +al O +.,). O + +Thus O +, O +YfiB B-protein +alone B-protein_state +represents O +an O +inactive B-protein_state +form O +that O +may O +only O +partially O +insert O +into O +the O +PG O +matrix O +. O + +By O +contrast O +, O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +( O +residues O +44 B-residue_range +– I-residue_range +168 I-residue_range +) O +has O +a O +stretched B-protein_state +conformation I-protein_state +of O +approximately O +55 O +Å O +in O +length O +. O + +In O +addition O +to O +the O +17 B-residue_range +preceding I-residue_range +intracellular I-residue_range +residues I-residue_range +( O +from O +the O +lipid O +acceptor O +Cys26 B-residue_range +to I-residue_range +Leu43 I-residue_range +), O +the O +length O +of O +the O +intracellular O +portion O +of O +active B-protein_state +YfiB B-protein +may O +extend O +over O +100 O +Å O +, O +assuming O +a O +fully B-protein_state +stretched I-protein_state +conformation I-protein_state +. O + +Provided O +that O +the O +diameter O +of O +the O +widest O +part O +of O +the O +YfiB B-protein +dimer B-oligomeric_state +is O +approximately O +64 O +Å O +, O +which O +is O +slightly O +smaller O +than O +the O +smallest O +diameter O +of O +the O +PG O +pore O +( O +70 O +Å O +) O +( O +Meroueh O +et O +al O +.,), O +the O +YfiB B-protein +dimer B-oligomeric_state +should O +be O +able O +to O +penetrate O +the O +PG O +layer O +. O + +Regulatory O +model O +of O +the O +YfiBNR B-complex_assembly +tripartite B-protein_state +system O +. O + +The O +periplasmic B-structure_element +domain I-structure_element +of O +YfiB B-protein +and O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +are O +depicted O +according O +to O +the O +crystal B-evidence +structures I-evidence +. O + +The O +lipid O +acceptor O +Cys26 B-residue_name_number +is O +indicated O +as O +blue O +ball O +. O + +The O +loop B-structure_element +connecting O +Cys26 B-residue_name_number +and O +Gly34 B-residue_name_number +of O +YfiB B-protein +is O +modeled O +. O + +The O +PAS B-structure_element +domain I-structure_element +of O +YfiN B-protein +is O +shown O +as O +pink O +oval O +. O + +Once O +activated B-protein_state +by O +certain O +cell O +stress O +, O +the O +dimeric B-oligomeric_state +YfiB B-protein +transforms O +from O +a O +compact B-protein_state +conformation I-protein_state +to O +a O +stretched B-protein_state +conformation I-protein_state +, O +allowing O +the O +periplasmic B-structure_element +domain I-structure_element +of O +the O +membrane B-protein_state +- I-protein_state +anchored I-protein_state +YfiB B-protein +to O +penetrate O +the O +cell O +wall O +and O +sequester O +the O +YfiR B-protein +dimer B-oligomeric_state +, O +thus O +relieving O +the O +repression O +of O +YfiN B-protein + +These O +results O +, O +together O +with O +our O +observation O +that O +activated B-protein_state +YfiB B-protein +has O +a O +much O +higher O +cell B-evidence +wall I-evidence +binding I-evidence +affinity I-evidence +, O +and O +previous O +mutagenesis O +data O +showing O +that O +( O +1 O +) O +both O +PG B-chemical +binding O +and O +membrane O +anchoring O +are O +required O +for O +YfiB B-protein +activity O +and O +( O +2 O +) O +activating O +mutations O +possessing O +an O +altered O +N O +- O +terminal O +loop B-structure_element +length O +are O +dominant O +over O +the O +loss O +of O +PG B-chemical +binding O +( O +Malone O +et O +al O +.,), O +suggest O +an O +updated O +regulatory O +model O +of O +the O +YfiBNR B-complex_assembly +system O +( O +Fig O +. O +7 O +). O + +In O +this O +model O +, O +in O +response O +to O +a O +particular O +cell O +stress O +that O +is O +yet O +to O +be O +identified O +, O +the O +dimeric B-oligomeric_state +YfiB B-protein +is O +activated B-protein_state +from O +a O +compact B-protein_state +, O +inactive B-protein_state +conformation B-protein_state +to O +a O +stretched B-protein_state +conformation I-protein_state +, O +which O +possesses O +increased O +PG B-chemical +binding O +affinity O +. O + +This O +allows O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +portion I-structure_element +of O +the O +membrane B-protein_state +- I-protein_state +anchored I-protein_state +YfiB B-protein +to O +reach O +, O +bind O +and O +penetrate O +the O +cell O +wall O +and O +sequester O +the O +YfiR B-protein +dimer B-oligomeric_state +. O + +The O +YfiBNR B-complex_assembly +system O +provides O +a O +good O +example O +of O +a O +delicate O +homeostatic O +system O +that O +integrates O +multiple O +signals O +to O +regulate O +the O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +level O +. O + +Homologs O +of O +the O +YfiBNR B-complex_assembly +system O +are O +functionally B-protein_state +conserved I-protein_state +in O +P B-species +. I-species +aeruginosa I-species +( O +Malone O +et O +al O +.,; O +Malone O +et O +al O +.,), O +E B-species +. I-species +coli I-species +( O +Hufnagel O +et O +al O +.,; O +Raterman O +et O +al O +.,; O +Sanchez O +- O +Torres O +et O +al O +.,), O +K B-species +. I-species +pneumonia I-species +( O +Huertas O +et O +al O +.,) O +and O +Y B-species +. I-species +pestis I-species +( O +Ren O +et O +al O +.,), O +where O +they O +affect O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +production O +and O +biofilm O +formation O +. O + +The O +mechanism O +by O +which O +activated B-protein_state +YfiB B-protein +relieves O +the O +repression O +of O +YfiN B-protein +may O +be O +applicable O +to O +the O +YfiBNR B-complex_assembly +system O +in O +other O +bacteria B-taxonomy_domain +and O +to O +analogous O +outside O +- O +in O +signaling O +for O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +production O +, O +which O +in O +turn O +may O +be O +relevant O +to O +the O +development O +of O +drugs O +that O +can O +circumvent O +complicated O +antibiotic O +resistance O +. O + +Hemi B-chemical +- I-chemical +methylated I-chemical +DNA I-chemical +opens O +a O +closed B-protein_state +conformation O +of O +UHRF1 B-protein +to O +facilitate O +its O +histone B-protein_type +recognition O + +UHRF1 B-protein +is O +an O +important O +epigenetic O +regulator O +for O +maintenance O +DNA O +methylation B-ptm +. O + +UHRF1 B-protein +recognizes O +hemi B-chemical +- I-chemical +methylated I-chemical +DNA I-chemical +( O +hm B-chemical +- I-chemical +DNA I-chemical +) O +and O +trimethylation B-ptm +of O +histone B-protein_type +H3K9 B-protein_type +( O +H3K9me3 B-protein_type +), O +but O +the O +regulatory O +mechanism O +remains O +unknown O +. O + +Here O +we O +show O +that O +UHRF1 B-protein +adopts O +a O +closed B-protein_state +conformation O +, O +in O +which O +a O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +( O +Spacer B-structure_element +) O +binds B-protein_state +to I-protein_state +the O +tandem B-structure_element +Tudor I-structure_element +domain I-structure_element +( O +TTD B-structure_element +) O +and O +inhibits O +H3K9me3 B-protein_type +recognition O +, O +whereas O +the O +SET B-structure_element +- I-structure_element +and I-structure_element +- I-structure_element +RING I-structure_element +- I-structure_element +associated I-structure_element +( O +SRA B-structure_element +) O +domain O +binds B-protein_state +to I-protein_state +the O +plant B-structure_element +homeodomain I-structure_element +( O +PHD B-structure_element +) O +and O +inhibits O +H3R2 B-site +recognition O +. O + +Hm B-chemical +- I-chemical +DNA I-chemical +impairs O +the O +intramolecular O +interactions O +and O +promotes O +H3K9me3 B-protein_type +recognition O +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +. O + +The O +Spacer B-structure_element +also O +facilitates O +UHRF1 B-complex_assembly +– I-complex_assembly +DNMT1 I-complex_assembly +interaction O +and O +enhances O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +SRA B-structure_element +. O + +When O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +binds B-protein_state +to I-protein_state +H3K9me3 B-protein_type +, O +SRA B-structure_element +- I-structure_element +Spacer I-structure_element +may O +exist O +in O +a O +dynamic O +equilibrium O +: O +either O +recognizes O +hm B-chemical +- I-chemical +DNA I-chemical +or O +recruits O +DNMT1 B-protein +to O +chromatin O +. O + +Our O +study O +reveals O +the O +mechanism O +for O +regulation O +of O +H3K9me3 B-protein_type +and O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +by O +URHF1 B-protein +. O + +UHRF1 B-protein +is O +involved O +in O +the O +maintenance O +of O +DNA O +methylation B-ptm +, O +but O +the O +regulatory O +mechanism O +of O +this O +epigenetic O +regulator O +is O +unclear O +. O + +Here O +, O +the O +authors O +show O +that O +it O +has O +a O +closed B-protein_state +conformation O +and O +are O +able O +to O +make O +conclusions O +about O +the O +mechanism O +of O +recognition O +of O +epigenetic O +marks O +. O + +DNA O +methylation B-ptm +is O +an O +important O +epigenetic O +modification O +for O +gene O +repression O +, O +X O +- O +chromosome O +inactivation O +, O +genome O +imprinting O +and O +maintenance O +of O +genome O +stability O +. O + +Mammalian B-taxonomy_domain +DNA O +methylation B-ptm +is O +established O +by O +de O +novo O +DNA B-protein_type +methyltransferases I-protein_type +DNMT3A B-protein +/ I-protein +3B I-protein +, O +and O +DNA O +methylation B-ptm +patterns O +are O +maintained O +by O +maintenance O +DNA B-protein +methyltransferase I-protein +1 I-protein +( O +DNMT1 B-protein +) O +during O +DNA O +replication O +. O + +Ubiquitin B-protein +- I-protein +like I-protein +, I-protein +containing I-protein +PHD I-protein +and I-protein +RING I-protein +fingers I-protein +domains I-protein +, I-protein +1 I-protein +( O +UHRF1 B-protein +, O +also O +known O +as O +ICBP90 B-protein +and O +NP95 B-protein +in O +mouse B-taxonomy_domain +) O +was O +shown O +to O +be O +essential O +for O +maintenance O +DNA O +methylation B-ptm +through O +recruiting O +DNMT1 B-protein +to O +replication O +forks O +in O +S O +phase O +of O +the O +cell O +cycle O +. O + +UHRF1 B-protein +is O +essential O +for O +S O +phase O +entry O +and O +is O +involved O +in O +heterochromatin O +formation O +. O + +UHRF1 B-protein +also O +plays O +an O +important O +role O +in O +promoting O +proliferation O +and O +is O +shown O +to O +be O +upregulated O +in O +a O +number O +of O +cancers O +, O +suggesting O +that O +UHRF1 B-protein +may O +serve O +as O +a O +potential O +drug O +target O +for O +therapeutic O +applications O +. O + +UHRF1 B-protein +is O +a O +multi O +- O +domain O +containing O +protein O +connecting O +histone B-protein_type +modification O +and O +DNA B-chemical +methylation B-ptm +. O + +As O +shown O +in O +Fig O +. O +1a O +, O +UHRF1 B-protein +is O +comprised O +of O +an O +N O +- O +terminal O +ubiquitin B-structure_element +- I-structure_element +like I-structure_element +domain I-structure_element +, O +followed O +by O +a O +tandem B-structure_element +Tudor I-structure_element +domain I-structure_element +( O +TTD B-structure_element +containing O +TTDN B-structure_element +and O +TTDC B-structure_element +sub O +- O +domains O +), O +a O +plant B-structure_element +homeodomain I-structure_element +( O +PHD B-structure_element +), O +a O +SET B-structure_element +- I-structure_element +and I-structure_element +- I-structure_element +RING I-structure_element +- I-structure_element +associated I-structure_element +( O +SRA B-structure_element +) O +domain O +, O +and O +a O +C O +- O +terminal O +really B-structure_element +interesting I-structure_element +new I-structure_element +gene I-structure_element +( O +RING B-structure_element +) O +domain O +. O + +We O +and O +other O +groups O +demonstrated O +that O +the O +TTD B-structure_element +and O +the O +PHD B-structure_element +coordinately O +recognize O +histone B-protein_type +H3K9me3 B-protein_type +, O +in O +which O +residue O +R2 B-residue_name_number +is O +recognized O +by O +the O +PHD B-structure_element +and O +tri B-ptm +- I-ptm +methylation I-ptm +of O +residue O +K9 B-residue_name_number +( O +K9me3 B-ptm +) O +is O +recognized O +by O +the O +TTD B-structure_element +. O + +The O +SRA B-structure_element +preferentially O +binds B-protein_state +to I-protein_state +hemi B-chemical +- I-chemical +methylated I-chemical +DNA I-chemical +( O +hm B-chemical +- I-chemical +DNA I-chemical +). O + +Recent O +studies O +show O +that O +the O +SRA B-structure_element +directly O +binds B-protein_state +to I-protein_state +replication B-structure_element +focus I-structure_element +targeting I-structure_element +sequence I-structure_element +( O +RFTS B-structure_element +) O +of O +DNMT1 B-protein +( O +RFTSDNMT1 B-protein +). O + +A O +spacer B-structure_element +region I-structure_element +( O +Fig O +. O +1a O +, O +designated O +Spacer B-structure_element +hereafter O +) O +connecting O +the O +SRA B-structure_element +and O +the O +RING B-structure_element +is O +rich O +in O +basic O +residues O +and O +predicted O +to O +be O +unstructured B-protein_state +for O +unknown O +function O +. O + +Recent O +study O +shows O +that O +phosphatidylinostiol B-chemical +phosphate I-chemical +PI5P B-chemical +binds B-protein_state +to I-protein_state +the O +Spacer B-structure_element +and O +induces O +a O +conformational O +change O +of O +UHRF1 B-protein +to O +allow O +the O +TTD B-structure_element +to O +recognize O +H3K9me3 B-protein_type +( O +ref O +.). O + +These O +studies O +indicate O +that O +UHRF1 B-protein +connects O +dynamic O +regulation O +of O +DNA B-chemical +methylation B-ptm +and O +H3K9me3 B-protein_type +, O +which O +are O +positively O +correlated O +in O +human B-species +genome O +. O + +However O +, O +how O +UHRF1 B-protein +regulates O +the O +recognition O +of O +these O +two O +repressive O +epigenetic O +marks O +and O +recruits O +DNMT1 B-protein +for O +chromatin O +localization O +remain O +largely O +unknown O +. O + +Here O +we O +report O +that O +UHRF1 B-protein +adopts O +a O +closed B-protein_state +conformation O +, O +in O +which O +the O +C O +- O +terminal O +Spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +and O +inhibits O +its O +recognition O +of O +H3K9me3 B-protein_type +, O +whereas O +the O +SRA B-structure_element +binds B-protein_state +to I-protein_state +the O +PHD B-structure_element +and O +inhibits O +its O +recognition O +of O +H3R2 B-site +( O +unmethylated B-protein_state +histone B-protein_type +H3 B-protein_type +at O +residue O +R2 B-residue_name_number +). O + +Upon O +binding B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +, O +UHRF1 B-protein +impairs O +the O +intramolecular O +interactions O +and O +promotes O +the O +H3K9me3 B-protein_type +recognition O +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +, O +which O +may O +further O +enhance O +its O +genomic O +localization O +. O + +As O +a O +result O +, O +UHRF1 B-protein +is O +locked O +in O +the O +open B-protein_state +conformation O +by O +the O +association O +of O +H3K9me3 B-protein_type +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +, O +and O +thus O +SRA B-structure_element +- I-structure_element +Spacer I-structure_element +either O +recognizes O +hm B-chemical +- I-chemical +DNA I-chemical +or O +recruits O +DNMT1 B-protein +for O +DNA B-chemical +methylation B-ptm +. O + +Therefore O +, O +UHRF1 B-protein +may O +engage O +in O +a O +sophisticated O +regulation O +for O +its O +chromatin O +localization O +and O +recruitment O +of O +DNMT1 B-protein +through O +a O +mechanism O +yet O +to O +be O +fully O +elucidated O +. O + +Our O +study O +reveals O +the O +mechanism O +for O +regulation O +of O +H3K9me3 B-protein_type +and O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +by O +UHRF1 B-protein +. O + +Hm B-chemical +- I-chemical +DNA I-chemical +facilitates O +histone B-protein_type +H3K9me3 B-protein_type +recognition O +by O +UHRF1 B-protein + +To O +investigate O +how O +UHRF1 B-protein +coordinates O +the O +recognition O +of O +H3K9me3 B-protein_type +and O +hm B-chemical +- I-chemical +DNA I-chemical +, O +we O +purified O +recombinant O +UHRF1 B-protein +( O +truncations O +and O +mutations O +) O +proteins O +from O +bacteria O +. O + +We O +first O +performed O +an O +in B-experimental_method +vitro I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +using O +biotinylated B-protein_state +histone B-protein_type +H3 B-protein_type +peptides O +and O +hm B-chemical +- I-chemical +DNA I-chemical +( O +Supplementary O +Table O +1 O +). O + +As O +shown O +in O +Fig O +. O +1b O +, O +hm B-chemical +- I-chemical +DNA I-chemical +largely O +enhanced O +the O +interaction O +between O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +unmethylated B-protein_state +histone B-protein_type +H3 B-protein_type +( O +H3K9me0 B-protein_type +) O +or O +H3K9me3 B-protein_type +peptide O +. O + +Compared O +with O +hm B-chemical +- I-chemical +DNA I-chemical +, O +um B-chemical +- I-chemical +DNA I-chemical +( O +unmethylated B-protein_state +DNA B-chemical +) O +or O +fm B-chemical +- I-chemical +DNA I-chemical +( O +fully B-protein_state +methylated I-protein_state +DNA B-chemical +) O +showed O +marginal O +effect O +on O +facilitating O +the O +interaction O +between O +UHRF1 B-protein +and O +histone B-protein_type +peptides O +, O +which O +is O +consistent O +with O +previous O +studies O +that O +UHRF1 B-protein +prefers O +hm B-chemical +- I-chemical +DNA I-chemical +for O +chromatin O +association O +( O +Supplementary O +Fig O +. O +1a O +). O + +In O +contrast O +, O +histone B-protein_type +peptides O +showed O +no O +enhancement O +on O +the O +interaction O +between O +hm B-chemical +- I-chemical +DNA I-chemical +and O +UHRF1 B-protein +( O +Fig O +. O +1c O +). O + +These O +results O +suggest O +that O +hm B-chemical +- I-chemical +DNA I-chemical +facilitates O +histone B-protein_type +recognition O +by O +UHRF1 B-protein +. O + +Our O +previous O +studies O +show O +that O +the O +PHD B-structure_element +recognizes O +H3K9me0 B-protein_type +and O +the O +TTD B-structure_element +and O +the O +PHD B-structure_element +together O +( O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +) O +coordinately O +recognize O +H3K9me3 B-protein_type +( O +refs O +.). O + +We O +noticed O +that O +the O +isolated B-protein_state +TTD B-structure_element +– I-structure_element +PHD I-structure_element +showed O +much O +higher O +(∼ O +31 O +- O +fold O +) O +binding B-evidence +affinity I-evidence +to O +H3K9me3 B-protein_type +peptide O +than O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +( O +Fig O +. O +1d O +and O +Supplementary O +Table O +2 O +), O +and O +the O +isolated O +PHD B-structure_element +showed O +much O +higher O +(∼ O +34 O +- O +fold O +) O +binding B-evidence +affinity I-evidence +to O +H3K9me0 B-protein_type +peptide O +than O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +( O +Fig O +. O +1e O +). O + +The O +gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +showed O +that O +UHRF1 B-protein +is O +a O +monomer B-oligomeric_state +in O +solution O +( O +Supplementary O +Fig O +. O +1b O +), O +indicating O +that O +the O +intramolecular O +( O +not O +intermolecular O +) O +interaction O +of O +UHRF1 B-protein +regulates O +histone B-protein_type +recognition O +. O + +These O +results O +suggest O +that O +UHRF1 B-protein +adopts O +an O +unfavourable O +conformation O +for O +histone B-protein_type +H3 B-protein_type +tails O +recognition O +, O +in O +which O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +might O +be O +blocked O +by O +other O +regions O +of O +UHRF1 B-protein +, O +and O +hm B-chemical +- I-chemical +DNA I-chemical +impairs O +this O +intramolecular O +interaction O +to O +facilitate O +its O +recognition O +of O +histone B-protein_type +H3 B-protein_type +tails O +. O + +Intramolecular O +interaction O +within O +UHRF1 B-protein + +To O +test O +above O +hypothesis O +, O +we O +performed O +glutathione B-experimental_method +S I-experimental_method +- I-experimental_method +transferase I-experimental_method +( I-experimental_method +GST I-experimental_method +) I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +using O +various O +truncations B-experimental_method +of O +UHRF1 B-protein +. O + +Interestingly O +, O +the O +TTD B-structure_element +directly O +bound B-protein_state +to I-protein_state +SRA B-structure_element +- I-structure_element +Spacer I-structure_element +but O +not O +the O +SRA B-structure_element +, O +suggesting O +that O +the O +Spacer B-structure_element +( O +residues O +587 B-residue_range +– I-residue_range +674 I-residue_range +) O +is O +important O +for O +the O +intramolecular O +interaction O +( O +Fig O +. O +2a O +). O + +The O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +( O +ITC B-experimental_method +) O +measurements O +show O +that O +the O +TTD B-structure_element +bound B-protein_state +to I-protein_state +the O +Spacer B-structure_element +( O +but O +not O +the O +SRA B-structure_element +) O +in O +a O +1 O +: O +1 O +stoichiometry O +with O +a O +binding B-evidence +affinity I-evidence +( O +KD B-evidence +) O +of O +1 O +. O +59 O +μM O +( O +Fig O +. O +2b O +). O + +The O +presence B-protein_state +of I-protein_state +the O +Spacer B-structure_element +markedly O +impaired O +the O +interaction O +between O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +and O +H3K9me3 B-protein_type +( O +Fig O +. O +2c O +). O + +The O +results O +indicate O +that O +the O +Spacer B-structure_element +directly O +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +and O +inhibits O +its O +interaction O +with O +H3K9me3 B-protein_type +. O + +The O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +also O +shows O +that O +the O +PHD B-structure_element +bound B-protein_state +to I-protein_state +the O +SRA B-structure_element +, O +which O +was O +further O +confirmed O +by O +the O +ITC B-experimental_method +measurements O +( O +KD B-evidence += O +26 O +. O +7 O +μM O +; O +Fig O +. O +2a O +, O +d O +). O + +Compared O +with O +the O +PHD B-structure_element +alone B-protein_state +, O +PHD B-structure_element +- I-structure_element +SRA I-structure_element +showed O +decreased O +binding B-evidence +affinity I-evidence +to O +H3K9me0 B-protein_type +peptide O +by O +a O +factor O +of O +eight O +( O +Fig O +. O +2e O +). O + +Pre B-experimental_method +- I-experimental_method +incubation I-experimental_method +of O +the O +SRA B-structure_element +also O +modestly O +impaired O +PHD B-structure_element +– O +H3K9me0 B-protein_type +interaction O +. O + +These O +results O +indicate O +that O +the O +SRA B-structure_element +directly O +binds B-protein_state +to I-protein_state +the O +PHD B-structure_element +and O +inhibits O +its O +binding B-evidence +affinity I-evidence +to O +H3K9me0 B-protein_type +. O + +Taken O +together O +, O +UHRF1 B-protein +seems O +to O +adopt O +a O +closed B-protein_state +form O +through O +intramolecular O +interactions O +( O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +and O +PHD B-structure_element +- I-structure_element +SRA I-structure_element +), O +which O +inhibit O +histone B-protein_type +H3 B-protein_type +tail O +recognition O +by O +UHRF1 B-protein +. O + +Overall O +structure B-evidence +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element + +To O +investigate O +the O +intramolecular O +interaction O +within O +UHRF1 B-protein +, O +we O +first O +mapped O +the O +minimal O +regions O +within O +the O +Spacer B-structure_element +for O +the O +interaction O +with O +the O +TTD B-structure_element +( O +Supplementary O +Fig O +. O +2a O +). O + +Internal O +deletions B-experimental_method +of O +the O +Spacer B-structure_element +, O +including O +SpacerΔ660 B-mutant +– I-mutant +664 I-mutant +, O +SpacerΔ665 B-mutant +– I-mutant +669 I-mutant +, O +SpacerΔ670 B-mutant +– I-mutant +674 I-mutant +and O +Spacer642 B-mutant +– I-mutant +674 I-mutant +, O +bound B-protein_state +to I-protein_state +the O +TTD B-structure_element +with O +comparable O +binding B-evidence +affinities I-evidence +to O +that O +of O +the O +Spacer B-structure_element +, O +whereas O +Spacer587 B-mutant +– I-mutant +641 I-mutant +showed O +no O +detectable O +interaction O +. O + +SpacerΔ642 B-mutant +– I-mutant +651 I-mutant +, O +SpacerΔ650 B-mutant +– I-mutant +654 I-mutant +and O +SpacerΔ655 B-mutant +– I-mutant +659 I-mutant +also O +decreased O +binding B-evidence +affinities I-evidence +, O +indicating O +that O +residues O +642 B-residue_range +– I-residue_range +674 I-residue_range +are O +important O +for O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +. O + +We O +next O +determined O +the O +solution B-evidence +structure I-evidence +of O +the O +TTD B-structure_element +( O +residues O +134 B-residue_range +– I-residue_range +285 I-residue_range +) O +bound B-protein_state +to I-protein_state +Spacer627 B-residue_range +– I-residue_range +674 I-residue_range +by O +conventional O +NMR B-experimental_method +techniques O +( O +Supplementary O +Table O +3 O +and O +Supplementary O +Fig O +. O +3a O +, O +b O +). O + +In O +the O +complex B-evidence +structure I-evidence +, O +each O +Tudor B-structure_element +domain I-structure_element +adopts O +a O +‘ B-structure_element +Royal I-structure_element +' I-structure_element +fold I-structure_element +containing O +a O +characteristic O +five B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +and O +the O +two O +Tudor B-structure_element +domains I-structure_element +tightly O +pack O +against O +each O +other O +with O +a O +buried O +area O +of O +573 O +Å2 O +( O +Fig O +. O +3a O +). O + +The O +TTD B-structure_element +adopts O +similar O +fold O +to O +that O +in O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +complex O +structure B-evidence +( O +PDB O +: O +4GY5 O +) O +with O +a O +root B-evidence +- I-evidence +mean I-evidence +- I-evidence +square I-evidence +deviation I-evidence +of O +1 O +. O +09 O +Å O +for O +128 O +Cα O +atoms O +, O +indicating O +that O +the O +Spacer B-structure_element +does O +not O +result O +in O +obvious O +conformational O +change O +of O +the O +TTD B-structure_element +( O +Fig O +. O +3b O +). O + +The O +Spacer B-structure_element +( O +residues O +643 B-residue_range +– I-residue_range +655 I-residue_range +were O +built O +in O +the O +model O +) O +adopts O +an O +extended B-protein_state +conformation I-protein_state +and O +binds B-protein_state +to I-protein_state +an O +acidic B-site +groove I-site +on O +the O +TTD B-structure_element +( O +Fig O +. O +3c O +). O + +The O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +is O +mediated O +by O +a O +number O +of O +hydrogen B-bond_interaction +bonds I-bond_interaction +( O +Fig O +. O +3d O +). O + +The O +side O +chain O +of O +residue O +K648 B-residue_name_number +forms O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +carbonyl O +oxygen O +atom O +of O +D189 B-residue_name_number +and O +side O +chain O +of O +D190 B-residue_name_number +of O +the O +TTD B-structure_element +. O + +The O +side O +chain O +of O +residue O +R649 B-residue_name_number +packs B-bond_interaction +against I-bond_interaction +an O +acidic O +surface O +mainly O +formed O +by O +residues O +D142 B-residue_name_number +and O +E153 B-residue_name_number +. O + +Residue O +S651 B-residue_name_number +forms O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +main O +chain O +of O +residues O +G236 B-residue_name_number +and O +W238 B-residue_name_number +. O + +The O +interaction O +is O +further O +supported O +by O +hydrogen B-bond_interaction +bonds I-bond_interaction +formed O +between O +residues O +K650 B-residue_name_number +, O +A652 B-residue_name_number +, O +G653 B-residue_name_number +and O +G654 B-residue_name_number +of O +the O +Spacer B-structure_element +and O +residues O +N228 B-residue_name_number +, O +G236 B-residue_name_number +and O +W238 B-residue_name_number +of O +the O +TTD B-structure_element +, O +respectively O +. O + +In O +support O +of O +above O +structural B-experimental_method +analyses I-experimental_method +, O +mutation B-experimental_method +D142A B-mutant +/ O +E153A B-mutant +of O +the O +TTD B-structure_element +abolished O +its O +interaction O +with O +the O +Spacer B-structure_element +( O +Fig O +. O +3e O +). O + +Mutations B-experimental_method +K648D B-mutant +and O +S651D B-mutant +of O +the O +Spacer B-structure_element +decreased O +their O +binding B-evidence +affinities I-evidence +to O +the O +TTD B-structure_element +, O +and O +mutation B-experimental_method +R649A B-mutant +of O +the O +Spacer B-structure_element +showed O +more O +significant O +decrease O +(∼ O +13 O +- O +fold O +) O +in O +the O +binding B-evidence +affinity I-evidence +( O +Fig O +. O +3f O +). O + +As O +negative O +control O +, O +mutations B-experimental_method +S639D B-mutant +and O +S666D B-mutant +of O +the O +Spacer B-structure_element +showed O +little O +effect O +on O +the O +interaction O +. O + +Interestingly O +, O +phosphorylation B-ptm +at O +residue O +S651 B-residue_name_number +of O +UHRF1 B-protein +was O +observed O +in O +previous O +mass B-experimental_method +- I-experimental_method +spectrometry I-experimental_method +analyses O +. O + +Compared O +with O +the O +unmodified B-protein_state +peptide O +of O +Spacer642 B-mutant +– I-mutant +664 I-mutant +, O +a O +phosphorylation B-ptm +at O +S651 B-residue_name_number +markedly O +decreased O +the O +binding B-evidence +affinity I-evidence +to O +the O +TTD B-structure_element +( O +Supplementary O +Fig O +. O +2b O +), O +suggesting O +that O +the O +phosphorylation B-ptm +may O +regulate O +the O +intramolecular O +interaction O +within O +UHRF1 B-protein +. O + +The O +spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +by O +competing O +with O +the O +linker B-structure_element + +Previous O +studies O +indicate O +that O +the O +TTD B-structure_element +binds B-protein_state +to I-protein_state +a O +linker B-structure_element +region I-structure_element +connecting O +the O +TTD B-structure_element +and O +PHD B-structure_element +( O +residues O +286 B-residue_range +– I-residue_range +306 I-residue_range +, O +designated O +Linker B-structure_element +, O +Fig O +. O +1a O +), O +and O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +interaction O +is O +essential O +for O +H3K9me3 B-protein_type +recognition O +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +. O + +Comparison B-experimental_method +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +and O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +( O +PDB O +: O +4GY5 O +) O +structures B-evidence +indicates O +that O +the O +Spacer B-structure_element +and O +the O +Linker B-structure_element +bind O +to O +the O +TTD B-structure_element +in O +a O +similar O +manner O +in O +the O +two O +complexes O +( O +Fig O +. O +3b O +). O + +In O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +structure B-evidence +, O +residues O +R295 B-residue_name_number +, O +R296 B-residue_name_number +and O +S298 B-residue_name_number +of O +the O +Linker B-structure_element +adopt O +almost O +identical O +conformation O +to O +residues O +K648 B-residue_name_number +, O +R649 B-residue_name_number +and O +S651 B-residue_name_number +of O +the O +Spacer B-structure_element +in O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +structure B-evidence +, O +respectively O +. O + +Similar O +intramolecular O +contacts O +( O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +and O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +) O +were O +observed O +in O +the O +two O +structures B-evidence +( O +Fig O +. O +3b O +, O +d O +and O +Supplementary O +Fig O +. O +4a O +). O + +Thus O +, O +the O +Spacer B-structure_element +may O +disrupt O +the O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +interaction O +and O +inhibits O +the O +recognition O +of O +H3K9me3 B-protein_type +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +. O + +To O +test O +this O +hypothesis O +, O +we O +first O +investigated O +the O +potential O +competition O +between O +the O +Linker B-structure_element +and O +the O +Spacer B-structure_element +for O +their O +interaction O +with O +the O +TTD B-structure_element +. O + +The O +ITC B-experimental_method +experiment O +shows O +that O +the O +Linker B-structure_element +peptide O +( O +289 B-residue_range +– I-residue_range +306 I-residue_range +) O +bound B-protein_state +to I-protein_state +the O +TTD B-structure_element +with O +a O +binding B-evidence +affinity I-evidence +of O +24 O +. O +04 O +μM O +( O +Supplementary O +Fig O +. O +4b O +), O +∼ O +15 O +- O +fold O +lower O +than O +that O +of O +the O +Spacer B-structure_element +peptide O +( O +KD B-evidence += O +1 O +. O +59 O +μM O +, O +Fig O +. O +3e O +). O + +The O +competitive B-experimental_method +ITC I-experimental_method +experiments O +show O +that O +TTD B-evidence +– I-evidence +Spacer I-evidence +binding I-evidence +affinity I-evidence +decreased O +by O +a O +factor O +of O +two O +in O +the O +presence B-protein_state +of I-protein_state +the O +Linker B-structure_element +, O +whereas O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +interaction O +was O +abolished O +in O +the O +presence B-protein_state +of I-protein_state +the O +Spacer B-structure_element +( O +Supplementary O +Fig O +. O +4c O +). O + +Compared O +with O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +( O +KD B-evidence += O +1 O +. O +48 O +μM O +), O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +decreased O +the O +binding B-evidence +affinity I-evidence +to O +the O +Spacer B-structure_element +( O +KD B-evidence += O +10 O +. O +68 O +μM O +), O +whereas O +mutation B-experimental_method +R295D B-mutant +/ O +R296D B-mutant +( O +within O +the O +Linker B-structure_element +and O +important O +for O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +interaction O +) O +of O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +showed O +minor O +decrease O +in O +the O +binding B-evidence +affinity I-evidence +( O +KD B-evidence += O +2 O +. O +69 O +μM O +; O +Fig O +. O +3g O +), O +indicating O +a O +competition O +between O +the O +Spacer B-structure_element +and O +the O +Linker B-structure_element +on O +the O +same O +binding B-site +site I-site +of O +the O +TTD B-structure_element +. O + +Notably O +, O +although O +the O +Linker B-structure_element +( O +in O +the O +context O +of O +TTD B-structure_element +- I-structure_element +PHD I-structure_element +) O +impairs O +the O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +to O +some O +extent O +, O +the O +isolated O +Spacer B-structure_element +could O +still O +bind O +to O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +with O +moderate O +binding B-evidence +affinity I-evidence +( O +KD B-evidence += O +10 O +. O +68 O +μM O +), O +supporting O +the O +existence O +of O +the O +intramolecular O +interaction O +within O +UHRF1 B-protein +. O + +To O +test O +whether O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +association O +exists O +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +, O +we O +used O +various O +truncations B-experimental_method +of O +UHRF1 B-protein +in O +the O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +. O + +As O +indicated O +in O +Fig O +. O +3h O +, O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +UHRF1ΔSRA B-mutant +showed O +no O +interaction O +with O +GST B-protein_state +- I-protein_state +tagged I-protein_state +TTD B-structure_element +, O +Linker B-structure_element +or O +Spacer B-structure_element +, O +suggesting O +that O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +in B-protein_state +- I-protein_state +cis I-protein_state +within O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +or O +UHRF1ΔSRA B-mutant +prohibits O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +complex O +formation O +in B-protein_state +- I-protein_state +trans I-protein_state +. O + +In O +contrast O +, O +UHRF1ΔTTD B-mutant +bound B-protein_state +to I-protein_state +GST B-experimental_method +- O +TTD B-structure_element +, O +and O +UHRF1Δ627 B-mutant +– I-mutant +674 I-mutant +bound B-protein_state +to I-protein_state +GST B-experimental_method +- O +Spacer B-structure_element +, O +indicating O +that O +lack O +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +in B-protein_state +- I-protein_state +cis I-protein_state +, O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +complex O +could O +form O +in B-protein_state +- I-protein_state +trans I-protein_state +, O +supporting O +that O +the O +TTD B-structure_element +binds B-protein_state +to I-protein_state +the O +Spacer B-structure_element +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +. O + +Moreover O +, O +GST B-experimental_method +- O +Linker B-structure_element +showed O +very O +weak O +if O +not O +undetectable O +interaction O +with O +wild B-protein_state +- I-protein_state +type I-protein_state +or O +deletions O +of O +UHRF1 B-protein +, O +suggesting O +that O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +interaction O +is O +much O +weaker O +than O +that O +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +. O + +Taken O +together O +, O +UHRF1 B-protein +adopts O +a O +closed B-protein_state +conformation O +, O +in O +which O +the O +Spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +through O +competing O +with O +the O +Linker B-structure_element +, O +and O +therefore O +inhibits O +H3K9me3 B-protein_type +recognition O +by O +UHRF1 B-protein +. O + +The O +spacer B-structure_element +inhibits O +H3K9me3 B-protein_type +recognition O +by O +the O +isolated O +TTD B-structure_element + +Our O +previous O +study O +indicates O +that O +H3K9me3 B-protein_type +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +in O +different O +manner O +in O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +( O +ref O +.) O +and O +TTD B-complex_assembly +- I-complex_assembly +H3K9me3 I-complex_assembly +( O +PDB O +: O +2L3R O +) O +structures B-evidence +. O + +Because O +the O +TTD B-structure_element +is O +always O +associated O +with O +the O +PHD B-structure_element +, O +whether O +the O +pattern O +of O +TTD B-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +interaction O +exists O +in O +vivo O +remains O +unknown O +. O + +Nevertheless O +, O +comparison B-experimental_method +of O +TTD B-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +and O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +structures B-evidence +indicates O +that O +H3K9me3 B-protein_type +and O +the O +Spacer B-structure_element +overlap O +on O +the O +surface O +of O +the O +TTD B-structure_element +( O +Supplementary O +Fig O +. O +4d O +), O +suggesting O +that O +the O +Spacer B-structure_element +might O +block O +the O +H3K9me3 B-protein_type +recognition O +by O +the O +isolated O +TTD B-structure_element +. O + +As O +shown O +in O +Supplementary O +Fig O +. O +4e O +, O +the O +Spacer B-structure_element +inhibited O +TTD B-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +interaction O +, O +whereas O +its O +TTD B-protein_state +- I-protein_state +binding I-protein_state +defective I-protein_state +mutants B-protein_state +of O +the O +Spacer B-structure_element +or O +the O +SRA B-structure_element +( O +a O +negative O +control O +) O +markedly O +decreased O +the O +inhibition O +. O + +We O +next O +tested O +whether O +such O +inhibition O +also O +occurs O +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +. O + +Compared O +with O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +, O +UHRF1Δ627 B-mutant +– I-mutant +674 I-mutant +enhanced O +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +by O +a O +factor O +of O +four O +( O +Supplementary O +Fig O +. O +4f O +). O + +The O +restoration O +of O +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +is O +not O +dramatic O +because O +the O +PHD B-structure_element +still O +binds B-protein_state +to I-protein_state +histone B-protein_type +H3 B-protein_type +in O +both O +proteins O +. O + +To O +exclude O +this O +effect O +, O +we O +performed O +the O +assay O +using O +UHRF1D334A B-mutant +, O +which O +abolishes B-protein_state +H3R2 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +PHD B-structure_element +. O + +UHRF1D334A B-mutant +showed O +undetectable O +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +, O +whereas O +UHRF1D334A B-mutant +& O +Δ627 B-mutant +– I-mutant +674 I-mutant +dramatically O +restored O +its O +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +( O +KD B-evidence += O +8 O +. O +69 O +μM O +; O +Supplementary O +Fig O +. O +4f O +), O +indicating O +that O +H3K9me3 B-protein_type +recognition O +by O +the O +TTD B-structure_element +is O +blocked O +by O +the O +Spacer B-structure_element +through O +competitive O +interaction O +with O +the O +TTD B-structure_element +. O + +Moreover O +, O +the O +R295D B-mutant +/ O +R296D B-mutant +mutant B-protein_state +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +showed O +decreased O +binding B-evidence +affinity I-evidence +to O +H3K9me3 B-protein_type +( O +eightfold O +lower O +than O +wild B-protein_state +type I-protein_state +), O +suggesting O +that O +mutation B-experimental_method +of O +R295D B-mutant +/ O +R296D B-mutant +favours O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +and O +therefore O +promotes O +UHRF1 B-protein +to O +exhibit O +a O +more O +stable O +closed B-protein_state +conformation O +( O +Supplementary O +Fig O +. O +4g O +). O + +Taken O +together O +, O +the O +Spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +and O +inhibits O +H3K9me3 B-protein_type +recognition O +by O +UHRF1 B-protein +through O +( O +i O +) O +disrupting O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +interaction O +, O +which O +is O +essential O +for O +H3K9me3 B-protein_type +recognition O +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +, O +( O +ii O +) O +prohibiting O +H3K9me3 B-protein_type +binding O +to O +the O +isolated O +TTD B-structure_element +. O + +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +complex O +inhibits O +TTD B-structure_element +– I-structure_element +spacer I-structure_element +interaction O + +Interestingly O +, O +pre B-experimental_method +- I-experimental_method +incubation I-experimental_method +of O +H3K9me3 B-protein_type +peptide O +completely O +blocked O +the O +interaction O +between O +the O +Spacer B-structure_element +and O +the O +TTD B-structure_element +alone B-protein_state +or O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +( O +Supplementary O +Fig O +. O +4h O +), O +whereas O +the O +presence B-protein_state +of I-protein_state +the O +Spacer B-structure_element +partially O +impaired O +the O +interaction O +between O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +and O +H3K9me3 B-protein_type +( O +Fig O +. O +2c O +). O + +The O +results O +are O +also O +consistent O +with O +the O +previous O +observation O +that O +the O +interaction O +between O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +and O +the O +Spacer B-structure_element +is O +much O +weaker O +( O +KD B-evidence += O +10 O +. O +68 O +μM O +, O +Fig O +. O +3g O +) O +than O +that O +between O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +and O +H3K9me3 B-protein_type +( O +KD B-evidence += O +0 O +. O +15 O +μM O +, O +Fig O +. O +1d O +). O + +These O +results O +suggest O +that O +once O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +binds B-protein_state +to I-protein_state +H3K9me3 B-protein_type +, O +UHRF1 B-protein +will O +be O +locked O +by O +H3K9me3 B-protein_type +and O +the O +Spacer B-structure_element +is O +unlikely O +to O +fold O +back O +for O +the O +intramolecular O +interaction O +. O + +Hm B-chemical +- I-chemical +DNA I-chemical +disrupts O +intramolecular O +interaction O +within O +UHRF1 B-protein + +To O +investigate O +whether O +hm B-chemical +- I-chemical +DNA I-chemical +could O +open B-protein_state +the O +closed B-protein_state +conformation O +of O +UHRF1 B-protein +, O +we O +first O +measured O +the O +intramolecular O +interaction O +using O +UHRF1 B-protein +truncations B-experimental_method +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +. O + +The O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +show O +that O +the O +PHD B-structure_element +bound B-protein_state +to I-protein_state +the O +SRA B-structure_element +and O +such O +interaction O +was O +impaired O +by O +the O +addition O +of O +hm B-chemical +- I-chemical +DNA I-chemical +( O +Fig O +. O +4a O +). O + +H3 B-experimental_method +peptide I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +show O +that O +hm B-chemical +- I-chemical +DNA I-chemical +only O +enhanced O +the O +H3K9me0 B-evidence +- I-evidence +binding I-evidence +affinities I-evidence +of O +UHRF1 B-protein +truncations B-experimental_method +containing O +PHD B-structure_element +- I-structure_element +SRA I-structure_element +, O +such O +as O +PHD B-structure_element +- I-structure_element +SRA I-structure_element +, O +TTD B-structure_element +- I-structure_element +PHD I-structure_element +- I-structure_element +SRA I-structure_element +, O +TTD B-structure_element +- I-structure_element +PHD I-structure_element +- I-structure_element +SRA I-structure_element +- I-structure_element +Spacer I-structure_element +, O +UHRF1ΔTTD B-mutant +and O +UHRF1ΔSpacer B-mutant +( O +Fig O +. O +4b O +). O + +The O +result O +indicates O +that O +hm B-chemical +- I-chemical +DNA I-chemical +disrupts O +PHD B-structure_element +– I-structure_element +SRA I-structure_element +interaction O +and O +facilitates O +H3K9me0 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +PHD B-structure_element +in O +a O +manner O +independent O +on O +the O +TTD B-structure_element +or O +the O +Spacer B-structure_element +. O + +Moreover O +, O +the O +TTD B-structure_element +or O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +bound B-protein_state +to I-protein_state +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +and O +the O +interaction O +was O +impaired O +by O +the O +addition O +of O +hm B-chemical +- I-chemical +DNA I-chemical +( O +Fig O +. O +4c O +). O + +The O +ITC B-experimental_method +measurements O +show O +that O +the O +presence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +markedly O +impaired O +the O +interaction O +between O +the O +TTD B-structure_element +and O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +( O +Supplementary O +Fig O +. O +5a O +). O + +However O +, O +the O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +was O +not O +affected O +by O +the O +presence B-protein_state +of I-protein_state +the O +hm B-chemical +- I-chemical +DNA I-chemical +, O +indicating O +that O +hm B-chemical +- I-chemical +DNA I-chemical +displaces O +the O +Spacer B-structure_element +from O +the O +TTD B-structure_element +in O +a O +SRA B-structure_element +- O +dependent O +manner O +( O +Supplementary O +Fig O +. O +5b O +). O + +To O +investigate O +whether O +hm B-chemical +- I-chemical +DNA I-chemical +disrupts O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +, O +we O +monitored O +the O +conformational O +changes O +of O +UHRF1 B-protein +using O +its O +histone B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +as O +read O +- O +out O +. O + +UHRF1D334A B-mutant +was O +used O +to O +exclude O +the O +effect O +of O +H3K9me0 B-protein_type +recognition O +by O +the O +PHD B-structure_element +. O + +As O +expected O +, O +all O +D334A B-mutant +- O +containing O +mutants B-protein_state +showed O +undetectable O +interaction O +with O +H3K9me0 B-protein_type +( O +Fig O +. O +4d O +). O + +UHRF1D334A B-mutant +bound B-protein_state +to I-protein_state +H3K9me3 B-protein_type +peptide O +in O +the O +presence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +, O +but O +showed O +no O +interaction O +in O +the O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +, O +which O +is O +consistent O +with O +the O +ITC B-experimental_method +experiments O +( O +Supplementary O +Fig O +. O +4f O +). O + +In O +contrast O +, O +UHRF1D334A B-mutant +& O +Δ627 B-mutant +– I-mutant +674 I-mutant +strongly O +bound B-protein_state +to I-protein_state +H3K9me3 B-protein_type +even O +in O +the O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +( O +Fig O +. O +4d O +), O +indicating O +that O +the O +deletion B-experimental_method +of O +the O +Spacer B-structure_element +releases O +otherwise O +blocked O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +for O +H3K9me3 B-protein_type +recognition O +. O + +The O +results O +further O +support O +the O +conclusion O +that O +the O +Spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +the O +intramolecular O +interactions O +are O +disrupted O +by O +hm B-chemical +- I-chemical +DNA I-chemical +. O + +We O +next O +performed O +similar O +peptide B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +using O +two O +mutants B-protein_state +( O +N228C B-mutant +/ O +G653C B-mutant +and O +R235C B-mutant +/ O +G654C B-mutant +) O +generated O +on O +UHRF1D334A B-mutant +. O + +Residues O +N228 B-residue_name_number +/ O +R235 B-residue_name_number +from O +the O +TTD B-structure_element +and O +G653 B-residue_name_number +/ O +G654 B-residue_name_number +from O +the O +Spacer B-structure_element +were O +chosen O +according O +to O +the O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +complex O +structure B-evidence +( O +Supplementary O +Fig O +. O +5c O +) O +so O +that O +the O +replaced O +Cysteine B-residue_name +residues O +( O +one O +from O +the O +TTD B-structure_element +and O +one O +from O +the O +Spacer B-structure_element +) O +are O +physically O +close O +enough O +to O +each O +other O +to O +form O +a O +disulphide B-ptm +bond I-ptm +in O +the O +absence B-protein_state +of I-protein_state +reducing O +reagent O +( O +dithiothreitol B-chemical +, O +DTT B-chemical +). O + +As O +shown O +in O +Fig O +. O +4d O +, O +hm B-chemical +- I-chemical +DNA I-chemical +largely O +enhanced O +the O +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinities I-evidence +of O +both O +mutants B-protein_state +in O +the O +presence B-protein_state +of I-protein_state +DTT B-chemical +, O +but O +not O +in O +the O +absence B-protein_state +of I-protein_state +DTT B-chemical +, O +indicating O +that O +the O +disulphide B-ptm +bond I-ptm +formation O +( O +in O +the O +absence B-protein_state +of I-protein_state +DTT B-chemical +) O +disallows O +hm B-chemical +- I-chemical +DNA I-chemical +to O +disrupt O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +for O +H3K9me3 B-protein_type +recognition O +. O + +As O +negative O +controls O +, O +H3K9me3 B-protein_type +recognition O +by O +UHRF1D334A B-mutant +or O +UHRF1D334A B-mutant +& O +Δ627 B-mutant +– I-mutant +674 I-mutant +is O +not O +affected O +by O +DTT B-chemical +. O + +The O +above O +results O +collectively O +demonstrate O +that O +( O +i O +) O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +adopts O +a O +closed B-protein_state +form O +, O +in O +which O +the O +Spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +and O +H3K9me3 B-protein_type +recognition O +is O +inhibited O +; O +( O +ii O +) O +hm B-chemical +- I-chemical +DNA I-chemical +displaces O +the O +Spacer B-structure_element +from O +the O +TTD B-structure_element +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +therefore O +largely O +enhances O +its O +histone B-protein_type +H3K9me3 B-protein_type +- O +binding O +activity O +in O +a O +manner O +independent O +on O +the O +PHD B-structure_element +( O +SRA B-structure_element +is O +required O +). O + +We O +have O +previously O +demonstrated O +that O +hm B-chemical +- I-chemical +DNA I-chemical +also O +disrupts O +PHD B-structure_element +– I-structure_element +SRA I-structure_element +interaction O +and O +facilitates O +H3K9me0 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +PHD B-structure_element +in O +a O +manner O +independent O +on O +the O +TTD B-structure_element +or O +the O +Spacer B-structure_element +. O + +Taken O +together O +, O +hm B-chemical +- I-chemical +DNA I-chemical +disrupts O +the O +intramolecular O +interactions O +within O +UHRF1 B-protein +, O +and O +therefore O +facilitates O +the O +coordinate O +recognition O +of O +H3K9me3 B-protein_type +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +. O + +The O +spacer B-structure_element +enhances O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +SRA B-structure_element + +To O +investigate O +how O +hm B-chemical +- I-chemical +DNA I-chemical +impairs O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +, O +we O +tested O +whether O +the O +Spacer B-structure_element +is O +involved O +in O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +by O +the O +SRA B-structure_element +, O +which O +is O +the O +only O +known O +domain O +for O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +within O +UHRF1 B-protein +. O + +In O +the O +electrophoretic B-experimental_method +mobility I-experimental_method +- I-experimental_method +shift I-experimental_method +assay I-experimental_method +, O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +showed O +higher O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +than O +the O +SRA B-structure_element +alone B-protein_state +( O +Supplementary O +Fig O +. O +6a O +). O + +ITC B-experimental_method +measurements O +show O +that O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +bound B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +with O +a O +much O +higher O +binding B-evidence +affinity I-evidence +( O +KD B-evidence += O +1 O +. O +75 O +μM O +) O +than O +the O +SRA B-structure_element +( O +KD B-evidence += O +25 O +. O +12 O +μM O +), O +whereas O +the O +Spacer B-structure_element +alone B-protein_state +showed O +no O +interaction O +with O +hm B-chemical +- I-chemical +DNA I-chemical +( O +Fig O +. O +5a O +). O + +In O +the O +fluorescence B-experimental_method +polarization I-experimental_method +( I-experimental_method +FP I-experimental_method +) O +measurements O +, O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +, O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +UHRF1ΔTTD B-mutant +showed O +comparable O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinities I-evidence +( O +Fig O +. O +5b O +and O +Supplementary O +Table O +4 O +), O +suggesting O +that O +UHRF1 B-protein +binds B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +no O +matter O +UHRF1 B-protein +adopts O +a O +closed B-protein_state +form O +or O +not O +. O + +In O +contrast O +, O +UHRF1ΔSRA B-mutant +abolished O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +, O +indicating O +that O +the O +SRA B-structure_element +is O +essential O +for O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +. O + +Compared O +with O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +, O +UHRF1Δ627 B-mutant +– I-mutant +674 I-mutant +decreased O +the O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +by O +a O +factor O +of O +14 O +( O +Fig O +. O +5b O +), O +further O +supporting O +that O +the O +Spacer B-structure_element +plays O +an O +important O +role O +in O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +. O + +In O +addition O +, O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinities I-evidence +of O +SRA B-structure_element +or O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +did O +not O +obviously O +vary O +upon O +the O +change O +of O +DNA O +lengths O +but O +did O +decrease O +with O +the O +increasing O +salt O +concentrations O +( O +Supplementary O +Fig O +. O +6b O +, O +c O +and O +Supplementary O +Table O +5 O +). O + +These O +results O +indicate O +that O +the O +Spacer B-structure_element +not O +only O +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +and O +inhibits O +H3K9me3 B-protein_type +recognition O +when O +UHRF1 B-protein +adopts O +closed B-protein_state +conformation O +, O +but O +also O +facilitates O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +by O +the O +SRA B-structure_element +when O +UHRF1 B-protein +binds B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +. O + +We O +next O +mapped O +the O +minimal O +region O +of O +the O +Spacer B-structure_element +for O +the O +enhancement O +of O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +. O + +SRA B-mutant +– I-mutant +Spacer I-mutant +- I-mutant +661 I-mutant +( O +residues O +414 B-residue_range +– I-residue_range +661 I-residue_range +) O +still O +maintained O +strong O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +comparable O +to O +that O +of O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +( O +residues O +414 B-residue_range +– I-residue_range +674 I-residue_range +), O +whereas O +SRA B-mutant +– I-mutant +Spacer I-mutant +- I-mutant +652 I-mutant +and O +SRA B-mutant +– I-mutant +Spacer I-mutant +- I-mutant +642 I-mutant +markedly O +decreased O +their O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinities I-evidence +( O +Fig O +. O +5c O +), O +indicating O +that O +residues O +642 B-residue_range +– I-residue_range +661 I-residue_range +are O +important O +for O +enhancing O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +SRA B-structure_element +. O + +This O +minimal O +region O +largely O +overlaps O +with O +the O +Spacer B-structure_element +region O +( O +643 B-residue_range +– I-residue_range +655 I-residue_range +) O +essential O +for O +TTD B-structure_element +interaction O +. O + +We O +also O +determined O +the O +crystal B-evidence +structure I-evidence +of O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +bound B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +at O +3 O +. O +15 O +Å O +resolution O +( O +Supplementary O +Table O +6 O +and O +Supplementary O +Fig O +. O +7a O +). O + +The O +structure B-evidence +shows O +that O +the O +SRA B-structure_element +binds B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +in O +a O +manner O +similar O +to O +that O +observed O +in O +the O +previously O +reported O +SRA B-complex_assembly +- I-complex_assembly +hm I-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +structures B-evidence +. O + +Intriguingly O +, O +no O +electron B-evidence +density I-evidence +was O +observed O +for O +the O +Spacer B-structure_element +. O + +A O +possible O +explanation O +is O +that O +the O +Spacer B-structure_element +facilitates O +SRA B-complex_assembly +– I-complex_assembly +hm I-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +interaction O +through O +nonspecific O +salt B-bond_interaction +bridge I-bond_interaction +contacts O +because O +DNA B-chemical +is O +rich O +in O +acidic O +groups O +and O +the O +Spacer B-structure_element +is O +rich O +in O +basic O +residues O +( O +Supplementary O +Fig O +. O +7b O +). O + +The O +nonspecific O +interaction O +is O +consistent O +with O +the O +previous O +observation O +that O +UHRF1 B-protein +has O +no O +DNA B-chemical +sequence O +selectivity O +besides O +hm B-chemical +- I-chemical +CpG I-chemical +dinucleotide I-chemical +. O + +The O +spacer B-structure_element +is O +important O +for O +PCH O +localization O +of O +UHRF1 B-protein + +To O +investigate O +the O +role O +of O +the O +Spacer B-structure_element +in O +the O +regulation O +of O +UHRF1 B-protein +function O +, O +we O +transiently B-experimental_method +overexpressed I-experimental_method +GFP B-protein_state +- I-protein_state +tagged I-protein_state +wild B-protein_state +type I-protein_state +or O +mutants B-protein_state +of O +UHRF1 B-protein +in O +NIH3T3 O +cells O +to O +determine O +their O +subcellular O +localization O +. O + +For O +the O +NIH3T3 O +cells O +expressing O +wild B-protein_state +- I-protein_state +type I-protein_state +UHRF1 B-protein +, O +most O +cells O +(∼ O +74 O +. O +6 O +%) O +showed O +a O +focal O +pattern O +of O +protein O +that O +is O +co O +- O +localized O +with O +4 B-chemical +, I-chemical +6 I-chemical +- I-chemical +diamidino I-chemical +- I-chemical +2 I-chemical +- I-chemical +phenylindole I-chemical +( O +DAPI B-chemical +) O +foci O +( O +Fig O +. O +5d O +), O +whereas O +the O +rest O +cells O +showed O +a O +diffuse O +nuclear O +staining O +pattern O +. O + +The O +result O +is O +consistent O +with O +the O +previous O +studies O +that O +UHRF1 B-protein +is O +mainly O +localized O +to O +highly B-protein_state +methylated I-protein_state +pericentromeric O +heterochromatin O +( O +PCH O +). O + +In O +contrast O +, O +for O +the O +cells O +expressing O +UHRF1Δ627 B-mutant +– I-mutant +674 I-mutant +, O +a O +spacer B-protein_state +deletion I-protein_state +mutant I-protein_state +with O +decreased O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +( O +Fig O +. O +5b O +), O +only O +∼ O +22 O +. O +1 O +% O +cells O +showed O +co O +- O +localization O +with O +DAPI B-chemical +. O + +Previous O +reports O +have O +shown O +that O +the O +H3K9me3 B-protein_type +recognition O +of O +UHRF1 B-protein +also O +plays O +an O +important O +role O +in O +its O +heterochromatin O +localization O +. O + +For O +example O +, O +UHRF1 B-protein +mutant B-protein_state +( O +within O +TTD B-structure_element +domain O +) O +lacking B-protein_state +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +largely O +reduces O +its O +co O +- O +localization O +with O +heterochromatin O +. O + +Because O +manipulation O +of O +endogenous O +hm B-chemical +- I-chemical +DNA I-chemical +in O +cells O +is O +technically O +challenging O +, O +we O +used O +UHRF1ΔSRA B-mutant +( O +lacks B-protein_state +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +but O +maintains O +closed B-protein_state +conformation O +, O +Figs O +3h O +and O +5b O +) O +to O +test O +whether O +closed B-protein_state +conformation O +of O +UHRF1 B-protein +exists O +in O +vivo O +. O + +In O +NIH3T3 O +cells O +, O +UHRF1ΔSRA B-mutant +largely O +decreased O +chromatin O +association O +( O +Fig O +. O +5d O +). O + +Only O +∼ O +4 O +. O +8 O +% O +cells O +expressing O +UHRF1ΔSRA B-mutant +showed O +an O +intermediate O +enrichment O +, O +but O +not O +characteristic O +focal O +pattern O +, O +at O +DAPI B-chemical +foci O +, O +whereas O +the O +majority O +of O +the O +cells O +showed O +a O +diffuse O +nuclear O +staining O +pattern O +. O + +The O +results O +suggest O +that O +UHRF1ΔSRA B-mutant +adopts O +closed B-protein_state +conformation O +so O +that O +H3K9me3 B-protein_type +recognition O +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +is O +blocked O +by O +the O +intramolecular O +interaction O +, O +and O +support O +the O +regulatory O +role O +of O +the O +Spacer B-structure_element +in O +PCH O +localization O +of O +UHRF1 B-protein +in O +vivo O +. O + +The O +spacer B-structure_element +facilitates O +UHRF1 B-complex_assembly +– I-complex_assembly +DNMT1 I-complex_assembly +interaction O + +Previous O +studies O +show O +that O +UHRF1 B-protein +recruits O +DNMT1 B-protein +to O +hm B-chemical +- I-chemical +DNA I-chemical +for O +maintenance O +DNA B-chemical +methylation B-ptm +through O +the O +interaction O +between O +the O +SRA B-structure_element +and O +RFTSDNMT1 B-protein +( O +refs O +). O + +We O +confirmed O +the O +direct O +interaction O +between O +RFTSDNMT1 B-protein +and O +the O +SRA B-structure_element +in O +a O +solution O +with O +low O +salt O +concentration O +( O +50 O +mM O +NaCl B-chemical +), O +but O +observed O +weak O +or O +undetectable O +interaction O +in O +a O +solution O +with O +higher O +salt O +concentrations O +( O +100 O +or O +150 O +mM O +NaCl B-chemical +) O +( O +Supplementary O +Fig O +. O +8a O +). O + +Compared O +with O +the O +SRA B-structure_element +, O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +exhibited O +stronger O +interaction O +with O +RFTSDNMT1 B-protein +. O + +In O +addition O +, O +RFTSDNMT1 B-protein +bound B-protein_state +to I-protein_state +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +with O +a O +binding B-evidence +affinity I-evidence +of O +7 O +. O +09 O +μM O +, O +but O +showed O +no O +detectable O +interaction O +with O +the O +SRA B-structure_element +( O +Supplementary O +Fig O +. O +8b O +). O + +Interestingly O +, O +the O +addition O +of O +hm B-chemical +- I-chemical +DNA I-chemical +abolished O +the O +interaction O +between O +RFTSDNMT1 B-protein +and O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +, O +suggesting O +that O +hm B-chemical +- I-chemical +DNA I-chemical +also O +regulates O +UHRF1 B-complex_assembly +– I-complex_assembly +DNMT1 I-complex_assembly +interaction O +( O +Supplementary O +Fig O +. O +8c O +). O + +These O +results O +indicate O +that O +the O +Spacer B-structure_element +facilitates O +the O +interaction O +between O +RFTSDNMT1 B-protein +and O +the O +SRA B-structure_element +, O +and O +the O +interaction O +is O +impaired O +by O +the O +presence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +. O + +We O +next O +tested O +whether O +the O +UHRF1 B-complex_assembly +– I-complex_assembly +DNMT1 I-complex_assembly +interaction O +is O +regulated O +by O +the O +conformational O +change O +of O +UHRF1 B-protein +. O + +Because O +the O +addition O +of O +hm B-chemical +- I-chemical +DNA I-chemical +disrupts O +the O +interaction O +between O +the O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +and O +RFTSDNMT1 B-protein +, O +we O +used O +various O +truncations B-experimental_method +to O +mimic O +open B-protein_state +and O +closed B-protein_state +forms O +of O +UHRF1 B-protein +. O + +In O +the O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +, O +only O +UHRF1ΔTTD B-mutant +bound B-protein_state +to I-protein_state +RFTSDNMT1 B-protein +, O +whereas O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +, O +UHRF1ΔSRA B-mutant +and O +UHRF1Δ627 B-mutant +– I-mutant +674 I-mutant +showed O +undetectable O +interaction O +( O +Fig O +. O +5e O +). O + +As O +the O +deletion B-experimental_method +of I-experimental_method +the O +TTD B-structure_element +allows O +UHRF1 B-protein +to O +adopt O +an O +open B-protein_state +conformation O +, O +the O +results O +suggest O +that O +RFTSDNMT1 B-protein +binds B-protein_state +to I-protein_state +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +when O +UHRF1 B-protein +adopts O +an O +open B-protein_state +conformation O +in O +the O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +. O + +In O +support O +of O +above O +observations O +, O +the O +addition B-experimental_method +of O +large O +amount O +of O +RFTSDNMT1 B-protein +impaired O +the O +interaction O +between O +UHRF1 B-protein +and O +hm B-chemical +- I-chemical +DNA I-chemical +( O +Supplementary O +Fig O +. O +8d O +), O +suggesting O +an O +existence O +of O +dynamic O +equilibrium O +between O +UHRF1 B-complex_assembly +– I-complex_assembly +hm I-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +and O +UHRF1 B-complex_assembly +– I-complex_assembly +DNMT1 I-complex_assembly +complexes O +. O + +According O +to O +the O +above O +results O +, O +we O +here O +proposed O +a O +working O +model O +for O +hm B-chemical +- I-chemical +DNA I-chemical +- O +mediated O +regulation O +of O +UHRF1 B-protein +conformation O +( O +Fig O +. O +5f O +). O + +In O +the O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +( O +A O +), O +UHRF1 B-protein +prefers O +a O +closed B-protein_state +conformation O +, O +in O +which O +the O +Spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +by O +competing O +with O +the O +Linker B-structure_element +and O +the O +SRA B-structure_element +binds B-protein_state +to I-protein_state +the O +PHD B-structure_element +. O + +As O +a O +result O +, O +the O +recognition O +of O +histone B-protein_type +H3K9me3 B-protein_type +by O +the O +TTD B-structure_element +is O +blocked O +by O +the O +Spacer B-structure_element +, O +and O +recognition O +of O +unmodified B-protein_state +histone B-protein_type +H3 B-protein_type +( O +H3R2 B-site +) O +by O +the O +PHD B-structure_element +is O +inhibited O +by O +the O +SRA B-structure_element +. O + +The O +interaction O +between O +UHRF1 B-protein +and O +DNMT1 B-protein +is O +also O +weak O +because O +the O +Spacer B-structure_element +is O +unable O +to O +facilitate O +the O +intermolecular O +interaction O +. O + +In O +the O +presence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +( O +B O +), O +UHRF1 B-protein +prefers O +an O +open B-protein_state +conformation O +, O +in O +which O +the O +SRA B-structure_element +binds B-protein_state +to I-protein_state +the O +hm B-chemical +- I-chemical +DNA I-chemical +; O +the O +Spacer B-structure_element +dissociates O +from O +the O +TTD B-structure_element +and O +facilitates O +the O +interaction O +between O +the O +SRA B-structure_element +and O +hm B-chemical +- I-chemical +DNA I-chemical +; O +the O +Linker B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +and O +allows O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +to O +recognize O +histone B-protein_type +H3K9me3 B-protein_type +. O + +When O +UHRF1 B-protein +adopts O +an O +open B-protein_state +conformation O +and O +has O +already O +bound B-protein_state +to I-protein_state +H3K9me3 B-protein_type +( O +B O +), O +the O +interaction O +between O +H3K9me3 B-protein_type +and O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +further O +prevents O +the O +Spacer B-structure_element +from O +folding O +back O +to O +interact O +with O +the O +TTD B-structure_element +, O +and O +therefore O +locks O +UHRF1 B-protein +in O +an O +open B-protein_state +conformation O +. O + +The O +association O +of O +UHRF1 B-protein +to O +the O +histone B-protein_type +may O +facilitate O +the O +ubiquitination B-ptm +of O +histone B-protein_type +tail O +( O +mediated O +by O +RING B-structure_element +domain O +) O +for O +DNMT1 B-protein +targeting O +. O + +Moreover O +, O +through O +a O +mechanism O +yet O +to O +be O +fully O +elucidated O +, O +DNMT1 B-protein +targets O +hm B-chemical +- I-chemical +DNA I-chemical +for O +maintenance O +DNA O +methylation B-ptm +, O +probably O +through O +interaction O +with O +the O +histone B-protein_type +ubiquitylation B-ptm +and O +/ O +or O +SRA B-structure_element +- I-structure_element +Spacer I-structure_element +. O + +The O +P B-evidence +( I-evidence +r I-evidence +) I-evidence +function I-evidence +obtained O +from O +small B-experimental_method +- I-experimental_method +angle I-experimental_method +X I-experimental_method +- I-experimental_method +ray I-experimental_method +scattering I-experimental_method +( O +SAXS B-experimental_method +) O +measurements O +of O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +SRA I-complex_assembly +– I-complex_assembly +Spacer I-complex_assembly +– I-complex_assembly +hm I-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +complex O +showed O +a O +broader O +distribution O +than O +that O +of O +the O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +SRA I-complex_assembly +– I-complex_assembly +Spacer I-complex_assembly +alone O +, O +supporting O +the O +proposed O +model O +that O +UHRF1 B-protein +adopts O +an O +open B-protein_state +conformation O +in O +the O +presence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +( O +Supplementary O +Fig O +. O +8e O +). O + +We O +have O +tried O +crystallizing B-experimental_method +more O +than O +three O +sub O +- O +constructs O +with B-protein_state +and O +without B-protein_state +DNA B-chemical +across O +over O +1 O +, O +200 O +crystallization O +conditions O +but O +failed O +to O +determine O +the O +structure B-evidence +of O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +SRA I-complex_assembly +– I-complex_assembly +Spacer I-complex_assembly +in O +the O +absence B-protein_state +or O +presence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +. O + +Getting O +these O +structures B-evidence +would O +greatly O +help O +for O +understanding O +the O +hm B-chemical +- I-chemical +DNA I-chemical +- O +mediated O +regulation O +of O +UHRF1 B-protein +. O + +In O +addition O +, O +this O +regulatory O +process O +should O +be O +further O +characterized O +using O +advanced O +techniques O +, O +such O +as O +single B-experimental_method +molecular I-experimental_method +measurement I-experimental_method +. O + +Our O +previous O +studies O +show O +that O +phosphorylation B-ptm +at O +S639 B-residue_name_number +within O +the O +Spacer B-structure_element +disrupts O +interaction O +between O +UHRF1 B-protein +and O +deubiquitylase B-protein_type +USP7 B-protein +and O +decreases O +UHRF1 B-protein +stability O +in O +the O +M O +phase O +of O +the O +cell O +cycle O +. O + +The O +Spacer B-structure_element +was O +predicted O +to O +contain O +two O +nuclear B-structure_element +localization I-structure_element +signals I-structure_element +, O +residues O +581 B-residue_range +– I-residue_range +600 I-residue_range +and O +648 B-residue_range +- I-residue_range +670 I-residue_range +( O +ref O +.). O + +In O +this O +report O +, O +we O +found O +that O +the O +Spacer B-structure_element +( O +i O +) O +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +in O +the O +closed B-protein_state +form O +of O +UHRF1 B-protein +and O +inhibits O +its O +interaction O +with O +H3K9me3 B-protein_type +; O +( O +ii O +) O +facilitates O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +by O +the O +SRA B-structure_element +and O +( O +iii O +) O +facilitates O +the O +interaction O +between O +the O +SRA B-structure_element +and O +RFTSDNMT1 B-protein +. O + +These O +findings O +together O +indicate O +that O +the O +Spacer B-structure_element +plays O +a O +very O +important O +role O +in O +the O +dynamic O +regulation O +of O +UHRF1 B-protein +. O + +When O +our O +manuscript O +was O +in O +preparation O +, O +Gelato O +et O +al O +. O +reported O +that O +binding O +of O +PI5P B-chemical +to O +the O +Spacer B-structure_element +opens O +the O +closed B-protein_state +conformation O +of O +UHRF1 B-protein +and O +increases O +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +TTD B-structure_element +. O + +The O +result O +suggests O +that O +PI5P B-chemical +may O +facilitate O +the O +conformational O +change O +of O +UHRF1 B-protein +induced O +by O +hm B-chemical +- I-chemical +DNA I-chemical +when O +UHRF1 B-protein +is O +recruited O +to O +chromatin O +. O + +In O +addition O +, O +mass B-experimental_method +- I-experimental_method +spectrometry I-experimental_method +analyses O +have O +identified O +several O +phosphorylation B-site +sites I-site +( O +S639 B-residue_name_number +, O +S651 B-residue_name_number +, O +S661 B-residue_name_number +) O +within O +the O +Spacer B-structure_element +, O +suggesting O +that O +post O +- O +translational O +modification O +may O +add O +another O +layer O +of O +regulation O +of O +UHRF1 B-protein +( O +refs O +). O + +It O +has O +been O +well O +characterized O +that O +the O +SRA B-structure_element +of O +UHRF1 B-protein +preferentially O +recognizes O +hm B-chemical +- I-chemical +DNA I-chemical +through O +a O +base O +- O +flipping O +mechanism O +. O + +Our O +study O +demonstrates O +that O +the O +Spacer B-structure_element +markedly O +enhances O +the O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +SRA B-structure_element +and O +the O +deletion B-experimental_method +of I-experimental_method +the O +Spacer B-structure_element +impairs O +heterochromatin O +localization O +of O +UHRF1 B-protein +, O +indicating O +that O +the O +Spacer B-structure_element +is O +essential O +for O +recognition O +of O +hm B-chemical +- I-chemical +DNA I-chemical +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +. O + +Interestingly O +, O +variant B-protein +in I-protein +methylation I-protein +1 I-protein +( O +VIM1 B-protein +, O +a O +UHRF1 B-protein +homologue O +in O +Arabidopsis B-taxonomy_domain +) O +contains O +an O +equivalent O +spacer B-structure_element +region O +, O +which O +was O +shown O +to O +be O +required O +for O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +by O +its O +SRA B-structure_element +domain O +, O +suggesting O +a O +conserved O +regulatory O +mechanism O +in O +SRA B-structure_element +domain O +- O +containing O +proteins O +. O + +Intriguingly O +, O +UHRF2 B-protein +( O +the O +only O +mammalian B-taxonomy_domain +homologue O +of O +UHRF1 B-protein +) O +and O +UHRF1 B-protein +show O +very O +high O +sequence O +similarities O +for O +all O +the O +domains O +but O +very O +low O +similarity O +for O +the O +Spacer B-structure_element +( O +Supplementary O +Fig O +. O +7c O +). O + +Thus O +, O +although O +UHRF2 B-protein +exhibits O +the O +histone O +- O +and O +hm O +- O +DNA O +- O +binding O +activities O +, O +the O +difference O +in O +the O +Spacer B-structure_element +region O +may O +contribute O +to O +the O +functional O +differences O +between O +UHRF1 B-protein +and O +UHRF2 B-protein +. O + +This O +is O +also O +consistent O +with O +previous O +finding O +that O +UHRF2 B-protein +is O +unable O +to O +replace O +UHRF1 B-protein +to O +maintain O +the O +DNA B-chemical +methylation B-ptm +. O + +One O +of O +the O +key O +questions O +in O +the O +field O +of O +DNA B-chemical +methylation B-ptm +is O +why O +UHRF1 B-protein +contains O +modules O +recognizing O +two O +repressive O +epigenetic O +marks O +: O +H3K9me3 B-protein_type +( O +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +) O +and O +hm B-chemical +- I-chemical +DNA I-chemical +( O +by O +the O +SRA B-structure_element +). O + +Previous O +studies O +show O +that O +chromatin O +localization O +of O +UHRF1 B-protein +is O +dependent O +on O +hm B-chemical +- I-chemical +DNA I-chemical +, O +whereas O +other O +studies O +indicate O +that O +histone B-protein_type +H3K9me3 B-protein_type +recognition O +and O +hm B-chemical +- I-chemical +DNA I-chemical +association O +are O +both O +required O +for O +UHRF1 B-protein +- O +mediated O +maintenance O +DNA B-chemical +methylation B-ptm +. O + +However O +, O +little O +is O +known O +about O +the O +crosstalk O +between O +these O +two O +epigenetic O +marks O +within O +UHRF1 B-protein +. O + +As O +shown O +in O +the O +proposed O +model O +, O +recognition O +of O +H3K9me3 B-protein_type +by O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +is O +blocked O +to O +avoid O +its O +miss O +- O +localization O +to O +unmethylated B-protein_state +genomic O +region O +, O +in O +which O +chromatin O +contains O +H3K9me3 B-protein_type +( O +KD B-evidence += O +4 O +. O +61 O +μM O +) O +or O +H3K9me0 B-protein_type +( O +KD B-evidence += O +25 O +. O +99 O +μM O +). O + +We O +have O +shown O +that O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +strongly O +bind O +to O +hm B-chemical +- I-chemical +DNA I-chemical +( O +0 O +. O +35 O +and O +0 O +. O +49 O +μM O +, O +respectively O +) O +and O +the O +Spacer B-structure_element +plays O +an O +important O +role O +in O +PCH O +localization O +( O +Fig O +. O +5d O +). O + +Therefore O +, O +genomic O +localization O +of O +UHRF1 B-protein +is O +primarily O +determined O +by O +its O +recognition O +of O +hm B-chemical +- I-chemical +DNA I-chemical +, O +which O +allows O +UHRF1 B-protein +to O +adopt O +an O +open B-protein_state +form O +and O +promotes O +its O +histone B-protein_type +tail O +recognition O +for O +proper O +genomic O +localization O +and O +function O +. O + +As O +a O +result O +, O +when O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +dissociates O +from O +hm B-chemical +- I-chemical +DNA I-chemical +and O +binds B-protein_state +to I-protein_state +DNMT1 B-protein +with O +a O +currently O +unknown O +mechanism O +, O +UHRF1 B-protein +may O +keep O +the O +complex O +associated O +with O +chromatin O +through O +the O +interaction O +between O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +and O +H3K9me3 B-protein_type +( O +or O +PHD B-structure_element +- O +H3 B-protein_type +), O +and O +make O +it O +possible O +for O +DNMT1 B-protein +to O +target O +proper O +DNA B-chemical +substrate O +for O +methylation B-ptm +. O + +This O +explanation O +agrees O +nicely O +with O +previous O +observations O +and O +clarifies O +the O +importance O +of O +coordinate O +recognition O +of O +H3K9me3 B-protein_type +and O +hm B-chemical +- I-chemical +DNA I-chemical +by O +UHRF1 B-protein +for O +maintenance O +DNA O +methylation B-ptm +. O + +UHRF1 B-protein +is O +essential O +for O +maintenance O +DNA B-chemical +methylation B-ptm +through O +recruiting O +DNMT1 B-protein +to O +DNA B-chemical +replication O +forks O +during O +S O +phase O +. O + +This O +function O +is O +probably O +induced O +by O +a O +direct O +interaction O +between O +the O +SRA B-structure_element +and O +RFTSDNMT1 B-protein +( O +refs O +) O +or O +interaction O +between O +DNMT1 B-protein +and O +ubiquitylation B-ptm +of O +histione B-protein_type +tail O +. O + +Recent O +study O +indicates O +that O +histone B-protein_type +tail O +association O +of O +UHRF1 B-protein +( O +by O +the O +PHD B-structure_element +domain O +) O +is O +required O +for O +histone B-protein_type +H3 B-protein_type +ubiquitylation B-ptm +, O +which O +is O +dependent O +on O +ubiquitin B-protein_type +ligase I-protein_type +activity O +of O +the O +RING B-structure_element +domain O +of O +UHRF1 B-protein +( O +ref O +.). O + +DNMT1 B-protein +binds B-protein_state +to I-protein_state +ubiquitylated B-protein_state +histone B-protein_type +H3 B-protein_type +and O +ubiquitylation B-ptm +is O +required O +for O +maintenance O +of O +DNA B-chemical +methylation B-ptm +in O +vivo O +. O + +In O +this O +study O +, O +we O +found O +that O +both O +TTD B-structure_element +and O +PHD B-structure_element +are O +regulated O +by O +hm B-chemical +- I-chemical +DNA I-chemical +to O +recognize O +histone B-protein_type +tail O +. O + +Thus O +, O +the O +closed B-protein_state +form O +UHRF1 B-protein +may O +prevent O +miss O +localization O +of O +URHF1 B-protein +, O +whereas O +only O +the O +UHRF1 B-protein +in O +open B-protein_state +conformation O +( O +induced O +by O +hm B-chemical +- I-chemical +DNA I-chemical +) O +could O +properly O +binds B-protein_state +to I-protein_state +histone B-protein_type +tail O +for O +ubiquitylation B-ptm +and O +subsequent O +DNA B-chemical +methylation B-ptm +. O + +Moreover O +, O +structural B-experimental_method +analyses I-experimental_method +of O +DNMT1 B-complex_assembly +– I-complex_assembly +DNA I-complex_assembly +and O +SRA B-complex_assembly +– I-complex_assembly +DNA I-complex_assembly +complexes O +also O +indicate O +that O +it O +is O +impossible O +for O +DNMT1 B-protein +to O +methylate O +the O +hm B-chemical +- I-chemical +DNA I-chemical +that O +UHRF1 B-protein +binds B-protein_state +to I-protein_state +because O +of O +steric O +hindrance O +. O + +In O +our O +in B-experimental_method +vitro I-experimental_method +assays I-experimental_method +, O +we O +could O +detect O +interaction O +between O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +and O +RFTSDNMT1 B-protein +, O +but O +not O +the O +interaction O +between O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +RFTSDNMT1 B-protein +( O +Supplementary O +Fig O +. O +8a O +, O +b O +and O +Fig O +. O +5e O +). O + +The O +results O +suggest O +that O +UHRF1 B-protein +adopts O +multiple O +conformations O +. O + +Binding O +of O +UHRF1 B-protein +to O +hm B-chemical +- I-chemical +DNA I-chemical +may O +serve O +as O +a O +switch O +for O +its O +recruitment O +of O +DNMT1 B-protein +. O + +The O +S O +phase O +- O +dependent O +interaction O +between O +UHRF1 B-protein +and O +DNMT1 B-protein +( O +refs O +) O +suggest O +that O +DNMT1 B-protein +may O +also O +undergo O +conformation O +changes O +so O +that O +RFTSDNMT1 B-protein +binds B-protein_state +to I-protein_state +UHRF1 B-protein +and O +the O +catalytic B-structure_element +domain I-structure_element +of O +DNMT1 B-protein +binds B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +for O +reaction O +. O + +Hm B-chemical +- I-chemical +DNA I-chemical +facilities O +histione B-protein_type +tails O +recognition O +by O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +. O + +( O +a O +) O +Colour O +- O +coded O +domain O +structure O +of O +human B-species +UHRF1 B-protein +. O + +Note O +that O +the O +conserved B-protein_state +motif O +( O +green O +background O +) O +of O +the O +Linker B-structure_element +( O +residues O +286 B-residue_range +– I-residue_range +306 I-residue_range +) O +and O +the O +Spacer B-structure_element +( O +residues O +587 B-residue_range +– I-residue_range +674 I-residue_range +) O +bind O +to O +the O +TTD B-structure_element +in O +a O +similar O +manner O +( O +Fig O +. O +3b O +). O +( O +b O +) O +Hm B-chemical +- I-chemical +DNA I-chemical +facilities O +histone B-protein_type +H3 B-protein_type +and O +H3K9me3 B-protein_type +recognition O +by O +UHRF1 B-protein +. O + +Purified O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +was O +incubated O +with O +biotinylated B-protein_state +H3 B-protein_type +( O +1 B-residue_range +– I-residue_range +21 I-residue_range +) O +or O +H3K9me3 B-protein_type +( O +1 B-residue_range +– I-residue_range +21 I-residue_range +) O +peptides O +in O +the O +presence O +or O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +( O +molar O +ratio O +UHRF1 B-protein +/ O +hm B-chemical +- I-chemical +DNA I-chemical += O +1 O +: O +2 O +). O + +The O +bound O +proteins O +were O +analysed O +in O +SDS B-experimental_method +– I-experimental_method +PAGE I-experimental_method +followed O +by O +Coomassie O +blue O +staining O +. O + +Sequences O +of O +the O +peptides O +are O +indicated O +in O +Supplementary O +Table O +1 O +. O +( O +c O +) O +Histone B-protein_type +peptides O +do O +not O +affect O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +UHRF1 B-protein +. O + +Full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +was O +incubated B-experimental_method +with I-experimental_method +biotinylated B-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +in O +the O +presence O +or O +absence B-protein_state +of I-protein_state +H3 B-protein_type +( O +1 B-residue_range +– I-residue_range +17 I-residue_range +) O +or O +H3K9me3 B-protein_type +( O +1 B-residue_range +– I-residue_range +17 I-residue_range +) O +peptides O +and O +analysed O +as O +in O +b O +. O +( O +d O +, O +e O +) O +Superimposed O +ITC B-experimental_method +enthalpy B-evidence +plots I-evidence +for O +binding O +of O +H3K9me3 B-protein_type +peptide O +( O +1 B-residue_range +– I-residue_range +17 I-residue_range +) O +to O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +and O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +( O +d O +), O +and O +H3 B-protein_type +peptide O +( O +1 B-residue_range +– I-residue_range +17 I-residue_range +) O +to O +the O +PHD B-structure_element +and O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +( O +e O +). O + +The O +estimated O +binding B-evidence +affinities I-evidence +( O +KD B-evidence +) O +are O +listed O +. O + +Intramolecular O +interactions O +inhibit O +histone B-protein_type +recognition O +by O +UHRF1 B-protein +. O + +( O +a O +) O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +for O +the O +intramolecular O +interactions O +. O + +The O +isolated O +domains O +of O +UHRF1 B-protein +were O +incubated B-experimental_method +with O +GST B-protein_state +- I-protein_state +tagged I-protein_state +TTD B-structure_element +or O +PHD B-structure_element +immobilized O +on O +glutathione O +resin O +. O + +The O +bound O +proteins O +were O +analysed O +by O +SDS B-experimental_method +– I-experimental_method +PAGE I-experimental_method +and O +Coomassie O +blue O +staining O +. O + +( O +b O +, O +d O +) O +Superimposed O +ITC B-experimental_method +enthalpy B-evidence +plots I-evidence +for O +the O +intramolecular O +interactions O +of O +isolated O +UHRF1 B-protein +domains O +. O + +The O +estimated O +binding B-evidence +affinities I-evidence +( O +KD B-evidence +) O +were O +listed O +. O + +ND O +, O +not O +detectable O +. O +( O +c O +) O +Superimposed O +ITC B-experimental_method +enthalpy B-evidence +plots I-evidence +for O +the O +binding O +of O +H3K9me3 B-protein_type +to O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +in O +the O +absence B-protein_state +or O +presence B-protein_state +of I-protein_state +the O +Spacer B-structure_element +( O +molar O +ratio O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +/ O +Spacer B-structure_element += O +1 O +: O +2 O +). O +( O +e O +) O +Superimposed O +ITC B-experimental_method +enthalpy B-evidence +plots I-evidence +for O +the O +binding O +of O +H3 B-protein_type +to O +PHD B-structure_element +– I-structure_element +SRA I-structure_element +or O +PHD B-structure_element +in O +the O +absence B-protein_state +or O +presence B-protein_state +of I-protein_state +the O +SRA B-structure_element +( O +molar O +ratio O +PHD B-structure_element +/ O +SRA B-structure_element += O +1 O +: O +1 O +or O +1 O +: O +2 O +). O + +NMR B-experimental_method +structure B-evidence +of O +the O +TTD B-structure_element +bound B-protein_state +to I-protein_state +the O +Spacer B-structure_element +. O + +( O +a O +) O +Ribbon O +representation O +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +structure B-evidence +. O + +N O +- O +and O +C O +- O +termini O +of O +the O +Spacer B-structure_element +are O +indicated O +. O + +The O +TTD B-structure_element +is O +coloured O +in O +green O +, O +and O +the O +Spacer B-structure_element +is O +coloured O +in O +yellow O +. O + +( O +b O +) O +Superimposition B-experimental_method +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +and O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +( O +4GY5 O +. O +PDB O +) O +structures B-evidence +shown O +in O +ribbon O +representations O +. O + +The O +TTD B-structure_element +is O +coloured O +in O +green O +and O +the O +Spacer B-structure_element +in O +yellow O +in O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +structure B-evidence +. O + +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +complex O +is O +coloured O +in O +grey O +, O +and O +the O +PHD B-structure_element +and O +H3K9me3 B-protein_type +are O +omitted O +for O +simplicity O +. O + +( O +c O +) O +Electrostatic O +potential O +surface O +representation O +of O +the O +TTD B-structure_element +with O +the O +Spacer B-structure_element +shown O +in O +ribbon O +representation O +. O + +The O +critical O +residues O +on O +the O +Spacer B-structure_element +for O +the O +interaction O +are O +shown O +in O +stick O +representation O +. O + +( O +d O +) O +Close O +- O +up O +view O +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +. O + +Hydrogen B-bond_interaction +bonds I-bond_interaction +are O +indicated O +as O +dashed O +lines O +. O + +( O +e O +– O +g O +) O +Superimposed O +ITC B-experimental_method +enthalpy B-evidence +plots I-evidence +for O +the O +interaction O +between O +the O +Spacer B-structure_element +and O +the O +TTD B-structure_element +( O +or O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +) O +with O +the O +estimated O +binding B-evidence +affinity I-evidence +( O +KD B-evidence +) O +indicated O +. O + +Wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +proteins O +for O +the O +measurements O +are O +indicated O +. O + +( O +h O +) O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +for O +the O +intramolecular O +interactions O +. 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O + +The O +SRA B-structure_element +was O +incubated B-experimental_method +with O +GST B-protein_state +- I-protein_state +tagged I-protein_state +PHD B-structure_element +in O +the O +presence B-protein_state +of I-protein_state +increasing O +concentrations O +of O +hm B-chemical +- I-chemical +DNA I-chemical +and O +immobilized O +on O +glutathione O +resin O +. O + +The O +bound O +proteins O +were O +analysed O +in O +SDS B-experimental_method +– I-experimental_method +PAGE I-experimental_method +and O +Coomassie B-experimental_method +blue I-experimental_method +staining I-experimental_method +( O +left O +) O +and O +quantified O +by O +band B-experimental_method +densitometry I-experimental_method +( O +right O +). O + +( O +b O +) O +Purified O +fragments O +of O +UHRF1 B-protein +were O +analysed O +by O +histone B-experimental_method +peptide I-experimental_method +( O +H3K9me0 B-protein_type +) O +pull B-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +as O +described O +in O +Fig O +. O +1b O +. O +( O +c O +) O +Hm B-chemical +- I-chemical +DNA I-chemical +impairs O +the O +intramolecular O +interaction O +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +. O + +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +was O +incubated B-experimental_method +with O +GST B-protein_state +- I-protein_state +tagged I-protein_state +TTD B-structure_element +– I-structure_element +PHD I-structure_element +or O +TTD B-structure_element +in O +the O +presence B-protein_state +of I-protein_state +increasing O +concentrations O +of O +hm B-chemical +- I-chemical +DNA I-chemical +and O +analysed O +in O +pull B-experimental_method +- I-experimental_method +down I-experimental_method +experiment I-experimental_method +as O +described O +in O +a O +. O +The O +quantified O +band B-experimental_method +densitometries I-experimental_method +are O +indicated O +below O +the O +Coomassie B-experimental_method +blue I-experimental_method +staining I-experimental_method +. 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O +( O +d O +) O +Subcellular O +localization O +of O +GFP B-protein_state +- I-protein_state +tagged I-protein_state +wild B-protein_state +- I-protein_state +type I-protein_state +or O +indicated O +mutants B-protein_state +of O +UHRF1 B-protein +in O +NIH3T3 O +cells O +. O + +The O +percentages O +of O +cells O +showing O +co O +- O +localization O +with O +DAPI B-chemical +foci O +were O +counted O +from O +at O +least O +100 O +cells O +and O +shown O +on O +the O +left O +of O +the O +corresponding O +representative O +confocal B-experimental_method +microscopy I-experimental_method +. O + +Scale O +bar O +, O +5 O +μm O +. 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O + +X B-evidence +- I-evidence +ray I-evidence +Crystallographic I-evidence +Structures I-evidence +of O +a O +Trimer B-oligomeric_state +, O +Dodecamer B-oligomeric_state +, O +and O +Annular B-site +Pore I-site +Formed O +by O +an O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +β B-structure_element +- I-structure_element +Hairpin I-structure_element + +High O +- O +resolution O +structures B-evidence +of O +oligomers B-oligomeric_state +formed O +by O +the O +β B-protein +- I-protein +amyloid I-protein +peptide I-protein +Aβ B-protein +are O +needed O +to O +understand O +the O +molecular O +basis O +of O +Alzheimer O +’ O +s O +disease O +and O +develop O +therapies O +. O + +This O +paper O +presents O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structures I-evidence +of O +oligomers B-oligomeric_state +formed O +by O +a O +20 B-residue_range +- I-residue_range +residue I-residue_range +peptide I-residue_range +segment I-residue_range +derived O +from O +Aβ B-protein +. O + +The O +development O +of O +a O +peptide O +in O +which O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +is O +stabilized O +as O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +is O +described O +, O +and O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structures I-evidence +of O +oligomers B-oligomeric_state +it O +forms O +are O +reported O +. O + +Two O +covalent O +constraints O +act O +in O +tandem O +to O +stabilize O +the O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +peptide O +in O +a O +hairpin B-structure_element +conformation O +: O +a O +δ B-protein_state +- I-protein_state +linked I-protein_state +ornithine B-residue_name +turn B-structure_element +connecting O +positions O +17 B-residue_number +and O +36 B-residue_number +to O +create O +a O +macrocycle O +and O +an O +intramolecular O +disulfide B-ptm +linkage I-ptm +between O +positions O +24 B-residue_number +and O +29 B-residue_number +. O + +An O +N O +- O +methyl O +group O +at O +position O +33 B-residue_number +blocks O +uncontrolled O +aggregation O +. O + +The O +peptide O +readily B-evidence +crystallizes I-evidence +as O +a O +folded B-protein_state +β B-structure_element +- I-structure_element +hairpin I-structure_element +, O +which O +assembles O +hierarchically O +in O +the O +crystal B-evidence +lattice I-evidence +. O + +Three O +β B-structure_element +- I-structure_element +hairpin I-structure_element +monomers B-oligomeric_state +assemble O +to O +form O +a O +triangular B-protein_state +trimer B-oligomeric_state +, O +four O +trimers B-oligomeric_state +assemble O +in O +a O +tetrahedral O +arrangement O +to O +form O +a O +dodecamer B-oligomeric_state +, O +and O +five O +dodecamers B-oligomeric_state +pack O +together O +to O +form O +an O +annular B-site +pore I-site +. O + +This O +hierarchical O +assembly O +provides O +a O +model O +, O +in O +which O +full B-protein_state +- I-protein_state +length I-protein_state +Aβ B-protein +transitions O +from O +an O +unfolded B-protein_state +monomer B-oligomeric_state +to O +a O +folded B-protein_state +β B-structure_element +- I-structure_element +hairpin I-structure_element +, O +which O +assembles O +to O +form O +oligomers B-oligomeric_state +that O +further O +pack O +to O +form O +an O +annular B-site +pore I-site +. 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O + +Many O +of O +these O +studies O +have O +reported O +the O +monomer B-oligomeric_state +subunit B-structure_element +as O +adopting O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +conformation O +, O +in O +which O +the O +hydrophobic O +central B-structure_element +and O +C B-structure_element +- I-structure_element +terminal I-structure_element +regions I-structure_element +form O +an O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +. O + +In O +2008 O +, O +Hoyer O +et O +al O +. O +reported O +the O +NMR B-experimental_method +structure B-evidence +of O +an O +Aβ B-protein +monomer B-oligomeric_state +bound B-protein_state +to I-protein_state +an O +artificial B-chemical +binding I-chemical +protein I-chemical +called O +an O +affibody B-chemical +( O +PDB O +2OTK O +). O + +The O +structure B-evidence +revealed O +that O +monomeric B-oligomeric_state +Aβ B-protein +forms O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +when O +bound B-protein_state +to I-protein_state +the O +affibody B-chemical +. O + +This O +Aβ B-protein +β B-structure_element +- I-structure_element +hairpin I-structure_element +encompasses O +residues O +17 B-residue_range +– I-residue_range +37 I-residue_range +and O +contains O +two O +β B-structure_element +- I-structure_element +strands I-structure_element +comprising O +Aβ17 B-protein +– B-residue_range +24 I-residue_range +and O +Aβ30 B-protein +– B-residue_range +37 I-residue_range +connected O +by O +an O +Aβ25 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +. O + +Sequestering O +Aβ B-protein +within O +the O +affibody B-chemical +prevents O +its O +fibrilization O +and O +reduces O +its O +neurotoxicity O +, O +providing O +evidence O +that O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +structure O +may O +contribute O +to O +the O +ability O +of O +Aβ B-protein +to O +form O +neurotoxic O +oligomers B-oligomeric_state +. O + +In O +a O +related O +study O +, O +Sandberg O +et O +al O +. O +constrained O +Aβ B-protein +in O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +conformation O +by O +mutating B-experimental_method +residues O +A21 B-residue_name_number +and O +A30 B-residue_name_number +to O +cysteine B-residue_name +and O +forming O +an O +intramolecular O +disulfide B-ptm +bond I-ptm +. O + +Locking O +Aβ B-protein +into O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +structure O +resulted O +in O +the O +formation O +Aβ B-protein +oligomers B-oligomeric_state +, O +which O +were O +observed O +by O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +( O +SEC B-experimental_method +) O +and O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +. O + +The O +oligomers B-oligomeric_state +with O +a O +molecular O +weight O +of O +∼ O +100 O +kDa O +that O +were O +isolated O +by O +SEC B-experimental_method +were O +toxic O +toward O +neuronally O +derived O +SH O +- O +SY5Y O +cells O +. O + +This O +study O +provides O +evidence O +for O +the O +role O +of O +β B-structure_element +- I-structure_element +hairpin I-structure_element +structure O +in O +Aβ B-protein +oligomerization O +and O +neurotoxicity O +. O + +Inspired O +by O +these O +β B-structure_element +- I-structure_element +hairpin I-structure_element +structures B-evidence +, O +our O +laboratory O +developed O +a O +macrocyclic O +β B-structure_element +- I-structure_element +sheet I-structure_element +peptide O +derived O +from O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +designed O +to O +mimic O +an O +Aβ B-protein +β B-structure_element +- I-structure_element +hairpin I-structure_element +and O +reported O +its O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +. O + +This O +peptide O +( O +peptide B-mutant +1 I-mutant +) O +consists O +of O +two O +β B-structure_element +- I-structure_element +strands I-structure_element +comprising O +Aβ17 B-protein +– B-residue_range +23 I-residue_range +and O +Aβ30 B-protein +– B-residue_range +36 I-residue_range +covalently O +linked O +by O +two O +δ B-protein_state +- I-protein_state +linked I-protein_state +ornithine B-residue_name +( O +δOrn B-structure_element +) O +β B-structure_element +- I-structure_element +turn I-structure_element +mimics O +. O + +The O +δOrn B-structure_element +that O +connects O +residues O +D23 B-residue_name_number +and O +A30 B-residue_name_number +replaces O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +. O + +The O +δOrn B-structure_element +that O +connects O +residues O +L17 B-residue_name_number +and O +V36 B-residue_name_number +enforces O +β B-structure_element +- I-structure_element +hairpin I-structure_element +structure O +. O + +We O +incorporated O +an O +N O +- O +methyl O +group O +at O +position O +G33 B-residue_name_number +to O +prevent O +uncontrolled O +aggregation O +and O +precipitation O +of O +the O +peptide O +. O + +To O +improve O +the O +solubility O +of O +the O +peptide O +we O +replaced B-experimental_method +M35 B-residue_name_number +with O +the O +hydrophilic O +isostere O +of O +methionine B-residue_name +, O +ornithine B-residue_name +( O +α B-protein_state +- I-protein_state +linked I-protein_state +) O +( O +Figure O +1B O +). O + +The O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +1 I-mutant +reveals O +that O +it O +folds O +to O +form O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +that O +assembles O +to O +form O +trimers B-oligomeric_state +and O +that O +the O +trimers B-oligomeric_state +further O +assemble O +to O +form O +hexamers B-oligomeric_state +and O +dodecamers B-oligomeric_state +. O + +( O +A O +) O +Cartoon O +illustrating O +the O +design O +of O +peptides B-chemical +1 I-chemical +and I-chemical +2 I-chemical +and O +their O +relationship O +to O +an O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +β B-structure_element +- I-structure_element +hairpin I-structure_element +. O + +( O +B O +) O +Chemical O +structure O +of O +peptide B-mutant +1 I-mutant +illustrating O +Aβ17 B-protein +– O +23 O +and O +Aβ30 B-protein +– O +36 O +, O +M35Orn O +, O +the O +N O +- O +methyl O +group O +, O +and O +the O +δ B-protein_state +- I-protein_state +linked I-protein_state +ornithine B-residue_name +turns B-structure_element +. O +( O +C O +) O +Chemical O +structure O +of O +peptide B-mutant +2 I-mutant +illustrating O +Aβ17 B-protein +– O +36 O +, O +the O +N O +- O +methyl O +group O +, O +the O +disulfide B-ptm +bond I-ptm +across O +positions O +24 B-residue_number +and O +29 B-residue_number +, O +and O +the O +δ B-protein_state +- I-protein_state +linked I-protein_state +ornithine B-residue_name +turn B-structure_element +. O + +Our O +design O +of O +peptide B-mutant +1 I-mutant +omitted O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +. O + +To O +visualize O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +, O +we O +performed O +replica B-experimental_method +- I-experimental_method +exchange I-experimental_method +molecular I-experimental_method +dynamics I-experimental_method +( O +REMD B-experimental_method +) O +simulations B-experimental_method +on O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +using O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +coordinates I-evidence +of O +Aβ17 B-protein +– B-residue_range +23 I-residue_range +and O +Aβ30 B-protein +– B-residue_range +36 I-residue_range +from O +peptide B-mutant +1 I-mutant +. O + +These O +studies O +provided O +a O +working O +model O +for O +a O +trimer B-oligomeric_state +of O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +β B-structure_element +- I-structure_element +hairpins I-structure_element +and O +demonstrated O +that O +the O +trimer B-oligomeric_state +should O +be O +capable O +of O +accommodating O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +. O + +In O +the O +current O +study O +we O +set O +out O +to O +restore B-experimental_method +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +, O +reintroduce B-experimental_method +the O +methionine B-residue_name +residue O +at O +position O +35 B-residue_number +, O +and O +determine O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structures I-evidence +of O +oligomers B-oligomeric_state +that O +form O +. O + +We O +designed O +peptide B-mutant +2 I-mutant +as O +a O +homologue O +of O +peptide B-mutant +1 I-mutant +that O +embodies O +these O +ideas O +. O + +Peptide B-mutant +2 I-mutant +contains O +a O +methionine B-residue_name +residue O +at O +position O +35 B-residue_number +and O +an O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +with O +residues O +24 B-residue_number +and O +29 B-residue_number +( O +Val B-residue_name +and O +Gly B-residue_name +) O +mutated B-experimental_method +to O +cysteine B-residue_name +and O +linked O +by O +a O +disulfide B-ptm +bond I-ptm +( O +Figure O +1C O +). O + +Here O +, O +we O +describe O +the O +development O +of O +peptide B-mutant +2 I-mutant +and O +report O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structures I-evidence +of O +the O +trimer B-oligomeric_state +, O +dodecamer B-oligomeric_state +, O +and O +annular B-site +pore I-site +observed O +within O +the O +crystal B-evidence +structure I-evidence +. O + +Development O +of O +Peptide B-mutant +2 I-mutant + +We O +developed O +peptide B-mutant +2 I-mutant +from O +peptide B-mutant +1 I-mutant +by O +an O +iterative O +process O +, O +in O +which O +we O +first O +attempted O +to O +restore O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +without O +a O +disulfide B-ptm +linkage I-ptm +. O + +We O +envisioned O +peptide B-mutant +3 I-mutant +as O +a O +homologue O +of O +peptide B-mutant +1 I-mutant +with O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +in O +place O +of O +the O +δOrn B-structure_element +that O +connects O +D23 B-residue_name_number +and O +A30 B-residue_name_number +and O +p B-chemical +- I-chemical +iodophenylalanine I-chemical +( O +FI B-chemical +) O +in O +place O +of O +F19 B-residue_name_number +. O + +We O +routinely O +use O +p B-chemical +- I-chemical +iodophenylalanine I-chemical +to O +determine O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +phases I-evidence +. O + +After O +determining O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +the O +p B-chemical +- I-chemical +iodophenylalanine I-chemical +variant O +we O +attempt O +to O +determine O +the O +structure B-evidence +of O +the O +native O +phenylalanine B-residue_name +compound O +by O +isomorphous B-experimental_method +replacement I-experimental_method +. O + +Upon O +synthesizing O +peptide B-mutant +3 I-mutant +, O +we O +found O +that O +it O +formed O +an O +amorphous O +precipitate O +in O +most O +crystallization O +conditions O +screened O +and O +failed O +to O +afford O +crystals B-evidence +in O +any O +condition O +. O + +We O +postulate O +that O +the O +loss O +of O +the O +δOrn B-structure_element +constraint O +leads O +to O +conformational O +heterogeneity O +that O +prevents O +peptide B-mutant +3 I-mutant +from O +crystallizing O +. O + +To O +address O +this O +issue O +, O +we O +next O +incorporated O +a O +disulfide B-ptm +bond I-ptm +between O +residues O +24 B-residue_number +and O +29 B-residue_number +as O +a O +conformational O +constraint O +that O +serves O +as O +a O +surrogate O +for O +δOrn B-structure_element +. O + +We O +designed O +peptide B-mutant +4 I-mutant +to O +embody O +this O +idea O +, O +mutating B-experimental_method +Val24 B-residue_name_number +and O +Gly29 B-residue_name_number +to O +cysteine B-residue_name +and O +forming O +an O +interstrand O +disulfide B-ptm +linkage I-ptm +. O + +We O +mutated B-experimental_method +these O +residues O +because O +they O +occupy O +the O +same O +position O +as O +the O +δOrn B-structure_element +that O +connects O +D23 B-residue_name_number +and O +A30 B-residue_name_number +in O +peptide B-mutant +1 I-mutant +. O + +Residues O +V24 B-residue_name_number +and O +G29 B-residue_name_number +form O +a O +non B-bond_interaction +- I-bond_interaction +hydrogen I-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +pair I-bond_interaction +, O +which O +can O +readily O +accommodate O +disulfide B-ptm +linkages I-ptm +in O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +. O + +Disulfide B-ptm +bonds I-ptm +across O +non B-bond_interaction +- I-bond_interaction +hydrogen I-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +pairs I-bond_interaction +stabilize O +β B-structure_element +- I-structure_element +hairpins I-structure_element +, O +while O +disulfide B-ptm +bonds I-ptm +across O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +pairs I-bond_interaction +do O +not O +. O + +Although O +the O +disulfide B-ptm +bond I-ptm +between O +positions O +24 B-residue_number +and O +29 B-residue_number +helps O +stabilize O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +, O +it O +does O +not O +alter O +the O +charge O +or O +substantially O +change O +the O +hydrophobicity O +of O +the O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +β B-structure_element +- I-structure_element +hairpin I-structure_element +. O + +We O +were O +gratified O +to O +find O +that O +peptide B-mutant +4 I-mutant +afforded O +crystals B-evidence +suitable O +for O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +As O +the O +next O +step O +in O +the O +iterative O +process O +, O +we O +determined B-experimental_method +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +this O +peptide O +( O +PDB O +5HOW O +). O + +After O +determining O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +4 I-mutant +we O +reintroduced B-experimental_method +the O +native O +phenylalanine B-residue_name +at O +position O +19 B-residue_number +and O +the O +methionine B-residue_name +at O +position O +35 B-residue_number +to O +afford O +peptide B-mutant +2 I-mutant +. O + +We O +completed O +the O +iterative O +process O +— O +from O +1 O +to O +3 O +to O +4 O +to O +2 O +— O +by O +successfully O +determining O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +2 I-mutant +( O +PDB O +5HOX O +and O +5HOY O +). O + +The O +following O +sections O +describe O +the O +synthesis O +of O +peptides B-mutant +2 I-mutant +– I-mutant +4 I-mutant +and O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +2 I-mutant +. O + +Synthesis O +of O +Peptides B-mutant +2 I-mutant +– I-mutant +4 I-mutant + +We O +synthesized O +peptides B-mutant +2 I-mutant +– I-mutant +4 I-mutant +by O +similar O +procedures O +to O +those O +we O +have O +developed O +for O +other O +macrocyclic O +peptides O +. O + +In O +synthesizing O +peptides B-mutant +2 I-mutant +and I-mutant +4 I-mutant +we O +formed O +the O +disulfide B-ptm +linkage I-ptm +after O +macrolactamization O +and O +deprotection O +of O +the O +acid O +- O +labile O +side O +chain O +protecting O +groups O +. O + +We O +used O +acid B-protein_state +- I-protein_state +stable I-protein_state +Acm B-protein_state +- I-protein_state +protected I-protein_state +cysteine B-residue_name +residues O +at O +positions O +24 B-residue_number +and O +29 B-residue_number +and O +removed O +the O +Acm O +groups O +by O +oxidation O +with O +I2 O +in O +aqueous O +acetic B-chemical +acid I-chemical +to O +afford O +the O +disulfide B-ptm +linkage I-ptm +. O + +Peptides B-mutant +2 I-mutant +– I-mutant +4 I-mutant +were O +purified O +by O +RP B-experimental_method +- I-experimental_method +HPLC I-experimental_method +. O + +Crystallization B-experimental_method +, O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +Crystallographic I-experimental_method +Data I-experimental_method +Collection I-experimental_method +, O +Data O +Processing O +, O +and O +Structure B-experimental_method +Determination I-experimental_method +of O +Peptides B-mutant +2 I-mutant +and I-mutant +4 I-mutant + +We O +screened B-experimental_method +crystallization I-experimental_method +conditions I-experimental_method +for O +peptide B-mutant +4 I-mutant +in O +a O +96 O +- O +well O +- O +plate O +format O +using O +three O +different O +Hampton O +Research O +crystallization O +kits O +( O +Crystal O +Screen O +, O +Index O +, O +and O +PEG O +/ O +Ion O +) O +with O +three O +ratios O +of O +peptide O +and O +mother O +liquor O +per O +condition O +( O +864 O +experiments O +). O + +Peptide B-mutant +4 I-mutant +afforded O +crystals B-evidence +in O +a O +single O +set O +of O +conditions O +containing O +HEPES O +buffer O +and O +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +— O +the O +same O +crystallization O +conditions O +that O +afforded O +crystals B-evidence +of O +peptide B-mutant +1 I-mutant +. O + +Peptide B-mutant +2 I-mutant +also O +afforded O +crystals B-evidence +in O +these O +conditions O +. O + +We O +further O +optimized O +these O +conditions O +to O +rapidly O +(∼ O +72 O +h O +) O +yield O +crystals B-evidence +suitable O +for O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +The O +optimized O +conditions O +consist O +of O +0 O +. O +1 O +M O +HEPES B-chemical +at O +pH O +6 O +. O +4 O +with O +31 O +% O +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +for O +peptide B-mutant +4 I-mutant +and O +0 O +. O +1 O +M O +HEPES B-chemical +pH O +7 O +. O +1 O +with O +29 O +% O +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +for O +peptide B-mutant +2 I-mutant +. O + +Crystal B-evidence +diffraction I-evidence +data I-evidence +for O +peptides B-mutant +4 I-mutant +and I-mutant +2 I-mutant +were O +collected O +in O +- O +house O +with O +a O +Rigaku O +MicroMax O +007HF O +X O +- O +ray O +diffractometer O +at O +1 O +. O +54 O +Å O +wavelength O +. O + +Crystal B-evidence +diffraction I-evidence +data I-evidence +for O +peptide B-mutant +2 I-mutant +were O +also O +collected O +at O +the O +Advanced O +Light O +Source O +at O +Lawrence O +Berkeley O +National O +Laboratory O +with O +a O +synchrotron O +source O +at O +1 O +. O +00 O +Å O +wavelength O +to O +achieve O +higher O +resolution O +. O + +Data O +from O +peptides B-mutant +4 I-mutant +and I-mutant +2 I-mutant +suitable O +for O +refinement O +at O +2 O +. O +30 O +Å O +were O +obtained O +from O +the O +diffractometer O +; O +data O +from O +peptide B-mutant +2 I-mutant +suitable O +for O +refinement O +at O +1 O +. O +90 O +Å O +were O +obtained O +from O +the O +synchrotron O +. O + +Data O +for O +peptides B-mutant +4 I-mutant +and I-mutant +2 I-mutant +were O +scaled O +and O +merged O +using O +XDS O +. O + +Phases B-evidence +for O +peptide B-mutant +4 I-mutant +were O +determined O +by O +single B-experimental_method +- I-experimental_method +wavelength I-experimental_method +anomalous I-experimental_method +diffraction I-experimental_method +( O +SAD B-experimental_method +) O +phasing B-experimental_method +by O +using O +the O +coordinates O +of O +the O +iodine B-evidence +anomalous I-evidence +signal I-evidence +from O +p B-chemical +- I-chemical +iodophenylalanine I-chemical +. O + +Phases B-evidence +for O +peptide B-mutant +2 I-mutant +were O +determined O +by O +isomorphous B-experimental_method +replacement I-experimental_method +of O +peptide B-mutant +4 I-mutant +. O + +The O +structures B-evidence +of O +peptides B-mutant +2 I-mutant +and I-mutant +4 I-mutant +were O +solved B-experimental_method +and O +refined O +in O +the O +P6122 O +space O +group O +. O + +The O +asymmetric O +unit O +of O +each O +peptide B-chemical +consists O +of O +six O +monomers B-oligomeric_state +, O +arranged O +as O +two O +trimers B-oligomeric_state +. O + +Peptides B-mutant +2 I-mutant +and I-mutant +4 I-mutant +form O +morphologically O +identical O +structures O +and O +assemblies O +in O +the O +crystal B-evidence +lattice I-evidence +. O + +X B-evidence +- I-evidence +ray I-evidence +Crystallographic I-evidence +Structure I-evidence +of O +Peptide B-mutant +2 I-mutant +and O +the O +Oligomers B-oligomeric_state +It O +Forms O + +The O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +2 I-mutant +reveals O +that O +it O +folds O +to O +form O +a O +twisted B-structure_element +β I-structure_element +- I-structure_element +hairpin I-structure_element +comprising O +two O +β B-structure_element +- I-structure_element +strands I-structure_element +connected O +by O +a O +loop B-structure_element +( O +Figure O +2A O +). O + +Eight O +residues O +make O +up O +each O +surface O +of O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +: O +L17 B-residue_name_number +, O +F19 B-residue_name_number +, O +A21 B-residue_name_number +, O +D23 B-residue_name_number +, O +A30 B-residue_name_number +, O +I32 B-residue_name_number +, O +L34 B-residue_name_number +, O +and O +V36 B-residue_name_number +make O +up O +one O +surface O +; O +V18 B-residue_name_number +, O +F20 B-residue_name_number +, O +E22 B-residue_name_number +, O +C24 B-residue_name_number +, O +C29 B-residue_name_number +, O +I31 B-residue_name_number +, O +G33 B-residue_name_number +, O +and O +M35 B-residue_name_number +make O +up O +the O +other O +surface O +. O + +The O +β B-structure_element +- I-structure_element +strands I-structure_element +of O +the O +monomers B-oligomeric_state +in O +the O +asymmetric O +unit O +are O +virtually O +identical O +, O +differing O +primarily O +in O +rotamers O +of O +F20 B-residue_name_number +, O +E22 B-residue_name_number +, O +C24 B-residue_name_number +, O +C29 B-residue_name_number +, O +I31 B-residue_name_number +, O +and O +M35 B-residue_name_number +( O +Figure O +S1 O +). O + +The O +disulfide B-ptm +linkages I-ptm +suffered O +radiation O +damage O +under O +synchrotron O +radiation O +. O + +We O +refined B-experimental_method +three O +of O +the O +β B-structure_element +- I-structure_element +hairpins I-structure_element +with O +intact B-protein_state +disulfide B-ptm +linkages I-ptm +and O +three O +with O +thiols O +to O +represent O +cleaved B-protein_state +disulfide B-ptm +linkages I-ptm +in O +the O +synchrotron O +data O +set O +( O +PDB O +5HOX O +). O + +No O +evidence O +for O +cleavage O +of O +the O +disulfides B-ptm +was O +observed O +in O +the O +refinement B-experimental_method +of O +the O +data O +set O +collected O +on O +the O +X O +- O +ray O +diffractometer O +, O +and O +we O +refined B-experimental_method +all O +disulfide B-ptm +linkages I-ptm +as O +intact B-protein_state +( O +PDB O +5HOY O +). O + +X O +- O +ray O +crystallographic O +structure O +of O +peptide B-mutant +2 I-mutant +( O +PDB O +5HOX O +, O +synchrotron O +data O +set O +). O +( O +A O +) O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +a O +representative O +β B-structure_element +- I-structure_element +hairpin I-structure_element +monomer B-oligomeric_state +formed O +by O +peptide B-mutant +2 I-mutant +. O +( O +B O +) O +Overlay B-experimental_method +of O +the O +six O +β B-structure_element +- I-structure_element +hairpin I-structure_element +monomers B-oligomeric_state +in O +the O +asymmetric O +unit O +. O + +The O +β B-structure_element +- I-structure_element +hairpins I-structure_element +are O +shown O +as O +cartoons O +to O +illustrate O +the O +differences O +in O +the O +Aβ25 B-protein +– B-residue_range +28 I-residue_range +loops B-structure_element +. O + +The O +Aβ25 B-protein +– B-residue_range +28 I-residue_range +loops B-structure_element +of O +the O +six O +monomers B-oligomeric_state +within O +the O +asymmetric O +unit O +vary O +substantially O +in O +backbone O +geometry O +and O +side O +chain O +rotamers O +( O +Figures O +2B O +and O +S1 O +). O + +The O +electron B-evidence +density I-evidence +for O +the O +loops B-structure_element +is O +weak O +and O +diffuse O +compared O +to O +the O +electron B-evidence +density I-evidence +for O +the O +β B-structure_element +- I-structure_element +strands I-structure_element +. O + +The O +B B-evidence +values I-evidence +for O +the O +loops B-structure_element +are O +large O +, O +indicating O +that O +the O +loops B-structure_element +are O +dynamic O +and O +not O +well O +ordered O +. O + +Thus O +, O +the O +differences O +in O +backbone O +geometry O +and O +side O +chain O +rotamers O +among O +the O +loops B-structure_element +are O +likely O +of O +little O +significance O +and O +should O +be O +interpreted O +with O +caution O +. O + +Peptide B-mutant +2 I-mutant +assembles O +into O +oligomers B-oligomeric_state +similar O +in O +morphology O +to O +those O +formed O +by O +peptide B-mutant +1 I-mutant +. O + +Like O +peptide B-mutant +1 I-mutant +, O +peptide B-mutant +2 I-mutant +forms O +a O +triangular B-protein_state +trimer B-oligomeric_state +, O +and O +four O +trimers B-oligomeric_state +assemble O +to O +form O +a O +dodecamer B-oligomeric_state +. O + +In O +the O +higher O +- O +order O +assembly O +of O +the O +dodecamers B-oligomeric_state +formed O +by O +peptide B-mutant +2 I-mutant +a O +new O +structure B-evidence +emerges O +, O +not O +seen O +in O +peptide B-mutant +1 I-mutant +, O +an O +annular B-site +pore I-site +consisting O +of O +five O +dodecamers B-oligomeric_state +. O + +Trimer B-oligomeric_state + +Peptide B-mutant +2 I-mutant +forms O +a O +trimer B-oligomeric_state +, O +much O +like O +that O +which O +we O +observed O +previously O +for O +peptide B-mutant +1 I-mutant +, O +in O +which O +three O +β B-structure_element +- I-structure_element +hairpins I-structure_element +assemble O +to O +form O +an O +equilateral B-structure_element +triangle I-structure_element +( O +Figure O +3A O +). O + +The O +trimer B-oligomeric_state +maintains O +all O +of O +the O +same O +stabilizing O +contacts O +as O +those O +of O +peptide B-mutant +1 I-mutant +. O + +Hydrogen B-bond_interaction +bonding I-bond_interaction +and O +hydrophobic B-bond_interaction +interactions I-bond_interaction +between O +residues O +on O +the O +β B-structure_element +- I-structure_element +strands I-structure_element +comprising O +Aβ17 B-protein +– B-residue_range +23 I-residue_range +and O +Aβ30 B-protein +– B-residue_range +36 I-residue_range +stabilize O +the O +core B-structure_element +of O +the O +trimer B-oligomeric_state +. O + +The O +disulfide B-ptm +bonds I-ptm +between O +residues O +24 B-residue_number +and O +29 B-residue_number +are O +adjacent O +to O +the O +structural B-structure_element +core I-structure_element +of O +the O +trimer B-oligomeric_state +and O +do O +not O +make O +any O +substantial O +intermolecular O +contacts O +. O + +Two O +crystallographically O +distinct O +trimers B-oligomeric_state +comprise O +the O +peptide B-chemical +portion O +of O +the O +asymmetric O +unit O +. O + +The O +two O +trimers B-oligomeric_state +are O +almost O +identical O +in O +structure O +, O +differing O +slightly O +among O +side O +chain O +rotamers O +and O +loop B-structure_element +conformations O +. O + +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +the O +trimer B-oligomeric_state +formed O +by O +peptide B-mutant +2 I-mutant +. O +( O +A O +) O +Triangular B-protein_state +trimer B-oligomeric_state +. O + +The O +three O +water B-chemical +molecules O +in O +the O +center O +hole O +of O +the O +trimer B-oligomeric_state +are O +shown O +as O +spheres O +. O +( O +B O +) O +Detailed O +view O +of O +the O +intermolecular O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +the O +main O +chains O +of O +V18 B-residue_name_number +and O +E22 B-residue_name_number +and O +δOrn B-structure_element +and O +C24 B-residue_name_number +, O +at O +the O +three O +corners O +of O +the O +triangular B-protein_state +trimer B-oligomeric_state +. O +( O +C O +) O +The O +F19 B-residue_name_number +face O +of O +the O +trimer B-oligomeric_state +, O +with O +key O +side O +chains O +shown O +as O +spheres O +. O +( O +D O +) O +The O +F20 B-residue_name_number +face O +of O +the O +trimer B-oligomeric_state +, O +with O +key O +side O +chains O +as O +spheres O +. O + +A O +network O +of O +18 O +intermolecular O +hydrogen B-bond_interaction +bonds I-bond_interaction +helps O +stabilize O +the O +trimer B-oligomeric_state +. O + +At O +the O +corners O +of O +the O +trimer B-oligomeric_state +, O +the O +pairs O +of O +β B-structure_element +- I-structure_element +hairpin I-structure_element +monomers B-oligomeric_state +form O +four O +hydrogen B-bond_interaction +bonds I-bond_interaction +: O +two O +between O +the O +main O +chains O +of O +V18 B-residue_name_number +and O +E22 B-residue_name_number +and O +two O +between O +δOrn B-structure_element +and O +the O +main O +chain O +of O +C24 B-residue_name_number +( O +Figure O +3B O +). O + +Three O +ordered O +water B-chemical +molecules O +fill O +the O +hole O +in O +the O +center O +of O +the O +trimer B-oligomeric_state +, O +hydrogen B-bond_interaction +bonding I-bond_interaction +to O +each O +other O +and O +to O +the O +main O +chain O +of O +F20 B-residue_name_number +( O +Figure O +3A O +). O + +Hydrophobic B-bond_interaction +contacts I-bond_interaction +between O +residues O +at O +the O +three O +corners O +of O +the O +trimer B-oligomeric_state +, O +where O +the O +β B-structure_element +- I-structure_element +hairpins I-structure_element +meet O +, O +further O +stabilize O +the O +trimer B-oligomeric_state +. O + +At O +each O +corner O +, O +the O +side O +chains O +of O +residues O +L17 B-residue_name_number +, O +F19 B-residue_name_number +, O +and O +V36 B-residue_name_number +of O +one O +β B-structure_element +- I-structure_element +hairpin I-structure_element +pack O +against O +the O +side O +chains O +of O +residues O +A21 B-residue_name_number +, O +I32 B-residue_name_number +, O +L34 B-residue_name_number +, O +and O +also O +D23 B-residue_name_number +of O +the O +adjacent O +β B-structure_element +- I-structure_element +hairpin I-structure_element +to O +create O +a O +hydrophobic B-site +cluster I-site +( O +Figure O +3C O +). O +The O +three O +hydrophobic B-site +clusters I-site +create O +a O +large O +hydrophobic B-site +surface I-site +on O +one O +face O +of O +the O +trimer B-oligomeric_state +. O + +The O +other O +face O +of O +the O +trimer B-oligomeric_state +displays O +a O +smaller O +hydrophobic B-site +surface I-site +, O +which O +includes O +the O +side O +chains O +of O +residues O +V18 B-residue_name_number +, O +F20 B-residue_name_number +, O +and O +I31 B-residue_name_number +of O +the O +three O +β B-structure_element +- I-structure_element +hairpins I-structure_element +( O +Figure O +3D O +). O + +In O +subsequent O +discussion O +, O +we O +designate O +the O +former O +surface O +the O +“ O +F19 B-residue_name_number +face O +” O +and O +the O +latter O +surface O +the O +“ O +F20 B-residue_name_number +face O +”. O + +Dodecamer B-oligomeric_state + +Four O +trimers B-oligomeric_state +assemble O +to O +form O +a O +dodecamer B-oligomeric_state +. O + +The O +four O +trimers B-oligomeric_state +arrange O +in O +a O +tetrahedral B-protein_state +fashion O +, O +creating O +a O +central B-site +cavity I-site +inside O +the O +dodecamer B-oligomeric_state +. O +Because O +each O +trimer B-oligomeric_state +is O +triangular B-protein_state +, O +the O +resulting O +arrangement O +resembles O +an O +octahedron B-protein_state +. O + +Each O +of O +the O +12 O +β B-structure_element +- I-structure_element +hairpins I-structure_element +constitutes O +an O +edge O +of O +the O +octahedron B-protein_state +, O +and O +the O +triangular B-protein_state +trimers B-oligomeric_state +occupy O +four O +of O +the O +eight O +faces O +of O +the O +octahedron B-protein_state +. O + +Figure O +4A O +illustrates O +the O +octahedral B-protein_state +shape O +of O +the O +dodecamer B-oligomeric_state +. O + +Figure O +4B O +illustrates O +the O +tetrahedral B-protein_state +arrangement O +of O +the O +four O +trimers B-oligomeric_state +. O + +X O +- O +ray O +crystallographic O +structure O +of O +the O +dodecamer B-oligomeric_state +formed O +by O +peptide B-mutant +2 I-mutant +. O +( O +A O +) O +View O +of O +the O +dodecamer B-oligomeric_state +that O +illustrates O +the O +octahedral B-protein_state +shape O +. O +( O +B O +) O +View O +of O +the O +dodecamer B-oligomeric_state +that O +illustrates O +the O +tetrahedral B-protein_state +arrangement O +of O +the O +four O +trimers B-oligomeric_state +that O +comprise O +the O +dodecamer B-oligomeric_state +. O +( O +C O +) O +View O +of O +two O +trimer B-oligomeric_state +subunits B-structure_element +from O +inside O +the O +cavity B-site +of O +the O +dodecamer B-oligomeric_state +. O + +Residues O +L17 B-residue_name_number +, O +L34 B-residue_name_number +, O +and O +V36 B-residue_name_number +are O +shown O +as O +spheres O +, O +illustrating O +the O +hydrophobic B-bond_interaction +packing I-bond_interaction +that O +occurs O +at O +the O +six O +vertices O +of O +the O +dodecamer B-oligomeric_state +. O +( O +D O +) O +Detailed O +view O +of O +one O +of O +the O +six O +vertices O +of O +the O +dodecamer B-oligomeric_state +. O + +The O +F19 B-residue_name_number +faces O +of O +the O +trimers B-oligomeric_state +line O +the O +interior O +of O +the O +dodecamer B-oligomeric_state +. O + +At O +the O +six O +vertices O +, O +hydrophobic B-bond_interaction +packing I-bond_interaction +between O +the O +side O +chains O +of O +L17 B-residue_name_number +, O +L34 B-residue_name_number +, O +and O +V36 B-residue_name_number +helps O +stabilize O +the O +dodecamer B-oligomeric_state +( O +Figures O +4C O +and O +D O +). O + +Salt O +bridges O +between O +the O +side O +chains O +of O +D23 B-residue_name_number +and O +δOrn B-structure_element +at O +the O +vertices O +further O +stabilize O +the O +dodecamer B-oligomeric_state +. O + +Each O +of O +the O +six O +vertices O +includes O +two O +Aβ25 B-protein +– B-residue_range +28 I-residue_range +loops B-structure_element +that O +extend O +past O +the O +core B-structure_element +of O +the O +dodecamer B-oligomeric_state +without O +making O +any O +substantial O +intermolecular O +contacts O +. O + +The O +exterior O +of O +the O +dodecamer B-oligomeric_state +displays O +four O +F20 B-residue_name_number +faces O +( O +Figure O +S3 O +). O + +In O +the O +crystal B-evidence +lattice I-evidence +, O +each O +F20 B-residue_name_number +face O +of O +one O +dodecamer B-oligomeric_state +packs O +against O +an O +F20 B-residue_name_number +face O +of O +another O +dodecamer B-oligomeric_state +. O + +Although O +the O +asymmetric O +unit O +comprises O +half O +a O +dodecamer B-oligomeric_state +, O +the O +crystal B-evidence +lattice I-evidence +may O +be O +thought O +of O +as O +being O +built O +of O +dodecamers B-oligomeric_state +. O + +The O +electron B-evidence +density I-evidence +map I-evidence +for O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +2 I-mutant +has O +long O +tubes O +of O +electron B-evidence +density I-evidence +inside O +the O +central B-site +cavity I-site +of O +the O +dodecamer B-oligomeric_state +. O + +The O +shape O +and O +length O +of O +the O +electron B-evidence +density I-evidence +is O +consistent O +with O +the O +structure B-evidence +of O +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +, O +which O +is O +an O +essential O +component O +of O +the O +crystallization O +conditions O +. O + +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +is O +a O +polypropylene O +glycol O +derivative O +with O +a O +2 O +- O +methoxyethoxy O +unit O +at O +one O +end O +and O +a O +2 O +- O +aminopropyl O +unit O +at O +the O +other O +end O +. O + +Although O +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +is O +a O +heterogeneous O +mixture O +with O +varying O +chain O +lengths O +and O +stereochemistry O +, O +we O +modeled O +a O +single O +stereoisomer O +with O +nine O +propylene O +glycol O +units O +( O +n O += O +9 O +) O +to O +fit O +the O +electron B-evidence +density I-evidence +. O + +The O +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +appears O +to O +stabilize O +the O +dodecamer B-oligomeric_state +by O +occupying O +the O +central B-site +cavity I-site +and O +making O +hydrophobic B-bond_interaction +contacts I-bond_interaction +with O +residues O +lining O +the O +cavity B-site +( O +Figure O +S3 O +). O + +In O +a O +dodecamer B-oligomeric_state +formed O +by O +full B-protein_state +- I-protein_state +length I-protein_state +Aβ B-protein +, O +the O +hydrophobic O +C O +- O +terminal O +residues O +( O +Aβ37 B-protein +– B-residue_range +40 I-residue_range +or O +Aβ37 B-protein +– B-residue_range +42 I-residue_range +) O +might O +play O +a O +similar O +role O +in O +filling O +the O +dodecamer B-oligomeric_state +and O +thus O +create O +a O +packed O +hydrophobic B-site +core I-site +within O +the O +central B-site +cavity I-site +of O +the O +dodecamer B-oligomeric_state +. O + +Annular B-site +Pore I-site + +Five O +dodecamers B-oligomeric_state +assemble O +to O +form O +an O +annular O +porelike B-structure_element +structure O +( O +Figure O +5A O +). O + +Hydrophobic B-bond_interaction +packing I-bond_interaction +between O +the O +F20 B-residue_name_number +faces O +of O +trimers B-oligomeric_state +displayed O +on O +the O +outer O +surface O +of O +each O +dodecamer B-oligomeric_state +stabilizes O +the O +porelike O +assembly O +. O + +Two O +morphologically O +distinct O +interactions O +between O +trimers B-oligomeric_state +occur O +at O +the O +interfaces B-site +of O +the O +five O +dodecamers B-oligomeric_state +: O +one O +in O +which O +the O +trimers B-oligomeric_state +are O +eclipsed B-protein_state +( O +Figure O +5B O +), O +and O +one O +in O +which O +the O +trimers B-oligomeric_state +are O +staggered B-protein_state +( O +Figure O +5C O +). O + +Hydrophobic B-bond_interaction +packing I-bond_interaction +between O +the O +side O +chains O +of O +F20 B-residue_name_number +, O +I31 B-residue_name_number +, O +and O +E22 B-residue_name_number +stabilizes O +these O +interfaces B-site +( O +Figure O +5D O +and O +E O +). O + +The O +annular B-site +pore I-site +contains O +three O +eclipsed B-protein_state +interfaces B-site +and O +two O +staggered B-protein_state +interfaces B-site +. O + +The O +eclipsed B-protein_state +interfaces B-site +occur O +between O +dodecamers B-structure_element +1 I-structure_element +and I-structure_element +2 I-structure_element +, O +1 B-structure_element +and I-structure_element +5 I-structure_element +, O +and O +3 B-structure_element +and I-structure_element +4 I-structure_element +, O +as O +shown O +in O +Figure O +5A O +. O + +The O +staggered B-protein_state +interfaces B-site +occur O +between O +dodecamers B-structure_element +2 I-structure_element +and I-structure_element +3 I-structure_element +and O +4 B-structure_element +and I-structure_element +5 I-structure_element +. O + +The O +annular B-site +pore I-site +is O +not O +completely O +flat O +, O +instead O +, O +adopting O +a O +slightly O +puckered O +shape O +, O +which O +accommodates O +the O +eclipsed B-protein_state +and O +staggered B-protein_state +interfaces B-site +. O + +Ten O +Aβ25 B-protein +– B-residue_range +28 I-residue_range +loops B-structure_element +from O +the O +vertices O +of O +the O +five O +dodecamers B-oligomeric_state +line O +the O +hole O +in O +the O +center O +of O +the O +pore B-site +. O + +The O +hydrophilic O +side O +chains O +of O +S26 B-residue_name_number +, O +N27 B-residue_name_number +, O +and O +K28 B-residue_name_number +decorate O +the O +hole O +. O + +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +the O +annular B-site +pore I-site +formed O +by O +peptide B-mutant +2 I-mutant +. O +( O +A O +) O +Annular B-structure_element +porelike I-structure_element +structure B-evidence +illustrating O +the O +relationship O +of O +the O +five O +dodecamers B-oligomeric_state +that O +form O +the O +pore B-site +( O +top O +view O +). O + +( O +B O +) O +Eclipsed B-site +interface I-site +between O +dodecamers B-structure_element +1 I-structure_element +and I-structure_element +2 I-structure_element +( O +side O +view O +). O + +The O +same O +eclipsed B-site +interface I-site +also O +occurs O +between O +dodecamers B-structure_element +1 I-structure_element +and I-structure_element +5 I-structure_element +and O +3 B-structure_element +and I-structure_element +4 I-structure_element +. O +( O +C O +) O +Staggered B-site +interface I-site +between O +dodecamers B-structure_element +2 I-structure_element +and I-structure_element +3 I-structure_element +( O +side O +view O +). O + +The O +same O +staggered B-site +interface I-site +also O +occurs O +between O +dodecamers O +4 O +and O +5 O +. O +( O +D O +) O +Eclipsed B-site +interface I-site +between O +dodecamers B-structure_element +1 I-structure_element +and I-structure_element +5 I-structure_element +( O +top O +view O +). O + +The O +annular B-site +pore I-site +is O +comparable O +in O +size O +to O +other O +large O +protein O +assemblies O +. O + +The O +diameter O +of O +the O +hole O +in O +the O +center O +of O +the O +pore B-site +is O +∼ O +2 O +nm O +. O + +The O +thickness O +of O +the O +pore B-site +is O +∼ O +5 O +nm O +, O +which O +is O +comparable O +to O +that O +of O +a O +lipid O +bilayer O +membrane O +. 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O + +The O +crystallographic B-evidence +assembly I-evidence +of O +peptide B-mutant +2 I-mutant +into O +a O +trimer B-oligomeric_state +, O +dodecamer B-oligomeric_state +, O +and O +annular B-site +pore I-site +provides O +a O +model O +for O +the O +assembly O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +Aβ B-protein +peptide O +to O +form O +oligomers B-oligomeric_state +. O + +In O +this O +model O +Aβ B-protein +folds O +to O +form O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +comprising O +the O +hydrophobic O +central B-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +regions I-structure_element +. 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O + +Fibrillar B-protein_state +and O +nonfibrillar B-protein_state +oligomers B-oligomeric_state +have O +structurally O +distinct O +characteristics O +, O +which O +are O +reflected O +in O +their O +reactivity O +with O +the O +fibril O +- O +specific O +OC O +antibody O +and O +the O +oligomer B-oligomeric_state +- O +specific O +A11 O +antibody O +. O + +Fibrillar B-protein_state +oligomers B-oligomeric_state +are O +recognized O +by O +the O +OC O +antibody O +but O +not O +the O +A11 O +antibody O +, O +whereas O +nonfibrillar B-protein_state +oligomers B-oligomeric_state +are O +recognized O +by O +the O +A11 O +antibody O +but O +not O +the O +OC O +antibody O +. O + +These O +criteria O +have O +been O +used O +to O +classify O +the O +Aβ B-protein +oligomers B-oligomeric_state +that O +accumulate O +in O +vivo O +. 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O + +Predictive O +features O +of O +ligand O +‐ O +specific O +signaling O +through O +the O +estrogen B-protein_type +receptor I-protein_type + +Some O +estrogen B-protein +receptor I-protein +‐ I-protein +α I-protein +( O +ERα B-protein +)‐ O +targeted O +breast O +cancer O +therapies O +such O +as O +tamoxifen B-chemical +have O +tissue O +‐ O +selective O +or O +cell O +‐ O +specific O +activities O +, O +while O +others O +have O +similar O +activities O +in O +different O +cell O +types O +. O + +To O +identify O +biophysical O +determinants O +of O +cell O +‐ O +specific O +signaling O +and O +breast O +cancer O +cell O +proliferation O +, O +we O +synthesized B-experimental_method +241 O +ERα B-protein +ligands O +based O +on O +19 O +chemical O +scaffolds O +, O +and O +compared O +ligand O +response O +using O +quantitative B-experimental_method +bioassays I-experimental_method +for O +canonical O +ERα B-protein +activities O +and O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +Ligands O +that O +regulate O +the O +dynamics O +and O +stability O +of O +the O +coactivator B-site +‐ I-site +binding I-site +site I-site +in O +the O +C O +‐ O +terminal O +ligand B-structure_element +‐ I-structure_element +binding I-structure_element +domain I-structure_element +, O +called O +activation B-structure_element +function I-structure_element +‐ I-structure_element +2 I-structure_element +( O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +), O +showed O +similar O +activity O +profiles O +in O +different O +cell O +types O +. O + +Such O +ligands O +induced O +breast O +cancer O +cell O +proliferation O +in O +a O +manner O +that O +was O +predicted O +by O +the O +canonical O +recruitment O +of O +the O +coactivators O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +and O +induction O +of O +the O +GREB1 B-protein +proliferative O +gene O +. O + +For O +some O +ligand O +series O +, O +a O +single O +inter B-evidence +‐ I-evidence +atomic I-evidence +distance I-evidence +in O +the O +ligand B-structure_element +‐ I-structure_element +binding I-structure_element +domain I-structure_element +predicted O +their O +proliferative O +effects O +. O + +In O +contrast O +, O +the O +N O +‐ O +terminal O +coactivator B-site +‐ I-site +binding I-site +site I-site +, O +activation B-structure_element +function I-structure_element +‐ I-structure_element +1 I-structure_element +( O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +), O +determined O +cell O +‐ O +specific O +signaling O +induced O +by O +ligands O +that O +used O +alternate O +mechanisms O +to O +control O +cell O +proliferation O +. O + +Thus O +, O +incorporating O +systems B-experimental_method +structural I-experimental_method +analyses I-experimental_method +with O +quantitative B-experimental_method +chemical I-experimental_method +biology I-experimental_method +reveals O +how O +ligands O +can O +achieve O +distinct O +allosteric O +signaling O +outcomes O +through O +ERα B-protein +. O + +Many O +drugs O +are O +small O +‐ O +molecule O +ligands O +of O +allosteric O +signaling O +proteins O +, O +including O +G B-protein_type +protein I-protein_type +‐ I-protein_type +coupled I-protein_type +receptors I-protein_type +( O +GPCRs B-protein_type +) O +and O +nuclear B-protein_type +receptors I-protein_type +such O +as O +ERα B-protein +. O + +Small O +‐ O +molecule O +ligands O +control O +receptor O +activity O +by O +modulating O +recruitment O +of O +effector O +enzymes O +to O +distal O +regions O +of O +the O +receptor O +, O +relative O +to O +the O +ligand B-site +‐ I-site +binding I-site +site I-site +. O + +For O +example O +, O +selective O +estrogen B-protein_type +receptor I-protein_type +modulators I-protein_type +( O +SERMs B-protein_type +) O +such O +as O +tamoxifen B-chemical +( O +Nolvadex B-chemical +®; I-chemical +AstraZeneca O +) O +or O +raloxifene B-chemical +( O +Evista B-chemical +®; I-chemical +Eli O +Lilly O +) O +( O +Fig O +1A O +) O +block O +the O +ERα B-protein +‐ O +mediated O +proliferative O +effects O +of O +the O +native O +estrogen B-chemical +, O +17β B-chemical +‐ I-chemical +estradiol I-chemical +( O +E2 B-chemical +), O +on O +breast O +cancer O +cells O +, O +but O +promote O +beneficial O +estrogenic O +effects O +on O +bone O +mineral O +density O +and O +adverse O +estrogenic O +effects O +such O +as O +uterine O +proliferation O +, O +fatty O +liver O +, O +or O +stroke O +( O +Frolik O +et O +al O +, O +1996 O +; O +Fisher O +et O +al O +, O +1998 O +; O +McDonnell O +et O +al O +, O +2002 O +; O +Jordan O +, O +2003 O +). O + +Allosteric O +control O +of O +ERα B-protein +activity O + +Chemical O +structures O +of O +some O +common O +ERα B-protein +ligands O +. O + +E2 B-chemical +‐ O +rings O +are O +numbered O +A O +‐ O +D O +. O +The O +E O +‐ O +ring O +is O +the O +common O +site O +of O +attachment O +for O +BSC O +found O +in O +many O +SERMS B-protein_type +. O + +ERα B-protein +domain O +organization O +lettered O +, O +A O +‐ O +F O +. O +DBD B-structure_element +, O +DNA B-structure_element +‐ I-structure_element +binding I-structure_element +domain I-structure_element +; O +LBD B-structure_element +, O +ligand B-structure_element +‐ I-structure_element +binding I-structure_element +domain I-structure_element +; O +AF B-structure_element +, O +activation B-structure_element +function I-structure_element + +Schematic O +illustration O +of O +the O +canonical O +ERα B-protein +signaling O +pathway O +. O + +Linear O +causality O +model O +for O +ERα B-protein +‐ O +mediated O +cell O +proliferation O +. O + +Branched O +causality O +model O +for O +ERα B-protein +‐ O +mediated O +cell O +proliferation O +. O + +ERα B-protein +contains O +structurally B-protein_state +conserved I-protein_state +globular B-structure_element +domains I-structure_element +of O +the O +nuclear B-protein_type +receptor I-protein_type +superfamily I-protein_type +, O +including O +a O +DNA B-structure_element +‐ I-structure_element +binding I-structure_element +domain I-structure_element +( O +DBD B-structure_element +) O +that O +is O +connected O +by O +a O +flexible B-protein_state +hinge B-structure_element +region I-structure_element +to O +the O +ligand B-structure_element +‐ I-structure_element +binding I-structure_element +domain I-structure_element +( O +LBD B-structure_element +), O +as O +well O +as O +unstructured B-protein_state +AB B-structure_element +and O +F B-structure_element +domains O +at O +its O +amino O +and O +carboxyl O +termini O +, O +respectively O +( O +Fig O +1B O +). O + +The O +LBD B-structure_element +contains O +a O +ligand O +‐ O +dependent O +coactivator B-site +‐ I-site +binding I-site +site I-site +called O +activation B-structure_element +function I-structure_element +‐ I-structure_element +2 I-structure_element +( O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +). O + +However O +, O +the O +agonist O +activity O +of O +SERMs B-protein_type +derives O +from O +activation B-structure_element +function I-structure_element +‐ I-structure_element +1 I-structure_element +( O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +)— O +a O +coactivator B-site +recruitment I-site +site I-site +located O +in O +the O +AB B-structure_element +domain O +( O +Berry O +et O +al O +, O +1990 O +; O +Shang O +& O +Brown O +, O +2002 O +; O +Abot O +et O +al O +, O +2013 O +). O + +AF B-structure_element +‐ I-structure_element +1 I-structure_element +and O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +bind O +distinct O +but O +overlapping O +sets O +of O +coregulators O +( O +Webb O +et O +al O +, O +1998 O +; O +Endoh O +et O +al O +, O +1999 O +; O +Delage O +‐ O +Mourroux O +et O +al O +, O +2000 O +; O +Yi O +et O +al O +, O +2015 O +). O + +AF B-structure_element +‐ I-structure_element +2 I-structure_element +binds O +the O +signature O +LxxLL B-structure_element +motif I-structure_element +peptides O +of O +coactivators O +such O +as O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +( O +also O +known O +as O +SRC B-protein +‐ I-protein +1 I-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +). O + +AF B-structure_element +‐ I-structure_element +1 I-structure_element +binds O +a O +separate O +surface O +on O +these O +coactivators O +( O +Webb O +et O +al O +, O +1998 O +; O +Yi O +et O +al O +, O +2015 O +). O + +Yet O +, O +it O +is O +unknown O +how O +different O +ERα B-protein +ligands O +control O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +through O +the O +LBD B-structure_element +, O +and O +whether O +this O +inter O +‐ O +domain O +communication O +is O +required O +for O +cell O +‐ O +specific O +signaling O +or O +anti O +‐ O +proliferative O +responses O +. O + +In O +the O +canonical O +model O +of O +the O +ERα B-protein +signaling O +pathway O +( O +Fig O +1C O +), O +E2 B-protein_state +‐ I-protein_state +bound I-protein_state +ERα B-protein +forms O +a O +homodimer B-oligomeric_state +that O +binds O +DNA O +at O +estrogen B-site +‐ I-site +response I-site +elements I-site +( O +EREs B-site +), O +recruits O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +( O +Metivier O +et O +al O +, O +2003 O +; O +Johnson O +& O +O O +' O +Malley O +, O +2012 O +), O +and O +activates O +the O +GREB1 B-protein +gene O +, O +which O +is O +required O +for O +proliferation O +of O +ERα B-protein +‐ O +positive O +breast O +cancer O +cells O +( O +Ghosh O +et O +al O +, O +2000 O +; O +Rae O +et O +al O +, O +2005 O +; O +Deschenes O +et O +al O +, O +2007 O +; O +Liu O +et O +al O +, O +2012 O +; O +Srinivasan O +et O +al O +, O +2013 O +). O + +However O +, O +ERα B-protein +‐ O +mediated O +proliferative O +responses O +vary O +in O +a O +ligand O +‐ O +dependent O +manner O +( O +Srinivasan O +et O +al O +, O +2013 O +); O +thus O +, O +it O +is O +not O +known O +whether O +this O +canonical O +model O +is O +widely O +applicable O +across O +diverse O +ERα B-protein +ligands O +. O + +Our O +long O +‐ O +term O +goal O +is O +to O +be O +able O +to O +predict O +proliferative O +or O +anti O +‐ O +proliferative O +activity O +of O +a O +ligand O +in O +different O +tissues O +from O +its O +crystal B-evidence +structure I-evidence +by O +identifying O +different O +structural O +perturbations O +that O +lead O +to O +specific O +signaling O +outcomes O +. O + +The O +simplest O +response O +model O +for O +ligand O +‐ O +specific O +proliferative O +effects O +is O +a O +linear O +causality O +model O +, O +where O +the O +degree O +of O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +determines O +GREB1 B-protein +expression O +, O +which O +in O +turn O +drives O +ligand O +‐ O +specific O +cell O +proliferation O +( O +Fig O +1D O +). O + +In O +this O +signaling O +model O +, O +multiple O +coregulator O +binding O +events O +and O +target O +genes O +( O +Won O +Jeong O +et O +al O +, O +2012 O +; O +Nwachukwu O +et O +al O +, O +2014 O +), O +LBD B-structure_element +conformation O +, O +nucleocytoplasmic O +shuttling O +, O +the O +occupancy O +and O +dynamics O +of O +DNA O +binding O +, O +and O +other O +biophysical O +features O +could O +contribute O +independently O +to O +cell O +proliferation O +( O +Lickwar O +et O +al O +, O +2012 O +). O + +To O +test O +these O +signaling O +models O +, O +we O +profiled O +a O +diverse O +library O +of O +ERα B-protein +ligands O +using O +systems O +biology O +approaches O +to O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +and O +chemical B-experimental_method +biology I-experimental_method +( O +Srinivasan O +et O +al O +, O +2013 O +), O +including O +a O +series O +of O +quantitative O +bioassays O +for O +ERα B-protein +function O +that O +were O +statistically O +robust O +and O +reproducible O +, O +based O +on O +the O +Z B-evidence +’‐ I-evidence +statistic I-evidence +( O +Fig O +EV1A O +and O +B O +; O +see O +Materials O +and O +Methods O +). O + +We O +also O +determined B-experimental_method +the O +structures B-evidence +of O +76 O +distinct O +ERα B-protein +LBD B-structure_element +complexes O +bound B-protein_state +to I-protein_state +different O +ligand O +types O +, O +which O +allowed O +us O +to O +understand O +how O +diverse O +ligand O +scaffolds O +distort O +the O +active B-protein_state +conformation O +of O +the O +ERα B-protein +LBD B-structure_element +. O + +Our O +findings O +here O +indicate O +that O +specific O +structural O +perturbations O +can O +be O +tied O +to O +ligand O +‐ O +selective O +domain O +usage O +and O +signaling O +patterns O +, O +thus O +providing O +a O +framework O +for O +structure O +‐ O +based O +design O +of O +improved O +breast O +cancer O +therapeutics O +, O +and O +understanding O +the O +different O +phenotypic O +effects O +of O +environmental O +estrogens B-chemical +. O + +High O +‐ O +throughput O +screens O +for O +ERα B-protein +ligand O +profiling O + +Summary O +of O +ligand B-experimental_method +screening I-experimental_method +assays I-experimental_method +used O +to O +measure O +ER O +‐ O +mediated O +activities O +. O + +ERE B-structure_element +, O +estrogen B-structure_element +‐ I-structure_element +response I-structure_element +element I-structure_element +; O +Luc B-experimental_method +, O +luciferase B-experimental_method +reporter I-experimental_method +gene I-experimental_method +; O +M2H B-experimental_method +, O +mammalian B-experimental_method +2 I-experimental_method +‐ I-experimental_method +hybrid I-experimental_method +; O +UAS B-structure_element +, O +upstream B-structure_element +‐ I-structure_element +activating I-structure_element +sequence I-structure_element +. O + +Strength O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +signaling O +does O +not O +determine O +cell O +‐ O +specific O +signaling O + +To O +compare O +ERα B-protein +signaling O +induced O +by O +diverse O +ligand O +types O +, O +we O +synthesized B-experimental_method +and I-experimental_method +assayed I-experimental_method +a O +library O +of O +241 O +ERα B-protein +ligands O +containing O +19 O +distinct O +molecular O +scaffolds O +. O + +These O +include O +15 O +indirect O +modulator O +series O +, O +which O +lack B-protein_state +a O +SERM B-protein_type +‐ I-protein_type +like I-protein_type +side O +chain O +and O +modulate O +coactivator O +binding O +indirectly O +from O +the O +ligand B-site +‐ I-site +binding I-site +pocket I-site +( O +Fig O +2A O +– O +E O +; O +Dataset O +EV1 O +) O +( O +Zheng O +et O +al O +, O +2012 O +) O +( O +Zhu O +et O +al O +, O +2012 O +) O +( O +Muthyala O +et O +al O +, O +2003 O +; O +Seo O +et O +al O +, O +2006 O +) O +( O +Srinivasan O +et O +al O +, O +2013 O +) O +( O +Wang O +et O +al O +, O +2012 O +) O +( O +Liao O +et O +al O +, O +2014 O +) O +( O +Min O +et O +al O +, O +2013 O +). O + +We O +also O +generated O +four O +direct O +modulator O +series O +with O +side O +chains O +designed O +to O +directly O +dislocate O +h12 B-structure_element +and O +thereby O +completely O +occlude O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +( O +Fig O +2C O +and O +E O +; O +Dataset O +EV1 O +) O +( O +Kieser O +et O +al O +, O +2010 O +). O + +Ligand B-experimental_method +profiling I-experimental_method +using O +our O +quantitative B-experimental_method +bioassays I-experimental_method +revealed O +a O +wide O +range O +of O +ligand O +‐ O +induced O +GREB1 B-protein +expression O +, O +reporter O +gene O +activities O +, O +ERα B-protein +‐ O +coactivator O +interactions O +, O +and O +proliferative O +effects O +on O +MCF O +‐ O +7 O +breast O +cancer O +cells O +( O +Figs O +EV1 O +and O +EV2A O +– O +J O +). O + +This O +wide O +variance O +enabled O +us O +to O +probe O +specific O +features O +of O +ERα B-protein +signaling O +using O +ligand B-experimental_method +class I-experimental_method +analyses I-experimental_method +, O +and O +identify O +signaling O +patterns O +shared O +by O +specific O +ligand O +series O +or O +scaffolds O +. O + +Classes O +of O +compounds O +in O +the O +ERα B-protein +ligand O +library O + +Structure B-evidence +of O +the O +E2 B-protein_state +‐ I-protein_state +bound I-protein_state +ERα B-protein +LBD B-structure_element +in B-protein_state +complex I-protein_state +with I-protein_state +an O +NCOA2 B-protein +peptide O +of O +( O +PDB O +1GWR O +). O + +Structural O +details O +of O +the O +ERα B-protein +LBD B-structure_element +bound B-protein_state +to I-protein_state +the O +indicated O +ligands O +. O + +Unlike O +E2 B-chemical +( O +PDB O +1GWR O +), O +TAM B-chemical +is O +a O +direct O +modulator O +with O +a O +BSC O +that O +dislocates O +h12 B-structure_element +to O +block O +the O +NCOA2 B-site +‐ I-site +binding I-site +site I-site +( O +PDB O +3ERT O +). O + +OBHS B-chemical +is O +an O +indirect O +modulator O +that O +dislocates O +the O +h11 B-structure_element +C O +‐ O +terminus O +to O +destabilize O +the O +h11 B-site +– I-site +h12 I-site +interface I-site +( O +PDB O +4ZN9 O +). O + +The O +ERα B-protein +ligand O +library O +contains O +241 O +ligands O +representing O +15 O +indirect O +modulator O +scaffolds O +, O +plus O +4 O +direct O +modulator O +scaffolds O +. O + +ERα B-protein +ligands O +induced O +a O +range O +of O +agonist O +activity O +profiles O + +To O +this O +end O +, O +we O +compared O +the O +average O +ligand O +‐ O +induced O +GREB1 B-protein +mRNA O +levels O +in O +MCF O +‐ O +7 O +cells O +and O +3 B-experimental_method +× I-experimental_method +ERE I-experimental_method +‐ I-experimental_method +Luc I-experimental_method +reporter O +gene O +activity O +in O +Ishikawa O +endometrial O +cancer O +cells O +( O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +) O +or O +in O +HepG2 O +cells O +transfected O +with O +wild B-protein_state +‐ I-protein_state +type I-protein_state +ERα B-protein +( O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +) O +( O +Figs O +3A O +and O +EV2A O +– O +C O +). O + +Direct O +modulators O +showed O +significant O +differences O +in O +average O +activity O +between O +cell O +types O +except O +OBHS B-chemical +‐ I-chemical +ASC I-chemical +analogs O +, O +which O +had O +similar O +low O +agonist O +activities O +in O +the O +three O +cell O +types O +. O + +While O +it O +was O +known O +that O +direct O +modulators O +such O +as O +tamoxifen B-chemical +drive O +cell O +‐ O +specific O +signaling O +, O +these O +experiments O +reveal O +that O +indirect O +modulators O +also O +drive O +cell O +‐ O +specific O +signaling O +, O +since O +eight O +of O +fourteen O +classes O +showed O +significant O +differences O +in O +average O +activity O +( O +Figs O +3A O +and O +EV2A O +– O +C O +). O + +Ligand O +‐ O +specific O +signaling O +underlies O +ERα B-protein +‐ O +mediated O +cell O +proliferation O + +( O +A O +) O +Ligand O +‐ O +specific O +ERα B-protein +activities O +in O +HepG2 O +, O +Ishikawa O +and O +MCF O +‐ O +7 O +cells O +. O + +The O +ligand O +‐ O +induced O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +and O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activities O +and O +GREB1 B-protein +mRNA O +levels O +are O +shown O +by O +scaffold O +( O +mean O ++ O +SD O +). O + +( O +B O +) O +Ligand O +class B-experimental_method +analysis I-experimental_method +of O +the O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +and O +ERα B-mutant +‐ I-mutant +ΔAB I-mutant +activities O +in O +HepG2 O +cells O +. O + +Significant O +sensitivity O +to O +AB B-structure_element +domain O +deletion O +was O +determined O +by O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +‐ I-experimental_method +test I-experimental_method +( O +n O += O +number O +of O +ligands O +per O +scaffold O +in O +Fig O +2 O +). O + +Correlation B-experimental_method +and I-experimental_method +regression I-experimental_method +analyses I-experimental_method +in O +a O +large O +test O +set O +. O + +In O +cluster O +1 O +, O +the O +first O +three O +comparisons O +( O +rows O +) O +showed O +significant O +positive O +correlations O +( O +F B-experimental_method +‐ I-experimental_method +test I-experimental_method +for O +nonzero O +slope O +, O +P B-evidence +≤ O +0 O +. O +05 O +). O + +In O +cluster O +2 O +, O +only O +one O +of O +these O +comparisons O +revealed O +a O +significant O +positive O +correlation O +, O +while O +none O +was O +significant O +in O +cluster O +3 O +. O ++, O +statistically O +significant O +correlations O +gained O +by O +deletion B-experimental_method +of O +the O +AB B-structure_element +or O +F B-structure_element +domains O +. O + +−, O +significant O +correlations O +lost O +upon O +deletion O +of O +AB B-structure_element +or O +F B-structure_element +domains O +. O + +Tamoxifen B-chemical +depends O +on O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +for O +its O +cell O +‐ O +specific O +activity O +( O +Sakamoto O +et O +al O +, O +2002 O +); O +therefore O +, O +we O +asked O +whether O +cell O +‐ O +specific O +signaling O +observed O +here O +is O +due O +to O +a O +similar O +dependence O +on O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +for O +activity O +( O +Fig O +EV1 O +). O + +To O +test O +this O +idea O +, O +we O +compared O +the O +average B-evidence +L I-evidence +‐ I-evidence +Luc I-evidence +activities I-evidence +of O +each O +scaffold O +in O +HepG2 O +cells O +co B-experimental_method +‐ I-experimental_method +transfected I-experimental_method +with O +wild B-protein_state +‐ I-protein_state +type I-protein_state +ERα B-protein +or O +with O +ERα B-protein +lacking B-protein_state +the I-protein_state +AB B-structure_element +domain O +( O +Figs O +1B O +and O +EV1 O +). O + +While O +E2 B-chemical +showed O +similar O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +and O +ERα B-mutant +‐ I-mutant +ΔAB I-mutant +activities O +, O +tamoxifen B-chemical +showed O +complete O +loss O +of O +activity O +without B-protein_state +the O +AB B-structure_element +domain O +( O +Fig O +EV1B O +). O + +Deletion B-experimental_method +of I-experimental_method +the O +AB B-structure_element +domain O +significantly O +reduced O +the O +average B-evidence +L I-evidence +‐ I-evidence +Luc I-evidence +activities I-evidence +of O +14 O +scaffolds O +( O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +‐ I-experimental_method +test I-experimental_method +, O +P B-evidence +≤ O +0 O +. O +05 O +) O +( O +Fig O +3B O +). O + +These O +“ O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +‐ O +sensitive O +” O +activities O +were O +exhibited O +by O +both O +direct O +and O +indirect O +modulators O +, O +and O +were O +not O +limited O +to O +scaffolds O +that O +showed O +cell O +‐ O +specific O +signaling O +( O +Fig O +3A O +and O +B O +). O + +Thus O +, O +the O +strength O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +signaling O +does O +not O +determine O +cell O +‐ O +specific O +signaling O +. O + +Identifying O +cell O +‐ O +specific O +signaling O +clusters O +in O +ERα B-protein +ligand O +classes O + +For O +each O +ligand O +class O +or O +scaffold O +, O +we O +calculated O +the O +Pearson B-evidence +' I-evidence +s I-evidence +correlation I-evidence +coefficient I-evidence +, O +r B-evidence +, O +for O +pairwise O +comparison O +of O +activity O +profiles O +in O +breast O +( O +GREB1 B-protein +), O +liver O +( O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +), O +and O +endometrial O +cells O +( O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +). O + +The O +value O +of O +r B-evidence +ranges O +from O +− O +1 O +to O +1 O +, O +and O +it O +defines O +the O +extent O +to O +which O +the O +data O +fit O +a O +straight O +line O +when O +compounds O +show O +similar O +agonist O +/ O +antagonist O +activity O +profiles O +between O +cell O +types O +( O +Fig O +EV3A O +). O + +We O +also O +calculated O +the O +coefficient B-evidence +of I-evidence +determination I-evidence +, O +r B-evidence +2 I-evidence +, O +which O +describes O +the O +percentage O +of O +variance O +in O +a O +dependent O +variable O +such O +as O +proliferation O +that O +can O +be O +predicted O +by O +an O +independent O +variable O +such O +as O +GREB1 B-protein +expression O +. O + +We O +present O +both O +calculations O +as O +r B-evidence +2 I-evidence +to O +readily O +compare O +signaling O +specificities O +using O +a O +heat O +map O +on O +which O +the O +red O +– O +yellow O +palette O +indicates O +significant O +positive O +correlations O +( O +P B-evidence +≤ O +0 O +. O +05 O +, O +F B-experimental_method +‐ I-experimental_method +test I-experimental_method +for O +nonzero O +slope O +), O +while O +the O +blue O +palette O +denotes O +negative O +correlations O +( O +Fig O +3C O +– O +F O +). O + +The O +side O +chain O +of O +OBHS B-chemical +‐ I-chemical +BSC I-chemical +analogs O +induces O +cell O +‐ O +specific O +signaling O + +Correlation O +analysis O +of O +OBHS B-chemical +versus O +OBHS B-chemical +‐ I-chemical +BSC I-chemical +activity O +across O +cell O +types O +. O + +Correlation O +analysis O +of O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-mutant +‐ I-mutant +ΔAB I-mutant +activity O +versus O +endogenous O +ERα B-protein +activity O +of O +OBHS B-chemical +analogs O +. O + +In O +panel O +( O +D O +), O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +activity O +from O +panel O +( O +B O +) O +is O +shown O +for O +comparison O +. O + +Correlation O +analysis O +of O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-mutant +‐ I-mutant +ΔF I-mutant +activity O +versus O +endogenous O +ERα B-protein +activities O +of O +OBHS B-chemical +analogs O +. O + +Correlation O +analysis O +of O +MCF O +‐ O +7 O +cell O +proliferation O +versus O +NCOA2 B-protein +/ I-protein +3 I-protein +recruitment O +or O +GREB1 B-protein +levels O +observed O +in O +response O +to O +( O +G O +) O +OBHS B-chemical +‐ I-chemical +N I-chemical +and O +( O +H O +) O +OBHS B-chemical +‐ I-chemical +BSC I-chemical +analogs O +. O + +Scaffolds O +in O +cluster O +1 O +exhibited O +strongly O +correlated O +GREB1 B-protein +levels O +, O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +and O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O +profiles O +across O +the O +three O +cell O +types O +( O +Fig O +3C O +lanes O +1 O +– O +4 O +), O +suggesting O +these O +ligands O +use O +similar O +ERα B-protein +signaling O +pathways O +in O +the O +breast O +, O +endometrial O +, O +and O +liver O +cell O +types O +. O + +This O +cluster O +includes O +WAY B-chemical +‐ I-chemical +C I-chemical +, O +OBHS B-chemical +, O +OBHS B-chemical +‐ I-chemical +N I-chemical +, O +and O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +, O +all O +of O +which O +are O +indirect O +modulators O +. O + +This O +cluster O +includes O +two O +classes O +of O +direct O +modulators O +( O +cyclofenil B-chemical +‐ I-chemical +ASC I-chemical +and O +WAY B-chemical +dimer I-chemical +), O +and O +six O +classes O +of O +indirect O +modulators O +( O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +, O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +, O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +2 I-chemical +and O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +, O +furan B-chemical +, O +and O +WAY B-chemical +‐ I-chemical +D I-chemical +). O + +For O +example O +, O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +, O +furan B-chemical +, O +and O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +2 I-chemical +drove O +positively O +correlated O +GREB1 B-protein +levels O +and O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +but O +not O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +activity O +( O +Fig O +3C O +lanes O +5 O +– O +7 O +). O + +In O +contrast O +, O +WAY B-chemical +dimer I-chemical +and O +WAY B-chemical +‐ I-chemical +D I-chemical +analogs O +drove O +positively O +correlated O +GREB1 B-protein +levels O +and O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +but O +not O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O +( O +Fig O +3C O +lanes O +8 O +and O +9 O +). O + +This O +cluster O +includes O +two O +direct O +modulator O +scaffolds O +( O +OBHS B-chemical +‐ I-chemical +ASC I-chemical +and O +OBHS B-chemical +‐ I-chemical +BSC I-chemical +), O +and O +five O +indirect O +modulator O +scaffolds O +( O +A B-chemical +‐ I-chemical +CD I-chemical +, O +cyclofenil B-chemical +, O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +, O +imine B-chemical +, O +and O +imidazopyridine B-chemical +). O + +These O +results O +suggest O +that O +addition O +of O +an O +extended O +side O +chain O +to O +an O +ERα B-protein +ligand O +scaffold O +is O +sufficient O +to O +induce O +cell O +‐ O +specific O +signaling O +, O +where O +the O +relative O +activity O +profiles O +of O +the O +individual O +ligands O +change O +between O +cell O +types O +. O + +This O +is O +demonstrated O +by O +directly O +comparing O +the O +signaling O +specificities O +of O +matched O +OBHS B-chemical +( O +indirect O +modulator O +, O +cluster O +1 O +) O +and O +OBHS B-chemical +‐ I-chemical +BSC I-chemical +analogs O +( O +direct O +modulator O +, O +cluster O +3 O +), O +which O +differ O +only O +in O +the O +basic O +side O +chain O +( O +Fig O +2E O +). O + +The O +activities O +of O +OBHS B-chemical +analogs O +were O +positively O +correlated O +across O +the O +three O +cell O +types O +, O +but O +the O +side O +chain O +of O +OBHS B-chemical +‐ I-chemical +BSC I-chemical +analogs O +was O +sufficient O +to O +abolish O +these O +correlations O +( O +Figs O +3C O +lanes O +1 O +and O +19 O +, O +and O +EV3A O +– O +C O +). O + +Thus O +, O +examining O +the O +correlated O +patterns O +of O +ERα B-protein +activity O +within O +each O +scaffold O +demonstrates O +that O +an O +extended O +side O +chain O +is O +not O +required O +for O +cell O +‐ O +specific O +signaling O +. O + +Modulation O +of O +signaling O +specificity O +by O +AF B-structure_element +‐ I-structure_element +1 I-structure_element + +To O +evaluate O +the O +role O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +and O +the O +F B-structure_element +domain O +in O +ERα B-protein +signaling O +specificity O +, O +we O +compared O +activity O +of O +truncated O +ERα B-protein +constructs O +in O +HepG2 O +liver O +cells O +with O +endogenous O +ERα B-protein +activity O +in O +the O +other O +cell O +types O +. O + +The O +positive O +correlation O +between O +the O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +and O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activities O +or O +GREB1 B-protein +levels O +induced O +by O +scaffolds O +in O +cluster O +1 O +was O +generally O +retained O +without O +the O +AB B-structure_element +domain O +, O +or O +the O +F B-structure_element +domain O +( O +Fig O +3D O +lanes O +1 O +– O +4 O +). O + +This O +demonstrates O +that O +the O +signaling O +specificities O +underlying O +these O +positive O +correlations O +are O +not O +modified O +by O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +. O + +OBHS B-chemical +analogs O +showed O +an O +average O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-mutant +‐ I-mutant +ΔAB I-mutant +activity O +of O +3 O +. O +2 O +% O +± O +3 O +( O +mean O ++ O +SEM O +) O +relative O +to O +E2 B-chemical +. O + +Despite O +this O +nearly O +complete O +lack O +of O +activity O +, O +the O +pattern O +of O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-mutant +‐ I-mutant +ΔAB I-mutant +activity O +was O +still O +highly O +correlated O +with O +the O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O +and O +GREB1 B-protein +expression O +( O +Fig O +EV3D O +and O +E O +), O +demonstrating O +that O +very O +small O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +activities O +can O +be O +amplified O +by O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +to O +produce O +robust O +signals O +. O + +Similarly O +, O +deletion B-experimental_method +of I-experimental_method +the O +F B-structure_element +domain O +did O +not O +abolish O +correlations O +between O +the O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +and O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +or O +GREB1 B-protein +levels O +induced O +by O +OBHS B-chemical +analogs O +( O +Fig O +EV3F O +). O + +These O +similar O +patterns O +of O +ligand O +activity O +in O +the O +wild B-protein_state +‐ I-protein_state +type I-protein_state +and O +deletion O +mutants B-protein_state +suggest O +that O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +and O +the O +F B-structure_element +domain O +purely O +amplify O +the O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +activities O +of O +ligands O +in O +cluster O +1 O +. O + +In O +contrast O +, O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +was O +a O +determinant O +of O +signaling O +specificity O +for O +scaffolds O +in O +cluster O +2 O +. O + +Deletion B-experimental_method +of I-experimental_method +the O +AB B-structure_element +or O +F B-structure_element +domain O +altered O +correlations O +for O +six O +of O +the O +eight O +scaffolds O +in O +this O +cluster O +( O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +, O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +, O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +, O +WAY B-chemical +‐ I-chemical +D I-chemical +, O +WAY B-chemical +dimer I-chemical +, O +and O +cyclofenil B-chemical +‐ I-chemical +ASC I-chemical +) O +( O +Fig O +3D O +lanes O +5 O +– O +12 O +). O + +Comparing O +Fig O +3C O +and O +D O +, O +the O ++ O +and O +− O +signs O +indicate O +where O +the O +deletion B-experimental_method +mutant I-experimental_method +assays I-experimental_method +led O +to O +a O +gain O +or O +loss O +of O +statically O +significant O +correlation O +, O +respectively O +. O + +Thus O +, O +in O +cluster O +2 O +, O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +substantially O +modulated O +the O +specificity O +of O +ligands O +with O +cell O +‐ O +specific O +activity O +( O +Fig O +3D O +lanes O +5 O +– O +12 O +). O + +For O +ligands O +in O +cluster O +3 O +, O +we O +could O +not O +eliminate O +a O +role O +for O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +in O +determining O +signaling O +specificity O +, O +since O +this O +cluster O +lacked O +positively O +correlated O +activity O +profiles O +( O +Fig O +3C O +), O +and O +deletion B-experimental_method +of I-experimental_method +the O +AB B-structure_element +or O +F B-structure_element +domain O +rarely O +induced O +such O +correlations O +( O +Fig O +3D O +), O +except O +for O +A B-chemical +‐ I-chemical +CD I-chemical +and O +OBHS B-chemical +‐ I-chemical +ASC I-chemical +analogs O +, O +where O +deletion B-experimental_method +of I-experimental_method +the O +AB B-structure_element +domain O +or O +F B-structure_element +domain O +led O +to O +positive O +correlations O +with O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O +and O +/ O +or O +GREB1 B-protein +levels O +( O +Fig O +3D O +lanes O +13 O +and O +18 O +). O + +Thus O +, O +ligands O +in O +cluster O +2 O +rely O +on O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +for O +both O +activity O +( O +Fig O +3B O +) O +and O +signaling O +specificity O +( O +Fig O +3D O +). O + +Ligand O +‐ O +specific O +control O +of O +GREB1 B-protein +expression O + +To O +determine O +whether O +ligand O +classes O +control O +expression O +of O +native O +ERα B-protein +target O +genes O +through O +the O +canonical O +linear O +signaling O +pathway O +, O +we O +performed O +pairwise B-experimental_method +linear I-experimental_method +regression I-experimental_method +analyses I-experimental_method +using O +ERα B-complex_assembly +– I-complex_assembly +NCOA1 I-complex_assembly +/ I-complex_assembly +2 I-complex_assembly +/ I-complex_assembly +3 I-complex_assembly +interactions O +in O +M2H B-experimental_method +assay I-experimental_method +as O +independent O +predictors O +of O +GREB1 B-protein +expression O +( O +the O +dependent O +variable O +) O +( O +Figs O +EV1 O +and O +EV2A O +, O +F O +– O +H O +). O + +In O +cluster O +1 O +, O +the O +recruitment O +of O +NCOA1 B-protein +and O +NCOA2 B-protein +was O +highest O +for O +WAY B-chemical +‐ I-chemical +C I-chemical +, O +followed O +by O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +, O +OBHS B-chemical +‐ I-chemical +N I-chemical +, O +and O +OBHS B-chemical +series O +, O +while O +for O +NCOA3 B-protein +, O +OBHS B-chemical +‐ I-chemical +N I-chemical +compounds O +induced O +the O +most O +recruitment O +and O +OBHS B-chemical +ligands O +were O +inverse O +agonists O +( O +Fig O +EV2F O +– O +H O +). O + +The O +average O +induction O +of O +GREB1 B-protein +by O +cluster O +1 O +ligands O +showed O +greater O +variance O +, O +with O +a O +range O +between O +~ O +25 O +and O +~ O +75 O +% O +for O +OBHS B-chemical +and O +a O +range O +from O +full O +agonist O +to O +inverse O +agonist O +for O +the O +others O +in O +cluster O +1 O +( O +Fig O +EV2A O +). O + +GREB1 B-protein +levels O +induced O +by O +OBHS B-chemical +analogs O +were O +determined O +by O +recruitment O +of O +NCOA1 B-protein +but O +not O +NCOA2 B-protein +/ I-protein +3 I-protein +( O +Fig O +3E O +lane O +1 O +), O +suggesting O +that O +there O +may O +be O +alternate O +or O +preferential O +use O +of O +these O +coactivators O +by O +different O +classes O +. O + +However O +, O +in O +cluster O +1 O +, O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +generally O +predicted O +GREB1 B-protein +levels O +( O +Fig O +3E O +lanes O +1 O +– O +4 O +), O +consistent O +with O +the O +canonical O +signaling O +model O +( O +Fig O +1D O +). O + +For O +clusters O +2 O +and O +3 O +, O +GREB1 B-protein +activity O +was O +generally O +not O +predicted O +by O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +. O + +Direct O +modulators O +showed O +low O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +( O +Fig O +EV2F O +– O +H O +), O +but O +only O +OBHS B-chemical +‐ I-chemical +ASC I-chemical +analogs O +had O +NCOA2 B-protein +recruitment O +profiles O +that O +predicted O +a O +full O +range O +of O +effects O +on O +GREB1 B-protein +levels O +( O +Figs O +3E O +lanes O +9 O +, O +11 O +, O +18 O +– O +19 O +, O +and O +EV2A O +). O + +The O +indirect O +modulators O +in O +clusters O +2 O +and O +3 O +stimulated O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +and O +GREB1 B-protein +expression O +with O +substantial O +variance O +( O +Figs O +3A O +and O +EV2F O +– O +H O +). O + +However O +, O +ligand O +‐ O +induced O +GREB1 B-protein +levels O +were O +generally O +not O +determined O +by O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +( O +Fig O +3E O +lanes O +5 O +– O +19 O +), O +consistent O +with O +an O +alternate O +causality O +model O +( O +Fig O +1E O +). O + +Out O +of O +11 O +indirect O +modulator O +series O +in O +cluster O +2 O +or O +3 O +, O +only O +the O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +class O +had O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +profiles O +that O +predicted O +GREB1 B-protein +levels O +( O +Fig O +3E O +lane O +12 O +). O + +These O +results O +suggest O +that O +compounds O +that O +show O +cell O +‐ O +specific O +signaling O +do O +not O +activate O +GREB1 B-protein +, O +or O +use O +coactivators O +other O +than O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +to O +control O +GREB1 B-protein +expression O +( O +Fig O +1E O +). O + +To O +determine O +mechanisms O +for O +ligand O +‐ O +dependent O +control O +of O +breast O +cancer O +cell O +proliferation O +, O +we O +performed O +linear B-experimental_method +regression I-experimental_method +analyses I-experimental_method +across O +the O +19 O +scaffolds O +using O +MCF O +‐ O +7 O +cell O +proliferation O +as O +the O +dependent O +variable O +, O +and O +the O +other O +activities O +as O +independent O +variables O +( O +Fig O +3F O +). O + +In O +cluster O +1 O +, O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +and O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activities O +, O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +, O +and O +GREB1 B-protein +levels O +generally O +predicted O +the O +proliferative O +response O +( O +Fig O +3F O +lanes O +2 O +– O +4 O +). O + +With O +the O +OBHS B-chemical +‐ I-chemical +N I-chemical +compounds O +, O +NCOA3 B-protein +and O +GREB1 B-protein +showed O +near O +perfect O +prediction O +of O +proliferation O +( O +Fig O +EV3G O +), O +with O +unexplained O +variance O +similar O +to O +the O +noise O +in O +the O +assays O +. O + +The O +lack O +of O +significant O +predictors O +for O +OBHS B-chemical +analogs O +( O +Fig O +3F O +lane O +1 O +) O +reflects O +their O +small O +range O +of O +proliferative O +effects O +on O +MCF O +‐ O +7 O +cells O +( O +Fig O +EV2I O +). O + +The O +significant O +correlations O +with O +GREB1 B-protein +expression O +and O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +observed O +in O +this O +cluster O +are O +consistent O +with O +the O +canonical O +signaling O +model O +( O +Fig O +1D O +), O +where O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +determines O +GREB1 B-protein +expression O +, O +which O +then O +drives O +proliferation O +. O + +Despite O +this O +phenotypic O +variance O +, O +proliferation O +was O +not O +generally O +predicted O +by O +correlated O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +and O +GREB1 B-protein +induction O +( O +Figs O +3F O +lanes O +5 O +– O +19 O +, O +and O +EV3H O +). O + +Out O +of O +15 O +ligand O +series O +in O +these O +clusters O +, O +only O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +induced O +a O +proliferative O +response O +that O +was O +predicted O +by O +GREB1 B-protein +levels O +, O +which O +were O +not O +determined O +by O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +( O +Fig O +3E O +and O +F O +lane O +10 O +). O + +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +, O +cyclofenil B-chemical +, O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +, O +and O +imidazopyridine B-chemical +analogs O +had O +NCOA1 B-protein +/ I-protein +3 I-protein +recruitment O +profiles O +that O +predicted O +their O +proliferative O +effects O +, O +without O +determining O +GREB1 B-protein +levels O +( O +Fig O +3E O +and O +F O +, O +lanes O +5 O +and O +14 O +– O +16 O +). O + +Similarly O +, O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +, O +cyclofenil B-chemical +‐ I-chemical +ASC I-chemical +, O +and O +OBHS B-chemical +‐ I-chemical +ASC I-chemical +had O +positively O +correlated O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +and O +GREB1 B-protein +levels O +, O +but O +none O +of O +these O +activities O +determined O +their O +proliferative O +effects O +( O +Fig O +3E O +and O +F O +lanes O +11 O +– O +12 O +and O +18 O +). O + +For O +ligands O +that O +show O +cell O +‐ O +specific O +signaling O +, O +ERα B-protein +‐ O +mediated O +recruitment O +of O +other O +coregulators O +and O +activation O +of O +other O +target O +genes O +likely O +determine O +their O +proliferative O +effects O +on O +MCF O +‐ O +7 O +cells O +. O + +NCOA3 B-protein +occupancy O +at O +GREB1 B-protein +did O +not O +predict O +the O +proliferative O +response O + +We O +also O +questioned O +whether O +promoter O +occupancy O +by O +coactivators O +is O +statistically O +robust O +and O +reproducible O +for O +ligand O +class O +analysis O +using O +a O +chromatin B-experimental_method +immunoprecipitation I-experimental_method +( I-experimental_method +ChIP I-experimental_method +)‐ I-experimental_method +based I-experimental_method +quantitative I-experimental_method +assay I-experimental_method +, I-experimental_method +and O +whether O +it O +has O +a O +better O +predictive O +power O +than O +the O +M2H B-experimental_method +assay I-experimental_method +. O + +ERα B-protein +and O +NCOA3 B-protein +cycle O +on O +and O +off O +the O +GREB1 B-protein +promoter O +( O +Nwachukwu O +et O +al O +, O +2014 O +). O + +Therefore O +, O +we O +first O +performed O +a O +time B-experimental_method +‐ I-experimental_method +course I-experimental_method +study I-experimental_method +, O +and O +found O +that O +E2 B-chemical +and O +the O +WAY B-chemical +‐ I-chemical +C I-chemical +analog O +, O +AAPII B-chemical +‐ I-chemical +151 I-chemical +‐ I-chemical +4 I-chemical +, O +induced O +recruitment O +of O +NCOA3 B-protein +to O +the O +GREB1 B-protein +promoter O +in O +a O +temporal O +cycle O +that O +peaked O +after O +45 O +min O +in O +MCF O +‐ O +7 O +cells O +( O +Fig O +4A O +). O + +At O +this O +time O +point O +, O +other O +WAY B-chemical +‐ I-chemical +C I-chemical +analogs O +also O +induced O +recruitment O +of O +NCOA3 B-protein +at O +this O +site O +to O +varying O +degrees O +( O +Fig O +4B O +). O + +The O +Z B-evidence +’ I-evidence +for O +this O +assay O +was O +0 O +. O +6 O +, O +showing O +statistical O +robustness O +( O +see O +Materials O +and O +Methods O +). O + +We O +prepared O +biological O +replicates O +with O +different O +cell O +passage O +numbers O +and O +separately O +prepared O +samples O +, O +which O +showed O +r B-evidence +2 I-evidence +of O +0 O +. O +81 O +, O +demonstrating O +high O +reproducibility O +( O +Fig O +4C O +). O + +NCOA3 B-protein +occupancy O +at O +GREB1 B-protein +is O +statistically O +robust O +but O +does O +not O +predict O +transcriptional O +activity O + +Kinetic B-experimental_method +ChIP I-experimental_method +assay I-experimental_method +examining O +recruitment O +of O +NCOA3 B-protein +to O +the O +GREB1 B-protein +gene O +in O +MCF O +‐ O +7 O +cells O +stimulated O +with O +E2 B-chemical +or O +the O +indicated O +WAY B-chemical +‐ I-chemical +C I-chemical +analog O +. O + +NCOA3 B-protein +occupancy O +at O +GREB1 B-protein +was O +compared O +by O +ChIP B-experimental_method +assay I-experimental_method +45 O +min O +after O +stimulation O +with O +vehicle O +, O +E2 B-chemical +, O +or O +the O +WAY B-chemical +‐ I-chemical +C I-chemical +analogs O +. O + +In O +panel O +( O +B O +), O +the O +average O +recruitment O +of O +two O +biological O +replicates O +are O +shown O +as O +mean O ++ O +SEM O +, O +and O +the O +Z B-evidence +‐ I-evidence +score I-evidence +is O +indicated O +. O + +In O +panel O +( O +C O +), O +correlation B-experimental_method +analysis I-experimental_method +was O +performed O +for O +two O +biological O +replicates O +. O + +Linear B-experimental_method +regression I-experimental_method +analyses I-experimental_method +comparing O +the O +ability O +of O +NCOA3 B-protein +recruitment O +, O +measured O +by O +ChIP B-experimental_method +or O +M2H B-experimental_method +, O +to O +predict O +other O +agonist O +activities O +of O +WAY B-chemical +‐ I-chemical +C I-chemical +analogs O +. O +* O +Significant O +positive O +correlation O +( O +F B-experimental_method +‐ I-experimental_method +test I-experimental_method +for O +nonzero O +slope O +, O +P B-evidence +‐ I-evidence +value I-evidence +). O + +The O +M2H B-experimental_method +assay I-experimental_method +for O +NCOA3 B-protein +recruitment O +broadly O +correlated O +with O +the O +other O +assays O +, O +and O +was O +predictive O +for O +GREB1 B-protein +expression O +and O +cell O +proliferation O +( O +Fig O +3E O +). O + +However O +, O +the O +ChIP B-experimental_method +assays I-experimental_method +for O +WAY B-chemical +‐ I-chemical +C I-chemical +‐ O +induced O +recruitment O +of O +NCOA3 B-protein +to O +the O +GREB1 B-protein +promoter O +did O +not O +correlate O +with O +any O +of O +the O +other O +WAY B-chemical +‐ I-chemical +C I-chemical +activity O +profiles O +( O +Fig O +4D O +), O +although O +the O +positive O +correlation O +between O +ChIP B-experimental_method +assays I-experimental_method +and O +NCOA3 B-protein +recruitment O +via O +M2H B-experimental_method +assay I-experimental_method +showed O +a O +trend O +toward O +significance O +with O +r B-evidence +2 I-evidence += O +0 O +. O +36 O +and O +P B-evidence += O +0 O +. O +09 O +( O +F B-experimental_method +‐ I-experimental_method +test I-experimental_method +for O +nonzero O +slope O +). O + +Thus O +, O +the O +simplified O +coactivator B-experimental_method +‐ I-experimental_method +binding I-experimental_method +assay I-experimental_method +showed O +much O +greater O +predictive O +power O +than O +the O +ChIP B-experimental_method +assay I-experimental_method +for O +ligand O +‐ O +specific O +effects O +on O +GREB1 B-protein +expression O +and O +cell O +proliferation O +. O + +ERβ B-protein +activity O +is O +not O +an O +independent O +predictor O +of O +cell O +‐ O +specific O +activity O + +One O +difference O +between O +MCF O +‐ O +7 O +breast O +cancer O +cells O +and O +Ishikawa O +endometrial O +cancer O +cells O +is O +the O +contribution O +of O +ERβ B-protein +to O +estrogenic O +response O +, O +as O +Ishikawa O +cells O +may O +express O +ERβ B-protein +( O +Bhat O +& O +Pezzuto O +, O +2001 O +). O + +When O +overexpressed B-experimental_method +in O +MCF O +‐ O +7 O +cells O +, O +ERβ B-protein +alters O +E2 B-chemical +‐ O +induced O +expression O +of O +only O +a O +subset O +of O +ERα B-protein +‐ O +target O +genes O +( O +Wu O +et O +al O +, O +2011 O +), O +raising O +the O +possibility O +that O +ligand O +‐ O +induced O +ERβ B-protein +activity O +may O +contribute O +to O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activities O +, O +and O +thus O +underlie O +the O +lack O +of O +correlation O +between O +the O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +and O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +activities O +or O +GREB1 B-protein +levels O +induced O +by O +cell O +‐ O +specific O +modulators O +in O +cluster O +2 O +and O +cluster O +3 O +( O +Fig O +3C O +). O + +To O +test O +this O +idea O +, O +we O +determined O +the O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERβ O +activity O +profiles O +of O +the O +ligands O +( O +Fig O +EV1 O +). O + +All O +direct O +modulator O +and O +two O +indirect O +modulator O +scaffolds O +( O +OBHS B-chemical +and O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +) O +lacked O +ERβ O +agonist O +activity O +. O + +For O +most O +scaffolds O +, O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERβ O +and O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activities O +were O +not O +correlated O +, O +except O +for O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +and O +cyclofenil B-chemical +analogs O +, O +which O +showed O +moderate O +but O +significant O +correlations O +( O +Fig O +EV4A O +). O + +Nevertheless O +, O +the O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activities O +of O +both O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +and O +cyclofenil B-chemical +analogs O +were O +better O +predicted O +by O +their O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +than O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERβ B-protein +activities O +( O +Fig O +EV4A O +and O +B O +). O + +ERβ B-protein +activity O +is O +not O +an O +independent O +predictor O +of O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O + +ERβ B-protein +activity O +in O +HepG2 O +cells O +rarely O +correlates O +with O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O +. O + +ERα B-protein +activity O +of O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +and O +cyclofenil B-chemical +analogs O +correlates O +with O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O +. O + +Data O +information O +: O +The O +r O +2 O +and O +P B-evidence +values I-evidence +for O +the O +indicated O +correlations O +are O +shown O +in O +both O +panels O +. O +* O +Significant O +positive O +correlation O +( O +F B-experimental_method +‐ I-experimental_method +test I-experimental_method +for O +nonzero O +slope O +, O +P B-evidence +‐ I-evidence +value I-evidence +) O + +To O +overcome O +barriers O +to O +crystallization B-experimental_method +of O +ERα B-protein +LBD B-structure_element +complexes O +, O +we O +developed O +a O +conformation B-experimental_method +‐ I-experimental_method +trapping I-experimental_method +X I-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +approach O +using O +the O +ERα B-mutant +‐ I-mutant +Y537S I-mutant +mutation O +( O +Nettles O +et O +al O +, O +2008 O +; O +Bruning O +et O +al O +, O +2010 O +; O +Srinivasan O +et O +al O +, O +2013 O +). O + +To O +further O +validate O +this O +approach O +, O +we O +solved B-experimental_method +the O +structure B-evidence +of O +the O +ERα B-mutant +‐ I-mutant +Y537S I-mutant +LBD B-structure_element +in B-protein_state +complex I-protein_state +with I-protein_state +diethylstilbestrol B-chemical +( O +DES B-chemical +), O +which O +bound O +identically O +in O +the O +wild B-protein_state +‐ I-protein_state +type I-protein_state +and O +ERα B-mutant +‐ I-mutant +Y537S I-mutant +LBDs B-structure_element +, O +demonstrating O +again O +that O +this O +surface O +mutation O +stabilizes O +h12 B-structure_element +dynamics O +to O +facilitate O +crystallization O +without O +changing O +ligand O +binding O +( O +Appendix O +Fig O +S1A O +and O +B O +) O +( O +Nettles O +et O +al O +, O +2008 O +; O +Bruning O +et O +al O +, O +2010 O +; O +Delfosse O +et O +al O +, O +2012 O +). O + +Using O +this O +approach O +, O +we O +solved B-experimental_method +76 O +ERα B-protein +LBD B-structure_element +structures B-evidence +in O +the O +active B-protein_state +conformation I-protein_state +and O +bound B-protein_state +to I-protein_state +ligands I-protein_state +studied O +here O +( O +Appendix O +Fig O +S1C O +). O + +Eleven O +of O +these O +structures B-evidence +have O +been O +published O +, O +while O +65 O +are O +new O +, O +including O +the O +DES B-protein_state +‐ I-protein_state +bound I-protein_state +ERα B-mutant +‐ I-mutant +Y537S I-mutant +LBD B-structure_element +. O + +We O +present O +57 O +of O +these O +new O +structures B-evidence +here O +( O +Dataset O +EV2 O +), O +while O +the O +remaining O +eight O +new O +structures B-evidence +bound B-protein_state +to I-protein_state +OBHS B-chemical +‐ I-chemical +N I-chemical +analogs O +will O +be O +published O +elsewhere O +( O +S O +. O +Srinivasan O +et O +al O +, O +in O +preparation O +). O + +Examining O +many O +closely O +related O +structures B-evidence +allows O +us O +to O +visualize O +subtle O +structural O +differences O +, O +in O +effect O +using O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +as O +a O +systems O +biology O +tool O +. O + +The O +indirect O +modulator O +scaffolds O +in O +cluster O +1 O +did O +not O +show O +cell O +‐ O +specific O +signaling O +( O +Fig O +3C O +), O +but O +shared O +common O +structural O +perturbations O +that O +we O +designed O +to O +modulate O +h12 B-structure_element +dynamics O +. O + +Based O +on O +our O +original O +OBHS B-chemical +structure B-evidence +, O +the O +OBHS B-chemical +, O +OBHS B-chemical +‐ I-chemical +N I-chemical +, O +and O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +compounds O +were O +modified O +with O +h11 B-structure_element +‐ O +directed O +pendant O +groups O +( O +Zheng O +et O +al O +, O +2012 O +; O +Zhu O +et O +al O +, O +2012 O +; O +Liao O +et O +al O +, O +2014 O +). O + +Superposing B-experimental_method +the O +LBDs B-structure_element +based O +on O +the O +class O +of O +bound O +ligands O +provides O +an O +ensemble O +view O +of O +the O +structural O +variance O +and O +clarifies O +what O +part O +of O +the O +ligand B-site +‐ I-site +binding I-site +pocket I-site +is O +differentially O +perturbed O +or O +targeted O +. O + +The O +24 O +structures B-evidence +containing O +OBHS B-chemical +, O +OBHS B-chemical +‐ I-chemical +N I-chemical +, O +or O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +showed O +structural O +diversity O +in O +the O +same O +part O +of O +the O +scaffolds O +( O +Figs O +5A O +and O +EV5A O +), O +and O +the O +same O +region O +of O +the O +LBD B-structure_element +— O +the O +C O +‐ O +terminal O +end O +of O +h11 B-structure_element +( O +Figs O +5B O +and O +C O +, O +and O +EV5B O +), O +which O +in O +turn O +nudges O +h12 B-structure_element +( O +Fig O +5C O +and O +D O +). O + +We O +observed O +that O +the O +OBHS B-chemical +‐ I-chemical +N I-chemical +analogs O +displaced O +h11 B-structure_element +along O +a O +vector O +away O +from O +Leu354 B-residue_name_number +in O +a O +region O +of O +h3 B-structure_element +that O +is O +unaffected O +by O +the O +ligands O +, O +and O +toward O +the O +dimer B-site +interface I-site +. O + +For O +the O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +, O +the O +displacement O +of O +h11 B-structure_element +was O +in O +a O +perpendicular O +direction O +, O +away O +from O +Ile424 B-residue_name_number +in O +h8 B-structure_element +and O +toward O +h12 B-structure_element +. O + +Remarkably O +, O +these O +individual O +inter B-evidence +‐ I-evidence +atomic I-evidence +distances I-evidence +showed O +a O +ligand O +class O +‐ O +specific O +ability O +to O +significantly O +predict O +proliferative O +effects O +( O +Fig O +5E O +and O +F O +), O +demonstrating O +the O +feasibility O +of O +developing O +a O +minimal O +set O +of O +activity O +predictors O +from O +crystal B-evidence +structures I-evidence +. O + +Structure B-experimental_method +‐ I-experimental_method +class I-experimental_method +analysis I-experimental_method +of O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +. O + +Triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +bound B-protein_state +to I-protein_state +the O +superposed B-experimental_method +crystal B-evidence +structures I-evidence +of O +the O +ERα B-protein +LBD B-structure_element +are O +shown O +. O + +Arrows O +indicate O +chemical O +variance O +in O +the O +orientation O +of O +the O +different O +h11 B-structure_element +‐ O +directed O +ligand O +side O +groups O +( O +PDB O +5DK9 O +, O +5DKB O +, O +5DKE O +, O +5DKG O +, O +5DKS O +, O +5DL4 O +, O +5DLR O +, O +5DMC O +, O +5DMF O +and O +5DP0 O +). O + +Triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +induce O +variance O +of O +ERα B-protein +conformations O +at O +the O +C O +‐ O +terminal O +region O +of O +h11 B-structure_element +. O + +Panel O +( O +B O +) O +shows O +the O +crystal B-evidence +structure I-evidence +of O +a O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analog O +‐ O +bound O +ERα B-protein +LBD B-structure_element +( O +PDB O +5DLR O +). O + +The O +h11 B-site +– I-site +h12 I-site +interface I-site +( O +circled O +) O +includes O +the O +C O +‐ O +terminal O +part O +of O +h11 B-structure_element +. O + +This O +region O +was O +expanded O +in O +panel O +( O +C O +), O +where O +the O +10 O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analog O +‐ O +bound O +ERα B-protein +LBD B-structure_element +structures B-evidence +( O +see O +Datasets O +EV1 O +and O +EV2 O +) O +were O +superposed B-experimental_method +to O +show O +variations O +in O +the O +h11 B-structure_element +C O +‐ O +terminus O +( O +PDB O +5DK9 O +, O +5DKB O +, O +5DKE O +, O +5DKG O +, O +5DKS O +, O +5DL4 O +, O +5DLR O +, O +5DMC O +, O +5DMF O +, O +and O +5DP0 O +). O + +ERα B-protein +LBDs B-structure_element +in B-protein_state +complex I-protein_state +with I-protein_state +diethylstilbestrol B-chemical +( O +DES B-chemical +) O +or O +a O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analog O +were O +superposed B-experimental_method +to O +show O +that O +the O +ligand O +‐ O +induced O +difference O +in O +h11 B-structure_element +conformation O +is O +transmitted O +to O +the O +C O +‐ O +terminus O +of O +h12 B-structure_element +( O +PDB O +4ZN7 O +, O +5DMC O +). O + +Inter B-evidence +‐ I-evidence +atomic I-evidence +distances I-evidence +predict O +the O +proliferative O +effects O +of O +specific O +ligand O +series O +. O + +Ile424 B-residue_name_number +– O +His524 B-residue_name_number +distance B-evidence +measured O +in O +the O +crystal B-evidence +structures I-evidence +correlates O +with O +the O +proliferative O +effect O +of O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +in O +MCF O +‐ O +7 O +cells O +. O + +In O +contrast O +, O +the O +Leu354 B-residue_name_number +– O +Leu525 B-residue_name_number +distance B-evidence +correlates O +with O +the O +proliferative O +effects O +of O +OBHS B-chemical +‐ I-chemical +N I-chemical +analogs O +in O +MCF O +‐ O +7 O +cells O +. O + +Structure B-experimental_method +‐ I-experimental_method +class I-experimental_method +analysis I-experimental_method +of O +WAY B-chemical +‐ I-chemical +C I-chemical +analogs O +. O + +WAY B-chemical +‐ I-chemical +C I-chemical +side O +groups O +subtly O +nudge O +h12 B-structure_element +Leu540 B-residue_name_number +. O + +ERα B-protein +LBD B-structure_element +structures B-evidence +bound B-protein_state +to I-protein_state +4 O +distinct O +WAY B-chemical +‐ I-chemical +C I-chemical +analogs O +were O +superposed B-experimental_method +( O +PDB O +4 O +IU7 O +, O +4IV4 O +, O +4IVW O +, O +4IW6 O +) O +( O +see O +Datasets O +EV1 O +and O +EV2 O +). O + +Structure B-experimental_method +‐ I-experimental_method +class I-experimental_method +analysis I-experimental_method +of O +indirect O +modulators O + +Structure B-experimental_method +‐ I-experimental_method +class I-experimental_method +analysis I-experimental_method +of O +indirect O +modulators O +in O +cluster O +1 O +. O + +Crystal B-evidence +structures I-evidence +of O +the O +ERα B-protein +LBD B-structure_element +bound B-protein_state +to I-protein_state +OBHS B-chemical +and O +OBHS B-chemical +‐ I-chemical +N I-chemical +analogs O +were O +superposed B-experimental_method +. O + +Arrows O +indicate O +chemical O +variance O +in O +the O +orientation O +of O +the O +different O +h11 B-structure_element +‐ O +directed O +ligand O +side O +groups O +. O + +Panel O +( O +B O +) O +shows O +the O +ligand O +‐ O +induced O +conformational O +variation O +at O +the O +C O +‐ O +terminal O +region O +of O +h11 B-structure_element +( O +OBHS B-chemical +: O +PDB O +4ZN9 O +, O +4ZNH O +, O +4ZNS O +, O +4ZNT O +, O +4ZNU O +, O +4ZNV O +, O +and O +4ZNW O +; O +OBHS B-chemical +‐ I-chemical +N I-chemical +: O +PDB O +4ZUB O +, O +4ZUC O +, O +4ZWH O +, O +4ZWK O +, O +5BNU O +, O +5BP6 O +, O +5BPR O +, O +and O +5BQ4 O +). O + +Structure B-experimental_method +‐ I-experimental_method +class I-experimental_method +analysis I-experimental_method +of O +indirect O +modulators O +in O +clusters O +2 O +and O +3 O +. O + +Crystal B-evidence +structures I-evidence +of O +the O +ERα B-protein +LBD B-structure_element +bound B-protein_state +to I-protein_state +ligands O +with O +cell O +‐ O +specific O +activities O +were O +superposed B-experimental_method +. O + +The O +bound O +ligands O +are O +shown O +, O +and O +arrows O +indicate O +considerable O +variation O +in O +the O +orientation O +of O +the O +different O +h3 B-structure_element +‐, O +h8 B-structure_element +‐, O +h11 B-structure_element +‐, O +or O +h12 B-structure_element +‐ O +directed O +ligand O +side O +groups O +. O + +As O +visualized O +in O +four O +LBD B-structure_element +structures B-evidence +( O +Srinivasan O +et O +al O +, O +2013 O +), O +WAY B-chemical +‐ I-chemical +C I-chemical +analogs O +were O +designed O +with O +small O +substitutions O +that O +slightly O +nudge O +h12 B-structure_element +Leu540 B-residue_name_number +, O +without O +exiting O +the O +ligand B-site +‐ I-site +binding I-site +pocket I-site +( O +Fig O +5G O +and O +H O +). O + +Therefore O +, O +changing O +h12 B-structure_element +dynamics O +maintains O +the O +canonical O +signaling O +pathway O +defined O +by O +E2 B-chemical +( O +Fig O +1D O +) O +to O +support O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +‐ O +driven O +signaling O +and O +recruit O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +for O +GREB1 B-protein +‐ O +stimulated O +proliferation O +. O + +Ligands O +with O +cell O +‐ O +specific O +activity O +alter O +the O +shape O +of O +the O +AF B-site +‐ I-site +2 I-site +surface I-site + +Direct O +modulators O +like O +tamoxifen B-chemical +drive O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +‐ O +dependent O +cell O +‐ O +specific O +activity O +by O +completely O +occluding O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +, O +but O +it O +is O +not O +known O +how O +indirect O +modulators O +produce O +cell O +‐ O +specific O +ERα B-protein +activity O +. O + +Therefore O +, O +we O +examined O +another O +50 O +LBD B-structure_element +structures B-evidence +containing O +ligands O +in O +clusters O +2 O +and O +3 O +. O + +These O +structures B-evidence +demonstrated O +that O +cell O +‐ O +specific O +activity O +derived O +from O +altering O +the O +shape O +of O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +without O +an O +extended O +side O +chain O +. O + +Ligands O +in O +cluster O +2 O +and O +cluster O +3 O +showed O +conformational O +heterogeneity O +in O +parts O +of O +the O +scaffold O +that O +were O +directed O +toward O +multiple O +regions O +of O +the O +receptor O +including O +h3 B-structure_element +, O +h8 B-structure_element +, O +h11 B-structure_element +, O +h12 B-structure_element +, O +and O +/ O +or O +the O +β B-structure_element +‐ I-structure_element +sheets I-structure_element +( O +Fig O +EV5C O +– O +G O +). O + +For O +instance O +, O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +2 I-chemical +and O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +analogs O +( O +Fig O +2 O +) O +had O +similar O +ERα B-protein +activity O +profiles O +in O +the O +different O +cell O +types O +( O +Fig O +EV2A O +– O +C O +), O +but O +the O +2 O +‐ O +versus O +3 O +‐ O +methyl O +substituted O +phenol O +rings O +altered O +the O +correlated O +signaling O +patterns O +in O +different O +cell O +types O +( O +Fig O +3B O +lanes O +7 O +and O +12 O +). O + +This O +difference O +in O +ligand O +positioning O +altered O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +via O +a O +shift O +in O +the O +N O +‐ O +terminus O +of O +h12 B-structure_element +, O +which O +directly O +contacts O +the O +coactivator O +. O + +This O +effect O +is O +evident O +in O +a O +single O +structure B-evidence +due O +to O +its O +1 O +Å O +magnitude O +( O +Fig O +6A O +and O +B O +). O + +The O +shifts O +in O +h12 B-structure_element +residues O +Asp538 B-residue_name_number +and O +Leu539 B-residue_name_number +led O +to O +rotation O +of O +the O +coactivator O +peptide O +( O +Fig O +6C O +). O + +Thus O +, O +cell O +‐ O +specific O +activity O +can O +stem O +from O +perturbation O +of O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +without O +an O +extended O +side O +chain O +, O +which O +presumably O +alters O +the O +receptor O +– O +coregulator O +interaction O +profile O +. O + +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +2 I-chemical +/ I-chemical +3 I-chemical +analogs O +subtly O +distort O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +. O + +Panel O +( O +A O +) O +shows O +the O +crystal B-evidence +structure I-evidence +of O +an O +S B-protein_state +‐ I-protein_state +OBHS I-protein_state +‐ I-protein_state +3 I-protein_state +‐ I-protein_state +bound I-protein_state +ERα B-protein +LBD B-structure_element +( O +PDB O +5DUH O +). O + +The O +h3 B-site +– I-site +h12 I-site +interface I-site +( O +circled O +) O +at O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +( O +pink O +) O +was O +expanded O +in O +panels O +( O +B O +, O +C O +). O + +The O +S B-protein_state +‐ I-protein_state +OBHS I-protein_state +‐ I-protein_state +2 I-protein_state +/ I-protein_state +3 I-protein_state +‐ I-protein_state +bound I-protein_state +ERα B-protein +LBDs B-structure_element +were O +superposed B-experimental_method +to O +show O +shifts O +in O +h3 B-structure_element +( O +panel O +B O +) O +and O +the O +NCOA2 B-protein +peptide O +docked O +at O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +( O +panel O +C O +). O + +Crystal B-evidence +structures I-evidence +show O +that O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +shift O +h3 B-structure_element +and O +h11 B-structure_element +further O +apart O +compared O +to O +an O +A O +‐ O +CD O +‐ O +ring O +estrogen B-chemical +( O +PDB O +4PPS O +, O +5DRM O +, O +5DRJ O +). O + +The O +2F O +o O +‐ O +F O +c O +electron O +density O +map O +and O +F O +o O +‐ O +F O +c O +difference O +map O +of O +a O +2 B-protein_state +, I-protein_state +5 I-protein_state +‐ I-protein_state +DTP I-protein_state +‐ I-protein_state +bound I-protein_state +structure B-evidence +( O +PDB O +5DRJ O +) O +were O +contoured O +at O +1 O +. O +0 O +sigma O +and O +± O +3 O +. O +0 O +sigma O +, O +respectively O +. O + +Average O +( O +mean O ++ O +SEM O +) O +α B-evidence +‐ I-evidence +carbon I-evidence +distance I-evidence +measured O +from O +h3 B-structure_element +Thr347 B-residue_name_number +to O +h11 B-structure_element +Leu525 B-residue_name_number +of O +A B-protein_state +‐ I-protein_state +CD I-protein_state +‐, I-protein_state +2 I-protein_state +, I-protein_state +5 I-protein_state +‐ I-protein_state +DTP I-protein_state +‐, I-protein_state +and I-protein_state +3 I-protein_state +, I-protein_state +4 I-protein_state +‐ I-protein_state +DTPD I-protein_state +‐ I-protein_state +bound I-protein_state +ERα B-protein +LBDs B-structure_element +. O + +* O +Two O +‐ O +tailed O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +‐ I-experimental_method +test I-experimental_method +, O +P B-evidence += O +0 O +. O +002 O +( O +PDB O +A B-chemical +‐ I-chemical +CD I-chemical +: O +5DI7 O +, O +5DID O +, O +5DIE O +, O +5DIG O +, O +and O +4PPS O +; O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +: O +4IWC O +, O +5DRM O +, O +and O +5DRJ O +; O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +: O +5DTV O +and O +5DU5 O +). O + +Crystal B-evidence +structures I-evidence +show O +that O +a O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +analog O +shifts O +h3 B-structure_element +( O +F B-structure_element +) O +and O +the O +NCOA2 B-protein +( O +G O +) O +peptide O +compared O +to O +an O +A B-chemical +‐ I-chemical +CD I-chemical +‐ O +ring O +estrogen B-chemical +( O +PDB O +4PPS O +, O +5DTV O +). O + +Hierarchical B-experimental_method +clustering I-experimental_method +of O +ligand O +‐ O +specific O +binding O +of O +154 O +interacting O +peptides O +to O +the O +ERα B-protein +LBD B-structure_element +was O +performed O +in O +triplicate O +by O +MARCoNI B-experimental_method +analysis I-experimental_method +. O + +The O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +showed O +perturbation O +of O +h11 B-structure_element +, O +as O +well O +as O +h3 B-structure_element +, O +which O +forms O +part O +of O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +. O + +These O +compounds O +bind O +the O +LBD B-structure_element +in O +an O +unusual O +fashion O +because O +they O +have O +a O +phenol O +‐ O +to O +‐ O +phenol O +length O +of O +~ O +12 O +Å O +, O +which O +is O +longer O +than O +steroids O +and O +other O +prototypical O +ERα B-protein +agonists O +that O +are O +~ O +10 O +Å O +in O +length O +. O + +One O +phenol O +pushed O +further O +toward O +h3 B-structure_element +( O +Fig O +6D O +), O +while O +the O +other O +phenol O +pushed O +toward O +the O +C O +‐ O +terminus O +of O +h11 B-structure_element +to O +a O +greater O +extent O +than O +A B-chemical +‐ I-chemical +CD I-chemical +‐ O +ring O +estrogens B-chemical +( O +Nwachukwu O +et O +al O +, O +2014 O +), O +which O +are O +close O +structural O +analogs O +of O +E2 B-chemical +that O +lack O +a O +B O +‐ O +ring O +( O +Fig O +2 O +). O + +To O +quantify O +this O +difference O +, O +we O +compared O +the O +distance B-evidence +between O +α O +‐ O +carbons O +at O +h3 B-structure_element +Thr347 B-residue_name_number +and O +h11 B-structure_element +Leu525 B-residue_name_number +in O +the O +set O +of O +structures B-evidence +containing O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +( O +n O += O +3 O +) O +or O +A B-chemical +‐ I-chemical +CD I-chemical +‐ O +ring O +analogs O +( O +n O += O +5 O +) O +( O +Fig O +6E O +). O + +We O +observed O +a O +difference O +of O +0 O +. O +4 O +Å O +that O +was O +significant O +( O +two O +‐ O +tailed O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +‐ I-experimental_method +test I-experimental_method +, O +P B-evidence += O +0 O +. O +002 O +) O +due O +to O +the O +very O +tight O +clustering O +of O +the O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +‐ O +induced O +LBD B-structure_element +conformation O +. O + +The O +shifts O +in O +h3 B-structure_element +suggest O +these O +compounds O +are O +positioned O +to O +alter O +coregulator O +preferences O +. O + +The O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +and O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +scaffolds O +are O +isomeric O +, O +but O +with O +aryl O +groups O +at O +obtuse O +and O +acute O +angles O +, O +respectively O +( O +Fig O +2 O +). O + +The O +crystal B-evidence +structure I-evidence +of O +ERα B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +a O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +is O +unknown O +; O +however O +, O +we O +solved B-experimental_method +two O +crystal B-evidence +structures I-evidence +of O +ERα B-protein +bound B-protein_state +to I-protein_state +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +analogs O +and O +one O +structure B-evidence +containing O +a O +furan B-chemical +ligand O +— O +all O +of O +which O +have O +a O +3 O +, O +4 O +‐ O +diaryl O +configuration O +( O +Fig O +2 O +; O +Datasets O +EV1 O +and O +EV2 O +). O + +In O +these O +structures B-evidence +, O +the O +A O +‐ O +ring O +mimetic O +of O +the O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +scaffold O +bound O +h3 B-structure_element +Glu353 B-residue_name_number +as O +expected O +, O +but O +the O +other O +phenol O +wrapped O +around O +h3 B-structure_element +to O +form O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +Thr347 B-residue_name_number +, O +indicating O +a O +change O +in O +binding O +epitopes O +in O +the O +ERα B-protein +ligand B-site +‐ I-site +binding I-site +pocket I-site +( O +Fig O +6F O +). O + +The O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +analogs O +also O +induced O +a O +shift O +in O +h3 B-structure_element +positioning O +, O +which O +translated O +again O +into O +a O +shift O +in O +the O +bound O +coactivator O +peptide O +( O +Fig O +6F O +). O + +Therefore O +, O +these O +indirect O +modulators O +, O +including O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +2 I-chemical +, O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +, O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +, O +and O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +analogs O +— O +all O +of O +which O +show O +cell O +‐ O +specific O +activity O +profiles O +— O +induced O +shifts O +in O +h3 B-structure_element +and O +h12 B-structure_element +that O +were O +transmitted O +to O +the O +coactivator O +peptide O +via O +an O +altered O +AF B-site +‐ I-site +2 I-site +surface I-site +. O + +To O +test O +whether O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +shows O +changes O +in O +shape O +in O +solution O +, O +we O +used O +the O +microarray B-experimental_method +assay I-experimental_method +for I-experimental_method +real I-experimental_method +‐ I-experimental_method +time I-experimental_method +coregulator I-experimental_method +– I-experimental_method +nuclear I-experimental_method +receptor I-experimental_method +interaction I-experimental_method +( O +MARCoNI B-experimental_method +) O +analysis O +( O +Aarts O +et O +al O +, O +2013 O +). O + +Here O +, O +the O +ligand O +‐ O +dependent O +interactions O +of O +the O +ERα B-protein +LBD B-structure_element +with O +over O +150 O +distinct O +LxxLL B-structure_element +motif I-structure_element +peptides O +were O +assayed O +to O +define O +structural O +fingerprints O +for O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +, O +in O +a O +manner O +similar O +to O +the O +use O +of O +phage B-experimental_method +display I-experimental_method +peptides I-experimental_method +as O +structural O +probes O +( O +Connor O +et O +al O +, O +2001 O +). O + +Despite O +the O +similar O +average O +activities O +of O +these O +ligand O +classes O +( O +Fig O +3A O +and O +B O +), O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +and O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +displayed O +remarkably O +different O +peptide O +recruitment O +patterns O +( O +Fig O +6H O +), O +consistent O +with O +the O +structural B-experimental_method +analyses I-experimental_method +. O + +Hierarchical B-experimental_method +clustering I-experimental_method +revealed O +that O +many O +of O +the O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +recapitulated O +most O +of O +the O +peptide O +recruitment O +and O +dismissal O +patterns O +observed O +with O +E2 B-chemical +( O +Fig O +6H O +). O + +However O +, O +there O +was O +a O +unique O +cluster O +of O +peptides O +that O +were O +recruited O +by O +E2 B-chemical +but O +not O +the O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +. O + +In O +contrast O +, O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +dismissed O +most O +of O +the O +peptides O +from O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +( O +Fig O +6H O +). O + +Thus O +, O +the O +isomeric O +attachment O +of O +diaryl O +groups O +to O +the O +thiophene B-chemical +core O +changed O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +from O +inside O +the O +ligand B-site +‐ I-site +binding I-site +pocket I-site +, O +as O +predicted O +by O +the O +crystal B-evidence +structures I-evidence +. O + +Together O +, O +these O +findings O +suggest O +that O +without O +an O +extended O +side O +chain O +, O +cell O +‐ O +specific O +activity O +stems O +from O +different O +coregulator O +recruitment O +profiles O +, O +due O +to O +unique O +ligand O +‐ O +induced O +conformations O +of O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +, O +in O +addition O +to O +differential O +usage O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +. O + +Indirect O +modulators O +in O +cluster O +1 O +avoid O +this O +by O +perturbing O +the O +h11 B-site +– I-site +h12 I-site +interface I-site +, O +and O +modulating O +the O +dynamics O +of O +h12 B-structure_element +without O +changing O +the O +shape O +of O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +when O +stabilized O +. O + +Our O +goal O +was O +to O +identify O +a O +minimal O +set O +of O +predictors O +that O +would O +link O +specific O +structural O +perturbations O +to O +ERα B-protein +signaling O +pathways O +that O +control O +cell O +‐ O +specific O +signaling O +and O +proliferation O +. O + +We O +found O +a O +very O +strong O +set O +of O +predictors O +, O +where O +ligands O +in O +cluster O +1 O +, O +defined O +by O +similar O +signaling O +across O +cell O +types O +, O +showed O +indirect O +modulation O +of O +h12 B-structure_element +dynamics O +via O +the O +h11 B-site +– I-site +12 I-site +interface I-site +or O +slight O +contact O +with O +h12 B-structure_element +. O + +This O +perturbation O +determined O +proliferation O +that O +correlated O +strongly O +with O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +activity O +, O +recruitment O +of O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +family O +members O +, O +and O +induction O +of O +the O +GREB1 B-protein +gene O +, O +consistent O +with O +the O +canonical O +ERα B-protein +signaling O +pathway O +( O +Fig O +1D O +). O + +For O +ligands O +in O +cluster O +1 O +, O +deletion B-experimental_method +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +reduced O +activity O +to O +varying O +degrees O +, O +but O +did O +not O +change O +the O +underlying O +signaling O +patterns O +established O +through O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +. O + +In O +contrast O +, O +an O +extended O +side O +chain O +designed O +to O +directly O +reposition O +h12 B-structure_element +and O +completely O +disrupt O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +results O +in O +cell O +‐ O +specific O +signaling O +. O + +Compared O +to O +cluster O +1 O +, O +the O +structural O +rules O +are O +less O +clear O +in O +clusters O +2 O +and O +3 O +, O +but O +a O +number O +of O +indirect O +modulator O +classes O +perturbed O +the O +LBD B-structure_element +conformation O +at O +the O +intersection O +of O +h3 B-structure_element +, O +the O +h12 B-structure_element +N O +‐ O +terminus O +, O +and O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +. O + +Ligands O +in O +these O +classes O +altered O +the O +shape O +of O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +to O +affect O +coregulator O +preferences O +. O + +For O +direct O +and O +indirect O +modulators O +in O +cluster O +2 O +or O +3 O +, O +the O +canonical O +ERα B-protein +signaling O +pathway O +involving O +recruitment O +of O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +and O +induction O +of O +GREB1 B-protein +did O +not O +generally O +predict O +their O +proliferative O +effects O +, O +indicating O +an O +alternate O +causal O +model O +( O +Fig O +1E O +). O + +These O +principles O +outlined O +above O +provide O +a O +structural O +basis O +for O +how O +the O +ligand B-site +– I-site +receptor I-site +interface I-site +leads O +to O +different O +signaling O +specificities O +through O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +and O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +. O + +It O +is O +noteworthy O +that O +regulation O +of O +h12 B-structure_element +dynamics O +indirectly O +through O +h11 B-structure_element +can O +virtually O +abolish O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +activity O +, O +and O +yet O +still O +drive O +robust O +transcriptional O +activity O +through O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +, O +as O +demonstrated O +with O +the O +OBHS B-chemical +series O +. O + +This O +finding O +can O +be O +explained O +by O +the O +fact O +that O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +contain O +distinct O +binding B-site +sites I-site +for O +interaction O +with O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +and O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +( O +McInerney O +et O +al O +, O +1996 O +; O +Webb O +et O +al O +, O +1998 O +), O +which O +allows O +ligands O +to O +nucleate O +ERα B-complex_assembly +– I-complex_assembly +NCOA1 I-complex_assembly +/ I-complex_assembly +2 I-complex_assembly +/ I-complex_assembly +3 I-complex_assembly +interaction O +through O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +, O +and O +reinforce O +this O +interaction O +with O +additional O +binding O +to O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +. O + +Completely O +blocking O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +with O +an O +extended O +side O +chain O +or O +altering O +the O +shape O +of O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +changes O +the O +preference O +away O +from O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +for O +determining O +GREB1 B-protein +levels O +and O +proliferation O +of O +breast O +cancer O +cells O +. O + +AF B-structure_element +‐ I-structure_element +2 I-structure_element +blockade O +also O +allows O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +to O +function O +independently O +, O +which O +is O +important O +since O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +drives O +tissue O +‐ O +selective O +effects O +in O +vivo O +. O + +This O +was O +demonstrated O +with O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +knockout O +mice O +that O +show O +E2 B-chemical +‐ O +dependent O +vascular O +protection O +, O +but O +not O +uterine O +proliferation O +, O +thus O +highlighting O +the O +role O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +in O +tissue O +‐ O +selective O +or O +cell O +‐ O +specific O +signaling O +( O +Billon O +‐ O +Gales O +et O +al O +, O +2009 O +; O +Abot O +et O +al O +, O +2013 O +). O + +Here O +, O +we O +examined O +many O +LBD B-structure_element +structures B-evidence +and O +tested O +several O +variables O +that O +were O +not O +predictive O +, O +including O +ERβ B-protein +activity O +, O +the O +strength O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +signaling O +, O +and O +NCOA3 B-protein +occupancy O +at O +the O +GREB1 B-protein +gene O +. O + +Similarly O +, O +we O +visualized O +structures B-evidence +to O +identify O +patterns O +. O + +For O +example O +, O +phage B-experimental_method +display I-experimental_method +was O +used O +to O +identify O +the O +androgen O +receptor O +interactome O +, O +which O +was O +cloned O +into O +an O +M2H B-experimental_method +library O +and O +used O +to O +identify O +clusters O +of O +ligand O +‐ O +selective O +interactions O +( O +Norris O +et O +al O +, O +2009 O +). O + +Also O +, O +we O +have O +used O +siRNA B-experimental_method +screening I-experimental_method +to O +identify O +a O +number O +of O +coregulators O +required O +for O +ERα B-protein +‐ O +mediated O +repression O +of O +the O +IL O +‐ O +6 O +gene O +( O +Nwachukwu O +et O +al O +, O +2014 O +). O + +If O +we O +calculated O +inter B-evidence +‐ I-evidence +atomic I-evidence +distance I-evidence +matrices I-evidence +containing O +4 O +, O +000 O +atoms O +per O +structure O +× O +76 O +ligand O +– O +receptor O +complexes O +, O +we O +would O +have O +3 O +× O +105 O +predictions O +. O + +We O +have O +identified O +atomic B-evidence +vectors I-evidence +for O +the O +OBHS B-chemical +‐ I-chemical +N I-chemical +and O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +classes O +that O +predict O +ligand O +response O +( O +Fig O +5E O +and O +F O +). O + +Indeed O +, O +the O +most O +anti O +‐ O +proliferative O +compound O +in O +the O +OBHS B-chemical +‐ I-chemical +N I-chemical +series O +had O +a O +fulvestrant O +‐ O +like O +profile O +across O +a O +battery O +of O +assays O +( O +S O +. O +Srinivasan O +et O +al O +, O +in O +preparation O +). O + +Secondly O +, O +our O +finding O +that O +WAY B-chemical +‐ I-chemical +C I-chemical +compounds O +do O +not O +rely O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +for O +signaling O +efficacy O +may O +derive O +from O +the O +slight O +contacts O +with O +h12 B-structure_element +observed O +in O +crystal B-evidence +structures I-evidence +( O +Figs O +3B O +and O +5H O +), O +unlike O +other O +compounds O +in O +cluster O +1 O +that O +dislocate O +h11 B-structure_element +and O +rely O +on O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +for O +signaling O +efficacy O +( O +Figs O +3B O +and O +5C O +, O +and O +EV5B O +). O + +Some O +of O +these O +ligands O +altered O +the O +shape O +of O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +by O +perturbing O +the O +h3 B-site +– I-site +h12 I-site +interface I-site +, O +thus O +providing O +a O +route O +to O +new O +SERM O +‐ O +like O +activity O +profiles O +by O +combining O +indirect O +and O +direct O +modulation O +of O +receptor O +structure O +. O + +Incorporation O +of O +statistical O +approaches O +to O +understand O +relationships O +between O +structure O +and O +signaling O +variables O +moves O +us O +toward O +predictive O +models O +for O +complex O +ERα B-protein +‐ O +mediated O +responses O +such O +as O +in O +vivo O +uterine O +proliferation O +or O +tumor O +growth O +, O +and O +more O +generally O +toward O +structure O +‐ O +based O +design O +for O +other O +allosteric O +drug O +targets O +including O +GPCRs B-protein_type +and O +other O +nuclear B-protein_type +receptors I-protein_type +. O + +Structure B-evidence +of O +a O +quinolone O +- O +stabilized O +cleavage O +complex O +of O +topoisomerase B-complex_assembly +IV I-complex_assembly +from O +Klebsiella B-species +pneumoniae I-species +and O +comparison O +with O +a O +related O +Streptococcus B-species +pneumoniae I-species +complex O + +Crystal B-evidence +structures I-evidence +of O +the O +cleavage O +complexes O +of O +topoisomerase B-complex_assembly +IV I-complex_assembly +from O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +( O +K B-species +. I-species +pneumoniae I-species +) O +and O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +( O +S B-species +. I-species +pneumoniae I-species +) O +bacterial B-taxonomy_domain +pathogens O +stabilized O +by O +the O +clinically O +important O +antibacterial O +drug O +levofloxacin B-chemical +are O +presented O +, O +analysed O +and O +compared O +. O + +For O +K B-species +. I-species +pneumoniae I-species +, O +this O +is O +the O +first O +high O +- O +resolution O +cleavage O +complex O +structure B-evidence +to O +be O +reported O +. O + +Klebsiella B-species +pneumoniae I-species +is O +a O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +bacterium I-taxonomy_domain +that O +is O +responsible O +for O +a O +range O +of O +common O +infections O +, O +including O +pulmonary O +pneumonia O +, O +bloodstream O +infections O +and O +meningitis O +. O + +Certain O +strains O +of O +Klebsiella B-taxonomy_domain +have O +become O +highly O +resistant O +to O +antibiotics O +. O + +Despite O +the O +vast O +amount O +of O +research O +carried O +out O +on O +this O +class O +of O +bacteria B-taxonomy_domain +, O +the O +molecular O +structure B-evidence +of O +its O +topoisomerase B-complex_assembly +IV I-complex_assembly +, O +a O +type B-protein_type +II I-protein_type +topoisomerase I-protein_type +essential O +for O +catalysing O +chromosomal O +segregation O +, O +had O +remained O +unknown O +. O + +In O +this O +paper O +, O +the O +structure B-evidence +of O +its O +DNA B-chemical +- O +cleavage O +complex O +is O +reported O +at O +3 O +. O +35 O +Å O +resolution O +. O + +The O +complex O +is O +comprised O +of O +ParC B-protein +breakage B-structure_element +- I-structure_element +reunion I-structure_element +and O +ParE B-protein +TOPRIM B-structure_element +domains O +of O +K B-species +. I-species +pneumoniae I-species +topoisomerase B-complex_assembly +IV I-complex_assembly +with O +DNA B-chemical +stabilized O +by O +levofloxacin B-chemical +, O +a O +broad O +- O +spectrum O +fluoroquinolone B-chemical +antimicrobial O +agent O +. O + +This O +complex O +is O +compared O +with O +a O +similar O +complex O +from O +Streptococcus B-species +pneumoniae I-species +, O +which O +has O +recently O +been O +solved O +. O + +Klebsiella B-taxonomy_domain +is O +a O +genus O +belonging O +to O +the O +Enterobacteriaceae B-taxonomy_domain +family O +of O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +bacilli I-taxonomy_domain +, O +which O +is O +divided O +into O +seven O +species O +with O +demonstrated O +similarities O +in O +DNA B-chemical +homology O +: O +K B-species +. I-species +pneumoniae I-species +, O +K B-species +. I-species +ozaenae I-species +, O +K B-species +. I-species +rhinoscleromatis I-species +, O +K B-species +. I-species +oxytoca I-species +, O +K B-species +. I-species +planticola I-species +, O +K B-species +. I-species +terrigena I-species +and O +K B-species +. I-species +ornithinolytica I-species +. O + +K B-species +. I-species +pneumoniae I-species +is O +the O +most O +medically O +important O +species O +of O +the O +genus O +owing O +to O +its O +high O +resistance O +to O +antibiotics O +. O + +Significant O +morbidity O +and O +mortality O +has O +been O +associated O +with O +an O +emerging O +, O +highly O +drug O +- O +resistant O +strain O +of O +K B-species +. I-species +pneumoniae I-species +characterized O +as O +producing O +the O +carbapenemase B-protein_type +enzyme O +( O +KPC O +- O +producing O +bacteria B-taxonomy_domain +; O +Nordmann O +et O +al O +., O +2009 O +). O + +However O +, O +common O +treatments O +( O +based O +on O +in B-experimental_method +vitro I-experimental_method +susceptibility I-experimental_method +testing I-experimental_method +) O +are O +the O +polymyxins B-chemical +, O +tigecycline B-chemical +and O +, O +less O +frequently O +, O +aminoglycoside B-chemical +antibiotics O +( O +Arnold O +et O +al O +., O +2011 O +). O + +Another O +effective O +strategy O +involves O +the O +limited O +use O +of O +certain O +antimicrobials O +, O +specifically O +fluoroquinolones B-chemical +and O +cephalo B-chemical +­ I-chemical +sporins I-chemical +( O +Gasink O +et O +al O +., O +2009 O +). O + +These O +include O +combinations O +of O +existing O +β O +- O +lactam O +antibiotics O +with O +new O +β B-protein_type +- I-protein_type +lactamase I-protein_type +inhibitors O +able O +to O +circumvent O +KPC O +resistance O +. O + +Neoglycosides B-chemical +are O +novel O +aminoglycosides B-chemical +that O +have O +activity O +against O +KPC O +- O +producing O +bacteria B-taxonomy_domain +that O +are O +also O +being O +developed O +( O +Chen O +et O +al O +., O +2012 O +). O + +Type B-protein_type +II I-protein_type +topoisomerase I-protein_type +enzymes I-protein_type +play O +important O +roles O +in O +prokaryotic B-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +DNA B-chemical +replication O +, O +recombination O +and O +transcription O +( O +Drlica O +et O +al O +., O +2008 O +; O +Laponogov O +et O +al O +., O +2013 O +; O +Lee O +et O +al O +., O +2013 O +; O +Nitiss O +, O +2009a O +, O +b O +; O +Schoeffler O +& O +Berger O +, O +2008 O +; O +Sissi O +& O +Palumbo O +, O +2009 O +; O +Vos O +et O +al O +., O +2011 O +; O +Wendorff O +et O +al O +., O +2012 O +; O +Wu O +et O +al O +., O +2011 O +, O +2013 O +). O + +In O +bacteria B-taxonomy_domain +, O +topoisomerase B-complex_assembly +IV I-complex_assembly +, O +a O +tetramer B-oligomeric_state +of O +two O +ParC B-protein +and O +two O +ParE B-protein +subunits O +, O +unlinks O +daughter O +chromosomes O +prior O +to O +cell O +division O +, O +whereas O +the O +related O +enzyme O +gyrase B-protein_type +, O +a O +GyrA2GyrB2 B-complex_assembly +tetramer B-oligomeric_state +, O +supercoils O +DNA B-chemical +and O +helps O +unwind O +DNA B-chemical +at O +replication O +forks O +. O + +Both O +enzymes O +act O +via O +a O +double O +- O +strand O +DNA B-chemical +break O +involving O +a O +cleavage O +complex O +and O +are O +targets O +for O +quinolone O +antimicrobials O +that O +act O +by O +trapping O +these O +enzymes O +at O +the O +DNA B-chemical +- O +cleavage O +stage O +and O +preventing O +strand O +re O +- O +joining O +( O +Drlica O +et O +al O +., O +2008 O +). O + +Levofloxacin B-chemical +is O +a O +broad O +- O +spectrum O +third O +- O +generation O +fluoro O +­ O +quinolone O +antibiotic O +. O + +It O +is O +active O +against O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +and O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +bacteria I-taxonomy_domain +and O +functions O +by O +inhibiting O +gyrase B-protein_type +and O +topoisomerase B-complex_assembly +IV I-complex_assembly +( O +Drlica O +& O +Zhao O +, O +1997 O +; O +Laponogov O +et O +al O +., O +2010 O +). O + +Acquiring O +a O +deep O +structural O +and O +functional O +understanding O +of O +the O +mode O +of O +action O +of O +fluoroquinolones B-chemical +( O +Tomašić O +& O +Mašič O +, O +2014 O +) O +and O +the O +development O +of O +new O +drugs O +targeted O +against O +topoisomerase B-complex_assembly +IV I-complex_assembly +and O +gyrase B-protein_type +from O +a O +wide O +range O +of O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +and O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +pathogenic O +bacteria B-taxonomy_domain +are O +highly O +active O +areas O +of O +current O +research O +directed O +at O +overcoming O +the O +vexed O +problem O +of O +drug O +resistance O +( O +Bax O +et O +al O +., O +2010 O +; O +Chan O +et O +al O +., O +2015 O +; O +Drlica O +et O +al O +., O +2014 O +; O +Mutsaev O +et O +al O +., O +2014 O +; O +Pommier O +, O +2013 O +; O +Srikannathasan O +et O +al O +., O +2015 O +). O + +Here O +, O +we O +report O +the O +first O +three O +- O +dimensional O +X B-evidence +- I-evidence +ray I-evidence +structure I-evidence +of O +a O +K B-species +. I-species +pneumoniae I-species +topoisomerase B-complex_assembly +IV I-complex_assembly +ParC B-complex_assembly +/ I-complex_assembly +ParE I-complex_assembly +cleavage O +complex O +with O +DNA B-chemical +stabilized O +by O +levofloxacin B-chemical +. O + +The O +crystal B-evidence +structure I-evidence +provides O +structural O +information O +on O +topoisomerase B-complex_assembly +IV I-complex_assembly +from O +K B-species +. I-species +pneumoniae I-species +, O +a O +pathogen O +for O +which O +drug O +resistance O +is O +a O +serious O +concern O +. O + +The O +structure B-evidence +of O +the O +ParC B-complex_assembly +/ I-complex_assembly +ParE I-complex_assembly +– O +DNA B-site +– I-site +levofloxacin I-site +binding I-site +site I-site +highlights O +the O +details O +of O +the O +cleavage O +- O +complex O +assembly O +that O +are O +essential O +for O +the O +rational O +design O +of O +Klebsiella B-taxonomy_domain +topoisomerase B-protein_type +inhibitors O +. O + +We O +have O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +the O +K B-species +. I-species +pneumoniae I-species +topoisomerase B-complex_assembly +IV I-complex_assembly +ParC B-complex_assembly +/ I-complex_assembly +ParE I-complex_assembly +breakage B-structure_element +- I-structure_element +reunion I-structure_element +domain O +( O +ParC55 B-protein +; O +residues O +1 B-residue_range +– I-residue_range +490 I-residue_range +) O +and O +ParE B-protein +TOPRIM B-structure_element +domain O +( O +ParE30 B-protein +; O +residues O +390 B-residue_range +– I-residue_range +631 I-residue_range +) O +with O +a O +precut O +34 O +bp O +DNA B-chemical +duplex O +( O +the O +E B-site +- I-site +site I-site +), O +stabilized O +by O +levofloxacin B-chemical +. O + +The O +X B-evidence +- I-evidence +ray I-evidence +crystal I-evidence +structure I-evidence +of O +the O +complex O +was O +determined O +to O +3 O +. O +35 O +Å O +resolution O +, O +revealing O +a O +closed B-protein_state +ParC55 B-protein +dimer B-oligomeric_state +flanked O +by O +two O +ParE30 B-protein +monomers B-oligomeric_state +( O +Figs O +. O +1 O +▸, O +2 O +▸ O +and O +3 O +▸). O + +The O +overall O +architecture O +of O +this O +complex O +is O +similar O +to O +that O +found O +for O +S B-species +. I-species +pneumoniae I-species +topoisomerase O +– O +DNA O +– O +drug O +complexes O +( O +Laponogov O +et O +al O +., O +2009 O +, O +2010 O +). O + +Residues O +6 B-residue_range +– I-residue_range +30 I-residue_range +of O +the O +N O +- O +terminal O +α B-structure_element +- I-structure_element +helix I-structure_element +α1 B-structure_element +of O +the O +ParC B-protein +subunit O +again O +embrace O +the O +ParE B-protein +subunit O +, O +‘ O +hugging O +’ O +the O +ParE B-protein +subunits O +close O +to O +either O +side O +of O +the O +ParC B-protein +dimer B-oligomeric_state +( O +Laponogov O +et O +al O +., O +2010 O +). O + +Deletion B-experimental_method +of I-experimental_method +this O +‘ O +arm B-structure_element +’ O +α1 B-structure_element +results O +in O +loss B-protein_state +of I-protein_state +DNA I-protein_state +- I-protein_state +cleavage I-protein_state +activity I-protein_state +( O +Laponogov O +et O +al O +., O +2007 O +) O +and O +is O +clearly O +very O +important O +in O +complex O +stability O +( O +Fig O +. O +3 O +▸). O + +This O +structural O +feature O +was O +absent O +in O +our O +original O +ParC55 B-protein +structure B-evidence +( O +Laponogov O +et O +al O +., O +2007 O +; O +Sohi O +et O +al O +., O +2008 O +). O + +The O +upper O +region O +of O +the O +topoisomerase B-protein_type +complex O +consists O +of O +the O +E B-protein +- I-protein +subunit I-protein +TOPRIM B-structure_element +metal I-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +formed O +of O +four O +parallel B-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +and O +the O +surrounding O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +The O +C B-protein +- I-protein +subunit I-protein +provides O +the O +WHD B-structure_element +( O +winged B-structure_element +- I-structure_element +helix I-structure_element +domain I-structure_element +; O +a O +CAP B-structure_element +- I-structure_element +like I-structure_element +structure I-structure_element +; O +McKay O +& O +Steitz O +, O +1981 O +) O +and O +the O +‘ O +tower B-structure_element +’ O +which O +form O +the O +U B-structure_element +groove I-structure_element +- O +shaped O +protein O +region O +into O +which O +the O +G B-structure_element +- I-structure_element +gate I-structure_element +DNA B-chemical +binds O +with O +an O +induced O +U O +- O +shaped O +bend O +. O + +The O +lower O +C B-structure_element +- I-structure_element +gate I-structure_element +region O +( O +Fig O +. O +3 O +▸) O +consists O +of O +the O +same O +disposition O +of O +pairs O +of O +two O +long B-structure_element +α I-structure_element +- I-structure_element +helices I-structure_element +terminated O +by O +a O +spanning O +short B-structure_element +α I-structure_element +- I-structure_element +helix I-structure_element +forming O +a O +30 O +Å O +wide O +DNA B-site +- I-site +accommodating I-site +cavity I-site +through O +which O +the O +T B-structure_element +- I-structure_element +gate I-structure_element +DNA B-chemical +passes O +as O +found O +in O +the O +S B-species +. I-species +pneumoniae I-species +complex O +. O + +Owing O +to O +the O +structural O +similarity O +, O +it O +appears O +that O +the O +topo B-complex_assembly +­ I-complex_assembly +isomerases I-complex_assembly +IV I-complex_assembly +from O +K B-species +. I-species +pneumoniae I-species +and O +S B-species +. I-species +pneumoniae I-species +are O +likely O +to O +follow O +a O +similar O +overall O +topoisomerase B-protein_type +catalytic O +cycle O +as O +shown O +in O +Fig O +. O +4 O +▸; O +we O +have O +confirmation O +of O +one O +intermediate O +from O +our O +recent O +structure B-evidence +of O +the O +full B-protein_state +complex I-protein_state +( O +the O +holoenzyme B-protein_state +less O +the O +CTD B-structure_element +β I-structure_element +- I-structure_element +pinwheel I-structure_element +domain O +) O +with O +the O +ATPase B-structure_element +domain I-structure_element +in O +the O +open B-protein_state +conformation O +( O +Laponogov O +et O +al O +., O +2013 O +). O + +The O +G B-structure_element +- I-structure_element +gate I-structure_element +DNA B-chemical +for O +the O +S B-species +. I-species +pneumoniae I-species +complex O +consists O +of O +an O +18 O +- O +base O +- O +pair O +E B-site +- I-site +site I-site +sequence O +( O +our O +designation O +for O +a O +DNA B-site +site I-site +which O +we O +first O +found O +from O +DNA B-experimental_method +- I-experimental_method +mapping I-experimental_method +studies I-experimental_method +; O +Leo O +et O +al O +., O +2005 O +; O +Arnoldi O +et O +al O +., O +2013 O +; O +Fig O +. O +1 O +▸). O + +The O +crystallized B-experimental_method +complex O +was O +formed O +by O +turning O +over O +the O +topoisomerase B-protein_type +tetramer B-oligomeric_state +in O +the O +presence B-protein_state +of I-protein_state +DNA B-chemical +and O +levofloxacin B-chemical +and O +crystallizing B-experimental_method +the O +product O +. O + +In O +contrast O +, O +the O +K B-species +. I-species +pneumoniae I-species +complex O +was O +formed O +by O +co B-experimental_method +- I-experimental_method +crystallizing I-experimental_method +the O +topoisomerase B-protein_type +tetramer B-oligomeric_state +complex O +in O +the O +presence B-protein_state +of I-protein_state +a O +34 O +- O +base O +- O +pair O +pre B-protein_state +- I-protein_state +cleaved I-protein_state +DNA B-chemical +in O +the O +presence B-protein_state +of I-protein_state +levofloxacin B-chemical +. O + +In O +both O +cases O +the O +DNA B-chemical +is O +bent O +into O +a O +U B-protein_state +- I-protein_state +form I-protein_state +and O +bound B-protein_state +snugly O +against O +the O +protein O +of O +the O +G B-structure_element +- I-structure_element +gate I-structure_element +. O + +We O +have O +been O +able O +to O +unambiguously O +read O +off O +the O +DNA B-chemical +sequences O +in O +the O +electron B-evidence +- I-evidence +density I-evidence +maps I-evidence +. O + +There O +is O +41 O +. O +6 O +% O +sequence O +identity O +and O +54 O +. O +4 O +% O +sequence O +homology O +between O +the O +ParE B-protein +subunit O +of O +K B-species +. I-species +pneumoniae I-species +and O +that O +of O +S B-species +. I-species +pneumoniae I-species +. O + +For O +the O +ParC B-protein +subunits O +, O +the O +figures O +are O +40 O +. O +8 O +identity O +and O +55 O +. O +6 O +% O +homology O +between O +the O +two O +organisms O +. O + +The O +sequence B-experimental_method +alignment I-experimental_method +is O +given O +in O +Supplementary O +Fig O +. O +S1 O +, O +with O +the O +key O +metal B-site +- I-site +binding I-site +residues I-site +and O +those O +which O +give O +rise O +to O +quinolone O +resistance O +highlighted O +. O + +The O +binding O +of O +levofloxacin B-chemical +in O +the O +K B-species +. I-species +pneumoniae I-species +complex O +is O +shown O +in O +Figs O +. O +2 O +▸, O +3 O +▸ O +and O +5 O +▸ O +and O +is O +hemi O +- O +intercalated O +into O +the O +DNA B-chemical +and O +stacked O +against O +the O +DNA B-chemical +bases O +at O +the O +cleavage B-site +site I-site +( O +positions O +− B-residue_number +1 I-residue_number +and O ++ B-residue_number +1 I-residue_number +of O +the O +four O +- O +base O +- O +pair O +staggered O +cut O +in O +the O +34 O +- O +mer O +DNA B-chemical +) O +which O +is O +similar O +to O +that O +found O +for O +the O +S B-species +. I-species +pneumoniae I-species +complex O +. O + +Fig O +. O +5 O +▸ O +presents O +side O +- O +by O +- O +side O +views O +of O +the O +K B-species +. I-species +pneumoniae I-species +and O +S B-species +. 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O + +The O +side O +chains O +surrounding O +them O +in O +ParE B-protein +are O +quite O +disordered O +and O +are O +more O +defined O +in O +K B-species +. I-species +pneumoniae I-species +( O +even O +though O +this O +complex O +is O +at O +lower O +resolution O +) O +than O +in O +S B-species +. I-species +pneumoniae I-species +. O + +There O +are O +no O +direct O +hydrogen B-bond_interaction +bonds I-bond_interaction +from O +the O +drug O +to O +these O +residues O +( O +although O +it O +is O +possible O +that O +some O +are O +formed O +through O +water B-chemical +, O +which O +cannot O +be O +observed O +at O +this O +resolution O +). O + +Obviously O +, O +the O +drug O +– O +ParE B-protein +interaction O +in O +this O +region O +is O +less O +strong O +compared O +with O +PD B-chemical +0305970 I-chemical +binding O +to O +the O +S B-species +. 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O +2 O +▸ O +and O +5 O +▸ O +and O +Supplementary O +Fig O +. O +2 O +▸). O + +For O +S B-species +. I-species +pneumoniae I-species +topoisomerase B-complex_assembly +IV I-complex_assembly +, O +one O +of O +the O +O O +atoms O +of O +the O +carboxyl O +of O +Asp83 B-residue_name_number +points O +towards O +the O +Mg2 B-chemical ++ I-chemical +ion O +and O +is O +within O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +distance O +( O +5 O +. O +04 O +Å O +) O +through O +an O +Mg2 B-chemical ++- I-chemical +coordinated O +water B-chemical +. O + +For O +K B-species +. I-species +pneumoniae I-species +both O +of O +the O +carboxyl O +O O +atoms O +are O +pointing O +towards O +the O +Mg2 B-chemical ++ I-chemical +ion O +at O +distances O +of O +4 O +. O +86 O +and O +4 O +. O +23 O +Å O +. O +These O +residues O +are O +ordered O +in O +only O +one O +of O +the O +two O +dimers B-oligomeric_state +in O +the O +K B-species +. 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O + +The O +inclusion O +of O +levofloxacin B-chemical +produced O +linear O +DNA B-chemical +in O +a O +dose O +- O +dependent O +and O +ATP B-chemical +- O +independent O +fashion O +. O + +Similar O +behaviour O +was O +observed O +for O +the O +S B-species +. I-species +pneumoniae I-species +topo B-complex_assembly +­ I-complex_assembly +isomerase I-complex_assembly +IV I-complex_assembly +ParE30 B-complex_assembly +- I-complex_assembly +ParC55 I-complex_assembly +fusion O +protein O +. O + +The O +CC25 B-evidence +( O +the O +drug O +concentration O +that O +converted O +25 O +% O +of O +the O +supercoiled O +DNA B-chemical +substrate O +to O +a O +linear O +form O +) O +was O +0 O +. O +5 O +µM O +for O +the O +Klebsiella B-taxonomy_domain +enzyme O +and O +1 O +µM O +for O +the O +pneumococcal B-taxonomy_domain +enzyme O +. O + +Interestingly O +, O +K B-species +. 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I-species +pneumoniae I-species +, O +it O +is O +likely O +to O +be O +gyrase B-protein_type +in O +the O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +K B-species +. I-species +pneumoniae I-species +. O + +In O +summary O +, O +we O +have O +determined O +the O +first O +structure B-evidence +of O +a O +quinolone B-chemical +– O +DNA B-chemical +cleavage O +complex O +involving O +a O +type B-protein_type +II I-protein_type +topo I-protein_type +­ I-protein_type +isomerase I-protein_type +from O +K B-species +. I-species +pneumoniae I-species +. O + +Given O +the O +current O +concerns O +about O +drug O +- O +resistant O +strains O +of O +Klebsiella B-taxonomy_domain +, O +the O +structure B-evidence +reported O +here O +provides O +key O +information O +in O +understanding O +the O +action O +of O +currently O +used O +quinolones B-chemical +and O +should O +aid O +in O +the O +development O +of O +other O +topoisomerase B-protein_type +- O +targeting O +therapeutics O +active O +against O +this O +major O +human B-species +pathogen O +. O + +Protein O +and O +DNA B-chemical +used O +in O +the O +co B-experimental_method +- I-experimental_method +crystallization I-experimental_method +experiment O +. O + +( O +a O +) O +Coloured O +diagram O +of O +the O +protein O +constructs O +used O +in O +crystallization B-experimental_method +. O + +( O +b O +) O +DNA B-chemical +sequences O +used O +in O +crystallization B-experimental_method +. O + +Chemical O +structure O +of O +levofloxacin B-chemical +( O +a O +) O +and O +its O +conformations O +observed O +within O +the O +active B-site +sites I-site +of O +S B-species +. I-species +pneumoniae I-species +topoisomerase B-complex_assembly +IV I-complex_assembly +( O +b O +) O +and O +K B-species +. I-species +pneumoniae I-species +topoisomerase B-complex_assembly +IV I-complex_assembly +( O +c O +, O +d O +). O + +Electron B-evidence +- I-evidence +density I-evidence +maps I-evidence +( O +2F O +obs O +− O +F O +calc O +) O +are O +shown O +as O +meshes O +for O +the O +drug O +molecules O +contoured O +at O +1 O +. O +5σ O +and O +are O +limited O +to O +a O +distance O +of O +2 O +. O +3 O +Å O +from O +the O +drug O +atoms O +. O + +Overall O +orthogonal O +views O +of O +the O +cleavage O +complex O +of O +topoisomerase B-complex_assembly +IV I-complex_assembly +from O +K B-species +. I-species +pneumoniae I-species +in O +surface O +( O +left O +) O +and O +cartoon O +( O +right O +) O +representations O +. O + +The O +ParC B-protein +subunit O +is O +in O +blue O +, O +ParE B-protein +is O +in O +yellow O +and O +DNA B-chemical +is O +in O +cyan O +. O + +The O +bound B-protein_state +quinolone B-chemical +molecules O +( O +levofloxacin B-chemical +) O +are O +in O +red O +and O +are O +shown O +using O +van O +der O +Waals O +representation O +. O + +Schematic O +representation O +of O +the O +catalytic O +cycle O +of O +type B-protein_type +II I-protein_type +topoisomerases I-protein_type +. O + +The O +ParC B-protein +N O +- O +terminal O +domain O +( O +ParC55 B-protein +) O +is O +in O +grey O +, O +the O +ParC B-protein +C O +- O +terminal O +β B-structure_element +-­ I-structure_element +pinwheel I-structure_element +domain I-structure_element +is O +in O +silver O +, O +the O +ParE B-protein +N O +- O +terminal O +ATPase B-structure_element +domain I-structure_element +is O +in O +red O +, O +the O +ParE B-protein +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +ParE30 B-protein +) O +is O +in O +yellow O +, O +the O +G B-structure_element +- I-structure_element +gate I-structure_element +DNA B-chemical +is O +in O +green O +and O +the O +T B-structure_element +- I-structure_element +segment I-structure_element +DNA B-chemical +is O +in O +purple O +. O + +Bound B-protein_state +ATP B-chemical +is O +indicated O +by O +pink O +circles O +in O +the O +ATPase B-structure_element +domains I-structure_element +( O +reproduced O +with O +permission O +from O +Fig O +. O +1 O +of O +Lapanogov O +et O +al O +., O +2013 O +). O + +Detailed O +views O +of O +the O +active B-site +sites I-site +of O +topoisomerase B-complex_assembly +IV I-complex_assembly +from O +S B-species +. I-species +pneumoniae I-species +and O +K B-species +. I-species +pneumoniae I-species +with O +quinolone B-chemical +molecules O +bound B-protein_state +. O + +The O +magnesium B-chemical +ions O +and O +their O +coordinating O +amino O +acids O +are O +shown O +in O +purple O +. O + +The O +active B-site +- I-site +site I-site +tyrosine B-residue_name +and O +arginine B-residue_name +are O +in O +orange O +. O + +The O +DNA B-chemical +is O +shown O +in O +silver O +/ O +cyan O +. O + +The O +ParC B-protein +and O +ParE B-protein +backbones O +are O +shown O +in O +blue O +and O +yellow O +, O +respectively O +. O + +Comparison O +of O +DNA B-chemical +cleavage O +by O +topoisomerase B-complex_assembly +IV I-complex_assembly +core O +ParE B-complex_assembly +- I-complex_assembly +ParC I-complex_assembly +fusion O +proteins O +from O +K B-species +. I-species +pneumoniae I-species +( O +KP B-species +) O +and O +S B-species +. I-species +pneumoniae I-species +( O +SP B-species +) O +promoted O +by O +levofloxacin B-chemical +. O + +Supercoiled O +plasmid O +pBR322 O +( O +400 O +ng O +) O +was O +incubated O +with O +topoisomerase B-complex_assembly +IV I-complex_assembly +proteins O +( O +400 O +ng O +) O +in O +the O +absence O +or O +presence B-protein_state +of I-protein_state +levofloxacin B-chemical +at O +the O +indicated O +concentrations O +. O + +After O +60 O +min O +incubation O +, O +samples O +were O +treated O +with O +SDS O +and O +proteinase O +K O +to O +remove O +proteins O +covalent O +bound O +to O +DNA B-chemical +, O +and O +the O +DNA B-chemical +products O +were O +examined O +by O +gel O +electrophoresis O +in O +1 O +% O +agarose O +. O + +Lane O +A O +, O +supercoiled O +pBR322 O +DNA B-chemical +; O +N O +, O +L O +and O +S O +, O +nicked O +, O +linear O +and O +supercoiled O +pBR322 O +, O +respectively O +. O + +Using O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +to O +Map O +Small O +Ligands O +on O +Dynamic O +Metabolic O +Enzymes O +: O +Studies O +with O +Glutamate B-protein_type +Dehydrogenase I-protein_type + +Cryo B-experimental_method +- I-experimental_method +electron I-experimental_method +microscopy I-experimental_method +( O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +) O +methods O +are O +now O +being O +used O +to O +determine O +structures B-evidence +at O +near O +- O +atomic O +resolution O +and O +have O +great O +promise O +in O +molecular O +pharmacology O +, O +especially O +in O +the O +context O +of O +mapping O +the O +binding O +of O +small O +- O +molecule O +ligands O +to O +protein O +complexes O +that O +display O +conformational O +flexibility O +. O + +We O +illustrate O +this O +here O +using O +glutamate B-protein_type +dehydrogenase I-protein_type +( O +GDH B-protein_type +), O +a O +336 O +- O +kDa O +metabolic O +enzyme O +that O +catalyzes O +the O +oxidative O +deamination O +of O +glutamate B-chemical +. O + +Dysregulation O +of O +GDH B-protein_type +leads O +to O +a O +variety O +of O +metabolic O +and O +neurologic O +disorders O +. O + +Here O +, O +we O +report O +near O +- O +atomic O +resolution O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +, O +at O +resolutions O +ranging O +from O +3 O +. O +2 O +Å O +to O +3 O +. O +6 O +Å O +for O +GDH B-protein_type +complexes O +, O +including O +complexes O +for O +which O +crystal B-evidence +structures I-evidence +are O +not O +available O +. O + +We O +show O +that O +the O +binding O +of O +the O +coenzyme O +NADH B-chemical +alone O +or O +in O +concert O +with O +GTP B-chemical +results O +in O +a O +binary O +mixture O +in O +which O +the O +enzyme O +is O +in O +either O +an O +“ O +open B-protein_state +” O +or O +“ O +closed B-protein_state +” O +state O +. O + +Whereas O +the O +structure B-evidence +of O +NADH B-chemical +in O +the O +active B-site +site I-site +is O +similar O +between O +the O +open B-protein_state +and O +closed B-protein_state +states O +, O +it O +is O +unexpectedly O +different O +at O +the O +regulatory B-site +site I-site +. O + +Our O +studies O +thus O +demonstrate O +that O +even O +in O +instances O +when O +there O +is O +considerable O +structural O +information O +available O +from O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +, O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +methods O +can O +provide O +useful O +complementary O +insights O +into O +regulatory O +mechanisms O +for O +dynamic O +protein O +complexes O +. O + +Recent O +advances O +in O +cryo B-experimental_method +- I-experimental_method +electron I-experimental_method +microscopy I-experimental_method +( O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +) O +allow O +determination O +of O +structures B-evidence +of O +small O +protein O +complexes O +and O +membrane O +proteins O +at O +near O +- O +atomic O +resolution O +, O +marking O +a O +critical O +shift O +in O +the O +structural O +biology O +field O +. O + +One O +specific O +area O +of O +broad O +general O +interest O +in O +drug O +discovery O +is O +the O +localization O +of O +bound O +ligands O +and O +cofactors O +under O +conditions O +in O +which O +efforts O +at O +crystallization B-experimental_method +have O +not O +been O +successful O +because O +of O +structural O +heterogeneity O +. O + +Recent O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +analyses O +have O +already O +demonstrated O +that O +it O +is O +now O +possible O +to O +use O +single B-experimental_method +- I-experimental_method +particle I-experimental_method +cryo I-experimental_method +- I-experimental_method +EM I-experimental_method +methods O +to O +localize O +small O +bound O +ligands O +or O +inhibitors O +on O +target O +proteins O +. O + +Here O +, O +we O +address O +this O +question O +using O +mammalian B-taxonomy_domain +glutamate B-protein_type +dehydrogenase I-protein_type +as O +an O +example O +. O + +Glutamate B-protein_type +dehydrogenase I-protein_type +( O +GDH B-protein_type +) O +is O +a O +highly B-protein_state +conserved I-protein_state +enzyme O +expressed O +in O +most O +organisms O +. O + +GDH B-protein_type +plays O +a O +central O +role O +in O +glutamate B-chemical +metabolism O +by O +catalyzing O +the O +reversible O +oxidative O +deamination O +of O +glutamate B-chemical +to O +generate O +α B-chemical +- I-chemical +ketoglutarate I-chemical +and O +ammonia B-chemical +, O +with O +the O +concomitant O +transfer O +of O +a O +pair O +of O +electrons O +to O +either O +NAD B-chemical ++ I-chemical +or O +NADP B-chemical ++. I-chemical + +Regulation O +of O +GDH B-protein_type +is O +tightly O +controlled O +through O +multiple O +allosteric O +mechanisms O +. O + +Extensive O +biochemical B-experimental_method +and I-experimental_method +crystallographic I-experimental_method +studies I-experimental_method +have O +characterized O +the O +enzymatic O +activity O +of O +GDH B-protein_type +and O +its O +modulation O +by O +a O +chemically O +diverse O +group O +of O +compounds O +such O +as O +nucleotides O +, O +amino O +acids O +, O +steroid O +hormones O +, O +antipsychotic O +drugs O +, O +and O +natural O +products O +. O + +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallographic I-experimental_method +studies I-experimental_method +have O +shown O +that O +the O +functional O +unit O +of O +GDH B-protein_type +is O +a O +homohexamer B-oligomeric_state +composed O +of O +a O +trimer B-oligomeric_state +of O +dimers B-oligomeric_state +, O +with O +a O +3 O +- O +fold O +axis O +and O +an O +equatorial O +plane O +that O +define O +its O +D3 O +symmetry O +( O +Fig O +. O +1A O +). O + +Each O +56 O +- O +kDa O +protomer B-oligomeric_state +consists O +of O +three O +domains O +. O + +The O +first O +is O +located O +near O +the O +dimer B-site +interface I-site +and O +forms O +the O +core O +of O +the O +hexamer B-oligomeric_state +. O + +The O +second O +, O +a O +nucleotide B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +( O +NBD B-structure_element +) O +with O +a O +Rossmann B-structure_element +fold I-structure_element +, O +defines O +one O +face O +of O +the O +catalytic B-site +cleft I-site +bounded O +by O +the O +core O +domain O +. O + +During O +the O +catalytic O +cycle O +, O +the O +NBD B-structure_element +executes O +a O +large O +movement O +, O +hinged O +around O +a O +“ B-structure_element +pivot I-structure_element +” I-structure_element +helix I-structure_element +, O +that O +closes O +the O +catalytic B-site +cleft I-site +, O +and O +drives O +a O +large O +conformational O +change O +in O +the O +hexamer B-oligomeric_state +from O +open B-protein_state +to O +closed B-protein_state +states O +( O +Fig O +. O +1B O +). O + +The O +third O +domain O +, O +dubbed O +the O +“ O +antenna B-structure_element +,” O +is O +an O +evolutionary O +acquisition O +in O +protista B-taxonomy_domain +and O +animals B-taxonomy_domain +. O + +Antennae B-structure_element +of O +adjacent O +protomers B-oligomeric_state +in O +each O +trimer B-oligomeric_state +intercalate O +to O +form O +a O +bundle O +, O +perpendicular O +to O +the O +pivot B-structure_element +helices I-structure_element +, O +that O +protrudes O +along O +the O +distal O +extremes O +of O +the O +3 O +- O +fold O +axis O +. O + +When O +a O +protomer B-oligomeric_state +undergoes O +a O +conformational O +change O +, O +the O +rotation O +of O +its O +pivot B-structure_element +helix I-structure_element +is O +transferred O +through O +the O +antenna B-structure_element +to O +the O +adjacent O +subunit B-structure_element +. O + +The O +influence O +of O +the O +antenna B-structure_element +, O +present O +only O +in O +protozoan B-taxonomy_domain +and O +metazoan B-taxonomy_domain +enzymes O +, O +has O +been O +proposed O +to O +explain O +its O +cooperative O +behavior O +, O +which O +is O +absent O +in O +bacterial B-taxonomy_domain +homologs O +. O + +Structure O +and O +quaternary O +conformational O +changes O +in O +GDH B-protein_type +. O +( O +A O +) O +Views O +of O +open B-protein_state +( O +PDB O +ID O +1NR7 O +) O +and O +closed B-protein_state +( O +PDB O +3MW9 O +) O +states O +of O +the O +GDH B-protein_type +hexamer B-oligomeric_state +, O +shown O +in O +ribbon O +representation O +perpendicular O +to O +the O +2 O +- O +fold O +symmetry O +axis O +( O +side O +view O +, O +top O +) O +and O +3 O +- O +fold O +symmetry O +axis O +( O +top O +view O +, O +bottom O +). O + +Only O +three O +protomers B-oligomeric_state +are O +shown O +in O +the O +top O +view O +for O +purposes O +of O +visual O +clarity O +. O + +The O +dashed O +lines O +and O +arrows O +, O +respectively O +, O +highlight O +the O +slight O +extension O +in O +length O +, O +and O +twist O +in O +shape O +that O +occurs O +with O +transition O +from O +open B-protein_state +to O +the O +closed B-protein_state +state O +. O + +The O +open B-protein_state +state O +shown O +is O +for O +unliganded B-protein_state +GDH B-protein_type +, O +whereas O +the O +closed B-protein_state +state O +has O +NADH B-chemical +, O +GTP B-chemical +, O +and O +glutamate B-chemical +bound B-protein_state +. O +( O +B O +) O +Superposition B-experimental_method +of O +structures B-evidence +for O +closed B-protein_state +and O +open B-protein_state +conformations O +, O +along O +with O +a O +series O +of O +possible O +intermediate O +conformations O +along O +the O +trajectory O +that O +serve O +to O +illustrate O +the O +extent O +of O +change O +in O +structure O +across O +different O +regions O +of O +the O +protein O +. O + +The O +transition O +between O +“ O +closed B-protein_state +” O +and O +“ O +open B-protein_state +” O +states O +of O +GDH B-protein_type +is O +modulated O +by O +two O +allosteric B-site +sites I-site +in O +each O +protomer B-oligomeric_state +( O +Fig O +. O +1A O +), O +which O +are O +differentially O +bound B-protein_state +by I-protein_state +GTP B-chemical +( O +an O +inhibitor O +) O +and O +ADP B-chemical +( O +an O +activator O +). O + +These O +allosteric O +modulators O +tightly O +control O +GDH B-protein_type +function O +in O +vivo O +. O + +In O +the O +first O +site O +, O +which O +sits O +next O +to O +the O +pivot B-structure_element +helix I-structure_element +at O +the O +base O +of O +the O +antenna B-structure_element +( O +the O +“ O +GTP B-site +binding I-site +site I-site +”), O +GTP B-chemical +binding O +is O +known O +to O +act O +as O +an O +inhibitor O +, O +preventing O +release O +of O +the O +reaction O +product O +from O +the O +catalytic B-site +site I-site +by O +stabilizing O +the O +closed B-protein_state +conformation O +of O +the O +catalytic B-site +cleft I-site +. O + +In O +the O +second O +“ O +regulatory B-site +site I-site +”, O +which O +is O +situated O +near O +the O +pivot B-structure_element +helix I-structure_element +between O +adjacent O +protomers B-oligomeric_state +, O +ADP B-chemical +acts O +as O +an O +activator O +of O +enzymatic O +activity O +, O +presumably O +by O +hastening O +the O +opening O +of O +the O +catalytic B-site +cleft I-site +that O +leads O +to O +the O +release O +of O +the O +reaction O +product O +. O + +Interestingly O +, O +it O +has O +also O +been O +shown O +that O +the O +coenzyme O +NADH B-chemical +can O +bind O +to O +the O +regulatory B-site +site I-site +( O +also O +bound B-protein_state +by I-protein_state +the O +activator O +ADP B-chemical +), O +exerting O +a O +converse O +, O +inhibitory O +effect O +on O +GDH B-protein_type +product O +release O +, O +although O +the O +role O +this O +may O +play O +in O +vivo O +is O +not O +entirely O +clear O +. O + +Although O +there O +are O +numerous O +crystal B-evidence +structures I-evidence +available O +for O +GDH B-protein_type +in B-protein_state +complex I-protein_state +with I-protein_state +cofactors O +and O +nucleotides O +, O +they O +are O +limited O +to O +the O +combinations O +that O +have O +been O +amenable O +to O +crystallization B-experimental_method +. O + +Nearly O +all O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +structures B-evidence +of O +mammalian B-taxonomy_domain +GDH B-protein_type +are O +in O +the O +closed B-protein_state +conformation O +, O +and O +the O +few O +structures B-evidence +that O +are O +in O +the O +open B-protein_state +conformation O +are O +at O +lower O +resolution O +( O +Table O +1 O +). O + +Of O +those O +structures B-evidence +in O +the O +closed B-protein_state +conformation O +, O +most O +include O +NAD B-chemical +[ I-chemical +P I-chemical +] I-chemical +H I-chemical +, O +GTP B-chemical +, O +and O +glutamate B-chemical +( O +or O +, O +alternately O +, O +NAD B-chemical ++, I-chemical +GTP B-chemical +, O +and O +α B-chemical +- I-chemical +ketoglutarate I-chemical +). O + +However O +, O +the O +effects O +of O +coenzyme O +and O +GTP B-chemical +, O +bound B-protein_state +alone I-protein_state +or O +in O +concert O +in O +the O +absence B-protein_state +of I-protein_state +glutamate B-chemical +, O +have O +not O +been O +analyzed O +by O +crystallographic O +methods O +. O + +Here O +, O +we O +report O +single B-experimental_method +- I-experimental_method +particle I-experimental_method +cryo I-experimental_method +- I-experimental_method +electron I-experimental_method +microscopy I-experimental_method +( O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +) O +studies O +that O +show O +that O +under O +these O +conditions O +enzyme O +complexes O +coexist O +in O +both O +closed B-protein_state +and O +open B-protein_state +conformations O +. O + +We O +show O +that O +the O +structures B-evidence +in O +both O +states O +can O +be O +resolved O +at O +near O +- O +atomic O +resolution O +, O +suggesting O +a O +molecular O +mechanism O +for O +synergistic O +inhibition O +of O +GDH B-protein_type +by O +NADH B-chemical +and O +GTP B-chemical +( O +see O +Table O +2 O +for O +detailed O +information O +on O +all O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +- O +derived O +structures B-evidence +that O +we O +report O +in O +this O +work O +). O + +X B-experimental_method +- I-experimental_method +ray I-experimental_method +structures B-evidence +of O +mammalian B-taxonomy_domain +GDH B-protein_type +reported O +in O +both O +the O +open B-protein_state +and O +closed B-protein_state +conformations O + +GDH B-protein +Ligands O +PDB O +ID O +Conformation O +Resolution O +WT B-protein_state +NADH B-chemical ++ O +GLU B-chemical ++ O +GTP B-chemical +3MW9 O +Closed B-protein_state +2 O +. O +4 O +WT B-protein_state +Glu B-chemical +, O +GTP B-chemical +, O +NADPH B-chemical +, O +and O +Bithionol O +3ETD O +Closed B-protein_state +2 O +. O +5 O +WT B-protein_state +Glu B-chemical +, O +NADPH B-chemical +, O +GTP B-chemical ++ O +GW5074 O +3ETG O +Closed B-protein_state +2 O +. O +5 O +WT B-protein_state +apo B-protein_state +1L1F O +Open B-protein_state +2 O +. O +7 O +WT B-protein_state +NADPH B-chemical +, O +glutamate B-chemical +, O +and O +GTP B-chemical +1HWZ O +Closed B-protein_state +2 O +. O +8 O +WT B-protein_state +NADPH B-chemical ++ O +GLU B-chemical ++ O +GTP B-chemical ++ O +Zinc B-chemical +3MVQ O +Closed B-protein_state +2 O +. O +94 O +WT B-protein_state +NADPH B-chemical +, O +Glu B-chemical +, O +GTP B-chemical +, O +Hexachlorophene O +3ETE O +Closed B-protein_state +3 O +WT B-protein_state +NAD B-chemical +, O +PO4 B-chemical +, O +and O +2 B-chemical +- I-chemical +oxoglutarate I-chemical +1HWY O +Closed B-protein_state +3 O +. O +2 O +WT B-protein_state +NADPH B-chemical ++ O +GLU B-chemical ++ O +Eu O +3MVO O +Closed B-protein_state +3 O +. O +23 O +R463A O +mutant B-protein_state +apo B-protein_state +1NR1 O +Open B-protein_state +3 O +. O +3 O +WT B-protein_state +apo B-protein_state +1NR7 O +Open B-protein_state +3 O +. O +3 O +WT B-protein_state +ADP B-chemical +1NQT O +Open B-protein_state +3 O +. O +5 O +WT B-protein_state +NADPH B-chemical +and O +Epicatechin O +- O +3 O +- O +gallate O +( O +Ecg O +) O +3QMU O +Open B-protein_state +3 O +. O +62 O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +of O +mammalian B-taxonomy_domain +GDH B-protein_type +determined O +for O +this O +study O + +GDH B-protein_type +Ligands O +EMDB O +ID O +PDB O +ID O +Conformation O +Resolution O +Particles O +WT B-protein_state +apo B-protein_state +EMD O +- O +6630 O +3JCZ O +Open B-protein_state +3 O +. O +26 O +22462 O +WT B-protein_state +GTP B-chemical +EMD O +- O +6631 O +3JD0 O +Open B-protein_state +3 O +. O +47 O +39439 O +WT B-protein_state +NADH B-chemical +EMD O +- O +6635 O +3JD2 O +Open B-protein_state +3 O +. O +27 O +34716 O +WT B-protein_state +NADH B-chemical +EMD O +- O +6634 O +3JD1 O +Closed B-protein_state +3 O +. O +27 O +34926 O +WT B-protein_state +NADH B-chemical ++ O +GTP B-chemical +EMD O +- O +6632 O +3JD3 O +Open B-protein_state +3 O +. O +55 O +14793 O +WT B-protein_state +NADH B-chemical ++ O +GTP B-chemical +EMD O +- O +6633 O +3JD4 O +Closed B-protein_state +3 O +. O +40 O +20429 O + +To O +explore O +the O +conformational O +landscape O +of O +apo B-protein_state +- O +GDH B-protein +, O +we O +first O +determined O +its O +structure B-evidence +in O +the O +absence B-protein_state +of I-protein_state +any O +added O +ligands O +( O +Supplemental O +Fig O +. O +1 O +, O +Fig O +. O +2 O +, O +A O +– O +C O +). O + +The O +density B-evidence +map I-evidence +, O +refined O +to O +an O +average O +resolution O +of O +∼ O +3 O +. O +0 O +Å O +( O +Supplemental O +Fig O +. O +2 O +), O +is O +in O +the O +open B-protein_state +conformation O +and O +closely O +matches O +the O +model O +of O +unliganded B-protein_state +GDH B-protein +derived O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +at O +3 O +. O +3 O +Å O +resolution O +( O +PDB O +ID O +1NR7 O +). O + +The O +variation O +in O +local O +resolution O +from O +the O +core O +to O +the O +periphery O +, O +as O +reported O +by O +ResMap B-experimental_method +( O +Supplemental O +Fig O +. O +3D O +), O +is O +consistent O +with O +the O +B B-evidence +- I-evidence +factor I-evidence +gradient I-evidence +observed O +in O +the O +crystal B-evidence +structure I-evidence +( O +Supplemental O +Fig O +. O +3A O +). O + +Extensive O +classification O +without O +imposing O +symmetry O +yielded O +only O +open B-protein_state +structures B-evidence +and O +failed O +to O +detect O +any O +closed B-protein_state +catalytic B-site +cleft I-site +in O +the O +unliganded B-protein_state +enzyme O +, O +suggesting O +that O +all O +six O +protomers B-oligomeric_state +are O +in O +the O +open B-protein_state +conformation O +. O + +Consistent O +with O +this O +conclusion O +, O +the O +loops B-structure_element +connecting O +the O +β B-structure_element +- I-structure_element +strands I-structure_element +of O +the O +Rossmann B-structure_element +fold I-structure_element +are O +well O +- O +defined O +( O +Fig O +. O +2B O +), O +implying O +that O +there O +is O +little O +movement O +at O +the O +NBD B-structure_element +, O +as O +the O +transition O +between O +closed B-protein_state +and O +open B-protein_state +states O +is O +associated O +with O +NBD B-structure_element +movement O +( O +Fig O +. O +1B O +). O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +of O +GDH B-protein +in O +unliganded B-protein_state +and O +NADH B-protein_state +- I-protein_state +bound I-protein_state +states O +. O +( O +A O +) O +Refined O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +map B-evidence +of O +unliganded B-protein_state +GDH B-protein +at O +∼ O +3 O +Å O +resolution O +. O + +( O +B O +, O +C O +) O +Illustration O +of O +density B-evidence +map I-evidence +in O +the O +regions O +that O +contain O +the O +Rossmann B-structure_element +nucleotide I-structure_element +binding I-structure_element +fold I-structure_element +( O +B O +), O +pivot B-structure_element +and I-structure_element +antenna I-structure_element +helices I-structure_element +( O +C O +) O +in O +the O +unliganded B-protein_state +GDH B-protein +map B-evidence +. O +( O +D O +) O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +- O +derived O +density B-evidence +maps I-evidence +for O +two O +coexisting O +conformations O +that O +are O +present O +when O +GDH B-protein +is O +bound B-protein_state +to I-protein_state +the O +cofactor O +NADH B-chemical +. O + +Each O +protomer B-oligomeric_state +is O +shown O +in O +a O +different O +color O +and O +densities B-evidence +for O +NADH B-chemical +bound B-protein_state +in I-protein_state +both O +regulatory B-site +( O +red O +) O +and O +catalytic B-site +( O +purple O +) O +sites B-site +on O +one O +protomer B-oligomeric_state +are O +indicated O +. O + +The O +overall O +quaternary O +structures O +of O +the O +two O +conformations O +are O +essentially O +the O +same O +as O +that O +of O +the O +open B-protein_state +and O +closed B-protein_state +states O +observed O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +When O +GDH B-protein +is O +bound B-protein_state +to I-protein_state +NADH B-chemical +, O +GTP B-chemical +, O +and O +glutamate B-chemical +, O +the O +enzyme O +adopts O +a O +closed B-protein_state +conformation O +; O +this O +“ O +abortive O +complex O +” O +has O +been O +determined O +to O +2 O +. O +4 O +- O +Å O +resolution O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +( O +PDB O +3MW9 O +). O + +However O +, O +crystal B-evidence +structures I-evidence +of O +GDH B-protein +bound B-protein_state +only I-protein_state +to I-protein_state +NADH B-chemical +or O +to B-protein_state +GTP B-chemical +have O +not O +yet O +been O +reported O +. O + +To O +test O +the O +effect O +of O +NADH B-chemical +binding O +on O +GDH B-protein +conformation O +in O +solution O +, O +we O +determined O +the O +structure B-evidence +of O +this O +binary O +complex O +using O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +methods O +combined O +with O +three B-experimental_method +- I-experimental_method +dimensional I-experimental_method +classification I-experimental_method +. O + +Two O +dominant O +conformational O +states O +, O +in O +an O +all O +open B-protein_state +or O +all O +closed B-protein_state +conformation O +were O +detected O +, O +segregated O +( O +Fig O +. O +2D O +), O +and O +further O +refined O +to O +near O +- O +atomic O +resolution O +(∼ O +3 O +. O +3 O +Å O +; O +Supplemental O +Fig O +. O +2 O +). O + +Densities B-evidence +for O +12 O +molecules O +of O +bound B-protein_state +NADH B-chemical +were O +identified O +in O +maps B-evidence +of O +both O +open B-protein_state +and O +closed B-protein_state +states O +( O +Supplemental O +Fig O +. O +4 O +). O + +The O +NADH B-protein_state +- I-protein_state +bound I-protein_state +closed B-protein_state +conformation O +matches O +the O +structure B-evidence +of O +the O +quaternary O +complex O +observed O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +, O +with O +the O +exception O +that O +density B-evidence +corresponding O +to O +GTP B-chemical +and O +glutamate B-chemical +was O +absent O +in O +the O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +- O +derived O +map B-evidence +. O + +Comparison O +of O +the O +NADH B-protein_state +- I-protein_state +bound I-protein_state +closed B-protein_state +conformation O +to O +the O +NADH B-protein_state +- I-protein_state +bound I-protein_state +open B-protein_state +conformation O +shows O +that O +, O +as O +expected O +, O +the O +catalytic B-site +cleft I-site +is O +closed B-protein_state +and O +the O +NBDs B-structure_element +are O +displaced O +toward O +the O +equatorial O +plane O +, O +accompanied O +by O +a O +rotation O +of O +the O +pivot B-structure_element +helix I-structure_element +by O +∼ O +7 O +°, O +concomitant O +with O +a O +large O +conformational O +change O +in O +the O +antennae B-structure_element +domains O +( O +Figs O +. O +1 O +and O +2D O +). O + +A O +comparison O +between O +NADH B-protein_state +- I-protein_state +bound I-protein_state +open B-protein_state +and O +closed B-protein_state +conformations O +also O +involves O +a O +displacement O +of O +helix B-structure_element +5 I-structure_element +( O +residues O +171 B-residue_range +– I-residue_range +186 I-residue_range +), O +as O +well O +as O +a O +tilt O +of O +the O +core O +β B-structure_element +- I-structure_element +sheets I-structure_element +relative O +to O +the O +equatorial O +plane O +of O +the O +enzyme O +( O +residues O +57 B-residue_range +– I-residue_range +97 I-residue_range +, O +122 B-residue_range +– I-residue_range +130 I-residue_range +) O +and O +α B-structure_element +- I-structure_element +helix I-structure_element +2 I-structure_element +( O +residues O +36 B-residue_range +– I-residue_range +54 I-residue_range +), O +and O +a O +bending O +of O +the O +N O +- O +terminal O +helix B-structure_element +. O + +Thus O +, O +closure O +of O +the O +catalytic B-site +cleft I-site +is O +accompanied O +by O +a O +quaternary O +structural O +change O +that O +can O +be O +described O +as O +a O +global O +bending O +of O +the O +structure B-evidence +about O +an O +axis O +that O +runs O +parallel O +to O +the O +pivot B-structure_element +helix I-structure_element +, O +accompanied O +by O +an O +expansion O +of O +the O +core O +( O +Figs O +. O +1A O +and O +2D O +). O + +Detailed O +analysis O +of O +the O +GDH B-complex_assembly +/ I-complex_assembly +NADH I-complex_assembly +structures B-evidence +shows O +that O +both O +the O +adenosine O +and O +nicotinamide O +moieties O +of O +NADH B-chemical +bind O +to O +the O +catalytic B-site +site I-site +within O +the O +NBD B-structure_element +in O +nearly O +the O +same O +orientation O +in O +both O +the O +open B-protein_state +and O +the O +closed B-protein_state +states O +, O +and O +display O +closely O +comparable O +interactions O +with O +the O +Rossmann B-structure_element +fold I-structure_element +( O +Fig O +. O +3 O +, O +A O +and O +B O +). O + +At O +the O +regulatory B-site +site I-site +, O +where O +either O +ADP B-chemical +can O +bind O +as O +an O +activator O +or O +NADH B-chemical +can O +bind O +as O +an O +inhibitor O +, O +the O +binding O +of O +the O +adenine O +moiety O +of O +NADH B-chemical +is O +nearly O +identical O +between O +the O +two O +conformers O +. O + +In O +the O +closed B-protein_state +state O +, O +the O +nicotinamide O +group O +is O +oriented O +toward O +the O +center O +of O +the O +hexamer B-oligomeric_state +, O +inserted O +into O +a O +narrow O +cavity B-site +between O +two O +adjacent O +subunits B-structure_element +in O +the O +trimer B-oligomeric_state +. O + +There O +are O +extensive O +interactions O +between O +NADH B-chemical +and O +the O +residues O +lining O +this O +cavity B-site +, O +which O +may O +explain O +the O +well O +- O +defined O +density B-evidence +of O +this O +portion O +of O +NADH B-chemical +in O +the O +closed B-protein_state +state O +. O + +In O +contrast O +, O +in O +the O +open B-protein_state +conformation O +, O +the O +cavity B-site +present O +in O +the O +closed B-protein_state +state O +becomes O +too O +narrow O +for O +the O +nicotinamide O +group O +; O +instead O +, O +the O +group O +is O +oriented O +in O +the O +opposite O +direction O +, O +parallel O +to O +the O +pivot B-structure_element +helix I-structure_element +with O +the O +amido O +group O +extending O +toward O +the O +C O +- O +terminal O +end O +of O +the O +helix B-structure_element +. O + +Detailed O +view O +of O +NADH B-chemical +conformation O +in O +catalytic B-site +and I-site +regulatory I-site +sites I-site +. O +( O +A O +, O +B O +) O +NADH B-chemical +density B-evidence +( O +purple O +) O +and O +interactions O +in O +the O +catalytic B-site +sites I-site +of O +closed B-protein_state +( O +A O +) O +and O +open B-protein_state +( O +B O +) O +states O +. O +( O +C O +, O +D O +) O +NADH B-chemical +density B-evidence +( O +red O +) O +and O +interactions O +in O +the O +regulatory B-site +sites I-site +of O +closed B-protein_state +( O +C O +) O +and O +open B-protein_state +( O +D O +) O +states O +. O + +Although O +there O +is O +a O +difference O +in O +orientation O +of O +the O +nicotinamide O +moiety O +between O +the O +closed B-protein_state +and O +open B-protein_state +states O +in O +the O +regulatory B-site +site I-site +, O +in O +both O +structures B-evidence +the O +adenine O +portion O +of O +NADH B-chemical +has O +a O +similar O +binding B-site +pocket I-site +and O +is O +located O +in O +almost O +exactly O +the O +same O +position O +as O +ADP B-chemical +, O +a O +potent O +activator O +of O +GDH B-protein +function O +( O +Supplemental O +Fig O +. O +5 O +). O + +In O +the O +open B-protein_state +state O +, O +the O +binding O +of O +ADP B-chemical +or O +NADH B-chemical +is O +further O +stabilized O +by O +His209 B-residue_name_number +, O +a O +residue O +that O +undergoes O +a O +large O +movement O +during O +the O +transition O +from O +open B-protein_state +to O +closed B-protein_state +conformation O +( O +Fig O +. O +3 O +, O +C O +and O +D O +). O + +In O +the O +open B-protein_state +conformation O +, O +the O +distance O +between O +His209 B-residue_name_number +and O +the O +α O +- O +phosphate O +of O +NADH B-chemical +is O +∼ O +4 O +. O +4 O +Å O +, O +which O +is O +comparable O +with O +the O +corresponding O +distance O +in O +the O +ADP B-protein_state +- I-protein_state +bound I-protein_state +conformation O +. O + +In O +the O +closed B-protein_state +conformation O +, O +however O +, O +this O +key O +histidine B-residue_name +residue O +is O +> O +10 O +. O +5 O +Å O +away O +from O +the O +nearest O +phosphate O +group O +on O +NADH B-chemical +, O +altering O +a O +critical O +stabilization O +point O +within O +the O +regulatory B-site +site I-site +. O + +This O +suggests O +that O +although O +the O +conformation O +of O +NADH B-chemical +in O +the O +open B-protein_state +state O +regulatory B-site +site I-site +more O +closely O +mimics O +the O +binding O +of O +ADP B-chemical +, O +the O +conformation O +of O +NADH B-chemical +in O +the O +closed B-protein_state +state O +regulatory B-site +site I-site +is O +significantly O +different O +; O +these O +differences O +may O +contribute O +to O +the O +opposite O +effects O +of O +NADH B-chemical +and O +ADP B-chemical +on O +GDH B-protein +enzymatic O +activity O +. O + +In O +the O +absence B-protein_state +of I-protein_state +NADH B-chemical +, O +GTP B-chemical +binds O +weakly O +to O +GDH B-protein +with O +a O +dissociation B-evidence +constant I-evidence +of O +∼ O +20 O +μM O +. O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +analysis O +of O +GDH B-protein +incubated B-protein_state +with I-protein_state +GTP B-chemical +resulted O +in O +a O +structure B-evidence +at O +an O +overall O +resolution O +of O +3 O +. O +5 O +Å O +, O +showing O +that O +it O +is O +in O +an O +open B-protein_state +conformation O +( O +Supplemental O +Fig O +. O +6 O +), O +with O +all O +NBDs B-structure_element +in O +the O +open B-protein_state +state O +. O + +The O +density B-evidence +for O +GTP B-chemical +is O +not O +very O +well O +defined O +, O +suggesting O +considerable O +wobble O +in O +the O +binding B-site +site I-site +. O + +Subtraction B-experimental_method +of O +the O +GTP B-protein_state +- I-protein_state +bound I-protein_state +map B-evidence +with O +that O +of O +the O +apo B-protein_state +state O +shows O +that O +GTP B-chemical +binding O +can O +nevertheless O +be O +visualized O +specifically O +in O +the O +GTP B-site +binding I-site +site I-site +( O +Supplemental O +Fig O +. O +6 O +). O + +Importantly O +, O +the O +binding O +of O +GTP B-chemical +alone O +does O +not O +appear O +to O +drive O +the O +transition O +from O +the O +open B-protein_state +to O +the O +closed B-protein_state +state O +of O +GDH B-protein +. O + +To O +further O +dissect O +the O +roles O +of O +NADH B-chemical +and O +GTP B-chemical +in O +the O +transition O +from O +the O +open B-protein_state +to O +closed B-protein_state +conformations O +, O +we O +next O +determined B-experimental_method +structures B-evidence +of O +GDH B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +both O +NADH B-chemical +and O +GTP B-chemical +, O +but O +without B-protein_state +glutamate B-chemical +. O + +When O +NADH B-chemical +and O +GTP B-chemical +are O +both O +present O +, O +classification B-experimental_method +reveals O +the O +presence B-protein_state +of I-protein_state +both O +closed B-protein_state +and O +open B-protein_state +GDH B-protein +conformations O +, O +similar O +to O +the O +condition O +when O +only O +NADH B-chemical +is O +present O +( O +Fig O +. O +4 O +, O +A O +and O +B O +). O + +Reconstruction B-experimental_method +without I-experimental_method +classification I-experimental_method +, O +however O +, O +yields O +a O +structure B-evidence +clearly O +in O +the O +closed B-protein_state +conformation O +, O +suggesting O +that O +, O +in O +coordination O +with O +NADH B-chemical +, O +GTP B-chemical +may O +further O +stabilize O +the O +closed B-protein_state +conformation O +. O + +The O +location O +of O +GTP B-chemical +in O +the O +open B-protein_state +and O +closed B-protein_state +states O +of O +the O +GDH B-complex_assembly +/ I-complex_assembly +NADH I-complex_assembly +/ I-complex_assembly +GTP I-complex_assembly +complex O +is O +similar O +to O +that O +in O +the O +crystal B-evidence +structure I-evidence +observed O +in O +the O +presence B-protein_state +of I-protein_state +NADH B-chemical +, O +GTP B-chemical +, O +and O +glutamate B-chemical +. O + +Likewise O +, O +the O +position O +of O +NADH B-chemical +in O +the O +open B-protein_state +and O +closed B-protein_state +states O +closely O +resembles O +the O +position O +of O +NADH B-chemical +in O +the O +GDH B-complex_assembly +/ I-complex_assembly +NADH I-complex_assembly +open B-protein_state +and O +closed B-protein_state +structures B-evidence +. O + +One O +key O +difference O +between O +the O +open B-protein_state +and O +closed B-protein_state +states O +of O +these O +structures B-evidence +is O +the O +position O +of O +the O +His209 B-residue_name_number +residue O +: O +As O +mentioned O +above O +, O +His209 B-residue_name_number +swings O +away O +from O +the O +adenine O +moiety O +of O +NADH B-chemical +in O +the O +closed B-protein_state +state O +. O + +When O +GTP B-chemical +is O +present O +in O +the O +GTP B-site +binding I-site +site I-site +, O +His209 B-residue_name_number +instead O +interacts O +with O +GTP B-chemical +, O +probably O +stabilizing O +the O +closed B-protein_state +conformation O +( O +Fig O +. O +4 O +, O +C O +and O +D O +). O + +Thus O +, O +GTP B-chemical +binding O +to O +GDH B-protein +appears O +synergistic O +with O +NADH B-chemical +and O +displaces O +the O +conformational O +landscape O +toward O +the O +closed B-protein_state +state O +. O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structure B-evidence +of O +GDH B-protein +bound B-protein_state +to I-protein_state +both O +NADH B-chemical +and O +GTP B-chemical +. O + +( O +A O +, O +B O +) O +Observation O +of O +co O +- O +existing O +open B-protein_state +( O +A O +) O +and O +closed B-protein_state +( O +B O +) O +conformations O +in O +the O +GDH B-complex_assembly +- I-complex_assembly +NADH I-complex_assembly +- I-complex_assembly +GTP I-complex_assembly +ternary O +complex O +. O + +Densities B-evidence +for O +GTP B-chemical +( O +yellow O +) O +as O +well O +as O +NADH B-chemical +bound B-protein_state +to I-protein_state +both O +catalytic B-site +( O +purple O +) O +and O +regulatory B-site +( O +red O +) O +sites B-site +in O +each O +protomer B-oligomeric_state +are O +shown O +. O + +( O +C O +, O +D O +) O +Detailed O +inspection O +of O +the O +interactions O +near O +the O +regulatory B-site +site I-site +show O +that O +the O +orientation O +of O +His209 B-residue_name_number +switches O +between O +the O +two O +states O +, O +which O +may O +allow O +interactions O +with O +bound B-protein_state +GTP B-chemical +in O +the O +closed B-protein_state +( O +D O +), O +but O +not O +open B-protein_state +( O +C O +) O +conformation O +. O + +Our O +structural B-experimental_method +studies I-experimental_method +thus O +establish O +that O +whether O +or O +not O +GTP B-chemical +is O +bound B-protein_state +, O +NADH B-chemical +binding O +is O +detectable O +at O +catalytic B-site +and I-site +regulatory I-site +sites I-site +, O +in O +both O +the O +open B-protein_state +and O +closed B-protein_state +conformational O +states O +. O + +Whereas O +the O +orientation O +in O +which O +NADH B-chemical +binds O +at O +the O +catalytic B-site +site I-site +is O +similar O +for O +both O +conformations O +, O +the O +orientation O +of O +the O +nicotinamide O +portion O +of O +NADH B-chemical +in O +the O +regulatory B-site +site I-site +is O +different O +between O +the O +open B-protein_state +and O +closed B-protein_state +conformations O +( O +Figs O +. O +3 O +and O +4 O +). O + +In O +the O +closed B-protein_state +state O +, O +the O +nicotinamide O +moiety O +is O +inserted O +into O +a O +well O +- O +defined O +cavity B-site +at O +the O +interface B-site +between O +two O +adjacent O +protomers B-oligomeric_state +in O +the O +trimer B-oligomeric_state +. O + +As O +mentioned O +above O +, O +this O +cavity B-site +is O +much O +narrower O +in O +the O +open B-protein_state +state O +, O +suggesting O +that O +this O +cavity B-site +may O +be O +unavailable O +to O +the O +NADH B-chemical +nicotinamide O +moiety O +when O +the O +enzyme O +is O +in O +the O +open B-protein_state +conformation O +. O + +These O +structural O +features O +provide O +a O +potential O +explanation O +of O +the O +weaker O +density B-evidence +for O +the O +nicotinamide O +moiety O +of O +NADH B-chemical +in O +the O +open B-protein_state +state O +, O +and O +may O +account O +for O +the O +higher O +reported O +affinity O +of O +NADH B-chemical +for O +the O +closed B-protein_state +state O +. O + +The O +role O +of O +the O +nicotinamide O +moiety O +in O +acting O +as O +a O +wedge O +that O +prevents O +the O +transition O +to O +the O +open B-protein_state +conformation O +also O +suggests O +a O +structural O +explanation O +of O +the O +mechanism O +by O +which O +NADH B-chemical +binding O +inhibits O +the O +activity O +of O +the O +enzyme O +by O +stabilizing O +the O +closed B-protein_state +conformation O +state O +. O + +The O +rapid O +emergence O +of O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +as O +a O +tool O +for O +near O +- O +atomic O +resolution O +structure B-experimental_method +determination I-experimental_method +provides O +new O +opportunities O +for O +complementing O +atomic O +resolution O +information O +from O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +, O +as O +illustrated O +here O +with O +GDH B-protein +. O + +Perhaps O +the O +most O +important O +contribution O +of O +these O +methods O +is O +the O +prospect O +that O +when O +there O +are O +discrete O +subpopulations O +present O +, O +the O +structure B-evidence +of O +each O +state O +can O +be O +determined O +at O +near O +- O +atomic O +resolution O +. O +What O +we O +demonstrate O +here O +with O +GDH B-protein +is O +that O +by O +employing O +three B-experimental_method +- I-experimental_method +dimensional I-experimental_method +image I-experimental_method +classification I-experimental_method +approaches I-experimental_method +, O +we O +not O +only O +can O +isolate O +distinct O +, O +coexisting O +conformations O +, O +but O +we O +can O +also O +localize O +small O +molecule O +ligands O +in O +each O +of O +these O +conformations O +. O + +Investigation O +of O +the O +Interaction O +between O +Cdc42 B-protein +and O +Its O +Effector O +TOCA1 B-protein + +Transducer B-protein +of I-protein +Cdc42 I-protein +- I-protein +dependent I-protein +actin I-protein +assembly I-protein +protein I-protein +1 I-protein +( O +TOCA1 B-protein +) O +is O +an O +effector O +of O +the O +Rho B-protein_type +family I-protein_type +small I-protein_type +G I-protein_type +protein I-protein_type +Cdc42 B-protein +. O + +It O +contains O +a O +membrane O +- O +deforming O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +as O +well O +as O +a O +Src B-structure_element +homology I-structure_element +3 I-structure_element +( O +SH3 B-structure_element +) O +domain O +and O +a O +G B-structure_element +protein I-structure_element +- I-structure_element +binding I-structure_element +homology I-structure_element +region I-structure_element +1 I-structure_element +( O +HR1 B-structure_element +) O +domain O +. O + +TOCA1 B-protein +binding O +to O +Cdc42 B-protein +leads O +to O +actin B-protein_type +rearrangements O +, O +which O +are O +thought O +to O +be O +involved O +in O +processes O +such O +as O +endocytosis O +, O +filopodia O +formation O +, O +and O +cell O +migration O +. O + +We O +have O +solved B-experimental_method +the O +structure B-evidence +of O +the O +HR1 B-structure_element +domain O +of O +TOCA1 B-protein +, O +providing O +the O +first O +structural B-evidence +data I-evidence +for O +this O +protein O +. O + +We O +have O +found O +that O +the O +TOCA1 B-protein +HR1 B-structure_element +, O +like O +the O +closely O +related O +CIP4 B-protein +HR1 B-structure_element +, O +has O +interesting O +structural O +features O +that O +are O +not O +observed O +in O +other O +HR1 B-structure_element +domains O +. O + +We O +have O +also O +investigated O +the O +binding O +of O +the O +TOCA B-protein +HR1 B-structure_element +domain O +to O +Cdc42 B-protein +and O +the O +potential O +ternary O +complex O +between O +Cdc42 B-protein +and O +the O +G B-site +protein I-site +- I-site +binding I-site +regions I-site +of O +TOCA1 B-protein +and O +a O +member O +of O +the O +Wiskott B-protein_type +- I-protein_type +Aldrich I-protein_type +syndrome I-protein_type +protein I-protein_type +family I-protein_type +, O +N B-protein +- I-protein +WASP I-protein +. O + +TOCA1 B-protein +binds O +Cdc42 B-protein +with O +micromolar O +affinity O +, O +in O +contrast O +to O +the O +nanomolar O +affinity O +of O +the O +N B-protein +- I-protein +WASP I-protein +G B-site +protein I-site +- I-site +binding I-site +region I-site +for O +Cdc42 B-protein +. O + +NMR B-experimental_method +experiments O +show O +that O +the O +Cdc42 B-site +- I-site +binding I-site +domain I-site +from O +N B-protein +- I-protein +WASP I-protein +is O +able O +to O +displace O +TOCA1 B-protein +HR1 B-structure_element +from O +Cdc42 B-protein +, O +whereas O +the O +N B-protein +- I-protein +WASP I-protein +domain O +but O +not O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +inhibits O +actin O +polymerization O +. O + +This O +suggests O +that O +TOCA1 B-protein +binding O +to O +Cdc42 B-protein +is O +an O +early O +step O +in O +the O +Cdc42 B-protein +- O +dependent O +pathways O +that O +govern O +actin O +dynamics O +, O +and O +the O +differential O +binding B-evidence +affinities I-evidence +of O +the O +effectors O +facilitate O +a O +handover O +from O +TOCA1 B-protein +to O +N B-protein +- I-protein +WASP I-protein +, O +which O +can O +then O +drive O +recruitment O +of O +the O +actin O +- O +modifying O +machinery O +. O + +The O +Ras B-protein_type +superfamily I-protein_type +of O +small B-protein_type +GTPases I-protein_type +comprises O +over O +150 O +members O +that O +regulate O +a O +multitude O +of O +cellular O +processes O +in O +eukaryotes B-taxonomy_domain +. O + +The O +superfamily O +can O +be O +divided O +into O +five O +families O +based O +on O +structural O +and O +functional O +similarities O +: O +Ras B-protein_type +, O +Rho B-protein_type +, O +Rab B-protein_type +, O +Arf B-protein_type +, O +and O +Ran B-protein_type +. O + +All O +members O +share O +a O +well O +defined O +core O +structure O +of O +∼ O +20 O +kDa O +known O +as O +the O +G B-structure_element +domain I-structure_element +, O +which O +is O +responsible O +for O +guanine B-chemical +nucleotide I-chemical +binding O +. O + +These O +molecular O +switches O +cycle O +between O +active B-protein_state +, O +GTP B-protein_state +- I-protein_state +bound I-protein_state +, O +and O +inactive B-protein_state +, O +GDP B-protein_state +- I-protein_state +bound I-protein_state +, O +states O +with O +the O +help O +of O +auxiliary O +proteins O +. O + +The O +guanine B-protein_type +nucleotide I-protein_type +exchange I-protein_type +factors I-protein_type +mediate O +formation O +of O +the O +active B-protein_state +state O +by O +promoting O +the O +dissociation O +of O +GDP B-chemical +, O +allowing O +GTP B-chemical +to O +bind O +. O + +The O +GTPase B-protein_type +- I-protein_type +activating I-protein_type +proteins I-protein_type +stimulate O +the O +rate O +of O +intrinsic O +GTP B-chemical +hydrolysis O +, O +mediating O +the O +return O +to O +the O +inactive B-protein_state +state O +( O +reviewed O +in O +Ref O +.). O + +The O +overall O +conformation O +of O +small B-protein_type +G I-protein_type +proteins I-protein_type +in O +the O +active B-protein_state +and O +inactive B-protein_state +states O +is O +similar O +, O +but O +they O +differ O +significantly O +in O +two O +main O +regions O +known O +as O +switch B-site +I I-site +and O +switch B-site +II I-site +. O + +These O +regions O +are O +responsible O +for O +“ O +sensing O +” O +the O +nucleotide O +state O +, O +with O +the O +GTP B-protein_state +- I-protein_state +bound I-protein_state +state O +showing O +greater O +rigidity O +and O +the O +GDP B-protein_state +- I-protein_state +bound I-protein_state +state O +adopting O +a O +more O +relaxed O +conformation O +( O +reviewed O +in O +Ref O +.). O + +In O +the O +active B-protein_state +state O +, O +G B-protein_type +proteins I-protein_type +bind O +to O +an O +array O +of O +downstream O +effectors O +, O +through O +which O +they O +exert O +their O +extensive O +roles O +within O +the O +cell O +. O + +The O +structures B-evidence +of O +more O +than O +60 O +small O +G B-protein_type +protein I-protein_type +· O +effector O +complexes O +have O +been O +solved B-experimental_method +, O +and O +, O +not O +surprisingly O +, O +the O +switch B-site +regions I-site +have O +been O +implicated O +in O +a O +large O +proportion O +of O +the O +G B-protein_type +protein I-protein_type +- O +effector O +interactions O +( O +reviewed O +in O +Ref O +.). O + +However O +, O +because O +each O +of O +the O +150 O +members O +of O +the O +superfamily O +interacts O +with O +multiple O +effectors O +, O +there O +are O +still O +a O +huge O +number O +of O +known O +G B-protein_type +protein I-protein_type +- O +effector O +interactions O +that O +have O +not O +yet O +been O +studied O +structurally O +. O + +The O +Rho B-protein_type +family I-protein_type +comprises O +20 O +members O +, O +of O +which O +three O +, O +RhoA B-protein +, O +Rac1 B-protein +, O +and O +Cdc42 B-protein +, O +have O +been O +relatively O +well O +studied O +. O + +RhoA B-protein +acts O +to O +rearrange O +existing O +actin O +structures O +to O +form O +stress O +fibers O +, O +whereas O +Rac1 B-protein +and O +Cdc42 B-protein +promote O +de O +novo O +actin O +polymerization O +to O +form O +lamellipodia O +and O +filopodia O +, O +respectively O +. O + +A O +number O +of O +RhoA B-protein +and O +Rac1 B-protein +effector O +proteins O +, O +including O +the O +formins O +and O +members O +of O +the O +protein B-protein_type +kinase I-protein_type +C I-protein_type +- I-protein_type +related I-protein_type +kinase I-protein_type +( O +PRK B-protein_type +) O +6 B-protein_type +family O +, O +along O +with O +Cdc42 B-protein +effectors O +, O +including O +the O +Wiskott B-protein_type +- I-protein_type +Aldrich I-protein_type +syndrome I-protein_type +( O +WASP B-protein_type +) O +family O +and O +the O +transducer O +of O +Cdc42 B-protein_type +- I-protein_type +dependent I-protein_type +actin I-protein_type +assembly I-protein_type +( O +TOCA B-protein_type +) O +family O +, O +have O +also O +been O +linked O +to O +the O +pathways O +that O +govern O +cytoskeletal O +dynamics O +. O + +Cdc42 B-protein +effectors O +, O +TOCA1 B-protein +and O +the O +ubiquitously O +expressed O +member O +of O +the O +WASP B-protein_type +family I-protein_type +, O +N B-protein +- I-protein +WASP I-protein +, O +have O +been O +implicated O +in O +the O +regulation O +of O +actin O +polymerization O +downstream O +of O +Cdc42 B-protein +and O +phosphatidylinositol B-chemical +4 I-chemical +, I-chemical +5 I-chemical +- I-chemical +bisphosphate I-chemical +( O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +). O + +N B-protein +- I-protein +WASP I-protein +exists O +in O +an O +autoinhibited B-protein_state +conformation I-protein_state +, O +which O +is O +released O +upon O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +and O +Cdc42 B-protein +binding O +or O +by O +other O +factors O +, O +such O +as O +phosphorylation O +. O + +Following O +their O +release O +, O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +regions I-structure_element +of O +N B-protein +- I-protein +WASP I-protein +are O +free O +to O +interact O +with O +G B-protein_type +- I-protein_type +actin I-protein_type +and O +a O +known O +nucleator O +of O +actin O +assembly O +, O +the O +Arp2 B-complex_assembly +/ I-complex_assembly +3 I-complex_assembly +complex O +. O + +The O +importance O +of O +TOCA1 B-protein +in O +actin O +polymerization O +has O +been O +demonstrated O +in O +a O +range O +of O +in O +vitro O +and O +in O +vivo O +studies O +, O +but O +the O +exact O +role O +of O +TOCA1 B-protein +in O +the O +many O +pathways O +involving O +actin O +assembly O +remains O +unclear O +. O + +The O +most O +widely O +studied O +role O +of O +TOCA1 B-protein +is O +in O +membrane O +invagination O +and O +endocytosis O +, O +although O +it O +has O +also O +been O +implicated O +in O +filopodia O +formation O +, O +neurite O +elongation O +, O +transcriptional O +reprogramming O +via O +nuclear O +actin B-protein_type +, O +and O +interaction O +with O +ZO B-protein +- I-protein +1 I-protein +at O +tight O +junctions O +. O + +TOCA1 B-protein +comprises O +an O +N O +- O +terminal O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +, O +a O +central B-structure_element +homology I-structure_element +region I-structure_element +1 I-structure_element +( O +HR1 B-structure_element +) O +domain O +, O +and O +a O +C O +- O +terminal O +SH3 B-structure_element +domain O +. O + +The O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +is O +a O +known O +dimerization O +, O +membrane O +- O +binding O +, O +and O +membrane O +- O +deforming O +module O +found O +in O +a O +number O +of O +cell O +signaling O +proteins O +. O + +The O +TOCA1 B-protein +SH3 B-structure_element +domain O +has O +many O +known O +binding O +partners O +, O +including O +N B-protein +- I-protein +WASP I-protein +and O +dynamin B-protein +. O + +The O +HR1 B-structure_element +domain O +has O +been O +directly O +implicated O +in O +the O +interaction O +between O +TOCA1 B-protein +and O +Cdc42 B-protein +, O +representing O +the O +first O +Cdc42 B-protein +- O +HR1 B-structure_element +domain O +interaction O +to O +be O +identified O +. O + +Other O +HR1 B-structure_element +domains O +studied O +so O +far O +, O +including O +those O +from O +the O +PRK B-protein_type +family I-protein_type +, O +have O +been O +found O +to O +bind O +their O +cognate O +Rho O +family O +G B-protein_type +protein I-protein_type +- O +binding O +partner O +with O +high O +specificity O +and O +affinities B-evidence +in O +the O +nanomolar O +range O +. O + +The O +structures B-evidence +of O +the O +PRK1 B-protein +HR1a B-structure_element +domain O +in O +complex B-protein_state +with I-protein_state +RhoA B-protein +and O +the O +HR1b B-structure_element +domain O +in O +complex B-protein_state +with I-protein_state +Rac1 B-protein +show O +that O +the O +HR1 B-structure_element +domain O +comprises O +an O +anti B-structure_element +- I-structure_element +parallel I-structure_element +coiled I-structure_element +- I-structure_element +coil I-structure_element +that O +interacts O +with O +its O +G B-protein_type +protein I-protein_type +binding O +partner O +via O +both O +helices B-structure_element +. O + +Both O +of O +the O +G B-site +protein I-site +switch I-site +regions I-site +are O +involved O +in O +the O +interaction O +. O + +The O +coiled B-structure_element +- I-structure_element +coil I-structure_element +fold I-structure_element +is O +shared O +by O +the O +HR1 B-structure_element +domain O +of O +the O +TOCA B-protein_type +family I-protein_type +protein I-protein_type +, O +CIP4 B-protein +, O +and O +, O +based O +on O +sequence O +homology O +, O +by O +TOCA1 B-protein +itself O +. O + +These O +HR1 B-structure_element +domains O +, O +however O +, O +show O +specificity O +for O +Cdc42 B-protein +, O +rather O +than O +RhoA B-protein +or O +Rac1 B-protein +. O + +How O +different O +HR1 B-structure_element +domain O +proteins O +distinguish O +their O +specific O +G B-protein_type +protein I-protein_type +partners O +remains O +only O +partially O +understood O +, O +and O +structural O +characterization O +of O +a O +novel O +G B-protein_type +protein I-protein_type +- O +HR1 B-structure_element +domain O +interaction O +would O +add O +to O +the O +growing O +body O +of O +information O +pertaining O +to O +these O +protein O +complexes O +. O + +Furthermore O +, O +the O +biological O +function O +of O +the O +interaction O +between O +TOCA1 B-protein +and O +Cdc42 B-protein +remains O +poorly O +understood O +, O +and O +so O +far O +there O +has O +been O +no O +biophysical O +or O +structural O +insight O +. O + +The O +interactions O +of O +TOCA1 B-protein +and O +N B-protein +- I-protein +WASP I-protein +with O +Cdc42 B-protein +as O +well O +as O +with O +each O +other O +have O +raised O +questions O +as O +to O +whether O +the O +two O +Cdc42 B-protein +effectors O +can O +interact O +with O +a O +single O +molecule O +of O +Cdc42 B-protein +simultaneously O +. O + +There O +is O +some O +evidence O +for O +a O +ternary O +complex O +between O +Cdc42 B-protein +, O +N B-protein +- I-protein +WASP I-protein +, O +and O +TOCA1 B-protein +, O +but O +there O +was O +no O +direct O +demonstration O +of O +simultaneous O +contacts O +between O +the O +two O +effectors O +and O +a O +single O +molecule O +of O +Cdc42 B-protein +. O + +Nonetheless O +, O +the O +substantial O +difference O +between O +the O +structures B-evidence +of O +the O +G B-site +protein I-site +- I-site +binding I-site +regions I-site +of O +the O +two O +effectors O +is O +intriguing O +and O +implies O +that O +they O +bind O +to O +Cdc42 B-protein +quite O +differently O +, O +providing O +motivation O +for O +investigating O +the O +possibility O +that O +Cdc42 B-protein +can O +bind O +both O +effectors O +concurrently O +. O + +WASP B-protein_type +interacts O +with O +Cdc42 B-protein +via O +a O +conserved B-protein_state +, O +unstructured B-structure_element +binding I-structure_element +motif I-structure_element +known O +as O +the O +Cdc42 B-structure_element +- I-structure_element +and I-structure_element +Rac I-structure_element +- I-structure_element +interactive I-structure_element +binding I-structure_element +region I-structure_element +( O +CRIB B-structure_element +), O +which O +forms O +an O +intermolecular B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +, O +expanding O +the O +anti O +- O +parallel O +β2 B-structure_element +and I-structure_element +β3 I-structure_element +strands I-structure_element +of O +Cdc42 B-protein +. O + +In O +contrast O +, O +the O +TOCA B-protein_type +family I-protein_type +proteins I-protein_type +are O +thought O +to O +interact O +via O +the O +HR1 B-structure_element +domain O +, O +which O +may O +form O +a O +triple B-structure_element +coiled I-structure_element +- I-structure_element +coil I-structure_element +with O +switch B-site +II I-site +of O +Rac1 B-protein +, O +like O +the O +HR1b B-structure_element +domain O +of O +PRK1 B-protein +. O + +Here O +, O +we O +present O +the O +solution B-experimental_method +NMR I-experimental_method +structure B-evidence +of O +the O +HR1 B-structure_element +domain O +of O +TOCA1 B-protein +, O +providing O +the O +first O +structural B-evidence +data I-evidence +for O +this O +protein O +. O + +We O +also O +present O +data O +pertaining O +to O +binding O +of O +the O +TOCA B-protein_type +HR1 B-structure_element +domain O +to O +Cdc42 B-protein +, O +which O +is O +the O +first O +biophysical O +description O +of O +an O +HR1 B-structure_element +domain O +binding O +this O +particular O +Rho B-protein_type +family I-protein_type +small I-protein_type +G I-protein_type +protein I-protein_type +. O + +Finally O +, O +we O +investigate O +the O +potential O +ternary O +complex O +between O +Cdc42 B-protein +and O +the O +G B-site +protein I-site +- I-site +binding I-site +regions I-site +of O +TOCA1 B-protein +and O +N B-protein +- I-protein +WASP I-protein +, O +contributing O +to O +our O +understanding O +of O +G B-protein_type +protein I-protein_type +- O +effector O +interactions O +as O +well O +as O +the O +roles O +of O +Cdc42 B-protein +, O +N B-protein +- I-protein +WASP I-protein +, O +and O +TOCA1 B-protein +in O +the O +pathways O +that O +govern O +actin B-protein_type +dynamics O +. O + +Cdc42 B-protein +- O +TOCA1 B-protein +Binding O + +TOCA1 B-protein +was O +identified O +in O +Xenopus B-taxonomy_domain +extracts O +as O +a O +protein O +necessary O +for O +Cdc42 B-protein +- O +dependent O +actin O +assembly O +and O +was O +shown O +to O +bind O +to O +Cdc42 B-complex_assembly +· I-complex_assembly +GTPγS I-complex_assembly +but O +not O +to O +Cdc42 B-complex_assembly +· I-complex_assembly +GDP I-complex_assembly +or O +to O +Rac1 B-protein +and O +RhoA B-protein +. O +Given O +its O +homology O +to O +other O +Rho B-site +family I-site +binding I-site +modules I-site +, O +it O +is O +likely O +that O +the O +HR1 B-structure_element +domain O +of O +TOCA1 B-protein +is O +sufficient O +to O +bind O +Cdc42 B-protein +. O + +The O +C B-species +. I-species +elegans I-species +TOCA1 B-protein +orthologues O +also O +bind O +to O +Cdc42 B-protein +via O +their O +consensus O +HR1 B-structure_element +domain O +. O + +The O +HR1 B-structure_element +domains O +from O +the O +PRK B-protein_type +family I-protein_type +bind O +their O +G B-protein_type +protein I-protein_type +partners O +with O +a O +high O +affinity O +, O +exhibiting O +a O +range O +of O +submicromolar O +dissociation B-evidence +constants I-evidence +( O +Kd B-evidence +) O +as O +low O +as O +26 O +nm O +. O + +A O +Kd B-evidence +in O +the O +nanomolar O +range O +was O +therefore O +expected O +for O +the O +interaction O +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +with O +Cdc42 B-protein +. O + +We O +generated O +an O +X B-species +. I-species +tropicalis I-species +TOCA1 B-protein +HR1 B-structure_element +domain O +construct O +encompassing O +residues O +330 B-residue_range +– I-residue_range +426 I-residue_range +. O + +This O +region O +comprises O +the O +complete O +HR1 B-structure_element +domain O +based O +on O +secondary O +structure O +predictions O +and O +sequence B-experimental_method +alignments I-experimental_method +with O +another O +TOCA B-protein_type +family I-protein_type +member O +, O +CIP4 B-protein +, O +whose O +structure B-evidence +has O +been O +determined O +. O + +The O +interaction O +between O +[ B-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +· I-complex_assembly +Cdc42 I-complex_assembly +and O +a O +C O +- O +terminally O +His B-protein_state +- I-protein_state +tagged I-protein_state +TOCA1 B-protein +HR1 B-structure_element +domain O +construct O +was O +investigated O +using O +SPA B-experimental_method +. O + +The O +binding B-evidence +isotherm I-evidence +for O +the O +interaction O +is O +shown O +in O +Fig O +. O +1A O +, O +together O +with O +the O +Cdc42 B-protein +- O +PAK B-protein +interaction O +as O +a O +positive O +control O +. O + +The O +binding O +of O +TOCA1 B-protein +HR1 B-structure_element +to O +Cdc42 B-protein +was O +unexpectedly O +weak O +, O +with O +a O +Kd B-evidence +of O +> O +1 O +μm O +. O + +It O +was O +not O +possible O +to O +estimate O +the O +Kd B-evidence +more O +accurately O +using O +direct O +SPA B-experimental_method +experiments O +, O +because O +saturation O +could O +not O +be O +reached O +due O +to O +nonspecific O +signal O +at O +higher O +protein O +concentrations O +. O + +The O +TOCA1 B-protein +HR1 B-structure_element +- O +Cdc42 B-protein +interaction O +is O +low O +affinity O +. O + +A O +, O +curves O +derived O +from O +direct B-experimental_method +binding I-experimental_method +assays I-experimental_method +in O +which O +the O +indicated O +concentrations O +of O +Cdc42Δ7Q61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +were O +incubated B-experimental_method +with O +30 O +nm O +GST B-mutant +- I-mutant +PAK I-mutant +or O +HR1 B-mutant +- I-mutant +His6 I-mutant +in O +SPAs B-experimental_method +. O + +The O +SPA B-experimental_method +signal O +was O +corrected O +by O +subtraction O +of O +control O +data O +with O +no O +GST B-mutant +- I-mutant +PAK I-mutant +or O +HR1 B-mutant +- I-mutant +His6 I-mutant +. O + +The O +data O +were O +fitted O +to O +a O +binding B-evidence +isotherm I-evidence +to O +give O +an O +apparent O +Kd B-evidence +and O +are O +expressed O +as O +a O +percentage O +of O +the O +maximum O +signal O +; O +B O +and O +C O +, O +competition B-experimental_method +SPA I-experimental_method +experiments O +were O +carried O +out O +with O +the O +indicated O +concentrations O +of O +ACK B-protein +GBD B-structure_element +( O +B O +) O +or O +HR1 B-structure_element +domain O +( O +C O +) O +titrated B-experimental_method +into O +30 O +nm O +GST B-mutant +- I-mutant +ACK I-mutant +and O +either O +30 O +nm O +Cdc42Δ7Q61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +or O +full B-protein_state +- I-protein_state +length I-protein_state +Cdc42Q61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +. O + +The O +Kd B-evidence +values O +derived O +for O +the O +ACK B-protein +GBD B-structure_element +with O +Cdc42Δ7 B-mutant +and O +full B-protein_state +- I-protein_state +length I-protein_state +Cdc42 B-protein +were O +0 O +. O +032 O +± O +0 O +. O +01 O +and O +0 O +. O +011 O +± O +0 O +. O +01 O +μm O +, O +respectively O +. O + +The O +Kd B-evidence +values O +derived O +for O +the O +TOCA1 B-protein +HR1 B-structure_element +with O +Cdc42Δ7 B-mutant +and O +full B-protein_state +- I-protein_state +length I-protein_state +Cdc42 B-protein +were O +6 O +. O +05 O +± O +1 O +. O +96 O +and O +5 O +. O +39 O +± O +1 O +. O +69 O +μm O +, O +respectively O +. O + +It O +was O +possible O +that O +the O +low O +affinity O +observed O +was O +due O +to O +negative O +effects O +of O +immobilization O +of O +the O +HR1 B-structure_element +domain O +, O +so O +other O +methods O +were O +employed O +, O +which O +utilized O +untagged B-protein_state +proteins O +. O + +Isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +was O +carried O +out O +, O +but O +no O +heat O +changes O +were O +observed O +at O +a O +range O +of O +concentrations O +and O +temperatures O +( O +data O +not O +shown O +), O +suggesting O +that O +the O +interaction O +is O +predominantly O +entropically O +driven O +. O + +Other O +G B-protein_type +protein I-protein_type +- O +HR1 B-structure_element +domain O +interactions O +have O +also O +failed O +to O +show O +heat O +changes O +in O +our O +hands O +. O +7 O +Infrared B-experimental_method +interferometry I-experimental_method +with O +immobilized B-protein_state +Cdc42 B-protein +was O +also O +attempted O +but O +was O +unsuccessful O +for O +both O +TOCA1 B-protein +HR1 B-structure_element +and O +for O +the O +positive O +control O +, O +ACK B-protein +. O + +The O +affinity B-evidence +was O +therefore O +determined O +using O +competition B-experimental_method +SPAs I-experimental_method +. O + +A O +complex O +of O +a O +GST B-experimental_method +fusion I-experimental_method +of O +the O +GBD B-structure_element +of O +ACK B-protein +, O +which O +binds O +with O +a O +high O +affinity O +to O +Cdc42 B-protein +, O +with O +radiolabeled O +[ B-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +· I-complex_assembly +Cdc42 I-complex_assembly +was O +preformed O +, O +and O +the O +effect O +of O +increasing B-experimental_method +concentrations I-experimental_method +of O +untagged B-protein_state +TOCA1 B-protein +HR1 B-structure_element +domain O +was O +examined O +. O + +Competition O +of O +GST B-mutant +- I-mutant +ACK I-mutant +GBD B-structure_element +bound B-protein_state +to I-protein_state +[ B-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +· I-complex_assembly +Cdc42 I-complex_assembly +by O +free B-protein_state +ACK B-protein +GBD B-structure_element +was O +used O +as O +a O +control O +and O +to O +establish O +the O +value O +of O +background O +counts O +when O +Cdc42 B-protein +is O +fully O +displaced O +. O + +The O +data O +were O +fitted O +to O +a O +binding B-evidence +isotherm I-evidence +describing O +competition O +. O + +Free B-protein_state +ACK B-protein +competed O +with O +itself O +with O +an O +affinity B-evidence +of O +32 O +nm O +, O +similar O +to O +the O +value O +obtained O +by O +direct B-experimental_method +binding I-experimental_method +of O +23 O +nm O +. O + +The O +TOCA1 B-protein +HR1 B-structure_element +domain O +also O +fully O +competed O +with O +the O +GST B-mutant +- I-mutant +ACK I-mutant +but O +bound B-protein_state +with O +an O +affinity B-evidence +of O +6 O +μm O +( O +Fig O +. O +1 O +, O +B O +and O +C O +), O +in O +agreement O +with O +the O +low O +affinity B-evidence +observed O +in O +the O +direct B-experimental_method +binding I-experimental_method +experiments I-experimental_method +. O + +The O +Cdc42 B-protein +construct O +used O +in O +the O +binding B-experimental_method +assays I-experimental_method +has O +seven B-residue_range +residues I-residue_range +deleted B-experimental_method +from O +the O +C O +terminus O +to O +facilitate O +purification O +. O + +These O +residues O +are O +not O +generally O +required O +for O +G B-protein_type +protein I-protein_type +- O +effector O +interactions O +, O +including O +the O +interaction O +between O +RhoA B-protein +and O +the O +PRK1 B-protein +HR1a B-structure_element +domain O +. O + +In O +contrast O +, O +the O +C O +terminus O +of O +Rac1 B-protein +contains O +a O +polybasic O +sequence O +, O +which O +is O +crucial O +for O +Rac1 B-protein +binding O +to O +the O +HR1b B-structure_element +domain O +from O +PRK1 B-protein +. O + +As O +the O +observed O +affinity B-evidence +between O +TOCA1 B-protein +HR1 B-structure_element +and O +Cdc42 B-protein +was O +much O +lower O +than O +expected O +, O +we O +reasoned O +that O +the O +C O +terminus O +of O +Cdc42 B-protein +might O +be O +necessary O +for O +a O +high O +affinity B-evidence +interaction O +. O + +The O +binding B-experimental_method +experiments I-experimental_method +were O +repeated O +with O +full B-protein_state +- I-protein_state +length I-protein_state +[ B-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +· I-complex_assembly +Cdc42 I-complex_assembly +, O +but O +the O +affinity B-evidence +of O +the O +HR1 B-structure_element +domain O +for O +full B-protein_state +- I-protein_state +length I-protein_state +Cdc42 B-protein +was O +similar O +to O +its O +affinity B-evidence +for O +truncated B-protein_state +Cdc42 B-protein +( O +Kd B-evidence +≈ O +5 O +μm O +; O +Fig O +. O +1C O +). O + +Thus O +, O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +Cdc42 B-protein +is O +not O +required O +for O +maximal O +binding O +of O +TOCA1 B-protein +HR1 B-structure_element +. O + +Another O +possible O +explanation O +for O +the O +low O +affinities B-evidence +observed O +was O +that O +the O +HR1 B-structure_element +domain O +alone B-protein_state +is O +not O +sufficient O +for O +maximal O +binding O +of O +the O +TOCA B-protein_type +proteins I-protein_type +to O +Cdc42 B-protein +and O +that O +the O +other O +domains O +are O +required O +. O + +Indeed O +, O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +downs I-experimental_method +performed O +with O +in O +vitro O +translated O +human B-species +TOCA1 B-protein +fragments O +had O +suggested O +that O +residues O +N O +- O +terminal O +to O +the O +HR1 B-structure_element +domain O +may O +be O +required O +to O +stabilize O +the O +HR1 B-structure_element +domain O +structure O +. O + +Furthermore O +, O +both O +BAR B-structure_element +and O +SH3 B-structure_element +domains O +have O +been O +implicated O +in O +interactions O +with O +small O +G B-protein_type +proteins I-protein_type +( O +e O +. O +g O +. O +the O +BAR B-structure_element +domain O +of O +Arfaptin2 B-protein +binds O +to O +Rac1 B-protein +and O +Arl1 B-protein +), O +while O +an O +SH3 B-structure_element +domain O +mediates O +the O +interaction O +between O +Rac1 B-protein +and O +the O +guanine B-protein +nucleotide I-protein +exchange I-protein +factor I-protein +, O +β B-protein +- I-protein +PIX I-protein +. O + +TOCA1 B-protein +dimerizes B-oligomeric_state +via O +its O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +, O +which O +could O +also O +affect O +Cdc42 B-protein +binding O +, O +for O +example O +by O +presenting O +two O +HR1 B-structure_element +domains O +for O +Cdc42 B-protein +interactions O +. O + +Various O +TOCA1 B-protein +fragments O +( O +Fig O +. O +2A O +) O +were O +therefore O +assessed O +for O +binding O +to O +full B-protein_state +- I-protein_state +length I-protein_state +Cdc42 B-protein +by O +direct O +SPA B-experimental_method +. O + +The O +isolated O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +showed O +no O +binding O +to O +full B-protein_state +- I-protein_state +length I-protein_state +Cdc42 B-protein +( O +Fig O +. O +2B O +). O + +Full B-protein_state +- I-protein_state +length I-protein_state +TOCA1 B-protein +and O +ΔSH3 B-mutant +TOCA1 B-protein +bound B-protein_state +with O +micromolar O +affinity O +( O +Fig O +. O +2B O +), O +in O +a O +similar O +manner O +to O +the O +isolated O +HR1 B-structure_element +domain O +( O +Fig O +. O +1A O +). O + +The O +HR1 B-mutant +- I-mutant +SH3 I-mutant +protein O +could O +not O +be O +purified O +to O +homogeneity O +as O +a O +fusion O +protein O +, O +so O +it O +was O +assayed O +in O +competition B-experimental_method +assays I-experimental_method +after O +cleavage O +of O +the O +His O +tag O +. O + +This O +construct O +competed O +with O +GST B-mutant +- I-mutant +ACK I-mutant +GBD B-structure_element +to O +give O +a O +similar O +affinity O +to O +the O +HR1 B-structure_element +domain O +alone B-protein_state +( O +Kd B-evidence += O +4 O +. O +6 O +± O +4 O +μm O +; O +Fig O +. O +2C O +). O + +Taken O +together O +, O +these O +data O +suggest O +that O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +sufficient O +for O +maximal O +binding O +and O +that O +this O +binding O +is O +of O +a O +relatively O +low O +affinity O +compared O +with O +many O +other O +Cdc42 B-protein +· O +effector O +complexes O +. O + +The O +Cdc42 B-complex_assembly +- I-complex_assembly +HR1 I-complex_assembly +interaction O +is O +of O +low O +affinity O +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +protein O +and O +in O +TOCA1 B-protein +paralogues O +. O + +A O +, O +diagram O +illustrating O +the O +TOCA1 B-protein +constructs O +assayed O +for O +Cdc42 B-protein +binding O +. O + +Domain O +boundaries O +are O +derived O +from O +secondary O +structure O +predictions O +; O +B O +, O +binding B-evidence +curves I-evidence +derived O +from O +direct B-experimental_method +binding I-experimental_method +assays I-experimental_method +, O +in O +which O +the O +indicated O +concentrations O +of O +Cdc42Δ7Q61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +were O +incubated B-experimental_method +with O +30 O +nm O +GST B-mutant +- I-mutant +ACK I-mutant +or O +His B-protein_state +- I-protein_state +tagged I-protein_state +TOCA1 B-protein +constructs O +, O +as O +indicated O +, O +in O +SPAs B-experimental_method +. O + +The O +SPA B-experimental_method +signal O +was O +corrected O +by O +subtraction O +of O +control O +data O +with O +no O +fusion O +protein O +. O + +The O +data O +were O +fitted O +to O +a O +binding B-evidence +isotherm I-evidence +to O +give O +an O +apparent O +Kd B-evidence +and O +are O +expressed O +as O +a O +percentage O +of O +the O +maximum O +signal O +. O + +C O +– O +E O +, O +representative O +examples O +of O +competition B-experimental_method +SPA I-experimental_method +experiments O +carried O +out O +with O +the O +indicated O +concentrations O +of O +the O +TOCA1 B-protein +HR1 B-mutant +- I-mutant +SH3 I-mutant +construct O +titrated B-experimental_method +into O +30 O +nm O +GST B-mutant +- I-mutant +ACK I-mutant +and O +30 O +nm O +Cdc42Δ7Q61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +( O +C O +) O +or O +HR1CIP4 B-structure_element +( O +D O +) O +or O +HR1FBP17 B-structure_element +( O +E O +) O +titrated B-experimental_method +into O +30 O +nm O +GST B-mutant +- I-mutant +ACK I-mutant +and O +30 O +nm O +Cdc42FLQ61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +. O + +The O +low O +affinity O +of O +the O +TOCA1 B-protein +HR1 B-structure_element +- O +Cdc42 B-protein +interaction O +raised O +the O +question O +of O +whether O +the O +other O +known O +Cdc42 B-protein +- O +binding O +TOCA B-protein_type +family I-protein_type +proteins I-protein_type +, O +FBP17 B-protein +and O +CIP4 B-protein +, O +also O +bind O +weakly O +. O + +The O +HR1 B-structure_element +domains O +from O +FBP17 B-protein +and O +CIP4 B-protein +were O +purified B-experimental_method +and O +assayed O +for O +Cdc42 B-protein +binding O +in O +competition B-experimental_method +SPAs I-experimental_method +, O +analogous O +to O +those O +carried O +out O +with O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +. O + +The O +affinities B-evidence +of O +both O +the O +FBP17 B-protein +and O +CIP4 B-protein +HR1 B-structure_element +domains O +were O +also O +in O +the O +low O +micromolar O +range O +( O +10 O +and O +5 O +μm O +, O +respectively O +) O +( O +Fig O +. O +2 O +, O +D O +and O +E O +), O +suggesting O +that O +low O +affinity O +interactions O +with O +Cdc42 B-protein +are O +a O +common O +feature O +within O +the O +TOCA B-protein_type +family I-protein_type +. O + +Structure B-evidence +of O +the O +TOCA1 B-protein +HR1 B-structure_element +Domain O + +Because O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +was O +sufficient O +for O +maximal O +Cdc42 B-protein +- O +binding O +, O +we O +used O +this O +construct O +for O +structural O +studies O +. O + +Initial O +experiments O +were O +performed O +with O +TOCA1 B-protein +residues O +324 B-residue_range +– I-residue_range +426 I-residue_range +, O +but O +we O +observed O +that O +the O +N O +terminus O +was O +cleaved O +during O +purification O +to O +yield O +a O +new O +N O +terminus O +at O +residue O +330 B-residue_number +( O +data O +not O +shown O +). O + +We O +therefore O +engineered O +a O +construct O +comprising O +residues O +330 B-residue_range +– I-residue_range +426 I-residue_range +to O +produce O +the O +minimal B-protein_state +, O +stable B-protein_state +HR1 B-structure_element +domain O +. O + +2 O +, O +778 O +non O +- O +degenerate O +NOE B-evidence +restraints I-evidence +were O +used O +in O +initial O +structure B-experimental_method +calculations I-experimental_method +( O +1 O +, O +791 O +unambiguous O +and O +987 O +ambiguous O +), O +derived O +from O +three O +- O +dimensional O +15N B-experimental_method +- I-experimental_method +separated I-experimental_method +NOESY I-experimental_method +and O +13C B-experimental_method +- I-experimental_method +separated I-experimental_method +NOESY I-experimental_method +experiments O +. O + +There O +were O +1 O +, O +845 O +unambiguous O +NOEs B-evidence +and O +757 O +ambiguous O +NOEs B-evidence +after O +eight O +iterations O +. O + +100 O +structures B-evidence +were O +calculated B-experimental_method +in O +the O +final O +iteration O +; O +the O +50 O +lowest O +energy O +structures B-evidence +were O +water O +- O +refined O +; O +and O +of O +these O +, O +the O +35 O +lowest O +energy O +structures B-evidence +were O +analyzed O +. O + +Table O +1 O +indicates O +that O +the O +HR1 B-structure_element +domain O +structure B-evidence +is O +well O +defined O +by O +the O +NMR B-experimental_method +data O +. O + +a O +< O +SA O +>, O +the O +average B-evidence +root I-evidence +mean I-evidence +square I-evidence +deviations I-evidence +for O +the O +ensemble O +± O +S O +. O +D O +. O + +b O +< O +SA O +> O +c O +, O +values O +for O +the O +structure B-evidence +that O +is O +closest O +to O +the O +mean O +. O + +The O +structure B-evidence +closest O +to O +the O +mean O +is O +shown O +in O +Fig O +. O +3A O +. O + +The O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +of O +the O +HR1 B-structure_element +domain O +interact O +to O +form O +an O +anti B-structure_element +- I-structure_element +parallel I-structure_element +coiled I-structure_element +- I-structure_element +coil I-structure_element +with O +a O +slight O +left O +- O +handed O +twist O +, O +reminiscent O +of O +the O +HR1 B-structure_element +domains O +of O +CIP4 B-protein +( O +PDB O +code O +2KE4 O +) O +and O +PRK1 B-protein +( O +PDB O +codes O +1CXZ O +and O +1URF O +). O + +A O +sequence B-experimental_method +alignment I-experimental_method +illustrating O +the O +secondary O +structure O +elements O +of O +the O +TOCA1 B-protein +and O +CIP4 B-protein +HR1 B-structure_element +domains O +and O +the O +HR1a B-structure_element +and O +HR1b B-structure_element +domains O +from O +PRK1 B-protein +is O +shown O +in O +Fig O +. O +3B O +. O + +The O +structure B-evidence +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +. O + +A O +, O +the O +backbone O +trace B-evidence +of O +the O +35 O +lowest O +energy O +structures B-evidence +of O +the O +HR1 B-structure_element +domain O +overlaid O +with O +the O +structure B-evidence +closest O +to O +the O +mean O +is O +shown O +alongside O +a O +schematic O +representation O +of O +the O +structure B-evidence +closest O +to O +the O +mean O +. O + +Flexible O +regions O +at O +the O +N O +and O +C O +termini O +( O +residues O +330 B-residue_range +– I-residue_range +333 I-residue_range +and O +421 B-residue_range +– I-residue_range +426 I-residue_range +) O +are O +omitted O +for O +clarity O +. O + +B O +, O +a O +sequence B-experimental_method +alignment I-experimental_method +of O +the O +HR1 B-structure_element +domains O +from O +TOCA1 B-protein +, O +CIP4 B-protein +, O +and O +PRK1 B-protein +. O + +The O +secondary O +structure O +was O +deduced O +using O +Stride B-experimental_method +, O +based O +on O +the O +Ramachandran B-evidence +angles I-evidence +, O +and O +is O +indicated O +as O +follows O +: O +gray O +, O +turn O +; O +yellow O +, O +α B-structure_element +- I-structure_element +helix I-structure_element +; O +blue O +, O +310 B-structure_element +helix I-structure_element +; O +white O +, O +coil O +. O + +C O +, O +a O +close O +- O +up O +of O +the O +N O +- O +terminal O +region O +of O +TOCA1 B-protein +HR1 B-structure_element +, O +indicating O +some O +of O +the O +NOEs B-evidence +defining O +its O +position O +with O +respect O +to O +the O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +Dotted O +lines O +, O +NOE B-evidence +restraints I-evidence +. O + +D O +, O +a O +close O +- O +up O +of O +the O +interhelix B-structure_element +loop I-structure_element +region O +showing O +some O +of O +the O +contacts O +between O +the O +loop B-structure_element +and O +helix B-structure_element +1 I-structure_element +. O + +In O +the O +HR1a B-structure_element +domain O +of O +PRK1 B-protein +, O +a O +region O +N O +- O +terminal O +to O +helix B-structure_element +1 I-structure_element +forms O +a O +short B-structure_element +α I-structure_element +- I-structure_element +helix I-structure_element +, O +which O +packs O +against O +both O +helices O +of O +the O +HR1 B-structure_element +domain O +. O + +This O +region O +of O +TOCA1 B-protein +HR1 B-structure_element +( O +residues O +334 B-residue_range +– I-residue_range +340 I-residue_range +) O +is O +well O +defined O +in O +the O +family O +of O +structures B-evidence +( O +Fig O +. O +3A O +) O +but O +does O +not O +form O +an O +α B-structure_element +- I-structure_element +helix I-structure_element +. O + +It O +instead O +forms O +a O +series O +of O +turns O +, O +defined O +by O +NOE B-evidence +restraints I-evidence +observed O +between O +residues O +separated O +by O +one O +( O +residues O +332 B-residue_range +– I-residue_range +334 I-residue_range +, O +333 B-residue_range +– I-residue_range +335 I-residue_range +, O +etc O +.) O +or O +two O +( O +residues O +337 B-residue_range +– I-residue_range +340 I-residue_range +) O +residues O +in O +the O +sequence O +and O +the O +φ B-evidence +and I-evidence +ψ I-evidence +angles I-evidence +, O +assessed O +using O +Stride B-experimental_method +. O + +These O +turns O +cause O +the O +chain O +to O +reverse O +direction O +, O +allowing O +the O +N O +- O +terminal O +segment O +( O +residues O +334 B-residue_range +– I-residue_range +340 I-residue_range +) O +to O +contact O +both O +helices O +of O +the O +HR1 B-structure_element +domain O +. O + +Long O +range O +NOEs B-evidence +were O +observed O +linking O +Leu B-residue_name_number +- I-residue_name_number +334 I-residue_name_number +, O +Glu B-residue_name_number +- I-residue_name_number +335 I-residue_name_number +, O +and O +Asp B-residue_name_number +- I-residue_name_number +336 I-residue_name_number +with O +Trp B-residue_name_number +- I-residue_name_number +413 I-residue_name_number +of O +helix B-structure_element +2 I-structure_element +, O +Leu B-residue_name_number +- I-residue_name_number +334 I-residue_name_number +with O +Lys B-residue_name_number +- I-residue_name_number +409 I-residue_name_number +of O +helix B-structure_element +2 I-structure_element +, O +and O +Phe B-residue_name_number +- I-residue_name_number +337 I-residue_name_number +and O +Ser B-residue_name_number +- I-residue_name_number +338 I-residue_name_number +with O +Arg B-residue_name_number +- I-residue_name_number +345 I-residue_name_number +, O +Arg B-residue_name_number +- I-residue_name_number +348 I-residue_name_number +, O +and O +Leu B-residue_name_number +- I-residue_name_number +349 I-residue_name_number +of O +helix B-structure_element +1 I-structure_element +. O + +The O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +of O +TOCA1 B-protein +HR1 B-structure_element +are O +separated O +by O +a O +long O +loop B-structure_element +of O +10 O +residues O +( O +residues O +380 B-residue_range +– I-residue_range +389 I-residue_range +) O +that O +contains O +two O +short B-structure_element +310 I-structure_element +helices I-structure_element +( O +residues O +381 B-residue_range +– I-residue_range +383 I-residue_range +and O +386 B-residue_range +– I-residue_range +389 I-residue_range +). O + +Interestingly O +, O +side O +chains O +of O +residues O +within O +the O +loop B-structure_element +region I-structure_element +point O +back O +toward O +helix B-structure_element +1 I-structure_element +; O +for O +example O +, O +there O +are O +numerous O +distinct O +NOEs O +between O +the O +side O +chains O +of O +Asn B-residue_name_number +- I-residue_name_number +380 I-residue_name_number +and O +Met B-residue_name_number +- I-residue_name_number +383 I-residue_name_number +of O +the O +loop B-structure_element +region I-structure_element +and O +Tyr B-residue_name_number +- I-residue_name_number +377 I-residue_name_number +and O +Val B-residue_name_number +- I-residue_name_number +376 I-residue_name_number +of O +helix B-structure_element +1 I-structure_element +( O +Fig O +. O +3D O +). O + +The O +backbone O +NH O +and O +CHα O +groups O +of O +Gly B-residue_name_number +- I-residue_name_number +384 I-residue_name_number +and O +Asp B-residue_name_number +- I-residue_name_number +385 I-residue_name_number +also O +show O +NOEs O +with O +the O +side O +chain O +of O +Tyr B-residue_name_number +- I-residue_name_number +377 I-residue_name_number +. O + +Mapping O +the O +TOCA1 B-protein +and O +Cdc42 B-site +Binding I-site +Interfaces I-site + +The O +HR1TOCA1 B-site +- I-site +Cdc42 I-site +interface I-site +was O +investigated O +using O +NMR B-experimental_method +spectroscopy I-experimental_method +. O + +A O +series O +of O +15N B-experimental_method +HSQC I-experimental_method +experiments O +was O +recorded O +on O +15N B-chemical +- O +labeled B-protein_state +TOCA1 B-protein +HR1 B-structure_element +domain O +in O +the O +presence B-protein_state +of I-protein_state +increasing B-experimental_method +concentrations I-experimental_method +of O +unlabeled B-protein_state +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +to O +map O +the O +Cdc42 B-site +- I-site +binding I-site +surface I-site +. O + +A O +comparison O +of O +the O +15N B-experimental_method +HSQC I-experimental_method +spectra B-evidence +of O +free B-protein_state +HR1 B-structure_element +and O +HR1 B-structure_element +in O +the O +presence B-protein_state +of I-protein_state +excess O +Cdc42 B-protein +shows O +that O +although O +some O +peaks O +were O +shifted O +, O +several O +were O +much O +broader O +in O +the O +complex O +, O +and O +a O +considerable O +subset O +had O +disappeared O +( O +Fig O +. O +4A O +). O + +This O +behavior O +cannot O +be O +explained O +by O +the O +increase O +in O +molecular O +mass O +( O +from O +12 O +to O +33 O +kDa O +) O +when O +Cdc42 B-protein +binds O +and O +is O +more O +likely O +to O +be O +due O +to O +conformational O +exchange O +. O + +Overall O +chemical B-experimental_method +shift I-experimental_method +perturbations I-experimental_method +( O +CSPs B-experimental_method +) O +were O +calculated O +for O +each O +residue O +, O +whereas O +those O +that O +had O +disappeared O +were O +assigned O +a O +shift O +change O +of O +0 O +. O +2 O +( O +Fig O +. O +4B O +). O + +A O +peak O +that O +disappeared O +or O +had O +a O +CSP B-experimental_method +above O +the O +mean O +CSP B-experimental_method +for O +the O +spectrum O +was O +considered O +to O +be O +significantly O +affected O +. O + +Mapping O +the O +binding B-site +surface I-site +of O +Cdc42 B-protein +onto O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +. O + +A O +, O +the O +15N B-experimental_method +HSQC I-experimental_method +of O +200 O +μm O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +shown O +in O +the O +free B-protein_state +form I-protein_state +( O +black O +) O +and O +in O +the O +presence B-protein_state +of I-protein_state +a O +4 O +- O +fold O +molar O +excess O +of O +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +( O +red O +). O + +B O +, O +CSPs B-experimental_method +were O +calculated O +as O +described O +under O +“ O +Experimental O +Procedures O +” O +and O +are O +shown O +for O +backbone O +and O +side O +chain O +NH O +groups O +. O + +The O +mean O +CSP B-experimental_method +is O +marked O +with O +a O +red O +line O +. O + +Residues O +that O +disappeared O +in O +the O +presence B-protein_state +of I-protein_state +Cdc42 B-protein +were O +assigned O +a O +CSP B-experimental_method +of O +0 O +. O +2 O +but O +were O +excluded O +when O +calculating O +the O +mean O +CSP B-experimental_method +and O +are O +indicated O +with O +open O +bars O +. O + +Those O +that O +were O +not O +traceable O +due O +to O +spectral O +overlap O +were O +assigned O +a O +CSP B-experimental_method +of O +zero O +and O +are O +marked O +with O +an O +asterisk O +below O +the O +bar O +. O + +Residues O +with O +affected O +side O +chain O +CSPs B-experimental_method +derived O +from O +13C B-experimental_method +HSQCs I-experimental_method +are O +marked O +with O +green O +asterisks O +above O +the O +bars O +. O + +C O +, O +a O +schematic O +representation O +of O +the O +HR1 B-structure_element +domain O +. O + +Residues O +with O +significantly O +affected O +backbone O +or O +side O +chain O +chemical O +shifts O +when O +Cdc42 B-protein_state +bound I-protein_state +and O +that O +are O +buried O +are O +colored O +dark O +blue O +, O +whereas O +those O +that O +are O +solvent B-protein_state +- I-protein_state +accessible I-protein_state +are O +colored O +yellow O +. O + +Residues O +with O +significantly O +affected O +backbone O +and O +side O +chain O +groups O +that O +are O +solvent B-protein_state +- I-protein_state +accessible I-protein_state +are O +colored O +red O +. O + +A O +close O +- O +up O +of O +the O +binding B-site +region I-site +is O +shown O +, O +with O +affected O +side O +chain O +heavy O +atoms O +shown O +as O +sticks O +. O + +D O +, O +the O +G B-site +protein I-site +- I-site +binding I-site +region I-site +is O +marked O +in O +red O +onto O +structures B-evidence +of O +the O +HR1 B-structure_element +domains O +as O +indicated O +. O + +15N B-experimental_method +HSQC I-experimental_method +shift I-experimental_method +mapping I-experimental_method +experiments O +report O +on O +changes O +to O +amide O +groups O +, O +which O +are O +mainly O +inaccessible O +because O +they O +are O +buried O +inside O +the O +helices B-structure_element +and O +are O +involved O +in O +hydrogen B-bond_interaction +bonds I-bond_interaction +. O + +Therefore O +, O +13C B-experimental_method +HSQC I-experimental_method +and O +methyl B-experimental_method +- I-experimental_method +selective I-experimental_method +SOFAST I-experimental_method +- I-experimental_method +HMQC I-experimental_method +experiments O +were O +also O +recorded O +on O +15N B-chemical +, O +13C B-chemical +- O +labeled B-protein_state +TOCA1 B-protein +HR1 B-structure_element +to O +yield O +more O +information O +on O +side O +chain O +involvement O +. O + +Side O +chains O +whose O +CH O +groups O +disappeared O +in O +the O +presence B-protein_state +of I-protein_state +Cdc42 B-protein +are O +marked O +on O +the O +graph O +in O +Fig O +. O +4B O +with O +green O +asterisks O +. O + +TOCA1 B-protein +residues O +whose O +signals O +were O +affected O +by O +Cdc42 B-protein +binding O +were O +mapped O +onto O +the O +structure B-evidence +of O +TOCA1 B-protein +HR1 B-structure_element +( O +Fig O +. O +4C O +). O + +The O +changes O +were O +localized O +to O +one O +end O +of O +the O +coiled B-structure_element +- I-structure_element +coil I-structure_element +, O +and O +the O +binding B-site +site I-site +appeared O +to O +include O +residues O +from O +both O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +the O +loop B-structure_element +region I-structure_element +that O +joins O +them O +. O + +The O +residues O +in O +the O +interhelical B-structure_element +loop I-structure_element +and O +helix B-structure_element +1 I-structure_element +that O +contact O +each O +other O +( O +Fig O +. O +3D O +) O +show O +shift O +changes O +in O +their O +backbone O +NH O +and O +side O +chains O +in O +the O +presence B-protein_state +of I-protein_state +Cdc42 B-protein +. O + +For O +example O +, O +the O +side O +chain O +of O +Asn B-residue_name_number +- I-residue_name_number +380 I-residue_name_number +and O +the O +backbones O +of O +Val B-residue_name_number +- I-residue_name_number +376 I-residue_name_number +and O +Tyr B-residue_name_number +- I-residue_name_number +377 I-residue_name_number +were O +significantly O +affected O +but O +are O +all O +buried O +in O +the O +free B-protein_state +TOCA1 B-protein +HR1 B-structure_element +structure B-evidence +, O +indicating O +that O +local O +conformational O +changes O +in O +the O +loop B-structure_element +may O +facilitate O +complex O +formation O +. O + +The O +chemical B-experimental_method +shift I-experimental_method +mapping I-experimental_method +data O +indicate O +that O +the O +G B-site +protein I-site +- I-site +binding I-site +region I-site +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +broadly O +similar O +to O +that O +of O +the O +CIP4 B-protein +and O +PRK1 B-protein +HR1 B-structure_element +domains O +( O +Figs O +. O +3B O +and O +4D O +). O + +The O +corresponding O +15N B-experimental_method +and O +13C B-experimental_method +NMR I-experimental_method +experiments O +were O +also O +recorded O +on O +15N B-chemical +- O +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +or O +15N B-chemical +/ O +13C B-chemical +- O +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +in O +the O +presence B-protein_state +of I-protein_state +unlabeled B-protein_state +HR1 B-structure_element +domain O +. O + +The O +overall O +CSP B-experimental_method +was O +calculated O +for O +each O +residue O +. O + +As O +was O +the O +case O +when O +labeled B-protein_state +HR1 B-structure_element +was O +observed O +, O +several O +peaks O +were O +shifted O +in O +the O +complex O +, O +but O +many O +disappeared O +, O +indicating O +exchange O +on O +an O +unfavorable O +, O +millisecond O +time O +scale O +( O +Fig O +. O +5A O +). O + +Detailed O +side O +chain O +data O +could O +not O +be O +obtained O +for O +all O +residues O +due O +to O +spectral O +overlap O +, O +but O +constant B-experimental_method +time I-experimental_method +13C I-experimental_method +HSQC I-experimental_method +and O +methyl B-experimental_method +- I-experimental_method +selective I-experimental_method +SOFAST I-experimental_method +- I-experimental_method +HMQC I-experimental_method +experiments O +provided O +further O +information O +on O +certain O +well O +resolved O +side O +chains O +( O +marked O +with O +green O +asterisks O +in O +Fig O +. O +5B O +). O + +Mapping O +the O +binding B-site +surface I-site +of O +the O +HR1 B-structure_element +domain O +onto O +Cdc42 B-protein +. O + +A O +, O +the O +15N B-experimental_method +HSQC I-experimental_method +of O +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +is O +shown O +in O +its O +free B-protein_state +form I-protein_state +( O +black O +) O +and O +in O +the O +presence B-protein_state +of I-protein_state +excess O +TOCA1 B-protein +HR1 B-structure_element +domain O +( O +1 O +: O +2 O +. O +2 O +, O +red O +). O + +B O +, O +CSPs B-experimental_method +are O +shown O +for O +backbone O +NH O +groups O +. O + +The O +red O +line O +indicates O +the O +mean O +CSP B-experimental_method +, O +plus O +one O +S O +. O +D O +. O +Residues O +that O +disappeared O +in O +the O +presence B-protein_state +of I-protein_state +Cdc42 B-protein +were O +assigned O +a O +CSP B-experimental_method +of O +0 O +. O +1 O +and O +are O +indicated O +with O +open O +bars O +. O + +Residues O +with O +disappeared O +peaks O +in O +13C B-experimental_method +HSQC I-experimental_method +experiments O +are O +marked O +on O +the O +chart O +with O +green O +asterisks O +. O + +C O +, O +the O +residues O +with O +significantly O +affected O +backbone O +and O +side O +chain O +groups O +are O +highlighted O +on O +an O +NMR B-experimental_method +structure B-evidence +of O +free B-protein_state +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +; O +those O +that O +are O +buried O +are O +colored O +dark O +blue O +, O +whereas O +those O +that O +are O +solvent B-protein_state +- I-protein_state +accessible I-protein_state +are O +colored O +red O +. O + +Residues O +with O +either O +side O +chain O +or O +backbone O +groups O +affected O +are O +colored O +blue O +if O +buried O +and O +yellow O +if O +solvent B-protein_state +- I-protein_state +accessible I-protein_state +. O + +Residues O +without O +information O +from O +shift B-experimental_method +mapping I-experimental_method +are O +colored O +gray O +. O + +The O +flexible B-protein_state +switch B-site +regions I-site +are O +circled O +. O + +As O +many O +of O +the O +peaks O +disappeared O +, O +the O +mean B-evidence +chemical I-evidence +shift I-evidence +change I-evidence +was O +relatively O +low O +, O +so O +a O +threshold O +of O +the O +mean O +plus O +one O +S O +. O +D O +. O +value O +was O +used O +to O +define O +a O +significant O +CSP B-experimental_method +. O + +Parts O +of O +the O +switch B-site +regions I-site +( O +Fig O +. O +5 O +, O +B O +and O +C O +) O +are O +invisible O +in O +NMR B-experimental_method +spectra B-evidence +recorded O +on O +free B-protein_state +Cdc42 B-protein +due O +to O +conformational O +exchange O +. O + +These O +switch B-site +regions I-site +become O +visible O +in O +Cdc42 B-protein +and O +other O +small O +G B-protein_type +protein I-protein_type +· O +effector O +complexes O +due O +to O +a O +decrease O +in O +conformational O +freedom O +upon O +complex O +formation O +. O + +The O +switch B-site +regions I-site +of O +Cdc42 B-protein +did O +not O +, O +however O +, O +become O +visible O +in O +the O +presence B-protein_state +of I-protein_state +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +. O + +Indeed O +, O +Ser B-residue_name_number +- I-residue_name_number +30 I-residue_name_number +of O +switch B-site +I I-site +and O +Arg B-residue_name_number +- I-residue_name_number +66 I-residue_name_number +, O +Arg B-residue_name_number +- I-residue_name_number +68 I-residue_name_number +, O +Leu B-residue_name_number +- I-residue_name_number +70 I-residue_name_number +, O +and O +Ser B-residue_name_number +- I-residue_name_number +71 I-residue_name_number +of O +switch B-site +II I-site +are O +visible O +in O +free B-protein_state +Cdc42 B-protein +but O +disappear O +in O +the O +presence B-protein_state +of I-protein_state +the O +HR1 B-structure_element +domain O +. O + +This O +suggests O +that O +the O +switch B-site +regions I-site +are O +not O +rigidified O +in O +the O +HR1 B-structure_element +complex O +and O +are O +still O +in O +conformational O +exchange O +. O + +Nevertheless O +, O +mapping O +of O +the O +affected O +residues O +onto O +the O +NMR B-experimental_method +structure B-evidence +of O +free B-protein_state +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +( O +Fig O +. O +5C O +) O +8 O +shows O +that O +, O +although O +they O +are O +relatively O +widespread O +compared O +with O +changes O +in O +the O +HR1 B-structure_element +domain O +, O +in O +general O +, O +they O +are O +on O +the O +face O +of O +the O +protein O +that O +includes O +the O +switches B-site +. O + +Although O +the O +binding B-site +interface I-site +may O +be O +overestimated O +, O +this O +suggests O +that O +the O +switch B-site +regions I-site +are O +involved O +in O +binding O +to O +TOCA1 B-protein +. O + +Modeling O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +HR1 I-complex_assembly +Complex O + +The O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1TOCA1 I-complex_assembly +complex O +was O +not O +amenable O +to O +full O +structural O +analysis O +due O +to O +the O +weak O +interaction O +and O +the O +extensive O +exchange O +broadening O +seen O +in O +the O +NMR B-experimental_method +experiments O +. O + +HADDOCK B-experimental_method +was O +therefore O +used O +to O +perform O +rigid B-experimental_method +body I-experimental_method +docking I-experimental_method +based O +on O +the O +structures B-evidence +of O +free B-protein_state +HR1 B-structure_element +domain O +and O +Cdc42 B-protein +and O +ambiguous O +interaction O +restraints O +derived O +from O +the O +titration B-experimental_method +experiments I-experimental_method +described O +above O +. O + +The O +orientation O +of O +the O +HR1 B-structure_element +domain O +with O +respect O +to O +Cdc42 B-protein +cannot O +be O +definitively O +concluded O +in O +the O +absence O +of O +unambiguous O +distance O +restraints O +; O +hence O +, O +HADDOCK B-experimental_method +produced O +a O +set O +of O +models O +in O +which O +the O +HR1 B-structure_element +domain O +contacts O +the O +same O +surface O +on O +Cdc42 B-protein +but O +is O +in O +various O +orientations O +with O +respect O +to O +Cdc42 B-protein +. O + +The O +cluster O +with O +the O +lowest O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +from O +the O +lowest O +energy O +structure B-evidence +is O +assumed O +to O +be O +the O +best O +model O +. O + +By O +these O +criteria O +, O +in O +the O +best O +model O +, O +the O +HR1 B-structure_element +domain O +is O +in O +a O +similar O +orientation O +to O +the O +HR1a B-structure_element +domain O +of O +PRK1 B-protein +bound B-protein_state +to I-protein_state +RhoA B-protein +and O +the O +HR1b B-structure_element +domain O +bound B-protein_state +to I-protein_state +Rac1 B-protein +. O + +A O +representative O +model O +from O +this O +cluster O +is O +shown O +in O +Fig O +. O +6A O +alongside O +the O +Rac1 B-complex_assembly +- I-complex_assembly +HR1b I-complex_assembly +structure B-evidence +( O +PDB O +code O +2RMK O +) O +in O +Fig O +. O +6B O +. O + +Model O +of O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1 I-complex_assembly +complex O +. O + +A O +, O +a O +representative O +model O +of O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1 I-complex_assembly +complex O +from O +the O +cluster O +closest O +to O +the O +lowest O +energy O +model O +produced O +using O +HADDOCK B-experimental_method +. O + +Residues O +of O +Cdc42 B-protein +that O +are O +affected O +in O +the O +presence B-protein_state +of I-protein_state +the O +HR1 B-structure_element +domain O +but O +are O +not O +in O +close O +proximity O +to O +it O +are O +colored O +in O +red O +and O +labeled O +. O + +B O +, O +structure B-evidence +of O +Rac1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +HR1b B-structure_element +domain O +of O +PRK1 B-protein +( O +PDB O +code O +2RMK O +). O + +C O +, O +sequence B-experimental_method +alignment I-experimental_method +of O +RhoA B-protein +, O +Cdc42 B-protein +and O +Rac1 B-protein +. O + +Contact O +residues O +of O +RhoA B-protein +and O +Rac1 B-protein +to O +PRK1 B-protein +HR1a B-structure_element +and O +HR1b B-structure_element +, O +respectively O +, O +are O +colored O +cyan O +. O + +Residues O +of O +Cdc42 B-protein +that O +disappear O +or O +show O +chemical O +shift O +changes O +in O +the O +presence B-protein_state +of I-protein_state +TOCA1 B-protein +are O +colored O +cyan O +if O +also O +identified O +as O +contacts O +in O +RhoA B-protein +and O +Rac1 B-protein +and O +yellow O +if O +they O +are O +not O +. O + +Residues O +equivalent O +to O +Rac1 B-protein +and O +RhoA B-protein +contact B-site +sites I-site +but O +that O +are O +invisible O +in O +free B-protein_state +Cdc42 B-protein +are O +gray O +. O + +D O +, O +regions O +of O +interest O +of O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1 I-complex_assembly +domain O +model O +. O + +The O +four O +lowest O +energy O +structures B-evidence +in O +the O +chosen O +HADDOCK B-experimental_method +cluster O +are O +shown O +overlaid O +, O +with O +the O +residues O +of O +interest O +shown O +as O +sticks O +and O +labeled O +. O + +Cdc42 O +is O +shown O +in O +cyan O +, O +and O +TOCA1 B-protein +is O +shown O +in O +purple O +. O + +A O +sequence B-experimental_method +alignment I-experimental_method +of O +RhoA B-protein +, O +Cdc42 B-protein +, O +and O +Rac1 B-protein +is O +shown O +in O +Fig O +. O +6C O +. O + +The O +RhoA B-protein +and O +Rac1 B-protein +contact O +residues O +in O +the O +switch B-site +regions I-site +are O +invisible O +in O +the O +spectra B-evidence +of O +Cdc42 B-protein +, O +but O +they O +are O +generally O +conserved B-protein_state +between O +all O +three O +G B-protein_type +proteins I-protein_type +. O + +Several O +Cdc42 B-protein +residues O +identified O +by O +chemical B-experimental_method +shift I-experimental_method +mapping I-experimental_method +are O +not O +in O +close O +contact O +in O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +model O +( O +Fig O +. O +6A O +). O + +Some O +of O +these O +can O +be O +rationalized O +; O +for O +example O +, O +Thr B-residue_name_number +- I-residue_name_number +24Cdc42 I-residue_name_number +, O +Leu B-residue_name_number +- I-residue_name_number +160Cdc42 I-residue_name_number +, O +and O +Lys B-residue_name_number +- I-residue_name_number +163Cdc42 I-residue_name_number +all O +pack O +behind O +switch B-site +I I-site +and O +are O +likely O +to O +be O +affected O +by O +conformational O +changes O +within O +the O +switch B-site +, O +while O +Glu B-residue_name_number +- I-residue_name_number +95Cdc42 I-residue_name_number +and O +Lys B-residue_name_number +- I-residue_name_number +96Cdc42 I-residue_name_number +are O +in O +the O +helix B-structure_element +behind O +switch B-site +II I-site +. O + +Other O +residues O +that O +are O +affected O +in O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +complex O +but O +that O +do O +not O +correspond O +to O +contact O +residues O +of O +RhoA B-protein +or O +Rac1 B-protein +( O +Fig O +. O +6C O +) O +include O +Gln B-residue_name_number +- I-residue_name_number +2Cdc42 I-residue_name_number +, O +Lys B-residue_name_number +- I-residue_name_number +16Cdc42 I-residue_name_number +, O +Thr B-residue_name_number +- I-residue_name_number +52Cdc42 I-residue_name_number +, O +and O +Arg B-residue_name_number +- I-residue_name_number +68Cdc42 I-residue_name_number +. O + +Lys B-residue_name_number +- I-residue_name_number +16Cdc42 I-residue_name_number +is O +unlikely O +to O +be O +a O +contact O +residue O +because O +it O +is O +involved O +in O +nucleotide O +binding O +, O +but O +the O +others O +may O +represent O +specific O +Cdc42 B-complex_assembly +- I-complex_assembly +TOCA1 I-complex_assembly +contacts O +. O + +Competition O +between O +N B-protein +- I-protein +WASP I-protein +and O +TOCA1 B-protein + +From O +the O +known O +interactions O +and O +effects O +of O +the O +proteins O +in O +biological O +systems O +, O +it O +has O +been O +suggested O +that O +TOCA1 B-protein +and O +N B-protein +- I-protein +WASP I-protein +could O +bind O +Cdc42 B-protein +simultaneously O +. O + +Studies O +in O +CHO O +cells O +indicated O +that O +a O +Cdc42 B-complex_assembly +· I-complex_assembly +N I-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +complex O +existed O +because O +FRET B-evidence +was O +observed O +between O +RFP B-chemical +- O +TOCA1 B-protein +and O +GFP B-chemical +- O +N B-protein +- I-protein +WASP I-protein +, O +and O +the O +efficiency O +was O +decreased O +when O +an O +N B-protein +- I-protein +WASP I-protein +mutant B-protein_state +was O +used O +that O +no O +longer O +binds O +Cdc42 B-protein +. O + +An O +overlay B-experimental_method +of O +the O +HADDOCK B-experimental_method +model B-evidence +of O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1TOCA1 I-complex_assembly +complex O +and O +the O +structure B-evidence +of O +Cdc42 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +GBD B-structure_element +of O +the O +N B-protein +- I-protein +WASP I-protein +homologue O +, O +WASP B-protein +( O +PDB O +code O +1CEE O +), O +shows O +that O +the O +HR1 B-structure_element +and O +GBD B-site +binding I-site +sites I-site +only O +partly O +overlap O +, O +and O +, O +therefore O +, O +a O +ternary O +complex O +remained O +possible O +( O +Fig O +. O +7A O +). O + +Interestingly O +, O +the O +presence B-protein_state +of I-protein_state +the O +TOCA1 B-protein +HR1 B-structure_element +would O +not O +prevent O +the O +core O +CRIB B-structure_element +of O +WASP B-protein +from O +binding O +to O +Cdc42 B-protein +, O +although O +the O +regions O +C O +- O +terminal O +to O +the O +CRIB B-structure_element +that O +are O +required O +for O +high O +affinity O +binding O +of O +WASP B-protein +would O +interfere O +sterically O +with O +the O +TOCA1 B-protein +HR1 B-structure_element +. O + +A O +basic O +region O +in O +WASP B-protein +including O +three O +lysines B-residue_name +( O +residues O +230 B-residue_range +– I-residue_range +232 I-residue_range +), O +N O +- O +terminal O +to O +the O +core O +CRIB B-structure_element +, O +has O +been O +implicated O +in O +an O +electrostatic O +steering O +mechanism O +, O +and O +these O +residues O +would O +be O +free O +to O +bind O +in O +the O +presence B-protein_state +of I-protein_state +TOCA1 B-protein +HR1 B-structure_element +( O +Fig O +. O +7A O +). O + +The O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +displaces O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +. O + +A O +, O +the O +model O +of O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +HR1 B-structure_element +domain O +complex O +overlaid O +with O +the O +Cdc42 B-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +structure B-evidence +. O + +Cdc42 O +is O +shown O +in O +green O +, O +and O +TOCA1 B-protein +is O +shown O +in O +purple O +. O + +The O +core O +CRIB B-structure_element +region O +of O +WASP B-protein +is O +shown O +in O +red O +, O +whereas O +its O +basic O +region O +is O +shown O +in O +orange O +and O +the O +C O +- O +terminal O +region O +required O +for O +maximal O +affinity O +is O +shown O +in O +cyan O +. O + +A O +semitransparent O +surface O +representation O +of O +Cdc42 B-protein +and O +WASP B-protein +is O +shown O +overlaid O +with O +the O +schematic O +. O + +B O +, O +competition B-experimental_method +SPA I-experimental_method +experiments O +carried O +out O +with O +indicated O +concentrations O +of O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +construct O +titrated B-experimental_method +into O +30 O +nm O +GST B-mutant +- I-mutant +ACK I-mutant +or O +GST B-mutant +- I-mutant +WASP I-mutant +GBD B-structure_element +and O +30 O +nm O +Cdc42Δ7Q61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +. O + +C O +, O +Selected O +regions O +of O +the O +15N B-experimental_method +HSQC I-experimental_method +of O +145 O +μm O +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +with O +the O +indicated O +ratios O +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +, O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +, O +or O +both O +, O +showing O +that O +the O +TOCA B-protein +HR1 B-structure_element +domain O +does O +not O +displace O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +. O + +D O +, O +selected O +regions O +of O +the O +15N B-experimental_method +HSQC I-experimental_method +of O +600 O +μm O +TOCA1 B-protein +HR1 B-structure_element +domain O +in B-protein_state +complex I-protein_state +with I-protein_state +Cdc42 B-protein +in O +the O +absence B-protein_state +and O +presence B-protein_state +of I-protein_state +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +, O +showing O +displacement O +of O +Cdc42 B-protein +from O +the O +HR1 B-structure_element +domain O +by O +N B-protein +- I-protein +WASP I-protein +. O + +An O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +construct O +was O +produced O +, O +and O +its O +affinity B-evidence +for O +Cdc42 B-protein +was O +measured O +by O +competition B-experimental_method +SPA I-experimental_method +( O +Fig O +. O +7B O +). O + +The O +Kd B-evidence +that O +was O +determined O +( O +37 O +nm O +) O +is O +consistent O +with O +the O +previously O +reported O +affinity B-evidence +. O + +Unlabeled B-protein_state +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +was O +titrated B-experimental_method +into O +15N B-chemical +- O +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +, O +and O +the O +backbone O +NH O +groups O +were O +monitored O +using O +HSQCs B-experimental_method +( O +Fig O +. O +7C O +). O + +Unlabeled B-protein_state +HR1TOCA1 B-structure_element +was O +then O +added O +to O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +N I-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +complex O +, O +and O +no O +changes O +were O +seen O +, O +suggesting O +that O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +was O +not O +displaced O +even O +in O +the O +presence B-protein_state +of I-protein_state +a O +5 O +- O +fold O +excess O +of O +HR1TOCA1 B-structure_element +. O + +These O +experiments O +were O +recorded O +at O +sufficiently O +high O +protein O +concentrations O +( O +145 O +μm O +Cdc42 B-protein +, O +145 O +μm O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +, O +725 O +μm O +TOCA1 B-protein +HR1 B-structure_element +domain O +) O +to O +be O +far O +in O +excess O +of O +the O +Kd B-evidence +values O +of O +the O +individual O +interactions O +( O +TOCA1 B-protein +Kd B-evidence +≈ O +5 O +μm O +, O +N B-protein +- I-protein +WASP I-protein +Kd B-evidence += O +37 O +nm O +). O + +A O +comparison O +of O +the O +HSQC B-experimental_method +experiments O +recorded O +on O +15N B-chemical +- O +Cdc42 B-protein +alone B-protein_state +, O +in O +the O +presence B-protein_state +of I-protein_state +TOCA1 B-protein +HR1 B-structure_element +, O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +, O +or O +both O +, O +shows O +that O +the O +spectra B-evidence +in O +the O +presence B-protein_state +of I-protein_state +N B-protein +- I-protein +WASP I-protein +and O +in O +the O +presence B-protein_state +of I-protein_state +both O +N B-protein +- I-protein +WASP I-protein +and O +TOCA1 B-protein +HR1 B-structure_element +are O +identical O +( O +Fig O +. O +7C O +). O + +Furthermore O +, O +15N B-chemical +- O +TOCA1 B-protein +HR1 B-structure_element +was O +monitored O +in O +the O +presence B-protein_state +of I-protein_state +unlabeled B-protein_state +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +( O +1 O +: O +1 O +) O +before O +and O +after O +the O +addition O +of O +0 O +. O +25 O +and O +1 O +. O +0 O +eq O +of O +unlabeled B-protein_state +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +. O + +The O +spectrum B-evidence +when O +N B-protein +- I-protein +WASP I-protein +and O +TOCA1 B-protein +were O +equimolar O +was O +identical O +to O +that O +of O +the O +free B-protein_state +HR1 B-structure_element +domain O +, O +whereas O +the O +spectrum B-evidence +in O +the O +presence B-protein_state +of I-protein_state +0 O +. O +25 O +eq O +of O +N B-protein +- I-protein +WASP I-protein +was O +intermediate O +between O +the O +TOCA1 B-protein +HR1 B-structure_element +free B-protein_state +and O +complex B-protein_state +spectra B-evidence +( O +Fig O +. O +7D O +). O + +When O +in O +fast O +exchange O +, O +the O +NMR B-experimental_method +signal O +represents O +a O +population O +- O +weighted O +average O +between O +free B-protein_state +and O +bound B-protein_state +states O +, O +so O +the O +intermediate O +spectrum B-evidence +indicates O +that O +the O +population O +comprises O +a O +mixture O +of O +free B-protein_state +and O +bound B-protein_state +HR1 B-structure_element +domain O +. O + +Again O +, O +the O +experiments O +were O +recorded O +on O +protein O +samples O +far O +in O +excess O +of O +the O +individual O +Kd B-evidence +values O +( O +600 O +μm O +each O +protein O +). O + +These O +data O +indicate O +that O +the O +HR1 B-structure_element +domain O +is O +displaced O +from O +Cdc42 B-protein +by O +N B-protein +- I-protein +WASP I-protein +and O +that O +a O +ternary O +complex O +comprising O +TOCA1 B-protein +HR1 B-structure_element +, O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +, O +and O +Cdc42 B-protein +is O +not O +formed O +. O + +Taken O +together O +, O +the O +data O +in O +Fig O +. O +7 O +, O +C O +and O +D O +, O +indicate O +unidirectional O +competition O +for O +Cdc42 B-protein +binding O +in O +which O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +displaces O +TOCA1 B-protein +HR1 B-structure_element +but O +not O +vice O +versa O +. O + +To O +extend O +these O +studies O +to O +a O +more O +complex O +system O +and O +to O +assess O +the O +ability O +of O +TOCA1 B-protein +HR1 B-structure_element +to O +compete O +with O +full B-protein_state +- I-protein_state +length I-protein_state +N B-protein +- I-protein +WASP I-protein +, O +pyrene B-experimental_method +actin I-experimental_method +assays I-experimental_method +were O +employed O +. O + +These O +assays O +, O +described O +in O +detail O +elsewhere O +, O +were O +carried O +out O +using O +pyrene B-chemical +actin I-chemical +- O +supplemented O +Xenopus B-taxonomy_domain +extracts O +into O +which O +exogenous O +TOCA1 B-protein +HR1 B-structure_element +domain O +or O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +was O +added O +, O +to O +assess O +their O +effects O +on O +actin B-protein_type +polymerization O +. O + +Actin B-protein_type +polymerization O +in O +all O +cases O +was O +initiated O +by O +the O +addition O +of O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +- O +containing O +liposomes O +. O + +Actin B-protein_type +polymerization O +triggered O +by O +the O +addition O +of O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +- O +containing O +liposomes O +has O +previously O +been O +shown O +to O +depend O +on O +TOCA1 B-protein +and O +N B-protein +- I-protein +WASP I-protein +. O + +Endogenous O +N B-protein +- I-protein +WASP I-protein +is O +present O +at O +∼ O +100 O +nm O +in O +Xenopus B-taxonomy_domain +extracts O +, O +whereas O +TOCA1 B-protein +is O +present O +at O +a O +10 O +- O +fold O +lower O +concentration O +than O +N B-protein +- I-protein +WASP I-protein +. O + +The O +addition B-experimental_method +of O +the O +isolated O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +significantly O +inhibited O +the O +polymerization O +of O +actin B-protein_type +at O +concentrations O +as O +low O +as O +100 O +nm O +and O +completely O +abolished O +polymerization O +at O +higher O +concentrations O +( O +Fig O +. O +8 O +). O + +The O +GBD B-structure_element +presumably O +acts O +as O +a O +dominant O +negative O +, O +sequestering O +endogenous O +Cdc42 B-protein +and O +preventing O +endogenous B-protein_state +full B-protein_state +- I-protein_state +length I-protein_state +N B-protein +- I-protein +WASP I-protein +from O +binding O +and O +becoming O +activated O +. O + +The O +addition B-experimental_method +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +to O +100 O +μm O +had O +no O +significant O +effect O +on O +the O +rate O +of O +actin B-protein_type +polymerization O +or O +maximum B-evidence +fluorescence I-evidence +. O + +This O +is O +consistent O +with O +endogenous B-protein_state +N B-protein +- I-protein +WASP I-protein +, O +activated O +by O +other O +components O +of O +the O +assay O +, O +outcompeting O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +for O +Cdc42 B-protein +binding O +. O + +Actin O +polymerization O +downstream O +of O +Cdc42 B-complex_assembly +· I-complex_assembly +N I-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +is O +inhibited B-protein_state +by O +excess O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +but O +not O +by O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +. O + +Fluorescence B-evidence +curves I-evidence +show O +actin O +polymerization O +in O +the O +presence B-protein_state +of I-protein_state +increasing B-experimental_method +concentrations I-experimental_method +of O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +or O +TOCA1 B-protein +HR1 B-structure_element +domain O +as O +indicated O +. O + +The O +Cdc42 B-protein +- O +TOCA1 B-protein +Interaction O + +The O +TOCA1 B-protein +HR1 B-structure_element +domain O +alone B-protein_state +is O +sufficient O +for O +Cdc42 B-protein +binding O +in O +vitro O +, O +yet O +the O +affinity B-evidence +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +for O +Cdc42 B-protein +is O +remarkably O +low O +( O +Kd B-evidence +≈ O +5 O +μm O +). O + +This O +is O +over O +100 O +times O +lower O +than O +that O +of O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +( O +Kd B-evidence += O +37 O +nm O +) O +and O +considerably O +lower O +than O +other O +known O +G B-protein_type +protein I-protein_type +- O +HR1 B-structure_element +domain O +interactions O +. O + +The O +polybasic O +tract O +within O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +Cdc42 B-protein +does O +not O +appear O +to O +be O +required O +for O +binding O +to O +TOCA1 B-protein +, O +which O +is O +in O +contrast O +to O +the O +interaction O +between O +Rac1 B-protein +and O +the O +HR1b B-structure_element +domain O +of O +PRK1 B-protein +but O +more O +similar O +to O +the O +PRK1 B-protein +HR1a B-structure_element +- O +RhoA B-protein +interaction O +. O + +A O +single O +binding B-site +interface I-site +on O +both O +the O +HR1 B-structure_element +domain O +and O +Cdc42 B-protein +can O +be O +concluded O +from O +the O +data O +presented O +here O +. O + +Furthermore O +, O +the O +interfaces B-site +are O +comparable O +with O +those O +of O +other O +G B-protein_type +protein I-protein_type +- O +HR1 B-structure_element +interactions O +( O +Fig O +. O +4 O +), O +and O +the O +lowest O +energy O +model B-evidence +produced O +in O +rigid B-experimental_method +body I-experimental_method +docking I-experimental_method +resembles O +previously O +studied O +G B-complex_assembly +protein I-complex_assembly +· I-complex_assembly +HR1 I-complex_assembly +complexes O +( O +Fig O +. O +6 O +). O + +It O +seems O +, O +therefore O +, O +that O +the O +interaction O +, O +despite O +its O +relatively O +low O +affinity O +, O +is O +specific O +and O +sterically O +similar O +to O +other O +HR1 B-structure_element +domain O +- O +G B-protein_type +protein I-protein_type +interactions O +. O + +The O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +a O +left O +- O +handed O +coiled B-structure_element +- I-structure_element +coil I-structure_element +comparable O +with O +other O +known O +HR1 B-structure_element +domains O +. O + +A O +short O +region O +N O +- O +terminal O +to O +the O +coiled B-structure_element +- I-structure_element +coil I-structure_element +exhibits O +a O +series O +of O +turns O +and O +contacts O +residues O +of O +both O +helices O +of O +the O +coiled B-structure_element +- I-structure_element +coil I-structure_element +( O +Fig O +. O +3 O +). O + +The O +corresponding O +sequence O +in O +CIP4 B-protein +also O +includes O +a O +series O +of O +turns O +but O +is O +flexible O +, O +whereas O +in O +the O +HR1a B-structure_element +domain O +of O +PRK1 B-protein +, O +the O +equivalent O +region O +adopts O +an O +α B-structure_element +- I-structure_element +helical I-structure_element +structure I-structure_element +that O +packs O +against O +the O +coiled B-structure_element +- I-structure_element +coil I-structure_element +. O + +The O +contacts B-bond_interaction +between O +the O +N O +- O +terminal O +region O +and O +the O +coiled B-structure_element +- I-structure_element +coil I-structure_element +are O +predominantly O +hydrophobic B-bond_interaction +in O +both O +cases O +, O +but O +sequence O +- O +specific O +contacts O +do O +not O +appear O +to O +be O +conserved O +. O + +This O +region O +is O +distant O +from O +the O +G B-site +protein I-site +- I-site +binding I-site +interface I-site +of O +the O +HR1 B-structure_element +domains O +, O +so O +the O +structural O +differences O +may O +relate O +to O +the O +structure O +and O +regulation O +of O +these O +domains O +rather O +than O +their O +G B-protein_type +protein I-protein_type +interactions O +. O + +The O +interhelical B-structure_element +loops I-structure_element +of O +TOCA1 B-protein +and O +CIP4 B-protein +differ O +from O +the O +same O +region O +in O +the O +HR1 B-structure_element +domains O +of O +PRK1 B-protein +in O +that O +they O +are O +longer O +and O +contain O +two O +short O +stretches O +of O +310 B-structure_element +- I-structure_element +helix I-structure_element +. O + +This O +region O +lies O +within O +the O +G B-site +protein I-site +- I-site +binding I-site +surface I-site +of O +all O +of O +the O +HR1 B-structure_element +domains O +( O +Fig O +. O +4D O +). O + +TOCA1 B-protein +and O +CIP4 B-protein +both O +bind O +weakly O +to O +Cdc42 B-protein +, O +whereas O +the O +HR1a B-structure_element +domain O +of O +PRK1 B-protein +binds O +tightly O +to O +RhoA B-protein +and O +Rac1 B-protein +, O +and O +the O +HR1b B-structure_element +domain O +binds O +to O +Rac1 B-protein +. O + +The O +structural O +features O +shared O +by O +TOCA1 B-protein +and O +CIP4 B-protein +may O +therefore O +be O +related O +to O +Cdc42 B-protein +binding O +specificity O +and O +the O +low O +affinities O +. O + +In O +free B-protein_state +TOCA1 B-protein +, O +the O +side O +chains O +of O +the O +interhelical B-structure_element +region I-structure_element +make O +extensive O +contacts O +with O +residues O +in O +helix B-structure_element +1 I-structure_element +. O + +Many O +of O +these O +residues O +are O +significantly O +affected O +in O +the O +presence B-protein_state +of I-protein_state +Cdc42 B-protein +, O +so O +it O +is O +likely O +that O +the O +conformation O +of O +this O +loop B-structure_element +is O +altered O +in O +the O +Cdc42 B-protein +complex O +. O + +These O +observations O +therefore O +provide O +a O +molecular O +mechanism O +whereby O +mutation B-experimental_method +of O +Met383 B-residue_name_number +- O +Gly384 B-residue_name_number +- O +Asp385 B-residue_name_number +to O +Ile383 B-residue_name_number +- O +Ser384 B-residue_name_number +- O +Thr385 B-residue_name_number +abolishes O +TOCA1 B-protein +binding O +to O +Cdc42 B-protein +. O + +The O +lowest O +energy O +model B-evidence +produced O +by O +HADDOCK B-experimental_method +using O +ambiguous O +interaction O +restraints O +from O +the O +titration B-evidence +data O +resembled O +the O +NMR B-experimental_method +structures B-evidence +of O +RhoA B-protein +and O +Rac1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +their O +HR1 B-structure_element +domain O +partners O +. O + +For O +example O +, O +Phe B-residue_name_number +- I-residue_name_number +56Cdc42 I-residue_name_number +, O +which O +is O +not O +visible O +in O +free B-protein_state +Cdc42 B-protein +or O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1TOCA1 I-complex_assembly +, O +is O +close O +to O +the O +TOCA1 B-protein +HR1 B-structure_element +( O +Fig O +. O +6A O +). O + +Phe B-residue_name_number +- I-residue_name_number +56Cdc42 I-residue_name_number +, O +which O +is O +a O +Trp B-residue_name +in O +both O +Rac1 B-protein +and O +RhoA B-protein +( O +Fig O +. O +6C O +), O +is O +thought O +to O +pack O +behind O +switch B-site +I I-site +when O +Cdc42 B-protein +interacts O +with O +ACK B-protein +, O +maintaining O +the O +switch O +in O +a O +binding O +- O +competent O +orientation O +. O + +This O +residue O +has O +also O +been O +identified O +as O +important O +for O +Cdc42 B-protein +- O +WASP B-protein +binding O +. O + +Phe B-residue_name_number +- I-residue_name_number +56Cdc42 I-residue_name_number +is O +therefore O +likely O +to O +be O +involved O +in O +the O +Cdc42 B-protein +- O +TOCA1 B-protein +interaction O +, O +probably O +by O +stabilizing O +the O +position O +of O +switch B-site +I I-site +. O + +Some O +residues O +that O +are O +affected O +in O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1TOCA1 I-complex_assembly +complex O +but O +do O +not O +correspond O +to O +contact O +residues O +of O +RhoA B-protein +or O +Rac1 B-protein +( O +Fig O +. O +6C O +) O +may O +contact O +HR1TOCA1 B-structure_element +directly O +( O +Fig O +. O +6D O +). O + +Gln B-residue_name_number +- I-residue_name_number +2Cdc42 I-residue_name_number +, O +which O +has O +also O +been O +identified O +as O +a O +contact O +residue O +in O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +ACK I-complex_assembly +complex O +, O +contacts O +Val B-residue_name_number +- I-residue_name_number +376TOCA1 I-residue_name_number +and O +Asn B-residue_name_number +- I-residue_name_number +380TOCA1 I-residue_name_number +in O +the O +model O +and O +disrupts O +the O +contacts O +between O +the O +interhelical B-structure_element +loop I-structure_element +and O +the O +first B-structure_element +helix I-structure_element +of O +the O +TOCA1 B-protein +coiled B-structure_element +- I-structure_element +coil I-structure_element +. O + +Thr B-residue_name_number +- I-residue_name_number +52Cdc42 I-residue_name_number +, O +which O +has O +also O +been O +identified O +as O +making O +minor O +contacts O +with O +ACK B-protein +, O +falls O +near O +the O +side O +chains O +of O +HR1TOCA1 B-structure_element +helix B-structure_element +1 I-structure_element +, O +particularly O +Lys B-residue_name_number +- I-residue_name_number +372TOCA1 I-residue_name_number +, O +whereas O +the O +equivalent O +position O +in O +Rac1 B-protein +is O +Asn B-residue_name_number +- I-residue_name_number +52Rac1 I-residue_name_number +. O + +N52T B-mutant +is O +one O +of O +a O +combination O +of O +seven O +residues O +found O +to O +confer O +ACK B-protein +binding O +on O +Rac1 B-protein +and O +so O +may O +represent O +a O +specific O +Cdc42 B-protein +- O +effector O +contact O +residue O +. O + +The O +position O +equivalent O +to O +Lys B-residue_name_number +- I-residue_name_number +372TOCA1 I-residue_name_number +in O +PRK1 B-protein +is O +Glu B-residue_name_number +- I-residue_name_number +58HR1a I-residue_name_number +or O +Gln B-residue_name_number +- I-residue_name_number +151HR1b I-residue_name_number +. O + +Thr B-residue_name_number +- I-residue_name_number +52Cdc42 I-residue_name_number +- O +Lys B-residue_name_number +- I-residue_name_number +372TOCA1 I-residue_name_number +may O +therefore O +represent O +a O +specific O +Cdc42 B-protein +- O +HR1TOCA1 B-structure_element +contact O +. O + +Arg B-residue_name_number +- I-residue_name_number +68Cdc42 I-residue_name_number +of O +switch B-site +II I-site +is O +positioned O +close O +to O +Glu B-residue_name_number +- I-residue_name_number +395TOCA1 I-residue_name_number +( O +Fig O +. O +6D O +), O +suggesting O +a O +direct O +electrostatic O +contact O +between O +switch B-site +II I-site +of O +Cdc42 B-protein +and O +helix B-structure_element +2 I-structure_element +of O +the O +HR1 B-structure_element +domain O +. O + +The O +equivalent O +Arg B-residue_name +in O +Rac1 B-protein +and O +RhoA B-protein +is O +pointing O +away O +from O +the O +HR1 B-structure_element +domains O +of O +PRK1 B-protein +. O + +The O +importance O +of O +this O +residue O +in O +the O +Cdc42 B-protein +- O +TOCA1 B-protein +interaction O +remains O +unclear O +, O +although O +its O +mutation B-experimental_method +reduces O +binding O +to O +RhoGAP B-protein +, O +suggesting O +that O +it O +can O +be O +involved O +in O +Cdc42 B-protein +interactions O +. O + +The O +solution B-evidence +structure I-evidence +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +presented O +here O +, O +along O +with O +the O +model O +of O +the O +HR1TOCA1 B-complex_assembly +· I-complex_assembly +Cdc42 I-complex_assembly +complex O +is O +consistent O +with O +a O +conserved O +mode O +of O +binding O +across O +the O +known O +HR1 B-structure_element +domain O +- O +Rho O +family O +interactions O +, O +despite O +their O +differing O +affinities O +. O + +The O +weak O +binding O +prevented O +detailed O +structural B-experimental_method +and I-experimental_method +thermodynamic I-experimental_method +studies I-experimental_method +of O +the O +complex O +. O + +Nonetheless O +, O +structural B-experimental_method +studies I-experimental_method +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +, O +combined O +with O +chemical B-experimental_method +shift I-experimental_method +mapping I-experimental_method +, O +have O +highlighted O +some O +potentially O +interesting O +differences O +between O +Cdc42 B-protein +- O +HR1TOCA1 B-structure_element +and O +RhoA B-protein +/ O +Rac1 B-protein +- O +HR1PRK1 B-structure_element +binding O +. O + +We O +have O +previously O +postulated O +that O +the O +inherent O +flexibility O +of O +HR1 B-structure_element +domains O +contributes O +to O +their O +ability O +to O +bind O +to O +different O +Rho B-protein_type +family I-protein_type +G I-protein_type +proteins I-protein_type +, O +with O +Rho O +- O +binding O +HR1 B-structure_element +domains O +displaying O +increased O +flexibility O +, O +reflected O +in O +their O +lower O +melting B-evidence +temperatures I-evidence +( O +Tm B-evidence +) O +and O +Rac B-protein_type +binders O +being O +more O +rigid O +. O + +The O +Tm B-evidence +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +61 O +. O +9 O +° O +C O +( O +data O +not O +shown O +), O +which O +is O +the O +highest O +Tm B-evidence +that O +we O +have O +measured O +for O +an O +HR1 B-structure_element +domain O +thus O +far O +. O + +As O +such O +, O +the O +ability O +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +to O +bind O +to O +Cdc42 B-protein +( O +a O +close O +relative O +of O +Rac1 B-protein +rather O +than O +RhoA B-protein +) O +fits O +this O +trend O +. O + +An O +investigation O +into O +the O +local O +motions O +, O +particularly O +in O +the O +G B-site +protein I-site +- I-site +binding I-site +regions I-site +, O +may O +offer O +further O +insight O +into O +the O +differential O +specificities O +and O +affinities O +of O +G B-protein_type +protein I-protein_type +- O +HR1 B-structure_element +domain O +interactions O +. O + +The O +low O +affinity O +of O +the O +Cdc42 B-protein +- O +HR1TOCA1 B-structure_element +interaction O +is O +consistent O +with O +a O +tightly O +spatially O +and O +temporally O +regulated O +pathway O +, O +requiring O +combinatorial O +signals O +leading O +to O +a O +series O +of O +coincident O +weak O +interactions O +that O +elicit O +full O +activation O +. O + +The O +HR1 B-structure_element +domains O +from O +other O +TOCA B-protein_type +family I-protein_type +members I-protein_type +, O +CIP4 B-protein +and O +FBP17 B-protein +, O +also O +bind O +at O +low O +micromolar O +affinities O +to O +Cdc42 B-protein +, O +so O +the O +low O +affinity O +interaction O +appears O +to O +be O +commonplace O +among O +this O +family O +of O +HR1 B-protein_type +domain I-protein_type +proteins I-protein_type +, O +in O +contrast O +to O +the O +PRK B-protein_type +family I-protein_type +. O + +The O +low O +affinity O +of O +the O +HR1TOCA1 B-structure_element +- O +Cdc42 B-protein +interaction O +in O +the O +context O +of O +the O +physiological O +concentration O +of O +TOCA1 B-protein +in O +Xenopus B-taxonomy_domain +extracts O +(∼ O +10 O +nm O +) O +suggests O +that O +binding O +between O +TOCA1 B-protein +and O +Cdc42 B-protein +is O +likely O +to O +occur O +in O +vivo O +only O +when O +TOCA1 B-protein +is O +at O +high O +local O +concentrations O +and O +membrane O +- O +localized O +and O +therefore O +in O +close O +proximity O +to O +activated B-protein_state +Cdc42 B-protein +. O + +Evidence O +suggests O +that O +the O +TOCA B-protein_type +family I-protein_type +of O +proteins O +are O +recruited O +to O +the O +membrane O +via O +an O +interaction O +between O +their O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +and O +specific O +signaling O +lipids O +. O + +For O +example O +, O +electrostatic B-bond_interaction +interactions I-bond_interaction +between O +the O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +and O +the O +membrane O +are O +required O +for O +TOCA1 B-protein +recruitment O +to O +membrane O +vesicles O +and O +tubules O +, O +and O +TOCA1 B-protein +- O +dependent O +actin O +polymerization O +is O +known O +to O +depend O +specifically O +on O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +. O + +Furthermore O +, O +the O +isolated B-experimental_method +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +of O +FBP17 B-protein +has O +been O +shown O +to O +induce O +membrane O +tubulation O +of O +brain O +liposomes O +and O +BAR B-structure_element +domain O +proteins O +that O +promote O +tubulation O +cluster O +on O +membranes O +at O +high O +densities O +. O + +Once O +at O +the O +membrane O +, O +high O +local O +concentrations O +of O +TOCA1 B-protein +could O +exceed O +the O +Kd B-evidence +of O +F B-structure_element +- I-structure_element +BAR I-structure_element +dimerization B-oligomeric_state +( O +likely O +to O +be O +comparable O +with O +that O +of O +the O +FCHo2 B-protein +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +( O +2 O +. O +5 O +μm O +)) O +and O +that O +of O +the O +Cdc42 B-protein +- O +HR1TOCA1 B-structure_element +interaction O +. O + +Cdc42 B-protein +- O +HR1TOCA1 B-structure_element +binding O +would O +then O +be O +favorable O +, O +as O +long O +as O +coincident O +activation O +of O +Cdc42 B-protein +had O +occurred O +, O +leading O +to O +stabilization O +of O +TOCA1 B-protein +at O +the O +membrane O +and O +downstream O +activation O +of O +N B-protein +- I-protein +WASP I-protein +. O + +It O +has O +been O +postulated O +that O +WASP B-protein_type +and O +N B-protein +- I-protein +WASP I-protein +exist O +in O +equilibrium O +between O +folded B-protein_state +( O +inactive B-protein_state +) O +and O +unfolded B-protein_state +( O +active B-protein_state +) O +forms O +, O +and O +the O +affinity B-evidence +of O +Cdc42 B-protein +for O +the O +unfolded B-protein_state +WASP B-protein_type +proteins O +is O +significantly O +enhanced O +. O + +The O +unfolded B-protein_state +, O +high O +affinity O +state O +of O +WASP B-protein_type +is O +represented O +by O +a O +short O +peptide B-chemical +, O +the O +GBD B-structure_element +, O +which O +binds O +with O +a O +low O +nanomolar O +affinity O +to O +Cdc42 B-protein +. O + +In O +contrast O +, O +the O +best O +estimate O +of O +the O +affinity B-evidence +of O +full B-protein_state +- I-protein_state +length I-protein_state +WASP B-protein_type +for O +Cdc42 B-protein +is O +low O +micromolar O +. O + +In O +the O +inactive B-protein_state +state O +of O +WASP B-protein_type +, O +the O +actin O +- O +and O +Arp2 B-complex_assembly +/ I-complex_assembly +3 I-complex_assembly +- O +binding O +VCA B-structure_element +domain O +contacts O +the O +GBD B-structure_element +, O +competing O +for O +Cdc42 B-protein +binding O +. O + +The O +high O +affinity O +of O +Cdc42 B-protein +for O +the O +unfolded B-protein_state +, O +active B-protein_state +form O +pushes O +the O +equilibrium O +in O +favor O +of O +( B-protein +N I-protein +-) I-protein +WASP I-protein +activation O +. O + +Binding O +of O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +to O +the O +basic O +region O +just O +N O +- O +terminal O +to O +the O +GBD B-structure_element +further O +favors O +the O +active B-protein_state +conformation O +. O + +A O +substantial O +body O +of O +data O +has O +illuminated O +the O +complex O +regulation O +of O +WASP B-protein_type +/ I-protein_type +N I-protein_type +- I-protein_type +WASP I-protein_type +proteins I-protein_type +, O +and O +current O +evidence O +suggests O +that O +these O +allosteric O +activation O +mechanisms O +and O +oligomerization O +combine O +to O +regulate O +WASP B-protein_type +activity O +, O +allowing O +the O +synchronization O +and O +integration O +of O +multiple O +potential O +activation O +signals O +( O +reviewed O +in O +Ref O +.). O + +We O +envisage O +that O +TOCA1 B-protein +is O +first O +recruited O +to O +the O +appropriate O +membrane O +in O +response O +to O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +via O +its O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +, O +where O +the O +local O +increase O +in O +concentration O +favors O +F B-structure_element +- I-structure_element +BAR I-structure_element +- O +mediated O +dimerization B-oligomeric_state +of O +TOCA1 B-protein +. O + +Cdc42 B-protein +is O +activated O +in O +response O +to O +co O +- O +incident O +signals O +and O +can O +then O +bind O +to O +TOCA1 B-protein +, O +further O +stabilizing O +TOCA1 B-protein +at O +the O +membrane O +. O + +TOCA1 B-protein +can O +then O +recruit O +N B-protein +- I-protein +WASP I-protein +via O +an O +interaction O +between O +its O +SH3 B-structure_element +domain O +and O +the O +N B-protein +- I-protein +WASP I-protein +proline B-structure_element +- I-structure_element +rich I-structure_element +region I-structure_element +. O + +The O +recruitment O +of O +N B-protein +- I-protein +WASP I-protein +alone B-protein_state +and O +of O +the O +N B-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +WIP I-complex_assembly +complex O +by O +TOCA1 B-protein +and O +FBP17 B-protein +has O +been O +demonstrated O +. O + +WIP B-protein +inhibits O +the O +activation O +of O +N B-protein +- I-protein +WASP I-protein +by O +Cdc42 B-protein +, O +an O +effect O +that O +is O +reversed O +by O +TOCA1 B-protein +. O + +It O +may O +therefore O +be O +envisaged O +that O +WIP B-protein +and O +TOCA1 B-protein +exert O +opposing O +allosteric O +effects O +on O +N B-protein +- I-protein +WASP I-protein +, O +with O +TOCA1 B-protein +favoring O +the O +unfolded B-protein_state +, O +active B-protein_state +conformation O +of O +N B-protein +- I-protein +WASP I-protein +and O +increasing O +its O +affinity O +for O +Cdc42 B-protein +. O + +TOCA1 B-protein +may O +also O +activate O +N B-protein +- I-protein +WASP I-protein +by O +effective O +oligomerization O +because O +clustering O +of O +TOCA1 B-protein +at O +the O +membrane O +following O +coincident O +interactions O +with O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +and O +Cdc42 B-protein +would O +in O +turn O +lead O +to O +clustering O +of O +N B-protein +- I-protein +WASP I-protein +, O +in O +addition O +to O +pushing O +the O +equilibrium O +toward O +the O +unfolded B-protein_state +, O +active B-protein_state +state O +. O + +In O +a O +cellular O +context O +, O +full B-protein_state +- I-protein_state +length I-protein_state +TOCA1 B-protein +and O +N B-protein +- I-protein +WASP I-protein +are O +likely O +to O +have O +similar O +affinities B-evidence +for O +active B-protein_state +Cdc42 B-protein +, O +but O +in O +the O +unfolded B-protein_state +, O +active B-protein_state +conformation O +, O +the O +affinity B-evidence +of O +N B-protein +- I-protein +WASP I-protein +for O +Cdc42 B-protein +dramatically O +increases O +. O + +Our O +binding B-evidence +data I-evidence +suggest O +that O +TOCA1 B-protein +HR1 B-structure_element +binding O +is O +not O +allosterically O +regulated O +, O +and O +our O +NMR B-experimental_method +data O +, O +along O +with O +the O +high O +stability B-protein_state +of O +TOCA1 B-protein +HR1 B-structure_element +, O +suggest O +that O +there O +is O +no O +widespread O +conformational O +change O +in O +the O +presence B-protein_state +of I-protein_state +Cdc42 B-protein +. O + +As O +full B-protein_state +- I-protein_state +length I-protein_state +TOCA1 B-protein +and O +the O +isolated B-protein_state +HR1 B-structure_element +domain O +bind O +Cdc42 B-protein +with O +similar O +affinities O +, O +the O +N B-protein +- I-protein +WASP I-protein +- O +Cdc42 B-protein +interaction O +will O +be O +favored O +because O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +can O +easily O +outcompete O +the O +TOCA1 B-protein +HR1 B-structure_element +for O +Cdc42 B-protein +. O + +A O +combination O +of O +allosteric O +activation O +by O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +, O +activated B-protein_state +Cdc42 B-protein +and O +TOCA1 B-protein +, O +and O +oligomeric O +activation O +implemented O +by O +TOCA1 B-protein +would O +lead O +to O +full B-protein_state +activation I-protein_state +of O +N B-protein +- I-protein +WASP I-protein +and O +downstream O +actin O +polymerization O +. O + +In O +such O +an O +array O +of O +molecules O +localized O +to O +a O +discrete O +region O +of O +the O +membrane O +, O +it O +is O +plausible O +that O +WASP B-protein +could O +bind O +to O +a O +second O +Cdc42 B-protein +molecule O +rather O +than O +displacing O +TOCA1 B-protein +from O +its O +cognate O +Cdc42 B-protein +. O + +Our O +NMR B-experimental_method +and O +affinity B-evidence +data I-evidence +, O +however O +, O +are O +consistent O +with O +displacement O +of O +the O +TOCA1 B-protein +HR1 B-structure_element +by O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +. O + +Furthermore O +, O +TOCA1 B-protein +is O +required O +for O +Cdc42 B-protein +- O +mediated O +activation O +of O +N B-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +WIP I-complex_assembly +, O +implying O +that O +it O +may O +not O +be O +possible O +for O +Cdc42 B-protein +to O +bind O +and O +activate O +N B-protein +- I-protein +WASP I-protein +prior O +to O +TOCA1 B-protein +- O +Cdc42 B-protein +binding O +. O + +The O +commonly O +used O +MGD B-mutant +→ I-mutant +IST I-mutant +( O +Cdc42 B-protein_state +- I-protein_state +binding I-protein_state +deficient I-protein_state +) O +mutant O +of O +TOCA1 B-protein +has O +a O +reduced O +ability O +to O +activate O +the O +N B-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +WIP I-complex_assembly +complex O +, O +further O +indicating O +the O +importance O +of O +the O +Cdc42 B-protein +- O +HR1TOCA1 B-structure_element +interaction O +prior O +to O +downstream O +activation O +of O +N B-protein +- I-protein +WASP I-protein +. O + +In O +light O +of O +this O +, O +we O +favor O +an O +“ O +effector O +handover O +” O +scheme O +whereby O +TOCA1 B-protein +interacts O +with O +Cdc42 B-protein +prior O +to O +N B-protein +- I-protein +WASP I-protein +activation O +, O +after O +which O +N B-protein +- I-protein +WASP I-protein +displaces O +TOCA1 B-protein +from O +its O +bound B-protein_state +Cdc42 B-protein +in O +order O +to O +be O +fully B-protein_state +activated I-protein_state +rather O +than O +binding O +a O +second O +Cdc42 B-protein +molecule O +. O + +Potentially O +, O +the O +TOCA1 B-protein +- O +Cdc42 B-protein +interaction O +functions O +to O +position O +N B-protein +- I-protein +WASP I-protein +and O +Cdc42 B-protein +such O +that O +they O +are O +poised O +to O +interact O +with O +high O +affinity O +. O + +The O +concomitant O +release O +of O +TOCA1 B-protein +from O +Cdc42 B-protein +while O +still O +bound B-protein_state +to I-protein_state +N B-protein +- I-protein +WASP I-protein +presumably O +enhances O +the O +ability O +of O +TOCA1 B-protein +to O +further O +activate O +N B-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +WIP I-complex_assembly +- O +induced O +actin O +polymerization O +. O + +There O +is O +an O +advantage O +to O +such O +an O +effector O +handover O +, O +in O +that O +N B-protein +- I-protein +WASP I-protein +would O +only O +be O +robustly O +recruited O +when O +F B-structure_element +- I-structure_element +BAR I-structure_element +domains O +are O +already O +present O +. O + +Hence O +, O +actin O +polymerization O +cannot O +occur O +until O +F B-structure_element +- I-structure_element +BAR I-structure_element +domains O +are O +poised O +for O +membrane O +distortion O +. O + +Our O +model O +of O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1TOCA1 I-complex_assembly +complex O +indicates O +a O +mechanism O +by O +which O +such O +a O +handover O +could O +take O +place O +( O +Fig O +. O +9 O +) O +because O +it O +shows O +that O +the O +effector B-site +binding I-site +sites I-site +only O +partially O +overlap O +on O +Cdc42 B-protein +. O + +The O +lysine B-residue_name +residues O +thought O +to O +be O +involved O +in O +an O +electrostatic O +steering O +mechanism O +in O +WASP B-protein +- O +Cdc42 B-protein +binding O +are O +conserved O +in O +N B-protein +- I-protein +WASP I-protein +and O +would O +be O +able O +to O +interact O +with O +Cdc42 B-protein +even O +when O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +already O +bound B-protein_state +. O + +It O +has O +been O +postulated O +that O +the O +initial O +interactions O +between O +this O +basic O +region O +and O +Cdc42 B-protein +could O +stabilize O +the O +active B-protein_state +conformation O +of O +WASP B-protein +, O +leading O +to O +high O +affinity O +binding O +between O +the O +core O +CRIB B-structure_element +and O +Cdc42 B-protein +. O + +The O +region O +C O +- O +terminal O +to O +the O +core O +CRIB B-structure_element +, O +required O +for O +maximal O +affinity O +binding O +, O +would O +then O +fully O +displace O +the O +TOCA1 B-protein +HR1 B-structure_element +. O + +A O +simplified O +model O +of O +the O +early O +stages O +of O +Cdc42 B-complex_assembly +· I-complex_assembly +N I-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +- O +dependent O +actin O +polymerization O +. O + +Step O +1 O +, O +TOCA1 B-protein +is O +recruited O +to O +the O +membrane O +via O +its O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +and O +/ O +or O +Cdc42 B-protein +interactions O +. O + +F O +- O +BAR O +oligomerization O +is O +expected O +to O +occur O +following O +membrane O +binding O +, O +but O +a O +single O +monomer B-oligomeric_state +is O +shown O +for O +clarity O +. O + +Step O +2 O +, O +N B-protein +- I-protein +WASP I-protein +exists O +in O +an O +inactive B-protein_state +, O +folded B-protein_state +conformation O +. O + +The O +TOCA1 B-protein +SH3 B-structure_element +domain O +interacts O +with O +N B-protein +- I-protein +WASP I-protein +, O +causing O +an O +activatory O +allosteric O +effect O +. O + +The O +HR1TOCA1 B-structure_element +- O +Cdc42 O +and O +SH3TOCA1 B-structure_element +- O +N O +- O +WASP O +interactions O +position O +Cdc42 B-protein +and O +N B-protein +- I-protein +WASP I-protein +for O +binding O +. O + +Step O +3 O +, O +electrostatic B-bond_interaction +interactions I-bond_interaction +between O +Cdc42 B-protein +and O +the O +basic O +region O +upstream O +of O +the O +CRIB B-structure_element +initiate O +Cdc42 B-complex_assembly +· I-complex_assembly +N I-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +binding O +. O + +Step O +4 O +, O +the O +core O +CRIB B-structure_element +binds O +with O +high O +affinity O +while O +the O +region O +C O +- O +terminal O +to O +the O +CRIB B-structure_element +displaces O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +and O +increases O +the O +affinity O +of O +the O +N B-protein +- I-protein +WASP I-protein +- O +Cdc42 O +interaction O +further O +. O + +The O +VCA B-structure_element +domain O +is O +released O +for O +downstream O +interactions O +, O +and O +actin O +polymerization O +proceeds O +. O + +WH1 O +, O +WASP B-structure_element +homology I-structure_element +1 I-structure_element +domain I-structure_element +; O +PP B-structure_element +, O +proline B-structure_element +- I-structure_element +rich I-structure_element +region I-structure_element +; O +VCA B-structure_element +, O +verprolin B-structure_element +homology I-structure_element +, I-structure_element +cofilin I-structure_element +homology I-structure_element +, I-structure_element +acidic I-structure_element +region I-structure_element +. O + +In O +conclusion O +, O +the O +data O +presented O +here O +show O +that O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +sufficient O +for O +Cdc42 B-protein +binding O +in O +vitro O +and O +that O +the O +interaction O +is O +of O +micromolar O +affinity O +, O +lower O +than O +that O +of O +other O +G B-protein_type +protein I-protein_type +- O +HR1 B-structure_element +domain O +interactions O +. O + +The O +analogous O +HR1 B-structure_element +domains O +from O +other O +TOCA1 B-protein_type +family I-protein_type +members O +, O +FBP17 B-protein +and O +CIP4 B-protein +, O +also O +exhibit O +micromolar O +affinity O +for O +Cdc42 B-protein +. O + +A O +role O +for O +the O +TOCA1 B-protein +-, O +FBP17 B-protein +-, O +and O +CIP4 B-protein +- O +Cdc42 B-protein +interactions O +in O +the O +recruitment O +of O +these O +proteins O +to O +the O +membrane O +therefore O +appears O +unlikely O +. O + +Instead O +, O +our O +findings O +agree O +with O +earlier O +suggestions O +that O +the O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +is O +responsible O +for O +membrane O +recruitment O +. O + +The O +role O +of O +the O +Cdc42 B-protein +- O +TOCA1 B-protein +interaction O +remains O +somewhat O +elusive O +, O +but O +it O +may O +serve O +to O +position O +activated B-protein_state +Cdc42 B-protein +and O +N B-protein +- I-protein +WASP I-protein +to O +allow O +full B-protein_state +activation I-protein_state +of O +N B-protein +- I-protein +WASP I-protein +and O +as O +such O +serve O +to O +couple O +F B-structure_element +- I-structure_element +BAR I-structure_element +- O +mediated O +membrane O +deformation O +with O +N B-protein +- I-protein +WASP I-protein +activation O +. O + +We O +envisage O +a O +complex O +interplay O +of O +equilibria O +between O +free B-protein_state +and O +bound B-protein_state +, O +active B-protein_state +and O +inactive B-protein_state +Cdc42 B-protein +, O +TOCA B-protein_type +family I-protein_type +, O +and O +WASP B-protein_type +family O +proteins O +, O +facilitating O +a O +tightly O +spatially O +and O +temporally O +regulated O +pathway O +requiring O +numerous O +simultaneous O +events O +in O +order O +to O +achieve O +appropriate O +and O +robust O +activation O +of O +the O +downstream O +pathway O +. O + +Our O +data O +are O +therefore O +easily O +reconciled O +with O +the O +dynamic O +instability O +models O +described O +in O +relation O +to O +the O +formation O +of O +endocytic O +vesicles O +and O +with O +the O +current O +data O +pertaining O +to O +the O +complex O +activation O +of O +WASP B-protein_type +/ O +N B-protein +- I-protein +WASP I-protein +pathways O +by O +allosteric O +and O +oligomeric O +effects O +. O + +It O +is O +clear O +from O +the O +data O +presented O +here O +that O +TOCA1 B-protein +and O +N B-protein +- I-protein +WASP I-protein +do O +not O +bind O +Cdc42 B-protein +simultaneously O +and O +that O +N B-protein +- I-protein +WASP I-protein +is O +likely O +to O +outcompete O +TOCA1 B-protein +for O +Cdc42 B-protein +binding O +. O + +We O +therefore O +postulate O +an O +effector O +handover O +mechanism O +based O +on O +current O +evidence O +surrounding O +WASP B-protein +/ O +N B-protein +- I-protein +WASP I-protein +activation O +and O +our O +model O +of O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1TOCA1 I-complex_assembly +complex O +. O + +The O +displacement O +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +from O +Cdc42 B-protein +by O +N B-protein +- I-protein +WASP I-protein +may O +represent O +a O +unidirectional O +step O +in O +the O +pathway O +of O +Cdc42 B-complex_assembly +· I-complex_assembly +N I-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +- O +dependent O +actin O +assembly O +. O + +The O +dynamic B-protein_state +organization O +of O +fungal B-taxonomy_domain +acetyl B-protein_type +- I-protein_type +CoA I-protein_type +carboxylase I-protein_type + +Acetyl B-protein_type +- I-protein_type +CoA I-protein_type +carboxylases I-protein_type +( O +ACCs B-protein_type +) O +catalyse O +the O +committed O +step O +in O +fatty O +- O +acid O +biosynthesis O +: O +the O +ATP B-chemical +- O +dependent O +carboxylation O +of O +acetyl B-chemical +- I-chemical +CoA I-chemical +to O +malonyl B-chemical +- I-chemical +CoA I-chemical +. O +They O +are O +important O +regulatory O +hubs O +for O +metabolic O +control O +and O +relevant O +drug O +targets O +for O +the O +treatment O +of O +the O +metabolic O +syndrome O +and O +cancer O +. O + +Eukaryotic B-taxonomy_domain +ACCs B-protein_type +are O +single B-protein_type +- I-protein_type +chain I-protein_type +multienzymes I-protein_type +characterized O +by O +a O +large O +, O +non B-protein_state +- I-protein_state +catalytic I-protein_state +central B-structure_element +domain I-structure_element +( O +CD B-structure_element +), O +whose O +role O +in O +ACC B-protein_type +regulation O +remains O +poorly O +characterized O +. O + +Here O +we O +report O +the O +crystal B-evidence +structure I-evidence +of O +the O +yeast B-taxonomy_domain +ACC B-protein_type +CD B-structure_element +, O +revealing O +a O +unique O +four O +- O +domain O +organization O +. O + +A O +regulatory B-structure_element +loop I-structure_element +, O +which O +is O +phosphorylated B-protein_state +at O +the O +key O +functional O +phosphorylation B-site +site I-site +of O +fungal B-taxonomy_domain +ACC B-protein_type +, O +wedges O +into O +a O +crevice O +between O +two O +domains O +of O +CD B-structure_element +. O + +Combining O +the O +yeast B-taxonomy_domain +CD B-structure_element +structure B-evidence +with O +intermediate O +and O +low O +- O +resolution O +data O +of O +larger B-mutant +fragments I-mutant +up O +to O +intact B-protein_state +ACCs B-protein_type +provides O +a O +comprehensive O +characterization O +of O +the O +dynamic B-protein_state +fungal B-taxonomy_domain +ACC B-protein_type +architecture O +. O + +In O +contrast O +to O +related O +carboxylases B-protein_type +, O +large O +- O +scale O +conformational O +changes O +are O +required O +for O +substrate O +turnover O +, O +and O +are O +mediated O +by O +the O +CD B-structure_element +under O +phosphorylation B-ptm +control O +. O + +Acetyl B-protein_type +- I-protein_type +CoA I-protein_type +carboxylases I-protein_type +are O +central O +regulatory O +hubs O +of O +fatty O +acid O +metabolism O +and O +are O +important O +targets O +for O +drug O +development O +in O +obesity O +and O +cancer O +. O + +Here O +, O +the O +authors O +demonstrate O +that O +the O +regulation O +of O +these O +highly B-protein_state +dynamic I-protein_state +enzymes B-protein_type +in O +fungi B-taxonomy_domain +is O +governed O +by O +a O +mechanism O +based O +on O +phosphorylation B-ptm +- O +dependent O +conformational O +variability O +. O + +Biotin B-protein_type +- I-protein_type +dependent I-protein_type +acetyl I-protein_type +- I-protein_type +CoA I-protein_type +carboxylases I-protein_type +( O +ACCs B-protein_type +) O +are O +essential O +enzymes O +that O +catalyse O +the O +ATP B-chemical +- O +dependent O +carboxylation O +of O +acetyl B-chemical +- I-chemical +CoA I-chemical +to O +malonyl B-chemical +- I-chemical +CoA I-chemical +. O +This O +reaction O +provides O +the O +committed O +activated O +substrate O +for O +the O +biosynthesis O +of O +fatty B-chemical +acids I-chemical +via O +fatty B-protein_type +- I-protein_type +acid I-protein_type +synthase I-protein_type +. O + +By O +catalysing O +this O +rate O +- O +limiting O +step O +in O +fatty O +- O +acid O +biosynthesis O +, O +ACC B-protein_type +plays O +a O +key O +role O +in O +anabolic O +metabolism O +. O + +ACC B-experimental_method +inhibition I-experimental_method +and I-experimental_method +knock I-experimental_method +- I-experimental_method +out I-experimental_method +studies I-experimental_method +show O +the O +potential O +of O +targeting O +ACC B-protein_type +for O +treatment O +of O +the O +metabolic O +syndrome O +. O + +Furthermore O +, O +elevated O +ACC B-protein_type +activity O +is O +observed O +in O +malignant O +tumours O +. O + +A O +direct O +link O +between O +ACC B-protein_type +and O +cancer O +is O +provided O +by O +cancer O +- O +associated O +mutations B-mutant +in O +the O +breast B-protein +cancer I-protein +susceptibility I-protein +gene I-protein +1 I-protein +( O +BRCA1 B-protein +), O +which O +relieve O +inhibitory O +interactions O +of O +BRCA1 B-protein +with O +ACC B-protein_type +. O + +Thus O +, O +ACC B-protein_type +is O +a O +relevant O +drug O +target O +for O +type O +2 O +diabetes O +and O +cancer O +. O + +Microbial B-taxonomy_domain +ACCs B-protein_type +are O +also O +the O +principal O +target O +of O +antifungal O +and O +antibiotic O +compounds O +, O +such O +as O +Soraphen B-chemical +A I-chemical +. O + +The O +principal O +functional O +protein O +components O +of O +ACCs B-protein_type +have O +been O +described O +already O +in O +the O +late O +1960s O +for O +Escherichia B-species +coli I-species +( O +E B-species +. I-species +coli I-species +) O +ACC B-protein_type +: O +Biotin B-protein_type +carboxylase I-protein_type +( O +BC B-protein_type +) O +catalyses O +the O +ATP B-chemical +- O +dependent O +carboxylation O +of O +a O +biotin B-chemical +moiety O +, O +which O +is O +covalently O +linked O +to O +the O +biotin B-protein_type +carboxyl I-protein_type +carrier I-protein_type +protein I-protein_type +( O +BCCP B-protein_type +). O + +Carboxyltransferase B-protein_type +( O +CT B-protein_type +) O +transfers O +the O +activated O +carboxyl B-chemical +group O +from O +carboxybiotin B-chemical +to O +acetyl B-chemical +- I-chemical +CoA I-chemical +to O +yield O +malonyl B-chemical +- I-chemical +CoA I-chemical +. O +Prokaryotic B-taxonomy_domain +ACCs B-protein_type +are O +transient B-protein_state +assemblies O +of O +individual O +BC B-protein_type +, O +CT B-protein_type +and O +BCCP B-protein_type +subunits O +. O + +Eukaryotic B-taxonomy_domain +ACCs B-protein_type +, O +instead O +, O +are O +multienzymes B-protein_type +, O +which O +integrate O +all O +functional O +components O +into O +a O +single O +polypeptide O +chain O +of O +∼ O +2 O +, O +300 O +amino O +acids O +. O + +Human B-species +ACC B-protein_type +occurs O +in O +two O +closely O +related O +isoforms B-protein_state +, O +ACC1 B-protein +and O +2 B-protein +, O +located O +in O +the O +cytosol O +and O +at O +the O +outer O +mitochondrial O +membrane O +, O +respectively O +. O + +In O +addition O +to O +the O +canonical O +ACC B-structure_element +components I-structure_element +, O +eukaryotic B-taxonomy_domain +ACCs B-protein_type +contain O +two O +non B-protein_state +- I-protein_state +catalytic I-protein_state +regions B-structure_element +, O +the O +large O +central B-structure_element +domain I-structure_element +( O +CD B-structure_element +) O +and O +the O +BC B-structure_element +– I-structure_element +CT I-structure_element +interaction I-structure_element +domain I-structure_element +( O +BT B-structure_element +). O + +The O +CD B-structure_element +comprises O +one O +- O +third O +of O +the O +protein O +and O +is O +a O +unique B-protein_state +feature I-protein_state +of I-protein_state +eukaryotic B-taxonomy_domain +ACCs B-protein_type +without O +homologues O +in O +other O +proteins O +. O + +The O +function O +of O +this O +domain O +remains O +poorly O +characterized O +, O +although O +phosphorylation B-ptm +of O +several O +serine B-residue_name +residues O +in O +the O +CD B-structure_element +regulates O +ACC B-protein_type +activity O +. O + +The O +BT B-structure_element +domain O +has O +been O +visualized O +in O +bacterial B-taxonomy_domain +carboxylases B-protein_type +, O +where O +it O +mediates O +contacts O +between O +α B-structure_element +- I-structure_element +and O +β B-structure_element +- I-structure_element +subunits I-structure_element +. O + +Structural B-experimental_method +studies I-experimental_method +on O +the O +functional O +architecture O +of O +intact B-protein_state +ACCs B-protein_type +have O +been O +hindered O +by O +their O +huge O +size O +and O +pronounced O +dynamics O +, O +as O +well O +as O +the O +transient B-protein_state +assembly O +mode O +of O +bacterial B-taxonomy_domain +ACCs B-protein_type +. O + +However O +, O +crystal B-evidence +structures I-evidence +of O +individual O +components O +or O +domains O +from O +prokaryotic B-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +ACCs B-protein_type +, O +respectively O +, O +have O +been O +solved O +. O + +The O +structure B-experimental_method +determination I-experimental_method +of O +the O +holoenzymes B-protein_state +of O +bacterial B-taxonomy_domain +biotin B-protein_type +- I-protein_type +dependent I-protein_type +carboxylases I-protein_type +, O +which O +lack B-protein_state +the O +characteristic O +CD B-structure_element +, O +such O +as O +the O +pyruvate B-protein_type +carboxylase I-protein_type +( O +PC B-protein_type +), O +propionyl B-protein_type +- I-protein_type +CoA I-protein_type +carboxylase I-protein_type +, O +3 B-protein_type +- I-protein_type +methyl I-protein_type +- I-protein_type +crotonyl I-protein_type +- I-protein_type +CoA I-protein_type +carboxylase I-protein_type +and O +a O +long B-protein_type +- I-protein_type +chain I-protein_type +acyl I-protein_type +- I-protein_type +CoA I-protein_type +carboxylase I-protein_type +revealed O +strikingly O +divergent O +architectures O +despite O +a O +general O +conservation O +of O +all O +functional O +components O +. O + +In O +these O +structures B-evidence +, O +the O +BC B-protein_type +and O +CT B-protein_type +active B-site +sites I-site +are O +at O +distances O +between O +40 O +and O +80 O +Å O +, O +such O +that O +substrate O +transfer O +could O +be O +mediated O +solely O +by O +the O +mobility O +of O +the O +flexibly B-protein_state +tethered I-protein_state +BCCP B-protein_type +. O + +Human B-species +ACC1 B-protein +is O +regulated B-protein_state +allosterically I-protein_state +, O +via O +specific O +protein O +– O +protein O +interactions O +, O +and O +by O +reversible O +phosphorylation B-ptm +. O + +Dynamic O +polymerization O +of O +human B-species +ACC1 B-protein +is O +linked O +to O +increased O +activity O +and O +is O +regulated B-protein_state +allosterically I-protein_state +by O +the O +activator O +citrate B-chemical +and O +the O +inhibitor O +palmitate B-chemical +, O +or O +by O +binding O +of O +the O +small O +protein O +MIG B-protein +- I-protein +12 I-protein +( O +ref O +.). O + +Human B-species +ACC1 B-protein +is O +further O +regulated O +by O +specific O +phosphorylation B-ptm +- O +dependent O +binding O +of O +BRCA1 B-protein +to O +Ser1263 B-residue_name_number +in O +the O +CD B-structure_element +. O + +BRCA1 B-protein +binds O +only O +to O +the O +phosphorylated B-protein_state +form O +of O +ACC1 B-protein +and O +prevents O +ACC B-protein_type +activation O +by O +phosphatase B-protein_type +- O +mediated O +dephosphorylation O +. O + +Furthermore O +, O +phosphorylation B-ptm +by O +AMP B-protein +- I-protein +activated I-protein +protein I-protein +kinase I-protein +( O +AMPK B-protein +) O +and O +cAMP B-protein +- I-protein +dependent I-protein +protein I-protein +kinase I-protein +( O +PKA B-protein +) O +leads O +to O +a O +decrease O +in O +ACC1 B-protein +activity O +. O + +AMPK B-protein +phosphorylates O +ACC1 B-protein +in O +vitro O +at O +Ser80 B-residue_name_number +, O +Ser1201 B-residue_name_number +and O +Ser1216 B-residue_name_number +and O +PKA B-protein +at O +Ser78 B-residue_name_number +and O +Ser1201 B-residue_name_number +. O + +However O +, O +regulatory O +effects O +on O +ACC1 B-protein +activity O +are O +mainly O +mediated O +by O +phosphorylation B-ptm +of O +Ser80 B-residue_name_number +and O +Ser1201 B-residue_name_number +( O +refs O +). O + +Phosphorylated B-protein_state +Ser80 B-residue_name_number +, O +which O +is O +highly B-protein_state +conserved I-protein_state +only O +in O +higher B-taxonomy_domain +eukaryotes I-taxonomy_domain +, O +presumably O +binds O +into O +the O +Soraphen B-site +A I-site +- I-site +binding I-site +pocket I-site +. O + +The O +regulatory O +Ser1201 B-residue_name_number +shows O +only O +moderate B-protein_state +conservation I-protein_state +across O +higher B-taxonomy_domain +eukaryotes I-taxonomy_domain +, O +while O +the O +phosphorylated B-protein_state +Ser1216 B-residue_name_number +is O +highly B-protein_state +conserved I-protein_state +across O +all O +eukaryotes B-taxonomy_domain +. O + +However O +, O +no O +effect O +of O +Ser1216 B-residue_name_number +phosphorylation B-ptm +on O +ACC B-protein_type +activity O +has O +been O +reported O +in O +higher B-taxonomy_domain +eukaryotes I-taxonomy_domain +. O + +For O +fungal B-taxonomy_domain +ACC B-protein_type +, O +neither O +spontaneous O +nor O +inducible O +polymerization O +has O +been O +detected O +despite O +considerable O +sequence O +conservation O +to O +human B-species +ACC1 B-protein +. O + +The O +BRCA1 B-protein +- O +interacting O +phosphoserine B-residue_name +position O +is O +not B-protein_state +conserved I-protein_state +in O +fungal B-taxonomy_domain +ACC B-protein_type +, O +and O +no O +other O +phospho O +- O +dependent O +protein O +– O +protein O +interactions O +of O +fungal B-taxonomy_domain +ACC B-protein_type +have O +been O +described O +. O + +In O +yeast B-taxonomy_domain +ACC B-protein_type +, O +phosphorylation B-site +sites I-site +have O +been O +identified O +at O +Ser2 B-residue_name_number +, O +Ser735 B-residue_name_number +, O +Ser1148 B-residue_name_number +, O +Ser1157 B-residue_name_number +and O +Ser1162 B-residue_name_number +( O +ref O +.). O + +Of O +these O +, O +only O +Ser1157 B-residue_name_number +is O +highly B-protein_state +conserved I-protein_state +in O +fungal B-taxonomy_domain +ACC B-protein_type +and O +aligns B-experimental_method +to I-experimental_method +Ser1216 B-residue_name_number +in O +human B-species +ACC1 B-protein +. O + +Its O +phosphorylation B-ptm +by O +the O +AMPK B-protein +homologue O +SNF1 B-protein +results O +in O +strongly O +reduced O +ACC B-protein_type +activity O +. O + +Despite O +the O +outstanding O +relevance O +of O +ACC B-protein_type +in O +primary O +metabolism O +and O +disease O +, O +the O +dynamic O +organization O +and O +regulation O +of O +the O +giant O +eukaryotic B-taxonomy_domain +, O +and O +in O +particular O +fungal B-taxonomy_domain +ACC B-protein_type +, O +remain O +poorly O +characterized O +. O + +Here O +we O +provide O +the O +structure B-evidence +of O +Saccharomyces B-species +cerevisiae I-species +( O +Sce B-species +) O +ACC B-protein_type +CD B-structure_element +, O +intermediate O +- O +and O +low O +- O +resolution O +structures B-evidence +of O +human B-species +( O +Hsa B-species +) O +ACC B-protein_type +CD B-structure_element +and O +larger B-mutant +fragments I-mutant +of O +fungal B-taxonomy_domain +ACC B-protein_type +from O +Chaetomium B-species +thermophilum I-species +( O +Cth B-species +; O +Fig O +. O +1a O +). O + +Integrating O +these O +data O +with O +small B-experimental_method +- I-experimental_method +angle I-experimental_method +X I-experimental_method +- I-experimental_method +ray I-experimental_method +scattering I-experimental_method +( O +SAXS B-experimental_method +) O +and O +electron B-experimental_method +microscopy I-experimental_method +( O +EM B-experimental_method +) O +observations O +yield O +a O +comprehensive O +representation O +of O +the O +dynamic O +structure O +and O +regulation O +of O +fungal B-taxonomy_domain +ACC B-protein_type +. O + +The O +organization O +of O +the O +yeast B-taxonomy_domain +ACC B-protein_type +CD B-structure_element + +First O +, O +we O +focused O +on O +structure B-experimental_method +determination I-experimental_method +of O +the O +82 O +- O +kDa O +CD B-structure_element +. O + +The O +crystal B-evidence +structure I-evidence +of O +the O +CD B-structure_element +of O +SceACC B-protein +( O +SceCD B-species +) O +was O +determined O +at O +3 O +. O +0 O +Å O +resolution O +by O +experimental B-experimental_method +phasing I-experimental_method +and O +refined B-experimental_method +to O +Rwork B-evidence +/ O +Rfree B-evidence += O +0 O +. O +20 O +/ O +0 O +. O +24 O +( O +Table O +1 O +). O + +The O +overall O +extent O +of O +the O +SceCD B-species +is O +70 O +by O +75 O +Å O +( O +Fig O +. O +1b O +and O +Supplementary O +Fig O +. O +1a O +, O +b O +), O +and O +the O +attachment O +points O +of O +the O +N O +- O +terminal O +26 B-structure_element +- I-structure_element +residue I-structure_element +linker I-structure_element +to O +the O +BCCP B-structure_element +domain O +and O +the O +C O +- O +terminal O +CT B-structure_element +domain O +are O +separated O +by O +46 O +Å O +( O +the O +N O +- O +and O +C O +termini O +are O +indicated O +with O +spheres O +in O +Fig O +. O +1b O +). O + +SceCD B-species +comprises O +four O +distinct O +domains O +, O +an O +N O +- O +terminal O +α B-structure_element +- I-structure_element +helical I-structure_element +domain I-structure_element +( O +CDN B-structure_element +), O +and O +a O +central O +four B-structure_element +- I-structure_element +helix I-structure_element +bundle I-structure_element +linker I-structure_element +domain I-structure_element +( O +CDL B-structure_element +), O +followed O +by O +two O +α B-structure_element +– I-structure_element +β I-structure_element +- I-structure_element +fold I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +domains I-structure_element +( O +CDC1 B-structure_element +/ O +CDC2 B-structure_element +). O + +CDN B-structure_element +adopts O +a O +letter O +C B-protein_state +shape I-protein_state +, O +where O +one O +of O +the O +ends O +is O +a O +regular B-structure_element +four I-structure_element +- I-structure_element +helix I-structure_element +bundle I-structure_element +( O +Nα3 B-structure_element +- I-structure_element +6 I-structure_element +), O +the O +other O +end O +is O +a O +helical B-structure_element +hairpin I-structure_element +( O +Nα8 B-structure_element +, I-structure_element +9 I-structure_element +) O +and O +the O +bridging B-structure_element +region I-structure_element +comprises O +six O +helices B-structure_element +( O +Nα1 B-structure_element +, I-structure_element +2 I-structure_element +, I-structure_element +7 I-structure_element +, I-structure_element +10 I-structure_element +– I-structure_element +12 I-structure_element +). O + +CDL B-structure_element +is O +composed O +of O +a O +small B-structure_element +, I-structure_element +irregular I-structure_element +four I-structure_element +- I-structure_element +helix I-structure_element +bundle I-structure_element +( O +Lα1 B-structure_element +– I-structure_element +4 I-structure_element +) O +and O +tightly O +interacts O +with O +the O +open O +face O +of O +CDC1 B-structure_element +via O +an O +interface B-site +of O +1 O +, O +300 O +Å2 O +involving O +helices B-structure_element +Lα3 B-structure_element +and O +Lα4 B-structure_element +. O + +CDL B-structure_element +does O +not O +interact O +with O +CDN B-structure_element +apart O +from O +the O +covalent O +linkage O +and O +forms O +only O +a O +small O +contact O +to O +CDC2 B-structure_element +via O +a O +loop B-structure_element +between O +Lα2 B-structure_element +/ I-structure_element +α3 I-structure_element +and O +the O +N O +- O +terminal O +end O +of O +Lα1 B-structure_element +, O +with O +an O +interface B-site +area O +of O +400 O +Å2 O +. O + +CDC1 B-structure_element +/ O +CDC2 B-structure_element +share O +a O +common O +fold O +; O +they O +are O +composed O +of O +six B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +flanked O +on O +one O +side O +by O +two O +long B-structure_element +, I-structure_element +bent I-structure_element +helices I-structure_element +inserted O +between O +strands B-structure_element +β3 B-structure_element +/ I-structure_element +β4 I-structure_element +and O +β4 B-structure_element +/ I-structure_element +β5 I-structure_element +. O + +CDC2 B-structure_element +is O +extended B-protein_state +at O +its O +C O +terminus O +by O +an O +additional O +β B-structure_element +- I-structure_element +strand I-structure_element +and O +an O +irregular B-structure_element +β I-structure_element +- I-structure_element +hairpin I-structure_element +. O + +On O +the O +basis O +of O +a O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +of O +main O +chain O +atom O +positions O +of O +2 O +. O +2 O +Å O +, O +CDC1 B-structure_element +/ O +CDC2 B-structure_element +are O +structurally O +more O +closely O +related O +to O +each O +other O +than O +to O +any O +other O +protein O +( O +Fig O +. O +1c O +); O +they O +may O +thus O +have O +evolved O +by O +duplication O +. O + +Close O +structural O +homologues O +could O +not O +be O +found O +for O +the O +CDN B-structure_element +or O +the O +CDC B-structure_element +domains O +. O + +A O +regulatory B-structure_element +loop I-structure_element +mediates O +interdomain O +interactions O + +To O +define O +the O +functional O +state O +of O +insect B-experimental_method +- I-experimental_method +cell I-experimental_method +- I-experimental_method +expressed I-experimental_method +ACC B-protein_type +variants O +, O +we O +employed O +mass B-experimental_method +spectrometry I-experimental_method +( O +MS B-experimental_method +) O +for O +phosphorylation B-experimental_method +site I-experimental_method +detection I-experimental_method +. O + +In O +insect B-experimental_method +- I-experimental_method +cell I-experimental_method +- I-experimental_method +expressed I-experimental_method +full B-protein_state +- I-protein_state +length I-protein_state +SceACC B-protein +, O +the O +highly B-protein_state +conserved I-protein_state +Ser1157 B-residue_name_number +is O +the O +only O +fully B-protein_state +occupied I-protein_state +phosphorylation B-site +site I-site +with O +functional O +relevance O +in O +S B-species +. I-species +cerevisiae I-species +. O + +Additional O +phosphorylation B-ptm +was O +detected O +for O +Ser2101 B-residue_name_number +and O +Tyr2179 B-residue_name_number +; O +however O +, O +these O +sites O +are O +neither B-protein_state +conserved I-protein_state +across O +fungal B-taxonomy_domain +ACC B-protein_type +nor B-protein_state +natively I-protein_state +phosphorylated I-protein_state +in O +yeast B-taxonomy_domain +. O + +MS B-experimental_method +analysis O +of O +dissolved B-experimental_method +crystals I-experimental_method +confirmed O +the O +phosphorylated B-protein_state +state O +of O +Ser1157 B-residue_name_number +also O +in O +SceCD B-species +crystals B-evidence +. O + +The O +SceCD B-species +structure B-evidence +thus O +authentically O +represents O +the O +state O +of O +SceACC B-protein +, O +where O +the O +enzyme B-protein +is O +inhibited B-protein_state +by O +SNF1 B-ptm +- I-ptm +dependent I-ptm +phosphorylation I-ptm +. O + +In O +the O +SceCD B-species +crystal B-evidence +structure I-evidence +, O +the O +phosphorylated B-protein_state +Ser1157 B-residue_name_number +resides O +in O +a O +regulatory B-structure_element +36 I-structure_element +- I-structure_element +amino I-structure_element +- I-structure_element +acid I-structure_element +loop I-structure_element +between O +strands B-structure_element +β2 B-structure_element +and O +β3 B-structure_element +of O +CDC1 B-structure_element +( O +Fig O +. O +1b O +, O +d O +), O +which O +contains O +two O +additional O +less B-protein_state +- I-protein_state +conserved I-protein_state +phosphorylation B-site +sites I-site +( O +Ser1148 B-residue_name_number +and O +Ser1162 B-residue_name_number +) O +confirmed O +in O +yeast B-taxonomy_domain +, O +but O +not O +occupied O +here O +. O + +This O +regulatory B-structure_element +loop I-structure_element +wedges O +between O +the O +CDC1 B-structure_element +and O +CDC2 B-structure_element +domains O +and O +provides O +the O +largest O +contribution O +to O +the O +interdomain B-site +interface I-site +. O + +The O +N O +- O +terminal O +region O +of O +the O +regulatory B-structure_element +loop I-structure_element +also O +directly O +contacts O +the O +C O +- O +terminal O +region O +of O +CDC2 B-structure_element +leading O +into O +CT B-structure_element +. O + +Phosphoserine B-residue_name_number +1157 I-residue_name_number +is O +tightly O +bound O +by O +two O +highly B-protein_state +conserved I-protein_state +arginines B-residue_name +( O +Arg1173 B-residue_name_number +and O +Arg1260 B-residue_name_number +) O +of O +CDC1 B-structure_element +( O +Fig O +. O +1d O +). O + +Already O +the O +binding O +of O +phosphorylated B-protein_state +Ser1157 B-residue_name_number +apparently O +stabilizes O +the O +regulatory B-structure_element +loop I-structure_element +conformation O +; O +the O +accessory O +phosphorylation B-site +sites I-site +Ser1148 B-residue_name_number +and O +Ser1162 B-residue_name_number +in O +the O +same B-structure_element +loop I-structure_element +may O +further O +modulate O +the O +strength O +of O +interaction O +between O +the O +regulatory B-structure_element +loop I-structure_element +and O +the O +CDC1 B-structure_element +and O +CDC2 B-structure_element +domains O +. O + +Phosphorylation B-ptm +of O +the O +regulatory B-structure_element +loop I-structure_element +thus O +determines O +interdomain O +interactions O +of O +CDC1 B-structure_element +and O +CDC2 B-structure_element +, O +suggesting O +that O +it O +may O +exert O +its O +regulatory O +function O +by O +modifying O +the O +overall O +structure O +and O +dynamics O +of O +the O +CD B-structure_element +. O + +The O +functional O +role O +of O +Ser1157 B-residue_name_number +was O +confirmed O +by O +an O +activity B-experimental_method +assay I-experimental_method +based O +on O +the O +incorporation O +of O +radioactive O +carbonate O +into O +acid O +non O +- O +volatile O +material O +. O + +Phosphorylated B-protein_state +SceACC B-protein +shows O +only O +residual O +activity O +( O +kcat B-evidence += O +0 O +. O +4 O +± O +0 O +. O +2 O +s O +− O +1 O +, O +s O +. O +d O +. O +based O +on O +five O +replicate O +measurements O +), O +which O +increases O +16 O +- O +fold O +( O +kcat B-evidence += O +6 O +. O +5 O +± O +0 O +. O +3 O +s O +− O +1 O +) O +after O +dephosphorylation O +with O +λ B-protein_type +protein I-protein_type +phosphatase I-protein_type +. O + +The O +values O +obtained O +for O +dephosphorylated B-protein_state +SceACC B-protein +are O +comparable O +to O +earlier O +measurements O +of O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +yeast B-taxonomy_domain +ACC B-protein_type +expressed B-experimental_method +in I-experimental_method +E B-species +. I-species +coli I-species +. O + +The O +variable O +CD B-structure_element +is O +conserved B-protein_state +between O +yeast B-taxonomy_domain +and O +human B-species + +To O +compare O +the O +organization O +of O +fungal B-taxonomy_domain +and O +human B-species +ACC B-protein_type +CD B-structure_element +, O +we O +determined B-experimental_method +the I-experimental_method +structure I-experimental_method +of O +a O +human B-species +ACC1 B-mutant +fragment I-mutant +that O +comprises O +the O +BT B-structure_element +and O +CD B-structure_element +domains O +( O +HsaBT B-mutant +- I-mutant +CD I-mutant +), O +but O +lacks B-protein_state +the O +mobile O +BCCP B-structure_element +in O +between O +( O +Fig O +. O +1a O +). O + +An O +experimentally B-evidence +phased I-evidence +map I-evidence +was O +obtained O +at O +3 O +. O +7 O +Å O +resolution O +for O +a O +cadmium B-chemical +- O +derivatized O +crystal O +and O +was O +interpreted O +by O +a O +poly O +- O +alanine O +model O +( O +Fig O +. O +1e O +and O +Table O +1 O +). O + +Each O +of O +the O +four O +CD B-structure_element +domains O +in O +HsaBT B-mutant +- I-mutant +CD I-mutant +individually O +resembles O +the O +corresponding O +SceCD B-species +domain O +; O +however O +, O +human B-species +and O +yeast B-taxonomy_domain +CDs B-structure_element +exhibit O +distinct O +overall O +structures B-evidence +. O + +In O +agreement O +with O +their O +tight O +interaction O +in O +SceCD B-species +, O +the O +relative O +spatial O +arrangement O +of O +CDL B-structure_element +and O +CDC1 B-structure_element +is O +preserved O +in O +HsaBT B-mutant +- I-mutant +CD I-mutant +, O +but O +the O +human B-species +CDL B-structure_element +/ O +CDC1 B-structure_element +didomain O +is O +tilted O +by O +30 O +° O +based O +on O +a O +superposition B-experimental_method +of O +human B-species +and O +yeast B-taxonomy_domain +CDC2 B-structure_element +( O +Supplementary O +Fig O +. O +1c O +). O + +As O +a O +result O +, O +the O +N O +terminus O +of O +CDL B-structure_element +at O +helix B-structure_element +Lα1 B-structure_element +, O +which O +connects O +to O +CDN B-structure_element +, O +is O +shifted O +by O +12 O +Å O +. O +Remarkably O +, O +CDN B-structure_element +of O +HsaBT B-mutant +- I-mutant +CD I-mutant +adopts O +a O +completely O +different O +orientation O +compared O +with O +SceCD B-species +. O + +With O +CDL B-structure_element +/ O +CDC1 B-structure_element +superposed B-experimental_method +, O +CDN B-structure_element +in O +HsaBT B-mutant +- I-mutant +CD I-mutant +is O +rotated O +by O +160 O +° O +around O +a O +hinge B-structure_element +at O +the O +connection O +of O +CDN B-structure_element +/ O +CDL B-structure_element +( O +Supplementary O +Fig O +. O +1d O +). O + +This O +rotation O +displaces O +the O +N O +terminus O +of O +CDN B-structure_element +in O +HsaBT B-mutant +- I-mutant +CD I-mutant +by O +51 O +Å O +compared O +with O +SceCD B-species +, O +resulting O +in O +a O +separation O +of O +the O +attachment O +points O +of O +the O +N O +- O +terminal O +linker B-structure_element +to O +the O +BCCP B-structure_element +domain I-structure_element +and O +the O +C O +- O +terminal O +CT B-structure_element +domain O +by O +67 O +Å O +( O +the O +attachment O +points O +are O +indicated O +with O +spheres O +in O +Fig O +. O +1e O +). O + +The O +BT B-structure_element +domain O +of O +HsaBT B-mutant +- I-mutant +CD I-mutant +consists O +of O +a O +helix B-structure_element +that O +is O +surrounded O +at O +its O +N O +terminus O +by O +an O +antiparallel B-structure_element +eight I-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +barrel I-structure_element +. O + +It O +resembles O +the O +BT B-structure_element +of O +propionyl B-protein_type +- I-protein_type +CoA I-protein_type +carboxylase I-protein_type +; O +only O +the O +four O +C O +- O +terminal O +strands B-structure_element +of I-structure_element +the I-structure_element +β I-structure_element +- I-structure_element +barrel I-structure_element +are O +slightly O +tilted O +. O + +On O +the O +basis O +of O +MS B-experimental_method +analysis O +of O +insect B-experimental_method +- I-experimental_method +cell I-experimental_method +- I-experimental_method +expressed I-experimental_method +human B-species +full B-protein_state +- I-protein_state +length I-protein_state +ACC B-protein_type +, O +Ser80 B-residue_name_number +shows O +the O +highest O +degree O +of O +phosphorylation B-ptm +( O +90 O +%). O + +Ser29 B-residue_name_number +and O +Ser1263 B-residue_name_number +, O +implicated O +in O +insulin B-ptm +- I-ptm +dependent I-ptm +phosphorylation I-ptm +and O +BRCA1 B-protein +binding O +, O +respectively O +, O +are O +phosphorylated B-protein_state +at O +intermediate O +levels O +( O +40 O +%). O + +The O +highly B-protein_state +conserved I-protein_state +Ser1216 B-residue_name_number +( O +corresponding O +to O +S B-species +. I-species +cerevisiae I-species +Ser1157 B-residue_name_number +), O +as O +well O +as O +Ser1201 B-residue_name_number +, O +both O +in O +the O +regulatory B-structure_element +loop I-structure_element +discussed O +above O +, O +are O +not B-protein_state +phosphorylated I-protein_state +. O + +However O +, O +residual O +phosphorylation B-ptm +levels O +were O +detected O +for O +Ser1204 B-residue_name_number +( O +7 O +%) O +and O +Ser1218 B-residue_name_number +( O +7 O +%) O +in O +the O +same B-structure_element +loop I-structure_element +. O + +MS B-experimental_method +analysis O +of O +the O +HsaBT B-mutant +- I-mutant +CD I-mutant +crystallization B-evidence +sample I-evidence +reveals O +partial O +proteolytic O +digestion O +of O +the O +regulatory B-structure_element +loop I-structure_element +. O + +Accordingly O +, O +most O +of O +this B-structure_element +loop I-structure_element +is O +not O +represented O +in O +the O +HsaBT B-mutant +- I-mutant +CD I-mutant +crystal B-evidence +structure I-evidence +. O + +The O +absence B-protein_state +of I-protein_state +the O +regulatory B-structure_element +loop I-structure_element +might O +be O +linked O +to O +the O +less B-protein_state +- I-protein_state +restrained I-protein_state +interface B-site +of O +CDL B-structure_element +/ O +CDC1 B-structure_element +and O +CDC2 B-structure_element +and O +altered O +relative O +orientations O +of O +these O +domains B-structure_element +. O + +Besides O +the O +regulatory B-structure_element +loop I-structure_element +, O +also O +the O +phosphopeptide B-site +target I-site +region I-site +for O +BRCA1 B-protein +interaction O +is O +not O +resolved O +presumably O +because O +of O +pronounced O +flexibility O +. O + +At O +the O +level O +of O +isolated B-experimental_method +yeast B-taxonomy_domain +and O +human B-species +CD B-structure_element +, O +the O +structural B-experimental_method +analysis I-experimental_method +indicates O +the O +presence O +of O +at O +least O +two O +hinges B-structure_element +, O +one O +with O +large O +- O +scale O +flexibility O +at O +the O +CDN B-structure_element +/ I-structure_element +CDL I-structure_element +connection I-structure_element +, O +and O +one O +with O +tunable O +plasticity O +between O +CDL B-structure_element +/ O +CDC1 B-structure_element +and O +CDC2 B-structure_element +, O +plausibly O +affected O +by O +phosphorylation B-ptm +in O +the O +regulatory B-structure_element +loop I-structure_element +region O +. O + +The O +integration O +of O +CD B-structure_element +into O +the O +fungal B-taxonomy_domain +ACC B-protein_type +multienzyme I-protein_type + +To O +further O +obtain O +insights O +into O +the O +functional O +architecture O +of O +fungal B-taxonomy_domain +ACC B-protein_type +, O +we O +characterized O +larger B-mutant +multidomain I-mutant +fragments I-mutant +up O +to O +the O +intact B-protein_state +enzymes B-protein +. O + +Using O +molecular B-experimental_method +replacement I-experimental_method +based O +on O +fungal B-taxonomy_domain +ACC B-protein_type +CD B-structure_element +and O +CT B-structure_element +models O +, O +we O +obtained O +structures B-evidence +of O +a O +variant B-mutant +comprising O +CthCT B-species +and O +CDC1 B-structure_element +/ O +CDC2 B-structure_element +in O +two B-evidence +crystal I-evidence +forms I-evidence +at O +resolutions O +of O +3 O +. O +6 O +and O +4 O +. O +5 O +Å O +( O +CthCD B-mutant +- I-mutant +CTCter1 I-mutant +/ I-mutant +2 I-mutant +), O +respectively O +, O +as O +well O +as O +of O +a O +CthCT B-species +linked O +to O +the O +entire O +CD B-structure_element +at O +7 O +. O +2 O +Å O +resolution O +( O +CthCD B-mutant +- I-mutant +CT I-mutant +; O +Figs O +1a O +and O +2 O +, O +Table O +1 O +). O + +No O +crystals O +diffracting O +to O +sufficient O +resolution O +were O +obtained O +for O +larger B-mutant +BC I-mutant +- I-mutant +containing I-mutant +fragments I-mutant +, O +or O +for O +full B-protein_state +- I-protein_state +length I-protein_state +Cth B-species +or O +SceACC B-protein +. O + +To O +improve B-experimental_method +crystallizability I-experimental_method +, O +we O +generated B-experimental_method +ΔBCCP B-mutant +variants I-mutant +of O +full B-protein_state +- I-protein_state +length I-protein_state +ACC B-protein_type +, O +which O +, O +based O +on O +SAXS B-experimental_method +analysis I-experimental_method +, O +preserve O +properties O +of O +intact B-protein_state +ACC B-protein_type +( O +Supplementary O +Table O +1 O +and O +Supplementary O +Fig O +. O +2a O +– O +c O +). O + +For O +CthΔBCCP B-mutant +, O +crystals B-evidence +diffracting O +to O +8 O +. O +4 O +Å O +resolution O +were O +obtained O +. O + +However O +, O +molecular B-experimental_method +replacement I-experimental_method +did O +not O +reveal O +a O +unique O +positioning O +of O +the O +BC B-structure_element +domain O +. O + +Owing O +to O +the O +limited O +resolution O +the O +discussion O +of O +structures B-evidence +of O +CthCD B-mutant +- I-mutant +CT I-mutant +and O +CthΔBCCP B-mutant +is O +restricted O +to O +the O +analysis O +of O +domain O +localization O +. O + +Still O +, O +these B-evidence +structures I-evidence +contribute O +considerably O +to O +the O +visualization O +of O +an O +intrinsically O +dynamic B-protein_state +fungal B-taxonomy_domain +ACC B-protein_type +. O + +In O +all O +these O +crystal B-evidence +structures I-evidence +, O +the O +CT B-structure_element +domains O +build O +a O +canonical O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +dimer B-oligomeric_state +, O +with O +active B-site +sites I-site +formed O +by O +contributions O +from O +both O +protomers B-oligomeric_state +( O +Fig O +. O +2 O +and O +Supplementary O +Fig O +. O +3a O +). O + +The O +connection B-structure_element +of O +CD B-structure_element +and O +CT B-structure_element +is O +provided O +by O +a O +10 B-residue_range +- I-residue_range +residue I-residue_range +peptide I-residue_range +stretch I-residue_range +, O +which O +links O +the O +N O +terminus O +of O +CT B-structure_element +to O +the O +irregular B-structure_element +β I-structure_element +- I-structure_element +hairpin I-structure_element +/ I-structure_element +β I-structure_element +- I-structure_element +strand I-structure_element +extension I-structure_element +of O +CDC2 B-structure_element +( O +Supplementary O +Fig O +. O +3b O +). O + +The O +connecting B-structure_element +region I-structure_element +is O +remarkably O +similar O +in O +isolated B-protein_state +CD B-structure_element +and O +CthCD B-mutant +- I-mutant +CTCter I-mutant +structures B-evidence +, O +indicating O +inherent O +conformational O +stability O +. O + +CD B-structure_element +/ O +CT B-structure_element +contacts O +are O +only O +formed O +in O +direct O +vicinity O +of O +the O +covalent O +linkage O +and O +involve O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +extension I-structure_element +of O +CDC2 B-structure_element +as O +well O +as O +the O +loop B-structure_element +between O +strands B-structure_element +β2 I-structure_element +/ I-structure_element +β3 I-structure_element +of O +the O +CT B-structure_element +N I-structure_element +- I-structure_element +lobe I-structure_element +, O +which O +contains O +a O +conserved B-protein_state +RxxGxN B-structure_element +motif I-structure_element +. O + +The O +neighbouring O +loop B-structure_element +on O +the O +CT B-structure_element +side O +( O +between O +CT B-structure_element +β1 B-structure_element +/ O +β2 B-structure_element +) O +is O +displaced O +by O +2 O +. O +5 O +Å O +compared O +to O +isolated B-protein_state +CT B-structure_element +structures B-evidence +( O +Supplementary O +Fig O +. O +3c O +). O + +On O +the O +basis O +of O +an O +interface O +area O +of O +∼ O +600 O +Å2 O +and O +its O +edge O +- O +to O +- O +edge O +connection O +characteristics O +, O +the O +interface B-site +between O +CT B-structure_element +and O +CD B-structure_element +might O +be O +classified O +as O +conformationally O +variable O +. O + +Indeed O +, O +the O +comparison O +of O +the O +positioning O +of O +eight O +instances O +of O +the O +C O +- O +terminal O +part O +of O +CD B-structure_element +relative O +to O +CT B-structure_element +in O +crystal B-evidence +structures I-evidence +determined B-experimental_method +here O +, O +reveals O +flexible O +interdomain O +linking O +( O +Fig O +. O +3a O +). O + +The O +CDC2 B-site +/ I-site +CT I-site +interface I-site +acts O +as O +a O +true B-structure_element +hinge I-structure_element +with O +observed O +rotation O +up O +to O +16 O +°, O +which O +results O +in O +a O +translocation O +of O +the O +distal O +end O +of O +CDC2 B-structure_element +by O +8 O +Å O +. O + +The O +interface B-site +between O +CDC2 B-structure_element +and O +CDL B-structure_element +/ O +CDC1 B-structure_element +, O +which O +is O +mediated O +by O +the O +phosphorylated B-protein_state +regulatory B-structure_element +loop I-structure_element +in O +the O +SceCD B-species +structure B-evidence +, O +is O +less O +variable O +than O +the O +CD B-structure_element +– I-structure_element +CT I-structure_element +junction I-structure_element +, O +and O +permits O +only O +limited O +rotation O +and O +tilting O +( O +Fig O +. O +3b O +). O + +Analysis O +of O +the O +impact O +of O +phosphorylation B-ptm +on O +the O +interface B-site +between O +CDC2 B-structure_element +and O +CDL B-structure_element +/ O +CDC1 B-structure_element +in O +CthACC B-mutant +variant I-mutant +structures B-evidence +is O +precluded O +by O +the O +limited O +crystallographic O +resolution O +. O + +However O +, O +MS B-experimental_method +analysis O +of O +CthCD B-mutant +- I-mutant +CT I-mutant +and O +CthΔBCCP B-mutant +constructs O +revealed O +between O +60 O +and O +70 O +% O +phosphorylation B-ptm +of O +Ser1170 B-residue_name_number +( O +corresponding O +to O +SceACC B-protein +Ser1157 B-residue_name_number +). O + +The O +CDN B-structure_element +domain O +positioning O +relative O +to O +CDL B-structure_element +/ O +CDC1 B-structure_element +is O +highly O +variable O +with O +three O +main O +orientations O +observed O +in O +the O +structures B-evidence +of O +SceCD B-species +and O +the O +larger B-mutant +CthACC I-mutant +fragments I-mutant +: O +CDN B-structure_element +tilts O +, O +resulting O +in O +a O +displacement O +of O +its O +N O +terminus O +by O +23 O +Å O +( O +Fig O +. O +4a O +, O +observed O +in O +both O +protomers B-oligomeric_state +of O +CthCD B-mutant +- I-mutant +CT I-mutant +and O +one O +protomer B-oligomeric_state +of O +CthΔBCCP B-mutant +, O +denoted O +as O +CthCD B-mutant +- I-mutant +CT1 I-mutant +/ I-mutant +2 I-mutant +and O +CthΔBCCP1 B-mutant +, O +respectively O +). O + +In O +addition O +, O +CDN B-structure_element +can O +rotate O +around O +hinges B-structure_element +in O +the O +connection O +between O +CDN B-structure_element +/ O +CDL B-structure_element +by O +70 O +° O +( O +Fig O +. O +4b O +, O +observed O +in O +the O +second O +protomer B-oligomeric_state +of O +CthΔBCCP B-mutant +, O +denoted O +as O +CthΔBCCP2 B-mutant +) O +and O +160 O +° O +( O +Fig O +. O +4c O +, O +observed O +in O +SceCD B-species +) O +leading O +to O +displacement O +of O +the O +anchor B-site +site I-site +for O +the O +BCCP B-structure_element +linker I-structure_element +by O +up O +to O +33 O +and O +40 O +Å O +, O +respectively O +. O + +Conformational O +variability O +in O +the O +CD B-structure_element +thus O +contributes O +considerably O +to O +variations O +in O +the O +spacing O +between O +the O +BC B-structure_element +and O +CT B-structure_element +domains O +, O +and O +may O +extend O +to O +distance O +variations O +beyond O +the O +mobility O +range O +of O +the O +flexibly B-protein_state +tethered I-protein_state +BCCP B-structure_element +. O + +On O +the O +basis O +of O +the O +occurrence O +of O +related O +conformational O +changes O +between O +fungal B-taxonomy_domain +and O +human B-species +ACC B-mutant +fragments I-mutant +, O +the O +observed O +set O +of O +conformations O +may O +well O +represent O +general O +states O +present O +in O +all O +eukaryotic B-taxonomy_domain +ACCs B-protein_type +. O + +Large O +- O +scale O +conformational O +variability O +of O +fungal B-taxonomy_domain +ACC B-protein_type + +To O +obtain O +a O +comprehensive O +view O +of O +fungal B-taxonomy_domain +ACC B-protein_type +dynamics O +in B-protein_state +solution I-protein_state +, O +we O +employed O +SAXS B-experimental_method +and O +EM B-experimental_method +. O + +SAXS B-experimental_method +analysis O +of O +CthACC B-protein +agrees O +with O +a O +dimeric B-oligomeric_state +state O +and O +an O +elongated B-protein_state +shape I-protein_state +with O +a O +maximum O +extent O +of O +350 O +Å O +( O +Supplementary O +Table O +1 O +). O + +The O +smooth O +appearance O +of O +scattering B-evidence +curves I-evidence +and O +derived B-evidence +distance I-evidence +distributions I-evidence +might O +indicate O +substantial O +interdomain O +flexibility O +( O +Supplementary O +Fig O +. O +2a O +– O +c O +). O + +Direct O +observation O +of O +individual O +full B-protein_state +- I-protein_state +length I-protein_state +CthACC B-protein +particles B-evidence +, O +according O +to O +MS B-experimental_method +results O +predominantly O +in O +a O +phosphorylated B-protein_state +low B-protein_state +- I-protein_state +activity I-protein_state +state I-protein_state +, O +in O +negative B-experimental_method +stain I-experimental_method +EM I-experimental_method +reveals O +a O +large O +set O +of O +conformations O +from O +rod B-protein_state +- I-protein_state +like I-protein_state +extended I-protein_state +to O +U B-protein_state +- I-protein_state +shaped I-protein_state +particles B-evidence +. O + +Class B-evidence +averages I-evidence +, O +obtained O +by O +maximum B-experimental_method +- I-experimental_method +likelihood I-experimental_method +- I-experimental_method +based I-experimental_method +two I-experimental_method +- I-experimental_method +dimensional I-experimental_method +( I-experimental_method +2D I-experimental_method +) I-experimental_method +classification I-experimental_method +, O +are O +focused O +on O +the O +dimeric B-oligomeric_state +CT B-structure_element +domain O +and O +the O +full B-protein_state +BC B-mutant +– I-mutant +BCCP I-mutant +– I-mutant +CD I-mutant +domain O +of O +only O +one O +protomer B-oligomeric_state +, O +due O +to O +the O +non O +- O +coordinated O +motions O +of O +the O +lateral O +BC B-structure_element +/ O +CD B-structure_element +regions O +relative O +to O +the O +CT B-structure_element +dimer B-oligomeric_state +. O + +They O +identify O +the O +connections O +between O +CDN B-structure_element +/ O +CDL B-structure_element +and O +between O +CDC2 B-structure_element +/ O +CT B-structure_element +as O +major O +contributors O +to O +conformational O +heterogeneity O +( O +Supplementary O +Fig O +. O +4a O +, O +b O +). O + +The O +flexibility O +in O +the O +CDC2 B-structure_element +/ I-structure_element +CT I-structure_element +hinge I-structure_element +appears O +substantially O +larger O +than O +the O +variations O +observed O +in O +the O +set O +of O +crystal B-evidence +structures I-evidence +. O + +The O +BC B-structure_element +domain O +is O +not O +completely O +disordered O +, O +but O +laterally O +attached O +to O +BT B-structure_element +/ O +CDN B-structure_element +in O +a O +generally B-protein_state +conserved I-protein_state +position I-protein_state +, O +albeit O +with O +increased O +flexibility O +. O + +Surprisingly O +, O +in O +both O +the O +linear B-protein_state +and I-protein_state +U I-protein_state +- I-protein_state +shaped I-protein_state +conformations I-protein_state +, O +the O +approximate O +distances O +between O +the O +BC B-structure_element +and O +CT B-structure_element +active B-site +sites I-site +would O +remain O +larger O +than O +110 O +Å O +. O +These O +observed O +distances O +are O +considerably O +larger O +than O +in O +static B-protein_state +structures B-evidence +of O +any O +other O +related O +biotin B-protein_type +- I-protein_type +dependent I-protein_type +carboxylase I-protein_type +. O + +Furthermore O +, O +based O +on O +an O +average O +length O +of O +the O +BCCP B-structure_element +– I-structure_element +CD I-structure_element +linker I-structure_element +in O +fungal B-taxonomy_domain +ACC B-protein_type +of O +26 B-residue_range +amino I-residue_range +acids I-residue_range +, O +mobility O +of O +the O +BCCP B-structure_element +alone O +would O +not O +be O +sufficient O +to O +bridge O +the O +active B-site +sites I-site +of O +BC B-structure_element +and O +CT B-structure_element +. O + +The O +most O +relevant O +candidate O +site O +for O +mediating O +such O +additional O +flexibility O +and O +permitting O +an O +extended O +set O +of O +conformations O +is O +the O +CDC1 B-site +/ I-site +CDC2 I-site +interface I-site +, O +which O +is O +rigidified O +by O +the O +Ser1157 B-residue_name_number +- O +phosphorylated B-protein_state +regulatory B-structure_element +loop I-structure_element +, O +as O +depicted O +in O +the O +SceCD B-species +crystal B-evidence +structure I-evidence +. O + +Altogether O +, O +the O +architecture O +of O +fungal B-taxonomy_domain +ACC B-protein_type +is O +based O +on O +the O +central O +dimeric B-oligomeric_state +CT B-structure_element +domain O +( O +Fig O +. O +4d O +). O + +The O +CD B-structure_element +consists O +of O +four O +distinct O +subdomains B-structure_element +and O +acts O +as O +a O +tether O +from O +the O +CT B-structure_element +to O +the O +mobile B-protein_state +BCCP B-structure_element +and O +an O +oriented B-protein_state +BC B-structure_element +domain O +. O + +The O +CD B-structure_element +has O +no O +direct O +role O +in O +substrate O +recognition O +or O +catalysis O +but O +contributes O +to O +the O +regulation O +of O +all O +eukaryotic B-taxonomy_domain +ACCs B-protein_type +. O + +In O +higher B-taxonomy_domain +eukaryotic I-taxonomy_domain +ACCs B-protein_type +, O +regulation O +via O +phosphorylation B-ptm +is O +achieved O +by O +combining O +the O +effects O +of O +phosphorylation B-ptm +at O +Ser80 B-residue_name_number +, O +Ser1201 B-residue_name_number +and O +Ser1263 B-residue_name_number +. O + +In O +fungal B-taxonomy_domain +ACC B-protein_type +, O +however O +, O +Ser1157 B-residue_name_number +in O +the O +regulatory B-structure_element +loop I-structure_element +of O +the O +CD B-structure_element +is O +the O +only O +phosphorylation B-site +site I-site +that O +has O +been O +demonstrated O +to O +be O +both O +phosphorylated B-protein_state +in O +vivo O +and O +involved O +in O +the O +regulation O +of O +ACC B-protein_type +activity O +. O + +In O +its O +phosphorylated B-protein_state +state O +, O +the O +regulatory B-structure_element +loop I-structure_element +containing O +Ser1157 B-residue_name_number +wedges O +between O +CDC1 B-structure_element +/ O +CDC2 B-structure_element +and O +presumably O +limits O +the O +conformational B-protein_state +freedom I-protein_state +at O +this O +interdomain B-site +interface I-site +. O + +However O +, O +flexibility O +at O +this O +hinge B-structure_element +may O +be O +required O +for O +full B-protein_state +ACC I-protein_state +activity I-protein_state +, O +as O +the O +distances O +between O +the O +BCCP B-structure_element +anchor I-structure_element +points I-structure_element +and O +the O +active B-site +sites I-site +of O +BC B-structure_element +and O +CT B-structure_element +observed O +here O +are O +such O +large O +that O +mobility O +of O +the O +BCCP B-structure_element +alone O +is O +not O +sufficient O +for O +substrate O +transfer O +. O + +The O +current O +data O +thus O +suggest O +that O +regulation O +of O +fungal B-taxonomy_domain +ACC B-protein_type +is O +mediated O +by O +controlling O +the O +dynamics O +of O +the O +unique B-protein_state +CD B-structure_element +, O +rather O +than O +directly O +affecting O +catalytic O +turnover O +at O +the O +active B-site +sites I-site +of O +BC B-structure_element +and O +CT B-structure_element +. O + +A O +comparison O +between O +fungal B-taxonomy_domain +and O +human B-species +ACC B-protein_type +will O +help O +to O +further O +discriminate O +mechanistic O +differences O +that O +contribute O +to O +the O +extended O +control O +and O +polymerization O +of O +human B-species +ACC B-protein_type +. O + +Most O +recently O +, O +a O +crystal B-evidence +structure I-evidence +of O +near B-protein_state +full I-protein_state +- I-protein_state +length I-protein_state +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +ACC B-protein_type +from O +S B-species +. I-species +cerevisae I-species +( O +lacking B-protein_state +only I-protein_state +21 B-residue_range +N O +- O +terminal O +amino O +acids O +, O +here O +denoted O +as O +flACC B-protein +) O +was O +published O +by O +Wei O +and O +Tong O +. O + +In O +flACC B-protein +, O +the O +ACC B-protein_type +dimer B-oligomeric_state +obeys O +twofold O +symmetry O +and O +assembles O +in O +a O +triangular B-protein_state +architecture I-protein_state +with O +dimeric B-oligomeric_state +BC B-structure_element +domains O +( O +Supplementary O +Fig O +. O +5a O +). O + +In O +their O +study O +, O +mutational B-experimental_method +data I-experimental_method +indicate O +a O +requirement O +for O +BC O +dimerization O +for O +catalytic O +activity O +. O + +The O +transition O +from O +the O +elongated B-protein_state +open I-protein_state +shape I-protein_state +, O +observed O +in O +our O +experiments O +, O +towards O +a O +compact B-protein_state +triangular I-protein_state +shape I-protein_state +is O +based O +on O +an O +intricate O +interplay O +of O +several O +hinge O +- O +bending O +motions O +in O +the O +CD B-structure_element +( O +Fig O +. O +4d O +). O + +Comparison B-experimental_method +of O +flACC B-protein +with O +our O +CthΔBCCP B-mutant +structure B-evidence +reveals O +the O +CDC2 B-structure_element +/ I-structure_element +CT I-structure_element +hinge I-structure_element +as O +a O +major O +contributor O +to O +conformational O +flexibility O +( O +Supplementary O +Fig O +. O +5b O +, O +c O +). O + +In O +flACC B-protein +, O +CDC2 B-structure_element +rotates O +∼ O +120 O +° O +with O +respect O +to O +the O +CT B-structure_element +domain O +. O + +A O +second B-structure_element +hinge I-structure_element +can O +be O +identified O +between O +CDC1 B-structure_element +/ O +CDC2 B-structure_element +. O + +On O +the O +basis O +of O +a O +superposition B-experimental_method +of O +CDC2 B-structure_element +, O +CDC1 B-structure_element +of O +the O +phosphorylated B-protein_state +SceCD B-species +is O +rotated O +by O +30 O +° O +relative O +to O +CDC1 B-structure_element +of O +the O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +flACC B-protein +( O +Supplementary O +Fig O +. O +5d O +), O +similar O +to O +what O +we O +have O +observed O +for O +the O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +HsaBT B-mutant +- I-mutant +CD I-mutant +( O +Supplementary O +Fig O +. O +1d O +). O + +When O +inspecting B-experimental_method +all O +individual O +protomer B-oligomeric_state +and O +fragment B-mutant +structures B-evidence +in O +their O +study O +, O +Wei O +and O +Tong O +also O +identify O +the O +CDN B-structure_element +/ I-structure_element +CDC1 I-structure_element +connection I-structure_element +as O +a O +highly B-protein_state +flexible I-protein_state +hinge B-structure_element +, O +in O +agreement O +with O +our O +observations O +. O + +The O +only O +bona O +fide O +regulatory B-protein_state +phophorylation B-site +site I-site +of O +fungal B-taxonomy_domain +ACC B-protein_type +in O +the O +regulatory B-structure_element +loop I-structure_element +is O +directly O +participating O +in O +CDC1 B-structure_element +/ O +CDC2 B-structure_element +domain O +interactions O +and O +thus O +stabilizes O +the O +hinge B-structure_element +conformation I-structure_element +. O + +In O +flACC B-protein +, O +the O +regulatory B-structure_element +loop I-structure_element +is O +mostly B-protein_state +disordered I-protein_state +, O +illustrating O +the O +increased O +flexibility O +due O +to O +the O +absence O +of O +the O +phosphoryl B-chemical +group O +. O + +Only O +in O +three O +out O +of O +eight O +observed O +protomers B-oligomeric_state +a O +short B-structure_element +peptide I-structure_element +stretch O +( O +including O +Ser1157 B-residue_name_number +) O +was O +modelled B-evidence +. O + +In O +those O +instances O +the O +Ser1157 B-residue_name_number +residue O +is O +located O +at O +a O +distance O +of O +14 O +– O +20 O +Å O +away O +from O +the O +location O +of O +the O +phosphorylated B-protein_state +serine B-residue_name +observed O +here O +, O +based O +on O +superposition B-experimental_method +of O +either O +CDC1 B-structure_element +or O +CDC2 B-structure_element +. O + +Applying B-experimental_method +the O +conformation O +of O +the O +CDC1 B-structure_element +/ I-structure_element +CDC2 I-structure_element +hinge I-structure_element +observed O +in O +SceCD B-species +on O +flACC B-protein +leads O +to O +CDN B-structure_element +sterically O +clashing O +with O +CDC2 B-structure_element +and O +BT B-structure_element +/ O +CDN B-structure_element +clashing O +with O +CT B-structure_element +( O +Supplementary O +Fig O +. O +6a O +, O +b O +). O + +Thus O +, O +in O +accordance O +with O +the O +results O +presented O +here O +, O +phosphorylation B-ptm +of O +Ser1157 B-residue_name_number +in O +SceACC B-protein +most O +likely O +limits O +flexibility O +in O +the O +CDC1 B-structure_element +/ I-structure_element +CDC2 I-structure_element +hinge I-structure_element +such O +that O +activation O +through O +BC B-structure_element +dimerization O +is O +not O +possible O +( O +Fig O +. O +4d O +), O +which O +however O +does O +not O +exclude O +intermolecular O +dimerization O +. O + +In O +addition O +, O +EM B-experimental_method +micrographs B-evidence +of O +phosphorylated B-protein_state +and O +dephosphorylated B-protein_state +SceACC B-protein +display O +for O +both O +samples O +mainly O +elongated B-protein_state +and I-protein_state +U I-protein_state +- I-protein_state +shaped I-protein_state +conformations I-protein_state +and O +reveal O +no O +apparent O +differences O +in O +particle B-evidence +shape I-evidence +distributions I-evidence +( O +Supplementary O +Fig O +. O +7 O +). O + +This O +implicates O +that O +the O +triangular B-protein_state +shape I-protein_state +with O +dimeric B-oligomeric_state +BC B-structure_element +domains O +has O +a O +low O +population O +also O +in O +the O +active B-protein_state +form I-protein_state +, O +even O +though O +a O +biasing O +influence O +of O +grid O +preparation O +cannot O +be O +excluded O +completely O +. O + +Large O +- O +scale O +conformational O +variability O +has O +also O +been O +observed O +in O +most O +other O +carrier B-protein_type +protein I-protein_type +- I-protein_type +based I-protein_type +multienzymes I-protein_type +, O +including O +polyketide B-protein_type +and I-protein_type +fatty I-protein_type +- I-protein_type +acid I-protein_type +synthases I-protein_type +( O +with O +the O +exception O +of O +fungal B-protein_type +- I-protein_type +type I-protein_type +fatty I-protein_type +- I-protein_type +acid I-protein_type +synthases I-protein_type +), O +non B-protein_type +- I-protein_type +ribosomal I-protein_type +peptide I-protein_type +synthetases I-protein_type +and O +the O +pyruvate B-protein_type +dehydrogenase I-protein_type +complexes I-protein_type +, O +although O +based O +on O +completely O +different O +architectures O +. O + +Together O +, O +this O +structural B-evidence +information I-evidence +suggests O +that O +variable O +carrier O +protein O +tethering O +is O +not O +sufficient O +for O +efficient O +substrate O +transfer O +and O +catalysis O +in O +any O +of O +these O +systems O +. O + +The O +determination B-experimental_method +of I-experimental_method +a I-experimental_method +set I-experimental_method +of I-experimental_method +crystal B-evidence +structures I-evidence +of O +SceACC B-protein +in O +two O +states O +, O +unphosphorylated B-protein_state +and O +phosphorylated B-protein_state +at O +the O +major B-site +regulatory I-site +site I-site +Ser1157 B-residue_name_number +, O +provides O +a O +unique O +depiction O +of O +multienzyme O +regulation O +by O +post O +- O +translational O +modification O +( O +Fig O +. O +4d O +). O + +The O +phosphorylated B-protein_state +regulatory B-structure_element +loop I-structure_element +binds O +to O +an O +allosteric B-site +site I-site +at O +the O +interface B-site +of O +two O +non B-protein_state +- I-protein_state +catalytic I-protein_state +domains O +and O +restricts O +conformational O +freedom O +at O +several O +hinges B-structure_element +in O +the O +dynamic B-protein_state +ACC B-protein_type +. O + +It O +disfavours O +the O +adoption O +of O +a O +rare B-protein_state +, I-protein_state +compact I-protein_state +conformation I-protein_state +, O +in O +which O +intramolecular O +dimerization O +of O +the O +BC B-structure_element +domains O +results O +in O +catalytic O +turnover O +. O + +The O +regulation O +of O +activity O +thus O +results O +from O +restrained O +large O +- O +scale O +conformational O +dynamics O +rather O +than O +a O +direct O +or O +indirect O +influence O +on O +active B-site +site I-site +structure I-site +. O + +To O +our O +best O +knowledge O +, O +ACC B-protein_type +is O +the O +first O +multienzyme B-protein_type +for O +which O +such O +a O +phosphorylation B-ptm +- O +dependent O +mechanical O +control O +mechanism O +has O +been O +visualized O +. O + +However O +, O +the O +example O +of O +ACC B-protein_type +now O +demonstrates O +the O +possibility O +of O +regulating O +activity O +by O +controlled O +dynamics O +of O +non B-structure_element +- I-structure_element +enzymatic I-structure_element +linker I-structure_element +regions I-structure_element +also O +in O +other O +families O +of O +carrier B-protein_type +- I-protein_type +dependent I-protein_type +multienzymes I-protein_type +. O + +The O +phosphorylated B-protein_state +central B-structure_element +domain I-structure_element +of O +yeast B-taxonomy_domain +ACC B-protein_type +. O + +( O +a O +) O +Schematic O +overview O +of O +the O +domain O +organization O +of O +eukaryotic B-taxonomy_domain +ACCs B-protein_type +. O + +Crystallized B-evidence +constructs I-evidence +are O +indicated O +. O + +( O +b O +) O +Cartoon O +representation O +of O +the O +SceCD B-species +crystal B-evidence +structure I-evidence +. O + +CDN B-structure_element +is O +linked O +by O +a O +four B-structure_element +- I-structure_element +helix I-structure_element +bundle I-structure_element +( O +CDL B-structure_element +) O +to O +two B-structure_element +α I-structure_element +– I-structure_element +β I-structure_element +- I-structure_element +fold I-structure_element +domains I-structure_element +( O +CDC1 B-structure_element +and O +CDC2 B-structure_element +). O + +The O +regulatory B-structure_element +loop I-structure_element +is O +shown O +as O +bold O +cartoon O +, O +and O +the O +phosphorylated B-protein_state +Ser1157 B-residue_name_number +is O +marked O +by O +a O +red O +triangle O +. O + +( O +c O +) O +Superposition B-experimental_method +of O +CDC1 B-structure_element +and O +CDC2 B-structure_element +reveals O +highly B-protein_state +conserved I-protein_state +folds B-structure_element +. O +( O +d O +) O +The O +regulatory B-structure_element +loop I-structure_element +with O +the O +phosphorylated B-protein_state +Ser1157 B-residue_name_number +is O +bound O +into O +a O +crevice O +between O +CDC1 B-structure_element +and O +CDC2 B-structure_element +, O +the O +conserved B-protein_state +residues O +Arg1173 B-residue_name_number +and O +Arg1260 B-residue_name_number +coordinate O +the O +phosphoryl B-chemical +- O +group O +. O + +( O +e O +) O +Structural O +overview O +of O +HsaBT B-mutant +- I-mutant +CD I-mutant +. O + +The O +attachment O +points O +to O +the O +N O +- O +terminal O +BCCP B-structure_element +domain O +and O +the O +C O +- O +terminal O +CT B-structure_element +domain O +are O +indicated O +with O +spheres O +. O + +Architecture O +of O +the O +CD B-structure_element +– O +CT B-structure_element +core O +of O +fungal B-taxonomy_domain +ACC B-protein_type +. O + +Cartoon O +representation O +of O +crystal B-evidence +structures I-evidence +of O +multidomain B-mutant +constructs I-mutant +of O +CthACC B-protein +. O + +One O +protomer B-oligomeric_state +is O +shown O +in O +colour O +and O +one O +in O +grey O +. O + +Individual O +domains O +are O +labelled O +; O +the O +active B-site +site I-site +of O +CT B-structure_element +and O +the O +position O +of O +the O +conserved B-protein_state +regulatory B-protein_state +phosphoserine B-site +site I-site +based O +on O +SceCD B-species +are O +indicated O +by O +an O +asterisk O +and O +a O +triangle O +, O +respectively O +. O + +Variability O +of O +the O +connections O +of O +CDC2 B-structure_element +to O +CT B-structure_element +and O +CDC1 B-structure_element +in O +fungal B-taxonomy_domain +ACC B-protein_type +. O + +( O +a O +) O +Hinge B-structure_element +properties O +of O +the O +CDC2 B-structure_element +– I-structure_element +CT I-structure_element +connection I-structure_element +analysed O +by O +a O +CT B-experimental_method +- I-experimental_method +based I-experimental_method +superposition I-experimental_method +of O +eight O +instances O +of O +the O +CDC2 B-mutant +- I-mutant +CT I-mutant +segment I-mutant +. O + +For O +clarity O +, O +only O +one O +protomer B-oligomeric_state +of O +CthCD B-mutant +- I-mutant +CTCter1 I-mutant +is O +shown O +in O +full O +colour O +as O +reference O +. O + +For O +other O +instances O +, O +CDC2 B-structure_element +domains O +are O +shown O +in O +transparent O +tube O +representation O +with O +only O +one O +helix O +each O +highlighted O +. O + +The O +range O +of O +hinge O +bending O +is O +indicated O +and O +the O +connection O +points O +between O +CDC2 B-structure_element +and O +CT B-structure_element +( O +blue O +) O +as O +well O +as O +between O +CDC1 B-structure_element +and O +CDC2 B-structure_element +( O +green O +and O +grey O +) O +are O +marked O +as O +spheres O +. O + +( O +b O +) O +The O +interdomain B-site +interface I-site +of O +CDC1 B-structure_element +and O +CDC2 B-structure_element +exhibits O +only O +limited O +plasticity O +. O + +Representation O +as O +in O +a O +, O +but O +the O +CDC1 B-structure_element +and O +CDC2 B-structure_element +are O +superposed B-experimental_method +based O +on O +CDC2 B-structure_element +. O + +One O +protomer B-oligomeric_state +of O +CthΔBCCP B-mutant +is O +shown O +in O +colour O +, O +the O +CDL B-structure_element +domains O +are O +omitted O +for O +clarity O +and O +the O +position O +of O +the O +phosphorylated B-protein_state +serine B-residue_name +based O +on O +SceCD B-species +is O +indicated O +with O +a O +red O +triangle O +. O + +The O +connection O +points O +from O +CDC1 B-structure_element +to O +CDC2 B-structure_element +and O +to O +CDL B-structure_element +are O +represented O +by O +green O +spheres O +. O + +The O +conformational O +dynamics O +of O +fungal B-taxonomy_domain +ACC B-protein_type +. O + +( O +a O +– O +c O +) O +Large O +- O +scale O +conformational O +variability O +of O +the O +CDN B-structure_element +domain O +relative O +to O +the O +CDL B-structure_element +/ O +CDC1 B-structure_element +domain O +. O + +CthCD B-mutant +- I-mutant +CT1 I-mutant +( O +in O +colour O +) O +serves O +as O +reference O +, O +the O +compared B-experimental_method +structures I-experimental_method +( O +as O +indicated O +, O +numbers O +after O +construct O +name O +differentiate O +between O +individual O +protomers B-oligomeric_state +) O +are O +shown O +in O +grey O +. O + +Domains O +other O +than O +CDN B-structure_element +and O +CDL B-structure_element +/ O +CDC1 B-structure_element +are O +omitted O +for O +clarity O +. O + +The O +domains O +are O +labelled O +and O +the O +distances O +between O +the O +N O +termini O +of O +CDN B-structure_element +( O +spheres O +) O +in O +the O +compared O +structures O +are O +indicated O +. O + +( O +d O +) O +Schematic O +model O +of O +fungal B-taxonomy_domain +ACC B-protein_type +showing O +the O +intrinsic O +, O +regulated O +flexibility O +of O +CD B-structure_element +in O +the O +phosphorylated B-protein_state +inhibited B-protein_state +or O +the O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +activated B-protein_state +state O +. O + +Flexibility O +of O +the O +CDC2 B-structure_element +/ O +CT B-structure_element +and O +CDN B-structure_element +/ O +CDL B-structure_element +hinges B-structure_element +is O +illustrated O +by O +arrows O +. O + +The O +Ser1157 B-residue_name_number +phosphorylation B-ptm +site O +and O +the O +regulatory B-structure_element +loop I-structure_element +are O +schematically O +indicated O +in O +magenta O +. O + +Crystal B-evidence +structure I-evidence +of O +SEL1L B-protein +: O +Insight O +into O +the O +roles O +of O +SLR B-structure_element +motifs O +in O +ERAD O +pathway O + +SEL1L B-protein +, O +a O +component O +of O +the O +ERAD O +machinery O +, O +plays O +an O +important O +role O +in O +selecting O +and O +transporting O +ERAD O +substrates O +for O +degradation O +. O + +We O +have O +determined O +the O +crystal B-evidence +structure I-evidence +of O +the O +mouse B-taxonomy_domain +SEL1L B-protein +central B-structure_element +domain I-structure_element +comprising O +five O +Sel1 B-structure_element +- I-structure_element +Like I-structure_element +Repeats I-structure_element +( O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +to I-structure_element +9 I-structure_element +; O +hereafter O +called O +SEL1Lcent B-structure_element +). O + +Strikingly O +, O +SEL1Lcent B-structure_element +forms O +a O +homodimer B-oligomeric_state +with O +two O +- O +fold O +symmetry O +in O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +manner O +. O + +Particularly O +, O +the O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +plays O +an O +important O +role O +in O +dimer B-oligomeric_state +formation O +by O +adopting O +a O +domain B-protein_state +- I-protein_state +swapped I-protein_state +structure O +and O +providing O +an O +extensive O +dimeric B-site +interface I-site +. O + +We O +identified O +that O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +forms O +a O +self B-oligomeric_state +- I-oligomeric_state +oligomer I-oligomeric_state +through O +the O +SEL1Lcent B-structure_element +domain O +in O +mammalian B-taxonomy_domain +cells O +. O + +Furthermore O +, O +we O +discovered O +that O +the O +SLR B-structure_element +- I-structure_element +C I-structure_element +, O +comprising O +SLR B-structure_element +motifs I-structure_element +10 I-structure_element +and I-structure_element +11 I-structure_element +, O +of O +SEL1L B-protein +directly O +interacts O +with O +the O +N O +- O +terminus O +luminal B-structure_element +loops I-structure_element +of O +HRD1 B-protein +. O + +Therefore O +, O +we O +propose O +that O +certain O +SLR B-structure_element +motifs O +of O +SEL1L B-protein +play O +a O +unique O +role O +in O +membrane O +bound O +ERAD O +machinery O +. O + +Protein O +quality O +control O +in O +the O +endoplasmic O +reticulum O +( O +ER O +) O +is O +essential O +for O +maintenance O +of O +cellular O +homeostasis O +in O +eukaryotes B-taxonomy_domain +and O +is O +implicated O +in O +many O +severe O +diseases O +. O + +Terminally O +misfolded O +proteins O +in O +the O +lumen O +or O +membrane O +of O +the O +ER O +are O +retrotranslocated O +into O +the O +cytosol O +, O +polyubiquitinated B-protein_state +, O +and O +degraded O +by O +the O +proteasome B-complex_assembly +. O + +The O +process O +is O +called O +ER O +- O +associated O +protein O +degradation O +( O +ERAD O +) O +and O +is O +conserved B-protein_state +in O +all O +eukaryotes B-taxonomy_domain +. O + +Accumulating O +studies O +have O +identified O +key O +components O +for O +ERAD O +, O +including O +HRD1 B-protein +, O +SEL1L B-protein +( O +Hrd3p B-protein +), O +Derlin B-protein +- I-protein +1 I-protein +, I-protein +- I-protein +2 I-protein +, I-protein +- I-protein +3 I-protein +( O +Der1p B-protein +), O +HERP B-protein +- I-protein +1 I-protein +, I-protein +- I-protein +2 I-protein +( O +Usa1p B-protein +), O +OS9 B-protein +( O +Yos9 B-protein +), O +XTP B-protein +- I-protein +B I-protein +, O +and O +Grp94 B-protein +, O +all O +of O +which O +are O +involved O +in O +the O +recognition O +and O +translocation O +of O +the O +ERAD O +substrates O +in O +yeast B-taxonomy_domain +and O +metazoans B-taxonomy_domain +. O + +Yeast B-taxonomy_domain +ERAD O +components O +, O +which O +have O +been O +extensively O +characterized O +through O +genetic B-experimental_method +and I-experimental_method +biochemical I-experimental_method +studies I-experimental_method +, O +are O +comparable O +with O +mammalian B-taxonomy_domain +ERAD O +components O +, O +sharing O +similar O +molecular O +functions O +and O +structural O +composition O +. O + +The O +HRD1 B-protein +E3 B-protein_type +ubiquitin I-protein_type +ligase I-protein_type +, O +which O +is O +embedded O +in O +the O +ER O +membrane O +, O +is O +involved O +in O +translocating O +ERAD O +substrates O +across O +the O +ER O +membrane O +and O +catalyzing O +substrate O +ubiquitination O +via O +its O +cytosolic O +RING B-structure_element +finger I-structure_element +domain I-structure_element +. O + +SEL1L B-protein +, O +the O +mammalian B-taxonomy_domain +homolog O +of O +Hrd3p B-protein +, O +associates O +with O +HRD1 B-protein +, O +mediates O +HRD1 B-protein +interactions O +with O +the O +ER O +luminal O +lectin B-protein_type +OS9 B-protein +, O +and O +recognizes O +substrates O +to O +be O +degraded O +. O + +In O +particular O +, O +SEL1L B-protein +is O +crucial O +for O +translocation O +of O +Class B-complex_assembly +I I-complex_assembly +major I-complex_assembly +histocompatibility I-complex_assembly +complex I-complex_assembly +( O +MHC B-complex_assembly +) O +heavy B-protein_type +chains I-protein_type +( O +HCs B-protein_type +). O + +Recent O +research O +based O +on O +the O +inducible O +Sel1l B-gene +knockout B-experimental_method +mouse I-experimental_method +model O +highlights O +the O +physiological O +functions O +of O +SEL1L B-protein +. O + +SEL1L B-protein +is O +required O +for O +ER O +homeostasis O +, O +which O +is O +essential O +for O +protein O +translation O +, O +pancreatic O +function O +, O +and O +cellular O +and O +organismal O +survival O +. O + +However O +, O +despite O +the O +functional O +importance O +of O +SEL1L B-protein +, O +the O +molecular O +structure B-evidence +of O +SEL1L B-protein +has O +not O +been O +solved O +. O + +Previous O +biochemical B-experimental_method +studies I-experimental_method +reveal O +that O +SEL1L B-protein +is O +a O +type B-protein_type +I I-protein_type +transmembrane I-protein_type +protein I-protein_type +and O +has O +a O +large O +luminal B-structure_element +domain I-structure_element +comprising O +sets O +of O +repeated B-structure_element +Sel1 I-structure_element +- I-structure_element +like I-structure_element +( O +SLR B-structure_element +) O +motifs O +. O + +The O +SLR B-structure_element +motif O +is O +a O +structural O +motif O +that O +closely O +resembles O +the O +tetratricopeptide B-structure_element +- I-structure_element +repeat I-structure_element +( O +TPR B-structure_element +) O +motif O +, O +which O +is O +a O +protein O +- O +protein O +interaction O +module O +. O + +Although O +there O +is O +evidence O +that O +the O +luminal B-structure_element +domain I-structure_element +of O +SEL1L B-protein +is O +involved O +in O +substrate O +recognition O +or O +in O +forming O +complexes O +with O +chaperones B-protein_type +, O +it O +is O +not O +known O +how O +the O +unique O +structure O +of O +the O +repeated O +SLR B-structure_element +motifs O +contributes O +to O +the O +molecular O +function O +of O +the O +HRD1 B-complex_assembly +- I-complex_assembly +SEL1L I-complex_assembly +E3 B-protein_type +ligase I-protein_type +complex O +and O +affects O +ERAD O +at O +the O +molecular O +level O +. O + +Furthermore O +, O +while O +repeated O +SLR B-structure_element +motifs O +are O +commonly O +found O +in O +tandem O +arrays O +, O +the O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +are O +, O +according O +to O +the O +primary O +structure O +prediction O +of O +SEL1L B-protein +, O +interspersed O +among O +other O +sequences O +in O +the O +luminal B-structure_element +domain I-structure_element +and O +form O +three O +SLR B-structure_element +domain O +clusters O +. O + +Therefore O +, O +the O +way O +in O +which O +these O +unique O +structural O +features O +of O +SEL1L B-protein +are O +related O +to O +its O +critical O +function O +in O +ERAD O +remains O +to O +be O +elucidated O +. O + +To O +clearly O +understand O +the O +biochemical O +role O +of O +the O +SLR B-structure_element +domains O +of O +SEL1L B-protein +in O +ERAD O +, O +we O +determined O +the O +crystal B-evidence +structure I-evidence +of O +the O +central O +SLR B-structure_element +domain O +of O +SEL1L B-protein +. O + +We O +found O +that O +the O +central B-structure_element +domain I-structure_element +of O +SEL1L B-protein +, O +comprising O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +through I-structure_element +9 I-structure_element +( O +SEL1Lcent B-structure_element +), O +forms O +a O +tight O +dimer B-oligomeric_state +with O +two O +- O +fold O +symmetry O +due O +to O +domain O +swapping O +of O +the O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +. O + +We O +also O +found O +that O +SLR B-structure_element +- I-structure_element +C I-structure_element +, O +consisting O +of O +SLR B-structure_element +motifs I-structure_element +10 I-structure_element +and I-structure_element +11 I-structure_element +, O +directly O +interacts O +with O +the O +N O +- O +terminus O +luminal B-structure_element +loop I-structure_element +of O +HRD1 B-protein +. O + +Based O +on O +these O +observations O +, O +we O +propose O +a O +model O +wherein O +the O +SLR B-structure_element +domains O +of O +SEL1L B-protein +contribute O +to O +the O +formation O +of O +stable B-protein_state +oligomers B-oligomeric_state +of O +the O +ERAD O +translocation O +machinery O +, O +which O +is O +indispensable O +for O +ERAD O +. O + +Structure B-experimental_method +Determination I-experimental_method +of O +SEL1Lcent B-structure_element + +The O +Mus B-species +musculus I-species +SEL1L B-protein +protein O +contains O +790 O +amino O +acids O +and O +has O +17 O +% O +sequence O +identity O +to O +its O +yeast B-taxonomy_domain +homolog O +, O +Hrd3p B-protein +. O + +Mouse B-taxonomy_domain +SEL1L B-protein +contains O +a O +fibronectin B-structure_element +type I-structure_element +II I-structure_element +domain I-structure_element +at O +the O +N O +- O +terminus O +, O +followed O +by O +11 O +SLR B-structure_element +motifs O +and O +a O +single O +transmembrane B-structure_element +domain I-structure_element +at O +the O +C O +- O +terminus O +( O +Fig O +. O +1A O +). O + +The O +11 O +SLR B-structure_element +motifs O +are O +located O +in O +the O +ER O +lumen O +and O +account O +for O +more O +than O +two O +thirds O +of O +the O +mass O +of O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +. O + +The O +SLR B-structure_element +motifs O +can O +be O +grouped O +into O +three O +regions O +due O +to O +the O +presence O +of O +linker B-structure_element +sequences I-structure_element +among O +the O +groups O +of O +SLR B-structure_element +motifs O +: O +SLR B-structure_element +- I-structure_element +N I-structure_element +( O +SLR B-structure_element +motifs I-structure_element +1 I-structure_element +to I-structure_element +4 I-structure_element +), O +SLR B-structure_element +- I-structure_element +M I-structure_element +( O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +to I-structure_element +9 I-structure_element +), O +and O +SLR B-structure_element +- I-structure_element +C I-structure_element +( O +SLR B-structure_element +motifs I-structure_element +10 I-structure_element +to I-structure_element +11 I-structure_element +) O +( O +Fig O +. O +1A O +). O + +Sequence B-experimental_method +alignment I-experimental_method +of O +the O +SLR B-structure_element +motifs O +revealed O +that O +there O +is O +a O +short O +linker B-structure_element +sequence I-structure_element +( O +residues O +336 B-residue_range +– I-residue_range +345 I-residue_range +) O +between O +SLR B-structure_element +- I-structure_element +N I-structure_element +and O +SLR B-structure_element +- I-structure_element +M I-structure_element +and O +a O +long O +linker B-structure_element +sequence I-structure_element +( O +residues O +528 B-residue_range +– I-residue_range +635 I-residue_range +) O +between O +SLR B-structure_element +- I-structure_element +M I-structure_element +and O +SLR B-structure_element +- I-structure_element +C I-structure_element +( O +Fig O +. O +1A O +). O + +We O +first O +tried O +to O +prepare O +the O +full B-protein_state +- I-protein_state +length I-protein_state +mouse B-taxonomy_domain +SEL1L B-protein +protein O +, O +excluding O +the O +transmembrane B-structure_element +domain I-structure_element +at O +the O +C O +- O +terminus O +( O +residues O +735 B-residue_range +– I-residue_range +755 I-residue_range +), O +by O +expression B-experimental_method +in I-experimental_method +bacteria I-experimental_method +. O + +However O +, O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +protein O +aggregated O +in O +solution O +and O +produced O +no O +soluble O +protein O +. O + +To O +identify O +a O +soluble O +form O +of O +SEL1L B-protein +, O +we O +generated O +serial B-experimental_method +truncation I-experimental_method +constructs I-experimental_method +of O +SEL1L B-protein +based O +on O +the O +predicted O +SLR B-structure_element +motifs O +and O +highly B-protein_state +conserved I-protein_state +regions O +across O +several O +different O +species O +. O + +Both O +SLR B-structure_element +- I-structure_element +N I-structure_element +( O +residues O +194 B-residue_range +– I-residue_range +343 I-residue_range +) O +and O +SLR B-structure_element +- I-structure_element +C I-structure_element +( O +residues O +639 B-residue_range +– I-residue_range +719 I-residue_range +) O +alone O +could O +be O +solubilized O +with O +an O +MBP B-experimental_method +tag I-experimental_method +at I-experimental_method +the I-experimental_method +N I-experimental_method +- I-experimental_method +terminus I-experimental_method +, O +but O +appeared O +to O +be O +polydisperse O +when O +analyzed O +by O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +. O + +However O +, O +the O +central B-structure_element +region I-structure_element +of O +SEL1L B-protein +, O +comprising O +residues O +337 B-residue_range +– I-residue_range +554 I-residue_range +, O +was O +soluble O +and O +homogenous O +in O +size O +, O +as O +determined O +by O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +. O + +To O +define O +compact O +domain O +boundaries O +for O +the O +central B-structure_element +region I-structure_element +of O +SEL1L B-protein +, O +we O +digested B-experimental_method +the I-experimental_method +protein I-experimental_method +with I-experimental_method +trypsin I-experimental_method +and O +analyzed O +the O +proteolysis O +products O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +and O +N B-experimental_method +- I-experimental_method +terminal I-experimental_method +sequencing I-experimental_method +. O + +The O +results O +of O +this O +preliminary O +biochemical O +analysis O +suggested O +that O +SEL1L B-protein +residues O +348 B-residue_range +– I-residue_range +533 I-residue_range +( O +SEL1Lcent B-structure_element +) O +would O +be O +amenable O +to O +structural B-experimental_method +analysis I-experimental_method +( O +Fig O +. O +1A O +). O + +Crystals B-evidence +of O +SEL1Lcent B-structure_element +grew O +in O +space O +group O +P21 O +with O +four O +copies O +of O +SEL1Lcent B-structure_element +( O +a O +total O +of O +82 O +kDa O +) O +in O +the O +asymmetric O +unit O +. O + +The O +structure B-evidence +was O +determined O +by O +the O +single B-experimental_method +- I-experimental_method +wavelength I-experimental_method +anomalous I-experimental_method +diffraction I-experimental_method +( O +SAD B-experimental_method +) O +method O +using O +selenium B-chemical +as O +the O +anomalous O +scatterer O +( O +Table O +1 O +and O +Methods O +). O + +The O +assignment O +of O +residues O +during O +model O +building O +was O +aided O +by O +the O +selenium B-chemical +atom O +positions O +, O +and O +the O +structure B-evidence +was O +refined O +with O +native O +data O +to O +2 O +. O +6 O +Å O +resolution O +with O +Rwork B-evidence +/ I-evidence +Rfree I-evidence +values O +of O +20 O +. O +7 O +/ O +27 O +. O +7 O +%. O + +Overall O +Structure B-evidence +of O +SEL1Lcent B-structure_element + +The O +mouse B-taxonomy_domain +SEL1Lcent B-structure_element +crystallized B-experimental_method +as O +a O +homodimer B-oligomeric_state +, O +and O +there O +were O +two O +homodimers B-oligomeric_state +in O +the O +crystal O +asymmetric O +unit O +( O +Fig O +. O +1B O +, O +C O +, O +Supplementary O +Fig O +. O +1 O +). O + +The O +two O +SEL1Lcent B-structure_element +molecules O +dimerize B-oligomeric_state +in O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +manner O +through O +a O +two B-site +- I-site +fold I-site +symmetry I-site +interface I-site +resulting O +in O +a O +cosmos O +- O +like O +shaped O +structure B-evidence +( O +Fig O +. O +1B O +). O + +The O +resulting O +structure B-evidence +resembles O +the O +yin O +- O +yang O +symbol O +with O +overall O +dimensions O +of O +60 O +× O +60 O +× O +25 O +Å O +, O +where O +a O +SEL1Lcent B-structure_element +monomer B-oligomeric_state +corresponds O +to O +half O +the O +symbol O +. O + +The O +dimer B-oligomeric_state +formation O +buries O +a O +surface O +area O +of O +1670 O +Å2 O +for O +each O +monomer B-oligomeric_state +, O +and O +no O +significant O +differences O +between O +the O +protomers B-oligomeric_state +were O +displayed O +( O +final O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +RMSD B-evidence +) O +of O +0 O +. O +6 O +Å O +for O +all O +Cα O +atoms O +). O + +Each O +protomer B-oligomeric_state +is O +composed O +of O +ten O +α B-structure_element +- I-structure_element +helices I-structure_element +, O +which O +form O +the O +five O +SLRs B-structure_element +, O +resulting O +in O +an O +elongated O +curved O +structure O +, O +confirming O +the O +primary O +structure O +prediction O +( O +Fig O +. O +1D O +). O + +The O +α B-structure_element +- I-structure_element +helices I-structure_element +subdivide O +the O +structure B-evidence +into O +five O +pairs O +( O +A B-structure_element +and O +B B-structure_element +) O +as O +shown O +in O +a O +number O +of O +TPRs B-structure_element +and O +SLRs B-structure_element +. O + +Helices B-structure_element +A I-structure_element +and I-structure_element +B I-structure_element +are O +14 O +and O +13 O +residues O +long O +, O +respectively O +, O +and O +the O +two O +helices B-structure_element +are O +connected O +by O +a O +short O +turn B-structure_element +and O +loop B-structure_element +( O +Fig O +. O +1D O +). O + +In O +addition O +, O +a O +longer O +loop B-structure_element +, O +consisting O +of O +approximately O +eight O +amino O +acids O +, O +is O +inserted O +between O +helix B-structure_element +B I-structure_element +of O +one O +SLR B-structure_element +and O +helix B-structure_element +A I-structure_element +of O +the O +next O +SLR B-structure_element +. O + +This O +arrangement O +is O +a O +unique O +feature O +for O +SLRs B-structure_element +among O +the O +major O +classes O +of O +repeats O +containing O +an O +α B-structure_element +- I-structure_element +solenoid I-structure_element +. O + +Starting O +from O +its O +N O +- O +terminus O +, O +the O +α B-structure_element +- I-structure_element +solenoid I-structure_element +of O +SEL1L B-protein +extends O +across O +a O +semi O +- O +circle O +in O +a O +right O +- O +handed O +superhelix O +fashion O +along O +the O +rotation O +axis O +of O +the O +yin B-structure_element +- I-structure_element +yang I-structure_element +circle I-structure_element +. O + +However O +, O +the O +last O +helix O +, O +9B B-structure_element +, O +at O +the O +C O +- O +terminus O +adopts O +a O +different O +conformation O +, O +lying O +parallel O +to O +the O +long O +axis O +of O +helix B-structure_element +9A I-structure_element +instead O +of O +forming O +an O +antiparallel O +SLR B-structure_element +. O + +This O +unique O +conformation O +of O +helix B-structure_element +9B I-structure_element +most O +likely O +contributes O +to O +formation O +of O +the O +dimer B-oligomeric_state +structure O +of O +SEL1Lcent B-structure_element +, O +as O +detailed O +below O +. O + +With O +the O +exception O +of O +the O +last O +SLR B-structure_element +, O +the O +four O +α B-structure_element +- I-structure_element +helix I-structure_element +pairs O +possess O +similar O +conformations O +, O +with O +RMSD B-evidence +values O +of O +0 O +. O +7 O +Å O +for O +all O +Cα O +atoms O +. O + +Although O +the O +sequence O +similarity O +for O +the O +pairwise B-experimental_method +alignments I-experimental_method +varies O +between O +25 O +% O +and O +35 O +%, O +all O +the O +residues O +present O +in O +the O +SLR B-structure_element +motifs O +are O +conserved B-protein_state +among O +the O +five O +pairs O +. O + +The O +SLR B-structure_element +domain O +of O +SLR B-structure_element +- I-structure_element +M I-structure_element +ends O +at O +residue O +524 B-residue_number +, O +and O +C O +- O +terminal O +amino O +acids O +525 B-residue_range +– I-residue_range +533 I-residue_range +of O +the O +protein O +are O +not O +visible O +in O +the O +electron B-evidence +density I-evidence +map I-evidence +, O +suggesting O +that O +this O +region O +is O +highly B-protein_state +flexible I-protein_state +. O + +Since O +no O +information O +regarding O +dimer B-oligomeric_state +formation O +by O +SEL1L B-protein +through O +its O +SLR B-structure_element +motifs O +is O +available O +, O +we O +tested O +whether O +the O +SEL1Lcent B-structure_element +dimer B-oligomeric_state +shown O +in O +our O +crystal B-evidence +structure I-evidence +is O +a O +biological O +unit O +. O + +First O +, O +we O +cross B-experimental_method +- I-experimental_method +linked I-experimental_method +SEL1Lcent B-structure_element +or O +a O +longer O +construct O +of O +SEL1L B-protein +( O +SEL1Llong B-mutant +, O +residues O +337 B-residue_range +– I-residue_range +554 I-residue_range +) O +using O +various O +concentrations O +of O +glutaraldehyde B-chemical +( O +GA B-chemical +) O +or O +dimethyl B-chemical +suberimidate I-chemical +( O +DMS B-chemical +) O +and O +analyzed O +the O +products O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +. O + +We O +detected O +bands O +at O +the O +mass O +of O +a O +dimer B-oligomeric_state +for O +both O +SEL1Lcent B-structure_element +and O +SEL1Llong B-mutant +when O +cross B-experimental_method +- I-experimental_method +linked I-experimental_method +with O +low O +concentrations O +of O +GA B-chemical +( O +0 O +. O +005 O +%) O +or O +DMS B-chemical +( O +0 O +. O +3 O +mM O +) O +( O +Supplementary O +Fig O +. O +2A O +, O +B O +). O + +Next O +, O +we O +conducted O +analytical B-experimental_method +ultracentrifugation I-experimental_method +of O +SEL1Lcent B-structure_element +. O + +Consistent O +with O +the O +cross B-experimental_method +- I-experimental_method +linking I-experimental_method +data O +, O +analytical B-experimental_method +ultracentrifugation I-experimental_method +revealed O +that O +the O +molecular B-evidence +weight I-evidence +of O +SEL1Lcent B-structure_element +corresponds O +to O +a O +dimer B-oligomeric_state +( O +Supplementary O +Fig O +. O +2C O +). O + +Taken O +together O +, O +these O +data O +indicate O +that O +some O +kind O +of O +dimer B-oligomeric_state +is O +formed O +in O +solution O +. O + +Dimer B-site +Interface I-site +of O +SEL1Lcent B-structure_element + +In O +contrast O +to O +a O +previously O +described O +SLR B-protein_type +motif I-protein_type +containing I-protein_type +proteins I-protein_type +that O +exist O +as O +monomers B-oligomeric_state +in O +solution O +, O +SEL1Lcent B-structure_element +forms O +an O +intimate O +two B-site +- I-site +fold I-site +homotypic I-site +dimer I-site +interface I-site +( O +Figs O +1B O +and O +2A O +). O + +The O +concave B-site +surface I-site +of O +each O +SEL1L B-protein +domain O +comprising O +helix B-structure_element +5A I-structure_element +to I-structure_element +9A I-structure_element +encircles O +its O +dimer B-oligomeric_state +counterpart O +in O +an O +interlocking O +clasp O +- O +like O +arrangement O +. O + +However O +, O +no O +interactions O +were O +seen O +between O +the O +two O +- O +fold O +- O +related O +protomers B-oligomeric_state +through O +the O +concave B-site +inner I-site +surfaces I-site +themselves O +. O + +Rather O +, O +the O +unique O +structure O +of O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +, O +consisting O +of O +two O +parallel O +helices O +( O +9A B-structure_element +and O +9B B-structure_element +), O +is O +located O +in O +the O +space O +generated O +by O +the O +concave B-site +surface I-site +and O +provides O +an O +extensive O +dimerization B-site +interface I-site +between O +the O +two O +- O +fold O +- O +related O +molecules O +( O +Fig O +. O +2A O +). O + +Helix B-structure_element +9B I-structure_element +from O +one O +protomer B-oligomeric_state +inserts O +into O +the O +empty O +space O +surrounded O +by O +the O +concave B-site +region I-site +in O +the O +other O +monomer B-oligomeric_state +, O +forming O +a O +domain B-protein_state +- I-protein_state +swapped I-protein_state +conformation O +. O + +Three O +major O +contact B-site +interfaces I-site +are O +involved O +in O +the O +interactions O +, O +and O +all O +interfaces B-site +are O +symmetrically O +related O +between O +the O +dimer B-oligomeric_state +subunits O +( O +Fig O +. O +2A O +). O + +Structure B-experimental_method +- I-experimental_method +based I-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +135 O +SEL1L B-protein +phylogenetic O +sequences O +using O +a O +ConSurf B-experimental_method +server I-experimental_method +revealed O +that O +the O +surface O +residues O +in O +the O +dimer B-site +interfaces I-site +were O +highly B-protein_state +conserved I-protein_state +among O +the O +SEL1L B-protein +orthologs O +( O +Fig O +. O +1E O +). O + +First O +, O +helix B-structure_element +9B I-structure_element +of O +each O +SEL1Lcent B-structure_element +subunit O +interacts O +with O +residues O +lining O +the O +inner B-site +groove I-site +from O +the O +SLR B-structure_element +α B-structure_element +- I-structure_element +helices I-structure_element +( O +5B B-structure_element +, O +6B B-structure_element +, O +7B B-structure_element +, O +and O +8B B-structure_element +) O +from O +its O +counterpart O +. O + +In O +this O +interface B-site +, O +Leu B-residue_name_number +516 I-residue_name_number +and O +Tyr B-residue_name_number +519 I-residue_name_number +on O +helix B-structure_element +9B I-structure_element +are O +located O +in O +the O +center O +, O +making O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +Trp B-residue_name_number +478 I-residue_name_number +on O +helix B-structure_element +8B I-structure_element +, O +Val B-residue_name_number +444 I-residue_name_number +on O +helix B-structure_element +7B I-structure_element +, O +Phe B-residue_name_number +411 I-residue_name_number +on O +helix B-structure_element +6B I-structure_element +, O +and O +Leu B-residue_name_number +380 I-residue_name_number +on O +helix B-structure_element +5B I-structure_element +from O +the O +SEL1Lcent B-structure_element +counterpart O +( O +Fig O +. O +2A O +, O +Interface B-site +1 I-site +detail O +). O + +In O +addition O +to O +hydrophobic B-bond_interaction +interactions I-bond_interaction +, O +the O +side O +chain O +hydroxyl O +group O +of O +Tyr B-residue_name_number +519 I-residue_name_number +and O +the O +main O +- O +chain O +oxygen O +of O +Ile B-residue_name_number +515 I-residue_name_number +form O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +the O +side O +chain O +of O +the O +conserved B-protein_state +Gln B-residue_name_number +377 I-residue_name_number +and O +His B-residue_name_number +381 I-residue_name_number +on O +helix B-structure_element +5B I-structure_element +of O +the O +two O +- O +fold O +- O +related O +protomer B-oligomeric_state +. O + +The O +side O +chain O +of O +Gln B-residue_name_number +523 I-residue_name_number +forms O +an O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +to O +the O +side O +chain O +of O +Asp B-residue_name_number +480 I-residue_name_number +on O +the O +two O +- O +fold O +- O +related O +protomer B-oligomeric_state +( O +Fig O +. O +2A O +, O +Interface B-site +1 I-site +detail O +). O + +Second O +, O +the O +residues O +from O +helix B-structure_element +9A I-structure_element +interact O +with O +the O +residues O +from O +helix B-structure_element +5A I-structure_element +of O +its O +counterpart O +in O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +orientation O +. O + +In O +this O +interface B-site +, O +the O +interacting O +residues O +on O +helix B-structure_element +9A I-structure_element +, O +including O +Leu B-residue_name_number +503 I-residue_name_number +, O +Tyr B-residue_name_number +499 I-residue_name_number +, O +and O +the O +aliphatic O +side O +chain O +of O +Lys B-residue_name_number +500 I-residue_name_number +, O +form O +an O +extensive O +network O +of O +van B-bond_interaction +der I-bond_interaction +Waals I-bond_interaction +contacts I-bond_interaction +with O +the O +hydrophobic O +residues O +of O +the O +counterpart O +helix B-structure_element +5A I-structure_element +, O +including O +Tyr B-residue_name_number +360 I-residue_name_number +, O +Leu B-residue_name_number +356 I-residue_name_number +, O +Tyr B-residue_name_number +359 I-residue_name_number +, O +and O +Leu B-residue_name_number +363 I-residue_name_number +. O + +In O +addition O +to O +hydrophobic B-bond_interaction +interactions I-bond_interaction +, O +the O +side O +chains O +of O +Asn B-residue_name_number +507 I-residue_name_number +and O +Ser B-residue_name_number +510 I-residue_name_number +on O +helix B-structure_element +9A I-structure_element +make O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +with O +highly B-protein_state +conserved I-protein_state +Arg B-residue_name_number +384 I-residue_name_number +in O +the O +loop B-structure_element +between O +helix B-structure_element +5B I-structure_element +and O +6A B-structure_element +from O +the O +two O +- O +fold O +- O +related O +protomer B-oligomeric_state +( O +Fig O +. O +2A O +, O +Interface O +2 O +detail O +). O + +Third O +, O +the O +helix B-structure_element +9B I-structure_element +from O +each O +protomer B-oligomeric_state +is O +involved O +in O +the O +dimer B-oligomeric_state +interaction O +by O +forming O +a O +two O +- O +fold O +antiparallel O +symmetry O +. O + +In O +particular O +, O +the O +side O +chains O +of O +hydrophobic O +residues O +, O +including O +Phe B-residue_name_number +518 I-residue_name_number +, O +Leu B-residue_name_number +521 I-residue_name_number +, O +and O +Met B-residue_name_number +524 I-residue_name_number +, O +are O +directed O +toward O +each O +other O +, O +where O +they O +make O +both O +inter O +- O +and O +intramolecular O +contacts O +( O +Fig O +. O +2A O +, O +Interface B-site +3 I-site +detail O +). O + +To O +further O +investigate O +the O +interactions O +observed O +in O +our O +crystal B-evidence +structure I-evidence +, O +we O +generated O +a O +C O +- O +terminal O +deletion B-protein_state +mutant I-protein_state +( O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +) O +lacking B-protein_state +SLR B-structure_element +motif I-structure_element +9 I-structure_element +( O +helix B-structure_element +9A I-structure_element +and O +9B B-structure_element +) O +from O +SEL1Lcent B-structure_element +for O +comparative O +analysis O +. O + +The O +deletion B-protein_state +mutant I-protein_state +and O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +SEL1Lcent B-structure_element +showed O +no O +difference O +in O +spectra B-evidence +by O +CD B-experimental_method +spectroscopy I-experimental_method +, O +indicating O +that O +the O +deletion B-experimental_method +of O +the O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +did O +not O +affect O +the O +secondary O +structure O +of O +SEL1Lcent B-structure_element +( O +Supplementary O +Fig O +. O +3 O +). O + +However O +, O +the O +mutant B-protein_state +behaved O +as O +a O +monomer B-oligomeric_state +in O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +and O +analytical B-experimental_method +ultracentrifugation I-experimental_method +experiments O +( O +Fig O +. O +2B O +, O +Supplementary O +Fig O +. O +2C O +). O + +Additionally O +, O +to O +further O +validate O +the O +key O +residues O +involved O +in O +dimer B-oligomeric_state +formation O +, O +we O +generated O +a O +triple B-protein_state +point I-protein_state +mutant I-protein_state +( O +Interface B-site +1 I-site +, O +I515A B-mutant +, O +L516A B-mutant +, O +and O +Y519A B-mutant +) O +of O +the O +hydrophobic O +residues O +that O +are O +involved O +in O +dimerization B-oligomeric_state +. O + +The O +triple B-protein_state +point I-protein_state +mutant I-protein_state +eluted O +at O +the O +monomer B-oligomeric_state +position O +upon O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +, O +while O +the O +negative O +control O +point B-protein_state +mutant I-protein_state +( O +Q460A B-mutant +) O +eluted O +at O +the O +same O +position O +as O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +. O + +Notably O +, O +a O +single B-experimental_method +- I-experimental_method +residue I-experimental_method +mutation I-experimental_method +( O +L521A B-mutant +in O +interface B-site +3 I-site +) O +abolished B-protein_state +the I-protein_state +dimerization I-protein_state +of O +SEL1Lcent B-structure_element +( O +Fig O +. O +2B O +). O + +Leu B-residue_name_number +521 I-residue_name_number +is O +located O +in O +the O +dimerization B-site +center I-site +of O +the O +antiparallel O +9B B-structure_element +helices I-structure_element +in O +the O +SEL1Lcent B-structure_element +dimer B-oligomeric_state +. O + +Taken O +together O +, O +these O +structural B-evidence +and I-evidence +biochemical I-evidence +data I-evidence +demonstrate O +that O +SEL1Lcent B-structure_element +exists O +as O +a O +dimer B-oligomeric_state +in O +solution O +and O +that O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +in O +SEL1Lcent B-structure_element +plays O +an O +important O +role O +in O +generating O +a O +two O +- O +fold O +dimerization B-site +interface I-site +. O + +The O +Two O +Glycine B-residue_name +Residues O +( O +G512 B-residue_name_number +and O +G513 B-residue_name_number +) O +Create O +a O +Hinge B-structure_element +for O +Domain O +Swapping O +of O +SLR B-structure_element +Motif I-structure_element +9 I-structure_element + +SLRs B-structure_element +of O +mouse B-taxonomy_domain +SEL1L B-protein +were O +predicted O +using O +the O +TPRpred B-experimental_method +server I-experimental_method +. O + +Based O +on O +the O +prediction O +, O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +contains O +a O +total O +of O +11 O +SLR B-structure_element +motifs O +, O +and O +our O +construct O +corresponds O +to O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +through I-structure_element +9 I-structure_element +. O + +Although O +amino O +acid O +sequences O +from O +helix B-structure_element +9A I-structure_element +and O +9B B-structure_element +correctly O +aligned O +with O +the O +regular O +SLR B-structure_element +repeats I-structure_element +and O +corresponded O +to O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +( O +Fig O +. O +3A O +), O +the O +structural O +arrangement O +of O +the O +two O +helices B-structure_element +deviated O +from O +the O +common O +structure O +for O +the O +SLR B-structure_element +motif O +. O + +According O +to O +our O +crystal B-evidence +structure I-evidence +, O +the O +central O +axis O +of O +helix B-structure_element +9B I-structure_element +is O +almost O +parallel O +to O +that O +of O +helix B-structure_element +9A I-structure_element +( O +Fig O +. O +3B O +). O + +However O +, O +this O +unusual O +conformation O +of O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +seems O +to O +be O +essential O +for O +dimer B-oligomeric_state +formation O +, O +as O +described O +earlier O +. O + +For O +this O +structural O +geometry O +, O +two O +adjacent O +residues O +, O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +, O +in O +SEL1L B-protein +confer O +flexibility O +at O +this O +position O +by O +adopting O +main O +- O +chain O +dihedral O +angles O +that O +are O +disallowed O +for O +non O +- O +glycine O +residues O +. O + +The O +phi O +and O +psi O +dihedrals O +are O +100 O +° O +and O +20 O +° O +for O +Gly B-residue_name_number +512 I-residue_name_number +, O +and O +110 O +° O +and O +− O +20 O +° O +for O +Gly B-residue_name_number +513 I-residue_name_number +, O +respectively O +( O +Fig O +. O +3C O +). O + +Gly B-residue_name_number +513 I-residue_name_number +is O +conserved B-protein_state +among O +other O +SLR B-structure_element +motifs O +in O +the O +SEL1Lcent B-structure_element +, O +but O +Gly B-residue_name_number +512 I-residue_name_number +is O +present O +only O +in O +the O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +of O +SEL1Lcent B-structure_element +( O +Fig O +. O +3A O +). O + +Thus O +, O +the O +Gly B-structure_element +- I-structure_element +Gly I-structure_element +residues O +generate O +an O +unusual O +sharp O +bend O +at O +the O +C O +- O +terminal O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +. O + +The O +involvement O +of O +a O +glycine B-residue_name +residue O +in O +forming O +a O +hinge B-structure_element +for O +domain O +swapping O +has O +been O +reported O +previously O +. O + +The O +significance O +of O +Gly B-residue_name_number +513 I-residue_name_number +is O +further O +highlighted O +by O +its O +absolute B-protein_state +conservation I-protein_state +among O +different O +species O +, O +including O +the O +budding B-taxonomy_domain +yeast I-taxonomy_domain +homolog O +Hrd3p B-protein +. O + +To O +further O +investigate O +the O +importance O +of O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +in O +the O +unusual O +SLR B-structure_element +motif O +geometry O +, O +we O +generated O +a O +point B-experimental_method +mutation I-experimental_method +( O +Gly B-mutant +to I-mutant +Ala I-mutant +), O +which O +restricts O +the O +flexibility O +. O +Although O +the O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +residues O +are O +closely O +surrounded O +by O +helix B-structure_element +9B I-structure_element +from O +the O +counter O +protomer B-oligomeric_state +, O +there O +is O +enough O +space O +for O +the O +side O +chain O +of O +alanine B-residue_name +, O +suggesting O +that O +no O +steric O +hindrance O +would O +be O +caused O +by O +the O +mutation B-experimental_method +( O +Fig O +. O +3C O +). O + +This O +means O +that O +the O +effect O +of O +the O +mutation B-experimental_method +is O +mainly O +to O +generate O +a O +more O +restricted O +geometry O +at O +the O +hinge B-structure_element +region O +. O + +G512A B-mutant +or O +G513A B-mutant +alone O +showed O +no O +differences O +from O +wild B-protein_state +- I-protein_state +type I-protein_state +in O +terms O +of O +the O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +elution O +profile O +( O +Fig O +. O +3D O +), O +suggesting O +that O +the O +restriction O +for O +single O +glycine B-residue_name +flexibility O +would O +not O +be O +enough O +to O +break O +the O +swapped O +structure O +of O +helix B-structure_element +9B I-structure_element +. O + +However O +, O +the O +double B-protein_state +mutant I-protein_state +( O +G512A B-mutant +/ O +G513A B-mutant +) O +eluted O +over O +a O +broad O +range O +and O +much O +earlier O +than O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +, O +suggesting O +that O +mutation B-experimental_method +of O +the O +residues O +involved O +in O +the O +hinge B-structure_element +linking O +helix B-structure_element +9A I-structure_element +and O +9B B-structure_element +significantly O +affected O +the O +geometry O +of O +helix B-structure_element +9B I-structure_element +in O +generating O +domain O +swapping O +, O +and O +eventually O +altered O +the O +overall O +oligomeric O +state O +of O +SEL1Lcent B-structure_element +into O +a O +polydisperse O +pattern O +( O +Fig O +. O +3D O +, O +Supplementary O +Fig O +. O +6 O +). O + +When O +the O +residues O +were O +mutated B-experimental_method +to I-experimental_method +lysine B-residue_name +( O +G512K B-mutant +/ O +G513K B-mutant +), O +the O +mutant B-protein_state +not O +only O +restricted O +the O +geometry O +of O +residues O +at O +the O +hinge B-structure_element +but O +also O +generated O +steric O +hindrance O +during O +interaction O +with O +the O +counter O +protomer B-oligomeric_state +of O +SEL1Lcent B-structure_element +, O +thereby O +inhibiting O +self O +- O +association O +of O +SEL1Lcent B-structure_element +completely O +. O + +The O +G512K B-mutant +/ O +G513K B-mutant +double B-protein_state +mutant I-protein_state +eluted O +at O +the O +monomer B-oligomeric_state +position O +in O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +( O +Fig O +. O +3D O +). O + +A O +previous O +study O +shows O +that O +induction O +of O +steric O +hindrance O +by O +mutation B-experimental_method +destabilizes O +the O +dimerization B-site +interface I-site +of O +a O +different O +protein O +, O +ClC B-protein_type +transporter I-protein_type +. O + +Collectively O +, O +these O +data O +suggest O +that O +the O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +at O +the O +connection O +between O +helix B-structure_element +9A I-structure_element +and O +9B B-structure_element +play O +a O +crucial O +role O +in O +forming O +the O +domain B-protein_state +- I-protein_state +swapped I-protein_state +conformation O +that O +enables O +dimer B-oligomeric_state +formation O +. O + +SEL1L B-protein +Forms O +Self B-oligomeric_state +- I-oligomeric_state +oligomers I-oligomeric_state +through O +SEL1Lcent B-structure_element +domain O +in O +vivo O + +Next O +, O +we O +examined O +if O +SEL1L B-protein +also O +forms O +self B-oligomeric_state +- I-oligomeric_state +oligomers I-oligomeric_state +in O +vivo O +using O +HEK293T O +cells O +. O + +We O +generated O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +- O +HA B-experimental_method +and O +SEL1L B-protein +- O +FLAG B-experimental_method +fusion B-experimental_method +constructs I-experimental_method +and O +co B-experimental_method +- I-experimental_method +transfected I-experimental_method +the O +constructs O +into O +HEK293T O +cells O +. O + +A O +co B-experimental_method +- I-experimental_method +immunoprecipitation I-experimental_method +assay I-experimental_method +using O +an O +anti O +- O +FLAG B-experimental_method +antibody O +followed O +by O +Western B-experimental_method +blot I-experimental_method +analysis O +using O +an O +anti O +- O +HA B-experimental_method +antibody O +showed O +that O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +forms O +self B-oligomeric_state +- I-oligomeric_state +oligomers I-oligomeric_state +in O +vivo O +( O +Fig O +. O +4A O +). O + +To O +further O +examine O +whether O +the O +SEL1Lcent B-structure_element +domain O +is O +sufficient O +to O +physically O +interact O +with O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +, O +we O +generated O +SEL1Lcent B-structure_element +and O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +deletion B-experimental_method +( O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +) O +construct O +, O +which O +were O +fused B-experimental_method +to I-experimental_method +the O +C O +- O +terminus O +of O +SEL1L B-protein +signal B-structure_element +peptides I-structure_element +. O + +Co B-experimental_method +- I-experimental_method +immunoprecipitation I-experimental_method +analysis I-experimental_method +showed O +that O +the O +SEL1Lcent B-structure_element +was O +sufficient O +to O +physically O +interact O +with O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +, O +while O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +failed O +to O +do O +so O +( O +Fig O +. O +4A O +). O + +Interestingly O +, O +however O +, O +the O +expression O +level O +of O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +was O +consistently O +lower O +than O +that O +of O +SEL1Lcent B-structure_element +( O +Fig O +. O +4A O +, O +B O +). O + +Semi B-experimental_method +- I-experimental_method +quantitative I-experimental_method +RT I-experimental_method +- I-experimental_method +PCR I-experimental_method +revealed O +no O +significant O +difference O +in O +transcriptional O +levels O +of O +the O +two O +constructs O +( O +data O +not O +shown O +). O + +We O +speculated O +that O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +could O +be O +secreted O +while O +the O +SEL1Lcent B-structure_element +is O +retained O +in O +the O +ER O +by O +association O +with O +the O +endogenous O +ERAD O +complex O +. O + +Indeed O +, O +immunoprecipitation B-experimental_method +followed O +by O +western B-experimental_method +blot I-experimental_method +analysis O +using O +the O +culture O +medium O +detected O +secreted O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +fragment O +, O +but O +not O +SEL1Lcent B-structure_element +( O +Fig O +. O +4B O +). O + +We O +next O +examined O +if O +the O +reason O +why O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +failed O +to O +bind O +to O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +may O +be O +because O +of O +the O +lower O +level O +of O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +in O +the O +ER O +lumen O +compared O +to O +SEL1Lcent B-structure_element +fragment O +. O + +In O +order O +to O +retain O +two O +SEL1L B-protein +fragments O +in O +the O +ER O +lumen O +, O +we O +added O +KDEL B-structure_element +ER B-structure_element +retention I-structure_element +sequence I-structure_element +to O +the O +C O +- O +terminus O +of O +both O +fragments O +. O + +Indeed O +, O +the O +addition O +of O +KDEL B-structure_element +peptide O +increased O +the O +level O +of O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +in O +the O +ER O +lumen O +( O +Fig O +. O +4D O +, O +E O +) O +and O +the O +immunostaining B-experimental_method +analysis O +showed O +both O +constructs O +were O +well O +localized O +to O +the O +ER O +( O +Fig O +. O +4C O +). O + +We O +further O +analyzed O +whether O +SEL1Lcent B-structure_element +may O +competitively O +inhibit O +the O +self O +- O +oligomerization O +of O +SEL1L B-protein +in O +vivo O +. O + +To O +this O +end O +, O +we O +co B-experimental_method +- I-experimental_method +transfected I-experimental_method +the O +differentially O +tagged B-protein_state +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +( O +SEL1L B-protein +- O +HA B-experimental_method +and O +SEL1L B-protein +- O +FLAG B-experimental_method +) O +and O +increasing B-experimental_method +doses I-experimental_method +of O +SEL1Lcent B-mutant +- I-mutant +KDEL I-mutant +, O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +- I-mutant +KDEL I-mutant +or O +SEL1Lcent B-mutant +( I-mutant +L521A I-mutant +)- I-mutant +KDEL I-mutant +, O +respectively O +. O + +Co B-experimental_method +- I-experimental_method +immunoprecipitation I-experimental_method +assay I-experimental_method +revealed O +that O +wild B-protein_state +- I-protein_state +type I-protein_state +SEL1Lcent B-mutant +- I-mutant +KDEL I-mutant +, O +indeed O +, O +competitively O +disrupted O +the O +self O +- O +association O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L O +( O +Fig O +. O +4E O +). O + +In O +contrast O +, O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +- I-mutant +KDEL I-mutant +and O +the O +single O +- O +residue O +mutation O +L521A B-mutant +in O +SEL1Lcent B-structure_element +did O +not O +competitively O +inhibit O +the O +self O +- O +association O +of O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +( O +Fig O +. O +4E O +, O +F O +). O + +These O +data O +suggest O +that O +the O +SEL1L B-protein +forms O +self B-oligomeric_state +- I-oligomeric_state +oligomers I-oligomeric_state +and O +the O +oligomerization O +is O +mediated O +by O +the O +SEL1Lcent B-structure_element +domain O +in O +vivo O +. O + +Structural B-experimental_method +Comparison I-experimental_method +of O +SEL1L B-protein +SLRs B-structure_element +with O +TPRs B-structure_element +or O +SLRs B-structure_element +of O +Other O +Proteins O + +Previous O +studies O +reveal O +that O +TPRs B-structure_element +and O +SLRs B-structure_element +have O +similar O +consensus O +sequences O +, O +suggesting O +that O +their O +three O +- O +dimensional O +structures O +are O +also O +similar O +. O + +The O +superposition B-experimental_method +of O +isolated O +TPRs B-structure_element +from O +Cdc23 B-protein +( O +S B-species +. I-species +pombe I-species +, O +cell B-protein +division I-protein +cycle I-protein +23 I-protein +homolog O +, O +PDB O +code O +3ZN3 O +) O +and O +SLRs B-structure_element +from O +HcpC B-protein +( O +Helicobacter B-protein +Cysteine I-protein +- I-protein +rich I-protein +Protein I-protein +C I-protein +, O +PDB O +code O +1OUV O +) O +yields O +RMSDs B-evidence +below O +1 O +Å O +, O +confirming O +that O +the O +isolated O +repeats O +are O +indeed O +similar O +. O + +This O +is O +relevant O +to O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +, O +as O +isolated O +SLR B-structure_element +motifs O +from O +SEL1Lcent B-structure_element +showed O +good O +structural B-experimental_method +alignment I-experimental_method +with O +isolated O +TPRs B-structure_element +( O +RMSD B-evidence +1 O +. O +6 O +Å O +for O +all O +Cα O +chains O +) O +from O +Cdc23N B-protein +- O +term O +and O +SLRs B-structure_element +( O +RMSD B-evidence +0 O +. O +6 O +Å O +for O +all O +Cα O +chains O +) O +from O +HcpC B-protein +( O +Fig O +. O +5A O +). O + +However O +, O +superimposing B-experimental_method +the O +structure B-evidence +of O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +to I-structure_element +9 I-structure_element +from O +SEL1Lcent B-structure_element +onto O +the O +overall O +Cdc23N B-protein +- O +term O +or O +full B-protein_state +- I-protein_state +length I-protein_state +HcpC B-protein +structures B-evidence +revealed O +that O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +to I-structure_element +9 I-structure_element +in O +SEL1Lcent B-structure_element +have O +a O +different O +superhelical O +structure O +than O +either O +Cdc23 B-protein +or O +HcpC B-protein +( O +RMSD B-evidence +values O +of O +> O +2 O +. O +5 O +Å O +for O +Cα O +atoms O +) O +( O +Fig O +. O +5B O +). O + +The O +differences O +may O +result O +from O +the O +differing O +numbers O +of O +residues O +in O +the O +loops B-structure_element +and O +differences O +in O +antiparallel B-structure_element +helix I-structure_element +packing O +. O + +Moreover O +, O +there O +are O +conserved B-protein_state +disulfide B-ptm +bonds I-ptm +in O +the O +SLR B-structure_element +motifs O +of O +HcpC B-protein +and O +HcpB B-protein +, O +but O +no O +such O +bonds O +are O +observed O +in O +SEL1Lcent B-structure_element +. O + +These O +factors O +contribute O +to O +the O +differences O +in O +the O +overall O +conformation O +of O +the O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +and O +other O +SLR B-protein_type +or I-protein_type +TPR I-protein_type +motif I-protein_type +- I-protein_type +containing I-protein_type +proteins I-protein_type +. O + +Another O +major O +difference O +in O +the O +structure B-evidence +of O +SLR B-structure_element +motifs O +between O +SEL1L B-protein +and O +HcpC B-protein +is O +the O +oligomeric O +state O +of O +proteins O +. O + +The O +TPR B-structure_element +motif O +is O +involved O +in O +the O +dimerization B-oligomeric_state +of O +proteins O +such O +as O +Cdc23 B-protein +, O +Cdc16 B-protein +, O +and O +Cdc27 B-protein +. O + +In O +particular O +, O +the O +N O +- O +terminal O +domain O +of O +Cdc23 B-protein +( O +Cdc23N B-protein +- O +term O +) O +has O +a O +TPR B-structure_element +- O +motif O +organization O +similar O +to O +that O +of O +the O +SLR B-structure_element +motif O +in O +SEL1Lcent B-structure_element +. O + +The O +seven O +TPR B-structure_element +motifs O +of O +Cdc23N B-protein +- O +term O +are O +assembled O +into O +a O +superhelical B-structure_element +structure I-structure_element +, O +generating O +a O +hollow O +surface O +and O +encircling O +its O +dimer B-oligomeric_state +counterpart O +in O +an O +interlocking O +clasp O +- O +like O +arrangement O +( O +Fig O +. O +5C O +). O + +The O +TPR B-structure_element +motif I-structure_element +1 I-structure_element +( O +TPR1 B-structure_element +) O +of O +each O +Cdc23N B-protein +- O +term O +subunit O +is O +located O +in O +the O +hollow O +surface O +of O +the O +counter O +subunit O +and O +interacts O +with O +residues O +lining O +the O +inner B-site +groove I-site +TPR B-structure_element +α B-structure_element +- I-structure_element +helices I-structure_element +, O +generating O +two O +- O +fold O +symmetry O +homotype O +interactions O +. O + +However O +, O +in O +this O +structure B-evidence +, O +a O +conformational O +change O +in O +the O +TPR B-structure_element +motif O +itself O +is O +not O +observed O +. O + +Self O +- O +association O +of O +HcpC B-protein +has O +not O +been O +reported O +, O +and O +there O +is O +no O +domain B-protein_state +- I-protein_state +swapped I-protein_state +structure O +in O +the O +SLR B-structure_element +motifs O +of O +HcpC B-protein +, O +in O +contrast O +to O +that O +observed O +in O +SEL1Lcent B-structure_element +. O + +Although O +SEL1L B-protein +contains O +a O +number O +of O +SLR B-structure_element +motifs O +comparable O +to O +HcpC B-protein +, O +the O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +are O +interrupted O +by O +other O +sequences O +, O +making O +three O +SLR B-structure_element +motif O +clusters O +( O +Fig O +. O +1A O +). O + +The O +interrupted O +SLR B-structure_element +motifs O +may O +be O +required O +for O +dimerization B-oligomeric_state +of O +SEL1Lcent B-structure_element +, O +as O +five O +SLR B-structure_element +motifs O +are O +more O +than O +enough O +to O +form O +the O +semicircle B-structure_element +of I-structure_element +the I-structure_element +yin I-structure_element +- I-structure_element +yang I-structure_element +symbol O +( O +Fig O +. O +1B O +). O + +Helix B-structure_element +5A I-structure_element +from O +SLR B-structure_element +motif I-structure_element +5 I-structure_element +meets O +helix B-structure_element +9A I-structure_element +from O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +of O +the O +counterpart O +SEL1L B-protein +. O + +If O +the O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +to I-structure_element +9 I-structure_element +were O +not O +isolated O +from O +other O +SLR B-structure_element +motifs O +, O +steric O +hindrance O +could O +interfere O +with O +dimerization B-oligomeric_state +of O +SEL1L B-protein +. O + +This O +is O +one O +of O +the O +biggest O +differences O +from O +TPRs B-structure_element +in O +Cdc23 B-protein +and O +from O +the O +SLRs B-structure_element +in O +HcpC B-protein +, O +where O +the O +motifs O +exist O +in O +tandem O +. O + +TPR B-structure_element +and O +SLR B-structure_element +motifs O +are O +generally O +involved O +in O +protein O +- O +protein O +interaction O +modules O +, O +and O +the O +sequences O +between O +the O +SLR B-structure_element +motifs O +of O +SEL1L B-protein +might O +actually O +facilitate O +the O +self O +- O +association O +of O +this O +protein O +. O + +SLR B-structure_element +- I-structure_element +C I-structure_element +of O +SEL1L B-protein +Binds O +HRD1 B-protein +N O +- O +terminus O +Luminal B-structure_element +Loop I-structure_element + +Based O +on O +the O +structural B-evidence +data I-evidence +presented O +herein O +, O +a O +possible O +arrangement O +of O +membrane O +- O +associated O +ERAD O +components O +in O +mammals B-taxonomy_domain +, O +highlighting O +the O +molecular O +functions O +of O +SLR B-structure_element +domains O +in O +SEL1L B-protein +, O +is O +shown O +in O +Fig O +. O +6C O +. O + +We O +suggest O +that O +the O +middle O +SLR B-structure_element +domains O +are O +involved O +in O +the O +dimerization B-oligomeric_state +of O +SEL1L B-protein +based O +on O +the O +crystal B-evidence +structure I-evidence +and O +biochemical O +data O +. O + +SLR B-structure_element +- I-structure_element +C I-structure_element +, O +which O +contains O +SLR B-structure_element +motifs I-structure_element +10 I-structure_element +to I-structure_element +11 I-structure_element +, O +might O +be O +involved O +in O +the O +interaction O +with O +HRD1 B-protein +. O + +Indirect O +evidence O +from O +a O +previous O +yeast B-taxonomy_domain +study O +shows O +that O +the O +circumscribed O +region O +of O +C O +- O +terminal O +Hrd3p B-protein +, O +specifically O +residues O +664 B-residue_range +– I-residue_range +695 I-residue_range +, O +forms O +contacts O +with O +the O +Hrd1 B-protein +luminal B-structure_element +loops I-structure_element +. O + +The O +Hrd3p B-protein +residues O +664 B-residue_range +– I-residue_range +695 I-residue_range +correspond O +to O +mouse B-taxonomy_domain +SEL1L B-protein +residues O +696 B-residue_range +– I-residue_range +727 I-residue_range +, O +which O +include O +the O +entire O +helix B-structure_element +11B I-structure_element +( O +residue O +697 B-residue_range +– I-residue_range +709 I-residue_range +) O +of O +SLR B-structure_element +motif I-structure_element +11 I-structure_element +and O +a O +well B-protein_state +- I-protein_state +conserved I-protein_state +adjacent O +region O +( O +Supplementary O +Fig O +. O +4 O +). O + +This O +observation O +is O +supported O +by O +the O +following O +: O +( O +1 O +) O +the O +meticulous O +range O +of O +SLR B-structure_element +motif I-structure_element +10 I-structure_element +to I-structure_element +11 I-structure_element +is O +newly O +established O +from O +a O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +SLR I-experimental_method +motif I-experimental_method +alignment I-experimental_method +, O +based O +on O +the O +present O +structure B-experimental_method +study I-experimental_method +, O +and O +( O +2 O +) O +the O +relatively O +high O +sequence B-protein_state +conservation I-protein_state +between O +mammalian B-taxonomy_domain +SEL1L B-protein +and O +yeast B-taxonomy_domain +Hrd3p B-protein +around O +SLR B-structure_element +motifs I-structure_element +10 I-structure_element +to I-structure_element +11 I-structure_element +, O +which O +contain O +contact O +regions O +with O +HRD1 B-protein +( O +Hrd1p B-protein +) O +( O +Supplementary O +Figs O +. O +4 O +and O +5 O +). O + +To O +address O +this O +hypothesis O +, O +we O +prepared O +constructs O +encoding O +mouse B-taxonomy_domain +HRD1 B-protein +luminal O +fragments O +fused B-experimental_method +to I-experimental_method +GST I-experimental_method +as O +shown O +in O +Fig O +. O +6A O +, O +and O +tested O +their O +ability O +to O +bind O +certain O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +. O + +The O +fusion O +proteins O +were O +immobilized O +on O +glutathione O +- O +Sepharose O +beads O +and O +probed O +for O +binding O +to O +SLR B-structure_element +- I-structure_element +N I-structure_element +, O +SLR B-structure_element +- I-structure_element +M I-structure_element +, O +SLR B-structure_element +- I-structure_element +C I-structure_element +, O +and O +monomer B-oligomeric_state +form O +of O +SLR B-structure_element +- I-structure_element +M I-structure_element +( O +SLR B-mutant +- I-mutant +ML521A I-mutant +). O + +Figure O +6B O +shows O +that O +the O +SLR B-structure_element +- I-structure_element +C I-structure_element +, O +consisting O +of O +SLR B-structure_element +motifs I-structure_element +10 I-structure_element +and I-structure_element +11 I-structure_element +, O +exclusively O +interacts O +with O +N O +- O +terminal O +luminal B-structure_element +loop I-structure_element +( O +residues O +21 B-residue_range +– I-residue_range +42 I-residue_range +) O +of O +HRD1 B-protein +. O + +The O +molecular O +functions O +of O +SLR B-structure_element +- I-structure_element +N I-structure_element +are O +unclear O +. O + +One O +possibility O +is O +that O +SLR B-structure_element +- I-structure_element +N I-structure_element +contributes O +to O +substrate O +recognition O +of O +proteins O +to O +be O +degraded O +because O +there O +are O +a O +couple O +of O +putative O +glycosylation B-site +sites I-site +within O +the O +SLR B-structure_element +- I-structure_element +N I-structure_element +domain O +( O +Fig O +. O +1A O +). O + +SEL1Lcent B-structure_element +contains O +a O +putative O +N B-site +- I-site +glycosylation I-site +site I-site +, O +Asn B-residue_name_number +427 I-residue_name_number +, O +which O +is O +highly B-protein_state +conserved I-protein_state +among O +different O +species O +and O +structurally O +exposed O +to O +the O +surface O +of O +the O +SEL1L B-protein +dimer B-oligomeric_state +according O +to O +the O +crystal B-evidence +structure I-evidence +( O +Fig O +. O +6C O +). O + +Many O +reports O +demonstrate O +that O +membrane O +- O +bound O +ERAD O +machinery O +proteins O +in O +yeast B-taxonomy_domain +, O +such O +as O +Hrd1p B-protein +, O +Der1p B-protein +, O +and O +Usa1p B-protein +, O +are O +involved O +in O +oligomerization O +of O +ERAD O +components O +. O + +The O +Hrd1p B-protein +complex O +forms O +dimers B-oligomeric_state +upon O +sucrose B-experimental_method +gradient I-experimental_method +sedimentation I-experimental_method +and O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +. O + +Previous O +data O +show O +that O +HA B-protein_state +- I-protein_state +epitope I-protein_state +- I-protein_state +tagged I-protein_state +Hrd3p B-protein +or O +Hrd1p B-protein +efficiently O +co O +- O +precipitate O +with O +unmodified B-protein_state +Hrd3p B-protein +and O +Hrd1p B-protein +, O +respectively O +, O +suggesting O +that O +both O +Hrd1p B-protein +and O +Hrd3p B-protein +homodimers B-oligomeric_state +are O +involved O +in O +self O +- O +association O +of O +the O +Hrd B-complex_assembly +complex O +. O + +Considering O +that O +the O +functional O +and O +structural O +composition O +of O +ERAD O +components O +are O +conserved O +in O +both O +yeast B-taxonomy_domain +and O +mammals B-taxonomy_domain +, O +we O +propose O +that O +the O +mammalian B-taxonomy_domain +ERAD O +components O +also O +form O +self O +- O +associating O +oligomers B-oligomeric_state +. O + +This O +hypothesis O +is O +supported O +by O +cross B-experimental_method +- I-experimental_method +linking I-experimental_method +data I-experimental_method +suggesting O +that O +human B-species +HRD1 B-protein +forms O +a O +homodimer B-oligomeric_state +. O + +Consistent O +with O +the O +previous O +data O +, O +our O +crystal B-evidence +structure I-evidence +and O +biochemical B-evidence +data I-evidence +demonstrate O +that O +mouse B-taxonomy_domain +SEL1Lcent B-structure_element +exists O +as O +a O +homodimer B-oligomeric_state +in O +the O +ER O +lumen O +via O +domain O +swapping O +of O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +. O + +We O +need O +to O +further O +test O +whether O +there O +are O +contacts O +involved O +in O +dimer B-oligomeric_state +formation O +in O +SEL1L B-protein +in O +addition O +to O +those O +in O +the O +SLR B-structure_element +- I-structure_element +M I-structure_element +region O +. O + +In O +yeast B-taxonomy_domain +, O +Usa1p B-protein +acts O +as O +a O +scaffold O +for O +Hrd1p B-protein +and O +Der1p B-protein +, O +in O +which O +the O +N O +- O +terminus O +of O +Usa1p B-protein +interacts O +with O +the O +C O +- O +terminal O +34 O +amino O +acids O +of O +Hrd1p B-protein +in O +the O +cytosol O +to O +induce O +oligomerization O +of O +Hrd1p B-protein +, O +which O +is O +essential O +for O +its O +activity O +. O + +However O +, O +metazoans B-taxonomy_domain +lack O +a O +clear O +Usa1p B-protein +homolog O +. O + +Although O +mammalian B-taxonomy_domain +HERP B-protein_type +has O +sequences O +and O +domains O +that O +are O +conserved B-protein_state +in I-protein_state +Usa1p B-protein +, O +the O +molecular O +function O +of O +HERP B-protein_type +is O +not O +clearly O +related O +to O +that O +of O +Usa1p B-protein +. O + +Rather O +, O +recent O +research O +shows O +that O +a O +transiently B-protein_state +expressed I-protein_state +HRD1 B-complex_assembly +- I-complex_assembly +SEL1L I-complex_assembly +complex O +alone O +associates O +with O +the O +ERAD O +lectins B-protein_type +OS9 B-protein +or O +XTP B-protein +- I-protein +B I-protein +and O +is O +sufficient O +to O +facilitate O +the O +retrotranslocation O +and O +degradation O +of O +the O +model O +ERAD O +substrate O +α B-protein +- I-protein +antitrypsin I-protein +null I-protein +Hong I-protein +- I-protein +Kong I-protein +( O +NHK B-protein +) O +and O +its O +variant O +, O +NHK B-mutant +- I-mutant +QQQ I-mutant +, O +which O +lacks B-protein_state +the O +N B-site +- I-site +glycosylation I-site +sites I-site +. O + +Assuming O +that O +the O +correct O +oligomerization O +of O +ERAD O +components O +may O +be O +critical O +for O +their O +function O +, O +we O +hypothesize O +that O +homodimer B-oligomeric_state +formation O +of O +SEL1L B-protein +in O +the O +ER O +lumen O +may O +stabilize O +oligomerization O +of O +the O +HRD B-complex_assembly +complex O +, O +given O +that O +SEL1L B-protein +forms O +a O +stoichiometric O +complex B-protein_state +with I-protein_state +HRD1 B-protein +. O + +This O +is O +further O +supported O +by O +our O +data O +showing O +that O +the O +SLR B-structure_element +- I-structure_element +C I-structure_element +of O +SEL1L B-protein +directly O +interacts O +with O +the O +luminal O +fragment O +of O +HRD1 B-protein +in O +the O +ER O +lumen O +. O + +Although O +the O +organization O +of O +membrane O +- O +bound O +HRD B-complex_assembly +complex O +components O +may O +be O +very O +similar O +between O +metazoans B-taxonomy_domain +and O +yeast B-taxonomy_domain +, O +the O +molecular O +details O +of O +interactions O +between O +the O +components O +may O +not O +necessarily O +be O +conserved O +. O + +In O +yeast B-taxonomy_domain +, O +it O +is O +unclear O +whether O +self O +- O +association O +of O +Hrd3p B-protein +is O +due O +to O +SLR B-structure_element +motifs O +because O +the O +sequence O +of O +Hrd3p B-protein +does O +not O +align O +precisely O +with O +the O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +. O + +Furthermore O +, O +we O +are O +uncertain O +whether O +self O +- O +association O +of O +Hrd3p B-protein +contributes O +to O +formation O +of O +the O +active B-protein_state +form O +of O +the O +Hrd1p B-protein +complex O +. O + +Recently O +, O +a O +truncated B-protein_state +version O +of O +Yos9 B-protein +was O +shown O +to O +form O +a O +dimer B-oligomeric_state +in O +the O +ER O +lumen O +and O +to O +contribute O +to O +the O +dimeric B-oligomeric_state +state O +of O +the O +Hrd1p B-protein +complex O +. O + +This O +interaction O +seems O +to O +be O +weak O +because O +direct O +Yos9 B-protein +- O +Yos9 B-protein +interactions O +were O +not O +detected O +in O +immunoprecipitation B-experimental_method +experiments I-experimental_method +from O +yeast B-taxonomy_domain +cell O +extracts O +containing O +different O +epitope B-protein_state +- I-protein_state +tagged I-protein_state +variants O +of O +Yos9 B-protein +. O + +However O +, O +the O +dimerization B-oligomeric_state +of O +Yos9 B-protein +could O +provide O +a O +higher O +stability O +for O +the O +Hrd1p B-protein +complex O +oligomer B-oligomeric_state +. O + +Likewise O +, O +the O +dimerization B-oligomeric_state +of O +SEL1L B-protein +might O +provide O +stability O +for O +the O +mammalian B-taxonomy_domain +HRD B-complex_assembly +oligomer B-oligomeric_state +complex O +. O + +Further O +cell O +biological O +studies O +are O +required O +to O +clarify O +whether O +SEL1L B-protein +( O +Hrd3p B-protein +) O +dimerization B-oligomeric_state +could O +be O +cooperative O +with O +the O +oligomerization O +of O +the O +HRD B-complex_assembly +complex O +. O + +Considering O +that O +it O +is O +very O +important O +for O +the O +function O +of O +the O +HRD B-complex_assembly +complex O +that O +the O +components O +assemble O +as O +oligomers B-oligomeric_state +, O +we O +believe O +that O +the O +self O +- O +association O +of O +SEL1L B-protein +strongly O +contributes O +to O +generating O +active B-protein_state +forms O +of O +the O +HRD B-complex_assembly +complex O +, O +even O +in O +the O +absence B-protein_state +of I-protein_state +Usa1p B-protein +, O +in O +metazoans B-taxonomy_domain +. O + +These O +findings O +should O +provide O +a O +foundation O +for O +molecular O +- O +level O +studies O +to O +understand O +the O +membrane O +- O +associated O +HRD B-complex_assembly +complex O +assembly O +in O +ERAD O +. O + +Crystal B-evidence +Structure I-evidence +of O +SEL1Lcent B-structure_element +. O + +( O +A O +) O +The O +diagram O +shows O +the O +domain O +structure O +of O +Mus B-species +musculus I-species +SEL1L B-protein +, O +as O +defined O +by O +proteolytic B-experimental_method +mapping I-experimental_method +and O +sequence B-experimental_method +/ I-experimental_method +structure I-experimental_method +analysis I-experimental_method +. O + +The O +11 O +SLR B-structure_element +motifs O +were O +divided O +into O +three O +groups O +( O +SLR B-structure_element +- I-structure_element +N I-structure_element +, O +SLR B-structure_element +- I-structure_element +M I-structure_element +, O +and O +SLR B-structure_element +- I-structure_element +C I-structure_element +) O +due O +to O +the O +presence O +of O +linker B-structure_element +sequences I-structure_element +that O +are O +not O +predicted O +SLR B-structure_element +motifs O +. O + +Putative O +N B-site +- I-site +glycosylation I-site +sites I-site +are O +indicated O +by O +black O +triangles O +. O + +We O +determined O +the O +crystal B-evidence +structure I-evidence +of O +the O +SLR B-structure_element +- I-structure_element +M I-structure_element +, O +residues O +348 B-residue_range +– I-residue_range +533 I-residue_range +. O +( O +B O +) O +Ribbon O +diagram O +of O +the O +biological O +unit O +of O +the O +SEL1Lcent B-structure_element +, O +viewed O +along O +the O +two O +- O +fold O +NCS O +axis O +. O + +The O +crystal B-evidence +structure I-evidence +was O +determined O +by O +SAD B-experimental_method +phasing I-experimental_method +using O +selenium B-chemical +as O +the O +anomalous O +scatterer O +and O +refined O +to O +2 O +. O +6 O +Å O +resolution O +( O +Table O +1 O +). O + +( O +C O +) O +SEL1Lcent B-structure_element +ribbon O +diagram O +rotated O +90 O +° O +around O +a O +horizontal O +axis O +relative O +to O +( O +B O +). O + +( O +D O +) O +One O +protomer B-oligomeric_state +of O +the O +SEL1Lcent B-structure_element +dimer B-oligomeric_state +. O + +Starting O +from O +the O +N O +- O +terminus O +, O +SEL1Lcent B-structure_element +has O +five O +SLR B-structure_element +motifs O +comprising O +ten O +α B-structure_element +helices I-structure_element +. O + +Each O +SLR B-structure_element +motif O +( O +from O +5 O +to O +9 O +) O +is O +indicated O +in O +a O +different O +color O +. O +( O +E O +) O +Evolutionary O +conservation O +of O +surface O +residues O +in O +SEL1Lcent B-structure_element +, O +calculated O +using O +ConSurf B-experimental_method +, O +from O +a O +structure B-experimental_method +- I-experimental_method +based I-experimental_method +alignment I-experimental_method +of O +135 O +SEL1L B-protein +sequences O +. O + +The O +surface O +is O +colored O +from O +red O +( O +high O +) O +to O +white O +( O +poor O +) O +according O +to O +the O +degree O +of O +conservation O +in O +the O +SEL1L B-protein +phylogenetic O +orthologs O +. O + +The O +ribbon O +diagram O +of O +the O +counterpart O +protomer B-oligomeric_state +is O +drawn O +to O +show O +the O +orientation O +of O +the O +SEL1Lcent B-structure_element +dimer B-oligomeric_state +. O + +Dimer B-site +Interface I-site +of O +SEL1Lcent B-structure_element +. O + +( O +A O +) O +The O +diagram O +on O +the O +left O +shows O +the O +SEL1Lcent B-structure_element +dimer B-oligomeric_state +viewed O +along O +the O +two O +- O +fold O +symmetry O +axis O +. O + +Three O +distinct O +contact B-site +regions I-site +are O +indicated O +with O +labeled O +boxes O +. O + +The O +close O +- O +up O +view O +on O +the O +right O +shows O +the O +residues O +of O +SEL1Lcent B-structure_element +that O +contribute O +to O +dimer B-oligomeric_state +formation O +via O +the O +three O +contact B-site +interfaces I-site +. O + +The O +yellow O +dotted O +lines O +indicate O +intermolecular O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +two O +protomers B-oligomeric_state +of O +SEL1Lcent B-structure_element +. O +( O +B O +) O +Size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +( O +SEC B-experimental_method +) O +analysis O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +dimeric B-site +interface I-site +SEL1Lcent B-structure_element +mutants B-protein_state +to O +compare O +the O +oligomeric O +states O +of O +the O +proteins O +. O + +The O +standard O +molecular O +masses O +for O +the O +SEC B-experimental_method +experiments O +( O +top O +) O +were O +obtained O +from O +the O +following O +proteins O +: O +aldolase O +, O +158 O +kDa O +; O +cobalbumin O +, O +75 O +kDa O +; O +ovalbumin O +, O +44 O +kDa O +; O +and O +carbonic O +anhydrase O +, O +29 O +kDa O +. O + +The O +elution O +fractions O +, O +indicated O +by O +the O +gray O +shading O +, O +were O +run O +on O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +and O +are O +shown O +below O +the O +gel B-evidence +- I-evidence +filtration I-evidence +elution I-evidence +profile I-evidence +. O + +The O +schematic O +diagrams O +representing O +the O +protein O +constructs O +used O +in O +the O +SEC B-experimental_method +are O +shown O +on O +the O +left O +of O +each O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +profile O +. O + +Domain O +Swapping O +for O +Dimerization B-oligomeric_state +of O +SEL1Lcent B-structure_element +. O + +( O +A O +) O +Sequence B-experimental_method +alignment I-experimental_method +of O +the O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +. O + +The O +11 O +SLR B-structure_element +motifs O +were O +aligned B-experimental_method +based O +on O +the O +present O +crystal B-evidence +structure I-evidence +of O +SEL1Lcent B-structure_element +. O + +The O +sequences O +of O +SEL1Lcent B-structure_element +included O +in O +the O +crystal B-evidence +structure I-evidence +are O +highlighted O +by O +the O +blue O +box O +. O + +The O +secondary O +structure O +elements O +are O +indicated O +above O +the O +sequences O +, O +with O +helices B-structure_element +depicted O +as O +cylinders O +. O + +The O +GG B-structure_element +sequence O +in O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +, O +which O +creates O +the O +hinge B-structure_element +for O +domain O +swapping O +( O +see O +text O +), O +is O +shaded O +yellow O +. O + +Stars O +below O +the O +sequences O +indicate O +the O +specific O +residues O +that O +commonly O +appear O +in O +SLRs B-structure_element +. O + +( O +B O +) O +Structure B-experimental_method +alignment I-experimental_method +of O +five O +SLR B-structure_element +motifs O +in O +SEL1Lcent B-structure_element +is O +shown O +to O +highlight O +the O +unusual O +geometry O +of O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +. O + +Each O +SLR B-structure_element +motif O +is O +shown O +in O +a O +different O +color O +. O + +In O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +, O +the O +axes O +for O +the O +two O +helices B-structure_element +are O +almost O +parallel O +, O +while O +the O +other O +SLR B-structure_element +motifs O +adopt O +an O +α B-structure_element +- I-structure_element +hairpin I-structure_element +structure O +. O +( O +C O +) O +Stereo O +view O +shows O +that O +the O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +residues O +are O +surrounded O +by O +neighboring O +residues O +from O +helix B-structure_element +9B I-structure_element +from O +the O +counterpart O +dimer B-oligomeric_state +. O + +The O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +residues O +are O +colored O +green O +and O +red O +, O +respectively O +. O +( O +D O +) O +The O +following O +point B-experimental_method +mutations I-experimental_method +were O +generated O +to O +check O +the O +effect O +of O +the O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +residues O +in O +terms O +of O +generating O +the O +hinge B-structure_element +of O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +: O +G512A B-mutant +, O +G513A B-mutant +, O +G512A B-mutant +/ O +G513A B-mutant +, O +and O +G512K B-mutant +/ O +G513K B-mutant +. O + +Size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +was O +conducted O +as O +described O +in O +Fig O +. O +2B O +. O + +SEL1L B-protein +forms O +self B-oligomeric_state +- I-oligomeric_state +oligomer I-oligomeric_state +mediated O +by O +the O +SEL1Lcent B-structure_element +domain O +in O +vivo O +. O + +( O +A O +) O +HEK293T O +cells O +were O +transfected O +with O +the O +indicated O +plasmid O +constructs O +and O +the O +lysates O +were O +immunoprecipitated B-experimental_method +with O +an O +anti O +- O +FLAG B-experimental_method +antibody O +followed O +by O +western B-experimental_method +blot I-experimental_method +analysis O +using O +an O +anti O +- O +HA B-experimental_method +antibody O +. O + +The O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +- O +FLAG B-experimental_method +was O +co B-experimental_method +- I-experimental_method +immunoprecipitated I-experimental_method +with O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +- O +HA B-experimental_method +. O + +Also O +, O +SEL1Lcent B-structure_element +was O +co B-experimental_method +- I-experimental_method +immunoprecipitated I-experimental_method +with O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +while O +the O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +deletion B-experimental_method +failed O +to O +do O +so O +. O +( O +B O +) O +The O +HEK293T O +cells O +were O +transfected O +with O +the O +indicated O +plasmid O +constructs O +and O +the O +cell O +lysate O +and O +culture O +media O +were O +analyzed O +by O +western B-experimental_method +blot I-experimental_method +analysis O +and O +immunoprecipitation B-experimental_method +respectively O +. O + +The O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +fragment O +was O +secreted O +to O +the O +culture O +media O +but O +the O +SEL1Lcent B-structure_element +was O +retained O +in O +the O +ER O +. O +( O +C O +) O +SEL1Lcent B-mutant +- I-mutant +FLAG I-mutant +- I-mutant +KDEL I-mutant +and O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +- I-mutant +FLAG I-mutant +- I-mutant +KDEL I-mutant +localized O +to O +the O +ER O +. O + +The O +SEL1L B-protein +fragments O +were O +stained O +in O +red O +. O +( O +D O +) O +HEK293T O +cells O +were O +transfected O +with O +the O +indicated O +plasmid O +constructs O +and O +the O +lysates O +were O +immunoprecipitated B-experimental_method +with O +an O +anti O +- O +HA B-experimental_method +antibody O +followed O +by O +Western B-experimental_method +blot I-experimental_method +analysis O +using O +an O +anti O +- O +FLAG B-experimental_method +antibody O +. O + +The O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +forms O +self B-oligomeric_state +- I-oligomeric_state +oligomers I-oligomeric_state +and O +the O +SEL1Lcent B-mutant +- I-mutant +FLAG I-mutant +- I-mutant +KDEL I-mutant +was O +co B-experimental_method +- I-experimental_method +immunoprecipitated I-experimental_method +with O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +- O +HA B-experimental_method +. O + +The O +red O +asterisk O +indicates O +the O +expected O +signal O +for O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +- I-mutant +FLAG I-mutant +- I-mutant +KDEL I-mutant +. O + +SEL1L348 B-mutant +– I-mutant +497 I-mutant +- I-mutant +FLAG I-mutant +- I-mutant +KDEL I-mutant +did O +not O +co O +- O +immunoprecipitate O +with O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +- O +HA B-experimental_method +. O + +The O +white O +asterisks O +indicate O +non O +- O +specific O +bands O +. O +( O +E O +) O +SEL1Lcent B-mutant +- I-mutant +HA I-mutant +- I-mutant +KDEL I-mutant +competitively O +inhibited O +self O +- O +oligomerization O +of O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +. O + +The O +indicated O +plasmid O +constructs O +were O +transfected O +and O +immunoprecipitation B-experimental_method +assay I-experimental_method +was O +performed O +using O +an O +anti O +- O +FLAG B-experimental_method +antibody O +followed O +by O +western B-experimental_method +blot I-experimental_method +analysis O +using O +an O +anti O +- O +HA B-experimental_method +antibody O +. O + +The O +red O +rectangle O +indicates O +competitively O +inhibited O +SEL1L B-protein +self O +- O +oligomer B-oligomeric_state +formation O +by O +the O +increasing O +doses O +of O +SEL1Lcent B-mutant +- I-mutant +HA I-mutant +- I-mutant +KDEL I-mutant +. O +( O +F O +) O +L521A B-mutant +point B-protein_state +mutant I-protein_state +in O +SEL1Lcent B-structure_element +did O +not O +inhibit O +the O +self O +- O +association O +of O +SEL1L B-protein +. O + +Comparison O +of O +SLR B-structure_element +in O +SEL1L B-protein +with O +TPR B-structure_element +or O +Other O +SLR B-protein_type +- I-protein_type +Containing I-protein_type +Proteins I-protein_type +. O + +( O +A O +) O +Ribbon O +diagram O +showing O +superimposition B-experimental_method +of O +an O +isolated O +TPR B-structure_element +motif O +from O +Cdc23 B-protein +and O +an O +SLR B-structure_element +motif O +from O +SEL1Lcent B-structure_element +( O +left O +), O +and O +SLR B-structure_element +motifs O +in O +HcpC B-protein +and O +SEL1Lcent B-structure_element +( O +right O +). O + +The O +SEL1L B-protein +, O +Cdc23 B-protein +, O +and O +HcpC B-protein +are O +colored O +magenta O +, O +green O +and O +cyan O +, O +respectively O +. O + +The O +red O +arrow O +indicates O +disulfide B-ptm +bonds I-ptm +in O +the O +HcpC B-protein +, O +and O +Cys B-residue_name +residues O +involved O +in O +disulfide B-ptm +bonding I-ptm +are O +shown O +by O +a O +yellow O +line O +. O +( O +B O +) O +Ribbon O +representation O +showing O +superimposition B-experimental_method +of O +Cdc23 B-protein +and O +SEL1Lcent B-structure_element +( O +left O +) O +or O +HcpC B-protein +and O +SEL1Lcent B-structure_element +( O +right O +) O +to O +compare O +the O +overall O +organization O +of O +the O +α B-structure_element +- I-structure_element +solenoid I-structure_element +domain I-structure_element +. O + +Both O +SEL1Lcent B-structure_element +schematics O +are O +identically O +oriented O +for O +comparison O +. O + +The O +Cα O +atoms O +of O +the O +residues O +in O +each O +α B-structure_element +- I-structure_element +solenoid I-structure_element +domain I-structure_element +are O +superimposed B-experimental_method +with O +a O +root B-evidence +- I-evidence +mean I-evidence +- I-evidence +squared I-evidence +deviation I-evidence +of O +3 O +. O +3 O +Å O +for O +Cdc23 B-protein +and O +SEL1Lcent B-structure_element +( O +left O +), O +and O +2 O +. O +5 O +Å O +for O +HcpC B-protein +and O +SEL1Lcent B-structure_element +( O +right O +). O + +SEL1Lcent B-structure_element +, O +Cdc23 B-protein +, O +and O +HcpC B-protein +are O +colored O +as O +in O +( O +A O +). O +( O +C O +) O +Ribbon O +diagram O +showing O +the O +overall O +structure B-evidence +of O +Cdc23N B-protein +- O +term O +( O +left O +) O +and O +SEL1Lcent B-structure_element +( O +right O +) O +to O +compare O +their O +similarities O +regarding O +dimer B-oligomeric_state +formation O +through O +domain O +swapping O +. O + +The O +Role O +of O +SLR B-structure_element +- I-structure_element +C I-structure_element +in O +ERAD O +machinery O +and O +Model O +for O +the O +Organization O +of O +Proteins O +in O +Membrane O +- O +Associated O +ERAD O +Components O +. O + +( O +A O +) O +Schematic O +diagram O +shows O +three O +HRD1 B-protein +fragment O +constructs O +used O +in O +the O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +experiment O +. O +( O +B O +) O +Pull B-experimental_method +- I-experimental_method +down I-experimental_method +experiments I-experimental_method +to O +examine O +the O +interactions O +between O +HRD B-complex_assembly +luminal B-structure_element +loops I-structure_element +and O +certain O +SLR B-structure_element +motifs O +of O +SEL1L B-protein +. O + +Fragments O +of O +the O +luminal B-structure_element +loop I-structure_element +of O +HRD1 B-protein +fused O +to O +GST B-chemical +were O +immobilized O +on O +glutathione O +sepharose O +beads O +and O +incubated O +with O +purified O +three O +clusters O +of O +SLR B-structure_element +motifs O +and O +monomer B-oligomeric_state +form O +of O +SLR B-structure_element +- I-structure_element +M I-structure_element +( O +SLR B-mutant +- I-mutant +ML521A I-mutant +, O +right O +panel O +) O +in O +SEL1L B-protein +. O + +Proteins O +were O +analyzed O +by O +12 O +% O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +and O +Coomassie O +blue O +staining O +. O + +( O +C O +) O +Schematic O +representation O +of O +the O +organization O +of O +metazoan B-taxonomy_domain +ERAD O +components O +in O +the O +ER O +membrane O +. O + +The O +11 O +SLR B-structure_element +motifs O +of O +SEL1L B-protein +were O +expressed O +with O +red O +cylinders O +and O +grouped O +into O +three O +parts O +( O +SLR B-structure_element +- I-structure_element +N I-structure_element +, O +SLR B-structure_element +- I-structure_element +M I-structure_element +, O +and O +SLR B-structure_element +- I-structure_element +C I-structure_element +) O +based O +on O +the O +sequence B-experimental_method +alignment I-experimental_method +across O +the O +motifs O +and O +the O +crystal B-evidence +structure I-evidence +presented O +herein O +. O + +We O +hypothesized O +that O +the O +interrupted O +SLR B-structure_element +motifs O +of O +SEL1L B-protein +have O +distinct O +functions O +such O +that O +the O +SLR B-structure_element +- I-structure_element +M I-structure_element +is O +important O +for O +dimer B-oligomeric_state +formation O +of O +the O +protein O +, O +and O +SLR B-structure_element +- I-structure_element +C I-structure_element +is O +involved O +in O +the O +interaction O +with O +HRD1 B-protein +in O +the O +ER O +lumen O +. O + +The O +surface O +representation O +of O +SEL1Lcent B-structure_element +is O +placed O +in O +the O +same O +orientation O +as O +that O +shown O +in O +the O +schematic O +model O +to O +show O +that O +the O +putative O +N B-site +- I-site +glycosylation I-site +site I-site +, O +residue O +N427 B-residue_name_number +( O +indicated O +in O +yellow O +), O +is O +exposed O +on O +the O +surface O +of O +the O +protein O +. O + +Crystal B-evidence +Structures I-evidence +of O +Putative O +Sugar B-protein_type +Kinases I-protein_type +from O +Synechococcus B-species +Elongatus I-species +PCC I-species +7942 I-species +and O +Arabidopsis B-species +Thaliana I-species + +The O +genome O +of O +the O +Synechococcus B-species +elongatus I-species +strain I-species +PCC I-species +7942 I-species +encodes O +a O +putative O +sugar B-protein_type +kinase I-protein_type +( O +SePSK B-protein +), O +which O +shares O +44 O +. O +9 O +% O +sequence O +identity O +with O +the O +xylulose B-protein +kinase I-protein +- I-protein +1 I-protein +( O +AtXK B-protein +- I-protein +1 I-protein +) O +from O +Arabidopsis B-species +thaliana I-species +. O + +Sequence B-experimental_method +alignment I-experimental_method +suggests O +that O +both O +kinases B-protein_type +belong O +to O +the O +ribulokinase B-protein_type +- I-protein_type +like I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +, O +a O +sub O +- O +family O +of O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +. O + +Here O +we O +solved B-experimental_method +the O +structures B-evidence +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +in O +both O +their O +apo B-protein_state +forms O +and O +in B-protein_state +complex I-protein_state +with I-protein_state +nucleotide B-chemical +substrates O +. O + +The O +two O +kinases O +exhibit O +nearly O +identical O +overall O +architecture O +, O +with O +both O +kinases B-protein_type +possessing O +ATP B-chemical +hydrolysis O +activity O +in O +the O +absence B-protein_state +of I-protein_state +substrates I-protein_state +. O + +In O +addition O +, O +our O +enzymatic B-experimental_method +assays I-experimental_method +suggested O +that O +SePSK B-protein +has O +the O +capability O +to O +phosphorylate O +D B-chemical +- I-chemical +ribulose I-chemical +. O + +In O +order O +to O +understand O +the O +catalytic O +mechanism O +of O +SePSK B-protein +, O +we O +solved B-experimental_method +the O +structure B-evidence +of O +SePSK B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +D B-chemical +- I-chemical +ribulose I-chemical +and O +found O +two O +potential O +substrate B-site +binding I-site +pockets I-site +in O +SePSK B-protein +. O + +Using O +mutation B-experimental_method +and I-experimental_method +activity I-experimental_method +analysis I-experimental_method +, O +we O +further O +verified O +the O +key O +residues O +important O +for O +its O +catalytic O +activity O +. O + +Moreover O +, O +our O +structural B-experimental_method +comparison I-experimental_method +with O +other O +family O +members O +suggests O +that O +there O +are O +major O +conformational O +changes O +in O +SePSK B-protein +upon O +substrate O +binding O +, O +facilitating O +the O +catalytic O +process O +. O + +Together O +, O +these O +results O +provide O +important O +information O +for O +a O +more O +detailed O +understanding O +of O +the O +cofactor O +and O +substrate O +binding O +mode O +as O +well O +as O +the O +catalytic O +mechanism O +of O +SePSK B-protein +, O +and O +possible O +similarities O +with O +its O +plant B-taxonomy_domain +homologue O +AtXK B-protein +- I-protein +1 I-protein +. O + +Carbohydrates B-chemical +are O +essential O +cellular O +compounds O +involved O +in O +the O +metabolic O +processes O +present O +in O +all O +organisms O +. O + +Phosphorylation B-ptm +is O +one O +of O +the O +various O +pivotal O +modifications O +of O +carbohydrates B-chemical +, O +and O +is O +catalyzed O +by O +specific O +sugar B-protein_type +kinases I-protein_type +. O + +These O +kinases B-protein_type +exhibit O +considerable O +differences O +in O +their O +folding O +pattern O +and O +substrate O +specificity O +. O + +Based O +on O +sequence B-experimental_method +analysis I-experimental_method +, O +they O +can O +be O +divided O +into O +four O +families O +, O +namely O +HSP B-protein_type +70_NBD I-protein_type +family I-protein_type +, O +FGGY B-protein_type +family I-protein_type +, O +Mer_B B-protein_type +like I-protein_type +family I-protein_type +and O +Parm_like B-protein_type +family I-protein_type +. O + +The O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +contain O +different O +types O +of O +sugar B-protein_type +kinases I-protein_type +, O +all O +of O +which O +possess O +different O +catalytic O +substrates O +with O +preferences O +for O +short O +- O +chained O +sugar B-chemical +substrates O +, O +ranging O +from O +triose B-chemical +to O +heptose B-chemical +. O + +These O +sugar B-chemical +substrates O +include O +L B-chemical +- I-chemical +ribulose I-chemical +, O +erythritol B-chemical +, O +L B-chemical +- I-chemical +fuculose I-chemical +, O +D B-chemical +- I-chemical +glycerol I-chemical +, O +D B-chemical +- I-chemical +gluconate I-chemical +, O +L B-chemical +- I-chemical +xylulose I-chemical +, O +D B-chemical +- I-chemical +ribulose I-chemical +, O +L B-chemical +- I-chemical +rhamnulose I-chemical +and O +D B-chemical +- I-chemical +xylulose I-chemical +. O + +Structures B-evidence +reported O +in O +the O +Protein O +Data O +Bank O +of O +the O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +exhibit O +a O +similar O +overall O +architecture O +containing O +two O +protein O +domains O +, O +one O +of O +which O +is O +responsible O +for O +the O +binding O +of O +substrate O +, O +while O +the O +second O +is O +used O +for O +binding O +cofactor O +ATP B-chemical +. O + +While O +the O +binding B-site +pockets I-site +for O +substrates O +are O +at O +the O +same O +position O +, O +each O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +uses O +different O +substrate B-site +- I-site +binding I-site +residues I-site +, O +resulting O +in O +high O +substrate O +specificity O +. O + +Synpcc7942_2462 B-gene +from O +the O +cyanobacteria B-taxonomy_domain +Synechococcus B-species +elongatus I-species +PCC I-species +7942 I-species +encodes O +a O +putative O +sugar B-protein_type +kinase I-protein_type +( O +SePSK B-protein +), O +and O +this O +kinase B-protein_type +contains O +426 B-residue_range +amino O +acids O +. O + +The O +At2g21370 B-gene +gene O +product O +from O +Arabidopsis B-species +thaliana I-species +, O +xylulose B-protein +kinase I-protein +- I-protein +1 I-protein +( O +AtXK B-protein +- I-protein +1 I-protein +), O +whose O +mature B-protein_state +form I-protein_state +contains O +436 B-residue_range +amino O +acids O +, O +is O +located O +in O +the O +chloroplast O +( O +ChloroP O +1 O +. O +1 O +Server O +). O + +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +display O +a O +sequence O +identity O +of O +44 O +. O +9 O +%, O +and O +belong O +to O +the O +ribulokinase B-protein_type +- I-protein_type +like I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +, O +a O +sub O +- O +family O +of O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +. O + +Members O +of O +this O +sub O +- O +family O +are O +responsible O +for O +the O +phosphorylation B-ptm +of O +sugars B-chemical +similar O +to O +L B-chemical +- I-chemical +ribulose I-chemical +and O +D B-chemical +- I-chemical +ribulose I-chemical +. O + +The O +sequence O +and O +the O +substrate O +specificity O +of O +ribulokinase B-protein_type +- I-protein_type +like I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +are O +different O +, O +but O +they O +share O +the O +common O +folding O +feature O +with O +two O +domains O +. O + +Domain B-structure_element +I I-structure_element +exhibits O +a O +ribonuclease B-structure_element +H I-structure_element +- I-structure_element +like I-structure_element +folding I-structure_element +pattern I-structure_element +, O +and O +is O +responsible O +for O +the O +substrate O +binding O +, O +while O +domain B-structure_element +II I-structure_element +possesses O +an O +actin B-structure_element +- I-structure_element +like I-structure_element +ATPase I-structure_element +domain I-structure_element +that O +binds O +cofactor O +ATP B-chemical +. O + +Two O +possible O +xylulose B-protein_type +kinases I-protein_type +( O +xylulose B-protein +kinase I-protein +- I-protein +1 I-protein +: O +XK B-protein +- I-protein +1 I-protein +and O +xylulose B-protein +kinase I-protein +- I-protein +2 I-protein +: O +XK B-protein +- I-protein +2 I-protein +) O +from O +Arabidopsis B-species +thaliana I-species +were O +previously O +proposed O +. O + +It O +was O +shown O +that O +XK B-protein +- I-protein +2 I-protein +( O +At5g49650 B-gene +) O +located O +in O +the O +cytosol O +is O +indeed O +xylulose B-protein_type +kinase I-protein_type +. O + +However O +, O +the O +function O +of O +XK B-protein +- I-protein +1 I-protein +( O +At2g21370 B-gene +) O +inside O +the O +chloroplast O +stroma O +has O +remained O +unknown O +. O + +SePSK B-protein +from O +Synechococcus B-species +elongatus I-species +strain I-species +PCC I-species +7942 I-species +is O +the O +homolog O +of O +AtXK B-protein +- I-protein +1 I-protein +, O +though O +its O +physiological O +function O +and O +substrates O +remain O +unclear O +. O + +In O +order O +to O +obtain O +functional O +and O +structural O +information O +about O +these O +two O +proteins O +, O +here O +we O +reported O +the O +crystal B-evidence +structures I-evidence +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +. O + +Our O +findings O +provide O +new O +details O +of O +the O +catalytic O +mechanism O +of O +SePSK B-protein +and O +lay O +the O +foundation O +for O +future O +studies O +into O +its O +homologs O +in O +eukaryotes B-taxonomy_domain +. O + +Overall O +structures B-evidence +of O +apo B-protein_state +- O +SePSK B-protein +and O +apo B-protein_state +- O +AtXK B-protein +- I-protein +1 I-protein + +The O +attempt O +to O +solve O +the O +SePSK B-protein +structure B-evidence +by O +molecular B-experimental_method +replacement I-experimental_method +method I-experimental_method +failed O +with O +ribulokinase B-protein +from O +Bacillus B-species +halodurans I-species +( O +PDB O +code O +: O +3QDK O +, O +15 O +. O +7 O +% O +sequence O +identity O +) O +as O +an O +initial O +model O +. O + +We O +therefore O +used O +single B-experimental_method +isomorphous I-experimental_method +replacement I-experimental_method +anomalous I-experimental_method +scattering I-experimental_method +method I-experimental_method +( O +SIRAS B-experimental_method +) O +for O +successful O +solution O +of O +the O +apo B-protein_state +- O +SePSK B-protein +structure B-evidence +at O +a O +resolution O +of O +2 O +. O +3 O +Å O +. O +Subsequently O +, O +the O +apo B-protein_state +- O +SePSK B-protein +structure B-evidence +was O +used O +as O +molecular B-experimental_method +replacement I-experimental_method +model I-experimental_method +to O +solve O +all O +other O +structures B-evidence +identified O +in O +this O +study O +. O + +Our O +structural B-experimental_method +analysis I-experimental_method +showed O +that O +apo B-protein_state +- O +SePSK B-protein +consists O +of O +one O +SePSK B-protein +protein O +molecule O +in O +an O +asymmetric O +unit O +. O + +The O +amino O +- O +acid O +residues O +were O +traced O +from O +Val2 B-residue_name_number +to O +His419 B-residue_name_number +, O +except O +for O +the O +Met1 B-residue_name_number +residue O +and O +the O +seven O +residues O +at O +the O +C O +- O +termini O +. O + +Apo B-protein_state +- O +SePSK B-protein +contains O +two O +domains O +referred O +to O +further O +on O +as O +domain B-structure_element +I I-structure_element +and O +domain B-structure_element +II I-structure_element +( O +Fig O +1A O +). O + +Domain B-structure_element +I I-structure_element +consists O +of O +non O +- O +contiguous O +portions O +of O +the O +polypeptide O +chains O +( O +aa O +. O + +2 B-residue_range +– I-residue_range +228 I-residue_range +and O +aa O +. O + +402 B-residue_range +– I-residue_range +419 I-residue_range +), O +exhibiting O +11 O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +11 O +β B-structure_element +- I-structure_element +sheets I-structure_element +. O + +Among O +all O +these O +structural O +elements O +, O +α4 B-structure_element +/ O +α5 B-structure_element +/ O +α11 B-structure_element +/ O +α18 B-structure_element +, O +β3 B-structure_element +/ O +β2 B-structure_element +/ O +β1 B-structure_element +/ O +β6 B-structure_element +/ O +β19 B-structure_element +/ O +β20 B-structure_element +/ O +β17 B-structure_element +and O +α21 B-structure_element +/ O +α32 B-structure_element +form O +three O +patches O +, O +referred O +to O +as O +A1 B-structure_element +, O +B1 B-structure_element +and O +A2 B-structure_element +, O +exhibiting O +the O +core B-structure_element +region I-structure_element +. O + +In O +addition O +, O +four O +β B-structure_element +- I-structure_element +sheets I-structure_element +( O +β7 B-structure_element +, O +β10 B-structure_element +, O +β12 B-structure_element +and O +β16 B-structure_element +) O +and O +five O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +α8 B-structure_element +, O +α9 B-structure_element +, O +α13 B-structure_element +, O +α14 B-structure_element +and O +α15 B-structure_element +) O +flank O +the O +left O +side O +of O +the O +core B-structure_element +region I-structure_element +. O + +Domain B-structure_element +II I-structure_element +is O +comprised O +of O +aa O +. O + +229 B-residue_range +– I-residue_range +401 I-residue_range +and O +classified O +into O +B2 B-structure_element +( O +β31 B-structure_element +/ O +β29 B-structure_element +/ O +β22 B-structure_element +/ O +β23 B-structure_element +/ O +β25 B-structure_element +/ O +β24 B-structure_element +) O +and O +A3 B-structure_element +( O +α26 B-structure_element +/ O +α27 B-structure_element +/ O +α28 B-structure_element +/ O +α30 B-structure_element +) O +( O +Fig O +1A O +and O +S1 O +Fig O +). O + +In O +the O +SePSK B-protein +structure B-evidence +, O +B1 B-structure_element +and O +B2 B-structure_element +are O +sandwiched O +by O +A1 B-structure_element +, O +A2 B-structure_element +and O +A3 B-structure_element +, O +and O +the O +whole O +structure B-evidence +shows O +the O +A1 B-structure_element +/ O +B1 B-structure_element +/ O +A2 B-structure_element +/ O +B2 B-structure_element +/ O +A3 B-structure_element +( O +α B-structure_element +/ O +β B-structure_element +/ O +α B-structure_element +/ O +β B-structure_element +/ O +α B-structure_element +) O +folding O +pattern O +, O +which O +is O +in O +common O +with O +other O +members O +of O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +( O +S2 O +Fig O +). O + +The O +overall O +folding O +of O +SePSK B-protein +resembles O +a O +clip O +, O +with O +A2 B-structure_element +of O +domain B-structure_element +I I-structure_element +acting O +as O +a O +hinge B-structure_element +region I-structure_element +. O + +Overall O +structures B-evidence +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +. O + +( O +A O +) O +Three O +- O +dimensional O +structure B-evidence +of O +apo B-protein_state +- O +SePSK B-protein +. O + +The O +secondary O +structural O +elements O +are O +indicated O +( O +α B-structure_element +- I-structure_element +helix I-structure_element +: O +cyan O +, O +β B-structure_element +- I-structure_element +sheet I-structure_element +: O +yellow O +). O + +( O +B O +) O +Three O +- O +dimensional O +structure B-evidence +of O +apo B-protein_state +- O +AtXK B-protein +- I-protein +1 I-protein +. O + +The O +secondary O +structural O +elements O +are O +indicated O +( O +α B-structure_element +- I-structure_element +helix I-structure_element +: O +green O +, O +β B-structure_element +- I-structure_element +sheet I-structure_element +: O +wheat O +). O + +Apo B-protein_state +- O +AtXK B-protein +- I-protein +1 I-protein +exhibits O +a O +folding O +pattern O +similar O +to O +that O +of O +SePSK B-protein +in O +line O +with O +their O +high O +sequence O +identity O +( O +Fig O +1B O +and O +S1 O +Fig O +). O + +However O +, O +superposition B-experimental_method +of O +structures B-evidence +of O +AtXK B-protein +- I-protein +1 I-protein +and O +SePSK B-protein +shows O +some O +differences O +, O +especially O +at O +the O +loop B-structure_element +regions I-structure_element +. O + +A O +considerable O +difference O +is O +found O +in O +the O +loop3 B-structure_element +linking O +β3 B-structure_element +and O +α4 B-structure_element +, O +which O +is O +stretched O +out O +in O +the O +AtXK B-protein +- I-protein +1 I-protein +structure B-evidence +, O +while O +in O +the O +SePSK B-protein +structure B-evidence +, O +it O +is O +bent O +back O +towards O +the O +inner O +part O +. O + +The O +corresponding O +residues O +between O +these O +two O +structures B-evidence +( O +SePSK B-protein +- O +Lys35 B-residue_name_number +and O +AtXK B-protein +- I-protein +1 I-protein +- O +Lys48 B-residue_name_number +) O +have O +a O +distance O +of O +15 O +. O +4 O +Å O +( O +S3 O +Fig O +). O + +Activity B-experimental_method +assays I-experimental_method +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein + +In O +order O +to O +understand O +the O +function O +of O +these O +two O +kinases O +, O +we O +performed O +structural B-experimental_method +comparison I-experimental_method +using O +Dali B-experimental_method +server I-experimental_method +. O + +The O +structures B-evidence +most O +closely O +related O +to O +SePSK B-protein +are O +xylulose B-protein_type +kinase I-protein_type +, O +glycerol B-protein_type +kinase I-protein_type +and O +ribulose B-protein_type +kinase I-protein_type +, O +implying O +that O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +might O +function O +similarly O +to O +these O +kinases B-protein_type +. O + +We O +first O +tested O +whether O +both O +enzymes O +possessed O +ATP B-chemical +hydrolysis O +activity O +in O +the O +absence B-protein_state +of I-protein_state +substrates O +. O + +As O +shown O +in O +Fig O +2A O +, O +both O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +exhibited O +ATP B-chemical +hydrolysis O +activity O +. O + +This O +finding O +is O +in O +agreement O +with O +a O +previous O +result O +showing O +that O +xylulose B-protein_type +kinase I-protein_type +( O +PDB O +code O +: O +2ITM O +) O +possessed O +ATP B-chemical +hydrolysis O +activity O +without O +adding O +substrate O +. O + +To O +further O +identify O +the O +actual O +substrate O +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +, O +five O +different O +sugar O +molecules O +, O +including O +D B-chemical +- I-chemical +ribulose I-chemical +, O +L B-chemical +- I-chemical +ribulose I-chemical +, O +D B-chemical +- I-chemical +xylulose I-chemical +, O +L B-chemical +- I-chemical +xylulose I-chemical +and O +Glycerol B-chemical +, O +were O +used O +in O +enzymatic B-experimental_method +activity I-experimental_method +assays I-experimental_method +. O + +As O +shown O +in O +Fig O +2B O +, O +the O +ATP B-chemical +hydrolysis O +activity O +of O +SePSK B-protein +greatly O +increased O +upon O +adding O +D B-chemical +- I-chemical +ribulose I-chemical +than O +adding O +other O +potential O +substrates O +, O +suggesting O +that O +it O +has O +D B-protein_type +- I-protein_type +ribulose I-protein_type +kinase I-protein_type +activity O +. O + +In O +contrary O +, O +limited O +increasing O +of O +ATP B-chemical +hydrolysis O +activity O +was O +detected O +for O +AtXK B-protein +- I-protein +1 I-protein +upon O +addition O +of O +D B-chemical +- I-chemical +ribulose I-chemical +( O +Fig O +2C O +), O +despite O +its O +structural O +similarity O +with O +SePSK B-protein +. O + +The O +enzymatic B-experimental_method +activity I-experimental_method +assays I-experimental_method +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +. O + +( O +A O +) O +The O +ATP B-chemical +hydrolysis O +activity O +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +. O + +Both O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +showed O +ATP B-chemical +hydrolysis O +activity O +in O +the O +absence B-protein_state +of I-protein_state +substrate O +. O + +While O +the O +ATP B-chemical +hydrolysis O +activity O +of O +SePSK B-protein +greatly O +increases O +upon O +addition O +of O +D B-chemical +- I-chemical +ribulose I-chemical +( O +DR B-chemical +). O + +( O +B O +) O +The O +ATP B-chemical +hydrolysis O +activity O +of O +SePSK B-protein +with O +addition O +of O +five O +different O +substrates O +. O + +The O +substrates O +are O +DR B-chemical +( O +D B-chemical +- I-chemical +ribulose I-chemical +), O +LR B-chemical +( O +L B-chemical +- I-chemical +ribulose I-chemical +), O +DX B-chemical +( O +D B-chemical +- I-chemical +xylulose I-chemical +), O +LX B-chemical +( O +L B-chemical +- I-chemical +xylulose I-chemical +) O +and O +GLY B-chemical +( O +Glycerol B-chemical +). O +( O +C O +) O +The O +ATP B-chemical +hydrolysis O +activity O +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +with O +or O +without O +D B-chemical +- I-chemical +ribulose I-chemical +. O +( O +D O +) O +The O +ATP B-chemical +hydrolysis O +activity O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +and O +single O +- O +site O +mutants O +of O +SePSK B-protein +. O + +Three O +single O +- O +site O +mutants O +of O +SePSK B-protein +are O +D8A B-mutant +- O +SePSK B-protein +, O +T11A B-mutant +- O +SePSK B-protein +and O +D221A B-mutant +- O +SePSK B-protein +. O + +The O +ATP B-chemical +hydrolysis O +activity O +measured O +via O +luminescent B-experimental_method +ADP I-experimental_method +- I-experimental_method +Glo I-experimental_method +assay I-experimental_method +( O +Promega O +). O + +To O +understand O +the O +catalytic O +mechanism O +of O +SePSK B-protein +, O +we O +performed O +structural B-experimental_method +comparisons I-experimental_method +among O +xylulose B-protein_type +kinase I-protein_type +, O +glycerol B-protein_type +kinase I-protein_type +, O +ribulose B-protein_type +kinase I-protein_type +and O +SePSK B-protein +. O + +Our O +results O +suggested O +that O +three O +conserved O +residues O +( O +D8 B-residue_name_number +, O +T11 B-residue_name_number +and O +D221 B-residue_name_number +of O +SePSK B-protein +) O +play O +an O +important O +role O +in O +SePSK B-protein +function O +. O + +Mutations B-experimental_method +of O +the O +corresponding O +residue O +in O +xylulose B-protein_type +kinase I-protein_type +and O +glycerol B-protein_type +kinase I-protein_type +from O +Escherichia B-species +coli I-species +greatly O +reduced O +their O +activity O +. O + +To O +identify O +the O +function O +of O +these O +three O +residues O +of O +SePSK B-protein +, O +we O +constructed O +D8A B-mutant +, O +T11A B-mutant +and O +D221A B-mutant +mutants B-protein_state +. O + +Using O +enzymatic B-experimental_method +activity I-experimental_method +assays I-experimental_method +, O +we O +found O +that O +all O +of O +these O +mutants O +exhibit O +much O +lower O +activity O +of O +ATP B-chemical +hydrolysis O +after O +adding O +D B-chemical +- I-chemical +ribulose I-chemical +than O +that O +of O +wild B-protein_state +type I-protein_state +, O +indicating O +the O +possibility O +that O +these O +three O +residues O +are O +involved O +in O +the O +catalytic O +process O +of O +phosphorylation B-ptm +D B-chemical +- I-chemical +ribulose I-chemical +and O +are O +vital O +for O +the O +function O +of O +SePSK B-protein +( O +Fig O +2D O +). O + +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +possess O +a O +similar O +ATP B-site +binding I-site +site I-site + +To O +obtain O +more O +detailed O +information O +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +in B-protein_state +complex I-protein_state +with I-protein_state +ATP B-chemical +, O +we O +soaked B-experimental_method +the O +apo B-protein_state +- O +crystals B-evidence +in O +the O +reservoir O +adding O +cofactor O +ATP B-chemical +, O +and O +obtained O +the O +structures B-evidence +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +bound B-protein_state +with I-protein_state +ATP B-chemical +at O +the O +resolution O +of O +2 O +. O +3 O +Å O +and O +1 O +. O +8 O +Å O +, O +respectively O +. O + +In O +both O +structures B-evidence +, O +a O +strong O +electron B-evidence +density I-evidence +was O +found O +in O +the O +conserved B-protein_state +ATP B-site +binding I-site +pocket I-site +, O +but O +can O +only O +be O +fitted O +with O +an O +ADP B-chemical +molecule O +( O +S4 O +Fig O +). O + +Thus O +the O +two O +structures B-evidence +were O +named O +ADP B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +and O +ADP B-complex_assembly +- I-complex_assembly +AtXK I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +, O +respectively O +. O + +The O +extremely O +weak O +electron B-evidence +densities I-evidence +of O +ATP O +γ O +- O +phosphate B-chemical +in O +both O +structures B-evidence +suggest O +that O +the O +γ O +- O +phosphate B-chemical +group O +of O +ATP B-chemical +is O +either O +flexible O +or O +hydrolyzed O +by O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +. O + +This O +result O +was O +consistent O +with O +our O +enzymatic B-experimental_method +activity I-experimental_method +assays I-experimental_method +where O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +showed O +ATP B-chemical +hydrolysis O +activity O +without O +adding O +any O +substrates O +( O +Fig O +2A O +and O +2C O +). O + +To O +avoid O +hydrolysis O +of O +ATP B-chemical +, O +we O +soaked B-experimental_method +the O +crystals B-evidence +of O +apo B-protein_state +- O +SePSK B-protein +and O +apo B-protein_state +- O +AtXK B-protein +- I-protein +1 I-protein +into O +the O +reservoir O +adding O +AMP B-chemical +- I-chemical +PNP I-chemical +. O + +However O +, O +we O +found O +that O +the O +electron B-evidence +densities I-evidence +of O +γ O +- O +phosphate B-chemical +group O +of O +AMP B-chemical +- I-chemical +PNP I-chemical +( O +AMP B-chemical +- I-chemical +PNP I-chemical +γ O +- O +phosphate B-chemical +) O +are O +still O +weak O +in O +the O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +and O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +AtXK I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +structures B-evidence +, O +suggesting O +high O +flexibility O +of O +ATP B-chemical +- O +γ O +- O +phosphate B-chemical +. O + +The O +γ O +- O +phosphate B-chemical +group O +of O +ATP B-chemical +is O +transferred O +to O +the O +sugar B-chemical +substrate O +during O +the O +reaction O +process O +, O +so O +this O +flexibility O +might O +be O +important O +for O +the O +ability O +of O +these O +kinases B-protein_type +. O + +The O +overall O +structures B-evidence +as O +well O +as O +the O +coordination O +modes O +of O +ADP B-chemical +and O +AMP B-chemical +- I-chemical +PNP I-chemical +in O +the O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +AtXK I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +, O +ADP B-complex_assembly +- I-complex_assembly +AtXK I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +, O +ADP B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +and O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +structures B-evidence +are O +nearly O +identical O +( O +S5 O +Fig O +), O +therefore O +the O +structure B-evidence +of O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +is O +used O +here O +to O +describe O +the O +structural O +details O +and O +to O +compare O +with O +those O +of O +other O +family O +members O +. O + +As O +shown O +in O +Fig O +3A O +, O +one O +SePSK B-protein +protein O +molecule O +is O +in O +an O +asymmetric O +unit O +with O +one O +AMP B-chemical +- I-chemical +PNP I-chemical +molecule O +. O + +The O +AMP B-chemical +- I-chemical +PNP I-chemical +is O +bound O +at O +the O +domain B-structure_element +II I-structure_element +, O +where O +it O +fits O +well O +inside O +a O +positively B-site +charged I-site +groove I-site +. O + +The O +AMP B-site +- I-site +PNP I-site +binding I-site +pocket I-site +consists O +of O +four B-structure_element +α I-structure_element +- I-structure_element +helices I-structure_element +( O +α26 B-structure_element +, O +α28 B-structure_element +, O +α27 B-structure_element +and O +α30 B-structure_element +) O +and O +forms O +a O +shape B-protein_state +resembling I-protein_state +a I-protein_state +half I-protein_state +- I-protein_state +fist I-protein_state +( O +Fig O +3A O +and O +3B O +). O + +The O +head O +group O +of O +the O +AMP B-chemical +- I-chemical +PNP I-chemical +is O +embedded O +in O +a O +pocket B-site +surrounded O +by O +Trp383 B-residue_name_number +, O +Asn380 B-residue_name_number +, O +Gly376 B-residue_name_number +and O +Gly377 B-residue_name_number +. O + +The O +purine O +ring O +of O +AMP B-chemical +- I-chemical +PNP I-chemical +is O +positioned O +in O +parallel O +to O +the O +indole O +ring O +of O +Trp383 B-residue_name_number +. O + +In O +addition O +, O +it O +is O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +with O +the O +side O +chain O +amide O +of O +Asn380 B-residue_name_number +( O +Fig O +3B O +). O + +The O +tail O +of O +AMP B-chemical +- I-chemical +PNP I-chemical +points O +to O +the O +hinge B-structure_element +region I-structure_element +of O +SePSK B-protein +, O +and O +its O +α O +- O +phosphate B-chemical +and O +β O +- O +phosphate B-chemical +groups O +are O +stabilized O +by O +Gly376 B-residue_name_number +and O +Ser243 B-residue_name_number +, O +respectively O +. O + +Together O +, O +this O +structure B-evidence +clearly O +shows O +that O +the O +AMP B-chemical +- I-chemical +PNP I-chemical +- O +β O +- O +phosphate B-chemical +is O +sticking O +out O +of O +the O +ATP B-site +binding I-site +pocket I-site +, O +thus O +the O +γ O +- O +phosphate B-chemical +group O +is O +at O +the O +empty O +space O +between O +domain B-structure_element +I I-structure_element +and O +domain B-structure_element +II I-structure_element +and O +is O +unconstrained O +in O +its O +movement O +by O +the O +protein O +. O + +Structure B-evidence +of O +SePSK B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +AMP B-chemical +- I-chemical +PNP I-chemical +. O + +( O +A O +) O +The O +electron B-evidence +density I-evidence +of O +AMP B-chemical +- I-chemical +PNP I-chemical +. O + +The O +SePSK B-protein +structure B-evidence +is O +shown O +in O +the O +electrostatic O +potential O +surface O +mode O +. O + +The O +AMP B-chemical +- I-chemical +PNP I-chemical +is O +depicted O +as O +sticks O +with O +its O +ǀFoǀ B-evidence +- I-evidence +ǀFcǀ I-evidence +map I-evidence +contoured O +at O +3 O +σ O +shown O +as O +cyan O +mesh O +. O + +( O +B O +) O +The O +AMP B-site +- I-site +PNP I-site +binding I-site +pocket I-site +. O + +The O +head O +of O +AMP B-chemical +- I-chemical +PNP I-chemical +is O +sandwiched B-bond_interaction +by I-bond_interaction +four O +residues O +( O +Leu293 B-residue_name_number +, O +Gly376 B-residue_name_number +, O +Gly377 B-residue_name_number +and O +Trp383 B-residue_name_number +). O + +The O +four O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +α26 B-structure_element +, O +α28 B-structure_element +, O +α27 B-structure_element +and O +α30 B-structure_element +) O +are O +labeled O +in O +red O +. O + +The O +AMP B-chemical +- I-chemical +PNP I-chemical +and O +coordinated O +residues O +are O +shown O +as O +sticks O +. O + +The O +potential O +substrate B-site +binding I-site +site I-site +in O +SePSK B-protein + +The O +results O +from O +our O +activity B-experimental_method +assays I-experimental_method +suggested O +that O +SePSK B-protein +has O +D B-protein_type +- I-protein_type +ribulose I-protein_type +kinase I-protein_type +activity O +. O + +To O +better O +understand O +the O +interaction O +pattern O +between O +SePSK B-protein +and O +D B-chemical +- I-chemical +ribulose I-chemical +, O +the O +apo B-protein_state +- O +SePSK B-protein +crystals B-experimental_method +were I-experimental_method +soaked I-experimental_method +into I-experimental_method +the O +reservoir B-experimental_method +with O +10 O +mM O +D B-chemical +- I-chemical +ribulose I-chemical +( O +RBL B-chemical +) O +and O +the O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +structure B-evidence +was O +solved B-experimental_method +. O + +As O +shown O +in O +S6 O +Fig O +, O +two O +residual O +electron B-evidence +densities I-evidence +are O +visible O +in O +domain B-structure_element +I I-structure_element +, O +which O +can O +be O +interpreted O +as O +two O +D B-chemical +- I-chemical +ribulose I-chemical +molecules O +with O +reasonable O +fit O +. O + +As O +shown O +in O +Fig O +4A O +, O +the O +nearest O +distance O +between O +the O +carbon O +skeleton O +of O +two O +D B-chemical +- I-chemical +ribulose I-chemical +molecules O +are O +approx O +. O + +7 O +. O +1 O +Å O +( O +RBL1 B-residue_name_number +- O +C4 O +and O +RBL2 B-residue_name_number +- O +C1 O +). O + +RBL1 B-residue_name_number +is O +located O +in O +the O +pocket B-site +consisting O +of O +α21 B-structure_element +and O +the O +loop B-structure_element +between O +β6 B-structure_element +and I-structure_element +β7 I-structure_element +. O + +The O +O4 O +and O +O5 O +of O +RBL1 B-residue_name_number +are O +coordinated B-bond_interaction +with I-bond_interaction +the O +side O +chain O +carboxyl O +group O +of O +Asp221 B-residue_name_number +. O + +Furthermore O +, O +the O +O2 O +of O +RBL1 B-residue_name_number +interacts B-bond_interaction +with I-bond_interaction +the O +main O +chain O +amide O +nitrogen O +of O +Ser72 B-residue_name_number +( O +Fig O +4B O +). O + +This O +pocket B-site +is O +at O +a O +similar O +position O +of O +substrate B-site +binding I-site +site I-site +of O +other O +sugar B-protein_type +kinase I-protein_type +, O +such O +as O +L B-protein +- I-protein +ribulokinase I-protein +( O +PDB O +code O +: O +3QDK O +) O +( O +S7 O +Fig O +). O + +However O +, O +structural B-experimental_method +comparison I-experimental_method +shows O +that O +the O +substrate O +ligating O +residues O +between O +the O +two O +structures B-evidence +are O +not B-protein_state +strictly I-protein_state +conserved I-protein_state +. O + +Based O +on O +the O +structures B-evidence +, O +the O +ligating O +residues O +of O +RBL1 B-residue_name_number +in O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +structure B-evidence +are O +Ser72 B-residue_name_number +, O +Asp221 B-residue_name_number +and O +Ser222 B-residue_name_number +, O +and O +the O +interacting O +residues O +of O +L B-chemical +- I-chemical +ribulose I-chemical +with O +L B-protein +- I-protein +ribulokinase I-protein +are O +Ala96 B-residue_name_number +, O +Lys208 B-residue_name_number +, O +Asp274 B-residue_name_number +and O +Glu329 B-residue_name_number +( O +S7 O +Fig O +). O + +Glu329 B-residue_name_number +in O +3QDK O +has O +no O +counterpart O +in O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +structure B-evidence +. O + +In O +addition O +, O +although O +Lys208 B-residue_name_number +of O +L B-protein +- I-protein +ribulokinase I-protein +has O +the O +corresponding O +residue O +( O +Lys163 B-residue_name_number +) O +in O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +structure B-evidence +, O +the O +hydrogen B-bond_interaction +bond I-bond_interaction +of O +Lys163 B-residue_name_number +is O +broken O +because O +of O +the O +conformational O +change O +of O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +α9 B-structure_element +and O +α13 B-structure_element +) O +of O +SePSK B-protein +. O + +The O +binding O +of O +D B-chemical +- I-chemical +ribulose I-chemical +( O +RBL B-chemical +) O +with O +SePSK B-protein +. O + +( O +A O +) O +The O +electrostatic B-evidence +potential I-evidence +surface I-evidence +map I-evidence +of O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +and O +a O +zoom O +- O +in O +view O +of O +RBL B-site +binding I-site +site I-site +. O + +The O +RBL1 B-residue_name_number +and O +RBL2 B-residue_name_number +are O +depicted O +as O +sticks O +. O +( O +B O +) O +Interaction O +of O +two O +D B-chemical +- I-chemical +ribulose I-chemical +molecules O +( O +RBL1 B-residue_name_number +and O +RBL2 B-residue_name_number +) O +with O +SePSK B-protein +. O + +The O +RBL B-chemical +molecules O +( O +carbon O +atoms O +colored O +yellow O +) O +and O +amino O +acid O +residues O +of O +SePSK B-protein +( O +carbon O +atoms O +colored O +green O +) O +involved O +in O +RBL B-chemical +interaction O +are O +shown O +as O +sticks O +. O + +The O +hydrogen B-bond_interaction +bonds I-bond_interaction +are O +indicated O +by O +the O +black O +dashed O +lines O +and O +the O +numbers O +near O +the O +dashed O +lines O +are O +the O +distances O +( O +Å O +). O +( O +C O +) O +The O +binding B-experimental_method +affinity I-experimental_method +assays I-experimental_method +of O +SePSK B-protein +with O +D B-chemical +- I-chemical +ribulose I-chemical +. O + +Single B-experimental_method +- I-experimental_method +cycle I-experimental_method +kinetic I-experimental_method +data I-experimental_method +are O +reflecting O +the O +interaction O +of O +SePSK B-protein +and O +D8A B-mutant +- O +SePSK B-protein +with O +D B-chemical +- I-chemical +ribulose I-chemical +. O + +It O +shows O +two O +experimental O +sensorgrams B-evidence +after O +minus O +the O +empty O +sensorgrams B-evidence +. O + +The O +original O +data O +is O +shown O +as O +black O +curve O +, O +and O +the O +fitted O +data O +is O +shown O +as O +different O +color O +( O +wild B-protein_state +type I-protein_state +SePSK B-protein +: O +red O +curve O +, O +D8A B-mutant +- O +SePSK B-protein +: O +green O +curve O +). O + +Dissociation B-evidence +rate I-evidence +constant I-evidence +of O +wild B-protein_state +type I-protein_state +and O +D8A B-mutant +- O +SePSK B-protein +are O +3 O +ms O +- O +1 O +and O +9 O +ms O +- O +1 O +, O +respectively O +. O + +The O +binding B-site +pocket I-site +of O +RBL2 B-residue_name_number +with O +relatively O +weak O +electron B-evidence +density I-evidence +is O +near O +the O +N O +- O +terminal O +region O +of O +SePSK B-protein +and O +is O +negatively O +charged O +. O + +The O +side O +chain O +of O +Asp8 B-residue_name_number +interacts B-bond_interaction +strongly I-bond_interaction +with I-bond_interaction +O3 O +and O +O4 O +of O +RBL2 B-residue_name_number +. O + +The O +hydroxyl O +group O +of O +Ser12 B-residue_name_number +coordinates B-bond_interaction +with I-bond_interaction +O2 O +of O +RBL2 B-residue_name_number +. O + +The O +backbone O +amide O +nitrogens O +of O +Gly13 B-residue_name_number +and O +Arg15 B-residue_name_number +also O +keep O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +RBL2 B-residue_name_number +( O +Fig O +4B O +). O + +Structural B-experimental_method +comparison I-experimental_method +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +showed O +that O +while O +the O +RBL1 B-site +binding I-site +pocket I-site +is O +conserved B-protein_state +, O +the O +RBL2 B-site +pocket I-site +is O +disrupted O +in O +AtXK B-protein +- I-protein +1 I-protein +structure B-evidence +, O +despite O +the O +fact O +that O +the O +residues O +interacting O +with O +RBL2 B-residue_name_number +are O +highly B-protein_state +conserved I-protein_state +between O +the O +two O +proteins O +. O + +In O +the O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +structure B-evidence +, O +a O +2 O +. O +6 O +Å O +hydrogen B-bond_interaction +bond I-bond_interaction +is O +present O +between O +RBL2 B-residue_name_number +and O +Ser12 B-residue_name_number +( O +Fig O +4B O +), O +while O +in O +the O +AtXK B-protein +- I-protein +1 I-protein +structure B-evidence +this O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +corresponding O +residue O +( O +Ser22 B-residue_name_number +) O +is O +broken O +. O + +This O +break O +is O +probably O +induced O +by O +the O +conformational O +change O +of O +the O +two O +β B-structure_element +- I-structure_element +sheets I-structure_element +( O +β1 B-structure_element +and O +β2 B-structure_element +), O +with O +the O +result O +that O +the O +linking B-structure_element +loop I-structure_element +( O +loop B-structure_element +1 I-structure_element +) O +is O +located O +further O +away O +from O +the O +RBL2 B-site +binding I-site +site I-site +. O + +This O +change O +might O +be O +the O +reason O +that O +AtXK B-protein +- I-protein +1 I-protein +only O +shows O +limited O +increasing O +in O +its O +ATP B-chemical +hydrolysis O +ability O +upon O +adding O +D B-chemical +- I-chemical +ribulose I-chemical +as O +a O +substrate O +after O +comparing O +with O +SePSK B-protein +( O +Fig O +2C O +). O + +Our O +SePSK B-protein +structure B-evidence +shows O +that O +the O +Asp8 B-residue_name_number +residue O +forms O +strong O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +RBL2 B-residue_name_number +( O +Fig O +4B O +). O + +In O +addition O +, O +our O +enzymatic B-experimental_method +assays I-experimental_method +indicated O +that O +Asp8 B-residue_name_number +is O +important O +for O +the O +activity O +of O +SePSK B-protein +( O +Fig O +2D O +). O + +To O +further O +verified O +this O +result O +, O +we O +measured O +the O +binding B-evidence +affinity I-evidence +for O +D B-chemical +- I-chemical +ribulose I-chemical +of O +both O +wild B-protein_state +type I-protein_state +( O +WT B-protein_state +) O +and O +D8A B-mutant +mutant B-protein_state +of O +SePSK B-protein +using O +a O +surface B-experimental_method +plasmon I-experimental_method +resonance I-experimental_method +method I-experimental_method +. O + +The O +results O +showed O +that O +the O +affinity B-evidence +of O +D8A B-mutant +- O +SePSK B-protein +with O +D B-chemical +- I-chemical +ribulose I-chemical +is O +weaker O +than O +that O +of O +WT B-protein_state +with O +a O +reduction O +of O +approx O +. O + +Dissociation B-evidence +rate I-evidence +constant I-evidence +( O +Kd B-evidence +) O +of O +wild B-protein_state +type I-protein_state +and O +D8A B-mutant +- O +SePSK B-protein +are O +3 O +ms O +- O +1 O +and O +9 O +ms O +- O +1 O +, O +respectively O +. O + +The O +results O +implied O +that O +the O +second B-site +RBL I-site +binding I-site +site I-site +plays O +a O +role O +in O +the O +D B-protein_type +- I-protein_type +ribulose I-protein_type +kinase I-protein_type +function O +of O +SePSK B-protein +. O + +However O +, O +considering O +the O +high O +concentration O +of O +D B-chemical +- I-chemical +ribulose I-chemical +used O +for O +crystal B-experimental_method +soaking I-experimental_method +, O +as O +well O +as O +the O +relatively O +weak O +electron B-evidence +density I-evidence +of O +RBL2 B-residue_name_number +, O +it O +is O +also O +possible O +that O +the O +second B-site +binding I-site +site I-site +of O +D B-chemical +- I-chemical +ribulose I-chemical +in O +SePSK B-protein +is O +an O +artifact O +. O + +Simulated O +conformational O +change O +of O +SePSK B-protein +during O +the O +catalytic O +process O + +It O +was O +reported O +earlier O +that O +the O +crossing O +angle O +between O +the O +domain B-structure_element +I I-structure_element +and O +domain B-structure_element +II I-structure_element +in O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +is O +different O +. O + +In O +addition O +, O +this O +difference O +may O +be O +caused O +by O +the O +binding O +of O +substrates O +and O +/ O +or O +ATP B-chemical +. O + +As O +reported O +previously O +, O +members O +of O +the O +sugar B-protein_type +kinase I-protein_type +family O +undergo O +a O +conformational O +change O +to O +narrow O +the O +crossing O +angle O +between O +two O +domains O +and O +reduce O +the O +distance O +between O +substrate O +and O +ATP B-chemical +in O +order O +to O +facilitate O +the O +catalytic O +reaction O +of O +phosphorylation B-ptm +of O +sugar O +substrates O +. O + +After O +comparing O +structures B-evidence +of O +apo B-protein_state +- O +SePSK B-protein +, O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +and O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +, O +we O +noticed O +that O +these O +structures B-evidence +presented O +here O +are O +similar O +. O + +Superposing B-experimental_method +the O +structures B-evidence +of O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +and O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +, O +the O +results O +show O +that O +the O +nearest O +distance O +between O +AMP B-chemical +- I-chemical +PNP I-chemical +γ O +- O +phosphate B-chemical +and O +RBL1 B-residue_name_number +/ O +RBL2 B-residue_name_number +is O +7 O +. O +5 O +Å O +( O +RBL1 B-residue_name_number +- O +O5 O +)/ O +6 O +. O +7 O +Å O +( O +RBL2 B-residue_name_number +- O +O1 O +) O +( O +S8 O +Fig O +). O + +This O +distance O +is O +too O +long O +to O +transfer O +the O +γ O +- O +phosphate B-chemical +group O +from O +ATP B-chemical +to O +the O +substrate O +. O + +Since O +the O +two O +domains O +of O +SePSK B-protein +are O +widely O +separated O +in O +this O +structure B-evidence +, O +we O +hypothesize O +that O +our O +structures B-evidence +of O +SePSK B-protein +represent O +its O +open B-protein_state +form O +, O +and O +that O +a O +conformational O +rearrangement O +must O +occur O +to O +switch O +to O +the O +closed B-protein_state +state O +in O +order O +to O +facilitate O +the O +catalytic O +process O +of O +phosphorylation B-ptm +of O +sugar O +substrates O +. O + +For O +studying O +such O +potential O +conformational O +change O +, O +a O +simulation B-experimental_method +on O +the O +Hingeprot B-experimental_method +Server I-experimental_method +was O +performed O +to O +predict O +the O +movement O +of O +different O +SePSK B-protein +domains O +. O + +The O +results O +showed O +that O +domain B-structure_element +I I-structure_element +and O +domain B-structure_element +II I-structure_element +are O +closer O +to O +each O +other O +with O +Ala228 B-residue_name_number +and O +Thr401 B-residue_name_number +in O +A2 B-structure_element +as O +Hinge B-structure_element +- I-structure_element +residues I-structure_element +. O + +Based O +on O +the O +above O +results O +, O +SePSK B-protein +is O +divided O +into O +two O +rigid O +parts O +. O + +The O +domain B-structure_element +I I-structure_element +of O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +( O +aa O +. O +1 B-residue_range +– I-residue_range +228 I-residue_range +, O +aa O +. O +402 B-residue_range +– I-residue_range +421 I-residue_range +) O +and O +the O +domain B-structure_element +II I-structure_element +of O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +( O +aa O +. O +229 B-residue_range +– I-residue_range +401 I-residue_range +) O +were O +superposed B-experimental_method +with O +structures B-evidence +, O +including O +apo B-protein_state +- O +AtXK B-protein +- I-protein +1 I-protein +, O +apo B-protein_state +- O +SePSK B-protein +, O +xylulose B-protein_type +kinase I-protein_type +from O +Lactobacillus B-species +acidophilus I-species +( O +PDB O +code O +: O +3LL3 O +) O +and O +the O +S58W B-mutant +mutant B-protein_state +form O +of O +glycerol B-protein_type +kinase I-protein_type +from O +Escherichia B-species +coli I-species +( O +PDB O +code O +: O +1GLJ O +). O + +The O +results O +of O +superposition B-experimental_method +displayed O +different O +crossing O +angle O +between O +these O +two O +domains O +. O + +After O +superposition B-experimental_method +, O +the O +distances O +of O +AMP B-chemical +- I-chemical +PNP I-chemical +γ O +- O +phosphate B-chemical +and O +the O +fifth O +hydroxyl O +group O +of O +RBL1 B-residue_name_number +are O +7 O +. O +9 O +Å O +( O +superposed B-experimental_method +with O +AtXK B-protein +- I-protein +1 I-protein +), O +7 O +. O +4 O +Å O +( O +superposed B-experimental_method +with O +SePSK B-protein +), O +6 O +. O +6 O +Å O +( O +superposed B-experimental_method +with O +3LL3 O +) O +and O +6 O +. O +1 O +Å O +( O +superposed B-experimental_method +with O +1GLJ O +). O + +Meanwhile O +, O +the O +distances O +of O +AMP B-chemical +- I-chemical +PNP I-chemical +γ O +- O +phosphate B-chemical +and O +the O +first O +hydroxyl O +group O +of O +RBL2 B-residue_name_number +are O +7 O +. O +2 O +Å O +( O +superposed B-experimental_method +with O +AtXK B-protein +- I-protein +1 I-protein +), O +6 O +. O +7 O +Å O +( O +superposed B-experimental_method +with O +SePSK B-protein +), O +3 O +. O +7 O +Å O +( O +superposed B-experimental_method +with O +3LL3 O +), O +until O +AMP B-chemical +- I-chemical +PNP I-chemical +γ O +- O +phosphate B-chemical +fully O +contacts O +RBL2 B-residue_name_number +after O +superposition B-experimental_method +with O +1GLJ O +( O +Fig O +5 O +). O + +This O +distance O +between O +RBL2 B-residue_name_number +and O +AMP B-chemical +- I-chemical +PNP I-chemical +- O +γ O +- O +phosphate B-chemical +is O +close O +enough O +to O +facilitate O +phosphate B-chemical +transferring O +. O + +Together O +, O +our O +superposition B-experimental_method +results O +provided O +snapshots O +of O +the O +conformational O +changes O +at O +different O +catalytic O +stages O +of O +SePSK B-protein +and O +potentially O +revealed O +the O +closed B-protein_state +form O +of O +SePSK B-protein +. O + +Simulated O +conformational O +change O +of O +SePSK B-protein +during O +the O +catalytic O +process O +. O + +The O +structures B-evidence +are O +shown O +as O +cartoon O +and O +the O +ligands O +are O +shown O +as O +sticks O +. O + +Domain B-structure_element +I I-structure_element +from O +D B-complex_assembly +- I-complex_assembly +ribulose I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +( O +green O +) O +and O +Domain B-structure_element +II I-structure_element +from O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +( O +cyan O +) O +are O +superposed B-experimental_method +with O +apo B-protein_state +- O +AtXK B-protein +- I-protein +1 I-protein +( O +1st O +), O +apo B-protein_state +- O +SePSK B-protein +( O +2nd O +), O +3LL3 O +( O +3rd O +) O +and O +1GLJ O +( O +4th O +), O +respectively O +. O + +The O +numbers O +near O +the O +black O +dashed O +lines O +show O +the O +distances O +( O +Å O +) O +between O +two O +nearest O +atoms O +of O +RBL B-chemical +and O +AMP B-chemical +- I-chemical +PNP I-chemical +. O + +In O +summary O +, O +our O +structural B-experimental_method +and I-experimental_method +enzymatic I-experimental_method +analyses I-experimental_method +provide O +evidence O +that O +SePSK B-protein +shows O +D B-protein_type +- I-protein_type +ribulose I-protein_type +kinase I-protein_type +activity O +, O +and O +exhibits O +the O +conserved O +features O +of O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +. O + +Three O +conserved B-site +residues O +in O +SePSK B-protein +were O +identified O +to O +be O +essential O +for O +this O +function O +. O + +Our O +results O +provide O +the O +detailed O +information O +about O +the O +interaction O +of O +SePSK B-protein +with O +ATP B-chemical +and O +substrates O +. O + +Moreover O +, O +structural B-experimental_method +superposition I-experimental_method +results O +enable O +us O +to O +visualize O +the O +conformational O +change O +of O +SePSK B-protein +during O +the O +catalytic O +process O +. O + +In O +conclusion O +, O +our O +results O +provide O +important O +information O +for O +a O +more O +detailed O +understanding O +of O +the O +mechanisms O +of O +SePSK B-protein +and O +other O +members O +of O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +. O + +Structural O +insights O +into O +the O +Escherichia B-species +coli I-species +lysine B-protein_type +decarboxylases I-protein_type +and O +molecular O +determinants O +of O +interaction O +with O +the O +AAA B-protein_type ++ I-protein_type +ATPase I-protein_type +RavA B-protein + +The O +inducible B-protein_state +lysine B-protein_type +decarboxylase I-protein_type +LdcI B-protein +is O +an O +important O +enterobacterial B-taxonomy_domain +acid B-protein_type +stress I-protein_type +response I-protein_type +enzyme I-protein_type +whereas O +LdcC B-protein +is O +its O +close O +paralogue O +thought O +to O +play O +mainly O +a O +metabolic O +role O +. O + +A O +unique O +macromolecular O +cage O +formed O +by O +two O +decamers B-oligomeric_state +of O +the O +Escherichia B-species +coli I-species +LdcI B-protein +and O +five O +hexamers B-oligomeric_state +of O +the O +AAA B-protein_type ++ I-protein_type +ATPase I-protein_type +RavA B-protein +was O +shown O +to O +counteract O +acid O +stress O +under O +starvation O +. O + +Previously O +, O +we O +proposed O +a O +pseudoatomic B-evidence +model I-evidence +of O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +based O +on O +its O +cryo B-experimental_method +- I-experimental_method +electron I-experimental_method +microscopy I-experimental_method +map B-evidence +and O +crystal B-evidence +structures I-evidence +of O +an O +inactive B-protein_state +LdcI B-protein +decamer B-oligomeric_state +and O +a O +RavA B-protein +monomer B-oligomeric_state +. O + +We O +now O +present O +cryo B-experimental_method +- I-experimental_method +electron I-experimental_method +microscopy I-experimental_method +3D B-evidence +reconstructions I-evidence +of O +the O +E B-species +. I-species +coli I-species +LdcI B-protein +and O +LdcC B-protein +, O +and O +an O +improved B-evidence +map I-evidence +of O +the O +LdcI B-protein +bound B-protein_state +to I-protein_state +the O +LARA B-structure_element +domain I-structure_element +of O +RavA B-protein +, O +at O +pH B-protein_state +optimal I-protein_state +for O +their O +enzymatic O +activity O +. O + +Comparison B-experimental_method +with O +each O +other O +and O +with O +available O +structures B-evidence +uncovers O +differences O +between O +LdcI B-protein +and O +LdcC B-protein +explaining O +why O +only O +the O +acid B-protein_type +stress I-protein_type +response I-protein_type +enzyme I-protein_type +is O +capable O +of O +binding O +RavA B-protein +. O +We O +identify O +interdomain O +movements O +associated O +with O +the O +pH B-protein_state +- I-protein_state +dependent I-protein_state +enzyme O +activation O +and O +with O +the O +RavA B-protein +binding O +. O + +Multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +coupled O +to O +a O +phylogenetic B-experimental_method +analysis I-experimental_method +reveals O +that O +certain O +enterobacteria B-taxonomy_domain +exert O +evolutionary O +pressure O +on O +the O +lysine B-protein_type +decarboxylase I-protein_type +towards O +the O +cage O +- O +like O +assembly O +with O +RavA B-protein +, O +implying O +that O +this O +complex O +may O +have O +an O +important O +function O +under O +particular O +stress O +conditions O +. O + +Enterobacterial B-taxonomy_domain +inducible B-protein_state +decarboxylases B-protein_type +of O +basic B-protein_state +amino B-chemical +acids I-chemical +lysine B-residue_name +, O +arginine B-residue_name +and O +ornithine B-residue_name +have O +a O +common O +evolutionary O +origin O +and O +belong O +to O +the O +α B-protein_type +- I-protein_type +family I-protein_type +of O +pyridoxal B-chemical +- I-chemical +5 I-chemical +′- I-chemical +phosphate I-chemical +( O +PLP B-chemical +)- O +dependent O +enzymes O +. O + +They O +counteract O +acid O +stress O +experienced O +by O +the O +bacterium B-taxonomy_domain +in O +the O +host O +digestive O +and O +urinary O +tract O +, O +and O +in O +particular O +in O +the O +extremely O +acidic O +stomach O +. O + +Each O +decarboxylase B-protein_type +is O +induced O +by O +an O +excess O +of O +the O +target O +amino B-chemical +acid I-chemical +and O +a O +specific O +range O +of O +extracellular O +pH O +, O +and O +works O +in O +conjunction O +with O +a O +cognate O +inner B-protein_type +membrane I-protein_type +antiporter I-protein_type +. O + +Decarboxylation O +of O +the O +amino B-chemical +acid I-chemical +into O +a O +polyamine B-chemical +is O +catalysed O +by O +a O +PLP B-chemical +cofactor O +in O +a O +multistep O +reaction O +that O +consumes O +a O +cytoplasmic O +proton B-chemical +and O +produces O +a O +CO2 B-chemical +molecule O +passively O +diffusing O +out O +of O +the O +cell O +, O +while O +the O +polyamine B-chemical +is O +excreted O +by O +the O +antiporter B-protein_type +in O +exchange O +for O +a O +new O +amino B-chemical +acid I-chemical +substrate O +. O + +Consequently O +, O +these O +enzymes O +buffer O +both O +the O +bacterial B-taxonomy_domain +cytoplasm O +and O +the O +local O +extracellular O +environment O +. O + +These O +amino B-protein_type +acid I-protein_type +decarboxylases I-protein_type +are O +therefore O +called O +acid O +stress O +inducible B-protein_state +or O +biodegradative B-protein_state +to O +distinguish O +them O +from O +their O +biosynthetic B-protein_state +lysine B-protein_type +and I-protein_type +ornithine I-protein_type +decarboxylase I-protein_type +paralogs O +catalysing O +the O +same O +reaction O +but O +responsible O +for O +the O +polyamine B-chemical +production O +at O +neutral B-protein_state +pH I-protein_state +. O + +Inducible B-protein_state +enterobacterial B-taxonomy_domain +amino B-protein_type +acid I-protein_type +decarboxylases I-protein_type +have O +been O +intensively O +studied O +since O +the O +early O +1940 O +because O +the O +ability O +of O +bacteria B-taxonomy_domain +to O +withstand O +acid O +stress O +can O +be O +linked O +to O +their O +pathogenicity O +in O +humans B-species +. O + +In O +particular O +, O +the O +inducible B-protein_state +lysine B-protein_type +decarboxylase I-protein_type +LdcI B-protein +( O +or O +CadA B-protein +) O +attracts O +attention O +due O +to O +its O +broad B-protein_state +pH I-protein_state +range I-protein_state +of O +activity O +and O +its O +capacity O +to O +promote O +survival O +and O +growth O +of O +pathogenic O +enterobacteria B-taxonomy_domain +such O +as O +Salmonella B-species +enterica I-species +serovar I-species +Typhimurium I-species +, O +Vibrio B-species +cholerae I-species +and O +Vibrio B-species +vulnificus I-species +under O +acidic O +conditions O +. O + +Furthermore O +, O +both O +LdcI B-protein +and O +the O +biosynthetic B-protein_state +lysine B-protein_type +decarboxylase I-protein_type +LdcC B-protein +of O +uropathogenic B-species +Escherichia I-species +coli I-species +( O +UPEC B-species +) O +appear O +to O +play O +an O +important O +role O +in O +increased O +resistance O +of O +this O +pathogen O +to O +nitrosative O +stress O +produced O +by O +nitric B-chemical +oxide I-chemical +and O +other O +damaging O +reactive O +nitrogen O +intermediates O +accumulating O +during O +the O +course O +of O +urinary O +tract O +infections O +( O +UTI O +). O + +This O +effect O +is O +attributed O +to O +cadaverine B-chemical +, O +the O +diamine O +produced O +by O +decarboxylation O +of O +lysine B-residue_name +by O +LdcI B-protein +and O +LdcC B-protein +, O +that O +was O +shown O +to O +enhance O +UPEC B-species +colonisation O +of O +the O +bladder O +. O + +In O +addition O +, O +the O +biosynthetic B-protein_state +E B-species +. I-species +coli I-species +lysine B-protein_type +decarboxylase I-protein_type +LdcC B-protein +, O +long O +thought O +to O +be O +constitutively O +expressed O +in O +low O +amounts O +, O +was O +demonstrated O +to O +be O +strongly O +upregulated O +by O +fluoroquinolones B-chemical +via O +their O +induction O +of O +RpoS B-protein +. O +A O +direct O +correlation O +between O +the O +level O +of O +cadaverine B-chemical +and O +the O +resistance O +of O +E B-species +. I-species +coli I-species +to O +these O +antibiotics O +commonly O +used O +as O +a O +first O +- O +line O +treatment O +of O +UTI O +could O +be O +established O +. O + +Both O +acid B-protein_state +pH I-protein_state +and O +cadaverine B-chemical +induce O +closure O +of O +outer O +membrane O +porins B-protein_type +thereby O +contributing O +to O +bacterial B-taxonomy_domain +protection O +from O +acid O +stress O +, O +but O +also O +from O +certain O +antibiotics O +, O +by O +reduction O +in O +membrane O +permeability O +. O + +The O +crystal B-evidence +structure I-evidence +of O +the O +E B-species +. I-species +coli I-species +LdcI B-protein +as O +well O +as O +its O +low O +resolution O +characterisation O +by O +electron B-experimental_method +microscopy I-experimental_method +( O +EM B-experimental_method +) O +showed O +that O +it O +is O +a O +decamer B-oligomeric_state +made O +of O +two O +pentameric B-oligomeric_state +rings B-structure_element +. O + +Each O +monomer B-oligomeric_state +is O +composed O +of O +three O +domains O +– O +an O +N O +- O +terminal O +wing B-structure_element +domain I-structure_element +( O +residues O +1 B-residue_range +– I-residue_range +129 I-residue_range +), O +a O +PLP B-structure_element +- I-structure_element +binding I-structure_element +core I-structure_element +domain I-structure_element +( O +residues O +130 B-residue_range +– I-residue_range +563 I-residue_range +), O +and O +a O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +CTD B-structure_element +, O +residues O +564 B-residue_range +– I-residue_range +715 I-residue_range +). O + +Monomers B-oligomeric_state +tightly O +associate O +via O +their O +core B-structure_element +domains I-structure_element +into O +2 B-protein_state +- I-protein_state +fold I-protein_state +symmetrical I-protein_state +dimers B-oligomeric_state +with O +two O +complete O +active B-site +sites I-site +, O +and O +further O +build O +a O +toroidal B-structure_element +D5 I-structure_element +- I-structure_element +symmetrical I-structure_element +structure I-structure_element +held O +by O +the O +wing B-structure_element +and O +core B-structure_element +domain I-structure_element +interactions O +around O +the O +central B-structure_element +pore I-structure_element +, O +with O +the O +CTDs B-structure_element +at O +the O +periphery O +. O + +Ten O +years O +ago O +we O +showed O +that O +the O +E B-species +. I-species +coli I-species +AAA B-protein_type ++ I-protein_type +ATPase I-protein_type +RavA B-protein +, O +involved O +in O +multiple O +stress O +response O +pathways O +, O +tightly O +interacted O +with O +LdcI B-protein +but O +was O +not O +capable O +of O +binding O +to O +LdcC B-protein +. O +We O +described O +how O +two O +double O +pentameric B-oligomeric_state +rings B-structure_element +of O +the O +LdcI B-protein +tightly O +associate O +with O +five O +hexameric B-oligomeric_state +rings B-structure_element +of O +RavA B-protein +to O +form O +a O +unique O +cage O +- O +like O +architecture O +that O +enables O +the O +bacterium B-taxonomy_domain +to O +withstand O +acid O +stress O +even O +under O +conditions O +of O +nutrient O +deprivation O +eliciting O +stringent O +response O +. O + +Furthermore O +, O +we O +recently O +solved B-experimental_method +the I-experimental_method +structure I-experimental_method +of O +the O +E B-species +. I-species +coli I-species +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +complex O +by O +cryo B-experimental_method +- I-experimental_method +electron I-experimental_method +microscopy I-experimental_method +( O +cryoEM B-experimental_method +) O +and O +combined O +it O +with O +the O +crystal B-evidence +structures I-evidence +of O +the O +individual O +proteins O +. O + +This O +allowed O +us O +to O +make O +a O +pseudoatomic B-evidence +model I-evidence +of O +the O +whole O +assembly O +, O +underpinned O +by O +a O +cryoEM B-experimental_method +map B-evidence +of O +the O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +complex O +( O +with O +LARA B-structure_element +standing O +for O +LdcI B-structure_element +associating I-structure_element +domain I-structure_element +of I-structure_element +RavA I-structure_element +), O +and O +to O +identify O +conformational O +rearrangements O +and O +specific O +elements O +essential O +for O +complex O +formation O +. O + +The O +main O +determinants O +of O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +assembly O +appeared O +to O +be O +the O +N O +- O +terminal O +loop B-structure_element +of O +the O +LARA B-structure_element +domain I-structure_element +of O +RavA B-protein +and O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +LdcI B-protein +. O + +In O +spite O +of O +this O +wealth O +of O +structural B-evidence +information I-evidence +, O +the O +fact O +that O +LdcC B-protein +does O +not O +interact O +with O +RavA B-protein +, O +although O +the O +two O +lysine B-protein_type +decarboxylases I-protein_type +are O +69 O +% O +identical O +and O +84 O +% O +similar O +, O +and O +the O +physiological O +significance O +of O +the O +absence O +of O +this O +interaction O +remained O +unexplored O +. O + +To O +solve O +this O +discrepancy O +, O +in O +the O +present O +work O +we O +provided O +a O +three O +- O +dimensional O +( O +3D O +) O +cryoEM B-experimental_method +reconstruction B-evidence +of O +LdcC B-protein +and O +compared O +it O +with O +the O +available O +LdcI B-protein +and O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +structures B-evidence +. O + +Given O +that O +the O +LdcI B-protein +crystal B-evidence +structures I-evidence +were O +obtained O +at O +high B-protein_state +pH I-protein_state +where O +the O +enzyme O +is O +inactive B-protein_state +( O +LdcIi B-protein +, O +pH B-protein_state +8 I-protein_state +. I-protein_state +5 I-protein_state +), O +whereas O +the O +cryoEM B-experimental_method +reconstructions B-evidence +of O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +and O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +were O +done O +at O +acidic B-protein_state +pH I-protein_state +optimal I-protein_state +for O +the O +enzymatic O +activity O +, O +for O +a O +meaningful O +comparison O +, O +we O +also O +produced O +a O +3D B-evidence +reconstruction I-evidence +of O +the O +LdcI B-protein +at O +active B-protein_state +pH I-protein_state +( O +LdcIa B-protein +, O +pH B-protein_state +6 I-protein_state +. I-protein_state +2 I-protein_state +). O + +This O +comparison O +pinpointed O +differences O +between O +the O +biodegradative B-protein_state +and O +the O +biosynthetic B-protein_state +lysine B-protein_type +decarboxylases I-protein_type +and O +brought O +to O +light O +interdomain O +movements O +associated O +to O +pH B-protein_state +- I-protein_state +dependent I-protein_state +enzyme O +activation O +and O +RavA B-protein +binding O +, O +notably O +at O +the O +predicted O +RavA B-site +binding I-site +site I-site +at O +the O +level O +of O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +LdcI B-protein +. O +Consequently O +, O +we O +tested O +the O +capacity O +of O +cage O +formation O +by O +LdcI B-mutant +- I-mutant +LdcC I-mutant +chimeras I-mutant +where O +we O +interchanged B-experimental_method +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheets I-structure_element +in O +question O +. O + +Finally O +, O +we O +performed O +multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +22 O +lysine B-protein_type +decarboxylases I-protein_type +from O +Enterobacteriaceae B-taxonomy_domain +containing O +the O +ravA B-gene +- I-gene +viaA I-gene +operon I-gene +in O +their O +genome O +. O + +Remarkably O +, O +this O +analysis O +revealed O +that O +several O +specific B-structure_element +residues I-structure_element +in O +the O +above O +- O +mentioned O +β B-structure_element +- I-structure_element +sheet I-structure_element +, O +independently O +of O +the O +rest O +of O +the O +protein O +sequence O +, O +are O +sufficient O +to O +define O +if O +a O +particular O +lysine B-protein_type +decarboxylase I-protein_type +should O +be O +classified O +as O +an O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +or O +an O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +”. O + +This O +fascinating O +parallelism O +between O +the O +propensity O +for O +RavA B-protein +binding O +and O +the O +genetic O +environment O +of O +an O +enterobacterial B-taxonomy_domain +lysine B-protein_type +decarboxylase I-protein_type +, O +as O +well O +as O +the O +high B-protein_state +degree I-protein_state +of I-protein_state +conservation I-protein_state +of O +this O +small B-structure_element +structural I-structure_element +motif I-structure_element +, O +emphasize O +the O +functional O +importance O +of O +the O +interaction O +between O +biodegradative B-protein_state +enterobacterial B-taxonomy_domain +lysine B-protein_type +decarboxylases I-protein_type +and O +the O +AAA B-protein_type ++ I-protein_type +ATPase I-protein_type +RavA B-protein +. O + +CryoEM B-experimental_method +3D B-evidence +reconstructions I-evidence +of O +LdcC B-protein +, O +LdcIa B-protein +and O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly + +In O +the O +frame O +of O +this O +work O +, O +we O +produced O +two O +novel O +subnanometer O +resolution O +cryoEM B-experimental_method +reconstructions B-evidence +of O +the O +E B-species +. I-species +coli I-species +lysine B-protein_type +decarboxylases I-protein_type +at O +pH B-protein_state +optimal I-protein_state +for O +their O +enzymatic O +activity O +– O +a O +5 O +. O +5 O +Å O +resolution O +cryoEM B-experimental_method +map B-evidence +of O +the O +LdcC B-protein +( O +pH B-protein_state +7 I-protein_state +. I-protein_state +5 I-protein_state +) O +for O +which O +no O +3D O +structural O +information O +has O +been O +previously O +available O +( O +Figs O +1A O +, O +B O +and O +S1 O +), O +and O +a O +6 O +. O +1 O +Å O +resolution O +cryoEM B-experimental_method +map B-evidence +of O +the O +LdcIa B-protein +, O +( O +pH B-protein_state +6 I-protein_state +. I-protein_state +2 I-protein_state +) O +( O +Figs O +1C O +, O +D O +and O +S2 O +). O + +In O +addition O +, O +we O +improved O +our O +earlier O +cryoEM B-experimental_method +map B-evidence +of O +the O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +complex O +from O +7 O +. O +5 O +Å O +to O +6 O +. O +2 O +Å O +resolution O +( O +Figs O +1E O +, O +F O +and O +S3 O +). O + +Based O +on O +these O +reconstructions B-evidence +, O +reliable O +pseudoatomic B-evidence +models I-evidence +of O +the O +three O +assemblies O +were O +obtained O +by O +flexible B-experimental_method +fitting I-experimental_method +of I-experimental_method +either O +the O +crystal B-evidence +structure I-evidence +of O +LdcIi B-protein +or O +a O +derived O +structural B-experimental_method +homology I-experimental_method +model I-experimental_method +of O +LdcC B-protein +( O +Table O +S1 O +). O + +Significant O +differences O +between O +these O +pseudoatomic B-evidence +models I-evidence +can O +be O +interpreted O +as O +movements O +between O +specific O +biological O +states O +of O +the O +proteins O +as O +described O +below O +. O + +The O +wing B-structure_element +domains I-structure_element +as O +a O +stable O +anchor O +at O +the O +center O +of O +the O +double B-structure_element +- I-structure_element +ring I-structure_element + +As O +a O +first O +step O +of O +a O +comparative O +analysis O +, O +we O +superimposed B-experimental_method +the O +three O +cryoEM B-experimental_method +reconstructions B-evidence +( O +LdcIa B-protein +, O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +and O +LdcC B-protein +) O +and O +the O +crystal B-evidence +structure I-evidence +of O +the O +LdcIi B-protein +decamer B-oligomeric_state +( O +Fig O +. O +2 O +and O +Movie O +S1 O +). O + +This O +superposition B-experimental_method +reveals O +that O +the O +densities B-evidence +lining O +the O +central B-structure_element +hole I-structure_element +of O +the O +toroid B-structure_element +are O +roughly O +at O +the O +same O +location O +, O +while O +the O +rest O +of O +the O +structure B-evidence +exhibits O +noticeable O +changes O +. O + +Specifically O +, O +at O +the O +center O +of O +the O +double B-structure_element +- I-structure_element +ring I-structure_element +the O +wing B-structure_element +domains I-structure_element +of O +the O +subunits O +provide O +the O +conserved B-protein_state +basis O +for O +the O +assembly O +with O +the O +lowest B-evidence +root I-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +RMSD B-evidence +) O +( O +between O +1 O +. O +4 O +and O +2 O +Å O +for O +the O +Cα O +atoms O +only O +), O +whereas O +the O +peripheral O +CTDs B-structure_element +containing O +the O +RavA B-site +binding I-site +interface I-site +manifest O +the O +highest O +RMSD B-evidence +( O +up O +to O +4 O +. O +2 O +Å O +) O +( O +Table O +S2 O +). O + +In O +addition O +, O +the O +wing B-structure_element +domains I-structure_element +of O +all O +structures B-evidence +are O +very O +similar O +, O +with O +the O +RMSD B-evidence +after O +optimal O +rigid O +body O +alignment O +( O +RMSDmin B-evidence +) O +less O +than O +1 O +. O +1 O +Å O +. O +Thus O +, O +taking O +the O +limited O +resolution O +of O +the O +cryoEM B-experimental_method +maps B-evidence +into O +account O +, O +we O +consider O +that O +the O +wing B-structure_element +domains I-structure_element +of O +all O +the O +four O +structures B-evidence +are O +essentially O +identical O +and O +that O +in O +the O +present O +study O +the O +RMSD B-evidence +of O +less O +than O +2 O +Å O +can O +serve O +as O +a O +baseline O +below O +which O +differences O +may O +be O +assumed O +as O +insignificant O +. O + +This O +preservation O +of O +the O +central B-structure_element +part I-structure_element +of O +the O +double O +- O +ring O +assembly O +may O +help O +the O +enzymes O +to O +maintain O +their O +decameric B-oligomeric_state +state O +upon O +activation O +and O +incorporation O +into O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +. O + +The O +core B-structure_element +domain I-structure_element +and O +the O +active B-site +site I-site +rearrangements O +upon O +pH B-protein_state +- I-protein_state +dependent I-protein_state +enzyme O +activation O +and O +LARA O +binding O + +Both O +visual B-experimental_method +inspection I-experimental_method +( O +Fig O +. O +2 O +) O +and O +RMSD B-experimental_method +calculations I-experimental_method +( O +Table O +S2 O +) O +show O +that O +globally O +the O +three O +structures B-evidence +at O +active B-protein_state +pH I-protein_state +( O +LdcIa B-protein +, O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +and O +LdcC B-protein +) O +are O +more O +similar O +to O +each O +other O +than O +to O +the O +structure O +determined O +at O +high B-protein_state +pH I-protein_state +conditions O +( O +LdcIi B-protein +). O + +The O +decameric B-oligomeric_state +enzyme O +is O +built O +of O +five O +dimers B-oligomeric_state +associating O +into O +a O +5 B-structure_element +- I-structure_element +fold I-structure_element +symmetrical I-structure_element +double I-structure_element +- I-structure_element +ring I-structure_element +( O +two O +monomers B-oligomeric_state +making O +a O +dimer B-oligomeric_state +are O +delineated O +in O +Fig O +. O +1 O +). O + +As O +common O +for O +the O +α B-protein_type +family I-protein_type +of O +the O +PLP B-protein_type +- I-protein_type +dependent I-protein_type +decarboxylases I-protein_type +, O +dimerization O +is O +required O +for O +the O +enzymatic O +activity O +because O +the O +active B-site +site I-site +is O +buried O +in O +the O +dimer B-site +interface I-site +( O +Fig O +. O +3A O +, O +B O +). O + +This O +interface B-site +is O +formed O +essentially O +by O +the O +core B-structure_element +domains I-structure_element +with O +some O +contribution O +of O +the O +CTDs B-structure_element +. O + +The O +core B-structure_element +domain I-structure_element +is O +built O +by O +the O +PLP B-structure_element +- I-structure_element +binding I-structure_element +subdomain I-structure_element +( O +PLP B-structure_element +- I-structure_element +SD I-structure_element +, O +residues O +184 B-residue_range +– I-residue_range +417 I-residue_range +) O +flanked O +by O +two O +smaller O +subdomains B-structure_element +rich O +in O +partly B-protein_state +disordered I-protein_state +loops B-structure_element +– O +the O +linker B-structure_element +region I-structure_element +( O +residues O +130 B-residue_range +– I-residue_range +183 I-residue_range +) O +and O +the O +subdomain B-structure_element +4 I-structure_element +( O +residues O +418 B-residue_range +– I-residue_range +563 I-residue_range +). O + +Zooming O +in O +the O +variations O +in O +the O +PLP B-structure_element +- I-structure_element +SD I-structure_element +shows O +that O +most O +of O +the O +structural O +changes O +concern O +displacements O +in O +the O +active B-site +site I-site +( O +Fig O +. O +3C O +– O +F O +). O + +The O +most O +conspicuous O +differences O +between O +the O +PLP B-structure_element +- I-structure_element +SDs I-structure_element +can O +be O +linked O +to O +the O +pH B-protein_state +- I-protein_state +dependent I-protein_state +activation O +of O +the O +enzymes O +. O + +The O +resolution O +of O +the O +cryoEM B-experimental_method +maps B-evidence +does O +not O +allow O +modeling O +the O +position O +of O +the O +PLP B-chemical +moiety O +and O +calls O +for O +caution O +in O +detailed O +mechanistic O +interpretations O +in O +terms O +of O +individual O +amino B-chemical +acids I-chemical +. O + +In O +particular O +, O +transition O +from O +LdcIi B-protein +to O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +involves O +~ O +3 O +. O +5 O +Å O +and O +~ O +4 O +. O +5 O +Å O +shifts O +away O +from O +the O +5 O +- O +fold O +axis O +in O +the O +active B-site +site I-site +α B-structure_element +- I-structure_element +helices I-structure_element +spanning O +residues O +218 B-residue_range +– I-residue_range +232 I-residue_range +and O +246 B-residue_range +– I-residue_range +254 I-residue_range +respectively O +( O +Fig O +. O +3C O +– O +E O +). O + +Between O +these O +two O +extremes O +, O +the O +PLP B-structure_element +- I-structure_element +SDs I-structure_element +of O +LdcIa B-protein +and O +LdcC B-protein +are O +similar O +both O +in O +the O +context O +of O +the O +decamer B-oligomeric_state +( O +Fig O +. O +3F O +) O +and O +in O +terms O +of O +RMSDmin B-evidence += O +0 O +. O +9 O +Å O +, O +which O +probably O +reflects O +the O +fact O +that O +, O +at O +the O +optimal B-protein_state +pH I-protein_state +, O +these O +lysine B-protein_type +decarboxylases I-protein_type +have O +a O +similar O +enzymatic O +activity O +. O + +In O +addition O +, O +our O +earlier O +biochemical B-experimental_method +observation I-experimental_method +that O +the O +enzymatic O +activity O +of O +LdcIa B-protein +is O +unaffected O +by O +RavA B-protein +binding O +is O +consistent O +with O +the O +relatively O +small O +changes O +undergone O +by O +the O +active B-site +site I-site +upon O +transition O +from O +LdcIa B-protein +to O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +. O + +Worthy O +of O +note O +, O +our O +previous O +comparison O +of O +the O +crystal B-evidence +structure I-evidence +of O +LdcIi B-protein +with O +that O +of O +the O +inducible B-protein_state +arginine B-protein_type +decarboxylase I-protein_type +AdiA B-protein +revealed O +high B-protein_state +conservation I-protein_state +of O +the O +PLP B-site +- I-site +coordinating I-site +residues I-site +and O +identified O +a O +patch B-site +of I-site +negatively I-site +charged I-site +residues I-site +lining O +the O +active B-site +site I-site +channel I-site +as O +a O +potential O +binding B-site +site I-site +for O +the O +target O +amino B-chemical +acid I-chemical +substrate O +( O +Figs O +S3 O +and O +S4 O +in O +ref O +.). O + +Rearrangements O +of O +the O +ppGpp B-site +binding I-site +pocket I-site +upon O +pH B-protein_state +- I-protein_state +dependent I-protein_state +enzyme O +activation O +and O +LARA B-structure_element +binding O + +An O +inhibitor O +of O +the O +LdcI B-protein +and O +LdcC B-protein +activity O +, O +the O +stringent B-chemical +response I-chemical +alarmone I-chemical +ppGpp B-chemical +, O +is O +known O +to O +bind O +at O +the O +interface B-site +between O +neighboring O +monomers B-oligomeric_state +within O +each O +ring B-structure_element +( O +Fig O +. O +S4 O +). O + +The O +ppGpp B-site +binding I-site +pocket I-site +is O +made O +up O +by O +residues O +from O +all O +domains O +and O +is O +located O +approximately O +30 O +Å O +away O +from O +the O +PLP B-chemical +moiety O +. O + +Whereas O +the O +crystal B-evidence +structure I-evidence +of O +the O +ppGpp B-complex_assembly +- I-complex_assembly +LdcIi I-complex_assembly +was O +solved B-experimental_method +to O +2 O +Å O +resolution O +, O +only O +a O +4 O +. O +1 O +Å O +resolution O +structure B-evidence +of O +the O +ppGpp B-protein_state +- I-protein_state +free I-protein_state +LdcIi B-protein +could O +be O +obtained O +. O + +At O +this O +resolution O +, O +the O +apo B-protein_state +- O +LdcIi B-protein +and O +ppGpp B-complex_assembly +- I-complex_assembly +LdcIi I-complex_assembly +structures B-evidence +( O +both O +solved O +at O +pH B-protein_state +8 I-protein_state +. I-protein_state +5 I-protein_state +) O +appeared O +indistinguishable O +except O +for O +the O +presence O +of O +ppGpp B-chemical +( O +Fig O +. O +S11 O +in O +ref O +. O +). O + +Thus O +, O +we O +speculated O +that O +inhibition O +of O +LdcI B-protein +by O +ppGpp B-chemical +would O +be O +accompanied O +by O +a O +transduction O +of O +subtle O +structural O +changes O +at O +the O +level O +of O +individual O +amino B-chemical +acid I-chemical +side O +chains O +between O +the O +ppGpp B-site +binding I-site +pocket I-site +and O +the O +active B-site +site I-site +of O +the O +enzyme O +. O + +All O +our O +current O +cryoEM B-experimental_method +reconstructions B-evidence +of O +the O +lysine B-protein_type +decarboxylases I-protein_type +were O +obtained O +in O +the O +absence B-protein_state +of I-protein_state +ppGpp B-chemical +in O +order O +to O +be O +closer O +to O +the O +active B-protein_state +state O +of O +the O +enzymes O +under O +study O +. O + +While O +differences O +in O +the O +ppGpp B-site +binding I-site +site I-site +could O +indeed O +be O +visualized O +( O +Fig O +. O +S4 O +), O +the O +level O +of O +resolution O +warns O +against O +speculations O +about O +their O +significance O +. O + +The O +fact O +that O +interaction O +with O +RavA B-protein +reduces O +the O +ppGpp B-chemical +affinity O +for O +LdcI B-protein +despite O +the O +long O +distance O +of O +~ O +30 O +Å O +between O +the O +LARA B-site +domain I-site +binding I-site +site I-site +and O +the O +closest O +ppGpp B-site +binding I-site +pocket I-site +( O +Fig O +. O +S5 O +) O +seems O +to O +favor O +an O +allosteric O +regulation O +mechanism O +. O + +Interestingly O +, O +although O +a O +number O +of O +ppGpp B-site +binding I-site +residues I-site +are O +strictly B-protein_state +conserved I-protein_state +between O +LdcI B-protein +and O +AdiA B-protein +that O +also O +forms O +decamers B-oligomeric_state +at O +low B-protein_state +pH I-protein_state +optimal I-protein_state +for O +its O +arginine B-protein_type +decarboxylase I-protein_type +activity O +, O +no O +ppGpp B-chemical +regulation O +of O +AdiA B-protein +could O +be O +demonstrated O +. O + +Swinging O +and O +stretching O +of O +the O +CTDs B-structure_element +upon O +pH B-protein_state +- I-protein_state +dependent I-protein_state +LdcI B-protein +activation O +and O +LARA B-structure_element +binding O + +Inspection O +of O +the O +superimposed B-experimental_method +decameric B-oligomeric_state +structures B-evidence +( O +Figs O +2 O +and O +S6 O +) O +suggests O +a O +depiction O +of O +the O +wing B-structure_element +domains I-structure_element +as O +an O +anchor O +around O +which O +the O +peripheral O +CTDs B-structure_element +swing O +. O + +This O +swinging O +movement O +seems O +to O +be O +mediated O +by O +the O +core B-structure_element +domains I-structure_element +and O +is O +accompanied O +by O +a O +stretching O +of O +the O +whole O +LdcI B-protein +subunits B-structure_element +attracted O +by O +the O +RavA B-protein +magnets O +. O + +Indeed O +, O +all O +CTDs B-structure_element +have O +very O +similar O +structures O +( O +RMSDmin B-evidence +< O +1 O +Å O +). O + +Yet O +the O +superposition B-experimental_method +of O +the O +decamers B-oligomeric_state +lays O +bare O +a O +progressive O +movement O +of O +the O +CTD B-structure_element +as O +a O +whole O +upon O +enzyme O +activation O +by O +pH O +and O +the O +binding O +of O +LARA B-structure_element +. O + +The O +LdcIi B-protein +monomer B-oligomeric_state +is O +the O +most B-protein_state +compact I-protein_state +, O +whereas O +LdcIa B-protein +and O +especially O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +gradually B-protein_state +extend I-protein_state +their O +CTDs B-structure_element +towards O +the O +LARA B-structure_element +domain I-structure_element +of O +RavA B-protein +( O +Figs O +2 O +and O +4 O +). O + +These O +small O +but O +noticeable O +swinging O +and O +stretching O +( O +up O +to O +~ O +4 O +Å O +) O +may O +be O +related O +to O +the O +incorporation O +of O +the O +LdcI B-protein +decamer B-oligomeric_state +into O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +. O + +The O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +a O +lysine B-protein_type +decarboxylase I-protein_type +as O +a O +major O +determinant O +of O +the O +interaction O +with O +RavA B-protein + +In O +our O +previous O +contribution O +, O +based O +on O +the O +fit O +of O +the O +LdcIi B-protein +and O +the O +LARA B-structure_element +crystal B-evidence +structures I-evidence +into O +the O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +cryoEM B-experimental_method +density B-evidence +, O +we O +predicted O +that O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +interaction O +should O +involve O +the O +C O +- O +terminal O +two B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +of O +the O +LdcI B-protein +. O +Our O +present O +cryoEM B-experimental_method +maps B-evidence +and O +pseudoatomic B-evidence +models I-evidence +provide O +first O +structure O +- O +based O +insights O +into O +the O +differences O +between O +the O +inducible B-protein_state +and O +the O +constitutive B-protein_state +lysine B-protein_type +decarboxylases I-protein_type +. O + +Therefore O +, O +we O +wanted O +to O +check O +the O +influence O +of O +the O +primary O +sequence O +of O +the O +two O +proteins O +in O +this O +region O +on O +their O +ability O +to O +interact O +with O +RavA B-protein +. O +To O +this O +end O +, O +we O +swapped B-experimental_method +the O +relevant O +β B-structure_element +- I-structure_element +sheets I-structure_element +of O +the O +two O +proteins O +and O +produced O +their O +chimeras B-mutant +, O +namely O +LdcIC B-mutant +( O +i O +. O +e O +. O +LdcI B-protein +with O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +LdcC B-protein +) O +and O +LdcCI B-mutant +( O +i O +. O +e O +. O +LdcC B-protein +with O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +LdcI B-protein +) O +( O +Fig O +. O +5A O +– O +C O +). O + +Both B-mutant +constructs I-mutant +could O +be O +purified O +and O +could O +form O +decamers B-oligomeric_state +visually O +indistinguishable O +from O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +proteins O +. O + +As O +expected O +, O +binding O +of O +LdcI B-protein +to O +RavA B-protein +was O +completely O +abolished O +by O +this O +procedure O +and O +no O +LdcIC B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +complex O +could O +be O +detected O +. O + +On O +the O +contrary O +, O +introduction B-experimental_method +of O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +LdcI B-protein +into O +LdcC B-protein +led O +to O +an O +assembly O +of O +the O +LdcCI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +complex O +. O + +On O +the O +negative B-experimental_method +stain I-experimental_method +EM I-experimental_method +grid I-experimental_method +, O +the O +chimeric B-protein_state +cages O +appeared O +less O +rigid O +than O +the O +native B-protein_state +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +, O +which O +probably O +means O +that O +the O +environment O +of O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +contributes O +to O +the O +efficiency O +of O +the O +interaction O +and O +the O +stability O +of O +the O +entire O +architecture O +( O +Fig O +. O +5D O +– O +F O +). O + +The O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +a O +lysine B-protein_type +decarboxylase I-protein_type +is O +a O +highly B-protein_state +conserved I-protein_state +signature O +allowing O +to O +distinguish O +between O +LdcI B-protein +and O +LdcC B-protein + +Alignment B-experimental_method +of I-experimental_method +the I-experimental_method +primary I-experimental_method +sequences I-experimental_method +of O +the O +E B-species +. I-species +coli I-species +LdcI B-protein +and O +LdcC B-protein +shows O +that O +some O +amino O +acid O +residues O +of O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +are O +the O +same O +in O +the O +two O +proteins O +, O +whereas O +others O +are O +notably O +different O +in O +chemical O +nature O +. O + +Importantly O +, O +most O +of O +the O +amino O +acid O +differences O +between O +the O +two O +enzymes O +are O +located O +in O +this O +very B-structure_element +region I-structure_element +. O + +Thus O +, O +to O +advance O +beyond O +our O +experimental O +confirmation O +of O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +as O +a O +major O +determinant O +of O +the O +capacity O +of O +a O +particular O +lysine B-protein_type +decarboxylase I-protein_type +to O +form O +a O +cage O +with O +RavA B-protein +, O +we O +set O +out O +to O +investigate O +whether O +certain B-structure_element +residues I-structure_element +in O +this O +β B-structure_element +- I-structure_element +sheet I-structure_element +are O +conserved B-protein_state +in O +lysine B-protein_type +decarboxylases I-protein_type +of O +different O +enterobacteria B-taxonomy_domain +that O +have O +the O +ravA B-gene +- I-gene +viaA I-gene +operon I-gene +in O +their O +genome O +. O + +We O +inspected B-experimental_method +the I-experimental_method +genetic I-experimental_method +environment I-experimental_method +of O +lysine B-protein_type +decarboxylases I-protein_type +from O +22 O +enterobacterial B-taxonomy_domain +species O +referenced O +in O +the O +NCBI O +database O +, O +corrected O +the O +gene O +annotation O +where O +necessary O +( O +Tables O +S3 O +and O +S4 O +), O +and O +performed O +multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +coupled O +to O +a O +phylogenetic B-experimental_method +analysis I-experimental_method +( O +see O +Methods O +). O + +First O +of O +all O +, O +consensus B-evidence +sequence I-evidence +for O +the O +entire O +lysine B-protein_type +decarboxylase I-protein_type +family O +was O +derived O +. O + +Second O +, O +the O +phylogenetic B-experimental_method +analysis I-experimental_method +clearly O +split O +the O +lysine B-protein_type +decarboxylases I-protein_type +into O +two O +groups O +( O +Fig O +. O +6A O +). O + +All O +lysine B-protein_type +decarboxylases I-protein_type +predicted O +to O +be O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +or O +biodegradable B-protein_state +based O +on O +their O +genetic O +environment O +, O +as O +for O +example O +their O +organization O +in O +an O +operon O +with O +a O +gene O +encoding O +the O +CadB B-protein +antiporter B-protein_type +( O +see O +Methods O +), O +were O +found O +in O +one O +group O +, O +whereas O +all O +enzymes B-protein_type +predicted O +as O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +or O +biosynthetic B-protein_state +partitioned O +into O +another O +group O +. O + +Thus O +, O +consensus B-evidence +sequences I-evidence +could O +also O +be O +determined O +for O +each O +of O +the O +two O +groups O +( O +Figs O +6B O +, O +C O +and O +S7 O +). O + +Inspection O +of O +these O +consensus B-evidence +sequences I-evidence +revealed O +important O +differences O +between O +the O +groups O +regarding O +charge O +, O +size O +and O +hydrophobicity O +of O +several O +residues O +precisely O +at O +the O +level O +of O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +that O +is O +responsible O +for O +the O +interaction O +with O +RavA B-protein +( O +Fig O +. O +6B O +– O +D O +). O + +For O +example O +, O +in O +our O +previous O +study O +, O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutations I-experimental_method +identified O +Y697 B-residue_name_number +as O +critically O +required O +for O +the O +RavA B-protein +binding O +. O + +Our O +current O +analysis O +shows O +that O +Y697 B-residue_name_number +is O +strictly B-protein_state +conserved I-protein_state +in O +the O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +group O +whereas O +the O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +enzymes O +always B-protein_state +have I-protein_state +a O +lysine B-residue_name +in O +this O +position O +; O +it O +also O +uncovers O +several O +other O +residues O +potentially O +essential O +for O +the O +interaction O +with O +RavA B-protein +which O +can O +now O +be O +addressed O +by O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +. O + +The O +third O +and O +most O +remarkable O +finding O +was O +that O +exactly O +the O +same O +separation O +into O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +and O +“ O +LdcC B-protein_type +”- I-protein_type +like I-protein_type +groups O +can O +be O +obtained O +based O +on O +a O +comparison O +of O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheets I-structure_element +only O +, O +without O +taking O +the O +rest O +of O +the O +primary O +sequence O +into O +account O +. O + +Therefore O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +emerges O +as O +being O +a O +highly B-protein_state +conserved I-protein_state +signature B-structure_element +sequence I-structure_element +, O +sufficient O +to O +unambiguously O +discriminate O +between O +the O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +and O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +enterobacterial B-taxonomy_domain +lysine B-protein_type +decarboxylases I-protein_type +independently O +of O +any O +other O +information O +( O +Figs O +6 O +and O +S7 O +). O + +Our O +structures B-evidence +show O +that O +this B-structure_element +motif I-structure_element +is O +not O +involved O +in O +the O +enzymatic O +activity O +or O +the O +oligomeric O +state O +of O +the O +proteins O +. O + +Thus O +, O +enterobacteria B-taxonomy_domain +identified O +here O +( O +Fig O +. O +6 O +, O +Table O +S4 O +) O +appear O +to O +exert O +evolutionary O +pressure O +on O +the O +biodegradative B-protein_state +lysine B-protein_type +decarboxylase I-protein_type +towards O +the O +RavA B-protein +binding O +. O + +One O +of O +the O +elucidated O +roles O +of O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +is O +to O +maintain O +LdcI B-protein +activity O +under O +conditions O +of O +enterobacterial B-taxonomy_domain +starvation O +by O +preventing O +LdcI B-protein +inhibition O +by O +the O +stringent B-chemical +response I-chemical +alarmone I-chemical +ppGpp B-chemical +. O + +Furthermore O +, O +the O +recently O +documented O +interaction O +of O +both O +LdcI B-protein +and O +RavA B-protein +with O +specific O +subunits B-structure_element +of O +the O +respiratory B-protein_type +complex I-protein_type +I I-protein_type +, O +together O +with O +the O +unanticipated O +link O +between O +RavA B-protein +and O +maturation O +of O +numerous O +iron B-protein_type +- I-protein_type +sulfur I-protein_type +proteins I-protein_type +, O +tend O +to O +suggest O +an O +additional O +intriguing O +function O +for O +this O +3 O +. O +5 O +MDa O +assembly O +. O + +The O +conformational O +rearrangements O +of O +LdcI B-protein +upon O +enzyme O +activation O +and O +RavA B-protein +binding O +revealed O +in O +this O +work O +, O +and O +our O +amazing O +finding O +that O +the O +molecular O +determinant O +of O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +interaction O +is O +the O +one O +that O +straightforwardly O +determines O +if O +a O +particular O +enterobacterial B-taxonomy_domain +lysine B-protein_type +decarboxylase I-protein_type +belongs O +to O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +or O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +proteins O +, O +should O +give O +a O +new O +impetus O +to O +functional O +studies O +of O +the O +unique O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +. O + +Besides O +, O +the O +structures B-evidence +and O +the O +pseudoatomic B-evidence +models I-evidence +of O +the O +active B-protein_state +ppGpp B-protein_state +- I-protein_state +free I-protein_state +states O +of O +both O +the O +biodegradative B-protein_state +and O +the O +biosynthetic B-protein_state +E B-species +. I-species +coli I-species +lysine B-protein_type +decarboxylases I-protein_type +offer O +an O +additional O +tool O +for O +analysis O +of O +their O +role O +in O +UPEC B-species +infectivity O +. O + +Together O +with O +the O +apo B-protein_state +- O +LdcI B-protein +and O +ppGpp B-complex_assembly +- I-complex_assembly +LdcIi I-complex_assembly +crystal B-evidence +structures I-evidence +, O +our O +cryoEM B-experimental_method +reconstructions B-evidence +provide O +a O +structural O +framework O +for O +future O +studies O +of O +structure O +- O +function O +relationships O +of O +lysine B-protein_type +decarboxylases I-protein_type +from O +other O +enterobacteria B-taxonomy_domain +and O +even O +of O +their O +homologues O +outside O +Enterobacteriaceae B-taxonomy_domain +. O +For O +example O +, O +the O +lysine B-protein_type +decarboxylase I-protein_type +of O +Eikenella B-species +corrodens I-species +is O +thought O +to O +play O +a O +major O +role O +in O +the O +periodontal O +disease O +and O +its O +inhibitors O +were O +shown O +to O +retard O +gingivitis O +development O +. O + +Finally O +, O +cadaverine B-chemical +being O +an O +important O +platform O +chemical O +for O +the O +production O +of O +industrial O +polymers O +such O +as O +nylon O +, O +structural O +information O +is O +valuable O +for O +optimisation O +of O +bacterial B-taxonomy_domain +lysine B-protein_type +decarboxylases I-protein_type +used O +for O +its O +production O +in O +biotechnology O +. O + +3D O +cryoEM B-experimental_method +reconstructions B-evidence +of O +LdcC B-protein +, O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +and O +LdcIa B-protein +. O + +( O +A O +, O +C O +, O +E O +) O +cryoEM B-experimental_method +map B-evidence +of O +the O +LdcC B-protein +( O +A O +), O +LdcIa B-protein +( O +C O +) O +and O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +( O +E O +) O +decamers B-oligomeric_state +with O +one O +protomer B-oligomeric_state +in O +light O +grey O +. O + +In O +the O +rest O +of O +the O +protomers B-oligomeric_state +, O +the O +wing B-structure_element +, O +core B-structure_element +and O +C B-structure_element +- I-structure_element +terminal I-structure_element +domains I-structure_element +are O +colored O +from O +light O +to O +dark O +in O +shades O +of O +green O +for O +LdcC B-protein +( O +A O +), O +pink O +for O +LdcIa B-protein +( O +C O +) O +and O +blue O +for O +LdcI B-protein +in O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +( O +E O +). O + +In O +( O +E O +), O +the O +LARA B-structure_element +domain I-structure_element +density O +is O +shown O +in O +dark O +grey O +. O + +Two O +monomers B-oligomeric_state +making O +a O +dimer B-oligomeric_state +are O +delineated O +. O + +Scale O +bar O +50 O +Å O +. O +( O +B O +, O +D O +, O +F O +) O +One O +protomer B-oligomeric_state +from O +the O +cryoEM B-experimental_method +map B-evidence +of O +the O +LdcC B-protein +( O +B O +), O +LdcIa B-protein +( O +D O +) O +and O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +( O +F O +) O +in O +light O +grey O +with O +the O +pseudoatomic B-evidence +model I-evidence +represented O +as O +cartoons O +and O +colored O +as O +the O +densities O +in O +( O +A O +, O +C O +, O +E O +). O + +Superposition B-experimental_method +of O +the O +pseudoatomic B-evidence +models I-evidence +of O +LdcC B-protein +, O +LdcI B-protein +from O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +and O +LdcIa B-protein +colored O +as O +in O +Fig O +. O +1 O +, O +and O +the O +crystal B-evidence +structure I-evidence +of O +LdcIi B-protein +in O +shades O +of O +yellow O +. O + +Only O +one O +of O +the O +two O +rings B-structure_element +of O +the O +double B-structure_element +toroid I-structure_element +is O +shown O +for O +clarity O +. O + +The O +dashed O +circle O +indicates O +the O +central O +region B-structure_element +that O +remains O +virtually O +unchanged O +between O +all O +the O +structures B-evidence +, O +while O +the O +periphery O +undergoes O +visible O +movements O +. O + +Conformational O +rearrangements O +in O +the O +enzyme O +active B-site +site I-site +. O + +( O +A O +) O +LdcIi B-protein +crystal B-evidence +structure I-evidence +, O +with O +one O +ring B-structure_element +represented O +as O +a O +grey O +surface O +and O +the O +second O +as O +a O +cartoon O +. O + +A O +monomer B-oligomeric_state +with O +its O +PLP B-chemical +cofactor O +is O +delineated O +. O + +The O +PLP B-chemical +moieties O +of O +the O +cartoon O +ring B-structure_element +are O +shown O +in O +red O +. O + +( O +B O +) O +The O +LdcIi B-protein +dimer B-oligomeric_state +extracted O +from O +the O +crystal B-evidence +structure I-evidence +of O +the O +decamer B-oligomeric_state +. O + +One O +monomer B-oligomeric_state +is O +colored O +in O +shades O +of O +yellow O +as O +in O +Figs O +1 O +and O +2 O +, O +while O +the O +monomer B-oligomeric_state +related O +by O +C2 O +symmetry O +is O +grey O +. O + +The O +PLP B-chemical +is O +red O +. O + +The O +active B-site +site I-site +is O +boxed O +. O + +Stretching O +of O +the O +LdcI B-protein +monomer B-oligomeric_state +upon O +pH B-protein_state +- I-protein_state +dependent I-protein_state +enzyme O +activation O +and O +LARA B-structure_element +binding O +. O + +( O +A O +– O +C O +) O +A O +slice O +through O +the O +pseudoatomic B-evidence +models I-evidence +of O +the O +LdcI B-protein +monomers B-oligomeric_state +extracted O +from O +the O +superimposed B-experimental_method +decamers B-oligomeric_state +( O +Fig O +. O +2 O +) O +The O +rectangle O +indicates O +the O +regions O +enlarged O +in O +( O +D O +– O +F O +). O + +( O +A O +) O +compares O +LdcIi B-protein +( O +yellow O +) O +and O +LdcIa B-protein +( O +pink O +), O +( O +B O +) O +compares O +LdcIa B-protein +( O +pink O +) O +and O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +( O +blue O +), O +and O +( O +C O +) O +compares O +LdcIi B-protein +( O +yellow O +), O +LdcIa B-protein +( O +pink O +) O +and O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +( O +blue O +) O +simultaneously O +in O +order O +to O +show O +the O +progressive O +stretching O +described O +in O +the O +text O +. O + +The O +cryoEM B-experimental_method +density B-evidence +of O +the O +LARA B-structure_element +domain I-structure_element +is O +represented O +as O +a O +grey O +surface O +to O +show O +the O +position O +of O +the O +binding B-site +site I-site +and O +the O +direction O +of O +the O +movement O +. O + +( O +D O +– O +F O +) O +Inserts O +zooming O +at O +the O +CTD B-structure_element +part O +in O +proximity O +of O +the O +LARA B-site +binding I-site +site I-site +. O + +Analysis O +of O +the O +LdcIC B-mutant +and O +LdcCI B-mutant +chimeras B-mutant +. O + +( O +A O +) O +A O +slice O +through O +the O +pseudoatomic B-evidence +models I-evidence +of O +the O +LdcIa B-protein +( O +purple O +) O +and O +LdcC B-protein +( O +green O +) O +monomers B-oligomeric_state +extracted O +from O +the O +superimposed B-experimental_method +decamers B-oligomeric_state +( O +Fig O +. O +2 O +). O +( O +B O +) O +The O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +in O +LdcIa B-protein +and O +LdcC B-protein +enlarged O +from O +( O +A O +, O +C O +) O +Exchanged O +primary O +sequences O +( O +capital O +letters O +) O +and O +their O +immediate O +vicinity O +( O +lower O +case O +letters O +) O +colored O +as O +in O +( O +A O +, O +B O +), O +with O +the O +corresponding O +secondary O +structure O +elements O +and O +the O +amino O +acid O +numbering O +shown O +. O + +( O +D O +, O +E O +) O +A O +gallery O +of O +negative O +stain O +EM O +images O +of O +( O +D O +) O +the O +wild B-protein_state +type I-protein_state +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +and O +( O +E O +) O +the O +LdcCI B-mutant +- I-mutant +RavA I-mutant +cage I-mutant +- I-mutant +like I-mutant +particles I-mutant +. O +( O +F O +) O +Some O +representative O +class O +averages O +of O +the O +LdcCI B-mutant +- I-mutant +RavA I-mutant +cage I-mutant +- I-mutant +like I-mutant +particles I-mutant +. O + +Sequence B-experimental_method +analysis I-experimental_method +of O +enterobacterial B-taxonomy_domain +lysine B-protein_type +decarboxylases I-protein_type +. O + +( O +A O +) O +Maximum B-evidence +likelihood I-evidence +tree I-evidence +with O +the O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +and O +the O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +groups O +highlighted O +in O +green O +and O +pink O +, O +respectively O +. O + +( O +B O +) O +Analysis O +of O +consensus O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +and O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +sequences O +around O +the O +first O +and O +second O +C O +- O +terminal O +β B-structure_element +- I-structure_element +strands I-structure_element +. O + +Numbering O +as O +in O +E B-species +. I-species +coli I-species +. O + +( O +C O +) O +Signature O +sequences O +of O +LdcI B-protein +and O +LdcC B-protein +in O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +. O + +Polarity O +differences O +are O +highlighted O +. O +( O +D O +) O +Position O +and O +nature O +of O +these O +differences O +at O +the O +surface O +of O +the O +respective O +cryoEM B-experimental_method +maps B-evidence +with O +the O +color O +code O +as O +in O +B O +. O +See O +also O +Fig O +. O +S7 O +and O +Tables O +S3 O +and O +S4 O +. 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O + +Relative O +to O +the O +open B-protein_state +bacterial B-taxonomy_domain +ammonium B-protein_type +transporters I-protein_type +, O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +Mep2 B-protein_type +exhibits O +shifts O +in O +cytoplasmic B-structure_element +loops I-structure_element +and O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +( O +CTR B-structure_element +) O +to O +occlude O +the O +cytoplasmic O +exit B-site +of O +the O +channel B-site +and O +to O +interact O +with O +His2 B-residue_name_number +of O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +. O + +The O +phosphorylation B-site +site I-site +in O +the O +CTR B-structure_element +is O +solvent B-protein_state +accessible I-protein_state +and O +located O +in O +a O +negatively B-site +charged I-site +pocket I-site +∼ O +30 O +Å O +away O +from O +the O +channel B-site +exit I-site +. O + +The O +crystal B-evidence +structure I-evidence +of O +phosphorylation B-protein_state +- I-protein_state +mimicking I-protein_state +Mep2 B-mutant +variants I-mutant +from O +C B-species +. I-species +albicans I-species +show O +large O +conformational O +changes O +in O +a O +conserved B-protein_state +and O +functionally O +important O +region O +of O +the O +CTR B-structure_element +. O + +The O +results O +allow O +us O +to O +propose O +a O +model O +for O +regulation O +of O +eukaryotic B-taxonomy_domain +ammonium B-chemical +transport O +by O +phosphorylation B-ptm +. 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O + +One O +of O +the O +most O +important O +unresolved O +questions O +in O +the O +field O +is O +how O +the O +transceptors B-protein_type +couple O +to O +downstream O +signalling O +pathways O +. O + +One O +hypothesis O +is O +that O +downstream O +signalling O +is O +dependent O +on O +a O +specific O +conformation O +of O +the O +transporter B-protein_type +. O + +Mep2 B-protein_type +( B-protein_type +methylammonium I-protein_type +( I-protein_type +MA I-protein_type +) I-protein_type +permease I-protein_type +) I-protein_type +proteins I-protein_type +are O +ammonium B-protein_type +transceptors I-protein_type +that O +are O +ubiquitous O +in O +fungi B-taxonomy_domain +. O + +They O +belong O +to O +the O +Amt B-protein_type +/ I-protein_type +Mep I-protein_type +/ I-protein_type +Rh I-protein_type +family I-protein_type +of I-protein_type +transporters I-protein_type +that O +are O +present O +in O +all B-taxonomy_domain +kingdoms I-taxonomy_domain +of I-taxonomy_domain +life I-taxonomy_domain +and O +they O +take O +up O +ammonium B-chemical +from O +the O +extracellular O +environment O +. O + +Fungi B-taxonomy_domain +typically O +have O +more O +than O +one O +Mep B-protein_type +paralogue O +, O +for O +example O +, O +Mep1 B-protein +- I-protein +3 I-protein +in O +S B-species +. I-species +cerevisiae I-species +. O + +Of O +these O +, O +only O +Mep2 B-protein_type +proteins I-protein_type +function O +as O +ammonium B-chemical +receptors O +/ O +sensors O +in O +fungal B-taxonomy_domain +development O +. O + +Under O +conditions O +of O +nitrogen O +limitation O +, O +Mep2 B-protein +initiates O +a O +signalling O +cascade O +that O +results O +in O +a O +switch O +from O +the O +yeast O +form O +to O +filamentous O +( O +pseudohyphal O +) O +growth O +that O +may O +be O +required O +for O +fungal B-taxonomy_domain +pathogenicity O +. O + +As O +is O +the O +case O +for O +other O +transceptors B-protein_type +, O +it O +is O +not O +clear O +how O +Mep2 B-protein +interacts O +with O +downstream O +signalling O +partners O +, O +but O +the O +protein O +kinase O +A O +and O +mitogen O +- O +activated O +protein O +kinase O +pathways O +have O +been O +proposed O +as O +downstream O +effectors O +of O +Mep2 B-protein +( O +refs O +). O + +Compared O +with O +Mep1 B-protein +and O +Mep3 B-protein +, O +Mep2 B-protein +is O +highly B-protein_state +expressed I-protein_state +and O +functions O +as O +a O +low O +- O +capacity O +, O +high O +- O +affinity O +transporter O +in O +the O +uptake O +of O +MA B-chemical +. O + +In O +addition O +, O +Mep2 B-protein +is O +also O +important O +for O +uptake O +of O +ammonium B-chemical +produced O +by O +growth O +on O +other O +nitrogen B-chemical +sources O +. O + +With O +the O +exception O +of O +the O +human B-species +RhCG B-protein +structure B-evidence +, O +no O +structural O +information O +is O +available O +for O +eukaryotic B-taxonomy_domain +ammonium B-protein_type +transporters I-protein_type +. O + +By O +contrast O +, O +several O +bacterial B-taxonomy_domain +Amt B-protein_type +orthologues O +have O +been O +characterized O +in O +detail O +via O +high O +- O +resolution O +crystal B-evidence +structures I-evidence +and O +a O +number O +of O +molecular B-experimental_method +dynamics I-experimental_method +( O +MD B-experimental_method +) O +studies O +. O + +All O +the O +solved O +structures B-evidence +including O +that O +of O +RhCG B-protein +are O +very O +similar O +, O +establishing O +the O +basic O +architecture O +of O +ammonium B-protein_type +transporters I-protein_type +. O + +The O +proteins O +form O +stable B-protein_state +trimers B-oligomeric_state +, O +with O +each O +monomer B-oligomeric_state +having O +11 O +transmembrane B-structure_element +( O +TM B-structure_element +) O +helices B-structure_element +and O +a O +central B-site +channel I-site +for O +the O +transport O +of O +ammonium B-chemical +. 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O + +Given O +that O +the O +modest O +resolution O +of O +the O +structure B-evidence +and O +the O +limited O +detergent O +stability O +of O +ScMep2 B-protein +would O +likely O +complicate O +structure B-experimental_method +– I-experimental_method +function I-experimental_method +studies I-experimental_method +, O +several O +other O +fungal B-taxonomy_domain +Mep2 B-protein_type +orthologues O +were O +subsequently O +overexpressed B-experimental_method +and I-experimental_method +screened I-experimental_method +for I-experimental_method +diffraction O +- O +quality O +crystals B-evidence +. O + +Of O +these O +, O +Mep2 B-protein +from O +C B-species +. I-species +albicans I-species +( O +CaMep2 B-protein +) O +showed O +superior O +stability O +in O +relatively O +harsh O +detergents O +such O +as O +nonyl O +- O +glucoside O +, O +allowing O +structure B-experimental_method +determination I-experimental_method +in O +two O +different O +crystal B-evidence +forms I-evidence +to O +high O +resolution O +( O +up O +to O +1 O +. O +5 O +Å O +). O + +Despite O +different O +crystal O +packing O +( O +Supplementary O +Table O +1 O +), O +the O +two O +CaMep2 B-protein +structures B-evidence +are O +identical O +to O +each O +other O +and O +very O +similar O +to O +ScMep2 B-protein +( O +Cα O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +. I-evidence + +( O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +)= O +0 O +. O +7 O +Å O +for O +434 O +residues O +), O +with O +the O +main O +differences O +confined O +to O +the O +N O +terminus O +and O +the O +CTR B-structure_element +( O +Fig O +. O +1 O +). O + +Electron B-evidence +density I-evidence +is O +visible O +for O +the O +entire O +polypeptide O +chains O +, O +with O +the O +exception O +of O +the O +C O +- O +terminal O +43 B-residue_range +( O +ScMep2 B-protein +) O +and O +25 B-residue_range +residues O +( O +CaMep2 B-protein +), O +which O +are O +poorly B-protein_state +conserved I-protein_state +and O +presumably O +disordered B-protein_state +. O + +Both O +Mep2 B-protein_type +proteins I-protein_type +show O +the O +archetypal O +trimeric B-oligomeric_state +assemblies O +in O +which O +each O +monomer B-oligomeric_state +consists O +of O +11 O +TM B-structure_element +helices I-structure_element +surrounding O +a O +central B-structure_element +pore I-structure_element +. O + +Important O +functional O +features O +such O +as O +the O +extracellular O +ammonium B-site +binding I-site +site I-site +, O +the O +Phe B-site +gate I-site +and O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +within O +the O +hydrophobic B-site +channel I-site +are O +all O +very O +similar O +to O +those O +present O +in O +the O +bacterial B-taxonomy_domain +transporters B-protein_type +and O +RhCG B-protein +. O + +In O +the O +remainder O +of O +the O +manuscript O +, O +we O +will O +specifically O +discuss O +CaMep2 B-protein +due O +to O +the O +superior O +resolution O +of O +the O +structure B-evidence +. O + +Unless O +specifically O +stated O +, O +the O +drawn O +conclusions O +also O +apply O +to O +ScMep2 B-protein +. O + +While O +the O +overall O +architecture O +of O +Mep2 B-protein +is O +similar O +to O +that O +of O +the O +prokaryotic B-taxonomy_domain +transporters B-protein_type +( O +Cα O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +. I-evidence +with O +Amt B-protein +- I-protein +1 I-protein += O +1 O +. O +4 O +Å O +for O +361 O +residues O +), O +there O +are O +large O +differences O +within O +the O +N O +terminus O +, O +intracellular B-structure_element +loops I-structure_element +( O +ICLs B-structure_element +) O +ICL1 B-structure_element +and O +ICL3 B-structure_element +, O +and O +the O +CTR B-structure_element +. O + +The O +N O +termini O +of O +the O +Mep2 B-protein_type +proteins I-protein_type +are O +∼ O +20 B-residue_range +– I-residue_range +25 I-residue_range +residues O +longer O +compared O +with O +their O +bacterial B-taxonomy_domain +counterparts O +( O +Figs O +1 O +and O +2 O +), O +substantially O +increasing O +the O +size O +of O +the O +extracellular B-structure_element +domain I-structure_element +. O + +Moreover O +, O +the O +N O +terminus O +of O +one O +monomer B-oligomeric_state +interacts O +with O +the O +extended O +extracellular B-structure_element +loop I-structure_element +ECL5 B-structure_element +of O +a O +neighbouring O +monomer B-oligomeric_state +. O + +Together O +with O +additional O +, O +smaller O +differences O +in O +other O +extracellular B-structure_element +loops I-structure_element +, O +these O +changes O +generate O +a O +distinct O +vestibule B-structure_element +leading O +to O +the O +ammonium B-site +binding I-site +site I-site +that O +is O +much O +more O +pronounced O +than O +in O +the O +bacterial B-taxonomy_domain +proteins O +. O + +The O +N O +- O +terminal O +vestibule B-structure_element +and O +the O +resulting O +inter O +- O +monomer B-oligomeric_state +interactions O +likely O +increase O +the O +stability O +of O +the O +Mep2 B-protein +trimer B-oligomeric_state +, O +in O +support O +of O +data O +for O +plant B-taxonomy_domain +AMT B-protein_type +proteins I-protein_type +. O + +However O +, O +given O +that O +an O +N O +- O +terminal O +deletion B-protein_state +mutant I-protein_state +( O +2 B-mutant +- I-mutant +27Δ I-mutant +) O +grows O +as O +well O +as O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +Mep2 B-protein +on O +minimal O +ammonium B-chemical +medium O +( O +Fig O +. O +3 O +and O +Supplementary O +Fig O +. O +1 O +), O +the O +importance O +of O +the O +N O +terminus O +for O +Mep2 B-protein +activity O +is O +not O +clear O +. O + +Mep2 B-protein +channels B-site +are O +closed B-protein_state +by O +a O +two O +- O +tier O +channel B-structure_element +block I-structure_element + +The O +largest O +differences O +between O +the O +Mep2 B-protein +structures B-evidence +and O +the O +other O +known O +ammonium B-protein_type +transporter I-protein_type +structures B-evidence +are O +located O +on O +the O +intracellular O +side O +of O +the O +membrane O +. O + +In O +the O +vicinity O +of O +the O +Mep2 B-protein +channel B-site +exit I-site +, O +the O +cytoplasmic O +end O +of O +TM2 B-structure_element +has O +unwound O +, O +generating O +a O +longer O +ICL1 B-structure_element +even O +though O +there O +are O +no O +insertions O +in O +this O +region O +compared O +to O +the O +bacterial B-taxonomy_domain +proteins O +( O +Figs O +2 O +and O +4 O +). O + +ICL1 B-structure_element +has O +also O +moved O +inwards O +relative O +to O +its O +position O +in O +the O +bacterial B-taxonomy_domain +Amts B-protein_type +. O + +The O +largest O +backbone O +movements O +of O +equivalent O +residues O +within O +ICL1 B-structure_element +are O +∼ O +10 O +Å O +, O +markedly O +affecting O +the O +conserved B-protein_state +basic B-protein_state +RxK B-structure_element +motif I-structure_element +( O +Fig O +. O +4 O +). O + +The O +head O +group O +of O +Arg54 B-residue_name_number +has O +moved O +∼ O +11 O +Å O +relative O +to O +that O +in O +Amt B-protein +- I-protein +1 I-protein +, O +whereas O +the O +shift O +of O +the O +head O +group O +of O +the O +variable O +Lys55 B-residue_name_number +residue O +is O +almost O +20 O +Å O +. O +The O +side O +chain O +of O +Lys56 B-residue_name_number +in O +the O +basic B-protein_state +motif B-structure_element +points O +in O +an O +opposite O +direction O +in O +the O +Mep2 B-protein +structures B-evidence +compared O +with O +that O +of O +, O +for O +example O +, O +Amt B-protein +- I-protein +1 I-protein +( O +Fig O +. O +4 O +). O + +In O +addition O +to O +changing O +the O +RxK B-structure_element +motif I-structure_element +, O +the O +movement O +of O +ICL1 B-structure_element +has O +another O +, O +crucial O +functional O +consequence O +. O + +At O +the O +C O +- O +terminal O +end O +of O +TM1 B-structure_element +, O +the O +side O +- O +chain O +hydroxyl O +group O +of O +the O +relatively B-protein_state +conserved I-protein_state +Tyr49 B-residue_name_number +( O +Tyr53 B-residue_name_number +in O +ScMep2 B-protein +) O +makes O +a O +strong O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +ɛ2 O +nitrogen O +atom O +of O +the O +absolutely B-protein_state +conserved I-protein_state +His342 B-residue_name_number +of O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +( O +His348 B-residue_name_number +in O +ScMep2 B-protein +), O +closing O +the O +channel B-site +( O +Figs O +4 O +and O +5 O +). O + +In O +bacterial B-taxonomy_domain +Amt B-protein_type +proteins I-protein_type +, O +this O +Tyr B-residue_name +side O +chain O +is O +rotated O +∼ O +4 O +Å O +away O +as O +a O +result O +of O +the O +different O +conformation O +of O +TM1 B-structure_element +, O +leaving O +the O +channel B-site +open B-protein_state +and O +the O +histidine B-residue_name +available O +for O +its O +putative O +role O +in O +substrate O +transport O +( O +Supplementary O +Fig O +. O +2 O +). O + +Compared O +with O +ICL1 B-structure_element +, O +the O +backbone O +conformational O +changes O +observed O +for O +the O +neighbouring O +ICL2 B-structure_element +are O +smaller O +, O +but O +large O +shifts O +are O +nevertheless O +observed O +for O +the O +conserved B-protein_state +residues O +Glu140 B-residue_name_number +and O +Arg141 B-residue_name_number +( O +Fig O +. O +4 O +). O + +Finally O +, O +the O +important O +ICL3 B-structure_element +linking O +the O +pseudo B-structure_element +- I-structure_element +symmetrical I-structure_element +halves I-structure_element +( O +TM1 B-structure_element +- I-structure_element +5 I-structure_element +and O +TM6 B-structure_element +- I-structure_element +10 I-structure_element +) O +of O +the O +transporter B-protein_type +is O +also O +shifted O +up O +to O +∼ O +10 O +Å O +and O +forms O +an O +additional O +barrier O +that O +closes O +the O +channel B-site +on O +the O +cytoplasmic O +side O +( O +Fig O +. O +5 O +). O + +This O +two O +- O +tier O +channel B-structure_element +block I-structure_element +likely O +ensures O +that O +very O +little O +ammonium B-chemical +transport O +will O +take O +place O +under O +nitrogen B-chemical +- O +sufficient O +conditions O +. O + +The O +closed B-protein_state +state O +of O +the O +channel B-site +might O +also O +explain O +why O +no B-evidence +density I-evidence +, O +which O +could O +correspond O +to O +ammonium B-chemical +( O +or O +water B-chemical +), O +is O +observed O +in O +the O +hydrophobic O +part O +of O +the O +Mep2 B-protein +channel B-site +close O +to O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +. O + +Significantly O +, O +this O +is O +also O +true O +for O +ScMep2 B-protein +, O +which O +was O +crystallized B-experimental_method +in O +the O +presence O +of O +0 O +. O +2 O +M O +ammonium B-chemical +ions O +( O +see O +Methods O +section O +). O + +The O +final O +region O +in O +Mep2 B-protein +that O +shows O +large O +differences O +compared O +with O +the O +bacterial B-taxonomy_domain +transporters B-protein_type +is O +the O +CTR B-structure_element +. O + +In O +Mep2 B-protein +, O +the O +CTR B-structure_element +has O +moved O +away O +and O +makes O +relatively O +few O +contacts O +with O +the O +main B-structure_element +body I-structure_element +of O +the O +transporter B-protein_type +, O +generating O +a O +more O +elongated B-protein_state +protein O +( O +Figs O +1 O +and O +4 O +). O + +By O +contrast O +, O +in O +the O +structures B-evidence +of O +bacterial B-taxonomy_domain +proteins O +, O +the O +CTR B-structure_element +is O +docked O +tightly O +onto O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +the O +transporters B-protein_type +( O +corresponding O +to O +TM1 B-structure_element +- I-structure_element +5 I-structure_element +), O +resulting O +in O +a O +more O +compact B-protein_state +structure B-evidence +. O + +This O +is O +illustrated O +by O +the O +positions O +of O +the O +five O +universally B-protein_state +conserved I-protein_state +residues O +within O +the O +CTR B-structure_element +, O +that O +is O +, O +Arg415 B-residue_name_number +( O +370 B-residue_number +), O +Glu421 B-residue_name_number +( O +376 B-residue_number +), O +Gly424 B-residue_name_number +( O +379 B-residue_number +), O +Asp426 B-residue_name_number +( O +381 B-residue_number +) O +and O +Tyr B-residue_name_number +435 I-residue_name_number +( O +390 B-residue_number +) O +in O +CaMep2 B-protein +( O +Amt B-protein +- I-protein +1 I-protein +) O +( O +Fig O +. O +2 O +). O + +These O +residues O +include O +those O +of O +the O +‘ B-structure_element +ExxGxD I-structure_element +' I-structure_element +motif I-structure_element +, O +which O +when O +mutated B-experimental_method +generate O +inactive B-protein_state +transporters B-protein_type +. O + +In O +Amt B-protein +- I-protein +1 I-protein +and O +other O +bacterial B-taxonomy_domain +ammonium B-protein_type +transporters I-protein_type +, O +these O +CTR B-structure_element +residues O +interact O +with O +residues O +within O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +the O +protein O +. O + +On O +one O +side O +, O +the O +Tyr390 B-residue_name_number +hydroxyl O +in O +Amt B-protein +- I-protein +1 I-protein +is O +hydrogen B-bond_interaction +bonded I-bond_interaction +with O +the O +side O +chain O +of O +the O +conserved B-protein_state +His185 B-residue_name_number +at O +the O +C O +- O +terminal O +end O +of O +loop B-structure_element +ICL3 B-structure_element +. O + +At O +the O +other O +end O +of O +ICL3 B-structure_element +, O +the O +backbone O +carbonyl O +groups O +of O +Gly172 B-residue_name_number +and O +Lys173 B-residue_name_number +are O +hydrogen B-bond_interaction +bonded I-bond_interaction +to O +the O +side O +chain O +of O +Arg370 B-residue_name_number +. O + +Similar O +interactions O +were O +also O +modelled B-experimental_method +in O +the O +active B-protein_state +, O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +plant B-taxonomy_domain +AtAmt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +structure B-evidence +( O +for O +example O +, O +Y467 B-residue_name_number +- O +H239 B-residue_name_number +and O +D458 B-residue_name_number +- O +K71 B-residue_name_number +). O + +The O +result O +of O +these O +interactions O +is O +that O +the O +CTR B-structure_element +‘ O +hugs O +' O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +the O +transporters B-protein_type +( O +Fig O +. O +4 O +). O + +Also O +noteworthy O +is O +Asp381 B-residue_name_number +, O +the O +side O +chain O +of O +which O +interacts O +strongly O +with O +the O +positive O +dipole O +on O +the O +N O +- O +terminal O +end O +of O +TM2 B-structure_element +. O + +This O +interaction O +in O +the O +centre O +of O +the O +protein O +may O +be O +particularly O +important O +to O +stabilize O +the O +open B-protein_state +conformations O +of O +ammonium B-protein_type +transporters I-protein_type +. O + +In O +the O +Mep2 B-protein +structures B-evidence +, O +none O +of O +the O +interactions O +mentioned O +above O +are O +present O +. O + +Phosphorylation B-site +target I-site +site I-site +is O +at O +the O +periphery O +of O +Mep2 B-protein + +Recently O +Boeckstaens O +et O +al O +. O +provided O +evidence O +that O +Ser457 B-residue_name_number +in O +ScMep2 B-protein +( O +corresponding O +to O +Ser453 B-residue_name_number +in O +CaMep2 B-protein +) O +is O +phosphorylated B-protein_state +by O +the O +TORC1 B-protein_type +effector I-protein_type +kinase I-protein_type +Npr1 B-protein +under O +nitrogen B-chemical +- O +limiting O +conditions O +. O + +In O +the O +absence B-protein_state +of I-protein_state +Npr1 B-protein +, O +plasmid B-experimental_method +- I-experimental_method +encoded I-experimental_method +WT B-protein_state +Mep2 B-protein +in O +a O +S B-species +. I-species +cerevisiae I-species +mep1 B-mutant +- I-mutant +3Δ I-mutant +strain O +( O +triple B-mutant +mepΔ I-mutant +) O +does O +not O +allow O +growth O +on O +low O +concentrations O +of O +ammonium B-chemical +, O +suggesting O +that O +the O +transporter B-protein_type +is O +inactive B-protein_state +( O +Fig O +. O +3 O +and O +Supplementary O +Fig O +. O +1 O +). O + +Conversely O +, O +the O +phosphorylation B-protein_state +- I-protein_state +mimicking I-protein_state +S457D B-mutant +variant O +is O +active B-protein_state +both O +in O +the O +triple B-mutant +mepΔ I-mutant +background O +and O +in O +a O +triple B-mutant +mepΔ I-mutant +npr1Δ I-mutant +strain O +( O +Fig O +. O +3 O +). O + +Mutation B-experimental_method +of O +other O +potential O +phosphorylation B-site +sites I-site +in O +the O +CTR B-structure_element +did O +not O +support O +growth O +in O +the O +npr1Δ B-mutant +background O +. O + +Collectively O +, O +these O +data O +suggest O +that O +phosphorylation B-ptm +of O +Ser457 B-residue_name_number +opens O +the O +Mep2 B-protein +channel B-site +to O +allow O +ammonium B-chemical +uptake O +. O + +Ser457 B-residue_name_number +is O +located O +in O +a O +part O +of O +the O +CTR B-structure_element +that O +is O +conserved B-protein_state +in O +a O +subgroup O +of O +Mep2 B-protein_type +proteins I-protein_type +, O +but O +which O +is O +not O +present O +in O +bacterial B-taxonomy_domain +proteins O +( O +Fig O +. O +2 O +). O + +This O +segment B-structure_element +( O +residues O +450 B-residue_range +– I-residue_range +457 I-residue_range +in O +ScMep2 B-protein +and O +446 B-residue_range +– I-residue_range +453 I-residue_range +in O +CaMep2 B-protein +) O +was O +dubbed O +an O +autoinhibitory B-structure_element +( I-structure_element +AI I-structure_element +) I-structure_element +region I-structure_element +based O +on O +the O +fact O +that O +its O +removal B-experimental_method +generates O +an O +active B-protein_state +transporter B-protein_type +in O +the O +absence B-protein_state +of I-protein_state +Npr1 B-protein +( O +Fig O +. O +3 O +). O + +Where O +is O +the O +AI B-structure_element +region I-structure_element +and O +the O +Npr1 B-protein +phosphorylation B-site +site I-site +located O +? O +Our O +structures B-evidence +reveal O +that O +surprisingly O +, O +the O +AI B-structure_element +region I-structure_element +is O +folded O +back O +onto O +the O +CTR B-structure_element +and O +is O +not O +located O +near O +the O +centre O +of O +the O +trimer B-oligomeric_state +as O +expected O +from O +the O +bacterial B-taxonomy_domain +structures B-evidence +( O +Fig O +. O +4 O +). O + +The O +AI B-structure_element +region I-structure_element +packs O +against O +the O +cytoplasmic O +ends O +of O +TM2 B-structure_element +and O +TM4 B-structure_element +, O +physically O +linking O +the O +main B-structure_element +body I-structure_element +of O +the O +transporter B-protein_type +with O +the O +CTR B-structure_element +via O +main O +chain O +interactions O +and O +side O +- O +chain O +interactions O +of O +Val447 B-residue_name_number +, O +Asp449 B-residue_name_number +, O +Pro450 B-residue_name_number +and O +Arg452 B-residue_name_number +( O +Fig O +. O +6 O +). O + +The O +AI B-structure_element +regions I-structure_element +have O +very O +similar O +conformations O +in O +CaMep2 B-protein +and O +ScMep2 B-protein +, O +despite O +considerable O +differences O +in O +the O +rest O +of O +the O +CTR B-structure_element +( O +Fig O +. O +6 O +). O + +Strikingly O +, O +the O +Npr1 B-site +target I-site +serine I-site +residue O +is O +located O +at O +the O +periphery O +of O +the O +trimer B-oligomeric_state +, O +far O +away O +(∼ O +30 O +Å O +) O +from O +any O +channel B-site +exit I-site +( O +Fig O +. O +6 O +). O + +Despite O +its O +location O +at O +the O +periphery O +of O +the O +trimer B-oligomeric_state +, O +the O +electron B-evidence +density I-evidence +for O +the O +serine B-residue_name +is O +well O +defined O +in O +both O +Mep2 B-protein +structures B-evidence +and O +corresponds O +to O +the O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +state O +( O +Fig O +. O +6 O +). O + +This O +makes O +sense O +since O +the O +proteins O +were O +expressed O +in O +rich O +medium O +and O +confirms O +the O +recent O +suggestion O +by O +Boeckstaens O +et O +al O +. O +that O +the O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +form O +of O +Mep2 B-protein +corresponds O +to O +the O +inactive B-protein_state +state O +. O + +For O +ScMep2 B-protein +, O +Ser457 B-residue_name_number +is O +the O +most O +C O +- O +terminal O +residue O +for O +which O +electron B-evidence +density I-evidence +is O +visible O +, O +indicating O +that O +the O +region O +beyond O +Ser457 B-residue_name_number +is O +disordered B-protein_state +. O + +In O +CaMep2 B-protein +, O +the O +visible O +part O +of O +the O +sequence O +extends O +for O +two O +residues O +beyond O +Ser453 B-residue_name_number +( O +Fig O +. O +6 O +). O + +The O +peripheral O +location O +and O +disorder B-protein_state +of O +the O +CTR B-structure_element +beyond O +the O +kinase B-site +target I-site +site I-site +should O +facilitate O +the O +phosphorylation B-ptm +by O +Npr1 B-protein +. O + +The O +disordered B-protein_state +part O +of O +the O +CTR B-structure_element +is O +not B-protein_state +conserved I-protein_state +in O +ammonium B-protein_type +transporters I-protein_type +( O +Fig O +. O +2 O +), O +suggesting O +that O +it O +is O +not O +important O +for O +transport O +. O + +Interestingly O +, O +a O +ScMep2 B-protein +457Δ B-mutant +truncation B-protein_state +mutant I-protein_state +in O +which O +a O +His O +- O +tag O +directly O +follows O +Ser457 B-residue_name_number +is O +highly O +expressed O +but O +has O +low B-protein_state +activity I-protein_state +( O +Fig O +. O +3 O +and O +Supplementary O +Fig O +. O +1b O +), O +suggesting O +that O +the O +His O +- O +tag O +interferes O +with O +phosphorylation B-ptm +by O +Npr1 B-protein +. O + +The O +same O +mutant B-mutant +lacking B-protein_state +the I-protein_state +His I-protein_state +- I-protein_state +tag I-protein_state +has O +WT B-protein_state +properties O +( O +Supplementary O +Fig O +. O +1b O +), O +confirming O +that O +the O +region O +following O +the O +phosphorylation B-site +site I-site +is O +dispensable O +for O +function O +. O + +Mep2 B-protein +lacking B-protein_state +the O +AI B-structure_element +region I-structure_element +is O +conformationally B-protein_state +heterogeneous I-protein_state + +Given O +that O +Ser457 B-residue_name_number +/ O +453 B-residue_number +is O +far O +from O +any O +channel B-site +exit I-site +( O +Fig O +. O +6 O +), O +the O +crucial O +question O +is O +how O +phosphorylation B-ptm +opens O +the O +Mep2 B-protein +channel B-site +to O +generate O +an O +active B-protein_state +transporter B-protein_type +. O + +Boeckstaens O +et O +al O +. O +proposed O +that O +phosphorylation B-ptm +does O +not O +affect O +channel O +activity O +directly O +, O +but O +instead O +relieves O +inhibition O +by O +the O +AI B-structure_element +region I-structure_element +. O + +The O +data O +behind O +this O +hypothesis O +is O +the O +observation O +that O +a O +ScMep2 B-protein +449 B-mutant +- I-mutant +485Δ I-mutant +deletion B-protein_state +mutant I-protein_state +lacking B-protein_state +the O +AI B-structure_element +region I-structure_element +is O +highly B-protein_state +active I-protein_state +in O +MA B-chemical +uptake O +both O +in O +the O +triple B-mutant +mepΔ I-mutant +and O +triple B-mutant +mepΔ I-mutant +npr1Δ I-mutant +backgrounds O +, O +implying O +that O +this O +Mep2 B-mutant +variant I-mutant +has O +a O +constitutively B-protein_state +open I-protein_state +channel B-site +. O + +We O +obtained O +a O +similar O +result O +for O +ammonium O +uptake O +by O +the O +446Δ B-mutant +mutant B-protein_state +( O +Fig O +. O +3 O +), O +supporting O +the O +data O +from O +Marini O +et O +al O +. O +We O +then O +constructed B-experimental_method +and I-experimental_method +purified I-experimental_method +the O +analogous O +CaMep2 B-protein +442Δ B-mutant +truncation B-protein_state +mutant I-protein_state +and O +determined B-experimental_method +the O +crystal B-evidence +structure I-evidence +using O +data O +to O +3 O +. O +4 O +Å O +resolution O +. O + +The O +structure B-evidence +shows O +that O +removal B-experimental_method +of I-experimental_method +the O +AI B-structure_element +region I-structure_element +markedly O +increases O +the O +dynamics O +of O +the O +cytoplasmic B-structure_element +parts I-structure_element +of O +the O +transporter B-protein_type +. O + +This O +is O +not O +unexpected O +given O +the O +fact O +that O +the O +AI B-structure_element +region I-structure_element +bridges O +the O +CTR B-structure_element +and O +the O +main B-structure_element +body I-structure_element +of O +Mep2 B-protein +( O +Fig O +. O +6 O +). O + +Density B-evidence +for O +ICL3 B-structure_element +and O +the O +CTR B-structure_element +beyond O +residue O +Arg415 B-residue_name_number +is O +missing O +in O +the O +442Δ B-mutant +mutant B-protein_state +, O +and O +the O +density B-evidence +for O +the O +other O +ICLs B-structure_element +including O +ICL1 B-structure_element +is O +generally O +poor O +with O +visible O +parts O +of O +the O +structure B-evidence +having O +high O +B O +- O +factors O +( O +Fig O +. O +7 O +). O + +Interestingly O +, O +however O +, O +the O +Tyr49 B-residue_name_number +- O +His342 B-residue_name_number +hydrogen B-bond_interaction +bond I-bond_interaction +that O +closes O +the O +channel O +in O +the O +WT B-protein_state +protein O +is O +still O +present O +( O +Fig O +. O +7 O +and O +Supplementary O +Fig O +. O +2 O +). O + +Why O +then O +does O +this O +mutant O +appear O +to O +be O +constitutively O +active B-protein_state +? O +We O +propose O +two O +possibilities O +. O + +The O +first O +one O +is O +that O +the O +open B-protein_state +state O +is O +disfavoured O +by O +crystallization B-experimental_method +because O +of O +lower O +stability O +or O +due O +to O +crystal O +packing O +constraints O +. O + +The O +second O +possibility O +is O +that O +the O +Tyr B-site +– I-site +His I-site +hydrogen I-site +bond I-site +has O +to O +be O +disrupted O +by O +the O +incoming O +substrate O +to O +open B-protein_state +the O +channel O +. O + +The O +latter O +model O +would O +fit O +well O +with O +the O +NH3 B-chemical +/ O +H B-chemical ++ I-chemical +symport O +model O +in O +which O +the O +proton O +is O +relayed O +by O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +. O + +The O +importance O +of O +the O +Tyr B-site +– I-site +His I-site +hydrogen I-site +bond I-site +is O +underscored O +by O +the O +fact O +that O +its O +removal B-experimental_method +in O +the O +ScMep2 B-protein +Y53A B-mutant +mutant B-protein_state +results O +in O +a O +constitutively B-protein_state +active I-protein_state +transporter B-protein_type +( O +Fig O +. O +3 O +). O + +Phosphorylation B-ptm +causes O +a O +conformational O +change O +in O +the O +CTR B-structure_element + +Do O +the O +Mep2 B-protein +structures B-evidence +provide O +any O +clues O +regarding O +the O +potential O +effect O +of O +phosphorylation B-ptm +? O + +The O +side O +- O +chain O +hydroxyl O +of O +Ser457 B-residue_name_number +/ O +453 B-residue_number +is O +located O +in O +a O +well O +- O +defined O +electronegative B-site +pocket I-site +that O +is O +solvent B-protein_state +accessible I-protein_state +( O +Fig O +. O +6 O +). O + +The O +closest O +atoms O +to O +the O +serine B-residue_name +hydroxyl O +group O +are O +the O +backbone O +carbonyl O +atoms O +of O +Asp419 B-residue_name_number +, O +Glu420 B-residue_name_number +and O +Glu421 B-residue_name_number +, O +which O +are O +3 O +– O +4 O +Å O +away O +. O + +We O +therefore O +predict O +that O +phosphorylation B-ptm +of O +Ser453 B-residue_name_number +will O +result O +in O +steric O +clashes O +as O +well O +as O +electrostatic O +repulsion O +, O +which O +in O +turn O +might O +cause O +substantial O +conformational O +changes O +within O +the O +CTR B-structure_element +. O + +To O +test O +this O +hypothesis O +, O +we O +determined B-experimental_method +the O +structure B-evidence +of O +the O +phosphorylation B-protein_state +- I-protein_state +mimicking I-protein_state +R452D B-mutant +/ I-mutant +S453D I-mutant +protein O +( O +hereafter O +termed O +‘ O +DD B-mutant +mutant I-mutant +'), O +using O +data O +to O +a O +resolution O +of O +2 O +. O +4 O +Å O +. O +The O +additional B-experimental_method +mutation I-experimental_method +of I-experimental_method +the O +arginine B-residue_name +preceding O +the O +phosphorylation B-site +site I-site +was O +introduced O +( O +i O +) O +to O +increase O +the O +negative O +charge O +density O +and O +make O +it O +more O +comparable O +to O +a O +phosphate B-chemical +at O +neutral O +pH O +, O +and O +( O +ii O +) O +to O +further O +destabilize O +the O +interactions O +of O +the O +AI B-structure_element +region I-structure_element +with O +the O +main B-structure_element +body I-structure_element +of O +the O +transporter B-protein_type +( O +Fig O +. O +6 O +). O + +The O +ammonium B-chemical +uptake O +activity O +of O +the O +S B-species +. I-species +cerevisiae I-species +version O +of O +the O +DD B-mutant +mutant I-mutant +is O +the O +same O +as O +that O +of O +WT B-protein_state +Mep2 B-protein +and O +the O +S453D B-mutant +mutant B-protein_state +, O +indicating O +that O +the O +mutations O +do O +not O +affect O +transporter O +functionality O +in O +the O +triple B-mutant +mepΔ I-mutant +background O +( O +Fig O +. O +3 O +). O + +Unexpectedly O +, O +the O +AI B-structure_element +segment I-structure_element +containing O +the O +mutated O +residues O +has O +only O +undergone O +a O +slight O +shift O +compared O +with O +the O +WT B-protein_state +protein O +( O +Fig O +. O +8 O +and O +Supplementary O +Fig O +. O +3 O +). O + +By O +contrast O +, O +the O +conserved B-protein_state +part O +of O +the O +CTR B-structure_element +has O +undergone O +a O +large O +conformational O +change O +involving O +formation O +of O +a O +12 B-structure_element +- I-structure_element +residue I-structure_element +- I-structure_element +long I-structure_element +α I-structure_element +- I-structure_element +helix I-structure_element +from O +Leu427 B-residue_range +to I-residue_range +Asp438 I-residue_range +. O + +In O +addition O +, O +residues O +Glu420 B-residue_range +- I-residue_range +Leu423 I-residue_range +including O +Glu421 B-residue_name_number +of O +the O +ExxGxD B-structure_element +motif I-structure_element +are O +now O +disordered B-protein_state +( O +Fig O +. O +8 O +and O +Supplementary O +Fig O +. O +3 O +). O + +This O +is O +the O +first O +time O +a O +large O +conformational O +change O +has O +been O +observed O +in O +an O +ammonium B-protein_type +transporter I-protein_type +as O +a O +result O +of O +a O +mutation B-experimental_method +, O +and O +confirms O +previous O +hypotheses O +that O +phosphorylation B-ptm +causes O +structural O +changes O +in O +the O +CTR B-structure_element +. O + +To O +exclude O +the O +possibility O +that O +the O +additional O +R452D B-mutant +mutation O +is O +responsible O +for O +the O +observed O +changes O +, O +we O +also O +determined B-experimental_method +the O +structure B-evidence +of O +the O +‘ O +single B-mutant +D I-mutant +' O +S453D B-mutant +mutant B-protein_state +. O + +As O +shown O +in O +Supplementary O +Fig O +. O +4 O +, O +the O +consequence O +of O +the O +single B-mutant +D I-mutant +mutation B-experimental_method +is O +very O +similar O +to O +that O +of O +the O +DD B-mutant +substitution I-mutant +, O +with O +conformational O +changes O +and O +increased O +dynamics O +confined O +to O +the O +conserved B-protein_state +part O +of O +the O +CTR B-structure_element +( O +Supplementary O +Fig O +. O +4 O +). O + +To O +supplement O +the O +crystal B-evidence +structures I-evidence +, O +we O +also O +performed O +modelling B-experimental_method +and O +MD B-experimental_method +studies O +of O +WT B-protein_state +CaMep2 B-protein +, O +the O +DD B-mutant +mutant I-mutant +and O +phosphorylated B-protein_state +protein O +( O +S453J B-mutant +). O + +In O +the O +WT B-protein_state +structure B-evidence +, O +the O +acidic O +residues O +Asp419 B-residue_name_number +, O +Glu420 B-residue_name_number +and O +Glu421 B-residue_name_number +are O +within O +hydrogen B-bond_interaction +bonding I-bond_interaction +distance O +of O +Ser453 B-residue_name_number +. O + +After O +200 O +ns O +of O +MD B-experimental_method +simulation B-experimental_method +, O +the O +interactions O +between O +these O +residues O +and O +Ser453 B-residue_name_number +remain O +intact O +. O + +The O +protein O +backbone O +has O +an O +average O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +. I-evidence +of O +only O +∼ O +3 O +Å O +during O +the O +200 O +- O +ns O +simulation B-experimental_method +, O +indicating O +that O +the O +protein O +is O +stable B-protein_state +. O + +There O +is O +flexibility O +in O +the O +side O +chains O +of O +the O +acidic O +residues O +so O +that O +they O +are O +able O +to O +form O +stable B-protein_state +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +Ser453 B-residue_name_number +. O + +In O +particular O +, O +persistent O +hydrogen B-bond_interaction +bonds I-bond_interaction +are O +observed O +between O +the O +Ser453 B-residue_name_number +hydroxyl O +group O +and O +the O +acidic O +group O +of O +Glu420 B-residue_name_number +, O +and O +also O +between O +the O +amine O +group O +of O +Ser453 B-residue_name_number +and O +the O +backbone O +carbonyl O +of O +Glu420 B-residue_name_number +( O +Supplementary O +Fig O +. O +5 O +). O + +The O +DD B-mutant +mutant I-mutant +is O +also O +stable B-protein_state +during O +the O +simulations B-experimental_method +, O +but O +the O +average O +backbone O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +of O +∼ O +3 O +. O +6 O +Å O +suggests O +slightly O +more O +conformational O +flexibility O +than O +WT B-protein_state +. O + +As O +the O +simulation B-experimental_method +proceeds O +, O +the O +side O +chains O +of O +the O +acidic O +residues O +move O +away O +from O +Asp452 B-residue_name_number +and O +Asp453 B-residue_name_number +, O +presumably O +to O +avoid O +electrostatic O +repulsion O +. O + +For O +example O +, O +the O +distance B-evidence +between O +the O +Asp453 B-residue_name_number +acidic O +oxygens O +and O +the O +Glu420 B-residue_name_number +acidic O +oxygens O +increases O +from O +∼ O +7 O +to O +> O +22 O +Å O +after O +200 O +ns O +simulations B-experimental_method +, O +and O +thus O +these O +residues O +are O +not O +interacting O +. O + +The O +protein O +is O +structurally B-protein_state +stable I-protein_state +throughout O +the O +simulation B-experimental_method +with O +little O +deviation O +in O +the O +other O +parts O +of O +the O +protein O +. O + +Finally O +, O +the O +S453J B-mutant +mutant B-protein_state +is O +also O +stable B-protein_state +throughout O +the O +200 O +- O +ns O +simulation B-experimental_method +and O +has O +an O +average O +backbone O +deviation O +of O +∼ O +3 O +. O +8 O +Å O +, O +which O +is O +similar O +to O +the O +DD B-mutant +mutant I-mutant +. O + +The O +movement O +of O +the O +acidic O +residues O +away O +from O +Arg452 B-residue_name_number +and O +Sep453 B-residue_name_number +is O +more O +pronounced O +in O +this O +simulation B-experimental_method +in O +comparison O +with O +the O +movement O +away O +from O +Asp452 B-residue_name_number +and O +Asp453 B-residue_name_number +in O +the O +DD B-mutant +mutant I-mutant +. O + +The O +distance B-evidence +between O +the O +phosphate B-chemical +of O +Sep453 B-residue_name_number +and O +the O +acidic O +oxygen O +atoms O +of O +Glu420 B-residue_name_number +is O +initially O +∼ O +11 O +Å O +, O +but O +increases O +to O +> O +30 O +Å O +after O +200 O +ns O +. O + +The O +short B-structure_element +helix I-structure_element +formed O +by O +residues O +Leu427 B-residue_range +to I-residue_range +Asp438 I-residue_range +unravels O +during O +the O +simulations B-experimental_method +to O +a O +disordered B-protein_state +state O +. O + +Thus O +, O +the O +MD B-experimental_method +simulations B-experimental_method +support O +the O +notion O +from O +the O +crystal B-evidence +structures I-evidence +that O +phosphorylation B-ptm +generates O +conformational O +changes O +in O +the O +conserved B-protein_state +part O +of O +the O +CTR B-structure_element +. O + +However O +, O +the O +conformational O +changes O +for O +the O +phosphomimetic B-mutant +mutants I-mutant +in O +the O +crystals B-evidence +are O +confined O +to O +the O +CTR B-structure_element +( O +Fig O +. O +8 O +), O +and O +the O +channels B-site +are O +still O +closed B-protein_state +( O +Supplementary O +Fig O +. O +2 O +). O + +One O +possible O +explanation O +is O +that O +the O +mutants B-mutant +do O +not O +accurately O +mimic O +a O +phosphoserine B-residue_name +, O +but O +the O +observation O +that O +the O +S453D B-mutant +and O +DD B-mutant +mutants I-mutant +are O +fully B-protein_state +active I-protein_state +in O +the O +absence B-protein_state +of I-protein_state +Npr1 B-protein +suggests O +that O +the O +mutations B-experimental_method +do O +mimic O +the O +effect O +of O +phosphorylation B-ptm +( O +Fig O +. O +3 O +). O + +The O +fact O +that O +the O +S453D B-mutant +structure B-evidence +was O +obtained O +in O +the O +presence O +of O +10 O +mM O +ammonium B-chemical +ions O +suggests O +that O +the O +crystallization B-experimental_method +process O +favours O +closed B-protein_state +states O +of O +the O +Mep2 B-protein +channels B-site +. O + +Knowledge O +about O +ammonium B-protein_type +transporter I-protein_type +structure B-evidence +has O +been O +obtained O +from O +experimental O +and O +theoretical O +studies O +on O +bacterial B-taxonomy_domain +family O +members O +. O + +In O +addition O +, O +a O +number O +of O +biochemical B-experimental_method +and I-experimental_method +genetic I-experimental_method +studies I-experimental_method +are O +available O +for O +bacterial B-taxonomy_domain +, O +fungal B-taxonomy_domain +and O +plant B-taxonomy_domain +proteins O +. O + +These O +efforts O +have O +advanced O +our O +knowledge O +considerably O +but O +have O +not O +yet O +yielded O +atomic O +- O +level O +answers O +to O +several O +important O +mechanistic O +questions O +, O +including O +how O +ammonium B-chemical +transport O +is O +regulated O +in O +eukaryotes B-taxonomy_domain +and O +the O +mechanism O +of O +ammonium B-chemical +signalling O +. O + +In O +Arabidopsis B-species +thaliana I-species +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +, O +phosphorylation B-ptm +of O +the O +CTR B-structure_element +residue O +T460 B-residue_name_number +under O +conditions O +of O +high O +ammonium B-chemical +inhibits O +transport O +activity O +, O +that O +is O +, O +the O +default O +( O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +) O +state O +of O +the O +plant B-taxonomy_domain +transporter B-protein_type +is O +open B-protein_state +. O + +Interestingly O +, O +phosphomimetic B-mutant +mutations I-mutant +introduced O +into O +one O +monomer B-oligomeric_state +inactivate O +the O +entire O +trimer B-oligomeric_state +, O +indicating O +that O +( O +i O +) O +heterotrimerization O +occurs O +and O +( O +ii O +) O +the O +CTR B-structure_element +mediates O +allosteric O +regulation O +of O +ammonium B-chemical +transport O +activity O +via O +phosphorylation B-ptm +. O + +Owing O +to O +the O +lack O +of O +structural O +information O +for O +plant B-taxonomy_domain +AMTs B-protein_type +, O +the O +details O +of O +channel B-site +closure O +and O +inter O +- O +monomer O +crosstalk O +are O +not O +yet O +clear O +. O + +Contrasting O +with O +the O +plant B-taxonomy_domain +transporters B-protein_type +, O +the O +inactive B-protein_state +states O +of O +Mep2 B-protein_type +proteins I-protein_type +under O +conditions O +of O +high O +ammonium B-chemical +are O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +, O +with O +channels B-site +that O +are O +closed B-protein_state +on O +the O +cytoplasmic O +side O +. O + +The O +reason O +why O +similar O +transporters B-protein_type +such O +as O +A B-species +. I-species +thaliana I-species +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +and O +Mep2 B-protein +are O +regulated O +in O +opposite O +ways O +by O +phosphorylation B-ptm +( O +inactivation B-protein_state +in O +plants B-taxonomy_domain +and O +activation B-protein_state +in O +fungi B-taxonomy_domain +) O +is O +not O +known O +. O + +In O +fungi B-taxonomy_domain +, O +preventing O +ammonium B-chemical +entry O +via O +channel O +closure O +in O +ammonium B-protein_type +transporters I-protein_type +would O +be O +one O +way O +to O +alleviate O +ammonium B-chemical +toxicity O +, O +in O +addition O +to O +ammonium B-chemical +excretion O +via O +Ato B-protein_type +transporters B-protein_type +and O +amino O +- O +acid O +secretion O +. O + +By O +determining O +the O +first O +structures B-evidence +of O +closed B-protein_state +ammonium B-protein_type +transporters I-protein_type +and O +comparing B-experimental_method +those O +structures B-evidence +with O +the O +permanently B-protein_state +open I-protein_state +bacterial B-taxonomy_domain +proteins O +, O +we O +demonstrate O +that O +Mep2 B-protein_type +channel B-site +closure O +is O +likely O +due O +to O +movements O +of O +the O +CTR B-structure_element +and O +ICL1 B-structure_element +and O +ICL3 B-structure_element +. O + +More O +specifically O +, O +the O +close O +interactions O +between O +the O +CTR B-structure_element +and O +ICL1 B-structure_element +/ O +ICL3 B-structure_element +present O +in O +open B-protein_state +transporters B-protein_type +are O +disrupted O +, O +causing O +ICL3 B-structure_element +to O +move O +outwards O +and O +block O +the O +channel B-site +( O +Figs O +4 O +and O +9a O +). O + +In O +addition O +, O +ICL1 B-structure_element +has O +shifted O +inwards O +to O +contribute O +to O +the O +channel B-site +closure O +by O +engaging O +His2 B-residue_name_number +from O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +via O +hydrogen B-bond_interaction +bonding I-bond_interaction +with O +a O +highly B-protein_state +conserved I-protein_state +tyrosine B-residue_name +hydroxyl O +group O +. O + +Upon O +phosphorylation B-ptm +by O +the O +Npr1 B-protein +kinase B-protein_type +in O +response O +to O +nitrogen B-chemical +limitation O +, O +the O +region O +around O +the O +conserved B-protein_state +ExxGxD B-structure_element +motif I-structure_element +undergoes O +a O +conformational O +change O +that O +opens O +the O +channel B-site +( O +Fig O +. O +9 O +). O + +Importantly O +, O +the O +structural B-evidence +similarities I-evidence +in O +the O +TM B-structure_element +parts I-structure_element +of O +Mep2 B-protein +and O +AfAmt B-protein +- I-protein +1 I-protein +( O +Fig O +. O +5a O +) O +suggest O +that O +channel B-site +opening O +/ O +closure O +does O +not O +require O +substantial O +changes O +in O +the O +residues O +lining O +the O +channel B-site +. O + +How O +exactly O +the O +channel B-site +opens O +and O +whether O +opening O +is O +intra O +- O +monomeric O +are O +still O +open B-protein_state +questions O +; O +it O +is O +possible O +that O +the O +change O +in O +the O +CTR B-structure_element +may O +disrupt O +its O +interactions O +with O +ICL3 B-structure_element +of O +the O +neighbouring O +monomer B-oligomeric_state +( O +Fig O +. O +9b O +), O +which O +could O +result O +in O +opening O +of O +the O +neighbouring O +channel B-site +via O +inward O +movement O +of O +its O +ICL3 B-structure_element +. O + +Owing O +to O +the O +crosstalk O +between O +monomers B-oligomeric_state +, O +a O +single O +phosphorylation B-ptm +event O +might O +lead O +to O +opening O +of O +the O +entire O +trimer B-oligomeric_state +, O +although O +this O +has O +not O +yet O +been O +tested O +( O +Fig O +. O +9b O +). O + +Whether O +or O +not O +Mep2 B-protein_type +channel B-site +opening O +requires O +, O +in O +addition O +to O +phosphorylation B-ptm +, O +disruption O +of O +the O +Tyr B-site +– I-site +His2 I-site +interaction I-site +by O +the O +ammonium B-chemical +substrate O +is O +not O +yet O +clear O +. O + +Is O +our O +model O +for O +opening O +and O +closing O +of O +Mep2 B-protein +channels B-site +valid O +for O +other O +eukaryotic B-taxonomy_domain +ammonium B-protein_type +transporters I-protein_type +? O +Our O +structural B-evidence +data I-evidence +support O +previous O +studies O +and O +clarify O +the O +central O +role O +of O +the O +CTR B-structure_element +and O +cytoplasmic B-structure_element +loops I-structure_element +in O +the O +transition O +between O +closed B-protein_state +and O +open B-protein_state +states O +. O + +However O +, O +even O +the O +otherwise O +highly O +similar O +Mep2 B-protein_type +proteins I-protein_type +of O +S B-species +. I-species +cerevisiae I-species +and O +C B-species +. I-species +albicans I-species +have O +different O +structures B-evidence +for O +their O +CTRs B-structure_element +( O +Fig O +. O +1 O +and O +Supplementary O +Fig O +. O +6 O +). O + +In O +addition O +, O +the O +AI B-structure_element +region I-structure_element +of O +the O +CTR B-structure_element +containing O +the O +Npr1 B-site +kinase I-site +site I-site +is O +conserved B-protein_state +in O +only O +a O +subset O +of O +fungal B-taxonomy_domain +transporters B-protein_type +, O +suggesting O +that O +the O +details O +of O +the O +structural O +changes O +underpinning O +regulation O +vary O +. O + +Nevertheless O +, O +given O +the O +central O +role O +of O +absolutely B-protein_state +conserved I-protein_state +residues O +within O +the O +ICL1 B-site +- I-site +ICL3 I-site +- I-site +CTR I-site +interaction I-site +network I-site +( O +Fig O +. O +4 O +), O +we O +propose O +that O +the O +structural O +basics O +of O +fungal B-taxonomy_domain +ammonium B-chemical +transporter O +activation O +are O +conserved B-protein_state +. O + +The O +fact O +that O +Mep2 B-protein_type +orthologues O +of O +distantly O +related O +fungi B-taxonomy_domain +are O +fully O +functional O +in O +ammonium B-chemical +transport O +and O +signalling O +in O +S B-species +. I-species +cerevisiae I-species +supports O +this O +notion O +. O + +It O +should O +also O +be O +noted O +that O +the O +tyrosine B-residue_name +residue O +interacting O +with O +His2 B-residue_name_number +is O +highly B-protein_state +conserved I-protein_state +in O +fungal B-taxonomy_domain +Mep2 B-protein_type +orthologues O +, O +suggesting O +that O +the O +Tyr B-site +– I-site +His2 I-site +hydrogen I-site +bond I-site +might O +be O +a O +general O +way O +to O +close B-protein_state +Mep2 B-protein_type +proteins I-protein_type +. O + +With O +regards O +to O +plant B-taxonomy_domain +AMTs B-protein_type +, O +it O +has O +been O +proposed O +that O +phosphorylation B-ptm +at O +T460 B-residue_name_number +generates O +conformational O +changes O +that O +would O +close O +the O +neighbouring O +pore B-site +via O +the O +C B-structure_element +terminus I-structure_element +. O + +This O +assumption O +was O +based O +partly O +on O +a O +homology B-experimental_method +model I-experimental_method +for O +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +based O +on O +the O +( O +open B-protein_state +) O +archaebacterial B-taxonomy_domain +AfAmt B-protein +- I-protein +1 I-protein +structure B-evidence +, O +which O +suggested O +that O +the O +C B-structure_element +terminus I-structure_element +of O +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +would O +extend O +further O +to O +the O +neighbouring O +monomer B-oligomeric_state +. O + +Our O +Mep2 B-protein +structures B-evidence +show O +that O +this O +assumption O +may O +not O +be O +correct O +( O +Fig O +. O +4 O +and O +Supplementary O +Fig O +. O +6 O +). O + +In O +addition O +, O +the O +considerable O +differences O +between O +structurally O +resolved O +CTR B-structure_element +domains O +means O +that O +the O +exact O +environment O +of O +T460 B-residue_name_number +in O +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +is O +also O +not O +known O +( O +Supplementary O +Fig O +. O +6 O +). O + +Based O +on O +the O +available O +structural B-evidence +information I-evidence +, O +we O +consider O +it O +more O +likely O +that O +phosphorylation O +- O +mediated O +pore O +closure O +in O +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +is O +intra O +- O +monomeric O +, O +via O +disruption O +of O +the O +interactions O +between O +the O +CTR B-structure_element +and O +ICL1 B-structure_element +/ O +ICL3 B-structure_element +( O +for O +example O +, O +Y467 B-residue_name_number +- O +H239 B-residue_name_number +and O +D458 B-residue_name_number +- O +K71 B-residue_name_number +). O + +There O +is O +generally O +no O +equivalent O +for O +CaMep2 B-protein +Tyr49 B-residue_name_number +in O +plant B-taxonomy_domain +AMTs B-protein_type +, O +indicating O +that O +a O +Tyr B-site +– I-site +His2 I-site +hydrogen I-site +bond I-site +as O +observed O +in O +Mep2 B-protein +may O +not O +contribute O +to O +the O +closed B-protein_state +state O +in O +plant B-taxonomy_domain +transporters B-protein_type +. O + +We O +propose O +that O +intra B-site +- I-site +monomeric I-site +CTR I-site +- I-site +ICL1 I-site +/ I-site +ICL3 I-site +interactions I-site +lie O +at O +the O +basis O +of O +regulation O +of O +both O +fungal B-taxonomy_domain +and O +plant B-taxonomy_domain +ammonium B-protein_type +transporters I-protein_type +; O +close O +interactions O +generate O +open B-protein_state +channels B-site +, O +whereas O +the O +lack B-protein_state +of I-protein_state +‘ O +intra O +-' O +interactions O +leads O +to O +inactive B-protein_state +states O +. O + +The O +need O +to O +regulate O +in O +opposite O +ways O +may O +be O +the O +reason O +why O +the O +phosphorylation B-site +sites I-site +are O +in O +different O +parts O +of O +the O +CTR B-structure_element +, O +that O +is O +, O +centrally O +located O +close O +to O +the O +ExxGxD B-structure_element +motif I-structure_element +in O +AMTs B-protein_type +and O +peripherally O +in O +Mep2 B-protein +. O + +In O +this O +way O +, O +phosphorylation B-ptm +can O +either O +lead O +to O +channel B-site +closing O +( O +in O +the O +case O +of O +AMTs B-protein_type +) O +or O +channel B-site +opening O +in O +the O +case O +of O +Mep2 B-protein +. O + +Our O +model O +also O +provides O +an O +explanation O +for O +the O +observation O +that O +certain B-mutant +mutations I-mutant +within O +the O +CTR B-structure_element +completely O +abolish O +transport O +activity O +. O + +An O +example O +of O +an O +inactivating O +residue O +is O +the O +glycine B-residue_name +of O +the O +ExxGxD B-structure_element +motif I-structure_element +of O +the O +CTR B-structure_element +. O + +Mutation B-experimental_method +of O +this O +residue O +( O +G393 B-residue_name_number +in O +EcAmtB B-protein +; O +G456 B-residue_name_number +in O +AtAmt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +) O +inactivates O +transporters B-protein_type +as O +diverse O +as O +Escherichia B-species +coli I-species +AmtB B-protein +and O +A B-species +. I-species +thaliana I-species +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +( O +refs O +). O + +Such O +mutations O +likely O +cause O +structural O +changes O +in O +the O +CTR B-structure_element +that O +prevent O +close O +contacts O +between O +the O +CTR B-structure_element +and O +ICL1 B-structure_element +/ O +ICL3 B-structure_element +, O +thereby O +stabilizing O +a O +closed B-protein_state +state O +that O +may O +be O +similar O +to O +that O +observed O +in O +Mep2 B-protein +. O + +Regulation O +and O +modulation O +of O +membrane O +transport O +by O +phosphorylation B-ptm +is O +known O +to O +occur O +in O +, O +for O +example O +, O +aquaporins B-protein_type +and O +urea B-protein_type +transporters I-protein_type +, O +and O +is O +likely O +to O +be O +a O +common O +theme O +for O +eukaryotic B-taxonomy_domain +channels B-protein_type +and O +transporters B-protein_type +. O + +Recently O +, O +phosphorylation B-ptm +was O +also O +shown O +to O +modulate O +substrate O +affinity O +in O +nitrate B-protein_type +transporters I-protein_type +. O + +With O +respect O +to O +ammonium B-chemical +transport O +, O +phosphorylation B-ptm +has O +thus O +far O +only O +been O +shown O +for O +A B-species +. I-species +thaliana I-species +AMTs B-protein_type +and O +for O +S B-species +. I-species +cerevisiae I-species +Mep2 B-protein +( O +refs O +). O + +However O +, O +the O +absence B-protein_state +of I-protein_state +GlnK B-protein_type +proteins I-protein_type +in O +eukaryotes B-taxonomy_domain +suggests O +that O +phosphorylation B-ptm +- O +based O +regulation O +of O +ammonium B-chemical +transport O +may O +be O +widespread O +. O + +With O +respect O +to O +Mep2 B-protein_type +- O +mediated O +signalling O +to O +induce O +pseudohyphal O +growth O +, O +two O +models O +have O +been O +put O +forward O +as O +to O +how O +this O +occurs O +and O +why O +it O +is O +specific O +to O +Mep2 B-protein_type +proteins I-protein_type +. O + +In O +one O +model O +, O +signalling O +is O +proposed O +to O +depend O +on O +the O +nature O +of O +the O +transported O +substrate O +, O +which O +might O +be O +different O +in O +certain O +subfamilies O +of O +ammonium B-protein_type +transporters I-protein_type +( O +for O +example O +, O +Mep1 B-protein +/ O +Mep3 B-protein +versus O +Mep2 B-protein +). O + +For O +example O +, O +NH3 B-chemical +uniport O +or O +symport O +of O +NH3 B-chemical +/ O +H B-chemical ++ I-chemical +might O +result O +in O +changes O +in O +local O +pH O +, O +but O +NH4 B-chemical ++ I-chemical +uniport O +might O +not O +, O +and O +this O +difference O +might O +determine O +signalling O +. O + +In O +the O +other O +model O +, O +signalling O +is O +thought O +to O +require O +a O +distinct O +conformation O +of O +the O +Mep2 B-protein +transporter B-protein_type +occurring O +during O +the O +transport O +cycle O +. O + +While O +the O +current O +study O +does O +not O +specifically O +address O +the O +mechanism O +of O +signalling O +underlying O +pseudohyphal O +growth O +, O +our O +structures B-evidence +do O +show O +that O +Mep2 B-protein_type +proteins I-protein_type +can O +assume O +different O +conformations O +. O + +It O +is O +clear O +that O +ammonium B-chemical +transport O +across O +biomembranes O +remains O +a O +fascinating O +and O +challenging O +field O +in O +large O +part O +due O +to O +the O +unique O +properties O +of O +the O +substrate O +. O + +Our O +Mep2 B-protein +structural O +work O +now O +provides O +a O +foundation O +for O +future O +studies O +to O +uncover O +the O +details O +of O +the O +structural O +changes O +that O +occur O +during O +eukaryotic B-taxonomy_domain +ammonium B-chemical +transport O +and O +signaling O +, O +and O +to O +assess O +the O +possibility O +to O +utilize O +small O +molecules O +to O +shut O +down O +ammonium B-chemical +sensing O +and O +downstream O +signalling O +pathways O +in O +pathogenic O +fungi B-taxonomy_domain +. O + +X B-evidence +- I-evidence +ray I-evidence +crystal I-evidence +structures I-evidence +of O +Mep2 B-protein +transceptors B-protein_type +. O + +( O +a O +) O +Monomer B-oligomeric_state +cartoon O +models O +viewed O +from O +the O +side O +for O +( O +left O +) O +A O +. O +fulgidus O +Amt B-protein +- I-protein +1 I-protein +( O +PDB O +ID O +2B2H O +), O +S B-species +. I-species +cerevisiae I-species +Mep2 B-protein +( O +middle O +) O +and O +C B-species +. I-species +albicans I-species +Mep2 B-protein +( O +right O +). O + +The O +region O +showing O +ICL1 B-structure_element +( O +blue O +), O +ICL3 B-structure_element +( O +green O +) O +and O +the O +CTR B-structure_element +( O +red O +) O +is O +boxed O +for O +comparison O +. O + +( O +b O +) O +CaMep2 B-protein +trimer B-oligomeric_state +viewed O +from O +the O +intracellular O +side O +( O +right O +). O + +One O +monomer B-oligomeric_state +is O +coloured O +as O +in O +a O +and O +one O +monomer B-oligomeric_state +is O +coloured O +by O +B O +- O +factor O +( O +blue O +, O +low O +; O +red O +; O +high O +). O + +The O +CTR B-structure_element +is O +boxed O +. O + +( O +c O +) O +Overlay B-experimental_method +of O +ScMep2 B-protein +( O +grey O +) O +and O +CaMep2 B-protein +( O +rainbow O +), O +illustrating O +the O +differences O +in O +the O +CTRs B-structure_element +. O + +Sequence B-evidence +conservation I-evidence +in O +ammonium B-protein_type +transporters I-protein_type +. O + +ClustalW B-experimental_method +alignment I-experimental_method +of O +CaMep2 B-protein +, O +ScMep2 B-protein +, O +A B-species +. I-species +fulgidus I-species +Amt B-protein +- I-protein +1 I-protein +, O +E O +. O +coli O +AmtB B-protein +and O +A B-species +. I-species +thaliana I-species +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +. O + +The O +secondary O +structure O +elements O +observed O +for O +CaMep2 B-protein +are O +indicated O +, O +with O +the O +numbers O +corresponding O +to O +the O +centre O +of O +the O +TM B-structure_element +segment I-structure_element +. O + +The O +conserved B-protein_state +RxK B-structure_element +motif I-structure_element +in O +ICL1 B-structure_element +is O +boxed O +in O +blue O +, O +the O +ER B-structure_element +motif I-structure_element +in O +ICL2 B-structure_element +in O +cyan O +, O +the O +conserved B-protein_state +ExxGxD B-structure_element +motif I-structure_element +of O +the O +CTR B-structure_element +in O +red O +and O +the O +AI B-structure_element +region I-structure_element +in O +yellow O +. O + +Coloured O +residues O +are O +functionally O +important O +and O +correspond O +to O +those O +of O +the O +Phe B-site +gate I-site +( O +blue O +), O +the O +binding B-site +site I-site +Trp B-residue_name +residue O +( O +magenta O +) O +and O +the O +twin O +- O +His O +motif O +( O +red O +). O + +The O +Npr1 B-site +kinase I-site +site I-site +in O +the O +AI B-structure_element +region I-structure_element +is O +highlighted O +pink O +. O + +The O +grey O +sequences O +at O +the O +C O +termini O +of O +CaMep2 B-protein +and O +ScMep2 B-protein +are O +not O +visible O +in O +the O +structures B-evidence +and O +are O +likely B-protein_state +disordered I-protein_state +. O + +Growth B-experimental_method +of O +ScMep2 B-mutant +variants I-mutant +on O +low O +ammonium O +medium O +. O + +( O +a O +) O +The O +triple B-mutant +mepΔ I-mutant +strain O +( O +black O +) O +and O +triple O +mepΔ O +npr1Δ O +strain O +( O +grey O +) O +containing O +plasmids O +expressing O +WT B-protein_state +and O +variant B-mutant +ScMep2 I-mutant +were O +grown B-experimental_method +on I-experimental_method +minimal I-experimental_method +medium I-experimental_method +containing O +1 O +mM O +ammonium B-chemical +sulphate I-chemical +. O + +The O +quantified O +cell B-evidence +density I-evidence +reflects O +logarithmic O +growth O +after O +24 O +h O +. O +Error O +bars O +are O +the O +s O +. O +d O +. O +for O +three O +replicates O +of O +each O +strain O +( O +b O +) O +The O +strains O +used O +in O +a O +were O +also O +serially O +diluted O +and O +spotted O +onto O +minimal O +agar O +plates O +containing O +glutamate B-chemical +( O +0 O +. O +1 O +%) O +or O +ammonium B-chemical +sulphate I-chemical +( O +1 O +mM O +), O +and O +grown O +for O +3 O +days O +at O +30 O +° O +C O +. O + +Structural O +differences O +between O +Mep2 B-protein +and O +bacterial B-taxonomy_domain +ammonium O +transporters O +. O + +( O +a O +) O +ICL1 B-structure_element +in O +AfAmt B-protein +- I-protein +1 I-protein +( O +light O +blue O +) O +and O +CaMep2 B-protein +( O +dark O +blue O +), O +showing O +unwinding O +and O +inward O +movement O +in O +the O +fungal B-taxonomy_domain +protein O +. O +( O +b O +) O +Stereo O +diagram O +viewed O +from O +the O +cytosol O +of O +ICL1 B-structure_element +, O +ICL3 B-structure_element +( O +green O +) O +and O +the O +CTR B-structure_element +( O +red O +) O +in O +AfAmt B-protein +- I-protein +1 I-protein +( O +light O +colours O +) O +and O +CaMep2 B-protein +( O +dark O +colours O +). O + +The O +side O +chains O +of O +residues O +in O +the O +RxK B-structure_element +motif I-structure_element +as O +well O +as O +those O +of O +Tyr49 B-residue_name_number +and O +His342 B-residue_name_number +are O +labelled O +. O + +The O +numbering O +is O +for O +CaMep2 B-protein +. O + +( O +c O +) O +Conserved B-protein_state +residues O +in O +ICL1 B-structure_element +- I-structure_element +3 I-structure_element +and O +the O +CTR B-structure_element +. O + +Views O +from O +the O +cytosol O +for O +CaMep2 B-protein +( O +left O +) O +and O +AfAmt B-protein +- I-protein +1 I-protein +, O +highlighting O +the O +large O +differences O +in O +conformation O +of O +the O +conserved B-protein_state +residues O +in O +ICL1 B-structure_element +( O +RxK O +motif O +; O +blue O +), O +ICL2 B-structure_element +( O +ER B-structure_element +motif I-structure_element +; O +cyan O +), O +ICL3 B-structure_element +( O +green O +) O +and O +the O +CTR B-structure_element +( O +red O +). O + +The O +labelled O +residues O +are O +analogous O +within O +both O +structures B-evidence +. O + +In O +b O +and O +c O +, O +the O +centre O +of O +the O +trimer B-oligomeric_state +is O +at O +top O +. O + +Channel O +closures O +in O +Mep2 B-protein +. O + +( O +a O +) O +Stereo O +superposition B-experimental_method +of O +AfAmt B-protein +- I-protein +1 I-protein +and O +CaMep2 B-protein +showing O +the O +residues O +of O +the O +Phe B-site +gate I-site +, O +His2 B-residue_name_number +of O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +and O +the O +tyrosine B-residue_name +residue O +Y49 B-residue_name_number +in O +TM1 B-structure_element +that O +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +His2 B-residue_name_number +in O +CaMep2 B-protein +. O +( O +b O +) O +Surface O +views O +from O +the O +side O +in O +rainbow O +colouring O +, O +showing O +the O +two O +- O +tier O +channel B-structure_element +block I-structure_element +( O +indicated O +by O +the O +arrows O +) O +in O +CaMep2 B-protein +. O + +The O +Npr1 B-protein +kinase B-protein_type +target O +Ser453 B-residue_name_number +is O +dephosphorylated B-protein_state +and O +located O +in O +an O +electronegative B-site +pocket I-site +. O + +( O +a O +) O +Stereoviews O +of O +CaMep2 B-protein +showing O +2Fo O +– O +Fc O +electron O +density O +( O +contoured O +at O +1 O +. O +0 O +σ O +) O +for O +CTR B-structure_element +residues O +Asp419 B-residue_range +- I-residue_range +Met422 I-residue_range +and O +for O +Tyr446 B-residue_range +- I-residue_range +Thr455 I-residue_range +of O +the O +AI B-structure_element +region I-structure_element +. O + +The O +phosphorylation B-ptm +target O +residue O +Ser453 B-residue_name_number +is O +labelled O +in O +bold O +. O + +( O +b O +) O +Overlay B-experimental_method +of O +the O +CTRs B-structure_element +of O +ScMep2 B-protein +( O +grey O +) O +and O +CaMep2 B-protein +( O +green O +), O +showing O +the O +similar O +electronegative O +environment O +surrounding O +the O +phosphorylation B-site +site I-site +( O +P O +). O + +The O +AI B-structure_element +regions I-structure_element +are O +coloured O +magenta O +. O + +( O +c O +) O +Cytoplasmic O +view O +of O +the O +Mep2 B-protein +trimer B-oligomeric_state +indicating O +the O +large O +distance O +between O +Ser453 B-residue_name_number +and O +the O +channel B-site +exits I-site +( O +circles O +; O +Ile52 B-residue_name_number +lining O +the O +channel B-site +exit I-site +is O +shown O +). O + +Effect O +of O +removal B-experimental_method +of O +the O +AI B-structure_element +region I-structure_element +on O +Mep2 B-protein +structure B-evidence +. O + +( O +a O +) O +Side O +views O +for O +WT B-protein_state +CaMep2 B-protein +( O +left O +) O +and O +the O +truncation B-protein_state +mutant I-protein_state +442Δ B-mutant +( O +right O +). O + +The O +latter O +is O +shown O +as O +a O +putty O +model O +according O +to O +B O +- O +factors O +to O +illustrate O +the O +disorder B-protein_state +in O +the O +protein O +on O +the O +cytoplasmic O +side O +. O + +Missing O +regions O +are O +labelled O +. O +( O +b O +) O +Stereo O +superpositions B-experimental_method +of O +WT B-protein_state +CaMep2 B-protein +and O +the O +truncation B-protein_state +mutant I-protein_state +. O + +2Fo O +– O +Fc O +electron O +density O +( O +contoured O +at O +1 O +. O +0 O +σ O +) O +for O +residues O +Tyr49 B-residue_name_number +and O +His342 B-residue_name_number +is O +shown O +for O +the O +truncation B-protein_state +mutant I-protein_state +. O + +Phosphorylation B-ptm +causes O +conformational O +changes O +in O +the O +CTR B-structure_element +. O + +( O +a O +) O +Cytoplasmic O +view O +of O +the O +DD B-mutant +mutant I-mutant +trimer B-oligomeric_state +, O +with O +WT B-protein_state +CaMep2 B-protein +superposed B-experimental_method +in O +grey O +for O +one O +of O +the O +monomers B-oligomeric_state +. O + +The O +arrow O +indicates O +the O +phosphorylation B-site +site I-site +. O + +The O +AI B-structure_element +region I-structure_element +is O +coloured O +magenta O +. O + +( O +b O +) O +Monomer B-oligomeric_state +side O +- O +view O +superposition B-experimental_method +of O +WT B-protein_state +CaMep2 B-protein +and O +the O +DD B-mutant +mutant I-mutant +, O +showing O +the O +conformational O +change O +and O +disorder O +around O +the O +ExxGxD B-structure_element +motif I-structure_element +. O + +Side O +chains O +for O +residues O +452 B-residue_number +and O +453 B-residue_number +are O +shown O +as O +stick O +models O +. O + +Schematic O +model O +for O +phosphorylation O +- O +based O +regulation O +of O +Mep2 B-protein +ammonium O +transporters O +. O + +( O +a O +) O +In O +the O +closed B-protein_state +, O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +state O +( O +i O +), O +the O +CTR B-structure_element +( O +magenta O +) O +and O +ICL3 B-structure_element +( O +green O +) O +are O +far O +apart O +with O +the O +latter O +blocking O +the O +intracellular O +channel B-site +exit I-site +( O +indicated O +with O +a O +hatched O +circle O +). O + +Upon O +phosphorylation B-ptm +and O +mimicked B-protein_state +by O +the O +CaMep2 B-protein +S453D B-mutant +and O +DD B-mutant +mutants I-mutant +( O +ii O +), O +the O +region O +around O +the O +ExxGxD B-structure_element +motif I-structure_element +undergoes O +a O +conformational O +change O +that O +results O +in O +the O +CTR B-structure_element +interacting O +with O +the O +inward O +- O +moving O +ICL3 B-structure_element +, O +opening O +the O +channel B-site +( O +full O +circle O +) O +( O +iii O +). O + +The O +open B-protein_state +- O +channel B-site +Mep2 B-protein +structure B-evidence +is O +represented O +by O +archaebacterial B-taxonomy_domain +Amt B-protein +- I-protein +1 I-protein +and O +shown O +in O +lighter O +colours O +consistent O +with O +Fig O +. O +4 O +. O + +As O +discussed O +in O +the O +text O +, O +similar O +structural O +arrangements O +may O +occur O +in O +plant B-taxonomy_domain +AMTs B-protein_type +. O + +In O +this O +case O +however O +, O +the O +open B-protein_state +channel B-site +corresponds O +to O +the O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +state O +; O +phosphorylation B-ptm +breaks O +the O +CTR O +– O +ICL3 O +interactions O +leading O +to O +channel B-site +closure O +. O +( O +b O +) O +Model O +based O +on O +AMT O +transporter O +analogy O +showing O +how O +phosphorylation B-ptm +of O +a O +Mep2 B-protein +monomer B-oligomeric_state +might O +allosterically O +open B-protein_state +channels B-site +in O +the O +entire O +trimer B-oligomeric_state +via O +disruption O +of O +the O +interactions O +between O +the O +CTR B-structure_element +and O +ICL3 B-structure_element +of O +a O +neighbouring O +monomer B-oligomeric_state +( O +arrow O +). O + +Template O +- O +dependent O +nucleotide O +addition O +in O +the O +reverse O +( O +3 O +′- O +5 O +′) O +direction O +by O +Thg1 B-protein_type +- I-protein_type +like I-protein_type +protein I-protein_type + +Structures B-evidence +of O +Thg1 B-protein_type +- I-protein_type +like I-protein_type +proteins I-protein_type +provide O +insight O +into O +the O +template O +- O +dependent O +nucleotide O +addition O +in O +the O +reverse O +( O +3 O +′- O +5 O +′) O +direction O +. O + +Thg1 B-protein_type +- I-protein_type +like I-protein_type +protein I-protein_type +( O +TLP B-protein_type +) O +catalyzes O +the O +addition O +of O +a O +nucleotide O +to O +the O +5 O +′- O +end O +of O +truncated O +transfer B-chemical +RNA I-chemical +( O +tRNA B-chemical +) O +species O +in O +a O +Watson O +- O +Crick O +template O +– O +dependent O +manner O +. O + +The O +reaction O +proceeds O +in O +two O +steps O +: O +the O +activation O +of O +the O +5 O +′- O +end O +by O +adenosine B-chemical +5 I-chemical +′- I-chemical +triphosphate I-chemical +( O +ATP B-chemical +)/ O +guanosine B-chemical +5 I-chemical +′- I-chemical +triphosphate I-chemical +( O +GTP B-chemical +), O +followed O +by O +nucleotide O +addition O +. O + +Structural B-experimental_method +analyses I-experimental_method +of O +the O +TLP B-protein_type +and O +its O +reaction O +intermediates O +have O +revealed O +the O +atomic O +detail O +of O +the O +template O +- O +dependent O +elongation O +reaction O +in O +the O +3 O +′- O +5 O +′ O +direction O +. O + +The O +enzyme O +creates O +two O +substrate B-site +binding I-site +sites I-site +for O +the O +first O +- O +and O +second O +- O +step O +reactions O +in O +the O +vicinity O +of O +one O +reaction B-site +center I-site +consisting O +of O +two O +Mg2 B-chemical ++ I-chemical +ions O +, O +and O +the O +two O +reactions O +are O +executed O +at O +the O +same O +reaction B-site +center I-site +in O +a O +stepwise O +fashion O +. O + +When O +the O +incoming O +nucleotide B-chemical +is O +bound B-protein_state +to I-protein_state +the O +second B-site +binding I-site +site I-site +with O +Watson B-bond_interaction +- I-bond_interaction +Crick I-bond_interaction +hydrogen I-bond_interaction +bonds I-bond_interaction +, O +the O +3 O +′- O +OH O +of O +the O +incoming O +nucleotide O +and O +the O +5 B-chemical +′- I-chemical +triphosphate I-chemical +of O +the O +tRNA B-chemical +are O +moved O +to O +the O +reaction B-site +center I-site +where O +the O +first O +reaction O +has O +occurred O +. O + +That O +the O +3 B-protein_type +′- I-protein_type +5 I-protein_type +′ I-protein_type +elongation I-protein_type +enzyme I-protein_type +performs O +this O +elaborate O +two O +- O +step O +reaction O +in O +one O +catalytic B-site +center I-site +suggests O +that O +these O +two O +reactions O +have O +been O +inseparable O +throughout O +the O +process O +of O +protein O +evolution O +. O + +Although O +TLP B-protein_type +and O +Thg1 B-protein +have O +similar O +tetrameric B-oligomeric_state +organization O +, O +the O +tRNA B-chemical +binding O +mode O +of O +TLP B-protein_type +is O +different O +from O +that O +of O +Thg1 B-protein +, O +a O +tRNAHis B-protein_type +- I-protein_type +specific I-protein_type +G I-protein_type +− I-protein_type +1 I-protein_type +addition I-protein_type +enzyme I-protein_type +. O + +Each O +tRNAHis B-chemical +binds O +to O +three O +of O +the O +four O +Thg1 B-protein +tetramer B-oligomeric_state +subunits B-structure_element +, O +whereas O +in O +TLP B-protein_type +, O +tRNA B-chemical +only O +binds O +to O +a O +dimer B-site +interface I-site +and O +the O +elongation O +reaction O +is O +terminated O +by O +measuring O +the O +accepter B-structure_element +stem I-structure_element +length O +through O +the O +flexible B-protein_state +β B-structure_element +- I-structure_element +hairpin I-structure_element +. O + +Furthermore O +, O +mutational B-experimental_method +analyses I-experimental_method +show O +that O +tRNAHis B-chemical +is O +bound B-protein_state +to I-protein_state +TLP B-protein_type +in O +a O +similar O +manner O +as O +Thg1 B-protein +, O +thus O +indicating O +that O +TLP B-protein_type +has O +a O +dual O +binding O +mode O +. O + +All O +polynucleotide O +chain O +elongation O +reactions O +, O +whether O +with O +DNA B-chemical +or O +RNA B-chemical +, O +proceed O +in O +the O +5 O +′- O +3 O +′ O +direction O +. O + +This O +reaction O +involves O +the O +nucleophilic O +attack O +of O +a O +3 O +′- O +OH O +of O +the O +terminal O +nucleotide O +in O +the O +elongating O +chain O +on O +the O +α O +- O +phosphate B-chemical +of O +an O +incoming O +nucleotide O +. O + +This O +elongation O +reaction O +of O +DNA B-chemical +/ O +RNA B-chemical +chains O +is O +in O +clear O +contrast O +to O +the O +elongation O +of O +protein O +chains O +in O +which O +the O +high O +energy O +of O +the O +incoming O +aminoacyl B-chemical +- I-chemical +tRNA I-chemical +is O +not O +used O +for O +its O +own O +addition O +but O +for O +the O +addition O +of O +the O +next O +monomer B-oligomeric_state +( O +termed O +head O +polymerization O +). O + +However O +, O +recent O +studies O +have O +shown O +that O +the O +Thg1 B-protein +/ O +Thg1 B-protein_type +- I-protein_type +like I-protein_type +protein I-protein_type +( O +TLP B-protein_type +) O +family O +of O +proteins O +extends O +tRNA B-chemical +nucleotide O +chains O +in O +the O +reverse O +( O +3 O +′- O +5 O +′) O +direction O +. O + +In O +this O +case O +, O +the O +5 O +′- O +end O +of O +tRNA B-chemical +is O +first O +activated O +using O +adenosine B-chemical +5 I-chemical +′- I-chemical +triphosphate I-chemical +( O +ATP B-chemical +)/ O +guanosine B-chemical +5 I-chemical +′- I-chemical +triphosphate I-chemical +( O +GTP B-chemical +), O +followed O +by O +nucleophilic O +attack O +of O +a O +3 O +′- O +OH O +on O +the O +incoming O +nucleotide O +[ O +nucleoside B-chemical +5 I-chemical +′- I-chemical +triphosphate I-chemical +( O +NTP B-chemical +)] O +to O +yield O +pppN B-chemical +- I-chemical +tRNA I-chemical +. O + +Thus O +, O +the O +energy O +in O +the O +triphosphate B-chemical +bond O +of O +the O +incoming O +nucleotide O +is O +not O +used O +for O +its O +own O +addition O +but O +is O +reserved O +for O +subsequent O +polymerization O +( O +that O +is O +, O +head O +polymerization O +) O +( O +Fig O +. O +1 O +). O + +Top O +: O +Reaction O +scheme O +of O +3 O +′- O +5 O +′ O +elongation O +by O +Thg1 B-protein +/ O +TLP B-protein_type +family O +proteins O +. O + +Bottom O +: O +Reaction O +scheme O +of O +5 O +′- O +3 O +′ O +elongation O +by O +DNA B-protein_type +/ I-protein_type +RNA I-protein_type +polymerases I-protein_type +. O + +In O +3 O +′- O +5 O +′ O +elongation O +by O +Thg1 B-protein +/ O +TLP B-protein_type +family O +proteins O +, O +the O +5 B-chemical +′- I-chemical +monophosphate I-chemical +of O +the O +tRNA B-chemical +is O +first O +activated O +by O +ATP B-chemical +/ O +GTP B-chemical +, O +followed O +by O +the O +actual O +elongation O +reaction O +. O + +In O +5 O +′- O +3 O +′ O +elongation O +by O +DNA B-protein_type +/ I-protein_type +RNA I-protein_type +polymerases I-protein_type +, O +the O +energy O +of O +the O +incoming O +nucleotide O +is O +used O +for O +its O +own O +addition O +( O +tail O +polymerization O +). O + +The O +best O +- O +characterized O +member O +of O +this O +family O +of O +proteins O +is O +eukaryotic B-taxonomy_domain +Thg1 B-protein +( O +tRNAHis B-protein_type +guanylyltransferase I-protein_type +), O +which O +catalyzes O +the O +nontemplated O +addition O +of O +a O +guanylate O +to O +the O +5 O +′- O +end O +of O +immature O +tRNAHis B-chemical +. O + +This O +guanosine B-chemical +at O +position O +− B-residue_number +1 I-residue_number +( O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +) O +of O +tRNAHis B-chemical +is O +a O +critical O +identity O +element O +for O +recognition O +by O +the O +histidyl B-protein_type +- I-protein_type +tRNA I-protein_type +synthase I-protein_type +. O + +Therefore O +, O +Thg1 B-protein +is O +essential O +to O +the O +fidelity O +of O +protein O +synthesis O +in O +eukaryotes B-taxonomy_domain +. O + +However O +, O +Thg1 B-protein +homologs O +or O +TLPs B-protein_type +are O +found O +in O +organisms O +in O +which O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +is O +genetically O +encoded O +, O +and O +thus O +, O +posttranscriptional O +modification O +is O +not O +required O +. O + +This O +finding O +suggests O +that O +TLPs B-protein_type +may O +have O +potential O +functions O +other O +than O +tRNAHis B-chemical +maturation O +. O + +TLPs B-protein_type +have O +been O +shown O +to O +catalyze O +5 O +′- O +end O +nucleotide O +addition O +to O +truncated O +tRNA B-chemical +species O +in O +vitro O +in O +a O +Watson O +- O +Crick O +template O +– O +dependent O +manner O +. O + +This O +function O +of O +TLPs B-protein_type +is O +not O +limited O +to O +tRNAHis B-chemical +but O +occurs O +efficiently O +with O +other O +tRNAs B-chemical +. O + +Furthermore O +, O +the O +yeast B-taxonomy_domain +homolog O +( O +Thg1p B-protein +) O +has O +been O +shown O +to O +interact O +with O +the O +replication O +origin O +recognition O +complex O +for O +DNA B-chemical +replication O +, O +and O +the O +plant B-taxonomy_domain +homolog O +( O +ICA1 B-protein +) O +was O +identified O +as O +a O +protein O +affecting O +the O +capacity O +to O +repair O +DNA B-chemical +damage O +. O + +These O +observations O +suggest O +that O +TLPs B-protein_type +may O +have O +more O +general O +functions O +in O +DNA B-chemical +/ O +RNA B-chemical +repair O +. O + +The O +3 O +′- O +5 O +′ O +addition O +reaction O +catalyzed O +by O +Thg1 B-protein +occurs O +through O +three O +reaction O +steps O +. O + +In O +the O +first O +step O +, O +the O +5 O +′- O +monophosphorylated O +tRNAHis B-chemical +, O +which O +is O +cleaved O +by O +ribonuclease B-protein_type +P I-protein_type +from O +pre B-chemical +- I-chemical +tRNAHis I-chemical +, O +is O +activated O +by O +ATP B-chemical +, O +creating O +a O +5 O +′- O +adenylylated O +tRNAHis B-chemical +intermediate O +. O + +In O +the O +second O +step O +, O +the O +3 O +′- O +OH O +of O +the O +incoming O +GTP B-chemical +attacks O +the O +activated O +intermediate O +, O +yielding O +pppG B-chemical +− I-chemical +1 I-chemical +- I-chemical +tRNAHis I-chemical +. O + +Finally O +, O +the O +pyrophosphate B-chemical +is O +removed O +, O +and O +mature O +pG B-chemical +− I-chemical +1 I-chemical +- I-chemical +tRNAHis I-chemical +is O +created O +. O + +The O +crystal B-evidence +structure I-evidence +of O +human B-species +Thg1 B-protein +( O +HsThg1 B-protein +) O +shows O +that O +its O +catalytic B-site +core I-site +shares O +structural O +homology O +with O +canonical O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +nucleotide I-protein_type +polymerases I-protein_type +, O +such O +as O +T7 B-protein_type +DNA I-protein_type +/ I-protein_type +RNA I-protein_type +polymerases I-protein_type +. O + +This O +finding O +suggests O +that O +3 B-protein_type +′- I-protein_type +5 I-protein_type +′ I-protein_type +elongation I-protein_type +enzymes I-protein_type +are O +related O +to O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +polymerases I-protein_type +and O +raises O +important O +questions O +on O +why O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +polymerases I-protein_type +predominate O +in O +nature O +. O + +The O +crystal B-evidence +structure I-evidence +of O +TLP B-protein_type +from O +Bacillus B-species +thuringiensis I-species +shows O +that O +it O +shares O +a O +similar O +tetrameric B-oligomeric_state +assembly O +and O +active B-site +- I-site +site I-site +architecture O +with O +HsThg1 B-protein +. O + +Furthermore O +, O +the O +structure B-evidence +of O +Candida B-species +albicans I-species +Thg1 B-protein +( O +CaThg1 B-protein +) O +complexed B-protein_state +with I-protein_state +tRNAHis B-chemical +reveals O +that O +the O +tRNA B-chemical +substrate O +accesses O +the O +reaction B-site +center I-site +from O +a O +direction O +opposite O +to O +that O +of O +canonical O +DNA B-protein_type +/ I-protein_type +RNA I-protein_type +polymerase I-protein_type +. O + +However O +, O +in O +this O +structural B-experimental_method +analysis I-experimental_method +, O +the O +5 O +′- O +end O +of O +tRNAHis B-chemical +was O +not O +activated O +and O +the O +second O +substrate O +( O +GTP B-chemical +) O +was O +not B-protein_state +bound I-protein_state +. O + +Here O +, O +we O +successfully O +solved B-experimental_method +the O +structure B-evidence +of O +TLP B-protein_type +from O +the O +methanogenic B-taxonomy_domain +archaeon I-taxonomy_domain +Methanosarcina B-species +acetivorans I-species +( O +MaTLP B-protein +) O +in B-protein_state +complex I-protein_state +with I-protein_state +ppptRNAPheΔ1 B-chemical +, O +which O +mimics O +the O +activated O +intermediate O +of O +the O +repair O +substrate O +. O + +Although O +TLP B-protein_type +and O +Thg1 B-protein +have O +similar O +tetrameric B-oligomeric_state +organization O +, O +the O +mode O +of O +tRNA B-chemical +binding O +is O +different O +in O +TLP B-protein_type +. O + +Furthermore O +, O +we O +obtained O +the O +structure B-evidence +in O +which O +the O +GTP B-chemical +analog O +( O +GDPNP B-chemical +) O +was O +inserted O +into O +this O +complex O +to O +form O +a O +Watson B-bond_interaction +- I-bond_interaction +Crick I-bond_interaction +base I-bond_interaction +pair I-bond_interaction +with O +C72 B-residue_name_number +at O +the O +3 O +′- O +end O +region O +of O +the O +tRNA B-chemical +. O + +On O +the O +basis O +of O +these O +structures B-evidence +, O +we O +discuss O +the O +reaction O +mechanism O +of O +template O +- O +dependent O +reverse O +( O +3 O +′- O +5 O +′) O +polymerization O +in O +comparison O +with O +canonical O +5 O +′- O +3 O +′ O +polymerization O +. O + +Anticodon O +- O +independent O +binding O +of O +ppptRNAPheΔ1 B-chemical +to O +MaTLP B-protein + +Previous O +biochemical B-experimental_method +experiments I-experimental_method +have O +suggested O +that O +ppptRNAPheΔ1 B-chemical +, O +in O +which O +the O +5 O +′- O +end O +of O +tRNAPhe B-chemical +was O +triphosphorylated O +and O +G1 B-residue_name_number +was O +deleted B-experimental_method +, O +can O +be O +an O +efficient O +substrate O +for O +the O +repair O +reaction O +( O +guanylyl O +transfer O +) O +of O +Thg1 B-protein +/ O +TLP B-protein_type +. O + +Therefore O +, O +we O +prepared O +a O +crystal B-evidence +of O +MaTLP B-protein +complexed B-protein_state +with I-protein_state +ppptRNAPheΔ1 B-chemical +and O +solved B-experimental_method +its O +structure B-evidence +to O +study O +the O +template O +- O +directed O +3 O +′- O +5 O +′ O +elongation O +reaction O +by O +TLP B-protein_type +( O +fig O +. O +S1 O +). O + +The O +crystal B-evidence +contained O +a O +dimer B-oligomeric_state +of O +TLP B-protein_type +( O +A B-structure_element +and O +B B-structure_element +molecules O +) O +and O +one O +tRNA B-chemical +in O +an O +asymmetric O +unit O +. O + +Two O +dimers B-oligomeric_state +in O +the O +crystal B-evidence +further O +assembled O +as O +a O +dimer B-oligomeric_state +of O +dimers B-oligomeric_state +by O +the O +crystallographic O +twofold O +axis O +( O +Fig O +. O +2 O +). O + +This O +tetrameric B-oligomeric_state +structure B-evidence +and O +4 O +: O +2 O +stoichiometry O +of O +the O +TLP B-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +complex O +are O +the O +same O +as O +those O +of O +the O +CaThg1 B-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +complex O +. O + +However O +, O +whereas O +the O +AB B-structure_element +and O +CD B-structure_element +dimers B-oligomeric_state +of O +tetrameric B-oligomeric_state +CaThg1 B-protein +play O +different O +roles O +, O +respectively O +recognizing O +the O +accepter B-structure_element +stem I-structure_element +and O +anticodon O +of O +tRNAHis B-chemical +, O +the O +AB B-structure_element +dimer B-oligomeric_state +and O +its O +symmetry O +mate O +( O +CD B-structure_element +dimer B-oligomeric_state +) O +on O +tetrameric B-oligomeric_state +MaTLP B-protein +independently O +bind O +one O +molecule O +of O +tRNA B-chemical +( O +fig O +. O +S2 O +), O +recognizing O +the O +tRNA B-chemical +accepter B-structure_element +stem I-structure_element +and O +elbow B-structure_element +region I-structure_element +. O + +Thus O +, O +consistent O +with O +the O +notion O +that O +MaTLP B-protein +is O +an O +anticodon B-protein_type +- I-protein_type +independent I-protein_type +repair I-protein_type +enzyme I-protein_type +, O +the O +anticodon O +was O +not O +recognized O +in O +the O +MaTLP B-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +complex O +, O +whereas O +the O +binding O +mode O +of O +CaThg1 B-protein +is O +for O +the O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +addition O +reaction O +, O +therefore O +the O +His B-residue_name +anticodon O +has O +to O +be O +recognized O +( O +see O +“ O +Dual O +binding O +mode O +for O +tRNA B-chemical +repair O +”). O + +Structure B-evidence +of O +the O +MaTLP B-protein +complex B-protein_state +with I-protein_state +ppptRNAPheΔ1 B-chemical +. O + +Left O +: O +One O +molecule O +of O +the O +tRNA B-chemical +substrate O +( O +ppptRNAPheΔ1 B-chemical +) O +is O +bound B-protein_state +to I-protein_state +the O +MaTLP B-protein +dimer B-oligomeric_state +. O + +The O +AB B-structure_element +and O +CD B-structure_element +dimers B-oligomeric_state +are O +further O +dimerized B-oligomeric_state +by O +the O +crystallographic O +twofold O +axis O +to O +form O +a O +tetrameric B-oligomeric_state +structure B-evidence +( O +dimer B-oligomeric_state +of O +dimers B-oligomeric_state +). O + +The O +CD B-structure_element +dimer B-oligomeric_state +is O +omitted O +for O +clarity O +. O + +The O +accepter B-structure_element +stem I-structure_element +of O +the O +tRNA B-chemical +is O +recognized O +by O +molecule O +A O +( O +yellow O +), O +and O +the O +elbow B-structure_element +region I-structure_element +by O +molecule O +B O +( O +blue O +). O + +The O +β B-structure_element +- I-structure_element +hairpin I-structure_element +region O +of O +molecule O +B O +is O +shown O +in O +red O +. O + +The O +elbow B-structure_element +region I-structure_element +of O +the O +tRNA B-chemical +substrate O +was O +recognized O +by O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +of O +molecule O +B O +of O +MaTLP B-protein +. O + +The O +N O +atoms O +in O +the O +side O +chain O +of O +R215 B-residue_name_number +in O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +region O +of O +MaTLP B-protein +were O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +phosphate B-chemical +groups O +of O +U55 B-residue_name_number +and O +G57 B-residue_name_number +. O + +The O +O O +atom O +on O +the O +S213 B-residue_name_number +side O +chain O +was O +also O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +phosphate B-chemical +moiety O +of O +G57 B-residue_name_number +of O +the O +tRNA B-chemical +( O +Fig O +. O +2 O +). O + +This O +β B-structure_element +- I-structure_element +hairpin I-structure_element +region O +was O +disordered B-protein_state +in O +the O +crystal B-evidence +structure I-evidence +of O +the O +CaThg1 B-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +complex O +. O + +The O +accepter B-structure_element +stem I-structure_element +of O +the O +tRNA B-chemical +substrate O +was O +recognized O +by O +molecule O +A O +of O +MaTLP B-protein +. O + +The O +N7 O +atom O +of O +G2 B-residue_name_number +at O +the O +5 O +′- O +end O +was O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +N O +atom O +of O +the O +R136 B-residue_name_number +side O +chain O +, O +whereas O +the O +α O +- O +phosphate B-chemical +was O +bonded O +to O +the O +N137 B-residue_name_number +side O +chain O +( O +Fig O +. O +2 O +). O + +R136 B-residue_name_number +was O +also O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +base O +of O +C72 B-residue_name_number +( O +the O +Watson O +- O +Crick O +bond O +partner O +of O +ΔG1 O +). O + +The O +triphosphate B-chemical +moiety O +at O +the O +5 O +′- O +end O +of O +the O +tRNA B-chemical +was O +bonded O +to O +the O +D21 B-residue_range +- I-residue_range +K26 I-residue_range +region O +. O + +These O +phosphates B-chemical +were O +also O +coordinated B-bond_interaction +to I-bond_interaction +two O +metal O +ions O +, O +presumably O +Mg2 B-chemical ++ I-chemical +( O +Mg2 B-chemical ++ I-chemical +A O +and O +Mg2 B-chemical ++ I-chemical +B O +) O +because O +they O +were O +observed O +at O +the O +same O +positions O +( O +figs O +. O +S3 O +and O +S4 O +) O +previously O +identified O +by O +CaThg1 B-protein +and O +HsThg1 B-protein +structures B-evidence +. O + +These O +ions O +were O +in O +turn O +coordinated B-bond_interaction +by I-bond_interaction +the O +O O +atoms O +of O +the O +side O +chains O +of O +D21 B-residue_name_number +and O +D69 B-residue_name_number +and O +the O +main O +- O +chain O +O O +of O +G22 B-residue_name_number +( O +fig O +. O +S3A O +). O + +Mutation B-experimental_method +of O +D29 B-residue_name_number +and O +D76 B-residue_name_number +in O +HsThg1 B-protein +( O +corresponding O +to O +D21 B-residue_name_number +and O +D69 B-residue_name_number +of O +MaTLP B-protein +) O +has O +been O +shown O +to O +markedly O +decrease O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +addition O +activity O +. O + +Template O +- O +dependent O +binding O +of O +the O +GTP B-chemical +analog O +to O +the O +MaTLP B-complex_assembly +- I-complex_assembly +ppptRNAPheΔ1 I-complex_assembly +complex O + +Here O +, O +we O +successfully O +obtained O +the O +structure B-evidence +of O +the O +ternary O +complex O +of O +MaTLP B-protein +, O +5 O +′- O +activated O +tRNA B-chemical +( O +ppptRNAPheΔ1 B-chemical +), O +and O +the O +GTP B-chemical +analog O +( O +GDPNP B-chemical +) O +( O +Fig O +. O +3 O +and O +fig O +. O +S4 O +) O +by O +soaking B-experimental_method +the O +MaTLP B-complex_assembly +- I-complex_assembly +ppptRNAPheΔ1 I-complex_assembly +complex O +crystal B-evidence +in O +a O +solution O +containing O +GDPNP B-chemical +. O + +The O +obtained O +structure B-evidence +showed O +that O +the O +guanine B-chemical +base O +of O +the O +incoming O +GDPNP B-chemical +formed O +Watson B-bond_interaction +- I-bond_interaction +Crick I-bond_interaction +hydrogen I-bond_interaction +bonds I-bond_interaction +with O +C72 B-residue_name_number +and O +accompanied O +base B-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +interactions I-bond_interaction +with O +G2 B-residue_name_number +of O +the O +tRNA B-chemical +( O +Fig O +. O +3B O +), O +whereas O +no O +interaction O +was O +observed O +between O +the O +guanine B-chemical +base O +and O +the O +enzyme O +. O + +The O +5 O +′- O +end O +( O +position O +2 O +) O +of O +the O +tRNA B-chemical +moved O +significantly O +( O +Fig O +. O +3C O +) O +due O +to O +the O +insertion O +of O +GDPNP B-chemical +. O + +Surprisingly O +, O +the O +5 B-chemical +′- I-chemical +triphosphate I-chemical +moiety O +after O +movement O +occupied O +the O +GTP B-chemical +/ O +ATP B-chemical +triphosphate B-chemical +position O +during O +the O +activation O +step O +( O +Fig O +. O +3D O +). O + +Together O +with O +the O +observation O +that O +the O +3 O +′- O +OH O +of O +the O +incoming O +GTP B-chemical +analog O +was O +within O +coordination O +distance O +( O +2 O +. O +8 O +Å O +) O +to O +Mg2 B-chemical ++ I-chemical +A O +( O +fig O +. O +S3B O +) O +and O +was O +able O +to O +execute O +a O +nucleophilic O +attack O +on O +the O +α O +- O +phosphate B-chemical +of O +the O +5 O +′- O +end O +, O +this O +structure B-evidence +indicates O +that O +the O +elongation O +reaction O +( O +second O +reaction O +) O +takes O +place O +at O +the O +same O +reaction B-site +center I-site +where O +the O +activation O +reaction O +( O +first O +reaction O +) O +occurs O +. O + +Structural O +change O +of O +the O +tRNA B-chemical +( O +ppptRNAPheΔ1 B-chemical +). O + +Structural O +change O +of O +the O +tRNA B-chemical +( O +ppptRNAPheΔ1 B-chemical +) O +accepter B-structure_element +stem I-structure_element +in O +MaTLP B-protein +caused O +by O +insertion O +of O +GDPNP B-chemical +. O +( O +A O +) O +Structure B-evidence +before O +GDPNP B-chemical +binding O +. O + +( O +B O +) O +Structure B-evidence +after O +GDPNP B-chemical +binding O +. O +( O +C O +) O +Superposition B-experimental_method +of O +the O +two O +structures B-evidence +showing O +movement O +of O +the O +5 O +′- O +end O +of O +the O +tRNA B-chemical +before O +( O +blue O +) O +and O +after O +( O +red O +) O +insertion O +of O +GDPNP B-chemical +. O +( O +D O +) O +Superposition B-experimental_method +of O +the O +5 O +′- O +end O +of O +the O +tRNA B-chemical +after O +GDPNP B-chemical +insertion O +( O +red O +) O +with O +GTP B-chemical +at O +the O +activation O +step O +( O +green O +), O +showing O +that O +both O +triphosphate B-chemical +moieties O +superpose O +well O +. O + +The O +triphosphate B-chemical +moiety O +of O +GDPNP B-chemical +was O +at O +the O +interface B-site +between O +molecules O +A B-structure_element +and O +B B-structure_element +and O +was O +recognized O +by O +the O +side O +chains O +of O +both O +molecules O +, O +including O +R19 B-residue_name_number +( O +molecule O +A B-structure_element +), O +R83 B-residue_name_number +( O +molecule O +B B-structure_element +), O +K86 B-residue_name_number +( O +molecule O +B B-structure_element +), O +and O +R114 B-residue_name_number +( O +molecule O +A B-structure_element +) O +( O +Fig O +. O +3B O +). O + +All O +of O +these O +residues O +are O +well B-protein_state +conserved I-protein_state +( O +fig O +. O +S5 O +), O +and O +mutation B-experimental_method +of O +corresponding O +residues O +in O +ScThg1 B-protein +( O +R27 B-residue_name_number +, O +R93 B-residue_name_number +, O +K96 B-residue_name_number +, O +and O +R133 B-residue_name_number +) O +decreased O +the O +catalytic O +efficiency O +of O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +addition O +. O + +The O +triphosphate B-chemical +of O +the O +GDPNP B-chemical +was O +also O +bonded O +to O +the O +third O +Mg2 B-chemical ++ I-chemical +( O +Mg2 B-chemical ++ I-chemical +C O +), O +which O +, O +unlike O +Mg2 B-chemical ++ I-chemical +A O +and O +Mg2 B-chemical ++ I-chemical +B O +, O +is O +not O +coordinated B-bond_interaction +by I-bond_interaction +the O +TLP B-protein_type +molecule O +( O +fig O +. O +S3B O +). O + +This O +triphosphate B-chemical +binding O +mode O +is O +the O +same O +as O +that O +for O +the O +second B-site +nucleotide I-site +binding I-site +site I-site +in O +Thg1 B-protein +. O + +However O +, O +in O +previous O +analyses O +, O +the O +base O +moiety O +at O +the O +second B-site +site I-site +was O +either O +invisible O +or O +far O +beyond O +the O +reaction O +distance O +of O +the O +phosphate B-chemical +, O +and O +therefore O +, O +flipping O +of O +the O +base O +was O +expected O +to O +occur O +. O + +tRNA B-experimental_method +binding I-experimental_method +and I-experimental_method +repair I-experimental_method +experiments I-experimental_method +of O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +mutants B-protein_state + +To O +confirm O +tRNA B-chemical +recognition O +by O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +, O +we O +created B-experimental_method +mutation I-experimental_method +variants I-experimental_method +with O +altered O +residues O +in O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +region O +. O + +Then O +, O +tRNA B-experimental_method +binding I-experimental_method +and I-experimental_method +enzymatic I-experimental_method +activities I-experimental_method +were I-experimental_method +measured I-experimental_method +. O + +β B-structure_element +- I-structure_element +Hairpin I-structure_element +deletion B-protein_state +variant I-protein_state +delR198 B-mutant +- I-mutant +R215 I-mutant +almost O +completely O +abolished O +the O +binding O +of O +tRNAPheΔ1 B-chemical +( O +fig O +. O +S6 O +). O + +Furthermore O +, O +the O +enzymatic O +activities O +of O +delR198 B-mutant +- I-mutant +R215 I-mutant +and O +delG202 B-mutant +- I-mutant +E210 I-mutant +were O +very O +weak O +( O +5 O +. O +2 O +and O +13 O +. O +5 O +%, O +respectively O +) O +compared O +with O +wild B-protein_state +type I-protein_state +, O +whereas O +mutations B-experimental_method +( O +N179A B-mutant +and O +F174A B-mutant +/ O +N179A B-mutant +/ O +R188A B-mutant +) O +on O +the O +anticodon B-site +recognition I-site +site I-site +[ O +deduced O +from O +the O +Thg1 B-complex_assembly +- I-complex_assembly +tRNAHis I-complex_assembly +complex O +structure B-evidence +] O +had O +no O +effect O +on O +the O +catalytic O +activity O +( O +Fig O +. O +4A O +). O + +Experiments O +using O +the O +tRNAHisΔ1 B-chemical +substrate O +gave O +similar O +results O +( O +Fig O +. O +4A O +). O + +All O +these O +results O +are O +consistent O +with O +the O +crystal B-evidence +structure I-evidence +and O +suggest O +that O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +plays O +an O +important O +role O +in O +anticodon O +- O +independent O +binding O +of O +the O +tRNA B-chemical +substrate O +. O + +Residues O +in O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +are O +not B-protein_state +well I-protein_state +conserved I-protein_state +, O +except O +for O +R215 B-residue_name_number +( O +fig O +. O +S5 O +). O + +Mutants B-protein_state +R215A B-mutant +and O +R215A B-mutant +/ O +S213A B-mutant +, O +in O +which O +the O +completely B-protein_state +conserved I-protein_state +R215 B-residue_name_number +was O +changed B-experimental_method +to O +alanine B-residue_name +, O +showed O +a O +moderate O +effect O +on O +the O +activity O +( O +27 O +. O +3 O +and O +16 O +. O +3 O +%, O +respectively O +). O + +Thus O +, O +specific O +interactions O +with O +the O +conserved B-protein_state +R215 B-residue_name_number +and O +van B-bond_interaction +der I-bond_interaction +Waals I-bond_interaction +contacts I-bond_interaction +to O +residues O +in O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +would O +be O +important O +for O +tRNA B-chemical +recognition O +. O + +Mutational B-experimental_method +analysis I-experimental_method +of O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +and O +anticodon B-site +binding I-site +region I-site +. O + +( O +A O +) O +Guanylylation O +of O +ppptRNAPheΔ1 B-chemical +and O +ppptRNAHisΔ1 B-chemical +by O +various O +TLP B-protein_type +mutants B-protein_state +. O + +The O +activity O +using O +[ B-chemical +α I-chemical +- I-chemical +32P I-chemical +] I-chemical +GTP I-chemical +, O +wild B-protein_state +- I-protein_state +type I-protein_state +MaTLP B-protein +, O +and O +ppptRNAPheΔ1 B-chemical +is O +denoted O +as O +100 O +. O +( O +B O +) O +Guanylylation O +of O +tRNAPheΔ1 B-chemical +, O +tRNAPhe B-chemical +, O +and O +tRNAHisΔ B-chemical +− I-chemical +1 I-chemical +by O +various O +TLP B-protein_type +mutants B-protein_state +. O + +The O +activity O +to O +tRNAPheΔ1 B-chemical +is O +about O +10 O +% O +of O +ppptRNAPheΔ1 B-chemical +. O + +Termination O +of O +the O +elongation O +reaction O +by O +measuring O +the O +accepter B-structure_element +stem I-structure_element + +TLPs B-protein_type +catalyze O +the O +Watson O +- O +Crick O +template O +– O +dependent O +elongation O +or O +repair O +reaction O +for O +5 O +′- O +end O +truncated O +tRNAPhe B-chemical +substrates O +lacking O +G1 B-residue_name_number +only O +( O +tRNAPheΔ1 B-chemical +), O +or O +lacking O +both O +G1 B-residue_name_number +and O +G2 B-residue_name_number +( O +tRNAPheΔ1 B-chemical +, I-chemical +2 I-chemical +), O +whereas O +they O +do O +not O +show O +any O +activity O +with O +intact O +tRNAPhe B-chemical +( O +thus O +, O +repair O +is O +unnecessary O +). O + +How O +TLP B-protein_type +distinguishes O +between O +tRNAs B-chemical +that O +need O +5 O +′- O +end O +repair O +from O +ones O +that O +do O +not O +, O +or O +in O +other O +words O +, O +how O +the O +elongation O +reaction O +is O +properly O +terminated O +, O +remains O +unknown O +. O + +The O +present O +structure B-evidence +of O +the O +MaTLP B-complex_assembly +- I-complex_assembly +ppptRNAPheΔ1 I-complex_assembly +complex O +shows O +that O +, O +unlike O +Thg1 B-protein +, O +the O +TLP B-protein_type +dimer B-oligomeric_state +binds O +one O +molecule O +of O +tRNA B-chemical +by O +recognizing O +the O +elbow B-structure_element +region I-structure_element +by O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +of O +molecule O +B B-structure_element +and O +the O +5 O +′- O +end O +by O +molecule O +A B-structure_element +. O +Therefore O +, O +we O +speculated O +that O +the O +flexible B-protein_state +nature O +of O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +enables O +the O +recognition O +of O +tRNA B-chemical +substrates O +with O +different O +accepter B-structure_element +stem I-structure_element +lengths O +. O + +To O +confirm O +this O +speculation O +, O +we O +used B-experimental_method +computer I-experimental_method +graphics I-experimental_method +to I-experimental_method +examine I-experimental_method +whether O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +region O +was O +able O +to O +bind O +tRNA B-chemical +substrates O +with O +different O +accepter B-structure_element +stem I-structure_element +lengths O +when O +the O +5 O +′- O +end O +was O +properly O +placed O +in O +the O +reaction B-site +site I-site +. O + +When O +the O +5 O +′- O +end O +was O +placed O +in O +the O +reaction O +site O +, O +the O +body O +of O +the O +tRNA B-chemical +molecule O +shifted O +in O +a O +manner O +dependent O +on O +the O +accepter B-structure_element +stem I-structure_element +length O +. O + +The O +tRNA B-chemical +body O +also O +rotated O +because O +of O +the O +helical O +nature O +of O +the O +accepter B-structure_element +stem I-structure_element +( O +fig O +. O +S7 O +). O + +This O +model O +structure B-evidence +showed O +that O +the O +accepter B-structure_element +stem I-structure_element +of O +intact O +tRNAPhe B-chemical +was O +too O +long O +for O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +to O +recognize O +its O +elbow B-structure_element +region I-structure_element +, O +whereas O +tRNAPheΔ1 B-chemical +and O +tRNAPheΔ1 B-chemical +, I-chemical +2 I-chemical +were O +recognized O +by O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +region O +( O +fig O +. O +S7 O +), O +which O +is O +consistent O +with O +previous O +experiments O +. O + +On O +the O +basis O +of O +these O +model O +structures B-evidence +, O +we O +concluded O +that O +the O +TLP B-protein_type +molecule O +can O +properly O +terminate O +elongation O +by O +measuring O +the O +accepter B-structure_element +stem I-structure_element +length O +of O +tRNA B-chemical +substrates O +. O + +Dual O +binding O +mode O +for O +tRNA B-chemical +repair O + +The O +present O +structural B-experimental_method +analysis I-experimental_method +revealed O +that O +although O +TLP B-protein_type +and O +Thg1 B-protein +have O +a O +similar O +tetrameric B-oligomeric_state +architecture O +, O +they O +have O +different O +binding O +modes O +for O +tRNAs B-chemical +: O +Thg1 B-protein +is O +bound B-protein_state +to I-protein_state +tRNAHis B-chemical +as O +a O +tetramer B-oligomeric_state +, O +whereas O +TLP B-protein_type +is O +bound B-protein_state +to I-protein_state +tRNAPhe B-chemical +as O +a O +dimer B-oligomeric_state +. O + +This O +difference O +in O +the O +tRNA B-chemical +binding O +modes O +is O +closely O +related O +to O +their O +enzymatic O +functions O +. O + +The O +tRNAHis B-protein_type +- I-protein_type +specific I-protein_type +G I-protein_type +− I-protein_type +1 I-protein_type +addition I-protein_type +enzyme I-protein_type +Thg1 B-protein +needs O +to O +recognize O +both O +the O +accepter B-structure_element +stem I-structure_element +and O +anticodon B-structure_element +of O +tRNAHis B-chemical +. O + +The O +tetrameric B-oligomeric_state +architecture O +of O +the O +Thg1 B-protein +molecule O +allows O +it O +to O +access O +both O +regions O +located O +at O +the O +opposite O +side O +of O +the O +tRNA B-chemical +molecule O +[ O +the O +AB B-structure_element +dimer B-oligomeric_state +recognizes O +the O +accepter B-structure_element +stem I-structure_element +and O +CD B-structure_element +dimer B-oligomeric_state +anticodon O +]. O + +In O +contrast O +, O +the O +binding O +mode O +of O +TLP B-protein_type +corresponds O +to O +the O +anticodon O +- O +independent O +repair O +reactions O +of O +5 O +′- O +truncated O +general O +tRNAs B-chemical +. O + +This O +binding O +mode O +is O +also O +suitable O +for O +the O +correct O +termination O +of O +the O +elongation O +or O +repair O +reaction O +by O +measuring O +the O +length O +of O +the O +accepter B-structure_element +stem I-structure_element +by O +the O +flexible B-protein_state +β B-structure_element +- I-structure_element +hairpin I-structure_element +. O + +Because O +tRNAHis B-chemical +requires O +an O +extra O +guanosine B-chemical +( O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +) O +at O +the O +5 O +′- O +end O +, O +the O +repair O +enzyme O +has O +to O +extend O +the O +5 O +′- O +end O +by O +one O +more O +nucleotide O +than O +other O +tRNAs B-chemical +. O + +TLP B-protein_type +has O +been O +shown O +to O +confer O +such O +catalytic O +activity O +on O +tRNAHisΔ B-chemical +− I-chemical +1 I-chemical +( O +Fig O +. O +4B O +). O + +Here O +, O +we O +showed O +that O +the O +TLP B-protein_type +mutants B-protein_state +, O +wherein O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +is O +truncated B-protein_state +and O +tRNAPheΔ1 B-chemical +binding O +ability O +is O +lost O +, O +can O +still O +bind O +to O +tRNAPhe B-chemical +( O +GUG B-chemical +) O +whose O +anticodon O +is O +changed O +to O +that O +for O +His B-residue_name +( O +fig O +. O +S6 O +, O +C O +, O +H O +, O +and O +I O +). O + +Also O +, O +the O +intact O +tRNAPhe B-chemical +, O +which O +is O +not O +recognized O +by O +TLP B-protein_type +( O +Fig O +. O +4B O +and O +fig O +. O +S6E O +), O +can O +be O +recognized O +when O +its O +anticodon O +is O +changed O +to O +that O +for O +His B-residue_name +( O +fig O +. O +S6D O +). O + +Furthermore O +, O +the O +TLP B-protein_type +variant B-protein_state +( O +F174A B-mutant +/ O +N179A B-mutant +/ O +R188A B-mutant +) O +whose O +anticodon B-site +recognition I-site +site I-site +[ O +deduced O +from O +the O +Thg1 B-complex_assembly +- I-complex_assembly +tRNAHis I-complex_assembly +complex O +structure B-evidence +] O +is O +disrupted O +has O +been O +shown O +to O +have O +a O +reduced O +catalytic O +activity O +to O +tRNAHisΔ B-chemical +− I-chemical +1 I-chemical +( O +Fig O +. O +4B O +). O + +All O +these O +experimental O +results O +indicate O +that O +TLP B-protein_type +recognizes O +and O +binds O +tRNAs B-chemical +carrying O +the O +His B-residue_name +anticodon O +in O +the O +same O +way O +that O +Thg1 B-protein +recognizes O +tRNAHis B-chemical +. O + +Thus O +, O +we O +concluded O +that O +TLP B-protein_type +has O +two O +tRNA B-chemical +binding O +modes O +that O +are O +selectively O +used O +, O +depending O +on O +both O +the O +length O +of O +the O +accepter B-structure_element +stem I-structure_element +and O +the O +anticodon B-structure_element +. O + +The O +elongation O +or O +repair O +reaction O +normally O +terminates O +when O +the O +5 O +′- O +end O +reaches O +position O +1 O +, O +but O +when O +the O +His B-residue_name +anticodon O +is O +present O +, O +TLP B-protein_type +binds O +the O +tRNA B-chemical +in O +the O +second O +mode O +by O +recognizing O +the O +anticodon O +to O +execute O +the O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +addition O +reaction O +. O + +By O +having O +two O +different O +binding O +modes O +, O +TLP B-protein_type +can O +manage O +this O +special O +feature O +of O +tRNAHis B-chemical +. O + +The O +Thg1 B-protein +/ O +TLP B-protein_type +family O +of O +proteins O +extends O +tRNA B-chemical +chains O +in O +the O +3 O +′- O +5 O +′ O +direction O +. O + +First O +, O +the O +5 B-chemical +′- I-chemical +phosphate I-chemical +is O +activated O +by O +GTP B-chemical +/ O +ATP B-chemical +. O + +Then O +, O +the O +activated O +phosphate B-chemical +is O +attacked O +by O +the O +incoming O +nucleotide O +, O +resulting O +in O +an O +extension O +by O +one O +nucleotide O +at O +the O +5 O +′- O +end O +. O + +Here O +, O +we O +successfully O +solved B-experimental_method +for O +the O +first O +time O +the O +intermediate O +structures B-evidence +of O +the O +template O +- O +dependent O +3 O +′- O +5 O +′ O +elongation O +complex O +of O +MaTLP B-protein +. O + +On O +the O +basis O +of O +these O +structures B-evidence +, O +we O +will O +discuss O +the O +3 O +′- O +5 O +′ O +addition O +reaction O +compared O +with O +canonical O +5 O +′- O +3 O +′ O +elongation O +by O +DNA B-protein_type +/ I-protein_type +RNA I-protein_type +polymerases I-protein_type +. O + +Figure O +5 O +is O +a O +schematic O +diagram O +of O +the O +3 O +′- O +5 O +′ O +addition O +reaction O +of O +TLP B-protein_type +. O + +This O +enzyme O +has O +two O +triphosphate B-site +binding I-site +sites I-site +and O +one O +reaction B-site +center I-site +at O +the O +position O +overlapping O +these O +two O +binding B-site +sites I-site +( O +Fig O +. O +5A O +). O + +In O +the O +first O +activation O +step O +, O +when O +GTP B-chemical +/ O +ATP B-chemical +is O +bound B-protein_state +to I-protein_state +site B-site +1 I-site +( O +Fig O +. O +5B O +), O +the O +5 B-chemical +′- I-chemical +phosphate I-chemical +of O +the O +tRNA B-chemical +is O +deprotonated O +by O +Mg2 B-chemical ++ I-chemical +A O +and O +attacks O +the O +α O +- O +phosphate B-chemical +of O +the O +GTP B-chemical +/ O +ATP B-chemical +, O +resulting O +in O +an O +activated O +intermediate O +( O +Fig O +. O +5C O +). O + +The O +structure B-evidence +of O +the O +MaTLP B-complex_assembly +- I-complex_assembly +ppptRNAPheΔ1 I-complex_assembly +complex O +, O +wherein O +β O +- O +and O +γ O +- O +phosphates B-chemical +coordinate B-bond_interaction +with I-bond_interaction +Mg2 B-chemical ++ I-chemical +A O +and O +Mg2 B-chemical ++ I-chemical +B O +, O +respectively O +( O +Figs O +. O +3A O +and O +5C O +′), O +may O +represent O +this O +activated O +intermediate O +. O + +Subsequent O +binding O +of O +an O +incoming O +nucleotide B-chemical +to O +site B-site +2 I-site +followed O +by O +formation O +of O +the O +Watson B-bond_interaction +- I-bond_interaction +Crick I-bond_interaction +base I-bond_interaction +pair I-bond_interaction +with O +a O +nucleotide B-chemical +in O +the O +template O +strand O +conveys O +the O +3 O +′- O +OH O +of O +the O +incoming O +nucleotide B-chemical +to O +the O +position O +of O +deprotonation O +by O +Mg2 B-chemical ++ I-chemical +A O +and O +the O +5 B-chemical +′- I-chemical +triphosphate I-chemical +of O +the O +tRNA B-chemical +to O +the O +reaction B-site +center I-site +( O +Figs O +. O +3B O +and O +5D O +). O + +Thus O +, O +the O +present O +structure B-evidence +shows O +that O +this O +3 B-protein_type +′- I-protein_type +5 I-protein_type +′ I-protein_type +elongation I-protein_type +enzyme I-protein_type +utilizes O +a O +reaction B-site +center I-site +homologous O +to O +that O +of O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +elongation I-protein_type +enzymes I-protein_type +for O +both O +activation O +and O +elongation O +in O +a O +stepwise O +fashion O +. O + +It O +should O +be O +noted O +that O +TLP B-protein_type +has O +evolved O +to O +allow O +the O +occurrence O +of O +these O +two O +elaborate O +reaction O +steps O +within O +one O +reaction B-site +center I-site +. O + +( O +A O +) O +The O +reaction B-site +center I-site +overlapped O +with O +two O +triphosphate B-site +binding I-site +sites I-site +. O + +A O +, O +B O +, O +and O +C O +( O +in O +green O +) O +represent O +binding B-site +sites I-site +for O +Mg2 B-chemical ++ I-chemical +A O +, O +Mg2 B-chemical ++ I-chemical +B O +, O +and O +Mg2 B-chemical ++ I-chemical +C O +. O +P B-site +( O +in O +blue O +) O +represents O +the O +phosphate B-site +binding I-site +sites I-site +; O +O O +− O +( O +in O +red O +) O +is O +the O +binding B-site +site I-site +for O +the O +deprotonated O +OH O +group O +. O + +Important O +TLP B-protein_type +residues O +for O +tRNA B-chemical +and O +Mg2 B-chemical ++ I-chemical +binding O +are O +also O +shown O +. O +( O +B O +) O +Structure B-evidence +of O +the O +activation O +complex O +( O +corresponding O +to O +fig O +. O +S8 O +). O + +GTP B-chemical +/ O +ATP B-chemical +binds O +to O +triphosphate B-site +binding I-site +site I-site +1 I-site +; O +the O +deprotonated O +OH O +group O +of O +the O +5 B-chemical +′- I-chemical +phosphate I-chemical +attacks O +the O +α O +- O +phosphate B-chemical +of O +GTP B-chemical +/ O +ATP B-chemical +, O +and O +PPi B-chemical +( O +inorganic B-chemical +pyrophosphate I-chemical +) O +is O +released O +. O +( O +C O +) O +Possible O +structure B-evidence +after O +the O +activation O +step O +as O +suggested O +from O +the O +structure B-evidence +of O +( O +C O +′). O + +( O +C O +′) O +Structure B-evidence +before O +the O +elongation O +reaction O +( O +corresponding O +to O +Fig O +. O +3A O +). O + +The O +5 B-chemical +′- I-chemical +triphosphate I-chemical +of O +the O +tRNA B-chemical +binds O +to O +the O +same O +site O +as O +for O +activation O +of O +the O +5 O +′- O +terminus O +of O +the O +tRNA B-chemical +in O +( O +B O +). O + +( O +D O +) O +Structure B-evidence +of O +initiation O +of O +the O +elongation O +reaction O +( O +corresponding O +to O +Fig O +. O +3B O +). O + +The O +base O +of O +the O +incoming O +GTP B-chemical +forms O +a O +Watson B-bond_interaction +- I-bond_interaction +Crick I-bond_interaction +hydrogen I-bond_interaction +bond I-bond_interaction +with O +the O +nucleotide B-chemical +at O +position O +72 B-residue_number +in O +the O +template O +chain O +and O +a O +base B-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +interaction I-bond_interaction +with O +a O +neighboring O +base O +( O +G2 B-residue_name_number +). O + +Movement O +of O +the O +5 O +′- O +terminal O +chain O +leaves O +the O +5 B-chemical +′- I-chemical +triphosphate I-chemical +of O +the O +tRNA B-chemical +in O +the O +same O +site O +as O +the O +activation O +step O +in O +( O +B O +). O + +The O +3 O +′- O +OH O +of O +the O +incoming O +GTP B-chemical +is O +deprotonated O +by O +Mg2 B-chemical ++ I-chemical +A O +and O +attacks O +the O +α O +- O +phosphate B-chemical +to O +form O +a O +covalent O +bond O +. O +( O +E O +) O +After O +the O +elongation O +reaction O +, O +the O +triphosphate B-chemical +of O +the O +new O +nucleotide B-chemical +is O +placed O +on O +site B-site +1 I-site +, O +as O +in O +( O +C O +′), O +and O +is O +ready O +for O +the O +next O +reaction O +. O + +Figure O +6 O +compares O +the O +3 O +′- O +5 O +′ O +and O +5 O +′- O +3 O +′ O +elongation O +mechanisms O +, O +showing O +the O +symmetrical O +nature O +of O +both O +elongation O +reactions O +using O +a O +similar O +reaction B-site +center I-site +composed O +of O +Mg2 B-chemical ++ I-chemical +A O +and O +Mg2 B-chemical ++ I-chemical +B O +in O +the O +conserved B-protein_state +catalytic B-site +core I-site +. O + +In O +TLP B-protein_type +, O +which O +carries O +out O +3 O +′- O +5 O +′ O +elongation O +, O +the O +3 O +′- O +OH O +of O +the O +incoming O +nucleotide O +attacks O +the O +5 O +′- O +activated O +phosphate B-chemical +of O +the O +tRNA B-chemical +to O +form O +a O +phosphodiester O +bond O +, O +whereas O +in O +the O +T7 B-protein +RNA I-protein +polymerase I-protein +, O +a O +representative O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +DNA I-protein_type +/ I-protein_type +RNA I-protein_type +polymerase I-protein_type +, O +the O +3 O +′- O +OH O +of O +the O +3 O +′- O +terminal O +nucleotide O +of O +the O +RNA B-chemical +attacks O +the O +activated O +phosphate B-chemical +of O +the O +incoming O +nucleotide O +to O +form O +a O +phosphodiester O +bond O +. O + +In O +these O +reactions O +, O +the O +roles O +of O +the O +two O +Mg B-chemical +ions O +are O +identical O +. O + +Mg2 B-chemical ++ I-chemical +A O +activates O +the O +3 O +′- O +OH O +of O +the O +incoming O +nucleotide O +in O +TLP B-protein_type +and O +the O +3 O +′- O +OH O +of O +the O +3 O +′- O +end O +of O +the O +RNA B-chemical +chain O +in O +T7 B-protein +RNA I-protein +polymerase I-protein +. O + +The O +role O +of O +Mg2 B-chemical ++ I-chemical +B O +is O +to O +position O +the O +5 B-chemical +′- I-chemical +triphosphate I-chemical +of O +the O +tRNA B-chemical +in O +TLP B-protein_type +and O +the O +incoming O +nucleotide O +in O +T7 B-protein +RNA I-protein +polymerase I-protein +. O + +These O +two O +Mg2 B-chemical ++ I-chemical +ions O +are O +coordinated B-bond_interaction +by I-bond_interaction +a O +conserved B-protein_state +Asp B-residue_name +( O +D21 B-residue_name_number +and O +D69 B-residue_name_number +in O +TLP B-protein_type +) O +in O +the O +conserved B-protein_state +catalytic B-site +core I-site +. O + +Structures B-evidence +of O +template O +- O +dependent O +nucleotide O +elongation O +in O +the O +3 O +′- O +5 O +′ O +and O +5 O +′- O +3 O +′ O +directions O +. O + +Symmetrical O +relationship O +between O +3 O +′- O +5 O +′ O +elongation O +by O +TLP B-protein_type +( O +this O +study O +) O +( O +left O +) O +and O +5 O +′- O +3 O +′ O +elongation O +by O +T7 B-protein +RNA I-protein +polymerase I-protein +[ O +Protein O +Data O +Bank O +( O +PDB O +) O +ID O +: O +1S76 O +] O +( O +right O +). O + +In O +the O +3 O +′- O +5 O +′ O +elongation O +reaction O +, O +the O +3 O +′- O +OH O +of O +the O +incoming O +nucleotide O +attacks O +the O +5 O +′- O +activated O +phosphate B-chemical +of O +the O +tRNA B-chemical +to O +form O +a O +phosphodiester O +bond O +, O +whereas O +in O +the O +5 O +′- O +3 O +′ O +elongation O +reaction O +, O +the O +3 O +′- O +OH O +of O +the O +3 O +′- O +terminal O +nucleotide O +of O +the O +RNA B-chemical +attacks O +the O +activated O +phosphate B-chemical +of O +the O +incoming O +nucleotide O +to O +form O +a O +phosphodiester O +bond O +. O + +Green O +spheres O +represent O +Mg2 B-chemical ++ I-chemical +ions O +. O + +Because O +the O +chemical O +roles O +of O +tRNA B-chemical +and O +the O +incoming O +nucleotide O +are O +reversed O +in O +these O +two O +reactions O +, O +these O +two O +substrates O +are O +inserted O +into O +a O +similar O +reaction B-site +center I-site +from O +opposite O +directions O +( O +Fig O +. O +6 O +). O + +However O +, O +from O +an O +energetic O +viewpoint O +, O +these O +two O +reactions O +are O +clearly O +different O +: O +Whereas O +the O +high O +energy O +of O +the O +incoming O +nucleotide O +is O +used O +for O +its O +own O +addition O +in O +DNA B-protein_type +/ I-protein_type +RNA I-protein_type +polymerases I-protein_type +, O +the O +high O +energy O +of O +the O +incoming O +nucleotide O +is O +used O +for O +subsequent O +addition O +in O +TLP B-protein_type +. O + +For O +this O +reason O +, O +TLP B-protein_type +requires O +a O +mechanism O +that O +activates O +the O +5 O +′- O +terminus O +of O +the O +tRNA B-chemical +during O +the O +initial O +step O +of O +the O +reaction O +. O + +Our O +analysis O +showed O +that O +the O +initial O +activation O +and O +subsequent O +elongation O +reactions O +occur O +sequentially O +at O +one O +reaction B-site +center I-site +. O + +In O +this O +case O +, O +the O +enzyme O +needs O +to O +create O +two O +substrate B-site +binding I-site +sites I-site +for O +two O +different O +reactions O +in O +the O +vicinities O +of O +one O +reaction B-site +center I-site +. O + +TLP B-protein_type +has O +successfully O +created O +such O +sites O +by O +utilizing O +a O +conformational O +change O +in O +the O +tRNA B-chemical +through O +Watson B-bond_interaction +- I-bond_interaction +Crick I-bond_interaction +base I-bond_interaction +pairing I-bond_interaction +( O +Fig O +. O +3 O +). O + +These O +structural O +features O +of O +the O +TLP B-protein_type +molecule O +suggest O +that O +development O +of O +an O +activation B-site +reaction I-site +site I-site +is O +a O +prerequisite O +for O +developing O +the O +3 B-protein_type +′- I-protein_type +5 I-protein_type +′ I-protein_type +elongation I-protein_type +enzyme I-protein_type +. O + +This O +is O +clearly O +more O +difficult O +than O +developing O +the O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +elongation I-protein_type +enzyme I-protein_type +, O +wherein O +the O +activation B-site +reaction I-site +site I-site +is O +not O +necessary O +, O +and O +which O +may O +be O +the O +primary O +reason O +why O +the O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +elongation I-protein_type +enzyme I-protein_type +has O +been O +exclusively O +developed O +. O + +Here O +, O +we O +established O +a O +structural O +basis O +for O +3 O +′- O +5 O +′ O +nucleotide O +elongation O +and O +showed O +that O +TLP B-protein_type +has O +evolved O +to O +acquire O +a O +two O +- O +step O +Watson O +- O +Crick O +template O +– O +dependent O +3 O +′- O +5 O +′ O +elongation O +reaction O +using O +the O +catalytic B-site +center I-site +homologous O +to O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +elongation I-protein_type +enzymes I-protein_type +. O + +The O +active B-site +site I-site +of O +this O +enzyme O +is O +created O +at O +the O +dimerization B-site +interface I-site +. O + +The O +dimerization O +also O +endows O +this O +protein O +with O +the O +ability O +to O +measure O +the O +length O +of O +the O +accepter B-structure_element +stem I-structure_element +of O +the O +tRNA B-chemical +substrate O +, O +so O +that O +the O +enzyme O +can O +properly O +terminate O +the O +elongation O +reaction O +. O + +Furthermore O +, O +the O +dual O +binding O +mode O +of O +this O +protein O +suggests O +that O +it O +has O +further O +evolved O +to O +cover O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +addition O +of O +tRNAHis B-chemical +by O +additional O +dimerization O +( O +dimer B-oligomeric_state +of O +dimers B-oligomeric_state +). O + +Thus O +, O +the O +present O +structural B-experimental_method +analysis I-experimental_method +is O +consistent O +with O +the O +scenario O +in O +which O +TLP B-protein_type +began O +as O +a O +5 O +′- O +end O +repair O +enzyme O +and O +evolved O +into O +a O +tRNAHis B-protein_type +- I-protein_type +specific I-protein_type +G I-protein_type +− I-protein_type +1 I-protein_type +addition I-protein_type +enzyme I-protein_type +. O + +The O +detailed O +molecular O +mechanism O +of O +the O +Thg1 B-protein +/ O +TLP B-protein_type +family O +established O +by O +our O +analysis O +will O +open O +up O +new O +perspectives O +in O +our O +understanding O +of O +3 O +′- O +5 O +′ O +versus O +5 O +′- O +3 O +′ O +polymerization O +and O +the O +molecular O +evolution O +of O +template B-protein_type +- I-protein_type +dependent I-protein_type +polymerases I-protein_type +. O + +Transcribed O +tRNAs B-chemical +were O +purified O +by O +a O +HiTrap O +DEAE O +FF O +column O +( O +GE O +Healthcare O +) O +as O +previously O +described O +. O + +Pooled O +tRNAs B-chemical +were O +precipitated O +with O +isopropanol O +and O +dissolved O +in O +buffer O +E O +[ O +20 O +mM O +Hepes O +- O +NaOH O +( O +pH O +7 O +. O +5 O +), O +100 O +mM O +NaCl O +, O +and O +10 O +mM O +MgCl2 O +]. O + +The O +highly O +conserved O +tRNAHis O +guanylyltransferase O +Thg1p B-protein +interacts O +with O +the O +origin O +recognition O +complex O +and O +is O +required O +for O +the O +G2 O +/ O +M O +phase O +transition O +in O +the O +yeast O +Saccharomyces O +cerevisiae O + +Structural O +diversity O +in O +a O +human B-species +antibody B-protein_type +germline O +library O + +To O +support O +antibody B-protein_type +therapeutic O +development O +, O +the O +crystal B-evidence +structures I-evidence +of O +a O +set O +of O +16 O +germline O +variants O +composed O +of O +4 O +different O +kappa B-structure_element +light I-structure_element +chains I-structure_element +paired O +with O +4 O +different O +heavy B-structure_element +chains I-structure_element +have O +been O +determined O +. O + +All O +four O +heavy B-structure_element +chains I-structure_element +of O +the O +antigen B-structure_element +- I-structure_element +binding I-structure_element +fragments I-structure_element +( O +Fabs B-structure_element +) O +have O +the O +same O +complementarity B-structure_element +- I-structure_element +determining I-structure_element +region I-structure_element +( O +CDR B-structure_element +) O +H3 B-structure_element +that O +was O +reported O +in O +an O +earlier O +Fab B-structure_element +structure B-evidence +. O + +The O +structure B-experimental_method +analyses I-experimental_method +include O +comparisons O +of O +the O +overall O +structures B-evidence +, O +canonical O +structures B-evidence +of O +the O +CDRs B-structure_element +and O +the O +VH B-complex_assembly +: I-complex_assembly +VL I-complex_assembly +packing B-bond_interaction +interactions I-bond_interaction +. O + +The O +CDR B-structure_element +conformations O +for O +the O +most O +part O +are O +tightly O +clustered O +, O +especially O +for O +the O +ones O +with O +shorter O +lengths O +. O + +The O +longer B-protein_state +CDRs B-structure_element +with O +tandem O +glycines B-residue_name +or O +serines B-residue_name +have O +more O +conformational O +diversity O +than O +the O +others O +. O + +CDR B-structure_element +H3 B-structure_element +, O +despite O +having O +the O +same O +amino O +acid O +sequence O +, O +exhibits O +the O +largest O +conformational O +diversity O +. O + +About O +half O +of O +the O +structures B-evidence +have O +CDR B-structure_element +H3 B-structure_element +conformations O +similar O +to O +that O +of O +the O +parent O +; O +the O +others O +diverge O +significantly O +. O + +One O +conclusion O +is O +that O +the O +CDR B-structure_element +H3 B-structure_element +conformations O +are O +influenced O +by O +both O +their O +amino O +acid O +sequence O +and O +their O +structural O +environment O +determined O +by O +the O +heavy B-structure_element +and O +light B-structure_element +chain I-structure_element +pairing O +. O + +The O +stem B-structure_element +regions I-structure_element +of O +14 O +of O +the O +variant O +pairs O +are O +in O +the O +‘ O +kinked B-protein_state +’ O +conformation O +, O +and O +only O +2 O +are O +in O +the O +extended B-protein_state +conformation O +. O + +The O +packing O +of O +the O +VH B-structure_element +and O +VL B-structure_element +domains O +is O +consistent O +with O +our O +knowledge O +of O +antibody B-protein_type +structure B-evidence +, O +and O +the O +tilt B-evidence +angles I-evidence +between O +these O +domains O +cover O +a O +range O +of O +11 O +degrees O +. O + +Two O +of O +16 O +structures B-evidence +showed O +particularly O +large O +variations O +in O +the O +tilt B-evidence +angles I-evidence +when O +compared O +with O +the O +other O +pairings O +. O + +The O +structures B-evidence +and O +their O +analyses O +provide O +a O +rich O +foundation O +for O +future O +antibody B-protein_type +modeling O +and O +engineering O +efforts O +. O + +At O +present O +, O +therapeutic O +antibodies B-protein_type +are O +the O +largest O +class O +of O +biotherapeutic O +proteins O +that O +are O +in O +clinical O +trials O +. O + +The O +use O +of O +monoclonal O +antibodies B-protein_type +as O +therapeutics O +began O +in O +the O +early O +1980s O +, O +and O +their O +composition O +has O +transitioned O +from O +murine B-taxonomy_domain +antibodies B-protein_type +to O +generally O +less O +immunogenic O +humanized O +and O +human B-species +antibodies B-protein_type +. O + +The O +technologies O +currently O +used O +to O +obtain O +human B-species +antibodies B-protein_type +include O +transgenic O +mice B-taxonomy_domain +containing O +human B-species +antibody B-protein_type +repertoires O +, O +cloning O +directly O +from O +human B-species +B O +cells O +, O +and O +in B-experimental_method +vitro I-experimental_method +selection I-experimental_method +from O +antibody B-experimental_method +libraries I-experimental_method +using O +various O +display O +technologies O +. O + +Once O +a O +candidate O +antibody B-protein_type +is O +identified O +, O +protein B-experimental_method +engineering I-experimental_method +is O +usually O +required O +to O +produce O +a O +molecule O +with O +the O +right O +biophysical O +and O +functional O +properties O +. O + +All O +engineering O +efforts O +are O +guided O +by O +our O +understanding O +of O +the O +atomic B-evidence +structures I-evidence +of O +antibodies B-protein_type +. O + +In O +such O +efforts O +, O +the O +crystal B-evidence +structure I-evidence +of O +the O +specific O +antibody B-protein_type +may O +not O +be O +available O +, O +but O +modeling O +can O +be O +used O +to O +guide O +the O +engineering O +efforts O +. O + +Today O +' O +s O +antibody B-protein_type +modeling O +approaches O +, O +which O +normally O +focus O +on O +the O +variable B-structure_element +region I-structure_element +, O +are O +being O +developed O +by O +the O +application O +of O +structural O +principles O +and O +insights O +that O +are O +evolving O +as O +our O +knowledge O +of O +antibody B-protein_type +structures B-evidence +continues O +to O +expand O +. O + +Our O +current O +structural O +knowledge O +of O +antibodies B-protein_type +is O +based O +on O +a O +multitude O +of O +studies O +that O +used O +many O +techniques O +to O +gain O +insight O +into O +the O +functional O +and O +structural O +properties O +of O +this O +class O +of O +macromolecule O +. O + +Five O +different O +antibody B-protein_type +isotypes O +occur O +, O +IgG B-protein +, O +IgD B-protein +, O +IgE B-protein +, O +IgA B-protein +and O +IgM B-protein +, O +and O +each O +isotype O +has O +a O +unique O +role O +in O +the O +adaptive O +immune O +system O +. O + +IgG B-protein +, O +IgD B-protein +and O +IgE B-protein +isotypes O +are O +composed O +of O +2 O +heavy B-structure_element +chains I-structure_element +( O +HCs B-structure_element +) O +and O +2 O +light B-structure_element +chains I-structure_element +( O +LCs B-structure_element +) O +linked O +through O +disulfide B-ptm +bonds I-ptm +, O +while O +IgA B-protein +and O +IgM B-protein +are O +double O +and O +quintuple O +versions O +of O +antibodies B-protein_type +, O +respectively O +. O + +Isotypes O +IgG B-protein +, O +IgD B-protein +and O +IgA B-protein +each O +have O +4 O +domains O +, O +one O +variable B-structure_element +( O +V B-structure_element +) O +and O +3 O +constant B-structure_element +( O +C B-structure_element +) O +domains O +, O +while O +IgE B-protein +and O +IgM B-protein +each O +have O +the O +same O +4 O +domains O +along O +with O +an O +additional O +C B-structure_element +domain I-structure_element +. O + +These O +multimeric O +forms O +are O +linked O +with O +an O +additional O +J B-structure_element +chain O +. O + +The O +LCs B-structure_element +that O +associate O +with O +the O +HCs B-structure_element +are O +divided O +into O +2 O +functionally O +indistinguishable O +classes O +, O +κ B-structure_element +and O +λ B-structure_element +. O + +Both O +κ B-structure_element +and O +λ B-structure_element +polypeptide O +chains O +are O +composed O +of O +a O +single O +V B-structure_element +domain I-structure_element +and O +a O +single O +C B-structure_element +domain I-structure_element +. O + +The O +heavy B-structure_element +and O +light B-structure_element +chains I-structure_element +are O +composed O +of O +structural B-structure_element +domains I-structure_element +that O +have O +∼ B-residue_range +110 I-residue_range +amino I-residue_range +acid I-residue_range +residues I-residue_range +. O + +These O +domains O +have O +a O +common O +folding O +pattern O +often O +referred O +to O +as O +the O +“ O +immunoglobulin B-structure_element +fold I-structure_element +,” O +formed O +by O +the O +packing O +together O +of O +2 O +anti B-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +. O + +All O +immunoglobulin B-protein_type +chains I-protein_type +have O +an O +N O +- O +terminal O +V B-structure_element +domain I-structure_element +followed O +by O +1 O +to O +4 O +C B-structure_element +domains I-structure_element +, O +depending O +upon O +the O +chain O +type O +. O + +In O +antibodies B-protein_type +, O +the O +heavy B-structure_element +and I-structure_element +light I-structure_element +chain I-structure_element +V B-structure_element +domains I-structure_element +pack O +together O +forming O +the O +antigen B-site +combining I-site +site I-site +. O + +This O +site O +, O +which O +interacts O +with O +the O +antigen O +( O +or O +target O +), O +is O +the O +focus O +of O +current O +antibody B-protein_type +modeling O +efforts O +. O + +This O +interaction B-site +site I-site +is O +composed O +of O +6 O +complementarity B-structure_element +- I-structure_element +determining I-structure_element +regions I-structure_element +( O +CDRs B-structure_element +) O +that O +were O +identified O +in O +early O +antibody B-experimental_method +amino I-experimental_method +acid I-experimental_method +sequence I-experimental_method +analyses I-experimental_method +to O +be O +hypervariable B-protein_state +in O +nature O +, O +and O +thus O +are O +responsible O +for O +the O +sequence O +and O +structural O +diversity O +of O +our O +antibody B-protein_type +repertoire O +. O + +The O +sequence O +diversity O +of O +the O +CDR B-structure_element +regions I-structure_element +presents O +a O +substantial O +challenge O +to O +antibody B-protein_type +modeling O +. O + +However O +, O +an O +initial O +structural B-experimental_method +analysis I-experimental_method +of O +the O +combining B-site +sites I-site +of O +the O +small O +set O +of O +structures B-evidence +of O +immunoglobulin O +fragments O +available O +in O +the O +1980s O +found O +that O +5 O +of O +the O +6 O +hypervariable B-structure_element +loops I-structure_element +or O +CDRs B-structure_element +had O +canonical O +structures O +( O +a O +limited O +set O +of O +main O +- O +chain O +conformations O +). O + +A O +CDR B-structure_element +canonical O +structure O +is O +defined O +by O +its O +length O +and O +conserved O +residues O +located O +in O +the O +hypervariable B-structure_element +loop I-structure_element +and O +framework B-structure_element +residues I-structure_element +( O +V B-structure_element +- I-structure_element +region I-structure_element +residues O +that O +are O +not O +part O +of O +the O +CDRs B-structure_element +). O + +Furthermore O +, O +studies O +of O +antibody B-protein_type +sequences O +revealed O +that O +the O +total O +number O +of O +canonical O +structures O +are O +limited O +for O +each O +CDR B-structure_element +, O +indicating O +possibly O +that O +antigen O +recognition O +may O +be O +affected O +by O +structural O +restrictions O +at O +the O +antigen B-site +- I-site +binding I-site +site I-site +. O + +Later O +studies O +found O +that O +the O +CDR B-structure_element +loop I-structure_element +length O +is O +the O +primary O +determining O +factor O +of O +antigen B-site +- I-site +binding I-site +site I-site +topography O +because O +it O +is O +the O +primary O +factor O +for O +determining O +a O +canonical O +structure O +. O + +Additional O +efforts O +have O +led O +to O +our O +current O +understanding O +that O +the O +LC B-structure_element +CDRs B-structure_element +L1 B-structure_element +, O +L2 B-structure_element +, O +and O +L3 B-structure_element +have O +preferred O +sets O +of O +canonical O +structures O +based O +on O +length O +and O +amino O +acid O +sequence O +composition O +. O + +This O +was O +also O +found O +to O +be O +the O +case O +for O +the O +H1 B-structure_element +and O +H2 B-structure_element +CDRs B-structure_element +. O + +Classification O +schemes O +for O +the O +canonical O +structures O +of O +these O +5 O +CDRs B-structure_element +have O +emerged O +and O +evolved O +as O +the O +number O +of O +depositions O +in O +the O +Protein O +Data O +Bank O +of O +Fab B-structure_element +fragments O +of O +antibodies B-protein_type +grow O +. O + +Recently O +, O +a O +comprehensive O +CDR B-structure_element +classification O +scheme O +was O +reported O +identifying O +72 O +clusters O +of O +conformations O +observed O +in O +antibody B-protein_type +structures B-evidence +. O + +The O +knowledge O +and O +predictability O +of O +these O +CDR B-structure_element +canonical O +structures B-evidence +have O +greatly O +advanced O +antibody B-protein_type +modeling O +efforts O +. O + +In O +contrast O +to O +CDRs B-structure_element +L1 B-structure_element +, O +L2 B-structure_element +, O +L3 B-structure_element +, O +H1 B-structure_element +and O +H2 B-structure_element +, O +no O +canonical O +structures B-evidence +have O +been O +observed O +for O +CDR B-structure_element +H3 B-structure_element +, O +which O +is O +the O +most O +variable O +in O +length O +and O +amino O +acid O +sequence O +. O + +Some O +clustering O +of O +conformations O +was O +observed O +for O +the O +shortest O +lengths O +; O +however O +, O +for O +the O +longer O +loops B-structure_element +, O +only O +the O +portions O +nearest O +the O +framework B-structure_element +( O +torso B-structure_element +, O +stem B-structure_element +or O +anchor B-structure_element +region I-structure_element +) O +were O +found O +to O +have O +defined O +conformations O +. O + +In O +the O +torso B-structure_element +region I-structure_element +, O +2 O +primary O +groups O +could O +be O +identified O +, O +which O +led O +to O +sequence O +- O +based O +rules O +that O +can O +predict O +with O +some O +degree O +of O +reliability O +the O +conformation O +of O +the O +stem B-structure_element +region I-structure_element +. O + +The O +“ O +kinked B-protein_state +” O +or O +“ O +bulged B-protein_state +” O +conformation O +is O +the O +most O +prevalent O +, O +but O +an O +“ O +extended B-protein_state +” O +or O +“ O +non B-protein_state +- I-protein_state +bulged I-protein_state +” O +conformation O +is O +also O +, O +but O +less O +frequently O +, O +observed O +. O + +The O +cataloging O +and O +development O +of O +the O +rules O +for O +predicting O +the O +conformation O +of O +the O +anchor B-structure_element +region I-structure_element +of O +CDR B-structure_element +H3 B-structure_element +continue O +to O +be O +refined O +, O +producing O +new O +insight O +into O +the O +CDR B-structure_element +H3 B-structure_element +conformations O +and O +new O +tools O +for O +antibody B-protein_type +engineering O +. O + +Current O +antibody B-protein_type +modeling O +approaches O +take O +advantage O +of O +the O +most O +recent O +advances O +in O +homology B-experimental_method +modeling I-experimental_method +, O +the O +evolving O +understanding O +of O +the O +CDR B-structure_element +canonical O +structures B-evidence +, O +the O +emerging O +rules O +for O +CDR B-structure_element +H3 B-structure_element +modeling O +and O +the O +growing O +body O +of O +antibody B-protein_type +structural O +data O +available O +from O +the O +PDB O +. O + +Recent O +antibody B-experimental_method +modeling I-experimental_method +assessments I-experimental_method +show O +continued O +improvement O +in O +the O +quality O +of O +the O +models O +being O +generated O +by O +a O +variety O +of O +modeling O +methods O +. O + +Although O +antibody B-protein_type +modeling O +is O +improving O +, O +the O +latest O +assessment O +revealed O +a O +number O +of O +challenges O +that O +need O +to O +be O +overcome O +to O +provide O +accurate O +3 O +- O +dimensional O +models O +of O +antibody B-protein_type +V B-structure_element +regions I-structure_element +, O +including O +accuracies O +in O +the O +modeling O +of O +CDR B-structure_element +H3 B-structure_element +. O + +The O +need O +for O +improvement O +in O +this O +area O +was O +also O +highlighted O +in O +a O +recent O +study O +reporting O +an O +approach O +and O +results O +that O +may O +influence O +future O +antibody B-protein_type +modeling O +efforts O +. O + +One O +important O +finding O +of O +the O +antibody B-experimental_method +modeling I-experimental_method +assessments I-experimental_method +was O +that O +errors O +in O +the O +structural O +templates O +that O +are O +used O +as O +the O +basis O +for O +homology B-experimental_method +models I-experimental_method +can O +propagate O +into O +the O +final O +models O +, O +producing O +inaccuracies O +that O +may O +negatively O +influence O +the O +predictive O +nature O +of O +the O +V B-structure_element +region I-structure_element +model O +. O + +To O +support O +antibody B-protein_type +engineering O +and O +therapeutic O +development O +efforts O +, O +a O +phage B-experimental_method +library I-experimental_method +was O +designed O +and O +constructed O +based O +on O +a O +limited O +number O +of O +scaffolds O +built O +with O +frequently O +used O +human B-species +germ O +- O +line O +IGV B-structure_element +and O +IGJ B-structure_element +gene O +segments O +that O +encode O +antigen B-site +combining I-site +sites I-site +suitable O +for O +recognition O +of O +peptides O +and O +proteins O +. O + +This O +Fab B-structure_element +library O +is O +composed O +of O +3 O +HC B-structure_element +germlines O +, O +IGHV1 B-mutant +- I-mutant +69 I-mutant +( O +H1 B-mutant +- I-mutant +69 I-mutant +), O +IGHV3 B-mutant +- I-mutant +23 I-mutant +( O +H3 B-mutant +- I-mutant +23 I-mutant +) O +and O +IGHV5 B-mutant +- I-mutant +51 I-mutant +( O +H5 B-mutant +- I-mutant +51 I-mutant +), O +and O +4 O +LC B-structure_element +germlines O +( O +all O +κ B-structure_element +), O +IGKV1 B-mutant +- I-mutant +39 I-mutant +( O +L1 B-mutant +- I-mutant +39 I-mutant +), O +IGKV3 B-mutant +- I-mutant +11 I-mutant +( O +L3 B-mutant +- I-mutant +11 I-mutant +), O +IGKV3 B-mutant +- I-mutant +20 I-mutant +( O +L3 B-mutant +- I-mutant +20 I-mutant +) O +and O +IGKV4 B-mutant +- I-mutant +1 I-mutant +( O +L4 B-mutant +- I-mutant +1 I-mutant +). O + +Selection O +of O +these O +genes O +was O +based O +on O +the O +high O +frequency O +of O +their O +use O +and O +their O +cognate O +canonical O +structures B-evidence +that O +were O +found O +binding O +to O +peptides O +and O +proteins O +, O +as O +well O +as O +their O +ability O +to O +be O +expressed B-experimental_method +in I-experimental_method +bacteria I-experimental_method +and O +displayed B-experimental_method +on I-experimental_method +filamentous I-experimental_method +phage I-experimental_method +. O + +The O +implementation O +of O +the O +library O +involves O +the O +diversification O +of O +the O +human B-species +germline O +genes O +to O +mimic O +that O +found O +in O +natural O +human B-species +libraries O +. O + +The O +crystal B-experimental_method +structure I-experimental_method +determinations I-experimental_method +and O +structural B-experimental_method +analyses I-experimental_method +of O +all O +germline O +Fabs B-structure_element +in O +the O +library O +described O +above O +along O +with O +the O +structures B-evidence +of O +a O +fourth O +HC B-structure_element +germline O +, O +IGHV3 B-mutant +- I-mutant +53 I-mutant +( O +H3 B-mutant +- I-mutant +53 I-mutant +), O +paired O +with O +the O +4 O +LCs B-structure_element +of O +the O +library O +have O +been O +carried O +out O +to O +support O +antibody B-protein_type +therapeutic O +development O +. O + +All O +16 O +HCs B-structure_element +of O +the O +Fabs B-structure_element +have O +the O +same O +CDR B-structure_element +H3 B-structure_element +that O +was O +reported O +in O +an O +earlier O +Fab B-structure_element +structure B-evidence +. O + +This O +is O +the O +first O +systematic O +study O +of O +the O +same O +VH B-structure_element +and O +VL B-structure_element +structures B-evidence +in O +the O +context O +of O +different O +pairings O +. O + +The O +structure O +analyses O +include O +comparisons O +of O +the O +overall O +structures B-evidence +, O +canonical O +structures B-evidence +of O +the O +L1 B-structure_element +, O +L2 B-structure_element +, O +L3 B-structure_element +, O +H1 B-structure_element +and O +H2 B-structure_element +CDRs B-structure_element +, O +the O +structures B-evidence +of O +all O +CDR B-structure_element +H3s B-structure_element +, O +and O +the O +VH B-complex_assembly +: I-complex_assembly +VL I-complex_assembly +packing B-bond_interaction +interactions I-bond_interaction +. O + +The O +structures B-evidence +and O +their O +analyses O +provide O +a O +foundation O +for O +future O +antibody B-protein_type +engineering O +and O +structure O +determination O +efforts O +. O + +Crystal B-evidence +structures I-evidence + +Crystal B-evidence +data I-evidence +, O +X B-evidence +- I-evidence +ray I-evidence +data I-evidence +, O +and O +refinement B-evidence +statistics I-evidence +. O + +( O +Continued O +) O +Crystal B-evidence +data I-evidence +, O +X B-evidence +- I-evidence +ray I-evidence +data I-evidence +, O +and O +refinement B-evidence +statistics I-evidence +. O + +The O +crystal B-evidence +structures I-evidence +of O +a O +germline B-experimental_method +library I-experimental_method +composed O +of O +16 O +Fabs B-structure_element +generated O +by O +combining O +4 O +HCs B-structure_element +( O +H1 B-mutant +- I-mutant +69 I-mutant +, O +H3 B-mutant +- I-mutant +23 I-mutant +, O +H3 B-mutant +- I-mutant +53 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +) O +and O +4 O +LCs B-structure_element +( O +L1 B-mutant +- I-mutant +39 I-mutant +, O +L3 B-mutant +- I-mutant +11 I-mutant +, O +L3 B-mutant +- I-mutant +20 I-mutant +and O +L4 B-mutant +- I-mutant +1 I-mutant +) O +have O +been O +determined O +. O + +The O +Fab B-structure_element +heavy O +and O +light B-structure_element +chain I-structure_element +sequences O +for O +the O +variants O +numbered O +according O +to O +Chothia O +are O +shown O +in O +Fig O +. O +S1 O +. O + +The O +four O +different O +HCs B-structure_element +all O +have O +the O +same O +CDR B-structure_element +H3 B-structure_element +sequence O +, O +ARYDGIYGELDF B-structure_element +. O + +Crystallization B-experimental_method +of O +the O +16 O +Fabs B-structure_element +was O +previously O +reported O +. O + +Three O +sets O +of O +the O +crystals B-evidence +were O +isomorphous O +with O +nearly O +identical O +unit O +cells O +( O +Table O +1 O +). O + +These O +include O +( O +1 O +) O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +in O +P212121 O +, O +( O +2 O +) O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +, O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +in O +P6522 O +, O +and O +( O +3 O +) O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +, O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +and O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +in O +P212121 O +. O + +Variations O +occur O +in O +the O +pH O +( O +buffer O +) O +and O +the O +additives O +, O +and O +, O +in O +group O +3 O +, O +PEG B-chemical +3350 I-chemical +is O +the O +precipitant O +for O +one O +variants O +while O +ammonium B-chemical +sulfate I-chemical +is O +the O +precipitant O +for O +the O +other O +two O +. O + +The O +similarity O +in O +the O +crystal B-evidence +forms I-evidence +is O +attributed O +in O +part O +to O +cross O +- O +seeding O +using O +the O +microseed B-experimental_method +matrix I-experimental_method +screening I-experimental_method +for O +groups O +2 O +and O +3 O +. O + +The O +crystal B-evidence +structures I-evidence +of O +the O +16 O +Fabs B-structure_element +have O +been O +determined O +at O +resolutions O +ranging O +from O +3 O +. O +3 O +Å O +to O +1 O +. O +65 O +Å O +( O +Table O +1 O +). O + +The O +number O +of O +Fab B-structure_element +molecules O +in O +the O +crystallographic O +asymmetric O +unit O +varies O +from O +1 O +( O +for O +12 O +Fabs B-structure_element +) O +to O +2 O +( O +for O +4 O +Fabs B-structure_element +). O + +Overall O +the O +structures B-evidence +are O +fairly O +complete O +, O +and O +, O +as O +can O +be O +expected O +, O +the O +models O +for O +the O +higher O +resolution O +structures B-evidence +are O +more O +complete O +than O +those O +for O +the O +lower O +resolution O +structures B-evidence +( O +Table O +S1 O +). O + +Invariably O +, O +the O +HCs B-structure_element +have O +more O +disorder B-protein_state +than O +the O +LCs B-structure_element +. O + +For O +the O +LC B-structure_element +, O +the O +disorder B-protein_state +is O +observed O +at O +2 O +of O +the O +C O +- O +terminal O +residues O +with O +few O +exceptions O +. O + +Apart O +from O +the O +C O +- O +terminus O +, O +only O +a O +few O +surface O +residues O +in O +LC B-structure_element +are O +disordered B-protein_state +. O + +The O +HCs B-structure_element +feature O +the O +largest O +number O +of O +disordered B-protein_state +residues O +, O +with O +the O +lower O +resolution O +structures B-evidence +having O +the O +most O +. O + +The O +C O +- O +terminal O +residues O +including O +the O +6xHis O +tags O +are O +disordered B-protein_state +in O +all O +16 O +structures B-evidence +. O + +In O +addition O +to O +these O +, O +2 O +primary O +disordered O +stretches O +of O +residues O +are O +observed O +in O +a O +number O +of O +structures B-evidence +( O +Table O +S1 O +). O + +One O +involves O +the O +loop B-structure_element +connecting O +the O +first O +2 O +β B-structure_element +- I-structure_element +strands I-structure_element +of O +the O +constant B-structure_element +domain I-structure_element +( O +in O +all O +Fabs B-structure_element +except O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +, O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +). O + +The O +other O +is O +located O +in O +CDR B-structure_element +H3 B-structure_element +( O +in O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +, O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +in O +one O +of O +2 O +copies O +of O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +). O + +CDR B-structure_element +H1 B-structure_element +and O +CDR B-structure_element +H2 B-structure_element +also O +show O +some O +degree O +of O +disorder B-protein_state +, O +but O +to O +a O +lesser O +extent O +. O + +CDR B-structure_element +canonical O +structures B-evidence + +Several O +CDR B-structure_element +definitions O +have O +evolved O +over O +decades O +of O +antibody B-protein_type +research O +. O + +Depending O +on O +the O +focus O +of O +the O +study O +, O +the O +CDR B-structure_element +boundaries O +differ O +slightly O +between O +various O +definitions O +. O + +In O +this O +work O +, O +we O +use O +the O +CDR B-structure_element +definition O +of O +North O +et O +al O +., O +which O +is O +similar O +to O +that O +of O +Martin O +with O +the O +following O +exceptions O +: O +1 O +) O +CDRs B-structure_element +H1 B-structure_element +and O +H3 B-structure_element +begin O +immediately O +after O +the O +Cys B-residue_name +; O +and O +2 O +) O +CDR B-structure_element +L2 B-structure_element +includes O +an O +additional O +residue O +at O +the O +N O +- O +terminal O +side O +, O +typically O +Tyr B-residue_name +. O + +CDR B-structure_element +H1 B-structure_element + +The O +superposition B-experimental_method +of O +CDR B-structure_element +H1 B-structure_element +backbones O +for O +all O +HC B-complex_assembly +: I-complex_assembly +LC I-complex_assembly +pairs O +with O +heavy B-structure_element +chains I-structure_element +: O +( O +A O +) O +H1 B-mutant +- I-mutant +69 I-mutant +, O +( O +B O +) O +H3 B-mutant +- I-mutant +23 I-mutant +, O +( O +C O +) O +H3 B-mutant +- I-mutant +53 I-mutant +and O +( O +D O +) O +H5 B-mutant +- I-mutant +51 I-mutant +. O + +CDRs B-structure_element +are O +defined O +using O +the O +Dunbrack O +convention O +[ O +12 O +]. O + +Assignments O +for O +2 O +copies O +of O +the O +Fab B-structure_element +in O +the O +asymmetric O +unit O +are O +given O +for O +5 O +structures B-evidence +. O + +No O +assignment O +( O +NA O +) O +for O +CDRs B-structure_element +with O +missing O +residues O +. O + +The O +four O +HCs B-structure_element +feature O +CDR B-structure_element +H1 B-structure_element +of O +the O +same O +length O +, O +and O +their O +sequences O +are O +highly O +similar O +( O +Table O +2 O +). O + +The O +CDR B-structure_element +H1 B-structure_element +backbone O +conformations O +for O +all O +variants O +for O +each O +of O +the O +HCs B-structure_element +are O +shown O +in O +Fig O +. O +1 O +. O + +Three O +of O +the O +HCs B-structure_element +, O +H3 B-mutant +- I-mutant +23 I-mutant +, O +H3 B-mutant +- I-mutant +53 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +, O +have O +the O +same O +canonical O +structure O +, O +H1 B-mutant +- I-mutant +13 I-mutant +- I-mutant +1 I-mutant +, O +and O +the O +backbone O +conformations O +are O +tightly O +clustered O +for O +each O +set O +of O +Fab B-structure_element +structures B-evidence +as O +reflected O +in O +the O +rmsd B-evidence +values I-evidence +( O +Fig O +. O +1B O +- O +D O +). O + +Some O +deviation O +is O +observed O +for O +H3 B-mutant +- I-mutant +53 I-mutant +, O +mostly O +due O +to O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +, O +which O +exhibits O +a O +significant O +degree O +of O +disorder O +in O +CDR B-structure_element +H1 B-structure_element +. O + +The O +electron B-evidence +density I-evidence +for O +the O +backbone O +is O +weak O +and O +discontinuous O +, O +and O +completely O +missing O +for O +several O +side O +chains O +. O + +The O +CDR B-structure_element +H1 B-structure_element +structures B-evidence +with O +H1 B-mutant +- I-mutant +69 I-mutant +shown O +in O +Fig O +. O +1A O +are O +quite O +variable O +, O +both O +for O +the O +structures B-evidence +with O +different O +LCs B-structure_element +and O +for O +the O +copies O +of O +the O +same O +Fab B-structure_element +in O +the O +asymmetric O +unit O +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +and O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +. O + +In O +total O +, O +6 O +independent O +Fab B-structure_element +structures B-evidence +produce O +5 O +different O +canonical O +structures B-evidence +, O +namely O +H1 B-mutant +- I-mutant +13 I-mutant +- I-mutant +1 I-mutant +, O +H1 B-mutant +- I-mutant +13 I-mutant +- I-mutant +3 I-mutant +, O +H1 B-mutant +- I-mutant +13 I-mutant +- I-mutant +4 I-mutant +, O +H1 B-mutant +- I-mutant +13 I-mutant +- I-mutant +6 I-mutant +and O +H1 B-mutant +- I-mutant +13 I-mutant +- I-mutant +10 I-mutant +. O + +A O +major O +difference O +of O +H1 B-mutant +- I-mutant +69 I-mutant +from O +the O +other O +germlines O +in O +the O +experimental O +data O +set O +is O +the O +presence O +of O +Gly B-residue_name +instead O +of O +Phe B-residue_name +or O +Tyr B-residue_name +at O +position O +27 B-residue_number +( O +residue O +5 O +of O +13 O +in O +CDR B-structure_element +H1 B-structure_element +). O + +Glycine B-residue_name +introduces O +the O +possibility O +of O +a O +higher O +degree O +of O +conformational O +flexibility O +that O +undoubtedly O +translates O +to O +the O +differences O +observed O +, O +and O +contributes O +to O +the O +elevated O +thermal O +parameters O +for O +the O +atoms O +in O +the O +amino O +acid O +residues O +in O +this O +region O +. O + +CDR B-structure_element +H2 B-structure_element + +The O +superposition B-experimental_method +of O +CDR B-structure_element +H2 B-structure_element +backbones O +for O +all O +HC B-complex_assembly +: I-complex_assembly +LC I-complex_assembly +pairs O +with O +heavy B-structure_element +chains I-structure_element +: O +( O +A O +) O +H1 B-mutant +- I-mutant +69 I-mutant +, O +( O +B O +) O +H3 B-mutant +- I-mutant +23 I-mutant +, O +( O +C O +) O +H3 B-mutant +- I-mutant +53 I-mutant +and O +( O +D O +) O +H5 B-mutant +- I-mutant +51 I-mutant +. O + +The O +canonical O +structures O +of O +CDR B-structure_element +H2 B-structure_element +have O +fairly O +consistent O +conformations O +( O +Table O +2 O +, O +Fig O +. O +2 O +). O + +Each O +of O +the O +4 O +HCs B-structure_element +adopts O +only O +one O +canonical O +structure O +regardless O +of O +the O +pairing O +LC B-structure_element +. O + +Germlines O +H1 B-mutant +- I-mutant +69 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +have O +the O +same O +canonical O +structure O +assignment O +H2 B-mutant +- I-mutant +10 I-mutant +- I-mutant +1 I-mutant +, O +H3 B-mutant +- I-mutant +23 I-mutant +has O +H2 B-mutant +- I-mutant +10 I-mutant +- I-mutant +2 I-mutant +, O +and O +H3 B-mutant +- I-mutant +53 I-mutant +has O +H2 B-mutant +- I-mutant +9 I-mutant +- I-mutant +3 I-mutant +. O + +The O +conformations O +for O +all O +of O +these O +CDR B-structure_element +H2s B-structure_element +are O +tightly O +clustered O +( O +Fig O +. O +2 O +). O + +In O +one O +case O +, O +in O +the O +second O +Fab B-structure_element +of O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +CDR B-structure_element +H2 B-structure_element +is O +partially B-protein_state +disordered I-protein_state +( O +Δ55 B-mutant +- I-mutant +60 I-mutant +). O + +Although O +three O +of O +the O +germlines O +have O +CDR B-structure_element +H2 B-structure_element +of O +the O +same O +length O +, O +10 B-residue_range +residues I-residue_range +, O +they O +adopt O +2 O +distinctively O +different O +conformations O +depending O +mostly O +on O +the O +residue O +at O +position O +71 B-residue_number +from O +the O +so O +- O +called O +CDR B-structure_element +H4 B-structure_element +. O + +Arg71 B-residue_name_number +in O +H3 B-mutant +- I-mutant +23 I-mutant +fills O +the O +space O +between O +CDRs B-structure_element +H2 B-structure_element +and O +H4 B-structure_element +, O +and O +defines O +the O +conformation O +of O +the O +tip O +of O +CDR B-structure_element +H2 B-structure_element +so O +that O +residue O +54 B-residue_number +points O +away O +from O +the O +antigen B-site +binding I-site +site I-site +. O + +Germlines O +H1 B-mutant +- I-mutant +69 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +are O +unique O +in O +the O +human B-species +repertoire O +in O +having O +an O +Ala B-residue_name +at O +position O +71 B-residue_number +that O +leaves O +enough O +space O +for O +H B-structure_element +- O +Pro52a B-residue_name_number +to O +pack O +deeper O +against O +CDR B-structure_element +H4 B-structure_element +so O +that O +the O +following O +residues O +53 B-residue_number +and O +54 B-residue_number +point O +toward O +the O +putative O +antigen O +. O + +Conformations O +of O +CDR B-structure_element +H2 B-structure_element +in O +H1 B-mutant +- I-mutant +69 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +, O +both O +of O +which O +have O +canonical O +structure O +H2 B-mutant +- I-mutant +10 I-mutant +- I-mutant +1 I-mutant +, O +show O +little O +deviation O +within O +each O +set O +of O +4 O +structures B-evidence +. O + +However O +, O +there O +is O +a O +significant O +shift O +of O +the O +CDR B-structure_element +as O +a O +rigid O +body O +when O +the O +2 O +sets O +are O +superimposed B-experimental_method +. O + +Most O +likely O +this O +is O +the O +result O +of O +interaction O +of O +CDR B-structure_element +H2 B-structure_element +with O +CDR B-structure_element +H1 B-structure_element +, O +namely O +with O +the O +residue O +at O +position O +33 B-residue_number +( O +residue O +11 O +of O +13 O +in O +CDR B-structure_element +H1 B-structure_element +). O + +Germline O +H1 B-mutant +- I-mutant +69 I-mutant +has O +Ala B-residue_name +at O +position O +33 B-residue_number +whereas O +in O +H5 B-mutant +- I-mutant +51 I-mutant +position O +33 B-residue_number +is O +occupied O +by O +a O +bulky O +Trp B-residue_name +, O +which O +stacks O +against O +H B-structure_element +- O +Tyr52 B-residue_name_number +and O +drives O +CDR B-structure_element +H2 B-structure_element +away O +from O +the O +center O +. O + +CDR B-structure_element +L1 B-structure_element + +The O +superposition B-experimental_method +of O +CDR B-structure_element +L1 B-structure_element +backbones O +for O +all O +HC B-complex_assembly +: I-complex_assembly +LC I-complex_assembly +pairs O +with O +light B-structure_element +chains I-structure_element +: O +( O +A O +) O +L1 B-mutant +- I-mutant +39 I-mutant +, O +( O +B O +) O +L3 B-mutant +- I-mutant +11 I-mutant +, O +( O +C O +) O +L3 B-mutant +- I-mutant +20 I-mutant +and O +( O +D O +) O +L4 B-mutant +- I-mutant +1 I-mutant +. O + +The O +four O +LC B-structure_element +CDRs B-structure_element +L1 B-structure_element +feature O +3 O +different O +lengths O +( O +11 B-residue_range +, O +12 B-residue_range +and O +17 B-residue_range +residues O +) O +having O +a O +total O +of O +4 O +different O +canonical O +structure O +assignments O +. O + +Of O +these O +LCs B-structure_element +, O +L1 B-mutant +- I-mutant +39 I-mutant +and O +L3 B-mutant +- I-mutant +11 I-mutant +have O +the O +same O +canonical O +structure O +, O +L1 B-mutant +- I-mutant +11 I-mutant +- I-mutant +1 I-mutant +, O +and O +superimpose B-experimental_method +very O +well O +( O +Fig O +. O +3A O +, O +B O +). O + +For O +the O +remaining O +2 O +, O +L3 B-mutant +- I-mutant +20 I-mutant +has O +2 O +different O +assignments O +, O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +1 I-mutant +and O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +2 I-mutant +, O +while O +L4 B-mutant +- I-mutant +1 I-mutant +has O +a O +single O +assignment O +, O +L1 B-mutant +- I-mutant +17 I-mutant +- I-mutant +1 I-mutant +. O + +L4 B-mutant +- I-mutant +1 I-mutant +has O +the O +longest O +CDR B-structure_element +L1 B-structure_element +, O +composed O +of O +17 B-residue_range +amino I-residue_range +acid I-residue_range +residues I-residue_range +( O +Fig O +. O +3D O +). O + +Despite O +this O +, O +the O +conformations O +are O +tightly O +clustered O +( O +rmsd B-evidence +is O +0 O +. O +20 O +Å O +). O + +The O +backbone O +conformations O +of O +the O +stem B-structure_element +regions I-structure_element +superimpose O +well O +. O + +Some O +changes O +in O +conformation O +occur O +between O +residues O +30a B-residue_number +and O +30f B-residue_number +( O +residues O +8 B-residue_number +and O +13 B-residue_number +of O +17 B-residue_number +in O +CDR B-structure_element +L1 B-structure_element +). O + +This O +is O +the O +tip O +of O +the O +loop B-structure_element +region I-structure_element +, O +which O +appears O +to O +have O +similar O +conformations O +that O +fan O +out O +the O +structures B-evidence +because O +of O +the O +slight O +differences O +in O +torsion O +angles O +in O +the O +backbone O +near O +Tyr30a B-residue_name_number +and O +Lys30f B-residue_name_number +. O + +L3 B-mutant +- I-mutant +20 I-mutant +is O +the O +most O +variable O +in O +CDR B-structure_element +L1 B-structure_element +among O +the O +4 O +germlines O +as O +indicated O +by O +an O +rmsd B-evidence +of O +0 O +. O +54 O +Å O +( O +Fig O +. O +3C O +). O + +Two O +structures B-evidence +, O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +are O +assigned O +to O +canonical O +structure O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +1 I-mutant +with O +virtually O +identical O +backbone O +conformations O +. O + +The O +third O +structure O +, O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +has O +CDR B-structure_element +L1 B-structure_element +as O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +2 I-mutant +, O +which O +deviates O +from O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +1 I-mutant +at O +residues O +29 B-residue_range +- I-residue_range +32 I-residue_range +, O +i O +. O +e O +., O +at O +the O +site O +of O +insertion O +with O +respect O +to O +the O +11 B-residue_range +- I-residue_range +residue I-residue_range +CDR B-structure_element +. O + +The O +fourth O +member O +of O +the O +set O +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +was O +crystallized B-experimental_method +with O +2 O +Fabs B-structure_element +in O +the O +asymmetric O +unit O +. O + +The O +conformation O +of O +CDR B-structure_element +L1 B-structure_element +in O +these O +2 O +Fabs B-structure_element +is O +slightly O +different O +, O +and O +both O +conformations O +fall O +somewhere O +between O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +1 I-mutant +and O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +2 I-mutant +. O + +This O +reflects O +the O +lack O +of O +accuracy O +in O +the O +structure B-evidence +due O +to O +low O +resolution O +of O +the O +X B-evidence +- I-evidence +ray I-evidence +data I-evidence +( O +3 O +. O +3 O +Å O +). O + +CDR B-structure_element +L2 B-structure_element + +The O +superposition B-experimental_method +of O +CDR B-structure_element +L2 B-structure_element +backbones O +for O +all O +HC B-complex_assembly +: I-complex_assembly +LC I-complex_assembly +pairs O +with O +light B-structure_element +chains I-structure_element +: O +( O +A O +) O +L1 B-mutant +- I-mutant +39 I-mutant +, O +( O +B O +) O +L3 B-mutant +- I-mutant +11 I-mutant +, O +( O +C O +) O +L3 B-mutant +- I-mutant +20 I-mutant +and O +( O +D O +) O +L4 B-mutant +- I-mutant +1 I-mutant +. O + +All O +four O +LCs B-structure_element +have O +CDR B-structure_element +L2 B-structure_element +of O +the O +same O +length O +and O +canonical O +structure O +, O +L2 B-mutant +- I-mutant +8 I-mutant +- I-mutant +1 I-mutant +( O +Table O +2 O +). O + +The O +CDR B-structure_element +L2 B-structure_element +conformations O +for O +each O +of O +the O +LCs B-structure_element +paired O +with O +the O +4 O +HCs B-structure_element +are O +clustered O +more O +tightly O +than O +any O +of O +the O +other O +CDRs B-structure_element +( O +rmsd B-evidence +values O +are O +in O +the O +range O +0 O +. O +09 O +- O +0 O +. O +16 O +Å O +), O +and O +all O +4 O +sets O +have O +virtually O +the O +same O +conformation O +despite O +the O +sequence O +diversity O +of O +the O +loop B-structure_element +. O + +CDR B-structure_element +L3 B-structure_element + +The O +superposition B-experimental_method +of O +CDR B-structure_element +L3 B-structure_element +backbones O +for O +all O +HC B-complex_assembly +: I-complex_assembly +LC I-complex_assembly +pairs O +with O +light B-structure_element +chains I-structure_element +: O +( O +A O +) O +L1 B-mutant +- I-mutant +39 I-mutant +, O +( O +B O +) O +L3 B-mutant +- I-mutant +11 I-mutant +, O +( O +C O +) O +L3 B-mutant +- I-mutant +20 I-mutant +and O +( O +D O +) O +L4 B-mutant +- I-mutant +1 I-mutant +. O + +As O +with O +CDR B-structure_element +L2 B-structure_element +, O +all O +4 O +LCs B-structure_element +have O +CDR B-structure_element +L3 B-structure_element +of O +the O +same O +length O +and O +canonical O +structure B-evidence +, O +L3 B-mutant +- I-mutant +9 I-mutant +- I-mutant +cis7 I-mutant +- I-mutant +1 I-mutant +( O +Table O +2 O +). O + +The O +conformations O +of O +CDR B-structure_element +L3 B-structure_element +for O +L1 B-mutant +- I-mutant +39 I-mutant +, O +L3 B-mutant +- I-mutant +11 I-mutant +, O +and O +particularly O +for O +L320 O +, O +are O +not O +as O +tightly O +clustered O +as O +those O +of O +L4 B-mutant +- I-mutant +1 I-mutant +( O +Fig O +. O +5 O +). O + +The O +slight O +conformational O +variability O +occurs O +in O +the O +region O +of O +amino O +acid O +residues O +90 B-residue_range +- I-residue_range +92 I-residue_range +, O +which O +is O +in O +contact O +with O +CDR B-structure_element +H3 B-structure_element +. O + +CDR B-structure_element +H3 B-structure_element +conformational O +diversity O + +As O +mentioned O +earlier O +, O +all O +16 O +Fabs B-structure_element +have O +the O +same O +CDR B-structure_element +H3 B-structure_element +, O +for O +which O +the O +amino O +acid O +sequence O +is O +derived O +from O +the O +anti O +- O +CCL2 O +antibody B-protein_type +CNTO B-chemical +888 I-chemical +. O + +The O +loop B-structure_element +and O +the O +2 O +β B-structure_element +- I-structure_element +strands I-structure_element +of O +the O +CDR B-structure_element +H3 B-structure_element +in O +this O +‘ O +parent O +’ O +structure B-evidence +are O +stabilized O +by O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +between O +the O +carbonyl O +oxygen O +and O +peptide O +nitrogen O +atoms O +in O +the O +2 O +strands O +. O + +An O +interesting O +feature O +of O +these O +CDR B-structure_element +H3 B-structure_element +structures B-evidence +is O +the O +presence O +of O +a O +water B-chemical +molecule O +that O +interacts O +with O +the O +peptide O +nitrogens O +and O +carbonyl O +oxygens O +near O +the O +bridging O +loop B-structure_element +connecting O +the O +2 O +β B-structure_element +- I-structure_element +strands I-structure_element +. O + +This O +water B-chemical +is O +present O +in O +both O +the O +bound B-protein_state +( O +4DN4 O +) O +and O +unbound B-protein_state +( O +4DN3 O +) O +forms O +of O +CNTO B-chemical +888 I-chemical +. O + +The O +stem B-structure_element +region I-structure_element +of O +CDR B-structure_element +H3 B-structure_element +in O +the O +parental O +Fab B-structure_element +is O +in O +a O +‘ O +kinked B-protein_state +’ O +conformation O +, O +in O +which O +the O +indole O +nitrogen O +of O +Trp103 B-residue_name_number +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +carbonyl O +oxygen O +of O +Leu100b B-residue_name_number +. O + +The O +carboxyl O +group O +of O +Asp101 B-residue_name_number +forms O +a O +salt B-bond_interaction +bridge I-bond_interaction +with O +Arg94 B-residue_name_number +. O + +Ribbon O +representations O +of O +( O +A O +) O +the O +superposition B-experimental_method +of O +all O +CDR B-structure_element +H3s B-structure_element +of O +the O +structures B-evidence +with O +complete O +backbone O +traces O +. O +( O +B O +) O +The O +CDR B-structure_element +H3s B-structure_element +rotated O +90 O +° O +about O +the O +y O +axis O +of O +the O +page O +. O + +The O +structure B-evidence +of O +each O +CDR B-structure_element +H3 B-structure_element +is O +represented O +with O +a O +different O +color O +. O + +Despite O +having O +the O +same O +amino O +acid O +sequence O +in O +all O +variants O +, O +CDR B-structure_element +H3 B-structure_element +has O +the O +highest O +degree O +of O +structural O +diversity O +and O +disorder O +of O +all O +of O +the O +CDRs B-structure_element +in O +the O +experimental O +set O +. O + +Three O +of O +the O +21 O +Fab B-structure_element +structures B-evidence +( O +including O +multiple O +copies O +in O +the O +asymmetric O +unit O +), O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +, O +H551 B-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +( O +one O +of O +the O +2 O +Fabs B-structure_element +), O +have O +missing B-protein_state +( O +disordered B-protein_state +) O +residues O +at O +the O +apex O +of O +the O +CDR B-structure_element +loop I-structure_element +. O + +Another O +four O +of O +the O +Fabs B-structure_element +, O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +, O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +, O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +have O +missing O +side O +- O +chain O +atoms O +. O + +The O +variations O +in O +CDR B-structure_element +H3 B-structure_element +conformation O +are O +illustrated O +in O +Fig O +. O +6 O +for O +the O +18 O +Fab B-structure_element +structures B-evidence +that O +have O +ordered O +backbone O +atoms O +. O + +A O +comparison O +of O +representatives O +of O +the O +“ O +kinked B-protein_state +” O +and O +“ O +extended B-protein_state +” O +structures B-evidence +. O + +( O +A O +) O +The O +“ O +kinked B-protein_state +” O +CDR B-structure_element +H3 B-structure_element +of O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +with O +purple O +carbon O +atoms O +and O +yellow O +dashed O +lines O +connecting O +the O +H O +- O +bond O +pairs O +for O +Leu100b B-residue_name_number +O O +and O +Trp103 B-residue_name_number +NE1 O +, O +Arg94 B-residue_name_number +NE O +and O +Asp101 B-residue_name_number +OD1 O +, O +and O +Arg94 B-residue_name_number +NH2 O +and O +Asp101 B-residue_name_number +OD2 O +. O + +( O +B O +) O +The O +“ O +extended B-protein_state +” O +CDR B-structure_element +H3 B-structure_element +of O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +with O +green O +carbon O +atoms O +and O +yellow O +dashed O +lines O +connecting O +the O +H O +- O +bond O +pairs O +for O +Asp101 B-residue_name_number +OD1 O +and O +OD2 O +and O +Trp103 B-residue_name_number +NE1 O +. 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O + +Pairing O +of O +different O +germlines O +yields O +antibodies B-protein_type +with O +various O +degrees O +of O +stability O +. O + +As O +indicated O +by O +the O +melting B-evidence +temperatures I-evidence +, O +germlines O +H1 B-mutant +- I-mutant +69 I-mutant +and O +H3 B-mutant +- I-mutant +23 I-mutant +for O +HC B-structure_element +and O +germline O +L1 B-mutant +- I-mutant +39 I-mutant +for O +LC B-structure_element +produce O +more O +stable B-protein_state +Fabs B-structure_element +compared O +to O +the O +other O +germlines O +in O +the O +experimental O +set O +. O + +One O +possible O +explanation O +of O +the O +clear O +preference O +of O +LC B-structure_element +germline O +L1 B-mutant +- I-mutant +39 I-mutant +is O +that O +CDR B-structure_element +L3 B-structure_element +has O +smaller O +residues O +at O +positions O +91 B-residue_number +and O +94 B-residue_number +, O +allowing O +for O +more O +room O +to O +accommodate O +CDR B-structure_element +H3 B-structure_element +. O + +Other O +germlines O +have O +bulky O +residues O +, O +Tyr B-residue_name +, O +Arg B-residue_name +and O +Trp B-residue_name +, O +at O +these O +positions O +, O +whereas O +L1 B-mutant +- I-mutant +39 I-mutant +has O +Ser B-residue_name +and O +Thr B-residue_name +. O + +Various O +combinations O +of O +germline O +sequences O +for O +VL B-structure_element +and O +VH B-structure_element +impose O +certain O +constraints O +on O +CDR B-structure_element +H3 B-structure_element +, O +which O +has O +to O +adapt O +to O +the O +environment O +. O + +A O +more O +compact B-protein_state +CDR B-structure_element +L3 B-structure_element +may O +be O +beneficial O +in O +this O +situation O +. O + +At O +the O +other O +end O +of O +the O +stability O +range O +is O +LC B-structure_element +germline O +L3 B-mutant +- I-mutant +20 I-mutant +, O +which O +yields O +antibodies B-protein_type +with O +the O +lowest O +Tms B-evidence +. O + +While O +pairings O +with O +H3 B-mutant +- I-mutant +53 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +may O +be O +safely O +called O +a O +mismatch O +, O +those O +with O +H1 B-mutant +- I-mutant +69 I-mutant +and O +H3 B-mutant +- I-mutant +23 I-mutant +have O +Tms B-evidence +about O +5 O +- O +6 O +° O +higher O +. O + +Curiously O +, O +the O +2 O +Fabs B-structure_element +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +deviate O +markedly O +in O +their O +tilt B-evidence +angles I-evidence +from O +the O +rest O +of O +the O +panel O +. O + +It O +is O +possible O +that O +by O +adopting O +extreme O +tilt B-evidence +angles I-evidence +the O +structure B-evidence +modulates O +CDR B-structure_element +H3 B-structure_element +and O +its O +environment O +, O +which O +apparently O +cannot O +be O +achieved O +solely O +by O +conformational O +rearrangement O +of O +the O +CDR B-structure_element +. O + +Note O +that O +most O +of O +the O +VH B-site +: I-site +VL I-site +interface I-site +residues O +are O +invariant O +; O +therefore O +, O +significant O +change O +of O +the O +tilt O +angle O +must O +come O +with O +a O +penalty O +in O +free O +energy O +. O + +Yet O +, O +for O +the O +2 O +antibodies B-protein_type +, O +the O +total O +gain O +in O +stability O +merits O +the O +domain O +repacking O +. O + +Overall O +, O +the O +stability O +of O +the O +Fab B-structure_element +, O +as O +measured O +by O +Tm B-evidence +, O +is O +a O +result O +of O +the O +mutual O +adjustment O +of O +the O +HC B-structure_element +and O +LC B-structure_element +variable B-structure_element +domains I-structure_element +and O +adjustment O +of O +CDR B-structure_element +H3 B-structure_element +to O +the O +VH B-site +: I-site +VL I-site +cleft I-site +. O + +The O +final O +conformation O +represents O +an O +energetic O +minimum O +; O +however O +, O +in O +most O +cases O +it O +is O +very O +shallow O +, O +so O +that O +a O +single O +mutation O +can O +cause O +a O +dramatic O +rearrangement O +of O +the O +structure B-evidence +. O + +In O +summary O +, O +the O +analysis O +of O +this O +structural B-experimental_method +library I-experimental_method +of O +germline O +variants O +composed O +of O +all O +pairs O +of O +4 O +HCs B-structure_element +and O +4LCs O +, O +all O +with O +the O +same O +CDR B-structure_element +H3 B-structure_element +, O +offers O +some O +unique O +insights O +into O +antibody B-protein_type +structure B-evidence +and O +how O +pairing O +and O +sequence O +may O +influence O +, O +or O +not O +, O +the O +canonical O +structures O +of O +the O +L1 B-structure_element +, O +L2 B-structure_element +, O +L3 B-structure_element +, O +H1 B-structure_element +and O +H2 B-structure_element +CDRs B-structure_element +. O + +Comparison O +of O +the O +CDR B-structure_element +H3s B-structure_element +reveals O +a O +large O +set O +of O +variants O +with O +conformations O +similar O +to O +the O +parent O +, O +while O +a O +second O +set O +has O +significant O +conformational O +variability O +, O +indicating O +that O +both O +the O +sequence O +and O +the O +structural O +context O +define O +the O +CDR B-structure_element +H3 B-structure_element +conformation O +. O + +Quite O +unexpectedly O +, O +2 O +of O +the O +variants O +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +, O +have O +the O +‘ O +extended B-protein_state +’ O +stem B-structure_element +region I-structure_element +differing O +from O +the O +other O +14 O +that O +have O +a O +‘ O +kinked B-protein_state +’ O +stem B-structure_element +region I-structure_element +. O + +These O +data O +reveal O +the O +difficulty O +of O +modeling O +CDR B-structure_element +H3 B-structure_element +accurately O +, O +as O +shown O +again O +in O +Antibody O +Modeling O +Assessment O +II O +. O + +Furthermore O +, O +antibody B-protein_type +CDRs B-structure_element +, O +H3 B-structure_element +in O +particular O +, O +may O +go O +through O +conformational O +changes O +upon O +binding O +their O +targets O +, O +making O +structural O +prediction O +for O +docking O +purposes O +an O +even O +more O +difficult O +task O +. O + +Fortunately O +, O +for O +most O +applications O +of O +antibody B-protein_type +modeling O +, O +such O +as O +engineering O +affinity O +and O +biophysical O +properties O +, O +an O +accurate O +CDR B-structure_element +H3 B-structure_element +structure B-evidence +is O +not O +always O +necessary O +. O + +For O +those O +applications O +where O +accurate O +CDR B-structure_element +structures B-evidence +are O +essential O +, O +such O +as O +docking O +, O +the O +results O +in O +this O +work O +demonstrate O +the O +importance O +of O +experimental O +structures B-evidence +. O + +With O +the O +recent O +advances O +in O +expression B-experimental_method +and I-experimental_method +crystallization I-experimental_method +methods I-experimental_method +, O +Fab B-structure_element +structures B-evidence +can O +be O +obtained O +rapidly O +. O + +The O +set O +of O +16 O +germline O +Fab B-structure_element +structures B-evidence +offers O +a O +unique O +dataset O +to O +facilitate O +software O +development O +for O +antibody B-protein_type +modeling O +. O + +The O +results O +essentially O +support O +the O +underlying O +idea O +of O +canonical O +structures B-evidence +, O +indicating O +that O +most O +CDRs B-structure_element +with O +germline O +sequences O +tend O +to O +adopt O +predefined O +conformations O +. O + +From O +this O +point O +of O +view O +, O +a O +novel O +approach O +to O +design O +combinatorial O +antibody B-protein_type +libraries O +would O +be O +to O +cover O +the O +range O +of O +CDR B-structure_element +conformations O +that O +may O +not O +necessarily O +coincide O +with O +the O +germline O +usage O +in O +the O +human B-species +repertoire O +. O + +This O +would O +insure O +more O +structural O +diversity O +, O +leading O +to O +a O +more O +diverse O +panel O +of O +antibodies B-protein_type +that O +would O +bind O +to O +a O +broad O +spectrum O +of O +targets O +. O + +Structure B-evidence +of O +the O +Response B-protein_type +Regulator I-protein_type +NsrR B-protein +from O +Streptococcus B-species +agalactiae I-species +, O +Which O +Is O +Involved O +in O +Lantibiotic B-chemical +Resistance O + +Lantibiotics B-chemical +are O +antimicrobial B-chemical +peptides I-chemical +produced O +by O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +bacteria I-taxonomy_domain +. O + +Interestingly O +, O +several O +clinically O +relevant O +and O +human B-species +pathogenic O +strains O +are O +inherently O +resistant O +towards O +lantibiotics B-chemical +. O + +The O +expression O +of O +the O +genes O +responsible O +for O +lantibiotic B-chemical +resistance O +is O +regulated O +by O +a O +specific O +two B-complex_assembly +- I-complex_assembly +component I-complex_assembly +system I-complex_assembly +consisting O +of O +a O +histidine B-protein_type +kinase I-protein_type +and O +a O +response B-protein_type +regulator I-protein_type +. O + +Here O +, O +we O +focused O +on O +a O +response B-protein_type +regulator I-protein_type +involved O +in O +lantibiotic B-chemical +resistance O +, O +NsrR B-protein +from O +Streptococcus B-species +agalactiae I-species +, O +and O +determined O +the O +crystal B-evidence +structures I-evidence +of O +its O +N O +- O +terminal O +receiver B-structure_element +domain I-structure_element +and O +C O +- O +terminal O +DNA B-structure_element +- I-structure_element +binding I-structure_element +effector I-structure_element +domain I-structure_element +. O + +The O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +exhibits O +a O +fold O +that O +classifies O +NsrR B-protein +as O +a O +member O +of O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +of O +regulators O +. O + +Amino O +acids O +involved O +in O +phosphorylation B-ptm +, O +dimerization O +, O +and O +DNA B-chemical +- O +binding O +were O +identified O +and O +demonstrated O +to O +be O +conserved B-protein_state +in O +lantibiotic B-protein_type +resistance I-protein_type +regulators I-protein_type +. O + +Finally O +, O +a O +model O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +in O +the O +active B-protein_state +and O +inactive B-protein_state +state O +provides O +insights O +into O +protein O +dimerization O +and O +DNA B-chemical +- O +binding O +. O + +This O +has O +led O +to O +the O +search O +for O +novel O +antibiotics O +that O +can O +be O +used O +as O +pharmaceuticals O +against O +human B-species +pathogenic O +bacteria B-taxonomy_domain +. O + +One O +of O +the O +potential O +antibiotic O +alternatives O +are O +lantibiotics B-chemical +. O + +Lantibiotics B-chemical +are O +small O +antimicrobial B-chemical +peptides I-chemical +( O +30 O +– O +50 O +amino O +acids O +in O +length O +), O +which O +are O +produced O +by O +several O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +bacterial I-taxonomy_domain +strains O +. O + +They O +are O +post O +- O +translationally O +modified O +and O +contain O +specific O +lanthionine B-chemical +/ O +methyl B-chemical +- I-chemical +lanthionine I-chemical +rings O +, O +which O +are O +crucial O +for O +their O +high O +antimicrobial O +activity O +. O + +Lantibiotics B-chemical +are O +for O +example O +highly O +effective O +against O +various O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +, O +human B-species +pathogenic O +bacteria B-taxonomy_domain +including O +Streptococcus B-species +pneumoniae I-species +and O +several O +methicillin B-species +- I-species +resistant I-species +Staphylococcus I-species +aureus I-species +( O +MRSA B-species +) O +strains O +. O + +The O +high O +potency O +of O +lantibiotics B-chemical +for O +medical O +usage O +has O +already O +been O +noticed O +, O +and O +several O +lantibiotics B-chemical +are O +already O +included O +in O +clinical O +trials O +. O + +Nisin B-chemical +is O +the O +most O +prominent O +member O +of O +the O +lantibiotic B-chemical +family O +and O +is O +able O +to O +inhibit O +cell O +growth O +, O +penetrates O +the O +membranes O +of O +various O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +bacteria I-taxonomy_domain +, O +and O +is O +characterized O +by O +five O +specific O +( O +methyl O +-) O +lanthionine O +rings O +, O +which O +are O +crucial O +for O +stability O +and O +activity O +in O +the O +nanomolar O +range O +. O + +Thus O +, O +the O +lantibiotic B-chemical +producer O +strains O +have O +an O +inbuilt O +self O +- O +protection O +mechanism O +( O +immunity O +) O +to O +prevent O +cell O +death O +caused O +due O +to O +the O +action O +of O +its O +cognate O +lantibiotic B-chemical +. O + +This O +immunity O +system O +consists O +of O +a O +membrane B-protein_type +– I-protein_type +associated I-protein_type +lipoprotein I-protein_type +( O +usually O +referred O +to O +as O +LanI B-protein_type +) O +and O +/ O +or O +an O +ABC B-protein_type +transporter I-protein_type +( O +termed O +as O +LanFEG B-protein_type +and O +comprising O +three O +subunits O +). O + +Although O +some O +lantibiotics B-chemical +such O +as O +Pep5 B-chemical +, O +epicidin B-chemical +, O +epilancin B-chemical +, O +and O +lactocin B-chemical +S I-chemical +only O +require O +LanI B-protein_type +for O +immunity O +, O +other O +lantibiotics B-chemical +with O +a O +dual O +mode O +of O +action O +involving O +pore O +formation O +and O +lipid O +II O +binding O +such O +as O +nisin B-chemical +, O +subtilin B-chemical +, O +epidermin B-chemical +, O +gallidermin B-chemical +, O +and O +lacticin B-chemical +3147 I-chemical +require O +additionally O +the O +presence O +of O +LanFEG B-protein_type +. O + +Examples O +for O +LanFEG B-protein_type +are O +NisI B-protein +and O +NisFEG B-protein +of O +the O +nisin B-chemical +system O +, O +SpaI B-protein +and O +SpaFEG B-protein +conferring O +immunity O +towards O +subtilin B-chemical +, O +and O +PepI B-protein +constituting O +the O +immunity O +system O +of O +Pep5 B-chemical +producing O +strains O +. O + +Structural B-evidence +data I-evidence +are O +reported O +for O +the O +immunity B-protein_type +proteins I-protein_type +NisI B-protein +from O +Lactococcus B-species +lactis I-species +, O +SpaI B-protein +from O +Bacillus B-species +subtilis I-species +and O +MlbQ B-protein +from O +the O +lantibiotic B-chemical +NAI B-chemical +- I-chemical +107 I-chemical +producer O +strain O +Microbispora B-species +ATCC I-species +PTA I-species +- I-species +5024 I-species +. O + +Recently O +, O +gene O +clusters O +were O +identified O +in O +certain O +clinically O +relevant O +human B-species +pathogenic O +strains O +such O +as O +Streptococcus B-species +agalactiae I-species +, O +S B-species +. I-species +aureus I-species +, O +and O +others O +that O +confer O +inherent O +resistance O +against O +specific O +lantibiotics B-chemical +such O +as O +nisin B-chemical +and O +resemble O +the O +genetic O +architecture O +of O +the O +lantibiotic O +immunity O +genes O +found O +in O +the O +producing O +strains O +. O + +Within O +these O +resistance O +operons O +, O +genes O +encoding O +for O +a O +membrane B-protein_type +- I-protein_type +associated I-protein_type +protease I-protein_type +and O +an O +ABC B-protein_type +transporter I-protein_type +were O +identified O +. O + +Expression O +of O +these O +proteins O +provides O +resistance O +against O +lantibiotics B-chemical +. O + +Recently O +, O +the O +structure B-evidence +of O +SaNSR B-protein +from O +S B-species +. I-species +agalactiae I-species +was O +solved O +which O +provides O +resistance O +against O +nisin B-chemical +by O +a O +protease O +activity O +. O + +Furthermore O +, O +the O +upregulation O +of O +these O +genes O +is O +mediated O +by O +a O +specific O +two B-complex_assembly +- I-complex_assembly +component I-complex_assembly +system I-complex_assembly +( O +TCS B-complex_assembly +) O +similar O +to O +the O +one O +found O +in O +lantibiotic B-chemical +producing O +strains O +, O +consisting O +of O +a O +sensor O +histidine B-protein_type +kinase I-protein_type +( O +HK B-protein_type +) O +and O +a O +response B-protein_type +regulator I-protein_type +( O +RR B-protein_type +), O +apparently O +mediate O +the O +expression O +of O +the O +resistance O +proteins O +: O +HK B-protein_type +senses O +the O +external O +lantibiotic B-chemical +and O +, O +upon O +receiving O +the O +stimuli O +, O +auto B-ptm +- I-ptm +phosphorylates I-ptm +at O +a O +conserved B-protein_state +histidine B-residue_name +residue O +within O +the O +cytosol O +; O +this O +high O +- O +energetic O +phosphoryl O +group O +is O +then O +transferred O +to O +the O +associated O +RR B-protein_type +inducing O +a O +conformational O +change O +there O +, O +which O +activates O +the O +RR B-protein_type +to O +evoke O +the O +cellular O +response O +. O + +Bacteria B-taxonomy_domain +have O +the O +ability O +to O +sense O +and O +survive O +various O +environmental O +stimuli O +through O +adaptive O +responses O +, O +which O +are O +regulated O +by O +TCSs B-complex_assembly +. O + +The O +absence B-protein_state +of I-protein_state +TCSs B-complex_assembly +within O +mammals B-taxonomy_domain +makes O +them O +unique O +targets O +for O +novel O +antimicrobial O +drugs O +. O + +The O +expression O +of O +the O +lantibiotic B-chemical +- O +resistance O +genes O +via O +TCS B-complex_assembly +is O +generally O +regulated O +by O +microorganism O +- O +specific O +lantibiotics B-chemical +, O +which O +act O +via O +external O +stimuli O +. O + +Some O +examples O +of O +TCS B-complex_assembly +are O +: O +BraRS B-protein +in O +S B-species +. I-species +aureus I-species +which O +is O +induced O +by O +bacitracin B-chemical +, O +nisin B-chemical +and O +nukacin B-chemical +- I-chemical +ISK I-chemical +- I-chemical +1 I-chemical +resistance O +, O +BceRS B-protein +in O +Bacillus B-taxonomy_domain +spp I-taxonomy_domain +. O +that O +is O +induced O +by O +actagardine B-chemical +and O +mersacidin B-chemical +resistance O +, O +LcrRS B-protein +in O +Streptococcus B-species +mutans I-species +induced O +by O +nukacin B-chemical +- I-chemical +ISK I-chemical +- I-chemical +1 I-chemical +and O +lacticin B-chemical +481 I-chemical +and O +LisRK B-protein +of O +Listeria B-species +monocytogenes I-species +induced O +by O +nisin B-chemical +resistance O +. O + +Furthermore O +, O +multiple O +lantibiotics B-chemical +can O +induce O +the O +TCS B-complex_assembly +CprRK B-protein +from O +Clostridium B-species +difficile I-species +, O +leading O +to O +the O +expression O +of O +the O +genes O +localized O +on O +the O +cpr B-gene +operon O +, O +resulting O +in O +resistance O +against O +several O +lantibiotics B-chemical +of O +which O +nisin B-chemical +, O +gallidermin B-chemical +, O +subtilin B-chemical +, O +and O +mutacin B-chemical +1140 I-chemical +are O +some O +examples O +. O + +Interestingly O +, O +the O +histidine B-protein_type +kinase I-protein_type +contains O +two B-structure_element +- I-structure_element +transmembrane I-structure_element +helices I-structure_element +but O +lacks O +an O +extracellular B-structure_element +sensory I-structure_element +domain I-structure_element +, O +and O +are O +therefore O +known O +as O +‘ B-protein_type +intramembrane I-protein_type +- I-protein_type +sensing I-protein_type +’ I-protein_type +histidine I-protein_type +kinases I-protein_type +. O + +It O +has O +been O +suggested O +that O +in O +addition O +to O +conferring O +general O +resistance O +against O +lantibiotics B-chemical +, O +the O +BceAB B-protein_type +- I-protein_type +type I-protein_type +transporters I-protein_type +assist O +in O +signalling O +as O +via O +the O +presence O +of O +a O +large O +extracellular B-structure_element +domain I-structure_element +within O +the O +transmembrane B-structure_element +segment I-structure_element +indicated O +by O +experimental O +evidence O +from O +various O +systems O +. O + +The O +recently O +discovered O +nsr B-gene +gene O +cluster O +of O +the O +human B-species +pathogen O +S B-species +. I-species +agalactiae I-species +encodes O +for O +the O +resistance B-protein_type +protein I-protein_type +NSR B-protein +and O +the O +ABC B-protein_type +transporter I-protein_type +NsrFP B-protein +, O +both O +conferring O +resistance O +against O +nisin B-chemical +. O + +Homologous O +operons O +have O +been O +identified O +in O +various O +human B-species +pathogenic O +strains O +such O +as O +Staphylococcus B-species +epidermis I-species +and O +Streptococcus B-species +ictaluri I-species +based O +on O +the O +high O +sequence O +identity O +of O +NSR B-protein +and O +NsrFP B-protein +. O + +In O +this O +gene O +cluster O +, O +the O +TCS B-complex_assembly +NsrRK B-protein +is O +responsible O +for O +the O +expression O +of O +the O +nsr B-gene +and O +nsrFP B-gene +genes O +. O + +The O +similarity O +of O +the O +TCS B-complex_assembly +within O +all O +the O +described O +nisin B-chemical +resistance O +operons O +suggests O +an O +expression O +specifically O +induced O +by O +nisin B-chemical +. O + +Thus O +, O +NsrRK B-protein +might O +be O +a O +useful O +target O +to O +combat O +inherently O +pathogenic O +lantibiotic B-chemical +- O +resistant O +strains O +. O + +Generally O +, O +RRs B-protein_type +consist O +of O +two O +distinct O +structural O +domains O +, O +a O +receiver B-structure_element +domain I-structure_element +( O +RD B-structure_element +) O +and O +an O +effector B-structure_element +domain I-structure_element +( O +ED B-structure_element +), O +that O +are O +separated O +from O +each O +other O +by O +a O +flexible B-protein_state +linker B-structure_element +. O + +RDs B-structure_element +contain O +a O +highly B-protein_state +conserved I-protein_state +aspartate B-residue_name +residue O +, O +which O +acts O +as O +a O +phosphoryl O +acceptor O +that O +becomes O +phosphorylated B-protein_state +by O +the O +kinase B-structure_element +domain I-structure_element +of O +the O +histidine B-protein_type +kinase I-protein_type +upon O +reception O +of O +an O +external O +signal O +. O + +The O +ED B-structure_element +is O +thereby O +activated O +and O +binds O +to O +the O +designated O +promoters O +, O +thus O +initiating O +transcription O +of O +the O +target O +genes O +. O + +The O +RRs B-protein_type +are O +classified O +into O +different O +subfamilies O +depending O +on O +the O +three O +- O +dimensional O +structure O +of O +their O +EDs B-structure_element +. O + +The O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +is O +the O +largest O +subgroup O +of O +RRs B-protein_type +and O +comprises O +approximately O +40 O +% O +of O +all O +response B-protein_type +regulators I-protein_type +in O +bacteria B-taxonomy_domain +. O + +All O +their O +members O +are O +characterized O +by O +a O +winged B-structure_element +helix I-structure_element +- I-structure_element +turn I-structure_element +- I-structure_element +helix I-structure_element +( O +wHTH B-structure_element +) O +motif O +. O + +Although O +numerous O +structures B-evidence +of O +the O +single O +domains O +are O +known O +, O +only O +a O +few O +structures B-evidence +of O +full B-protein_state +- I-protein_state +length I-protein_state +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +- I-protein_type +type I-protein_type +RRs I-protein_type +have O +been O +determined O +: O +RegX3 B-protein +( O +PDB O +code O +: O +2OQR O +), O +MtrA B-protein +( O +PDB O +code O +: O +2GWR O +), O +PrrA B-protein +( O +PDB O +code O +: O +1YS6 O +) O +and O +PhoP B-protein +( O +PDB O +code O +: O +3R0J O +) O +from O +Mycobacterium B-species +tuberculosis I-species +; O +DrrB B-protein +( O +PDB O +code O +: O +1P2F O +) O +and O +DrrD B-protein +( O +PDB O +code O +: O +1KGS O +) O +from O +Thermotoga B-species +maritima I-species +; O +and O +KdpE B-protein +from O +Escherichia B-species +coli I-species +( O +PDB O +code O +: O +4KNY O +). O + +The O +various O +structures B-evidence +of O +RRs B-protein_type +reveal O +that O +in O +addition O +to O +being O +in O +either O +“ O +inactive B-protein_state +” O +or O +“ O +active B-protein_state +” O +state O +, O +the O +RRs B-protein_type +can O +also O +exist O +in O +two O +distinct O +conformations O +: O +“ O +open B-protein_state +” O +and O +“ O +closed B-protein_state +”. O + +MtrA B-protein +and O +PrrA B-protein +exhibit O +a O +very B-protein_state +compact I-protein_state +, O +closed B-protein_state +structure B-evidence +with O +the O +DNA B-structure_element +- I-structure_element +binding I-structure_element +sequence I-structure_element +, O +called O +recognition B-structure_element +helix I-structure_element +, O +of O +the O +ED B-structure_element +being O +inaccessible O +to O +DNA B-chemical +. O + +The O +structures B-evidence +of O +DrrD B-protein +and O +DrrB B-protein +exist O +in O +an O +open B-protein_state +conformation O +, O +here O +the O +recognition B-structure_element +helix I-structure_element +is O +fully B-protein_state +exposed I-protein_state +, O +suggesting O +that O +RRs B-protein_type +are O +flexible B-protein_state +in O +solution O +and O +can O +adopt O +multiple O +conformations O +. O + +Here O +, O +we O +describe O +the O +crystal B-evidence +structures I-evidence +of O +the O +N O +- O +terminal O +RD B-structure_element +and O +the O +C O +- O +terminal O +ED B-structure_element +of O +the O +lantibiotic B-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +RR I-protein_type +NsrR B-protein +from O +S B-species +. I-species +agalactiae I-species +. O + +NsrR B-protein +is O +part O +of O +the O +nisin B-chemical +resistance O +operon O +. O + +The O +expression O +of O +the O +genes O +of O +this O +operon O +is O +induced O +by O +a O +TCS B-complex_assembly +consisting O +of O +the O +HK B-protein_type +NsrK B-protein +and O +the O +RR B-protein_type +NsrR B-protein +. O +Based O +on O +the O +crystal B-evidence +structures I-evidence +of O +both O +the O +domains O +, O +modeling O +was O +employed O +to O +shed O +light O +on O +the O +putative O +DNA B-protein_state +- I-protein_state +bound I-protein_state +state O +of O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +. O + +NsrR B-protein +was O +expressed B-experimental_method +and I-experimental_method +purified I-experimental_method +as O +described O +, O +resulting O +in O +a O +homogenous O +protein O +as O +observed O +by O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +( O +Fig O +1A O +), O +with O +a O +yield O +of O +2 O +mg O +per O +liter O +of O +cell O +culture O +. O + +By O +calibrating O +the O +column O +with O +proteins O +of O +known O +molecular O +weight O +the O +NsrR B-protein +full B-protein_state +length I-protein_state +protein O +elutes O +as O +a O +dimer B-oligomeric_state +. O + +The O +purified O +NsrR B-protein +protein O +has O +a O +theoretical O +molecular B-evidence +mass I-evidence +of O +27 O +. O +7 O +kDa O +and O +was O +> O +98 O +% O +pure O +as O +assessed O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +( O +Fig O +1B O +, O +indicated O +by O +*). O + +Surprisingly O +, O +over O +time O +NsrR B-protein +degraded O +into O +two O +distinct O +fragments O +as O +visible O +on O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +analysis O +using O +the O +same O +purified O +protein O +sample O +after O +one O +week O +( O +Fig O +1C O +, O +indicated O +by O +** O +and O +***, O +respectively O +). O + +This O +was O +also O +observed O +by O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +where O +a O +peak O +at O +an O +elution O +time O +of O +18 O +min O +appeared O +( O +Fig O +1A O +). O + +Both O +bands O +were O +subjected O +to O +mass B-experimental_method +spectrometry I-experimental_method +analysis I-experimental_method +. O + +The O +analysis O +revealed O +that O +the O +larger O +fragment O +(**) O +represents O +the O +N O +- O +terminal O +receiver B-structure_element +domain I-structure_element +( O +residues O +1 B-residue_range +– I-residue_range +119 I-residue_range +; O +referred O +to O +as O +NsrR B-protein +- O +RD B-structure_element +) O +whereas O +the O +smaller O +fragment O +(***) O +contained O +the O +C O +- O +terminal O +DNA B-structure_element +- I-structure_element +binding I-structure_element +effector I-structure_element +domain I-structure_element +of O +NsrR B-protein +( O +residues O +129 B-residue_range +– I-residue_range +243 I-residue_range +including O +21 O +amino O +acids O +derived O +from O +the O +expression O +tag O +; O +referred O +to O +as O +NsrR B-protein +- O +ED B-structure_element +) O +( O +Fig O +1C O +). O + +Residues O +120 B-residue_range +– I-residue_range +128 I-residue_range +form O +the O +linker B-structure_element +connecting O +the O +RD B-structure_element +and O +ED B-structure_element +. O + +Such O +a O +cleavage O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +RR B-protein_type +into O +two O +specific O +domains O +is O +not O +unusual O +and O +has O +been O +previously O +reported O +for O +other O +RRs B-protein_type +as O +well O +. O + +Mass B-experimental_method +spectrometry I-experimental_method +analysis I-experimental_method +did O +not O +reveal O +the O +presence O +of O +any O +specific O +protease O +in O +the O +purified O +NsrR B-protein +sample O +. O + +Furthermore O +, O +addition O +of O +a O +protease O +inhibitor O +, O +such O +as O +PMSF B-chemical +( O +Phenylmethylsulfonyl B-chemical +fluoride I-chemical +) O +and O +AEBSF B-chemical +{ O +4 B-chemical +-( I-chemical +2 I-chemical +- I-chemical +Aminoethyl I-chemical +) I-chemical +benzenesulfonyl I-chemical +fluoride I-chemical +hydrochloride I-chemical +}, O +even O +at O +high O +concentrations O +, O +did O +not O +inhibit O +proteolysis O +( O +data O +not O +shown O +). O + +Purification B-experimental_method +of O +NsrR B-protein +and O +SDS B-experimental_method +PAGE I-experimental_method +analysis O +of O +purified O +NsrR B-protein +directly O +and O +one O +week O +after O +purification O +. O + +( O +a O +) O +Elution B-evidence +profile I-evidence +of O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +step O +of O +NsrR B-protein +. O +The O +y O +- O +axis O +represents O +the O +UV O +absorption O +of O +the O +protein O +at O +280 O +nm O +, O +while O +the O +x O +- O +axis O +represents O +the O +elution O +volume O +. O + +The O +bold O +line O +represents O +the O +chromatogram B-evidence +of O +freshly O +purified O +NsrR B-protein +while O +the O +dashed O +line O +shows O +the O +chromatogram B-evidence +of O +the O +same O +NsrR B-protein +protein O +after O +one O +week O +. O + +( O +b O +) O +Freshly O +purified O +NsrR B-protein +protein O +, O +and O +( O +c O +) O +NsrR B-protein +protein O +after O +one O +week O +. O + +Lanes O +: O +M O +represents O +the O +PAGE O +Ruler O +Unstained O +Ladder O +; O +1 O +: O +NsrR B-protein +after O +a O +two O +- O +step O +purification O +; O +2 O +: O +NsrR B-protein +one O +week O +after O +purification O +. O + +* O +corresponds O +to O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +protein O +at O +27 O +kDa O +, O +while O +** O +and O +*** O +correspond O +to O +the O +NsrR B-protein +- O +RD B-structure_element +and O +NsrR B-protein +- O +ED B-structure_element +domain O +at O +around O +13 O +kDa O +, O +respectively O +. O + +Since O +formation O +of O +the O +crystals B-evidence +took O +around O +one O +month O +, O +it O +is O +not O +surprising O +that O +this O +cleavage O +also O +occurred O +in O +the O +crystallization O +drop O +. O + +NsrR B-protein +was O +crystallized B-experimental_method +yielding O +two O +crystal O +forms O +, O +which O +were O +distinguishable O +by O +visual O +inspection O +. O + +Initially O +, O +we O +tried O +to O +solve O +the O +structure B-evidence +of O +NsrR B-protein +by O +molecular B-experimental_method +replacement I-experimental_method +, O +which O +was O +not O +successful O +. O + +Therefore O +, O +we O +tried O +heavy B-experimental_method +atom I-experimental_method +phasing I-experimental_method +using O +a O +platinum B-chemical +compound O +. O + +This O +succeeded O +for O +the O +rectangular O +plate O +- O +shaped O +crystals B-evidence +. O + +After O +the O +structure B-evidence +was O +solved O +, O +it O +became O +evident O +that O +these O +crystals B-evidence +contained O +two O +monomers B-oligomeric_state +of O +the O +ED B-structure_element +of O +NsrR B-protein +in O +the O +asymmetric O +unit O +. O + +We O +also O +tried O +to O +solve O +the O +structure B-evidence +of O +the O +thin O +plate O +- O +shaped O +crystals B-evidence +with O +this O +template O +, O +but O +the O +resulting O +model O +generated O +was O +not O +sufficient O +. O + +Therefore O +, O +we O +thought O +that O +these O +crystals B-evidence +contained O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +of O +NsrR B-protein +and O +successfully O +phased O +this O +dataset O +using O +molecular B-experimental_method +replacement I-experimental_method +with O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +of O +PhoB B-protein +( O +PDB O +code O +: O +1B00 O +; O +as O +a O +template O +. O + +This O +approach O +revealed O +that O +this O +crystal O +form O +indeed O +contained O +two O +monomers B-oligomeric_state +of O +the O +RD B-structure_element +of O +NsrR B-protein +in O +the O +asymmetric O +unit O +. O + +Since O +both O +crystals O +forms O +were O +obtained O +in O +the O +same O +drop O +it O +is O +not O +surprising O +that O +, O +when O +dissolving O +several O +crystals B-evidence +and O +performing O +subsequent O +mass B-experimental_method +- I-experimental_method +spectrometry I-experimental_method +to O +identify O +the O +protein O +in O +the O +crystals B-evidence +, O +it O +yielded O +peptide O +fragments O +throughout O +the O +NsrR B-protein +sequence O +. O + +In O +summary O +, O +the O +two O +crystal B-evidence +forms I-evidence +contained O +one O +of O +the O +two O +domains O +, O +respectively O +, O +such O +that O +both O +domains O +were O +successfully O +crystallized B-experimental_method +. O + +We O +determined O +the O +crystal B-evidence +structures I-evidence +of O +NsrR B-protein +- O +RD B-structure_element +and O +NsrR B-protein +- O +ED B-structure_element +separately O +. O + +However O +, O +a O +part O +of O +the O +linker B-structure_element +region I-structure_element +( O +residues O +120 B-residue_range +– I-residue_range +128 I-residue_range +; O +120RRSQQFIQQ128 B-structure_element +; O +underlined O +are O +the O +amino O +acid O +residues O +not O +visible O +in O +either O +domain O +) O +could O +not O +be O +traced O +in O +the O +electron B-evidence +density I-evidence +. O + +Overall O +structure B-evidence +of O +the O +N O +- O +terminal O +NsrR B-protein +receiver B-structure_element +domain I-structure_element +( O +NsrR B-protein +- O +RD B-structure_element +) O + +The O +structure B-evidence +of O +the O +NsrR B-protein +- O +RD B-structure_element +was O +determined O +at O +a O +resolution O +of O +1 O +. O +8 O +Å O +( O +Table O +1 O +). O + +The O +Rwork B-evidence +and O +Rfree B-evidence +values O +after O +refinement O +were O +0 O +. O +17 O +and O +0 O +. O +22 O +, O +respectively O +. O + +Ramachandran B-evidence +validation I-evidence +revealed O +that O +all O +residues O +( O +100 O +%, O +236 O +amino O +acids O +) O +were O +in O +the O +preferred O +or O +allowed O +regions O +. O + +The O +structure B-evidence +contained O +many O +ethylene B-chemical +glycol I-chemical +molecules O +arising O +from O +the O +cryo O +- O +protecting O +procedure O +. O + +The O +asymmetric O +unit O +contains O +two O +copies O +of O +NsrR B-protein +- O +RD B-structure_element +. O + +Although O +the O +entire O +N O +- O +terminal O +receiver B-structure_element +domain I-structure_element +is O +composed O +of O +residues O +Met1 B-residue_range +- I-residue_range +Leu119 I-residue_range +, O +only O +residues O +Asn4 B-residue_range +to I-residue_range +Arg121 I-residue_range +of O +chain B-structure_element +A I-structure_element +( O +including O +residues O +Arg120 B-residue_name_number +and O +Arg121 B-residue_name_number +of O +the O +linker B-structure_element +) O +and O +Gln5 B-residue_range +to I-residue_range +Ser122 I-residue_range +of O +chain B-structure_element +B I-structure_element +( O +including O +residues O +Arg120 B-residue_range +until I-residue_range +Ser122 I-residue_range +of O +the O +linker B-structure_element +) O +could O +be O +traced O +in O +the O +electron B-evidence +density I-evidence +of O +NsrR B-protein +- O +RD B-structure_element +. O + +For O +Asn85 B-residue_name_number +, O +Asp86 B-residue_name_number +, O +and O +Glu87 B-residue_name_number +of O +chain B-structure_element +A I-structure_element +, O +poor O +electron B-evidence +density I-evidence +was O +observed O +for O +the O +side O +chains O +and O +, O +thus O +, O +these O +side O +chains O +were O +deleted O +during O +refinement O +and O +are O +not O +present O +in O +the O +final O +structure B-evidence +. O + +Since O +the O +two O +monomers B-oligomeric_state +of O +NsrR B-protein +- O +RD B-structure_element +were O +virtually O +identical O +( O +rmsd B-evidence +of O +0 O +. O +6 O +Å O +over O +116 O +Cα O +atoms O +for O +the O +two O +monomers B-oligomeric_state +). O + +Therefore O +, O +the O +overall O +structure B-evidence +is O +described O +for O +monomer B-oligomeric_state +A B-structure_element +only O +. O + +NsrR B-protein +- O +RD B-structure_element +structurally O +adopts O +a O +αβ B-structure_element +doubly I-structure_element +- I-structure_element +wound I-structure_element +fold I-structure_element +previously O +observed O +in O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +type I-protein_type +regulators I-protein_type +. O + +Five O +β B-structure_element +- I-structure_element +strands I-structure_element +( O +β1 B-structure_element +- I-structure_element +β5 I-structure_element +) O +are O +arranged O +in O +a O +parallel O +fashion O +constituting O +the O +central O +core O +of O +the O +structure B-evidence +, O +which O +is O +surrounded O +by O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +α1 B-structure_element +and O +α5 B-structure_element +) O +on O +one O +and O +three O +helices B-structure_element +( O +α2 B-structure_element +, O +α3 B-structure_element +, O +α4 B-structure_element +) O +on O +the O +other O +side O +( O +Fig O +2 O +). O + +The O +NsrR B-protein +- O +RD B-structure_element +structure B-evidence +shows O +a O +β1 B-structure_element +- I-structure_element +α1 I-structure_element +- I-structure_element +β2 I-structure_element +- I-structure_element +α2 I-structure_element +- I-structure_element +β3 I-structure_element +- I-structure_element +α3 I-structure_element +- I-structure_element +β4 I-structure_element +- I-structure_element +α4 I-structure_element +- I-structure_element +β5 I-structure_element +- I-structure_element +α5 I-structure_element +topology O +as O +also O +observed O +for O +other O +RRs B-protein_type +. O + +Structure B-evidence +of O +NsrR B-protein +- O +RD B-structure_element +. O + +Cartoon O +representation O +of O +the O +helices B-structure_element +( O +α1 B-structure_element +– I-structure_element +α5 I-structure_element +) O +and O +β B-structure_element +- I-structure_element +sheets I-structure_element +( O +β1 B-structure_element +- I-structure_element +β5 I-structure_element +). O + +Structural O +areas O +with O +the O +highest O +variations O +to O +the O +receiver B-structure_element +domains I-structure_element +of O +DrrB B-protein +( O +pink O +, O +1P2F O +), O +MtrA B-protein +( O +grey O +, O +2GWR O +), O +and O +PhoB B-protein +( O +blue O +, O +1B00 O +) O +are O +marked O +in O +separate O +boxes O +. O + +Comparison B-experimental_method +with O +structures B-evidence +of O +other O +receiver B-structure_element +domains I-structure_element + +NsrR B-protein +belongs O +to O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +family I-protein_type +of O +RRs B-protein_type +. O + +The O +receiver B-structure_element +domain I-structure_element +of O +NsrR B-protein +was O +superimposed B-experimental_method +with O +other O +structurally O +characterized O +receiver B-structure_element +domains I-structure_element +from O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +family I-protein_type +, O +such O +as O +DrrB B-protein +, O +KdpE B-protein +, O +MtrA B-protein +, O +and O +the O +crystal B-evidence +structure I-evidence +of O +only O +the O +receiver B-structure_element +domain I-structure_element +of O +PhoB B-protein +. O +The O +rmsd B-evidence +of O +the O +overlays B-experimental_method +and O +the O +corresponding O +PDB O +codes O +used O +are O +highlighted O +in O +Table O +2 O +. O + +Superimposition B-experimental_method +of O +the O +structures B-evidence +revealed O +that O +helix B-structure_element +α4 B-structure_element +is O +slightly O +rotated O +outward O +in O +NsrR B-protein +- O +RD B-structure_element +( O +Fig O +2 O +). O + +In O +receiver B-structure_element +domains I-structure_element +of O +response B-protein_type +regulators I-protein_type +, O +helix B-structure_element +α4 B-structure_element +has O +been O +shown O +to O +be O +a O +crucial O +part O +of O +the O +dimerization B-site +interface I-site +. O + +Furthermore O +, O +helix B-structure_element +α4 B-structure_element +in O +NsrR B-protein +is O +shorter O +than O +in O +other O +RRs B-protein_type +. O + +The O +first B-structure_element +helical I-structure_element +turn I-structure_element +is O +unwound B-protein_state +and O +adopts O +an O +unstructured B-protein_state +region O +( O +see O +Fig O +2 O +). O + +A O +slightly O +outward O +rotation O +or O +unwinding O +of O +helix B-structure_element +α4 B-structure_element +has O +been O +observed O +in O +the O +structures B-evidence +of O +other O +RD B-structure_element +of O +regulators O +. O + +For O +example O +, O +the O +structure B-evidence +of O +BaeR B-protein +and O +RegX3 B-protein +displayed O +a O +completely O +unwound B-protein_state +helix B-structure_element +α4 B-structure_element +. O + +In O +the O +structure B-evidence +of O +DrrD B-protein +, O +helix B-structure_element +α4 B-structure_element +is O +only O +partially O +displaced O +. O + +In O +the O +receiver B-structure_element +domain I-structure_element +of O +NsrR B-protein +, O +helix B-structure_element +α4 B-structure_element +is O +also O +partially O +displaced O +but O +in O +a O +different O +direction O +( O +S1 O +Fig O +). O + +Inspection O +of O +the O +crystal O +contacts O +revealed O +no O +major O +interactions O +in O +this O +region O +that O +could O +have O +influenced O +the O +orientation O +of O +helix B-structure_element +α4 B-structure_element +. O + +Furthermore O +, O +NsrR B-protein +is O +crystallized B-experimental_method +as O +a O +monomer B-oligomeric_state +, O +and O +investigation O +of O +the O +symmetry O +- O +related O +molecules O +did O +not O +reveal O +a O +functional O +dimer B-oligomeric_state +within O +the O +crystal B-evidence +. O + +This O +could O +explain O +the O +flexibility O +and O +thereby O +the O +different O +orientation O +of O +helix B-structure_element +α4 B-structure_element +in O +NsrR B-protein +. O + +The O +structures B-evidence +of O +the O +RD B-structure_element +and O +ED B-structure_element +domains O +of O +NsrR B-protein +aligned B-experimental_method +to O +other O +response B-protein_type +regulators I-protein_type +. O + +The O +rmsd B-evidence +values O +of O +the O +superimpositions B-experimental_method +of O +the O +structures B-evidence +of O +NsrR B-protein +- O +RD B-structure_element +and O +NsrR B-protein +- O +ED B-structure_element +with O +the O +available O +structures B-evidence +of O +members O +of O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +are O +highlighted O +. O +* O +Seq O +ID O +(%) O +corresponds O +to O +the O +full B-protein_state +- I-protein_state +length I-protein_state +protein O +sequence O +. O + +Based O +on O +the O +Dali B-experimental_method +server I-experimental_method +, O +the O +NsrR B-protein +- O +RD B-structure_element +domain O +is O +structurally O +closely O +related O +to O +KdpE B-protein +( O +PDB O +code O +: O +4KNY O +) O +from O +E B-species +. I-species +coli I-species +, O +displaying O +a O +sequence O +identity O +of O +28 O +%. O + +This O +structural O +homology O +is O +also O +reflected O +by O +the O +low O +rmsd B-evidence +of O +1 O +. O +9 O +Å O +over O +117 O +Cα O +atoms O +after O +superimposition B-experimental_method +of O +the O +receiver B-structure_element +domains I-structure_element +of O +NsrR B-protein +and O +KdpE B-protein +( O +Table O +2 O +). O + +Furthermore O +, O +the O +orientation O +of O +the O +helix B-structure_element +α4 B-structure_element +in O +NsrR B-protein +is O +close O +to O +that O +present O +in O +KdpE B-protein +( O +S1 O +Fig O +). O + +Active B-site +site I-site +residues O +and O +dimerization O + +All O +RRs B-protein_type +contain O +a O +highly B-protein_state +conserved I-protein_state +aspartate B-residue_name +residue O +in O +the O +active B-site +site I-site +( O +Fig O +3 O +; O +shown O +in O +red O +). O + +Phosphorylation B-ptm +of O +this O +aspartate B-residue_name +residue O +induces O +a O +conformational O +change O +leading O +to O +the O +activation O +of O +the O +effector B-structure_element +domain I-structure_element +that O +binds O +DNA B-chemical +and O +regulates O +the O +transcription O +of O +target O +genes O +. O + +This O +site O +of O +phosphorylation B-ptm +is O +conserved B-protein_state +throughout O +the O +family O +of O +response B-protein_type +regulators I-protein_type +, O +including O +the O +lantibiotic B-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +RRs I-protein_type +such O +as O +BraR B-protein +from O +L B-species +. I-species +monocytogenes I-species +, O +BceR B-protein +from O +Bacillus B-species +subtilis I-species +, O +CprR B-protein +from O +C B-species +. I-species +difficile I-species +, O +GraR B-protein +from O +S B-species +. I-species +aureus I-species +, O +LcrR B-protein +from O +S B-species +. I-species +mutans I-species +, O +LisR B-protein +, O +and O +VirR B-protein +from O +L B-species +. I-species +monocytogenes I-species +( O +Fig O +3 O +). O + +Sequence B-experimental_method +alignment I-experimental_method +of O +NsrR B-protein +protein O +with O +other O +response B-protein_type +regulators I-protein_type +. O + +A O +sequence B-experimental_method +alignment I-experimental_method +of O +NsrR B-protein +with O +RRs B-protein_type +belonging O +to O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +( O +marked O +in O +grey O +) O +and O +RRs B-protein_type +involved O +in O +lantibiotic B-chemical +resistance O +( O +black O +) O +is O +shown O +. O + +The O +active B-site +site I-site +aspartate B-residue_name +residue O +( O +highlighted O +in O +red O +), O +the O +residues O +forming O +the O +acidic B-site +pocket I-site +surrounding O +it O +( O +highlighted O +in O +pink O +), O +the O +switch B-site +residues I-site +( O +highlighted O +in O +blue O +), O +the O +conserved B-protein_state +lysine B-residue_name +residue O +( O +highlighted O +in O +green O +), O +the O +highly B-protein_state +conserved I-protein_state +residues O +of O +the O +linker B-structure_element +region I-structure_element +( O +colored O +in O +purple O +), O +the O +residues O +involved O +in O +dimer B-site +interface I-site +of O +receiver B-structure_element +domain I-structure_element +( O +highlighted O +in O +yellow O +), O +residues O +involved O +in O +interdomain O +interactions O +( O +shown O +in O +orange O +boxes O +and O +in O +cyan O +) O +and O +the O +residues O +involved O +in O +interaction O +with O +DNA B-chemical +( O +colored O +in O +blue O +) O +are O +shown O +. O + +The O +linker B-structure_element +region I-structure_element +of O +the O +known O +structures B-evidence +is O +underlined O +within O +the O +sequence O +. O + +The O +putative O +phosphorylation B-site +site I-site +of O +NsrR B-protein +is O +Asp55 B-residue_name_number +, O +which O +is O +localized O +at O +the O +end O +of O +strand B-structure_element +β3 B-structure_element +( O +Fig O +3 O +, O +shown O +in O +red O +; O +Fig O +4 O +) O +and O +lies O +within O +an O +acidic O +environment O +composed O +of O +the O +side O +chains O +of O +Glu12 B-residue_name_number +and O +Asp13 B-residue_name_number +( O +Fig O +3 O +, O +highlighted O +in O +pink O +). O + +This O +pocket B-site +is O +similar O +to O +the O +acidic B-protein_state +active B-site +site I-site +observed O +within O +most O +structures B-evidence +of O +RRs B-protein_type +such O +as O +PhoB B-protein +from O +E B-species +. I-species +coli I-species +, O +PhoP B-protein +from O +M B-species +. I-species +tuberculosis I-species +, O +and O +DivK B-protein +from O +Caulobacter B-species +crescentus I-species +. O + +In O +NsrR B-protein +, O +Glu12 B-residue_name_number +, O +Asp13 B-residue_name_number +, O +and O +Asp55 B-residue_name_number +are O +in O +close O +proximity O +of O +a O +highly B-protein_state +conserved I-protein_state +Lys104 B-residue_name_number +residue O +( O +highlighted O +in O +green O +in O +Fig O +3 O +). O + +Location O +of O +the O +highly B-protein_state +conserved I-protein_state +Asp55 B-residue_name_number +and O +inactive B-protein_state +state O +conformation O +of O +the O +key O +switch B-site +residues I-site +, O +Ser82 B-residue_name_number +and O +Phe101 B-residue_name_number +in O +NsrR B-protein +- O +RD B-structure_element +. O + +NsrR B-protein +( O +represented O +in O +yellow O +) O +displays O +a O +geometry O +representing O +the O +inactive B-protein_state +state O +as O +deduced O +from O +the O +inactive B-protein_state +state O +structure B-evidence +of O +PhoB B-protein +( O +shown O +in O +brown O +, O +PDB O +code O +1B00 O +) O +( O +a O +). O + +The O +inactive B-protein_state +conformation O +of O +NsrR B-protein +differs O +from O +the O +active B-protein_state +state O +structure B-evidence +of O +PhoB B-protein +( O +light O +blue O +, O +PDB O +code O +1ZES O +) O +( O +b O +) O +in O +the O +orientation O +of O +the O +corresponding O +switch B-site +residues I-site +, O +Ser82 B-residue_name_number +and O +Phe101 B-residue_name_number +, O +which O +adopt O +a O +conformation O +pointing O +away O +from O +the O +active B-site +site I-site +( O +Asp55 B-residue_name_number +in O +NsrR B-protein +). O + +A O +divalent O +metal O +ion O +is O +usually O +bound O +in O +this O +acidic O +environment O +and O +is O +essential O +for O +phosphorylation B-ptm +and O +de B-ptm +- I-ptm +phosphorylation I-ptm +of O +RRs B-protein_type +. O + +In O +some O +RRs B-protein_type +like O +CheY B-protein +, O +Mg2 B-chemical ++ I-chemical +is O +observed O +in O +the O +structure B-evidence +, O +bound B-protein_state +near O +the O +phosphorylation B-site +site I-site +. O + +In O +the O +KdpE B-protein +regulator B-protein_type +from O +E B-species +. I-species +coli I-species +that O +is O +involved O +in O +osmoregulation O +, O +a O +divalent O +calcium B-chemical +ion O +is O +present O +. O + +However O +, O +the O +structure B-evidence +of O +NsrR B-protein +- O +RD B-structure_element +did O +not O +contain O +any O +divalent O +ion O +. O + +Instead O +, O +a O +water B-chemical +molecule O +is O +present O +, O +which O +interacts O +with O +Glu12 B-residue_name_number +of O +the O +acidic B-site +pocket I-site +, O +Lys104 B-residue_name_number +, O +and O +another O +water B-chemical +molecule O +in O +the O +vicinity O +. O + +Within O +the O +β4 B-structure_element +- I-structure_element +α4 I-structure_element +loop I-structure_element +and O +in O +β5 B-structure_element +of O +the O +RD B-structure_element +of O +RRs B-protein_type +, O +specific O +amino O +acids O +are O +crucial O +for O +signal O +transduction O +from O +the O +RD B-structure_element +to O +the O +ED B-structure_element +via O +conformational O +changes O +that O +are O +a O +consequence O +of O +phosphorylation B-ptm +of O +the O +RD B-structure_element +. O + +These O +amino O +acids O +are O +Ser B-residue_name +/ O +Thr B-residue_name +and O +Phe B-residue_name +/ O +Tyr B-residue_name +located O +at O +the O +end O +of O +β4 B-structure_element +and O +before O +β5 B-structure_element +, O +respectively O +, O +and O +designated O +as O +“ O +signature B-site +switch I-site +residues I-site +”. O + +As O +seen O +in O +the O +alignment B-experimental_method +( O +Fig O +3 O +, O +highlighted O +in O +blue O +), O +these O +signature O +residues O +( O +Ser B-residue_name +/ O +Thr B-residue_name +and O +Phe B-residue_name +/ O +Tyr B-residue_name +) O +are O +highly B-protein_state +conserved I-protein_state +in O +the O +lantibiotic B-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +RRs I-protein_type +. O + +The O +orientation O +of O +the O +side O +chains O +of O +these O +residues O +determines O +whether O +the O +RD B-structure_element +is O +in O +an O +active B-protein_state +or O +inactive B-protein_state +state O +. O + +In O +the O +inactive B-protein_state +state O +, O +the O +phenylalanine B-residue_name +or O +tyrosine B-residue_name +residue O +faces O +away O +from O +the O +active B-site +site I-site +, O +and O +the O +corresponding O +serine B-residue_name +or O +threonine B-residue_name +residue O +adopts O +an O +outward B-protein_state +- I-protein_state +facing I-protein_state +conformation O +as O +well O +( O +Fig O +4A O +). O + +In O +contrast O +, O +the O +switch B-site +residues I-site +face O +towards O +the O +active B-site +site I-site +in O +the O +active B-protein_state +state O +conformation O +( O +Fig O +4B O +). O + +By O +sequence B-experimental_method +alignment I-experimental_method +with O +other O +lantibiotic B-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +RRs I-protein_type +, O +these O +“ O +signature B-site +switch I-site +residues I-site +” O +are O +identified O +as O +Ser82 B-residue_name_number +and O +Phe101 B-residue_name_number +in O +NsrR B-protein +( O +see O +above O +). O + +Although O +some O +RRs B-protein_type +such O +as O +KdpE B-protein +, O +BraR B-protein +, O +BceR B-protein +, O +GraR B-protein +, O +and O +VirR B-protein +contain O +a O +serine B-residue_name +residue O +as O +the O +first B-site +switch I-site +residue I-site +, O +the O +others O +possess O +a O +threonine B-residue_name +instead O +. O + +Furthermore O +, O +the O +second B-site +switch I-site +residue I-site +is O +mostly O +a O +tyrosine B-residue_name +, O +with O +NsrR B-protein +, O +BraR B-protein +, O +and O +BceR B-protein +being O +the O +only O +exceptions O +containing O +a O +phenylalanine B-residue_name +at O +that O +position O +. O + +A O +comparison O +of O +the O +NsrR B-protein +- O +RD B-structure_element +structure B-evidence +with O +the O +available O +structures B-evidence +of O +PhoB B-protein +( O +Fig O +4 O +) O +in O +the O +active B-protein_state +( O +PDB O +code O +: O +1ZES O +) O +and O +inactive B-protein_state +( O +PDB O +code O +: O +1B00 O +) O +states O +demonstrates O +that O +Ser82 B-residue_name_number +( O +NsrR B-protein +- O +RD B-structure_element +) O +is O +oriented O +away O +from O +the O +active B-site +site I-site +Asp55 B-residue_name_number +, O +and O +that O +Phe101 B-residue_name_number +is O +also O +in O +an O +outward B-protein_state +conformation O +suggesting O +an O +inactive B-protein_state +state O +of O +the O +NsrR B-protein +- O +RD B-structure_element +( O +Fig O +4A O +). O + +As O +mentioned O +above O +, O +RRs B-protein_type +contain O +a O +phosphorylation B-protein_state +- I-protein_state +activated I-protein_state +switch B-site +and O +normally O +exist O +in O +equilibrium O +between O +the O +active B-protein_state +and O +inactive B-protein_state +conformations O +. O + +Phosphorylation B-ptm +shifts O +the O +equilibrium O +towards O +the O +active B-protein_state +conformation O +and O +induces O +the O +formation O +of O +rotationally O +symmetric O +dimers B-oligomeric_state +on O +the O +α4 B-site +- I-site +β5 I-site +- I-site +α5 I-site +interface I-site +of O +RDs B-structure_element +. O + +It O +has O +been O +suggested O +that O +dimerization O +is O +crucial O +for O +DNA B-chemical +- O +binding O +of O +RRs B-protein_type +of O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +. O + +The O +RD B-structure_element +domain O +of O +NsrR B-protein +was O +crystallized B-experimental_method +with O +two O +separate O +monomers B-oligomeric_state +in O +the O +asymmetric O +unit O +. O + +Therefore O +, O +we O +performed O +a O +DALI B-experimental_method +search I-experimental_method +and O +focused O +on O +RD B-structure_element +domains O +that O +were O +structurally O +determined O +as O +functional B-protein_state +dimers B-oligomeric_state +. O + +In O +this O +context O +, O +the O +dimer B-oligomeric_state +of O +full B-protein_state +- I-protein_state +length I-protein_state +KdpE B-protein +from O +E B-species +. I-species +coli I-species +( O +Z B-evidence +- I-evidence +score I-evidence +18 O +. O +8 O +, O +rmsd B-evidence +1 O +. O +9 O +Å O +over O +117 O +Cα O +atoms O +) O +( O +PDB O +code O +: O +4KNY O +) O +and O +the O +structure B-evidence +of O +the O +functional B-protein_state +dimer B-oligomeric_state +of O +the O +RD B-structure_element +of O +KdpE B-protein +from O +E B-species +. I-species +coli I-species +( O +PDB O +code O +: O +1ZH2 O +) O +represent O +the O +most O +structurally O +related O +structures B-evidence +. O + +We O +aligned B-experimental_method +NsrR B-protein +- O +RD B-structure_element +on O +both O +monomers B-oligomeric_state +of O +the O +RD B-structure_element +of O +KdpE B-protein +. O +Since O +helix B-structure_element +α4 B-structure_element +of O +NsrR B-protein +- O +RD B-structure_element +is O +orientated O +slightly O +different O +when O +compared O +with O +other O +structures B-evidence +of O +RDs B-structure_element +( O +Fig O +2 O +), O +helix B-structure_element +α4 B-structure_element +and O +the O +N O +- O +terminal O +loop B-structure_element +of O +one O +monomer B-oligomeric_state +were O +clashing O +with O +the O +second O +monomer B-oligomeric_state +( O +S2A O +Fig O +). O + +Therefore O +, O +helix B-structure_element +α4 B-structure_element +and O +the O +N O +- O +terminal O +loop B-structure_element +were O +shifted O +to O +the O +position O +of O +KdpE B-protein +by O +primarily O +modifying O +backbone O +torsion O +angles O +in O +the O +region O +immediately O +C O +- O +terminal O +to O +helix B-structure_element +α4 B-structure_element +. O + +Afterwards O +, O +helix B-structure_element +α4 B-structure_element +and O +the O +adjacent O +loops B-structure_element +were O +energy B-experimental_method +minimized I-experimental_method +with I-experimental_method +the I-experimental_method +MAB I-experimental_method +force I-experimental_method +field I-experimental_method +as O +implemented O +in O +the O +program O +Moloc O +; O +all O +other O +atoms O +of O +NsrR B-protein +- O +RD B-structure_element +were O +kept O +fixed O +. O + +The O +result O +is O +highlighted O +in O +S2B O +Fig O +. O +The O +energy B-protein_state +minimized I-protein_state +structure B-evidence +of O +NsrR B-protein +- O +RD B-structure_element +was O +then O +superimposed B-experimental_method +on O +the O +dimeric B-oligomeric_state +structure B-evidence +of O +KdpE B-protein +. O + +The O +putative O +functional O +dimer B-oligomeric_state +of O +NsrR B-protein +- O +RD B-structure_element +is O +depicted O +in O +Fig O +5 O +. O + +The O +dimeric B-site +interface I-site +is O +formed O +by O +α4 B-structure_element +- I-structure_element +β5 I-structure_element +- I-structure_element +α5 I-structure_element +of O +RD B-structure_element +( O +Fig O +5A O +), O +as O +previously O +observed O +in O +other O +RRs B-protein_type +. O + +In O +KdpE B-protein +, O +a O +network O +of O +salt B-bond_interaction +bridges I-bond_interaction +and O +other O +electrostatic B-bond_interaction +interactions I-bond_interaction +stabilize O +the O +interface B-site +within O +a O +single O +monomer B-oligomeric_state +as O +well O +as O +between O +the O +monomers B-oligomeric_state +. O + +Majority O +of O +these O +interactions O +involve O +residues O +that O +are O +highly B-protein_state +conserved I-protein_state +within O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +of O +RRs B-protein_type +. O + +In O +addition O +, O +the O +dimeric B-site +interface I-site +of O +KdpE B-protein +is O +characterized O +by O +hydrophobic B-site +patch I-site +formed O +by O +residues O +Ile88 B-residue_name_number +( O +α4 B-structure_element +), O +Leu91 B-residue_name_number +( O +α4 B-structure_element +), O +Ala110 B-residue_name_number +( O +α5 B-structure_element +), O +and O +Val114 B-residue_name_number +( O +α5 B-structure_element +). O + +Structurally O +, O +a O +similar O +set O +of O +residues O +is O +also O +found O +in O +NsrR B-protein +: O +Leu94 B-residue_name_number +( O +α4 B-structure_element +), O +Val110 B-residue_name_number +( O +α5 B-structure_element +) O +and O +Ala113 B-residue_name_number +( O +α5 B-structure_element +), O +respectively O +( O +depicted O +as O +spheres O +in O +Fig O +5B O +), O +which O +are O +conserved B-protein_state +to O +some O +extent O +on O +sequence O +level O +( O +highlighted O +in O +yellow O +; O +Fig O +3 O +). O + +Functional O +dimer B-oligomeric_state +orientation O +of O +the O +RDs B-structure_element +of O +NsrR B-protein +. O + +Dimeric B-oligomeric_state +structure B-evidence +of O +the O +RD B-structure_element +of O +NsrR B-protein +aligned O +to O +the O +structure B-evidence +of O +KdpE B-protein +( O +PDB O +code O +1ZH2 O +, O +not O +shown O +). O + +( O +a O +) O +The O +two O +monomers B-oligomeric_state +of O +NsrR B-protein +as O +functional O +dimers B-oligomeric_state +are O +represented O +in O +a O +cartoon O +representation O +displayed O +in O +cyan O +and O +yellow O +colors O +. O + +( O +b O +) O +Zoom O +- O +in O +of O +the O +dimeric B-site +interface I-site +mediated O +by O +α4 B-structure_element +- I-structure_element +β5 I-structure_element +- I-structure_element +α5 I-structure_element +. O + +The O +monomer B-oligomeric_state +- O +monomer B-oligomeric_state +interactions O +are O +facilitated O +by O +hydrophobic O +residues O +( O +displayed O +as O +spheres O +), O +inter O +- O +and O +intra O +- O +domain O +interactions O +( O +displayed O +as O +sticks O +). O + +Conserved O +intermolecular O +electrostatic B-bond_interaction +interactions I-bond_interaction +further O +stabilize O +the O +monomer B-oligomeric_state +- O +monomer B-oligomeric_state +interaction O +of O +KdpE B-protein +and O +are O +formed O +between O +Asp97 B-residue_name_number +( O +β5 B-structure_element +) O +and O +Arg111 B-residue_name_number +( O +α5 B-structure_element +), O +Asp96 B-residue_name_number +( O +α4 B-structure_element +– I-structure_element +β5 I-structure_element +loop I-structure_element +) O +and O +Arg118 B-residue_name_number +( O +α5 B-structure_element +), O +and O +Asp92 B-residue_name_number +( O +α4 B-structure_element +) O +and O +Arg113 B-residue_name_number +( O +α5 B-structure_element +). O + +Some O +of O +these O +interactions O +can O +also O +be O +identified O +in O +the O +dimeric B-oligomeric_state +model O +of O +NsrR B-protein +- O +RD B-structure_element +. O + +Here O +, O +Asp100 B-residue_name_number +( O +β5 B-structure_element +) O +and O +Lys114 B-residue_name_number +( O +α5 B-structure_element +) O +form O +an O +interaction O +within O +one O +monomer B-oligomeric_state +, O +and O +an O +intermolecular O +interaction O +can O +be O +observed O +between O +Asn95 B-residue_name_number +( O +α4 B-structure_element +) O +of O +one O +monomer B-oligomeric_state +with O +Thr116 B-residue_name_number +( O +α5 B-structure_element +) O +of O +the O +other O +monomer B-oligomeric_state +( O +Fig O +3 O +, O +shown O +in O +cyan O +). O + +Asp99 B-residue_name_number +( O +α4 B-structure_element +– I-structure_element +β5 I-structure_element +loop I-structure_element +; O +Fig O +3 O +, O +shown O +in O +cyan O +) O +points O +toward O +the O +side O +chain O +of O +Arg121 B-residue_name_number +. O + +This O +interaction O +is O +also O +observed O +in O +KdpE B-protein +( O +Asp96 B-residue_name_number +( O +α4 B-structure_element +– I-structure_element +β5 I-structure_element +loop I-structure_element +) O +and O +Arg118 B-residue_name_number +( O +α5 B-structure_element +)). O + +In O +KdpE B-protein +, O +Arg111 B-residue_name_number +is O +additionally O +stabilized O +by O +another O +intra O +- O +molecular O +salt B-bond_interaction +bridge I-bond_interaction +with O +Glu107 B-residue_name_number +( O +α5 B-structure_element +). O + +Interestingly O +, O +in O +NsrR B-protein +- O +RD B-structure_element +this O +amino O +acid O +corresponds O +to O +Val110 B-residue_name_number +( O +highlighted O +in O +yellow O +in O +Fig O +3 O +). O + +As O +observed O +in O +this O +alignment B-experimental_method +, O +the O +above O +- O +mentioned O +arginine B-residue_name +residue O +( O +Arg111 B-residue_name_number +in O +KdpE B-protein +) O +is O +either O +an O +arginine B-residue_name +or O +a O +lysine B-residue_name +residue O +( O +Lys114 B-residue_name_number +in O +NsrR B-protein +) O +in O +all O +RRs B-protein_type +used O +in O +the O +alignment B-experimental_method +( O +Fig O +3 O +, O +shown O +in O +cyan O +). O + +Interestingly O +, O +whenever O +an O +arginine B-residue_name +is O +present O +at O +this O +position O +( O +Arg111 B-residue_name_number +in O +KdpE B-protein +), O +a O +glutamate B-residue_name +( O +Glu107 B-residue_name_number +in O +KdpE B-protein +) O +is O +present O +as O +well O +, O +presumably O +stabilizing O +the O +arginine B-residue_name +side O +chain O +. O + +However O +, O +when O +a O +lysine B-residue_name +is O +present O +at O +this O +position O +, O +the O +glutamate B-residue_name +is O +exchanged O +to O +a O +hydrophobic O +residue O +contributing O +to O +the O +hydrophobic B-site +patch I-site +described O +above O +. O + +Additionally O +, O +it O +has O +been O +shown O +for O +PhoB B-protein +from O +E B-species +. I-species +coli I-species +and O +PhoP B-protein +from O +B B-species +. I-species +subtilis I-species +that O +mutating B-experimental_method +the O +corresponding O +residues O +involved O +in O +dimerisation O +( O +residues O +Asp100 B-residue_name_number +, O +Val110 B-residue_name_number +and O +Lys114 B-residue_name_number +in O +NsrR B-protein +) O +results O +in O +monomeric B-oligomeric_state +form O +of O +response B-protein_type +regulator I-protein_type +which O +has O +lost B-protein_state +the I-protein_state +ability I-protein_state +to I-protein_state +dimerize I-protein_state +as O +well O +as O +display O +reduced O +DNA B-chemical +binding O +capabilities O +. O + +Overall O +Structure B-evidence +of O +C O +- O +terminal O +DNA B-structure_element +- I-structure_element +binding I-structure_element +effector I-structure_element +domain I-structure_element +of O +NsrR B-protein + +The O +structure B-evidence +of O +NsrR B-protein +- O +ED B-structure_element +from O +S B-species +. I-species +agalactiae I-species +was O +determined O +using O +experimental O +phases O +from O +a O +single B-experimental_method +- I-experimental_method +wavelength I-experimental_method +anomalous I-experimental_method +dispersion I-experimental_method +dataset I-experimental_method +from O +the O +rectangular O +plate O +- O +shaped O +crystal O +derivatized O +with O +platinum B-chemical +at O +a O +resolution O +of O +1 O +. O +6 O +Å O +in O +space O +group O +P21212 O +. O + +The O +Rwork B-evidence +and O +Rfree B-evidence +values O +after O +refinement O +were O +0 O +. O +18 O +and O +0 O +. O +22 O +, O +respectively O +. O + +Ramachandran B-evidence +validation I-evidence +was O +done O +using O +MolProbity O +. O + +The O +latter O +is O +Glu128 B-residue_name_number +( O +last O +residue O +of O +the O +linker B-structure_element +region I-structure_element +) O +of O +chain B-structure_element +B I-structure_element +that O +is O +involved O +in O +crystal O +contacts O +and O +, O +therefore O +, O +likely O +adopts O +an O +unfavorable O +conformation O +. O + +The O +structure B-evidence +contained O +a O +few O +ethylene B-chemical +glycol I-chemical +molecules O +introduced O +by O +the O +cryo O +- O +protecting O +procedure O +. O + +The O +C O +- O +terminal O +effector B-structure_element +DNA I-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +of O +NsrR B-protein +is O +about O +13 O +kDa O +in O +size O +and O +consists O +of O +residues O +129 B-residue_range +– I-residue_range +243 I-residue_range +( O +including O +21 O +amino O +acid O +residues O +of O +the O +expression O +tag O +). O + +Monomer B-oligomeric_state +A B-structure_element +contains O +residue O +129 B-residue_range +– I-residue_range +224 I-residue_range +and O +monomer B-oligomeric_state +B B-structure_element +contain O +residues O +128 B-residue_range +– I-residue_range +225 I-residue_range +. O + +For O +Asp147 B-residue_name_number +of O +chain B-structure_element +A I-structure_element +and O +Glu174 B-residue_name_number +of O +chain B-structure_element +B I-structure_element +, O +poor O +electron B-evidence +density I-evidence +was O +observed O +for O +the O +side O +chains O +and O +, O +thus O +, O +these O +side O +chains O +were O +removed O +during O +refinement O +. O + +The O +asymmetric O +unit O +contains O +two O +copies O +of O +NsrR B-protein +- O +ED B-structure_element +related O +by O +two O +- O +fold O +rotational O +symmetry O +. O + +An O +overlay B-experimental_method +revealed O +that O +both O +monomers B-oligomeric_state +display O +high O +similarity O +in O +their O +overall O +structure B-evidence +with O +an O +rmsd B-evidence +of O +0 O +. O +5 O +Å O +over O +95 O +Cα O +atoms O +. O + +We O +therefore O +describe O +for O +the O +overall O +structure B-evidence +only O +monomer B-oligomeric_state +A B-structure_element +. O + +The O +ED B-structure_element +domain O +of O +NsrR B-protein +consists O +of O +six O +β B-structure_element +- I-structure_element +strands I-structure_element +and O +three O +α B-structure_element +- I-structure_element +helices I-structure_element +in O +a O +β6 B-structure_element +- I-structure_element +β7 I-structure_element +- I-structure_element +β8 I-structure_element +- I-structure_element +β9 I-structure_element +- I-structure_element +α6 I-structure_element +- I-structure_element +α7 I-structure_element +- I-structure_element +α8 I-structure_element +- I-structure_element +β10 I-structure_element +- I-structure_element +β11 I-structure_element +topology O +( O +the O +secondary O +structure O +elements O +are O +counted O +in O +continuation O +of O +those O +of O +the O +RD B-structure_element +). O + +The O +effector B-structure_element +domain I-structure_element +starts O +with O +a O +4 B-structure_element +- I-structure_element +stranded I-structure_element +antiparallel I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +, O +followed O +by O +three O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +eventually O +ends O +in O +a O +C O +- O +terminal O +β B-structure_element +- I-structure_element +hairpin I-structure_element +( O +Fig O +6 O +). O + +The O +two O +β B-structure_element +- I-structure_element +sheets I-structure_element +sandwich O +the O +three O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +Structure B-evidence +of O +the O +C O +- O +terminal O +effector B-structure_element +domain I-structure_element +of O +NsrR B-protein +. O + +Cartoon O +representation O +of O +the O +C O +- O +terminal O +effector B-structure_element +domain I-structure_element +of O +NsrR B-protein +( O +green O +; O +recognition B-structure_element +helix I-structure_element +in O +cyan O +). O + +The O +structural O +areas O +with O +the O +highest O +variations O +compared O +to O +the O +effector B-structure_element +domains I-structure_element +of O +DrrB B-protein +( O +pink O +, O +1P2F O +), O +MtrA B-protein +( O +grey O +, O +2GWR O +), O +and O +PhoB B-protein +( O +blue O +, O +1GXQ O +) O +are O +marked O +. O + +The O +transactivation B-structure_element +loop I-structure_element +of O +MtrA B-protein +is O +missing B-protein_state +in O +the O +structure B-evidence +, O +therefore O +, O +the O +two O +termini O +are O +connected O +by O +a O +dashed O +line O +. O + +The O +characteristic O +feature O +of O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +of O +RRs B-protein_type +is O +a O +winged B-structure_element +helix I-structure_element +- I-structure_element +turn I-structure_element +- I-structure_element +helix I-structure_element +( O +wHTH B-structure_element +) O +fold O +that O +is O +adopted O +by O +the O +α7 B-structure_element +- I-structure_element +loop I-structure_element +- I-structure_element +α8 I-structure_element +segment I-structure_element +in O +full B-protein_state +- I-protein_state +length I-protein_state +and O +single O +effector B-structure_element +domain I-structure_element +structures B-evidence +of O +RRs B-protein_type +of O +this O +subfamily O +. O + +The O +structure B-evidence +of O +NsrR B-protein +- O +ED B-structure_element +also O +contains O +such O +a O +wHTH B-structure_element +motif O +built O +up O +by O +helices B-structure_element +α7 B-structure_element +and O +α8 B-structure_element +( O +Fig O +6 O +). O + +The O +second O +helix B-structure_element +of O +the O +wHTH B-structure_element +motif O +is O +important O +for O +DNA B-chemical +- O +binding O +and O +, O +therefore O +, O +is O +termed O +“ O +recognition B-structure_element +helix I-structure_element +” O +( O +shown O +in O +cyan O +in O +Fig O +6 O +). O + +Furthermore O +, O +a O +helix B-structure_element +within O +the O +HTH B-structure_element +motif O +, O +named O +“ O +positioning B-structure_element +helix I-structure_element +”, O +is O +important O +for O +proper O +orientation O +and O +positioning O +of O +the O +loop B-structure_element +between O +these O +two O +helices O +and O +is O +referred O +to O +as O +“ O +transactivation B-structure_element +loop I-structure_element +” O +( O +also O +called O +α B-structure_element +- I-structure_element +loop I-structure_element +; O +Fig O +6 O +). O + +In O +the O +structure B-evidence +of O +NsrR B-protein +- O +ED B-structure_element +, O +helix B-structure_element +α8 B-structure_element +is O +identified O +as O +the O +recognition B-structure_element +helix I-structure_element +, O +α7 B-structure_element +as O +the O +positioning B-structure_element +helix I-structure_element +, O +and O +the O +loop B-structure_element +region I-structure_element +between O +helices O +α7 B-structure_element +- I-structure_element +α8 I-structure_element +as O +transactivation B-structure_element +loop I-structure_element +as O +observed O +in O +other O +RRs B-protein_type +( O +Fig O +6 O +). O + +The O +16 O +- O +residue O +long O +, O +solvent B-protein_state +- I-protein_state +exposed I-protein_state +recognition B-structure_element +helix I-structure_element +α8 B-structure_element +of O +NsrR B-protein +- O +ED B-structure_element +contains O +four O +positively O +charged O +residues O +that O +can O +potentially O +interact O +with O +DNA B-chemical +. O + +These O +are O +Arg198 B-residue_name_number +, O +Arg200 B-residue_name_number +, O +Lys201 B-residue_name_number +, O +and O +Lys202 B-residue_name_number +. O + +When O +comparing O +the O +sequence O +of O +NsrR B-protein +with O +PhoB B-protein +, O +KdpE B-protein +, O +and O +MtrA B-protein +, O +the O +alignment B-experimental_method +( O +Fig O +3 O +, O +colored O +in O +blue O +) O +emphasizes O +the O +variations O +at O +these O +positions O +, O +except O +for O +Arg200 B-residue_name_number +, O +which O +is O +conserved B-protein_state +throughout O +the O +lantibiotic B-protein_type +resistance I-protein_type +RRs I-protein_type +. O + +Additionally O +, O +Lys202 B-residue_name_number +is O +also O +highly B-protein_state +conserved I-protein_state +throughout O +the O +family O +of O +RRs B-protein_type +except O +PhoB B-protein +, O +clearly O +reflecting O +differences O +in O +the O +sequences O +of O +DNA B-chemical +to O +be O +bound O +. O + +Comparison O +with O +structures B-evidence +of O +other O +effector B-structure_element +domains I-structure_element + +We O +performed O +a O +DALI B-experimental_method +search I-experimental_method +to O +identify O +structurally O +related O +proteins O +to O +NsrR B-protein +- O +ED B-structure_element +. O + +Here O +the O +structure B-evidence +of O +the O +effector B-structure_element +domain I-structure_element +of O +PhoB B-protein +from O +E B-species +. I-species +coli I-species +( O +PDB O +code O +: O +1GXQ O +) O +( O +Z B-evidence +- I-evidence +score I-evidence +of O +13 O +. O +7 O +) O +is O +structurally O +the O +most O +closely O +related O +. O + +Similar O +to O +the O +PhoB B-protein +effector B-structure_element +domain I-structure_element +, O +a O +9 O +- O +residues O +long O +loop B-structure_element +( O +amino O +acid O +182 B-residue_range +– I-residue_range +189 I-residue_range +) O +is O +also O +present O +in O +the O +structure B-evidence +of O +NsrR B-protein +- O +ED B-structure_element +that O +connects O +helices B-structure_element +α7 B-structure_element +and O +α8 B-structure_element +. O + +The O +rmsd B-evidence +between O +the O +three O +helices O +of O +the O +effector B-structure_element +domain I-structure_element +( O +including O +the O +two O +helices B-structure_element +forming O +the O +wHTH B-structure_element +motif O +) O +of O +PhoB B-protein +and O +NsrR B-protein +- O +ED B-structure_element +is O +1 O +. O +6 O +Å O +over O +47 O +Cα O +atoms O +, O +clearly O +indicating O +that O +NsrR B-protein +belongs O +to O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +family I-protein_type +of I-protein_type +RRs I-protein_type +. O + +Therefore O +, O +we O +superimposed B-experimental_method +the O +Cα O +traces O +of O +the O +effector B-structure_element +domain I-structure_element +of O +NsrR B-protein +( O +NsrR B-protein +- O +ED B-structure_element +) O +with O +other O +previously O +determined O +effector B-structure_element +domains I-structure_element +from O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +family I-protein_type +such O +as O +DrrB B-protein +, O +MtrA B-protein +and O +of O +only O +the O +effector B-structure_element +domain I-structure_element +structure B-evidence +of O +PhoB B-protein +from O +E B-species +. I-species +coli I-species +. O + +Overall O +, O +the O +structures B-evidence +are O +very O +similar O +with O +rmsd B-evidence +’ O +s O +ranging O +from O +1 O +. O +7 O +to O +2 O +. O +6 O +Å O +( O +Table O +2 O +). O + +The O +highest O +variations O +( O +Fig O +6 O +) O +are O +visible O +in O +in O +the O +loop B-structure_element +regions O +α7 B-structure_element +- I-structure_element +α8 I-structure_element +, O +which O +corresponds O +to O +the O +transactivation B-structure_element +loop I-structure_element +. O + +In O +many O +RRs B-protein_type +this O +transactivation B-structure_element +loop I-structure_element +along O +with O +the O +recognition B-structure_element +helix I-structure_element +α8 B-structure_element +, O +form O +inter O +- O +domain O +contacts O +in O +the O +inactive B-protein_state +state O +and O +are O +only O +exposed O +upon O +activation O +of O +the O +RRs B-protein_type +via O +a O +conformational O +change O +where O +the O +N O +- O +and O +C O +- O +terminal O +domains O +move O +away O +from O +each O +other O +. O + +Linker B-structure_element +region I-structure_element + +The O +linkers B-structure_element +that O +connect O +the O +RDs B-structure_element +and O +EDs B-structure_element +in O +response B-protein_type +regulators I-protein_type +are O +highly B-protein_state +variable I-protein_state +with O +respect O +to O +both O +length O +and O +sequence O +. O + +The O +exact O +boundaries O +of O +these O +linkers B-structure_element +are O +difficult O +to O +predict O +from O +sequence B-experimental_method +alignments I-experimental_method +in O +the O +absence B-protein_state +of I-protein_state +structural O +information O +of O +the O +distinct O +RR B-protein_type +. O + +Linker B-structure_element +lengths O +in O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +proteins I-protein_type +of O +unknown O +structure O +have O +been O +estimated O +by O +comparing O +the O +number O +of O +residues O +between O +conserved O +landmark O +residues O +in O +the O +regulatory B-structure_element +and I-structure_element +effector I-structure_element +domains I-structure_element +to O +those O +from O +structurally O +characterized O +family O +members O +. O + +Similar O +to O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +family I-protein_type +, O +the O +lantibiotic B-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +family I-protein_type +of I-protein_type +response I-protein_type +regulators I-protein_type +also O +displays O +diverse O +linker B-structure_element +regions I-structure_element +, O +which O +are O +recognized O +in O +sequence B-experimental_method +alignments I-experimental_method +by O +the O +introduction O +of O +gaps O +( O +Fig O +3 O +). O + +Interestingly O +, O +two O +arginine B-residue_name +residues O +( O +Arg120 B-residue_name_number +and O +Arg121 B-residue_name_number +in O +NsrR B-protein +; O +Fig O +3 O +, O +shown O +in O +purple O +) O +at O +the O +end O +of O +the O +RDs B-structure_element +seem O +to O +be O +strictly B-protein_state +conserved I-protein_state +throughout O +the O +family O +of O +response B-protein_type +regulators I-protein_type +in O +both O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +and I-protein_type +lantibiotic I-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +RRs I-protein_type +, O +indicating O +a O +conserved B-protein_state +similarity O +. O + +As O +seen O +in O +the O +structures B-evidence +of O +MtrA B-protein +and O +KdpE B-protein +, O +this O +arginine B-residue_name +residue O +residing O +at O +the O +end O +of O +α5 B-structure_element +participates O +in O +the O +active B-protein_state +state O +dimer B-site +interface I-site +of O +the O +RD B-structure_element +through O +a O +salt B-bond_interaction +bridge I-bond_interaction +interaction O +with O +an O +aspartate B-residue_name +residue O +. O + +This O +aspartate B-residue_name +residue O +is O +identified O +in O +NsrR B-protein +as O +Asp99 B-residue_name_number +( O +see O +above O +). O + +Arginine B-residue_name_number +121 I-residue_name_number +of O +NsrR B-protein +points O +towards O +this O +Asp99 B-residue_name_number +residue O +however O +, O +the O +distance O +for O +a O +salt B-bond_interaction +bridge I-bond_interaction +interaction O +is O +too O +large O +. O + +Although O +we O +aimed O +at O +crystallizing B-experimental_method +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +, O +this O +endeavor O +failed O +due O +to O +proteolytic O +cleavage O +within O +the O +linker B-structure_element +region I-structure_element +during O +the O +time O +period O +of O +crystallization B-experimental_method +. O + +Nonetheless O +, O +the O +structures B-evidence +of O +NsrR B-protein +- O +RD B-structure_element +and O +NsrR B-protein +- O +ED B-structure_element +together O +provide O +the O +required O +structural O +knowledge O +to O +predict O +the O +linker B-structure_element +region I-structure_element +that O +joins O +the O +receiver B-structure_element +and I-structure_element +effector I-structure_element +domains I-structure_element +. O + +The O +linker B-structure_element +region I-structure_element +of O +NsrR B-protein +consists O +of O +approximately O +nine O +residues O +( O +Fig O +3 O +), O +comprising O +120RRSQQFIQQ128 B-structure_element +( O +underlined O +residues O +are O +neither O +present O +in O +the O +structure B-evidence +of O +RD B-structure_element +nor O +in O +ED B-structure_element +of O +NsrR B-protein +) O +and O +contains O +two O +positively O +charged O +amino O +acids O +. O + +DNA B-chemical +- O +binding O +mode O +of O +NsrR B-protein +using O +a O +full B-protein_state +- I-protein_state +length I-protein_state +model O + +Since O +the O +structures B-evidence +of O +both O +domains O +of O +NsrR B-protein +were O +determined O +, O +we O +used O +this O +structural B-evidence +information I-evidence +together O +with O +the O +available O +crystal B-evidence +structures I-evidence +of O +related O +proteins O +to O +create O +a O +model O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +in O +its O +active B-protein_state +and O +inactive B-protein_state +state O +. O + +To O +achieve O +this O +, O +we O +first O +carefully O +analyzed O +the O +outcome O +of O +the O +Dali B-experimental_method +search I-experimental_method +for O +each O +domain O +and O +identified O +structurally O +highly O +similar O +proteins O +( O +based O +on O +Z B-evidence +- I-evidence +scores I-evidence +and O +rmsd B-evidence +values O +) O +and O +choose O +the O +full B-protein_state +- I-protein_state +length I-protein_state +structures B-evidence +previously O +reported O +. O + +This O +resulted O +in O +a O +list O +of O +possible O +templates O +for O +modeling O +the O +full B-protein_state +- I-protein_state +length I-protein_state +structure B-evidence +of O +NsrR B-protein +( O +Table O +2 O +). O + +In O +solution O +, O +RRs B-protein_type +exist O +in O +equilibrium O +between O +the O +active B-protein_state +and O +inactive B-protein_state +state O +, O +which O +is O +shifted O +towards O +the O +active B-protein_state +state O +upon O +phosphorylation B-ptm +of O +the O +ED B-structure_element +. O + +This O +results O +in O +oligomerization O +of O +the O +RR B-protein_type +and O +a O +higher O +affinity O +towards O +DNA B-chemical +. O + +Based O +on O +the O +above O +- O +mentioned O +criteria O +, O +the O +structure B-evidence +of O +MtrA B-protein +from O +M B-species +. I-species +tuberculosis I-species +, O +crystallized B-experimental_method +in O +an O +inactive B-protein_state +and O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +state O +, O +seemed O +best O +suited O +for O +modeling O +purposes O +. O + +Furthermore O +, O +the O +linker B-structure_element +between O +the O +two O +domains O +of O +MtrA B-protein +contains O +nine O +amino O +acids O +and O +is O +of O +similar O +length O +as O +the O +linker B-structure_element +of O +NsrR B-protein +. O +We O +aligned B-experimental_method +the O +NsrR B-protein +- O +RD B-structure_element +and O +- O +ED B-structure_element +to O +the O +corresponding O +MtrA B-protein +domains O +and O +evaluated O +the O +structure B-evidence +. O + +This O +mimics O +the O +closed B-protein_state +inactive B-protein_state +conformation O +of O +NsrR B-protein +( O +Fig O +7A O +; O +the O +missing B-protein_state +linker B-structure_element +is O +represented O +as O +dotted O +line O +). O + +Model O +of O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +in O +its O +inactive B-protein_state +state O +and O +active B-protein_state +state O +. O + +The O +RD B-structure_element +domain O +of O +NsrR B-protein +is O +highlighted O +in O +yellow O +and O +the O +ED B-structure_element +domain O +in O +green O +with O +the O +“ O +recognition B-structure_element +helix I-structure_element +” O +colored O +in O +cyan O +. O +( O +a O +) O +Inactive B-protein_state +state O +conformation O +: O +Both O +domains O +of O +NsrR B-protein +were O +aligned B-experimental_method +to O +the O +structure B-evidence +of O +MtrA B-protein +( O +not O +shown O +), O +which O +adopts O +a O +closed B-protein_state +inactive B-protein_state +conformation O +, O +to O +obtain O +a O +model O +of O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +. O +Phe101 B-residue_name_number +and O +Asp187 B-residue_name_number +stabilize O +this O +closed B-protein_state +conformation O +. O + +The O +missing B-protein_state +linker B-structure_element +is O +represented O +by O +a O +dotted O +line O +. O + +( O +b O +) O +Active B-protein_state +state O +conformation O +: O +A O +model O +of O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +in O +active B-protein_state +conformation O +based O +on O +the O +alignment B-experimental_method +of O +both O +the O +domains O +of O +NsrR B-protein +to O +the O +structure B-evidence +of O +DNA B-protein_state +bound I-protein_state +structure B-evidence +of O +KdpE B-protein +( O +PDB O +code O +: O +4KNY O +), O +adopting O +an O +active B-protein_state +open B-protein_state +conformation O +, O +where O +the O +other O +molecule O +of O +NsrR B-protein +is O +shown O +in O +shades O +of O +blue O +with O +the O +recognition B-structure_element +helix I-structure_element +colored O +in O +green O +. O + +In O +MtrA B-protein +, O +the O +two O +domains O +interact O +via O +the O +α4 B-site +- I-site +β5 I-site +- I-site +α5 I-site +interface I-site +of O +the O +receiver B-structure_element +domain I-structure_element +and O +the O +end O +of O +α7 B-structure_element +, O +α7 B-structure_element +- I-structure_element +α8 I-structure_element +loop I-structure_element +and O +α8 B-structure_element +of O +the O +effector B-structure_element +domain I-structure_element +. O + +Both O +interfaces B-site +have O +been O +shown O +to O +form O +functionally O +important O +contact O +areas O +in O +the O +active B-protein_state +state O +within O +members O +of O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +. O + +In O +our O +model O +of O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +, O +a O +similar O +orientation O +between O +the O +domains O +is O +observed O +, O +contributing O +to O +the O +inter O +- O +domain O +interactions O +. O + +The O +inactive B-protein_state +conformation O +of O +MtrA B-protein +is O +supported O +by O +the O +orientation O +of O +the O +side O +chain O +of O +Tyr102 B-residue_name_number +, O +which O +points O +away O +from O +the O +active B-protein_state +Asp56 B-residue_name_number +residue O +, O +while O +the O +side O +chain O +of O +Tyr102 B-residue_name_number +interacts O +with O +Asp190 B-residue_name_number +of O +the O +RD B-structure_element +of O +MtrA B-protein +, O +thereby O +stabilizing O +its O +closed B-protein_state +conformation O +. O + +In O +the O +model O +of O +NsrR B-protein +, O +similar O +amino O +acids O +are O +present O +, O +Phe101 B-residue_name_number +( O +switch B-site +residue I-site +) O +and O +Asp188 B-residue_name_number +( O +Fig O +3 O +, O +represented O +by O +orange O +boxes O +) O +forming O +a O +likewise O +similar O +network O +of O +interaction O +. O + +Next O +, O +we O +were O +interested O +in O +the O +active B-protein_state +conformation O +of O +the O +NsrR B-protein +protein O +adopting O +an O +active B-protein_state +“ O +open B-protein_state +” O +conformation O +in O +the O +dimeric B-oligomeric_state +state O +. O + +We O +compared B-experimental_method +and I-experimental_method +aligned I-experimental_method +the O +NsrR B-protein +- O +RD B-structure_element +and O +ED B-structure_element +on O +the O +dimeric B-oligomeric_state +structure B-evidence +of O +KdpE B-protein +that O +was O +solved B-experimental_method +in O +the O +DNA B-protein_state +- I-protein_state +bound I-protein_state +state O +( O +Fig O +7B O +). O + +Also O +the O +linker B-structure_element +region I-structure_element +of O +KdpE B-protein +is O +of O +similar O +length O +as O +of O +NsrR B-protein +, O +which O +suggests O +that O +the O +distance O +in O +the O +DNA B-protein_state +- I-protein_state +bound I-protein_state +state O +between O +the O +RD B-structure_element +and O +ED B-structure_element +of O +NsrR B-protein +will O +be O +similar O +to O +that O +in O +the O +KdpE B-protein +active B-protein_state +dimer B-oligomeric_state +. O + +We O +superimposed B-experimental_method +the O +ED B-structure_element +of O +NsrR B-protein +with O +the O +DNA B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +of O +KdpE B-protein +resulting O +in O +a O +reasonably O +well O +- O +aligned O +structure B-evidence +( O +rmsd B-evidence +of O +2 O +. O +6Å O +over O +86 O +Cα O +atoms O +; O +Table O +2 O +). O + +As O +a O +result O +, O +a O +highly B-site +positive I-site +groove I-site +is O +created O +by O +the O +two O +ED B-structure_element +domains O +of O +NsrR B-protein +which O +likely O +represents O +the O +DNA B-site +binding I-site +site I-site +as O +observed O +in O +KdpE B-protein +. O +A O +prediction O +of O +the O +putative O +promoter O +sequence O +that O +NsrR B-protein +binds O +via O +the O +BPROM O +online O +server O +was O +performed O +( O +S3 O +Fig O +). O + +A O +promoter O +region O +was O +identified O +upstream O +of O +the O +nsr B-gene +operon O +. O + +However O +, O +the O +regulation O +of O +the O +predicted O +promoter O +and O +the O +DNA B-chemical +binding O +by O +NsrR B-protein +has O +to O +be O +confirmed O +. O + +In O +numerous O +pathogenic O +bacteria B-taxonomy_domain +such O +as O +S B-species +. I-species +agalactiae I-species +, O +S B-species +. I-species +aureus I-species +, O +and O +C B-species +. I-species +difficile I-species +that O +apparently O +do O +not O +produce O +a O +lantibiotic B-chemical +, O +a O +gene O +cluster O +is O +present O +to O +provide O +resistance O +against O +lantibiotics B-chemical +such O +as O +nisin B-chemical +, O +nukacin B-chemical +ISK I-chemical +- I-chemical +1 I-chemical +, O +lacticin B-chemical +481 I-chemical +gallidermin B-chemical +, O +actagardine B-chemical +, O +or O +mersacidin B-chemical +. O + +The O +structure B-evidence +of O +the O +response B-protein_type +regulator I-protein_type +NsrR B-protein +from O +S B-species +. I-species +agalactiae I-species +presented O +in O +this O +study O +is O +the O +first O +structural O +information O +available O +for O +the O +subgroup O +of O +lantibiotic B-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +RRs I-protein_type +. O + +Visualizing O +chaperone B-protein_type +- O +assisted O +protein O +folding O + +Challenges O +in O +determining O +the O +structures B-evidence +of O +heterogeneous O +and O +dynamic O +protein O +complexes O +have O +greatly O +hampered O +past O +efforts O +to O +obtain O +a O +mechanistic O +understanding O +of O +many O +important O +biological O +processes O +. O + +One O +such O +process O +is O +chaperone B-protein_type +- O +assisted O +protein O +folding O +, O +where O +obtaining O +structural O +ensembles O +of O +chaperone B-protein_type +: O +substrate O +complexes O +would O +ultimately O +reveal O +how O +chaperones B-protein_type +help O +proteins O +fold O +into O +their O +native O +state O +. O + +To O +address O +this O +problem O +, O +we O +devised O +a O +novel O +structural O +biology O +approach O +based O +on O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +, O +termed O +Residual B-experimental_method +Electron I-experimental_method +and I-experimental_method +Anomalous I-experimental_method +Density I-experimental_method +( O +READ B-experimental_method +). O + +READ B-experimental_method +enabled O +us O +to O +visualize O +even O +sparsely O +populated O +conformations O +of O +the O +substrate O +protein O +immunity B-protein +protein I-protein +7 I-protein +( O +Im7 B-protein +) O +in B-protein_state +complex I-protein_state +with I-protein_state +the O +E B-species +. I-species +coli I-species +chaperone B-protein_type +Spy B-protein +. O + +This O +study O +resulted O +in O +a O +series O +of O +snapshots O +depicting O +the O +various O +folding O +states O +of O +Im7 B-protein +while O +bound B-protein_state +to I-protein_state +Spy B-protein +. O + +The O +ensemble O +shows O +that O +Spy B-protein_state +- I-protein_state +associated I-protein_state +Im7 B-protein +samples O +conformations O +ranging O +from O +unfolded B-protein_state +to O +partially O +folded B-protein_state +and O +native B-protein_state +- O +like O +states O +, O +and O +reveals O +how O +a O +substrate O +can O +explore O +its O +folding O +landscape O +while O +bound B-protein_state +to I-protein_state +a O +chaperone B-protein_type +. O + +High O +- O +resolution O +structural B-evidence +models I-evidence +of O +protein O +- O +protein O +interactions O +are O +critical O +for O +obtaining O +mechanistic O +insights O +into O +biological O +processes O +. O + +However O +, O +many O +protein O +- O +protein O +interactions O +are O +highly B-protein_state +dynamic I-protein_state +, O +making O +it O +difficult O +to O +obtain O +high O +- O +resolution O +data O +. O + +Particularly O +challenging O +are O +interactions O +of O +intrinsically B-protein_state +or I-protein_state +conditionally I-protein_state +disordered I-protein_state +sections O +of O +proteins O +with O +their O +partner O +proteins O +. O + +Recent O +advances O +in O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +and O +NMR B-experimental_method +spectroscopy I-experimental_method +continue O +to O +improve O +our O +ability O +to O +analyze O +biomolecules O +that O +exist O +in O +multiple O +conformations O +. O + +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +has O +historically O +provided O +valuable O +information O +on O +small O +- O +scale O +conformational O +changes O +, O +but O +observing O +large O +- O +amplitude O +heterogeneous O +conformational O +changes O +often O +falls O +beyond O +the O +reach O +of O +current O +crystallographic O +techniques O +. O + +NMR B-experimental_method +can O +theoretically O +be O +used O +to O +determine O +heterogeneous O +ensembles O +, O +but O +in O +practice O +, O +this O +proves O +to O +be O +very O +challenging O +. O + +It O +is O +clear O +that O +molecular O +chaperones B-protein_type +aid O +in O +protein O +folding O +. O + +Structural O +characterization O +of O +chaperone B-protein_type +- O +assisted O +protein O +folding O +likely O +would O +help O +bring O +clarity O +to O +this O +question O +. O + +Structural B-evidence +models I-evidence +of O +chaperone B-protein_type +- O +substrate O +complexes O +have O +recently O +begun O +to O +provide O +information O +as O +to O +how O +a O +chaperone B-protein_type +can O +recognize O +its O +substrate O +. O + +However O +, O +the O +impact O +that O +chaperones B-protein_type +have O +on O +their O +substrates O +, O +and O +how O +these O +interactions O +affect O +the O +folding O +process O +remain O +largely O +unknown O +. O + +For O +most O +chaperones B-protein_type +, O +it O +is O +still O +unclear O +whether O +the O +chaperone B-protein_type +actively O +participates O +in O +and O +affects O +the O +folding O +of O +the O +substrate O +proteins O +, O +or O +merely O +provides O +a O +suitable O +microenvironment O +enabling O +the O +substrate O +to O +fold O +on O +its O +own O +. O + +This O +is O +a O +truly O +fundamental O +question O +in O +the O +chaperone B-protein_type +field O +, O +and O +one O +that O +has O +eluded O +the O +community O +largely O +because O +of O +the O +highly B-protein_state +dynamic I-protein_state +nature O +of O +the O +chaperone B-protein_type +- O +substrate O +complexes O +. O + +To O +address O +this O +question O +, O +we O +investigated O +the O +ATP B-protein_state +- I-protein_state +independent I-protein_state +Escherichia B-species +coli I-species +periplasmic O +chaperone B-protein_type +Spy B-protein +. O + +Spy B-protein +prevents O +protein O +aggregation O +and O +aids O +in O +protein O +folding O +under O +various O +stress O +conditions O +, O +including O +treatment O +with O +tannin B-chemical +and O +butanol B-chemical +. O + +We O +originally O +discovered O +Spy B-protein +by O +its O +ability O +to O +stabilize O +the O +protein O +- O +folding O +model O +Im7 B-protein +in O +vivo O +and O +recently O +demonstrated O +that O +Im7 B-protein +folds O +while O +associated O +with O +Spy B-protein +. O + +The O +crystal B-evidence +structure I-evidence +of O +Spy B-protein +revealed O +that O +it O +forms O +a O +thin O +α O +- O +helical O +homodimeric B-oligomeric_state +cradle B-site +. O + +Crosslinking B-experimental_method +and I-experimental_method +genetic I-experimental_method +experiments I-experimental_method +suggested O +that O +Spy B-protein +interacts O +with O +substrates O +somewhere O +on O +its O +concave O +side O +. O + +By O +using O +a O +novel O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +- O +based O +approach O +to O +model O +disorder O +in O +crystal B-evidence +structures I-evidence +, O +we O +have O +now O +determined O +the O +high O +- O +resolution O +ensemble B-evidence +of O +the O +dynamic B-protein_state +Spy B-complex_assembly +: I-complex_assembly +Im7 I-complex_assembly +complex O +. O + +This O +work O +provides O +a O +detailed O +view O +of O +chaperone B-protein_type +- O +mediated O +protein O +folding O +and O +shows O +how O +substrates O +like O +Im7 B-protein +find O +their O +native O +fold O +while O +bound B-protein_state +to I-protein_state +their O +chaperones B-protein_type +. O + +Crystallizing B-experimental_method +the O +Spy B-complex_assembly +: I-complex_assembly +Im7 I-complex_assembly +complex O + +We O +reasoned O +that O +to O +obtain O +crystals B-evidence +of O +complexes O +between O +Spy B-protein +( O +domain O +boundaries O +in O +Supplementary O +Fig O +. O +1 O +) O +and O +its O +substrate O +proteins O +, O +our O +best O +approach O +was O +to O +identify O +crystallization B-experimental_method +conditions I-experimental_method +that O +yielded O +Spy B-protein +crystals B-evidence +in O +the O +presence B-protein_state +of I-protein_state +protein O +substrates O +but O +not O +in O +their O +absence B-protein_state +. O + +We O +therefore O +screened B-experimental_method +crystallization B-experimental_method +conditions I-experimental_method +for O +Spy B-protein +with O +four O +different O +substrate O +proteins O +: O +a O +fragment O +of O +the O +largely O +unfolded B-protein_state +bovine B-taxonomy_domain +α B-chemical +- I-chemical +casein I-chemical +protein O +, O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +E B-species +. I-species +coli I-species +Im7 B-protein +, O +an O +unfolded B-protein_state +variant O +of O +Im7 B-protein +( O +L18A B-mutant +L19A B-mutant +L37A B-mutant +), O +and O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +Im7 B-protein +( O +Im76 B-mutant +- I-mutant +45 I-mutant +), O +which O +encompasses O +the O +entire O +Spy B-structure_element +- I-structure_element +binding I-structure_element +portion I-structure_element +of O +Im7 B-protein +. O + +We O +found O +conditions O +in O +which O +all O +four O +substrates O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +with B-protein_state +Spy B-protein +, O +but O +in O +which O +Spy B-protein +alone B-protein_state +did O +not O +yield O +crystals B-evidence +. O + +Subsequent O +crystal B-experimental_method +washing I-experimental_method +and I-experimental_method +dissolution I-experimental_method +experiments O +confirmed O +the O +presence O +of O +the O +substrates O +in O +the O +co B-experimental_method +- I-experimental_method +crystals I-experimental_method +( O +Supplementary O +Fig O +. O +2 O +). O + +The O +crystals B-evidence +diffracted O +to O +~ O +1 O +. O +8 O +Å O +resolution O +. O + +We O +used O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +selenomethionine B-chemical +crystals B-evidence +for O +phasing O +with O +single B-experimental_method +- I-experimental_method +wavelength I-experimental_method +anomalous I-experimental_method +diffraction I-experimental_method +( O +SAD B-experimental_method +) O +experiments O +, O +and O +used O +this O +solution O +to O +build O +the O +well O +- O +ordered O +Spy B-protein +portions O +of O +all O +four O +complexes O +. O + +However O +, O +modeling O +of O +the O +substrate O +in O +the O +complex O +proved O +to O +be O +a O +substantial O +challenge O +, O +as O +the O +electron B-evidence +density I-evidence +of O +the O +substrate O +was O +discontinuous O +and O +fragmented O +. O + +Even O +the O +minimal B-structure_element +binding I-structure_element +portion I-structure_element +of O +Im7 B-protein +( O +Im76 B-mutant +- I-mutant +45 I-mutant +) O +showed O +highly O +dispersed O +electron B-evidence +density I-evidence +( O +Fig O +. O +1a O +). O + +We O +hypothesized O +that O +the O +fragmented O +density B-evidence +was O +due O +to O +multiple O +, O +partially O +occupied O +conformations O +of O +the O +substrate O +bound O +within O +the O +crystal B-evidence +. O + +Such O +residual O +density O +is O +typically O +not O +considered O +usable O +by O +traditional O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +methods O +. O + +Thus O +, O +we O +developed O +a O +new O +approach O +to O +interpret O +the O +chaperone B-protein_state +- I-protein_state +bound I-protein_state +substrate O +in O +multiple O +conformations O +. O + +READ B-experimental_method +: O +a O +strategy O +to O +visualize O +heterogeneous O +and O +dynamic O +biomolecules O + +To O +determine O +the O +structure B-evidence +of O +the O +substrate O +portion O +of O +these O +Spy B-protein +: O +substrate O +complexes O +, O +we O +conceived O +of O +an O +approach O +that O +we O +term O +READ B-experimental_method +, O +for O +Residual B-experimental_method +Electron I-experimental_method +and I-experimental_method +Anomalous I-experimental_method +Density I-experimental_method +. O + +We O +split O +this O +approach O +into O +five O +steps O +: O +( O +1 O +) O +By O +using O +a O +well O +- O +diffracting O +Spy B-protein +: O +substrate O +co B-evidence +- I-evidence +crystal I-evidence +, O +we O +first O +determined O +the O +structure B-evidence +of O +the O +folded B-protein_state +domain B-structure_element +of O +Spy B-protein +and O +obtained O +high O +quality O +residual B-evidence +electron I-evidence +density I-evidence +within O +the O +dynamic B-protein_state +regions O +of O +the O +substrate O +. O + +( O +2 O +) O +We O +then O +labeled O +individual O +residues O +in O +the O +flexible B-protein_state +regions O +of O +the O +substrate O +with O +the O +strong O +anomalous O +scatterer O +iodine B-chemical +, O +which O +serves O +to O +locate O +these O +residues O +in O +three O +- O +dimensional O +space O +using O +their O +anomalous B-evidence +density I-evidence +. O + +( O +3 O +) O +We O +performed O +molecular B-experimental_method +dynamics I-experimental_method +( O +MD B-experimental_method +) O +simulations B-experimental_method +to O +generate O +a O +pool O +of O +energetically O +reasonable O +conformations O +of O +the O +dynamic B-protein_state +complex O +and O +( O +4 O +) O +applied O +a O +sample B-experimental_method +- I-experimental_method +and I-experimental_method +- I-experimental_method +select I-experimental_method +algorithm I-experimental_method +to O +determine O +the O +minimal O +set O +of O +substrate O +conformations O +that O +fit O +both O +the O +residual B-evidence +and I-evidence +anomalous I-evidence +density I-evidence +. O + +Importantly O +, O +even O +though O +we O +only O +labeled O +a O +subset O +of O +the O +residues O +in O +the O +flexible B-protein_state +regions O +of O +the O +substrate O +with O +iodine B-chemical +, O +the O +residual B-evidence +electron I-evidence +density I-evidence +can O +provide O +spatial O +information O +on O +many O +of O +the O +other O +flexible B-protein_state +residues O +. O + +The O +electron B-evidence +density I-evidence +then O +allowed O +us O +to O +connect O +the O +labeled O +residues O +of O +the O +substrate O +by O +confining O +the O +protein O +chain O +within O +regions O +of O +detectable O +density B-evidence +. O + +In O +this O +way O +, O +the O +two O +forms O +of O +data O +together O +were O +able O +to O +describe O +multiple O +conformations O +of O +the O +substrate O +within O +the O +crystal B-evidence +. O + +As O +described O +in O +detail O +below O +, O +we O +developed O +the O +READ B-experimental_method +method O +to O +uncover O +the O +ensemble O +of O +conformations O +that O +the O +Spy B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +of O +Im7 B-protein +( O +i O +. O +e O +., O +Im76 B-mutant +- I-mutant +45 I-mutant +) O +adopts O +while O +bound B-protein_state +to I-protein_state +Spy B-protein +. O + +However O +, O +we O +believe O +that O +READ B-experimental_method +will O +prove O +generally O +applicable O +to O +visualizing O +heterogeneous O +and O +dynamic O +complexes O +that O +have O +previously O +escaped O +detailed O +structural O +analysis O +. O + +Collecting O +READ B-experimental_method +data O +for O +the O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +complex O + +To O +apply O +the O +READ B-experimental_method +technique I-experimental_method +to O +the O +folding O +mechanism O +employed O +by O +the O +chaperone B-protein_type +Spy B-protein +, O +we O +selected O +Im76 B-mutant +- I-mutant +45 I-mutant +for O +further O +investigation O +because O +NMR B-experimental_method +data O +suggested O +that O +Im76 B-mutant +- I-mutant +45 I-mutant +could O +recapitulate O +unfolded B-protein_state +, O +partially O +folded B-protein_state +, O +and O +native O +- O +like O +states O +of O +Im7 B-protein +( O +Supplementary O +Fig O +. O +3 O +). O + +Moreover O +, O +binding B-experimental_method +experiments I-experimental_method +indicated O +that O +Im76 B-mutant +- I-mutant +45 I-mutant +comprises O +the O +entire O +Spy B-site +- I-site +binding I-site +region I-site +. O + +To O +introduce O +the O +anomalous O +scatterer O +iodine B-chemical +, O +we O +replaced B-experimental_method +eight O +Im76 B-mutant +- I-mutant +45 I-mutant +residues O +with O +the O +non O +- O +canonical O +amino O +acid O +4 B-chemical +- I-chemical +iodophenylalanine I-chemical +( O +pI B-chemical +- I-chemical +Phe I-chemical +). O + +Its O +strong O +anomalous B-evidence +scattering I-evidence +allowed O +us O +to O +track O +the O +positions O +of O +these O +individual O +Im76 B-mutant +- I-mutant +45 I-mutant +residues O +one O +at O +a O +time O +, O +potentially O +even O +if O +the O +residue O +was O +found O +in O +several O +locations O +in O +the O +same O +crystal B-evidence +. O + +We O +then O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +Spy B-protein +and O +the O +eight O +Im76 B-mutant +- I-mutant +45 I-mutant +peptides O +, O +each O +of O +which O +harbored O +an O +individual O +pI B-chemical +- I-chemical +Phe I-chemical +substitution B-experimental_method +at O +one O +distinct O +position O +, O +and O +collected B-experimental_method +anomalous B-evidence +data I-evidence +for O +all O +eight O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +complexes O +( O +Fig O +. O +1B O +, O +Supplementary O +Table O +1 O +Supplementary O +Dataset O +1 O +, O +and O +Supplementary O +Table O +2 O +). O + +Consistent O +with O +our O +electron B-evidence +density I-evidence +map I-evidence +, O +we O +found O +that O +the O +majority O +of O +anomalous B-evidence +signals I-evidence +emerged O +in O +the O +cradle B-site +of O +Spy B-protein +, O +implying O +that O +this O +is O +the O +likely O +Im7 B-protein +substrate B-site +binding I-site +site I-site +. O + +Consistent O +with O +the O +fragmented O +density B-evidence +, O +however O +, O +we O +observed O +multiple O +iodine B-chemical +positions O +for O +seven O +of O +the O +eight O +substituted O +residues O +. O + +Together O +, O +these O +results O +indicated O +that O +the O +Im7 B-protein +substrate O +binds O +Spy B-protein +in O +multiple O +conformations O +. O + +READ B-experimental_method +sample B-experimental_method +- I-experimental_method +and I-experimental_method +- I-experimental_method +select I-experimental_method +procedure O + +To O +determine O +the O +structural O +ensemble O +that O +Im76 B-mutant +- I-mutant +45 I-mutant +adopts O +while O +bound B-protein_state +to I-protein_state +Spy B-protein +, O +we O +combined O +the O +residual B-evidence +electron I-evidence +density I-evidence +and O +the O +anomalous B-evidence +signals I-evidence +from O +our O +pI B-chemical +- I-chemical +Phe I-chemical +substituted O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +complexes O +. O + +To O +generate O +an O +accurate O +depiction O +of O +the O +chaperone B-protein_type +- O +substrate O +interactions O +, O +we O +devised O +a O +selection O +protocol O +based O +on O +a O +sample B-experimental_method +- I-experimental_method +and I-experimental_method +- I-experimental_method +select I-experimental_method +procedure O +employed O +in O +NMR B-experimental_method +spectroscopy I-experimental_method +. O + +During O +each O +round O +of O +the O +selection O +, O +a O +genetic B-experimental_method +algorithm I-experimental_method +alters O +the O +ensemble O +and O +its O +agreement O +to O +the O +experimental O +data O +is O +re O +- O +evaluated O +. O + +If O +successful O +, O +the O +selection O +identifies O +the O +smallest O +group O +of O +specific O +conformations O +that O +best O +fits O +the O +residual B-evidence +electron I-evidence +density I-evidence +and O +anomalous B-evidence +signals I-evidence +. O + +The O +READ B-experimental_method +sample B-experimental_method +- I-experimental_method +and I-experimental_method +- I-experimental_method +select I-experimental_method +algorithm I-experimental_method +is O +diagrammed O +in O +Fig O +. O +2 O +. O + +Prior O +to O +performing O +the O +selection O +, O +we O +generated O +a O +large O +and O +diverse O +pool O +of O +chaperone B-protein_type +- O +substrate O +complexes O +using O +coarse B-experimental_method +- I-experimental_method +grained I-experimental_method +MD I-experimental_method +simulations I-experimental_method +in O +a O +pseudo B-experimental_method +- I-experimental_method +crystal I-experimental_method +environment I-experimental_method +( O +Fig O +. O +2 O +and O +Supplementary O +Fig O +. O +4 O +). O + +The O +coarse B-experimental_method +- I-experimental_method +grained I-experimental_method +simulations I-experimental_method +are O +based O +on O +a O +single O +- O +residue O +resolution O +model O +for O +protein O +folding O +and O +were O +extended O +here O +to O +describe O +Spy B-complex_assembly +- I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +binding O +events O +( O +Online O +Methods O +). O + +The O +initial O +conditions O +of O +the O +binding B-experimental_method +simulations I-experimental_method +are O +not O +biased O +toward O +a O +particular O +conformation O +of O +the O +substrate O +or O +any O +specific O +chaperone B-protein_type +- O +substrate O +interaction O +( O +Online O +Methods O +). O + +Im76 B-mutant +- I-mutant +45 I-mutant +binds O +and O +unbinds O +to O +Spy B-protein +throughout O +the O +simulations B-experimental_method +. O + +This O +strategy O +allows O +a O +wide O +range O +of O +substrate O +conformations O +to O +interact O +with O +the O +chaperone B-protein_type +. O + +From O +the O +MD B-experimental_method +simulations B-experimental_method +, O +we O +extracted O +~ O +10 O +, O +000 O +diverse O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +complexes O +to O +be O +used O +by O +the O +ensuing O +selection O +. O + +Each O +complex O +within O +this O +pool O +comprises O +one O +Spy B-protein +dimer B-oligomeric_state +bound B-protein_state +to I-protein_state +a O +single O +Im76 B-mutant +- I-mutant +45 I-mutant +substrate O +. O + +This O +pool O +was O +then O +used O +by O +the O +selection O +algorithm O +to O +identify O +the O +minimal O +ensemble O +that O +best O +satisfies O +both O +the O +residual B-evidence +electron I-evidence +and I-evidence +anomalous I-evidence +crystallographic I-evidence +data I-evidence +. O + +The O +anomalous B-evidence +scattering I-evidence +portion O +of O +the O +selection O +uses O +our O +basic O +knowledge O +of O +pI B-chemical +- I-chemical +Phe I-chemical +geometry O +: O +the O +iodine B-chemical +is O +separated O +from O +its O +respective O +Cα O +atom O +in O +each O +coarse O +- O +grained O +conformer O +by O +6 O +. O +5 O +Å O +. O +The O +selection O +then O +picks O +ensembles O +that O +best O +reproduce O +the O +collection O +of O +iodine B-chemical +anomalous B-evidence +signals I-evidence +. O + +Simultaneously O +, O +it O +uses O +the O +residual B-evidence +electron I-evidence +density I-evidence +to O +help O +choose O +ensembles O +. O + +To O +make O +the O +electron B-experimental_method +density I-experimental_method +selection I-experimental_method +practical O +, O +we O +needed O +to O +develop O +a O +method O +to O +rapidly O +evaluate O +the O +agreement O +between O +the O +selected O +sub O +- O +ensembles O +and O +the O +experimental O +electron B-evidence +density I-evidence +on O +- O +the O +- O +fly O +during O +the O +selection O +procedure O +. O + +To O +accomplish O +this O +task O +, O +we O +generated O +a O +compressed O +version O +of O +the O +experimental O +2mFo B-evidence +− I-evidence +DFc I-evidence +electron I-evidence +density I-evidence +map I-evidence +for O +use O +in O +the O +selection O +. O + +This O +process O +provided O +us O +with O +a O +target O +map B-evidence +that O +the O +ensuing O +selection O +tried O +to O +recapitulate O +. O + +To O +reduce O +the O +extent O +of O +3D O +space O +to O +be O +explored O +, O +this O +compressed O +map B-evidence +was O +created O +by O +only O +using O +density B-evidence +from O +regions O +of O +space O +significantly O +sampled O +by O +Im76 B-mutant +- I-mutant +45 I-mutant +in O +the O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +MD B-experimental_method +simulations B-experimental_method +. O + +For O +each O +of O +the O +~ O +10 O +, O +000 O +complexes O +in O +the O +coarse B-experimental_method +- I-experimental_method +grained I-experimental_method +MD B-experimental_method +pool O +, O +the O +electron B-evidence +density I-evidence +at O +the O +Cα O +positions O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +was O +extracted O +and O +used O +to O +construct O +an O +electron B-evidence +density I-evidence +map I-evidence +( O +Online O +Methods O +). O + +These O +individual O +electron B-evidence +density I-evidence +maps I-evidence +from O +the O +separate O +conformers O +could O +then O +be O +combined O +( O +Fig O +. O +2 O +) O +and O +compared O +to O +the O +averaged O +experimental O +electron B-evidence +density I-evidence +map I-evidence +as O +part O +of O +the O +selection O +algorithm O +. O + +This O +approach O +allowed O +us O +to O +simultaneously O +use O +both O +the O +iodine B-chemical +anomalous B-evidence +signals I-evidence +and O +the O +residual B-evidence +electron I-evidence +density I-evidence +in O +the O +selection O +procedure O +. O + +The O +selection O +resulted O +in O +small O +ensembles O +from O +the O +MD B-experimental_method +pool O +that O +best O +fit O +the O +READ B-experimental_method +data O +( O +Fig O +. O +1c O +, O +d O +). O + +Before O +analyzing O +the O +details O +of O +the O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +complex O +, O +we O +first O +engaged O +in O +a O +series O +of O +validation O +tests O +to O +verify O +the O +ensemble O +and O +selection O +procedure O +( O +Supplementary O +Note O +1 O +, O +Figures O +1c O +, O +d O +, O +Supplemental O +Figures O +5 O +- O +7 O +). O + +Of O +note O +, O +the O +final O +six O +- O +membered O +ensemble O +was O +the O +largest O +ensemble O +that O +could O +simultaneously O +decrease O +the O +RFree B-evidence +and O +pass O +the O +10 B-experimental_method +- I-experimental_method +fold I-experimental_method +cross I-experimental_method +- I-experimental_method +validation I-experimental_method +test I-experimental_method +. O + +This O +ensemble O +depicts O +the O +conformations O +that O +the O +substrate O +Im76 B-mutant +- I-mutant +45 I-mutant +adopts O +while O +bound B-protein_state +to I-protein_state +the O +chaperone B-protein_type +Spy B-protein +( O +Fig O +. O +3 O +Supplementary O +Movie O +1 O +, O +and O +Table O +1 O +). O + +Folding O +and O +interactions O +of O +Im7 B-protein +while O +bound B-protein_state +to I-protein_state +Spy B-protein + +Our O +results O +showed O +that O +by O +using O +this O +novel O +READ B-experimental_method +approach O +, O +we O +were O +able O +to O +obtain O +structural O +information O +about O +the O +dynamic O +interaction O +of O +a O +chaperone B-protein_type +with O +its O +substrate O +protein O +. O + +We O +were O +particularly O +interested O +in O +finding O +answers O +to O +one O +of O +the O +most O +fundamental O +questions O +in O +chaperone B-protein_type +biology O +— O +how O +does O +chaperone B-protein_type +binding O +affect O +substrate O +structure O +and O +vice O +versa O +. O + +By O +analyzing O +the O +individual O +structures B-evidence +of O +the O +six O +- O +member O +ensemble O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +bound B-protein_state +to I-protein_state +Spy B-protein +, O +we O +observed O +that O +Im76 B-mutant +- I-mutant +45 I-mutant +takes O +on O +several O +different O +conformations O +while O +bound B-protein_state +. O + +We O +found O +these O +conformations O +to O +be O +highly O +heterogeneous O +and O +to O +include O +unfolded B-protein_state +, O +partially B-protein_state +folded I-protein_state +, O +and O +native B-protein_state +- I-protein_state +like I-protein_state +states O +( O +Fig O +. O +3 O +). O + +The O +ensemble O +primarily O +encompasses O +Im76 B-mutant +- I-mutant +45 I-mutant +laying O +diagonally O +within O +the O +Spy B-protein +cradle B-site +in O +several O +different O +orientations O +, O +but O +some O +conformations O +traverse O +as O +far O +as O +the O +tips O +or O +even O +extend O +over O +the O +side O +of O +the O +cradle B-site +( O +Figs O +. O +3 O +, O +4a O +). O + +We O +constructed O +a O +contact B-evidence +map I-evidence +of O +the O +complex O +, O +which O +shows O +the O +frequency O +of O +interactions O +for O +chaperone B-protein_type +- O +substrate O +residue O +pairs O +( O +Fig O +. O +4 O +). O + +We O +found O +that O +the O +primary O +interaction B-site +sites I-site +on O +Spy B-protein +reside O +at O +the O +N O +and O +C O +termini O +( O +Arg122 B-residue_name_number +, O +Thr124 B-residue_name_number +, O +and O +Phe29 B-residue_name_number +) O +as O +well O +as O +on O +the O +concave O +face O +of O +the O +chaperone B-protein_type +( O +Arg61 B-residue_name_number +, O +Arg43 B-residue_name_number +, O +Lys47 B-residue_name_number +, O +His96 B-residue_name_number +, O +and O +Met46 B-residue_name_number +). O + +The O +Spy B-site +- I-site +contacting I-site +residues I-site +comprise O +a O +mixture O +of O +charged O +, O +polar O +, O +and O +hydrophobic O +residues O +. O + +Surprisingly O +, O +we O +noted O +that O +in O +the O +ensemble O +, O +Im76 B-mutant +- I-mutant +45 I-mutant +interacts O +with O +only O +38 O +% O +of O +the O +hydrophobic O +residues O +in O +the O +Spy B-protein +cradle B-site +, O +but O +interacts O +with O +61 O +% O +of O +the O +hydrophilic O +residues O +in O +the O +cradle B-site +. O + +This O +mixture O +suggests O +the O +importance O +of O +both O +electrostatic O +and O +hydrophobic O +components O +in O +binding O +the O +Im76 B-mutant +- I-mutant +45 I-mutant +ensemble O +. O + +With O +respect O +to O +the O +substrate O +, O +we O +observed O +that O +nearly O +every O +residue O +in O +Im76 B-mutant +- I-mutant +45 I-mutant +is O +in O +contact O +with O +Spy B-protein +( O +Fig O +. O +4a O +). O + +However O +, O +we O +did O +notice O +that O +despite O +this O +uniformity O +, O +regions O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +preferentially O +interact O +with O +different O +regions O +in O +Spy B-protein +( O +Fig O +. O +4b O +). O + +For O +example O +, O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +Im76 B-mutant +- I-mutant +45 I-mutant +binds O +more O +consistently O +in O +the O +Spy B-protein +cradle B-site +, O +whereas O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +predominantly O +binds O +to O +the O +outer O +edges O +of O +Spy B-protein +’ O +s O +concave B-site +surface I-site +. O + +Not O +unexpectedly O +, O +we O +found O +that O +as O +Im76 B-mutant +- I-mutant +45 I-mutant +progresses O +from O +the O +unfolded B-protein_state +to O +the O +native B-protein_state +state O +, O +its O +interactions O +with O +Spy B-protein +shift O +accordingly O +. O + +Whereas O +the O +least B-protein_state +- I-protein_state +folded I-protein_state +Im76 B-mutant +- I-mutant +45 I-mutant +pose O +in O +the O +ensemble O +forms O +the O +most O +hydrophobic O +contacts O +with O +Spy B-protein +( O +Fig O +. O +3 O +), O +the O +two O +most B-protein_state +- I-protein_state +folded I-protein_state +conformations O +form O +the O +fewest O +hydrophobic O +contacts O +( O +Fig O +. O +3 O +). O + +This O +shift O +in O +contacts O +is O +likely O +due O +to O +hydrophobic O +residues O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +preferentially O +forming O +intra O +- O +molecular O +contacts O +upon O +folding O +( O +i O +. O +e O +., O +hydrophobic O +collapse O +), O +effectively O +removing O +themselves O +from O +the O +interaction B-site +sites I-site +. O + +The O +diversity O +of O +conformations O +and O +binding B-site +sites I-site +observed O +here O +emphasizes O +the O +dynamic O +and O +heterogeneous O +nature O +of O +the O +chaperone B-protein_type +- O +substrate O +ensemble O +. O + +Although O +we O +do O +not O +yet O +have O +time O +resolution O +data O +of O +these O +various O +snapshots O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +, O +this O +ensemble O +illustrates O +how O +a O +substrate O +samples O +its O +folding O +landscape O +while O +bound B-protein_state +to I-protein_state +a O +chaperone B-protein_type +. O + +Spy B-protein +changes O +conformation O +upon O +substrate O +binding O + +Comparing O +the O +structure B-evidence +of O +Spy B-protein +in O +its O +substrate B-protein_state +- I-protein_state +bound I-protein_state +and O +apo B-protein_state +states O +revealed O +that O +the O +Spy B-protein +dimer B-oligomeric_state +also O +undergoes O +significant O +conformational O +changes O +upon O +substrate O +binding O +( O +Fig O +. O +5a O +and O +Supplementary O +Movie O +2 O +). O + +Upon O +substrate O +binding O +, O +the O +Spy B-protein +dimer B-oligomeric_state +twists O +9 O +° O +about O +its O +center O +relative O +to O +its O +apo B-protein_state +form O +. O + +This O +twist O +yields O +asymmetry O +and O +results O +in O +substantially O +different O +interaction O +patterns O +in O +the O +two O +Spy B-protein +monomers B-oligomeric_state +( O +Fig O +. O +4b O +). O + +It O +is O +possible O +that O +this O +twist O +serves O +to O +increase O +heterogeneity O +in O +Spy B-protein +by O +providing O +more O +binding O +poses O +. O + +Additionally O +, O +we O +observed O +that O +the O +linker B-structure_element +region I-structure_element +( O +residues O +47 B-residue_range +– I-residue_range +57 I-residue_range +) O +of O +Spy B-protein +, O +which O +participates O +in O +substrate O +interaction O +, O +becomes O +mostly O +disordered B-protein_state +upon O +binding O +the O +substrate O +. O + +This O +increased O +disorder O +might O +explain O +how O +Spy B-protein +is O +able O +to O +recognize O +and O +bind O +different O +substrates O +and O +/ O +or O +differing O +conformations O +of O +the O +same O +substrate O +. O + +Importantly O +, O +we O +observed O +the O +same O +structural O +changes O +in O +Spy B-protein +regardless O +of O +which O +of O +the O +four O +substrates O +was O +bound O +( O +Fig O +. O +5b O +, O +Table O +1 O +). O + +The O +RMSD B-evidence +between O +the O +well B-protein_state +- I-protein_state +folded I-protein_state +sections O +of O +Spy B-protein +in O +the O +four O +chaperone B-protein_type +- O +substrate O +complexes O +was O +very O +small O +, O +less O +than O +0 O +. O +3 O +Å O +. O +Combined O +with O +competition B-experimental_method +experiments I-experimental_method +showing O +that O +the O +substrates O +compete O +in O +solution O +for O +Spy B-protein +binding O +( O +Fig O +. O +5c O +and O +Supplementary O +Fig O +. O +8 O +), O +we O +conclude O +that O +all O +the O +tested O +substrates O +share O +the O +same O +overall O +Spy B-site +binding I-site +site I-site +. O + +To O +shed O +light O +on O +how O +chaperones B-protein_type +interact O +with O +their O +substrates O +, O +we O +developed O +a O +novel O +structural O +biology O +method O +( O +READ B-experimental_method +) O +and O +applied O +it O +to O +determine O +a O +conformational B-evidence +ensemble I-evidence +of O +the O +chaperone B-protein_type +Spy B-protein +bound B-protein_state +to I-protein_state +substrate I-protein_state +. O + +As O +a O +substrate O +, O +we O +used O +Im76 B-mutant +- I-mutant +45 I-mutant +, O +the O +chaperone B-structure_element +- I-structure_element +interacting I-structure_element +portion I-structure_element +of O +the O +protein O +- O +folding O +model O +protein O +Im7 B-protein +. O + +In O +the O +chaperone B-protein_state +- I-protein_state +bound I-protein_state +ensemble O +, O +Im76 B-mutant +- I-mutant +45 I-mutant +samples O +unfolded B-protein_state +, O +partially O +folded B-protein_state +, O +and O +native B-protein_state +- O +like O +states O +. O + +The O +ensemble O +provides O +an O +unprecedented O +description O +of O +the O +conformations O +that O +a O +substrate O +assumes O +while O +exploring O +its O +chaperone B-protein_type +- O +associated O +folding O +landscape O +. O + +This O +substrate O +- O +chaperone B-protein_type +ensemble O +helps O +accomplish O +the O +longstanding O +goal O +of O +obtaining O +a O +detailed O +view O +of O +how O +a O +chaperone B-protein_type +aids O +protein O +folding O +. O + +We O +recently O +showed O +that O +Im7 B-protein +can O +fold O +while O +remaining O +continuously B-protein_state +bound I-protein_state +to I-protein_state +Spy B-protein +. O + +The O +high O +- O +resolution O +ensemble B-evidence +obtained O +here O +now O +provides O +insight O +into O +exactly O +how O +this O +occurs O +. O + +The O +structures B-evidence +of O +our O +ensemble B-evidence +agree O +well O +with O +lower O +- O +resolution O +crosslinking O +data O +, O +which O +indicate O +that O +chaperone B-protein_type +- O +substrate O +interactions O +primarily O +occur O +on O +the O +concave B-site +surface I-site +of O +Spy B-protein +. O + +The O +ensemble B-evidence +suggests O +a O +model O +in O +which O +Spy B-protein +provides O +an O +amphipathic B-site +surface I-site +that O +allows O +substrate O +proteins O +to O +assume O +different O +conformations O +while O +bound B-protein_state +to I-protein_state +the O +chaperone B-protein_type +. O + +This O +model O +is O +consistent O +with O +previous O +studies O +postulating O +that O +the O +flexible O +binding O +of O +chaperones B-protein_type +allows O +for O +substrate O +protein O +folding O +. O + +The O +amphipathic O +concave B-site +surface I-site +of O +Spy B-protein +likely O +facilitates O +this O +flexible O +binding O +and O +may O +be O +a O +crucial O +feature O +for O +Spy B-protein +and O +potentially O +other O +chaperones B-protein_type +, O +allowing O +them O +to O +bind O +multiple O +conformations O +of O +many O +different O +substrates O +. O + +In O +contrast O +to O +Spy B-protein +’ O +s O +binding B-site +hotspots I-site +, O +Im76 B-mutant +- I-mutant +45 I-mutant +displays O +substantially O +less O +specificity O +in O +its O +binding B-site +sites I-site +. O + +Nearly O +all O +Im76 B-mutant +- I-mutant +45 I-mutant +residues O +come O +in O +contact O +with O +Spy B-protein +. O + +Unfolded B-protein_state +substrate O +conformers O +interact O +with O +Spy B-protein +through O +both O +hydrophobic B-bond_interaction +and I-bond_interaction +hydrophilic I-bond_interaction +interactions I-bond_interaction +, O +whereas O +the O +binding O +of O +native B-protein_state +- I-protein_state +like I-protein_state +states O +is O +mainly O +hydrophilic O +. O + +This O +trend O +suggests O +that O +complex O +formation O +between O +an O +ATP B-protein_state +- I-protein_state +independent I-protein_state +chaperone B-protein_type +and O +its O +unfolded B-protein_state +substrate O +may O +initially O +involve O +hydrophobic B-bond_interaction +interactions I-bond_interaction +, O +effectively O +shielding O +the O +exposed O +aggregation O +- O +sensitive O +hydrophobic B-site +regions I-site +in O +the O +substrate O +. O + +Once O +the O +substrate O +begins O +to O +fold O +within O +this O +protected O +environment O +, O +it O +progressively O +buries O +its O +own O +hydrophobic O +residues O +, O +and O +its O +interactions O +with O +the O +chaperone B-protein_type +shift O +towards O +becoming O +more O +electrostatic O +. O + +Notably O +, O +the O +most O +frequent O +contacts O +between O +Spy B-protein +and O +Im76 B-mutant +- I-mutant +45 I-mutant +are O +charge B-bond_interaction +- I-bond_interaction +charge I-bond_interaction +interactions I-bond_interaction +. O + +The O +negatively O +charged O +Im7 B-protein +residues O +Glu21 B-residue_name_number +, O +Asp32 B-residue_name_number +, O +and O +Asp35 B-residue_name_number +reside O +on O +the O +surface O +of O +Im7 B-protein +and O +form O +interactions O +with O +Spy B-protein +’ O +s O +positively O +charged O +cradle B-site +in O +both O +the O +unfolded B-protein_state +and O +native B-protein_state +- I-protein_state +like I-protein_state +states O +. O + +Residues O +Asp32 B-residue_name_number +and O +Asp35 B-residue_name_number +are O +close O +to O +each O +other O +in O +the O +folded B-protein_state +state O +of O +Im7 B-protein +. O + +This O +proximity O +likely O +causes O +electrostatic O +repulsion O +that O +destabilizes O +Im7 B-protein +’ O +s O +native B-protein_state +state O +. O + +Interaction O +with O +Spy B-protein +’ O +s O +positively O +- O +charged O +residues O +likely O +relieves O +the O +charge O +repulsion O +between O +Asp32 B-residue_name_number +and O +Asp35 B-residue_name_number +, O +promoting O +their O +compaction O +into O +a O +helical B-protein_state +conformation I-protein_state +. O + +As O +inter O +- O +molecular O +hydrophobic B-bond_interaction +interactions I-bond_interaction +between O +Spy B-protein +and O +the O +substrate O +become O +progressively O +replaced O +by O +intra O +- O +molecular O +interactions O +within O +the O +substrate O +, O +the O +affinity O +between O +chaperone B-protein_type +and O +substrates O +could O +decrease O +, O +eventually O +leading O +to O +release O +of O +the O +folded B-protein_state +client O +protein O +. O + +Recently O +, O +we O +employed O +a O +genetic B-experimental_method +selection I-experimental_method +system I-experimental_method +to O +improve O +the O +chaperone B-protein_type +activity O +of O +Spy B-protein +. O + +This O +selection O +resulted O +in O +“ O +Super O +Spy B-protein +” O +variants B-protein_state +that O +were O +more O +effective O +at O +both O +preventing O +aggregation O +and O +promoting O +protein O +folding O +. O + +In O +conjunction O +with O +our O +bound B-protein_state +Im76 B-mutant +- I-mutant +45 I-mutant +ensemble B-evidence +, O +these O +mutants O +now O +allowed O +us O +to O +investigate O +structural O +features O +important O +to O +chaperone B-protein_type +function O +. O + +Previous O +analysis O +revealed O +that O +the O +Super O +Spy B-protein +variants B-protein_state +either O +bound B-protein_state +Im7 B-protein +tighter O +than O +WT B-protein_state +Spy B-protein +, O +increased O +chaperone B-protein_type +flexibility O +as O +measured O +via O +H B-experimental_method +/ I-experimental_method +D I-experimental_method +exchange I-experimental_method +, O +or O +both O +. O + +Our O +ensemble B-evidence +revealed O +that O +two O +of O +the O +Super O +Spy B-protein +mutations B-protein_state +( O +H96L B-mutant +and O +Q100L B-mutant +) O +form O +part O +of O +the O +chaperone B-site +contact I-site +surface I-site +that O +binds O +to O +Im76 B-mutant +- I-mutant +45 I-mutant +( O +Fig O +. O +4a O +). O + +Moreover O +, O +our O +co B-evidence +- I-evidence +structure I-evidence +suggests O +that O +the O +L32P B-mutant +substitution O +, O +which O +increases O +Spy B-protein +’ O +s O +flexibility O +, O +could O +operate O +by O +unhinging O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +helix I-structure_element +and O +effectively O +expanding O +the O +size O +of O +the O +disordered B-protein_state +linker B-structure_element +. O + +This O +possibility O +is O +supported O +by O +the O +Spy B-protein +: O +substrate O +structures B-evidence +, O +in O +which O +the O +linker B-structure_element +region I-structure_element +becomes O +more O +flexible O +compared O +to O +the O +apo B-protein_state +state O +( O +Fig O +. O +6a O +). O + +By O +sampling O +multiple O +conformations O +, O +this O +linker B-structure_element +region I-structure_element +may O +allow O +diverse O +substrate O +conformations O +to O +be O +accommodated O +. O + +Other O +Super O +Spy B-protein +mutations B-protein_state +( O +F115I B-mutant +and O +F115L B-mutant +) O +caused O +increased O +flexibility O +but O +not O +tighter O +substrate O +binding O +. O + +This O +residue O +does O +not O +directly O +contact O +Im76 B-mutant +- I-mutant +45 I-mutant +in O +our O +READ B-experimental_method +- O +derived O +ensemble B-evidence +. O + +Instead O +, O +when O +Spy B-protein +is O +bound B-protein_state +to I-protein_state +substrate O +, O +F115 B-residue_name_number +engages O +in O +close O +CH O +⋯ O +π O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +Tyr104 B-residue_name_number +( O +Fig O +. O +6b O +). O + +This O +interaction O +presumably O +reduces O +the O +mobility O +of O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +helix I-structure_element +. O + +The O +F115I B-mutant +/ O +L B-mutant +substitutions O +would O +replace O +these O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +hydrophobic B-bond_interaction +interactions I-bond_interaction +that O +have O +little O +angular O +dependence O +. O + +As O +a O +result O +, O +the O +C O +- O +terminus O +, O +and O +possibly O +also O +the O +flexible B-protein_state +linker B-structure_element +, O +is O +likely O +to O +become O +more O +flexible O +and O +thus O +more O +accommodating O +of O +different O +conformations O +of O +substrates O +. O + +Overall O +, O +comparison O +of O +our O +ensemble B-evidence +to O +the O +Super O +Spy B-protein +variants B-protein_state +provides O +specific O +examples O +to O +corroborate O +the O +importance O +of O +conformational O +flexibility O +in O +chaperone B-protein_type +- O +substrate O +interactions O +. O + +Despite O +extensive O +studies O +, O +exactly O +how O +complex O +chaperone B-protein_type +machines O +help O +proteins O +fold O +remains O +controversial O +. O + +Our O +study O +indicates O +that O +the O +chaperone B-protein_type +Spy B-protein +employs O +a O +simple O +surface O +binding O +approach O +that O +allows O +the O +substrate O +to O +explore O +various O +conformations O +and O +form O +transiently O +favorable O +interactions O +while O +being O +protected O +from O +aggregation O +. O + +We O +speculate O +that O +many O +other O +chaperones B-protein_type +could O +utilize O +a O +similar O +strategy O +. O + +ATP B-chemical +and O +co O +- O +chaperone B-protein_type +dependencies O +may O +have O +emerged O +later O +through O +evolution O +to O +better O +modulate O +and O +control O +chaperone B-protein_type +action O +. O + +In O +addition O +to O +insights O +into O +chaperone B-protein_type +function O +, O +this O +work O +presents O +a O +new O +method O +for O +determining O +heterogeneous O +structural O +ensembles O +via O +a O +hybrid O +methodology O +of O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +and O +computational B-experimental_method +modeling I-experimental_method +. O + +Heterogeneous O +dynamic O +complexes O +or O +disordered B-protein_state +regions O +of O +single O +proteins O +, O +once O +considered O +solely O +approachable O +by O +NMR B-experimental_method +spectroscopy I-experimental_method +, O +can O +now O +be O +visualized O +through O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +Crystallographic O +data O +and O +ensemble O +selection O +. O +( O +a O +) O +2mFo B-evidence +− I-evidence +DFc I-evidence +omit I-evidence +map I-evidence +of O +residual O +Im76 B-mutant +- I-mutant +45 I-mutant +and O +flexible B-structure_element +linker I-structure_element +electron B-evidence +density I-evidence +contoured O +at O +0 O +. O +5 O +σ O +. O + +This O +is O +the O +residual O +density B-evidence +that O +is O +used O +in O +the O +READ B-experimental_method +selection O +. O + +( O +b O +) O +Composites O +of O +iodine B-chemical +positions O +detected O +from O +anomalous B-evidence +signals I-evidence +using O +pI B-chemical +- I-chemical +Phe I-chemical +substitutions B-experimental_method +, O +colored O +and O +numbered O +by O +sequence O +. O + +Multiple O +iodine B-chemical +positions O +were O +detected O +for O +most O +residues O +. O + +Agreement O +to O +the O +residual O +Im76 B-mutant +- I-mutant +45 I-mutant +electron B-evidence +density I-evidence +( O +c O +) O +and O +anomalous B-evidence +iodine I-evidence +signals I-evidence +( O +d O +) O +for O +ensembles O +of O +varying O +size O +generated O +by O +randomly O +choosing O +from O +the O +MD B-experimental_method +pool O +( O +blue O +) O +and O +from O +the O +selection O +procedure O +( O +black O +). O + +The O +cost B-evidence +function I-evidence +, O +χ2 B-evidence +, O +decreases O +as O +the O +agreement O +to O +the O +experimental O +data O +increases O +and O +is O +defined O +in O +the O +Online O +Methods O +. O + +Flowchart O +of O +the O +READ B-experimental_method +sample B-experimental_method +- I-experimental_method +and I-experimental_method +- I-experimental_method +select I-experimental_method +process O +. O + +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +ensemble O +, O +arranged O +by O +RMSD B-evidence +to O +native B-protein_state +state O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +. O +Although O +the O +six O +- O +membered O +ensemble O +from O +the O +READ B-experimental_method +selection O +should O +be O +considered O +only O +as O +an O +ensemble O +, O +for O +clarity O +, O +the O +individual O +conformers O +are O +shown O +separately O +here O +. O + +Spy B-protein +is O +depicted O +as O +a O +gray O +surface O +and O +the O +Im76 B-mutant +- I-mutant +45 I-mutant +conformer O +is O +shown O +as O +orange O +balls O +. 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O + +Shown O +below O +each O +ensemble O +member O +is O +the O +RMSD B-evidence +of O +each O +conformer O +to O +the O +native B-protein_state +state O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +, O +as O +well O +as O +the O +percentage O +of O +contacts O +between O +Im76 B-mutant +- I-mutant +45 I-mutant +and O +Spy B-protein +that O +are O +hydrophobic O +. O + +Contact B-evidence +maps I-evidence +of O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +complex O +. O + +( O +a O +) O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +contact B-evidence +map I-evidence +projected O +onto O +the O +bound B-protein_state +Spy B-protein +dimer B-oligomeric_state +( O +above O +) O +and O +Im76 B-mutant +- I-mutant +45 I-mutant +( O +below O +) O +structures B-evidence +. O + +For O +clarity O +, O +Im76 B-mutant +- I-mutant +45 I-mutant +is O +represented O +with O +a O +single O +conformation O +. O + +The O +frequency O +plotted O +is O +calculated O +as O +the O +average O +contact B-evidence +frequency I-evidence +from O +Spy B-protein +to O +every O +residue O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +and O +vice O +- O +versa O +. O + +As O +the O +residues O +involved O +in O +contacts O +are O +more O +evenly O +distributed O +in O +Im76 B-mutant +- I-mutant +45 I-mutant +compared O +to O +Spy B-protein +, O +its O +contact B-evidence +map I-evidence +was O +amplified O +. O +( O +b O +) O +Detailed O +contact B-evidence +maps I-evidence +of O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +. O + +Contacts O +to O +the O +two O +Spy B-protein +monomers B-oligomeric_state +are O +depicted O +separately O +. 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O +( O +b O +) O +Overlay B-experimental_method +of O +WT B-protein_state +Spy B-protein +bound B-protein_state +to I-protein_state +Im76 B-mutant +- I-mutant +45 I-mutant +( O +green O +), O +H96L B-mutant +Spy B-protein +bound B-protein_state +to I-protein_state +Im7 B-protein +L18A B-mutant +L19 B-mutant +AL13A I-mutant +( O +blue O +), O +H96L B-mutant +Spy B-protein +bound B-protein_state +to I-protein_state +WT B-protein_state +Im7 B-protein +( O +yellow O +), O +and O +WT B-protein_state +Spy B-protein +bound B-protein_state +to I-protein_state +casein B-chemical +( O +salmon O +). O +( O +c O +) O +Competition B-experimental_method +assay I-experimental_method +showing O +Im76 B-mutant +- I-mutant +45 I-mutant +competes O +with O +Im7 B-protein +L18A B-mutant +L19A B-mutant +L37A B-mutant +H40W B-mutant +for O +the O +same O +binding B-site +site I-site +on O +Spy B-protein +( O +further O +substrate B-experimental_method +competition I-experimental_method +assays I-experimental_method +are O +shown O +in O +Supplementary O +Fig O +. O +8 O +). O + +Flexibility O +of O +Spy B-protein +linker B-structure_element +region I-structure_element +and O +effect O +of O +Super O +Spy B-protein +mutants O +. O +( O +a O +) O +The O +Spy B-protein +linker B-structure_element +region I-structure_element +adopts O +one O +dominant O +conformation O +in O +its O +apo B-protein_state +state O +( O +PDB O +ID O +3039 O +, O +gray O +), O +but O +expands O +and O +adopts O +multiple O +conformations O +in O +bound B-protein_state +states O +( O +green O +). O + +( O +b O +) O +F115 B-residue_name_number +and O +L32 B-residue_name_number +tether O +Spy B-protein +’ O +s O +linker B-structure_element +region I-structure_element +to O +its O +cradle B-site +, O +decreasing O +Spy B-protein +activity O +by O +limiting O +linker B-structure_element +region I-structure_element +flexibility O +. O + +The O +Super O +Spy B-protein +mutants O +F115L B-mutant +, O +F115I B-mutant +, O +and O +L32P B-mutant +are O +proposed O +to O +gain O +activity O +by O +increasing O +the O +flexibility O +or O +size O +of O +this O +linker B-structure_element +region I-structure_element +. O + +L32 B-residue_name_number +, O +F115 B-residue_name_number +, O +and O +Y104 B-residue_name_number +are O +rendered O +in O +purple O +to O +illustrate O +residues O +that O +are O +most O +affected O +by O +Super O +Spy B-protein +mutations B-protein_state +; O +CH O +⋯ O +π O +hydrogen B-bond_interaction +bonds I-bond_interaction +are O +depicted O +by O +orange O +dashes O +. O + +Reversal O +of O +DNA B-chemical +damage O +induced O +Topoisomerase B-protein_type +2 I-protein_type +DNA B-chemical +– O +protein O +crosslinks O +by O +Tdp2 B-protein + +Mammalian B-taxonomy_domain +Tyrosyl B-protein +- I-protein +DNA I-protein +phosphodiesterase I-protein +2 I-protein +( O +Tdp2 B-protein +) O +reverses O +Topoisomerase B-protein_type +2 I-protein_type +( O +Top2 B-protein_type +) O +DNA B-chemical +– O +protein O +crosslinks O +triggered O +by O +Top2 B-protein_type +engagement O +of O +DNA B-chemical +damage O +or O +poisoning O +by O +anticancer O +drugs O +. O + +Tdp2 B-protein +deficiencies O +are O +linked O +to O +neurological O +disease O +and O +cellular O +sensitivity O +to O +Top2 B-protein_type +poisons O +. O + +Herein O +, O +we O +report O +X B-evidence +- I-evidence +ray I-evidence +crystal I-evidence +structures I-evidence +of O +ligand B-protein_state +- I-protein_state +free I-protein_state +Tdp2 B-protein +and O +Tdp2 B-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +complexes O +with O +alkylated O +and O +abasic O +DNA B-chemical +that O +unveil O +a O +dynamic B-protein_state +Tdp2 B-protein +active B-structure_element +site I-structure_element +lid I-structure_element +and O +deep O +substrate B-site +binding I-site +trench I-site +well O +- O +suited O +for O +engaging O +the O +diverse O +DNA B-chemical +damage O +triggers O +of O +abortive O +Top2 B-protein_type +reactions O +. O + +Modeling O +of O +a O +proposed O +Tdp2 B-protein +reaction O +coordinate O +, O +combined O +with O +mutagenesis B-experimental_method +and O +biochemical B-experimental_method +studies I-experimental_method +support O +a O +single O +Mg2 B-chemical ++- I-chemical +ion O +mechanism O +assisted O +by O +a O +phosphotyrosyl B-site +- I-site +arginine I-site +cation I-site +- I-site +π I-site +interface I-site +. O + +We O +further O +identify O +a O +Tdp2 B-protein +active B-site +site I-site +SNP O +that O +ablates B-protein_state +Tdp2 B-protein +Mg2 B-chemical ++ I-chemical +binding O +and O +catalytic O +activity O +, O +impairs O +Tdp2 B-protein +mediated O +NHEJ O +of O +tyrosine B-residue_name +blocked O +termini O +, O +and O +renders O +cells O +sensitive O +to O +the O +anticancer O +agent O +etoposide B-chemical +. O + +Collectively O +, O +our O +results O +provide O +a O +structural O +mechanism O +for O +Tdp2 B-protein +engagement O +of O +heterogeneous O +DNA B-chemical +damage O +that O +causes O +Top2 B-protein_type +poisoning O +, O +and O +indicate O +that O +evaluation O +of O +Tdp2 B-protein +status O +may O +be O +an O +important O +personalized O +medicine O +biomarker O +informing O +on O +individual O +sensitivities O +to O +chemotherapeutic O +Top2 B-protein_type +poisons O +. O + +Nuclear O +DNA B-chemical +compaction O +and O +the O +action O +of O +DNA B-chemical +and O +RNA B-protein_type +polymerases I-protein_type +create O +positive O +and O +negative O +DNA B-chemical +supercoiling O +— O +over O +- O +and O +under O +- O +winding O +of O +DNA B-chemical +strands O +, O +respectively O +— O +and O +the O +linking O +together O +( O +catenation O +) O +of O +DNA B-chemical +strands O +. O + +Topoisomerases B-protein_type +relieve O +topological O +DNA B-chemical +strain O +and O +entanglement O +to O +facilitate O +critical O +nuclear O +DNA B-chemical +transactions O +including O +DNA B-chemical +replication O +, O +transcription O +and O +cell O +division O +. O + +The O +mammalian B-taxonomy_domain +type B-protein_type +II I-protein_type +topoisomerases I-protein_type +Top2α B-protein +and O +Top2β B-protein +enzymes O +generate O +transient O +, O +reversible O +DNA B-chemical +double O +strand O +breaks O +( O +DSBs O +) O +to O +drive O +topological O +transactions O +. O + +Reversibility O +of O +Top2 B-protein_type +DNA B-chemical +cleavage O +reactions O +is O +facilitated O +by O +formation O +of O +covalent O +enzyme O +phosphotyrosyl B-ptm +linkages I-ptm +between O +the O +5 B-chemical +′- I-chemical +phosphate I-chemical +ends O +of O +the O +incised O +duplex O +and O +an O +active B-site +site I-site +Top2 B-protein_type +tyrosine B-residue_name +, O +resulting O +in O +Top2 B-protein_type +cleavage O +complexes O +( O +Top2cc B-complex_assembly +). O + +The O +Top2cc B-complex_assembly +protein O +– O +DNA B-chemical +adduct O +is O +a O +unique O +threat O +to O +genomic O +integrity O +which O +must O +be O +resolved O +to O +prevent O +catastrophic O +Top2cc B-complex_assembly +collisions O +with O +the O +cellular O +replication O +and O +transcription O +machineries O +. O + +To O +promote O +cancer O +cell O +death O +, O +Top2 B-protein_type +reactions O +are O +‘ O +poisoned O +’ O +by O +keystone O +pharmacological O +anticancer O +agents O +like O +etoposide B-chemical +, O +teniposide B-chemical +and O +doxorubicin B-chemical +. O + +Importantly O +, O +Top2 B-protein_type +is O +also O +poisoned O +when O +it O +engages O +abundant O +endogenous O +DNA B-chemical +damage O +not O +limited O +to O +but O +including O +ribonucleotides O +, O +abasic O +sites O +and O +alkylation O +damage O +such O +as O +exocyclic O +DNA B-chemical +adducts O +arising O +from O +bioactivation O +of O +the O +vinyl O +chloride O +carcinogen O +( O +Figure O +1A O +). O + +In O +the O +case O +of O +DNA B-chemical +damage O +- O +triggered O +Top2cc B-complex_assembly +, O +compound O +DNA B-chemical +lesions O +arise O +that O +consist O +of O +the O +instigating O +lesion O +, O +and O +a O +DNA B-chemical +DSB O +bearing O +a O +bulky O +terminal O +5 O +′- O +linked O +Top2 B-protein_type +DNA B-chemical +– O +protein O +crosslink O +. O + +The O +chemical O +complexity O +of O +DNA B-chemical +damage O +- O +derived O +Top2cc B-complex_assembly +necessitates O +that O +DNA B-chemical +repair O +machinery O +dedicated O +to O +resolving O +these O +lesions O +recognizes O +both O +DNA B-chemical +and O +protein O +, O +whilst O +accommodating O +diverse O +chemical O +structures O +that O +trap O +Top2cc B-complex_assembly +. O + +Precisely O +how O +the O +cellular O +DNA B-chemical +repair O +machinery O +navigates O +these O +complex O +lesions O +is O +an O +important O +aspect O +of O +Top2cc B-complex_assembly +repair O +that O +has O +not O +yet O +been O +explored O +. O + +Tdp2 B-protein +processes O +phosphotyrosyl B-ptm +linkages I-ptm +in O +diverse O +DNA B-chemical +damage O +contexts O +. O + +( O +A O +) O +Unrepaired O +DNA B-chemical +damage O +and O +repair O +intermediates O +such O +as O +bulky O +DNA B-chemical +adducts O +, O +ribonucleotides O +or O +abasic O +sites O +can O +poison O +Top2 B-protein_type +and O +trap O +Top2 B-protein_type +cleavage O +complex O +( O +Top2cc B-complex_assembly +), O +resulting O +in O +a O +DSB O +with O +a O +5 O +′– O +Top2 B-protein_type +protein O +adduct O +linked O +by O +a O +phosphotyrosine B-residue_name +bond O +. O + +Tdp2 B-protein +hydrolyzes O +the O +5 O +′– O +phosphotyrosine B-residue_name +adduct O +derived O +from O +poisoned O +Top2 B-protein_type +leaving O +DNA B-chemical +ends O +with O +a O +5 B-chemical +′- I-chemical +phosphate I-chemical +, O +which O +facilitates O +DNA B-chemical +end O +joining O +through O +the O +NHEJ O +pathway O +. O + +( O +B O +) O +DNA B-chemical +oligonucleotide O +substrates O +synthesized O +by O +EDC O +- O +imidazole O +coupling O +and O +used O +in O +Tdp2 B-experimental_method +enzyme I-experimental_method +assays I-experimental_method +contain O +deoxyadenine B-chemical +( O +dA B-chemical +), O +Ethenoadenine B-chemical +( O +ϵA B-chemical +) O +or O +an O +abasic B-site +site I-site +( O +THF B-chemical +) O +and O +a O +5 O +′– O +nitrophenol O +moiety O +. O + +Phosphotyrosyl B-ptm +bond O +hydrolysis O +catalyzed O +by O +mTdp2cat B-structure_element +releases O +p B-chemical +- I-chemical +nitrophenol I-chemical +, O +which O +is O +detected O +by O +measuring O +absorbance O +at O +415 O +nm O +. O +( O +C O +) O +mTdp2cat B-structure_element +reaction B-evidence +rates I-evidence +on O +p B-chemical +– I-chemical +nitrophenol I-chemical +modified O +DNA B-chemical +substrates O +shown O +in O +panel O +B O +. O +Rates O +are O +reported O +as O +molecules O +of O +PNP B-chemical +s O +− O +1 O +produced O +by O +mTdp2cat B-structure_element +. O + +P B-evidence +- I-evidence +values I-evidence +calculated O +using O +two O +- O +tailed O +t B-experimental_method +- I-experimental_method +test I-experimental_method +; O +error O +bars O +, O +s O +. O +d O +. O +n O += O +4 O +, O +n O +. O +s O +. O += O +not O +statistically O +significant O +. O +( O +D O +) O +Structure B-evidence +of O +mTdp2cat B-structure_element +bound B-protein_state +to I-protein_state +5 B-chemical +′- I-chemical +phosphate I-chemical +DNA I-chemical +( O +product O +complex O +) O +containing O +ϵA B-chemical +( O +yellow O +). O + +DNA B-site +binding I-site +β2Hβ I-site +– I-site +grasp I-site +( O +tan O +) O +and O +cap O +elements O +engage O +the O +5 O +′- O +nucleotide O +as O +well O +as O +the O ++ O +2 O +and O ++ O +3 O +nucleotides O +( O +blue O +) O +of O +substrate O +DNA B-chemical +. O + +PDB O +entry O +5HT2 O +is O +displayed O +, O +also O +see O +Table O +1 O +. O +( O +E O +) O +Structure B-evidence +of O +mTdp2cat B-structure_element +bound B-protein_state +to I-protein_state +5 B-chemical +′- I-chemical +phosphate I-chemical +DNA I-chemical +( O +product O +complex O +) O +containing O +THF B-chemical +( O +yellow O +). O + +PDB O +entry O +5INK O +is O +displayed O +, O +also O +see O +Table O +1 O +. O +( O +F O +) O +Structure B-evidence +of O +mTdp2cat B-structure_element +in O +the O +absence B-protein_state +of I-protein_state +DNA B-chemical +showing O +the O +extended B-protein_state +3 B-structure_element +- I-structure_element +helix I-structure_element +loop I-structure_element +( O +tan O +) O +open B-protein_state +- O +conformation O +of O +the O +DNA B-site +- I-site +binding I-site +grasp I-site +as O +seen O +in O +monomer B-oligomeric_state +E B-structure_element +of O +the O +apo B-protein_state +structure B-evidence +. O + +Tyrosyl B-protein +DNA I-protein +phosphodiesterase I-protein +2 I-protein +( O +Tdp2 B-protein +) O +directly O +hydrolyzes O +5 B-ptm +′- I-ptm +phosphotyrosyl I-ptm +( O +5 B-ptm +′- I-ptm +Y I-ptm +) O +linkages B-ptm +, O +and O +is O +a O +key O +modulator O +of O +cellular O +resistance O +to O +chemotherapeutic O +Top2 B-protein_type +poisons O +. O + +Tdp2 B-protein +knockdown B-experimental_method +sensitizes O +A549 O +lung O +cancer O +cells O +to O +etoposide B-chemical +, O +and O +increases O +formation O +of O +nuclear O +γH2AX O +foci O +, O +a O +marker O +of O +DSBs O +, O +underlining O +the O +importance O +of O +Tdp2 B-protein +in O +cellular O +Top2cc B-complex_assembly +repair O +. O + +Tdp2 B-protein +is O +overexpressed O +in O +lung O +cancers O +, O +is O +transcriptionally O +up O +- O +regulated O +in O +mutant B-protein_state +p53 B-protein +cells O +and O +mediates O +mutant B-protein_state +p53 B-protein +gain O +of O +function O +phenotypes O +, O +which O +can O +lead O +to O +acquisition O +of O +therapy O +resistance O +during O +cancer O +progression O +. O + +The O +importance O +of O +Tdp2 B-protein +in O +mediating O +topoisomerase B-protein_type +biology O +is O +further O +underlined O +by O +the O +facts O +that O +human B-species +TDP2 B-protein +inactivating O +mutations O +are O +found O +in O +individuals O +with O +intellectual O +disabilities O +, O +seizures O +and O +ataxia O +, O +and O +at O +the O +cellular O +level O +, O +loss B-protein_state +of I-protein_state +Tdp2 B-protein +inhibits O +Top2β B-protein +- O +dependent O +transcription O +. O + +It O +is O +possible O +that O +TDP2 B-protein +single O +nucleotide O +polymorphisms O +( O +SNPs O +) O +encode O +mutations O +that O +impact O +Tdp2 B-protein +function O +, O +but O +the O +molecular O +underpinnings O +for O +such O +Tdp2 B-protein +deficiencies O +are O +not O +understood O +. O + +Previously O +we O +reported O +high O +- O +resolution O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystal B-evidence +structures I-evidence +of O +the O +minimal B-protein_state +catalytically I-protein_state +active I-protein_state +endonuclease B-structure_element +/ I-structure_element +exonuclease I-structure_element +/ I-structure_element +phosphatase I-structure_element +( O +EEP B-structure_element +) O +domain O +of O +mouse B-taxonomy_domain +Tdp2 B-protein +( O +mTdp2cat B-structure_element +) O +bound B-protein_state +to I-protein_state +a O +DNA B-chemical +substrate O +mimic O +, O +and O +a O +5 B-protein_state +′- I-protein_state +phosphorylated I-protein_state +reaction O +product O +. O + +However O +, O +important O +questions O +regarding O +the O +mechanism O +of O +Tdp2 B-protein +engagement O +and O +processing O +of O +DNA B-chemical +damage O +remain O +. O + +First O +, O +it O +is O +unclear O +if O +Tdp2 B-protein +processes O +phosphotyrosyl B-ptm +linkages I-ptm +in O +the O +context O +of O +DNA B-chemical +damage O +that O +triggers O +Top2cc B-complex_assembly +, O +and O +if O +so O +, O +how O +the O +enzyme O +can O +accommodate O +such O +complex O +DNA B-chemical +damage O +within O +its O +active B-site +site I-site +. O + +Based O +on O +metal B-protein_state +- I-protein_state +bound I-protein_state +Tdp2 B-protein +structures B-evidence +, O +we O +also O +proposed O +a O +single O +Mg2 B-chemical ++ I-chemical +mediated O +catalytic O +mechanism O +, O +but O +this O +mechanism O +requires O +further O +scrutiny O +and O +characterization O +. O + +Herein O +, O +we O +report O +an O +integrated O +structure B-experimental_method +- I-experimental_method +function I-experimental_method +study I-experimental_method +of O +the O +Tdp2 B-protein +reaction O +mechanism O +, O +including O +a O +description O +of O +new O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +structures B-evidence +of O +ligand B-protein_state +- I-protein_state +free I-protein_state +Tdp2 B-protein +, O +and O +Tdp2 B-protein +bound B-protein_state +to I-protein_state +abasic O +and O +alkylated O +( O +1 B-chemical +- I-chemical +N6 I-chemical +- I-chemical +etheno I-chemical +- I-chemical +adenine I-chemical +) O +DNA B-chemical +damage O +. O + +Our O +integrated O +results O +from O +structural B-experimental_method +analysis I-experimental_method +, O +mutagenesis B-experimental_method +, O +functional B-experimental_method +assays I-experimental_method +and O +quanyum B-experimental_method +mechanics I-experimental_method +/ I-experimental_method +molecular I-experimental_method +mechanics I-experimental_method +( O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +) O +modeling B-experimental_method +of O +the O +Tdp2 B-protein +reaction O +coordinate O +describe O +in O +detail O +how O +Tdp2 B-protein +mediates O +a O +single O +- O +metal O +ion O +tyrosyl B-protein_type +DNA I-protein_type +phosphodiesterase I-protein_type +reaction O +capable O +of O +acting O +on O +diverse O +DNA B-chemical +end O +damage O +. O + +We O +further O +establish O +that O +DNA B-chemical +damage O +binding O +in O +the O +Tdp2 B-protein +active B-site +site I-site +is O +linked O +to O +conformational O +change O +and O +binding O +of O +metal O +cofactor O +. O + +Finally O +, O +we O +characterize O +a O +Tdp2 B-protein +SNP O +that O +ablates B-protein_state +the O +Tdp2 B-protein +single B-site +metal I-site +binding I-site +site I-site +and O +Tdp2 B-protein +substrate O +induced O +conformational O +changes O +, O +and O +confers O +Top2 B-protein_type +drug O +sensitivity O +in O +mammalian B-taxonomy_domain +cells O +. O + +Tdp2 B-protein +processing O +of O +compound O +DNA B-chemical +damage O + +Two O +potent O +Top2 B-protein_type +poisons O +include O +bulky O +alkylated O +DNA B-chemical +helix O +- O +distorting O +DNA B-chemical +base O +adducts O +( O +e O +. O +g O +. O +1 B-chemical +- I-chemical +N6 I-chemical +- I-chemical +ethenoadenine I-chemical +, O +ϵA B-chemical +) O +and O +abundant O +abasic O +sites O +( O +Figure O +1A O +). O + +Whether O +Tdp2 B-protein +processes O +phosphotyrosyl B-ptm +linkages I-ptm +within O +these O +diverse O +structural O +contexts O +is O +not O +known O +. O + +To O +test O +this O +, O +we O +adapted O +an O +EDC B-experimental_method +coupling I-experimental_method +method I-experimental_method +to O +generate O +5 O +′- O +terminal O +p B-chemical +- I-chemical +nitrophenol I-chemical +( O +PNP B-chemical +) O +modified O +oligonucleotides O +that O +also O +harbored O +DNA B-chemical +damage O +at O +the O +5 O +′- O +nucleotide O +position O +( O +see O +Materials O +and O +Methods O +). O + +We O +then O +evaluated O +the O +ability O +of O +a O +recombinant O +purified O +mouse B-taxonomy_domain +Tdp2 B-protein +catalytic B-structure_element +domain I-structure_element +( O +mTdp2cat B-structure_element +) O +to O +release O +PNP B-chemical +( O +a O +structural O +mimic O +of O +a O +topoisomerase B-protein_type +tyrosine B-residue_name +) O +from O +the O +5 O +′- O +terminus O +of O +compound O +damaged O +DNA B-chemical +substrates O +using O +a O +colorimetric B-experimental_method +assay I-experimental_method +( O +Figure O +1B O +). O + +We O +observe O +robust O +Tdp2 B-protein +- O +dependent O +release O +of O +PNP B-chemical +from O +5 O +′- O +modified O +oligonucleotides O +in O +the O +context O +of O +dA B-chemical +- I-chemical +PNP I-chemical +, O +ϵA B-chemical +- I-chemical +PNP I-chemical +or O +the O +abasic O +- O +site O +analog O +tetrahydrofuran B-chemical +spacer I-chemical +( O +THF B-chemical +) O +( O +Figure O +1C O +). O + +Thus O +, O +Tdp2 B-protein +efficiently O +cleaves O +phosphotyrosyl B-ptm +linkages I-ptm +in O +the O +context O +of O +a O +compound O +5 O +′ O +lesions O +composed O +of O +abasic O +or O +bulky O +DNA B-chemical +base O +adduct O +DNA B-chemical +damage O +. O + +To O +understand O +the O +molecular O +basis O +for O +Tdp2 B-protein +processing O +of O +Top2cc B-complex_assembly +in O +the O +context O +of O +DNA B-chemical +damage O +, O +we O +crystallized B-experimental_method +and I-experimental_method +determined I-experimental_method +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystal B-evidence +structures I-evidence +of O +mTdp2cat B-structure_element +bound B-protein_state +to I-protein_state +5 B-chemical +′- I-chemical +phosphate I-chemical +DNA I-chemical +( O +product O +complex O +) O +with O +a O +5 B-chemical +′- I-chemical +ϵA I-chemical +at O +1 O +. O +43 O +Å O +resolution O +( O +PDB O +entry O +5HT2 O +) O +and O +the O +abasic O +DNA B-chemical +damage O +mimic O +5 B-chemical +′- I-chemical +THF I-chemical +at O +2 O +. O +15 O +Å O +resolution O +( O +PDB O +entry O +5INK O +; O +Figure O +1D O +and O +E O +, O +Table O +1 O +). O + +In O +these O +Tdp2 B-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +complex O +structures B-evidence +, O +mTdp2cat B-structure_element +adopts O +a O +mixed B-structure_element +α I-structure_element +- I-structure_element +β I-structure_element +fold I-structure_element +typified O +by O +a O +central O +12 B-structure_element +- I-structure_element +stranded I-structure_element +anti I-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +sandwich I-structure_element +enveloped O +by O +several O +helical O +elements O +that O +mold O +the O +Tdp2 B-protein +active B-site +site I-site +. O + +One O +half O +of O +the O +molecule O +contributes O +to O +formation O +of O +the O +walls O +of O +the O +DNA B-site +- I-site +binding I-site +cleft I-site +that O +embraces O +the O +terminal O +position O +of O +the O +damaged O +DNA B-chemical +substrate O +. O + +In O +the O +DNA B-protein_state +lesion I-protein_state +- I-protein_state +bound I-protein_state +state O +, O +two O +key O +DNA B-chemical +binding O +elements O +, O +the O +β B-structure_element +- I-structure_element +2 I-structure_element +- I-structure_element +helix I-structure_element +- I-structure_element +β I-structure_element +( O +β2Hβ B-structure_element +) O +‘ O +grasp B-structure_element +’, O +and O +‘ O +helical B-structure_element +cap I-structure_element +’ O +mold O +the O +substrate B-site +binding I-site +trench I-site +and O +direct O +the O +ssDNA B-chemical +of O +a O +5 O +′- O +overhang O +substrate O +into O +the O +active B-site +site I-site +. O + +A O +comparison O +to O +an O +additional O +new O +structure B-evidence +of O +DNA B-protein_state +- I-protein_state +free I-protein_state +Tdp2 B-protein +( O +apo B-protein_state +state O +, O +Figure O +1F O +) O +shows O +that O +this O +loop B-structure_element +is O +conformationally B-protein_state +mobile I-protein_state +and O +important O +for O +engaging O +DNA B-chemical +substrates O +. O + +The O +mode O +of O +engagement O +of O +the O +5 O +′- O +nucleobase O +of O +the O +bulky O +ϵA B-chemical +adduct O +describes O +a O +mechanism O +for O +Tdp2 B-protein +to O +bind O +5 B-protein_state +′- I-protein_state +tyrosylated I-protein_state +substrates O +that O +contain O +diverse O +forms O +of O +DNA B-chemical +damage O +. O + +The O +5 B-chemical +′- I-chemical +ϵA I-chemical +nucleobase O +is O +recognized O +by O +an O +extended O +Tdp2 B-protein +van B-site +Der I-site +Waals I-site +interaction I-site +surface I-site +, O +referred O +to O +here O +as O +the O +‘ O +hydrophobic B-site +wall I-site +’ O +that O +is O +assembled O +with O +the O +sidechains O +of O +residues O +Leu315 B-residue_name_number +and O +Ile317 B-residue_name_number +( O +Figure O +2A O +and O +B O +). O + +Structures B-evidence +of O +mTdp2cat B-structure_element +bound B-protein_state +to I-protein_state +DNA B-chemical +damage O +that O +triggers O +Top2 B-protein_type +poisoning O +. O + +( O +A O +) O +Structure B-evidence +of O +mTdp2cat B-structure_element +bound B-protein_state +to I-protein_state +5 B-chemical +′- I-chemical +phosphate I-chemical +DNA I-chemical +( O +product O +complex O +) O +containing O +ϵA B-chemical +( O +yellow O +), O +Mg2 B-chemical ++ I-chemical +( O +magenta O +) O +and O +its O +inner O +- O +sphere O +waters B-chemical +( O +gray O +). O + +mTdp2cat B-structure_element +is O +colored O +by O +electrostatic O +surface O +potential O +( O +red O += O +negative O +, O +blue O += O +positive O +, O +gray O += O +neutral O +/ O +hydrophobic O +). O + +( O +B O +) O +σ B-evidence +- I-evidence +A I-evidence +weighted I-evidence +2Fo I-evidence +- I-evidence +Fc I-evidence +electron I-evidence +density I-evidence +map I-evidence +( O +at O +1 O +. O +43 O +Å O +resolution O +, O +contoured O +at O +2 O +. O +0 O +σ O +) O +for O +the O +ϵA B-chemical +DNA I-chemical +complex O +. O + +The O +ϵA B-chemical +nucleotide O +is O +shown O +in O +yellow O +and O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +from O +the O +ϵA B-chemical +O4 O +′ O +to O +inner O +- O +sphere O +water B-chemical +is O +shown O +as O +gray O +dashes O +. O +( O +C O +) O +Structure B-evidence +of O +mTdp2cat B-structure_element +bound B-protein_state +to I-protein_state +5 B-chemical +′- I-chemical +phosphate I-chemical +DNA I-chemical +( O +product O +complex O +) O +containing O +THF B-chemical +( O +yellow O +), O +Mg2 B-chemical ++ I-chemical +( O +magenta O +) O +and O +its O +inner O +- O +sphere O +waters B-chemical +( O +gray O +). O + +mTdp2cat B-structure_element +is O +colored O +with O +red O +( O +electronegative O +), O +blue O +( O +electropositive O +) O +and O +gray O +( O +hydrophobic O +) O +electrostatic O +surface O +potential O +displayed O +. O + +PDB O +entry O +5INK O +is O +displayed O +. O +( O +D O +) O +σ B-evidence +- I-evidence +A I-evidence +weighted I-evidence +2Fo I-evidence +- I-evidence +Fc I-evidence +electron I-evidence +density I-evidence +map I-evidence +( O +at O +2 O +. O +15 O +Å O +resolution O +, O +contoured O +at O +2 O +. O +0 O +σ O +) O +for O +THF B-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +complex O +. O + +The O +THF B-chemical +is O +shown O +in O +yellow O +and O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +from O +the O +THF B-chemical +O4 O +′ O +to O +inner O +- O +sphere O +water B-chemical +is O +shown O +as O +gray O +dashes O +. O + +For O +comparison O +, O +we O +also O +determined B-experimental_method +a O +structure B-evidence +of O +an O +undamaged O +5 B-chemical +′- I-chemical +adenine I-chemical +( O +5 B-chemical +′- I-chemical +dA I-chemical +) O +bound B-protein_state +to I-protein_state +Tdp2 B-protein +at O +1 O +. O +55 O +Å O +( O +PDB O +entry O +5INL O +). O + +A O +structural B-experimental_method +overlay I-experimental_method +of O +damaged O +and O +undamaged O +nucleotides O +shows O +no O +major O +distortions O +to O +nucleotide O +planarity O +between O +different O +bound B-protein_state +sequences O +and O +DNA B-chemical +damage O +( O +compare O +ϵA B-chemical +, O +dA B-chemical +and O +dC B-chemical +, O +Supplementary O +Figure O +S1A O +– O +D O +). O + +Therefore O +, O +structurally O +diverse O +undamaged O +or O +alkylated O +bases O +( O +e O +. O +g O +. O +ϵG B-chemical +, O +ϵT B-chemical +) O +could O +likely O +be O +accommodated O +in O +the O +Tdp2 B-protein +active B-site +site I-site +via O +planar B-bond_interaction +base I-bond_interaction +stacking I-bond_interaction +with O +the O +active B-site +site I-site +facing O +hydrophobic B-site +wall I-site +of O +the O +β2Hβ B-structure_element +motif O +. O + +Likewise O +, O +the O +abasic B-chemical +deoxyribose I-chemical +analog O +THF B-chemical +substrate O +binds O +similar O +to O +the O +alkylated O +and O +non O +- O +alkylated O +substrates O +, O +but O +with O +a O +slight O +alteration O +in O +the O +approach O +of O +the O +5 O +′- O +terminus O +( O +Figure O +2C O +). O + +Interestingly O +, O +in O +the O +absence B-protein_state +of I-protein_state +a O +nucleobase O +, O +O4 O +′ O +of O +the O +THF B-chemical +ring O +adopts O +a O +close O +approach O +( O +2 O +. O +8 O +Å O +) O +to O +a O +water B-chemical +molecule O +that O +directly O +participates O +in O +the O +outer O +sphere O +single O +Mg2 B-chemical ++ I-chemical +ion B-bond_interaction +coordination I-bond_interaction +shell I-bond_interaction +( O +Figure O +2D O +). O + +These O +collective O +differences O +may O +explain O +the O +slight O +, O +but O +statistically O +significant O +elevated O +activity O +on O +the O +THF B-chemical +substrate O +( O +Figure O +1C O +). O + +Structural O +plasticity O +in O +the O +Tdp2 B-protein +DNA B-site +binding I-site +trench I-site + +An O +intriguing O +feature O +of O +the O +DNA B-protein_state +- I-protein_state +damage I-protein_state +bound I-protein_state +conformation O +of O +the O +Tdp2 B-protein +active B-site +site I-site +is O +an O +underlying O +network O +of O +protein O +– O +water B-chemical +– O +protein O +contacts O +that O +span O +a O +gap O +between O +the O +catalytic B-site +core I-site +and O +the O +DNA B-site +binding I-site +β2Hβ I-site +- I-site +grasp I-site +( O +Supplementary O +Figure O +S2 O +). O + +In O +this O +arrangement O +, O +six O +solvent O +molecules O +form O +a O +channel O +under O +the O +β2Hβ B-site +- I-site +grasp I-site +, O +ending O +with O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +peptide O +backbone O +of O +the O +Mg2 B-chemical ++ I-chemical +ligand O +Asp358 B-residue_name_number +. O + +The O +paucity O +of O +hydrophobic B-bond_interaction +interactions I-bond_interaction +stabilizing O +the O +β2Hβ B-structure_element +DNA B-protein_state +- I-protein_state +bound I-protein_state +conformation O +suggests O +that O +conformational O +plasticity O +in O +the O +β2Hβ B-structure_element +might O +be O +a O +feature O +of O +DNA B-chemical +damage O +and O +metal O +cofactor O +engagement O +. O + +To O +test O +this O +hypothesis O +, O +we O +crystallized B-experimental_method +Tdp2 B-protein +in O +the O +absence B-protein_state +of I-protein_state +DNA B-chemical +and O +determined O +a O +DNA B-protein_state +free I-protein_state +Tdp2 B-protein +structure B-evidence +to O +2 O +. O +4 O +Å O +resolution O +( O +PDB O +entry O +5INM O +; O +Figures O +1F O +and O +3A O +). O + +Conformational O +plasticity O +in O +the O +Tdp2 B-protein +active B-site +site I-site +. O + +( O +A O +) O +The O +open B-protein_state +, O +3 B-structure_element +- I-structure_element +helix I-structure_element +conformation O +( O +tan O +) O +of O +flexible B-protein_state +active B-structure_element +- I-structure_element +site I-structure_element +loop I-structure_element +observed O +in O +monomer B-oligomeric_state +E B-structure_element +of O +the O +DNA B-protein_state +- I-protein_state +free I-protein_state +mTdp2cat B-structure_element +structure B-evidence +( O +PDB O +entry O +5INM O +) O +is O +supported O +by O +T309 B-residue_name_number +( O +green O +), O +which O +packs O +against O +the O +EEP B-structure_element +core O +. O + +The O +β2Hβ B-site +docking I-site +pocket I-site +( O +circled O +) O +is O +unoccupied O +and O +residues O +N312 B-residue_name_number +, O +N314 B-residue_name_number +and O +L315 B-residue_name_number +( O +orange O +) O +are O +solvent B-protein_state +- I-protein_state +exposed I-protein_state +. O + +Wall O +- O +eyed O +stereo O +view O +is O +displayed O +. O +( O +B O +) O +The O +closed B-protein_state +β2Hβ B-structure_element +conformation O +in O +the O +mTdp2cat B-complex_assembly +– I-complex_assembly +DNA I-complex_assembly +product O +structure B-evidence +containing O +5 B-chemical +′- I-chemical +ϵA I-chemical +( O +yellow O +, O +PDB O +entry O +5HT2 O +). O +T309 B-residue_name_number +( O +green O +) O +is O +an O +integral O +part O +of O +the O +β2Hβ B-site +DNA I-site +- I-site +binding I-site +grasp I-site +( O +tan O +) O +and O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +backbone O +of O +Y321 B-residue_name_number +, O +while O +N314 B-residue_name_number +( O +orange O +) O +occupies O +the O +β2Hβ B-site +docking I-site +pocket I-site +. O + +Wall O +- O +eyed O +stereo O +view O +is O +displayed O +. O +( O +C O +) O +Alignment O +of O +active B-structure_element +site I-structure_element +loop I-structure_element +conformers O +observed O +in O +the O +5 O +promoters B-oligomeric_state +of O +the O +DNA B-protein_state +- I-protein_state +free I-protein_state +mTdp2cat B-structure_element +( O +PDB O +entry O +5INM O +, O +see O +Table O +1 O +) O +crystallographic O +asymmetric O +unit O +( O +left O +) O +and O +sequence B-experimental_method +alignment I-experimental_method +showing O +residues O +not O +observed O +in O +the O +electron B-evidence +density I-evidence +as O +‘∼’ O +( O +right O +). O +( O +D O +) O +Limited B-experimental_method +trypsin I-experimental_method +proteolysis I-experimental_method +probes O +the O +solvent O +accessibility O +of O +the O +flexible B-protein_state +active B-structure_element +- I-structure_element +site I-structure_element +loop I-structure_element +. O + +mTdp2cat B-structure_element +WT B-protein_state +( O +lanes O +1 O +– O +13 O +) O +or O +mTdp2cat B-structure_element +D358N B-mutant +( O +lanes O +14 O +– O +26 O +) O +were O +incubated O +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +Mg2 B-chemical ++ I-chemical +and O +/ O +or O +a O +12 O +nt O +self O +annealing O +, O +5 O +′- O +phosphorylated O +DNA B-chemical +( O +substrate O +‘ O +12 O +nt O +’ O +in O +Supplementary O +Table O +S1 O +), O +then O +reacted O +with O +0 O +. O +6 O +, O +1 O +. O +7 O +or O +5 O +ng O +μl O +− O +1 O +of O +trypsin O +. O + +Reactions O +were O +separated O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +and O +proteins O +visualized O +by O +staining O +with O +coomassie O +blue O +. O +( O +E O +) O +Limited B-experimental_method +chymotrypsin I-experimental_method +proteolysis I-experimental_method +probes O +the O +solvent O +accessibility O +of O +the O +flexible B-protein_state +active B-structure_element +- I-structure_element +site I-structure_element +loop I-structure_element +. O + +Experiments O +performed O +as O +in O +panel O +D O +for O +mTdp2cat B-structure_element +WT B-protein_state +( O +lanes O +27 O +– O +39 O +) O +or O +mTdp2cat B-structure_element +D358N B-mutant +( O +lanes O +40 O +– O +52 O +), O +but O +with O +chymotrypsin B-experimental_method +instead O +of O +trypsin B-experimental_method +. O + +This O +crystal O +form O +contains O +5 O +Tdp2 B-protein +protein O +molecules O +in O +the O +asymmetric O +unit O +, O +with O +variations O +in O +active B-site +site I-site +Mg2 B-chemical ++ I-chemical +occupancy O +and O +substrate B-structure_element +binding I-structure_element +loops I-structure_element +observed O +for O +the O +individual O +protomers B-oligomeric_state +. O + +The O +most O +striking O +feature O +of O +the O +DNA B-protein_state +ligand I-protein_state +- I-protein_state +free I-protein_state +state O +is O +that O +the O +active B-site +site I-site +β2Hβ I-site +- I-site +grasp I-site +can O +adopt O +alternative O +structures O +that O +are O +distinct O +from O +the O +DNA B-protein_state +- I-protein_state +bound I-protein_state +, O +closed B-protein_state +β2Hβ B-site +DNA I-site +binding I-site +grasp I-site +( O +Figure O +3A O +and O +B O +). O + +In O +one O +monomer B-oligomeric_state +( O +chain B-structure_element +‘ I-structure_element +E I-structure_element +’), I-structure_element +the O +grasp B-structure_element +adopts O +an O +‘ O +open B-protein_state +’ O +3 B-structure_element +- I-structure_element +helix I-structure_element +loop I-structure_element +conformation O +that O +projects O +away O +from O +the O +EEP B-structure_element +catalytic B-site +core I-site +. O + +Two O +monomers B-oligomeric_state +have O +variable O +disordered B-protein_state +states O +for O +which O +much O +of O +the O +DNA B-structure_element +binding I-structure_element +loop I-structure_element +is O +not O +visible O +in O +the O +electron B-evidence +density I-evidence +. O + +The O +remaining O +two O +molecules O +in O +the O +DNA B-protein_state +- I-protein_state +free I-protein_state +crystal B-evidence +form I-evidence +are O +closed B-protein_state +β2Hβ B-structure_element +conformers O +similar O +to O +the O +DNA B-protein_state +bound I-protein_state +structures B-evidence +( O +Figure O +3C O +). O + +Thus O +, O +we O +posit O +that O +Tdp2 B-protein +DNA B-chemical +binding O +conformationally O +selects O +the O +closed B-protein_state +form O +of O +the O +β2Hβ B-site +grasp I-site +, O +rather O +than O +inducing O +closure O +upon O +binding O +. O + +A O +detailed O +analysis O +of O +the O +extended B-protein_state +3 B-structure_element +- I-structure_element +helix I-structure_element +conformation O +shows O +that O +the O +substrate B-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +is O +able O +to O +undergo O +metamorphic O +structural O +changes O +. O + +In O +this O +open B-protein_state +form O +, O +residues O +Asn312 B-residue_range +- I-residue_range +Leu315 I-residue_range +are O +distal O +from O +the O +active B-site +site I-site +and O +solvent B-protein_state +- I-protein_state +exposed I-protein_state +( O +orange O +sticks O +, O +Figure O +3A O +), O +while O +Thr309 B-residue_name_number +( O +green O +surface O +, O +Figure O +3A O +) O +packs O +into O +a O +shallow O +pocket B-site +of O +the O +EEP B-structure_element +core O +to O +anchor O +the O +loop B-structure_element +. O + +Burial O +of O +Thr309 B-residue_name_number +is O +enabled O +by O +an O +unusual O +main O +chain O +cis B-bond_interaction +– I-bond_interaction +peptide I-bond_interaction +bond I-bond_interaction +between O +Asp308 B-residue_name_number +- O +Thr309 B-residue_name_number +and O +disassembly O +of O +the O +short B-structure_element +antiparallel I-structure_element +beta I-structure_element +- I-structure_element +strand I-structure_element +of O +the O +β2Hβ B-structure_element +fold O +. O + +By O +comparison O +, O +the O +closed B-protein_state +β2Hβ B-site +grasp I-site +conformer O +is O +stabilized O +by O +Asn312 B-residue_name_number +and O +Asn314 B-residue_name_number +binding O +into O +two O +β2Hβ B-site +docking I-site +pockets I-site +, O +and O +Leu315 B-residue_name_number +engagement O +of O +the O +5 O +′- O +terminal O +nucleobase O +( O +Figure O +3B O +). O + +To O +transition O +into O +the O +closed B-protein_state +β2Hβ B-structure_element +conformation O +, O +Thr309 B-residue_name_number +disengages O +from O +the O +EEP B-structure_element +domain O +pocket B-site +, O +flips O +peptide O +backbone O +conformation O +cis O +to O +trans O +, O +and O +is O +integral O +to O +the O +β2Hβ B-structure_element +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +. O + +Stabilization O +of O +the O +closed B-protein_state +β2Hβ B-site +- I-site +grasp I-site +conformation O +is O +linked O +to O +the O +active B-site +site I-site +through O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +Trp307 B-residue_name_number +and O +the O +Mg2 B-site ++ I-site +coordinating I-site +residue I-site +Asp358 B-residue_name_number +. O + +Accordingly O +, O +in O +the O +DNA B-protein_state +free I-protein_state +structure B-evidence +, O +we O +observe O +a O +trend O +where O +the O +2 O +closed B-protein_state +monomers B-oligomeric_state +have O +an O +ordered O +Mg2 B-chemical ++ I-chemical +ion O +in O +their O +active B-site +sites I-site +, O +while O +the O +monomers B-oligomeric_state +with O +open B-protein_state +conformations O +have O +a O +poorly O +ordered O +or O +vacant O +metal B-site +binding I-site +site I-site +. O + +Overall O +, O +these O +observations O +suggest O +that O +engagement O +of O +diverse O +damaged O +DNA B-chemical +ends O +is O +enabled O +by O +an O +elaborate O +substrate O +selected O +stabilization O +of O +the O +β2Hβ B-site +DNA I-site +binding I-site +grasp I-site +, O +and O +these O +rearrangements O +are O +coordinated O +with O +Mg2 B-chemical ++ I-chemical +binding O +in O +the O +Tdp2 B-protein +active B-site +site I-site +. O + +To O +evaluate O +Mg2 B-chemical ++ I-chemical +and O +DNA B-chemical +- O +dependent O +Tdp2 B-protein +structural O +states O +in O +solution O +, O +we O +probed O +mTdp2cat B-structure_element +conformations O +using O +limited B-experimental_method +trypsin I-experimental_method +and I-experimental_method +chymotrypsin I-experimental_method +proteolysis I-experimental_method +( O +Figure O +3C O +– O +E O +). O + +In B-protein_state +the I-protein_state +absence I-protein_state +of I-protein_state +DNA B-chemical +or O +Mg2 B-chemical ++, I-chemical +mTdp2cat B-structure_element +is O +efficiently O +cleaved O +in O +the O +metamorphic O +DNA B-site +binding I-site +grasp I-site +at O +one O +site O +by O +trypsin B-experimental_method +( O +Arg316 B-residue_name_number +), O +or O +at O +two O +positions O +by O +chymotrypsin B-experimental_method +( O +Trp307 B-residue_name_number +and O +Leu315 B-residue_name_number +). O + +By O +comparison O +, O +Mg2 B-chemical ++, I-chemical +and O +to O +a O +greater O +extent O +Mg2 B-chemical ++/ I-chemical +DNA B-chemical +mixtures O +( O +compare O +Figure O +3 O +, O +lanes O +4 O +, O +7 O +and O +13 O +) O +protect O +mTdp2cat B-structure_element +from O +proteolytic O +cleavage O +. O + +Interestingly O +, O +addition O +of O +Mg2 B-chemical ++ I-chemical +alone O +protects O +against O +proteolysis O +as O +well O +. O + +This O +is O +consistent O +with O +Mg2 B-chemical ++ I-chemical +stabilizing O +the O +closed B-protein_state +conformation O +of O +the O +β2Hβ B-site +- I-site +grasp I-site +through O +an O +extended O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +network I-bond_interaction +with O +Asp358 B-residue_name_number +and O +the O +indole O +ring O +of O +the O +β2Hβ B-site +- I-site +grasp I-site +residue O +Trp307 B-residue_name_number +( O +also O +discussion O +below O +on O +Tdp2 B-protein +active B-site +site I-site +SNPs O +). O + +To O +assess O +structural O +conservation O +of O +Tdp2 B-protein +conformational O +changes O +between O +human B-species +and O +mouse B-taxonomy_domain +Tdp2 B-protein +, O +we O +also O +determined B-experimental_method +a O +3 O +. O +2 O +Å O +resolution O +structure B-evidence +of O +the O +human B-species +Tdp2cat B-structure_element +domain O +bound B-protein_state +to I-protein_state +a O +DNA B-chemical +5 I-chemical +′- I-chemical +PO4 I-chemical +terminus O +product O +complex O +( O +PDB O +entry O +5INO O +). O + +Comparisons O +of O +the O +human B-species +hTdp2cat B-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +complex O +structure B-evidence +to O +the O +mTdp2cat B-structure_element +DNA B-protein_state +bound I-protein_state +state O +show O +a O +high O +level O +of O +conservation O +of O +the O +DNA B-protein_state +- I-protein_state +bound I-protein_state +conformations O +( O +Supplementary O +Figure O +S3A O +). O + +Moreover O +, O +similar O +to O +mTdp2cat B-structure_element +, O +proteolytic O +protection O +of O +the O +hTdp2cat B-structure_element +substrate B-structure_element +binding I-structure_element +loop I-structure_element +occurs O +with O +addition O +of O +Mg2 B-chemical ++ I-chemical +and O +DNA B-chemical +( O +Supplementary O +Figure O +S3B O +). O + +Thus O +, O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +structures B-evidence +and O +limited B-experimental_method +proteolysis I-experimental_method +analysis I-experimental_method +indicate O +that O +DNA B-chemical +- O +and O +metal O +- O +induced O +conformational O +changes O +are O +a O +conserved B-protein_state +feature O +of O +the O +vertebrate B-taxonomy_domain +Tdp2 B-protein +- O +substrate O +interaction O +. O + +Tdp2 B-protein +metal O +ion O +dependence O + +Consistently O +in O +high O +- O +resolution O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +structural I-experimental_method +analyses I-experimental_method +we O +, O +and O +others O +observe O +a O +single O +Mg2 B-chemical ++ I-chemical +metal O +bound B-protein_state +in I-protein_state +the O +Tdp2 B-protein +active B-site +site I-site +. O + +This O +includes O +the O +DNA B-protein_state +- I-protein_state +free I-protein_state +( O +Figure O +3A O +), O +DNA B-protein_state +damage I-protein_state +bound I-protein_state +( O +Figure O +3B O +) O +and O +reaction B-protein_state +product I-protein_state +- I-protein_state +bound I-protein_state +crystal B-evidence +forms I-evidence +of O +mouse B-taxonomy_domain +, O +( O +PDB O +entry O +4GZ1 O +), O +D B-species +. I-species +rerio I-species +( O +PDB O +entry O +4FPV O +) O +and O +C B-species +. I-species +elegans I-species +Tdp2 B-protein +( O +PDB O +entry O +4FVA O +). O + +However O +, O +previous O +biochemical B-experimental_method +analysis I-experimental_method +has O +suggested O +an O +alternative O +two O +- O +metal O +ion O +mechanism O +for O +the O +Tdp2 B-protein +- O +phosphotyrosyl B-protein_type +phosphodiesterase I-protein_type +reaction O +. O + +In O +these O +experiments O +, O +at O +limiting O +Mg2 B-chemical ++ I-chemical +concentrations O +, O +Ca2 B-chemical ++ I-chemical +addition O +to O +Tdp2 B-protein +reactions O +stimulated O +activity O +. O + +While O +this O +work O +was O +suggestive O +of O +a O +two O +metal O +ion O +mechanism O +for O +phosphotyrosyl B-ptm +bond O +cleavage O +by O +Tdp2 B-protein +, O +we O +note O +that O +second O +metal O +ion O +titrations O +can O +be O +influenced O +by O +metal B-site +ion I-site +binding I-site +sites I-site +outside O +of O +the O +active B-site +site I-site +. O + +In O +fact O +, O +divalent B-chemical +metals I-chemical +have O +been O +observed O +in O +the O +Tdp2 B-protein +protein O +– O +DNA B-chemical +complexes O +( O +PDB O +entry O +4GZ2 O +) O +distal O +to O +the O +active B-site +center I-site +, O +and O +we O +propose O +this O +might O +account O +for O +varied O +results O +in O +different O +studies O +. O + +To O +further O +probe O +the O +metal O +ion O +dependence O +of O +the O +Tdp2 B-protein +phosphodiesterase B-protein_type +reaction O +, O +we O +performed O +metal B-experimental_method +ion I-experimental_method +binding I-experimental_method +assays I-experimental_method +, O +determined O +crystal B-evidence +structures I-evidence +in O +the O +presence B-protein_state +of I-protein_state +varied O +divalent B-chemical +metals I-chemical +( O +Mn2 B-chemical ++ I-chemical +and O +Ca2 B-chemical ++), I-chemical +and O +analyzed O +metal O +ion O +dependence O +of O +the O +Tdp2 B-protein +phosphotyrosyl B-protein_type +phosphodiesterase I-protein_type +reaction O +( O +Figure O +4 O +). O + +Metal O +cofactor O +interactions O +with O +Tdp2 B-protein +. O +( O +A O +) O +Intrinsic B-evidence +tryptophan I-evidence +fluorescence I-evidence +of O +mTdp2cat B-structure_element +was O +used O +to O +monitor O +a O +conformational O +response O +to O +divalent O +metal O +ion O +binding O +. O + +Either O +Mg2 B-chemical ++ I-chemical +or O +Ca2 B-chemical ++ I-chemical +were O +titrated B-experimental_method +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +5 B-chemical +′- I-chemical +P I-chemical +DNA I-chemical +, O +and O +the O +tryptophan B-evidence +fluorescence I-evidence +was O +monitored O +with O +an O +excitation O +wavelength O +of O +280 O +nm O +and O +emission O +wavelength O +of O +350 O +nm O +using O +10 O +nm O +band O +pass O +filters O +. O + +Both O +Mg2 B-chemical ++ I-chemical +and O +Ca2 B-chemical ++ I-chemical +induce O +a O +conformational O +change O +which O +elicits O +an O +increase O +in O +tryptophan B-evidence +fluorescence I-evidence +of O +mTdp2cat B-structure_element +in O +the O +presence B-protein_state +and O +absence B-protein_state +of I-protein_state +DNA B-chemical +, O +while O +D358N B-mutant +active B-site +site I-site +mutant B-protein_state +of O +mTdp2cat B-structure_element +is O +unresponsive B-protein_state +to O +Mg2 B-chemical ++. I-chemical +( O +B O +) O +mTdp2cat B-structure_element +activity O +assayed O +on O +a O +T5PNP B-chemical +substrate O +as O +a O +function O +of O +Mg2 B-chemical ++ I-chemical +and O +Ca2 B-chemical ++ I-chemical +concentration O +. O + +PNP B-chemical +release O +( O +monitored O +by O +absorbance O +at O +415 O +nm O +) O +as O +a O +function O +of O +Mg2 B-chemical ++ I-chemical +concentration O +and O +in O +the O +absence B-protein_state +or O +presence B-protein_state +of I-protein_state +1 O +or O +10 O +mM O +Ca2 B-chemical ++ I-chemical +is O +shown O +; O +error O +bars O +, O +s O +. O +d O +. O +n O += O +4 O +. O +( O +C O +) O +σ B-evidence +- I-evidence +A I-evidence +weighted I-evidence +2Fo I-evidence +- I-evidence +Fc I-evidence +electron I-evidence +density I-evidence +map I-evidence +( O +blue O +) O +and O +model B-evidence +- I-evidence +phased I-evidence +anomalous I-evidence +difference I-evidence +Fourier I-evidence +( O +magenta O +) O +maps B-evidence +for O +the O +mTdp2cat B-complex_assembly +– I-complex_assembly +DNA I-complex_assembly +– I-complex_assembly +Mn2 I-complex_assembly ++ I-complex_assembly +complex O +( O +PDB O +entry O +5INP O +) O +show O +a O +single O +Mn2 B-chemical ++ I-chemical +( O +cyan O +) O +is O +bound O +with O +expected O +octahedral O +coordination O +geometry O +. O + +A O +53σ O +peak O +in O +the O +anomalous B-evidence +difference I-evidence +Fourier I-evidence +map I-evidence +( O +data O +collected O +at O +λ O += O +1 O +. O +5418 O +Å O +) O +supports O +Mn2 B-chemical ++ I-chemical +as O +the O +identity O +of O +this O +atom O +. O + +( O +D O +) O +Comparison O +of O +Ca2 B-chemical ++ I-chemical +( O +green O +Ca2 B-chemical ++ I-chemical +ion O +, O +orange O +DNA B-chemical +) O +( O +PDB O +entry O +5INQ O +), O +and O +Mg2 B-chemical ++ I-chemical +( O +magenta O +Mg2 B-chemical ++ I-chemical +ion O +, O +yellow O +DNA B-chemical +) O +( O +PDB O +entry O +4GZ1 O +) O +mTdp2cat B-complex_assembly +– I-complex_assembly +DNA I-complex_assembly +structures B-evidence +shows O +that O +Ca2 B-chemical ++ I-chemical +distorts O +the O +5 B-site +′- I-site +phosphate I-site +binding I-site +mode I-site +. O + +Our O +proteolysis B-experimental_method +results O +indicate O +a O +Mg2 B-chemical ++- I-chemical +dependent O +Tdp2 B-protein +conformational O +response O +to O +metal O +binding O +. O + +The O +Tdp2 B-protein +active B-site +site I-site +has O +three O +tryptophan B-residue_name +residues O +within O +10 O +Å O +of O +the O +metal B-site +binding I-site +center I-site +, O +so O +we O +assayed O +intrinsic B-evidence +tryptophan I-evidence +fluorescence I-evidence +to O +detect O +metal O +- O +induced O +conformational O +changes O +in O +mTdp2cat B-structure_element +. O + +These O +data O +were O +an O +excellent O +fit O +to O +a O +single O +- O +site O +binding O +model O +both O +in O +the O +presence B-protein_state +and O +absence B-protein_state +of I-protein_state +DNA B-chemical +( O +Figure O +4A O +). O + +This O +analysis O +revealed O +Mg2 B-chemical ++ I-chemical +Kd B-evidence +values O +in O +the O +sub O +- O +millimolar O +range O +and O +Hill B-evidence +coefficients I-evidence +which O +were O +consistent O +with O +a O +single O +metal B-site +binding I-site +site I-site +both O +in O +the O +presence B-protein_state +and O +absence B-protein_state +of I-protein_state +DNA B-chemical +( O +Supplementary O +Table O +S2 O +). O + +We O +then O +measured O +effects O +of O +metal O +ion O +concentrations O +on O +Tdp2 B-protein +cleavage O +of O +p B-chemical +- I-chemical +nitrophenyl I-chemical +- I-chemical +thymidine I-chemical +- I-chemical +5 I-chemical +′- I-chemical +phosphate I-chemical +by O +mTdp2cat B-structure_element +. O + +This O +small O +molecule O +substrate O +is O +not O +expected O +to O +be O +influenced O +by O +metal O +– O +DNA B-chemical +coordination O +outside O +of O +the O +active B-site +site I-site +. O + +Inclusion O +of O +ultrapure O +Ca2 B-chemical ++ I-chemical +( O +1 O +mM O +or O +10 O +mM O +) O +results O +in O +a O +dose O +- O +dependent O +inhibition O +but O +not O +stimulation O +Tdp2 B-protein +activity O +, O +even O +in O +conditions O +of O +limiting O +Mg2 B-chemical ++ I-chemical +( O +Figure O +4B O +). O + +We O +performed O +the O +same O +titrations B-experimental_method +with O +human B-species +hTdp2FL B-protein +and O +hTdp2cat B-structure_element +( O +Supplementary O +Figure O +S4 O +), O +and O +find O +similar O +stimulation O +of O +activity O +by O +Mg2 B-chemical ++ I-chemical +and O +inhibition O +by O +Ca2 B-chemical ++. I-chemical + +Overall O +, O +these O +metal B-experimental_method +binding I-experimental_method +analyses I-experimental_method +are O +consistent O +with O +a O +single O +metal O +ion O +mediated O +reaction O +. O + +To O +further O +evaluate O +the O +structural O +influence O +of O +divalent O +cations O +on O +the O +Tdp2 B-protein +active B-site +site I-site +, O +we O +determined O +crystal B-evidence +structures I-evidence +by O +soaking B-experimental_method +crystals I-experimental_method +with O +metal O +cofactors O +that O +either O +support B-protein_state +( O +Mn2 B-chemical ++) I-chemical +or O +inhibit B-protein_state +( O +Ca2 B-chemical ++, I-chemical +Figure O +4B O +) O +the O +Tdp2 B-protein +reaction O +( O +PDB O +entries O +5INP O +and O +5INQ O +). O + +Anomalous B-evidence +difference I-evidence +Fourier I-evidence +maps I-evidence +of O +the O +Tdp2 B-complex_assembly +– I-complex_assembly +DNA I-complex_assembly +– I-complex_assembly +Mn2 I-complex_assembly ++ I-complex_assembly +complex O +show O +a O +single O +binding B-site +site I-site +for O +Mn2 B-chemical ++ I-chemical +in O +each O +Tdp2 B-protein +active B-site +site I-site +( O +Figure O +4C O +), O +with O +octahedral B-bond_interaction +coordination I-bond_interaction +and O +bond O +lengths O +typical O +for O +Mn2 B-chemical ++ I-chemical +ligands O +( O +Supplementary O +Table O +S3 O +). O + +The O +Mn2 B-chemical ++ I-chemical +ion O +is O +positioned O +in O +the O +Tdp2 B-protein +active B-site +site I-site +similar O +to O +the O +Mg2 B-protein_state ++- I-protein_state +bound I-protein_state +complex O +( O +Figure O +2C O +), O +which O +is O +consistent O +with O +the O +ability O +of O +Mn2 B-chemical ++ I-chemical +to O +support O +robust O +Tdp2 B-protein +catalytic O +activity O +. O + +In O +contrast O +, O +while O +co B-evidence +- I-evidence +complex I-evidence +structures I-evidence +with O +Ca2 B-chemical ++ I-chemical +also O +show O +a O +single O +metal O +ion O +, O +Ca2 B-chemical ++ I-chemical +binds O +in O +a O +slightly O +different O +position O +, O +shifted O +∼ O +1 O +Å O +from O +the O +Mg2 B-site ++ I-site +site I-site +. O + +Although O +Ca2 B-chemical ++ I-chemical +is O +also O +octahedrally B-bond_interaction +coordinated I-bond_interaction +, O +longer O +bond O +lengths O +for O +the O +Ca2 B-chemical ++ I-chemical +ligands O +( O +Supplementary O +Table O +S3 O +) O +shift O +the O +Ca2 B-chemical ++ I-chemical +ion O +relative O +to O +the O +Mg2 B-site ++ I-site +ion I-site +site I-site +. O + +Interestingly O +, O +bi B-bond_interaction +- I-bond_interaction +dentate I-bond_interaction +inner I-bond_interaction +sphere I-bond_interaction +metal I-bond_interaction +contacts I-bond_interaction +from O +the O +Ca2 B-chemical ++ I-chemical +ion O +to O +Glu162 B-residue_name_number +distort O +the O +active B-site +site I-site +phosphate I-site +- I-site +binding I-site +mode I-site +, O +and O +dislodge O +the O +5 B-chemical +′- I-chemical +PO4 I-chemical +out O +of O +the O +Tdp2 B-protein +active B-site +site I-site +( O +Figure O +4D O +). O + +Together O +with O +results O +showing O +that O +under O +the O +conditions O +examined O +here O +, O +Ca2 B-chemical ++ I-chemical +inhibits O +rather O +than O +stimulates O +the O +Tdp2 B-protein +reaction O +, O +the O +divalent B-protein_state +metal I-protein_state +bound I-protein_state +Tdp2 B-protein +structures B-evidence +provide O +a O +mechanism O +for O +Ca2 B-chemical ++- I-chemical +mediated O +inhibition O +of O +the O +Tdp2 B-protein +reaction O +. O + +Modeling O +the O +Tdp2 B-protein +reaction O +coordinate O + +Next O +, O +to O +examine O +the O +feasibility O +of O +our O +proposed O +single O +Mg2 B-chemical ++ I-chemical +mechanism O +, O +we O +simulated B-experimental_method +the O +Tdp2 B-protein +reaction O +coordinate O +with O +hybrid B-experimental_method +QM I-experimental_method +/ I-experimental_method +MM I-experimental_method +modeling I-experimental_method +using O +Tdp2 B-protein +substrate B-protein_state +analog I-protein_state +- I-protein_state +and O +product B-protein_state +- I-protein_state +bound I-protein_state +structures B-evidence +as O +guides O +. O + +Previous O +structural B-experimental_method +analyses I-experimental_method +showed O +that O +the O +superposition B-experimental_method +of O +a O +DNA B-chemical +substrate O +mimic O +( O +5 B-chemical +′- I-chemical +aminohexanol I-chemical +) O +and O +product O +( O +5 B-chemical +′- I-chemical +PO4 I-chemical +) O +complexes O +delineates O +a O +probable O +Tdp2 B-protein +reaction O +trajectory O +characterized O +by O +inversion O +of O +stereochemistry O +about O +the O +adducted O +5 O +′- O +phosphorus O +. O + +In O +this O +scheme O +( O +Figure O +5A O +), O +a O +candidate O +nucleophilic O +water B-chemical +that O +is O +strongly O +hydrogen B-bond_interaction +bonded I-bond_interaction +to O +Asp272 B-residue_name_number +and O +Asn274 B-residue_name_number +, O +is O +well O +positioned O +for O +the O +in O +- O +line O +nucleophilic O +attack O +∼ O +180 O +° O +opposite O +of O +the O +P O +– O +O O +bond O +of O +the O +5 O +′- O +Tyr O +adduct O +. O + +Structure O +- O +function O +analysis O +of O +the O +Tdp2 B-protein +reaction O +mechanism O +. O + +( O +A O +) O +Proposed O +mechanism O +for O +hydrolysis O +of O +phosphotyrosine B-residue_name +bond O +by O +Tdp2 B-protein +. O + +Residues O +in O +green O +form O +the O +binding B-site +- I-site +site I-site +for O +the O +5 B-residue_name +′- I-residue_name +tyrosine I-residue_name +( O +red O +) O +and O +phosphate B-chemical +, O +yellow O +bind O +the O +5 O +′ O +nucleotide O +and O +blue O +bind O +nucleotides O +2 O +– O +3 O +. O + +Residue O +numbers O +shown O +are O +for O +the O +mTdp2 B-protein +homolog O +. O +( O +B O +) O +Free B-evidence +energy I-evidence +during O +the O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +simulation I-experimental_method +as O +a O +function O +of O +distance O +between O +the O +nucleophilic O +water B-chemical +and O +5 O +′- O +phosphorus O +atom O +. O + +Reaction O +proceeds O +from O +right O +to O +left O +. O +( O +C O +) O +Models O +for O +the O +mTdp2cat B-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +complex O +during O +the O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +reaction I-experimental_method +path I-experimental_method +simulation I-experimental_method +showing O +the O +substrate O +( O +left O +, O +tan O +), O +transition O +state O +intermediate O +( O +center O +, O +cyan O +) O +and O +product O +( O +right O +, O +pink O +) O +states O +. O + +Residue O +numbers O +shown O +are O +for O +the O +mTdp2 B-protein +homolog O +. O +( O +D O +) O +Electrostatic B-evidence +surface I-evidence +potential I-evidence +calculated O +for O +5 B-residue_name +′- I-residue_name +phosphotyrosine I-residue_name +in O +isolation O +( O +upper O +panel O +) O +and O +in O +the O +presence B-protein_state +of I-protein_state +a O +cation B-bond_interaction +– I-bond_interaction +π I-bond_interaction +interaction I-bond_interaction +with O +the O +guanidinium O +group O +of O +Arg216 B-residue_name_number +( O +lower O +panel O +) O +shows O +electron O +- O +withdrawing O +effect O +of O +this O +interaction O +. O + +Electrostatic B-evidence +potential I-evidence +color O +gradient O +extends O +from O +positive O +( O +red O +) O +through O +neutral O +( O +gray O +), O +to O +negative O +( O +blue O +). O +( O +E O +) O +Bar O +graph O +displaying O +the O +relative O +activity O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +human B-species +MBP B-experimental_method +- O +hTdp2cat B-structure_element +fusion B-experimental_method +proteins I-experimental_method +on O +the O +three O +substrates O +. O + +Release O +of O +PNP B-chemical +from O +PNP B-chemical +phosphate B-chemical +and O +T5PNP B-chemical +was O +detected O +as O +an O +increase O +in O +absorbance O +at O +415 O +nm O +. O + +Reaction B-evidence +rates I-evidence +are O +expressed O +as O +the O +percent O +of O +activity O +relative O +to O +wildtype B-protein_state +MBP B-experimental_method +- O +hTdp2cat B-structure_element +; O +error O +bars O +, O +s O +. O +d O +. O + +Mutants O +of O +hTdp2 B-protein +( O +black O +) O +and O +the O +equivalent O +residue O +in O +mTdp2 B-protein +( O +tan O +) O +are O +indicated O +. O + +We O +examined O +the O +energy O +profile O +of O +the O +nucleophilic O +attack O +of O +the O +water B-chemical +molecule O +by O +using O +the O +distance O +between O +the O +water B-chemical +oxygen O +and O +the O +P O +atom O +on O +the O +phosphate O +moiety O +as O +the O +sole O +reaction O +coordinate O +in O +the O +present O +calculation O +( O +Figure O +5B O +and O +C O +). O + +A O +starting O +model O +was O +generated O +from O +atomic O +coordinates O +of O +the O +mTdp2cat B-structure_element +5 B-chemical +′– I-chemical +aminohexanol I-chemical +substrate O +analog O +structure B-evidence +( O +PDB O +4GZ0 O +) O +with O +a O +tyrosine B-residue_name +replacing O +the O +5 B-chemical +′- I-chemical +aminohexanol I-chemical +then O +adding O +the O +Mg2 B-chemical ++ I-chemical +and O +inner O +- O +sphere O +waters B-chemical +from O +the O +mTdp2 B-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +product O +structure B-evidence +( O +PDB O +, O +4GZ1 O +), O +and O +running O +an O +initial O +round O +of O +molecular B-experimental_method +dynamics I-experimental_method +simulation I-experimental_method +( O +10 O +ns O +) O +to O +allow O +the O +system O +to O +reach O +an O +equilibrium O +. O + +After O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +optimization I-experimental_method +of O +this O +model O +( O +Figure O +5C O +, O +‘ O +i O +- O +substrate O +’), O +the O +O O +– O +P O +distance O +is O +3 O +. O +4 O +Å O +, O +which O +is O +in O +agreement O +with O +the O +range O +of O +distances O +observed O +in O +the O +mTdp2cat B-structure_element +5 B-chemical +′- I-chemical +aminohexanol I-chemical +substrate O +analog O +structure B-evidence +( O +3 O +. O +2 O +– O +3 O +. O +4 O +Å O +). O + +Here O +, O +the O +water B-chemical +proton O +and O +the O +neighboring O +O O +of O +Asp272 B-residue_name_number +participates O +in O +a O +strong O +hydrogen B-bond_interaction +bond I-bond_interaction +( O +distance O +of O +1 O +. O +58 O +Å O +) O +and O +the O +phosphotyrosyl B-ptm +O O +– O +P O +distance O +is O +stretched O +to O +1 O +. O +77 O +Å O +, O +which O +is O +0 O +. O +1 O +Å O +beyond O +an O +equilibrium O +bond O +length O +. O + +In O +the O +subsequent O +two O +steps O +of O +the O +simulation B-experimental_method +, O +as O +the O +water B-chemical +- O +phosphate O +O O +– O +P O +distance O +reduces O +to O +1 O +. O +98 O +Å O +, O +a O +key O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +the O +nucleophilic O +water B-chemical +and O +Asp272 B-residue_name_number +shortens O +to O +1 O +. O +38 O +Å O +as O +the O +water B-chemical +H O +– O +O O +bond O +approaches O +the O +point O +of O +dissociation O +. O + +The O +second O +proton O +on O +the O +water B-chemical +nucleophile O +maintains O +a O +strong O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +Asn274 B-residue_name_number +throughout O +the O +reaction O +, O +implicating O +this O +residue O +in O +orienting O +the O +water B-chemical +nucleophile O +during O +the O +reaction O +. O + +Concomitant O +with O +this O +, O +the O +phosphotyrosyl B-ptm +O O +– O +P O +bond O +weakens O +( O +d O += O +1 O +. O +89 O +Å O +), O +and O +the O +formation O +of O +the O +penta O +- O +covalent O +transition O +state O +( O +Figure O +5C O +‘ O +ii O +- O +transition O +state O +’) O +is O +observed O +. O + +The O +final O +steps O +show O +inversion O +of O +stereochemistry O +at O +the O +phosphate B-chemical +, O +along O +with O +lengthening O +and O +breaking O +of O +the O +phosphotyrosyl B-ptm +O O +– O +P O +bond O +. O + +Product O +formation O +is O +coupled O +to O +a O +transfer O +of O +a O +proton O +from O +the O +nucleophillic O +water B-chemical +to O +Asp272 B-residue_name_number +, O +consistent O +with O +the O +proposed O +function O +for O +this O +residue O +as O +the O +catalytic O +base O +. O + +Of O +note O +, O +both O +nitrogens O +of O +the O +imidazole O +side O +chain O +of O +His B-residue_name_number +359 I-residue_name_number +require O +protonation O +for O +stability O +of O +the O +simulation B-experimental_method +. O + +Asp B-residue_name_number +326 I-residue_name_number +makes O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +to O +N O +∂ O +1 O +of O +His359 B-residue_name_number +, O +suggesting O +that O +this O +salt B-bond_interaction +bridge I-bond_interaction +could O +stabilize O +the O +protonated B-protein_state +form O +of O +His359 B-residue_name_number +as O +has O +been O +demonstrated O +for O +the O +analogous O +Asp B-residue_name +- O +His B-residue_name +pair O +in O +the O +EEP B-structure_element +domain O +of O +APE1 B-protein +, O +which O +elevates O +the O +pKa B-evidence +of O +this O +His B-residue_name +above O +8 O +. O +0 O +. O +In O +our O +model O +, O +the O +transition O +state O +contains O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +the O +doubly B-protein_state +protonated I-protein_state +His359 B-residue_name_number +and O +the O +phosphate B-chemical +oxygen O +that O +also O +coordinates O +with O +the O +single O +catalytic O +Mg2 B-chemical ++, I-chemical +while O +the O +second O +His359 B-residue_name_number +imidazole O +proton O +maintains O +a O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +with O +the O +Asp326 B-residue_name_number +residue O +throughout O +the O +reaction O +. O + +In O +the O +final O +optimized O +structure B-evidence +, O +the O +observed O +product O +state O +( O +Figure O +5C O +, O +‘ O +iii O +- O +product O +’) O +is O +found O +in O +a O +conformation O +that O +is O +7 O +. O +4 O +kcal O +mol O +− O +1 O +more O +stable O +than O +the O +initial O +reactive O +state O +( O +Figure O +5B O +). O + +The O +tyrosine B-residue_name +oxy O +- O +anion O +product O +is O +coordinated B-bond_interaction +to I-bond_interaction +the O +Mg2 B-chemical ++ I-chemical +ion O +with O +a O +2 O +. O +0 O +Å O +distance O +, O +which O +is O +the O +shortest O +of O +the O +six O +Mg2 B-chemical ++ I-chemical +ligands O +( O +including O +three O +water B-chemical +molecules O +, O +one O +of O +the O +free O +oxygens O +on O +the O +phosphate B-chemical +group O +and O +the O +Glu162 B-residue_name_number +residue O +), O +indicating O +the O +single O +Mg2 B-chemical ++ I-chemical +greatly O +stabilizes O +the O +product O +oxy O +- O +anion O +. O + +An O +additional O +striking O +feature O +gleaned O +from O +the O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +modeling I-experimental_method +is O +the O +putative O +binding O +mode O +of O +the O +Top2 B-protein_type +tyrosine B-residue_name +- O +leaving O +group O +. O + +A O +trio O +of O +conserved B-protein_state +residues O +( O +Tyr B-residue_name_number +188 I-residue_name_number +, O +Arg B-residue_name_number +216 I-residue_name_number +and O +Ser B-residue_name_number +239 I-residue_name_number +) O +forms O +the O +walls O +of O +a O +conserved B-protein_state +Top2 B-protein_type +tyrosine B-site +binding I-site +pocket I-site +. O + +We O +propose O +this O +cation B-bond_interaction +– I-bond_interaction +π I-bond_interaction +interaction I-bond_interaction +further O +contributes O +to O +tuned O +stabilization O +of O +the O +negatively O +charged O +phenolate O +reaction O +product O +. O + +Consistent O +with O +this O +, O +analysis O +of O +electrostatic B-evidence +potential I-evidence +of O +the O +phosphotyrosyl B-ptm +moiety O +using O +Gaussian O +09 O +. O +D01 O +in O +the O +presence B-protein_state +and O +absence B-protein_state +of I-protein_state +the O +Arg216 B-residue_name_number +guanidinium O +reveals O +Arg216 B-residue_name_number +is O +strongly O +electron O +withdrawing O +( O +Figure O +5D O +). O + +We O +further O +examined O +the O +contribution O +of O +this O +cation B-bond_interaction +– I-bond_interaction +π I-bond_interaction +interaction I-bond_interaction +to O +the O +reaction O +chemistry O +by O +moving O +the O +guanidinium O +group O +of O +Arg216 B-residue_name_number +from O +the O +QM B-experimental_method +system O +to O +the O +MM B-experimental_method +system O +as O +either O +a O ++ O +1 O +or O +∼ O +0 O +charge O +species O +, O +and O +re O +- O +computed O +energy B-evidence +penalties I-evidence +for O +each O +step O +in O +the O +reaction O +coordinate O +( O +Supplementary O +Figure O +S5A O +). O + +Removing O +Arg216 B-residue_name_number +from O +the O +quantum O +subsystem O +incurs O +an O +∼ O +2 O +kcal O +mol O +− O +1 O +penalty O +in O +the O +transition O +state O +and O +product O +complex O +. O + +Removing O +the O ++ O +1 O +charge O +on O +the O +Arg216 B-residue_name_number +has O +a O +minimal O +impact O +on O +the O +transition O +state O +, O +but O +incurs O +an O +additional O +∼ O +2 O +kcal O +mol O +− O +1 O +penalty O +in O +the O +product O +complex O +. O + +Altogether O +, O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +modeling I-experimental_method +identifies O +new O +determinants O +of O +the O +Tdp2 B-protein +reaction O +, O +and O +demonstrates O +our O +proposed O +single O +Mg2 B-chemical ++ I-chemical +catalyzed O +reaction O +model O +is O +a O +viable O +mechanism O +for O +Tdp2 B-protein +- O +catalyzed O +5 B-residue_name +′- I-residue_name +phosphotyrosine I-residue_name +bond O +hydrolysis O +. O + +Tdp2 B-protein +mutational B-experimental_method +analysis I-experimental_method + +To O +test O +the O +aspects O +of O +the O +Tdp2 B-protein +reaction O +mechanism O +described O +here O +derived O +from O +high O +- O +resolution O +mouse B-taxonomy_domain +Tdp2 B-protein +crystal B-evidence +structures I-evidence +( O +denoted O +with O +superscript O +numbering O +‘ O +m O +’ O +for O +numbering O +of O +the O +mouse B-taxonomy_domain +protein O +), O +we O +engineered B-experimental_method +and I-experimental_method +purified I-experimental_method +thirteen O +human B-species +MBP B-experimental_method +- O +hTdp2cat B-structure_element +mutant B-protein_state +proteins O +( O +denoted O +with O +superscript O +numbering O +and O +‘ O +h O +’ O +for O +the O +human B-species +protein O +) O +and O +assayed O +the O +impacts O +of O +mutations B-experimental_method +on O +Tdp2 B-protein +catalytic O +activity O +using O +three O +in O +vitro O +reporter O +substrates O +including O +a O +tyrosylated B-protein_state +DNA B-chemical +substrate O +( O +5 B-ptm +′- I-ptm +Y I-ptm +), O +p B-chemical +- I-chemical +nitrophenyl I-chemical +phosphate I-chemical +( O +PNPP B-chemical +) O +and O +thymidine B-chemical +5 I-chemical +′- I-chemical +monophosphate I-chemical +p I-chemical +- I-chemical +nitrophenyl I-chemical +ester I-chemical +( O +T5PNP B-chemical +) O +( O +Figure O +5E O +, O +Supplementary O +Figures O +S5B O +and O +S5C O +). O + +By O +analyzing O +activities O +on O +this O +nested O +set O +of O +chemically O +related O +substrates O +we O +aimed O +to O +dissect O +structure O +- O +activity O +relationships O +of O +Tdp2 B-protein +catalysis O +. O + +For O +example O +, O +mutations B-experimental_method +impacting O +Tdp2 B-protein +active B-site +site I-site +chemistry O +and O +phosphotyrosyl B-ptm +bond O +cleavage O +should O +similarly O +affect O +catalysis O +on O +all O +three O +substrates O +, O +but O +mutants O +impacting O +DNA B-chemical +damage O +binding O +might O +only O +impair O +catalysis O +on O +5 B-ptm +′- I-ptm +Y I-ptm +and O +T5PNP B-chemical +but O +not O +PNPP B-chemical +that O +lacks O +a O +nucleobase O +. O + +Structural B-evidence +results I-evidence +and O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +modeling I-experimental_method +indicate O +mAsp272 B-residue_name_number +activates O +a O +water B-chemical +molecule O +for O +in O +- O +line O +nucleophilic O +attack O +of O +the O +scissile O +phosphotyrosyl B-ptm +linkage I-ptm +. O + +To O +test O +if O +this O +proposed O +Lewis O +base O +is O +critical O +for O +reaction O +chemistry O +we O +mutated B-experimental_method +it O +to B-experimental_method +a O +His B-residue_name +, O +which O +could O +alternatively O +support O +metal O +binding O +, O +as O +well O +as O +bulky O +hydrophobic O +residues O +( O +Leu B-residue_name +and O +Met B-residue_name +) O +that O +we O +predict O +would O +block O +the O +water B-site +- I-site +binding I-site +site I-site +. O + +Similar O +to O +a O +previously O +characterized O +hD262N B-mutant +mutation O +, O +all O +three O +substitutions B-experimental_method +ablate O +activity O +, O +supporting O +essential O +roles O +for O +hAsp262 B-residue_name_number +( O +mAsp272 B-residue_name_number +) O +in O +catalysis O +. O + +Next O +, O +we O +mutated B-experimental_method +key O +elements O +of O +the O +mobile O +loop O +( O +β2Hβ B-site +hydrophobic I-site +wall I-site +, O +Figure O +2A O +and O +C O +). O + +Mutations B-experimental_method +hI307A B-mutant +, O +hL305A B-mutant +, O +hL305F B-mutant +and O +hL305W B-mutant +all O +impaired O +catalysis O +on O +both O +nucleotide O +- O +containing O +substrates O +(< O +50 O +% O +activity O +). O + +The O +hL305W B-mutant +substitution O +that O +we O +expect O +to O +have O +the O +most O +distorting O +impact O +on O +conformation O +of O +the O +β2Hβ B-site +hydrophobic I-site +wall I-site +also O +has O +the O +largest O +impact O +on O +catalysis O +of O +the O +DNA B-chemical +substrate O +5 B-ptm +′- I-ptm +Y I-ptm +. O +By O +comparison O +, O +as O +predicted O +by O +our O +model O +where O +β2Hβ B-structure_element +dictates O +key O +interactions O +with O +undamaged O +and O +damaged O +nucleobases O +, O +all O +of O +these O +substitutions B-experimental_method +have O +little O +impact O +on O +PNPP B-chemical +(> O +90 O +% O +activity O +). O + +Third O +, O +we O +altered O +properties O +of O +the O +proposed O +enzyme B-site +substrate I-site +cation I-site +– I-site +π I-site +interface I-site +. O + +No O +activity O +was O +detected O +for O +a O +mutant B-protein_state +that O +removes O +the O +positive O +charge O +at O +this O +position O +( O +hR206A B-mutant +). O + +The O +precise O +geometry O +of O +this O +pocket B-site +is O +also O +critical O +for O +catalysis O +as O +replacement B-experimental_method +of O +hArg206 B-residue_name_number +( O +mArg216 B-residue_name_number +) O +with O +a O +lysine B-residue_name +also O +results O +in O +a O +profound O +decrease O +in O +catalysis O +(< O +5 O +% O +activity O +on O +5 B-ptm +′- I-ptm +Y I-ptm +, O +no O +detectable O +activity O +on O +T5PNP B-chemical +or O +PNPP B-chemical +). O + +Similarly O +, O +mutation B-experimental_method +of O +hTyr178 B-residue_name_number +that O +structurally O +scaffolds O +the O +hArg206 B-residue_name_number +( O +mArg216 B-residue_name_number +) O +guanidinium O +also O +significantly O +impacts O +activity O +, O +with O +Y178F B-mutant +and O +Y178W B-mutant +having O +< O +25 O +% O +activity O +on O +all O +substrates O +. O + +Fourth O +, O +we O +evaluated O +roles O +for O +the O +hHis351 B-residue_name_number +– O +hAsp316 B-residue_name_number +( O +mAsp326 B-residue_name_number +– O +mHis359 B-residue_name_number +) O +transition O +state O +stabilization O +charge O +pair O +. O + +We O +found O +that O +mutations B-experimental_method +that O +removed B-experimental_method +the O +charge O +yet O +retained O +the O +ability O +to O +hydrogen B-bond_interaction +bond I-bond_interaction +( O +hH351Q B-mutant +) O +or O +should O +abrogate O +the O +elevated O +pKa B-evidence +of O +the O +Histidine B-residue_name +( O +hD316N B-mutant +) O +had O +severe O +impacts O +on O +catalysis O +. O + +Thus O +altogether O +, O +our O +mutational O +data O +support O +key O +roles O +for O +the O +active B-site +site I-site +Lewis O +base O +aspartate B-residue_name +, O +mobile B-protein_state +substrate B-structure_element +engagement I-structure_element +loops I-structure_element +, O +enzyme O +– O +substrate O +cation B-bond_interaction +– I-bond_interaction +π I-bond_interaction +interactions I-bond_interaction +, O +and O +active B-site +site I-site +transition O +state O +stabilizing O +charge B-bond_interaction +interaction I-bond_interaction +in O +supporting O +Tdp2 B-protein +catalysis O +. O + +A O +Tdp2 B-protein +active B-site +site I-site +single O +nucleotide O +polymorphism O +impairs O +Tdp2 B-protein +function O + +Recently O +, O +it O +was O +found O +that O +inactivation O +of O +TDP2 B-protein +by O +a O +splice O +- O +site O +mutation O +is O +associated O +with O +neurological O +disease O +and O +confers O +hypersensitivity O +to O +Top2 B-protein_type +poisons O +. O + +We O +considered O +whether O +human B-species +SNPs O +causing O +missense O +mutations O +might O +also O +impact O +Tdp2 B-protein +DNA B-chemical +– O +protein O +crosslink O +repair O +functions O +established O +here O +as O +well O +as O +Tdp2 B-protein +- O +mediated O +NHEJ O +of O +blocked O +DNA B-chemical +termini O +. O + +We O +identified O +two O +SNPs O +in O +human B-species +TDP2 B-protein +curated O +in O +the O +NCBI O +SNP O +database O +that O +result O +in O +missense O +mutations O +within O +the O +DNA B-site +processing I-site +active I-site +site I-site +: O +rs199602263 B-gene +( O +minor O +allele O +frequency O +0 O +. O +0002 O +), O +which O +substitutes O +hAsp350 B-residue_name_number +for O +Asn B-residue_name +, O +and O +rs77273535 B-gene +( O +minor O +allele O +frequency O +0 O +. O +004 O +, O +which O +substitutes O +hIle307 B-residue_name_number +for O +Val B-residue_name +) O +( O +Figure O +6A O +). O + +We O +show O +the O +hD350N B-mutant +substitution B-experimental_method +severely O +impairs O +activity O +on O +all O +substrates O +tested O +in O +vitro O +, O +whereas O +hI307V B-mutant +only O +has O +a O +mild O +impact O +on O +catalysis O +( O +Figure O +6B O +– O +D O +). O + +To O +better O +understand O +the O +basis O +for O +the O +D350N B-mutant +catalytic O +defect O +, O +we O +analyzed O +the O +structural O +environment O +of O +this O +substitution O +based O +on O +the O +high O +- O +resolution O +structures B-evidence +of O +mTdp2cat B-structure_element +( O +Figure O +6A O +). O + +Interestingly O +, O +the O +Tdp2 B-protein +single O +Mg2 B-chemical ++ I-chemical +ion O +octahedral B-bond_interaction +coordination I-bond_interaction +shell I-bond_interaction +also O +involves O +an O +extended O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +network I-bond_interaction +mediated O +by O +hAsp350 B-residue_name_number +( O +mAsp358 B-residue_name_number +) O +that O +stabilizes O +the O +DNA B-protein_state +- I-protein_state +bound I-protein_state +conformation O +of O +the O +β2Hβ B-structure_element +substrate I-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +through O +hydrogen B-bond_interaction +bonding I-bond_interaction +to O +mTrp307 B-residue_name_number +. O + +Here O +, O +hAsp350 B-residue_name_number +( O +mAsp358 B-residue_name_number +) O +serves O +as O +a O +structural O +nexus O +linking O +active B-site +site I-site +metal O +binding O +to O +substrate B-structure_element +binding I-structure_element +loop I-structure_element +conformations O +. O + +Tdp2 B-protein +SNPs O +impair O +function O +. O +( O +A O +) O +Active B-site +site I-site +residues O +mutated O +by O +TDP2 B-protein +SNPs O +. O + +D350N B-mutant +( O +mTdp2 B-protein +D358N B-mutant +) O +and O +I307V B-mutant +( O +mTdp2 B-protein +I317V B-mutant +) O +substitutions B-experimental_method +are O +mapped O +onto O +the O +Tdp2 B-protein +active B-site +site I-site +of O +the O +high O +- O +resolution O +mTdp2cat B-structure_element +structure B-evidence +( O +4GZ1 O +). O + +( O +B O +) O +Coomassie O +blue O +stained O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +gel O +of O +purified O +WT B-protein_state +and O +mutant B-protein_state +MBP B-experimental_method +- O +hTdp2cat B-structure_element +proteins O +used O +for O +assays O +in O +panels O +C O +and O +D O +. O +( O +C O +) O +Activity O +of O +WT B-protein_state +and O +mutant B-protein_state +MBP B-experimental_method +- O +hTdp2cat B-structure_element +proteins O +on O +a O +5 B-chemical +′– I-chemical +phosphotyrosyl I-chemical +– I-chemical +DNA I-chemical +oligonucleotides I-chemical +with O +3 O +′- O +fluorescein B-chemical +label O +. O + +Samples O +were O +withdrawn O +from O +reactions O +, O +neutralized O +with O +TBE O +- O +urea O +loading O +dye O +at O +the O +indicated O +timepoints O +, O +and O +electrophoresed O +on O +a O +20 O +% O +TBE B-experimental_method +- I-experimental_method +urea I-experimental_method +PAGE I-experimental_method +. O + +( O +D O +) O +Relative O +activity O +of O +WT B-protein_state +and O +indicated O +mutant B-protein_state +human B-species +MBP B-experimental_method +- O +hTdp2cat B-structure_element +fusion O +proteins O +on O +three O +model O +Tdp2 B-protein +substrates O +. O + +Quantification O +of O +percent O +MBP B-experimental_method +- O +hTdp2cat B-structure_element +activity O +relative O +to O +WT B-protein_state +protein O +for O +the O +5 B-chemical +′- I-chemical +Y I-chemical +DNA I-chemical +oligonucleotide I-chemical +substrate O +( O +blue O +bars O +), O +T5PNP B-chemical +( O +red O +bars O +) O +and O +PNPP B-chemical +( O +green O +bars O +) O +is O +displayed O +. O + +Release O +of O +PNP B-chemical +from O +PNP B-chemical +phosphate I-chemical +( O +PNPP B-chemical +) O +and O +was O +detected O +as O +an O +increase O +in O +absorbance O +at O +415 O +nm O +, O +whereas O +the O +5 B-ptm +′- I-ptm +Y I-ptm +substrate O +is O +quantification O +of O +activity O +in O +a O +gel B-experimental_method +based I-experimental_method +assay I-experimental_method +shown O +in O +Figure O +6C O +. O + +To O +define O +the O +molecular O +basis O +for O +the O +hD350N B-mutant +( O +mD358N B-mutant +) O +defect O +, O +we O +crystallized B-experimental_method +and I-experimental_method +determined I-experimental_method +the O +structure B-evidence +of O +the O +DNA B-protein_state +- I-protein_state +free I-protein_state +form O +of O +the O +mD358N B-mutant +protein O +to O +2 O +. O +8Å O +resolution O +( O +PDB O +entry O +5INN O +). O + +This O +structure B-evidence +shows O +the O +D358N B-mutant +mutation B-experimental_method +disrupts O +the O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +Asp358 B-residue_name_number +and O +Trp307 B-residue_name_number +, O +shifts O +the O +position O +of O +Asn358 B-residue_name_number +and O +destabilizes O +Trp307 B-residue_name_number +. O + +Consequently O +, O +poor O +electron B-evidence +density I-evidence +is O +visible O +for O +the O +β2Hβ B-structure_element +loop I-structure_element +which O +is O +mostly O +disordered B-protein_state +( O +Supplementary O +Figure O +S6 O +). O + +Although O +Mg2 B-chemical ++ I-chemical +is O +present O +at O +the O +same O +concentration O +as O +the O +WT B-protein_state +- O +mTdpcat B-protein +crystals B-evidence +( O +10 O +mM O +), O +we O +find O +the O +metal B-site +site I-site +is O +unoccupied B-protein_state +in O +the O +mD358N B-mutant +crystals B-evidence +. O + +Therefore O +, O +metal O +- O +regulated O +opening O +/ O +closure O +of O +the O +active B-site +site I-site +may O +modulate O +Tdp2 B-protein +activity O +, O +and O +D350N B-mutant +is O +sufficient O +to O +block O +both O +metal O +binding O +and O +conformational O +change O +. O + +In O +support O +of O +this O +, O +we O +also O +find O +that O +hD350N B-mutant +( O +mD358N B-mutant +) O +impairs O +Mg2 B-chemical ++ I-chemical +binding O +as O +measured O +by O +intrinsic B-evidence +tryptophan I-evidence +fluorescence I-evidence +( O +Figure O +4A O +), O +and O +abrogates O +Mg2 B-chemical ++- I-chemical +stimulated O +active B-site +site I-site +conformational O +changes O +detected O +by O +trypsin O +and O +chymotrypsin O +sensitivity O +of O +the O +Tdp2 B-protein +metamorphic O +loop B-structure_element +( O +Figure O +3D O +). O + +Tdp2 B-protein +facilitates O +NHEJ O +repair O +of O +5 B-residue_name +′- I-residue_name +phosphotyrosine I-residue_name +adducted O +DSBs O + +Overall O +, O +our O +Tdp2 B-protein +structure B-experimental_method +/ I-experimental_method +activity I-experimental_method +studies I-experimental_method +reveal O +a O +tuned O +, O +5 B-ptm +′- I-ptm +detyrosylation I-ptm +DNA B-chemical +end O +processing O +activity O +and O +it O +has O +been O +demonstrated O +that O +Tdp2 B-protein +could O +enable O +repair O +of O +Top2 B-protein_type +damage O +by O +the O +non O +- O +homologous O +end O +- O +joining O +( O +NHEJ O +) O +pathway O +. O + +Accordingly O +, O +we O +demonstrate O +here O +that O +5 B-protein_state +′- I-protein_state +tyrosylated I-protein_state +ends O +are O +sufficient O +to O +severely O +impair O +an O +in O +vitro O +reconstituted O +mammalian B-taxonomy_domain +NHEJ O +reaction O +( O +Figure O +7A O +, O +lanes O +3 O +and O +6 O +), O +unless O +supplemented O +with O +catalytic O +quantities O +of O +hTdp2FL B-protein +( O +Figure O +7A O +, O +lane O +8 O +). O + +Interestingly O +, O +hTdp2cat B-structure_element +is O +slightly O +more O +effective O +than O +hTdp2FL B-protein +in O +promoting O +NHEJ O +of O +adducted O +ends O +, O +while O +a O +catalytically B-protein_state +deficient I-protein_state +E152Q B-mutant +mutant B-protein_state +was O +inactive B-protein_state +in O +this O +assay O +, O +supporting O +the O +notion O +that O +Tdp2 B-protein +catalytic O +activity O +is O +required O +to O +support O +NHEJ O +of O +phosphotyrosyl B-ptm +blocked O +DSBs O +( O +Supplementary O +Figure O +S7A O +). O + +We O +confirmed O +that O +efficient O +joining O +of O +the O +same O +tyrosine B-residue_name +- O +adducted O +substrate O +in O +cells O +( O +Figure O +7B O +) O +was O +dependent O +on O +both O +NHEJ O +( O +reduced O +over O +10 O +- O +fold O +in O +ligase O +IV O +deficient O +HCT O +116 O +cells O +; O +Supplementary O +Figure O +S7B O +), O +and O +Tdp2 B-protein +( O +reduced O +5 O +- O +fold O +in O +Tdp2 B-protein +deficient O +MEFs O +; O +Figure O +7C O +). O + +Moreover O +, O +products O +with O +error O +( O +i O +. O +e O +. O +junctions O +have O +missing O +sequence O +flanking O +the O +adducted O +terminus O +) O +are O +twice O +as O +frequent O +in O +cells O +deficient O +in O +Tdp2 B-protein +( O +Figure O +7D O +). O + +Therefore O +, O +in O +accord O +with O +previous O +work O +, O +joining O +of O +tyrosine B-residue_name +adducted O +ends O +after O +Tdp2 B-protein +- O +mediated O +detyrosylation B-ptm +is O +both O +more O +efficient O +and O +more O +accurate O +than O +joining O +after O +endonucleolytic O +excision O +( O +e O +. O +g O +. O +mediated O +by O +Artemis B-protein +or O +the O +Mre11 B-complex_assembly +/ I-complex_assembly +Rad50 I-complex_assembly +/ I-complex_assembly +Nbs1 I-complex_assembly +complex O +). O + +Effects O +of O +Tdp2 B-protein +active B-site +site I-site +SNP O +- O +encoded O +mutants O +on O +cellular O +Tdp2 B-protein +functions O +. O +( O +A O +) O +Cy5 B-chemical +labeled O +substrates O +with O +5 B-chemical +′- I-chemical +phosphate I-chemical +termini O +( O +Lanes O +1 O +– O +4 O +) O +or O +5 B-protein_state +′- I-protein_state +tyrosylated I-protein_state +termini O +( O +Lanes O +5 O +– O +9 O +) O +were O +incubated O +with O +Ku B-protein +, O +the O +NHEJ B-protein_type +ligase I-protein_type +( O +XRCC4 B-protein +, O +ligase B-protein +IV I-protein +and O +XLF B-protein +; O +X O +- O +L O +- O +X O +) O +and O +1 O +nM O +hTdp2FL B-protein +as O +indicated O +(+) O +for O +5 O +min O +at O +37 O +° O +C O +. O + +Concatemer O +ligation O +products O +were O +detected O +by O +5 O +% O +native B-experimental_method +PAGE I-experimental_method +. O + +( O +B O +) O +Workflow O +diagram O +of O +cellular B-experimental_method +end I-experimental_method +joining I-experimental_method +assays I-experimental_method +. O + +DNA B-chemical +substrates O +with O +5 B-residue_name +′- I-residue_name +phosphotyrosine I-residue_name +adducts O +and O +4 O +nucleotide O +5 O +′ O +overhangs O +were O +electroporated O +into O +cultured O +mammalian B-taxonomy_domain +cells O +. O + +After O +1 O +h O +, O +DNA B-chemical +was O +recovered O +from O +cells O +and O +repair O +efficiency O +by O +qPCR B-experimental_method +or O +sequencing B-experimental_method +as O +indicated O +. O +( O +C O +) O +qPCR B-experimental_method +assessment O +of O +cellular O +end O +joining O +efficiency O +of O +the O +tyrosylated B-protein_state +substrate O +comparing O +results O +from O +wildtype B-protein_state +MEF O +cells O +to O +Tdp2 B-protein +−/− O +cells O +and O +Tdp2 B-protein +−/− O +cells O +complemented O +with O +wildtype B-protein_state +or O +the O +noted O +hTDP2FL B-protein +variants O +; O +Joining O +efficiency O +shown O +is O +the O +ratio O +of O +junctions O +recovered O +relative O +to O +WT B-protein_state +cells O +. O + +Error O +bars O +, O +s O +. O +d O +, O +n O += O +3 O +. O +( O +D O +) O +Junctions O +recovered O +from O +cellular B-experimental_method +end I-experimental_method +- I-experimental_method +joining I-experimental_method +assays I-experimental_method +in O +the O +noted O +cell O +types O +were O +characterized O +by O +sequencing B-experimental_method +to O +assess O +the O +end B-evidence +- I-evidence +joining I-evidence +error I-evidence +rate I-evidence +. O + +Error O +bars O +, O +s O +. O +d O +, O +n O += O +3 O +. O +( O +E O +) O +Clonogenic B-experimental_method +survival I-experimental_method +assay I-experimental_method +of O +WT B-protein_state +, O +Tdp2 B-protein +knockout O +and O +complemented O +MEF O +cells O +after O +treatment O +with O +indicated O +concentrations O +of O +etoposide B-chemical +for O +3 O +h O +; O +error O +bars O +, O +s O +. O +d O +, O +n O += O +3 O +. O + +We O +next O +compared O +the O +ability O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +hTdp2FL B-protein +variants O +to O +complement O +Tdp2 B-protein +deficient O +mouse B-taxonomy_domain +embryonic O +fibroblasts O +( O +Supplementary O +Figure O +S7C O +). O + +Joining O +of O +extrachromosomal O +DNA B-chemical +with O +phosphotyrosine B-residue_name +blocked O +ends O +, O +both O +in O +terms O +of O +efficiency O +( O +Figure O +7C O +) O +and O +fidelity O +( O +Figure O +7D O +), O +was O +indistinguishable O +comparing O +MEFs O +from O +a O +wild B-protein_state +- I-protein_state +type I-protein_state +mouse B-taxonomy_domain +, O +MEFs O +from O +a O +Tdp2 B-protein +-/- O +mouse B-taxonomy_domain +overexpressing O +wild B-protein_state +- I-protein_state +type I-protein_state +human B-species +Tdp2 B-protein +, O +and O +Tdp2 B-protein +-/- O +MEFs O +overexpressing O +the O +I307V B-mutant +variant B-protein_state +human B-species +Tdp2 B-protein +. O + +In O +contrast O +, O +joining O +of O +5 O +′ O +phosphotyrosine O +- O +blocked O +ends O +was O +reduced O +5 O +- O +fold O +in O +Tdp2 B-protein +-/- O +MEFs O +, O +and O +an O +equivalent O +defect O +was O +observed O +in O +Tdp2 B-protein +-/- O +MEFs O +overexpressing O +Tdp2 B-protein +D350N B-mutant +. O + +Moreover O +, O +the O +frequency O +of O +inaccurate O +repair O +was O +2 O +- O +fold O +higher O +in O +both O +Tdp2 B-protein +deficient O +cells O +and O +Tdp2 B-protein +deficient O +cells O +overexpressing O +D350N B-mutant +, O +relative O +to O +cells O +expressing O +wild B-protein_state +type I-protein_state +Tdp2 B-protein +or O +hTdp2 B-protein +I307V B-mutant +( O +Figure O +7D O +). O + +Expression O +of O +wild B-protein_state +type I-protein_state +or O +I307V B-mutant +human B-species +Tdp2 B-protein +in O +Tdp2 B-protein +-/- O +MEFs O +was O +also O +sufficient O +to O +confer O +levels O +of O +resistance O +to O +etoposide B-chemical +comparable O +to O +the O +matched O +wild B-protein_state +- I-protein_state +type I-protein_state +MEF O +line O +, O +while O +overexpression B-experimental_method +of O +human B-species +D350N B-mutant +Tdp2 B-protein +had O +no O +apparent O +complementation O +activity O +( O +Figure O +7E O +). O + +The O +rare O +D350N B-mutant +variant B-protein_state +is O +thus O +inactive B-protein_state +by O +all O +metrics O +analyzed O +. O + +By O +comparison O +the O +more O +frequent O +I307V B-mutant +has O +only O +mild O +effects O +on O +in O +vitro O +activity O +, O +and O +no O +detectable O +impact O +on O +cellular O +assays O +. O + +Top2 B-protein_type +chemotherapeutic O +agents O +remain O +frontline O +treatments O +, O +and O +exposure O +to O +the O +chemical O +and O +damaged O +DNA B-chemical +triggers O +of O +Top2 B-protein_type +- O +DNA B-chemical +protein O +crosslink O +formation O +are O +unavoidable O +. O + +Understanding O +how O +cells O +cope O +with O +complex O +DNA B-chemical +breaks O +bearing O +topoisomerase O +– O +DNA B-chemical +protein O +crosslinks O +is O +key O +to O +deciphering O +individual O +responses O +to O +chemotherapeutic O +outcomes O +and O +genotoxic O +agents O +that O +poison O +Top2 B-protein_type +. O + +Together O +with O +mutagenesis B-experimental_method +and O +functional B-experimental_method +assays I-experimental_method +, O +our O +new O +Tdp2 B-protein +structures B-evidence +in B-protein_state +the I-protein_state +absence I-protein_state +of I-protein_state +ligands B-chemical +and O +in B-protein_state +complex I-protein_state +with I-protein_state +DNA B-chemical +damage O +reveal O +four O +novel O +facets O +of O +Tdp2 B-protein +DNA B-chemical +- O +protein O +conjugate O +processing O +: O +( O +i O +) O +The O +Tdp2 B-protein +active B-site +site I-site +is O +well O +- O +suited O +for O +accommodating O +a O +variety O +of O +DNA B-chemical +structures O +including O +abasic O +and O +bulky O +alkylated O +DNA B-chemical +lesions O +that O +trigger O +Top2 B-protein_type +poisoning O +, O +( O +ii O +) O +High O +- O +resolution O +structural B-experimental_method +analysis I-experimental_method +coupled O +with O +mutational B-experimental_method +studies I-experimental_method +and O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +molecular I-experimental_method +modeling I-experimental_method +of O +the O +Tdp2 B-protein +reaction O +coordinate O +support O +a O +single O +metal O +- O +ion O +mechanism O +for O +the O +diverse O +clade O +of O +EEP B-structure_element +domain O +catalyzed O +phosphoryl B-protein_type +hydrolase I-protein_type +reactions O +, O +( O +iii O +) O +The O +Tdp2 B-protein +active B-site +site I-site +is O +conformationally B-protein_state +plastic I-protein_state +, O +and O +undergoes O +intricate O +rearrangements O +upon O +DNA B-chemical +and O +Mg2 B-chemical ++ I-chemical +cofactor O +binding O +and O +( O +iv O +) O +Naturally O +occurring O +Tdp2 B-protein +variants O +undermine O +Tdp2 B-protein +active B-site +site I-site +chemistry O +, O +cellular O +and O +biochemical O +activities O +. O + +This O +mechanistic O +dissection O +of O +Tdp2 B-protein +interactions O +with O +damaged O +DNA B-chemical +and O +metal O +cofactor O +provides O +a O +detailed O +molecular O +understanding O +of O +the O +mechanism O +of O +Tdp2 B-protein +DNA B-chemical +protein O +crosslink O +processing O +. O + +Tdp2 B-protein +was O +originally O +identified O +as O +a O +protein O +conferring O +resistance O +to O +both O +Top1 B-protein_type +and O +Top2 B-protein_type +anti O +- O +cancer O +drugs O +, O +however O +it O +is O +hypothesized O +that O +the O +predominant O +natural O +source O +of O +substrates O +for O +Tdp2 B-protein +are O +likely O +the O +potent O +DNA B-chemical +damage O +triggers O +of O +Top2 B-protein_type +poisoning O +and O +Top2 B-protein_type +DNA B-chemical +protein O +crosslinks O +encountered O +during O +transcription O +. O + +The O +properties O +of O +complex O +DNA B-chemical +strand O +breaks O +bearing O +Top2 B-protein_type +- O +DNA B-chemical +protein O +crosslinks O +necessitate O +that O +Tdp2 B-protein +accommodates O +both O +damaged O +nucleic O +acid O +as O +well O +as O +the O +topoisomerase B-protein_type +protein O +in O +its O +active B-site +site I-site +for O +catalysis O +. O + +The O +Tdp2 B-protein +substrate B-site +interaction I-site +groove I-site +facilitates O +DNA B-chemical +- O +protein O +conjugate O +recognition O +in O +two O +important O +ways O +. O + +First O +, O +the O +nucleic B-site +acid I-site +binding I-site +trench I-site +is O +assembled O +by O +a O +dynamic B-protein_state +β2Hβ B-structure_element +DNA I-structure_element +damage I-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +that O +is O +capable O +of O +recognizing O +and O +processing O +diverse O +phosphotyrosyl B-ptm +linkages I-ptm +even O +in O +the O +context O +of O +bulky O +adducts O +such O +as O +ϵA B-chemical +. O +This O +is O +achieved O +by O +binding O +of O +nucleic O +acid O +‘ O +bases O +out O +’ O +by O +an O +extended O +base B-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +hydrophobic B-site +wall I-site +of O +the O +β2Hβ B-structure_element +- I-structure_element +loop I-structure_element +. O + +Secondly O +, O +our O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +analysis O +further O +highlights O +an O +enzyme O +– O +substrate O +cation B-bond_interaction +– I-bond_interaction +π I-bond_interaction +interaction I-bond_interaction +as O +an O +additional O +key O +feature O +of O +the O +Tdp2 B-protein +protein O +– O +DNA B-chemical +crosslink O +binding O +and O +reversal O +. O + +The O +strictly B-protein_state +conserved I-protein_state +active B-site +site I-site +Arg216 B-residue_name_number +appears O +optimally O +positioned O +to O +stabilize O +a O +delocalized O +charge O +on O +the O +phenolate O +product O +of O +the O +phosphotyrosyl B-ptm +cleavage O +reaction O +through O +molecular O +orbital O +overlap O +and O +polarization O +of O +the O +leaving O +group O +. O + +To O +our O +knowledge O +, O +this O +is O +the O +first O +proposed O +example O +of O +a O +substrate B-site +cation I-site +– I-site +π I-site +interface I-site +exploited O +to O +promote O +a O +phosphoryl O +- O +transfer O +reaction O +. O + +This O +unique O +feature O +likely O +provides O +an O +additional O +level O +of O +substrate O +- O +specificity O +for O +Tdp2 B-protein +by O +restricting O +activity O +to O +hydrolysis O +of O +aromatic O +adducts O +characteristic O +of O +Top2cc B-complex_assembly +, O +picornaviral B-taxonomy_domain +protein O +– O +RNA B-chemical +and O +Hepatitis B-taxonomy_domain +B I-taxonomy_domain +Virus I-taxonomy_domain +( O +HBV B-taxonomy_domain +) O +protein O +– O +DNA B-chemical +processing O +intermediates O +. O + +By O +comparison O +, O +other O +EEP B-structure_element +nucleases B-protein_type +such O +as O +Ape1 B-protein +and O +Ape2 B-protein +have O +evolved O +robust O +DNA B-chemical +damage O +specific O +endonucleolytic O +and O +exonucleolytic O +activities O +not O +shared O +with O +Tdp2 B-protein +. O + +The O +dynamic O +nature O +of O +the O +Tdp2 B-protein +active B-site +site I-site +presents O +opportunities O +for O +enzyme O +regulation O +. O + +However O +, O +whether O +additional O +protein O +factors O +can O +bind O +to O +Tdp2 B-protein +and O +modulate O +assembly O +/ O +disassembly O +of O +the O +Tdp2 B-protein +β2Hβ B-structure_element +- I-structure_element +loop I-structure_element +is O +unknown O +. O + +We O +hypothesize O +that O +binding O +of O +the O +Top2 B-protein_type +protein O +component O +of O +a O +DNA B-chemical +– O +protein O +crosslink O +and O +/ O +or O +other O +protein O +- O +regulated O +assembly O +of O +the O +Tdp2 B-protein +active B-site +site I-site +might O +also O +serve O +to O +regulate O +Tdp2 B-protein +activity O +to O +restrict O +it O +from O +misplaced O +Top2 B-protein_type +processing O +events O +, O +such O +that O +it O +cleaves O +only O +topologically O +trapped O +or O +poisoned O +Top2 B-protein_type +molecules O +when O +needed O +. O + +Furthermore O +, O +high O +- O +resolution O +structures B-evidence +of O +mouse B-taxonomy_domain +( O +Figures O +3 O +and O +4 O +) O +and O +C B-species +. I-species +elegans I-species +Tdp2 B-protein +show O +that O +a O +single O +metal O +ion O +typifies O +the O +Tdp2 B-protein +active B-site +site I-site +from O +worms B-taxonomy_domain +to O +man B-taxonomy_domain +. O + +Herein O +, O +we O +report O +five O +additional O +lines O +of O +evidence O +from O +metal O +binding O +detected O +by O +intrinsic B-experimental_method +tryptophan I-experimental_method +fluorescence I-experimental_method +, O +crystallographic B-experimental_method +analysis I-experimental_method +of O +varied O +metal O +cofactor O +complexes O +, O +mutagenesis B-experimental_method +, O +Ca2 B-experimental_method ++ I-experimental_method +inhibition I-experimental_method +studies I-experimental_method +and O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +analysis I-experimental_method +that O +all O +support O +a O +feasible O +single O +Mg2 B-chemical ++ I-chemical +mediated O +Tdp2 B-protein +catalytic O +mechanism O +. O + +Etoposide B-chemical +and O +other O +Top2 B-protein_type +poisons O +remain O +front O +line O +anti O +- O +cancer O +drugs O +, O +and O +Tdp2 B-protein +frameshift O +mutations O +in O +the O +human B-species +population O +confer O +hypersensitivity O +to O +Top2 B-protein_type +poisons O +including O +etoposide B-chemical +and O +doxyrubicin B-chemical +. O + +Given O +Tdp2 B-protein +variation O +in O +the O +human B-species +population O +, O +links O +to O +neurological O +disease O +and O +viral O +pathogenesis O +, O +our O +finding O +that O +TDP2 B-protein +SNPs O +ablate O +catalytic O +activity O +has O +probable O +implications O +for O +modulation O +of O +cancer O +chemotherapy O +, O +susceptibility O +to O +environmentally O +linked O +Top2 B-protein_type +poisons O +, O +and O +viral B-taxonomy_domain +infection O +. O + +Lastly O +, O +Tdp2 B-protein +inhibitors O +may O +synergize O +or O +potentiate O +cytotoxic O +effects O +of O +current O +anticancer O +treatments O +that O +target O +Tdp2 B-protein +. O + +Thus O +, O +we O +anticipate O +this O +atomic O +- O +level O +and O +mechanistic O +definition O +of O +the O +molecular O +determinants O +of O +Tdp2 B-protein +catalysis O +and O +conformational O +changes O +driven O +by O +DNA B-chemical +– O +protein O +and O +protein O +– O +protein O +interactions O +will O +foster O +unique O +strategies O +for O +the O +development O +of O +Tdp2 B-protein +targeted O +small O +molecule O +interventions O +. O + +Mechanism O +of O +extracellular O +ion O +exchange O +and O +binding B-site +- I-site +site I-site +occlusion O +in O +the O +sodium B-protein_type +- I-protein_type +calcium I-protein_type +exchanger I-protein_type + +Na B-protein_type ++/ I-protein_type +Ca2 I-protein_type ++ I-protein_type +exchangers I-protein_type +utilize O +the O +Na B-chemical ++ I-chemical +electrochemical O +gradient O +across O +the O +plasma O +membrane O +to O +extrude O +intracellular O +Ca2 B-chemical ++, I-chemical +and O +play O +a O +central O +role O +in O +Ca2 B-chemical ++ I-chemical +homeostasis O +. O + +Here O +, O +we O +elucidate O +their O +mechanisms O +of O +extracellular O +ion O +recognition O +and O +exchange O +through O +a O +structural B-experimental_method +analysis I-experimental_method +of O +the O +exchanger B-protein_type +from O +Methanococcus B-species +jannaschii I-species +( O +NCX_Mj B-protein +) O +bound B-protein_state +to I-protein_state +Na B-chemical ++, I-chemical +Ca2 B-chemical ++ I-chemical +or O +Sr2 B-chemical ++ I-chemical +in O +various O +occupancies O +and O +in O +an O +apo B-protein_state +state O +. O + +This O +analysis O +defines O +the O +binding O +mode O +and O +relative O +affinity O +of O +these O +ions O +, O +establishes O +the O +structural O +basis O +for O +the O +anticipated O +3Na O ++: B-chemical +1Ca2 O ++ B-chemical +exchange O +stoichiometry O +, O +and O +reveals O +the O +conformational O +changes O +at O +the O +onset O +of O +the O +alternating O +- O +access O +transport O +mechanism O +. O + +An O +independent O +analysis O +of O +the O +dynamics O +and O +conformational B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscape I-evidence +of O +NCX_Mj B-protein +in O +different O +ion B-protein_state +- I-protein_state +occupancy I-protein_state +states O +, O +based O +on O +enhanced B-experimental_method +- I-experimental_method +sampling I-experimental_method +molecular I-experimental_method +- I-experimental_method +dynamics I-experimental_method +simulations I-experimental_method +, O +demonstrates O +that O +the O +crystal B-evidence +structures I-evidence +reflect O +mechanistically O +relevant O +, O +interconverting O +conformations O +. O + +These O +calculations B-experimental_method +also O +reveal O +the O +mechanism O +by O +which O +the O +outward B-protein_state +- O +to O +- O +inward B-protein_state +transition O +is O +controlled O +by O +the O +ion O +- O +occupancy O +state O +, O +thereby O +explaining O +the O +emergence O +of O +strictly O +- O +coupled O +Na B-chemical ++/ I-chemical +Ca2 B-chemical ++ I-chemical +antiport O +. O + +Na B-protein_type ++/ I-protein_type +Ca2 I-protein_type ++ I-protein_type +exchangers I-protein_type +( O +NCX B-protein_type +) O +play O +physiologically O +essential O +roles O +in O +Ca2 B-chemical ++ I-chemical +signaling O +and O +homeostasis O +. O + +NCX B-protein_type +catalyzes O +the O +uphill O +extrusion O +of O +intracellular O +Ca2 B-chemical ++ I-chemical +across O +the O +cell O +membrane O +, O +by O +coupling O +this O +process O +to O +the O +downhill O +permeation O +of O +Na B-chemical ++ I-chemical +into O +the O +cell O +, O +with O +a O +3 O +Na B-chemical ++ I-chemical +to O +1 O +Ca2 B-chemical ++ I-chemical +stoichiometry O +. O + +The O +mechanism O +of O +NCX B-protein_type +proteins O +is O +therefore O +highly O +likely O +to O +be O +consistent O +with O +the O +alternating O +- O +access O +model O +of O +secondary O +- O +active O +transport O +. O + +The O +basic O +functional O +unit O +for O +ion O +transport O +in O +NCX B-protein_type +consists O +of O +ten O +membrane B-structure_element +- I-structure_element +spanning I-structure_element +segments I-structure_element +, O +comprising O +two O +homologous O +halves B-structure_element +. O + +Each O +of O +these O +halves B-structure_element +contains O +a O +highly B-protein_state +conserved I-protein_state +region O +, O +referred O +to O +as O +α B-structure_element +- I-structure_element +repeat I-structure_element +, O +known O +to O +be O +important O +for O +ion O +binding O +and O +translocation O +; O +in O +eukaryotic B-taxonomy_domain +NCX B-protein_type +, O +the O +two O +halves B-structure_element +are O +connected O +by O +a O +large O +intracellular B-structure_element +regulatory I-structure_element +domain I-structure_element +, O +which O +is O +absent B-protein_state +in O +microbial B-taxonomy_domain +NCX B-protein_type +( O +Supplementary O +Fig O +. O +1 O +). O + +Despite O +a O +long O +history O +of O +physiological O +and O +functional O +studies O +, O +the O +molecular O +mechanism O +of O +NCX B-protein_type +has O +been O +elusive O +, O +owing O +to O +the O +lack O +of O +structural O +information O +. O + +Our O +recent O +atomic O +- O +resolution O +structure B-evidence +of O +NCX_Mj B-protein +from O +Methanococcus B-species +jannaschii I-species +provided O +the O +first O +view O +of O +the O +basic O +functional O +unit O +of O +an O +NCX B-protein_type +protein O +. O + +This O +structure B-evidence +shows O +the O +exchanger B-protein_type +in O +an O +outward B-protein_state +- I-protein_state +facing I-protein_state +conformation O +and O +reveals O +four O +putative O +ion B-site +- I-site +binding I-site +sites I-site +, O +denominated O +internal B-site +( O +Sint B-site +), O +external B-site +( O +Sext B-site +), O +Ca2 B-site ++- I-site +binding I-site +( O +SCa B-site +) O +and O +middle B-site +( O +Smid B-site +), O +clustered O +in O +the O +center O +of O +the O +protein O +and O +occluded B-protein_state +from I-protein_state +the O +solvent O +( O +Fig O +. O +1a O +- O +b O +). O + +With O +similar O +ion O +exchange O +properties O +to O +those O +of O +its O +eukaryotic B-taxonomy_domain +counterparts O +, O +NCX_Mj B-protein +provides O +a O +compelling O +model O +system O +to O +investigate O +the O +structural O +basis O +for O +the O +specificity O +, O +stoichiometry O +and O +mechanism O +of O +the O +ion O +- O +exchange O +reaction O +catalyzed O +by O +NCX B-protein_type +. O + +In O +this O +study O +, O +we O +set O +out O +to O +determine O +the O +structures B-evidence +of O +outward B-protein_state +- I-protein_state +facing I-protein_state +wild B-protein_state +- I-protein_state +type I-protein_state +NCX_Mj B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +Na B-chemical ++, I-chemical +Ca2 B-chemical ++ I-chemical +and O +Sr2 B-chemical ++, I-chemical +at O +various O +concentrations O +. O + +These O +structures B-evidence +reveal O +the O +mode O +of O +recognition O +of O +these O +ions O +, O +their O +relative O +affinities O +, O +and O +the O +mechanism O +of O +extracellular O +ion O +exchange O +, O +for O +a O +well O +- O +defined O +, O +functional O +conformation O +in O +a O +membrane O +- O +like O +environment O +. O + +An O +independent O +analysis O +based O +on O +molecular B-experimental_method +- I-experimental_method +dynamics I-experimental_method +simulations I-experimental_method +demonstrates O +that O +the O +structures B-evidence +capture O +mechanistically O +relevant O +states O +. O + +These O +calculations B-experimental_method +also O +reveal O +how O +the O +ion O +occupancy O +state O +of O +the O +outward B-protein_state +- I-protein_state +facing I-protein_state +exchanger B-protein_type +determines O +the O +feasibility O +of O +the O +transition O +to O +the O +inward B-protein_state +- I-protein_state +facing I-protein_state +conformation O +, O +thereby O +addressing O +a O +key O +outstanding O +question O +in O +secondary O +- O +active O +transport O +, O +namely O +how O +the O +transported O +substrates O +control O +the O +alternating O +- O +access O +mechanism O +. O + +Extracellular O +Na B-chemical ++ I-chemical +binding O + +The O +assignment O +of O +the O +four O +central B-site +binding I-site +sites I-site +identified O +in O +the O +previously O +reported O +NCX_Mj B-protein +structure B-evidence +was O +hampered O +by O +the O +presence O +of O +both O +Na B-chemical ++ I-chemical +and O +Ca2 B-chemical ++ I-chemical +in O +the O +protein O +crystals B-evidence +. O + +To O +conclusively O +clarify O +this O +assignment O +, O +we O +first O +set O +out O +to O +examine O +the O +Na B-chemical ++ I-chemical +occupancy O +of O +these O +sites O +without O +Ca2 B-chemical ++. I-chemical + +Crystals B-evidence +were O +grown O +in O +150 O +mM O +NaCl B-chemical +using O +the O +lipidic B-experimental_method +cubic I-experimental_method +phase I-experimental_method +( O +LCP B-experimental_method +) O +technique O +. O + +The O +crystallization O +solutions O +around O +the O +LCP B-experimental_method +droplets O +were O +then O +slowly O +replaced O +by O +solutions O +containing O +different O +concentrations O +of O +NaCl B-chemical +and O +EGTA B-chemical +( O +Methods O +). O + +X B-experimental_method +- I-experimental_method +ray I-experimental_method +diffraction I-experimental_method +of O +these O +soaked O +crystals B-evidence +revealed O +a O +Na B-chemical ++- I-chemical +dependent O +variation O +in O +the O +electron B-evidence +- I-evidence +density I-evidence +distribution I-evidence +at O +sites O +Sext B-site +, O +SCa B-site +and O +Sint B-site +, O +indicating O +a O +Na B-chemical ++ I-chemical +occupancy O +change O +( O +Fig O +. O +1c O +). O + +Occupancy B-experimental_method +refinement I-experimental_method +indicated O +two O +Na B-chemical ++ I-chemical +ions O +bind O +to O +Sint B-site +and O +SCa B-site +at O +low O +Na B-chemical ++ I-chemical +concentrations O +( O +Fig O +. O +1c O +), O +with O +a O +slight O +preference O +for O +Sint B-site +( O +Table O +1 O +). O + +Binding O +of O +a O +third O +Na B-chemical ++ I-chemical +to O +Sext B-site +occurs O +at O +higher O +concentrations O +, O +as O +no O +density B-evidence +was O +observed O +there O +at O +10 O +mM O +Na B-chemical ++ I-chemical +or O +lower O +( O +Fig O +. O +1c O +); O +Sext B-site +is O +however O +partially O +occupied O +at O +20 O +mM O +Na B-chemical ++, I-chemical +and O +fully O +occupied O +at O +150 O +mM O +( O +Fig O +. O +1c O +). O + +The O +Na B-chemical ++ I-chemical +occupation O +at O +SCa B-site +, O +compounded O +with O +the O +expected O +3Na O ++: B-chemical +1Ca2 O ++ B-chemical +stoichiometry O +, O +implies O +our O +previous O +assignment O +of O +the O +Smid B-site +site O +must O +be O +re O +- O +evaluated O +. O + +Indeed O +, O +two O +observations O +indicate O +that O +a O +water B-chemical +molecule O +rather O +than O +a O +Na B-chemical ++ I-chemical +ion O +occupies O +Smid B-site +, O +as O +was O +predicted O +in O +a O +recent O +simulation B-experimental_method +study O +. O + +First O +, O +the O +electron B-evidence +density I-evidence +at O +Smid B-site +does O +not O +depend O +significantly O +on O +the O +Na B-chemical ++ I-chemical +concentration O +. O + +Second O +, O +the O +protein O +coordination O +geometry O +at O +Smid B-site +is O +clearly O +suboptimal O +for O +Na B-chemical ++ I-chemical +( O +Supplementary O +Fig O +. O +1d O +). O + +The O +water B-chemical +molecule O +at O +Smid B-site +forms O +hydrogen B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +with O +the O +highly B-protein_state +conserved I-protein_state +Glu54 B-residue_name_number +and O +Glu213 B-residue_name_number +( O +Supplementary O +Fig O +. O +1d O +), O +stabilizing O +their O +orientation O +to O +properly O +coordinate B-bond_interaction +multiple O +Na B-chemical ++ I-chemical +ions O +at O +Sext B-site +, O +SCa B-site +and O +Sint B-site +. O + +It O +can O +be O +inferred O +from O +this O +assignment O +that O +Glu54 B-residue_name_number +and O +Glu213 B-residue_name_number +are O +ionized O +, O +while O +Asp240 B-residue_name_number +, O +which O +flanks O +Smid B-site +( O +and O +is O +replaced O +by O +Asn B-residue_name +in O +eukaryotic B-taxonomy_domain +NCX B-protein_type +) O +would O +be O +protonated B-protein_state +, O +as O +indicated O +by O +the O +abovementioned O +simulation B-experimental_method +study O +. O + +Na B-chemical ++- I-chemical +dependent O +conformational O +change O + +The O +NCX_Mj B-protein +structures B-evidence +in O +various O +Na B-chemical ++ I-chemical +concentrations O +also O +reveal O +that O +Na B-chemical ++ I-chemical +binding O +to O +Sext B-site +is O +coupled O +to O +a O +subtle O +but O +important O +conformational O +change O +( O +Fig O +. O +2 O +). O + +When O +Na B-chemical ++ I-chemical +binds O +to O +Sext B-site +at O +high B-protein_state +concentrations O +, O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +TM7 B-structure_element +is O +bent O +into O +two O +short B-structure_element +helices I-structure_element +, O +TM7a B-structure_element +and O +TM7b B-structure_element +( O +Fig O +. O +2a O +). O + +TM7b B-structure_element +occludes O +the O +four O +central B-site +binding I-site +sites I-site +from O +the O +external O +solution O +, O +with O +the O +backbone O +carbonyl O +of O +Ala206 B-residue_name_number +coordinating B-bond_interaction +the O +Na B-chemical ++ I-chemical +ion O +( O +Fig O +. O +2b O +- O +d O +). O + +However O +, O +when O +Sext B-site +becomes O +empty B-protein_state +at O +low B-protein_state +Na B-chemical ++ I-chemical +concentrations O +, O +TM7a B-structure_element +and O +TM7b B-structure_element +become O +a O +continuous O +straight O +helix B-structure_element +( O +Fig O +. O +2a O +), O +and O +the O +carbonyl O +group O +of O +Ala206 B-residue_name_number +retracts O +away O +( O +Fig O +. O +2b O +- O +d O +). O + +TM7a B-structure_element +also O +forms O +hydrophobic B-bond_interaction +contacts I-bond_interaction +with O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +TM6 B-structure_element +. O + +These O +contacts O +are O +absent O +in O +the O +structure B-evidence +with O +Na B-chemical ++ I-chemical +at O +Sext B-site +, O +in O +which O +there O +is O +an O +open O +gap O +between O +the O +two O +helices B-structure_element +( O +Fig O +. O +2b O +). O + +This O +difference O +is O +noteworthy O +because O +TM6 B-structure_element +and O +TM1 B-structure_element +are O +believed O +to O +undergo O +a O +sliding O +motion O +, O +relative O +to O +the O +rest O +of O +the O +protein O +, O +when O +the O +transporter B-protein_type +switches O +to O +the O +inward B-protein_state +- I-protein_state +facing I-protein_state +conformation O +. O + +The O +straightening O +of O +TM7ab B-structure_element +also O +opens O +up O +a O +passageway O +from O +the O +external O +solution O +to O +Sext B-site +and O +Smid B-site +, O +while O +SCa B-site +and O +Sint B-site +remain O +occluded B-protein_state +( O +Fig O +. O +2d O +). O + +Thus O +, O +the O +structures B-evidence +at O +high B-protein_state +and O +low B-protein_state +Na B-chemical ++ I-chemical +concentrations O +represent O +the O +outward B-protein_state +- I-protein_state +facing I-protein_state +occluded B-protein_state +and O +partially B-protein_state +open I-protein_state +states O +, O +respectively O +. O + +This O +conformational O +change O +is O +dependent O +on O +the O +Na B-chemical ++ I-chemical +occupancy O +of O +Sext B-site +and O +occurs O +when O +Na B-chemical ++ I-chemical +already O +occupies O +Sint B-site +and O +SCa B-site +. O + +Our O +crystallographic B-experimental_method +titration I-experimental_method +experiment I-experimental_method +indicates O +that O +the O +K1 B-evidence +/ I-evidence +2 I-evidence +of O +this O +Na B-chemical ++- I-chemical +driven O +conformational O +transition O +is O +~ O +20 O +mM O +. O +At O +this O +concentration O +, O +Sext B-site +is O +partially B-protein_state +occupied I-protein_state +and O +the O +NCX_Mj B-protein +crystal B-evidence +is O +a O +mixture O +of O +both O +the O +occluded B-protein_state +and O +partially B-protein_state +open I-protein_state +conformations O +. O + +This O +structurally O +- O +derived O +Na B-evidence ++ I-evidence +affinity I-evidence +agrees O +well O +with O +the O +external O +Na B-chemical ++ I-chemical +concentration O +required O +for O +NCX B-protein_type +activation O +in O +eukaryotes B-taxonomy_domain +. O + +The O +finding O +that O +the O +Na B-chemical ++ I-chemical +occupancy O +change O +from O +2 O +to O +3 O +ions O +coincides O +with O +a O +conformational O +change O +of O +the O +transporter B-protein_type +also O +provides O +a O +rationale O +to O +the O +Hill B-evidence +coefficient I-evidence +of O +the O +Na B-chemical ++- I-chemical +dependent O +activation O +process O +in O +eukaryotic B-taxonomy_domain +NCX B-protein_type +. O + +Extracellular O +Ca2 B-chemical ++ I-chemical +and O +Sr2 B-chemical ++ I-chemical +binding O +and O +their O +competition O +with O +Na B-chemical ++ I-chemical + +To O +determine O +how O +Ca2 B-chemical ++ I-chemical +binds O +to O +NCX_Mj B-protein +and O +competes O +with O +Na B-chemical ++, I-chemical +we O +first O +titrated B-experimental_method +the I-experimental_method +crystals I-experimental_method +with O +Sr2 B-chemical ++ I-chemical +( O +Methods O +). O + +Sr2 B-chemical ++ I-chemical +is O +transported O +by O +NCX B-protein_type +similarly O +to O +Ca2 B-chemical ++ I-chemical +, O +and O +is O +distinguishable O +from O +Na B-chemical ++ I-chemical +by O +its O +greater O +electron B-evidence +- I-evidence +density I-evidence +intensity I-evidence +. O + +Protein B-experimental_method +crystals I-experimental_method +soaked I-experimental_method +with O +10 O +mM O +Sr2 B-chemical ++ I-chemical +and O +2 O +. O +5 O +mM O +Na B-chemical ++ I-chemical +revealed O +a O +strong O +electron B-evidence +- I-evidence +density I-evidence +peak I-evidence +at O +site O +SCa B-site +, O +indicating O +binding O +of O +a O +single O +Sr2 B-chemical ++ I-chemical +ion O +( O +Fig O +. O +3a O +). O + +The O +Sr2 B-protein_state ++- I-protein_state +loaded I-protein_state +NCX_Mj B-protein +structure B-evidence +adopts O +the O +partially B-protein_state +open I-protein_state +conformation O +observed O +at O +low O +Na B-chemical ++ I-chemical +concentrations O +. O + +Binding O +of O +Sr2 B-chemical ++, I-chemical +however O +, O +excludes O +Na B-chemical ++ I-chemical +entirely O +. O + +Crystal B-experimental_method +titrations I-experimental_method +with O +decreasing B-experimental_method +Sr2 B-chemical ++ I-chemical +or O +increasing B-experimental_method +Na B-chemical ++ I-chemical +demonstrated O +that O +Sr2 B-chemical ++ I-chemical +binds O +to O +the O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +with O +low O +affinity O +, O +and O +that O +it O +can O +be O +out O +- O +competed O +by O +Na B-chemical ++ I-chemical +even O +at O +low O +concentrations O +( O +Supplementary O +Note O +1 O +and O +Supplementary O +Fig O +. O +2a O +- O +b O +). O + +Thus O +, O +in O +100 O +mM O +Na B-chemical ++ I-chemical +and O +10 O +mM O +Sr2 B-chemical ++, I-chemical +Na B-chemical ++ I-chemical +completely O +replaced O +Sr2 B-chemical ++ I-chemical +( O +Fig O +. O +3a O +) O +and O +reverted O +NCX_Mj B-protein +to O +the O +Na B-protein_state ++- I-protein_state +loaded I-protein_state +, O +fully B-protein_state +occluded I-protein_state +state O +. O + +Similar O +titration B-experimental_method +experiments I-experimental_method +showed O +that O +Ca2 B-chemical ++ I-chemical +and O +Sr2 B-chemical ++ I-chemical +binding O +to O +NCX_Mj B-protein +are O +not O +exactly O +alike O +The O +electron B-evidence +density I-evidence +distribution I-evidence +from O +crystals B-experimental_method +soaked I-experimental_method +in I-experimental_method +high B-protein_state +Ca2 B-chemical ++ I-chemical +and O +low B-protein_state +Na B-chemical ++, I-chemical +indicates O +that O +Ca2 B-chemical ++ I-chemical +can O +bind O +to O +Smid B-site +as O +well O +as O +SCa B-site +, O +with O +a O +preference O +for O +SCa B-site +( O +Fig O +. O +3b O +). O + +Binding O +of O +Ca2 B-chemical ++ I-chemical +to O +both O +sites O +simultaneously O +is O +highly O +improbable O +due O +to O +their O +close O +proximity O +, O +and O +at O +least O +one O +water B-chemical +molecule O +can O +be O +discerned O +coordinating B-bond_interaction +the O +ion O +( O +Fig O +. O +3b O +). O + +The O +partial B-protein_state +Ca2 B-chemical ++ I-chemical +occupancy B-protein_state +at O +Smid B-site +is O +likely O +caused O +by O +Asp240 B-residue_name_number +, O +which O +flanks O +this O +site O +and O +can O +in O +principle O +coordinate B-bond_interaction +Ca2 B-chemical ++. I-chemical + +Previous O +functional B-experimental_method +and I-experimental_method +computational I-experimental_method +studies I-experimental_method +, O +however O +, O +indicate O +Asp240 B-residue_name_number +becomes O +protonated B-protein_state +during O +transport O +. O + +Indeed O +, O +in O +most O +NCX B-protein_type +proteins O +Asp240 B-residue_name_number +is O +substituted B-experimental_method +by O +Asn B-residue_name +, O +which O +would O +likely O +weaken O +or O +abrogate O +Ca2 B-chemical ++ I-chemical +binding O +to O +Smid B-site +. O + +SCa B-site +is O +therefore O +the O +functional O +Ca2 B-site ++ I-site +site I-site +. O + +Similarly O +to O +Sr2 B-chemical ++, I-chemical +Ca2 B-chemical ++ I-chemical +binds O +with O +low O +affinity B-evidence +to O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +and O +can O +be O +readily O +displaced O +by O +Na B-chemical ++ I-chemical +( O +Supplementary O +Note O +1 O +and O +Supplementary O +Fig O +. O +2c O +). O + +This O +finding O +is O +consistent O +with O +physiological B-evidence +and I-evidence +biochemical I-evidence +data I-evidence +for O +both O +eukaryotic B-taxonomy_domain +NCX B-protein_type +and O +NCX_Mj B-protein +indicating O +that O +the O +apparent O +Ca2 B-evidence ++ I-evidence +affinity I-evidence +is O +much O +lower O +on O +the O +extracellular O +than O +the O +cytoplasmic O +side O +. O + +Specifically O +, O +our O +crystallographic B-experimental_method +titration I-experimental_method +assay I-experimental_method +indicates O +Ca2 B-chemical ++ I-chemical +binds O +with O +sub O +- O +millimolar O +affinity B-evidence +, O +in O +good O +agreement O +with O +the O +external O +apparent O +Ca2 B-evidence ++ I-evidence +affinities I-evidence +deduced O +functionally O +for O +cardiac O +NCX B-protein_type +( O +Km B-evidence +~ O +0 O +. O +32 O +mM O +) O +and O +NCX_Mj B-protein +( O +Km B-evidence +~ O +0 O +. O +175 O +mM O +). O + +Taken O +together O +, O +these O +crystal B-experimental_method +titration I-experimental_method +experiments I-experimental_method +demonstrate O +that O +the O +four O +binding B-site +sites I-site +in O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +exhibit O +different O +specificity O +: O +Sint B-site +and O +Sext B-site +are O +Na B-chemical ++ I-chemical +specific O +whereas O +SCa B-site +, O +previously O +hypothesized O +to O +be O +Ca2 B-chemical ++ I-chemical +specific O +, O +can O +also O +bind O +Na B-chemical ++, I-chemical +confirming O +our O +earlier O +simulation B-experimental_method +study O +, O +as O +well O +as O +Sr2 B-chemical ++; I-chemical +Smid B-site +can O +also O +transiently O +accommodate O +Ca2 B-chemical ++ I-chemical +but O +during O +transport O +Smid B-site +is O +most O +likely O +occupied O +by O +water B-chemical +. O + +The O +ion B-site +- I-site +binding I-site +sites I-site +in O +NCX_Mj B-protein +can O +therefore O +accommodate O +up O +to O +three O +Na B-chemical ++ I-chemical +ions O +or O +a O +single O +divalent O +ion O +, O +and O +occupancy O +by O +Na B-chemical ++ I-chemical +and O +Ca2 B-chemical ++ I-chemical +( O +or O +Sr2 B-chemical ++) I-chemical +are O +mutually O +exclusive O +, O +as O +was O +deduced O +for O +eukaryotic B-taxonomy_domain +exchangers B-protein_type +. O + +A O +structure B-evidence +of O +NCX_Mj B-protein +without B-protein_state +Na B-chemical ++ I-chemical +or O +Ca2 B-chemical ++ I-chemical +bound B-protein_state + +An O +apo B-protein_state +state O +of O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +is O +likely O +to O +exist O +transiently O +in O +physiological O +conditions O +, O +despite O +the O +high O +amounts O +of O +extracellular O +Na B-chemical ++ I-chemical +(~ O +150 O +mM O +) O +and O +Ca2 B-chemical ++ I-chemical +(~ O +2 O +mM O +). O + +We O +were O +able O +to O +determine O +an O +apo B-protein_state +- O +state O +structure B-evidence +of O +NCX_Mj B-protein +, O +by O +crystallizing B-experimental_method +the O +protein O +at O +lower B-protein_state +pH I-protein_state +and O +in O +the O +absence B-protein_state +of I-protein_state +Na B-chemical ++ I-chemical +( O +Methods O +). O + +This O +structure B-evidence +is O +similar O +to O +the O +partially B-protein_state +open I-protein_state +structure B-evidence +with O +two O +Na B-chemical ++ I-chemical +or O +either O +one O +Ca2 B-chemical ++ I-chemical +or O +one O +Sr2 B-chemical ++ I-chemical +ion O +, O +with O +two O +noticeable O +differences O +. O + +First O +, O +TM7ab B-structure_element +along O +with O +the O +extracellular B-structure_element +half I-structure_element +of O +the O +TM6 B-structure_element +and O +TM1 B-structure_element +swing O +further O +away O +from O +the O +protein O +core O +( O +Fig O +. O +3c O +), O +resulting O +in O +a O +slightly O +wider O +passageway O +into O +the O +binding B-site +sites I-site +. O + +Second O +, O +Glu54 B-residue_name_number +and O +Glu213 B-residue_name_number +side O +chains O +rotate O +away O +from O +the O +binding B-site +sites I-site +and O +appear O +to O +form O +hydrogen B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +with O +residues O +involved O +in O +ion B-bond_interaction +coordination I-bond_interaction +in O +the O +fully B-protein_state +Na I-protein_state ++- I-protein_state +loaded I-protein_state +structure B-evidence +( O +Fig O +. O +3d O +). O + +Although O +the O +binding B-site +sites I-site +are O +thus O +fully B-protein_state +accessible I-protein_state +to O +the O +external O +solution O +( O +Fig O +. O +3e O +), O +the O +lack O +of O +electron B-evidence +density I-evidence +therein O +indicates O +no O +ions O +or O +ordered O +solvent O +molecules O +. O + +This O +apo B-protein_state +structure B-evidence +might O +therefore O +represent O +the O +unloaded B-protein_state +, O +open B-protein_state +state O +of O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +. O + +Alternatively O +, O +this O +structure B-evidence +might O +capture O +a O +fully B-protein_state +protonated I-protein_state +state O +of O +the O +transporter B-protein_type +, O +to O +which O +Na B-chemical ++ I-chemical +and O +Ca2 B-chemical ++ I-chemical +cannot O +bind O +. O + +Such O +interpretation O +would O +be O +consistent O +with O +the O +computer B-experimental_method +simulations I-experimental_method +reported O +below O +. O + +Indeed O +, O +transport B-experimental_method +assays I-experimental_method +of O +NCX_Mj B-protein +have O +shown O +that O +even O +in O +the O +presence B-protein_state +of I-protein_state +Na B-chemical ++ I-chemical +or O +Ca2 B-chemical ++, I-chemical +low B-protein_state +pH I-protein_state +inactivates B-protein_state +the O +transport O +cycle O +. O + +Ion O +occupancy O +determines O +the O +free O +- O +energy O +landscape O +of O +NCX_Mj B-protein + +That O +secondary B-protein_type +- I-protein_type +active I-protein_type +transporters I-protein_type +are O +able O +to O +harness O +an O +electrochemical O +gradient O +of O +one O +substrate O +to O +power O +the O +uphill O +transport O +of O +another O +relies O +on O +a O +seemingly O +simple O +principle O +: O +they O +must O +not O +transition O +between O +outward B-protein_state +- I-protein_state +and O +inward B-protein_state +- I-protein_state +open I-protein_state +conformations O +unless O +in O +two O +precise O +substrate O +occupancy O +states O +. O + +NCX B-protein_type +must O +be O +loaded O +either O +with O +3 O +Na B-chemical ++ I-chemical +or O +1 O +Ca2 B-chemical ++, I-chemical +and O +therefore O +functions O +as O +an O +antiporter B-protein_type +; O +symporters B-protein_type +, O +by O +contrast O +, O +undergo O +the O +alternating O +- O +access O +transition O +only O +when O +all O +substrates O +and O +coupling O +ions O +are O +concurrently O +bound B-protein_state +, O +or O +in O +the O +apo B-protein_state +state O +. O + +To O +examine O +this O +central O +question O +, O +we O +sought O +to O +characterize O +the O +conformational B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscape I-evidence +of O +NCX_Mj B-protein +and O +to O +examine O +its O +dependence O +on O +the O +ion O +- O +occupancy O +state O +, O +using O +molecular B-experimental_method +dynamics I-experimental_method +( O +MD B-experimental_method +) O +simulations B-experimental_method +. O + +This O +computational O +analysis O +was O +based O +solely O +on O +the O +published O +structure B-evidence +of O +NCX_Mj B-protein +, O +independently O +of O +the O +crystallographic B-experimental_method +studies I-experimental_method +described O +above O +. O + +As O +it O +happens O +, O +the O +results O +confirm O +that O +the O +structures B-evidence +now O +available O +are O +representing O +interconverting O +states O +of O +the O +functional O +cycle O +of O +NCX_Mj B-protein +, O +while O +revealing O +how O +the O +alternating O +- O +access O +mechanism O +is O +controlled O +by O +the O +ion O +- O +occupancy O +state O +. O + +A O +series O +of O +exploratory O +MD B-experimental_method +simulations I-experimental_method +was O +initially O +carried O +out O +to O +examine O +what O +features O +of O +the O +NCX_Mj B-protein +structure B-evidence +might O +depend O +on O +the O +ion B-site +- I-site +binding I-site +sites I-site +occupancy O +. O + +Specifically O +, O +we O +first O +simulated B-experimental_method +the O +outward B-protein_state +- I-protein_state +occluded I-protein_state +form O +, O +in O +the O +ion O +configuration O +we O +previously O +predicted O +, O +now O +confirmed O +by O +the O +high B-protein_state +- I-protein_state +Na I-protein_state ++ I-protein_state +crystal B-evidence +structure I-evidence +described O +above O +( O +Fig O +. O +1b O +). O + +That O +is O +, O +Na B-chemical ++ I-chemical +ions O +occupy O +Sext B-site +, O +SCa B-site +, O +and O +Sint B-site +, O +while O +D240 B-residue_name_number +is O +protonated B-protein_state +and O +a O +water B-chemical +molecule O +occupies O +Smid B-site +. O + +The O +Na B-chemical ++ I-chemical +ion O +at O +Sext B-site +was O +then O +relocated O +from O +the O +site O +to O +the O +bulk O +solution O +( O +Methods O +), O +and O +this O +system O +was O +then O +allowed O +to O +evolve O +freely O +in O +time O +. O + +The O +Na B-chemical ++ I-chemical +ions O +at O +SCa B-site +and O +Sint B-site +were O +displaced O +subsequently O +, O +and O +an O +analogous O +simulation B-experimental_method +was O +then O +carried O +out O +. O + +These O +initial O +simulations B-experimental_method +revealed O +noticeable O +changes O +in O +the O +transporter B-protein_type +, O +consistent O +with O +those O +observed O +in O +the O +new O +crystal B-evidence +structures I-evidence +. O + +The O +most O +notable O +change O +upon O +displacement O +of O +Na B-chemical ++ I-chemical +from O +Sext B-site +was O +the O +straightening O +of O +TM7ab B-structure_element +( O +Fig O +. O +4a O +). O + +When O +3 O +Na B-chemical ++ I-chemical +ions O +are O +bound B-protein_state +, O +TM7ab B-structure_element +primarily O +folds O +as O +two O +distinct O +, O +non O +- O +collinear O +α B-structure_element +- I-structure_element +helical I-structure_element +fragments I-structure_element +, O +owing O +to O +the O +loss O +of O +the O +backbone O +carbonyl O +- O +amide O +hydrogen B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +between O +F202 B-residue_name_number +and O +A206 B-residue_name_number +, O +and O +T203 B-residue_name_number +and O +F207 B-residue_name_number +( O +Fig O +. O +4b O +). O + +This O +distortion O +occludes O +Sext B-site +from O +the O +exterior O +( O +Fig O +. O +4d O +, O +4h O +- O +i O +) O +and O +appears O +to O +be O +induced O +by O +the O +Na B-chemical ++ I-chemical +ion O +itself O +, O +which O +pulls O +the O +carbonyl O +group O +of O +A206 B-residue_name_number +into O +its O +coordination O +sphere O +( O +Fig O +. O +4g O +). O + +With O +Sext B-site +empty B-protein_state +, O +however O +, O +TM7ab B-structure_element +forms O +a O +canonical O +α B-structure_element +- I-structure_element +helix I-structure_element +( O +Fig O +. O +4a O +- O +b O +, O +4g O +), O +thereby O +creating O +an O +opening O +between O +TM3 B-structure_element +and O +TM7 B-structure_element +, O +which O +in O +turn O +allows O +water B-chemical +molecules O +from O +the O +external O +solution O +to O +reach O +into O +Sext B-site +( O +Fig O +. O +4e O +, O +4h O +- O +i O +), O +i O +. O +e O +. O +the O +transporter B-protein_type +is O +no B-protein_state +longer I-protein_state +occluded I-protein_state +. O + +Displacement O +of O +Na B-chemical ++ I-chemical +from O +SCa B-site +and O +Sint B-site +induces O +further O +changes O +( O +Fig O +. O +4c O +). O + +The O +most O +noticeable O +is O +an O +increased O +separation O +between O +TM7 B-structure_element +and O +TM2 B-structure_element +( O +Fig O +. O +4f O +), O +previously O +brought O +together O +by O +concurrent O +backbone O +interactions O +with O +the O +Na B-chemical ++ I-chemical +ion O +at O +SCa B-site +( O +Fig O +. O +4d O +- O +e O +). O + +TM1 B-structure_element +and O +TM6 B-structure_element +also O +slide O +further O +towards O +the O +membrane O +center O +, O +relative O +to O +the O +outward B-protein_state +- I-protein_state +occluded I-protein_state +state O +( O +Fig O +. O +4c O +). O + +Together O +, O +these O +changes O +open O +a O +second O +aqueous B-site +channel I-site +leading O +directly O +into O +SCa B-site +and O +Sint B-site +( O +Fig O +. O +4f O +, O +Fig O +. O +4h O +- O +i O +). O + +The O +transporter B-protein_type +thus O +becomes O +fully B-protein_state +outward I-protein_state +- I-protein_state +open I-protein_state +. O + +To O +more O +rigorously O +characterize O +the O +influence O +of O +the O +ion O +- O +occupancy O +state O +on O +the O +conformational O +dynamics O +of O +the O +exchanger B-protein_type +, O +we O +carried O +out O +a O +series O +of O +enhanced O +- O +sampling O +MD B-experimental_method +calculations I-experimental_method +designed O +to O +reversibly O +simulate O +the O +transition O +between O +the O +outward B-protein_state +- I-protein_state +occluded I-protein_state +and O +fully B-protein_state +outward I-protein_state +- I-protein_state +open I-protein_state +states O +, O +and O +thus O +quantify O +the O +free B-evidence +- I-evidence +energy I-evidence +landscape I-evidence +encompassing O +these O +states O +( O +Methods O +). O + +As O +above O +, O +we O +initially O +examined O +three O +occupancy O +states O +, O +namely O +with O +Na B-chemical ++ I-chemical +in O +Sext B-site +, O +SCa B-site +and O +Sint B-site +, O +with O +Na B-chemical ++ I-chemical +only O +at O +SCa B-site +and O +Sint B-site +, O +and O +without B-protein_state +Na B-chemical ++. I-chemical + +These O +calculations B-experimental_method +demonstrate O +that O +the O +Na B-chemical ++ I-chemical +occupancy O +state O +of O +the O +transporter B-protein_type +has O +a O +profound O +effect O +on O +its O +conformational B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscape I-evidence +. O + +When O +all O +Na B-site ++ I-site +sites I-site +are O +occupied O +, O +the O +global O +free B-evidence +- I-evidence +energy I-evidence +minimum I-evidence +corresponds O +to O +a O +conformation O +in O +which O +the O +ions O +are O +maximally O +coordinated O +by O +the O +protein O +( O +Fig O +. O +5a O +, O +5c O +); O +TM7ab B-structure_element +is O +bent O +and O +packs O +closely O +with O +TM2 B-structure_element +and O +TM3 B-structure_element +, O +and O +so O +the O +binding B-site +sites I-site +are O +occluded O +from O +the O +solvent O +( O +Fig O +. O +5b O +). O + +At O +a O +small O +energetic O +cost O +, O +however O +, O +the O +transporter B-protein_type +can O +adopt O +a O +metastable B-protein_state +‘ O +half B-protein_state +- I-protein_state +open I-protein_state +’ O +conformation O +in O +which O +TM7ab B-structure_element +is O +completely O +straight O +and O +Sext B-site +is O +open B-protein_state +to O +the O +exterior O +( O +Fig O +. O +5a O +, O +5b O +). O + +The O +Na B-chemical ++ I-chemical +ion O +at O +Sext B-site +remains O +fully B-protein_state +coordinated I-protein_state +, O +but O +an O +ordered O +water B-chemical +molecule O +now O +mediates O +its O +interaction O +with O +A206 B-residue_name_number +: O +O O +, O +relieving O +the O +strain O +on O +the O +F202 B-residue_name_number +: O +O O +– O +A206 B-residue_name_number +: O +N O +hydrogen B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +( O +Fig O +. O +5c O +). O + +This O +semi B-protein_state +- I-protein_state +open I-protein_state +conformation O +is O +nearly O +identical O +to O +that O +found O +to O +be O +the O +most O +probable O +when O +Na B-chemical ++ I-chemical +occupies O +only O +SCa B-site +and O +Sint B-site +( O +2 O +× O +Na B-chemical ++, I-chemical +Fig O +. O +5a O +), O +demonstrating O +that O +binding O +( O +or O +release O +) O +of O +Na B-chemical ++ I-chemical +to O +Sext B-site +occurs O +in O +this O +metastable B-protein_state +conformation O +. O + +Interestingly O +, O +this O +doubly O +occupied O +state O +can O +also O +access O +conformations O +in O +which O +the O +second O +aqueous B-site +channel I-site +mentioned O +above O +, O +i O +. O +e O +. O +leading O +to O +SCa B-site +between O +TM7 B-structure_element +and O +TM2 B-structure_element +and O +over O +the O +gating B-structure_element +helices I-structure_element +TM1 B-structure_element +and O +TM6 B-structure_element +, O +also O +becomes O +open B-protein_state +( O +Fig O +. O +5b O +- O +c O +). O + +Crucially O +, O +though O +, O +the O +free B-evidence +- I-evidence +energy I-evidence +landscape I-evidence +for O +this O +partially B-protein_state +occupied I-protein_state +state O +demonstrates O +that O +the O +occluded B-protein_state +conformation O +is O +no O +longer O +energetically O +feasible O +( O +Fig O +. O +5a O +). O + +Displacement O +of O +the O +two O +remaining O +Na B-chemical ++ I-chemical +ions O +from O +SCa B-site +and O +Sint B-site +further O +reshapes O +the O +free B-evidence +- I-evidence +energy I-evidence +landscape I-evidence +of O +the O +transporter B-protein_type +( O +No O +ions O +, O +Fig O +. O +5a O +), O +which O +now O +can O +only O +adopt O +a O +fully B-protein_state +open I-protein_state +state O +featuring O +the O +two O +aqueous B-site +channels I-site +( O +Fig O +. O +5b O +- O +c O +). O + +The O +transition O +to O +the O +occluded B-protein_state +state O +in O +this O +apo B-protein_state +state O +is O +again O +energetically O +unfeasible O +. O + +From O +a O +mechanistic O +standpoint O +, O +it O +is O +satisfying O +to O +observe O +how O +the O +open B-protein_state +and O +semi B-protein_state +- I-protein_state +open I-protein_state +states O +are O +each O +compatible O +with O +two O +different O +Na B-chemical ++ I-chemical +occupancies O +, O +explaining O +how O +sequential O +Na B-chemical ++ I-chemical +binding O +to O +energetically O +accessible O +conformations O +( O +prior O +to O +those O +binding O +events O +) O +progressively O +reshape O +the O +free B-evidence +- I-evidence +energy I-evidence +landscape I-evidence +of O +the O +transporter B-protein_type +; O +by O +contrast O +, O +the O +occluded B-protein_state +conformation O +is O +forbidden O +unless O +the O +Na B-protein_state ++ I-protein_state +occupancy I-protein_state +is I-protein_state +complete I-protein_state +. O + +This O +processivity O +is O +logical O +since O +three O +Na B-chemical ++ I-chemical +ions O +are O +involved O +, O +but O +also O +implies O +that O +in O +the O +Ca2 B-protein_state ++- I-protein_state +bound I-protein_state +state O +, O +which O +includes O +a O +single O +ion O +, O +the O +transporter B-protein_type +ought O +to O +be O +able O +to O +access O +all O +three O +major O +conformations O +, O +i O +. O +e O +. O +the O +outward B-protein_state +- I-protein_state +open I-protein_state +state O +, O +in O +order O +to O +release O +( O +or O +re O +- O +bind O +) O +Ca2 B-chemical ++, I-chemical +but O +also O +the O +occluded B-protein_state +conformation O +, O +and O +thus O +the O +semi B-protein_state +- I-protein_state +open I-protein_state +intermediate O +, O +in O +order O +to O +transition O +to O +the O +inward B-protein_state +- I-protein_state +open I-protein_state +state O +. O + +By O +contrast O +, O +occupancy O +by O +H B-chemical ++, I-chemical +which O +as O +mentioned O +are O +not O +transported O +, O +might O +be O +compatible O +with O +a O +semi B-protein_state +- I-protein_state +open I-protein_state +state O +as O +well O +as O +with O +the O +fully B-protein_state +open I-protein_state +conformation O +, O +but O +should O +not O +be O +conducive O +to O +occlusion O +. O + +To O +assess O +this O +hypothesis O +, O +we O +carried O +out O +enhanced B-experimental_method +- I-experimental_method +sampling I-experimental_method +simulations I-experimental_method +for O +the O +Ca2 B-protein_state ++ I-protein_state +and O +H B-protein_state ++- I-protein_state +bound I-protein_state +states O +of O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +analogous O +to O +those O +described O +above O +for O +Na B-chemical ++ I-chemical +( O +see O +Supplementary O +Note O +2 O +and O +Supplementary O +Fig O +. O +3 O +- O +4 O +for O +details O +on O +how O +the O +structures B-evidence +of O +the O +Ca2 B-protein_state ++- I-protein_state +bound I-protein_state +state O +was O +predicted O +). O + +The O +calculated B-experimental_method +free B-evidence +- I-evidence +energy I-evidence +landscape I-evidence +for O +Ca2 B-protein_state ++- I-protein_state +bound I-protein_state +NCX_Mj B-protein +confirms O +the O +hypothesis O +outlined O +above O +( O +1 O +× O +Ca2 B-chemical ++, I-chemical +Fig O +. O +6a O +): O +consistent O +with O +the O +fact O +that O +NCX_Mj B-protein +transports O +a O +single O +Ca2 B-chemical ++, I-chemical +the O +occluded B-protein_state +, O +dehydrated B-protein_state +conformation O +is O +one O +of O +the O +major O +energetic O +minima O +, O +but O +clearly O +the O +exchanger B-protein_type +can O +also O +adopt O +the O +semi B-protein_state +- I-protein_state +open I-protein_state +and O +open B-protein_state +states O +that O +would O +be O +required O +for O +Ca2 B-chemical ++ I-chemical +release O +and O +Na B-chemical ++ I-chemical +entry O +, O +via O +either O +of O +the O +aqueous B-site +access I-site +channels I-site +that O +lead O +to O +Sext B-site +and O +SCa B-site +( O +Fig O +. O +6b O +- O +c O +). O + +By O +contrast O +, O +protonation B-protein_state +of O +Glu54 B-residue_name_number +and O +Glu213 B-residue_name_number +makes O +the O +occluded B-protein_state +conformation O +energetically O +unfeasible O +, O +consistent O +with O +the O +fact O +that O +NCX_Mj B-protein +does O +not O +transport O +protons B-chemical +; O +in O +this O +H B-protein_state ++- I-protein_state +bound I-protein_state +state O +, O +though O +, O +the O +exchanger B-protein_type +can O +adopt O +the O +semi B-protein_state +- I-protein_state +open I-protein_state +conformation O +captured O +in O +the O +low B-protein_state +pH I-protein_state +, O +apo B-protein_state +crystal B-evidence +structure I-evidence +( O +2 O +× O +H B-chemical ++, I-chemical +Fig O +. O +6a O +- O +c O +). O + +Taken O +together O +, O +this O +systematic B-experimental_method +computational I-experimental_method +analysis I-experimental_method +of O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +clearly O +demonstrates O +that O +the O +alternating O +- O +access O +and O +ion O +- O +recognition O +mechanisms O +in O +this O +Na B-protein_type ++/ I-protein_type +Ca2 I-protein_type ++ I-protein_type +exchanger I-protein_type +are O +coupled O +through O +the O +influence O +that O +the O +bound O +ions O +have O +on O +the O +free B-evidence +- I-evidence +energy I-evidence +landscape I-evidence +of O +the O +protein O +, O +which O +in O +turn O +determines O +whether O +or O +not O +the O +occluded B-protein_state +conformation O +is O +energetically O +feasible O +. O + +This O +occluded B-protein_state +conformation O +, O +which O +is O +a O +necessary O +intermediate O +between O +the O +outward B-protein_state +and O +inward B-protein_state +- I-protein_state +open I-protein_state +states O +, O +and O +which O +entails O +the O +internal O +dehydration B-protein_state +of O +the O +protein O +, O +is O +only O +attainable O +upon O +complete B-protein_state +occupancy I-protein_state +of O +the O +binding B-site +sites I-site +. O + +The O +alternating O +- O +access O +hypothesis O +implicitly O +dictates O +that O +the O +switch O +between O +outward B-protein_state +- O +and O +inward B-protein_state +- I-protein_state +open I-protein_state +conformations O +of O +a O +given O +secondary B-protein_state +- I-protein_state +active I-protein_state +transporter B-protein_type +must O +not O +occur O +unless O +the O +appropriate O +type O +and O +number O +of O +substrates O +are O +recognized O +. O + +It O +is O +however O +also O +non O +- O +trivial O +: O +antiporters B-protein_type +, O +for O +example O +, O +do O +not O +undergo O +the O +alternating O +- O +access O +transition O +without O +a O +cargo O +, O +but O +this O +is O +precisely O +how O +membrane B-protein_type +symporters I-protein_type +reset O +their O +transport O +cycles O +. O + +Similarly O +puzzling O +is O +that O +a O +given O +antiporter B-protein_type +will O +undergo O +this O +transition O +upon O +recognition O +of O +substrates O +of O +different O +charge O +, O +size O +and O +number O +. O + +Yet O +, O +when O +multiple O +species O +are O +to O +be O +co O +- O +translocated O +, O +by O +either O +an O +antiporter B-protein_type +or O +a O +symporter B-protein_type +, O +partial O +occupancies O +must O +not O +be O +conducive O +to O +the O +alternating B-site +- I-site +access I-site +switch I-site +. O + +Here O +, O +we O +have O +provided O +novel O +insights O +into O +this O +intriguing O +mechanism O +of O +conformational O +control O +through O +structural B-experimental_method +studies I-experimental_method +and O +quantitative B-experimental_method +molecular I-experimental_method +simulations I-experimental_method +of O +a O +Na B-protein_type ++/ I-protein_type +Ca2 I-protein_type ++ I-protein_type +exchanger I-protein_type +. O + +Specifically O +, O +our O +studies O +of O +NCX_Mj B-protein +reveal O +the O +mechanism O +of O +forward O +ion O +exchange O +( O +Fig O +. O +7 O +). O + +The O +internal O +symmetry O +of O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +and O +the O +inward B-protein_state +- I-protein_state +facing I-protein_state +crystal B-evidence +structures I-evidence +of O +several O +Ca2 B-protein_type ++/ I-protein_type +H I-protein_type ++ I-protein_type +exchangers I-protein_type +indicate O +that O +the O +alternating O +- O +access O +mechanism O +of O +NCX B-protein_type +proteins O +entails O +a O +sliding O +motion O +of O +TM1 B-structure_element +and O +TM6 B-structure_element +relative O +to O +the O +rest O +of O +the O +transporter B-protein_type +. O + +Here O +, O +we O +demonstrate O +that O +conformational O +changes O +in O +the O +extracellular B-structure_element +region I-structure_element +of O +the O +TM2 B-structure_element +- I-structure_element +TM3 I-structure_element +and O +TM7 B-structure_element +- I-structure_element +TM8 I-structure_element +bundle I-structure_element +precede O +and O +are O +necessary O +for O +the O +transition O +, O +and O +are O +associated O +with O +ion O +recognition O +and O +/ O +or O +release O +. O + +The O +most O +apparent O +of O +these O +changes O +involves O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +TM7 B-structure_element +( O +TM7ab B-structure_element +); O +together O +with O +more O +subtle O +displacements O +in O +TM2 B-structure_element +and O +TM3 B-structure_element +, O +this O +change O +in O +TM7ab B-structure_element +correlates O +with O +the O +opening O +and O +closing O +of O +two O +distinct O +aqueous B-site +channels I-site +leading O +into O +the O +ion B-site +- I-site +binding I-site +sites I-site +from O +the O +extracellular O +solution O +. O + +Interestingly O +, O +the O +bending O +of O +TM7 B-structure_element +associated O +with O +the O +occlusion O +of O +the O +ion B-site +- I-site +binding I-site +sites I-site +also O +unlocks O +its O +interaction O +with O +TM6 B-structure_element +, O +and O +thus O +enables O +TM6 B-structure_element +and O +TM1 B-structure_element +to O +freely O +slide O +to O +the O +inward B-protein_state +- I-protein_state +facing I-protein_state +conformation O +. O + +The O +crystal B-evidence +structures I-evidence +of O +NCX_Mj B-protein +reported O +here O +, O +with O +either O +Na B-chemical ++, I-chemical +Ca2 B-chemical ++, I-chemical +Sr2 B-chemical ++ I-chemical +or O +H B-chemical ++ I-chemical +bound B-protein_state +, O +capture O +the O +exchanger B-protein_type +in O +different O +conformational O +states O +. O + +These O +states O +can O +only O +represent O +a O +subset O +among O +all O +possible O +, O +but O +they O +ought O +to O +reflect O +inherent O +preferences O +of O +the O +transporter B-protein_type +, O +modulated O +by O +the O +experimental O +conditions O +. O + +For O +example O +, O +in O +the O +crystal B-evidence +of O +NCX_Mj B-protein +in O +LCP B-experimental_method +, O +the O +extracellular B-structure_element +half I-structure_element +of O +the O +gating B-structure_element +helices I-structure_element +( O +TM6 B-structure_element +and O +TM1 B-structure_element +) O +form O +a O +lattice O +contact O +, O +which O +might O +ultimately O +restrict O +the O +degree O +of O +opening O +of O +the O +ion B-site +- I-site +binding I-site +sites I-site +in O +some O +cases O +( O +e O +. O +g O +. O +in O +the O +apo B-protein_state +, O +low B-protein_state +pH I-protein_state +structure B-evidence +). O + +Nonetheless O +, O +the O +calculated B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscapes I-evidence +, O +derived O +without O +knowledge O +of O +the O +experimental O +data O +, O +reassuringly O +confirm O +that O +the O +crystallized B-evidence +structures I-evidence +correspond O +to O +mechanistically O +relevant O +, O +interconverting O +states O +. O + +The O +simulations B-experimental_method +also O +demonstrate O +how O +this O +landscape O +is O +drastically O +re O +- O +shaped O +upon O +each O +ion O +- O +binding O +event O +. O + +Indeed O +, O +we O +show O +that O +it O +is O +the O +presence O +or O +absence O +of O +the O +occluded B-protein_state +state O +in O +this O +landscape O +that O +explains O +the O +antiport O +function O +of O +NCX_Mj B-protein +and O +its O +3Na O ++: B-chemical +1Ca2 O ++ B-chemical +stoichiometry O +. O + +We O +posit O +that O +a O +similar O +principle O +might O +govern O +the O +alternating O +- O +access O +mechanism O +in O +other O +transporters B-protein_type +; O +that O +is O +, O +we O +anticipate O +that O +for O +both O +symporters B-protein_type +and O +antiporters B-protein_type +, O +it O +is O +the O +feasibility O +of O +the O +occluded B-protein_state +state O +, O +encoded O +in O +the O +protein B-evidence +conformational I-evidence +free I-evidence +- I-evidence +energy I-evidence +landscape I-evidence +and O +its O +dependence O +on O +substrate O +binding O +, O +that O +ultimately O +explains O +their O +specific O +coupling O +mechanisms O +. O + +In O +multiple O +ways O +, O +our O +findings O +provide O +an O +explanation O +for O +, O +existing O +functional O +, O +biochemical O +and O +biophysical O +data O +for O +both O +NCX_Mj B-protein +and O +its O +eukaryotic B-taxonomy_domain +homologues O +. O + +The O +striking O +quantitative O +agreement O +between O +the O +ion B-evidence +- I-evidence +binding I-evidence +affinities I-evidence +inferred O +from O +our O +crystallographic B-experimental_method +titrations I-experimental_method +and O +the O +Km B-evidence +and O +K1 B-evidence +/ I-evidence +2 I-evidence +values I-evidence +previously O +deduced O +from O +functional B-experimental_method +assays I-experimental_method +has O +been O +discussed O +above O +. O + +Consistent O +with O +that O +finding O +, O +mutations O +that O +have O +been O +shown O +to O +inactivate O +or O +diminish O +the O +transport O +activity O +of O +NCX_Mj B-protein +and O +cardiac O +NCX B-protein_type +perfectly O +map O +to O +the O +first O +ion O +- O +coordination O +shell O +in O +our O +NCX_Mj B-protein +structures B-evidence +( O +Supplementary O +Fig O +. O +4c O +- O +d O +). O + +The O +crystallographic B-evidence +data I-evidence +also O +provides O +the O +long O +- O +sought O +structural O +basis O +for O +the O +‘ O +two O +- O +site O +’ O +model O +proposed O +to O +describe O +competitive O +cation O +binding O +in O +eukaryotic B-taxonomy_domain +NCX B-protein_type +, O +underscoring O +the O +relevance O +of O +these O +studies O +of O +NCX_Mj B-protein +as O +a O +prototypical O +Na B-protein_type ++/ I-protein_type +Ca2 I-protein_type ++ I-protein_type +exchanger I-protein_type +. O + +Specifically O +, O +our O +crystal B-experimental_method +titrations I-experimental_method +suggest O +that O +, O +during O +forward O +Na B-chemical ++/ I-chemical +Ca2 B-chemical ++ I-chemical +exchange O +, O +sites O +Sint B-site +and O +SCa B-site +, O +which O +Ca2 B-chemical ++ I-chemical +and O +Na B-chemical ++ I-chemical +compete O +for O +, O +can O +be O +grouped O +into O +one O +; O +Na B-chemical ++ I-chemical +binding O +to O +these O +sites O +does O +not O +require O +high O +Na B-chemical ++ I-chemical +concentrations O +, O +and O +two O +Na B-chemical ++ I-chemical +ions O +along O +with O +a O +water B-chemical +molecule O +( O +at O +Smid B-site +) O +are O +sufficient O +to O +displace O +Ca2 B-chemical ++, I-chemical +explaining O +the O +Hill B-evidence +coefficient I-evidence +of O +~ O +2 O +for O +Na B-chemical ++- I-chemical +dependent O +inhibition O +of O +Ca2 B-chemical ++ I-chemical +fluxes O +. O + +The O +Sext B-site +site O +, O +by O +contrast O +, O +might O +be O +thought O +as O +an O +activation B-site +site I-site +for O +inward O +Na B-chemical ++ I-chemical +translocation O +, O +since O +this O +is O +where O +the O +third O +Na B-chemical ++ I-chemical +ion O +binds O +at O +high O +Na B-chemical ++ I-chemical +concentration O +, O +enabling O +the O +transition O +to O +the O +occluded B-protein_state +state O +. O + +Interestingly O +, O +binding O +of O +Ca2 B-chemical ++ I-chemical +to O +Smid B-site +appears O +to O +be O +also O +possible O +, O +but O +available O +evidence O +indicates O +that O +this O +event O +transiently O +blocks O +the O +exchange O +cycle O +. O + +Indeed O +, O +structures B-evidence +of O +NCX_Mj B-protein +bound B-protein_state +to I-protein_state +Cd2 B-chemical ++ I-chemical +or O +Mn2 B-chemical ++, I-chemical +both O +of O +which O +inhibit O +transport O +, O +show O +these O +ions O +at O +Smid B-site +; O +by O +contrast O +, O +Sr2 B-chemical ++ I-chemical +binds O +only O +to O +SCa B-site +, O +and O +accordingly O +, O +is O +transported O +by O +NCX B-protein_type +similarly O +to O +calcium B-chemical +. O + +Lastly O +, O +our O +theory O +that O +occlusion O +of O +NCX_Mj B-protein +is O +selectively O +induced O +upon O +Ca2 B-chemical ++ I-chemical +or O +Na B-chemical ++ I-chemical +recognition O +is O +consonant O +with O +a O +recent O +analysis O +of O +the O +rate O +of O +hydrogen B-experimental_method +- I-experimental_method +deuterium I-experimental_method +exchange I-experimental_method +( O +HDX B-experimental_method +) O +in O +NCX_Mj B-protein +, O +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +these O +ions O +, O +in O +conditions O +that O +favor O +outward B-protein_state +- I-protein_state +facing I-protein_state +conformations O +. O + +Specifically O +, O +saturating O +amounts O +of O +Ca2 B-chemical ++ I-chemical +or O +Na B-chemical ++ I-chemical +resulted O +in O +a O +noticeable O +slowdown O +in O +the O +HDX B-evidence +rate I-evidence +for O +extracellular O +portions O +of O +the O +α B-structure_element +- I-structure_element +repeat I-structure_element +helices I-structure_element +. O + +We O +interpret O +these O +observations O +as O +reflecting O +that O +the O +solvent O +accessibility O +of O +the O +protein O +interior O +is O +diminished O +upon O +ion O +recognition O +, O +consistent O +with O +our O +finding O +that O +opening O +and O +closing O +of O +extracellular O +aqueous O +pathways O +to O +the O +ion B-site +- I-site +binding I-site +sites I-site +depend O +on O +ion O +occupancy O +state O +. O + +In O +addition O +, O +the O +increased O +compactness O +of O +the O +protein O +tertiary O +structure O +in O +the O +occluded B-protein_state +state O +would O +also O +slow O +down O +the O +dynamics O +of O +the O +secondary O +- O +structure O +elements O +, O +and O +thus O +further O +reduce O +the O +HDX B-evidence +rate I-evidence +. O + +Our O +data O +would O +also O +explain O +the O +observation O +that O +the O +reduction O +in O +the O +HDX B-evidence +rate I-evidence +is O +comparable O +for O +Na B-chemical ++ I-chemical +and O +Ca2 B-chemical ++, I-chemical +as O +well O +as O +the O +finding O +that O +the O +degree O +of O +deuterium O +incorporation O +remains O +non O +- O +negligible O +even O +under O +saturating O +ion O +concentrations O +. O + +As O +the O +calculated B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscapes I-evidence +show O +, O +Na B-chemical ++ I-chemical +and O +Ca2 B-chemical ++ I-chemical +induce O +the O +occlusion O +of O +the O +transporter B-protein_type +in O +a O +comparable O +manner O +, O +and O +yet O +the O +ion B-protein_state +- I-protein_state +bound I-protein_state +states O +retain O +the O +ability O +to O +explore O +conformations O +that O +are O +partially O +or O +fully B-protein_state +open I-protein_state +to O +the O +extracellular O +solution O +, O +precisely O +so O +as O +to O +be O +able O +to O +unload O +and O +re O +- O +load O +the O +substrates O +. O + +Na B-chemical ++ I-chemical +binding O +to O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +. O + +( O +a O +) O +Overall O +structure B-evidence +of O +native B-protein_state +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +from O +crystals B-experimental_method +grown I-experimental_method +in O +150 O +mM O +Na B-chemical ++. I-chemical + +Colored O +spheres O +represent O +the O +bound O +Na B-chemical ++ I-chemical +( O +green O +) O +and O +water B-chemical +( O +red O +). O + +( O +b O +) O +Structural O +details O +and O +definition O +of O +the O +four O +central B-site +binding I-site +sites I-site +. O + +The O +electron B-evidence +density I-evidence +( O +grey O +mesh O +, O +1 O +. O +9 O +Å O +Fo B-evidence +- I-evidence +Fc I-evidence +ion I-evidence +omit I-evidence +map I-evidence +contoured O +at O +4σ O +) O +at O +Smid B-site +was O +modeled O +as O +water B-chemical +( O +red O +sphere O +) O +and O +those O +at O +Sext B-site +, O +SCa B-site +and O +Sint B-site +as O +Na B-chemical ++ I-chemical +ions O +( O +green O +spheres O +). O + +Further O +details O +are O +shown O +in O +Supplementary O +Fig O +. O +1 O +. O +( O +c O +) O +Concentration O +- O +dependent O +change O +in O +Na B-chemical ++ I-chemical +occupancy O +( O +see O +also O +Table O +1 O +). O + +All O +Fo B-evidence +– I-evidence +Fc I-evidence +ion I-evidence +- I-evidence +omit I-evidence +maps I-evidence +are O +calculated O +to O +2 O +. O +4 O +Å O +and O +contoured O +at O +3σ O +for O +comparison O +. O + +The O +displacement O +of O +A206 B-residue_name_number +reflects O +the O +[ O +Na B-chemical ++]- I-chemical +dependent O +conformational O +change O +from O +the O +partially B-protein_state +open I-protein_state +to O +the O +occluded B-protein_state +state O +( O +observed O +at O +low O +and O +high O +Na B-chemical ++ I-chemical +concentrations O +, O +respectively O +). O + +At O +20 O +mM O +Na B-chemical ++, I-chemical +both O +conformations O +co O +- O +exist O +. O + +No O +significant O +changes O +were O +observed O +in O +the O +side O +- O +chains O +involved O +in O +ion O +or O +water B-chemical +coordination O +at O +the O +SCa B-site +, O +Sint B-site +and O +Smid B-site +sites O +. O + +Na B-chemical ++- I-chemical +occupancy O +dependent O +conformational O +change O +in O +NCX_Mj B-protein +. O + +( O +a O +) O +Superimposition B-experimental_method +of O +the O +NCX_Mj B-protein +crystal B-evidence +structures I-evidence +obtained O +in O +high O +( O +100 O +mM O +, O +cyan O +cylinders O +) O +and O +low O +( O +10 O +mM O +, O +brown O +cylinders O +) O +Na B-chemical ++ I-chemical +concentrations O +. O + +( O +b O +) O +Close O +- O +up O +view O +of O +the O +interface B-site +between O +TM6 B-structure_element +and O +TM7ab B-structure_element +in O +the O +NCX_Mj B-protein +structures B-evidence +obtained O +at O +high O +and O +low O +Na B-chemical ++ I-chemical +concentrations O +( O +top O +and O +lower O +panels O +, O +respectively O +). O + +Residues O +forming O +van O +- O +der O +- O +Waals O +contacts O +in O +the O +structure B-evidence +at O +low B-protein_state +Na B-chemical ++ I-chemical +concentration O +are O +shown O +in O +detail O +. O + +( O +c O +) O +Close O +- O +up O +view O +of O +the O +Na B-site ++- I-site +binding I-site +sites I-site +. O + +The O +vacant O +Sext B-site +site O +in O +the O +structure B-evidence +at O +low B-protein_state +Na B-chemical ++ I-chemical +concentration O +is O +indicated O +with O +a O +white O +sphere O +. O + +Residues O +surrounding O +this O +site O +are O +also O +indicated O +; O +note O +A206 B-residue_name_number +( O +labeled O +in O +red O +) O +coordinates B-bond_interaction +Na B-chemical ++ I-chemical +at O +Sext B-site +via O +its O +backbone O +carbonyl O +oxygen O +. O + +( O +d O +) O +Extracellular O +solvent O +accessibility O +of O +the O +ion B-site +binding I-site +sites I-site +in O +the O +structures B-evidence +at O +high B-protein_state +and O +low B-protein_state +[ O +Na B-chemical ++]. I-chemical + +Putative O +solvent B-site +channels I-site +are O +represented O +as O +light O +- O +purple O +surfaces O +. O + +Divalent O +cation O +binding O +and O +apo B-protein_state +structure B-evidence +of O +NCX_Mj B-protein +. O +( O +a O +) O +A O +single O +Sr2 B-chemical ++ I-chemical +( O +dark O +blue O +sphere O +) O +binds O +at O +SCa B-site +in O +crystals B-experimental_method +titrated I-experimental_method +with O +10 O +mM O +Sr2 B-chemical ++ I-chemical +and O +2 O +. O +5 O +mM O +Na B-chemical ++ I-chemical +( O +see O +also O +Supplementary O +Fig O +. O +2 O +). O + +Residues O +involved O +in O +Sr2 B-chemical ++ I-chemical +coordination O +are O +labeled O +. O + +There O +are O +no O +significant O +changes O +in O +the O +side O +- O +chains O +involved O +in O +ion O +coordination O +, O +relative O +to O +the O +Na B-protein_state ++- I-protein_state +bound I-protein_state +state O +. O + +T50 B-residue_name_number +and O +T209 B-residue_name_number +( O +labeled O +in O +red O +) O +coordinate B-bond_interaction +Sr2 B-chemical ++ I-chemical +through O +their O +backbone O +carbonyl O +- O +oxygen O +atoms O +. O + +High O +Na B-chemical ++ I-chemical +concentration O +( O +100 O +mM O +) O +completely O +displaces O +Sr2 B-chemical ++ I-chemical +and O +reverts O +NCX_Mj B-protein +to O +the O +occluded B-protein_state +state O +( O +right O +panel O +). O + +The O +contour O +level O +of O +the O +Fo B-evidence +– I-evidence +Fc I-evidence +omit I-evidence +map I-evidence +of O +the O +structure B-evidence +at O +high O +Na B-chemical ++ I-chemical +concentration O +was O +lowered O +( O +to O +4σ O +) O +so O +as O +to O +visualize O +the O +density B-evidence +from O +Na B-chemical ++ I-chemical +ions O +and O +H2O B-chemical +. O + +( O +b O +) O +Ca2 B-chemical ++ I-chemical +( O +tanned O +spheres O +) O +binds O +either O +to O +SCa B-site +or O +Smid B-site +in O +crystals B-experimental_method +titrated I-experimental_method +with O +10 O +mM O +Ca2 B-chemical ++ I-chemical +and O +2 O +. O +5 O +mM O +Na B-chemical ++ I-chemical +( O +see O +also O +Supplementary O +Fig O +. O +2 O +). O + +The O +relative O +occupancies O +are O +55 O +% O +and O +45 O +%, O +respectively O +. O +( O +c O +) O +Superimposition B-experimental_method +of O +NCX_Mj B-protein +structures B-evidence +obtained O +at O +low O +Na B-chemical ++ I-chemical +concentration O +( O +10 O +mM O +) O +and O +pH O +6 O +. O +5 O +( O +brown O +) O +and O +in O +the O +absence B-protein_state +of I-protein_state +Na B-chemical ++ I-chemical +and O +pH B-protein_state +4 I-protein_state +( O +light O +green O +), O +referred O +to O +as O +apo B-protein_state +state O +. O +( O +d O +) O +Close O +- O +up O +view O +of O +the O +ion B-site +- I-site +binding I-site +sites I-site +in O +the O +apo B-protein_state +( O +or O +high B-protein_state +H I-protein_state ++) I-protein_state +state O +. O + +The O +side O +chains O +of O +E54 B-residue_name_number +and O +E213 B-residue_name_number +from O +the O +low B-protein_state +Na I-protein_state ++ I-protein_state +structure B-evidence +are O +also O +shown O +( O +light O +brown O +) O +for O +comparison O +. O + +White O +spheres O +indicate O +the O +location O +Sint B-site +, O +Smid B-site +SCa B-site +. O +( O +e O +) O +Extracellular O +solvent O +accessibility O +of O +the O +ion B-site +- I-site +binding I-site +sites I-site +in O +apo B-protein_state +NCX_Mj B-protein +. O + +Spontaneous O +changes O +in O +the O +structure B-evidence +of O +outward B-protein_state +- I-protein_state +occluded I-protein_state +, O +fully B-protein_state +Na I-protein_state ++- I-protein_state +occupied I-protein_state +NCX_Mj B-protein +( O +PDB O +code O +3V5U O +) O +upon O +sequential O +displacement O +of O +Na B-chemical ++. I-chemical + +( O +a O +) O +Representative O +simulation B-experimental_method +snapshots O +of O +NCX_Mj B-protein +( O +Methods O +) O +with O +Na B-chemical ++ I-chemical +bound B-protein_state +at I-protein_state +Sext B-site +, O +SCa B-site +and O +Sint B-site +( O +orange O +cartoons O +, O +green O +spheres O +) O +and O +with O +Na B-chemical ++ I-chemical +bound B-protein_state +only I-protein_state +at I-protein_state +SCa B-site +and O +Sint B-site +( O +marine O +cartoons O +, O +yellow O +spheres O +) O +( O +b O +) O +Close O +- O +up O +of O +the O +backbone O +of O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +TM7 B-structure_element +( O +TM7ab B-structure_element +), O +in O +the O +same O +Na B-chemical ++ I-chemical +occupancy O +states O +depicted O +in O +( O +a O +). O + +( O +c O +) O +Representative O +simulation B-evidence +snapshots I-evidence +( O +same O +as O +above O +) O +with O +Na B-chemical ++ I-chemical +bound B-protein_state +at I-protein_state +SCa B-site +and O +Sint B-site +( O +marine O +cartoons O +, O +yellow O +spheres O +) O +and O +without B-protein_state +any O +Na B-chemical ++ I-chemical +bound B-protein_state +( O +grey O +cartoons O +). O + +( O +d O +) O +Close O +- O +up O +of O +the O +ion B-site +- I-site +binding I-site +region I-site +in O +the O +fully B-protein_state +Na I-protein_state ++- I-protein_state +occupied I-protein_state +state O +. O + +Approximate O +distances O +between O +TM2 B-structure_element +, O +TM3 B-structure_element +and O +TM7 B-structure_element +are O +indicated O +in O +Å O +. O +( O +e O +) O +Close O +- O +up O +of O +the O +ion B-site +- I-site +binding I-site +region I-site +in O +the O +partially B-protein_state +Na I-protein_state ++- I-protein_state +occupied I-protein_state +state O +. O + +( O +f O +) O +Close O +- O +up O +of O +the O +ion B-site +- I-site +binding I-site +region I-site +in O +the O +Na B-protein_state ++- I-protein_state +free I-protein_state +state O +. O +( O +g O +- O +i O +) O +Analytical O +descriptors O +of O +the O +changes O +just O +described O +, O +calculated O +from O +the O +simulations B-experimental_method +of O +each O +Na B-protein_state ++- I-protein_state +occupancy I-protein_state +state O +depicted O +in O +panels O +( O +a O +- O +f O +). O + +These O +descriptors O +were O +employed O +as O +collective O +variables O +in O +the O +Bias B-experimental_method +- I-experimental_method +Exchange I-experimental_method +Metadynamics I-experimental_method +simulations I-experimental_method +( O +Methods O +). O + +( O +g O +) O +Probability B-evidence +distributions I-evidence +of O +an O +analytical O +descriptor O +of O +the O +backbone O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +pattern O +in O +TM7ab B-structure_element +( O +Eq O +. O +2 O +). O +( O +h O +) O +Mean O +value O +( O +with O +standard O +deviation O +) O +of O +a O +quantitative O +descriptor O +of O +the O +solvent O +accessibility O +of O +the O +Sext B-site +site O +( O +Eq O +. O +1 O +). O +( O +i O +) O +Mean O +value O +( O +with O +standard O +deviation O +) O +of O +a O +quantitative O +descriptor O +of O +the O +solvent O +accessibility O +of O +the O +SCa B-site +site O +( O +Eq O +. O +1 O +). O + +Thermodynamic O +basis O +for O +the O +proposed O +mechanism O +of O +substrate O +control O +of O +the O +alternating O +- O +access O +transition O +of O +NCX B-protein_type +. O +( O +a O +) O +Calculated B-evidence +conformational I-evidence +free I-evidence +- I-evidence +energy I-evidence +landscapes I-evidence +for O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +, O +for O +two O +different O +Na B-chemical ++- I-chemical +occupancy O +states O +, O +and O +for O +a O +state O +with O +no B-protein_state +ions I-protein_state +bound I-protein_state +. O + +The O +free B-evidence +energy I-evidence +is O +plotted O +as O +a O +function O +of O +two O +coordinates O +, O +each O +describing O +the O +degree O +of O +opening O +of O +the O +aqueous B-site +channels I-site +leading O +to O +the O +Sext B-site +and O +SCa B-site +sites O +, O +respectively O +( O +see O +Methods O +). O + +Black O +circles O +map O +the O +X B-evidence +- I-evidence +ray I-evidence +structures I-evidence +of O +NCX_Mj B-protein +obtained O +at O +high B-protein_state +and O +low B-protein_state +Na B-chemical ++ I-chemical +concentration O +, O +as O +well O +as O +that O +at O +low B-protein_state +pH I-protein_state +, O +reported O +in O +this O +study O +. O + +( O +b O +) O +Density B-evidence +isosurfaces I-evidence +for O +water B-chemical +molecules O +within O +12 O +Å O +of O +the O +ion B-site +- I-site +binding I-site +region I-site +( O +grey O +volumes O +), O +for O +each O +of O +the O +major O +conformational B-evidence +free I-evidence +- I-evidence +energy I-evidence +minima I-evidence +in O +each O +ion O +- O +occupancy O +state O +. O + +Na B-chemical ++ I-chemical +ions O +are O +shown O +as O +green O +spheres O +. O + +The O +two O +inverted B-structure_element +- I-structure_element +topology I-structure_element +repeats I-structure_element +in O +the O +transporter B-protein_type +structure B-evidence +( O +transparent O +cartoons O +) O +are O +colored O +differently O +( O +TM1 B-structure_element +- I-structure_element +5 I-structure_element +, O +orange O +; O +TM6 B-structure_element +- I-structure_element +10 I-structure_element +, O +marine O +). O + +( O +c O +) O +Close O +- O +up O +views O +of O +the O +ion B-site +- I-site +binding I-site +region I-site +in O +the O +same O +conformational B-evidence +free I-evidence +- I-evidence +energy I-evidence +minima I-evidence +. O + +Key O +residues O +involved O +in O +Na B-chemical ++ I-chemical +and O +water B-chemical +coordination O +( O +W O +) O +are O +highlighted O +( O +sticks O +, O +black O +lines O +). O + +The O +water B-evidence +- I-evidence +density I-evidence +maps I-evidence +in O +( O +b O +) O +is O +shown O +here O +as O +a O +grey O +mesh O +. O + +Note O +D240 B-residue_name_number +is O +protonated O +, O +while O +E54 B-residue_name_number +and O +E213 B-residue_name_number +are O +ionized O +. O + +Thermodynamic O +basis O +for O +the O +proposed O +mechanism O +of O +substrate O +control O +of O +the O +alternating O +- O +access O +transition O +of O +NCX B-protein_type +. O +( O +a O +) O +Calculated B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscapes I-evidence +for O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +, O +for O +the O +Ca2 B-chemical ++ I-chemical +and O +the O +fully B-protein_state +protonated I-protein_state +state O +. O + +The O +free B-evidence +energy I-evidence +is O +plotted O +as O +in O +Fig O +. O +5 O +. O + +For O +Ca2 B-chemical ++, I-chemical +a O +map B-evidence +is O +shown O +in O +which O +a O +correction O +for O +the O +charge O +- O +transfer O +between O +the O +ion O +and O +the O +protein O +is O +introduced O +, O +alongside O +the O +uncorrected O +map B-evidence +( O +see O +Supplementary O +Notes O +3 O +- O +4 O +and O +Supplementary O +Fig O +. O +5 O +- O +6 O +). O + +The O +uncorrected O +map B-evidence +overstabilizes O +the O +open B-protein_state +state O +relative O +to O +the O +semi B-protein_state +- I-protein_state +open I-protein_state +and O +occluded B-protein_state +because O +it O +also O +overestimates O +the O +cost O +of O +dehydration O +of O +the O +ion O +, O +once O +it O +is O +bound B-protein_state +to I-protein_state +the O +protein O +( O +this O +effect O +is O +negligible O +for O +Na B-chemical ++). I-chemical + +Black O +circles O +map O +the O +crystal B-evidence +structures I-evidence +obtained O +at O +high O +Ca2 B-chemical ++ I-chemical +concentration O +and O +at O +low B-protein_state +pH I-protein_state +( O +or O +high B-protein_state +H I-protein_state ++) I-protein_state +reported O +in O +this O +study O +. O + +( O +b O +) O +Water B-evidence +- I-evidence +density I-evidence +isosurfaces I-evidence +analogous O +to O +those O +in O +Fig O +. O +5 O +are O +shown O +for O +each O +of O +the O +major O +conformational O +free B-evidence +- I-evidence +energy I-evidence +minima I-evidence +in O +the O +free B-evidence +- I-evidence +energy I-evidence +maps I-evidence +. O + +The O +Ca2 B-chemical ++ I-chemical +ion O +is O +shown O +as O +a O +red O +sphere O +; O +the O +protein O +is O +shown O +as O +in O +Fig O +. O +5 O +. O +( O +c O +) O +Close O +- O +up O +views O +of O +the O +ion B-site +- I-site +binding I-site +region I-site +in O +the O +same O +conformational B-evidence +free I-evidence +- I-evidence +energy I-evidence +minima I-evidence +. O + +Key O +residues O +involved O +in O +Ca2 B-chemical ++ I-chemical +and O +water B-chemical +coordination O +( O +W O +) O +are O +highlighted O +( O +sticks O +, O +black O +lines O +). O + +The O +water B-evidence +- I-evidence +density I-evidence +maps I-evidence +in O +( O +b O +) O +are O +shown O +here O +as O +a O +grey O +mesh O +. O + +In O +the O +occluded B-protein_state +state O +with O +Ca2 B-chemical ++ I-chemical +bound B-protein_state +, O +helix B-structure_element +TM7ab B-structure_element +bends O +in O +the O +same O +way O +as O +in O +the O +fully B-protein_state +occupied I-protein_state +Na B-chemical ++ I-chemical +state O +, O +as O +the O +carbonyl O +of O +Ala206 B-residue_name_number +forms O +a O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +interaction I-bond_interaction +with O +Ser210 B-residue_name_number +. O + +Structural O +mechanism O +of O +extracellular O +forward O +ion O +exchange O +in O +NCX B-protein_type +. O + +The O +carbonyl O +groups O +of O +Ala47 B-residue_name_number +( O +on O +TM2b B-structure_element +) O +and O +Ala206 B-residue_name_number +( O +on O +TM7b B-structure_element +), O +and O +the O +side O +chains O +of O +Glu54 B-residue_name_number +( O +on O +TM2c B-structure_element +) O +and O +Glu213 B-residue_name_number +( O +on O +TM7c B-structure_element +) O +are O +highlighted O +; O +these O +are O +four O +of O +the O +key O +residues O +for O +ion O +chelation O +and O +conformational O +changes O +. O + +The O +green O +open O +cylinders O +represent O +the O +gating B-structure_element +helices I-structure_element +TM1 B-structure_element +and O +TM6 B-structure_element +. O + +Asterisks O +mark O +the O +states O +whose O +crystal B-evidence +structures I-evidence +have O +been O +determined O +in O +this O +study O +. O + +These O +states O +and O +their O +connectivity O +can O +also O +be O +deduced O +from O +the O +calculated B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscapes I-evidence +, O +which O +also O +reveal O +a O +Ca2 B-protein_state ++- I-protein_state +loaded I-protein_state +outward B-protein_state +- I-protein_state +facing I-protein_state +occluded B-protein_state +state O +, O +and O +an O +unloaded B-protein_state +, O +fully B-protein_state +open I-protein_state +state O +. O + +An O +extended B-protein_state +U2AF65 B-structure_element +– I-structure_element +RNA I-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +recognizes O +the O +3 B-site +′ I-site +splice I-site +site I-site +signal O + +How O +the O +essential O +pre B-protein_type +- I-protein_type +mRNA I-protein_type +splicing I-protein_type +factor I-protein_type +U2AF65 B-protein +recognizes O +the O +polypyrimidine B-chemical +( O +Py B-chemical +) O +signals O +of O +the O +major O +class O +of O +3 B-site +′ I-site +splice I-site +sites I-site +in O +human B-species +gene O +transcripts O +remains O +incompletely O +understood O +. O + +We O +determined B-experimental_method +four I-experimental_method +structures I-experimental_method +of O +an O +extended B-protein_state +U2AF65 B-structure_element +– I-structure_element +RNA I-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +bound B-protein_state +to I-protein_state +Py B-chemical +- I-chemical +tract I-chemical +oligonucleotides I-chemical +at O +resolutions O +between O +2 O +. O +0 O +and O +1 O +. O +5 O +Å O +. O +These O +structures B-evidence +together O +with O +RNA B-experimental_method +binding I-experimental_method +and I-experimental_method +splicing I-experimental_method +assays I-experimental_method +reveal O +unforeseen O +roles O +for O +U2AF65 B-protein +inter B-site +- I-site +domain I-site +residues I-site +in O +recognizing O +a O +contiguous B-structure_element +, O +nine O +- O +nucleotide B-chemical +Py B-chemical +tract I-chemical +. O + +The O +U2AF65 B-protein +linker B-structure_element +residues O +between O +the O +dual O +RNA B-structure_element +recognition I-structure_element +motifs I-structure_element +( O +RRMs B-structure_element +) O +recognize O +the O +central O +nucleotide B-chemical +, O +whereas O +the O +N O +- O +and O +C O +- O +terminal O +RRM B-structure_element +extensions I-structure_element +recognize O +the O +3 B-site +′ I-site +terminus I-site +and O +third B-residue_number +nucleotide B-chemical +. O + +Single B-experimental_method +- I-experimental_method +molecule I-experimental_method +FRET I-experimental_method +experiments O +suggest O +that O +conformational O +selection O +and O +induced O +fit O +of O +the O +U2AF65 B-protein +RRMs B-structure_element +are O +complementary O +mechanisms O +for O +Py B-chemical +- I-chemical +tract I-chemical +association O +. O + +Altogether O +, O +these O +results O +advance O +the O +mechanistic O +understanding O +of O +molecular O +recognition O +for O +a O +major O +class O +of O +splice B-site +site I-site +signals O +. O + +The O +pre B-protein_type +- I-protein_type +mRNA I-protein_type +splicing I-protein_type +factor I-protein_type +U2AF65 B-protein +recognizes O +3 B-site +′ I-site +splice I-site +sites I-site +in O +human B-species +gene O +transcripts O +, O +but O +the O +details O +are O +not O +fully O +understood O +. O + +Here O +, O +the O +authors O +report O +U2AF65 B-protein +structures B-evidence +and O +single B-experimental_method +molecule I-experimental_method +FRET I-experimental_method +that O +reveal O +mechanistic O +insights O +into O +splice B-site +site I-site +recognition O +. O + +The O +differential O +skipping O +or O +inclusion O +of O +alternatively O +spliced O +pre B-structure_element +- I-structure_element +mRNA I-structure_element +regions I-structure_element +is O +a O +major O +source O +of O +diversity O +for O +nearly O +all O +human B-species +gene O +transcripts O +. O + +The O +splice B-site +sites I-site +are O +marked O +by O +relatively O +short B-structure_element +consensus I-structure_element +sequences I-structure_element +and O +are O +regulated O +by O +additional O +pre B-structure_element +- I-structure_element +mRNA I-structure_element +motifs I-structure_element +( O +reviewed O +in O +ref O +.). O + +At O +the O +3 B-site +′ I-site +splice I-site +site I-site +of O +the O +major O +intron O +class O +, O +these O +include O +a O +polypyrimidine B-chemical +( I-chemical +Py I-chemical +) I-chemical +tract I-chemical +comprising O +primarily O +Us B-residue_name +or O +Cs B-residue_name +, O +which O +is O +preceded O +by O +a O +branch B-site +point I-site +sequence I-site +( O +BPS B-site +) O +that O +ultimately O +serves O +as O +the O +nucleophile O +in O +the O +splicing O +reaction O +and O +an O +AG B-chemical +- I-chemical +dinucleotide I-chemical +at O +the O +3 B-site +′ I-site +splice I-site +site I-site +junction O +. O + +Disease O +- O +causing O +mutations O +often O +compromise O +pre B-chemical +- I-chemical +mRNA I-chemical +splicing O +( O +reviewed O +in O +refs O +), O +yet O +a O +priori O +predictions O +of O +splice B-site +sites I-site +and O +the O +consequences O +of O +their O +mutations O +are O +challenged O +by O +the O +brevity O +and O +degeneracy O +of O +known O +splice B-site +site I-site +sequences O +. O + +High O +- O +resolution O +structures B-evidence +of O +intact B-protein_state +splicing B-complex_assembly +factor I-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complexes O +would O +offer O +key O +insights O +regarding O +the O +juxtaposition O +of O +the O +distinct O +splice B-site +site I-site +consensus O +sequences O +and O +their O +relationship O +to O +disease O +- O +causing O +point O +mutations O +. O + +The O +early O +- O +stage O +pre B-protein_type +- I-protein_type +mRNA I-protein_type +splicing I-protein_type +factor I-protein_type +U2AF65 B-protein +is O +essential O +for O +viability O +in O +vertebrates B-taxonomy_domain +and O +other O +model O +organisms O +( O +for O +example O +, O +ref O +.). O + +A O +tightly O +controlled O +assembly B-complex_assembly +among O +U2AF65 B-protein +, O +the O +pre B-chemical +- I-chemical +mRNA I-chemical +, O +and O +partner O +proteins O +sequentially O +identifies O +the O +3 B-site +′ I-site +splice I-site +site I-site +and O +promotes O +association O +of O +the O +spliceosome B-complex_assembly +, O +which O +ultimately O +accomplishes O +the O +task O +of O +splicing O +. O + +Initially O +U2AF65 B-protein +recognizes O +the O +Py B-chemical +- I-chemical +tract I-chemical +splice B-site +site I-site +signal O +. O + +In O +turn O +, O +the O +ternary B-complex_assembly +complex I-complex_assembly +of O +U2AF65 B-protein +with O +SF1 B-protein +and O +U2AF35 B-protein +identifies O +the O +surrounding O +BPS B-site +and O +3 B-site +′ I-site +splice I-site +site I-site +junctions O +. O + +Subsequently O +U2AF65 B-protein +recruits O +the O +U2 B-complex_assembly +small I-complex_assembly +nuclear I-complex_assembly +ribonucleoprotein I-complex_assembly +particle I-complex_assembly +( O +snRNP B-complex_assembly +) O +and O +ultimately O +dissociates O +from O +the O +active B-protein_state +spliceosome B-complex_assembly +. O + +Biochemical B-experimental_method +characterizations I-experimental_method +of O +U2AF65 B-protein +demonstrated O +that O +tandem O +RNA B-structure_element +recognition I-structure_element +motifs I-structure_element +( O +RRM1 B-structure_element +and O +RRM2 B-structure_element +) O +recognize O +the O +Py B-chemical +tract I-chemical +( O +Fig O +. O +1a O +). O + +Milestone O +crystal B-evidence +structures I-evidence +of O +the O +core B-protein_state +U2AF65 B-protein +RRM1 B-structure_element +and O +RRM2 B-structure_element +connected O +by O +a O +shortened B-protein_state +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +( O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +) O +detailed O +a O +subset O +of O +nucleotide O +interactions O +with O +the O +individual O +U2AF65 B-protein +RRMs B-structure_element +. O + +A O +subsequent O +NMR B-experimental_method +structure B-evidence +characterized O +the O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +arrangement O +of O +the O +minimal B-protein_state +U2AF65 B-protein +RRM1 B-structure_element +and O +RRM2 B-structure_element +connected O +by O +a O +linker B-structure_element +of O +natural B-protein_state +length I-protein_state +( O +U2AF651 B-mutant +, I-mutant +2 I-mutant +), O +yet O +depended O +on O +the O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +crystal B-evidence +structures I-evidence +for O +RNA B-chemical +interactions O +and O +an O +ab O +initio O +model O +for O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +conformation O +. O + +As O +such O +, O +the O +molecular O +mechanisms O +for O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +by O +the O +intact B-protein_state +U2AF65 B-structure_element +– I-structure_element +RNA I-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +remained O +unknown O +. O + +Here O +, O +we O +use O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +and O +biochemical B-experimental_method +studies I-experimental_method +to O +reveal O +new O +roles O +in O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +for O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +and O +key O +residues O +surrounding O +the O +core B-protein_state +U2AF65 B-protein +RRMs B-structure_element +. O + +We O +use O +single B-experimental_method +- I-experimental_method +molecule I-experimental_method +Förster I-experimental_method +resonance I-experimental_method +energy I-experimental_method +transfer I-experimental_method +( O +smFRET B-experimental_method +) O +to O +characterize O +the O +conformational B-evidence +dynamics I-evidence +of O +this O +extended B-protein_state +U2AF65 B-structure_element +– I-structure_element +RNA I-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +during O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +. O + +Cognate O +U2AF65 B-protein +– O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +requires O +RRM B-structure_element +extensions I-structure_element + +The O +RNA B-evidence +affinity I-evidence +of O +the O +minimal B-protein_state +U2AF651 B-mutant +, I-mutant +2 I-mutant +domain O +comprising O +the O +core B-protein_state +RRM1 B-structure_element +– O +RRM2 B-structure_element +folds B-structure_element +( O +U2AF651 B-mutant +, I-mutant +2 I-mutant +, O +residues O +148 B-residue_range +– I-residue_range +336 I-residue_range +) O +is O +relatively O +weak O +compared O +with O +full B-protein_state +- I-protein_state +length I-protein_state +U2AF65 B-protein +( O +Fig O +. O +1a O +, O +b O +; O +Supplementary O +Fig O +. O +1 O +). O + +Historically O +, O +this O +difference O +was O +attributed O +to O +the O +U2AF65 B-protein +arginine B-structure_element +– I-structure_element +serine I-structure_element +rich I-structure_element +domain I-structure_element +, O +which O +contacts O +pre B-complex_assembly +- I-complex_assembly +mRNA I-complex_assembly +– I-complex_assembly +U2 I-complex_assembly +snRNA I-complex_assembly +duplexes I-complex_assembly +outside O +of O +the O +Py B-chemical +tract I-chemical +. O + +We O +noticed O +that O +the O +RNA B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +U2AF651 B-mutant +, I-mutant +2 I-mutant +domain O +was O +greatly O +enhanced O +by O +the O +addition B-experimental_method +of I-experimental_method +seven I-experimental_method +and I-experimental_method +six I-experimental_method +residues I-experimental_method +at O +the O +respective O +N O +and O +C O +termini O +of O +the O +minimal B-protein_state +RRM1 B-structure_element +and O +RRM2 B-structure_element +( O +U2AF651 B-mutant +, I-mutant +2L I-mutant +, O +residues O +141 B-residue_range +– I-residue_range +342 I-residue_range +; O +Fig O +. O +1a O +). O + +In O +a O +fluorescence B-experimental_method +anisotropy I-experimental_method +assay I-experimental_method +for O +binding O +a O +representative O +Py B-chemical +tract I-chemical +derived O +from O +the O +well O +- O +characterized O +splice B-site +site I-site +of O +the O +adenovirus B-gene +major I-gene +late I-gene +promoter I-gene +( O +AdML B-gene +), O +the O +RNA B-evidence +affinity I-evidence +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +increased O +by O +100 O +- O +fold O +relative O +to O +U2AF651 B-mutant +, I-mutant +2 I-mutant +to O +comparable O +levels O +as O +full B-protein_state +- I-protein_state +length I-protein_state +U2AF65 B-protein +( O +Fig O +. O +1b O +; O +Supplementary O +Fig O +. O +1a O +– O +d O +). O + +Likewise O +, O +both O +U2AF651 B-mutant +, I-mutant +2L I-mutant +and O +full B-protein_state +- I-protein_state +length I-protein_state +U2AF65 B-protein +showed O +similar O +sequence B-evidence +specificity I-evidence +for O +U B-structure_element +- I-structure_element +rich I-structure_element +stretches I-structure_element +in O +the O +5 B-site +′- I-site +region I-site +of O +the O +Py B-chemical +tract I-chemical +and O +promiscuity O +for O +C B-structure_element +- I-structure_element +rich I-structure_element +regions I-structure_element +in O +the O +3 B-site +′- I-site +region I-site +( O +Fig O +. O +1c O +, O +Supplementary O +Fig O +. O +1e O +– O +h O +). O + +U2AF65 B-protein_state +- I-protein_state +bound I-protein_state +Py B-chemical +tract I-chemical +comprises O +nine O +contiguous B-structure_element +nucleotides B-chemical + +To O +investigate O +the O +structural O +basis O +for O +cognate O +U2AF65 B-protein +recognition O +of O +a O +contiguous B-structure_element +Py B-chemical +tract I-chemical +, O +we O +determined B-experimental_method +four O +crystal B-evidence +structures I-evidence +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +bound B-protein_state +to I-protein_state +Py B-chemical +- I-chemical +tract I-chemical +oligonucleotides I-chemical +( O +Fig O +. O +2a O +; O +Table O +1 O +). O + +By O +sequential B-experimental_method +boot I-experimental_method +strapping I-experimental_method +( O +Methods O +), O +we O +optimized O +the O +oligonucleotide B-chemical +length O +, O +the O +position O +of O +a O +Br B-chemical +- I-chemical +dU I-chemical +, O +and O +the O +identity O +of O +the O +terminal O +nucleotide B-chemical +( O +rU B-residue_name +, O +dU B-residue_name +and O +rC B-residue_name +) O +to O +achieve O +full O +views O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +bound B-protein_state +to I-protein_state +contiguous B-structure_element +Py B-chemical +tracts I-chemical +at O +up O +to O +1 O +. O +5 O +Å O +resolution O +. O + +The O +protein O +and O +oligonucleotide B-chemical +conformations O +are O +nearly O +identical O +among O +the O +four O +new O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +( O +Supplementary O +Fig O +. O +2a O +). O + +The O +U2AF651 B-mutant +, I-mutant +2L I-mutant +RRM1 B-structure_element +and O +RRM2 B-structure_element +associate O +with O +the O +Py B-chemical +tract I-chemical +in O +a O +parallel B-protein_state +, O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +arrangement O +( O +shown O +for O +representative O +structure O +iv O +in O +Fig O +. O +2b O +, O +c O +; O +Supplementary O +Movie O +1 O +). O + +An O +extended B-protein_state +conformation I-protein_state +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +traverses O +across O +the O +α B-structure_element +- I-structure_element +helical I-structure_element +surface I-structure_element +of O +RRM1 B-structure_element +and O +the O +central O +β B-structure_element +- I-structure_element +strands I-structure_element +of O +RRM2 B-structure_element +and O +is O +well O +defined O +in O +the O +electron B-evidence +density I-evidence +( O +Fig O +. O +2b O +). O + +The O +extensions B-structure_element +at O +the O +N O +terminus O +of O +RRM1 B-structure_element +and O +C O +terminus O +of O +RRM2 B-structure_element +adopt O +well O +- O +ordered O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +Both O +RRM1 B-structure_element +/ O +RRM2 B-structure_element +extensions B-structure_element +and O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +directly O +recognize O +the O +bound B-protein_state +oligonucleotide B-chemical +. O + +We O +compare O +the O +global O +conformation O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +with O +the O +prior O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +crystal B-evidence +structure I-evidence +and O +U2AF651 B-mutant +, I-mutant +2 I-mutant +NMR B-experimental_method +structure B-evidence +in O +the O +Supplementary O +Discussion O +and O +Supplementary O +Fig O +. O +2 O +. O + +The O +discovery O +of O +nine O +U2AF65 B-site +- I-site +binding I-site +sites I-site +for O +contiguous B-structure_element +Py B-chemical +- I-chemical +tract I-chemical +nucleotides I-chemical +was O +unexpected O +. O + +Based O +on O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +structures B-evidence +, O +we O +originally O +hypothesized O +that O +the O +U2AF65 B-protein +RRMs B-structure_element +would O +bind O +the O +minimal B-protein_state +seven O +nucleotides B-chemical +observed O +in O +these O +structures B-evidence +. O + +Surprisingly O +, O +the O +RRM2 B-structure_element +extension I-structure_element +/ O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +contribute O +new O +central O +nucleotide B-site +- I-site +binding I-site +sites I-site +near O +the O +RRM1 B-site +/ I-site +RRM2 I-site +junction I-site +and O +the O +RRM1 B-structure_element +extension I-structure_element +recognizes O +the O +3 O +′- O +terminal O +nucleotide B-chemical +( O +Fig O +. O +2c O +; O +Supplementary O +Movie O +1 O +). O + +The O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +characterize O +ribose B-chemical +( O +r B-chemical +) O +nucleotides B-chemical +at O +all O +of O +the O +binding B-site +sites I-site +except O +the O +seventh B-residue_number +and O +eighth B-residue_number +deoxy B-chemical +-( I-chemical +d I-chemical +) I-chemical +U I-chemical +, O +which O +are O +likely O +to O +lack O +2 O +′- O +hydroxyl O +contacts O +based O +on O +the O +RNA B-protein_state +- I-protein_state +bound I-protein_state +dU2AF651 B-mutant +, I-mutant +2 I-mutant +structure B-evidence +. O + +Qualitatively O +, O +a O +subset O +of O +the O +U2AF651 B-site +, I-site +2L I-site +- I-site +nucleotide I-site +- I-site +binding I-site +sites I-site +( O +sites B-site +1 I-site +– I-site +3 I-site +and O +7 B-site +– I-site +9 I-site +) O +share O +similar O +locations O +to O +those O +of O +the O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +structures B-evidence +( O +Supplementary O +Figs O +2c O +, O +d O +and O +3 O +). O + +Yet O +, O +only O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +interactions O +at O +sites B-site +1 I-site +and I-site +7 I-site +are O +nearly O +identical O +to O +those O +of O +the O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +structures B-evidence +( O +Supplementary O +Fig O +. O +3a O +, O +f O +). O + +In O +striking O +departures O +from O +prior O +partial O +views O +, O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +reveal O +three O +unanticipated O +nucleotide B-site +- I-site +binding I-site +sites I-site +at O +the O +centre O +of O +the O +Py B-chemical +tract I-chemical +, O +as O +well O +as O +numerous O +new O +interactions O +that O +underlie O +cognate O +recognition O +of O +the O +Py B-chemical +tract I-chemical +( O +Fig O +. O +3a O +– O +h O +). O + +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +interacts O +with O +the O +Py B-chemical +tract I-chemical + +The O +U2AF651 B-mutant +, I-mutant +2L I-mutant +RRM2 B-structure_element +, O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +and O +RRM1 B-structure_element +concomitantly O +recognize O +the O +three O +central O +nucleotides B-chemical +of O +the O +Py B-chemical +tract I-chemical +, O +which O +are O +likely O +to O +coordinate O +the O +conformational O +arrangement O +of O +these O +disparate O +portions O +of O +the O +protein O +. O + +Residues O +in O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +comprise O +a O +centrally O +located O +binding B-site +site I-site +for O +the O +fifth B-residue_number +nucleotide B-chemical +on O +the O +RRM2 B-site +surface I-site +and O +abutting O +the O +RRM1 B-site +/ I-site +RRM2 I-site +interface I-site +( O +Fig O +. O +3d O +). O + +The O +backbone O +amide O +of O +the O +linker B-structure_element +V254 B-residue_name_number +and O +the O +carbonyl O +of O +T252 B-residue_name_number +engage O +in O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +rU5 B-residue_name_number +- O +O4 O +and O +- O +N3H O +atoms O +. O + +In O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +strand I-structure_element +of O +RRM1 B-structure_element +, O +the O +side O +chains O +of O +K225 B-residue_name_number +and O +R227 B-residue_name_number +donate O +additional O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +rU5 B-residue_name_number +- O +O2 O +lone O +pair O +electrons O +. O + +The O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +also O +participates O +in O +the O +preceding O +rU4 B-site +- I-site +binding I-site +site I-site +, O +where O +the O +V254 B-residue_name_number +backbone O +carbonyl O +and O +D256 B-residue_name_number +carboxylate O +position O +the O +K260 B-residue_name_number +side O +chain O +to O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +rU4 B-residue_name_number +- O +O4 O +( O +Fig O +. O +3c O +). O + +Otherwise O +, O +the O +rU4 B-residue_name_number +nucleotide B-chemical +packs O +against O +F304 B-residue_name_number +in O +the O +signature O +ribonucleoprotein B-structure_element +consensus I-structure_element +motif I-structure_element +( I-structure_element +RNP I-structure_element +)- I-structure_element +2 I-structure_element +of O +RRM2 B-structure_element +. O + +At O +the O +opposite O +side O +of O +the O +central O +fifth B-residue_number +nucleotide B-chemical +, O +the O +sixth B-residue_number +rU6 B-residue_name_number +nucleotide B-chemical +is O +located O +at O +the O +inter B-site +- I-site +RRM1 I-site +/ I-site +RRM2 I-site +interface I-site +( O +Fig O +. O +3e O +; O +Supplementary O +Movie O +1 O +). O + +This O +nucleotide B-chemical +twists O +to O +face O +away O +from O +the O +U2AF65 B-protein +linker B-structure_element +and O +instead O +inserts O +the O +rU6 B-residue_name_number +- O +uracil B-residue_name +into O +a O +sandwich O +between O +the O +β2 B-structure_element +/ I-structure_element +β3 I-structure_element +loops I-structure_element +of O +RRM1 B-structure_element +and O +RRM2 B-structure_element +. O + +The O +rU6 B-residue_name_number +base O +edge O +is O +relatively O +solvent B-protein_state +exposed I-protein_state +; O +accordingly O +, O +the O +rU6 B-residue_name_number +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +U2AF65 B-protein +are O +water B-chemical +mediated O +apart O +from O +a O +single O +direct O +interaction O +by O +the O +RRM1 B-structure_element +- O +N196 B-residue_name_number +side O +chain O +. O + +We O +tested B-experimental_method +the I-experimental_method +contribution I-experimental_method +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +interactions O +with O +the O +new O +central O +nucleotide B-chemical +to O +Py B-evidence +- I-evidence +tract I-evidence +affinity I-evidence +( O +Fig O +. O +3i O +; O +Supplementary O +Fig O +. O +4a O +, O +b O +). O + +Mutagenesis B-experimental_method +of O +either O +V254 B-residue_name_number +in O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +to O +proline B-residue_name +or O +RRM1 B-structure_element +– O +R227 B-residue_name_number +to O +alanine B-residue_name +, O +which O +remove O +the O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +fifth B-residue_number +uracil B-residue_name +- O +O4 O +or O +- O +O2 O +, O +reduced O +the O +affinities B-evidence +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +for O +the O +representative O +AdML B-gene +Py B-chemical +tract I-chemical +by O +four O +- O +or O +five O +- O +fold O +, O +respectively O +. O + +The O +energetic O +penalties O +due O +to O +these O +mutations O +( O +ΔΔG B-evidence +0 O +. O +8 O +– O +0 O +. O +9 O +kcal O +mol O +− O +1 O +) O +are O +consistent O +with O +the O +loss O +of O +each O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +rU5 B-residue_name_number +base O +and O +support O +the O +relevance O +of O +the O +central O +nucleotide O +interactions O +observed O +in O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +. O + +U2AF65 B-protein +RRM B-structure_element +extensions I-structure_element +interact O +with O +the O +Py B-chemical +tract I-chemical + +The O +N B-structure_element +- I-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +extensions I-structure_element +of O +the O +U2AF65 B-protein +RRM1 B-structure_element +and O +RRM2 B-structure_element +directly O +contact O +the O +bound B-protein_state +Py B-chemical +tract I-chemical +. O + +Rather O +than O +interacting O +with O +a O +new O +5 O +′- O +terminal O +nucleotide B-chemical +as O +we O +had O +hypothesized O +, O +the O +C O +- O +terminal O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +RRM2 B-structure_element +instead O +folds O +across O +one O +surface O +of O +rU3 B-residue_name_number +in O +the O +third B-site +binding I-site +site I-site +( O +Fig O +. O +3b O +). O + +There O +, O +a O +salt B-bond_interaction +bridge I-bond_interaction +between O +the O +K340 B-residue_name_number +side O +chain O +and O +nucleotide B-chemical +phosphate O +, O +as O +well O +as O +G338 B-residue_name_number +- O +base O +stacking B-bond_interaction +and O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +the O +backbone O +amide O +of O +G338 B-residue_name_number +and O +the O +rU3 B-residue_name_number +- O +O4 O +, O +secure O +the O +RRM2 B-structure_element +extension I-structure_element +. O + +Indirectly O +, O +the O +additional O +contacts O +with O +the O +third B-residue_number +nucleotide B-chemical +shift O +the O +rU2 B-residue_name_number +nucleotide B-chemical +in O +the O +second B-site +binding I-site +site I-site +closer O +to O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +strand I-structure_element +of O +RRM2 B-structure_element +. O + +Consequently O +, O +the O +U2AF651 B-protein_state +, I-protein_state +2L I-protein_state +- I-protein_state +bound I-protein_state +rU2 B-residue_name_number +- O +O4 O +and O +- O +N3H O +form O +dual O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +K329 B-residue_name_number +backbone O +atoms O +( O +Fig O +. O +3a O +), O +rather O +than O +a O +single O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +K329 B-residue_name_number +side O +chain O +as O +in O +the O +prior O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +structure B-evidence +( O +Supplementary O +Fig O +. O +3b O +). O + +At O +the O +N O +terminus O +, O +the O +α B-structure_element +- I-structure_element +helical I-structure_element +extension I-structure_element +of O +U2AF65 B-protein +RRM1 B-structure_element +positions O +the O +Q147 B-residue_name_number +side O +chain O +to O +bridge O +the O +eighth B-residue_number +and O +ninth B-residue_number +nucleotides B-chemical +at O +the O +3 B-site +′ I-site +terminus I-site +of O +the O +Py B-chemical +tract I-chemical +( O +Fig O +. O +3f O +– O +h O +). O + +The O +Q147 B-residue_name_number +residue O +participates O +in O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +- O +N3H O +of O +the O +eighth B-residue_number +uracil B-residue_name +and O +- O +O2 O +of O +the O +ninth B-residue_number +pyrimidine B-chemical +. O + +The O +adjacent O +R146 B-residue_name_number +guanidinium O +group O +donates O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +3 O +′- O +terminal O +ribose B-chemical +- O +O2 O +′ O +and O +O3 O +′ O +atoms O +, O +where O +it O +could O +form O +a O +salt B-bond_interaction +bridge I-bond_interaction +with O +a O +phospho O +- O +diester O +group O +in O +the O +context O +of O +a O +longer O +pre B-chemical +- I-chemical +mRNA I-chemical +. O + +Consistent O +with O +loss O +of O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +ninth B-residue_number +pyrimidine B-chemical +- O +O2 O +( O +ΔΔG B-evidence +1 O +. O +0 O +kcal O +mol O +− O +1 O +), O +mutation B-experimental_method +of O +the O +Q147 B-residue_name_number +to O +an O +alanine B-residue_name +reduced O +U2AF651 B-evidence +, I-evidence +2L I-evidence +affinity I-evidence +for O +the O +AdML B-gene +Py B-chemical +tract I-chemical +by O +five O +- O +fold O +( O +Fig O +. O +3i O +; O +Supplementary O +Fig O +. O +4c O +). O + +We O +compare B-experimental_method +U2AF65 B-protein +interactions O +with O +uracil B-residue_name +relative O +to O +cytosine B-residue_name +pyrimidines B-chemical +at O +the O +ninth B-site +binding I-site +site I-site +in O +Fig O +. O +3g O +, O +h O +and O +the O +Supplementary O +Discussion O +. O + +Versatile O +primary O +sequence O +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element + +The O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +reveal O +that O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +mediates O +an O +extensive B-site +interface I-site +with O +the O +second O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +RRM1 B-structure_element +, O +the O +β2 B-structure_element +/ I-structure_element +β3 I-structure_element +strands I-structure_element +of O +RRM2 B-structure_element +and O +the O +N O +- O +terminal O +α B-structure_element +- I-structure_element +helical I-structure_element +extension I-structure_element +of O +RRM1 B-structure_element +. O + +Altogether O +, O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +residues O +( O +R228 B-residue_range +– I-residue_range +K260 I-residue_range +) O +bury O +2 O +, O +800 O +Å2 O +of O +surface O +area O +in O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +holo B-protein_state +- I-protein_state +protein I-protein_state +, O +suggestive O +of O +a O +cognate B-site +interface I-site +compared O +with O +1 O +, O +900 O +Å2 O +for O +a O +typical O +protein O +– O +protein O +complex O +. O + +The O +path O +of O +the O +linker B-structure_element +initiates O +at O +P229 B-residue_name_number +following O +the O +core B-protein_state +RRM1 B-structure_element +β B-structure_element +- I-structure_element +strand I-structure_element +, O +in O +a O +kink B-structure_element +that O +is O +positioned O +by O +intra B-bond_interaction +- I-bond_interaction +molecular I-bond_interaction +stacking I-bond_interaction +among O +the O +consecutive O +R228 B-residue_name_number +, O +Y232 B-residue_name_number +and O +P234 B-residue_name_number +side O +chains O +( O +Fig O +. O +4a O +, O +lower O +right O +). O + +A O +second B-structure_element +kink I-structure_element +at O +P236 B-residue_name_number +, O +coupled O +with O +respective O +packing O +of O +the O +L235 B-residue_name_number +and O +M238 B-residue_name_number +side O +chains O +on O +the O +N O +- O +terminal O +α B-structure_element +- I-structure_element +helical I-structure_element +RRM1 I-structure_element +extension I-structure_element +and O +the O +core B-protein_state +RRM1 B-structure_element +α2 B-structure_element +- I-structure_element +helix I-structure_element +, O +reverses O +the O +direction O +of O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +towards O +the O +RRM1 B-site +/ I-site +RRM2 I-site +interface I-site +and O +away O +from O +the O +RNA B-site +- I-site +binding I-site +site I-site +. O + +In O +the O +neighbouring O +apical O +region O +of O +the O +linker B-structure_element +, O +the O +V244 B-residue_name_number +and O +V246 B-residue_name_number +side O +chains O +pack O +in O +a O +hydrophobic B-site +pocket I-site +between O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +of O +the O +core B-protein_state +RRM1 B-structure_element +. O + +The O +adjacent O +V249 B-residue_name_number +and O +V250 B-residue_name_number +are O +notable O +for O +their O +respective O +interactions O +that O +connect O +RRM1 B-structure_element +and O +RRM2 B-structure_element +at O +this O +distal O +interface B-site +from O +the O +RNA B-site +- I-site +binding I-site +site I-site +( O +Fig O +. O +4a O +, O +top O +). O + +A O +third B-structure_element +kink I-structure_element +stacks B-bond_interaction +P247 B-residue_name_number +and O +G248 B-residue_name_number +with O +Y245 B-residue_name_number +and O +re O +- O +orients O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +the O +linker B-structure_element +towards O +the O +RRM2 B-structure_element +and O +bound B-protein_state +RNA B-chemical +. O + +At O +the O +RNA B-chemical +surface O +, O +the O +key O +V254 B-residue_name_number +that O +recognizes O +the O +fifth B-residue_number +uracil B-residue_name +is O +secured O +via O +hydrophobic B-bond_interaction +contacts I-bond_interaction +between O +its O +side O +chain O +and O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +surface I-structure_element +of O +RRM2 B-structure_element +, O +chiefly O +the O +consensus O +RNP1 B-structure_element +- O +F304 B-residue_name_number +residue O +that O +stacks B-bond_interaction +with O +the O +fourth B-residue_number +uracil B-residue_name +( O +Fig O +. O +4a O +, O +lower O +left O +). O + +Few O +direct O +contacts O +are O +made O +between O +the O +remaining O +residues O +of O +the O +linker B-structure_element +and O +the O +U2AF65 B-protein +RRM2 B-structure_element +; O +instead O +, O +the O +C O +- O +terminal O +conformation O +of O +the O +linker B-structure_element +appears O +primarily O +RNA B-chemical +mediated O +( O +Fig O +. O +3c O +, O +d O +). O + +We O +investigated O +whether O +the O +observed O +contacts O +between O +the O +RRMs B-structure_element +and O +linker B-structure_element +were O +critical O +for O +RNA O +binding O +by O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +mutagenesis I-experimental_method +( O +Fig O +. O +4b O +). O + +We O +titrated B-experimental_method +these O +mutant B-protein_state +U2AF651 B-mutant +, I-mutant +2L I-mutant +proteins O +into O +fluorescein B-chemical +- O +labelled O +AdML B-gene +Py B-chemical +- I-chemical +tract I-chemical +RNA I-chemical +and O +fit O +the O +fluorescence B-evidence +anisotropy I-evidence +changes I-evidence +to O +obtain O +the O +apparent O +equilibrium B-evidence +affinities I-evidence +( O +Supplementary O +Fig O +. O +4d O +– O +h O +). O + +We O +introduced O +glycine B-residue_name +substitutions B-experimental_method +to O +maximally O +reduce O +the O +buried O +surface O +area O +without O +directly O +interfering O +with O +its O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +backbone O +atoms O +and O +the O +base O +. O + +First O +, O +we O +replaced B-experimental_method +V249 B-residue_name_number +and O +V250 B-residue_name_number +at O +the O +RRM1 B-site +/ I-site +RRM2 I-site +interface I-site +and O +V254 B-residue_name_number +at O +the O +bound B-protein_state +RNA B-chemical +site O +with O +glycine B-residue_name +( O +3Gly B-mutant +). O + +However O +, O +the O +resulting O +decrease O +in O +the O +AdML B-gene +RNA B-evidence +affinity I-evidence +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +- I-mutant +3Gly I-mutant +mutant B-protein_state +relative O +to O +wild B-protein_state +- I-protein_state +type I-protein_state +protein B-protein +was O +not O +significant O +( O +Fig O +. O +4b O +). O + +In O +parallel O +, O +we O +replaced B-experimental_method +five O +linker B-structure_element +residues I-structure_element +( O +S251 B-residue_name_number +, O +T252 B-residue_name_number +, O +V253 B-residue_name_number +, O +V254 B-residue_name_number +and O +P255 B-residue_name_number +) O +at O +the O +fifth B-site +nucleotide I-site +- I-site +binding I-site +site I-site +with O +glycines B-residue_name +( O +5Gly B-mutant +) O +and O +also O +found O +that O +the O +RNA B-evidence +affinity I-evidence +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +- I-mutant +5Gly I-mutant +mutant B-protein_state +likewise O +decreased O +only O +slightly O +relative O +to O +wild B-protein_state +- I-protein_state +type I-protein_state +protein B-protein +. O + +A O +more O +conservative B-experimental_method +substitution I-experimental_method +of O +these O +five O +residues O +( O +251 B-residue_range +– I-residue_range +255 I-residue_range +) O +with O +an O +unrelated O +sequence O +capable O +of O +backbone O +- O +mediated O +hydrogen B-bond_interaction +bonds I-bond_interaction +( O +STVVP B-mutant +> I-mutant +NLALA I-mutant +) O +confirmed O +the O +subtle O +impact O +of O +this O +versatile O +inter B-structure_element +- I-structure_element +RRM I-structure_element +sequence I-structure_element +on O +affinity B-evidence +for O +the O +AdML B-gene +Py B-chemical +tract I-chemical +. O + +Finally O +, O +to O +ensure O +that O +these O +selective O +mutations O +were O +sufficient O +to O +disrupt O +the O +linker B-structure_element +/ O +RRM B-structure_element +contacts O +, O +we O +substituted B-experimental_method +glycine B-residue_name +for O +the O +majority O +of O +buried O +hydrophobic O +residues O +in O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +( O +including O +M144 B-residue_name_number +, O +L235 B-residue_name_number +, O +M238 B-residue_name_number +, O +V244 B-residue_name_number +, O +V246 B-residue_name_number +, O +V249 B-residue_name_number +, O +V250 B-residue_name_number +, O +S251 B-residue_name_number +, O +T252 B-residue_name_number +, O +V253 B-residue_name_number +, O +V254 B-residue_name_number +, O +P255 B-residue_name_number +; O +called O +12Gly B-mutant +). O + +Despite O +12 B-experimental_method +concurrent I-experimental_method +mutations I-experimental_method +, O +the O +AdML B-gene +RNA B-evidence +affinity I-evidence +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +- I-mutant +12Gly I-mutant +variant B-protein_state +was O +reduced O +by O +only O +three O +- O +fold O +relative O +to O +the O +unmodified B-protein_state +protein B-protein +( O +Fig O +. O +4b O +), O +which O +is O +less O +than O +the O +penalty O +of O +the O +V254P B-mutant +mutation O +that O +disrupts O +the O +rU5 B-residue_name_number +hydrogen B-bond_interaction +bond I-bond_interaction +( O +Fig O +. O +3d O +, O +i O +). O + +To O +test O +the O +interplay O +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +with O +its O +N O +- O +and O +C O +- O +terminal O +RRM B-structure_element +extensions I-structure_element +, O +we O +constructed B-experimental_method +an O +internal O +linker B-experimental_method +deletion I-experimental_method +of O +20 B-residue_range +- I-residue_range +residues I-residue_range +within O +the O +extended B-protein_state +RNA B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +( O +dU2AF651 B-mutant +, I-mutant +2L I-mutant +). O + +We O +found O +that O +the O +affinity B-evidence +of O +dU2AF651 B-mutant +, I-mutant +2L I-mutant +for O +the O +AdML B-gene +RNA B-chemical +was O +significantly O +reduced O +relative O +to O +U2AF651 B-mutant +, I-mutant +2L I-mutant +( O +four O +- O +fold O +, O +Figs O +1b O +and O +4b O +; O +Supplementary O +Fig O +. O +4i O +). O + +Yet O +, O +it O +is O +well O +known O +that O +the O +linker B-experimental_method +deletion I-experimental_method +in O +the O +context O +of O +the O +minimal B-protein_state +RRM1 B-structure_element +– O +RRM2 B-structure_element +boundaries O +has O +no O +detectable O +effect O +on O +the O +RNA B-evidence +affinities I-evidence +of O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +compared O +with O +U2AF651 B-mutant +, I-mutant +2 I-mutant +( O +refs O +; O +Figs O +1b O +and O +4b O +; O +Supplementary O +Fig O +. O +4j O +). O + +The O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +suggest O +that O +an O +extended B-protein_state +conformation I-protein_state +of O +the O +truncated B-protein_state +dU2AF651 B-mutant +, I-mutant +2 I-mutant +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +would O +suffice O +to O +connect O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +RRM1 B-structure_element +C O +terminus O +to O +the O +N O +terminus O +of O +RRM2 B-structure_element +( O +24 O +Å O +distance O +between O +U2AF651 B-mutant +, I-mutant +2L I-mutant +R227 B-residue_name_number +- O +Cα O +– O +H259 B-residue_name_number +- O +Cα O +atoms O +), O +which O +agrees O +with O +the O +greater O +RNA B-evidence +affinities I-evidence +of O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +and O +U2AF651 B-mutant +, I-mutant +2 I-mutant +dual B-protein_state +RRMs B-structure_element +compared O +with O +the O +individual B-protein_state +U2AF65 B-protein +RRMs B-structure_element +. O + +However O +, O +stretching O +of O +the O +truncated B-protein_state +dU2AF651 B-mutant +, I-mutant +2L I-mutant +linker B-structure_element +to O +connect O +the O +RRM B-structure_element +termini I-structure_element +is O +expected O +to O +disrupt O +its O +nucleotide O +interactions O +. O + +Likewise O +, O +deletion B-experimental_method +of O +the O +N O +- O +terminal O +RRM1 B-structure_element +extension I-structure_element +in O +the O +shortened B-protein_state +constructs O +would O +remove O +packing O +interactions O +that O +position O +the O +linker B-structure_element +in O +a O +kinked B-structure_element +turn I-structure_element +following O +P229 B-residue_name_number +( O +Fig O +. O +4a O +), O +consistent O +with O +the O +lower O +RNA B-evidence +affinities I-evidence +of O +dU2AF651 B-mutant +, I-mutant +2L I-mutant +, O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +and O +U2AF651 B-mutant +, I-mutant +2 I-mutant +compared O +with O +U2AF651 B-mutant +, I-mutant +2L I-mutant +. O + +To O +further O +test O +cooperation O +among O +the O +U2AF65 B-protein +RRM B-structure_element +extensions I-structure_element +and O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +for O +RNA O +recognition O +, O +we O +tested O +the O +impact O +of O +a O +triple O +Q147A B-mutant +/ O +V254P B-mutant +/ O +R227A B-mutant +mutation B-experimental_method +( O +U2AF651 B-mutant +, I-mutant +2L I-mutant +- I-mutant +3Mut I-mutant +) O +for O +RNA O +binding O +( O +Fig O +. O +4b O +; O +Supplementary O +Fig O +. O +4d O +). O + +Notably O +, O +the O +Q147A B-mutant +/ O +V254P B-mutant +/ O +R227A B-mutant +mutation B-experimental_method +reduced O +the O +RNA B-evidence +affinity I-evidence +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +- I-mutant +3Mut I-mutant +protein O +by O +30 O +- O +fold O +more O +than O +would O +be O +expected O +based O +on O +simple O +addition O +of O +the O +ΔΔG B-evidence +' O +s O +for O +the O +single O +mutations O +. O + +This O +difference O +indicates O +that O +the O +linearly B-protein_state +distant I-protein_state +regions B-structure_element +of O +the O +U2AF65 B-protein +primary O +sequence O +, O +including O +Q147 B-residue_name_number +in O +the O +N O +- O +terminal O +RRM1 B-structure_element +extension I-structure_element +and O +R227 B-residue_name_number +/ O +V254 B-residue_name_number +in O +the O +N O +-/ O +C O +- O +terminal O +linker B-structure_element +regions I-structure_element +at O +the O +fifth B-site +nucleotide I-site +site I-site +, O +cooperatively O +recognize O +the O +Py B-chemical +tract I-chemical +. O + +Altogether O +, O +we O +conclude O +that O +the O +conformation O +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +is O +key O +for O +recognizing O +RNA B-chemical +and O +is O +positioned O +by O +the O +RRM B-structure_element +extension I-structure_element +but O +otherwise O +relatively O +independent O +of O +the O +side O +chain O +composition O +. O + +The O +non O +- O +additive O +effects O +of O +the O +Q147A B-mutant +/ O +V254P B-mutant +/ O +R227A B-mutant +triple B-experimental_method +mutation I-experimental_method +, O +coupled O +with O +the O +context O +- O +dependent O +penalties O +of O +an O +internal O +U2AF65 B-protein +linker B-experimental_method +deletion I-experimental_method +, O +highlights O +the O +importance O +of O +the O +structural O +interplay O +among O +the O +U2AF65 B-protein +linker B-structure_element +and O +the O +N B-structure_element +- I-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +extensions I-structure_element +flanking O +the O +core B-protein_state +RRMs B-structure_element +. O + +Importance O +of O +U2AF65 B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +contacts O +for O +pre B-chemical +- I-chemical +mRNA I-chemical +splicing O + +We O +proceeded O +to O +test O +the O +importance O +of O +new O +U2AF65 B-complex_assembly +– I-complex_assembly +Py I-complex_assembly +- I-complex_assembly +tract I-complex_assembly +interactions O +for O +splicing O +of O +a O +model O +pre B-chemical +- I-chemical +mRNA I-chemical +substrate O +in O +a O +human B-species +cell O +line O +( O +Fig O +. O +5 O +; O +Supplementary O +Fig O +. O +5 O +). O + +As O +a O +representative O +splicing O +substrate O +, O +we O +utilized O +a O +well O +- O +characterized O +minigene B-chemical +splicing I-chemical +reporter I-chemical +( O +called O +pyPY B-chemical +) O +comprising O +a O +weak O +( O +that O +is O +, O +degenerate O +, O +py B-chemical +) O +and O +strong O +( O +that O +is O +, O +U B-structure_element +- I-structure_element +rich I-structure_element +, O +PY B-chemical +) O +polypyrimidine B-chemical +tracts I-chemical +preceding O +two O +alternative O +splice B-site +sites I-site +( O +Fig O +. O +5a O +). O + +When O +transfected B-experimental_method +into O +HEK293T O +cells O +containing O +only O +endogenous B-protein_state +U2AF65 B-protein +, O +the O +PY B-site +splice I-site +site I-site +is O +used O +and O +the O +remaining O +transcript O +remains O +unspliced O +. O + +When O +co B-experimental_method +- I-experimental_method +transfected I-experimental_method +with O +an O +expression B-experimental_method +plasmid I-experimental_method +for O +wild B-protein_state +- I-protein_state +type I-protein_state +U2AF65 B-protein +, O +use O +of O +the O +py B-site +splice I-site +site I-site +significantly O +increases O +( O +by O +more O +than O +five O +- O +fold O +) O +and O +as O +documented O +converts O +a O +fraction O +of O +the O +unspliced O +to O +spliced O +transcript O +. O + +The O +strong O +PY B-site +splice I-site +site I-site +is O +insensitive O +to O +added O +U2AF65 B-protein +, O +suggesting O +that O +endogenous B-protein_state +U2AF65 B-protein +levels O +are O +sufficient O +to O +saturate O +this O +site O +( O +Supplementary O +Fig O +. O +5b O +). O + +We O +introduced O +the O +triple B-experimental_method +mutation I-experimental_method +( O +V254P B-mutant +/ O +R227A B-mutant +/ O +Q147A B-mutant +) O +that O +significantly O +reduced O +U2AF651 B-mutant +, I-mutant +2L I-mutant +association O +with O +the O +Py B-chemical +tract I-chemical +( O +Fig O +. O +4b O +) O +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +U2AF65 B-protein +( O +U2AF65 B-mutant +- I-mutant +3Mut I-mutant +). O + +Co B-experimental_method +- I-experimental_method +transfection I-experimental_method +of O +the O +U2AF65 B-mutant +- I-mutant +3Mut I-mutant +with O +the O +pyPY B-chemical +splicing O +substrate O +significantly O +reduced O +splicing O +of O +the O +weak O +‘ B-site +py I-site +' I-site +splice I-site +site I-site +relative O +to O +wild B-protein_state +- I-protein_state +type I-protein_state +U2AF65 B-protein +( O +Fig O +. O +5b O +, O +c O +). O + +We O +conclude O +that O +the O +Py B-chemical +- I-chemical +tract I-chemical +interactions O +with O +these O +residues O +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +and O +RRM B-structure_element +extensions I-structure_element +are O +important O +for O +splicing O +as O +well O +as O +for O +binding O +a O +representative O +of O +the O +major B-structure_element +U2 I-structure_element +- I-structure_element +class I-structure_element +of I-structure_element +splice I-structure_element +sites I-structure_element +. O + +Sparse O +inter B-structure_element +- I-structure_element +RRM I-structure_element +contacts O +underlie O +apo B-protein_state +- O +U2AF65 B-protein +dynamics O + +The O +direct O +interface B-site +between O +U2AF651 B-mutant +, I-mutant +2L I-mutant +RRM1 B-structure_element +and O +RRM2 B-structure_element +is O +minor O +, O +burying O +265 O +Å2 O +of O +solvent O +accessible O +surface O +area O +compared O +with O +570 O +Å2 O +on O +average O +for O +a O +crystal O +packing O +interface O +. O + +A O +handful O +of O +inter B-structure_element +- I-structure_element +RRM I-structure_element +hydrogen B-bond_interaction +bonds I-bond_interaction +are O +apparent O +between O +the O +side O +chains O +of O +RRM1 B-structure_element +- O +N155 B-residue_name_number +and O +RRM2 B-structure_element +- O +K292 B-residue_name_number +, O +RRM1 B-structure_element +- O +N155 B-residue_name_number +and O +RRM2 B-structure_element +- O +D272 B-residue_name_number +as O +well O +as O +the O +backbone O +atoms O +of O +RRM1 B-structure_element +- O +G221 B-residue_name_number +and O +RRM2 B-structure_element +- O +D273 B-residue_name_number +( O +Fig O +. O +4c O +). O + +This O +minor O +U2AF65 B-protein +RRM1 B-site +/ I-site +RRM2 I-site +interface I-site +, O +coupled O +with O +the O +versatile O +sequence O +of O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +, O +highlighted O +the O +potential O +role O +for O +inter B-structure_element +- I-structure_element +RRM I-structure_element +conformational O +dynamics O +in O +U2AF65 B-protein +- O +splice O +site O +recognition O +. O + +Paramagnetic B-experimental_method +resonance I-experimental_method +enhancement I-experimental_method +( O +PRE B-experimental_method +) O +measurements O +previously O +had O +suggested O +a O +predominant O +back B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +back I-protein_state +, O +or O +‘ O +closed B-protein_state +' O +conformation O +of O +the O +apo B-protein_state +- O +U2AF651 B-mutant +, I-mutant +2 I-mutant +RRM1 B-structure_element +and O +RRM2 B-structure_element +in O +equilibrium O +with O +a O +minor O +‘ O +open B-protein_state +' O +conformation O +resembling O +the O +RNA B-protein_state +- I-protein_state +bound I-protein_state +inter B-structure_element +- I-structure_element +RRM I-structure_element +arrangement O +. O + +Yet O +, O +small B-experimental_method +- I-experimental_method +angle I-experimental_method +X I-experimental_method +- I-experimental_method +ray I-experimental_method +scattering I-experimental_method +( O +SAXS B-experimental_method +) O +data O +indicated O +that O +both O +the O +minimal B-protein_state +U2AF651 B-mutant +, I-mutant +2 I-mutant +and O +longer O +constructs O +comprise O +a O +highly B-protein_state +diverse I-protein_state +continuum I-protein_state +of I-protein_state +conformations I-protein_state +in O +the O +absence B-protein_state +of I-protein_state +RNA B-chemical +that O +includes O +the O +‘ O +closed B-protein_state +' O +and O +‘ O +open B-protein_state +' O +conformations O +. O + +To O +complement O +the O +static O +portraits O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structure B-evidence +that O +we O +had O +determined O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +, O +we O +used O +smFRET B-experimental_method +to O +characterize O +the O +probability B-evidence +distribution I-evidence +functions I-evidence +and O +time O +dependence O +of O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +conformational O +dynamics O +in O +solution O +. O + +The O +inter B-structure_element +- I-structure_element +RRM I-structure_element +dynamics O +of O +U2AF65 B-protein +were O +followed O +using O +FRET B-experimental_method +between O +fluorophores B-chemical +attached O +to O +RRM1 B-structure_element +and O +RRM2 B-structure_element +( O +Fig O +. O +6a O +, O +b O +, O +Methods O +). O + +The O +positions O +of O +single O +cysteine B-residue_name +mutations B-experimental_method +for O +fluorophore B-chemical +attachment O +( O +A181C B-mutant +in O +RRM1 B-structure_element +and O +Q324C B-mutant +in O +RRM2 B-structure_element +) O +were O +chosen O +based O +on O +inspection O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +and O +the O +‘ O +closed B-protein_state +' O +model O +of O +apo B-protein_state +- O +U2AF651 B-mutant +, I-mutant +2 I-mutant +. O + +Criteria O +included O +( O +i O +) O +residue O +locations O +that O +are O +distant O +from O +and O +hence O +not O +expected O +to O +interfere O +with O +the O +RRM B-complex_assembly +/ I-complex_assembly +RNA I-complex_assembly +or O +inter B-site +- I-site +RRM I-site +interfaces I-site +, O +( O +ii O +) O +inter O +- O +dye O +distances O +( O +50 O +Å O +for O +U2AF651 B-complex_assembly +, I-complex_assembly +2L I-complex_assembly +– I-complex_assembly +Py I-complex_assembly +tract I-complex_assembly +and O +30 O +Å O +for O +the O +closed B-protein_state +apo B-protein_state +- O +model O +) O +that O +are O +expected O +to O +be O +near O +the O +Förster B-experimental_method +radius I-experimental_method +( I-experimental_method +Ro I-experimental_method +) I-experimental_method +for O +the O +Cy3 B-chemical +/ O +Cy5 B-chemical +pair O +( O +56 O +Å O +), O +where O +changes O +in O +the O +efficiency O +of O +energy O +transfer O +are O +most O +sensitive O +to O +distance O +, O +and O +( O +iii O +) O +FRET B-evidence +efficiencies I-evidence +that O +are O +calculated O +to O +be O +significantly O +greater O +for O +the O +‘ O +closed B-protein_state +' O +apo B-protein_state +- O +model O +as O +opposed O +to O +the O +‘ O +open B-protein_state +' O +RNA B-protein_state +- I-protein_state +bound I-protein_state +structures B-evidence +( O +by O +∼ O +30 O +%). O + +The O +FRET B-evidence +efficiencies I-evidence +of O +either O +of O +these O +structurally O +characterized O +conformations O +also O +are O +expected O +to O +be O +significantly O +greater O +than O +elongated B-protein_state +U2AF65 B-protein +conformations O +that O +lack B-protein_state +inter O +- O +RRM B-structure_element +contacts O +. O + +Double O +- O +cysteine B-residue_name +variant B-protein_state +of O +U2AF651 B-mutant +, I-mutant +2 I-mutant +was O +modified B-experimental_method +with O +equimolar O +amount O +of O +Cy3 B-chemical +and O +Cy5 B-chemical +. O + +Only O +traces B-evidence +that O +showed O +single O +photobleaching O +events O +for O +both O +donor O +and O +acceptor O +dyes O +and O +anti O +- O +correlated O +changes O +in O +acceptor O +and O +donor O +fluorescence O +were O +included O +in O +smFRET B-experimental_method +data O +analysis O +. O + +We O +first O +characterized O +the O +conformational O +dynamics O +spectrum O +of O +U2AF65 B-protein +in O +the O +absence B-protein_state +of I-protein_state +RNA B-chemical +( O +Fig O +. O +6c O +, O +d O +; O +Supplementary O +Fig O +. O +7a O +, O +b O +). O + +The O +double O +- O +labelled O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +protein O +was O +tethered B-protein_state +to O +a O +slide O +via O +biotin B-chemical +- I-chemical +NTA I-chemical +/ I-chemical +Ni I-chemical ++ I-chemical +2 I-chemical +resin I-chemical +. O + +Virtually O +no O +fluorescent O +molecules O +were O +detected O +in O +the O +absence B-protein_state +of I-protein_state +biotin B-chemical +- I-chemical +NTA I-chemical +/ I-chemical +Ni I-chemical ++ I-chemical +2 I-chemical +, O +which O +demonstrates O +the O +absence B-protein_state +of I-protein_state +detectable O +non O +- O +specific O +binding O +of O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +to O +the O +slide O +. O + +The O +FRET B-evidence +distribution I-evidence +histogram I-evidence +built O +from O +more O +than O +a O +thousand O +traces B-evidence +of O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +in O +the O +absence B-protein_state +of I-protein_state +ligand B-chemical +showed O +an O +extremely O +broad O +distribution O +centred O +at O +a O +FRET B-evidence +efficiency I-evidence +of O +∼ O +0 O +. O +4 O +( O +Fig O +. O +6d O +). O + +Approximately O +40 O +% O +of O +the O +smFRET B-experimental_method +traces B-evidence +showed O +apparent O +transitions O +between O +multiple O +FRET B-evidence +values I-evidence +( O +for O +example O +, O +Fig O +. O +6c O +). O + +Despite O +the O +large O +width O +of O +the O +FRET B-evidence +- I-evidence +distribution I-evidence +histogram I-evidence +, O +the O +majority O +( O +80 O +%) O +of O +traces B-evidence +that O +showed O +fluctuations O +sampled O +only O +two O +distinct O +FRET B-evidence +states I-evidence +( O +for O +example O +, O +Supplementary O +Fig O +. O +7a O +). O + +Approximately O +70 O +% O +of O +observed O +fluctuations O +were O +interchanges O +between O +the O +∼ O +0 O +. O +65 O +and O +∼ O +0 O +. O +45 O +FRET B-evidence +values I-evidence +( O +Supplementary O +Fig O +. O +7b O +). O + +We O +cannot O +exclude O +a O +possibility O +that O +tethering O +of O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +to O +the O +microscope O +slide O +introduces O +structural O +heterogeneity O +into O +the O +protein O +and O +, O +thus O +, O +contributes O +to O +the O +breadth O +of O +the O +FRET B-evidence +distribution I-evidence +histogram I-evidence +. O + +However O +, O +the O +presence O +of O +repetitive O +fluctuations O +between O +particular O +FRET B-evidence +values I-evidence +supports O +the O +hypothesis O +that O +RNA B-protein_state +- I-protein_state +free I-protein_state +U2AF65 B-protein +samples O +several O +distinct O +conformations O +. O + +This O +result O +is O +consistent O +with O +the O +broad O +ensembles O +of O +extended B-protein_state +solution O +conformations O +that O +best O +fit O +the O +SAXS B-experimental_method +data O +collected O +for O +U2AF651 B-mutant +, I-mutant +2 I-mutant +as O +well O +as O +for O +a O +longer O +construct O +( O +residues O +136 B-residue_range +– I-residue_range +347 I-residue_range +). O + +We O +conclude O +that O +weak O +contacts O +between O +the O +U2AF65 B-protein +RRM1 B-structure_element +and O +RRM2 B-structure_element +permit O +dissociation O +of O +these O +RRMs B-structure_element +in O +the O +absence B-protein_state +of I-protein_state +RNA B-chemical +. O + +U2AF65 B-protein +conformational O +selection O +and O +induced O +fit O +by O +bound B-protein_state +RNA B-chemical + +We O +next O +used O +smFRET B-experimental_method +to O +probe O +the O +conformational O +selection O +of O +distinct O +inter B-structure_element +- I-structure_element +RRM I-structure_element +arrangements O +following O +association O +of O +U2AF65 B-protein +with O +the O +AdML B-gene +Py B-chemical +- I-chemical +tract I-chemical +prototype O +. O + +Addition O +of O +the O +AdML B-gene +RNA B-chemical +to O +tethered B-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +selectively O +increases O +a O +fraction O +of O +molecules O +showing O +an O +∼ O +0 O +. O +45 O +apparent O +FRET B-evidence +efficiency I-evidence +, O +suggesting O +that O +RNA O +binding O +stabilizes O +a O +single O +conformation O +, O +which O +corresponds O +to O +the O +0 O +. O +45 O +FRET B-evidence +state I-evidence +( O +Fig O +. O +6e O +, O +f O +). O + +To O +assess O +the O +possible O +contributions O +of O +RNA B-protein_state +- I-protein_state +free I-protein_state +conformations O +of O +U2AF65 B-protein +and O +/ O +or O +structural O +heterogeneity O +introduced O +by O +tethering B-experimental_method +of O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +to O +the O +slide O +to O +the O +observed O +distribution B-evidence +of I-evidence +FRET I-evidence +values I-evidence +, O +we O +reversed B-experimental_method +the I-experimental_method +immobilization I-experimental_method +scheme I-experimental_method +. O + +We O +tethered B-protein_state +the O +AdML B-gene +RNA B-chemical +to O +the O +slide O +via O +a O +biotinylated B-chemical +oligonucleotide I-chemical +DNA I-chemical +handle O +and O +added B-experimental_method +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +in O +the O +absence B-protein_state +of I-protein_state +biotin B-chemical +- I-chemical +NTA I-chemical +resin I-chemical +( O +Fig O +. O +6g O +, O +h O +; O +Supplementary O +Fig O +. O +7c O +– O +g O +). O + +A O +0 O +. O +45 O +FRET B-evidence +value I-evidence +was O +again O +predominant O +, O +indicating O +a O +similar O +RNA B-protein_state +- I-protein_state +bound I-protein_state +conformation O +and O +structural O +dynamics O +for O +the O +untethered B-protein_state +and O +tethered B-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +). O + +We O +examined O +the O +effect O +on O +U2AF651 B-mutant +, I-mutant +2L I-mutant +conformations O +of O +purine B-experimental_method +interruptions I-experimental_method +that O +often O +occur O +in O +relatively O +degenerate O +human B-species +Py B-chemical +tracts I-chemical +. O + +We O +introduced B-experimental_method +an O +rArA B-chemical +purine B-chemical +dinucleotide I-chemical +within O +a O +variant O +of O +the O +AdML B-gene +Py B-chemical +tract I-chemical +( O +detailed O +in O +Methods O +). O + +Insertion B-experimental_method +of O +adenine B-chemical +nucleotides I-chemical +decreased O +binding B-evidence +affinity I-evidence +of O +U2AF65 B-protein +to O +RNA B-chemical +by O +approximately O +five O +- O +fold O +. O + +Nevertheless O +, O +in O +the O +presence O +of O +saturating O +concentrations O +of O +rArA B-chemical +- O +interrupted O +RNA B-chemical +slide B-protein_state +- I-protein_state +tethered I-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +showed O +a O +prevalent O +∼ O +0 O +. O +45 O +apparent O +FRET B-evidence +value I-evidence +( O +Fig O +. O +6i O +, O +j O +), O +which O +was O +also O +predominant O +in O +the O +presence O +of O +continuous O +Py B-chemical +tract I-chemical +. O + +Therefore O +, O +RRM1 B-structure_element +- O +to O +- O +RRM2 B-structure_element +distance O +remains O +similar O +regardless O +of O +whether O +U2AF65 B-protein +is O +bound B-protein_state +to I-protein_state +interrupted O +or O +continuous O +Py B-chemical +tract I-chemical +. O + +The O +inter B-evidence +- I-evidence +fluorophore I-evidence +distances I-evidence +derived O +from O +the O +observed O +0 O +. O +45 O +FRET B-evidence +state I-evidence +agree O +with O +the O +distances O +between O +the O +α O +- O +carbon O +atoms O +of O +the O +respective O +residues O +in O +the O +crystal B-evidence +structures I-evidence +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +bound B-protein_state +to I-protein_state +Py B-chemical +- I-chemical +tract I-chemical +oligonucleotides I-chemical +. O + +It O +should O +be O +noted O +that O +inferring O +distances O +from O +FRET B-evidence +values I-evidence +is O +prone O +to O +significant O +error O +because O +of O +uncertainties O +in O +the O +determination O +of O +fluorophore O +orientation O +factor O +κ2 O +and O +Förster O +radius O +R0 O +, O +the O +parameters O +used O +in O +distance O +calculations O +. O + +Nevertheless O +, O +the O +predominant O +0 O +. O +45 O +FRET B-evidence +state I-evidence +in O +the O +presence O +of O +RNA B-chemical +agrees O +with O +the O +Py B-protein_state +- I-protein_state +tract I-protein_state +- I-protein_state +bound I-protein_state +crystal B-evidence +structure I-evidence +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +. O + +Importantly O +, O +the O +majority O +of O +traces B-evidence +(∼ O +70 O +%) O +of O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +bound B-protein_state +to I-protein_state +the O +slide O +- O +tethered O +RNA B-chemical +lacked O +FRET O +fluctuations O +and O +predominately O +exhibited O +a O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +( O +for O +example O +, O +Fig O +. O +6g O +). O + +The O +remaining O +∼ O +30 O +% O +of O +traces B-evidence +for O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +bound B-protein_state +to I-protein_state +the O +slide O +- O +tethered O +RNA B-chemical +showed O +fluctuations O +between O +distinct O +FRET B-evidence +values I-evidence +. O + +The O +majority O +of O +traces B-evidence +that O +show O +fluctuations O +began O +at O +high O +( O +0 O +. O +65 O +– O +0 O +. O +8 O +) O +FRET B-evidence +value I-evidence +and O +transitioned O +to O +a O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +( O +Supplementary O +Fig O +. O +7c O +– O +g O +). O + +Hidden B-experimental_method +Markov I-experimental_method +modelling I-experimental_method +analysis I-experimental_method +of O +smFRET B-experimental_method +traces B-evidence +suggests O +that O +RNA B-protein_state +- I-protein_state +bound I-protein_state +U2AF651 B-mutant +, I-mutant +2L I-mutant +can O +sample O +at O +least O +two O +other O +conformations O +corresponding O +to O +∼ O +0 O +. O +7 O +– O +0 O +. O +8 O +and O +∼ O +0 O +. O +3 O +FRET B-evidence +values I-evidence +in O +addition O +to O +the O +predominant O +conformation O +corresponding O +to O +the O +0 O +. O +45 O +FRET B-evidence +state I-evidence +. O + +Although O +a O +compact O +conformation O +( O +or O +multiple O +conformations O +) O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +corresponding O +to O +∼ O +0 O +. O +7 O +– O +0 O +. O +8 O +FRET B-evidence +values I-evidence +can O +bind O +RNA B-chemical +, O +on O +RNA B-chemical +binding O +, O +these O +compact B-protein_state +conformations O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +transition O +into O +a O +more O +stable O +structural O +state O +that O +corresponds O +to O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +and O +is O +likely O +similar O +to O +the O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +inter B-structure_element +- I-structure_element +RRM I-structure_element +- O +arrangement O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +crystal B-evidence +structures I-evidence +. O + +Thus O +, O +the O +sequence O +of O +structural O +rearrangements O +of O +U2AF65 B-protein +observed O +in O +smFRET B-experimental_method +traces B-evidence +( O +Supplementary O +Fig O +. O +7c O +– O +g O +) O +suggests O +that O +a O +‘ O +conformational O +selection O +' O +mechanism O +of O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +( O +that O +is O +, O +RNA O +ligand O +stabilization O +of O +a O +pre B-protein_state +- I-protein_state +configured I-protein_state +U2AF65 B-protein +conformation O +) O +is O +complemented O +by O +‘ O +induced O +fit O +' O +( O +that O +is O +, O +RNA O +- O +induced O +rearrangement O +of O +the O +U2AF65 B-protein +RRMs B-structure_element +to O +achieve O +the O +final O +‘ O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +' O +conformation O +), O +as O +discussed O +below O +. O + +The O +U2AF65 B-protein +structures B-evidence +and O +analyses B-evidence +presented O +here O +represent O +a O +successful O +step O +towards O +defining O +a O +molecular O +map O +of O +the O +3 B-site +′ I-site +splice I-site +site I-site +. O + +Several O +observations O +indicate O +that O +the O +numerous O +intramolecular O +contacts O +, O +here O +revealed O +among O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +and O +RRM1 B-structure_element +, O +RRM2 B-structure_element +, O +and O +the O +N O +- O +terminal O +RRM1 B-structure_element +extension I-structure_element +, O +synergistically O +coordinate O +U2AF65 B-protein +– O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +. O + +Truncation B-experimental_method +of O +U2AF65 B-protein +to O +the O +core B-protein_state +RRM1 B-structure_element +– I-structure_element +RRM2 I-structure_element +region I-structure_element +reduces O +its O +RNA B-evidence +affinity I-evidence +by O +100 O +- O +fold O +. O + +Likewise O +, O +deletion B-experimental_method +of O +20 B-residue_range +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +residues I-structure_element +significantly O +reduces O +U2AF65 B-protein +– O +RNA B-chemical +binding O +only O +when O +introduced O +in O +the O +context O +of O +the O +longer B-protein_state +U2AF651 B-mutant +, I-mutant +2L I-mutant +construct O +comprising O +the O +RRM B-structure_element +extensions I-structure_element +, O +which O +in O +turn O +position O +the O +linker B-structure_element +for O +RNA B-chemical +interactions O +. O + +Notably O +, O +a O +triple B-protein_state +mutation I-protein_state +of O +three O +residues O +( O +V254P B-mutant +, O +Q147A B-mutant +and O +R227A B-mutant +) O +in O +the O +respective O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +, O +N B-structure_element +- I-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +extensions I-structure_element +non O +- O +additively O +reduce O +RNA B-evidence +binding I-evidence +by O +150 O +- O +fold O +. O + +Altogether O +, O +these O +data O +indicate O +that O +interactions O +among O +the O +U2AF65 B-protein +RRM1 B-structure_element +/ O +RRM2 B-structure_element +, O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +, O +N B-structure_element +- I-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +extensions I-structure_element +are O +mutually O +inter O +- O +dependent O +for O +cognate O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +. O + +The O +implications O +of O +this O +finding O +for O +U2AF65 B-protein +conservation O +and O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +are O +detailed O +in O +the O +Supplementary O +Discussion O +. O + +Recently O +, O +high B-experimental_method +- I-experimental_method +throughput I-experimental_method +sequencing I-experimental_method +studies I-experimental_method +have O +shown O +that O +somatic O +mutations O +in O +pre B-protein_type +- I-protein_type +mRNA I-protein_type +splicing I-protein_type +factors I-protein_type +occur O +in O +the O +majority O +of O +patients O +with O +myelodysplastic O +syndrome O +( O +MDS O +). O + +MDS O +- O +relevant O +mutations O +are O +common O +in O +the O +small B-protein_state +U2AF B-protein_type +subunit I-protein_type +( O +U2AF35 B-protein +, O +or O +U2AF1 B-protein +), O +yet O +such O +mutations O +are O +rare O +in O +the O +large B-protein_state +U2AF65 B-protein +subunit O +( O +also O +called O +U2AF2 B-protein +)— O +possibly O +due O +to O +the O +selective O +versus O +nearly O +universal O +requirements O +of O +these O +factors O +for O +splicing O +. O + +A O +confirmed O +somatic O +mutation O +of O +U2AF65 B-protein +in O +patients O +with O +MDS O +, O +L187V B-mutant +, O +is O +located O +on O +a O +solvent B-site +- I-site +exposed I-site +surface I-site +of O +RRM1 B-structure_element +that O +is O +distinct O +from O +the O +RNA B-site +interface I-site +( O +Fig O +. O +7a O +). O + +This O +L187 B-residue_name_number +surface O +is O +oriented O +towards O +the O +N O +terminus O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +construct O +, O +where O +it O +is O +expected O +to O +abut O +the O +U2AF35 B-site +- I-site +binding I-site +site I-site +in O +the O +context O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +U2AF B-protein +heterodimer B-oligomeric_state +. O + +Likewise O +, O +an O +unconfirmed O +M144I B-mutant +mutation O +reported O +by O +the O +same O +group O +corresponds O +to O +the O +N O +- O +terminal O +residue O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +, O +which O +is O +separated O +by O +only O +∼ O +20 O +residues O +from O +the O +U2AF35 B-site +- I-site +binding I-site +site I-site +. O + +As O +such O +, O +we O +suggest O +that O +the O +MDS O +- O +relevant O +U2AF65 B-protein +mutations O +contribute O +to O +MDS O +progression O +indirectly O +, O +by O +destabilizing O +a O +relevant O +conformation O +of O +the O +conjoined O +U2AF35 B-protein +subunit O +rather O +than O +affecting O +U2AF65 B-protein +functions O +in O +RNA B-chemical +binding O +or O +spliceosome B-complex_assembly +recruitment O +per O +se O +. O + +Our O +smFRET B-experimental_method +results O +agree O +with O +prior O +NMR B-experimental_method +/ O +PRE B-experimental_method +evidence O +for O +multi O +- O +domain O +conformational O +selection O +as O +one O +mechanistic O +basis O +for O +U2AF65 B-protein +– O +RNA B-chemical +association O +( O +Fig O +. O +7b O +). O + +An O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +is O +likely O +to O +correspond O +to O +the O +U2AF65 B-protein +conformation O +visualized O +in O +our O +U2AF651 B-mutant +, I-mutant +2L I-mutant +crystal B-evidence +structures I-evidence +, O +in O +which O +the O +RRM1 B-structure_element +and O +RRM2 B-structure_element +bind O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +to O +the O +Py B-chemical +- I-chemical +tract I-chemical +oligonucleotide I-chemical +. O + +The O +lesser O +0 O +. O +65 O +– O +0 O +. O +8 O +and O +0 O +. O +2 O +– O +0 O +. O +3 O +FRET B-evidence +values I-evidence +in O +the O +untethered B-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +experiment O +could O +correspond O +to O +respective O +variants O +of O +the O +‘ O +closed B-protein_state +', O +back B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +back I-protein_state +U2AF65 B-protein +conformations O +characterized O +by O +NMR B-experimental_method +/ O +PRE B-experimental_method +data O +, O +or O +to O +extended B-protein_state +U2AF65 B-protein +conformations O +, O +in O +which O +the O +intramolecular O +RRM1 B-structure_element +/ O +RRM2 B-structure_element +interactions O +have O +dissociated O +the O +protein B-protein +is O +bound B-protein_state +to I-protein_state +RNA B-chemical +via O +single B-protein_state +RRMs B-structure_element +. O + +An O +increased O +prevalence O +of O +the O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +following O +U2AF65 B-protein +– O +RNA B-chemical +binding O +, O +coupled O +with O +the O +apparent O +absence B-protein_state +of I-protein_state +transitions O +in O +many O +∼ O +0 O +. O +45 O +- O +value O +single O +molecule O +traces B-evidence +( O +for O +example O +, O +Fig O +. O +6e O +), O +suggests O +a O +population O +shift O +in O +which O +RNA B-chemical +binds O +to O +( O +and O +draws O +the O +equilibrium O +towards O +) O +a O +pre B-protein_state +- I-protein_state +configured I-protein_state +inter B-structure_element +- I-structure_element +RRM I-structure_element +proximity O +that O +most O +often O +corresponds O +to O +the O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +. O + +Notably O +, O +our O +smFRET B-experimental_method +results O +reveal O +that O +U2AF65 B-protein +– O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +can O +be O +characterized O +by O +an O +‘ O +extended O +conformational O +selection O +' O +model O +( O +Fig O +. O +7b O +). O + +Examples O +of O +‘ O +extended B-protein_state +conformational O +selection O +' O +during O +ligand O +binding O +have O +been O +characterized O +for O +a O +growing O +number O +of O +macromolecules O +( O +for O +example O +, O +adenylate B-protein_type +kinase I-protein_type +, O +LAO B-protein_type +- I-protein_type +binding I-protein_type +protein I-protein_type +, O +poly B-protein_type +- I-protein_type +ubiquitin I-protein_type +, O +maltose B-protein_type +- I-protein_type +binding I-protein_type +protein I-protein_type +and O +the O +preQ1 B-protein_type +riboswitch I-protein_type +, O +among O +others O +). O + +Here O +, O +the O +majority O +of O +changes O +in O +smFRET B-experimental_method +traces B-evidence +for O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +bound B-protein_state +to I-protein_state +slide O +- O +tethered O +RNA B-chemical +began O +at O +high O +( O +0 O +. O +65 O +– O +0 O +. O +8 O +) O +FRET B-evidence +value I-evidence +and O +transition O +to O +the O +predominant O +0 O +. O +45 O +FRET B-evidence +value I-evidence +( O +Supplementary O +Fig O +. O +7c O +– O +g O +). O + +These O +transitions O +could O +correspond O +to O +rearrangement O +from O +the O +‘ O +closed B-protein_state +' O +NMR B-experimental_method +/ O +PRE B-experimental_method +- O +based O +U2AF65 B-protein +conformation O +in O +which O +the O +RNA B-site +- I-site +binding I-site +surface I-site +of O +only O +a O +single B-protein_state +RRM B-structure_element +is O +exposed O +and O +available O +for O +RNA O +binding O +, O +to O +the O +structural O +state O +seen O +in O +the O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +, O +RNA B-protein_state +- I-protein_state +bound I-protein_state +crystal B-evidence +structure I-evidence +. O + +As O +such O +, O +the O +smFRET B-experimental_method +approach O +reconciles O +prior O +inconsistencies O +between O +two O +major O +conformations O +that O +were O +detected O +by O +NMR B-experimental_method +/ O +PRE B-experimental_method +experiments O +and O +a O +broad O +ensemble O +of O +diverse O +inter B-structure_element +- I-structure_element +RRM I-structure_element +arrangements O +that O +fit O +the O +SAXS B-experimental_method +data O +for O +the O +apo B-protein_state +- O +protein B-protein +. O + +Similar O +interdisciplinary O +structural O +approaches O +are O +likely O +to O +illuminate O +whether O +similar O +mechanistic O +bases O +for O +RNA O +binding O +are O +widespread O +among O +other O +members O +of O +the O +vast O +multi O +- O +RRM B-structure_element +family O +. O + +The O +finding O +that O +U2AF65 B-protein +recognizes O +a O +nine O +base O +pair O +Py B-chemical +tract I-chemical +contributes O +to O +an O +elusive O +‘ O +code O +' O +for O +predicting O +splicing O +patterns O +from O +primary O +sequences O +in O +the O +post O +- O +genomic O +era O +( O +reviewed O +in O +ref O +.). O + +Based O +on O +( O +i O +) O +similar O +RNA B-evidence +affinities I-evidence +of O +U2AF65 B-protein +and O +U2AF651 B-mutant +, I-mutant +2L I-mutant +, O +( O +ii O +) O +indistinguishable O +conformations O +among O +four O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +in O +two O +different O +crystal O +packing O +arrangements O +and O +( O +iii O +) O +penalties B-evidence +of O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +mutations I-experimental_method +in O +RNA B-experimental_method +binding I-experimental_method +and I-experimental_method +splicing I-experimental_method +assays I-experimental_method +, O +we O +suggest O +that O +the O +extended B-protein_state +inter B-structure_element +- I-structure_element +RRM I-structure_element +regions I-structure_element +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +underlie O +cognate O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +by O +the O +full B-protein_state +- I-protein_state +length I-protein_state +U2AF65 B-protein +protein O +. O + +Further O +research O +will O +be O +needed O +to O +understand O +the O +roles O +of O +SF1 B-protein +and O +U2AF35 B-protein +subunits O +in O +the O +conformational O +equilibria O +underlying O +U2AF65 B-protein +association O +with O +Py B-chemical +tracts I-chemical +. O + +Moreover O +, O +structural O +differences O +among O +U2AF65 B-protein +homologues O +and O +paralogues O +may O +regulate O +splice B-site +site I-site +selection O +. O + +Ultimately O +, O +these O +guidelines O +will O +assist O +the O +identification O +of O +3 B-site +′ I-site +splice I-site +sites I-site +and O +the O +relationship O +of O +disease O +- O +causing O +mutations O +to O +penalties O +for O +U2AF65 B-protein +association O +. O + +The O +intact B-protein_state +U2AF65 B-protein +RRM1 B-structure_element +/ O +RRM2 B-structure_element +- O +containing O +domain O +and O +flanking O +residues O +are O +required O +for O +binding O +contiguous B-structure_element +Py B-chemical +tracts I-chemical +. O + +( O +a O +) O +Domain O +organization O +of O +full B-protein_state +- I-protein_state +length I-protein_state +( O +fl B-protein_state +) O +U2AF65 B-protein +and O +constructs O +used O +for O +RNA B-chemical +binding O +and O +structural O +experiments O +. O + +An O +internal O +deletion O +( O +d B-mutant +, O +Δ B-mutant +) O +of O +residues O +238 B-residue_range +– I-residue_range +257 I-residue_range +removes O +a O +portion O +of O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +from O +the O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +and O +dU2AF651 B-mutant +, I-mutant +2L I-mutant +constructs O +. O + +( O +b O +) O +Comparison O +of O +the O +apparent O +equilibrium B-evidence +affinities I-evidence +of O +various O +U2AF65 B-protein +constructs O +for O +binding O +the O +prototypical O +AdML B-gene +Py B-chemical +tract I-chemical +( O +5 B-chemical +′- I-chemical +CCCUUUUUUUUCC I-chemical +- I-chemical +3 I-chemical +′). I-chemical + +The O +flU2AF65 B-protein +protein O +includes O +a O +heterodimerization B-structure_element +domain I-structure_element +of O +the O +U2AF35 B-protein +subunit O +to O +promote O +solubility O +and O +folding O +. O + +The O +apparent O +equilibrium B-evidence +dissociation I-evidence +constants I-evidence +( O +KD B-evidence +) O +for O +binding O +the O +AdML B-gene +13mer O +are O +as O +follows O +: O +flU2AF65 B-protein +, O +30 O +± O +3 O +nM O +; O +U2AF651 B-mutant +, I-mutant +2L I-mutant +, O +35 O +± O +6 O +nM O +; O +U2AF651 B-mutant +, I-mutant +2 I-mutant +, O +3 O +, O +600 O +± O +300 O +nM O +. O +( O +c O +) O +Comparison O +of O +the O +RNA B-evidence +sequence I-evidence +specificities I-evidence +of O +flU2AF65 B-protein +and O +U2AF651 B-mutant +, I-mutant +2L I-mutant +constructs O +binding O +C B-structure_element +- I-structure_element +rich I-structure_element +Py B-chemical +tracts I-chemical +with O +4U O +' O +s O +embedded O +in O +either O +the O +5 O +′- O +( O +light O +grey O +fill O +) O +or O +3 O +′- O +( O +dark O +grey O +fill O +) O +regions O +. O + +The O +KD B-evidence +' O +s O +for O +binding O +5 B-chemical +′- I-chemical +CCUUUUCCCCCCC I-chemical +- I-chemical +3 I-chemical +′ I-chemical +are O +: O +flU2AF65 B-protein +, O +41 O +± O +2 O +nM O +; O +U2AF651 B-mutant +, I-mutant +2L I-mutant +, O +31 O +± O +3 O +nM O +. O +The O +KD B-evidence +' O +s O +for O +binding O +5 B-chemical +′- I-chemical +CCCCCCCUUUUCC I-chemical +- I-chemical +3 I-chemical +′ I-chemical +are O +: O +flU2AF65 B-protein +, O +414 O +± O +12 O +nM O +; O +U2AF651 B-mutant +, I-mutant +2L I-mutant +, O +417 O +± O +10 O +nM O +. O +Bar O +graphs O +are O +hatched O +to O +match O +the O +constructs O +shown O +in O +a O +. O +The O +average B-evidence +apparent I-evidence +equilibrium I-evidence +affinity I-evidence +( O +KA B-evidence +) O +and O +s O +. O +e O +. O +m O +. O +for O +three O +independent O +titrations O +are O +plotted O +. O + +The O +purified O +protein O +and O +average B-evidence +fitted I-evidence +fluorescence I-evidence +anisotropy I-evidence +RNA I-evidence +- I-evidence +binding I-evidence +curves I-evidence +are O +shown O +in O +Supplementary O +Fig O +. O +1 O +. O + +RRM B-structure_element +, O +RNA B-structure_element +recognition I-structure_element +motif I-structure_element +; O +RS B-structure_element +, O +arginine B-structure_element +- I-structure_element +serine I-structure_element +rich I-structure_element +; O +UHM B-structure_element +, O +U2AF B-structure_element +homology I-structure_element +motif I-structure_element +; O +ULM B-structure_element +, O +U2AF B-structure_element +ligand I-structure_element +motif I-structure_element +. O + +Structures B-evidence +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +recognizing O +a O +contiguous B-structure_element +Py B-chemical +tract I-chemical +. O + +( O +a O +) O +Alignment B-experimental_method +of O +oligonucleotide B-chemical +sequences O +that O +were O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +in O +the O +indicated O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +. O + +The O +regions O +of O +RRM1 B-structure_element +, O +RRM2 B-structure_element +and O +linker B-structure_element +contacts O +are O +indicated O +above O +by O +respective O +black O +and O +blue O +arrows O +from O +N O +- O +to O +C O +- O +terminus O +. O + +For O +clarity O +, O +we O +consistently O +number O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +nucleotide B-site +- I-site +binding I-site +sites I-site +from O +one O +to O +nine O +, O +although O +in O +some O +cases O +the O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +oligonucleotide B-chemical +comprises O +eight O +nucleotides B-chemical +and O +as O +such O +leaves O +the O +first B-site +binding I-site +site I-site +empty O +. O + +The O +prior O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +nucleotide B-site +- I-site +binding I-site +sites I-site +are O +given O +in O +parentheses O +( O +site O +4 O +' O +interacts O +with O +dU2AF65 B-mutant +RRM1 B-structure_element +and O +RRM2 B-structure_element +by O +crystallographic O +symmetry O +). O + +( O +b O +) O +Stereo O +views O +of O +a O +‘ O +kicked O +' O +2 B-evidence +| I-evidence +Fo I-evidence +|−| I-evidence +Fc I-evidence +| I-evidence +electron I-evidence +density I-evidence +map I-evidence +contoured O +at O +1σ O +for O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +, O +N O +- O +and O +C O +- O +terminal O +residues O +( O +blue O +) O +or O +bound O +oligonucleotide B-chemical +of O +a O +representative O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structure O +( O +structure O +iv O +, O +bound B-protein_state +to I-protein_state +5 O +′-( O +P O +) O +rUrUrUdUrUrU O +( O +BrdU O +) O +dUrC O +) O +( O +magenta O +). O + +Crystallographic O +statistics O +are O +given O +in O +Table O +1 O +and O +the O +overall O +conformations O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +and O +prior O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +/ O +U2AF651 B-mutant +, I-mutant +2 I-mutant +structures B-evidence +are O +compared O +in O +Supplementary O +Fig O +. O +2 O +. O + +BrdU B-chemical +, O +5 B-chemical +- I-chemical +bromo I-chemical +- I-chemical +deoxy I-chemical +- I-chemical +uridine I-chemical +; O +d B-chemical +, O +deoxy B-chemical +- I-chemical +ribose I-chemical +; O +P B-chemical +-, I-chemical +5 B-chemical +′- I-chemical +phosphorylation I-chemical +; O +r B-chemical +, O +ribose B-chemical +. O + +Representative O +views O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +interactions O +with O +each O +new O +nucleotide B-chemical +of O +the O +bound B-protein_state +Py B-chemical +tract I-chemical +. O + +New O +residues O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +are O +coloured O +a O +darker O +shade O +of O +blue O +, O +apart O +from O +residues O +that O +were O +tested O +by O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +, O +which O +are O +coloured O +yellow O +. O + +The O +nucleotide B-site +- I-site +binding I-site +sites I-site +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +and O +prior O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +structure B-evidence +are O +compared O +in O +Supplementary O +Fig O +. O +3a O +– O +h O +. O +The O +first B-site +and I-site +seventh I-site +U2AF651 I-site +, I-site +2L I-site +- I-site +binding I-site +sites I-site +are O +unchanged O +from O +the O +prior O +dU2AF651 B-complex_assembly +, I-complex_assembly +2 I-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +structure B-evidence +and O +are O +portrayed O +in O +Supplementary O +Fig O +. O +3a O +, O +f O +. O +The O +four O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +are O +similar O +with O +the O +exception O +of O +pH O +- O +dependent O +variations O +at O +the O +ninth B-site +site I-site +that O +are O +detailed O +in O +Supplementary O +Fig O +. O +3i O +, O +j O +. O +The O +representative O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structure B-evidence +shown O +has O +the O +highest O +resolution O +and O +/ O +or O +ribose B-chemical +nucleotide I-chemical +at O +the O +given O +site O +: O +( O +a O +) O +rU2 B-residue_name_number +of O +structure O +iv O +; O +( O +b O +) O +rU3 B-residue_name_number +of O +structure O +iii O +; O +( O +c O +) O +rU4 B-residue_name_number +of O +structure O +i O +; O +( O +d O +) O +rU5 B-residue_name_number +of O +structure O +iii O +; O +( O +e O +) O +rU6 B-residue_name_number +of O +structure O +ii O +; O +( O +f O +) O +dU8 B-residue_name_number +of O +structure O +iii O +; O +( O +g O +) O +dU9 B-residue_name_number +of O +structure O +iii O +; O +( O +h O +) O +rC9 B-residue_name_number +of O +structure O +iv O +. O + +( O +i O +) O +Bar O +graph O +of O +apparent O +equilibrium B-evidence +affinities I-evidence +( O +KA B-evidence +) O +of O +the O +wild B-protein_state +type I-protein_state +( O +blue O +) O +and O +the O +indicated O +mutant B-protein_state +( O +yellow O +) O +U2AF651 B-mutant +, I-mutant +2L I-mutant +proteins O +binding O +the O +AdML B-gene +Py B-chemical +tract I-chemical +( O +5 B-chemical +′- I-chemical +CCCUUUUUUUUCC I-chemical +- I-chemical +3 I-chemical +′). I-chemical + +The O +apparent O +equilibrium B-evidence +dissociation I-evidence +constants I-evidence +( O +KD B-evidence +) O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +mutant B-protein_state +proteins O +are O +: O +wild B-protein_state +type I-protein_state +( O +WT B-protein_state +), O +35 O +± O +6 O +nM O +; O +R227A B-mutant +, O +166 O +± O +2 O +nM O +; O +V254P B-mutant +, O +137 O +± O +10 O +nM O +; O +Q147A B-mutant +, O +171 O +± O +21 O +nM O +. O +The O +average O +KA B-evidence +and O +s O +. O +e O +. O +m O +. O +for O +three O +independent O +titrations O +are O +plotted O +. O + +The O +average O +fitted O +fluorescence O +anisotropy O +RNA B-evidence +- I-evidence +binding I-evidence +curves I-evidence +are O +shown O +in O +Supplementary O +Fig O +. O +4a O +– O +c O +. O + +The O +U2AF65 B-protein +linker B-structure_element +/ O +RRM B-structure_element +and O +inter O +- O +RRM B-structure_element +interactions O +. O + +( O +a O +) O +Contacts O +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +with O +the O +RRMs B-structure_element +. O + +A O +semi O +- O +transparent O +space O +- O +filling O +surface O +is O +shown O +for O +the O +RRM1 B-structure_element +( O +green O +) O +and O +RRM2 B-structure_element +( O +light O +blue O +). O + +Residues O +V249 B-residue_name_number +, O +V250 B-residue_name_number +, O +V254 B-residue_name_number +( O +yellow O +) O +are O +mutated B-experimental_method +to O +V249G B-mutant +/ O +V250G B-mutant +/ O +V254G B-mutant +in O +the O +3Gly B-mutant +mutant I-mutant +; O +residues O +S251 B-residue_name_number +, O +T252 B-residue_name_number +, O +V253 B-residue_name_number +, O +P255 B-residue_name_number +( O +red O +) O +along O +with O +V254 B-residue_name_number +are O +mutated B-experimental_method +to O +S251G B-mutant +/ O +T252G B-mutant +/ O +V253G B-mutant +/ O +V254G B-mutant +/ O +P255G B-mutant +in O +the O +5Gly B-mutant +mutant I-mutant +or O +to O +S251N B-mutant +/ O +T252L B-mutant +/ O +V253A B-mutant +/ O +V254L B-mutant +/ O +P255A B-mutant +in O +the O +NLALA B-mutant +mutant I-mutant +; O +residues O +M144 B-residue_name_number +, O +L235 B-residue_name_number +, O +M238 B-residue_name_number +, O +V244 B-residue_name_number +, O +V246 B-residue_name_number +( O +orange O +) O +along O +with O +V249 B-residue_name_number +, O +V250 B-residue_name_number +, O +S251 B-residue_name_number +, O +T252 B-residue_name_number +, O +V253 B-residue_name_number +, O +V254 B-residue_name_number +, O +P255 B-residue_name_number +are O +mutated B-experimental_method +to O +M144G B-mutant +/ O +L235G B-mutant +/ O +M238G B-mutant +/ O +V244G B-mutant +/ O +V246G B-mutant +/ O +V249G B-mutant +/ O +V250G B-mutant +/ O +S251G B-mutant +/ O +T252G B-mutant +/ O +V253G B-mutant +/ O +V254G B-mutant +/ O +P255G B-mutant +in O +the O +12Gly B-mutant +mutant I-mutant +. O + +Other O +linker B-structure_element +residues O +are O +coloured O +either O +dark O +blue O +for O +new O +residues O +in O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structure O +or O +light O +blue O +for O +the O +remaining O +inter B-structure_element +- I-structure_element +RRM I-structure_element +residues O +. O + +The O +central O +panel O +shows O +an O +overall O +view O +with O +stick O +diagrams O +for O +mutated O +residues O +; O +boxed O +regions O +are O +expanded O +to O +show O +the O +C O +- O +terminal O +( O +bottom O +left O +) O +and O +central B-structure_element +linker I-structure_element +regions I-structure_element +( O +top O +) O +at O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +interfaces I-structure_element +, O +and O +N O +- O +terminal O +linker O +region O +contacts O +with O +RRM1 B-structure_element +( O +bottom O +right O +). O + +( O +b O +) O +Bar O +graph O +of O +apparent O +equilibrium B-evidence +affinities I-evidence +( O +KA B-evidence +) O +for O +the O +AdML B-gene +Py B-chemical +tract I-chemical +( O +5 B-chemical +′- I-chemical +CCCUUUUUUUUCC I-chemical +- I-chemical +3 I-chemical +′) I-chemical +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +blue O +) O +U2AF651 B-mutant +, I-mutant +2L I-mutant +protein O +compared O +with O +mutations O +of O +the O +residues O +shown O +in O +a O +: O +3Gly B-mutant +( O +yellow O +), O +5Gly B-mutant +( O +red O +), O +NLALA B-mutant +( O +hatched O +red O +), O +12Gly B-mutant +( O +orange O +) O +and O +the O +linker B-experimental_method +deletions I-experimental_method +dU2AF651 B-mutant +, I-mutant +2 I-mutant +in O +the O +minimal B-protein_state +RRM1 B-structure_element +– I-structure_element +RRM2 I-structure_element +region I-structure_element +( O +residues O +148 B-residue_range +– I-residue_range +237 I-residue_range +, O +258 B-residue_range +– I-residue_range +336 I-residue_range +) O +or O +dU2AF651 B-mutant +, I-mutant +2L I-mutant +( O +residues O +141 B-residue_range +– I-residue_range +237 I-residue_range +, O +258 B-residue_range +– I-residue_range +342 I-residue_range +). O + +The O +apparent O +equilibrium B-evidence +dissociation I-evidence +constants I-evidence +( O +KD B-evidence +) O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +mutant B-protein_state +proteins O +are O +: O +wild B-protein_state +type I-protein_state +( O +WT B-protein_state +), O +35 O +± O +6 O +nM O +; O +3Gly B-mutant +, O +47 O +± O +4 O +nM O +; O +5Gly B-mutant +, O +61 O +± O +3 O +nM O +; O +12Gly B-mutant +, O +88 O +± O +21 O +nM O +; O +NLALA B-mutant +, O +45 O +± O +3 O +nM O +; O +dU2AF651 B-mutant +, I-mutant +2L I-mutant +, O +123 O +± O +5 O +nM O +; O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +, O +5000 O +± O +100 O +nM O +; O +3Mut B-mutant +, O +5630 O +± O +70 O +nM O +. O +The O +average O +KA B-evidence +and O +s O +. O +e O +. O +m O +. O +for O +three O +independent O +titrations O +are O +plotted O +. O + +The O +fitted O +fluorescence O +anisotropy O +RNA B-evidence +- I-evidence +binding I-evidence +curves I-evidence +are O +shown O +in O +Supplementary O +Fig O +. O +4d O +– O +j O +. O +( O +c O +) O +Close O +view O +of O +the O +U2AF65 B-protein +RRM1 B-site +/ I-site +RRM2 I-site +interface I-site +following O +a O +two O +- O +fold O +rotation O +about O +the O +x O +- O +axis O +relative O +to O +a O +. O + +U2AF65 B-protein +inter O +- O +domain O +residues O +are O +important O +for O +splicing O +a O +representative O +pre B-chemical +- I-chemical +mRNA I-chemical +substrate O +in O +human B-species +cells O +. O + +( O +a O +) O +Schematic O +diagram O +of O +the O +pyPY B-chemical +reporter O +minigene O +construct O +comprising O +two O +alternative O +splice B-site +sites I-site +preceded O +by O +either O +the O +weak O +IgM O +Py B-chemical +tract I-chemical +( O +py B-chemical +) O +or O +the O +strong O +AdML B-gene +Py B-chemical +tract I-chemical +( O +PY B-chemical +) O +( O +sequences O +inset O +). O + +( O +b O +) O +Representative O +RT B-experimental_method +- I-experimental_method +PCR I-experimental_method +of O +pyPY B-chemical +transcripts O +from O +HEK293T O +cells O +co B-experimental_method +- I-experimental_method +transfected I-experimental_method +with O +constructs O +encoding O +the O +pyPY B-chemical +minigene O +and O +either O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +U2AF65 B-protein +or O +a O +triple O +U2AF65 B-protein +mutant B-protein_state +( O +3Mut B-mutant +) O +of O +Q147A B-mutant +, O +R227A B-mutant +and O +V254P B-mutant +residues O +. O +( O +c O +) O +A O +bar O +graph O +of O +the O +average O +percentage O +of O +the O +py B-chemical +- O +spliced O +mRNA B-chemical +relative O +to O +total O +detected O +pyPY B-chemical +transcripts O +( O +spliced O +and O +unspliced O +) O +for O +the O +corresponding O +gel O +lanes O +( O +black O +, O +no O +U2AF65 B-protein +added O +; O +white O +, O +WT B-protein_state +U2AF65 B-protein +; O +grey O +, O +3Mut B-mutant +U2AF65 B-protein +). O + +Protein B-experimental_method +overexpression I-experimental_method +and O +qRT B-experimental_method +- I-experimental_method +PCR I-experimental_method +results O +are O +shown O +in O +Supplementary O +Fig O +. O +5 O +. O + +RNA O +binding O +stabilizes O +the O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +conformation O +of O +U2AF65 B-protein +RRMs B-structure_element +. O + +( O +a O +, O +b O +) O +Views O +of O +FRET B-experimental_method +pairs O +chosen O +to O +follow O +the O +relative O +movement O +of O +RRM1 B-structure_element +and O +RRM2 B-structure_element +on O +the O +crystal B-evidence +structure I-evidence +of O +‘ O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +' O +U2AF651 B-mutant +, I-mutant +2L I-mutant +RRMs B-structure_element +bound B-protein_state +to I-protein_state +a O +Py B-chemical +- I-chemical +tract I-chemical +oligonucleotide I-chemical +( O +a O +, O +representative O +structure O +iv O +) O +or O +‘ O +closed B-protein_state +' O +NMR B-experimental_method +/ O +PRE B-experimental_method +- O +based O +model O +of O +U2AF651 B-mutant +, I-mutant +2 I-mutant +( O +b O +, O +PDB O +ID O +2YH0 O +) O +in O +identical O +orientations O +of O +RRM2 B-structure_element +. O + +The O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +proteins O +were O +doubly O +labelled O +at O +A181C B-mutant +/ O +Q324C B-mutant +such O +that O +a O +mixture O +of O +Cy3 B-chemical +/ O +Cy5 B-chemical +fluorophores B-chemical +are O +expected O +to O +be O +present O +at O +each O +site O +. O + +( O +c O +– O +f O +, O +i O +, O +j O +) O +The O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +protein O +was O +immobilized O +on O +the O +microscope O +slide O +via O +biotin B-chemical +- I-chemical +NTA I-chemical +/ I-chemical +Ni I-chemical ++ I-chemical +2 I-chemical +( O +orange O +line O +) O +on O +a O +neutravidin O +( O +black O +X O +)- O +biotin O +- O +PEG O +( O +orange O +triangle O +)- O +treated O +surface O +and O +imaged O +either O +in O +the O +absence B-protein_state +of I-protein_state +ligands B-chemical +( O +c O +, O +d O +), O +in O +the O +presence O +of O +5 O +μM O +AdML B-gene +Py B-chemical +- I-chemical +tract I-chemical +RNA I-chemical +( O +5 B-chemical +′- I-chemical +CCUUUUUUUUCC I-chemical +- I-chemical +3 I-chemical +′) I-chemical +( O +e O +, O +f O +), O +or O +in O +the O +presence O +of O +10 O +μM O +adenosine B-residue_name +- O +interrupted O +variant O +RNA B-chemical +( O +5 B-chemical +′- I-chemical +CUUUUUAAUUUCCA I-chemical +- I-chemical +3 I-chemical +′) I-chemical +( O +i O +, O +j O +). O + +The O +untethered B-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +protein O +( O +1 O +nM O +) O +was O +added O +to O +AdML B-gene +RNA B-chemical +– I-chemical +polyethylene I-chemical +- I-chemical +glycol I-chemical +- I-chemical +linker I-chemical +– I-chemical +DNA I-chemical +oligonucleotide I-chemical +( O +10 O +nM O +), O +which O +was O +immobilized O +on O +the O +microscope O +slide O +by O +annealing O +with O +a O +complementary O +biotinyl B-chemical +- I-chemical +DNA I-chemical +oligonucleotide I-chemical +( O +black O +vertical O +line O +). O + +Typical O +single B-experimental_method +- I-experimental_method +molecule I-experimental_method +FRET I-experimental_method +traces B-evidence +( O +c O +, O +e O +, O +g O +, O +i O +) O +show O +fluorescence O +intensities O +from O +Cy3 B-chemical +( O +green O +) O +and O +Cy5 B-chemical +( O +red O +) O +and O +the O +calculated B-evidence +apparent I-evidence +FRET I-evidence +efficiency I-evidence +( O +blue O +). O + +Additional O +traces B-evidence +for O +untethered B-protein_state +, O +RNA B-protein_state +- I-protein_state +bound I-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +are O +shown O +in O +Supplementary O +Fig O +. O +7c O +, O +d O +. O +Histograms B-evidence +( O +d O +, O +f O +, O +h O +, O +j O +) O +show O +the O +distribution B-evidence +of I-evidence +FRET I-evidence +values I-evidence +in O +RNA B-protein_state +- I-protein_state +free I-protein_state +, O +slide B-protein_state +- I-protein_state +tethered I-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +( O +d O +); O +AdML B-gene +RNA B-protein_state +- I-protein_state +bound I-protein_state +, O +slide B-protein_state +- I-protein_state +tethered I-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +( O +f O +); O +AdML B-gene +RNA B-protein_state +- I-protein_state +bound I-protein_state +, O +untethered B-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +( O +h O +) O +and O +adenosine O +- O +interrupted O +RNA B-protein_state +- I-protein_state +bound I-protein_state +, O +slide B-protein_state +- I-protein_state +tethered I-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +( O +j O +). O + +N O +is O +the O +number O +of O +single O +- O +molecule O +traces B-evidence +compiled O +for O +each O +histogram B-evidence +. O + +Schematic O +models O +of O +U2AF65 B-protein +recognizing O +the O +Py B-chemical +tract I-chemical +. O + +( O +a O +) O +Diagram O +of O +the O +U2AF65 B-protein +, O +SF1 B-protein +and O +U2AF35 B-protein +splicing O +factors O +bound B-protein_state +to I-protein_state +the O +consensus O +elements O +of O +the O +3 B-site +′ I-site +splice I-site +site I-site +. O + +A O +surface O +representation O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +is O +shown O +bound B-protein_state +to I-protein_state +nine O +nucleotides B-chemical +( O +nt O +); O +the O +relative O +distances O +and O +juxtaposition O +of O +the O +branch B-site +point I-site +sequence I-site +( O +BPS B-site +) O +and O +consensus O +AG B-chemical +dinucleotide I-chemical +at O +the O +3 B-site +′ I-site +splice I-site +site I-site +are O +unknown O +. O + +MDS O +- O +relevant O +mutated O +residues O +of O +U2AF65 B-protein +are O +shown O +as O +yellow O +spheres O +( O +L187 B-residue_name_number +and O +M144 B-residue_name_number +). O + +( O +b O +) O +Following O +binding O +to O +the O +Py B-chemical +- I-chemical +tract I-chemical +RNA I-chemical +, O +a O +conformation O +corresponding O +to O +high B-evidence +FRET I-evidence +and O +consistent O +with O +the O +‘ O +closed B-protein_state +', O +back B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +back I-protein_state +apo B-protein_state +- O +U2AF65 B-protein +model O +resulting O +from O +PRE B-experimental_method +/ O +NMR B-experimental_method +characterization O +( O +PDB O +ID O +2YH0 O +) O +often O +transitions O +to O +a O +conformation O +corresponding O +to O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +, O +which O +is O +consistent O +with O +‘ O +open B-protein_state +', O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +RRMs B-structure_element +such O +as O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +crystal B-evidence +structures I-evidence +. O + +Alternatively O +, O +a O +conformation O +of O +U2AF65 B-protein +corresponding O +to O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +can O +directly O +bind O +to O +RNA B-chemical +; O +RNA B-chemical +binding O +stabilizes O +the O +‘ O +open B-protein_state +', O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +conformation O +and O +thus O +shifts O +the O +U2AF65 B-protein +population O +towards O +the O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +. O + +RRM1 B-structure_element +, O +green O +; O +RRM2 B-structure_element +, O +pale O +blue O +; O +RRM B-structure_element +extensions I-structure_element +/ O +linker B-structure_element +, O +blue O +. O + +RNA B-chemical +protects O +a O +nucleoprotein B-complex_assembly +complex O +against O +radiation O +damage O + +Systematic O +analysis O +of O +radiation O +damage O +within O +a O +protein B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complex O +over O +a O +large O +dose O +range O +( O +1 O +. O +3 O +– O +25 O +MGy O +) O +reveals O +significant O +differential O +susceptibility O +of O +RNA B-chemical +and O +protein O +. O + +A O +new O +method O +of O +difference B-experimental_method +electron I-experimental_method +- I-experimental_method +density I-experimental_method +quantification I-experimental_method +is O +presented O +. O + +Radiation O +damage O +during O +macromolecular B-experimental_method +X I-experimental_method +- I-experimental_method +ray I-experimental_method +crystallographic I-experimental_method +data I-experimental_method +collection I-experimental_method +is O +still O +the O +main O +impediment O +for O +many O +macromolecular B-experimental_method +structure I-experimental_method +determinations I-experimental_method +. O + +Although O +this O +has O +been O +well O +characterized O +within O +protein O +crystals B-evidence +, O +far O +less O +is O +known O +about O +specific O +damage O +effects O +within O +the O +larger O +class O +of O +nucleoprotein O +complexes O +. O + +Here O +, O +a O +methodology O +has O +been O +developed O +whereby O +per B-evidence +- I-evidence +atom I-evidence +density I-evidence +changes I-evidence +could O +be O +quantified O +with O +increasing O +dose O +over O +a O +wide O +( O +1 O +. O +3 O +– O +25 O +. O +0 O +MGy O +) O +range O +and O +at O +higher O +resolution O +( O +1 O +. O +98 O +Å O +) O +than O +the O +previous O +systematic O +specific O +damage O +study O +on O +a O +protein O +– O +DNA B-chemical +complex O +. O + +Specific O +damage O +manifestations O +were O +determined O +within O +the O +large O +trp B-protein_type +RNA I-protein_type +- I-protein_type +binding I-protein_type +attenuation I-protein_type +protein I-protein_type +( O +TRAP B-complex_assembly +) O +bound B-protein_state +to I-protein_state +a O +single O +- O +stranded O +RNA B-chemical +that O +forms O +a O +belt O +around O +the O +protein O +. O + +Over O +a O +large O +dose O +range O +, O +the O +RNA B-chemical +was O +found O +to O +be O +far O +less O +susceptible O +to O +radiation O +- O +induced O +chemical O +changes O +than O +the O +protein O +. O + +The O +availability O +of O +two O +TRAP B-complex_assembly +molecules O +in O +the O +asymmetric O +unit O +, O +of O +which O +only O +one O +contained O +bound B-protein_state +RNA B-chemical +, O +allowed O +a O +controlled O +investigation O +into O +the O +exact O +role O +of O +RNA B-chemical +binding O +in O +protein O +specific O +damage O +susceptibility O +. O + +The O +11 O +- O +fold O +symmetry O +within O +each O +TRAP B-complex_assembly +ring B-structure_element +permitted O +statistically O +significant O +analysis O +of O +the O +Glu B-residue_name +and O +Asp B-residue_name +damage O +patterns O +, O +with O +RNA B-chemical +binding O +unexpectedly O +being O +observed O +to O +protect O +these O +otherwise O +highly O +sensitive O +residues O +within O +the O +11 O +RNA B-site +- I-site +binding I-site +pockets I-site +distributed O +around O +the O +outside O +of O +the O +protein O +molecule O +. O + +Additionally O +, O +the O +method O +enabled O +a O +quantification O +of O +the O +reduction O +in O +radiation O +- O +induced O +Lys B-residue_name +and O +Phe B-residue_name +disordering O +upon O +RNA B-chemical +binding O +directly O +from O +the O +electron B-evidence +density I-evidence +. O + +With O +the O +wide O +use O +of O +high O +- O +flux O +third O +- O +generation O +synchrotron O +sources O +, O +radiation O +damage O +( O +RD O +) O +has O +once O +again O +become O +a O +dominant O +reason O +for O +the O +failure O +of O +structure B-experimental_method +determination I-experimental_method +using O +macromolecular B-experimental_method +crystallography I-experimental_method +( O +MX B-experimental_method +) O +in O +experiments O +conducted O +both O +at O +room O +temperature O +and O +under O +cryocooled O +conditions O +( O +100 O +K O +). O + +Significant O +progress O +has O +been O +made O +in O +recent O +years O +in O +understanding O +the O +inevitable O +manifestations O +of O +X O +- O +ray O +- O +induced O +RD O +within O +protein O +crystals B-evidence +, O +and O +there O +is O +now O +a O +body O +of O +literature O +on O +possible O +strategies O +to O +mitigate O +the O +effects O +of O +RD O +( O +e O +. O +g O +. O +Zeldin O +, O +Brockhauser O +et O +al O +., O +2013 O +; O +Bourenkov O +& O +Popov O +, O +2010 O +). O + +However O +, O +there O +is O +still O +no O +general O +consensus O +within O +the O +field O +on O +how O +to O +minimize O +RD O +during O +MX B-experimental_method +data O +collection O +, O +and O +debates O +on O +the O +dependence O +of O +RD O +progression O +on O +incident O +X O +- O +ray O +energy O +( O +Shimizu O +et O +al O +., O +2007 O +; O +Liebschner O +et O +al O +., O +2015 O +) O +and O +the O +efficacy O +of O +radical O +scavengers O +( O +Allan O +et O +al O +., O +2013 O +) O +have O +yet O +to O +be O +resolved O +. O + +Global O +radiation O +damage O +is O +observed O +within O +reciprocal O +space O +as O +the O +overall O +decay O +of O +the O +summed O +intensity O +of O +reflections O +detected O +within O +the O +diffraction B-evidence +pattern I-evidence +as O +dose O +increases O +( O +Garman O +, O +2010 O +; O +Murray O +& O +Garman O +, O +2002 O +). O + +Dose O +is O +defined O +as O +the O +absorbed O +energy O +per O +unit O +mass O +of O +crystal O +in O +grays O +( O +Gy O +; O +1 O +Gy O += O +1 O +J O +kg O +− O +1 O +), O +and O +is O +the O +metric O +against O +which O +damage O +progression O +should O +be O +monitored O +during O +MX B-experimental_method +data O +collection O +, O +as O +opposed O +to O +time O +. O + +At O +100 O +K O +, O +an O +experimental O +dose O +limit O +of O +30 O +MGy O +has O +been O +recommended O +as O +an O +upper O +limit O +beyond O +which O +the O +biological O +information O +derived O +from O +any O +macromolecular O +crystal B-evidence +may O +be O +compromised O +( O +Owen O +et O +al O +., O +2006 O +). O + +Specific B-experimental_method +radiation I-experimental_method +damage I-experimental_method +( O +SRD B-experimental_method +) O +is O +observed O +in O +the O +real B-evidence +- I-evidence +space I-evidence +electron I-evidence +density I-evidence +, O +and O +has O +been O +detected O +at O +much O +lower O +doses O +than O +any O +observable O +decay O +in O +the O +intensity O +of O +reflections O +. O + +Indeed O +, O +the O +C O +— O +Se B-chemical +bond O +in O +selenomethionine B-chemical +, O +the O +stability O +of O +which O +is O +key O +for O +the O +success O +of O +experimental O +phasing O +methods O +, O +can O +be O +cleaved O +at O +a O +dose O +as O +low O +as O +2 O +MGy O +for O +a O +crystal B-evidence +maintained O +at O +100 O +K O +( O +Holton O +, O +2007 O +). O + +SRD O +has O +been O +well O +characterized O +in O +a O +large O +range O +of O +proteins O +, O +and O +is O +seen O +to O +follow O +a O +reproducible O +order O +: O +metallo O +- O +centre O +reduction O +, O +disulfide B-ptm +- I-ptm +bond I-ptm +cleavage O +, O +acidic O +residue O +decarboxylation O +and O +methionine O +methylthio O +cleavage O +( O +Ravelli O +& O +McSweeney O +, O +2000 O +; O +Burmeister O +, O +2000 O +; O +Weik O +et O +al O +., O +2000 O +; O +Yano O +et O +al O +., O +2005 O +). O + +There O +are O +a O +number O +of O +cases O +where O +SRD O +manifestations O +have O +compromised O +the O +biological O +information O +extracted O +from O +MX B-experimental_method +- I-experimental_method +determined I-experimental_method +structures B-evidence +at O +much O +lower O +doses O +than O +the O +recommended O +30 O +MGy O +limit O +, O +leading O +to O +false O +structural O +interpretations O +of O +protein O +mechanisms O +. O + +Active B-site +- I-site +site I-site +residues I-site +appear O +to O +be O +particularly O +susceptible O +, O +particularly O +for O +photosensitive O +proteins O +and O +in O +instances O +where O +chemical O +strain O +is O +an O +intrinsic O +feature O +of O +the O +reaction O +mechanism O +. O + +For O +instance O +, O +structure B-experimental_method +determination I-experimental_method +of O +the O +purple O +membrane O +protein O +bacterio B-protein_type +­ I-protein_type +rhodopsin I-protein_type +required O +careful O +corrections O +for O +radiation O +- O +induced O +structural O +changes O +before O +the O +correct O +photosensitive O +intermediate O +states O +could O +be O +isolated O +( O +Matsui O +et O +al O +., O +2002 O +). O + +The O +significant O +chemical O +strain O +required O +for O +catalysis O +within O +the O +active B-site +site I-site +of O +phosphoserine B-protein_type +aminotransferase I-protein_type +has O +been O +observed O +to O +diminish O +during O +X O +- O +ray O +exposure O +( O +Dubnovitsky O +et O +al O +., O +2005 O +). O + +Since O +the O +majority O +of O +SRD B-experimental_method +studies I-experimental_method +to O +date O +have O +focused O +on O +proteins O +, O +much O +less O +is O +known O +about O +the O +effects O +of O +X O +- O +ray O +irradiation O +on O +the O +wider O +class O +of O +crystalline O +nucleoprotein B-complex_assembly +complexes O +or O +how O +to O +correct O +for O +such O +radiation O +- O +induced O +structural O +changes O +. O + +Understanding O +RD O +to O +such O +complexes O +is O +crucial O +, O +since O +DNA B-chemical +is O +rarely O +naked O +within O +a O +cell O +, O +instead O +dynamically O +interacting O +with O +proteins O +, O +facilitating O +replication O +, O +transcription O +, O +modification O +and O +DNA B-chemical +repair O +. O + +As O +of O +early O +2016 O +, O +> O +5400 O +nucleoprotein B-complex_assembly +complex O +structures B-evidence +have O +been O +deposited O +within O +the O +PDB O +, O +with O +91 O +% O +solved O +by O +MX B-experimental_method +. O + +It O +is O +essential O +to O +understand O +how O +these O +increasingly O +complex O +macromolecular O +structures B-evidence +are O +affected O +by O +the O +radiation O +used O +to O +solve O +them O +. O + +Nucleoproteins B-complex_assembly +also O +represent O +one O +of O +the O +main O +targets O +of O +radiotherapy O +, O +and O +an O +insight O +into O +the O +damage O +mechanisms O +induced O +by O +X O +- O +ray O +irradiation O +could O +inform O +innovative O +treatments O +. O + +Investigations O +on O +sub O +- O +ionization O +- O +level O +LEEs O +( O +0 O +– O +15 O +eV O +) O +interacting O +with O +both O +dried O +and O +aqueous O +oligonucleotides O +( O +Alizadeh O +& O +Sanche O +, O +2014 O +; O +Simons O +, O +2006 O +) O +concluded O +that O +resonant O +electron O +attachment O +to O +DNA B-chemical +bases O +and O +the O +sugar O +- O +phosphate O +backbone O +could O +lead O +to O +the O +preferential O +cleavage O +of O +strong O +(∼ O +4 O +eV O +, O +385 O +kJ O +mol O +− O +1 O +) O +sugar O +- O +phosphate O +C O +— O +O O +covalent O +bonds O +within O +the O +DNA B-chemical +backbone O +and O +then O +base O +- O +sugar O +N1 O +— O +C O +bonds O +, O +eventually O +leading O +to O +single O +- O +strand O +breakages O +( O +SSBs O +; O +Ptasińska O +& O +Sanche O +, O +2007 O +). O + +Electrons O +have O +been O +shown O +to O +be O +mobile O +at O +77 O +K O +by O +electron B-experimental_method +spin I-experimental_method +resonance I-experimental_method +spectroscopy I-experimental_method +studies O +( O +Symons O +, O +1997 O +; O +Jones O +et O +al O +., O +1987 O +), O +with O +rapid O +electron O +quantum O +tunnelling O +and O +positive O +hole O +migration O +along O +the O +protein O +backbone O +and O +through O +stacked O +DNA B-chemical +bases O +indicated O +as O +a O +dominant O +mechanism O +by O +which O +oxidative O +and O +reductive O +damage O +localizes O +at O +distances O +from O +initial O +ionization B-site +sites I-site +at O +100 O +K O +( O +O O +’ O +Neill O +et O +al O +., O +2002 O +). O + +The O +investigation O +of O +naturally O +forming O +nucleoprotein O +complexes O +circumvents O +the O +inherent O +challenges O +in O +making O +controlled O +comparisons O +of O +damage O +mechanisms O +between O +protein O +and O +nucleic O +acids O +crystallized B-experimental_method +separately O +. O +Recently O +, O +for O +a O +well O +characterized O +bacterial B-taxonomy_domain +protein O +– O +DNA B-chemical +complex O +( O +C B-complex_assembly +. I-complex_assembly +Esp1396I I-complex_assembly +; O +PDB O +entry O +3clc O +; O +resolution O +2 O +. O +8 O +Å O +; O +McGeehan O +et O +al O +., O +2008 O +) O +it O +was O +concluded O +that O +over O +a O +wide O +dose O +range O +( O +2 O +. O +1 O +– O +44 O +. O +6 O +MGy O +) O +the O +protein O +was O +far O +more O +susceptible O +to O +SRD O +than O +the O +DNA B-chemical +within O +the O +crystal B-evidence +( O +Bury O +et O +al O +., O +2015 O +). O + +Only O +at O +doses O +above O +20 O +MGy O +were O +precursors O +of O +phosphodiester O +- O +bond O +cleavage O +observed O +within O +AT B-structure_element +- I-structure_element +rich I-structure_element +regions I-structure_element +of O +the O +35 O +- O +mer O +DNA B-chemical +. O + +For O +crystalline O +complexes O +such O +as O +C B-complex_assembly +. I-complex_assembly +Esp1396I I-complex_assembly +, O +whether O +the O +protein O +is O +intrinsically O +more O +susceptible O +to O +X O +- O +ray O +- O +induced O +damage O +or O +whether O +the O +protein O +scavenges O +electrons O +to O +protect O +the O +DNA B-chemical +remains O +unclear O +in O +the O +absence O +of O +a O +non O +- O +nucleic O +acid O +- O +bound O +protein O +control O +obtained O +under O +exactly O +the O +same O +crystallization O +and O +data O +- O +collection O +conditions O +. O + +To O +monitor O +the O +effects O +of O +nucleic O +acid O +binding O +on O +protein O +damage O +susceptibility O +, O +a O +crystal B-evidence +containing O +two O +protein O +molecules O +per O +asymmetric O +unit O +, O +only O +one O +of O +which O +was O +bound B-protein_state +to I-protein_state +RNA B-chemical +, O +is O +reported O +here O +( O +Fig O +. O +1 O +▸). O + +Using O +newly O +developed O +methodology O +, O +we O +present O +a O +controlled B-experimental_method +SRD I-experimental_method +investigation O +at O +1 O +. O +98 O +Å O +resolution O +using O +a O +large O +(∼ O +91 O +kDa O +) O +crystalline O +protein B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complex O +: O +trp B-protein_type +RNA I-protein_type +- I-protein_type +binding I-protein_type +attenuation I-protein_type +protein I-protein_type +( O +TRAP B-complex_assembly +) O +bound B-protein_state +to I-protein_state +a O +53 O +bp O +RNA B-chemical +sequence O +( B-chemical +GAGUU I-chemical +) I-chemical +10GAG I-chemical +( O +PDB O +entry O +1gtf O +; O +Hopcroft O +et O +al O +., O +2002 O +). O + +TRAP B-complex_assembly +consists O +of O +11 O +identical O +subunits B-structure_element +assembled O +into O +a O +ring B-structure_element +with O +11 O +- O +fold O +rotational O +symmetry O +. O + +It O +binds O +with O +high O +affinity O +( O +K B-evidence +d I-evidence +≃ O +1 O +. O +0 O +nM O +) O +to O +RNA B-chemical +segments O +containing O +11 O +GAG B-structure_element +/ I-structure_element +UAG I-structure_element +triplets I-structure_element +separated O +by O +two O +or O +three O +spacer B-structure_element +nucleotides I-structure_element +( O +Elliott O +et O +al O +., O +2001 O +) O +to O +regulate O +the O +transcription O +of O +tryptophan B-chemical +biosynthetic O +genes O +in O +Bacillus B-species +subtilis I-species +( O +Antson O +et O +al O +., O +1999 O +). O + +In O +this O +structure B-evidence +, O +the O +bases O +of O +the O +G1 B-chemical +- I-chemical +A2 I-chemical +- I-chemical +G3 I-chemical +nucleotides O +form O +direct O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +protein O +, O +unlike O +the O +U4 B-chemical +- I-chemical +U5 I-chemical +nucleotides O +, O +which O +appear O +to O +be O +more O +flexible O +. O + +Ten O +successive O +1 O +. O +98 O +Å O +resolution O +MX B-experimental_method +data O +sets O +were O +collected O +from O +the O +same O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +crystal B-evidence +to O +analyse O +X O +- O +ray O +- O +induced O +structural O +changes O +over O +a O +large O +dose O +range O +( O +d O +1 O += O +1 O +. O +3 O +MGy O +to O +d O +10 O += O +25 O +. O +0 O +MGy O +). O + +To O +avoid O +the O +previous O +necessity O +for O +visual O +inspection O +of O +electron B-evidence +- I-evidence +density I-evidence +maps I-evidence +to O +detect O +SRD B-site +sites I-site +, O +a O +computational O +approach O +was O +designed O +to O +quantify O +the O +electron B-evidence +- I-evidence +density I-evidence +change I-evidence +for O +each O +refined O +atom O +with O +increasing O +dose O +, O +thus O +providing O +a O +rapid O +systematic O +method O +for O +SRD O +study O +on O +such O +large O +multimeric O +complexes O +. O + +By O +employing O +the O +high O +11 O +- O +fold O +structural O +symmetry O +within O +each O +TRAP B-complex_assembly +macromolecule O +, O +this O +approach O +permitted O +a O +thorough O +statistical O +quantification O +of O +the O +RD O +effects O +of O +RNA B-chemical +binding O +to O +TRAP B-complex_assembly +. O + +Per B-experimental_method +- I-experimental_method +atom I-experimental_method +quantification I-experimental_method +of I-experimental_method +electron I-experimental_method +density I-experimental_method + +To O +quantify O +the O +exact O +effects O +of O +nucleic O +acid O +binding O +to O +a O +protein O +on O +SRD O +susceptibility O +, O +a O +high O +- O +throughput O +and O +automated O +pipeline O +was O +created O +to O +systematically O +calculate O +the O +electron B-evidence +- I-evidence +density I-evidence +change I-evidence +for O +every O +refined O +atom O +within O +the O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +structure B-evidence +as O +a O +function O +of O +dose O +. O + +This O +provides O +an O +atom O +- O +specific O +quantification O +of O +density B-evidence +– I-evidence +dose I-evidence +dynamics I-evidence +, O +which O +was O +previously O +lacking O +within O +the O +field O +. O + +Previous O +studies O +have O +characterized O +SRD B-site +sites I-site +by O +reporting O +magnitudes O +of O +F B-evidence +obs I-evidence +( I-evidence +d I-evidence +n I-evidence +) I-evidence +− I-evidence +F I-evidence +obs I-evidence +( I-evidence +d I-evidence +1 I-evidence +) I-evidence +Fourier I-evidence +difference I-evidence +map I-evidence +peaks I-evidence +in O +terms O +of O +the O +sigma B-evidence +( O +σ B-evidence +) O +contour O +level O +( O +the O +number O +of O +standard B-evidence +deviations I-evidence +from O +the O +mean B-evidence +map I-evidence +electron I-evidence +- I-evidence +density I-evidence +value I-evidence +) O +at O +which O +peaks O +become O +visible O +. O + +However O +, O +these O +σ B-evidence +levels O +depend O +on O +the O +standard B-evidence +deviation I-evidence +values O +of O +the O +map B-evidence +, O +which O +can O +deviate O +between O +data O +sets O +, O +and O +are O +thus O +unsuitable O +for O +quantitative O +comparison O +of O +density B-evidence +between O +different O +dose O +data O +sets O +. O + +Instead O +, O +we O +use O +here O +a O +maximum B-evidence +density I-evidence +- I-evidence +loss I-evidence +metric I-evidence +( O +D B-evidence +loss I-evidence +), O +which O +is O +the O +per O +- O +atom O +equivalent O +of O +the O +magnitude O +of O +these O +negative B-evidence +Fourier I-evidence +difference I-evidence +map I-evidence +peaks I-evidence +in O +units O +of O +e O +Å O +− O +3 O +. O + +Large O +positive O +D B-evidence +loss I-evidence +values O +indicate O +radiation O +- O +induced O +atomic O +disordering O +reproducibly O +throughout O +the O +unit O +cells O +with O +respect O +to O +the O +initial O +low O +- O +dose O +data O +set O +. O + +For O +each O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +data O +set O +, O +the O +D B-evidence +loss I-evidence +metric I-evidence +successfully O +identified O +the O +recognized O +forms O +of O +protein O +SRD B-experimental_method +( O +Fig O +. O +2 O +▸ O +a O +), O +with O +clear O +Glu B-residue_name +and O +Asp B-residue_name +side O +- O +chain O +decarboxylation O +even O +in O +the O +first O +difference B-evidence +map I-evidence +calculated O +( O +3 O +. O +9 O +MGy O +; O +Fig O +. O +3 O +▸ O +a O +). O + +The O +main O +sequence O +of O +TRAP B-complex_assembly +does O +not O +contain O +any O +Trp B-residue_name +and O +Cys B-residue_name +residues O +( O +and O +thus O +contains O +no O +disulfide O +bonds O +). O + +The O +substrate O +Trp B-chemical +amino O +- O +acid O +ligands O +also O +exhibited O +disordering O +of O +the O +free O +terminal O +carboxyl O +groups O +at O +higher O +doses O +( O +Fig O +. O +2 O +▸ O +a O +); O +however O +, O +no O +clear O +Fourier B-evidence +difference I-evidence +peaks I-evidence +could O +be O +observed O +visually O +. O + +Even O +for O +radiation O +- O +insensitive O +residues O +( O +e O +. O +g O +. O +Gly B-residue_name +) O +the O +average O +D B-evidence +loss I-evidence +increases O +with O +dose O +: O +this O +is O +the O +effect O +of O +global O +radiation O +damage O +, O +since O +as O +dose O +increases O +the O +electron B-evidence +density I-evidence +associated O +with O +each O +refined O +atom O +becomes O +weaker O +as O +the O +atomic O +occupancy O +decreases O +( O +Fig O +. O +2 O +▸ O +b O +). O + +Only O +Glu B-residue_name +and O +Asp B-residue_name +residues O +exhibit O +a O +rate O +of O +D B-evidence +loss I-evidence +increase O +that O +consistently O +exceeds O +the O +average O +decay O +( O +Fig O +. O +2 O +▸ O +b O +, O +dashed O +line O +) O +at O +each O +dose O +. O + +The O +rate O +of O +D B-evidence +loss I-evidence +( O +attributed O +to O +side O +- O +chain O +decarboxylation O +) O +was O +consistently O +larger O +for O +Glu B-residue_name +compared O +with O +Asp B-residue_name +residues O +over O +the O +large O +dose O +range O +( O +Fig O +. O +2 O +▸ O +b O +and O +Supplementary O +Fig O +. O +S3 O +); O +this O +observation O +is O +consistent O +with O +our O +calculations O +on O +model O +systems O +( O +see O +above O +) O +that O +suggest O +that O +, O +without O +considering O +differential O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +environments O +, O +CO2 B-chemical +loss O +is O +more O +exothermic O +by O +around O +8 O +kJ O +mol O +− O +1 O +from O +oxidized B-protein_state +Glu B-residue_name +residues O +than O +from O +their O +Asp B-residue_name +counterparts O +. O + +RNA B-chemical +is O +less O +susceptible O +to O +electron B-evidence +- I-evidence +density I-evidence +loss O +than O +protein O +within O +the O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complex O + +Visual B-experimental_method +inspection I-experimental_method +of I-experimental_method +Fourier B-evidence +difference I-evidence +maps I-evidence +illustrated O +the O +clear O +lack O +of O +RNA B-chemical +electron B-evidence +- I-evidence +density I-evidence +degradation I-evidence +with O +increasing O +dose O +compared O +with O +the O +obvious O +protein O +damage O +manifestations O +( O +Figs O +. O +3 O +▸ O +b O +and O +3 O +▸ O +c O +). O + +Only O +at O +the O +highest O +doses O +investigated O +(> O +20 O +MGy O +) O +was O +density O +loss O +observed O +at O +the O +RNA B-chemical +phosphate O +and O +C O +— O +O O +bonds O +of O +the O +phosphodiester O +backbone O +. O + +However O +, O +the O +median O +D B-evidence +loss I-evidence +was O +lower O +by O +a O +factor O +of O +> O +2 O +for O +RNA B-chemical +P O +atoms O +than O +for O +Glu B-residue_name +and O +Asp B-residue_name +side O +- O +chain O +groups O +at O +25 O +. O +0 O +MGy O +( O +Supplementary O +Fig O +. O +S4 O +), O +and O +furthermore O +could O +not O +be O +numerically O +distinguished O +from O +Gly B-residue_name +Cα O +atoms O +within O +TRAP B-complex_assembly +, O +which O +are O +not O +radiation O +- O +sensitive O +at O +the O +doses O +tested O +here O +( O +Supplementary O +Fig O +. O +S3 O +). O + +RNA B-chemical +binding O +protects O +radiation O +- O +sensitive O +residues O + +For O +the O +large O +number O +of O +acidic O +residues O +per O +TRAP B-complex_assembly +ring B-structure_element +( O +four O +Asp B-residue_name +and O +six O +Glu B-residue_name +residues O +per O +protein O +monomer B-oligomeric_state +), O +a O +strong O +dependence O +of O +decarboxylation O +susceptibility O +on O +local O +environment O +was O +observed O +( O +Fig O +. O +4 O +▸). O + +For O +each O +Glu B-residue_name +Cδ O +or O +Asp B-residue_name +Cγ O +atom O +, O +D B-evidence +loss I-evidence +provided O +a O +direct O +measure O +of O +the O +rate O +of O +side O +- O +chain O +carboxyl O +- O +group O +disordering O +and O +subsequent O +decarboxylation O +. O + +For O +acidic O +residues O +with O +no O +differing O +interactions O +between O +nonbound B-protein_state +and O +bound B-protein_state +TRAP B-complex_assembly +( O +Fig O +. O +4 O +▸ O +a O +), O +similar O +damage O +was O +apparent O +between O +the O +two O +rings O +within O +the O +asymmetric O +unit O +, O +as O +expected O +. O + +However O +, O +TRAP B-complex_assembly +residues O +directly O +on O +the O +RNA B-site +- I-site +binding I-site +interfaces I-site +exhibited O +greater O +damage O +accumulation O +in O +nonbound B-protein_state +TRAP B-complex_assembly +( O +Fig O +. O +4 O +▸ O +b O +), O +and O +for O +residues O +at O +the O +ring B-site +– I-site +ring I-site +interfaces I-site +( O +where O +crystal O +contacts O +were O +detected O +) O +bound B-protein_state +TRAP B-complex_assembly +exhibited O +enhanced O +SRD O +accumulation O +( O +Fig O +. O +4 O +▸ O +c O +). O + +Three O +acidic O +residues O +( O +Glu36 B-residue_name_number +, O +Asp39 B-residue_name_number +and O +Glu42 B-residue_name_number +) O +are O +involved O +in O +RNA B-chemical +interactions O +within O +each O +of O +the O +11 O +TRAP B-complex_assembly +ring B-structure_element +subunits B-structure_element +, O +and O +Fig O +. O +5 O +▸ O +shows O +their O +density B-evidence +changes I-evidence +with O +increasing O +dose O +. O + +Hotelling B-experimental_method +’ I-experimental_method +s I-experimental_method +T I-experimental_method +- I-experimental_method +squared I-experimental_method +test I-experimental_method +( O +the O +multivariate O +counterpart O +of O +Student B-experimental_method +’ I-experimental_method +s I-experimental_method +t I-experimental_method +- I-experimental_method +test I-experimental_method +) O +was O +used O +to O +reject O +the O +null O +hypothesis O +that O +the O +means O +of O +the O +D B-evidence +loss I-evidence +metric I-evidence +were O +equal O +for O +the O +bound B-protein_state +and O +nonbound B-protein_state +groups O +in O +Fig O +. O +5 O +▸. O + +A O +significant O +reduction O +in O +D B-evidence +loss I-evidence +is O +seen O +for O +Glu36 B-residue_name_number +in O +RNA B-protein_state +- I-protein_state +bound I-protein_state +compared O +with O +nonbound B-protein_state +TRAP B-complex_assembly +, O +indicative O +of O +a O +lower O +rate O +of O +side O +- O +chain O +decarboxylation O +( O +Fig O +. O +5 O +▸ O +a O +; O +p O += O +6 O +. O +06 O +× O +10 O +− O +5 O +). O + +For O +each O +TRAP B-complex_assembly +ring B-structure_element +subunit B-structure_element +, O +the O +Glu36 B-residue_name_number +side O +- O +chain O +carboxyl O +group O +accepts O +a O +pair O +of O +hydrogen B-bond_interaction +bonds I-bond_interaction +from O +the O +two O +N O +atoms O +of O +the O +G3 B-residue_name_number +RNA B-chemical +base O +. O + +In O +our O +analysis O +, O +Asp39 B-residue_name_number +in O +the O +TRAP B-complex_assembly +–( I-complex_assembly +GAGUU I-complex_assembly +) I-complex_assembly +10GAG I-complex_assembly +structure B-evidence +appears O +to O +exhibit O +two O +distinct O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +G1 B-residue_name_number +base O +within O +each O +of O +the O +11 O +TRAP B-site +– I-site +RNA I-site +interfaces I-site +, O +as O +does O +Glu36 B-residue_name_number +to O +G3 B-residue_name_number +; O +however O +, O +the O +reduction O +in O +density B-evidence +disordering O +upon O +RNA B-chemical +binding O +is O +far O +less O +significant O +for O +Asp39 B-residue_name_number +than O +for O +Glu36 B-residue_name_number +( O +Fig O +. O +5 O +▸ O +b O +, O +p O += O +0 O +. O +0925 O +). O + +RNA B-chemical +binding O +reduces O +radiation O +- O +induced O +disorder O +on O +the O +atomic O +scale O + +One O +oxygen O +( O +O O +∊ O +1 O +) O +of O +Glu42 B-residue_name_number +appears O +to O +form O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +to O +a O +nearby O +water B-chemical +within O +each O +TRAP B-site +RNA I-site +- I-site +binding I-site +pocket I-site +, O +with O +the O +other O +( O +O O +∊ O +2 O +) O +being O +involved O +in O +a O +salt B-bond_interaction +- I-bond_interaction +bridge I-bond_interaction +interaction O +with O +Arg58 B-residue_name_number +( O +Hopcroft O +et O +al O +., O +2002 O +; O +Antson O +et O +al O +., O +1999 O +). O + +Salt B-bond_interaction +- I-bond_interaction +bridge I-bond_interaction +interactions O +have O +previously O +been O +suggested O +to O +reduce O +the O +glutamate B-residue_name +decarboxylation O +rate O +within O +the O +large O +(∼ O +62 O +. O +4 O +kDa O +) O +myrosinase B-protein_type +protein O +structure B-evidence +( O +Burmeister O +, O +2000 O +). O + +A O +significant O +difference O +was O +observed O +between O +the O +D B-evidence +loss I-evidence +dynamics I-evidence +for O +the O +nonbound B-protein_state +/ O +bound B-protein_state +Glu42 B-residue_name_number +O O +∊ O +1 O +atoms O +( O +Fig O +. O +5 O +▸ O +c O +; O +p O += O +0 O +. O +007 O +) O +but O +not O +for O +the O +Glu42 B-residue_name_number +O O +∊ O +2 O +atoms O +( O +Fig O +. O +5 O +▸ O +d O +; O +p O += O +0 O +. O +239 O +), O +indicating O +that O +the O +stabilizing O +strength O +of O +this O +salt B-bond_interaction +- I-bond_interaction +bridge I-bond_interaction +interaction O +was O +conserved O +upon O +RNA B-chemical +binding O +and O +that O +the O +water B-chemical +- O +mediated O +hydrogen B-bond_interaction +bond I-bond_interaction +had O +a O +greater O +relative O +susceptibility O +to O +atomic O +disordering O +in O +the O +absence B-protein_state +of I-protein_state +RNA B-chemical +. O + +The O +density B-evidence +- I-evidence +change I-evidence +dynamics I-evidence +were O +statistically O +indistinguishable O +between O +bound B-protein_state +and O +nonbound B-protein_state +TRAP B-complex_assembly +for O +each O +Glu42 B-residue_name_number +carboxyl O +group O +Cδ O +atom O +( O +p O += O +0 O +. O +435 O +), O +indicating O +that O +upon O +RNA B-chemical +binding O +the O +conserved O +salt B-bond_interaction +- I-bond_interaction +bridge I-bond_interaction +interaction O +ultimately O +dictated O +the O +overall O +Glu42 B-residue_name_number +decarboxylation O +rate O +. O + +The O +RNA B-chemical +- O +stabilizing O +effect O +was O +not O +restricted O +to O +radiation O +- O +sensitive O +acidic O +residues O +. O + +The O +side O +chain O +of O +Phe32 B-residue_name_number +stacks O +against O +the O +G3 B-residue_name_number +base O +within O +the O +11 O +TRAP B-site +RNA I-site +- I-site +binding I-site +interfaces I-site +( O +Antson O +et O +al O +., O +1999 O +). O + +With O +increasing O +dose O +, O +the O +D B-evidence +loss I-evidence +associated O +with O +the O +Phe32 B-residue_name_number +side O +chain O +was O +significantly O +reduced O +upon O +RNA B-chemical +binding O +( O +Fig O +. O +5 O +▸ O +e O +; O +Phe32 B-residue_name_number +Cζ O +; O +p O += O +0 O +. O +0014 O +), O +an O +indication O +that O +radiation O +- O +induced O +conformation O +disordering O +of O +Phe32 B-residue_name_number +had O +been O +reduced O +. O + +The O +extended O +aliphatic O +Lys37 B-residue_name_number +side O +chain O +stacks O +against O +the O +nearby O +G1 B-residue_name_number +base O +, O +making O +a O +series O +of O +nonpolar B-bond_interaction +contacts I-bond_interaction +within O +each O +RNA B-site +- I-site +binding I-site +interface I-site +. O + +The O +D B-evidence +loss I-evidence +for O +Lys37 B-residue_name_number +side O +- O +chain O +atoms O +was O +also O +reduced O +when O +stacked B-bond_interaction +against O +the O +G1 B-residue_name_number +base O +( O +Fig O +. O +5 O +▸ O +f O +; O +p O += O +0 O +. O +0243 O +for O +Lys37 B-residue_name_number +C O +∊ O +atoms O +). O + +Representative O +Phe32 B-residue_name_number +and O +Lys37 B-residue_name_number +atoms O +were O +selected O +to O +illustrate O +these O +trends O +. O + +Here O +, O +MX B-experimental_method +radiation O +- O +induced O +specific O +structural O +changes O +within O +the O +large O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +assembly O +over O +a O +large O +dose O +range O +( O +1 O +. O +3 O +– O +25 O +. O +0 O +MGy O +) O +have O +been O +analysed O +using O +a O +high O +- O +throughput O +quantitative O +approach O +, O +providing O +a O +measure O +of O +the O +electron B-evidence +- I-evidence +density I-evidence +distribution I-evidence +for O +each O +refined O +atom O +with O +increasing O +dose O +, O +D B-evidence +loss I-evidence +. O + +Compared O +with O +previous O +studies O +, O +the O +results O +provide O +a O +further O +step O +in O +the O +detailed O +characterization O +of O +SRD O +effects O +in O +MX B-experimental_method +. O + +Our O +method O +­ O +ology O +, O +which O +eliminated O +tedious O +and O +error O +- O +prone O +visual O +inspection O +, O +permitted O +the O +determination O +on O +a O +per O +- O +atom O +basis O +of O +the O +most O +damaged O +sites O +, O +as O +characterized O +by O +F B-evidence +obs I-evidence +( I-evidence +d I-evidence +n I-evidence +) I-evidence +− I-evidence +F I-evidence +obs I-evidence +( I-evidence +d I-evidence +1 I-evidence +) I-evidence +Fourier I-evidence +difference I-evidence +map I-evidence +peaks I-evidence +between O +successive O +data O +sets O +collected O +from O +the O +same O +crystal B-evidence +. O + +Here O +, O +it O +provided O +the O +precision O +required O +to O +quantify O +the O +role O +of O +RNA B-chemical +in O +the O +damage O +susceptibilities O +of O +equivalent O +atoms O +between O +RNA B-protein_state +- I-protein_state +bound I-protein_state +and O +nonbound B-protein_state +TRAP B-complex_assembly +, O +but O +it O +is O +applicable O +to O +any O +MX B-experimental_method +SRD O +study O +. O + +The O +RNA B-chemical +was O +found O +to O +be O +substantially O +more O +radiation B-protein_state +- I-protein_state +resistant I-protein_state +than O +the O +protein O +, O +even O +at O +the O +highest O +doses O +investigated O +(∼ O +25 O +. O +0 O +MGy O +), O +which O +is O +in O +strong O +concurrence O +with O +our O +previous O +SRD B-experimental_method +investigation I-experimental_method +of O +the O +C B-complex_assembly +. I-complex_assembly +Esp1396I I-complex_assembly +protein O +– O +DNA B-chemical +complex O +( O +Bury O +et O +al O +., O +2015 O +). O + +Consistent O +with O +that O +study O +, O +at O +high O +doses O +of O +above O +∼ O +20 O +MGy O +, O +F B-evidence +obs I-evidence +( I-evidence +d I-evidence +n I-evidence +) I-evidence +− I-evidence +F I-evidence +obs I-evidence +( I-evidence +d I-evidence +1 I-evidence +) I-evidence +map I-evidence +density I-evidence +was O +detected O +around O +P O +, O +O3 O +′ O +and O +O5 O +′ O +atoms O +of O +the O +RNA B-chemical +backbone O +, O +with O +no O +significant O +difference B-evidence +density I-evidence +localized O +to O +RNA B-chemical +ribose O +and O +basic O +subunits B-structure_element +. O + +RNA B-chemical +backbone O +disordering O +thus O +appears O +to O +be O +the O +main O +radiation O +- O +induced O +effect O +in O +RNA B-chemical +, O +with O +the O +protein O +– O +base O +interactions O +maintained O +even O +at O +high O +doses O +(> O +20 O +MGy O +). O + +The O +U4 B-residue_name_number +phosphate B-chemical +exhibited O +marginally O +larger O +D B-evidence +loss I-evidence +values O +above O +20 O +MGy O +than O +G1 B-residue_name_number +, O +A2 B-residue_name_number +and O +G3 B-residue_name_number +( O +Supplementary O +Fig O +. O +S4 O +). O + +Since O +U4 B-residue_name_number +is O +the O +only O +refined O +nucleotide O +not O +to O +exhibit O +significant O +base O +– O +protein O +interactions O +around O +TRAP B-complex_assembly +( O +with O +a O +water B-chemical +- O +mediated O +hydrogen B-bond_interaction +bond I-bond_interaction +detected O +in O +only O +three O +of O +the O +11 O +subunits B-structure_element +and O +a O +single O +Arg58 B-residue_name_number +hydrogen B-bond_interaction +bond I-bond_interaction +suggested O +in O +a O +further O +four O +subunits B-structure_element +), O +this O +increased O +U4 B-residue_name_number +D B-evidence +loss I-evidence +can O +be O +explained O +owing O +to O +its O +greater O +flexibility O +. O + +At O +25 O +. O +0 O +MGy O +, O +the O +magnitude O +of O +the O +RNA B-chemical +backbone O +D B-evidence +loss I-evidence +was O +of O +the O +same O +order O +as O +for O +the O +radiation O +- O +insensitive O +Gly B-residue_name +Cα O +atoms O +and O +on O +average O +less O +than O +half O +that O +of O +the O +acidic O +residues O +of O +the O +protein O +( O +Supplementary O +Fig O +. O +S3 O +). O + +Consequently O +, O +no O +clear O +single O +- O +strand O +breaks O +could O +be O +located O +, O +and O +since O +RNA B-chemical +- O +binding O +within O +the O +current O +TRAP B-complex_assembly +–( I-complex_assembly +GAGUU I-complex_assembly +) I-complex_assembly +10GAG I-complex_assembly +complex O +is O +mediated O +predominantly O +through O +base O +– O +protein O +interactions O +, O +the O +biological O +integrity O +of O +the O +RNA B-chemical +complex O +was O +dictated O +by O +the O +rate O +at O +which O +protein O +decarboxylation O +occurred O +. O + +RNA B-chemical +interacting O +with O +TRAP B-complex_assembly +was O +shown O +to O +offer O +significant O +protection O +against O +radiation O +- O +induced O +structural O +changes O +. O + +Both O +Glu36 B-residue_name_number +and O +Asp39 B-residue_name_number +bind O +directly O +to O +RNA B-chemical +, O +each O +through O +two O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +guanine B-chemical +bases O +( O +G3 B-residue_name_number +and O +G1 B-residue_name_number +, O +respectively O +). O + +However O +, O +compared O +with O +Asp39 B-residue_name_number +, O +Glu36 B-residue_name_number +is O +strikingly O +less O +decarboxylated O +when O +bound B-protein_state +to I-protein_state +RNA B-chemical +( O +Fig O +. O +4 O +▸). O + +This O +is O +in O +good O +agreement O +with O +previous O +mutagenesis B-experimental_method +and I-experimental_method +nucleoside I-experimental_method +analogue I-experimental_method +studies I-experimental_method +( O +Elliott O +et O +al O +., O +2001 O +), O +which O +indicated O +that O +the O +G1 B-residue_name_number +nucleotide O +does O +not O +bind O +to O +TRAP B-complex_assembly +as O +strongly O +as O +do O +A2 B-residue_name_number +and O +G3 B-residue_name_number +, O +and O +plays O +little O +role O +in O +the O +high O +RNA B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +TRAP B-complex_assembly +( O +K B-evidence +d I-evidence +≃ O +1 O +. O +1 O +± O +0 O +. O +4 O +nM O +). O + +For O +Glu36 B-residue_name_number +and O +Asp39 B-residue_name_number +, O +no O +direct O +quantitative O +correlation O +could O +be O +established O +between O +hydrogen B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +length O +and O +D B-evidence +loss I-evidence +( O +linear B-evidence +R I-evidence +2 I-evidence +of O +< O +0 O +. O +23 O +for O +all O +doses O +; O +Supplementary O +Fig O +. O +S5 O +). O + +Thus O +, O +another O +factor O +must O +be O +responsible O +for O +this O +clear O +reduction O +in O +Glu36 B-residue_name_number +CO2 O +decarboxyl O +­ O +ation O +in O +RNA B-protein_state +- I-protein_state +bound I-protein_state +TRAP B-complex_assembly +. O + +The O +Glu36 B-residue_name_number +carboxyl O +side O +chain O +also O +potentially O +forms O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +His34 B-residue_name_number +and O +Lys56 B-residue_name_number +, O +but O +since O +these O +interactions O +are O +conserved B-protein_state +irrespective O +of O +G3 B-residue_name_number +nucleotide O +binding O +, O +this O +cannot O +directly O +account O +for O +the O +stabilization O +effect O +on O +Glu36 B-residue_name_number +in O +RNA B-protein_state +- I-protein_state +bound I-protein_state +TRAP B-complex_assembly +. O + +When O +bound B-protein_state +to I-protein_state +RNA B-chemical +, O +the O +average O +solvent O +- O +accessible O +area O +of O +the O +Glu36 B-residue_name_number +side O +- O +chain O +O O +atoms O +is O +reduced O +from O +∼ O +15 O +to O +0 O +Å2 O +. O + +We O +propose O +that O +with O +no O +solvent O +accessibility O +Glu36 B-residue_name_number +decarboxylation O +is O +inhibited O +, O +since O +the O +CO2 B-evidence +- I-evidence +formation I-evidence +rate I-evidence +K I-evidence +2 I-evidence +is O +greatly O +reduced O +, O +and O +suggest O +that O +steric O +hindrance O +prevents O +each O +radicalized O +Glu36 B-residue_name_number +CO2 O +group O +from O +achieving O +the O +planar O +conformation O +required O +for O +complete O +dissociation O +from O +TRAP B-complex_assembly +. O + +The O +electron B-evidence +- I-evidence +recombination I-evidence +rate I-evidence +K I-evidence +− I-evidence +1 I-evidence +remains O +high O +, O +however O +, O +owing O +to O +rapid O +electron O +migration O +through O +the O +protein B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complex O +to O +refill O +the O +Glu36 B-residue_name_number +positive B-site +hole I-site +( O +the O +precursor O +for O +Glu B-residue_name +decarboxylation O +). O + +Upon O +RNA B-chemical +binding O +, O +the O +Asp39 B-residue_name_number +side O +- O +chain O +carboxyl O +group O +solvent O +- O +accessible O +area O +changes O +from O +∼ O +75 O +to O +35 O +Å2 O +, O +still O +allowing O +a O +high O +CO2 B-chemical +- O +formation O +rate B-evidence +K I-evidence +2 I-evidence +. O + +The O +prevalence O +of O +radical O +attack O +from O +solvent O +channels O +surrounding O +the O +protein O +in O +the O +crystal B-evidence +is O +a O +questionable O +cause O +, O +considering O +previous O +observations O +indicating O +that O +the O +strongly O +oxidizing O +hydroxyl O +radical O +is O +immobile O +at O +100 O +K O +( O +Allan O +et O +al O +., O +2013 O +; O +Owen O +et O +al O +., O +2012 O +). O + +By O +comparing O +equivalent O +acidic O +residues O +with B-protein_state +and O +without B-protein_state +RNA B-chemical +, O +we O +have O +now O +deconvoluted O +the O +role O +of O +solvent O +accessibility O +from O +other O +local O +protein O +environment O +factors O +, O +and O +thus O +propose O +a O +suitable O +mechanism O +by O +which O +exceptionally O +low O +solvent O +accessibility O +can O +reduce O +the O +rate O +of O +decarboxylation O +. O + +Apart O +from O +these O +RNA B-site +- I-site +binding I-site +interfaces I-site +, O +RNA B-chemical +binding O +was O +seen O +to O +enhance O +decarboxylation O +for O +residues O +Glu50 B-residue_name_number +, O +Glu71 B-residue_name_number +and O +Glu73 B-residue_name_number +, O +all O +of O +which O +are O +involved O +in O +crystal O +contacts O +between O +TRAP B-complex_assembly +rings B-structure_element +( O +Fig O +. O +4 O +▸ O +c O +). O + +However O +, O +for O +each O +of O +these O +residues O +the O +exact O +crystal O +contacts O +are O +not O +preserved O +between O +bound B-protein_state +and O +nonbound B-protein_state +TRAP B-complex_assembly +or O +even O +between O +monomers O +within O +one O +TRAP B-complex_assembly +ring B-structure_element +. O + +For O +example O +, O +in O +bound B-protein_state +TRAP B-complex_assembly +, O +Glu73 B-residue_name_number +hydrogen O +- O +bonds O +to O +a O +nearby O +lysine B-residue_name +on O +each O +of O +the O +11 O +subunits B-structure_element +, O +whereas O +in O +nonbound B-protein_state +TRAP B-complex_assembly +no O +such O +interaction O +exists O +and O +Glu73 B-residue_name_number +interacts O +with O +a O +variable O +number O +of O +refined O +waters B-chemical +in O +each O +subunit B-structure_element +. O + +Radiation O +- O +induced O +side O +- O +chain O +conformational O +changes O +have O +been O +poorly O +characterized O +in O +previous O +SRD B-experimental_method +investigations I-experimental_method +owing O +to O +their O +strong O +dependence O +on O +packing O +density O +and O +geometric O +strain O +. O + +Such O +structural O +changes O +are O +known O +to O +have O +significant O +roles O +within O +enzymatic O +pathways O +, O +and O +experimenters O +must O +be O +aware O +of O +these O +possible O +confounding O +factors O +when O +assigning O +true O +functional O +mechanisms O +using O +MX B-experimental_method +. O + +Our O +results O +show O +that O +RNA B-chemical +binding O +to O +TRAP B-complex_assembly +physically O +stabilizes O +non O +- O +acidic O +residues O +within O +the O +TRAP B-complex_assembly +macromolecule O +, O +most O +notably O +Lys37 B-residue_name_number +and O +Phe32 B-residue_name_number +, O +which O +stack O +against O +the O +G1 B-residue_name_number +and O +G3 B-residue_name_number +bases O +, O +respectively O +. O + +It O +has O +been O +suggested O +( O +Burmeister O +, O +2000 O +) O +that O +Tyr B-residue_name +residues O +can O +lose O +their O +aromatic O +– O +OH O +group O +owing O +to O +radiation O +- O +induced O +effects O +; O +however O +, O +no O +energetically O +favourable O +pathway O +for O +– O +OH O +cleavage O +exists O +and O +this O +has O +not O +been O +detected O +in O +aqueous O +radiation O +- O +chemistry O +studies O +. O + +In O +TRAP B-complex_assembly +, O +D B-evidence +loss I-evidence +increased O +at O +a O +similar O +rate O +for O +both O +the O +Tyr B-residue_name +O O +atoms O +and O +aromatic O +ring B-structure_element +atoms O +, O +suggesting O +that O +full O +ring B-structure_element +conformational O +disordering O +is O +more O +likely O +. O + +Indeed O +, O +no O +convincing O +reproducible O +Fourier B-evidence +difference I-evidence +peaks I-evidence +above O +the O +background O +map B-evidence +noise O +were O +observed O +around O +any O +Tyr B-residue_name +terminal O +– O +OH O +groups O +. O + +The O +RNA B-chemical +- O +stabilization O +effects O +on O +protein O +are O +observed O +at O +short O +ranges O +and O +are O +restricted O +to O +within O +the O +RNA B-site +- I-site +binding I-site +interfaces I-site +around O +the O +TRAP B-complex_assembly +ring B-structure_element +. O + +For O +example O +, O +Asp17 B-residue_name_number +is O +located O +∼ O +6 O +. O +8 O +Å O +from O +the O +G1 B-residue_name_number +base O +, O +outside O +the O +RNA B-site +- I-site +binding I-site +interfaces I-site +, O +and O +has O +indistinguishable O +Cγ O +atom O +D O +loss B-evidence +dose I-evidence +- I-evidence +dynamics I-evidence +between O +RNA B-protein_state +- I-protein_state +bound I-protein_state +and O +nonbound B-protein_state +TRAP B-complex_assembly +( O +p O +> O +0 O +. O +9 O +). O + +An O +increase O +in O +the O +dose O +at O +which O +functionally O +important O +residues O +remain O +intact O +has O +biological O +ramifications O +for O +understanding O +the O +mechanisms O +at O +which O +ionizing O +radiation O +damage O +is O +mitigated O +within O +naturally O +forming O +DNA B-complex_assembly +– I-complex_assembly +protein I-complex_assembly +and O +RNA B-complex_assembly +– I-complex_assembly +protein I-complex_assembly +complexes O +. O + +Observations O +of O +lower O +protein O +radiation O +- O +sensitivity O +in O +DNA B-protein_state +- I-protein_state +bound I-protein_state +forms O +have O +been O +recorded O +in O +solution O +at O +RT O +at O +much O +lower O +doses O +(∼ O +1 O +kGy O +) O +than O +those O +used O +for O +typical O +MX B-experimental_method +experiments O +[ O +e O +. O +g O +. O +an O +oestrogen O +response O +element O +– O +receptor O +complex O +( O +Stísová O +et O +al O +., O +2006 O +) O +and O +a O +DNA B-protein_type +glycosylase I-protein_type +and O +its O +abasic B-site +DNA I-site +target I-site +site I-site +( O +Gillard O +et O +al O +., O +2004 O +)]. O + +In O +these O +studies O +, O +the O +main O +damaging O +species O +is O +predicted O +to O +be O +the O +oxidizing O +hydroxyl O +radical O +produced O +through O +solvent O +irradiation O +, O +which O +is O +known O +to O +add O +to O +double O +covalent O +bonds O +within O +both O +DNA B-chemical +and O +RNA B-chemical +bases O +to O +induce O +strand O +breaks O +and O +base O +modification O +( O +Spotheim O +- O +Maurizot O +& O +Davídková O +, O +2011 O +; O +Chance O +et O +al O +., O +1997 O +). O + +It O +was O +suggested O +that O +physical O +screening O +of O +DNA B-chemical +by O +protein O +shielded O +the O +DNA B-site +– I-site +protein I-site +interaction I-site +sites I-site +from O +radical O +damage O +, O +yielding O +an O +extended O +life O +- O +dose O +for O +the O +nucleoprotein O +complex O +compared O +with O +separate O +protein O +and O +DNA B-chemical +constituents O +at O +RT O +. O + +However O +, O +in O +the O +current O +MX B-experimental_method +study O +at O +100 O +K O +, O +the O +main O +damaging O +species O +are O +believed O +to O +be O +migrating O +LEEs O +and O +holes O +produced O +directly O +within O +the O +protein B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +components O +or O +in O +closely O +associated O +solvent O +. O + +The O +results O +presented O +here O +suggest O +that O +biologically O +relevant O +nucleoprotein B-complex_assembly +complexes O +also O +exhibit O +prolonged O +life O +- O +doses O +under O +the O +effect O +of O +LEE O +- O +induced O +structural O +changes O +, O +involving O +direct O +physical O +protection O +of O +key O +RNA B-site +- I-site +binding I-site +residues I-site +. O + +Such O +reduced O +radiation O +- O +sensitivity O +in O +this O +case O +ensures O +that O +the O +interacting O +protein O +remains O +bound B-protein_state +long O +enough O +to O +the O +RNA B-chemical +to O +complete O +its O +function O +, O +even O +whilst O +exposed O +to O +ionizing O +radiation O +. O + +Within O +the O +nonbound B-protein_state +TRAP B-complex_assembly +macromolecule O +, O +the O +acidic O +residues O +within O +the O +unoccupied O +RNA B-site +- I-site +binding I-site +interfaces I-site +( O +Asp39 B-residue_name_number +, O +Glu36 B-residue_name_number +, O +Glu42 B-residue_name_number +) O +are O +notably O +amongst O +the O +most O +susceptible O +residues O +within O +the O +asymmetric O +unit O +( O +Fig O +. O +4 O +▸). O + +When O +exposed O +to O +X O +- O +rays O +, O +these O +residues O +will O +be O +preferentially O +damaged O +by O +X O +- O +rays O +and O +subsequently O +reduce O +the O +affinity O +with O +which O +TRAP B-complex_assembly +binds O +to O +RNA B-chemical +. O + +Within O +the O +cellular O +environment O +, O +this O +mechanism O +could O +reduce O +the O +risk O +that O +radiation O +- O +damaged O +proteins O +might O +bind O +to O +RNA B-chemical +, O +thus O +avoiding O +the O +detrimental O +introduction O +of O +incorrect O +DNA B-chemical +- O +repair O +, O +transcriptional O +and O +base O +- O +modification O +pathways O +. O + +The O +TRAP B-complex_assembly +–( I-complex_assembly +GAGUU I-complex_assembly +) I-complex_assembly +10GAG I-complex_assembly +complex O +asymmetric O +unit O +( O +PDB O +entry O +1gtf O +; O +Hopcroft O +et O +al O +., O +2002 O +). O + +Bound B-protein_state +tryptophan B-chemical +ligands O +are O +represented O +as O +coloured O +spheres O +. O + +RNA B-chemical +is O +shown O +is O +yellow O +. O + +( O +a O +) O +Electron O +- O +density O +loss O +sites O +as O +indicated O +by O +D O +loss O +in O +the O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complex O +crystal B-evidence +by O +residue O +/ O +nucleotide O +type O +for O +five O +doses O +[ O +sites O +determined O +above O +the O +4 O +× O +average O +D O +loss O +threshold O +, O +calculated O +over O +the O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +structure B-evidence +for O +the O +first O +difference B-evidence +map I-evidence +: O +F O +obs O +( O +d O +2 O +) O +− O +F O +obs O +( O +d O +1 O +)]. O + +( O +b O +) O +Average O +D O +loss O +for O +each O +residue O +/ O +nucleotide O +type O +with O +respect O +to O +the O +DWD B-evidence +( O +diffraction B-evidence +- I-evidence +weighted I-evidence +dose I-evidence +; O +Zeldin O +, O +Brock O +­ O +hauser O +et O +al O +., O +2013 O +). O + +Only O +a O +subset O +of O +key O +TRAP B-complex_assembly +residue O +types O +are O +included O +. O + +The O +average O +D O +loss O +( O +calculated O +over O +the O +whole O +TRAP B-complex_assembly +asymmetric O +unit O +) O +is O +shown O +at O +each O +dose O +( O +dashed O +line O +). O + +In O +( O +a O +) O +clear O +difference B-evidence +density I-evidence +is O +observed O +around O +the O +Glu42 B-residue_name_number +carboxyl O +side O +chain O +in O +chain O +H O +, O +within O +the O +lowest B-evidence +dose I-evidence +difference I-evidence +map I-evidence +at O +d O +2 O += O +3 O +. O +9 O +MGy O +. O + +Radiation O +- O +induced O +protein O +disordering O +is O +evident O +across O +the O +large O +dose O +range O +( O +b O +, O +c O +); O +in O +comparison O +, O +no O +clear O +deterioration O +of O +the O +RNA B-chemical +density B-evidence +was O +observed O +. O + +D O +loss O +calculated O +for O +all O +side O +- O +chain O +carboxyl O +group O +Glu B-residue_name +Cδ O +and O +Asp B-residue_name +Cγ O +atoms O +within O +the O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complex O +for O +a O +dose O +of O +19 O +. O +3 O +MGy O +( O +d O +8 O +). O + +Residues O +have O +been O +grouped O +by O +amino O +- O +acid O +number O +, O +and O +split O +into O +bound B-protein_state +and O +nonbound B-protein_state +groupings O +, O +with O +each O +bar O +representing O +the O +mean O +calculated O +over O +11 O +equivalent O +atoms O +around O +a O +TRAP B-complex_assembly +ring B-structure_element +. O + +D O +loss O +against O +dose O +for O +( O +a O +) O +Glu36 B-residue_name_number +Cδ O +, O +( O +b O +) O +Asp39 B-residue_name_number +Cγ O +, O +( O +c O +) O +Glu42 B-residue_name_number +O O +∊ O +1 O +, O +( O +d O +) O +Glu42 B-residue_name_number +O O +∊ O +2 O +, O +( O +e O +) O +Phe32 B-residue_name_number +Cζ O +and O +( O +f O +) O +Lys37 B-residue_name_number +C O +∊ O +atoms O +. O + +95 O +% O +CI O +are O +included O +for O +each O +set O +of O +11 O +equivalent O +atoms O +grouped O +as O +bound B-protein_state +/ O +nonbound B-protein_state +. O + +RNA B-site +- I-site +binding I-site +interface I-site +interactions O +are O +shown O +for O +TRAP B-complex_assembly +chain O +N O +, O +with O +the O +F O +obs O +( O +d O +7 O +) O +− O +F O +obs O +( O +d O +1 O +) O +Fourier O +difference O +map O +( O +dose O +16 O +. O +7 O +MGy O +) O +overlaid O +and O +contoured O +at O +a O +± O +4σ O +level O +. O + +A O +conserved O +motif O +in O +JNK B-protein_type +/ I-protein_type +p38 I-protein_type +- I-protein_type +specific I-protein_type +MAPK I-protein_type +phosphatases I-protein_type +as O +a O +determinant O +for O +JNK1 B-protein +recognition O +and O +inactivation O + +Mitogen B-protein_type +- I-protein_type +activated I-protein_type +protein I-protein_type +kinases I-protein_type +( O +MAPKs B-protein_type +), O +important O +in O +a O +large O +array O +of O +signalling O +pathways O +, O +are O +tightly O +controlled O +by O +a O +cascade O +of O +protein B-protein_type +kinases I-protein_type +and O +by O +MAPK B-protein_type +phosphatases I-protein_type +( O +MKPs B-protein_type +). O + +MAPK B-protein_type +signalling O +efficiency O +and O +specificity O +is O +modulated O +by O +protein O +– O +protein O +interactions O +between O +individual O +MAPKs B-protein_type +and O +the O +docking B-structure_element +motifs I-structure_element +in O +cognate O +binding O +partners O +. O + +Two O +types O +of O +docking O +interactions O +have O +been O +identified O +: O +D B-structure_element +- I-structure_element +motif I-structure_element +- O +mediated O +interaction O +and O +FXF B-site +- I-site +docking I-site +interaction I-site +. O + +Here O +we O +report O +the O +crystal B-evidence +structure I-evidence +of O +JNK1 B-protein +bound B-protein_state +to I-protein_state +the O +catalytic B-structure_element +domain I-structure_element +of O +MKP7 B-protein +at O +2 O +. O +4 O +- O +Å O +resolution O +, O +providing O +high O +- O +resolution O +structural O +insight O +into O +the O +FXF B-site +- I-site +docking I-site +interaction I-site +. O + +The O +285FNFL288 B-structure_element +segment I-structure_element +in O +MKP7 B-protein +directly O +binds O +to O +a O +hydrophobic B-site +site I-site +on O +JNK1 B-protein +that O +is O +near O +the O +MAPK B-protein_type +insertion O +and O +helix B-structure_element +αG B-structure_element +. O +Biochemical B-experimental_method +studies I-experimental_method +further O +reveal O +that O +this O +highly B-protein_state +conserved I-protein_state +structural B-structure_element +motif I-structure_element +is O +present O +in O +all O +members O +of O +the O +MKP B-protein_type +family I-protein_type +, O +and O +the O +interaction O +mode O +is O +universal O +and O +critical O +for O +the O +MKP B-protein_type +- O +MAPK B-protein_type +recognition O +and O +biological O +function O +. O + +The O +important O +MAPK B-protein_type +family I-protein_type +of O +signalling O +proteins O +is O +controlled O +by O +MAPK B-protein_type +phosphatases I-protein_type +( O +MKPs B-protein_type +). O + +Here O +, O +the O +authors O +report O +the O +structure B-evidence +of O +MKP7 B-protein +bound B-protein_state +to I-protein_state +JNK1 B-protein +and O +characterise O +the O +conserved B-protein_state +MKP B-protein_type +- O +MAPK B-protein_type +interaction O +. O + +The O +mitogen B-protein_type +- I-protein_type +activated I-protein_type +protein I-protein_type +kinases I-protein_type +( O +MAPKs B-protein_type +) O +are O +central O +components O +of O +the O +signal O +- O +transduction O +pathways O +, O +which O +mediate O +the O +cellular O +response O +to O +a O +variety O +of O +extracellular O +stimuli O +, O +ranging O +from O +growth O +factors O +to O +environmental O +stresses O +. O + +The O +MAPK B-protein_type +signalling O +pathways O +are O +evolutionally O +highly O +conserved O +. O + +The O +basic O +assembly O +of O +MAPK B-protein_type +pathways O +is O +a O +three O +- O +tier O +kinase B-protein_type +module O +that O +establishes O +a O +sequential O +activation O +cascade O +: O +a O +MAPK B-protein_type +kinase I-protein_type +kinase I-protein_type +activates O +a O +MAPK B-protein_type +kinase I-protein_type +, O +which O +in O +turn O +activates O +a O +MAPK B-protein_type +. O + +The O +three O +best O +- O +characterized O +MAPK B-protein_type +signalling O +pathways O +are O +mediated O +by O +the O +kinases B-protein_type +extracellular B-protein_type +signal I-protein_type +- I-protein_type +regulated I-protein_type +kinase I-protein_type +( O +ERK B-protein_type +), O +c B-protein_type +- I-protein_type +Jun I-protein_type +N I-protein_type +- I-protein_type +terminal I-protein_type +kinase I-protein_type +( O +JNK B-protein_type +) O +and O +p38 B-protein_type +. O + +The O +ERK B-protein_type +pathway O +is O +activated O +by O +various O +mitogens O +and O +phorbol O +esters O +, O +whereas O +the O +JNK B-protein_type +and O +p38 B-protein_type +pathways O +are O +stimulated O +mainly O +by O +environmental O +stress O +and O +inflammatory O +cytokines B-protein_type +. O + +The O +MAPKs B-protein_type +are O +activated O +by O +MAPK B-protein_type +kinases I-protein_type +that O +phosphorylate O +the O +MAPKs B-protein_type +at O +conserved B-protein_state +threonine B-residue_name +and O +tyrosine B-residue_name +residues O +within O +their O +activation B-structure_element +loop I-structure_element +. O + +After O +activation O +, O +each O +MAPK B-protein_type +phosphorylates O +a O +distinct O +set O +of O +protein O +substrates O +, O +which O +act O +as O +the O +critical O +effectors O +that O +enable O +cells O +to O +mount O +the O +appropriate O +responses O +to O +varied O +stimuli O +. O + +MAPKs B-protein_type +lie O +at O +the O +bottom O +of O +conserved O +three O +- O +component O +phosphorylation O +cascades O +and O +utilize O +docking O +interactions O +to O +link O +module O +components O +and O +bind O +substrates O +. O + +Two O +types O +of O +docking B-structure_element +motifs I-structure_element +have O +been O +identified O +in O +MAPK B-protein_type +substrates O +and O +cognate O +proteins O +: O +kinase B-structure_element +- I-structure_element +interacting I-structure_element +motif I-structure_element +( O +D B-structure_element +- I-structure_element +motif I-structure_element +) O +and O +FXF B-structure_element +- I-structure_element +motif I-structure_element +( O +also O +called O +DEF B-structure_element +motif I-structure_element +, O +docking B-site +site I-site +for O +ERK B-protein_type +FXF B-structure_element +). O + +The O +best O +- O +studied O +docking O +interactions O +are O +those O +between O +MAP B-protein_type +kinases I-protein_type +and O +‘ O +D B-structure_element +- I-structure_element +motifs I-structure_element +', O +which O +consists O +of O +two O +or O +more O +basic O +residues O +followed O +by O +a O +short B-structure_element +linker I-structure_element +and O +a O +cluster O +of O +hydrophobic O +residues O +. O + +The O +D B-site +- I-site +motif I-site +- I-site +docking I-site +site I-site +( O +D B-site +- I-site +site I-site +) O +in O +MAPKs B-protein_type +is O +situated O +in O +a O +noncatalytic B-site +region I-site +opposite O +of O +the O +kinase B-protein_type +catalytic B-site +pocket I-site +and O +is O +comprised O +of O +a O +highly B-site +acidic I-site +patch I-site +and O +a O +hydrophobic B-site +groove I-site +. O + +D B-structure_element +- I-structure_element +motifs I-structure_element +are O +found O +in O +many O +MAPK B-protein_type +- I-protein_type +interacting I-protein_type +proteins I-protein_type +, O +including O +substrates O +, O +activating O +kinases B-protein_type +and O +inactivating O +phosphatases B-protein_type +, O +as O +well O +as O +scaffolding O +proteins O +. O + +A O +second B-structure_element +docking I-structure_element +motif I-structure_element +for O +MAPKs B-protein_type +consists O +of O +two O +Phe B-residue_name +residues O +separated O +by O +one O +residue O +( O +FXF B-structure_element +- I-structure_element +motif I-structure_element +). O + +This O +motif O +has O +been O +observed O +in O +several O +MAPK B-protein_type +substrates O +. O + +The O +FXF B-site +- I-site +motif I-site +- I-site +binding I-site +site I-site +of O +ERK2 B-protein +has O +been O +mapped O +to O +a O +hydrophobic B-site +pocket I-site +formed O +between O +the O +P B-site ++ I-site +1 I-site +site I-site +, O +αG B-structure_element +helix I-structure_element +and O +the O +MAPK B-structure_element +insert I-structure_element +. O + +However O +, O +the O +generality O +and O +mechanism O +of O +the O +FXF B-structure_element +- O +mediated O +interaction O +is O +unclear O +. O + +The O +physiological O +outcome O +of O +MAPK B-protein_type +signalling O +depends O +on O +both O +the O +magnitude O +and O +the O +duration O +of O +kinase O +activation O +. O + +Downregulation O +of O +MAPK B-protein_type +activity O +can O +be O +achieved O +through O +direct O +dephosphorylation O +of O +the O +phospho B-residue_name +- I-residue_name +threonine I-residue_name +and I-residue_name +/ I-residue_name +or I-residue_name +tyrosine I-residue_name +residues O +by O +various O +serine B-protein_type +/ I-protein_type +threonine I-protein_type +phosphatases I-protein_type +, O +tyrosine B-protein_type +phosphatases I-protein_type +and O +dual B-protein_type +- I-protein_type +specificity I-protein_type +phosphatases I-protein_type +( O +DUSPs B-protein_type +) O +termed O +MKPs B-protein_type +. O + +MKPs B-protein_type +constitute O +a O +group O +of O +DUSPs B-protein_type +that O +are O +characterized O +by O +their O +ability O +to O +dephosphorylate O +both O +phosphotyrosine B-residue_name +and O +phosphoserine B-residue_name +/ O +phospho B-residue_name +- I-residue_name +threonine I-residue_name +residues O +within O +a O +substrate O +. O + +Dysregulated O +expression O +of O +MKPs B-protein_type +has O +been O +associated O +with O +pathogenesis O +of O +various O +diseases O +, O +and O +understanding O +their O +precise O +recognition O +mechanism O +presents O +an O +important O +challenge O +and O +opportunity O +for O +drug O +development O +. O + +Here O +, O +we O +present O +the O +crystal B-evidence +structure I-evidence +of O +JNK1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +catalytic B-structure_element +domain I-structure_element +of O +MKP7 B-protein +. O + +This O +structure B-evidence +reveals O +the O +molecular O +mechanism O +underlying O +the O +docking O +interaction O +between O +MKP7 B-protein +and O +JNK1 B-protein +. O + +In O +the O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +complex O +, O +a O +hydrophobic B-structure_element +motif I-structure_element +( O +285FNFL288 B-structure_element +) O +that O +initiates O +the O +helix B-structure_element +α5 B-structure_element +in O +the O +MKP7 B-protein +catalytic B-structure_element +domain I-structure_element +directly O +binds O +to O +the O +FXF B-site +- I-site +motif I-site +- I-site +binding I-site +site I-site +on O +JNK1 B-protein +, O +providing O +the O +structural O +insight O +into O +the O +classic O +FXF B-site +- I-site +type I-site +docking I-site +interaction I-site +. O + +Biochemical B-experimental_method +and I-experimental_method +modelling I-experimental_method +studies I-experimental_method +further O +demonstrate O +that O +the O +molecular O +interactions O +mediate O +this O +key O +element O +for O +substrate O +recognition O +are O +highly O +conserved O +among O +all O +MKP B-protein_type +- I-protein_type +family I-protein_type +members I-protein_type +. O + +Thus O +, O +our O +study O +reveals O +a O +hitherto O +unrecognized O +interaction O +mode O +for O +encoding O +complex O +target O +specificity O +among O +MAPK B-protein_type +isoforms I-protein_type +. O + +Interaction O +of O +JNK1 B-protein +with O +the O +MKP7 B-protein +catalytic B-structure_element +domain I-structure_element + +DUSPs B-protein_type +belong O +to O +the O +protein B-protein_type +- I-protein_type +tyrosine I-protein_type +phosphatases I-protein_type +( O +PTPase B-protein_type +) O +superfamily O +, O +which O +is O +defined O +by O +the O +PTPase B-protein_type +- O +signature O +motif O +CXXGXXR B-structure_element +. O + +MKPs B-protein_type +represent O +a O +distinct O +subfamily O +within O +a O +larger O +group O +of O +DUSPs B-protein_type +. O + +In O +mammalian B-taxonomy_domain +cells O +, O +the O +MKP B-protein_type +subfamily I-protein_type +includes O +10 O +distinct O +catalytically B-protein_state +active I-protein_state +MKPs B-protein_type +. O + +All O +MKPs B-protein_type +contain O +a O +highly B-protein_state +conserved I-protein_state +C O +- O +terminal O +catalytic B-structure_element +domain I-structure_element +( O +CD B-structure_element +) O +and O +an O +N O +- O +terminal O +kinase B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +( O +KBD B-structure_element +). O + +The O +KBD B-structure_element +is O +homologous O +to O +the O +rhodanese B-protein_type +family I-protein_type +and O +contains O +an O +intervening O +cluster O +of O +basic O +amino O +acids O +, O +which O +has O +been O +suggested O +to O +be O +important O +for O +interacting O +with O +the O +target O +MAPKs B-protein_type +. O + +On O +the O +basis O +of O +sequence O +similarity O +, O +substrate O +specificity O +and O +predominant O +subcellular O +localization O +, O +the O +MKP B-protein_type +family I-protein_type +can O +be O +further O +divided O +into O +three O +groups O +( O +Fig O +. O +1 O +). O + +Biochemical B-experimental_method +and I-experimental_method +structural I-experimental_method +studies I-experimental_method +have O +revealed O +that O +the O +KBD B-structure_element +of O +MKPs B-protein_type +is O +critical O +for O +MKP3 B-protein +docking O +to O +ERK2 B-protein +, O +and O +MKP5 B-protein +binding O +to O +p38α B-protein +, O +although O +their O +binding O +mechanisms O +are O +completely O +different O +. O + +However O +, O +it O +is O +unknown O +if O +other O +MAPKs B-protein_type +can O +interact O +with O +the O +KBD B-structure_element +of O +their O +cognate O +phosphatases B-protein_type +in O +the O +same O +manner O +as O +observed O +for O +recognition O +of O +ERK2 B-protein +and O +p38α B-protein +by O +their O +MKPs B-protein_type +, O +or O +whether O +they O +recognize O +distinct O +docking B-structure_element +motifs I-structure_element +of O +MKPs B-protein_type +. O + +MKP7 B-protein +, O +the O +biggest O +molecule O +in O +the O +MKP B-protein_type +family I-protein_type +, O +selectively O +inactivates O +JNK B-protein_type +and O +p38 B-protein_type +following O +stress O +activation O +. O + +In O +addition O +to O +the O +CD B-structure_element +and O +KBD B-structure_element +, O +MKP7 B-protein +has O +a O +long O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +that O +contains O +both O +nuclear O +localization O +and O +export O +sequences O +by O +which O +MKP7 B-protein +shuttles O +between O +the O +nucleus O +and O +the O +cytoplasm O +( O +Fig O +. O +2a O +). O + +To O +quantitatively O +assess O +the O +contribution O +of O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +to O +the O +MKP7 B-protein +- O +catalysed O +JNK1 B-protein +dephosphorylation B-ptm +, O +we O +first O +measured O +the O +kinetic B-evidence +parameters O +of O +the O +C O +- O +terminal O +truncation B-experimental_method +of O +MKP7 B-protein +( O +MKP7ΔC304 B-mutant +, O +residues O +5 B-residue_range +– I-residue_range +303 I-residue_range +) O +and O +MKP7 B-protein +- O +CD B-structure_element +( O +residues O +156 B-residue_range +– I-residue_range +301 I-residue_range +) O +towards O +phosphorylated B-protein_state +JNK1 B-protein +( O +pJNK1 B-protein_state +). O + +Figure O +2b O +shows O +the O +variation B-evidence +of I-evidence +initial I-evidence +rates I-evidence +of O +the O +MKP7ΔC304 B-mutant +and O +MKP7 B-protein +- O +CD B-structure_element +- O +catalysed O +reaction O +with O +the O +concentration O +of O +phospho B-protein_state +- O +JNK1 B-protein +. O +Because O +the O +concentrations O +of O +MKP7 B-protein +and O +pJNK1 B-protein_state +were O +comparable O +in O +the O +reaction O +, O +the O +assumption O +that O +the O +free O +- O +substrate O +concentration O +is O +equal O +to O +the O +total O +substrate O +concentration O +is O +not O +valid O +. O + +Thus O +, O +the O +kinetic B-evidence +data I-evidence +were O +analysed O +using O +the O +general O +initial B-evidence +velocity I-evidence +equation I-evidence +, O +taking O +substrate O +depletion O +into O +account O +: O + +The O +kcat B-evidence +and O +Km B-evidence +of O +the O +MKP7 B-protein +- O +CD B-structure_element +( O +0 O +. O +028 O +s O +− O +1 O +and O +0 O +. O +26 O +μM O +) O +so O +determined O +were O +nearly O +identical O +to O +those O +of O +MKP7ΔC304 B-mutant +( O +0 O +. O +029 O +s O +− O +1 O +and O +0 O +. O +27 O +μM O +), O +indicating O +that O +the O +MKP7 B-protein +- O +KBD B-structure_element +has O +no O +effect O +on O +enzyme O +catalysis O +. O + +We O +next O +examined O +the O +interaction O +of O +JNK1 B-protein +with O +the O +CD B-structure_element +and O +KBD B-structure_element +of O +MKP7 B-protein +by O +gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +. O + +When O +3 O +molar O +equivalents O +of O +CD B-structure_element +were O +mixed O +with O +1 O +molar O +equivalent O +of O +JNK1 B-protein +, O +a O +significant O +amount O +of O +CD B-structure_element +co O +- O +migrated O +with O +JNK1 B-protein +to O +earlier O +fractions O +, O +and O +the O +excess O +amount O +of O +CD B-structure_element +was O +eluted O +from O +the O +size O +exclusion O +column O +as O +a O +monomer B-oligomeric_state +, O +indicating O +stable O +complex O +formation O +( O +Fig O +. O +2c O +). O + +In O +contrast O +, O +no O +KBD B-complex_assembly +– I-complex_assembly +JNK1 I-complex_assembly +complex O +was O +detected O +when O +3 O +molar O +equivalents O +of O +KBD B-structure_element +were O +mixed O +with O +1 O +molar O +equivalent O +of O +JNK1 B-protein +. O + +To O +further O +confirm O +the O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +interaction O +, O +we O +performed O +a O +pull B-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +using O +the O +purified O +proteins O +. O + +As O +shown O +in O +Fig O +. O +2d O +, O +the O +CD B-structure_element +of O +MKP7 B-protein +can O +be O +pulled O +down O +by O +JNK1 B-protein +, O +while O +the O +KBD B-structure_element +failed O +to O +bind O +to O +the O +counterpart O +protein O +. O + +Taken O +together O +, O +our O +data O +indicate O +that O +the O +CD B-structure_element +of O +MKP7 B-protein +, O +but O +not O +the O +KBD B-structure_element +domain O +, O +is O +responsible O +for O +JNK B-protein_type +substrate O +- O +binding O +and O +enzymatic O +specificity O +. O + +Crystal B-evidence +structure I-evidence +of O +JNK1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +MKP7 B-protein +- O +CD B-structure_element + +To O +understand O +the O +molecular O +basis O +of O +JNK1 B-protein +recognition O +by O +MKP7 B-protein +, O +we O +determined O +the O +crystal B-evidence +structure I-evidence +of O +unphosphorylated B-protein_state +JNK1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +MKP7 B-protein +- O +CD B-structure_element +( O +Fig O +. O +3a O +, O +Supplementary O +Fig O +. O +1a O +and O +Table O +1 O +). O + +In O +the O +complex O +, O +JNK1 B-protein +has O +its O +characteristic O +bilobal O +structure O +comprising O +an O +N B-structure_element +- I-structure_element +terminal I-structure_element +lobe I-structure_element +rich O +in O +β B-structure_element +- I-structure_element +sheet I-structure_element +and O +a O +C B-structure_element +- I-structure_element +terminal I-structure_element +lobe I-structure_element +that O +is O +mostly O +α B-structure_element +- I-structure_element +helical I-structure_element +. O + +The O +overall O +folding O +of O +MKP7 B-protein +- O +CD B-structure_element +is O +typical O +of O +DUSPs B-protein_type +, O +with O +a O +central O +twisted B-structure_element +five I-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +surrounded O +by O +six O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +One O +side O +of O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +is O +covered O +with O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +the O +other O +is O +covered O +with O +four O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +Fig O +. O +3b O +). O + +The O +catalytic B-structure_element +domain I-structure_element +of O +MKP7 B-protein +interacts O +with O +JNK1 B-protein +through O +a O +contiguous O +surface O +area O +that O +is O +remote O +from O +the O +active B-site +site I-site +. O + +MKP7 B-protein +- O +CD B-structure_element +is O +positioned O +onto O +the O +JNK1 B-protein +molecule O +so O +that O +the O +active B-site +site I-site +of O +the O +phosphatase B-protein_type +faces O +towards O +the O +activation B-structure_element +segment I-structure_element +. O + +In O +an O +alignment B-experimental_method +of O +the O +structure B-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +with O +that O +of O +VHR B-protein +, O +an O +atypical O +‘ O +MKP B-protein_type +' O +consisting O +of O +only O +a O +catalytic B-structure_element +domain I-structure_element +, O +119 O +of O +147 O +MKP7 B-protein +- O +CD B-structure_element +residues O +could O +be O +superimposed B-experimental_method +with O +a O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +. I-evidence +( O +root B-evidence +mean I-evidence +squared I-evidence +deviation I-evidence +) O +of O +1 O +. O +05 O +Å O +( O +Fig O +. O +3c O +). O + +The O +most O +striking O +difference O +is O +that O +helix B-structure_element +α0 B-structure_element +and O +loop B-structure_element +α0 B-structure_element +– I-structure_element +β1 I-structure_element +of O +VHR B-protein +are O +absent O +in O +MKP7 B-protein +- O +CD B-structure_element +. O + +Another O +region O +that O +cannot O +be O +aligned O +with O +VHR B-protein +is O +found O +in O +loop B-structure_element +β3 B-structure_element +– I-structure_element +β4 I-structure_element +. O + +This O +loop B-structure_element +is O +shortened O +by O +nine O +residues O +in O +MKP7 B-protein +- O +CD B-structure_element +compared O +with O +that O +in O +VHR B-protein +. O + +Since O +helix B-structure_element +α0 B-structure_element +and O +the O +following O +loop B-structure_element +α0 B-structure_element +– I-structure_element +β1 I-structure_element +are O +known O +for O +a O +substrate B-site +- I-site +recognition I-site +motif I-site +of O +VHR B-protein +and O +other O +phosphatases B-protein_type +, O +the O +absence O +of O +these O +moieties O +implicates O +a O +different O +substrate O +- O +binding O +mode O +of O +MKP7 B-protein +. O + +The O +active B-site +site I-site +of O +MKP7 B-protein +consists O +of O +the O +phosphate B-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +( O +P B-structure_element +- I-structure_element +loop I-structure_element +, O +Cys244 B-residue_name_number +- O +Leu245 B-residue_name_number +- O +Ala246 B-residue_name_number +- O +Gly247 B-residue_name_number +- O +Ile248 B-residue_name_number +- O +Ser249 B-residue_name_number +- O +Arg250 B-residue_name_number +), O +and O +Asp213 B-residue_name_number +in O +the O +general B-structure_element +acid I-structure_element +loop I-structure_element +( O +Fig O +. O +3b O +and O +Supplementary O +Fig O +. O +1b O +). O + +The O +MKP7 B-protein +- O +CD B-structure_element +structure B-evidence +near O +the O +active B-site +site I-site +exhibits O +a O +typical O +active B-protein_state +conformation I-protein_state +as O +found O +in O +VHR B-protein +and O +other O +PTPs B-protein_type +. O + +The O +catalytic B-site +residue I-site +, O +Cys244 B-residue_name_number +, O +is O +located O +just O +after O +strand B-structure_element +β5 B-structure_element +and O +optimally O +positioned O +for O +nucleophilic O +attack O +. O + +Asp213 B-residue_name_number +in O +MKP7 B-protein +also O +adopts O +a O +position O +similar O +to O +that O +of O +Asp92 B-residue_name_number +in O +VHR B-protein +( O +Supplementary O +Fig O +. O +1c O +), O +indicating O +that O +Asp213 B-residue_name_number +is O +likely O +to O +function O +as O +the O +general O +acid O +in O +MKP7 B-protein +. O + +We O +also O +observed O +the O +binding O +of O +a O +chloride B-chemical +ion O +in O +the O +active B-site +site I-site +of O +MKP7 B-protein +- O +CD B-structure_element +. O + +It O +is O +located O +3 O +. O +36 O +Å O +from O +the O +Cys244 B-residue_name_number +side O +chain O +and O +makes O +electrostatic B-bond_interaction +interactions I-bond_interaction +with O +the O +dipole O +moment O +of O +helix B-structure_element +α3 B-structure_element +and O +with O +several O +main O +- O +chain O +amide O +groups O +. O + +The O +side O +chain O +of O +strictly B-protein_state +conserved I-protein_state +Arg250 B-residue_name_number +is O +oriented O +towards O +the O +negatively O +charged O +chloride B-chemical +, O +similar O +to O +the O +canonical O +phosphate B-structure_element +- I-structure_element +coordinating I-structure_element +conformation I-structure_element +. O + +Thus O +this O +chloride B-chemical +ion O +is O +a O +mimic O +for O +the O +phosphate B-chemical +group O +of O +the O +substrate O +, O +as O +revealed O +by O +a O +comparison O +with O +the O +structure B-evidence +of O +PTP1B B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +phosphotyrosine B-residue_name +( O +Supplementary O +Fig O +. O +1d O +). O + +Although O +the O +catalytically O +important O +residues O +in O +MKP7 B-protein +- O +CD B-structure_element +are O +well O +aligned O +with O +those O +in O +VHR B-protein +, O +the O +residues O +in O +the O +P B-structure_element +- I-structure_element +loop I-structure_element +of O +MKP7 B-protein +are O +smaller O +and O +have O +a O +more O +hydrophobic O +character O +than O +those O +of O +VHR B-protein +( O +Cys124 B-residue_name_number +- O +Arg125 B-residue_name_number +- O +Glu126 B-residue_name_number +- O +Gly127 B-residue_name_number +- O +Tyr128 B-residue_name_number +- O +Gly129 B-residue_name_number +- O +Arg130 B-residue_name_number +; O +Fig O +. O +3b O +, O +c O +). O + +The O +difference O +in O +the O +polarity O +/ O +hydrophobicity O +of O +the O +surface O +may O +also O +point O +to O +the O +origin O +of O +the O +differences O +in O +the O +substrate O +- O +recognition O +mechanism O +for O +these O +two O +phosphatases B-protein_type +( O +Supplementary O +Fig O +. O +1e O +, O +f O +). O + +In O +the O +complex O +, O +MKP7 B-protein +- O +CD B-structure_element +and O +JNK1 B-protein +form O +extensive O +protein O +– O +protein O +interactions O +involving O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +helices I-structure_element +of O +MKP7 B-protein +- O +CD B-structure_element +and O +C B-structure_element +- I-structure_element +lobe I-structure_element +of O +JNK1 B-protein +( O +Fig O +. O +3d O +, O +e O +). O + +As O +a O +result O +, O +the O +buried O +solvent O +- O +accessible O +surface O +area O +is O +∼ O +1 O +, O +315 O +Å O +. O +In O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +, O +JNK1 B-protein +has O +an O +insertion O +after O +the O +helix B-structure_element +αG B-structure_element +. O +This O +insertion O +consists O +of O +two O +helices B-structure_element +( O +α1L14 B-structure_element +and O +α2L14 B-structure_element +) O +that O +are O +common O +to O +all O +members O +of O +the O +MAPK B-protein_type +family I-protein_type +. O + +The O +interactive B-site +surface I-site +in O +JNK1 B-protein +, O +formed O +by O +the O +helices B-structure_element +αG B-structure_element +and O +α2L14 B-structure_element +, O +displays O +a O +hydrophobic B-site +region I-site +, O +centred O +at O +Trp234 B-residue_name_number +( O +Fig O +. O +3d O +). O + +The O +MKP7 B-site +- I-site +docking I-site +region I-site +includes O +two O +helices B-structure_element +, O +α4 B-structure_element +and O +α5 B-structure_element +, O +and O +the O +general B-structure_element +acid I-structure_element +loop I-structure_element +. O + +The O +aromatic O +ring O +of O +Phe285 B-residue_name_number +on O +MKP7 B-protein +α5 B-structure_element +- I-structure_element +helix I-structure_element +is O +nestled O +in O +a O +hydrophobic B-site +pocket I-site +on O +JNK1 B-protein +, O +formed O +by O +side O +chains O +of O +Ile197 B-residue_name_number +, O +Leu198 B-residue_name_number +, O +Ile231 B-residue_name_number +, O +Trp234 B-residue_name_number +, O +Val256 B-residue_name_number +, O +Tyr259 B-residue_name_number +, O +Val260 B-residue_name_number +and O +the O +aliphatic O +portion O +of O +His230 B-residue_name_number +( O +Fig O +. O +3d O +, O +f O +and O +Supplementary O +Fig O +. O +1g O +). O + +In O +addition O +, O +there O +are O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +Ser282 B-residue_name_number +and O +Asn286 B-residue_name_number +of O +MKP7 B-protein +and O +His230 B-residue_name_number +and O +Thr255 B-residue_name_number +of O +JNK1 B-protein +, O +and O +the O +main O +chain O +of O +Phe215 B-residue_name_number +in O +the O +general B-structure_element +acid I-structure_element +loop I-structure_element +of O +MKP7 B-protein +is O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +side O +chain O +of O +Gln253 B-residue_name_number +in O +JNK1 B-protein +. O + +The O +second B-site +interactive I-site +area I-site +involves O +the O +α4 B-structure_element +helix I-structure_element +of O +MKP7 B-protein +and O +charged O +/ O +polar O +residues O +of O +JNK1 B-protein +( O +Fig O +. O +3e O +). O + +The O +carboxylate O +of O +Asp268 B-residue_name_number +in O +MKP7 B-protein +forms O +a O +salt B-bond_interaction +bridge I-bond_interaction +with O +side O +chain O +of O +Arg263 B-residue_name_number +in O +JNK1 B-protein +, O +and O +Lys275 B-residue_name_number +of O +MKP7 B-protein +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +and O +a O +salt B-bond_interaction +bridge I-bond_interaction +with O +Thr228 B-residue_name_number +and O +Asp229 B-residue_name_number +of O +JNK1 B-protein +, O +respectively O +. O + +Mutational B-experimental_method +analysis I-experimental_method +of O +the O +JNK1 B-site +– I-site +MKP7 I-site +docking I-site +interface I-site + +To O +assess O +the O +importance O +of O +the O +aforementioned O +interactions O +, O +we O +generated O +a O +series O +of O +point B-experimental_method +mutations I-experimental_method +on O +the O +MKP7 B-protein +- O +CD B-structure_element +and O +examined O +their O +effect O +on O +the O +MKP7 B-protein +- O +catalysed O +JNK1 B-protein +dephosphorylation B-ptm +( O +Fig O +. O +4a O +). O + +When O +the O +hydrophobic O +residues O +Phe285 B-residue_name_number +and O +Phe287 B-residue_name_number +on O +the O +α5 B-structure_element +of O +MKP7 B-protein +- O +CD B-structure_element +were O +replaced B-experimental_method +by O +Asp B-residue_name +or O +Ala B-residue_name +, O +their O +phosphatase O +activities O +for O +JNK1 B-protein +dephosphorylation B-ptm +decreased O +∼ O +10 O +- O +fold O +. O + +In O +comparison O +, O +replacement B-experimental_method +of O +the O +other O +residues O +( O +Phe215 B-residue_name_number +, O +Asp268 B-residue_name_number +, O +Lys275 B-residue_name_number +, O +Ser282 B-residue_name_number +, O +Asn286 B-residue_name_number +and O +Leu292 B-residue_name_number +) O +with O +an O +Ala B-residue_name +or O +Asp B-residue_name +individually O +led O +to O +a O +modest O +decrease O +in O +catalytic O +efficiencies O +, O +suggesting O +that O +this O +position O +may O +only O +affect O +some O +selectivity O +of O +MKP B-protein_type +. O + +Mutation B-experimental_method +of O +Leu288 B-residue_name_number +markedly O +reduced O +its O +solubility O +when O +expressed O +in O +Escherichia B-species +coli I-species +, O +resulting O +in O +the O +insoluble O +aggregation O +of O +the O +mutant B-protein_state +protein O +. O + +Gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +further O +confirmed O +the O +key O +role O +of O +Phe285 B-residue_name_number +in O +the O +MKP7 B-protein +– O +JNK1 B-protein +interaction O +: O +no O +F285D B-complex_assembly +– I-complex_assembly +JNK1 I-complex_assembly +complex O +was O +detected O +when O +3 O +molar O +equivalents O +of O +MKP7 B-protein +- O +CD B-structure_element +( O +F285D B-mutant +) O +were O +mixed O +with O +1 O +molar O +equivalent O +of O +JNK1 B-protein +( O +Fig O +. O +4b O +). O + +Interestingly O +, O +mutation B-experimental_method +of O +Phe287 B-residue_name_number +results O +in O +a O +considerable O +loss O +of O +activity O +against O +pJNK1 B-protein_state +without O +altering O +the O +affinity B-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +for O +JNK1 B-protein +( O +Supplementary O +Fig O +. O +2a O +). O + +We O +also O +generated O +a O +series O +of O +point B-experimental_method +mutations I-experimental_method +in O +the O +JNK1 B-protein +and O +assessed O +the O +effect O +on O +JNK1 B-protein +binding O +using O +the O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +( O +Fig O +. O +4c O +). O + +Substitution B-experimental_method +at O +Asp229 B-residue_name_number +, O +Trp234 B-residue_name_number +, O +Thr255 B-residue_name_number +, O +Val256 B-residue_name_number +, O +Tyr259 B-residue_name_number +and O +Val260 B-residue_name_number +significantly O +reduced O +the O +binding B-evidence +affinity I-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +for O +JNK B-protein_type +. O + +To O +determine O +whether O +the O +deficiencies O +in O +their O +abilities O +to O +bind O +partner O +proteins O +or O +carry O +out O +catalytic O +function O +are O +owing O +to O +misfolding O +of O +the O +purified O +mutant B-protein_state +proteins O +, O +we O +also O +examined O +the O +folding O +properties O +of O +the O +JNK1 B-protein +and O +MKP7 B-protein +mutants B-protein_state +with O +circular B-experimental_method +dichroism I-experimental_method +. O + +The O +spectra B-evidence +of O +these O +mutants B-protein_state +are O +similar O +to O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +proteins O +, O +indicating O +that O +these O +mutants B-protein_state +fold O +as O +well O +as O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +proteins O +( O +Fig O +. O +4d O +, O +e O +). O + +Taken O +together O +, O +these O +results O +are O +consistent O +with O +the O +present O +crystallographic B-evidence +model I-evidence +, O +which O +reveal O +the O +hydrophobic B-bond_interaction +contacts I-bond_interaction +between O +the O +MKP7 B-protein +catalytic B-structure_element +domain I-structure_element +and O +JNK1 B-protein +have O +a O +predominant O +role O +in O +the O +enzyme O +– O +substrate O +interaction O +, O +and O +hydrophobic O +residue O +Phe285 B-residue_name_number +in O +the O +MKP7 B-protein +- O +CD B-structure_element +is O +a O +key O +residue O +for O +its O +high O +- O +affinity O +binding O +to O +JNK1 B-protein +. O + +It O +has O +previously O +been O +reported O +that O +several O +cytosolic O +and O +inducible O +nuclear O +MKPs B-protein_type +undergo O +catalytic O +activation O +upon O +interaction O +with O +the O +MAPK B-protein_type +substrates O +. O + +This O +allosteric O +activation O +of O +MKP3 B-protein +has O +been O +well O +- O +documented O +in O +vitro O +using O +pNPP B-chemical +, O +a O +small O +- O +molecule O +phosphotyrosine B-residue_name +analogue O +of O +its O +normal O +substrate O +. O + +We O +then O +assayed O +pNPPase B-protein_type +activities O +of O +MKP7ΔC304 B-mutant +and O +MKP7 B-protein +- O +CD B-structure_element +in O +the O +presence B-protein_state +of I-protein_state +JNK1 B-protein +. O + +Incubation B-experimental_method +of O +MKP7 B-protein +with O +JNK1 B-protein +did O +not O +markedly O +stimulate O +the O +phosphatase B-protein_type +activity O +, O +which O +is O +consistent O +with O +previous O +results O +that O +MKP7 B-protein +solely O +possesses O +the O +intrinsic O +activity O +( O +Supplementary O +Fig O +. O +2b O +). O + +The O +small O +pNPP B-chemical +molecule O +binds O +directly O +at O +the O +enzyme O +active B-site +site I-site +and O +can O +be O +used O +to O +probe O +the O +reaction O +mechanism O +of O +protein B-protein_type +phosphatases I-protein_type +. O + +We O +therefore O +examined O +the O +effects O +of O +the O +MKP7 B-protein +- O +CD B-structure_element +mutants B-protein_state +on O +their O +pNPPase B-protein_type +activities O +. O + +As O +shown O +in O +Fig O +. O +4f O +, O +all O +the O +mutants B-protein_state +, O +except O +F287D B-mutant +/ I-mutant +A I-mutant +, O +showed O +little O +or O +no O +activity O +change O +compared O +with O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +MKP7 B-protein +- O +CD B-structure_element +. O + +In O +the O +JNK1 B-complex_assembly +/ I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +complex O +structure B-evidence +, O +Phe287 B-residue_name_number +of O +MKP7 B-protein +does O +not O +make O +contacts O +with O +JNK1 B-protein +substrate O +. O + +It O +penetrates O +into O +a O +pocket B-site +formed O +by O +residues O +from O +the O +P B-structure_element +- I-structure_element +loop I-structure_element +and O +general B-structure_element +acid I-structure_element +loop I-structure_element +and O +forms O +hydrophobic B-bond_interaction +contacts I-bond_interaction +with O +the O +aliphatic O +portions O +of O +side O +chains O +of O +Arg250 B-residue_name_number +, O +Glu217 B-residue_name_number +and O +Ile219 B-residue_name_number +, O +suggesting O +that O +Phe287 B-residue_name_number +in O +MKP7 B-protein +would O +play O +a O +similar O +role O +to O +that O +of O +its O +structural O +counterpart O +in O +the O +PTPs B-protein_type +( O +Gln266 B-residue_name_number +in O +PTP1B B-protein +) O +and O +VHR B-protein +( O +Phe166 B-residue_name_number +in O +VHR B-protein +) O +in O +the O +precise O +alignment O +of O +active B-site +- I-site +site I-site +residues I-site +in O +MKP7 B-protein +with O +respect O +to O +the O +substrate O +for O +efficient O +catalysis O +( O +Supplementary O +Fig O +. O +2c O +). O + +Kinase B-protein +- I-protein +associated I-protein +phosphatase I-protein +( O +KAP B-protein +), O +a O +member O +of O +the O +DUSP B-protein_type +family I-protein_type +, O +plays O +a O +crucial O +role O +in O +cell O +cycle O +regulation O +by O +dephosphorylating O +the O +pThr160 B-ptm +residue O +of O +CDK2 B-protein +( O +cyclin B-protein +- I-protein +dependent I-protein +kinase I-protein +2 I-protein +). O + +The O +crystal B-evidence +structure I-evidence +of O +the O +CDK2 B-complex_assembly +/ I-complex_assembly +KAP I-complex_assembly +complex O +has O +been O +determined O +at O +3 O +. O +0 O +Å O +( O +Fig O +. O +5a O +). O + +The O +interface B-site +between O +these O +two O +proteins O +consists O +of O +three O +discontinuous O +contact O +regions O +. O + +Biochemical O +results O +suggested O +that O +the O +affinity O +and O +specificity O +between O +KAP B-protein +and O +CDK2 B-protein +results O +from O +the O +recognition B-site +site I-site +comprising O +CDK2 B-protein +residues O +from O +the O +αG B-structure_element +helix I-structure_element +and O +L14 B-structure_element +loop I-structure_element +and O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +helical I-structure_element +region I-structure_element +of O +KAP B-protein +( O +Fig O +. O +5b O +). O + +There O +is O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +the O +main O +- O +chain O +nitrogen O +of O +Ile183 B-residue_name_number +( O +KAP B-protein +) O +and O +side O +chain O +oxygen O +of O +Glu208 B-residue_name_number +( O +CDK2 B-protein +), O +and O +salt O +bridges O +between O +Lys184 B-residue_name_number +of O +KAP B-protein +and O +Asp235 B-residue_name_number +of O +CDK2 B-protein +. O + +Structural B-experimental_method +analysis I-experimental_method +and O +sequence B-experimental_method +alignment I-experimental_method +reveal O +that O +one O +of O +the O +few O +differences O +between O +MKP7 B-protein +- O +CD B-structure_element +and O +KAP B-protein +in O +the O +substrate B-site +- I-site +binding I-site +region I-site +is O +the O +presence O +of O +the O +motif O +FNFL B-structure_element +in O +MKP7 B-protein +- O +CD B-structure_element +, O +which O +corresponds O +to O +IKQY B-structure_element +in O +KAP B-protein +( O +Fig O +. O +5c O +). O + +The O +substitution B-experimental_method +of O +the O +two O +hydrophobic O +residues O +with O +charged O +/ O +polar O +residues O +( O +F285I B-mutant +/ O +N286K B-mutant +) O +seriously O +disrupts O +the O +hydrophobic B-bond_interaction +interaction I-bond_interaction +required O +for O +MKP7 B-protein +binding O +on O +JNK1 B-protein +( O +Fig O +. O +4a O +). O + +In O +addition O +, O +His230 B-residue_name_number +and O +Val256 B-residue_name_number +in O +JNK1 B-protein +are O +replaced O +by O +the O +negatively O +charged O +residues O +Glu208 B-residue_name_number +and O +Asp235 B-residue_name_number +in O +CDK2 B-protein +( O +Fig O +. O +5d O +), O +and O +the O +charge O +distribution O +on O +the O +CDK2 B-protein +interactive B-site +surface I-site +is O +quite O +different O +from O +that O +of O +JNK B-protein_type +. O + +These O +data O +indicated O +that O +a O +unique O +hydrophobic B-site +pocket I-site +formed O +between O +the O +MAPK B-structure_element +insert I-structure_element +and O +αG B-structure_element +helix I-structure_element +plays O +a O +major O +role O +in O +the O +substrate O +recognition O +by O +MKPs B-protein_type +. O + +F B-site +- I-site +site I-site +interaction O +is O +crucial O +for O +JNK1 B-protein +inactivation O +in O +vivo O + +JNK B-protein_type +is O +activated O +following O +cellular O +exposure O +to O +a O +number O +of O +acute O +stimuli O +such O +as O +anisomycin B-chemical +, O +H2O2 B-chemical +, O +ultraviolet O +light O +, O +sorbitol B-chemical +, O +DNA O +- O +damaging O +agents O +and O +several O +strong O +apoptosis O +inducers O +( O +etoposide B-chemical +, O +cisplatin B-chemical +and O +taxol B-chemical +). O + +To O +assess O +the O +effects O +of O +MKP7 B-protein +and O +its O +mutants B-protein_state +on O +the O +activation O +of O +endogenous O +JNK B-protein_type +in O +vivo O +, O +HEK293T O +cells O +were O +transfected O +with O +blank O +vector O +or O +with O +HA B-protein_state +- I-protein_state +tagged I-protein_state +constructs O +for O +full B-protein_state +- I-protein_state +length I-protein_state +MKP7 B-protein +, O +MKP7ΔC304 B-mutant +and O +MKP7 B-protein +- O +CD B-structure_element +or O +MKP7 B-protein +mutants B-protein_state +, O +and O +stimulated O +with O +ultraviolet O +or O +etoposide B-chemical +treatment O +. O + +As O +shown O +in O +Fig O +. O +6a O +– O +c O +, O +immunobloting B-experimental_method +showed O +similar O +expression O +levels O +for O +the O +different O +MKP7 B-protein +constructs O +in O +all O +the O +cells O +. O + +Overexpressed B-experimental_method +full B-protein_state +- I-protein_state +length I-protein_state +MKP7 B-protein +, O +MKP7ΔC304 B-mutant +and O +MKP7 B-protein +- O +CD B-structure_element +significantly O +reduced O +the O +endogenous O +level O +of O +phosphorylated B-protein_state +JNK B-protein_type +compared O +with O +vector O +- O +transfected O +cells O +. O + +Parallel O +experiments O +showed O +clearly O +that O +the O +D B-structure_element +- I-structure_element +motif I-structure_element +mutants B-protein_state +( O +R56A B-mutant +/ O +R57A B-mutant +and O +V63A B-mutant +/ O +I65A B-mutant +) O +dephosphorylated B-protein_state +JNK B-protein_type +as O +did O +the O +wild B-protein_state +type I-protein_state +under O +the O +same O +conditions O +, O +further O +confirming O +that O +the O +MKP7 B-protein +- O +KBD B-structure_element +is O +not O +required O +for O +the O +JNK B-protein_type +inactivation O +in O +vivo O +. O + +Consistent O +with O +the O +in O +vitro O +data O +, O +the O +level O +of O +phosphorylated B-protein_state +JNK B-protein_type +was O +not O +or O +little O +altered O +in O +MKP7 B-protein +FXF B-structure_element +- I-structure_element +motif I-structure_element +mutants B-protein_state +( O +F285D B-mutant +, O +F287D B-mutant +and O +L288D B-mutant +)- O +transfected O +cells O +, O +and O +the O +MKP7 B-protein +D268A B-mutant +and O +N286A B-mutant +mutants B-protein_state +retained O +the O +ability O +to O +reduce O +the O +phosphorylation O +levels O +of O +JNK B-protein_type +. O + +We O +next O +tested O +in O +vivo O +interactions O +between O +JNK1 B-protein +mutants B-protein_state +and O +full B-protein_state +- I-protein_state +length I-protein_state +MKP7 B-protein +by O +coimmunoprecipitation B-experimental_method +experiments I-experimental_method +under O +unstimulated O +conditions O +. O + +When O +co B-experimental_method +- I-experimental_method +expressed I-experimental_method +in O +HEK293T O +cells O +, O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +HA O +)- O +JNK1 B-protein +was O +readily O +precipitated O +with O +( O +Myc O +)- O +MKP7 B-protein +( O +Fig O +. O +6d O +), O +indicating O +that O +MKP7 B-protein +binds O +dephosphorylated B-protein_state +JNK1 B-protein +protein O +in O +vivo O +. O + +In O +agreement O +with O +the O +in B-experimental_method +vitro I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +results O +, O +the O +mutants B-protein_state +D229A B-mutant +, O +W234D B-mutant +and O +Y259D B-mutant +were O +not O +co O +- O +precipitated O +with O +MKP7 B-protein +, O +and O +the O +I231D B-mutant +mutant B-protein_state +had O +only O +little O +effect O +on O +the O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +interaction O +( O +Fig O +. O +6d O +and O +Supplementary O +Fig O +. O +3a O +). O + +Activation O +of O +the O +JNK B-protein_type +signalling O +pathway O +is O +frequently O +associated O +with O +apoptotic O +cell O +death O +, O +and O +inhibition O +of O +JNK B-protein_type +can O +prevent O +apoptotic O +death O +of O +multiple O +cells O +. O + +To O +examine O +whether O +the O +inhibition O +of O +JNK B-protein_type +activity O +by O +MKP7 B-protein +would O +provide O +protections O +against O +the O +apoptosis O +, O +we O +analysed O +the O +rate O +of O +apoptosis O +in O +ultraviolet O +- O +irradiated O +cells O +transfected O +with O +MKP7 B-protein +( O +wild B-protein_state +type I-protein_state +or O +mutants B-protein_state +) O +by O +flow B-experimental_method +cytometry I-experimental_method +. O + +The O +results O +showed O +similar O +apoptotic O +rates O +between O +cells O +transfected O +with O +blank O +vector O +or O +with O +MKP7 B-protein +( O +wild B-protein_state +type I-protein_state +or O +mutants B-protein_state +) O +under O +unstimulated O +conditions O +( O +Supplementary O +Fig O +. O +3b O +), O +while O +ultraviolet O +- O +irradiation O +significantly O +increased O +apoptotic O +rate O +in O +cells O +transfected O +with O +blank O +vector O +( O +Fig O +. O +6e O +). O + +Expressions B-experimental_method +of O +wild B-protein_state +- I-protein_state +type I-protein_state +MKP7 B-protein +, O +MKP7ΔC304 B-mutant +and O +MKP7 B-protein +- O +CD B-structure_element +significantly O +decreased O +the O +proportion O +of O +apoptotic O +cells O +after O +ultraviolet O +treatment O +. O + +Moreover O +, O +treatment O +of O +cells O +expressing O +MKP7 B-protein +- O +KBD B-structure_element +mutants B-protein_state +( O +R56A B-mutant +/ O +R57A B-mutant +and O +V63A B-mutant +/ O +I65A B-mutant +) O +decreased O +the O +apoptosis O +rates O +to O +a O +similar O +extent O +as O +MKP7 B-protein +wild B-protein_state +type I-protein_state +did O +. O + +In O +contrast O +, O +cells O +transfected O +with O +the O +MKP7 B-protein +FXF B-structure_element +- I-structure_element +motif I-structure_element +mutants B-protein_state +( O +F285D B-mutant +, O +F287D B-mutant +and O +L288D B-mutant +) O +showed O +little O +protective O +effect O +after O +ultraviolet O +treatment O +and O +similar O +levels O +of O +apoptosis O +rates O +were O +detected O +to O +cells O +transfected O +with O +control O +vectors O +( O +Fig O +. O +6e O +, O +f O +). O + +Taken O +together O +, O +our O +results O +suggested O +that O +FXF B-structure_element +- I-structure_element +motif I-structure_element +- O +mediated O +, O +rather O +than O +KBD B-structure_element +- O +mediated O +, O +interaction O +is O +essential O +for O +MKP7 B-protein +to O +block O +ultraviolet O +- O +induced O +apoptosis O +. O + +A O +similar O +docking O +mechanism O +for O +JNK1 B-protein +recognition O +by O +MKP5 B-protein + +MKP5 B-protein +belongs O +to O +the O +same O +subfamily O +as O +MKP7 B-protein +. O + +MKP5 B-protein +is O +unique O +among O +the O +MKPs B-protein_type +in O +possessing O +an O +additional O +domain O +of O +unknown O +function O +at O +the O +N O +- O +terminus O +( O +Fig O +. O +7a O +). O + +The O +KBD B-structure_element +of O +MKP5 B-protein +interacts O +with O +the O +D B-site +- I-site +site I-site +of O +p38α B-protein +to O +mediate O +the O +enzyme O +– O +substrate O +interaction O +. O + +Deletion B-experimental_method +of I-experimental_method +the O +KBD B-structure_element +in O +MKP5 B-protein +leads O +to O +a O +280 O +- O +fold O +increase O +in O +Km B-evidence +for O +p38α B-protein +substrate O +. O + +In O +contrast O +to O +p38α B-protein +substrate O +, O +deletion B-experimental_method +of I-experimental_method +the O +MKP5 B-protein +- O +KBD B-structure_element +had O +little O +effects O +on O +the O +kinetic O +parameters O +for O +the O +JNK1 B-protein +dephosphorylation O +, O +indicating O +that O +the O +KBD B-structure_element +of O +MKP5 B-protein +is O +not O +required O +for O +the O +JNK1 B-protein +dephosphorylation O +( O +Fig O +. O +7b O +). O + +The O +substrate B-evidence +specificity I-evidence +constant I-evidence +kcat B-evidence +/ I-evidence +Km I-evidence +value O +for O +MKP5 B-protein +- O +CD B-structure_element +was O +calculated O +as O +1 O +. O +0 O +× O +105 O +M O +− O +1 O +s O +− O +1 O +, O +which O +is O +very O +close O +to O +that O +of O +MKP7 B-protein +- O +CD B-structure_element +( O +1 O +. O +07 O +× O +105 O +M O +− O +1 O +s O +− O +1 O +). O + +The O +crystal B-evidence +structure I-evidence +of O +human B-species +MKP5 B-protein +- O +CD B-structure_element +has O +been O +determined O +. O + +Comparisons O +between O +catalytic B-structure_element +domains I-structure_element +structures B-evidence +of O +MKP5 B-protein +and O +MKP7 B-protein +reveal O +that O +the O +overall O +folds O +of O +the O +two O +proteins O +are O +highly O +similar O +, O +with O +only O +a O +few O +regions O +exhibiting O +small O +deviations O +( O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +. I-evidence +of O +0 O +. O +79 O +Å O +; O +Fig O +. O +7c O +). O + +Given O +the O +distinct O +interaction O +mode O +revealed O +by O +the O +crystal B-evidence +structure I-evidence +of O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +, O +one O +obvious O +question O +is O +whether O +this O +is O +a O +general O +mechanism O +used O +by O +all O +members O +of O +the O +JNK B-protein_type +- I-protein_type +specific I-protein_type +MKPs I-protein_type +. O + +To O +address O +this O +issue O +, O +we O +first O +examined O +the O +docking O +ability O +of O +JNK1 B-protein +to O +the O +KBD B-structure_element +and O +CD B-structure_element +of O +MKP5 B-protein +using O +gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +and O +pull B-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +. O + +It O +can O +be O +seen O +from O +gel B-experimental_method +filtration I-experimental_method +experiments I-experimental_method +that O +JNK1 B-protein +can O +forms O +a O +stable B-protein_state +heterodimer B-oligomeric_state +with O +MKP5 B-protein +- O +CD B-structure_element +in O +solution O +, O +but O +no O +detectable O +interaction O +was O +found O +with O +the O +KBD B-structure_element +domain O +( O +Fig O +. O +7d O +). O + +Pull B-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +also O +confirmed O +the O +protein O +– O +protein O +interactions O +observed O +above O +. O + +The O +catalytic B-structure_element +domain I-structure_element +of O +MKP5 B-protein +, O +but O +not O +its O +KBD B-structure_element +, O +was O +able O +to O +pull O +- O +down O +a O +detectable O +amount O +of O +JNK1 B-protein +( O +Fig O +. O +7e O +), O +implicating O +a O +different O +substrate O +- O +recognition O +mechanisms O +for O +p38 B-protein_type +and O +JNK B-protein_type +MAPKs B-protein_type +. O + +To O +further O +test O +our O +hypothesis O +, O +we O +generated O +forms O +of O +MKP5 B-protein +- O +CD B-structure_element +bearing O +mutations B-experimental_method +corresponding O +to O +the O +changes O +we O +made O +on O +MKP7 B-protein +- O +CD B-structure_element +on O +the O +basis O +of O +sequence B-experimental_method +and I-experimental_method +structural I-experimental_method +alignment I-experimental_method +and O +examined O +their O +effects O +on O +the O +phosphatase B-protein_type +activity O +. O + +As O +shown O +in O +Fig O +. O +7f O +, O +the O +T432A B-mutant +and O +L449F B-mutant +MKP5 B-protein +mutant B-protein_state +showed O +little O +or O +no O +difference O +in O +phosphatase O +activity O +, O +whereas O +the O +other O +mutants B-protein_state +showed O +reduced O +specific O +activities O +of O +MKP5 B-protein +. O + +As O +in O +the O +case O +of O +MKP7 B-protein +, O +all O +the O +mutants B-protein_state +, O +except O +F451D B-mutant +/ I-mutant +A I-mutant +, O +showed O +no O +pNPPase B-protein_type +activity O +changes O +compared O +with O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +MKP5 B-protein +- O +CD B-structure_element +( O +Fig O +. O +7g O +), O +and O +the O +point B-experimental_method +mutations I-experimental_method +in O +JNK1 B-protein +also O +reduced O +the O +binding B-evidence +affinity I-evidence +of O +MKP5 B-protein +- O +CD B-structure_element +for O +JNK1 B-protein +( O +Fig O +. O +7h O +). O + +In O +addition O +, O +there O +were O +no O +significant O +differences O +in O +the O +CD B-evidence +spectra I-evidence +between O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +proteins O +, O +indicating O +that O +the O +overall O +structures B-evidence +of O +these O +mutants B-protein_state +did O +not O +change O +significantly O +from O +that O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +MKP5 B-protein +protein O +( O +Supplementary O +Fig O +. O +4a O +). O + +Taken O +together O +, O +our O +results O +suggest O +that O +MKP5 B-protein +binds O +JNK1 B-protein +in O +a O +docking O +mode O +similar O +to O +that O +in O +the O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +complex O +, O +and O +the O +detailed O +interaction O +model O +can O +be O +generated O +using O +molecular B-experimental_method +dynamics I-experimental_method +simulation I-experimental_method +based O +on O +the O +structure B-evidence +of O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +complex O +( O +Supplementary O +Fig O +. O +4b O +, O +c O +). O + +In O +this O +model O +, O +the O +MKP5 B-protein +- O +CD B-structure_element +adopts O +a O +conformation O +nearly O +identical O +to O +that O +in O +its O +unbound B-protein_state +form O +, O +suggesting O +that O +the O +conformation O +of O +the O +catalytic B-structure_element +domain I-structure_element +undergoes O +little O +change O +, O +if O +any O +at O +all O +, O +upon O +JNK1 B-protein +binding O +. O + +In O +particular O +, O +Leu449 B-residue_name_number +of O +MKP5 B-protein +, O +which O +is O +equivalent O +to O +the O +key O +residue O +Phe285 B-residue_name_number +of O +MKP7 B-protein +, O +buried O +deeply O +within O +the O +hydrophobic B-site +pocket I-site +of O +JNK1 B-protein +in O +the O +same O +way O +as O +Phe285 B-residue_name_number +in O +the O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +complex O +( O +Supplementary O +Fig O +. O +4d O +). O + +Despite O +the O +strong O +similarities O +between O +JNK1 B-protein +– O +MKP5 B-protein +- O +CD B-structure_element +and O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +, O +however O +, O +there O +are O +differences O +. O + +The O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +interaction O +is O +better O +and O +more O +extensive O +. O + +Asp268 B-residue_name_number +of O +MKP7 B-protein +- O +CD B-structure_element +forms O +salt B-bond_interaction +bridge I-bond_interaction +with O +JNK1 B-protein +Arg263 B-residue_name_number +, O +whereas O +the O +corresponding O +residue O +Thr432 B-residue_name_number +in O +MKP5 B-protein +- O +CD B-structure_element +may O +not O +interact O +with O +JNK1 B-protein +. O + +In O +addition O +, O +the O +key O +interacting O +residues O +of O +MKP7 B-protein +- O +CD B-structure_element +, O +Phe215 B-residue_name_number +, O +Leu267 B-residue_name_number +and O +Leu288 B-residue_name_number +, O +are O +replaced O +by O +less O +hydrophobic O +residues O +, O +Asn379 B-residue_name_number +, O +Met431 B-residue_name_number +and O +Met452 B-residue_name_number +in O +MKP5 B-protein +- O +CD B-structure_element +( O +Fig O +. O +5c O +), O +respectively O +, O +which O +may O +result O +in O +weaker O +hydrophobic B-bond_interaction +interactions I-bond_interaction +between O +MKP5 B-protein +- O +CD B-structure_element +and O +JNK1 B-protein +. O + +This O +is O +consistent O +with O +the O +experimental O +observation O +showing O +that O +JNK1 B-protein +binds O +to O +MKP7 B-protein +- O +CD B-structure_element +much O +more O +tightly O +than O +MKP5 B-protein +- O +CD B-structure_element +( O +Km O +value O +of O +MKP5 B-protein +- O +CD B-structure_element +for O +pJNK1 B-protein_state +substrate O +is O +∼ O +20 O +- O +fold O +higher O +than O +that O +of O +MKP7 B-protein +- O +CD B-structure_element +). O + +The O +MAPKs B-protein_type +p38 B-protein_type +, O +ERK B-protein_type +and O +JNK B-protein_type +, O +are O +central O +to O +evolutionarily O +conserved O +signalling O +pathways O +that O +are O +present O +in O +all O +eukaryotic B-taxonomy_domain +cells O +. O + +Each O +MAPK B-protein_type +cascade O +is O +activated O +in O +response O +to O +a O +diverse O +array O +of O +extracellular O +signals O +and O +culminates O +in O +the O +dual B-ptm +- I-ptm +phosphorylation I-ptm +of O +a O +threonine B-residue_name +and O +a O +tyrosine B-residue_name +residue O +in O +the O +MAPK B-structure_element +- I-structure_element +activation I-structure_element +loop I-structure_element +. O + +The O +propagation O +of O +MAPK B-protein_type +signals O +is O +attenuated O +through O +the O +actions O +of O +the O +MKPs B-protein_type +. O + +Most O +studies O +have O +focused O +on O +the O +dephosphorylation O +of O +MAPKs B-protein_type +by O +phosphatases B-protein_type +containing O +the O +‘ O +kinase B-structure_element +- I-structure_element +interaction I-structure_element +motif I-structure_element +' O +( O +D B-structure_element +- I-structure_element +motif I-structure_element +), O +including O +a O +group O +of O +DUSPs B-protein_type +( O +MKPs B-protein_type +) O +and O +a O +distinct O +subfamily O +of O +tyrosine B-protein_type +phosphatases I-protein_type +( O +HePTP B-protein +, O +STEP B-protein +and O +PTP B-protein +- I-protein +SL I-protein +). O + +Crystal B-evidence +structures I-evidence +of O +ERK2 B-protein +bound B-protein_state +with I-protein_state +the O +D B-structure_element +- I-structure_element +motif I-structure_element +sequences O +derived O +from O +MKP3 B-protein +and O +HePTP B-protein +have O +been O +reported O +. O + +These O +structures B-evidence +revealed O +that O +linear B-structure_element +docking I-structure_element +motifs I-structure_element +in O +interacting O +proteins O +bind O +to O +a O +common O +docking B-site +site I-site +on O +MAPKs B-protein_type +outside O +the O +kinase B-protein_type +active B-site +site I-site +. O + +The O +particular O +amino O +acids O +and O +their O +spacing O +within O +D B-structure_element +- I-structure_element +motif I-structure_element +sequences O +and O +amino O +acid O +composition O +of O +the O +docking B-site +sites I-site +on O +MAPKs B-protein_type +appear O +to O +determine O +the O +specificity O +of O +D B-structure_element +- I-structure_element +motifs I-structure_element +for O +individual O +MAPKs B-protein_type +. O + +Recently O +, O +the O +crystal B-evidence +structure I-evidence +of O +a O +complex O +between O +the O +KBD B-structure_element +of O +MKP5 B-protein +and O +p38α B-protein +has O +been O +obtained O +. O + +This O +complex O +has O +revealed O +a O +distinct O +interaction O +mode O +for O +MKP5 B-protein +. O + +The O +KBD B-structure_element +of O +MKP5 B-protein +binds O +to O +p38α B-protein +in O +the O +opposite O +polypeptide O +direction O +compared O +with O +how O +the O +D B-structure_element +- I-structure_element +motif I-structure_element +of O +MKP3 B-protein +binds O +to O +ERK2 B-protein +. O + +In O +contrast O +to O +the O +canonical O +D B-site +- I-site +motif I-site +- I-site +binding I-site +mode I-site +, O +separate O +helices B-structure_element +, O +α2 B-structure_element +and O +α3 B-structure_element +′, I-structure_element +in O +the O +KBD B-structure_element +of O +MKP5 B-protein +engage O +the O +p38α B-site +- I-site +docking I-site +site I-site +. O + +Further O +structural B-experimental_method +and I-experimental_method +biochemical I-experimental_method +studies I-experimental_method +indicate O +that O +KBD B-structure_element +of O +MKP7 B-protein +may O +interact O +with O +p38α B-protein +in O +a O +similar O +manner O +to O +that O +of O +MKP5 B-protein +. O + +In O +contrast O +to O +MKP5 B-protein +, O +removal B-experimental_method +of I-experimental_method +the O +KBD B-structure_element +domain O +from O +MKP7 B-protein +does O +not O +drastically O +affect O +enzyme O +catalysis O +, O +and O +the O +kinetic O +parameters O +of O +MKP7 B-protein +- O +CD B-structure_element +for O +p38α B-protein +substrate O +are O +very O +similar O +to O +those O +for O +JNK1 B-protein +substrate O +. O + +Taken O +together O +, O +these O +results O +suggest O +that O +MKP7 B-protein +utilizes O +a O +bipartite O +recognition O +mechanism O +to O +achieve O +the O +efficiency O +and O +fidelity O +of O +p38α B-protein +signalling O +. O + +The O +MKP7 B-protein +- O +KBD B-structure_element +docks O +to O +the O +D B-site +- I-site +site I-site +located O +on O +the O +back O +side O +of O +the O +p38α B-protein +catalytic B-site +pocket I-site +for O +high O +- O +affinity O +association O +, O +whereas O +the O +interaction O +of O +the O +MKP7 B-protein +- O +CD B-structure_element +with O +another O +p38α B-protein +structural O +region O +, O +which O +is O +close O +to O +the O +activation B-structure_element +loop I-structure_element +, O +may O +not O +only O +stabilize O +binding O +but O +also O +provide O +contacts O +crucial O +for O +organizing O +the O +MKP7 B-protein +active B-site +site I-site +with O +respect O +to O +the O +phosphoreceptor O +in O +the O +p38α B-protein +substrate O +for O +efficient O +dephosphorylation O +. O + +In O +addition O +to O +the O +canonical O +D B-site +- I-site +site I-site +, O +the O +MAPK B-protein_type +ERK2 B-protein +contains O +a O +second B-site +binding I-site +site I-site +utilized O +by O +transcription O +factor O +substrates O +and O +phosphatases B-protein_type +, O +the O +FXF B-site +- I-site +motif I-site +- I-site +binding I-site +site I-site +( O +also O +called O +F B-site +- I-site +site I-site +), O +that O +is O +exposed O +in O +active B-protein_state +ERK2 B-protein +and O +the O +D B-structure_element +- I-structure_element +motif I-structure_element +peptide O +- O +induced O +conformation O +of O +MAPKs B-protein_type +. O + +This O +hydrophobic B-site +site I-site +was O +first O +identified O +by O +changes B-evidence +in I-evidence +deuterium I-evidence +exchange I-evidence +profiles I-evidence +, O +and O +is O +near O +the O +MAPK B-structure_element +insertion I-structure_element +and O +helix B-structure_element +αG B-structure_element +. O +Interestingly O +, O +many O +of O +the O +equivalent O +residues O +in O +JNK1 B-protein +, O +important O +for O +MKP7 B-protein +- O +CD B-structure_element +recognition O +, O +are O +also O +used O +for O +substrate O +binding O +by O +ERK2 B-protein +( O +ref O +.), O +indicating O +that O +this O +site O +is O +overlapped O +with O +the O +DEF B-site +- I-site +site I-site +previously O +identified O +in O +ERK2 B-protein +( O +Fig O +. O +5d O +). O + +MKP3 B-protein +is O +highly O +specific O +in O +dephosphorylating O +and O +inactivating O +ERK2 B-protein +, O +and O +the O +phosphatase O +activity O +of O +the O +MKP3 B-protein +- O +catalysed O +pNPP B-chemical +reaction O +can O +be O +markedly O +increased O +in O +the O +presence B-protein_state +of I-protein_state +ERK2 B-protein +( O +refs O +). O + +Sequence B-experimental_method +alignment I-experimental_method +of O +all O +MKPs B-protein_type +reveals O +a O +high O +degree O +of O +conservation O +of O +residues O +surrounding O +the O +interacting B-site +region I-site +observed O +in O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +complex O +( O +Supplementary O +Fig O +. O +5 O +). O + +Therefore O +, O +it O +is O +tempting O +to O +speculate O +that O +the O +catalytic B-structure_element +domain I-structure_element +of O +MKP3 B-protein +may O +bind O +to O +ERK2 B-protein +in O +a O +manner O +analogous O +to O +the O +way O +by O +which O +MKP7 B-protein +- O +CD B-structure_element +binds O +to O +JNK1 B-protein +. O + +A O +comprehensive O +examination O +of O +the O +molecular O +basis O +of O +the O +specific O +ERK2 B-protein +recognition O +by O +MKP3 B-protein +is O +underway O +. O + +The O +ongoing O +work O +demonstrates O +that O +although O +the O +overall O +interaction O +modes O +are O +similar O +between O +the O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +and O +ERK2 B-complex_assembly +– I-complex_assembly +MKP3 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +complexes O +, O +the O +ERK2 B-complex_assembly +– I-complex_assembly +MKP3 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +interaction O +is O +less O +extensive O +and O +helix B-structure_element +α4 B-structure_element +from O +MKP3 B-protein +- O +CD B-structure_element +does O +not O +interact O +directly O +with O +ERK2 B-protein +. O + +The O +FXF B-structure_element +- I-structure_element +motif I-structure_element +- O +mediated O +interaction O +is O +critical O +for O +both O +pERK2 B-protein_state +inactivation O +and O +ERK2 B-protein +- O +induced O +MKP3 B-protein +activation O +( O +manuscript O +in O +preparation O +). O + +In O +summary O +, O +we O +have O +resolved O +the O +structure B-evidence +of O +JNK1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +catalytic B-structure_element +domain I-structure_element +of O +MKP7 B-protein +. O + +This O +structure B-evidence +reveals O +an O +FXF B-site +- I-site +docking I-site +interaction I-site +mode I-site +between O +MAPK B-protein_type +and O +MKP B-protein_type +. O + +Results O +from O +biochemical B-experimental_method +characterization I-experimental_method +of O +the O +Phe285 B-residue_name_number +and O +Phe287 B-residue_name_number +MKP7 B-protein +mutants B-protein_state +combined O +with O +structural B-evidence +information I-evidence +support O +the O +conclusion O +that O +the O +two O +Phe B-residue_name +residues O +serve O +different O +roles O +in O +the O +catalytic O +reaction O +. O + +Phe285 B-residue_name_number +is O +essential O +for O +JNK1 B-protein +substrate O +binding O +, O +whereas O +Phe287 B-residue_name_number +plays O +a O +role O +for O +the O +precise O +alignment O +of O +active B-site +- I-site +site I-site +residues I-site +, O +which O +are O +important O +for O +transition O +- O +state O +stabilization O +. O + +This O +newly O +identified O +FXF B-structure_element +- I-structure_element +type I-structure_element +motif I-structure_element +is O +present O +in O +all O +MKPs B-protein_type +, O +except O +that O +the O +residue O +at O +the O +first O +position O +in O +MKP5 B-protein +is O +an O +equivalent O +hydrophobic O +leucine B-residue_name +residue O +( O +see O +also O +Fig O +. O +7f O +, O +g O +), O +suggesting O +that O +these O +two O +Phe B-residue_name +residues O +would O +play O +a O +similar O +role O +in O +facilitating O +substrate O +recognition O +and O +catalysis O +, O +respectively O +. O + +An O +important O +feature O +of O +MKP B-protein_type +– O +JNK1 B-protein +interactions O +is O +that O +MKP7 B-protein +or O +MKP5 B-protein +only O +interact O +with O +the O +F B-site +- I-site +site I-site +of O +JNK1 B-protein +. O + +One O +possible O +explanation O +is O +that O +JNK1 B-protein +needs O +to O +use O +the O +D B-site +- I-site +site I-site +to O +interact O +with O +JIP B-protein +- I-protein +1 I-protein +, O +a O +scaffold O +protein O +for O +JNK B-protein_type +signalling O +. O + +The O +N O +- O +terminal O +JNK B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +of O +JIP B-protein +- I-protein +1 I-protein +interacts O +with O +the O +D B-site +- I-site +site I-site +on O +JNK B-protein_type +and O +this O +interaction O +is O +required O +for O +JIP B-protein +- I-protein +1 I-protein +- O +mediated O +enhancement O +of O +JNK B-protein_type +activation O +. O + +In O +addition O +, O +JIP B-protein +- I-protein +1 I-protein +can O +also O +associate O +with O +MKP7 B-protein +via O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +MKP7 B-protein +( O +ref O +.). O + +When O +MKP7 B-protein +is O +bound B-protein_state +to I-protein_state +JIP B-protein +- I-protein +1 I-protein +, O +it O +reduces O +JNK B-protein_type +activation O +, O +leading O +to O +reduced O +phosphorylation O +of O +the O +JNK B-protein_type +target O +c B-protein_type +- I-protein_type +Jun I-protein_type +. O + +Thus O +, O +our O +biochemical B-evidence +and I-evidence +structural I-evidence +data I-evidence +allow O +us O +to O +present O +a O +model O +for O +the O +JNK1 B-complex_assembly +– I-complex_assembly +JIP I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +ternary O +complex O +and O +provide O +an O +important O +insight O +into O +the O +assembly O +and O +function O +of O +JNK B-protein_type +signalling O +modules O +( O +Supplementary O +Fig O +. O +6 O +). O + +Domain O +structures B-evidence +of O +ten O +human B-species +MKPs B-protein_type +and O +the O +atypical O +VHR B-protein +. O + +On O +the O +basis O +of O +sequence O +similarity O +, O +protein O +structure B-evidence +, O +substrate O +specificity O +and O +subcellular O +localization O +, O +the O +ten O +members O +of O +MKP B-protein_type +family I-protein_type +can O +be O +divided O +into O +three O +groups O +. O + +The O +first O +subfamily O +comprises O +MKP1 B-protein +, O +MKP2 B-protein +, O +PAC1 B-protein +and O +hVH3 B-protein +, O +which O +are O +inducible B-protein_state +nuclear B-protein_type +phosphatases I-protein_type +and O +can O +dephosphorylate O +ERK B-protein_type +( O +and O +JNK B-protein_type +, O +p38 B-protein_type +) O +MAPKs B-protein_type +. O + +The O +second O +subfamily O +contains O +MKP3 B-protein +, O +MKP4 B-protein +and O +MKPX B-protein +, O +which O +are O +cytoplasmic O +ERK B-protein_type +- I-protein_type +specific I-protein_type +MKPs I-protein_type +. O + +The O +third O +subfamily O +comprises O +MKP5 B-protein +, O +MKP7 B-protein +and O +hVH5 B-protein +, O +which O +were O +located O +in O +both O +nucleus O +and O +cytoplasm O +, O +and O +selectively O +inactivate O +JNK B-protein_type +and O +p38 B-protein_type +. O + +All O +MKPs B-protein_type +contain O +both O +the O +CD B-structure_element +and O +KBD B-structure_element +domains O +, O +whereas O +VHR B-protein +, O +an O +atypical O +MKP B-protein_type +, O +only O +contains O +a O +highly B-protein_state +conserved I-protein_state +catalytic B-structure_element +domain I-structure_element +. O + +In O +addition O +to O +the O +CD B-structure_element +and O +KBD B-structure_element +, O +MKP7 B-protein +contains O +a O +unique O +long O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +that O +contains O +NES B-structure_element +, O +NLS B-structure_element +and O +PEST B-structure_element +motifs I-structure_element +, O +which O +has O +no O +effect O +on O +the O +binding O +ability O +and O +phosphatase O +activity O +of O +MKP7 B-protein +toward O +MAPKs B-protein_type +. O + +NES B-structure_element +, O +nuclear B-structure_element +export I-structure_element +signal I-structure_element +; O +NLS B-structure_element +, O +nuclear B-structure_element +localization I-structure_element +signal I-structure_element +; O +PEST B-structure_element +, O +C B-structure_element +- I-structure_element +terminal I-structure_element +sequence I-structure_element +rich I-structure_element +in O +prolines B-residue_name +, O +glutamates B-residue_name +, O +serines B-residue_name +and O +threonines B-residue_name +. O + +MKP7 B-protein +- O +CD B-structure_element +is O +crucial O +for O +JNK1 B-protein +binding O +and O +enzyme O +catalysis O +. O + +( O +a O +) O +Domain O +organization O +of O +human B-species +MKP7 B-protein +and O +JNK1 B-protein +. O + +The O +KBD B-structure_element +and O +CD B-structure_element +of O +MKP7 B-protein +are O +shown O +in O +green O +and O +blue O +, O +and O +the O +N B-structure_element +- I-structure_element +lobe I-structure_element +and O +C B-structure_element +- I-structure_element +lobe I-structure_element +of O +JNK1 B-protein +are O +coloured O +in O +lemon O +and O +yellow O +, O +respectively O +. O + +The O +colour O +scheme O +is O +the O +same O +in O +the O +following O +figures O +unless O +indicated O +otherwise O +. O +( O +b O +) O +Plots B-evidence +of I-evidence +initial I-evidence +velocity I-evidence +of O +the O +MKP7 B-protein +- O +catalysed O +reaction O +versus O +phospho B-ptm +- O +JNK1 B-protein +concentration O +. O + +The O +error O +bars O +represent O +s O +. O +e O +. O +m O +. O +( O +c O +) O +Gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +for O +interaction O +of O +JNK1 B-protein +with O +MKP7 B-protein +- O +CD B-structure_element +and O +MKP7 B-protein +- O +KBD B-structure_element +. O + +( O +d O +) O +GST B-experimental_method +- I-experimental_method +mediated I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +for O +interaction O +of O +JNK1 B-protein +with O +MKP7 B-protein +- O +CD B-structure_element +and O +MKP7 B-protein +- O +KBD B-structure_element +. O + +The O +top O +panel O +shows O +the O +relative O +affinities B-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +and O +MKP7 B-protein +- O +KBD B-structure_element +to O +JNK1 B-protein +, O +with O +the O +affinity B-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +defined O +as O +100 O +%; O +the O +middle O +panel O +is O +the O +electrophoretic O +pattern O +of O +MKP7 B-protein +and O +JNK1 B-protein +after O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +. O + +The O +protein O +amounts O +of O +MKP7 B-protein +used O +are O +shown O +at O +the O +bottom O +. O + +Structure B-evidence +of O +JNK1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +MKP7 B-protein +- O +CD B-structure_element +. O + +( O +a O +) O +Ribbon O +diagram O +of O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +complex O +in O +two O +views O +related O +by O +a O +45 O +° O +rotation O +around O +a O +vertical O +axis O +. O +( O +b O +) O +Structure B-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +with O +its O +active B-site +site I-site +highlight O +in O +cyan O +. O + +The O +2Fo B-evidence +− I-evidence +Fc I-evidence +omit I-evidence +map I-evidence +( O +contoured O +at O +1 O +. O +5σ O +) O +for O +the O +P B-structure_element +- I-structure_element +loop I-structure_element +of O +MKP7 B-protein +- O +CD B-structure_element +is O +shown O +at O +inset O +of O +b O +. O +( O +c O +) O +Structure B-evidence +of O +VHR B-protein +with O +its O +active B-site +site I-site +highlighted O +in O +marine O +blue O +. O +( O +d O +) O +Close O +- O +up O +view O +of O +the O +JNK1 B-site +– I-site +MKP7 I-site +interface I-site +showing O +interacting O +amino O +acids O +of O +JNK1 B-protein +( O +orange O +) O +and O +MKP7 B-protein +- O +CD B-structure_element +( O +cyan O +). O + +The O +JNK1 B-protein +is O +shown O +in O +surface O +representation O +coloured O +according O +to O +electrostatic O +potential O +( O +positive O +, O +blue O +; O +negative O +, O +red O +). O + +( O +e O +) O +Interaction B-site +networks I-site +mainly O +involving O +helices B-structure_element +α4 B-structure_element +and O +α5 B-structure_element +from O +MKP7 B-protein +- O +CD B-structure_element +, O +and O +αG B-structure_element +and O +α2L14 B-structure_element +of O +JNK1 B-protein +. O + +MKP7 B-protein +- O +CD B-structure_element +is O +shown O +in O +surface O +representation O +coloured O +according O +to O +electrostatic O +potential O +( O +positive O +, O +blue O +; O +negative O +, O +red O +). O + +Blue O +dashed O +lines O +represent O +polar B-bond_interaction +interactions I-bond_interaction +. O + +( O +f O +) O +The O +2Fo B-evidence +− I-evidence +Fc I-evidence +omit I-evidence +map I-evidence +( O +contoured O +at O +1 O +. O +5σ O +) O +clearly O +shows O +electron B-evidence +density I-evidence +for O +the O +285FNFL288 B-structure_element +segment I-structure_element +of O +MKP7 B-protein +- O +CD B-structure_element +. O + +Mutational B-experimental_method +analysis I-experimental_method +on O +interactions O +between O +MKP7 B-protein +- O +CD B-structure_element +and O +JNK1 B-protein +. O + +( O +a O +) O +Effects O +of O +mutations O +in O +MKP7 B-protein +- O +CD B-structure_element +on O +the O +JNK1 B-protein +dephosphorylation B-ptm +( O +mean O +± O +s O +. O +e O +. O +m O +., O +n O += O +3 O +). O + +Residues O +involved O +in O +hydrophobic B-bond_interaction +and I-bond_interaction +hydrophilic I-bond_interaction +contacts I-bond_interaction +are O +coloured O +in O +red O +and O +blue O +, O +respectively O +. O +( O +b O +) O +Gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +for O +interaction O +of O +JNK1 B-protein +with O +MKP7 B-protein +- O +CD B-structure_element +mutant B-protein_state +F285D B-mutant +. O + +Mutant B-protein_state +F285D B-mutant +and O +JNK1 B-protein +were O +eluted O +as O +monomers B-oligomeric_state +, O +with O +the O +molecular O +masses O +of O +∼ O +17 O +and O +44 O +kDa O +, O +respectively O +. O + +However O +, O +in O +contrast O +to O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +MKP7 B-protein +- O +CD B-structure_element +, O +mutant B-protein_state +F285D B-mutant +did O +not O +co O +- O +migrate O +with O +JNK1 B-protein +. O + +( O +c O +) O +Pull B-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +of O +MKP7 B-protein +- O +CD B-structure_element +by O +GST B-protein_state +- I-protein_state +tagged I-protein_state +JNK1 B-protein +mutants B-protein_state +. O + +The O +top O +panel O +shows O +the O +relative O +affinities B-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +to O +JNK1 B-protein +mutants B-protein_state +, O +with O +the O +affinity B-evidence +of O +wild B-protein_state +- I-protein_state +type I-protein_state +JNK1 B-protein +defined O +as O +100 O +%, O +the O +middle O +panel O +is O +the O +electrophoretic O +pattern O +of O +MKP7 B-protein +- O +CD B-structure_element +and O +JNK1 B-protein +mutants B-protein_state +after O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +. O + +The O +protein O +amounts O +of O +MKP7 B-protein +- O +CD B-structure_element +used O +are O +shown O +at O +the O +bottom O +. O +( O +d O +) O +Circular B-experimental_method +dichroism I-experimental_method +spectra B-evidence +for O +MKP7 B-protein +- O +CD B-structure_element +wild B-protein_state +type I-protein_state +and O +mutants B-protein_state +. O + +Measurements O +were O +averaged O +for O +three O +scans O +. O +( O +e O +) O +Circular B-experimental_method +dichroism I-experimental_method +spectra B-evidence +for O +JNK1 B-protein +wild B-protein_state +type I-protein_state +and O +mutants B-protein_state +. O + +( O +f O +) O +Effects O +of O +mutations B-experimental_method +in O +MKP7 B-protein +- O +CD B-structure_element +on O +the O +pNPP B-chemical +hydrolysis O +reaction O +( O +mean O +± O +s O +. O +e O +. O +m O +., O +n O += O +3 O +). O + +Comparison O +of O +CDK2 B-complex_assembly +- I-complex_assembly +KAP I-complex_assembly +and O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +. O + +( O +a O +) O +Superposition B-experimental_method +of O +the O +complex O +structures B-evidence +of O +CDK2 B-complex_assembly +- I-complex_assembly +KAP I-complex_assembly +( O +PDB O +1FQ1 O +) O +and O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +. O + +The O +N B-structure_element +- I-structure_element +lobe I-structure_element +and O +C B-structure_element +- I-structure_element +lobe I-structure_element +of O +CDK2 B-protein +are O +coloured O +in O +grey O +and O +pink O +, O +respectively O +, O +and O +KAP B-protein +is O +coloured O +in O +green O +. O + +The O +interactions O +between O +these O +two O +proteins O +consist O +of O +three O +discontinuous O +contact B-site +regions I-site +, O +centred O +at O +the O +multiple O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +the O +pThr160 B-ptm +of O +CDK2 B-protein +and O +the O +active B-site +site I-site +of O +KAP B-protein +( O +region B-structure_element +I I-structure_element +). O + +Interestingly O +, O +the O +recognition O +of O +CDK2 B-protein +by O +KAP B-protein +is O +augmented O +by O +a O +similar O +interface B-site +as O +that O +observed O +in O +the O +complex O +of O +JNK1 B-protein +and O +MKP7 B-protein +- O +CD B-structure_element +( O +region B-structure_element +II I-structure_element +). O + +( O +b O +) O +Interactions O +networks O +at O +the O +auxiliary B-structure_element +region I-structure_element +II I-structure_element +mainly O +involving O +helix B-structure_element +α7 B-structure_element +from O +KAP B-protein +and O +the O +αG B-structure_element +helix I-structure_element +and O +following O +L14 B-structure_element +loop I-structure_element +of O +CDK2 B-protein +. O + +The O +CDK2 B-protein +is O +shown O +in O +surface O +representation O +coloured O +according O +to O +the O +electrostatic O +potential O +( O +positive O +, O +blue O +; O +negative O +, O +red O +). O + +Residues O +of O +KAP B-protein +and O +CDK2 B-protein +are O +highlighted O +as O +green O +and O +red O +sticks O +, O +respectively O +. O + +One O +remarkable O +difference O +between O +these O +two O +kinase O +- O +phosphatase O +complexes O +is O +that O +helix B-structure_element +α6 B-structure_element +of O +KAP B-protein +( O +corresponding O +to O +helix B-structure_element +α4 B-structure_element +of O +MKP7 B-protein +- O +CD B-structure_element +) O +plays O +little O +, O +if O +any O +, O +role O +in O +the O +formation O +of O +a O +stable B-protein_state +heterodimer B-oligomeric_state +of O +CDK2 B-protein +and O +KAP B-protein +. O +( O +c O +) O +Sequence B-experimental_method +alignment I-experimental_method +of O +the O +JNK B-site +- I-site +interacting I-site +regions I-site +on O +MKPs B-protein_type +. O + +Residues O +of O +MKP7 B-protein +- O +CD B-structure_element +involved O +in O +JNK1 B-protein +recognition O +are O +indicated O +by O +cyan O +asterisks O +, O +and O +the O +conserved B-protein_state +FXF B-structure_element +- I-structure_element +motif I-structure_element +is O +highlighted O +in O +cyan O +. O + +The O +secondary O +structure O +assignments O +of O +MKP7 B-protein +- O +CD B-structure_element +and O +KAP B-protein +are O +shown O +above O +and O +below O +each O +sequence O +. O + +( O +d O +) O +Sequence B-experimental_method +alignment I-experimental_method +of O +the O +F B-structure_element +- I-structure_element +site I-structure_element +regions I-structure_element +on O +MAPKs B-protein_type +. O + +Residues O +of O +JNK1 B-protein +involved O +in O +recognition O +of O +MKP7 B-protein +are O +indicated O +by O +orange O +asterisks O +, O +and O +those O +forming O +the O +F B-site +- I-site +site I-site +are O +highlighted O +in O +yellow O +. O + +FXF B-structure_element +- I-structure_element +motif I-structure_element +is O +critical O +for O +controlling O +the O +phosphorylation B-ptm +of O +JNK B-protein_type +and O +ultraviolet O +- O +induced O +apoptosis O +. O + +( O +a O +– O +c O +) O +FXF B-structure_element +- I-structure_element +motif I-structure_element +is O +essential O +for O +the O +dephosphorylation O +of O +JNK B-protein_type +by O +MKP7 B-protein +. O + +HEK293T O +cells O +were O +infected O +with O +lentiviruses B-taxonomy_domain +expressing O +MKP7 B-protein +and O +its O +mutants B-protein_state +( O +1 O +. O +0 O +μg O +). O + +After O +36 O +h O +infection O +, O +cells O +were O +untreated O +in O +a O +, O +stimulated O +with O +30 O +μM O +etoposide B-chemical +for O +3 O +h O +in O +b O +or O +irradiated O +with O +25 O +J O +m O +− O +2 O +ultraviolet O +light O +at O +30 O +min O +before O +lysis O +in O +c O +. O +Whole O +- O +cell O +extracts O +were O +then O +immunoblotted O +with O +antibody O +indicated O +. O + +Shown O +is O +a O +typical O +immunoblot O +for O +phosphorylated B-protein_state +JNK B-protein_type +from O +three O +independent O +experiments O +. O + +( O +d O +) O +F B-site +- I-site +site I-site +is O +required O +for O +JNK1 B-protein +to O +interact O +with O +MKP7 B-protein +. O + +HEK293T O +cells O +were O +co B-experimental_method +- I-experimental_method +transfected I-experimental_method +with O +MKP7 B-protein +full B-protein_state +- I-protein_state +length I-protein_state +( O +1 O +. O +0 O +μg O +) O +and O +JNK1 B-protein +( O +wild B-protein_state +type I-protein_state +or O +mutants B-protein_state +as O +indicated O +, O +1 O +. O +0 O +μg O +). O + +Whole O +- O +cell O +extracts O +were O +then O +immunoprecipitated B-experimental_method +with O +antibody O +against O +Myc O +for O +MKP7 B-protein +; O +immunobloting O +was O +carried O +out O +with O +antibodies O +indicated O +. O + +IP B-experimental_method +, O +immunoprecipitation B-experimental_method +; O +TCL O +, O +total O +cell O +lysate O +. O + +( O +e O +) O +Effect O +of O +MKP7 B-protein +( O +wild B-protein_state +type I-protein_state +or O +mutants B-protein_state +) O +expression O +on O +ultraviolet O +- O +induced O +apoptosis O +. O + +HeLa O +cells O +were O +infected O +with O +lentiviruses B-taxonomy_domain +expressing O +MKP7 B-protein +full B-protein_state +- I-protein_state +length I-protein_state +and O +its O +mutants B-protein_state +. O + +Cells O +were O +then O +subjected O +to O +flow B-experimental_method +cytometry I-experimental_method +analysis O +. O + +Apoptotic O +cells O +were O +determined O +by O +Annexin B-chemical +- I-chemical +V I-chemical +- I-chemical +APC I-chemical +/ O +PI B-chemical +staining O +. O + +The O +results O +using O +Annexin B-chemical +- I-chemical +V I-chemical +stain O +for O +membrane O +phosphatidylserine O +eversion O +, O +combined O +with O +propidium B-chemical +iodide I-chemical +( O +PI B-chemical +) O +uptake O +to O +evaluate O +cells O +whose O +membranes O +had O +been O +compromised O +. O + +Staining O +with O +both O +Annexin B-chemical +- I-chemical +V I-chemical +and O +PI B-chemical +indicate O +apoptosis O +( O +upper O +right O +quadrant O +). O + +( O +f O +) O +Statistical O +analysis O +of O +apoptotic O +cells O +( O +mean O +± O +s O +. O +e O +. O +m O +., O +n O += O +3 O +), O +* B-evidence +P I-evidence +< O +0 O +. O +05 O +, O +*** B-evidence +P I-evidence +< O +0 O +. O +001 O +( O +ANOVA B-experimental_method +followed O +by O +Tukey B-experimental_method +' I-experimental_method +s I-experimental_method +test I-experimental_method +). O + +MKP5 B-protein +- O +CD B-structure_element +is O +crucial O +for O +JNK1 B-protein +binding O +and O +enzyme O +catalysis O +. O + +( O +a O +) O +Domain O +organization O +of O +human B-species +MKP5 B-protein +. O + +The O +KBD B-structure_element +and O +CD B-structure_element +of O +MKP5 B-protein +are O +shown O +in O +brown O +and O +grey O +, O +respectively O +. O +( O +b O +) O +Plots B-evidence +of I-evidence +initial I-evidence +velocity I-evidence +of O +the O +MKP5 B-protein +- O +catalysed O +reaction O +versus O +phospho B-protein_state +- O +JNK1 B-protein +concentration O +. O + +The O +solid O +lines O +are O +best O +- O +fitting O +results O +according O +to O +the O +Michaelis O +– O +Menten O +equation O +with O +Km B-evidence +and O +kcat B-evidence +values O +indicated O +. O + +The O +error O +bars O +represent O +s O +. O +e O +. O +m O +. O +( O +c O +) O +Structural B-experimental_method +comparison I-experimental_method +of O +the O +JNK B-site +- I-site +interacting I-site +residues I-site +on O +MKP5 B-protein +- O +CD B-structure_element +( O +PDB O +1ZZW O +) O +and O +MKP7 B-protein +- O +CD B-structure_element +. O + +The O +corresponding O +residues O +on O +MKP5 B-protein +are O +depicted O +as O +orange O +sticks O +, O +and O +MKP5 B-protein +residues O +numbers O +are O +in O +parentheses O +. O + +( O +d O +) O +Gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +for O +interaction O +of O +JNK1 B-protein +with O +MKP5 B-protein +- O +CD B-structure_element +and O +MKP5 B-protein +- O +KBD B-structure_element +. O +( O +e O +) O +GST B-experimental_method +- I-experimental_method +mediated I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +for O +interaction O +of O +JNK1 B-protein +with O +MKP5 B-protein +- O +CD B-structure_element +and O +MKP5 B-protein +- O +KBD B-structure_element +. O + +The O +panels O +are O +arranged O +the O +same O +as O +in O +Fig O +. O +2d O +. O +( O +f O +) O +Effects O +of O +mutations B-experimental_method +in O +MKP5 B-protein +- O +CD B-structure_element +on O +the O +JNK1 B-protein +dephosphorylation O +( O +mean O +± O +s O +. O +e O +. O +m O +., O +n O += O +3 O +). O + +( O +g O +) O +Effects O +of O +mutations B-experimental_method +in O +MKP5 B-protein +- O +CD B-structure_element +on O +the O +pNPP B-chemical +hydrolysis O +reaction O +( O +mean O +± O +s O +. O +e O +. O +m O +., O +n O += O +3 O +). O + +( O +h O +) O +Pull B-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +of O +MKP5 B-protein +- O +CD B-structure_element +by O +GST B-protein_state +- I-protein_state +tagged I-protein_state +JNK1 B-protein +mutants B-protein_state +. O + +Mechanistic O +insight O +into O +a O +peptide B-protein_type +hormone I-protein_type +signaling O +complex O +mediating O +floral O +organ O +abscission O + +Plants B-taxonomy_domain +constantly O +renew O +during O +their O +life O +cycle O +and O +thus O +require O +to O +shed O +senescent O +and O +damaged O +organs O +. O + +Floral O +abscission O +is O +controlled O +by O +the O +leucine B-protein_type +- I-protein_type +rich I-protein_type +repeat I-protein_type +receptor I-protein_type +kinase I-protein_type +( O +LRR B-protein_type +- I-protein_type +RK I-protein_type +) O +HAESA B-protein +and O +the O +peptide B-protein_type +hormone I-protein_type +IDA B-protein +. O + +It O +is O +unknown O +how O +expression O +of O +IDA B-protein +in O +the O +abscission O +zone O +leads O +to O +HAESA B-protein +activation O +. O + +Here O +we O +show O +that O +IDA B-protein +is O +sensed O +directly O +by O +the O +HAESA B-protein +ectodomain B-structure_element +. O + +Crystal B-evidence +structures I-evidence +of O +HAESA B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +IDA B-protein +reveal O +a O +hormone B-site +binding I-site +pocket I-site +that O +accommodates O +an O +active B-protein_state +dodecamer B-structure_element +peptide B-chemical +. O + +A O +central O +hydroxyproline B-residue_name +residue O +anchors O +IDA B-protein +to O +the O +receptor O +. O + +The O +HAESA B-protein +co B-protein_type +- I-protein_type +receptor I-protein_type +SERK1 B-protein +, O +a O +positive O +regulator O +of O +the O +floral O +abscission O +pathway O +, O +allows O +for O +high O +- O +affinity O +sensing O +of O +the O +peptide B-protein_type +hormone I-protein_type +by O +binding O +to O +an O +Arg B-structure_element +- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +in O +IDA B-protein +. O + +This O +sequence O +pattern O +is O +conserved B-protein_state +among O +diverse O +plant B-taxonomy_domain +peptides B-chemical +, O +suggesting O +that O +plant B-taxonomy_domain +peptide B-protein_type +hormone I-protein_type +receptors I-protein_type +may O +share O +a O +common O +ligand O +binding O +mode O +and O +activation O +mechanism O +. O + +Plants B-taxonomy_domain +can O +shed O +their O +leaves O +, O +flowers O +or O +other O +organs O +when O +they O +no O +longer O +need O +them O +. O +But O +how O +does O +a O +leaf O +or O +a O +flower O +know O +when O +to O +let O +go O +? O +A O +receptor B-protein_type +protein I-protein_type +called O +HAESA B-protein +is O +found O +on O +the O +surface O +of O +the O +cells O +that O +surround O +a O +future O +break O +point O +on O +the O +plant O +. O +When O +its O +time O +to O +shed O +an O +organ O +, O +a O +hormone B-chemical +called O +IDA B-protein +instructs O +HAESA B-protein +to O +trigger O +the O +shedding O +process O +. O + +However O +, O +the O +molecular O +details O +of O +how O +IDA B-protein +triggers O +organ O +shedding O +are O +not O +clear O +. O + +The O +shedding O +of O +floral O +organs O +( O +or O +leaves O +) O +can O +be O +easily O +studied O +in O +a O +model O +plant B-taxonomy_domain +called O +Arabidopsis B-taxonomy_domain +. O + +Santiago O +et O +al O +. O +used O +protein B-experimental_method +biochemistry I-experimental_method +, O +structural B-experimental_method +biology I-experimental_method +and O +genetics B-experimental_method +to O +uncover O +how O +the O +IDA B-protein +hormone B-chemical +activates O +HAESA B-protein +. O + +The O +experiments O +show O +that O +IDA B-protein +binds B-protein_state +directly I-protein_state +to I-protein_state +a O +canyon B-protein_state +shaped I-protein_state +pocket B-site +in O +HAESA B-protein +that O +extends O +out O +from O +the O +surface O +of O +the O +cell O +. O + +IDA B-protein +binding O +to O +HAESA B-protein +allows O +another O +receptor B-protein_type +protein I-protein_type +called O +SERK1 B-protein +to B-protein_state +bind I-protein_state +to I-protein_state +HAESA B-protein +, O +which O +results O +in O +the O +release O +of O +signals O +inside O +the O +cell O +that O +trigger O +the O +shedding O +of O +organs O +. O + +The O +next O +step O +following O +on O +from O +this O +work O +is O +to O +understand O +what O +signals O +are O +produced O +when O +IDA B-protein +activates O +HAESA B-protein +. O + +Another O +challenge O +will O +be O +to O +find O +out O +where O +IDA B-protein +is O +produced O +in O +the O +plant B-taxonomy_domain +and O +what O +causes O +it O +to O +accumulate O +in O +specific O +places O +in O +preparation O +for O +organ O +shedding O +. O + +The O +HAESA B-protein +ectodomain B-structure_element +folds O +into O +a O +superhelical B-structure_element +assembly I-structure_element +of O +21 O +leucine B-structure_element +- I-structure_element +rich I-structure_element +repeats I-structure_element +. O + +( O +A O +) O +SDS B-experimental_method +PAGE I-experimental_method +analysis O +of O +the O +purified O +Arabidopsis B-species +thaliana I-species +HAESA B-protein +ectodomain B-structure_element +( O +residues O +20 B-residue_range +– I-residue_range +620 I-residue_range +) O +obtained O +by O +secreted B-experimental_method +expression I-experimental_method +in I-experimental_method +insect I-experimental_method +cells I-experimental_method +. O + +The O +calculated O +molecular O +mass O +is O +65 O +. O +7 O +kDa O +, O +the O +actual O +molecular O +mass O +obtained O +by O +mass B-experimental_method +spectrometry I-experimental_method +is O +74 O +, O +896 O +Da O +, O +accounting O +for O +the O +N B-chemical +- I-chemical +glycans I-chemical +. O +( O +B O +) O +Ribbon O +diagrams O +showing O +front O +( O +left O +panel O +) O +and O +side O +views O +( O +right O +panel O +) O +of O +the O +isolated O +HAESA B-protein +LRR B-structure_element +domain I-structure_element +. O + +The O +N O +- O +( O +residues O +20 B-residue_range +– I-residue_range +88 I-residue_range +) O +and O +C O +- O +terminal O +( O +residues O +593 B-residue_range +– I-residue_range +615 I-residue_range +) O +capping B-structure_element +domains I-structure_element +are O +shown O +in O +yellow O +, O +the O +central O +21 O +LRR B-structure_element +motifs I-structure_element +are O +in O +blue O +and O +disulphide B-ptm +bonds I-ptm +are O +highlighted O +in O +green O +( O +in O +bonds O +representation O +). O +( O +C O +) O +Structure B-experimental_method +based I-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +the O +21 O +leucine B-structure_element +- I-structure_element +rich I-structure_element +repeats I-structure_element +in O +HAESA B-protein +with O +the O +plant B-taxonomy_domain +LRR B-structure_element +consensus O +sequence O +shown O +for O +comparison O +. O + +Conserved B-protein_state +hydrophobic B-protein_state +residues B-structure_element +are O +shaded O +in O +gray O +, O +N B-site +- I-site +glycosylation I-site +sites I-site +visible O +in O +our O +structures B-evidence +are O +highlighted O +in O +blue O +, O +cysteine B-residue_name +residues O +involved O +in O +disulphide B-ptm +bridge I-ptm +formation O +in O +green O +. O +( O +D O +) O +Asn B-ptm +- I-ptm +linked I-ptm +glycans I-ptm +mask O +the O +N O +- O +terminal O +portion O +of O +the O +HAESA B-protein +ectodomain B-structure_element +. O + +Oligomannose B-chemical +core O +structures O +( O +containing O +two O +N B-chemical +- I-chemical +actylglucosamines I-chemical +and O +three O +terminal O +mannose B-chemical +units O +) O +as O +found O +in O +Trichoplusia B-species +ni I-species +cells O +and O +in O +plants B-taxonomy_domain +were O +modeled O +onto O +the O +seven O +glycosylation B-site +sites I-site +observed O +in O +our O +HAESA B-protein +structures B-evidence +, O +to O +visualize O +the O +surface O +areas O +potentially O +not O +masked O +by O +carbohydrate B-chemical +. O + +The O +HAESA B-protein +ectodomain B-structure_element +is O +shown O +in O +blue O +( O +in O +surface O +representation O +), O +the O +glycan B-chemical +structures O +are O +shown O +in O +yellow O +. O + +Hydrophobic B-bond_interaction +contacts I-bond_interaction +and O +a O +hydrogen B-site +- I-site +bond I-site +network I-site +mediate O +the O +interaction O +between O +HAESA B-protein +and O +the O +peptide B-protein_type +hormone I-protein_type +IDA B-protein +. O + +( O +A O +) O +Details O +of O +the O +IDA B-site +binding I-site +pocket I-site +. O + +HAESA B-protein +is O +shown O +in O +blue O +( O +ribbon O +diagram O +), O +the O +C O +- O +terminal O +Arg B-structure_element +- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +( O +left O +panel O +), O +the O +central O +Hyp B-structure_element +anchor I-structure_element +( O +center O +) O +and O +the O +N O +- O +terminal O +Pro B-structure_element +- I-structure_element +rich I-structure_element +motif I-structure_element +in O +IDA B-protein +( O +right O +panel O +) O +are O +shown O +in O +yellow O +( O +in O +bonds O +representation O +). O + +HAESA B-site +interface I-site +residues I-site +are O +shown O +as O +sticks O +, O +selected O +hydrogen B-bond_interaction +bond I-bond_interaction +interactions I-bond_interaction +are O +denoted O +as O +dotted O +lines O +( O +in O +magenta O +). O +( O +B O +) O +View O +of O +the O +complete O +IDA B-protein +( O +in O +bonds O +representation O +, O +in O +yellow O +) O +binding B-site +pocket I-site +in O +HAESA B-protein +( O +surface O +view O +, O +in O +blue O +). O + +Orientation O +as O +in O +( O +A O +). O +( O +C O +) O +Structure B-experimental_method +based I-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +leucine B-structure_element +- I-structure_element +rich I-structure_element +repeats I-structure_element +in O +HAESA B-protein +with O +the O +plant B-taxonomy_domain +LRR B-structure_element +consensus B-evidence +sequence I-evidence +shown O +for O +comparison O +. O + +Residues O +mediating O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +the O +IDA B-chemical +peptide I-chemical +are O +highlighted O +in O +blue O +, O +residues O +contributing O +to O +hydrogen B-bond_interaction +bond I-bond_interaction +interactions I-bond_interaction +and O +/ O +or O +salt B-bond_interaction +bridges I-bond_interaction +are O +shown O +in O +red O +. O + +The O +IDA B-site +binding I-site +pocket I-site +covers O +LRRs B-structure_element +2 I-structure_element +– I-structure_element +14 I-structure_element +and O +all O +residues O +originate O +from O +the O +inner O +surface O +of O +the O +HAESA B-protein +superhelix B-structure_element +. O + +The O +IDA B-complex_assembly +- I-complex_assembly +HAESA I-complex_assembly +and O +SERK1 B-complex_assembly +- I-complex_assembly +HAESA I-complex_assembly +complex O +interfaces B-site +are O +conserved B-protein_state +among O +HAESA B-protein +and O +HAESA B-protein_type +- I-protein_type +like I-protein_type +proteins I-protein_type +from O +different O +plant B-taxonomy_domain +species O +. O + +Structure B-experimental_method +- I-experimental_method +based I-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +the O +HAESA B-protein_type +family I-protein_type +members I-protein_type +: O +Arabidopsis B-species +thaliana I-species +HAESA B-protein +( O +Uniprot O +( O +http O +:// O +www O +. O +uniprot O +. O +org O +) O +ID O +P47735 O +), O +Arabidopsis B-species +thaliana I-species +HSL2 B-protein +( O +Uniprot O +ID O +C0LGX3 O +), O +Capsella B-species +rubella I-species +HAESA B-protein +( O +Uniprot O +ID O +R0F2U6 O +), O +Citrus B-species +clementina I-species +HSL2 B-protein +( O +Uniprot O +ID O +V4U227 O +), O +Vitis B-species +vinifera I-species +HAESA B-protein +( O +Uniprot O +ID O +F6HM39 O +). O + +The O +alignment O +includes O +a O +secondary O +structure O +assignment O +calculated O +with O +the O +program O +DSSP O +and O +colored O +according O +to O +Figure O +1 O +, O +with O +the O +N O +- O +and O +C O +- O +terminal O +caps B-structure_element +and O +the O +21 O +LRR B-structure_element +motifs I-structure_element +indicated O +in O +orange O +and O +blue O +, O +respectively O +. O + +Cysteine B-residue_name +residues O +engaged O +in O +disulphide B-ptm +bonds I-ptm +are O +depicted O +in O +green O +. O + +HAESA B-protein +residues O +interacting O +with O +the O +IDA B-chemical +peptide I-chemical +and O +/ O +or O +the O +SERK1 B-protein +co B-protein_type +- I-protein_type +receptor I-protein_type +kinase I-protein_type +ectodomain B-structure_element +are O +highlighted O +in O +blue O +and O +orange O +, O +respectively O +. O + +The O +peptide B-protein_type +hormone I-protein_type +IDA B-protein +binds O +to O +the O +HAESA B-protein +LRR B-structure_element +ectodomain I-structure_element +. O + +( O +A O +) O +Multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +selected O +IDA B-protein_type +family I-protein_type +members I-protein_type +. O + +The O +conserved B-protein_state +PIP B-structure_element +motif I-structure_element +is O +highlighted O +in O +yellow O +, O +the O +central O +Hyp B-residue_name +in O +blue O +. O + +The O +PKGV B-structure_element +motif I-structure_element +present O +in O +our O +N B-protein_state +- I-protein_state +terminally I-protein_state +extended I-protein_state +IDA B-chemical +peptide I-chemical +is O +highlighted O +in O +red O +. O +( O +B O +) O +Isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +of O +the O +HAESA B-protein +ectodomain B-structure_element +vs O +. O +IDA B-protein +and O +including O +the O +synthetic B-protein_state +peptide B-chemical +sequence O +. O + +( O +C O +) O +Structure O +of O +the O +HAESA B-complex_assembly +– I-complex_assembly +IDA I-complex_assembly +complex O +with O +HAESA B-protein +shown O +in O +blue O +( O +ribbon O +diagram O +). O + +IDA B-protein +( O +in O +bonds O +representation O +, O +surface O +view O +included O +) O +is O +depicted O +in O +yellow O +. O + +The O +peptide B-site +binding I-site +pocket I-site +covers O +HAESA B-protein +LRRs B-structure_element +2 I-structure_element +– I-structure_element +14 I-structure_element +. O +( O +D O +) O +Close O +- O +up O +view O +of O +the O +entire O +IDA B-protein +( O +in O +yellow O +) O +peptide B-site +binding I-site +site I-site +in O +HAESA B-protein +( O +in O +blue O +). O + +Details O +of O +the O +interactions O +between O +the O +central O +Hyp B-structure_element +anchor I-structure_element +in O +IDA B-protein +and O +the O +C O +- O +terminal O +Arg B-structure_element +- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +with O +HAESA B-protein +are O +highlighted O +in O +( O +E O +) O +and O +( O +F O +), O +respectively O +. O + +Hydrogren O +bonds O +are O +depicted O +as O +dotted O +lines O +( O +in O +magenta O +), O +a O +water B-chemical +molecule O +is O +shown O +as O +a O +red O +sphere O +. O + +During O +their O +growth O +, O +development O +and O +reproduction O +plants B-taxonomy_domain +use O +cell O +separation O +processes O +to O +detach O +no O +- O +longer O +required O +, O +damaged O +or O +senescent O +organs O +. O + +Abscission O +of O +floral O +organs O +in O +Arabidopsis B-taxonomy_domain +is O +a O +model O +system O +to O +study O +these O +cell O +separation O +processes O +in O +molecular O +detail O +. O + +The O +LRR B-structure_element +- I-structure_element +RKs I-structure_element +HAESA B-protein +( O +greek O +: O +to O +adhere O +to O +) O +and O +HAESA B-protein +- I-protein +LIKE I-protein +2 I-protein +( O +HSL2 B-protein +) O +redundantly O +control O +floral O +abscission O +. O + +Loss O +- O +of O +- O +function O +of O +the O +secreted O +small O +protein O +INFLORESCENCE B-protein +DEFICIENT I-protein +IN I-protein +ABSCISSION I-protein +( O +IDA B-protein +) O +causes O +floral O +organs O +to O +remain O +attached O +while O +its O +over O +- O +expression O +leads O +to O +premature O +shedding O +. O + +Full B-protein_state +- I-protein_state +length I-protein_state +IDA B-protein +is O +proteolytically B-ptm +processed I-ptm +and O +a O +conserved B-protein_state +stretch B-residue_range +of I-residue_range +20 I-residue_range +amino I-residue_range +- I-residue_range +acids I-residue_range +( O +termed O +EPIP B-structure_element +) O +can O +rescue O +the O +IDA B-protein +loss O +- O +of O +- O +function O +phenotype O +( O +Figure O +1A O +). O + +It O +has O +been O +demonstrated O +that O +a O +dodecamer B-structure_element +peptide B-chemical +within O +EPIP B-structure_element +is O +able O +to O +activate O +HAESA B-protein +and O +HSL2 B-protein +in O +transient B-experimental_method +assays I-experimental_method +in O +tobacco B-taxonomy_domain +cells O +. O + +This B-structure_element +sequence I-structure_element +motif I-structure_element +is O +highly B-protein_state +conserved I-protein_state +among O +IDA B-protein_type +family I-protein_type +members I-protein_type +( O +IDA B-protein_type +- I-protein_type +LIKE I-protein_type +PROTEINS I-protein_type +, O +IDLs B-protein_type +) O +and O +contains O +a O +central O +Pro B-residue_name +residue O +, O +presumed O +to O +be O +post B-protein_state +- I-protein_state +translationally I-protein_state +modified I-protein_state +to O +hydroxyproline B-residue_name +( O +Hyp B-residue_name +; O +Figure O +1A O +). O + +The O +available O +genetic O +and O +biochemical O +evidence O +suggests O +that O +IDA B-protein +and O +HAESA B-protein +together O +control O +floral O +abscission O +, O +but O +it O +is O +poorly O +understood O +if O +IDA B-protein +is O +directly O +sensed O +by O +the O +receptor B-protein_type +kinase I-protein_type +HAESA B-protein +and O +how O +IDA B-protein +binding O +at O +the O +cell O +surface O +would O +activate O +the O +receptor O +. O + +IDA B-protein +directly O +binds O +to O +the O +LRR B-structure_element +domain I-structure_element +of O +HAESA B-protein + +Active B-protein_state +IDA B-protein_type +- I-protein_type +family I-protein_type +peptide I-protein_type +hormones I-protein_type +are O +hydroxyprolinated B-protein_state +dodecamers B-structure_element +. O + +Close O +- O +up O +views O +of O +( O +A O +) O +IDA B-protein +, O +( O +B O +) O +the O +N B-protein_state +- I-protein_state +terminally I-protein_state +extended I-protein_state +PKGV B-mutant +- I-mutant +IDA I-mutant +and O +( O +C O +) O +IDL1 B-protein +bound B-protein_state +to I-protein_state +the O +HAESA B-protein +hormone B-site +binding I-site +pocket I-site +( O +in O +bonds O +representation O +, O +in O +yellow O +) O +and O +including O +simulated B-experimental_method +annealing I-experimental_method +2Fo B-evidence +– I-evidence +Fc I-evidence +omit I-evidence +electron I-evidence +density I-evidence +maps I-evidence +contoured O +at O +1 O +. O +0 O +σ O +. O + +Note O +that O +Pro58IDA B-residue_name_number +and O +Leu67IDA B-residue_name_number +are O +the O +first O +residues O +defined O +by O +electron B-evidence +density I-evidence +when O +bound B-protein_state +to I-protein_state +the O +HAESA B-protein +ectodomain B-structure_element +. O +( O +D O +) O +Table O +summaries O +for O +equilibrium B-evidence +dissociation I-evidence +constants I-evidence +( O +Kd B-evidence +), O +binding B-evidence +enthalpies I-evidence +( O +ΔH B-evidence +), O +binding B-evidence +entropies I-evidence +( O +ΔS B-evidence +) O +and O +stoichoimetries O +( O +N O +) O +for O +different O +IDA B-chemical +peptides I-chemical +binding O +to O +the O +HAESA B-protein +ectodomain B-structure_element +( O +± O +fitting O +errors O +; O +n O +. O +d O +. O + +no O +detectable O +binding O +). O +( O +E O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +active B-protein_state +IDA B-protein +( O +in O +bonds O +representation O +, O +in O +gray O +) O +and O +IDL1 B-chemical +peptide I-chemical +( O +in O +yellow O +) O +hormones O +bound B-protein_state +to I-protein_state +the O +HAESA B-protein +ectodomain B-structure_element +. O + +Root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +.) I-evidence +is O +1 O +. O +0 O +Å O +comparing O +100 O +corresponding O +atoms O +. O + +The O +receptor B-protein_type +kinase I-protein_type +SERK1 B-protein +acts O +as O +a O +HAESA B-protein_type +co I-protein_type +- I-protein_type +receptor I-protein_type +and O +promotes O +high O +- O +affinity O +IDA B-protein +sensing O +. O + +( O +A O +) O +Petal B-experimental_method +break I-experimental_method +- I-experimental_method +strength I-experimental_method +assays I-experimental_method +measure O +the O +force O +( O +expressed O +in O +gram O +equivalents O +) O +required O +to O +remove O +the O +petals O +from O +the O +flower O +of O +serk B-gene +mutant B-protein_state +plants B-taxonomy_domain +compared O +to O +haesa B-gene +/ O +hsl2 B-gene +mutant B-protein_state +and O +Col O +- O +0 O +wild B-protein_state +- I-protein_state +type I-protein_state +flowers O +. O + +Petal O +break O +- O +strength O +was O +found O +significantly O +increased O +in O +almost O +all O +positions O +( O +indicated O +with O +a O +*) O +for O +haesa B-gene +/ O +hsl2 B-gene +and O +serk1 B-gene +- I-gene +1 I-gene +mutant B-protein_state +plants B-taxonomy_domain +with O +respect O +to O +the O +Col O +- O +0 O +control O +. O + +( O +B O +) O +Analytical B-experimental_method +size I-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +. O + +The O +HAESA B-protein +LRR B-structure_element +domain I-structure_element +elutes O +as O +a O +monomer B-oligomeric_state +( O +black O +dotted O +line O +), O +as O +does O +the O +isolated O +SERK1 B-protein +ectodomain B-structure_element +( O +blue O +dotted O +line O +). O + +A O +HAESA B-complex_assembly +– I-complex_assembly +IDA I-complex_assembly +– I-complex_assembly +SERK1 I-complex_assembly +complex O +elutes O +as O +an O +apparent O +heterodimer B-oligomeric_state +( O +red O +line O +), O +while O +a O +mixture O +of O +HAESA B-protein +and O +SERK1 B-protein +yields O +two O +isolated O +peaks O +that O +correspond O +to O +monomeric B-oligomeric_state +HAESA B-protein +and O +SERK1 B-protein +, O +respectively O +( O +black O +line O +). O + +Void O +( O +V0 O +) O +volume O +and O +total O +volume O +( O +Vt O +) O +are O +shown O +, O +together O +with O +elution O +volumes O +for O +molecular O +mass O +standards O +( O +A O +, O +Thyroglobulin B-protein +, O +669 O +, O +000 O +Da O +; O +B O +, O +Ferritin B-protein +, O +440 O +, O +00 O +Da O +, O +C O +, O +Aldolase B-protein +, O +158 O +, O +000 O +Da O +; O +D O +, O +Conalbumin B-protein +, O +75 O +, O +000 O +Da O +; O +E O +, O +Ovalbumin B-protein +, O +44 O +, O +000 O +Da O +; O +F O +, O +Carbonic B-protein +anhydrase I-protein +, O +29 O +, O +000 O +Da O +). O + +A O +SDS B-experimental_method +PAGE I-experimental_method +of O +the O +peak O +fractions O +is O +shown O +alongside O +. O + +Purified O +HAESA B-protein +and O +SERK1 B-protein +are O +~ O +75 O +and O +~ O +28 O +kDa O +, O +respectively O +. O +( O +C O +) O +Isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +of O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +Hyp64 B-ptm +→ I-ptm +Pro I-ptm +IDA B-protein +versus O +the O +HAESA B-protein +and O +SERK1 B-protein +ectodomains B-structure_element +. O + +The O +titration B-experimental_method +of O +IDA B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +versus O +the O +isolated O +HAESA B-protein +ectodomain B-structure_element +from O +Figure O +1B O +is O +shown O +for O +comparison O +( O +red O +line O +; O +n O +. O +d O +. O + +no O +detectable O +binding O +) O +( O +D O +) O +Analytical B-experimental_method +size I-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +in O +the O +presence B-protein_state +of I-protein_state +the O +IDA B-protein +Hyp64 B-ptm +→ I-ptm +Pro I-ptm +mutant B-protein_state +peptide B-chemical +reveals O +no O +complex O +formation O +between O +HAESA B-protein +and O +SERK1 B-protein +ectodomains B-structure_element +. O + +( O +E O +) O +In B-experimental_method +vitro I-experimental_method +kinase I-experimental_method +assays I-experimental_method +of O +the O +HAESA B-protein +and O +SERK1 B-protein +kinase B-structure_element +domains I-structure_element +. O + +Wild B-protein_state +- I-protein_state +type I-protein_state +HAESA B-protein +and O +SERK1 B-protein +kinase B-structure_element +domains I-structure_element +( O +KDs B-structure_element +) O +exhibit O +auto O +- O +phosphorylation O +activities O +( O +lanes O +1 O ++ O +3 O +). O + +Mutant B-protein_state +( O +m O +) O +versions O +, O +which O +carry O +point B-experimental_method +mutations I-experimental_method +in O +their O +active B-site +sites I-site +( O +Asp837HAESA B-mutant +→ I-mutant +Asn I-mutant +, O +Asp447SERK1 B-mutant +→ I-mutant +Asn I-mutant +) O +possess O +no O +autophosphorylation O +activity O +( O +lanes O +2 O ++ O +4 O +). O + +Transphosphorylation O +activity O +from O +the O +active B-protein_state +kinase O +to O +the O +mutated B-protein_state +form O +can O +be O +observed O +in O +both O +directions O +( O +lanes O +5 O ++ O +6 O +). O + +We O +purified B-experimental_method +the O +HAESA B-protein +ectodomain B-structure_element +( O +residues O +20 B-residue_range +– I-residue_range +620 I-residue_range +) O +from O +baculovirus B-experimental_method +- I-experimental_method +infected I-experimental_method +insect I-experimental_method +cells I-experimental_method +( O +Figure O +1 O +— O +figure O +supplement O +1A O +, O +see O +Materials O +and O +methods O +) O +and O +quantified O +the O +interaction O +of O +the O +~ O +75 O +kDa O +glycoprotein B-protein_type +with O +synthetic B-protein_state +IDA B-chemical +peptides I-chemical +using O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +( O +ITC B-experimental_method +). O + +A O +Hyp B-protein_state +- I-protein_state +modified I-protein_state +dodecamer B-structure_element +comprising O +the O +highly B-protein_state +conserved I-protein_state +PIP B-structure_element +motif I-structure_element +in O +IDA B-protein +( O +Figure O +1A O +) O +interacts O +with O +HAESA B-protein +with O +1 O +: O +1 O +stoichiometry O +( O +N O +) O +and O +with O +a O +dissociation B-evidence +constant I-evidence +( O +Kd B-evidence +) O +of O +~ O +20 O +μM O +( O +Figure O +1B O +). O + +We O +next O +determined O +crystal B-evidence +structures I-evidence +of O +the O +apo B-protein_state +HAESA B-protein +ectodomain B-structure_element +and O +of O +a O +HAESA B-complex_assembly +- I-complex_assembly +IDA I-complex_assembly +complex O +, O +at O +1 O +. O +74 O +and O +1 O +. O +86 O +Å O +resolution O +, O +respectively O +( O +Figure O +1C O +; O +Figure O +1 O +— O +figure O +supplement O +1B O +– O +D O +; O +Tables O +1 O +, O +2 O +). O + +IDA B-protein +binds O +in O +a O +completely B-protein_state +extended I-protein_state +conformation I-protein_state +along O +the O +inner O +surface O +of O +the O +HAESA B-protein +ectodomain B-structure_element +, O +covering O +LRRs B-structure_element +2 I-structure_element +– I-structure_element +14 I-structure_element +( O +Figure O +1C O +, O +D O +, O +Figure O +1 O +— O +figure O +supplement O +2 O +). O + +The O +central O +Hyp64IDA B-ptm +is O +buried O +in O +a O +specific O +pocket B-site +formed O +by O +HAESA B-protein +LRRs B-structure_element +8 I-structure_element +– I-structure_element +10 I-structure_element +, O +with O +its O +hydroxyl O +group O +establishing O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +strictly B-protein_state +conserved I-protein_state +Glu266HAESA B-residue_name_number +and O +with O +a O +water B-chemical +molecule O +, O +which O +in O +turn O +is O +coordinated O +by O +the O +main O +chain O +oxygens O +of O +Phe289HAESA B-residue_name_number +and O +Ser311HAESA B-residue_name_number +( O +Figure O +1E O +; O +Figure O +1 O +— O +figure O +supplement O +3 O +). O + +The O +restricted O +size O +of O +the O +Hyp B-site +pocket I-site +suggests O +that O +IDA B-protein +does O +not O +require O +arabinosylation B-ptm +of O +Hyp64IDA B-ptm +for O +activity O +in O +vivo O +, O +a O +modification O +that O +has O +been O +reported O +for O +Hyp B-residue_name +residues O +in O +plant B-taxonomy_domain +CLE B-protein_type +peptide I-protein_type +hormones I-protein_type +. O + +The O +C O +- O +terminal O +Arg B-structure_element +- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +in O +IDA B-protein +maps O +to O +a O +cavity B-site +formed O +by O +HAESA B-protein +LRRs B-structure_element +11 I-structure_element +– I-structure_element +14 I-structure_element +( O +Figure O +1D O +, O +F O +). O + +The O +COO O +- O +group O +of O +Asn69IDA B-residue_name_number +is O +in O +direct O +contact O +with O +Arg407HAESA B-residue_name_number +and O +Arg409HAESA B-residue_name_number +and O +HAESA B-protein +cannot O +bind O +a O +C B-protein_state +- I-protein_state +terminally I-protein_state +extended I-protein_state +IDA B-mutant +- I-mutant +SFVN I-mutant +peptide O +( O +Figures O +1D O +, O +F O +, O +2D O +). O + +This O +suggests O +that O +the O +conserved B-protein_state +Asn69IDA B-residue_name_number +may O +constitute O +the O +very O +C O +- O +terminus O +of O +the O +mature B-protein_state +IDA B-chemical +peptide I-chemical +in O +planta B-taxonomy_domain +and O +that O +active B-protein_state +IDA B-protein +is O +generated O +by O +proteolytic O +processing O +from O +a O +longer O +pre O +- O +protein O +. O + +Mutation B-experimental_method +of O +Arg417HSL2 B-residue_name_number +( O +which O +corresponds O +to O +Arg409HAESA B-residue_name_number +) O +causes O +a O +loss O +- O +of O +- O +function O +phenotype O +in O +HSL2 B-protein +, O +which O +indicates O +that O +the O +peptide B-site +binding I-site +pockets I-site +in O +different O +HAESA B-protein_type +receptors I-protein_type +have O +common O +structural O +and O +sequence O +features O +. O + +Indeed O +, O +we O +find O +many O +of O +the O +residues O +contributing O +to O +the O +formation O +of O +the O +IDA B-site +binding I-site +surface I-site +in O +HAESA B-protein +to O +be O +conserved B-protein_state +in O +HSL2 B-protein +and O +in O +other O +HAESA B-protein_type +- I-protein_type +type I-protein_type +receptors I-protein_type +in O +different O +plant B-taxonomy_domain +species O +( O +Figure O +1 O +— O +figure O +supplement O +3 O +). O + +A O +N O +- O +terminal O +Pro B-structure_element +- I-structure_element +rich I-structure_element +motif I-structure_element +in O +IDA B-protein +makes O +contacts O +with O +LRRs B-structure_element +2 I-structure_element +– I-structure_element +6 I-structure_element +of O +the O +receptor O +( O +Figure O +1D O +, O +Figure O +1 O +— O +figure O +supplement O +2A O +– O +C O +). O + +Other O +hydrophobic B-bond_interaction +and I-bond_interaction +polar I-bond_interaction +interactions I-bond_interaction +are O +mediated O +by O +Ser62IDA B-residue_name_number +, O +Ser65IDA B-residue_name_number +and O +by O +backbone O +atoms O +along O +the O +IDA B-chemical +peptide I-chemical +( O +Figure O +1D O +, O +Figure O +1 O +— O +figure O +supplement O +2A O +– O +C O +). O + +HAESA B-protein +specifically O +senses O +IDA B-protein_type +- I-protein_type +family I-protein_type +dodecamer B-structure_element +peptides B-chemical + +We O +next O +investigated O +whether O +HAESA B-protein +binds O +N B-protein_state +- I-protein_state +terminally I-protein_state +extended I-protein_state +versions O +of O +IDA B-protein +. O + +We O +obtained O +a O +structure B-evidence +of O +HAESA B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +a O +PKGV B-mutant +- I-mutant +IDA I-mutant +peptide B-chemical +at O +1 O +. O +94 O +Å O +resolution O +( O +Table O +2 O +). O + +In O +this O +structure B-evidence +, O +no O +additional O +electron B-evidence +density I-evidence +accounts O +for O +the O +PKGV B-structure_element +motif I-structure_element +at O +the O +IDA B-protein +N O +- O +terminus O +( O +Figure O +2A O +, O +B O +). O + +Consistently O +, O +PKGV B-mutant +- I-mutant +IDA I-mutant +and O +IDA B-protein +have O +similar O +binding B-evidence +affinities I-evidence +in O +our O +ITC B-experimental_method +assays I-experimental_method +, O +further O +indicating O +that O +HAESA B-protein +senses O +a O +dodecamer B-structure_element +peptide B-chemical +comprising O +residues O +58 B-residue_range +- I-residue_range +69IDA I-residue_range +( O +Figure O +2D O +). O + +We O +next O +tested O +if O +HAESA B-protein +binds O +other O +IDA B-chemical +peptide I-chemical +family I-chemical +members I-chemical +. O + +IDL1 B-protein +, O +which O +can O +rescue O +IDA B-protein +loss O +- O +of O +- O +function O +mutants O +when O +introduced O +in O +abscission O +zone O +cells O +, O +can O +also O +be O +sensed O +by O +HAESA B-protein +, O +albeit O +with O +lower O +affinity B-evidence +( O +Figure O +2D O +). O + +A O +2 O +. O +56 O +Å O +co B-evidence +- I-evidence +crystal I-evidence +structure I-evidence +with O +IDL1 B-protein +reveals O +that O +different O +IDA B-protein_type +family I-protein_type +members I-protein_type +use O +a O +common O +binding O +mode O +to O +interact O +with O +HAESA B-protein_type +- I-protein_type +type I-protein_type +receptors I-protein_type +( O +Figure O +2A O +– O +C O +, O +E O +, O +Table O +2 O +). O + +We O +do O +not O +detect O +interaction O +between O +HAESA B-protein +and O +a O +synthetic B-protein_state +peptide B-chemical +missing B-protein_state +the I-protein_state +C I-protein_state +- I-protein_state +terminal I-protein_state +Asn69IDA B-residue_name_number +( O +ΔN69 B-mutant +), O +highlighting O +the O +importance O +of O +the O +polar B-bond_interaction +interactions I-bond_interaction +between O +the O +IDA B-protein +carboxy O +- O +terminus O +and O +Arg407HAESA B-residue_name_number +/ O +Arg409HAESA B-residue_name_number +( O +Figures O +1F O +, O +2D O +). O + +Replacing B-experimental_method +Hyp64IDA B-ptm +, O +which O +is O +common O +to O +all O +IDLs B-protein_type +, O +with O +proline B-residue_name +impairs O +the O +interaction O +with O +the O +receptor O +, O +as O +does O +the O +Lys66IDA B-mutant +/ I-mutant +Arg67IDA I-mutant +→ I-mutant +Ala I-mutant +double B-protein_state +- I-protein_state +mutant I-protein_state +discussed O +below O +( O +Figure O +1A O +, O +2D O +). O + +Notably O +, O +HAESA B-protein +can O +discriminate O +between O +IDLs B-protein_type +and O +functionally B-protein_state +unrelated I-protein_state +dodecamer B-structure_element +peptides B-chemical +with O +Hyp B-ptm +modifications I-ptm +, O +such O +as O +CLV3 B-protein +( O +Figures O +2D O +, O +7 O +). O + +The O +co B-protein_type +- I-protein_type +receptor I-protein_type +kinase I-protein_type +SERK1 B-protein +allows O +for O +high O +- O +affinity O +IDA O +sensing O + +Our O +binding B-experimental_method +assays I-experimental_method +reveal O +that O +IDA B-chemical +family I-chemical +peptides I-chemical +are O +sensed O +by O +the O +isolated B-protein_state +HAESA B-protein +ectodomain B-structure_element +with O +relatively O +weak O +binding B-evidence +affinities I-evidence +( O +Figures O +1B O +, O +2A O +– O +D O +). O + +It O +has O +been O +recently O +reported O +that O +SOMATIC B-protein_type +EMBRYOGENESIS I-protein_type +RECEPTOR I-protein_type +KINASES I-protein_type +( O +SERKs B-protein_type +) O +are O +positive O +regulators O +of O +floral O +abscission O +and O +can O +interact O +with O +HAESA B-protein +and O +HSL2 B-protein +in O +an O +IDA O +- O +dependent O +manner O +. O + +As O +all O +five O +SERK B-protein_type +family I-protein_type +members I-protein_type +appear O +to O +be O +expressed O +in O +the O +Arabidopsis B-taxonomy_domain +abscission O +zone O +, O +we O +quantified O +their O +relative O +contribution O +to O +floral O +abscission O +in O +Arabidopsis B-taxonomy_domain +using O +a O +petal B-experimental_method +break I-experimental_method +- I-experimental_method +strength I-experimental_method +assay I-experimental_method +. O + +Our O +experiments O +suggest O +that O +among O +the O +SERK B-protein_type +family I-protein_type +members I-protein_type +, O +SERK1 B-protein +is O +a O +positive O +regulator O +of O +floral O +abscission O +. O + +We O +found O +that O +the O +force O +required O +to O +remove O +the O +petals O +of O +serk1 B-gene +- I-gene +1 I-gene +mutants B-protein_state +is O +significantly O +higher O +than O +that O +needed O +for O +wild B-protein_state +- I-protein_state +type I-protein_state +plants B-taxonomy_domain +, O +as O +previously O +observed O +for O +haesa B-gene +/ O +hsl2 B-gene +mutants B-protein_state +, O +and O +that O +floral O +abscission O +is O +delayed O +in O +serk1 B-gene +- I-gene +1 I-gene +( O +Figure O +3A O +). O + +The O +serk2 B-gene +- I-gene +2 I-gene +, O +serk3 B-gene +- I-gene +1 I-gene +, O +serk4 B-gene +- I-gene +1 I-gene +and O +serk5 B-gene +- I-gene +1 I-gene +mutant B-protein_state +lines O +showed O +a O +petal O +break O +- O +strength O +profile O +not O +significantly O +different O +from O +wild B-protein_state +- I-protein_state +type I-protein_state +plants B-taxonomy_domain +. O + +Possibly O +because O +SERKs B-protein_type +have O +additional O +roles O +in O +plant O +development O +such O +as O +in O +pollen O +formation O +and O +brassinosteroid O +signaling O +, O +we O +found O +that O +higher O +- O +order O +SERK O +mutants O +exhibit O +pleiotropic O +phenotypes O +in O +the O +flower O +, O +rendering O +their O +analysis O +and O +comparison O +by O +quantitative B-experimental_method +petal I-experimental_method +break I-experimental_method +- I-experimental_method +strength I-experimental_method +assays I-experimental_method +difficult O +. O + +We O +thus O +focused O +on O +analyzing O +the O +contribution O +of O +SERK1 B-protein +to O +HAESA B-protein +ligand O +sensing O +and O +receptor O +activation O +. O + +In O +vitro O +, O +the O +LRR B-structure_element +ectodomain I-structure_element +of O +SERK1 B-protein +( O +residues O +24 B-residue_range +– I-residue_range +213 I-residue_range +) O +forms O +stable B-protein_state +, O +IDA B-protein_state +- I-protein_state +dependent I-protein_state +heterodimeric B-oligomeric_state +complexes B-protein_state +with I-protein_state +HAESA B-protein +in O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +experiments O +( O +Figure O +3B O +). O + +We O +next O +quantified O +the O +contribution O +of O +SERK1 B-protein +to O +IDA B-protein +recognition O +by O +HAESA B-protein +. O + +We O +found O +that O +HAESA B-protein +senses O +IDA B-protein +with O +a O +~ O +60 O +fold O +higher O +binding B-evidence +affinity I-evidence +in O +the O +presence B-protein_state +of I-protein_state +SERK1 B-protein +, O +suggesting O +that O +SERK1 B-protein +is O +involved O +in O +the O +specific O +recognition O +of O +the O +peptide B-protein_type +hormone I-protein_type +( O +Figure O +3C O +). O + +We O +next O +titrated B-experimental_method +SERK1 B-protein +into O +a O +solution O +containing O +only O +the O +HAESA B-protein +ectodomain B-structure_element +. O + +In O +this O +case O +, O +there O +was O +no O +detectable O +interaction O +between O +receptor O +and O +co O +- O +receptor O +, O +while O +in O +the O +presence B-protein_state +of I-protein_state +IDA B-protein +, O +SERK1 B-protein +strongly O +binds O +HAESA B-protein +with O +a O +dissociation B-evidence +constant I-evidence +in O +the O +mid O +- O +nanomolar O +range O +( O +Figure O +3C O +). O + +This O +suggests O +that O +IDA B-protein +itself O +promotes O +receptor O +– O +co O +- O +receptor O +association O +, O +as O +previously O +described O +for O +the O +steroid B-chemical +hormone I-chemical +brassinolide B-chemical +and O +for O +other O +LRR B-complex_assembly +- I-complex_assembly +RK I-complex_assembly +complexes O +. O + +Importantly O +, O +hydroxyprolination B-ptm +of O +IDA B-protein +is O +critical O +for O +HAESA B-complex_assembly +- I-complex_assembly +IDA I-complex_assembly +- I-complex_assembly +SERK1 I-complex_assembly +complex O +formation O +( O +Figure O +3C O +, O +D O +). O + +Our O +calorimetry B-experimental_method +experiments O +now O +reveal O +that O +SERKs B-protein_type +may O +render O +HAESA B-protein +, O +and O +potentially O +other O +receptor B-protein_type +kinases I-protein_type +, O +competent O +for O +high O +- O +affinity O +sensing O +of O +their O +cognate O +ligands O +. O + +Upon O +IDA B-protein +binding O +at O +the O +cell O +surface O +, O +the O +kinase B-structure_element +domains I-structure_element +of O +HAESA B-protein +and O +SERK1 B-protein +, O +which O +have O +been O +shown O +to O +be O +active B-protein_state +protein B-protein_type +kinases I-protein_type +, O +may O +interact O +in O +the O +cytoplasm O +to O +activate O +each O +other O +. O + +Consistently O +, O +the O +HAESA B-protein +kinase B-structure_element +domain I-structure_element +can O +transphosphorylate O +SERK1 B-protein +and O +vice O +versa O +in O +in O +vitro O +transphosphorylation B-experimental_method +assays I-experimental_method +( O +Figure O +3E O +). O + +Together O +, O +our O +genetic B-experimental_method +and I-experimental_method +biochemical I-experimental_method +experiments I-experimental_method +implicate O +SERK1 B-protein +as O +a O +HAESA B-protein_type +co I-protein_type +- I-protein_type +receptor I-protein_type +in O +the O +Arabidopsis B-taxonomy_domain +abscission O +zone O +. O + +SERK1 B-protein +senses O +a O +conserved B-protein_state +motif B-structure_element +in O +IDA B-chemical +family I-chemical +peptides I-chemical + +Crystal B-evidence +structure I-evidence +of O +a O +HAESA B-complex_assembly +– I-complex_assembly +IDA I-complex_assembly +– I-complex_assembly +SERK1 I-complex_assembly +signaling O +complex O +. O + +( O +A O +) O +Overview O +of O +the O +ternary O +complex O +with O +HAESA B-protein +in O +blue O +( O +surface O +representation O +), O +IDA B-protein +in O +yellow O +( O +bonds O +representation O +) O +and O +SERK1 B-protein +in O +orange O +( O +surface O +view O +). O +( O +B O +) O +The O +HAESA B-protein +ectodomain B-structure_element +undergoes O +a O +conformational O +change O +upon O +SERK1 B-protein +co O +- O +receptor O +binding O +. O + +Shown O +are O +Cα O +traces O +of O +a O +structural B-experimental_method +superposition I-experimental_method +of O +the O +unbound B-protein_state +( O +yellow O +) O +and O +SERK1 B-protein_state +- I-protein_state +bound I-protein_state +( O +blue O +) O +HAESA B-protein +ectodomains B-structure_element +( O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +. I-evidence +is O +1 O +. O +5 O +Å O +between O +572 O +corresponding O +Cα O +atoms O +). O + +SERK1 B-protein +( O +in O +orange O +) O +and O +IDA B-protein +( O +in O +red O +) O +are O +shown O +alongside O +. O + +The O +conformational O +change O +in O +the O +C O +- O +terminal O +LRRs B-structure_element +and O +capping B-structure_element +domain I-structure_element +is O +indicated O +by O +an O +arrow O +. O +( O +C O +) O +SERK1 B-protein +forms O +an O +integral O +part O +of O +the O +receptor O +' O +s O +peptide B-site +binding I-site +pocket I-site +. O + +The O +N O +- O +terminal O +capping B-structure_element +domain I-structure_element +of O +SERK1 B-protein +( O +in O +orange O +) O +directly O +contacts O +the O +C O +- O +terminal O +part O +of O +IDA B-protein +( O +in O +yellow O +, O +in O +bonds O +representation O +) O +and O +the O +receptor B-protein_type +HAESA B-protein +( O +in O +blue O +). O + +Polar B-bond_interaction +contacts I-bond_interaction +of O +SERK1 B-protein +with O +IDA B-protein +are O +shown O +in O +magenta O +, O +with O +the O +HAESA B-protein +LRR B-structure_element +domain I-structure_element +in O +gray O +. O +( O +D O +) O +Details O +of O +the O +zipper B-structure_element +- I-structure_element +like I-structure_element +SERK1 B-site +- I-site +HAESA I-site +interface I-site +. O + +Ribbon O +diagrams O +of O +HAESA B-protein +( O +in O +blue O +) O +and O +SERK1 B-protein +( O +in O +orange O +) O +are O +shown O +with O +selected O +interface B-site +residues I-site +( O +in O +bonds O +representation O +). O + +Polar B-bond_interaction +interactions I-bond_interaction +are O +highlighted O +as O +dotted O +lines O +( O +in O +magenta O +). O + +To O +understand O +in O +molecular O +terms O +how O +SERK1 B-protein +contributes O +to O +high O +- O +affinity O +IDA B-protein +recognition O +, O +we O +solved O +a O +2 O +. O +43 O +Å O +crystal B-evidence +structure I-evidence +of O +the O +ternary O +HAESA B-complex_assembly +– I-complex_assembly +IDA I-complex_assembly +– I-complex_assembly +SERK1 I-complex_assembly +complex O +( O +Figure O +4A O +, O +Table O +2 O +). O + +HAESA B-protein +LRRs B-structure_element +16 I-structure_element +– I-structure_element +21 I-structure_element +and O +its O +C O +- O +terminal O +capping B-structure_element +domain I-structure_element +undergo O +a O +conformational O +change O +upon O +SERK1 B-protein +binding O +( O +Figure O +4B O +). O + +The O +SERK1 B-protein +ectodomain B-structure_element +interacts O +with O +the O +IDA B-site +peptide I-site +binding I-site +site I-site +using O +a O +loop B-structure_element +region I-structure_element +( O +residues O +51 B-residue_range +- I-residue_range +59SERK1 I-residue_range +) O +from O +its O +N O +- O +terminal O +cap B-structure_element +( O +Figure O +4A O +, O +C O +). O + +SERK1 B-protein +loop B-structure_element +residues O +establish O +multiple O +hydrophobic B-bond_interaction +and I-bond_interaction +polar I-bond_interaction +contacts I-bond_interaction +with O +Lys66IDA B-residue_name_number +and O +the O +C O +- O +terminal O +Arg B-structure_element +- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +in O +IDA B-protein +( O +Figure O +4C O +). O + +SERK1 B-protein +LRRs B-structure_element +1 I-structure_element +– I-structure_element +5 I-structure_element +and O +its O +C O +- O +terminal O +capping B-structure_element +domain I-structure_element +form O +an O +additional O +zipper B-structure_element +- I-structure_element +like I-structure_element +interface B-site +with O +residues O +originating O +from O +HAESA B-protein +LRRs B-structure_element +15 I-structure_element +– I-structure_element +21 I-structure_element +and O +from O +the O +HAESA B-protein +C O +- O +terminal O +cap B-structure_element +( O +Figure O +4D O +). O + +SERK1 B-protein +binds O +HAESA B-protein +using O +these O +two O +distinct O +interaction B-site +surfaces I-site +( O +Figure O +1 O +— O +figure O +supplement O +3 O +), O +with O +the O +N B-structure_element +- I-structure_element +cap I-structure_element +of O +the O +SERK1 B-protein +LRR B-structure_element +domain I-structure_element +partially O +covering O +the O +IDA B-site +peptide I-site +binding I-site +cleft I-site +. O + +The O +IDA B-protein +C B-structure_element +- I-structure_element +terminal I-structure_element +motif I-structure_element +is O +required O +for O +HAESA B-complex_assembly +- I-complex_assembly +SERK1 I-complex_assembly +complex O +formation O +and O +for O +IDA O +bioactivity O +. O + +( O +A O +) O +Size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +experiments O +similar O +to O +Figure O +3B O +, O +D O +reveal O +that O +IDA B-protein +mutant B-protein_state +peptides B-chemical +targeting O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +motif I-structure_element +do O +not O +form O +biochemically B-protein_state +stable I-protein_state +HAESA B-complex_assembly +- I-complex_assembly +IDA I-complex_assembly +- I-complex_assembly +SERK1 I-complex_assembly +complexes O +. O + +Deletion B-experimental_method +of O +the O +C O +- O +terminal O +Asn69IDA B-residue_name_number +completely O +inhibits B-protein_state +complex O +formation O +. O + +Purified B-experimental_method +HAESA B-protein +and O +SERK1 B-protein +are O +~ O +75 O +and O +~ O +28 O +kDa O +, O +respectively O +. O + +Left O +panel O +: O +IDA B-mutant +K66A I-mutant +/ I-mutant +R67A I-mutant +; O +center O +: O +IDA B-mutant +ΔN69 I-mutant +, O +right O +panel O +: O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +of O +peak O +fractions O +. O + +Note O +that O +the O +HAESA B-protein +and O +SERK1 B-protein +input O +lanes O +have O +already O +been O +shown O +in O +Figure O +3D O +. O +( O +B O +) O +Isothermal B-evidence +titration I-evidence +thermographs I-evidence +of O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +IDA B-chemical +peptides I-chemical +titrated B-experimental_method +into O +a O +HAESA B-protein +- O +SERK1 B-protein +mixture O +in O +the O +cell O +. O + +Table O +summaries O +for O +calorimetric B-evidence +binding I-evidence +constants I-evidence +and O +stoichoimetries O +for O +different O +IDA B-chemical +peptides I-chemical +binding O +to O +the O +HAESA B-protein +– O +SERK1 B-protein +ectodomain B-structure_element +mixture O +( O +± O +fitting O +errors O +; O +n O +. O +d O +. O + +( O +C O +) O +Quantitative O +petal B-experimental_method +break I-experimental_method +- I-experimental_method +strength I-experimental_method +assay I-experimental_method +for O +Col O +- O +0 O +wild B-protein_state +- I-protein_state +type I-protein_state +flowers O +and O +35S B-gene +:: O +IDA B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +and O +35S B-gene +:: O +IDA B-mutant +K66A I-mutant +/ I-mutant +R67A I-mutant +mutant B-protein_state +flowers O +. O + +35S B-gene +:: O +IDA B-protein +plants B-taxonomy_domain +showed O +significantly O +increased O +abscission O +compared O +to O +Col O +- O +0 O +controls O +in O +inflorescence O +positions O +2 O +and O +3 O +( O +a O +). O + +Up O +to O +inflorescence O +position O +4 O +, O +petal O +break O +in O +35S B-gene +:: O +IDA B-mutant +K66A I-mutant +/ I-mutant +R67A I-mutant +mutant B-protein_state +plants B-taxonomy_domain +was O +significantly O +increased O +compared O +to O +both O +Col O +- O +0 O +control O +plants B-taxonomy_domain +( O +b O +) O +and O +35S B-gene +:: O +IDA B-protein +plants B-taxonomy_domain +( O +c O +) O +( O +D O +) O +Normalized O +expression O +levels O +( O +relative O +expression O +± O +standard O +error O +; O +ida O +: O +- O +0 O +. O +02 O +± O +0 O +. O +001 O +; O +Col O +- O +0 O +: O +1 O +± O +0 O +. O +11 O +; O +35S B-gene +:: O +IDA B-protein +124 O +± O +0 O +. O +75 O +; O +35S B-gene +:: O +IDA B-mutant +K66A I-mutant +/ I-mutant +R67A I-mutant +: O +159 O +± O +0 O +. O +58 O +) O +of O +IDA B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +transcripts O +in O +the O +35S B-experimental_method +promoter I-experimental_method +over I-experimental_method +- I-experimental_method +expression I-experimental_method +lines I-experimental_method +analyzed O +in O +( O +C O +). O +( O +E O +) O +Magnified O +view O +of O +representative O +abscission O +zones O +from O +35S B-gene +:: O +IDA B-protein +, O +Col O +- O +0 O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +35S B-gene +:: O +IDA B-mutant +K66A I-mutant +/ I-mutant +R67A I-mutant +double B-protein_state +- I-protein_state +mutant I-protein_state +T3 B-experimental_method +transgenic I-experimental_method +lines I-experimental_method +. O + +15 O +out O +of O +15 O +35S B-gene +:: O +IDA B-protein +plants B-taxonomy_domain +, O +0 O +out O +of O +15 O +Col O +- O +0 O +plants B-taxonomy_domain +and O +0 O +out O +of O +15 O +35S B-gene +:: O +IDA B-mutant +K66A I-mutant +/ I-mutant +R67A I-mutant +double B-protein_state +- I-protein_state +mutant I-protein_state +plants B-taxonomy_domain +, O +showed O +an O +enlarged O +abscission O +zone O +, O +respectively O +( O +3 O +independent O +lines O +were O +analyzed O +). O + +The O +four O +C O +- O +terminal O +residues O +in O +IDA B-protein +( O +Lys66IDA B-residue_range +- I-residue_range +Asn69IDA I-residue_range +) O +are O +conserved B-protein_state +among O +IDA B-protein_type +family I-protein_type +members I-protein_type +and O +are O +in O +direct O +contact O +with O +SERK1 B-protein +( O +Figures O +1A O +, O +4C O +). O + +We O +thus O +assessed O +their O +contribution O +to O +HAESA B-complex_assembly +– I-complex_assembly +SERK1 I-complex_assembly +complex O +formation O +. O + +Deletion B-experimental_method +of O +the O +buried O +Asn69IDA B-residue_name_number +completely B-protein_state +inhibits I-protein_state +receptor O +– O +co O +- O +receptor O +complex O +formation O +and O +HSL2 O +activation O +( O +Figure O +5A O +, O +B O +). O + +A O +synthetic B-protein_state +Lys66IDA B-mutant +/ I-mutant +Arg67IDA I-mutant +→ I-mutant +Ala I-mutant +mutant B-protein_state +peptide B-chemical +( O +IDA B-mutant +K66A I-mutant +/ I-mutant +R66A I-mutant +) O +showed O +a O +10 O +fold O +reduced O +binding B-evidence +affinity I-evidence +when O +titrated B-experimental_method +in O +a O +HAESA B-protein +/ O +SERK1 B-protein +protein O +solution O +( O +Figures O +5A O +, O +B O +, O +2D O +). O + +We O +over B-experimental_method +- I-experimental_method +expressed I-experimental_method +full B-protein_state +- I-protein_state +length I-protein_state +wild B-protein_state +- I-protein_state +type I-protein_state +IDA B-protein +or O +this O +Lys66IDA B-mutant +/ I-mutant +Arg67IDA I-mutant +→ I-mutant +Ala I-mutant +double B-protein_state +- I-protein_state +mutant I-protein_state +to O +similar O +levels O +in O +Col O +- O +0 O +Arabidopsis B-taxonomy_domain +plants B-taxonomy_domain +( O +Figure O +5D O +). O + +We O +found O +that O +over B-experimental_method +- I-experimental_method +expression I-experimental_method +of O +wild B-protein_state +- I-protein_state +type I-protein_state +IDA B-protein +leads O +to O +early O +floral O +abscission O +and O +an O +enlargement O +of O +the O +abscission O +zone O +( O +Figure O +5C O +– O +E O +). O + +In O +contrast O +, O +over B-experimental_method +- I-experimental_method +expression I-experimental_method +of O +the O +IDA B-mutant +Lys66IDA I-mutant +/ I-mutant +Arg67IDA I-mutant +→ I-mutant +Ala I-mutant +double B-protein_state +mutant I-protein_state +significantly O +delays O +floral O +abscission O +when O +compared O +to O +wild B-protein_state +- I-protein_state +type I-protein_state +control O +plants B-taxonomy_domain +, O +suggesting O +that O +the O +mutant B-protein_state +IDA B-chemical +peptide I-chemical +has O +reduced O +activity O +in O +planta B-taxonomy_domain +( O +Figure O +5C O +– O +E O +). O + +Comparison O +of O +35S B-gene +:: O +IDA B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +plants B-taxonomy_domain +further O +indicates O +that O +mutation B-experimental_method +of O +Lys66IDA B-mutant +/ I-mutant +Arg67IDA I-mutant +→ I-mutant +Ala I-mutant +may O +cause O +a O +weak O +dominant O +negative O +effect O +( O +Figure O +5C O +– O +E O +). O + +In O +agreement O +with O +our O +structures B-evidence +and O +biochemical B-experimental_method +assays I-experimental_method +, O +this O +experiment O +suggests O +a O +role O +of O +the O +conserved B-protein_state +IDA B-protein +C O +- O +terminus O +in O +the O +control O +of O +floral O +abscission O +. O + +In O +contrast O +to O +animal B-taxonomy_domain +LRR B-protein_type +receptors I-protein_type +, O +plant B-taxonomy_domain +LRR B-structure_element +- I-structure_element +RKs I-structure_element +harbor O +spiral B-protein_state +- I-protein_state +shaped I-protein_state +ectodomains B-structure_element +and O +thus O +they O +require O +shape B-protein_state +- I-protein_state +complementary I-protein_state +co B-protein_type +- I-protein_type +receptor I-protein_type +proteins I-protein_type +for O +receptor O +activation O +. O + +For O +a O +rapidly O +growing O +number O +of O +plant B-taxonomy_domain +signaling O +pathways O +, O +SERK B-protein_type +proteins I-protein_type +act O +as O +these O +essential O +co B-protein_type +- I-protein_type +receptors I-protein_type +(; O +). O + +SERK1 O +has O +been O +previously O +reported O +as O +a O +positive O +regulator O +in O +plant B-taxonomy_domain +embryogenesis O +, O +male O +sporogenesis O +, O +brassinosteroid O +signaling O +and O +in O +phytosulfokine O +perception O +. O + +Recent O +findings O +by O +and O +our O +mechanistic O +studies O +now O +also O +support O +a O +positive O +role O +for O +SERK1 B-protein +in O +floral O +abscission O +. O + +As O +serk1 B-gene +- I-gene +1 I-gene +mutant B-protein_state +plants B-taxonomy_domain +show O +intermediate O +abscission O +phenotypes O +when O +compared O +to O +haesa B-gene +/ O +hsl2 O +mutants B-protein_state +, O +SERK1 B-protein +likely O +acts O +redundantly O +with O +other O +SERKs B-protein_type +in O +the O +abscission O +zone O +( O +Figure O +3A O +). O + +It O +has O +been O +previously O +suggested O +that O +SERK1 B-protein +can O +inhibit O +cell O +separation O +. O + +However O +our O +results O +show O +that O +SERK1 B-protein +also O +can O +activate O +this O +process O +upon O +IDA B-protein +sensing O +, O +indicating O +that O +SERKs B-protein_type +may O +fulfill O +several O +different O +functions O +in O +the O +course O +of O +the O +abscission O +process O +. O + +While O +the O +sequence O +of O +the O +mature B-protein_state +IDA B-chemical +peptide I-chemical +has O +not O +been O +experimentally O +determined O +in O +planta B-taxonomy_domain +, O +our O +HAESA B-complex_assembly +- I-complex_assembly +IDA I-complex_assembly +complex O +structures B-evidence +and O +calorimetry B-evidence +assays I-evidence +suggest O +that O +active B-protein_state +IDLs B-protein_type +are O +hydroxyprolinated B-protein_state +dodecamers B-structure_element +. O + +It O +will O +be O +thus O +interesting O +to O +see O +if O +proteolytic O +processing O +of O +full B-protein_state +- I-protein_state +length I-protein_state +IDA B-protein +in O +vivo O +is O +regulated O +in O +a O +cell O +- O +type O +or O +tissue O +- O +specific O +manner O +. O + +The O +central O +Hyp B-residue_name +residue O +in O +IDA B-protein +is O +found O +buried O +in O +the O +HAESA B-protein +peptide B-site +binding I-site +surface I-site +and O +thus O +this O +post O +- O +translational O +modification O +may O +regulate O +IDA B-protein +bioactivity O +. O + +Our O +comparative B-experimental_method +structural I-experimental_method +and I-experimental_method +biochemical I-experimental_method +analysis I-experimental_method +further O +suggests O +that O +IDLs B-protein_type +share O +a O +common O +receptor O +binding O +mode O +, O +but O +may O +preferably O +bind O +to O +HAESA B-protein +, O +HSL1 B-protein +or O +HSL2 B-protein +in O +different O +plant B-taxonomy_domain +tissues O +and O +organs O +. O + +In O +our O +quantitative B-experimental_method +biochemical I-experimental_method +assays I-experimental_method +, O +the O +presence B-protein_state +of I-protein_state +SERK1 B-protein +dramatically O +increases O +the O +HAESA B-protein +binding O +specificity O +and O +affinity O +for O +IDA B-protein +. O + +This O +observation O +is O +consistent O +with O +our O +complex O +structure B-evidence +in O +which O +receptor O +and O +co O +- O +receptor O +together O +form O +the O +IDA B-site +binding I-site +pocket I-site +. O + +The O +fact O +that O +SERK1 B-protein +specifically O +interacts O +with O +the O +very O +C O +- O +terminus O +of O +IDLs B-protein_type +may O +allow O +for O +the O +rational O +design O +of O +peptide B-chemical +hormone I-chemical +antagonists I-chemical +, O +as O +previously O +demonstrated O +for O +the O +brassinosteroid O +pathway O +. O + +Importantly O +, O +our O +calorimetry B-experimental_method +assays I-experimental_method +reveal O +that O +the O +SERK1 B-protein +ectodomain B-structure_element +binds B-protein_state +HAESA B-protein +with O +nanomolar O +affinity O +, O +but O +only O +in O +the O +presence B-protein_state +of I-protein_state +IDA B-protein +( O +Figure O +3C O +). O + +This O +ligand O +- O +induced O +formation O +of O +a O +receptor O +– O +co O +- O +receptor O +complex O +may O +allow O +the O +HAESA B-protein +and O +SERK1 B-protein +kinase B-structure_element +domains I-structure_element +to O +efficiently O +trans O +- O +phosphorylate O +and O +activate O +each O +other O +in O +the O +cytoplasm O +. O + +It O +is O +of O +note O +that O +our O +reported O +binding B-evidence +affinities I-evidence +for O +IDA B-protein +and O +SERK1 B-protein +have O +been O +measured O +using O +synthetic B-protein_state +peptides B-chemical +and O +the O +isolated B-experimental_method +HAESA B-protein +and O +SERK1 B-protein +ectodomains B-structure_element +, O +and O +thus O +might O +differ O +in O +the O +context O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +, O +membrane B-protein_state +- I-protein_state +embedded I-protein_state +signaling O +complex O +. O + +SERK1 B-protein +uses O +partially O +overlapping O +surface O +areas O +to O +activate O +different O +plant B-taxonomy_domain +signaling B-protein_type +receptors I-protein_type +. O + +( O +A O +) O +Structural B-experimental_method +comparison I-experimental_method +of O +plant B-taxonomy_domain +steroid B-chemical +and O +peptide B-protein_type +hormone I-protein_type +membrane B-protein_type +signaling I-protein_type +complexes I-protein_type +. O + +Left O +panel O +: O +Ribbon O +diagram O +of O +HAESA B-protein +( O +in O +blue O +), O +SERK1 B-protein +( O +in O +orange O +) O +and O +IDA B-protein +( O +in O +bonds O +and O +surface O +represention O +). O + +Right O +panel O +: O +Ribbon O +diagram O +of O +the O +plant B-taxonomy_domain +steroid B-protein_type +receptor I-protein_type +BRI1 B-protein +( O +in O +blue O +) O +bound B-protein_state +to I-protein_state +brassinolide B-chemical +( O +in O +gray O +, O +in O +bonds O +representation O +) O +and O +to O +SERK1 B-protein +, O +shown O +in O +the O +same O +orientation O +( O +PDB O +- O +ID O +. O +4lsx O +). O + +( O +B O +) O +View O +of O +the O +inner O +surface O +of O +the O +SERK1 B-protein +LRR B-structure_element +domain I-structure_element +( O +PDB O +- O +ID O +4lsc O +, O +surface O +representation O +, O +in O +gray O +). O + +A O +ribbon O +diagram O +of O +SERK1 B-protein +in O +the O +same O +orientation O +is O +shown O +alongside O +. O + +Residues O +interacting O +with O +the O +HAESA B-protein +or O +BRI1 B-protein +LRR B-structure_element +domains I-structure_element +are O +shown O +in O +orange O +or O +magenta O +, O +respectively O +. O + +Comparison B-experimental_method +of O +our O +HAESA B-complex_assembly +– I-complex_assembly +IDA I-complex_assembly +– I-complex_assembly +SERK1 I-complex_assembly +structure B-evidence +with O +the O +brassinosteroid O +receptor O +signaling O +complex O +, O +where O +SERK1 B-protein +also O +acts O +as O +co B-protein_type +- I-protein_type +receptor I-protein_type +, O +reveals O +an O +overall O +conserved B-protein_state +mode O +of O +SERK1 B-protein +binding O +, O +while O +the O +ligand B-site +binding I-site +pockets I-site +map O +to O +very O +different O +areas O +in O +the O +corresponding O +receptors O +( O +LRRs B-structure_element +2 I-structure_element +– I-structure_element +14 I-structure_element +; O +HAESA B-protein +; O +LRRs B-structure_element +21 I-structure_element +– I-structure_element +25 I-structure_element +, O +BRI1 B-protein +) O +and O +may O +involve O +an O +island O +domain O +( O +BRI1 B-protein +) O +or O +not O +( O +HAESA B-protein +) O +( O +Figure O +6A O +). O + +Several O +residues O +in O +the O +SERK1 B-protein +N O +- O +terminal O +capping B-structure_element +domain I-structure_element +( O +Thr59SERK1 B-residue_name_number +, O +Phe61SERK1 B-residue_name_number +) O +and O +the O +LRR B-site +inner I-site +surface I-site +( O +Asp75SERK1 B-residue_name_number +, O +Tyr101SERK1 B-residue_name_number +, O +SER121SERK1 B-residue_name_number +, O +Phe145SERK1 B-residue_name_number +) O +contribute O +to O +the O +formation O +of O +both O +complexes O +( O +Figures O +4C O +, O +D O +, O +6B O +). O + +In O +addition O +, O +residues O +53 B-residue_range +- I-residue_range +55SERK1 I-residue_range +from O +the O +SERK1 B-protein +N O +- O +terminal O +cap B-structure_element +mediate O +specific O +interactions O +with O +the O +IDA B-chemical +peptide I-chemical +( O +Figures O +4C O +, O +6B O +). O + +These O +residues O +are O +not O +involved O +in O +the O +sensing O +of O +the O +steroid B-chemical +hormone I-chemical +brassinolide B-chemical +. O + +In O +both O +cases O +however O +, O +the O +co O +- O +receptor O +completes O +the O +hormone B-site +binding I-site +pocket I-site +. O + +This O +fact O +together O +with O +the O +largely O +overlapping O +SERK1 B-site +binding I-site +surfaces I-site +in O +HAESA B-protein +and O +BRI1 B-protein +allows O +us O +to O +speculate O +that O +SERK1 B-protein +may O +promote O +high O +- O +affinity O +peptide B-protein_type +hormone I-protein_type +and O +brassinosteroid O +sensing O +by O +simply O +slowing O +down O +dissociation O +of O +the O +ligand O +from O +its O +cognate O +receptor O +. O + +Different O +plant B-taxonomy_domain +peptide B-protein_type +hormone I-protein_type +families I-protein_type +contain O +a O +C O +- O +terminal O +( B-structure_element +Arg I-structure_element +)- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +, O +which O +in O +IDA B-protein +represents O +the O +co B-site +- I-site +receptor I-site +recognition I-site +site I-site +. O + +Structure B-experimental_method +- I-experimental_method +guided I-experimental_method +multiple I-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +IDA B-protein +and O +IDA B-chemical +- I-chemical +like I-chemical +peptides I-chemical +with O +other O +plant B-taxonomy_domain +peptide B-protein_type +hormone I-protein_type +families I-protein_type +, O +including O +CLAVATA3 B-protein_type +– I-protein_type +EMBRYO I-protein_type +SURROUNDING I-protein_type +REGION I-protein_type +- I-protein_type +RELATED I-protein_type +( O +CLV3 B-protein_type +/ I-protein_type +CLE I-protein_type +), O +ROOT B-protein_type +GROWTH I-protein_type +FACTOR I-protein_type +– I-protein_type +GOLVEN I-protein_type +( O +RGF B-protein_type +/ I-protein_type +GLV I-protein_type +), O +PRECURSOR B-protein_type +GENE I-protein_type +PROPEP1 I-protein_type +( O +PEP1 B-protein_type +) O +from O +Arabidopsis B-species +thaliana I-species +. O + +The O +conserved B-protein_state +( B-structure_element +Arg I-structure_element +)- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +is O +highlighted O +in O +red O +, O +the O +central O +Hyp B-residue_name +residue O +in O +IDLs B-protein_type +and O +CLEs B-protein_type +is O +marked O +in O +blue O +. O + +Our O +experiments O +reveal O +that O +SERK1 B-protein +recognizes O +a O +C O +- O +terminal O +Arg B-structure_element +- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +in O +IDA B-protein +. O + +Importantly O +, O +this B-structure_element +motif I-structure_element +can O +also O +be O +found O +in O +other O +peptide B-protein_type +hormone I-protein_type +families I-protein_type +( O +Figure O +7 O +). O + +Among O +these O +are O +the O +CLE B-chemical +peptides I-chemical +regulating O +stem O +cell O +maintenance O +in O +the O +shoot O +and O +the O +root O +. O + +It O +is O +interesting O +to O +note O +, O +that O +CLEs B-protein_type +in O +their O +mature B-protein_state +form I-protein_state +are O +also O +hydroxyprolinated B-protein_state +dodecamers B-structure_element +, O +which O +bind O +to O +a O +surface B-site +area I-site +in O +the O +BARELY B-protein_type +ANY I-protein_type +MERISTEM I-protein_type +1 I-protein_type +receptor I-protein_type +that O +would O +correspond O +to O +part O +of O +the O +IDA B-site +binding I-site +cleft I-site +in O +HAESA B-protein +. O + +Diverse O +plant B-taxonomy_domain +peptide B-protein_type +hormones I-protein_type +may O +thus O +also O +bind O +their O +LRR B-protein_type +- I-protein_type +RK I-protein_type +receptors I-protein_type +in O +an O +extended B-protein_state +conformation I-protein_state +along O +the O +inner O +surface O +of O +the O +LRR B-structure_element +domain I-structure_element +and O +may O +also O +use O +small B-protein_state +, O +shape B-protein_state +- I-protein_state +complementary I-protein_state +co B-protein_type +- I-protein_type +receptors I-protein_type +for O +high O +- O +affinity O +ligand O +binding O +and O +receptor O +activation O +. O + +Ensemble O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +uncovers O +inchworm B-protein_state +- O +like O +translocation O +of O +a O +viral B-taxonomy_domain +IRES B-site +through O +the O +ribosome B-complex_assembly + +Internal B-site +ribosome I-site +entry I-site +sites I-site +( O +IRESs B-site +) O +mediate O +cap O +- O +independent O +translation O +of O +viral B-taxonomy_domain +mRNAs B-chemical +. O + +Using O +electron B-experimental_method +cryo I-experimental_method +- I-experimental_method +microscopy I-experimental_method +of O +a O +single O +specimen O +, O +we O +present O +five O +ribosome B-complex_assembly +structures B-evidence +formed O +with O +the O +Taura B-species +syndrome I-species +virus I-species +IRES B-site +and O +translocase B-protein_type +eEF2 B-complex_assembly +• I-complex_assembly +GTP I-complex_assembly +bound B-protein_state +with I-protein_state +sordarin B-chemical +. O + +The O +structures B-evidence +suggest O +a O +trajectory O +of O +IRES B-site +translocation O +, O +required O +for O +translation O +initiation B-protein_state +, O +and O +provide O +an O +unprecedented O +view O +of O +eEF2 B-protein +dynamics O +. O + +The O +IRES B-site +rearranges O +from O +extended B-protein_state +to O +bent B-protein_state +to O +extended B-protein_state +conformations O +. O + +This O +inchworm B-protein_state +- O +like O +movement O +is O +coupled O +with O +ribosomal O +inter O +- O +subunit O +rotation O +and O +40S B-complex_assembly +head B-structure_element +swivel O +. O + +eEF2 B-protein +, O +attached O +to O +the O +60S B-complex_assembly +subunit B-structure_element +, O +slides O +along O +the O +rotating O +40S B-complex_assembly +subunit B-structure_element +to O +enter O +the O +A B-site +site I-site +. O + +Its O +diphthamide B-ptm +- O +bearing O +tip O +at O +domain O +IV B-structure_element +separates O +the O +tRNA B-structure_element +- I-structure_element +mRNA I-structure_element +- I-structure_element +like I-structure_element +pseudoknot I-structure_element +I I-structure_element +( O +PKI B-structure_element +) O +of O +the O +IRES B-site +from O +the O +decoding B-site +center I-site +. O + +This O +unlocks O +40S B-complex_assembly +domains O +, O +facilitating O +head B-structure_element +swivel O +and O +biasing O +IRES B-site +translocation O +via O +hitherto O +- O +elusive O +intermediates O +with O +PKI B-structure_element +captured O +between O +the O +A B-site +and I-site +P I-site +sites I-site +. O + +The O +structures B-evidence +suggest O +missing O +links O +in O +our O +understanding O +of O +tRNA B-chemical +translocation O +. O + +Virus B-taxonomy_domain +propagation O +relies O +on O +the O +host O +translational O +apparatus O +. O + +To O +efficiently O +compete O +with O +host O +mRNAs B-chemical +and O +engage O +in O +translation O +under O +stress O +, O +some O +viral B-taxonomy_domain +mRNAs B-chemical +undergo O +cap O +- O +independent O +translation O +. O + +To O +this O +end O +, O +internal B-site +ribosome I-site +entry I-site +site I-site +( O +IRES B-site +) O +RNAs B-chemical +are O +employed O +( O +reviewed O +in O +. O + +An O +IRES B-site +is O +located O +at O +the O +5 B-structure_element +’ I-structure_element +untranslated I-structure_element +region I-structure_element +of O +the O +viral B-taxonomy_domain +mRNA B-chemical +, O +preceding O +an O +open B-structure_element +reading I-structure_element +frame I-structure_element +( O +ORF B-structure_element +). O + +To O +initiate O +translation O +, O +a O +structured B-protein_state +IRES B-site +RNA B-chemical +interacts O +with O +the O +40S B-complex_assembly +subunit B-structure_element +or O +the O +80S B-complex_assembly +ribosome I-complex_assembly +, O +resulting O +in O +precise O +positioning O +of O +the O +downstream O +start O +codon O +in O +the O +small B-protein_state +40S B-complex_assembly +subunit B-structure_element +. O + +The O +canonical O +scenario O +of O +cap O +- O +dependent O +and O +IRES B-site +- O +dependent O +initiation O +involves O +positioning O +of O +the O +AUG O +start O +codon O +and O +the O +initiator O +tRNAMet B-chemical +in O +the O +ribosomal O +peptidyl B-site +- I-site +tRNA I-site +( I-site +P I-site +) I-site +site I-site +, O +facilitated O +by O +interaction O +with O +initiation B-protein_type +factors I-protein_type +. O + +Subsequent O +binding O +of O +an O +elongator O +aminoacyl B-chemical +- I-chemical +tRNA I-chemical +to O +the O +ribosomal O +A B-site +site I-site +transitions O +the O +initiation B-complex_assembly +complex I-complex_assembly +into O +the O +elongation O +cycle O +of O +translation O +. O + +Upon O +peptide O +bond O +formation O +, O +the O +two O +tRNAs B-chemical +and O +their O +respective O +mRNA B-chemical +codons O +translocate O +from O +the O +A B-site +and I-site +P I-site +to O +P B-site +and I-site +E I-site +( I-site +exit I-site +) I-site +sites I-site +, O +freeing O +the O +A B-site +site I-site +for O +the O +next O +elongator O +tRNA B-chemical +. O + +An O +unusual O +strategy O +of O +initiation B-protein_state +is O +used O +by O +intergenic B-structure_element +- I-structure_element +region I-structure_element +( O +IGR B-structure_element +) O +IRESs B-site +found O +in O +Dicistroviridae B-species +arthropod I-species +- O +infecting O +viruses B-taxonomy_domain +. O + +These O +include O +shrimp B-taxonomy_domain +- O +infecting O +Taura B-species +syndrome I-species +virus I-species +( O +TSV B-species +), O +and O +insect B-taxonomy_domain +viruses O +Plautia B-species +stali I-species +intestine I-species +virus I-species +( O +PSIV B-species +) O +and O +Cricket B-species +paralysis I-species +virus I-species +( O +CrPV B-species +). O + +The O +IGR B-structure_element +IRES B-site +mRNAs B-chemical +do O +not O +contain O +an O +AUG O +start O +codon O +. O + +The O +IGR B-structure_element +- O +IRES B-site +- O +driven O +initiation B-protein_state +does O +not O +involve O +initiator O +tRNAMet B-chemical +and O +initiation B-protein_state +factors O +. O + +As O +such O +, O +this O +group O +of O +IRESs B-site +represents O +the O +most O +streamlined O +mechanism O +of O +eukaryotic B-taxonomy_domain +translation O +initiation B-protein_state +. O + +A O +recent O +demonstration O +of O +bacterial B-taxonomy_domain +translation O +initiation B-protein_state +by O +an O +IGR B-structure_element +IRES B-site +indicates O +that O +the O +IRESs B-site +take O +advantage O +of O +conserved O +structural O +and O +dynamic O +properties O +of O +the O +ribosome B-complex_assembly +. O + +Early O +electron B-experimental_method +cryo I-experimental_method +- I-experimental_method +microscopy I-experimental_method +( O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +) O +studies O +have O +found O +that O +the O +CrPV B-species +IRES B-site +packs O +in O +the O +ribosome B-complex_assembly +intersubunit B-site +space I-site +. O + +Recent O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +of O +ribosome B-protein_state +- I-protein_state +bound I-protein_state +TSV B-species +IRES B-site +and O +CrPV B-species +IRES B-site +revealed O +that O +IGR B-structure_element +IRESs B-site +position O +the O +ORF B-structure_element +by O +mimicking O +a O +translating O +ribosome B-complex_assembly +bound B-protein_state +with I-protein_state +tRNA B-chemical +and O +mRNA B-chemical +. O + +The O +~ O +200 O +- O +nt O +IRES B-site +RNAs B-chemical +span O +from O +the O +A B-site +site I-site +beyond O +the O +E B-site +site I-site +. O + +A O +conserved B-protein_state +tRNA B-structure_element +- I-structure_element +mRNA I-structure_element +– I-structure_element +like I-structure_element +structural I-structure_element +element I-structure_element +of O +pseudoknot B-structure_element +I I-structure_element +( O +PKI B-structure_element +) O +interacts O +with O +the O +decoding B-site +center I-site +in O +the O +A B-site +site I-site +of O +the O +40S B-complex_assembly +subunit B-structure_element +. 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O + +PKI B-structure_element +of O +IGR B-structure_element +IRESs B-site +therefore O +mimics O +an O +A B-site +- I-site +site I-site +elongator O +tRNA B-chemical +interacting O +with O +an O +mRNA B-chemical +sense O +codon O +, O +but O +not O +a O +P B-site +- I-site +site I-site +initiator O +tRNAMet B-chemical +and O +the O +AUG O +start O +codon O +. O + +How O +this O +non O +- O +canonical O +initiation B-protein_state +complex O +transitions O +to O +the O +elongation O +step O +is O +not O +fully O +understood O +. O + +For O +a O +cognate O +aminoacyl B-chemical +- I-chemical +tRNA I-chemical +to O +bind O +the O +first O +viral B-taxonomy_domain +mRNA B-chemical +codon O +, O +PKI B-structure_element +has O +to O +be O +translocated O +from O +the O +A B-site +site I-site +, O +so O +that O +the O +first O +codon O +can O +be O +presented O +in O +the O +A B-site +site I-site +. O + +A O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structure B-evidence +of O +the O +ribosome B-complex_assembly +bound B-protein_state +with I-protein_state +a O +CrPV B-species +IRES B-site +and O +release B-protein_type +factor I-protein_type +eRF1 B-protein +occupying O +the O +A B-site +site I-site +provided O +insight O +into O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +state O +. O + +In O +this O +structure B-evidence +, O +PKI B-structure_element +is O +positioned O +in O +the O +P B-site +site I-site +and O +the O +first O +mRNA B-chemical +codon O +is O +located O +in O +the O +A B-site +site I-site +. O + +How O +the O +large B-protein_state +IRES B-site +RNA B-chemical +translocates O +within O +the O +ribosome B-complex_assembly +, O +allowing O +PKI B-structure_element +translocation O +from O +the O +A B-site +to I-site +P I-site +site I-site +is O +not O +known O +. 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O +Pre B-protein_state +- I-protein_state +translocation I-protein_state +tRNA B-protein_state +- I-protein_state +bound I-protein_state +ribosomes B-complex_assembly +contain O +a O +peptidyl B-chemical +- I-chemical +and I-chemical +deacyl I-chemical +- I-chemical +tRNA I-chemical +, O +both O +base O +- O +paired O +to O +mRNA B-chemical +codons O +in O +the O +A B-site +and I-site +P I-site +sites I-site +( O +termed O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +). O + +Translocation O +of O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +involves O +two O +major O +large O +- O +scale O +ribosome B-complex_assembly +rearrangements O +( O +Figure O +1 O +— O +figure O +supplement O +1 O +) O +( O +reviewed O +in O +). 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O +Sub B-experimental_method +- I-experimental_method +classification I-experimental_method +of O +each O +class O +did O +not O +yield O +additional O +classes O +, O +but O +helped O +improve O +density B-evidence +in O +the O +PKI B-structure_element +region O +of O +class O +III O +( O +estimated O +resolution O +and O +percentage O +of O +particles B-experimental_method +in O +the O +sub B-experimental_method +- I-experimental_method +classified I-experimental_method +reconstruction B-evidence +are O +shown O +in O +parentheses O +). O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +density B-evidence +of O +Structures B-evidence +I I-evidence +- I-evidence +V I-evidence +. O + +In O +panels O +( O +a O +- O +e O +), O +the O +maps B-evidence +are O +segmented O +and O +colored O +as O +in O +Figure O +1 O +. 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O +( O +k O +- O +o O +) O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +density B-evidence +for O +the O +TSV B-species +IRES B-site +( O +red O +model O +) O +and O +eEF2 B-protein +( O +green O +model O +) O +in O +Structures B-evidence +I I-evidence +, I-evidence +II I-evidence +, I-evidence +III I-evidence +, I-evidence +IV I-evidence +and I-evidence +V I-evidence +. O +( O +p O +) O +Fourier B-evidence +shell I-evidence +correlation I-evidence +( O +FSC B-evidence +) O +curves B-evidence +for O +Structures B-evidence +I I-evidence +- I-evidence +V I-evidence +. O +The O +horizontal O +axis O +is O +labeled O +with O +spatial O +frequency O +Å O +- O +1 O +and O +with O +Å O +. O +The O +resolutions O +stated O +in O +the O +text O +correspond O +to O +an O +FSC B-evidence +threshold O +value O +of O +0 O +. O +143 O +, O +shown O +as O +a O +dotted O +line O +, O +for O +the O +FREALIGN B-experimental_method +- O +derived O +FSC B-evidence +(' O +Part_FSC O +'). O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +of O +the O +80S B-complex_assembly +• I-complex_assembly +TSV I-complex_assembly +IRES I-complex_assembly +bound B-protein_state +with I-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +• I-complex_assembly +sordarin I-complex_assembly +. O + +( O +a O +) O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +. O +In O +all O +panels O +, O +the O +large B-structure_element +ribosomal I-structure_element +subunit I-structure_element +is O +shown O +in O +cyan O +; O +the O +small B-structure_element +subunit I-structure_element +in O +light O +yellow O +( O +head B-structure_element +) O +and O +wheat O +- O +yellow O +( O +body B-structure_element +); O +the O +TSV B-species +IRES B-site +in O +red O +, O +eEF2 B-protein +in O +green O +. O + +Nucleotides O +C1274 B-residue_name_number +, O +U1191 B-residue_name_number +of O +the O +40S B-complex_assembly +head B-structure_element +and O +G904 B-residue_name_number +of O +the O +platform B-site +( O +C1054 B-residue_name_number +, O +G966 B-residue_name_number +and O +G693 B-residue_name_number +in O +E B-species +. I-species +coli I-species +16S B-chemical +rRNA I-chemical +) O +are O +shown O +in O +black O +to O +denote O +the O +A B-site +, I-site +P I-site +and I-site +E I-site +sites I-site +, O +respectively O +. 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O +( O +4 O +) O +What O +, O +if O +any O +, O +is O +the O +mechanistic O +role O +of O +40S B-complex_assembly +head B-structure_element +rotation O +in O +IRES B-site +translocation O +? O + +We O +used O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +to O +visualize O +80S B-complex_assembly +• I-complex_assembly +TSV I-complex_assembly +IRES I-complex_assembly +complexes O +formed O +in O +the O +presence B-protein_state +of I-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +GTP I-complex_assembly +and O +the O +translation O +inhibitor O +sordarin B-chemical +, O +which O +stabilizes O +eEF2 B-protein +on O +the O +ribosome B-complex_assembly +. O + +Although O +the O +mechanism O +of O +sordarin B-chemical +action O +is O +not O +fully O +understood O +, O +the O +inhibitor O +does O +not O +affect O +the O +conformation O +of O +eEF2 B-complex_assembly +• I-complex_assembly +GDPNP I-complex_assembly +on O +the O +ribosome B-complex_assembly +, O +rendering O +it O +an O +excellent O +tool O +in O +translocation O +studies O +. O + +Maximum B-experimental_method +- I-experimental_method +likelihood I-experimental_method +classification I-experimental_method +using O +FREALIGN B-experimental_method +identified O +five O +IRES B-protein_state +- I-protein_state +eEF2 I-protein_state +- I-protein_state +bound I-protein_state +ribosome B-complex_assembly +structures B-evidence +within O +a O +single O +sample O +( O +Figures O +1 O +and O +2 O +). O + +The O +structures B-evidence +differ O +in O +the O +positions O +and O +conformations O +of O +ribosomal O +subunits O +( O +Figures O +1b O +and O +2 O +), O +IRES B-site +RNA B-chemical +( O +Figures O +3 O +and O +4 O +) O +and O +eEF2 B-protein +( O +Figures O +5 O +and O +6 O +). O + +This O +ensemble O +of O +structures B-evidence +allowed O +us O +to O +reconstruct O +a O +sequence O +of O +steps O +in O +IRES B-site +translocation O +induced O +by O +eEF2 B-protein +. O + +We O +used O +single B-experimental_method +- I-experimental_method +particle I-experimental_method +cryo I-experimental_method +- I-experimental_method +EM I-experimental_method +and O +maximum B-experimental_method +- I-experimental_method +likelihood I-experimental_method +image I-experimental_method +classification I-experimental_method +in O +FREALIGN B-experimental_method +to O +obtain O +three O +- O +dimensional O +density B-evidence +maps I-evidence +from O +a O +single O +specimen O +. O + +The O +translocation O +complex O +was O +formed O +using O +S B-species +. I-species +cerevisiae I-species +80S B-complex_assembly +ribosomes I-complex_assembly +, O +Taura B-species +syndrome I-species +virus I-species +IRES B-site +, O +and O +S B-species +. I-species +cerevisiae I-species +eEF2 B-protein +in O +the O +presence B-protein_state +of I-protein_state +GTP B-chemical +and O +the O +eEF2 B-protein +- O +binding O +translation O +inhibitor O +sordarin B-chemical +. 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O +2 O +Å O +, O +sufficient O +to O +locate O +IRES B-site +domains O +and O +to O +resolve O +individual O +residues O +in O +the O +core O +regions O +of O +the O +ribosome B-complex_assembly +and O +eEF2 B-protein +( O +Figures O +3c O +, O +d O +, O +and O +5f O +, O +h O +; O +see O +also O +Figure O +1 O +— O +figure O +supplement O +2 O +and O +Figure O +5 O +— O +figure O +supplement O +2 O +), O +including O +the O +post O +- O +translational O +modification O +diphthamide B-ptm +699 I-ptm +( O +Figure O +3c O +). O + +Large O +- O +scale O +rearrangements O +in O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +, O +coupled O +with O +the O +movement O +of O +PKI B-structure_element +from O +the O +A B-site +to I-site +P I-site +site I-site +and O +eEF2 B-protein +entry O +into O +the O +A B-site +site I-site +. 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O + +We O +numbered O +the O +structures B-evidence +from I-evidence +I I-evidence +to I-evidence +V I-evidence +, O +according O +to O +the O +position O +of O +the O +tRNA B-complex_assembly +- I-complex_assembly +mRNA I-complex_assembly +- O +like O +PKI B-structure_element +on O +the O +40S B-complex_assembly +subunit B-structure_element +( O +Figure O +2 O +— O +source O +data O +1 O +). O + +Specifically O +, O +PKI B-structure_element +is O +partially O +withdrawn O +from O +the O +A B-site +site I-site +in O +Structure B-evidence +I I-evidence +, O +and O +fully B-protein_state +translocated I-protein_state +to O +the O +P B-site +site I-site +in O +Structure B-evidence +V I-evidence +( O +Figure O +4 O +; O +see O +also O +Figure O +3 O +— O +figure O +supplement O +1 O +). 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O + +Structure B-evidence +I I-evidence +comprises O +the O +most B-protein_state +rotated I-protein_state +ribosome B-complex_assembly +conformation O +(~ O +10 O +°), O +characteristic O +of O +pre B-protein_state +- I-protein_state +translocation I-protein_state +hybrid B-protein_state +- I-protein_state +tRNA I-protein_state +states O +. O + +From O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +, O +the O +body B-structure_element +of O +the O +small B-structure_element +subunit I-structure_element +undergoes O +backward O +( O +reverse O +) O +rotation O +( O +Figure O +2b O +; O +see O +also O +Figure O +1 O +— O +figure O +supplement O +2 O +and O +Figure O +2 O +— O +figure O +supplement O +1 O +). O + +Structures B-evidence +II I-evidence +and I-evidence +III I-evidence +are O +in O +mid B-protein_state +- I-protein_state +rotation I-protein_state +conformations O +(~ O +5 O +°). O + +Structure B-evidence +IV I-evidence +adopts O +a O +slightly B-protein_state +rotated I-protein_state +conformation O +(~ O +1 O +°). O + +Structure B-evidence +V I-evidence +is O +in O +a O +nearly O +non B-protein_state +- I-protein_state +rotated I-protein_state +conformation O +( O +0 O +. O +5 O +°), O +very O +similar O +to O +that O +of O +post B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +complexes O +. O + +Thus O +, O +intersubunit O +rotation O +of O +~ O +9 O +° O +from O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +covers O +a O +nearly O +complete O +range O +of O +relative O +subunit B-structure_element +positions O +, O +similar O +to O +what O +was O +reported O +for O +tRNA B-protein_state +- I-protein_state +bound I-protein_state +yeast B-taxonomy_domain +, O +bacterial B-taxonomy_domain +and O +mammalian B-taxonomy_domain +ribosomes B-complex_assembly +. O + +40S B-complex_assembly +head B-structure_element +swivel O + +The O +pattern O +of O +40S B-complex_assembly +head B-structure_element +swivel O +between O +the O +structures B-evidence +is O +different O +from O +that O +of O +intersubunit O +rotation O +( O +Figures O +2c O +and O +d O +; O +see O +also O +Figure O +2 O +— O +source O +data O +1 O +). O + +As O +with O +the O +intersubunit O +rotation O +, O +the O +small O +head B-structure_element +swivel O +(~ O +1 O +°) O +in O +the O +non B-protein_state +- I-protein_state +rotated I-protein_state +Structure B-evidence +V I-evidence +is O +closest O +to O +that O +in O +the O +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +post B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +. O + +However O +in O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +intermediates O +( O +from O +Structure B-evidence +I I-evidence +to I-evidence +IV I-evidence +), O +the O +beak O +of O +the O +head B-structure_element +domain O +first O +turns O +toward O +the O +large B-structure_element +subunit I-structure_element +and O +then O +backs O +off O +( O +Figure O +2 O +— O +figure O +supplement O +1 O +). O + +The O +head B-structure_element +samples O +a O +mid B-protein_state +- I-protein_state +swiveled I-protein_state +position O +in O +Structure B-evidence +I I-evidence +( O +12 O +°), O +then O +a O +highly B-protein_state +- I-protein_state +swiveled I-protein_state +position O +in O +Structures B-evidence +II I-evidence +and I-evidence +III I-evidence +( O +17 O +°) O +and O +a O +less B-protein_state +swiveled I-protein_state +position O +in O +Structure B-evidence +IV I-evidence +( O +14 O +°). O + +The O +maximum O +head B-structure_element +swivel O +is O +observed O +in O +the O +mid B-protein_state +- I-protein_state +rotated I-protein_state +complexes O +II B-evidence +and I-evidence +III I-evidence +, O +in O +which O +PKI B-structure_element +transitions O +from O +the O +A B-site +to I-site +P I-site +site I-site +, O +while O +eEF2 B-protein +occupies O +the O +A B-site +site I-site +partially O +. O + +By O +comparison O +, O +the O +similarly O +mid B-protein_state +- I-protein_state +rotated I-protein_state +( O +4 O +°) O +80S B-complex_assembly +• I-complex_assembly +TSV I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +complex O +, O +in O +the O +absence B-protein_state +of I-protein_state +eEF2 B-protein +, O +adopts O +a O +mid B-protein_state +- I-protein_state +swiveled I-protein_state +position O +(~ O +10 O +°) O +( O +Figure O +2c O +). O + +These O +observations O +suggest O +that O +eEF2 B-protein +is O +necessary O +for O +inducing O +or O +stabilizing O +the O +large O +head B-structure_element +swivel O +of O +the O +40S B-complex_assembly +subunit B-structure_element +characteristic O +for O +IRES B-site +translocation O +intermediates O +. O + +IRES B-site +rearrangements O + +Comparison O +of O +the O +TSV B-species +IRES B-site +and O +eEF2 B-protein +positions O +in O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +. O + +( O +a O +) O +Positions O +of O +the O +IRES B-site +and O +eEF2 B-protein +in O +the O +initiation B-protein_state +, O +pre B-protein_state +- I-protein_state +translocation I-protein_state +( O +I B-evidence +) O +and O +post B-protein_state +- I-protein_state +translocation I-protein_state +( O +V B-evidence +) O +states O +, O +relative O +to O +the O +body B-structure_element +of O +the O +40S B-complex_assembly +subunit B-structure_element +( O +not O +shown O +) O +( O +b O +) O +Positions O +of O +the O +IRES B-site +and O +eEF2 B-protein +in O +the O +initiation B-protein_state +state O +( O +INIT B-complex_assembly +) O +and O +intermediate O +steps O +of O +translocation O +( O +II B-evidence +, I-evidence +III I-evidence +and I-evidence +IV I-evidence +), O +relative O +to O +the O +body B-structure_element +of O +the O +40S B-complex_assembly +subunit B-structure_element +( O +not O +shown O +). O + +Superpositions O +were O +obtained O +by O +structural B-experimental_method +alignments I-experimental_method +of O +the O +18S B-chemical +rRNAs I-chemical +excluding O +the O +head B-structure_element +domains O +( O +nt O +1150 B-residue_range +– I-residue_range +1620 I-residue_range +). O + +Positions O +of O +the O +IRES B-site +relative O +to O +proteins O +uS7 B-protein +, O +uS11 B-protein +and O +eS25 B-protein +. O + +( O +a O +) O +Intra O +- O +IRES B-site +rearrangements O +from O +the O +80S B-complex_assembly +* I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +structure B-evidence +( O +INIT B-complex_assembly +; O +PDB O +3J6Y O +,) O +to O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +. O +For O +each O +structure B-evidence +( O +shown O +in O +red O +), O +the O +conformation O +from O +a O +preceding O +structure B-evidence +is O +shown O +in O +light O +red O +for O +comparison O +. O + +Superpositions B-experimental_method +were O +obtained O +by O +structural B-experimental_method +alignments I-experimental_method +of O +18S B-chemical +rRNA I-chemical +. O + +( O +b O +) O +Positions O +of O +the O +IRES B-site +and O +eEF2 B-protein +relative O +to O +those O +of O +classical O +P B-site +- I-site +and I-site +E I-site +- I-site +site I-site +tRNAs B-chemical +in O +the O +80S B-complex_assembly +• I-complex_assembly +tRNA I-complex_assembly +complex O +. O +( O +c O +) O +Positions O +of O +the O +IRES B-site +relative O +to O +proteins O +uS11 B-protein +( O +40S B-site +platform I-site +) O +and O +uS7 B-protein +and O +eS25 B-protein +( O +40S B-complex_assembly +head B-structure_element +), O +which O +interact O +with O +the O +5 B-structure_element +′ I-structure_element +domain I-structure_element +of O +the O +IRES B-site +in O +the O +initiation B-protein_state +state O +( O +left O +panel O +). O + +In O +all O +panels O +, O +superpositions B-experimental_method +were O +obtained O +by O +structural B-experimental_method +alignments I-experimental_method +of O +the O +18S B-chemical +rRNAs I-chemical +. O + +Ribosomal O +proteins O +of O +the O +initiation B-protein_state +state O +are O +shown O +in O +gray O +for O +comparison O +. O + +Positions O +of O +the O +L1stalk B-structure_element +, O +tRNA B-chemical +and O +TSV B-species +IRES B-site +relative O +to O +proteins O +uS7 B-protein +and O +eS25 B-protein +, O +in O +80S B-complex_assembly +• I-complex_assembly +tRNA I-complex_assembly +structures B-evidence +and O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +structures B-evidence +I I-evidence +and I-evidence +V I-evidence +( O +this O +work O +). O + +The O +view O +shows O +the O +vicinity O +of O +the O +ribosomal O +E B-site +site I-site +. O + +Loop B-structure_element +1 I-structure_element +. I-structure_element +1 I-structure_element +and O +stem B-structure_element +loops I-structure_element +4 I-structure_element +and I-structure_element +5 I-structure_element +of O +the O +IRES B-site +are O +labeled O +. O + +Interactions O +of O +the O +stem B-structure_element +loops I-structure_element +4 I-structure_element +and I-structure_element +5 I-structure_element +of O +the O +TSV B-species +with O +proteins O +uS7 B-protein +and O +eS25 B-protein +. O + +Position O +and O +interactions O +of O +loop B-structure_element +3 I-structure_element +( O +variable B-structure_element +loop I-structure_element +region I-structure_element +) O +of O +the O +PKI B-structure_element +domain O +in O +Structure B-evidence +V I-evidence +( O +this O +work O +) O +resembles O +those O +of O +the O +anticodon B-structure_element +stem I-structure_element +loop I-structure_element +of O +the O +E B-site +- I-site +site I-site +tRNA B-chemical +( O +blue O +) O +in O +the O +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +. O + +Positions O +of O +tRNAs B-chemical +and O +the O +TSV B-species +IRES B-site +relative O +to O +the O +A B-structure_element +- I-structure_element +site I-structure_element +finger I-structure_element +( O +blue O +, O +nt O +1008 B-residue_range +– I-residue_range +1043 I-residue_range +of O +25S B-chemical +rRNA I-chemical +) O +and O +the O +P B-site +site I-site +of O +the O +large B-structure_element +subunit I-structure_element +, O +comprising O +helix B-structure_element +84 I-structure_element +of O +25S B-chemical +rRNA I-chemical +( O +nt O +. O + +2668 B-residue_range +– I-residue_range +2687 I-residue_range +) O +and O +protein O +uL5 B-protein +( O +collectively O +labeled O +as O +central B-structure_element +protuberance I-structure_element +, O +CP B-structure_element +, O +in O +the O +upper O +- O +row O +first O +figure O +, O +and O +individually O +labeled O +in O +the O +lower O +- O +row O +first O +figure O +). O + +Structures B-evidence +of O +translocation O +complexes O +of O +the O +bacterial B-taxonomy_domain +70S B-complex_assembly +ribosome I-complex_assembly +bound B-protein_state +with I-protein_state +two O +tRNAs B-chemical +and O +yeast B-taxonomy_domain +80S B-complex_assembly +complexes B-protein_state +with I-protein_state +tRNAs B-chemical +are O +shown O +in O +the O +upper O +row O +and O +labeled O +. O + +Structures B-evidence +of O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +complexes O +in O +the O +absence B-protein_state +of I-protein_state +eEF2 B-protein +( O +INIT B-complex_assembly +; O +PDB O +3J6Y O +,) O +and O +in O +the O +presence B-protein_state +of I-protein_state +eEF2 B-protein +( O +this O +work O +) O +are O +shown O +in O +the O +lower O +row O +and O +labeled O +. O + +Interactions O +of O +the O +TSV B-species +IRES B-site +with O +uL5 B-protein +and O +eL42 B-protein +. O + +Structures B-evidence +of O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +complexes O +in O +the O +absence B-protein_state +of I-protein_state +eEF2 B-protein +( O +INIT B-complex_assembly +; O +PDB O +3J6Y O +,) O +and O +in O +the O +presence B-protein_state +of I-protein_state +eEF2 B-protein +( O +this O +work O +) O +are O +shown O +in O +the O +upper O +row O +and O +labeled O +. O + +Structures B-evidence +of O +the O +80S B-complex_assembly +complexes B-protein_state +with I-protein_state +tRNAs B-chemical +are O +shown O +in O +the O +lower O +row O +in O +a O +view O +similar O +to O +that O +for O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +complex O +. O + +Positions O +of O +the O +IRES B-site +relative O +to O +eEF2 B-protein +and O +elements O +of O +the O +ribosome B-complex_assembly +in O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +. O + +( O +a O +) O +Secondary O +structure B-evidence +of O +the O +TSV B-species +IRES B-site +. O + +The O +TSV B-species +IRES B-site +comprises O +two O +domains O +: O +the O +5 B-structure_element +' I-structure_element +domain I-structure_element +( O +blue O +) O +and O +the O +PKI B-structure_element +domain O +( O +red O +). O + +The O +open B-structure_element +reading I-structure_element +frame I-structure_element +( O +gray O +) O +is O +immediately O +following O +pseudoknot B-structure_element +I I-structure_element +( O +PKI B-structure_element +). O + +( O +b O +) O +Three O +- O +dimensional O +structure B-evidence +of O +the O +TSV B-species +IRES B-site +( O +Structure B-evidence +II I-evidence +). O + +Pseudoknots O +and O +stem B-structure_element +loops I-structure_element +are O +labeled O +and O +colored O +as O +in O +( O +a O +). O + +( O +c O +) O +Positions O +of O +the O +IRES B-site +and O +eEF2 B-protein +on O +the O +small B-structure_element +subunit I-structure_element +in O +Structures B-evidence +I I-evidence +to I-evidence +V I-evidence +. O +The O +initiation B-protein_state +- O +state O +IRES B-site +is O +shown O +in O +gray O +. O + +The O +insert O +shows O +density O +for O +interaction O +of O +diphthamide O +699 O +( B-protein +eEF2 O +; O +green O +) O +with O +the O +codon O +- O +anticodon O +- O +like O +helix O +( B-structure_element +PKI O +; O +red O +) O +in O +Structure O +V O +. O +( O +d O +and O +e O +) O +Density O +of O +the O +P O +site O +in O +Structure O +V O +shows O +that O +interactions O +of O +PKI O +with O +the O +18S O +rRNA O +nucleotides O +( O +c O +) O +are O +nearly O +identical O +to O +those O +in O +the O +P O +site O +of O +the O +2tRNA O +• O +mRNA O +- O +bound O +70S O +ribosome O +( O +d O +). O + +In O +each O +structure B-evidence +, O +the O +TSV B-species +IRES B-site +adopts O +a O +distinct O +conformation O +in O +the O +intersubunit O +space O +of O +the O +ribosome B-complex_assembly +( O +Figures O +3 O +and O +4 O +). O + +The O +IRES B-site +( O +nt O +6758 B-residue_range +– I-residue_range +6952 I-residue_range +) O +consists O +of O +two O +globular O +parts O +( O +Figure O +3a O +): O +the O +5 B-structure_element +’- I-structure_element +region I-structure_element +( O +domains O +I B-structure_element +and O +II B-structure_element +, O +nt O +6758 B-residue_range +– I-residue_range +6888 I-residue_range +) O +and O +the O +PKI B-structure_element +domain O +( O +domain O +III B-structure_element +, O +nt O +6889 B-residue_range +– I-residue_range +6952 I-residue_range +). O + +We O +collectively O +term O +domains O +I B-structure_element +and O +II B-structure_element +the O +5 B-structure_element +’ I-structure_element +domain I-structure_element +. O + +The O +PKI B-structure_element +domain O +comprises O +PKI B-structure_element +and O +stem B-structure_element +loop I-structure_element +3 I-structure_element +( O +SL3 B-structure_element +), O +which O +stacks O +on O +top O +of O +the O +stem O +of O +PKI B-structure_element +. O + +The O +6953GCU O +triplet O +immediately O +following O +the O +PKI B-structure_element +domain O +is O +the O +first O +codon O +of O +the O +open B-structure_element +reading I-structure_element +frame I-structure_element +. O + +In O +the O +eEF2 B-protein_state +- I-protein_state +free I-protein_state +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +complex O +( O +INIT B-complex_assembly +), O +the O +bulk O +of O +the O +5 B-structure_element +’- I-structure_element +domain I-structure_element +( O +nt O +. O + +6758 B-residue_range +– I-residue_range +6888 I-residue_range +) O +binds O +near O +the O +E B-site +site I-site +, O +contacting O +the O +ribosome B-complex_assembly +mostly O +by O +means O +of O +three O +protruding O +structural O +elements O +: O +the O +L1 B-structure_element +. I-structure_element +1 I-structure_element +region I-structure_element +and O +stem B-structure_element +loops I-structure_element +4 I-structure_element +and I-structure_element +5 I-structure_element +( O +SL4 B-structure_element +and O +SL5 B-structure_element +). O + +In O +Structures B-evidence +I I-evidence +to I-evidence +IV I-evidence +, O +these O +contacts O +remain O +as O +in O +the O +initiation B-complex_assembly +complex I-complex_assembly +( O +Figure O +1a O +). O + +Specifically O +, O +the O +L1 B-structure_element +. I-structure_element +1 I-structure_element +region I-structure_element +interacts O +with O +the O +L1 B-structure_element +stalk I-structure_element +of O +the O +large B-structure_element +subunit I-structure_element +, O +while O +SL4 B-structure_element +and O +SL5 B-structure_element +bind O +at O +the O +side O +of O +the O +40S B-complex_assembly +head B-structure_element +and O +interact O +with O +proteins O +uS7 B-protein +, O +uS11 B-protein +and O +eS25 B-protein +( O +Figure O +3 O +— O +figure O +supplement O +2 O +and O +Figure O +3 O +— O +figure O +supplement O +3 O +; O +ribosomal O +proteins O +are O +termed O +according O +to O +). O + +In O +Structures B-evidence +I I-evidence +- I-evidence +IV I-evidence +, O +the O +minor B-site +groove I-site +of O +SL4 B-structure_element +( O +at O +nt O +6840 B-residue_range +– I-residue_range +6846 I-residue_range +) O +binds O +next O +to O +an O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +uS7 B-protein +, O +which O +is O +rich O +in O +positively O +charged O +residues O +( O +K212 B-residue_name_number +, O +K213 B-residue_name_number +, O +R219 B-residue_name_number +and O +K222 B-residue_name_number +). O + +The O +tip O +of O +SL4 B-structure_element +binds O +in O +the O +vicinity O +of O +R157 B-residue_name_number +in O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +of O +uS7 B-protein +and O +of O +Y58 B-residue_name_number +in O +uS11 B-protein +. O + +The O +minor B-site +groove I-site +of O +SL5 B-structure_element +( O +at O +nt O +6862 B-residue_range +– I-residue_range +6868 I-residue_range +) O +contacts O +the O +positively O +charged O +region O +of O +eS25 B-protein +( O +R49 B-residue_name_number +, O +R58 B-residue_name_number +and O +R68 B-residue_name_number +) O +( O +Figure O +3 O +— O +figure O +supplement O +4 O +). O + +In O +Structure B-evidence +V I-evidence +, O +however O +, O +the O +density B-evidence +for O +SL5 B-structure_element +is O +missing O +suggesting O +that O +SL5 B-structure_element +is O +mobile B-protein_state +, O +while O +weak O +SL4 B-structure_element +density B-evidence +suggests O +that O +SL4 B-structure_element +is O +shifted O +along O +the O +surface O +of O +uS7 B-protein +, O +~ O +20 O +Å O +away O +from O +its O +initial O +position O +( O +Figure O +3 O +— O +figure O +supplement O +2c O +). O + +The O +L1 B-structure_element +. I-structure_element +1 I-structure_element +region I-structure_element +remains O +in O +contact O +with O +the O +L1 B-structure_element +stalk I-structure_element +( O +Figure O +3 O +— O +figure O +supplement O +3 O +). O + +Inchworm B-protein_state +- O +like O +translocation O +of O +the O +TSV B-species +IRES B-site +. O + +Conformations O +and O +positions O +of O +the O +IRES B-site +in O +the O +initiation B-protein_state +state O +and O +in O +Structures B-evidence +I I-evidence +- I-evidence +V I-evidence +are O +shown O +relative O +to O +those O +of O +the O +A B-site +-, I-site +P I-site +- I-site +and I-site +E I-site +- I-site +site I-site +tRNAs B-chemical +. O + +The O +view O +was O +obtained O +by O +structural B-experimental_method +alignment I-experimental_method +of O +the O +body B-structure_element +domains O +of O +18S B-chemical +rRNAs I-chemical +of O +the O +corresponding O +80S B-complex_assembly +structures B-evidence +. O + +Distances O +between O +nucleotides O +6848 B-residue_number +and O +6913 B-residue_number +in O +SL4 B-structure_element +and O +PKI B-structure_element +, O +respectively O +, O +are O +shown O +( O +see O +also O +Figure O +2 O +— O +source O +data O +1 O +). O + +The O +shape O +of O +the O +IRES B-site +changes O +considerably O +from O +the O +initiation B-protein_state +state O +to O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +, O +from O +an O +extended B-protein_state +to O +compact B-protein_state +to O +extended B-protein_state +conformation O +( O +Figure O +4 O +; O +see O +also O +Figure O +3 O +— O +figure O +supplement O +2a O +). O + +Because O +in O +Structures B-evidence +I I-evidence +to I-evidence +IV I-evidence +the O +PKI B-structure_element +domain O +shifts O +toward O +the O +P B-site +site I-site +, O +while O +the O +5 O +’ O +remains O +unchanged O +near O +the O +E B-site +site I-site +, O +the O +distance O +between O +the O +domains O +shortens O +( O +Figure O +4 O +). O + +In O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +state O +, O +the O +A B-protein_state +- I-protein_state +site I-protein_state +- I-protein_state +bound I-protein_state +PKI B-structure_element +is O +separated O +from O +SL4 B-structure_element +by O +almost O +50 O +Å O +( O +Figure O +4 O +). O + +In O +Structures B-evidence +I I-evidence +and I-evidence +II I-evidence +, O +the O +PKI B-structure_element +is O +partially O +retracted O +from O +the O +A B-site +site I-site +and O +the O +distance O +from O +SL4 B-structure_element +shortens O +to O +~ O +35 O +Å O +. O +As O +PKI B-structure_element +moves O +toward O +the O +P B-site +site I-site +in O +Structures B-evidence +III I-evidence +and I-evidence +IV I-evidence +, O +the O +PKI B-structure_element +domain O +approaches O +to O +within O +~ O +25 O +Å O +of O +SL4 B-structure_element +. O + +Because O +the O +5 B-structure_element +’- I-structure_element +domain I-structure_element +in O +the O +following O +structure B-evidence +( I-evidence +V I-evidence +) I-evidence +moves O +by O +~ O +20 O +Å O +along O +the O +40S B-complex_assembly +head B-structure_element +, O +the O +IRES B-site +returns O +to O +an O +extended B-protein_state +conformation O +(~ O +45 O +Å O +) O +that O +is O +similar O +to O +that O +in O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +complex O +. O + +Rearrangements O +of O +the O +IRES B-site +involve O +restructuring O +of O +several O +interactions O +with O +the O +ribosome B-complex_assembly +. O + +In O +Structure B-evidence +I I-evidence +, O +SL3 B-structure_element +of O +the O +PKI B-structure_element +domain O +is O +positioned O +between O +the O +A B-structure_element +- I-structure_element +site I-structure_element +finger I-structure_element +( O +nt O +1008 B-residue_range +– I-residue_range +1043 I-residue_range +of O +25S B-chemical +rRNA I-chemical +) O +and O +the O +P B-site +site I-site +of O +the O +60S B-complex_assembly +subunit B-structure_element +, O +comprising O +helix B-structure_element +84 I-structure_element +of O +25S B-chemical +rRNA I-chemical +( O +nt O +. O + +2668 B-residue_range +– I-residue_range +2687 I-residue_range +) O +and O +protein O +uL5 B-protein +( O +Figure O +3 O +— O +figure O +supplement O +6 O +). O + +This O +position O +of O +SL3 B-structure_element +is O +~ O +25 O +Å O +away O +from O +that O +in O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +state O +, O +in O +which O +PKI B-structure_element +and O +SL3 B-structure_element +closely O +mimic O +the O +ASL B-structure_element +and O +elbow B-structure_element +of O +the O +A B-site +- I-site +site I-site +tRNA B-chemical +, O +respectively O +. O + +As O +such O +, O +the O +transition O +from O +the O +initiation B-protein_state +state O +to O +Structure B-evidence +I I-evidence +involves O +repositioning O +of O +SL3 B-structure_element +around O +the O +A B-structure_element +- I-structure_element +site I-structure_element +finger I-structure_element +, O +resembling O +the O +transition O +between O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +A B-site +/ I-site +P I-site +and O +A B-site +/ I-site +P I-site +* I-site +tRNA B-chemical +. O + +The O +second O +set O +of O +major O +structural O +changes O +involves O +interaction O +of O +the O +P B-site +site I-site +region I-site +of O +the O +large B-structure_element +subunit I-structure_element +with O +the O +hinge B-structure_element +point I-structure_element +of O +the O +IRES B-site +bending O +between O +the O +5 B-structure_element +´ I-structure_element +domain I-structure_element +and O +the O +PKI B-structure_element +domain O +( O +nt O +. O +6886 B-residue_range +– I-residue_range +6890 I-residue_range +). O + +In O +the O +highly B-protein_state +bent I-protein_state +Structures B-evidence +III I-evidence +and I-evidence +IV I-evidence +, O +the O +hinge B-structure_element +region I-structure_element +interacts O +with O +the O +universally B-protein_state +conserved I-protein_state +uL5 B-protein +and O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +tail I-structure_element +of O +eL42 B-protein +( O +Figure O +3 O +— O +figure O +supplement O +7 O +). O + +However O +, O +in O +the O +extended B-protein_state +conformations O +, O +these O +parts O +of O +the O +IRES B-site +and O +the O +60S B-complex_assembly +subunit B-structure_element +are O +separated O +by O +more O +than O +10 O +Å O +, O +suggesting O +that O +an O +interaction O +between O +them O +stabilizes O +the O +bent B-protein_state +conformations O +but O +not O +the O +extended B-protein_state +ones O +. O + +Another O +local O +rearrangement O +concerns O +loop B-structure_element +3 I-structure_element +, O +also O +known O +as O +the O +variable B-structure_element +loop I-structure_element +region I-structure_element +, O +which O +connects O +the O +ASL B-structure_element +- I-structure_element +and I-structure_element +mRNA I-structure_element +- I-structure_element +like I-structure_element +parts I-structure_element +of O +PKI B-structure_element +. O + +This O +loop B-structure_element +is O +poorly O +resolved O +in O +Structures B-evidence +I I-evidence +through I-evidence +IV I-evidence +, O +suggesting O +conformational O +flexibility O +in O +agreement O +with O +structural B-experimental_method +studies I-experimental_method +of O +the O +isolated B-protein_state +PKI B-structure_element +and O +biochemical B-experimental_method +studies I-experimental_method +of O +unbound B-protein_state +IRESs B-site +. O + +In O +Structure B-evidence +V I-evidence +, O +loop B-structure_element +3 I-structure_element +is O +bound B-protein_state +in I-protein_state +the O +40S B-complex_assembly +E B-site +site I-site +and O +the O +backbone O +of O +loop B-structure_element +3 I-structure_element +near O +the O +codon B-structure_element +- I-structure_element +like I-structure_element +part I-structure_element +of O +PKI B-structure_element +( O +at O +nt O +. O + +6945 B-residue_range +– I-residue_range +6946 I-residue_range +) O +interacts O +with O +R148 B-residue_name_number +and O +R157 B-residue_name_number +in O +β B-structure_element +- I-structure_element +hairpin I-structure_element +of O +uS7 B-protein +. O + +The O +interaction O +of O +loop B-structure_element +3 I-structure_element +backbone O +with O +uS7 B-protein +resembles O +that O +of O +the O +anticodon B-structure_element +- I-structure_element +stem I-structure_element +loop I-structure_element +of O +E B-site +- I-site +site I-site +tRNA B-chemical +in O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +structure B-evidence +( O +Figure O +3 O +— O +figure O +supplement O +5 O +). O + +Ordering O +of O +loop B-structure_element +3 I-structure_element +suggests O +that O +this O +flexible O +region O +contributes O +to O +the O +stabilization O +of O +the O +PKI B-structure_element +domain O +in O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +state O +. O + +This O +interpretation O +is O +consistent O +with O +the O +recent O +observation O +that O +alterations O +in O +loop B-structure_element +3 I-structure_element +of O +the O +CrPV B-species +IRES B-site +result O +in O +decreased O +efficiency O +of O +translocation O +. O + +eEF2 B-protein +structures B-evidence + +Elements O +of O +the O +80S B-complex_assembly +ribosome I-complex_assembly +that O +contact O +eEF2 B-protein +in O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +. O + +The O +view O +and O +colors O +are O +as O +in O +Figure O +5b O +: O +eEF2 B-protein +is O +shown O +in O +green O +, O +IRES B-site +RNA B-chemical +in O +red O +, O +40S B-complex_assembly +subunit B-structure_element +elements O +in O +orange O +, O +60S B-complex_assembly +in O +cyan O +/ O +teal O +. O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +density B-evidence +of O +the O +GTPase B-structure_element +region I-structure_element +in O +Structures B-evidence +I I-evidence +and I-evidence +II I-evidence +. O + +The O +switch B-structure_element +loop I-structure_element +I I-structure_element +in O +Structure B-evidence +I I-evidence +is O +shown O +in O +blue O +. O + +The O +putative O +position O +of O +the O +switch B-structure_element +loop I-structure_element +I I-structure_element +, O +unresolved O +in O +the O +density B-evidence +of O +Structure B-evidence +II I-evidence +, O +is O +shown O +with O +a O +dashed O +line O +. O + +Colors O +for O +the O +ribosome B-complex_assembly +and O +eEF2 B-protein +are O +as O +in O +Figure O +1 O +. O + +Conformations O +and O +interactions O +of O +eEF2 B-protein +. O + +( O +a O +) O +Conformations O +of O +eEF2 B-protein +in O +Structures B-evidence +I I-evidence +- I-evidence +V I-evidence +and O +domain O +organization O +of O +eEF2 B-protein +are O +shown O +. O + +Roman O +numerals O +denote O +eEF2 B-protein +domains O +. O + +Superposition B-experimental_method +was O +obtained O +by O +structural B-experimental_method +alignment I-experimental_method +of O +domains O +I B-structure_element +and O +II B-structure_element +. O + +( O +b O +) O +Elements O +of O +the O +80S B-complex_assembly +ribosome I-complex_assembly +in O +Structures B-evidence +I I-evidence +and I-evidence +V I-evidence +that O +contact O +eEF2 B-protein +. O + +eEF2 B-protein +is O +shown O +in O +green O +, O +IRES B-site +RNA B-chemical +in O +red O +, O +40S B-complex_assembly +subunit B-structure_element +elements O +in O +orange O +, O +60S B-complex_assembly +in O +cyan O +/ O +teal O +. O + +( O +c O +) O +Comparison O +of O +conformations O +of O +eEF2 B-complex_assembly +• I-complex_assembly +sordarin I-complex_assembly +in O +Structure B-evidence +I I-evidence +( O +light O +green O +) O +with O +those O +of O +free B-protein_state +apo B-protein_state +- O +eEF2 B-protein +( O +magenta O +) O +and O +eEF2 B-complex_assembly +• I-complex_assembly +sordarin I-complex_assembly +( O +teal O +). O + +( O +d O +) O +Interactions O +of O +the O +GTPase B-structure_element +domains I-structure_element +with O +the O +40S B-complex_assembly +and O +60S B-complex_assembly +subunits B-structure_element +in O +Structure B-evidence +I I-evidence +( O +colored O +in O +green O +/ O +blue O +, O +eEF2 B-protein +; O +orange O +, O +40S B-complex_assembly +; O +cyan O +/ O +teal O +, O +60S B-complex_assembly +) O +and O +in O +Structure B-evidence +II I-evidence +( O +gray O +). O + +Switch B-structure_element +loop I-structure_element +I I-structure_element +( O +SWI B-structure_element +) O +in O +Structure B-evidence +I I-evidence +is O +in O +blue O +; O +dashed O +line O +shows O +the O +putative O +location O +of O +unresolved O +switch B-structure_element +loop I-structure_element +I I-structure_element +in O +Structure B-evidence +II I-evidence +. O + +Superposition B-experimental_method +was O +obtained O +by O +structural B-experimental_method +alignment I-experimental_method +of O +the O +25S B-chemical +rRNAs I-chemical +. O + +( O +e O +) O +Comparison O +of O +the O +GTP B-protein_state +- I-protein_state +like I-protein_state +conformation O +of O +eEF2 B-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +in O +Structure B-evidence +I I-evidence +( O +light O +green O +) O +with O +those O +of O +70S B-protein_state +- I-protein_state +bound I-protein_state +elongation B-protein_type +factors I-protein_type +EF B-complex_assembly +- I-complex_assembly +Tu I-complex_assembly +• I-complex_assembly +GDPCP I-complex_assembly +( O +teal O +) O +and O +EF B-complex_assembly +- I-complex_assembly +G I-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +• I-complex_assembly +fusidic I-complex_assembly +acid I-complex_assembly +( O +magenta O +; O +fusidic O +acid O +not O +shown O +). O +( O +f O +) O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +density B-evidence +showing O +guanosine B-chemical +diphosphate I-chemical +bound B-protein_state +in I-protein_state +the O +GTPase B-site +center I-site +( O +green O +) O +next O +to O +the O +sarcin B-structure_element +- I-structure_element +ricin I-structure_element +loop I-structure_element +of O +25S B-chemical +rRNA I-chemical +( O +cyan O +) O +of O +Structure B-evidence +II I-evidence +. O +( O +g O +) O +Comparison O +of O +the O +sordarin B-site +- I-site +binding I-site +sites I-site +in O +the O +ribosome B-protein_state +- I-protein_state +bound I-protein_state +( O +light O +green O +; O +Structure B-evidence +II I-evidence +) O +and O +isolated O +eEF2 B-protein +( O +teal O +). O + +( O +h O +) O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +density B-evidence +showing O +the O +sordarin B-site +- I-site +binding I-site +pocket I-site +of O +eEF2 B-protein +( O +Structure B-evidence +II I-evidence +). O + +Sordarin B-chemical +is O +shown O +in O +pink O +with O +oxygen O +atoms O +in O +red O +. O + +Elongation B-protein_type +factor I-protein_type +eEF2 B-protein +in O +all O +five O +structures B-evidence +is O +bound B-protein_state +with I-protein_state +GDP B-chemical +and O +sordarin B-chemical +( O +Figure O +5 O +). O + +The O +elongation B-protein_type +factor I-protein_type +consists O +of O +three O +dynamic O +superdomains B-structure_element +: O +an O +N O +- O +terminal O +globular O +superdomain B-structure_element +formed O +by O +the O +G B-structure_element +( I-structure_element +GTPase I-structure_element +) I-structure_element +domain I-structure_element +( O +domain O +I B-structure_element +) O +and O +domain O +II B-structure_element +; O +a O +linker B-structure_element +domain I-structure_element +III I-structure_element +; O +and O +a O +C O +- O +terminal O +superdomain B-structure_element +comprising O +domains O +IV B-structure_element +and O +V B-structure_element +( O +Figure O +5a O +). O + +Domain O +IV B-structure_element +extends O +from O +the O +main O +body B-structure_element +and O +is O +critical O +for O +translocation O +catalyzed O +by O +eEF2 B-protein +or O +EF B-protein +- I-protein +G I-protein +. O +ADP B-ptm +- I-ptm +ribosylation I-ptm +of O +eEF2 B-protein +at O +the O +tip O +of O +domain O +IV B-structure_element +or O +deletion B-experimental_method +of O +domain O +IV B-structure_element +from O +EF B-protein +- I-protein +G I-protein +abrogate O +translocation O +. O + +In O +post B-protein_state +- I-protein_state +translocation I-protein_state +- O +like O +80S B-complex_assembly +• I-complex_assembly +tRNA I-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +complexes O +, O +domain O +IV B-structure_element +binds O +in O +the O +40S B-complex_assembly +A B-site +site I-site +, O +suggesting O +direct O +involvement O +of O +domain O +IV B-structure_element +in O +translocation O +of O +tRNA B-chemical +from O +the O +A B-site +to I-site +P I-site +site I-site +. O + +GDP B-chemical +in O +our O +structures B-evidence +is O +bound B-protein_state +in I-protein_state +the O +GTPase B-site +center I-site +( O +Figures O +5d O +, O +e O +and O +f O +) O +and O +sordarin B-chemical +is O +sandwiched O +between O +the O +β B-structure_element +- I-structure_element +platforms I-structure_element +of O +domains O +III B-structure_element +and O +V B-structure_element +( O +Figures O +5g O +and O +h O +), O +as O +in O +the O +structure B-evidence +of O +free B-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +sordarin I-complex_assembly +complex O +. O + +The O +global O +conformations O +of O +eEF2 B-protein +( O +Figure O +5a O +) O +are O +similar O +in O +these O +structures B-evidence +( O +all O +- O +atom O +RMSD B-evidence +≤ O +2 O +Å O +), O +but O +the O +positions O +of O +eEF2 B-protein +relative O +to O +the O +40S B-complex_assembly +subunit B-structure_element +differ O +substantially O +as O +a O +result O +of O +40S B-complex_assembly +subunit B-structure_element +rotation O +( O +Figure O +2 O +— O +source O +data O +1 O +). O + +From O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +, O +eEF2 B-protein +is O +rigidly O +attached O +to O +the O +GTPase B-site +- I-site +associated I-site +center I-site +of O +the O +60S B-complex_assembly +subunit B-structure_element +. O + +The O +GTPase B-site +- I-site +associated I-site +center I-site +comprises O +the O +P B-structure_element +stalk I-structure_element +( O +L11 B-structure_element +and O +L7 B-structure_element +/ O +L12 B-structure_element +stalk B-structure_element +in O +bacteria B-taxonomy_domain +) O +and O +the O +sarcin B-structure_element +- I-structure_element +ricin I-structure_element +loop I-structure_element +( O +SRL B-structure_element +, O +nt O +3012 B-residue_range +– I-residue_range +3042 I-residue_range +). O + +The O +tips O +of O +25S B-chemical +rRNA I-chemical +helices B-structure_element +43 I-structure_element +and I-structure_element +44 I-structure_element +of O +the O +P B-structure_element +stalk I-structure_element +( O +nucleotides O +G1242 B-residue_name_number +and O +A1270 B-residue_name_number +, O +respectively O +) O +stack B-bond_interaction +on O +V754 B-residue_name_number +and O +Y744 B-residue_name_number +of O +domain O +V B-structure_element +. O +An O +αββ B-structure_element +motif I-structure_element +of O +the O +eukaryote B-taxonomy_domain +- O +specific O +protein O +P0 B-protein +( O +aa O +126 B-residue_range +– I-residue_range +154 I-residue_range +) O +packs O +in O +the O +crevice O +between O +the O +long B-structure_element +α I-structure_element +- I-structure_element +helix I-structure_element +D I-structure_element +( O +aa O +172 B-residue_range +– I-residue_range +188 I-residue_range +) O +of O +the O +GTPase B-structure_element +domain I-structure_element +and O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +region I-structure_element +( O +aa O +246 B-residue_range +– I-residue_range +263 I-residue_range +) O +of O +the O +GTPase B-structure_element +domain I-structure_element +insert I-structure_element +( O +or O +G B-structure_element +’ I-structure_element +insert I-structure_element +) O +of O +eEF2 B-protein +( O +secondary O +- O +structure O +nomenclatures O +for O +eEF2 B-protein +and O +EF B-protein +- I-protein +G I-protein +are O +the O +same O +). O + +Although O +the O +P B-structure_element +/ I-structure_element +L11 I-structure_element +stalk I-structure_element +is O +known O +to O +be O +dynamic O +, O +its O +position O +remains O +unchanged O +from O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +: O +all O +- O +atom O +root B-evidence +- I-evidence +mean I-evidence +- I-evidence +square I-evidence +differences I-evidence +for O +the O +25S B-chemical +rRNA I-chemical +of O +the O +P B-structure_element +stalk I-structure_element +( O +nt O +1223 B-residue_range +– I-residue_range +1286 I-residue_range +) O +are O +within O +2 O +. O +5 O +Å O +. O +However O +, O +with O +respect O +to O +its O +position O +in O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +complex O +in O +the O +absence B-protein_state +of I-protein_state +eEF2 B-protein +and O +in O +the O +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +, O +the O +P B-structure_element +stalk I-structure_element +is O +shifted O +by O +~ O +13 O +Å O +toward O +the O +A B-site +site I-site +( O +Figure O +2d O +). O + +The O +sarcin B-structure_element +- I-structure_element +ricin I-structure_element +loop I-structure_element +interacts O +with O +the O +GTP B-site +- I-site +binding I-site +site I-site +of O +eEF2 B-protein +( O +Figures O +5d O +and O +f O +). O + +While O +the O +overall O +mode O +of O +this O +interaction O +is O +similar O +to O +that O +seen O +in O +70S B-complex_assembly +• I-complex_assembly +EF I-complex_assembly +- I-complex_assembly +G I-complex_assembly +crystal B-evidence +structures I-evidence +, O +there O +is O +an O +important O +local O +difference O +between O +Structure B-evidence +I I-evidence +and O +Structures B-evidence +II I-evidence +- I-evidence +V I-evidence +in O +switch B-structure_element +loop I-structure_element +I I-structure_element +, O +as O +discussed O +below O +. O + +Repositioning O +( O +sliding O +) O +of O +the O +positively B-site +- I-site +charged I-site +cluster I-site +of O +domain O +IV B-structure_element +of O +eEF2 B-protein +over O +the O +phosphate O +backbone O +( O +red O +) O +of O +the O +18S B-structure_element +helices I-structure_element +33 I-structure_element +and I-structure_element +34 I-structure_element +. O + +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +are O +shown O +. O + +Electrostatic O +surface O +of O +eEF2 B-protein +is O +shown O +; O +negatively O +and O +positively O +charged O +regions O +are O +shown O +in O +red O +and O +blue O +, O +respectively O +. O + +The O +view O +was O +obtained O +by O +structural B-experimental_method +alignment I-experimental_method +of O +the O +18S B-chemical +rRNAs I-chemical +. O + +Interactions O +of O +eEF2 B-protein +with O +the O +40S B-complex_assembly +subunit B-structure_element +. O + +( O +a O +) O +eEF2 B-protein +( O +green O +) O +interacts O +only O +with O +the O +body B-structure_element +in O +Structure B-evidence +I I-evidence +( O +eEF2 B-protein +domains O +are O +labeled O +with O +roman O +numerals O +in O +white O +), O +and O +with O +both O +the O +head B-structure_element +and O +body B-structure_element +in O +Structures B-evidence +II I-evidence +through I-evidence +V I-evidence +. O +Colors O +are O +as O +in O +Figure O +1 O +, O +except O +for O +the O +40S B-complex_assembly +structural O +elements O +that O +contact O +eEF2 B-protein +, O +which O +are O +labeled O +and O +shown O +in O +purple O +. O +( O +b O +) O +Entry O +of O +eEF2 B-protein +into O +the O +40S B-complex_assembly +A B-site +site I-site +, O +from O +Structure B-evidence +I I-evidence +through I-evidence +V I-evidence +. O +Distances O +to O +the O +A B-site +- I-site +site I-site +accommodated O +eEF2 B-protein +( O +Structure B-evidence +V I-evidence +) O +are O +shown O +. O + +The O +view O +was O +obtained O +by O +superpositions B-experimental_method +of O +the O +body B-structure_element +domains O +of O +18S B-chemical +rRNAs I-chemical +. O + +( O +c O +) O +Rearrangements O +, O +from O +Structure B-evidence +I I-evidence +through I-evidence +V I-evidence +, O +of O +a O +positively O +charged O +cluster O +of O +eEF2 B-protein +( O +K613 B-residue_name_number +, O +R617 B-residue_name_number +and O +R631 B-residue_name_number +) O +positioned O +over O +the O +phosphate O +backbone O +of O +18S B-structure_element +helices I-structure_element +33 I-structure_element +and I-structure_element +34 I-structure_element +, O +suggesting O +a O +role O +of O +electrostatic B-bond_interaction +interactions I-bond_interaction +in O +eEF2 B-protein +diffusion O +over O +the O +40S B-complex_assembly +surface O +. O + +( O +d O +) O +Shift O +of O +the O +tip O +of O +domain O +III B-structure_element +of O +eEF2 B-protein +, O +interacting O +with O +uS12 B-protein +upon O +reverse O +subunit B-structure_element +rotation O +from O +Structure B-evidence +I I-evidence +to I-evidence +Structure I-evidence +V I-evidence +. O +Structure B-evidence +I I-evidence +colored O +as O +in O +Figure O +1 O +, O +except O +uS12 B-protein +, O +which O +is O +in O +purple O +; O +Structure B-evidence +V I-evidence +is O +in O +gray O +. O + +There O +are O +two O +modest O +but O +noticeable O +domain O +rearrangements O +between O +Structures B-evidence +I I-evidence +and I-evidence +V I-evidence +. O +Unlike O +in O +free B-protein_state +eEF2 B-protein +, O +which O +can O +sample O +large O +movements O +of O +at O +least O +50 O +Å O +of O +the O +C O +- O +terminal O +superdomain B-structure_element +relative O +to O +the O +N O +- O +terminal O +superdomain B-structure_element +( O +Figure O +5c O +), O +eEF2 B-protein +undergoes O +moderate O +repositioning O +of O +domain O +IV B-structure_element +(~ O +3 O +Å O +; O +Figure O +5a O +) O +and O +domain O +III B-structure_element +(~ O +5 O +Å O +; O +Figure O +6d O +). O + +This O +limited O +flexibility O +of O +the O +ribosome B-protein_state +- I-protein_state +bound I-protein_state +eEF2 B-protein +is O +likely O +the O +result O +of O +simultaneous O +fixation O +of O +eEF2 B-protein +superdomains B-structure_element +, O +via O +domains O +I B-structure_element +and O +V B-structure_element +, O +by O +the O +GTPase B-site +- I-site +associated I-site +center I-site +of O +the O +large B-structure_element +subunit I-structure_element +. O + +Domain O +IV B-structure_element +of O +eEF2 B-protein +binds O +at O +the O +40S B-complex_assembly +A B-site +site I-site +in O +Structures B-evidence +I I-evidence +to I-evidence +V I-evidence +but O +the O +mode O +of O +interaction O +differs O +in O +each O +complex O +( O +Figure O +6 O +). O + +Because O +eEF2 B-protein +is O +rigidly O +attached O +to O +the O +60S B-complex_assembly +subunit B-structure_element +and O +does O +not O +undergo O +large O +inter O +- O +subunit B-structure_element +rearrangements O +, O +gradual O +entry O +of O +domain O +IV B-structure_element +into O +the O +A B-site +site I-site +between O +Structures B-evidence +I I-evidence +and I-evidence +V I-evidence +is O +due O +to O +40S B-complex_assembly +subunit B-structure_element +rotation O +and O +head B-structure_element +swivel O +. O + +eEF2 B-protein +settles O +into O +the O +A B-site +site I-site +from O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +, O +as O +the O +tip O +of O +domain O +IV B-structure_element +shifts O +by O +~ O +10 O +Å O +relative O +to O +the O +body B-structure_element +and O +by O +~ O +20 O +Å O +relative O +to O +the O +swiveling O +head B-structure_element +. O + +Modest O +intra O +- O +eEF2 B-protein +shifts O +of O +domain O +IV B-structure_element +between O +Structures B-evidence +I I-evidence +to I-evidence +V I-evidence +outline O +a O +stochastic O +trajectory O +( O +Figure O +5a O +), O +consistent O +with O +local O +adjustments O +of O +the O +domain O +in O +the O +A B-site +site I-site +. O + +At O +the O +central O +region O +of O +eEF2 B-protein +, O +domains O +II B-structure_element +and O +III B-structure_element +contact O +the O +40S B-complex_assembly +body B-structure_element +( O +mainly O +at O +nucleotides O +48 B-residue_range +– I-residue_range +52 I-residue_range +and O +429 B-residue_range +– I-residue_range +432 I-residue_range +of O +18S B-chemical +rRNA I-chemical +helix B-structure_element +5 I-structure_element +and O +uS12 B-protein +). O + +From O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +, O +these O +central O +domains O +migrate O +by O +~ O +10 O +Å O +along O +the O +40S B-complex_assembly +surface O +( O +Figure O +6c O +). O + +Comparison O +of O +eEF2 B-protein +conformations O +reveals O +that O +in O +Structure B-evidence +V I-evidence +, O +domain O +III B-structure_element +is O +displaced O +as O +a O +result O +of O +interaction O +with O +uS12 B-protein +, O +as O +discussed O +below O +. O + +In O +summary O +, O +between O +Structures B-evidence +I I-evidence +and I-evidence +V I-evidence +, O +a O +step O +- O +wise O +translocation O +of O +PKI B-structure_element +by O +~ O +15 O +Å O +from O +the O +A B-site +to I-site +P I-site +site I-site +- O +within O +the O +40S B-complex_assembly +subunit B-structure_element +– O +occurs O +simultaneously O +with O +the O +~ O +11 O +Å O +side O +- O +way O +entry O +of O +domain O +IV B-structure_element +into O +the O +A B-site +site I-site +coupled O +with O +~ O +3 O +to O +5 O +Å O +inter O +- O +domain O +rearrangements O +in O +eEF2 B-protein +. O + +These O +shifts O +occur O +during O +the O +reverse O +rotation O +of O +the O +40S B-complex_assembly +body B-structure_element +coupled O +with O +the O +forward O +- O +then O +- O +reverse O +head B-structure_element +swivel O +. O + +To O +elucidate O +the O +detailed O +structural O +mechanism O +of O +IRES B-site +translocation O +and O +the O +roles O +of O +eEF2 B-protein +and O +ribosome B-complex_assembly +rearrangements O +, O +we O +describe O +in O +the O +following O +sections O +the O +interactions O +of O +PKI B-structure_element +and O +eEF2 B-protein +with O +the O +ribosomal O +A B-site +and I-site +P I-site +sites I-site +in O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +( O +Figure O +2g O +; O +see O +also O +Figure O +1 O +— O +figure O +supplement O +1 O +). O + +Structure B-evidence +I I-evidence +represents O +a O +pre B-protein_state +- I-protein_state +translocation I-protein_state +IRES B-site +and O +initial O +entry O +of O +eEF2 B-protein +in O +a O +GTP B-chemical +- O +like O +state O + +In O +the O +fully B-protein_state +rotated I-protein_state +Structure B-evidence +I I-evidence +, O +PKI B-structure_element +is O +shifted O +toward O +the O +P B-site +site I-site +by O +~ O +3 O +Å O +relative O +to O +its O +position O +in O +the O +initiation B-complex_assembly +complex I-complex_assembly +but O +maintains O +interactions O +with O +the O +partially B-protein_state +swiveled I-protein_state +head B-structure_element +. O + +At O +the O +head B-structure_element +, O +C1274 B-residue_name_number +of O +the O +18S B-chemical +rRNA I-chemical +( O +C1054 B-residue_name_number +in O +E B-species +. I-species +coli I-species +) O +base O +pairs O +with O +the O +first O +nucleotide O +of O +the O +ORF B-structure_element +immediately O +downstream O +of O +PKI B-structure_element +. O + +The O +C1274 B-residue_name_number +: O +G6953 B-residue_name_number +base O +pair O +provides O +a O +stacking B-site +platform I-site +for O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +– I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +. O + +We O +therefore O +define O +C1274 B-residue_name_number +as O +the O +foundation O +of O +the O +' O +head B-structure_element +A B-site +site I-site +'. O + +Accordingly O +, O +we O +use O +U1191 B-residue_name_number +( O +G966 B-residue_name_number +in O +E B-species +. I-species +coli I-species +) O +and O +C1637 B-residue_name_number +( O +C1400 B-residue_name_number +in O +E B-species +. I-species +coli I-species +) O +as O +the O +reference O +points O +of O +the O +' O +head B-structure_element +P B-site +site I-site +' O +and O +' O +body B-structure_element +P B-site +site I-site +' O +( O +Figure O +2g O +), O +respectively O +, O +because O +these O +nucleotides O +form O +a O +stacking O +foundation O +for O +the O +fully B-protein_state +translocated I-protein_state +mRNA B-structure_element +- I-structure_element +tRNA I-structure_element +helix I-structure_element +in O +tRNA B-protein_state +- I-protein_state +bound I-protein_state +structures B-evidence +and O +in O +our O +post B-protein_state +- I-protein_state +translocation I-protein_state +Structure B-evidence +V I-evidence +discussed O +below O +. O + +Interactions O +of O +the O +residues O +at O +the O +eEF2 B-protein +tip O +with O +the O +decoding B-site +center I-site +of O +the O +IRES B-protein_state +- I-protein_state +bound I-protein_state +ribosome B-complex_assembly +. O + +Key O +elements O +of O +the O +decoding B-site +center I-site +of O +the O +' O +locked B-protein_state +' O +initiation B-protein_state +structure B-evidence +, O +' O +unlocked B-protein_state +' O +Structure B-evidence +I I-evidence +, O +and O +post B-protein_state +- I-protein_state +translocation I-protein_state +Structure B-evidence +V I-evidence +( O +this O +work O +) O +are O +shown O +. O + +The O +histidine B-site +- I-site +diphthamide I-site +tip I-site +of O +eEF2 B-protein +is O +shown O +in O +green O +. O + +The O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +- I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +is O +shown O +in O +red O +, O +the O +downstream O +first O +codon O +of O +the O +ORF B-structure_element +in O +magenta O +. O + +Nucleotides O +of O +the O +18S B-chemical +rRNA I-chemical +body B-structure_element +are O +in O +orange O +and O +head B-structure_element +in O +yellow O +; O +25S B-chemical +rRNA I-chemical +nucleotide O +A2256 B-residue_name_number +is O +blue O +. O + +A B-site +and I-site +P I-site +sites I-site +are O +schematically O +demarcated O +by O +dotted O +lines O +. O + +The O +interaction O +of O +PKI B-structure_element +with O +the O +40S B-complex_assembly +body B-structure_element +is O +substantially O +rearranged O +relative O +to O +that O +in O +the O +initiation B-protein_state +state O +. O + +In O +the O +latter O +, O +PKI B-structure_element +is O +stabilized O +by O +interactions O +with O +the O +universally B-protein_state +conserved I-protein_state +decoding B-site +- I-site +center I-site +nucleotides O +G577 B-residue_name_number +, O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +(' O +body B-structure_element +A B-site +site I-site +'), O +as O +in O +the O +A B-site +- I-site +site I-site +tRNA B-protein_state +bound I-protein_state +complexes O +. O + +In O +Structure B-evidence +I I-evidence +, O +PKI B-structure_element +does O +not O +contact O +these O +nucleotides O +( O +Figures O +2g O +and O +7 O +). O + +The O +position O +of O +eEF2 B-protein +on O +the O +40S B-complex_assembly +subunit B-structure_element +of O +Structure B-evidence +I I-evidence +is O +markedly O +distinct O +from O +those O +in O +Structures B-evidence +II I-evidence +to I-evidence +V I-evidence +. O +The O +translocase B-protein_type +interacts O +with O +the O +40S B-complex_assembly +body B-structure_element +but O +does O +not O +contact O +the O +head B-structure_element +( O +Figures O +5b O +and O +6a O +; O +Figure O +5 O +— O +figure O +supplement O +1 O +). O + +Domain O +IV B-structure_element +is O +partially O +engaged O +with O +the O +body B-structure_element +A B-site +site I-site +. O + +The O +tip O +of O +domain O +IV B-structure_element +is O +wedged O +between O +PKI B-structure_element +and O +decoding B-site +- I-site +center I-site +nucleotides O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +, O +which O +are O +bulged O +out O +of O +h44 O +. O + +This O +tip O +contains O +the O +histidine B-site +- I-site +diphthamide I-site +triad I-site +( O +H583 B-residue_name_number +, O +H694 B-residue_name_number +and O +Diph699 B-ptm +), O +which O +interacts O +with O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +- I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +and O +A1756 B-residue_name_number +( O +Figure O +7 O +). O + +Histidines B-residue_name_number +583 I-residue_name_number +and I-residue_name_number +694 I-residue_name_number +interact O +with O +the O +phosphate O +backbone O +of O +the O +anticodon B-structure_element +- I-structure_element +like I-structure_element +strand I-structure_element +( O +at O +G6907 B-residue_name_number +and O +C6908 B-residue_name_number +). O + +Diphthamide B-ptm +is O +a O +unique O +posttranslational O +modification O +conserved B-protein_state +in O +archaeal B-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +EF2 B-protein +( O +at O +residue O +699 B-residue_number +in O +S B-species +. I-species +cerevisiae I-species +) O +and O +involves O +addition O +of O +a O +~ O +7 O +- O +Å O +long O +3 O +- O +carboxyamido O +- O +3 O +-( O +trimethylamino O +)- O +propyl O +moiety O +to O +the O +histidine B-residue_name +imidazole O +ring O +at O +CE1 O +. O + +The O +trimethylamino O +end O +of O +Diph699 B-ptm +packs O +over O +A1756 B-residue_name_number +( O +Figure O +7 O +). O + +The O +opposite O +surface O +of O +the O +tail O +is O +oriented O +toward O +the O +minor B-site +- I-site +groove I-site +side O +of O +the O +second O +base O +pair O +of O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +helix I-structure_element +( O +G6906 B-residue_name_number +: O +C6951 B-residue_name_number +). O + +Thus O +, O +in O +comparison O +with O +the O +initiation B-protein_state +state O +, O +the O +histidine B-site +- I-site +diphthamide I-site +tip I-site +of O +eEF2 B-protein +replaces O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +– I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +. O + +The O +splitting O +of O +the O +interaction O +of O +A1755 B-residue_name_number +- O +A1756 B-residue_name_number +and O +PKI B-structure_element +is O +achieved O +by O +providing O +the O +histidine B-site +- I-site +diphthamine I-site +tip I-site +as O +a O +binding O +partner O +for O +both O +A1756 B-residue_name_number +and O +the O +minor B-site +groove I-site +of O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +helix I-structure_element +( O +Figure O +7 O +). O + +Unlike O +in O +Structures B-evidence +II I-evidence +to I-evidence +V I-evidence +, O +the O +conformation O +of O +the O +eEF2 B-protein +GTPase B-site +center I-site +in O +Structure B-evidence +I I-evidence +resembles O +that O +of O +a O +GTP B-protein_state +- I-protein_state +bound I-protein_state +translocase B-protein_type +( O +Figure O +5e O +). O + +In O +translational B-protein_type +GTPases I-protein_type +, O +switch B-structure_element +loops I-structure_element +I I-structure_element +and I-structure_element +II I-structure_element +are O +involved O +in O +the O +GTPase B-protein_type +activity O +( O +reviewed O +in O +). O + +Switch B-structure_element +loop I-structure_element +II I-structure_element +( O +aa O +105 B-residue_range +– I-residue_range +110 I-residue_range +), O +which O +carries O +the O +catalytic B-protein_state +H108 B-residue_name_number +( O +H92 B-residue_name_number +in O +E B-species +. I-species +coli I-species +EF B-protein +- I-protein +G I-protein +; O +is O +well O +resolved O +in O +all O +five O +structures B-evidence +. O + +The O +histidine B-residue_name +resides O +next O +to O +the O +backbone O +of O +G3028 B-residue_name_number +of O +the O +sarcin B-structure_element +- I-structure_element +ricin I-structure_element +loop I-structure_element +and O +near O +the O +diphosphate O +of O +GDP B-chemical +( O +Figure O +5e O +). O + +By O +contrast O +, O +switch B-structure_element +loop I-structure_element +I I-structure_element +( O +aa O +50 B-residue_range +– I-residue_range +70 I-residue_range +in O +S B-species +. I-species +cerevisiae I-species +eEF2 B-protein +) O +is O +resolved O +only O +in O +Structure B-evidence +I I-evidence +( O +Figure O +5 O +— O +figure O +supplement O +2 O +). O + +The O +N O +- O +terminal O +part O +of O +the O +loop B-structure_element +( O +aa O +50 B-residue_range +– I-residue_range +60 I-residue_range +) O +is O +sandwiched O +between O +the O +tip O +of O +helix B-structure_element +14 I-structure_element +( O +415CAAA418 B-structure_element +) O +of O +the O +18S B-chemical +rRNA I-chemical +of O +the O +40S B-complex_assembly +subunit B-structure_element +and O +helix B-structure_element +A I-structure_element +( O +aa O +32 B-residue_range +– I-residue_range +42 I-residue_range +) O +of O +eEF2 B-protein +( O +Figure O +5d O +). O + +Bulged B-protein_state +A416 B-residue_name_number +interacts O +with O +the O +switch B-structure_element +loop I-structure_element +in O +the O +vicinity O +of O +D53 B-residue_name_number +. O + +Next O +to O +GDP B-chemical +, O +the O +C O +- O +terminal O +part O +of O +the O +switch B-structure_element +loop I-structure_element +( O +aa O +61 B-residue_range +– I-residue_range +67 I-residue_range +) O +adopts O +a O +helical B-protein_state +fold I-protein_state +. O + +As O +such O +, O +the O +conformations O +of O +SWI B-structure_element +and O +the O +GTPase B-site +center I-site +in O +general O +are O +similar O +to O +those O +observed O +in O +ribosome B-protein_state +- I-protein_state +bound I-protein_state +EF B-protein +- I-protein +Tu I-protein +and O +EF B-protein +- I-protein +G I-protein +in O +the O +presence B-protein_state +of I-protein_state +GTP B-chemical +analogs O +. O + +Structure B-evidence +II I-evidence +reveals O +PKI B-structure_element +between O +the O +body B-structure_element +A B-site +and I-site +P I-site +sites I-site +and O +eEF2 B-protein +partially O +advanced O +into O +the O +A B-site +site I-site + +In O +Structure B-evidence +II I-evidence +, O +relative O +to O +Structure B-evidence +I I-evidence +, O +PKI B-structure_element +is O +further O +shifted O +along O +the O +40S B-complex_assembly +body B-structure_element +, O +traversing O +~ O +4 O +Å O +toward O +the O +P B-site +site I-site +( O +Figures O +2e O +, O +f O +, O +and O +g O +), O +while O +stacking B-bond_interaction +on O +C1274 B-residue_name_number +at O +the O +head B-structure_element +A B-site +site I-site +. O + +Thus O +, O +the O +intermediate O +position O +of O +PKI B-structure_element +is O +possible O +due O +to O +a O +large O +swivel O +of O +the O +head B-structure_element +relative O +to O +the O +body B-structure_element +, O +which O +brings O +the O +head B-structure_element +A B-site +site I-site +close O +to O +the O +body B-structure_element +P B-site +site I-site +. O + +Domain O +IV B-structure_element +of O +eEF2 B-protein +is O +further O +entrenched O +in O +the O +A B-site +site I-site +by O +~ O +3 O +Å O +relative O +to O +the O +body B-structure_element +and O +~ O +8 O +Å O +relative O +to O +the O +head B-structure_element +, O +preserving O +its O +interactions O +with O +PKI B-structure_element +. O + +The O +decoding B-site +center I-site +residues O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +are O +rearranged O +to O +pack O +inside O +helix B-structure_element +44 I-structure_element +, O +making O +room O +for O +eEF2 B-protein +. O + +This O +conformation O +of O +decoding B-site +center I-site +residues O +is O +also O +observed O +in O +the O +absence B-protein_state +of I-protein_state +A B-site +- I-site +site I-site +ligands O +. O + +The O +head B-site +interface I-site +of O +domain O +IV B-structure_element +interacts O +with O +the O +40S B-complex_assembly +head B-structure_element +( O +Figure O +6a O +). O + +Here O +, O +a O +positively B-site +charged I-site +surface I-site +of O +eEF2 B-protein +, O +formed O +by O +K613 B-residue_name_number +, O +R617 B-residue_name_number +and O +R631 B-residue_name_number +contacts O +the O +phosphate O +backbone O +of O +helix B-structure_element +33 I-structure_element +( O +Figures O +6c O +; O +see O +also O +Figure O +6 O +— O +figure O +supplement O +1 O +). O + +Structure B-evidence +III I-evidence +represents O +a O +highly B-protein_state +bent I-protein_state +IRES B-site +with O +PKI B-structure_element +captured O +between O +the O +head B-structure_element +A B-site +and I-site +P I-site +sites I-site + +Consistent O +with O +the O +similar O +head B-structure_element +swivels O +in O +Structure B-evidence +III I-evidence +and O +Structure B-evidence +II I-evidence +, O +relative O +positions O +of O +the O +40S B-complex_assembly +head B-structure_element +A B-site +site I-site +and O +body B-structure_element +P B-site +site I-site +remain O +as O +in O +Structure B-evidence +II I-evidence +. O + +Among O +the O +five O +structures B-evidence +, O +the O +PKI B-structure_element +domain O +is O +least O +ordered O +in O +Structure B-evidence +III I-evidence +and O +lacks O +density B-evidence +for O +SL3 B-structure_element +. O + +The O +map B-evidence +allows O +placement O +of O +PKI B-structure_element +at O +the O +body B-structure_element +P B-site +site I-site +( O +Figure O +1 O +— O +figure O +supplement O +3 O +). O + +Thus O +, O +in O +Structure B-evidence +III I-evidence +, O +PKI B-structure_element +has O +translocated O +along O +the O +40S B-complex_assembly +body B-structure_element +, O +but O +the O +head B-structure_element +remains O +fully B-protein_state +swiveled I-protein_state +so O +that O +PKI B-structure_element +is O +between O +the O +head B-structure_element +A B-site +and I-site +P I-site +sites I-site +. O + +Lower O +resolution O +of O +the O +map B-evidence +in O +this O +region O +suggests O +that O +PKI B-structure_element +is O +somewhat O +destabilized O +in O +the O +vicinity O +of O +the O +body B-structure_element +P B-site +site I-site +in O +the O +absence B-protein_state +of I-protein_state +stacking B-bond_interaction +with O +the O +foundations O +of O +the O +head B-structure_element +A B-site +site I-site +( O +C1274 B-residue_name_number +) O +or O +P B-site +site I-site +( O +U1191 B-residue_name_number +). O + +The O +position O +of O +eEF2 B-protein +is O +similar O +to O +that O +in O +Structure B-evidence +II I-evidence +. O + +Structure B-evidence +IV I-evidence +represents O +a O +highly B-protein_state +bent I-protein_state +IRES B-site +with O +PKI B-structure_element +partially O +accommodated O +in O +the O +P B-site +site I-site + +In O +Structure B-evidence +IV I-evidence +, O +the O +40S B-complex_assembly +subunit B-structure_element +is O +almost O +non B-protein_state +- I-protein_state +rotated I-protein_state +relative O +to O +the O +60S B-complex_assembly +subunit B-structure_element +, O +and O +the O +40S B-complex_assembly +head B-structure_element +is O +mid B-protein_state +- I-protein_state +swiveled I-protein_state +. O + +Unwinding O +of O +the O +head B-structure_element +moves O +the O +head B-structure_element +P B-site +- I-site +site I-site +residue O +U1191 B-residue_name_number +and O +body B-structure_element +P B-site +- I-site +site I-site +residue O +C1637 B-residue_name_number +closer O +together O +, O +resulting O +in O +a O +partially O +restored O +40S B-complex_assembly +P B-site +site I-site +. O + +Whereas O +C1637 B-residue_name_number +forms O +a O +stacking B-site +platform I-site +for O +the O +last O +base O +pair O +of O +PKI B-structure_element +, O +U1191 B-residue_name_number +does O +not O +yet O +stack B-bond_interaction +on O +PKI B-structure_element +because O +the O +head B-structure_element +remains O +partially O +swiveled O +. O + +This O +renders O +PKI B-structure_element +partially O +accommodated O +in O +the O +P B-site +site I-site +( O +Figure O +2g O +). O + +Unwinding O +of O +the O +40S B-complex_assembly +head B-structure_element +also O +positions O +the O +head B-structure_element +A B-site +site I-site +closer O +to O +the O +body B-structure_element +A B-site +site I-site +. O + +This O +results O +in O +rearrangements O +of O +eEF2 B-protein +interactions O +with O +the O +head B-structure_element +, O +allowing O +eEF2 B-protein +to O +advance O +further O +into O +the O +A B-site +site I-site +. O + +To O +this O +end O +, O +the O +head B-site +- I-site +interacting I-site +interface I-site +of O +domain O +IV B-structure_element +slides O +along O +the O +surface O +of O +the O +head B-structure_element +by O +5 O +Å O +. O +Helix B-structure_element +A I-structure_element +of O +domain O +IV B-structure_element +is O +positioned O +next O +to O +the O +backbone O +of O +h34 B-structure_element +, O +with O +positively O +charged O +residues O +K613 B-residue_name_number +, O +R617 B-residue_name_number +and O +R631 B-residue_name_number +rearranged O +from O +the O +backbone O +of O +h33 B-structure_element +( O +Figure O +6c O +; O +see O +also O +Figure O +6 O +— O +figure O +supplement O +1 O +). O + +Structure B-evidence +V I-evidence +represents O +an O +extended B-protein_state +IRES B-site +with O +PKI B-structure_element +fully O +accommodated O +in O +the O +P B-site +site I-site +and O +domain O +IV B-structure_element +of O +eEF2 B-protein +in O +the O +A B-site +site I-site + +In O +the O +nearly B-protein_state +non I-protein_state +- I-protein_state +rotated I-protein_state +and O +non B-protein_state +- I-protein_state +swiveled I-protein_state +ribosome B-complex_assembly +conformation O +in O +Structure B-evidence +V I-evidence +closely O +resembling O +that O +of O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +, O +PKI B-structure_element +is O +fully O +accommodated O +in O +the O +P B-site +site I-site +. O + +The O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +– I-structure_element +like I-structure_element +helix I-structure_element +is O +stacked O +on O +P B-site +- I-site +site I-site +residues O +U1191 B-residue_name_number +and O +C1637 B-residue_name_number +( O +Figure O +3d O +), O +analogous O +to O +stacking B-bond_interaction +of O +the O +tRNA B-complex_assembly +- I-complex_assembly +mRNA I-complex_assembly +helix B-structure_element +( O +Figure O +3e O +). O + +A O +notable O +conformational O +change O +in O +eEF2 B-protein +from O +that O +in O +the O +preceding O +Structures B-evidence +is O +visible O +in O +the O +position O +of O +domain O +III B-structure_element +, O +which O +contacts O +uS12 B-protein +( O +Figure O +6d O +). O + +In O +Structure B-evidence +V I-evidence +, O +protein O +uS12 B-protein +is O +shifted O +along O +with O +the O +40S B-complex_assembly +body B-structure_element +as O +a O +result O +of O +intersubunit O +rotation O +. O + +In O +this O +position O +, O +uS12 B-protein +forms O +extensive O +interactions O +with O +eEF2 B-protein +domains O +II B-structure_element +and O +III B-structure_element +. O + +Specifically O +, O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +tail I-structure_element +of O +uS12 B-protein +packs O +against O +the O +β B-structure_element +- I-structure_element +barrel I-structure_element +of O +domain O +II B-structure_element +, O +while O +the O +β B-structure_element +- I-structure_element +barrel I-structure_element +of O +uS12 B-protein +packs O +against O +helix B-structure_element +A I-structure_element +of O +domain O +III B-structure_element +. O + +This O +shifts O +the O +tip O +of O +helix B-structure_element +A I-structure_element +of O +domain O +III B-structure_element +( O +at O +aa O +500 B-residue_number +) O +by O +~ O +5 O +Å O +( O +relative O +to O +that O +in O +Structure B-evidence +I I-evidence +) O +toward O +domain O +I B-structure_element +. O +Although O +domain O +III B-structure_element +remains O +in O +contact O +with O +domain O +V B-structure_element +, O +the O +shift O +occurs O +in O +the O +direction O +that O +could O +eventually O +disconnect O +the O +β B-structure_element +- I-structure_element +platforms I-structure_element +of O +these O +domains O +. O + +Domain O +IV B-structure_element +of O +eEF2 B-protein +is O +fully O +accommodated O +in O +the O +A B-site +site I-site +. O + +The O +first O +codon O +of O +the O +open B-structure_element +reading I-structure_element +frame I-structure_element +is O +also O +positioned O +in O +the O +A B-site +site I-site +, O +with O +bases O +exposed O +toward O +eEF2 B-protein +( O +Figure O +7 O +), O +resembling O +the O +conformations O +of O +the O +A B-site +- I-site +site I-site +codons O +in O +EF B-protein_state +- I-protein_state +G I-protein_state +- I-protein_state +bound I-protein_state +70S B-complex_assembly +complexes O +. O + +As O +in O +the O +preceding O +Structures B-evidence +, O +the O +histidine B-site +- I-site +diphthamide I-site +tip I-site +is O +bound B-protein_state +in I-protein_state +the O +minor B-site +groove I-site +of O +the O +P B-site +- I-site +site I-site +codon B-structure_element +- I-structure_element +anticodon I-structure_element +helix I-structure_element +. O + +Diph699 B-ptm +slightly O +rearranges O +, O +relative O +to O +that O +in O +Structure B-evidence +I I-evidence +( O +Figure O +7 O +), O +and O +interacts O +with O +four O +out O +of O +six O +codon O +- O +anticodon O +nucleotides O +. O + +The O +imidazole O +moiety O +stacks O +on O +G6907 B-residue_name_number +( O +corresponding O +to O +nt O +36 O +in O +the O +tRNA B-chemical +anticodon O +) O +and O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +O2 O +’ O +of O +G6906 B-residue_name_number +( O +nt O +35 O +of O +tRNA B-chemical +). O + +The O +amide O +at O +the O +diphthamide B-ptm +end O +interacts O +with O +N2 O +of O +G6906 B-residue_name_number +and O +O2 O +and O +O2 O +’ O +of O +C6951 B-residue_name_number +( O +corresponding O +to O +nt O +2 O +of O +the O +codon O +). O + +The O +trimethylamino O +- O +group O +is O +positioned O +over O +the O +ribose O +of O +C6952 B-residue_name_number +( O +codon O +nt O +3 O +). O + +IRES B-site +translocation O +mechanism O + +Animation O +showing O +the O +transition O +from O +the O +initiation B-protein_state +80S B-complex_assembly +• I-complex_assembly +TSV I-complex_assembly +IRES I-complex_assembly +structures B-evidence +( O +Koh O +et O +al O +., O +2014 O +) O +to O +eEF2 B-protein_state +- I-protein_state +bound I-protein_state +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +( O +this O +work O +). O + +Four O +views O +( O +scenes O +) O +are O +shown O +: O +( O +1 O +) O +A O +view O +down O +the O +intersubunit O +space O +, O +with O +the O +head B-structure_element +of O +the O +40S B-complex_assembly +subunit B-structure_element +oriented O +toward O +a O +viewer O +, O +as O +in O +Figure O +1a O +; O +( O +2 O +) O +A O +view O +at O +the O +solvent O +side O +of O +the O +40S B-complex_assembly +subunit B-structure_element +, O +with O +the O +40S B-complex_assembly +head B-structure_element +shown O +at O +the O +top O +, O +as O +in O +Figure O +2 O +— O +figure O +supplement O +1 O +; O +( O +3 O +) O +A O +view O +down O +at O +the O +subunit O +interface O +of O +the O +40S B-complex_assembly +subunit B-structure_element +; O +( O +4 O +) O +A O +close O +- O +up O +view O +of O +the O +decoding B-site +center I-site +( O +A B-site +site I-site +) O +and O +the O +P B-site +site I-site +, O +as O +in O +Figure O +2g O +. O +Each O +scene O +is O +shown O +twice O +. O + +In O +scenes O +1 O +, O +2 O +and O +3 O +, O +nucleotides O +C1274 B-residue_name_number +, O +U1191 B-residue_name_number +of O +the O +40S B-complex_assembly +head B-structure_element +and O +G904 B-residue_name_number +of O +the O +40S B-site +platform I-site +are O +shown O +in O +black O +to O +denote O +the O +A B-site +, I-site +P I-site +and I-site +E I-site +sites I-site +, O +respectively O +. O + +In O +scene O +4 O +, O +C1274 B-residue_name_number +and O +U1191 B-residue_name_number +are O +labeled O +and O +shown O +in O +yellow O +; O +G577 B-residue_name_number +, O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +of O +the O +40S B-complex_assembly +body B-structure_element +A B-site +site I-site +and O +C1637 B-residue_name_number +of O +the O +body B-structure_element +P B-site +site I-site +are O +labeled O +and O +shown O +in O +orange O +. O + +In O +this O +work O +we O +have O +captured O +the O +structures B-evidence +of O +the O +TSV B-species +IRES B-site +, O +whose O +PKI B-structure_element +samples O +positions O +between O +the O +A B-site +and I-site +P I-site +sites I-site +( O +Structures B-evidence +I I-evidence +– I-evidence +IV I-evidence +), O +as O +well O +as O +in O +the O +P B-site +site I-site +( O +Structure B-evidence +V I-evidence +). O + +We O +propose O +that O +together O +with O +the O +previously O +reported O +initiation B-protein_state +state O +, O +these O +structures B-evidence +represent O +the O +trajectory O +of O +eEF2 B-protein +- O +induced O +IRES B-site +translocation O +( O +shown O +as O +an O +animation O +in O +http O +:// O +labs O +. O +umassmed O +. O +edu O +/ O +korostelevlab O +/ O +msc O +/ O +iresmovie O +. O +gif O +and O +Video O +1 O +). O + +Our O +structures B-evidence +reveal O +previously O +unseen O +intermediate O +states O +of O +eEF2 B-protein +or O +EF B-protein +- I-protein +G I-protein +engagement O +with O +the O +A B-site +site I-site +, O +providing O +the O +structural O +basis O +for O +the O +mechanism O +of O +translocase B-protein_type +action O +. O + +Furthermore O +, O +they O +provide O +insight O +into O +the O +mechanism O +of O +eEF2 B-complex_assembly +• I-complex_assembly +GTP I-complex_assembly +association O +with O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +and O +eEF2 B-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +dissociation O +from O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +, O +also O +delineating O +the O +mechanism O +of O +translation O +inhibition O +by O +the O +antifungal O +drug O +sordarin B-chemical +. O + +In O +summary O +, O +the O +reported O +ensemble O +of O +structures B-evidence +substantially O +enhances O +our O +understanding O +of O +the O +translocation O +mechanism O +, O +including O +that O +of O +tRNAs B-chemical +as O +discussed O +below O +. O + +Translocation O +of O +the O +TSV B-species +IRES B-site +on O +the O +40S B-complex_assembly +subunit B-structure_element +globally O +resembles O +a O +step O +of O +an O +inchworm B-protein_state +( O +Figure O +4 O +; O +see O +also O +Figure O +3 O +— O +figure O +supplement O +2 O +). O + +At O +the O +start O +( O +initiation B-protein_state +state O +), O +the O +IRES B-site +adopts O +an O +extended B-protein_state +conformation O +( O +extended B-protein_state +inchworm I-protein_state +). O + +The O +front B-structure_element +' I-structure_element +legs I-structure_element +' O +( O +SL4 B-structure_element +and O +SL5 B-structure_element +) O +of O +the O +5 B-structure_element +’- I-structure_element +domain I-structure_element +( O +front B-structure_element +end I-structure_element +) O +are O +attached O +to O +the O +40S B-complex_assembly +head B-structure_element +proteins O +uS7 B-protein +, O +uS11 B-protein +and O +eS25 B-protein +( O +Figure O +3 O +— O +figure O +supplement O +2 O +). O + +PKI B-structure_element +, O +representing O +the O +hind B-structure_element +end I-structure_element +, O +is O +bound B-protein_state +in I-protein_state +the O +A B-site +site I-site +. O + +In O +the O +first O +sub O +- O +step O +( O +Structures B-evidence +I I-evidence +to I-evidence +IV I-evidence +), O +the O +hind B-structure_element +end I-structure_element +advances O +from O +the O +A B-site +to I-site +the I-site +P I-site +site I-site +and O +approaches O +the O +front B-structure_element +end I-structure_element +, O +which O +remains O +attached O +to O +the O +40S B-complex_assembly +surface O +. O + +This O +shortens O +the O +distance O +between O +PKI B-structure_element +and O +SL4 B-structure_element +by O +up O +to O +20 O +Å O +relative O +to O +the O +initiating O +IRES B-site +structure B-evidence +, O +resulting O +in O +a O +bent B-protein_state +IRES B-site +conformation O +( O +bent B-protein_state +inchworm I-protein_state +). O + +Finally O +( O +Structures B-evidence +IV I-evidence +to I-evidence +V I-evidence +), O +as O +the O +hind B-structure_element +end I-structure_element +is O +accommodated O +in O +the O +P B-site +site I-site +, O +the O +front B-structure_element +' I-structure_element +legs I-structure_element +' I-structure_element +advance O +by O +departing O +from O +their O +initial B-site +binding I-site +sites I-site +. O + +This O +converts O +the O +IRES B-site +into O +an O +extended B-protein_state +conformation O +, O +rendering O +the O +inchworm B-protein_state +prepared O +for O +the O +next O +translocation O +step O +. O + +Notably O +, O +at O +all O +steps O +, O +the O +head B-structure_element +of O +the O +IRES B-site +inchworm B-protein_state +( O +L1 B-structure_element +. I-structure_element +1 I-structure_element +region I-structure_element +) O +is O +supported O +by O +the O +mobile B-protein_state +L1 B-structure_element +stalk I-structure_element +. O + +In O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +CrPV B-species +IRES B-site +structure B-evidence +, O +the O +5 B-structure_element +’- I-structure_element +domain I-structure_element +similarly O +protrudes O +between O +the O +subunits O +and O +interacts O +with O +the O +L1 B-structure_element +stalk I-structure_element +, O +as O +in O +the O +initiation B-protein_state +state O +for O +this O +IRES B-site +. O + +This O +underlines O +structural O +similarity O +for O +the O +TSV B-species +and O +CrPV B-species +IRES B-site +translocation O +mechanisms O +. O + +Upon O +translocation O +, O +the O +GCU O +start O +codon O +is O +positioned O +in O +the O +A B-site +site I-site +( O +Structure B-evidence +V I-evidence +), O +ready O +for O +interaction O +with O +Ala B-chemical +- I-chemical +tRNAAla I-chemical +upon O +eEF2 B-protein +departure O +. O + +Recent O +studies O +have O +shown O +that O +in O +some O +cases O +a O +fraction O +of O +IGR B-structure_element +IRES B-site +- O +driven O +translation O +results O +from O +an O +alternative O +reading O +frame O +, O +which O +is O +shifted O +by O +one O +nucleotide O +relative O +to O +the O +normal O +ORF B-structure_element +. O + +One O +of O +the O +mechanistic O +scenarios O +( O +discussed O +in O +) O +involves O +binding O +of O +the O +first O +aminoacyl B-chemical +- I-chemical +tRNA I-chemical +to O +the O +post B-protein_state +- I-protein_state +translocated I-protein_state +IRES B-site +mRNA B-chemical +frame O +shifted O +by O +one O +nucleotide O +( O +predominantly O +a O ++ O +1 O +frame O +shift O +). O + +In O +our O +structures B-evidence +, O +the O +IRES B-site +presents O +to O +the O +decoding B-site +center I-site +a O +pre B-protein_state +- I-protein_state +translocated I-protein_state +or O +fully B-protein_state +translocated I-protein_state +ORF B-structure_element +, O +rather O +than O +a O ++ O +1 O +( O +more O +translocated O +) O +ORF B-structure_element +, O +suggesting O +that O +eEF2 B-protein +does O +not O +induce O +a O +highly O +populated O +fraction O +of O ++ O +1 O +shifted O +IRES B-site +mRNAs B-chemical +. O + +It O +is O +likely O +that O +alternative O +frame O +setting O +occurs O +following O +eEF2 B-protein +release O +and O +that O +this O +depends O +on O +transient O +displacement O +of O +the O +start O +codon O +in O +the O +decoding B-site +center I-site +, O +allowing O +binding O +of O +the O +corresponding O +amino B-chemical +acyl I-chemical +- I-chemical +tRNA I-chemical +to O +an O +off O +- O +frame O +codon O +. O + +Further O +structural B-experimental_method +studies I-experimental_method +involving O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +• I-complex_assembly +tRNA I-complex_assembly +complexes O +are O +necessary O +to O +understand O +the O +mechanisms O +underlying O +alternative O +reading O +frame O +selection O +. O + +The O +presence B-protein_state +of I-protein_state +several O +translocation O +complexes O +in O +a O +single O +sample O +suggests O +that O +the O +structures B-evidence +represent O +equilibrium O +states O +of O +forward O +and O +reverse O +translocation O +of O +the O +IRES B-site +, O +which O +interconvert O +among O +each O +other O +. O + +This O +is O +consistent O +with O +the O +observations O +that O +the O +intergenic O +IRESs B-site +are O +prone O +to O +reverse O +translocation O +. O + +Specifically O +, O +biochemical B-experimental_method +toe I-experimental_method +- I-experimental_method +printing I-experimental_method +studies I-experimental_method +in O +the O +presence B-protein_state +of I-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +GTP I-complex_assembly +identified O +IRES B-site +in O +a O +non B-protein_state +- I-protein_state +translocated I-protein_state +position O +unless O +eEF1a B-complex_assembly +• I-complex_assembly +aa I-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +is O +also O +present O +. O + +These O +findings O +indicate O +that O +IRES B-site +translocation O +by O +eEF2 B-protein +is O +futile O +: O +the O +IRES B-site +returns O +to O +the O +A B-site +site I-site +upon O +releasing O +eEF2 B-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +unless O +an O +amino B-chemical +- I-chemical +acyl I-chemical +tRNA I-chemical +enters O +the O +A B-site +site I-site +and O +blocks O +IRES B-site +back O +- O +translocation O +. O + +This O +contrasts O +with O +the O +post B-protein_state +- I-protein_state +translocated I-protein_state +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +, O +in O +which O +the O +classical O +P B-site +and I-site +E I-site +- I-site +site I-site +tRNAs B-chemical +are O +stabilized O +in O +the O +non B-protein_state +- I-protein_state +rotated I-protein_state +ribosome B-complex_assembly +after O +translocase B-protein_type +release O +. O + +Thus O +, O +the O +meta O +- O +stability O +of O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +IRES B-site +is O +likely O +due O +to O +the O +absence B-protein_state +of I-protein_state +stabilizing O +structural O +features O +present O +in O +the O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +. O + +In O +the O +initiation B-protein_state +state O +, O +the O +IRES B-site +resembles O +a O +pre B-protein_state +- I-protein_state +translocation I-protein_state +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +reduced O +to O +the O +A B-site +/ I-site +P I-site +- O +tRNA B-chemical +anticodon B-structure_element +- I-structure_element +stem I-structure_element +loop I-structure_element +and O +elbow B-structure_element +in O +the O +A B-site +site I-site +and O +the O +P B-site +/ I-site +E I-site +- O +tRNA B-chemical +elbow B-structure_element +contacting O +the O +L1 B-structure_element +stalk I-structure_element +. O + +Because O +the O +anticodon B-structure_element +- I-structure_element +stem I-structure_element +loop I-structure_element +of O +the O +A B-site +- O +tRNA B-chemical +is O +sufficient O +for O +translocation O +completion O +, O +we O +ascribe O +the O +meta O +- O +stability O +of O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +IRES B-site +to O +the O +absence B-protein_state +of I-protein_state +the O +P B-site +/ I-site +E I-site +- O +tRNA B-chemical +elements O +, O +either O +the O +ASL B-structure_element +or O +the O +acceptor O +arm O +, O +or O +both O +. O + +Furthermore O +, O +interactions O +of O +SL4 B-structure_element +and O +SL5 B-structure_element +with O +the O +40S B-complex_assembly +subunit B-structure_element +likely O +contribute O +to O +stabilization O +of O +pre B-protein_state +- I-protein_state +translocation I-protein_state +structures B-evidence +. O + +Partitioned O +roles O +of O +40S B-complex_assembly +subunit B-structure_element +rearrangements O + +Our O +structures B-evidence +delineate O +the O +mechanistic O +functions O +for O +intersubunit O +rotation O +and O +head B-structure_element +swivel O +in O +translocation O +. O + +Specifically O +, O +intersubunit O +rotation O +allows O +eEF2 B-protein +entry O +into O +the O +A B-site +site I-site +, O +while O +the O +head B-structure_element +swivel O +mediates O +PKI B-structure_element +translocation O +. O + +Various O +degrees O +of O +intersubunit O +rotation O +have O +been O +observed O +in O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +studies I-experimental_method +of O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +complexes O +. O + +This O +suggests O +that O +the O +subunits B-structure_element +are O +capable O +of O +spontaneous O +rotation O +, O +as O +is O +the O +case O +for O +tRNA B-protein_state +- I-protein_state +bound I-protein_state +pre B-protein_state +- I-protein_state +translocation I-protein_state +complexes O +. O + +The O +pre B-protein_state +- I-protein_state +translocation I-protein_state +Structure B-evidence +I I-evidence +with O +eEF2 B-protein +least O +advanced O +into O +the O +A B-site +site I-site +adopts O +a O +fully B-protein_state +rotated I-protein_state +conformation I-protein_state +. O + +Reverse O +intersubunit O +rotation O +from O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +shifts O +the O +translocation B-site +tunnel I-site +( O +the O +tunnel B-site +between O +the O +A B-site +, I-site +P I-site +and I-site +E I-site +sites I-site +) O +toward O +eEF2 B-protein +, O +which O +is O +rigidly O +attached O +to O +the O +60S B-complex_assembly +subunit B-structure_element +. O + +This O +allows O +eEF2 B-protein +to O +move O +into O +the O +A B-site +site I-site +. O + +As O +such O +, O +reverse O +intersubunit O +rotation O +facilitates O +full O +docking O +of O +eEF2 B-protein +in O +the O +A B-site +site I-site +. O + +Because O +the O +histidine B-site +- I-site +diphthamide I-site +tip I-site +of O +eEF2 B-protein +( O +H583 B-residue_name_number +, O +H694 B-residue_name_number +and O +Diph699 B-ptm +) O +attaches O +to O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +- I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +, O +eEF2 B-protein +appears O +to O +directly O +force O +PKI B-structure_element +out O +of O +the O +A B-site +site I-site +. O + +The O +head B-structure_element +swivel O +allows O +gradual O +translocation O +of O +PKI B-structure_element +to O +the O +P B-site +site I-site +, O +first O +with O +respect O +to O +the O +body B-structure_element +and O +then O +to O +the O +head B-structure_element +. O + +The O +fully B-protein_state +swiveled I-protein_state +conformations O +of O +Structures B-evidence +II I-evidence +and I-evidence +III I-evidence +represent O +the O +mid O +- O +point O +of O +translocation O +, O +in O +which O +PKI B-structure_element +relocates O +between O +the O +head B-structure_element +A B-site +site I-site +and O +body B-structure_element +P B-site +site I-site +. O + +We O +note O +that O +such O +mid O +- O +states O +have O +not O +been O +observed O +for O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +, O +but O +their O +formation O +can O +explain O +the O +formation O +of O +subsequent O +pe B-protein_state +/ I-protein_state +E I-protein_state +hybrid I-protein_state +and O +ap B-protein_state +/ I-protein_state +P I-protein_state +chimeric I-protein_state +structures B-evidence +( O +Figure O +1 O +— O +figure O +supplement O +1 O +). O + +Reverse O +swivel O +from O +Structure B-evidence +III I-evidence +to I-evidence +V I-evidence +brings O +the O +head B-structure_element +to O +the O +non B-protein_state +- I-protein_state +swiveled I-protein_state +position O +, O +restoring O +the O +A B-site +and I-site +P I-site +sites I-site +on O +the O +small B-structure_element +subunit I-structure_element +. O + +The O +functions O +of O +eEF2 B-protein +in O +translocation O + +To O +our O +knowledge O +, O +our O +work O +provides O +the O +first O +high O +- O +resolution O +view O +of O +the O +dynamics O +of O +a O +ribosomal B-protein_type +translocase I-protein_type +that O +is O +inferred O +from O +an O +ensemble O +of O +structures B-evidence +sampled O +under O +uniform O +conditions O +. O + +The O +structures B-evidence +, O +therefore O +, O +offer O +a O +unique O +opportunity O +to O +address O +the O +role O +of O +the O +elongation B-protein_type +factors I-protein_type +during O +translocation O +. O + +Translocases B-protein_type +are O +efficient O +enzymes O +. O + +While O +the O +ribosome B-complex_assembly +itself O +has O +the O +capacity O +to O +translocate O +in O +the O +absence B-protein_state +of I-protein_state +the O +translocase B-protein_type +, O +spontaneous O +translocation O +is O +slow O +. O + +EF B-protein +- I-protein +G I-protein +enhances O +the O +translocation O +rate O +by O +several O +orders O +of O +magnitude O +, O +aided O +by O +an O +additional O +2 O +- O +to O +50 O +- O +fold O +boost O +from O +GTP B-chemical +hydrolysis O +. O + +Due O +to O +the O +lack O +of O +structures B-evidence +of O +translocation O +intermediates O +, O +the O +mechanistic O +role O +of O +eEF2 B-protein +/ O +EF B-protein +- I-protein +G I-protein +is O +not O +fully O +understood O +. O + +The O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +structures B-evidence +reported O +here O +suggest O +two O +main O +roles O +for O +eEF2 B-protein +in O +translocation O +. O + +As O +discussed O +above O +, O +the O +first O +role O +is O +to O +directly O +shift O +PKI B-structure_element +out O +of O +the O +A B-site +site I-site +upon O +spontaneous O +reverse O +intersubunit O +rotation O +. O + +In O +our O +structures B-evidence +, O +the O +tip O +of O +domain O +IV B-structure_element +docks O +next O +to O +PKI B-structure_element +, O +with O +diphthamide B-ptm +699 I-ptm +fit O +into O +the O +minor B-site +groove I-site +of O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +- I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +( O +Figure O +7 O +). O + +This O +arrangement O +rationalizes O +inactivation O +of O +eEF2 B-protein +by O +diphtheria B-protein_type +toxin I-protein_type +, O +which O +catalyzes O +ADP B-ptm +- I-ptm +ribosylation I-ptm +of O +the O +diphthamide B-ptm +( O +reviewed O +in O +). O + +The O +enzyme O +ADP B-ptm +- I-ptm +ribosylates I-ptm +the O +NE2 O +atom O +of O +the O +imidazole O +ring O +, O +which O +in O +our O +structures B-evidence +interacts O +with O +the O +first O +two O +residues O +of O +the O +anticodon B-structure_element +- I-structure_element +like I-structure_element +strand I-structure_element +of O +PKI B-structure_element +. O + +The O +bulky O +ADP B-chemical +- O +ribosyl O +moiety O +at O +this O +position O +would O +disrupt O +the O +interaction O +, O +rendering O +eEF2 B-protein +unable O +to O +bind O +to O +the O +A B-site +site I-site +and O +/ O +or O +stalled O +on O +ribosomes B-complex_assembly +in O +a O +non O +- O +productive O +conformation O +. O + +As O +eEF2 B-protein +shifts O +PKI B-structure_element +toward O +the O +P B-site +site I-site +in O +the O +course O +of O +reverse O +intersubunit O +rotation O +, O +the O +60S B-protein_state +- I-protein_state +attached I-protein_state +translocase B-protein_type +migrates O +along O +the O +surface O +of O +the O +40S B-complex_assembly +subunit B-structure_element +, O +guided O +by O +electrostatic B-bond_interaction +interactions I-bond_interaction +. O + +Positively B-site +- I-site +charged I-site +patches I-site +of O +domains O +II B-structure_element +and O +III B-structure_element +( O +R391 B-residue_name_number +, O +K394 B-residue_name_number +, O +R433 B-residue_name_number +, O +R510 B-residue_name_number +) O +and O +IV B-structure_element +( O +K613 B-residue_name_number +, O +R617 B-residue_name_number +, O +R609 B-residue_name_number +, O +R631 B-residue_name_number +, O +K651 B-residue_name_number +) O +slide O +over O +rRNA B-chemical +of O +the O +40S B-complex_assembly +body B-structure_element +( O +h5 B-structure_element +) O +and O +head B-structure_element +( O +h18 B-structure_element +and O +h33 B-structure_element +/ O +h34 B-structure_element +), O +respectively O +. O + +The O +Structures B-evidence +reveal O +hopping O +of O +the O +positive O +clusters O +over O +rRNA B-chemical +helices B-structure_element +. O + +For O +example O +, O +between O +Structures B-evidence +II I-evidence +and I-evidence +V I-evidence +, O +the O +K613 B-residue_name_number +/ O +R617 B-residue_name_number +/ O +R631 B-residue_name_number +cluster O +of O +domain O +IV B-structure_element +hops O +by O +~ O +19 O +Å O +( O +for O +Cα O +of O +R617 B-residue_name_number +) O +from O +the O +phosphate O +backbone O +of O +h33 B-structure_element +( O +at O +nt O +1261 B-residue_range +– I-residue_range +1264 I-residue_range +) O +to O +that O +of O +the O +neighboring O +h34 B-structure_element +( O +at O +nt O +1442 B-residue_range +– I-residue_range +1445 I-residue_range +). O + +Thus O +, O +sliding O +of O +eEF2 B-protein +involves O +reorganization O +of O +electrostatic B-bond_interaction +, I-bond_interaction +perhaps I-bond_interaction +isoenergetic I-bond_interaction +interactions I-bond_interaction +, O +echoing O +those O +implied O +in O +extraordinarily O +fast O +ribosome B-complex_assembly +inactivation O +rates O +by O +the O +small O +- O +protein O +ribotoxins O +and O +in O +fast O +protein O +association O +and O +diffusion O +along O +DNA O +. O + +Comparison B-experimental_method +of O +our O +structures B-evidence +with O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +structure B-evidence +reveals O +the O +structural O +basis O +for O +the O +second O +key O +function O +of O +the O +translocase B-protein_type +: O +' O +unlocking O +' O +of O +intrasubunit O +rearrangements O +that O +are O +required O +for O +step O +- O +wise O +translocation O +of O +PKI B-structure_element +on O +the O +small B-structure_element +subunit I-structure_element +. O + +The O +unlocking O +model O +of O +the O +ribosome B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +pre B-protein_state +- I-protein_state +translocation I-protein_state +complex O +has O +been O +proposed O +decades O +ago O +and O +functional O +requirement O +of O +the O +translocase B-protein_type +in O +this O +process O +has O +been O +implicated O +. O + +However O +, O +the O +structural O +and O +mechanistic O +definitions O +of O +the O +locked B-protein_state +and O +unlocked B-protein_state +states O +have O +remained O +unclear O +, O +ranging O +from O +the O +globally O +distinct O +ribosome B-complex_assembly +conformations O +to O +unknown O +local O +rearrangements O +, O +e O +. O +g O +. O +those O +in O +the O +decoding B-site +center I-site +. O + +FRET B-evidence +data I-evidence +indicate O +that O +translocation O +of O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +on O +the O +70S B-complex_assembly +ribosome I-complex_assembly +requires O +a O +forward O +- O +and O +- O +reverse O +head B-structure_element +swivel O +, O +which O +may O +be O +related O +to O +the O +unlocking O +phenomenon O +. O + +Whereas O +intersubunit O +rotation O +of O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +complex O +occurs O +spontaneously O +, O +the O +head B-structure_element +swivel O +is O +induced O +by O +the O +eEF2 B-protein +/ O +EF B-protein +- I-protein +G I-protein +translocase B-protein_type +, O +consistent O +with O +requirement O +of O +eEF2 B-protein +for O +unlocking O +. O + +Structural B-experimental_method +studies I-experimental_method +revealed O +large O +head B-structure_element +swivels O +in O +various O +70S B-complex_assembly +• I-complex_assembly +tRNA I-complex_assembly +• I-complex_assembly +EF I-complex_assembly +- I-complex_assembly +G I-complex_assembly +and O +80S B-complex_assembly +• I-complex_assembly +tRNA I-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +complexes O +, O +but O +not O +in O +' O +locked B-protein_state +' O +complexes B-protein_state +with I-protein_state +the O +A B-site +site I-site +occupied O +by O +the O +tRNA B-chemical +in O +the O +absence B-protein_state +of I-protein_state +the O +translocase B-protein_type +. O + +Our O +structures B-evidence +suggest O +that O +eEF2 B-protein +induces O +head B-structure_element +swivel O +by O +' O +unlocking O +' O +the O +head B-structure_element +- O +body B-structure_element +interactions O +( O +Figure O +7 O +). O + +Binding O +of O +the O +ASL B-structure_element +to O +the O +A B-site +site I-site +is O +known O +from O +structural B-experimental_method +studies I-experimental_method +of O +bacterial B-taxonomy_domain +ribosomes B-complex_assembly +to O +result O +in O +' O +domain B-protein_state +closure I-protein_state +' O +of O +the O +small B-structure_element +subunit I-structure_element +, O +i O +. O +e O +. O +closer O +association O +of O +the O +head B-structure_element +, O +shoulder B-structure_element +and O +body B-structure_element +domains O +. O + +The O +domain O +closure O +' O +locks O +' O +cognate O +tRNA B-chemical +in O +the O +A B-site +site I-site +via O +stacking B-bond_interaction +on O +the O +head B-structure_element +A B-site +site I-site +( O +C1274 B-residue_name_number +in O +S B-species +. I-species +cerevisiae I-species +or O +C1054 B-residue_name_number +in O +E B-species +. I-species +coli I-species +) O +and O +interactions O +with O +the O +body B-structure_element +A B-site +- I-site +site I-site +nucleotides O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +( O +A1492 B-residue_name_number +and O +A1493 B-residue_name_number +in O +E B-species +. I-species +coli I-species +). O + +This O +' O +locked B-protein_state +' O +state O +is O +identical O +to O +that O +observed O +for O +PKI B-structure_element +in O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +structures B-evidence +in O +the O +absence B-protein_state +of I-protein_state +eEF2 B-protein +. O + +Structure B-evidence +I I-evidence +demonstrates O +that O +at O +an O +early O +pre B-protein_state +- I-protein_state +translocation I-protein_state +step O +, O +the O +histidine B-site +- I-site +diphthamide I-site +tip I-site +of O +eEF2 B-protein +is O +wedged O +between O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +and O +PKI B-structure_element +. O + +This O +destabilization O +allows O +PKI B-structure_element +to O +detach O +from O +the O +body B-structure_element +A B-site +site I-site +upon O +spontaneous O +reverse O +40S B-complex_assembly +body B-structure_element +rotation O +, O +while O +maintaining O +interactions O +with O +the O +head B-structure_element +A B-site +site I-site +. O + +Destabilization O +of O +the O +head B-protein_state +- I-protein_state +bound I-protein_state +PKI B-structure_element +at O +the O +body B-structure_element +A B-site +site I-site +thus O +allows O +mobility O +of O +the O +head B-structure_element +relative O +to O +the O +body B-structure_element +. O + +The O +histidine B-ptm +- I-ptm +diphthamide I-ptm +- O +induced O +disengagement O +of O +PKI B-structure_element +from O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +therefore O +provides O +the O +structural O +definition O +for O +the O +' O +unlocking O +' O +mode O +of O +eEF2 B-protein +action O +. O + +In O +summary O +, O +our O +structures B-evidence +are O +consistent O +with O +a O +model O +of O +eEF2 B-protein +- O +induced O +translocation O +in O +which O +both O +PKI B-structure_element +and O +eEF2 B-protein +passively O +migrate O +into O +the O +P B-site +and I-site +A I-site +site I-site +, O +respectively O +, O +during O +spontaneous O +40S B-complex_assembly +body B-structure_element +rotation O +and O +head B-structure_element +swivel O +, O +the O +latter O +being O +allowed O +by O +' O +unlocking O +' O +of O +the O +A B-site +site I-site +by O +eEF2 B-protein +. O + +Observation O +of O +different O +PKI B-structure_element +conformations O +sampling O +a O +range O +of O +positions O +between O +the O +A B-site +and I-site +P I-site +sites I-site +in O +the O +presence B-protein_state +of I-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +implies O +that O +thermal O +fluctuations O +of O +the O +40S B-complex_assembly +head B-structure_element +domain O +are O +sufficient O +for O +translocation O +along O +the O +energetically O +flat O +trajectory O +. O + +Insights O +into O +eEF2 B-protein +association O +with O +and O +dissociation O +from O +the O +ribosome B-complex_assembly + +The O +conformational O +rearrangements O +in O +eEF2 B-protein +from O +Structure B-evidence +I I-evidence +through O +Structure B-evidence +V I-evidence +provide O +insights O +into O +the O +mechanisms O +of O +eEF2 B-protein +association O +with O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +and O +dissociation O +from O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +. O + +In O +all O +five O +structures B-evidence +, O +the O +GTPase B-structure_element +domain I-structure_element +is O +attached O +to O +the O +P B-structure_element +stalk I-structure_element +and O +the O +sarcin B-structure_element +- I-structure_element +ricin I-structure_element +loop I-structure_element +. O + +In O +the O +fully B-protein_state +- I-protein_state +rotated I-protein_state +pre B-protein_state +- I-protein_state +translocation I-protein_state +- O +like O +Structure B-evidence +I I-evidence +, O +an O +additional O +interaction O +exists O +. O + +Here O +, O +switch B-structure_element +loop I-structure_element +I I-structure_element +interacts O +with O +helix B-structure_element +14 I-structure_element +( O +415CAAA418 B-structure_element +) O +of O +the O +18S B-chemical +rRNA I-chemical +. O + +This O +stabilization O +renders O +the O +GTPase B-site +center I-site +to O +adopt O +a O +GTP B-protein_state +- I-protein_state +bound I-protein_state +conformation O +, O +similar O +to O +those O +observed O +in O +other O +translational B-protein_type +GTPases I-protein_type +in O +the O +presence B-protein_state +of I-protein_state +GTP B-chemical +analogs O +and O +in O +the O +80S B-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +complex O +bound B-protein_state +with I-protein_state +a O +transition O +- O +state O +mimic O +GDP B-complex_assembly +• I-complex_assembly +AlF4 I-complex_assembly +–. I-complex_assembly +The O +switch B-structure_element +loop I-structure_element +contacts O +the O +base O +of O +A416 B-residue_name_number +( O +invariable B-protein_state +A344 B-residue_name_number +in O +E B-species +. I-species +coli I-species +and O +A463 B-residue_name_number +in O +H B-species +. I-species +sapiens I-species +). O + +Mutations B-experimental_method +of O +residues O +flanking O +A344 B-residue_name_number +in O +E B-species +. I-species +coli I-species +16S B-chemical +rRNA I-chemical +modestly O +inhibit O +translation O +but O +do O +not O +specifically O +affect O +EF B-protein +- I-protein +G I-protein +- O +mediated O +translocation O +. O + +However O +, O +the O +effect O +of O +A344 B-residue_name_number +mutation B-experimental_method +on O +translation O +was O +not O +addressed O +in O +that O +study O +, O +leaving O +the O +question O +open O +whether O +this O +residue O +is O +critical O +for O +eEF2 B-protein +/ O +EF B-protein +- I-protein +G I-protein +function O +. O + +The O +interaction O +between O +h14 B-structure_element +and O +switch B-structure_element +loop I-structure_element +I I-structure_element +is O +not O +resolved O +in O +Structures B-evidence +II I-evidence +to I-evidence +V I-evidence +, O +in O +all O +of O +which O +the O +small B-structure_element +subunit I-structure_element +is O +partially B-protein_state +rotated I-protein_state +or O +non B-protein_state +- I-protein_state +rotated I-protein_state +, O +so O +that O +helix B-structure_element +14 I-structure_element +is O +placed O +at O +least O +6 O +Å O +farther O +from O +eEF2 B-protein +( O +Figure O +5d O +). O + +We O +conclude O +that O +unlike O +other O +conformations O +of O +the O +ribosome B-complex_assembly +, O +the O +fully B-protein_state +rotated I-protein_state +40S B-complex_assembly +subunit B-structure_element +of O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +provides O +an O +interaction B-site +surface I-site +, O +complementing O +the O +P B-structure_element +stalk I-structure_element +and O +SRL B-structure_element +, O +for O +binding O +of O +the O +GTP B-protein_state +- I-protein_state +bound I-protein_state +translocase B-protein_type +. O + +This O +structural O +basis O +rationalizes O +the O +observation O +of O +transient O +stabilization O +of O +the O +rotated B-protein_state +70S B-complex_assembly +ribosome I-complex_assembly +upon O +EF B-complex_assembly +- I-complex_assembly +G I-complex_assembly +• I-complex_assembly +GTP I-complex_assembly +binding O +and O +prior O +to O +translocation O +. O + +The O +least B-protein_state +rotated I-protein_state +conformation O +of O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +Structure B-evidence +V I-evidence +suggests O +conformational O +changes O +that O +may O +trigger O +eEF2 B-protein +release O +from O +the O +ribosome B-complex_assembly +at O +the O +end O +of O +translocation O +. O + +The O +most O +pronounced O +inter O +- O +domain O +rearrangement O +in O +eEF2 B-protein +involves O +movement O +of O +domain O +III B-structure_element +. O + +In O +the O +rotated B-protein_state +or O +mid B-protein_state +- I-protein_state +rotated I-protein_state +Structures B-evidence +I I-evidence +through I-evidence +III I-evidence +, O +this O +domain O +remains O +rigidly O +associated O +with O +domain O +V B-structure_element +and O +the O +N O +- O +terminal O +superdomain B-structure_element +and O +does O +not O +undergo O +noticeable O +rearrangements O +. O + +In O +Structure B-evidence +V I-evidence +, O +however O +, O +the O +tip O +of O +helix B-structure_element +A I-structure_element +of O +domain O +III B-structure_element +is O +displaced O +toward O +domain O +I B-structure_element +by O +~ O +5 O +Å O +relative O +to O +that O +in O +mid B-protein_state +- I-protein_state +rotated I-protein_state +or O +fully B-protein_state +rotated I-protein_state +structures B-evidence +. O + +This O +displacement O +is O +caused O +by O +the O +8 O +Å O +movement O +of O +the O +40S B-complex_assembly +body B-structure_element +protein O +uS12 B-protein +upon O +reverse O +intersubunit O +rotation O +from O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +( O +Figure O +6d O +). O + +We O +propose O +that O +the O +shift O +of O +domain O +III B-structure_element +by O +uS12 B-protein +initiates O +intra O +- O +domain O +rearrangements O +in O +eEF2 B-protein +, O +which O +unstack O +the O +β B-structure_element +- I-structure_element +platform I-structure_element +of O +domain O +III B-structure_element +from O +that O +of O +domain O +V B-structure_element +. O +This O +would O +result O +in O +a O +conformation O +characteristic O +of O +free B-protein_state +eEF2 B-protein +and O +EF B-protein +- I-protein +G I-protein +in O +which O +the O +β B-structure_element +- I-structure_element +platforms I-structure_element +are O +nearly O +perpendicular O +. O + +As O +we O +discuss O +below O +, O +Structure B-evidence +V I-evidence +captures O +a O +' O +pre B-protein_state +- I-protein_state +unstacking I-protein_state +' O +state O +due O +to O +stabilization O +of O +the O +interface B-site +between O +domains O +III B-structure_element +and O +V B-structure_element +by O +sordarin B-chemical +. O + +Sordarin B-chemical +stabilizes O +GDP B-protein_state +- I-protein_state +bound I-protein_state +eEF2 B-protein +on O +the O +ribosome B-complex_assembly + +Sordarin B-chemical +is O +a O +potent O +antifungal O +antibiotic O +that O +inhibits O +translation O +. O + +Based O +on O +biochemical B-experimental_method +experiments I-experimental_method +, O +two O +alternative O +mechanisms O +of O +action O +were O +proposed O +: O +sordarin B-chemical +either O +prevents O +eEF2 B-protein +departure O +by O +inhibiting O +GTP B-chemical +hydrolysis O +or O +acts O +after O +GTP B-chemical +hydrolysis O +. O + +Although O +our O +complex O +was O +assembled O +using O +eEF2 B-complex_assembly +• I-complex_assembly +GTP I-complex_assembly +, O +density B-evidence +maps I-evidence +clearly O +show O +GDP B-chemical +and O +Mg2 B-chemical ++ I-chemical +in O +each O +structure B-evidence +( O +Figure O +5g O +). O + +Our O +structures B-evidence +therefore O +indicate O +that O +sordarin B-chemical +stalls O +eEF2 B-protein +on O +the O +ribosome B-complex_assembly +in O +the O +GDP B-protein_state +- I-protein_state +bound I-protein_state +form O +, O +i O +. O +e O +. O +following O +GTP B-chemical +hydrolysis O +and O +phosphate O +release O +. O + +The O +mechanism O +of O +stalling O +is O +suggested O +by O +comparison O +of O +pre B-protein_state +- I-protein_state +translocation I-protein_state +and O +post B-protein_state +- I-protein_state +translocation I-protein_state +structures B-evidence +in O +our O +ensemble O +. O + +In O +all O +five O +structures B-evidence +, O +sordarin B-chemical +is O +bound B-protein_state +between O +domains O +III B-structure_element +and O +V B-structure_element +of O +eEF2 B-protein +, O +stabilized O +by O +hydrophobic B-bond_interaction +interactions I-bond_interaction +identical O +to O +those O +in O +the O +isolated B-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +sordarin I-complex_assembly +complex O +( O +Figures O +5g O +and O +h O +). O + +In O +the O +nearly B-protein_state +non I-protein_state +- I-protein_state +rotated I-protein_state +post B-protein_state +- I-protein_state +translocation I-protein_state +Structure B-evidence +V I-evidence +, O +the O +tip O +of O +domain O +III B-structure_element +is O +shifted O +, O +however O +the O +interface B-site +between O +domains O +III B-structure_element +and O +V B-structure_element +remains O +unchanged O +, O +suggesting O +strong O +stabilization O +of O +this O +interface B-site +by O +sordarin B-chemical +. O + +We O +note O +that O +Structure B-evidence +V I-evidence +is O +slightly O +more O +rotated O +than O +the O +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +in O +the O +absence B-protein_state +of I-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +sordarin I-complex_assembly +, O +implying O +that O +sordarin B-chemical +interferes O +with O +the O +final O +stages O +of O +reverse O +rotation O +of O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +. O + +We O +propose O +that O +sordarin B-chemical +acts O +to O +prevent O +full O +reverse O +rotation O +and O +release O +of O +eEF2 B-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +by O +stabilizing O +the O +interdomain B-site +interface I-site +and O +thus O +blocking O +uS12 B-protein +- O +induced O +disengagement O +of O +domain O +III B-structure_element +from O +domain O +V B-structure_element +. O + +Implications O +for O +tRNA B-chemical +and O +mRNA B-chemical +translocation O +during O +translation O + +Because O +translocation O +of O +tRNA B-chemical +must O +involve O +large O +- O +scale O +dynamics O +, O +this O +step O +has O +long O +been O +regarded O +as O +the O +most O +puzzling O +step O +of O +translation O +. O + +Intersubunit O +rearrangements O +and O +tRNA B-chemical +hybrid B-protein_state +states O +have O +been O +proposed O +to O +play O +key O +roles O +half O +a O +century O +ago O +. O + +Despite O +an O +impressive O +body B-structure_element +of O +biochemical B-evidence +, I-evidence +fluorescence I-evidence +and I-evidence +structural I-evidence +data I-evidence +accumulated O +since O +then O +, O +translocation O +remains O +the O +least O +understood O +step O +of O +elongation O +. O + +The O +structural O +understanding O +of O +ribosome B-complex_assembly +and O +tRNA B-chemical +dynamics O +has O +been O +greatly O +aided O +by O +a O +wealth O +of O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +and O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +( O +reviewed O +in O +). O + +However O +, O +visualization O +of O +the O +eEF2 B-protein +/ O +EF B-protein +- I-protein +G I-protein +- O +induced O +translocation O +is O +confined O +to O +very O +early O +pre B-protein_state +- I-protein_state +EF I-protein_state +- I-protein_state +G I-protein_state +- I-protein_state +entry I-protein_state +states O +and O +late O +( O +almost B-protein_state +translocated I-protein_state +or O +fully B-protein_state +translocated I-protein_state +) O +states O +, O +leaving O +most O +of O +the O +path O +from O +the O +A B-site +to I-site +the I-site +P I-site +site I-site +uncharacterized O +( O +Figure O +1 O +— O +figure O +supplement O +1 O +). O + +Our O +study O +provides O +new O +insights O +into O +the O +structural O +understanding O +of O +tRNA B-chemical +translocation O +. O + +First O +, O +we O +propose O +that O +tRNA B-chemical +and O +IRES B-site +translocations O +occur O +via O +the O +same O +general O +trajectory O +. O + +This O +is O +evident O +from O +the O +fact O +that O +ribosome B-complex_assembly +rearrangements O +in O +translocation O +are O +inherent O +to O +the O +ribosome B-complex_assembly +and O +likely O +occur O +in O +similar O +ways O +in O +both O +cases O +. O + +Furthermore O +, O +the O +step O +- O +wise O +coupling O +of O +ribosome B-complex_assembly +dynamics O +with O +IRES B-site +translocation O +is O +overall O +consistent O +with O +that O +observed O +for O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +translocation O +in O +solution O +. O + +For O +example O +, O +fluorescence B-experimental_method +and I-experimental_method +biochemical I-experimental_method +studies I-experimental_method +revealed O +that O +the O +early O +pre B-protein_state +- I-protein_state +translocation I-protein_state +EF B-protein_state +- I-protein_state +G I-protein_state +- I-protein_state +bound I-protein_state +ribosomes B-complex_assembly +are O +fully B-protein_state +rotated I-protein_state +and O +translocation O +of O +the O +tRNA B-complex_assembly +- I-complex_assembly +mRNA I-complex_assembly +complex O +occurs O +during O +reverse O +rotation O +of O +the O +small B-structure_element +subunit I-structure_element +, O +coupled O +with O +head B-structure_element +swivel O +. O + +The O +sequence O +of O +ribosome B-complex_assembly +rearrangements O +during O +IRES B-site +translocation O +also O +agrees O +with O +that O +inferred O +from O +70S B-complex_assembly +• I-complex_assembly +EF I-complex_assembly +- I-complex_assembly +G I-complex_assembly +structures B-evidence +, O +including O +those O +in O +which O +the O +A B-site +- I-site +to I-site +- I-site +P I-site +- I-site +site I-site +translocating O +tRNA B-chemical +was O +not O +present O +. O + +Specifically O +, O +an O +earlier O +translocation O +intermediate O +ribosome B-complex_assembly +( O +TIpre O +) O +was O +proposed O +to O +adopt O +a O +rotated B-protein_state +( O +7 O +– O +9 O +°) O +body B-structure_element +and O +a O +partly B-protein_state +rotated I-protein_state +head B-structure_element +( O +5 O +– O +7 O +. O +5 O +°), O +in O +agreement O +with O +the O +conformation O +of O +our O +Structure B-evidence +I I-evidence +. O +The O +most B-protein_state +swiveled I-protein_state +head B-structure_element +( O +18 O +– O +21 O +°) O +was O +observed O +in O +a O +mid B-protein_state +- I-protein_state +rotated I-protein_state +ribosome B-complex_assembly +( O +3 O +– O +5 O +°) O +of O +a O +later O +translocation O +intermediate O +TIpost O +, O +similar O +to O +the O +conformation O +of O +our O +Structure B-evidence +III I-evidence +. O + +Overall O +, O +these O +correlations O +suggest O +that O +the O +intermediate O +locations O +of O +the O +elusive O +A B-site +- I-site +to I-site +- I-site +P I-site +- I-site +site I-site +translocating O +tRNA B-chemical +are O +similar O +to O +those O +of O +PKI B-structure_element +in O +our O +structures B-evidence +. O + +Second O +, O +the O +structures B-evidence +clarify O +the O +structural O +basis O +of O +the O +often O +- O +used O +but O +structurally O +undefined O +terms O +' O +locking O +' O +and O +' O +unlocking O +' O +with O +respect O +to O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +complex O +( O +Figure O +6f O +). O + +We O +deem O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +complex O +locked B-protein_state +, O +because O +the O +A B-protein_state +- I-protein_state +site I-protein_state +bound I-protein_state +ASL O +- O +mRNA B-chemical +is O +stabilized O +by O +interactions O +with O +the O +decoding B-site +center I-site +. O + +These O +interactions O +are O +maintained O +for O +the O +classical B-protein_state +- O +and O +hybrid B-protein_state +- O +state O +tRNAs B-chemical +in O +the O +spontaneously O +sampled O +non B-protein_state +- I-protein_state +rotated I-protein_state +and O +rotated B-protein_state +ribosomes B-complex_assembly +, O +respectively O +. O + +Unlocking O +involves O +separation O +of O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +helix I-structure_element +from O +the O +decoding B-site +center I-site +residues O +by O +the O +protruding O +tip O +of O +eEF2 B-protein +/ O +EF B-protein +- I-protein +G I-protein +( O +Figure O +7 O +), O +occurring O +in O +the O +fully B-protein_state +rotated I-protein_state +ribosome B-complex_assembly +at O +an O +early O +pre B-protein_state +- I-protein_state +translocation I-protein_state +step O +. O + +This O +unlatches O +the O +head B-structure_element +, O +allowing O +creation O +of O +hitherto O +elusive O +intermediate O +tRNA B-chemical +positions O +during O +spontaneous O +reverse O +body B-structure_element +rotation O +. O + +Third O +, O +our O +findings O +uncover O +a O +new O +role O +of O +the O +head B-structure_element +swivel O +. O + +Previous O +studies O +showed O +that O +this O +movement O +widens O +the O +constriction B-site +(' O +gate B-site +') O +between O +the O +P B-site +and I-site +E I-site +sites I-site +, O +thus O +allowing O +the O +P B-site +- O +tRNA B-chemical +passage O +to O +the O +E B-site +site I-site +. O + +In O +addition O +to O +the O +' O +gate B-site +- O +opening O +' O +role O +, O +we O +now O +show O +that O +the O +head B-structure_element +swivel O +brings O +the O +head B-structure_element +A B-site +site I-site +to O +the O +body B-structure_element +P B-site +site I-site +, O +allowing O +a O +step O +- O +wise O +conveying O +of O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +helix I-structure_element +between O +the O +A B-site +and I-site +P I-site +sites I-site +. O + +Finally O +, O +the O +similar O +populations O +of O +particles B-experimental_method +( O +within O +a O +2X O +range O +) O +in O +our O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +reconstructions B-evidence +( O +Figure O +1 O +— O +figure O +supplement O +2 O +) O +suggest O +that O +the O +intermediate O +translocation O +states O +sample O +several O +energetically O +similar O +and O +interconverting O +conformations O +. O + +This O +is O +consistent O +with O +the O +idea O +of O +a O +rather O +flat O +energy O +landscape O +of O +translocation O +, O +suggested O +by O +recent O +work O +that O +measured O +mechanical O +work O +produced O +by O +the O +ribosome B-complex_assembly +during O +translocation O +. O + +Our O +findings O +implicate O +, O +however O +, O +that O +the O +energy O +landscape O +is O +not O +completely O +flat O +and O +contains O +local O +minima O +for O +transient O +positions O +of O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +helix I-structure_element +between O +the O +A B-site +and I-site +P I-site +sites I-site +. O + +The O +shift O +of O +the O +PKI B-structure_element +with O +respect O +to O +the O +body B-structure_element +occurs O +during O +forward O +head B-structure_element +swivel O +in O +two O +major O +sub O +- O +steps O +of O +~ O +4 O +Å O +each O +( O +initiation B-complex_assembly +complex I-complex_assembly +to O +I B-evidence +, O +and O +I B-evidence +to O +II B-evidence +), O +after O +which O +PKI B-structure_element +undergoes O +small O +shifts O +to O +settle O +in O +the O +body B-structure_element +P B-site +site I-site +in O +Structures B-evidence +III I-evidence +, I-evidence +IV I-evidence +and I-evidence +V I-evidence +( O +Figure O +2 O +— O +source O +data O +1 O +). O + +Movement O +of O +PKI B-structure_element +relative O +to O +the O +head B-structure_element +occurs O +during O +the O +subsequent O +reverse O +swivel O +in O +three O +3 O +– O +7 O +Å O +sub O +- O +steps O +( O +II B-evidence +to I-evidence +III I-evidence +to I-evidence +IV I-evidence +to I-evidence +V I-evidence +). O + +We O +note O +that O +four O +of O +our O +near O +- O +atomic O +resolution O +maps B-evidence +comprised O +~ O +30 O +, O +000 O +particles B-experimental_method +each O +, O +the O +minimum O +number O +required O +for O +a O +near B-evidence +- I-evidence +atomic I-evidence +- I-evidence +resolution I-evidence +reconstruction I-evidence +of O +the O +ribosome B-complex_assembly +. O + +Translation O +of O +viral B-taxonomy_domain +mRNA B-chemical + +Our O +work O +sheds O +light O +on O +the O +dynamic O +mechanism O +of O +cap O +- O +independent O +translation O +by O +IGR B-structure_element +IRESs B-site +, O +tightly O +coupled O +with O +the O +universally B-protein_state +conserved I-protein_state +dynamic O +properties O +of O +the O +ribosome B-complex_assembly +. O + +The O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +demonstrate O +that O +the O +TSV B-species +IRES B-site +structurally O +and O +dynamically O +represents O +a O +chimera O +of O +the O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +translocating O +complex O +( O +A B-complex_assembly +/ I-complex_assembly +P I-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +• I-complex_assembly +P I-complex_assembly +/ I-complex_assembly +E I-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +). O + +Like O +in O +the O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +translocating O +complex O +in O +which O +the O +two O +tRNAs B-chemical +move O +independently O +of O +each O +other O +, O +the O +PKI B-structure_element +domain O +moves O +relative O +to O +the O +5 B-structure_element +´- I-structure_element +domain I-structure_element +, O +causing O +the O +IRES B-site +to O +undergo O +an O +inchworm B-protein_state +- O +walk O +translocation O +. O + +A O +large O +structural O +difference O +between O +the O +IRES B-site +and O +the O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +exists O +, O +however O +, O +in O +that O +the O +IRES B-site +lacks B-protein_state +three O +out O +of O +six O +tRNA B-structure_element +- I-structure_element +like I-structure_element +domains I-structure_element +involved O +in O +tRNA B-chemical +translocation O +. O + +This O +difference O +likely O +accounts O +for O +the O +inefficient O +translocation O +of O +the O +IRES B-site +, O +which O +is O +difficult O +to O +stabilize O +in O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +state O +and O +therefore O +is O +prone O +to O +reverse O +translocation O +. O + +Although O +structurally O +handicapped O +, O +the O +TSV B-species +IRES B-site +manages O +to O +translocate O +by O +employing O +ribosome B-complex_assembly +dynamics O +that O +are O +remarkably O +similar O +to O +that O +in O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +translocation O +. O + +The O +uniformity O +of O +ribosome B-complex_assembly +dynamics O +underscores O +the O +idea O +that O +translocation O +is O +an O +inherent O +and O +structurally O +- O +optimized O +property O +of O +the O +ribosome B-complex_assembly +, O +supported O +also O +by O +translocation O +activity O +in O +the O +absence B-protein_state +of I-protein_state +the O +elongation B-protein_type +factor I-protein_type +. O + +This O +property O +is O +rendered O +by O +the O +relative O +mobility O +of O +the O +three O +major O +building O +blocks O +, O +the O +60S B-complex_assembly +subunit B-structure_element +and O +the O +40S B-complex_assembly +head B-structure_element +and O +body B-structure_element +, O +assisted O +by O +ligand B-structure_element +- I-structure_element +interacting I-structure_element +extensions I-structure_element +including O +the O +L1 B-structure_element +stalk I-structure_element +and O +the O +P B-structure_element +stalk I-structure_element +. O + +Intergenic O +IRESs B-site +, O +in O +turn O +, O +represent O +a O +striking O +example O +of O +convergent O +molecular O +evolution O +. O + +Viral B-taxonomy_domain +mRNAs B-chemical +have O +evolved O +to O +adopt O +an O +atypical O +structure B-evidence +to O +employ O +the O +inherent O +ribosome B-complex_assembly +dynamics O +, O +to O +be O +able O +to O +hijack O +the O +host O +translational O +machinery O +in O +a O +simple O +fashion O +. O + +Ensemble O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method + +Our O +current O +understanding O +of O +macromolecular O +machines O +, O +such O +as O +the O +ribosome B-complex_assembly +, O +is O +often O +limited O +by O +a O +gap O +between O +biophysical B-experimental_method +/ I-experimental_method +biochemical I-experimental_method +studies I-experimental_method +and O +structural B-experimental_method +studies I-experimental_method +. O + +For O +example O +, O +Förster B-experimental_method +resonance I-experimental_method +energy I-experimental_method +transfer I-experimental_method +can O +provide O +insight O +into O +the O +macromolecular O +dynamics O +of O +an O +assembly O +at O +the O +single O +- O +molecule O +level O +but O +is O +limited O +to O +specifically O +labeled O +locations O +within O +the O +assembly O +. O + +High O +- O +resolution O +crystal B-evidence +structures I-evidence +, O +on O +the O +other O +hand O +, O +can O +provide O +static O +images O +of O +an O +assembly O +, O +and O +the O +structural O +dynamics O +can O +only O +be O +inferred O +by O +comparing O +structures B-evidence +that O +are O +usually O +obtained O +in O +different O +experiments O +and O +under O +different O +, O +often O +non O +- O +native O +, O +conditions O +. O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +offers O +the O +possibility O +of O +obtaining O +integrated O +information O +of O +both O +structure B-evidence +and O +dynamics O +as O +demonstrated O +in O +lower O +- O +resolution O +studies O +of O +bacterial B-taxonomy_domain +ribosome B-complex_assembly +complexes O +. O + +This O +is O +presumably O +one O +of O +the O +reasons O +why O +most O +recent O +studies O +of O +ribosome B-complex_assembly +complexes O +have O +focused O +on O +a O +single O +high O +- O +resolution O +structure B-evidence +despite O +the O +non O +- O +uniform O +local O +resolution O +of O +the O +maps B-evidence +that O +likely O +reflects O +structural O +heterogeneity O +. O + +The O +computational O +efficiency O +of O +FREALIGN B-experimental_method +has O +allowed O +us O +to O +classify O +a O +relatively O +large O +dataset O +( O +1 O +. O +1 O +million O +particles B-experimental_method +) O +into O +15 O +classes O +( O +Figure O +1 O +— O +figure O +supplement O +2 O +) O +and O +obtain O +eight O +near O +- O +atomic O +- O +resolution O +structures B-evidence +from O +it O +. O + +The O +classification O +, O +which O +followed O +an O +initial O +alignment O +of O +all O +particles B-experimental_method +to O +a O +single O +reference O +, O +required O +about O +130 O +, O +000 O +CPU O +hours O +or O +about O +five O +to O +six O +full O +days O +on O +a O +1000 O +- O +CPU O +cluster O +. O + +Therefore O +, O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +has O +the O +potential O +to O +become O +a O +standard O +tool O +for O +uncovering O +detailed O +dynamic O +pathways O +of O +complex O +macromolecular O +machines O +. O + +A O +unified O +mechanism O +for O +proteolysis O +and O +autocatalytic B-ptm +activation I-ptm +in O +the O +20S B-complex_assembly +proteasome I-complex_assembly + +Biogenesis O +of O +the O +20S B-complex_assembly +proteasome I-complex_assembly +is O +tightly O +regulated O +. O + +The O +N O +- O +terminal O +propeptides B-structure_element +protecting O +the O +active B-site +- I-site +site I-site +threonines B-residue_name +are O +autocatalytically B-ptm +released O +only O +on O +completion O +of O +assembly O +. O + +However O +, O +the O +trigger O +for O +the O +self O +- O +activation O +and O +the O +reason O +for O +the O +strict B-protein_state +conservation I-protein_state +of O +threonine B-residue_name +as O +the O +active O +site O +nucleophile O +remain O +enigmatic O +. O + +Here O +we O +use O +mutagenesis B-experimental_method +, O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +and O +biochemical B-experimental_method +assays I-experimental_method +to O +suggest O +that O +Lys33 B-residue_name_number +initiates O +nucleophilic O +attack O +of O +the O +propeptide B-structure_element +by O +deprotonating O +the O +Thr1 B-residue_name_number +hydroxyl O +group O +and O +that O +both O +residues O +together O +with O +Asp17 B-residue_name_number +are O +part O +of O +a O +catalytic B-site +triad I-site +. O + +Substitution B-experimental_method +of O +Thr1 B-residue_name_number +by O +Cys B-residue_name +disrupts O +the O +interaction O +with O +Lys33 B-residue_name_number +and O +inactivates B-protein_state +the O +proteasome B-complex_assembly +. O + +Although O +a O +Thr1Ser B-mutant +mutant B-protein_state +is O +active B-protein_state +, O +it O +is O +less O +efficient O +compared O +with O +wild B-protein_state +type I-protein_state +because O +of O +the O +unfavourable O +orientation O +of O +Ser1 B-residue_name_number +towards O +incoming O +substrates O +. O + +This O +work O +provides O +insights O +into O +the O +basic O +mechanism O +of O +proteolysis O +and O +propeptide B-ptm +autolysis I-ptm +, O +as O +well O +as O +the O +evolutionary O +pressures O +that O +drove O +the O +proteasome B-complex_assembly +to O +become O +a O +threonine B-protein_type +protease I-protein_type +. O + +The O +proteasome B-complex_assembly +, O +an O +essential O +molecular O +machine O +, O +is O +a O +threonine B-protein_type +protease I-protein_type +, O +but O +the O +evolution O +and O +the O +components O +of O +its O +proteolytic O +centre O +are O +unclear O +. O + +Here O +, O +the O +authors O +use O +structural O +biology O +and O +biochemistry O +to O +investigate O +the O +role O +of O +proteasome B-complex_assembly +active B-site +site I-site +residues O +on O +maturation O +and O +activity O +. O + +The O +20S B-complex_assembly +proteasome I-complex_assembly +core I-complex_assembly +particle I-complex_assembly +( O +CP B-complex_assembly +) O +is O +the O +key O +non B-protein_type +- I-protein_type +lysosomal I-protein_type +protease I-protein_type +of O +eukaryotic B-taxonomy_domain +cells O +. O + +Its O +seven O +different O +α B-protein +and O +seven O +different O +β B-protein +subunits I-protein +assemble O +into O +four O +heptameric B-oligomeric_state +rings B-structure_element +that O +are O +stacked O +on O +each O +other O +to O +form O +a O +hollow B-structure_element +cylinder I-structure_element +. O + +While O +the O +inactive B-protein_state +α B-protein +subunits I-protein +build O +the O +two O +outer O +rings B-structure_element +, O +the O +β B-protein +subunits I-protein +form O +the O +inner O +rings B-structure_element +. O + +Only O +three O +out O +of O +the O +seven O +different O +β B-protein +subunits I-protein +, O +namely O +β1 B-protein +, O +β2 B-protein +and O +β5 B-protein +, O +bear O +N O +- O +terminal O +proteolytic B-site +active I-site +centres I-site +, O +and O +before O +CP B-complex_assembly +maturation O +these O +are O +protected O +by O +propeptides B-structure_element +. O + +In O +the O +last O +stage O +of O +CP B-complex_assembly +biogenesis O +, O +the O +prosegments B-structure_element +are O +autocatalytically B-ptm +removed I-ptm +through O +nucleophilic O +attack O +by O +the O +active B-site +site I-site +residue I-site +Thr1 B-residue_name_number +on O +the O +preceding O +peptide O +bond O +involving O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +). I-residue_name_number + +Release O +of O +the O +propeptides B-structure_element +creates O +a O +functionally O +active B-protein_state +CP B-complex_assembly +that O +cleaves O +proteins O +into O +short O +peptides O +. O + +Although O +the O +chemical O +nature O +of O +the O +substrate B-site +- I-site +binding I-site +channel I-site +and O +hence O +substrate O +preferences O +are O +unique O +to O +each O +of O +the O +distinct O +active B-protein_state +β B-protein +subunits I-protein +, O +all O +active B-site +sites I-site +employ O +an O +identical O +reaction O +mechanism O +to O +hydrolyse O +peptide O +bonds O +. O + +Nucleophilic O +attack O +of O +Thr1Oγ B-residue_name_number +on O +the O +carbonyl O +carbon O +atom O +of O +the O +scissile O +peptide O +bond O +creates O +a O +first O +cleavage O +product O +and O +a O +covalent O +acyl O +- O +enzyme O +intermediate O +. O + +Hydrolysis O +of O +this O +complex B-complex_assembly +by O +the O +addition O +of O +a O +nucleophilic O +water B-chemical +molecule O +regenerates O +the O +enzyme B-complex_assembly +and O +releases O +the O +second O +peptide B-chemical +fragment O +. O + +The O +proteasome B-complex_assembly +belongs O +to O +the O +family O +of O +N B-protein_type +- I-protein_type +terminal I-protein_type +nucleophilic I-protein_type +( I-protein_type +Ntn I-protein_type +) I-protein_type +hydrolases I-protein_type +, O +and O +the O +free B-protein_state +N O +- O +terminal O +amine O +group O +of O +Thr1 B-residue_name_number +was O +proposed O +to O +deprotonate O +the O +Thr1 B-residue_name_number +hydroxyl O +group O +to O +generate O +a O +nucleophilic O +Thr1Oγ B-residue_name_number +for O +peptide O +- O +bond O +cleavage O +. O + +This O +mechanism O +, O +however O +, O +cannot O +explain O +autocatalytic B-ptm +precursor I-ptm +processing I-ptm +because O +in O +the O +immature B-protein_state +active B-site +sites I-site +, O +Thr1N B-residue_name_number +is O +part O +of O +the O +peptide O +bond O +with O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +), I-residue_name_number +the O +bond O +that O +needs O +to O +be O +hydrolysed O +. O + +An O +alternative O +candidate O +for O +deprotonating O +the O +Thr1 B-residue_name_number +hydroxyl O +group O +is O +the O +side O +chain O +of O +Lys33 B-residue_name_number +as O +it O +is O +within O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +distance O +to O +Thr1OH B-residue_name_number +( O +2 O +. O +7 O +Å O +). O + +In O +principle O +it O +could O +function O +as O +the O +general O +base O +during O +both O +autocatalytic B-ptm +removal I-ptm +of O +the O +propeptide B-structure_element +and O +protein O +substrate O +cleavage O +. O + +Here O +we O +provide O +experimental O +evidences O +for O +this O +distinct O +view O +of O +the O +proteasome B-complex_assembly +active B-site +- I-site +site I-site +mechanism O +. O + +Data O +from O +biochemical B-experimental_method +and I-experimental_method +structural I-experimental_method +analyses I-experimental_method +of O +proteasome O +variants O +with O +mutations O +in O +the O +β5 B-protein +propeptide B-structure_element +and O +the O +active B-site +site I-site +strongly O +support O +the O +model O +and O +deliver O +novel O +insights O +into O +the O +structural O +constraints O +required O +for O +the O +autocatalytic B-ptm +activation I-ptm +of O +the O +proteasome B-complex_assembly +. O + +Furthermore O +, O +we O +determine O +the O +advantages O +of O +Thr B-residue_name +over O +Cys B-residue_name +or O +Ser B-residue_name +as O +the O +active O +- O +site O +nucleophile O +using O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +together O +with O +activity B-experimental_method +and I-experimental_method +inhibition I-experimental_method +assays I-experimental_method +. O + +Inactivation O +of O +proteasome B-complex_assembly +subunits B-protein +by O +T1A B-mutant +mutations B-experimental_method + +Proteasome B-complex_assembly +- O +mediated O +degradation O +of O +cell O +- O +cycle O +regulators O +and O +potentially O +toxic O +misfolded O +proteins O +is O +required O +for O +the O +viability O +of O +eukaryotic B-taxonomy_domain +cells O +. O + +Inactivation O +of O +the O +active B-site +site I-site +Thr1 B-residue_name_number +by O +mutation B-experimental_method +to I-experimental_method +Ala B-residue_name +has O +been O +used O +to O +study O +substrate O +specificity O +and O +the O +hierarchy O +of O +the O +proteasome B-complex_assembly +active B-site +sites I-site +. O + +Yeast B-taxonomy_domain +strains O +carrying O +the O +single O +mutations O +β1 B-mutant +- I-mutant +T1A I-mutant +or O +β2 B-mutant +- I-mutant +T1A I-mutant +, O +or O +both O +, O +are O +viable O +, O +even O +though O +one O +or O +two O +of O +the O +three O +distinct O +catalytic B-protein_state +β B-protein +subunits I-protein +are O +disabled B-protein_state +and O +carry B-protein_state +remnants I-protein_state +of I-protein_state +their O +N O +- O +terminal O +propeptides B-structure_element +( O +Table O +1 O +). O + +These O +results O +indicate O +that O +the O +β1 B-protein +and O +β2 B-protein +proteolytic O +activities O +are O +not O +essential O +for O +cell O +survival O +. O + +By O +contrast O +, O +the O +T1A B-mutant +mutation O +in O +subunit O +β5 B-protein +has O +been O +reported O +to O +be O +lethal O +or O +nearly O +so O +. O + +Viability O +is O +restored O +if O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +subunit O +has O +its O +propeptide B-structure_element +( O +pp B-chemical +) O +deleted B-experimental_method +but I-experimental_method +expressed I-experimental_method +separately I-experimental_method +in O +trans B-protein_state +( O +β5 B-mutant +- I-mutant +T1A I-mutant +pp B-chemical +trans B-protein_state +), O +although O +substantial O +phenotypic O +impairment O +remains O +( O +Table O +1 O +). O + +Our O +present O +crystallographic B-experimental_method +analysis I-experimental_method +of O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +pp B-chemical +trans B-protein_state +mutant B-protein_state +demonstrates O +that O +the O +mutation B-experimental_method +per O +se O +does O +not O +structurally O +alter O +the O +catalytic B-site +active I-site +site I-site +and O +that O +the O +trans B-experimental_method +- I-experimental_method +expressed I-experimental_method +β5 B-protein +propeptide B-structure_element +is O +not B-protein_state +bound I-protein_state +in O +the O +β5 B-protein +substrate B-site +- I-site +binding I-site +channel I-site +( O +Supplementary O +Fig O +. O +1a O +). O + +The O +extremely O +weak O +growth O +of O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +mutant B-protein_state +pp B-chemical +cis B-protein_state +described O +by O +Chen O +and O +Hochstrasser O +compared O +with O +the O +inviability O +reported O +by O +Heinemeyer O +et O +al O +. O +prompted O +us O +to O +analyse O +this O +discrepancy O +. O + +Sequencing B-experimental_method +of I-experimental_method +the I-experimental_method +plasmids I-experimental_method +, O +testing O +them O +in O +both O +published O +yeast B-taxonomy_domain +strain O +backgrounds O +and O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +revealed O +that O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +mutant B-protein_state +pp B-chemical +cis B-protein_state +is O +viable O +, O +but O +suffers O +from O +a O +marked O +growth O +defect O +that O +requires O +extended O +incubation O +of O +4 O +– O +5 O +days O +for O +initial O +colony O +formation O +( O +Table O +1 O +and O +Supplementary O +Methods O +). O + +We O +also O +identified O +an O +additional O +point O +mutation O +K81R B-mutant +in O +subunit O +β5 B-protein +that O +was O +present O +in O +the O +allele O +used O +in O +ref O +.. O +This B-experimental_method +single I-experimental_method +amino I-experimental_method +- I-experimental_method +acid I-experimental_method +exchange I-experimental_method +is O +located O +at O +the O +interface B-site +of O +the O +subunits O +α4 B-protein +, O +β4 B-protein +and O +β5 B-protein +( O +Supplementary O +Fig O +. O +1b O +) O +and O +might O +weakly O +promote O +CP B-complex_assembly +assembly O +by O +enhancing O +inter O +- O +subunit O +contacts O +. O + +The O +slightly O +better O +growth O +of O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +mutant B-protein_state +allowed O +us O +to O +solve O +the O +crystal B-evidence +structure I-evidence +of O +a O +yeast B-taxonomy_domain +proteasome B-complex_assembly +( O +yCP B-complex_assembly +) O +with O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +mutation O +, O +which O +is O +discussed O +in O +the O +following O +section O +( O +for O +details O +see O +Supplementary O +Note O +1 O +). O + +Propeptide B-structure_element +conformation O +and O +triggering O +of O +autolysis B-ptm + +In O +the O +final O +steps O +of O +proteasome B-complex_assembly +biogenesis O +, O +the O +propeptides B-structure_element +are O +autocatalytically B-ptm +cleaved I-ptm +from O +the O +mature B-protein_state +β B-protein +- I-protein +subunit I-protein +domains I-protein +. O + +For O +subunit O +β1 B-protein +, O +this O +process O +was O +previously O +inferred O +to O +require O +that O +the O +propeptide B-structure_element +residue O +at O +position O +(- B-residue_number +2 I-residue_number +) I-residue_number +of O +the O +subunit O +precursor O +occupies O +the O +S1 B-site +specificity I-site +pocket I-site +of O +the O +substrate B-site +- I-site +binding I-site +channel I-site +formed O +by O +amino O +acid O +45 B-residue_number +( O +for O +details O +see O +Supplementary O +Note O +2 O +). O + +Furthermore O +, O +it O +was O +observed O +that O +the O +prosegment B-structure_element +forms O +an O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +in O +the O +active B-site +site I-site +, O +and O +that O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +adopts O +a O +γ B-structure_element +- I-structure_element +turn I-structure_element +conformation I-structure_element +, O +which O +by O +definition O +is O +characterized O +by O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +Leu B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +O O +and O +Thr1NH B-residue_name_number +( O +ref O +.). O + +Here O +we O +again O +analysed O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +mutant B-protein_state +crystallographically B-experimental_method +but O +in O +addition O +determined O +the O +structures B-evidence +of O +the O +β2 B-mutant +- I-mutant +T1A I-mutant +single O +and O +β1 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +β2 I-mutant +- I-mutant +T1A I-mutant +double O +mutants O +( O +Protein O +Data O +Bank O +( O +PDB O +) O +entry O +codes O +are O +provided O +in O +Supplementary O +Table O +1 O +). O + +In O +subunit O +β1 B-protein +, O +we O +found O +that O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +indeed O +forms O +a O +sharp B-structure_element +turn I-structure_element +, O +which O +relaxes O +on O +prosegment B-ptm +cleavage I-ptm +( O +Fig O +. O +1a O +and O +Supplementary O +Fig O +. O +2a O +). O + +However O +, O +the O +γ B-structure_element +- I-structure_element +turn I-structure_element +conformation I-structure_element +and O +the O +associated O +hydrogen B-bond_interaction +bond I-bond_interaction +initially O +proposed O +is O +for O +geometric O +and O +chemical O +reasons O +inappropriate O +and O +would O +not O +perfectly O +position O +the O +carbonyl O +carbon O +atom O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +for O +nucleophilic O +attack O +by O +Thr1 B-residue_name_number +. O + +Regarding O +the O +β2 B-protein +propeptide B-structure_element +, O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +occupies O +the O +S1 B-site +pocket I-site +but O +is O +less O +deeply O +anchored O +compared O +with O +Leu B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +in O +β1 B-protein +, O +which O +might O +be O +due O +to O +the O +rather O +large O +β2 B-protein +- O +S1 B-site +pocket I-site +created O +by O +Gly45 B-residue_name_number +. O + +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +positions O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +O O +via O +hydrogen B-bond_interaction +bonding I-bond_interaction +(∼ O +2 O +. O +8 O +Å O +) O +in O +a O +perfect O +trajectory O +for O +the O +nucleophilic O +attack O +by O +Thr1Oγ B-residue_name_number +( O +Fig O +. O +1b O +and O +Supplementary O +Fig O +. O +2b O +). O + +Next O +, O +we O +examined O +the O +position O +of O +the O +β5 B-protein +propeptide B-structure_element +in O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +mutant B-protein_state +. O + +Surprisingly O +, O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +is O +completely O +extended O +and O +forces O +the O +histidine B-residue_name +side O +chain O +at O +position O +(- B-residue_number +2 I-residue_number +) I-residue_number +to O +occupy O +the O +S2 B-site +instead O +of O +the O +S1 B-site +pocket I-site +, O +thereby O +disrupting O +the O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +. O + +Nonetheless O +, O +the O +carbonyl O +carbon O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +would O +be O +ideally O +placed O +for O +nucleophilic O +attack O +by O +Thr1Oγ B-residue_name_number +( O +Fig O +. O +1c O +and O +Supplementary O +Fig O +. O +2c O +, O +d O +). O + +As O +the O +K81R B-mutant +mutation O +is O +located O +far O +from O +the O +active B-site +site I-site +( O +Thr1Cα B-residue_name_number +– O +Arg81Cα B-residue_name_number +: O +24 O +Å O +), O +any O +influence O +on O +propeptide B-structure_element +conformation O +can O +be O +excluded O +. O + +Instead O +, O +the O +plasticity O +of O +the O +β5 B-protein +S1 B-site +pocket I-site +caused O +by O +the O +rotational O +flexibility O +of O +Met45 B-residue_name_number +might O +prevent O +stable O +accommodation O +of O +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +in O +the O +S1 B-site +site I-site +and O +thus O +also O +promote O +its O +immediate O +release O +after O +autolysis B-ptm +. O + +Processing O +of O +β O +- O +subunit O +precursors O +requires O +deprotonation O +of O +Thr1OH B-residue_name_number +; O +however O +, O +the O +general O +base O +initiating O +autolysis B-ptm +is O +unknown O +. O + +Remarkably O +, O +eukaryotic B-taxonomy_domain +proteasomal O +β5 B-protein +subunits O +bear O +a O +His B-residue_name +residue O +in O +position O +(- B-residue_number +2 I-residue_number +) I-residue_number +of O +the O +propeptide B-structure_element +( O +Supplementary O +Fig O +. O +3a O +). O + +As O +histidine B-residue_name +commonly O +functions O +as O +a O +proton O +shuttle O +in O +the O +catalytic B-site +triads I-site +of O +serine B-protein_type +proteases I-protein_type +, O +we O +investigated O +the O +role O +of O +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +in O +processing O +of O +the O +β5 B-protein +propeptide B-structure_element +by O +exchanging B-experimental_method +it I-experimental_method +for I-experimental_method +Asn B-residue_name +, O +Lys B-residue_name +, O +Phe B-residue_name +and O +Ala B-residue_name +. O +All O +yeast B-taxonomy_domain +mutants O +were O +viable O +at O +30 O +° O +C O +, O +but O +suffered O +from O +growth O +defects O +at O +37 O +° O +C O +with O +the O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +N I-mutant +and O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +F I-mutant +mutants O +being O +most O +affected O +( O +Supplementary O +Fig O +. O +3b O +and O +Table O +1 O +). O + +In O +agreement O +, O +the O +chymotrypsin O +- O +like O +( O +ChT O +- O +L O +) O +activity O +of O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +N I-mutant +and O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +F I-mutant +mutant B-protein_state +yCPs B-complex_assembly +was O +impaired O +in O +situ O +and O +in O +vitro O +( O +Supplementary O +Fig O +. O +3c O +). O + +Structural B-experimental_method +analyses I-experimental_method +revealed O +that O +the O +propeptides B-structure_element +of O +all O +mutant B-protein_state +yCPs B-complex_assembly +shared O +residual O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +densities I-evidence +. O + +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +and O +Phe B-residue_name +/ O +Lys B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +were O +visualized O +at O +low O +occupancy O +, O +while O +Ala B-residue_name +/ O +Asn B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +could O +not O +be O +assigned O +. O + +This O +observation O +indicates O +a O +mixture O +of O +processed B-protein_state +and O +unprocessed B-protein_state +β5 B-protein +subunits O +and O +partially O +impaired O +autolysis B-ptm +, O +thereby O +excluding O +any O +essential O +role O +of O +residue O +(- B-residue_number +2 I-residue_number +) I-residue_number +as O +the O +general O +base O +. O + +Next O +, O +we O +examined O +the O +effect O +of O +residue O +(- B-residue_number +2 I-residue_number +) I-residue_number +on O +the O +orientation O +of O +the O +propeptide B-structure_element +by O +creating B-experimental_method +mutants I-experimental_method +that I-experimental_method +combine I-experimental_method +the O +T1A B-mutant +( O +K81R B-mutant +) O +mutation B-experimental_method +( I-experimental_method +s I-experimental_method +) I-experimental_method +with O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +L I-mutant +, O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +T I-mutant +or O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +A I-mutant +substitutions B-experimental_method +. O + +Leu B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +is O +encoded O +in O +the O +yeast B-taxonomy_domain +β1 B-protein +subunit O +precursor O +( O +Supplementary O +Fig O +. O +3a O +); O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +is O +generally O +part O +of O +β2 B-protein +- O +propeptides B-structure_element +( O +Supplementary O +Fig O +. O +3a O +); O +and O +Ala B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +was O +expected O +to O +fit O +the O +β5 B-protein +- O +S1 B-site +pocket I-site +without O +inducing O +conformational O +changes O +of O +Met45 B-residue_name_number +, O +allowing O +it O +to O +accommodate O +‘ O +β1 O +- O +like O +' O +propeptide O +positioning O +. O + +As O +expected O +from O +β5 B-mutant +- I-mutant +T1A I-mutant +mutants O +, O +the O +yeasts B-taxonomy_domain +show O +severe O +growth O +phenotypes O +, O +with O +minor O +variations O +( O +Supplementary O +Fig O +. O +4a O +and O +Table O +1 O +). O + +We O +determined O +crystal B-evidence +structures I-evidence +of O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +L I-mutant +- I-mutant +T1A I-mutant +, O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +T I-mutant +- I-mutant +T1A I-mutant +and O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +A I-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +mutants O +( O +Supplementary O +Table O +1 O +). O + +For O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +A I-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +variant O +, O +only O +the O +residues O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +and O +Ala B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +could O +be O +visualized O +, O +indicating O +that O +Ala B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +leads O +to O +insufficient O +stabilization O +of O +the O +propeptide B-structure_element +in O +the O +substrate B-site +- I-site +binding I-site +channel I-site +( O +Supplementary O +Fig O +. O +4d O +). O + +By O +contrast O +, O +the O +prosegments B-structure_element +of O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +L I-mutant +- I-mutant +T1A I-mutant +and O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +T I-mutant +- I-mutant +T1A I-mutant +mutants O +were O +significantly O +better O +resolved O +in O +the O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +- I-evidence +density I-evidence +maps I-evidence +yet O +not O +at O +full O +occupancy O +( O +Supplementary O +Fig O +. O +4b O +, O +c O +and O +Supplementary O +Table O +1 O +), O +suggesting O +that O +the O +natural O +propeptide B-structure_element +bearing O +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +is O +most O +favourable O +. O + +Nevertheless O +, O +both O +Leu B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +and O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +were O +found O +to O +occupy O +the O +S1 B-site +specificity I-site +pocket I-site +formed O +by O +Met45 B-residue_name_number +( O +Fig O +. O +2a O +, O +b O +and O +Supplementary O +Fig O +. O +4f O +– O +h O +). O + +This O +result O +proves O +that O +the O +naturally O +occurring O +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +of O +the O +β5 B-protein +propeptide B-structure_element +does O +not O +stably O +fit O +into O +the O +S1 B-site +site I-site +. O + +Since O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +adopts O +the O +same O +position O +in O +both O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +and O +mutant B-protein_state +β5 B-protein +propeptides B-structure_element +, O +and O +since O +in O +all O +cases O +its O +carbonyl O +carbon O +is O +perfectly O +placed O +for O +nucleophilic O +attack O +by O +Thr1Oγ B-residue_name_number +( O +Fig O +. O +2b O +), O +we O +propose O +that O +neither O +binding O +of O +residue O +(- B-residue_number +2 I-residue_number +) I-residue_number +to O +the O +S1 B-site +pocket I-site +nor O +formation O +of O +the O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +is O +essential O +for O +autolysis B-ptm +of O +the O +propeptide B-structure_element +. O + +Next O +, O +we O +determined O +the O +crystal B-evidence +structure I-evidence +of O +a O +chimeric B-protein_state +yCP B-complex_assembly +having O +the O +yeast B-taxonomy_domain +β1 B-protein +- O +propeptide B-structure_element +replaced B-experimental_method +by I-experimental_method +its O +β5 B-protein +counterpart B-structure_element +. O + +Although O +we O +observed O +fragments O +of O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +density I-evidence +in O +the O +β1 B-protein +active B-site +site I-site +, O +the O +data O +were O +not O +interpretable O +. O + +Bearing O +in O +mind O +that O +in O +contrast O +to O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +in O +β2 B-protein +, O +Leu B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +in O +subunit O +β1 B-protein +is O +not B-protein_state +conserved I-protein_state +among O +species O +( O +Supplementary O +Fig O +. O +3a O +), O +we O +created B-experimental_method +a O +β2 B-mutant +- I-mutant +T I-mutant +(- I-mutant +2 I-mutant +) I-mutant +V I-mutant +proteasome B-complex_assembly +mutant B-protein_state +. O + +As O +proven O +by O +the O +β2 B-mutant +- I-mutant +T1A I-mutant +crystal B-evidence +structures I-evidence +, O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +O O +. O +Although O +this O +interaction O +was O +not O +observed O +for O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +T I-mutant +- I-mutant +T1A I-mutant +mutant B-protein_state +( O +Fig O +. O +2c O +and O +Supplementary O +Fig O +. O +4c O +, O +i O +), O +exchange B-experimental_method +of O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +by O +Val B-residue_name +in O +β2 B-protein +, O +a O +conservative O +mutation O +regarding O +size O +but O +drastic O +with O +respect O +to O +polarity O +, O +was O +found O +to O +inhibit O +maturation O +of O +this O +subunit O +( O +Fig O +. O +2d O +and O +Supplementary O +Fig O +. O +4e O +, O +j O +). O + +Notably O +, O +the O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +- I-evidence +density I-evidence +map I-evidence +displays O +a O +different O +orientation O +for O +the O +β2 B-protein +propeptide B-structure_element +than O +has O +been O +observed O +for O +the O +β2 B-mutant +- I-mutant +T1A I-mutant +proteasome B-complex_assembly +. O + +In O +particular O +, O +Val B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +is O +displaced O +from O +the O +S1 B-site +site I-site +and O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +is O +severely O +shifted O +( O +movement O +of O +the O +carbonyl O +oxygen O +atom O +of O +3 O +. O +8 O +Å O +), O +thereby O +preventing O +nucleophilic O +attack O +of O +Thr1 B-residue_name_number +( O +Fig O +. O +2d O +and O +Supplementary O +Fig O +. O +4j O +, O +k O +). O + +These O +results O +further O +confirm O +that O +correct O +positioning O +of O +the O +active B-site +- I-site +site I-site +residues I-site +and O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +is O +decisive O +for O +the O +maturation O +of O +the O +proteasome B-complex_assembly +. O + +The O +active B-site +site I-site +of O +the O +proteasome B-complex_assembly + +Proton O +shuttling O +from O +the O +proteasomal O +active B-site +site I-site +Thr1OH B-residue_name_number +to O +Thr1NH2 B-residue_name_number +via O +a O +nucleophilic O +water B-chemical +molecule O +was O +suggested O +to O +initiate O +peptide O +- O +bond O +hydrolysis O +. O + +However O +, O +in O +the O +immature B-protein_state +particle B-complex_assembly +Thr1NH2 B-residue_name_number +is O +blocked O +by O +the O +propeptide B-structure_element +and O +cannot O +activate O +Thr1Oγ B-residue_name_number +. O + +Instead O +, O +Lys33NH2 B-residue_name_number +, O +which O +is O +in O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +distance O +to O +Thr1Oγ B-residue_name_number +( O +2 O +. O +7 O +Å O +) O +in O +all O +catalytically B-protein_state +active I-protein_state +β B-protein +subunits I-protein +( O +Fig O +. O +3a O +, O +b O +), O +was O +proposed O +to O +serve O +as O +the O +proton O +acceptor O +. O + +A O +proposed O +catalytic B-site +tetrad I-site +model O +involving O +Thr1OH B-residue_name_number +, O +Thr1NH2 B-residue_name_number +, O +Lys33NH2 B-residue_name_number +and O +Asp17Oδ B-residue_name_number +, O +as O +well O +as O +a O +nucleophilic O +water B-chemical +molecule O +as O +the O +proton O +shuttle O +appeared O +to O +accommodate O +all O +possible O +views O +of O +the O +proteasomal O +active B-site +site I-site +. O + +Twenty O +years O +later O +, O +with O +a O +plethora O +of O +yCP B-complex_assembly +X B-evidence +- I-evidence +ray I-evidence +structures I-evidence +in O +hand O +, O +we O +decided O +to O +re O +- O +analyse O +the O +active B-site +site I-site +of O +the O +proteasome B-complex_assembly +and O +to O +resolve O +the O +uncertainty O +regarding O +the O +nature O +of O +the O +general O +base O +. O + +Mutation B-experimental_method +of O +β5 B-protein +- O +Lys33 B-residue_name_number +to O +Ala B-residue_name +causes O +a O +strongly O +deleterious O +phenotype O +, O +and O +previous O +structural B-experimental_method +and I-experimental_method +biochemical I-experimental_method +analyses I-experimental_method +confirmed O +that O +this O +is O +caused O +by O +failure O +of O +propeptide B-ptm +cleavage I-ptm +, O +and O +consequently O +, O +lack O +of O +ChT O +- O +L O +activity O +( O +Fig O +. O +4a O +, O +Supplementary O +Fig O +. O +3b O +and O +Table O +1 O +; O +for O +details O +see O +Supplementary O +Note O +1 O +). O + +The O +phenotype O +of O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +mutant B-protein_state +was O +however O +less O +pronounced O +than O +for O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +yeast B-taxonomy_domain +( O +Fig O +. O +4a O +). O + +This O +discrepancy O +in O +growth O +was O +traced O +to O +an O +additional O +point O +mutation O +L B-mutant +(- I-mutant +49 I-mutant +) I-mutant +S I-mutant +in O +the O +β5 B-protein +- O +propeptide B-structure_element +of O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +mutant B-protein_state +( O +see O +also O +Supplementary O +Note O +1 O +). O + +Structural B-experimental_method +comparison I-experimental_method +of O +the O +β5 B-mutant +- I-mutant +L I-mutant +(- I-mutant +49 I-mutant +) I-mutant +S I-mutant +- I-mutant +K33A I-mutant +and O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +active B-site +sites I-site +revealed O +that O +mutation B-experimental_method +of O +Lys33 B-residue_name_number +to O +Ala B-residue_name +creates O +a O +cavity O +that O +is O +filled O +with O +Thr1 B-residue_name_number +and O +the O +remnant O +propeptide B-structure_element +. O + +This O +structural O +alteration O +destroys O +active B-site +- I-site +site I-site +integrity O +and O +abolishes O +catalytic O +activity O +of O +the O +β5 B-protein +active B-site +site I-site +( O +Supplementary O +Fig O +. O +5a O +). O + +Additional O +proof O +for O +the O +key O +function O +of O +Lys33 B-residue_name_number +was O +obtained O +from O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +mutant B-protein_state +, O +with O +the O +propeptide B-structure_element +expressed B-experimental_method +separately I-experimental_method +from O +the O +main O +subunit O +( O +pp B-chemical +trans B-protein_state +). O + +The O +Thr1 B-residue_name_number +N O +terminus O +of O +this O +mutant B-protein_state +is O +not O +blocked O +by O +the O +propeptide B-structure_element +, O +yet O +its O +catalytic O +activity O +is O +reduced O +by O +∼ O +83 O +% O +( O +Supplementary O +Fig O +. O +6b O +). O + +Consistent O +with O +this O +, O +the O +crystal B-evidence +structure I-evidence +of O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +pp B-chemical +trans B-protein_state +mutant B-protein_state +in B-protein_state +complex I-protein_state +with I-protein_state +carfilzomib B-chemical +only O +showed O +partial O +occupancy O +of O +the O +ligand O +at O +the O +β5 B-protein +active B-site +sites I-site +( O +Supplementary O +Fig O +. O +5b O +and O +Supplementary O +Table O +1 O +). O + +Since O +no O +acetylation B-ptm +of O +the O +Thr1 B-residue_name_number +N O +terminus O +was O +observed O +for O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +pp B-chemical +trans B-protein_state +apo B-protein_state +crystal B-evidence +structure I-evidence +, O +the O +reduced O +reactivity O +towards O +substrates O +and O +inhibitors O +indicates O +that O +Lys33NH2 B-residue_name_number +, O +rather O +than O +Thr1NH2 B-residue_name_number +, O +deprotonates O +and O +activates O +Thr1OH B-residue_name_number +. O + +Furthermore O +, O +the O +crystal B-evidence +structure I-evidence +of O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +pp B-chemical +trans B-protein_state +mutant B-protein_state +without B-protein_state +inhibitor I-protein_state +revealed O +that O +Thr1Oγ B-residue_name_number +strongly O +coordinates B-bond_interaction +a O +well O +- O +defined O +water B-chemical +molecule O +(∼ O +2 O +Å O +; O +Fig O +. O +3c O +and O +Supplementary O +Fig O +. O +5c O +, O +d O +). O + +This O +water B-chemical +hydrogen B-bond_interaction +bonds I-bond_interaction +also O +to O +Arg19O B-residue_name_number +(∼ O +3 O +. O +0 O +Å O +) O +and O +Asp17Oδ B-residue_name_number +(∼ O +3 O +. O +0 O +Å O +), O +and O +thereby O +presumably O +enables O +residual O +activity O +of O +the O +mutant B-protein_state +. O + +Remarkably O +, O +the O +solvent O +molecule O +occupies O +the O +position O +normally O +taken O +by O +Lys33NH2 B-residue_name_number +in O +the O +WT B-protein_state +proteasome B-complex_assembly +structure B-evidence +( O +Fig O +. O +3c O +), O +further O +corroborating O +the O +essential O +role O +of O +Lys33 B-residue_name_number +as O +the O +general O +base O +for O +autolysis B-ptm +and O +proteolysis O +. O + +Conservative B-experimental_method +substitution I-experimental_method +of O +Lys33 B-residue_name_number +by O +Arg B-residue_name +delays O +autolysis B-ptm +of O +the O +β5 B-protein +precursor O +and O +impairs O +yeast B-taxonomy_domain +growth O +( O +for O +details O +see O +Supplementary O +Note O +1 O +). O + +While O +Thr1 B-residue_name_number +occupies O +the O +same O +position O +as O +in O +WT B-protein_state +yCPs B-complex_assembly +, O +Arg33 B-residue_name_number +is O +unable O +to O +hydrogen B-bond_interaction +bond I-bond_interaction +to O +Asp17 B-residue_name_number +, O +thereby O +inactivating O +the O +β5 B-protein +active B-site +site I-site +( O +Supplementary O +Fig O +. O +5e O +). O + +The O +conservative B-experimental_method +mutation I-experimental_method +of O +Asp17 B-residue_name_number +to O +Asn B-residue_name +in O +subunit O +β5 B-protein +of O +the O +yCP B-complex_assembly +also O +provokes O +a O +severe O +growth O +defect O +( O +Supplementary O +Note O +1 O +, O +Supplementary O +Fig O +. O +6a O +and O +Table O +1 O +). O + +Notably O +, O +only O +with O +the O +additional O +point O +mutation O +L B-mutant +(- I-mutant +49 I-mutant +) I-mutant +S I-mutant +present O +in O +the O +β5 B-protein +propeptide B-structure_element +could O +we O +purify O +a O +small O +amount O +of O +the O +β5 B-mutant +- I-mutant +D17N I-mutant +mutant B-protein_state +yCP B-complex_assembly +. O + +As O +determined O +by O +crystallographic B-experimental_method +analysis I-experimental_method +, O +this O +mutant B-protein_state +β5 B-protein +subunit O +was O +partially B-protein_state +processed I-protein_state +( O +Table O +1 O +) O +but O +displayed O +impaired O +reactivity O +towards O +the O +proteasome B-complex_assembly +inhibitor O +carfilzomib B-chemical +compared O +with O +the O +subunits O +β1 B-protein +and O +β2 B-protein +, O +and O +with O +WT B-protein_state +β5 B-protein +( O +Supplementary O +Fig O +. O +7a O +). O + +In O +contrast O +to O +the O +cis B-protein_state +- O +construct O +, O +expression B-experimental_method +of O +the O +β5 B-protein +propeptide B-structure_element +in O +trans B-protein_state +allowed O +straightforward O +isolation B-experimental_method +and O +crystallization B-experimental_method +of O +the O +D17N B-mutant +mutant B-protein_state +proteasome B-complex_assembly +. O + +The O +ChT O +- O +L O +activity O +of O +the O +β5 B-mutant +- I-mutant +D17N I-mutant +pp B-chemical +in O +trans B-protein_state +CP B-complex_assembly +towards O +the O +canonical O +β5 B-protein +model O +substrates O +N B-chemical +- I-chemical +succinyl I-chemical +- I-chemical +Leu I-chemical +- I-chemical +Leu I-chemical +- I-chemical +Val I-chemical +- I-chemical +Tyr I-chemical +- I-chemical +7 I-chemical +- I-chemical +amino I-chemical +- I-chemical +4 I-chemical +- I-chemical +methylcoumarin I-chemical +( O +Suc B-chemical +- I-chemical +LLVY I-chemical +- I-chemical +AMC I-chemical +) O +and O +carboxybenzyl B-chemical +- I-chemical +Gly I-chemical +- I-chemical +Gly I-chemical +- I-chemical +Leu I-chemical +- I-chemical +para I-chemical +- I-chemical +nitroanilide I-chemical +( O +Z B-chemical +- I-chemical +GGL I-chemical +- I-chemical +pNA I-chemical +) O +was O +severely O +reduced O +( O +Supplementary O +Fig O +. O +6b O +), O +confirming O +that O +Asp17 B-residue_name_number +is O +of O +fundamental O +importance O +for O +the O +catalytic O +activity O +of O +the O +mature B-protein_state +proteasome B-complex_assembly +. O + +Even O +though O +the O +β5 B-mutant +- I-mutant +D17N I-mutant +pp B-chemical +trans B-protein_state +yCP B-complex_assembly +crystal B-evidence +structure I-evidence +appeared O +identical O +to O +the O +WT B-protein_state +yCP B-complex_assembly +( O +Supplementary O +Fig O +. O +7b O +), O +the O +co B-evidence +- I-evidence +crystal I-evidence +structure I-evidence +with O +the O +α B-chemical +′, I-chemical +β I-chemical +′ I-chemical +epoxyketone I-chemical +inhibitor O +carfilzomib B-chemical +visualized O +only O +partial O +occupancy O +of O +the O +ligand O +in O +the O +β5 B-protein +active B-site +site I-site +( O +Supplementary O +Fig O +. O +7a O +). O + +This O +observation O +is O +consistent O +with O +a O +strongly O +reduced O +reactivity O +of O +β5 B-protein +- O +Thr1 B-residue_name_number +and O +the O +crystal B-evidence +structure I-evidence +of O +the O +β5 B-mutant +- I-mutant +D17N I-mutant +pp B-chemical +cis B-protein_state +mutant B-protein_state +in B-protein_state +complex I-protein_state +with I-protein_state +carfilzomib B-chemical +. O + +Autolysis B-ptm +and O +residual O +catalytic O +activity O +of O +the O +β5 B-mutant +- I-mutant +D17N I-mutant +mutants O +may O +originate O +from O +the O +carbonyl O +group O +of O +Asn17 B-residue_name_number +, O +which O +albeit O +to O +a O +lower O +degree O +still O +can O +polarize O +Lys33 B-residue_name_number +for O +the O +activation O +of O +Thr1 B-residue_name_number +. O + +In O +agreement O +, O +an O +E17A B-mutant +mutant B-protein_state +in O +the O +proteasomal O +β B-protein +- I-protein +subunit I-protein +of O +the O +archaeon B-taxonomy_domain +Thermoplasma B-species +acidophilum I-species +prevents O +autolysis B-ptm +and O +catalysis O +. O + +Strikingly O +, O +although O +the O +X B-evidence +- I-evidence +ray I-evidence +data I-evidence +on O +the O +β5 B-mutant +- I-mutant +D17N I-mutant +mutant B-protein_state +with O +the O +propeptide B-structure_element +expressed B-experimental_method +in O +cis B-protein_state +and O +in O +trans B-protein_state +looked O +similar O +, O +there O +was O +a O +pronounced O +difference O +in O +their O +growth O +phenotypes O +observed O +( O +Supplementary O +Fig O +. O +6a O +and O +Supplementary O +Fig O +. O +7b O +). O + +On O +the O +basis O +of O +these O +results O +, O +we O +propose O +that O +CPs B-complex_assembly +from O +all O +domains O +of O +life O +use O +a O +catalytic B-site +triad I-site +consisting O +of O +Thr1 B-residue_name_number +, O +Lys33 B-residue_name_number +and O +Asp B-residue_name +/ O +Glu17 B-residue_name_number +for O +both O +autocatalytic B-ptm +precursor I-ptm +processing I-ptm +and O +proteolysis O +( O +Fig O +. O +3d O +). O + +This O +model O +is O +also O +consistent O +with O +the O +fact O +that O +no O +defined O +water B-chemical +molecule O +is O +observed O +in O +the O +mature B-protein_state +WT B-protein_state +proteasomal O +active B-site +site I-site +that O +could O +shuttle O +the O +proton O +from O +Thr1Oγ B-residue_name_number +to O +Thr1NH2 B-residue_name_number +. O + +To O +explore O +this O +active B-site +- I-site +site I-site +model O +further O +, O +we O +exchanged B-experimental_method +the I-experimental_method +conserved I-experimental_method +Asp166 B-residue_name_number +residue O +for O +Asn B-residue_name +in O +the O +yeast B-taxonomy_domain +β5 B-protein +subunit O +. O + +Asp166Oδ B-residue_name_number +is O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +Thr1NH2 B-residue_name_number +via O +Ser129OH B-residue_name_number +and O +Ser169OH B-residue_name_number +, O +and O +therefore O +was O +proposed O +to O +be O +involved O +in O +catalysis O +. O + +The O +β5 B-mutant +- I-mutant +D166N I-mutant +pp B-chemical +cis B-protein_state +yeast B-taxonomy_domain +mutant B-protein_state +is O +significantly O +impaired O +in O +growth O +and O +its O +ChT O +- O +L O +activity O +is O +drastically O +reduced O +( O +Supplementary O +Fig O +. O +6a O +, O +b O +and O +Table O +1 O +). O + +X B-evidence +- I-evidence +ray I-evidence +data I-evidence +on O +the O +β5 B-mutant +- I-mutant +D166N I-mutant +mutant B-protein_state +indicate O +that O +the O +β5 B-protein +propeptide B-structure_element +is O +hydrolysed O +, O +but O +due O +to O +reorientation O +of O +Ser129OH B-residue_name_number +, O +the O +interaction O +with O +Asn166Oδ B-residue_name_number +is O +disrupted O +( O +Supplementary O +Fig O +. O +8a O +). O + +Instead O +, O +a O +water B-chemical +molecule O +is O +bound B-protein_state +to I-protein_state +Ser129OH B-residue_name_number +and O +Thr1NH2 B-residue_name_number +( O +Supplementary O +Fig O +. O +8b O +), O +which O +may O +enable O +precursor B-ptm +processing I-ptm +. O + +The O +hydrogen B-bond_interaction +bonds I-bond_interaction +involving O +Ser169OH B-residue_name_number +are O +intact O +and O +may O +account O +for O +residual O +substrate O +turnover O +. O + +Soaking B-experimental_method +the O +β5 B-mutant +- I-mutant +D166N I-mutant +crystals B-experimental_method +with O +carfilzomib B-chemical +and O +MG132 B-chemical +resulted O +in O +covalent O +modification O +of O +Thr1 B-residue_name_number +at O +high O +occupancy O +( O +Supplementary O +Fig O +. O +8c O +). O + +In O +the O +carfilzomib B-complex_assembly +complex I-complex_assembly +structure B-evidence +, O +Thr1Oγ B-residue_name_number +and O +Thr1N B-residue_name_number +incorporate O +into O +a O +morpholine O +ring O +structure O +and O +Ser129 B-residue_name_number +adopts O +its O +WT B-protein_state +- O +like O +orientation O +. O + +In O +the O +MG132 B-protein_state +- I-protein_state +bound I-protein_state +state I-protein_state +, O +Thr1N B-residue_name_number +is O +unmodified B-protein_state +, O +and O +we O +again O +observe O +that O +Ser129 B-residue_name_number +is O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +a O +water B-chemical +molecule O +instead O +of O +Asn166 B-residue_name_number +. O + +Whereas O +Asn B-residue_name +can O +to O +some O +degree O +replace O +Asp166 B-residue_name_number +due O +to O +its O +carbonyl O +group O +in O +the O +side O +chain O +, O +Ala B-residue_name +at O +this O +position O +was O +found O +to O +prevent O +both O +autolysis B-ptm +and O +catalysis O +. O + +These O +results O +suggest O +that O +Asp166 B-residue_name_number +and O +Ser129 B-residue_name_number +function O +as O +a O +proton O +shuttle O +and O +affect O +the O +protonation O +state O +of O +Thr1N B-residue_name_number +during O +autolysis B-ptm +and O +catalysis O +. O + +Substitution B-experimental_method +of O +the O +active B-site +- I-site +site I-site +Thr1 B-residue_name_number +by O +Cys B-residue_name + +Mutation B-experimental_method +of O +Thr1 B-residue_name_number +to O +Cys B-residue_name +inactivates O +the O +20S B-complex_assembly +proteasome I-complex_assembly +from O +the O +archaeon B-taxonomy_domain +T B-species +. I-species +acidophilum I-species +. O + +In O +yeast B-taxonomy_domain +, O +this O +mutation B-experimental_method +causes O +a O +strong O +growth O +defect O +( O +Fig O +. O +4a O +and O +Table O +1 O +), O +although O +the O +propeptide B-structure_element +is O +hydrolysed O +, O +as O +shown O +here O +by O +its O +X B-evidence +- I-evidence +ray I-evidence +structure I-evidence +. O + +In O +one O +of O +the O +two O +β5 B-protein +subunits O +, O +however O +, O +we O +found O +the O +cleaved B-protein_state +propeptide B-structure_element +still B-protein_state +bound I-protein_state +in O +the O +substrate B-site +- I-site +binding I-site +channel I-site +( O +Fig O +. O +4c O +). O + +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +occupies O +the O +S2 B-site +pocket I-site +like O +observed O +for O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +mutant B-protein_state +, O +but O +in O +contrast O +to O +the O +latter O +, O +the O +propeptide B-structure_element +in O +the O +T1C B-mutant +mutant B-protein_state +adopts O +an O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +conformation O +as O +known O +from O +inhibitors O +like O +MG132 B-chemical +( O +Fig O +. O +4c O +– O +e O +and O +Supplementary O +Fig O +. O +9b O +). O + +On O +the O +basis O +of O +the O +phenotype O +of O +the O +T1C B-mutant +mutant B-protein_state +and O +the O +propeptide B-structure_element +remnant O +identified O +in O +its O +active B-site +site I-site +, O +we O +suppose O +that O +autolysis B-ptm +is O +retarded O +and O +may O +not O +have O +been O +completed O +before O +crystallization B-experimental_method +. O + +Owing O +to O +the O +unequal O +positions O +of O +the O +two O +β5 B-protein +subunits O +within O +the O +CP B-complex_assembly +in O +the O +crystal O +lattice O +, O +maturation O +and O +propeptide B-structure_element +displacement O +may O +occur O +at O +different O +timescales O +in O +the O +two O +subunits O +. O + +Despite O +propeptide B-ptm +hydrolysis I-ptm +, O +the O +β5 B-mutant +- I-mutant +T1C I-mutant +active B-site +site I-site +is O +catalytically B-protein_state +inactive I-protein_state +( O +Fig O +. O +4b O +and O +Supplementary O +Fig O +. O +9a O +). O + +In O +agreement O +, O +soaking B-experimental_method +crystals I-experimental_method +with O +the O +CP B-complex_assembly +inhibitors O +bortezomib B-chemical +or O +carfilzomib B-chemical +modifies O +only O +the O +β1 B-protein +and O +β2 B-protein +active B-site +sites I-site +, O +while O +leaving O +the O +β5 B-mutant +- I-mutant +T1C I-mutant +proteolytic B-site +centres I-site +unmodified B-protein_state +even O +though O +they O +are O +only O +partially O +occupied O +by O +the O +cleaved B-protein_state +propeptide B-structure_element +remnant O +. O + +Moreover O +, O +the O +structural B-evidence +data I-evidence +reveal O +that O +the O +thiol O +group O +of O +Cys1 B-residue_name_number +is O +rotated O +by O +74 O +° O +with O +respect O +to O +the O +hydroxyl O +side O +chain O +of O +Thr1 B-residue_name_number +( O +Fig O +. O +4f O +and O +Supplementary O +Fig O +. O +9b O +). O + +Consequently O +, O +the O +hydrogen B-bond_interaction +bond I-bond_interaction +bridging O +the O +active O +- O +site O +nucleophile O +and O +Lys33 B-residue_name_number +in O +WT B-protein_state +CPs B-complex_assembly +is O +broken O +with O +Cys1 B-residue_name_number +. O + +Notably O +, O +the O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +- I-evidence +density I-evidence +map I-evidence +of O +the O +T1C B-mutant +mutant B-protein_state +also O +indicates O +that O +Lys33NH2 B-residue_name_number +is O +disordered B-protein_state +. O + +Together O +, O +these O +observations O +suggest O +that O +efficient O +peptide O +- O +bond O +hydrolysis O +requires O +that O +Lys33NH2 B-residue_name_number +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +active O +site O +nucleophile O +. O + +The O +benefit O +of O +Thr B-residue_name +over O +Ser B-residue_name +as O +the O +active O +- O +site O +nucleophile O + +All O +proteasomes B-complex_assembly +strictly B-protein_state +employ I-protein_state +threonine B-residue_name +as O +the O +active B-site +- I-site +site I-site +residue I-site +instead O +of O +serine B-residue_name +. O + +To O +investigate O +the O +reason O +for O +this O +singularity O +, O +we O +analysed O +a O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +, O +which O +is O +viable O +but O +suffers O +from O +growth O +defects O +( O +Fig O +. O +4a O +and O +Table O +1 O +). O + +Activity B-experimental_method +assays I-experimental_method +with O +the O +β5 B-protein +- O +specific O +substrate O +Suc B-chemical +- I-chemical +LLVY I-chemical +- I-chemical +AMC I-chemical +demonstrated O +that O +the O +ChT O +- O +L O +activity O +of O +the O +T1S B-mutant +mutant B-protein_state +is O +reduced O +by O +40 O +– O +45 O +% O +compared O +with O +WT B-protein_state +proteasomes B-complex_assembly +depending O +on O +the O +incubation O +temperature O +( O +Fig O +. O +4b O +and O +Supplementary O +Fig O +. O +9c O +). O + +By O +contrast O +, O +turnover O +of O +the O +substrate O +Z B-chemical +- I-chemical +GGL I-chemical +- I-chemical +pNA I-chemical +, O +used O +to O +monitor O +ChT O +- O +L O +activity O +in O +situ O +but O +in O +a O +less O +quantitative O +fashion O +, O +is O +not O +detectably O +impaired O +( O +Supplementary O +Fig O +. O +9a O +). O + +Crystal B-evidence +structure I-evidence +analysis O +of O +the O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +confirmed O +precursor B-ptm +processing I-ptm +( O +Fig O +. O +4g O +), O +and O +ligand B-complex_assembly +- I-complex_assembly +complex I-complex_assembly +structures B-evidence +with O +bortezomib B-chemical +and O +carfilzomib B-chemical +unambiguously O +corroborated O +the O +reactivity O +of O +Ser1 B-residue_name_number +( O +Fig O +. O +5 O +). O + +However O +, O +the O +apo B-protein_state +crystal B-evidence +structure I-evidence +revealed O +that O +Ser1Oγ B-residue_name_number +is O +turned O +away O +from O +the O +substrate B-site +- I-site +binding I-site +channel I-site +( O +Fig O +. O +4g O +). O + +Compared O +with O +Thr1Oγ B-residue_name_number +in O +WT B-protein_state +CP B-complex_assembly +structures B-evidence +, O +Ser1Oγ B-residue_name_number +is O +rotated O +by O +60 O +°. O + +Because O +both O +conformations O +of O +Ser1Oγ B-residue_name_number +are O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +Lys33NH2 B-residue_name_number +( O +Fig O +. O +4h O +), O +the O +relay O +system O +is O +capable O +of O +hydrolysing O +peptide O +substrates O +, O +albeit O +at O +lower O +rates O +compared O +with O +Thr1 B-residue_name_number +. O + +The O +active B-site +- I-site +site I-site +residue I-site +Thr1 B-residue_name_number +is O +fixed O +in O +its O +position O +, O +as O +its O +methyl O +group O +is O +engaged O +in O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +Thr3 B-residue_name_number +and O +Ala46 B-residue_name_number +( O +Fig O +. O +4h O +). O + +Consequently O +, O +the O +hydroxyl O +group O +of O +Thr1 B-residue_name_number +requires O +no O +reorientation O +before O +substrate O +cleavage O +and O +is O +thus O +more O +catalytically O +efficient O +than O +Ser1 B-residue_name_number +. O + +In O +agreement O +, O +at O +an O +elevated O +growing O +temperature O +of O +37 O +° O +C O +the O +T1S B-mutant +mutant B-protein_state +is O +unable O +to O +grow O +( O +Fig O +. O +4a O +). O + +In O +vitro O +, O +the O +mutant B-protein_state +proteasome B-complex_assembly +is O +less O +susceptible O +to O +proteasome B-complex_assembly +inhibition O +by O +bortezomib B-chemical +( O +3 O +. O +7 O +- O +fold O +) O +and O +carfilzomib B-chemical +( O +1 O +. O +8 O +- O +fold O +; O +Fig O +. O +5 O +). O + +Nevertheless O +, O +inhibitor B-complex_assembly +complex I-complex_assembly +structures B-evidence +indicate O +identical O +binding O +modes O +compared O +with O +the O +WT B-protein_state +yCP B-complex_assembly +structures B-evidence +, O +with B-protein_state +the I-protein_state +same I-protein_state +inhibitors I-protein_state +. O + +Notably O +, O +the O +affinity B-evidence +of O +the O +tetrapeptide O +carfilzomib B-chemical +is O +less O +impaired O +, O +as O +it O +is O +better O +stabilized O +in O +the O +substrate B-site +- I-site +binding I-site +channel I-site +than O +the O +dipeptide O +bortezomib B-chemical +, O +which O +lacks O +a O +defined O +P3 O +site O +and O +has O +only O +a O +few O +interactions O +with O +the O +surrounding O +protein O +. O + +Hence O +, O +the O +mean B-evidence +residence I-evidence +time I-evidence +of O +carfilzomib B-chemical +at O +the O +active B-site +site I-site +is O +prolonged O +and O +the O +probability O +to O +covalently O +react O +with O +Ser1 B-residue_name_number +is O +increased O +. O + +Considered O +together O +, O +these O +results O +provide O +a O +plausible O +explanation O +for O +the O +invariance O +of O +threonine B-residue_name +as O +the O +active O +- O +site O +nucleophile O +in O +proteasomes B-complex_assembly +in O +all O +three O +domains O +of O +life O +, O +as O +well O +as O +in O +proteasome B-protein_type +- I-protein_type +like I-protein_type +proteases I-protein_type +such O +as O +HslV B-protein +( O +ref O +.). O + +The O +20S B-complex_assembly +proteasome I-complex_assembly +CP B-complex_assembly +is O +the O +major O +non B-protein_type +- I-protein_type +lysosomal I-protein_type +protease I-protein_type +in O +eukaryotic B-taxonomy_domain +cells O +, O +and O +its O +assembly O +is O +highly O +organized O +. O + +The O +β B-protein +- I-protein +subunit I-protein +propeptides B-structure_element +, O +particularly O +that O +of O +β5 B-protein +, O +are O +key O +factors O +that O +help O +drive O +proper O +assembly O +of O +the O +CP B-complex_assembly +complex O +. O + +In O +addition O +, O +they O +prevent O +irreversible O +inactivation O +of O +the O +Thr1 B-residue_name_number +N O +terminus O +by O +N B-ptm +- I-ptm +acetylation I-ptm +. O + +By O +contrast O +, O +the O +prosegments B-structure_element +of O +β B-protein +subunits I-protein +are O +dispensable O +for O +archaeal B-taxonomy_domain +proteasome B-complex_assembly +assembly O +, O +at O +least O +when O +heterologously B-experimental_method +expressed I-experimental_method +in O +Escherichia B-species +coli I-species +. O + +In O +eukaryotes B-taxonomy_domain +, O +deletion O +of O +or O +failure O +to O +cleave O +the O +β1 B-protein +and O +β2 B-protein +propeptides B-structure_element +is O +well O +tolerated O +. O + +However O +, O +removal B-experimental_method +of I-experimental_method +the O +β5 B-protein +prosegment B-structure_element +or O +any O +interference O +with O +its O +cleavage O +causes O +severe O +phenotypic O +defects O +. O + +These O +observations O +highlight O +the O +unique O +function O +and O +importance O +of O +the O +β5 B-protein +propeptide B-structure_element +as O +well O +as O +the O +β5 B-protein +active B-site +site I-site +for O +maturation O +and O +function O +of O +the O +eukaryotic B-taxonomy_domain +CP B-complex_assembly +. O + +Here O +we O +have O +described O +the O +atomic B-evidence +structures I-evidence +of O +various O +β5 B-mutant +- I-mutant +T1A I-mutant +mutants O +, O +which O +allowed O +for O +the O +first O +time O +visualization O +of O +the O +residual O +β5 B-protein +propeptide B-structure_element +. O + +Depending O +on O +the O +(- B-residue_number +2 I-residue_number +) I-residue_number +residue O +we O +observed O +various O +propeptide B-structure_element +conformations O +, O +but O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +is O +in O +all O +structures B-evidence +perfectly O +located O +for O +the O +nucleophilic O +attack O +by O +Thr1Oγ B-residue_name_number +, O +although O +it O +does O +not O +adopt O +the O +tight B-structure_element +turn I-structure_element +observed O +for O +the O +prosegment B-structure_element +of O +subunit O +β1 B-protein +. O + +From O +these O +data O +we O +conclude O +that O +only O +the O +positioning O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +and O +Thr1 B-residue_name_number +as O +well O +as O +the O +integrity O +of O +the O +proteasomal O +active B-site +site I-site +are O +required O +for O +autolysis B-ptm +. O + +In O +this O +regard O +, O +inappropriate O +N B-ptm +- I-ptm +acetylation I-ptm +of O +the O +Thr1 B-residue_name_number +N O +terminus O +cannot O +be O +removed O +by O +Thr1Oγ B-residue_name_number +due O +to O +the O +rotational O +freedom O +and O +flexibility O +of O +the O +acetyl O +group O +. O + +The O +propeptide B-structure_element +needs O +some O +anchoring O +in O +the O +substrate B-site +- I-site +binding I-site +channel I-site +to O +properly O +position O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +), I-residue_name_number +but O +this O +seems O +to O +be O +independent O +of O +the O +orientation O +of O +residue O +(- B-residue_number +2 I-residue_number +). I-residue_number + +Autolytic O +activation O +of O +the O +CP B-complex_assembly +constitutes O +one O +of O +the O +final O +steps O +of O +proteasome O +biogenesis O +, O +but O +the O +trigger O +for O +propeptide B-ptm +cleavage I-ptm +had O +remained O +enigmatic O +. O + +On O +the O +basis O +of O +the O +numerous O +CP B-complex_assembly +: I-complex_assembly +ligand I-complex_assembly +complexes O +solved O +during O +the O +past O +18 O +years O +and O +in O +the O +current O +study O +, O +we O +provide O +a O +revised O +interpretation O +of O +proteasome B-complex_assembly +active B-site +- I-site +site I-site +architecture I-site +. O + +We O +propose O +a O +catalytic B-site +triad I-site +for O +the O +active B-site +site I-site +of O +the O +CP B-complex_assembly +consisting O +of O +residues O +Thr1 B-residue_name_number +, O +Lys33 B-residue_name_number +and O +Asp B-residue_name +/ O +Glu17 B-residue_name_number +, O +which O +are O +conserved O +among O +all O +proteolytically O +active O +eukaryotic B-taxonomy_domain +, O +bacterial B-taxonomy_domain +and O +archaeal B-taxonomy_domain +proteasome B-complex_assembly +subunits O +. O + +Lys33NH2 B-residue_name_number +is O +expected O +to O +act O +as O +the O +proton O +acceptor O +during O +autocatalytic B-ptm +removal I-ptm +of O +the O +propeptides B-structure_element +, O +as O +well O +as O +during O +substrate O +proteolysis O +, O +while O +Asp17Oδ B-residue_name_number +orients O +Lys33NH2 B-residue_name_number +and O +makes O +it O +more O +prone O +to O +protonation O +by O +raising O +its O +pKa O +( O +hydrogen B-bond_interaction +bond I-bond_interaction +distance O +: O +Lys33NH3 B-residue_name_number ++– O +Asp17Oδ B-residue_name_number +: O +2 O +. O +9 O +Å O +). O + +Analogously O +to O +the O +proteasome B-complex_assembly +, O +a O +Thr B-site +– I-site +Lys I-site +– I-site +Asp I-site +triad I-site +is O +also O +found O +in O +L B-protein_type +- I-protein_type +asparaginase I-protein_type +. O + +Thus O +, O +specific O +protein O +surroundings O +can O +significantly O +alter O +the O +chemical O +properties O +of O +amino O +acids O +such O +as O +Lys B-residue_name +to O +function O +as O +an O +acid O +– O +base O +catalyst O +. O + +In O +this O +new O +view O +of O +the O +proteasomal O +active B-site +site I-site +, O +the O +positively O +charged O +Thr1NH3 B-residue_name_number ++- O +terminus O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +amide O +nitrogen O +of O +incoming O +peptide O +substrates O +and O +stabilizes O +as O +well O +as O +activates O +them O +for O +the O +endoproteolytic B-ptm +cleavage I-ptm +by O +Thr1Oγ B-residue_name_number +( O +Fig O +. O +3d O +). O + +Consistent O +with O +this O +model O +, O +the O +positively O +charged O +Thr1 B-residue_name_number +N O +terminus O +is O +engaged O +in O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +inhibitory O +compounds O +like O +fellutamide B-chemical +B I-chemical +( O +ref O +.), O +α B-chemical +- I-chemical +ketoamides I-chemical +, O +homobelactosin B-chemical +C I-chemical +( O +ref O +.) O +and O +salinosporamide B-chemical +A I-chemical +( O +ref O +.). O + +Furthermore O +, O +opening O +of O +the O +β O +- O +lactone O +compound O +omuralide B-chemical +by O +Thr1 B-residue_name_number +creates O +a O +C3 O +- O +hydroxyl O +group O +, O +whose O +proton O +originates O +from O +Thr1NH3 B-residue_name_number ++. O + +The O +resulting O +uncharged O +Thr1NH2 B-residue_name_number +is O +hydrogen B-bond_interaction +- I-bond_interaction +bridged I-bond_interaction +to O +the O +C3 O +- O +OH O +group O +. O + +In O +agreement O +, O +acetylation B-ptm +of O +the O +Thr1 B-residue_name_number +N O +terminus O +irreversibly O +blocks O +hydrolytic O +activity O +, O +and O +binding O +of O +substrates O +is O +prevented O +for O +steric O +reasons O +. O + +By O +acting O +as O +a O +proton O +donor O +during O +catalysis O +, O +the O +Thr1 B-residue_name_number +N O +terminus O +may O +also O +favour O +cleavage O +of O +substrate O +peptide O +bonds O +( O +Fig O +. O +3d O +). O + +Cleavage O +of O +the O +scissile O +peptide O +bond O +requires O +protonation O +of O +the O +emerging O +free O +amine O +, O +and O +in O +the O +proteasome B-complex_assembly +, O +the O +Thr1 B-residue_name_number +amine O +group O +is O +likely O +to O +assume O +this O +function O +. O + +Analogously O +, O +Thr1NH3 B-residue_name_number ++ O +might O +promote O +the O +bivalent O +reaction O +mode O +of O +epoxyketone O +inhibitors O +by O +protonating O +the O +epoxide O +moiety O +to O +create O +a O +positively O +charged O +trivalent O +oxygen O +atom O +that O +is O +subsequently O +nucleophilically O +attacked O +by O +Thr1NH2 B-residue_name_number +. O + +During O +autolysis B-ptm +the O +Thr1 B-residue_name_number +N O +terminus O +is O +engaged O +in O +a O +hydroxyoxazolidine O +ring O +intermediate O +( O +Fig O +. O +3d O +), O +which O +is O +unstable O +and O +short O +- O +lived O +. O + +Breakdown O +of O +this O +tetrahedral O +transition O +state O +releases O +the O +Thr1 B-residue_name_number +N O +terminus O +that O +is O +protonated O +by O +aspartic B-residue_name_number +acid I-residue_name_number +166 I-residue_name_number +via O +Ser129OH B-residue_name_number +to O +yield O +Thr1NH3 B-residue_name_number ++. O + +The O +residues O +Ser129 B-residue_name_number +and O +Asp166 B-residue_name_number +are O +expected O +to O +increase O +the O +pKa O +value O +of O +Thr1N B-residue_name_number +, O +thereby O +favouring O +its O +charged O +state O +. O + +Consistent O +with O +playing O +an O +essential O +role O +in O +proton O +shuttling O +, O +the O +mutation B-experimental_method +D166A B-mutant +prevents O +autolysis B-ptm +of O +the O +archaeal B-taxonomy_domain +CP B-complex_assembly +and O +the O +exchange B-experimental_method +D166N B-mutant +impairs O +catalytic O +activity O +of O +the O +yeast B-taxonomy_domain +CP B-complex_assembly +about O +60 O +%. O + +The O +mutation B-experimental_method +D166N B-mutant +lowers O +the O +pKa O +of O +Thr1N B-residue_name_number +, O +which O +is O +thus O +more O +likely O +to O +exist O +in O +the O +uncharged O +deprotonated O +state O +( O +Thr1NH2 B-residue_name_number +). O + +This O +interpretation O +agrees O +with O +the O +strongly O +reduced O +catalytic O +activity O +of O +the O +β5 B-mutant +- I-mutant +D166N I-mutant +mutant B-protein_state +on O +the O +one O +hand O +, O +and O +the O +ability O +to O +react O +readily O +with O +carfilzomib B-chemical +on O +the O +other O +. O + +Hence O +, O +the O +proteasome B-complex_assembly +can O +be O +viewed O +as O +having O +a O +second B-site +triad I-site +that O +is O +essential O +for O +efficient O +proteolysis O +. O + +While O +Lys33NH2 B-residue_name_number +and O +Asp17Oδ B-residue_name_number +are O +required O +to O +deprotonate O +the O +Thr1 B-residue_name_number +hydroxyl O +side O +chain O +, O +Ser129OH B-residue_name_number +and O +Asp166OH B-residue_name_number +serve O +to O +protonate O +the O +N O +- O +terminal O +amine O +group O +of O +Thr1 B-residue_name_number +. O + +In O +accord O +with O +the O +proposed O +Thr1 B-residue_name_number +– O +Lys33 B-residue_name_number +– O +Asp17 B-residue_name_number +catalytic B-site +triad I-site +, O +crystallographic B-evidence +data I-evidence +on O +the O +proteolytically B-protein_state +inactive I-protein_state +β5 B-mutant +- I-mutant +T1C I-mutant +mutant B-protein_state +demonstrate O +that O +the O +interaction O +of O +Lys33NH2 B-residue_name_number +and O +Cys1 B-residue_name_number +is O +broken O +. O + +However O +, O +owing O +to O +Cys B-residue_name +being O +a O +strong O +nucleophile O +, O +the O +propeptide B-structure_element +can O +still O +be O +cleaved B-protein_state +off O +over O +time O +. O + +While O +only O +one O +single O +turnover O +is O +necessary O +for O +autolysis B-ptm +, O +continuous O +enzymatic O +activity O +is O +required O +for O +significant O +and O +detectable O +substrate O +hydrolysis O +. O + +Notably O +, O +in O +the O +Ntn B-protein_type +hydrolase I-protein_type +penicillin B-protein_type +acylase I-protein_type +, O +substitution B-experimental_method +of O +the O +catalytic B-protein_state +N O +- O +terminal O +Ser B-residue_name +residue O +by O +Cys B-residue_name +also O +inactivates B-protein_state +the O +enzyme B-protein_type +but O +still O +enables O +precursor B-ptm +processing I-ptm +. O + +To O +investigate O +why O +the O +CP B-complex_assembly +specifically O +employs O +threonine B-residue_name +as O +its O +active B-site +- I-site +site I-site +residue I-site +, O +we O +used O +a O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +of O +the O +yCP B-complex_assembly +and O +characterized O +it O +biochemically B-experimental_method +and I-experimental_method +structurally I-experimental_method +. O + +Activity B-experimental_method +assays I-experimental_method +with O +the O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +revealed O +reduced O +turnover O +of O +Suc B-chemical +- I-chemical +LLVY I-chemical +- I-chemical +AMC I-chemical +. O + +We O +also O +observed O +slightly O +lower O +affinity O +of O +the O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +yCP B-complex_assembly +for O +the O +Food O +and O +Drug O +Administration O +- O +approved O +proteasome B-complex_assembly +inhibitors O +bortezomib B-chemical +and O +carfilzomib B-chemical +. O + +Structural B-evidence +analyses I-evidence +support O +these O +findings O +with O +the O +T1S B-mutant +mutant B-protein_state +and O +provide O +an O +explanation O +for O +the O +strict B-protein_state +use I-protein_state +of I-protein_state +Thr B-residue_name +residues O +in O +proteasomes B-complex_assembly +. O + +Thr1 B-residue_name_number +is O +well O +anchored O +in O +the O +active B-site +site I-site +by O +hydrophobic B-bond_interaction +interactions I-bond_interaction +of O +its O +Cγ O +methyl O +group O +with O +Ala46 B-residue_name_number +( O +Cβ O +), O +Lys33 B-residue_name_number +( O +carbon O +side O +chain O +) O +and O +Thr3 B-residue_name_number +( O +Cγ O +). O + +Notably O +, O +proteolytically B-protein_state +active I-protein_state +proteasome B-complex_assembly +subunits O +from O +archaea B-taxonomy_domain +, O +yeast B-taxonomy_domain +and O +mammals B-taxonomy_domain +, O +including O +constitutive O +, O +immuno O +- O +and O +thymoproteasome O +subunits O +, O +either O +encode O +Thr B-residue_name +or O +Ile B-residue_name +at O +position O +3 B-residue_number +, O +indicating O +the O +importance O +of O +the O +Cγ O +for O +fixing O +the O +position O +of O +the O +nucleophilic O +Thr1 B-residue_name_number +. O + +In O +contrast O +to O +Thr1 B-residue_name_number +, O +the O +hydroxyl O +group O +of O +Ser1 B-residue_name_number +occupies O +the O +position O +of O +the O +Thr1 B-residue_name_number +methyl O +side O +chain O +in O +the O +WT B-protein_state +enzyme B-complex_assembly +, O +which O +requires O +its O +reorientation O +relative O +to O +the O +substrate O +to O +allow O +cleavage O +( O +Fig O +. O +4g O +, O +h O +). O + +Notably O +, O +in O +the O +threonine B-protein_type +aspartase I-protein_type +Taspase1 B-protein +, O +mutation B-experimental_method +of O +the O +active B-site +- I-site +site I-site +Thr234 B-residue_name_number +to O +Ser B-residue_name +also O +places O +the O +side O +chain O +in O +the O +position O +of O +the O +methyl O +group O +of O +Thr234 B-residue_name_number +in O +the O +WT B-protein_state +, O +thereby O +reducing O +catalytic O +activity O +. O + +Similarly O +, O +although O +the O +serine B-residue_name +mutant B-protein_state +is O +active B-protein_state +, O +threonine B-residue_name +is O +more O +efficient O +in O +the O +context O +of O +the O +proteasome B-complex_assembly +active B-site +site I-site +. O + +The O +greater O +suitability O +of O +threonine B-residue_name +for O +the O +proteasome B-complex_assembly +active B-site +site I-site +, O +which O +has O +been O +noted O +in O +biochemical O +as O +well O +as O +in O +kinetic O +studies O +, O +constitutes O +a O +likely O +reason O +for O +the O +conservation B-protein_state +of O +the O +Thr1 B-residue_name_number +residue O +in O +all O +proteasomes B-complex_assembly +from O +bacteria B-taxonomy_domain +to O +eukaryotes B-taxonomy_domain +. O + +Conformation O +of O +proteasomal O +propeptides B-structure_element +. O + +( O +a O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +and O +the O +matured B-protein_state +WT B-protein_state +β1 B-protein +active B-site +- I-site +site I-site +Thr1 B-residue_name_number +. O + +Only O +the O +residues O +(- B-residue_range +5 I-residue_range +) I-residue_range +to I-residue_range +(- I-residue_range +1 I-residue_range +) I-residue_range +of O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +are O +displayed O +. O + +The O +major O +determinant O +of O +the O +S1 B-site +specificity I-site +pocket I-site +, O +residue O +45 B-residue_number +, O +is O +depicted O +. O + +Note O +the O +tight O +conformation O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +and O +Ala1 B-residue_name_number +before O +propeptide B-structure_element +removal O +( O +G B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +turn O +; O +cyan O +double O +arrow O +) O +compared O +with O +the O +relaxed O +, O +processed B-protein_state +WT B-protein_state +active B-site +- I-site +site I-site +Thr1 B-residue_name_number +( O +red O +double O +arrow O +). O + +The O +black O +arrow O +indicates O +the O +attack O +of O +Thr1Oγ B-residue_name_number +onto O +the O +carbonyl O +carbon O +atom O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +). I-residue_name_number + +( O +b O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +and O +the O +β2 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +highlights O +subtle O +differences O +in O +their O +conformations O +, O +but O +illustrates O +that O +Ala1 B-residue_name_number +and O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +match O +well O +. O + +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +OH O +is O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +O O +(∼ O +2 O +. O +8 O +Å O +; O +black O +dashed O +line O +). O + +( O +c O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +, O +the O +β2 B-mutant +- I-mutant +T1A I-mutant +and O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +propeptide B-structure_element +remnants O +depict O +their O +differences O +in O +conformation O +. O + +While O +residue O +(- B-residue_number +2 I-residue_number +) I-residue_number +of O +the O +β1 B-protein +and O +β2 B-protein +prosegments B-structure_element +fit O +the O +S1 B-site +pocket I-site +, O +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +of O +the O +β5 B-protein +propeptide B-structure_element +occupies O +the O +S2 B-site +pocket I-site +. O + +Nonetheless O +, O +in O +all O +mutants O +the O +carbonyl O +carbon O +atom O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +is O +ideally O +placed O +for O +the O +nucleophilic O +attack O +by O +Thr1Oγ B-residue_name_number +. O + +The O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +OH O +and O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +O O +(∼ O +2 O +. O +8 O +Å O +) O +is O +indicated O +by O +a O +black O +dashed O +line O +. O + +Mutations B-experimental_method +of O +residue O +(- B-residue_number +2 I-residue_number +) I-residue_number +and O +their O +influence O +on O +propeptide B-structure_element +conformation O +and O +autolysis B-ptm +. O + +( O +a O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +and O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +L I-mutant +- I-mutant +T1A I-mutant +mutant B-protein_state +propeptide B-structure_element +. O + +The O +(- B-residue_number +2 I-residue_number +) I-residue_number +residues O +of O +both O +prosegments B-structure_element +point O +into O +the O +S1 B-site +pocket I-site +. O + +( O +b O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β5 B-protein +propeptides B-structure_element +in O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +L I-mutant +- I-mutant +T1A I-mutant +, O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +T I-mutant +- I-mutant +T1A I-mutant +, O +β5 B-mutant +-( I-mutant +H I-mutant +- I-mutant +2 I-mutant +) I-mutant +A I-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +and O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +mutant B-protein_state +proteasomes B-complex_assembly +. O + +While O +the O +residues O +(- B-residue_range +2 I-residue_range +) I-residue_range +to I-residue_range +(- I-residue_range +4 I-residue_range +) I-residue_range +vary O +in O +their O +conformation O +, O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +and O +Ala1 B-residue_name_number +are O +located O +in O +all O +structures B-evidence +at O +the O +same O +positions O +. O + +( O +c O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β2 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +and O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +T I-mutant +- I-mutant +T1A I-mutant +mutant B-protein_state +propeptide B-structure_element +. O + +The O +(- B-residue_number +2 I-residue_number +) I-residue_number +residues O +of O +both O +prosegments B-structure_element +point O +into O +the O +S1 B-site +pocket I-site +, O +but O +only O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +OH O +of O +β2 B-protein +forms O +a O +hydrogen B-bond_interaction +bridge I-bond_interaction +to O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +O O +( O +black O +dashed O +line O +). O + +( O +d O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +matured B-protein_state +β2 B-protein +active B-site +site I-site +, O +the O +WT B-protein_state +β2 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +and O +the O +β2 B-mutant +- I-mutant +T I-mutant +(- I-mutant +2 I-mutant +) I-mutant +V I-mutant +mutant B-protein_state +propeptide B-structure_element +. O + +Notably O +, O +Val B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +of O +the O +latter O +does O +not O +occupy O +the O +S1 B-site +pocket I-site +, O +thereby O +changing O +the O +orientation O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +and O +preventing O +nucleophilic O +attack O +of O +Thr1Oγ B-residue_name_number +on O +the O +carbonyl O +carbon O +atom O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +). I-residue_name_number + +Architecture O +and O +proposed O +reaction O +mechanism O +of O +the O +proteasomal O +active B-site +site I-site +. O + +( O +a O +) O +Hydrogen B-site +- I-site +bonding I-site +network I-site +at O +the O +mature B-protein_state +WT B-protein_state +β5 B-protein +proteasomal O +active B-site +site I-site +( O +dotted O +lines O +). O + +Thr1OH B-residue_name_number +is O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +Lys33NH2 B-residue_name_number +( O +2 O +. O +7 O +Å O +), O +which O +in O +turn O +interacts O +with O +Asp17Oδ B-residue_name_number +. O + +The O +Thr1 B-residue_name_number +N O +terminus O +is O +engaged O +in O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +Ser129Oγ B-residue_name_number +, O +the O +carbonyl O +oxygen O +of O +residue O +168 B-residue_number +, O +Ser169Oγ B-residue_name_number +and O +Asp166Oδ B-residue_name_number +. O +( O +b O +) O +The O +orientations O +of O +the O +active B-site +- I-site +site I-site +residues I-site +involved O +in O +hydrogen B-bond_interaction +bonding I-bond_interaction +are O +strictly B-protein_state +conserved I-protein_state +in O +each O +proteolytic B-site +centre I-site +, O +as O +shown O +by O +superposition B-experimental_method +of O +the O +β B-protein +subunits I-protein +. O + +( O +c O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +WT B-protein_state +β5 B-protein +and O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +pp B-chemical +trans B-protein_state +mutant B-protein_state +active B-site +site I-site +. O + +In O +the O +latter O +, O +a O +water B-chemical +molecule O +( O +red O +sphere O +) O +is O +found O +at O +the O +position O +where O +in O +the O +WT B-protein_state +structure O +the O +side O +chain O +amine O +group O +of O +Lys33 B-residue_name_number +is O +located O +. O + +Similarly O +to O +Lys33 B-residue_name_number +, O +the O +water B-chemical +molecule O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +Arg19O B-residue_name_number +, O +Asp17Oδ B-residue_name_number +and O +Thr1OH B-residue_name_number +. O + +Note O +, O +the O +strong O +interaction O +with O +the O +water B-chemical +molecule O +causes O +a O +minor O +shift O +of O +Thr1 B-residue_name_number +, O +while O +all O +other O +active B-site +- I-site +site I-site +residues I-site +remain O +in O +place O +. O + +( O +d O +) O +Proposed O +chemical O +reaction O +mechanism O +for O +autocatalytic B-ptm +precursor I-ptm +processing I-ptm +and O +proteolysis O +in O +the O +proteasome B-complex_assembly +. O + +The O +active B-site +- I-site +site I-site +Thr1 B-residue_name_number +is O +depicted O +in O +blue O +, O +the O +propeptide B-structure_element +segment O +and O +the O +peptide O +substrate O +are O +coloured O +in O +green O +, O +whereas O +the O +scissile O +peptide O +bond O +is O +highlighted O +in O +red O +. O + +Autolysis B-ptm +( O +left O +set O +of O +structures O +) O +is O +initiated O +by O +deprotonation O +of O +Thr1OH B-residue_name_number +via O +Lys33NH2 B-residue_name_number +and O +the O +formation O +of O +a O +tetrahedral O +transition O +state O +. O + +The O +strictly B-protein_state +conserved I-protein_state +oxyanion O +hole O +Gly47NH B-residue_name_number +stabilizing O +the O +negatively O +charged O +intermediate O +is O +illustrated O +as O +a O +semicircle O +. O + +Collapse O +of O +the O +transition O +state O +frees O +the O +Thr1 B-residue_name_number +N O +terminus O +( O +by O +completing O +an O +N O +- O +to O +- O +O O +acyl O +shift O +of O +the O +propeptide B-structure_element +), O +which O +is O +subsequently O +protonated O +by O +Asp166OH B-residue_name_number +via O +Ser129OH B-residue_name_number +. O + +Next O +, O +Thr1NH2 B-residue_name_number +polarizes O +a O +water B-chemical +molecule O +for O +the O +nucleophilic O +attack O +of O +the O +acyl O +- O +enzyme O +intermediate O +. O + +On O +hydrolysis O +of O +the O +latter O +, O +the O +active B-site +- I-site +site I-site +Thr1 B-residue_name_number +is O +ready O +for O +catalysis O +( O +right O +set O +of O +structures O +). O + +The O +charged O +Thr1 B-residue_name_number +N O +terminus O +may O +engage O +in O +the O +orientation O +of O +the O +amide O +moiety O +and O +donate O +a O +proton O +to O +the O +emerging O +N O +terminus O +of O +the O +C O +- O +terminal O +cleavage O +product O +. O + +The O +resulting O +deprotonated O +Thr1NH2 B-residue_name_number +finally O +activates O +a O +water B-chemical +molecule O +for O +hydrolysis O +of O +the O +acyl O +- O +enzyme O +. O + +The O +proteasome B-complex_assembly +favours O +threonine B-residue_name +as O +the O +active O +- O +site O +nucleophile O +. O + +( O +a O +) O +Growth B-experimental_method +tests I-experimental_method +by I-experimental_method +serial I-experimental_method +dilution I-experimental_method +of O +WT B-protein_state +and O +pre2 O +( O +β5 B-protein +) O +mutant B-protein_state +yeast B-taxonomy_domain +cultures O +reveal O +growth O +defects O +of O +the O +active B-site +- I-site +site I-site +mutants B-experimental_method +under O +the O +indicated O +conditions O +after O +2 O +days O +( O +2 O +d O +) O +of O +incubation O +. O + +( O +b O +) O +Purified O +WT B-protein_state +and O +mutant B-protein_state +proteasomes B-complex_assembly +were O +tested O +for O +their O +chymotrypsin O +- O +like O +activity O +( O +β5 B-protein +) O +using O +the O +substrate O +Suc B-chemical +- I-chemical +LLVY I-chemical +- I-chemical +AMC I-chemical +. O + +( O +c O +) O +Illustration O +of O +the O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +- I-evidence +density I-evidence +map I-evidence +( O +blue O +mesh O +contoured O +at O +1σ O +) O +for O +the O +β5 B-mutant +- I-mutant +T1C I-mutant +propeptide B-structure_element +fragment O +. O + +The O +prosegment B-structure_element +is O +cleaved B-protein_state +but O +still B-protein_state +bound I-protein_state +in O +the O +substrate B-site +- I-site +binding I-site +channel I-site +. O + +Notably O +, O +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +does O +not O +occupy O +the O +S1 B-site +pocket I-site +formed O +by O +Met45 B-residue_name_number +, O +similar O +to O +what O +was O +observed O +for O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +mutant B-protein_state +. O + +( O +d O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +and O +the O +β5 B-mutant +- I-mutant +T1C I-mutant +mutant B-protein_state +subunits O +onto O +the O +WT B-protein_state +β5 B-protein +subunit O +. O +( O +e O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β5 B-mutant +- I-mutant +T1C I-mutant +propeptide B-structure_element +onto O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +active B-site +site I-site +( O +blue O +) O +and O +the O +WT B-protein_state +β5 B-protein +active B-site +site I-site +in B-protein_state +complex I-protein_state +with I-protein_state +the O +proteasome B-complex_assembly +inhibitor O +MG132 B-chemical +( O +ref O +.). O + +The O +inhibitor B-chemical +as O +well O +as O +the O +propeptides B-structure_element +adopt O +similar O +conformations O +in O +the O +substrate B-site +- I-site +binding I-site +channel I-site +. O + +( O +f O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +WT B-protein_state +β5 B-protein +and O +β5 B-mutant +- I-mutant +T1C I-mutant +mutant B-protein_state +active B-site +sites I-site +illustrates O +the O +different O +orientations O +of O +the O +hydroxyl O +group O +of O +Thr1 B-residue_name_number +and O +the O +thiol O +side O +chain O +of O +Cys1 B-residue_name_number +. O + +( O +g O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +WT B-protein_state +β5 B-protein +and O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +active B-site +sites I-site +reveals O +different O +orientations O +of O +the O +hydroxyl O +groups O +of O +Thr1 B-residue_name_number +and O +Ser1 B-residue_name_number +, O +respectively O +. O + +The O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +- I-evidence +density I-evidence +map I-evidence +for O +Ser1 B-residue_name_number +( O +blue O +mesh O +contoured O +at O +1σ O +) O +is O +illustrated O +. O + +( O +h O +) O +The O +methyl O +group O +of O +Thr1 B-residue_name_number +is O +anchored O +by O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +Ala46Cβ B-residue_name_number +and O +Thr3Cγ B-residue_name_number +. O + +Ser1 B-residue_name_number +lacks B-protein_state +this O +stabilization O +and O +is O +therefore O +rotated O +by O +60 O +°. O + +Inhibition O +of O +WT B-protein_state +and O +mutant B-protein_state +β5 B-mutant +- I-mutant +T1S I-mutant +proteasomes B-complex_assembly +by O +bortezomib B-chemical +and O +carfilzomib B-chemical +. O + +Inhibition B-experimental_method +assays I-experimental_method +( O +left O +panel O +). O + +Purified O +yeast B-taxonomy_domain +proteasomes B-complex_assembly +were O +tested O +for O +the O +susceptibility O +of O +their O +ChT O +- O +L O +( O +β5 B-protein +) O +activity O +to O +inhibition O +by O +bortezomib B-chemical +and O +carfilzomib B-chemical +using O +the O +substrate O +Suc B-chemical +- I-chemical +LLVY I-chemical +- I-chemical +AMC I-chemical +. O + +IC50 B-evidence +values I-evidence +were O +determined O +in O +triplicate O +; O +s O +. O +d O +.' O +s O +are O +indicated O +by O +error O +bars O +. O + +Note O +that O +IC50 B-evidence +values I-evidence +depend O +on O +time O +and O +enzyme O +concentration O +. O + +Proteasomes B-complex_assembly +( O +final O +concentration O +: O +66 O +nM O +) O +were O +incubated O +with O +inhibitor O +for O +45 O +min O +before O +substrate O +addition O +( O +final O +concentration O +: O +200 O +μM O +). O + +Structures B-evidence +of O +the O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +in O +complex B-complex_assembly +with I-complex_assembly +both I-complex_assembly +ligands I-complex_assembly +( O +green O +) O +prove O +the O +reactivity O +of O +Ser1 B-residue_name_number +( O +right O +panel O +). O + +The O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +- I-evidence +density I-evidence +maps I-evidence +( O +blue O +mesh O +) O +for O +Ser1 B-residue_name_number +( O +brown O +) O +and O +the O +covalently O +bound O +ligands O +( O +green O +; O +only O +the O +P1 B-site +site I-site +( O +Leu1 B-residue_name_number +) O +is O +shown O +) O +are O +contoured O +at O +1σ O +. O + +The O +WT B-protein_state +proteasome B-complex_assembly +: I-complex_assembly +inhibitor I-complex_assembly +complex I-complex_assembly +structures B-evidence +( O +inhibitor O +in O +grey O +; O +Thr1 B-residue_name_number +in O +black O +) O +are O +superimposed B-experimental_method +and O +demonstrate O +that O +mutation B-experimental_method +of O +Thr1 B-residue_name_number +to O +Ser B-residue_name +does O +not O +affect O +the O +binding O +mode O +of O +bortezomib B-chemical +or O +carfilzomib B-chemical +. O + +The O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +is O +a O +reader O +of O +histone B-protein_type +crotonylation B-ptm + +The O +discovery O +of O +new O +histone B-protein_type +modifications O +is O +unfolding O +at O +startling O +rates O +, O +however O +, O +the O +identification O +of O +effectors O +capable O +of O +interpreting O +these O +modifications O +has O +lagged O +behind O +. O + +Here O +we O +report O +the O +YEATS B-structure_element +domain I-structure_element +as O +an O +effective O +reader O +of O +histone B-protein_type +lysine B-residue_name +crotonylation B-ptm +– O +an O +epigenetic O +signature O +associated O +with O +active O +transcription O +. O + +We O +show O +that O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +engages O +crotonyllysine B-residue_name +via O +a O +unique O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +mechanism O +and O +that O +other O +YEATS B-structure_element +domains I-structure_element +have O +crotonyllysine B-residue_name +binding O +activity O +. O + +Crotonylation B-ptm +of O +lysine B-residue_name +residues O +( O +crotonyllysine B-residue_name +, O +Kcr B-residue_name +) O +has O +emerged O +as O +one O +of O +the O +fundamental O +histone B-protein_type +post O +- O +translational O +modifications O +( O +PTMs O +) O +found O +in O +mammalian B-taxonomy_domain +chromatin O +. O + +The O +crotonyllysine B-residue_name +mark O +on O +histone B-protein_type +H3K18 B-protein_type +is O +produced O +by O +p300 B-protein +, O +a O +histone B-protein_type +acetyltransferase I-protein_type +also O +responsible O +for O +acetylation B-ptm +of O +histones O +. O + +Owing O +to O +some O +differences O +in O +their O +genomic O +distribution O +, O +the O +crotonyllysine B-residue_name +and O +acetyllysine B-residue_name +( O +Kac B-residue_name +) O +modifications O +have O +been O +linked O +to O +distinct O +functional O +outcomes O +. O + +p300 B-protein +- O +catalyzed O +histone B-protein_type +crotonylation B-ptm +, O +which O +is O +likely O +metabolically O +regulated O +, O +stimulates O +transcription O +to O +a O +greater O +degree O +than O +p300 B-protein +- O +catalyzed O +acetylation B-ptm +. O + +The O +discovery O +of O +individual O +biological O +roles O +for O +the O +crotonyllysine B-residue_name +and O +acetyllysine B-residue_name +marks O +suggests O +that O +these O +PTMs O +can O +be O +read O +by O +distinct O +readers O +. O + +While O +a O +number O +of O +acetyllysine B-residue_name +readers O +have O +been O +identified O +and O +characterized O +, O +a O +specific O +reader O +of O +the O +crotonyllysine B-residue_name +mark O +remains O +unknown O +( O +reviewed O +in O +). O + +A O +recent O +survey O +of O +bromodomains B-structure_element +( O +BDs B-structure_element +) O +demonstrates O +that O +only O +one O +BD B-structure_element +associates O +very O +weakly O +with O +a O +crotonylated B-protein_state +peptide O +, O +however O +it O +binds O +more O +tightly O +to O +acetylated B-protein_state +peptides O +, O +inferring O +that O +bromodomains B-structure_element +do O +not O +possess O +physiologically O +relevant O +crotonyllysine B-residue_name +binding O +activity O +. O + +The O +family O +of O +acetyllysine B-residue_name +readers O +has O +been O +expanded O +with O +the O +discovery O +that O +the O +YEATS B-structure_element +( O +Yaf9 B-protein +, O +ENL B-protein +, O +AF9 B-protein +, O +Taf14 B-protein +, O +Sas5 B-protein +) O +domains O +of O +human B-species +AF9 B-protein +and O +yeast B-taxonomy_domain +Taf14 B-protein +are O +capable O +of O +recognizing O +the O +histone B-protein_type +mark O +H3K9ac B-protein_type +. O + +The O +acetyllysine B-residue_name +binding O +function O +of O +the O +AF9 B-protein +YEATS B-structure_element +domain I-structure_element +is O +essential O +for O +the O +recruitment O +of O +the O +histone B-protein_type +methyltransferase I-protein_type +DOT1L B-protein +to O +H3K9ac B-protein_type +- O +containing O +chromatin O +and O +for O +DOT1L B-protein +- O +mediated O +H3K79 B-protein_type +methylation B-ptm +and O +transcription O +. O + +Similarly O +, O +activation O +of O +a O +subset O +of O +genes O +and O +DNA O +damage O +repair O +in O +yeast B-taxonomy_domain +require O +the O +acetyllysine B-residue_name +binding O +activity O +of O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +. O + +Consistent O +with O +its O +role O +in O +gene O +regulation O +, O +Taf14 B-protein +was O +identified O +as O +a O +core O +component O +of O +the O +transcription O +factor O +complexes O +TFIID B-complex_assembly +and O +TFIIF B-complex_assembly +. O + +However O +, O +Taf14 B-protein +is O +also O +found O +in O +a O +number O +of O +chromatin O +- O +remodeling O +complexes O +( O +i O +. O +e O +., O +INO80 B-complex_assembly +, O +SWI B-complex_assembly +/ I-complex_assembly +SNF I-complex_assembly +and O +RSC B-complex_assembly +) O +and O +the O +histone B-protein_type +acetyltransferase I-protein_type +complex O +NuA3 B-complex_assembly +, O +indicating O +a O +multifaceted O +role O +of O +Taf14 B-protein +in O +transcriptional O +regulation O +and O +chromatin O +biology O +. O + +In O +this O +study O +, O +we O +identified O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +as O +a O +reader O +of O +crotonyllysine B-residue_name +that O +binds O +to O +histone B-protein_type +H3 B-protein_type +crotonylated B-protein_state +at O +lysine B-residue_name_number +9 I-residue_name_number +( O +H3K9cr B-protein_type +) O +via O +a O +distinctive O +binding O +mechanism O +. O + +We O +found O +that O +H3K9cr B-protein_type +is O +present O +in O +yeast B-taxonomy_domain +and O +is O +dynamically O +regulated O +. O + +To O +elucidate O +the O +molecular O +basis O +for O +recognition O +of O +the O +H3K9cr B-protein_type +mark O +, O +we O +obtained O +a O +crystal B-evidence +structure I-evidence +of O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +in B-protein_state +complex I-protein_state +with I-protein_state +H3K9cr5 B-chemical +- I-chemical +13 I-chemical +( O +residues O +5 B-residue_range +– I-residue_range +13 I-residue_range +of O +H3 B-protein_type +) O +peptide O +( O +Fig O +. O +1 O +, O +Supplementary O +Results O +, O +Supplementary O +Fig O +. O +1 O +and O +Supplementary O +Table O +1 O +). O + +The O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +adopts O +an O +immunoglobin B-structure_element +- I-structure_element +like I-structure_element +β I-structure_element +sandwich I-structure_element +fold I-structure_element +containing O +eight O +anti B-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +strands I-structure_element +linked O +by O +short O +loops B-structure_element +that O +form O +a O +binding B-site +site I-site +for O +H3K9cr B-protein_type +( O +Fig O +. O +1b O +). O + +The O +H3K9cr B-protein_type +peptide O +lays O +in O +an O +extended B-protein_state +conformation I-protein_state +in O +an O +orientation O +orthogonal O +to O +the O +β B-structure_element +strands I-structure_element +and O +is O +stabilized O +through O +an O +extensive O +network O +of O +direct O +and O +water B-chemical +- O +mediated O +hydrogen B-bond_interaction +bonds I-bond_interaction +and O +a O +salt B-bond_interaction +bridge I-bond_interaction +( O +Fig O +. O +1c O +). O + +The O +most O +striking O +feature O +of O +the O +crotonyllysine B-residue_name +recognition O +mechanism O +is O +the O +unique O +coordination O +of O +crotonylated B-protein_state +lysine B-residue_name +residue O +. O + +The O +fully O +extended O +side O +chain O +of O +K9cr B-ptm +transverses O +the O +narrow O +tunnel O +, O +crossing O +the O +β B-structure_element +sandwich I-structure_element +at O +right O +angle O +in O +a O +corkscrew O +- O +like O +manner O +( O +Fig O +. O +1b O +and O +Supplementary O +Figure O +1b O +). O + +The O +planar O +crotonyl B-chemical +group O +is O +inserted O +between O +Trp81 B-residue_name_number +and O +Phe62 B-residue_name_number +of O +the O +protein O +, O +the O +aromatic O +rings O +of O +which O +are O +positioned O +strictly O +parallel O +to O +each O +other O +and O +at O +equal O +distance O +from O +the O +crotonyl B-chemical +group O +, O +yielding O +a O +novel O +aromatic O +- O +amide O +/ O +aliphatic O +- O +aromatic O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +system O +that O +, O +to O +our O +knowledge O +, O +has O +not O +been O +reported O +previously O +for O +any O +protein O +- O +protein O +interaction O +( O +Fig O +. O +1d O +and O +Supplementary O +Fig O +. O +1c O +). O + +The O +side O +chain O +of O +Trp81 B-residue_name_number +appears O +to O +adopt O +two O +conformations O +, O +one O +of O +which O +provides O +maximum O +π B-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +with O +the O +alkene O +functional O +group O +while O +the O +other O +rotamer O +affords O +maximum O +π B-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +with O +the O +amide O +π O +electrons O +( O +Supplementary O +Fig O +. O +1c O +). O + +The O +dual O +conformation O +of O +Trp81 B-residue_name_number +is O +likely O +due O +to O +the O +conjugated O +nature O +of O +the O +C O += O +C O +and O +C O += O +O O +π O +- O +orbitals O +within O +the O +crotonyl B-chemical +functional O +group O +. O + +In O +addition O +to O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +π I-bond_interaction +stacking I-bond_interaction +, O +the O +crotonyl B-chemical +group O +is O +stabilized O +by O +a O +set O +of O +hydrogen B-bond_interaction +bonds I-bond_interaction +and O +electrostatic B-bond_interaction +interactions I-bond_interaction +. O + +The O +π B-bond_interaction +bond I-bond_interaction +conjugation O +of O +the O +crotonyl B-chemical +group O +gives O +rise O +to O +a O +dipole O +moment O +of O +the O +alkene O +moiety O +, O +resulting O +in O +a O +partial O +positive O +charge O +on O +the O +β O +- O +carbon O +( O +Cβ O +) O +and O +a O +partial O +negative O +charge O +on O +the O +α O +- O +carbon O +( O +Cα O +). O + +This O +provides O +the O +capability O +for O +the O +alkene O +moiety O +to O +form O +electrostatic B-bond_interaction +contacts I-bond_interaction +, O +as O +Cα O +and O +Cβ O +lay O +within O +electrostatic B-bond_interaction +interaction I-bond_interaction +distances O +of O +the O +carbonyl O +oxygen O +of O +Gln79 B-residue_name_number +and O +of O +the O +hydroxyl O +group O +of O +Thr61 B-residue_name_number +, O +respectively O +. O + +The O +hydroxyl O +group O +of O +Thr61 B-residue_name_number +also O +participates O +in O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +amide O +nitrogen O +of O +the O +K9cr B-ptm +side O +chain O +( O +Fig O +. O +1d O +). O + +The O +fixed O +position O +of O +the O +Thr61 B-residue_name_number +hydroxyl O +group O +, O +which O +facilitates O +interactions O +with O +both O +the O +amide O +and O +Cα O +of O +K9cr B-ptm +, O +is O +achieved O +through O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +imidazole O +ring O +of O +His59 B-residue_name_number +. O + +Extra O +stabilization O +of O +K9cr B-ptm +is O +attained O +by O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +formed O +between O +its O +carbonyl O +oxygen O +and O +the O +backbone O +nitrogen O +of O +Trp81 B-residue_name_number +, O +as O +well O +as O +a O +water B-chemical +- O +mediated O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +backbone O +carbonyl O +group O +of O +Gly82 B-residue_name_number +( O +Fig O +1d O +). O + +This O +distinctive O +mechanism O +was O +corroborated O +through O +mapping O +the O +Taf14 B-protein +YEATS B-site +- I-site +H3K9cr I-site +binding I-site +interface I-site +in O +solution O +using O +NMR B-experimental_method +chemical I-experimental_method +shift I-experimental_method +perturbation I-experimental_method +analysis I-experimental_method +( O +Supplementary O +Fig O +. O +2a O +, O +b O +). O + +Binding O +of O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +to O +H3K9cr B-protein_type +is O +robust O +. O + +The O +dissociation B-evidence +constant I-evidence +( O +Kd B-evidence +) O +for O +the O +Taf14 B-complex_assembly +YEATS I-complex_assembly +- I-complex_assembly +H3K9cr5 I-complex_assembly +- I-complex_assembly +13 I-complex_assembly +complex O +was O +found O +to O +be O +9 O +. O +5 O +μM O +, O +as O +measured O +by O +fluorescence B-experimental_method +spectroscopy I-experimental_method +( O +Supplementary O +Fig O +. O +2c O +). O + +This O +value O +is O +in O +the O +range O +of O +binding B-evidence +affinities I-evidence +exhibited O +by O +the O +majority O +of O +histone O +readers O +, O +thus O +attesting O +to O +the O +physiological O +relevance O +of O +the O +H3K9cr B-protein_type +recognition O +by O +Taf14 B-protein +. O + +To O +determine O +whether O +H3K9cr B-protein_type +is O +present O +in O +yeast B-taxonomy_domain +, O +we O +generated O +whole B-experimental_method +cell I-experimental_method +extracts I-experimental_method +from O +logarithmically O +growing O +yeast B-taxonomy_domain +cells O +and O +subjected O +them O +to O +Western B-experimental_method +blot I-experimental_method +analysis I-experimental_method +using O +antibodies O +directed O +towards O +H3K9cr B-protein_type +, O +H3K9ac B-protein_type +and O +H3 B-protein_type +( O +Fig O +. O +2a O +, O +b O +, O +Supplementary O +Fig O +. O +3 O +and O +Supplementary O +Table O +2 O +). O + +Both O +H3K9cr B-protein_type +and O +H3K9ac B-protein_type +were O +detected O +in O +yeast B-taxonomy_domain +histones B-protein_type +; O +to O +our O +knowledge O +, O +this O +is O +the O +first O +report O +of O +H3K9cr B-protein_type +occurring O +in O +yeast B-taxonomy_domain +. O + +We O +next O +asked O +if O +H3K9cr B-protein_type +is O +regulated O +by O +the O +actions O +of O +histone B-protein_type +acetyltransferases I-protein_type +( O +HATs B-protein_type +) O +and O +histone B-protein_type +deacetylases I-protein_type +( O +HDACs B-protein_type +). O + +Towards O +this O +end O +, O +we O +probed O +extracts O +derived O +from O +yeast B-taxonomy_domain +cells O +in O +which O +major O +yeast B-taxonomy_domain +HATs B-protein_type +( O +HAT1 B-protein +, O +Gcn5 B-protein +, O +and O +Rtt109 B-protein +) O +or O +HDACs B-protein_type +( O +Rpd3 B-protein +, O +Hos1 B-protein +, O +and O +Hos2 B-protein +) O +were O +deleted B-experimental_method +. O + +As O +shown O +in O +Figure O +2a O +, O +b O +and O +Supplementary O +Fig O +. O +3e O +, O +H3K9cr B-protein_type +levels O +were O +abolished O +or O +reduced O +considerably O +in O +the O +HAT B-protein_type +deletion B-experimental_method +strains O +, O +whereas O +they O +were O +dramatically O +increased O +in O +the O +HDAC B-protein_type +deletion B-experimental_method +strains O +. O + +Furthermore O +, O +fluctuations O +in O +the O +H3K9cr B-protein_type +levels O +were O +more O +substantial O +than O +fluctuations O +in O +the O +corresponding O +H3K9ac B-protein_type +levels O +. O + +Together O +, O +these O +results O +reveal O +that O +H3K9cr B-protein_type +is O +a O +dynamic O +mark O +of O +chromatin O +in O +yeast B-taxonomy_domain +and O +suggest O +an O +important O +role O +for O +this O +modification O +in O +transcription O +as O +it O +is O +regulated O +by O +HATs B-protein_type +and O +HDACs B-protein_type +. O + +We O +have O +previously O +shown O +that O +among O +acetylated B-protein_state +histone B-protein_type +marks O +, O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +prefers O +acetylated B-protein_state +H3K9 B-protein_type +( O +also O +see O +Supplementary O +Fig O +. O +3b O +), O +however O +it O +binds O +to O +H3K9cr B-protein_type +tighter O +. O + +The O +selectivity O +of O +Taf14 B-protein +towards O +crotonyllysine B-residue_name +was O +substantiated O +by O +1H B-experimental_method +, I-experimental_method +15N I-experimental_method +HSQC I-experimental_method +experiments O +, O +in O +which O +either O +H3K9cr5 B-chemical +- I-chemical +13 I-chemical +or O +H3K9ac5 B-chemical +- I-chemical +13 I-chemical +peptide O +was O +titrated B-experimental_method +into O +the O +15N B-protein_state +- I-protein_state +labeled I-protein_state +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +( O +Fig O +. O +2c O +and O +Supplementary O +Fig O +. O +4a O +, O +b O +). O + +Binding O +of O +H3K9cr B-protein_type +induced O +resonance B-evidence +changes I-evidence +in O +slow O +exchange O +regime O +on O +the O +NMR B-experimental_method +time O +scale O +, O +indicative O +of O +strong O +interaction O +. O + +In O +contrast O +, O +binding O +of O +H3K9ac B-protein_type +resulted O +in O +an O +intermediate O +exchange O +, O +which O +is O +characteristic O +of O +a O +weaker O +association O +. O + +Furthermore O +, O +crosspeaks B-evidence +of O +Gly80 B-residue_name_number +and O +Trp81 B-residue_name_number +of O +the O +YEATS B-structure_element +domain I-structure_element +were O +uniquely O +perturbed O +by O +H3K9cr B-protein_type +and O +H3K9ac B-protein_type +, O +indicating O +a O +different O +chemical O +environment O +in O +the O +respective O +crotonyllysine B-site +and I-site +acetyllysine I-site +binding I-site +pockets I-site +( O +Supplementary O +Fig O +. O +4a O +). O + +These O +differences O +support O +our O +model O +that O +Trp81 B-residue_name_number +adopts O +two O +conformations O +upon O +complex O +formation O +with O +the O +H3K9cr B-protein_type +mark O +as O +compared O +to O +H3K9ac B-protein_type +( O +Supplementary O +Figs O +. O +1c O +, O +d O +and O +4c O +). O + +One O +of O +the O +conformations O +, O +characterized O +by O +the O +π O +stacking O +involving O +two O +aromatic O +residues O +and O +the O +alkene O +group O +, O +is O +observed O +only O +in O +the O +YEATS B-complex_assembly +- I-complex_assembly +H3K9cr I-complex_assembly +complex O +. O + +To O +establish O +whether O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +is O +able O +to O +recognize O +other O +recently O +identified O +acyllysine B-residue_name +marks O +, O +we O +performed O +solution B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +using O +H3 B-protein_type +peptides O +acetylated B-protein_state +, O +propionylated B-protein_state +, O +butyrylated B-protein_state +, O +and O +crotonylated B-protein_state +at O +lysine B-residue_name_number +9 I-residue_name_number +( O +residues O +1 B-residue_range +– I-residue_range +20 I-residue_range +of O +H3 B-protein_type +). O + +As O +shown O +in O +Figure O +2d O +and O +Supplementary O +Fig O +. O +5a O +, O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +binds O +more O +strongly O +to O +H3K9cr1 B-chemical +- I-chemical +20 I-chemical +, O +as O +compared O +to O +other O +acylated B-protein_state +histone O +peptides O +. O + +The O +preference O +for O +H3K9cr B-protein_type +over O +H3K9ac B-protein_type +, O +H3K9pr B-protein_type +and O +H3K9bu B-protein_type +was O +supported O +by O +1H B-experimental_method +, I-experimental_method +15N I-experimental_method +HSQC I-experimental_method +titration I-experimental_method +experiments I-experimental_method +. O + +Addition O +of O +H3K9ac1 B-chemical +- I-chemical +20 I-chemical +, O +H3K9pr1 B-chemical +- I-chemical +20 I-chemical +, O +and O +H3K9bu1 B-chemical +- I-chemical +20 I-chemical +peptides O +caused O +chemical B-evidence +shift I-evidence +perturbations I-evidence +in O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +in O +intermediate O +exchange O +regime O +, O +implying O +that O +these O +interactions O +are O +weaker O +compared O +to O +the O +interaction O +with O +the O +H3K9cr1 B-chemical +- I-chemical +20 I-chemical +peptide O +( O +Supplementary O +Fig O +. O +5b O +). O + +We O +concluded O +that O +H3K9cr B-protein_type +is O +the O +preferred O +target O +of O +this O +domain O +. O + +From O +comparative B-experimental_method +structural I-experimental_method +analysis I-experimental_method +of O +the O +YEATS O +complexes O +, O +Gly80 B-residue_name_number +emerged O +as O +candidate O +residue O +potentially O +responsible O +for O +the O +preference O +for O +crotonyllysine B-residue_name +. O + +In O +attempt O +to O +generate O +a O +mutant O +capable O +of O +accommodating O +a O +short O +acetyl O +moiety O +but O +discriminating O +against O +a O +longer O +, O +planar O +crotonyl B-chemical +moiety O +, O +we O +mutated B-protein_state +Gly80 B-residue_name_number +to O +more O +bulky O +residues O +, O +however O +all O +mutants B-protein_state +of I-protein_state +Gly80 B-residue_name_number +lost O +their O +binding O +activities O +towards O +either O +acylated B-protein_state +peptide O +, O +suggesting O +that O +Gly80 B-residue_name_number +is O +absolutely O +required O +for O +the O +interaction O +. O + +In O +contrast O +, O +mutation B-experimental_method +of O +Val24 B-residue_name_number +, O +a O +residue O +located O +on O +another O +side O +of O +Trp81 B-residue_name_number +, O +had O +no O +effect O +on O +binding O +( O +Fig O +. O +2d O +and O +Supplementary O +Fig O +. O +5a O +, O +c O +). O + +To O +determine O +if O +the O +binding O +to O +crotonyllysine B-residue_name +is O +conserved B-protein_state +, O +we O +tested O +human B-species +YEATS B-structure_element +domains I-structure_element +by O +pull B-experimental_method +- I-experimental_method +down I-experimental_method +experiments I-experimental_method +using O +singly O +and O +multiply O +acetylated B-protein_state +, O +propionylated B-protein_state +, O +butyrylated B-protein_state +, O +and O +crotonylated B-protein_state +histone B-protein_type +peptides O +( O +Supplementary O +Fig O +. O +6 O +). O + +We O +found O +that O +all O +YEATS B-structure_element +domains I-structure_element +tested O +are O +capable O +of O +binding O +to O +crotonyllysine B-residue_name +peptides O +, O +though O +they O +display O +variable O +preferences O +for O +the O +acyl O +moieties O +. O + +While O +YEATS2 B-protein +and O +ENL B-protein +showed O +selectivity O +for O +the O +crotonylated B-protein_state +peptides O +, O +GAS41 B-protein +and O +AF9 B-protein +bound O +acylated B-protein_state +peptides O +almost O +equally O +well O +. O + +Unlike O +the O +YEATS B-structure_element +domain I-structure_element +, O +a O +known O +acetyllysine B-protein_type +reader I-protein_type +, O +bromodomain B-structure_element +, O +does O +not O +recognize O +crotonyllysine B-residue_name +. O + +We O +assayed O +a O +large O +set O +of O +BDs B-structure_element +in O +pull B-experimental_method +- I-experimental_method +down I-experimental_method +experiments I-experimental_method +and O +found O +that O +this O +module O +is O +highly O +specific O +for O +acetyllysine B-residue_name +and O +propionyllysine B-residue_name +containing O +peptides O +( O +Supplementary O +Fig O +. O +7 O +). O + +However O +, O +bromodomains B-structure_element +did O +not O +interact O +( O +or O +associated O +very O +weakly O +) O +with O +longer O +acyl O +modifications O +, O +including O +crotonyllysine B-residue_name +, O +as O +in O +the O +case O +of O +BDs B-structure_element +of O +TAF1 B-protein +and O +BRD2 B-protein +, O +supporting O +recent O +reports O +. O + +These O +results O +demonstrate O +that O +the O +YEATS B-structure_element +domain I-structure_element +is O +currently O +the O +sole O +reader O +of O +crotonyllysine B-residue_name +. O + +In O +conclusion O +, O +we O +have O +identified O +the O +YEATS B-structure_element +domain I-structure_element +of O +Taf14 B-protein +as O +the O +first O +reader O +of O +histone B-protein_type +crotonylation B-ptm +. O + +The O +unique O +and O +previously O +unobserved O +aromatic O +- O +amide O +/ O +aliphatic O +- O +aromatic O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +mechanism O +facilitates O +the O +specific O +recognition O +of O +the O +crotonyl B-chemical +moiety O +. O + +We O +further O +demonstrate O +that O +H3K9cr B-protein_type +exists O +in O +yeast B-taxonomy_domain +and O +is O +dynamically O +regulated O +by O +HATs B-protein_type +and O +HDACs B-protein_type +. O + +As O +we O +previously O +showed O +the O +importance O +of O +acyllysine B-residue_name +binding O +by O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +for O +the O +DNA O +damage O +response O +and O +gene O +transcription O +, O +it O +will O +be O +essential O +in O +the O +future O +to O +define O +the O +physiological O +role O +of O +crotonyllysine B-residue_name +recognition O +and O +to O +differentiate O +the O +activities O +of O +Taf14 B-protein +that O +are O +due O +to O +binding O +to O +crotonyllysine B-residue_name +and O +acetyllysine B-residue_name +modifications O +. O + +Furthermore O +, O +the O +functional O +significance O +of O +crotonyllysine B-residue_name +recognition O +by O +other O +YEATS B-protein_type +proteins O +will O +be O +of O +great O +importance O +to O +elucidate O +and O +compare O +. O + +The O +structural O +mechanism O +for O +the O +recognition O +of O +H3K9cr B-protein_type + +( O +a O +) O +Chemical O +structure O +of O +crotonyllysine B-residue_name +. O +( O +b O +) O +The O +crystal B-evidence +structure I-evidence +of O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +( O +wheat O +) O +in B-protein_state +complex I-protein_state +with I-protein_state +the O +H3K9cr5 B-chemical +- I-chemical +13 I-chemical +peptide O +( O +green O +). O +( O +c O +) O +H3K9cr B-protein_type +is O +stabilized O +via O +an O +extensive O +network O +of O +intermolecular O +electrostatic B-bond_interaction +and I-bond_interaction +polar I-bond_interaction +interactions I-bond_interaction +with O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +. O + +( O +d O +) O +The O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +π I-bond_interaction +stacking I-bond_interaction +mechanism O +involving O +the O +alkene O +moiety O +of O +crotonyllysine B-residue_name +. O + +H3K9cr B-protein_type +is O +a O +selective O +target O +of O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element + +( O +a O +, O +b O +) O +Western B-experimental_method +blot I-experimental_method +analysis O +comparing O +the O +levels O +of O +H3K9cr B-protein_type +and O +H3K9ac B-protein_type +in O +wild B-protein_state +type I-protein_state +( O +WT B-protein_state +), O +HAT B-protein_type +deletion O +, O +or O +HDAC B-protein_type +deletion B-experimental_method +yeast B-taxonomy_domain +strains O +. O + +Total O +H3 B-protein_type +was O +used O +as O +a O +loading O +control O +. O + +( O +c O +) O +Superimposed O +1H B-experimental_method +, I-experimental_method +15N I-experimental_method +HSQC I-experimental_method +spectra B-evidence +of O +Taf14 B-protein +YEATS B-structure_element +recorded O +as O +H3K9cr5 B-chemical +- I-chemical +13 I-chemical +and O +H3K9ac5 B-chemical +- I-chemical +13 I-chemical +peptides O +were O +titrated B-experimental_method +in O +. O + +Spectra B-evidence +are O +color O +coded O +according O +to O +the O +protein O +: O +peptide O +molar O +ratio O +. O + +( O +d O +) O +Western B-experimental_method +blot I-experimental_method +analyses O +of O +peptide B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +using O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutated B-protein_state +Taf14 B-protein +YEATS B-structure_element +domains I-structure_element +and O +indicated O +peptides O +. O + +Structure B-evidence +of O +the O +GAT B-structure_element +domain O +of O +the O +endosomal O +adapter B-protein_type +protein I-protein_type +Tom1 B-protein + +Cellular O +homeostasis O +requires O +correct O +delivery O +of O +cell B-protein_type +- I-protein_type +surface I-protein_type +receptor I-protein_type +proteins O +( O +cargo O +) O +to O +their O +target O +subcellular O +compartments O +. O + +The O +adapter B-protein_type +proteins I-protein_type +Tom1 B-protein +and O +Tollip B-protein +are O +involved O +in O +sorting O +of O +ubiquitinated B-ptm +cargo O +in O +endosomal O +compartments O +. O + +Recruitment O +of O +Tom1 B-protein +to O +the O +endosomal O +compartments O +is O +mediated O +by O +its O +GAT B-structure_element +domain O +’ O +s O +association O +to O +Tollip B-protein +’ O +s O +Tom1 B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +( O +TBD B-structure_element +). O + +In O +this O +data O +article O +, O +we O +report O +the O +solution B-experimental_method +NMR I-experimental_method +- O +derived O +structure B-evidence +of O +the O +Tom1 B-protein +GAT B-structure_element +domain O +. O + +The O +estimated O +protein O +structure B-evidence +exhibits O +a O +bundle O +of O +three O +helical O +elements O +. O + +We O +compare B-experimental_method +the O +Tom1 B-protein +GAT B-structure_element +structure B-evidence +with O +those O +structures B-evidence +corresponding O +to O +the O +Tollip B-protein +TBD B-protein_state +- I-protein_state +and O +ubiquitin B-protein_state +- I-protein_state +bound I-protein_state +states O +. O + +Subject O +area O +Biology O +More O +specific O +subject O +area O +Structural O +biology O +Type O +of O +data O +Table O +, O +text O +file O +, O +graph O +, O +figures O +How O +data O +was O +acquired O +Circular B-experimental_method +dichroism I-experimental_method +and O +NMR B-experimental_method +. O + +NMR B-experimental_method +data O +was O +recorded O +using O +a O +Bruker O +800 O +MHz O +Data O +format O +PDB O +format O +text O +file O +. O + +Analyzed O +by O +CS B-experimental_method +- I-experimental_method +Rosetta I-experimental_method +, O +Protein B-experimental_method +Structure I-experimental_method +Validation I-experimental_method +Server I-experimental_method +( O +PSVS B-experimental_method +), O +NMRPipe B-experimental_method +, O +NMRDraw B-experimental_method +, O +and O +PyMol O +Experimental O +factors O +Recombinant O +human B-species +Tom1 B-protein +GAT B-structure_element +domain O +was O +purified O +to O +homogeneity O +before O +use O +Experimental O +features O +Solution B-evidence +structure I-evidence +of O +Tom1 B-protein +GAT B-structure_element +was O +determined O +from O +NMR B-experimental_method +chemical B-evidence +shift I-evidence +data O +Data O +source O +location O +Virginia O +and O +Colorado O +, O +United O +States O +. O + +Tom1 O +GAT B-structure_element +structural O +data O +is O +publicly O +available O +in O +the O +RCSB O +Protein O +Data O +Bank O +( O +http O +:// O +www O +. O +rscb O +. O +org O +/) O +under O +the O +accession O +number O +PDB O +: O +2n9d O + +The O +Tom1 B-protein +GAT B-structure_element +domain O +solution B-evidence +structure I-evidence +will O +provide O +additional O +tools O +for O +modulating O +its O +biological O +function O +. O + +Tom1 B-protein +GAT B-structure_element +can O +adopt O +distinct O +conformations O +upon O +ligand O +binding O +. O + +A O +conformational O +response O +of O +the O +Tom1 B-protein +GAT B-structure_element +domain O +upon O +Tollip B-protein +TBD B-structure_element +binding O +can O +serve O +as O +an O +example O +to O +explain O +mutually O +exclusive O +ligand O +binding O +events O +. O + +Analysis O +of O +the O +far B-experimental_method +- I-experimental_method +UV I-experimental_method +circular I-experimental_method +dichroism I-experimental_method +( O +CD B-experimental_method +) O +spectrum B-evidence +of O +the O +Tom B-protein +1 I-protein +GAT B-structure_element +domain O +( O +Fig O +. O +1 O +) O +predicts O +58 O +. O +7 O +% O +α B-structure_element +- I-structure_element +helix I-structure_element +, O +3 O +% O +β B-structure_element +- I-structure_element +strand I-structure_element +, O +15 O +. O +5 O +% O +turn O +, O +and O +22 O +. O +8 O +% O +disordered O +regions O +. O + +The O +Tom1 B-protein +GAT B-structure_element +structural B-evidence +restraints I-evidence +yielded O +ten O +helical O +structures B-evidence +( O +Fig O +. O +2A O +, O +B O +) O +with O +a O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +RMSD B-evidence +) O +of O +0 O +. O +9 O +Å O +for O +backbone O +and O +1 O +. O +3 O +Å O +for O +all O +heavy O +atoms O +( O +Table O +1 O +) O +and O +estimated O +the O +presence O +of O +three O +helices O +spanning O +residues O +Q216 B-residue_range +- I-residue_range +E240 I-residue_range +( O +α B-structure_element +- I-structure_element +helix I-structure_element +1 I-structure_element +), O +P248 B-residue_range +- I-residue_range +Q274 I-residue_range +( O +α B-structure_element +- I-structure_element +helix I-structure_element +2 I-structure_element +), O +and O +E278 B-residue_range +- I-residue_range +T306 I-residue_range +( O +α B-structure_element +- I-structure_element +helix I-structure_element +3 I-structure_element +). O + +Unlike O +ubiquitin B-chemical +binding O +, O +data O +suggest O +that O +conformational O +changes O +of O +the O +Tom1 B-protein +GAT B-structure_element +α B-structure_element +- I-structure_element +helices I-structure_element +1 I-structure_element +and I-structure_element +2 I-structure_element +occur O +upon O +Tollip B-protein +TBD B-structure_element +binding O +( O +Fig O +. O +3A O +, O +B O +). O + +Representative O +far B-experimental_method +- I-experimental_method +UV I-experimental_method +CD I-experimental_method +spectrum B-evidence +of O +the O +His B-experimental_method +- I-experimental_method +Tom1 B-protein +GAT B-structure_element +domain O +. O + +( O +A O +) O +Stereo O +view O +displaying O +the O +best O +- O +fit O +backbone B-experimental_method +superposition I-experimental_method +of O +the O +refined O +structures B-evidence +for O +the O +Tom1 B-protein +GAT B-structure_element +domain O +. O + +Helices O +are O +shown O +in O +orange O +, O +whereas O +loops O +are O +colored O +in O +green O +. O +( O +B O +) O +Ribbon O +illustration O +of O +the O +Tom1 B-protein +GAT B-structure_element +domain O +. O + +( O +A O +) O +Two O +views O +of O +the O +superimposed B-experimental_method +structures I-experimental_method +of O +the O +Tom1 B-protein +GAT B-structure_element +domain O +in O +the O +free B-protein_state +state O +( O +gray O +) O +with O +that O +in O +the O +Tollip B-protein +TBD B-protein_state +- I-protein_state +bound I-protein_state +state O +( O +red O +). O +( O +B O +) O +Two O +views O +of O +the O +superimposed B-experimental_method +structures I-experimental_method +of O +the O +Tom1 B-protein +GAT B-structure_element +domain O +( O +gray O +) O +with O +that O +in O +the O +Ub B-protein_state +- I-protein_state +bound I-protein_state +state O +( O +green O +). O + +NMR B-experimental_method +and O +refinement B-evidence +statistics I-evidence +for O +the O +Tom1 B-protein +GAT B-structure_element +domain O +. O + +NMR B-experimental_method +structural B-evidence +statistics I-evidence +for O +lowest O +energy O +conformers O +of O +Tom1 B-protein +GAT B-structure_element +using O +PSVS B-experimental_method +. O + +deviations O +were O +obtained O +by O +superimposing B-experimental_method +residues O +215 B-residue_range +– I-residue_range +309 I-residue_range +of O +Tom1 B-protein +GAT B-structure_element +among O +10 O +lowest O +energy O +refined O +structures B-evidence +. O + +Haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +/ O +Sigma B-protein +- I-protein +2 I-protein +receptor O +facilitates O +cancer O +proliferation O +and O +chemoresistance O + +Progesterone B-protein +- I-protein +receptor I-protein +membrane I-protein +component I-protein +1 I-protein +( O +PGRMC1 B-protein +/ O +Sigma B-protein +- I-protein +2 I-protein +receptor I-protein +) O +is O +a O +haem B-protein_type +- I-protein_type +containing I-protein_type +protein I-protein_type +that O +interacts O +with O +epidermal B-protein_type +growth I-protein_type +factor I-protein_type +receptor I-protein_type +( O +EGFR B-protein_type +) O +and O +cytochromes B-protein_type +P450 I-protein_type +to O +regulate O +cancer O +proliferation O +and O +chemoresistance O +; O +its O +structural O +basis O +remains O +unknown O +. O + +Here O +crystallographic B-experimental_method +analyses I-experimental_method +of O +the O +PGRMC1 B-protein +cytosolic B-structure_element +domain I-structure_element +at O +1 O +. O +95 O +Å O +resolution O +reveal O +that O +it O +forms O +a O +stable B-protein_state +dimer B-oligomeric_state +through O +stacking B-bond_interaction +interactions I-bond_interaction +of O +two O +protruding O +haem B-chemical +molecules O +. O + +The O +haem B-chemical +iron B-chemical +is O +five B-bond_interaction +- I-bond_interaction +coordinated I-bond_interaction +by I-bond_interaction +Tyr113 B-residue_name_number +, O +and O +the O +open O +surface B-site +of O +the O +haem B-chemical +mediates O +dimerization B-oligomeric_state +. O + +Carbon B-chemical +monoxide I-chemical +( O +CO B-chemical +) O +interferes O +with O +PGRMC1 B-protein +dimerization B-oligomeric_state +by O +binding O +to O +the O +sixth B-site +coordination I-site +site I-site +of O +the O +haem B-chemical +. O + +Haem B-chemical +- O +mediated O +PGRMC1 B-protein +dimerization B-oligomeric_state +is O +required O +for O +interactions O +with O +EGFR B-protein_type +and O +cytochromes B-protein_type +P450 I-protein_type +, O +cancer O +proliferation O +and O +chemoresistance O +against O +anti O +- O +cancer O +drugs O +; O +these O +events O +are O +attenuated O +by O +either O +CO B-chemical +or O +haem B-chemical +deprivation O +in O +cancer O +cells O +. O + +This O +study O +demonstrates O +protein O +dimerization B-oligomeric_state +via O +haem B-bond_interaction +– I-bond_interaction +haem I-bond_interaction +stacking I-bond_interaction +, O +which O +has O +not O +been O +seen O +in O +eukaryotes B-taxonomy_domain +, O +and O +provides O +insights O +into O +its O +functional O +significance O +in O +cancer O +. O + +PGRMC1 B-protein +binds O +to O +EGFR B-protein_type +and O +cytochromes B-protein_type +P450 I-protein_type +, O +and O +is O +known O +to O +be O +involved O +in O +cancer O +proliferation O +and O +in O +drug O +resistance O +. O + +Here O +, O +the O +authors O +determine O +the O +structure B-evidence +of O +the O +cytosolic B-structure_element +domain I-structure_element +of O +PGRMC1 B-protein +, O +which O +forms O +a O +dimer B-oligomeric_state +via O +haem B-bond_interaction +– I-bond_interaction +haem I-bond_interaction +stacking I-bond_interaction +, O +and O +propose O +how O +this O +interaction O +could O +be O +involved O +in O +its O +function O +. O + +Much O +attention O +has O +been O +paid O +to O +the O +roles O +of O +haem B-chemical +- O +iron B-chemical +in O +cancer O +development O +. O + +Increased O +dietary O +intake O +of O +haem B-chemical +is O +a O +risk O +factor O +for O +several O +types O +of O +cancer O +. O + +Previous O +studies O +showed O +that O +deprivation B-protein_state +of I-protein_state +iron B-chemical +or O +haem B-chemical +suppresses O +tumourigenesis O +. O + +On O +the O +other O +hand O +, O +carbon B-chemical +monoxide I-chemical +( O +CO B-chemical +), O +the O +gaseous O +mediator O +generated O +by O +oxidative O +degradation O +of O +haem B-chemical +via O +haem B-protein_type +oxygenase I-protein_type +( O +HO B-protein_type +), O +inhibits O +tumour O +growth O +. O + +Thus O +, O +a O +tenuous O +balance O +between O +free O +haem B-chemical +and O +CO B-chemical +plays O +key O +roles O +in O +cancer O +development O +and O +chemoresistance O +, O +although O +the O +underlying O +mechanisms O +are O +not O +fully O +understood O +. O + +To O +gain O +insight O +into O +the O +underlying O +mechanisms O +, O +we O +took O +chemical O +biological O +approaches O +using O +affinity B-experimental_method +nanobeads I-experimental_method +carrying O +haem B-chemical +and O +identified O +progesterone B-protein +- I-protein +receptor I-protein +membrane I-protein +component I-protein +1 I-protein +( O +PGRMC1 B-protein +) O +as O +a O +haem B-chemical +- O +binding O +protein O +from O +mouse B-taxonomy_domain +liver O +extracts O +( O +Supplementary O +Fig O +. O +1 O +). O + +PGRMC1 B-protein +is O +a O +member O +of O +the O +membrane B-protein_type +- I-protein_type +associated I-protein_type +progesterone I-protein_type +receptor I-protein_type +( O +MAPR B-protein_type +) O +family O +with O +a O +cytochrome B-structure_element +b5 I-structure_element +- I-structure_element +like I-structure_element +haem B-site +- I-site +binding I-site +region I-site +, O +and O +is O +known O +to O +be O +highly B-protein_state +expressed I-protein_state +in O +various O +types O +of O +cancers O +. O + +PGRMC1 B-protein +is O +anchored O +to O +the O +cell O +membrane O +through O +the O +N O +- O +terminal O +transmembrane B-structure_element +helix I-structure_element +and O +interacts O +with O +epidermal B-protein_type +growth I-protein_type +factor I-protein_type +receptor I-protein_type +( O +EGFR B-protein_type +) O +and O +cytochromes B-protein_type +P450 I-protein_type +( O +ref O +). O + +While O +PGRMC1 B-protein +is O +implicated O +in O +cell O +proliferation O +and O +cholesterol O +biosynthesis O +, O +the O +structural O +basis O +on O +which O +PGRMC1 B-protein +exerts O +its O +function O +remains O +largely O +unknown O +. O + +Here O +we O +show O +that O +PGRMC1 B-protein +exhibits O +a O +unique O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +. O + +The O +dimer B-oligomeric_state +binds O +to O +EGFR B-protein_type +and O +cytochromes B-protein_type +P450 I-protein_type +to O +enhance O +tumour O +cell O +proliferation O +and O +chemoresistance O +. O + +The O +dimer B-oligomeric_state +is O +dissociated O +to O +monomers B-oligomeric_state +by O +physiological O +levels O +of O +CO B-chemical +, O +suggesting O +that O +PGRMC1 B-protein +serves O +as O +a O +CO B-chemical +- O +sensitive O +molecular O +switch O +regulating O +cancer O +cell O +proliferation O +. O + +X B-evidence +- I-evidence +ray I-evidence +crystal I-evidence +structure I-evidence +of O +PGRMC1 B-protein + +We O +solved B-experimental_method +the O +crystal B-evidence +structure I-evidence +of O +the O +haem B-protein_state +- I-protein_state +bound I-protein_state +PGRMC1 B-protein +cytosolic B-structure_element +domain I-structure_element +( O +a O +. O +a O +. O +72 B-residue_range +– I-residue_range +195 I-residue_range +) O +at O +1 O +. O +95 O +Å O +resolution O +( O +Supplementary O +Fig O +. O +2 O +). O + +In O +the O +presence B-protein_state +of I-protein_state +haem B-chemical +, O +PGRMC1 B-protein +forms O +a O +dimeric B-oligomeric_state +structure O +largely O +through O +hydrophobic B-bond_interaction +interactions I-bond_interaction +between O +the O +haem B-chemical +moieties O +of O +two O +monomers B-oligomeric_state +( O +Fig O +. O +1a O +, O +Table O +1 O +and O +Supplementary O +Fig O +. O +3 O +; O +a O +stereo O +- O +structural O +image O +is O +shown O +in O +Supplementary O +Fig O +4 O +). O + +While O +the O +overall O +fold O +of O +PGRMC1 B-protein +is O +similar O +to O +that O +of O +canonical O +cytochrome B-protein_type +b5 I-protein_type +, O +their O +haem B-chemical +irons O +are O +coordinated O +differently O +. O + +In O +cytochrome B-protein_type +b5 I-protein_type +, O +the O +haem B-chemical +iron B-chemical +is O +six B-bond_interaction +- I-bond_interaction +coordinated I-bond_interaction +by I-bond_interaction +two O +axial O +histidine B-residue_name +residues O +. O + +These O +histidines B-residue_name +are O +missing B-protein_state +in O +PGRMC1 B-protein +, O +and O +the O +haem B-chemical +iron B-chemical +is O +five B-bond_interaction +- I-bond_interaction +coordinated I-bond_interaction +by I-bond_interaction +Tyr113 B-residue_name_number +( O +Y113 B-residue_name_number +) O +alone B-protein_state +( O +Fig O +. O +1b O +and O +Supplementary O +Fig O +. O +3 O +). O + +A O +homologous B-structure_element +helix I-structure_element +that O +holds O +haem B-chemical +in O +cytochrome B-protein_type +b5 I-protein_type +is O +longer O +, O +shifts O +away O +from O +haem B-chemical +, O +and O +does O +not O +form O +a O +coordinate O +bond O +in O +PGRMC1 B-protein +( O +Fig O +. O +1c O +). O + +Consequently O +, O +the O +five O +- O +coordinated O +haem B-chemical +of O +PGRMC1 B-protein +has O +an O +open O +surface B-site +that O +allows O +its O +dimerization B-oligomeric_state +through O +hydrophobic B-bond_interaction +haem I-bond_interaction +– I-bond_interaction +haem I-bond_interaction +stacking I-bond_interaction +. O + +Contrary O +to O +our O +finding O +, O +Kaluka O +et O +al O +. O +recently O +reported O +that O +Tyr164 B-residue_name_number +of O +PGRMC1 B-protein +is O +the O +axial O +ligand O +of O +haem B-chemical +because O +mutation B-experimental_method +of O +this O +residue O +impairs O +haem B-chemical +binding O +. O + +Our O +structural B-evidence +data I-evidence +revealed O +that O +Tyr164 B-residue_name_number +and O +a O +few O +other O +residues O +such O +as O +Tyr107 B-residue_name_number +and O +Lys163 B-residue_name_number +are O +in O +fact O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +haem B-chemical +propionates O +. O + +This O +is O +consistent O +with O +observations O +by O +Min O +et O +al O +. O +that O +Tyr B-residue_name_number +107 I-residue_name_number +and O +Tyr113 B-residue_name_number +of O +PGRMC1 B-protein +are O +involved O +in O +binding O +with O +haem B-chemical +. O + +These O +amino O +acid O +residues O +are O +conserved B-protein_state +among O +MAPR B-protein_type +family O +members O +( O +Supplementary O +Fig O +. O +5a O +), O +suggesting O +that O +these O +proteins O +share O +the O +ability O +to O +exhibit O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +. O + +PGRMC1 B-protein +exhibits O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +in O +solution O + +In O +the O +PGRMC1 B-protein +crystal B-evidence +, O +two O +different O +types O +of O +crystal O +contacts O +( O +chain O +A O +– O +A O +″ O +and O +A O +– O +B O +) O +were O +observed O +in O +addition O +to O +the O +haem B-chemical +- O +mediated O +dimer B-oligomeric_state +( O +chain O +A O +– O +A O +′) O +( O +Supplementary O +Figs O +3 O +and O +6a O +). O + +To O +confirm O +that O +haem B-chemical +- O +assisted O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +occurs O +in O +solution O +, O +we O +analysed O +the O +structure B-evidence +of O +apo B-protein_state +- O +and O +haem B-protein_state +- I-protein_state +bound I-protein_state +PGMRC1 B-protein +by O +two B-experimental_method +- I-experimental_method +dimensional I-experimental_method +nuclear I-experimental_method +magnetic I-experimental_method +resonance I-experimental_method +( O +NMR B-experimental_method +) O +using O +heteronuclear B-experimental_method +single I-experimental_method +- I-experimental_method +quantum I-experimental_method +coherence I-experimental_method +and I-experimental_method +transverse I-experimental_method +relaxation I-experimental_method +- I-experimental_method +optimized I-experimental_method +spectroscopy I-experimental_method +( O +Supplementary O +Figs O +6b O +and O +7 O +). O + +NMR B-experimental_method +signals O +from O +some O +amino O +acid O +residues O +of O +PGRMC1 B-protein +disappeared O +due O +to O +the O +paramagnetic O +relaxation O +effect O +of O +haem B-chemical +( O +Supplementary O +Figs O +6b O +); O +these O +residues O +were O +located O +in O +the O +haem B-site +- I-site +binding I-site +region I-site +. O + +When O +chemical B-evidence +shifts I-evidence +of O +apo B-protein_state +- O +and O +haem B-protein_state +- I-protein_state +bound I-protein_state +forms O +of O +PGMRC1 B-protein +were O +compared O +, O +some O +amino O +acid O +residues O +close O +to O +those O +which O +disappeared O +because O +of O +the O +paramagnetic O +relaxation O +effect O +of O +haem B-chemical +exhibit O +notable O +chemical O +shifts O +( O +Supplementary O +Fig O +. O +6a O +, O +b O +; O +dark O +yellow O +). O + +However O +, O +at O +the O +interfaces B-site +of O +the O +other O +possible O +dimeric B-oligomeric_state +structures B-evidence +( O +Supplementary O +Fig O +. O +6a O +, O +chain O +A O +– O +A O +″; O +cyan O +and O +chain O +A O +– O +B O +; O +violet O +), O +no O +significant O +difference O +was O +observed O +. O + +Furthermore O +, O +free B-evidence +energy I-evidence +of I-evidence +dissociation I-evidence +predicted O +by O +PISA B-experimental_method +suggested O +that O +the O +haem B-chemical +- O +mediated O +dimer B-oligomeric_state +is O +stable B-protein_state +in O +solution O +while O +the O +other O +potential O +interactions O +are O +not O +. O + +We O +also O +attempted O +to O +predict O +the O +secondary O +structure O +of O +PGRMC1 B-protein +through O +NMR B-experimental_method +data O +by O +calculating O +with O +TALOS B-experimental_method ++ I-experimental_method +program I-experimental_method +( O +Supplementary O +Fig O +. O +8 O +); O +the O +prediction O +suggested O +that O +the O +overall O +secondary O +structure O +is O +comparable O +between O +apo B-protein_state +- O +and O +haem B-protein_state +- I-protein_state +bound I-protein_state +forms O +of O +PGRMC1 B-protein +in O +solution O +. O + +We O +analysed O +the O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +the O +PGRMC1 B-protein +cytosolic B-structure_element +domain I-structure_element +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +) O +in O +solution O +( O +Fig O +. O +2 O +and O +Table O +2 O +). O + +Mass B-experimental_method +spectrometry I-experimental_method +( O +MS B-experimental_method +) O +analyses O +under O +non B-experimental_method +- I-experimental_method +denaturing I-experimental_method +condition I-experimental_method +demonstrated O +that O +the O +apo B-protein_state +- O +monomer B-oligomeric_state +PGRMC1 B-protein +resulted O +in O +dimerization B-oligomeric_state +by O +binding O +with O +haem B-chemical +( O +Fig O +. O +2a O +). O + +It O +should O +be O +noted O +that O +a O +disulfide B-ptm +bond I-ptm +between O +two O +Cys129 B-residue_name_number +residues O +is O +observed O +in O +the O +crystal B-evidence +of O +PGRMC1 B-protein +( O +Fig O +. O +1a O +), O +while O +Cys129 B-residue_name_number +is O +not B-protein_state +conserved I-protein_state +among O +the O +MAPR B-protein_type +family O +proteins O +( O +Supplementary O +Fig O +. O +5a O +). O + +This O +observation O +led O +us O +to O +examine O +whether O +or O +not O +the O +disulfide B-ptm +bond I-ptm +contributes O +to O +PGRMC1 B-protein +dimerization B-oligomeric_state +. O + +MS B-experimental_method +analyses O +under O +non B-experimental_method +- I-experimental_method +denaturing I-experimental_method +conditions I-experimental_method +clearly O +showed O +that O +the O +Cys129Ser B-mutant +( O +C129S B-mutant +) O +mutant B-protein_state +is O +dimerized B-protein_state +in O +the O +presence B-protein_state +of I-protein_state +haem B-chemical +, O +indicating O +that O +the O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +occurs O +independently O +of O +the O +disulfide B-ptm +bond I-ptm +formation O +via O +Cys129 B-residue_name_number +( O +Fig O +. O +2a O +). O + +Supporting O +this O +, O +MS B-experimental_method +analyses O +under O +denaturing B-experimental_method +conditions I-experimental_method +showed O +that O +haem B-chemical +- O +mediated O +PGRMC1 B-protein +dimer B-oligomeric_state +is O +completely O +dissociated O +into O +monomer B-oligomeric_state +, O +indicating O +that O +dimerization B-oligomeric_state +of O +this O +kind O +is O +not O +mediated O +by O +any O +covalent O +bond O +such O +as O +disulfide B-ptm +bond I-ptm +( O +Supplementary O +Fig O +. O +9 O +). O + +We O +also O +analysed O +the O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +by O +diffusion B-experimental_method +- I-experimental_method +ordered I-experimental_method +NMR I-experimental_method +spectroscopy I-experimental_method +( O +DOSY B-experimental_method +) O +analyses O +( O +Table O +2 O +, O +Supplementary O +Fig O +. O +10 O +). O + +The O +results O +suggested O +that O +the O +hydrodynamic B-evidence +radius I-evidence +of O +haem B-protein_state +- I-protein_state +bound I-protein_state +PGRMC1 B-protein +is O +larger O +than O +that O +of O +apo B-protein_state +- O +PGRMC1 B-protein +. O + +To O +further O +evaluate O +changes O +in O +molecular O +weights O +in O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +, O +sedimentation B-experimental_method +velocity I-experimental_method +analytical I-experimental_method +ultracentrifugation I-experimental_method +( O +SV B-experimental_method +- I-experimental_method +AUC I-experimental_method +) O +analysis O +was O +carried O +out O +. O + +Whereas O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +wt B-protein_state +) O +apo B-protein_state +- O +PGRMC1 B-protein +appeared O +at O +a O +1 O +. O +9 O +S O +peak O +as O +monomer B-oligomeric_state +, O +the O +haem B-chemical +- O +binding O +PGRMC1 B-protein +was O +converted O +into O +dimer B-oligomeric_state +at O +a O +3 O +. O +1 O +S O +peak O +( O +Fig O +. O +2b O +). O + +Similarly O +, O +the O +C129S B-mutant +mutant B-protein_state +of O +PGRMC1 B-protein +converted O +from O +monomer B-oligomeric_state +to O +dimer B-oligomeric_state +by O +binding O +to O +haem B-chemical +( O +Fig O +. O +2b O +). O + +SV B-experimental_method +- I-experimental_method +AUC I-experimental_method +analyses O +also O +allowed O +us O +to O +examine O +the O +stability O +of O +haem B-chemical +/ O +PGRMC1 B-protein +dimer B-oligomeric_state +. O + +To O +this O +end O +, O +we O +used O +different O +concentrations O +( O +3 O +. O +5 O +– O +147 O +μmol O +l O +− O +1 O +) O +of O +haem B-protein_state +- I-protein_state +bound I-protein_state +PGRMC1 B-protein +protein O +( O +a O +. O +a O +. O +72 B-residue_range +– I-residue_range +195 I-residue_range +), O +which O +were O +identical O +to O +that O +used O +in O +the O +crystallographic B-experimental_method +analysis I-experimental_method +. O + +The O +sedimentation B-evidence +coefficients I-evidence +calculated O +on O +the O +basis O +of O +the O +crystal B-evidence +structure I-evidence +were O +1 O +. O +71 O +S O +for O +monomer B-oligomeric_state +and O +2 O +. O +56 O +S O +for O +dimer B-oligomeric_state +( O +Supplementary O +Fig O +. O +11 O +, O +upper O +panel O +). O + +The O +results O +showed O +that O +the O +PGRMC1 B-protein +dimer B-oligomeric_state +is O +not O +dissociated O +into O +monomer B-oligomeric_state +at O +all O +concentrations O +examined O +( O +Supplementary O +Fig O +. O +11 O +, O +lower O +panel O +), O +suggesting O +that O +the O +Kd B-evidence +value O +of O +haem B-chemical +- O +mediated O +dimer B-oligomeric_state +of O +PGRMC1 B-protein +is O +under O +3 O +. O +5 O +μmol O +l O +− O +1 O +. O + +A O +value O +of O +this O +kind O +implies O +that O +the O +PGRMC1 B-protein +dimer B-oligomeric_state +is O +more O +stable O +than O +other O +dimers B-oligomeric_state +of O +extracellular B-structure_element +domain I-structure_element +of O +membrane B-protein_type +proteins I-protein_type +such O +as O +Toll B-protein +like I-protein +receptor I-protein +9 I-protein +( O +dimerization B-oligomeric_state +Kd B-evidence +of O +20 O +μmol O +l O +− O +1 O +) O +( O +ref O +.) O +and O +plexin B-protein +A2 I-protein +receptor I-protein +( O +dimerization B-oligomeric_state +Kd B-evidence +higher O +than O +300 O +μmol O +l O +− O +1 O +) O +( O +ref O +.). O + +The O +current O +analytical O +data O +confirmed O +that O +apo B-protein_state +- O +PGRMC1 B-protein +monomer B-oligomeric_state +converts O +into O +dimer B-oligomeric_state +by O +binding O +to O +haem B-chemical +in O +solution O +( O +Table O +2 O +). O + +We O +also O +showed O +by O +haem B-experimental_method +titration I-experimental_method +experiments I-experimental_method +that O +haem B-chemical +binding O +to O +PGRMC1 B-protein +was O +of O +low O +affinity O +with O +a O +Kd B-evidence +value O +of O +50 O +nmol O +l O +− O +1 O +; O +this O +is O +comparable O +with O +that O +of O +iron B-protein +regulatory I-protein +protein I-protein +2 I-protein +, O +which O +is O +known O +to O +be O +regulated O +by O +intracellular O +levels O +of O +haem B-chemical +( O +Fig O +. O +2c O +and O +Supplementary O +Table O +1 O +). O + +These O +results O +raised O +the O +possibility O +that O +the O +function O +of O +PGRMC1 B-protein +is O +regulated O +by O +intracellular O +haem B-chemical +concentrations O +. O + +CO B-chemical +inhibits O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein + +Crystallographic B-experimental_method +analyses I-experimental_method +revealed O +that O +Tyr113 B-residue_name_number +of O +PGRMC1 B-protein +is O +an O +axial O +ligand O +for O +haem B-chemical +and O +contributes O +to O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +( O +Fig O +. O +1a O +). O + +Analysis O +of O +UV B-evidence +- I-evidence +visible I-evidence +spectra I-evidence +revealed O +that O +the O +heme B-chemical +of O +PGRMC1 B-protein +is O +reducible O +from O +ferric B-protein_state +to O +ferrous B-protein_state +state O +, O +thus O +allowing O +CO B-chemical +binding O +( O +Fig O +. O +3a O +). O + +Furthermore O +, O +the O +UV B-evidence +- I-evidence +visible I-evidence +spectrum I-evidence +of O +the O +wild B-protein_state +type I-protein_state +PGRMC1 B-protein +was O +the O +same O +as O +that O +of O +the O +C129S B-mutant +mutant B-protein_state +of O +PGRMC1 B-protein +, O +and O +the O +R B-evidence +/ I-evidence +Z I-evidence +ratio I-evidence +determined O +by O +the O +intensities O +between O +the O +Soret O +band O +( O +394 O +nm O +) O +peak O +and O +the O +274 O +- O +nm O +peak O +showed O +that O +these O +proteins O +were O +fully B-protein_state +loaded I-protein_state +with I-protein_state +haem B-chemical +( O +Supplementary O +Fig O +. O +12 O +). O + +Analysis O +of O +the O +ferric B-protein_state +form O +of O +PGRMC1 B-protein +using O +resonance B-experimental_method +Raman I-experimental_method +spectroscopy I-experimental_method +( O +Supplementary O +Fig O +. O +13 O +) O +showed O +that O +the O +relative O +intensity O +of O +oxidation O +and O +spin O +state O +marker O +bands O +( O +ν4 O +and O +ν3 O +) O +is O +close O +to O +1 O +. O +0 O +, O +which O +is O +consistent O +with O +it O +being O +a O +haem B-chemical +protein O +with O +a O +proximal O +Tyr B-residue_name +coordination O +. O + +A O +specific O +Raman B-evidence +shift I-evidence +peaking O +at O +vFe O +– O +CO O += O +500 O +cm O +− O +1 O +demonstrated O +that O +the O +CO B-protein_state +- I-protein_state +bound I-protein_state +haem B-chemical +of O +PGRMC1 B-protein +is O +six O +- O +coordinated O +( O +Supplementary O +Fig O +. O +13 O +). O + +Since O +PGRMC1 B-protein +dimerization B-oligomeric_state +involves O +the O +open O +surface B-site +of O +haem B-chemical +on O +the O +opposite O +side O +of O +the O +axial O +Tyr113 B-residue_name_number +, O +no O +space O +for O +CO B-chemical +binding O +is O +available O +in O +the O +dimeric B-oligomeric_state +structure B-evidence +( O +Fig O +. O +3b O +). O + +This O +prompted O +us O +to O +ask O +if O +CO B-chemical +binding O +to O +haem B-chemical +causes O +dissociation O +of O +the O +PGRMC1 B-protein +dimer B-oligomeric_state +. O + +Analysis O +by O +gel B-experimental_method +filtration I-experimental_method +chromatography I-experimental_method +revealed O +that O +the O +relative O +molecular O +sizes O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +the O +C129S B-mutant +mutant B-protein_state +of O +PGRMC1 B-protein +are O +increased O +by O +adding O +haem B-chemical +to O +apo B-protein_state +- O +PGRMC1 B-protein +regardless O +of O +the O +oxidation O +state O +of O +the O +iron B-chemical +( O +Fig O +. O +3c O +), O +which O +is O +in O +agreement O +with O +the O +results O +in O +Table O +1 O +. O + +CO B-chemical +application O +to O +ferrous B-protein_state +PGRMC1 B-protein +abolished O +the O +haem B-chemical +- O +dependent O +increase O +in O +its O +molecular O +size O +. O + +Under O +this O +reducing O +condition O +in O +the O +presence B-protein_state +of I-protein_state +dithionite B-chemical +, O +analyses O +of O +UV B-evidence +- I-evidence +visible I-evidence +spectra I-evidence +indicated O +that O +CO B-chemical +- O +binding O +with O +haem B-complex_assembly +- I-complex_assembly +PGRMC1 I-complex_assembly +is O +stable B-protein_state +, O +showing O +only O +20 O +% O +reduction O +of O +the O +absorbance O +at O +412 O +nm O +within O +2 O +h O +( O +Supplementary O +Fig O +. O +14 O +). O + +Furthermore O +, O +the O +Tyr113Phe B-mutant +( O +Y113F B-mutant +) O +mutant B-protein_state +of O +PGRMC1 B-protein +was O +not O +responsive O +to O +haem B-chemical +. O + +These O +results O +suggest O +that O +CO B-chemical +favours O +the O +six O +- O +coordinate O +form O +of O +haem B-chemical +and O +interferes O +with O +the O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +. O + +To O +examine O +the O +inhibitory O +effects O +of O +CO B-chemical +on O +haem B-chemical +- O +mediated O +PGRMC1 B-protein +dimerization B-oligomeric_state +, O +SV B-experimental_method +- I-experimental_method +AUC I-experimental_method +analysis O +was O +carried O +out O +. O + +The O +peak O +corresponding O +to O +the O +haem B-chemical +/ O +PGRMC1 B-protein +dimer B-oligomeric_state +was O +detected O +under O +reducing O +conditions O +in O +the O +presence B-protein_state +of I-protein_state +dithionite B-chemical +( O +Supplementary O +Fig O +. O +15 O +, O +middle O +panel O +). O + +Under O +these O +circumstances O +, O +CO B-chemical +application O +induced O +dissociation O +of O +the O +haem B-chemical +- O +mediated O +dimers B-oligomeric_state +of O +PGRMC1 B-protein +to O +generate O +a O +peak O +of O +monomers B-oligomeric_state +( O +Supplementary O +Fig O +. O +15 O +, O +lower O +panel O +). O + +These O +observations O +raised O +the O +transition O +model O +for O +structural O +regulation O +of O +PGRMC1 B-protein +in O +response O +to O +haem B-chemical +( O +Fig O +. O +3d O +). O + +As O +mentioned O +above O +, O +apo B-protein_state +- O +PGRMC1 B-protein +exists O +as O +monomer B-oligomeric_state +. O + +By O +binding O +with O +haem B-chemical +( O +binding O +Kd B-evidence += O +50 O +nmol O +l O +− O +1 O +), O +PGRMC1 B-protein +forms O +a O +stable B-protein_state +dimer B-oligomeric_state +( O +dimerization B-oligomeric_state +Kd B-evidence +<< O +3 O +. O +5 O +μmol O +l O +− O +1 O +) O +through O +stacking B-bond_interaction +of O +the O +two O +open O +surfaces B-site +of O +the O +five O +- O +coordinated O +haem B-chemical +molecules O +in O +each O +monomer B-oligomeric_state +. O + +CO B-chemical +induces O +the O +dissociation O +of O +the O +haem B-chemical +- O +mediated O +dimer B-oligomeric_state +of O +PGRMC1 B-protein +by O +interfering O +with O +the O +haem B-site +- I-site +stacking I-site +interface I-site +via O +formation O +of O +the O +six O +- O +coordinated O +CO B-complex_assembly +- I-complex_assembly +haem I-complex_assembly +- I-complex_assembly +PGRMC1 I-complex_assembly +complex O +. O + +Such O +a O +dynamic O +structural O +regulation O +led O +us O +to O +further O +examine O +the O +regulation O +of O +PGRMC1 B-protein +functions O +in O +cancer O +cells O +. O + +PGRMC1 B-protein +dimerization B-oligomeric_state +is O +required O +for O +binding O +to O +EGFR B-protein_type + +Because O +PGRMC1 B-protein +is O +known O +to O +interact O +with O +EGFR B-protein_type +and O +to O +accelerate O +tumour O +progression O +, O +we O +examined O +the O +effect O +of O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +on O +its O +interaction O +with O +EGFR B-protein_type +by O +using O +purified O +proteins O +. O + +As O +shown O +in O +Fig O +. O +4a O +, O +the O +cytosolic B-structure_element +domain I-structure_element +of O +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +, O +but O +not O +the O +Y113F B-mutant +mutant B-protein_state +, O +interacted O +with O +purified O +EGFR B-protein_type +in O +a O +haem B-chemical +- O +dependent O +manner O +. O + +This O +interaction O +was O +disrupted O +by O +the O +ruthenium B-chemical +- O +based O +CO B-chemical +- O +releasing O +molecule O +, O +CORM3 B-chemical +, O +but O +not O +by O +RuCl3 B-chemical +as O +a O +control O +reagent O +( O +Fig O +. O +4b O +). O + +We O +further O +analysed O +the O +intracellular O +interaction O +between O +PGRMC1 B-protein +and O +EGFR B-protein_type +. O + +FLAG B-protein_state +- I-protein_state +tagged I-protein_state +PGRMC1 B-protein +ectopically B-experimental_method +expressed I-experimental_method +in O +human B-species +colon O +cancer O +HCT116 O +cells O +was O +immunoprecipitated B-experimental_method +with O +anti O +- O +FLAG O +antibody O +, O +and O +co B-experimental_method +- I-experimental_method +immunoprecipitated I-experimental_method +EGFR B-protein_type +and O +endogenous B-protein_state +PGRMC1 B-protein +binding O +to O +FLAG O +- O +PGRMC1 B-protein +were O +detected O +by O +Western B-experimental_method +blotting I-experimental_method +( O +Fig O +. O +4c O +). O + +The O +C129S B-mutant +mutant B-protein_state +of O +PGRMC1 B-protein +also O +interacted O +with O +endogenous B-protein_state +PGRMC1 B-protein +and O +EGFR B-protein_type +( O +Supplementary O +Fig O +. O +16 O +). O + +Whereas O +FLAG B-protein_state +- I-protein_state +tagged I-protein_state +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +interacted O +with O +endogenous B-protein_state +PGRMC1 B-protein +and O +EGFR B-protein_type +, O +the O +Y113F B-mutant +mutant B-protein_state +did O +not O +. O + +We O +also O +examined O +the O +effect O +of O +succinylacetone B-chemical +( O +SA B-chemical +), O +an O +inhibitor O +of O +haem B-chemical +biosynthesis O +( O +Fig O +. O +4d O +). O + +As O +expected O +, O +SA B-chemical +significantly O +reduced B-protein_state +PGRMC1 B-protein +dimerization B-oligomeric_state +and O +its O +interaction O +with O +EGFR B-protein_type +( O +Fig O +. O +4e O +), O +indicating O +that O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGMRC1 B-protein +is O +critical O +for O +its O +binding O +to O +EGFR B-protein_type +. O + +PGRMC1 B-protein +dimer B-oligomeric_state +facilitates O +EGFR B-protein_type +- O +mediated O +cancer O +growth O + +Next O +, O +we O +investigated O +the O +functional O +significance O +of O +PGRMC1 B-protein +dimerization B-oligomeric_state +in O +EGFR B-protein_type +signaling O +. O + +EGF B-protein_type +- O +induced O +phosphorylations B-ptm +of O +EGFR B-protein_type +and O +its O +downstream O +targets O +AKT B-protein_type +and O +ERK B-protein_type +were O +decreased O +by O +PGRMC1 B-protein +knockdown B-protein_state +( O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +) O +( O +Fig O +. O +4f O +). O + +Similarly O +, O +EGFR B-protein_type +signaling O +was O +suppressed O +by O +treatment O +of O +HCT116 O +cells O +with O +SA B-chemical +( O +Fig O +. O +4g O +) O +or O +CORM3 B-chemical +( O +Fig O +. O +4h O +). O + +These O +results O +suggested O +that O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +is O +critical O +for O +EGFR B-protein_type +signaling O +. O + +To O +further O +investigate O +the O +role O +of O +the O +dimerized B-protein_state +form O +of O +PGRMC1 B-protein +in O +cancer O +proliferation O +, O +we O +performed O +PGRMC1 B-protein +knockdown B-experimental_method +- I-experimental_method +rescue I-experimental_method +experiments I-experimental_method +using O +FLAG B-protein_state +- I-protein_state +tagged I-protein_state +wild B-protein_state +- I-protein_state +type I-protein_state +and O +Y113F B-mutant +PGRMC1 B-protein +expression B-experimental_method +vectors I-experimental_method +, O +in O +which O +silent B-experimental_method +mutations I-experimental_method +were O +introduced B-experimental_method +into O +the O +nucleotide O +sequence O +targeted O +by O +shRNA B-chemical +( O +Fig O +. O +5a O +). O + +While O +proliferation O +of O +HCT116 O +cells O +was O +not O +affected O +by O +knocking B-experimental_method +down I-experimental_method +PGRMC1 B-protein +, O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +were O +more O +sensitive O +to O +the O +EGFR B-protein_type +inhibitor O +erlotinib B-chemical +than O +control O +HCT116 O +cells O +, O +and O +the O +knockdown O +effect O +was O +reversed O +by O +co B-experimental_method +- I-experimental_method +expression I-experimental_method +of O +shRNA B-protein_state +- I-protein_state +resistant I-protein_state +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +but O +not O +of O +the O +Y113F B-mutant +mutant B-protein_state +( O +Fig O +. O +5b O +). O + +Chemosensitivity O +enhancement O +by O +two O +different O +shRNAs B-chemical +to O +PGRMC1 B-protein +was O +seen O +also O +in O +HCT116 O +cells O +and O +human B-species +hepatoma O +HuH7 O +cells O +( O +Supplementary O +Fig O +. O +17 O +). O + +Furthermore O +, O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +inhibited O +spheroid O +formation O +of O +HCT116 O +cells O +in O +culture O +, O +and O +this O +inhibition O +was O +reversed O +by O +co B-experimental_method +- I-experimental_method +expression I-experimental_method +of O +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +but O +not O +of O +the O +Y113F B-mutant +mutant B-protein_state +( O +Fig O +. O +5c O +and O +Supplementary O +Fig O +. O +18 O +). O + +Thus O +, O +PGRMC1 B-protein +dimerization B-oligomeric_state +is O +important O +for O +cancer O +cell O +proliferation O +and O +chemoresistance O +. O + +We O +examined O +the O +role O +of O +PGRMC1 B-protein +in O +metastatic O +progression O +by O +xenograft B-experimental_method +transplantation I-experimental_method +assays I-experimental_method +using O +super O +- O +immunodeficient O +NOD O +/ O +scid O +/ O +γnull O +( O +NOG O +) O +mice O +. O + +Ten O +days O +after O +intra B-experimental_method +- I-experimental_method +splenic I-experimental_method +implantation I-experimental_method +of O +HCT116 O +cells O +that O +were O +genetically O +tagged O +with O +a O +fluorescent O +protein O +Venus O +, O +the O +group O +implanted O +with O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +showed O +a O +significant O +decrease O +of O +liver O +metastasis O +in O +comparison O +with O +the O +control O +group O +( O +Fig O +. O +5d O +). O + +Interaction O +of O +PGRMC1 B-protein +dimer B-oligomeric_state +with O +cytochromes B-protein_type +P450 I-protein_type + +Since O +PGRMC1 B-protein +has O +been O +shown O +to O +interact O +with O +cytochromes B-protein_type +P450 I-protein_type +( O +ref O +), O +we O +investigated O +whether O +the O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +is O +necessary O +for O +their O +interactions O +. O + +Recombinant O +CYP1A2 B-protein +and O +CYP3A4 B-protein +including O +a O +microsomal O +formulation O +containing O +cytochrome B-protein_type +b5 I-protein_type +and O +cytochrome B-protein +P450 I-protein +reductase I-protein +, O +drug O +- O +metabolizing O +cytochromes B-protein_type +P450 I-protein_type +, O +interacted O +with O +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +, O +but O +not O +with O +the O +Y113F B-mutant +mutant B-protein_state +, O +in O +a O +haem B-chemical +- O +dependent O +manner O +( O +Fig O +. O +6a O +, O +b O +). O + +Moreover O +, O +the O +interaction O +of O +PGRMC1 B-protein +with O +CYP1A2 B-protein +was O +blocked O +by O +CORM3 B-chemical +under O +reducing O +conditions O +( O +Fig O +. O +6c O +), O +indicating O +that O +PGRMC1 B-protein +dimerization B-oligomeric_state +is O +necessary O +for O +its O +interaction O +with O +cytochromes B-protein_type +P450 I-protein_type +. O + +Doxorubicin B-chemical +is O +an O +anti O +- O +cancer O +reagent O +that O +is O +metabolized O +into O +inactive O +doxorubicinol B-chemical +by O +CYP2D6 B-protein +and O +CYP3A4 B-protein +( O +Fig O +. O +6d O +). O + +PGRMC1 B-mutant +- I-mutant +KD I-mutant +significantly O +suppressed O +the O +conversion O +of O +doxorubicin B-chemical +to O +doxorubicinol B-chemical +( O +Fig O +. O +6d O +) O +and O +increased O +sensitivity O +to O +doxorubicin B-chemical +( O +Fig O +. O +6e O +). O + +Enhanced O +doxorubicin B-chemical +sensitivity O +was O +modestly O +but O +significantly O +induced O +by O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +. O + +This O +effect O +was O +reversed O +by O +co B-experimental_method +- I-experimental_method +expression I-experimental_method +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +but O +not O +of O +the O +Y113F B-mutant +mutant B-protein_state +, O +suggesting O +that O +PGRMC1 B-protein +enhances O +doxorubicin B-chemical +resistance O +of O +cancer O +cells O +by O +facilitating O +its O +degradation O +via O +cytochromes B-protein_type +P450 I-protein_type +. O + +To O +gain O +further O +insight O +into O +the O +interaction O +between O +PGRMC1 B-protein +and O +cytochromes B-protein_type +P450 I-protein_type +, O +surface B-experimental_method +plasmon I-experimental_method +resonance I-experimental_method +analyses I-experimental_method +were O +conducted O +using O +recombinant O +CYP51 B-protein +and O +PGRMC1 B-protein +. O + +This O +was O +based O +on O +a O +previous O +study O +showing O +that O +PGRMC1 B-protein +binds O +to O +CYP51 B-protein +and O +enhances O +cholesterol O +biosynthesis O +by O +CYP51 B-protein +( O +refs O +). O + +CYP51 B-protein +interacted O +with O +PGRMC1 B-protein +in O +a O +concentration O +- O +dependent O +manner O +in O +the O +presence B-protein_state +of I-protein_state +haem B-chemical +, O +but O +not O +in O +its O +absence B-protein_state +( O +Supplementary O +Fig O +. O +19 O +), O +suggesting O +the O +requirement O +for O +the O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +. O + +The O +Kd B-evidence +value O +of O +PGRMC1 B-protein +binding O +to O +CYP51 B-protein +was O +in O +a O +micromolar O +range O +and O +comparable O +with O +those O +of O +other O +haem B-chemical +proteins O +, O +such O +as O +cytochrome B-protein +P450 I-protein +reductase I-protein +and O +neuroglobin B-protein +/ O +Gαi1 B-protein +( O +ref O +.), O +suggesting O +that O +haem B-chemical +- O +dependent O +PGRMC1 B-protein +interaction O +with O +CYP51 B-protein +is O +biologically O +relevant O +. O + +In O +this O +study O +, O +we O +showed O +that O +PGRMC1 B-protein +dimerizes B-oligomeric_state +by O +stacking B-bond_interaction +interactions I-bond_interaction +of O +haem B-chemical +molecules O +from O +each O +monomer B-oligomeric_state +. O + +Recently O +, O +Lucas O +et O +al O +. O +reported O +that O +translationally B-protein_type +- I-protein_type +controlled I-protein_type +tumour I-protein_type +protein I-protein_type +was O +dimerized B-protein_state +by O +binding O +with O +haem B-chemical +, O +but O +its O +structural O +basis O +remains O +unclear O +. O + +This O +is O +the O +report O +showing O +crystallographic O +evidence O +that O +indicates O +roles O +of O +the O +direct O +haem B-bond_interaction +– I-bond_interaction +haem I-bond_interaction +stacking I-bond_interaction +in O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +in O +eukaryotes B-taxonomy_domain +, O +although O +a O +few O +examples O +are O +known O +in O +bacteria B-taxonomy_domain +. O + +Sequence B-experimental_method +alignments I-experimental_method +show O +that O +haem B-site +- I-site +binding I-site +residues I-site +( O +Tyr113 B-residue_name_number +, O +Tyr107 B-residue_name_number +, O +Lys163 B-residue_name_number +and O +Tyr164 B-residue_name_number +) O +in O +PGRMC1 B-protein +are O +conserved B-protein_state +among O +MAPR B-protein_type +proteins O +( O +Supplementary O +Fig O +. O +5 O +). O + +In O +the O +current O +study O +, O +the O +Y113 B-residue_name_number +residue O +plays O +a O +crucial O +role O +for O +the O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +and O +resultant O +regulation O +of O +cancer O +proliferation O +and O +chemoresistance O +( O +Figs O +5c O +and O +6e O +). O + +Since O +the O +Y113 B-residue_name_number +residue O +is O +involved O +in O +the O +putative O +consensus B-structure_element +motif I-structure_element +of O +phosphorylation B-ptm +by O +tyrosine B-protein_type +kinases I-protein_type +such O +as O +Abl B-protein_type +and O +Lck B-protein_type +, O +we O +investigated O +whether O +phosphorylated B-protein_state +Y113 B-residue_name_number +is O +present O +in O +HCT116 O +cells O +by O +ESI B-experimental_method +- I-experimental_method +MS I-experimental_method +analysis O +. O + +Recently O +, O +Peluso O +et O +al O +. O +reported O +that O +PGRMC1 B-protein +binds O +to O +PGRMC2 B-protein +, O +suggesting O +that O +MAPR B-protein_type +family O +members O +may O +also O +undergo O +haem B-chemical +- O +mediated O +heterodimerization O +. O + +We O +showed O +that O +the O +haem B-chemical +- O +mediated O +dimer B-oligomeric_state +of O +PGRMC1 B-protein +enables O +interaction O +with O +different O +subclasses O +of O +cytochromes B-protein_type +P450 I-protein_type +( O +CYP B-protein_type +) O +( O +Fig O +. O +6 O +). O + +While O +the O +effects O +of O +PGRMC1 B-protein +on O +cholesterol O +synthesis O +mediated O +by O +CYP51 B-protein +have O +been O +well O +documented O +in O +yeast B-taxonomy_domain +and O +human B-species +cells O +, O +it O +has O +not O +been O +clear O +whether O +drug O +- O +metabolizing O +CYP B-protein_type +activities O +are O +regulated O +by O +PGRMC1 B-protein +. O + +Szczesna O +- O +Skorupa O +and O +Kemper O +reported O +that O +PGRMC1 B-protein +exhibited O +an O +inhibitory O +effect O +on O +CYP3A4 B-protein +drug O +metabolizing O +activity O +by O +competitively O +binding O +with O +cytochrome B-protein +P450 I-protein +reductase I-protein +( O +CPR B-protein +) O +in O +HEK293 O +or O +HepG2 O +cells O +. O + +On O +the O +other O +hand O +, O +Oda O +et O +al O +. O +reported O +that O +PGRMC1 B-protein +had O +no O +effect O +to O +CYP2E1 B-protein +and O +CYP3A4 B-protein +activities O +in O +HepG2 O +cell O +. O + +Several O +other O +groups O +showed O +that O +PGRMC1 B-protein +enhanced O +chemoresistance O +in O +several O +cancer O +cells O +such O +as O +uterine O +sarcoma O +, O +breast O +cancer O +, O +endometrial O +tumour O +and O +ovarian O +cancer O +; O +however O +, O +no O +evidence O +of O +PGRMC1 B-protein +- O +dependent O +regulation O +of O +CYP B-protein_type +activity O +was O +provided O +. O + +Our O +results O +showed O +that O +PGRMC1 B-protein +contributes O +to O +enhancement O +of O +the O +doxorubicin B-chemical +metabolism O +, O +which O +is O +mediated O +by O +CYP2D6 B-protein +or O +CYP3A4 B-protein +in O +human B-species +colon O +cancer O +HCT116 O +cells O +( O +Fig O +. O +6d O +). O + +While O +the O +effects O +of O +structural O +diversity O +of O +CYP B-protein_type +family O +proteins O +and O +interactions O +with O +different O +xenobiotic O +substrates O +should O +further O +be O +examined O +, O +the O +current O +results O +suggest O +that O +the O +interaction O +of O +drug O +- O +metabolizing O +CYPs B-protein_type +with O +the O +haem B-chemical +- O +mediated O +dimer B-oligomeric_state +of O +PGRMC1 B-protein +plays O +a O +crucial O +role O +in O +regulating O +their O +activities O +. O + +We O +showed O +that O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +enhances O +proliferation O +and O +chemoresistance O +of O +cancer O +cells O +through O +binding O +to O +and O +regulating O +EGFR B-protein_type +and O +cytochromes B-protein_type +P450 I-protein_type +( O +illustrated O +in O +Fig O +. O +7 O +). O + +Since O +the O +haem B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +PGRMC1 B-protein +is O +lower O +than O +those O +of O +constitutive B-protein_state +haem B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +such O +as O +myoglobin B-protein +, O +PGMRC1 B-protein +is O +probably O +interconverted O +between O +apo B-protein_state +- O +monomer B-oligomeric_state +and O +haem B-protein_state +- I-protein_state +bound I-protein_state +dimer B-oligomeric_state +forms O +in O +response O +to O +changes O +in O +the O +intracellular O +haem B-chemical +concentration O +. O + +Considering O +microenvironments O +in O +and O +around O +malignant O +tumours O +, O +the O +haem B-chemical +concentration O +in O +cancer O +cells O +is O +likely O +to O +be O +elevated O +through O +multiple O +mechanisms O +, O +such O +as O +( O +i O +) O +an O +increased O +intake O +of O +haem B-chemical +, O +( O +ii O +) O +mutation O +of O +enzymes O +in O +TCA O +cycle O +( O +for O +example O +, O +fumarate B-protein_type +hydratase I-protein_type +) O +that O +increases O +the O +level O +of O +succinyl B-chemical +CoA I-chemical +, O +a O +substrate O +for O +haem B-chemical +biosynthesis O +and O +( O +iii O +) O +metastasis O +to O +haem B-chemical +- O +rich O +organs O +such O +as O +liver O +, O +brain O +and O +bone O +marrow O +. O + +Moreover O +, O +exposure O +of O +cancer O +cells O +to O +stimuli O +such O +as O +hypoxia O +, O +radiation O +and O +chemotherapy O +causes O +cell O +damages O +and O +leads O +to O +protein O +degradation O +, O +resulting O +in O +increased O +levels O +of O +TCA O +cycle O +intermediates O +and O +in O +an O +enhanced O +haem B-chemical +biosynthesis O +. O + +On O +the O +other O +hand O +, O +excessive O +haem B-chemical +induces O +HO B-protein +- I-protein +1 I-protein +, O +the O +enzyme O +that O +oxidatively O +degrades O +haem B-chemical +and O +generates O +CO B-chemical +. O + +Thus O +, O +HO B-protein +- I-protein +1 I-protein +induction O +in O +cancer O +cells O +may O +inhibit O +the O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +through O +the O +production O +of O +CO B-chemical +and O +thereby O +suppress O +tumour O +progression O +. O + +This O +idea O +is O +consistent O +with O +the O +observation O +that O +HO B-protein +- I-protein +1 I-protein +induction O +or O +CO B-chemical +inhibits O +tumour O +growth O +. O + +Besides O +the O +regulatory O +roles O +of O +PGRMC1 B-protein +/ O +Sigma B-protein +- I-protein +2 I-protein +receptor O +in O +proliferation O +and O +chemoresistance O +in O +cancer O +cells O +( O +ref O +.), O +recent O +reports O +show O +that O +PGRMC1 B-protein +is O +able O +to O +bind O +to O +amyloid B-protein +beta I-protein +oligomer B-oligomeric_state +to O +enhance O +its O +neurotoxicity O +. O + +Furthermore O +, O +Sigma B-protein +- I-protein +2 I-protein +ligand O +- O +binding O +is O +decreased O +in O +transgenic O +amyloid O +beta O +deposition O +model O +APP O +/ O +PS1 O +female O +mice O +. O + +These O +results O +suggest O +a O +possible O +involvement O +of O +PGRMC1 B-protein +in O +Alzheimer O +' O +s O +disease O +. O + +The O +roles O +of O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +in O +the O +functional O +regulation O +of O +its O +target O +proteins O +deserve O +further O +studies O +to O +find O +evidence O +that O +therapeutic O +interventions O +to O +interfere O +with O +the O +function O +of O +the O +dimer B-oligomeric_state +may O +control O +varied O +disease O +conditions O +. O + +Alzheimer O +' O +s O +therapeutics O +targeting O +amyloid O +beta O +1 O +- O +42 O +oligomers O +II O +: O +Sigma O +- O +2 O +/ O +PGRMC1 O +receptors O +mediate O +Abeta O +42 O +oligomer B-oligomeric_state +binding O +and O +synaptotoxicity O + +X B-evidence +- I-evidence +ray I-evidence +crystal I-evidence +structure I-evidence +of O +PGRMC1 B-protein +. O + +( O +a O +) O +Structure O +of O +the O +PGRMC1 B-protein +dimer B-oligomeric_state +formed O +through O +stacked O +haems B-chemical +. O + +Two O +PGRMC1 B-protein +subunits B-structure_element +( O +blue O +and O +green O +ribbons O +) O +dimerize B-oligomeric_state +via O +stacking B-bond_interaction +of O +the O +haem B-chemical +molecules O +. O + +( O +b O +) O +Haem B-chemical +coordination B-bond_interaction +of O +PGRMC1 B-protein +with O +Tyr113 B-residue_name_number +. O + +Comparison O +of O +PGRMC1 B-protein +( O +blue O +) O +and O +cytochrome B-protein_type +b5 I-protein_type +( O +yellow O +, O +ID O +: O +3NER O +). O +( O +c O +) O +PGRMC1 B-protein +has O +a O +longer O +helix B-structure_element +( O +a O +. O +a O +. O +147 B-residue_range +– I-residue_range +163 I-residue_range +), O +which O +is O +shifted O +away O +from O +the O +haem B-chemical +( O +arrow O +). O + +PGRCM1 B-protein +is O +dimerized B-protein_state +by O +binding O +with O +haem B-chemical +. O + +( O +a O +) O +Mass B-experimental_method +spectrometric I-experimental_method +analyses O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +wt B-protein_state +) O +PGRMC1 B-protein +or O +the O +C129S B-mutant +mutant B-protein_state +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +haem B-chemical +under O +non O +- O +denaturing O +condition O +. O + +Both O +proteins O +had O +identical O +lengths O +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +). O + +( O +b O +) O +SV B-experimental_method +- I-experimental_method +AUC I-experimental_method +analyses O +of O +the O +wt B-protein_state +- O +PGRMC1 B-protein +and O +the O +C129S B-mutant +mutant B-protein_state +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +) O +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +haem B-chemical +. O + +SV B-experimental_method +- I-experimental_method +AUC I-experimental_method +experiments O +were O +performed O +with O +1 O +. O +5 O +mg O +ml O +− O +1 O +of O +PGRMC1 B-protein +proteins O +. O + +The O +major O +peak O +with O +sedimentation B-evidence +coefficient I-evidence +S20 B-evidence +, I-evidence +w I-evidence +of O +1 O +. O +9 O +∼ O +2 O +. O +0 O +S O +( O +monomer B-oligomeric_state +) O +or O +3 O +. O +1 O +S O +( O +dimer B-oligomeric_state +) O +was O +detected O +. O + +( O +c O +) O +Difference B-evidence +absorption I-evidence +spectra I-evidence +of O +PGRMC1 B-protein +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +) O +titrated B-experimental_method +with I-experimental_method +haem B-chemical +( O +left O +panel O +). O + +The O +titration B-evidence +curve I-evidence +of O +haem B-chemical +to O +PGRMC1 B-protein +( O +right O +panel O +). O + +The O +absorbance B-evidence +difference I-evidence +at O +400 O +nm O +is O +plotted O +against O +the O +haem B-chemical +concentration O +. O + +Carbon B-chemical +monoxide I-chemical +inhibits O +haem B-chemical +- O +dependent O +PGRMC1 B-protein +dimerization B-oligomeric_state +. O + +( O +a O +) O +UV B-evidence +- I-evidence +visible I-evidence +absorption I-evidence +spectra I-evidence +of O +PGRMC1 B-protein +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +). O + +Measurements O +were O +performed O +in O +the O +presence B-protein_state +of I-protein_state +the O +oxidized B-protein_state +form O +of O +haem B-chemical +( O +ferric B-protein_state +), O +the O +reduced B-protein_state +form O +of O +haem B-chemical +( O +ferrous B-protein_state +) O +and O +the O +reduced B-protein_state +form O +of O +haem B-chemical +plus O +CO B-chemical +gas O +( O +ferrous B-protein_state ++ O +CO B-chemical +). O + +( O +b O +) O +Close O +- O +up O +view O +of O +haem B-bond_interaction +stacking I-bond_interaction +. O + +( O +c O +) O +Gel B-experimental_method +- I-experimental_method +filtration I-experimental_method +chromatography I-experimental_method +analyses O +of O +PGRMC1 B-protein +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +) O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +wt B-protein_state +) O +and O +the O +Y113F B-mutant +or O +C129S B-mutant +mutant B-protein_state +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +haem B-chemical +, O +dithionite B-chemical +and O +/ O +or O +CO B-chemical +. O +( O +d O +) O +Transition O +model O +for O +structural O +regulation O +of O +PGRMC1 B-protein +in O +response O +to O +haem B-chemical +and O +CO B-chemical +. O + +Haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +is O +necessary O +for O +tumour O +proliferation O +mediated O +by O +EGFR B-protein_type +signalling O +. O + +( O +a O +) O +FLAG O +- O +PGRMC1 B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +( O +wt B-protein_state +) O +and O +Y113F B-mutant +mutant B-protein_state +proteins O +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +), O +in O +either O +apo B-protein_state +- O +or O +haem B-protein_state +- I-protein_state +bound I-protein_state +form O +, O +were O +incubated B-experimental_method +with O +purified O +EGFR B-protein_type +and O +co B-experimental_method +- I-experimental_method +immunoprecipitated I-experimental_method +with O +anti O +- O +FLAG O +antibody O +- O +conjugated O +beads O +. O + +Input O +and O +bound O +proteins O +were O +detected O +by O +Western B-experimental_method +blotting I-experimental_method +. O + +( O +b O +) O +In B-experimental_method +vitro I-experimental_method +binding I-experimental_method +assay I-experimental_method +was O +performed O +as O +in O +( O +a O +) O +using O +haem B-protein_state +- I-protein_state +bound I-protein_state +FLAG O +- O +PGRMC1 B-protein +wt B-protein_state +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +) O +and O +purified O +EGFR B-protein_type +with O +or O +without O +treatment O +of O +RuCl3 B-chemical +and O +CORM3 B-chemical +. O + +( O +c O +) O +FLAG O +- O +PGRMC1 B-protein +wt B-protein_state +or O +Y113F B-mutant +( O +full B-protein_state +length I-protein_state +) O +was O +over B-experimental_method +- I-experimental_method +expressed I-experimental_method +in O +HCT116 O +cells O +and O +immunoprecipitated B-experimental_method +with O +anti O +- O +FLAG O +antibody O +- O +conjugated O +beads O +. O + +Co B-experimental_method +- I-experimental_method +immunoprecipitated I-experimental_method +proteins O +( O +FLAG O +- O +PGRMC1 B-protein +, O +endogenous B-protein_state +PGRMC1 B-protein +and O +EGFR B-protein_type +) O +were O +detected O +with O +Western B-experimental_method +blotting I-experimental_method +by O +using O +anti O +- O +PGRMC1 B-protein +or O +anti O +- O +EGFR B-protein_type +antibody O +. O + +( O +d O +) O +HCT116 O +cells O +were O +treated O +with O +or O +without O +250 O +μmol O +l O +− O +1 O +of O +succinylacetone B-chemical +( O +SA B-chemical +) O +for O +48 O +h O +. O +The O +intracellular O +haem B-chemical +was O +extracted O +and O +quantified O +by O +reverse B-experimental_method +- I-experimental_method +phase I-experimental_method +HPLC I-experimental_method +. O + +of O +four O +separate O +experiments O +. O +** O +P O +< O +0 O +. O +01 O +using O +unpaired O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +- I-experimental_method +test I-experimental_method +. O +( O +e O +) O +Co B-experimental_method +- I-experimental_method +immunoprecipitation I-experimental_method +assay I-experimental_method +was O +performed O +as O +in O +( O +c O +) O +with O +or O +without O +SA B-chemical +treatment O +in O +HCT116 O +cells O +. O + +( O +f O +) O +HCT116 O +cells O +expressing O +control O +shRNA B-chemical +or O +those O +knocking B-experimental_method +down I-experimental_method +PGRMC1 B-protein +( O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +) O +were O +treated O +with O +EGF B-protein_type +or O +left O +untreated O +, O +and O +components O +of O +the O +EGFR B-protein_type +signaling O +pathway O +were O +detected O +by O +Western B-experimental_method +blotting I-experimental_method +. O + +( O +g O +, O +h O +) O +HCT116 O +cells O +were O +treated O +with O +or O +without O +EGF B-protein_type +, O +SA B-chemical +, O +RuCl3 B-chemical +and O +CORM3 B-chemical +as O +indicated O +, O +and O +components O +of O +the O +EGFR B-protein_type +signaling O +pathway O +were O +detected O +by O +Western B-experimental_method +blotting I-experimental_method +. O + +Haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +accelerates O +tumour O +growth O +through O +the O +EGFR B-protein_type +signaling O +pathway O +. O + +( O +a O +) O +Nucleotide O +sequences O +of O +PGRMC1 B-protein +targeted O +by O +shRNA B-chemical +and O +of O +the O +shRNA B-protein_state +- I-protein_state +resistant I-protein_state +full B-protein_state +length I-protein_state +PGRMC1 B-protein +expression O +vector O +. O + +Stable O +PGRMC1 B-mutant +- I-mutant +knockdown I-mutant +( O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +) O +HCT116 O +cells O +were O +transiently B-experimental_method +transfected I-experimental_method +with O +the O +shRNA B-protein_state +- I-protein_state +resistant I-protein_state +expression B-experimental_method +vector I-experimental_method +of O +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +( O +wt B-protein_state +) O +or O +the O +Y113F B-mutant +mutant B-protein_state +( O +Y113F B-mutant +). O + +( O +b O +) O +Erlotinib B-chemical +was O +added O +to O +HCT116 O +( O +control O +) O +cells O +, O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +or O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +expressing O +shRNA B-protein_state +- I-protein_state +resistant I-protein_state +PGRMC1 B-protein +wt B-protein_state +or O +Y113F B-mutant +, O +and O +cell O +viability O +was O +examined O +by O +MTT B-experimental_method +assay I-experimental_method +. O + +of O +four O +separate O +experiments O +. O +* B-evidence +P I-evidence +< O +0 O +. O +01 O +using O +ANOVA B-experimental_method +with O +Fischer B-experimental_method +' I-experimental_method +s I-experimental_method +LSD I-experimental_method +test I-experimental_method +. O + +( O +c O +) O +Spheroid O +formation O +in O +control O +and O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +HCT116 O +cells O +. O + +The O +graph O +represents O +mean O +± O +s O +. O +e O +. O +of O +each O +spheroid O +size O +. O +* B-evidence +P I-evidence +< O +0 O +. O +01 O +using O +ANOVA B-experimental_method +with O +Fischer B-experimental_method +' I-experimental_method +s I-experimental_method +LSD I-experimental_method +test I-experimental_method +. O + +Scale O +bar O +: O +0 O +. O +1 O +mm O +. O +( O +d O +) O +Tumour O +- O +bearing O +livers O +of O +NOG O +mice O +at O +10 O +days O +after O +intrasplenic B-experimental_method +injection I-experimental_method +of O +HCT116 O +( O +control O +) O +or O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +. O + +of O +10 O +separate O +experiments O +. O +* B-evidence +P I-evidence +< O +0 O +. O +05 O +using O +unpaired O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +- I-experimental_method +test I-experimental_method +. O + +Haem B-chemical +- O +dependent O +PGRMC1 B-protein +dimerization B-oligomeric_state +enhances O +tumour O +chemoresistance O +through O +interaction O +with O +cytochromes B-protein_type +P450 I-protein_type +. O + +( O +a O +, O +b O +) O +FLAG O +- O +PGRMC1 B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +( O +wt B-protein_state +) O +and O +Y113F B-mutant +mutant B-protein_state +proteins O +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +), O +in O +either O +apo B-protein_state +or O +haem B-protein_state +- I-protein_state +bound I-protein_state +form O +, O +were O +incubated B-experimental_method +with O +CYP1A2 B-protein +( O +a O +) O +or O +CYP3A4 B-protein +( O +b O +) O +and O +immunoprecipitated B-experimental_method +with O +anti O +- O +FLAG O +antibody O +- O +conjugated O +beads O +. O + +( O +c O +) O +Binding B-experimental_method +assay I-experimental_method +was O +performed O +as O +in O +( O +a O +) O +using O +haem B-protein_state +- I-protein_state +bound I-protein_state +FLAG O +- O +PGRMC1 B-protein +wt B-protein_state +and O +CYP1A2 B-protein +with O +or O +without O +RuCl3 B-chemical +and O +CORM3 B-chemical +. O + +( O +d O +) O +Schematic O +illustration O +of O +doxorubicin B-chemical +metabolism O +is O +shown O +on O +the O +left O +. O + +Doxorubicin B-chemical +was O +incubated B-experimental_method +with O +HCT116 O +cells O +expressing O +control O +shRNA B-chemical +or O +shPGRMC1 B-chemical +( O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +), O +and O +the O +doxorubicinol B-chemical +/ O +doxorubicin B-chemical +ratios O +in O +cell O +pellets O +were O +determined O +using O +LC B-experimental_method +- I-experimental_method +MS I-experimental_method +. O + +of O +four O +separate O +experiments O +. O +** O +P B-evidence +< O +0 O +. O +01 O +versus O +control O +using O +unpaired O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +- I-experimental_method +test I-experimental_method +. O +( O +e O +) O +Indicated O +amounts O +of O +doxorubicin B-chemical +were O +added O +to O +HCT116 O +( O +control O +) O +cells O +, O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +, O +or O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +expressing O +shRNA B-protein_state +- I-protein_state +resistant I-protein_state +full B-protein_state +- I-protein_state +length I-protein_state +PGRMC1 B-protein +wt B-protein_state +or O +Y113F B-mutant +, O +and O +cell O +viability O +was O +examined O +by O +MTT B-experimental_method +assay I-experimental_method +. O + +Schematic O +diagram O +for O +the O +regulation O +of O +PGRMC1 B-protein +functions O +. O + +Apo B-protein_state +- O +PGRMC1 B-protein +exists O +as O +an O +inactive B-protein_state +monomer B-oligomeric_state +. O + +On O +binding B-protein_state +to I-protein_state +haem B-chemical +, O +PGRMC1 B-protein +forms O +a O +dimer B-oligomeric_state +through O +stacking B-bond_interaction +interactions I-bond_interaction +between O +the O +haem B-chemical +moieties O +, O +which O +enables O +PGRMC1 B-protein +to O +interact O +with O +EGFR B-protein_type +and O +cytochromes B-protein_type +P450 I-protein_type +, O +leading O +to O +an O +enhanced O +proliferation O +and O +chemoresistance O +of O +cancer O +cells O +. O + +CO B-chemical +interferes O +with O +the O +stacking B-bond_interaction +interactions I-bond_interaction +of O +the O +haems B-chemical +and O +thereby O +inhibits O +PGRMC1 B-protein +functions O +. O + +PGRMC1 O +proteins O +exhibit O +haem O +- O +dependent O +dimerization B-oligomeric_state +in O +solution O +. O + +Apo O +form O +Haem O +- O +bound O +form O +Mass O +( O +Da O +) O +Mass O +( O +Da O +) O +aPGRMC1 O +wt O +( O +a O +. O +a O +. O +44 O +– O +195 O +) O +ESI O +- O +MS O +— O +17 O +, O +844 O +. O +14 O +— O +36 O +, O +920 O +. O +19 O +Theoretical O +17 O +, O +843 O +. O +65 O +36 O +, O +918 O +. O +06 O +Hydrodynamic O +radius O +10 O +− O +9 O +( O +m O +) O +MW O +( O +kDa O +) O +Hydrodynamic O +radius O +10 O +− O +9 O +( O +m O +) O +MW O +( O +kDa O +) O +DOSY O +2 O +. O +04 O +– O +2 O +. O +15 O +20 O +2 O +. O +94 O +– O +3 O +. O +02 O +42 O +S20 O +, O +w O +( O +S O +) O +MW O +( O +kDa O +) O +S20 O +, O +w O +( O +S O +) O +MW O +( O +kDa O +) O +SV O +- O +AUC O +1 O +. O +9 O +17 O +. O +6 O +3 O +. O +1 O +35 O +. O +5 O +bPGRMC1 O +C129S B-mutant +( O +a O +. O +a O +. O +44 O +– O +195 O +) O +ESI O +- O +MS O +— O +17 O +, O +827 O +. O +91 O +— O +36 O +, O +887 O +. O +07 O +Theoretical O +17 O +, O +827 O +. O +59 O +36 O +, O +885 O +. O +6 O +S20 O +, O +w O +( O +S O +) O +MW O +( O +kDa O +) O +S20 O +, O +w O +( O +S O +) O +MW O +( O +kDa O +) O +SV O +- O +AUC O +2 O +. O +0 O +18 O +. O +1 O +3 O +. O +1 O +35 O +. O +8 O + +Differences O +in O +molecular O +weights O +of O +the O +wild O +- O +type O +( O +wt O +; O +a O +) O +and O +the O +C129S B-mutant +mutant O +( O +b O +) O +PGRMC1 O +proteins O +in O +the O +absence O +( O +apo O +form O +) O +or O +the O +presence O +of O +haem O +( O +haem O +- O +bound O +form O +). O + +The O +protein O +sizes O +of O +the O +wt O +and O +C129S B-mutant +PGRMC1 O +cytosolic O +domains O +( O +a O +. O +a O +. O +44 O +– O +195 O +) O +in O +the O +presence O +or O +absence O +of O +haem O +were O +estimated O +by O +ESI O +- O +MS O +, O +DOSY O +and O +SV O +- O +AUC O +. O + +Hotspot O +autoimmune O +T B-protein_type +cell I-protein_type +receptor I-protein_type +binding O +underlies O +pathogen O +and O +insulin B-chemical +peptide O +cross O +- O +reactivity O + +However O +, O +the O +mechanisms O +that O +allow O +the O +clonal O +T B-complex_assembly +cell I-complex_assembly +antigen I-complex_assembly +receptor I-complex_assembly +( O +TCR B-complex_assembly +) O +to O +functionally O +engage O +multiple O +peptide B-complex_assembly +– I-complex_assembly +major I-complex_assembly +histocompatibility I-complex_assembly +complexes I-complex_assembly +( O +pMHC B-complex_assembly +) O +are O +unclear O +. O + +Here O +, O +we O +studied O +multiligand O +discrimination O +by O +a O +human B-species +, O +preproinsulin B-protein +reactive O +, O +MHC B-complex_assembly +class O +- O +I O +– O +restricted O +CD8 O ++ O +T O +cell O +clone O +( O +1E6 O +) O +that O +can O +recognize O +over O +1 O +million O +different O +peptides O +. O + +We O +generated O +high O +- O +resolution O +structures B-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +bound B-protein_state +to I-protein_state +7 O +altered B-chemical +peptide I-chemical +ligands I-chemical +, O +including O +a O +pathogen O +- O +derived O +peptide O +that O +was O +an O +order O +of O +magnitude O +more O +potent O +than O +the O +natural O +self O +- O +peptide O +. O + +Evaluation O +of O +these O +structures B-evidence +demonstrated O +that O +binding O +was O +stabilized O +through O +a O +conserved O +lock O +- O +and O +- O +key O +– O +like O +minimal O +binding O +footprint O +that O +enables O +1E6 B-complex_assembly +TCR I-complex_assembly +to O +tolerate O +vast O +numbers O +of O +substitutions O +outside O +of O +this O +so O +- O +called O +hotspot O +. O + +Highly O +potent O +antigens O +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +engaged O +with O +a O +strong O +antipathogen B-evidence +- I-evidence +like I-evidence +binding I-evidence +affinity I-evidence +; O +this O +engagement O +was O +governed O +though O +an O +energetic O +switch O +from O +an O +enthalpically O +to O +entropically O +driven O +interaction O +compared O +with O +the O +natural O +autoimmune O +ligand O +. O + +T O +cells O +perform O +an O +essential O +role O +in O +adaptive O +immunity O +by O +interrogating O +the O +host O +proteome O +for O +anomalies O +, O +classically O +by O +recognizing O +peptides O +bound O +in O +major B-complex_assembly +histocompatibility I-complex_assembly +( O +MHC B-complex_assembly +) O +molecules O +at O +the O +cell O +surface O +. O + +Recent O +data O +supports O +the O +notion O +that O +, O +to O +perform O +this O +role O +, O +the O +highly B-protein_state +variable I-protein_state +αβ B-complex_assembly +T I-complex_assembly +cell I-complex_assembly +antigen I-complex_assembly +receptor I-complex_assembly +( O +TCR B-complex_assembly +) O +must O +be O +able O +to O +recognize O +thousands O +, O +if O +not O +millions O +, O +of O +different O +peptide O +ligands O +. O + +This O +ability O +is O +required O +to O +enable O +the O +estimated O +25 O +million O +distinct O +TCRs B-complex_assembly +expressed O +in O +humans B-species +to O +provide O +effective O +immune O +coverage O +against O +all O +possible O +foreign O +peptide O +antigens O +. O + +Several O +mechanisms O +, O +by O +which O +TCRs B-complex_assembly +could O +bind O +to O +a O +large O +number O +of O +different O +peptide B-complex_assembly +- I-complex_assembly +MHC I-complex_assembly +( O +pMHC B-complex_assembly +), O +have O +been O +proposed O +. O + +Structures B-evidence +of O +unligated B-protein_state +and O +ligated B-protein_state +TCRs B-complex_assembly +have O +shown O +that O +the O +TCR B-complex_assembly +complementarity B-structure_element +determining I-structure_element +region I-structure_element +( O +CDR B-structure_element +) O +loops B-structure_element +can O +be O +flexible O +, O +perhaps O +enabling O +peptide O +binding O +using O +different O +loop B-structure_element +conformations O +. O + +Both O +MHC B-complex_assembly +and O +peptide B-chemical +have O +also O +been O +shown O +to O +undergo O +structural O +changes O +upon O +TCR B-complex_assembly +binding O +, O +mediating O +an O +induced O +fit O +between O +the O +TCR B-complex_assembly +and O +pMHC B-complex_assembly +. O + +Other O +studies O +, O +mainly O +in O +the O +murine B-taxonomy_domain +system O +, O +have O +demonstrated O +that O +the O +same O +TCR B-complex_assembly +can O +interact O +with O +different O +pMHCs B-complex_assembly +using O +a O +common O +or O +divergent O +modality O +. O + +Recent O +studies O +in O +model O +murine B-taxonomy_domain +systems O +demonstrate O +that O +TCR B-complex_assembly +cross O +- O +reactivity O +can O +be O +governed O +by O +recognition O +of O +a O +conserved O +region O +in O +the O +peptide O +that O +allows O +tolerance O +of O +peptide O +sequence O +variation O +outside O +of O +this O +hotspot O +. O + +We O +recently O +reported O +that O +the O +1E6 O +human B-species +CD8 O ++ O +T O +cell O +clone O +— O +which O +mediates O +the O +destruction O +of O +β O +cells O +through O +the O +recognition O +of O +a O +major O +, O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +– O +restricted O +, O +preproinsulin B-protein +signal B-structure_element +peptide I-structure_element +( O +ALWGPDPAAA15 B-chemical +– I-chemical +24 I-chemical +) O +— O +can O +recognize O +upwards O +of O +1 O +million O +different O +peptides O +. O + +CD8 O ++ O +T O +cells O +that O +recognize O +HLA B-complex_assembly +- I-complex_assembly +A I-complex_assembly +* I-complex_assembly +0201 I-complex_assembly +– I-complex_assembly +ALWGPDPAAA I-complex_assembly +have O +been O +shown O +to O +populate O +insulitic O +lesions O +in O +patients O +with O +type O +1 O +diabetes O +( O +T1D O +). O + +We O +demonstrated O +that O +the O +TCR B-complex_assembly +from O +the O +1E6 O +T O +cell O +clone O +bound B-protein_state +to I-protein_state +HLA B-complex_assembly +- I-complex_assembly +A I-complex_assembly +* I-complex_assembly +0201 I-complex_assembly +– I-complex_assembly +ALWGPDPAAA I-complex_assembly +using O +a O +limited O +footprint O +and O +very O +weak O +binding B-evidence +affinity I-evidence +. O + +This O +first O +experimental O +evidence O +of O +a O +high O +level O +of O +CD8 O ++ O +T O +cell O +cross O +- O +reactivity O +in O +a O +human B-species +autoimmune O +disease O +system O +hinted O +toward O +molecular O +mimicry O +by O +a O +more O +potent O +pathogenic O +peptide O +as O +a O +potential O +mechanism O +leading O +to O +β O +cell O +destruction O +. O + +Here O +, O +we O +solved B-experimental_method +the O +structure B-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +with O +7 O +altered B-chemical +peptide I-chemical +ligands I-chemical +( O +APLs B-chemical +) O +determined O +by O +our O +previously O +published O +combinatorial B-experimental_method +peptide I-experimental_method +library I-experimental_method +( I-experimental_method +CPL I-experimental_method +) I-experimental_method +screening I-experimental_method +, O +2 O +of O +which O +mapped O +within O +human B-species +pathogens O +. O + +These O +APLs B-chemical +differed O +from O +the O +natural O +preproinsulin B-protein +peptide O +by O +up O +to O +7 O +of O +10 O +residues O +. O + +We O +also O +solved B-experimental_method +the O +structure B-evidence +of O +each O +unligated B-protein_state +APL B-chemical +to O +investigate O +whether O +structural O +changes O +occurred O +before O +or O +after O +binding O +— O +which O +, O +combined O +with O +an O +in O +- O +depth O +cellular B-experimental_method +and I-experimental_method +biophysical I-experimental_method +analysis I-experimental_method +of O +the O +1E6 O +interaction O +with O +each O +APL B-chemical +, O +demonstrated O +the O +molecular O +mechanism O +mediating O +the O +high O +level O +of O +cross O +- O +reactivity O +exhibited O +by O +this O +preproinsulin B-protein +- O +reactive O +human B-species +CD8 O ++ O +T O +cell O +clone O +. O + +The O +1E6 O +T O +cell O +clone O +recognizes O +APLs B-chemical +across O +a O +large O +dynamic O +range O +. O + +We O +have O +previously O +demonstrated O +that O +the O +1E6 O +T O +cell O +clone O +can O +recognize O +over O +1 O +million O +different O +peptides O +with O +a O +potency O +comparable O +with O +, O +or O +better O +than O +, O +the O +cognate O +preproinsulin B-protein +peptide O +ALWGPDPAAA B-chemical +. O + +From O +this O +large O +functional O +scan O +, O +we O +selected O +7 O +different O +APLs B-chemical +that O +activated O +the O +1E6 O +T O +cell O +clone O +across O +a O +wide O +( O +4 O +- O +log O +) O +functional O +range O +( O +Table O +1 O +). O + +Two O +of O +these O +peptides O +, O +MVWGPDPLYV B-chemical +and O +RQFGPDWIVA B-chemical +( O +bold O +text O +signifies O +amino O +acids O +that O +are O +different O +from O +the O +index O +preproinsulin B-protein +– O +derived O +sequence O +), O +are O +contained O +within O +the O +proteomes O +of O +the O +human B-species +pathogens O +Bacteroides B-species +fragilis I-species +/ I-species +thetaiotaomicron I-species +and O +Clostridium B-species +asparagiforme I-species +, O +respectively O +. O + +Competitive B-experimental_method +functional I-experimental_method +testing I-experimental_method +revealed O +that O +the O +preproinsulin B-protein +- O +derived O +sequence O +ALWGPDPAAA B-chemical +was O +one O +of O +the O +least O +potent O +targets O +for O +1E6 O +, O +with O +only O +the O +MVWGPDPLYV B-chemical +and O +YLGGPDFPTI B-chemical +demonstrating O +a O +similar O +low O +- O +activity O +profile O +in O +MIP B-protein +- I-protein +1β I-protein +secretion O +and O +target O +killing O +assays O +( O +Figure O +1 O +, O +A O +and O +B O +). O + +The O +RQFGPDWIVA B-chemical +sequence O +( O +present O +in O +C B-species +. I-species +asparagiforme I-species +) O +activated O +the O +1E6 O +T O +cell O +with O +around O +1 O +log O +– O +greater O +potency O +compared O +with O +ALWGPDPAAA B-chemical +. O + +At O +the O +other O +end O +of O +the O +spectrum O +, O +the O +RQFGPDFPTI B-chemical +peptide O +stimulated O +MIP B-protein +- I-protein +1β I-protein +release O +and O +killing O +by O +1E6 O +at O +an O +exogenous O +peptide O +concentration O +2 O +– O +3 O +logs O +lower O +compared O +with O +ALWGPDPAAA B-chemical +. O + +The O +pattern O +of O +peptide O +potency O +was O +closely O +mirrored O +by O +pMHC B-complex_assembly +tetramer B-experimental_method +staining I-experimental_method +experiments O +( O +Figure O +1C O +and O +plots O +shown O +in O +Supplemental O +Figure O +1 O +; O +supplemental O +material O +available O +online O +with O +this O +article O +; O +doi O +: O +10 O +. O +1172 O +/ O +JCI85679DS1 O +). O + +Here O +, O +the O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +tetramer B-oligomeric_state +stained O +1E6 O +with O +the O +greatest O +MFI O +, O +gradually O +decreasing O +to O +the O +weakest O +tetramers B-oligomeric_state +: O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +and O +- O +YLGGPDFPTI B-chemical +. O + +To O +parallel O +the O +functional O +analysis O +, O +we O +also O +performed O +thermal B-experimental_method +melt I-experimental_method +( O +Tm B-evidence +) O +experiments O +using O +synchrotron B-experimental_method +radiation I-experimental_method +circular I-experimental_method +dichroism I-experimental_method +( O +SRCD B-experimental_method +) O +to O +investigate O +the O +stability O +of O +each O +APL B-chemical +( O +Figure O +1D O +). O + +The O +range O +of O +Tm B-evidence +was O +between O +49 O +. O +4 O +° O +C O +( O +RQFGPDWIVA B-chemical +) O +and O +60 O +. O +3 O +° O +C O +( O +YQFGPDFPIA B-chemical +), O +with O +an O +average O +approximately O +55 O +° O +C O +, O +similar O +to O +our O +previous O +findings O +. O + +This O +pattern O +of O +stability O +did O +not O +correlate O +with O +the O +T O +cell O +activation O +or O +tetramer B-experimental_method +staining I-experimental_method +experiments O +, O +indicating O +that O +peptide O +binding O +to O +the O +MHC B-complex_assembly +do O +not O +explain O +ligand O +potency O +. O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +can O +bind O +peptides O +with O +strong O +antipathogen O +- O +like O +affinities O +. O + +We O +, O +and O +others O +, O +have O +previously O +demonstrated O +that O +antipathogenic O +TCRs B-complex_assembly +tend O +to O +bind O +with O +stronger O +affinity B-evidence +compared O +with O +self O +- O +reactive O +TCRs B-complex_assembly +, O +likely O +a O +consequence O +of O +the O +deletion O +of O +T O +cells O +with O +high O +- O +affinity B-evidence +self O +- O +reactive O +TCR B-complex_assembly +during O +thymic O +selection O +. O + +In O +accordance O +with O +this O +trend O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +bound B-protein_state +the O +natural O +preproinsulin B-protein +peptide O +, O +ALWGPDPAAA B-chemical +, O +with O +the O +weakest O +affinity B-evidence +currently O +published O +for O +a O +human B-species +CD8 O ++ O +T O +cell O +– O +derived O +TCR B-complex_assembly +with O +a O +biologically O +relevant O +ligand O +( O +KD B-evidence +> O +200 O +μM O +; O +KD B-evidence +, O +equilibrium B-evidence +binding I-evidence +constant I-evidence +). O + +Surface B-experimental_method +plasmon I-experimental_method +resonance I-experimental_method +( O +SPR B-experimental_method +) O +analysis O +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +– O +pMHC B-complex_assembly +interaction O +for O +all O +7 O +APLs B-chemical +( O +Figure O +2 O +, O +A O +– O +H O +) O +demonstrated O +that O +stronger O +binding B-evidence +affinity I-evidence +( O +represented O +as O +ΔG B-evidence +°, I-evidence +kcal O +/ O +mol O +) O +correlated O +well O +with O +the O +EC50 B-evidence +values O +( O +peptide O +concentration O +required O +to O +reach O +half O +- O +maximal O +1E6 O +T O +cell O +killing O +) O +for O +each O +ligand O +, O +demonstrated O +by O +a O +Pearson B-experimental_method +’ I-experimental_method +s I-experimental_method +correlation I-experimental_method +analysis I-experimental_method +value O +of O +0 O +. O +8 O +( O +P O += O +0 O +. O +01 O +) O +( O +Figure O +2I O +). O + +It O +should O +be O +noted O +that O +this O +correlation O +, O +although O +consistent O +with O +the O +T O +cell O +killing O +experiments O +, O +uses O +only O +approximate O +affinities B-evidence +calculated O +for O +the O +2 O +weakest O +ligands O +. O + +First O +, O +the O +1E6 O +T O +cell O +could O +still O +functionally O +respond O +to O +peptide O +when O +the O +TCR B-evidence +binding I-evidence +affinity I-evidence +was O +extremely O +weak O +, O +e O +. O +g O +., O +the O +1E6 B-evidence +TCR I-evidence +binding I-evidence +affinity I-evidence +for O +the O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +peptide O +was O +KD B-evidence += O +~ O +600 O +μM O +. O +Second O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +bound B-protein_state +to I-protein_state +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +with O +KD B-evidence += O +0 O +. O +5 O +μM O +, O +equivalent O +to O +the O +binding B-evidence +affinity I-evidence +of O +the O +very O +strongest O +antipathogen O +TCRs B-complex_assembly +. O + +Third O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +bound B-protein_state +to I-protein_state +A2 B-chemical +- I-chemical +RQFGPDWIVA I-chemical +peptide O +, O +within O +the O +C B-species +. I-species +asparagiforme I-species +proteome O +, O +with O +approximately O +4 O +- O +fold O +stronger O +affinity B-evidence +than O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +, O +demonstrating O +the O +potential O +for O +a O +pathogen O +- O +derived O +antigen O +to O +initiate O +a O +response O +to O +the O +self O +- O +derived O +sequence O +. O + +Finally O +, O +these O +data O +demonstrate O +the O +largest O +range O +of O +binding B-evidence +affinities I-evidence +reported O +for O +a O +natural O +, O +endogenous B-protein_state +human B-species +TCR B-complex_assembly +of O +more O +than O +3 O +logs O +of O +magnitude O +( O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +vs O +. O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +). O + +To O +confirm O +the O +affinity B-evidence +spread O +detected O +by O +SPR B-experimental_method +, O +and O +to O +evaluate O +whether O +experiments O +performed O +using O +soluble O +molecules O +were O +biologically O +relevant O +to O +events O +at O +the O +T O +cell O +surface O +, O +we O +determined O +the O +effective O +2D B-evidence +affinity I-evidence +of O +each O +APL B-chemical +using O +an O +adhesion B-experimental_method +frequency I-experimental_method +assay I-experimental_method +in O +which O +a O +human B-species +rbc O +coated O +in O +pMHC B-complex_assembly +acted O +as O +an O +adhesion O +sensor O +. O + +In O +agreement O +with O +SPR B-experimental_method +experiments O +, O +the O +range O +of O +2D B-evidence +affinities I-evidence +we O +detected O +differed O +by O +around O +3 O +logs O +, O +with O +the O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +generating O +the O +weakest O +2D B-evidence +affinity I-evidence +( O +2 O +. O +6 O +× O +10 O +– O +5 O +AcKa B-evidence +μm4 O +) O +and O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +the O +strongest O +( O +4 O +. O +5 O +× O +10 O +– O +2 O +AcKa B-evidence +μm4 O +) O +( O +Figure O +2J O +). O + +As O +with O +the O +3D B-evidence +affinity I-evidence +measurements O +, O +the O +2D B-evidence +affinity I-evidence +measurements O +correlated O +well O +with O +the O +EC50 B-evidence +values O +for O +each O +ligand O +( O +Figure O +2K O +) O +demonstrating O +a O +strong O +correlation O +( O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence += O +0 O +. O +8 O +, O +P B-evidence += O +0 O +. O +01 O +) O +between O +T O +cell O +antigen O +sensitivity O +and O +TCR B-evidence +binding I-evidence +affinity I-evidence +. O + +Of O +note O +, O +these O +data O +demonstrate O +a O +close O +agreement O +between O +the O +3D B-evidence +affinity I-evidence +values O +generated O +using O +SPR B-experimental_method +and O +2D B-evidence +affinity I-evidence +values O +generated O +using O +adhesion O +frequency O +assays O +. O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +uses O +a O +consensus O +binding O +mode O +to O +engage O +multiple O +APLs B-chemical +. O + +Our O +previous O +structure B-evidence +of O +the O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +ALWGPDPAAA I-complex_assembly +complex O +demonstrated O +a O +limited O +binding B-site +footprint I-site +between O +the O +TCR B-complex_assembly +and O +pMHC B-complex_assembly +. O + +The O +low O +number O +of O +contacts O +between O +the O +2 O +molecules O +most O +likely O +contributed O +to O +the O +weak O +binding B-evidence +affinity I-evidence +of O +the O +interaction O +. O + +In O +order O +to O +examine O +the O +mechanism O +by O +which O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +engaged O +a O +wide O +range O +of O +peptides O +with O +divergent O +binding B-evidence +affinities I-evidence +, O +we O +solved B-experimental_method +the O +structure B-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +in B-protein_state +complex I-protein_state +with I-protein_state +all O +7 O +APLs B-chemical +used O +in O +Figure O +2 O +. O + +All O +structures B-evidence +were O +solved B-experimental_method +in O +space O +group O +P1 O +to O +2 O +– O +3 O +Å O +resolution O +with O +crystallographic O +Rwork B-evidence +/ I-evidence +Rfree I-evidence +ratios I-evidence +within O +accepted O +limits O +as O +shown O +in O +the O +theoretically O +expected O +distribution O +( O +ref O +. O +and O +Supplemental O +Table O +1 O +). O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +used O +a O +very O +similar O +overall O +binding O +modality O +to O +engage O +all O +of O +the O +APLs B-chemical +, O +with O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +ranging O +between O +0 O +. O +81 O +and O +1 O +. O +12 O +Å2 O +( O +compared O +with O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +ALWGPDPAAA I-complex_assembly +). O + +The O +relatively O +broad O +range O +of O +buried O +surface O +areas O +( O +1 O +, O +670 O +– O +1 O +, O +920 O +Å2 O +) O +did O +not O +correlate O +well O +with O +TCR B-evidence +binding I-evidence +affinity I-evidence +( O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence += O +0 O +. O +45 O +, O +P B-evidence += O +0 O +. O +2 O +). O + +The O +surface B-evidence +complementarity I-evidence +values I-evidence +( O +0 O +. O +52 O +– O +0 O +. O +7 O +) O +correlated O +slightly O +with O +affinity B-evidence +( O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence += O +0 O +. O +7 O +, O +P B-evidence += O +0 O +. O +05 O +) O +but O +could O +not O +explain O +all O +differences O +in O +binding O +( O +Figure O +3A O +and O +Table O +2 O +). O + +The O +TCR B-complex_assembly +CDR B-structure_element +loops I-structure_element +were O +in O +a O +very O +similar O +position O +in O +all O +complexes O +, O +apart O +from O +some O +slight O +deviations O +in O +the O +TCR B-complex_assembly +β B-structure_element +- I-structure_element +chain I-structure_element +( O +Figure O +3B O +); O +the O +peptides O +were O +all O +presented O +in O +a O +similar O +conformation O +( O +Figure O +3C O +); O +and O +there O +was O +minimal O +variation O +in O +crossing O +angles O +of O +the O +TCR B-complex_assembly +( O +42 O +. O +3 O +°– O +45 O +. O +6 O +°) O +( O +Figure O +3D O +). O + +Overall O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +used O +a O +canonical O +binding O +mode O +to O +engage O +each O +APL B-chemical +with O +the O +TCR B-complex_assembly +α B-structure_element +- I-structure_element +chain I-structure_element +positioned O +over O +the O +MHC B-complex_assembly +class I-complex_assembly +I I-complex_assembly +( O +MHCI B-complex_assembly +) O +α2 B-structure_element +- I-structure_element +helix I-structure_element +and O +the O +TCR B-complex_assembly +β B-structure_element +- I-structure_element +chain I-structure_element +over O +the O +MHCI B-complex_assembly +α B-structure_element +- I-structure_element +1 I-structure_element +helix I-structure_element +, O +straddling O +the O +peptide O +cargo O +. O + +However O +, O +subtle O +differences O +in O +the O +respective O +interfaces B-site +were O +apparent O +( O +discussed O +below O +) O +and O +resulted O +in O +altered O +binding B-evidence +affinities I-evidence +of O +the O +respective O +complexes O +. O + +Interactions O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +different O +APLs B-chemical +are O +focused O +around O +a O +conserved B-protein_state +GPD B-structure_element +peptide I-structure_element +motif I-structure_element +. O + +We O +next O +performed O +an O +in O +- O +depth O +atomic B-experimental_method +analysis I-experimental_method +of O +the O +contacts O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +each O +APL B-chemical +to O +determine O +the O +structural O +basis O +for O +the O +altered O +T O +cell O +peptide O +sensitivities O +and O +TCR B-evidence +binding I-evidence +affinities I-evidence +( O +Table O +2 O +). O + +Concomitant O +with O +our O +global O +analysis O +of O +1E6 B-complex_assembly +TCR I-complex_assembly +binding O +to O +the O +APLs B-chemical +, O +we O +observed O +a O +common O +interaction O +element O +, O +consistent O +with O +our O +previous O +findings O +, O +that O +utilized O +TCR B-complex_assembly +residues O +Tyr97α B-residue_name_number +and O +Trp97β B-residue_name_number +, O +forming O +an O +aromatic B-structure_element +cap I-structure_element +over O +a O +central O +GPD B-structure_element +motif I-structure_element +that O +was O +present O +in O +all O +of O +the O +APLs B-chemical +( O +Figure O +4 O +). O + +Interactions O +between O +these O +2 O +TCR B-complex_assembly +and O +3 O +peptide O +residues O +accounted O +for O +41 O +%– O +50 O +% O +of O +the O +total O +contacts O +across O +all O +complexes O +( O +Table O +2 O +), O +demonstrating O +the O +conserved B-protein_state +peptide O +centric O +binding O +mode O +utilized O +by O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +. O + +This O +fixed O +anchoring O +between O +the O +2 O +molecules O +was O +important O +for O +stabilization O +of O +the O +TCR B-complex_assembly +- I-complex_assembly +pMHC I-complex_assembly +complex O +, O +as O +— O +although O +other O +peptides O +without O +the O +‘ B-structure_element +GDP I-structure_element +’ I-structure_element +motif I-structure_element +were O +tested O +and O +shown O +to O +activate O +the O +1E6 O +T O +cell O +clone O +— O +we O +were O +unable O +to O +measure O +robust O +affinities B-evidence +using O +SPR B-experimental_method +( O +data O +not O +shown O +). O + +These O +data O +support O +the O +requirement O +for O +a O +conserved B-protein_state +interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +the O +GPD B-structure_element +motif I-structure_element +, O +as O +we O +observed O +in O +our O +previously O +published O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +ALWGPDPAAA I-complex_assembly +structure B-evidence +. O + +Focused O +hotspot O +binding O +around O +a O +conserved B-protein_state +GPD B-structure_element +motif I-structure_element +enables O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +to O +tolerate O +peptide O +degeneracy O +. O + +Although O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +formed O +a O +similar O +overall O +interaction O +with O +each O +APL B-chemical +, O +the O +stabilization O +between O +the O +TCR B-complex_assembly +and O +the O +GPD B-structure_element +motif I-structure_element +enabled O +fine O +differences O +in O +the O +contact B-site +network I-site +with O +both O +the O +peptide B-chemical +and O +MHC B-site +surface I-site +that O +allowed O +discrimination O +between O +each O +ligand O +( O +Figure O +5 O +). O + +For O +example O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +made O +only O +47 O +peptide O +contacts O +with O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +( O +KD B-evidence += O +~ O +600 O +μM O +) O +compared O +with O +63 O +and O +57 O +contacts O +with O +A2 B-chemical +- I-chemical +YQFGPDFPIA I-chemical +( O +KD B-evidence += O +7 O +. O +4 O +μM O +) O +and O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +( O +KD B-evidence += O +0 O +. O +5 O +μM O +), O +respectively O +. O + +Although O +the O +number O +of O +peptide O +contacts O +was O +a O +good O +predictor O +of O +TCR B-evidence +binding I-evidence +affinity I-evidence +for O +some O +of O +the O +APLs B-chemical +, O +for O +others O +, O +the O +correlation O +was O +poor O +( O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence += O +0 O +. O +045 O +, O +P O += O +0 O +. O +92 O +), O +possibly O +because O +of O +different O +resolutions O +for O +each O +complex O +structure B-evidence +. O + +For O +example O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +made O +64 O +peptide O +contacts O +with O +A2 B-chemical +- I-chemical +YLGGPDFPTI I-chemical +( O +KD B-evidence += O +~ O +400 O +μM O +) O +compared O +with O +43 O +contacts O +with O +A2 B-chemical +- I-chemical +RQWGPDPAAV I-chemical +( O +KD B-evidence += O +7 O +. O +8 O +μM O +). O + +The O +most O +important O +peptide O +modification O +in O +terms O +of O +generating O +new O +contacts O +was O +peptide O +position O +1 B-residue_number +. O + +The O +stronger O +ligands O +all O +encoded O +larger O +side O +chains O +( O +Arg B-residue_name +or O +Tyr B-residue_name +) O +at O +peptide O +position O +1 B-residue_number +( O +Figure O +5 O +, O +E O +– O +H O +), O +enabling O +interactions O +with O +1E6 O +that O +were O +not O +present O +in O +the O +weaker O +APLs B-chemical +that O +lacked O +large O +side O +chains O +in O +this O +position O +( O +Figure O +5 O +, O +A O +, O +C O +, O +and O +D O +). O + +We O +have O +previously O +shown O +that O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +uses O +a O +rigid O +lock O +- O +and O +- O +key O +mechanism O +during O +binding O +to O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +. O + +These O +data O +demonstrated O +that O +the O +unligated B-protein_state +structure B-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +was O +virtually O +identical O +to O +its O +ligated B-protein_state +counterparts O +. O + +In O +order O +to O +determine O +whether O +any O +of O +the O +APLs B-chemical +required O +an O +induced O +fit O +mechanism O +during O +binding O +that O +could O +explain O +the O +difference O +in O +free B-evidence +binding I-evidence +energy I-evidence +( O +ΔG B-evidence +) O +between O +each O +complex O +( O +Table O +2 O +), O +we O +solved B-experimental_method +the O +unligated B-protein_state +structures B-evidence +of O +all O +7 O +APLs B-chemical +( O +the O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +structure B-evidence +has O +been O +previously O +published O +and O +was O +used O +in O +this O +comparison O +, O +ref O +.) O +( O +Figure O +6 O +and O +Supplemental O +Table O +2 O +). O + +The O +unligated B-protein_state +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +( O +KD B-evidence += O +~ O +600 O +μM O +) O +structure B-evidence +revealed O +that O +the O +side O +chain O +Tyr9 B-residue_name_number +swung O +around O +8 O +Å O +in O +the O +complex O +structure B-evidence +, O +subsequently O +making O +contacts O +with O +TCR B-complex_assembly +residues O +Asp30β B-residue_name_number +and O +Asn51β B-residue_name_number +( O +Figure O +6A O +and O +Figure O +5A O +, O +respectively O +). O + +This O +movement O +could O +result O +in O +an O +entropic O +penalty O +contributing O +to O +the O +weak O +TCR B-evidence +binding I-evidence +affinity I-evidence +we O +observed O +for O +this O +ligand O +. O + +Additional O +small O +movements O +in O +the O +Cα O +backbone O +of O +the O +peptide O +around O +peptide O +residue O +Asp6 B-residue_name_number +were O +apparent O +in O +the O +A2 B-chemical +- I-chemical +YLGGPDFPTI I-chemical +( O +KD B-evidence += O +~ O +400 O +μM O +), O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +( O +KD B-evidence += O +~ O +208 O +μM O +), O +and O +A2 B-chemical +- I-chemical +RQFGPDWIVA I-chemical +( O +KD B-evidence += O +44 O +. O +4 O +μM O +) O +structures B-evidence +( O +Figure O +6 O +, O +B O +, O +C O +, O +and O +E O +). O + +The O +unligated B-protein_state +structures B-evidence +of O +A2 B-chemical +- I-chemical +AQWGPDAAA I-chemical +, O +A2 B-chemical +- I-chemical +RQWGPDPAAV I-chemical +, O +A2 B-chemical +- I-chemical +YQFGPDFPIA I-chemical +, O +and O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +were O +virtually O +identical O +when O +in B-protein_state +complex I-protein_state +with I-protein_state +1E6 O +( O +Figure O +6 O +, O +D O +and O +F O +– O +H O +). O + +Apart O +from O +the O +case O +of O +A2 B-chemical +- I-chemical +AQWGPDAAA I-chemical +( O +KD B-evidence += O +61 O +. O +9 O +μM O +), O +these O +observations O +support O +the O +conclusion O +that O +the O +higher O +- O +affinity B-evidence +ligands O +required O +less O +conformational O +melding O +during O +binding O +, O +which O +could O +be O +energetically O +beneficial O +( O +lower O +entopic O +cost O +) O +during O +ligation O +with O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +. O + +Peptide O +modifications O +alter O +the O +interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +the O +MHC B-site +surface I-site +. O + +In O +addition O +to O +changes O +between O +the O +TCR B-complex_assembly +and O +peptide O +component O +, O +we O +also O +observed O +that O +different O +APLs B-chemical +had O +different O +knock O +- O +on O +effects O +between O +the O +TCR B-complex_assembly +and O +MHC B-complex_assembly +. O + +MHC B-complex_assembly +residue O +Arg65 B-residue_name_number +that O +forms O +part O +of O +the O +MHC B-site +restriction I-site +triad I-site +( O +Arg65 B-residue_name_number +, O +Ala69 B-residue_name_number +, O +and O +Gln155 B-residue_name_number +) O +played O +a O +central O +role O +in O +TCR B-complex_assembly +- O +MHC B-complex_assembly +contacts O +, O +with O +Gln155 B-residue_name_number +playing O +a O +less O +important O +role O +and O +Ala69 B-residue_name_number +playing O +no O +role O +in O +binding O +at O +the O +interface B-site +( O +Figure O +7 O +). O + +Generally O +, O +the O +weaker O +- O +affinity B-evidence +APLs B-chemical +made O +fewer O +contacts O +with O +the O +MHC B-complex_assembly +surface O +( O +27 O +– O +29 O +interactions O +) O +compared O +with O +the O +stronger O +- O +affinity B-evidence +APLs B-chemical +( O +29 O +– O +35 O +contacts O +), O +consistent O +with O +a O +better O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence +value I-evidence +( O +0 O +. O +55 O +) O +compared O +with O +TCR B-complex_assembly +- O +peptide O +interactions O +versus O +affinity B-evidence +( O +0 O +. O +045 O +). O + +For O +instance O +, O +contacts O +were O +made O +between O +TCR B-complex_assembly +residue O +Val53β B-residue_name_number +and O +MHC B-complex_assembly +residue O +Gln72 B-residue_name_number +in O +all O +APLs B-chemical +except O +for O +in O +the O +weakest O +affinity B-evidence +ligand O +pair O +, O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +MVWGPDPLYV I-complex_assembly +, O +in O +which O +a O +subtle O +change O +in O +TCR B-complex_assembly +conformation O +— O +probably O +mediated O +by O +different O +peptide O +contacts O +— O +abrogated O +this O +interaction O +( O +Figure O +7A O +). O + +An O +energetic O +switch O +from O +unfavorable O +to O +favorable O +entropy B-evidence +( O +order O +- O +to O +- O +disorder O +) O +correlates O +with O +antigen O +potency O +. O + +Our O +analysis O +of O +the O +contact B-site +network I-site +provided O +some O +clues O +that O +could O +explain O +the O +different O +antigen O +potencies O +and O +binding B-evidence +affinities I-evidence +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +the O +different O +APLs B-chemical +. O + +For O +example O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +bound B-protein_state +to I-protein_state +A2 B-chemical +- I-chemical +RQWGPDPAAV I-chemical +with O +the O +third O +strongest O +affinity B-evidence +( O +KD B-evidence += O +7 O +. O +8 O +μM O +) O +but O +made O +fewer O +contacts O +than O +with O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +( O +KD B-evidence += O +~ O +208 O +μM O +) O +( O +Table O +2 O +). O + +Thus O +, O +we O +performed O +an O +in O +- O +depth O +thermodynamic B-experimental_method +analysis I-experimental_method +of O +6 O +of O +the O +ligands O +under O +investigation O +( O +Figure O +8 O +and O +Supplemental O +Table O +3 O +). O + +The O +weak O +binding B-evidence +affinity I-evidence +between O +1E6 O +and O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +and O +A2 B-chemical +- I-chemical +YLGGPDFPTI I-chemical +generated O +thermodynamic O +data O +that O +were O +not O +robust O +enough O +to O +gain O +insight O +into O +the O +enthalpic B-evidence +( O +ΔH B-evidence +°) I-evidence +and O +entropic B-evidence +( O +TΔS B-evidence +°) I-evidence +changes O +that O +contributed O +to O +the O +different O +binding B-evidence +affinities I-evidence +/ O +potencies O +for O +each O +APL B-chemical +. O + +The O +overall O +free B-evidence +binding I-evidence +energies I-evidence +( O +ΔG B-evidence +°) I-evidence +were O +between O +– O +4 O +. O +4 O +and O +– O +8 O +. O +6 O +kcal O +/ O +mol O +, O +reflecting O +the O +wide O +range O +of O +TCR B-evidence +binding I-evidence +affinities I-evidence +we O +observed O +for O +the O +different O +APLs B-chemical +. O + +The O +enthalpic O +contribution O +in O +each O +complex O +did O +not O +follow O +a O +clear O +trend O +with O +affinity B-evidence +, O +with O +all O +but O +the O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQFGPDFPTI I-complex_assembly +interaction O +( O +ΔH B-evidence +° I-evidence += O +6 O +. O +3 O +kcal O +/ O +mol O +) O +generating O +an O +energetically O +favorable O +enthalpy B-evidence +value O +( O +ΔH B-evidence +° I-evidence += O +– O +3 O +. O +7 O +to O +– O +11 O +. O +4 O +kcal O +/ O +mol O +); O +this O +indicated O +a O +net O +gain O +in O +electrostatic O +interactions O +during O +complex O +formation O +. O + +However O +, O +there O +was O +a O +clear O +switch O +in O +entropy B-evidence +between O +the O +weaker O +- O +affinity B-evidence +and O +stronger O +- O +affinity B-evidence +ligands O +, O +indicated O +by O +a O +strong O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence +value I-evidence +between O +entropy B-evidence +and O +affinity B-evidence +( O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence +value I-evidence +0 O +. O +93 O +, O +P B-evidence += O +0 O +. O +007 O +). O + +For O +instance O +, O +the O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +, O +A2 B-chemical +- I-chemical +AQWGPDAAA I-chemical +, O +and O +A2 B-chemical +- I-chemical +RQFGPDWIVA I-chemical +( O +KD B-evidence += O +~ O +208 O +μM O +, O +KD B-evidence += O +61 O +. O +9 O +μM O +, O +and O +KD B-evidence += O +44 O +. O +4 O +μM O +, O +respectively O +) O +were O +all O +entropically O +unfavorable O +( O +TΔS B-evidence +° I-evidence += O +– O +2 O +. O +9 O +to O +– O +5 O +. O +6 O +kcal O +/ O +mol O +), O +indicating O +a O +net O +change O +from O +disorder O +to O +order O +. O + +Conversely O +, O +the O +stronger O +- O +affinity B-evidence +ligands O +A2 B-chemical +- I-chemical +RQWGPDPAAV I-chemical +( O +KD B-evidence += O +7 O +. O +8 O +μM O +), O +A2 B-chemical +- I-chemical +YQFGPDFPIA I-chemical +( O +KD B-evidence += O +7 O +. O +4 O +μM O +), O +and O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +( O +KD B-evidence += O +0 O +. O +5 O +μM O +) O +exhibited O +favorable O +entropy B-evidence +( O +TΔS B-evidence +° I-evidence += O +2 O +. O +2 O +to O +14 O +. O +9 O +kcal O +/ O +mol O +), O +indicating O +an O +order O +- O +to O +- O +disorder O +change O +during O +binding O +, O +possibly O +through O +the O +expulsion O +of O +ordered O +water O +molecules O +. O + +Furthermore O +, O +the O +structures O +of O +the O +unligated B-protein_state +pMHCs B-complex_assembly +demonstrated O +that O +, O +for O +these O +stronger O +- O +affinity B-evidence +ligands O +, O +there O +was O +less O +conformational O +difference O +between O +the O +TCR B-complex_assembly +ligated B-protein_state +pMHCs B-complex_assembly +compared O +with O +the O +weaker O +- O +affinity B-evidence +ligands O +( O +Figure O +6 O +). O + +The O +potential O +requirement O +for O +a O +larger O +degree O +of O +induced O +fit O +during O +binding O +to O +these O +weaker O +- O +affinity B-evidence +ligands O +is O +consistent O +with O +the O +larger O +entropic O +penalties O +observed O +for O +these O +interactions O +. O + +Potential O +epitopes O +for O +1E6 B-complex_assembly +TCR I-complex_assembly +occur O +commonly O +in O +the O +viral B-taxonomy_domain +proteome O +. O + +We O +searched O +a O +database O +of O +over O +1 O +, O +924 O +, O +572 O +unique O +decamer O +peptides B-chemical +from O +the O +proteome O +of O +viral B-taxonomy_domain +pathogens O +that O +are O +known O +, O +or O +strongly O +suspected O +, O +to O +infect O +humans B-species +. O + +Three O +hundred O +forty O +- O +two O +of O +these O +decamers O +conformed O +to O +the O +motif O +xxxGPDxxxx B-structure_element +. O + +Of O +these O +, O +53 O +peptides O +contained O +the O +motif O +xOxGPDxxxO B-structure_element +, O +where O +O O +is O +one O +of O +the O +hydrophobic O +amino O +acid O +residues O +A B-residue_name +, O +V B-residue_name +, O +I B-residue_name +, O +L B-residue_name +, O +M B-residue_name +, O +Y B-residue_name +, O +F B-residue_name +, O +and O +W B-residue_name +that O +might O +allow O +binding O +to O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +( O +Supplemental O +Table O +4 O +). O + +Thus O +, O +there O +are O +many O +pathogen O +- O +encoded O +peptides O +that O +could O +act O +as O +agonists O +for O +the O +1E6 O +T O +cell O +beyond O +the O +MVWGPDPLYV B-chemical +and O +RQFGPDWIVA B-chemical +sequences O +studied O +here O +. O + +Extension O +of O +these O +analyses O +to O +include O +the O +larger O +genomes O +of O +bacterial B-taxonomy_domain +pathogens O +would O +be O +expected O +to O +considerably O +increase O +these O +numbers O +. O + +The O +binding B-evidence +affinity I-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +interaction O +with O +A2 B-chemical +- I-chemical +RQFGPDWIVA I-chemical +is O +considerably O +higher O +than O +with O +the O +disease O +- O +implicated O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +sequence O +( O +KD B-evidence += O +44 O +. O +4 O +μM O +and O +KD B-evidence +> O +200 O +μM O +, O +respectively O +), O +highlighting O +how O +a O +pathogen O +- O +derived O +sequence O +might O +be O +capable O +of O +priming O +a O +1E6 O +- O +like O +T O +cell O +. O + +T O +cell O +antigen O +discrimination O +is O +governed O +by O +an O +interaction O +between O +the O +clonally O +expressed O +TCR B-complex_assembly +and O +pMHC B-complex_assembly +, O +mediated O +by O +the O +chemical O +characteristics O +of O +the O +interacting O +molecules O +. O + +It O +has O +recently O +become O +clear O +that O +TCR B-complex_assembly +cross O +- O +reactivity O +with O +large O +numbers O +of O +different O +pMHC B-complex_assembly +ligands O +is O +essential O +to O +plug O +holes O +in O +T O +cell O +immune O +coverage O +that O +pathogens O +could O +exploit O +. O + +Flexibility O +at O +the O +interface B-site +between O +the O +TCR B-complex_assembly +and O +pMHC B-complex_assembly +, O +demonstrated O +in O +various O +studies O +, O +has O +been O +suggested O +as O +a O +mechanism O +mediating O +T O +cell O +cross O +- O +reactivity O +with O +multiple O +distinct O +epitopes O +. O + +This O +notion O +is O +attractive O +because O +the O +CDR B-structure_element +loops I-structure_element +, O +which O +form O +the O +TCR B-site +antigen I-site +- I-site +binding I-site +site I-site +, O +are O +usually O +the O +most O +flexible O +part O +of O +the O +TCR B-complex_assembly +and O +have O +the O +ability O +to O +mold O +around O +differently O +shaped O +ligands O +. O + +Focused O +binding O +around O +a O +minimal O +peptide O +motif O +has O +also O +been O +implicated O +as O +an O +alternative O +mechanism O +enabling O +TCR B-complex_assembly +cross O +- O +reactivity O +. O + +Notably O +among O +these O +studies O +, O +Garcia O +and O +colleagues O +recently O +used O +the O +alloreactive B-protein_state +murine B-taxonomy_domain +TCR B-complex_assembly +- O +MHC B-complex_assembly +pair O +of O +the O +42F3 B-protein +TCR B-complex_assembly +and O +H2 B-protein +- I-protein +Ld I-protein +to O +demonstrate O +recognition O +of O +a O +large O +number O +of O +different O +peptides O +via O +conserved B-protein_state +hotspot B-site +contacts O +with O +prominent O +up O +- O +facing O +peptide O +residues O +. O + +Sethi O +and O +colleagues O +recently O +demonstrated O +that O +the O +MHCII B-protein_type +- O +restricted O +Hy B-protein +. I-protein +1B11 I-protein +TCR B-complex_assembly +, O +which O +was O +isolated O +from O +a O +patient O +with O +multiple O +sclerosis O +, O +could O +anchor O +into O +a O +deep B-site +pocket I-site +formed O +from O +peptide O +residues O +2 B-residue_number +, O +3 B-residue_number +, O +and O +5 B-residue_number +( O +from O +MBP85 B-protein +– I-protein +99 I-protein +bound B-protein_state +to I-protein_state +HLA B-protein +- I-protein +DQ1 I-protein +). O + +This O +motif O +was O +conserved B-protein_state +in O +at O +least O +2 O +potential O +foreign O +peptides O +, O +originating O +from O +Herpes B-species +simplex I-species +virus I-species +and O +Pseudomonas B-species +aeruginosa I-species +, O +enabling O +TCR B-complex_assembly +recognition O +of O +foreign O +epitopes O +. O + +First O +, O +we O +currently O +know O +nothing O +about O +how O +human B-species +MHCI B-complex_assembly +– O +restricted O +TCRs B-complex_assembly +mediate O +cross O +- O +reactivity O +in O +the O +context O +of O +a O +clinically O +relevant O +model O +of O +autoimmunity O +, O +thought O +to O +be O +a O +major O +pathway O +of O +disease O +initiation O +in O +several O +autoimmune O +diseases O +. O + +Second O +, O +molecular O +studies O +have O +not O +yet O +revealed O +a O +broad O +set O +of O +rules O +that O +determine O +TCR B-complex_assembly +cross O +- O +reactivity O +because O +, O +with O +the O +exception O +of O +the O +allo B-protein_state +– O +TCR B-complex_assembly +- I-complex_assembly +MHC I-complex_assembly +pair O +of O +the O +42F3 B-protein +TCR B-complex_assembly +and O +H2 B-protein +- I-protein +Ld I-protein +that O +did O +not O +encounter O +each O +other O +during O +T O +cell O +development O +, O +studies O +have O +been O +limited O +to O +structures B-evidence +of O +a O +TCR B-complex_assembly +with O +only O +2 O +or O +3 O +different O +ligands O +. O + +Here O +, O +we O +investigated O +a O +highly O +cross O +- O +reactive O +MHCI B-complex_assembly +- O +restricted O +TCR B-complex_assembly +isolated O +from O +a O +patient O +with O +T1D O +that O +recognizes O +an O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +– O +restricted O +preproinsulin B-protein +signal B-structure_element +peptide I-structure_element +( O +ALWGPDPAAA15 B-chemical +– I-chemical +24 I-chemical +). O + +Human B-species +CD8 O ++ O +T O +cell O +clones O +expressing O +TCRs B-complex_assembly +with O +this O +specificity O +mediate O +the O +destruction O +of O +β O +cells O +, O +have O +been O +found O +in O +islets O +early O +in O +infection O +, O +and O +are O +proposed O +to O +be O +a O +major O +driver O +of O +disease O +. O + +We O +solved B-experimental_method +the O +structure B-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +with O +7 O +APLs B-chemical +to O +enable O +a O +comprehensive O +analysis O +of O +the O +molecular O +basis O +of O +TCR B-complex_assembly +degeneracy O +. O + +Overall O +, O +the O +difference O +in O +antigen O +potency O +correlated O +well O +with O +the O +binding B-evidence +energy I-evidence +( O +ΔG B-evidence +° I-evidence +kcal O +/ O +mol O +) O +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +for O +the O +different O +epitopes O +, O +which O +ranged O +from O +values O +of O +ΔG B-evidence +° I-evidence += O +~– O +4 O +. O +4 O +to O +– O +8 O +. O +6 O +kcal O +/ O +mol O +( O +calculated O +from O +3D B-evidence +affinity I-evidence +data O +) O +or O +2D B-evidence +affinity I-evidence +values O +of O +AcKa B-evidence += O +2 O +. O +5 O +× O +10 O +– O +5 O +to O +4 O +. O +4 O +× O +10 O +– O +2 O +μm4 O +. O + +The O +weaker O +end O +of O +this O +spectrum O +extends O +our O +understanding O +of O +the O +limits O +in O +which O +T O +cells O +can O +functionally O +operate O +in O +terms O +of O +TCR B-evidence +3D I-evidence +binding I-evidence +affinity I-evidence +and O +is O +in O +line O +with O +the O +types O +of O +very O +low O +affinity B-evidence +, O +yet O +fully O +functional O +self O +- O +reactive O +CD8 O ++ O +T O +cells O +we O +have O +observed O +in O +tumor O +- O +infiltrating O +lymphocytes O +. O + +Previous O +studies O +of O +autoreactive O +TCRs B-complex_assembly +have O +shown O +that O +their O +binding O +mode O +is O +generally O +atypical O +, O +either O +due O +to O +an O +unusual O +binding O +manner O +, O +weak O +TCR B-evidence +binding I-evidence +affinity I-evidence +, O +an O +unstable B-protein_state +pMHC B-complex_assembly +, O +or O +a O +combination O +of O +these O +factors O +. O + +Our O +data O +demonstrate O +the O +potential O +for O +an O +autoreactive O +TCR B-complex_assembly +to O +bind O +with O +a O +conventional O +binding O +mode O +to O +a O +stable B-protein_state +pMHC B-complex_assembly +with O +antipathogen O +- O +like O +affinity B-evidence +( O +KD B-evidence += O +0 O +. O +5 O +μM O +) O +depending O +on O +the O +peptide O +sequence O +. O + +Our O +structural B-experimental_method +analysis I-experimental_method +revealed O +that O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +bound B-protein_state +with O +a O +conserved B-protein_state +conformation I-protein_state +across O +all O +APLs B-chemical +investigated O +. O + +This O +binding O +orientation O +was O +mediated O +through O +a O +focused O +interaction O +with O +TCR B-complex_assembly +residues O +Tyr97α B-residue_name_number +and O +Trp97β B-residue_name_number +that O +formed O +an O +aromatic B-structure_element +cap I-structure_element +over O +a O +central O +‘ B-structure_element +GDP I-structure_element +’ I-structure_element +motif I-structure_element +that O +was O +common O +to O +all O +APLs B-chemical +. O + +We O +have O +previously O +demonstrated O +the O +importance O +of O +the O +GPD B-structure_element +motif I-structure_element +using O +a O +peptide B-experimental_method +library I-experimental_method +scan I-experimental_method +, O +as O +well O +as O +a O +CPL B-experimental_method +scan I-experimental_method +approach O +. O + +Although O +the O +1E6 O +T O +cell O +was O +able O +to O +activate O +weakly O +with O +peptides O +that O +lacked B-protein_state +this O +motif O +, O +we O +were O +unable O +to O +robustly O +measure O +binding B-evidence +affinities I-evidence +or O +generate O +complex O +structures B-evidence +with O +these O +ligands O +, O +highlighting O +the O +central O +role O +of O +this O +interaction O +during O +1E6 O +T O +cell O +antigen O +recognition O +. O + +This O +hotspot O +binding O +, O +defined O +as O +a O +localized O +cluster O +of O +interactions O +that O +dominate O +binding O +energy O +during O +protein O +- O +protein O +interactions O +, O +has O +been O +previously O +shown O +to O +contribute O +to O +TCR B-complex_assembly +recognition O +of O +MHC B-complex_assembly +as O +a O +mechanism O +that O +tunes O +T O +cell O +cross O +- O +reactivity O +by O +providing O +fixed O +anchor O +points O +that O +enable O +TCRs B-complex_assembly +to O +tolerate O +a O +variable O +peptide O +cargo O +. O + +Alternatively O +, O +interactions O +between O +the O +TCR B-complex_assembly +and O +peptide O +have O +been O +shown O +to O +dominate O +the O +energetic O +landscape O +during O +ligand O +engagement O +, O +ensuring O +that O +T O +cells O +retain O +peptide O +specificity O +. O + +The O +binding O +mechanism O +utilized O +by O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +during O +pMHC B-complex_assembly +recognition O +is O +consistent O +with O +both O +of O +these O +models O +. O + +Ligand O +engagement O +is O +dominated O +by O +peptide O +interactions O +, O +but O +hotspot O +- O +like O +interactions O +with O +the O +central O +GPD B-structure_element +motif I-structure_element +enable O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +to O +tolerate O +peptide O +residues O +that O +vary O +outside O +of O +this O +region O +, O +explaining O +how O +T O +cells O +expressing O +this O +TCR B-complex_assembly +may O +cross O +- O +react O +with O +a O +large O +number O +of O +different O +peptides O +. O + +These O +findings O +are O +also O +analogous O +to O +the O +observed O +binding O +mode O +of O +the O +Hy B-protein +. I-protein +1B11 I-protein +TCR B-complex_assembly +, O +in O +which O +one O +aromatic O +residue O +of O +the O +TCR B-complex_assembly +CDR3α B-structure_element +loop I-structure_element +anchored O +into O +a O +pocket O +created O +by O +a O +conserved O +peptide O +motif O +. O + +In O +both O +of O +these O +examples O +, O +self O +- O +recognition O +is O +mediated O +by O +TCR B-complex_assembly +residues O +with O +aromatic O +side O +chains O +. O + +Combined O +with O +evidence O +demonstrating O +that O +aromatic O +side O +chains O +are O +conserved O +in O +the O +CDR2 B-structure_element +loops I-structure_element +of O +TCRs B-complex_assembly +from O +many O +species O +, O +we O +speculate O +that O +these O +aromatic O +residues O +could O +impart O +a O +level O +of O +“ O +stickiness O +” O +to O +TCRs B-complex_assembly +, O +which O +might O +be O +enriched O +in O +an O +autoimmune O +setting O +when O +the O +TCR B-complex_assembly +often O +binds O +in O +a O +nonoptimal O +fashion O +. O + +Despite O +some O +weak O +statistical O +correlation O +between O +the O +surface B-evidence +complementarity I-evidence +( O +SC B-evidence +) O +and O +affinity B-evidence +, O +closer O +inspection O +of O +the O +interface B-site +revealed O +no O +obvious O +structural O +signature O +that O +could O +definitively O +explain O +the O +differences O +in O +antigen B-evidence +potency I-evidence +and O +TCR B-evidence +binding I-evidence +strength I-evidence +between O +the O +different O +ligands O +. O + +However O +, O +similar O +to O +our O +findings O +in O +other O +systems O +, O +modifications O +to O +residues O +outside O +of O +the O +canonical O +central B-structure_element +peptide I-structure_element +bulge I-structure_element +were O +important O +for O +generating O +new O +interactions O +. O + +For O +example O +, O +all O +of O +the O +stronger O +ligands O +encoded O +larger O +side O +chains O +( O +Arg B-residue_name +or O +Tyr B-residue_name +) O +at O +peptide O +position O +1 B-residue_number +that O +enabled O +new O +interactions O +with O +1E6 O +not O +present O +with O +the O +Ala B-residue_name +at O +this O +position O +in O +the O +natural O +preproinsulin B-protein +peptide B-chemical +. O + +These O +data O +also O +explain O +our O +previous O +findings O +that O +alteration O +of O +the O +anchor B-structure_element +residue I-structure_element +at O +peptide O +position O +2 B-residue_number +( O +Leu B-mutant +- I-mutant +Gln I-mutant +) O +has O +a O +direct O +effect O +on O +1E6 B-evidence +TCR I-evidence +binding I-evidence +affinity I-evidence +because O +our O +structural B-experimental_method +analysis I-experimental_method +demonstrated O +that O +1E6 O +made O +3 O +additional O +bonds O +with O +A2 B-chemical +- I-chemical +AQWGPDPAAA I-chemical +compared O +with O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +, O +consistent O +with O +the O +> O +3 O +- O +fold O +stronger O +binding B-evidence +affinity I-evidence +. O + +We O +have O +recently O +demonstrated O +how O +a O +suboptimal O +position O +2 B-residue_number +anchor B-structure_element +in O +a O +melanoma O +- O +derived O +antigen O +can O +improve O +TCR B-complex_assembly +binding O +through O +a O +similar O +mechanism O +. O + +These O +results O +challenge O +the O +notion O +that O +the O +most O +potent O +peptide O +antigens O +exhibit O +the O +greatest O +pMHC B-complex_assembly +stability O +and O +have O +implications O +for O +the O +design O +of O +anchor O +residue O +– O +modified O +heteroclitic O +peptides O +for O +vaccination O +. O + +Early O +thermodynamic B-experimental_method +analysis I-experimental_method +of O +TCR B-complex_assembly +- I-complex_assembly +pMHC I-complex_assembly +interactions O +suggested O +a O +common O +energetic O +signature O +, O +driven O +by O +favorable O +enthalpy B-evidence +( O +generally O +mediated O +through O +an O +increase O +in O +electrostatic O +interactions O +) O +and O +unfavorable O +entropy B-evidence +( O +changes O +from O +disorder O +to O +order O +). O + +These O +parameters O +aligned O +well O +with O +structural B-evidence +data I-evidence +, O +demonstrating O +that O +TCRs B-complex_assembly +engaged O +pMHC B-complex_assembly +using O +an O +induced O +fit O +binding O +mode O +. O + +However O +, O +more O +recent O +data O +have O +shown O +that O +TCRs B-complex_assembly +can O +utilize O +a O +range O +of O +energetic O +strategies O +during O +pMHC B-complex_assembly +binding O +, O +currently O +with O +no O +obvious O +pattern O +in O +terms O +of O +TCR B-evidence +affinity I-evidence +, O +binding O +mechanism O +, O +or O +specificity O +( O +pathogen O +, O +cancer O +, O +or O +self O +- O +ligands O +). O + +Although O +no O +energetic O +signature O +appears O +to O +exist O +for O +different O +TCRs B-complex_assembly +, O +we O +used O +thermodynamic B-experimental_method +analysis I-experimental_method +here O +to O +explore O +whether O +changes O +in O +energetics O +could O +help O +explain O +ligand O +discrimination O +by O +a O +single O +TCR B-complex_assembly +. O + +This O +analysis O +demonstrated O +a O +strong O +relationship O +( O +according O +to O +the O +Pearson B-experimental_method +’ I-experimental_method +s I-experimental_method +correlation I-experimental_method +analysis I-experimental_method +) O +between O +the O +energetic O +signature O +used O +by O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +the O +sensitivity O +of O +the O +1E6 O +T O +cell O +clone O +to O +different O +APLs B-chemical +. O + +The O +weaker O +APL B-chemical +ligands O +were O +characterized O +by O +favorable O +enthalpy B-evidence +and O +unfavorable O +entropy B-evidence +, O +whereas O +the O +stronger O +ligands O +progressively O +shifted O +to O +favorable O +entropy B-evidence +. O + +These O +differences O +were O +consistent O +with O +a O +greater O +degree O +of O +movement O +between O +the O +unligated B-protein_state +and O +ligated B-protein_state +pMHCs B-complex_assembly +for O +the O +weaker O +ligands O +, O +suggesting O +a O +greater O +requirement O +for O +disorder O +- O +to O +- O +order O +changes O +during O +TCR B-complex_assembly +binding O +. O + +Thus O +, O +the O +enhanced O +antigen O +potency O +was O +probably O +mediated O +through O +a O +shift O +from O +an O +induced O +fit O +to O +a O +lock O +- O +and O +- O +key O +interaction O +between O +the O +stronger O +ligands O +( O +less O +requirement O +for O +energetically O +unfavorable O +disorder O +- O +to O +- O +order O +changes O +), O +resulting O +in O +a O +more O +energetically O +favorable O +ΔG B-evidence +value I-evidence +. O + +Importantly O +, O +the O +preproinsulin B-protein +- O +derived O +epitope O +was O +one O +of O +the O +least O +potent O +peptides O +, O +demonstrating O +that O +the O +1E6 O +T O +cell O +clone O +had O +the O +ability O +to O +respond O +to O +different O +peptide O +sequences O +with O +far O +greater O +potency O +. O + +The O +RQFGPDWIVA B-chemical +peptide O +, O +which O +was O +substantially O +more O +potent O +than O +the O +preproinsulin B-protein +peptide O +, O +is O +within O +the O +proteome O +of O +a O +common O +human B-species +pathogen O +( O +C B-species +. I-species +asparagiforme I-species +), O +demonstrating O +the O +potential O +for O +an O +encounter O +between O +a O +naive O +1E6 O +- O +like O +T O +cell O +and O +a O +foreign O +peptide O +with O +a O +more O +potent O +ligand O +that O +might O +then O +break O +self O +- O +tolerance O +. O + +Indeed O +, O +we O +found O +over O +50 O +decamer O +peptides O +from O +the O +proteome O +of O +likely O +, O +or O +known O +, O +human B-species +viral B-taxonomy_domain +pathogens O +alone O +that O +contained O +both O +the O +conserved B-protein_state +central O +GPD B-structure_element +motif I-structure_element +and O +anchor B-structure_element +residues I-structure_element +at O +positions O +2 B-residue_number +and O +10 B-residue_number +that O +would O +enable O +binding O +to O +HLA B-protein +- I-protein +A I-protein +* I-protein +02 I-protein +: I-protein +01 I-protein +. O + +Further O +experiments O +will O +be O +required O +to O +determine O +whether O +any O +naturally O +presented O +, O +human B-species +pathogen O +– O +derived O +peptides O +act O +as O +active O +ligands O +for O +1E6 O +, O +but O +our O +work O +presented O +here O +demonstrates O +that O +it O +is O +at O +least O +feasible O +for O +an O +autoimmune O +TCR B-complex_assembly +to O +bind O +to O +a O +different O +peptide O +sequence O +that O +could O +be O +present O +in O +a O +pathogen O +proteome O +with O +substantially O +higher O +affinity B-evidence +and O +potency O +than O +the O +interaction O +it O +might O +use O +to O +attack O +self O +- O +tissue O +. O + +In O +summary O +, O +this O +investigation O +into O +the O +molecular O +basis O +of O +T O +cell O +cross O +- O +reactivity O +using O +a O +clinically O +relevant O +cytotoxic O +CD8 O ++ O +T O +cell O +clone O +that O +kills O +human B-species +pancreatic O +β O +cells O +provides O +answers O +to O +a O +number O +of O +previously O +outstanding O +questions O +. O + +First O +, O +our O +data O +shows O +that O +a O +single O +TCR B-complex_assembly +has O +the O +potential O +to O +functionally O +( O +assessed O +through O +T O +cell O +activation O +) O +bind O +to O +different O +ligands O +with O +affinities B-evidence +ranging O +across O +3 O +orders O +of O +magnitude O +. O + +Second O +, O +this O +is O +the O +first O +example O +in O +which O +ligands O +have O +been O +identified O +and O +characterized O +for O +a O +human B-species +autoreactive O +TCR B-complex_assembly +that O +are O +substantially O +more O +potent O +than O +the O +natural O +self O +- O +ligand O +, O +demonstrating O +the O +potential O +for O +a O +pathogenic O +ligand O +to O +break O +self O +- O +tolerance O +and O +prime O +self O +- O +reactive O +T O +cells O +. O + +Third O +, O +this O +first O +structural B-experimental_method +analysis I-experimental_method +of O +a O +cross O +- O +reactive O +human B-species +MHCI B-complex_assembly +– O +restricted O +autoimmune O +TCR B-complex_assembly +showed O +that O +degeneracy O +was O +mediated O +through O +TCR B-complex_assembly +- I-complex_assembly +pMHC I-complex_assembly +anchoring O +by O +a O +conserved B-protein_state +minimal B-structure_element +binding I-structure_element +peptide I-structure_element +motif I-structure_element +. O + +Finally O +, O +TCR B-complex_assembly +ligand O +discrimination O +was O +characterized O +by O +an O +energetic O +shift O +from O +an O +enthalpically O +to O +entropically O +driven O +interaction O +. O + +Our O +demonstration O +of O +the O +molecular O +mechanism O +governing O +cross O +- O +reactivity O +by O +this O +preproinsulin B-protein +reactive O +human B-species +CD8 O ++ O +T O +cell O +clone O +supports O +the O +notion O +first O +put O +forward O +by O +Wucherpfennig O +and O +Strominger O +that O +molecular O +mimicry O +could O +mediate O +autoimmunity O +and O +has O +far O +- O +reaching O +implications O +for O +the O +complex O +nature O +of O +T O +cell O +antigen O +discrimination O +. O + +The O +1E6 O +T O +cell O +clone O +reacts O +with O +a O +broad O +sensitivity O +range O +to O +APLs B-chemical +. O + +( O +A O +and O +B O +) O +The O +1E6 O +T O +cell O +clone O +was O +tested O +in O +a O +peptide B-experimental_method +dilution I-experimental_method +assay I-experimental_method +, O +in O +triplicate O +, O +with O +MVWGPDPLYV B-chemical +( O +gray O +), O +YLGGPDFPTI B-chemical +( O +red O +), O +ALWGPDPAAA B-chemical +( O +blue O +), O +AQWGPDPAAA B-chemical +( O +green O +), O +RQFGPDWIVA B-chemical +( O +dark O +blue O +), O +RQWGPDPAAV B-chemical +( O +purple O +), O +YQFGPDFPTA B-chemical +( O +yellow O +), O +and O +RQFGPDFPTI B-chemical +( O +cyan O +) O +peptides O +presented O +by O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +– O +expressing O +C1R O +cells O +for O +release O +of O +MIP B-protein +- I-protein +1β I-protein +( O +A O +) O +and O +killing O +( O +B O +). O + +( O +C O +) O +The O +1E6 O +T O +cell O +clone O +was O +stained O +, O +in O +duplicate O +, O +with O +tetramers B-oligomeric_state +composed O +of O +each O +APL B-chemical +( O +colored O +as O +above O +) O +presented O +by O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +. O +( O +D O +) O +The O +stability O +of O +each O +APL B-chemical +( O +colored O +as O +above O +) O +was O +tested O +, O +in O +duplicate O +, O +using O +CD B-experimental_method +by O +recording O +the O +peak O +at O +218 O +nm O +absorbance O +from O +5 O +° O +C O +– O +90 O +° O +C O +. O + +Tm B-evidence +values O +were O +calculated O +using O +a O +Boltzmann B-experimental_method +fit I-experimental_method +to I-experimental_method +each I-experimental_method +set I-experimental_method +of I-experimental_method +data I-experimental_method +. O + +3D B-experimental_method +and I-experimental_method +2D I-experimental_method +binding I-experimental_method +analysis I-experimental_method +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +with O +A2 B-chemical +- I-chemical +ALW I-chemical +and O +the O +APLs B-chemical +. O + +( O +A O +– O +H O +) O +Binding B-evidence +affinity I-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +interaction O +at O +25 O +° O +C O +using O +SPR B-experimental_method +. O + +Eight O +serial O +dilutions O +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +were O +measured O +( O +shown O +in O +the O +inset O +); O +representative O +data O +from O +3 O +independent O +experiments O +are O +plotted O +. O + +The O +equilibrium B-evidence +binding I-evidence +constant I-evidence +( O +KD B-evidence +) O +values O +were O +calculated O +using O +a O +nonlinear B-experimental_method +curve I-experimental_method +fit I-experimental_method +( O +y O += O +[ O +P1x O +]/[ O +P2 O ++ O +X O +]). O + +In O +order O +to O +calculate O +each O +response O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +was O +also O +injected O +over O +a O +control O +sample O +( O +HLA B-complex_assembly +- I-complex_assembly +A I-complex_assembly +* I-complex_assembly +0201 I-complex_assembly +– I-complex_assembly +ILAKFLHWL I-complex_assembly +) O +that O +was O +deducted O +from O +the O +experimental O +data O +. O + +( O +A O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +MVWGPDPLYV I-complex_assembly +( O +approximate O +value O +); O +( O +B O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +YLGGPDFPTI I-complex_assembly +( O +approximate O +value O +); O +( O +C O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +ALWGPDPAAA I-complex_assembly +; O +( O +D O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +AQWGPDPAAA I-complex_assembly +; O +( O +E O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQFGPDWIVA I-complex_assembly +; O +( O +F O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQWGPDPAAV I-complex_assembly +; O +( O +G O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +YQFGPDFPTA I-complex_assembly +; O +and O +( O +H O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQFGPDFPTI I-complex_assembly +. O +( O +I O +) O +ΔG B-evidence +values I-evidence +, O +calculated O +from O +SPR B-experimental_method +experiments O +, O +plotted O +against O +1 O +/ O +EC50 B-evidence +( O +the O +reciprocal O +peptide O +concentration O +required O +to O +reach O +half O +- O +maximal O +1E6 O +T O +cell O +killing O +) O +showing O +Pearson B-experimental_method +’ I-experimental_method +s I-experimental_method +coefficient I-experimental_method +analysis I-experimental_method +( O +r B-evidence +) O +and O +P B-evidence +value O +( O +including O +approximate O +values O +from O +A O +and O +B O +). O + +( O +J O +) O +Effective B-evidence +2D I-evidence +affinity I-evidence +( O +AcKa B-evidence +) O +calculated O +using O +adhesion B-experimental_method +frequency I-experimental_method +assays I-experimental_method +, O +using O +at O +least O +5 O +cell O +pairs O +, O +and O +calculated O +as O +an O +average O +of O +100 O +cell O +cell O +contacts O +. O + +( O +K O +) O +Effective B-evidence +2D I-evidence +affinity I-evidence +plotted O +against O +1 O +/ O +EC50 B-evidence +showing O +Pearson B-experimental_method +’ I-experimental_method +s I-experimental_method +coefficient I-experimental_method +analysis I-experimental_method +( O +r B-evidence +) O +and O +P B-evidence +value I-evidence +. O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +uses O +a O +conserved O +binding O +mode O +to O +engage O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +and O +the O +APLs B-chemical +. O + +( O +A O +) O +Superposition B-experimental_method +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +multicolored O +illustration O +) O +in B-protein_state +complex I-protein_state +with I-protein_state +all O +7 O +APLs B-chemical +( O +multicolored O +sticks O +) O +and O +the O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +ligand O +using O +the O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +( O +gray O +illustration O +) O +molecule O +to O +align B-experimental_method +all O +of O +the O +structures B-evidence +. O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +each O +peptide O +are O +colored O +according O +to O +the O +APL B-chemical +used O +in O +the O +complex O +as O +in O +Figure O +1 O +. O +( O +B O +) O +Position O +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +CDR B-structure_element +loops I-structure_element +( O +multicolored O +lines O +) O +in O +each O +complex O +. O + +The O +ALWGPDPAAA B-chemical +peptide O +( O +green O +sticks O +) O +is O +shown O +in O +the O +HLA B-site +- I-site +A I-site +* I-site +0201 I-site +binding I-site +groove I-site +( O +gray O +surface O +). O +( O +C O +) O +The O +Cα O +backbone O +conformation O +of O +each O +APL B-chemical +( O +multicolored O +illustration O +) O +in O +the O +context O +of O +the O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +α1 B-structure_element +helices I-structure_element +( O +gray O +illustration O +). O +( O +D O +) O +Crossing B-evidence +angle I-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +multicolored O +lines O +) O +calculated O +using O +previously O +published O +parameters O +in O +the O +context O +of O +the O +ALWGPDPAAA B-chemical +peptide O +( O +green O +sticks O +) O +bound B-protein_state +in I-protein_state +the O +HLA B-site +- I-site +A I-site +* I-site +0201 I-site +binding I-site +groove I-site +( O +gray O +surface O +). O + +A O +conserved O +interaction O +with O +a O +GPD B-structure_element +motif I-structure_element +underpins O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +interaction O +with O +the O +APLs B-chemical +. O + +Interaction O +between O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +gray O +illustration O +) O +residues O +Tyr97α B-residue_name_number +and O +Tyr97β B-residue_name_number +( O +the O +position O +of O +these O +side O +chains O +in O +the O +TCR B-complex_assembly +in B-protein_state +complex I-protein_state +with I-protein_state +all O +7 O +APLs B-chemical +, O +and O +the O +previously O +reported O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +epitope O +, O +is O +shown O +in O +multicolored O +sticks O +; O +ref O +.) O +and O +the O +GPD B-structure_element +peptide I-structure_element +motif I-structure_element +( O +the O +position O +of O +these O +side O +chains O +in O +all O +7 O +APLs B-chemical +and O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +in B-protein_state +complex I-protein_state +with I-protein_state +the O +1E6 B-complex_assembly +TCR I-complex_assembly +is O +shown O +in O +multicolored O +sticks O +). O + +The O +rest O +of O +the O +peptide O +, O +and O +the O +MHCα1 B-complex_assembly +helix B-structure_element +, O +are O +shown O +as O +a O +gray O +illustration O +. O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +makes O +distinct O +peptide O +contacts O +with O +peripheral O +APL B-chemical +residues O +. O + +Interactions O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +peptide O +residues O +outside O +of O +the O +conserved B-protein_state +GPD B-structure_element +motif I-structure_element +. O + +The O +MHCα1 B-complex_assembly +helix B-structure_element +is O +shown O +in O +gray O +illustrations O +. O + +Hydrogen B-bond_interaction +bonds I-bond_interaction +are O +shown O +as O +red O +dotted O +lines O +; O +van B-bond_interaction +der I-bond_interaction +Waals I-bond_interaction +( I-bond_interaction +vdW I-bond_interaction +) I-bond_interaction +contacts I-bond_interaction +are O +shown O +as O +black O +dotted O +lines O +. O + +Boxes O +show O +total O +contacts O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +each O +peptide O +ligand O +. O + +( O +A O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +black O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +( O +black O +illustration O +and O +sticks O +). O +( O +B O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +red O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +YLGGPDFPTI I-chemical +( O +red O +illustration O +and O +sticks O +). O +( O +C O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +blue O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +( O +blue O +illustration O +and O +sticks O +) O +reproduced O +from O +previous O +published O +data O +. O + +( O +D O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +green O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +AQWGPDPAAA I-chemical +( O +green O +illustration O +and O +sticks O +). O +( O +E O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +dark O +blue O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +RQFGPDWIVA I-chemical +( O +dark O +blue O +illustration O +and O +sticks O +). O +( O +F O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +purple O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +RQWGPDPAAV I-chemical +( O +purple O +illustration O +and O +sticks O +). O +( O +G O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +yellow O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +YQFGPDFPTA I-chemical +( O +yellow O +illustration O +and O +sticks O +). O +( O +H O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +cyan O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +( O +cyan O +illustration O +and O +sticks O +). O + +Comparison O +of O +ligated B-protein_state +and O +unligated B-protein_state +APLs B-chemical +. O + +Superposition B-experimental_method +of O +each O +APL B-chemical +in O +unligated B-protein_state +form O +and O +ligated B-protein_state +to O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +. O + +All O +unligated B-protein_state +pMHCs B-complex_assembly +are O +shown O +as O +light O +green O +illustrations O +. O + +Peptide O +sequences O +are O +shown O +underneath O +each O +structure B-evidence +aligned O +with O +the O +peptide O +structure B-evidence +. O + +( O +A O +) O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +( O +black O +sticks O +). O + +A O +large O +conformational O +shift O +was O +observed O +for O +Tyr8 B-residue_name_number +in O +the O +ligated B-protein_state +versus O +unligated B-protein_state +states O +( O +black O +circle O +). O +( O +B O +) O +A2 B-chemical +- I-chemical +YLGGPDFPTI I-chemical +( O +red O +sticks O +). O +( O +C O +) O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +( O +blue O +sticks O +) O +reproduced O +from O +previous O +published O +data O +. O +( O +D O +) O +A2 B-chemical +- I-chemical +AQWGPDPAAA I-chemical +( O +green O +sticks O +). O +( O +E O +) O +A2 B-chemical +- I-chemical +RQFGPDWIVA I-chemical +( O +dark O +blue O +sticks O +). O +( O +F O +) O +A2 B-chemical +- I-chemical +RQWGPDPAAV I-chemical +( O +purple O +sticks O +). O +( O +G O +) O +A2 B-chemical +- I-chemical +YQFGPDFPTA I-chemical +( O +yellow O +sticks O +). O +( O +H O +) O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +( O +cyan O +sticks O +). O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +makes O +distinct O +peptide O +contacts O +with O +the O +MHC B-site +surface I-site +depending O +on O +the O +peptide O +cargo O +. O + +Interactions O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +the O +MHC B-complex_assembly +α1 B-structure_element +helix I-structure_element +residues O +Arg65 B-residue_name_number +, O +Lys66 B-residue_name_number +, O +and O +Gln72 B-residue_name_number +. O + +Hydrogen B-bond_interaction +bonds I-bond_interaction +are O +shown O +as O +red O +dotted O +lines O +; O +vdW B-bond_interaction +contacts O +are O +shown O +as O +black O +dotted O +lines O +. O + +MHCα1 B-complex_assembly +helix B-structure_element +are O +shown O +in O +gray O +illustrations O +. O + +Boxes O +show O +total O +contacts O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +these O +key O +residues O +( O +green O +boxes O +are O +MHC B-complex_assembly +residues O +; O +white O +boxes O +are O +TCR B-complex_assembly +residues O +). O + +Thermodynamic B-experimental_method +analysis I-experimental_method +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +with O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +and O +the O +APLs B-chemical +. O + +Eight O +serial O +dilutions O +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +were O +injected O +, O +in O +duplicate O +, O +over O +each O +immobilized O +APL B-chemical +and O +A2 B-chemical +- I-chemical +ALW I-chemical +at O +5 O +° O +C O +, O +13 O +° O +C O +, O +18 O +° O +C O +, O +25 O +° O +C O +, O +30 O +° O +C O +, O +and O +37 O +° O +C O +. O + +The O +equilibrium B-evidence +binding I-evidence +constant I-evidence +( O +KD B-evidence +) O +values O +were O +calculated O +using O +a O +nonlinear B-experimental_method +curve I-experimental_method +fit I-experimental_method +( O +y O += O +[ O +P1x O +]/[ O +P2 O ++ O +X O +]), O +and O +thermodynamic O +parameters O +were O +calculated O +according O +to O +the O +Gibbs B-experimental_method +- I-experimental_method +Helmholtz I-experimental_method +equation I-experimental_method +( O +ΔG B-evidence +° I-evidence += O +ΔH B-evidence +− O +TΔS B-evidence +°). I-evidence + +The O +binding B-evidence +free I-evidence +energies I-evidence +, O +ΔG B-evidence +° I-evidence +( O +ΔG B-evidence +° I-evidence += O +RTlnKD O +), O +were O +plotted O +against O +temperature O +( O +K O +) O +using O +nonlinear B-experimental_method +regression I-experimental_method +to O +fit O +the O +3 O +- O +parameters O +van B-experimental_method +’ I-experimental_method +t I-experimental_method +Hoff I-experimental_method +equation I-experimental_method +( O +RT B-evidence +ln I-evidence +KD I-evidence += O +ΔH B-evidence +° I-evidence +– O +TΔS B-evidence +° I-evidence ++ O +ΔCp B-evidence +°[ I-evidence +T O +- O +T0 O +] O +– O +TΔCp B-evidence +° I-evidence +ln O +[ O +T O +/ O +T0 O +] O +with O +T0 O += O +298 O +K O +). O + +( O +A O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +ALWGPDPAAA I-complex_assembly +; O +( O +B O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +AQWGPDPAAA I-complex_assembly +; O +( O +C O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQFGPDWIVA I-complex_assembly +; O +( O +D O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQWGPDPAAV I-complex_assembly +, O +( O +E O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +YQFGPDFPTA I-complex_assembly +; O +and O +( O +F O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQFGPDFPTI I-complex_assembly +. O + +1E6 B-complex_assembly +TCR I-complex_assembly +- I-complex_assembly +pMHC I-complex_assembly +contacts O +, O +affinity B-experimental_method +measurements I-experimental_method +and O +thermodynamics B-experimental_method + +The O +immunity B-protein_type +- I-protein_type +related I-protein_type +GTPase I-protein_type +Irga6 B-protein +dimerizes B-oligomeric_state +in O +a O +parallel B-protein_state +head I-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +head I-protein_state +fashion O + +The O +immunity B-protein_type +- I-protein_type +related I-protein_type +GTPases I-protein_type +( O +IRGs B-protein_type +) O +constitute O +a O +powerful O +cell O +- O +autonomous O +resistance O +system O +against O +several O +intracellular O +pathogens O +. O + +Irga6 B-protein +is O +a O +dynamin B-protein_type +- I-protein_type +like I-protein_type +protein I-protein_type +that O +oligomerizes O +at O +the O +parasitophorous O +vacuolar O +membrane O +( O +PVM O +) O +of O +Toxoplasma B-species +gondii I-species +leading O +to O +its O +vesiculation O +. O + +Based O +on O +a O +previous O +biochemical B-experimental_method +analysis I-experimental_method +, O +it O +has O +been O +proposed O +that O +the O +GTPase B-structure_element +domains I-structure_element +of O +Irga6 B-protein +dimerize B-oligomeric_state +in O +an O +antiparallel B-protein_state +fashion O +during O +oligomerization O +. O + +We O +determined B-experimental_method +the O +crystal B-evidence +structure I-evidence +of O +an O +oligomerization B-protein_state +- I-protein_state +impaired I-protein_state +Irga6 B-protein +mutant B-protein_state +bound B-protein_state +to I-protein_state +a O +non O +- O +hydrolyzable O +GTP B-chemical +analog O +. O + +Contrary O +to O +the O +previous O +model O +, O +the O +structure B-evidence +shows O +that O +the O +GTPase B-structure_element +domains I-structure_element +dimerize B-oligomeric_state +in O +a O +parallel B-protein_state +fashion O +. O + +The O +nucleotides B-chemical +in O +the O +center O +of O +the O +interface B-site +participate O +in O +dimerization O +by O +forming O +symmetric O +contacts O +with O +each O +other O +and O +with O +the O +switch B-site +I I-site +region O +of O +the O +opposing O +Irga6 B-protein +molecule O +. O + +The O +latter O +contact O +appears O +to O +activate O +GTP B-chemical +hydrolysis O +by O +stabilizing O +the O +position O +of O +the O +catalytic B-protein_state +glutamate B-residue_name_number +106 I-residue_name_number +in O +switch B-site +I I-site +close O +to O +the O +active B-site +site I-site +. O + +Further O +dimerization O +contacts O +involve O +switch B-site +II I-site +, O +the O +G4 B-structure_element +helix I-structure_element +and O +the O +trans B-structure_element +stabilizing I-structure_element +loop I-structure_element +. O + +The O +Irga6 B-protein +structure B-evidence +features O +a O +parallel B-protein_state +GTPase B-structure_element +domain I-structure_element +dimer B-oligomeric_state +, O +which O +appears O +to O +be O +a O +unifying O +feature O +of O +all O +dynamin B-protein_type +and O +septin B-protein_type +superfamily O +members O +. O + +This O +study O +contributes O +important O +insights O +into O +the O +assembly O +and O +catalytic O +mechanisms O +of O +IRG B-protein_type +proteins O +as O +prerequisite O +to O +understand O +their O +anti O +- O +microbial O +action O +. O + +Immunity B-protein_type +- I-protein_type +related I-protein_type +GTPases I-protein_type +( O +IRGs B-protein_type +) O +comprise O +a O +family O +of O +dynamin B-protein_type +- I-protein_type +related I-protein_type +cell I-protein_type +- I-protein_type +autonomous I-protein_type +resistance I-protein_type +proteins I-protein_type +targeting O +intracellular O +pathogens O +, O +such O +as O +Mycobacterium B-species +tuberculosis I-species +, O +Mycobacterium B-species +avium I-species +, O +Listeria B-species +monocytogenes I-species +, O +Trypanosoma B-species +cruzi I-species +, O +and O +Toxoplasma B-species +gondii I-species +. O + +In O +mice B-taxonomy_domain +, O +the O +23 O +IRG B-protein_type +members O +are O +induced O +by O +interferons B-protein_type +, O +whereas O +the O +single O +human B-species +homologue O +is O +constitutively O +expressed O +in O +some O +tissues O +, O +especially O +in O +testis O +. O + +In O +non O +- O +infected O +cells O +, O +most O +IRGs B-protein_type +are O +largely O +cytosolic O +. O + +In O +this O +way O +, O +IRGs B-protein_type +contribute O +to O +the O +release O +of O +the O +pathogen O +into O +the O +cytoplasm O +and O +its O +subsequent O +destruction O +. O + +Irga6 B-protein +, O +one O +of O +the O +effector O +IRG B-protein_type +proteins O +, O +localizes O +to O +the O +intact O +parasitophorous O +vacuole O +membrane O +( O +PVM O +) O +and O +, O +after O +disruption O +of O +the O +PVM O +, O +is O +found O +associated O +with O +vesicular O +accumulations O +, O +presumably O +derived O +from O +the O +PVM O +. O + +A O +myristoylation B-site +site I-site +at O +Gly2 B-residue_name_number +is O +necessary O +for O +the O +recruitment O +to O +the O +PVM O +but O +not O +for O +the O +weak O +constitutive O +binding O +to O +the O +ER O +membrane O +. O + +An O +internally O +oriented O +antibody O +epitope O +on O +helix B-structure_element +A I-structure_element +between O +positions O +20 B-residue_range +and I-residue_range +24 I-residue_range +was O +demonstrated O +to O +be O +accessible O +in O +the O +GTP B-protein_state +-, I-protein_state +but O +not O +in O +the O +GDP B-protein_state +- I-protein_state +bound I-protein_state +state O +. O + +This O +indicates O +large O +- O +scale O +structural O +changes O +upon O +GTP B-chemical +binding O +that O +probably O +include O +exposure O +of O +the O +myristoyl O +group O +, O +enhancing O +binding O +to O +the O +PVM O +. O + +Biochemical B-experimental_method +studies I-experimental_method +indicated O +that O +Irga6 B-protein +hydrolyses O +GTP B-chemical +in O +a O +cooperative O +manner O +and O +forms O +GTP B-protein_state +- I-protein_state +dependent I-protein_state +oligomers B-oligomeric_state +in O +vitro O +and O +in O +vivo O +. O + +Crystal B-evidence +structures I-evidence +of O +Irga6 B-protein +in O +various O +nucleotide B-protein_state +- I-protein_state +loaded I-protein_state +states O +revealed O +the O +basic O +architecture O +of O +IRG B-protein_type +proteins O +, O +including O +a O +GTPase B-structure_element +domain I-structure_element +and O +a O +composite B-structure_element +helical I-structure_element +domain I-structure_element +. O + +These O +studies O +additionally O +showed O +a O +dimerization B-site +interface I-site +in O +the O +nucleotide B-protein_state +- I-protein_state +free I-protein_state +protein O +as O +well O +as O +in O +all O +nucleotide B-protein_state +- I-protein_state +bound I-protein_state +states O +. O + +It O +involves O +a O +GTPase B-site +domain I-site +surface I-site +, O +which O +is O +located O +at O +the O +opposite O +side O +of O +the O +nucleotide O +, O +and O +an O +interface B-site +in O +the O +helical B-structure_element +domain I-structure_element +, O +with O +a O +water B-chemical +- O +filled O +gap O +between O +the O +two O +contact B-site +surfaces I-site +. O + +Mutagenesis B-experimental_method +of O +the O +contact B-site +surfaces I-site +suggests O +that O +this O +"""" O +backside B-site +"""" O +interface B-site +is O +not O +required O +for O +GTP B-chemical +- O +dependent O +oligomerization O +or O +cooperative O +hydrolysis O +, O +despite O +an O +earlier O +suggestion O +to O +the O +contrary O +. O + +Extensive O +biochemical B-experimental_method +studies I-experimental_method +suggested O +that O +GTP B-chemical +- O +induced O +oligomerization O +of O +Irga6 B-protein +requires O +an O +interface B-site +in O +the O +GTPase B-structure_element +domain I-structure_element +across O +the O +nucleotide B-site +- I-site +binding I-site +site I-site +. O + +Recent O +structural B-experimental_method +studies I-experimental_method +indicated O +that O +a O +' O +G B-site +interface I-site +' O +is O +typical O +of O +dynamin B-protein_type +superfamily O +members O +, O +such O +as O +dynamin B-protein_type +, O +MxA B-protein +, O +the O +guanylate B-protein +binding I-protein +protein I-protein +- I-protein +1 I-protein +( O +GBP B-protein +- I-protein +1 I-protein +), O +atlastin B-protein_type +and O +the O +bacterial B-taxonomy_domain +dynamin B-protein_type +- I-protein_type +like I-protein_type +proteins I-protein_type +( O +BDLP B-protein_type +). O + +For O +several O +of O +these O +proteins O +, O +formation O +of O +the O +G B-site +interface I-site +was O +shown O +to O +trigger O +GTP B-chemical +hydrolysis O +by O +inducing O +rearrangements O +of O +catalytic O +residues O +in O +cis O +. O + +In O +dynamin B-protein_type +, O +the O +G B-site +interface I-site +includes O +residues O +in O +the O +phosphate B-structure_element +binding I-structure_element +loop I-structure_element +, O +the O +two O +switch B-site +regions I-site +, O +the O +' O +trans B-structure_element +stabilizing I-structure_element +loop I-structure_element +' O +and O +the O +' O +G4 B-structure_element +loop I-structure_element +'. O + +For O +Irga6 B-protein +, O +it O +was O +demonstrated O +that O +besides O +residues O +in O +the O +switch B-site +I I-site +and O +switch B-site +II I-site +regions O +, O +the O +3 O +'- O +OH O +group O +of O +the O +ribose O +participates O +in O +this O +interface B-site +. O + +Since O +the O +signal B-protein_type +recognition I-protein_type +particle I-protein_type +GTPase I-protein_type +and O +its O +homologous O +receptor B-protein_type +( O +called O +FfH B-protein +and O +FtsY B-protein +in O +bacteria B-taxonomy_domain +) O +also O +employ O +the O +3 O +'- O +OH O +ribose O +group O +to O +dimerize B-oligomeric_state +in O +an O +anti B-protein_state +- I-protein_state +parallel I-protein_state +orientation O +therefore O +activating O +its O +GTPase B-protein_type +, O +an O +analogous O +dimerization O +model O +was O +proposed O +for O +Irga6 B-protein +. O + +However O +, O +the O +crystal B-evidence +structure I-evidence +of O +Irga6 B-protein +in O +the O +presence B-protein_state +of I-protein_state +the O +non O +- O +hydrolyzable O +GTP B-chemical +analogue O +5 B-chemical +'- I-chemical +guanylyl I-chemical +imidodiphosphate I-chemical +( O +GMPPNP B-chemical +) O +showed O +only O +subtle O +differences O +relative O +to O +the O +apo B-protein_state +or O +GDP B-protein_state +- I-protein_state +bound I-protein_state +protein O +and O +did O +not O +reveal O +a O +new O +dimer B-site +interface I-site +associated O +with O +the O +GTPase B-structure_element +domain I-structure_element +. O + +This O +structure B-evidence +was O +obtained O +by O +soaking B-experimental_method +GMPPNP B-chemical +in O +nucleotide B-protein_state +- I-protein_state +free I-protein_state +crystals B-evidence +of O +Irga6 B-protein +, O +an O +approach O +which O +may O +have O +interfered O +with O +nucleotide O +- O +induced O +domain O +rearrangements O +. O + +To O +clarify O +the O +dimerization O +mode O +via O +the O +G B-site +interface I-site +, O +we O +determined B-experimental_method +the O +GMPPNP B-protein_state +- I-protein_state +bound I-protein_state +crystal B-evidence +structure I-evidence +of O +a O +non B-protein_state +- I-protein_state +oligomerizing I-protein_state +Irga6 B-protein +variant B-protein_state +. O + +The O +structure B-evidence +revealed O +that O +Irga6 B-protein +can O +dimerize B-oligomeric_state +via O +the O +G B-site +interface I-site +in O +a O +parallel B-protein_state +head I-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +head I-protein_state +fashion O +. O + +Our O +data O +suggest O +that O +a O +parallel B-protein_state +dimerization O +mode O +may O +be O +a O +unifying O +feature O +in O +all O +dynamin B-protein_type +and O +septin B-protein_type +superfamily O +proteins O +. O + +Previous O +results O +indicated O +that O +Irga6 B-protein +mutations B-experimental_method +in O +a O +loosely O +defined O +surface B-site +region I-site +( O +the O +"""" O +secondary B-site +patch I-site +""")," O +which O +is O +distant O +from O +the O +G B-site +- I-site +interface I-site +and O +only O +slightly O +overlapping O +with O +the O +backside B-site +interface I-site +( O +see O +below O +), O +individually O +reduced O +GTP B-chemical +- O +dependent O +oligomerization O +. O + +A O +combination B-experimental_method +of I-experimental_method +four I-experimental_method +of I-experimental_method +these I-experimental_method +mutations I-experimental_method +( O +R31E B-mutant +, O +K32E B-mutant +, O +K176E B-mutant +, O +and O +K246E B-mutant +) O +essentially O +eliminated B-protein_state +GTP B-chemical +- O +dependent O +assembly O +( O +Additional O +file O +1 O +: O +Figure O +S1 O +) O +and O +allowed O +crystallization B-experimental_method +of O +Irga6 B-protein +in O +the O +presence B-protein_state +of I-protein_state +GMPPNP B-chemical +. O + +Crystals B-evidence +diffracted O +to O +3 O +. O +2 O +Å O +resolution O +and O +displayed O +one O +exceptionally O +long O +unit O +cell O +axis O +of O +1289 O +Å O +( O +Additional O +file O +1 O +: O +Table O +S1 O +). O + +The O +structure B-evidence +was O +solved O +by O +molecular B-experimental_method +replacement I-experimental_method +and O +refined O +to O +Rwork B-evidence +/ O +Rfree B-evidence +of O +29 O +. O +7 O +%/ O +31 O +. O +7 O +% O +( O +Additional O +file O +1 O +: O +Table O +S2 O +). O + +The O +asymmetric O +unit O +contained O +seven O +Irga6 B-protein +molecules O +that O +were O +arranged O +in O +a O +helical O +pattern O +along O +the O +long O +cell O +axis O +( O +Additional O +file O +1 O +: O +Figure O +S2 O +). O + +Structure B-evidence +of O +the O +Irga6 B-protein +dimer B-oligomeric_state +. O + +a O +Schematic O +view O +of O +the O +domain O +architecture O +of O +mouse B-taxonomy_domain +Irga6 B-protein +. O + +b O +Ribbon O +- O +type O +representation O +of O +the O +Irga6 B-protein +dimer B-oligomeric_state +. O + +The O +nucleotide O +and O +Mg2 B-chemical ++ I-chemical +ion O +( O +green O +) O +are O +shown O +in O +sphere O +representation O +. O + +The O +GTPase B-structure_element +domain I-structure_element +dimer B-oligomeric_state +is O +boxed O +. O + +Secondary O +structure O +was O +numbered O +according O +to O +ref O +.. O +c O +Top O +view O +on O +the O +GTPase B-structure_element +domain I-structure_element +dimer B-oligomeric_state +. O + +d O +Magnification O +of O +the O +contact B-site +sites I-site +. O + +e O +Superposition B-experimental_method +of O +different O +switch B-site +I I-site +conformations O +in O +the O +asymmetric O +unit O +; O +the O +same O +colors O +as O +in O +Additional O +file O +1 O +: O +Figure O +S2 O +are O +used O +for O +the O +switch B-site +I I-site +regions O +of O +the O +individual O +subunits O +. O + +Switch B-site +I I-site +residues O +of O +subunit O +A B-structure_element +( O +yellow O +) O +involved O +in O +ribose O +binding O +are O +labelled O +and O +shown O +in O +stick O +representation O +. O + +Irga6 B-protein +immunity B-protein +- I-protein +related I-protein +GTPase I-protein +6 I-protein + +Like O +other O +dynamin B-protein_type +superfamily O +members O +, O +the O +GTPase B-structure_element +domain I-structure_element +of O +Irga6 B-protein +comprises O +a O +canonical O +GTPase B-structure_element +domain I-structure_element +fold O +, O +with O +a O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +surrounded O +by O +helices B-structure_element +on O +both O +sides O +( O +Fig O +. O +1a O +- O +c O +). O + +The O +helical B-structure_element +domain I-structure_element +is O +a O +bipartite O +structure O +composed O +of O +helices B-structure_element +αA B-structure_element +- I-structure_element +C I-structure_element +at O +the O +N O +- O +terminus O +and O +helix B-structure_element +αF B-structure_element +- I-structure_element +L I-structure_element +at O +the O +C O +- O +terminus O +of O +the O +GTPase B-structure_element +domain I-structure_element +. O + +Overall O +, O +the O +seven O +molecules O +in O +the O +asymmetric O +unit O +are O +very O +similar O +to O +each O +other O +, O +with O +root B-evidence +mean I-evidence +square I-evidence +deviations I-evidence +( O +rmsd B-evidence +) O +ranging O +from O +0 O +. O +32 O +– O +0 O +. O +45 O +Å O +over O +all O +Cα O +atoms O +. O + +The O +structures B-evidence +of O +the O +seven O +molecules O +also O +agree O +well O +with O +the O +previously O +determined O +structure B-evidence +of O +native B-protein_state +GMPPNP B-protein_state +- I-protein_state +bound I-protein_state +Irga6 B-protein +( O +PDB O +: O +1TQ6 O +; O +rmsd B-evidence +of O +1 O +. O +00 O +- O +1 O +. O +13 O +Å O +over O +all O +Cα O +atoms O +). O + +The O +seven O +Irga6 B-protein +molecules O +in O +the O +asymmetric O +unit O +form O +various O +higher O +order O +contacts O +in O +the O +crystals B-evidence +. O + +Within O +the O +asymmetric O +unit O +, O +six O +molecules O +dimerize B-oligomeric_state +via O +the O +symmetric O +backside B-site +dimer I-site +interface I-site +( O +buried O +surface O +area O +930 O +Å2 O +), O +and O +the O +remaining O +seventh O +molecule O +forms O +the O +same O +type O +of O +interaction O +with O +its O +symmetry O +mate O +of O +the O +adjacent O +asymmetric O +unit O +( O +Additional O +file O +1 O +: O +Figure O +S2a O +, O +b O +, O +Figure O +S3 O +). O + +This O +indicates O +that O +the O +introduced O +mutations B-experimental_method +in O +the O +secondary B-site +patch I-site +, O +from O +which O +only O +Lys176 B-residue_name_number +is O +part O +of O +the O +backside B-site +interface I-site +, O +do O +, O +in O +fact O +, O +not O +prevent O +this O +interaction O +. O + +Another O +assembly B-site +interface I-site +with O +a O +buried O +surface O +area O +of O +450 O +Å2 O +, O +which O +we O +call O +the O +“ O +tertiary B-site +patch I-site +”, O +was O +formed O +via O +two O +interaction B-site +sites I-site +in O +the O +helical B-structure_element +domains I-structure_element +( O +Additional O +file O +1 O +: O +Figure O +S2c O +, O +d O +, O +S3 O +). O + +In O +this O +interface B-site +, O +helices B-structure_element +αK B-structure_element +from O +two O +adjacent O +molecules O +form O +a O +hydrogen B-bond_interaction +bonding I-bond_interaction +network I-bond_interaction +involving O +residues O +373 B-residue_range +- I-residue_range +376 I-residue_range +. O + +Furthermore O +, O +two O +adjacent O +helices B-structure_element +αA B-structure_element +form O +hydrophobic B-bond_interaction +contacts I-bond_interaction +. O + +It O +was O +previously O +shown O +that O +the O +double B-protein_state +mutation I-protein_state +L372R B-mutant +/ O +A373R B-mutant +did O +not O +prevent O +GTP B-chemical +- O +induced O +assembly O +, O +so O +there O +is O +currently O +no O +evidence O +supporting O +an O +involvement O +of O +this O +interface B-site +in O +higher O +- O +order O +oligomerization O +. O + +Strikingly O +, O +molecule O +A B-structure_element +of O +one O +asymmetric O +unit O +assembled O +with O +an O +equivalent O +molecule O +of O +the O +adjacent O +asymmetric O +unit O +via O +the O +G B-site +- I-site +interface I-site +in O +a O +symmetric O +parallel B-protein_state +fashion O +via O +a O +470 O +Å2 O +interface O +. O + +This O +assembly O +results O +in O +a O +butterfly B-protein_state +- I-protein_state +shaped I-protein_state +Irga6 B-protein +dimer B-oligomeric_state +in O +which O +the O +helical B-structure_element +domains I-structure_element +protrude O +in O +parallel B-protein_state +orientations O +( O +Fig O +. O +1b O +, O +Additional O +file O +1 O +: O +Figure O +S3 O +). O + +In O +contrast O +, O +the O +other O +six O +molecules O +in O +the O +asymmetric O +unit O +do O +not O +assemble O +via O +the O +G B-site +interface I-site +. O + +The O +G B-site +interface I-site +in O +molecule O +A O +can O +be O +subdivided O +into O +three O +distinct O +contact B-site +sites I-site +( O +Fig O +. O +1c O +, O +d O +). O + +Contact B-site +site I-site +I I-site +is O +formed O +between O +R159 B-residue_name_number +and O +K161 B-residue_name_number +in O +the O +trans B-structure_element +stabilizing I-structure_element +loops I-structure_element +, O +and O +S132 B-residue_name_number +in O +the O +switch B-site +II I-site +regions O +of O +the O +opposing O +molecules O +. O + +Contact B-site +site I-site +II I-site +features O +polar B-bond_interaction +and I-bond_interaction +hydrophobic I-bond_interaction +interactions I-bond_interaction +formed O +by O +switch B-site +I I-site +( O +V104 B-residue_name_number +, O +V107 B-residue_name_number +) O +with O +a O +helix B-structure_element +following O +the O +guanine B-structure_element +specificity I-structure_element +motif I-structure_element +( O +G4 B-structure_element +helix I-structure_element +, O +K184 B-residue_name_number +and O +S187 B-residue_name_number +) O +and O +the O +trans B-structure_element +stabilizing I-structure_element +loop I-structure_element +( O +T158 B-residue_name_number +) O +of O +the O +opposing O +GTPase B-structure_element +domain I-structure_element +. O + +In O +contact B-site +site I-site +III I-site +, O +G103 B-residue_name_number +of O +switch B-site +I I-site +interacts O +via O +its O +main O +chain O +nitrogen O +with O +the O +exocyclic O +2 O +’- O +OH O +and O +3 O +’- O +OH O +groups O +of O +the O +opposing O +ribose B-chemical +in O +trans O +, O +whereas O +the O +two O +opposing O +exocyclic O +3 O +’- O +OH O +group O +of O +the O +ribose B-chemical +form O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +each O +other O +. O + +Via O +the O +ribose B-chemical +contact O +, O +switch B-site +I I-site +is O +pulled O +towards O +the O +opposing O +nucleotide B-chemical +( O +Fig O +. O +1e O +). O + +In O +turn O +, O +E106 B-residue_name_number +of O +switch B-site +I I-site +reorients O +towards O +the O +nucleotide B-chemical +and O +now O +participates O +in O +the O +coordination B-bond_interaction +of I-bond_interaction +the O +Mg2 B-chemical ++ I-chemical +ion O +( O +Fig O +. O +1e O +, O +Additional O +file O +1 O +: O +Figure O +S4 O +). O + +E106 B-residue_name_number +was O +previously O +shown O +to O +be O +essential O +for O +catalysis O +, O +and O +the O +observed O +interactions O +in O +contact B-site +site I-site +III I-site +explain O +how O +dimerization O +via O +the O +ribose B-chemical +is O +directly O +coupled O +to O +the O +activation O +of O +GTP B-chemical +hydrolysis O +. O + +The O +G B-site +interface I-site +is O +in O +full O +agreement O +with O +previously O +published O +biochemical O +data O +that O +indicate O +crucial O +roles O +of O +E77 B-residue_name_number +, O +G103 B-residue_name_number +, O +E106 B-residue_name_number +, O +S132 B-residue_name_number +, O +R159 B-residue_name_number +, O +K161 B-residue_name_number +, O +K162 B-residue_name_number +, O +D164 B-residue_name_number +, O +N191 B-residue_name_number +, O +and O +K196 B-residue_name_number +for O +oligomerization O +and O +oligomerization O +- O +induced O +GTP B-chemical +hydrolysis O +. O + +All O +of O +these O +residues O +directly O +participate O +in O +contacts O +( O +G103 B-residue_name_number +, O +S132 B-residue_name_number +, O +R159 B-residue_name_number +, O +and O +K161 B-residue_name_number +) O +or O +are O +in O +direct O +vicinity O +to O +the O +interface B-site +( O +E77 B-residue_name_number +, O +E106 B-residue_name_number +, O +K162 B-residue_name_number +, O +D164 B-residue_name_number +, O +and O +N191 B-residue_name_number +). O + +Residues O +E77 B-residue_name_number +, O +K162 B-residue_name_number +, O +and O +D164 B-residue_name_number +appear O +to O +orient O +the O +trans B-structure_element +stabilizing I-structure_element +loop I-structure_element +which O +is O +involved O +in O +interface B-site +formation O +in O +contact B-site +site I-site +II I-site +. O + +In O +the O +earlier O +model O +of O +an O +anti B-protein_state +- I-protein_state +parallel I-protein_state +G B-site +interface I-site +, O +it O +was O +not O +possible O +to O +position O +the O +side O +chain O +of O +R159 B-residue_name_number +to O +avoid O +steric O +conflict O +. O + +In O +the O +present O +structure B-evidence +, O +the O +side O +- O +chain O +of O +R159 B-residue_name_number +projects O +laterally O +along O +the O +G B-site +interface I-site +and O +, O +therefore O +, O +does O +not O +cause O +a O +steric O +conflict O +. O + +A O +conserved O +dimerization O +mode O +via O +the O +G B-site +interface I-site +in O +dynamin B-protein_type +and O +septin B-protein_type +GTPases I-protein_type +. O + +The O +overall O +architecture O +of O +the O +parallel B-protein_state +GTPase B-structure_element +domain I-structure_element +dimer B-oligomeric_state +of O +Irga6 B-protein +is O +related O +to O +that O +of O +other O +dynamin B-protein_type +and O +septin B-protein_type +superfamily O +proteins O +. O + +The O +following O +structures B-evidence +are O +shown O +in O +cylinder O +representations O +, O +in O +similar O +orientations O +of O +their O +GTPase B-structure_element +domains I-structure_element +: O +a O +the O +GMPPNP B-protein_state +- I-protein_state +bound I-protein_state +Irga6 B-protein +dimer B-oligomeric_state +, O +b O +the O +GDP O +- O +AlF4 O +-- O +bound O +dynamin B-protein +1 I-protein +GTPase B-structure_element +- I-structure_element +minimal I-structure_element +BSE O +construct O +[ O +pdb O +2X2E O +], O +c O +the O +GDP B-protein_state +- I-protein_state +bound I-protein_state +atlastin B-protein +1 I-protein +dimer B-oligomeric_state +[ O +pdb O +3Q5E O +], O +d O +the O +GDP B-protein_state +- I-protein_state +AlF3 I-protein_state +- I-protein_state +bound I-protein_state +GBP1 B-protein +GTPase B-structure_element +domain I-structure_element +dimer B-oligomeric_state +[ O +pdb O +2B92 O +], O +e O +the O +BDLP B-protein_type +dimer B-oligomeric_state +bound B-protein_state +to I-protein_state +GDP B-chemical +[ O +pdb O +2J68 O +] O +and O +f O +the O +GTP B-protein_state +- I-protein_state +bound I-protein_state +GIMAP2 B-protein +dimer B-oligomeric_state +[ O +pdb O +2XTN O +]. O + +The O +GTPase B-structure_element +domains I-structure_element +of O +the O +left O +molecules O +are O +shown O +in O +orange O +, O +helical B-structure_element +domains I-structure_element +or O +extensions O +in O +blue O +. O + +Nucleotide O +, O +Mg2 B-chemical ++ I-chemical +( O +green O +) O +and O +AlF4 O +- O +are O +shown O +in O +sphere O +representation O +, O +the O +buried O +interface B-site +sizes O +per O +molecule O +are O +indicated O +on O +the O +right O +. O + +Irga6 B-protein +immunity B-protein +- I-protein +related I-protein +GTPase I-protein +6 I-protein +, O +GMPPNP B-chemical +5 B-chemical +'- I-chemical +guanylyl I-chemical +imidodiphosphate I-chemical +, O +GTP B-chemical +guanosine B-chemical +- I-chemical +triphosphate I-chemical +, O +BDLP B-protein_type +bacterial B-taxonomy_domain +dynamin B-protein_type +like I-protein_type +protein I-protein_type +, O +GIMAP2 B-protein +, O +GTPase B-protein +of I-protein +immunity I-protein +associated I-protein +protein I-protein +2 I-protein + +The O +buried O +surface O +area O +per O +molecule O +( O +BSA O +) O +of O +the O +G B-site +interface I-site +in O +Irga6 B-protein +is O +relatively O +small O +( O +470 O +Å2 O +) O +compared O +to O +that O +of O +other O +dynamin B-protein_type +superfamily O +members O +, O +such O +as O +dynamin B-protein_type +( O +BSA O +: O +1400 O +Å2 O +), O +atlastin B-protein_type +( O +BSA O +: O +820 O +Å2 O +), O +GBP B-protein +- I-protein +1 I-protein +( O +BSA O +: O +2060 O +Å2 O +), O +BDLP B-protein_type +( O +BSA O +: O +2300 O +Å2 O +) O +or O +the O +septin B-protein +- I-protein +related I-protein +GTPase I-protein +of I-protein +immunity I-protein +associated I-protein +protein I-protein +2 I-protein +( O +GIMAP2 B-protein +) O +( O +BSA O +: O +590 O +Å2 O +) O +( O +Fig O +. O +2 O +). O + +However O +, O +the O +relative O +orientations O +of O +the O +GTPase B-structure_element +domains I-structure_element +in O +these O +dimers B-oligomeric_state +are O +strikingly O +similar O +, O +and O +the O +same O +elements O +, O +such O +as O +switch B-site +I I-site +, O +switch B-site +II I-site +, O +the O +trans B-structure_element +activating I-structure_element +and I-structure_element +G4 I-structure_element +loops I-structure_element +are O +involved O +in O +the O +parallel B-protein_state +dimerization O +mode O +in O +all O +of O +these O +GTPase B-protein_type +families O +. O + +IRG B-protein_type +proteins O +are O +crucial O +mediators O +of O +the O +innate O +immune O +response O +in O +mice B-taxonomy_domain +against O +a O +specific O +subset O +of O +intracellular O +pathogens O +, O +all O +of O +which O +enter O +the O +cell O +to O +form O +a O +membrane O +- O +bounded O +vacuole O +without O +engagement O +of O +the O +phagocytic O +machinery O +. O + +As O +members O +of O +the O +dynamin B-protein_type +superfamily O +, O +IRGs B-protein_type +oligomerize O +at O +cellular O +membranes O +in O +response O +to O +GTP B-chemical +binding O +. O + +Oligomerization O +and O +oligomerization O +- O +induced O +GTP B-chemical +hydrolysis O +are O +thought O +to O +induce O +membrane O +remodeling O +events O +ultimately O +leading O +to O +disruption O +of O +the O +PVM O +. O + +Recent O +structural B-experimental_method +and I-experimental_method +mechanistic I-experimental_method +analyses I-experimental_method +have O +begun O +to O +unravel O +the O +molecular O +basis O +for O +the O +membrane O +- O +remodeling O +activity O +and O +mechano O +- O +chemical O +function O +of O +some O +members O +( O +reviewed O +in O +). O + +For O +example O +, O +for O +dynamin B-protein_type +and O +atlastin B-protein_type +, O +it O +was O +shown O +that O +GTP B-chemical +binding O +and O +/ O +or O +hydrolysis O +leads O +to O +dimerization O +of O +the O +GTPase B-structure_element +domains I-structure_element +and O +to O +the O +reorientation O +of O +the O +adjacent O +helical B-structure_element +domains I-structure_element +. O + +However O +, O +for O +other O +dynamin B-protein_type +superfamily O +members O +such O +as O +IRGs B-protein_type +, O +the O +molecular O +basis O +for O +GTP B-chemical +hydrolysis O +and O +the O +exact O +role O +of O +the O +mechano O +- O +chemical O +function O +are O +still O +unclear O +. O + +Our O +structural B-experimental_method +analysis I-experimental_method +of O +an O +oligomerization B-protein_state +- I-protein_state +and I-protein_state +GTPase I-protein_state +- I-protein_state +defective I-protein_state +Irga6 B-protein +mutant B-protein_state +indicates O +that O +Irga6 B-protein +dimerizes B-oligomeric_state +via O +the O +G B-site +interface I-site +in O +a O +parallel B-protein_state +orientation O +. O + +Only O +one O +of O +the O +seven O +Irga6 B-protein +molecules O +in O +the O +asymmetric O +unit O +formed O +this O +contact O +pointing O +to O +a O +low O +affinity O +interaction O +via O +the O +G B-site +interface I-site +, O +which O +is O +in O +agreement O +with O +its O +small O +size O +. O + +In O +the O +crystals B-evidence +, O +dimerization O +via O +the O +G B-site +interface I-site +is O +promoted O +by O +the O +high O +protein O +concentrations O +which O +may O +mimic O +a O +situation O +when O +Irga6 B-protein +oligomerizes O +on O +a O +membrane O +surface O +. O + +Such O +a O +low O +affinity O +interaction O +mode O +may O +allow O +reversibility O +of O +oligomerization O +following O +GTP B-chemical +hydrolysis O +. O + +Similar O +low O +affinity O +G B-site +interface I-site +interactions O +were O +reported O +for O +dynamin B-protein_type +and O +MxA B-protein +. O + +The O +dimerization O +mode O +is O +strikingly O +different O +from O +the O +previously O +proposed O +anti B-protein_state +- I-protein_state +parallel I-protein_state +model O +that O +was O +based O +on O +the O +crystal B-evidence +structure I-evidence +of O +the O +signal B-protein_type +recognition I-protein_type +particle I-protein_type +GTPase I-protein_type +, O +SRP54 B-protein +and O +its O +homologous O +receptor O +. O + +However O +, O +the O +G B-site +dimer I-site +interface I-site +is O +reminiscent O +of O +the O +GTPase B-structure_element +domain I-structure_element +dimers B-oligomeric_state +observed O +for O +several O +other O +dynamin B-protein_type +superfamily O +members O +, O +such O +as O +dynamin B-protein_type +, O +GBP1 B-protein +, O +atlastin B-protein_type +, O +and O +BDLP B-protein_type +. O + +It O +was O +recently O +shown O +that O +septin B-protein_type +and O +septin B-protein_type +- I-protein_type +related I-protein_type +GTPases I-protein_type +, O +such O +as O +the O +Tocs B-protein_type +GTPases I-protein_type +or O +GTPases B-protein_type +of I-protein_type +immunity I-protein_type +related I-protein_type +proteins I-protein_type +( O +GIMAPs B-protein_type +), O +also O +employ O +a O +GTP B-chemical +- O +dependent O +parallel B-protein_state +dimerization O +mode O +. O + +Based O +on O +phylogenetic B-experimental_method +and I-experimental_method +structural I-experimental_method +analysis I-experimental_method +, O +these O +observations O +suggest O +that O +dynamin B-protein_type +and O +septin B-protein_type +superfamilies O +are O +derived O +from O +a O +common O +ancestral O +membrane B-protein_type +- I-protein_type +associated I-protein_type +GTPase I-protein_type +that O +featured O +a O +GTP B-chemical +- O +dependent O +parallel B-protein_state +dimerization O +mode O +. O + +Importantly O +, O +our O +analysis O +indicates O +that O +IRGs B-protein_type +are O +not O +outliers O +, O +but O +bona O +- O +fide O +representatives O +of O +the O +dynamin B-protein_type +superfamily O +. O + +Whereas O +the O +overall O +dimerization O +mode O +is O +similar O +in O +septin B-protein_type +and O +dynamin B-protein_type +GTPases I-protein_type +, O +family O +- O +specific O +differences O +in O +the O +G B-site +interface I-site +and O +the O +oligomerization B-site +interfaces I-site +exist O +. O + +For O +example O +, O +the O +involvement O +of O +the O +2 O +’ O +and O +3 O +’- O +OH O +groups O +of O +the O +ribose B-chemical +in O +the O +dimerization B-site +interface I-site +of O +Irga6 B-protein +has O +not O +been O +observed O +for O +other O +dynamin B-protein_type +and O +septin B-protein_type +superfamily O +members O +. O + +The O +surface O +- O +exposed O +location O +of O +the O +ribose O +in O +the O +Irga6 B-protein +structure B-evidence +, O +with O +a O +wide B-protein_state +- I-protein_state +open I-protein_state +nucleotide B-site +- I-site +binding I-site +pocket I-site +, O +facilitates O +its O +engagement O +in O +the O +dimerization B-site +interface I-site +. O + +This O +contact O +, O +in O +turn O +, O +appears O +to O +activate O +GTP B-chemical +hydrolysis O +by O +inducing O +rearrangements O +in O +switch B-site +I I-site +and O +the O +positioning O +of O +the O +catalytic B-protein_state +E106 B-residue_name_number +. O + +During O +dimerization O +of O +GBP1 B-protein +, O +an O +arginine B-structure_element +finger I-structure_element +from O +the O +P B-structure_element +loop I-structure_element +reorients O +towards O +the O +nucleotide B-chemical +in O +cis O +to O +trigger O +GTP B-chemical +hydrolysis O +. O + +In O +dynamin B-protein_type +, O +the O +corresponding O +serine B-residue_name +residue O +coordinates O +a O +sodium B-chemical +ion O +that O +is O +crucial O +for O +GTP B-chemical +hydrolysis O +. O + +Irga6 B-protein +bears O +Gly79 B-residue_name_number +at O +this O +position O +, O +which O +in O +the O +dimerizing B-oligomeric_state +molecule O +A B-structure_element +appears O +to O +approach O +the O +bridging O +imido O +group O +of O +GMPPNP B-chemical +via O +a O +main O +chain O +hydrogen B-bond_interaction +bond I-bond_interaction +. O + +Higher O +resolution O +structures B-evidence +of O +the O +Irga6 B-protein +dimer B-oligomeric_state +in O +the O +presence B-protein_state +of I-protein_state +a O +transition O +state O +analogue O +are O +required O +to O +show O +whether O +Gly79 B-residue_name_number +directly O +participates O +in O +GTP B-chemical +hydrolysis O +or O +whether O +it O +may O +also O +position O +a O +catalytic O +cation O +. O + +In O +dynamin B-protein_type +, O +further O +assembly B-site +sites I-site +are O +provided O +by O +the O +helical B-structure_element +domains I-structure_element +which O +assemble O +in O +a O +criss O +- O +cross O +fashion O +to O +form O +a O +helical B-structure_element +filament I-structure_element +. O + +In O +dynamin B-protein_type +- I-protein_type +related I-protein_type +Eps15 I-protein_type +homology I-protein_type +domain I-protein_type +containing I-protein_type +proteins I-protein_type +( O +EHDs B-protein_type +), O +a O +second B-site +assembly I-site +interface I-site +is O +present O +in O +the O +GTPase B-structure_element +domain I-structure_element +. O + +For O +Irga6 B-protein +, O +additional O +interfaces B-site +in O +the O +helical B-structure_element +domain I-structure_element +are O +presumably O +involved O +in O +oligomerization O +, O +such O +as O +the O +secondary B-site +patch I-site +residues O +whose O +mutation B-experimental_method +prevented O +oligomerization O +in O +the O +crystallized B-evidence +mutant B-protein_state +. O + +Further O +structural B-experimental_method +studies I-experimental_method +, O +especially O +electron B-experimental_method +microscopy I-experimental_method +analysis I-experimental_method +of O +the O +Irga6 B-protein +oligomers B-oligomeric_state +, O +are O +required O +to O +clarify O +the O +assembly O +mode O +via O +the O +helical B-structure_element +domains I-structure_element +and O +to O +show O +how O +these O +interfaces B-site +cooperate O +with O +the O +G B-site +interface I-site +to O +mediate O +the O +regulated O +assembly O +on O +a O +membrane O +surface O +. O + +Notably O +, O +we O +did O +not O +observe O +major O +rearrangements O +of O +the O +helical B-structure_element +domain I-structure_element +versus O +the O +GTPase B-structure_element +domain I-structure_element +in O +the O +Irga6 B-protein +molecules O +that O +dimerized B-protein_state +via O +the O +G B-site +interface I-site +. O + +In O +a O +manner O +similar O +to O +BDLP B-protein_type +, O +such O +large O +- O +scale O +conformational O +changes O +may O +be O +induced O +by O +membrane O +binding O +. O + +Our O +structural B-experimental_method +analysis I-experimental_method +and O +the O +identification O +of O +the O +G B-site +- I-site +interface I-site +paves O +the O +way O +for O +determining O +the O +specific O +assembly O +of O +Irga6 B-protein +into O +a O +membrane O +- O +associated O +scaffold O +as O +the O +prerequisite O +to O +understand O +its O +action O +as O +an O +anti O +- O +parasitic O +machine O +. O + +Our O +study O +indicates O +that O +Irg B-protein_type +proteins O +dimerize B-oligomeric_state +via O +the O +G B-site +interface I-site +in O +a O +parallel B-protein_state +head I-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +head I-protein_state +fashion O +thereby O +facilitating O +GTPase B-protein_type +activation O +. O + +These O +findings O +contribute O +to O +a O +molecular O +understanding O +of O +the O +anti O +- O +parasitic O +action O +of O +the O +Irg B-protein_type +protein O +family O +and O +suggest O +that O +Irgs B-protein_type +are O +bona O +- O +fide O +members O +of O +the O +dynamin B-protein_type +superfamily O +. O + diff --git a/annotation_IOB/dev.tsv b/annotation_IOB/dev.tsv new file mode 100644 index 0000000000000000000000000000000000000000..4079fbfc7e4ab6c705dec84b69c3262fec520b0b --- /dev/null +++ b/annotation_IOB/dev.tsv @@ -0,0 +1,40483 @@ +The O +human B-species +gut O +microbiota B-taxonomy_domain +influences O +the O +course O +of O +human B-species +development O +and O +health O +, O +playing O +key O +roles O +in O +immune O +stimulation O +, O +intestinal O +cell O +proliferation O +, O +and O +metabolic O +balance O +. O + +The O +ability O +to O +acquire O +energy O +from O +carbohydrates B-chemical +of O +dietary O +or O +host O +origin O +is O +central O +to O +the O +adaptation O +of O +human B-species +gut O +bacterial B-taxonomy_domain +species O +to O +their O +niche O +. O + +Xyloglucan B-chemical +and O +the O +Bacteroides B-species +ovatus I-species +xyloglucan B-gene +utilization I-gene +locus I-gene +( O +XyGUL B-gene +). O +( O +A O +) O +Representative O +structures B-evidence +of O +common O +xyloglucans B-chemical +using O +the O +Consortium O +for O +Functional O +Glycomics O +Symbol O +Nomenclature O +( O +http O +:// O +www O +. O +functionalglycomics O +. O +org O +/ O +static O +/ O +consortium O +/ O +Nomenclature O +. O +shtml O +). O + +Whereas O +our O +previous O +study O +focused O +on O +the O +characterization O +of O +the O +linkage O +specificity O +of O +these O +GHs B-protein_type +, O +a O +key O +outstanding O +question O +regarding O +this O +locus O +is O +how O +XyG B-chemical +recognition O +is O +mediated O +at O +the O +cell O +surface O +. O + +Here O +, O +the O +SGBPs B-protein_type +very O +likely O +work O +in O +concert O +with O +the O +cell B-protein_type +- I-protein_type +surface I-protein_type +- I-protein_type +localized I-protein_type +endo I-protein_type +- I-protein_type +xyloglucanase I-protein_type +B B-species +. I-species +ovatus I-species +GH5 B-protein +( O +BoGH5 B-protein +) O +to O +recruit O +and O +cleave O +XyG B-chemical +for O +subsequent O +periplasmic O +import O +via O +the O +SusC B-protein_type +- I-protein_type +like I-protein_type +TBDT I-protein_type +of O +the O +XyGUL B-gene +( O +Fig O +. O +1B O +and O +C O +). O + +In O +our O +initial O +study O +focused O +on O +the O +functional O +characterization O +of O +the O +glycoside B-protein_type +hydrolases I-protein_type +of O +the O +XyGUL B-gene +, O +we O +reported O +preliminary O +affinity B-experimental_method +PAGE I-experimental_method +and O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +( O +ITC B-experimental_method +) O +data O +indicating O +that O +both O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +are O +competent O +xyloglucan B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +( O +affinity B-evidence +constant I-evidence +[ O +Ka B-evidence +] O +values O +of O +3 O +. O +74 O +× O +105 O +M O +− O +1 O +and O +4 O +. O +98 O +× O +104 O +M O +− O +1 O +, O +respectively O +[ O +23 O +]). O + +Together O +, O +these O +results O +highlight O +the O +high O +specificities O +of O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +for O +XyG B-chemical +, O +which O +is O +concordant O +with O +their O +association O +with O +XyG B-protein_type +- I-protein_type +specific I-protein_type +GHs I-protein_type +in O +the O +XyGUL B-gene +, O +as O +well O +as O +transcriptomic O +analysis O +indicating O +that O +B B-species +. I-species +ovatus I-species +has O +discrete O +PUL B-gene +for O +MLG B-chemical +, O +GM B-chemical +, O +and O +GGM B-chemical +( O +11 O +). O + +The O +apo B-protein_state +structure B-evidence +is O +color O +ramped O +from O +blue O +to O +red O +. O + +The O +approximate O +length O +of O +each O +glycan B-site +- I-site +binding I-site +site I-site +is O +displayed O +, O +colored O +to O +match O +the O +protein B-evidence +structures I-evidence +. O +( O +E O +) O +Stereo O +view O +of O +the O +xyloglucan B-site +- I-site +binding I-site +site I-site +of O +SGBP B-protein +- I-protein +A I-protein +, O +displaying O +all O +residues O +within O +4 O +Å O +of O +the O +ligand O +. O + +Potential O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +interactions I-bond_interaction +are O +shown O +as O +dashed O +lines O +, O +and O +the O +distance O +is O +shown O +in O +angstroms O +. O + +Dissection O +of O +the O +individual O +contribution O +of O +these O +residues O +reveals O +that O +the O +W82A B-mutant +mutant B-protein_state +displays O +a O +significant O +4 O +. O +9 O +- O +fold O +decrease O +in O +the O +Ka B-evidence +value O +for O +XyG B-chemical +, O +while O +the O +W306A B-mutant +substitution B-experimental_method +completely O +abolishes B-protein_state +XyG I-protein_state +binding I-protein_state +. O + +Prolines B-residue_name +between O +domains O +are O +indicated O +as O +spheres O +. O + +The O +backbone O +is O +flat O +, O +with O +less O +of O +the O +“ O +twisted O +- O +ribbon O +” O +geometry O +observed O +in O +some O +cello B-chemical +- I-chemical +and I-chemical +xylogluco I-chemical +- I-chemical +oligosaccharides I-chemical +. O + +Hoping O +to O +achieve O +a O +higher O +- O +resolution O +view O +of O +the O +SGBP B-protein +- I-protein +B I-protein +– O +xyloglucan B-chemical +interaction O +, O +we O +solved B-experimental_method +the O +crystal B-evidence +structure I-evidence +of O +the O +fused B-mutant +CD I-mutant +domains I-mutant +in B-protein_state +complex I-protein_state +with I-protein_state +XyGO2 B-chemical +( O +1 O +. O +57 O +Å O +, O +Rwork B-evidence += O +15 O +. O +6 O +%, O +Rfree B-evidence += O +17 O +. O +1 O +%, O +residues O +230 B-residue_range +to I-residue_range +489 I-residue_range +) O +( O +Table O +2 O +). O + +While O +this O +may O +occur O +for O +a O +number O +of O +reasons O +in O +crystal B-evidence +structures I-evidence +, O +it O +is O +likely O +that O +the O +poor O +ligand O +density O +even O +at O +higher O +resolution O +is O +due O +to O +movement O +or O +multiple O +orientations O +of O +the O +sugar B-chemical +averaged O +throughout O +the O +lattice O +. O + +The O +similarity O +of O +the O +glycan B-chemical +specificity O +of O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +presents O +an O +intriguing O +conundrum O +regarding O +their O +individual O +roles O +in O +XyG B-chemical +utilization O +by O +B B-species +. I-species +ovatus I-species +. O + +The O +ΔSGBP B-mutant +- I-mutant +A I-mutant +( O +ΔBacova_02651 B-mutant +) O +strain O +( O +cf O +. O + +The O +specific O +glycan B-chemical +signal O +that O +upregulates O +BoXyGUL B-gene +is O +currently O +unknown O +. O + +From O +our O +present O +data O +, O +we O +cannot O +eliminate O +the O +possibility O +that O +the O +glycan B-chemical +binding O +by O +SGBP B-protein +- I-protein +A I-protein +enhances O +transcriptional O +activation O +of O +the O +XyGUL B-gene +. O + +Beyond O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +, O +we O +speculated O +that O +the O +catalytically B-protein_state +feeble I-protein_state +endo B-protein_type +- I-protein_type +xyloglucanase I-protein_type +GH9 B-protein +, O +which O +is O +expendable O +for O +growth O +in O +the O +presence O +of O +GH5 B-protein +, O +might O +also O +play O +a O +role O +in O +glycan B-chemical +binding O +to O +the O +cell O +surface O +. O + +We O +hypothesize O +that O +during O +exponential O +growth O +the O +essential O +role O +of O +SGBP B-protein +- I-protein +A I-protein +extends O +beyond O +glycan B-chemical +recognition O +, O +perhaps O +due O +to O +a O +critical O +interaction O +with O +the O +TBDT B-protein_type +. O + +A O +particularly O +understudied O +aspect O +of O +glycan B-chemical +utilization O +is O +the O +mechanism O +of O +import O +via O +TBDTs B-protein_type +( O +SusC B-protein +homologs O +) O +( O +Fig O +. O +1 O +), O +which O +are O +ubiquitous O +and O +defining O +components O +of O +all O +PUL B-gene +. O + +Similarly O +, O +the O +deletion O +of O +BT1762 B-gene +encoding O +a O +fructan B-protein_type +- I-protein_type +targeting I-protein_type +SusD I-protein_type +- I-protein_type +like I-protein_type +protein I-protein_type +in O +B B-species +. I-species +thetaiotaomicron I-species +did O +not O +result O +in O +a O +dramatic O +loss O +of O +growth O +on O +fructans B-chemical +. O + +Furthermore O +, O +considering O +the O +broader O +distribution O +of O +TBDTs B-protein_type +in O +PUL B-gene +lacking O +SGBPs B-protein_type +( O +sometimes O +known O +as O +carbohydrate B-gene +utilization I-gene +containing I-gene +TBDT I-gene +[ I-gene +CUT I-gene +] I-gene +loci I-gene +; O +see O +reference O +and O +reviewed O +in O +reference O +) O +across O +bacterial B-taxonomy_domain +phyla O +, O +it O +appears O +that O +the O +intimate O +biophysical O +association O +of O +these O +substrate O +- O +transport O +and O +- O +binding O +proteins O +is O +the O +result O +of O +specific O +evolution O +within O +the O +Bacteroidetes B-taxonomy_domain +. O + +Equally O +intriguing O +is O +the O +observation O +that O +while O +SusD B-protein_type +- I-protein_type +like I-protein_type +proteins I-protein_type +such O +as O +SGBP B-protein +- I-protein +A I-protein +share O +moderate O +primary O +and O +high O +tertiary O +structural O +conservation O +, O +the O +genes O +for O +the O +SGBPs B-protein_type +encoded O +immediately O +downstream O +( O +Fig O +. O +1B O +[ O +sometimes O +referred O +to O +as O +“ O +susE O +positioned O +”]) O +encode O +glycan B-protein_type +- I-protein_type +binding I-protein_type +lipoproteins I-protein_type +with O +little O +or O +no O +sequence O +or O +structural O +conservation O +, O +even O +among O +syntenic O +PUL B-gene +that O +target O +the O +same O +polysaccharide B-chemical +. O + +Because O +the O +intestinal O +ecosystem O +is O +a O +dense O +consortium O +of O +bacteria B-taxonomy_domain +that O +must O +compete O +for O +their O +nutrients O +, O +these O +multimodular O +SGBPs B-protein_type +may O +reflect O +ongoing O +evolutionary O +experiments O +to O +enhance O +glycan B-chemical +uptake O +efficiency O +. O + +Whether O +organisms O +that O +express O +longer O +SGBPs B-protein_type +, O +extending O +further O +above O +the O +cell O +surface O +toward O +the O +extracellular O +environment O +, O +are O +better O +equipped O +to O +compete O +for O +available O +carbohydrates B-chemical +is O +presently O +unknown O +. O + +Monoclonal O +antibodies B-protein_type +inhibiting O +IL B-protein +- I-protein +17A I-protein +signaling O +have O +demonstrated O +remarkable O +efficacy O +, O +but O +an O +oral O +therapy O +is O +still O +lacking O +. O + +Tested O +in O +primary O +human B-species +cells O +, O +HAP B-chemical +blocked O +the O +production O +of O +multiple O +inflammatory O +cytokines B-protein_type +. O + +These O +polypeptides O +form O +covalent B-protein_state +homodimers B-oligomeric_state +, O +and O +IL B-protein +- I-protein +17A I-protein +and O +IL B-protein +- I-protein +17F I-protein +also O +form O +an O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17F I-complex_assembly +hetereodimer B-oligomeric_state +. O + +In O +these O +structures B-evidence +, O +both O +IL B-protein +- I-protein +17A I-protein +and O +IL B-protein +- I-protein +17F I-protein +adopt O +a O +cysteine B-structure_element +- I-structure_element +knot I-structure_element +fold O +with O +two O +intramolecular O +disulfides B-ptm +and O +two O +interchain O +disulfide B-ptm +bonds I-ptm +that O +covalently O +link O +two O +monomers B-oligomeric_state +. O + +Identification O +of O +IL B-protein +- I-protein +17A I-protein +peptide O +inhibitors O + +Peptides O +specifically O +binding O +to O +human B-species +IL B-protein +- I-protein +17A I-protein +were O +identified O +from O +phage B-experimental_method +panning I-experimental_method +using O +cyclic B-experimental_method +and I-experimental_method +linear I-experimental_method +peptide I-experimental_method +libraries I-experimental_method +( O +Supplementary O +Figure O +S1 O +). O + +The O +positive O +binding O +supernatants O +were O +tested O +for O +the O +ability O +to O +block O +biotinylated B-protein_state +IL B-protein +- I-protein +17A I-protein +signaling O +through O +IL B-protein +- I-protein +17RA I-protein +in O +an O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +competition B-experimental_method +ELISA I-experimental_method +assay I-experimental_method +where O +unlabeled O +IL B-protein +- I-protein +17A I-protein +was O +used O +as O +positive O +control O +to O +inhibit O +biotinylated B-protein_state +IL B-protein +- I-protein +17A I-protein +binding O +. O + +An O +alanine B-experimental_method +scan I-experimental_method +of O +peptide B-chemical +2 I-chemical +, O +an O +analogue O +of O +1 B-chemical +with O +a O +lysine B-residue_name +to O +arginine B-residue_name +substitution B-experimental_method +at O +position O +14 B-residue_number +, O +was O +initiated O +. O + +Modifications O +at O +positions O +2 B-residue_number +and O +14 B-residue_number +were O +shown O +to O +display O +improvement O +in O +binding B-evidence +affinity I-evidence +( O +data O +not O +shown O +). O + +In O +this O +work O +, O +32 B-chemical +– I-chemical +34 I-chemical +are O +capped B-protein_state +by O +protective O +acetyl O +group O +and O +reflect O +the O +same O +inactivity O +as O +reported O +. O + +Peptide B-chemical +45 I-chemical +, O +dimerized B-oligomeric_state +via O +attachment O +of O +a O +PEG21 B-chemical +spacer O +at O +position O +14 B-residue_number +( O +Supplementary O +Scheme O +S1 O +and O +Figure O +S3 O +), O +was O +the O +most O +potent O +with O +cellular O +IC50 B-evidence +of O +0 O +. O +1 O +nM O +. O +This O +significant O +improvement O +in O +antagonism O +was O +not O +seen O +in O +the O +peptide O +monomer B-oligomeric_state +functionalized O +with O +a O +PEG21 B-chemical +group O +at O +position O +14 B-residue_number +as O +peptide B-chemical +48 I-chemical +had O +an O +IC50 B-evidence +of O +21 O +nM O +( O +Supplementary O +Scheme O +S2 O +). O + +To O +further O +characterize O +the O +interaction O +of O +HAP B-chemical +with O +IL B-protein +- I-protein +17A I-protein +, O +we O +set O +out O +to O +determine O +its O +in O +vitro O +binding B-evidence +affinity I-evidence +, O +specificity O +and O +kinetic B-evidence +profile I-evidence +using O +Surface B-experimental_method +Plasmon I-experimental_method +Resonance I-experimental_method +( O +SPR B-experimental_method +) O +methods O +( O +Fig O +. O +1A O +). O + +HAP B-chemical +blocks O +IL B-protein +- I-protein +17A I-protein +signaling O +in O +a O +human B-species +primary O +cell O +assay O + +In O +patients O +, O +the O +concentration O +of O +IL B-protein +- I-protein +17A I-protein +in O +psoriatic O +lesions O +is O +reported O +to O +be O +0 O +. O +01 O +ng O +/ O +ml O +, O +well O +below O +the O +EC50 O +( O +5 O +– O +10ng O +/ O +ml O +) O +of O +IL B-protein +- I-protein +17A I-protein +induced O +IL B-protein_type +- I-protein_type +8 I-protein_type +production O +in O +vitro O +. O + +It O +is O +known O +that O +an O +antibody B-protein_type +antigen B-structure_element +- I-structure_element +binding I-structure_element +fragment I-structure_element +( O +Fab B-structure_element +) O +can O +be O +used O +as O +crystallization O +chaperones O +in O +crystallizing O +difficult O +targets O +. O + +Furthermore O +, O +since O +it O +binds O +to O +an O +area O +far O +away O +from O +that O +of O +HAP B-chemical +( O +see O +below O +), O +this O +Fab B-structure_element +should O +have O +minimum O +effects O +on O +HAP B-chemical +binding O +conformation O +. O + +Crystals B-evidence +of O +Fab B-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +ternary O +complex O +were O +obtained O +readily O +in O +crystallization B-experimental_method +screens I-experimental_method +. O + +Crystallization B-experimental_method +of O +IL B-protein +- I-protein +17A I-protein +and O +its O +binding O +partners O +was O +accomplished O +using O +two O +forms O +of O +IL B-protein +- I-protein +17A I-protein +. O + +Both O +structures B-evidence +were O +solved O +by O +molecular B-experimental_method +replacement I-experimental_method +. O + +The O +C O +- O +terminal O +8 B-residue_range +residues I-residue_range +of O +the O +HAP B-chemical +that O +are O +ordered O +in O +the O +structure B-evidence +, O +7ADLWDWIN B-chemical +, O +form O +an O +amphipathic B-protein_state +α B-structure_element +- I-structure_element +helix I-structure_element +interacting O +with O +the O +second O +IL B-protein +- I-protein +17A I-protein +monomer B-oligomeric_state +. O + +Pro6 B-residue_name_number +of O +HAP B-chemical +makes O +a O +transition O +between O +the O +N O +- O +terminal O +β B-structure_element +- I-structure_element +strand I-structure_element +and O +the O +C O +- O +terminal O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +HAP B-chemical +. O + +Conformational O +changes O +in O +region B-structure_element +I I-structure_element +induced O +by O +HAP B-chemical +binding O +alone O +may O +allosterically O +affect O +IL B-protein +- I-protein +17RA I-protein +binding O +, O +but O +more O +importantly O +, O +the O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +HAP B-chemical +directly O +competes O +with O +IL B-protein +- I-protein +17RA I-protein +for O +binding O +to O +IL B-protein +- I-protein +17A I-protein +( O +Fig O +. O +3 O +). O + +However O +, O +it O +mimics O +the O +β B-structure_element +- I-structure_element +strand I-structure_element +0 I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +. O + +Conformational O +changes O +of O +IL B-protein +- I-protein +17A I-protein +are O +needed O +for O +both O +HAP B-chemical +and O +IL B-protein +- I-protein +17RA I-protein +to O +bind O +to O +that O +region O +. O + +During O +IL B-protein +- I-protein +17A I-protein +signaling O +, O +IL B-protein +- I-protein +17A I-protein +binds O +to O +one O +copy O +of O +IL B-protein +- I-protein +17RA I-protein +and O +one O +copy O +of O +IL B-protein +- I-protein +17RC I-protein +. O + +HAP B-chemical +, O +with O +only O +15 B-residue_range +residues I-residue_range +, O +can O +achieve O +almost O +the O +same O +binding B-evidence +affinity I-evidence +as O +the O +much O +larger O +IL B-protein +- I-protein +17RA I-protein +molecule O +, O +indicating O +a O +more O +efficient O +way O +of O +binding O +to O +IL B-protein +- I-protein +17A I-protein +. O + +As O +demonstrated O +by O +the O +crystal B-evidence +structure I-evidence +, O +binding O +of O +the O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +HAP B-chemical +should O +be O +sufficient O +for O +preventing O +IL B-protein +- I-protein +17RA I-protein +binding O +to O +IL B-protein +- I-protein +17A I-protein +. O + +Theoretically O +, O +it O +is O +possible O +to O +design O +chemicals O +such O +as O +stapled O +α O +- O +helical O +peptides O +to O +block O +α B-structure_element +- I-structure_element +helix I-structure_element +- O +mediated O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +interactions O +. O + +( O +A O +) O +HAP B-chemical +binds O +at O +region B-structure_element +I I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +. O + +Polar B-bond_interaction +interactions I-bond_interaction +are O +shown O +in O +dashes O +. O + +Notice O +that O +the O +Trp B-site +binding I-site +pocket I-site +for O +W12 B-residue_name_number +of O +HAP B-chemical +or O +W31 B-residue_name_number +of O +IL B-protein +- I-protein +17RA I-protein +is O +missing O +in O +the O +apo B-protein_state +structure B-evidence +. O + +It O +is O +therefore O +important O +to O +understand O +the O +mechanisms O +which O +regulate O +nadA B-gene +expression O +levels O +, O +which O +are O +predominantly O +controlled O +by O +the O +transcriptional B-protein_type +regulator I-protein_type +NadR B-protein +( O +Neisseria B-protein +adhesin I-protein +A I-protein +Regulator I-protein +) O +both O +in O +vitro O +and O +in O +vivo O +. O + +NadR B-protein +binds O +the O +nadA B-gene +promoter O +and O +represses O +gene O +transcription O +. O + +Serogroup B-taxonomy_domain +B I-taxonomy_domain +meningococcus I-taxonomy_domain +( O +MenB B-species +) O +causes O +fatal O +sepsis O +and O +invasive O +meningococcal B-taxonomy_domain +disease O +, O +particularly O +in O +young O +children O +and O +adolescents O +, O +as O +highlighted O +by O +recent O +MenB B-species +outbreaks O +in O +universities O +of O +the O +United O +States O +and O +Canada O +. O + +The O +amount O +of O +NadA B-protein +exposed O +on O +the O +meningococcal B-taxonomy_domain +surface O +also O +influences O +the O +antibody O +- O +mediated O +serum O +bactericidal O +response O +measured O +in O +vitro O +. O + +Although O +additional O +factors O +influence O +nadA B-gene +expression O +, O +we O +focused O +on O +its O +regulation O +by O +NadR B-protein +, O +the O +major O +mediator O +of O +NadA B-protein +phase O +variable O +expression O +. O + +However O +, O +the O +homologous O +archeal B-taxonomy_domain +Sulfolobus B-species +tokodaii I-species +protein O +ST1710 B-protein +presented O +essentially O +the O +same O +structure B-evidence +in O +ligand B-protein_state +- I-protein_state +free I-protein_state +and O +salicylate B-protein_state +- I-protein_state +bound I-protein_state +forms O +, O +apparently O +contrasting O +the O +mechanism O +proposed O +for O +MTH313 B-protein +. O + +Despite O +these O +apparent O +differences O +, O +MTH313 B-protein +and O +ST1710 B-protein +bind O +salicylate B-chemical +in O +approximately O +the O +same O +site O +, O +between O +their O +dimerization B-structure_element +and I-structure_element +DNA I-structure_element +- I-structure_element +binding I-structure_element +domains I-structure_element +. O + +We O +obtained O +detailed O +new O +insights O +into O +ligand O +specificity O +, O +how O +the O +ligand O +allosterically O +influences O +the O +DNA O +- O +binding O +ability O +of O +NadR B-protein +, O +and O +the O +regulation O +of O +nadA B-gene +expression O +, O +thus O +also O +providing O +a O +deeper O +structural O +understanding O +of O +the O +ligand O +- O +responsive O +MarR B-protein_type +super O +- O +family O +. O + +NadR B-protein +is O +dimeric B-oligomeric_state +and O +is O +stabilized O +by O +specific O +hydroxyphenylacetate B-chemical +ligands O + +Recombinant O +NadR B-protein +was O +produced O +in O +E B-species +. I-species +coli I-species +using O +an O +expression B-experimental_method +construct I-experimental_method +prepared O +from O +N B-species +. I-species +meningitidis I-species +serogroup I-species +B I-species +strain I-species +MC58 I-species +. O + +Since O +ligand O +- O +binding O +often O +increases O +protein O +stability O +, O +we O +also O +investigated O +the O +effect O +of O +various O +HPAs B-chemical +( O +Fig O +1A O +) O +on O +the O +melting B-evidence +temperature I-evidence +( O +Tm B-evidence +) O +of O +NadR B-protein +. O +As O +a O +control O +of O +specificity O +, O +we O +also O +tested O +salicylate B-chemical +, O +a O +known O +ligand O +of O +some O +MarR B-protein_type +proteins O +previously O +reported O +to O +increase O +the O +Tm B-evidence +of O +ST1710 B-protein +and O +MTH313 B-protein +. O + +3 B-chemical +- I-chemical +HPA I-chemical +70 O +. O +0 O +± O +0 O +. O +1 O +2 O +. O +7 O +2 O +. O +7 O +± O +0 O +. O +1 O +4 B-chemical +- I-chemical +HPA I-chemical +70 O +. O +7 O +± O +0 O +. O +1 O +3 O +. O +3 O +1 O +. O +5 O +± O +0 O +. O +1 O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +71 O +. O +3 O +± O +0 O +. O +2 O +3 O +. O +9 O +1 O +. O +1 O +± O +0 O +. O +1 O + +To O +further O +investigate O +the O +binding O +of O +HPAs B-chemical +to O +NadR B-protein +, O +we O +used O +surface B-experimental_method +plasmon I-experimental_method +resonance I-experimental_method +( O +SPR B-experimental_method +). O + +Data O +collection O +and O +refinement O +statistics O +for O +NadR B-protein +structures B-evidence +. O + +In O +the O +apo B-protein_state +- O +NadR B-protein +crystals B-evidence +, O +the O +two O +homodimers B-oligomeric_state +were O +related O +by O +a O +rotation O +of O +~ O +90 O +°; O +the O +observed O +association O +of O +the O +two O +dimers B-oligomeric_state +was O +presumably O +merely O +an O +effect O +of O +crystal O +packing O +, O +since O +the O +interface B-site +between O +the O +two O +homodimers B-oligomeric_state +is O +small O +(< O +550 O +Å2 O +of O +buried O +surface O +area O +), O +and O +is O +not O +predicted O +to O +be O +physiologically O +relevant O +by O +the O +PISA O +software O +. O + +Helices B-structure_element +α3 B-structure_element +and O +α4 B-structure_element +form O +a O +helix B-structure_element +- I-structure_element +turn I-structure_element +- I-structure_element +helix I-structure_element +motif I-structure_element +, O +followed O +by O +the O +“ O +wing B-structure_element +motif I-structure_element +” O +comprised O +of O +two O +short B-structure_element +antiparallel I-structure_element +β I-structure_element +- I-structure_element +strands I-structure_element +( O +β1 B-structure_element +- I-structure_element +β2 I-structure_element +) O +linked O +by O +a O +relatively O +long O +and O +flexible O +loop B-structure_element +. O + +Interestingly O +, O +in O +the O +α4 B-structure_element +- I-structure_element +β2 I-structure_element +region I-structure_element +, O +the O +stretch O +of O +residues O +from O +R64 B-residue_range +- I-residue_range +R91 I-residue_range +presents O +seven O +positively O +- O +charged O +side O +chains O +, O +all O +available O +for O +potential O +interactions O +with O +DNA B-chemical +. O + +Using O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +, O +a O +panel O +of O +eight O +mutant B-protein_state +NadR B-protein +proteins O +was O +prepared O +( O +including O +mutations O +H7A B-mutant +, O +S9A B-mutant +, O +N11A B-mutant +, O +D112A B-mutant +, O +R114A B-mutant +, O +Y115A B-mutant +, O +K126A B-mutant +, O +L130K B-mutant +and O +L133K B-mutant +), O +sufficient O +to O +explore O +the O +entire O +dimer B-site +interface I-site +. O + +It O +is O +notable O +that O +L130 B-residue_name_number +is O +usually O +present O +as O +Leu B-residue_name +, O +or O +an O +alternative O +bulky O +hydrophobic O +amino O +acid O +( O +e O +. O +g O +. O +Phe B-residue_name +, O +Val B-residue_name +), O +in O +many O +MarR B-protein_type +family O +proteins O +, O +suggesting O +a O +conserved B-protein_state +role O +in O +stabilizing O +the O +dimer B-site +interface I-site +. O + +The O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +structure B-evidence +revealed O +the O +ligand B-site +- I-site +binding I-site +site I-site +nestled O +between O +the O +dimerization B-structure_element +and I-structure_element +DNA I-structure_element +- I-structure_element +binding I-structure_element +domains I-structure_element +( O +Fig O +2 O +). O + +The O +binding B-site +pocket I-site +was O +almost O +entirely O +filled O +by O +4 B-chemical +- I-chemical +HPA I-chemical +and O +one O +water B-chemical +molecule O +, O +although O +there O +also O +remained O +a O +small O +tunnel B-site +2 O +- O +4Å O +in O +diameter O +and O +5 O +- O +6Å O +long O +leading O +from O +the O +pocket B-site +( O +proximal O +to O +the O +4 O +- O +hydroxyl O +position O +) O +to O +the O +protein O +surface O +. O + +Green O +and O +blue O +ribbons O +depict O +NadR B-protein +chains B-structure_element +A I-structure_element +and I-structure_element +B I-structure_element +, O +respectively O +. O + +Residues O +AsnA11 B-residue_name_number +and O +ArgB18 B-residue_name_number +likely O +make O +indirect O +yet O +local O +contributions O +to O +ligand O +binding O +, O +mainly O +by O +stabilizing O +the O +position O +of O +AspB36 B-residue_name_number +. O + +List O +of O +4 B-chemical +- I-chemical +HPA I-chemical +atoms O +bound O +to O +NadR B-protein +via O +ionic B-bond_interaction +interactions I-bond_interaction +and O +/ O +or O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +. O + +4 B-chemical +- I-chemical +HPA I-chemical +atom O +NadR B-protein +residue O +/ O +atom O +Distance O +( O +Å O +) O +O2 O +TrpB39 B-residue_name_number +/ O +NE1 O +2 O +. O +83 O +O2 O +ArgB43 B-residue_name_number +/ O +NH1 O +2 O +. O +76 O +O1 O +ArgB43 B-residue_name_number +/ O +NH1 O +3 O +. O +84 O +O1 O +SerA9 B-residue_name_number +/ O +OG O +2 O +. O +75 O +O1 O +TyrB115 B-residue_name_number +/ O +OH O +2 O +. O +50 O +O2 O +Water B-chemical +(* O +Ser9 B-residue_name_number +/ O +Asn11 B-residue_name_number +) O +2 O +. O +88 O +OH O +AspB36 B-residue_name_number +/ O +OD1 O +/ O +OD2 O +3 O +. O +6 O +/ O +3 O +. O +7 O + +* O +Bond O +distance O +between O +the O +ligand O +carboxylate O +group O +and O +the O +water B-chemical +molecule O +, O +which O +in O +turn O +makes O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +to O +the O +SerA9 B-residue_name_number +and O +AsnA11 B-residue_name_number +side O +chains O +. O + +In O +SPR B-experimental_method +, O +the O +signal O +measured O +is O +proportional O +to O +the O +total O +molecular O +mass O +proximal O +to O +the O +sensor O +surface O +; O +consequently O +, O +if O +the O +molecular O +weights O +of O +the O +interactors O +are O +known O +, O +then O +the O +stoichiometry O +of O +the O +resulting O +complex O +can O +be O +determined O +. O + +The O +stoichiometry O +of O +the O +NadR B-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +interactions O +was O +determined O +using O +Eq O +1 O +( O +see O +Materials O +and O +Methods O +), O +and O +revealed O +stoichiometries B-evidence +of O +1 O +. O +13 O +for O +4 B-chemical +- I-chemical +HPA I-chemical +, O +1 O +. O +02 O +for O +3 B-chemical +- I-chemical +HPA I-chemical +, O +and O +1 O +. O +21 O +for O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +, O +strongly O +suggesting O +that O +one O +NadR B-protein +dimer B-oligomeric_state +bound B-protein_state +to I-protein_state +1 O +HPA B-chemical +analyte O +molecule O +. O + +To O +explore O +the O +molecular O +basis O +of O +asymmetry O +in O +holo B-protein_state +- O +NadR B-protein +, O +we O +superposed B-experimental_method +its O +ligand B-protein_state +- I-protein_state +free I-protein_state +monomer B-oligomeric_state +( O +chain B-structure_element +A I-structure_element +) O +onto O +the O +ligand B-protein_state +- I-protein_state +occupied I-protein_state +monomer B-oligomeric_state +( O +chain B-structure_element +B I-structure_element +). O + +Indeed O +, O +we O +noted O +interesting O +differences O +in O +the O +side O +chains O +of O +Met22 B-residue_name_number +, O +Phe25 B-residue_name_number +and O +Arg43 B-residue_name_number +, O +which O +in O +monomer B-oligomeric_state +B B-structure_element +are O +used O +to O +contact O +the O +ligand O +while O +in O +monomer B-oligomeric_state +A B-structure_element +they O +partially O +occupied O +the O +pocket B-site +and O +collectively O +reduced O +its O +volume O +significantly O +. O + +In O +contrast O +, O +the O +apo B-protein_state +- O +form O +Met22 B-residue_name_number +and O +Phe25 B-residue_name_number +residues O +were O +still O +encroaching O +the O +spaces O +of O +the O +4 O +- O +hydroxyl O +group O +and O +the O +phenyl O +ring O +of O +the O +ligand O +, O +respectively O +( O +Fig O +5C O +). O + +The O +‘ O +outward B-protein_state +’ O +position O +of O +Arg43 B-residue_name_number +generated O +an O +open B-protein_state +apo B-protein_state +- O +form O +pocket B-site +with O +volume O +approximately O +380Å3 O +. O + +Taken O +together O +, O +these O +observations O +suggest O +that O +Arg43 B-residue_name_number +is O +a O +major O +determinant O +of O +ligand O +binding O +, O +and O +that O +its O +‘ O +inward B-protein_state +’ O +position O +inhibits O +the O +binding O +of O +4 B-chemical +- I-chemical +HPA I-chemical +to O +the O +empty O +pocket B-site +of O +holo B-protein_state +- O +NadR B-protein +. O + +The O +inner O +conformer O +is O +the O +one O +that O +would O +display O +major O +clashes O +if O +4 B-chemical +- I-chemical +HPA I-chemical +were O +present O +. O +( O +C O +) O +Comparison O +of O +the O +empty O +pocket B-site +from O +holo B-protein_state +- O +NadR B-protein +( O +green O +residues O +) O +with O +the O +four O +empty O +pockets B-site +of O +apo B-protein_state +- O +NadR B-protein +( O +grey O +residues O +), O +shows O +that O +in O +the O +absence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +the O +Arg43 B-residue_name_number +side O +chain O +is O +always O +observed O +in O +the O +‘ O +outward B-protein_state +’ O +conformation O +. O + +The O +broad O +spectral O +dispersion O +and O +the O +number O +of O +peaks O +observed O +, O +which O +is O +close O +to O +the O +number O +of O +expected O +backbone O +amide O +N O +- O +H O +groups O +for O +this O +polypeptide O +, O +confirmed O +that O +apo B-protein_state +- O +NadR B-protein +is O +well B-protein_state +- I-protein_state +folded I-protein_state +under O +these O +conditions O +and O +exhibits O +one O +conformation O +appreciable O +on O +the O +NMR B-experimental_method +timescale O +, O +i O +. O +e O +. O +in O +the O +NMR B-experimental_method +experiments O +at O +25 O +° O +C O +, O +two O +or O +more O +distinct O +conformations O +of O +apo B-protein_state +- O +NadR B-protein +monomers B-oligomeric_state +were O +not O +readily O +apparent O +. O + +( O +B O +, O +C O +) O +Overlay B-experimental_method +of O +selected O +regions O +of O +the O +1H B-experimental_method +- I-experimental_method +15N I-experimental_method +TROSY I-experimental_method +- I-experimental_method +HSQC I-experimental_method +spectra B-evidence +acquired O +at O +25 O +° O +C O +of O +apo B-protein_state +- O +NadR B-protein +( O +cyan O +) O +and O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +( O +red O +) O +superimposed B-experimental_method +with O +the O +spectra B-evidence +acquired O +at O +10 O +° O +C O +of O +apo B-protein_state +- O +NadR B-protein +( O +blue O +) O +and O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +( O +green O +). O + +Considering O +the O +small O +size O +, O +fast O +diffusion O +and O +relatively O +low O +binding B-evidence +affinity I-evidence +of O +4 B-chemical +- I-chemical +HPA I-chemical +, O +it O +would O +not O +be O +surprising O +if O +the O +ligand O +associates O +and O +dissociates O +rapidly O +on O +the O +NMR B-experimental_method +time O +scale O +, O +resulting O +in O +only O +one O +set O +of O +peaks O +whose O +chemical O +shifts O +represent O +the O +average O +environment O +of O +the O +bound B-protein_state +and O +unbound B-protein_state +states O +. O + +Interestingly O +, O +by O +cooling O +the O +samples O +to O +10 O +° O +C O +, O +we O +observed O +that O +a O +number O +of O +those O +peaks O +strongly O +affected O +by O +4 B-chemical +- I-chemical +HPA I-chemical +( O +and O +therefore O +likely O +to O +be O +in O +the O +ligand B-site +- I-site +binding I-site +site I-site +) O +demonstrated O +evidence O +of O +peak O +splitting O +, O +i O +. O +e O +. O +a O +tendency O +to O +become O +two O +distinct O +peaks O +rather O +than O +one O +single O +peak O +( O +Fig O +6B O +and O +6C O +). O + +Similarly O +, O +the O +entire O +holo B-protein_state +- O +homodimer B-oligomeric_state +could O +be O +closely B-experimental_method +superposed I-experimental_method +onto O +each O +of O +the O +apo B-protein_state +- O +homodimers B-oligomeric_state +, O +showing O +rmsd B-evidence +values O +of O +1 O +. O +29Å O +and O +1 O +. O +31Å O +, O +and O +with O +more O +notable O +differences O +in O +the O +α6 B-structure_element +helix I-structure_element +positions O +( O +Fig O +7B O +). O + +Structural B-experimental_method +comparisons I-experimental_method +of O +NadR B-protein +and O +modelling O +of O +interactions O +with O +DNA B-chemical +. O + +However O +, O +structural B-experimental_method +comparisons I-experimental_method +revealed O +that O +the O +shift O +of O +holo B-protein_state +- O +NadR B-protein +helix B-structure_element +α4 B-structure_element +induced O +by O +the O +presence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +was O +also O +accompanied O +by O +several O +changes O +at O +the O +holo B-protein_state +dimer B-site +interface I-site +, O +while O +such O +extensive O +structural O +differences O +were O +not O +observed O +in O +the O +apo B-protein_state +dimer B-site +interfaces I-site +, O +particularly O +notable O +when O +comparing O +the O +α6 B-structure_element +helices I-structure_element +( O +S3 O +Fig O +). O + +Interestingly O +, O +OhrR B-protein +contacts O +ohrA B-gene +across O +22 O +base O +pairs O +( O +bp O +), O +and O +similarly O +the O +main O +NadR B-protein +target B-site +sites I-site +identified O +in O +the O +nadA B-gene +promoter O +( O +the O +operators O +Op O +I O +and O +Op O +II O +) O +both O +span O +22 O +bp O +. O + +When O +aligned B-experimental_method +with O +OhrR B-protein +, O +the O +apo B-protein_state +- O +homodimer B-oligomeric_state +CD B-structure_element +presented O +yet O +another O +different O +intermediate O +conformation O +( O +rmsd B-evidence +2 O +. O +9Å O +), O +apparently O +not O +ideally O +pre O +- O +configured O +for O +DNA B-chemical +binding O +, O +but O +which O +in O +solution O +can O +presumably O +readily O +adopt O +the O +AB B-structure_element +conformation O +due O +to O +the O +intrinsic O +flexibility O +described O +above O +. O + +Western B-experimental_method +blot I-experimental_method +analyses O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +strain O +( O +lanes O +1 O +– O +2 O +) O +or O +isogenic O +nadR B-gene +knockout O +strains O +( O +ΔNadR B-mutant +) O +complemented O +to O +express O +the O +indicated O +NadR B-protein +WT B-protein_state +or O +mutant B-protein_state +proteins O +( O +lanes O +3 O +– O +12 O +) O +or O +not O +complemented O +( O +lanes O +13 O +– O +14 O +), O +grown O +in O +the O +presence O +( O +even O +lanes O +) O +or O +absence O +( O +odd O +lanes O +) O +of O +5mM O +4 B-chemical +- I-chemical +HPA I-chemical +, O +showing O +NadA B-protein +and O +NadR B-protein +expression O +. O + +The O +H7A B-mutant +, O +S9A B-mutant +and O +F25A B-mutant +mutants O +efficiently O +repress O +nadA B-gene +expression O +but O +are O +less O +ligand O +- O +responsive O +than O +WT B-protein_state +NadR B-protein +. O +The O +N11A B-mutant +mutant B-protein_state +does O +not O +efficiently O +repress O +nadA B-gene +expression O +either O +in O +presence O +or O +absence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +. O +( O +The O +protein O +abundance O +levels O +of O +the O +meningococcal B-taxonomy_domain +factor B-protein +H I-protein +binding I-protein +protein I-protein +( O +fHbp B-protein +) O +were O +used O +as O +a O +gel O +loading O +control O +). O + +NadA B-protein +is O +a O +surface O +- O +exposed O +meningococcal B-taxonomy_domain +protein O +contributing O +to O +pathogenesis O +, O +and O +is O +one O +of O +three O +main O +antigens O +present O +in O +the O +vaccine O +Bexsero O +. O + +We O +confirmed O +this O +stoichiometry O +in O +solution O +using O +SPR B-experimental_method +methods O +. O + +Structural B-experimental_method +analyses I-experimental_method +suggested O +that O +‘ O +inward B-protein_state +’ O +side O +chain O +positions O +of O +Met22 B-residue_name_number +, O +Phe25 B-residue_name_number +and O +especially O +Arg43 B-residue_name_number +precluded O +binding O +of O +a O +second O +ligand O +molecule O +. O + +In O +the O +S B-species +. I-species +tokodaii I-species +protein O +ST1710 B-protein +, O +salicylate B-chemical +binds O +to O +the O +same O +position O +in O +each O +monomer B-oligomeric_state +of O +the O +dimer B-oligomeric_state +, O +in O +a O +site O +equivalent O +to O +the O +putative O +biologically O +relevant O +site O +of O +MTH313 B-protein +( O +Fig O +10B O +). O + +Unlike O +other O +MarR B-protein_type +family O +proteins O +which O +revealed O +multiple O +ligand O +binding O +interactions O +, O +we O +observed O +only O +1 O +molecule O +of O +4 B-chemical +- I-chemical +HPA I-chemical +bound B-protein_state +to I-protein_state +NadR B-protein +, O +suggesting O +a O +more O +specific O +and O +less O +promiscuous O +interaction O +. O + +NadR B-protein +shows O +a O +ligand B-site +binding I-site +site I-site +distinct O +from O +other O +MarR B-protein_type +homologues O +. O + +( O +B O +) O +A O +structural B-experimental_method +alignment I-experimental_method +of O +MTH313 B-protein +chain B-structure_element +A I-structure_element +and O +ST1710 B-protein +( O +pink O +) O +( O +Cα O +rmsd B-evidence +2 O +. O +3Å O +), O +shows O +that O +they O +bind O +salicylate B-chemical +in O +equivalent O +sites O +( O +differing O +by O +only O +~ O +3Å O +) O +and O +with O +the O +same O +orientation O +. O + +Alternatively O +, O +it O +is O +possible O +that O +other O +MarR B-protein_type +homologues O +( O +e O +. O +g O +. O +NMB1585 B-protein +) O +may O +have O +no O +extant O +functional O +binding B-site +pocket I-site +and O +thus O +may O +have O +lost O +the O +ability O +to O +respond O +to O +a O +ligand O +, O +acting O +instead O +as O +constitutive O +DNA B-chemical +- O +binding O +regulatory O +proteins O +. O + +The O +noted O +flexibility O +may O +also O +explain O +how O +NadR B-protein +can O +adapt O +to O +bind O +various O +DNA B-chemical +target O +sequences O +with O +slightly O +different O +structural O +features O +. O + +Like O +other O +nuclear B-protein_type +hormone I-protein_type +receptors I-protein_type +, O +RORγ B-protein +’ O +s O +helix12 B-structure_element +which O +makes O +up O +the O +C O +- O +termini O +of O +the O +LBD B-structure_element +is O +an O +essential O +part O +of O +the O +coactivator B-site +binding I-site +pocket I-site +and O +is O +commonly O +referred O +to O +as O +the O +activation B-structure_element +function I-structure_element +helix I-structure_element +2 I-structure_element +( O +AF2 B-structure_element +). O + +FRET B-evidence +results I-evidence +for O +agonist B-protein_state +BIO592 B-chemical +( O +a O +) O +and O +Inverse B-protein_state +Agonist I-protein_state +BIO399 B-chemical +( O +b O +) O + +a O +The O +ternary B-evidence +structure I-evidence +of O +RORγ518 B-protein +BIO592 B-chemical +and O +EBI96 B-chemical +. O + +b O +RORγ B-protein +AF2 B-structure_element +helix I-structure_element +in O +the O +agonist B-protein_state +conformation O +. O + +The O +structure B-evidence +of O +the O +ternary O +complex O +had O +features O +similar O +to O +other O +ROR B-protein_type +agonist B-protein_state +coactivator O +structures B-evidence +in O +a O +transcriptionally B-protein_state +active I-protein_state +canonical B-protein_state +three I-protein_state +layer I-protein_state +helix I-protein_state +fold I-protein_state +with O +the O +AF2 B-structure_element +helix I-structure_element +in O +the O +agonist B-protein_state +conformation O +. O + +The O +agonist B-protein_state +conformation O +is O +stabilized O +by O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +His479 B-residue_name_number +and O +Tyr502 B-residue_name_number +, O +in O +addition O +to O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +interactions I-bond_interaction +between O +His479 B-residue_name_number +, O +Tyr502 B-residue_name_number +and O +Phe506 B-residue_name_number +( O +Fig O +. O +2b O +). O + +Electron B-evidence +density I-evidence +for O +the O +coactivator O +peptide O +EBI96 B-chemical +was O +observed O +for O +residues O +EFPYLLSLLG B-structure_element +which O +formed O +a O +α B-structure_element +- I-structure_element +helix I-structure_element +stabilized O +through O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +the O +coactivator B-site +binding I-site +pocket I-site +on O +RORγ B-protein +( O +Fig O +. O +2c O +). O + +b O +Benzoxazinone B-chemical +ring O +system O +of O +agonist B-protein_state +BIO592 B-chemical +packing O +against O +His479 B-residue_name_number +of O +RORγ B-protein +stabilizing O +agonist B-protein_state +conformation O +of O +the O +AF2 B-structure_element +helix I-structure_element + +BIO592 B-chemical +bound B-protein_state +in I-protein_state +a O +collapsed B-protein_state +conformational O +state O +in O +the O +LBS B-site +of O +RORγ B-protein +with O +the O +xylene B-chemical +ring O +positioned O +at O +the O +bottom O +of O +the O +pocket B-site +making O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +Val376 B-residue_name_number +, O +Phe378 B-residue_name_number +, O +Phe388 B-residue_name_number +and O +Phe401 B-residue_name_number +, O +with O +the O +ethyl B-chemical +- I-chemical +benzoxazinone I-chemical +ring O +making O +several O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +Trp317 B-residue_name_number +, O +Leu324 B-residue_name_number +, O +Met358 B-residue_name_number +, O +Leu391 B-residue_name_number +, O +Ile B-residue_name_number +400 I-residue_name_number +and O +His479 B-residue_name_number +( O +Fig O +. O +3a O +, O +Additional O +file O +3 O +). O + +Hydrophobic B-bond_interaction +interaction I-bond_interaction +between O +the O +ethyl O +group O +of O +the O +benzoxazinone B-chemical +and O +His479 B-residue_name_number +reinforce O +the O +His479 B-residue_name_number +sidechain O +position O +for O +making O +the O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +Tyr502 B-residue_name_number +thereby O +stabilizing O +the O +agonist B-protein_state +conformation O +( O +Fig O +. O +3b O +). O + +However O +, O +in O +the O +presence B-protein_state +of I-protein_state +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +, O +the O +proteolytic B-evidence +pattern I-evidence +showed O +significantly O +less O +protection O +, O +albeit O +the O +products O +were O +more O +heterogeneous O +( O +majority O +ending O +at O +494 B-residue_number +/ O +495 B-residue_number +), O +indicating O +the O +destabilization O +of O +the O +AF2 B-structure_element +helix I-structure_element +compared O +to O +either O +the O +APO B-protein_state +or O +ternary B-protein_state +agonist I-protein_state +complex I-protein_state +( O +Fig O +. O +4 O +, O +Additional O +file O +5 O +). O + +BIO399 B-chemical +binds O +to O +the O +ligand B-site +binding I-site +site I-site +of O +RORγ B-protein +adopting O +a O +collapsed B-protein_state +conformation O +as O +seen O +with O +BIO592 B-chemical +where O +the O +two O +compounds O +superimpose B-experimental_method +with O +an O +RMSD B-evidence +of O +0 O +. O +72 O +Å O +( O +Fig O +. O +5b O +). O + +a O +Overlay B-experimental_method +of O +RORγ B-protein +structures B-evidence +bound B-protein_state +to I-protein_state +BIO596 B-chemical +( O +Green O +), O +BIO399 B-chemical +( O +Cyan O +) O +and O +T0901317 B-chemical +( O +Pink O +). O + +We O +hypothesize O +that O +since O +the O +Met358 B-residue_name_number +sidechain O +conformation O +in O +the O +T0901317 B-chemical +RORγ B-protein +structure B-evidence +is O +not O +in O +the O +BIO399 B-chemical +conformation O +, O +this O +difference O +could O +account O +for O +the O +10 O +- O +fold O +reduction O +in O +the O +inverse O +agonism O +for O +T0901317 B-chemical +compared O +to O +BIO399 B-chemical +in O +the O +FRET B-experimental_method +assay I-experimental_method +. O + +The O +inverse B-protein_state +agonist I-protein_state +activity O +for O +these O +compounds O +has O +been O +attributed O +to O +orientating O +Trp317 B-residue_name_number +to O +clash O +with O +Tyr502 B-residue_name_number +or O +a O +direct O +inverse B-protein_state +agonist I-protein_state +hydrogen B-bond_interaction +bonding I-bond_interaction +event O +with O +His479 B-residue_name_number +, O +both O +of O +which O +would O +perturb O +the O +agonist B-protein_state +conformation O +of O +RORγ B-protein +. O + +GAL4 B-experimental_method +cell I-experimental_method +assay I-experimental_method +selectivity O +profile O +for O +BIO399 B-chemical +toward O +RORα B-protein +and O +RORβ B-protein +in O +GAL4 B-protein + +Furthermore O +, O +RORα B-protein +contains O +two O +phenylalanine B-residue_name +residues O +in O +its O +LBS B-site +whereas O +RORβ B-protein +and O +γ B-protein +have O +a O +leucine B-residue_name +in O +the O +same O +position O +( O +Fig O +. O +6b O +). O + +In O +metabolism O +, O +molecules O +with O +“ O +high O +- O +energy O +” O +bonds O +( O +e O +. O +g O +., O +ATP B-chemical +and O +Acetyl B-chemical +~ I-chemical +CoA I-chemical +) O +are O +critical O +for O +both O +catabolic O +and O +anabolic O +processes O +. O + +The O +facets O +of O +the O +shell B-structure_element +are O +composed O +primarily O +of O +hexamers B-oligomeric_state +that O +are O +typically O +perforated O +by O +pores B-site +lined O +with O +highly B-protein_state +conserved I-protein_state +, O +polar B-protein_state +residues B-structure_element +that O +presumably O +function O +as O +the O +conduits O +for O +metabolites O +into O +and O +out O +of O +the O +shell B-structure_element +. O + +Substrates O +and O +cofactors O +involving O +the O +PTAC B-protein_type +reaction O +are O +shown O +in O +red O +; O +other O +substrates O +and O +enzymes O +are O +shown O +in O +black O +, O +and O +other O +cofactors O +are O +shown O +in O +gray O +. O + +The O +activities O +of O +core O +enzymes O +are O +not O +confined O +to O +BMC B-complex_assembly +- O +associated O +functions O +: O +aldehyde B-protein_type +and I-protein_type +alcohol I-protein_type +dehydrogenases I-protein_type +are O +utilized O +in O +diverse O +metabolic O +reactions O +, O +and O +PTAC B-protein_type +catalyzes O +a O +key O +biochemical O +reaction O +in O +the O +process O +of O +obtaining O +energy O +during O +fermentation O +. O + +This O +occurs O +, O +for O +example O +, O +during O +acetoclastic O +methanogenesis O +in O +the O +archaeal B-taxonomy_domain +Methanosarcina B-taxonomy_domain +species I-taxonomy_domain +. O + +Another O +distinctive O +feature O +of O +BMC B-protein_state +- I-protein_state +associated I-protein_state +PduL B-protein_type +homologs O +is O +an O +N O +- O +terminal O +encapsulation B-structure_element +peptide I-structure_element +( O +EP B-structure_element +) O +that O +is O +thought O +to O +“ O +target O +” O +proteins O +for O +encapsulation O +by O +the O +BMC B-complex_assembly +shell B-structure_element +. O + +EPs B-structure_element +are O +frequently O +found O +on O +BMC B-protein_type +- I-protein_type +associated I-protein_type +proteins I-protein_type +and O +have O +been O +shown O +to O +interact O +with O +shell O +proteins O +. O + +Here O +we O +report O +high O +- O +resolution O +crystal B-evidence +structures I-evidence +of O +a O +PduL B-protein_type +- I-protein_type +type I-protein_type +PTAC I-protein_type +in O +both O +CoA B-protein_state +- I-protein_state +and O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +forms O +, O +completing O +our O +understanding O +of O +the O +structural O +basis O +of O +catalysis O +by O +the O +metabolosome B-complex_assembly +common O +core O +enzymes O +. O + +β B-structure_element +- I-structure_element +Barrel I-structure_element +1 I-structure_element +consists O +of O +the O +N O +- O +terminal O +β B-structure_element +strand I-structure_element +and O +β B-structure_element +strands I-structure_element +from O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +the O +polypeptide O +chain O +( O +β1 B-structure_element +, O +β10 B-structure_element +- I-structure_element +β14 I-structure_element +; O +residues O +37 B-residue_range +– I-residue_range +46 I-residue_range +and O +155 B-residue_range +– I-residue_range +224 I-residue_range +). O + +Primary O +structure O +conservation O +of O +the O +PduL B-protein_type +protein O +family O +. O + +Sequence O +logo O +calculated O +from O +the O +multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +PduL B-protein_type +homologs O +( O +see O +Materials O +and O +Methods O +), O +but O +not B-protein_state +including I-protein_state +putative O +EP B-structure_element +sequences O +. O + +The O +sequences O +aligning O +to O +the O +PF06130 B-structure_element +domain O +( O +determined O +by O +BLAST O +) O +are O +highlighted O +in O +red O +and O +blue O +. O + +The O +position O +numbers O +shown O +correspond O +to O +the O +residue O +numbering O +of O +rPduL B-protein +; O +note O +that O +some O +positions O +in O +the O +logo O +represent O +gaps O +in O +the O +rPduL B-protein +sequence O +. O + +The O +asterisk O +and O +double O +arrow O +marks O +the O +location O +of O +the O +π B-bond_interaction +– I-bond_interaction +π I-bond_interaction +interaction I-bond_interaction +between O +F116 B-residue_name_number +and O +the O +CoA B-chemical +base O +of O +the O +other O +dimer B-oligomeric_state +chain O +. O + +Size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +of O +PduL B-protein_type +homologs O +. O + +( O +a O +)–( O +c O +): O +Chromatograms B-evidence +of O +sPduL B-protein +( O +a O +), O +rPduL B-protein +( O +b O +), O +and O +pPduL B-protein +( O +c O +) O +with O +( O +orange O +) O +or O +without O +( O +blue O +) O +the O +predicted O +EP B-structure_element +, O +post O +- O +nickel B-experimental_method +affinity I-experimental_method +purification I-experimental_method +, O +applied O +over O +a O +preparative O +size O +exclusion O +column O +( O +see O +Materials O +and O +Methods O +). O + +The O +second O +( O +Zn2 B-chemical +) O +is O +in O +octahedral O +coordination O +by O +three O +conserved B-protein_state +histidine B-residue_name +residues O +( O +His157 B-residue_name_number +, O +His159 B-residue_name_number +and O +His204 B-residue_name_number +) O +as O +well O +as O +three O +water B-chemical +molecules O +( O +Fig O +4a O +). O + +When O +the O +crystals B-experimental_method +were I-experimental_method +soaked I-experimental_method +in O +a O +sodium B-chemical +phosphate I-chemical +solution O +for O +2 O +d O +prior O +to O +data O +collection O +, O +the O +CoA B-chemical +dissociates O +, O +and O +density B-evidence +for O +a O +phosphate B-chemical +molecule O +is O +visible O +at O +the O +active B-site +site I-site +( O +Table O +2 O +, O +Fig O +4b O +). O + +Oligomeric O +States O +of O +PduL B-protein_type +Orthologs O +Are O +Influenced O +by O +the O +EP B-structure_element + +Given O +the O +diversity O +of O +signature O +enzymes O +( O +Table O +1 O +), O +it O +is O +plausible O +that O +PduL B-protein_type +orthologs O +may O +adopt O +different O +oligomeric O +states O +that O +reflect O +the O +differences O +in O +the O +proteins O +being O +packaged O +with O +them O +in O +the O +organelle O +lumen O +. O + +pPduLΔEP B-mutant +eluted O +as O +two O +smaller O +forms O +, O +possibly O +corresponding O +to O +a O +trimer B-oligomeric_state +and O +a O +monomer B-oligomeric_state +. O + +Homologs O +of O +the O +predominant O +cofactor O +utilizer O +( O +aldehyde B-protein_type +dehydrogenase I-protein_type +) O +and O +NAD B-chemical ++ I-chemical +regenerator O +( O +alcohol B-protein_type +dehydrogenase I-protein_type +) O +have O +been O +structurally O +characterized O +, O +but O +until O +now O +structural O +information O +was O +lacking O +for O +PduL B-protein_type +, O +which O +recycles O +CoA B-chemical +in O +the O +organelle O +lumen O +. O + +Refined O +domain O +assignment O +based O +on O +our O +structure B-evidence +should O +be O +able O +to O +predict O +domains O +of O +PF06130 B-structure_element +homologs O +much O +more O +accurately O +. O + +Implications O +for O +Metabolosome B-complex_assembly +Core O +Assembly O + +Free O +CoA B-chemical +and O +NAD B-chemical ++/ I-chemical +H B-chemical +could O +potentially O +be O +bound O +to O +the O +enzymes O +as O +the O +core O +assembles O +and O +is O +encapsulated O +. O + +The O +free O +CoA B-protein_state +- I-protein_state +bound I-protein_state +form O +is O +presumably O +poised O +for O +attack O +upon O +an O +acyl B-chemical +- I-chemical +phosphate I-chemical +, O +indicating O +that O +the O +enzyme O +initially O +binds O +CoA B-chemical +as O +opposed O +to O +acyl B-chemical +- I-chemical +phosphate I-chemical +. O + +The O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +structure B-evidence +indicates O +that O +in O +the O +opposite O +reaction O +direction O +phosphate B-chemical +is O +bound O +first O +, O +and O +then O +an O +acyl B-chemical +- I-chemical +CoA I-chemical +enters O +. O + +There O +could O +be O +some O +intrinsic O +biochemical O +difference O +between O +the O +two O +enzymes O +that O +renders O +PduL B-protein_type +a O +more O +attractive O +candidate O +for O +encapsulation O +in O +a O +BMC B-complex_assembly +— O +for O +example O +, O +PduL B-protein_type +might O +be O +more O +amenable O +to O +tight O +packaging O +, O +or O +is O +better O +suited O +for O +the O +chemical O +microenvironment O +formed O +within O +the O +lumen O +of O +the O +BMC B-complex_assembly +, O +which O +can O +be O +quite O +different O +from O +the O +cytosol O +. O + +The O +two O +crystal B-evidence +structures I-evidence +that O +we O +report O +here O +for O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +with O +resolutions O +of O +1 O +. O +2 O +Å O +and O +2 O +. O +0 O +Å O +, O +respectively O +), O +and O +data O +derived O +from O +extensive O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +experiments O +targeting O +evolutionarily B-protein_state +highly I-protein_state +conserved I-protein_state +residues O +within O +the O +extended O +EctC B-protein_type +protein I-protein_type +family O +, O +provide O +a O +first O +view O +into O +the O +architecture O +of O +the O +catalytic B-site +core I-site +of O +the O +ectoine B-protein_type +synthase I-protein_type +. O + +The O +( O +Sa B-species +) O +EctC B-protein +protein O +was O +overproduced O +and O +isolated O +with O +good O +yields O +( O +30 O +– O +40 O +mg O +L O +- O +1 O +of O +culture O +) O +and O +purity O +( O +S2a O +Fig O +). O + +Biochemical O +properties O +of O +the O +ectoine B-protein_type +synthase I-protein_type + +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +has O +so O +far O +not O +been O +considered O +as O +a O +substrate O +for O +EctC B-protein +, O +but O +microorganisms B-taxonomy_domain +that O +use O +ectoine B-chemical +as O +a O +nutrient O +produce O +it O +as O +an O +intermediate O +during O +catabolism O +. O + +The O +stimulation O +of O +EctC B-protein +enzyme O +activity O +by O +salts O +has O +previously O +also O +been O +observed O +for O +other O +ectoine B-protein_type +synthases I-protein_type +. O + +Since O +variations O +of O +the O +above O +- O +described O +metal B-structure_element +- I-structure_element +binding I-structure_element +motif I-structure_element +occur O +frequently O +, O +we O +experimentally O +investigated O +the O +presence O +and O +nature O +of O +the O +metal B-chemical +that O +might O +be O +contained O +in O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +by O +inductive B-experimental_method +- I-experimental_method +coupled I-experimental_method +plasma I-experimental_method +mass I-experimental_method +spectrometry I-experimental_method +( O +ICP B-experimental_method +- I-experimental_method +MS I-experimental_method +). O + +We O +note O +in O +this O +context O +, O +that O +the O +values O +obtained O +for O +the O +iron B-chemical +content O +of O +the O +( O +Sa B-species +) O +EctC B-protein +proteins O +varied O +by O +approximately O +10 O +to O +20 O +% O +between O +the O +two O +methods O +. O + +Dependency O +of O +the O +ectoine B-protein_type +synthase I-protein_type +activity O +on O +metals O +. O + +We O +then O +took O +such O +an O +inactivated B-protein_state +enzyme O +preparation O +, O +removed O +the O +EDTA B-chemical +by O +dialysis B-experimental_method +, O +and O +added O +stoichiometric O +amounts O +( O +10 O +μM O +) O +of O +various O +metals O +to O +the O +( O +Sa B-species +) O +EctC B-protein +enzyme O +. O + +Hence O +, O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +is O +a O +newly O +recognized O +substrate O +for O +ectoine B-protein_type +synthase I-protein_type +. O + +The O +Km B-evidence +dropped O +fife O +- O +fold O +from O +4 O +. O +9 O +± O +0 O +. O +5 O +mM O +to O +25 O +. O +4 O +± O +2 O +. O +9 O +mM O +, O +and O +the O +catalytic B-evidence +efficiency I-evidence +was O +reduced O +from O +1 O +. O +47 O +mM O +- O +1 O +s O +- O +1 O +to O +0 O +. O +02 O +mM O +- O +1 O +s O +- O +1 O +, O +a O +73 O +- O +fold O +decrease O +. O + +Finally O +, O +a O +monomer B-oligomeric_state +of O +this O +structure B-evidence +was O +used O +as O +a O +template O +for O +molecular B-experimental_method +replacement I-experimental_method +to O +phase O +the O +high O +- O +resolution O +( O +1 O +. O +2 O +Å O +) O +dataset O +of O +crystal O +form O +A O +, O +which O +was O +subsequently O +refined O +to O +a O +final O +Rcryst B-evidence +of O +12 O +. O +4 O +% O +and O +an O +Rfree B-evidence +of O +14 O +. O +9 O +% O +( O +S1 O +Table O +). O + +This O +structure B-evidence +adopts O +an O +open B-protein_state +conformation O +with O +respect O +to O +the O +typical O +fold O +of O +cupin B-structure_element +barrels I-structure_element +and O +is O +therefore O +termed O +in O +the O +following O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +( O +Fig O +4b O +). O + +Interestingly O +, O +the O +three O +other O +monomers B-oligomeric_state +present O +in O +the O +asymmetric O +unit O +all O +range O +from O +Met B-residue_range +- I-residue_range +1 I-residue_range +to I-residue_range +Glu I-residue_range +- I-residue_range +115 I-residue_range +and O +adopt O +a O +conformation O +similar O +to O +the O +“ O +open B-protein_state +” O +EctC B-protein +structure B-evidence +. O + +The O +structure B-evidence +of O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +protein O +consists O +of O +11 O +β B-structure_element +- I-structure_element +strands I-structure_element +( O +β1 B-structure_element +- I-structure_element +β11 I-structure_element +) O +and O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +α B-structure_element +- I-structure_element +I I-structure_element +and O +α B-structure_element +- I-structure_element +II I-structure_element +) O +( O +Fig O +4a O +). O + +Our O +data O +classify O +EctC B-protein +, O +in O +addition O +to O +the O +polyketide B-protein_type +cyclase I-protein_type +RemF B-protein +, O +as O +the O +second O +known O +cupin B-protein_type +- I-protein_type +related I-protein_type +enzyme O +that O +catalyze O +a O +cyclocondensation O +reaction O +. O + +Analysis O +of O +the O +EctC B-protein +dimer B-site +interface I-site +as O +observed O +in O +the O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structure I-evidence + +It O +is O +worth O +mentioning O +that O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +is O +located O +next O +to O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +, O +which O +in O +all O +likelihood O +involved O +in O +metal B-chemical +binding O +( O +see O +below O +). O + +In O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +, O +both O +proline B-residue_name +residues O +are O +visible O +in O +the O +electron B-evidence +density I-evidence +; O +however O +, O +almost O +directly O +after O +Pro B-residue_name_number +- I-residue_name_number +110 I-residue_name_number +, O +the O +electron B-evidence +density I-evidence +is O +drastically O +diminished O +caused O +by O +the O +flexibility O +of O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +. O + +Since O +these O +proline B-residue_name +residues O +are O +followed O +by O +the O +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +, O +the O +interaction O +of O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +with O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +will O +likely O +play O +a O +substantial O +role O +in O +spatially O +orienting O +this O +very O +flexible O +part O +of O +the O +protein O +. O + +The O +interaction O +between O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +and O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +is O +only O +visible O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +where O +the O +partially B-protein_state +extended I-protein_state +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +is O +resolved O +in O +the O +electron B-evidence +density I-evidence +. O + +( O +b O +) O +An O +overlay B-experimental_method +of O +the O +“ O +open B-protein_state +” O +( O +colored O +in O +light O +blue O +) O +and O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +colored O +in O +green O +) O +structure B-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +The O +putative O +iron B-site +binding I-site +site I-site +of O +( O +Sa B-species +) O +EctC B-protein + +In O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +of O +( O +Sa B-species +) O +EctC B-protein +, O +each O +of O +the O +four O +monomers B-oligomeric_state +in O +the O +asymmetric O +unit O +contains O +a O +relative O +strong O +electron B-evidence +density I-evidence +positioned O +within O +the O +cupin B-structure_element +barrel I-structure_element +. O + +Of O +note O +is O +the O +different O +spatial O +arrangement O +of O +the O +side O +- O +chain O +of O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +( O +located O +in O +a O +loop B-structure_element +after O +the O +end O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +) O +in O +the O +“ O +open B-protein_state +” O +and O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structures B-evidence +. O + +It O +becomes O +apparent O +from O +an O +overlay B-experimental_method +of O +the O +“ O +open B-protein_state +” O +and O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structures I-evidence +that O +the O +side O +- O +chain O +of O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +rotates O +away O +from O +the O +position O +of O +the O +presumptive O +iron B-chemical +, O +whereas O +the O +side O +- O +chains O +of O +those O +residues O +that O +probably O +contacting O +the O +metal B-chemical +directly O +[ O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +and O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +], O +remain O +in O +place O +( O +Fig O +6a O +and O +6b O +). O + +We O +also O +replaced B-experimental_method +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +with O +either O +a O +Phe B-residue_name +or O +a O +Trp B-residue_name +residue O +and O +both O +mutant B-protein_state +proteins O +largely O +lost O +their O +catalytic O +activity O +and O +iron B-chemical +content O +( O +Table O +1 O +) O +despite O +the O +fact O +that O +these O +substitutions O +were O +conservative O +. O + +Since O +we O +used O +PEG B-chemical +molecules O +in O +the O +crystallization O +conditions O +, O +the O +observed O +density B-evidence +might O +stem O +from O +an O +ordered O +part O +of O +a O +PEG B-chemical +molecule O +, O +or O +low O +molecular O +weight O +PEG B-chemical +species O +that O +might O +have O +been O +present O +in O +the O +PEG B-chemical +preparation O +used O +in O +our O +experiments O +. O + +Despite O +these O +notable O +limitations O +, O +we O +considered O +the O +serendipitously O +trapped O +compound O +as O +a O +mock O +ligand O +that O +might O +provide O +useful O +insights O +into O +the O +spatial O +positioning O +of O +the O +true O +EctC B-protein +substrate O +and O +those O +residues O +that O +coordinate O +it O +within O +the O +ectoine B-protein_type +synthase I-protein_type +active B-site +site I-site +. O + +The O +electron B-evidence +density I-evidence +was O +calculated O +as O +an O +omit B-evidence +map I-evidence +and O +contoured O +at O +1 O +. O +0 O +σ O +. O + +We O +also O +calculated O +an O +omit B-evidence +map I-evidence +and O +the O +electron B-evidence +density I-evidence +reappeared O +( O +Fig O +7b O +). O + +These O +correspond O +to O +amino O +acids O +Thr B-residue_name_number +- I-residue_name_number +40 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +, O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +, O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Gly B-residue_name_number +- I-residue_name_number +64 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +- O +Leu B-residue_name_number +- I-residue_name_number +87 I-residue_name_number +, O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +, O +Phe B-residue_name_number +- I-residue_name_number +107 I-residue_name_number +, O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +, O +Gly B-residue_name_number +- I-residue_name_number +113 I-residue_name_number +, O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +, O +and O +His B-residue_name_number +- I-residue_name_number +117 I-residue_name_number +in O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +2 O +). O + +Each O +of O +these O +mutant B-protein_state +( O +Sa B-species +) O +EctC B-protein +proteins O +was O +overproduced O +in O +E B-species +. I-species +coli I-species +and O +purified O +by O +affinity B-experimental_method +chromatography I-experimental_method +; O +they O +all O +yielded O +pure O +and O +stable O +protein O +preparations O +. O + +We O +replaced B-experimental_method +each O +of O +these O +residues O +with O +an O +Ala B-residue_name +residue O +and O +found O +that O +none O +of O +them O +had O +an O +influence O +on O +the O +iron B-chemical +content O +of O +the O +mutant B-protein_state +proteins O +. O + +Each O +of O +these O +residues O +is O +evolutionarily B-protein_state +highly I-protein_state +conserved I-protein_state +. O + +His B-residue_name_number +- I-residue_name_number +117 I-residue_name_number +is O +a O +strictly B-protein_state +conserved I-protein_state +residue O +and O +its O +substitution B-experimental_method +by O +an O +Ala B-residue_name +residue O +results O +in O +a O +drop O +of O +enzyme O +activity O +( O +down O +to O +44 O +%) O +and O +an O +iron B-chemical +content O +of O +83 O +% O +( O +Table O +1 O +). O + +As O +an O +internal O +control O +for O +our O +mutagenesis B-experimental_method +experiments I-experimental_method +, O +we O +also O +substituted B-experimental_method +Thr B-residue_name_number +- I-residue_name_number +41 I-residue_name_number +and O +His B-residue_name_number +- I-residue_name_number +51 I-residue_name_number +, O +two O +residues O +that O +are O +not B-protein_state +evolutionarily I-protein_state +conserved I-protein_state +in O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +with O +Ala B-residue_name +residues O +. O + +Hence O +, O +the O +active B-site +site I-site +of O +ectoine B-protein_type +synthase I-protein_type +must O +possess O +a O +certain O +degree O +of O +structural O +plasticity O +, O +a O +notion O +that O +is O +supported O +by O +the O +report O +on O +the O +EctC B-protein +- O +catalyzed O +formation O +of O +the O +synthetic O +compatible O +solute O +ADPC B-chemical +through O +the O +cyclic O +condensation O +of O +two O +glutamine B-chemical +molecules O +. O + +We O +assumed O +that O +its O +location O +and O +mode O +of O +binding O +gives O +, O +in O +all O +likelihood O +, O +clues O +as O +to O +the O +position O +of O +the O +true O +substrate O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +within O +the O +EctC B-protein +active B-site +site I-site +. O + +This O +probably O +worked O +to O +the O +detriment O +of O +our O +efforts O +in O +solving O +crystal B-evidence +structures I-evidence +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +( O +Sa B-species +) O +EctC B-protein +protein O +in B-protein_state +complex I-protein_state +with I-protein_state +either O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +or O +ectoine B-chemical +. O + +Finally O +, O +our O +results O +provide O +a O +structural O +framework O +for O +understanding O +the O +effects O +of O +ADAR B-protein_type +mutations O +associated O +with O +human B-species +disease O +. O + +Two O +different O +enzymes O +carry O +out O +A O +to O +I O +editing O +in O +humans B-species +; O +ADAR1 B-protein +and O +ADAR2 B-protein +. O + +Furthermore O +, O +how O +an O +ADAR B-protein_type +deaminase B-structure_element +domain I-structure_element +contributes O +to O +editing B-site +site I-site +selectivity O +is O +also O +unknown O +, O +since O +no O +structures B-evidence +of O +ADAR B-complex_assembly +deaminase I-complex_assembly +domain I-complex_assembly +- I-complex_assembly +RNA I-complex_assembly +complexes O +have O +been O +reported O +. O + +In O +addition O +, O +an O +inositol B-chemical +hexakisphosphate I-chemical +( O +IHP B-chemical +) O +molecule O +was O +found O +buried O +in O +the O +core O +of O +the O +protein O +hydrogen B-bond_interaction +bonded I-bond_interaction +to O +numerous O +conserved O +polar O +residues O +. O + +The O +8 B-chemical +- I-chemical +azanebularine I-chemical +is O +flipped B-protein_state +out I-protein_state +of O +the O +helix B-structure_element +and O +bound B-protein_state +into I-protein_state +the O +active B-site +site I-site +as O +its O +covalent O +hydrate O +where O +it O +interacts O +with O +several O +amino O +acids O +including O +V351 B-residue_name_number +, O +T375 B-residue_name_number +, O +K376 B-residue_name_number +, O +E396 B-residue_name_number +and O +R455 B-residue_name_number +( O +Fig O +. O +3a O +, O +Supplementary O +Fig O +. O +3a O +). O + +The O +R455 B-residue_name_number +side O +chain O +ion B-bond_interaction +pairs I-bond_interaction +with O +the O +5 O +’- O +phosphodiester O +of O +8 B-chemical +- I-chemical +azanebularine I-chemical +while O +the O +K376 B-residue_name_number +side O +chain O +contacts O +its O +3 O +’- O +phosphodiester O +. O + +ADARs B-protein_type +use O +a O +unique O +mechanism O +to O +modify O +duplex B-structure_element +RNA I-structure_element + +However O +, O +unlike O +the O +case O +of O +the O +DNA B-protein_type +MTase I-protein_type +that O +approaches O +the O +DNA B-chemical +from O +the O +major B-site +groove I-site +, O +the O +ADAR2 B-protein +loop B-structure_element +approaches O +the O +duplex B-structure_element +from O +the O +minor B-site +groove I-site +side O +. O + +The O +DNA B-chemical +B B-structure_element +- I-structure_element +form I-structure_element +helix I-structure_element +has O +groove O +widths O +and O +depths O +that O +would O +prevent O +productive O +interactions O +with O +ADAR B-protein_type +. O + +ADARs B-protein_type +have O +a O +preference O +for O +editing O +adenosines B-residue_name +with O +5 O +’ O +nearest O +neighbor O +U B-residue_name +( O +or O +A B-residue_name +) O +and O +3 O +’ O +nearest O +neighbor O +G B-residue_name +. O +The O +ADAR2 B-protein +flipping B-structure_element +loop I-structure_element +occupies O +the O +minor B-site +groove I-site +spanning O +the O +three O +base O +pairs O +that O +include O +the O +nearest O +neighbor O +nucleotides O +flanking O +the O +edited O +base O +( O +Figs O +. O +3b O +, O +3c O +). O + +These O +observations O +suggest O +that O +hADAR2 B-protein +’ O +s O +5 O +’ O +nearest O +neighbor O +preference O +for O +U B-residue_name +( O +or O +A B-residue_name +) O +is O +due O +to O +a O +destabilizing O +clash O +with O +the O +protein O +backbone O +at O +G489 B-residue_name_number +that O +results O +from O +the O +presence O +of O +an O +amino O +group O +in O +the O +minor B-site +groove I-site +at O +this O +location O +for O +sequences O +with O +5 O +’ O +nearest O +neighbor O +G B-residue_name +or O +C B-residue_name +. O +However O +, O +the O +observed O +clash O +is O +not O +severe O +and O +the O +enzyme O +would O +be O +able O +to O +accommodate O +G B-residue_name +or O +C B-residue_name +5 O +’ O +nearest O +neighbors O +by O +slight O +structural O +perturbations O +, O +explaining O +why O +this O +sequence O +preference O +is O +not O +an O +absolute O +requirement O +. O + +In O +addition O +, O +the O +substrate O +with O +a O +3 O +’ O +I B-residue_name +displayed O +a O +reduced B-evidence +deamination I-evidence +rate I-evidence +compared O +to O +the O +substrate O +with O +a O +3 O +’ O +G B-residue_name +suggesting O +the O +observed O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +to O +the O +2 O +- O +amino O +group O +contributes O +to O +the O +3 O +’ O +nearest O +neighbor O +selectivity O +( O +Fig O +. O +5f O +). O + +Mutation B-experimental_method +of O +any O +of O +these O +residues O +to O +alanine B-residue_name +( O +G593A B-mutant +, O +K594A B-mutant +, O +R348A B-mutant +) O +substantially O +reduces O +editing O +activity O +( O +Fig O +. O +6c O +). O + +Base O +flipping O +is O +a O +well O +- O +characterized O +mechanism O +by O +which O +nucleic B-protein_type +acid I-protein_type +modifying I-protein_type +enzymes I-protein_type +gain O +access O +to O +sites O +of O +reaction O +that O +are O +otherwise O +buried O +in O +base O +- O +paired O +structures B-evidence +. O + +However O +, O +these O +nucleotides O +are O +located O +in O +a O +highly B-protein_state +distorted I-protein_state +and O +dynamic B-protein_state +duplex B-structure_element +region I-structure_element +containing O +several O +mismatches O +and O +are O +predisposed O +to O +undergo O +this O +conformational O +change O +. O + +Other O +RNA B-protein_type +modification I-protein_type +enzymes I-protein_type +are O +known O +that O +flip O +nucleotides O +out O +of O +loops O +, O +even O +from O +base O +pairs O +in O +loop O +regions O +( O +pseudoU B-protein_type +synthetase I-protein_type +, O +tRNA B-protein_type +transglycosylase I-protein_type +, O +and O +restrictocin B-protein +bound B-protein_state +to I-protein_state +sarcin B-structure_element +/ I-structure_element +ricin I-structure_element +loop I-structure_element +of O +28S B-chemical +rRNA I-chemical +) O +( O +Supplementary O +Fig O +. O +5b O +). O + +Our O +use O +of O +8 B-chemical +- I-chemical +azanebularine I-chemical +, O +with O +its O +high O +propensity O +to O +form O +a O +covalent O +hydrate O +, O +allowed O +us O +to O +capture O +a O +true O +mimic O +of O +the O +tetrahedral O +intermediate O +and O +reveal O +the O +interactions O +between O +the O +deaminase B-protein_type +active B-site +site I-site +and O +the O +reactive O +nucleotide O +. O + +Several O +other O +base B-protein_type +- I-protein_type +flipping I-protein_type +enzymes I-protein_type +stabilize O +the O +altered O +nucleic O +acid O +conformation O +by O +intercalation O +of O +an O +amino O +acid O +side O +chain O +into O +the O +space O +vacated O +by O +the O +flipped B-protein_state +out I-protein_state +base B-chemical +. O + +The O +latter O +interaction O +requires O +E488 B-residue_name_number +to O +be O +protonated B-protein_state +. O + +However O +, O +since O +dsRBDs B-structure_element +are O +known O +to O +bind O +promiscuously O +with O +duplex B-structure_element +RNA I-structure_element +, O +it O +is O +possible O +that O +the O +S258 B-residue_name_number +- O +3 O +’ O +G B-residue_name +interaction O +found O +in O +a O +complex O +lacking B-protein_state +the I-protein_state +deaminase B-structure_element +domain I-structure_element +is O +not O +relevant O +to O +catalysis O +at O +the O +editing B-site +site I-site +. O + +In O +summary O +, O +the O +structures B-evidence +described O +here O +establish O +human B-species +ADAR2 B-protein +as O +a O +base O +- O +flipping O +enzyme O +that O +uses O +a O +unique O +mechanism O +well O +suited O +for O +modifying O +duplex B-structure_element +RNA I-structure_element +. O + +b O +, O +View O +of O +structure O +along O +the O +dsRNA B-chemical +helical O +axis O +. O + +Other O +ADAR B-protein_type +- O +induced O +changes O +in O +RNA B-chemical +conformation O + +c O +, O +Unusual O +“ O +wobble O +” O +A13 B-residue_name_number +’- O +U11 B-residue_name_number +interaction O +in O +the O +hADAR2d B-complex_assembly +WT I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +U I-complex_assembly +complex O +shown O +in O +stick O +with O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +indicated O +with O +yellow O +dashes O +and O +distances O +shown O +in O +Å O +. O +The O +position O +of O +this O +base O +pair O +in O +the O +hADAR2d B-complex_assembly +E488Q I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +C I-complex_assembly +duplex O +is O +shown O +in O +wire O +with O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +shown O +with O +gray O +dashes O +. O + +f O +, O +Comparison O +of O +deamination B-evidence +rate I-evidence +constants I-evidence +by O +hADAR2d B-mutant +at O +the O +editing B-site +site I-site +adenosine B-residue_name +( O +red O +) O +for O +duplexes O +bearing O +different O +3 O +’ O +nearest O +neighbors O +. O + +Interestingly O +, O +mutations B-experimental_method +blocking O +PIN B-structure_element +oligomerization O +had O +no O +RNase B-protein_type +activity O +, O +indicating O +that O +both O +oligomerization O +and O +NTD B-structure_element +binding O +are O +crucial O +for O +RNase B-protein_type +activity O +in O +vitro O +. O + +Recently O +, O +the O +crystal B-evidence +structure I-evidence +of O +the O +Regnase B-protein +- I-protein +1 I-protein +PIN B-structure_element +domain O +derived O +from O +Homo B-species +sapiens I-species +was O +reported O +. O + +In O +addition O +, O +Regnase B-protein +- I-protein +1 I-protein +has O +been O +predicted O +to O +possess O +other O +domains O +in O +the O +N B-structure_element +- I-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +regions I-structure_element +. O + +However O +, O +the O +structure B-evidence +and O +function O +of O +the O +ZF B-structure_element +domain O +, O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +NTD B-structure_element +) O +and O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +CTD B-structure_element +) O +of O +Regnase B-protein +- I-protein +1 I-protein +have O +not O +been O +solved O +. O + +Contribution O +of O +each O +domain O +of O +Regnase B-protein +- I-protein +1 I-protein +to O +the O +mRNA B-chemical +binding O +activity O + +Upon O +addition O +of O +a O +larger O +amount O +of O +Regnase B-protein +- I-protein +1 I-protein +, O +the O +fluorescence B-evidence +of O +free B-protein_state +RNA B-chemical +decreased O +, O +indicating O +that O +Regnase B-protein +- I-protein +1 I-protein +bound B-protein_state +to I-protein_state +the O +RNA B-chemical +. O + +Mutation B-experimental_method +of O +Arg215 B-residue_name_number +, O +whose O +side O +chain O +faces O +to O +the O +opposite O +side O +of O +the O +oligomeric B-site +surface I-site +, O +to O +Glu B-residue_name +preserved O +the O +monomer B-oligomeric_state +/ O +dimer B-oligomeric_state +equilibrium O +, O +similar O +to O +the O +wild B-protein_state +type I-protein_state +. O + +These O +results O +indicate O +that O +the O +PIN B-structure_element +domain O +forms O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomer B-oligomeric_state +in O +solution O +similar O +to O +the O +crystal B-evidence +structure I-evidence +. O + +The O +side O +chains O +of O +these O +residues O +point O +away O +from O +the O +catalytic B-site +center I-site +on O +the O +same O +molecule O +( O +Fig O +. O +2b O +). O + +Therefore O +, O +we O +concluded O +that O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +PIN B-structure_element +dimerization O +, O +together O +with O +the O +NTD B-structure_element +, O +are O +required O +for O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O +in O +vitro O +. O + +R214 B-residue_name_number +is O +an O +important O +residue O +for O +dimer B-oligomeric_state +formation O +as O +shown O +in O +Fig O +. O +2 O +, O +therefore O +, O +R214A B-mutant +in O +the O +secondary B-protein_state +PIN B-structure_element +cannot O +dimerize O +. O + +Although O +the O +function O +of O +the O +CTD B-structure_element +remains O +elusive O +, O +we O +revealed O +the O +functions O +of O +the O +NTD B-structure_element +, O +PIN B-structure_element +, O +and O +ZF B-structure_element +domains O +. O + +Our O +NMR B-experimental_method +experiments O +confirmed O +direct O +binding O +of O +the O +ZF B-structure_element +domain O +to O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +with O +a O +Kd B-evidence +of O +10 O +± O +1 O +. O +1 O +μM O +. O +Furthermore O +, O +an O +in B-experimental_method +vitro I-experimental_method +gel I-experimental_method +shift I-experimental_method +assay I-experimental_method +indicated O +that O +Regnase B-protein +- I-protein +1 I-protein +containing O +the O +ZF B-structure_element +domain O +enhanced O +target O +mRNA B-chemical +- O +binding O +, O +but O +the O +protein O +- O +RNA B-chemical +complex O +remained O +in O +the O +bottom O +of O +the O +well O +without O +entering O +into O +the O +polyacrylamide O +gel O +. O + +Due O +to O +this O +limitation O +, O +it O +is O +difficult O +to O +perform O +further O +structural B-experimental_method +analyses I-experimental_method +of O +mRNA B-complex_assembly +- I-complex_assembly +Regnase I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +complexes O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +or O +NMR B-experimental_method +. O + +While O +further O +analyses O +are O +necessary O +to O +prove O +this O +point O +, O +our O +preliminary O +docking B-experimental_method +and I-experimental_method +molecular I-experimental_method +dynamics I-experimental_method +simulations I-experimental_method +indicate O +that O +NTD B-structure_element +- O +binding O +rearranges O +the O +catalytic B-site +residues I-site +of O +the O +PIN B-structure_element +domain O +toward O +an O +active B-protein_state +conformation O +suitable O +for O +binding O +Mg2 B-chemical ++. I-chemical + +In O +this O +context O +, O +it O +is O +interesting O +that O +, O +in O +response O +to O +TCR O +stimulation O +, O +Malt1 B-protein +cleaves O +Regnase B-protein +- I-protein +1 I-protein +at O +R111 B-residue_name_number +to O +control O +immune O +responses O +in O +vivo O +. O + +This O +result O +is O +consistent O +with O +a O +model O +in O +which O +the O +NTD B-structure_element +acts O +as O +an O +enhancer O +, O +and O +cleavage O +of O +the O +linker B-structure_element +lowers O +enzymatic O +activity O +dramatically O +. O + +We O +incorporated O +information O +from O +the O +cleavage B-site +site I-site +of O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +in O +vitro O +is O +indicated O +by O +denaturing O +polyacrylamide B-experimental_method +gel I-experimental_method +electrophoresis I-experimental_method +( O +Supplementary O +Fig O +. O +7a O +, O +b O +). O + +The O +overall O +model O +of O +regulation O +of O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O +through O +domain O +- O +domain O +interactions O +in O +vitro O +is O +summarized O +in O +Fig O +. O +6 O +. O + +A O +fully B-protein_state +active I-protein_state +catalytic B-site +center I-site +can O +be O +formed O +only O +when O +the O +NTD B-structure_element +associates O +with O +the O +oligomer B-oligomeric_state +surface O +of O +the O +PIN B-structure_element +domain O +, O +which O +terminates O +the O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomer B-oligomeric_state +formation O +in O +one O +direction O +( O +primary B-protein_state +PIN B-structure_element +), O +and O +forms O +a O +functional B-protein_state +dimer B-oligomeric_state +together O +with O +the O +neighboring O +PIN B-structure_element +( O +secondary B-protein_state +PIN B-structure_element +). O + +( O +a O +) O +Domain O +architecture O +of O +Regnase B-protein +- I-protein +1 I-protein +. O +( O +b O +) O +Solution B-evidence +structure I-evidence +of O +the O +NTD B-structure_element +. O +( O +c O +) O +Crystal B-evidence +structure I-evidence +of O +the O +PIN B-structure_element +domain O +. O + +( O +e O +) O +Solution B-evidence +structure I-evidence +of O +the O +CTD B-structure_element +. O + +( O +g O +) O +Binding O +of O +Regnase B-protein +- I-protein +1 I-protein +and O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +was O +plotted O +. O + +( O +h O +) O +In B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assay I-experimental_method +of O +Regnase B-protein +- I-protein +1 I-protein +to O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +. O + +The O +residues O +with O +significant B-evidence +chemical I-evidence +shift I-evidence +changes I-evidence +were O +labeled O +in O +the O +overlaid B-experimental_method +spectra B-evidence +( O +left O +) O +and O +colored O +red O +, O +yellow O +, O +or O +green O +on O +the O +surface O +and O +ribbon O +structure O +of O +the O +NTD B-structure_element +. O + +Heterodimer O +formation O +by O +combination O +of O +the O +Regnase B-protein +- I-protein +1 I-protein +basic O +residue O +mutants B-protein_state +and O +the O +DDNN B-mutant +mutant B-protein_state +restored O +the O +RNase B-protein_type +activity O +. O + +Structure O +‐ O +activity O +relationship O +of O +the O +peptide B-structure_element +binding I-structure_element +‐ I-structure_element +motif I-structure_element +mediating O +the O +BRCA2 B-complex_assembly +: I-complex_assembly +RAD51 I-complex_assembly +protein O +– O +protein O +interaction O + +We O +have O +tabulated O +the O +effects O +of O +mutation B-experimental_method +of O +this O +sequence O +, O +across O +a O +variety O +of O +experimental O +methods O +and O +from O +relevant O +mutations O +observed O +in O +the O +clinic O +. O + +Both O +BRCA2 B-protein +and O +RAD51 B-protein +together O +are O +vital O +for O +helping O +to O +repair O +and O +maintain O +a O +high O +fidelity O +in O +DNA O +replication O +. O + +However O +, O +whilst O +the O +identification O +of O +highly B-protein_state +conserved I-protein_state +residues O +may O +be O +a O +good O +starting O +point O +for O +identifying O +hot B-site +‐ I-site +spots I-site +, O +experimental O +validation O +by O +mutation B-experimental_method +of O +these O +sequences O +is O +vital O +. O + +The O +mutation O +, O +relevant O +peptide O +context O +, O +resulting O +FxxA B-structure_element +motif O +sequence O +and O +experimental O +technique O +for O +each O +entry O +is O +given O +. O + +The O +wild B-protein_state +‐ I-protein_state +type I-protein_state +FxxA B-structure_element +sequence O +is O +indicated O +in O +parenthesis O +. O + +Wild B-protein_state +‐ I-protein_state +type I-protein_state +human B-species +RAD51 B-protein +, O +however O +, O +is O +a O +heterogeneous O +mixture O +of O +oligomers B-oligomeric_state +and O +when O +monomerised B-oligomeric_state +by O +mutation B-experimental_method +, O +is O +highly B-protein_state +unstable I-protein_state +. O + +One O +RAD51 B-protein +( O +green O +cartoon O +) O +interacts O +with O +another O +molecule O +of O +RAD51 B-protein +( O +grey O +and O +pink O +surface O +) O +via O +the O +FxxA B-site +pocket I-site +indicated O +by O +the O +dashed O +blue O +box O +. O + +A O +summary O +of O +the O +peptide O +sequence O +, O +PDB O +codes O +and O +K B-evidence +D I-evidence +data O +measured O +by O +ITC B-experimental_method +with O +the O +corresponding O +ΔH B-evidence +and O +TΔS B-evidence +values O +are O +collated O +in O +Table O +2 O +. O + +Interestingly O +, O +it O +was O +found O +that O +a O +proline B-residue_name +at O +this O +position O +improved O +the O +affinity B-evidence +almost O +threefold O +, O +to O +113 O +μm O +( O +Table O +2 O +, O +entry O +6 O +). O + +This O +beneficial O +mutation B-experimental_method +was O +incorporated O +with O +another O +previously O +identified O +variant O +to O +produce O +the O +peptide O +WHPA B-structure_element +. O + +Perhaps O +surprisingly O +, O +it O +was O +accommodated O +and O +the O +affinity B-evidence +dropped O +only O +by O +twofold O +as O +compared O +to O +FHTA B-structure_element +. O + +The O +effect O +of O +simply O +removing B-experimental_method +the O +β O +‐ O +carbon O +of O +alanine B-residue_name +, O +by O +mutation B-experimental_method +to I-experimental_method +glycine B-residue_name +( O +FHTG B-structure_element +), O +produced O +an O +approximately O +sixfold O +drop O +in O +binding B-evidence +affinity I-evidence +( O +Table O +2 O +, O +entry O +8 O +). O + +This O +is O +in O +line O +with O +the O +observation O +that O +alanine B-residue_name +is O +not B-protein_state +100 I-protein_state +% I-protein_state +conserved I-protein_state +and O +some O +archeal B-taxonomy_domain +RadA B-protein_type +proteins I-protein_type +contain O +a O +glycine B-residue_name +in O +the O +place O +of O +alanine B-residue_name +23 O +. O + +Structures B-evidence +of O +the O +key O +tetrapeptides B-chemical +were O +solved O +by O +soaking B-experimental_method +into I-experimental_method +crystals B-evidence +of O +a O +humanised B-protein_state +form O +of O +RAD51 B-protein +, O +HumRadA1 B-mutant +, O +which O +we O +have O +previously O +reported O +as O +a O +convenient O +surrogate O +system O +for O +RAD51 B-protein +crystallography B-experimental_method +15 O +. O + +All O +structures B-evidence +are O +of O +high O +resolution O +( O +1 O +. O +2 O +– O +1 O +. O +7 O +Å O +) O +and O +the O +electron B-evidence +density I-evidence +for O +the O +peptide O +was O +clearly O +visible O +after O +the O +first O +refinement O +using O +unliganded B-protein_state +RadA B-protein +coordinates O +( O +Fig O +. O +S1 O +). O + +Some O +of O +the O +SAR O +observed O +in O +the O +binding B-experimental_method +analysis I-experimental_method +can O +be O +interpreted O +in O +terms O +of O +these O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystal B-evidence +structures I-evidence +. O + +The O +thermodynamic B-evidence +data I-evidence +of O +peptide O +binding O +are O +also O +shown O +in O +Table O +2 O +. O +Although O +we O +have O +both O +thermodynamic B-evidence +data I-evidence +and O +high O +‐ O +quality O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +structural B-evidence +information I-evidence +for O +some O +of O +the O +mutant B-protein_state +peptides B-chemical +, O +we O +do O +not O +attempt O +to O +interpret O +differences O +in O +thermodynamic B-evidence +profiles I-evidence +between O +ligands O +, O +that O +is O +, O +to O +analyse O +ΔΔH B-evidence +and O +ΔΔS B-evidence +. O + +As O +ΔS B-evidence +is O +derived O +from O +ΔG B-evidence +by O +subtracting O +ΔH B-evidence +, O +errors O +in O +ΔH B-evidence +will O +be O +correlated O +with O +errors O +in O +ΔS B-evidence +, O +giving O +rise O +to O +a O +‘ O +phantom O +’ O +enthalpy O +– O +entropy O +compensation O +. O + +Figure O +3A O +shows O +the O +binding O +pose O +of O +BRC4 B-chemical +when O +bound B-protein_state +to I-protein_state +RAD51 B-protein +and O +the O +intrapeptide O +hydrogen B-bond_interaction +bonds I-bond_interaction +that O +are O +made O +by O +BRC4 B-chemical +. O + +While O +Phe1524 B-residue_name_number +and O +Ala1527 B-residue_name_number +are O +buried O +in O +hydrophobic B-site +pockets I-site +on O +the O +surface O +, O +His1525 B-residue_name_number +is O +close O +enough O +to O +form O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +carbonyl O +of O +Thr1520 B-residue_name_number +, O +but O +the O +rotamer O +of O +His1525 B-residue_name_number +, O +supported O +by O +clearly O +positioned O +water B-chemical +molecules O +, O +is O +not O +compatible O +with O +hydrogen B-bond_interaction +bonding I-bond_interaction +. O + +( O +A O +) O +Highlight O +of O +intra O +‐ O +BRC4 B-chemical +interactions O +when O +bound B-protein_state +to I-protein_state +RAD51 B-protein +( O +omitted O +for O +clarity O +) O +( O +PDB O +: O +1n0w O +), O +with O +key O +residues O +shown O +in O +colour O +. O +( O +B O +) O +Intrapeptide O +interactions O +from O +oligomerisation B-structure_element +epitope I-structure_element +of O +S B-species +. I-species +cerevisiae I-species +RAD51 B-protein +when O +bound B-protein_state +to I-protein_state +next O +RAD51 B-protein +in O +the O +filament O +( O +PDB O +: O +1szp O +). O + +Thr1526 B-residue_name_number +makes O +no O +direct O +interactions O +with O +the O +RAD51 B-protein +protein O +, O +but O +instead O +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +network I-bond_interaction +with O +the O +highly B-protein_state +conserved I-protein_state +S1528 B-residue_name_number +and O +K1530 B-residue_name_number +( O +Fig O +. O +1C O +). O + +The O +conserved B-protein_state +threonine B-residue_name +residue O +at O +the O +third O +position O +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +peptide O +backbone O +amide O +, O +which O +forms O +the O +base O +of O +an O +α B-structure_element +‐ I-structure_element +helix I-structure_element +. O + +The O +reason O +for O +this O +disconnection O +is O +suggested O +to O +be O +that O +threonine B-residue_name +plays O +a O +role O +in O +stabilising O +the O +β B-structure_element +‐ I-structure_element +turn I-structure_element +in O +the O +BRC B-structure_element +repeats I-structure_element +, O +which O +is O +absent O +in O +the O +tetrapeptides B-chemical +studied O +. O + +The O +differences O +in O +ΔG B-evidence +for O +different O +peptide O +variants O +relative O +to O +FHTA B-structure_element +are O +shown O +in O +the O +bar O +chart O +with O +colouring O +matching O +with O +the O +structural B-experimental_method +overlay I-experimental_method +below O +. O +( O +C O +) O +Overlay B-experimental_method +of O +tetrapeptide B-chemical +structures B-evidence +, O +with O +wild B-protein_state +‐ I-protein_state +type I-protein_state +FHTA B-structure_element +peptide O +across O +the O +figure O +for O +reference O +and O +truncated O +segments O +of O +mutated O +residues O +shown O +in O +each O +panel O +. O + +Here O +, O +we O +describe O +the O +first O +crystal B-evidence +structure I-evidence +of O +a O +C11 B-protein_type +protein O +from O +the O +human B-species +gut O +bacterium B-taxonomy_domain +, O +Parabacteroides B-species +merdae I-species +( O +PmC11 B-protein +), O +determined O +to O +1 O +. O +7 O +- O +Å O +resolution O +. O + +Biochemical B-experimental_method +and I-experimental_method +kinetic I-experimental_method +analysis I-experimental_method +revealed O +Lys147 B-residue_name_number +to O +be O +an O +intramolecular B-site +processing I-site +site I-site +at O +which O +cleavage B-ptm +is O +required O +for O +full B-protein_state +activation I-protein_state +of O +the O +enzyme B-protein +, O +suggesting O +an O +autoinhibitory O +mechanism O +for O +self O +- O +preservation O +. O + +PmC11 B-protein +has O +an O +acidic B-site +binding I-site +pocket I-site +and O +a O +preference O +for O +basic O +substrates O +, O +and O +accepts O +substrates O +with O +Arg B-residue_name +and O +Lys B-residue_name +in O +P1 B-residue_number +and O +does O +not O +require O +Ca2 B-chemical ++ I-chemical +for O +activity O +. O + +Clan B-protein_type +CD I-protein_type +families I-protein_type +are O +typically O +described O +using O +the O +name O +of O +their O +archetypal O +, O +or O +founding O +, O +member O +and O +also O +given O +an O +identification O +number O +preceded O +by O +a O +“ O +C O +,” O +to O +denote O +cysteine B-protein_type +peptidase I-protein_type +. O + +Clostripain B-protein +has O +been O +described O +as O +an O +arginine B-protein_type +- I-protein_type +specific I-protein_type +peptidase I-protein_type +with O +a O +requirement O +for O +Ca2 B-chemical ++ I-chemical +and O +loss O +of O +an O +internal B-structure_element +nonapeptide I-structure_element +for O +full B-protein_state +activation I-protein_state +; O +lack O +of O +structural O +information O +on O +the O +family O +appears O +to O +have O +prohibited O +further O +investigation O +. O + +A O +single B-ptm +cleavage I-ptm +was O +observed O +in O +the O +polypeptide O +chain O +at O +Lys147 B-residue_name_number +( O +Fig O +. O +1 O +, O +A O +and O +B O +), O +where O +both O +ends O +of O +the O +cleavage B-site +site I-site +are O +fully O +visible O +and O +well O +ordered O +in O +the O +electron B-evidence +density I-evidence +. O + +The O +secondary O +structure O +of O +PmC11 B-protein +from O +the O +crystal B-evidence +structure I-evidence +is O +mapped O +onto O +its O +sequence O +with O +the O +position O +of O +the O +PmC11 B-protein +catalytic B-site +dyad I-site +, O +autocatalytic B-site +cleavage I-site +site I-site +( O +Lys147 B-residue_name_number +), O +and O +S1 B-site +binding I-site +pocket I-site +Asp B-residue_name +( O +Asp177 B-residue_name_number +) O +highlighted O +by O +a O +red O +star O +, O +a O +red O +downturned O +triangle O +, O +and O +a O +red O +upturned O +triangle O +, O +respectively O +. O + +B O +, O +topology O +diagram O +of O +PmC11 B-protein +colored O +as O +in O +A O +except O +that O +additional O +( O +non O +- O +core O +) O +β B-structure_element +- I-structure_element +strands I-structure_element +are O +in O +yellow O +. O + +Helices O +found O +on O +either O +side O +of O +the O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +are O +shown O +above O +and O +below O +the O +sheet B-structure_element +, O +respectively O +. O + +His133 B-residue_name_number +and O +Cys179 B-residue_name_number +were O +found O +at O +locations O +structurally O +homologous O +to O +the O +caspase B-protein_type +catalytic B-site +dyad I-site +, O +and O +other O +clan B-protein_type +CD I-protein_type +structures B-evidence +, O +at O +the O +C O +termini O +of O +strands B-structure_element +β5 B-structure_element +and O +β6 B-structure_element +, O +respectively O +( O +Figs O +. O +1 O +, O +A O +and O +B O +, O +and O +2A O +). O + +Summary O +of O +PDBeFOLD B-experimental_method +superposition I-experimental_method +of O +structures O +found O +to O +be O +most O +similar O +to O +PmC11 B-protein +in O +the O +PBD O +based O +on O +DaliLite B-experimental_method + +The O +overall O +structure B-evidence +of O +PmC11 B-protein +is O +shown O +in O +gray O +, O +looking O +down O +into O +the O +catalytic B-site +site I-site +with O +the O +catalytic B-site +dyad I-site +in O +red O +. O + +A O +single O +lane O +of O +20 O +μg O +of O +active B-protein_state +PmC11 B-protein +( O +labeled O +20 O +) O +is O +shown O +for O +comparison O +. O + +The O +two O +ends O +of O +the O +cleavage B-site +site I-site +are O +remarkably O +well O +ordered O +in O +the O +crystal B-evidence +structure I-evidence +and O +displaced O +from O +one O +another O +by O +19 O +. O +5 O +Å O +( O +Fig O +. O +2A O +). O + +Consequently O +, O +it O +appears O +feasible O +that O +the O +helix B-structure_element +attached O +to O +Lys147 B-residue_name_number +( O +α3 B-structure_element +) O +could O +be O +responsible O +for O +steric O +autoinhibition O +of O +PmC11 B-protein +when O +Lys147 B-residue_name_number +is O +covalently O +bonded O +to O +Ala148 B-residue_name_number +. O + +These O +studies O +revealed O +that O +there O +was O +no O +apparent O +cleavage O +of O +PmC11C179A B-mutant +by O +the O +active B-protein_state +enzyme O +at B-experimental_method +low I-experimental_method +concentrations I-experimental_method +of O +PmC11 B-protein +and O +that O +only O +limited O +cleavage O +was O +observed O +when O +the O +ratio O +of O +active B-protein_state +enzyme O +( O +PmC11 B-protein +: O +PmC11C179A B-mutant +) O +was O +increased B-experimental_method +to I-experimental_method +∼ I-experimental_method +1 I-experimental_method +: I-experimental_method +10 I-experimental_method +and I-experimental_method +1 I-experimental_method +: I-experimental_method +4 I-experimental_method +, O +with O +complete O +cleavage O +observed O +at O +a O +ratio B-experimental_method +of I-experimental_method +1 I-experimental_method +: I-experimental_method +1 I-experimental_method +( O +Fig O +. O +2E O +). O + +This O +cleavage B-ptm +subsequently O +allows O +movement O +of O +the O +region O +containing O +Lys147 B-residue_name_number +and O +the O +active B-site +site I-site +to O +open B-protein_state +up O +for O +substrate O +access O +. O + +The O +cleavage O +of O +Bz B-chemical +- I-chemical +R I-chemical +- I-chemical +AMC I-chemical +by O +PmC11 B-protein +was O +measured O +in O +the O +presence O +of O +the O +cations O +Ca2 B-chemical ++, I-chemical +Mn2 B-chemical ++, I-chemical +Zn2 B-chemical ++, I-chemical +Co2 B-chemical ++, I-chemical +Cu2 B-chemical ++, I-chemical +Mg2 B-chemical ++, I-chemical +and O +Fe3 B-chemical ++ I-chemical +with O +EGTA B-chemical +as O +a O +negative O +control O +, O +and O +relative B-experimental_method +fluorescence I-experimental_method +measured I-experimental_method +against I-experimental_method +time I-experimental_method +( O +min O +). O + +The O +addition B-experimental_method +of I-experimental_method +cations I-experimental_method +produced O +no O +improvement O +in O +activity O +of O +PmC11 B-protein +when O +compared O +in O +the O +presence O +of O +EGTA B-chemical +, O +suggesting O +that O +PmC11 B-protein +does O +not O +require O +metal O +ions O +for O +proteolytic O +activity O +. O + +A O +multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +revealed O +that O +most O +of O +the O +secondary B-structure_element +structural I-structure_element +elements I-structure_element +are O +conserved B-protein_state +between O +the O +two O +enzymes O +, O +although O +they O +are O +only O +∼ O +23 O +% O +identical O +( O +Fig O +. O +1A O +). O + +Nevertheless O +, O +PmC11 B-protein +may O +be O +a O +good O +model O +for O +the O +core O +structure O +of O +clostripain B-protein +. O + +The O +primary B-experimental_method +structural I-experimental_method +alignment I-experimental_method +also O +shows O +that O +the O +catalytic B-site +dyad I-site +in O +PmC11 B-protein +is O +structurally B-protein_state +conserved I-protein_state +in O +clostripain B-protein +( O +Fig O +. O +1A O +). O + +Interestingly O +, O +Arg190 B-residue_name_number +was O +found O +to O +align O +with O +Lys147 B-residue_name_number +in O +PmC11 B-protein +. O + +The O +caspases B-protein_type +and O +gingipain B-protein +- I-protein +R I-protein +both O +undergo O +intermolecular B-ptm +( I-ptm +trans I-ptm +) I-ptm +cleavage I-ptm +and O +legumain B-protein +and O +MARTX B-protein +- I-protein +CPD I-protein +are O +reported O +to O +perform O +intramolecular B-ptm +( I-ptm +cis I-ptm +) I-ptm +cleavage I-ptm +. O + +Eukaryotic B-taxonomy_domain +ribosome O +biogenesis O +is O +highly O +complex O +and O +requires O +a O +large O +number O +of O +non O +- O +ribosomal O +proteins O +and O +small B-chemical +non I-chemical +- I-chemical +coding I-chemical +RNAs I-chemical +in O +addition O +to O +ribosomal B-chemical +RNAs I-chemical +( O +rRNAs B-chemical +) O +and O +proteins O +. O + +While O +in O +humans B-species +the O +18S B-chemical +rRNA I-chemical +base O +modifications O +are O +highly B-protein_state +conserved I-protein_state +, O +only O +three O +of O +the O +yeast B-taxonomy_domain +base O +modifications O +catalyzed O +by O +ScRrp8 B-protein +/ O +HsNML B-protein +, O +ScRcm1 B-protein +/ O +HsNSUN5 B-protein +and O +ScNop2 B-protein +/ O +HsNSUN1 B-protein +are O +preserved O +in O +the O +corresponding O +human B-species +28S B-chemical +rRNA I-chemical +. O + +Tsr3 B-protein +is O +necessary O +for O +acp B-chemical +modification O +of O +18S B-chemical +rRNA I-chemical +in O +yeast B-taxonomy_domain +and O +human B-species +. O +( O +A O +) O +Hypermodified B-protein_state +nucleotide B-chemical +m1acp3Ψ B-chemical +is O +synthesized O +in O +three O +steps O +: O +pseudouridylation B-ptm +catalyzed O +by O +snoRNP35 B-complex_assembly +, O +N1 B-ptm +- I-ptm +methylation I-ptm +catalyzed O +by O +methyltransferase B-protein_type +Nep1 B-protein +and O +N3 O +- O +acp B-chemical +modification O +catalyzed O +by O +Tsr3 B-protein +. O + +( O +C O +) O +14C B-chemical +- I-chemical +acp I-chemical +labeling O +of O +18S B-chemical +rRNAs I-chemical +. O + +In O +a O +recent O +bioinformatic O +study O +, O +the O +uncharacterized O +yeast B-taxonomy_domain +gene O +YOR006c B-gene +was O +predicted O +to O +be O +involved O +in O +ribosome O +biogenesis O +. O + +The O +S B-species +. I-species +cerevisiae I-species +18S B-protein_type +rRNA I-protein_type +acp I-protein_type +transferase I-protein_type +was O +identified O +in O +a O +systematic O +genetic O +screen O +where O +numerous O +deletion O +mutants O +from O +the O +EUROSCARF O +strain O +collection O +( O +www O +. O +euroscarf O +. O +de O +) O +were O +analyzed O +by O +HPLC B-experimental_method +for O +alterations O +in O +18S B-chemical +rRNA I-chemical +base O +modifications O +. O + +No O +radioactive O +labeling O +was O +detected O +in O +the O +18S B-mutant +U1191A I-mutant +mutant B-protein_state +which O +served O +as O +a O +control O +for O +the O +specificity O +of O +the O +14C B-chemical +- I-chemical +aminocarboxypropyl I-chemical +incorporation O +. O + +The O +Tsr3 B-protein +protein O +is O +highly B-protein_state +conserved I-protein_state +in O +yeast B-taxonomy_domain +and O +humans B-species +( O +50 O +% O +identity O +). O + +Similar O +to O +yeast B-taxonomy_domain +, O +siRNA B-experimental_method +- I-experimental_method +mediated I-experimental_method +depletion I-experimental_method +of O +the O +Ψ1248 B-protein_type +N1 I-protein_type +- I-protein_type +methyltransferase I-protein_type +Nep1 B-protein +/ O +Emg1 B-protein +had O +no O +influence O +on O +the O +primer B-evidence +extension I-evidence +arrest I-evidence +( O +Figure O +1E O +). O + +Although O +the O +acp B-chemical +modification O +of O +18S B-chemical +rRNA I-chemical +is O +highly B-protein_state +conserved I-protein_state +in O +eukaryotes B-taxonomy_domain +, O +yeast B-taxonomy_domain +Δtsr3 B-mutant +mutants O +showed O +only O +a O +minor O +growth O +defect O +. O + +In O +polysome B-evidence +profiles I-evidence +, O +a O +reduced O +level O +of O +80S B-complex_assembly +ribosomes I-complex_assembly +and O +a O +strong O +signal O +for O +free O +60S B-complex_assembly +subunits O +was O +observed O +in O +line O +with O +the O +40S B-complex_assembly +subunit O +deficiency O +( O +Supplementary O +Figure O +S2G O +). O + +Structure B-evidence +of O +Tsr3 B-protein + +N O +- O +terminal O +truncations B-experimental_method +of O +up O +to O +45 B-residue_range +aa I-residue_range +and O +C O +- O +terminal O +truncations B-experimental_method +of O +up O +to O +76 B-residue_range +aa I-residue_range +mediated O +acp B-chemical +modification O +as O +efficiently O +as O +the O +full B-protein_state +- I-protein_state +length I-protein_state +protein O +and O +no O +significant O +increased O +levels O +of O +20S B-chemical +pre I-chemical +- I-chemical +RNA I-chemical +were O +detected O +. O + +The O +loop B-structure_element +connecting O +β2 B-structure_element +and O +β3 B-structure_element +contains O +a O +single O +turn O +of O +a O +310 B-structure_element +- I-structure_element +helix I-structure_element +. O +Helices B-structure_element +α1 B-structure_element +and O +α2 B-structure_element +are O +located O +on O +one O +side O +of O +the O +five B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +while O +α3 B-structure_element +packs O +against O +the O +opposite O +β B-structure_element +- I-structure_element +sheet I-structure_element +surface O +. O + +Structure B-experimental_method +predictions I-experimental_method +suggested O +that O +Tsr3 B-protein +might O +contain O +a O +so O +- O +called O +RLI B-structure_element +domain I-structure_element +which O +contains O +a O +‘ O +bacterial B-structure_element +like I-structure_element +’ I-structure_element +ferredoxin I-structure_element +fold I-structure_element +and O +binds O +two O +iron O +- O +sulfur O +clusters O +through O +eight O +conserved B-protein_state +cysteine B-residue_name +residues O +. O + +The O +closest O +structural O +homolog O +identified O +in O +a O +DALI B-experimental_method +search I-experimental_method +is O +the O +tRNA B-protein_type +methyltransferase I-protein_type +Trm10 B-protein +( O +DALI B-evidence +Z I-evidence +- I-evidence +score I-evidence +6 O +. O +8 O +) O +which O +methylates O +the O +N1 O +nitrogen O +of O +G9 B-residue_name_number +/ O +A9 B-residue_name_number +in O +many O +archaeal B-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +tRNAs B-chemical +by O +using O +SAM B-chemical +as O +the O +methyl O +group O +donor O +. O + +A O +notable O +exception O +is O +Trm10 B-protein +. O + +Gel B-experimental_method +filtration I-experimental_method +experiments O +with O +both O +VdTsr3 B-protein +and O +SsTsr3 B-protein +( O +Figure O +4E O +) O +showed O +that O +both O +proteins O +are O +monomeric B-oligomeric_state +in O +solution O +thereby O +extending O +the O +structural O +similarities O +to O +Trm10 B-protein +. O + +This O +enzyme O +, O +Tyw2 B-protein +, O +is O +part O +of O +the O +biosynthesis O +pathway O +of O +wybutosine B-chemical +nucleotides I-chemical +in O +tRNAs B-chemical +. O + +Instead O +, O +Tyw2 B-protein +has O +a O +fold O +typical O +for O +the O +class B-protein_type +- I-protein_type +I I-protein_type +- I-protein_type +or I-protein_type +Rossmann I-protein_type +- I-protein_type +fold I-protein_type +class I-protein_type +of I-protein_type +methyltransferases I-protein_type +( O +Supplementary O +Figure O +S5B O +). O + +A O +W66A B-mutant +- O +mutant B-protein_state +of O +SsTsr3 B-protein +( O +W73 B-residue_name_number +in O +VdTsr3 B-protein +) O +does O +not O +bind O +SAM B-chemical +. O + +( O +F O +) O +Primer B-experimental_method +extension I-experimental_method +( O +upper O +left O +) O +shows O +a O +strongly O +reduced O +acp B-chemical +modification O +of O +yeast B-taxonomy_domain +18S B-chemical +rRNA I-chemical +in O +Δtsr3 B-mutant +cells O +expressing O +Tsr3 B-mutant +- I-mutant +S62D I-mutant +, O +- B-mutant +E111A I-mutant +or O +– B-mutant +W114A I-mutant +. O + +S B-chemical +- I-chemical +adenosylhomocysteine I-chemical +which O +lacks O +the O +methyl O +group O +of O +SAM B-chemical +binds O +with O +significantly O +lower O +affinity B-evidence +( O +KD B-evidence += O +55 O +. O +5 O +μM O +) O +( O +Figure O +5D O +). O + +However O +, O +the O +mutation B-experimental_method +of O +the O +corresponding O +residue O +of O +ScTsr3 B-protein +( O +E111A B-mutant +) O +leads O +to O +a O +significant O +decrease O +of O +the O +acp B-protein_type +transferase I-protein_type +activity O +in O +vivo O +( O +Figure O +5F O +). O + +In O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +, O +the O +surface O +exposed O +α B-structure_element +- I-structure_element +helices I-structure_element +α5 B-structure_element +and O +α7 B-structure_element +carry O +a O +significant O +amount O +of O +positively O +charged O +amino O +acids O +. O + +The O +m1acp3Ψ B-chemical +base O +is O +located O +at O +the O +tip O +of O +helix B-structure_element +31 I-structure_element +on O +the O +18S B-chemical +rRNA I-chemical +( O +Supplementary O +Figure O +S1B O +) O +which O +, O +together O +with O +helices B-structure_element +18 I-structure_element +, I-structure_element +24 I-structure_element +, I-structure_element +34 I-structure_element +and I-structure_element +44 I-structure_element +, O +contribute O +to O +building O +the O +decoding O +center O +of O +the O +small O +ribosomal O +subunit O +. O + +This O +suggests O +that O +enzymes O +with O +a O +SAM B-protein_type +- I-protein_type +dependent I-protein_type +acp I-protein_type +transferase I-protein_type +activity O +might O +have O +evolved O +from O +SAM B-protein_type +- I-protein_type +dependent I-protein_type +methyltransferases I-protein_type +by O +slight O +modifications O +of O +the O +SAM B-site +- I-site +binding I-site +pocket I-site +. O + +In O +contrast O +, O +for O +several O +Tsr3 B-protein +mutants O +( O +SAM B-protein_state +- I-protein_state +binding I-protein_state +and O +cysteine B-protein_state +mutations I-protein_state +) O +we O +found O +a O +systematic O +correlation O +between O +the O +loss O +of O +acp B-chemical +modification O +and O +the O +efficiency O +of O +18S B-chemical +rRNA I-chemical +maturation O +. O + +After O +structural O +changes O +, O +possibly O +driven O +by O +GTP B-chemical +hydrolysis O +, O +which O +go O +together O +with O +the O +formation O +of O +the O +decoding B-site +site I-site +, O +the O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +becomes O +accessible O +for O +Nob1 B-protein +cleavage O +at O +site B-site +D I-site +. O +This O +also O +involves O +joining O +of O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +and O +60S B-complex_assembly +subunits I-complex_assembly +to O +80S B-complex_assembly +- I-complex_assembly +like I-complex_assembly +particles I-complex_assembly +in O +a O +translation O +- O +like O +cycle O +promoted O +by O +eIF5B B-protein +. O + +Finally O +, O +termination B-protein_type +factor I-protein_type +Rli1 B-protein +, O +an O +ATPase B-protein_type +, O +promotes O +the O +dissociation O +of O +assembly O +factors O +and O +the O +80S B-complex_assembly +- I-complex_assembly +like I-complex_assembly +complex I-complex_assembly +dissociates O +and O +releases O +the O +mature B-protein_state +40S B-complex_assembly +subunit I-complex_assembly +. O + +Early O +cytoplasmic O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +subunits I-complex_assembly +still O +containing O +the O +ribosome B-protein_type +assembly I-protein_type +factors I-protein_type +Tsr1 B-protein +, O +Ltv1 B-protein +, O +Enp1 B-protein +and O +Rio2 B-protein +were O +not O +or O +only O +partially O +acp B-protein_state +modified I-protein_state +. O + +Therefore O +, O +Rio2 B-protein +either O +blocks O +the O +access O +of O +Tsr3 B-protein +to O +helix B-structure_element +31 I-structure_element +, O +and O +acp B-chemical +modification O +can O +only O +occur O +after O +Rio2 B-protein +is O +released O +, O +or O +the O +acp B-chemical +modification O +of O +m1Ψ1191 B-residue_name_number +and O +putative O +subsequent O +conformational O +changes O +of O +20S B-chemical +rRNA I-chemical +weaken O +the O +binding O +of O +Rio2 B-protein +to O +helix B-structure_element +31 I-structure_element +and O +support O +its O +release O +from O +the O +pre B-chemical +- I-chemical +rRNA I-chemical +. O + +Structural B-experimental_method +analyses I-experimental_method +revealed O +that O +in O +contrast O +to O +the O +compact B-protein_state +conformation I-protein_state +of O +the O +dimeric B-oligomeric_state +YfiB B-protein +alone B-protein_state +, O +YfiBL43P B-mutant +adopts O +a O +stretched B-protein_state +conformation I-protein_state +allowing O +activated B-protein_state +YfiB B-protein +to O +penetrate O +the O +peptidoglycan B-chemical +( O +PG B-chemical +) O +layer O +and O +access O +YfiR B-protein +. O +YfiBL43P B-mutant +shows O +a O +more O +compact O +PG B-site +- I-site +binding I-site +pocket I-site +and O +much O +higher O +PG B-evidence +binding I-evidence +affinity I-evidence +than O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +, O +suggesting O +a O +tight O +correlation O +between O +PG O +binding O +and O +YfiB B-protein +activation O +. O + +Bis B-chemical +-( I-chemical +3 I-chemical +’- I-chemical +5 I-chemical +’)- I-chemical +cyclic I-chemical +dimeric I-chemical +GMP I-chemical +( O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +) O +is O +a O +ubiquitous O +second O +messenger O +that O +bacteria B-taxonomy_domain +use O +to O +facilitate O +behavioral O +adaptations O +to O +their O +ever O +- O +changing O +environment O +. O + +Intriguingly O +, O +studies O +in O +diverse O +species O +have O +revealed O +that O +a O +single O +bacterium B-taxonomy_domain +can O +have O +dozens O +of O +DGCs B-protein_type +and O +PDEs B-protein_type +( O +Hickman O +et O +al O +.,; O +Kirillina O +et O +al O +.,; O +Kulasakara O +et O +al O +.,; O +Tamayo O +et O +al O +.,). O + +After O +the O +sequestration O +of O +YfiR B-protein +by O +YfiB B-protein +, O +the O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +produced O +by O +activated B-protein_state +YfiN B-protein +increases O +the O +biosynthesis O +of O +the O +Pel B-chemical +and O +Psl B-chemical +EPSs B-chemical +, O +resulting O +in O +the O +appearance O +of O +the O +SCV O +phenotype O +, O +which O +indicates O +enhanced O +biofilm O +formation O +( O +Malone O +et O +al O +.,). O + +In O +the O +present O +study O +, O +we O +solved O +the O +crystal B-evidence +structures I-evidence +of O +an O +N O +- O +terminal O +truncated B-protein_state +form O +of O +YfiB B-protein +( O +34 B-residue_range +– I-residue_range +168 I-residue_range +) O +and O +YfiR B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +an O +active B-protein_state +mutant B-protein_state +YfiBL43P B-mutant +. O + +Moreover O +, O +we O +found O +that O +Vitamin B-chemical +B6 I-chemical +( O +VB6 B-chemical +) O +or O +L B-chemical +- I-chemical +Trp I-chemical +can O +bind O +YfiR B-protein +with O +an O +affinity B-evidence +in O +the O +ten O +millimolar O +range O +. O + +We O +obtained O +two O +crystal B-evidence +forms I-evidence +of O +YfiB B-protein +( O +residues O +34 B-residue_range +– I-residue_range +168 I-residue_range +, O +lacking B-protein_state +the O +signal B-structure_element +peptide I-structure_element +from O +residues O +1 B-residue_range +– I-residue_range +26 I-residue_range +and O +periplasmic O +residues O +27 B-residue_range +– I-residue_range +33 I-residue_range +), O +crystal O +forms O +I O +and O +II O +, O +belonging O +to O +space O +groups O +P21 O +and O +P41 O +, O +respectively O +. O + +Two O +dimeric B-oligomeric_state +types O +of O +YfiB B-protein +dimer B-oligomeric_state +. O +( O +A O +– O +C O +) O +The O +“ O +head B-protein_state +to I-protein_state +head I-protein_state +” O +dimer B-oligomeric_state +. O + +( O +A O +) O +and O +( O +E O +) O +indicate O +the O +front O +views O +of O +the O +two O +dimers B-oligomeric_state +, O +( O +B O +) O +and O +( O +F O +) O +indicate O +the O +top O +views O +of O +the O +two O +dimers B-oligomeric_state +, O +and O +( O +C O +) O +and O +( O +D O +) O +indicate O +the O +details O +of O +the O +two O +dimeric B-site +interfaces I-site + +The O +crystal B-evidence +structure I-evidence +of O +YfiB B-protein +monomer B-oligomeric_state +consists O +of O +a O +five B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +( O +β1 B-structure_element +- I-structure_element +2 I-structure_element +- I-structure_element +5 I-structure_element +- I-structure_element +3 I-structure_element +- I-structure_element +4 I-structure_element +) O +flanked O +by O +five B-structure_element +α I-structure_element +- I-structure_element +helices I-structure_element +( O +α1 B-structure_element +– I-structure_element +5 I-structure_element +) O +on O +one O +side O +. O + +The O +dimeric B-oligomeric_state +interaction B-bond_interaction +is I-bond_interaction +mainly I-bond_interaction +hydrophilic I-bond_interaction +, O +occurring O +among O +the O +main O +- O +chain O +and O +side O +- O +chain O +atoms O +of O +N68 B-residue_name_number +, O +L69 B-residue_name_number +, O +D70 B-residue_name_number +and O +R71 B-residue_name_number +on O +the O +α2 B-structure_element +- I-structure_element +α3 I-structure_element +loops I-structure_element +and O +R116 B-residue_name_number +and O +S120 B-residue_name_number +on O +the O +α4 B-structure_element +helices I-structure_element +of O +both O +molecules O +, O +resulting O +in O +a O +complex O +hydrogen B-site +bond I-site +network I-site +( O +Fig O +. O +2D O +– O +F O +). O + +The O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +interaction O + +The O +YfiR B-protein +molecules O +are O +shown O +in O +green O +and O +magenta O +. O + +It O +is O +likely O +that O +these O +residues O +may O +be O +involved O +in O +the O +conformational O +changes O +of O +YfiB B-protein +that O +are O +related O +to O +YfiR B-protein +sequestration O +( O +Fig O +. O +3C O +). O + +Additionally O +, O +three O +hydrophobic B-site +anchoring I-site +sites I-site +exist O +in O +region B-structure_element +I I-structure_element +. O +The O +residues O +F48 B-residue_name_number +and O +W55 B-residue_name_number +of O +YfiB B-protein +are O +inserted O +into O +the O +hydrophobic B-site +cores I-site +mainly O +formed O +by O +the O +main O +chain O +and O +side O +chain O +carbon O +atoms O +of O +residues O +S57 B-residue_name_number +/ O +Q88 B-residue_name_number +/ O +A89 B-residue_name_number +/ O +N90 B-residue_name_number +and O +R60 B-residue_name_number +/ O +R175 B-residue_name_number +/ O +H177 B-residue_name_number +of O +YfiR B-protein +, O +respectively O +; O +and O +F57 B-residue_name_number +of O +YfiB B-protein +is O +inserted O +into O +the O +hydrophobic B-site +pocket I-site +formed O +by O +L166 B-residue_name_number +/ O +I169 B-residue_name_number +/ O +V176 B-residue_name_number +/ O +P178 B-residue_name_number +/ O +L181 B-residue_name_number +of O +YfiR B-protein +( O +Fig O +. O +3D O +- O +I O +( O +ii O +)). O + +For O +simplicity O +, O +we O +only O +discuss O +the O +“ O +head B-protein_state +to I-protein_state +head I-protein_state +” O +dimer B-oligomeric_state +in O +the O +following O +text O +. O + +PG B-protein_type +- I-protein_type +associated I-protein_type +lipoprotein I-protein_type +( O +Pal B-protein_type +) O +is O +highly B-protein_state +conserved I-protein_state +in O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +bacteria I-taxonomy_domain +and O +anchors O +to O +the O +outer O +membrane O +through O +an O +N O +- O +terminal O +lipid O +attachment O +and O +to O +PG B-chemical +layer O +through O +its O +periplasmic B-structure_element +domain I-structure_element +, O +which O +is O +implicated O +in O +maintaining O +outer O +membrane O +integrity O +. O + +Interestingly O +, O +superposition B-experimental_method +of O +apo B-protein_state +YfiB B-protein +with O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +revealed O +that O +the O +PG B-site +- I-site +binding I-site +region I-site +is O +largely O +altered O +mainly O +due O +to O +different B-protein_state +conformation I-protein_state +of O +the O +N68 B-residue_name_number +containing O +loop B-structure_element +. O + +The O +results O +indicated O +that O +the O +PG B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +YfiBL43P B-mutant +is O +65 O +. O +5 O +μmol O +/ O +L O +, O +which O +is O +about O +16 O +- O +fold O +stronger O +than O +that O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +( O +Kd B-evidence += O +1 O +. O +1 O +mmol O +/ O +L O +) O +( O +Fig O +. O +4E O +– O +F O +). O + +Calculation O +using O +the O +ConSurf B-experimental_method +Server I-experimental_method +( O +http O +:// O +consurf O +. O +tau O +. O +ac O +. O +il O +/), O +which O +estimates O +the O +evolutionary B-evidence +conservation I-evidence +of O +amino O +acid O +positions O +and O +visualizes O +information O +on O +the O +structure B-site +surface I-site +, O +revealed O +a O +conserved B-site +surface I-site +on O +YfiR B-protein +that O +contributes O +to O +the O +interaction O +with O +YfiB B-protein +( O +Fig O +. O +3E O +and O +3F O +). O + +Previous O +studies O +indicated O +that O +YfiR B-protein +constitutes O +a O +YfiB B-protein +- O +independent O +sensing O +device O +that O +can O +activate O +YfiN B-protein +in O +response O +to O +the O +redox O +status O +of O +the O +periplasm O +, O +and O +we O +have O +reported O +YfiR B-protein +structures B-evidence +in O +both O +the O +non B-protein_state +- I-protein_state +oxidized I-protein_state +and O +the O +oxidized B-protein_state +states O +earlier O +, O +revealing O +that O +the O +Cys145 B-residue_name_number +- O +Cys152 B-residue_name_number +disulfide B-ptm +bond I-ptm +plays O +an O +essential O +role O +in O +maintaining O +the O +correct O +folding O +of O +YfiR B-protein +( O +Yang O +et O +al O +.,). O + +Interestingly O +, O +at O +a O +concentration O +higher O +than O +8 O +mmol O +/ O +L O +, O +VB6 B-chemical +lost O +its O +ability O +to O +inhibit O +biofilm O +formation O +, O +implying O +that O +the O +VB6 B-chemical +- O +involving O +regulatory O +mechanism O +is O +highly O +complicated O +and O +remains O +to O +be O +further O +investigated O +. O + +Of O +note O +, O +both O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +have O +been O +reported O +to O +correlate O +with O +biofilm O +formation O +in O +certain O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +bacteria I-taxonomy_domain +( O +Grubman O +et O +al O +.,; O +Shimazaki O +et O +al O +.,). O + +Based O +on O +our O +results O +, O +we O +concluded O +that O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +can O +bind O +to O +YfiR B-protein +, O +however O +, O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +alone B-protein_state +may O +have O +little O +effects O +in O +interrupting O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +interaction O +, O +the O +mechanism O +by O +which O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +inhibits O +biofilm O +formation O +remains O +unclear O +and O +requires O +further O +investigation O +. O + +Our O +structural B-experimental_method +data I-experimental_method +analysis I-experimental_method +shows O +that O +the O +activated B-protein_state +YfiB B-protein +has O +an O +N B-structure_element +- I-structure_element +terminal I-structure_element +portion I-structure_element +that O +is O +largely O +altered O +, O +adopting O +a O +stretched B-protein_state +conformation I-protein_state +compared O +with O +the O +compact B-protein_state +conformation I-protein_state +of O +the O +apo B-protein_state +YfiB B-protein +. O +The O +apo B-protein_state +YfiB B-protein +structure B-evidence +constructed O +beginning O +at O +residue O +34 B-residue_number +has O +a O +compact B-protein_state +conformation I-protein_state +of O +approximately O +45 O +Å O +in O +length O +. O + +The O +loop B-structure_element +connecting O +Cys26 B-residue_name_number +and O +Gly34 B-residue_name_number +of O +YfiB B-protein +is O +modeled O +. O + +This O +allows O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +portion I-structure_element +of O +the O +membrane B-protein_state +- I-protein_state +anchored I-protein_state +YfiB B-protein +to O +reach O +, O +bind O +and O +penetrate O +the O +cell O +wall O +and O +sequester O +the O +YfiR B-protein +dimer B-oligomeric_state +. O + +Our O +study O +reveals O +the O +mechanism O +for O +regulation O +of O +H3K9me3 B-protein_type +and O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +by O +URHF1 B-protein +. O + +However O +, O +how O +UHRF1 B-protein +regulates O +the O +recognition O +of O +these O +two O +repressive O +epigenetic O +marks O +and O +recruits O +DNMT1 B-protein +for O +chromatin O +localization O +remain O +largely O +unknown O +. O + +Therefore O +, O +UHRF1 B-protein +may O +engage O +in O +a O +sophisticated O +regulation O +for O +its O +chromatin O +localization O +and O +recruitment O +of O +DNMT1 B-protein +through O +a O +mechanism O +yet O +to O +be O +fully O +elucidated O +. O + +To O +test O +above O +hypothesis O +, O +we O +performed O +glutathione B-experimental_method +S I-experimental_method +- I-experimental_method +transferase I-experimental_method +( I-experimental_method +GST I-experimental_method +) I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +using O +various O +truncations B-experimental_method +of O +UHRF1 B-protein +. O + +The O +presence B-protein_state +of I-protein_state +the O +Spacer B-structure_element +markedly O +impaired O +the O +interaction O +between O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +and O +H3K9me3 B-protein_type +( O +Fig O +. O +2c O +). O + +The O +results O +indicate O +that O +the O +Spacer B-structure_element +directly O +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +and O +inhibits O +its O +interaction O +with O +H3K9me3 B-protein_type +. O + +Pre B-experimental_method +- I-experimental_method +incubation I-experimental_method +of O +the O +SRA B-structure_element +also O +modestly O +impaired O +PHD B-structure_element +– O +H3K9me0 B-protein_type +interaction O +. O + +SpacerΔ642 B-mutant +– I-mutant +651 I-mutant +, O +SpacerΔ650 B-mutant +– I-mutant +654 I-mutant +and O +SpacerΔ655 B-mutant +– I-mutant +659 I-mutant +also O +decreased O +binding B-evidence +affinities I-evidence +, O +indicating O +that O +residues O +642 B-residue_range +– I-residue_range +674 I-residue_range +are O +important O +for O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +. O + +Mutations B-experimental_method +K648D B-mutant +and O +S651D B-mutant +of O +the O +Spacer B-structure_element +decreased O +their O +binding B-evidence +affinities I-evidence +to O +the O +TTD B-structure_element +, O +and O +mutation B-experimental_method +R649A B-mutant +of O +the O +Spacer B-structure_element +showed O +more O +significant O +decrease O +(∼ O +13 O +- O +fold O +) O +in O +the O +binding B-evidence +affinity I-evidence +( O +Fig O +. O +3f O +). O + +Thus O +, O +the O +Spacer B-structure_element +may O +disrupt O +the O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +interaction O +and O +inhibits O +the O +recognition O +of O +H3K9me3 B-protein_type +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +. O + +Notably O +, O +although O +the O +Linker B-structure_element +( O +in O +the O +context O +of O +TTD B-structure_element +- I-structure_element +PHD I-structure_element +) O +impairs O +the O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +to O +some O +extent O +, O +the O +isolated O +Spacer B-structure_element +could O +still O +bind O +to O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +with O +moderate O +binding B-evidence +affinity I-evidence +( O +KD B-evidence += O +10 O +. O +68 O +μM O +), O +supporting O +the O +existence O +of O +the O +intramolecular O +interaction O +within O +UHRF1 B-protein +. O + +To O +test O +whether O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +association O +exists O +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +, O +we O +used O +various O +truncations B-experimental_method +of O +UHRF1 B-protein +in O +the O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +. O + +Taken O +together O +, O +UHRF1 B-protein +adopts O +a O +closed B-protein_state +conformation O +, O +in O +which O +the O +Spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +through O +competing O +with O +the O +Linker B-structure_element +, O +and O +therefore O +inhibits O +H3K9me3 B-protein_type +recognition O +by O +UHRF1 B-protein +. O + +Because O +the O +TTD B-structure_element +is O +always O +associated O +with O +the O +PHD B-structure_element +, O +whether O +the O +pattern O +of O +TTD B-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +interaction O +exists O +in O +vivo O +remains O +unknown O +. O + +Nevertheless O +, O +comparison B-experimental_method +of O +TTD B-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +and O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +structures B-evidence +indicates O +that O +H3K9me3 B-protein_type +and O +the O +Spacer B-structure_element +overlap O +on O +the O +surface O +of O +the O +TTD B-structure_element +( O +Supplementary O +Fig O +. O +4d O +), O +suggesting O +that O +the O +Spacer B-structure_element +might O +block O +the O +H3K9me3 B-protein_type +recognition O +by O +the O +isolated O +TTD B-structure_element +. O + +We O +next O +tested O +whether O +such O +inhibition O +also O +occurs O +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +. O + +Taken O +together O +, O +the O +Spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +and O +inhibits O +H3K9me3 B-protein_type +recognition O +by O +UHRF1 B-protein +through O +( O +i O +) O +disrupting O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +interaction O +, O +which O +is O +essential O +for O +H3K9me3 B-protein_type +recognition O +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +, O +( O +ii O +) O +prohibiting O +H3K9me3 B-protein_type +binding O +to O +the O +isolated O +TTD B-structure_element +. O + +Residues O +N228 B-residue_name_number +/ O +R235 B-residue_name_number +from O +the O +TTD B-structure_element +and O +G653 B-residue_name_number +/ O +G654 B-residue_name_number +from O +the O +Spacer B-structure_element +were O +chosen O +according O +to O +the O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +complex O +structure B-evidence +( O +Supplementary O +Fig O +. O +5c O +) O +so O +that O +the O +replaced O +Cysteine B-residue_name +residues O +( O +one O +from O +the O +TTD B-structure_element +and O +one O +from O +the O +Spacer B-structure_element +) O +are O +physically O +close O +enough O +to O +each O +other O +to O +form O +a O +disulphide B-ptm +bond I-ptm +in O +the O +absence B-protein_state +of I-protein_state +reducing O +reagent O +( O +dithiothreitol B-chemical +, O +DTT B-chemical +). O + +These O +results O +indicate O +that O +the O +Spacer B-structure_element +not O +only O +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +and O +inhibits O +H3K9me3 B-protein_type +recognition O +when O +UHRF1 B-protein +adopts O +closed B-protein_state +conformation O +, O +but O +also O +facilitates O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +by O +the O +SRA B-structure_element +when O +UHRF1 B-protein +binds B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +. O + +The O +structure B-evidence +shows O +that O +the O +SRA B-structure_element +binds B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +in O +a O +manner O +similar O +to O +that O +observed O +in O +the O +previously O +reported O +SRA B-complex_assembly +- I-complex_assembly +hm I-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +structures B-evidence +. O + +To O +investigate O +the O +role O +of O +the O +Spacer B-structure_element +in O +the O +regulation O +of O +UHRF1 B-protein +function O +, O +we O +transiently B-experimental_method +overexpressed I-experimental_method +GFP B-protein_state +- I-protein_state +tagged I-protein_state +wild B-protein_state +type I-protein_state +or O +mutants B-protein_state +of O +UHRF1 B-protein +in O +NIH3T3 O +cells O +to O +determine O +their O +subcellular O +localization O +. O + +For O +example O +, O +UHRF1 B-protein +mutant B-protein_state +( O +within O +TTD B-structure_element +domain O +) O +lacking B-protein_state +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +largely O +reduces O +its O +co O +- O +localization O +with O +heterochromatin O +. O + +In O +the O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +, O +only O +UHRF1ΔTTD B-mutant +bound B-protein_state +to I-protein_state +RFTSDNMT1 B-protein +, O +whereas O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +, O +UHRF1ΔSRA B-mutant +and O +UHRF1Δ627 B-mutant +– I-mutant +674 I-mutant +showed O +undetectable O +interaction O +( O +Fig O +. O +5e O +). O + +In O +addition O +, O +this O +regulatory O +process O +should O +be O +further O +characterized O +using O +advanced O +techniques O +, O +such O +as O +single B-experimental_method +molecular I-experimental_method +measurement I-experimental_method +. O + +Intriguingly O +, O +UHRF2 B-protein +( O +the O +only O +mammalian B-taxonomy_domain +homologue O +of O +UHRF1 B-protein +) O +and O +UHRF1 B-protein +show O +very O +high O +sequence O +similarities O +for O +all O +the O +domains O +but O +very O +low O +similarity O +for O +the O +Spacer B-structure_element +( O +Supplementary O +Fig O +. O +7c O +). O + +One O +of O +the O +key O +questions O +in O +the O +field O +of O +DNA B-chemical +methylation B-ptm +is O +why O +UHRF1 B-protein +contains O +modules O +recognizing O +two O +repressive O +epigenetic O +marks O +: O +H3K9me3 B-protein_type +( O +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +) O +and O +hm B-chemical +- I-chemical +DNA I-chemical +( O +by O +the O +SRA B-structure_element +). O + +Previous O +studies O +show O +that O +chromatin O +localization O +of O +UHRF1 B-protein +is O +dependent O +on O +hm B-chemical +- I-chemical +DNA I-chemical +, O +whereas O +other O +studies O +indicate O +that O +histone B-protein_type +H3K9me3 B-protein_type +recognition O +and O +hm B-chemical +- I-chemical +DNA I-chemical +association O +are O +both O +required O +for O +UHRF1 B-protein +- O +mediated O +maintenance O +DNA B-chemical +methylation B-ptm +. O + +Therefore O +, O +genomic O +localization O +of O +UHRF1 B-protein +is O +primarily O +determined O +by O +its O +recognition O +of O +hm B-chemical +- I-chemical +DNA I-chemical +, O +which O +allows O +UHRF1 B-protein +to O +adopt O +an O +open B-protein_state +form O +and O +promotes O +its O +histone B-protein_type +tail O +recognition O +for O +proper O +genomic O +localization O +and O +function O +. O + +Recent O +study O +indicates O +that O +histone B-protein_type +tail O +association O +of O +UHRF1 B-protein +( O +by O +the O +PHD B-structure_element +domain O +) O +is O +required O +for O +histone B-protein_type +H3 B-protein_type +ubiquitylation B-ptm +, O +which O +is O +dependent O +on O +ubiquitin B-protein_type +ligase I-protein_type +activity O +of O +the O +RING B-structure_element +domain O +of O +UHRF1 B-protein +( O +ref O +.). O + +Moreover O +, O +structural B-experimental_method +analyses I-experimental_method +of O +DNMT1 B-complex_assembly +– I-complex_assembly +DNA I-complex_assembly +and O +SRA B-complex_assembly +– I-complex_assembly +DNA I-complex_assembly +complexes O +also O +indicate O +that O +it O +is O +impossible O +for O +DNMT1 B-protein +to O +methylate O +the O +hm B-chemical +- I-chemical +DNA I-chemical +that O +UHRF1 B-protein +binds B-protein_state +to I-protein_state +because O +of O +steric O +hindrance O +. O + +The O +S O +phase O +- O +dependent O +interaction O +between O +UHRF1 B-protein +and O +DNMT1 B-protein +( O +refs O +) O +suggest O +that O +DNMT1 B-protein +may O +also O +undergo O +conformation O +changes O +so O +that O +RFTSDNMT1 B-protein +binds B-protein_state +to I-protein_state +UHRF1 B-protein +and O +the O +catalytic B-structure_element +domain I-structure_element +of O +DNMT1 B-protein +binds B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +for O +reaction O +. O + +The O +bound O +proteins O +were O +analysed O +in O +SDS B-experimental_method +– I-experimental_method +PAGE I-experimental_method +followed O +by O +Coomassie O +blue O +staining O +. O + +Sequences O +of O +the O +peptides O +are O +indicated O +in O +Supplementary O +Table O +1 O +. O +( O +c O +) O +Histone B-protein_type +peptides O +do O +not O +affect O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +UHRF1 B-protein +. O + +Intramolecular O +interactions O +inhibit O +histone B-protein_type +recognition O +by O +UHRF1 B-protein +. O + +The O +estimated O +binding B-evidence +affinities I-evidence +( O +KD B-evidence +) O +were O +listed O +. O + +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +complex O +is O +coloured O +in O +grey O +, O +and O +the O +PHD B-structure_element +and O +H3K9me3 B-protein_type +are O +omitted O +for O +simplicity O +. O + +The O +bound O +proteins O +were O +analysed O +in O +SDS B-experimental_method +– I-experimental_method +PAGE I-experimental_method +and O +Coomassie B-experimental_method +blue I-experimental_method +staining I-experimental_method +( O +left O +) O +and O +quantified O +by O +band B-experimental_method +densitometry I-experimental_method +( O +right O +). O + +( O +d O +) O +Histone B-experimental_method +peptide I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +using O +UHRF1 B-protein +mutants B-protein_state +as O +indicated O +. O + +The O +estimated O +binding B-evidence +affinities I-evidence +( O +KD B-evidence +) O +are O +listed O +above O +. O +( O +d O +) O +Subcellular O +localization O +of O +GFP B-protein_state +- I-protein_state +tagged I-protein_state +wild B-protein_state +- I-protein_state +type I-protein_state +or O +indicated O +mutants B-protein_state +of O +UHRF1 B-protein +in O +NIH3T3 O +cells O +. O + +The O +percentages O +of O +cells O +showing O +co O +- O +localization O +with O +DAPI B-chemical +foci O +were O +counted O +from O +at O +least O +100 O +cells O +and O +shown O +on O +the O +left O +of O +the O +corresponding O +representative O +confocal B-experimental_method +microscopy I-experimental_method +. O + +This O +paper O +presents O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structures I-evidence +of O +oligomers B-oligomeric_state +formed O +by O +a O +20 B-residue_range +- I-residue_range +residue I-residue_range +peptide I-residue_range +segment I-residue_range +derived O +from O +Aβ B-protein +. O + +The O +development O +of O +a O +peptide O +in O +which O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +is O +stabilized O +as O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +is O +described O +, O +and O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structures I-evidence +of O +oligomers B-oligomeric_state +it O +forms O +are O +reported O +. O + +An O +N O +- O +methyl O +group O +at O +position O +33 B-residue_number +blocks O +uncontrolled O +aggregation O +. O + +The O +peptide O +readily B-evidence +crystallizes I-evidence +as O +a O +folded B-protein_state +β B-structure_element +- I-structure_element +hairpin I-structure_element +, O +which O +assembles O +hierarchically O +in O +the O +crystal B-evidence +lattice I-evidence +. O + +Treatment O +of O +the O +mixture O +of O +low O +molecular O +weight O +oligomers B-oligomeric_state +with O +hexafluoroisopropanol B-chemical +resulted O +in O +the O +dissociation O +of O +the O +putative O +dodecamers B-oligomeric_state +, O +nonamers B-oligomeric_state +, O +and O +hexamers B-oligomeric_state +into O +trimers B-oligomeric_state +and O +monomers B-oligomeric_state +, O +suggesting O +that O +trimers B-oligomeric_state +may O +be O +the O +building O +block O +of O +the O +dodecamers B-oligomeric_state +, O +nonamers B-oligomeric_state +, O +and O +hexamers B-oligomeric_state +. O + +The O +sizes O +of O +APFs B-complex_assembly +prepared O +in O +vitro O +vary O +among O +different O +studies O +. O + +Quist O +et O +al O +. O +observed O +APFs B-complex_assembly +with O +an O +outer O +diameter O +of O +16 O +nm O +embedded O +in O +a O +lipid O +bilayer O +. O + +This O +Aβ B-protein +β B-structure_element +- I-structure_element +hairpin I-structure_element +encompasses O +residues O +17 B-residue_range +– I-residue_range +37 I-residue_range +and O +contains O +two O +β B-structure_element +- I-structure_element +strands I-structure_element +comprising O +Aβ17 B-protein +– B-residue_range +24 I-residue_range +and O +Aβ30 B-protein +– B-residue_range +37 I-residue_range +connected O +by O +an O +Aβ25 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +. O + +Locking O +Aβ B-protein +into O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +structure O +resulted O +in O +the O +formation O +Aβ B-protein +oligomers B-oligomeric_state +, O +which O +were O +observed O +by O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +( O +SEC B-experimental_method +) O +and O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +. O + +We O +mutated B-experimental_method +these O +residues O +because O +they O +occupy O +the O +same O +position O +as O +the O +δOrn B-structure_element +that O +connects O +D23 B-residue_name_number +and O +A30 B-residue_name_number +in O +peptide B-mutant +1 I-mutant +. O + +After O +determining O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +4 I-mutant +we O +reintroduced B-experimental_method +the O +native O +phenylalanine B-residue_name +at O +position O +19 B-residue_number +and O +the O +methionine B-residue_name +at O +position O +35 B-residue_number +to O +afford O +peptide B-mutant +2 I-mutant +. O + +Peptides B-mutant +2 I-mutant +– I-mutant +4 I-mutant +were O +purified O +by O +RP B-experimental_method +- I-experimental_method +HPLC I-experimental_method +. O + +The O +optimized O +conditions O +consist O +of O +0 O +. O +1 O +M O +HEPES B-chemical +at O +pH O +6 O +. O +4 O +with O +31 O +% O +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +for O +peptide B-mutant +4 I-mutant +and O +0 O +. O +1 O +M O +HEPES B-chemical +pH O +7 O +. O +1 O +with O +29 O +% O +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +for O +peptide B-mutant +2 I-mutant +. O + +The O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +2 I-mutant +reveals O +that O +it O +folds O +to O +form O +a O +twisted B-structure_element +β I-structure_element +- I-structure_element +hairpin I-structure_element +comprising O +two O +β B-structure_element +- I-structure_element +strands I-structure_element +connected O +by O +a O +loop B-structure_element +( O +Figure O +2A O +). O + +The O +disulfide B-ptm +linkages I-ptm +suffered O +radiation O +damage O +under O +synchrotron O +radiation O +. O + +Two O +crystallographically O +distinct O +trimers B-oligomeric_state +comprise O +the O +peptide B-chemical +portion O +of O +the O +asymmetric O +unit O +. O + +A O +network O +of O +18 O +intermolecular O +hydrogen B-bond_interaction +bonds I-bond_interaction +helps O +stabilize O +the O +trimer B-oligomeric_state +. O + +Hydrophobic B-bond_interaction +contacts I-bond_interaction +between O +residues O +at O +the O +three O +corners O +of O +the O +trimer B-oligomeric_state +, O +where O +the O +β B-structure_element +- I-structure_element +hairpins I-structure_element +meet O +, O +further O +stabilize O +the O +trimer B-oligomeric_state +. O + +Each O +of O +the O +12 O +β B-structure_element +- I-structure_element +hairpins I-structure_element +constitutes O +an O +edge O +of O +the O +octahedron B-protein_state +, O +and O +the O +triangular B-protein_state +trimers B-oligomeric_state +occupy O +four O +of O +the O +eight O +faces O +of O +the O +octahedron B-protein_state +. O + +Residues O +L17 B-residue_name_number +, O +L34 B-residue_name_number +, O +and O +V36 B-residue_name_number +are O +shown O +as O +spheres O +, O +illustrating O +the O +hydrophobic B-bond_interaction +packing I-bond_interaction +that O +occurs O +at O +the O +six O +vertices O +of O +the O +dodecamer B-oligomeric_state +. O +( O +D O +) O +Detailed O +view O +of O +one O +of O +the O +six O +vertices O +of O +the O +dodecamer B-oligomeric_state +. O + +The O +exterior O +of O +the O +dodecamer B-oligomeric_state +displays O +four O +F20 B-residue_name_number +faces O +( O +Figure O +S3 O +). O + +Two O +morphologically O +distinct O +interactions O +between O +trimers B-oligomeric_state +occur O +at O +the O +interfaces B-site +of O +the O +five O +dodecamers B-oligomeric_state +: O +one O +in O +which O +the O +trimers B-oligomeric_state +are O +eclipsed B-protein_state +( O +Figure O +5B O +), O +and O +one O +in O +which O +the O +trimers B-oligomeric_state +are O +staggered B-protein_state +( O +Figure O +5C O +). O + +The O +annular B-site +pore I-site +contains O +three O +eclipsed B-protein_state +interfaces B-site +and O +two O +staggered B-protein_state +interfaces B-site +. O + +Ten O +Aβ25 B-protein +– B-residue_range +28 I-residue_range +loops B-structure_element +from O +the O +vertices O +of O +the O +five O +dodecamers B-oligomeric_state +line O +the O +hole O +in O +the O +center O +of O +the O +pore B-site +. O + +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +the O +annular B-site +pore I-site +formed O +by O +peptide B-mutant +2 I-mutant +. O +( O +A O +) O +Annular B-structure_element +porelike I-structure_element +structure B-evidence +illustrating O +the O +relationship O +of O +the O +five O +dodecamers B-oligomeric_state +that O +form O +the O +pore B-site +( O +top O +view O +). O + +The O +same O +staggered B-site +interface I-site +also O +occurs O +between O +dodecamers O +4 O +and O +5 O +. O +( O +D O +) O +Eclipsed B-site +interface I-site +between O +dodecamers B-structure_element +1 I-structure_element +and I-structure_element +5 I-structure_element +( O +top O +view O +). O + +Rather O +, O +the O +crystal B-evidence +lattice I-evidence +is O +composed O +of O +conjoined O +annular B-site +pores I-site +in O +which O +all O +four O +F20 B-residue_name_number +faces O +on O +the O +surface O +of O +each O +dodecamer B-oligomeric_state +contact O +F20 B-residue_name_number +faces O +on O +other O +dodecamers B-oligomeric_state +( O +Figure O +S4 O +). O + +In O +this O +model O +Aβ B-protein +folds O +to O +form O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +comprising O +the O +hydrophobic O +central B-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +regions I-structure_element +. O + +Three O +β B-structure_element +- I-structure_element +hairpins I-structure_element +assemble O +to O +form O +a O +trimer B-oligomeric_state +, O +and O +four O +trimers B-oligomeric_state +assemble O +to O +form O +a O +dodecamer B-oligomeric_state +. O + +Three O +β B-structure_element +- I-structure_element +hairpin I-structure_element +monomers B-oligomeric_state +assemble O +to O +form O +a O +triangular B-protein_state +trimer B-oligomeric_state +. O + +Four O +triangular B-protein_state +trimers B-oligomeric_state +assemble O +to O +form O +a O +dodecamer B-oligomeric_state +. O + +The O +molecular O +weights O +shown O +correspond O +to O +an O +Aβ42 B-protein +monomer B-oligomeric_state +(∼ O +4 O +. O +5 O +kDa O +), O +an O +Aβ42 B-protein +trimer B-oligomeric_state +(∼ O +13 O +. O +5 O +kDa O +), O +an O +Aβ42 B-protein +dodecamer B-oligomeric_state +(∼ O +54 O +kDa O +), O +and O +an O +Aβ42 B-protein +annular B-site +pore I-site +composed O +of O +five O +dodecamers B-oligomeric_state +(∼ O +270 O +kDa O +). O + +Fibrillar B-protein_state +and O +nonfibrillar B-protein_state +oligomers B-oligomeric_state +have O +structurally O +distinct O +characteristics O +, O +which O +are O +reflected O +in O +their O +reactivity O +with O +the O +fibril O +- O +specific O +OC O +antibody O +and O +the O +oligomer B-oligomeric_state +- O +specific O +A11 O +antibody O +. O + +The O +varying O +sizes O +of O +APFs B-complex_assembly +formed O +by O +full B-protein_state +- I-protein_state +length I-protein_state +Aβ B-protein +might O +result O +from O +differences O +in O +the O +number O +of O +oligomer B-oligomeric_state +subunits B-structure_element +comprising O +each O +APF B-complex_assembly +. O + +Although O +the O +annular B-site +pore I-site +formed O +by O +peptide B-mutant +2 I-mutant +contains O +five O +dodecamer B-oligomeric_state +subunits B-structure_element +, O +pores B-site +containing O +fewer O +or O +more O +subunits B-structure_element +can O +easily O +be O +envisioned O +. O + +Surface O +views O +of O +the O +annular B-site +pore I-site +formed O +by O +peptide B-mutant +2 I-mutant +. O +( O +A O +) O +Top O +view O +. O + +This O +reactivity O +suggests O +that O +peptide B-mutant +2 I-mutant +forms O +oligomers B-oligomeric_state +in O +solution O +that O +share O +structural O +similarities O +to O +the O +nonfibrillar B-protein_state +oligomers B-oligomeric_state +formed O +by O +full B-protein_state +- I-protein_state +length I-protein_state +Aβ B-protein +. O + +To O +identify O +biophysical O +determinants O +of O +cell O +‐ O +specific O +signaling O +and O +breast O +cancer O +cell O +proliferation O +, O +we O +synthesized B-experimental_method +241 O +ERα B-protein +ligands O +based O +on O +19 O +chemical O +scaffolds O +, O +and O +compared O +ligand O +response O +using O +quantitative B-experimental_method +bioassays I-experimental_method +for O +canonical O +ERα B-protein +activities O +and O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +Many O +drugs O +are O +small O +‐ O +molecule O +ligands O +of O +allosteric O +signaling O +proteins O +, O +including O +G B-protein_type +protein I-protein_type +‐ I-protein_type +coupled I-protein_type +receptors I-protein_type +( O +GPCRs B-protein_type +) O +and O +nuclear B-protein_type +receptors I-protein_type +such O +as O +ERα B-protein +. O + +Chemical O +structures O +of O +some O +common O +ERα B-protein +ligands O +. O + +AF B-structure_element +‐ I-structure_element +1 I-structure_element +binds O +a O +separate O +surface O +on O +these O +coactivators O +( O +Webb O +et O +al O +, O +1998 O +; O +Yi O +et O +al O +, O +2015 O +). O + +We O +also O +generated O +four O +direct O +modulator O +series O +with O +side O +chains O +designed O +to O +directly O +dislocate O +h12 B-structure_element +and O +thereby O +completely O +occlude O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +( O +Fig O +2C O +and O +E O +; O +Dataset O +EV1 O +) O +( O +Kieser O +et O +al O +, O +2010 O +). O + +ERα B-protein +ligands O +induced O +a O +range O +of O +agonist O +activity O +profiles O + +Correlation O +analysis O +of O +OBHS B-chemical +versus O +OBHS B-chemical +‐ I-chemical +BSC I-chemical +activity O +across O +cell O +types O +. O + +These O +results O +suggest O +that O +addition O +of O +an O +extended O +side O +chain O +to O +an O +ERα B-protein +ligand O +scaffold O +is O +sufficient O +to O +induce O +cell O +‐ O +specific O +signaling O +, O +where O +the O +relative O +activity O +profiles O +of O +the O +individual O +ligands O +change O +between O +cell O +types O +. O + +Modulation O +of O +signaling O +specificity O +by O +AF B-structure_element +‐ I-structure_element +1 I-structure_element + +The O +positive O +correlation O +between O +the O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +and O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activities O +or O +GREB1 B-protein +levels O +induced O +by O +scaffolds O +in O +cluster O +1 O +was O +generally O +retained O +without O +the O +AB B-structure_element +domain O +, O +or O +the O +F B-structure_element +domain O +( O +Fig O +3D O +lanes O +1 O +– O +4 O +). O + +OBHS B-chemical +analogs O +showed O +an O +average O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-mutant +‐ I-mutant +ΔAB I-mutant +activity O +of O +3 O +. O +2 O +% O +± O +3 O +( O +mean O ++ O +SEM O +) O +relative O +to O +E2 B-chemical +. O + +These O +similar O +patterns O +of O +ligand O +activity O +in O +the O +wild B-protein_state +‐ I-protein_state +type I-protein_state +and O +deletion O +mutants B-protein_state +suggest O +that O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +and O +the O +F B-structure_element +domain O +purely O +amplify O +the O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +activities O +of O +ligands O +in O +cluster O +1 O +. O + +In O +contrast O +, O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +was O +a O +determinant O +of O +signaling O +specificity O +for O +scaffolds O +in O +cluster O +2 O +. O + +Comparing O +Fig O +3C O +and O +D O +, O +the O ++ O +and O +− O +signs O +indicate O +where O +the O +deletion B-experimental_method +mutant I-experimental_method +assays I-experimental_method +led O +to O +a O +gain O +or O +loss O +of O +statically O +significant O +correlation O +, O +respectively O +. O + +These O +results O +suggest O +that O +compounds O +that O +show O +cell O +‐ O +specific O +signaling O +do O +not O +activate O +GREB1 B-protein +, O +or O +use O +coactivators O +other O +than O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +to O +control O +GREB1 B-protein +expression O +( O +Fig O +1E O +). O + +Out O +of O +15 O +ligand O +series O +in O +these O +clusters O +, O +only O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +induced O +a O +proliferative O +response O +that O +was O +predicted O +by O +GREB1 B-protein +levels O +, O +which O +were O +not O +determined O +by O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +( O +Fig O +3E O +and O +F O +lane O +10 O +). O + +However O +, O +the O +ChIP B-experimental_method +assays I-experimental_method +for O +WAY B-chemical +‐ I-chemical +C I-chemical +‐ O +induced O +recruitment O +of O +NCOA3 B-protein +to O +the O +GREB1 B-protein +promoter O +did O +not O +correlate O +with O +any O +of O +the O +other O +WAY B-chemical +‐ I-chemical +C I-chemical +activity O +profiles O +( O +Fig O +4D O +), O +although O +the O +positive O +correlation O +between O +ChIP B-experimental_method +assays I-experimental_method +and O +NCOA3 B-protein +recruitment O +via O +M2H B-experimental_method +assay I-experimental_method +showed O +a O +trend O +toward O +significance O +with O +r B-evidence +2 I-evidence += O +0 O +. O +36 O +and O +P B-evidence += O +0 O +. O +09 O +( O +F B-experimental_method +‐ I-experimental_method +test I-experimental_method +for O +nonzero O +slope O +). O + +For O +most O +scaffolds O +, O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERβ O +and O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activities O +were O +not O +correlated O +, O +except O +for O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +and O +cyclofenil B-chemical +analogs O +, O +which O +showed O +moderate O +but O +significant O +correlations O +( O +Fig O +EV4A O +). O + +Triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +induce O +variance O +of O +ERα B-protein +conformations O +at O +the O +C O +‐ O +terminal O +region O +of O +h11 B-structure_element +. O + +ERα B-protein +LBDs B-structure_element +in B-protein_state +complex I-protein_state +with I-protein_state +diethylstilbestrol B-chemical +( O +DES B-chemical +) O +or O +a O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analog O +were O +superposed B-experimental_method +to O +show O +that O +the O +ligand O +‐ O +induced O +difference O +in O +h11 B-structure_element +conformation O +is O +transmitted O +to O +the O +C O +‐ O +terminus O +of O +h12 B-structure_element +( O +PDB O +4ZN7 O +, O +5DMC O +). O + +The O +bound O +ligands O +are O +shown O +, O +and O +arrows O +indicate O +considerable O +variation O +in O +the O +orientation O +of O +the O +different O +h3 B-structure_element +‐, O +h8 B-structure_element +‐, O +h11 B-structure_element +‐, O +or O +h12 B-structure_element +‐ O +directed O +ligand O +side O +groups O +. O + +Ligands O +with O +cell O +‐ O +specific O +activity O +alter O +the O +shape O +of O +the O +AF B-site +‐ I-site +2 I-site +surface I-site + +Direct O +modulators O +like O +tamoxifen B-chemical +drive O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +‐ O +dependent O +cell O +‐ O +specific O +activity O +by O +completely O +occluding O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +, O +but O +it O +is O +not O +known O +how O +indirect O +modulators O +produce O +cell O +‐ O +specific O +ERα B-protein +activity O +. O + +For O +instance O +, O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +2 I-chemical +and O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +analogs O +( O +Fig O +2 O +) O +had O +similar O +ERα B-protein +activity O +profiles O +in O +the O +different O +cell O +types O +( O +Fig O +EV2A O +– O +C O +), O +but O +the O +2 O +‐ O +versus O +3 O +‐ O +methyl O +substituted O +phenol O +rings O +altered O +the O +correlated O +signaling O +patterns O +in O +different O +cell O +types O +( O +Fig O +3B O +lanes O +7 O +and O +12 O +). O + +The O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +and O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +scaffolds O +are O +isomeric O +, O +but O +with O +aryl O +groups O +at O +obtuse O +and O +acute O +angles O +, O +respectively O +( O +Fig O +2 O +). O + +Hierarchical B-experimental_method +clustering I-experimental_method +revealed O +that O +many O +of O +the O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +recapitulated O +most O +of O +the O +peptide O +recruitment O +and O +dismissal O +patterns O +observed O +with O +E2 B-chemical +( O +Fig O +6H O +). O + +Together O +, O +these O +findings O +suggest O +that O +without O +an O +extended O +side O +chain O +, O +cell O +‐ O +specific O +activity O +stems O +from O +different O +coregulator O +recruitment O +profiles O +, O +due O +to O +unique O +ligand O +‐ O +induced O +conformations O +of O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +, O +in O +addition O +to O +differential O +usage O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +. O + +Our O +goal O +was O +to O +identify O +a O +minimal O +set O +of O +predictors O +that O +would O +link O +specific O +structural O +perturbations O +to O +ERα B-protein +signaling O +pathways O +that O +control O +cell O +‐ O +specific O +signaling O +and O +proliferation O +. O + +Ligands O +in O +these O +classes O +altered O +the O +shape O +of O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +to O +affect O +coregulator O +preferences O +. O + +It O +is O +noteworthy O +that O +regulation O +of O +h12 B-structure_element +dynamics O +indirectly O +through O +h11 B-structure_element +can O +virtually O +abolish O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +activity O +, O +and O +yet O +still O +drive O +robust O +transcriptional O +activity O +through O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +, O +as O +demonstrated O +with O +the O +OBHS B-chemical +series O +. O + +Also O +, O +we O +have O +used O +siRNA B-experimental_method +screening I-experimental_method +to O +identify O +a O +number O +of O +coregulators O +required O +for O +ERα B-protein +‐ O +mediated O +repression O +of O +the O +IL O +‐ O +6 O +gene O +( O +Nwachukwu O +et O +al O +, O +2014 O +). O + +If O +we O +calculated O +inter B-evidence +‐ I-evidence +atomic I-evidence +distance I-evidence +matrices I-evidence +containing O +4 O +, O +000 O +atoms O +per O +structure O +× O +76 O +ligand O +– O +receptor O +complexes O +, O +we O +would O +have O +3 O +× O +105 O +predictions O +. O + +We O +have O +identified O +atomic B-evidence +vectors I-evidence +for O +the O +OBHS B-chemical +‐ I-chemical +N I-chemical +and O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +classes O +that O +predict O +ligand O +response O +( O +Fig O +5E O +and O +F O +). O + +Significant O +morbidity O +and O +mortality O +has O +been O +associated O +with O +an O +emerging O +, O +highly O +drug O +- O +resistant O +strain O +of O +K B-species +. I-species +pneumoniae I-species +characterized O +as O +producing O +the O +carbapenemase B-protein_type +enzyme O +( O +KPC O +- O +producing O +bacteria B-taxonomy_domain +; O +Nordmann O +et O +al O +., O +2009 O +). O + +In O +bacteria B-taxonomy_domain +, O +topoisomerase B-complex_assembly +IV I-complex_assembly +, O +a O +tetramer B-oligomeric_state +of O +two O +ParC B-protein +and O +two O +ParE B-protein +subunits O +, O +unlinks O +daughter O +chromosomes O +prior O +to O +cell O +division O +, O +whereas O +the O +related O +enzyme O +gyrase B-protein_type +, O +a O +GyrA2GyrB2 B-complex_assembly +tetramer B-oligomeric_state +, O +supercoils O +DNA B-chemical +and O +helps O +unwind O +DNA B-chemical +at O +replication O +forks O +. O + +Levofloxacin B-chemical +is O +a O +broad O +- O +spectrum O +third O +- O +generation O +fluoro O +­ O +quinolone O +antibiotic O +. O + +The O +upper O +region O +of O +the O +topoisomerase B-protein_type +complex O +consists O +of O +the O +E B-protein +- I-protein +subunit I-protein +TOPRIM B-structure_element +metal I-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +formed O +of O +four O +parallel B-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +and O +the O +surrounding O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +There O +is O +41 O +. O +6 O +% O +sequence O +identity O +and O +54 O +. O +4 O +% O +sequence O +homology O +between O +the O +ParE B-protein +subunit O +of O +K B-species +. I-species +pneumoniae I-species +and O +that O +of O +S B-species +. I-species +pneumoniae I-species +. O + +Interestingly O +, O +for O +S B-species +. I-species +pneumoniae I-species +we O +observe O +only O +one O +possible O +orientation O +of O +the O +C7 O +groups O +in O +both O +sub O +­ O +units O +, O +while O +for O +K B-species +. I-species +pneumoniae I-species +we O +can O +see O +two O +: O +one O +with O +the O +same O +orientation O +as O +in O +S B-species +. I-species +pneumoniae I-species +and O +other O +rotated O +180 O +° O +away O +. O + +They O +both O +exist O +within O +the O +same O +crystal B-evidence +in O +the O +two O +different O +dimers B-oligomeric_state +in O +the O +asymmetric O +unit O +. O + +Similar O +behaviour O +was O +observed O +for O +the O +S B-species +. I-species +pneumoniae I-species +topo B-complex_assembly +­ I-complex_assembly +isomerase I-complex_assembly +IV I-complex_assembly +ParE30 B-complex_assembly +- I-complex_assembly +ParC55 I-complex_assembly +fusion O +protein O +. O + +Given O +the O +current O +concerns O +about O +drug O +- O +resistant O +strains O +of O +Klebsiella B-taxonomy_domain +, O +the O +structure B-evidence +reported O +here O +provides O +key O +information O +in O +understanding O +the O +action O +of O +currently O +used O +quinolones B-chemical +and O +should O +aid O +in O +the O +development O +of O +other O +topoisomerase B-protein_type +- O +targeting O +therapeutics O +active O +against O +this O +major O +human B-species +pathogen O +. O + +Bound B-protein_state +ATP B-chemical +is O +indicated O +by O +pink O +circles O +in O +the O +ATPase B-structure_element +domains I-structure_element +( O +reproduced O +with O +permission O +from O +Fig O +. O +1 O +of O +Lapanogov O +et O +al O +., O +2013 O +). O + +After O +60 O +min O +incubation O +, O +samples O +were O +treated O +with O +SDS O +and O +proteinase O +K O +to O +remove O +proteins O +covalent O +bound O +to O +DNA B-chemical +, O +and O +the O +DNA B-chemical +products O +were O +examined O +by O +gel O +electrophoresis O +in O +1 O +% O +agarose O +. O + +We O +illustrate O +this O +here O +using O +glutamate B-protein_type +dehydrogenase I-protein_type +( O +GDH B-protein_type +), O +a O +336 O +- O +kDa O +metabolic O +enzyme O +that O +catalyzes O +the O +oxidative O +deamination O +of O +glutamate B-chemical +. O + +Here O +, O +we O +report O +near O +- O +atomic O +resolution O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +, O +at O +resolutions O +ranging O +from O +3 O +. O +2 O +Å O +to O +3 O +. O +6 O +Å O +for O +GDH B-protein_type +complexes O +, O +including O +complexes O +for O +which O +crystal B-evidence +structures I-evidence +are O +not O +available O +. O + +The O +first O +is O +located O +near O +the O +dimer B-site +interface I-site +and O +forms O +the O +core O +of O +the O +hexamer B-oligomeric_state +. O + +In O +contrast O +, O +in O +the O +open B-protein_state +conformation O +, O +the O +cavity B-site +present O +in O +the O +closed B-protein_state +state O +becomes O +too O +narrow O +for O +the O +nicotinamide O +group O +; O +instead O +, O +the O +group O +is O +oriented O +in O +the O +opposite O +direction O +, O +parallel O +to O +the O +pivot B-structure_element +helix I-structure_element +with O +the O +amido O +group O +extending O +toward O +the O +C O +- O +terminal O +end O +of O +the O +helix B-structure_element +. O + +In O +the O +open B-protein_state +conformation O +, O +the O +distance O +between O +His209 B-residue_name_number +and O +the O +α O +- O +phosphate O +of O +NADH B-chemical +is O +∼ O +4 O +. O +4 O +Å O +, O +which O +is O +comparable O +with O +the O +corresponding O +distance O +in O +the O +ADP B-protein_state +- I-protein_state +bound I-protein_state +conformation O +. O + +Importantly O +, O +the O +binding O +of O +GTP B-chemical +alone O +does O +not O +appear O +to O +drive O +the O +transition O +from O +the O +open B-protein_state +to O +the O +closed B-protein_state +state O +of O +GDH B-protein +. O + +When O +NADH B-chemical +and O +GTP B-chemical +are O +both O +present O +, O +classification B-experimental_method +reveals O +the O +presence B-protein_state +of I-protein_state +both O +closed B-protein_state +and O +open B-protein_state +GDH B-protein +conformations O +, O +similar O +to O +the O +condition O +when O +only O +NADH B-chemical +is O +present O +( O +Fig O +. O +4 O +, O +A O +and O +B O +). O + +When O +GTP B-chemical +is O +present O +in O +the O +GTP B-site +binding I-site +site I-site +, O +His209 B-residue_name_number +instead O +interacts O +with O +GTP B-chemical +, O +probably O +stabilizing O +the O +closed B-protein_state +conformation O +( O +Fig O +. O +4 O +, O +C O +and O +D O +). O + +These O +structural O +features O +provide O +a O +potential O +explanation O +of O +the O +weaker O +density B-evidence +for O +the O +nicotinamide O +moiety O +of O +NADH B-chemical +in O +the O +open B-protein_state +state O +, O +and O +may O +account O +for O +the O +higher O +reported O +affinity O +of O +NADH B-chemical +for O +the O +closed B-protein_state +state O +. O + +It O +contains O +a O +membrane O +- O +deforming O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +as O +well O +as O +a O +Src B-structure_element +homology I-structure_element +3 I-structure_element +( O +SH3 B-structure_element +) O +domain O +and O +a O +G B-structure_element +protein I-structure_element +- I-structure_element +binding I-structure_element +homology I-structure_element +region I-structure_element +1 I-structure_element +( O +HR1 B-structure_element +) O +domain O +. O + +TOCA1 B-protein +binding O +to O +Cdc42 B-protein +leads O +to O +actin B-protein_type +rearrangements O +, O +which O +are O +thought O +to O +be O +involved O +in O +processes O +such O +as O +endocytosis O +, O +filopodia O +formation O +, O +and O +cell O +migration O +. O + +The O +structures B-evidence +of O +more O +than O +60 O +small O +G B-protein_type +protein I-protein_type +· O +effector O +complexes O +have O +been O +solved B-experimental_method +, O +and O +, O +not O +surprisingly O +, O +the O +switch B-site +regions I-site +have O +been O +implicated O +in O +a O +large O +proportion O +of O +the O +G B-protein_type +protein I-protein_type +- O +effector O +interactions O +( O +reviewed O +in O +Ref O +.). O + +The O +TOCA1 B-protein +SH3 B-structure_element +domain O +has O +many O +known O +binding O +partners O +, O +including O +N B-protein +- I-protein +WASP I-protein +and O +dynamin B-protein +. O + +The O +structures B-evidence +of O +the O +PRK1 B-protein +HR1a B-structure_element +domain O +in O +complex B-protein_state +with I-protein_state +RhoA B-protein +and O +the O +HR1b B-structure_element +domain O +in O +complex B-protein_state +with I-protein_state +Rac1 B-protein +show O +that O +the O +HR1 B-structure_element +domain O +comprises O +an O +anti B-structure_element +- I-structure_element +parallel I-structure_element +coiled I-structure_element +- I-structure_element +coil I-structure_element +that O +interacts O +with O +its O +G B-protein_type +protein I-protein_type +binding O +partner O +via O +both O +helices B-structure_element +. O + +Both O +of O +the O +G B-site +protein I-site +switch I-site +regions I-site +are O +involved O +in O +the O +interaction O +. O + +The O +coiled B-structure_element +- I-structure_element +coil I-structure_element +fold I-structure_element +is O +shared O +by O +the O +HR1 B-structure_element +domain O +of O +the O +TOCA B-protein_type +family I-protein_type +protein I-protein_type +, O +CIP4 B-protein +, O +and O +, O +based O +on O +sequence O +homology O +, O +by O +TOCA1 B-protein +itself O +. O + +The O +data O +were O +fitted O +to O +a O +binding B-evidence +isotherm I-evidence +to O +give O +an O +apparent O +Kd B-evidence +and O +are O +expressed O +as O +a O +percentage O +of O +the O +maximum O +signal O +; O +B O +and O +C O +, O +competition B-experimental_method +SPA I-experimental_method +experiments O +were O +carried O +out O +with O +the O +indicated O +concentrations O +of O +ACK B-protein +GBD B-structure_element +( O +B O +) O +or O +HR1 B-structure_element +domain O +( O +C O +) O +titrated B-experimental_method +into O +30 O +nm O +GST B-mutant +- I-mutant +ACK I-mutant +and O +either O +30 O +nm O +Cdc42Δ7Q61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +or O +full B-protein_state +- I-protein_state +length I-protein_state +Cdc42Q61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +. O + +The O +Kd B-evidence +values O +derived O +for O +the O +TOCA1 B-protein +HR1 B-structure_element +with O +Cdc42Δ7 B-mutant +and O +full B-protein_state +- I-protein_state +length I-protein_state +Cdc42 B-protein +were O +6 O +. O +05 O +± O +1 O +. O +96 O +and O +5 O +. O +39 O +± O +1 O +. O +69 O +μm O +, O +respectively O +. O + +It O +was O +possible O +that O +the O +low O +affinity O +observed O +was O +due O +to O +negative O +effects O +of O +immobilization O +of O +the O +HR1 B-structure_element +domain O +, O +so O +other O +methods O +were O +employed O +, O +which O +utilized O +untagged B-protein_state +proteins O +. O + +Other O +G B-protein_type +protein I-protein_type +- O +HR1 B-structure_element +domain O +interactions O +have O +also O +failed O +to O +show O +heat O +changes O +in O +our O +hands O +. O +7 O +Infrared B-experimental_method +interferometry I-experimental_method +with O +immobilized B-protein_state +Cdc42 B-protein +was O +also O +attempted O +but O +was O +unsuccessful O +for O +both O +TOCA1 B-protein +HR1 B-structure_element +and O +for O +the O +positive O +control O +, O +ACK B-protein +. O + +Full B-protein_state +- I-protein_state +length I-protein_state +TOCA1 B-protein +and O +ΔSH3 B-mutant +TOCA1 B-protein +bound B-protein_state +with O +micromolar O +affinity O +( O +Fig O +. O +2B O +), O +in O +a O +similar O +manner O +to O +the O +isolated O +HR1 B-structure_element +domain O +( O +Fig O +. O +1A O +). O + +The O +data O +were O +fitted O +to O +a O +binding B-evidence +isotherm I-evidence +to O +give O +an O +apparent O +Kd B-evidence +and O +are O +expressed O +as O +a O +percentage O +of O +the O +maximum O +signal O +. O + +The O +affinities B-evidence +of O +both O +the O +FBP17 B-protein +and O +CIP4 B-protein +HR1 B-structure_element +domains O +were O +also O +in O +the O +low O +micromolar O +range O +( O +10 O +and O +5 O +μm O +, O +respectively O +) O +( O +Fig O +. O +2 O +, O +D O +and O +E O +), O +suggesting O +that O +low O +affinity O +interactions O +with O +Cdc42 B-protein +are O +a O +common O +feature O +within O +the O +TOCA B-protein_type +family I-protein_type +. O + +100 O +structures B-evidence +were O +calculated B-experimental_method +in O +the O +final O +iteration O +; O +the O +50 O +lowest O +energy O +structures B-evidence +were O +water O +- O +refined O +; O +and O +of O +these O +, O +the O +35 O +lowest O +energy O +structures B-evidence +were O +analyzed O +. O + +The O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +of O +the O +HR1 B-structure_element +domain O +interact O +to O +form O +an O +anti B-structure_element +- I-structure_element +parallel I-structure_element +coiled I-structure_element +- I-structure_element +coil I-structure_element +with O +a O +slight O +left O +- O +handed O +twist O +, O +reminiscent O +of O +the O +HR1 B-structure_element +domains O +of O +CIP4 B-protein +( O +PDB O +code O +2KE4 O +) O +and O +PRK1 B-protein +( O +PDB O +codes O +1CXZ O +and O +1URF O +). O + +The O +structure B-evidence +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +. O + +Long O +range O +NOEs B-evidence +were O +observed O +linking O +Leu B-residue_name_number +- I-residue_name_number +334 I-residue_name_number +, O +Glu B-residue_name_number +- I-residue_name_number +335 I-residue_name_number +, O +and O +Asp B-residue_name_number +- I-residue_name_number +336 I-residue_name_number +with O +Trp B-residue_name_number +- I-residue_name_number +413 I-residue_name_number +of O +helix B-structure_element +2 I-structure_element +, O +Leu B-residue_name_number +- I-residue_name_number +334 I-residue_name_number +with O +Lys B-residue_name_number +- I-residue_name_number +409 I-residue_name_number +of O +helix B-structure_element +2 I-structure_element +, O +and O +Phe B-residue_name_number +- I-residue_name_number +337 I-residue_name_number +and O +Ser B-residue_name_number +- I-residue_name_number +338 I-residue_name_number +with O +Arg B-residue_name_number +- I-residue_name_number +345 I-residue_name_number +, O +Arg B-residue_name_number +- I-residue_name_number +348 I-residue_name_number +, O +and O +Leu B-residue_name_number +- I-residue_name_number +349 I-residue_name_number +of O +helix B-structure_element +1 I-structure_element +. O + +Residues O +that O +disappeared O +in O +the O +presence B-protein_state +of I-protein_state +Cdc42 B-protein +were O +assigned O +a O +CSP B-experimental_method +of O +0 O +. O +2 O +but O +were O +excluded O +when O +calculating O +the O +mean O +CSP B-experimental_method +and O +are O +indicated O +with O +open O +bars O +. O + +Side O +chains O +whose O +CH O +groups O +disappeared O +in O +the O +presence B-protein_state +of I-protein_state +Cdc42 B-protein +are O +marked O +on O +the O +graph O +in O +Fig O +. O +4B O +with O +green O +asterisks O +. O + +The O +corresponding O +15N B-experimental_method +and O +13C B-experimental_method +NMR I-experimental_method +experiments O +were O +also O +recorded O +on O +15N B-chemical +- O +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +or O +15N B-chemical +/ O +13C B-chemical +- O +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +in O +the O +presence B-protein_state +of I-protein_state +unlabeled B-protein_state +HR1 B-structure_element +domain O +. O + +Mapping O +the O +binding B-site +surface I-site +of O +the O +HR1 B-structure_element +domain O +onto O +Cdc42 B-protein +. O + +Residues O +with O +disappeared O +peaks O +in O +13C B-experimental_method +HSQC I-experimental_method +experiments O +are O +marked O +on O +the O +chart O +with O +green O +asterisks O +. O + +Residues O +with O +either O +side O +chain O +or O +backbone O +groups O +affected O +are O +colored O +blue O +if O +buried O +and O +yellow O +if O +solvent B-protein_state +- I-protein_state +accessible I-protein_state +. O + +The O +switch B-site +regions I-site +of O +Cdc42 B-protein +did O +not O +, O +however O +, O +become O +visible O +in O +the O +presence B-protein_state +of I-protein_state +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +. O + +Residues O +of O +Cdc42 B-protein +that O +disappear O +or O +show O +chemical O +shift O +changes O +in O +the O +presence B-protein_state +of I-protein_state +TOCA1 B-protein +are O +colored O +cyan O +if O +also O +identified O +as O +contacts O +in O +RhoA B-protein +and O +Rac1 B-protein +and O +yellow O +if O +they O +are O +not O +. O + +D O +, O +regions O +of O +interest O +of O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1 I-complex_assembly +domain O +model O +. O + +D O +, O +selected O +regions O +of O +the O +15N B-experimental_method +HSQC I-experimental_method +of O +600 O +μm O +TOCA1 B-protein +HR1 B-structure_element +domain O +in B-protein_state +complex I-protein_state +with I-protein_state +Cdc42 B-protein +in O +the O +absence B-protein_state +and O +presence B-protein_state +of I-protein_state +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +, O +showing O +displacement O +of O +Cdc42 B-protein +from O +the O +HR1 B-structure_element +domain O +by O +N B-protein +- I-protein +WASP I-protein +. O + +An O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +construct O +was O +produced O +, O +and O +its O +affinity B-evidence +for O +Cdc42 B-protein +was O +measured O +by O +competition B-experimental_method +SPA I-experimental_method +( O +Fig O +. O +7B O +). O + +A O +comparison O +of O +the O +HSQC B-experimental_method +experiments O +recorded O +on O +15N B-chemical +- O +Cdc42 B-protein +alone B-protein_state +, O +in O +the O +presence B-protein_state +of I-protein_state +TOCA1 B-protein +HR1 B-structure_element +, O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +, O +or O +both O +, O +shows O +that O +the O +spectra B-evidence +in O +the O +presence B-protein_state +of I-protein_state +N B-protein +- I-protein +WASP I-protein +and O +in O +the O +presence B-protein_state +of I-protein_state +both O +N B-protein +- I-protein +WASP I-protein +and O +TOCA1 B-protein +HR1 B-structure_element +are O +identical O +( O +Fig O +. O +7C O +). O + +Actin B-protein_type +polymerization O +triggered O +by O +the O +addition O +of O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +- O +containing O +liposomes O +has O +previously O +been O +shown O +to O +depend O +on O +TOCA1 B-protein +and O +N B-protein +- I-protein +WASP I-protein +. O + +The O +contacts B-bond_interaction +between O +the O +N O +- O +terminal O +region O +and O +the O +coiled B-structure_element +- I-structure_element +coil I-structure_element +are O +predominantly O +hydrophobic B-bond_interaction +in O +both O +cases O +, O +but O +sequence O +- O +specific O +contacts O +do O +not O +appear O +to O +be O +conserved O +. O + +Some O +residues O +that O +are O +affected O +in O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1TOCA1 I-complex_assembly +complex O +but O +do O +not O +correspond O +to O +contact O +residues O +of O +RhoA B-protein +or O +Rac1 B-protein +( O +Fig O +. O +6C O +) O +may O +contact O +HR1TOCA1 B-structure_element +directly O +( O +Fig O +. O +6D O +). O + +Thr B-residue_name_number +- I-residue_name_number +52Cdc42 I-residue_name_number +, O +which O +has O +also O +been O +identified O +as O +making O +minor O +contacts O +with O +ACK B-protein +, O +falls O +near O +the O +side O +chains O +of O +HR1TOCA1 B-structure_element +helix B-structure_element +1 I-structure_element +, O +particularly O +Lys B-residue_name_number +- I-residue_name_number +372TOCA1 I-residue_name_number +, O +whereas O +the O +equivalent O +position O +in O +Rac1 B-protein +is O +Asn B-residue_name_number +- I-residue_name_number +52Rac1 I-residue_name_number +. O + +The O +importance O +of O +this O +residue O +in O +the O +Cdc42 B-protein +- O +TOCA1 B-protein +interaction O +remains O +unclear O +, O +although O +its O +mutation B-experimental_method +reduces O +binding O +to O +RhoGAP B-protein +, O +suggesting O +that O +it O +can O +be O +involved O +in O +Cdc42 B-protein +interactions O +. O + +An O +investigation O +into O +the O +local O +motions O +, O +particularly O +in O +the O +G B-site +protein I-site +- I-site +binding I-site +regions I-site +, O +may O +offer O +further O +insight O +into O +the O +differential O +specificities O +and O +affinities O +of O +G B-protein_type +protein I-protein_type +- O +HR1 B-structure_element +domain O +interactions O +. O + +WIP B-protein +inhibits O +the O +activation O +of O +N B-protein +- I-protein +WASP I-protein +by O +Cdc42 B-protein +, O +an O +effect O +that O +is O +reversed O +by O +TOCA1 B-protein +. O + +A O +combination O +of O +allosteric O +activation O +by O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +, O +activated B-protein_state +Cdc42 B-protein +and O +TOCA1 B-protein +, O +and O +oligomeric O +activation O +implemented O +by O +TOCA1 B-protein +would O +lead O +to O +full B-protein_state +activation I-protein_state +of O +N B-protein +- I-protein +WASP I-protein +and O +downstream O +actin O +polymerization O +. O + +Furthermore O +, O +TOCA1 B-protein +is O +required O +for O +Cdc42 B-protein +- O +mediated O +activation O +of O +N B-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +WIP I-complex_assembly +, O +implying O +that O +it O +may O +not O +be O +possible O +for O +Cdc42 B-protein +to O +bind O +and O +activate O +N B-protein +- I-protein +WASP I-protein +prior O +to O +TOCA1 B-protein +- O +Cdc42 B-protein +binding O +. O + +There O +is O +an O +advantage O +to O +such O +an O +effector O +handover O +, O +in O +that O +N B-protein +- I-protein +WASP I-protein +would O +only O +be O +robustly O +recruited O +when O +F B-structure_element +- I-structure_element +BAR I-structure_element +domains O +are O +already O +present O +. O + +The O +region O +C O +- O +terminal O +to O +the O +core O +CRIB B-structure_element +, O +required O +for O +maximal O +affinity O +binding O +, O +would O +then O +fully O +displace O +the O +TOCA1 B-protein +HR1 B-structure_element +. O + +We O +envisage O +a O +complex O +interplay O +of O +equilibria O +between O +free B-protein_state +and O +bound B-protein_state +, O +active B-protein_state +and O +inactive B-protein_state +Cdc42 B-protein +, O +TOCA B-protein_type +family I-protein_type +, O +and O +WASP B-protein_type +family O +proteins O +, O +facilitating O +a O +tightly O +spatially O +and O +temporally O +regulated O +pathway O +requiring O +numerous O +simultaneous O +events O +in O +order O +to O +achieve O +appropriate O +and O +robust O +activation O +of O +the O +downstream O +pathway O +. O + +Our O +data O +are O +therefore O +easily O +reconciled O +with O +the O +dynamic O +instability O +models O +described O +in O +relation O +to O +the O +formation O +of O +endocytic O +vesicles O +and O +with O +the O +current O +data O +pertaining O +to O +the O +complex O +activation O +of O +WASP B-protein_type +/ O +N B-protein +- I-protein +WASP I-protein +pathways O +by O +allosteric O +and O +oligomeric O +effects O +. O + +Acetyl B-protein_type +- I-protein_type +CoA I-protein_type +carboxylases I-protein_type +( O +ACCs B-protein_type +) O +catalyse O +the O +committed O +step O +in O +fatty O +- O +acid O +biosynthesis O +: O +the O +ATP B-chemical +- O +dependent O +carboxylation O +of O +acetyl B-chemical +- I-chemical +CoA I-chemical +to O +malonyl B-chemical +- I-chemical +CoA I-chemical +. O +They O +are O +important O +regulatory O +hubs O +for O +metabolic O +control O +and O +relevant O +drug O +targets O +for O +the O +treatment O +of O +the O +metabolic O +syndrome O +and O +cancer O +. O + +Biotin B-protein_type +- I-protein_type +dependent I-protein_type +acetyl I-protein_type +- I-protein_type +CoA I-protein_type +carboxylases I-protein_type +( O +ACCs B-protein_type +) O +are O +essential O +enzymes O +that O +catalyse O +the O +ATP B-chemical +- O +dependent O +carboxylation O +of O +acetyl B-chemical +- I-chemical +CoA I-chemical +to O +malonyl B-chemical +- I-chemical +CoA I-chemical +. O +This O +reaction O +provides O +the O +committed O +activated O +substrate O +for O +the O +biosynthesis O +of O +fatty B-chemical +acids I-chemical +via O +fatty B-protein_type +- I-protein_type +acid I-protein_type +synthase I-protein_type +. O + +The O +principal O +functional O +protein O +components O +of O +ACCs B-protein_type +have O +been O +described O +already O +in O +the O +late O +1960s O +for O +Escherichia B-species +coli I-species +( O +E B-species +. I-species +coli I-species +) O +ACC B-protein_type +: O +Biotin B-protein_type +carboxylase I-protein_type +( O +BC B-protein_type +) O +catalyses O +the O +ATP B-chemical +- O +dependent O +carboxylation O +of O +a O +biotin B-chemical +moiety O +, O +which O +is O +covalently O +linked O +to O +the O +biotin B-protein_type +carboxyl I-protein_type +carrier I-protein_type +protein I-protein_type +( O +BCCP B-protein_type +). O + +The O +CD B-structure_element +comprises O +one O +- O +third O +of O +the O +protein O +and O +is O +a O +unique B-protein_state +feature I-protein_state +of I-protein_state +eukaryotic B-taxonomy_domain +ACCs B-protein_type +without O +homologues O +in O +other O +proteins O +. O + +The O +function O +of O +this O +domain O +remains O +poorly O +characterized O +, O +although O +phosphorylation B-ptm +of O +several O +serine B-residue_name +residues O +in O +the O +CD B-structure_element +regulates O +ACC B-protein_type +activity O +. O + +In O +these O +structures B-evidence +, O +the O +BC B-protein_type +and O +CT B-protein_type +active B-site +sites I-site +are O +at O +distances O +between O +40 O +and O +80 O +Å O +, O +such O +that O +substrate O +transfer O +could O +be O +mediated O +solely O +by O +the O +mobility O +of O +the O +flexibly B-protein_state +tethered I-protein_state +BCCP B-protein_type +. O + +For O +fungal B-taxonomy_domain +ACC B-protein_type +, O +neither O +spontaneous O +nor O +inducible O +polymerization O +has O +been O +detected O +despite O +considerable O +sequence O +conservation O +to O +human B-species +ACC1 B-protein +. O + +Despite O +the O +outstanding O +relevance O +of O +ACC B-protein_type +in O +primary O +metabolism O +and O +disease O +, O +the O +dynamic O +organization O +and O +regulation O +of O +the O +giant O +eukaryotic B-taxonomy_domain +, O +and O +in O +particular O +fungal B-taxonomy_domain +ACC B-protein_type +, O +remain O +poorly O +characterized O +. O + +Integrating O +these O +data O +with O +small B-experimental_method +- I-experimental_method +angle I-experimental_method +X I-experimental_method +- I-experimental_method +ray I-experimental_method +scattering I-experimental_method +( O +SAXS B-experimental_method +) O +and O +electron B-experimental_method +microscopy I-experimental_method +( O +EM B-experimental_method +) O +observations O +yield O +a O +comprehensive O +representation O +of O +the O +dynamic O +structure O +and O +regulation O +of O +fungal B-taxonomy_domain +ACC B-protein_type +. O + +CDL B-structure_element +is O +composed O +of O +a O +small B-structure_element +, I-structure_element +irregular I-structure_element +four I-structure_element +- I-structure_element +helix I-structure_element +bundle I-structure_element +( O +Lα1 B-structure_element +– I-structure_element +4 I-structure_element +) O +and O +tightly O +interacts O +with O +the O +open O +face O +of O +CDC1 B-structure_element +via O +an O +interface B-site +of O +1 O +, O +300 O +Å2 O +involving O +helices B-structure_element +Lα3 B-structure_element +and O +Lα4 B-structure_element +. O + +In O +insect B-experimental_method +- I-experimental_method +cell I-experimental_method +- I-experimental_method +expressed I-experimental_method +full B-protein_state +- I-protein_state +length I-protein_state +SceACC B-protein +, O +the O +highly B-protein_state +conserved I-protein_state +Ser1157 B-residue_name_number +is O +the O +only O +fully B-protein_state +occupied I-protein_state +phosphorylation B-site +site I-site +with O +functional O +relevance O +in O +S B-species +. I-species +cerevisiae I-species +. O + +Additional O +phosphorylation B-ptm +was O +detected O +for O +Ser2101 B-residue_name_number +and O +Tyr2179 B-residue_name_number +; O +however O +, O +these O +sites O +are O +neither B-protein_state +conserved I-protein_state +across O +fungal B-taxonomy_domain +ACC B-protein_type +nor B-protein_state +natively I-protein_state +phosphorylated I-protein_state +in O +yeast B-taxonomy_domain +. O + +On O +the O +basis O +of O +MS B-experimental_method +analysis O +of O +insect B-experimental_method +- I-experimental_method +cell I-experimental_method +- I-experimental_method +expressed I-experimental_method +human B-species +full B-protein_state +- I-protein_state +length I-protein_state +ACC B-protein_type +, O +Ser80 B-residue_name_number +shows O +the O +highest O +degree O +of O +phosphorylation B-ptm +( O +90 O +%). O + +Ser29 B-residue_name_number +and O +Ser1263 B-residue_name_number +, O +implicated O +in O +insulin B-ptm +- I-ptm +dependent I-ptm +phosphorylation I-ptm +and O +BRCA1 B-protein +binding O +, O +respectively O +, O +are O +phosphorylated B-protein_state +at O +intermediate O +levels O +( O +40 O +%). O + +Accordingly O +, O +most O +of O +this B-structure_element +loop I-structure_element +is O +not O +represented O +in O +the O +HsaBT B-mutant +- I-mutant +CD I-mutant +crystal B-evidence +structure I-evidence +. O + +Besides O +the O +regulatory B-structure_element +loop I-structure_element +, O +also O +the O +phosphopeptide B-site +target I-site +region I-site +for O +BRCA1 B-protein +interaction O +is O +not O +resolved O +presumably O +because O +of O +pronounced O +flexibility O +. O + +On O +the O +basis O +of O +an O +interface O +area O +of O +∼ O +600 O +Å2 O +and O +its O +edge O +- O +to O +- O +edge O +connection O +characteristics O +, O +the O +interface B-site +between O +CT B-structure_element +and O +CD B-structure_element +might O +be O +classified O +as O +conformationally O +variable O +. O + +SAXS B-experimental_method +analysis O +of O +CthACC B-protein +agrees O +with O +a O +dimeric B-oligomeric_state +state O +and O +an O +elongated B-protein_state +shape I-protein_state +with O +a O +maximum O +extent O +of O +350 O +Å O +( O +Supplementary O +Table O +1 O +). O + +The O +CD B-structure_element +has O +no O +direct O +role O +in O +substrate O +recognition O +or O +catalysis O +but O +contributes O +to O +the O +regulation O +of O +all O +eukaryotic B-taxonomy_domain +ACCs B-protein_type +. O + +However O +, O +flexibility O +at O +this O +hinge B-structure_element +may O +be O +required O +for O +full B-protein_state +ACC I-protein_state +activity I-protein_state +, O +as O +the O +distances O +between O +the O +BCCP B-structure_element +anchor I-structure_element +points I-structure_element +and O +the O +active B-site +sites I-site +of O +BC B-structure_element +and O +CT B-structure_element +observed O +here O +are O +such O +large O +that O +mobility O +of O +the O +BCCP B-structure_element +alone O +is O +not O +sufficient O +for O +substrate O +transfer O +. O + +A O +second B-structure_element +hinge I-structure_element +can O +be O +identified O +between O +CDC1 B-structure_element +/ O +CDC2 B-structure_element +. O + +Large O +- O +scale O +conformational O +variability O +has O +also O +been O +observed O +in O +most O +other O +carrier B-protein_type +protein I-protein_type +- I-protein_type +based I-protein_type +multienzymes I-protein_type +, O +including O +polyketide B-protein_type +and I-protein_type +fatty I-protein_type +- I-protein_type +acid I-protein_type +synthases I-protein_type +( O +with O +the O +exception O +of O +fungal B-protein_type +- I-protein_type +type I-protein_type +fatty I-protein_type +- I-protein_type +acid I-protein_type +synthases I-protein_type +), O +non B-protein_type +- I-protein_type +ribosomal I-protein_type +peptide I-protein_type +synthetases I-protein_type +and O +the O +pyruvate B-protein_type +dehydrogenase I-protein_type +complexes I-protein_type +, O +although O +based O +on O +completely O +different O +architectures O +. O + +The O +phosphorylated B-protein_state +regulatory B-structure_element +loop I-structure_element +binds O +to O +an O +allosteric B-site +site I-site +at O +the O +interface B-site +of O +two O +non B-protein_state +- I-protein_state +catalytic I-protein_state +domains O +and O +restricts O +conformational O +freedom O +at O +several O +hinges B-structure_element +in O +the O +dynamic B-protein_state +ACC B-protein_type +. O + +CDN B-structure_element +is O +linked O +by O +a O +four B-structure_element +- I-structure_element +helix I-structure_element +bundle I-structure_element +( O +CDL B-structure_element +) O +to O +two B-structure_element +α I-structure_element +– I-structure_element +β I-structure_element +- I-structure_element +fold I-structure_element +domains I-structure_element +( O +CDC1 B-structure_element +and O +CDC2 B-structure_element +). O + +( O +c O +) O +Superposition B-experimental_method +of O +CDC1 B-structure_element +and O +CDC2 B-structure_element +reveals O +highly B-protein_state +conserved I-protein_state +folds B-structure_element +. O +( O +d O +) O +The O +regulatory B-structure_element +loop I-structure_element +with O +the O +phosphorylated B-protein_state +Ser1157 B-residue_name_number +is O +bound O +into O +a O +crevice O +between O +CDC1 B-structure_element +and O +CDC2 B-structure_element +, O +the O +conserved B-protein_state +residues O +Arg1173 B-residue_name_number +and O +Arg1260 B-residue_name_number +coordinate O +the O +phosphoryl B-chemical +- O +group O +. O + +Variability O +of O +the O +connections O +of O +CDC2 B-structure_element +to O +CT B-structure_element +and O +CDC1 B-structure_element +in O +fungal B-taxonomy_domain +ACC B-protein_type +. O + +For O +other O +instances O +, O +CDC2 B-structure_element +domains O +are O +shown O +in O +transparent O +tube O +representation O +with O +only O +one O +helix O +each O +highlighted O +. O + +Representation O +as O +in O +a O +, O +but O +the O +CDC1 B-structure_element +and O +CDC2 B-structure_element +are O +superposed B-experimental_method +based O +on O +CDC2 B-structure_element +. O + +( O +a O +– O +c O +) O +Large O +- O +scale O +conformational O +variability O +of O +the O +CDN B-structure_element +domain O +relative O +to O +the O +CDL B-structure_element +/ O +CDC1 B-structure_element +domain O +. O + +CthCD B-mutant +- I-mutant +CT1 I-mutant +( O +in O +colour O +) O +serves O +as O +reference O +, O +the O +compared B-experimental_method +structures I-experimental_method +( O +as O +indicated O +, O +numbers O +after O +construct O +name O +differentiate O +between O +individual O +protomers B-oligomeric_state +) O +are O +shown O +in O +grey O +. O + +Strikingly O +, O +SEL1Lcent B-structure_element +forms O +a O +homodimer B-oligomeric_state +with O +two O +- O +fold O +symmetry O +in O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +manner O +. O + +Particularly O +, O +the O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +plays O +an O +important O +role O +in O +dimer B-oligomeric_state +formation O +by O +adopting O +a O +domain B-protein_state +- I-protein_state +swapped I-protein_state +structure O +and O +providing O +an O +extensive O +dimeric B-site +interface I-site +. O + +In O +particular O +, O +SEL1L B-protein +is O +crucial O +for O +translocation O +of O +Class B-complex_assembly +I I-complex_assembly +major I-complex_assembly +histocompatibility I-complex_assembly +complex I-complex_assembly +( O +MHC B-complex_assembly +) O +heavy B-protein_type +chains I-protein_type +( O +HCs B-protein_type +). O + +Although O +there O +is O +evidence O +that O +the O +luminal B-structure_element +domain I-structure_element +of O +SEL1L B-protein +is O +involved O +in O +substrate O +recognition O +or O +in O +forming O +complexes O +with O +chaperones B-protein_type +, O +it O +is O +not O +known O +how O +the O +unique O +structure O +of O +the O +repeated O +SLR B-structure_element +motifs O +contributes O +to O +the O +molecular O +function O +of O +the O +HRD1 B-complex_assembly +- I-complex_assembly +SEL1L I-complex_assembly +E3 B-protein_type +ligase I-protein_type +complex O +and O +affects O +ERAD O +at O +the O +molecular O +level O +. O + +The O +11 O +SLR B-structure_element +motifs O +are O +located O +in O +the O +ER O +lumen O +and O +account O +for O +more O +than O +two O +thirds O +of O +the O +mass O +of O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +. O + +Sequence B-experimental_method +alignment I-experimental_method +of O +the O +SLR B-structure_element +motifs O +revealed O +that O +there O +is O +a O +short O +linker B-structure_element +sequence I-structure_element +( O +residues O +336 B-residue_range +– I-residue_range +345 I-residue_range +) O +between O +SLR B-structure_element +- I-structure_element +N I-structure_element +and O +SLR B-structure_element +- I-structure_element +M I-structure_element +and O +a O +long O +linker B-structure_element +sequence I-structure_element +( O +residues O +528 B-residue_range +– I-residue_range +635 I-residue_range +) O +between O +SLR B-structure_element +- I-structure_element +M I-structure_element +and O +SLR B-structure_element +- I-structure_element +C I-structure_element +( O +Fig O +. O +1A O +). O + +To O +identify O +a O +soluble O +form O +of O +SEL1L B-protein +, O +we O +generated O +serial B-experimental_method +truncation I-experimental_method +constructs I-experimental_method +of O +SEL1L B-protein +based O +on O +the O +predicted O +SLR B-structure_element +motifs O +and O +highly B-protein_state +conserved I-protein_state +regions O +across O +several O +different O +species O +. O + +Helices B-structure_element +A I-structure_element +and I-structure_element +B I-structure_element +are O +14 O +and O +13 O +residues O +long O +, O +respectively O +, O +and O +the O +two O +helices B-structure_element +are O +connected O +by O +a O +short O +turn B-structure_element +and O +loop B-structure_element +( O +Fig O +. O +1D O +). O + +The O +concave B-site +surface I-site +of O +each O +SEL1L B-protein +domain O +comprising O +helix B-structure_element +5A I-structure_element +to I-structure_element +9A I-structure_element +encircles O +its O +dimer B-oligomeric_state +counterpart O +in O +an O +interlocking O +clasp O +- O +like O +arrangement O +. O + +Leu B-residue_name_number +521 I-residue_name_number +is O +located O +in O +the O +dimerization B-site +center I-site +of O +the O +antiparallel O +9B B-structure_element +helices I-structure_element +in O +the O +SEL1Lcent B-structure_element +dimer B-oligomeric_state +. O + +For O +this O +structural O +geometry O +, O +two O +adjacent O +residues O +, O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +, O +in O +SEL1L B-protein +confer O +flexibility O +at O +this O +position O +by O +adopting O +main O +- O +chain O +dihedral O +angles O +that O +are O +disallowed O +for O +non O +- O +glycine O +residues O +. O + +Gly B-residue_name_number +513 I-residue_name_number +is O +conserved B-protein_state +among O +other O +SLR B-structure_element +motifs O +in O +the O +SEL1Lcent B-structure_element +, O +but O +Gly B-residue_name_number +512 I-residue_name_number +is O +present O +only O +in O +the O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +of O +SEL1Lcent B-structure_element +( O +Fig O +. O +3A O +). O + +This O +means O +that O +the O +effect O +of O +the O +mutation B-experimental_method +is O +mainly O +to O +generate O +a O +more O +restricted O +geometry O +at O +the O +hinge B-structure_element +region O +. O + +The O +G512K B-mutant +/ O +G513K B-mutant +double B-protein_state +mutant I-protein_state +eluted O +at O +the O +monomer B-oligomeric_state +position O +in O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +( O +Fig O +. O +3D O +). O + +To O +further O +examine O +whether O +the O +SEL1Lcent B-structure_element +domain O +is O +sufficient O +to O +physically O +interact O +with O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +, O +we O +generated O +SEL1Lcent B-structure_element +and O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +deletion B-experimental_method +( O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +) O +construct O +, O +which O +were O +fused B-experimental_method +to I-experimental_method +the O +C O +- O +terminus O +of O +SEL1L B-protein +signal B-structure_element +peptides I-structure_element +. O + +In O +contrast O +, O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +- I-mutant +KDEL I-mutant +and O +the O +single O +- O +residue O +mutation O +L521A B-mutant +in O +SEL1Lcent B-structure_element +did O +not O +competitively O +inhibit O +the O +self O +- O +association O +of O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +( O +Fig O +. O +4E O +, O +F O +). O + +The O +fusion O +proteins O +were O +immobilized O +on O +glutathione O +- O +Sepharose O +beads O +and O +probed O +for O +binding O +to O +SLR B-structure_element +- I-structure_element +N I-structure_element +, O +SLR B-structure_element +- I-structure_element +M I-structure_element +, O +SLR B-structure_element +- I-structure_element +C I-structure_element +, O +and O +monomer B-oligomeric_state +form O +of O +SLR B-structure_element +- I-structure_element +M I-structure_element +( O +SLR B-mutant +- I-mutant +ML521A I-mutant +). O + +One O +possibility O +is O +that O +SLR B-structure_element +- I-structure_element +N I-structure_element +contributes O +to O +substrate O +recognition O +of O +proteins O +to O +be O +degraded O +because O +there O +are O +a O +couple O +of O +putative O +glycosylation B-site +sites I-site +within O +the O +SLR B-structure_element +- I-structure_element +N I-structure_element +domain O +( O +Fig O +. O +1A O +). O + +Consistent O +with O +the O +previous O +data O +, O +our O +crystal B-evidence +structure I-evidence +and O +biochemical B-evidence +data I-evidence +demonstrate O +that O +mouse B-taxonomy_domain +SEL1Lcent B-structure_element +exists O +as O +a O +homodimer B-oligomeric_state +in O +the O +ER O +lumen O +via O +domain O +swapping O +of O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +. O + +Rather O +, O +recent O +research O +shows O +that O +a O +transiently B-protein_state +expressed I-protein_state +HRD1 B-complex_assembly +- I-complex_assembly +SEL1L I-complex_assembly +complex O +alone O +associates O +with O +the O +ERAD O +lectins B-protein_type +OS9 B-protein +or O +XTP B-protein +- I-protein +B I-protein +and O +is O +sufficient O +to O +facilitate O +the O +retrotranslocation O +and O +degradation O +of O +the O +model O +ERAD O +substrate O +α B-protein +- I-protein +antitrypsin I-protein +null I-protein +Hong I-protein +- I-protein +Kong I-protein +( O +NHK B-protein +) O +and O +its O +variant O +, O +NHK B-mutant +- I-mutant +QQQ I-mutant +, O +which O +lacks B-protein_state +the O +N B-site +- I-site +glycosylation I-site +sites I-site +. O + +In O +yeast B-taxonomy_domain +, O +it O +is O +unclear O +whether O +self O +- O +association O +of O +Hrd3p B-protein +is O +due O +to O +SLR B-structure_element +motifs O +because O +the O +sequence O +of O +Hrd3p B-protein +does O +not O +align O +precisely O +with O +the O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +. O + +Furthermore O +, O +we O +are O +uncertain O +whether O +self O +- O +association O +of O +Hrd3p B-protein +contributes O +to O +formation O +of O +the O +active B-protein_state +form O +of O +the O +Hrd1p B-protein +complex O +. O + +This O +interaction O +seems O +to O +be O +weak O +because O +direct O +Yos9 B-protein +- O +Yos9 B-protein +interactions O +were O +not O +detected O +in O +immunoprecipitation B-experimental_method +experiments I-experimental_method +from O +yeast B-taxonomy_domain +cell O +extracts O +containing O +different O +epitope B-protein_state +- I-protein_state +tagged I-protein_state +variants O +of O +Yos9 B-protein +. O + +However O +, O +the O +dimerization B-oligomeric_state +of O +Yos9 B-protein +could O +provide O +a O +higher O +stability O +for O +the O +Hrd1p B-protein +complex O +oligomer B-oligomeric_state +. O + +Crystal B-evidence +Structure I-evidence +of O +SEL1Lcent B-structure_element +. O + +The O +11 O +SLR B-structure_element +motifs O +were O +divided O +into O +three O +groups O +( O +SLR B-structure_element +- I-structure_element +N I-structure_element +, O +SLR B-structure_element +- I-structure_element +M I-structure_element +, O +and O +SLR B-structure_element +- I-structure_element +C I-structure_element +) O +due O +to O +the O +presence O +of O +linker B-structure_element +sequences I-structure_element +that O +are O +not O +predicted O +SLR B-structure_element +motifs O +. O + +( O +A O +) O +The O +diagram O +on O +the O +left O +shows O +the O +SEL1Lcent B-structure_element +dimer B-oligomeric_state +viewed O +along O +the O +two O +- O +fold O +symmetry O +axis O +. O + +The O +elution O +fractions O +, O +indicated O +by O +the O +gray O +shading O +, O +were O +run O +on O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +and O +are O +shown O +below O +the O +gel B-evidence +- I-evidence +filtration I-evidence +elution I-evidence +profile I-evidence +. O + +The O +sequences O +of O +SEL1Lcent B-structure_element +included O +in O +the O +crystal B-evidence +structure I-evidence +are O +highlighted O +by O +the O +blue O +box O +. O + +In O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +, O +the O +axes O +for O +the O +two O +helices B-structure_element +are O +almost O +parallel O +, O +while O +the O +other O +SLR B-structure_element +motifs O +adopt O +an O +α B-structure_element +- I-structure_element +hairpin I-structure_element +structure O +. O +( O +C O +) O +Stereo O +view O +shows O +that O +the O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +residues O +are O +surrounded O +by O +neighboring O +residues O +from O +helix B-structure_element +9B I-structure_element +from O +the O +counterpart O +dimer B-oligomeric_state +. O + +The O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +residues O +are O +colored O +green O +and O +red O +, O +respectively O +. O +( O +D O +) O +The O +following O +point B-experimental_method +mutations I-experimental_method +were O +generated O +to O +check O +the O +effect O +of O +the O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +residues O +in O +terms O +of O +generating O +the O +hinge B-structure_element +of O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +: O +G512A B-mutant +, O +G513A B-mutant +, O +G512A B-mutant +/ O +G513A B-mutant +, O +and O +G512K B-mutant +/ O +G513K B-mutant +. O + +The O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +fragment O +was O +secreted O +to O +the O +culture O +media O +but O +the O +SEL1Lcent B-structure_element +was O +retained O +in O +the O +ER O +. O +( O +C O +) O +SEL1Lcent B-mutant +- I-mutant +FLAG I-mutant +- I-mutant +KDEL I-mutant +and O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +- I-mutant +FLAG I-mutant +- I-mutant +KDEL I-mutant +localized O +to O +the O +ER O +. O + +The O +SEL1L B-protein +fragments O +were O +stained O +in O +red O +. O +( O +D O +) O +HEK293T O +cells O +were O +transfected O +with O +the O +indicated O +plasmid O +constructs O +and O +the O +lysates O +were O +immunoprecipitated B-experimental_method +with O +an O +anti O +- O +HA B-experimental_method +antibody O +followed O +by O +Western B-experimental_method +blot I-experimental_method +analysis O +using O +an O +anti O +- O +FLAG B-experimental_method +antibody O +. O + +The O +red O +asterisk O +indicates O +the O +expected O +signal O +for O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +- I-mutant +FLAG I-mutant +- I-mutant +KDEL I-mutant +. O + +( O +A O +) O +Ribbon O +diagram O +showing O +superimposition B-experimental_method +of O +an O +isolated O +TPR B-structure_element +motif O +from O +Cdc23 B-protein +and O +an O +SLR B-structure_element +motif O +from O +SEL1Lcent B-structure_element +( O +left O +), O +and O +SLR B-structure_element +motifs O +in O +HcpC B-protein +and O +SEL1Lcent B-structure_element +( O +right O +). O + +The O +red O +arrow O +indicates O +disulfide B-ptm +bonds I-ptm +in O +the O +HcpC B-protein +, O +and O +Cys B-residue_name +residues O +involved O +in O +disulfide B-ptm +bonding I-ptm +are O +shown O +by O +a O +yellow O +line O +. O +( O +B O +) O +Ribbon O +representation O +showing O +superimposition B-experimental_method +of O +Cdc23 B-protein +and O +SEL1Lcent B-structure_element +( O +left O +) O +or O +HcpC B-protein +and O +SEL1Lcent B-structure_element +( O +right O +) O +to O +compare O +the O +overall O +organization O +of O +the O +α B-structure_element +- I-structure_element +solenoid I-structure_element +domain I-structure_element +. O + +Fragments O +of O +the O +luminal B-structure_element +loop I-structure_element +of O +HRD1 B-protein +fused O +to O +GST B-chemical +were O +immobilized O +on O +glutathione O +sepharose O +beads O +and O +incubated O +with O +purified O +three O +clusters O +of O +SLR B-structure_element +motifs O +and O +monomer B-oligomeric_state +form O +of O +SLR B-structure_element +- I-structure_element +M I-structure_element +( O +SLR B-mutant +- I-mutant +ML521A I-mutant +, O +right O +panel O +) O +in O +SEL1L B-protein +. O + +Proteins O +were O +analyzed O +by O +12 O +% O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +and O +Coomassie O +blue O +staining O +. O + +In O +order O +to O +understand O +the O +catalytic O +mechanism O +of O +SePSK B-protein +, O +we O +solved B-experimental_method +the O +structure B-evidence +of O +SePSK B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +D B-chemical +- I-chemical +ribulose I-chemical +and O +found O +two O +potential O +substrate B-site +binding I-site +pockets I-site +in O +SePSK B-protein +. O + +Using O +mutation B-experimental_method +and I-experimental_method +activity I-experimental_method +analysis I-experimental_method +, O +we O +further O +verified O +the O +key O +residues O +important O +for O +its O +catalytic O +activity O +. O + +Together O +, O +these O +results O +provide O +important O +information O +for O +a O +more O +detailed O +understanding O +of O +the O +cofactor O +and O +substrate O +binding O +mode O +as O +well O +as O +the O +catalytic O +mechanism O +of O +SePSK B-protein +, O +and O +possible O +similarities O +with O +its O +plant B-taxonomy_domain +homologue O +AtXK B-protein +- I-protein +1 I-protein +. O + +Carbohydrates B-chemical +are O +essential O +cellular O +compounds O +involved O +in O +the O +metabolic O +processes O +present O +in O +all O +organisms O +. O + +The O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +contain O +different O +types O +of O +sugar B-protein_type +kinases I-protein_type +, O +all O +of O +which O +possess O +different O +catalytic O +substrates O +with O +preferences O +for O +short O +- O +chained O +sugar B-chemical +substrates O +, O +ranging O +from O +triose B-chemical +to O +heptose B-chemical +. O + +Synpcc7942_2462 B-gene +from O +the O +cyanobacteria B-taxonomy_domain +Synechococcus B-species +elongatus I-species +PCC I-species +7942 I-species +encodes O +a O +putative O +sugar B-protein_type +kinase I-protein_type +( O +SePSK B-protein +), O +and O +this O +kinase B-protein_type +contains O +426 B-residue_range +amino O +acids O +. O + +Our O +structural B-experimental_method +analysis I-experimental_method +showed O +that O +apo B-protein_state +- O +SePSK B-protein +consists O +of O +one O +SePSK B-protein +protein O +molecule O +in O +an O +asymmetric O +unit O +. O + +Domain B-structure_element +II I-structure_element +is O +comprised O +of O +aa O +. O + +This O +finding O +is O +in O +agreement O +with O +a O +previous O +result O +showing O +that O +xylulose B-protein_type +kinase I-protein_type +( O +PDB O +code O +: O +2ITM O +) O +possessed O +ATP B-chemical +hydrolysis O +activity O +without O +adding O +substrate O +. O + +Both O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +showed O +ATP B-chemical +hydrolysis O +activity O +in O +the O +absence B-protein_state +of I-protein_state +substrate O +. O + +To O +understand O +the O +catalytic O +mechanism O +of O +SePSK B-protein +, O +we O +performed O +structural B-experimental_method +comparisons I-experimental_method +among O +xylulose B-protein_type +kinase I-protein_type +, O +glycerol B-protein_type +kinase I-protein_type +, O +ribulose B-protein_type +kinase I-protein_type +and O +SePSK B-protein +. O + +The O +extremely O +weak O +electron B-evidence +densities I-evidence +of O +ATP O +γ O +- O +phosphate B-chemical +in O +both O +structures B-evidence +suggest O +that O +the O +γ O +- O +phosphate B-chemical +group O +of O +ATP B-chemical +is O +either O +flexible O +or O +hydrolyzed O +by O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +. O + +The O +SePSK B-protein +structure B-evidence +is O +shown O +in O +the O +electrostatic O +potential O +surface O +mode O +. O + +However O +, O +structural B-experimental_method +comparison I-experimental_method +shows O +that O +the O +substrate O +ligating O +residues O +between O +the O +two O +structures B-evidence +are O +not B-protein_state +strictly I-protein_state +conserved I-protein_state +. O + +Based O +on O +the O +structures B-evidence +, O +the O +ligating O +residues O +of O +RBL1 B-residue_name_number +in O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +structure B-evidence +are O +Ser72 B-residue_name_number +, O +Asp221 B-residue_name_number +and O +Ser222 B-residue_name_number +, O +and O +the O +interacting O +residues O +of O +L B-chemical +- I-chemical +ribulose I-chemical +with O +L B-protein +- I-protein +ribulokinase I-protein +are O +Ala96 B-residue_name_number +, O +Lys208 B-residue_name_number +, O +Asp274 B-residue_name_number +and O +Glu329 B-residue_name_number +( O +S7 O +Fig O +). O + +The O +RBL B-chemical +molecules O +( O +carbon O +atoms O +colored O +yellow O +) O +and O +amino O +acid O +residues O +of O +SePSK B-protein +( O +carbon O +atoms O +colored O +green O +) O +involved O +in O +RBL B-chemical +interaction O +are O +shown O +as O +sticks O +. O + +This O +break O +is O +probably O +induced O +by O +the O +conformational O +change O +of O +the O +two O +β B-structure_element +- I-structure_element +sheets I-structure_element +( O +β1 B-structure_element +and O +β2 B-structure_element +), O +with O +the O +result O +that O +the O +linking B-structure_element +loop I-structure_element +( O +loop B-structure_element +1 I-structure_element +) O +is O +located O +further O +away O +from O +the O +RBL2 B-site +binding I-site +site I-site +. O + +This O +change O +might O +be O +the O +reason O +that O +AtXK B-protein +- I-protein +1 I-protein +only O +shows O +limited O +increasing O +in O +its O +ATP B-chemical +hydrolysis O +ability O +upon O +adding O +D B-chemical +- I-chemical +ribulose I-chemical +as O +a O +substrate O +after O +comparing O +with O +SePSK B-protein +( O +Fig O +2C O +). O + +However O +, O +considering O +the O +high O +concentration O +of O +D B-chemical +- I-chemical +ribulose I-chemical +used O +for O +crystal B-experimental_method +soaking I-experimental_method +, O +as O +well O +as O +the O +relatively O +weak O +electron B-evidence +density I-evidence +of O +RBL2 B-residue_name_number +, O +it O +is O +also O +possible O +that O +the O +second B-site +binding I-site +site I-site +of O +D B-chemical +- I-chemical +ribulose I-chemical +in O +SePSK B-protein +is O +an O +artifact O +. O + +Superposing B-experimental_method +the O +structures B-evidence +of O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +and O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +, O +the O +results O +show O +that O +the O +nearest O +distance O +between O +AMP B-chemical +- I-chemical +PNP I-chemical +γ O +- O +phosphate B-chemical +and O +RBL1 B-residue_name_number +/ O +RBL2 B-residue_name_number +is O +7 O +. O +5 O +Å O +( O +RBL1 B-residue_name_number +- O +O5 O +)/ O +6 O +. O +7 O +Å O +( O +RBL2 B-residue_name_number +- O +O1 O +) O +( O +S8 O +Fig O +). O + +This O +distance O +is O +too O +long O +to O +transfer O +the O +γ O +- O +phosphate B-chemical +group O +from O +ATP B-chemical +to O +the O +substrate O +. O + +The O +results O +showed O +that O +domain B-structure_element +I I-structure_element +and O +domain B-structure_element +II I-structure_element +are O +closer O +to O +each O +other O +with O +Ala228 B-residue_name_number +and O +Thr401 B-residue_name_number +in O +A2 B-structure_element +as O +Hinge B-structure_element +- I-structure_element +residues I-structure_element +. O + +Together O +, O +our O +superposition B-experimental_method +results O +provided O +snapshots O +of O +the O +conformational O +changes O +at O +different O +catalytic O +stages O +of O +SePSK B-protein +and O +potentially O +revealed O +the O +closed B-protein_state +form O +of O +SePSK B-protein +. O + +Previously O +, O +we O +proposed O +a O +pseudoatomic B-evidence +model I-evidence +of O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +based O +on O +its O +cryo B-experimental_method +- I-experimental_method +electron I-experimental_method +microscopy I-experimental_method +map B-evidence +and O +crystal B-evidence +structures I-evidence +of O +an O +inactive B-protein_state +LdcI B-protein +decamer B-oligomeric_state +and O +a O +RavA B-protein +monomer B-oligomeric_state +. O + +We O +now O +present O +cryo B-experimental_method +- I-experimental_method +electron I-experimental_method +microscopy I-experimental_method +3D B-evidence +reconstructions I-evidence +of O +the O +E B-species +. I-species +coli I-species +LdcI B-protein +and O +LdcC B-protein +, O +and O +an O +improved B-evidence +map I-evidence +of O +the O +LdcI B-protein +bound B-protein_state +to I-protein_state +the O +LARA B-structure_element +domain I-structure_element +of O +RavA B-protein +, O +at O +pH B-protein_state +optimal I-protein_state +for O +their O +enzymatic O +activity O +. O + +They O +counteract O +acid O +stress O +experienced O +by O +the O +bacterium B-taxonomy_domain +in O +the O +host O +digestive O +and O +urinary O +tract O +, O +and O +in O +particular O +in O +the O +extremely O +acidic O +stomach O +. O + +In O +addition O +, O +the O +biosynthetic B-protein_state +E B-species +. I-species +coli I-species +lysine B-protein_type +decarboxylase I-protein_type +LdcC B-protein +, O +long O +thought O +to O +be O +constitutively O +expressed O +in O +low O +amounts O +, O +was O +demonstrated O +to O +be O +strongly O +upregulated O +by O +fluoroquinolones B-chemical +via O +their O +induction O +of O +RpoS B-protein +. O +A O +direct O +correlation O +between O +the O +level O +of O +cadaverine B-chemical +and O +the O +resistance O +of O +E B-species +. I-species +coli I-species +to O +these O +antibiotics O +commonly O +used O +as O +a O +first O +- O +line O +treatment O +of O +UTI O +could O +be O +established O +. O + +Monomers B-oligomeric_state +tightly O +associate O +via O +their O +core B-structure_element +domains I-structure_element +into O +2 B-protein_state +- I-protein_state +fold I-protein_state +symmetrical I-protein_state +dimers B-oligomeric_state +with O +two O +complete O +active B-site +sites I-site +, O +and O +further O +build O +a O +toroidal B-structure_element +D5 I-structure_element +- I-structure_element +symmetrical I-structure_element +structure I-structure_element +held O +by O +the O +wing B-structure_element +and O +core B-structure_element +domain I-structure_element +interactions O +around O +the O +central B-structure_element +pore I-structure_element +, O +with O +the O +CTDs B-structure_element +at O +the O +periphery O +. O + +This O +allowed O +us O +to O +make O +a O +pseudoatomic B-evidence +model I-evidence +of O +the O +whole O +assembly O +, O +underpinned O +by O +a O +cryoEM B-experimental_method +map B-evidence +of O +the O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +complex O +( O +with O +LARA B-structure_element +standing O +for O +LdcI B-structure_element +associating I-structure_element +domain I-structure_element +of I-structure_element +RavA I-structure_element +), O +and O +to O +identify O +conformational O +rearrangements O +and O +specific O +elements O +essential O +for O +complex O +formation O +. O + +To O +solve O +this O +discrepancy O +, O +in O +the O +present O +work O +we O +provided O +a O +three O +- O +dimensional O +( O +3D O +) O +cryoEM B-experimental_method +reconstruction B-evidence +of O +LdcC B-protein +and O +compared O +it O +with O +the O +available O +LdcI B-protein +and O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +structures B-evidence +. O + +Given O +that O +the O +LdcI B-protein +crystal B-evidence +structures I-evidence +were O +obtained O +at O +high B-protein_state +pH I-protein_state +where O +the O +enzyme O +is O +inactive B-protein_state +( O +LdcIi B-protein +, O +pH B-protein_state +8 I-protein_state +. I-protein_state +5 I-protein_state +), O +whereas O +the O +cryoEM B-experimental_method +reconstructions B-evidence +of O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +and O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +were O +done O +at O +acidic B-protein_state +pH I-protein_state +optimal I-protein_state +for O +the O +enzymatic O +activity O +, O +for O +a O +meaningful O +comparison O +, O +we O +also O +produced O +a O +3D B-evidence +reconstruction I-evidence +of O +the O +LdcI B-protein +at O +active B-protein_state +pH I-protein_state +( O +LdcIa B-protein +, O +pH B-protein_state +6 I-protein_state +. I-protein_state +2 I-protein_state +). O + +This O +comparison O +pinpointed O +differences O +between O +the O +biodegradative B-protein_state +and O +the O +biosynthetic B-protein_state +lysine B-protein_type +decarboxylases I-protein_type +and O +brought O +to O +light O +interdomain O +movements O +associated O +to O +pH B-protein_state +- I-protein_state +dependent I-protein_state +enzyme O +activation O +and O +RavA B-protein +binding O +, O +notably O +at O +the O +predicted O +RavA B-site +binding I-site +site I-site +at O +the O +level O +of O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +LdcI B-protein +. O +Consequently O +, O +we O +tested O +the O +capacity O +of O +cage O +formation O +by O +LdcI B-mutant +- I-mutant +LdcC I-mutant +chimeras I-mutant +where O +we O +interchanged B-experimental_method +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheets I-structure_element +in O +question O +. O + +Remarkably O +, O +this O +analysis O +revealed O +that O +several O +specific B-structure_element +residues I-structure_element +in O +the O +above O +- O +mentioned O +β B-structure_element +- I-structure_element +sheet I-structure_element +, O +independently O +of O +the O +rest O +of O +the O +protein O +sequence O +, O +are O +sufficient O +to O +define O +if O +a O +particular O +lysine B-protein_type +decarboxylase I-protein_type +should O +be O +classified O +as O +an O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +or O +an O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +”. O + +As O +common O +for O +the O +α B-protein_type +family I-protein_type +of O +the O +PLP B-protein_type +- I-protein_type +dependent I-protein_type +decarboxylases I-protein_type +, O +dimerization O +is O +required O +for O +the O +enzymatic O +activity O +because O +the O +active B-site +site I-site +is O +buried O +in O +the O +dimer B-site +interface I-site +( O +Fig O +. O +3A O +, O +B O +). O + +The O +core B-structure_element +domain I-structure_element +is O +built O +by O +the O +PLP B-structure_element +- I-structure_element +binding I-structure_element +subdomain I-structure_element +( O +PLP B-structure_element +- I-structure_element +SD I-structure_element +, O +residues O +184 B-residue_range +– I-residue_range +417 I-residue_range +) O +flanked O +by O +two O +smaller O +subdomains B-structure_element +rich O +in O +partly B-protein_state +disordered I-protein_state +loops B-structure_element +– O +the O +linker B-structure_element +region I-structure_element +( O +residues O +130 B-residue_range +– I-residue_range +183 I-residue_range +) O +and O +the O +subdomain B-structure_element +4 I-structure_element +( O +residues O +418 B-residue_range +– I-residue_range +563 I-residue_range +). O + +The O +resolution O +of O +the O +cryoEM B-experimental_method +maps B-evidence +does O +not O +allow O +modeling O +the O +position O +of O +the O +PLP B-chemical +moiety O +and O +calls O +for O +caution O +in O +detailed O +mechanistic O +interpretations O +in O +terms O +of O +individual O +amino B-chemical +acids I-chemical +. O + +Between O +these O +two O +extremes O +, O +the O +PLP B-structure_element +- I-structure_element +SDs I-structure_element +of O +LdcIa B-protein +and O +LdcC B-protein +are O +similar O +both O +in O +the O +context O +of O +the O +decamer B-oligomeric_state +( O +Fig O +. O +3F O +) O +and O +in O +terms O +of O +RMSDmin B-evidence += O +0 O +. O +9 O +Å O +, O +which O +probably O +reflects O +the O +fact O +that O +, O +at O +the O +optimal B-protein_state +pH I-protein_state +, O +these O +lysine B-protein_type +decarboxylases I-protein_type +have O +a O +similar O +enzymatic O +activity O +. O + +The O +ppGpp B-site +binding I-site +pocket I-site +is O +made O +up O +by O +residues O +from O +all O +domains O +and O +is O +located O +approximately O +30 O +Å O +away O +from O +the O +PLP B-chemical +moiety O +. O + +All O +our O +current O +cryoEM B-experimental_method +reconstructions B-evidence +of O +the O +lysine B-protein_type +decarboxylases I-protein_type +were O +obtained O +in O +the O +absence B-protein_state +of I-protein_state +ppGpp B-chemical +in O +order O +to O +be O +closer O +to O +the O +active B-protein_state +state O +of O +the O +enzymes O +under O +study O +. O + +This O +swinging O +movement O +seems O +to O +be O +mediated O +by O +the O +core B-structure_element +domains I-structure_element +and O +is O +accompanied O +by O +a O +stretching O +of O +the O +whole O +LdcI B-protein +subunits B-structure_element +attracted O +by O +the O +RavA B-protein +magnets O +. O + +Yet O +the O +superposition B-experimental_method +of O +the O +decamers B-oligomeric_state +lays O +bare O +a O +progressive O +movement O +of O +the O +CTD B-structure_element +as O +a O +whole O +upon O +enzyme O +activation O +by O +pH O +and O +the O +binding O +of O +LARA B-structure_element +. O + +These O +small O +but O +noticeable O +swinging O +and O +stretching O +( O +up O +to O +~ O +4 O +Å O +) O +may O +be O +related O +to O +the O +incorporation O +of O +the O +LdcI B-protein +decamer B-oligomeric_state +into O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +. O + +The O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +a O +lysine B-protein_type +decarboxylase I-protein_type +as O +a O +major O +determinant O +of O +the O +interaction O +with O +RavA B-protein + +Thus O +, O +to O +advance O +beyond O +our O +experimental O +confirmation O +of O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +as O +a O +major O +determinant O +of O +the O +capacity O +of O +a O +particular O +lysine B-protein_type +decarboxylase I-protein_type +to O +form O +a O +cage O +with O +RavA B-protein +, O +we O +set O +out O +to O +investigate O +whether O +certain B-structure_element +residues I-structure_element +in O +this O +β B-structure_element +- I-structure_element +sheet I-structure_element +are O +conserved B-protein_state +in O +lysine B-protein_type +decarboxylases I-protein_type +of O +different O +enterobacteria B-taxonomy_domain +that O +have O +the O +ravA B-gene +- I-gene +viaA I-gene +operon I-gene +in O +their O +genome O +. O + +We O +inspected B-experimental_method +the I-experimental_method +genetic I-experimental_method +environment I-experimental_method +of O +lysine B-protein_type +decarboxylases I-protein_type +from O +22 O +enterobacterial B-taxonomy_domain +species O +referenced O +in O +the O +NCBI O +database O +, O +corrected O +the O +gene O +annotation O +where O +necessary O +( O +Tables O +S3 O +and O +S4 O +), O +and O +performed O +multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +coupled O +to O +a O +phylogenetic B-experimental_method +analysis I-experimental_method +( O +see O +Methods O +). O + +Finally O +, O +cadaverine B-chemical +being O +an O +important O +platform O +chemical O +for O +the O +production O +of O +industrial O +polymers O +such O +as O +nylon O +, O +structural O +information O +is O +valuable O +for O +optimisation O +of O +bacterial B-taxonomy_domain +lysine B-protein_type +decarboxylases I-protein_type +used O +for O +its O +production O +in O +biotechnology O +. O + +( O +A O +, O +C O +, O +E O +) O +cryoEM B-experimental_method +map B-evidence +of O +the O +LdcC B-protein +( O +A O +), O +LdcIa B-protein +( O +C O +) O +and O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +( O +E O +) O +decamers B-oligomeric_state +with O +one O +protomer B-oligomeric_state +in O +light O +grey O +. O + +The O +dashed O +circle O +indicates O +the O +central O +region B-structure_element +that O +remains O +virtually O +unchanged O +between O +all O +the O +structures B-evidence +, O +while O +the O +periphery O +undergoes O +visible O +movements O +. O + +( O +D O +– O +F O +) O +Inserts O +zooming O +at O +the O +CTD B-structure_element +part O +in O +proximity O +of O +the O +LARA B-site +binding I-site +site I-site +. O + +( O +D O +, O +E O +) O +A O +gallery O +of O +negative O +stain O +EM O +images O +of O +( O +D O +) O +the O +wild B-protein_state +type I-protein_state +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +and O +( O +E O +) O +the O +LdcCI B-mutant +- I-mutant +RavA I-mutant +cage I-mutant +- I-mutant +like I-mutant +particles I-mutant +. O +( O +F O +) O +Some O +representative O +class O +averages O +of O +the O +LdcCI B-mutant +- I-mutant +RavA I-mutant +cage I-mutant +- I-mutant +like I-mutant +particles I-mutant +. O + +Polarity O +differences O +are O +highlighted O +. O +( O +D O +) O +Position O +and O +nature O +of O +these O +differences O +at O +the O +surface O +of O +the O +respective O +cryoEM B-experimental_method +maps B-evidence +with O +the O +color O +code O +as O +in O +B O +. O +See O +also O +Fig O +. O +S7 O +and O +Tables O +S3 O +and O +S4 O +. O + +The O +phosphorylation B-site +site I-site +in O +the O +CTR B-structure_element +is O +solvent B-protein_state +accessible I-protein_state +and O +located O +in O +a O +negatively B-site +charged I-site +pocket I-site +∼ O +30 O +Å O +away O +from O +the O +channel B-site +exit I-site +. O + +A O +common O +feature O +of O +transceptors B-protein_type +is O +that O +they O +are O +induced O +when O +cells O +are O +starved O +for O +their O +substrate O +. O + +Fungi B-taxonomy_domain +typically O +have O +more O +than O +one O +Mep B-protein_type +paralogue O +, O +for O +example O +, O +Mep1 B-protein +- I-protein +3 I-protein +in O +S B-species +. I-species +cerevisiae I-species +. O + +As O +is O +the O +case O +for O +other O +transceptors B-protein_type +, O +it O +is O +not O +clear O +how O +Mep2 B-protein +interacts O +with O +downstream O +signalling O +partners O +, O +but O +the O +protein O +kinase O +A O +and O +mitogen O +- O +activated O +protein O +kinase O +pathways O +have O +been O +proposed O +as O +downstream O +effectors O +of O +Mep2 B-protein +( O +refs O +). O + +By O +contrast O +, O +several O +bacterial B-taxonomy_domain +Amt B-protein_type +orthologues O +have O +been O +characterized O +in O +detail O +via O +high O +- O +resolution O +crystal B-evidence +structures I-evidence +and O +a O +number O +of O +molecular B-experimental_method +dynamics I-experimental_method +( O +MD B-experimental_method +) O +studies O +. O + +A O +highly B-protein_state +conserved I-protein_state +pair O +of O +channel B-site +- O +lining O +histidine B-residue_name +residues O +dubbed O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +may O +serve O +as O +a O +proton O +relay O +system O +while O +NH3 B-chemical +moves O +through O +the O +channel B-site +during O +NH3 B-chemical +/ O +H B-chemical ++ I-chemical +symport O +. O + +Together O +with O +a O +structure B-evidence +of O +a O +C O +- O +terminal O +Mep2 B-mutant +variant I-mutant +lacking B-protein_state +the O +segment B-structure_element +containing O +the O +phosphorylation B-site +site I-site +, O +the O +results O +allow O +us O +to O +propose O +a O +structural O +model O +for O +phosphorylation O +- O +based O +regulation O +of O +eukaryotic B-taxonomy_domain +ammonium B-chemical +transport O +. O + +The O +N O +termini O +of O +the O +Mep2 B-protein_type +proteins I-protein_type +are O +∼ O +20 B-residue_range +– I-residue_range +25 I-residue_range +residues O +longer O +compared O +with O +their O +bacterial B-taxonomy_domain +counterparts O +( O +Figs O +1 O +and O +2 O +), O +substantially O +increasing O +the O +size O +of O +the O +extracellular B-structure_element +domain I-structure_element +. O + +The O +largest O +differences O +between O +the O +Mep2 B-protein +structures B-evidence +and O +the O +other O +known O +ammonium B-protein_type +transporter I-protein_type +structures B-evidence +are O +located O +on O +the O +intracellular O +side O +of O +the O +membrane O +. O + +ICL1 B-structure_element +has O +also O +moved O +inwards O +relative O +to O +its O +position O +in O +the O +bacterial B-taxonomy_domain +Amts B-protein_type +. O + +The O +head O +group O +of O +Arg54 B-residue_name_number +has O +moved O +∼ O +11 O +Å O +relative O +to O +that O +in O +Amt B-protein +- I-protein +1 I-protein +, O +whereas O +the O +shift O +of O +the O +head O +group O +of O +the O +variable O +Lys55 B-residue_name_number +residue O +is O +almost O +20 O +Å O +. O +The O +side O +chain O +of O +Lys56 B-residue_name_number +in O +the O +basic B-protein_state +motif B-structure_element +points O +in O +an O +opposite O +direction O +in O +the O +Mep2 B-protein +structures B-evidence +compared O +with O +that O +of O +, O +for O +example O +, O +Amt B-protein +- I-protein +1 I-protein +( O +Fig O +. O +4 O +). O + +This O +is O +illustrated O +by O +the O +positions O +of O +the O +five O +universally B-protein_state +conserved I-protein_state +residues O +within O +the O +CTR B-structure_element +, O +that O +is O +, O +Arg415 B-residue_name_number +( O +370 B-residue_number +), O +Glu421 B-residue_name_number +( O +376 B-residue_number +), O +Gly424 B-residue_name_number +( O +379 B-residue_number +), O +Asp426 B-residue_name_number +( O +381 B-residue_number +) O +and O +Tyr B-residue_name_number +435 I-residue_name_number +( O +390 B-residue_number +) O +in O +CaMep2 B-protein +( O +Amt B-protein +- I-protein +1 I-protein +) O +( O +Fig O +. O +2 O +). O + +These O +residues O +include O +those O +of O +the O +‘ B-structure_element +ExxGxD I-structure_element +' I-structure_element +motif I-structure_element +, O +which O +when O +mutated B-experimental_method +generate O +inactive B-protein_state +transporters B-protein_type +. O + +Despite O +its O +location O +at O +the O +periphery O +of O +the O +trimer B-oligomeric_state +, O +the O +electron B-evidence +density I-evidence +for O +the O +serine B-residue_name +is O +well O +defined O +in O +both O +Mep2 B-protein +structures B-evidence +and O +corresponds O +to O +the O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +state O +( O +Fig O +. O +6 O +). O + +The O +same O +mutant B-mutant +lacking B-protein_state +the I-protein_state +His I-protein_state +- I-protein_state +tag I-protein_state +has O +WT B-protein_state +properties O +( O +Supplementary O +Fig O +. O +1b O +), O +confirming O +that O +the O +region O +following O +the O +phosphorylation B-site +site I-site +is O +dispensable O +for O +function O +. O + +Mep2 B-protein +lacking B-protein_state +the O +AI B-structure_element +region I-structure_element +is O +conformationally B-protein_state +heterogeneous I-protein_state + +This O +is O +not O +unexpected O +given O +the O +fact O +that O +the O +AI B-structure_element +region I-structure_element +bridges O +the O +CTR B-structure_element +and O +the O +main B-structure_element +body I-structure_element +of O +Mep2 B-protein +( O +Fig O +. O +6 O +). O + +As O +shown O +in O +Supplementary O +Fig O +. O +4 O +, O +the O +consequence O +of O +the O +single B-mutant +D I-mutant +mutation B-experimental_method +is O +very O +similar O +to O +that O +of O +the O +DD B-mutant +substitution I-mutant +, O +with O +conformational O +changes O +and O +increased O +dynamics O +confined O +to O +the O +conserved B-protein_state +part O +of O +the O +CTR B-structure_element +( O +Supplementary O +Fig O +. O +4 O +). O + +As O +the O +simulation B-experimental_method +proceeds O +, O +the O +side O +chains O +of O +the O +acidic O +residues O +move O +away O +from O +Asp452 B-residue_name_number +and O +Asp453 B-residue_name_number +, O +presumably O +to O +avoid O +electrostatic O +repulsion O +. O + +One O +possible O +explanation O +is O +that O +the O +mutants B-mutant +do O +not O +accurately O +mimic O +a O +phosphoserine B-residue_name +, O +but O +the O +observation O +that O +the O +S453D B-mutant +and O +DD B-mutant +mutants I-mutant +are O +fully B-protein_state +active I-protein_state +in O +the O +absence B-protein_state +of I-protein_state +Npr1 B-protein +suggests O +that O +the O +mutations B-experimental_method +do O +mimic O +the O +effect O +of O +phosphorylation B-ptm +( O +Fig O +. O +3 O +). O + +In O +addition O +, O +a O +number O +of O +biochemical B-experimental_method +and I-experimental_method +genetic I-experimental_method +studies I-experimental_method +are O +available O +for O +bacterial B-taxonomy_domain +, O +fungal B-taxonomy_domain +and O +plant B-taxonomy_domain +proteins O +. O + +However O +, O +even O +the O +otherwise O +highly O +similar O +Mep2 B-protein_type +proteins I-protein_type +of O +S B-species +. I-species +cerevisiae I-species +and O +C B-species +. I-species +albicans I-species +have O +different O +structures B-evidence +for O +their O +CTRs B-structure_element +( O +Fig O +. O +1 O +and O +Supplementary O +Fig O +. O +6 O +). O + +In O +addition O +, O +the O +AI B-structure_element +region I-structure_element +of O +the O +CTR B-structure_element +containing O +the O +Npr1 B-site +kinase I-site +site I-site +is O +conserved B-protein_state +in O +only O +a O +subset O +of O +fungal B-taxonomy_domain +transporters B-protein_type +, O +suggesting O +that O +the O +details O +of O +the O +structural O +changes O +underpinning O +regulation O +vary O +. O + +In O +addition O +, O +the O +considerable O +differences O +between O +structurally O +resolved O +CTR B-structure_element +domains O +means O +that O +the O +exact O +environment O +of O +T460 B-residue_name_number +in O +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +is O +also O +not O +known O +( O +Supplementary O +Fig O +. O +6 O +). O + +( O +a O +) O +Monomer B-oligomeric_state +cartoon O +models O +viewed O +from O +the O +side O +for O +( O +left O +) O +A O +. O +fulgidus O +Amt B-protein +- I-protein +1 I-protein +( O +PDB O +ID O +2B2H O +), O +S B-species +. I-species +cerevisiae I-species +Mep2 B-protein +( O +middle O +) O +and O +C B-species +. I-species +albicans I-species +Mep2 B-protein +( O +right O +). O + +One O +monomer B-oligomeric_state +is O +coloured O +as O +in O +a O +and O +one O +monomer B-oligomeric_state +is O +coloured O +by O +B O +- O +factor O +( O +blue O +, O +low O +; O +red O +; O +high O +). O + +The O +conserved B-protein_state +RxK B-structure_element +motif I-structure_element +in O +ICL1 B-structure_element +is O +boxed O +in O +blue O +, O +the O +ER B-structure_element +motif I-structure_element +in O +ICL2 B-structure_element +in O +cyan O +, O +the O +conserved B-protein_state +ExxGxD B-structure_element +motif I-structure_element +of O +the O +CTR B-structure_element +in O +red O +and O +the O +AI B-structure_element +region I-structure_element +in O +yellow O +. O + +The O +Npr1 B-site +kinase I-site +site I-site +in O +the O +AI B-structure_element +region I-structure_element +is O +highlighted O +pink O +. O + +( O +a O +) O +ICL1 B-structure_element +in O +AfAmt B-protein +- I-protein +1 I-protein +( O +light O +blue O +) O +and O +CaMep2 B-protein +( O +dark O +blue O +), O +showing O +unwinding O +and O +inward O +movement O +in O +the O +fungal B-taxonomy_domain +protein O +. O +( O +b O +) O +Stereo O +diagram O +viewed O +from O +the O +cytosol O +of O +ICL1 B-structure_element +, O +ICL3 B-structure_element +( O +green O +) O +and O +the O +CTR B-structure_element +( O +red O +) O +in O +AfAmt B-protein +- I-protein +1 I-protein +( O +light O +colours O +) O +and O +CaMep2 B-protein +( O +dark O +colours O +). O + +The O +side O +chains O +of O +residues O +in O +the O +RxK B-structure_element +motif I-structure_element +as O +well O +as O +those O +of O +Tyr49 B-residue_name_number +and O +His342 B-residue_name_number +are O +labelled O +. O + +( O +a O +) O +Stereoviews O +of O +CaMep2 B-protein +showing O +2Fo O +– O +Fc O +electron O +density O +( O +contoured O +at O +1 O +. O +0 O +σ O +) O +for O +CTR B-structure_element +residues O +Asp419 B-residue_range +- I-residue_range +Met422 I-residue_range +and O +for O +Tyr446 B-residue_range +- I-residue_range +Thr455 I-residue_range +of O +the O +AI B-structure_element +region I-structure_element +. O + +Phosphorylation B-ptm +causes O +conformational O +changes O +in O +the O +CTR B-structure_element +. O + +The O +arrow O +indicates O +the O +phosphorylation B-site +site I-site +. O + +In O +this O +case O +however O +, O +the O +open B-protein_state +channel B-site +corresponds O +to O +the O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +state O +; O +phosphorylation B-ptm +breaks O +the O +CTR O +– O +ICL3 O +interactions O +leading O +to O +channel B-site +closure O +. O +( O +b O +) O +Model O +based O +on O +AMT O +transporter O +analogy O +showing O +how O +phosphorylation B-ptm +of O +a O +Mep2 B-protein +monomer B-oligomeric_state +might O +allosterically O +open B-protein_state +channels B-site +in O +the O +entire O +trimer B-oligomeric_state +via O +disruption O +of O +the O +interactions O +between O +the O +CTR B-structure_element +and O +ICL3 B-structure_element +of O +a O +neighbouring O +monomer B-oligomeric_state +( O +arrow O +). O + +Furthermore O +, O +mutational B-experimental_method +analyses I-experimental_method +show O +that O +tRNAHis B-chemical +is O +bound B-protein_state +to I-protein_state +TLP B-protein_type +in O +a O +similar O +manner O +as O +Thg1 B-protein +, O +thus O +indicating O +that O +TLP B-protein_type +has O +a O +dual O +binding O +mode O +. O + +In O +3 O +′- O +5 O +′ O +elongation O +by O +Thg1 B-protein +/ O +TLP B-protein_type +family O +proteins O +, O +the O +5 B-chemical +′- I-chemical +monophosphate I-chemical +of O +the O +tRNA B-chemical +is O +first O +activated O +by O +ATP B-chemical +/ O +GTP B-chemical +, O +followed O +by O +the O +actual O +elongation O +reaction O +. O + +Furthermore O +, O +the O +structure B-evidence +of O +Candida B-species +albicans I-species +Thg1 B-protein +( O +CaThg1 B-protein +) O +complexed B-protein_state +with I-protein_state +tRNAHis B-chemical +reveals O +that O +the O +tRNA B-chemical +substrate O +accesses O +the O +reaction B-site +center I-site +from O +a O +direction O +opposite O +to O +that O +of O +canonical O +DNA B-protein_type +/ I-protein_type +RNA I-protein_type +polymerase I-protein_type +. O + +Therefore O +, O +we O +prepared O +a O +crystal B-evidence +of O +MaTLP B-protein +complexed B-protein_state +with I-protein_state +ppptRNAPheΔ1 B-chemical +and O +solved B-experimental_method +its O +structure B-evidence +to O +study O +the O +template O +- O +directed O +3 O +′- O +5 O +′ O +elongation O +reaction O +by O +TLP B-protein_type +( O +fig O +. O +S1 O +). O + +( O +B O +) O +Structure B-evidence +after O +GDPNP B-chemical +binding O +. O +( O +C O +) O +Superposition B-experimental_method +of O +the O +two O +structures B-evidence +showing O +movement O +of O +the O +5 O +′- O +end O +of O +the O +tRNA B-chemical +before O +( O +blue O +) O +and O +after O +( O +red O +) O +insertion O +of O +GDPNP B-chemical +. O +( O +D O +) O +Superposition B-experimental_method +of O +the O +5 O +′- O +end O +of O +the O +tRNA B-chemical +after O +GDPNP B-chemical +insertion O +( O +red O +) O +with O +GTP B-chemical +at O +the O +activation O +step O +( O +green O +), O +showing O +that O +both O +triphosphate B-chemical +moieties O +superpose O +well O +. O + +The O +triphosphate B-chemical +moiety O +of O +GDPNP B-chemical +was O +at O +the O +interface B-site +between O +molecules O +A B-structure_element +and O +B B-structure_element +and O +was O +recognized O +by O +the O +side O +chains O +of O +both O +molecules O +, O +including O +R19 B-residue_name_number +( O +molecule O +A B-structure_element +), O +R83 B-residue_name_number +( O +molecule O +B B-structure_element +), O +K86 B-residue_name_number +( O +molecule O +B B-structure_element +), O +and O +R114 B-residue_name_number +( O +molecule O +A B-structure_element +) O +( O +Fig O +. O +3B O +). O + +On O +the O +basis O +of O +these O +structures B-evidence +, O +we O +will O +discuss O +the O +3 O +′- O +5 O +′ O +addition O +reaction O +compared O +with O +canonical O +5 O +′- O +3 O +′ O +elongation O +by O +DNA B-protein_type +/ I-protein_type +RNA I-protein_type +polymerases I-protein_type +. O + +In O +the O +first O +activation O +step O +, O +when O +GTP B-chemical +/ O +ATP B-chemical +is O +bound B-protein_state +to I-protein_state +site B-site +1 I-site +( O +Fig O +. O +5B O +), O +the O +5 B-chemical +′- I-chemical +phosphate I-chemical +of O +the O +tRNA B-chemical +is O +deprotonated O +by O +Mg2 B-chemical ++ I-chemical +A O +and O +attacks O +the O +α O +- O +phosphate B-chemical +of O +the O +GTP B-chemical +/ O +ATP B-chemical +, O +resulting O +in O +an O +activated O +intermediate O +( O +Fig O +. O +5C O +). O + +( O +D O +) O +Structure B-evidence +of O +initiation O +of O +the O +elongation O +reaction O +( O +corresponding O +to O +Fig O +. O +3B O +). O + +In O +these O +reactions O +, O +the O +roles O +of O +the O +two O +Mg B-chemical +ions O +are O +identical O +. O + +The O +role O +of O +Mg2 B-chemical ++ I-chemical +B O +is O +to O +position O +the O +5 B-chemical +′- I-chemical +triphosphate I-chemical +of O +the O +tRNA B-chemical +in O +TLP B-protein_type +and O +the O +incoming O +nucleotide O +in O +T7 B-protein +RNA I-protein +polymerase I-protein +. O + +Structures B-evidence +of O +template O +- O +dependent O +nucleotide O +elongation O +in O +the O +3 O +′- O +5 O +′ O +and O +5 O +′- O +3 O +′ O +directions O +. O + +Furthermore O +, O +the O +dual O +binding O +mode O +of O +this O +protein O +suggests O +that O +it O +has O +further O +evolved O +to O +cover O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +addition O +of O +tRNAHis B-chemical +by O +additional O +dimerization O +( O +dimer B-oligomeric_state +of O +dimers B-oligomeric_state +). O + +Thus O +, O +the O +present O +structural B-experimental_method +analysis I-experimental_method +is O +consistent O +with O +the O +scenario O +in O +which O +TLP B-protein_type +began O +as O +a O +5 O +′- O +end O +repair O +enzyme O +and O +evolved O +into O +a O +tRNAHis B-protein_type +- I-protein_type +specific I-protein_type +G I-protein_type +− I-protein_type +1 I-protein_type +addition I-protein_type +enzyme I-protein_type +. O + +Pooled O +tRNAs B-chemical +were O +precipitated O +with O +isopropanol O +and O +dissolved O +in O +buffer O +E O +[ O +20 O +mM O +Hepes O +- O +NaOH O +( O +pH O +7 O +. O +5 O +), O +100 O +mM O +NaCl O +, O +and O +10 O +mM O +MgCl2 O +]. O + +The O +longer B-protein_state +CDRs B-structure_element +with O +tandem O +glycines B-residue_name +or O +serines B-residue_name +have O +more O +conformational O +diversity O +than O +the O +others O +. O + +One O +conclusion O +is O +that O +the O +CDR B-structure_element +H3 B-structure_element +conformations O +are O +influenced O +by O +both O +their O +amino O +acid O +sequence O +and O +their O +structural O +environment O +determined O +by O +the O +heavy B-structure_element +and O +light B-structure_element +chain I-structure_element +pairing O +. O + +In O +such O +efforts O +, O +the O +crystal B-evidence +structure I-evidence +of O +the O +specific O +antibody B-protein_type +may O +not O +be O +available O +, O +but O +modeling O +can O +be O +used O +to O +guide O +the O +engineering O +efforts O +. O + +These O +multimeric O +forms O +are O +linked O +with O +an O +additional O +J B-structure_element +chain O +. O + +The O +LCs B-structure_element +that O +associate O +with O +the O +HCs B-structure_element +are O +divided O +into O +2 O +functionally O +indistinguishable O +classes O +, O +κ B-structure_element +and O +λ B-structure_element +. O + +The O +heavy B-structure_element +and O +light B-structure_element +chains I-structure_element +are O +composed O +of O +structural B-structure_element +domains I-structure_element +that O +have O +∼ B-residue_range +110 I-residue_range +amino I-residue_range +acid I-residue_range +residues I-residue_range +. O + +These O +domains O +have O +a O +common O +folding O +pattern O +often O +referred O +to O +as O +the O +“ O +immunoglobulin B-structure_element +fold I-structure_element +,” O +formed O +by O +the O +packing O +together O +of O +2 O +anti B-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +. O + +The O +“ O +kinked B-protein_state +” O +or O +“ O +bulged B-protein_state +” O +conformation O +is O +the O +most O +prevalent O +, O +but O +an O +“ O +extended B-protein_state +” O +or O +“ O +non B-protein_state +- I-protein_state +bulged I-protein_state +” O +conformation O +is O +also O +, O +but O +less O +frequently O +, O +observed O +. O + +Recent O +antibody B-experimental_method +modeling I-experimental_method +assessments I-experimental_method +show O +continued O +improvement O +in O +the O +quality O +of O +the O +models O +being O +generated O +by O +a O +variety O +of O +modeling O +methods O +. O + +The O +need O +for O +improvement O +in O +this O +area O +was O +also O +highlighted O +in O +a O +recent O +study O +reporting O +an O +approach O +and O +results O +that O +may O +influence O +future O +antibody B-protein_type +modeling O +efforts O +. O + +One O +important O +finding O +of O +the O +antibody B-experimental_method +modeling I-experimental_method +assessments I-experimental_method +was O +that O +errors O +in O +the O +structural O +templates O +that O +are O +used O +as O +the O +basis O +for O +homology B-experimental_method +models I-experimental_method +can O +propagate O +into O +the O +final O +models O +, O +producing O +inaccuracies O +that O +may O +negatively O +influence O +the O +predictive O +nature O +of O +the O +V B-structure_element +region I-structure_element +model O +. O + +This O +Fab B-structure_element +library O +is O +composed O +of O +3 O +HC B-structure_element +germlines O +, O +IGHV1 B-mutant +- I-mutant +69 I-mutant +( O +H1 B-mutant +- I-mutant +69 I-mutant +), O +IGHV3 B-mutant +- I-mutant +23 I-mutant +( O +H3 B-mutant +- I-mutant +23 I-mutant +) O +and O +IGHV5 B-mutant +- I-mutant +51 I-mutant +( O +H5 B-mutant +- I-mutant +51 I-mutant +), O +and O +4 O +LC B-structure_element +germlines O +( O +all O +κ B-structure_element +), O +IGKV1 B-mutant +- I-mutant +39 I-mutant +( O +L1 B-mutant +- I-mutant +39 I-mutant +), O +IGKV3 B-mutant +- I-mutant +11 I-mutant +( O +L3 B-mutant +- I-mutant +11 I-mutant +), O +IGKV3 B-mutant +- I-mutant +20 I-mutant +( O +L3 B-mutant +- I-mutant +20 I-mutant +) O +and O +IGKV4 B-mutant +- I-mutant +1 I-mutant +( O +L4 B-mutant +- I-mutant +1 I-mutant +). O + +The O +structure O +analyses O +include O +comparisons O +of O +the O +overall O +structures B-evidence +, O +canonical O +structures B-evidence +of O +the O +L1 B-structure_element +, O +L2 B-structure_element +, O +L3 B-structure_element +, O +H1 B-structure_element +and O +H2 B-structure_element +CDRs B-structure_element +, O +the O +structures B-evidence +of O +all O +CDR B-structure_element +H3s B-structure_element +, O +and O +the O +VH B-complex_assembly +: I-complex_assembly +VL I-complex_assembly +packing B-bond_interaction +interactions I-bond_interaction +. O + +In O +addition O +to O +these O +, O +2 O +primary O +disordered O +stretches O +of O +residues O +are O +observed O +in O +a O +number O +of O +structures B-evidence +( O +Table O +S1 O +). O + +The O +other O +is O +located O +in O +CDR B-structure_element +H3 B-structure_element +( O +in O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +, O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +in O +one O +of O +2 O +copies O +of O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +). O + +The O +CDR B-structure_element +H1 B-structure_element +backbone O +conformations O +for O +all O +variants O +for O +each O +of O +the O +HCs B-structure_element +are O +shown O +in O +Fig O +. O +1 O +. O + +The O +superposition B-experimental_method +of O +CDR B-structure_element +H2 B-structure_element +backbones O +for O +all O +HC B-complex_assembly +: I-complex_assembly +LC I-complex_assembly +pairs O +with O +heavy B-structure_element +chains I-structure_element +: O +( O +A O +) O +H1 B-mutant +- I-mutant +69 I-mutant +, O +( O +B O +) O +H3 B-mutant +- I-mutant +23 I-mutant +, O +( O +C O +) O +H3 B-mutant +- I-mutant +53 I-mutant +and O +( O +D O +) O +H5 B-mutant +- I-mutant +51 I-mutant +. O + +Arg71 B-residue_name_number +in O +H3 B-mutant +- I-mutant +23 I-mutant +fills O +the O +space O +between O +CDRs B-structure_element +H2 B-structure_element +and O +H4 B-structure_element +, O +and O +defines O +the O +conformation O +of O +the O +tip O +of O +CDR B-structure_element +H2 B-structure_element +so O +that O +residue O +54 B-residue_number +points O +away O +from O +the O +antigen B-site +binding I-site +site I-site +. O + +Some O +changes O +in O +conformation O +occur O +between O +residues O +30a B-residue_number +and O +30f B-residue_number +( O +residues O +8 B-residue_number +and O +13 B-residue_number +of O +17 B-residue_number +in O +CDR B-structure_element +L1 B-structure_element +). O + +Two O +structures B-evidence +, O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +are O +assigned O +to O +canonical O +structure O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +1 I-mutant +with O +virtually O +identical O +backbone O +conformations O +. O + +The O +conformation O +of O +CDR B-structure_element +L1 B-structure_element +in O +these O +2 O +Fabs B-structure_element +is O +slightly O +different O +, O +and O +both O +conformations O +fall O +somewhere O +between O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +1 I-mutant +and O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +2 I-mutant +. O + +As O +with O +CDR B-structure_element +L2 B-structure_element +, O +all O +4 O +LCs B-structure_element +have O +CDR B-structure_element +L3 B-structure_element +of O +the O +same O +length O +and O +canonical O +structure B-evidence +, O +L3 B-mutant +- I-mutant +9 I-mutant +- I-mutant +cis7 I-mutant +- I-mutant +1 I-mutant +( O +Table O +2 O +). O + +As O +mentioned O +earlier O +, O +all O +16 O +Fabs B-structure_element +have O +the O +same O +CDR B-structure_element +H3 B-structure_element +, O +for O +which O +the O +amino O +acid O +sequence O +is O +derived O +from O +the O +anti O +- O +CCL2 O +antibody B-protein_type +CNTO B-chemical +888 I-chemical +. O + +Ribbon O +representations O +of O +( O +A O +) O +the O +superposition B-experimental_method +of O +all O +CDR B-structure_element +H3s B-structure_element +of O +the O +structures B-evidence +with O +complete O +backbone O +traces O +. O +( O +B O +) O +The O +CDR B-structure_element +H3s B-structure_element +rotated O +90 O +° O +about O +the O +y O +axis O +of O +the O +page O +. O + +A O +comparison O +of O +representatives O +of O +the O +“ O +kinked B-protein_state +” O +and O +“ O +extended B-protein_state +” O +structures B-evidence +. O + +( O +A O +) O +The O +“ O +kinked B-protein_state +” O +CDR B-structure_element +H3 B-structure_element +of O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +with O +purple O +carbon O +atoms O +and O +yellow O +dashed O +lines O +connecting O +the O +H O +- O +bond O +pairs O +for O +Leu100b B-residue_name_number +O O +and O +Trp103 B-residue_name_number +NE1 O +, O +Arg94 B-residue_name_number +NE O +and O +Asp101 B-residue_name_number +OD1 O +, O +and O +Arg94 B-residue_name_number +NH2 O +and O +Asp101 B-residue_name_number +OD2 O +. O + +The O +VH B-structure_element +and O +VL B-structure_element +domains O +have O +a O +β B-structure_element +- I-structure_element +sandwich I-structure_element +structure I-structure_element +( O +also O +often O +referred O +as O +a O +Greek B-structure_element +key I-structure_element +motif I-structure_element +) O +and O +each O +is O +composed O +of O +a O +4 B-structure_element +- I-structure_element +stranded I-structure_element +and I-structure_element +a I-structure_element +5 I-structure_element +- I-structure_element +stranded I-structure_element +antiparallel I-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +. O + +VH B-site +: I-site +VL I-site +interface I-site +amino O +acid O +residue O +interactions O + +They O +include O +: O +1 O +) O +a O +bidentate O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +L B-structure_element +- O +Gln38 B-residue_name_number +and O +H B-structure_element +- O +Gln39 B-residue_name_number +; O +2 O +) O +H B-structure_element +- O +Leu45 B-residue_name_number +in O +a O +hydrophobic B-site +pocket I-site +between O +L B-structure_element +- O +Phe98 B-residue_name_number +, O +L B-structure_element +- O +Tyr87 B-residue_name_number +and O +L B-structure_element +- O +Pro44 B-residue_name_number +; O +3 O +) O +L B-structure_element +- O +Pro44 B-residue_name_number +stacked O +against O +H B-structure_element +- O +Trp103 B-residue_name_number +; O +and O +4 O +) O +L B-structure_element +- O +Ala43 B-residue_name_number +opposite O +the O +face O +of O +H B-structure_element +- O +Tyr91 B-residue_name_number +( O +Fig O +. O +8 O +). O + +These O +core O +interactions O +provide O +enough O +stability O +to O +the O +VH B-complex_assembly +: I-complex_assembly +VL I-complex_assembly +dimer B-oligomeric_state +so O +that O +additional O +VH B-site +- I-site +VL I-site +contacts I-site +can O +tolerate O +amino O +acid O +sequence O +variations O +in O +CDRs B-structure_element +H3 B-structure_element +and O +L3 B-structure_element +that O +form O +part O +of O +the O +VH B-site +: I-site +VL I-site +interface I-site +. O + +In O +most O +of O +the O +structures B-evidence +, O +it O +has O +the O +χ2 B-evidence +angle O +of O +∼ O +80 O +°, O +while O +the O +ring O +is O +flipped O +over O +( O +χ2 B-evidence += O +− O +100 O +°) O +in O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +: I-complex_assembly +11 I-complex_assembly +and O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +. O + +Apparently O +, O +residues O +flanking O +CDR B-structure_element +H3 B-structure_element +in O +the O +2 O +VH B-complex_assembly +: I-complex_assembly +VL I-complex_assembly +pairings O +are O +inconsistent O +with O +any O +stable B-protein_state +conformation O +of O +CDR B-structure_element +H3 B-structure_element +, O +which O +translates O +into O +a O +less O +restricted O +conformational O +space O +for O +some O +of O +them O +, O +including O +H B-structure_element +- O +Trp47 B-residue_name_number +. O + +Residues O +in O +CDR B-structure_element +H3 B-structure_element +are O +missing O +: O +YGE B-structure_element +in O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +, O +GIY B-structure_element +in O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +. O + +Melting B-evidence +temperatures I-evidence +for O +the O +16 O +Fabs B-structure_element +. O + +Colors O +: O +blue O +( O +Tm B-evidence +< O +70 O +° O +C O +), O +green O +( O +70 O +° O +C O +< O +Tm B-evidence +< O +73 O +° O +C O +), O +yellow O +( O +73 O +° O +C O +< O +Tm B-evidence +< O +78 O +° O +C O +), O +orange O +( O +Tm B-evidence +> O +78 O +° O +C O +). O + +It O +appears O +that O +for O +each O +given O +LC B-structure_element +, O +the O +Fabs B-structure_element +with O +germlines O +H1 B-mutant +- I-mutant +69 I-mutant +and O +H3 B-mutant +- I-mutant +23 I-mutant +are O +substantially O +more O +stable B-protein_state +than O +those O +with O +germlines O +H3 B-mutant +- I-mutant +53 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +. O + +No O +electron B-evidence +density I-evidence +is O +observed O +for O +a O +number O +of O +side O +chains O +in O +CDRs B-structure_element +H3 B-structure_element +and O +L3 B-structure_element +in O +all O +Fabs B-structure_element +with O +germline O +H3 B-mutant +- I-mutant +53 I-mutant +, O +which O +indicates O +loose O +packing O +of O +the O +variable B-structure_element +domains I-structure_element +. O + +Of O +the O +4 O +HCs B-structure_element +, O +H1 B-mutant +- I-mutant +69 I-mutant +has O +the O +greatest O +number O +of O +canonical O +structure O +assignments O +( O +Table O +2 O +). O + +As O +mentioned O +in O +the O +Results O +section O +, O +this O +data O +set O +is O +composed O +of O +21 O +Fabs B-structure_element +, O +since O +5 O +of O +the O +16 O +variants O +have O +2 O +Fab B-structure_element +copies O +in O +the O +asymmetric O +unit O +. O + +For O +the O +18 O +Fabs B-structure_element +with O +complete O +backbone O +atoms O +for O +CDR B-structure_element +H3 B-structure_element +, O +10 O +have O +conformations O +similar O +to O +that O +of O +the O +parent O +, O +while O +the O +others O +have O +significantly O +different O +conformations O +( O +Fig O +. O +6 O +). O + +Thus O +, O +it O +is O +likely O +that O +the O +CDR B-structure_element +H3 B-structure_element +conformation O +is O +dependent O +upon O +2 O +dominating O +factors O +: O +1 O +) O +amino O +acid O +sequence O +; O +and O +2 O +) O +VH B-structure_element +and O +VL B-structure_element +context O +. O + +Interestingly O +, O +as O +described O +earlier O +, O +these O +2 O +pairs O +differ O +in O +the O +stem B-structure_element +regions I-structure_element +with O +the O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +pair O +in O +the O +‘ O +extended B-protein_state +’ O +conformation O +and O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +pair O +in O +the O +‘ O +kinked B-protein_state +’ O +conformation O +. O + +The O +CDR B-structure_element +H3 B-structure_element +conformational B-experimental_method +analysis I-experimental_method +shows O +that O +, O +for O +each O +set O +of O +variants O +of O +one O +HC B-structure_element +paired O +with O +the O +4 O +different O +LCs B-structure_element +, O +both O +“ O +parental O +” O +and O +“ O +non O +- O +parental O +” O +conformations O +are O +observed O +. O + +The O +same O +variability O +is O +observed O +for O +the O +sets O +of O +variants O +composed O +of O +one O +LC B-structure_element +paired O +with O +each O +of O +the O +4 O +HCs B-structure_element +. O + +This O +finding O +supports O +the O +hypothesis O +of O +Weitzner O +et O +al O +. O +that O +the O +H3 B-structure_element +conformation O +is O +controlled O +both O +by O +its O +sequence O +and O +its O +environment O +. O + +Two O +variants O +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +have O +the O +largest O +differences O +in O +the O +tilt O +angles O +compared O +to O +other O +variants O +as O +seen O +in O +Table O +3 O +. O + +One O +of O +the O +variants O +, O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +has O +the O +CDR B-structure_element +H3 B-structure_element +conformation O +similar O +to O +the O +parent O +, O +but O +the O +other O +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +is O +different O +. O + +Pairing O +of O +different O +germlines O +yields O +antibodies B-protein_type +with O +various O +degrees O +of O +stability O +. O + +Note O +that O +most O +of O +the O +VH B-site +: I-site +VL I-site +interface I-site +residues O +are O +invariant O +; O +therefore O +, O +significant O +change O +of O +the O +tilt O +angle O +must O +come O +with O +a O +penalty O +in O +free O +energy O +. O + +The O +final O +conformation O +represents O +an O +energetic O +minimum O +; O +however O +, O +in O +most O +cases O +it O +is O +very O +shallow O +, O +so O +that O +a O +single O +mutation O +can O +cause O +a O +dramatic O +rearrangement O +of O +the O +structure B-evidence +. O + +These O +data O +reveal O +the O +difficulty O +of O +modeling O +CDR B-structure_element +H3 B-structure_element +accurately O +, O +as O +shown O +again O +in O +Antibody O +Modeling O +Assessment O +II O +. O + +With O +the O +recent O +advances O +in O +expression B-experimental_method +and I-experimental_method +crystallization I-experimental_method +methods I-experimental_method +, O +Fab B-structure_element +structures B-evidence +can O +be O +obtained O +rapidly O +. O + +Finally O +, O +a O +model O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +in O +the O +active B-protein_state +and O +inactive B-protein_state +state O +provides O +insights O +into O +protein O +dimerization O +and O +DNA B-chemical +- O +binding O +. O + +One O +of O +the O +potential O +antibiotic O +alternatives O +are O +lantibiotics B-chemical +. O + +Thus O +, O +the O +lantibiotic B-chemical +producer O +strains O +have O +an O +inbuilt O +self O +- O +protection O +mechanism O +( O +immunity O +) O +to O +prevent O +cell O +death O +caused O +due O +to O +the O +action O +of O +its O +cognate O +lantibiotic B-chemical +. O + +Recently O +, O +the O +structure B-evidence +of O +SaNSR B-protein +from O +S B-species +. I-species +agalactiae I-species +was O +solved O +which O +provides O +resistance O +against O +nisin B-chemical +by O +a O +protease O +activity O +. O + +The O +expression O +of O +the O +lantibiotic B-chemical +- O +resistance O +genes O +via O +TCS B-complex_assembly +is O +generally O +regulated O +by O +microorganism O +- O +specific O +lantibiotics B-chemical +, O +which O +act O +via O +external O +stimuli O +. O + +Interestingly O +, O +the O +histidine B-protein_type +kinase I-protein_type +contains O +two B-structure_element +- I-structure_element +transmembrane I-structure_element +helices I-structure_element +but O +lacks O +an O +extracellular B-structure_element +sensory I-structure_element +domain I-structure_element +, O +and O +are O +therefore O +known O +as O +‘ B-protein_type +intramembrane I-protein_type +- I-protein_type +sensing I-protein_type +’ I-protein_type +histidine I-protein_type +kinases I-protein_type +. O + +All O +their O +members O +are O +characterized O +by O +a O +winged B-structure_element +helix I-structure_element +- I-structure_element +turn I-structure_element +- I-structure_element +helix I-structure_element +( O +wHTH B-structure_element +) O +motif O +. O + +The O +structures B-evidence +of O +DrrD B-protein +and O +DrrB B-protein +exist O +in O +an O +open B-protein_state +conformation O +, O +here O +the O +recognition B-structure_element +helix I-structure_element +is O +fully B-protein_state +exposed I-protein_state +, O +suggesting O +that O +RRs B-protein_type +are O +flexible B-protein_state +in O +solution O +and O +can O +adopt O +multiple O +conformations O +. O + +NsrR B-protein +was O +expressed B-experimental_method +and I-experimental_method +purified I-experimental_method +as O +described O +, O +resulting O +in O +a O +homogenous O +protein O +as O +observed O +by O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +( O +Fig O +1A O +), O +with O +a O +yield O +of O +2 O +mg O +per O +liter O +of O +cell O +culture O +. O + +The O +purified O +NsrR B-protein +protein O +has O +a O +theoretical O +molecular B-evidence +mass I-evidence +of O +27 O +. O +7 O +kDa O +and O +was O +> O +98 O +% O +pure O +as O +assessed O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +( O +Fig O +1B O +, O +indicated O +by O +*). O + +This O +was O +also O +observed O +by O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +where O +a O +peak O +at O +an O +elution O +time O +of O +18 O +min O +appeared O +( O +Fig O +1A O +). O + +NsrR B-protein +was O +crystallized B-experimental_method +yielding O +two O +crystal O +forms O +, O +which O +were O +distinguishable O +by O +visual O +inspection O +. O + +After O +the O +structure B-evidence +was O +solved O +, O +it O +became O +evident O +that O +these O +crystals B-evidence +contained O +two O +monomers B-oligomeric_state +of O +the O +ED B-structure_element +of O +NsrR B-protein +in O +the O +asymmetric O +unit O +. O + +The O +Rwork B-evidence +and O +Rfree B-evidence +values O +after O +refinement O +were O +0 O +. O +17 O +and O +0 O +. O +22 O +, O +respectively O +. O + +Although O +the O +entire O +N O +- O +terminal O +receiver B-structure_element +domain I-structure_element +is O +composed O +of O +residues O +Met1 B-residue_range +- I-residue_range +Leu119 I-residue_range +, O +only O +residues O +Asn4 B-residue_range +to I-residue_range +Arg121 I-residue_range +of O +chain B-structure_element +A I-structure_element +( O +including O +residues O +Arg120 B-residue_name_number +and O +Arg121 B-residue_name_number +of O +the O +linker B-structure_element +) O +and O +Gln5 B-residue_range +to I-residue_range +Ser122 I-residue_range +of O +chain B-structure_element +B I-structure_element +( O +including O +residues O +Arg120 B-residue_range +until I-residue_range +Ser122 I-residue_range +of O +the O +linker B-structure_element +) O +could O +be O +traced O +in O +the O +electron B-evidence +density I-evidence +of O +NsrR B-protein +- O +RD B-structure_element +. O + +The O +NsrR B-protein +- O +RD B-structure_element +structure B-evidence +shows O +a O +β1 B-structure_element +- I-structure_element +α1 I-structure_element +- I-structure_element +β2 I-structure_element +- I-structure_element +α2 I-structure_element +- I-structure_element +β3 I-structure_element +- I-structure_element +α3 I-structure_element +- I-structure_element +β4 I-structure_element +- I-structure_element +α4 I-structure_element +- I-structure_element +β5 I-structure_element +- I-structure_element +α5 I-structure_element +topology O +as O +also O +observed O +for O +other O +RRs B-protein_type +. O + +The O +receiver B-structure_element +domain I-structure_element +of O +NsrR B-protein +was O +superimposed B-experimental_method +with O +other O +structurally O +characterized O +receiver B-structure_element +domains I-structure_element +from O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +family I-protein_type +, O +such O +as O +DrrB B-protein +, O +KdpE B-protein +, O +MtrA B-protein +, O +and O +the O +crystal B-evidence +structure I-evidence +of O +only O +the O +receiver B-structure_element +domain I-structure_element +of O +PhoB B-protein +. O +The O +rmsd B-evidence +of O +the O +overlays B-experimental_method +and O +the O +corresponding O +PDB O +codes O +used O +are O +highlighted O +in O +Table O +2 O +. O + +This O +could O +explain O +the O +flexibility O +and O +thereby O +the O +different O +orientation O +of O +helix B-structure_element +α4 B-structure_element +in O +NsrR B-protein +. O + +Based O +on O +the O +Dali B-experimental_method +server I-experimental_method +, O +the O +NsrR B-protein +- O +RD B-structure_element +domain O +is O +structurally O +closely O +related O +to O +KdpE B-protein +( O +PDB O +code O +: O +4KNY O +) O +from O +E B-species +. I-species +coli I-species +, O +displaying O +a O +sequence O +identity O +of O +28 O +%. O + +The O +putative O +phosphorylation B-site +site I-site +of O +NsrR B-protein +is O +Asp55 B-residue_name_number +, O +which O +is O +localized O +at O +the O +end O +of O +strand B-structure_element +β3 B-structure_element +( O +Fig O +3 O +, O +shown O +in O +red O +; O +Fig O +4 O +) O +and O +lies O +within O +an O +acidic O +environment O +composed O +of O +the O +side O +chains O +of O +Glu12 B-residue_name_number +and O +Asp13 B-residue_name_number +( O +Fig O +3 O +, O +highlighted O +in O +pink O +). O + +A O +divalent O +metal O +ion O +is O +usually O +bound O +in O +this O +acidic O +environment O +and O +is O +essential O +for O +phosphorylation B-ptm +and O +de B-ptm +- I-ptm +phosphorylation I-ptm +of O +RRs B-protein_type +. O + +In O +contrast O +, O +the O +switch B-site +residues I-site +face O +towards O +the O +active B-site +site I-site +in O +the O +active B-protein_state +state O +conformation O +( O +Fig O +4B O +). O + +A O +comparison O +of O +the O +NsrR B-protein +- O +RD B-structure_element +structure B-evidence +with O +the O +available O +structures B-evidence +of O +PhoB B-protein +( O +Fig O +4 O +) O +in O +the O +active B-protein_state +( O +PDB O +code O +: O +1ZES O +) O +and O +inactive B-protein_state +( O +PDB O +code O +: O +1B00 O +) O +states O +demonstrates O +that O +Ser82 B-residue_name_number +( O +NsrR B-protein +- O +RD B-structure_element +) O +is O +oriented O +away O +from O +the O +active B-site +site I-site +Asp55 B-residue_name_number +, O +and O +that O +Phe101 B-residue_name_number +is O +also O +in O +an O +outward B-protein_state +conformation O +suggesting O +an O +inactive B-protein_state +state O +of O +the O +NsrR B-protein +- O +RD B-structure_element +( O +Fig O +4A O +). O + +Phosphorylation B-ptm +shifts O +the O +equilibrium O +towards O +the O +active B-protein_state +conformation O +and O +induces O +the O +formation O +of O +rotationally O +symmetric O +dimers B-oligomeric_state +on O +the O +α4 B-site +- I-site +β5 I-site +- I-site +α5 I-site +interface I-site +of O +RDs B-structure_element +. O + +It O +has O +been O +suggested O +that O +dimerization O +is O +crucial O +for O +DNA B-chemical +- O +binding O +of O +RRs B-protein_type +of O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +. O + +The O +putative O +functional O +dimer B-oligomeric_state +of O +NsrR B-protein +- O +RD B-structure_element +is O +depicted O +in O +Fig O +5 O +. O + +Majority O +of O +these O +interactions O +involve O +residues O +that O +are O +highly B-protein_state +conserved I-protein_state +within O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +of O +RRs B-protein_type +. O + +Structurally O +, O +a O +similar O +set O +of O +residues O +is O +also O +found O +in O +NsrR B-protein +: O +Leu94 B-residue_name_number +( O +α4 B-structure_element +), O +Val110 B-residue_name_number +( O +α5 B-structure_element +) O +and O +Ala113 B-residue_name_number +( O +α5 B-structure_element +), O +respectively O +( O +depicted O +as O +spheres O +in O +Fig O +5B O +), O +which O +are O +conserved B-protein_state +to O +some O +extent O +on O +sequence O +level O +( O +highlighted O +in O +yellow O +; O +Fig O +3 O +). O + +Here O +, O +Asp100 B-residue_name_number +( O +β5 B-structure_element +) O +and O +Lys114 B-residue_name_number +( O +α5 B-structure_element +) O +form O +an O +interaction O +within O +one O +monomer B-oligomeric_state +, O +and O +an O +intermolecular O +interaction O +can O +be O +observed O +between O +Asn95 B-residue_name_number +( O +α4 B-structure_element +) O +of O +one O +monomer B-oligomeric_state +with O +Thr116 B-residue_name_number +( O +α5 B-structure_element +) O +of O +the O +other O +monomer B-oligomeric_state +( O +Fig O +3 O +, O +shown O +in O +cyan O +). O + +Ramachandran B-evidence +validation I-evidence +was O +done O +using O +MolProbity O +. O + +Monomer B-oligomeric_state +A B-structure_element +contains O +residue O +129 B-residue_range +– I-residue_range +224 I-residue_range +and O +monomer B-oligomeric_state +B B-structure_element +contain O +residues O +128 B-residue_range +– I-residue_range +225 I-residue_range +. O + +The O +two O +β B-structure_element +- I-structure_element +sheets I-structure_element +sandwich O +the O +three O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +Structure B-evidence +of O +the O +C O +- O +terminal O +effector B-structure_element +domain I-structure_element +of O +NsrR B-protein +. O + +Overall O +, O +the O +structures B-evidence +are O +very O +similar O +with O +rmsd B-evidence +’ O +s O +ranging O +from O +1 O +. O +7 O +to O +2 O +. O +6 O +Å O +( O +Table O +2 O +). O + +This O +resulted O +in O +a O +list O +of O +possible O +templates O +for O +modeling O +the O +full B-protein_state +- I-protein_state +length I-protein_state +structure B-evidence +of O +NsrR B-protein +( O +Table O +2 O +). O + +This O +mimics O +the O +closed B-protein_state +inactive B-protein_state +conformation O +of O +NsrR B-protein +( O +Fig O +7A O +; O +the O +missing B-protein_state +linker B-structure_element +is O +represented O +as O +dotted O +line O +). O + +The O +RD B-structure_element +domain O +of O +NsrR B-protein +is O +highlighted O +in O +yellow O +and O +the O +ED B-structure_element +domain O +in O +green O +with O +the O +“ O +recognition B-structure_element +helix I-structure_element +” O +colored O +in O +cyan O +. O +( O +a O +) O +Inactive B-protein_state +state O +conformation O +: O +Both O +domains O +of O +NsrR B-protein +were O +aligned B-experimental_method +to O +the O +structure B-evidence +of O +MtrA B-protein +( O +not O +shown O +), O +which O +adopts O +a O +closed B-protein_state +inactive B-protein_state +conformation O +, O +to O +obtain O +a O +model O +of O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +. O +Phe101 B-residue_name_number +and O +Asp187 B-residue_name_number +stabilize O +this O +closed B-protein_state +conformation O +. O + +READ B-experimental_method +enabled O +us O +to O +visualize O +even O +sparsely O +populated O +conformations O +of O +the O +substrate O +protein O +immunity B-protein +protein I-protein +7 I-protein +( O +Im7 B-protein +) O +in B-protein_state +complex I-protein_state +with I-protein_state +the O +E B-species +. I-species +coli I-species +chaperone B-protein_type +Spy B-protein +. O + +This O +study O +resulted O +in O +a O +series O +of O +snapshots O +depicting O +the O +various O +folding O +states O +of O +Im7 B-protein +while O +bound B-protein_state +to I-protein_state +Spy B-protein +. O + +Particularly O +challenging O +are O +interactions O +of O +intrinsically B-protein_state +or I-protein_state +conditionally I-protein_state +disordered I-protein_state +sections O +of O +proteins O +with O +their O +partner O +proteins O +. O + +Structural O +characterization O +of O +chaperone B-protein_type +- O +assisted O +protein O +folding O +likely O +would O +help O +bring O +clarity O +to O +this O +question O +. O + +To O +address O +this O +question O +, O +we O +investigated O +the O +ATP B-protein_state +- I-protein_state +independent I-protein_state +Escherichia B-species +coli I-species +periplasmic O +chaperone B-protein_type +Spy B-protein +. O + +We O +used O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +selenomethionine B-chemical +crystals B-evidence +for O +phasing O +with O +single B-experimental_method +- I-experimental_method +wavelength I-experimental_method +anomalous I-experimental_method +diffraction I-experimental_method +( O +SAD B-experimental_method +) O +experiments O +, O +and O +used O +this O +solution O +to O +build O +the O +well O +- O +ordered O +Spy B-protein +portions O +of O +all O +four O +complexes O +. O + +Moreover O +, O +binding B-experimental_method +experiments I-experimental_method +indicated O +that O +Im76 B-mutant +- I-mutant +45 I-mutant +comprises O +the O +entire O +Spy B-site +- I-site +binding I-site +region I-site +. O + +Consistent O +with O +the O +fragmented O +density B-evidence +, O +however O +, O +we O +observed O +multiple O +iodine B-chemical +positions O +for O +seven O +of O +the O +eight O +substituted O +residues O +. O + +Simultaneously O +, O +it O +uses O +the O +residual B-evidence +electron I-evidence +density I-evidence +to O +help O +choose O +ensembles O +. O + +By O +analyzing O +the O +individual O +structures B-evidence +of O +the O +six O +- O +member O +ensemble O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +bound B-protein_state +to I-protein_state +Spy B-protein +, O +we O +observed O +that O +Im76 B-mutant +- I-mutant +45 I-mutant +takes O +on O +several O +different O +conformations O +while O +bound B-protein_state +. O + +Surprisingly O +, O +we O +noted O +that O +in O +the O +ensemble O +, O +Im76 B-mutant +- I-mutant +45 I-mutant +interacts O +with O +only O +38 O +% O +of O +the O +hydrophobic O +residues O +in O +the O +Spy B-protein +cradle B-site +, O +but O +interacts O +with O +61 O +% O +of O +the O +hydrophilic O +residues O +in O +the O +cradle B-site +. O + +This O +mixture O +suggests O +the O +importance O +of O +both O +electrostatic O +and O +hydrophobic O +components O +in O +binding O +the O +Im76 B-mutant +- I-mutant +45 I-mutant +ensemble O +. O + +Whereas O +the O +least B-protein_state +- I-protein_state +folded I-protein_state +Im76 B-mutant +- I-mutant +45 I-mutant +pose O +in O +the O +ensemble O +forms O +the O +most O +hydrophobic O +contacts O +with O +Spy B-protein +( O +Fig O +. O +3 O +), O +the O +two O +most B-protein_state +- I-protein_state +folded I-protein_state +conformations O +form O +the O +fewest O +hydrophobic O +contacts O +( O +Fig O +. O +3 O +). O + +Spy B-protein +changes O +conformation O +upon O +substrate O +binding O + +As O +inter O +- O +molecular O +hydrophobic B-bond_interaction +interactions I-bond_interaction +between O +Spy B-protein +and O +the O +substrate O +become O +progressively O +replaced O +by O +intra O +- O +molecular O +interactions O +within O +the O +substrate O +, O +the O +affinity O +between O +chaperone B-protein_type +and O +substrates O +could O +decrease O +, O +eventually O +leading O +to O +release O +of O +the O +folded B-protein_state +client O +protein O +. O + +This O +interaction O +presumably O +reduces O +the O +mobility O +of O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +helix I-structure_element +. O + +The O +F115I B-mutant +/ O +L B-mutant +substitutions O +would O +replace O +these O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +hydrophobic B-bond_interaction +interactions I-bond_interaction +that O +have O +little O +angular O +dependence O +. O + +As O +a O +result O +, O +the O +C O +- O +terminus O +, O +and O +possibly O +also O +the O +flexible B-protein_state +linker B-structure_element +, O +is O +likely O +to O +become O +more O +flexible O +and O +thus O +more O +accommodating O +of O +different O +conformations O +of O +substrates O +. O + +( O +b O +) O +Composites O +of O +iodine B-chemical +positions O +detected O +from O +anomalous B-evidence +signals I-evidence +using O +pI B-chemical +- I-chemical +Phe I-chemical +substitutions B-experimental_method +, O +colored O +and O +numbered O +by O +sequence O +. O + +The O +frequency O +plotted O +is O +calculated O +as O +the O +average O +contact B-evidence +frequency I-evidence +from O +Spy B-protein +to O +every O +residue O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +and O +vice O +- O +versa O +. O + +Flexibility O +of O +Spy B-protein +linker B-structure_element +region I-structure_element +and O +effect O +of O +Super O +Spy B-protein +mutants O +. O +( O +a O +) O +The O +Spy B-protein +linker B-structure_element +region I-structure_element +adopts O +one O +dominant O +conformation O +in O +its O +apo B-protein_state +state O +( O +PDB O +ID O +3039 O +, O +gray O +), O +but O +expands O +and O +adopts O +multiple O +conformations O +in O +bound B-protein_state +states O +( O +green O +). O + +( O +b O +) O +F115 B-residue_name_number +and O +L32 B-residue_name_number +tether O +Spy B-protein +’ O +s O +linker B-structure_element +region I-structure_element +to O +its O +cradle B-site +, O +decreasing O +Spy B-protein +activity O +by O +limiting O +linker B-structure_element +region I-structure_element +flexibility O +. O + +L32 B-residue_name_number +, O +F115 B-residue_name_number +, O +and O +Y104 B-residue_name_number +are O +rendered O +in O +purple O +to O +illustrate O +residues O +that O +are O +most O +affected O +by O +Super O +Spy B-protein +mutations B-protein_state +; O +CH O +⋯ O +π O +hydrogen B-bond_interaction +bonds I-bond_interaction +are O +depicted O +by O +orange O +dashes O +. O + +Herein O +, O +we O +report O +X B-evidence +- I-evidence +ray I-evidence +crystal I-evidence +structures I-evidence +of O +ligand B-protein_state +- I-protein_state +free I-protein_state +Tdp2 B-protein +and O +Tdp2 B-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +complexes O +with O +alkylated O +and O +abasic O +DNA B-chemical +that O +unveil O +a O +dynamic B-protein_state +Tdp2 B-protein +active B-structure_element +site I-structure_element +lid I-structure_element +and O +deep O +substrate B-site +binding I-site +trench I-site +well O +- O +suited O +for O +engaging O +the O +diverse O +DNA B-chemical +damage O +triggers O +of O +abortive O +Top2 B-protein_type +reactions O +. O + +Nuclear O +DNA B-chemical +compaction O +and O +the O +action O +of O +DNA B-chemical +and O +RNA B-protein_type +polymerases I-protein_type +create O +positive O +and O +negative O +DNA B-chemical +supercoiling O +— O +over O +- O +and O +under O +- O +winding O +of O +DNA B-chemical +strands O +, O +respectively O +— O +and O +the O +linking O +together O +( O +catenation O +) O +of O +DNA B-chemical +strands O +. O + +To O +promote O +cancer O +cell O +death O +, O +Top2 B-protein_type +reactions O +are O +‘ O +poisoned O +’ O +by O +keystone O +pharmacological O +anticancer O +agents O +like O +etoposide B-chemical +, O +teniposide B-chemical +and O +doxorubicin B-chemical +. O + +The O +chemical O +complexity O +of O +DNA B-chemical +damage O +- O +derived O +Top2cc B-complex_assembly +necessitates O +that O +DNA B-chemical +repair O +machinery O +dedicated O +to O +resolving O +these O +lesions O +recognizes O +both O +DNA B-chemical +and O +protein O +, O +whilst O +accommodating O +diverse O +chemical O +structures O +that O +trap O +Top2cc B-complex_assembly +. O + +Tdp2 B-protein +processes O +phosphotyrosyl B-ptm +linkages I-ptm +in O +diverse O +DNA B-chemical +damage O +contexts O +. O + +Tdp2 B-protein +hydrolyzes O +the O +5 O +′– O +phosphotyrosine B-residue_name +adduct O +derived O +from O +poisoned O +Top2 B-protein_type +leaving O +DNA B-chemical +ends O +with O +a O +5 B-chemical +′- I-chemical +phosphate I-chemical +, O +which O +facilitates O +DNA B-chemical +end O +joining O +through O +the O +NHEJ O +pathway O +. O + +Phosphotyrosyl B-ptm +bond O +hydrolysis O +catalyzed O +by O +mTdp2cat B-structure_element +releases O +p B-chemical +- I-chemical +nitrophenol I-chemical +, O +which O +is O +detected O +by O +measuring O +absorbance O +at O +415 O +nm O +. O +( O +C O +) O +mTdp2cat B-structure_element +reaction B-evidence +rates I-evidence +on O +p B-chemical +– I-chemical +nitrophenol I-chemical +modified O +DNA B-chemical +substrates O +shown O +in O +panel O +B O +. O +Rates O +are O +reported O +as O +molecules O +of O +PNP B-chemical +s O +− O +1 O +produced O +by O +mTdp2cat B-structure_element +. O + +However O +, O +important O +questions O +regarding O +the O +mechanism O +of O +Tdp2 B-protein +engagement O +and O +processing O +of O +DNA B-chemical +damage O +remain O +. O + +Finally O +, O +we O +characterize O +a O +Tdp2 B-protein +SNP O +that O +ablates B-protein_state +the O +Tdp2 B-protein +single B-site +metal I-site +binding I-site +site I-site +and O +Tdp2 B-protein +substrate O +induced O +conformational O +changes O +, O +and O +confers O +Top2 B-protein_type +drug O +sensitivity O +in O +mammalian B-taxonomy_domain +cells O +. O + +To O +test O +this O +, O +we O +adapted O +an O +EDC B-experimental_method +coupling I-experimental_method +method I-experimental_method +to O +generate O +5 O +′- O +terminal O +p B-chemical +- I-chemical +nitrophenol I-chemical +( O +PNP B-chemical +) O +modified O +oligonucleotides O +that O +also O +harbored O +DNA B-chemical +damage O +at O +the O +5 O +′- O +nucleotide O +position O +( O +see O +Materials O +and O +Methods O +). O + +mTdp2cat B-structure_element +is O +colored O +with O +red O +( O +electronegative O +), O +blue O +( O +electropositive O +) O +and O +gray O +( O +hydrophobic O +) O +electrostatic O +surface O +potential O +displayed O +. O + +A O +structural B-experimental_method +overlay I-experimental_method +of O +damaged O +and O +undamaged O +nucleotides O +shows O +no O +major O +distortions O +to O +nucleotide O +planarity O +between O +different O +bound B-protein_state +sequences O +and O +DNA B-chemical +damage O +( O +compare O +ϵA B-chemical +, O +dA B-chemical +and O +dC B-chemical +, O +Supplementary O +Figure O +S1A O +– O +D O +). O + +Likewise O +, O +the O +abasic B-chemical +deoxyribose I-chemical +analog O +THF B-chemical +substrate O +binds O +similar O +to O +the O +alkylated O +and O +non O +- O +alkylated O +substrates O +, O +but O +with O +a O +slight O +alteration O +in O +the O +approach O +of O +the O +5 O +′- O +terminus O +( O +Figure O +2C O +). O + +An O +intriguing O +feature O +of O +the O +DNA B-protein_state +- I-protein_state +damage I-protein_state +bound I-protein_state +conformation O +of O +the O +Tdp2 B-protein +active B-site +site I-site +is O +an O +underlying O +network O +of O +protein O +– O +water B-chemical +– O +protein O +contacts O +that O +span O +a O +gap O +between O +the O +catalytic B-site +core I-site +and O +the O +DNA B-site +binding I-site +β2Hβ I-site +- I-site +grasp I-site +( O +Supplementary O +Figure O +S2 O +). O + +The O +β2Hβ B-site +docking I-site +pocket I-site +( O +circled O +) O +is O +unoccupied O +and O +residues O +N312 B-residue_name_number +, O +N314 B-residue_name_number +and O +L315 B-residue_name_number +( O +orange O +) O +are O +solvent B-protein_state +- I-protein_state +exposed I-protein_state +. O + +Wall O +- O +eyed O +stereo O +view O +is O +displayed O +. O +( O +C O +) O +Alignment O +of O +active B-structure_element +site I-structure_element +loop I-structure_element +conformers O +observed O +in O +the O +5 O +promoters B-oligomeric_state +of O +the O +DNA B-protein_state +- I-protein_state +free I-protein_state +mTdp2cat B-structure_element +( O +PDB O +entry O +5INM O +, O +see O +Table O +1 O +) O +crystallographic O +asymmetric O +unit O +( O +left O +) O +and O +sequence B-experimental_method +alignment I-experimental_method +showing O +residues O +not O +observed O +in O +the O +electron B-evidence +density I-evidence +as O +‘∼’ O +( O +right O +). O +( O +D O +) O +Limited B-experimental_method +trypsin I-experimental_method +proteolysis I-experimental_method +probes O +the O +solvent O +accessibility O +of O +the O +flexible B-protein_state +active B-structure_element +- I-structure_element +site I-structure_element +loop I-structure_element +. O + +Reactions O +were O +separated O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +and O +proteins O +visualized O +by O +staining O +with O +coomassie O +blue O +. O +( O +E O +) O +Limited B-experimental_method +chymotrypsin I-experimental_method +proteolysis I-experimental_method +probes O +the O +solvent O +accessibility O +of O +the O +flexible B-protein_state +active B-structure_element +- I-structure_element +site I-structure_element +loop I-structure_element +. O + +Burial O +of O +Thr309 B-residue_name_number +is O +enabled O +by O +an O +unusual O +main O +chain O +cis B-bond_interaction +– I-bond_interaction +peptide I-bond_interaction +bond I-bond_interaction +between O +Asp308 B-residue_name_number +- O +Thr309 B-residue_name_number +and O +disassembly O +of O +the O +short B-structure_element +antiparallel I-structure_element +beta I-structure_element +- I-structure_element +strand I-structure_element +of O +the O +β2Hβ B-structure_element +fold O +. O + +To O +transition O +into O +the O +closed B-protein_state +β2Hβ B-structure_element +conformation O +, O +Thr309 B-residue_name_number +disengages O +from O +the O +EEP B-structure_element +domain O +pocket B-site +, O +flips O +peptide O +backbone O +conformation O +cis O +to O +trans O +, O +and O +is O +integral O +to O +the O +β2Hβ B-structure_element +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +. O + +Accordingly O +, O +in O +the O +DNA B-protein_state +free I-protein_state +structure B-evidence +, O +we O +observe O +a O +trend O +where O +the O +2 O +closed B-protein_state +monomers B-oligomeric_state +have O +an O +ordered O +Mg2 B-chemical ++ I-chemical +ion O +in O +their O +active B-site +sites I-site +, O +while O +the O +monomers B-oligomeric_state +with O +open B-protein_state +conformations O +have O +a O +poorly O +ordered O +or O +vacant O +metal B-site +binding I-site +site I-site +. O + +Overall O +, O +these O +observations O +suggest O +that O +engagement O +of O +diverse O +damaged O +DNA B-chemical +ends O +is O +enabled O +by O +an O +elaborate O +substrate O +selected O +stabilization O +of O +the O +β2Hβ B-site +DNA I-site +binding I-site +grasp I-site +, O +and O +these O +rearrangements O +are O +coordinated O +with O +Mg2 B-chemical ++ I-chemical +binding O +in O +the O +Tdp2 B-protein +active B-site +site I-site +. O + +While O +this O +work O +was O +suggestive O +of O +a O +two O +metal O +ion O +mechanism O +for O +phosphotyrosyl B-ptm +bond O +cleavage O +by O +Tdp2 B-protein +, O +we O +note O +that O +second O +metal O +ion O +titrations O +can O +be O +influenced O +by O +metal B-site +ion I-site +binding I-site +sites I-site +outside O +of O +the O +active B-site +site I-site +. O + +This O +analysis O +revealed O +Mg2 B-chemical ++ I-chemical +Kd B-evidence +values O +in O +the O +sub O +- O +millimolar O +range O +and O +Hill B-evidence +coefficients I-evidence +which O +were O +consistent O +with O +a O +single O +metal B-site +binding I-site +site I-site +both O +in O +the O +presence B-protein_state +and O +absence B-protein_state +of I-protein_state +DNA B-chemical +( O +Supplementary O +Table O +S2 O +). O + +In O +contrast O +, O +while O +co B-evidence +- I-evidence +complex I-evidence +structures I-evidence +with O +Ca2 B-chemical ++ I-chemical +also O +show O +a O +single O +metal O +ion O +, O +Ca2 B-chemical ++ I-chemical +binds O +in O +a O +slightly O +different O +position O +, O +shifted O +∼ O +1 O +Å O +from O +the O +Mg2 B-site ++ I-site +site I-site +. O + +Residue O +numbers O +shown O +are O +for O +the O +mTdp2 B-protein +homolog O +. O +( O +D O +) O +Electrostatic B-evidence +surface I-evidence +potential I-evidence +calculated O +for O +5 B-residue_name +′- I-residue_name +phosphotyrosine I-residue_name +in O +isolation O +( O +upper O +panel O +) O +and O +in O +the O +presence B-protein_state +of I-protein_state +a O +cation B-bond_interaction +– I-bond_interaction +π I-bond_interaction +interaction I-bond_interaction +with O +the O +guanidinium O +group O +of O +Arg216 B-residue_name_number +( O +lower O +panel O +) O +shows O +electron O +- O +withdrawing O +effect O +of O +this O +interaction O +. O + +Here O +, O +the O +water B-chemical +proton O +and O +the O +neighboring O +O O +of O +Asp272 B-residue_name_number +participates O +in O +a O +strong O +hydrogen B-bond_interaction +bond I-bond_interaction +( O +distance O +of O +1 O +. O +58 O +Å O +) O +and O +the O +phosphotyrosyl B-ptm +O O +– O +P O +distance O +is O +stretched O +to O +1 O +. O +77 O +Å O +, O +which O +is O +0 O +. O +1 O +Å O +beyond O +an O +equilibrium O +bond O +length O +. O + +Product O +formation O +is O +coupled O +to O +a O +transfer O +of O +a O +proton O +from O +the O +nucleophillic O +water B-chemical +to O +Asp272 B-residue_name_number +, O +consistent O +with O +the O +proposed O +function O +for O +this O +residue O +as O +the O +catalytic O +base O +. O + +By O +analyzing O +activities O +on O +this O +nested O +set O +of O +chemically O +related O +substrates O +we O +aimed O +to O +dissect O +structure O +- O +activity O +relationships O +of O +Tdp2 B-protein +catalysis O +. O + +Structural B-evidence +results I-evidence +and O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +modeling I-experimental_method +indicate O +mAsp272 B-residue_name_number +activates O +a O +water B-chemical +molecule O +for O +in O +- O +line O +nucleophilic O +attack O +of O +the O +scissile O +phosphotyrosyl B-ptm +linkage I-ptm +. O + +Mutations B-experimental_method +hI307A B-mutant +, O +hL305A B-mutant +, O +hL305F B-mutant +and O +hL305W B-mutant +all O +impaired O +catalysis O +on O +both O +nucleotide O +- O +containing O +substrates O +(< O +50 O +% O +activity O +). O + +Quantification O +of O +percent O +MBP B-experimental_method +- O +hTdp2cat B-structure_element +activity O +relative O +to O +WT B-protein_state +protein O +for O +the O +5 B-chemical +′- I-chemical +Y I-chemical +DNA I-chemical +oligonucleotide I-chemical +substrate O +( O +blue O +bars O +), O +T5PNP B-chemical +( O +red O +bars O +) O +and O +PNPP B-chemical +( O +green O +bars O +) O +is O +displayed O +. O + +Accordingly O +, O +we O +demonstrate O +here O +that O +5 B-protein_state +′- I-protein_state +tyrosylated I-protein_state +ends O +are O +sufficient O +to O +severely O +impair O +an O +in O +vitro O +reconstituted O +mammalian B-taxonomy_domain +NHEJ O +reaction O +( O +Figure O +7A O +, O +lanes O +3 O +and O +6 O +), O +unless O +supplemented O +with O +catalytic O +quantities O +of O +hTdp2FL B-protein +( O +Figure O +7A O +, O +lane O +8 O +). O + +DNA B-chemical +substrates O +with O +5 B-residue_name +′- I-residue_name +phosphotyrosine I-residue_name +adducts O +and O +4 O +nucleotide O +5 O +′ O +overhangs O +were O +electroporated O +into O +cultured O +mammalian B-taxonomy_domain +cells O +. O + +Error O +bars O +, O +s O +. O +d O +, O +n O += O +3 O +. O +( O +E O +) O +Clonogenic B-experimental_method +survival I-experimental_method +assay I-experimental_method +of O +WT B-protein_state +, O +Tdp2 B-protein +knockout O +and O +complemented O +MEF O +cells O +after O +treatment O +with O +indicated O +concentrations O +of O +etoposide B-chemical +for O +3 O +h O +; O +error O +bars O +, O +s O +. O +d O +, O +n O += O +3 O +. O + +Top2 B-protein_type +chemotherapeutic O +agents O +remain O +frontline O +treatments O +, O +and O +exposure O +to O +the O +chemical O +and O +damaged O +DNA B-chemical +triggers O +of O +Top2 B-protein_type +- O +DNA B-chemical +protein O +crosslink O +formation O +are O +unavoidable O +. O + +Together O +with O +mutagenesis B-experimental_method +and O +functional B-experimental_method +assays I-experimental_method +, O +our O +new O +Tdp2 B-protein +structures B-evidence +in B-protein_state +the I-protein_state +absence I-protein_state +of I-protein_state +ligands B-chemical +and O +in B-protein_state +complex I-protein_state +with I-protein_state +DNA B-chemical +damage O +reveal O +four O +novel O +facets O +of O +Tdp2 B-protein +DNA B-chemical +- O +protein O +conjugate O +processing O +: O +( O +i O +) O +The O +Tdp2 B-protein +active B-site +site I-site +is O +well O +- O +suited O +for O +accommodating O +a O +variety O +of O +DNA B-chemical +structures O +including O +abasic O +and O +bulky O +alkylated O +DNA B-chemical +lesions O +that O +trigger O +Top2 B-protein_type +poisoning O +, O +( O +ii O +) O +High O +- O +resolution O +structural B-experimental_method +analysis I-experimental_method +coupled O +with O +mutational B-experimental_method +studies I-experimental_method +and O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +molecular I-experimental_method +modeling I-experimental_method +of O +the O +Tdp2 B-protein +reaction O +coordinate O +support O +a O +single O +metal O +- O +ion O +mechanism O +for O +the O +diverse O +clade O +of O +EEP B-structure_element +domain O +catalyzed O +phosphoryl B-protein_type +hydrolase I-protein_type +reactions O +, O +( O +iii O +) O +The O +Tdp2 B-protein +active B-site +site I-site +is O +conformationally B-protein_state +plastic I-protein_state +, O +and O +undergoes O +intricate O +rearrangements O +upon O +DNA B-chemical +and O +Mg2 B-chemical ++ I-chemical +cofactor O +binding O +and O +( O +iv O +) O +Naturally O +occurring O +Tdp2 B-protein +variants O +undermine O +Tdp2 B-protein +active B-site +site I-site +chemistry O +, O +cellular O +and O +biochemical O +activities O +. O + +We O +hypothesize O +that O +binding O +of O +the O +Top2 B-protein_type +protein O +component O +of O +a O +DNA B-chemical +– O +protein O +crosslink O +and O +/ O +or O +other O +protein O +- O +regulated O +assembly O +of O +the O +Tdp2 B-protein +active B-site +site I-site +might O +also O +serve O +to O +regulate O +Tdp2 B-protein +activity O +to O +restrict O +it O +from O +misplaced O +Top2 B-protein_type +processing O +events O +, O +such O +that O +it O +cleaves O +only O +topologically O +trapped O +or O +poisoned O +Top2 B-protein_type +molecules O +when O +needed O +. O + +Etoposide B-chemical +and O +other O +Top2 B-protein_type +poisons O +remain O +front O +line O +anti O +- O +cancer O +drugs O +, O +and O +Tdp2 B-protein +frameshift O +mutations O +in O +the O +human B-species +population O +confer O +hypersensitivity O +to O +Top2 B-protein_type +poisons O +including O +etoposide B-chemical +and O +doxyrubicin B-chemical +. O + +Here O +, O +we O +elucidate O +their O +mechanisms O +of O +extracellular O +ion O +recognition O +and O +exchange O +through O +a O +structural B-experimental_method +analysis I-experimental_method +of O +the O +exchanger B-protein_type +from O +Methanococcus B-species +jannaschii I-species +( O +NCX_Mj B-protein +) O +bound B-protein_state +to I-protein_state +Na B-chemical ++, I-chemical +Ca2 B-chemical ++ I-chemical +or O +Sr2 B-chemical ++ I-chemical +in O +various O +occupancies O +and O +in O +an O +apo B-protein_state +state O +. O + +The O +basic O +functional O +unit O +for O +ion O +transport O +in O +NCX B-protein_type +consists O +of O +ten O +membrane B-structure_element +- I-structure_element +spanning I-structure_element +segments I-structure_element +, O +comprising O +two O +homologous O +halves B-structure_element +. O + +With O +similar O +ion O +exchange O +properties O +to O +those O +of O +its O +eukaryotic B-taxonomy_domain +counterparts O +, O +NCX_Mj B-protein +provides O +a O +compelling O +model O +system O +to O +investigate O +the O +structural O +basis O +for O +the O +specificity O +, O +stoichiometry O +and O +mechanism O +of O +the O +ion O +- O +exchange O +reaction O +catalyzed O +by O +NCX B-protein_type +. O + +An O +independent O +analysis O +based O +on O +molecular B-experimental_method +- I-experimental_method +dynamics I-experimental_method +simulations I-experimental_method +demonstrates O +that O +the O +structures B-evidence +capture O +mechanistically O +relevant O +states O +. O + +Extracellular O +Na B-chemical ++ I-chemical +binding O + +The O +Na B-chemical ++ I-chemical +occupation O +at O +SCa B-site +, O +compounded O +with O +the O +expected O +3Na O ++: B-chemical +1Ca2 O ++ B-chemical +stoichiometry O +, O +implies O +our O +previous O +assignment O +of O +the O +Smid B-site +site O +must O +be O +re O +- O +evaluated O +. O + +However O +, O +when O +Sext B-site +becomes O +empty B-protein_state +at O +low B-protein_state +Na B-chemical ++ I-chemical +concentrations O +, O +TM7a B-structure_element +and O +TM7b B-structure_element +become O +a O +continuous O +straight O +helix B-structure_element +( O +Fig O +. O +2a O +), O +and O +the O +carbonyl O +group O +of O +Ala206 B-residue_name_number +retracts O +away O +( O +Fig O +. O +2b O +- O +d O +). O + +This O +structurally O +- O +derived O +Na B-evidence ++ I-evidence +affinity I-evidence +agrees O +well O +with O +the O +external O +Na B-chemical ++ I-chemical +concentration O +required O +for O +NCX B-protein_type +activation O +in O +eukaryotes B-taxonomy_domain +. O + +Sr2 B-chemical ++ I-chemical +is O +transported O +by O +NCX B-protein_type +similarly O +to O +Ca2 B-chemical ++ I-chemical +, O +and O +is O +distinguishable O +from O +Na B-chemical ++ I-chemical +by O +its O +greater O +electron B-evidence +- I-evidence +density I-evidence +intensity I-evidence +. O + +The O +Sr2 B-protein_state ++- I-protein_state +loaded I-protein_state +NCX_Mj B-protein +structure B-evidence +adopts O +the O +partially B-protein_state +open I-protein_state +conformation O +observed O +at O +low O +Na B-chemical ++ I-chemical +concentrations O +. O + +This O +finding O +is O +consistent O +with O +physiological B-evidence +and I-evidence +biochemical I-evidence +data I-evidence +for O +both O +eukaryotic B-taxonomy_domain +NCX B-protein_type +and O +NCX_Mj B-protein +indicating O +that O +the O +apparent O +Ca2 B-evidence ++ I-evidence +affinity I-evidence +is O +much O +lower O +on O +the O +extracellular O +than O +the O +cytoplasmic O +side O +. O + +Although O +the O +binding B-site +sites I-site +are O +thus O +fully B-protein_state +accessible I-protein_state +to O +the O +external O +solution O +( O +Fig O +. O +3e O +), O +the O +lack O +of O +electron B-evidence +density I-evidence +therein O +indicates O +no O +ions O +or O +ordered O +solvent O +molecules O +. O + +Alternatively O +, O +this O +structure B-evidence +might O +capture O +a O +fully B-protein_state +protonated I-protein_state +state O +of O +the O +transporter B-protein_type +, O +to O +which O +Na B-chemical ++ I-chemical +and O +Ca2 B-chemical ++ I-chemical +cannot O +bind O +. O + +Ion O +occupancy O +determines O +the O +free O +- O +energy O +landscape O +of O +NCX_Mj B-protein + +As O +it O +happens O +, O +the O +results O +confirm O +that O +the O +structures B-evidence +now O +available O +are O +representing O +interconverting O +states O +of O +the O +functional O +cycle O +of O +NCX_Mj B-protein +, O +while O +revealing O +how O +the O +alternating O +- O +access O +mechanism O +is O +controlled O +by O +the O +ion O +- O +occupancy O +state O +. O + +Specifically O +, O +we O +first O +simulated B-experimental_method +the O +outward B-protein_state +- I-protein_state +occluded I-protein_state +form O +, O +in O +the O +ion O +configuration O +we O +previously O +predicted O +, O +now O +confirmed O +by O +the O +high B-protein_state +- I-protein_state +Na I-protein_state ++ I-protein_state +crystal B-evidence +structure I-evidence +described O +above O +( O +Fig O +. O +1b O +). O + +That O +is O +, O +Na B-chemical ++ I-chemical +ions O +occupy O +Sext B-site +, O +SCa B-site +, O +and O +Sint B-site +, O +while O +D240 B-residue_name_number +is O +protonated B-protein_state +and O +a O +water B-chemical +molecule O +occupies O +Smid B-site +. O + +The O +Na B-chemical ++ I-chemical +ion O +at O +Sext B-site +was O +then O +relocated O +from O +the O +site O +to O +the O +bulk O +solution O +( O +Methods O +), O +and O +this O +system O +was O +then O +allowed O +to O +evolve O +freely O +in O +time O +. O + +The O +most O +noticeable O +is O +an O +increased O +separation O +between O +TM7 B-structure_element +and O +TM2 B-structure_element +( O +Fig O +. O +4f O +), O +previously O +brought O +together O +by O +concurrent O +backbone O +interactions O +with O +the O +Na B-chemical ++ I-chemical +ion O +at O +SCa B-site +( O +Fig O +. O +4d O +- O +e O +). O + +TM1 B-structure_element +and O +TM6 B-structure_element +also O +slide O +further O +towards O +the O +membrane O +center O +, O +relative O +to O +the O +outward B-protein_state +- I-protein_state +occluded I-protein_state +state O +( O +Fig O +. O +4c O +). O + +This O +semi B-protein_state +- I-protein_state +open I-protein_state +conformation O +is O +nearly O +identical O +to O +that O +found O +to O +be O +the O +most O +probable O +when O +Na B-chemical ++ I-chemical +occupies O +only O +SCa B-site +and O +Sint B-site +( O +2 O +× O +Na B-chemical ++, I-chemical +Fig O +. O +5a O +), O +demonstrating O +that O +binding O +( O +or O +release O +) O +of O +Na B-chemical ++ I-chemical +to O +Sext B-site +occurs O +in O +this O +metastable B-protein_state +conformation O +. O + +This O +occluded B-protein_state +conformation O +, O +which O +is O +a O +necessary O +intermediate O +between O +the O +outward B-protein_state +and O +inward B-protein_state +- I-protein_state +open I-protein_state +states O +, O +and O +which O +entails O +the O +internal O +dehydration B-protein_state +of O +the O +protein O +, O +is O +only O +attainable O +upon O +complete B-protein_state +occupancy I-protein_state +of O +the O +binding B-site +sites I-site +. O + +For O +example O +, O +in O +the O +crystal B-evidence +of O +NCX_Mj B-protein +in O +LCP B-experimental_method +, O +the O +extracellular B-structure_element +half I-structure_element +of O +the O +gating B-structure_element +helices I-structure_element +( O +TM6 B-structure_element +and O +TM1 B-structure_element +) O +form O +a O +lattice O +contact O +, O +which O +might O +ultimately O +restrict O +the O +degree O +of O +opening O +of O +the O +ion B-site +- I-site +binding I-site +sites I-site +in O +some O +cases O +( O +e O +. O +g O +. O +in O +the O +apo B-protein_state +, O +low B-protein_state +pH I-protein_state +structure B-evidence +). O + +The O +simulations B-experimental_method +also O +demonstrate O +how O +this O +landscape O +is O +drastically O +re O +- O +shaped O +upon O +each O +ion O +- O +binding O +event O +. O + +We O +posit O +that O +a O +similar O +principle O +might O +govern O +the O +alternating O +- O +access O +mechanism O +in O +other O +transporters B-protein_type +; O +that O +is O +, O +we O +anticipate O +that O +for O +both O +symporters B-protein_type +and O +antiporters B-protein_type +, O +it O +is O +the O +feasibility O +of O +the O +occluded B-protein_state +state O +, O +encoded O +in O +the O +protein B-evidence +conformational I-evidence +free I-evidence +- I-evidence +energy I-evidence +landscape I-evidence +and O +its O +dependence O +on O +substrate O +binding O +, O +that O +ultimately O +explains O +their O +specific O +coupling O +mechanisms O +. O + +Specifically O +, O +saturating O +amounts O +of O +Ca2 B-chemical ++ I-chemical +or O +Na B-chemical ++ I-chemical +resulted O +in O +a O +noticeable O +slowdown O +in O +the O +HDX B-evidence +rate I-evidence +for O +extracellular O +portions O +of O +the O +α B-structure_element +- I-structure_element +repeat I-structure_element +helices I-structure_element +. O + +Na B-chemical ++ I-chemical +binding O +to O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +. O + +( O +a O +) O +Overall O +structure B-evidence +of O +native B-protein_state +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +from O +crystals B-experimental_method +grown I-experimental_method +in O +150 O +mM O +Na B-chemical ++. I-chemical + +No O +significant O +changes O +were O +observed O +in O +the O +side O +- O +chains O +involved O +in O +ion O +or O +water B-chemical +coordination O +at O +the O +SCa B-site +, O +Sint B-site +and O +Smid B-site +sites O +. O + +For O +Ca2 B-chemical ++, I-chemical +a O +map B-evidence +is O +shown O +in O +which O +a O +correction O +for O +the O +charge O +- O +transfer O +between O +the O +ion O +and O +the O +protein O +is O +introduced O +, O +alongside O +the O +uncorrected O +map B-evidence +( O +see O +Supplementary O +Notes O +3 O +- O +4 O +and O +Supplementary O +Fig O +. O +5 O +- O +6 O +). O + +Asterisks O +mark O +the O +states O +whose O +crystal B-evidence +structures I-evidence +have O +been O +determined O +in O +this O +study O +. O + +We O +use O +single B-experimental_method +- I-experimental_method +molecule I-experimental_method +Förster I-experimental_method +resonance I-experimental_method +energy I-experimental_method +transfer I-experimental_method +( O +smFRET B-experimental_method +) O +to O +characterize O +the O +conformational B-evidence +dynamics I-evidence +of O +this O +extended B-protein_state +U2AF65 B-structure_element +– I-structure_element +RNA I-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +during O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +. O + +Both O +RRM1 B-structure_element +/ O +RRM2 B-structure_element +extensions B-structure_element +and O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +directly O +recognize O +the O +bound B-protein_state +oligonucleotide B-chemical +. O + +The O +discovery O +of O +nine O +U2AF65 B-site +- I-site +binding I-site +sites I-site +for O +contiguous B-structure_element +Py B-chemical +- I-chemical +tract I-chemical +nucleotides I-chemical +was O +unexpected O +. O + +Surprisingly O +, O +the O +RRM2 B-structure_element +extension I-structure_element +/ O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +contribute O +new O +central O +nucleotide B-site +- I-site +binding I-site +sites I-site +near O +the O +RRM1 B-site +/ I-site +RRM2 I-site +junction I-site +and O +the O +RRM1 B-structure_element +extension I-structure_element +recognizes O +the O +3 O +′- O +terminal O +nucleotide B-chemical +( O +Fig O +. O +2c O +; O +Supplementary O +Movie O +1 O +). O + +We O +tested B-experimental_method +the I-experimental_method +contribution I-experimental_method +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +interactions O +with O +the O +new O +central O +nucleotide B-chemical +to O +Py B-evidence +- I-evidence +tract I-evidence +affinity I-evidence +( O +Fig O +. O +3i O +; O +Supplementary O +Fig O +. O +4a O +, O +b O +). O + +U2AF65 B-protein +RRM B-structure_element +extensions I-structure_element +interact O +with O +the O +Py B-chemical +tract I-chemical + +Indirectly O +, O +the O +additional O +contacts O +with O +the O +third B-residue_number +nucleotide B-chemical +shift O +the O +rU2 B-residue_name_number +nucleotide B-chemical +in O +the O +second B-site +binding I-site +site I-site +closer O +to O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +strand I-structure_element +of O +RRM2 B-structure_element +. O + +A O +second B-structure_element +kink I-structure_element +at O +P236 B-residue_name_number +, O +coupled O +with O +respective O +packing O +of O +the O +L235 B-residue_name_number +and O +M238 B-residue_name_number +side O +chains O +on O +the O +N O +- O +terminal O +α B-structure_element +- I-structure_element +helical I-structure_element +RRM1 I-structure_element +extension I-structure_element +and O +the O +core B-protein_state +RRM1 B-structure_element +α2 B-structure_element +- I-structure_element +helix I-structure_element +, O +reverses O +the O +direction O +of O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +towards O +the O +RRM1 B-site +/ I-site +RRM2 I-site +interface I-site +and O +away O +from O +the O +RNA B-site +- I-site +binding I-site +site I-site +. O + +At O +the O +RNA B-chemical +surface O +, O +the O +key O +V254 B-residue_name_number +that O +recognizes O +the O +fifth B-residue_number +uracil B-residue_name +is O +secured O +via O +hydrophobic B-bond_interaction +contacts I-bond_interaction +between O +its O +side O +chain O +and O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +surface I-structure_element +of O +RRM2 B-structure_element +, O +chiefly O +the O +consensus O +RNP1 B-structure_element +- O +F304 B-residue_name_number +residue O +that O +stacks B-bond_interaction +with O +the O +fourth B-residue_number +uracil B-residue_name +( O +Fig O +. O +4a O +, O +lower O +left O +). O + +Despite O +12 B-experimental_method +concurrent I-experimental_method +mutations I-experimental_method +, O +the O +AdML B-gene +RNA B-evidence +affinity I-evidence +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +- I-mutant +12Gly I-mutant +variant B-protein_state +was O +reduced O +by O +only O +three O +- O +fold O +relative O +to O +the O +unmodified B-protein_state +protein B-protein +( O +Fig O +. O +4b O +), O +which O +is O +less O +than O +the O +penalty O +of O +the O +V254P B-mutant +mutation O +that O +disrupts O +the O +rU5 B-residue_name_number +hydrogen B-bond_interaction +bond I-bond_interaction +( O +Fig O +. O +3d O +, O +i O +). O + +To O +further O +test O +cooperation O +among O +the O +U2AF65 B-protein +RRM B-structure_element +extensions I-structure_element +and O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +for O +RNA O +recognition O +, O +we O +tested O +the O +impact O +of O +a O +triple O +Q147A B-mutant +/ O +V254P B-mutant +/ O +R227A B-mutant +mutation B-experimental_method +( O +U2AF651 B-mutant +, I-mutant +2L I-mutant +- I-mutant +3Mut I-mutant +) O +for O +RNA O +binding O +( O +Fig O +. O +4b O +; O +Supplementary O +Fig O +. O +4d O +). O + +The O +non O +- O +additive O +effects O +of O +the O +Q147A B-mutant +/ O +V254P B-mutant +/ O +R227A B-mutant +triple B-experimental_method +mutation I-experimental_method +, O +coupled O +with O +the O +context O +- O +dependent O +penalties O +of O +an O +internal O +U2AF65 B-protein +linker B-experimental_method +deletion I-experimental_method +, O +highlights O +the O +importance O +of O +the O +structural O +interplay O +among O +the O +U2AF65 B-protein +linker B-structure_element +and O +the O +N B-structure_element +- I-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +extensions I-structure_element +flanking O +the O +core B-protein_state +RRMs B-structure_element +. O + +Sparse O +inter B-structure_element +- I-structure_element +RRM I-structure_element +contacts O +underlie O +apo B-protein_state +- O +U2AF65 B-protein +dynamics O + +The O +direct O +interface B-site +between O +U2AF651 B-mutant +, I-mutant +2L I-mutant +RRM1 B-structure_element +and O +RRM2 B-structure_element +is O +minor O +, O +burying O +265 O +Å2 O +of O +solvent O +accessible O +surface O +area O +compared O +with O +570 O +Å2 O +on O +average O +for O +a O +crystal O +packing O +interface O +. O + +A O +handful O +of O +inter B-structure_element +- I-structure_element +RRM I-structure_element +hydrogen B-bond_interaction +bonds I-bond_interaction +are O +apparent O +between O +the O +side O +chains O +of O +RRM1 B-structure_element +- O +N155 B-residue_name_number +and O +RRM2 B-structure_element +- O +K292 B-residue_name_number +, O +RRM1 B-structure_element +- O +N155 B-residue_name_number +and O +RRM2 B-structure_element +- O +D272 B-residue_name_number +as O +well O +as O +the O +backbone O +atoms O +of O +RRM1 B-structure_element +- O +G221 B-residue_name_number +and O +RRM2 B-structure_element +- O +D273 B-residue_name_number +( O +Fig O +. O +4c O +). O + +We O +first O +characterized O +the O +conformational O +dynamics O +spectrum O +of O +U2AF65 B-protein +in O +the O +absence B-protein_state +of I-protein_state +RNA B-chemical +( O +Fig O +. O +6c O +, O +d O +; O +Supplementary O +Fig O +. O +7a O +, O +b O +). O + +A O +0 O +. O +45 O +FRET B-evidence +value I-evidence +was O +again O +predominant O +, O +indicating O +a O +similar O +RNA B-protein_state +- I-protein_state +bound I-protein_state +conformation O +and O +structural O +dynamics O +for O +the O +untethered B-protein_state +and O +tethered B-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +). O + +Therefore O +, O +RRM1 B-structure_element +- O +to O +- O +RRM2 B-structure_element +distance O +remains O +similar O +regardless O +of O +whether O +U2AF65 B-protein +is O +bound B-protein_state +to I-protein_state +interrupted O +or O +continuous O +Py B-chemical +tract I-chemical +. O + +The O +inter B-evidence +- I-evidence +fluorophore I-evidence +distances I-evidence +derived O +from O +the O +observed O +0 O +. O +45 O +FRET B-evidence +state I-evidence +agree O +with O +the O +distances O +between O +the O +α O +- O +carbon O +atoms O +of O +the O +respective O +residues O +in O +the O +crystal B-evidence +structures I-evidence +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +bound B-protein_state +to I-protein_state +Py B-chemical +- I-chemical +tract I-chemical +oligonucleotides I-chemical +. O + +Thus O +, O +the O +sequence O +of O +structural O +rearrangements O +of O +U2AF65 B-protein +observed O +in O +smFRET B-experimental_method +traces B-evidence +( O +Supplementary O +Fig O +. O +7c O +– O +g O +) O +suggests O +that O +a O +‘ O +conformational O +selection O +' O +mechanism O +of O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +( O +that O +is O +, O +RNA O +ligand O +stabilization O +of O +a O +pre B-protein_state +- I-protein_state +configured I-protein_state +U2AF65 B-protein +conformation O +) O +is O +complemented O +by O +‘ O +induced O +fit O +' O +( O +that O +is O +, O +RNA O +- O +induced O +rearrangement O +of O +the O +U2AF65 B-protein +RRMs B-structure_element +to O +achieve O +the O +final O +‘ O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +' O +conformation O +), O +as O +discussed O +below O +. O + +Several O +observations O +indicate O +that O +the O +numerous O +intramolecular O +contacts O +, O +here O +revealed O +among O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +and O +RRM1 B-structure_element +, O +RRM2 B-structure_element +, O +and O +the O +N O +- O +terminal O +RRM1 B-structure_element +extension I-structure_element +, O +synergistically O +coordinate O +U2AF65 B-protein +– O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +. O + +The O +lesser O +0 O +. O +65 O +– O +0 O +. O +8 O +and O +0 O +. O +2 O +– O +0 O +. O +3 O +FRET B-evidence +values I-evidence +in O +the O +untethered B-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +experiment O +could O +correspond O +to O +respective O +variants O +of O +the O +‘ O +closed B-protein_state +', O +back B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +back I-protein_state +U2AF65 B-protein +conformations O +characterized O +by O +NMR B-experimental_method +/ O +PRE B-experimental_method +data O +, O +or O +to O +extended B-protein_state +U2AF65 B-protein +conformations O +, O +in O +which O +the O +intramolecular O +RRM1 B-structure_element +/ O +RRM2 B-structure_element +interactions O +have O +dissociated O +the O +protein B-protein +is O +bound B-protein_state +to I-protein_state +RNA B-chemical +via O +single B-protein_state +RRMs B-structure_element +. O + +Notably O +, O +our O +smFRET B-experimental_method +results O +reveal O +that O +U2AF65 B-protein +– O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +can O +be O +characterized O +by O +an O +‘ O +extended O +conformational O +selection O +' O +model O +( O +Fig O +. O +7b O +). O + +Based O +on O +( O +i O +) O +similar O +RNA B-evidence +affinities I-evidence +of O +U2AF65 B-protein +and O +U2AF651 B-mutant +, I-mutant +2L I-mutant +, O +( O +ii O +) O +indistinguishable O +conformations O +among O +four O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +in O +two O +different O +crystal O +packing O +arrangements O +and O +( O +iii O +) O +penalties B-evidence +of O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +mutations I-experimental_method +in O +RNA B-experimental_method +binding I-experimental_method +and I-experimental_method +splicing I-experimental_method +assays I-experimental_method +, O +we O +suggest O +that O +the O +extended B-protein_state +inter B-structure_element +- I-structure_element +RRM I-structure_element +regions I-structure_element +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +underlie O +cognate O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +by O +the O +full B-protein_state +- I-protein_state +length I-protein_state +U2AF65 B-protein +protein O +. O + +RRM B-structure_element +, O +RNA B-structure_element +recognition I-structure_element +motif I-structure_element +; O +RS B-structure_element +, O +arginine B-structure_element +- I-structure_element +serine I-structure_element +rich I-structure_element +; O +UHM B-structure_element +, O +U2AF B-structure_element +homology I-structure_element +motif I-structure_element +; O +ULM B-structure_element +, O +U2AF B-structure_element +ligand I-structure_element +motif I-structure_element +. O + +( O +b O +) O +Stereo O +views O +of O +a O +‘ O +kicked O +' O +2 B-evidence +| I-evidence +Fo I-evidence +|−| I-evidence +Fc I-evidence +| I-evidence +electron I-evidence +density I-evidence +map I-evidence +contoured O +at O +1σ O +for O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +, O +N O +- O +and O +C O +- O +terminal O +residues O +( O +blue O +) O +or O +bound O +oligonucleotide B-chemical +of O +a O +representative O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structure O +( O +structure O +iv O +, O +bound B-protein_state +to I-protein_state +5 O +′-( O +P O +) O +rUrUrUdUrUrU O +( O +BrdU O +) O +dUrC O +) O +( O +magenta O +). O + +New O +residues O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +are O +coloured O +a O +darker O +shade O +of O +blue O +, O +apart O +from O +residues O +that O +were O +tested O +by O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +, O +which O +are O +coloured O +yellow O +. O + +The O +nucleotide B-site +- I-site +binding I-site +sites I-site +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +and O +prior O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +structure B-evidence +are O +compared O +in O +Supplementary O +Fig O +. O +3a O +– O +h O +. O +The O +first B-site +and I-site +seventh I-site +U2AF651 I-site +, I-site +2L I-site +- I-site +binding I-site +sites I-site +are O +unchanged O +from O +the O +prior O +dU2AF651 B-complex_assembly +, I-complex_assembly +2 I-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +structure B-evidence +and O +are O +portrayed O +in O +Supplementary O +Fig O +. O +3a O +, O +f O +. O +The O +four O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +are O +similar O +with O +the O +exception O +of O +pH O +- O +dependent O +variations O +at O +the O +ninth B-site +site I-site +that O +are O +detailed O +in O +Supplementary O +Fig O +. O +3i O +, O +j O +. O +The O +representative O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structure B-evidence +shown O +has O +the O +highest O +resolution O +and O +/ O +or O +ribose B-chemical +nucleotide I-chemical +at O +the O +given O +site O +: O +( O +a O +) O +rU2 B-residue_name_number +of O +structure O +iv O +; O +( O +b O +) O +rU3 B-residue_name_number +of O +structure O +iii O +; O +( O +c O +) O +rU4 B-residue_name_number +of O +structure O +i O +; O +( O +d O +) O +rU5 B-residue_name_number +of O +structure O +iii O +; O +( O +e O +) O +rU6 B-residue_name_number +of O +structure O +ii O +; O +( O +f O +) O +dU8 B-residue_name_number +of O +structure O +iii O +; O +( O +g O +) O +dU9 B-residue_name_number +of O +structure O +iii O +; O +( O +h O +) O +rC9 B-residue_name_number +of O +structure O +iv O +. O + +Other O +linker B-structure_element +residues O +are O +coloured O +either O +dark O +blue O +for O +new O +residues O +in O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structure O +or O +light O +blue O +for O +the O +remaining O +inter B-structure_element +- I-structure_element +RRM I-structure_element +residues O +. O + +( O +b O +) O +Representative O +RT B-experimental_method +- I-experimental_method +PCR I-experimental_method +of O +pyPY B-chemical +transcripts O +from O +HEK293T O +cells O +co B-experimental_method +- I-experimental_method +transfected I-experimental_method +with O +constructs O +encoding O +the O +pyPY B-chemical +minigene O +and O +either O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +U2AF65 B-protein +or O +a O +triple O +U2AF65 B-protein +mutant B-protein_state +( O +3Mut B-mutant +) O +of O +Q147A B-mutant +, O +R227A B-mutant +and O +V254P B-mutant +residues O +. O +( O +c O +) O +A O +bar O +graph O +of O +the O +average O +percentage O +of O +the O +py B-chemical +- O +spliced O +mRNA B-chemical +relative O +to O +total O +detected O +pyPY B-chemical +transcripts O +( O +spliced O +and O +unspliced O +) O +for O +the O +corresponding O +gel O +lanes O +( O +black O +, O +no O +U2AF65 B-protein +added O +; O +white O +, O +WT B-protein_state +U2AF65 B-protein +; O +grey O +, O +3Mut B-mutant +U2AF65 B-protein +). O + +The O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +proteins O +were O +doubly O +labelled O +at O +A181C B-mutant +/ O +Q324C B-mutant +such O +that O +a O +mixture O +of O +Cy3 B-chemical +/ O +Cy5 B-chemical +fluorophores B-chemical +are O +expected O +to O +be O +present O +at O +each O +site O +. O + +Typical O +single B-experimental_method +- I-experimental_method +molecule I-experimental_method +FRET I-experimental_method +traces B-evidence +( O +c O +, O +e O +, O +g O +, O +i O +) O +show O +fluorescence O +intensities O +from O +Cy3 B-chemical +( O +green O +) O +and O +Cy5 B-chemical +( O +red O +) O +and O +the O +calculated B-evidence +apparent I-evidence +FRET I-evidence +efficiency I-evidence +( O +blue O +). O + +( O +a O +) O +Diagram O +of O +the O +U2AF65 B-protein +, O +SF1 B-protein +and O +U2AF35 B-protein +splicing O +factors O +bound B-protein_state +to I-protein_state +the O +consensus O +elements O +of O +the O +3 B-site +′ I-site +splice I-site +site I-site +. O + +A O +surface O +representation O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +is O +shown O +bound B-protein_state +to I-protein_state +nine O +nucleotides B-chemical +( O +nt O +); O +the O +relative O +distances O +and O +juxtaposition O +of O +the O +branch B-site +point I-site +sequence I-site +( O +BPS B-site +) O +and O +consensus O +AG B-chemical +dinucleotide I-chemical +at O +the O +3 B-site +′ I-site +splice I-site +site I-site +are O +unknown O +. O + +The O +availability O +of O +two O +TRAP B-complex_assembly +molecules O +in O +the O +asymmetric O +unit O +, O +of O +which O +only O +one O +contained O +bound B-protein_state +RNA B-chemical +, O +allowed O +a O +controlled O +investigation O +into O +the O +exact O +role O +of O +RNA B-chemical +binding O +in O +protein O +specific O +damage O +susceptibility O +. O + +However O +, O +there O +is O +still O +no O +general O +consensus O +within O +the O +field O +on O +how O +to O +minimize O +RD O +during O +MX B-experimental_method +data O +collection O +, O +and O +debates O +on O +the O +dependence O +of O +RD O +progression O +on O +incident O +X O +- O +ray O +energy O +( O +Shimizu O +et O +al O +., O +2007 O +; O +Liebschner O +et O +al O +., O +2015 O +) O +and O +the O +efficacy O +of O +radical O +scavengers O +( O +Allan O +et O +al O +., O +2013 O +) O +have O +yet O +to O +be O +resolved O +. O + +At O +100 O +K O +, O +an O +experimental O +dose O +limit O +of O +30 O +MGy O +has O +been O +recommended O +as O +an O +upper O +limit O +beyond O +which O +the O +biological O +information O +derived O +from O +any O +macromolecular O +crystal B-evidence +may O +be O +compromised O +( O +Owen O +et O +al O +., O +2006 O +). O + +Specific B-experimental_method +radiation I-experimental_method +damage I-experimental_method +( O +SRD B-experimental_method +) O +is O +observed O +in O +the O +real B-evidence +- I-evidence +space I-evidence +electron I-evidence +density I-evidence +, O +and O +has O +been O +detected O +at O +much O +lower O +doses O +than O +any O +observable O +decay O +in O +the O +intensity O +of O +reflections O +. O + +Indeed O +, O +the O +C O +— O +Se B-chemical +bond O +in O +selenomethionine B-chemical +, O +the O +stability O +of O +which O +is O +key O +for O +the O +success O +of O +experimental O +phasing O +methods O +, O +can O +be O +cleaved O +at O +a O +dose O +as O +low O +as O +2 O +MGy O +for O +a O +crystal B-evidence +maintained O +at O +100 O +K O +( O +Holton O +, O +2007 O +). O + +The O +significant O +chemical O +strain O +required O +for O +catalysis O +within O +the O +active B-site +site I-site +of O +phosphoserine B-protein_type +aminotransferase I-protein_type +has O +been O +observed O +to O +diminish O +during O +X O +- O +ray O +exposure O +( O +Dubnovitsky O +et O +al O +., O +2005 O +). O + +Nucleoproteins B-complex_assembly +also O +represent O +one O +of O +the O +main O +targets O +of O +radiotherapy O +, O +and O +an O +insight O +into O +the O +damage O +mechanisms O +induced O +by O +X O +- O +ray O +irradiation O +could O +inform O +innovative O +treatments O +. O + +Instead O +, O +we O +use O +here O +a O +maximum B-evidence +density I-evidence +- I-evidence +loss I-evidence +metric I-evidence +( O +D B-evidence +loss I-evidence +), O +which O +is O +the O +per O +- O +atom O +equivalent O +of O +the O +magnitude O +of O +these O +negative B-evidence +Fourier I-evidence +difference I-evidence +map I-evidence +peaks I-evidence +in O +units O +of O +e O +Å O +− O +3 O +. O + +Salt B-bond_interaction +- I-bond_interaction +bridge I-bond_interaction +interactions O +have O +previously O +been O +suggested O +to O +reduce O +the O +glutamate B-residue_name +decarboxylation O +rate O +within O +the O +large O +(∼ O +62 O +. O +4 O +kDa O +) O +myrosinase B-protein_type +protein O +structure B-evidence +( O +Burmeister O +, O +2000 O +). O + +A O +significant O +difference O +was O +observed O +between O +the O +D B-evidence +loss I-evidence +dynamics I-evidence +for O +the O +nonbound B-protein_state +/ O +bound B-protein_state +Glu42 B-residue_name_number +O O +∊ O +1 O +atoms O +( O +Fig O +. O +5 O +▸ O +c O +; O +p O += O +0 O +. O +007 O +) O +but O +not O +for O +the O +Glu42 B-residue_name_number +O O +∊ O +2 O +atoms O +( O +Fig O +. O +5 O +▸ O +d O +; O +p O += O +0 O +. O +239 O +), O +indicating O +that O +the O +stabilizing O +strength O +of O +this O +salt B-bond_interaction +- I-bond_interaction +bridge I-bond_interaction +interaction O +was O +conserved O +upon O +RNA B-chemical +binding O +and O +that O +the O +water B-chemical +- O +mediated O +hydrogen B-bond_interaction +bond I-bond_interaction +had O +a O +greater O +relative O +susceptibility O +to O +atomic O +disordering O +in O +the O +absence B-protein_state +of I-protein_state +RNA B-chemical +. O + +The O +RNA B-chemical +- O +stabilizing O +effect O +was O +not O +restricted O +to O +radiation O +- O +sensitive O +acidic O +residues O +. O + +The O +side O +chain O +of O +Phe32 B-residue_name_number +stacks O +against O +the O +G3 B-residue_name_number +base O +within O +the O +11 O +TRAP B-site +RNA I-site +- I-site +binding I-site +interfaces I-site +( O +Antson O +et O +al O +., O +1999 O +). O + +Consistent O +with O +that O +study O +, O +at O +high O +doses O +of O +above O +∼ O +20 O +MGy O +, O +F B-evidence +obs I-evidence +( I-evidence +d I-evidence +n I-evidence +) I-evidence +− I-evidence +F I-evidence +obs I-evidence +( I-evidence +d I-evidence +1 I-evidence +) I-evidence +map I-evidence +density I-evidence +was O +detected O +around O +P O +, O +O3 O +′ O +and O +O5 O +′ O +atoms O +of O +the O +RNA B-chemical +backbone O +, O +with O +no O +significant O +difference B-evidence +density I-evidence +localized O +to O +RNA B-chemical +ribose O +and O +basic O +subunits B-structure_element +. O + +At O +25 O +. O +0 O +MGy O +, O +the O +magnitude O +of O +the O +RNA B-chemical +backbone O +D B-evidence +loss I-evidence +was O +of O +the O +same O +order O +as O +for O +the O +radiation O +- O +insensitive O +Gly B-residue_name +Cα O +atoms O +and O +on O +average O +less O +than O +half O +that O +of O +the O +acidic O +residues O +of O +the O +protein O +( O +Supplementary O +Fig O +. O +S3 O +). O + +Upon O +RNA B-chemical +binding O +, O +the O +Asp39 B-residue_name_number +side O +- O +chain O +carboxyl O +group O +solvent O +- O +accessible O +area O +changes O +from O +∼ O +75 O +to O +35 O +Å2 O +, O +still O +allowing O +a O +high O +CO2 B-chemical +- O +formation O +rate B-evidence +K I-evidence +2 I-evidence +. O + +However O +, O +for O +each O +of O +these O +residues O +the O +exact O +crystal O +contacts O +are O +not O +preserved O +between O +bound B-protein_state +and O +nonbound B-protein_state +TRAP B-complex_assembly +or O +even O +between O +monomers O +within O +one O +TRAP B-complex_assembly +ring B-structure_element +. O + +For O +example O +, O +in O +bound B-protein_state +TRAP B-complex_assembly +, O +Glu73 B-residue_name_number +hydrogen O +- O +bonds O +to O +a O +nearby O +lysine B-residue_name +on O +each O +of O +the O +11 O +subunits B-structure_element +, O +whereas O +in O +nonbound B-protein_state +TRAP B-complex_assembly +no O +such O +interaction O +exists O +and O +Glu73 B-residue_name_number +interacts O +with O +a O +variable O +number O +of O +refined O +waters B-chemical +in O +each O +subunit B-structure_element +. O + +It O +has O +been O +suggested O +( O +Burmeister O +, O +2000 O +) O +that O +Tyr B-residue_name +residues O +can O +lose O +their O +aromatic O +– O +OH O +group O +owing O +to O +radiation O +- O +induced O +effects O +; O +however O +, O +no O +energetically O +favourable O +pathway O +for O +– O +OH O +cleavage O +exists O +and O +this O +has O +not O +been O +detected O +in O +aqueous O +radiation O +- O +chemistry O +studies O +. O + +Indeed O +, O +no O +convincing O +reproducible O +Fourier B-evidence +difference I-evidence +peaks I-evidence +above O +the O +background O +map B-evidence +noise O +were O +observed O +around O +any O +Tyr B-residue_name +terminal O +– O +OH O +groups O +. O + +Within O +the O +nonbound B-protein_state +TRAP B-complex_assembly +macromolecule O +, O +the O +acidic O +residues O +within O +the O +unoccupied O +RNA B-site +- I-site +binding I-site +interfaces I-site +( O +Asp39 B-residue_name_number +, O +Glu36 B-residue_name_number +, O +Glu42 B-residue_name_number +) O +are O +notably O +amongst O +the O +most O +susceptible O +residues O +within O +the O +asymmetric O +unit O +( O +Fig O +. O +4 O +▸). O + +In O +( O +a O +) O +clear O +difference B-evidence +density I-evidence +is O +observed O +around O +the O +Glu42 B-residue_name_number +carboxyl O +side O +chain O +in O +chain O +H O +, O +within O +the O +lowest B-evidence +dose I-evidence +difference I-evidence +map I-evidence +at O +d O +2 O += O +3 O +. O +9 O +MGy O +. O + +The O +important O +MAPK B-protein_type +family I-protein_type +of O +signalling O +proteins O +is O +controlled O +by O +MAPK B-protein_type +phosphatases I-protein_type +( O +MKPs B-protein_type +). O + +The O +best O +- O +studied O +docking O +interactions O +are O +those O +between O +MAP B-protein_type +kinases I-protein_type +and O +‘ O +D B-structure_element +- I-structure_element +motifs I-structure_element +', O +which O +consists O +of O +two O +or O +more O +basic O +residues O +followed O +by O +a O +short B-structure_element +linker I-structure_element +and O +a O +cluster O +of O +hydrophobic O +residues O +. O + +Here O +, O +we O +present O +the O +crystal B-evidence +structure I-evidence +of O +JNK1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +catalytic B-structure_element +domain I-structure_element +of O +MKP7 B-protein +. O + +Thus O +, O +the O +kinetic B-evidence +data I-evidence +were O +analysed O +using O +the O +general O +initial B-evidence +velocity I-evidence +equation I-evidence +, O +taking O +substrate O +depletion O +into O +account O +: O + +To O +further O +confirm O +the O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +interaction O +, O +we O +performed O +a O +pull B-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +using O +the O +purified O +proteins O +. O + +To O +understand O +the O +molecular O +basis O +of O +JNK1 B-protein +recognition O +by O +MKP7 B-protein +, O +we O +determined O +the O +crystal B-evidence +structure I-evidence +of O +unphosphorylated B-protein_state +JNK1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +MKP7 B-protein +- O +CD B-structure_element +( O +Fig O +. O +3a O +, O +Supplementary O +Fig O +. O +1a O +and O +Table O +1 O +). O + +This O +loop B-structure_element +is O +shortened O +by O +nine O +residues O +in O +MKP7 B-protein +- O +CD B-structure_element +compared O +with O +that O +in O +VHR B-protein +. O + +Although O +the O +catalytically O +important O +residues O +in O +MKP7 B-protein +- O +CD B-structure_element +are O +well O +aligned O +with O +those O +in O +VHR B-protein +, O +the O +residues O +in O +the O +P B-structure_element +- I-structure_element +loop I-structure_element +of O +MKP7 B-protein +are O +smaller O +and O +have O +a O +more O +hydrophobic O +character O +than O +those O +of O +VHR B-protein +( O +Cys124 B-residue_name_number +- O +Arg125 B-residue_name_number +- O +Glu126 B-residue_name_number +- O +Gly127 B-residue_name_number +- O +Tyr128 B-residue_name_number +- O +Gly129 B-residue_name_number +- O +Arg130 B-residue_name_number +; O +Fig O +. O +3b O +, O +c O +). O + +In O +the O +complex O +, O +MKP7 B-protein +- O +CD B-structure_element +and O +JNK1 B-protein +form O +extensive O +protein O +– O +protein O +interactions O +involving O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +helices I-structure_element +of O +MKP7 B-protein +- O +CD B-structure_element +and O +C B-structure_element +- I-structure_element +lobe I-structure_element +of O +JNK1 B-protein +( O +Fig O +. O +3d O +, O +e O +). O + +As O +shown O +in O +Fig O +. O +4f O +, O +all O +the O +mutants B-protein_state +, O +except O +F287D B-mutant +/ I-mutant +A I-mutant +, O +showed O +little O +or O +no O +activity O +change O +compared O +with O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +MKP7 B-protein +- O +CD B-structure_element +. O + +In O +addition O +, O +His230 B-residue_name_number +and O +Val256 B-residue_name_number +in O +JNK1 B-protein +are O +replaced O +by O +the O +negatively O +charged O +residues O +Glu208 B-residue_name_number +and O +Asp235 B-residue_name_number +in O +CDK2 B-protein +( O +Fig O +. O +5d O +), O +and O +the O +charge O +distribution O +on O +the O +CDK2 B-protein +interactive B-site +surface I-site +is O +quite O +different O +from O +that O +of O +JNK B-protein_type +. O + +Parallel O +experiments O +showed O +clearly O +that O +the O +D B-structure_element +- I-structure_element +motif I-structure_element +mutants B-protein_state +( O +R56A B-mutant +/ O +R57A B-mutant +and O +V63A B-mutant +/ O +I65A B-mutant +) O +dephosphorylated B-protein_state +JNK B-protein_type +as O +did O +the O +wild B-protein_state +type I-protein_state +under O +the O +same O +conditions O +, O +further O +confirming O +that O +the O +MKP7 B-protein +- O +KBD B-structure_element +is O +not O +required O +for O +the O +JNK B-protein_type +inactivation O +in O +vivo O +. O + +Consistent O +with O +the O +in O +vitro O +data O +, O +the O +level O +of O +phosphorylated B-protein_state +JNK B-protein_type +was O +not O +or O +little O +altered O +in O +MKP7 B-protein +FXF B-structure_element +- I-structure_element +motif I-structure_element +mutants B-protein_state +( O +F285D B-mutant +, O +F287D B-mutant +and O +L288D B-mutant +)- O +transfected O +cells O +, O +and O +the O +MKP7 B-protein +D268A B-mutant +and O +N286A B-mutant +mutants B-protein_state +retained O +the O +ability O +to O +reduce O +the O +phosphorylation O +levels O +of O +JNK B-protein_type +. O + +We O +next O +tested O +in O +vivo O +interactions O +between O +JNK1 B-protein +mutants B-protein_state +and O +full B-protein_state +- I-protein_state +length I-protein_state +MKP7 B-protein +by O +coimmunoprecipitation B-experimental_method +experiments I-experimental_method +under O +unstimulated O +conditions O +. O + +When O +co B-experimental_method +- I-experimental_method +expressed I-experimental_method +in O +HEK293T O +cells O +, O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +HA O +)- O +JNK1 B-protein +was O +readily O +precipitated O +with O +( O +Myc O +)- O +MKP7 B-protein +( O +Fig O +. O +6d O +), O +indicating O +that O +MKP7 B-protein +binds O +dephosphorylated B-protein_state +JNK1 B-protein +protein O +in O +vivo O +. O + +In O +contrast O +, O +cells O +transfected O +with O +the O +MKP7 B-protein +FXF B-structure_element +- I-structure_element +motif I-structure_element +mutants B-protein_state +( O +F285D B-mutant +, O +F287D B-mutant +and O +L288D B-mutant +) O +showed O +little O +protective O +effect O +after O +ultraviolet O +treatment O +and O +similar O +levels O +of O +apoptosis O +rates O +were O +detected O +to O +cells O +transfected O +with O +control O +vectors O +( O +Fig O +. O +6e O +, O +f O +). O + +MKP5 B-protein +is O +unique O +among O +the O +MKPs B-protein_type +in O +possessing O +an O +additional O +domain O +of O +unknown O +function O +at O +the O +N O +- O +terminus O +( O +Fig O +. O +7a O +). O + +Deletion B-experimental_method +of I-experimental_method +the O +KBD B-structure_element +in O +MKP5 B-protein +leads O +to O +a O +280 O +- O +fold O +increase O +in O +Km B-evidence +for O +p38α B-protein +substrate O +. O + +Comparisons O +between O +catalytic B-structure_element +domains I-structure_element +structures B-evidence +of O +MKP5 B-protein +and O +MKP7 B-protein +reveal O +that O +the O +overall O +folds O +of O +the O +two O +proteins O +are O +highly O +similar O +, O +with O +only O +a O +few O +regions O +exhibiting O +small O +deviations O +( O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +. I-evidence +of O +0 O +. O +79 O +Å O +; O +Fig O +. O +7c O +). O + +Pull B-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +also O +confirmed O +the O +protein O +– O +protein O +interactions O +observed O +above O +. O + +As O +shown O +in O +Fig O +. O +7f O +, O +the O +T432A B-mutant +and O +L449F B-mutant +MKP5 B-protein +mutant B-protein_state +showed O +little O +or O +no O +difference O +in O +phosphatase O +activity O +, O +whereas O +the O +other O +mutants B-protein_state +showed O +reduced O +specific O +activities O +of O +MKP5 B-protein +. O + +Taken O +together O +, O +our O +results O +suggest O +that O +MKP5 B-protein +binds O +JNK1 B-protein +in O +a O +docking O +mode O +similar O +to O +that O +in O +the O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +complex O +, O +and O +the O +detailed O +interaction O +model O +can O +be O +generated O +using O +molecular B-experimental_method +dynamics I-experimental_method +simulation I-experimental_method +based O +on O +the O +structure B-evidence +of O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +complex O +( O +Supplementary O +Fig O +. O +4b O +, O +c O +). O + +The O +MKP7 B-protein +- O +KBD B-structure_element +docks O +to O +the O +D B-site +- I-site +site I-site +located O +on O +the O +back O +side O +of O +the O +p38α B-protein +catalytic B-site +pocket I-site +for O +high O +- O +affinity O +association O +, O +whereas O +the O +interaction O +of O +the O +MKP7 B-protein +- O +CD B-structure_element +with O +another O +p38α B-protein +structural O +region O +, O +which O +is O +close O +to O +the O +activation B-structure_element +loop I-structure_element +, O +may O +not O +only O +stabilize O +binding O +but O +also O +provide O +contacts O +crucial O +for O +organizing O +the O +MKP7 B-protein +active B-site +site I-site +with O +respect O +to O +the O +phosphoreceptor O +in O +the O +p38α B-protein +substrate O +for O +efficient O +dephosphorylation O +. O + +In O +addition O +to O +the O +canonical O +D B-site +- I-site +site I-site +, O +the O +MAPK B-protein_type +ERK2 B-protein +contains O +a O +second B-site +binding I-site +site I-site +utilized O +by O +transcription O +factor O +substrates O +and O +phosphatases B-protein_type +, O +the O +FXF B-site +- I-site +motif I-site +- I-site +binding I-site +site I-site +( O +also O +called O +F B-site +- I-site +site I-site +), O +that O +is O +exposed O +in O +active B-protein_state +ERK2 B-protein +and O +the O +D B-structure_element +- I-structure_element +motif I-structure_element +peptide O +- O +induced O +conformation O +of O +MAPKs B-protein_type +. O + +MKP7 B-protein +- O +CD B-structure_element +is O +shown O +in O +surface O +representation O +coloured O +according O +to O +electrostatic O +potential O +( O +positive O +, O +blue O +; O +negative O +, O +red O +). O + +Mutant B-protein_state +F285D B-mutant +and O +JNK1 B-protein +were O +eluted O +as O +monomers B-oligomeric_state +, O +with O +the O +molecular O +masses O +of O +∼ O +17 O +and O +44 O +kDa O +, O +respectively O +. O + +One O +remarkable O +difference O +between O +these O +two O +kinase O +- O +phosphatase O +complexes O +is O +that O +helix B-structure_element +α6 B-structure_element +of O +KAP B-protein +( O +corresponding O +to O +helix B-structure_element +α4 B-structure_element +of O +MKP7 B-protein +- O +CD B-structure_element +) O +plays O +little O +, O +if O +any O +, O +role O +in O +the O +formation O +of O +a O +stable B-protein_state +heterodimer B-oligomeric_state +of O +CDK2 B-protein +and O +KAP B-protein +. O +( O +c O +) O +Sequence B-experimental_method +alignment I-experimental_method +of O +the O +JNK B-site +- I-site +interacting I-site +regions I-site +on O +MKPs B-protein_type +. O + +( O +d O +) O +Sequence B-experimental_method +alignment I-experimental_method +of O +the O +F B-structure_element +- I-structure_element +site I-structure_element +regions I-structure_element +on O +MAPKs B-protein_type +. O + +HEK293T O +cells O +were O +infected O +with O +lentiviruses B-taxonomy_domain +expressing O +MKP7 B-protein +and O +its O +mutants B-protein_state +( O +1 O +. O +0 O +μg O +). O + +HEK293T O +cells O +were O +co B-experimental_method +- I-experimental_method +transfected I-experimental_method +with O +MKP7 B-protein +full B-protein_state +- I-protein_state +length I-protein_state +( O +1 O +. O +0 O +μg O +) O +and O +JNK1 B-protein +( O +wild B-protein_state +type I-protein_state +or O +mutants B-protein_state +as O +indicated O +, O +1 O +. O +0 O +μg O +). O + +MKP5 B-protein +- O +CD B-structure_element +is O +crucial O +for O +JNK1 B-protein +binding O +and O +enzyme O +catalysis O +. O + +The O +solid O +lines O +are O +best O +- O +fitting O +results O +according O +to O +the O +Michaelis O +– O +Menten O +equation O +with O +Km B-evidence +and O +kcat B-evidence +values O +indicated O +. O + +( O +h O +) O +Pull B-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +of O +MKP5 B-protein +- O +CD B-structure_element +by O +GST B-protein_state +- I-protein_state +tagged I-protein_state +JNK1 B-protein +mutants B-protein_state +. O + +The O +next O +step O +following O +on O +from O +this O +work O +is O +to O +understand O +what O +signals O +are O +produced O +when O +IDA B-protein +activates O +HAESA B-protein +. O + +The O +IDA B-site +binding I-site +pocket I-site +covers O +LRRs B-structure_element +2 I-structure_element +– I-structure_element +14 I-structure_element +and O +all O +residues O +originate O +from O +the O +inner O +surface O +of O +the O +HAESA B-protein +superhelix B-structure_element +. O + +The O +conserved B-protein_state +PIP B-structure_element +motif I-structure_element +is O +highlighted O +in O +yellow O +, O +the O +central O +Hyp B-residue_name +in O +blue O +. O + +IDA B-protein +( O +in O +bonds O +representation O +, O +surface O +view O +included O +) O +is O +depicted O +in O +yellow O +. O + +The O +peptide B-site +binding I-site +pocket I-site +covers O +HAESA B-protein +LRRs B-structure_element +2 I-structure_element +– I-structure_element +14 I-structure_element +. O +( O +D O +) O +Close O +- O +up O +view O +of O +the O +entire O +IDA B-protein +( O +in O +yellow O +) O +peptide B-site +binding I-site +site I-site +in O +HAESA B-protein +( O +in O +blue O +). O + +Full B-protein_state +- I-protein_state +length I-protein_state +IDA B-protein +is O +proteolytically B-ptm +processed I-ptm +and O +a O +conserved B-protein_state +stretch B-residue_range +of I-residue_range +20 I-residue_range +amino I-residue_range +- I-residue_range +acids I-residue_range +( O +termed O +EPIP B-structure_element +) O +can O +rescue O +the O +IDA B-protein +loss O +- O +of O +- O +function O +phenotype O +( O +Figure O +1A O +). O + +Close O +- O +up O +views O +of O +( O +A O +) O +IDA B-protein +, O +( O +B O +) O +the O +N B-protein_state +- I-protein_state +terminally I-protein_state +extended I-protein_state +PKGV B-mutant +- I-mutant +IDA I-mutant +and O +( O +C O +) O +IDL1 B-protein +bound B-protein_state +to I-protein_state +the O +HAESA B-protein +hormone B-site +binding I-site +pocket I-site +( O +in O +bonds O +representation O +, O +in O +yellow O +) O +and O +including O +simulated B-experimental_method +annealing I-experimental_method +2Fo B-evidence +– I-evidence +Fc I-evidence +omit I-evidence +electron I-evidence +density I-evidence +maps I-evidence +contoured O +at O +1 O +. O +0 O +σ O +. O + +no O +detectable O +binding O +). O +( O +E O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +active B-protein_state +IDA B-protein +( O +in O +bonds O +representation O +, O +in O +gray O +) O +and O +IDL1 B-chemical +peptide I-chemical +( O +in O +yellow O +) O +hormones O +bound B-protein_state +to I-protein_state +the O +HAESA B-protein +ectodomain B-structure_element +. O + +The O +titration B-experimental_method +of O +IDA B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +versus O +the O +isolated O +HAESA B-protein +ectodomain B-structure_element +from O +Figure O +1B O +is O +shown O +for O +comparison O +( O +red O +line O +; O +n O +. O +d O +. O + +Mutant B-protein_state +( O +m O +) O +versions O +, O +which O +carry O +point B-experimental_method +mutations I-experimental_method +in O +their O +active B-site +sites I-site +( O +Asp837HAESA B-mutant +→ I-mutant +Asn I-mutant +, O +Asp447SERK1 B-mutant +→ I-mutant +Asn I-mutant +) O +possess O +no O +autophosphorylation O +activity O +( O +lanes O +2 O ++ O +4 O +). O + +The O +COO O +- O +group O +of O +Asn69IDA B-residue_name_number +is O +in O +direct O +contact O +with O +Arg407HAESA B-residue_name_number +and O +Arg409HAESA B-residue_name_number +and O +HAESA B-protein +cannot O +bind O +a O +C B-protein_state +- I-protein_state +terminally I-protein_state +extended I-protein_state +IDA B-mutant +- I-mutant +SFVN I-mutant +peptide O +( O +Figures O +1D O +, O +F O +, O +2D O +). O + +This O +suggests O +that O +the O +conserved B-protein_state +Asn69IDA B-residue_name_number +may O +constitute O +the O +very O +C O +- O +terminus O +of O +the O +mature B-protein_state +IDA B-chemical +peptide I-chemical +in O +planta B-taxonomy_domain +and O +that O +active B-protein_state +IDA B-protein +is O +generated O +by O +proteolytic O +processing O +from O +a O +longer O +pre O +- O +protein O +. O + +Replacing B-experimental_method +Hyp64IDA B-ptm +, O +which O +is O +common O +to O +all O +IDLs B-protein_type +, O +with O +proline B-residue_name +impairs O +the O +interaction O +with O +the O +receptor O +, O +as O +does O +the O +Lys66IDA B-mutant +/ I-mutant +Arg67IDA I-mutant +→ I-mutant +Ala I-mutant +double B-protein_state +- I-protein_state +mutant I-protein_state +discussed O +below O +( O +Figure O +1A O +, O +2D O +). O + +Our O +binding B-experimental_method +assays I-experimental_method +reveal O +that O +IDA B-chemical +family I-chemical +peptides I-chemical +are O +sensed O +by O +the O +isolated B-protein_state +HAESA B-protein +ectodomain B-structure_element +with O +relatively O +weak O +binding B-evidence +affinities I-evidence +( O +Figures O +1B O +, O +2A O +– O +D O +). O + +It O +has O +been O +recently O +reported O +that O +SOMATIC B-protein_type +EMBRYOGENESIS I-protein_type +RECEPTOR I-protein_type +KINASES I-protein_type +( O +SERKs B-protein_type +) O +are O +positive O +regulators O +of O +floral O +abscission O +and O +can O +interact O +with O +HAESA B-protein +and O +HSL2 B-protein +in O +an O +IDA O +- O +dependent O +manner O +. O + +In O +vitro O +, O +the O +LRR B-structure_element +ectodomain I-structure_element +of O +SERK1 B-protein +( O +residues O +24 B-residue_range +– I-residue_range +213 I-residue_range +) O +forms O +stable B-protein_state +, O +IDA B-protein_state +- I-protein_state +dependent I-protein_state +heterodimeric B-oligomeric_state +complexes B-protein_state +with I-protein_state +HAESA B-protein +in O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +experiments O +( O +Figure O +3B O +). O + +We O +next O +titrated B-experimental_method +SERK1 B-protein +into O +a O +solution O +containing O +only O +the O +HAESA B-protein +ectodomain B-structure_element +. O + +Our O +calorimetry B-experimental_method +experiments O +now O +reveal O +that O +SERKs B-protein_type +may O +render O +HAESA B-protein +, O +and O +potentially O +other O +receptor B-protein_type +kinases I-protein_type +, O +competent O +for O +high O +- O +affinity O +sensing O +of O +their O +cognate O +ligands O +. O + +Together O +, O +our O +genetic B-experimental_method +and I-experimental_method +biochemical I-experimental_method +experiments I-experimental_method +implicate O +SERK1 B-protein +as O +a O +HAESA B-protein_type +co I-protein_type +- I-protein_type +receptor I-protein_type +in O +the O +Arabidopsis B-taxonomy_domain +abscission O +zone O +. O + +SERK1 B-protein +loop B-structure_element +residues O +establish O +multiple O +hydrophobic B-bond_interaction +and I-bond_interaction +polar I-bond_interaction +contacts I-bond_interaction +with O +Lys66IDA B-residue_name_number +and O +the O +C O +- O +terminal O +Arg B-structure_element +- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +in O +IDA B-protein +( O +Figure O +4C O +). O + +Deletion B-experimental_method +of O +the O +C O +- O +terminal O +Asn69IDA B-residue_name_number +completely O +inhibits B-protein_state +complex O +formation O +. O + +35S B-gene +:: O +IDA B-protein +plants B-taxonomy_domain +showed O +significantly O +increased O +abscission O +compared O +to O +Col O +- O +0 O +controls O +in O +inflorescence O +positions O +2 O +and O +3 O +( O +a O +). O + +It O +is O +of O +note O +that O +our O +reported O +binding B-evidence +affinities I-evidence +for O +IDA B-protein +and O +SERK1 B-protein +have O +been O +measured O +using O +synthetic B-protein_state +peptides B-chemical +and O +the O +isolated B-experimental_method +HAESA B-protein +and O +SERK1 B-protein +ectodomains B-structure_element +, O +and O +thus O +might O +differ O +in O +the O +context O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +, O +membrane B-protein_state +- I-protein_state +embedded I-protein_state +signaling O +complex O +. O + +( O +A O +) O +Structural B-experimental_method +comparison I-experimental_method +of O +plant B-taxonomy_domain +steroid B-chemical +and O +peptide B-protein_type +hormone I-protein_type +membrane B-protein_type +signaling I-protein_type +complexes I-protein_type +. O + +In O +addition O +, O +residues O +53 B-residue_range +- I-residue_range +55SERK1 I-residue_range +from O +the O +SERK1 B-protein +N O +- O +terminal O +cap B-structure_element +mediate O +specific O +interactions O +with O +the O +IDA B-chemical +peptide I-chemical +( O +Figures O +4C O +, O +6B O +). O + +Structure B-experimental_method +- I-experimental_method +guided I-experimental_method +multiple I-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +IDA B-protein +and O +IDA B-chemical +- I-chemical +like I-chemical +peptides I-chemical +with O +other O +plant B-taxonomy_domain +peptide B-protein_type +hormone I-protein_type +families I-protein_type +, O +including O +CLAVATA3 B-protein_type +– I-protein_type +EMBRYO I-protein_type +SURROUNDING I-protein_type +REGION I-protein_type +- I-protein_type +RELATED I-protein_type +( O +CLV3 B-protein_type +/ I-protein_type +CLE I-protein_type +), O +ROOT B-protein_type +GROWTH I-protein_type +FACTOR I-protein_type +– I-protein_type +GOLVEN I-protein_type +( O +RGF B-protein_type +/ I-protein_type +GLV I-protein_type +), O +PRECURSOR B-protein_type +GENE I-protein_type +PROPEP1 I-protein_type +( O +PEP1 B-protein_type +) O +from O +Arabidopsis B-species +thaliana I-species +. O + +The O +conserved B-protein_state +( B-structure_element +Arg I-structure_element +)- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +is O +highlighted O +in O +red O +, O +the O +central O +Hyp B-residue_name +residue O +in O +IDLs B-protein_type +and O +CLEs B-protein_type +is O +marked O +in O +blue O +. O + +Among O +these O +are O +the O +CLE B-chemical +peptides I-chemical +regulating O +stem O +cell O +maintenance O +in O +the O +shoot O +and O +the O +root O +. O + +It O +is O +interesting O +to O +note O +, O +that O +CLEs B-protein_type +in O +their O +mature B-protein_state +form I-protein_state +are O +also O +hydroxyprolinated B-protein_state +dodecamers B-structure_element +, O +which O +bind O +to O +a O +surface B-site +area I-site +in O +the O +BARELY B-protein_type +ANY I-protein_type +MERISTEM I-protein_type +1 I-protein_type +receptor I-protein_type +that O +would O +correspond O +to O +part O +of O +the O +IDA B-site +binding I-site +cleft I-site +in O +HAESA B-protein +. O + +Diverse O +plant B-taxonomy_domain +peptide B-protein_type +hormones I-protein_type +may O +thus O +also O +bind O +their O +LRR B-protein_type +- I-protein_type +RK I-protein_type +receptors I-protein_type +in O +an O +extended B-protein_state +conformation I-protein_state +along O +the O +inner O +surface O +of O +the O +LRR B-structure_element +domain I-structure_element +and O +may O +also O +use O +small B-protein_state +, O +shape B-protein_state +- I-protein_state +complementary I-protein_state +co B-protein_type +- I-protein_type +receptors I-protein_type +for O +high O +- O +affinity O +ligand O +binding O +and O +receptor O +activation O +. O + +The O +structures B-evidence +suggest O +missing O +links O +in O +our O +understanding O +of O +tRNA B-chemical +translocation O +. O + +The O +canonical O +scenario O +of O +cap O +- O +dependent O +and O +IRES B-site +- O +dependent O +initiation O +involves O +positioning O +of O +the O +AUG O +start O +codon O +and O +the O +initiator O +tRNAMet B-chemical +in O +the O +ribosomal O +peptidyl B-site +- I-site +tRNA I-site +( I-site +P I-site +) I-site +site I-site +, O +facilitated O +by O +interaction O +with O +initiation B-protein_type +factors I-protein_type +. O + +Subsequent O +binding O +of O +an O +elongator O +aminoacyl B-chemical +- I-chemical +tRNA I-chemical +to O +the O +ribosomal O +A B-site +site I-site +transitions O +the O +initiation B-complex_assembly +complex I-complex_assembly +into O +the O +elongation O +cycle O +of O +translation O +. O + +The O +IGR B-structure_element +- O +IRES B-site +- O +driven O +initiation B-protein_state +does O +not O +involve O +initiator O +tRNAMet B-chemical +and O +initiation B-protein_state +factors O +. O + +The O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +- I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +is O +stabilized O +by O +interactions O +with O +the O +universally B-protein_state +conserved I-protein_state +decoding B-site +- I-site +center I-site +nucleotides O +G577 B-residue_name_number +, O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +( O +G530 B-residue_name_number +, O +A1492 B-residue_name_number +and O +A1493 B-residue_name_number +in O +E B-species +. I-species +coli I-species +16S O +ribosomal O +RNA B-chemical +, O +or O +rRNA B-chemical +). O + +For O +a O +cognate O +aminoacyl B-chemical +- I-chemical +tRNA I-chemical +to O +bind O +the O +first O +viral B-taxonomy_domain +mRNA B-chemical +codon O +, O +PKI B-structure_element +has O +to O +be O +translocated O +from O +the O +A B-site +site I-site +, O +so O +that O +the O +first O +codon O +can O +be O +presented O +in O +the O +A B-site +site I-site +. O + +Intersubunit O +rotation O +occurs O +spontaneously O +upon O +peptidyl O +transfer O +, O +and O +is O +coupled O +with O +formation O +of O +hybrid B-protein_state +tRNA B-chemical +states O +. O + +( O +a O +) O +Structures B-evidence +of O +bacterial B-taxonomy_domain +70S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +translocation O +complexes O +, O +ordered O +according O +to O +the O +position O +of O +the O +translocating O +A B-site +-> I-site +P I-site +tRNA B-chemical +( O +orange O +). O + +The O +large O +ribosomal O +subunit B-structure_element +is O +shown O +in O +cyan O +; O +the O +small B-structure_element +subunit I-structure_element +in O +light O +yellow O +( O +head B-structure_element +) O +and O +wheat O +- O +yellow O +( O +body B-structure_element +); O +the O +TSV B-species +IRES B-site +in O +red O +, O +eEF2 B-protein +in O +green O +. O + +( O +a O +) O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +. O +In O +all O +panels O +, O +the O +large B-structure_element +ribosomal I-structure_element +subunit I-structure_element +is O +shown O +in O +cyan O +; O +the O +small B-structure_element +subunit I-structure_element +in O +light O +yellow O +( O +head B-structure_element +) O +and O +wheat O +- O +yellow O +( O +body B-structure_element +); O +the O +TSV B-species +IRES B-site +in O +red O +, O +eEF2 B-protein +in O +green O +. O + +Although O +the O +mechanism O +of O +sordarin B-chemical +action O +is O +not O +fully O +understood O +, O +the O +inhibitor O +does O +not O +affect O +the O +conformation O +of O +eEF2 B-complex_assembly +• I-complex_assembly +GDPNP I-complex_assembly +on O +the O +ribosome B-complex_assembly +, O +rendering O +it O +an O +excellent O +tool O +in O +translocation O +studies O +. O + +( O +a O +) O +Rotational O +states O +of O +the O +40S B-complex_assembly +subunit B-structure_element +in O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +structure B-evidence +( O +INIT B-complex_assembly +; O +PDB O +3J6Y O +) O +and O +in O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +Structures B-evidence +I I-evidence +, I-evidence +II I-evidence +, I-evidence +III I-evidence +, I-evidence +IV I-evidence +and I-evidence +V I-evidence +( O +this O +work O +). O + +The O +sizes O +of O +the O +arrows O +correspond O +to O +the O +extent O +of O +the O +head B-structure_element +swivel O +( O +yellow O +) O +and O +subunit B-structure_element +rotation O +( O +black O +). O + +Our O +structures B-evidence +represent O +hitherto O +uncharacterized O +translocation O +complexes O +of O +the O +TSV B-species +IRES B-site +captured O +within O +globally O +distinct O +80S B-complex_assembly +conformations O +( O +Figures O +1b O +and O +2 O +). O + +Changes O +in O +ribosome B-complex_assembly +conformation O +and O +eEF2 B-protein +positions O +are O +coupled O +with O +IRES B-site +movement O +through O +the O +ribosome B-complex_assembly + +Using O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +S B-species +. I-species +cerevisiae I-species +80S B-complex_assembly +ribosome I-complex_assembly +bound B-protein_state +with I-protein_state +the O +P B-site +and I-site +E I-site +site I-site +tRNAs B-chemical +as O +a O +reference O +( O +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +), O +in O +which O +both O +the O +subunit B-structure_element +rotation O +and O +the O +head B-structure_element +- O +body B-structure_element +swivel O +are O +0 O +°, O +we O +found O +that O +the O +ribosome B-complex_assembly +adopts O +four O +globally O +distinct O +conformations O +in O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +( O +Figure O +1b O +; O +see O +also O +Figure O +1 O +— O +figure O +supplement O +1 O +and O +Figure O +2 O +— O +source O +data O +1 O +). O + +Structure B-evidence +V I-evidence +is O +in O +a O +nearly O +non B-protein_state +- I-protein_state +rotated I-protein_state +conformation O +( O +0 O +. O +5 O +°), O +very O +similar O +to O +that O +of O +post B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +complexes O +. O + +However O +in O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +intermediates O +( O +from O +Structure B-evidence +I I-evidence +to I-evidence +IV I-evidence +), O +the O +beak O +of O +the O +head B-structure_element +domain O +first O +turns O +toward O +the O +large B-structure_element +subunit I-structure_element +and O +then O +backs O +off O +( O +Figure O +2 O +— O +figure O +supplement O +1 O +). O + +By O +comparison O +, O +the O +similarly O +mid B-protein_state +- I-protein_state +rotated I-protein_state +( O +4 O +°) O +80S B-complex_assembly +• I-complex_assembly +TSV I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +complex O +, O +in O +the O +absence B-protein_state +of I-protein_state +eEF2 B-protein +, O +adopts O +a O +mid B-protein_state +- I-protein_state +swiveled I-protein_state +position O +(~ O +10 O +°) O +( O +Figure O +2c O +). O + +The O +view O +shows O +the O +vicinity O +of O +the O +ribosomal O +E B-site +site I-site +. O + +Structures B-evidence +of O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +complexes O +in O +the O +absence B-protein_state +of I-protein_state +eEF2 B-protein +( O +INIT B-complex_assembly +; O +PDB O +3J6Y O +,) O +and O +in O +the O +presence B-protein_state +of I-protein_state +eEF2 B-protein +( O +this O +work O +) O +are O +shown O +in O +the O +lower O +row O +and O +labeled O +. O + +Positions O +of O +the O +IRES B-site +relative O +to O +eEF2 B-protein +and O +elements O +of O +the O +ribosome B-complex_assembly +in O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +. O + +Pseudoknots O +and O +stem B-structure_element +loops I-structure_element +are O +labeled O +and O +colored O +as O +in O +( O +a O +). O + +We O +collectively O +term O +domains O +I B-structure_element +and O +II B-structure_element +the O +5 B-structure_element +’ I-structure_element +domain I-structure_element +. O + +The O +view O +was O +obtained O +by O +structural B-experimental_method +alignment I-experimental_method +of O +the O +body B-structure_element +domains O +of O +18S B-chemical +rRNAs I-chemical +of O +the O +corresponding O +80S B-complex_assembly +structures B-evidence +. O + +Distances O +between O +nucleotides O +6848 B-residue_number +and O +6913 B-residue_number +in O +SL4 B-structure_element +and O +PKI B-structure_element +, O +respectively O +, O +are O +shown O +( O +see O +also O +Figure O +2 O +— O +source O +data O +1 O +). O + +Rearrangements O +of O +the O +IRES B-site +involve O +restructuring O +of O +several O +interactions O +with O +the O +ribosome B-complex_assembly +. O + +However O +, O +in O +the O +extended B-protein_state +conformations O +, O +these O +parts O +of O +the O +IRES B-site +and O +the O +60S B-complex_assembly +subunit B-structure_element +are O +separated O +by O +more O +than O +10 O +Å O +, O +suggesting O +that O +an O +interaction O +between O +them O +stabilizes O +the O +bent B-protein_state +conformations O +but O +not O +the O +extended B-protein_state +ones O +. O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +density B-evidence +of O +the O +GTPase B-structure_element +region I-structure_element +in O +Structures B-evidence +I I-evidence +and I-evidence +II I-evidence +. O + +Superposition B-experimental_method +was O +obtained O +by O +structural B-experimental_method +alignment I-experimental_method +of O +the O +25S B-chemical +rRNAs I-chemical +. O + +Sordarin B-chemical +is O +shown O +in O +pink O +with O +oxygen O +atoms O +in O +red O +. O + +The O +GTPase B-site +- I-site +associated I-site +center I-site +comprises O +the O +P B-structure_element +stalk I-structure_element +( O +L11 B-structure_element +and O +L7 B-structure_element +/ O +L12 B-structure_element +stalk B-structure_element +in O +bacteria B-taxonomy_domain +) O +and O +the O +sarcin B-structure_element +- I-structure_element +ricin I-structure_element +loop I-structure_element +( O +SRL B-structure_element +, O +nt O +3012 B-residue_range +– I-residue_range +3042 I-residue_range +). O + +The O +view O +was O +obtained O +by O +structural B-experimental_method +alignment I-experimental_method +of O +the O +18S B-chemical +rRNAs I-chemical +. O + +( O +a O +) O +eEF2 B-protein +( O +green O +) O +interacts O +only O +with O +the O +body B-structure_element +in O +Structure B-evidence +I I-evidence +( O +eEF2 B-protein +domains O +are O +labeled O +with O +roman O +numerals O +in O +white O +), O +and O +with O +both O +the O +head B-structure_element +and O +body B-structure_element +in O +Structures B-evidence +II I-evidence +through I-evidence +V I-evidence +. O +Colors O +are O +as O +in O +Figure O +1 O +, O +except O +for O +the O +40S B-complex_assembly +structural O +elements O +that O +contact O +eEF2 B-protein +, O +which O +are O +labeled O +and O +shown O +in O +purple O +. O +( O +b O +) O +Entry O +of O +eEF2 B-protein +into O +the O +40S B-complex_assembly +A B-site +site I-site +, O +from O +Structure B-evidence +I I-evidence +through I-evidence +V I-evidence +. O +Distances O +to O +the O +A B-site +- I-site +site I-site +accommodated O +eEF2 B-protein +( O +Structure B-evidence +V I-evidence +) O +are O +shown O +. O + +There O +are O +two O +modest O +but O +noticeable O +domain O +rearrangements O +between O +Structures B-evidence +I I-evidence +and I-evidence +V I-evidence +. O +Unlike O +in O +free B-protein_state +eEF2 B-protein +, O +which O +can O +sample O +large O +movements O +of O +at O +least O +50 O +Å O +of O +the O +C O +- O +terminal O +superdomain B-structure_element +relative O +to O +the O +N O +- O +terminal O +superdomain B-structure_element +( O +Figure O +5c O +), O +eEF2 B-protein +undergoes O +moderate O +repositioning O +of O +domain O +IV B-structure_element +(~ O +3 O +Å O +; O +Figure O +5a O +) O +and O +domain O +III B-structure_element +(~ O +5 O +Å O +; O +Figure O +6d O +). O + +Domain O +IV B-structure_element +of O +eEF2 B-protein +binds O +at O +the O +40S B-complex_assembly +A B-site +site I-site +in O +Structures B-evidence +I I-evidence +to I-evidence +V I-evidence +but O +the O +mode O +of O +interaction O +differs O +in O +each O +complex O +( O +Figure O +6 O +). O + +Because O +eEF2 B-protein +is O +rigidly O +attached O +to O +the O +60S B-complex_assembly +subunit B-structure_element +and O +does O +not O +undergo O +large O +inter O +- O +subunit B-structure_element +rearrangements O +, O +gradual O +entry O +of O +domain O +IV B-structure_element +into O +the O +A B-site +site I-site +between O +Structures B-evidence +I I-evidence +and I-evidence +V I-evidence +is O +due O +to O +40S B-complex_assembly +subunit B-structure_element +rotation O +and O +head B-structure_element +swivel O +. O + +Modest O +intra O +- O +eEF2 B-protein +shifts O +of O +domain O +IV B-structure_element +between O +Structures B-evidence +I I-evidence +to I-evidence +V I-evidence +outline O +a O +stochastic O +trajectory O +( O +Figure O +5a O +), O +consistent O +with O +local O +adjustments O +of O +the O +domain O +in O +the O +A B-site +site I-site +. O + +We O +therefore O +define O +C1274 B-residue_name_number +as O +the O +foundation O +of O +the O +' O +head B-structure_element +A B-site +site I-site +'. O + +Accordingly O +, O +we O +use O +U1191 B-residue_name_number +( O +G966 B-residue_name_number +in O +E B-species +. I-species +coli I-species +) O +and O +C1637 B-residue_name_number +( O +C1400 B-residue_name_number +in O +E B-species +. I-species +coli I-species +) O +as O +the O +reference O +points O +of O +the O +' O +head B-structure_element +P B-site +site I-site +' O +and O +' O +body B-structure_element +P B-site +site I-site +' O +( O +Figure O +2g O +), O +respectively O +, O +because O +these O +nucleotides O +form O +a O +stacking O +foundation O +for O +the O +fully B-protein_state +translocated I-protein_state +mRNA B-structure_element +- I-structure_element +tRNA I-structure_element +helix I-structure_element +in O +tRNA B-protein_state +- I-protein_state +bound I-protein_state +structures B-evidence +and O +in O +our O +post B-protein_state +- I-protein_state +translocation I-protein_state +Structure B-evidence +V I-evidence +discussed O +below O +. O + +The O +interaction O +of O +PKI B-structure_element +with O +the O +40S B-complex_assembly +body B-structure_element +is O +substantially O +rearranged O +relative O +to O +that O +in O +the O +initiation B-protein_state +state O +. O + +Diphthamide B-ptm +is O +a O +unique O +posttranslational O +modification O +conserved B-protein_state +in O +archaeal B-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +EF2 B-protein +( O +at O +residue O +699 B-residue_number +in O +S B-species +. I-species +cerevisiae I-species +) O +and O +involves O +addition O +of O +a O +~ O +7 O +- O +Å O +long O +3 O +- O +carboxyamido O +- O +3 O +-( O +trimethylamino O +)- O +propyl O +moiety O +to O +the O +histidine B-residue_name +imidazole O +ring O +at O +CE1 O +. O + +Switch B-structure_element +loop I-structure_element +II I-structure_element +( O +aa O +105 B-residue_range +– I-residue_range +110 I-residue_range +), O +which O +carries O +the O +catalytic B-protein_state +H108 B-residue_name_number +( O +H92 B-residue_name_number +in O +E B-species +. I-species +coli I-species +EF B-protein +- I-protein +G I-protein +; O +is O +well O +resolved O +in O +all O +five O +structures B-evidence +. O + +Bulged B-protein_state +A416 B-residue_name_number +interacts O +with O +the O +switch B-structure_element +loop I-structure_element +in O +the O +vicinity O +of O +D53 B-residue_name_number +. O + +Next O +to O +GDP B-chemical +, O +the O +C O +- O +terminal O +part O +of O +the O +switch B-structure_element +loop I-structure_element +( O +aa O +61 B-residue_range +– I-residue_range +67 I-residue_range +) O +adopts O +a O +helical B-protein_state +fold I-protein_state +. O + +As O +such O +, O +the O +conformations O +of O +SWI B-structure_element +and O +the O +GTPase B-site +center I-site +in O +general O +are O +similar O +to O +those O +observed O +in O +ribosome B-protein_state +- I-protein_state +bound I-protein_state +EF B-protein +- I-protein +Tu I-protein +and O +EF B-protein +- I-protein +G I-protein +in O +the O +presence B-protein_state +of I-protein_state +GTP B-chemical +analogs O +. O + +The O +head B-site +interface I-site +of O +domain O +IV B-structure_element +interacts O +with O +the O +40S B-complex_assembly +head B-structure_element +( O +Figure O +6a O +). O + +Structure B-evidence +III I-evidence +represents O +a O +highly B-protein_state +bent I-protein_state +IRES B-site +with O +PKI B-structure_element +captured O +between O +the O +head B-structure_element +A B-site +and I-site +P I-site +sites I-site + +The O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +– I-structure_element +like I-structure_element +helix I-structure_element +is O +stacked O +on O +P B-site +- I-site +site I-site +residues O +U1191 B-residue_name_number +and O +C1637 B-residue_name_number +( O +Figure O +3d O +), O +analogous O +to O +stacking B-bond_interaction +of O +the O +tRNA B-complex_assembly +- I-complex_assembly +mRNA I-complex_assembly +helix B-structure_element +( O +Figure O +3e O +). O + +As O +in O +the O +preceding O +Structures B-evidence +, O +the O +histidine B-site +- I-site +diphthamide I-site +tip I-site +is O +bound B-protein_state +in I-protein_state +the O +minor B-site +groove I-site +of O +the O +P B-site +- I-site +site I-site +codon B-structure_element +- I-structure_element +anticodon I-structure_element +helix I-structure_element +. O + +Animation O +showing O +the O +transition O +from O +the O +initiation B-protein_state +80S B-complex_assembly +• I-complex_assembly +TSV I-complex_assembly +IRES I-complex_assembly +structures B-evidence +( O +Koh O +et O +al O +., O +2014 O +) O +to O +eEF2 B-protein_state +- I-protein_state +bound I-protein_state +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +( O +this O +work O +). O + +In O +scene O +4 O +, O +C1274 B-residue_name_number +and O +U1191 B-residue_name_number +are O +labeled O +and O +shown O +in O +yellow O +; O +G577 B-residue_name_number +, O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +of O +the O +40S B-complex_assembly +body B-structure_element +A B-site +site I-site +and O +C1637 B-residue_name_number +of O +the O +body B-structure_element +P B-site +site I-site +are O +labeled O +and O +shown O +in O +orange O +. O + +In O +this O +work O +we O +have O +captured O +the O +structures B-evidence +of O +the O +TSV B-species +IRES B-site +, O +whose O +PKI B-structure_element +samples O +positions O +between O +the O +A B-site +and I-site +P I-site +sites I-site +( O +Structures B-evidence +I I-evidence +– I-evidence +IV I-evidence +), O +as O +well O +as O +in O +the O +P B-site +site I-site +( O +Structure B-evidence +V I-evidence +). O + +In O +summary O +, O +the O +reported O +ensemble O +of O +structures B-evidence +substantially O +enhances O +our O +understanding O +of O +the O +translocation O +mechanism O +, O +including O +that O +of O +tRNAs B-chemical +as O +discussed O +below O +. O + +In O +the O +first O +sub O +- O +step O +( O +Structures B-evidence +I I-evidence +to I-evidence +IV I-evidence +), O +the O +hind B-structure_element +end I-structure_element +advances O +from O +the O +A B-site +to I-site +the I-site +P I-site +site I-site +and O +approaches O +the O +front B-structure_element +end I-structure_element +, O +which O +remains O +attached O +to O +the O +40S B-complex_assembly +surface O +. O + +Notably O +, O +at O +all O +steps O +, O +the O +head B-structure_element +of O +the O +IRES B-site +inchworm B-protein_state +( O +L1 B-structure_element +. I-structure_element +1 I-structure_element +region I-structure_element +) O +is O +supported O +by O +the O +mobile B-protein_state +L1 B-structure_element +stalk I-structure_element +. O + +Upon O +translocation O +, O +the O +GCU O +start O +codon O +is O +positioned O +in O +the O +A B-site +site I-site +( O +Structure B-evidence +V I-evidence +), O +ready O +for O +interaction O +with O +Ala B-chemical +- I-chemical +tRNAAla I-chemical +upon O +eEF2 B-protein +departure O +. O + +In O +our O +structures B-evidence +, O +the O +IRES B-site +presents O +to O +the O +decoding B-site +center I-site +a O +pre B-protein_state +- I-protein_state +translocated I-protein_state +or O +fully B-protein_state +translocated I-protein_state +ORF B-structure_element +, O +rather O +than O +a O ++ O +1 O +( O +more O +translocated O +) O +ORF B-structure_element +, O +suggesting O +that O +eEF2 B-protein +does O +not O +induce O +a O +highly O +populated O +fraction O +of O ++ O +1 O +shifted O +IRES B-site +mRNAs B-chemical +. O + +The O +presence B-protein_state +of I-protein_state +several O +translocation O +complexes O +in O +a O +single O +sample O +suggests O +that O +the O +structures B-evidence +represent O +equilibrium O +states O +of O +forward O +and O +reverse O +translocation O +of O +the O +IRES B-site +, O +which O +interconvert O +among O +each O +other O +. O + +These O +findings O +indicate O +that O +IRES B-site +translocation O +by O +eEF2 B-protein +is O +futile O +: O +the O +IRES B-site +returns O +to O +the O +A B-site +site I-site +upon O +releasing O +eEF2 B-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +unless O +an O +amino B-chemical +- I-chemical +acyl I-chemical +tRNA I-chemical +enters O +the O +A B-site +site I-site +and O +blocks O +IRES B-site +back O +- O +translocation O +. O + +Various O +degrees O +of O +intersubunit O +rotation O +have O +been O +observed O +in O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +studies I-experimental_method +of O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +complexes O +. O + +The O +pre B-protein_state +- I-protein_state +translocation I-protein_state +Structure B-evidence +I I-evidence +with O +eEF2 B-protein +least O +advanced O +into O +the O +A B-site +site I-site +adopts O +a O +fully B-protein_state +rotated I-protein_state +conformation I-protein_state +. O + +Translocases B-protein_type +are O +efficient O +enzymes O +. O + +The O +Structures B-evidence +reveal O +hopping O +of O +the O +positive O +clusters O +over O +rRNA B-chemical +helices B-structure_element +. O + +Whereas O +intersubunit O +rotation O +of O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +complex O +occurs O +spontaneously O +, O +the O +head B-structure_element +swivel O +is O +induced O +by O +the O +eEF2 B-protein +/ O +EF B-protein +- I-protein +G I-protein +translocase B-protein_type +, O +consistent O +with O +requirement O +of O +eEF2 B-protein +for O +unlocking O +. O + +Structural B-experimental_method +studies I-experimental_method +revealed O +large O +head B-structure_element +swivels O +in O +various O +70S B-complex_assembly +• I-complex_assembly +tRNA I-complex_assembly +• I-complex_assembly +EF I-complex_assembly +- I-complex_assembly +G I-complex_assembly +and O +80S B-complex_assembly +• I-complex_assembly +tRNA I-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +complexes O +, O +but O +not O +in O +' O +locked B-protein_state +' O +complexes B-protein_state +with I-protein_state +the O +A B-site +site I-site +occupied O +by O +the O +tRNA B-chemical +in O +the O +absence B-protein_state +of I-protein_state +the O +translocase B-protein_type +. O + +This O +' O +locked B-protein_state +' O +state O +is O +identical O +to O +that O +observed O +for O +PKI B-structure_element +in O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +structures B-evidence +in O +the O +absence B-protein_state +of I-protein_state +eEF2 B-protein +. O + +Observation O +of O +different O +PKI B-structure_element +conformations O +sampling O +a O +range O +of O +positions O +between O +the O +A B-site +and I-site +P I-site +sites I-site +in O +the O +presence B-protein_state +of I-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +implies O +that O +thermal O +fluctuations O +of O +the O +40S B-complex_assembly +head B-structure_element +domain O +are O +sufficient O +for O +translocation O +along O +the O +energetically O +flat O +trajectory O +. O + +In O +all O +five O +structures B-evidence +, O +the O +GTPase B-structure_element +domain I-structure_element +is O +attached O +to O +the O +P B-structure_element +stalk I-structure_element +and O +the O +sarcin B-structure_element +- I-structure_element +ricin I-structure_element +loop I-structure_element +. O + +The O +least B-protein_state +rotated I-protein_state +conformation O +of O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +Structure B-evidence +V I-evidence +suggests O +conformational O +changes O +that O +may O +trigger O +eEF2 B-protein +release O +from O +the O +ribosome B-complex_assembly +at O +the O +end O +of O +translocation O +. O + +Sordarin B-chemical +is O +a O +potent O +antifungal O +antibiotic O +that O +inhibits O +translation O +. O + +Although O +our O +complex O +was O +assembled O +using O +eEF2 B-complex_assembly +• I-complex_assembly +GTP I-complex_assembly +, O +density B-evidence +maps I-evidence +clearly O +show O +GDP B-chemical +and O +Mg2 B-chemical ++ I-chemical +in O +each O +structure B-evidence +( O +Figure O +5g O +). O + +Because O +translocation O +of O +tRNA B-chemical +must O +involve O +large O +- O +scale O +dynamics O +, O +this O +step O +has O +long O +been O +regarded O +as O +the O +most O +puzzling O +step O +of O +translation O +. O + +Intersubunit O +rearrangements O +and O +tRNA B-chemical +hybrid B-protein_state +states O +have O +been O +proposed O +to O +play O +key O +roles O +half O +a O +century O +ago O +. O + +Second O +, O +the O +structures B-evidence +clarify O +the O +structural O +basis O +of O +the O +often O +- O +used O +but O +structurally O +undefined O +terms O +' O +locking O +' O +and O +' O +unlocking O +' O +with O +respect O +to O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +complex O +( O +Figure O +6f O +). O + +Previous O +studies O +showed O +that O +this O +movement O +widens O +the O +constriction B-site +(' O +gate B-site +') O +between O +the O +P B-site +and I-site +E I-site +sites I-site +, O +thus O +allowing O +the O +P B-site +- O +tRNA B-chemical +passage O +to O +the O +E B-site +site I-site +. O + +In O +addition O +to O +the O +' O +gate B-site +- O +opening O +' O +role O +, O +we O +now O +show O +that O +the O +head B-structure_element +swivel O +brings O +the O +head B-structure_element +A B-site +site I-site +to O +the O +body B-structure_element +P B-site +site I-site +, O +allowing O +a O +step O +- O +wise O +conveying O +of O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +helix I-structure_element +between O +the O +A B-site +and I-site +P I-site +sites I-site +. O + +Movement O +of O +PKI B-structure_element +relative O +to O +the O +head B-structure_element +occurs O +during O +the O +subsequent O +reverse O +swivel O +in O +three O +3 O +– O +7 O +Å O +sub O +- O +steps O +( O +II B-evidence +to I-evidence +III I-evidence +to I-evidence +IV I-evidence +to I-evidence +V I-evidence +). O + +Our O +work O +sheds O +light O +on O +the O +dynamic O +mechanism O +of O +cap O +- O +independent O +translation O +by O +IGR B-structure_element +IRESs B-site +, O +tightly O +coupled O +with O +the O +universally B-protein_state +conserved I-protein_state +dynamic O +properties O +of O +the O +ribosome B-complex_assembly +. O + +This O +difference O +likely O +accounts O +for O +the O +inefficient O +translocation O +of O +the O +IRES B-site +, O +which O +is O +difficult O +to O +stabilize O +in O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +state O +and O +therefore O +is O +prone O +to O +reverse O +translocation O +. O + +Intergenic O +IRESs B-site +, O +in O +turn O +, O +represent O +a O +striking O +example O +of O +convergent O +molecular O +evolution O +. O + +Ensemble O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method + +While O +the O +inactive B-protein_state +α B-protein +subunits I-protein +build O +the O +two O +outer O +rings B-structure_element +, O +the O +β B-protein +subunits I-protein +form O +the O +inner O +rings B-structure_element +. O + +Only O +three O +out O +of O +the O +seven O +different O +β B-protein +subunits I-protein +, O +namely O +β1 B-protein +, O +β2 B-protein +and O +β5 B-protein +, O +bear O +N O +- O +terminal O +proteolytic B-site +active I-site +centres I-site +, O +and O +before O +CP B-complex_assembly +maturation O +these O +are O +protected O +by O +propeptides B-structure_element +. O + +In O +the O +last O +stage O +of O +CP B-complex_assembly +biogenesis O +, O +the O +prosegments B-structure_element +are O +autocatalytically B-ptm +removed I-ptm +through O +nucleophilic O +attack O +by O +the O +active B-site +site I-site +residue I-site +Thr1 B-residue_name_number +on O +the O +preceding O +peptide O +bond O +involving O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +). I-residue_name_number + +Although O +the O +chemical O +nature O +of O +the O +substrate B-site +- I-site +binding I-site +channel I-site +and O +hence O +substrate O +preferences O +are O +unique O +to O +each O +of O +the O +distinct O +active B-protein_state +β B-protein +subunits I-protein +, O +all O +active B-site +sites I-site +employ O +an O +identical O +reaction O +mechanism O +to O +hydrolyse O +peptide O +bonds O +. O + +Data O +from O +biochemical B-experimental_method +and I-experimental_method +structural I-experimental_method +analyses I-experimental_method +of O +proteasome O +variants O +with O +mutations O +in O +the O +β5 B-protein +propeptide B-structure_element +and O +the O +active B-site +site I-site +strongly O +support O +the O +model O +and O +deliver O +novel O +insights O +into O +the O +structural O +constraints O +required O +for O +the O +autocatalytic B-ptm +activation I-ptm +of O +the O +proteasome B-complex_assembly +. O + +Inactivation O +of O +the O +active B-site +site I-site +Thr1 B-residue_name_number +by O +mutation B-experimental_method +to I-experimental_method +Ala B-residue_name +has O +been O +used O +to O +study O +substrate O +specificity O +and O +the O +hierarchy O +of O +the O +proteasome B-complex_assembly +active B-site +sites I-site +. O + +These O +results O +indicate O +that O +the O +β1 B-protein +and O +β2 B-protein +proteolytic O +activities O +are O +not O +essential O +for O +cell O +survival O +. O + +By O +contrast O +, O +the O +T1A B-mutant +mutation O +in O +subunit O +β5 B-protein +has O +been O +reported O +to O +be O +lethal O +or O +nearly O +so O +. O + +Our O +present O +crystallographic B-experimental_method +analysis I-experimental_method +of O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +pp B-chemical +trans B-protein_state +mutant B-protein_state +demonstrates O +that O +the O +mutation B-experimental_method +per O +se O +does O +not O +structurally O +alter O +the O +catalytic B-site +active I-site +site I-site +and O +that O +the O +trans B-experimental_method +- I-experimental_method +expressed I-experimental_method +β5 B-protein +propeptide B-structure_element +is O +not B-protein_state +bound I-protein_state +in O +the O +β5 B-protein +substrate B-site +- I-site +binding I-site +channel I-site +( O +Supplementary O +Fig O +. O +1a O +). O + +The O +extremely O +weak O +growth O +of O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +mutant B-protein_state +pp B-chemical +cis B-protein_state +described O +by O +Chen O +and O +Hochstrasser O +compared O +with O +the O +inviability O +reported O +by O +Heinemeyer O +et O +al O +. O +prompted O +us O +to O +analyse O +this O +discrepancy O +. O + +In O +subunit O +β1 B-protein +, O +we O +found O +that O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +indeed O +forms O +a O +sharp B-structure_element +turn I-structure_element +, O +which O +relaxes O +on O +prosegment B-ptm +cleavage I-ptm +( O +Fig O +. O +1a O +and O +Supplementary O +Fig O +. O +2a O +). O + +Regarding O +the O +β2 B-protein +propeptide B-structure_element +, O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +occupies O +the O +S1 B-site +pocket I-site +but O +is O +less O +deeply O +anchored O +compared O +with O +Leu B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +in O +β1 B-protein +, O +which O +might O +be O +due O +to O +the O +rather O +large O +β2 B-protein +- O +S1 B-site +pocket I-site +created O +by O +Gly45 B-residue_name_number +. O + +As O +histidine B-residue_name +commonly O +functions O +as O +a O +proton O +shuttle O +in O +the O +catalytic B-site +triads I-site +of O +serine B-protein_type +proteases I-protein_type +, O +we O +investigated O +the O +role O +of O +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +in O +processing O +of O +the O +β5 B-protein +propeptide B-structure_element +by O +exchanging B-experimental_method +it I-experimental_method +for I-experimental_method +Asn B-residue_name +, O +Lys B-residue_name +, O +Phe B-residue_name +and O +Ala B-residue_name +. O +All O +yeast B-taxonomy_domain +mutants O +were O +viable O +at O +30 O +° O +C O +, O +but O +suffered O +from O +growth O +defects O +at O +37 O +° O +C O +with O +the O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +N I-mutant +and O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +F I-mutant +mutants O +being O +most O +affected O +( O +Supplementary O +Fig O +. O +3b O +and O +Table O +1 O +). O + +Nevertheless O +, O +both O +Leu B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +and O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +were O +found O +to O +occupy O +the O +S1 B-site +specificity I-site +pocket I-site +formed O +by O +Met45 B-residue_name_number +( O +Fig O +. O +2a O +, O +b O +and O +Supplementary O +Fig O +. O +4f O +– O +h O +). O + +Bearing O +in O +mind O +that O +in O +contrast O +to O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +in O +β2 B-protein +, O +Leu B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +in O +subunit O +β1 B-protein +is O +not B-protein_state +conserved I-protein_state +among O +species O +( O +Supplementary O +Fig O +. O +3a O +), O +we O +created B-experimental_method +a O +β2 B-mutant +- I-mutant +T I-mutant +(- I-mutant +2 I-mutant +) I-mutant +V I-mutant +proteasome B-complex_assembly +mutant B-protein_state +. O + +Instead O +, O +Lys33NH2 B-residue_name_number +, O +which O +is O +in O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +distance O +to O +Thr1Oγ B-residue_name_number +( O +2 O +. O +7 O +Å O +) O +in O +all O +catalytically B-protein_state +active I-protein_state +β B-protein +subunits I-protein +( O +Fig O +. O +3a O +, O +b O +), O +was O +proposed O +to O +serve O +as O +the O +proton O +acceptor O +. O + +In O +contrast O +to O +the O +cis B-protein_state +- O +construct O +, O +expression B-experimental_method +of O +the O +β5 B-protein +propeptide B-structure_element +in O +trans B-protein_state +allowed O +straightforward O +isolation B-experimental_method +and O +crystallization B-experimental_method +of O +the O +D17N B-mutant +mutant B-protein_state +proteasome B-complex_assembly +. O + +The O +ChT O +- O +L O +activity O +of O +the O +β5 B-mutant +- I-mutant +D17N I-mutant +pp B-chemical +in O +trans B-protein_state +CP B-complex_assembly +towards O +the O +canonical O +β5 B-protein +model O +substrates O +N B-chemical +- I-chemical +succinyl I-chemical +- I-chemical +Leu I-chemical +- I-chemical +Leu I-chemical +- I-chemical +Val I-chemical +- I-chemical +Tyr I-chemical +- I-chemical +7 I-chemical +- I-chemical +amino I-chemical +- I-chemical +4 I-chemical +- I-chemical +methylcoumarin I-chemical +( O +Suc B-chemical +- I-chemical +LLVY I-chemical +- I-chemical +AMC I-chemical +) O +and O +carboxybenzyl B-chemical +- I-chemical +Gly I-chemical +- I-chemical +Gly I-chemical +- I-chemical +Leu I-chemical +- I-chemical +para I-chemical +- I-chemical +nitroanilide I-chemical +( O +Z B-chemical +- I-chemical +GGL I-chemical +- I-chemical +pNA I-chemical +) O +was O +severely O +reduced O +( O +Supplementary O +Fig O +. O +6b O +), O +confirming O +that O +Asp17 B-residue_name_number +is O +of O +fundamental O +importance O +for O +the O +catalytic O +activity O +of O +the O +mature B-protein_state +proteasome B-complex_assembly +. O + +This O +observation O +is O +consistent O +with O +a O +strongly O +reduced O +reactivity O +of O +β5 B-protein +- O +Thr1 B-residue_name_number +and O +the O +crystal B-evidence +structure I-evidence +of O +the O +β5 B-mutant +- I-mutant +D17N I-mutant +pp B-chemical +cis B-protein_state +mutant B-protein_state +in B-protein_state +complex I-protein_state +with I-protein_state +carfilzomib B-chemical +. O + +On O +the O +basis O +of O +these O +results O +, O +we O +propose O +that O +CPs B-complex_assembly +from O +all O +domains O +of O +life O +use O +a O +catalytic B-site +triad I-site +consisting O +of O +Thr1 B-residue_name_number +, O +Lys33 B-residue_name_number +and O +Asp B-residue_name +/ O +Glu17 B-residue_name_number +for O +both O +autocatalytic B-ptm +precursor I-ptm +processing I-ptm +and O +proteolysis O +( O +Fig O +. O +3d O +). O + +Soaking B-experimental_method +the O +β5 B-mutant +- I-mutant +D166N I-mutant +crystals B-experimental_method +with O +carfilzomib B-chemical +and O +MG132 B-chemical +resulted O +in O +covalent O +modification O +of O +Thr1 B-residue_name_number +at O +high O +occupancy O +( O +Supplementary O +Fig O +. O +8c O +). O + +These O +results O +suggest O +that O +Asp166 B-residue_name_number +and O +Ser129 B-residue_name_number +function O +as O +a O +proton O +shuttle O +and O +affect O +the O +protonation O +state O +of O +Thr1N B-residue_name_number +during O +autolysis B-ptm +and O +catalysis O +. O + +Despite O +propeptide B-ptm +hydrolysis I-ptm +, O +the O +β5 B-mutant +- I-mutant +T1C I-mutant +active B-site +site I-site +is O +catalytically B-protein_state +inactive I-protein_state +( O +Fig O +. O +4b O +and O +Supplementary O +Fig O +. O +9a O +). O + +Moreover O +, O +the O +structural B-evidence +data I-evidence +reveal O +that O +the O +thiol O +group O +of O +Cys1 B-residue_name_number +is O +rotated O +by O +74 O +° O +with O +respect O +to O +the O +hydroxyl O +side O +chain O +of O +Thr1 B-residue_name_number +( O +Fig O +. O +4f O +and O +Supplementary O +Fig O +. O +9b O +). O + +Crystal B-evidence +structure I-evidence +analysis O +of O +the O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +confirmed O +precursor B-ptm +processing I-ptm +( O +Fig O +. O +4g O +), O +and O +ligand B-complex_assembly +- I-complex_assembly +complex I-complex_assembly +structures B-evidence +with O +bortezomib B-chemical +and O +carfilzomib B-chemical +unambiguously O +corroborated O +the O +reactivity O +of O +Ser1 B-residue_name_number +( O +Fig O +. O +5 O +). O + +Because O +both O +conformations O +of O +Ser1Oγ B-residue_name_number +are O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +Lys33NH2 B-residue_name_number +( O +Fig O +. O +4h O +), O +the O +relay O +system O +is O +capable O +of O +hydrolysing O +peptide O +substrates O +, O +albeit O +at O +lower O +rates O +compared O +with O +Thr1 B-residue_name_number +. O + +In O +agreement O +, O +at O +an O +elevated O +growing O +temperature O +of O +37 O +° O +C O +the O +T1S B-mutant +mutant B-protein_state +is O +unable O +to O +grow O +( O +Fig O +. O +4a O +). O + +These O +observations O +highlight O +the O +unique O +function O +and O +importance O +of O +the O +β5 B-protein +propeptide B-structure_element +as O +well O +as O +the O +β5 B-protein +active B-site +site I-site +for O +maturation O +and O +function O +of O +the O +eukaryotic B-taxonomy_domain +CP B-complex_assembly +. O + +On O +the O +basis O +of O +the O +numerous O +CP B-complex_assembly +: I-complex_assembly +ligand I-complex_assembly +complexes O +solved O +during O +the O +past O +18 O +years O +and O +in O +the O +current O +study O +, O +we O +provide O +a O +revised O +interpretation O +of O +proteasome B-complex_assembly +active B-site +- I-site +site I-site +architecture I-site +. O + +In O +this O +new O +view O +of O +the O +proteasomal O +active B-site +site I-site +, O +the O +positively O +charged O +Thr1NH3 B-residue_name_number ++- O +terminus O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +amide O +nitrogen O +of O +incoming O +peptide O +substrates O +and O +stabilizes O +as O +well O +as O +activates O +them O +for O +the O +endoproteolytic B-ptm +cleavage I-ptm +by O +Thr1Oγ B-residue_name_number +( O +Fig O +. O +3d O +). O + +Consistent O +with O +this O +model O +, O +the O +positively O +charged O +Thr1 B-residue_name_number +N O +terminus O +is O +engaged O +in O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +inhibitory O +compounds O +like O +fellutamide B-chemical +B I-chemical +( O +ref O +.), O +α B-chemical +- I-chemical +ketoamides I-chemical +, O +homobelactosin B-chemical +C I-chemical +( O +ref O +.) O +and O +salinosporamide B-chemical +A I-chemical +( O +ref O +.). O + +In O +agreement O +, O +acetylation B-ptm +of O +the O +Thr1 B-residue_name_number +N O +terminus O +irreversibly O +blocks O +hydrolytic O +activity O +, O +and O +binding O +of O +substrates O +is O +prevented O +for O +steric O +reasons O +. O + +Activity B-experimental_method +assays I-experimental_method +with O +the O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +revealed O +reduced O +turnover O +of O +Suc B-chemical +- I-chemical +LLVY I-chemical +- I-chemical +AMC I-chemical +. O + +The O +greater O +suitability O +of O +threonine B-residue_name +for O +the O +proteasome B-complex_assembly +active B-site +site I-site +, O +which O +has O +been O +noted O +in O +biochemical O +as O +well O +as O +in O +kinetic O +studies O +, O +constitutes O +a O +likely O +reason O +for O +the O +conservation B-protein_state +of O +the O +Thr1 B-residue_name_number +residue O +in O +all O +proteasomes B-complex_assembly +from O +bacteria B-taxonomy_domain +to O +eukaryotes B-taxonomy_domain +. O + +Conformation O +of O +proteasomal O +propeptides B-structure_element +. O + +Note O +the O +tight O +conformation O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +and O +Ala1 B-residue_name_number +before O +propeptide B-structure_element +removal O +( O +G B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +turn O +; O +cyan O +double O +arrow O +) O +compared O +with O +the O +relaxed O +, O +processed B-protein_state +WT B-protein_state +active B-site +- I-site +site I-site +Thr1 B-residue_name_number +( O +red O +double O +arrow O +). O + +( O +b O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β5 B-protein +propeptides B-structure_element +in O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +L I-mutant +- I-mutant +T1A I-mutant +, O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +T I-mutant +- I-mutant +T1A I-mutant +, O +β5 B-mutant +-( I-mutant +H I-mutant +- I-mutant +2 I-mutant +) I-mutant +A I-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +and O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +mutant B-protein_state +proteasomes B-complex_assembly +. O + +( O +d O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +matured B-protein_state +β2 B-protein +active B-site +site I-site +, O +the O +WT B-protein_state +β2 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +and O +the O +β2 B-mutant +- I-mutant +T I-mutant +(- I-mutant +2 I-mutant +) I-mutant +V I-mutant +mutant B-protein_state +propeptide B-structure_element +. O + +( O +c O +) O +Illustration O +of O +the O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +- I-evidence +density I-evidence +map I-evidence +( O +blue O +mesh O +contoured O +at O +1σ O +) O +for O +the O +β5 B-mutant +- I-mutant +T1C I-mutant +propeptide B-structure_element +fragment O +. O + +Ser1 B-residue_name_number +lacks B-protein_state +this O +stabilization O +and O +is O +therefore O +rotated O +by O +60 O +°. O + +The O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +- I-evidence +density I-evidence +maps I-evidence +( O +blue O +mesh O +) O +for O +Ser1 B-residue_name_number +( O +brown O +) O +and O +the O +covalently O +bound O +ligands O +( O +green O +; O +only O +the O +P1 B-site +site I-site +( O +Leu1 B-residue_name_number +) O +is O +shown O +) O +are O +contoured O +at O +1σ O +. O + +Crotonylation B-ptm +of O +lysine B-residue_name +residues O +( O +crotonyllysine B-residue_name +, O +Kcr B-residue_name +) O +has O +emerged O +as O +one O +of O +the O +fundamental O +histone B-protein_type +post O +- O +translational O +modifications O +( O +PTMs O +) O +found O +in O +mammalian B-taxonomy_domain +chromatin O +. O + +While O +a O +number O +of O +acetyllysine B-residue_name +readers O +have O +been O +identified O +and O +characterized O +, O +a O +specific O +reader O +of O +the O +crotonyllysine B-residue_name +mark O +remains O +unknown O +( O +reviewed O +in O +). O + +The O +acetyllysine B-residue_name +binding O +function O +of O +the O +AF9 B-protein +YEATS B-structure_element +domain I-structure_element +is O +essential O +for O +the O +recruitment O +of O +the O +histone B-protein_type +methyltransferase I-protein_type +DOT1L B-protein +to O +H3K9ac B-protein_type +- O +containing O +chromatin O +and O +for O +DOT1L B-protein +- O +mediated O +H3K79 B-protein_type +methylation B-ptm +and O +transcription O +. O + +In O +this O +study O +, O +we O +identified O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +as O +a O +reader O +of O +crotonyllysine B-residue_name +that O +binds O +to O +histone B-protein_type +H3 B-protein_type +crotonylated B-protein_state +at O +lysine B-residue_name_number +9 I-residue_name_number +( O +H3K9cr B-protein_type +) O +via O +a O +distinctive O +binding O +mechanism O +. O + +The O +planar O +crotonyl B-chemical +group O +is O +inserted O +between O +Trp81 B-residue_name_number +and O +Phe62 B-residue_name_number +of O +the O +protein O +, O +the O +aromatic O +rings O +of O +which O +are O +positioned O +strictly O +parallel O +to O +each O +other O +and O +at O +equal O +distance O +from O +the O +crotonyl B-chemical +group O +, O +yielding O +a O +novel O +aromatic O +- O +amide O +/ O +aliphatic O +- O +aromatic O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +system O +that O +, O +to O +our O +knowledge O +, O +has O +not O +been O +reported O +previously O +for O +any O +protein O +- O +protein O +interaction O +( O +Fig O +. O +1d O +and O +Supplementary O +Fig O +. O +1c O +). O + +In O +addition O +to O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +π I-bond_interaction +stacking I-bond_interaction +, O +the O +crotonyl B-chemical +group O +is O +stabilized O +by O +a O +set O +of O +hydrogen B-bond_interaction +bonds I-bond_interaction +and O +electrostatic B-bond_interaction +interactions I-bond_interaction +. O + +The O +fixed O +position O +of O +the O +Thr61 B-residue_name_number +hydroxyl O +group O +, O +which O +facilitates O +interactions O +with O +both O +the O +amide O +and O +Cα O +of O +K9cr B-ptm +, O +is O +achieved O +through O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +imidazole O +ring O +of O +His59 B-residue_name_number +. O + +Binding O +of O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +to O +H3K9cr B-protein_type +is O +robust O +. O + +Binding O +of O +H3K9cr B-protein_type +induced O +resonance B-evidence +changes I-evidence +in O +slow O +exchange O +regime O +on O +the O +NMR B-experimental_method +time O +scale O +, O +indicative O +of O +strong O +interaction O +. O + +To O +establish O +whether O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +is O +able O +to O +recognize O +other O +recently O +identified O +acyllysine B-residue_name +marks O +, O +we O +performed O +solution B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +using O +H3 B-protein_type +peptides O +acetylated B-protein_state +, O +propionylated B-protein_state +, O +butyrylated B-protein_state +, O +and O +crotonylated B-protein_state +at O +lysine B-residue_name_number +9 I-residue_name_number +( O +residues O +1 B-residue_range +– I-residue_range +20 I-residue_range +of O +H3 B-protein_type +). O + +We O +concluded O +that O +H3K9cr B-protein_type +is O +the O +preferred O +target O +of O +this O +domain O +. O + +In O +contrast O +, O +mutation B-experimental_method +of O +Val24 B-residue_name_number +, O +a O +residue O +located O +on O +another O +side O +of O +Trp81 B-residue_name_number +, O +had O +no O +effect O +on O +binding O +( O +Fig O +. O +2d O +and O +Supplementary O +Fig O +. O +5a O +, O +c O +). O + +As O +we O +previously O +showed O +the O +importance O +of O +acyllysine B-residue_name +binding O +by O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +for O +the O +DNA O +damage O +response O +and O +gene O +transcription O +, O +it O +will O +be O +essential O +in O +the O +future O +to O +define O +the O +physiological O +role O +of O +crotonyllysine B-residue_name +recognition O +and O +to O +differentiate O +the O +activities O +of O +Taf14 B-protein +that O +are O +due O +to O +binding O +to O +crotonyllysine B-residue_name +and O +acetyllysine B-residue_name +modifications O +. O + +NMR B-experimental_method +data O +was O +recorded O +using O +a O +Bruker O +800 O +MHz O +Data O +format O +PDB O +format O +text O +file O +. O + +Tom1 O +GAT B-structure_element +structural O +data O +is O +publicly O +available O +in O +the O +RCSB O +Protein O +Data O +Bank O +( O +http O +:// O +www O +. O +rscb O +. O +org O +/) O +under O +the O +accession O +number O +PDB O +: O +2n9d O + +NMR B-experimental_method +and O +refinement B-evidence +statistics I-evidence +for O +the O +Tom1 B-protein +GAT B-structure_element +domain O +. O + +PGRMC1 B-protein +binds O +to O +EGFR B-protein_type +and O +cytochromes B-protein_type +P450 I-protein_type +, O +and O +is O +known O +to O +be O +involved O +in O +cancer O +proliferation O +and O +in O +drug O +resistance O +. O + +Previous O +studies O +showed O +that O +deprivation B-protein_state +of I-protein_state +iron B-chemical +or O +haem B-chemical +suppresses O +tumourigenesis O +. O + +The O +dimer B-oligomeric_state +binds O +to O +EGFR B-protein_type +and O +cytochromes B-protein_type +P450 I-protein_type +to O +enhance O +tumour O +cell O +proliferation O +and O +chemoresistance O +. O + +However O +, O +at O +the O +interfaces B-site +of O +the O +other O +possible O +dimeric B-oligomeric_state +structures B-evidence +( O +Supplementary O +Fig O +. O +6a O +, O +chain O +A O +– O +A O +″; O +cyan O +and O +chain O +A O +– O +B O +; O +violet O +), O +no O +significant O +difference O +was O +observed O +. O + +We O +analysed O +the O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +the O +PGRMC1 B-protein +cytosolic B-structure_element +domain I-structure_element +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +) O +in O +solution O +( O +Fig O +. O +2 O +and O +Table O +2 O +). O + +The O +C129S B-mutant +mutant B-protein_state +of O +PGRMC1 B-protein +also O +interacted O +with O +endogenous B-protein_state +PGRMC1 B-protein +and O +EGFR B-protein_type +( O +Supplementary O +Fig O +. O +16 O +). O + +Similarly O +, O +EGFR B-protein_type +signaling O +was O +suppressed O +by O +treatment O +of O +HCT116 O +cells O +with O +SA B-chemical +( O +Fig O +. O +4g O +) O +or O +CORM3 B-chemical +( O +Fig O +. O +4h O +). O + +We O +examined O +the O +role O +of O +PGRMC1 B-protein +in O +metastatic O +progression O +by O +xenograft B-experimental_method +transplantation I-experimental_method +assays I-experimental_method +using O +super O +- O +immunodeficient O +NOD O +/ O +scid O +/ O +γnull O +( O +NOG O +) O +mice O +. O + +This O +effect O +was O +reversed O +by O +co B-experimental_method +- I-experimental_method +expression I-experimental_method +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +but O +not O +of O +the O +Y113F B-mutant +mutant B-protein_state +, O +suggesting O +that O +PGRMC1 B-protein +enhances O +doxorubicin B-chemical +resistance O +of O +cancer O +cells O +by O +facilitating O +its O +degradation O +via O +cytochromes B-protein_type +P450 I-protein_type +. O + +In O +the O +current O +study O +, O +the O +Y113 B-residue_name_number +residue O +plays O +a O +crucial O +role O +for O +the O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +and O +resultant O +regulation O +of O +cancer O +proliferation O +and O +chemoresistance O +( O +Figs O +5c O +and O +6e O +). O + +Recently O +, O +Peluso O +et O +al O +. O +reported O +that O +PGRMC1 B-protein +binds O +to O +PGRMC2 B-protein +, O +suggesting O +that O +MAPR B-protein_type +family O +members O +may O +also O +undergo O +haem B-chemical +- O +mediated O +heterodimerization O +. O + +While O +the O +effects O +of O +structural O +diversity O +of O +CYP B-protein_type +family O +proteins O +and O +interactions O +with O +different O +xenobiotic O +substrates O +should O +further O +be O +examined O +, O +the O +current O +results O +suggest O +that O +the O +interaction O +of O +drug O +- O +metabolizing O +CYPs B-protein_type +with O +the O +haem B-chemical +- O +mediated O +dimer B-oligomeric_state +of O +PGRMC1 B-protein +plays O +a O +crucial O +role O +in O +regulating O +their O +activities O +. O + +Considering O +microenvironments O +in O +and O +around O +malignant O +tumours O +, O +the O +haem B-chemical +concentration O +in O +cancer O +cells O +is O +likely O +to O +be O +elevated O +through O +multiple O +mechanisms O +, O +such O +as O +( O +i O +) O +an O +increased O +intake O +of O +haem B-chemical +, O +( O +ii O +) O +mutation O +of O +enzymes O +in O +TCA O +cycle O +( O +for O +example O +, O +fumarate B-protein_type +hydratase I-protein_type +) O +that O +increases O +the O +level O +of O +succinyl B-chemical +CoA I-chemical +, O +a O +substrate O +for O +haem B-chemical +biosynthesis O +and O +( O +iii O +) O +metastasis O +to O +haem B-chemical +- O +rich O +organs O +such O +as O +liver O +, O +brain O +and O +bone O +marrow O +. O + +Furthermore O +, O +Sigma B-protein +- I-protein +2 I-protein +ligand O +- O +binding O +is O +decreased O +in O +transgenic O +amyloid O +beta O +deposition O +model O +APP O +/ O +PS1 O +female O +mice O +. O + +These O +results O +suggest O +a O +possible O +involvement O +of O +PGRMC1 B-protein +in O +Alzheimer O +' O +s O +disease O +. O + +Comparison O +of O +PGRMC1 B-protein +( O +blue O +) O +and O +cytochrome B-protein_type +b5 I-protein_type +( O +yellow O +, O +ID O +: O +3NER O +). O +( O +c O +) O +PGRMC1 B-protein +has O +a O +longer O +helix B-structure_element +( O +a O +. O +a O +. O +147 B-residue_range +– I-residue_range +163 I-residue_range +), O +which O +is O +shifted O +away O +from O +the O +haem B-chemical +( O +arrow O +). O + +SV B-experimental_method +- I-experimental_method +AUC I-experimental_method +experiments O +were O +performed O +with O +1 O +. O +5 O +mg O +ml O +− O +1 O +of O +PGRMC1 B-protein +proteins O +. O + +The O +major O +peak O +with O +sedimentation B-evidence +coefficient I-evidence +S20 B-evidence +, I-evidence +w I-evidence +of O +1 O +. O +9 O +∼ O +2 O +. O +0 O +S O +( O +monomer B-oligomeric_state +) O +or O +3 O +. O +1 O +S O +( O +dimer B-oligomeric_state +) O +was O +detected O +. O + +( O +b O +) O +Close O +- O +up O +view O +of O +haem B-bond_interaction +stacking I-bond_interaction +. O + +( O +a O +) O +FLAG O +- O +PGRMC1 B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +( O +wt B-protein_state +) O +and O +Y113F B-mutant +mutant B-protein_state +proteins O +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +), O +in O +either O +apo B-protein_state +- O +or O +haem B-protein_state +- I-protein_state +bound I-protein_state +form O +, O +were O +incubated B-experimental_method +with O +purified O +EGFR B-protein_type +and O +co B-experimental_method +- I-experimental_method +immunoprecipitated I-experimental_method +with O +anti O +- O +FLAG O +antibody O +- O +conjugated O +beads O +. O + +of O +10 O +separate O +experiments O +. O +* B-evidence +P I-evidence +< O +0 O +. O +05 O +using O +unpaired O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +- I-experimental_method +test I-experimental_method +. O + +CO B-chemical +interferes O +with O +the O +stacking B-bond_interaction +interactions I-bond_interaction +of O +the O +haems B-chemical +and O +thereby O +inhibits O +PGRMC1 B-protein +functions O +. O + +Recent O +data O +supports O +the O +notion O +that O +, O +to O +perform O +this O +role O +, O +the O +highly B-protein_state +variable I-protein_state +αβ B-complex_assembly +T I-complex_assembly +cell I-complex_assembly +antigen I-complex_assembly +receptor I-complex_assembly +( O +TCR B-complex_assembly +) O +must O +be O +able O +to O +recognize O +thousands O +, O +if O +not O +millions O +, O +of O +different O +peptide O +ligands O +. O + +We O +recently O +reported O +that O +the O +1E6 O +human B-species +CD8 O ++ O +T O +cell O +clone O +— O +which O +mediates O +the O +destruction O +of O +β O +cells O +through O +the O +recognition O +of O +a O +major O +, O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +– O +restricted O +, O +preproinsulin B-protein +signal B-structure_element +peptide I-structure_element +( O +ALWGPDPAAA15 B-chemical +– I-chemical +24 I-chemical +) O +— O +can O +recognize O +upwards O +of O +1 O +million O +different O +peptides O +. O + +From O +this O +large O +functional O +scan O +, O +we O +selected O +7 O +different O +APLs B-chemical +that O +activated O +the O +1E6 O +T O +cell O +clone O +across O +a O +wide O +( O +4 O +- O +log O +) O +functional O +range O +( O +Table O +1 O +). O + +As O +with O +the O +3D B-evidence +affinity I-evidence +measurements O +, O +the O +2D B-evidence +affinity I-evidence +measurements O +correlated O +well O +with O +the O +EC50 B-evidence +values O +for O +each O +ligand O +( O +Figure O +2K O +) O +demonstrating O +a O +strong O +correlation O +( O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence += O +0 O +. O +8 O +, O +P B-evidence += O +0 O +. O +01 O +) O +between O +T O +cell O +antigen O +sensitivity O +and O +TCR B-evidence +binding I-evidence +affinity I-evidence +. O + +The O +low O +number O +of O +contacts O +between O +the O +2 O +molecules O +most O +likely O +contributed O +to O +the O +weak O +binding B-evidence +affinity I-evidence +of O +the O +interaction O +. O + +In O +order O +to O +examine O +the O +mechanism O +by O +which O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +engaged O +a O +wide O +range O +of O +peptides O +with O +divergent O +binding B-evidence +affinities I-evidence +, O +we O +solved B-experimental_method +the O +structure B-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +in B-protein_state +complex I-protein_state +with I-protein_state +all O +7 O +APLs B-chemical +used O +in O +Figure O +2 O +. O + +The O +relatively O +broad O +range O +of O +buried O +surface O +areas O +( O +1 O +, O +670 O +– O +1 O +, O +920 O +Å2 O +) O +did O +not O +correlate O +well O +with O +TCR B-evidence +binding I-evidence +affinity I-evidence +( O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence += O +0 O +. O +45 O +, O +P B-evidence += O +0 O +. O +2 O +). O + +Although O +the O +number O +of O +peptide O +contacts O +was O +a O +good O +predictor O +of O +TCR B-evidence +binding I-evidence +affinity I-evidence +for O +some O +of O +the O +APLs B-chemical +, O +for O +others O +, O +the O +correlation O +was O +poor O +( O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence += O +0 O +. O +045 O +, O +P O += O +0 O +. O +92 O +), O +possibly O +because O +of O +different O +resolutions O +for O +each O +complex O +structure B-evidence +. O + +The O +stronger O +ligands O +all O +encoded O +larger O +side O +chains O +( O +Arg B-residue_name +or O +Tyr B-residue_name +) O +at O +peptide O +position O +1 B-residue_number +( O +Figure O +5 O +, O +E O +– O +H O +), O +enabling O +interactions O +with O +1E6 O +that O +were O +not O +present O +in O +the O +weaker O +APLs B-chemical +that O +lacked O +large O +side O +chains O +in O +this O +position O +( O +Figure O +5 O +, O +A O +, O +C O +, O +and O +D O +). O + +These O +data O +demonstrated O +that O +the O +unligated B-protein_state +structure B-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +was O +virtually O +identical O +to O +its O +ligated B-protein_state +counterparts O +. O + +The O +unligated B-protein_state +structures B-evidence +of O +A2 B-chemical +- I-chemical +AQWGPDAAA I-chemical +, O +A2 B-chemical +- I-chemical +RQWGPDPAAV I-chemical +, O +A2 B-chemical +- I-chemical +YQFGPDFPIA I-chemical +, O +and O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +were O +virtually O +identical O +when O +in B-protein_state +complex I-protein_state +with I-protein_state +1E6 O +( O +Figure O +6 O +, O +D O +and O +F O +– O +H O +). O + +Peptide O +modifications O +alter O +the O +interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +the O +MHC B-site +surface I-site +. O + +An O +energetic O +switch O +from O +unfavorable O +to O +favorable O +entropy B-evidence +( O +order O +- O +to O +- O +disorder O +) O +correlates O +with O +antigen O +potency O +. O + +The O +weak O +binding B-evidence +affinity I-evidence +between O +1E6 O +and O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +and O +A2 B-chemical +- I-chemical +YLGGPDFPTI I-chemical +generated O +thermodynamic O +data O +that O +were O +not O +robust O +enough O +to O +gain O +insight O +into O +the O +enthalpic B-evidence +( O +ΔH B-evidence +°) I-evidence +and O +entropic B-evidence +( O +TΔS B-evidence +°) I-evidence +changes O +that O +contributed O +to O +the O +different O +binding B-evidence +affinities I-evidence +/ O +potencies O +for O +each O +APL B-chemical +. O + +Flexibility O +at O +the O +interface B-site +between O +the O +TCR B-complex_assembly +and O +pMHC B-complex_assembly +, O +demonstrated O +in O +various O +studies O +, O +has O +been O +suggested O +as O +a O +mechanism O +mediating O +T O +cell O +cross O +- O +reactivity O +with O +multiple O +distinct O +epitopes O +. O + +This O +motif O +was O +conserved B-protein_state +in O +at O +least O +2 O +potential O +foreign O +peptides O +, O +originating O +from O +Herpes B-species +simplex I-species +virus I-species +and O +Pseudomonas B-species +aeruginosa I-species +, O +enabling O +TCR B-complex_assembly +recognition O +of O +foreign O +epitopes O +. O + +Second O +, O +molecular O +studies O +have O +not O +yet O +revealed O +a O +broad O +set O +of O +rules O +that O +determine O +TCR B-complex_assembly +cross O +- O +reactivity O +because O +, O +with O +the O +exception O +of O +the O +allo B-protein_state +– O +TCR B-complex_assembly +- I-complex_assembly +MHC I-complex_assembly +pair O +of O +the O +42F3 B-protein +TCR B-complex_assembly +and O +H2 B-protein +- I-protein +Ld I-protein +that O +did O +not O +encounter O +each O +other O +during O +T O +cell O +development O +, O +studies O +have O +been O +limited O +to O +structures B-evidence +of O +a O +TCR B-complex_assembly +with O +only O +2 O +or O +3 O +different O +ligands O +. O + +Although O +the O +1E6 O +T O +cell O +was O +able O +to O +activate O +weakly O +with O +peptides O +that O +lacked B-protein_state +this O +motif O +, O +we O +were O +unable O +to O +robustly O +measure O +binding B-evidence +affinities I-evidence +or O +generate O +complex O +structures B-evidence +with O +these O +ligands O +, O +highlighting O +the O +central O +role O +of O +this O +interaction O +during O +1E6 O +T O +cell O +antigen O +recognition O +. O + +The O +binding O +mechanism O +utilized O +by O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +during O +pMHC B-complex_assembly +recognition O +is O +consistent O +with O +both O +of O +these O +models O +. O + +Combined O +with O +evidence O +demonstrating O +that O +aromatic O +side O +chains O +are O +conserved O +in O +the O +CDR2 B-structure_element +loops I-structure_element +of O +TCRs B-complex_assembly +from O +many O +species O +, O +we O +speculate O +that O +these O +aromatic O +residues O +could O +impart O +a O +level O +of O +“ O +stickiness O +” O +to O +TCRs B-complex_assembly +, O +which O +might O +be O +enriched O +in O +an O +autoimmune O +setting O +when O +the O +TCR B-complex_assembly +often O +binds O +in O +a O +nonoptimal O +fashion O +. O + +Early O +thermodynamic B-experimental_method +analysis I-experimental_method +of O +TCR B-complex_assembly +- I-complex_assembly +pMHC I-complex_assembly +interactions O +suggested O +a O +common O +energetic O +signature O +, O +driven O +by O +favorable O +enthalpy B-evidence +( O +generally O +mediated O +through O +an O +increase O +in O +electrostatic O +interactions O +) O +and O +unfavorable O +entropy B-evidence +( O +changes O +from O +disorder O +to O +order O +). O + +This O +analysis O +demonstrated O +a O +strong O +relationship O +( O +according O +to O +the O +Pearson B-experimental_method +’ I-experimental_method +s I-experimental_method +correlation I-experimental_method +analysis I-experimental_method +) O +between O +the O +energetic O +signature O +used O +by O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +the O +sensitivity O +of O +the O +1E6 O +T O +cell O +clone O +to O +different O +APLs B-chemical +. O + +These O +differences O +were O +consistent O +with O +a O +greater O +degree O +of O +movement O +between O +the O +unligated B-protein_state +and O +ligated B-protein_state +pMHCs B-complex_assembly +for O +the O +weaker O +ligands O +, O +suggesting O +a O +greater O +requirement O +for O +disorder O +- O +to O +- O +order O +changes O +during O +TCR B-complex_assembly +binding O +. O + +Importantly O +, O +the O +preproinsulin B-protein +- O +derived O +epitope O +was O +one O +of O +the O +least O +potent O +peptides O +, O +demonstrating O +that O +the O +1E6 O +T O +cell O +clone O +had O +the O +ability O +to O +respond O +to O +different O +peptide O +sequences O +with O +far O +greater O +potency O +. O + +Finally O +, O +TCR B-complex_assembly +ligand O +discrimination O +was O +characterized O +by O +an O +energetic O +shift O +from O +an O +enthalpically O +to O +entropically O +driven O +interaction O +. O + +( O +C O +) O +The O +1E6 O +T O +cell O +clone O +was O +stained O +, O +in O +duplicate O +, O +with O +tetramers B-oligomeric_state +composed O +of O +each O +APL B-chemical +( O +colored O +as O +above O +) O +presented O +by O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +. O +( O +D O +) O +The O +stability O +of O +each O +APL B-chemical +( O +colored O +as O +above O +) O +was O +tested O +, O +in O +duplicate O +, O +using O +CD B-experimental_method +by O +recording O +the O +peak O +at O +218 O +nm O +absorbance O +from O +5 O +° O +C O +– O +90 O +° O +C O +. O + +( O +A O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +MVWGPDPLYV I-complex_assembly +( O +approximate O +value O +); O +( O +B O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +YLGGPDFPTI I-complex_assembly +( O +approximate O +value O +); O +( O +C O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +ALWGPDPAAA I-complex_assembly +; O +( O +D O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +AQWGPDPAAA I-complex_assembly +; O +( O +E O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQFGPDWIVA I-complex_assembly +; O +( O +F O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQWGPDPAAV I-complex_assembly +; O +( O +G O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +YQFGPDFPTA I-complex_assembly +; O +and O +( O +H O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQFGPDFPTI I-complex_assembly +. O +( O +I O +) O +ΔG B-evidence +values I-evidence +, O +calculated O +from O +SPR B-experimental_method +experiments O +, O +plotted O +against O +1 O +/ O +EC50 B-evidence +( O +the O +reciprocal O +peptide O +concentration O +required O +to O +reach O +half O +- O +maximal O +1E6 O +T O +cell O +killing O +) O +showing O +Pearson B-experimental_method +’ I-experimental_method +s I-experimental_method +coefficient I-experimental_method +analysis I-experimental_method +( O +r B-evidence +) O +and O +P B-evidence +value O +( O +including O +approximate O +values O +from O +A O +and O +B O +). O + +( O +A O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +black O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +( O +black O +illustration O +and O +sticks O +). O +( O +B O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +red O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +YLGGPDFPTI I-chemical +( O +red O +illustration O +and O +sticks O +). O +( O +C O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +blue O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +( O +blue O +illustration O +and O +sticks O +) O +reproduced O +from O +previous O +published O +data O +. O + +Superposition B-experimental_method +of O +each O +APL B-chemical +in O +unligated B-protein_state +form O +and O +ligated B-protein_state +to O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +. O + +Interactions O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +the O +MHC B-complex_assembly +α1 B-structure_element +helix I-structure_element +residues O +Arg65 B-residue_name_number +, O +Lys66 B-residue_name_number +, O +and O +Gln72 B-residue_name_number +. O + +Hydrogen B-bond_interaction +bonds I-bond_interaction +are O +shown O +as O +red O +dotted O +lines O +; O +vdW B-bond_interaction +contacts O +are O +shown O +as O +black O +dotted O +lines O +. O + +Further O +dimerization O +contacts O +involve O +switch B-site +II I-site +, O +the O +G4 B-structure_element +helix I-structure_element +and O +the O +trans B-structure_element +stabilizing I-structure_element +loop I-structure_element +. O + +Extensive O +biochemical B-experimental_method +studies I-experimental_method +suggested O +that O +GTP B-chemical +- O +induced O +oligomerization O +of O +Irga6 B-protein +requires O +an O +interface B-site +in O +the O +GTPase B-structure_element +domain I-structure_element +across O +the O +nucleotide B-site +- I-site +binding I-site +site I-site +. O + +Since O +the O +signal B-protein_type +recognition I-protein_type +particle I-protein_type +GTPase I-protein_type +and O +its O +homologous O +receptor B-protein_type +( O +called O +FfH B-protein +and O +FtsY B-protein +in O +bacteria B-taxonomy_domain +) O +also O +employ O +the O +3 O +'- O +OH O +ribose O +group O +to O +dimerize B-oligomeric_state +in O +an O +anti B-protein_state +- I-protein_state +parallel I-protein_state +orientation O +therefore O +activating O +its O +GTPase B-protein_type +, O +an O +analogous O +dimerization O +model O +was O +proposed O +for O +Irga6 B-protein +. O + +The O +structure B-evidence +revealed O +that O +Irga6 B-protein +can O +dimerize B-oligomeric_state +via O +the O +G B-site +interface I-site +in O +a O +parallel B-protein_state +head I-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +head I-protein_state +fashion O +. O + +Our O +data O +suggest O +that O +a O +parallel B-protein_state +dimerization O +mode O +may O +be O +a O +unifying O +feature O +in O +all O +dynamin B-protein_type +and O +septin B-protein_type +superfamily O +proteins O +. O + +Crystals B-evidence +diffracted O +to O +3 O +. O +2 O +Å O +resolution O +and O +displayed O +one O +exceptionally O +long O +unit O +cell O +axis O +of O +1289 O +Å O +( O +Additional O +file O +1 O +: O +Table O +S1 O +). O + +The O +structure B-evidence +was O +solved O +by O +molecular B-experimental_method +replacement I-experimental_method +and O +refined O +to O +Rwork B-evidence +/ O +Rfree B-evidence +of O +29 O +. O +7 O +%/ O +31 O +. O +7 O +% O +( O +Additional O +file O +1 O +: O +Table O +S2 O +). O + +Secondary O +structure O +was O +numbered O +according O +to O +ref O +.. O +c O +Top O +view O +on O +the O +GTPase B-structure_element +domain I-structure_element +dimer B-oligomeric_state +. O + +The O +structures B-evidence +of O +the O +seven O +molecules O +also O +agree O +well O +with O +the O +previously O +determined O +structure B-evidence +of O +native B-protein_state +GMPPNP B-protein_state +- I-protein_state +bound I-protein_state +Irga6 B-protein +( O +PDB O +: O +1TQ6 O +; O +rmsd B-evidence +of O +1 O +. O +00 O +- O +1 O +. O +13 O +Å O +over O +all O +Cα O +atoms O +). O + +This O +indicates O +that O +the O +introduced O +mutations B-experimental_method +in O +the O +secondary B-site +patch I-site +, O +from O +which O +only O +Lys176 B-residue_name_number +is O +part O +of O +the O +backside B-site +interface I-site +, O +do O +, O +in O +fact O +, O +not O +prevent O +this O +interaction O +. O + +Strikingly O +, O +molecule O +A B-structure_element +of O +one O +asymmetric O +unit O +assembled O +with O +an O +equivalent O +molecule O +of O +the O +adjacent O +asymmetric O +unit O +via O +the O +G B-site +- I-site +interface I-site +in O +a O +symmetric O +parallel B-protein_state +fashion O +via O +a O +470 O +Å2 O +interface O +. O + +Contact B-site +site I-site +II I-site +features O +polar B-bond_interaction +and I-bond_interaction +hydrophobic I-bond_interaction +interactions I-bond_interaction +formed O +by O +switch B-site +I I-site +( O +V104 B-residue_name_number +, O +V107 B-residue_name_number +) O +with O +a O +helix B-structure_element +following O +the O +guanine B-structure_element +specificity I-structure_element +motif I-structure_element +( O +G4 B-structure_element +helix I-structure_element +, O +K184 B-residue_name_number +and O +S187 B-residue_name_number +) O +and O +the O +trans B-structure_element +stabilizing I-structure_element +loop I-structure_element +( O +T158 B-residue_name_number +) O +of O +the O +opposing O +GTPase B-structure_element +domain I-structure_element +. O + +The O +GTPase B-structure_element +domains I-structure_element +of O +the O +left O +molecules O +are O +shown O +in O +orange O +, O +helical B-structure_element +domains I-structure_element +or O +extensions O +in O +blue O +. O + +Our O +structural B-experimental_method +analysis I-experimental_method +of O +an O +oligomerization B-protein_state +- I-protein_state +and I-protein_state +GTPase I-protein_state +- I-protein_state +defective I-protein_state +Irga6 B-protein +mutant B-protein_state +indicates O +that O +Irga6 B-protein +dimerizes B-oligomeric_state +via O +the O +G B-site +interface I-site +in O +a O +parallel B-protein_state +orientation O +. O + +In O +the O +crystals B-evidence +, O +dimerization O +via O +the O +G B-site +interface I-site +is O +promoted O +by O +the O +high O +protein O +concentrations O +which O +may O +mimic O +a O +situation O +when O +Irga6 B-protein +oligomerizes O +on O +a O +membrane O +surface O +. O + +Irga6 B-protein +bears O +Gly79 B-residue_name_number +at O +this O +position O +, O +which O +in O +the O +dimerizing B-oligomeric_state +molecule O +A B-structure_element +appears O +to O +approach O +the O +bridging O +imido O +group O +of O +GMPPNP B-chemical +via O +a O +main O +chain O +hydrogen B-bond_interaction +bond I-bond_interaction +. O + +For O +Irga6 B-protein +, O +additional O +interfaces B-site +in O +the O +helical B-structure_element +domain I-structure_element +are O +presumably O +involved O +in O +oligomerization O +, O +such O +as O +the O +secondary B-site +patch I-site +residues O +whose O +mutation B-experimental_method +prevented O +oligomerization O +in O +the O +crystallized B-evidence +mutant B-protein_state +. O + diff --git a/annotation_IOB/test.tsv b/annotation_IOB/test.tsv new file mode 100644 index 0000000000000000000000000000000000000000..eb6bc804382bdd58c5d9062238c4532d5ae41f1a --- /dev/null +++ b/annotation_IOB/test.tsv @@ -0,0 +1,40231 @@ +The O +Bacteroidetes B-taxonomy_domain +are O +dominant O +bacteria B-taxonomy_domain +in O +the O +human B-species +gut O +that O +are O +responsible O +for O +the O +digestion O +of O +the O +complex B-chemical +polysaccharides I-chemical +that O +constitute O +“ O +dietary O +fiber O +.” O +Although O +this O +symbiotic O +relationship O +has O +been O +appreciated O +for O +decades O +, O +little O +is O +currently O +known O +about O +how O +Bacteroidetes B-taxonomy_domain +seek O +out O +and O +bind O +plant B-taxonomy_domain +cell O +wall O +polysaccharides B-chemical +as O +a O +necessary O +first O +step O +in O +their O +metabolism O +. O + +Here O +, O +we O +provide O +the O +first O +biochemical B-experimental_method +, I-experimental_method +crystallographic I-experimental_method +, I-experimental_method +and I-experimental_method +genetic I-experimental_method +insight I-experimental_method +into O +how O +two O +surface B-protein_type +glycan I-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +from O +the O +complex O +Bacteroides B-species +ovatus I-species +xyloglucan B-gene +utilization I-gene +locus I-gene +( O +XyGUL B-gene +) O +enable O +recognition O +and O +uptake O +of O +this O +ubiquitous O +vegetable B-taxonomy_domain +polysaccharide B-chemical +. O + +More O +importantly O +, O +this O +makes O +diet O +a O +tractable O +way O +to O +manipulate O +the O +abundance O +and O +metabolic O +output O +of O +the O +microbiota B-taxonomy_domain +toward O +improved O +human B-species +health O +. O + +The O +archetypal O +PUL B-gene +- O +encoded O +system O +is O +the O +starch B-complex_assembly +utilization I-complex_assembly +system I-complex_assembly +( O +Sus B-complex_assembly +) O +( O +Fig O +. O +1B O +) O +of O +Bacteroides B-species +thetaiotaomicron I-species +. O + +The O +location O +of O +SGBP B-protein +- I-protein +A I-protein +/ O +B B-protein +is O +presented O +in O +this O +work O +; O +the O +location O +of O +GH5 B-protein +has O +been O +empirically O +determined O +, O +and O +the O +enzymes O +have O +been O +placed O +based O +upon O +their O +predicted O +cellular O +location O +. O + +We O +recently O +reported O +the O +detailed O +molecular O +characterization O +of O +a O +PUL B-gene +that O +confers O +the O +ability O +of O +the O +human B-species +gut O +commensal O +B B-species +. I-species +ovatus I-species +ATCC I-species +8483 I-species +to O +grow O +on O +a O +prominent O +family O +of O +plant B-taxonomy_domain +cell O +wall O +glycans B-chemical +, O +the O +xyloglucans B-chemical +( O +XyG B-chemical +). O + +As O +the O +Sus B-complex_assembly +SGBPs B-protein_type +remain O +the O +only O +structurally O +characterized O +cohort O +to O +date O +, O +we O +therefore O +wondered O +whether O +such O +glycan B-chemical +binding O +and O +function O +are O +extended O +to O +other O +PUL B-gene +that O +target O +more O +complex O +and O +heterogeneous O +polysaccharides B-chemical +, O +such O +as O +XyG B-chemical +. O + +These O +data O +extend O +our O +current O +understanding O +of O +the O +Sus O +- O +like O +glycan B-chemical +uptake O +paradigm O +within O +the O +Bacteroidetes B-taxonomy_domain +and O +reveals O +how O +the O +complex O +dietary O +polysaccharide B-chemical +xyloglucan B-chemical +is O +recognized O +at O +the O +cell O +surface O +. O + +Similarly O +, O +SGBP B-protein +- I-protein +B I-protein +also O +bound B-protein_state +to I-protein_state +XyG B-chemical +and O +XyGO2 B-chemical +with O +approximately O +equal O +affinities B-evidence +, O +although O +in O +both O +cases O +, O +Ka B-evidence +values O +were O +nearly O +10 O +- O +fold O +lower O +than O +those O +for O +SGBP B-protein +- I-protein +A I-protein +. O +Also O +in O +contrast O +to O +SGBP B-protein +- I-protein +A I-protein +, O +SGBP B-protein +- I-protein +B I-protein +also O +bound B-protein_state +to I-protein_state +XyGO1 B-chemical +, O +yet O +the O +affinity B-evidence +for O +this O +minimal B-structure_element +repeating I-structure_element +unit I-structure_element +was O +poor O +, O +with O +a O +Ka B-evidence +value O +of O +ca O +. O +1 O +order O +of O +magnitude O +lower O +than O +for O +XyG B-chemical +and O +XyGO2 B-chemical +. O + +As O +anticipated O +by O +sequence O +similarity O +, O +the O +high O +- O +resolution O +tertiary O +structure B-evidence +of O +apo B-protein_state +- O +SGBP B-protein +- I-protein +A I-protein +( O +1 O +. O +36 O +Å O +, O +Rwork B-evidence += O +14 O +. O +7 O +%, O +Rfree B-evidence += O +17 O +. O +4 O +%, O +residues O +28 B-residue_range +to I-residue_range +546 I-residue_range +) O +( O +Table O +2 O +) O +displays O +the O +canonical O +“ B-structure_element +SusD I-structure_element +- I-structure_element +like I-structure_element +” I-structure_element +protein I-structure_element +fold I-structure_element +dominated O +by O +four O +tetratrico B-structure_element +- I-structure_element +peptide I-structure_element +repeat I-structure_element +( O +TPR B-structure_element +) O +motifs O +that O +cradle O +the O +rest O +of O +the O +structure B-evidence +( O +Fig O +. O +4A O +). O + +Cocrystallization B-experimental_method +of O +SGBP B-protein +- I-protein +A I-protein +with O +XyGO2 B-chemical +generated O +a O +substrate B-complex_assembly +complex I-complex_assembly +structure B-evidence +( O +2 O +. O +3 O +Å O +, O +Rwork B-evidence += O +21 O +. O +8 O +%, O +Rfree B-evidence += O +24 O +. O +8 O +%, O +residues O +36 B-residue_range +to I-residue_range +546 I-residue_range +) O +( O +Fig O +. O +4A O +and O +B O +; O +Table O +2 O +) O +that O +revealed O +the O +distinct O +binding B-site +- I-site +site I-site +architecture O +of O +the O +XyG B-protein_type +binding I-protein_type +protein I-protein_type +. O + +Seven O +of O +the O +eight O +backbone O +glucosyl B-chemical +residues O +of O +XyGO2 B-chemical +could O +be O +convincingly O +modeled O +in O +the O +ligand B-evidence +electron I-evidence +density I-evidence +, O +and O +only O +two O +α B-chemical +( I-chemical +1 I-chemical +→ I-chemical +6 I-chemical +)- I-chemical +linked I-chemical +xylosyl I-chemical +residues O +were O +observed O +( O +Fig O +. O +4B O +; O +cf O +. O + +The O +functional O +importance O +of O +this O +platform B-site +is O +underscored O +by O +the O +observation O +that O +the O +W82A B-mutant +W283A B-mutant +W306A B-mutant +mutant B-protein_state +of O +SGBP B-protein +- I-protein +A I-protein +, O +designated O +SGBP B-mutant +- I-mutant +A I-mutant +*, I-mutant +is O +completely B-protein_state +devoid I-protein_state +of I-protein_state +XyG I-protein_state +affinity I-protein_state +( O +Table O +3 O +; O +see O +Fig O +. O +S4 O +in O +the O +supplemental O +material O +). O + +Protein O +name O +Ka B-evidence +ΔG B-evidence +( O +kcal O +⋅ O +mol O +− O +1 O +) O +ΔH B-evidence +( O +kcal O +⋅ O +mol O +− O +1 O +) O +TΔS B-evidence +( O +kcal O +⋅ O +mol O +− O +1 O +) O +Fold O +changeb O +M O +− O +1 O +SGBP B-protein +- I-protein +A I-protein +( O +W82A B-mutant +W283A B-mutant +W306A B-mutant +) O +ND O +NB O +NB O +NB O +NB O +SGBP B-protein +- I-protein +A I-protein +( O +W82A B-mutant +) O +c O +4 O +. O +9 O +9 O +. O +1 O +× O +104 O +− O +6 O +. O +8 O +− O +6 O +. O +3 O +0 O +. O +5 O +SGBP B-protein +- I-protein +A I-protein +( O +W306 B-residue_name_number +) O +ND O +NB O +NB O +NB O +NB O +SGBP B-protein +- I-protein +B I-protein +( O +230 B-residue_range +– I-residue_range +489 I-residue_range +) O +0 O +. O +7 O +( O +8 O +. O +6 O +± O +0 O +. O +20 O +) O +× O +104 O +− O +6 O +. O +7 O +− O +14 O +. O +9 O +± O +0 O +. O +1 O +− O +8 O +. O +2 O +SGBP B-protein +- I-protein +B I-protein +( O +Y363A B-mutant +) O +19 O +. O +7 O +( O +2 O +. O +9 O +± O +0 O +. O +10 O +) O +× O +103 O +− O +4 O +. O +7 O +− O +18 O +. O +1 O +± O +0 O +. O +1 O +− O +13 O +. O +3 O +SGBP B-protein +- I-protein +B I-protein +( O +W364A B-mutant +) O +ND O +Weak O +Weak O +Weak O +Weak O +SGBP B-protein +- I-protein +B I-protein +( O +F414A B-mutant +) O +3 O +. O +2 O +( O +1 O +. O +80 O +± O +0 O +. O +03 O +) O +× O +104 O +− O +5 O +. O +8 O +− O +11 O +. O +4 O +± O +0 O +. O +1 O +− O +5 O +. O +6 O + +Binding O +thermodynamics O +are O +based O +on O +the O +concentration O +of O +the O +binding O +unit O +, O +XyGO2 B-chemical +. O + +Domains O +A B-structure_element +, O +B B-structure_element +, O +and O +C B-structure_element +display O +similar O +β B-structure_element +- I-structure_element +sandwich I-structure_element +folds I-structure_element +; O +domains O +B B-structure_element +( O +residues O +134 B-residue_range +to I-residue_range +230 I-residue_range +) O +and O +C B-structure_element +( O +residues O +231 B-residue_range +to I-residue_range +313 I-residue_range +) O +can O +be O +superimposed B-experimental_method +onto O +domain O +A B-structure_element +( O +residues O +34 B-residue_range +to I-residue_range +133 I-residue_range +) O +with O +RMSDs B-evidence +of O +1 O +. O +1 O +and O +1 O +. O +2 O +Å O +, O +respectively O +, O +for O +47 O +atom O +pairs O +( O +23 O +% O +and O +16 O +% O +sequence O +identity O +, O +respectively O +). O + +While O +there O +is O +no O +substrate O +- O +complexed O +structure O +of O +Bacova_04391 B-protein +available O +, O +the O +binding B-site +site I-site +is O +predicted O +to O +include O +W241 B-residue_name_number +and O +Y404 B-residue_name_number +, O +which O +are O +proximal O +to O +the O +XyGO B-site +binding I-site +site I-site +in O +SGBP B-protein +- I-protein +B I-protein +. O +However O +, O +the O +opposing B-protein_state +, I-protein_state +clamp I-protein_state +- I-protein_state +like I-protein_state +arrangement I-protein_state +of O +these B-structure_element +residues I-structure_element +in O +Bacova_04391 B-protein +is O +clearly O +distinct O +from O +the O +planar B-site +surface I-site +arrangement I-site +of O +the O +residues B-structure_element +that O +interact O +with O +XyG B-chemical +in O +SGBP B-protein +- I-protein +B I-protein +( O +described O +below O +). O + +Inspection O +of O +the O +tertiary O +structure B-evidence +indicates O +that O +domains O +C B-structure_element +and O +D B-structure_element +are O +effectively O +inseparable O +, O +with O +a O +contact O +interface O +of O +396 O +Å2 O +. O + +Despite O +the O +lack O +of O +sequence O +and O +structural O +conservation O +, O +a O +similarly O +positioned O +proline B-residue_name +joins O +the O +Ig B-structure_element +- I-structure_element +like I-structure_element +domains I-structure_element +of O +the O +xylan O +- O +binding O +Bacova_04391 B-protein +and O +the O +starch B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +SusE B-protein +and O +SusF B-protein +. O +We O +speculate O +that O +this O +is O +a O +biologically O +important O +adaptation O +that O +serves O +to O +project O +the O +glycan B-site +binding I-site +site I-site +of O +these O +proteins O +far O +from O +the O +membrane O +surface O +. O + +In O +these O +growth B-experimental_method +experiments I-experimental_method +, O +overnight O +cultures O +of O +strains O +grown O +on O +minimal O +medium O +plus O +glucose B-chemical +were O +back O +- O +diluted O +1 O +: O +100 O +- O +fold O +into O +minimal O +medium O +containing O +5 O +mg O +/ O +ml O +of O +the O +reported O +carbohydrate B-chemical +. O + +Complementation B-experimental_method +of O +the O +ΔSGBP B-mutant +- I-mutant +A I-mutant +strain O +( O +ΔSGBP B-mutant +- I-mutant +A I-mutant +:: O +SGBP B-protein +- I-protein +A I-protein +) O +restores O +growth O +to O +wild B-protein_state +- I-protein_state +type I-protein_state +rates O +on O +xyloglucan B-chemical +and O +XyGO1 B-chemical +, O +yet O +the O +calculated O +rate O +of O +the O +complemented O +strain O +is O +~ O +72 O +% O +that O +of O +the O +WT B-protein_state +Δtdk B-mutant +strain O +on O +XyGO2 B-chemical +; O +similar O +results O +were O +obtained O +for O +the O +SGBP B-protein +- I-protein +B I-protein +complemented O +strain O +despite O +the O +fact O +that O +the O +growth O +curves O +do O +not O +appear O +much O +different O +( O +see O +Fig O +. O +S8C O +and O +F O +). O + +Growth O +was O +measured O +over O +time O +in O +minimal O +medium O +containing O +( O +A O +) O +XyG B-chemical +, O +( O +B O +) O +XyGO2 B-chemical +, O +( O +C O +) O +XyGO1 B-chemical +, O +( O +D O +) O +glucose B-chemical +, O +and O +( O +E O +) O +xylose B-chemical +. O + +In O +panel O +F O +, O +the O +growth O +rate O +of O +each O +strain O +on O +the O +five O +carbon O +sources O +is O +displayed O +, O +and O +in O +panel O +G O +, O +the O +normalized O +lag B-evidence +time I-evidence +of O +each O +culture O +, O +relative O +to O +its O +growth O +on O +glucose B-chemical +, O +is O +displayed O +. O + +Intriguingly O +, O +the O +ΔSGBP B-mutant +- I-mutant +B I-mutant +strain O +( O +ΔBacova_02650 B-mutant +) O +( O +cf O +. O + +Fig O +. O +1B O +) O +exhibited O +a O +minor O +growth O +defect O +on O +both O +XyG B-chemical +and O +XyGO2 B-chemical +, O +with O +rates O +84 O +. O +6 O +% O +and O +93 O +. O +9 O +% O +that O +of O +the O +WT B-protein_state +Δtdk B-mutant +strain O +. O + +However O +, O +growth O +of O +the O +ΔSGBP B-mutant +- I-mutant +B I-mutant +strain O +on O +XyGO1 B-chemical +was O +54 O +. O +2 O +% O +the O +rate O +of O +the O +parental O +strain O +, O +despite O +the O +fact O +that O +SGBP B-protein +- I-protein +B I-protein +binds O +this O +substrate O +ca O +. O + +Taken O +together O +, O +the O +data O +indicate O +that O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +functionally O +complement O +each O +other O +in O +the O +capture O +of O +XyG B-chemical +polysaccharide B-chemical +, O +while O +SGBP B-protein +- I-protein +B I-protein +may O +allow O +B B-species +. I-species +ovatus I-species +to O +scavenge O +smaller O +XyGOs B-chemical +liberated O +by O +other O +gut O +commensals O +. O + +It O +may O +then O +be O +that O +only O +after O +a O +sufficient O +amount O +of O +glycan B-chemical +is O +processed O +and O +imported O +by O +the O +cell O +is O +XyGUL B-gene +upregulated O +and O +exponential O +growth O +on O +the O +glycan B-chemical +can O +begin O +. O + +Likewise O +, O +such O +cognate O +interactions O +between O +homologous O +protein O +pairs O +such O +as O +SGBP B-protein +- I-protein +A I-protein +and O +its O +TBDT B-protein_type +may O +underlie O +our O +observation O +that O +a O +ΔSGBP B-mutant +- I-mutant +A I-mutant +mutant B-protein_state +cannot O +grow O +on O +xyloglucan B-chemical +. O + +Thus O +, O +understanding O +glycan B-chemical +capture O +at O +the O +cell O +surface O +is O +fundamental O +to O +explaining O +, O +and O +eventually O +predicting O +, O +how O +the O +carbohydrate O +content O +of O +the O +diet O +shapes O +the O +gut O +community O +structure O +as O +well O +as O +its O +causative O +health O +effects O +. O + +PUL B-gene +- O +encoded O +TBDTs B-protein_type +in O +Bacteroidetes B-taxonomy_domain +are O +larger O +than O +the O +well O +- O +characterized O +iron B-protein_type +- I-protein_type +targeting I-protein_type +TBDTs I-protein_type +from O +many O +Proteobacteria B-taxonomy_domain +and O +are O +further O +distinguished O +as O +the O +only O +known O +glycan B-protein_type +- I-protein_type +importing I-protein_type +TBDTs I-protein_type +coexpressed O +with O +an O +SGBP B-protein_type +. O + +Our O +observation O +here O +that O +the O +physical O +presence O +of O +the O +SusD B-protein +homolog O +SGBP B-protein +- I-protein +A I-protein +, O +independent O +of O +XyG B-chemical +- O +binding O +ability O +, O +is O +both O +necessary O +and O +sufficient O +for O +XyG B-chemical +utilization O +further O +supports O +a O +model O +of O +glycan B-chemical +import O +whereby O +the O +SusC B-protein_type +- I-protein_type +like I-protein_type +TBDTs I-protein_type +and O +the O +SusD B-protein_type +- I-protein_type +like I-protein_type +SGBPs I-protein_type +must O +be O +intimately O +associated O +to O +support O +glycan B-chemical +uptake O +( O +Fig O +. O +1C O +). O + +A O +molecular O +understanding O +of O +glycan B-chemical +uptake O +by O +human B-species +gut O +bacteria B-taxonomy_domain +is O +therefore O +central O +to O +the O +development O +of O +strategies O +to O +improve O +human B-species +health O +through O +manipulation O +of O +the O +microbiota B-taxonomy_domain +. O + +A O +high B-chemical +affinity I-chemical +IL I-chemical +- I-chemical +17A I-chemical +peptide I-chemical +antagonist I-chemical +( O +HAP B-chemical +) O +of O +15 B-residue_range +residues I-residue_range +was O +identified O +through O +phage B-experimental_method +- I-experimental_method +display I-experimental_method +screening I-experimental_method +followed O +by O +saturation B-experimental_method +mutagenesis I-experimental_method +optimization I-experimental_method +and O +amino B-experimental_method +acid I-experimental_method +substitutions I-experimental_method +. O + +The O +family O +of O +IL B-protein_type +- I-protein_type +17 I-protein_type +cytokines I-protein_type +and O +receptors O +consists O +of O +six O +polypeptides O +, O +IL B-protein +- I-protein +17A I-protein +- I-protein +F I-protein +, O +and O +five O +receptors O +, O +IL B-protein +- I-protein +17RA I-protein +- I-protein +E I-protein +. O +IL B-protein +- I-protein +17A I-protein +is O +secreted O +from O +activated O +Th17 O +cells O +, O +and O +several O +innate O +immune O +T O +cell O +types O +including O +macrophages O +, O +neutrophils O +, O +natural O +killer O +cells O +, O +and O +dendritic O +cells O +. O + +There O +has O +been O +active O +research O +in O +identifying O +orally O +available O +chemical O +entities O +that O +would O +functionally O +antagonize O +IL B-protein +- I-protein +17A I-protein +- O +mediated O +signaling O +. O + +Since O +IL B-protein +- I-protein +17RA I-protein +is O +a O +shared O +receptor B-protein_type +for O +at O +least O +IL B-protein +- I-protein +17A I-protein +, O +IL B-protein +- I-protein +17F I-protein +, O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17F I-complex_assembly +and O +IL B-protein +- I-protein +17E I-protein +, O +we O +chose O +to O +seek O +IL B-protein +- I-protein +17A I-protein +- O +specific O +inhibitors O +that O +may O +have O +more O +defined O +pharmacological O +responses O +than O +IL B-protein +- I-protein +17RA I-protein +inhibitors O +. O + +Positive B-experimental_method +phage I-experimental_method +pools I-experimental_method +were O +then O +sub B-experimental_method +- I-experimental_method +cloned I-experimental_method +into O +a O +maltose B-experimental_method +- I-experimental_method +binding I-experimental_method +protein I-experimental_method +( I-experimental_method +MBP I-experimental_method +) I-experimental_method +fusion I-experimental_method +system I-experimental_method +. O + +Sequences O +identified O +from O +phage B-experimental_method +clones I-experimental_method +were O +chemically B-experimental_method +synthesized I-experimental_method +( O +Supplementary O +Table O +1 O +) O +and O +tested O +for O +inhibition O +of O +IL B-protein +- I-protein +17A I-protein +binding O +to O +IL B-protein +- I-protein +17RA I-protein +( O +Table O +1 O +). O + +In O +particular O +, O +at O +position O +5 B-residue_number +( O +13 B-chemical +), O +substitution B-experimental_method +of O +methionine B-residue_name +with O +alanine B-residue_name +resulted O +in O +a O +seven O +fold O +improvement O +in O +potency O +( O +80 O +nM O +versus O +11 O +nM O +respectively O +). O + +Since O +the O +replacement B-experimental_method +of O +methionine B-residue_name +at O +position O +5 B-residue_number +with O +alanine B-residue_name +was O +beneficial O +, O +the O +additional O +hydrophobic O +amino O +acids O +isoleucine B-residue_name +( O +24 B-chemical +), O +leucine B-residue_name +( O +25 B-chemical +) O +and O +valine B-residue_name +( O +26 B-chemical +) O +were O +evaluated O +and O +an O +additional O +two O +- O +three O +fold O +improvement O +in O +binding O +was O +observed O +for O +the O +valine B-residue_name +and O +isoleucine B-residue_name +replacements B-experimental_method +in O +comparison O +with O +alanine B-residue_name +. O + +Dimerization O +of O +HAP B-chemical +can O +further O +increase O +its O +potency O + +Orthogonal O +assays O +to O +confirm O +HAP B-chemical +antagonism O + +The O +relatively O +high O +IC50 B-evidence +values O +in O +this O +assay O +( O +Table O +3 O +) O +are O +probably O +due O +to O +the O +high O +IL B-protein +- I-protein +17A I-protein +concentration O +( O +100 O +ng O +/ O +ml O +) O +needed O +for O +detection O +of O +IL B-protein_type +- I-protein_type +6 I-protein_type +. O + +Crystallization B-experimental_method +and I-experimental_method +structure I-experimental_method +determination I-experimental_method + +Crystals B-evidence +of O +the O +Fab B-complex_assembly +/ I-complex_assembly +truncated I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O +diffracted O +to O +2 O +. O +2 O +Å O +, O +and O +the O +Fab B-complex_assembly +/ I-complex_assembly +full I-complex_assembly +length I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O +diffracted O +to O +3 O +. O +0 O +Å O +( O +Supplementary O +Table O +S3 O +). O + +Two O +copies O +of O +HAP B-chemical +bind O +to O +the O +N O +- O +terminal O +of O +the O +cytokine B-protein_type +dimer B-oligomeric_state +, O +also O +symmetrically O +, O +and O +each O +HAP B-chemical +molecule O +also O +interacts O +with O +both O +IL B-protein +- I-protein +17A I-protein +monomers B-oligomeric_state +( O +Fig O +. O +2 O +). O + +Inhibition O +mechanism O +of O +IL B-protein +- I-protein +17A I-protein +signaling O +by O +HAP B-chemical + +Structure O +basis O +for O +the O +observed O +SAR B-experimental_method +of O +peptides O + +The O +C O +- O +terminal O +Asn14 B-residue_name_number +and O +Lys15 B-residue_name_number +of O +HAP B-chemical +are O +not O +directly O +involved O +in O +interactions O +with O +IL B-protein +- I-protein +17A I-protein +, O +and O +this O +is O +reflected O +in O +the O +gradual O +reduction O +in O +activity O +caused O +by O +C O +- O +terminal O +truncations B-experimental_method +( O +35 B-chemical +and O +36 B-chemical +, O +Table O +2 O +). O + +For O +example O +, O +inspection O +of O +the O +published O +IL B-protein +- I-protein +17F I-protein +crystal B-evidence +structure I-evidence +( O +PDB O +code O +1JPY O +) O +revealed O +a O +pocket B-site +of O +IL B-protein +- I-protein +17F I-protein +similar O +to O +that O +of O +IL B-protein +- I-protein +17A I-protein +for O +W12 B-residue_name_number +of O +HAP B-chemical +binding O +, O +but O +it O +is O +occupied O +by O +a O +Phe B-structure_element +- I-structure_element +Phe I-structure_element +motif I-structure_element +at O +the O +N O +- O +terminal O +peptide O +of O +IL B-protein +- I-protein +17F I-protein +. O + +We O +have O +also O +determined B-experimental_method +the O +complex B-evidence +structure I-evidence +of O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +, O +which O +provides O +the O +structural O +basis O +for O +HAP B-chemical +’ O +s O +antagonism O +to O +IL B-protein +- I-protein +17A I-protein +signaling O +. O + +Since O +apo B-protein_state +IL B-protein +- I-protein +17A I-protein +is O +a O +homodimer B-oligomeric_state +with O +2 O +fold O +symmetry O +, O +IL B-protein +- I-protein +17RA I-protein +potentially O +can O +bind O +to O +either O +face O +of O +the O +IL B-protein +- I-protein +17A I-protein +dimer B-oligomeric_state +. O + +The O +interaction O +of O +IL B-protein +- I-protein +17A I-protein +with O +IL B-protein +- I-protein +17RA I-protein +has O +an O +extensive O +interface B-site +, O +covering O +~ O +2 O +, O +200 O +Å2 O +surface O +area O +of O +IL B-protein +- I-protein +17A I-protein +. O + +One O +way O +of O +further O +improving O +HAP B-chemical +’ O +s O +potency O +is O +by O +dimerization O +. O + +KD B-evidence +determined O +by O +the O +standard O +equation O +, O +KD B-evidence += O +kd B-evidence +/ O +ka B-evidence +. O +( O +B O +) O +HAP B-chemical +inhibits O +SPR B-experimental_method +signaling O +of O +IL B-protein +- I-protein +17A I-protein +binding O +to O +immobilized B-protein_state +IL B-protein +- I-protein +17RA I-protein +. O + +Overall O +structure B-evidence +of O +the O +Fab B-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O +in O +ribbon O +presentation O +. O + +( O +C O +) O +As O +a O +comparison O +, O +the O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +complex O +was O +shown O +with O +IL B-protein +- I-protein +17A I-protein +in O +the O +same O +orientation O +. O + +ELISA B-experimental_method +competition I-experimental_method +activity I-experimental_method +of O +peptide O +analogues O +of O +1 O +. O + +The O +amount O +of O +NadA B-protein +on O +the O +bacterial B-taxonomy_domain +surface O +is O +of O +direct O +relevance O +in O +the O +constant O +battle O +of O +host O +- O +pathogen O +interactions O +: O +it O +influences O +the O +ability O +of O +the O +pathogen O +to O +engage O +human B-species +cell O +surface O +- O +exposed O +receptors O +and O +, O +conversely O +, O +the O +bacterial B-taxonomy_domain +susceptibility O +to O +the O +antibody O +- O +mediated O +immune O +response O +. O + +NadR B-protein +also O +mediates O +ligand O +- O +dependent O +regulation O +of O +many O +other O +meningococcal B-taxonomy_domain +genes O +, O +for O +example O +the O +highly O +- O +conserved O +multiple O +adhesin O +family O +( O +maf O +) O +genes O +, O +which O +encode O +proteins O +emerging O +with O +important O +roles O +in O +host O +- O +pathogen O +interactions O +, O +immune O +evasion O +and O +niche O +adaptation O +. O + +The O +abundance O +of O +surface O +- O +exposed O +NadA B-protein +is O +regulated O +by O +the O +ligand B-protein_type +- I-protein_type +responsive I-protein_type +transcriptional I-protein_type +repressor I-protein_type +NadR B-protein +. O +Here O +, O +we O +present O +functional B-evidence +, I-evidence +biochemical I-evidence +and I-evidence +high I-evidence +- I-evidence +resolution I-evidence +structural I-evidence +data I-evidence +on O +NadR B-protein +. O +Our O +studies O +provide O +detailed O +insights O +into O +how O +small O +molecule O +ligands O +, O +such O +as O +hydroxyphenylacetate B-chemical +derivatives O +, O +found O +in O +relevant O +host O +niches O +, O +modulate O +the O +structure O +and O +activity O +of O +NadR B-protein +, O +by O +‘ O +conformational O +selection O +’ O +of O +inactive B-protein_state +forms O +. O + +The O +DNA O +- O +binding O +activity O +of O +NadR B-protein +is O +attenuated O +in O +vitro O +upon O +addition O +of O +various O +hydroxyphenylacetate B-chemical +( O +HPA B-chemical +) O +derivatives O +, O +including O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +Moreover O +, O +these O +findings O +are O +important O +because O +the O +activity O +of O +NadR B-protein +impacts O +the O +potential O +coverage O +provided O +by O +anti O +- O +NadA B-protein +antibodies O +elicited O +by O +the O +Bexsero O +vaccine O +and O +influences O +host O +- O +bacteria B-taxonomy_domain +interactions O +that O +contribute O +to O +meningococcal B-taxonomy_domain +pathogenesis O +. O + +In O +analytical B-experimental_method +size I-experimental_method +- I-experimental_method +exclusion I-experimental_method +high I-experimental_method +- I-experimental_method +performance I-experimental_method +liquid I-experimental_method +chromatography I-experimental_method +( O +SE B-experimental_method +- I-experimental_method +HPLC I-experimental_method +) O +experiments O +coupled O +with O +multi B-experimental_method +- I-experimental_method +angle I-experimental_method +laser I-experimental_method +light I-experimental_method +scattering I-experimental_method +( O +MALLS B-experimental_method +), O +NadR B-protein +presented O +a O +single O +species O +with O +an O +absolute O +molecular O +mass O +of O +35 O +kDa O +( O +S1 O +Fig O +). O + +( O +A O +) O +Molecular O +structures O +of O +3 B-chemical +- I-chemical +HPA I-chemical +( O +MW O +152 O +. O +2 O +), O +4 B-chemical +- I-chemical +HPA I-chemical +( O +MW O +152 O +. O +2 O +), O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +( O +MW O +186 O +. O +6 O +) O +and O +salicylic B-chemical +acid I-chemical +( O +MW O +160 O +. O +1 O +). O +( O +B O +) O +DSC B-experimental_method +profiles B-evidence +, O +colored O +as O +follows O +: O +apo B-protein_state +- O +NadR B-protein +( O +violet O +), O +NadR B-complex_assembly ++ I-complex_assembly +salicylate I-complex_assembly +( O +red O +), O +NadR B-complex_assembly ++ I-complex_assembly +3 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +( O +green O +), O +NadR B-complex_assembly ++ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +( O +blue O +), O +NadR B-complex_assembly ++ I-complex_assembly +3Cl I-complex_assembly +, I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +( O +pink O +). O + +All O +DSC B-experimental_method +profiles B-evidence +are O +representative O +of O +triplicate O +experiments O +. O + +However O +, O +steady B-experimental_method +- I-experimental_method +state I-experimental_method +SPR I-experimental_method +analyses O +of O +the O +NadR B-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +interactions O +allowed O +determination O +of O +the O +equilibrium B-evidence +dissociation I-evidence +constants I-evidence +( O +KD B-evidence +) O +( O +Table O +1 O +and O +S2 O +Fig O +). O + +The O +interactions O +of O +4 B-chemical +- I-chemical +HPA I-chemical +and O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +with O +NadR B-protein +exhibited O +KD B-evidence +values O +of O +1 O +. O +5 O +mM O +and O +1 O +. O +1 O +mM O +, O +respectively O +. O + +To O +fully O +characterize O +the O +NadR B-protein +/ O +HPA B-chemical +interactions O +, O +we O +sought O +to O +determine O +crystal B-evidence +structures I-evidence +of O +NadR B-protein +in O +ligand B-protein_state +- I-protein_state +bound I-protein_state +( O +holo B-protein_state +) O +and O +ligand B-protein_state +- I-protein_state +free I-protein_state +( O +apo B-protein_state +) O +forms O +. O + +The O +map B-evidence +is O +contoured O +at O +1σ O +and O +the O +figure O +was O +prepared O +with O +a O +density B-evidence +mesh I-evidence +carve O +factor O +of O +1 O +. O +7 O +, O +using O +Pymol O +( O +www O +. O +pymol O +. O +org O +). O + +Only O +the O +mutation O +L130K B-mutant +has O +a O +noteworthy O +effect O +on O +the O +oligomeric O +state O +, O +inducing O +a O +second O +peak O +with O +a O +longer O +retention O +time O +and O +a O +second O +peak O +maximum O +at O +18 O +. O +6 O +min O +. O + +To O +a O +much O +lesser O +extent O +, O +the O +L133K B-mutant +mutation O +also O +appears O +to O +induce O +a O +‘ O +shoulder O +’ O +to O +the O +main O +peak O +, O +suggesting O +very O +weak O +ability O +to O +disrupt O +the O +dimer B-oligomeric_state +. O +( O +D O +) O +SE B-experimental_method +- I-experimental_method +HPLC I-experimental_method +/ I-experimental_method +MALLS I-experimental_method +analyses O +of O +the O +L130K B-mutant +mutant B-protein_state +, O +shows O +20 O +% O +dimer B-oligomeric_state +and O +80 O +% O +monomer B-oligomeric_state +. O + +The O +ligand O +showed O +a O +different O +position O +and O +orientation O +compared O +to O +salicylate B-chemical +complexed B-protein_state +with I-protein_state +MTH313 B-protein +and O +ST1710 B-protein +( O +see O +Discussion O +). O + +At O +the O +other O +‘ O +end O +’ O +of O +the O +ligand O +, O +the O +4 O +- O +hydroxyl O +group O +was O +proximal O +to O +AspB36 B-residue_name_number +, O +with O +which O +it O +may O +establish O +an O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +( O +see O +bond O +distances O +in O +Table O +3 O +). O + +The O +water B-chemical +molecule O +observed O +in O +the O +pocket O +was O +bound O +by O +the O +carboxylate O +group O +and O +the O +side O +chains O +of O +SerA9 B-residue_name_number +and O +AsnA11 B-residue_name_number +. O + +In O +addition O +to O +the O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +involving O +the O +carboxylate O +and O +hydroxyl O +groups O +of O +4 B-chemical +- I-chemical +HPA I-chemical +, O +binding O +of O +the O +phenyl O +moiety O +appeared O +to O +be O +stabilized O +by O +several O +van B-bond_interaction +der I-bond_interaction +Waals I-bond_interaction +’ I-bond_interaction +contacts I-bond_interaction +, O +particularly O +those O +involving O +the O +hydrophobic O +side O +chain O +atoms O +of O +LeuB21 B-residue_name_number +, O +MetB22 B-residue_name_number +, O +PheB25 B-residue_name_number +, O +LeuB29 B-residue_name_number +and O +ValB111 B-residue_name_number +( O +Fig O +4A O +). O + +The O +presence O +of O +a O +single O +hydroxyl O +group O +at O +position O +2 O +, O +as O +in O +2 B-chemical +- I-chemical +HPA I-chemical +, O +rather O +than O +at O +position O +4 O +, O +would O +eliminate O +the O +possibility O +of O +favorable O +interactions O +with O +AspB36 B-residue_name_number +, O +resulting O +in O +the O +lack O +of O +NadR B-protein +regulation O +by O +2 B-chemical +- I-chemical +HPA I-chemical +described O +previously O +. O + +Firstly O +, O +NadR B-protein +is O +expected O +to O +be O +covalently O +immobilized O +on O +the O +sensor O +chip O +as O +a O +dimer B-oligomeric_state +in O +random O +orientations O +, O +since O +it O +is O +a O +stable B-protein_state +dimer B-oligomeric_state +in O +solution O +and O +has O +sixteen O +lysines B-residue_name +well O +- O +distributed O +around O +its O +surface O +, O +all O +able O +to O +act O +as O +potential O +sites O +for O +amine O +coupling O +to O +the O +chip O +, O +and O +none O +of O +which O +are O +close O +to O +the O +ligand B-site +- I-site +binding I-site +pocket I-site +. O + +Secondly O +, O +the O +HPA B-chemical +analytes O +are O +all O +very O +small O +( O +MW O +150 O +– O +170 O +, O +Fig O +1A O +) O +and O +therefore O +are O +expected O +to O +be O +able O +to O +diffuse O +readily O +into O +all O +potential O +binding B-site +sites I-site +, O +irrespective O +of O +the O +random O +orientations O +of O +the O +immobilized O +NadR B-protein +dimers B-oligomeric_state +on O +the O +chip O +. O + +The O +crystallographic B-evidence +data I-evidence +, O +supported O +by O +the O +SPR B-experimental_method +studies O +of O +binding B-evidence +stoichiometry I-evidence +, O +revealed O +the O +lack O +of O +a O +second O +4 B-chemical +- I-chemical +HPA I-chemical +molecule O +in O +the O +homodimer B-oligomeric_state +, O +suggesting O +negative O +co O +- O +operativity O +, O +a O +phenomenon O +previously O +described O +for O +the O +MTH313 B-protein +/ O +salicylate B-chemical +interaction O +and O +for O +other O +MarR B-protein_type +family O +proteins O +. O + +However O +, O +since O +residues O +of O +helix B-structure_element +α6 B-structure_element +were O +not O +directly O +involved O +in O +ligand O +binding O +, O +an O +explanation O +for O +the O +lack O +of O +4 B-chemical +- I-chemical +HPA I-chemical +in O +monomer B-oligomeric_state +A B-structure_element +did O +not O +emerge O +by O +analyzing O +only O +these O +backbone O +atom O +positions O +, O +suggesting O +that O +a O +more O +complex O +series O +of O +allosteric O +events O +may O +occur O +. O + +Specifically O +, O +upon O +analysis O +with O +the O +CASTp B-experimental_method +software O +, O +the O +pocket B-site +in O +chain B-structure_element +B I-structure_element +containing O +the O +4 B-chemical +- I-chemical +HPA I-chemical +exhibited O +a O +total O +volume O +of O +approximately O +370 O +Å3 O +, O +while O +the O +pocket B-site +in O +chain B-structure_element +A I-structure_element +was O +occupied O +by O +these O +three O +side O +chains O +that O +adopted O +‘ O +inward B-protein_state +’ O +positions O +and O +thereby O +divided O +the O +space O +into O +a O +few O +much O +smaller O +pockets O +, O +each O +with O +volume O +< O +50 O +Å3 O +, O +evidently O +rendering O +chain B-structure_element +A I-structure_element +unfavorable O +for O +ligand O +binding O +. O + +Although O +more O +comprehensive O +NMR B-experimental_method +experiments O +and O +full O +chemical O +shift O +assignment O +of O +the O +spectra B-evidence +would O +be O +required O +to O +precisely O +define O +this O +multi O +- O +state O +behavior O +, O +the O +NMR B-experimental_method +data O +clearly O +demonstrate O +that O +NadR B-protein +exhibits O +conformational O +flexibility O +which O +is O +modulated O +by O +4 B-chemical +- I-chemical +HPA I-chemical +in O +solution O +. O + +( O +A O +) O +The O +holo B-protein_state +- O +homodimer B-oligomeric_state +structure B-evidence +is O +shown O +as O +green O +and O +blue O +cartoons O +, O +for O +chain B-structure_element +A I-structure_element +and I-structure_element +B I-structure_element +, O +respectively O +, O +while O +the O +two O +homodimers B-oligomeric_state +of O +apo B-protein_state +- O +NadR B-protein +are O +both O +cyan O +and O +pale O +blue O +for O +chains O +A B-structure_element +/ I-structure_element +C I-structure_element +and O +B B-structure_element +/ I-structure_element +D I-structure_element +, O +respectively O +. O + +The O +three O +homodimers B-oligomeric_state +( O +chains O +AB B-structure_element +holo B-protein_state +, O +AB B-structure_element +apo B-protein_state +, O +and O +CD B-structure_element +apo B-protein_state +) O +were O +overlaid B-experimental_method +by O +structural B-experimental_method +alignment I-experimental_method +exclusively O +of O +all O +heavy O +atoms O +in O +residues O +R64 B-residue_range +- I-residue_range +A77 I-residue_range +( O +shown O +in O +red O +, O +with O +side O +chain O +sticks O +) O +of O +chains O +A B-structure_element +holo B-protein_state +, O +A B-structure_element +apo B-protein_state +, O +and O +C B-structure_element +apo B-protein_state +, O +belonging O +to O +helix B-structure_element +α4 B-structure_element +( O +left O +). O + +Thus O +, O +the O +apo B-protein_state +- O +homodimer B-oligomeric_state +AB B-structure_element +presented O +the O +DNA B-structure_element +- I-structure_element +binding I-structure_element +helices I-structure_element +in O +a O +conformation O +similar O +to O +that O +observed O +in O +the O +protein O +: O +DNA O +complex O +of O +OhrR B-complex_assembly +: I-complex_assembly +ohrA I-complex_assembly +from O +Bacillus B-species +subtilis I-species +( O +Fig O +8C O +). O + +This O +mutagenesis B-experimental_method +data O +revealed O +that O +NadR B-protein +residues O +His7 B-residue_name_number +, O +Ser9 B-residue_name_number +, O +Asn11 B-residue_name_number +and O +Phe25 B-residue_name_number +play O +key O +roles O +in O +the O +ligand O +- O +mediated O +regulation O +of O +NadR B-protein +; O +they O +are O +each O +involved O +in O +the O +controlled O +de O +- O +repression O +of O +the O +nadA B-gene +promoter O +and O +synthesis O +of O +NadA B-protein +in O +response O +to O +4 B-chemical +- I-chemical +HPA I-chemical +in O +vivo O +. O + +Given O +the O +importance O +of O +NadR B-protein +- O +mediated O +regulation O +of O +NadA B-protein +levels O +in O +the O +contexts O +of O +meningococcal B-taxonomy_domain +pathogenesis O +, O +we O +sought O +to O +characterize O +NadR B-protein +, O +and O +its O +interaction O +with O +ligands O +, O +at O +atomic O +resolution O +. O + +While O +some O +flexibility O +of O +helix B-structure_element +α4 B-structure_element +was O +also O +observed O +in O +the O +two O +apo B-protein_state +- O +structures B-evidence +, O +concomitant O +changes O +in O +the O +dimer B-site +interfaces I-site +were O +not O +observed O +, O +possibly O +due O +to O +the O +absence B-protein_state +of I-protein_state +ligand I-protein_state +. O + +The O +latter O +may O +influence O +the O +surface O +abundance O +or O +secretion O +of O +maf O +proteins O +, O +an O +emerging O +class O +of O +highly B-protein_state +conserved I-protein_state +meningococcal B-taxonomy_domain +putative O +adhesins O +and O +toxins O +with O +many O +important O +roles O +. O + +Further O +work O +is O +required O +to O +investigate O +how O +the O +two O +different O +promoter O +types O +influence O +the O +ligand O +- O +responsiveness O +of O +NadR B-protein +during O +bacterial B-taxonomy_domain +infection O +and O +may O +provide O +insights O +into O +the O +regulatory O +mechanisms O +occurring O +during O +these O +host O +- O +pathogen O +interactions O +. O + +Structure O +of O +an O +OhrR O +- O +ohrA B-gene +operator O +complex O +reveals O +the O +DNA O +binding O +mechanism O +of O +the O +MarR O +family O + +Structural O +determinant O +for O +inducing O +RORgamma B-protein +specific O +inverse O +agonism O +triggered O +by O +a O +synthetic O +benzoxazinone B-chemical +ligand O + +Our O +goal O +was O +to O +develop O +a O +RORγ B-protein +specific O +inverse B-protein_state +agonist I-protein_state +that O +would O +help O +down O +regulate O +pro O +- O +inflammatory O +gene O +transcription O +by O +disrupting O +the O +protein O +protein O +interaction O +with O +coactivator O +proteins O +as O +a O +therapeutic O +agent O +. O + +Using O +an O +in B-experimental_method +vivo I-experimental_method +reporter I-experimental_method +assay I-experimental_method +, O +we O +show O +that O +the O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +displayed O +specificity O +for O +RORγ B-protein +over O +ROR B-protein_type +sub O +- O +family O +members O +α B-protein +and O +β B-protein +. O + +The O +synthetic O +benzoxazinone B-chemical +ligands O +identified O +in O +our O +FRET B-experimental_method +assay I-experimental_method +have O +an O +agonist B-protein_state +( O +BIO592 B-chemical +) O +or O +inverse B-protein_state +agonist I-protein_state +( O +BIO399 B-chemical +) O +effect O +by O +stabilizing O +or O +destabilizing O +the O +agonist B-protein_state +conformation O +of O +RORγ B-protein +. O + +Our O +structural B-experimental_method +investigation I-experimental_method +of O +the O +BIO592 B-chemical +agonist B-protein_state +and O +BIO399 B-chemical +inverse B-protein_state +agonist I-protein_state +structures B-evidence +identified O +residue O +Met358 B-residue_name_number +on O +RORγ B-protein +as O +the O +trigger O +for O +RORγ B-protein +specific O +inverse O +agonism O +. O + +Retinoid B-protein +- I-protein +related I-protein +orphan I-protein +receptor I-protein +gamma I-protein +( O +RORγ B-protein +) O +is O +a O +transcription B-protein_type +factor I-protein_type +belonging O +to O +a O +sub O +- O +family O +of O +nuclear B-protein_type +receptors I-protein_type +that O +includes O +two O +closely O +related O +members O +RORα B-protein +and O +RORβ B-protein +. O + +Here O +we O +present O +the O +identification O +of O +two O +synthetic O +benzoxazinone B-chemical +RORγ B-protein +ligands O +, O +a O +weak O +agonist B-protein_state +BIO592 B-chemical +( O +Fig O +. O +1a O +) O +and O +an O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +( O +Fig O +. O +1b O +) O +which O +were O +identified O +using O +a O +Fluorescence B-experimental_method +Resonance I-experimental_method +Energy I-experimental_method +transfer I-experimental_method +( I-experimental_method +FRET I-experimental_method +) I-experimental_method +based I-experimental_method +assay I-experimental_method +that O +monitored O +coactivator O +peptide O +recruitment O +. O + +Using O +partial B-experimental_method +proteolysis I-experimental_method +in O +combination O +with O +mass B-experimental_method +spectrometry I-experimental_method +analysis O +we O +demonstrate O +that O +the O +AF2 B-structure_element +helix I-structure_element +of O +RORγ B-protein +destabilizes O +upon O +BIO399 B-chemical +( O +inverse B-protein_state +agonist I-protein_state +) O +binding O +. O + +Using O +a O +FRET B-experimental_method +based I-experimental_method +assay I-experimental_method +we O +discovered O +agonist B-protein_state +BIO592 B-chemical +( O +Fig O +. O +1a O +) O +which O +increased O +the O +coactivator O +peptide O +TRAP220 B-chemical +recruitment O +to O +RORγ B-protein +( O +EC50 B-evidence +0f O +58nM O +and O +Emax B-evidence +of O +130 O +%) O +and O +a O +potent O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +( O +Fig O +. O +1b O +) O +which O +inhibited O +coactivator O +recruitment O +( O +IC50 B-evidence +: O +4 O +. O +7nM O +). O + +c O +EBI96 B-chemical +coactivator O +peptide O +bound B-protein_state +in I-protein_state +the O +coactivator B-site +pocket I-site +of O +RORγ B-protein + +Specific O +proteolytic O +positions O +on O +RORγ518 B-protein +when O +treated B-experimental_method +with I-experimental_method +Actinase B-protein +E I-protein +alone O +( O +Green O +) O +or O +in O +the O +presence B-protein_state +of I-protein_state +BIO399 B-chemical +( O +Red O +) O +and O +shared O +proteolytic B-site +sites I-site +( O +Yellow O +) O + +Several O +rounds O +of O +cocrystallization B-experimental_method +attempts O +with O +RORγ518 B-protein +or O +other O +RORγ B-protein +AF2 B-structure_element +helix I-structure_element +containing O +constructs O +complexed B-protein_state +with I-protein_state +BIO399 B-chemical +had O +not O +produced O +crystals B-evidence +. O + +We O +reasoned O +that O +if O +we O +could O +remove O +the O +unfolded B-protein_state +AF2 B-structure_element +helix I-structure_element +using O +proteolysis B-experimental_method +we O +could O +produce O +a O +binary O +complex O +more O +amenable O +to O +crystallization B-experimental_method +. O + +The O +aeRORγ493 B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +BIO399 I-complex_assembly +structure B-evidence +diverged O +at O +the O +c O +- O +terminal O +end O +of O +Helix B-structure_element +11 I-structure_element +from O +the O +RORγ518 B-complex_assembly +BIO592 I-complex_assembly +EBI96 I-complex_assembly +structure B-evidence +, O +where O +helix B-structure_element +11 I-structure_element +unwinds O +into O +a O +random O +coil O +after O +residue O +L475 B-residue_name_number +. O + +BIO399 B-chemical +and O +Inverse B-protein_state +agonist I-protein_state +T0901317 B-chemical +bind O +in O +a O +collapsed B-protein_state +conformation O +distinct O +from O +other O +RORγ B-protein +Inverse O +Agonists O +Cocrystal B-evidence +structures I-evidence + +However O +, O +the O +inverse O +agonism O +trigger O +of O +BIO399 B-chemical +, O +residue O +Met358 B-residue_name_number +, O +is O +a O +leucine B-residue_name +in O +both O +RORα B-protein +and O +β B-protein +. O + +The O +Structural O +Basis O +of O +Coenzyme B-chemical +A I-chemical +Recycling O +in O +a O +Bacterial B-taxonomy_domain +Organelle O + +The O +majority O +of O +catabolic B-protein_state +BMCs B-complex_assembly +( O +metabolosomes B-complex_assembly +) O +compartmentalize O +a O +common O +core O +of O +enzymes O +to O +metabolize O +compounds O +via O +a O +toxic O +and O +/ O +or O +volatile O +aldehyde B-chemical +intermediate O +. O + +Accordingly O +, O +PduL B-protein_type +and O +Pta B-protein_type +exemplify O +functional O +, O +but O +not O +structural O +, O +convergent O +evolution O +. O + +This O +enzyme O +, O +PduL B-protein_type +, O +is O +exclusively B-protein_state +associated O +with O +organelles O +called O +bacterial B-taxonomy_domain +microcompartments B-complex_assembly +, O +which O +are O +used O +to O +catabolize O +various O +compounds O +. O + +The O +aldehyde B-chemical +is O +subsequently O +converted O +into O +an O +acyl B-chemical +- I-chemical +CoA I-chemical +by O +aldehyde B-protein_type +dehydrogenase I-protein_type +, O +which O +uses O +NAD B-chemical ++ I-chemical +and O +CoA B-chemical +as O +cofactors O +. O + +NAD B-chemical ++ I-chemical +is O +recycled O +via O +alcohol B-protein_type +dehydrogenase I-protein_type +, O +and O +CoA B-chemical +is O +recycled O +via O +phosphotransacetylase B-protein_type +( O +PTAC B-protein_type +) O +( O +Fig O +1 O +). O + +They O +can O +also O +work O +in O +the O +reverse O +direction O +to O +activate O +acetate B-chemical +to O +the O +CoA B-chemical +- I-chemical +thioester I-chemical +. O + +The O +canonical O +PTAC B-protein_type +, O +Pta B-protein_type +, O +is O +an O +ancient O +enzyme O +found O +in O +some O +eukaryotes B-taxonomy_domain +and O +archaea B-taxonomy_domain +, O +and O +widespread O +among O +the O +bacteria B-taxonomy_domain +; O +90 O +% O +of O +the O +bacterial B-taxonomy_domain +genomes O +in O +the O +Integrated O +Microbial O +Genomes O +database O +contain O +a O +gene O +encoding O +the O +PTA_PTB B-protein_type +phosphotransacylase I-protein_type +( O +Pfam O +domain O +PF01515 B-structure_element +). O + +The O +primary O +structure O +of O +PduL B-protein_type +homologs O +is O +subdivided O +into O +two O +PF06130 B-structure_element +domains O +, O +each O +roughly O +80 B-residue_range +residues I-residue_range +in I-residue_range +length I-residue_range +. O + +Structure B-experimental_method +Determination I-experimental_method +of O +PduL B-protein_type + +Remarkably O +, O +after O +removing B-experimental_method +the O +N O +- O +terminal O +putative O +EP B-structure_element +( O +27 B-residue_range +amino I-residue_range +acids I-residue_range +), O +most O +of O +the O +sPduLΔEP B-mutant +protein O +was O +in O +the O +soluble O +fraction O +upon O +cell O +lysis O +. O + +A O +CoA B-chemical +cofactor O +as O +well O +as O +two O +metal O +ions O +are O +clearly O +resolved O +in O +the O +density B-evidence +( O +for O +omit B-evidence +maps I-evidence +of O +CoA B-chemical +see O +S2 O +Fig O +). O + +Distances O +between O +atom O +centers O +are O +indicated O +in O +Å O +. O +( O +a O +) O +Coenzyme B-chemical +A I-chemical +containing O +, O +( O +b O +) O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +structure B-evidence +. O + +( O +d O +)–( O +f O +): O +Chromatograms B-evidence +of O +sPduL B-protein +( O +d O +), O +rPduL B-protein +( O +e O +), O +and O +pPduL B-protein +( O +f O +) O +post O +- O +preparative O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +with O +different O +size O +fractions O +separated O +, O +applied O +over O +an O +analytical O +size O +exclusion O +column O +( O +see O +Materials O +and O +Methods O +). O + +The O +phosphate B-chemical +contacts O +both O +zinc B-chemical +atoms O +( O +Fig O +4b O +) O +and O +replaces O +the O +coordination O +by O +CoA B-chemical +at O +Zn1 B-chemical +; O +the O +coordination O +for O +Zn2 B-chemical +changes O +from O +octahedral O +with O +three O +bound O +waters B-chemical +to O +tetrahedral O +with O +a O +phosphate B-chemical +ion O +as O +one O +of O +the O +ligands O +( O +Fig O +4b O +). O + +The O +two O +zinc B-chemical +atoms O +are O +slightly O +closer O +together O +in O +the O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +form O +( O +5 O +. O +8 O +Å O +vs O +6 O +. O +3 O +Å O +), O +possibly O +due O +to O +the O +bridging O +effect O +of O +the O +phosphate B-chemical +. O + +rPduL B-protein +full B-protein_state +length I-protein_state +runs O +as O +Mw B-evidence += O +140 O +. O +3 O +kDa O ++/− O +1 O +. O +2 O +% O +and O +Mn B-evidence += O +140 O +. O +5 O +kDa O ++/− O +1 O +. O +2 O +%. O + +Moreover O +, O +the O +PduL B-protein_type +crystal B-evidence +structures I-evidence +offer O +a O +clue O +as O +to O +how O +required O +cofactors O +enter O +the O +BMC B-complex_assembly +lumen O +during O +assembly O +. O + +The O +native O +substrate O +for O +the O +forward O +reaction O +of O +rPduL B-protein +and O +pPduL B-protein +, O +propionyl B-chemical +- I-chemical +CoA I-chemical +, O +most O +likely O +binds O +to O +the O +enzyme O +in O +the O +same O +way O +at O +the O +observed O +nucleotide B-chemical +and O +pantothenic B-chemical +acid I-chemical +moiety O +, O +but O +the O +propionyl O +group O +in O +the O +CoA B-chemical +- I-chemical +thioester I-chemical +might O +point O +in O +a O +different O +direction O +. O + +Indeed O +, O +in O +the O +majority O +of O +PduLs B-protein_type +encoded O +in O +pvm B-gene +loci I-gene +, O +Gln77 B-residue_name_number +is O +substituted O +by O +either O +a O +Tyr B-residue_name +or O +Phe B-residue_name +, O +whereas O +it O +is O +typically O +a O +Gln B-residue_name +or O +Glu B-residue_name +in O +PduLs B-protein_type +in O +all O +other O +BMC B-complex_assembly +types O +that O +degrade O +acetyl B-chemical +- I-chemical +or O +propionyl B-chemical +- I-chemical +CoA I-chemical +. O +A O +comparison B-experimental_method +of O +the O +PduL B-protein_type +active B-site +site I-site +to O +that O +of O +the O +functionally O +identical O +Pta B-protein_type +suggests O +that O +the O +two O +enzymes O +have O +distinctly O +different O +mechanisms O +. O + +The O +two O +high O +- O +resolution O +crystal B-evidence +structures I-evidence +presented O +here O +will O +serve O +as O +the O +foundation O +for O +mechanistic O +studies O +on O +this O +noncanonical O +PTAC B-protein_type +enzyme O +to O +determine O +how O +the O +dimetal B-site +active I-site +site I-site +functions O +to O +catalyze O +both O +forward O +and O +reverse O +reactions O +. O + +A O +detailed O +understanding O +of O +the O +underlying O +principles O +governing O +the O +assembly O +and O +internal O +structural O +organization O +of O +BMCs B-complex_assembly +is O +a O +requisite O +for O +synthetic O +biologists O +to O +design O +custom O +nanoreactors O +that O +use O +BMC B-complex_assembly +architectures O +as O +a O +template O +. O + +Furthermore O +, O +given O +the O +growing O +number O +of O +metabolosomes B-complex_assembly +implicated O +in O +pathogenesis O +, O +the O +PduL B-protein_type +structure B-evidence +will O +be O +useful O +in O +the O +development O +of O +therapeutics O +. O + +EctC B-protein +forms O +a O +dimer B-oligomeric_state +with O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +arrangement O +, O +both O +in O +solution O +and O +in O +the O +crystal B-evidence +structure I-evidence +. O + +We O +show O +for O +the O +first O +time O +that O +ectoine B-protein_type +synthase I-protein_type +harbors O +a O +catalytically O +important O +metal B-chemical +co O +- O +factor O +; O +metal B-experimental_method +depletion I-experimental_method +and I-experimental_method +reconstitution I-experimental_method +experiments I-experimental_method +suggest O +that O +EctC B-protein +is O +probably O +an O +iron B-protein_state +- I-protein_state +dependent I-protein_state +enzyme O +. O + +Structure B-experimental_method +- I-experimental_method +guided I-experimental_method +site I-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +experiments O +targeting O +amino O +acid O +residues O +that O +are O +evolutionarily B-protein_state +highly I-protein_state +conserved I-protein_state +among O +the O +extended O +EctC B-protein_type +protein I-protein_type +family I-protein_type +, O +including O +those O +forming O +the O +presumptive O +iron B-site +- I-site +binding I-site +site I-site +, O +were O +conducted O +to O +functionally O +analyze O +the O +properties O +of O +the O +resulting O +EctC B-protein +variants O +. O + +This O +stereospecific O +chemical O +modification O +of O +ectoine B-chemical +( O +Fig O +1 O +) O +is O +catalyzed O +by O +the O +ectoine B-protein_type +hydroxylase I-protein_type +( O +EctD B-protein_type +) O +( O +EC O +1 O +. O +14 O +. O +11 O +), O +a O +member O +of O +the O +non B-protein_type +- I-protein_type +heme I-protein_type +containing I-protein_type +iron I-protein_type +( I-protein_type +II I-protein_type +) I-protein_type +and I-protein_type +2 I-protein_type +- I-protein_type +oxoglutarate I-protein_type +- I-protein_type +dependent I-protein_type +dioxygenase I-protein_type +superfamily I-protein_type +. O + +Scheme O +of O +the O +ectoine B-chemical +and O +5 B-chemical +- I-chemical +hydroxyectoine I-chemical +biosynthetic O +pathway O +. O + +The O +EctC B-protein +protein O +forms O +a O +dimer B-oligomeric_state +in O +solution O +and O +our O +structural B-experimental_method +analysis I-experimental_method +identifies O +it O +as O +a O +member O +of O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +( O +Sa B-species +) O +EctC B-protein +is O +a O +highly O +salt O +- O +tolerant O +enzyme O +since O +it O +exhibited O +substantial O +enzyme O +activity O +even O +at O +NaCl B-chemical +and O +KCl B-chemical +concentrations O +of O +1 O +M O +in O +the O +assay O +buffer O +( O +S3c O +and O +S3d O +Fig O +). O + +The O +ectoine B-protein_type +synthase I-protein_type +is O +a O +metal B-protein_type +- I-protein_type +containing I-protein_type +protein I-protein_type + +The O +amino O +acid O +sequences O +of O +20 O +selected O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +are O +compared O +. O + +A O +metal B-chemical +cofactor O +is O +important O +for O +the O +catalytic O +activity O +of O +EctC B-protein + +To O +address O +these O +questions O +, O +we O +incubated B-experimental_method +the O +( O +Sa B-species +) O +EctC B-protein +enzyme O +with B-experimental_method +increasing I-experimental_method +concentrations I-experimental_method +of O +the O +metal B-chemical +chelator O +ethylene B-chemical +- I-chemical +diamine I-chemical +- I-chemical +tetraacetic I-chemical +- I-chemical +acid I-chemical +( O +EDTA B-chemical +) O +and O +subsequently O +assayed O +ectoine B-protein_type +synthase I-protein_type +activity O +. O + +The O +EctC B-protein +- O +catalyzed O +ring O +- O +closure O +of O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +to O +form O +ectoine B-chemical +exhibited O +Michaelis B-experimental_method +- I-experimental_method +Menten I-experimental_method +- I-experimental_method +kinetics I-experimental_method +with O +an O +apparent O +Km B-evidence +of O +4 O +. O +9 O +± O +0 O +. O +5 O +mM O +, O +a O +vmax B-evidence +of O +25 O +. O +0 O +± O +0 O +. O +8 O +U O +/ O +mg O +and O +a O +kcat B-evidence +of O +7 O +. O +2 O +s O +- O +1 O +( O +S4a O +Fig O +). O + +( O +Sa B-species +) O +EctC B-protein +catalyzed O +this O +reaction O +with O +Michaelis B-experimental_method +- I-experimental_method +Menten I-experimental_method +- I-experimental_method +kinetics I-experimental_method +exhibiting O +an O +apparent O +Km B-evidence +of O +25 O +. O +4 O +± O +2 O +. O +9 O +mM O +, O +a O +vmax B-evidence +of O +24 O +. O +6 O +± O +1 O +. O +0 O +U O +/ O +mg O +and O +a O +kcat B-evidence +0 O +. O +6 O +s O +- O +1 O +( O +S4b O +Fig O +). O + +However O +, O +two O +crystal B-evidence +forms I-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +in O +the O +absence B-protein_state +of I-protein_state +the O +substrate O +were O +obtained O +. O + +Overall O +fold O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O + +The O +β B-structure_element +- I-structure_element +strands I-structure_element +are O +numbered O +β1 B-structure_element +- I-structure_element +β11 I-structure_element +and O +the O +helices B-structure_element +α B-structure_element +- I-structure_element +I I-structure_element +to I-structure_element +α I-structure_element +- I-structure_element +II I-structure_element +. O + +The O +entrance O +to O +the O +active B-site +site I-site +of O +the O +ectoine B-protein_type +synthase I-protein_type +is O +marked O +. O + +( O +c O +) O +Overlay B-experimental_method +of O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +and O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structures B-evidence +. O + +Hence O +, O +( O +Sa B-species +) O +EctC B-protein +adopts O +an O +overall O +bowl O +shape O +in O +which O +one O +side O +is O +opened O +towards O +the O +solvent O +( O +Fig O +4a O +to O +4c O +). O + +The O +formation O +of O +this O +α B-structure_element +- I-structure_element +II I-structure_element +helix I-structure_element +induces O +a O +reorientation O +and O +shift O +of O +a O +long O +unstructured B-protein_state +loop B-structure_element +( O +as O +observed O +in O +the O +“ O +open B-protein_state +” O +structure B-evidence +) O +connecting O +β4 B-structure_element +and O +β6 B-structure_element +, O +resulting O +in O +the O +formation O +of O +the O +stable B-protein_state +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +as O +observed O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +state O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +4a O +). O + +Both O +the O +SEC B-experimental_method +analysis O +and O +the O +HPLC B-experimental_method +- I-experimental_method +MALS I-experimental_method +experiments O +( O +S2b O +Fig O +) O +have O +shown O +that O +the O +ectoine B-protein_type +synthase I-protein_type +from O +S B-species +. I-species +alaskensis I-species +is O +a O +dimer B-oligomeric_state +in O +solution O +. O + +The O +crystal B-evidence +structure I-evidence +of O +this O +protein O +reflects O +this O +quaternary O +arrangement O +. O + +As O +calculated O +with O +PDBePISA B-experimental_method +, O +the O +surface O +area O +buried O +upon O +dimer B-oligomeric_state +formation O +is O +1462 O +Å2 O +, O +which O +is O +20 O +. O +5 O +% O +of O +the O +total O +accessible O +surface O +of O +a O +monomer B-oligomeric_state +of O +this O +protein O +. O + +In O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +, O +one O +monomer B-oligomeric_state +is O +present O +in O +the O +asymmetric O +unit O +. O + +We O +therefore O +inspected O +the O +crystal O +packing B-experimental_method +and O +analyzed O +the O +monomer B-oligomeric_state +- O +monomer B-oligomeric_state +interactions O +with O +symmetry O +related O +molecules O +to O +elucidate O +whether O +a O +physiologically O +relevant O +dimer B-oligomeric_state +could O +be O +deduced O +from O +this O +crystal B-evidence +form I-evidence +as O +well O +. O + +These O +additional O +amino O +acids O +fold O +into O +a O +small B-structure_element +helix I-structure_element +, O +which O +seals O +the O +open B-protein_state +cavity B-site +of O +the O +cupin B-structure_element +- I-structure_element +fold I-structure_element +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +4a O +). O + +As O +a O +result O +, O +the O +newly O +formed O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +is O +reoriented O +and O +moved O +by O +2 O +. O +4 O +Å O +within O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +( O +Fig O +4a O +to O +4c O +). O + +Therefore O +the O +sealing O +of O +the O +cupin B-structure_element +fold I-structure_element +, O +as O +described O +above O +, O +seem O +to O +have O +an O +indirect O +influence O +on O +the O +architecture O +of O +the O +postulated O +iron B-site +- I-site +binding I-site +site I-site +. O + +In O +the O +“ O +open B-protein_state +” O +structure B-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +, O +this O +interaction O +does O +not O +occur O +since O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +is O +rotated O +outwards O +( O +Fig O +6a O +and O +6b O +). O + +Hence O +, O +one O +might O +speculate O +that O +this O +missing O +interaction O +might O +be O +responsible O +for O +the O +flexibility O +of O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +in O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +and O +consequently O +results O +in O +less O +well O +defined O +electron B-evidence +density I-evidence +in O +this O +region O +. O + +These O +distances O +are O +to O +long O +when O +compared O +to O +other O +iron B-site +binding I-site +sites I-site +, O +a O +fact O +that O +might O +be O +caused O +by O +the O +absence B-protein_state +of I-protein_state +the O +proper O +substrate O +in O +the O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structure I-evidence +. O + +Since O +both O +the O +refinement O +and O +the O +distance O +did O +not O +clearly O +identify O +an O +iron B-chemical +molecule O +, O +we O +decided O +to O +conservatively O +place O +a O +water B-chemical +molecule O +at O +this O +position O +. O + +Only O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +is O +slightly O +rotated O +inwards O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +, O +most O +likely O +due O +to O +formation O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +as O +described O +above O +. O + +Taken O +together O +, O +this O +observations O +indicate O +, O +that O +the O +architecture O +of O +the O +presumptive O +iron B-site +- I-site +binding I-site +site I-site +is O +pre O +- O +set O +for O +the O +binding O +of O +the O +catalytically O +important O +metal B-chemical +by O +the O +ectoine B-protein_type +synthase I-protein_type +. O + +This O +is O +in O +contrast O +to O +the O +high O +- O +resolution O +“ O +open B-protein_state +” O +structure B-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +where O +no O +additional O +electron B-evidence +density I-evidence +was O +observed O +after O +refinement O +. O + +When O +analyzing O +the O +interactions O +of O +this O +compound O +within O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +, O +we O +found O +that O +it O +is O +bound B-protein_state +via O +interactions O +with O +Trp B-residue_name_number +- I-residue_name_number +21 I-residue_name_number +and O +Ser B-residue_name_number +- I-residue_name_number +23 I-residue_name_number +of O +β B-structure_element +- I-structure_element +sheet I-structure_element +β3 B-structure_element +, O +Thr B-residue_name_number +- I-residue_name_number +40 I-residue_name_number +located O +in O +β B-structure_element +- I-structure_element +sheet I-structure_element +β4 B-structure_element +, O +and O +Cys B-residue_name_number +- I-residue_name_number +105 I-residue_name_number +and O +Phe B-residue_name_number +- I-residue_name_number +107 I-residue_name_number +, O +which O +are O +both O +part O +of O +β B-structure_element +- I-structure_element +sheet I-structure_element +β11 B-structure_element +. O + +As O +described O +above O +, O +the O +side O +chains O +of O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +and O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +are O +probably O +involved O +in O +iron B-chemical +binding O +( O +Table O +1 O +and O +Fig O +6a O +). O + +However O +, O +the O +Cys B-mutant +- I-mutant +105 I-mutant +/ I-mutant +Ala I-mutant +variant B-protein_state +was O +practically O +catalytically B-protein_state +inactive I-protein_state +while O +largely O +maintaining O +its O +iron B-chemical +content O +( O +Table O +1 O +). O + +We O +observed O +two O +amino B-experimental_method +acid I-experimental_method +substitutions I-experimental_method +that O +simultaneously O +strongly O +affected O +enzyme O +activity O +and O +iron B-chemical +content O +; O +these O +were O +the O +Tyr B-mutant +- I-mutant +52 I-mutant +/ I-mutant +Ala I-mutant +and O +the O +His B-mutant +- I-mutant +55 I-mutant +/ I-mutant +Ala I-mutant +( O +Sa B-species +) O +EctC B-protein +protein O +variants O +( O +Table O +1 O +). O + +The O +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +is O +held O +in O +its O +position O +via O +an O +interaction O +of O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +with O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +, O +where O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +in O +turn O +interacts O +with O +Pro B-residue_name_number +- I-residue_name_number +110 I-residue_name_number +( O +Fig O +6a O +and O +6b O +). O + +The O +Glu B-mutant +- I-mutant +115 I-mutant +/ I-mutant +Ala I-mutant +mutant B-protein_state +possessed O +wild B-protein_state +- I-protein_state +type I-protein_state +levels O +of O +iron B-chemical +, O +whereas O +the O +iron B-chemical +content O +of O +the O +His B-mutant +- I-mutant +55 I-mutant +/ I-mutant +Ala I-mutant +substitutions O +dropped O +to O +15 O +% O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +level O +( O +Table O +1 O +). O + +As O +a O +consequence O +of O +the O +structural O +relatedness O +of O +EctC B-protein +and O +RemF B-protein +and O +the O +type O +of O +chemical O +reaction O +these O +two O +enzymes O +catalyze O +, O +is O +now O +understandable O +why O +bona O +fide O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +are O +frequently O +( O +mis O +)- O +annotated O +in O +microbial B-taxonomy_domain +genome O +sequences O +as O +“ O +RemF B-protein_type +- I-protein_type +like I-protein_type +” O +proteins O +. O + +Except O +for O +some O +cupin B-protein_type +- I-protein_type +related I-protein_type +proteins I-protein_type +that O +seem O +to O +function O +as O +metallo B-protein_type +- I-protein_type +chaperones I-protein_type +, O +the O +bound B-protein_state +metal B-chemical +is O +typically O +an O +essential O +part O +of O +the O +active B-site +sites I-site +. O + +The O +architecture O +of O +the O +metal B-site +center I-site +of O +ectoine B-protein_type +synthase I-protein_type +seems O +to O +be O +subjected O +to O +considerable O +evolutionary O +constraints O +. O + +This O +set O +of O +data O +and O +the O +fact O +that O +the O +targeted O +residues O +are O +strongly B-protein_state +conserved I-protein_state +among O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +( O +Fig O +2 O +) O +is O +consistent O +with O +their O +potential O +role O +in O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +binding O +or O +enzyme O +catalysis O +. O + +Because O +microbial B-taxonomy_domain +ectoine B-chemical +producers O +can O +colonize O +ecological O +niches O +with O +rather O +different O +physicochemical O +attributes O +, O +it O +seems O +promising O +to O +exploit O +this O +considerable O +biodiversity O +to O +identify O +EctC B-protein_type +proteins I-protein_type +with O +enhanced O +protein O +stability O +. O + +Structures B-evidence +of O +human B-species +ADAR2 B-protein +bound B-protein_state +to I-protein_state +dsRNA B-chemical +reveal O +base O +- O +flipping O +mechanism O +and O +basis O +for O +site O +selectivity O + +We O +then O +evaluated O +the O +importance O +of O +protein O +- O +RNA B-chemical +contacts O +using O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +mutagenesis I-experimental_method +and O +RNA B-experimental_method +- I-experimental_method +modification I-experimental_method +experiments I-experimental_method +coupled O +with O +adenosine B-experimental_method +deamination I-experimental_method +kinetics I-experimental_method +. O + +For O +trapping O +hADAR2d B-mutant +bound B-protein_state +to I-protein_state +RNA B-chemical +for O +crystallography B-experimental_method +, O +we O +incorporated O +8 B-chemical +- I-chemical +azanebularine I-chemical +into O +duplex B-structure_element +RNAs I-structure_element +shown O +recently O +to O +be O +excellent O +substrates O +for O +deamination O +by O +hADAR2d B-mutant +( O +for O +duplex O +sequence O +see O +Fig O +. O +1c O +) O +( O +for O +characterization O +of O +protein O +– O +RNA B-chemical +complex O +see O +Supplementary O +Fig O +. O +1 O +). O + +In O +each O +of O +these O +complexes O +, O +the O +protein O +binds O +the O +RNA B-chemical +on O +one O +face O +of O +the O +duplex O +over O +~ O +20 O +bp O +using O +a O +positively O +charged O +surface O +near O +the O +zinc B-site +- I-site +containing I-site +active I-site +site I-site +( O +Fig O +. O +2 O +, O +Supplementary O +Fig O +. O +2a O +). O + +The O +side O +chain O +of O +E396 B-residue_name_number +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +purine B-chemical +N1 O +and O +O6 O +. O + +Lastly O +, O +the O +side O +chain O +of O +V351 B-residue_name_number +provides O +a O +hydrophobic B-site +surface I-site +for O +interaction O +with O +the O +nucleobase O +of O +the O +edited B-protein_state +nucleotide B-chemical +. O + +The O +side O +chain O +of O +this O +amino O +acid O +penetrates O +the O +helix O +where O +it O +occupies O +the O +space O +vacated O +by O +the O +flipped B-protein_state +out I-protein_state +base B-chemical +and O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +the O +complementary O +strand O +orphaned B-protein_state +base B-chemical +and O +to O +the O +2 O +’ O +hydroxyl O +of O +the O +nucleotide O +immediately O +5 O +’ O +to O +the O +editing B-site +site I-site +( O +Figs O +. O +3b O +, O +3c O +). O + +In O +the O +four O +structures B-evidence +reported O +here O +, O +three O +different O +combinations O +of O +helix O +- O +penetrating O +residue O +and O +orphan B-protein_state +base B-chemical +are O +observed O +( O +i O +. O +e O +. O +E488 B-residue_name_number ++ O +U B-residue_name +, O +E488 B-residue_name_number ++ O +C B-residue_name +and O +Q488 B-residue_name_number ++ O +C B-residue_name +) O +and O +all O +three O +combinations O +show O +the O +same O +side O +chain O +and O +base O +positions O +( O +Figs O +. O +3b O +, O +3c O +, O +Supplementary O +Fig O +. O +4a O +for O +overlay B-experimental_method +of O +all O +three O +). O + +In O +the O +complex B-protein_state +with I-protein_state +hADAR2d B-mutant +WT B-protein_state +and O +the O +Bdf2 B-chemical +- I-chemical +U I-chemical +duplex I-chemical +, O +a O +similar O +interaction O +is O +observed O +with O +the O +E488 B-residue_name_number +backbone O +NH O +hydrogen B-bond_interaction +bonded I-bond_interaction +to O +the O +uracil B-residue_name +O2 O +and O +the O +E488 B-residue_name_number +side O +chain O +H B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +uracil B-residue_name +N3H O +( O +Fig O +. O +3c O +). O + +The O +flipping B-structure_element +loop I-structure_element +in O +ADAR2 B-protein +( O +i O +. O +e O +. O +aa487 O +– B-residue_range +489 I-residue_range +) O +also O +has O +the O +helix O +- O +penetrating O +residue O +flanked O +by O +glycines B-residue_name +. O + +The O +ADAR B-protein_type +- O +induced O +distortion O +in O +RNA B-chemical +conformation O +results O +in O +a O +kink B-structure_element +in O +the O +RNA B-chemical +strand O +opposite O +the O +editing B-site +site I-site +( O +Fig O +. O +4b O +). O + +As O +described O +above O +, O +the O +base O +pair O +including O +the O +5 O +’ O +nearest O +neighbor O +U B-residue_name +( O +U11 B-residue_name_number +- O +A13 B-residue_name_number +’ O +in O +the O +Bdf2 B-chemical +duplex O +) O +is O +shifted O +from O +the O +position O +it O +would O +occupy O +in O +a O +typical O +A B-structure_element +- I-structure_element +form I-structure_element +helix I-structure_element +to O +accommodate O +the O +loop B-structure_element +( O +Fig O +. O +4a O +). O + +Modeling O +a O +G B-structure_element +- I-structure_element +C I-structure_element +or I-structure_element +C I-structure_element +- I-structure_element +G I-structure_element +pair I-structure_element +at O +this O +position O +( O +i O +. O +e O +. O +5 O +’ O +G B-residue_name +or O +5 O +’ O +C B-residue_name +) O +suggests O +a O +2 O +- O +amino O +group O +in O +the O +minor B-site +groove I-site +would O +clash O +with O +the O +protein O +at O +G489 B-residue_name_number +( O +Fig O +. O +5a O +, O +Supplementary O +Fig O +. O +7c O +). O + +Indeed O +, O +replacing O +the O +U B-structure_element +- I-structure_element +A I-structure_element +pair I-structure_element +adjacent O +to O +the O +editing B-site +site I-site +with O +a O +C B-structure_element +- I-structure_element +G I-structure_element +pair I-structure_element +in O +the O +Gli1 B-protein +duplex O +substrate O +resulted O +in O +an O +80 O +% O +reduction O +in O +the O +rate O +of O +hADAR2d B-mutant +- O +catalyzed O +deamination O +( O +Figs O +. O +5b O +, O +5c O +). O + +To O +determine O +whether O +this O +effect O +arises O +from O +an O +increase O +in O +local O +duplex O +stability O +from O +the O +C O +- O +G O +for O +U O +- O +A O +substitution O +or O +from O +the O +presence O +of O +the O +2 O +- O +amino O +group O +, O +we O +replaced O +the O +U B-structure_element +- I-structure_element +A I-structure_element +pair I-structure_element +with O +a O +U B-structure_element +- I-structure_element +2 I-structure_element +- I-structure_element +aminopurine I-structure_element +( I-structure_element +2AP I-structure_element +) I-structure_element +pair I-structure_element +. O + +RNA B-structure_element +- I-structure_element +binding I-structure_element +loops I-structure_element +of O +the O +ADAR B-protein_type +catalytic B-structure_element +domain I-structure_element + +In O +addition O +, O +mutation B-experimental_method +of O +G593 B-residue_name_number +to O +glutamic B-residue_name +acid I-residue_name +( O +G593E B-mutant +) O +resulted O +in O +a O +nearly O +two O +orders O +of O +magnitude O +reduction O +in O +rate O +, O +consistent O +with O +proximity O +of O +this O +residue O +to O +the O +negatively O +charged O +phosphodiester O +backbone O +of O +the O +RNA B-chemical +( O +Fig O +. O +6c O +). O + +This O +loop B-structure_element +binds O +the O +RNA B-structure_element +duplex I-structure_element +contacting O +the O +minor B-site +groove I-site +near O +the O +editing B-site +site I-site +and O +inserting O +into O +the O +adjacent O +major B-site +groove I-site +( O +Fig O +. O +6e O +). O + +Thus O +, O +this O +system O +is O +not O +illustrative O +of O +base O +flipping O +from O +a O +normal B-protein_state +duplex O +and O +does O +not O +involve O +an O +enzyme O +that O +must O +carryout O +a O +chemical O +reaction O +on O +the O +flipped B-protein_state +out I-protein_state +nucleotide B-chemical +. O + +Thus O +, O +the O +E488 B-residue_name_number +side O +chain O +directly O +contacts O +each O +orphan B-protein_state +base B-chemical +, O +likely O +by O +accepting O +an O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +from O +uracil B-residue_name +N3H O +or O +by O +donating O +an O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +to O +cytidine B-residue_name +N3 O +. O + +Aicardi O +- O +Goutieres O +Syndrome O +( O +AGS O +) O +and O +Dyschromatosis O +Symmetrica O +Hereditaria O +( O +DSH O +) O +are O +human B-species +diseases O +caused O +by O +mutations O +in O +the O +human B-species +ADAR1 B-protein +gene O +and O +several O +of O +the O +disease O +- O +associated O +mutations O +are O +found O +in O +the O +deaminase B-structure_element +domain I-structure_element +. O + +An O +arginine B-residue_name +at O +this O +position O +would O +preclude O +close O +approach O +of O +the O +flipping B-structure_element +loop I-structure_element +to O +the O +RNA B-chemical +, O +preventing O +E1008 B-residue_name_number +insertion O +and O +base O +flipping O +into O +the O +active B-site +site I-site +( O +Supplementary O +Fig O +. O +8b O +). O + +This O +is O +consistent O +with O +the O +observation O +that O +the O +G1007R B-mutant +mutation O +in O +hADAR1 B-protein +inhibits O +RNA B-chemical +editing O +activity O +. O + +In O +addition O +, O +this O +work O +provides O +a O +basis O +for O +understanding O +the O +role O +of O +the O +ADAR B-protein_type +catalytic B-structure_element +domain I-structure_element +in O +determining O +editing B-site +site I-site +selectivity O +and O +additional O +structural O +context O +to O +evaluate O +the O +impact O +of O +ADAR B-protein_type +mutations O +associated O +with O +human B-species +disease O +. O + +Human B-species +ADAR2 B-protein +and O +modified O +RNAs B-chemical +for O +crystallography B-experimental_method + +A O +transparent O +surface O +is O +shown O +for O +the O +hADAR2d B-mutant +protein O +. O + +c O +, O +Summary O +of O +the O +contacts O +between O +hADAR2d B-mutant +E488Q B-mutant +and O +the O +Bdf2 B-chemical +- I-chemical +C I-chemical +RNA I-chemical +duplex I-chemical +. O + +b O +, O +Orphan B-protein_state +nucleotide B-chemical +recognition O +in O +the O +hADAR2d B-complex_assembly +E488Q I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +C I-complex_assembly +complex O +. O + +a O +, O +The O +minor B-site +groove I-site +edge O +of O +the O +U11 B-residue_name_number +- O +A13 B-residue_name_number +’ O +base O +pair O +from O +the O +Bdf2 B-chemical +duplex I-chemical +approaches O +G489 B-residue_name_number +; O +model O +with O +a O +C B-structure_element +- I-structure_element +G I-structure_element +pair I-structure_element +at O +this O +position O +suggests O +a O +clash O +with O +the O +G B-residue_name +2 O +- O +amino O +group O +b O +, O +RNA B-structure_element +duplex I-structure_element +substrates O +prepared O +with O +different O +5 O +’ O +nearest O +neighbor O +nucleotides O +adjacent O +to O +editing B-site +site I-site +indicated O +in O +red O +( O +2AP B-structure_element += O +2 B-structure_element +- I-structure_element +aminopurine I-structure_element +). O + +Regnase B-protein +- I-protein +1 I-protein +is O +an O +RNase B-protein_type +that O +directly O +cleaves O +mRNAs B-chemical +of O +inflammatory O +genes O +such O +as O +IL B-protein_type +- I-protein_type +6 I-protein_type +and O +IL B-protein_type +- I-protein_type +12p40 I-protein_type +, O +and O +negatively O +regulates O +cellular O +inflammatory O +responses O +. O + +Regnase B-protein +- I-protein +1 I-protein +is O +a O +member O +of O +Regnase B-protein_type +family I-protein_type +and O +is O +composed O +of O +a O +PilT B-structure_element +N I-structure_element +- I-structure_element +terminus I-structure_element +like I-structure_element +( O +PIN B-structure_element +) O +domain O +followed O +by O +a O +CCCH B-structure_element +- I-structure_element +type I-structure_element +zinc I-structure_element +– I-structure_element +finger I-structure_element +( O +ZF B-structure_element +) O +domain O +, O +which O +are O +conserved B-protein_state +among O +Regnase B-protein_type +family I-protein_type +members I-protein_type +. O + +The O +structure B-evidence +combined O +with O +functional O +analyses O +revealed O +that O +four O +catalytically O +important O +Asp B-residue_name +residues O +form O +the O +catalytic B-site +center I-site +and O +stabilize O +Mg2 B-chemical ++ I-chemical +binding O +that O +is O +crucial O +for O +RNase B-protein_type +activity O +. O + +Our O +data O +revealed O +that O +the O +catalytic O +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +is O +regulated O +through O +both O +intra O +and O +intermolecular O +domain O +interactions O +in O +vitro O +. O + +In O +order O +to O +characterize O +the O +role O +of O +each O +domain O +in O +the O +RNase B-protein_type +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +, O +we O +performed O +an O +in B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assay I-experimental_method +using O +fluorescently B-protein_state +5 I-protein_state +′- I-protein_state +labeled I-protein_state +RNA B-chemical +corresponding O +to O +nucleotides O +82 O +– O +106 O +of O +the O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +3 B-structure_element +′ I-structure_element +UTR I-structure_element +( O +Fig O +. O +1g O +). O + +Regnase B-protein +- I-protein +1 I-protein +constructs O +consisting O +of O +NTD B-mutant +- I-mutant +PIN I-mutant +- I-mutant +ZF I-mutant +completely O +cleaved O +the O +target O +mRNA B-chemical +and O +generated O +the O +cleaved O +products O +. O + +During O +purification B-experimental_method +by O +gel B-experimental_method +filtration I-experimental_method +, O +the O +PIN B-structure_element +domain O +exhibited O +extremely O +asymmetric O +elution O +peaks O +in O +a O +concentration O +dependent O +manner O +( O +Fig O +. O +2a O +). O + +Likewise O +, O +upon O +addition B-experimental_method +of I-experimental_method +the O +PIN B-structure_element +domain O +, O +NMR B-experimental_method +signals O +derived O +from O +R56 B-residue_name_number +, O +L58 B-residue_range +- I-residue_range +G59 I-residue_range +, O +and O +V86 B-residue_range +- I-residue_range +H88 I-residue_range +in O +the O +NTD B-structure_element +exhibited O +large O +chemical O +shift O +changes O +and O +residues O +D53 B-residue_name_number +, O +F55 B-residue_name_number +, O +K57 B-residue_name_number +, O +Y60 B-residue_range +- I-residue_range +S61 I-residue_range +, O +V68 B-residue_name_number +, O +T80 B-residue_range +- I-residue_range +G83 I-residue_range +, O +L85 B-residue_name_number +, O +and O +G89 B-residue_name_number +of O +the O +NTD B-structure_element +as O +well O +as O +side O +chain O +amide O +signals O +of O +N79 B-residue_name_number +exhibited O +small O +but O +appreciable O +chemical O +shift O +changes O +( O +Fig O +. O +3b O +and O +Supplementary O +Fig O +. O +5 O +). O + +Our O +mutational B-experimental_method +experiments I-experimental_method +indicated O +that O +the O +observed O +dimer B-oligomeric_state +is O +functional O +and O +that O +the O +role O +of O +the O +secondary B-protein_state +PIN B-structure_element +domain O +is O +to O +position O +Regnase B-protein +- I-protein +1 I-protein +- O +unique O +RNA B-site +binding I-site +residues I-site +near O +the O +active B-site +site I-site +of O +the O +primary B-protein_state +PIN B-structure_element +domain O +. O + +If O +this O +model O +is O +correct O +, O +then O +we O +reasoned O +that O +a O +catalytically B-protein_state +inactive I-protein_state +PIN B-structure_element +and O +a O +PIN B-structure_element +lacking B-protein_state +the O +putative O +RNA B-site +- I-site +binding I-site +residues I-site +ought O +to O +be O +inactive B-protein_state +in O +isolation O +but O +become O +active B-protein_state +when O +mixed O +together O +. O + +In O +order O +to O +test O +this O +hypothesis O +, O +we O +performed O +in B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assays I-experimental_method +using O +combinations O +of O +Regnase B-protein +- I-protein +1 I-protein +mutants B-protein_state +that O +had O +no O +or O +decreased O +RNase B-protein_type +activities O +by O +themselves O +( O +Fig O +. O +5 O +). O + +Consistently O +, O +when O +we O +compared O +the O +fluorescence B-evidence +intensity I-evidence +of O +the O +uncleaved B-protein_state +Regnase B-protein +- I-protein +1 I-protein +mRNA B-chemical +, O +basic O +residue O +mutants B-protein_state +K184A B-mutant +and O +R214A B-mutant +were O +rescued O +upon O +addition O +of O +the O +DDNN B-mutant +mutant B-protein_state +( O +Fig O +. O +5c O +). O + +According O +to O +the O +proposed O +model O +, O +an O +R214A B-mutant +PIN B-structure_element +domain O +can O +only O +form O +a O +dimer B-oligomeric_state +when O +the O +DDNN B-mutant +PIN B-structure_element +acts O +as O +the O +secondary B-protein_state +PIN B-structure_element +. O + +This O +inconsistency O +might O +be O +due O +to O +difference O +in O +the O +analytical O +methods O +and O +/ O +or O +protein O +concentrations O +used O +in O +each O +experiment O +, O +since O +the O +oligomer B-oligomeric_state +formation O +of O +PIN B-structure_element +was O +dependent O +on O +the O +protein O +concentration O +in O +our O +study O +. O + +Since O +the O +NMR B-experimental_method +spectra B-evidence +of O +monomeric B-oligomeric_state +mutants B-protein_state +overlaps O +with O +those O +of O +the O +oligomeric O +forms O +, O +it O +is O +unlikely O +that O +the O +tertiary O +structure O +of O +the O +monomeric B-oligomeric_state +mutants B-protein_state +were O +affected O +by O +the O +mutations O +. O +( O +Supplementary O +Fig O +. O +4b O +, O +c O +). O + +Based O +on O +these O +observations O +, O +we O +concluded O +that O +PIN B-structure_element +- O +PIN B-structure_element +dimer B-oligomeric_state +formation O +is O +critical O +for O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O +in O +vitro O +. O + +Within O +the O +crystal B-evidence +structure I-evidence +of O +the O +PIN B-structure_element +dimer B-oligomeric_state +, O +the O +Regnase B-protein +- I-protein +1 I-protein +specific O +basic O +regions O +in O +both O +the O +“ O +primary B-protein_state +” O +and O +“ O +secondary B-protein_state +” O +PINs B-structure_element +are O +located O +around O +the O +catalytic B-site +site I-site +of O +the O +primary O +PIN B-structure_element +( O +Supplementary O +Fig O +. O +6 O +). O + +( O +d O +) O +Solution B-evidence +structure I-evidence +of O +the O +ZF B-structure_element +domain O +. O + +( O +f O +) O +In B-experimental_method +vitro I-experimental_method +gel I-experimental_method +shift I-experimental_method +binding I-experimental_method +assay I-experimental_method +between O +Regnase B-protein +- I-protein +1 I-protein +and O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +. O + +Fluorescence B-evidence +intensity I-evidence +of O +the O +uncleaved B-protein_state +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +was O +indicated O +as O +the O +percentage O +against O +that O +in O +the O +absence B-protein_state +of I-protein_state +Regnase B-protein +- I-protein +1 I-protein +. O + +( O +a O +) O +Gel B-experimental_method +filtration I-experimental_method +analyses I-experimental_method +of O +the O +PIN B-structure_element +domain O +. O + +( O +b O +) O +Dimer B-oligomeric_state +structure B-evidence +of O +the O +PIN B-structure_element +domain O +. O + +S62 B-residue_name_number +was O +colored O +gray O +and O +excluded O +from O +the O +analysis O +, O +due O +to O +low O +signal O +intensity O +. O + +( O +c O +) O +Docking O +model O +of O +the O +NTD B-structure_element +and O +the O +PIN B-structure_element +domain O +. O + +Residues O +in O +close O +proximity O +(< O +5 O +Å O +) O +to O +each O +other O +in O +the O +docking B-evidence +structure I-evidence +were O +colored O +yellow O +. O + +Critical O +residues O +in O +the O +PIN B-structure_element +domain O +for O +the O +RNase B-protein_type +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +. O + +The O +mutations O +whose O +RNase B-protein_type +activities O +were O +restored O +in O +the O +presence B-protein_state +of I-protein_state +DDNN B-mutant +mutant B-protein_state +were O +colored O +in O +red O +or O +yellow O +on O +the O +primary O +PIN B-structure_element +. O + +RAD51 B-protein +is O +a O +recombinase B-protein_type +involved O +in O +the O +homologous O +recombination O +of O +double O +‐ O +strand O +breaks O +in O +DNA O +. O + +RAD51 B-protein +interacts O +with O +BRCA2 B-protein +, O +and O +is O +thought O +to O +localise O +RAD51 B-protein +to O +sites O +of O +DNA O +damage O +2 O +, O +3 O +. O + +The O +ability O +of O +BRC3 B-chemical +to O +interact O +with O +RAD51 B-protein +nucleoprotein O +filaments O +is O +disrupted O +when O +threonine B-residue_name +is O +mutated B-experimental_method +to O +an O +alanine B-residue_name +3 O +. O + +The O +BRC5 B-chemical +repeat O +in O +humans B-species +has O +serine B-residue_name +in O +the O +place O +of O +alanine B-residue_name +, O +and O +is O +thought O +to O +be O +a O +nonbinding B-structure_element +repeat I-structure_element +12 O +. O + +Affinities B-evidence +of O +peptides O +were O +measured O +directly O +using O +Isothermal B-experimental_method +Titration I-experimental_method +Calorimetry I-experimental_method +( O +ITC B-experimental_method +) O +and O +the O +structures B-evidence +of O +many O +of O +the O +peptides O +bound B-protein_state +to I-protein_state +humanised B-protein_state +RadA B-protein +were O +determined O +by O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +In O +this O +context O +, O +we O +have O +previously O +reported O +the O +use O +of O +stable B-protein_state +monomeric B-oligomeric_state +forms O +of O +RAD51 B-protein +, O +humanised B-protein_state +from O +Pyrococcus B-species +furiosus I-species +homologue O +RadA B-protein +, O +for O +ITC B-experimental_method +experiments O +and O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +8 O +, O +15 O +. O + +The O +residue O +makes O +no O +interactions O +with O +the O +RAD51 B-protein +protein O +, O +but O +may O +make O +an O +internal O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +Thr1520 B-residue_name_number +in O +the O +context O +of O +BRC4 B-chemical +, O +Fig O +. O +3A O +. O + +FPTA B-structure_element +was O +also O +tested O +, O +but O +was O +found O +to O +have O +no O +affinity B-evidence +for O +the O +protein O +( O +Table O +2 O +, O +entry O +5 O +). O + +For O +example O +, O +an O +overlay B-experimental_method +of O +the O +bound O +poses O +of O +the O +ligands O +FHTA B-structure_element +and O +FHPA B-structure_element +( O +Fig O +. O +2B O +) O +reveals O +a O +high O +similarity O +in O +the O +binding O +modes O +, O +indicating O +that O +the O +conformational O +rigidity O +conferred O +by O +the O +proline B-residue_name +is O +compatible O +with O +the O +FHTA B-structure_element +‐ O +binding O +mode O +, O +and O +a O +reduction O +in O +an O +entropic B-evidence +penalty I-evidence +of O +binding O +may O +be O +the O +source O +of O +the O +improvement O +in O +affinity B-evidence +. O + +Only O +one O +structure B-evidence +of O +BRC4 B-chemical +is O +published O +in B-protein_state +complex I-protein_state +with I-protein_state +human B-species +RAD51 B-protein +( O +PDB O +: O +1n0w O +). O + +Either O +a O +threonine B-residue_name +or O +serine B-residue_name +is O +most O +commonly O +found O +in O +the O +third O +position O +of O +the O +FxxA B-structure_element +motif O +. O + +The O +high B-protein_state +degree I-protein_state +of I-protein_state +conservation I-protein_state +of O +these O +three O +residues O +suggests O +an O +important O +possible O +role O +in O +facilitating O +a O +turn O +and O +stabilising O +the O +conformation O +of O +the O +peptide O +as O +it O +continues O +its O +way O +to O +a O +second O +interaction B-site +site I-site +on O +the O +side O +of O +RAD51 B-protein +. O + +Two O +residues O +in O +the O +FxxA B-structure_element +motif O +, O +phenylalanine B-residue_name +and O +alanine B-residue_name +, O +are O +highly B-protein_state +conserved I-protein_state +( O +Fig O +4a O +). O + +Phenylalanine B-residue_name +mutated B-experimental_method +to I-experimental_method +tryptophan B-residue_name +, O +in O +the O +context O +of O +the O +tetrapeptide B-chemical +improved O +potency O +, O +contrary O +to O +the O +reported O +result O +of O +comparable O +activity O +in O +the O +context O +of O +BRC4 B-chemical +12 O +. O + +These O +studies O +also O +revealed O +a O +well O +ordered O +break O +in O +the O +polypeptide O +chain O +at O +Lys147 B-residue_name_number +, O +resulting O +in O +a O +large O +conformational O +rearrangement O +close O +to O +the O +active B-site +site I-site +. O + +Cysteine B-protein_type +peptidases I-protein_type +play O +crucial O +roles O +in O +the O +virulence O +of O +bacterial B-taxonomy_domain +and O +other O +eukaryotic B-taxonomy_domain +pathogens O +. O + +However O +, O +despite O +these O +similarities O +, O +clan B-protein_type +CD I-protein_type +forms O +a O +functionally O +diverse O +group O +of O +enzymes O +: O +the O +overall O +structural O +diversity O +between O +( O +and O +at O +times O +within O +) O +the O +various O +families O +provides O +these O +peptidases B-protein_type +with O +a O +wide O +variety O +of O +substrate O +specificities O +and O +activation O +mechanisms O +. O + +The O +archetypal O +and O +arguably O +most O +notable O +family O +in O +the O +clan O +is O +that O +of O +the O +mammalian B-taxonomy_domain +caspases B-protein_type +( O +C14a B-protein_type +), O +although O +clan B-protein_type +CD I-protein_type +members O +are O +distributed O +throughout O +the O +entire O +phylogenetic O +kingdom O +and O +are O +often O +required O +in O +fundamental O +biological O +processes O +. O + +The O +structure B-evidence +also O +includes O +two O +short O +β B-structure_element +- I-structure_element +hairpins I-structure_element +( O +βA B-structure_element +– I-structure_element +βB I-structure_element +and O +βD B-structure_element +– I-structure_element +βE I-structure_element +) O +and O +a O +small B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +( O +βC B-structure_element +– I-structure_element +βF I-structure_element +), O +which O +is O +formed O +from O +two O +distinct O +regions O +of O +the O +sequence O +( O +βC B-structure_element +precedes O +α11 B-structure_element +, O +α12 B-structure_element +and O +β9 B-structure_element +, O +whereas O +βF B-structure_element +follows O +the O +βD B-structure_element +- I-structure_element +βE I-structure_element +hairpin B-structure_element +) O +in O +the O +middle O +of O +the O +CTD B-structure_element +( O +Fig O +. O +1B O +). O + +Crystal B-evidence +structure I-evidence +of O +a O +C11 B-protein_type +peptidase I-protein_type +from O +P B-species +. I-species +merdae I-species +. O + +The O +N O +and O +C O +termini O +( O +N O +and O +C O +) O +of O +PmC11 B-protein +along O +with O +the O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +( O +1 O +– O +9 O +), O +helix B-structure_element +α5 B-structure_element +, O +and O +helices B-structure_element +α8 B-structure_element +, O +α11 B-structure_element +, O +and O +α13 B-structure_element +from O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +, O +are O +all O +labeled O +. O + +Of O +the O +interacting O +secondary O +structure O +elements O +, O +α5 B-structure_element +is O +perhaps O +the O +most O +interesting O +. O + +PmC11 B-protein +is O +, O +as O +expected O +, O +most O +structurally O +similar O +to O +other O +members O +of O +clan B-protein_type +CD I-protein_type +with O +the O +top O +hits O +in O +a O +search O +of O +known O +structures B-evidence +being O +caspase B-protein +- I-protein +7 I-protein +, O +gingipain B-protein +- I-protein +K I-protein +, O +and O +legumain B-protein +( O +PBD O +codes O +4hq0 O +, O +4tkx O +, O +and O +4aw9 O +, O +respectively O +) O +( O +Table O +2 O +). O + +E O +, O +intermolecular B-ptm +processing I-ptm +of O +PmC11C179A B-mutant +by O +PmC11 B-protein +. O + +Inactive O +PmC11C179A B-mutant +was O +not O +processed O +to O +a O +major O +extent O +by O +active B-protein_state +PmC11 B-protein +until O +around O +a O +ratio O +of O +1 O +: O +4 O +( O +5 O +μg O +of O +active B-protein_state +PmC11 B-protein +). O + +The O +position O +of O +the O +catalytic B-site +dyad I-site +, O +one O +potential O +key B-site +substrate I-site +binding I-site +residue I-site +Asp177 B-residue_name_number +, O +and O +the O +ends O +of O +the O +cleavage B-site +site I-site +Lys147 B-residue_name_number +and O +Ala148 B-residue_name_number +are O +indicated O +. O + +Other O +than O +its O +more O +extended B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +, O +PmC11 B-protein +differs O +most O +significantly O +from O +other O +clan B-protein_type +CD I-protein_type +members O +at O +its O +C O +terminus O +, O +where O +the O +CTD B-structure_element +contains O +a O +further O +seven O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +four O +β B-structure_element +- I-structure_element +strands I-structure_element +after O +β8 B-structure_element +. O + +To O +investigate O +whether O +processing O +is O +a O +result O +of O +intra O +- O +or O +intermolecular O +cleavage O +, O +the O +PmC11C179A B-mutant +mutant B-protein_state +was O +incubated B-experimental_method +with I-experimental_method +increasing I-experimental_method +concentrations I-experimental_method +of O +processed B-protein_state +and O +activated B-protein_state +PmC11 B-protein +. O + +Collectively O +, O +these O +data O +suggest O +that O +the O +pro B-protein_state +- I-protein_state +form I-protein_state +of O +PmC11 B-protein +is O +autoinhibited B-protein_state +by O +a O +section O +of O +L5 B-structure_element +blocking O +access O +to O +the O +active B-site +site I-site +, O +prior O +to O +intramolecular B-ptm +cleavage I-ptm +at O +Lys147 B-residue_name_number +. O + +These O +results O +confirm O +that O +PmC11 B-protein +accepts O +substrates O +containing O +Arg B-residue_name +or O +Lys B-residue_name +in O +P1 B-residue_number +with O +a O +possible O +preference O +for O +Arg B-residue_name +. O + +Because O +PmC11 B-protein +recognizes O +basic O +substrates O +, O +the O +tetrapeptide O +inhibitor O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +was O +tested O +as O +an O +enzyme O +inhibitor O +and O +was O +found O +to O +inhibit B-protein_state +both O +the O +autoprocessing B-ptm +and O +activity O +of O +PmC11 B-protein +( O +Fig O +. O +3A O +). O + +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +was O +also O +shown O +to O +bind O +to O +the O +enzyme O +: O +a O +size B-evidence +- I-evidence +shift I-evidence +was O +observed O +, O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +analysis O +, O +in O +the O +larger O +processed O +product O +of O +PmC11 B-protein +suggesting O +that O +the O +inhibitor B-protein_state +bound I-protein_state +to O +the O +active B-site +site I-site +( O +Fig O +. O +3B O +). O + +Asp177 B-residue_name_number +is O +highly B-protein_state +conserved I-protein_state +throughout O +the O +clan B-protein_type +CD I-protein_type +C11 I-protein_type +peptidases I-protein_type +and O +is O +thought O +to O +be O +primarily O +responsible O +for O +substrate O +specificity O +of O +the O +clan B-protein_type +CD I-protein_type +enzymes I-protein_type +, O +as O +also O +illustrated O +from O +the O +proximity O +of O +these O +residues O +relative O +to O +the O +inhibitor O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +when O +PmC11 B-protein +is O +overlaid B-experimental_method +on O +the O +MALT1 B-protein +- I-protein +P I-protein +structure B-evidence +( O +Fig O +. O +3C O +). O + +Cleavage O +of O +Bz B-chemical +- I-chemical +R I-chemical +- I-chemical +AMC I-chemical +by O +PmC11 B-protein +was O +measured O +in O +a O +fluorometric B-experimental_method +activity I-experimental_method +assay I-experimental_method +with O +(+, O +purple O +) O +and O +without O +(−, O +red O +) O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +. O + +B O +, O +gel B-experimental_method +- I-experimental_method +shift I-experimental_method +assay I-experimental_method +reveals O +that O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +binds O +to O +PmC11 B-protein +. O + +Furthermore O +, O +Cu2 B-chemical ++, I-chemical +Fe2 B-chemical ++, I-chemical +and O +Zn2 B-chemical ++ I-chemical +appear O +to O +inhibit B-protein_state +PmC11 B-protein +. O + +Comparison O +with O +Clostripain B-protein + +In O +addition O +, O +the O +predicted O +primary O +S1 B-site +- I-site +binding I-site +residue I-site +in O +PmC11 B-protein +Asp177 B-residue_name_number +also O +overlays B-experimental_method +with O +the O +residue O +predicted O +to O +be O +the O +P1 B-site +specificity I-site +determining I-site +residue I-site +in O +clostripain B-protein +( O +Asp229 B-residue_name_number +, O +Fig O +. O +1A O +). O + +This O +is O +also O +the O +case O +in O +PmC11 B-protein +, O +although O +the O +cleavage B-ptm +loop B-structure_element +is O +structurally O +different O +to O +that O +found O +in O +the O +caspases B-protein_type +and O +follows O +the O +catalytic B-protein_state +His B-residue_name +( O +Fig O +. O +1A O +), O +as O +opposed O +to O +the O +Cys B-residue_name +in O +the O +caspases B-protein_type +. O + +The O +PmC11 B-protein +structure B-evidence +should O +provide O +a O +good O +basis O +for O +structural B-experimental_method +modeling I-experimental_method +and O +, O +given O +the O +importance O +of O +other O +clan B-protein_type +CD I-protein_type +enzymes I-protein_type +, O +this O +work O +should O +also O +advance O +the O +exploration O +of O +these O +peptidases B-protein_type +and O +potentially O +identify O +new O +biologically O +important O +substrates O +. O + +Ribosome B-protein_type +biogenesis I-protein_type +factor I-protein_type +Tsr3 B-protein +is O +the O +aminocarboxypropyl B-protein_type +transferase I-protein_type +responsible O +for O +18S B-chemical +rRNA I-chemical +hypermodification O +in O +yeast B-taxonomy_domain +and O +humans B-species + +Here O +we O +identify O +the O +cytoplasmic O +ribosome O +biogenesis O +protein O +Tsr3 B-protein +as O +the O +responsible O +enzyme O +in O +yeast B-taxonomy_domain +and O +human B-species +cells O +. O + +This O +unique O +SAM B-site +binding I-site +mode I-site +explains O +why O +Tsr3 B-protein +transfers O +the O +acp B-chemical +and O +not O +the O +methyl O +group O +of O +SAM B-chemical +to O +its O +substrate O +. O + +Defects O +of O +rRNA B-chemical +modification O +enzymes O +often O +lead O +to O +disturbed O +ribosome O +biogenesis O +or O +functionally O +impaired O +ribosomes O +, O +although O +the O +lack O +of O +individual O +rRNA B-chemical +modifications O +often O +has O +no O +or O +only O +a O +slight O +influence O +on O +the O +cell O +. O + +Methylation B-ptm +can O +only O +occur O +once O +pseudouridylation B-ptm +has O +taken O +place O +, O +as O +the O +latter O +reaction O +generates O +the O +substrate O +for O +the O +former O +. O + +Hypermodified B-protein_state +m1acp3Ψ B-chemical +elutes O +at O +7 O +. O +4 O +min O +( O +wild B-protein_state +type I-protein_state +, O +left O +profile O +) O +and O +is O +missing O +in O +Δtsr3 B-mutant +( O +middle O +profile O +) O +and O +Δnep1 B-mutant +Δnop6 I-mutant +mutants O +( O +right O +profile O +). O + +Upper O +lanes O +show O +the O +ethidium B-chemical +bromide I-chemical +staining O +of O +the O +18S B-chemical +rRNAs I-chemical +for O +quantification O +. O + +Archaeal B-taxonomy_domain +Tyw2 B-protein +has O +a O +structure B-evidence +very O +similar O +to O +Rossmann B-protein_type +- I-protein_type +fold I-protein_type +( I-protein_type +class I-protein_type +I I-protein_type +) I-protein_type +RNA I-protein_type +- I-protein_type +methyltransferases I-protein_type +, O +but O +its O +distinctive O +SAM B-site +- I-site +binding I-site +mode I-site +enables O +the O +transfer O +of O +the O +acp B-chemical +group O +instead O +of O +the O +methyl O +group O +of O +the O +cofactor O +. O + +On O +this O +basis O +, O +YOR006C B-gene +was O +renamed O +‘ O +Twenty B-protein +S I-protein +rRNA I-protein +accumulation I-protein +3 I-protein +′ O +( O +TSR3 B-protein +). O + +In O +contrast O +, O +the O +only O +other O +structurally O +characterized O +acp B-protein_type +transferase I-protein_type +enzyme O +Tyw2 B-protein +belongs O +to O +the O +Rossmann B-protein_type +- I-protein_type +fold I-protein_type +class I-protein_type +of I-protein_type +methyltransferase I-protein_type +proteins I-protein_type +. O + +By O +comparison O +, O +treating O +cells O +with O +siRNA B-chemical +545 O +, O +which O +only O +reduced O +the O +TSR3 B-protein +mRNA O +to O +20 O +%, O +did O +not O +markedly O +reduced O +the O +acp B-chemical +signal O +. O + +This O +suggests O +that O +low O +residual O +levels O +of O +HsTsr3 B-protein +are O +sufficient O +to O +modify O +the O +RNA B-chemical +. O + +Similar O +to O +a O +temperature O +- O +sensitive O +nep1 B-gene +mutant B-protein_state +, O +the O +Δtsr3 B-mutant +deletion O +caused O +hypersensitivity O +to O +paromomycin B-chemical +and O +, O +to O +a O +lesser O +extent O +, O +to O +hygromycin B-chemical +B I-chemical +( O +Figure O +2B O +), O +but O +not O +to O +G418 B-chemical +or O +cycloheximide B-chemical +( O +data O +not O +shown O +). O + +In O +accordance O +with O +the O +synthetic O +sick O +growth O +phenotype O +the O +paromomycin B-chemical +and O +hygromycin B-chemical +B I-chemical +hypersensitivity O +further O +increased O +in O +a O +Δtsr3 B-mutant +Δsnr35 I-mutant +recombination O +strain O +( O +Figure O +2B O +). O + +In O +a O +yeast B-taxonomy_domain +Δtsr3 B-mutant +strain O +as O +well O +as O +in O +the O +Δtsr3 B-mutant +Δsnr35 I-mutant +recombinant O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +accumulated O +significantly O +and O +the O +level O +of O +mature O +18S B-chemical +rRNA I-chemical +was O +reduced O +( O +Supplementary O +Figures O +S2C O +and O +S3D O +), O +as O +reported O +previously O +. O + +However O +, O +these O +archaeal B-taxonomy_domain +homologs O +are O +significantly O +smaller O +than O +ScTsr3 B-protein +(∼ O +190 O +aa O +in O +archaea B-taxonomy_domain +vs O +. O +313 O +aa O +in O +yeast B-taxonomy_domain +) O +due O +to O +shortened O +N O +- O +and O +C O +- O +termini O +( O +Supplementary O +Figure O +S1A O +). O + +Even O +a O +Tsr3 B-protein +fragment O +with O +a O +90 B-residue_range +aa I-residue_range +C O +- O +terminal O +truncation O +showed O +a O +residual O +primer O +extension O +stop O +, O +whereas O +N O +- O +terminal O +truncations O +exceeding O +46 B-residue_range +aa I-residue_range +almost O +completely O +abolished O +the O +primer O +extension O +arrest O +( O +Figure O +3B O +). O + +( O +B O +) O +Primer B-experimental_method +extension I-experimental_method +analysis I-experimental_method +of O +18S B-chemical +rRNA I-chemical +acp B-chemical +modification O +in O +yeast B-taxonomy_domain +cells O +expressing O +the O +indicated O +TSR3 B-protein +fragments O +. O + +While O +for O +S B-species +. I-species +solfataricus I-species +the O +existence O +of O +a O +modified O +nucleotide B-chemical +of O +unknown O +chemical O +composition O +in O +the O +loop B-structure_element +capping I-structure_element +helix I-structure_element +31 I-structure_element +of O +its O +16S B-chemical +rRNA I-chemical +has O +been O +demonstrated O +, O +no O +information O +regarding O +rRNA O +modifications O +is O +yet O +available O +for O +V B-species +. I-species +distributa I-species +. O + +The O +structure B-evidence +of O +VdTsr3 B-protein +was O +solved O +ab O +initio O +, O +by O +single B-experimental_method +- I-experimental_method +wavelength I-experimental_method +anomalous I-experimental_method +diffraction I-experimental_method +phasing I-experimental_method +( O +Se B-experimental_method +- I-experimental_method +SAD I-experimental_method +) O +with O +Se B-chemical +containing O +derivatives O +( O +selenomethionine B-chemical +and O +seleno B-chemical +- I-chemical +substituted I-chemical +SAM I-chemical +). O + +Thus O +, O +the O +VdTsr3 B-protein +structure B-evidence +contains O +a O +deep B-structure_element +trefoil I-structure_element +knot I-structure_element +. O + +β B-structure_element +- I-structure_element +strands I-structure_element +are O +colored O +in O +crimson O +whereas O +α B-structure_element +- I-structure_element +helices I-structure_element +in O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +are O +colored O +light O +blue O +and O +α B-structure_element +- I-structure_element +helices I-structure_element +in O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +are O +colored O +dark O +blue O +. O + +A O +red O +arrow O +marks O +the O +location O +of O +the O +topological B-structure_element +knot I-structure_element +in O +the O +structure B-evidence +. O +( O +B O +) O +Secondary O +structure O +representation O +of O +the O +VdTsr3 B-protein +structure B-evidence +. O + +However O +, O +there O +are O +no O +structural O +similarities O +between O +Tsr3 B-protein +and O +Tyw2 B-protein +, O +which O +contains O +an O +all B-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +of O +a O +different O +topology O +and O +no O +knot B-structure_element +structure I-structure_element +. O + +SAM B-chemical +- O +binding O +by O +Tsr3 B-protein +. O + +Bound B-protein_state +SAM B-chemical +was O +modelled O +based O +on O +the O +X B-evidence +- I-evidence +ray I-evidence +structure I-evidence +of O +the O +Trm10 B-complex_assembly +/ I-complex_assembly +SAH I-complex_assembly +- O +complex O +( O +pdb4jwf O +). O + +3xHA B-protein_state +tagged I-protein_state +Tsr3 B-protein +mutants B-protein_state +are O +expressed O +comparable O +to O +the O +wild B-protein_state +type I-protein_state +as O +shown O +by O +western B-experimental_method +blot I-experimental_method +( O +lower O +left O +). O + +VdTsr3 B-protein +could O +not O +be O +used O +in O +these O +experiments O +since O +we O +could O +not O +purify O +it O +in O +a O +stable B-protein_state +SAM B-protein_state +- I-protein_state +free I-protein_state +form O +. O + +The O +side O +chain O +of O +D70 B-residue_name_number +( O +VdTsr3 B-protein +) O +located O +in O +β4 B-structure_element +is O +∼ O +5 O +Å O +away O +from O +the O +SAM B-chemical +sulfur O +atom O +. O + +A O +second O +cluster O +of O +positively O +charged O +residues O +is O +found O +in O +or O +near O +helix B-structure_element +α3 B-structure_element +( O +K74 B-residue_name_number +, O +R75 B-residue_name_number +, O +K82 B-residue_name_number +, O +R85 B-residue_name_number +and O +K87 B-residue_name_number +). O + +A O +triple B-experimental_method +mutation I-experimental_method +of O +the O +conserved B-protein_state +positively O +charged O +residues O +R60 B-residue_name_number +, O +K65 B-residue_name_number +and O +R131 B-residue_name_number +to O +A B-residue_name +in O +ScTsr3 B-protein +resulted O +in O +a O +protein O +with O +a O +significantly O +impaired O +acp B-protein_type +transferase I-protein_type +activity O +in O +vivo O +( O +Figure O +6D O +) O +in O +line O +with O +an O +important O +functional O +role O +for O +these O +positively O +charged O +residues O +. O + +For O +S B-species +. I-species +solfataricus I-species +the O +chemical O +identity O +of O +the O +hypermodified B-protein_state +nucleotide B-chemical +is O +not O +known O +but O +the O +existence O +of O +NEP1 B-protein +and O +TSR3 B-protein +homologs O +suggest O +that O +it O +is O +indeed O +N1 B-chemical +- I-chemical +methyl I-chemical +- I-chemical +N3 I-chemical +- I-chemical +acp I-chemical +- I-chemical +pseudouridine I-chemical +. O + +The O +formation O +of O +1 B-chemical +- I-chemical +methyl I-chemical +- I-chemical +3 I-chemical +-( I-chemical +3 I-chemical +- I-chemical +amino I-chemical +- I-chemical +3 I-chemical +- I-chemical +carboxypropyl I-chemical +)- I-chemical +pseudouridine I-chemical +( O +m1acp3Ψ B-chemical +) O +is O +very O +complex O +requiring O +three O +successive O +modification O +reactions O +involving O +one O +H B-structure_element +/ I-structure_element +ACA I-structure_element +snoRNP B-complex_assembly +( O +snR35 B-protein +) O +and O +two O +protein O +enzymes O +( O +Nep1 B-protein +/ O +Emg1 B-protein +and O +Tsr3 B-protein +). O + +This O +makes O +it O +unique O +in O +eukaryotic B-taxonomy_domain +rRNA B-chemical +modification O +. O + +A O +similar O +modification O +( O +acp3U B-chemical +) O +was O +identified O +in O +Haloferax B-species +volcanii I-species +and O +corresponding O +modified O +nucleotides B-chemical +were O +also O +shown O +to O +occur O +in O +other O +archaea B-taxonomy_domain +. O + +As O +shown O +here O +TSR3 B-protein +encodes O +the O +transferase O +catalyzing O +the O +acp B-chemical +modification O +as O +the O +last O +step O +in O +the O +biosynthesis O +of O +m1acp3Ψ B-chemical +in O +yeast B-taxonomy_domain +and O +human B-species +cells O +. O + +In O +contrast O +, O +in O +the O +structurally O +closely O +related O +RNA B-protein_type +methyltransferase I-protein_type +Trm10 B-protein +the O +methyl O +group O +of O +the O +cofactor O +SAM B-chemical +is O +accessible O +whereas O +its O +acp B-chemical +side O +chain O +is O +buried O +inside O +the O +protein O +. O + +In O +response O +to O +cell O +stress O +, O +YfiB B-protein +in O +the O +outer O +membrane O +can O +sequester O +the O +periplasmic O +protein O +YfiR B-protein +, O +releasing O +its O +inhibition O +of O +YfiN B-protein +on O +the O +inner O +membrane O +and O +thus O +provoking O +the O +diguanylate O +cyclase O +activity O +of O +YfiN B-protein +to O +induce O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +production O +. O + +Based O +on O +the O +structural B-evidence +and I-evidence +biochemical I-evidence +data I-evidence +, O +we O +propose O +an O +updated O +regulatory O +model O +of O +the O +YfiBNR B-complex_assembly +system O +. O + +The O +functional O +role O +of O +a O +number O +of O +downstream O +effectors O +of O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +has O +been O +characterized O +as O +affecting O +exopolysaccharide B-chemical +( O +EPS B-chemical +) O +production O +, O +transcription O +, O +motility O +, O +and O +surface O +attachment O +( O +Caly O +et O +al O +.,; O +Camilli O +and O +Bassler O +,; O +Ha O +and O +O O +’ O +Toole O +,; O +Pesavento O +and O +Hengge O +,). O + +Recently O +, O +Malone O +and O +coworkers O +identified O +the O +tripartite B-protein_state +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +signaling O +module O +system O +YfiBNR B-complex_assembly +( O +also O +known O +as O +AwsXRO B-complex_assembly +( O +Beaumont O +et O +al O +.,; O +Giddens O +et O +al O +.,) O +or O +Tbp B-complex_assembly +( O +Ueda O +and O +Wood O +,)) O +by O +genetic B-experimental_method +screening I-experimental_method +for O +mutants O +that O +displayed O +SCV O +phenotypes O +in O +P B-species +. I-species +aeruginosa I-species +PAO1 I-species +( O +Malone O +et O +al O +.,; O +Malone O +et O +al O +.,). O + +Together O +with O +functional O +data O +, O +these O +results O +provide O +new O +mechanistic O +insights O +into O +how O +activated B-protein_state +YfiB B-protein +sequesters O +YfiR B-protein +and O +releases O +the O +suppression O +of O +YfiN B-protein +. O +These O +findings O +may O +facilitate O +the O +development O +and O +optimization O +of O +anti O +- O +biofilm O +drugs O +for O +the O +treatment O +of O +chronic O +infections O +. O + +Overall O +structure B-evidence +of O +YfiB B-protein + +Each O +crystal O +form O +contains O +three O +different O +dimeric B-oligomeric_state +types O +of O +YfiB B-protein +, O +two O +of O +which O +are O +present O +in O +both O +, O +suggesting O +that O +the O +rest O +of O +the O +dimeric B-oligomeric_state +types O +may O +result O +from O +crystal O +packing O +. O + +The O +dimerization O +occurs O +mainly O +via O +hydrophobic B-bond_interaction +interactions I-bond_interaction +formed O +by O +A37 B-residue_name_number +and O +I40 B-residue_name_number +on O +the O +α1 B-structure_element +helices I-structure_element +, O +L50 B-residue_name_number +on O +the O +β1 B-structure_element +strands I-structure_element +, O +and O +W55 B-residue_name_number +on O +the O +β2 B-structure_element +strands I-structure_element +of O +both O +molecules O +, O +making O +a O +hydrophobic B-site +interacting I-site +core I-site +( O +Fig O +. O +2A O +– O +C O +). O + +Two O +interacting O +regions O +are O +highlighted O +by O +red O +rectangles O +. O +( O +B O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +apo B-protein_state +YfiB B-protein +and O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +. O + +To O +gain O +structural O +insights O +into O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +interaction O +, O +we O +co B-experimental_method +- I-experimental_method +expressed I-experimental_method +YfiB B-protein +( O +residues O +34 B-residue_range +– I-residue_range +168 I-residue_range +) O +and O +YfiR B-protein +( O +residues O +35 B-residue_range +– I-residue_range +190 I-residue_range +, O +lacking B-protein_state +the O +signal B-structure_element +peptide I-structure_element +), O +but O +failed O +to O +obtain O +the O +complex O +, O +in O +accordance O +with O +a O +previous O +report O +in O +which O +no B-protein_state +stable I-protein_state +complex O +of O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +was O +observed O +( O +Malone O +et O +al O +.,). O + +The O +N O +- O +terminal O +structural O +conformation O +of O +YfiBL43P B-mutant +, O +from O +the O +foremost O +N O +- O +terminus O +to O +residue O +D70 B-residue_name_number +, O +is O +significantly O +altered O +compared O +with O +that O +of O +the O +apo B-protein_state +YfiB B-protein +. O +The O +majority O +of O +the O +α1 B-structure_element +helix I-structure_element +( O +residues O +34 B-residue_range +– I-residue_range +43 I-residue_range +) O +is O +invisible O +on O +the O +electron B-evidence +density I-evidence +map I-evidence +, O +and O +the O +α2 B-structure_element +helix I-structure_element +and O +β1 B-structure_element +and O +β2 B-structure_element +strands I-structure_element +are O +rearranged O +to O +form O +a O +long O +loop B-structure_element +containing O +two O +short O +α B-structure_element +- I-structure_element +helix I-structure_element +turns I-structure_element +( O +Fig O +. O +3B O +and O +3C O +), O +thus O +embracing O +the O +YfiR B-protein +dimer B-oligomeric_state +. O + +Therefore O +, O +it O +is O +possible O +that O +both O +dimeric B-oligomeric_state +types O +might O +exist O +in O +solution O +. O + +( O +C O +) O +Close O +- O +up O +view O +showing O +the O +key O +residues O +of O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +interacting O +with O +a O +sulfate B-chemical +ion O +. O + +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +is O +shown O +in O +cyan O +; O +the O +sulfate B-chemical +ion O +, O +in O +green O +; O +and O +the O +water B-chemical +molecule O +, O +in O +yellow O +. O +( O +D O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +PG B-site +- I-site +binding I-site +sites I-site +of O +apo B-protein_state +YfiB B-protein +and O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +, O +the O +key O +residues O +are O +shown O +in O +stick O +. O + +In O +the O +Pal B-complex_assembly +/ I-complex_assembly +PG I-complex_assembly +- I-complex_assembly +P I-complex_assembly +complex O +structure B-evidence +, O +the O +m B-chemical +- I-chemical +Dap5 I-chemical +ϵ I-chemical +- I-chemical +carboxylate I-chemical +group O +interacts O +with O +the O +side O +- O +chain O +atoms O +of O +D71 B-residue_name_number +and O +the O +main O +- O +chain O +amide O +of O +D37 B-residue_name_number +( O +Fig O +. O +4B O +). O + +In O +addition O +, O +sequence B-experimental_method +alignment I-experimental_method +of O +YfiB B-protein +with O +Pal B-protein_type +and O +the O +periplasmic B-structure_element +domain I-structure_element +of O +OmpA B-protein_type +( O +proteins O +containing O +PG B-site +- I-site +binding I-site +site I-site +) O +showed O +that O +N68 B-residue_name_number +and O +D102 B-residue_name_number +are O +highly B-protein_state +conserved I-protein_state +( O +Fig O +. O +4G O +, O +blue O +stars O +), O +suggesting O +that O +these O +residues O +contribute O +to O +the O +PG O +- O +binding O +ability O +of O +YfiB B-protein +. O + +Therefore O +, O +we O +proposed O +that O +the O +PG B-chemical +- O +binding O +ability O +of O +inactive B-protein_state +YfiB B-protein +might O +be O +weaker O +than O +that O +of O +active B-protein_state +YfiB B-protein +. O +To O +validate O +this O +, O +we O +performed O +a O +microscale B-experimental_method +thermophoresis I-experimental_method +( O +MST B-experimental_method +) O +assay O +to O +measure O +the O +binding B-evidence +affinities I-evidence +of O +PG B-chemical +to O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +and O +YfiBL43P B-mutant +, O +respectively O +. O + +Interestingly O +, O +these O +residues O +are O +part O +of O +the O +conserved B-site +surface I-site +of O +YfiR B-protein +( O +Fig O +. O +3G O +). O + +Collectively O +, O +a O +part O +of O +the O +YfiB B-site +- I-site +YfiR I-site +interface I-site +overlaps O +with O +the O +proposed O +YfiR B-site +- I-site +YfiN I-site +interface I-site +, O +suggesting O +alteration O +in O +the O +association O +- O +disassociation O +equilibrium O +of O +YfiR B-protein +- O +YfiN B-protein +and O +hence O +the O +ability O +of O +YfiB B-protein +to O +sequester O +YfiR B-protein +. O + +However O +, O +whether O +YfiR B-protein +is O +involved O +in O +other O +regulatory O +mechanisms O +is O +still O +an O +open O +question O +. O + +Overall O +Structures B-evidence +of O +VB6 B-protein_state +- I-protein_state +bound I-protein_state +and O +Trp B-protein_state +- I-protein_state +bound I-protein_state +YfiR B-protein +. O +( O +A O +) O +Superposition B-experimental_method +of O +the O +overall O +structures B-evidence +of O +VB6 B-protein_state +- I-protein_state +bound I-protein_state +and O +Trp B-protein_state +- I-protein_state +bound I-protein_state +YfiR B-protein +. O +( O +B O +) O +Close O +- O +up O +views O +showing O +the O +key O +residues O +of O +YfiR B-protein +that O +bind O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +. O + +In O +parallel O +, O +to O +better O +understand O +the O +putative O +functional O +role O +of O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +, O +yfiB B-gene +was O +deleted B-experimental_method +in O +a O +PAO1 B-species +wild B-protein_state +- I-protein_state +type I-protein_state +strain O +, O +and O +a O +construct B-experimental_method +expressing I-experimental_method +the O +YfiBL43P B-mutant +mutant B-protein_state +was O +transformed B-experimental_method +into I-experimental_method +the O +PAO1 B-species +ΔyfiB B-mutant +strain O +to O +trigger O +YfiBL43P B-mutant +- O +induced O +biofilm O +formation O +. O + +By O +contrast O +, O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +( O +residues O +44 B-residue_range +– I-residue_range +168 I-residue_range +) O +has O +a O +stretched B-protein_state +conformation I-protein_state +of O +approximately O +55 O +Å O +in O +length O +. O + +Provided O +that O +the O +diameter O +of O +the O +widest O +part O +of O +the O +YfiB B-protein +dimer B-oligomeric_state +is O +approximately O +64 O +Å O +, O +which O +is O +slightly O +smaller O +than O +the O +smallest O +diameter O +of O +the O +PG O +pore O +( O +70 O +Å O +) O +( O +Meroueh O +et O +al O +.,), O +the O +YfiB B-protein +dimer B-oligomeric_state +should O +be O +able O +to O +penetrate O +the O +PG O +layer O +. O + +Regulatory O +model O +of O +the O +YfiBNR B-complex_assembly +tripartite B-protein_state +system O +. O + +Once O +activated B-protein_state +by O +certain O +cell O +stress O +, O +the O +dimeric B-oligomeric_state +YfiB B-protein +transforms O +from O +a O +compact B-protein_state +conformation I-protein_state +to O +a O +stretched B-protein_state +conformation I-protein_state +, O +allowing O +the O +periplasmic B-structure_element +domain I-structure_element +of O +the O +membrane B-protein_state +- I-protein_state +anchored I-protein_state +YfiB B-protein +to O +penetrate O +the O +cell O +wall O +and O +sequester O +the O +YfiR B-protein +dimer B-oligomeric_state +, O +thus O +relieving O +the O +repression O +of O +YfiN B-protein + +These O +results O +, O +together O +with O +our O +observation O +that O +activated B-protein_state +YfiB B-protein +has O +a O +much O +higher O +cell B-evidence +wall I-evidence +binding I-evidence +affinity I-evidence +, O +and O +previous O +mutagenesis O +data O +showing O +that O +( O +1 O +) O +both O +PG B-chemical +binding O +and O +membrane O +anchoring O +are O +required O +for O +YfiB B-protein +activity O +and O +( O +2 O +) O +activating O +mutations O +possessing O +an O +altered O +N O +- O +terminal O +loop B-structure_element +length O +are O +dominant O +over O +the O +loss O +of O +PG B-chemical +binding O +( O +Malone O +et O +al O +.,), O +suggest O +an O +updated O +regulatory O +model O +of O +the O +YfiBNR B-complex_assembly +system O +( O +Fig O +. O +7 O +). O + +The O +mechanism O +by O +which O +activated B-protein_state +YfiB B-protein +relieves O +the O +repression O +of O +YfiN B-protein +may O +be O +applicable O +to O +the O +YfiBNR B-complex_assembly +system O +in O +other O +bacteria B-taxonomy_domain +and O +to O +analogous O +outside O +- O +in O +signaling O +for O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +production O +, O +which O +in O +turn O +may O +be O +relevant O +to O +the O +development O +of O +drugs O +that O +can O +circumvent O +complicated O +antibiotic O +resistance O +. O + +UHRF1 B-protein +recognizes O +hemi B-chemical +- I-chemical +methylated I-chemical +DNA I-chemical +( O +hm B-chemical +- I-chemical +DNA I-chemical +) O +and O +trimethylation B-ptm +of O +histone B-protein_type +H3K9 B-protein_type +( O +H3K9me3 B-protein_type +), O +but O +the O +regulatory O +mechanism O +remains O +unknown O +. O + +UHRF1 B-protein +also O +plays O +an O +important O +role O +in O +promoting O +proliferation O +and O +is O +shown O +to O +be O +upregulated O +in O +a O +number O +of O +cancers O +, O +suggesting O +that O +UHRF1 B-protein +may O +serve O +as O +a O +potential O +drug O +target O +for O +therapeutic O +applications O +. O + +Here O +we O +report O +that O +UHRF1 B-protein +adopts O +a O +closed B-protein_state +conformation O +, O +in O +which O +the O +C O +- O +terminal O +Spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +and O +inhibits O +its O +recognition O +of O +H3K9me3 B-protein_type +, O +whereas O +the O +SRA B-structure_element +binds B-protein_state +to I-protein_state +the O +PHD B-structure_element +and O +inhibits O +its O +recognition O +of O +H3R2 B-site +( O +unmethylated B-protein_state +histone B-protein_type +H3 B-protein_type +at O +residue O +R2 B-residue_name_number +). O + +As O +a O +result O +, O +UHRF1 B-protein +is O +locked O +in O +the O +open B-protein_state +conformation O +by O +the O +association O +of O +H3K9me3 B-protein_type +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +, O +and O +thus O +SRA B-structure_element +- I-structure_element +Spacer I-structure_element +either O +recognizes O +hm B-chemical +- I-chemical +DNA I-chemical +or O +recruits O +DNMT1 B-protein +for O +DNA B-chemical +methylation B-ptm +. O + +To O +investigate O +how O +UHRF1 B-protein +coordinates O +the O +recognition O +of O +H3K9me3 B-protein_type +and O +hm B-chemical +- I-chemical +DNA I-chemical +, O +we O +purified O +recombinant O +UHRF1 B-protein +( O +truncations O +and O +mutations O +) O +proteins O +from O +bacteria O +. O + +These O +results O +suggest O +that O +hm B-chemical +- I-chemical +DNA I-chemical +facilitates O +histone B-protein_type +recognition O +by O +UHRF1 B-protein +. O + +The O +gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +showed O +that O +UHRF1 B-protein +is O +a O +monomer B-oligomeric_state +in O +solution O +( O +Supplementary O +Fig O +. O +1b O +), O +indicating O +that O +the O +intramolecular O +( O +not O +intermolecular O +) O +interaction O +of O +UHRF1 B-protein +regulates O +histone B-protein_type +recognition O +. O + +These O +results O +suggest O +that O +UHRF1 B-protein +adopts O +an O +unfavourable O +conformation O +for O +histone B-protein_type +H3 B-protein_type +tails O +recognition O +, O +in O +which O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +might O +be O +blocked O +by O +other O +regions O +of O +UHRF1 B-protein +, O +and O +hm B-chemical +- I-chemical +DNA I-chemical +impairs O +this O +intramolecular O +interaction O +to O +facilitate O +its O +recognition O +of O +histone B-protein_type +H3 B-protein_type +tails O +. O + +The O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +( O +ITC B-experimental_method +) O +measurements O +show O +that O +the O +TTD B-structure_element +bound B-protein_state +to I-protein_state +the O +Spacer B-structure_element +( O +but O +not O +the O +SRA B-structure_element +) O +in O +a O +1 O +: O +1 O +stoichiometry O +with O +a O +binding B-evidence +affinity I-evidence +( O +KD B-evidence +) O +of O +1 O +. O +59 O +μM O +( O +Fig O +. O +2b O +). O + +Compared O +with O +the O +PHD B-structure_element +alone B-protein_state +, O +PHD B-structure_element +- I-structure_element +SRA I-structure_element +showed O +decreased O +binding B-evidence +affinity I-evidence +to O +H3K9me0 B-protein_type +peptide O +by O +a O +factor O +of O +eight O +( O +Fig O +. O +2e O +). O + +These O +results O +indicate O +that O +the O +SRA B-structure_element +directly O +binds B-protein_state +to I-protein_state +the O +PHD B-structure_element +and O +inhibits O +its O +binding B-evidence +affinity I-evidence +to O +H3K9me0 B-protein_type +. O + +The O +Spacer B-structure_element +( O +residues O +643 B-residue_range +– I-residue_range +655 I-residue_range +were O +built O +in O +the O +model O +) O +adopts O +an O +extended B-protein_state +conformation I-protein_state +and O +binds B-protein_state +to I-protein_state +an O +acidic B-site +groove I-site +on O +the O +TTD B-structure_element +( O +Fig O +. O +3c O +). O + +Comparison B-experimental_method +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +and O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +( O +PDB O +: O +4GY5 O +) O +structures B-evidence +indicates O +that O +the O +Spacer B-structure_element +and O +the O +Linker B-structure_element +bind O +to O +the O +TTD B-structure_element +in O +a O +similar O +manner O +in O +the O +two O +complexes O +( O +Fig O +. O +3b O +). O + +The O +ITC B-experimental_method +experiment O +shows O +that O +the O +Linker B-structure_element +peptide O +( O +289 B-residue_range +– I-residue_range +306 I-residue_range +) O +bound B-protein_state +to I-protein_state +the O +TTD B-structure_element +with O +a O +binding B-evidence +affinity I-evidence +of O +24 O +. O +04 O +μM O +( O +Supplementary O +Fig O +. O +4b O +), O +∼ O +15 O +- O +fold O +lower O +than O +that O +of O +the O +Spacer B-structure_element +peptide O +( O +KD B-evidence += O +1 O +. O +59 O +μM O +, O +Fig O +. O +3e O +). O + +As O +shown O +in O +Fig O +. O +4d O +, O +hm B-chemical +- I-chemical +DNA I-chemical +largely O +enhanced O +the O +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinities I-evidence +of O +both O +mutants B-protein_state +in O +the O +presence B-protein_state +of I-protein_state +DTT B-chemical +, O +but O +not O +in O +the O +absence B-protein_state +of I-protein_state +DTT B-chemical +, O +indicating O +that O +the O +disulphide B-ptm +bond I-ptm +formation O +( O +in O +the O +absence B-protein_state +of I-protein_state +DTT B-chemical +) O +disallows O +hm B-chemical +- I-chemical +DNA I-chemical +to O +disrupt O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +for O +H3K9me3 B-protein_type +recognition O +. O + +The O +above O +results O +collectively O +demonstrate O +that O +( O +i O +) O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +adopts O +a O +closed B-protein_state +form O +, O +in O +which O +the O +Spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +and O +H3K9me3 B-protein_type +recognition O +is O +inhibited O +; O +( O +ii O +) O +hm B-chemical +- I-chemical +DNA I-chemical +displaces O +the O +Spacer B-structure_element +from O +the O +TTD B-structure_element +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +therefore O +largely O +enhances O +its O +histone B-protein_type +H3K9me3 B-protein_type +- O +binding O +activity O +in O +a O +manner O +independent O +on O +the O +PHD B-structure_element +( O +SRA B-structure_element +is O +required O +). O + +The O +results O +suggest O +that O +UHRF1ΔSRA B-mutant +adopts O +closed B-protein_state +conformation O +so O +that O +H3K9me3 B-protein_type +recognition O +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +is O +blocked O +by O +the O +intramolecular O +interaction O +, O +and O +support O +the O +regulatory O +role O +of O +the O +Spacer B-structure_element +in O +PCH O +localization O +of O +UHRF1 B-protein +in O +vivo O +. O + +Getting O +these O +structures B-evidence +would O +greatly O +help O +for O +understanding O +the O +hm B-chemical +- I-chemical +DNA I-chemical +- O +mediated O +regulation O +of O +UHRF1 B-protein +. O + +These O +findings O +together O +indicate O +that O +the O +Spacer B-structure_element +plays O +a O +very O +important O +role O +in O +the O +dynamic O +regulation O +of O +UHRF1 B-protein +. O + +When O +our O +manuscript O +was O +in O +preparation O +, O +Gelato O +et O +al O +. O +reported O +that O +binding O +of O +PI5P B-chemical +to O +the O +Spacer B-structure_element +opens O +the O +closed B-protein_state +conformation O +of O +UHRF1 B-protein +and O +increases O +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +TTD B-structure_element +. O + +Interestingly O +, O +variant B-protein +in I-protein +methylation I-protein +1 I-protein +( O +VIM1 B-protein +, O +a O +UHRF1 B-protein +homologue O +in O +Arabidopsis B-taxonomy_domain +) O +contains O +an O +equivalent O +spacer B-structure_element +region O +, O +which O +was O +shown O +to O +be O +required O +for O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +by O +its O +SRA B-structure_element +domain O +, O +suggesting O +a O +conserved O +regulatory O +mechanism O +in O +SRA B-structure_element +domain O +- O +containing O +proteins O +. O + +As O +shown O +in O +the O +proposed O +model O +, O +recognition O +of O +H3K9me3 B-protein_type +by O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +is O +blocked O +to O +avoid O +its O +miss O +- O +localization O +to O +unmethylated B-protein_state +genomic O +region O +, O +in O +which O +chromatin O +contains O +H3K9me3 B-protein_type +( O +KD B-evidence += O +4 O +. O +61 O +μM O +) O +or O +H3K9me0 B-protein_type +( O +KD B-evidence += O +25 O +. O +99 O +μM O +). O + +This O +function O +is O +probably O +induced O +by O +a O +direct O +interaction O +between O +the O +SRA B-structure_element +and O +RFTSDNMT1 B-protein +( O +refs O +) O +or O +interaction O +between O +DNMT1 B-protein +and O +ubiquitylation B-ptm +of O +histione B-protein_type +tail O +. O + +In O +our O +in B-experimental_method +vitro I-experimental_method +assays I-experimental_method +, O +we O +could O +detect O +interaction O +between O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +and O +RFTSDNMT1 B-protein +, O +but O +not O +the O +interaction O +between O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +RFTSDNMT1 B-protein +( O +Supplementary O +Fig O +. O +8a O +, O +b O +and O +Fig O +. O +5e O +). O + +The O +results O +suggest O +that O +UHRF1 B-protein +adopts O +multiple O +conformations O +. O + +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +was O +incubated B-experimental_method +with O +GST B-protein_state +- I-protein_state +tagged I-protein_state +TTD B-structure_element +– I-structure_element +PHD I-structure_element +or O +TTD B-structure_element +in O +the O +presence B-protein_state +of I-protein_state +increasing O +concentrations O +of O +hm B-chemical +- I-chemical +DNA I-chemical +and O +analysed O +in O +pull B-experimental_method +- I-experimental_method +down I-experimental_method +experiment I-experimental_method +as O +described O +in O +a O +. O +The O +quantified O +band B-experimental_method +densitometries I-experimental_method +are O +indicated O +below O +the O +Coomassie B-experimental_method +blue I-experimental_method +staining I-experimental_method +. O + +This O +hierarchical O +assembly O +provides O +a O +model O +, O +in O +which O +full B-protein_state +- I-protein_state +length I-protein_state +Aβ B-protein +transitions O +from O +an O +unfolded B-protein_state +monomer B-oligomeric_state +to O +a O +folded B-protein_state +β B-structure_element +- I-structure_element +hairpin I-structure_element +, O +which O +assembles O +to O +form O +oligomers B-oligomeric_state +that O +further O +pack O +to O +form O +an O +annular B-site +pore I-site +. O + +In O +Alzheimer O +’ O +s O +disease O +, O +monomeric B-oligomeric_state +Aβ B-protein +aggregates O +to O +form O +soluble O +low O +molecular O +weight O +oligomers B-oligomeric_state +, O +such O +as O +dimers B-oligomeric_state +, O +trimers B-oligomeric_state +, O +tetramers B-oligomeric_state +, O +hexamers B-oligomeric_state +, O +nonamers B-oligomeric_state +, O +and O +dodecamers B-oligomeric_state +, O +as O +well O +as O +high O +molecular O +weight O +aggregates O +, O +such O +as O +annular B-complex_assembly +protofibrils I-complex_assembly +. O + +Mouse B-taxonomy_domain +models O +for O +Alzheimer O +’ O +s O +disease O +have O +helped O +shape O +our O +current O +understanding O +about O +the O +Aβ B-protein +oligomerization O +that O +precedes O +neurodegeneration O +. O + +Purified O +Aβ B-complex_assembly +* I-complex_assembly +56 I-complex_assembly +injected B-experimental_method +intercranially I-experimental_method +into O +healthy O +rats B-taxonomy_domain +was O +found O +to O +impair O +memory O +, O +providing O +evidence O +that O +this O +Aβ B-protein +oligomer B-oligomeric_state +may O +cause O +memory O +loss O +in O +Alzheimer O +’ O +s O +disease O +. O + +The O +approach O +of O +isolating O +and O +characterizing O +Aβ B-protein +oligomers B-oligomeric_state +has O +not O +provided O +any O +high O +- O +resolution O +structures B-evidence +of O +Aβ B-protein +oligomers B-oligomeric_state +. O + +The O +structure B-evidence +revealed O +that O +monomeric B-oligomeric_state +Aβ B-protein +forms O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +when O +bound B-protein_state +to I-protein_state +the O +affibody B-chemical +. O + +In O +the O +current O +study O +we O +set O +out O +to O +restore B-experimental_method +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +, O +reintroduce B-experimental_method +the O +methionine B-residue_name +residue O +at O +position O +35 B-residue_number +, O +and O +determine O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structures I-evidence +of O +oligomers B-oligomeric_state +that O +form O +. O + +We O +routinely O +use O +p B-chemical +- I-chemical +iodophenylalanine I-chemical +to O +determine O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +phases I-evidence +. O + +Upon O +synthesizing O +peptide B-mutant +3 I-mutant +, O +we O +found O +that O +it O +formed O +an O +amorphous O +precipitate O +in O +most O +crystallization O +conditions O +screened O +and O +failed O +to O +afford O +crystals B-evidence +in O +any O +condition O +. O + +To O +address O +this O +issue O +, O +we O +next O +incorporated O +a O +disulfide B-ptm +bond I-ptm +between O +residues O +24 B-residue_number +and O +29 B-residue_number +as O +a O +conformational O +constraint O +that O +serves O +as O +a O +surrogate O +for O +δOrn B-structure_element +. O + +We O +used O +acid B-protein_state +- I-protein_state +stable I-protein_state +Acm B-protein_state +- I-protein_state +protected I-protein_state +cysteine B-residue_name +residues O +at O +positions O +24 B-residue_number +and O +29 B-residue_number +and O +removed O +the O +Acm O +groups O +by O +oxidation O +with O +I2 O +in O +aqueous O +acetic B-chemical +acid I-chemical +to O +afford O +the O +disulfide B-ptm +linkage I-ptm +. O + +Peptide B-mutant +2 I-mutant +also O +afforded O +crystals B-evidence +in O +these O +conditions O +. O + +Crystal B-evidence +diffraction I-evidence +data I-evidence +for O +peptide B-mutant +2 I-mutant +were O +also O +collected O +at O +the O +Advanced O +Light O +Source O +at O +Lawrence O +Berkeley O +National O +Laboratory O +with O +a O +synchrotron O +source O +at O +1 O +. O +00 O +Å O +wavelength O +to O +achieve O +higher O +resolution O +. O + +Data O +for O +peptides B-mutant +4 I-mutant +and I-mutant +2 I-mutant +were O +scaled O +and O +merged O +using O +XDS O +. O + +No O +evidence O +for O +cleavage O +of O +the O +disulfides B-ptm +was O +observed O +in O +the O +refinement B-experimental_method +of O +the O +data O +set O +collected O +on O +the O +X O +- O +ray O +diffractometer O +, O +and O +we O +refined B-experimental_method +all O +disulfide B-ptm +linkages I-ptm +as O +intact B-protein_state +( O +PDB O +5HOY O +). O + +The O +trimer B-oligomeric_state +maintains O +all O +of O +the O +same O +stabilizing O +contacts O +as O +those O +of O +peptide B-mutant +1 I-mutant +. O + +Hydrogen B-bond_interaction +bonding I-bond_interaction +and O +hydrophobic B-bond_interaction +interactions I-bond_interaction +between O +residues O +on O +the O +β B-structure_element +- I-structure_element +strands I-structure_element +comprising O +Aβ17 B-protein +– B-residue_range +23 I-residue_range +and O +Aβ30 B-protein +– B-residue_range +36 I-residue_range +stabilize O +the O +core B-structure_element +of O +the O +trimer B-oligomeric_state +. O + +The O +disulfide B-ptm +bonds I-ptm +between O +residues O +24 B-residue_number +and O +29 B-residue_number +are O +adjacent O +to O +the O +structural B-structure_element +core I-structure_element +of O +the O +trimer B-oligomeric_state +and O +do O +not O +make O +any O +substantial O +intermolecular O +contacts O +. O + +Three O +ordered O +water B-chemical +molecules O +fill O +the O +hole O +in O +the O +center O +of O +the O +trimer B-oligomeric_state +, O +hydrogen B-bond_interaction +bonding I-bond_interaction +to O +each O +other O +and O +to O +the O +main O +chain O +of O +F20 B-residue_name_number +( O +Figure O +3A O +). O + +Figure O +4A O +illustrates O +the O +octahedral B-protein_state +shape O +of O +the O +dodecamer B-oligomeric_state +. O + +The O +electron B-evidence +density I-evidence +map I-evidence +for O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +2 I-mutant +has O +long O +tubes O +of O +electron B-evidence +density I-evidence +inside O +the O +central B-site +cavity I-site +of O +the O +dodecamer B-oligomeric_state +. O + +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +is O +a O +polypropylene O +glycol O +derivative O +with O +a O +2 O +- O +methoxyethoxy O +unit O +at O +one O +end O +and O +a O +2 O +- O +aminopropyl O +unit O +at O +the O +other O +end O +. O + +Annular B-site +Pore I-site + +The O +staggered B-protein_state +interfaces B-site +occur O +between O +dodecamers B-structure_element +2 I-structure_element +and I-structure_element +3 I-structure_element +and O +4 B-structure_element +and I-structure_element +5 I-structure_element +. O + +The O +same O +eclipsed B-site +interface I-site +also O +occurs O +between O +dodecamers B-structure_element +1 I-structure_element +and I-structure_element +5 I-structure_element +and O +3 B-structure_element +and I-structure_element +4 I-structure_element +. O +( O +C O +) O +Staggered B-site +interface I-site +between O +dodecamers B-structure_element +2 I-structure_element +and I-structure_element +3 I-structure_element +( O +side O +view O +). O + +The O +annular B-site +pore I-site +is O +comparable O +in O +size O +to O +other O +large O +protein O +assemblies O +. O + +At O +this O +point O +, O +we O +can O +only O +speculate O +whether O +the O +trimer B-oligomeric_state +and O +dodecamer B-oligomeric_state +formed O +by O +peptide B-mutant +2 I-mutant +share O +structural O +similarities O +to O +Aβ B-protein +trimers B-oligomeric_state +and O +Aβ B-complex_assembly +* I-complex_assembly +56 I-complex_assembly +, O +as O +little O +is O +known O +about O +the O +structure B-evidence +of O +Aβ B-protein +trimers B-oligomeric_state +and O +Aβ B-complex_assembly +* I-complex_assembly +56 I-complex_assembly +. O + +The O +difficulty O +in O +studying O +the O +oligomers B-oligomeric_state +formed O +in O +solution O +may O +reflect O +the O +propensity O +of O +the O +dodecamer B-oligomeric_state +to O +assemble O +on O +all O +four O +F20 B-residue_name_number +faces O +. O + +The O +crystallographically B-evidence +observed I-evidence +trimer B-oligomeric_state +recapitulates O +the O +Aβ B-protein +trimers B-oligomeric_state +that O +are O +observed O +even O +before O +the O +onset O +of O +symptoms O +in O +Alzheimer O +’ O +s O +disease O +. O + +Our O +approach O +of O +constraining O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +into O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +conformation O +and O +blocking O +aggregation O +with O +an O +N O +- O +methyl O +group O +has O +allowed O +us O +to O +crystallize B-experimental_method +a O +large O +fragment O +of O +what O +is O +generally O +considered O +to O +be O +an O +uncrystallizable O +peptide O +. O + +Predictive O +features O +of O +ligand O +‐ O +specific O +signaling O +through O +the O +estrogen B-protein_type +receptor I-protein_type + +For O +some O +ligand O +series O +, O +a O +single O +inter B-evidence +‐ I-evidence +atomic I-evidence +distance I-evidence +in O +the O +ligand B-structure_element +‐ I-structure_element +binding I-structure_element +domain I-structure_element +predicted O +their O +proliferative O +effects O +. O + +In O +contrast O +, O +the O +N O +‐ O +terminal O +coactivator B-site +‐ I-site +binding I-site +site I-site +, O +activation B-structure_element +function I-structure_element +‐ I-structure_element +1 I-structure_element +( O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +), O +determined O +cell O +‐ O +specific O +signaling O +induced O +by O +ligands O +that O +used O +alternate O +mechanisms O +to O +control O +cell O +proliferation O +. O + +Thus O +, O +incorporating O +systems B-experimental_method +structural I-experimental_method +analyses I-experimental_method +with O +quantitative B-experimental_method +chemical I-experimental_method +biology I-experimental_method +reveals O +how O +ligands O +can O +achieve O +distinct O +allosteric O +signaling O +outcomes O +through O +ERα B-protein +. O + +ERα B-protein +domain O +organization O +lettered O +, O +A O +‐ O +F O +. O +DBD B-structure_element +, O +DNA B-structure_element +‐ I-structure_element +binding I-structure_element +domain I-structure_element +; O +LBD B-structure_element +, O +ligand B-structure_element +‐ I-structure_element +binding I-structure_element +domain I-structure_element +; O +AF B-structure_element +, O +activation B-structure_element +function I-structure_element + +Linear O +causality O +model O +for O +ERα B-protein +‐ O +mediated O +cell O +proliferation O +. O + +Yet O +, O +it O +is O +unknown O +how O +different O +ERα B-protein +ligands O +control O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +through O +the O +LBD B-structure_element +, O +and O +whether O +this O +inter O +‐ O +domain O +communication O +is O +required O +for O +cell O +‐ O +specific O +signaling O +or O +anti O +‐ O +proliferative O +responses O +. O + +Our O +long O +‐ O +term O +goal O +is O +to O +be O +able O +to O +predict O +proliferative O +or O +anti O +‐ O +proliferative O +activity O +of O +a O +ligand O +in O +different O +tissues O +from O +its O +crystal B-evidence +structure I-evidence +by O +identifying O +different O +structural O +perturbations O +that O +lead O +to O +specific O +signaling O +outcomes O +. O + +In O +this O +signaling O +model O +, O +multiple O +coregulator O +binding O +events O +and O +target O +genes O +( O +Won O +Jeong O +et O +al O +, O +2012 O +; O +Nwachukwu O +et O +al O +, O +2014 O +), O +LBD B-structure_element +conformation O +, O +nucleocytoplasmic O +shuttling O +, O +the O +occupancy O +and O +dynamics O +of O +DNA O +binding O +, O +and O +other O +biophysical O +features O +could O +contribute O +independently O +to O +cell O +proliferation O +( O +Lickwar O +et O +al O +, O +2012 O +). O + +We O +also O +determined B-experimental_method +the O +structures B-evidence +of O +76 O +distinct O +ERα B-protein +LBD B-structure_element +complexes O +bound B-protein_state +to I-protein_state +different O +ligand O +types O +, O +which O +allowed O +us O +to O +understand O +how O +diverse O +ligand O +scaffolds O +distort O +the O +active B-protein_state +conformation O +of O +the O +ERα B-protein +LBD B-structure_element +. O + +To O +compare O +ERα B-protein +signaling O +induced O +by O +diverse O +ligand O +types O +, O +we O +synthesized B-experimental_method +and I-experimental_method +assayed I-experimental_method +a O +library O +of O +241 O +ERα B-protein +ligands O +containing O +19 O +distinct O +molecular O +scaffolds O +. O + +Structure B-evidence +of O +the O +E2 B-protein_state +‐ I-protein_state +bound I-protein_state +ERα B-protein +LBD B-structure_element +in B-protein_state +complex I-protein_state +with I-protein_state +an O +NCOA2 B-protein +peptide O +of O +( O +PDB O +1GWR O +). O + +The O +ERα B-protein +ligand O +library O +contains O +241 O +ligands O +representing O +15 O +indirect O +modulator O +scaffolds O +, O +plus O +4 O +direct O +modulator O +scaffolds O +. O + +To O +test O +this O +idea O +, O +we O +compared O +the O +average B-evidence +L I-evidence +‐ I-evidence +Luc I-evidence +activities I-evidence +of O +each O +scaffold O +in O +HepG2 O +cells O +co B-experimental_method +‐ I-experimental_method +transfected I-experimental_method +with O +wild B-protein_state +‐ I-protein_state +type I-protein_state +ERα B-protein +or O +with O +ERα B-protein +lacking B-protein_state +the I-protein_state +AB B-structure_element +domain O +( O +Figs O +1B O +and O +EV1 O +). O + +Deletion B-experimental_method +of I-experimental_method +the O +AB B-structure_element +domain O +significantly O +reduced O +the O +average B-evidence +L I-evidence +‐ I-evidence +Luc I-evidence +activities I-evidence +of O +14 O +scaffolds O +( O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +‐ I-experimental_method +test I-experimental_method +, O +P B-evidence +≤ O +0 O +. O +05 O +) O +( O +Fig O +3B O +). O + +This O +cluster O +includes O +two O +direct O +modulator O +scaffolds O +( O +OBHS B-chemical +‐ I-chemical +ASC I-chemical +and O +OBHS B-chemical +‐ I-chemical +BSC I-chemical +), O +and O +five O +indirect O +modulator O +scaffolds O +( O +A B-chemical +‐ I-chemical +CD I-chemical +, O +cyclofenil B-chemical +, O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +, O +imine B-chemical +, O +and O +imidazopyridine B-chemical +). O + +Thus O +, O +examining O +the O +correlated O +patterns O +of O +ERα B-protein +activity O +within O +each O +scaffold O +demonstrates O +that O +an O +extended O +side O +chain O +is O +not O +required O +for O +cell O +‐ O +specific O +signaling O +. O + +To O +evaluate O +the O +role O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +and O +the O +F B-structure_element +domain O +in O +ERα B-protein +signaling O +specificity O +, O +we O +compared O +activity O +of O +truncated O +ERα B-protein +constructs O +in O +HepG2 O +liver O +cells O +with O +endogenous O +ERα B-protein +activity O +in O +the O +other O +cell O +types O +. O + +Thus O +, O +in O +cluster O +2 O +, O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +substantially O +modulated O +the O +specificity O +of O +ligands O +with O +cell O +‐ O +specific O +activity O +( O +Fig O +3D O +lanes O +5 O +– O +12 O +). O + +Thus O +, O +ligands O +in O +cluster O +2 O +rely O +on O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +for O +both O +activity O +( O +Fig O +3B O +) O +and O +signaling O +specificity O +( O +Fig O +3D O +). O + +The O +average O +induction O +of O +GREB1 B-protein +by O +cluster O +1 O +ligands O +showed O +greater O +variance O +, O +with O +a O +range O +between O +~ O +25 O +and O +~ O +75 O +% O +for O +OBHS B-chemical +and O +a O +range O +from O +full O +agonist O +to O +inverse O +agonist O +for O +the O +others O +in O +cluster O +1 O +( O +Fig O +EV2A O +). O + +The O +significant O +correlations O +with O +GREB1 B-protein +expression O +and O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +observed O +in O +this O +cluster O +are O +consistent O +with O +the O +canonical O +signaling O +model O +( O +Fig O +1D O +), O +where O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +determines O +GREB1 B-protein +expression O +, O +which O +then O +drives O +proliferation O +. O + +Despite O +this O +phenotypic O +variance O +, O +proliferation O +was O +not O +generally O +predicted O +by O +correlated O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +and O +GREB1 B-protein +induction O +( O +Figs O +3F O +lanes O +5 O +– O +19 O +, O +and O +EV3H O +). O + +NCOA3 B-protein +occupancy O +at O +GREB1 B-protein +did O +not O +predict O +the O +proliferative O +response O + +NCOA3 B-protein +occupancy O +at O +GREB1 B-protein +is O +statistically O +robust O +but O +does O +not O +predict O +transcriptional O +activity O + +In O +panel O +( O +C O +), O +correlation B-experimental_method +analysis I-experimental_method +was O +performed O +for O +two O +biological O +replicates O +. O + +The O +M2H B-experimental_method +assay I-experimental_method +for O +NCOA3 B-protein +recruitment O +broadly O +correlated O +with O +the O +other O +assays O +, O +and O +was O +predictive O +for O +GREB1 B-protein +expression O +and O +cell O +proliferation O +( O +Fig O +3E O +). O + +ERβ B-protein +activity O +is O +not O +an O +independent O +predictor O +of O +cell O +‐ O +specific O +activity O + +ERα B-protein +activity O +of O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +and O +cyclofenil B-chemical +analogs O +correlates O +with O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O +. O + +Examining O +many O +closely O +related O +structures B-evidence +allows O +us O +to O +visualize O +subtle O +structural O +differences O +, O +in O +effect O +using O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +as O +a O +systems O +biology O +tool O +. O + +The O +24 O +structures B-evidence +containing O +OBHS B-chemical +, O +OBHS B-chemical +‐ I-chemical +N I-chemical +, O +or O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +showed O +structural O +diversity O +in O +the O +same O +part O +of O +the O +scaffolds O +( O +Figs O +5A O +and O +EV5A O +), O +and O +the O +same O +region O +of O +the O +LBD B-structure_element +— O +the O +C O +‐ O +terminal O +end O +of O +h11 B-structure_element +( O +Figs O +5B O +and O +C O +, O +and O +EV5B O +), O +which O +in O +turn O +nudges O +h12 B-structure_element +( O +Fig O +5C O +and O +D O +). O + +Triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +bound B-protein_state +to I-protein_state +the O +superposed B-experimental_method +crystal B-evidence +structures I-evidence +of O +the O +ERα B-protein +LBD B-structure_element +are O +shown O +. O + +Panel O +( O +A O +) O +shows O +the O +crystal B-evidence +structure I-evidence +of O +an O +S B-protein_state +‐ I-protein_state +OBHS I-protein_state +‐ I-protein_state +3 I-protein_state +‐ I-protein_state +bound I-protein_state +ERα B-protein +LBD B-structure_element +( O +PDB O +5DUH O +). O + +Hierarchical B-experimental_method +clustering I-experimental_method +of O +ligand O +‐ O +specific O +binding O +of O +154 O +interacting O +peptides O +to O +the O +ERα B-protein +LBD B-structure_element +was O +performed O +in O +triplicate O +by O +MARCoNI B-experimental_method +analysis I-experimental_method +. O + +One O +phenol O +pushed O +further O +toward O +h3 B-structure_element +( O +Fig O +6D O +), O +while O +the O +other O +phenol O +pushed O +toward O +the O +C O +‐ O +terminus O +of O +h11 B-structure_element +to O +a O +greater O +extent O +than O +A B-chemical +‐ I-chemical +CD I-chemical +‐ O +ring O +estrogens B-chemical +( O +Nwachukwu O +et O +al O +, O +2014 O +), O +which O +are O +close O +structural O +analogs O +of O +E2 B-chemical +that O +lack O +a O +B O +‐ O +ring O +( O +Fig O +2 O +). O + +Despite O +the O +similar O +average O +activities O +of O +these O +ligand O +classes O +( O +Fig O +3A O +and O +B O +), O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +and O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +displayed O +remarkably O +different O +peptide O +recruitment O +patterns O +( O +Fig O +6H O +), O +consistent O +with O +the O +structural B-experimental_method +analyses I-experimental_method +. O + +Thus O +, O +the O +isomeric O +attachment O +of O +diaryl O +groups O +to O +the O +thiophene B-chemical +core O +changed O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +from O +inside O +the O +ligand B-site +‐ I-site +binding I-site +pocket I-site +, O +as O +predicted O +by O +the O +crystal B-evidence +structures I-evidence +. O + +This O +perturbation O +determined O +proliferation O +that O +correlated O +strongly O +with O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +activity O +, O +recruitment O +of O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +family O +members O +, O +and O +induction O +of O +the O +GREB1 B-protein +gene O +, O +consistent O +with O +the O +canonical O +ERα B-protein +signaling O +pathway O +( O +Fig O +1D O +). O + +This O +finding O +can O +be O +explained O +by O +the O +fact O +that O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +contain O +distinct O +binding B-site +sites I-site +for O +interaction O +with O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +and O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +( O +McInerney O +et O +al O +, O +1996 O +; O +Webb O +et O +al O +, O +1998 O +), O +which O +allows O +ligands O +to O +nucleate O +ERα B-complex_assembly +– I-complex_assembly +NCOA1 I-complex_assembly +/ I-complex_assembly +2 I-complex_assembly +/ I-complex_assembly +3 I-complex_assembly +interaction O +through O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +, O +and O +reinforce O +this O +interaction O +with O +additional O +binding O +to O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +. O + +For O +K B-species +. I-species +pneumoniae I-species +, O +this O +is O +the O +first O +high O +- O +resolution O +cleavage O +complex O +structure B-evidence +to O +be O +reported O +. O + +This O +complex O +is O +compared O +with O +a O +similar O +complex O +from O +Streptococcus B-species +pneumoniae I-species +, O +which O +has O +recently O +been O +solved O +. O + +Acquiring O +a O +deep O +structural O +and O +functional O +understanding O +of O +the O +mode O +of O +action O +of O +fluoroquinolones B-chemical +( O +Tomašić O +& O +Mašič O +, O +2014 O +) O +and O +the O +development O +of O +new O +drugs O +targeted O +against O +topoisomerase B-complex_assembly +IV I-complex_assembly +and O +gyrase B-protein_type +from O +a O +wide O +range O +of O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +and O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +pathogenic O +bacteria B-taxonomy_domain +are O +highly O +active O +areas O +of O +current O +research O +directed O +at O +overcoming O +the O +vexed O +problem O +of O +drug O +resistance O +( O +Bax O +et O +al O +., O +2010 O +; O +Chan O +et O +al O +., O +2015 O +; O +Drlica O +et O +al O +., O +2014 O +; O +Mutsaev O +et O +al O +., O +2014 O +; O +Pommier O +, O +2013 O +; O +Srikannathasan O +et O +al O +., O +2015 O +). O + +In O +both O +cases O +the O +DNA B-chemical +is O +bent O +into O +a O +U B-protein_state +- I-protein_state +form I-protein_state +and O +bound B-protein_state +snugly O +against O +the O +protein O +of O +the O +G B-structure_element +- I-structure_element +gate I-structure_element +. O + +The O +sequence B-experimental_method +alignment I-experimental_method +is O +given O +in O +Supplementary O +Fig O +. O +S1 O +, O +with O +the O +key O +metal B-site +- I-site +binding I-site +residues I-site +and O +those O +which O +give O +rise O +to O +quinolone O +resistance O +highlighted O +. O + +The O +side O +chains O +surrounding O +them O +in O +ParE B-protein +are O +quite O +disordered O +and O +are O +more O +defined O +in O +K B-species +. I-species +pneumoniae I-species +( O +even O +though O +this O +complex O +is O +at O +lower O +resolution O +) O +than O +in O +S B-species +. I-species +pneumoniae I-species +. O + +There O +are O +no O +direct O +hydrogen B-bond_interaction +bonds I-bond_interaction +from O +the O +drug O +to O +these O +residues O +( O +although O +it O +is O +possible O +that O +some O +are O +formed O +through O +water B-chemical +, O +which O +cannot O +be O +observed O +at O +this O +resolution O +). O + +The O +CC25 B-evidence +( O +the O +drug O +concentration O +that O +converted O +25 O +% O +of O +the O +supercoiled O +DNA B-chemical +substrate O +to O +a O +linear O +form O +) O +was O +0 O +. O +5 O +µM O +for O +the O +Klebsiella B-taxonomy_domain +enzyme O +and O +1 O +µM O +for O +the O +pneumococcal B-taxonomy_domain +enzyme O +. O + +Moreover O +, O +although O +topoisomerase B-complex_assembly +IV I-complex_assembly +is O +primarily O +the O +target O +of O +levofloxacin B-chemical +in O +S B-species +. I-species +pneumoniae I-species +, O +it O +is O +likely O +to O +be O +gyrase B-protein_type +in O +the O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +K B-species +. I-species +pneumoniae I-species +. O + +The O +magnesium B-chemical +ions O +and O +their O +coordinating O +amino O +acids O +are O +shown O +in O +purple O +. O + +The O +active B-site +- I-site +site I-site +tyrosine B-residue_name +and O +arginine B-residue_name +are O +in O +orange O +. O + +The O +ParC B-protein +and O +ParE B-protein +backbones O +are O +shown O +in O +blue O +and O +yellow O +, O +respectively O +. O + +Comparison O +of O +DNA B-chemical +cleavage O +by O +topoisomerase B-complex_assembly +IV I-complex_assembly +core O +ParE B-complex_assembly +- I-complex_assembly +ParC I-complex_assembly +fusion O +proteins O +from O +K B-species +. I-species +pneumoniae I-species +( O +KP B-species +) O +and O +S B-species +. I-species +pneumoniae I-species +( O +SP B-species +) O +promoted O +by O +levofloxacin B-chemical +. O + +Lane O +A O +, O +supercoiled O +pBR322 O +DNA B-chemical +; O +N O +, O +L O +and O +S O +, O +nicked O +, O +linear O +and O +supercoiled O +pBR322 O +, O +respectively O +. O + +Using O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +to O +Map O +Small O +Ligands O +on O +Dynamic O +Metabolic O +Enzymes O +: O +Studies O +with O +Glutamate B-protein_type +Dehydrogenase I-protein_type + +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallographic I-experimental_method +studies I-experimental_method +have O +shown O +that O +the O +functional O +unit O +of O +GDH B-protein_type +is O +a O +homohexamer B-oligomeric_state +composed O +of O +a O +trimer B-oligomeric_state +of O +dimers B-oligomeric_state +, O +with O +a O +3 O +- O +fold O +axis O +and O +an O +equatorial O +plane O +that O +define O +its O +D3 O +symmetry O +( O +Fig O +. O +1A O +). O + +Each O +56 O +- O +kDa O +protomer B-oligomeric_state +consists O +of O +three O +domains O +. O + +Structure O +and O +quaternary O +conformational O +changes O +in O +GDH B-protein_type +. O +( O +A O +) O +Views O +of O +open B-protein_state +( O +PDB O +ID O +1NR7 O +) O +and O +closed B-protein_state +( O +PDB O +3MW9 O +) O +states O +of O +the O +GDH B-protein_type +hexamer B-oligomeric_state +, O +shown O +in O +ribbon O +representation O +perpendicular O +to O +the O +2 O +- O +fold O +symmetry O +axis O +( O +side O +view O +, O +top O +) O +and O +3 O +- O +fold O +symmetry O +axis O +( O +top O +view O +, O +bottom O +). O + +The O +dashed O +lines O +and O +arrows O +, O +respectively O +, O +highlight O +the O +slight O +extension O +in O +length O +, O +and O +twist O +in O +shape O +that O +occurs O +with O +transition O +from O +open B-protein_state +to O +the O +closed B-protein_state +state O +. O + +The O +open B-protein_state +state O +shown O +is O +for O +unliganded B-protein_state +GDH B-protein_type +, O +whereas O +the O +closed B-protein_state +state O +has O +NADH B-chemical +, O +GTP B-chemical +, O +and O +glutamate B-chemical +bound B-protein_state +. O +( O +B O +) O +Superposition B-experimental_method +of O +structures B-evidence +for O +closed B-protein_state +and O +open B-protein_state +conformations O +, O +along O +with O +a O +series O +of O +possible O +intermediate O +conformations O +along O +the O +trajectory O +that O +serve O +to O +illustrate O +the O +extent O +of O +change O +in O +structure O +across O +different O +regions O +of O +the O +protein O +. O + +These O +allosteric O +modulators O +tightly O +control O +GDH B-protein_type +function O +in O +vivo O +. O + +Although O +there O +are O +numerous O +crystal B-evidence +structures I-evidence +available O +for O +GDH B-protein_type +in B-protein_state +complex I-protein_state +with I-protein_state +cofactors O +and O +nucleotides O +, O +they O +are O +limited O +to O +the O +combinations O +that O +have O +been O +amenable O +to O +crystallization B-experimental_method +. O + +When O +GDH B-protein +is O +bound B-protein_state +to I-protein_state +NADH B-chemical +, O +GTP B-chemical +, O +and O +glutamate B-chemical +, O +the O +enzyme O +adopts O +a O +closed B-protein_state +conformation O +; O +this O +“ O +abortive O +complex O +” O +has O +been O +determined O +to O +2 O +. O +4 O +- O +Å O +resolution O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +( O +PDB O +3MW9 O +). O + +However O +, O +crystal B-evidence +structures I-evidence +of O +GDH B-protein +bound B-protein_state +only I-protein_state +to I-protein_state +NADH B-chemical +or O +to B-protein_state +GTP B-chemical +have O +not O +yet O +been O +reported O +. O + +Comparison O +of O +the O +NADH B-protein_state +- I-protein_state +bound I-protein_state +closed B-protein_state +conformation O +to O +the O +NADH B-protein_state +- I-protein_state +bound I-protein_state +open B-protein_state +conformation O +shows O +that O +, O +as O +expected O +, O +the O +catalytic B-site +cleft I-site +is O +closed B-protein_state +and O +the O +NBDs B-structure_element +are O +displaced O +toward O +the O +equatorial O +plane O +, O +accompanied O +by O +a O +rotation O +of O +the O +pivot B-structure_element +helix I-structure_element +by O +∼ O +7 O +°, O +concomitant O +with O +a O +large O +conformational O +change O +in O +the O +antennae B-structure_element +domains O +( O +Figs O +. O +1 O +and O +2D O +). O + +A O +comparison O +between O +NADH B-protein_state +- I-protein_state +bound I-protein_state +open B-protein_state +and O +closed B-protein_state +conformations O +also O +involves O +a O +displacement O +of O +helix B-structure_element +5 I-structure_element +( O +residues O +171 B-residue_range +– I-residue_range +186 I-residue_range +), O +as O +well O +as O +a O +tilt O +of O +the O +core O +β B-structure_element +- I-structure_element +sheets I-structure_element +relative O +to O +the O +equatorial O +plane O +of O +the O +enzyme O +( O +residues O +57 B-residue_range +– I-residue_range +97 I-residue_range +, O +122 B-residue_range +– I-residue_range +130 I-residue_range +) O +and O +α B-structure_element +- I-structure_element +helix I-structure_element +2 I-structure_element +( O +residues O +36 B-residue_range +– I-residue_range +54 I-residue_range +), O +and O +a O +bending O +of O +the O +N O +- O +terminal O +helix B-structure_element +. O + +Although O +there O +is O +a O +difference O +in O +orientation O +of O +the O +nicotinamide O +moiety O +between O +the O +closed B-protein_state +and O +open B-protein_state +states O +in O +the O +regulatory B-site +site I-site +, O +in O +both O +structures B-evidence +the O +adenine O +portion O +of O +NADH B-chemical +has O +a O +similar O +binding B-site +pocket I-site +and O +is O +located O +in O +almost O +exactly O +the O +same O +position O +as O +ADP B-chemical +, O +a O +potent O +activator O +of O +GDH B-protein +function O +( O +Supplemental O +Fig O +. O +5 O +). O + +This O +suggests O +that O +although O +the O +conformation O +of O +NADH B-chemical +in O +the O +open B-protein_state +state O +regulatory B-site +site I-site +more O +closely O +mimics O +the O +binding O +of O +ADP B-chemical +, O +the O +conformation O +of O +NADH B-chemical +in O +the O +closed B-protein_state +state O +regulatory B-site +site I-site +is O +significantly O +different O +; O +these O +differences O +may O +contribute O +to O +the O +opposite O +effects O +of O +NADH B-chemical +and O +ADP B-chemical +on O +GDH B-protein +enzymatic O +activity O +. O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structure B-evidence +of O +GDH B-protein +bound B-protein_state +to I-protein_state +both O +NADH B-chemical +and O +GTP B-chemical +. O + +( O +C O +, O +D O +) O +Detailed O +inspection O +of O +the O +interactions O +near O +the O +regulatory B-site +site I-site +show O +that O +the O +orientation O +of O +His209 B-residue_name_number +switches O +between O +the O +two O +states O +, O +which O +may O +allow O +interactions O +with O +bound B-protein_state +GTP B-chemical +in O +the O +closed B-protein_state +( O +D O +), O +but O +not O +open B-protein_state +( O +C O +) O +conformation O +. O + +Our O +structural B-experimental_method +studies I-experimental_method +thus O +establish O +that O +whether O +or O +not O +GTP B-chemical +is O +bound B-protein_state +, O +NADH B-chemical +binding O +is O +detectable O +at O +catalytic B-site +and I-site +regulatory I-site +sites I-site +, O +in O +both O +the O +open B-protein_state +and O +closed B-protein_state +conformational O +states O +. O + +The O +role O +of O +the O +nicotinamide O +moiety O +in O +acting O +as O +a O +wedge O +that O +prevents O +the O +transition O +to O +the O +open B-protein_state +conformation O +also O +suggests O +a O +structural O +explanation O +of O +the O +mechanism O +by O +which O +NADH B-chemical +binding O +inhibits O +the O +activity O +of O +the O +enzyme O +by O +stabilizing O +the O +closed B-protein_state +conformation O +state O +. O + +We O +have O +solved B-experimental_method +the O +structure B-evidence +of O +the O +HR1 B-structure_element +domain O +of O +TOCA1 B-protein +, O +providing O +the O +first O +structural B-evidence +data I-evidence +for O +this O +protein O +. O + +All O +members O +share O +a O +well O +defined O +core O +structure O +of O +∼ O +20 O +kDa O +known O +as O +the O +G B-structure_element +domain I-structure_element +, O +which O +is O +responsible O +for O +guanine B-chemical +nucleotide I-chemical +binding O +. O + +The O +overall O +conformation O +of O +small B-protein_type +G I-protein_type +proteins I-protein_type +in O +the O +active B-protein_state +and O +inactive B-protein_state +states O +is O +similar O +, O +but O +they O +differ O +significantly O +in O +two O +main O +regions O +known O +as O +switch B-site +I I-site +and O +switch B-site +II I-site +. O + +However O +, O +because O +each O +of O +the O +150 O +members O +of O +the O +superfamily O +interacts O +with O +multiple O +effectors O +, O +there O +are O +still O +a O +huge O +number O +of O +known O +G B-protein_type +protein I-protein_type +- O +effector O +interactions O +that O +have O +not O +yet O +been O +studied O +structurally O +. O + +The O +most O +widely O +studied O +role O +of O +TOCA1 B-protein +is O +in O +membrane O +invagination O +and O +endocytosis O +, O +although O +it O +has O +also O +been O +implicated O +in O +filopodia O +formation O +, O +neurite O +elongation O +, O +transcriptional O +reprogramming O +via O +nuclear O +actin B-protein_type +, O +and O +interaction O +with O +ZO B-protein +- I-protein +1 I-protein +at O +tight O +junctions O +. O + +How O +different O +HR1 B-structure_element +domain O +proteins O +distinguish O +their O +specific O +G B-protein_type +protein I-protein_type +partners O +remains O +only O +partially O +understood O +, O +and O +structural O +characterization O +of O +a O +novel O +G B-protein_type +protein I-protein_type +- O +HR1 B-structure_element +domain O +interaction O +would O +add O +to O +the O +growing O +body O +of O +information O +pertaining O +to O +these O +protein O +complexes O +. O + +We O +also O +present O +data O +pertaining O +to O +binding O +of O +the O +TOCA B-protein_type +HR1 B-structure_element +domain O +to O +Cdc42 B-protein +, O +which O +is O +the O +first O +biophysical O +description O +of O +an O +HR1 B-structure_element +domain O +binding O +this O +particular O +Rho B-protein_type +family I-protein_type +small I-protein_type +G I-protein_type +protein I-protein_type +. O + +The O +data O +were O +fitted O +to O +a O +binding B-evidence +isotherm I-evidence +describing O +competition O +. O + +As O +the O +observed O +affinity B-evidence +between O +TOCA1 B-protein +HR1 B-structure_element +and O +Cdc42 B-protein +was O +much O +lower O +than O +expected O +, O +we O +reasoned O +that O +the O +C O +terminus O +of O +Cdc42 B-protein +might O +be O +necessary O +for O +a O +high O +affinity B-evidence +interaction O +. O + +Thus O +, O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +Cdc42 B-protein +is O +not O +required O +for O +maximal O +binding O +of O +TOCA1 B-protein +HR1 B-structure_element +. O + +The O +SPA B-experimental_method +signal O +was O +corrected O +by O +subtraction O +of O +control O +data O +with O +no O +fusion O +protein O +. O + +The O +low O +affinity O +of O +the O +TOCA1 B-protein +HR1 B-structure_element +- O +Cdc42 B-protein +interaction O +raised O +the O +question O +of O +whether O +the O +other O +known O +Cdc42 B-protein +- O +binding O +TOCA B-protein_type +family I-protein_type +proteins I-protein_type +, O +FBP17 B-protein +and O +CIP4 B-protein +, O +also O +bind O +weakly O +. O + +Initial O +experiments O +were O +performed O +with O +TOCA1 B-protein +residues O +324 B-residue_range +– I-residue_range +426 I-residue_range +, O +but O +we O +observed O +that O +the O +N O +terminus O +was O +cleaved O +during O +purification O +to O +yield O +a O +new O +N O +terminus O +at O +residue O +330 B-residue_number +( O +data O +not O +shown O +). O + +We O +therefore O +engineered O +a O +construct O +comprising O +residues O +330 B-residue_range +– I-residue_range +426 I-residue_range +to O +produce O +the O +minimal B-protein_state +, O +stable B-protein_state +HR1 B-structure_element +domain O +. O + +A O +, O +the O +backbone O +trace B-evidence +of O +the O +35 O +lowest O +energy O +structures B-evidence +of O +the O +HR1 B-structure_element +domain O +overlaid O +with O +the O +structure B-evidence +closest O +to O +the O +mean O +is O +shown O +alongside O +a O +schematic O +representation O +of O +the O +structure B-evidence +closest O +to O +the O +mean O +. O + +D O +, O +a O +close O +- O +up O +of O +the O +interhelix B-structure_element +loop I-structure_element +region O +showing O +some O +of O +the O +contacts O +between O +the O +loop B-structure_element +and O +helix B-structure_element +1 I-structure_element +. O + +Overall O +chemical B-experimental_method +shift I-experimental_method +perturbations I-experimental_method +( O +CSPs B-experimental_method +) O +were O +calculated O +for O +each O +residue O +, O +whereas O +those O +that O +had O +disappeared O +were O +assigned O +a O +shift O +change O +of O +0 O +. O +2 O +( O +Fig O +. O +4B O +). O + +Mapping O +the O +binding B-site +surface I-site +of O +Cdc42 B-protein +onto O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +. O + +TOCA1 B-protein +residues O +whose O +signals O +were O +affected O +by O +Cdc42 B-protein +binding O +were O +mapped O +onto O +the O +structure B-evidence +of O +TOCA1 B-protein +HR1 B-structure_element +( O +Fig O +. O +4C O +). O + +As O +was O +the O +case O +when O +labeled B-protein_state +HR1 B-structure_element +was O +observed O +, O +several O +peaks O +were O +shifted O +in O +the O +complex O +, O +but O +many O +disappeared O +, O +indicating O +exchange O +on O +an O +unfavorable O +, O +millisecond O +time O +scale O +( O +Fig O +. O +5A O +). O + +A O +, O +the O +15N B-experimental_method +HSQC I-experimental_method +of O +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +is O +shown O +in O +its O +free B-protein_state +form I-protein_state +( O +black O +) O +and O +in O +the O +presence B-protein_state +of I-protein_state +excess O +TOCA1 B-protein +HR1 B-structure_element +domain O +( O +1 O +: O +2 O +. O +2 O +, O +red O +). O + +The O +flexible B-protein_state +switch B-site +regions I-site +are O +circled O +. O + +Although O +the O +binding B-site +interface I-site +may O +be O +overestimated O +, O +this O +suggests O +that O +the O +switch B-site +regions I-site +are O +involved O +in O +binding O +to O +TOCA1 B-protein +. O + +The O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1TOCA1 I-complex_assembly +complex O +was O +not O +amenable O +to O +full O +structural O +analysis O +due O +to O +the O +weak O +interaction O +and O +the O +extensive O +exchange O +broadening O +seen O +in O +the O +NMR B-experimental_method +experiments O +. O + +The O +cluster O +with O +the O +lowest O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +from O +the O +lowest O +energy O +structure B-evidence +is O +assumed O +to O +be O +the O +best O +model O +. O + +By O +these O +criteria O +, O +in O +the O +best O +model O +, O +the O +HR1 B-structure_element +domain O +is O +in O +a O +similar O +orientation O +to O +the O +HR1a B-structure_element +domain O +of O +PRK1 B-protein +bound B-protein_state +to I-protein_state +RhoA B-protein +and O +the O +HR1b B-structure_element +domain O +bound B-protein_state +to I-protein_state +Rac1 B-protein +. O + +Model O +of O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1 I-complex_assembly +complex O +. O + +C O +, O +sequence B-experimental_method +alignment I-experimental_method +of O +RhoA B-protein +, O +Cdc42 B-protein +and O +Rac1 B-protein +. O + +Some O +of O +these O +can O +be O +rationalized O +; O +for O +example O +, O +Thr B-residue_name_number +- I-residue_name_number +24Cdc42 I-residue_name_number +, O +Leu B-residue_name_number +- I-residue_name_number +160Cdc42 I-residue_name_number +, O +and O +Lys B-residue_name_number +- I-residue_name_number +163Cdc42 I-residue_name_number +all O +pack O +behind O +switch B-site +I I-site +and O +are O +likely O +to O +be O +affected O +by O +conformational O +changes O +within O +the O +switch B-site +, O +while O +Glu B-residue_name_number +- I-residue_name_number +95Cdc42 I-residue_name_number +and O +Lys B-residue_name_number +- I-residue_name_number +96Cdc42 I-residue_name_number +are O +in O +the O +helix B-structure_element +behind O +switch B-site +II I-site +. O + +Lys B-residue_name_number +- I-residue_name_number +16Cdc42 I-residue_name_number +is O +unlikely O +to O +be O +a O +contact O +residue O +because O +it O +is O +involved O +in O +nucleotide O +binding O +, O +but O +the O +others O +may O +represent O +specific O +Cdc42 B-complex_assembly +- I-complex_assembly +TOCA1 I-complex_assembly +contacts O +. O + +Studies O +in O +CHO O +cells O +indicated O +that O +a O +Cdc42 B-complex_assembly +· I-complex_assembly +N I-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +complex O +existed O +because O +FRET B-evidence +was O +observed O +between O +RFP B-chemical +- O +TOCA1 B-protein +and O +GFP B-chemical +- O +N B-protein +- I-protein +WASP I-protein +, O +and O +the O +efficiency O +was O +decreased O +when O +an O +N B-protein +- I-protein +WASP I-protein +mutant B-protein_state +was O +used O +that O +no O +longer O +binds O +Cdc42 B-protein +. O + +A O +basic O +region O +in O +WASP B-protein +including O +three O +lysines B-residue_name +( O +residues O +230 B-residue_range +– I-residue_range +232 I-residue_range +), O +N O +- O +terminal O +to O +the O +core O +CRIB B-structure_element +, O +has O +been O +implicated O +in O +an O +electrostatic O +steering O +mechanism O +, O +and O +these O +residues O +would O +be O +free O +to O +bind O +in O +the O +presence B-protein_state +of I-protein_state +TOCA1 B-protein +HR1 B-structure_element +( O +Fig O +. O +7A O +). O + +The O +core O +CRIB B-structure_element +region O +of O +WASP B-protein +is O +shown O +in O +red O +, O +whereas O +its O +basic O +region O +is O +shown O +in O +orange O +and O +the O +C O +- O +terminal O +region O +required O +for O +maximal O +affinity O +is O +shown O +in O +cyan O +. O + +Unlabeled B-protein_state +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +was O +titrated B-experimental_method +into O +15N B-chemical +- O +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +, O +and O +the O +backbone O +NH O +groups O +were O +monitored O +using O +HSQCs B-experimental_method +( O +Fig O +. O +7C O +). O + +These O +experiments O +were O +recorded O +at O +sufficiently O +high O +protein O +concentrations O +( O +145 O +μm O +Cdc42 B-protein +, O +145 O +μm O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +, O +725 O +μm O +TOCA1 B-protein +HR1 B-structure_element +domain O +) O +to O +be O +far O +in O +excess O +of O +the O +Kd B-evidence +values O +of O +the O +individual O +interactions O +( O +TOCA1 B-protein +Kd B-evidence +≈ O +5 O +μm O +, O +N B-protein +- I-protein +WASP I-protein +Kd B-evidence += O +37 O +nm O +). O + +The O +spectrum B-evidence +when O +N B-protein +- I-protein +WASP I-protein +and O +TOCA1 B-protein +were O +equimolar O +was O +identical O +to O +that O +of O +the O +free B-protein_state +HR1 B-structure_element +domain O +, O +whereas O +the O +spectrum B-evidence +in O +the O +presence B-protein_state +of I-protein_state +0 O +. O +25 O +eq O +of O +N B-protein +- I-protein +WASP I-protein +was O +intermediate O +between O +the O +TOCA1 B-protein +HR1 B-structure_element +free B-protein_state +and O +complex B-protein_state +spectra B-evidence +( O +Fig O +. O +7D O +). O + +Actin B-protein_type +polymerization O +in O +all O +cases O +was O +initiated O +by O +the O +addition O +of O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +- O +containing O +liposomes O +. O + +The O +GBD B-structure_element +presumably O +acts O +as O +a O +dominant O +negative O +, O +sequestering O +endogenous O +Cdc42 B-protein +and O +preventing O +endogenous B-protein_state +full B-protein_state +- I-protein_state +length I-protein_state +N B-protein +- I-protein +WASP I-protein +from O +binding O +and O +becoming O +activated O +. O + +This O +is O +over O +100 O +times O +lower O +than O +that O +of O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +( O +Kd B-evidence += O +37 O +nm O +) O +and O +considerably O +lower O +than O +other O +known O +G B-protein_type +protein I-protein_type +- O +HR1 B-structure_element +domain O +interactions O +. O + +The O +polybasic O +tract O +within O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +Cdc42 B-protein +does O +not O +appear O +to O +be O +required O +for O +binding O +to O +TOCA1 B-protein +, O +which O +is O +in O +contrast O +to O +the O +interaction O +between O +Rac1 B-protein +and O +the O +HR1b B-structure_element +domain O +of O +PRK1 B-protein +but O +more O +similar O +to O +the O +PRK1 B-protein +HR1a B-structure_element +- O +RhoA B-protein +interaction O +. O + +The O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +a O +left O +- O +handed O +coiled B-structure_element +- I-structure_element +coil I-structure_element +comparable O +with O +other O +known O +HR1 B-structure_element +domains O +. O + +This O +region O +is O +distant O +from O +the O +G B-site +protein I-site +- I-site +binding I-site +interface I-site +of O +the O +HR1 B-structure_element +domains O +, O +so O +the O +structural O +differences O +may O +relate O +to O +the O +structure O +and O +regulation O +of O +these O +domains O +rather O +than O +their O +G B-protein_type +protein I-protein_type +interactions O +. O + +N52T B-mutant +is O +one O +of O +a O +combination O +of O +seven O +residues O +found O +to O +confer O +ACK B-protein +binding O +on O +Rac1 B-protein +and O +so O +may O +represent O +a O +specific O +Cdc42 B-protein +- O +effector O +contact O +residue O +. O + +Nonetheless O +, O +structural B-experimental_method +studies I-experimental_method +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +, O +combined O +with O +chemical B-experimental_method +shift I-experimental_method +mapping I-experimental_method +, O +have O +highlighted O +some O +potentially O +interesting O +differences O +between O +Cdc42 B-protein +- O +HR1TOCA1 B-structure_element +and O +RhoA B-protein +/ O +Rac1 B-protein +- O +HR1PRK1 B-structure_element +binding O +. O + +Evidence O +suggests O +that O +the O +TOCA B-protein_type +family I-protein_type +of O +proteins O +are O +recruited O +to O +the O +membrane O +via O +an O +interaction O +between O +their O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +and O +specific O +signaling O +lipids O +. O + +A O +simplified O +model O +of O +the O +early O +stages O +of O +Cdc42 B-complex_assembly +· I-complex_assembly +N I-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +- O +dependent O +actin O +polymerization O +. O + +The O +HR1TOCA1 B-structure_element +- O +Cdc42 O +and O +SH3TOCA1 B-structure_element +- O +N O +- O +WASP O +interactions O +position O +Cdc42 B-protein +and O +N B-protein +- I-protein +WASP I-protein +for O +binding O +. O + +In O +conclusion O +, O +the O +data O +presented O +here O +show O +that O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +sufficient O +for O +Cdc42 B-protein +binding O +in O +vitro O +and O +that O +the O +interaction O +is O +of O +micromolar O +affinity O +, O +lower O +than O +that O +of O +other O +G B-protein_type +protein I-protein_type +- O +HR1 B-structure_element +domain O +interactions O +. O + +Eukaryotic B-taxonomy_domain +ACCs B-protein_type +are O +single B-protein_type +- I-protein_type +chain I-protein_type +multienzymes I-protein_type +characterized O +by O +a O +large O +, O +non B-protein_state +- I-protein_state +catalytic I-protein_state +central B-structure_element +domain I-structure_element +( O +CD B-structure_element +), O +whose O +role O +in O +ACC B-protein_type +regulation O +remains O +poorly O +characterized O +. O + +Combining O +the O +yeast B-taxonomy_domain +CD B-structure_element +structure B-evidence +with O +intermediate O +and O +low O +- O +resolution O +data O +of O +larger B-mutant +fragments I-mutant +up O +to O +intact B-protein_state +ACCs B-protein_type +provides O +a O +comprehensive O +characterization O +of O +the O +dynamic B-protein_state +fungal B-taxonomy_domain +ACC B-protein_type +architecture O +. O + +ACC B-experimental_method +inhibition I-experimental_method +and I-experimental_method +knock I-experimental_method +- I-experimental_method +out I-experimental_method +studies I-experimental_method +show O +the O +potential O +of O +targeting O +ACC B-protein_type +for O +treatment O +of O +the O +metabolic O +syndrome O +. O + +BRCA1 B-protein +binds O +only O +to O +the O +phosphorylated B-protein_state +form O +of O +ACC1 B-protein +and O +prevents O +ACC B-protein_type +activation O +by O +phosphatase B-protein_type +- O +mediated O +dephosphorylation O +. O + +The O +crystal B-evidence +structure I-evidence +of O +the O +CD B-structure_element +of O +SceACC B-protein +( O +SceCD B-species +) O +was O +determined O +at O +3 O +. O +0 O +Å O +resolution O +by O +experimental B-experimental_method +phasing I-experimental_method +and O +refined B-experimental_method +to O +Rwork B-evidence +/ O +Rfree B-evidence += O +0 O +. O +20 O +/ O +0 O +. O +24 O +( O +Table O +1 O +). O + +A O +regulatory B-structure_element +loop I-structure_element +mediates O +interdomain O +interactions O + +To O +define O +the O +functional O +state O +of O +insect B-experimental_method +- I-experimental_method +cell I-experimental_method +- I-experimental_method +expressed I-experimental_method +ACC B-protein_type +variants O +, O +we O +employed O +mass B-experimental_method +spectrometry I-experimental_method +( O +MS B-experimental_method +) O +for O +phosphorylation B-experimental_method +site I-experimental_method +detection I-experimental_method +. O + +The O +SceCD B-species +structure B-evidence +thus O +authentically O +represents O +the O +state O +of O +SceACC B-protein +, O +where O +the O +enzyme B-protein +is O +inhibited B-protein_state +by O +SNF1 B-ptm +- I-ptm +dependent I-ptm +phosphorylation I-ptm +. O + +Already O +the O +binding O +of O +phosphorylated B-protein_state +Ser1157 B-residue_name_number +apparently O +stabilizes O +the O +regulatory B-structure_element +loop I-structure_element +conformation O +; O +the O +accessory O +phosphorylation B-site +sites I-site +Ser1148 B-residue_name_number +and O +Ser1162 B-residue_name_number +in O +the O +same B-structure_element +loop I-structure_element +may O +further O +modulate O +the O +strength O +of O +interaction O +between O +the O +regulatory B-structure_element +loop I-structure_element +and O +the O +CDC1 B-structure_element +and O +CDC2 B-structure_element +domains O +. O + +However O +, O +residual O +phosphorylation B-ptm +levels O +were O +detected O +for O +Ser1204 B-residue_name_number +( O +7 O +%) O +and O +Ser1218 B-residue_name_number +( O +7 O +%) O +in O +the O +same B-structure_element +loop I-structure_element +. O + +To O +further O +obtain O +insights O +into O +the O +functional O +architecture O +of O +fungal B-taxonomy_domain +ACC B-protein_type +, O +we O +characterized O +larger B-mutant +multidomain I-mutant +fragments I-mutant +up O +to O +the O +intact B-protein_state +enzymes B-protein +. O + +Using O +molecular B-experimental_method +replacement I-experimental_method +based O +on O +fungal B-taxonomy_domain +ACC B-protein_type +CD B-structure_element +and O +CT B-structure_element +models O +, O +we O +obtained O +structures B-evidence +of O +a O +variant B-mutant +comprising O +CthCT B-species +and O +CDC1 B-structure_element +/ O +CDC2 B-structure_element +in O +two B-evidence +crystal I-evidence +forms I-evidence +at O +resolutions O +of O +3 O +. O +6 O +and O +4 O +. O +5 O +Å O +( O +CthCD B-mutant +- I-mutant +CTCter1 I-mutant +/ I-mutant +2 I-mutant +), O +respectively O +, O +as O +well O +as O +of O +a O +CthCT B-species +linked O +to O +the O +entire O +CD B-structure_element +at O +7 O +. O +2 O +Å O +resolution O +( O +CthCD B-mutant +- I-mutant +CT I-mutant +; O +Figs O +1a O +and O +2 O +, O +Table O +1 O +). O + +Owing O +to O +the O +limited O +resolution O +the O +discussion O +of O +structures B-evidence +of O +CthCD B-mutant +- I-mutant +CT I-mutant +and O +CthΔBCCP B-mutant +is O +restricted O +to O +the O +analysis O +of O +domain O +localization O +. O + +However O +, O +MS B-experimental_method +analysis O +of O +CthCD B-mutant +- I-mutant +CT I-mutant +and O +CthΔBCCP B-mutant +constructs O +revealed O +between O +60 O +and O +70 O +% O +phosphorylation B-ptm +of O +Ser1170 B-residue_name_number +( O +corresponding O +to O +SceACC B-protein +Ser1157 B-residue_name_number +). O + +Class B-evidence +averages I-evidence +, O +obtained O +by O +maximum B-experimental_method +- I-experimental_method +likelihood I-experimental_method +- I-experimental_method +based I-experimental_method +two I-experimental_method +- I-experimental_method +dimensional I-experimental_method +( I-experimental_method +2D I-experimental_method +) I-experimental_method +classification I-experimental_method +, O +are O +focused O +on O +the O +dimeric B-oligomeric_state +CT B-structure_element +domain O +and O +the O +full B-protein_state +BC B-mutant +– I-mutant +BCCP I-mutant +– I-mutant +CD I-mutant +domain O +of O +only O +one O +protomer B-oligomeric_state +, O +due O +to O +the O +non O +- O +coordinated O +motions O +of O +the O +lateral O +BC B-structure_element +/ O +CD B-structure_element +regions O +relative O +to O +the O +CT B-structure_element +dimer B-oligomeric_state +. O + +They O +identify O +the O +connections O +between O +CDN B-structure_element +/ O +CDL B-structure_element +and O +between O +CDC2 B-structure_element +/ O +CT B-structure_element +as O +major O +contributors O +to O +conformational O +heterogeneity O +( O +Supplementary O +Fig O +. O +4a O +, O +b O +). O + +The O +most O +relevant O +candidate O +site O +for O +mediating O +such O +additional O +flexibility O +and O +permitting O +an O +extended O +set O +of O +conformations O +is O +the O +CDC1 B-site +/ I-site +CDC2 I-site +interface I-site +, O +which O +is O +rigidified O +by O +the O +Ser1157 B-residue_name_number +- O +phosphorylated B-protein_state +regulatory B-structure_element +loop I-structure_element +, O +as O +depicted O +in O +the O +SceCD B-species +crystal B-evidence +structure I-evidence +. O + +The O +CD B-structure_element +consists O +of O +four O +distinct O +subdomains B-structure_element +and O +acts O +as O +a O +tether O +from O +the O +CT B-structure_element +to O +the O +mobile B-protein_state +BCCP B-structure_element +and O +an O +oriented B-protein_state +BC B-structure_element +domain O +. O + +In O +higher B-taxonomy_domain +eukaryotic I-taxonomy_domain +ACCs B-protein_type +, O +regulation O +via O +phosphorylation B-ptm +is O +achieved O +by O +combining O +the O +effects O +of O +phosphorylation B-ptm +at O +Ser80 B-residue_name_number +, O +Ser1201 B-residue_name_number +and O +Ser1263 B-residue_name_number +. O + +A O +comparison O +between O +fungal B-taxonomy_domain +and O +human B-species +ACC B-protein_type +will O +help O +to O +further O +discriminate O +mechanistic O +differences O +that O +contribute O +to O +the O +extended O +control O +and O +polymerization O +of O +human B-species +ACC B-protein_type +. O + +In O +flACC B-protein +, O +CDC2 B-structure_element +rotates O +∼ O +120 O +° O +with O +respect O +to O +the O +CT B-structure_element +domain O +. O + +On O +the O +basis O +of O +a O +superposition B-experimental_method +of O +CDC2 B-structure_element +, O +CDC1 B-structure_element +of O +the O +phosphorylated B-protein_state +SceCD B-species +is O +rotated O +by O +30 O +° O +relative O +to O +CDC1 B-structure_element +of O +the O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +flACC B-protein +( O +Supplementary O +Fig O +. O +5d O +), O +similar O +to O +what O +we O +have O +observed O +for O +the O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +HsaBT B-mutant +- I-mutant +CD I-mutant +( O +Supplementary O +Fig O +. O +1d O +). O + +When O +inspecting B-experimental_method +all O +individual O +protomer B-oligomeric_state +and O +fragment B-mutant +structures B-evidence +in O +their O +study O +, O +Wei O +and O +Tong O +also O +identify O +the O +CDN B-structure_element +/ I-structure_element +CDC1 I-structure_element +connection I-structure_element +as O +a O +highly B-protein_state +flexible I-protein_state +hinge B-structure_element +, O +in O +agreement O +with O +our O +observations O +. O + +It O +disfavours O +the O +adoption O +of O +a O +rare B-protein_state +, I-protein_state +compact I-protein_state +conformation I-protein_state +, O +in O +which O +intramolecular O +dimerization O +of O +the O +BC B-structure_element +domains O +results O +in O +catalytic O +turnover O +. O + +( O +e O +) O +Structural O +overview O +of O +HsaBT B-mutant +- I-mutant +CD I-mutant +. O + +Cartoon O +representation O +of O +crystal B-evidence +structures I-evidence +of O +multidomain B-mutant +constructs I-mutant +of O +CthACC B-protein +. O + +( O +b O +) O +The O +interdomain B-site +interface I-site +of O +CDC1 B-structure_element +and O +CDC2 B-structure_element +exhibits O +only O +limited O +plasticity O +. O + +We O +identified O +that O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +forms O +a O +self B-oligomeric_state +- I-oligomeric_state +oligomer I-oligomeric_state +through O +the O +SEL1Lcent B-structure_element +domain O +in O +mammalian B-taxonomy_domain +cells O +. O + +The O +process O +is O +called O +ER O +- O +associated O +protein O +degradation O +( O +ERAD O +) O +and O +is O +conserved B-protein_state +in O +all O +eukaryotes B-taxonomy_domain +. O + +SEL1L B-protein +is O +required O +for O +ER O +homeostasis O +, O +which O +is O +essential O +for O +protein O +translation O +, O +pancreatic O +function O +, O +and O +cellular O +and O +organismal O +survival O +. O + +Based O +on O +these O +observations O +, O +we O +propose O +a O +model O +wherein O +the O +SLR B-structure_element +domains O +of O +SEL1L B-protein +contribute O +to O +the O +formation O +of O +stable B-protein_state +oligomers B-oligomeric_state +of O +the O +ERAD O +translocation O +machinery O +, O +which O +is O +indispensable O +for O +ERAD O +. O + +The O +SLR B-structure_element +motifs O +can O +be O +grouped O +into O +three O +regions O +due O +to O +the O +presence O +of O +linker B-structure_element +sequences I-structure_element +among O +the O +groups O +of O +SLR B-structure_element +motifs O +: O +SLR B-structure_element +- I-structure_element +N I-structure_element +( O +SLR B-structure_element +motifs I-structure_element +1 I-structure_element +to I-structure_element +4 I-structure_element +), O +SLR B-structure_element +- I-structure_element +M I-structure_element +( O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +to I-structure_element +9 I-structure_element +), O +and O +SLR B-structure_element +- I-structure_element +C I-structure_element +( O +SLR B-structure_element +motifs I-structure_element +10 I-structure_element +to I-structure_element +11 I-structure_element +) O +( O +Fig O +. O +1A O +). O + +We O +first O +tried O +to O +prepare O +the O +full B-protein_state +- I-protein_state +length I-protein_state +mouse B-taxonomy_domain +SEL1L B-protein +protein O +, O +excluding O +the O +transmembrane B-structure_element +domain I-structure_element +at O +the O +C O +- O +terminus O +( O +residues O +735 B-residue_range +– I-residue_range +755 I-residue_range +), O +by O +expression B-experimental_method +in I-experimental_method +bacteria I-experimental_method +. O + +Both O +SLR B-structure_element +- I-structure_element +N I-structure_element +( O +residues O +194 B-residue_range +– I-residue_range +343 I-residue_range +) O +and O +SLR B-structure_element +- I-structure_element +C I-structure_element +( O +residues O +639 B-residue_range +– I-residue_range +719 I-residue_range +) O +alone O +could O +be O +solubilized O +with O +an O +MBP B-experimental_method +tag I-experimental_method +at I-experimental_method +the I-experimental_method +N I-experimental_method +- I-experimental_method +terminus I-experimental_method +, O +but O +appeared O +to O +be O +polydisperse O +when O +analyzed O +by O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +. O + +Starting O +from O +its O +N O +- O +terminus O +, O +the O +α B-structure_element +- I-structure_element +solenoid I-structure_element +of O +SEL1L B-protein +extends O +across O +a O +semi O +- O +circle O +in O +a O +right O +- O +handed O +superhelix O +fashion O +along O +the O +rotation O +axis O +of O +the O +yin B-structure_element +- I-structure_element +yang I-structure_element +circle I-structure_element +. O + +However O +, O +the O +last O +helix O +, O +9B B-structure_element +, O +at O +the O +C O +- O +terminus O +adopts O +a O +different O +conformation O +, O +lying O +parallel O +to O +the O +long O +axis O +of O +helix B-structure_element +9A I-structure_element +instead O +of O +forming O +an O +antiparallel O +SLR B-structure_element +. O + +Helix B-structure_element +9B I-structure_element +from O +one O +protomer B-oligomeric_state +inserts O +into O +the O +empty O +space O +surrounded O +by O +the O +concave B-site +region I-site +in O +the O +other O +monomer B-oligomeric_state +, O +forming O +a O +domain B-protein_state +- I-protein_state +swapped I-protein_state +conformation O +. O + +In O +this O +interface B-site +, O +Leu B-residue_name_number +516 I-residue_name_number +and O +Tyr B-residue_name_number +519 I-residue_name_number +on O +helix B-structure_element +9B I-structure_element +are O +located O +in O +the O +center O +, O +making O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +Trp B-residue_name_number +478 I-residue_name_number +on O +helix B-structure_element +8B I-structure_element +, O +Val B-residue_name_number +444 I-residue_name_number +on O +helix B-structure_element +7B I-structure_element +, O +Phe B-residue_name_number +411 I-residue_name_number +on O +helix B-structure_element +6B I-structure_element +, O +and O +Leu B-residue_name_number +380 I-residue_name_number +on O +helix B-structure_element +5B I-structure_element +from O +the O +SEL1Lcent B-structure_element +counterpart O +( O +Fig O +. O +2A O +, O +Interface B-site +1 I-site +detail O +). O + +Second O +, O +the O +residues O +from O +helix B-structure_element +9A I-structure_element +interact O +with O +the O +residues O +from O +helix B-structure_element +5A I-structure_element +of O +its O +counterpart O +in O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +orientation O +. O + +However O +, O +the O +mutant B-protein_state +behaved O +as O +a O +monomer B-oligomeric_state +in O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +and O +analytical B-experimental_method +ultracentrifugation I-experimental_method +experiments O +( O +Fig O +. O +2B O +, O +Supplementary O +Fig O +. O +2C O +). O + +SLRs B-structure_element +of O +mouse B-taxonomy_domain +SEL1L B-protein +were O +predicted O +using O +the O +TPRpred B-experimental_method +server I-experimental_method +. O + +Although O +amino O +acid O +sequences O +from O +helix B-structure_element +9A I-structure_element +and O +9B B-structure_element +correctly O +aligned O +with O +the O +regular O +SLR B-structure_element +repeats I-structure_element +and O +corresponded O +to O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +( O +Fig O +. O +3A O +), O +the O +structural O +arrangement O +of O +the O +two O +helices B-structure_element +deviated O +from O +the O +common O +structure O +for O +the O +SLR B-structure_element +motif O +. O + +Thus O +, O +the O +Gly B-structure_element +- I-structure_element +Gly I-structure_element +residues O +generate O +an O +unusual O +sharp O +bend O +at O +the O +C O +- O +terminal O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +. O + +G512A B-mutant +or O +G513A B-mutant +alone O +showed O +no O +differences O +from O +wild B-protein_state +- I-protein_state +type I-protein_state +in O +terms O +of O +the O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +elution O +profile O +( O +Fig O +. O +3D O +), O +suggesting O +that O +the O +restriction O +for O +single O +glycine B-residue_name +flexibility O +would O +not O +be O +enough O +to O +break O +the O +swapped O +structure O +of O +helix B-structure_element +9B I-structure_element +. O + +We O +generated O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +- O +HA B-experimental_method +and O +SEL1L B-protein +- O +FLAG B-experimental_method +fusion B-experimental_method +constructs I-experimental_method +and O +co B-experimental_method +- I-experimental_method +transfected I-experimental_method +the O +constructs O +into O +HEK293T O +cells O +. O + +Indeed O +, O +immunoprecipitation B-experimental_method +followed O +by O +western B-experimental_method +blot I-experimental_method +analysis O +using O +the O +culture O +medium O +detected O +secreted O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +fragment O +, O +but O +not O +SEL1Lcent B-structure_element +( O +Fig O +. O +4B O +). O + +To O +this O +end O +, O +we O +co B-experimental_method +- I-experimental_method +transfected I-experimental_method +the O +differentially O +tagged B-protein_state +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +( O +SEL1L B-protein +- O +HA B-experimental_method +and O +SEL1L B-protein +- O +FLAG B-experimental_method +) O +and O +increasing B-experimental_method +doses I-experimental_method +of O +SEL1Lcent B-mutant +- I-mutant +KDEL I-mutant +, O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +- I-mutant +KDEL I-mutant +or O +SEL1Lcent B-mutant +( I-mutant +L521A I-mutant +)- I-mutant +KDEL I-mutant +, O +respectively O +. O + +Co B-experimental_method +- I-experimental_method +immunoprecipitation I-experimental_method +assay I-experimental_method +revealed O +that O +wild B-protein_state +- I-protein_state +type I-protein_state +SEL1Lcent B-mutant +- I-mutant +KDEL I-mutant +, O +indeed O +, O +competitively O +disrupted O +the O +self O +- O +association O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L O +( O +Fig O +. O +4E O +). O + +This O +is O +one O +of O +the O +biggest O +differences O +from O +TPRs B-structure_element +in O +Cdc23 B-protein +and O +from O +the O +SLRs B-structure_element +in O +HcpC B-protein +, O +where O +the O +motifs O +exist O +in O +tandem O +. O + +Indirect O +evidence O +from O +a O +previous O +yeast B-taxonomy_domain +study O +shows O +that O +the O +circumscribed O +region O +of O +C O +- O +terminal O +Hrd3p B-protein +, O +specifically O +residues O +664 B-residue_range +– I-residue_range +695 I-residue_range +, O +forms O +contacts O +with O +the O +Hrd1 B-protein +luminal B-structure_element +loops I-structure_element +. O + +This O +hypothesis O +is O +supported O +by O +cross B-experimental_method +- I-experimental_method +linking I-experimental_method +data I-experimental_method +suggesting O +that O +human B-species +HRD1 B-protein +forms O +a O +homodimer B-oligomeric_state +. O + +However O +, O +metazoans B-taxonomy_domain +lack O +a O +clear O +Usa1p B-protein +homolog O +. O + +Assuming O +that O +the O +correct O +oligomerization O +of O +ERAD O +components O +may O +be O +critical O +for O +their O +function O +, O +we O +hypothesize O +that O +homodimer B-oligomeric_state +formation O +of O +SEL1L B-protein +in O +the O +ER O +lumen O +may O +stabilize O +oligomerization O +of O +the O +HRD B-complex_assembly +complex O +, O +given O +that O +SEL1L B-protein +forms O +a O +stoichiometric O +complex B-protein_state +with I-protein_state +HRD1 B-protein +. O + +Recently O +, O +a O +truncated B-protein_state +version O +of O +Yos9 B-protein +was O +shown O +to O +form O +a O +dimer B-oligomeric_state +in O +the O +ER O +lumen O +and O +to O +contribute O +to O +the O +dimeric B-oligomeric_state +state O +of O +the O +Hrd1p B-protein +complex O +. O + +Putative O +N B-site +- I-site +glycosylation I-site +sites I-site +are O +indicated O +by O +black O +triangles O +. O + +The O +schematic O +diagrams O +representing O +the O +protein O +constructs O +used O +in O +the O +SEC B-experimental_method +are O +shown O +on O +the O +left O +of O +each O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +profile O +. O + +( O +A O +) O +Sequence B-experimental_method +alignment I-experimental_method +of O +the O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +. O + +The O +11 O +SLR B-structure_element +motifs O +of O +SEL1L B-protein +were O +expressed O +with O +red O +cylinders O +and O +grouped O +into O +three O +parts O +( O +SLR B-structure_element +- I-structure_element +N I-structure_element +, O +SLR B-structure_element +- I-structure_element +M I-structure_element +, O +and O +SLR B-structure_element +- I-structure_element +C I-structure_element +) O +based O +on O +the O +sequence B-experimental_method +alignment I-experimental_method +across O +the O +motifs O +and O +the O +crystal B-evidence +structure I-evidence +presented O +herein O +. O + +Crystal B-evidence +Structures I-evidence +of O +Putative O +Sugar B-protein_type +Kinases I-protein_type +from O +Synechococcus B-species +Elongatus I-species +PCC I-species +7942 I-species +and O +Arabidopsis B-species +Thaliana I-species + +The O +genome O +of O +the O +Synechococcus B-species +elongatus I-species +strain I-species +PCC I-species +7942 I-species +encodes O +a O +putative O +sugar B-protein_type +kinase I-protein_type +( O +SePSK B-protein +), O +which O +shares O +44 O +. O +9 O +% O +sequence O +identity O +with O +the O +xylulose B-protein +kinase I-protein +- I-protein +1 I-protein +( O +AtXK B-protein +- I-protein +1 I-protein +) O +from O +Arabidopsis B-species +thaliana I-species +. O + +The O +At2g21370 B-gene +gene O +product O +from O +Arabidopsis B-species +thaliana I-species +, O +xylulose B-protein +kinase I-protein +- I-protein +1 I-protein +( O +AtXK B-protein +- I-protein +1 I-protein +), O +whose O +mature B-protein_state +form I-protein_state +contains O +436 B-residue_range +amino O +acids O +, O +is O +located O +in O +the O +chloroplast O +( O +ChloroP O +1 O +. O +1 O +Server O +). O + +Two O +possible O +xylulose B-protein_type +kinases I-protein_type +( O +xylulose B-protein +kinase I-protein +- I-protein +1 I-protein +: O +XK B-protein +- I-protein +1 I-protein +and O +xylulose B-protein +kinase I-protein +- I-protein +2 I-protein +: O +XK B-protein +- I-protein +2 I-protein +) O +from O +Arabidopsis B-species +thaliana I-species +were O +previously O +proposed O +. O + +The O +attempt O +to O +solve O +the O +SePSK B-protein +structure B-evidence +by O +molecular B-experimental_method +replacement I-experimental_method +method I-experimental_method +failed O +with O +ribulokinase B-protein +from O +Bacillus B-species +halodurans I-species +( O +PDB O +code O +: O +3QDK O +, O +15 O +. O +7 O +% O +sequence O +identity O +) O +as O +an O +initial O +model O +. O + +Among O +all O +these O +structural O +elements O +, O +α4 B-structure_element +/ O +α5 B-structure_element +/ O +α11 B-structure_element +/ O +α18 B-structure_element +, O +β3 B-structure_element +/ O +β2 B-structure_element +/ O +β1 B-structure_element +/ O +β6 B-structure_element +/ O +β19 B-structure_element +/ O +β20 B-structure_element +/ O +β17 B-structure_element +and O +α21 B-structure_element +/ O +α32 B-structure_element +form O +three O +patches O +, O +referred O +to O +as O +A1 B-structure_element +, O +B1 B-structure_element +and O +A2 B-structure_element +, O +exhibiting O +the O +core B-structure_element +region I-structure_element +. O + +( O +A O +) O +Three O +- O +dimensional O +structure B-evidence +of O +apo B-protein_state +- O +SePSK B-protein +. O + +( O +B O +) O +Three O +- O +dimensional O +structure B-evidence +of O +apo B-protein_state +- O +AtXK B-protein +- I-protein +1 I-protein +. O + +In O +contrary O +, O +limited O +increasing O +of O +ATP B-chemical +hydrolysis O +activity O +was O +detected O +for O +AtXK B-protein +- I-protein +1 I-protein +upon O +addition O +of O +D B-chemical +- I-chemical +ribulose I-chemical +( O +Fig O +2C O +), O +despite O +its O +structural O +similarity O +with O +SePSK B-protein +. O + +While O +the O +ATP B-chemical +hydrolysis O +activity O +of O +SePSK B-protein +greatly O +increases O +upon O +addition O +of O +D B-chemical +- I-chemical +ribulose I-chemical +( O +DR B-chemical +). O + +The O +ATP B-chemical +hydrolysis O +activity O +measured O +via O +luminescent B-experimental_method +ADP I-experimental_method +- I-experimental_method +Glo I-experimental_method +assay I-experimental_method +( O +Promega O +). O + +Our O +results O +suggested O +that O +three O +conserved O +residues O +( O +D8 B-residue_name_number +, O +T11 B-residue_name_number +and O +D221 B-residue_name_number +of O +SePSK B-protein +) O +play O +an O +important O +role O +in O +SePSK B-protein +function O +. O + +Using O +enzymatic B-experimental_method +activity I-experimental_method +assays I-experimental_method +, O +we O +found O +that O +all O +of O +these O +mutants O +exhibit O +much O +lower O +activity O +of O +ATP B-chemical +hydrolysis O +after O +adding O +D B-chemical +- I-chemical +ribulose I-chemical +than O +that O +of O +wild B-protein_state +type I-protein_state +, O +indicating O +the O +possibility O +that O +these O +three O +residues O +are O +involved O +in O +the O +catalytic O +process O +of O +phosphorylation B-ptm +D B-chemical +- I-chemical +ribulose I-chemical +and O +are O +vital O +for O +the O +function O +of O +SePSK B-protein +( O +Fig O +2D O +). O + +Thus O +the O +two O +structures B-evidence +were O +named O +ADP B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +and O +ADP B-complex_assembly +- I-complex_assembly +AtXK I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +, O +respectively O +. O + +As O +shown O +in O +Fig O +3A O +, O +one O +SePSK B-protein +protein O +molecule O +is O +in O +an O +asymmetric O +unit O +with O +one O +AMP B-chemical +- I-chemical +PNP I-chemical +molecule O +. O + +The O +AMP B-chemical +- I-chemical +PNP I-chemical +is O +bound O +at O +the O +domain B-structure_element +II I-structure_element +, O +where O +it O +fits O +well O +inside O +a O +positively B-site +charged I-site +groove I-site +. O + +( O +B O +) O +The O +AMP B-site +- I-site +PNP I-site +binding I-site +pocket I-site +. O + +7 O +. O +1 O +Å O +( O +RBL1 B-residue_name_number +- O +C4 O +and O +RBL2 B-residue_name_number +- O +C1 O +). O + +Furthermore O +, O +the O +O2 O +of O +RBL1 B-residue_name_number +interacts B-bond_interaction +with I-bond_interaction +the O +main O +chain O +amide O +nitrogen O +of O +Ser72 B-residue_name_number +( O +Fig O +4B O +). O + +The O +original O +data O +is O +shown O +as O +black O +curve O +, O +and O +the O +fitted O +data O +is O +shown O +as O +different O +color O +( O +wild B-protein_state +type I-protein_state +SePSK B-protein +: O +red O +curve O +, O +D8A B-mutant +- O +SePSK B-protein +: O +green O +curve O +). O + +The O +side O +chain O +of O +Asp8 B-residue_name_number +interacts B-bond_interaction +strongly I-bond_interaction +with I-bond_interaction +O3 O +and O +O4 O +of O +RBL2 B-residue_name_number +. O + +Structural B-experimental_method +comparison I-experimental_method +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +showed O +that O +while O +the O +RBL1 B-site +binding I-site +pocket I-site +is O +conserved B-protein_state +, O +the O +RBL2 B-site +pocket I-site +is O +disrupted O +in O +AtXK B-protein +- I-protein +1 I-protein +structure B-evidence +, O +despite O +the O +fact O +that O +the O +residues O +interacting O +with O +RBL2 B-residue_name_number +are O +highly B-protein_state +conserved I-protein_state +between O +the O +two O +proteins O +. O + +Our O +SePSK B-protein +structure B-evidence +shows O +that O +the O +Asp8 B-residue_name_number +residue O +forms O +strong O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +RBL2 B-residue_name_number +( O +Fig O +4B O +). O + +In O +addition O +, O +this O +difference O +may O +be O +caused O +by O +the O +binding O +of O +substrates O +and O +/ O +or O +ATP B-chemical +. O + +The O +results O +of O +superposition B-experimental_method +displayed O +different O +crossing O +angle O +between O +these O +two O +domains O +. O + +Meanwhile O +, O +the O +distances O +of O +AMP B-chemical +- I-chemical +PNP I-chemical +γ O +- O +phosphate B-chemical +and O +the O +first O +hydroxyl O +group O +of O +RBL2 B-residue_name_number +are O +7 O +. O +2 O +Å O +( O +superposed B-experimental_method +with O +AtXK B-protein +- I-protein +1 I-protein +), O +6 O +. O +7 O +Å O +( O +superposed B-experimental_method +with O +SePSK B-protein +), O +3 O +. O +7 O +Å O +( O +superposed B-experimental_method +with O +3LL3 O +), O +until O +AMP B-chemical +- I-chemical +PNP I-chemical +γ O +- O +phosphate B-chemical +fully O +contacts O +RBL2 B-residue_name_number +after O +superposition B-experimental_method +with O +1GLJ O +( O +Fig O +5 O +). O + +In O +summary O +, O +our O +structural B-experimental_method +and I-experimental_method +enzymatic I-experimental_method +analyses I-experimental_method +provide O +evidence O +that O +SePSK B-protein +shows O +D B-protein_type +- I-protein_type +ribulose I-protein_type +kinase I-protein_type +activity O +, O +and O +exhibits O +the O +conserved O +features O +of O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +. O + +Three O +conserved B-site +residues O +in O +SePSK B-protein +were O +identified O +to O +be O +essential O +for O +this O +function O +. O + +The O +inducible B-protein_state +lysine B-protein_type +decarboxylase I-protein_type +LdcI B-protein +is O +an O +important O +enterobacterial B-taxonomy_domain +acid B-protein_type +stress I-protein_type +response I-protein_type +enzyme I-protein_type +whereas O +LdcC B-protein +is O +its O +close O +paralogue O +thought O +to O +play O +mainly O +a O +metabolic O +role O +. O + +Decarboxylation O +of O +the O +amino B-chemical +acid I-chemical +into O +a O +polyamine B-chemical +is O +catalysed O +by O +a O +PLP B-chemical +cofactor O +in O +a O +multistep O +reaction O +that O +consumes O +a O +cytoplasmic O +proton B-chemical +and O +produces O +a O +CO2 B-chemical +molecule O +passively O +diffusing O +out O +of O +the O +cell O +, O +while O +the O +polyamine B-chemical +is O +excreted O +by O +the O +antiporter B-protein_type +in O +exchange O +for O +a O +new O +amino B-chemical +acid I-chemical +substrate O +. O + +Each O +monomer B-oligomeric_state +is O +composed O +of O +three O +domains O +– O +an O +N O +- O +terminal O +wing B-structure_element +domain I-structure_element +( O +residues O +1 B-residue_range +– I-residue_range +129 I-residue_range +), O +a O +PLP B-structure_element +- I-structure_element +binding I-structure_element +core I-structure_element +domain I-structure_element +( O +residues O +130 B-residue_range +– I-residue_range +563 I-residue_range +), O +and O +a O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +CTD B-structure_element +, O +residues O +564 B-residue_range +– I-residue_range +715 I-residue_range +). O + +Furthermore O +, O +we O +recently O +solved B-experimental_method +the I-experimental_method +structure I-experimental_method +of O +the O +E B-species +. I-species +coli I-species +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +complex O +by O +cryo B-experimental_method +- I-experimental_method +electron I-experimental_method +microscopy I-experimental_method +( O +cryoEM B-experimental_method +) O +and O +combined O +it O +with O +the O +crystal B-evidence +structures I-evidence +of O +the O +individual O +proteins O +. O + +In O +addition O +, O +the O +wing B-structure_element +domains I-structure_element +of O +all O +structures B-evidence +are O +very O +similar O +, O +with O +the O +RMSD B-evidence +after O +optimal O +rigid O +body O +alignment O +( O +RMSDmin B-evidence +) O +less O +than O +1 O +. O +1 O +Å O +. O +Thus O +, O +taking O +the O +limited O +resolution O +of O +the O +cryoEM B-experimental_method +maps B-evidence +into O +account O +, O +we O +consider O +that O +the O +wing B-structure_element +domains I-structure_element +of O +all O +the O +four O +structures B-evidence +are O +essentially O +identical O +and O +that O +in O +the O +present O +study O +the O +RMSD B-evidence +of O +less O +than O +2 O +Å O +can O +serve O +as O +a O +baseline O +below O +which O +differences O +may O +be O +assumed O +as O +insignificant O +. O + +This O +interface B-site +is O +formed O +essentially O +by O +the O +core B-structure_element +domains I-structure_element +with O +some O +contribution O +of O +the O +CTDs B-structure_element +. O + +Zooming O +in O +the O +variations O +in O +the O +PLP B-structure_element +- I-structure_element +SD I-structure_element +shows O +that O +most O +of O +the O +structural O +changes O +concern O +displacements O +in O +the O +active B-site +site I-site +( O +Fig O +. O +3C O +– O +F O +). O + +The O +most O +conspicuous O +differences O +between O +the O +PLP B-structure_element +- I-structure_element +SDs I-structure_element +can O +be O +linked O +to O +the O +pH B-protein_state +- I-protein_state +dependent I-protein_state +activation O +of O +the O +enzymes O +. O + +In O +particular O +, O +transition O +from O +LdcIi B-protein +to O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +involves O +~ O +3 O +. O +5 O +Å O +and O +~ O +4 O +. O +5 O +Å O +shifts O +away O +from O +the O +5 O +- O +fold O +axis O +in O +the O +active B-site +site I-site +α B-structure_element +- I-structure_element +helices I-structure_element +spanning O +residues O +218 B-residue_range +– I-residue_range +232 I-residue_range +and O +246 B-residue_range +– I-residue_range +254 I-residue_range +respectively O +( O +Fig O +. O +3C O +– O +E O +). O + +An O +inhibitor O +of O +the O +LdcI B-protein +and O +LdcC B-protein +activity O +, O +the O +stringent B-chemical +response I-chemical +alarmone I-chemical +ppGpp B-chemical +, O +is O +known O +to O +bind O +at O +the O +interface B-site +between O +neighboring O +monomers B-oligomeric_state +within O +each O +ring B-structure_element +( O +Fig O +. O +S4 O +). O + +Interestingly O +, O +although O +a O +number O +of O +ppGpp B-site +binding I-site +residues I-site +are O +strictly B-protein_state +conserved I-protein_state +between O +LdcI B-protein +and O +AdiA B-protein +that O +also O +forms O +decamers B-oligomeric_state +at O +low B-protein_state +pH I-protein_state +optimal I-protein_state +for O +its O +arginine B-protein_type +decarboxylase I-protein_type +activity O +, O +no O +ppGpp B-chemical +regulation O +of O +AdiA B-protein +could O +be O +demonstrated O +. O + +All O +lysine B-protein_type +decarboxylases I-protein_type +predicted O +to O +be O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +or O +biodegradable B-protein_state +based O +on O +their O +genetic O +environment O +, O +as O +for O +example O +their O +organization O +in O +an O +operon O +with O +a O +gene O +encoding O +the O +CadB B-protein +antiporter B-protein_type +( O +see O +Methods O +), O +were O +found O +in O +one O +group O +, O +whereas O +all O +enzymes B-protein_type +predicted O +as O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +or O +biosynthetic B-protein_state +partitioned O +into O +another O +group O +. O + +Our O +structures B-evidence +show O +that O +this B-structure_element +motif I-structure_element +is O +not O +involved O +in O +the O +enzymatic O +activity O +or O +the O +oligomeric O +state O +of O +the O +proteins O +. O + +Only O +one O +of O +the O +two O +rings B-structure_element +of O +the O +double B-structure_element +toroid I-structure_element +is O +shown O +for O +clarity O +. O + +( O +B O +) O +The O +LdcIi B-protein +dimer B-oligomeric_state +extracted O +from O +the O +crystal B-evidence +structure I-evidence +of O +the O +decamer B-oligomeric_state +. O + +The O +PLP B-chemical +is O +red O +. O + +( O +A O +) O +Maximum B-evidence +likelihood I-evidence +tree I-evidence +with O +the O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +and O +the O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +groups O +highlighted O +in O +green O +and O +pink O +, O +respectively O +. O + +( O +B O +) O +Analysis O +of O +consensus O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +and O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +sequences O +around O +the O +first O +and O +second O +C O +- O +terminal O +β B-structure_element +- I-structure_element +strands I-structure_element +. O + +( O +C O +) O +Signature O +sequences O +of O +LdcI B-protein +and O +LdcC B-protein +in O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +. O + +Mep2 B-protein_type +proteins I-protein_type +are O +fungal B-taxonomy_domain +transceptors B-protein_type +that O +play O +an O +important O +role O +as O +ammonium B-chemical +sensors O +in O +fungal B-taxonomy_domain +development O +. O + +Mep2 B-protein_type +activity O +is O +tightly O +regulated O +by O +phosphorylation B-ptm +, O +but O +how O +this O +is O +achieved O +at O +the O +molecular O +level O +is O +not O +clear O +. O + +Relative O +to O +the O +open B-protein_state +bacterial B-taxonomy_domain +ammonium B-protein_type +transporters I-protein_type +, O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +Mep2 B-protein_type +exhibits O +shifts O +in O +cytoplasmic B-structure_element +loops I-structure_element +and O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +( O +CTR B-structure_element +) O +to O +occlude O +the O +cytoplasmic O +exit B-site +of O +the O +channel B-site +and O +to O +interact O +with O +His2 B-residue_name_number +of O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +. O + +One O +of O +the O +most O +important O +unresolved O +questions O +in O +the O +field O +is O +how O +the O +transceptors B-protein_type +couple O +to O +downstream O +signalling O +pathways O +. O + +Mep2 B-protein_type +( B-protein_type +methylammonium I-protein_type +( I-protein_type +MA I-protein_type +) I-protein_type +permease I-protein_type +) I-protein_type +proteins I-protein_type +are O +ammonium B-protein_type +transceptors I-protein_type +that O +are O +ubiquitous O +in O +fungi B-taxonomy_domain +. O + +Compared O +with O +Mep1 B-protein +and O +Mep3 B-protein +, O +Mep2 B-protein +is O +highly B-protein_state +expressed I-protein_state +and O +functions O +as O +a O +low O +- O +capacity O +, O +high O +- O +affinity O +transporter O +in O +the O +uptake O +of O +MA B-chemical +. O + +All O +the O +solved O +structures B-evidence +including O +that O +of O +RhCG B-protein +are O +very O +similar O +, O +establishing O +the O +basic O +architecture O +of O +ammonium B-protein_type +transporters I-protein_type +. O + +Where O +earlier O +studies O +favoured O +the O +transport O +of O +ammonia B-chemical +gas O +, O +recent O +data O +and O +theoretical O +considerations O +suggest O +that O +Amt B-protein_type +/ I-protein_type +Mep I-protein_type +proteins I-protein_type +are O +instead O +active B-protein_state +, O +electrogenic B-protein_type +transporters I-protein_type +of O +either O +NH4 B-chemical ++ I-chemical +( O +uniport O +) O +or O +NH3 B-chemical +/ O +H B-chemical ++ I-chemical +( O +symport O +). O + +Ammonium B-chemical +transport O +is O +tightly O +regulated O +. O + +The O +structures B-evidence +are O +similar O +to O +each O +other O +but O +show O +considerable O +differences O +to O +all O +other O +ammonium B-protein_type +transporter I-protein_type +structures B-evidence +. O + +The O +Mep2 B-protein +protein O +of O +S B-species +. I-species +cerevisiae I-species +( O +ScMep2 B-protein +) O +was O +overexpressed B-experimental_method +in O +S B-species +. I-species +cerevisiae I-species +in O +high O +yields O +, O +enabling O +structure B-experimental_method +determination I-experimental_method +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +using O +data O +to O +3 O +. O +2 O +Å O +resolution O +by O +molecular B-experimental_method +replacement I-experimental_method +( O +MR B-experimental_method +) O +with O +the O +archaebacterial B-taxonomy_domain +Amt B-protein +- I-protein +1 I-protein +structure B-evidence +( O +see O +Methods O +section O +). O + +Given O +that O +the O +modest O +resolution O +of O +the O +structure B-evidence +and O +the O +limited O +detergent O +stability O +of O +ScMep2 B-protein +would O +likely O +complicate O +structure B-experimental_method +– I-experimental_method +function I-experimental_method +studies I-experimental_method +, O +several O +other O +fungal B-taxonomy_domain +Mep2 B-protein_type +orthologues O +were O +subsequently O +overexpressed B-experimental_method +and I-experimental_method +screened I-experimental_method +for I-experimental_method +diffraction O +- O +quality O +crystals B-evidence +. O + +Mep2 B-protein +channels B-site +are O +closed B-protein_state +by O +a O +two O +- O +tier O +channel B-structure_element +block I-structure_element + +In O +Mep2 B-protein +, O +the O +CTR B-structure_element +has O +moved O +away O +and O +makes O +relatively O +few O +contacts O +with O +the O +main B-structure_element +body I-structure_element +of O +the O +transporter B-protein_type +, O +generating O +a O +more O +elongated B-protein_state +protein O +( O +Figs O +1 O +and O +4 O +). O + +On O +one O +side O +, O +the O +Tyr390 B-residue_name_number +hydroxyl O +in O +Amt B-protein +- I-protein +1 I-protein +is O +hydrogen B-bond_interaction +bonded I-bond_interaction +with O +the O +side O +chain O +of O +the O +conserved B-protein_state +His185 B-residue_name_number +at O +the O +C O +- O +terminal O +end O +of O +loop B-structure_element +ICL3 B-structure_element +. O + +At O +the O +other O +end O +of O +ICL3 B-structure_element +, O +the O +backbone O +carbonyl O +groups O +of O +Gly172 B-residue_name_number +and O +Lys173 B-residue_name_number +are O +hydrogen B-bond_interaction +bonded I-bond_interaction +to O +the O +side O +chain O +of O +Arg370 B-residue_name_number +. O + +The O +result O +of O +these O +interactions O +is O +that O +the O +CTR B-structure_element +‘ O +hugs O +' O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +the O +transporters B-protein_type +( O +Fig O +. O +4 O +). O + +Conversely O +, O +the O +phosphorylation B-protein_state +- I-protein_state +mimicking I-protein_state +S457D B-mutant +variant O +is O +active B-protein_state +both O +in O +the O +triple B-mutant +mepΔ I-mutant +background O +and O +in O +a O +triple B-mutant +mepΔ I-mutant +npr1Δ I-mutant +strain O +( O +Fig O +. O +3 O +). O + +Mutation B-experimental_method +of O +other O +potential O +phosphorylation B-site +sites I-site +in O +the O +CTR B-structure_element +did O +not O +support O +growth O +in O +the O +npr1Δ B-mutant +background O +. O + +This O +segment B-structure_element +( O +residues O +450 B-residue_range +– I-residue_range +457 I-residue_range +in O +ScMep2 B-protein +and O +446 B-residue_range +– I-residue_range +453 I-residue_range +in O +CaMep2 B-protein +) O +was O +dubbed O +an O +autoinhibitory B-structure_element +( I-structure_element +AI I-structure_element +) I-structure_element +region I-structure_element +based O +on O +the O +fact O +that O +its O +removal B-experimental_method +generates O +an O +active B-protein_state +transporter B-protein_type +in O +the O +absence B-protein_state +of I-protein_state +Npr1 B-protein +( O +Fig O +. O +3 O +). O + +The O +AI B-structure_element +region I-structure_element +packs O +against O +the O +cytoplasmic O +ends O +of O +TM2 B-structure_element +and O +TM4 B-structure_element +, O +physically O +linking O +the O +main B-structure_element +body I-structure_element +of O +the O +transporter B-protein_type +with O +the O +CTR B-structure_element +via O +main O +chain O +interactions O +and O +side O +- O +chain O +interactions O +of O +Val447 B-residue_name_number +, O +Asp449 B-residue_name_number +, O +Pro450 B-residue_name_number +and O +Arg452 B-residue_name_number +( O +Fig O +. O +6 O +). O + +The O +peripheral O +location O +and O +disorder B-protein_state +of O +the O +CTR B-structure_element +beyond O +the O +kinase B-site +target I-site +site I-site +should O +facilitate O +the O +phosphorylation B-ptm +by O +Npr1 B-protein +. O + +The O +disordered B-protein_state +part O +of O +the O +CTR B-structure_element +is O +not B-protein_state +conserved I-protein_state +in O +ammonium B-protein_type +transporters I-protein_type +( O +Fig O +. O +2 O +), O +suggesting O +that O +it O +is O +not O +important O +for O +transport O +. O + +The O +data O +behind O +this O +hypothesis O +is O +the O +observation O +that O +a O +ScMep2 B-protein +449 B-mutant +- I-mutant +485Δ I-mutant +deletion B-protein_state +mutant I-protein_state +lacking B-protein_state +the O +AI B-structure_element +region I-structure_element +is O +highly B-protein_state +active I-protein_state +in O +MA B-chemical +uptake O +both O +in O +the O +triple B-mutant +mepΔ I-mutant +and O +triple B-mutant +mepΔ I-mutant +npr1Δ I-mutant +backgrounds O +, O +implying O +that O +this O +Mep2 B-mutant +variant I-mutant +has O +a O +constitutively B-protein_state +open I-protein_state +channel B-site +. O + +Interestingly O +, O +however O +, O +the O +Tyr49 B-residue_name_number +- O +His342 B-residue_name_number +hydrogen B-bond_interaction +bond I-bond_interaction +that O +closes O +the O +channel O +in O +the O +WT B-protein_state +protein O +is O +still O +present O +( O +Fig O +. O +7 O +and O +Supplementary O +Fig O +. O +2 O +). O + +We O +therefore O +predict O +that O +phosphorylation B-ptm +of O +Ser453 B-residue_name_number +will O +result O +in O +steric O +clashes O +as O +well O +as O +electrostatic O +repulsion O +, O +which O +in O +turn O +might O +cause O +substantial O +conformational O +changes O +within O +the O +CTR B-structure_element +. O + +By O +contrast O +, O +the O +conserved B-protein_state +part O +of O +the O +CTR B-structure_element +has O +undergone O +a O +large O +conformational O +change O +involving O +formation O +of O +a O +12 B-structure_element +- I-structure_element +residue I-structure_element +- I-structure_element +long I-structure_element +α I-structure_element +- I-structure_element +helix I-structure_element +from O +Leu427 B-residue_range +to I-residue_range +Asp438 I-residue_range +. O + +This O +is O +the O +first O +time O +a O +large O +conformational O +change O +has O +been O +observed O +in O +an O +ammonium B-protein_type +transporter I-protein_type +as O +a O +result O +of O +a O +mutation B-experimental_method +, O +and O +confirms O +previous O +hypotheses O +that O +phosphorylation B-ptm +causes O +structural O +changes O +in O +the O +CTR B-structure_element +. O + +After O +200 O +ns O +of O +MD B-experimental_method +simulation B-experimental_method +, O +the O +interactions O +between O +these O +residues O +and O +Ser453 B-residue_name_number +remain O +intact O +. O + +Finally O +, O +the O +S453J B-mutant +mutant B-protein_state +is O +also O +stable B-protein_state +throughout O +the O +200 O +- O +ns O +simulation B-experimental_method +and O +has O +an O +average O +backbone O +deviation O +of O +∼ O +3 O +. O +8 O +Å O +, O +which O +is O +similar O +to O +the O +DD B-mutant +mutant I-mutant +. O + +The O +distance B-evidence +between O +the O +phosphate B-chemical +of O +Sep453 B-residue_name_number +and O +the O +acidic O +oxygen O +atoms O +of O +Glu420 B-residue_name_number +is O +initially O +∼ O +11 O +Å O +, O +but O +increases O +to O +> O +30 O +Å O +after O +200 O +ns O +. O + +Thus O +, O +the O +MD B-experimental_method +simulations B-experimental_method +support O +the O +notion O +from O +the O +crystal B-evidence +structures I-evidence +that O +phosphorylation B-ptm +generates O +conformational O +changes O +in O +the O +conserved B-protein_state +part O +of O +the O +CTR B-structure_element +. O + +In O +Arabidopsis B-species +thaliana I-species +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +, O +phosphorylation B-ptm +of O +the O +CTR B-structure_element +residue O +T460 B-residue_name_number +under O +conditions O +of O +high O +ammonium B-chemical +inhibits O +transport O +activity O +, O +that O +is O +, O +the O +default O +( O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +) O +state O +of O +the O +plant B-taxonomy_domain +transporter B-protein_type +is O +open B-protein_state +. O + +Owing O +to O +the O +lack O +of O +structural O +information O +for O +plant B-taxonomy_domain +AMTs B-protein_type +, O +the O +details O +of O +channel B-site +closure O +and O +inter O +- O +monomer O +crosstalk O +are O +not O +yet O +clear O +. O + +Upon O +phosphorylation B-ptm +by O +the O +Npr1 B-protein +kinase B-protein_type +in O +response O +to O +nitrogen B-chemical +limitation O +, O +the O +region O +around O +the O +conserved B-protein_state +ExxGxD B-structure_element +motif I-structure_element +undergoes O +a O +conformational O +change O +that O +opens O +the O +channel B-site +( O +Fig O +. O +9 O +). O + +Our O +Mep2 B-protein +structures B-evidence +show O +that O +this O +assumption O +may O +not O +be O +correct O +( O +Fig O +. O +4 O +and O +Supplementary O +Fig O +. O +6 O +). O + +In O +this O +way O +, O +phosphorylation B-ptm +can O +either O +lead O +to O +channel B-site +closing O +( O +in O +the O +case O +of O +AMTs B-protein_type +) O +or O +channel B-site +opening O +in O +the O +case O +of O +Mep2 B-protein +. O + +Such O +mutations O +likely O +cause O +structural O +changes O +in O +the O +CTR B-structure_element +that O +prevent O +close O +contacts O +between O +the O +CTR B-structure_element +and O +ICL1 B-structure_element +/ O +ICL3 B-structure_element +, O +thereby O +stabilizing O +a O +closed B-protein_state +state O +that O +may O +be O +similar O +to O +that O +observed O +in O +Mep2 B-protein +. O + +Recently O +, O +phosphorylation B-ptm +was O +also O +shown O +to O +modulate O +substrate O +affinity O +in O +nitrate B-protein_type +transporters I-protein_type +. O + +X B-evidence +- I-evidence +ray I-evidence +crystal I-evidence +structures I-evidence +of O +Mep2 B-protein +transceptors B-protein_type +. O + +ClustalW B-experimental_method +alignment I-experimental_method +of O +CaMep2 B-protein +, O +ScMep2 B-protein +, O +A B-species +. I-species +fulgidus I-species +Amt B-protein +- I-protein +1 I-protein +, O +E O +. O +coli O +AmtB B-protein +and O +A B-species +. I-species +thaliana I-species +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +. O + +The O +grey O +sequences O +at O +the O +C O +termini O +of O +CaMep2 B-protein +and O +ScMep2 B-protein +are O +not O +visible O +in O +the O +structures B-evidence +and O +are O +likely B-protein_state +disordered I-protein_state +. O + +In O +b O +and O +c O +, O +the O +centre O +of O +the O +trimer B-oligomeric_state +is O +at O +top O +. O + +That O +the O +3 B-protein_type +′- I-protein_type +5 I-protein_type +′ I-protein_type +elongation I-protein_type +enzyme I-protein_type +performs O +this O +elaborate O +two O +- O +step O +reaction O +in O +one O +catalytic B-site +center I-site +suggests O +that O +these O +two O +reactions O +have O +been O +inseparable O +throughout O +the O +process O +of O +protein O +evolution O +. O + +However O +, O +recent O +studies O +have O +shown O +that O +the O +Thg1 B-protein +/ O +Thg1 B-protein_type +- I-protein_type +like I-protein_type +protein I-protein_type +( O +TLP B-protein_type +) O +family O +of O +proteins O +extends O +tRNA B-chemical +nucleotide O +chains O +in O +the O +reverse O +( O +3 O +′- O +5 O +′) O +direction O +. O + +In O +5 O +′- O +3 O +′ O +elongation O +by O +DNA B-protein_type +/ I-protein_type +RNA I-protein_type +polymerases I-protein_type +, O +the O +energy O +of O +the O +incoming O +nucleotide O +is O +used O +for O +its O +own O +addition O +( O +tail O +polymerization O +). O + +This O +guanosine B-chemical +at O +position O +− B-residue_number +1 I-residue_number +( O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +) O +of O +tRNAHis B-chemical +is O +a O +critical O +identity O +element O +for O +recognition O +by O +the O +histidyl B-protein_type +- I-protein_type +tRNA I-protein_type +synthase I-protein_type +. O + +This O +finding O +suggests O +that O +TLPs B-protein_type +may O +have O +potential O +functions O +other O +than O +tRNAHis B-chemical +maturation O +. O + +This O +finding O +suggests O +that O +3 B-protein_type +′- I-protein_type +5 I-protein_type +′ I-protein_type +elongation I-protein_type +enzymes I-protein_type +are O +related O +to O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +polymerases I-protein_type +and O +raises O +important O +questions O +on O +why O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +polymerases I-protein_type +predominate O +in O +nature O +. O + +The O +O O +atom O +on O +the O +S213 B-residue_name_number +side O +chain O +was O +also O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +phosphate B-chemical +moiety O +of O +G57 B-residue_name_number +of O +the O +tRNA B-chemical +( O +Fig O +. O +2 O +). O + +The O +N7 O +atom O +of O +G2 B-residue_name_number +at O +the O +5 O +′- O +end O +was O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +N O +atom O +of O +the O +R136 B-residue_name_number +side O +chain O +, O +whereas O +the O +α O +- O +phosphate B-chemical +was O +bonded O +to O +the O +N137 B-residue_name_number +side O +chain O +( O +Fig O +. O +2 O +). O + +The O +obtained O +structure B-evidence +showed O +that O +the O +guanine B-chemical +base O +of O +the O +incoming O +GDPNP B-chemical +formed O +Watson B-bond_interaction +- I-bond_interaction +Crick I-bond_interaction +hydrogen I-bond_interaction +bonds I-bond_interaction +with O +C72 B-residue_name_number +and O +accompanied O +base B-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +interactions I-bond_interaction +with O +G2 B-residue_name_number +of O +the O +tRNA B-chemical +( O +Fig O +. O +3B O +), O +whereas O +no O +interaction O +was O +observed O +between O +the O +guanine B-chemical +base O +and O +the O +enzyme O +. O + +Surprisingly O +, O +the O +5 B-chemical +′- I-chemical +triphosphate I-chemical +moiety O +after O +movement O +occupied O +the O +GTP B-chemical +/ O +ATP B-chemical +triphosphate B-chemical +position O +during O +the O +activation O +step O +( O +Fig O +. O +3D O +). O + +All O +of O +these O +residues O +are O +well B-protein_state +conserved I-protein_state +( O +fig O +. O +S5 O +), O +and O +mutation B-experimental_method +of O +corresponding O +residues O +in O +ScThg1 B-protein +( O +R27 B-residue_name_number +, O +R93 B-residue_name_number +, O +K96 B-residue_name_number +, O +and O +R133 B-residue_name_number +) O +decreased O +the O +catalytic O +efficiency O +of O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +addition O +. O + +( O +A O +) O +Guanylylation O +of O +ppptRNAPheΔ1 B-chemical +and O +ppptRNAHisΔ1 B-chemical +by O +various O +TLP B-protein_type +mutants B-protein_state +. O + +The O +activity O +using O +[ B-chemical +α I-chemical +- I-chemical +32P I-chemical +] I-chemical +GTP I-chemical +, O +wild B-protein_state +- I-protein_state +type I-protein_state +MaTLP B-protein +, O +and O +ppptRNAPheΔ1 B-chemical +is O +denoted O +as O +100 O +. O +( O +B O +) O +Guanylylation O +of O +tRNAPheΔ1 B-chemical +, O +tRNAPhe B-chemical +, O +and O +tRNAHisΔ B-chemical +− I-chemical +1 I-chemical +by O +various O +TLP B-protein_type +mutants B-protein_state +. O + +The O +tRNAHis B-protein_type +- I-protein_type +specific I-protein_type +G I-protein_type +− I-protein_type +1 I-protein_type +addition I-protein_type +enzyme I-protein_type +Thg1 B-protein +needs O +to O +recognize O +both O +the O +accepter B-structure_element +stem I-structure_element +and O +anticodon B-structure_element +of O +tRNAHis B-chemical +. O + +TLP B-protein_type +has O +been O +shown O +to O +confer O +such O +catalytic O +activity O +on O +tRNAHisΔ B-chemical +− I-chemical +1 I-chemical +( O +Fig O +. O +4B O +). O + +Thus O +, O +we O +concluded O +that O +TLP B-protein_type +has O +two O +tRNA B-chemical +binding O +modes O +that O +are O +selectively O +used O +, O +depending O +on O +both O +the O +length O +of O +the O +accepter B-structure_element +stem I-structure_element +and O +the O +anticodon B-structure_element +. O + +This O +enzyme O +has O +two O +triphosphate B-site +binding I-site +sites I-site +and O +one O +reaction B-site +center I-site +at O +the O +position O +overlapping O +these O +two O +binding B-site +sites I-site +( O +Fig O +. O +5A O +). O + +( O +A O +) O +The O +reaction B-site +center I-site +overlapped O +with O +two O +triphosphate B-site +binding I-site +sites I-site +. O + +Movement O +of O +the O +5 O +′- O +terminal O +chain O +leaves O +the O +5 B-chemical +′- I-chemical +triphosphate I-chemical +of O +the O +tRNA B-chemical +in O +the O +same O +site O +as O +the O +activation O +step O +in O +( O +B O +). O + +These O +two O +Mg2 B-chemical ++ I-chemical +ions O +are O +coordinated B-bond_interaction +by I-bond_interaction +a O +conserved B-protein_state +Asp B-residue_name +( O +D21 B-residue_name_number +and O +D69 B-residue_name_number +in O +TLP B-protein_type +) O +in O +the O +conserved B-protein_state +catalytic B-site +core I-site +. O + +However O +, O +from O +an O +energetic O +viewpoint O +, O +these O +two O +reactions O +are O +clearly O +different O +: O +Whereas O +the O +high O +energy O +of O +the O +incoming O +nucleotide O +is O +used O +for O +its O +own O +addition O +in O +DNA B-protein_type +/ I-protein_type +RNA I-protein_type +polymerases I-protein_type +, O +the O +high O +energy O +of O +the O +incoming O +nucleotide O +is O +used O +for O +subsequent O +addition O +in O +TLP B-protein_type +. O + +TLP B-protein_type +has O +successfully O +created O +such O +sites O +by O +utilizing O +a O +conformational O +change O +in O +the O +tRNA B-chemical +through O +Watson B-bond_interaction +- I-bond_interaction +Crick I-bond_interaction +base I-bond_interaction +pairing I-bond_interaction +( O +Fig O +. O +3 O +). O + +Structural O +diversity O +in O +a O +human B-species +antibody B-protein_type +germline O +library O + +The O +structures B-evidence +and O +their O +analyses O +provide O +a O +rich O +foundation O +for O +future O +antibody B-protein_type +modeling O +and O +engineering O +efforts O +. O + +Our O +current O +structural O +knowledge O +of O +antibodies B-protein_type +is O +based O +on O +a O +multitude O +of O +studies O +that O +used O +many O +techniques O +to O +gain O +insight O +into O +the O +functional O +and O +structural O +properties O +of O +this O +class O +of O +macromolecule O +. O + +IgG B-protein +, O +IgD B-protein +and O +IgE B-protein +isotypes O +are O +composed O +of O +2 O +heavy B-structure_element +chains I-structure_element +( O +HCs B-structure_element +) O +and O +2 O +light B-structure_element +chains I-structure_element +( O +LCs B-structure_element +) O +linked O +through O +disulfide B-ptm +bonds I-ptm +, O +while O +IgA B-protein +and O +IgM B-protein +are O +double O +and O +quintuple O +versions O +of O +antibodies B-protein_type +, O +respectively O +. O + +This O +site O +, O +which O +interacts O +with O +the O +antigen O +( O +or O +target O +), O +is O +the O +focus O +of O +current O +antibody B-protein_type +modeling O +efforts O +. O + +This O +interaction B-site +site I-site +is O +composed O +of O +6 O +complementarity B-structure_element +- I-structure_element +determining I-structure_element +regions I-structure_element +( O +CDRs B-structure_element +) O +that O +were O +identified O +in O +early O +antibody B-experimental_method +amino I-experimental_method +acid I-experimental_method +sequence I-experimental_method +analyses I-experimental_method +to O +be O +hypervariable B-protein_state +in O +nature O +, O +and O +thus O +are O +responsible O +for O +the O +sequence O +and O +structural O +diversity O +of O +our O +antibody B-protein_type +repertoire O +. O + +A O +CDR B-structure_element +canonical O +structure O +is O +defined O +by O +its O +length O +and O +conserved O +residues O +located O +in O +the O +hypervariable B-structure_element +loop I-structure_element +and O +framework B-structure_element +residues I-structure_element +( O +V B-structure_element +- I-structure_element +region I-structure_element +residues O +that O +are O +not O +part O +of O +the O +CDRs B-structure_element +). O + +Classification O +schemes O +for O +the O +canonical O +structures O +of O +these O +5 O +CDRs B-structure_element +have O +emerged O +and O +evolved O +as O +the O +number O +of O +depositions O +in O +the O +Protein O +Data O +Bank O +of O +Fab B-structure_element +fragments O +of O +antibodies B-protein_type +grow O +. O + +Some O +clustering O +of O +conformations O +was O +observed O +for O +the O +shortest O +lengths O +; O +however O +, O +for O +the O +longer O +loops B-structure_element +, O +only O +the O +portions O +nearest O +the O +framework B-structure_element +( O +torso B-structure_element +, O +stem B-structure_element +or O +anchor B-structure_element +region I-structure_element +) O +were O +found O +to O +have O +defined O +conformations O +. O + +Current O +antibody B-protein_type +modeling O +approaches O +take O +advantage O +of O +the O +most O +recent O +advances O +in O +homology B-experimental_method +modeling I-experimental_method +, O +the O +evolving O +understanding O +of O +the O +CDR B-structure_element +canonical O +structures B-evidence +, O +the O +emerging O +rules O +for O +CDR B-structure_element +H3 B-structure_element +modeling O +and O +the O +growing O +body O +of O +antibody B-protein_type +structural O +data O +available O +from O +the O +PDB O +. O + +All O +16 O +HCs B-structure_element +of O +the O +Fabs B-structure_element +have O +the O +same O +CDR B-structure_element +H3 B-structure_element +that O +was O +reported O +in O +an O +earlier O +Fab B-structure_element +structure B-evidence +. O + +This O +is O +the O +first O +systematic O +study O +of O +the O +same O +VH B-structure_element +and O +VL B-structure_element +structures B-evidence +in O +the O +context O +of O +different O +pairings O +. O + +Crystallization B-experimental_method +of O +the O +16 O +Fabs B-structure_element +was O +previously O +reported O +. O + +Three O +sets O +of O +the O +crystals B-evidence +were O +isomorphous O +with O +nearly O +identical O +unit O +cells O +( O +Table O +1 O +). O + +These O +include O +( O +1 O +) O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +in O +P212121 O +, O +( O +2 O +) O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +, O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +in O +P6522 O +, O +and O +( O +3 O +) O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +, O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +and O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +in O +P212121 O +. O + +No O +assignment O +( O +NA O +) O +for O +CDRs B-structure_element +with O +missing O +residues O +. O + +A O +major O +difference O +of O +H1 B-mutant +- I-mutant +69 I-mutant +from O +the O +other O +germlines O +in O +the O +experimental O +data O +set O +is O +the O +presence O +of O +Gly B-residue_name +instead O +of O +Phe B-residue_name +or O +Tyr B-residue_name +at O +position O +27 B-residue_number +( O +residue O +5 O +of O +13 O +in O +CDR B-structure_element +H1 B-structure_element +). O + +The O +superposition B-experimental_method +of O +CDR B-structure_element +L1 B-structure_element +backbones O +for O +all O +HC B-complex_assembly +: I-complex_assembly +LC I-complex_assembly +pairs O +with O +light B-structure_element +chains I-structure_element +: O +( O +A O +) O +L1 B-mutant +- I-mutant +39 I-mutant +, O +( O +B O +) O +L3 B-mutant +- I-mutant +11 I-mutant +, O +( O +C O +) O +L3 B-mutant +- I-mutant +20 I-mutant +and O +( O +D O +) O +L4 B-mutant +- I-mutant +1 I-mutant +. O + +For O +the O +remaining O +2 O +, O +L3 B-mutant +- I-mutant +20 I-mutant +has O +2 O +different O +assignments O +, O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +1 I-mutant +and O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +2 I-mutant +, O +while O +L4 B-mutant +- I-mutant +1 I-mutant +has O +a O +single O +assignment O +, O +L1 B-mutant +- I-mutant +17 I-mutant +- I-mutant +1 I-mutant +. O + +The O +third O +structure O +, O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +has O +CDR B-structure_element +L1 B-structure_element +as O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +2 I-mutant +, O +which O +deviates O +from O +L1 B-mutant +- I-mutant +12 I-mutant +- I-mutant +1 I-mutant +at O +residues O +29 B-residue_range +- I-residue_range +32 I-residue_range +, O +i O +. O +e O +., O +at O +the O +site O +of O +insertion O +with O +respect O +to O +the O +11 B-residue_range +- I-residue_range +residue I-residue_range +CDR B-structure_element +. O + +The O +stem B-structure_element +region I-structure_element +of O +CDR B-structure_element +H3 B-structure_element +in O +the O +parental O +Fab B-structure_element +is O +in O +a O +‘ O +kinked B-protein_state +’ O +conformation O +, O +in O +which O +the O +indole O +nitrogen O +of O +Trp103 B-residue_name_number +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +carbonyl O +oxygen O +of O +Leu100b B-residue_name_number +. O + +The O +structure B-evidence +of O +each O +CDR B-structure_element +H3 B-structure_element +is O +represented O +with O +a O +different O +color O +. O + +Despite O +having O +the O +same O +amino O +acid O +sequence O +in O +all O +variants O +, O +CDR B-structure_element +H3 B-structure_element +has O +the O +highest O +degree O +of O +structural O +diversity O +and O +disorder O +of O +all O +of O +the O +CDRs B-structure_element +in O +the O +experimental O +set O +. O + +In O +10 O +of O +the O +18 O +Fab B-structure_element +structures B-evidence +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +( O +2 O +Fabs B-structure_element +), O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +, O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +( O +2 O +Fabs B-structure_element +), O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +, O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +, O +the O +CDRs B-structure_element +have O +similar O +conformations O +to O +that O +found O +in O +4DN3 O +. O + +The O +stem B-structure_element +regions I-structure_element +in O +these O +3 O +cases O +are O +in O +the O +‘ O +kinked B-protein_state +’ O +conformation O +consistent O +with O +that O +observed O +for O +4DN3 O +. O + +The O +domain O +packing O +of O +the O +variants O +was O +assessed O +by O +computing O +the O +domain B-site +interface I-site +interactions O +, O +the O +VH B-complex_assembly +: I-complex_assembly +VL I-complex_assembly +tilt B-evidence +angles I-evidence +, O +the O +buried O +surface O +area O +and O +surface O +complementarity O +. O + +The O +VH B-structure_element +residues O +are O +in O +blue O +, O +the O +VL B-structure_element +residues O +are O +in O +orange O +. O + +The O +VH B-site +: I-site +VL I-site +interface I-site +is O +pseudosymmetric B-protein_state +, O +and O +involves O +2 O +stretches O +of O +the O +polypeptide O +chain O +from O +each O +domain O +, O +namely O +CDR3 B-structure_element +and O +the O +framework B-structure_element +region I-structure_element +between O +CDRs B-structure_element +1 I-structure_element +and I-structure_element +2 I-structure_element +. O + +These O +stretches O +form O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +hairpins I-structure_element +within O +the O +internal O +5 B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +. O + +The O +second O +approach O +used O +for O +comparing O +tilt B-evidence +angles I-evidence +involved O +computing O +the O +difference B-evidence +in O +the O +tilt B-evidence +angles I-evidence +between O +all O +pairs O +of O +structures B-evidence +. O + +Indeed O +, O +this O +Fab B-structure_element +has O +the O +largest O +twist B-evidence +angle I-evidence +HC2 B-structure_element +within O +the O +experimental O +set O +that O +exceeds O +the O +mean O +value O +by O +2 O +. O +5 O +standard O +deviations O +( O +Table O +S2 O +). O + +One O +of O +the O +2 O +structures B-evidence +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +has O +its O +CDR B-structure_element +H3 B-structure_element +in O +the O +‘ O +extended B-protein_state +’ O +conformation O +; O +the O +other O +structure O +has O +it O +in O +the O +‘ O +kinked B-protein_state +’ O +conformation O +. O + +Some O +side O +chain O +atoms O +in O +CDR B-structure_element +H3 B-structure_element +are O +missing O +. O + +Having O +all O +16 O +VH B-complex_assembly +: I-complex_assembly +VL I-complex_assembly +pairs O +with O +the O +same O +CDR B-structure_element +H3 B-structure_element +provides O +some O +insights O +into O +why O +molecular O +modeling O +efforts O +of O +CDR B-structure_element +H3 B-structure_element +have O +proven O +so O +difficult O +. O + +This O +subset O +also O +has O +2 O +structures B-evidence +with O +2 O +Fab B-structure_element +copies O +in O +the O +asymmetric O +unit O +. O + +Thus O +, O +no O +patterns O +of O +conformational O +preference O +for O +a O +particular O +HC B-structure_element +or O +LC B-structure_element +emerge O +to O +shed O +any O +direct O +light O +on O +what O +drives O +the O +conformational O +differences O +. O + +At O +the O +other O +end O +of O +the O +stability O +range O +is O +LC B-structure_element +germline O +L3 B-mutant +- I-mutant +20 I-mutant +, O +which O +yields O +antibodies B-protein_type +with O +the O +lowest O +Tms B-evidence +. O + +Yet O +, O +for O +the O +2 O +antibodies B-protein_type +, O +the O +total O +gain O +in O +stability O +merits O +the O +domain O +repacking O +. O + +Quite O +unexpectedly O +, O +2 O +of O +the O +variants O +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +, O +have O +the O +‘ O +extended B-protein_state +’ O +stem B-structure_element +region I-structure_element +differing O +from O +the O +other O +14 O +that O +have O +a O +‘ O +kinked B-protein_state +’ O +stem B-structure_element +region I-structure_element +. O + +For O +those O +applications O +where O +accurate O +CDR B-structure_element +structures B-evidence +are O +essential O +, O +such O +as O +docking O +, O +the O +results O +in O +this O +work O +demonstrate O +the O +importance O +of O +experimental O +structures B-evidence +. O + +Interestingly O +, O +several O +clinically O +relevant O +and O +human B-species +pathogenic O +strains O +are O +inherently O +resistant O +towards O +lantibiotics B-chemical +. O + +The O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +exhibits O +a O +fold O +that O +classifies O +NsrR B-protein +as O +a O +member O +of O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +of O +regulators O +. O + +They O +are O +post O +- O +translationally O +modified O +and O +contain O +specific O +lanthionine B-chemical +/ O +methyl B-chemical +- I-chemical +lanthionine I-chemical +rings O +, O +which O +are O +crucial O +for O +their O +high O +antimicrobial O +activity O +. O + +Examples O +for O +LanFEG B-protein_type +are O +NisI B-protein +and O +NisFEG B-protein +of O +the O +nisin B-chemical +system O +, O +SpaI B-protein +and O +SpaFEG B-protein +conferring O +immunity O +towards O +subtilin B-chemical +, O +and O +PepI B-protein +constituting O +the O +immunity O +system O +of O +Pep5 B-chemical +producing O +strains O +. O + +Furthermore O +, O +the O +upregulation O +of O +these O +genes O +is O +mediated O +by O +a O +specific O +two B-complex_assembly +- I-complex_assembly +component I-complex_assembly +system I-complex_assembly +( O +TCS B-complex_assembly +) O +similar O +to O +the O +one O +found O +in O +lantibiotic B-chemical +producing O +strains O +, O +consisting O +of O +a O +sensor O +histidine B-protein_type +kinase I-protein_type +( O +HK B-protein_type +) O +and O +a O +response B-protein_type +regulator I-protein_type +( O +RR B-protein_type +), O +apparently O +mediate O +the O +expression O +of O +the O +resistance O +proteins O +: O +HK B-protein_type +senses O +the O +external O +lantibiotic B-chemical +and O +, O +upon O +receiving O +the O +stimuli O +, O +auto B-ptm +- I-ptm +phosphorylates I-ptm +at O +a O +conserved B-protein_state +histidine B-residue_name +residue O +within O +the O +cytosol O +; O +this O +high O +- O +energetic O +phosphoryl O +group O +is O +then O +transferred O +to O +the O +associated O +RR B-protein_type +inducing O +a O +conformational O +change O +there O +, O +which O +activates O +the O +RR B-protein_type +to O +evoke O +the O +cellular O +response O +. O + +The O +recently O +discovered O +nsr B-gene +gene O +cluster O +of O +the O +human B-species +pathogen O +S B-species +. I-species +agalactiae I-species +encodes O +for O +the O +resistance B-protein_type +protein I-protein_type +NSR B-protein +and O +the O +ABC B-protein_type +transporter I-protein_type +NsrFP B-protein +, O +both O +conferring O +resistance O +against O +nisin B-chemical +. O + +The O +ED B-structure_element +is O +thereby O +activated O +and O +binds O +to O +the O +designated O +promoters O +, O +thus O +initiating O +transcription O +of O +the O +target O +genes O +. O + +Although O +numerous O +structures B-evidence +of O +the O +single O +domains O +are O +known O +, O +only O +a O +few O +structures B-evidence +of O +full B-protein_state +- I-protein_state +length I-protein_state +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +- I-protein_type +type I-protein_type +RRs I-protein_type +have O +been O +determined O +: O +RegX3 B-protein +( O +PDB O +code O +: O +2OQR O +), O +MtrA B-protein +( O +PDB O +code O +: O +2GWR O +), O +PrrA B-protein +( O +PDB O +code O +: O +1YS6 O +) O +and O +PhoP B-protein +( O +PDB O +code O +: O +3R0J O +) O +from O +Mycobacterium B-species +tuberculosis I-species +; O +DrrB B-protein +( O +PDB O +code O +: O +1P2F O +) O +and O +DrrD B-protein +( O +PDB O +code O +: O +1KGS O +) O +from O +Thermotoga B-species +maritima I-species +; O +and O +KdpE B-protein +from O +Escherichia B-species +coli I-species +( O +PDB O +code O +: O +4KNY O +). O + +By O +calibrating O +the O +column O +with O +proteins O +of O +known O +molecular O +weight O +the O +NsrR B-protein +full B-protein_state +length I-protein_state +protein O +elutes O +as O +a O +dimer B-oligomeric_state +. O + +Surprisingly O +, O +over O +time O +NsrR B-protein +degraded O +into O +two O +distinct O +fragments O +as O +visible O +on O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +analysis O +using O +the O +same O +purified O +protein O +sample O +after O +one O +week O +( O +Fig O +1C O +, O +indicated O +by O +** O +and O +***, O +respectively O +). O + +Such O +a O +cleavage O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +RR B-protein_type +into O +two O +specific O +domains O +is O +not O +unusual O +and O +has O +been O +previously O +reported O +for O +other O +RRs B-protein_type +as O +well O +. O + +The O +bold O +line O +represents O +the O +chromatogram B-evidence +of O +freshly O +purified O +NsrR B-protein +while O +the O +dashed O +line O +shows O +the O +chromatogram B-evidence +of O +the O +same O +NsrR B-protein +protein O +after O +one O +week O +. O + +We O +also O +tried O +to O +solve O +the O +structure B-evidence +of O +the O +thin O +plate O +- O +shaped O +crystals B-evidence +with O +this O +template O +, O +but O +the O +resulting O +model O +generated O +was O +not O +sufficient O +. O + +We O +determined O +the O +crystal B-evidence +structures I-evidence +of O +NsrR B-protein +- O +RD B-structure_element +and O +NsrR B-protein +- O +ED B-structure_element +separately O +. O + +Ramachandran B-evidence +validation I-evidence +revealed O +that O +all O +residues O +( O +100 O +%, O +236 O +amino O +acids O +) O +were O +in O +the O +preferred O +or O +allowed O +regions O +. O + +Cartoon O +representation O +of O +the O +helices B-structure_element +( O +α1 B-structure_element +– I-structure_element +α5 I-structure_element +) O +and O +β B-structure_element +- I-structure_element +sheets I-structure_element +( O +β1 B-structure_element +- I-structure_element +β5 I-structure_element +). O + +Structural O +areas O +with O +the O +highest O +variations O +to O +the O +receiver B-structure_element +domains I-structure_element +of O +DrrB B-protein +( O +pink O +, O +1P2F O +), O +MtrA B-protein +( O +grey O +, O +2GWR O +), O +and O +PhoB B-protein +( O +blue O +, O +1B00 O +) O +are O +marked O +in O +separate O +boxes O +. O + +Furthermore O +, O +NsrR B-protein +is O +crystallized B-experimental_method +as O +a O +monomer B-oligomeric_state +, O +and O +investigation O +of O +the O +symmetry O +- O +related O +molecules O +did O +not O +reveal O +a O +functional O +dimer B-oligomeric_state +within O +the O +crystal B-evidence +. O + +The O +rmsd B-evidence +values O +of O +the O +superimpositions B-experimental_method +of O +the O +structures B-evidence +of O +NsrR B-protein +- O +RD B-structure_element +and O +NsrR B-protein +- O +ED B-structure_element +with O +the O +available O +structures B-evidence +of O +members O +of O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +are O +highlighted O +. O +* O +Seq O +ID O +(%) O +corresponds O +to O +the O +full B-protein_state +- I-protein_state +length I-protein_state +protein O +sequence O +. O + +This O +site O +of O +phosphorylation B-ptm +is O +conserved B-protein_state +throughout O +the O +family O +of O +response B-protein_type +regulators I-protein_type +, O +including O +the O +lantibiotic B-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +RRs I-protein_type +such O +as O +BraR B-protein +from O +L B-species +. I-species +monocytogenes I-species +, O +BceR B-protein +from O +Bacillus B-species +subtilis I-species +, O +CprR B-protein +from O +C B-species +. I-species +difficile I-species +, O +GraR B-protein +from O +S B-species +. I-species +aureus I-species +, O +LcrR B-protein +from O +S B-species +. I-species +mutans I-species +, O +LisR B-protein +, O +and O +VirR B-protein +from O +L B-species +. I-species +monocytogenes I-species +( O +Fig O +3 O +). O + +The O +linker B-structure_element +region I-structure_element +of O +the O +known O +structures B-evidence +is O +underlined O +within O +the O +sequence O +. O + +This O +pocket B-site +is O +similar O +to O +the O +acidic B-protein_state +active B-site +site I-site +observed O +within O +most O +structures B-evidence +of O +RRs B-protein_type +such O +as O +PhoB B-protein +from O +E B-species +. I-species +coli I-species +, O +PhoP B-protein +from O +M B-species +. I-species +tuberculosis I-species +, O +and O +DivK B-protein +from O +Caulobacter B-species +crescentus I-species +. O + +The O +inactive B-protein_state +conformation O +of O +NsrR B-protein +differs O +from O +the O +active B-protein_state +state O +structure B-evidence +of O +PhoB B-protein +( O +light O +blue O +, O +PDB O +code O +1ZES O +) O +( O +b O +) O +in O +the O +orientation O +of O +the O +corresponding O +switch B-site +residues I-site +, O +Ser82 B-residue_name_number +and O +Phe101 B-residue_name_number +, O +which O +adopt O +a O +conformation O +pointing O +away O +from O +the O +active B-site +site I-site +( O +Asp55 B-residue_name_number +in O +NsrR B-protein +). O + +In O +some O +RRs B-protein_type +like O +CheY B-protein +, O +Mg2 B-chemical ++ I-chemical +is O +observed O +in O +the O +structure B-evidence +, O +bound B-protein_state +near O +the O +phosphorylation B-site +site I-site +. O + +In O +the O +KdpE B-protein +regulator B-protein_type +from O +E B-species +. I-species +coli I-species +that O +is O +involved O +in O +osmoregulation O +, O +a O +divalent O +calcium B-chemical +ion O +is O +present O +. O + +Within O +the O +β4 B-structure_element +- I-structure_element +α4 I-structure_element +loop I-structure_element +and O +in O +β5 B-structure_element +of O +the O +RD B-structure_element +of O +RRs B-protein_type +, O +specific O +amino O +acids O +are O +crucial O +for O +signal O +transduction O +from O +the O +RD B-structure_element +to O +the O +ED B-structure_element +via O +conformational O +changes O +that O +are O +a O +consequence O +of O +phosphorylation B-ptm +of O +the O +RD B-structure_element +. O + +These O +amino O +acids O +are O +Ser B-residue_name +/ O +Thr B-residue_name +and O +Phe B-residue_name +/ O +Tyr B-residue_name +located O +at O +the O +end O +of O +β4 B-structure_element +and O +before O +β5 B-structure_element +, O +respectively O +, O +and O +designated O +as O +“ O +signature B-site +switch I-site +residues I-site +”. O + +Although O +some O +RRs B-protein_type +such O +as O +KdpE B-protein +, O +BraR B-protein +, O +BceR B-protein +, O +GraR B-protein +, O +and O +VirR B-protein +contain O +a O +serine B-residue_name +residue O +as O +the O +first B-site +switch I-site +residue I-site +, O +the O +others O +possess O +a O +threonine B-residue_name +instead O +. O + +The O +RD B-structure_element +domain O +of O +NsrR B-protein +was O +crystallized B-experimental_method +with O +two O +separate O +monomers B-oligomeric_state +in O +the O +asymmetric O +unit O +. O + +Afterwards O +, O +helix B-structure_element +α4 B-structure_element +and O +the O +adjacent O +loops B-structure_element +were O +energy B-experimental_method +minimized I-experimental_method +with I-experimental_method +the I-experimental_method +MAB I-experimental_method +force I-experimental_method +field I-experimental_method +as O +implemented O +in O +the O +program O +Moloc O +; O +all O +other O +atoms O +of O +NsrR B-protein +- O +RD B-structure_element +were O +kept O +fixed O +. O + +The O +result O +is O +highlighted O +in O +S2B O +Fig O +. O +The O +energy B-protein_state +minimized I-protein_state +structure B-evidence +of O +NsrR B-protein +- O +RD B-structure_element +was O +then O +superimposed B-experimental_method +on O +the O +dimeric B-oligomeric_state +structure B-evidence +of O +KdpE B-protein +. O + +In O +addition O +, O +the O +dimeric B-site +interface I-site +of O +KdpE B-protein +is O +characterized O +by O +hydrophobic B-site +patch I-site +formed O +by O +residues O +Ile88 B-residue_name_number +( O +α4 B-structure_element +), O +Leu91 B-residue_name_number +( O +α4 B-structure_element +), O +Ala110 B-residue_name_number +( O +α5 B-structure_element +), O +and O +Val114 B-residue_name_number +( O +α5 B-structure_element +). O + +Dimeric B-oligomeric_state +structure B-evidence +of O +the O +RD B-structure_element +of O +NsrR B-protein +aligned O +to O +the O +structure B-evidence +of O +KdpE B-protein +( O +PDB O +code O +1ZH2 O +, O +not O +shown O +). O + +( O +a O +) O +The O +two O +monomers B-oligomeric_state +of O +NsrR B-protein +as O +functional O +dimers B-oligomeric_state +are O +represented O +in O +a O +cartoon O +representation O +displayed O +in O +cyan O +and O +yellow O +colors O +. O + +Overall O +Structure B-evidence +of O +C O +- O +terminal O +DNA B-structure_element +- I-structure_element +binding I-structure_element +effector I-structure_element +domain I-structure_element +of O +NsrR B-protein + +The O +asymmetric O +unit O +contains O +two O +copies O +of O +NsrR B-protein +- O +ED B-structure_element +related O +by O +two O +- O +fold O +rotational O +symmetry O +. O + +The O +structure B-evidence +of O +NsrR B-protein +- O +ED B-structure_element +also O +contains O +such O +a O +wHTH B-structure_element +motif O +built O +up O +by O +helices B-structure_element +α7 B-structure_element +and O +α8 B-structure_element +( O +Fig O +6 O +). O + +In O +the O +structure B-evidence +of O +NsrR B-protein +- O +ED B-structure_element +, O +helix B-structure_element +α8 B-structure_element +is O +identified O +as O +the O +recognition B-structure_element +helix I-structure_element +, O +α7 B-structure_element +as O +the O +positioning B-structure_element +helix I-structure_element +, O +and O +the O +loop B-structure_element +region I-structure_element +between O +helices O +α7 B-structure_element +- I-structure_element +α8 I-structure_element +as O +transactivation B-structure_element +loop I-structure_element +as O +observed O +in O +other O +RRs B-protein_type +( O +Fig O +6 O +). O + +The O +rmsd B-evidence +between O +the O +three O +helices O +of O +the O +effector B-structure_element +domain I-structure_element +( O +including O +the O +two O +helices B-structure_element +forming O +the O +wHTH B-structure_element +motif O +) O +of O +PhoB B-protein +and O +NsrR B-protein +- O +ED B-structure_element +is O +1 O +. O +6 O +Å O +over O +47 O +Cα O +atoms O +, O +clearly O +indicating O +that O +NsrR B-protein +belongs O +to O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +family I-protein_type +of I-protein_type +RRs I-protein_type +. O + +The O +exact O +boundaries O +of O +these O +linkers B-structure_element +are O +difficult O +to O +predict O +from O +sequence B-experimental_method +alignments I-experimental_method +in O +the O +absence B-protein_state +of I-protein_state +structural O +information O +of O +the O +distinct O +RR B-protein_type +. O + +Linker B-structure_element +lengths O +in O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +proteins I-protein_type +of O +unknown O +structure O +have O +been O +estimated O +by O +comparing O +the O +number O +of O +residues O +between O +conserved O +landmark O +residues O +in O +the O +regulatory B-structure_element +and I-structure_element +effector I-structure_element +domains I-structure_element +to O +those O +from O +structurally O +characterized O +family O +members O +. O + +As O +seen O +in O +the O +structures B-evidence +of O +MtrA B-protein +and O +KdpE B-protein +, O +this O +arginine B-residue_name +residue O +residing O +at O +the O +end O +of O +α5 B-structure_element +participates O +in O +the O +active B-protein_state +state O +dimer B-site +interface I-site +of O +the O +RD B-structure_element +through O +a O +salt B-bond_interaction +bridge I-bond_interaction +interaction O +with O +an O +aspartate B-residue_name +residue O +. O + +Although O +we O +aimed O +at O +crystallizing B-experimental_method +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +, O +this O +endeavor O +failed O +due O +to O +proteolytic O +cleavage O +within O +the O +linker B-structure_element +region I-structure_element +during O +the O +time O +period O +of O +crystallization B-experimental_method +. O + +DNA B-chemical +- O +binding O +mode O +of O +NsrR B-protein +using O +a O +full B-protein_state +- I-protein_state +length I-protein_state +model O + +Since O +the O +structures B-evidence +of O +both O +domains O +of O +NsrR B-protein +were O +determined O +, O +we O +used O +this O +structural B-evidence +information I-evidence +together O +with O +the O +available O +crystal B-evidence +structures I-evidence +of O +related O +proteins O +to O +create O +a O +model O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +in O +its O +active B-protein_state +and O +inactive B-protein_state +state O +. O + +Model O +of O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +in O +its O +inactive B-protein_state +state O +and O +active B-protein_state +state O +. O + +In O +numerous O +pathogenic O +bacteria B-taxonomy_domain +such O +as O +S B-species +. I-species +agalactiae I-species +, O +S B-species +. I-species +aureus I-species +, O +and O +C B-species +. I-species +difficile I-species +that O +apparently O +do O +not O +produce O +a O +lantibiotic B-chemical +, O +a O +gene O +cluster O +is O +present O +to O +provide O +resistance O +against O +lantibiotics B-chemical +such O +as O +nisin B-chemical +, O +nukacin B-chemical +ISK I-chemical +- I-chemical +1 I-chemical +, O +lacticin B-chemical +481 I-chemical +gallidermin B-chemical +, O +actagardine B-chemical +, O +or O +mersacidin B-chemical +. O + +NMR B-experimental_method +can O +theoretically O +be O +used O +to O +determine O +heterogeneous O +ensembles O +, O +but O +in O +practice O +, O +this O +proves O +to O +be O +very O +challenging O +. O + +However O +, O +the O +impact O +that O +chaperones B-protein_type +have O +on O +their O +substrates O +, O +and O +how O +these O +interactions O +affect O +the O +folding O +process O +remain O +largely O +unknown O +. O + +Spy B-protein +prevents O +protein O +aggregation O +and O +aids O +in O +protein O +folding O +under O +various O +stress O +conditions O +, O +including O +treatment O +with O +tannin B-chemical +and O +butanol B-chemical +. O + +We O +originally O +discovered O +Spy B-protein +by O +its O +ability O +to O +stabilize O +the O +protein O +- O +folding O +model O +Im7 B-protein +in O +vivo O +and O +recently O +demonstrated O +that O +Im7 B-protein +folds O +while O +associated O +with O +Spy B-protein +. O + +Such O +residual O +density O +is O +typically O +not O +considered O +usable O +by O +traditional O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +methods O +. O + +To O +determine O +the O +structure B-evidence +of O +the O +substrate O +portion O +of O +these O +Spy B-protein +: O +substrate O +complexes O +, O +we O +conceived O +of O +an O +approach O +that O +we O +term O +READ B-experimental_method +, O +for O +Residual B-experimental_method +Electron I-experimental_method +and I-experimental_method +Anomalous I-experimental_method +Density I-experimental_method +. O + +We O +split O +this O +approach O +into O +five O +steps O +: O +( O +1 O +) O +By O +using O +a O +well O +- O +diffracting O +Spy B-protein +: O +substrate O +co B-evidence +- I-evidence +crystal I-evidence +, O +we O +first O +determined O +the O +structure B-evidence +of O +the O +folded B-protein_state +domain B-structure_element +of O +Spy B-protein +and O +obtained O +high O +quality O +residual B-evidence +electron I-evidence +density I-evidence +within O +the O +dynamic B-protein_state +regions O +of O +the O +substrate O +. O + +We O +then O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +Spy B-protein +and O +the O +eight O +Im76 B-mutant +- I-mutant +45 I-mutant +peptides O +, O +each O +of O +which O +harbored O +an O +individual O +pI B-chemical +- I-chemical +Phe I-chemical +substitution B-experimental_method +at O +one O +distinct O +position O +, O +and O +collected B-experimental_method +anomalous B-evidence +data I-evidence +for O +all O +eight O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +complexes O +( O +Fig O +. O +1B O +, O +Supplementary O +Table O +1 O +Supplementary O +Dataset O +1 O +, O +and O +Supplementary O +Table O +2 O +). O + +During O +each O +round O +of O +the O +selection O +, O +a O +genetic B-experimental_method +algorithm I-experimental_method +alters O +the O +ensemble O +and O +its O +agreement O +to O +the O +experimental O +data O +is O +re O +- O +evaluated O +. O + +This O +strategy O +allows O +a O +wide O +range O +of O +substrate O +conformations O +to O +interact O +with O +the O +chaperone B-protein_type +. O + +The O +ensemble O +primarily O +encompasses O +Im76 B-mutant +- I-mutant +45 I-mutant +laying O +diagonally O +within O +the O +Spy B-protein +cradle B-site +in O +several O +different O +orientations O +, O +but O +some O +conformations O +traverse O +as O +far O +as O +the O +tips O +or O +even O +extend O +over O +the O +side O +of O +the O +cradle B-site +( O +Figs O +. O +3 O +, O +4a O +). O + +The O +Spy B-site +- I-site +contacting I-site +residues I-site +comprise O +a O +mixture O +of O +charged O +, O +polar O +, O +and O +hydrophobic O +residues O +. O + +For O +example O +, O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +Im76 B-mutant +- I-mutant +45 I-mutant +binds O +more O +consistently O +in O +the O +Spy B-protein +cradle B-site +, O +whereas O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +predominantly O +binds O +to O +the O +outer O +edges O +of O +Spy B-protein +’ O +s O +concave B-site +surface I-site +. O + +Although O +we O +do O +not O +yet O +have O +time O +resolution O +data O +of O +these O +various O +snapshots O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +, O +this O +ensemble O +illustrates O +how O +a O +substrate O +samples O +its O +folding O +landscape O +while O +bound B-protein_state +to I-protein_state +a O +chaperone B-protein_type +. O + +Additionally O +, O +we O +observed O +that O +the O +linker B-structure_element +region I-structure_element +( O +residues O +47 B-residue_range +– I-residue_range +57 I-residue_range +) O +of O +Spy B-protein +, O +which O +participates O +in O +substrate O +interaction O +, O +becomes O +mostly O +disordered B-protein_state +upon O +binding O +the O +substrate O +. O + +In O +the O +chaperone B-protein_state +- I-protein_state +bound I-protein_state +ensemble O +, O +Im76 B-mutant +- I-mutant +45 I-mutant +samples O +unfolded B-protein_state +, O +partially O +folded B-protein_state +, O +and O +native B-protein_state +- O +like O +states O +. O + +The O +ensemble O +provides O +an O +unprecedented O +description O +of O +the O +conformations O +that O +a O +substrate O +assumes O +while O +exploring O +its O +chaperone B-protein_type +- O +associated O +folding O +landscape O +. O + +The O +high O +- O +resolution O +ensemble B-evidence +obtained O +here O +now O +provides O +insight O +into O +exactly O +how O +this O +occurs O +. O + +The O +ensemble B-evidence +suggests O +a O +model O +in O +which O +Spy B-protein +provides O +an O +amphipathic B-site +surface I-site +that O +allows O +substrate O +proteins O +to O +assume O +different O +conformations O +while O +bound B-protein_state +to I-protein_state +the O +chaperone B-protein_type +. O + +Residues O +Asp32 B-residue_name_number +and O +Asp35 B-residue_name_number +are O +close O +to O +each O +other O +in O +the O +folded B-protein_state +state O +of O +Im7 B-protein +. O + +Our O +study O +indicates O +that O +the O +chaperone B-protein_type +Spy B-protein +employs O +a O +simple O +surface O +binding O +approach O +that O +allows O +the O +substrate O +to O +explore O +various O +conformations O +and O +form O +transiently O +favorable O +interactions O +while O +being O +protected O +from O +aggregation O +. O + +Crystallographic O +data O +and O +ensemble O +selection O +. O +( O +a O +) O +2mFo B-evidence +− I-evidence +DFc I-evidence +omit I-evidence +map I-evidence +of O +residual O +Im76 B-mutant +- I-mutant +45 I-mutant +and O +flexible B-structure_element +linker I-structure_element +electron B-evidence +density I-evidence +contoured O +at O +0 O +. O +5 O +σ O +. O + +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +ensemble O +, O +arranged O +by O +RMSD B-evidence +to O +native B-protein_state +state O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +. O +Although O +the O +six O +- O +membered O +ensemble O +from O +the O +READ B-experimental_method +selection O +should O +be O +considered O +only O +as O +an O +ensemble O +, O +for O +clarity O +, O +the O +individual O +conformers O +are O +shown O +separately O +here O +. O + +Dashed O +lines O +connect O +non O +- O +contiguous O +segments O +of O +the O +Im76 B-mutant +- I-mutant +45 I-mutant +substrate O +. O + +( O +a O +) O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +contact B-evidence +map I-evidence +projected O +onto O +the O +bound B-protein_state +Spy B-protein +dimer B-oligomeric_state +( O +above O +) O +and O +Im76 B-mutant +- I-mutant +45 I-mutant +( O +below O +) O +structures B-evidence +. O + +For O +clarity O +, O +Im76 B-mutant +- I-mutant +45 I-mutant +is O +represented O +with O +a O +single O +conformation O +. O + +As O +the O +residues O +involved O +in O +contacts O +are O +more O +evenly O +distributed O +in O +Im76 B-mutant +- I-mutant +45 I-mutant +compared O +to O +Spy B-protein +, O +its O +contact B-evidence +map I-evidence +was O +amplified O +. O +( O +b O +) O +Detailed O +contact B-evidence +maps I-evidence +of O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +. O + +( O +a O +) O +Overlay B-experimental_method +of O +apo B-protein_state +Spy B-protein +( O +PDB O +ID O +: O +3O39 O +, O +gray O +) O +and O +bound B-protein_state +Spy B-protein +( O +green O +). O +( O +b O +) O +Overlay B-experimental_method +of O +WT B-protein_state +Spy B-protein +bound B-protein_state +to I-protein_state +Im76 B-mutant +- I-mutant +45 I-mutant +( O +green O +), O +H96L B-mutant +Spy B-protein +bound B-protein_state +to I-protein_state +Im7 B-protein +L18A B-mutant +L19 B-mutant +AL13A I-mutant +( O +blue O +), O +H96L B-mutant +Spy B-protein +bound B-protein_state +to I-protein_state +WT B-protein_state +Im7 B-protein +( O +yellow O +), O +and O +WT B-protein_state +Spy B-protein +bound B-protein_state +to I-protein_state +casein B-chemical +( O +salmon O +). O +( O +c O +) O +Competition B-experimental_method +assay I-experimental_method +showing O +Im76 B-mutant +- I-mutant +45 I-mutant +competes O +with O +Im7 B-protein +L18A B-mutant +L19A B-mutant +L37A B-mutant +H40W B-mutant +for O +the O +same O +binding B-site +site I-site +on O +Spy B-protein +( O +further O +substrate B-experimental_method +competition I-experimental_method +assays I-experimental_method +are O +shown O +in O +Supplementary O +Fig O +. O +8 O +). O + +Reversal O +of O +DNA B-chemical +damage O +induced O +Topoisomerase B-protein_type +2 I-protein_type +DNA B-chemical +– O +protein O +crosslinks O +by O +Tdp2 B-protein + +Mammalian B-taxonomy_domain +Tyrosyl B-protein +- I-protein +DNA I-protein +phosphodiesterase I-protein +2 I-protein +( O +Tdp2 B-protein +) O +reverses O +Topoisomerase B-protein_type +2 I-protein_type +( O +Top2 B-protein_type +) O +DNA B-chemical +– O +protein O +crosslinks O +triggered O +by O +Top2 B-protein_type +engagement O +of O +DNA B-chemical +damage O +or O +poisoning O +by O +anticancer O +drugs O +. O + +( O +B O +) O +DNA B-chemical +oligonucleotide O +substrates O +synthesized O +by O +EDC O +- O +imidazole O +coupling O +and O +used O +in O +Tdp2 B-experimental_method +enzyme I-experimental_method +assays I-experimental_method +contain O +deoxyadenine B-chemical +( O +dA B-chemical +), O +Ethenoadenine B-chemical +( O +ϵA B-chemical +) O +or O +an O +abasic B-site +site I-site +( O +THF B-chemical +) O +and O +a O +5 O +′– O +nitrophenol O +moiety O +. O + +P B-evidence +- I-evidence +values I-evidence +calculated O +using O +two O +- O +tailed O +t B-experimental_method +- I-experimental_method +test I-experimental_method +; O +error O +bars O +, O +s O +. O +d O +. O +n O += O +4 O +, O +n O +. O +s O +. O += O +not O +statistically O +significant O +. O +( O +D O +) O +Structure B-evidence +of O +mTdp2cat B-structure_element +bound B-protein_state +to I-protein_state +5 B-chemical +′- I-chemical +phosphate I-chemical +DNA I-chemical +( O +product O +complex O +) O +containing O +ϵA B-chemical +( O +yellow O +). O + +DNA B-site +binding I-site +β2Hβ I-site +– I-site +grasp I-site +( O +tan O +) O +and O +cap O +elements O +engage O +the O +5 O +′- O +nucleotide O +as O +well O +as O +the O ++ O +2 O +and O ++ O +3 O +nucleotides O +( O +blue O +) O +of O +substrate O +DNA B-chemical +. O + +Our O +integrated O +results O +from O +structural B-experimental_method +analysis I-experimental_method +, O +mutagenesis B-experimental_method +, O +functional B-experimental_method +assays I-experimental_method +and O +quanyum B-experimental_method +mechanics I-experimental_method +/ I-experimental_method +molecular I-experimental_method +mechanics I-experimental_method +( O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +) O +modeling B-experimental_method +of O +the O +Tdp2 B-protein +reaction O +coordinate O +describe O +in O +detail O +how O +Tdp2 B-protein +mediates O +a O +single O +- O +metal O +ion O +tyrosyl B-protein_type +DNA I-protein_type +phosphodiesterase I-protein_type +reaction O +capable O +of O +acting O +on O +diverse O +DNA B-chemical +end O +damage O +. O + +To O +understand O +the O +molecular O +basis O +for O +Tdp2 B-protein +processing O +of O +Top2cc B-complex_assembly +in O +the O +context O +of O +DNA B-chemical +damage O +, O +we O +crystallized B-experimental_method +and I-experimental_method +determined I-experimental_method +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystal B-evidence +structures I-evidence +of O +mTdp2cat B-structure_element +bound B-protein_state +to I-protein_state +5 B-chemical +′- I-chemical +phosphate I-chemical +DNA I-chemical +( O +product O +complex O +) O +with O +a O +5 B-chemical +′- I-chemical +ϵA I-chemical +at O +1 O +. O +43 O +Å O +resolution O +( O +PDB O +entry O +5HT2 O +) O +and O +the O +abasic O +DNA B-chemical +damage O +mimic O +5 B-chemical +′- I-chemical +THF I-chemical +at O +2 O +. O +15 O +Å O +resolution O +( O +PDB O +entry O +5INK O +; O +Figure O +1D O +and O +E O +, O +Table O +1 O +). O + +( O +A O +) O +Structure B-evidence +of O +mTdp2cat B-structure_element +bound B-protein_state +to I-protein_state +5 B-chemical +′- I-chemical +phosphate I-chemical +DNA I-chemical +( O +product O +complex O +) O +containing O +ϵA B-chemical +( O +yellow O +), O +Mg2 B-chemical ++ I-chemical +( O +magenta O +) O +and O +its O +inner O +- O +sphere O +waters B-chemical +( O +gray O +). O + +The O +THF B-chemical +is O +shown O +in O +yellow O +and O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +from O +the O +THF B-chemical +O4 O +′ O +to O +inner O +- O +sphere O +water B-chemical +is O +shown O +as O +gray O +dashes O +. O + +Structural O +plasticity O +in O +the O +Tdp2 B-protein +DNA B-site +binding I-site +trench I-site + +The O +paucity O +of O +hydrophobic B-bond_interaction +interactions I-bond_interaction +stabilizing O +the O +β2Hβ B-structure_element +DNA B-protein_state +- I-protein_state +bound I-protein_state +conformation O +suggests O +that O +conformational O +plasticity O +in O +the O +β2Hβ B-structure_element +might O +be O +a O +feature O +of O +DNA B-chemical +damage O +and O +metal O +cofactor O +engagement O +. O + +Conformational O +plasticity O +in O +the O +Tdp2 B-protein +active B-site +site I-site +. O + +Wall O +- O +eyed O +stereo O +view O +is O +displayed O +. O +( O +B O +) O +The O +closed B-protein_state +β2Hβ B-structure_element +conformation O +in O +the O +mTdp2cat B-complex_assembly +– I-complex_assembly +DNA I-complex_assembly +product O +structure B-evidence +containing O +5 B-chemical +′- I-chemical +ϵA I-chemical +( O +yellow O +, O +PDB O +entry O +5HT2 O +). O +T309 B-residue_name_number +( O +green O +) O +is O +an O +integral O +part O +of O +the O +β2Hβ B-site +DNA I-site +- I-site +binding I-site +grasp I-site +( O +tan O +) O +and O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +backbone O +of O +Y321 B-residue_name_number +, O +while O +N314 B-residue_name_number +( O +orange O +) O +occupies O +the O +β2Hβ B-site +docking I-site +pocket I-site +. O + +Experiments O +performed O +as O +in O +panel O +D O +for O +mTdp2cat B-structure_element +WT B-protein_state +( O +lanes O +27 O +– O +39 O +) O +or O +mTdp2cat B-structure_element +D358N B-mutant +( O +lanes O +40 O +– O +52 O +), O +but O +with O +chymotrypsin B-experimental_method +instead O +of O +trypsin B-experimental_method +. O + +Thus O +, O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +structures B-evidence +and O +limited B-experimental_method +proteolysis I-experimental_method +analysis I-experimental_method +indicate O +that O +DNA B-chemical +- O +and O +metal O +- O +induced O +conformational O +changes O +are O +a O +conserved B-protein_state +feature O +of O +the O +vertebrate B-taxonomy_domain +Tdp2 B-protein +- O +substrate O +interaction O +. O + +However O +, O +previous O +biochemical B-experimental_method +analysis I-experimental_method +has O +suggested O +an O +alternative O +two O +- O +metal O +ion O +mechanism O +for O +the O +Tdp2 B-protein +- O +phosphotyrosyl B-protein_type +phosphodiesterase I-protein_type +reaction O +. O + +Either O +Mg2 B-chemical ++ I-chemical +or O +Ca2 B-chemical ++ I-chemical +were O +titrated B-experimental_method +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +5 B-chemical +′- I-chemical +P I-chemical +DNA I-chemical +, O +and O +the O +tryptophan B-evidence +fluorescence I-evidence +was O +monitored O +with O +an O +excitation O +wavelength O +of O +280 O +nm O +and O +emission O +wavelength O +of O +350 O +nm O +using O +10 O +nm O +band O +pass O +filters O +. O + +PNP B-chemical +release O +( O +monitored O +by O +absorbance O +at O +415 O +nm O +) O +as O +a O +function O +of O +Mg2 B-chemical ++ I-chemical +concentration O +and O +in O +the O +absence B-protein_state +or O +presence B-protein_state +of I-protein_state +1 O +or O +10 O +mM O +Ca2 B-chemical ++ I-chemical +is O +shown O +; O +error O +bars O +, O +s O +. O +d O +. O +n O += O +4 O +. O +( O +C O +) O +σ B-evidence +- I-evidence +A I-evidence +weighted I-evidence +2Fo I-evidence +- I-evidence +Fc I-evidence +electron I-evidence +density I-evidence +map I-evidence +( O +blue O +) O +and O +model B-evidence +- I-evidence +phased I-evidence +anomalous I-evidence +difference I-evidence +Fourier I-evidence +( O +magenta O +) O +maps B-evidence +for O +the O +mTdp2cat B-complex_assembly +– I-complex_assembly +DNA I-complex_assembly +– I-complex_assembly +Mn2 I-complex_assembly ++ I-complex_assembly +complex O +( O +PDB O +entry O +5INP O +) O +show O +a O +single O +Mn2 B-chemical ++ I-chemical +( O +cyan O +) O +is O +bound O +with O +expected O +octahedral O +coordination O +geometry O +. O + +These O +data O +were O +an O +excellent O +fit O +to O +a O +single O +- O +site O +binding O +model O +both O +in O +the O +presence B-protein_state +and O +absence B-protein_state +of I-protein_state +DNA B-chemical +( O +Figure O +4A O +). O + +Overall O +, O +these O +metal B-experimental_method +binding I-experimental_method +analyses I-experimental_method +are O +consistent O +with O +a O +single O +metal O +ion O +mediated O +reaction O +. O + +Altogether O +, O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +modeling I-experimental_method +identifies O +new O +determinants O +of O +the O +Tdp2 B-protein +reaction O +, O +and O +demonstrates O +our O +proposed O +single O +Mg2 B-chemical ++ I-chemical +catalyzed O +reaction O +model O +is O +a O +viable O +mechanism O +for O +Tdp2 B-protein +- O +catalyzed O +5 B-residue_name +′- I-residue_name +phosphotyrosine I-residue_name +bond O +hydrolysis O +. O + +To O +test O +the O +aspects O +of O +the O +Tdp2 B-protein +reaction O +mechanism O +described O +here O +derived O +from O +high O +- O +resolution O +mouse B-taxonomy_domain +Tdp2 B-protein +crystal B-evidence +structures I-evidence +( O +denoted O +with O +superscript O +numbering O +‘ O +m O +’ O +for O +numbering O +of O +the O +mouse B-taxonomy_domain +protein O +), O +we O +engineered B-experimental_method +and I-experimental_method +purified I-experimental_method +thirteen O +human B-species +MBP B-experimental_method +- O +hTdp2cat B-structure_element +mutant B-protein_state +proteins O +( O +denoted O +with O +superscript O +numbering O +and O +‘ O +h O +’ O +for O +the O +human B-species +protein O +) O +and O +assayed O +the O +impacts O +of O +mutations B-experimental_method +on O +Tdp2 B-protein +catalytic O +activity O +using O +three O +in O +vitro O +reporter O +substrates O +including O +a O +tyrosylated B-protein_state +DNA B-chemical +substrate O +( O +5 B-ptm +′- I-ptm +Y I-ptm +), O +p B-chemical +- I-chemical +nitrophenyl I-chemical +phosphate I-chemical +( O +PNPP B-chemical +) O +and O +thymidine B-chemical +5 I-chemical +′- I-chemical +monophosphate I-chemical +p I-chemical +- I-chemical +nitrophenyl I-chemical +ester I-chemical +( O +T5PNP B-chemical +) O +( O +Figure O +5E O +, O +Supplementary O +Figures O +S5B O +and O +S5C O +). O + +We O +found O +that O +mutations B-experimental_method +that O +removed B-experimental_method +the O +charge O +yet O +retained O +the O +ability O +to O +hydrogen B-bond_interaction +bond I-bond_interaction +( O +hH351Q B-mutant +) O +or O +should O +abrogate O +the O +elevated O +pKa B-evidence +of O +the O +Histidine B-residue_name +( O +hD316N B-mutant +) O +had O +severe O +impacts O +on O +catalysis O +. O + +Interestingly O +, O +the O +Tdp2 B-protein +single O +Mg2 B-chemical ++ I-chemical +ion O +octahedral B-bond_interaction +coordination I-bond_interaction +shell I-bond_interaction +also O +involves O +an O +extended O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +network I-bond_interaction +mediated O +by O +hAsp350 B-residue_name_number +( O +mAsp358 B-residue_name_number +) O +that O +stabilizes O +the O +DNA B-protein_state +- I-protein_state +bound I-protein_state +conformation O +of O +the O +β2Hβ B-structure_element +substrate I-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +through O +hydrogen B-bond_interaction +bonding I-bond_interaction +to O +mTrp307 B-residue_name_number +. O + +Samples O +were O +withdrawn O +from O +reactions O +, O +neutralized O +with O +TBE O +- O +urea O +loading O +dye O +at O +the O +indicated O +timepoints O +, O +and O +electrophoresed O +on O +a O +20 O +% O +TBE B-experimental_method +- I-experimental_method +urea I-experimental_method +PAGE I-experimental_method +. O + +( O +D O +) O +Relative O +activity O +of O +WT B-protein_state +and O +indicated O +mutant B-protein_state +human B-species +MBP B-experimental_method +- O +hTdp2cat B-structure_element +fusion O +proteins O +on O +three O +model O +Tdp2 B-protein +substrates O +. O + +Although O +Mg2 B-chemical ++ I-chemical +is O +present O +at O +the O +same O +concentration O +as O +the O +WT B-protein_state +- O +mTdpcat B-protein +crystals B-evidence +( O +10 O +mM O +), O +we O +find O +the O +metal B-site +site I-site +is O +unoccupied B-protein_state +in O +the O +mD358N B-mutant +crystals B-evidence +. O + +Therefore O +, O +metal O +- O +regulated O +opening O +/ O +closure O +of O +the O +active B-site +site I-site +may O +modulate O +Tdp2 B-protein +activity O +, O +and O +D350N B-mutant +is O +sufficient O +to O +block O +both O +metal O +binding O +and O +conformational O +change O +. O + +In O +support O +of O +this O +, O +we O +also O +find O +that O +hD350N B-mutant +( O +mD358N B-mutant +) O +impairs O +Mg2 B-chemical ++ I-chemical +binding O +as O +measured O +by O +intrinsic B-evidence +tryptophan I-evidence +fluorescence I-evidence +( O +Figure O +4A O +), O +and O +abrogates O +Mg2 B-chemical ++- I-chemical +stimulated O +active B-site +site I-site +conformational O +changes O +detected O +by O +trypsin O +and O +chymotrypsin O +sensitivity O +of O +the O +Tdp2 B-protein +metamorphic O +loop B-structure_element +( O +Figure O +3D O +). O + +Error O +bars O +, O +s O +. O +d O +, O +n O += O +3 O +. O +( O +D O +) O +Junctions O +recovered O +from O +cellular B-experimental_method +end I-experimental_method +- I-experimental_method +joining I-experimental_method +assays I-experimental_method +in O +the O +noted O +cell O +types O +were O +characterized O +by O +sequencing B-experimental_method +to O +assess O +the O +end B-evidence +- I-evidence +joining I-evidence +error I-evidence +rate I-evidence +. O + +By O +comparison O +the O +more O +frequent O +I307V B-mutant +has O +only O +mild O +effects O +on O +in O +vitro O +activity O +, O +and O +no O +detectable O +impact O +on O +cellular O +assays O +. O + +Tdp2 B-protein +was O +originally O +identified O +as O +a O +protein O +conferring O +resistance O +to O +both O +Top1 B-protein_type +and O +Top2 B-protein_type +anti O +- O +cancer O +drugs O +, O +however O +it O +is O +hypothesized O +that O +the O +predominant O +natural O +source O +of O +substrates O +for O +Tdp2 B-protein +are O +likely O +the O +potent O +DNA B-chemical +damage O +triggers O +of O +Top2 B-protein_type +poisoning O +and O +Top2 B-protein_type +DNA B-chemical +protein O +crosslinks O +encountered O +during O +transcription O +. O + +The O +mechanism O +of O +NCX B-protein_type +proteins O +is O +therefore O +highly O +likely O +to O +be O +consistent O +with O +the O +alternating O +- O +access O +model O +of O +secondary O +- O +active O +transport O +. O + +Our O +recent O +atomic O +- O +resolution O +structure B-evidence +of O +NCX_Mj B-protein +from O +Methanococcus B-species +jannaschii I-species +provided O +the O +first O +view O +of O +the O +basic O +functional O +unit O +of O +an O +NCX B-protein_type +protein O +. O + +These O +structures B-evidence +reveal O +the O +mode O +of O +recognition O +of O +these O +ions O +, O +their O +relative O +affinities O +, O +and O +the O +mechanism O +of O +extracellular O +ion O +exchange O +, O +for O +a O +well O +- O +defined O +, O +functional O +conformation O +in O +a O +membrane O +- O +like O +environment O +. O + +The O +water B-chemical +molecule O +at O +Smid B-site +forms O +hydrogen B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +with O +the O +highly B-protein_state +conserved I-protein_state +Glu54 B-residue_name_number +and O +Glu213 B-residue_name_number +( O +Supplementary O +Fig O +. O +1d O +), O +stabilizing O +their O +orientation O +to O +properly O +coordinate B-bond_interaction +multiple O +Na B-chemical ++ I-chemical +ions O +at O +Sext B-site +, O +SCa B-site +and O +Sint B-site +. O + +When O +Na B-chemical ++ I-chemical +binds O +to O +Sext B-site +at O +high B-protein_state +concentrations O +, O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +TM7 B-structure_element +is O +bent O +into O +two O +short B-structure_element +helices I-structure_element +, O +TM7a B-structure_element +and O +TM7b B-structure_element +( O +Fig O +. O +2a O +). O + +These O +contacts O +are O +absent O +in O +the O +structure B-evidence +with O +Na B-chemical ++ I-chemical +at O +Sext B-site +, O +in O +which O +there O +is O +an O +open O +gap O +between O +the O +two O +helices B-structure_element +( O +Fig O +. O +2b O +). O + +This O +difference O +is O +noteworthy O +because O +TM6 B-structure_element +and O +TM1 B-structure_element +are O +believed O +to O +undergo O +a O +sliding O +motion O +, O +relative O +to O +the O +rest O +of O +the O +protein O +, O +when O +the O +transporter B-protein_type +switches O +to O +the O +inward B-protein_state +- I-protein_state +facing I-protein_state +conformation O +. O + +Thus O +, O +in O +100 O +mM O +Na B-chemical ++ I-chemical +and O +10 O +mM O +Sr2 B-chemical ++, I-chemical +Na B-chemical ++ I-chemical +completely O +replaced O +Sr2 B-chemical ++ I-chemical +( O +Fig O +. O +3a O +) O +and O +reverted O +NCX_Mj B-protein +to O +the O +Na B-protein_state ++- I-protein_state +loaded I-protein_state +, O +fully B-protein_state +occluded I-protein_state +state O +. O + +Similar O +titration B-experimental_method +experiments I-experimental_method +showed O +that O +Ca2 B-chemical ++ I-chemical +and O +Sr2 B-chemical ++ I-chemical +binding O +to O +NCX_Mj B-protein +are O +not O +exactly O +alike O +The O +electron B-evidence +density I-evidence +distribution I-evidence +from O +crystals B-experimental_method +soaked I-experimental_method +in I-experimental_method +high B-protein_state +Ca2 B-chemical ++ I-chemical +and O +low B-protein_state +Na B-chemical ++, I-chemical +indicates O +that O +Ca2 B-chemical ++ I-chemical +can O +bind O +to O +Smid B-site +as O +well O +as O +SCa B-site +, O +with O +a O +preference O +for O +SCa B-site +( O +Fig O +. O +3b O +). O + +An O +apo B-protein_state +state O +of O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +is O +likely O +to O +exist O +transiently O +in O +physiological O +conditions O +, O +despite O +the O +high O +amounts O +of O +extracellular O +Na B-chemical ++ I-chemical +(~ O +150 O +mM O +) O +and O +Ca2 B-chemical ++ I-chemical +(~ O +2 O +mM O +). O + +That O +secondary B-protein_type +- I-protein_type +active I-protein_type +transporters I-protein_type +are O +able O +to O +harness O +an O +electrochemical O +gradient O +of O +one O +substrate O +to O +power O +the O +uphill O +transport O +of O +another O +relies O +on O +a O +seemingly O +simple O +principle O +: O +they O +must O +not O +transition O +between O +outward B-protein_state +- I-protein_state +and O +inward B-protein_state +- I-protein_state +open I-protein_state +conformations O +unless O +in O +two O +precise O +substrate O +occupancy O +states O +. O + +To O +examine O +this O +central O +question O +, O +we O +sought O +to O +characterize O +the O +conformational B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscape I-evidence +of O +NCX_Mj B-protein +and O +to O +examine O +its O +dependence O +on O +the O +ion O +- O +occupancy O +state O +, O +using O +molecular B-experimental_method +dynamics I-experimental_method +( O +MD B-experimental_method +) O +simulations B-experimental_method +. O + +With O +Sext B-site +empty B-protein_state +, O +however O +, O +TM7ab B-structure_element +forms O +a O +canonical O +α B-structure_element +- I-structure_element +helix I-structure_element +( O +Fig O +. O +4a O +- O +b O +, O +4g O +), O +thereby O +creating O +an O +opening O +between O +TM3 B-structure_element +and O +TM7 B-structure_element +, O +which O +in O +turn O +allows O +water B-chemical +molecules O +from O +the O +external O +solution O +to O +reach O +into O +Sext B-site +( O +Fig O +. O +4e O +, O +4h O +- O +i O +), O +i O +. O +e O +. O +the O +transporter B-protein_type +is O +no B-protein_state +longer I-protein_state +occluded I-protein_state +. O + +Displacement O +of O +Na B-chemical ++ I-chemical +from O +SCa B-site +and O +Sint B-site +induces O +further O +changes O +( O +Fig O +. O +4c O +). O + +These O +calculations B-experimental_method +demonstrate O +that O +the O +Na B-chemical ++ I-chemical +occupancy O +state O +of O +the O +transporter B-protein_type +has O +a O +profound O +effect O +on O +its O +conformational B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscape I-evidence +. O + +At O +a O +small O +energetic O +cost O +, O +however O +, O +the O +transporter B-protein_type +can O +adopt O +a O +metastable B-protein_state +‘ O +half B-protein_state +- I-protein_state +open I-protein_state +’ O +conformation O +in O +which O +TM7ab B-structure_element +is O +completely O +straight O +and O +Sext B-site +is O +open B-protein_state +to O +the O +exterior O +( O +Fig O +. O +5a O +, O +5b O +). O + +Similarly O +puzzling O +is O +that O +a O +given O +antiporter B-protein_type +will O +undergo O +this O +transition O +upon O +recognition O +of O +substrates O +of O +different O +charge O +, O +size O +and O +number O +. O + +Yet O +, O +when O +multiple O +species O +are O +to O +be O +co O +- O +translocated O +, O +by O +either O +an O +antiporter B-protein_type +or O +a O +symporter B-protein_type +, O +partial O +occupancies O +must O +not O +be O +conducive O +to O +the O +alternating B-site +- I-site +access I-site +switch I-site +. O + +Nonetheless O +, O +the O +calculated B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscapes I-evidence +, O +derived O +without O +knowledge O +of O +the O +experimental O +data O +, O +reassuringly O +confirm O +that O +the O +crystallized B-evidence +structures I-evidence +correspond O +to O +mechanistically O +relevant O +, O +interconverting O +states O +. O + +Specifically O +, O +our O +crystal B-experimental_method +titrations I-experimental_method +suggest O +that O +, O +during O +forward O +Na B-chemical ++/ I-chemical +Ca2 B-chemical ++ I-chemical +exchange O +, O +sites O +Sint B-site +and O +SCa B-site +, O +which O +Ca2 B-chemical ++ I-chemical +and O +Na B-chemical ++ I-chemical +compete O +for O +, O +can O +be O +grouped O +into O +one O +; O +Na B-chemical ++ I-chemical +binding O +to O +these O +sites O +does O +not O +require O +high O +Na B-chemical ++ I-chemical +concentrations O +, O +and O +two O +Na B-chemical ++ I-chemical +ions O +along O +with O +a O +water B-chemical +molecule O +( O +at O +Smid B-site +) O +are O +sufficient O +to O +displace O +Ca2 B-chemical ++, I-chemical +explaining O +the O +Hill B-evidence +coefficient I-evidence +of O +~ O +2 O +for O +Na B-chemical ++- I-chemical +dependent O +inhibition O +of O +Ca2 B-chemical ++ I-chemical +fluxes O +. O + +Interestingly O +, O +binding O +of O +Ca2 B-chemical ++ I-chemical +to O +Smid B-site +appears O +to O +be O +also O +possible O +, O +but O +available O +evidence O +indicates O +that O +this O +event O +transiently O +blocks O +the O +exchange O +cycle O +. O + +The O +electron B-evidence +density I-evidence +( O +grey O +mesh O +, O +1 O +. O +9 O +Å O +Fo B-evidence +- I-evidence +Fc I-evidence +ion I-evidence +omit I-evidence +map I-evidence +contoured O +at O +4σ O +) O +at O +Smid B-site +was O +modeled O +as O +water B-chemical +( O +red O +sphere O +) O +and O +those O +at O +Sext B-site +, O +SCa B-site +and O +Sint B-site +as O +Na B-chemical ++ I-chemical +ions O +( O +green O +spheres O +). O + +The O +displacement O +of O +A206 B-residue_name_number +reflects O +the O +[ O +Na B-chemical ++]- I-chemical +dependent O +conformational O +change O +from O +the O +partially B-protein_state +open I-protein_state +to O +the O +occluded B-protein_state +state O +( O +observed O +at O +low O +and O +high O +Na B-chemical ++ I-chemical +concentrations O +, O +respectively O +). O + +Residues O +forming O +van O +- O +der O +- O +Waals O +contacts O +in O +the O +structure B-evidence +at O +low B-protein_state +Na B-chemical ++ I-chemical +concentration O +are O +shown O +in O +detail O +. O + +The O +vacant O +Sext B-site +site O +in O +the O +structure B-evidence +at O +low B-protein_state +Na B-chemical ++ I-chemical +concentration O +is O +indicated O +with O +a O +white O +sphere O +. O + +Residues O +involved O +in O +Sr2 B-chemical ++ I-chemical +coordination O +are O +labeled O +. O + +( O +d O +) O +Close O +- O +up O +of O +the O +ion B-site +- I-site +binding I-site +region I-site +in O +the O +fully B-protein_state +Na I-protein_state ++- I-protein_state +occupied I-protein_state +state O +. O + +These O +descriptors O +were O +employed O +as O +collective O +variables O +in O +the O +Bias B-experimental_method +- I-experimental_method +Exchange I-experimental_method +Metadynamics I-experimental_method +simulations I-experimental_method +( O +Methods O +). O + +Thermodynamic O +basis O +for O +the O +proposed O +mechanism O +of O +substrate O +control O +of O +the O +alternating O +- O +access O +transition O +of O +NCX B-protein_type +. O +( O +a O +) O +Calculated B-evidence +conformational I-evidence +free I-evidence +- I-evidence +energy I-evidence +landscapes I-evidence +for O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +, O +for O +two O +different O +Na B-chemical ++- I-chemical +occupancy O +states O +, O +and O +for O +a O +state O +with O +no B-protein_state +ions I-protein_state +bound I-protein_state +. O + +The O +uncorrected O +map B-evidence +overstabilizes O +the O +open B-protein_state +state O +relative O +to O +the O +semi B-protein_state +- I-protein_state +open I-protein_state +and O +occluded B-protein_state +because O +it O +also O +overestimates O +the O +cost O +of O +dehydration O +of O +the O +ion O +, O +once O +it O +is O +bound B-protein_state +to I-protein_state +the O +protein O +( O +this O +effect O +is O +negligible O +for O +Na B-chemical ++). I-chemical + +How O +the O +essential O +pre B-protein_type +- I-protein_type +mRNA I-protein_type +splicing I-protein_type +factor I-protein_type +U2AF65 B-protein +recognizes O +the O +polypyrimidine B-chemical +( O +Py B-chemical +) O +signals O +of O +the O +major O +class O +of O +3 B-site +′ I-site +splice I-site +sites I-site +in O +human B-species +gene O +transcripts O +remains O +incompletely O +understood O +. O + +Single B-experimental_method +- I-experimental_method +molecule I-experimental_method +FRET I-experimental_method +experiments O +suggest O +that O +conformational O +selection O +and O +induced O +fit O +of O +the O +U2AF65 B-protein +RRMs B-structure_element +are O +complementary O +mechanisms O +for O +Py B-chemical +- I-chemical +tract I-chemical +association O +. O + +The O +pre B-protein_type +- I-protein_type +mRNA I-protein_type +splicing I-protein_type +factor I-protein_type +U2AF65 B-protein +recognizes O +3 B-site +′ I-site +splice I-site +sites I-site +in O +human B-species +gene O +transcripts O +, O +but O +the O +details O +are O +not O +fully O +understood O +. O + +Milestone O +crystal B-evidence +structures I-evidence +of O +the O +core B-protein_state +U2AF65 B-protein +RRM1 B-structure_element +and O +RRM2 B-structure_element +connected O +by O +a O +shortened B-protein_state +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +( O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +) O +detailed O +a O +subset O +of O +nucleotide O +interactions O +with O +the O +individual O +U2AF65 B-protein +RRMs B-structure_element +. O + +In O +a O +fluorescence B-experimental_method +anisotropy I-experimental_method +assay I-experimental_method +for O +binding O +a O +representative O +Py B-chemical +tract I-chemical +derived O +from O +the O +well O +- O +characterized O +splice B-site +site I-site +of O +the O +adenovirus B-gene +major I-gene +late I-gene +promoter I-gene +( O +AdML B-gene +), O +the O +RNA B-evidence +affinity I-evidence +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +increased O +by O +100 O +- O +fold O +relative O +to O +U2AF651 B-mutant +, I-mutant +2 I-mutant +to O +comparable O +levels O +as O +full B-protein_state +- I-protein_state +length I-protein_state +U2AF65 B-protein +( O +Fig O +. O +1b O +; O +Supplementary O +Fig O +. O +1a O +– O +d O +). O + +The O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +characterize O +ribose B-chemical +( O +r B-chemical +) O +nucleotides B-chemical +at O +all O +of O +the O +binding B-site +sites I-site +except O +the O +seventh B-residue_number +and O +eighth B-residue_number +deoxy B-chemical +-( I-chemical +d I-chemical +) I-chemical +U I-chemical +, O +which O +are O +likely O +to O +lack O +2 O +′- O +hydroxyl O +contacts O +based O +on O +the O +RNA B-protein_state +- I-protein_state +bound I-protein_state +dU2AF651 B-mutant +, I-mutant +2 I-mutant +structure B-evidence +. O + +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +interacts O +with O +the O +Py B-chemical +tract I-chemical + +Otherwise O +, O +the O +rU4 B-residue_name_number +nucleotide B-chemical +packs O +against O +F304 B-residue_name_number +in O +the O +signature O +ribonucleoprotein B-structure_element +consensus I-structure_element +motif I-structure_element +( I-structure_element +RNP I-structure_element +)- I-structure_element +2 I-structure_element +of O +RRM2 B-structure_element +. O + +At O +the O +opposite O +side O +of O +the O +central O +fifth B-residue_number +nucleotide B-chemical +, O +the O +sixth B-residue_number +rU6 B-residue_name_number +nucleotide B-chemical +is O +located O +at O +the O +inter B-site +- I-site +RRM1 I-site +/ I-site +RRM2 I-site +interface I-site +( O +Fig O +. O +3e O +; O +Supplementary O +Movie O +1 O +). O + +Versatile O +primary O +sequence O +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element + +However O +, O +stretching O +of O +the O +truncated B-protein_state +dU2AF651 B-mutant +, I-mutant +2L I-mutant +linker B-structure_element +to O +connect O +the O +RRM B-structure_element +termini I-structure_element +is O +expected O +to O +disrupt O +its O +nucleotide O +interactions O +. O + +Likewise O +, O +deletion B-experimental_method +of O +the O +N O +- O +terminal O +RRM1 B-structure_element +extension I-structure_element +in O +the O +shortened B-protein_state +constructs O +would O +remove O +packing O +interactions O +that O +position O +the O +linker B-structure_element +in O +a O +kinked B-structure_element +turn I-structure_element +following O +P229 B-residue_name_number +( O +Fig O +. O +4a O +), O +consistent O +with O +the O +lower O +RNA B-evidence +affinities I-evidence +of O +dU2AF651 B-mutant +, I-mutant +2L I-mutant +, O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +and O +U2AF651 B-mutant +, I-mutant +2 I-mutant +compared O +with O +U2AF651 B-mutant +, I-mutant +2L I-mutant +. O + +Notably O +, O +the O +Q147A B-mutant +/ O +V254P B-mutant +/ O +R227A B-mutant +mutation B-experimental_method +reduced O +the O +RNA B-evidence +affinity I-evidence +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +- I-mutant +3Mut I-mutant +protein O +by O +30 O +- O +fold O +more O +than O +would O +be O +expected O +based O +on O +simple O +addition O +of O +the O +ΔΔG B-evidence +' O +s O +for O +the O +single O +mutations O +. O + +As O +a O +representative O +splicing O +substrate O +, O +we O +utilized O +a O +well O +- O +characterized O +minigene B-chemical +splicing I-chemical +reporter I-chemical +( O +called O +pyPY B-chemical +) O +comprising O +a O +weak O +( O +that O +is O +, O +degenerate O +, O +py B-chemical +) O +and O +strong O +( O +that O +is O +, O +U B-structure_element +- I-structure_element +rich I-structure_element +, O +PY B-chemical +) O +polypyrimidine B-chemical +tracts I-chemical +preceding O +two O +alternative O +splice B-site +sites I-site +( O +Fig O +. O +5a O +). O + +Paramagnetic B-experimental_method +resonance I-experimental_method +enhancement I-experimental_method +( O +PRE B-experimental_method +) O +measurements O +previously O +had O +suggested O +a O +predominant O +back B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +back I-protein_state +, O +or O +‘ O +closed B-protein_state +' O +conformation O +of O +the O +apo B-protein_state +- O +U2AF651 B-mutant +, I-mutant +2 I-mutant +RRM1 B-structure_element +and O +RRM2 B-structure_element +in O +equilibrium O +with O +a O +minor O +‘ O +open B-protein_state +' O +conformation O +resembling O +the O +RNA B-protein_state +- I-protein_state +bound I-protein_state +inter B-structure_element +- I-structure_element +RRM I-structure_element +arrangement O +. O + +Approximately O +40 O +% O +of O +the O +smFRET B-experimental_method +traces B-evidence +showed O +apparent O +transitions O +between O +multiple O +FRET B-evidence +values I-evidence +( O +for O +example O +, O +Fig O +. O +6c O +). O + +Nevertheless O +, O +the O +predominant O +0 O +. O +45 O +FRET B-evidence +state I-evidence +in O +the O +presence O +of O +RNA B-chemical +agrees O +with O +the O +Py B-protein_state +- I-protein_state +tract I-protein_state +- I-protein_state +bound I-protein_state +crystal B-evidence +structure I-evidence +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +. O + +The O +majority O +of O +traces B-evidence +that O +show O +fluctuations O +began O +at O +high O +( O +0 O +. O +65 O +– O +0 O +. O +8 O +) O +FRET B-evidence +value I-evidence +and O +transitioned O +to O +a O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +( O +Supplementary O +Fig O +. O +7c O +– O +g O +). O + +Altogether O +, O +these O +data O +indicate O +that O +interactions O +among O +the O +U2AF65 B-protein +RRM1 B-structure_element +/ O +RRM2 B-structure_element +, O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +, O +N B-structure_element +- I-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +extensions I-structure_element +are O +mutually O +inter O +- O +dependent O +for O +cognate O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +. O + +Our O +smFRET B-experimental_method +results O +agree O +with O +prior O +NMR B-experimental_method +/ O +PRE B-experimental_method +evidence O +for O +multi O +- O +domain O +conformational O +selection O +as O +one O +mechanistic O +basis O +for O +U2AF65 B-protein +– O +RNA B-chemical +association O +( O +Fig O +. O +7b O +). O + +An O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +is O +likely O +to O +correspond O +to O +the O +U2AF65 B-protein +conformation O +visualized O +in O +our O +U2AF651 B-mutant +, I-mutant +2L I-mutant +crystal B-evidence +structures I-evidence +, O +in O +which O +the O +RRM1 B-structure_element +and O +RRM2 B-structure_element +bind O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +to O +the O +Py B-chemical +- I-chemical +tract I-chemical +oligonucleotide I-chemical +. O + +As O +such O +, O +the O +smFRET B-experimental_method +approach O +reconciles O +prior O +inconsistencies O +between O +two O +major O +conformations O +that O +were O +detected O +by O +NMR B-experimental_method +/ O +PRE B-experimental_method +experiments O +and O +a O +broad O +ensemble O +of O +diverse O +inter B-structure_element +- I-structure_element +RRM I-structure_element +arrangements O +that O +fit O +the O +SAXS B-experimental_method +data O +for O +the O +apo B-protein_state +- O +protein B-protein +. O + +Similar O +interdisciplinary O +structural O +approaches O +are O +likely O +to O +illuminate O +whether O +similar O +mechanistic O +bases O +for O +RNA O +binding O +are O +widespread O +among O +other O +members O +of O +the O +vast O +multi O +- O +RRM B-structure_element +family O +. O + +The O +prior O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +nucleotide B-site +- I-site +binding I-site +sites I-site +are O +given O +in O +parentheses O +( O +site O +4 O +' O +interacts O +with O +dU2AF65 B-mutant +RRM1 B-structure_element +and O +RRM2 B-structure_element +by O +crystallographic O +symmetry O +). O + +( O +i O +) O +Bar O +graph O +of O +apparent O +equilibrium B-evidence +affinities I-evidence +( O +KA B-evidence +) O +of O +the O +wild B-protein_state +type I-protein_state +( O +blue O +) O +and O +the O +indicated O +mutant B-protein_state +( O +yellow O +) O +U2AF651 B-mutant +, I-mutant +2L I-mutant +proteins O +binding O +the O +AdML B-gene +Py B-chemical +tract I-chemical +( O +5 B-chemical +′- I-chemical +CCCUUUUUUUUCC I-chemical +- I-chemical +3 I-chemical +′). I-chemical + +The O +average O +fitted O +fluorescence O +anisotropy O +RNA B-evidence +- I-evidence +binding I-evidence +curves I-evidence +are O +shown O +in O +Supplementary O +Fig O +. O +4a O +– O +c O +. O + +Protein B-experimental_method +overexpression I-experimental_method +and O +qRT B-experimental_method +- I-experimental_method +PCR I-experimental_method +results O +are O +shown O +in O +Supplementary O +Fig O +. O +5 O +. O + +( O +c O +– O +f O +, O +i O +, O +j O +) O +The O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +protein O +was O +immobilized O +on O +the O +microscope O +slide O +via O +biotin B-chemical +- I-chemical +NTA I-chemical +/ I-chemical +Ni I-chemical ++ I-chemical +2 I-chemical +( O +orange O +line O +) O +on O +a O +neutravidin O +( O +black O +X O +)- O +biotin O +- O +PEG O +( O +orange O +triangle O +)- O +treated O +surface O +and O +imaged O +either O +in O +the O +absence B-protein_state +of I-protein_state +ligands B-chemical +( O +c O +, O +d O +), O +in O +the O +presence O +of O +5 O +μM O +AdML B-gene +Py B-chemical +- I-chemical +tract I-chemical +RNA I-chemical +( O +5 B-chemical +′- I-chemical +CCUUUUUUUUCC I-chemical +- I-chemical +3 I-chemical +′) I-chemical +( O +e O +, O +f O +), O +or O +in O +the O +presence O +of O +10 O +μM O +adenosine B-residue_name +- O +interrupted O +variant O +RNA B-chemical +( O +5 B-chemical +′- I-chemical +CUUUUUAAUUUCCA I-chemical +- I-chemical +3 I-chemical +′) I-chemical +( O +i O +, O +j O +). O + +N O +is O +the O +number O +of O +single O +- O +molecule O +traces B-evidence +compiled O +for O +each O +histogram B-evidence +. O + +Schematic O +models O +of O +U2AF65 B-protein +recognizing O +the O +Py B-chemical +tract I-chemical +. O + +Alternatively O +, O +a O +conformation O +of O +U2AF65 B-protein +corresponding O +to O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +can O +directly O +bind O +to O +RNA B-chemical +; O +RNA B-chemical +binding O +stabilizes O +the O +‘ O +open B-protein_state +', O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +conformation O +and O +thus O +shifts O +the O +U2AF65 B-protein +population O +towards O +the O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +. O + +Specific O +damage O +manifestations O +were O +determined O +within O +the O +large O +trp B-protein_type +RNA I-protein_type +- I-protein_type +binding I-protein_type +attenuation I-protein_type +protein I-protein_type +( O +TRAP B-complex_assembly +) O +bound B-protein_state +to I-protein_state +a O +single O +- O +stranded O +RNA B-chemical +that O +forms O +a O +belt O +around O +the O +protein O +. O + +Over O +a O +large O +dose O +range O +, O +the O +RNA B-chemical +was O +found O +to O +be O +far O +less O +susceptible O +to O +radiation O +- O +induced O +chemical O +changes O +than O +the O +protein O +. O + +The O +11 O +- O +fold O +symmetry O +within O +each O +TRAP B-complex_assembly +ring B-structure_element +permitted O +statistically O +significant O +analysis O +of O +the O +Glu B-residue_name +and O +Asp B-residue_name +damage O +patterns O +, O +with O +RNA B-chemical +binding O +unexpectedly O +being O +observed O +to O +protect O +these O +otherwise O +highly O +sensitive O +residues O +within O +the O +11 O +RNA B-site +- I-site +binding I-site +pockets I-site +distributed O +around O +the O +outside O +of O +the O +protein O +molecule O +. O + +Global O +radiation O +damage O +is O +observed O +within O +reciprocal O +space O +as O +the O +overall O +decay O +of O +the O +summed O +intensity O +of O +reflections O +detected O +within O +the O +diffraction B-evidence +pattern I-evidence +as O +dose O +increases O +( O +Garman O +, O +2010 O +; O +Murray O +& O +Garman O +, O +2002 O +). O + +For O +instance O +, O +structure B-experimental_method +determination I-experimental_method +of O +the O +purple O +membrane O +protein O +bacterio B-protein_type +­ I-protein_type +rhodopsin I-protein_type +required O +careful O +corrections O +for O +radiation O +- O +induced O +structural O +changes O +before O +the O +correct O +photosensitive O +intermediate O +states O +could O +be O +isolated O +( O +Matsui O +et O +al O +., O +2002 O +). O + +As O +of O +early O +2016 O +, O +> O +5400 O +nucleoprotein B-complex_assembly +complex O +structures B-evidence +have O +been O +deposited O +within O +the O +PDB O +, O +with O +91 O +% O +solved O +by O +MX B-experimental_method +. O + +TRAP B-complex_assembly +consists O +of O +11 O +identical O +subunits B-structure_element +assembled O +into O +a O +ring B-structure_element +with O +11 O +- O +fold O +rotational O +symmetry O +. O + +The O +substrate O +Trp B-chemical +amino O +- O +acid O +ligands O +also O +exhibited O +disordering O +of O +the O +free O +terminal O +carboxyl O +groups O +at O +higher O +doses O +( O +Fig O +. O +2 O +▸ O +a O +); O +however O +, O +no O +clear O +Fourier B-evidence +difference I-evidence +peaks I-evidence +could O +be O +observed O +visually O +. O + +The O +rate O +of O +D B-evidence +loss I-evidence +( O +attributed O +to O +side O +- O +chain O +decarboxylation O +) O +was O +consistently O +larger O +for O +Glu B-residue_name +compared O +with O +Asp B-residue_name +residues O +over O +the O +large O +dose O +range O +( O +Fig O +. O +2 O +▸ O +b O +and O +Supplementary O +Fig O +. O +S3 O +); O +this O +observation O +is O +consistent O +with O +our O +calculations O +on O +model O +systems O +( O +see O +above O +) O +that O +suggest O +that O +, O +without O +considering O +differential O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +environments O +, O +CO2 B-chemical +loss O +is O +more O +exothermic O +by O +around O +8 O +kJ O +mol O +− O +1 O +from O +oxidized B-protein_state +Glu B-residue_name +residues O +than O +from O +their O +Asp B-residue_name +counterparts O +. O + +For O +the O +large O +number O +of O +acidic O +residues O +per O +TRAP B-complex_assembly +ring B-structure_element +( O +four O +Asp B-residue_name +and O +six O +Glu B-residue_name +residues O +per O +protein O +monomer B-oligomeric_state +), O +a O +strong O +dependence O +of O +decarboxylation O +susceptibility O +on O +local O +environment O +was O +observed O +( O +Fig O +. O +4 O +▸). O + +For O +each O +Glu B-residue_name +Cδ O +or O +Asp B-residue_name +Cγ O +atom O +, O +D B-evidence +loss I-evidence +provided O +a O +direct O +measure O +of O +the O +rate O +of O +side O +- O +chain O +carboxyl O +- O +group O +disordering O +and O +subsequent O +decarboxylation O +. O + +For O +acidic O +residues O +with O +no O +differing O +interactions O +between O +nonbound B-protein_state +and O +bound B-protein_state +TRAP B-complex_assembly +( O +Fig O +. O +4 O +▸ O +a O +), O +similar O +damage O +was O +apparent O +between O +the O +two O +rings O +within O +the O +asymmetric O +unit O +, O +as O +expected O +. O + +However O +, O +TRAP B-complex_assembly +residues O +directly O +on O +the O +RNA B-site +- I-site +binding I-site +interfaces I-site +exhibited O +greater O +damage O +accumulation O +in O +nonbound B-protein_state +TRAP B-complex_assembly +( O +Fig O +. O +4 O +▸ O +b O +), O +and O +for O +residues O +at O +the O +ring B-site +– I-site +ring I-site +interfaces I-site +( O +where O +crystal O +contacts O +were O +detected O +) O +bound B-protein_state +TRAP B-complex_assembly +exhibited O +enhanced O +SRD O +accumulation O +( O +Fig O +. O +4 O +▸ O +c O +). O + +Three O +acidic O +residues O +( O +Glu36 B-residue_name_number +, O +Asp39 B-residue_name_number +and O +Glu42 B-residue_name_number +) O +are O +involved O +in O +RNA B-chemical +interactions O +within O +each O +of O +the O +11 O +TRAP B-complex_assembly +ring B-structure_element +subunits B-structure_element +, O +and O +Fig O +. O +5 O +▸ O +shows O +their O +density B-evidence +changes I-evidence +with O +increasing O +dose O +. O + +Here O +, O +MX B-experimental_method +radiation O +- O +induced O +specific O +structural O +changes O +within O +the O +large O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +assembly O +over O +a O +large O +dose O +range O +( O +1 O +. O +3 O +– O +25 O +. O +0 O +MGy O +) O +have O +been O +analysed O +using O +a O +high O +- O +throughput O +quantitative O +approach O +, O +providing O +a O +measure O +of O +the O +electron B-evidence +- I-evidence +density I-evidence +distribution I-evidence +for O +each O +refined O +atom O +with O +increasing O +dose O +, O +D B-evidence +loss I-evidence +. O + +Here O +, O +it O +provided O +the O +precision O +required O +to O +quantify O +the O +role O +of O +RNA B-chemical +in O +the O +damage O +susceptibilities O +of O +equivalent O +atoms O +between O +RNA B-protein_state +- I-protein_state +bound I-protein_state +and O +nonbound B-protein_state +TRAP B-complex_assembly +, O +but O +it O +is O +applicable O +to O +any O +MX B-experimental_method +SRD O +study O +. O + +Since O +U4 B-residue_name_number +is O +the O +only O +refined O +nucleotide O +not O +to O +exhibit O +significant O +base O +– O +protein O +interactions O +around O +TRAP B-complex_assembly +( O +with O +a O +water B-chemical +- O +mediated O +hydrogen B-bond_interaction +bond I-bond_interaction +detected O +in O +only O +three O +of O +the O +11 O +subunits B-structure_element +and O +a O +single O +Arg58 B-residue_name_number +hydrogen B-bond_interaction +bond I-bond_interaction +suggested O +in O +a O +further O +four O +subunits B-structure_element +), O +this O +increased O +U4 B-residue_name_number +D B-evidence +loss I-evidence +can O +be O +explained O +owing O +to O +its O +greater O +flexibility O +. O + +For O +Glu36 B-residue_name_number +and O +Asp39 B-residue_name_number +, O +no O +direct O +quantitative O +correlation O +could O +be O +established O +between O +hydrogen B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +length O +and O +D B-evidence +loss I-evidence +( O +linear B-evidence +R I-evidence +2 I-evidence +of O +< O +0 O +. O +23 O +for O +all O +doses O +; O +Supplementary O +Fig O +. O +S5 O +). O + +The O +Glu36 B-residue_name_number +carboxyl O +side O +chain O +also O +potentially O +forms O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +His34 B-residue_name_number +and O +Lys56 B-residue_name_number +, O +but O +since O +these O +interactions O +are O +conserved B-protein_state +irrespective O +of O +G3 B-residue_name_number +nucleotide O +binding O +, O +this O +cannot O +directly O +account O +for O +the O +stabilization O +effect O +on O +Glu36 B-residue_name_number +in O +RNA B-protein_state +- I-protein_state +bound I-protein_state +TRAP B-complex_assembly +. O + +We O +propose O +that O +with O +no O +solvent O +accessibility O +Glu36 B-residue_name_number +decarboxylation O +is O +inhibited O +, O +since O +the O +CO2 B-evidence +- I-evidence +formation I-evidence +rate I-evidence +K I-evidence +2 I-evidence +is O +greatly O +reduced O +, O +and O +suggest O +that O +steric O +hindrance O +prevents O +each O +radicalized O +Glu36 B-residue_name_number +CO2 O +group O +from O +achieving O +the O +planar O +conformation O +required O +for O +complete O +dissociation O +from O +TRAP B-complex_assembly +. O + +Such O +reduced O +radiation O +- O +sensitivity O +in O +this O +case O +ensures O +that O +the O +interacting O +protein O +remains O +bound B-protein_state +long O +enough O +to O +the O +RNA B-chemical +to O +complete O +its O +function O +, O +even O +whilst O +exposed O +to O +ionizing O +radiation O +. O + +RNA B-chemical +is O +shown O +is O +yellow O +. O + +( O +b O +) O +Average O +D O +loss O +for O +each O +residue O +/ O +nucleotide O +type O +with O +respect O +to O +the O +DWD B-evidence +( O +diffraction B-evidence +- I-evidence +weighted I-evidence +dose I-evidence +; O +Zeldin O +, O +Brock O +­ O +hauser O +et O +al O +., O +2013 O +). O + +Only O +a O +subset O +of O +key O +TRAP B-complex_assembly +residue O +types O +are O +included O +. O + +Mitogen B-protein_type +- I-protein_type +activated I-protein_type +protein I-protein_type +kinases I-protein_type +( O +MAPKs B-protein_type +), O +important O +in O +a O +large O +array O +of O +signalling O +pathways O +, O +are O +tightly O +controlled O +by O +a O +cascade O +of O +protein B-protein_type +kinases I-protein_type +and O +by O +MAPK B-protein_type +phosphatases I-protein_type +( O +MKPs B-protein_type +). O + +Two O +types O +of O +docking O +interactions O +have O +been O +identified O +: O +D B-structure_element +- I-structure_element +motif I-structure_element +- O +mediated O +interaction O +and O +FXF B-site +- I-site +docking I-site +interaction I-site +. O + +The O +285FNFL288 B-structure_element +segment I-structure_element +in O +MKP7 B-protein +directly O +binds O +to O +a O +hydrophobic B-site +site I-site +on O +JNK1 B-protein +that O +is O +near O +the O +MAPK B-protein_type +insertion O +and O +helix B-structure_element +αG B-structure_element +. O +Biochemical B-experimental_method +studies I-experimental_method +further O +reveal O +that O +this O +highly B-protein_state +conserved I-protein_state +structural B-structure_element +motif I-structure_element +is O +present O +in O +all O +members O +of O +the O +MKP B-protein_type +family I-protein_type +, O +and O +the O +interaction O +mode O +is O +universal O +and O +critical O +for O +the O +MKP B-protein_type +- O +MAPK B-protein_type +recognition O +and O +biological O +function O +. O + +The O +MAPKs B-protein_type +are O +activated O +by O +MAPK B-protein_type +kinases I-protein_type +that O +phosphorylate O +the O +MAPKs B-protein_type +at O +conserved B-protein_state +threonine B-residue_name +and O +tyrosine B-residue_name +residues O +within O +their O +activation B-structure_element +loop I-structure_element +. O + +MAPKs B-protein_type +lie O +at O +the O +bottom O +of O +conserved O +three O +- O +component O +phosphorylation O +cascades O +and O +utilize O +docking O +interactions O +to O +link O +module O +components O +and O +bind O +substrates O +. O + +Downregulation O +of O +MAPK B-protein_type +activity O +can O +be O +achieved O +through O +direct O +dephosphorylation O +of O +the O +phospho B-residue_name +- I-residue_name +threonine I-residue_name +and I-residue_name +/ I-residue_name +or I-residue_name +tyrosine I-residue_name +residues O +by O +various O +serine B-protein_type +/ I-protein_type +threonine I-protein_type +phosphatases I-protein_type +, O +tyrosine B-protein_type +phosphatases I-protein_type +and O +dual B-protein_type +- I-protein_type +specificity I-protein_type +phosphatases I-protein_type +( O +DUSPs B-protein_type +) O +termed O +MKPs B-protein_type +. O + +DUSPs B-protein_type +belong O +to O +the O +protein B-protein_type +- I-protein_type +tyrosine I-protein_type +phosphatases I-protein_type +( O +PTPase B-protein_type +) O +superfamily O +, O +which O +is O +defined O +by O +the O +PTPase B-protein_type +- O +signature O +motif O +CXXGXXR B-structure_element +. O + +The O +KBD B-structure_element +is O +homologous O +to O +the O +rhodanese B-protein_type +family I-protein_type +and O +contains O +an O +intervening O +cluster O +of O +basic O +amino O +acids O +, O +which O +has O +been O +suggested O +to O +be O +important O +for O +interacting O +with O +the O +target O +MAPKs B-protein_type +. O + +As O +shown O +in O +Fig O +. O +2d O +, O +the O +CD B-structure_element +of O +MKP7 B-protein +can O +be O +pulled O +down O +by O +JNK1 B-protein +, O +while O +the O +KBD B-structure_element +failed O +to O +bind O +to O +the O +counterpart O +protein O +. O + +In O +the O +complex O +, O +JNK1 B-protein +has O +its O +characteristic O +bilobal O +structure O +comprising O +an O +N B-structure_element +- I-structure_element +terminal I-structure_element +lobe I-structure_element +rich O +in O +β B-structure_element +- I-structure_element +sheet I-structure_element +and O +a O +C B-structure_element +- I-structure_element +terminal I-structure_element +lobe I-structure_element +that O +is O +mostly O +α B-structure_element +- I-structure_element +helical I-structure_element +. O + +In O +an O +alignment B-experimental_method +of O +the O +structure B-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +with O +that O +of O +VHR B-protein +, O +an O +atypical O +‘ O +MKP B-protein_type +' O +consisting O +of O +only O +a O +catalytic B-structure_element +domain I-structure_element +, O +119 O +of O +147 O +MKP7 B-protein +- O +CD B-structure_element +residues O +could O +be O +superimposed B-experimental_method +with O +a O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +. I-evidence +( O +root B-evidence +mean I-evidence +squared I-evidence +deviation I-evidence +) O +of O +1 O +. O +05 O +Å O +( O +Fig O +. O +3c O +). O + +Since O +helix B-structure_element +α0 B-structure_element +and O +the O +following O +loop B-structure_element +α0 B-structure_element +– I-structure_element +β1 I-structure_element +are O +known O +for O +a O +substrate B-site +- I-site +recognition I-site +motif I-site +of O +VHR B-protein +and O +other O +phosphatases B-protein_type +, O +the O +absence O +of O +these O +moieties O +implicates O +a O +different O +substrate O +- O +binding O +mode O +of O +MKP7 B-protein +. O + +We O +also O +observed O +the O +binding O +of O +a O +chloride B-chemical +ion O +in O +the O +active B-site +site I-site +of O +MKP7 B-protein +- O +CD B-structure_element +. O + +Thus O +this O +chloride B-chemical +ion O +is O +a O +mimic O +for O +the O +phosphate B-chemical +group O +of O +the O +substrate O +, O +as O +revealed O +by O +a O +comparison O +with O +the O +structure B-evidence +of O +PTP1B B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +phosphotyrosine B-residue_name +( O +Supplementary O +Fig O +. O +1d O +). O + +The O +aromatic O +ring O +of O +Phe285 B-residue_name_number +on O +MKP7 B-protein +α5 B-structure_element +- I-structure_element +helix I-structure_element +is O +nestled O +in O +a O +hydrophobic B-site +pocket I-site +on O +JNK1 B-protein +, O +formed O +by O +side O +chains O +of O +Ile197 B-residue_name_number +, O +Leu198 B-residue_name_number +, O +Ile231 B-residue_name_number +, O +Trp234 B-residue_name_number +, O +Val256 B-residue_name_number +, O +Tyr259 B-residue_name_number +, O +Val260 B-residue_name_number +and O +the O +aliphatic O +portion O +of O +His230 B-residue_name_number +( O +Fig O +. O +3d O +, O +f O +and O +Supplementary O +Fig O +. O +1g O +). O + +Interestingly O +, O +mutation B-experimental_method +of O +Phe287 B-residue_name_number +results O +in O +a O +considerable O +loss O +of O +activity O +against O +pJNK1 B-protein_state +without O +altering O +the O +affinity B-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +for O +JNK1 B-protein +( O +Supplementary O +Fig O +. O +2a O +). O + +Incubation B-experimental_method +of O +MKP7 B-protein +with O +JNK1 B-protein +did O +not O +markedly O +stimulate O +the O +phosphatase B-protein_type +activity O +, O +which O +is O +consistent O +with O +previous O +results O +that O +MKP7 B-protein +solely O +possesses O +the O +intrinsic O +activity O +( O +Supplementary O +Fig O +. O +2b O +). O + +Biochemical O +results O +suggested O +that O +the O +affinity O +and O +specificity O +between O +KAP B-protein +and O +CDK2 B-protein +results O +from O +the O +recognition B-site +site I-site +comprising O +CDK2 B-protein +residues O +from O +the O +αG B-structure_element +helix I-structure_element +and O +L14 B-structure_element +loop I-structure_element +and O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +helical I-structure_element +region I-structure_element +of O +KAP B-protein +( O +Fig O +. O +5b O +). O + +There O +is O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +the O +main O +- O +chain O +nitrogen O +of O +Ile183 B-residue_name_number +( O +KAP B-protein +) O +and O +side O +chain O +oxygen O +of O +Glu208 B-residue_name_number +( O +CDK2 B-protein +), O +and O +salt O +bridges O +between O +Lys184 B-residue_name_number +of O +KAP B-protein +and O +Asp235 B-residue_name_number +of O +CDK2 B-protein +. O + +JNK B-protein_type +is O +activated O +following O +cellular O +exposure O +to O +a O +number O +of O +acute O +stimuli O +such O +as O +anisomycin B-chemical +, O +H2O2 B-chemical +, O +ultraviolet O +light O +, O +sorbitol B-chemical +, O +DNA O +- O +damaging O +agents O +and O +several O +strong O +apoptosis O +inducers O +( O +etoposide B-chemical +, O +cisplatin B-chemical +and O +taxol B-chemical +). O + +Expressions B-experimental_method +of O +wild B-protein_state +- I-protein_state +type I-protein_state +MKP7 B-protein +, O +MKP7ΔC304 B-mutant +and O +MKP7 B-protein +- O +CD B-structure_element +significantly O +decreased O +the O +proportion O +of O +apoptotic O +cells O +after O +ultraviolet O +treatment O +. O + +As O +in O +the O +case O +of O +MKP7 B-protein +, O +all O +the O +mutants B-protein_state +, O +except O +F451D B-mutant +/ I-mutant +A I-mutant +, O +showed O +no O +pNPPase B-protein_type +activity O +changes O +compared O +with O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +MKP5 B-protein +- O +CD B-structure_element +( O +Fig O +. O +7g O +), O +and O +the O +point B-experimental_method +mutations I-experimental_method +in O +JNK1 B-protein +also O +reduced O +the O +binding B-evidence +affinity I-evidence +of O +MKP5 B-protein +- O +CD B-structure_element +for O +JNK1 B-protein +( O +Fig O +. O +7h O +). O + +In O +this O +model O +, O +the O +MKP5 B-protein +- O +CD B-structure_element +adopts O +a O +conformation O +nearly O +identical O +to O +that O +in O +its O +unbound B-protein_state +form O +, O +suggesting O +that O +the O +conformation O +of O +the O +catalytic B-structure_element +domain I-structure_element +undergoes O +little O +change O +, O +if O +any O +at O +all O +, O +upon O +JNK1 B-protein +binding O +. O + +The O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +interaction O +is O +better O +and O +more O +extensive O +. O + +These O +structures B-evidence +revealed O +that O +linear B-structure_element +docking I-structure_element +motifs I-structure_element +in O +interacting O +proteins O +bind O +to O +a O +common O +docking B-site +site I-site +on O +MAPKs B-protein_type +outside O +the O +kinase B-protein_type +active B-site +site I-site +. O + +In O +contrast O +to O +the O +canonical O +D B-site +- I-site +motif I-site +- I-site +binding I-site +mode I-site +, O +separate O +helices B-structure_element +, O +α2 B-structure_element +and O +α3 B-structure_element +′, I-structure_element +in O +the O +KBD B-structure_element +of O +MKP5 B-protein +engage O +the O +p38α B-site +- I-site +docking I-site +site I-site +. O + +Sequence B-experimental_method +alignment I-experimental_method +of O +all O +MKPs B-protein_type +reveals O +a O +high O +degree O +of O +conservation O +of O +residues O +surrounding O +the O +interacting B-site +region I-site +observed O +in O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +complex O +( O +Supplementary O +Fig O +. O +5 O +). O + +In O +summary O +, O +we O +have O +resolved O +the O +structure B-evidence +of O +JNK1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +catalytic B-structure_element +domain I-structure_element +of O +MKP7 B-protein +. O + +In O +addition O +, O +JIP B-protein +- I-protein +1 I-protein +can O +also O +associate O +with O +MKP7 B-protein +via O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +MKP7 B-protein +( O +ref O +.). O + +When O +MKP7 B-protein +is O +bound B-protein_state +to I-protein_state +JIP B-protein +- I-protein +1 I-protein +, O +it O +reduces O +JNK B-protein_type +activation O +, O +leading O +to O +reduced O +phosphorylation O +of O +the O +JNK B-protein_type +target O +c B-protein_type +- I-protein_type +Jun I-protein_type +. O + +Thus O +, O +our O +biochemical B-evidence +and I-evidence +structural I-evidence +data I-evidence +allow O +us O +to O +present O +a O +model O +for O +the O +JNK1 B-complex_assembly +– I-complex_assembly +JIP I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +ternary O +complex O +and O +provide O +an O +important O +insight O +into O +the O +assembly O +and O +function O +of O +JNK B-protein_type +signalling O +modules O +( O +Supplementary O +Fig O +. O +6 O +). O + +The O +colour O +scheme O +is O +the O +same O +in O +the O +following O +figures O +unless O +indicated O +otherwise O +. O +( O +b O +) O +Plots B-evidence +of I-evidence +initial I-evidence +velocity I-evidence +of O +the O +MKP7 B-protein +- O +catalysed O +reaction O +versus O +phospho B-ptm +- O +JNK1 B-protein +concentration O +. O + +( O +d O +) O +GST B-experimental_method +- I-experimental_method +mediated I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +for O +interaction O +of O +JNK1 B-protein +with O +MKP7 B-protein +- O +CD B-structure_element +and O +MKP7 B-protein +- O +KBD B-structure_element +. O + +Structure B-evidence +of O +JNK1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +MKP7 B-protein +- O +CD B-structure_element +. O + +( O +a O +) O +Ribbon O +diagram O +of O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +complex O +in O +two O +views O +related O +by O +a O +45 O +° O +rotation O +around O +a O +vertical O +axis O +. O +( O +b O +) O +Structure B-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +with O +its O +active B-site +site I-site +highlight O +in O +cyan O +. O + +The O +2Fo B-evidence +− I-evidence +Fc I-evidence +omit I-evidence +map I-evidence +( O +contoured O +at O +1 O +. O +5σ O +) O +for O +the O +P B-structure_element +- I-structure_element +loop I-structure_element +of O +MKP7 B-protein +- O +CD B-structure_element +is O +shown O +at O +inset O +of O +b O +. O +( O +c O +) O +Structure B-evidence +of O +VHR B-protein +with O +its O +active B-site +site I-site +highlighted O +in O +marine O +blue O +. O +( O +d O +) O +Close O +- O +up O +view O +of O +the O +JNK1 B-site +– I-site +MKP7 I-site +interface I-site +showing O +interacting O +amino O +acids O +of O +JNK1 B-protein +( O +orange O +) O +and O +MKP7 B-protein +- O +CD B-structure_element +( O +cyan O +). O + +( O +a O +) O +Effects O +of O +mutations O +in O +MKP7 B-protein +- O +CD B-structure_element +on O +the O +JNK1 B-protein +dephosphorylation B-ptm +( O +mean O +± O +s O +. O +e O +. O +m O +., O +n O += O +3 O +). O + +However O +, O +in O +contrast O +to O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +MKP7 B-protein +- O +CD B-structure_element +, O +mutant B-protein_state +F285D B-mutant +did O +not O +co O +- O +migrate O +with O +JNK1 B-protein +. O + +( O +f O +) O +Effects O +of O +mutations B-experimental_method +in O +MKP7 B-protein +- O +CD B-structure_element +on O +the O +pNPP B-chemical +hydrolysis O +reaction O +( O +mean O +± O +s O +. O +e O +. O +m O +., O +n O += O +3 O +). O + +FXF B-structure_element +- I-structure_element +motif I-structure_element +is O +critical O +for O +controlling O +the O +phosphorylation B-ptm +of O +JNK B-protein_type +and O +ultraviolet O +- O +induced O +apoptosis O +. O + +After O +36 O +h O +infection O +, O +cells O +were O +untreated O +in O +a O +, O +stimulated O +with O +30 O +μM O +etoposide B-chemical +for O +3 O +h O +in O +b O +or O +irradiated O +with O +25 O +J O +m O +− O +2 O +ultraviolet O +light O +at O +30 O +min O +before O +lysis O +in O +c O +. O +Whole O +- O +cell O +extracts O +were O +then O +immunoblotted O +with O +antibody O +indicated O +. O + +Apoptotic O +cells O +were O +determined O +by O +Annexin B-chemical +- I-chemical +V I-chemical +- I-chemical +APC I-chemical +/ O +PI B-chemical +staining O +. O + +Floral O +abscission O +is O +controlled O +by O +the O +leucine B-protein_type +- I-protein_type +rich I-protein_type +repeat I-protein_type +receptor I-protein_type +kinase I-protein_type +( O +LRR B-protein_type +- I-protein_type +RK I-protein_type +) O +HAESA B-protein +and O +the O +peptide B-protein_type +hormone I-protein_type +IDA B-protein +. O + +Crystal B-evidence +structures I-evidence +of O +HAESA B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +IDA B-protein +reveal O +a O +hormone B-site +binding I-site +pocket I-site +that O +accommodates O +an O +active B-protein_state +dodecamer B-structure_element +peptide B-chemical +. O + +The O +HAESA B-protein +co B-protein_type +- I-protein_type +receptor I-protein_type +SERK1 B-protein +, O +a O +positive O +regulator O +of O +the O +floral O +abscission O +pathway O +, O +allows O +for O +high O +- O +affinity O +sensing O +of O +the O +peptide B-protein_type +hormone I-protein_type +by O +binding O +to O +an O +Arg B-structure_element +- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +in O +IDA B-protein +. O + +This O +sequence O +pattern O +is O +conserved B-protein_state +among O +diverse O +plant B-taxonomy_domain +peptides B-chemical +, O +suggesting O +that O +plant B-taxonomy_domain +peptide B-protein_type +hormone I-protein_type +receptors I-protein_type +may O +share O +a O +common O +ligand O +binding O +mode O +and O +activation O +mechanism O +. O + +Another O +challenge O +will O +be O +to O +find O +out O +where O +IDA B-protein +is O +produced O +in O +the O +plant B-taxonomy_domain +and O +what O +causes O +it O +to O +accumulate O +in O +specific O +places O +in O +preparation O +for O +organ O +shedding O +. O + +( O +A O +) O +SDS B-experimental_method +PAGE I-experimental_method +analysis O +of O +the O +purified O +Arabidopsis B-species +thaliana I-species +HAESA B-protein +ectodomain B-structure_element +( O +residues O +20 B-residue_range +– I-residue_range +620 I-residue_range +) O +obtained O +by O +secreted B-experimental_method +expression I-experimental_method +in I-experimental_method +insect I-experimental_method +cells I-experimental_method +. O + +Residues O +mediating O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +the O +IDA B-chemical +peptide I-chemical +are O +highlighted O +in O +blue O +, O +residues O +contributing O +to O +hydrogen B-bond_interaction +bond I-bond_interaction +interactions I-bond_interaction +and O +/ O +or O +salt B-bond_interaction +bridges I-bond_interaction +are O +shown O +in O +red O +. O + +The O +alignment O +includes O +a O +secondary O +structure O +assignment O +calculated O +with O +the O +program O +DSSP O +and O +colored O +according O +to O +Figure O +1 O +, O +with O +the O +N O +- O +and O +C O +- O +terminal O +caps B-structure_element +and O +the O +21 O +LRR B-structure_element +motifs I-structure_element +indicated O +in O +orange O +and O +blue O +, O +respectively O +. O + +Cysteine B-residue_name +residues O +engaged O +in O +disulphide B-ptm +bonds I-ptm +are O +depicted O +in O +green O +. O + +HAESA B-protein +residues O +interacting O +with O +the O +IDA B-chemical +peptide I-chemical +and O +/ O +or O +the O +SERK1 B-protein +co B-protein_type +- I-protein_type +receptor I-protein_type +kinase I-protein_type +ectodomain B-structure_element +are O +highlighted O +in O +blue O +and O +orange O +, O +respectively O +. O + +The O +PKGV B-structure_element +motif I-structure_element +present O +in O +our O +N B-protein_state +- I-protein_state +terminally I-protein_state +extended I-protein_state +IDA B-chemical +peptide I-chemical +is O +highlighted O +in O +red O +. O +( O +B O +) O +Isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +of O +the O +HAESA B-protein +ectodomain B-structure_element +vs O +. O +IDA B-protein +and O +including O +the O +synthetic B-protein_state +peptide B-chemical +sequence O +. O + +( O +C O +) O +Structure O +of O +the O +HAESA B-complex_assembly +– I-complex_assembly +IDA I-complex_assembly +complex O +with O +HAESA B-protein +shown O +in O +blue O +( O +ribbon O +diagram O +). O + +We O +purified B-experimental_method +the O +HAESA B-protein +ectodomain B-structure_element +( O +residues O +20 B-residue_range +– I-residue_range +620 I-residue_range +) O +from O +baculovirus B-experimental_method +- I-experimental_method +infected I-experimental_method +insect I-experimental_method +cells I-experimental_method +( O +Figure O +1 O +— O +figure O +supplement O +1A O +, O +see O +Materials O +and O +methods O +) O +and O +quantified O +the O +interaction O +of O +the O +~ O +75 O +kDa O +glycoprotein B-protein_type +with O +synthetic B-protein_state +IDA B-chemical +peptides I-chemical +using O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +( O +ITC B-experimental_method +). O + +We O +obtained O +a O +structure B-evidence +of O +HAESA B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +a O +PKGV B-mutant +- I-mutant +IDA I-mutant +peptide B-chemical +at O +1 O +. O +94 O +Å O +resolution O +( O +Table O +2 O +). O + +We O +next O +tested O +if O +HAESA B-protein +binds O +other O +IDA B-chemical +peptide I-chemical +family I-chemical +members I-chemical +. O + +We O +do O +not O +detect O +interaction O +between O +HAESA B-protein +and O +a O +synthetic B-protein_state +peptide B-chemical +missing B-protein_state +the I-protein_state +C I-protein_state +- I-protein_state +terminal I-protein_state +Asn69IDA B-residue_name_number +( O +ΔN69 B-mutant +), O +highlighting O +the O +importance O +of O +the O +polar B-bond_interaction +interactions I-bond_interaction +between O +the O +IDA B-protein +carboxy O +- O +terminus O +and O +Arg407HAESA B-residue_name_number +/ O +Arg409HAESA B-residue_name_number +( O +Figures O +1F O +, O +2D O +). O + +We O +found O +that O +the O +force O +required O +to O +remove O +the O +petals O +of O +serk1 B-gene +- I-gene +1 I-gene +mutants B-protein_state +is O +significantly O +higher O +than O +that O +needed O +for O +wild B-protein_state +- I-protein_state +type I-protein_state +plants B-taxonomy_domain +, O +as O +previously O +observed O +for O +haesa B-gene +/ O +hsl2 B-gene +mutants B-protein_state +, O +and O +that O +floral O +abscission O +is O +delayed O +in O +serk1 B-gene +- I-gene +1 I-gene +( O +Figure O +3A O +). O + +The O +serk2 B-gene +- I-gene +2 I-gene +, O +serk3 B-gene +- I-gene +1 I-gene +, O +serk4 B-gene +- I-gene +1 I-gene +and O +serk5 B-gene +- I-gene +1 I-gene +mutant B-protein_state +lines O +showed O +a O +petal O +break O +- O +strength O +profile O +not O +significantly O +different O +from O +wild B-protein_state +- I-protein_state +type I-protein_state +plants B-taxonomy_domain +. O + +In O +this O +case O +, O +there O +was O +no O +detectable O +interaction O +between O +receptor O +and O +co O +- O +receptor O +, O +while O +in O +the O +presence B-protein_state +of I-protein_state +IDA B-protein +, O +SERK1 B-protein +strongly O +binds O +HAESA B-protein +with O +a O +dissociation B-evidence +constant I-evidence +in O +the O +mid O +- O +nanomolar O +range O +( O +Figure O +3C O +). O + +SERK1 B-protein +senses O +a O +conserved B-protein_state +motif B-structure_element +in O +IDA B-chemical +family I-chemical +peptides I-chemical + +The O +conformational O +change O +in O +the O +C O +- O +terminal O +LRRs B-structure_element +and O +capping B-structure_element +domain I-structure_element +is O +indicated O +by O +an O +arrow O +. O +( O +C O +) O +SERK1 B-protein +forms O +an O +integral O +part O +of O +the O +receptor O +' O +s O +peptide B-site +binding I-site +pocket I-site +. O + +Ribbon O +diagrams O +of O +HAESA B-protein +( O +in O +blue O +) O +and O +SERK1 B-protein +( O +in O +orange O +) O +are O +shown O +with O +selected O +interface B-site +residues I-site +( O +in O +bonds O +representation O +). O + +To O +understand O +in O +molecular O +terms O +how O +SERK1 B-protein +contributes O +to O +high O +- O +affinity O +IDA B-protein +recognition O +, O +we O +solved O +a O +2 O +. O +43 O +Å O +crystal B-evidence +structure I-evidence +of O +the O +ternary O +HAESA B-complex_assembly +– I-complex_assembly +IDA I-complex_assembly +– I-complex_assembly +SERK1 I-complex_assembly +complex O +( O +Figure O +4A O +, O +Table O +2 O +). O + +SERK1 B-protein +LRRs B-structure_element +1 I-structure_element +– I-structure_element +5 I-structure_element +and O +its O +C O +- O +terminal O +capping B-structure_element +domain I-structure_element +form O +an O +additional O +zipper B-structure_element +- I-structure_element +like I-structure_element +interface B-site +with O +residues O +originating O +from O +HAESA B-protein +LRRs B-structure_element +15 I-structure_element +– I-structure_element +21 I-structure_element +and O +from O +the O +HAESA B-protein +C O +- O +terminal O +cap B-structure_element +( O +Figure O +4D O +). O + +Deletion B-experimental_method +of O +the O +buried O +Asn69IDA B-residue_name_number +completely B-protein_state +inhibits I-protein_state +receptor O +– O +co O +- O +receptor O +complex O +formation O +and O +HSL2 O +activation O +( O +Figure O +5A O +, O +B O +). O + +Comparison O +of O +35S B-gene +:: O +IDA B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +plants B-taxonomy_domain +further O +indicates O +that O +mutation B-experimental_method +of O +Lys66IDA B-mutant +/ I-mutant +Arg67IDA I-mutant +→ I-mutant +Ala I-mutant +may O +cause O +a O +weak O +dominant O +negative O +effect O +( O +Figure O +5C O +– O +E O +). O + +In O +contrast O +to O +animal B-taxonomy_domain +LRR B-protein_type +receptors I-protein_type +, O +plant B-taxonomy_domain +LRR B-structure_element +- I-structure_element +RKs I-structure_element +harbor O +spiral B-protein_state +- I-protein_state +shaped I-protein_state +ectodomains B-structure_element +and O +thus O +they O +require O +shape B-protein_state +- I-protein_state +complementary I-protein_state +co B-protein_type +- I-protein_type +receptor I-protein_type +proteins I-protein_type +for O +receptor O +activation O +. O + +As O +serk1 B-gene +- I-gene +1 I-gene +mutant B-protein_state +plants B-taxonomy_domain +show O +intermediate O +abscission O +phenotypes O +when O +compared O +to O +haesa B-gene +/ O +hsl2 O +mutants B-protein_state +, O +SERK1 B-protein +likely O +acts O +redundantly O +with O +other O +SERKs B-protein_type +in O +the O +abscission O +zone O +( O +Figure O +3A O +). O + +Our O +comparative B-experimental_method +structural I-experimental_method +and I-experimental_method +biochemical I-experimental_method +analysis I-experimental_method +further O +suggests O +that O +IDLs B-protein_type +share O +a O +common O +receptor O +binding O +mode O +, O +but O +may O +preferably O +bind O +to O +HAESA B-protein +, O +HSL1 B-protein +or O +HSL2 B-protein +in O +different O +plant B-taxonomy_domain +tissues O +and O +organs O +. O + +Several O +residues O +in O +the O +SERK1 B-protein +N O +- O +terminal O +capping B-structure_element +domain I-structure_element +( O +Thr59SERK1 B-residue_name_number +, O +Phe61SERK1 B-residue_name_number +) O +and O +the O +LRR B-site +inner I-site +surface I-site +( O +Asp75SERK1 B-residue_name_number +, O +Tyr101SERK1 B-residue_name_number +, O +SER121SERK1 B-residue_name_number +, O +Phe145SERK1 B-residue_name_number +) O +contribute O +to O +the O +formation O +of O +both O +complexes O +( O +Figures O +4C O +, O +D O +, O +6B O +). O + +This O +fact O +together O +with O +the O +largely O +overlapping O +SERK1 B-site +binding I-site +surfaces I-site +in O +HAESA B-protein +and O +BRI1 B-protein +allows O +us O +to O +speculate O +that O +SERK1 B-protein +may O +promote O +high O +- O +affinity O +peptide B-protein_type +hormone I-protein_type +and O +brassinosteroid O +sensing O +by O +simply O +slowing O +down O +dissociation O +of O +the O +ligand O +from O +its O +cognate O +receptor O +. O + +Ensemble O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +uncovers O +inchworm B-protein_state +- O +like O +translocation O +of O +a O +viral B-taxonomy_domain +IRES B-site +through O +the O +ribosome B-complex_assembly + +This O +unlocks O +40S B-complex_assembly +domains O +, O +facilitating O +head B-structure_element +swivel O +and O +biasing O +IRES B-site +translocation O +via O +hitherto O +- O +elusive O +intermediates O +with O +PKI B-structure_element +captured O +between O +the O +A B-site +and I-site +P I-site +sites I-site +. O + +Virus B-taxonomy_domain +propagation O +relies O +on O +the O +host O +translational O +apparatus O +. O + +A O +recent O +demonstration O +of O +bacterial B-taxonomy_domain +translation O +initiation B-protein_state +by O +an O +IGR B-structure_element +IRES B-site +indicates O +that O +the O +IRESs B-site +take O +advantage O +of O +conserved O +structural O +and O +dynamic O +properties O +of O +the O +ribosome B-complex_assembly +. O + +Nucleotides O +C1274 B-residue_name_number +, O +U1191 B-residue_name_number +of O +the O +40S B-complex_assembly +head B-structure_element +and O +G904 B-residue_name_number +of O +the O +platform B-structure_element +( O +corresponding O +to O +C1054 B-residue_name_number +, O +G966 B-residue_name_number +and O +G693 B-residue_name_number +in O +E B-species +. I-species +coli I-species +16S B-chemical +rRNA I-chemical +) O +are O +shown O +in O +black O +to O +denote O +the O +A B-site +, I-site +P I-site +and I-site +E I-site +sites I-site +, O +respectively O +. O + +The O +extents O +of O +the O +40S B-complex_assembly +subunit B-structure_element +rotation O +and O +head B-structure_element +swivel O +relative O +to O +their O +positions O +in O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +structure B-evidence +are O +shown O +with O +arrows O +. O + +In O +panels O +( O +a O +- O +e O +), O +the O +maps B-evidence +are O +segmented O +and O +colored O +as O +in O +Figure O +1 O +. O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +of O +the O +80S B-complex_assembly +• I-complex_assembly +TSV I-complex_assembly +IRES I-complex_assembly +bound B-protein_state +with I-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +• I-complex_assembly +sordarin I-complex_assembly +. O + +Unsupervised B-experimental_method +cryo I-experimental_method +- I-experimental_method +EM I-experimental_method +data I-experimental_method +classification I-experimental_method +was O +combined O +with O +the O +use O +of O +three B-experimental_method +- I-experimental_method +dimensional I-experimental_method +and I-experimental_method +two I-experimental_method +- I-experimental_method +dimensional I-experimental_method +masking I-experimental_method +around O +the O +ribosomal O +A B-site +site I-site +( O +Figure O +1 O +— O +figure O +supplement O +2 O +). O + +The O +views O +were O +obtained O +by O +structural B-experimental_method +alignment I-experimental_method +of O +the O +25S B-chemical +rRNAs I-chemical +; O +the O +sarcin B-structure_element +- I-structure_element +ricin I-structure_element +loop I-structure_element +( O +SRL B-structure_element +) O +of O +25S B-chemical +rRNA I-chemical +is O +shown O +in O +gray O +for O +reference O +. O + +Conformation O +of O +the O +non B-protein_state +- I-protein_state +swiveled I-protein_state +40S B-complex_assembly +subunit B-structure_element +in O +the O +S B-species +. I-species +cerevisiae I-species +80S B-complex_assembly +ribosome I-complex_assembly +bound B-protein_state +with I-protein_state +two O +tRNAs B-chemical +is O +shown O +for O +reference O +( O +blue O +). O + +Structure B-evidence +I I-evidence +comprises O +the O +most B-protein_state +rotated I-protein_state +ribosome B-complex_assembly +conformation O +(~ O +10 O +°), O +characteristic O +of O +pre B-protein_state +- I-protein_state +translocation I-protein_state +hybrid B-protein_state +- I-protein_state +tRNA I-protein_state +states O +. O + +Structure B-evidence +IV I-evidence +adopts O +a O +slightly B-protein_state +rotated I-protein_state +conformation O +(~ O +1 O +°). O + +Thus O +, O +intersubunit O +rotation O +of O +~ O +9 O +° O +from O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +covers O +a O +nearly O +complete O +range O +of O +relative O +subunit B-structure_element +positions O +, O +similar O +to O +what O +was O +reported O +for O +tRNA B-protein_state +- I-protein_state +bound I-protein_state +yeast B-taxonomy_domain +, O +bacterial B-taxonomy_domain +and O +mammalian B-taxonomy_domain +ribosomes B-complex_assembly +. O + +40S B-complex_assembly +head B-structure_element +swivel O + +As O +with O +the O +intersubunit O +rotation O +, O +the O +small O +head B-structure_element +swivel O +(~ O +1 O +°) O +in O +the O +non B-protein_state +- I-protein_state +rotated I-protein_state +Structure B-evidence +V I-evidence +is O +closest O +to O +that O +in O +the O +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +post B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +. O + +The O +head B-structure_element +samples O +a O +mid B-protein_state +- I-protein_state +swiveled I-protein_state +position O +in O +Structure B-evidence +I I-evidence +( O +12 O +°), O +then O +a O +highly B-protein_state +- I-protein_state +swiveled I-protein_state +position O +in O +Structures B-evidence +II I-evidence +and I-evidence +III I-evidence +( O +17 O +°) O +and O +a O +less B-protein_state +swiveled I-protein_state +position O +in O +Structure B-evidence +IV I-evidence +( O +14 O +°). O + +Superpositions O +were O +obtained O +by O +structural B-experimental_method +alignments I-experimental_method +of O +the O +18S B-chemical +rRNAs I-chemical +excluding O +the O +head B-structure_element +domains O +( O +nt O +1150 B-residue_range +– I-residue_range +1620 I-residue_range +). O + +Interactions O +of O +the O +stem B-structure_element +loops I-structure_element +4 I-structure_element +and I-structure_element +5 I-structure_element +of O +the O +TSV B-species +with O +proteins O +uS7 B-protein +and O +eS25 B-protein +. O + +Position O +and O +interactions O +of O +loop B-structure_element +3 I-structure_element +( O +variable B-structure_element +loop I-structure_element +region I-structure_element +) O +of O +the O +PKI B-structure_element +domain O +in O +Structure B-evidence +V I-evidence +( O +this O +work O +) O +resembles O +those O +of O +the O +anticodon B-structure_element +stem I-structure_element +loop I-structure_element +of O +the O +E B-site +- I-site +site I-site +tRNA B-chemical +( O +blue O +) O +in O +the O +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +. O + +Positions O +of O +tRNAs B-chemical +and O +the O +TSV B-species +IRES B-site +relative O +to O +the O +A B-structure_element +- I-structure_element +site I-structure_element +finger I-structure_element +( O +blue O +, O +nt O +1008 B-residue_range +– I-residue_range +1043 I-residue_range +of O +25S B-chemical +rRNA I-chemical +) O +and O +the O +P B-site +site I-site +of O +the O +large B-structure_element +subunit I-structure_element +, O +comprising O +helix B-structure_element +84 I-structure_element +of O +25S B-chemical +rRNA I-chemical +( O +nt O +. O + +Interactions O +of O +the O +TSV B-species +IRES B-site +with O +uL5 B-protein +and O +eL42 B-protein +. O + +Structures B-evidence +of O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +complexes O +in O +the O +absence B-protein_state +of I-protein_state +eEF2 B-protein +( O +INIT B-complex_assembly +; O +PDB O +3J6Y O +,) O +and O +in O +the O +presence B-protein_state +of I-protein_state +eEF2 B-protein +( O +this O +work O +) O +are O +shown O +in O +the O +upper O +row O +and O +labeled O +. O + +6758 B-residue_range +– I-residue_range +6888 I-residue_range +) O +binds O +near O +the O +E B-site +site I-site +, O +contacting O +the O +ribosome B-complex_assembly +mostly O +by O +means O +of O +three O +protruding O +structural O +elements O +: O +the O +L1 B-structure_element +. I-structure_element +1 I-structure_element +region I-structure_element +and O +stem B-structure_element +loops I-structure_element +4 I-structure_element +and I-structure_element +5 I-structure_element +( O +SL4 B-structure_element +and O +SL5 B-structure_element +). O + +The O +minor B-site +groove I-site +of O +SL5 B-structure_element +( O +at O +nt O +6862 B-residue_range +– I-residue_range +6868 I-residue_range +) O +contacts O +the O +positively O +charged O +region O +of O +eS25 B-protein +( O +R49 B-residue_name_number +, O +R58 B-residue_name_number +and O +R68 B-residue_name_number +) O +( O +Figure O +3 O +— O +figure O +supplement O +4 O +). O + +Conformations O +and O +positions O +of O +the O +IRES B-site +in O +the O +initiation B-protein_state +state O +and O +in O +Structures B-evidence +I I-evidence +- I-evidence +V I-evidence +are O +shown O +relative O +to O +those O +of O +the O +A B-site +-, I-site +P I-site +- I-site +and I-site +E I-site +- I-site +site I-site +tRNAs B-chemical +. O + +In O +Structure B-evidence +I I-evidence +, O +SL3 B-structure_element +of O +the O +PKI B-structure_element +domain O +is O +positioned O +between O +the O +A B-structure_element +- I-structure_element +site I-structure_element +finger I-structure_element +( O +nt O +1008 B-residue_range +– I-residue_range +1043 I-residue_range +of O +25S B-chemical +rRNA I-chemical +) O +and O +the O +P B-site +site I-site +of O +the O +60S B-complex_assembly +subunit B-structure_element +, O +comprising O +helix B-structure_element +84 I-structure_element +of O +25S B-chemical +rRNA I-chemical +( O +nt O +. O + +In O +Structure B-evidence +V I-evidence +, O +loop B-structure_element +3 I-structure_element +is O +bound B-protein_state +in I-protein_state +the O +40S B-complex_assembly +E B-site +site I-site +and O +the O +backbone O +of O +loop B-structure_element +3 I-structure_element +near O +the O +codon B-structure_element +- I-structure_element +like I-structure_element +part I-structure_element +of O +PKI B-structure_element +( O +at O +nt O +. O + +The O +interaction O +of O +loop B-structure_element +3 I-structure_element +backbone O +with O +uS7 B-protein +resembles O +that O +of O +the O +anticodon B-structure_element +- I-structure_element +stem I-structure_element +loop I-structure_element +of O +E B-site +- I-site +site I-site +tRNA B-chemical +in O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +structure B-evidence +( O +Figure O +3 O +— O +figure O +supplement O +5 O +). O + +Ordering O +of O +loop B-structure_element +3 I-structure_element +suggests O +that O +this O +flexible O +region O +contributes O +to O +the O +stabilization O +of O +the O +PKI B-structure_element +domain O +in O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +state O +. O + +Colors O +for O +the O +ribosome B-complex_assembly +and O +eEF2 B-protein +are O +as O +in O +Figure O +1 O +. O + +Switch B-structure_element +loop I-structure_element +I I-structure_element +( O +SWI B-structure_element +) O +in O +Structure B-evidence +I I-evidence +is O +in O +blue O +; O +dashed O +line O +shows O +the O +putative O +location O +of O +unresolved O +switch B-structure_element +loop I-structure_element +I I-structure_element +in O +Structure B-evidence +II I-evidence +. O + +Elongation B-protein_type +factor I-protein_type +eEF2 B-protein +in O +all O +five O +structures B-evidence +is O +bound B-protein_state +with I-protein_state +GDP B-chemical +and O +sordarin B-chemical +( O +Figure O +5 O +). O + +GDP B-chemical +in O +our O +structures B-evidence +is O +bound B-protein_state +in I-protein_state +the O +GTPase B-site +center I-site +( O +Figures O +5d O +, O +e O +and O +f O +) O +and O +sordarin B-chemical +is O +sandwiched O +between O +the O +β B-structure_element +- I-structure_element +platforms I-structure_element +of O +domains O +III B-structure_element +and O +V B-structure_element +( O +Figures O +5g O +and O +h O +), O +as O +in O +the O +structure B-evidence +of O +free B-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +sordarin I-complex_assembly +complex O +. O + +From O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +, O +eEF2 B-protein +is O +rigidly O +attached O +to O +the O +GTPase B-site +- I-site +associated I-site +center I-site +of O +the O +60S B-complex_assembly +subunit B-structure_element +. O + +The O +tips O +of O +25S B-chemical +rRNA I-chemical +helices B-structure_element +43 I-structure_element +and I-structure_element +44 I-structure_element +of O +the O +P B-structure_element +stalk I-structure_element +( O +nucleotides O +G1242 B-residue_name_number +and O +A1270 B-residue_name_number +, O +respectively O +) O +stack B-bond_interaction +on O +V754 B-residue_name_number +and O +Y744 B-residue_name_number +of O +domain O +V B-structure_element +. O +An O +αββ B-structure_element +motif I-structure_element +of O +the O +eukaryote B-taxonomy_domain +- O +specific O +protein O +P0 B-protein +( O +aa O +126 B-residue_range +– I-residue_range +154 I-residue_range +) O +packs O +in O +the O +crevice O +between O +the O +long B-structure_element +α I-structure_element +- I-structure_element +helix I-structure_element +D I-structure_element +( O +aa O +172 B-residue_range +– I-residue_range +188 I-residue_range +) O +of O +the O +GTPase B-structure_element +domain I-structure_element +and O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +region I-structure_element +( O +aa O +246 B-residue_range +– I-residue_range +263 I-residue_range +) O +of O +the O +GTPase B-structure_element +domain I-structure_element +insert I-structure_element +( O +or O +G B-structure_element +’ I-structure_element +insert I-structure_element +) O +of O +eEF2 B-protein +( O +secondary O +- O +structure O +nomenclatures O +for O +eEF2 B-protein +and O +EF B-protein +- I-protein +G I-protein +are O +the O +same O +). O + +While O +the O +overall O +mode O +of O +this O +interaction O +is O +similar O +to O +that O +seen O +in O +70S B-complex_assembly +• I-complex_assembly +EF I-complex_assembly +- I-complex_assembly +G I-complex_assembly +crystal B-evidence +structures I-evidence +, O +there O +is O +an O +important O +local O +difference O +between O +Structure B-evidence +I I-evidence +and O +Structures B-evidence +II I-evidence +- I-evidence +V I-evidence +in O +switch B-structure_element +loop I-structure_element +I I-structure_element +, O +as O +discussed O +below O +. O + +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +are O +shown O +. O + +The O +view O +was O +obtained O +by O +superpositions B-experimental_method +of O +the O +body B-structure_element +domains O +of O +18S B-chemical +rRNAs I-chemical +. O + +At O +the O +head B-structure_element +, O +C1274 B-residue_name_number +of O +the O +18S B-chemical +rRNA I-chemical +( O +C1054 B-residue_name_number +in O +E B-species +. I-species +coli I-species +) O +base O +pairs O +with O +the O +first O +nucleotide O +of O +the O +ORF B-structure_element +immediately O +downstream O +of O +PKI B-structure_element +. O + +Key O +elements O +of O +the O +decoding B-site +center I-site +of O +the O +' O +locked B-protein_state +' O +initiation B-protein_state +structure B-evidence +, O +' O +unlocked B-protein_state +' O +Structure B-evidence +I I-evidence +, O +and O +post B-protein_state +- I-protein_state +translocation I-protein_state +Structure B-evidence +V I-evidence +( O +this O +work O +) O +are O +shown O +. O + +The O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +- I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +is O +shown O +in O +red O +, O +the O +downstream O +first O +codon O +of O +the O +ORF B-structure_element +in O +magenta O +. O + +Nucleotides O +of O +the O +18S B-chemical +rRNA I-chemical +body B-structure_element +are O +in O +orange O +and O +head B-structure_element +in O +yellow O +; O +25S B-chemical +rRNA I-chemical +nucleotide O +A2256 B-residue_name_number +is O +blue O +. O + +In O +the O +latter O +, O +PKI B-structure_element +is O +stabilized O +by O +interactions O +with O +the O +universally B-protein_state +conserved I-protein_state +decoding B-site +- I-site +center I-site +nucleotides O +G577 B-residue_name_number +, O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +(' O +body B-structure_element +A B-site +site I-site +'), O +as O +in O +the O +A B-site +- I-site +site I-site +tRNA B-protein_state +bound I-protein_state +complexes O +. O + +Histidines B-residue_name_number +583 I-residue_name_number +and I-residue_name_number +694 I-residue_name_number +interact O +with O +the O +phosphate O +backbone O +of O +the O +anticodon B-structure_element +- I-structure_element +like I-structure_element +strand I-structure_element +( O +at O +G6907 B-residue_name_number +and O +C6908 B-residue_name_number +). O + +The O +N O +- O +terminal O +part O +of O +the O +loop B-structure_element +( O +aa O +50 B-residue_range +– I-residue_range +60 I-residue_range +) O +is O +sandwiched O +between O +the O +tip O +of O +helix B-structure_element +14 I-structure_element +( O +415CAAA418 B-structure_element +) O +of O +the O +18S B-chemical +rRNA I-chemical +of O +the O +40S B-complex_assembly +subunit B-structure_element +and O +helix B-structure_element +A I-structure_element +( O +aa O +32 B-residue_range +– I-residue_range +42 I-residue_range +) O +of O +eEF2 B-protein +( O +Figure O +5d O +). O + +Among O +the O +five O +structures B-evidence +, O +the O +PKI B-structure_element +domain O +is O +least O +ordered O +in O +Structure B-evidence +III I-evidence +and O +lacks O +density B-evidence +for O +SL3 B-structure_element +. O + +Thus O +, O +in O +Structure B-evidence +III I-evidence +, O +PKI B-structure_element +has O +translocated O +along O +the O +40S B-complex_assembly +body B-structure_element +, O +but O +the O +head B-structure_element +remains O +fully B-protein_state +swiveled I-protein_state +so O +that O +PKI B-structure_element +is O +between O +the O +head B-structure_element +A B-site +and I-site +P I-site +sites I-site +. O + +The O +position O +of O +eEF2 B-protein +is O +similar O +to O +that O +in O +Structure B-evidence +II I-evidence +. O + +To O +this O +end O +, O +the O +head B-site +- I-site +interacting I-site +interface I-site +of O +domain O +IV B-structure_element +slides O +along O +the O +surface O +of O +the O +head B-structure_element +by O +5 O +Å O +. O +Helix B-structure_element +A I-structure_element +of O +domain O +IV B-structure_element +is O +positioned O +next O +to O +the O +backbone O +of O +h34 B-structure_element +, O +with O +positively O +charged O +residues O +K613 B-residue_name_number +, O +R617 B-residue_name_number +and O +R631 B-residue_name_number +rearranged O +from O +the O +backbone O +of O +h33 B-structure_element +( O +Figure O +6c O +; O +see O +also O +Figure O +6 O +— O +figure O +supplement O +1 O +). O + +In O +the O +nearly B-protein_state +non I-protein_state +- I-protein_state +rotated I-protein_state +and O +non B-protein_state +- I-protein_state +swiveled I-protein_state +ribosome B-complex_assembly +conformation O +in O +Structure B-evidence +V I-evidence +closely O +resembling O +that O +of O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +, O +PKI B-structure_element +is O +fully O +accommodated O +in O +the O +P B-site +site I-site +. O + +In O +this O +position O +, O +uS12 B-protein +forms O +extensive O +interactions O +with O +eEF2 B-protein +domains O +II B-structure_element +and O +III B-structure_element +. O + +Domain O +IV B-structure_element +of O +eEF2 B-protein +is O +fully O +accommodated O +in O +the O +A B-site +site I-site +. O + +The O +first O +codon O +of O +the O +open B-structure_element +reading I-structure_element +frame I-structure_element +is O +also O +positioned O +in O +the O +A B-site +site I-site +, O +with O +bases O +exposed O +toward O +eEF2 B-protein +( O +Figure O +7 O +), O +resembling O +the O +conformations O +of O +the O +A B-site +- I-site +site I-site +codons O +in O +EF B-protein_state +- I-protein_state +G I-protein_state +- I-protein_state +bound I-protein_state +70S B-complex_assembly +complexes O +. O + +Diph699 B-ptm +slightly O +rearranges O +, O +relative O +to O +that O +in O +Structure B-evidence +I I-evidence +( O +Figure O +7 O +), O +and O +interacts O +with O +four O +out O +of O +six O +codon O +- O +anticodon O +nucleotides O +. O + +The O +imidazole O +moiety O +stacks O +on O +G6907 B-residue_name_number +( O +corresponding O +to O +nt O +36 O +in O +the O +tRNA B-chemical +anticodon O +) O +and O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +O2 O +’ O +of O +G6906 B-residue_name_number +( O +nt O +35 O +of O +tRNA B-chemical +). O + +In O +scenes O +1 O +, O +2 O +and O +3 O +, O +nucleotides O +C1274 B-residue_name_number +, O +U1191 B-residue_name_number +of O +the O +40S B-complex_assembly +head B-structure_element +and O +G904 B-residue_name_number +of O +the O +40S B-site +platform I-site +are O +shown O +in O +black O +to O +denote O +the O +A B-site +, I-site +P I-site +and I-site +E I-site +sites I-site +, O +respectively O +. O + +Translocation O +of O +the O +TSV B-species +IRES B-site +on O +the O +40S B-complex_assembly +subunit B-structure_element +globally O +resembles O +a O +step O +of O +an O +inchworm B-protein_state +( O +Figure O +4 O +; O +see O +also O +Figure O +3 O +— O +figure O +supplement O +2 O +). O + +Finally O +( O +Structures B-evidence +IV I-evidence +to I-evidence +V I-evidence +), O +as O +the O +hind B-structure_element +end I-structure_element +is O +accommodated O +in O +the O +P B-site +site I-site +, O +the O +front B-structure_element +' I-structure_element +legs I-structure_element +' I-structure_element +advance O +by O +departing O +from O +their O +initial B-site +binding I-site +sites I-site +. O + +This O +converts O +the O +IRES B-site +into O +an O +extended B-protein_state +conformation O +, O +rendering O +the O +inchworm B-protein_state +prepared O +for O +the O +next O +translocation O +step O +. O + +Recent O +studies O +have O +shown O +that O +in O +some O +cases O +a O +fraction O +of O +IGR B-structure_element +IRES B-site +- O +driven O +translation O +results O +from O +an O +alternative O +reading O +frame O +, O +which O +is O +shifted O +by O +one O +nucleotide O +relative O +to O +the O +normal O +ORF B-structure_element +. O + +Specifically O +, O +biochemical B-experimental_method +toe I-experimental_method +- I-experimental_method +printing I-experimental_method +studies I-experimental_method +in O +the O +presence B-protein_state +of I-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +GTP I-complex_assembly +identified O +IRES B-site +in O +a O +non B-protein_state +- I-protein_state +translocated I-protein_state +position O +unless O +eEF1a B-complex_assembly +• I-complex_assembly +aa I-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +is O +also O +present O +. O + +Thus O +, O +the O +meta O +- O +stability O +of O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +IRES B-site +is O +likely O +due O +to O +the O +absence B-protein_state +of I-protein_state +stabilizing O +structural O +features O +present O +in O +the O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +. O + +Reverse O +intersubunit O +rotation O +from O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +shifts O +the O +translocation B-site +tunnel I-site +( O +the O +tunnel B-site +between O +the O +A B-site +, I-site +P I-site +and I-site +E I-site +sites I-site +) O +toward O +eEF2 B-protein +, O +which O +is O +rigidly O +attached O +to O +the O +60S B-complex_assembly +subunit B-structure_element +. 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O + +Destabilization O +of O +the O +head B-protein_state +- I-protein_state +bound I-protein_state +PKI B-structure_element +at O +the O +body B-structure_element +A B-site +site I-site +thus O +allows O +mobility O +of O +the O +head B-structure_element +relative O +to O +the O +body B-structure_element +. O + +Here O +, O +switch B-structure_element +loop I-structure_element +I I-structure_element +interacts O +with O +helix B-structure_element +14 I-structure_element +( O +415CAAA418 B-structure_element +) O +of O +the O +18S B-chemical +rRNA I-chemical +. 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O +8 O +Å O +) O +in O +a O +perfect O +trajectory O +for O +the O +nucleophilic O +attack O +by O +Thr1Oγ B-residue_name_number +( O +Fig O +. O +1b O +and O +Supplementary O +Fig O +. O +2b O +). O + +Next O +, O +we O +examined O +the O +position O +of O +the O +β5 B-protein +propeptide B-structure_element +in O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +mutant B-protein_state +. O + +Processing O +of O +β O +- O +subunit O +precursors O +requires O +deprotonation O +of O +Thr1OH B-residue_name_number +; O +however O +, O +the O +general O +base O +initiating O +autolysis B-ptm +is O +unknown O +. O + +Structural B-experimental_method +analyses I-experimental_method +revealed O +that O +the O +propeptides B-structure_element +of O +all O +mutant B-protein_state +yCPs B-complex_assembly +shared O +residual O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +densities I-evidence +. O + +Although O +we O +observed O +fragments O +of O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +density I-evidence +in O +the O +β1 B-protein +active B-site +site I-site +, O +the O +data O +were O +not O +interpretable O +. O + +Notably O +, O +the O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +- I-evidence +density I-evidence +map I-evidence +displays O +a O +different O +orientation O +for O +the O +β2 B-protein +propeptide B-structure_element +than O +has O +been O +observed O +for O +the O +β2 B-mutant +- I-mutant +T1A I-mutant +proteasome B-complex_assembly +. O + +The O +active B-site +site I-site +of O +the O +proteasome B-complex_assembly + +Mutation B-experimental_method +of O +β5 B-protein +- O +Lys33 B-residue_name_number +to O +Ala B-residue_name +causes O +a O +strongly O +deleterious O +phenotype O +, O +and O +previous O +structural B-experimental_method +and I-experimental_method +biochemical I-experimental_method +analyses I-experimental_method +confirmed O +that O +this O +is O +caused O +by O +failure O +of O +propeptide B-ptm +cleavage I-ptm +, O +and O +consequently O +, O +lack O +of O +ChT O +- O +L O +activity O +( O +Fig O +. O +4a O +, O +Supplementary O +Fig O +. O +3b O +and O +Table O +1 O +; O +for O +details O +see O +Supplementary O +Note O +1 O +). O + +The O +phenotype O +of O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +mutant B-protein_state +was O +however O +less O +pronounced O +than O +for O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +yeast B-taxonomy_domain +( O +Fig O +. O +4a O +). O + +Structural B-experimental_method +comparison I-experimental_method +of O +the O +β5 B-mutant +- I-mutant +L I-mutant +(- I-mutant +49 I-mutant +) I-mutant +S I-mutant +- I-mutant +K33A I-mutant +and O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +active B-site +sites I-site +revealed O +that O +mutation B-experimental_method +of O +Lys33 B-residue_name_number +to O +Ala B-residue_name +creates O +a O +cavity O +that O +is O +filled O +with O +Thr1 B-residue_name_number +and O +the O +remnant O +propeptide B-structure_element +. O + +Furthermore O +, O +the O +crystal B-evidence +structure I-evidence +of O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +pp B-chemical +trans B-protein_state +mutant B-protein_state +without B-protein_state +inhibitor I-protein_state +revealed O +that O +Thr1Oγ B-residue_name_number +strongly O +coordinates B-bond_interaction +a O +well O +- O +defined O +water B-chemical +molecule O +(∼ O +2 O +Å O +; O +Fig O +. O +3c O +and O +Supplementary O +Fig O +. O +5c O +, O +d O +). O + +This O +water B-chemical +hydrogen B-bond_interaction +bonds I-bond_interaction +also O +to O +Arg19O B-residue_name_number +(∼ O +3 O +. O +0 O +Å O +) O +and O +Asp17Oδ B-residue_name_number +(∼ O +3 O +. O +0 O +Å O +), O +and O +thereby O +presumably O +enables O +residual O +activity O +of O +the O +mutant B-protein_state +. O + +Even O +though O +the O +β5 B-mutant +- I-mutant +D17N I-mutant +pp B-chemical +trans B-protein_state +yCP B-complex_assembly +crystal B-evidence +structure I-evidence +appeared O +identical O +to O +the O +WT B-protein_state +yCP B-complex_assembly +( O +Supplementary O +Fig O +. O +7b O +), O +the O +co B-evidence +- I-evidence +crystal I-evidence +structure I-evidence +with O +the O +α B-chemical +′, I-chemical +β I-chemical +′ I-chemical +epoxyketone I-chemical +inhibitor O +carfilzomib B-chemical +visualized O +only O +partial O +occupancy O +of O +the O +ligand O +in O +the O +β5 B-protein +active B-site +site I-site +( O +Supplementary O +Fig O +. O +7a O +). O + +In O +agreement O +, O +an O +E17A B-mutant +mutant B-protein_state +in O +the O +proteasomal O +β B-protein +- I-protein +subunit I-protein +of O +the O +archaeon B-taxonomy_domain +Thermoplasma B-species +acidophilum I-species +prevents O +autolysis B-ptm +and O +catalysis O +. O + +The O +β5 B-mutant +- I-mutant +D166N I-mutant +pp B-chemical +cis B-protein_state +yeast B-taxonomy_domain +mutant B-protein_state +is O +significantly O +impaired O +in O +growth O +and O +its O +ChT O +- O +L O +activity O +is O +drastically O +reduced O +( O +Supplementary O +Fig O +. O +6a O +, O +b O +and O +Table O +1 O +). O + +X B-evidence +- I-evidence +ray I-evidence +data I-evidence +on O +the O +β5 B-mutant +- I-mutant +D166N I-mutant +mutant B-protein_state +indicate O +that O +the O +β5 B-protein +propeptide B-structure_element +is O +hydrolysed O +, O +but O +due O +to O +reorientation O +of O +Ser129OH B-residue_name_number +, O +the O +interaction O +with O +Asn166Oδ B-residue_name_number +is O +disrupted O +( O +Supplementary O +Fig O +. O +8a O +). O + +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +occupies O +the O +S2 B-site +pocket I-site +like O +observed O +for O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +mutant B-protein_state +, O +but O +in O +contrast O +to O +the O +latter O +, O +the O +propeptide B-structure_element +in O +the O +T1C B-mutant +mutant B-protein_state +adopts O +an O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +conformation O +as O +known O +from O +inhibitors O +like O +MG132 B-chemical +( O +Fig O +. O +4c O +– O +e O +and O +Supplementary O +Fig O +. O +9b O +). O + +Owing O +to O +the O +unequal O +positions O +of O +the O +two O +β5 B-protein +subunits O +within O +the O +CP B-complex_assembly +in O +the O +crystal O +lattice O +, O +maturation O +and O +propeptide B-structure_element +displacement O +may O +occur O +at O +different O +timescales O +in O +the O +two O +subunits O +. O + +Notably O +, O +the O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +- I-evidence +density I-evidence +map I-evidence +of O +the O +T1C B-mutant +mutant B-protein_state +also O +indicates O +that O +Lys33NH2 B-residue_name_number +is O +disordered B-protein_state +. O + +However O +, O +the O +apo B-protein_state +crystal B-evidence +structure I-evidence +revealed O +that O +Ser1Oγ B-residue_name_number +is O +turned O +away O +from O +the O +substrate B-site +- I-site +binding I-site +channel I-site +( O +Fig O +. O +4g O +). O + +The O +20S B-complex_assembly +proteasome I-complex_assembly +CP B-complex_assembly +is O +the O +major O +non B-protein_type +- I-protein_type +lysosomal I-protein_type +protease I-protein_type +in O +eukaryotic B-taxonomy_domain +cells O +, O +and O +its O +assembly O +is O +highly O +organized O +. O + +By O +contrast O +, O +the O +prosegments B-structure_element +of O +β B-protein +subunits I-protein +are O +dispensable O +for O +archaeal B-taxonomy_domain +proteasome B-complex_assembly +assembly O +, O +at O +least O +when O +heterologously B-experimental_method +expressed I-experimental_method +in O +Escherichia B-species +coli I-species +. O + +Here O +we O +have O +described O +the O +atomic B-evidence +structures I-evidence +of O +various O +β5 B-mutant +- I-mutant +T1A I-mutant +mutants O +, O +which O +allowed O +for O +the O +first O +time O +visualization O +of O +the O +residual O +β5 B-protein +propeptide B-structure_element +. O + +From O +these O +data O +we O +conclude O +that O +only O +the O +positioning O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +and O +Thr1 B-residue_name_number +as O +well O +as O +the O +integrity O +of O +the O +proteasomal O +active B-site +site I-site +are O +required O +for O +autolysis B-ptm +. O + +The O +propeptide B-structure_element +needs O +some O +anchoring O +in O +the O +substrate B-site +- I-site +binding I-site +channel I-site +to O +properly O +position O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +), I-residue_name_number +but O +this O +seems O +to O +be O +independent O +of O +the O +orientation O +of O +residue O +(- B-residue_number +2 I-residue_number +). I-residue_number + +Lys33NH2 B-residue_name_number +is O +expected O +to O +act O +as O +the O +proton O +acceptor O +during O +autocatalytic B-ptm +removal I-ptm +of O +the O +propeptides B-structure_element +, O +as O +well O +as O +during O +substrate O +proteolysis O +, O +while O +Asp17Oδ B-residue_name_number +orients O +Lys33NH2 B-residue_name_number +and O +makes O +it O +more O +prone O +to O +protonation O +by O +raising O +its O +pKa O +( O +hydrogen B-bond_interaction +bond I-bond_interaction +distance O +: O +Lys33NH3 B-residue_name_number ++– O +Asp17Oδ B-residue_name_number +: O +2 O +. O +9 O +Å O +). O + +Furthermore O +, O +opening O +of O +the O +β O +- O +lactone O +compound O +omuralide B-chemical +by O +Thr1 B-residue_name_number +creates O +a O +C3 O +- O +hydroxyl O +group O +, O +whose O +proton O +originates O +from O +Thr1NH3 B-residue_name_number ++. O + +By O +acting O +as O +a O +proton O +donor O +during O +catalysis O +, O +the O +Thr1 B-residue_name_number +N O +terminus O +may O +also O +favour O +cleavage O +of O +substrate O +peptide O +bonds O +( O +Fig O +. O +3d O +). O + +During O +autolysis B-ptm +the O +Thr1 B-residue_name_number +N O +terminus O +is O +engaged O +in O +a O +hydroxyoxazolidine O +ring O +intermediate O +( O +Fig O +. O +3d O +), O +which O +is O +unstable O +and O +short O +- O +lived O +. O + +The O +mutation B-experimental_method +D166N B-mutant +lowers O +the O +pKa O +of O +Thr1N B-residue_name_number +, O +which O +is O +thus O +more O +likely O +to O +exist O +in O +the O +uncharged O +deprotonated O +state O +( O +Thr1NH2 B-residue_name_number +). O + +We O +also O +observed O +slightly O +lower O +affinity O +of O +the O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +yCP B-complex_assembly +for O +the O +Food O +and O +Drug O +Administration O +- O +approved O +proteasome B-complex_assembly +inhibitors O +bortezomib B-chemical +and O +carfilzomib B-chemical +. O + +Thr1 B-residue_name_number +is O +well O +anchored O +in O +the O +active B-site +site I-site +by O +hydrophobic B-bond_interaction +interactions I-bond_interaction +of O +its O +Cγ O +methyl O +group O +with O +Ala46 B-residue_name_number +( O +Cβ O +), O +Lys33 B-residue_name_number +( O +carbon O +side O +chain O +) O +and O +Thr3 B-residue_name_number +( O +Cγ O +). O + +In O +contrast O +to O +Thr1 B-residue_name_number +, O +the O +hydroxyl O +group O +of O +Ser1 B-residue_name_number +occupies O +the O +position O +of O +the O +Thr1 B-residue_name_number +methyl O +side O +chain O +in O +the O +WT B-protein_state +enzyme B-complex_assembly +, O +which O +requires O +its O +reorientation O +relative O +to O +the O +substrate O +to O +allow O +cleavage O +( O +Fig O +. O +4g O +, O +h O +). O + +Notably O +, O +in O +the O +threonine B-protein_type +aspartase I-protein_type +Taspase1 B-protein +, O +mutation B-experimental_method +of O +the O +active B-site +- I-site +site I-site +Thr234 B-residue_name_number +to O +Ser B-residue_name +also O +places O +the O +side O +chain O +in O +the O +position O +of O +the O +methyl O +group O +of O +Thr234 B-residue_name_number +in O +the O +WT B-protein_state +, O +thereby O +reducing O +catalytic O +activity O +. O + +Only O +the O +residues O +(- B-residue_range +5 I-residue_range +) I-residue_range +to I-residue_range +(- I-residue_range +1 I-residue_range +) I-residue_range +of O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +are O +displayed O +. O + +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +OH O +is O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +O O +(∼ O +2 O +. O +8 O +Å O +; O +black O +dashed O +line O +). O + +Mutations B-experimental_method +of O +residue O +(- B-residue_number +2 I-residue_number +) I-residue_number +and O +their O +influence O +on O +propeptide B-structure_element +conformation O +and O +autolysis B-ptm +. O + +( O +a O +) O +Hydrogen B-site +- I-site +bonding I-site +network I-site +at O +the O +mature B-protein_state +WT B-protein_state +β5 B-protein +proteasomal O +active B-site +site I-site +( O +dotted O +lines O +). O + +( O +c O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +WT B-protein_state +β5 B-protein +and O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +pp B-chemical +trans B-protein_state +mutant B-protein_state +active B-site +site I-site +. O + +Note O +, O +the O +strong O +interaction O +with O +the O +water B-chemical +molecule O +causes O +a O +minor O +shift O +of O +Thr1 B-residue_name_number +, O +while O +all O +other O +active B-site +- I-site +site I-site +residues I-site +remain O +in O +place O +. O + +Notably O +, O +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +does O +not O +occupy O +the O +S1 B-site +pocket I-site +formed O +by O +Met45 B-residue_name_number +, O +similar O +to O +what O +was O +observed O +for O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +mutant B-protein_state +. O + +( O +f O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +WT B-protein_state +β5 B-protein +and O +β5 B-mutant +- I-mutant +T1C I-mutant +mutant B-protein_state +active B-site +sites I-site +illustrates O +the O +different O +orientations O +of O +the O +hydroxyl O +group O +of O +Thr1 B-residue_name_number +and O +the O +thiol O +side O +chain O +of O +Cys1 B-residue_name_number +. O + +( O +g O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +WT B-protein_state +β5 B-protein +and O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +active B-site +sites I-site +reveals O +different O +orientations O +of O +the O +hydroxyl O +groups O +of O +Thr1 B-residue_name_number +and O +Ser1 B-residue_name_number +, O +respectively O +. O + +A O +recent O +survey O +of O +bromodomains B-structure_element +( O +BDs B-structure_element +) O +demonstrates O +that O +only O +one O +BD B-structure_element +associates O +very O +weakly O +with O +a O +crotonylated B-protein_state +peptide O +, O +however O +it O +binds O +more O +tightly O +to O +acetylated B-protein_state +peptides O +, O +inferring O +that O +bromodomains B-structure_element +do O +not O +possess O +physiologically O +relevant O +crotonyllysine B-residue_name +binding O +activity O +. O + +To O +elucidate O +the O +molecular O +basis O +for O +recognition O +of O +the O +H3K9cr B-protein_type +mark O +, O +we O +obtained O +a O +crystal B-evidence +structure I-evidence +of O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +in B-protein_state +complex I-protein_state +with I-protein_state +H3K9cr5 B-chemical +- I-chemical +13 I-chemical +( O +residues O +5 B-residue_range +– I-residue_range +13 I-residue_range +of O +H3 B-protein_type +) O +peptide O +( O +Fig O +. O +1 O +, O +Supplementary O +Results O +, O +Supplementary O +Fig O +. O +1 O +and O +Supplementary O +Table O +1 O +). O + +The O +H3K9cr B-protein_type +peptide O +lays O +in O +an O +extended B-protein_state +conformation I-protein_state +in O +an O +orientation O +orthogonal O +to O +the O +β B-structure_element +strands I-structure_element +and O +is O +stabilized O +through O +an O +extensive O +network O +of O +direct O +and O +water B-chemical +- O +mediated O +hydrogen B-bond_interaction +bonds I-bond_interaction +and O +a O +salt B-bond_interaction +bridge I-bond_interaction +( O +Fig O +. O +1c O +). O + +The O +hydroxyl O +group O +of O +Thr61 B-residue_name_number +also O +participates O +in O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +amide O +nitrogen O +of O +the O +K9cr B-ptm +side O +chain O +( O +Fig O +. O +1d O +). O + +To O +determine O +whether O +H3K9cr B-protein_type +is O +present O +in O +yeast B-taxonomy_domain +, O +we O +generated O +whole B-experimental_method +cell I-experimental_method +extracts I-experimental_method +from O +logarithmically O +growing O +yeast B-taxonomy_domain +cells O +and O +subjected O +them O +to O +Western B-experimental_method +blot I-experimental_method +analysis I-experimental_method +using O +antibodies O +directed O +towards O +H3K9cr B-protein_type +, O +H3K9ac B-protein_type +and O +H3 B-protein_type +( O +Fig O +. O +2a O +, O +b O +, O +Supplementary O +Fig O +. O +3 O +and O +Supplementary O +Table O +2 O +). O + +We O +next O +asked O +if O +H3K9cr B-protein_type +is O +regulated O +by O +the O +actions O +of O +histone B-protein_type +acetyltransferases I-protein_type +( O +HATs B-protein_type +) O +and O +histone B-protein_type +deacetylases I-protein_type +( O +HDACs B-protein_type +). O + +Unlike O +the O +YEATS B-structure_element +domain I-structure_element +, O +a O +known O +acetyllysine B-protein_type +reader I-protein_type +, O +bromodomain B-structure_element +, O +does O +not O +recognize O +crotonyllysine B-residue_name +. O + +The O +unique O +and O +previously O +unobserved O +aromatic O +- O +amide O +/ O +aliphatic O +- O +aromatic O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +mechanism O +facilitates O +the O +specific O +recognition O +of O +the O +crotonyl B-chemical +moiety O +. O + +( O +d O +) O +The O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +π I-bond_interaction +stacking I-bond_interaction +mechanism O +involving O +the O +alkene O +moiety O +of O +crotonyllysine B-residue_name +. O + +H3K9cr B-protein_type +is O +a O +selective O +target O +of O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element + +( O +a O +, O +b O +) O +Western B-experimental_method +blot I-experimental_method +analysis O +comparing O +the O +levels O +of O +H3K9cr B-protein_type +and O +H3K9ac B-protein_type +in O +wild B-protein_state +type I-protein_state +( O +WT B-protein_state +), O +HAT B-protein_type +deletion O +, O +or O +HDAC B-protein_type +deletion B-experimental_method +yeast B-taxonomy_domain +strains O +. O + +Structure B-evidence +of O +the O +GAT B-structure_element +domain O +of O +the O +endosomal O +adapter B-protein_type +protein I-protein_type +Tom1 B-protein + +Analyzed O +by O +CS B-experimental_method +- I-experimental_method +Rosetta I-experimental_method +, O +Protein B-experimental_method +Structure I-experimental_method +Validation I-experimental_method +Server I-experimental_method +( O +PSVS B-experimental_method +), O +NMRPipe B-experimental_method +, O +NMRDraw B-experimental_method +, O +and O +PyMol O +Experimental O +factors O +Recombinant O +human B-species +Tom1 B-protein +GAT B-structure_element +domain O +was O +purified O +to O +homogeneity O +before O +use O +Experimental O +features O +Solution B-evidence +structure I-evidence +of O +Tom1 B-protein +GAT B-structure_element +was O +determined O +from O +NMR B-experimental_method +chemical B-evidence +shift I-evidence +data O +Data O +source O +location O +Virginia O +and O +Colorado O +, O +United O +States O +. O + +The O +Tom1 B-protein +GAT B-structure_element +structural B-evidence +restraints I-evidence +yielded O +ten O +helical O +structures B-evidence +( O +Fig O +. O +2A O +, O +B O +) O +with O +a O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +RMSD B-evidence +) O +of O +0 O +. O +9 O +Å O +for O +backbone O +and O +1 O +. O +3 O +Å O +for O +all O +heavy O +atoms O +( O +Table O +1 O +) O +and O +estimated O +the O +presence O +of O +three O +helices O +spanning O +residues O +Q216 B-residue_range +- I-residue_range +E240 I-residue_range +( O +α B-structure_element +- I-structure_element +helix I-structure_element +1 I-structure_element +), O +P248 B-residue_range +- I-residue_range +Q274 I-residue_range +( O +α B-structure_element +- I-structure_element +helix I-structure_element +2 I-structure_element +), O +and O +E278 B-residue_range +- I-residue_range +T306 I-residue_range +( O +α B-structure_element +- I-structure_element +helix I-structure_element +3 I-structure_element +). O + +Unlike O +ubiquitin B-chemical +binding O +, O +data O +suggest O +that O +conformational O +changes O +of O +the O +Tom1 B-protein +GAT B-structure_element +α B-structure_element +- I-structure_element +helices I-structure_element +1 I-structure_element +and I-structure_element +2 I-structure_element +occur O +upon O +Tollip B-protein +TBD B-structure_element +binding O +( O +Fig O +. O +3A O +, O +B O +). O + +NMR B-experimental_method +structural B-evidence +statistics I-evidence +for O +lowest O +energy O +conformers O +of O +Tom1 B-protein +GAT B-structure_element +using O +PSVS B-experimental_method +. O + +The O +dimer B-oligomeric_state +is O +dissociated O +to O +monomers B-oligomeric_state +by O +physiological O +levels O +of O +CO B-chemical +, O +suggesting O +that O +PGRMC1 B-protein +serves O +as O +a O +CO B-chemical +- O +sensitive O +molecular O +switch O +regulating O +cancer O +cell O +proliferation O +. O + +While O +the O +overall O +fold O +of O +PGRMC1 B-protein +is O +similar O +to O +that O +of O +canonical O +cytochrome B-protein_type +b5 I-protein_type +, O +their O +haem B-chemical +irons O +are O +coordinated O +differently O +. O + +Our O +structural B-evidence +data I-evidence +revealed O +that O +Tyr164 B-residue_name_number +and O +a O +few O +other O +residues O +such O +as O +Tyr107 B-residue_name_number +and O +Lys163 B-residue_name_number +are O +in O +fact O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +haem B-chemical +propionates O +. O + +When O +chemical B-evidence +shifts I-evidence +of O +apo B-protein_state +- O +and O +haem B-protein_state +- I-protein_state +bound I-protein_state +forms O +of O +PGMRC1 B-protein +were O +compared O +, O +some O +amino O +acid O +residues O +close O +to O +those O +which O +disappeared O +because O +of O +the O +paramagnetic O +relaxation O +effect O +of O +haem B-chemical +exhibit O +notable O +chemical O +shifts O +( O +Supplementary O +Fig O +. O +6a O +, O +b O +; O +dark O +yellow O +). O + +Similarly O +, O +the O +C129S B-mutant +mutant B-protein_state +of O +PGRMC1 B-protein +converted O +from O +monomer B-oligomeric_state +to O +dimer B-oligomeric_state +by O +binding O +to O +haem B-chemical +( O +Fig O +. O +2b O +). O + +SV B-experimental_method +- I-experimental_method +AUC I-experimental_method +analyses O +also O +allowed O +us O +to O +examine O +the O +stability O +of O +haem B-chemical +/ O +PGRMC1 B-protein +dimer B-oligomeric_state +. O + +To O +this O +end O +, O +we O +used O +different O +concentrations O +( O +3 O +. O +5 O +– O +147 O +μmol O +l O +− O +1 O +) O +of O +haem B-protein_state +- I-protein_state +bound I-protein_state +PGRMC1 B-protein +protein O +( O +a O +. O +a O +. O +72 B-residue_range +– I-residue_range +195 I-residue_range +), O +which O +were O +identical O +to O +that O +used O +in O +the O +crystallographic B-experimental_method +analysis I-experimental_method +. O + +We O +also O +showed O +by O +haem B-experimental_method +titration I-experimental_method +experiments I-experimental_method +that O +haem B-chemical +binding O +to O +PGRMC1 B-protein +was O +of O +low O +affinity O +with O +a O +Kd B-evidence +value O +of O +50 O +nmol O +l O +− O +1 O +; O +this O +is O +comparable O +with O +that O +of O +iron B-protein +regulatory I-protein +protein I-protein +2 I-protein +, O +which O +is O +known O +to O +be O +regulated O +by O +intracellular O +levels O +of O +haem B-chemical +( O +Fig O +. O +2c O +and O +Supplementary O +Table O +1 O +). O + +Under O +these O +circumstances O +, O +CO B-chemical +application O +induced O +dissociation O +of O +the O +haem B-chemical +- O +mediated O +dimers B-oligomeric_state +of O +PGRMC1 B-protein +to O +generate O +a O +peak O +of O +monomers B-oligomeric_state +( O +Supplementary O +Fig O +. O +15 O +, O +lower O +panel O +). O + +This O +interaction O +was O +disrupted O +by O +the O +ruthenium B-chemical +- O +based O +CO B-chemical +- O +releasing O +molecule O +, O +CORM3 B-chemical +, O +but O +not O +by O +RuCl3 B-chemical +as O +a O +control O +reagent O +( O +Fig O +. O +4b O +). O + +To O +further O +investigate O +the O +role O +of O +the O +dimerized B-protein_state +form O +of O +PGRMC1 B-protein +in O +cancer O +proliferation O +, O +we O +performed O +PGRMC1 B-protein +knockdown B-experimental_method +- I-experimental_method +rescue I-experimental_method +experiments I-experimental_method +using O +FLAG B-protein_state +- I-protein_state +tagged I-protein_state +wild B-protein_state +- I-protein_state +type I-protein_state +and O +Y113F B-mutant +PGRMC1 B-protein +expression B-experimental_method +vectors I-experimental_method +, O +in O +which O +silent B-experimental_method +mutations I-experimental_method +were O +introduced B-experimental_method +into O +the O +nucleotide O +sequence O +targeted O +by O +shRNA B-chemical +( O +Fig O +. O +5a O +). O + +While O +proliferation O +of O +HCT116 O +cells O +was O +not O +affected O +by O +knocking B-experimental_method +down I-experimental_method +PGRMC1 B-protein +, O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +were O +more O +sensitive O +to O +the O +EGFR B-protein_type +inhibitor O +erlotinib B-chemical +than O +control O +HCT116 O +cells O +, O +and O +the O +knockdown O +effect O +was O +reversed O +by O +co B-experimental_method +- I-experimental_method +expression I-experimental_method +of O +shRNA B-protein_state +- I-protein_state +resistant I-protein_state +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +but O +not O +of O +the O +Y113F B-mutant +mutant B-protein_state +( O +Fig O +. O +5b O +). O + +Interaction O +of O +PGRMC1 B-protein +dimer B-oligomeric_state +with O +cytochromes B-protein_type +P450 I-protein_type + +We O +showed O +that O +the O +haem B-chemical +- O +mediated O +dimer B-oligomeric_state +of O +PGRMC1 B-protein +enables O +interaction O +with O +different O +subclasses O +of O +cytochromes B-protein_type +P450 I-protein_type +( O +CYP B-protein_type +) O +( O +Fig O +. O +6 O +). O + +Thus O +, O +HO B-protein +- I-protein +1 I-protein +induction O +in O +cancer O +cells O +may O +inhibit O +the O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +through O +the O +production O +of O +CO B-chemical +and O +thereby O +suppress O +tumour O +progression O +. O + +( O +b O +) O +Haem B-chemical +coordination B-bond_interaction +of O +PGRMC1 B-protein +with O +Tyr113 B-residue_name_number +. O + +( O +a O +) O +UV B-evidence +- I-evidence +visible I-evidence +absorption I-evidence +spectra I-evidence +of O +PGRMC1 B-protein +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +). O + +Measurements O +were O +performed O +in O +the O +presence B-protein_state +of I-protein_state +the O +oxidized B-protein_state +form O +of O +haem B-chemical +( O +ferric B-protein_state +), O +the O +reduced B-protein_state +form O +of O +haem B-chemical +( O +ferrous B-protein_state +) O +and O +the O +reduced B-protein_state +form O +of O +haem B-chemical +plus O +CO B-chemical +gas O +( O +ferrous B-protein_state ++ O +CO B-chemical +). O + +( O +c O +) O +Gel B-experimental_method +- I-experimental_method +filtration I-experimental_method +chromatography I-experimental_method +analyses O +of O +PGRMC1 B-protein +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +) O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +wt B-protein_state +) O +and O +the O +Y113F B-mutant +or O +C129S B-mutant +mutant B-protein_state +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +haem B-chemical +, O +dithionite B-chemical +and O +/ O +or O +CO B-chemical +. O +( O +d O +) O +Transition O +model O +for O +structural O +regulation O +of O +PGRMC1 B-protein +in O +response O +to O +haem B-chemical +and O +CO B-chemical +. O + +Input O +and O +bound O +proteins O +were O +detected O +by O +Western B-experimental_method +blotting I-experimental_method +. O + +( O +b O +) O +In B-experimental_method +vitro I-experimental_method +binding I-experimental_method +assay I-experimental_method +was O +performed O +as O +in O +( O +a O +) O +using O +haem B-protein_state +- I-protein_state +bound I-protein_state +FLAG O +- O +PGRMC1 B-protein +wt B-protein_state +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +) O +and O +purified O +EGFR B-protein_type +with O +or O +without O +treatment O +of O +RuCl3 B-chemical +and O +CORM3 B-chemical +. O + +Co B-experimental_method +- I-experimental_method +immunoprecipitated I-experimental_method +proteins O +( O +FLAG O +- O +PGRMC1 B-protein +, O +endogenous B-protein_state +PGRMC1 B-protein +and O +EGFR B-protein_type +) O +were O +detected O +with O +Western B-experimental_method +blotting I-experimental_method +by O +using O +anti O +- O +PGRMC1 B-protein +or O +anti O +- O +EGFR B-protein_type +antibody O +. O + +( O +f O +) O +HCT116 O +cells O +expressing O +control O +shRNA B-chemical +or O +those O +knocking B-experimental_method +down I-experimental_method +PGRMC1 B-protein +( O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +) O +were O +treated O +with O +EGF B-protein_type +or O +left O +untreated O +, O +and O +components O +of O +the O +EGFR B-protein_type +signaling O +pathway O +were O +detected O +by O +Western B-experimental_method +blotting I-experimental_method +. O + +Haem B-chemical +- O +dependent O +PGRMC1 B-protein +dimerization B-oligomeric_state +enhances O +tumour O +chemoresistance O +through O +interaction O +with O +cytochromes B-protein_type +P450 I-protein_type +. O + +Doxorubicin B-chemical +was O +incubated B-experimental_method +with O +HCT116 O +cells O +expressing O +control O +shRNA B-chemical +or O +shPGRMC1 B-chemical +( O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +), O +and O +the O +doxorubicinol B-chemical +/ O +doxorubicin B-chemical +ratios O +in O +cell O +pellets O +were O +determined O +using O +LC B-experimental_method +- I-experimental_method +MS I-experimental_method +. O + +of O +four O +separate O +experiments O +. O +** O +P B-evidence +< O +0 O +. O +01 O +versus O +control O +using O +unpaired O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +- I-experimental_method +test I-experimental_method +. O +( O +e O +) O +Indicated O +amounts O +of O +doxorubicin B-chemical +were O +added O +to O +HCT116 O +( O +control O +) O +cells O +, O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +, O +or O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +expressing O +shRNA B-protein_state +- I-protein_state +resistant I-protein_state +full B-protein_state +- I-protein_state +length I-protein_state +PGRMC1 B-protein +wt B-protein_state +or O +Y113F B-mutant +, O +and O +cell O +viability O +was O +examined O +by O +MTT B-experimental_method +assay I-experimental_method +. O + +PGRMC1 O +proteins O +exhibit O +haem O +- O +dependent O +dimerization B-oligomeric_state +in O +solution O +. O + +T O +cells O +perform O +an O +essential O +role O +in O +adaptive O +immunity O +by O +interrogating O +the O +host O +proteome O +for O +anomalies O +, O +classically O +by O +recognizing O +peptides O +bound O +in O +major B-complex_assembly +histocompatibility I-complex_assembly +( O +MHC B-complex_assembly +) O +molecules O +at O +the O +cell O +surface O +. O + +Structures B-evidence +of O +unligated B-protein_state +and O +ligated B-protein_state +TCRs B-complex_assembly +have O +shown O +that O +the O +TCR B-complex_assembly +complementarity B-structure_element +determining I-structure_element +region I-structure_element +( O +CDR B-structure_element +) O +loops B-structure_element +can O +be O +flexible O +, O +perhaps O +enabling O +peptide O +binding O +using O +different O +loop B-structure_element +conformations O +. O + +Here O +, O +the O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +tetramer B-oligomeric_state +stained O +1E6 O +with O +the O +greatest O +MFI O +, O +gradually O +decreasing O +to O +the O +weakest O +tetramers B-oligomeric_state +: O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +and O +- O +YLGGPDFPTI B-chemical +. O + +The O +range O +of O +Tm B-evidence +was O +between O +49 O +. O +4 O +° O +C O +( O +RQFGPDWIVA B-chemical +) O +and O +60 O +. O +3 O +° O +C O +( O +YQFGPDFPIA B-chemical +), O +with O +an O +average O +approximately O +55 O +° O +C O +, O +similar O +to O +our O +previous O +findings O +. O + +In O +accordance O +with O +this O +trend O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +bound B-protein_state +the O +natural O +preproinsulin B-protein +peptide O +, O +ALWGPDPAAA B-chemical +, O +with O +the O +weakest O +affinity B-evidence +currently O +published O +for O +a O +human B-species +CD8 O ++ O +T O +cell O +– O +derived O +TCR B-complex_assembly +with O +a O +biologically O +relevant O +ligand O +( O +KD B-evidence +> O +200 O +μM O +; O +KD B-evidence +, O +equilibrium B-evidence +binding I-evidence +constant I-evidence +). O + +Surface B-experimental_method +plasmon I-experimental_method +resonance I-experimental_method +( O +SPR B-experimental_method +) O +analysis O +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +– O +pMHC B-complex_assembly +interaction O +for O +all O +7 O +APLs B-chemical +( O +Figure O +2 O +, O +A O +– O +H O +) O +demonstrated O +that O +stronger O +binding B-evidence +affinity I-evidence +( O +represented O +as O +ΔG B-evidence +°, I-evidence +kcal O +/ O +mol O +) O +correlated O +well O +with O +the O +EC50 B-evidence +values O +( O +peptide O +concentration O +required O +to O +reach O +half O +- O +maximal O +1E6 O +T O +cell O +killing O +) O +for O +each O +ligand O +, O +demonstrated O +by O +a O +Pearson B-experimental_method +’ I-experimental_method +s I-experimental_method +correlation I-experimental_method +analysis I-experimental_method +value O +of O +0 O +. O +8 O +( O +P O += O +0 O +. O +01 O +) O +( O +Figure O +2I O +). O + +It O +should O +be O +noted O +that O +this O +correlation O +, O +although O +consistent O +with O +the O +T O +cell O +killing O +experiments O +, O +uses O +only O +approximate O +affinities B-evidence +calculated O +for O +the O +2 O +weakest O +ligands O +. O + +Finally O +, O +these O +data O +demonstrate O +the O +largest O +range O +of O +binding B-evidence +affinities I-evidence +reported O +for O +a O +natural O +, O +endogenous B-protein_state +human B-species +TCR B-complex_assembly +of O +more O +than O +3 O +logs O +of O +magnitude O +( O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +vs O +. O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +). O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +uses O +a O +consensus O +binding O +mode O +to O +engage O +multiple O +APLs B-chemical +. O + +Our O +previous O +structure B-evidence +of O +the O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +ALWGPDPAAA I-complex_assembly +complex O +demonstrated O +a O +limited O +binding B-site +footprint I-site +between O +the O +TCR B-complex_assembly +and O +pMHC B-complex_assembly +. O + +The O +surface B-evidence +complementarity I-evidence +values I-evidence +( O +0 O +. O +52 O +– O +0 O +. O +7 O +) O +correlated O +slightly O +with O +affinity B-evidence +( O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence += O +0 O +. O +7 O +, O +P B-evidence += O +0 O +. O +05 O +) O +but O +could O +not O +explain O +all O +differences O +in O +binding O +( O +Figure O +3A O +and O +Table O +2 O +). O + +The O +TCR B-complex_assembly +CDR B-structure_element +loops I-structure_element +were O +in O +a O +very O +similar O +position O +in O +all O +complexes O +, O +apart O +from O +some O +slight O +deviations O +in O +the O +TCR B-complex_assembly +β B-structure_element +- I-structure_element +chain I-structure_element +( O +Figure O +3B O +); O +the O +peptides O +were O +all O +presented O +in O +a O +similar O +conformation O +( O +Figure O +3C O +); O +and O +there O +was O +minimal O +variation O +in O +crossing O +angles O +of O +the O +TCR B-complex_assembly +( O +42 O +. O +3 O +°– O +45 O +. O +6 O +°) O +( O +Figure O +3D O +). O + +Focused O +hotspot O +binding O +around O +a O +conserved B-protein_state +GPD B-structure_element +motif I-structure_element +enables O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +to O +tolerate O +peptide O +degeneracy O +. O + +In O +addition O +to O +changes O +between O +the O +TCR B-complex_assembly +and O +peptide O +component O +, O +we O +also O +observed O +that O +different O +APLs B-chemical +had O +different O +knock O +- O +on O +effects O +between O +the O +TCR B-complex_assembly +and O +MHC B-complex_assembly +. O + +Generally O +, O +the O +weaker O +- O +affinity B-evidence +APLs B-chemical +made O +fewer O +contacts O +with O +the O +MHC B-complex_assembly +surface O +( O +27 O +– O +29 O +interactions O +) O +compared O +with O +the O +stronger O +- O +affinity B-evidence +APLs B-chemical +( O +29 O +– O +35 O +contacts O +), O +consistent O +with O +a O +better O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence +value I-evidence +( O +0 O +. O +55 O +) O +compared O +with O +TCR B-complex_assembly +- O +peptide O +interactions O +versus O +affinity B-evidence +( O +0 O +. O +045 O +). O + +For O +example O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +bound B-protein_state +to I-protein_state +A2 B-chemical +- I-chemical +RQWGPDPAAV I-chemical +with O +the O +third O +strongest O +affinity B-evidence +( O +KD B-evidence += O +7 O +. O +8 O +μM O +) O +but O +made O +fewer O +contacts O +than O +with O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +( O +KD B-evidence += O +~ O +208 O +μM O +) O +( O +Table O +2 O +). O + +Thus O +, O +we O +performed O +an O +in O +- O +depth O +thermodynamic B-experimental_method +analysis I-experimental_method +of O +6 O +of O +the O +ligands O +under O +investigation O +( O +Figure O +8 O +and O +Supplemental O +Table O +3 O +). O + +However O +, O +there O +was O +a O +clear O +switch O +in O +entropy B-evidence +between O +the O +weaker O +- O +affinity B-evidence +and O +stronger O +- O +affinity B-evidence +ligands O +, O +indicated O +by O +a O +strong O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence +value I-evidence +between O +entropy B-evidence +and O +affinity B-evidence +( O +Pearson B-evidence +’ I-evidence +s I-evidence +correlation I-evidence +value I-evidence +0 O +. O +93 O +, O +P B-evidence += O +0 O +. O +007 O +). O + +Conversely O +, O +the O +stronger O +- O +affinity B-evidence +ligands O +A2 B-chemical +- I-chemical +RQWGPDPAAV I-chemical +( O +KD B-evidence += O +7 O +. O +8 O +μM O +), O +A2 B-chemical +- I-chemical +YQFGPDFPIA I-chemical +( O +KD B-evidence += O +7 O +. O +4 O +μM O +), O +and O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +( O +KD B-evidence += O +0 O +. O +5 O +μM O +) O +exhibited O +favorable O +entropy B-evidence +( O +TΔS B-evidence +° I-evidence += O +2 O +. O +2 O +to O +14 O +. O +9 O +kcal O +/ O +mol O +), O +indicating O +an O +order O +- O +to O +- O +disorder O +change O +during O +binding O +, O +possibly O +through O +the O +expulsion O +of O +ordered O +water O +molecules O +. O + +The O +binding B-evidence +affinity I-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +interaction O +with O +A2 B-chemical +- I-chemical +RQFGPDWIVA I-chemical +is O +considerably O +higher O +than O +with O +the O +disease O +- O +implicated O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +sequence O +( O +KD B-evidence += O +44 O +. O +4 O +μM O +and O +KD B-evidence +> O +200 O +μM O +, O +respectively O +), O +highlighting O +how O +a O +pathogen O +- O +derived O +sequence O +might O +be O +capable O +of O +priming O +a O +1E6 O +- O +like O +T O +cell O +. O + +Focused O +binding O +around O +a O +minimal O +peptide O +motif O +has O +also O +been O +implicated O +as O +an O +alternative O +mechanism O +enabling O +TCR B-complex_assembly +cross O +- O +reactivity O +. O + +Ligand O +engagement O +is O +dominated O +by O +peptide O +interactions O +, O +but O +hotspot O +- O +like O +interactions O +with O +the O +central O +GPD B-structure_element +motif I-structure_element +enable O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +to O +tolerate O +peptide O +residues O +that O +vary O +outside O +of O +this O +region O +, O +explaining O +how O +T O +cells O +expressing O +this O +TCR B-complex_assembly +may O +cross O +- O +react O +with O +a O +large O +number O +of O +different O +peptides O +. O + +These O +data O +also O +explain O +our O +previous O +findings O +that O +alteration O +of O +the O +anchor B-structure_element +residue I-structure_element +at O +peptide O +position O +2 B-residue_number +( O +Leu B-mutant +- I-mutant +Gln I-mutant +) O +has O +a O +direct O +effect O +on O +1E6 B-evidence +TCR I-evidence +binding I-evidence +affinity I-evidence +because O +our O +structural B-experimental_method +analysis I-experimental_method +demonstrated O +that O +1E6 O +made O +3 O +additional O +bonds O +with O +A2 B-chemical +- I-chemical +AQWGPDPAAA I-chemical +compared O +with O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +, O +consistent O +with O +the O +> O +3 O +- O +fold O +stronger O +binding B-evidence +affinity I-evidence +. O + +Further O +experiments O +will O +be O +required O +to O +determine O +whether O +any O +naturally O +presented O +, O +human B-species +pathogen O +– O +derived O +peptides O +act O +as O +active O +ligands O +for O +1E6 O +, O +but O +our O +work O +presented O +here O +demonstrates O +that O +it O +is O +at O +least O +feasible O +for O +an O +autoimmune O +TCR B-complex_assembly +to O +bind O +to O +a O +different O +peptide O +sequence O +that O +could O +be O +present O +in O +a O +pathogen O +proteome O +with O +substantially O +higher O +affinity B-evidence +and O +potency O +than O +the O +interaction O +it O +might O +use O +to O +attack O +self O +- O +tissue O +. O + +3D B-experimental_method +and I-experimental_method +2D I-experimental_method +binding I-experimental_method +analysis I-experimental_method +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +with O +A2 B-chemical +- I-chemical +ALW I-chemical +and O +the O +APLs B-chemical +. O + +The O +equilibrium B-evidence +binding I-evidence +constant I-evidence +( O +KD B-evidence +) O +values O +were O +calculated O +using O +a O +nonlinear B-experimental_method +curve I-experimental_method +fit I-experimental_method +( O +y O += O +[ O +P1x O +]/[ O +P2 O ++ O +X O +]). O + +All O +unligated B-protein_state +pMHCs B-complex_assembly +are O +shown O +as O +light O +green O +illustrations O +. O + +Boxes O +show O +total O +contacts O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +these O +key O +residues O +( O +green O +boxes O +are O +MHC B-complex_assembly +residues O +; O +white O +boxes O +are O +TCR B-complex_assembly +residues O +). O + +We O +determined B-experimental_method +the O +crystal B-evidence +structure I-evidence +of O +an O +oligomerization B-protein_state +- I-protein_state +impaired I-protein_state +Irga6 B-protein +mutant B-protein_state +bound B-protein_state +to I-protein_state +a O +non O +- O +hydrolyzable O +GTP B-chemical +analog O +. O + +This O +study O +contributes O +important O +insights O +into O +the O +assembly O +and O +catalytic O +mechanisms O +of O +IRG B-protein_type +proteins O +as O +prerequisite O +to O +understand O +their O +anti O +- O +microbial O +action O +. O + +Mutagenesis B-experimental_method +of O +the O +contact B-site +surfaces I-site +suggests O +that O +this O +"""" O +backside B-site +"""" O +interface B-site +is O +not O +required O +for O +GTP B-chemical +- O +dependent O +oligomerization O +or O +cooperative O +hydrolysis O +, O +despite O +an O +earlier O +suggestion O +to O +the O +contrary O +. O + +For O +several O +of O +these O +proteins O +, O +formation O +of O +the O +G B-site +interface I-site +was O +shown O +to O +trigger O +GTP B-chemical +hydrolysis O +by O +inducing O +rearrangements O +of O +catalytic O +residues O +in O +cis O +. O + +However O +, O +the O +crystal B-evidence +structure I-evidence +of O +Irga6 B-protein +in O +the O +presence B-protein_state +of I-protein_state +the O +non O +- O +hydrolyzable O +GTP B-chemical +analogue O +5 B-chemical +'- I-chemical +guanylyl I-chemical +imidodiphosphate I-chemical +( O +GMPPNP B-chemical +) O +showed O +only O +subtle O +differences O +relative O +to O +the O +apo B-protein_state +or O +GDP B-protein_state +- I-protein_state +bound I-protein_state +protein O +and O +did O +not O +reveal O +a O +new O +dimer B-site +interface I-site +associated O +with O +the O +GTPase B-structure_element +domain I-structure_element +. O + +Structure B-evidence +of O +the O +Irga6 B-protein +dimer B-oligomeric_state +. O + +b O +Ribbon O +- O +type O +representation O +of O +the O +Irga6 B-protein +dimer B-oligomeric_state +. O + +Irga6 B-protein +immunity B-protein +- I-protein +related I-protein +GTPase I-protein +6 I-protein + +Within O +the O +asymmetric O +unit O +, O +six O +molecules O +dimerize B-oligomeric_state +via O +the O +symmetric O +backside B-site +dimer I-site +interface I-site +( O +buried O +surface O +area O +930 O +Å2 O +), O +and O +the O +remaining O +seventh O +molecule O +forms O +the O +same O +type O +of O +interaction O +with O +its O +symmetry O +mate O +of O +the O +adjacent O +asymmetric O +unit O +( O +Additional O +file O +1 O +: O +Figure O +S2a O +, O +b O +, O +Figure O +S3 O +). O + +In O +turn O +, O +E106 B-residue_name_number +of O +switch B-site +I I-site +reorients O +towards O +the O +nucleotide B-chemical +and O +now O +participates O +in O +the O +coordination B-bond_interaction +of I-bond_interaction +the O +Mg2 B-chemical ++ I-chemical +ion O +( O +Fig O +. O +1e O +, O +Additional O +file O +1 O +: O +Figure O +S4 O +). O + +The O +following O +structures B-evidence +are O +shown O +in O +cylinder O +representations O +, O +in O +similar O +orientations O +of O +their O +GTPase B-structure_element +domains I-structure_element +: O +a O +the O +GMPPNP B-protein_state +- I-protein_state +bound I-protein_state +Irga6 B-protein +dimer B-oligomeric_state +, O +b O +the O +GDP O +- O +AlF4 O +-- O +bound O +dynamin B-protein +1 I-protein +GTPase B-structure_element +- I-structure_element +minimal I-structure_element +BSE O +construct O +[ O +pdb O +2X2E O +], O +c O +the O +GDP B-protein_state +- I-protein_state +bound I-protein_state +atlastin B-protein +1 I-protein +dimer B-oligomeric_state +[ O +pdb O +3Q5E O +], O +d O +the O +GDP B-protein_state +- I-protein_state +AlF3 I-protein_state +- I-protein_state +bound I-protein_state +GBP1 B-protein +GTPase B-structure_element +domain I-structure_element +dimer B-oligomeric_state +[ O +pdb O +2B92 O +], O +e O +the O +BDLP B-protein_type +dimer B-oligomeric_state +bound B-protein_state +to I-protein_state +GDP B-chemical +[ O +pdb O +2J68 O +] O +and O +f O +the O +GTP B-protein_state +- I-protein_state +bound I-protein_state +GIMAP2 B-protein +dimer B-oligomeric_state +[ O +pdb O +2XTN O +]. O + +Nucleotide O +, O +Mg2 B-chemical ++ I-chemical +( O +green O +) O +and O +AlF4 O +- O +are O +shown O +in O +sphere O +representation O +, O +the O +buried O +interface B-site +sizes O +per O +molecule O +are O +indicated O +on O +the O +right O +. O + +For O +example O +, O +for O +dynamin B-protein_type +and O +atlastin B-protein_type +, O +it O +was O +shown O +that O +GTP B-chemical +binding O +and O +/ O +or O +hydrolysis O +leads O +to O +dimerization O +of O +the O +GTPase B-structure_element +domains I-structure_element +and O +to O +the O +reorientation O +of O +the O +adjacent O +helical B-structure_element +domains I-structure_element +. O + +Only O +one O +of O +the O +seven O +Irga6 B-protein +molecules O +in O +the O +asymmetric O +unit O +formed O +this O +contact O +pointing O +to O +a O +low O +affinity O +interaction O +via O +the O +G B-site +interface I-site +, O +which O +is O +in O +agreement O +with O +its O +small O +size O +. O + +Based O +on O +phylogenetic B-experimental_method +and I-experimental_method +structural I-experimental_method +analysis I-experimental_method +, O +these O +observations O +suggest O +that O +dynamin B-protein_type +and O +septin B-protein_type +superfamilies O +are O +derived O +from O +a O +common O +ancestral O +membrane B-protein_type +- I-protein_type +associated I-protein_type +GTPase I-protein_type +that O +featured O +a O +GTP B-chemical +- O +dependent O +parallel B-protein_state +dimerization O +mode O +. O + +During O +dimerization O +of O +GBP1 B-protein +, O +an O +arginine B-structure_element +finger I-structure_element +from O +the O +P B-structure_element +loop I-structure_element +reorients O +towards O +the O +nucleotide B-chemical +in O +cis O +to O +trigger O +GTP B-chemical +hydrolysis O +. O + diff --git a/annotation_IOB/train.tsv b/annotation_IOB/train.tsv new file mode 100644 index 0000000000000000000000000000000000000000..9b479e284bbf2df59f3341703ec0f26b558f9995 --- /dev/null +++ b/annotation_IOB/train.tsv @@ -0,0 +1,183950 @@ +Molecular O +Dissection O +of O +Xyloglucan B-chemical +Recognition O +in O +a O +Prominent O +Human B-species +Gut O +Symbiont O + +Polysaccharide B-gene +utilization I-gene +loci I-gene +( O +PUL B-gene +) O +within O +the O +genomes O +of O +resident O +human B-species +gut O +Bacteroidetes B-taxonomy_domain +are O +central O +to O +the O +metabolism O +of O +the O +otherwise O +indigestible O +complex O +carbohydrates B-chemical +known O +as O +“ O +dietary O +fiber O +.” O +However O +, O +functional O +characterization O +of O +PUL B-gene +lags O +significantly O +behind O +sequencing O +efforts O +, O +which O +limits O +physiological O +understanding O +of O +the O +human B-species +- O +bacterial B-taxonomy_domain +symbiosis O +. O + +In O +particular O +, O +the O +molecular O +basis O +of O +complex B-chemical +polysaccharide I-chemical +recognition O +, O +an O +essential O +prerequisite O +to O +hydrolysis O +by O +cell O +surface O +glycosidases B-protein_type +and O +subsequent O +metabolism O +, O +is O +generally O +poorly O +understood O +. O + +Here O +, O +we O +present O +the O +biochemical B-experimental_method +, I-experimental_method +structural I-experimental_method +, I-experimental_method +and I-experimental_method +reverse I-experimental_method +genetic I-experimental_method +characterization I-experimental_method +of O +two O +unique O +cell B-protein_type +surface I-protein_type +glycan I-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +( O +SGBPs B-protein_type +) O +encoded O +by O +a O +xyloglucan B-gene +utilization I-gene +locus I-gene +( O +XyGUL B-gene +) O +from O +Bacteroides B-species +ovatus I-species +, O +which O +are O +integral O +to O +growth O +on O +this O +key O +dietary O +vegetable B-taxonomy_domain +polysaccharide B-chemical +. O + +Biochemical B-experimental_method +analysis I-experimental_method +reveals O +that O +these O +outer B-protein_type +membrane I-protein_type +- I-protein_type +anchored I-protein_type +proteins I-protein_type +are O +in O +fact O +exquisitely O +specific O +for O +the O +highly O +branched O +xyloglucan B-chemical +( O +XyG B-chemical +) O +polysaccharide B-chemical +. O + +The O +crystal B-evidence +structure I-evidence +of O +SGBP B-protein +- I-protein +A I-protein +, O +a O +SusD B-protein +homolog O +, O +with O +a O +bound B-protein_state +XyG B-chemical +tetradecasaccharide B-chemical +reveals O +an O +extended O +carbohydrate B-site +- I-site +binding I-site +platform I-site +that O +primarily O +relies O +on O +recognition O +of O +the O +β B-chemical +- I-chemical +glucan I-chemical +backbone O +. O + +The O +unique O +, O +tetra B-structure_element +- I-structure_element +modular I-structure_element +structure B-evidence +of O +SGBP B-protein +- I-protein +B I-protein +is O +comprised O +of O +tandem B-structure_element +Ig I-structure_element +- I-structure_element +like I-structure_element +folds I-structure_element +, O +with O +XyG B-chemical +binding O +mediated O +at O +the O +distal O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +. O + +Despite O +displaying O +similar O +affinities B-evidence +for O +XyG B-chemical +, O +reverse B-experimental_method +- I-experimental_method +genetic I-experimental_method +analysis I-experimental_method +reveals O +that O +SGBP B-protein +- I-protein +B I-protein +is O +only O +required O +for O +the O +efficient O +capture O +of O +smaller O +oligosaccharides B-chemical +, O +whereas O +the O +presence O +of O +SGBP B-protein +- I-protein +A I-protein +is O +more O +critical O +than O +its O +carbohydrate B-chemical +- O +binding O +ability O +for O +growth O +on O +XyG B-chemical +. O +Together O +, O +these O +data O +demonstrate O +that O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +play O +complementary O +, O +specialized O +roles O +in O +carbohydrate B-chemical +capture O +by O +B B-species +. I-species +ovatus I-species +and O +elaborate O +a O +model O +of O +how O +vegetable B-taxonomy_domain +xyloglucans B-chemical +are O +accessed O +by O +the O +Bacteroidetes B-taxonomy_domain +. O + +Our O +combined O +analysis O +illuminates O +new O +fundamental O +aspects O +of O +complex B-chemical +polysaccharide I-chemical +recognition O +, O +cleavage O +, O +and O +import O +at O +the O +Bacteroidetes B-taxonomy_domain +cell O +surface O +that O +may O +facilitate O +the O +development O +of O +prebiotics O +to O +target O +this O +phylum O +of O +gut O +bacteria B-taxonomy_domain +. O + +This O +microbial B-taxonomy_domain +community O +is O +largely O +bacterial B-taxonomy_domain +, O +with O +the O +Bacteroidetes B-taxonomy_domain +, O +Firmicutes B-taxonomy_domain +, O +and O +Actinobacteria B-taxonomy_domain +comprising O +the O +dominant O +phyla O +. O + +However O +, O +there O +is O +a O +paucity O +of O +data O +regarding O +how O +the O +vast O +array O +of O +complex B-chemical +carbohydrate I-chemical +structures O +are O +selectively O +recognized O +and O +imported O +by O +members O +of O +the O +microbiota B-taxonomy_domain +, O +a O +critical O +process O +that O +enables O +these O +organisms O +to O +thrive O +in O +the O +competitive O +gut O +environment O +. O + +The O +human B-species +gut O +bacteria B-taxonomy_domain +Bacteroidetes B-taxonomy_domain +share O +a O +profound O +capacity O +for O +dietary O +glycan B-chemical +degradation O +, O +with O +many O +species O +containing O +> O +250 O +predicted O +carbohydrate O +- O +active O +enzymes O +( O +CAZymes O +), O +compared O +to O +50 O +to O +100 O +within O +many O +Firmicutes B-taxonomy_domain +and O +only O +17 O +in O +the O +human B-species +genome O +devoted O +toward O +carbohydrate O +utilization O +. O + +A O +remarkable O +feature O +of O +the O +Bacteroidetes B-taxonomy_domain +is O +the O +packaging O +of O +genes O +for O +carbohydrate O +catabolism O +into O +discrete O +polysaccharide B-gene +utilization I-gene +loci I-gene +( O +PUL B-gene +), O +which O +are O +transcriptionally O +regulated O +by O +specific O +substrate O +signatures O +. O + +The O +Sus B-complex_assembly +includes O +a O +lipid B-protein_state +- I-protein_state +anchored I-protein_state +, O +outer O +membrane O +endo B-protein_type +- I-protein_type +amylase I-protein_type +, O +SusG B-protein +; O +a O +TonB B-protein_type +- I-protein_type +dependent I-protein_type +transporter I-protein_type +( O +TBDT B-protein_type +), O +SusC B-protein +, O +which O +imports O +oligosaccharides B-chemical +with O +the O +help O +of O +an O +associated O +starch B-protein_type +- I-protein_type +binding I-protein_type +protein I-protein_type +, O +SusD B-protein +; O +two O +additional O +carbohydrate B-protein_type +- I-protein_type +binding I-protein_type +lipoproteins I-protein_type +, O +SusE B-protein +and O +SusF B-protein +; O +and O +two O +periplasmic O +exo B-protein_type +- I-protein_type +glucosidases I-protein_type +, O +SusA B-protein +and O +SusB B-protein +, O +which O +generate O +glucose B-chemical +for O +transport O +into O +the O +cytoplasm O +. O + +The O +importance O +of O +PUL B-gene +as O +a O +successful O +evolutionary O +strategy O +is O +underscored O +by O +the O +observation O +that O +Bacteroidetes B-taxonomy_domain +such O +as O +B B-species +. I-species +thetaiotaomicron I-species +and O +Bacteroides B-species +ovatus I-species +devote O +~ O +18 O +% O +of O +their O +genomes O +to O +these O +systems O +. O + +Moving O +beyond O +seminal O +genomic O +and O +transcriptomic O +analyses O +, O +the O +current O +state O +- O +of O +- O +the O +- O +art O +PUL B-gene +characterization O +involves O +combined O +reverse B-experimental_method +- I-experimental_method +genetic I-experimental_method +, I-experimental_method +biochemical I-experimental_method +, I-experimental_method +and I-experimental_method +structural I-experimental_method +studies I-experimental_method +to O +illuminate O +the O +molecular O +details O +of O +PUL B-gene +function O +. O + +Cleavage O +sites O +for O +BoXyGUL B-gene +glycosidases B-protein_type +( O +GHs B-protein_type +) O +are O +indicated O +for O +solanaceous B-taxonomy_domain +xyloglucan B-chemical +. O +( O +B O +) O +BtSus B-gene +and O +BoXyGUL B-gene +. O +( O +C O +) O +Localization O +of O +BoXyGUL B-gene +- O +encoded O +proteins O +in O +cellular O +membranes O +and O +concerted O +modes O +of O +action O +in O +the O +degradation O +of O +xyloglucans B-chemical +to O +monosaccharides O +. O + +XyG B-chemical +variants O +( O +Fig O +. O +1A O +) O +constitute O +up O +to O +25 O +% O +of O +the O +dry O +weight O +of O +common O +vegetables B-taxonomy_domain +. O + +Analogous O +to O +the O +Sus B-gene +locus I-gene +, O +the O +xyloglucan B-gene +utilization I-gene +locus I-gene +( O +XyGUL B-gene +) O +encodes O +a O +cohort O +of O +carbohydrate B-protein_type +- I-protein_type +binding I-protein_type +, I-protein_type +- I-protein_type +hydrolyzing I-protein_type +, I-protein_type +and I-protein_type +- I-protein_type +importing I-protein_type +proteins I-protein_type +( O +Fig O +. O +1B O +and O +C O +). O + +The O +number O +of O +glycoside B-protein_type +hydrolases I-protein_type +( O +GHs B-protein_type +) O +encoded O +by O +the O +XyGUL B-gene +is O +, O +however O +, O +more O +expansive O +than O +that O +by O +the O +Sus B-gene +locus I-gene +( O +Fig O +. O +1B O +), O +which O +reflects O +the O +greater O +complexity O +of O +glycosidic O +linkages O +found O +in O +XyG B-chemical +vis O +- O +à O +- O +vis O +starch B-chemical +. O + +In O +the O +archetypal O +starch B-complex_assembly +utilization I-complex_assembly +system I-complex_assembly +of O +B B-species +. I-species +thetaiotaomicron I-species +, O +starch O +binding O +to O +the O +cell O +surface O +is O +mediated O +at O +eight O +distinct O +starch B-site +- I-site +binding I-site +sites I-site +distributed O +among O +four O +surface B-protein_type +glycan I-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +( O +SGBPs B-protein_type +): O +two O +within O +the O +amylase B-protein_type +SusG B-protein +, O +one O +within O +SusD B-protein +, O +two O +within O +SusE B-protein +, O +and O +three O +within O +SusF B-protein +. O +The O +functional O +redundancy O +of O +many O +of O +these O +sites O +is O +high O +: O +whereas O +SusD B-protein +is O +essential O +for O +growth O +on O +starch B-chemical +, O +combined O +mutations O +of O +the O +SusE B-protein +, O +SusF B-protein +, O +and O +SusG B-protein +binding B-site +sites I-site +are O +required O +to O +impair O +growth O +on O +the O +polysaccharide B-chemical +. O + +Bacteroidetes B-taxonomy_domain +PUL B-gene +ubiquitously O +encode O +homologs O +of O +SusC B-protein +and O +SusD B-protein +, O +as O +well O +as O +proteins O +whose O +genes O +are O +immediately O +downstream O +of O +susD B-gene +, O +akin O +to O +susE B-gene +/ I-gene +F I-gene +, O +and O +these O +are O +typically O +annotated O +as O +“ O +putative B-protein_state +lipoproteins B-protein_type +”. O + +The O +genes O +coding O +for O +these O +proteins O +, O +sometimes O +referred O +to O +as O +“ O +susE B-gene +/ I-gene +F I-gene +positioned O +,” O +display O +products O +with O +a O +wide O +variation O +in O +amino O +acid O +sequence O +and O +which O +have O +little O +or O +no O +homology O +to O +other O +PUL B-gene +- O +encoded O +proteins O +or O +known O +carbohydrate B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +. O + +We O +describe O +here O +the O +detailed O +functional B-experimental_method +and I-experimental_method +structural I-experimental_method +characterization I-experimental_method +of O +the O +noncatalytic B-protein_state +SGBPs B-protein_type +encoded O +by O +Bacova_02651 B-gene +and O +Bacova_02650 B-gene +of O +the O +XyGUL B-gene +, O +here O +referred O +to O +as O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +, O +to O +elucidate O +their O +molecular O +roles O +in O +carbohydrate O +acquisition O +in O +vivo O +. O + +Combined O +biochemical B-experimental_method +, I-experimental_method +structural I-experimental_method +, I-experimental_method +and I-experimental_method +reverse I-experimental_method +- I-experimental_method +genetic I-experimental_method +approaches I-experimental_method +clearly O +illuminate O +the O +distinct O +, O +yet O +complementary O +, O +functions O +that O +these O +two O +proteins O +play O +in O +XyG B-chemical +recognition O +as O +it O +impacts O +the O +physiology O +of O +B B-species +. I-species +ovatus I-species +. O + +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +are O +cell B-protein_type +- I-protein_type +surface I-protein_type +- I-protein_type +localized I-protein_type +, I-protein_type +xyloglucan I-protein_type +- I-protein_type +specific I-protein_type +binding I-protein_type +proteins I-protein_type +. O + +SGBP B-protein +- I-protein +A I-protein +, O +encoded O +by O +the O +XyGUL B-gene +locus O +tag O +Bacova_02651 B-gene +( O +Fig O +. O +1B O +), O +shares O +26 O +% O +amino O +acid O +sequence O +identity O +( O +40 O +% O +similarity O +) O +with O +its O +homolog O +, O +B B-species +. I-species +thetaiotaomicron I-species +SusD B-protein +, O +and O +similar O +homology O +with O +the O +SusD B-protein_type +- I-protein_type +like I-protein_type +proteins I-protein_type +encoded O +within O +syntenic O +XyGUL B-gene +identified O +in O +our O +earlier O +work O +. O + +In O +contrast O +, O +SGBP B-protein +- I-protein +B I-protein +, O +encoded O +by O +locus O +tag O +Bacova_02650 B-gene +, O +displays O +little O +sequence O +similarity O +to O +the O +products O +of O +similarly O +positioned O +genes O +in O +syntenic O +XyGUL B-gene +nor O +to O +any O +other O +gene O +product O +among O +the O +diversity O +of O +Bacteroidetes B-taxonomy_domain +PUL B-gene +. O + +Whereas O +sequence O +similarity O +among O +SusC B-protein +/ O +SusD B-protein +homolog O +pairs O +often O +serves O +as O +a O +hallmark O +for O +PUL B-gene +identification O +, O +the O +sequence O +similarities O +of O +downstream O +genes O +encoding O +SGBPs B-protein_type +are O +generally O +too O +low O +to O +allow O +reliable O +bioinformatic O +classification O +of O +their O +products O +into O +protein O +families O +, O +let O +alone O +prediction O +of O +function O +. O + +Hence O +, O +there O +is O +a O +critical O +need O +for O +the O +elucidation O +of O +detailed O +structure O +- O +function O +relationships O +among O +PUL B-gene +SGBPs B-protein_type +, O +in O +light O +of O +the O +manifold O +glycan B-chemical +structures O +in O +nature O +. O + +Immunofluorescence B-experimental_method +of O +formaldehyde O +- O +fixed O +, O +nonpermeabilized O +cells O +grown O +in O +minimal O +medium O +with O +XyG B-chemical +as O +the O +sole O +carbon O +source O +to O +induce O +XyGUL B-gene +expression O +, O +reveals O +that O +both O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +are O +presented O +on O +the O +cell O +surface O +by O +N O +- O +terminal O +lipidation B-ptm +, O +as O +predicted O +by O +signal O +peptide O +analysis O +with O +SignalP O +( O +Fig O +. O +2 O +). O + +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +visualized O +by O +immunofluorescence B-experimental_method +. O + +Formalin O +- O +fixed O +, O +nonpermeabilized O +B B-species +. I-species +ovatus I-species +cells O +were O +grown O +in O +minimal O +medium O +plus O +XyG B-chemical +, O +probed O +with O +custom O +rabbit O +antibodies O +to O +SGBP B-protein +- I-protein +A I-protein +or O +SGBP B-protein +- I-protein +B I-protein +, O +and O +then O +stained O +with O +Alexa O +Fluor O +488 O +goat O +anti O +- O +rabbit O +IgG O +. O +( O +A O +) O +Overlay B-experimental_method +of O +bright B-evidence +- I-evidence +field I-evidence +and I-evidence +FITC I-evidence +images I-evidence +of O +B B-species +. I-species +ovatus I-species +cells O +labeled O +with O +anti O +- O +SGBP O +- O +A O +. O +( O +B O +) O +Overlay B-experimental_method +of O +bright B-evidence +- I-evidence +field I-evidence +and I-evidence +FITC I-evidence +images I-evidence +of O +B B-species +. I-species +ovatus I-species +cells O +labeled O +with O +anti O +- O +SGBP O +- O +B O +. O +( O +C O +) O +Bright B-evidence +- I-evidence +field I-evidence +image I-evidence +of O +ΔSGBP B-mutant +- I-mutant +B I-mutant +cells O +labeled O +with O +anti O +- O +SGBP O +- O +B O +antibodies O +. O + +( O +D O +) O +FITC B-evidence +images I-evidence +of O +ΔSGBP B-mutant +- I-mutant +B I-mutant +cells O +labeled O +with O +anti O +- O +SGBP O +- O +B O +antibodies O +. O + +Cells O +lacking B-protein_state +SGBP B-protein +- I-protein +A I-protein +( O +ΔSGBP B-mutant +- I-mutant +A I-mutant +) O +do O +not O +grow O +on O +XyG B-chemical +and O +therefore O +could O +not O +be O +tested O +in O +parallel O +. O + +Additional O +affinity B-experimental_method +PAGE I-experimental_method +analysis O +( O +Fig O +. O +3 O +) O +demonstrates O +that O +SGBP B-protein +- I-protein +A I-protein +also O +has O +moderate O +affinity O +for O +the O +artificial O +soluble O +cellulose O +derivative O +hydroxyethyl B-chemical +cellulose I-chemical +[ O +HEC B-chemical +; O +a O +β B-chemical +( I-chemical +1 I-chemical +→ I-chemical +4 I-chemical +)- I-chemical +glucan I-chemical +] O +and O +limited O +affinity O +for O +mixed B-chemical +- I-chemical +linkage I-chemical +β I-chemical +( I-chemical +1 I-chemical +→ I-chemical +3 I-chemical +)/ I-chemical +β I-chemical +( I-chemical +1 I-chemical +→ I-chemical +4 I-chemical +)- I-chemical +glucan I-chemical +( O +MLG B-chemical +) O +and O +glucomannan B-chemical +( O +GM B-chemical +; O +mixed O +glucosyl B-chemical +and O +mannosyl B-chemical +backbone O +), O +which O +together O +indicate O +general O +binding O +to O +polysaccharide B-chemical +backbone O +residues O +and O +major O +contributions O +from O +side O +- O +chain O +recognition O +. O + +In O +contrast O +, O +SGBP B-protein +- I-protein +B I-protein +bound O +to O +HEC B-chemical +more O +weakly O +than O +SGBP B-protein +- I-protein +A I-protein +and O +did O +not O +bind O +to O +MLG B-chemical +or O +GM B-chemical +. O + +Neither O +SGBP B-protein_type +recognized O +galactomannan B-chemical +( O +GGM B-chemical +), O +starch B-chemical +, O +carboxymethylcellulose B-chemical +, O +or O +mucin B-chemical +( O +see O +Fig O +. O +S1 O +in O +the O +supplemental O +material O +). O + +Notably O +, O +the O +absence O +of O +carbohydrate B-site +- I-site +binding I-site +modules I-site +in O +the O +GHs B-protein_type +encoded O +by O +the O +XyGUL B-gene +implies O +that O +noncatalytic O +recognition O +of O +xyloglucan B-chemical +is O +mediated O +entirely O +by O +SGBP B-protein +- I-protein +A I-protein +and O +- B-protein +B I-protein +. O + +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +preferentially O +bind O +xyloglucan B-chemical +. O + +Affinity B-experimental_method +electrophoresis I-experimental_method +( O +10 O +% O +acrylamide O +) O +of O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +with O +BSA B-protein +as O +a O +control O +protein O +. O + +All O +samples O +were O +loaded O +on O +the O +same O +gel O +next O +to O +the O +BSA B-protein +controls O +; O +thin O +black O +lines O +indicate O +where O +intervening O +lanes O +were O +removed O +from O +the O +final O +image O +for O +both O +space O +and O +clarity O +. O + +The O +percentage O +of O +polysaccharide B-chemical +incorporated O +into O +each O +native O +gel O +is O +displayed O +. O + +The O +vanguard O +endo B-protein_type +- I-protein_type +xyloglucanase I-protein_type +of O +the O +XyGUL B-gene +, O +BoGH5 B-protein +, O +preferentially O +cleaves O +the O +polysaccharide B-chemical +at O +unbranched O +glucosyl B-chemical +residues O +to O +generate O +xylogluco B-chemical +- I-chemical +oligosaccharides I-chemical +( O +XyGOs B-chemical +) O +comprising O +a O +Glc4 B-structure_element +backbone I-structure_element +with O +variable B-structure_element +side I-structure_element +- I-structure_element +chain I-structure_element +galactosylation I-structure_element +( O +XyGO1 B-chemical +) O +( O +Fig O +. O +1A O +; O +n O += O +1 O +) O +as O +the O +limit O +of O +digestion O +products O +in O +vitro O +; O +controlled B-experimental_method +digestion I-experimental_method +and I-experimental_method +fractionation I-experimental_method +by O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +allow O +the O +production O +of O +higher O +- O +order O +oligosaccharides B-chemical +( O +e O +. O +g O +., O +XyGO2 B-chemical +) O +( O +Fig O +. O +1A O +; O +n O += O +2 O +). O + +ITC B-experimental_method +demonstrates O +that O +SGBP B-protein +- I-protein +A I-protein +binds O +to O +XyG B-chemical +polysaccharide B-chemical +and O +XyGO2 B-chemical +( O +based O +on O +a O +Glc8 B-structure_element +backbone I-structure_element +) O +with O +essentially O +equal O +affinities B-evidence +, O +while O +no O +binding O +of O +XyGO1 B-chemical +( O +Glc4 B-structure_element +backbone I-structure_element +) O +was O +detectable O +( O +Table O +1 O +; O +see O +Fig O +. O +S2 O +and O +S3 O +in O +the O +supplemental O +material O +). O + +Together O +, O +these O +data O +clearly O +suggest O +that O +polysaccharide B-chemical +binding O +of O +both O +SGBPs B-protein_type +is O +fulfilled O +by O +a O +dimer B-oligomeric_state +of O +the O +minimal B-structure_element +repeat I-structure_element +, O +corresponding O +to O +XyGO2 B-chemical +( O +cf O +. O + +The O +observation O +by O +affinity B-experimental_method +PAGE I-experimental_method +that O +these O +proteins O +specifically O +recognize O +XyG B-chemical +is O +further O +substantiated O +by O +their O +lack O +of O +binding O +for O +the O +undecorated O +oligosaccharide B-chemical +cellotetraose B-chemical +( O +Table O +1 O +; O +see O +Fig O +. O +S3 O +). O + +Furthermore O +, O +SGBP B-protein +- I-protein +A I-protein +binds O +cellohexaose B-chemical +with O +~ O +770 O +- O +fold O +weaker O +affinity B-evidence +than O +XyG B-chemical +, O +while O +SGBP B-protein +- I-protein +B I-protein +displays O +no O +detectable O +binding O +to O +this O +linear O +hexasaccharide B-chemical +. O + +To O +provide O +molecular O +- O +level O +insight O +into O +how O +the O +XyGUL B-gene +SGBPs B-protein_type +equip O +B B-species +. I-species +ovatus I-species +to O +specifically O +harvest O +XyG B-chemical +from O +the O +gut O +environment O +, O +we O +performed O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +analysis O +of O +both O +SGBP B-protein +- I-protein +A I-protein +and O +SGPB B-protein +- I-protein +B I-protein +in O +oligosaccharide B-complex_assembly +- I-complex_assembly +complex I-complex_assembly +forms I-complex_assembly +. O + +Summary O +of O +thermodynamic O +parameters O +for O +wild B-protein_state +- I-protein_state +type I-protein_state +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +obtained O +by O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +at O +25 O +° O +Ca O + +Carbohydrate O +Ka O +( O +M O +− O +1 O +) O +ΔG B-evidence +( O +kcal O +⋅ O +mol O +− O +1 O +) O +ΔH O +( O +kcal O +⋅ O +mol O +− O +1 O +) O +TΔS O +( O +kcal O +⋅ O +mol O +− O +1 O +) O +SGBP O +- O +A O +SGBP O +- O +B O +SGBP O +- O +A O +SGBP O +- O +B O +SGBP O +- O +A O +SGBP O +- O +B O +SGBP O +- O +A O +SGBP O +- O +B O +XyGb O +( O +4 O +. O +4 O +± O +0 O +. O +1 O +) O +× O +105 O +( O +5 O +. O +7 O +± O +0 O +. O +2 O +) O +× O +104 O +− O +7 O +. O +7 O +− O +6 O +. O +5 O +− O +14 O +± O +3 O +− O +14 O +± O +2 O +− O +6 O +. O +5 O +− O +7 O +. O +6 O +XyGO2c O +3 O +. O +0 O +× O +105 O +2 O +. O +0 O +× O +104 O +− O +7 O +. O +5 O +− O +5 O +. O +9 O +− O +17 O +. O +2 O +− O +17 O +. O +6 O +− O +9 O +. O +7 O +− O +11 O +. O +7 O +XyGO1 O +NBd O +( O +2 O +. O +4 O +± O +0 O +. O +1 O +) O +× O +103 O +NB O +− O +4 O +. O +6 O +NB O +− O +4 O +. O +4 O +± O +0 O +. O +2 O +NB O +0 O +. O +2 O +Cellohexaose O +568 O +. O +0 O +± O +291 O +. O +0 O +NB O +− O +3 O +. O +8 O +NB O +− O +16 O +± O +8 O +NB O +− O +12 O +. O +7 O +NB O +Cellotetraose O +NB O +NB O +NB O +NB O +NB O +NB O +NB O +NB O + +SGBP B-protein +- I-protein +A I-protein +is O +a O +SusD B-protein +homolog O +with O +an O +extensive O +glycan B-site +- I-site +binding I-site +platform I-site +. O + +Specifically O +, O +SGBP B-protein +- I-protein +A I-protein +overlays B-experimental_method +B B-species +. I-species +thetaiotaomicron I-species +SusD B-protein +( O +BtSusD B-protein +) O +with O +a O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +RMSD B-evidence +) O +value O +of O +2 O +. O +2 O +Å O +for O +363 O +Cα O +pairs O +, O +which O +is O +notable O +given O +the O +26 O +% O +amino O +acid O +identity O +( O +40 O +% O +similarity O +) O +between O +these O +homologs O +( O +Fig O +. O +4C O +). O + +The O +SGBP B-complex_assembly +- I-complex_assembly +A I-complex_assembly +: I-complex_assembly +XyGO2 I-complex_assembly +complex O +superimposes B-experimental_method +closely O +with O +the O +apo B-protein_state +structure B-evidence +( O +RMSD B-evidence +of O +0 O +. O +6 O +Å O +) O +and O +demonstrates O +that O +no O +major O +conformational O +change O +occurs O +upon O +substrate O +binding O +; O +small O +deviations O +in O +the O +orientation O +of O +several O +surface O +loops O +are O +likely O +the O +result O +of O +differential O +crystal O +packing O +. O + +It O +is O +particularly O +notable O +that O +although O +the O +location O +of O +the O +ligand B-site +- I-site +binding I-site +site I-site +is O +conserved B-protein_state +between O +SGBP B-protein +- I-protein +A I-protein +and O +SusD B-protein +, O +that O +of O +SGBP B-protein +- I-protein +A I-protein +displays O +an O +~ O +29 O +- O +Å O +- O +long O +aromatic B-site +platform I-site +to O +accommodate O +the O +extended O +, O +linear O +XyG B-chemical +chain O +( O +see O +reference O +for O +a O +review O +of O +XyG B-chemical +secondary O +structure O +), O +versus O +the O +shorter O +, O +~ O +18 O +- O +Å O +- O +long O +, O +site B-site +within O +SusD B-protein +that O +complements O +the O +helical O +conformation O +of O +amylose B-chemical +( O +Fig O +. O +4C O +and O +D O +). O + +Molecular O +structure B-evidence +of O +SGBP B-protein +- I-protein +A I-protein +( O +Bacova_02651 B-gene +). O +( O +A O +) O +Overlay B-experimental_method +of O +SGBP B-protein +- I-protein +A I-protein +from O +the O +apo B-protein_state +( O +rainbow O +) O +and O +XyGO2 B-chemical +( O +gray O +) O +structures B-evidence +. O + +An O +omit B-evidence +map I-evidence +( O +2σ O +) O +for O +XyGO2 B-chemical +( O +orange O +and O +red O +sticks O +) O +is O +displayed O +. O + +( O +B O +) O +Close O +- O +up O +view O +of O +the O +omit B-evidence +map I-evidence +as O +in O +panel O +A O +, O +rotated O +90 O +° O +clockwise O +. O + +( O +C O +) O +Overlay B-experimental_method +of O +the O +Cα O +backbones O +of O +SGBP B-protein +- I-protein +A I-protein +( O +black O +) O +with O +XyGO2 B-chemical +( O +orange O +and O +red O +spheres O +) O +and O +BtSusD B-protein +( O +blue O +) O +with O +maltoheptaose B-chemical +( O +pink O +and O +red O +spheres O +), O +highlighting O +the O +conservation O +of O +the O +glycan B-site +- I-site +binding I-site +site I-site +location O +. O + +( O +D O +) O +Close O +- O +up O +of O +the O +SGBP B-protein +- I-protein +A I-protein +( O +black O +and O +orange O +) O +and O +SusD B-protein +( O +blue O +and O +pink O +) O +glycan B-site +- I-site +binding I-site +sites I-site +. O + +The O +backbone O +glucose B-chemical +residues O +are O +numbered O +from O +the O +nonreducing O +end O +; O +xylose B-chemical +residues O +are O +labeled O +X1 B-residue_name_number +and O +X2 B-residue_name_number +. O + +Indeed O +, O +the O +electron B-evidence +density I-evidence +for O +the O +ligand O +suggests O +some O +disorder O +, O +which O +may O +arise O +from O +multiple O +oligosaccharide B-chemical +orientations O +along O +the O +binding B-site +site I-site +. O + +Three O +aromatic O +residues O +— O +W82 B-residue_name_number +, O +W283 B-residue_name_number +, O +W306 B-residue_name_number +— O +comprise O +the O +flat B-site +platform I-site +that O +stacks B-bond_interaction +along O +the O +naturally O +twisted O +β B-chemical +- I-chemical +glucan I-chemical +backbone O +( O +Fig O +. O +4E O +). O + +Contrasting O +with O +the O +clear O +importance O +of O +these O +hydrophobic B-bond_interaction +interactions I-bond_interaction +, O +there O +are O +remarkably O +few O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +interactions I-bond_interaction +with O +the O +ligand B-chemical +, O +which O +are O +provided O +by O +R65 B-residue_name_number +, O +N83 B-residue_name_number +, O +and O +S308 B-residue_name_number +, O +which O +are O +proximal O +to O +Glc5 B-residue_name_number +and O +Glc3 B-residue_name_number +. O + +Most O +surprising O +in O +light O +of O +the O +saccharide B-evidence +- I-evidence +binding I-evidence +data I-evidence +, O +however O +, O +was O +a O +lack O +of O +extensive O +recognition O +of O +the O +XyG B-chemical +side O +chains O +; O +only O +Y84 B-residue_name_number +appeared O +to O +provide O +a O +hydrophobic B-site +interface I-site +for O +a O +xylosyl B-chemical +residue O +( O +Xyl1 B-residue_name_number +). O + +Summary O +of O +thermodynamic O +parameters O +for O +site O +- O +directed O +mutants O +of O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +obtained O +by O +ITC B-experimental_method +with O +XyG B-chemical +at O +25 O +° O +Ca O + +Weak O +binding O +represents O +a O +Ka B-evidence +of O +< O +500 O +M O +− O +1 O +. O + +Ka B-evidence +fold O +change O += O +Ka B-evidence +of O +wild B-protein_state +- I-protein_state +type I-protein_state +protein O +/ O +Ka B-evidence +of O +mutant O +protein O +for O +xyloglucan B-chemical +binding O +. O + +SGBP B-protein +- I-protein +B I-protein +has O +a O +multimodular O +structure O +with O +a O +single O +, O +C O +- O +terminal O +glycan B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +. O + +The O +crystal B-evidence +structure I-evidence +of O +full B-protein_state +- I-protein_state +length I-protein_state +SGBP B-protein +- I-protein +B I-protein +in B-protein_state +complex I-protein_state +with I-protein_state +XyGO2 B-chemical +( O +2 O +. O +37 O +Å O +, O +Rwork B-evidence += O +19 O +. O +9 O +%, O +Rfree B-evidence += O +23 O +. O +9 O +%, O +residues O +34 B-residue_range +to I-residue_range +489 I-residue_range +) O +( O +Table O +2 O +) O +revealed O +an O +extended O +structure B-evidence +composed O +of O +three O +tandem B-structure_element +immunoglobulin I-structure_element +( I-structure_element +Ig I-structure_element +)- I-structure_element +like I-structure_element +domains I-structure_element +( O +domains O +A B-structure_element +, O +B B-structure_element +, O +and O +C B-structure_element +) O +followed O +at O +the O +C O +terminus O +by O +a O +novel O +xyloglucan B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +( O +domain O +D B-structure_element +) O +( O +Fig O +. O +5A O +). O + +These B-structure_element +domains I-structure_element +also O +display O +similarity O +to O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sandwich I-structure_element +domains I-structure_element +of O +many O +GH13 B-protein_type +enzymes I-protein_type +, O +including O +the O +cyclodextrin B-protein_type +glucanotransferase I-protein_type +of O +Geobacillus B-species +stearothermophilus I-species +( O +Fig O +. O +5B O +). O + +Such B-structure_element +domains I-structure_element +are O +not O +typically O +involved O +in O +carbohydrate B-chemical +binding O +. O + +Indeed O +, O +visual B-experimental_method +inspection I-experimental_method +of O +the O +SGBP B-protein +- I-protein +B I-protein +structure B-evidence +, O +as O +well O +as O +individual O +production O +of O +the O +A B-structure_element +and O +B B-structure_element +domains O +and O +affinity B-experimental_method +PAGE I-experimental_method +analysis O +( O +see O +Fig O +. O +S5 O +in O +the O +supplemental O +material O +), O +indicates O +that O +these O +domains O +do O +not O +contribute O +to O +XyG B-chemical +capture O +. O + +On O +the O +other O +hand O +, O +production B-experimental_method +of O +the O +fused B-mutant +domains I-mutant +C I-mutant +and I-mutant +D I-mutant +in O +tandem O +( O +SGBP B-protein +- I-protein +B I-protein +residues O +230 B-residue_range +to I-residue_range +489 I-residue_range +) O +retains O +complete O +binding O +of O +xyloglucan B-chemical +in O +vitro O +, O +with O +the O +observed O +slight O +increase O +in O +affinity O +likely O +arising O +from O +a O +reduced O +potential O +for O +steric O +hindrance O +of O +the O +smaller O +protein O +construct O +during O +polysaccharide B-chemical +interactions O +( O +Table O +3 O +). O + +While O +neither O +the O +full B-protein_state +- I-protein_state +length I-protein_state +protein O +nor O +domain O +D B-structure_element +displays O +structural O +homology O +to O +known O +XyG B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +, O +the O +topology O +of O +SGBP B-protein +- I-protein +B I-protein +resembles O +the O +xylan B-protein_type +- I-protein_type +binding I-protein_type +protein I-protein_type +Bacova_04391 B-protein +( O +PDB O +3ORJ O +) O +encoded O +within O +a O +xylan B-chemical +- O +targeting O +PUL B-gene +of O +B B-species +. I-species +ovatus I-species +( O +Fig O +. O +5C O +). O + +The O +structure B-experimental_method +- I-experimental_method +based I-experimental_method +alignment I-experimental_method +of O +these O +proteins O +reveals O +17 O +% O +sequence O +identity O +, O +with O +a O +core O +RMSD B-evidence +of O +3 O +. O +6 O +Å O +for O +253 O +aligned O +residues O +. O + +Multimodular O +structure O +of O +SGBP B-protein +- I-protein +B I-protein +( O +Bacova_02650 B-gene +). O +( O +A O +) O +Full B-protein_state +- I-protein_state +length I-protein_state +structure B-evidence +of O +SGBP B-protein +- I-protein +B I-protein +, O +color O +coded O +by O +domain O +as O +indicated O +. O + +An O +omit B-evidence +map I-evidence +( O +2σ O +) O +for O +XyGO2 B-chemical +is O +displayed O +to O +highlight O +the O +location O +of O +the O +glycan B-site +- I-site +binding I-site +site I-site +. O + +( O +B O +) O +Overlay O +of O +SGBP B-protein +- I-protein +B I-protein +domains O +A B-structure_element +, O +B B-structure_element +, O +and O +C B-structure_element +( O +colored O +as O +in O +panel O +A O +), O +with O +a O +C O +- O +terminal O +Ig B-structure_element +- I-structure_element +like I-structure_element +domain I-structure_element +of O +the O +G B-species +. I-species +stearothermophilus I-species +cyclodextrin B-protein_type +glucanotransferase I-protein_type +( O +PDB O +1CYG O +[ O +residues O +375 B-residue_range +to I-residue_range +493 I-residue_range +]) O +in O +green O +. O +( O +C O +) O +Cα O +overlay B-experimental_method +of O +SGBP B-protein +- I-protein +B I-protein +( O +gray O +) O +and O +Bacova_04391 B-protein +( O +PDB O +3ORJ O +) O +( O +pink O +). O + +( O +D O +) O +Close O +- O +up O +omit B-evidence +map I-evidence +for O +the O +XyGO2 B-chemical +ligand O +, O +contoured O +at O +2σ O +. O +( O +E O +) O +Stereo O +view O +of O +the O +xyloglucan B-site +- I-site +binding I-site +site I-site +of O +SGBP B-protein +- I-protein +B I-protein +, O +displaying O +all O +residues O +within O +4 O +Å O +of O +the O +ligand O +. O + +The O +backbone O +glucose B-chemical +residues O +are O +numbered O +from O +the O +nonreducing O +end O +, O +xylose B-chemical +residues O +are O +shown O +as O +X1 B-residue_name_number +, O +X2 B-residue_name_number +, O +and O +X3 B-residue_name_number +, O +potential O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +interactions I-bond_interaction +are O +shown O +as O +dashed O +lines O +, O +and O +the O +distance O +is O +shown O +in O +angstroms O +. O + +Domains O +A B-structure_element +, O +B B-structure_element +, O +and O +C B-structure_element +do O +not O +pack O +against O +each O +other O +. O + +Moreover O +, O +the O +five B-structure_element +- I-structure_element +residue I-structure_element +linkers I-structure_element +between O +these O +first O +three O +domains O +all O +feature O +a O +proline B-residue_name +as O +the O +middle B-structure_element +residue I-structure_element +, O +suggesting O +significant O +conformational O +rigidity O +( O +Fig O +. O +5A O +). O + +Any O +mobility O +of O +SGBP B-protein +- I-protein +B I-protein +on O +the O +surface O +of O +the O +cell O +( O +beyond O +lateral O +diffusion O +within O +the O +membrane O +) O +is O +likely O +imparted O +by O +the O +eight B-structure_element +- I-structure_element +residue I-structure_element +linker I-structure_element +that O +spans O +the O +predicted O +lipidated B-protein_state +Cys B-residue_name +( O +C28 B-residue_name_number +) O +and O +the O +first B-structure_element +β I-structure_element +- I-structure_element +strand I-structure_element +of O +domain O +A B-structure_element +. O +Other O +outer B-protein_type +membrane I-protein_type +proteins I-protein_type +from O +various O +Sus B-complex_assembly +- I-complex_assembly +like I-complex_assembly +systems I-complex_assembly +possess O +a O +similar O +10 B-structure_element +- I-structure_element +to I-structure_element +20 I-structure_element +- I-structure_element +amino I-structure_element +- I-structure_element +acid I-structure_element +flexible I-structure_element +linker I-structure_element +between O +the O +lipidated B-protein_state +Cys B-residue_name +that O +tethers O +the O +protein O +to O +the O +outside O +the O +cell O +and O +the O +first O +secondary O +structure O +element O +. O + +Analogously O +, O +the O +outer B-protein_state +membrane I-protein_state +- I-protein_state +anchored I-protein_state +endo B-protein_type +- I-protein_type +xyloglucanase I-protein_type +BoGH5 B-protein +of O +the O +XyGUL B-gene +contains O +a O +100 B-structure_element +- I-structure_element +amino I-structure_element +- I-structure_element +acid I-structure_element +, I-structure_element +all I-structure_element +- I-structure_element +β I-structure_element +- I-structure_element +strand I-structure_element +, O +N B-structure_element +- I-structure_element +terminal I-structure_element +module I-structure_element +and O +flexible B-structure_element +linker I-structure_element +that O +imparts O +conformational O +flexibility O +and O +distances O +the O +catalytic B-structure_element +module I-structure_element +from O +the O +cell O +surface O +. O + +XyG B-chemical +binds B-protein_state +to I-protein_state +domain O +D B-structure_element +of O +SGBP B-protein +- I-protein +B I-protein +at O +the O +concave B-site +interface I-site +of O +the O +top O +β B-structure_element +- I-structure_element +sheet I-structure_element +, O +with O +binding O +mediated O +by O +loops B-structure_element +connecting O +the O +β B-structure_element +- I-structure_element +strands I-structure_element +. O + +Six O +glucosyl B-chemical +residues O +, O +comprising O +the O +main O +chain O +, O +and O +three O +branching O +xylosyl B-chemical +residues O +of O +XyGO2 B-chemical +can O +be O +modeled O +in O +the O +density B-evidence +( O +Fig O +. O +5D O +; O +cf O +. O + +The O +aromatic B-site +platform I-site +created O +by O +W330 B-residue_name_number +, O +W364 B-residue_name_number +, O +and O +Y363 B-residue_name_number +spans O +four O +glucosyl B-chemical +residues O +, O +compared O +to O +the O +longer B-protein_state +platform B-site +of O +SGBP B-protein +- I-protein +A I-protein +, O +which O +supports O +six O +glucosyl B-chemical +residues O +( O +Fig O +. O +5E O +). O + +The O +Y363A B-mutant +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutant I-experimental_method +of O +SGBP B-protein +- I-protein +B I-protein +displays O +a O +20 O +- O +fold O +decrease O +in O +the O +Ka B-evidence +for O +XyG B-chemical +, O +while O +the O +W364A B-mutant +mutant B-protein_state +lacks B-protein_state +XyG I-protein_state +binding I-protein_state +( O +Table O +3 O +; O +see O +Fig O +. O +S6 O +in O +the O +supplemental O +material O +). O + +There O +are O +no O +additional O +contacts O +between O +the O +protein O +and O +the O +β B-chemical +- I-chemical +glucan I-chemical +backbone O +and O +surprisingly O +few O +interactions O +with O +the O +side O +- O +chain O +xylosyl B-chemical +residues O +, O +despite O +that O +fact O +that O +ITC B-experimental_method +data O +demonstrate O +that O +SGBP B-protein +- I-protein +B I-protein +does O +not O +measurably O +bind O +the O +cellohexaose B-chemical +( O +Table O +1 O +). O + +F414 B-residue_name_number +stacks B-bond_interaction +with O +the O +xylosyl B-chemical +residue O +of O +Glc3 B-residue_name_number +, O +while O +Q407 B-residue_name_number +is O +positioned O +for O +hydrogen B-bond_interaction +bonding I-bond_interaction +with O +the O +O4 O +of O +xylosyl B-chemical +residue O +Xyl1 B-residue_name_number +. O + +Surprisingly O +, O +an O +F414A B-mutant +mutant B-protein_state +of O +SGBP B-protein +- I-protein +B I-protein +displays O +only O +a O +mild O +3 O +- O +fold O +decrease O +in O +the O +Ka B-evidence +value O +for O +XyG B-chemical +, O +again O +suggesting O +that O +glycan B-chemical +recognition O +is O +primarily O +mediated O +via O +contact O +with O +the O +β O +- O +glucan O +backbone O +( O +Table O +3 O +; O +see O +Fig O +. O +S6 O +). O + +Additional O +residues B-structure_element +surrounding O +the O +binding B-site +site I-site +, O +including O +Y369 B-residue_name_number +and O +E412 B-residue_name_number +, O +may O +contribute O +to O +the O +recognition O +of O +more O +highly O +decorated O +XyG B-chemical +, O +but O +precisely O +how O +this O +is O +mediated O +is O +presently O +unclear O +. O + +The O +CD B-structure_element +domains I-structure_element +of O +the O +truncated B-protein_state +and O +full B-protein_state +- I-protein_state +length I-protein_state +proteins O +superimpose B-experimental_method +with O +a O +0 O +. O +4 O +- O +Å O +RMSD B-evidence +of O +the O +Cα O +backbone O +, O +with O +no O +differences O +in O +the O +position O +of O +any O +of O +the O +glycan B-site +- I-site +binding I-site +residues I-site +( O +see O +Fig O +. O +S7A O +in O +the O +supplemental O +material O +). O + +While O +density B-evidence +is O +observed O +for O +XyGO2 B-chemical +, O +the O +ligand O +could O +not O +be O +unambiguously O +modeled O +into O +this O +density B-evidence +to O +achieve O +a O +reasonable O +fit O +between O +the O +X B-evidence +- I-evidence +ray I-evidence +data I-evidence +and O +the O +known O +stereochemistry O +of O +the O +sugar O +( O +see O +Fig O +. O +S7B O +and O +C O +). O + +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +have O +distinct O +, O +coordinated O +functions O +in O +vivo O +. O + +To O +disentangle O +the O +functions O +of O +SGBP B-protein +- I-protein +A I-protein +and O +SGBP B-protein +- I-protein +B I-protein +in O +XyG B-chemical +recognition O +and O +uptake O +, O +we O +created O +individual O +in B-experimental_method +- I-experimental_method +frame I-experimental_method +deletion I-experimental_method +and I-experimental_method +complementation I-experimental_method +mutant I-experimental_method +strains O +of O +B B-species +. I-species +ovatus I-species +. O + +Growth O +on O +glucose B-chemical +displayed O +the O +shortest O +lag B-evidence +time I-evidence +for O +each O +strain O +, O +and O +so O +lag B-evidence +times I-evidence +were O +normalized O +for O +each O +carbohydrate B-chemical +by O +subtracting O +the O +lag B-evidence +time I-evidence +of O +that O +strain O +in O +glucose B-chemical +( O +Fig O +. O +6 O +; O +see O +Fig O +. O +S8 O +in O +the O +supplemental O +material O +). O + +A O +strain O +in O +which O +the O +entire O +XyGUL B-gene +is O +deleted B-experimental_method +displays O +a O +lag B-evidence +of O +24 O +. O +5 O +h O +during O +growth O +on O +glucose B-chemical +compared O +to O +the O +isogenic O +parental O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +Δtdk B-mutant +strain O +, O +for O +which O +exponential O +growth O +lags B-evidence +for O +19 O +. O +8 O +h O +( O +see O +Fig O +. O +S8D O +). O + +It O +is O +unknown O +whether O +this O +is O +because O +cultures O +were O +not O +normalized O +by O +the O +starting O +optical O +density O +( O +OD O +) O +or O +viable O +cells O +or O +reflects O +a O +minor O +defect O +for O +glucose B-chemical +utilization O +. O + +The O +former O +seems O +more O +likely O +as O +the O +growth O +rates O +are O +nearly O +identical O +for O +these O +strains O +on O +glucose B-chemical +and O +xylose B-chemical +. O + +The O +ΔXyGUL B-mutant +and O +WT B-protein_state +Δtdk B-mutant +strains O +display O +normalized O +lag B-evidence +times I-evidence +on O +xylose B-chemical +within O +experimental O +error O +, O +and O +curiously O +some O +of O +the O +mutant O +and O +complemented O +strains O +display O +a O +nominally O +shorter O +lag B-evidence +time I-evidence +on O +xylose B-chemical +than O +the O +WT B-protein_state +Δtdk B-mutant +strain O +. O + +The O +reason O +for O +this O +observation O +on O +XyGO2 B-chemical +is O +unclear O +, O +as O +the O +ΔSGBP B-mutant +- I-mutant +B I-mutant +mutant B-protein_state +does O +not O +have O +a O +significantly O +different O +growth O +rate O +from O +the O +WT B-protein_state +on O +XyGO2 B-chemical +. O + +Growth O +of O +select O +XyGUL B-gene +mutants O +on O +xyloglucan B-chemical +and O +oligosaccharides B-chemical +. O + +B B-species +. I-species +ovatus I-species +mutants O +were O +created O +in O +a O +thymidine B-mutant +kinase I-mutant +deletion I-mutant +( O +Δtdk B-mutant +) O +mutant O +as O +described O +previously O +. O + +SGBP B-mutant +- I-mutant +A I-mutant +* I-mutant +denotes O +the O +Bacova_02651 B-gene +( O +W82A B-mutant +W283A B-mutant +W306A B-mutant +) O +allele O +, O +and O +the O +GH9 B-protein +gene O +is O +Bacova_02649 B-gene +. O + +Solid O +bars O +indicate O +conditions O +that O +are O +not O +statistically O +significant O +from O +the O +WT B-protein_state +Δtdk B-mutant +cultures O +grown O +on O +the O +indicated O +carbohydrate B-chemical +, O +while O +open O +bars O +indicate O +a O +P O +value O +of O +< O +0 O +. O +005 O +compared O +to O +the O +WT B-protein_state +Δtdk B-mutant +strain O +. O + +Conditions O +denoted O +by O +the O +same O +letter O +( O +b O +, O +c O +, O +or O +d O +) O +are O +not O +statistically O +significant O +from O +each O +other O +but O +are O +significantly O +different O +from O +the O +condition O +labeled O +“ O +a O +.” O +Complementation O +of O +ΔSGBP B-mutant +- I-mutant +A I-mutant +and O +ΔSBGP B-mutant +- I-mutant +B I-mutant +was O +performed O +by O +allelic O +exchange O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +genes O +back O +into O +the O +genome O +for O +expression O +via O +the O +native O +promoter O +: O +these O +growth O +curves O +, O +quantified O +rates O +and O +lag B-evidence +times I-evidence +are O +displayed O +in O +Fig O +. O +S8 O +in O +the O +supplemental O +material O +. O + +Fig O +. O +1B O +) O +was O +completely O +incapable O +of O +growth O +on O +XyG B-chemical +, O +XyGO1 B-chemical +, O +and O +XyGO2 B-chemical +, O +indicating O +that O +SGBP B-protein +- I-protein +A I-protein +is O +essential O +for O +XyG B-chemical +utilization O +( O +Fig O +. O +6 O +). O + +This O +result O +mirrors O +our O +previous O +data O +for O +the O +canonical O +Sus B-complex_assembly +of O +B B-species +. I-species +thetaiotaomicron I-species +, O +which O +revealed O +that O +a O +homologous O +ΔsusD B-mutant +mutant B-protein_state +is O +unable O +to O +grow O +on O +starch B-chemical +or O +malto B-chemical +- I-chemical +oligosaccharides I-chemical +, O +despite O +normal O +cell O +surface O +expression O +of O +all O +other O +PUL B-gene +- O +encoded O +proteins O +. O + +More O +recently O +, O +we O +demonstrated O +that O +this O +phenotype O +is O +due O +to O +the O +loss O +of O +the O +physical O +presence O +of O +SusD B-protein +; O +complementation B-experimental_method +of O +ΔsusD B-mutant +with O +SusD B-mutant +*, I-mutant +a O +triple B-protein_state +site I-protein_state +- I-protein_state +directed I-protein_state +mutant I-protein_state +( O +W96A B-mutant +W320A B-mutant +Y296A B-mutant +) O +that O +ablates B-protein_state +glycan I-protein_state +binding I-protein_state +, O +restores O +B B-species +. I-species +thetaiotaomicron I-species +growth O +on O +malto B-chemical +- I-chemical +oligosaccharides I-chemical +and O +starch B-chemical +when O +sus B-gene +transcription O +is O +induced O +by O +maltose B-chemical +addition O +. O + +Similarly O +, O +the O +function O +of O +SGBP B-protein +- I-protein +A I-protein +extends O +beyond O +glycan B-chemical +binding O +. O + +Complementation B-experimental_method +of O +ΔSGBP B-mutant +- I-mutant +A I-mutant +with O +the O +SGBP B-mutant +- I-mutant +A I-mutant +* I-mutant +( O +W82A B-mutant +W283A B-mutant +W306A B-mutant +) O +variant O +, O +which O +does O +not B-protein_state +bind I-protein_state +XyG B-chemical +, O +supports O +growth O +on O +XyG B-chemical +and O +XyGOs B-chemical +( O +Fig O +. O +6 O +; O +ΔSGBP B-mutant +- I-mutant +A I-mutant +:: O +SGBP B-mutant +- I-mutant +A I-mutant +*), I-mutant +with O +growth O +rates O +that O +are O +~ O +70 O +% O +that O +of O +the O +WT B-protein_state +. O + +In O +previous O +studies O +, O +we O +observed O +that O +carbohydrate B-chemical +binding O +by O +SusD B-protein +enhanced O +the O +sensitivity O +of O +the O +cells O +to O +limiting O +concentrations O +of O +malto O +- O +oligosaccharides O +by O +several O +orders O +of O +magnitude O +, O +such O +that O +the O +addition O +of O +0 O +. O +5 O +g O +/ O +liter O +maltose B-chemical +was O +required O +to O +restore O +growth O +of O +the O +ΔsusD B-mutant +:: O +SusD B-mutant +* I-mutant +strain O +on O +starch B-chemical +, O +which O +nonetheless O +occurred O +following O +an O +extended O +lag B-evidence +phase I-evidence +. O + +In O +contrast O +, O +the O +ΔSGBP B-mutant +- I-mutant +A I-mutant +:: O +SGBP B-mutant +- I-mutant +A I-mutant +* I-mutant +strain O +does O +not O +display O +an O +extended O +lag B-evidence +time I-evidence +on O +any O +of O +the O +xyloglucan B-chemical +substrates O +compared O +to O +the O +WT B-protein_state +( O +Fig O +. O +6 O +). O + +However O +, O +the O +modest O +rate O +defect O +displayed O +by O +the O +SGBP B-protein +- I-protein +A I-protein +:: O +SGBP B-mutant +- I-mutant +A I-mutant +* I-mutant +strain O +suggests O +that O +recognition O +of O +XyG B-chemical +and O +product O +import O +is O +somewhat O +less O +efficient O +in O +these O +cells O +. O + +10 O +- O +fold O +more O +weakly O +than O +XyGO2 B-chemical +and O +XyG B-chemical +( O +Fig O +. O +6 O +; O +Table O +1 O +). O + +As O +such O +, O +the O +data O +suggest O +that O +SGBP B-protein +- I-protein +A I-protein +can O +compensate O +for O +the O +loss O +of O +function O +of O +SGBP B-protein +- I-protein +B I-protein +on O +longer O +oligo B-chemical +- I-chemical +and I-chemical +polysaccharides I-chemical +, O +while O +SGBP B-protein +- I-protein +B I-protein +may O +adapt O +the O +cell O +to O +recognize O +smaller O +oligosaccharides B-chemical +efficiently O +. O + +Indeed O +, O +a O +double B-protein_state +mutant I-protein_state +, O +consisting O +of O +a O +crippled B-protein_state +SGBP B-protein +- I-protein +A I-protein +and O +a O +deletion B-experimental_method +of I-experimental_method +SGBP B-protein +- I-protein +B I-protein +( O +ΔSGBP B-mutant +- I-mutant +A I-mutant +:: O +SGBP B-mutant +- I-mutant +A I-mutant +*/ I-mutant +ΔSGBP B-mutant +- I-mutant +B I-mutant +), O +exhibits O +an O +extended O +lag B-evidence +time I-evidence +on O +both O +XyG B-chemical +and O +XyGO2 B-chemical +, O +as O +well O +as O +XyGO1 B-chemical +. O + +This O +additional O +role O +of O +SGBP B-protein +- I-protein +B I-protein +is O +especially O +notable O +in O +the O +context O +of O +studies O +on O +BtSusE B-protein +and O +BtSusF B-protein +( O +positioned O +similarly O +in O +the O +archetypal O +Sus B-gene +locus I-gene +) O +( O +Fig O +. O +1B O +), O +for O +which O +growth O +defects O +on O +starch B-chemical +or O +malto B-chemical +- I-chemical +oligosaccharides I-chemical +have O +never O +been O +observed O +. O + +However O +, O +combined B-experimental_method +deletion I-experimental_method +of I-experimental_method +the I-experimental_method +genes I-experimental_method +encoding I-experimental_method +GH9 B-protein +( O +encoded O +by O +Bacova_02649 B-gene +) O +and O +SGBP B-protein +- I-protein +B I-protein +does O +not O +exacerbate O +the O +growth O +defect O +on O +XyGO1 B-chemical +( O +Fig O +. O +6 O +; O +ΔSGBP B-mutant +- I-mutant +B I-mutant +/ O +ΔGH9 B-mutant +). O + +The O +necessity O +of O +SGBP B-protein +- I-protein +B I-protein +is O +elevated O +in O +the O +SGBP B-mutant +- I-mutant +A I-mutant +* I-mutant +strain O +, O +as O +the O +ΔSGBP B-mutant +- I-mutant +A I-mutant +:: O +SGBP B-mutant +- I-mutant +A I-mutant +*/ I-mutant +ΔSGBP B-mutant +- I-mutant +B I-mutant +mutant B-protein_state +displays O +an O +extended O +lag B-evidence +during O +growth O +on O +XyG B-chemical +and O +xylogluco B-chemical +- I-chemical +oligosaccharides I-chemical +, O +while O +growth O +rate O +differences O +are O +more O +subtle O +. O + +The O +precise O +reason O +for O +this O +lag B-evidence +is O +unclear O +, O +but O +recapitulating O +our O +findings O +on O +the O +role O +of O +SusD B-protein +in O +malto B-chemical +- I-chemical +oligosaccharide I-chemical +sensing O +in O +B B-species +. I-species +thetaiotaomicron I-species +, O +this O +extended O +lag B-evidence +may O +be O +due O +to O +inefficient O +import O +and O +thus O +sensing O +of O +xyloglucan B-chemical +in O +the O +environment O +in O +the O +absence O +of O +glycan B-chemical +binding O +by O +essential O +SGBPs B-protein_type +. O + +Our O +previous O +work O +demonstrates O +that O +B B-species +. I-species +ovatus I-species +cells O +grown O +in O +minimal O +medium O +plus O +glucose B-chemical +express O +low O +levels O +of O +the O +XyGUL B-gene +transcript O +. O + +Thus O +, O +in O +our O +experiments O +, O +we O +presume O +that O +each O +strain O +, O +initially O +grown O +in O +glucose B-chemical +, O +expresses O +low O +levels O +of O +the O +XyGUL B-gene +transcript O +and O +thus O +low O +levels O +of O +the O +XyGUL B-gene +- O +encoded O +surface O +proteins O +, O +including O +the O +vanguard O +GH5 B-protein +. O + +Presumably O +without O +glycan B-chemical +binding O +by O +the O +SGBPs B-protein_type +, O +the O +GH5 B-protein +protein O +cannot O +efficiently O +process O +xyloglucan B-chemical +, O +and O +/ O +or O +the O +lack O +of O +SGBP B-protein_type +function O +prevents O +efficient O +capture O +and O +import O +of O +the O +processed O +oligosaccharides B-chemical +. O + +In O +the O +BtSus B-gene +, O +SusD B-protein +and O +the O +TBDT B-protein_type +SusC B-protein +interact O +, O +and O +we O +speculate O +that O +this O +interaction O +is O +necessary O +for O +glycan B-chemical +uptake O +, O +as O +suggested O +by O +the O +fact O +that O +a O +ΔsusD B-mutant +mutant B-protein_state +cannot O +grow O +on O +starch B-chemical +, O +but O +a O +ΔsusD B-mutant +:: O +SusD B-mutant +* I-mutant +strain O +regains O +this O +ability O +if O +a O +transcriptional B-protein_type +activator I-protein_type +of O +the O +sus B-gene +operon I-gene +is O +supplied O +. O + +However O +, O +unlike O +the O +Sus B-complex_assembly +, O +in O +which O +elimination B-experimental_method +of I-experimental_method +SusE B-protein +and O +SusF B-protein +does O +not O +affect O +growth O +on O +starch B-chemical +, O +SGBP B-protein +- I-protein +B I-protein +appears O +to O +have O +a O +dedicated O +role O +in O +growth O +on O +small O +xylogluco B-chemical +- I-chemical +oligosaccharides I-chemical +. O + +The O +ability O +of O +gut O +- O +adapted O +microorganisms B-taxonomy_domain +to O +thrive O +in O +the O +gastrointestinal O +tract O +is O +critically O +dependent O +upon O +their O +ability O +to O +efficiently O +recognize O +, O +cleave O +, O +and O +import O +glycans B-chemical +. O + +The O +human B-species +gut O +, O +in O +particular O +, O +is O +a O +densely O +packed O +ecosystem O +with O +hundreds O +of O +species O +, O +in O +which O +there O +is O +potential O +for O +both O +competition O +and O +synergy O +in O +the O +utilization O +of O +different O +substrates O +. O + +Recent O +work O +has O +elucidated O +that O +Bacteroidetes B-taxonomy_domain +cross O +- O +feed O +during O +growth O +on O +many O +glycans B-chemical +; O +the O +glycoside B-protein_type +hydrolases I-protein_type +expressed O +by O +one O +species O +liberate O +oligosaccharides B-chemical +for O +consumption O +by O +other O +members O +of O +the O +community O +. O + +Here O +, O +we O +demonstrate O +that O +the O +surface B-protein_type +glycan I-protein_type +binding I-protein_type +proteins I-protein_type +encoded O +within O +the O +BoXyGUL B-gene +play O +unique O +and O +essential O +roles O +in O +the O +acquisition O +of O +the O +ubiquitous O +and O +abundant O +vegetable B-taxonomy_domain +polysaccharide B-chemical +xyloglucan B-chemical +. O + +Yet O +, O +a O +number O +of O +questions O +remain O +regarding O +the O +molecular O +interplay O +of O +SGBPs B-protein_type +with O +their O +cotranscribed O +cohort O +of O +glycoside B-protein_type +hydrolases I-protein_type +and O +TonB B-protein_type +- I-protein_type +dependent I-protein_type +transporters I-protein_type +. O + +A O +direct O +interaction O +between O +the O +BtSusC B-protein +TBDT B-protein_type +and O +the O +SusD B-protein +SGBP B-protein_type +has O +been O +previously O +demonstrated O +, O +as O +has O +an O +interaction O +between O +the O +homologous O +components O +encoded O +by O +an O +N O +- O +glycan B-chemical +- O +scavenging O +PUL B-gene +of O +Capnocytophaga B-species +canimorsus I-species +. O + +It O +is O +yet O +presently O +unclear O +whether O +this O +interaction O +is O +static O +or O +dynamic O +and O +to O +what O +extent O +the O +association O +of O +cognate O +TBDT B-protein_type +/ O +SGBPs B-protein_type +is O +dependent O +upon O +the O +structure O +of O +the O +carbohydrate B-chemical +to O +be O +imported O +. O + +On O +the O +other O +hand O +, O +there O +is O +clear O +evidence O +for O +independent O +TBDTs B-protein_type +in O +Bacteroidetes B-taxonomy_domain +that O +do O +not O +require O +SGBP B-protein_type +association O +for O +activity O +. O + +For O +example O +, O +it O +was O +recently O +demonstrated O +that O +expression O +of O +nanO B-gene +, O +which O +encodes O +a O +SusC B-protein_type +- I-protein_type +like I-protein_type +TBDT I-protein_type +as O +part O +of O +a O +sialic O +- O +acid O +- O +targeting O +PUL B-gene +from O +B B-species +. I-species +fragilis I-species +, O +restored O +growth O +on O +this O +monosaccharide B-chemical +in O +a O +mutant O +strain O +of O +E B-species +. I-species +coli I-species +. O + +In O +this O +instance O +, O +coexpression O +of O +the O +susD B-gene +- O +like O +gene O +nanU B-gene +was O +not O +required O +, O +nor O +did O +the O +expression O +of O +the O +nanU B-gene +gene O +enhance O +growth O +kinetics O +. O + +Thus O +, O +the O +strict O +dependence O +on O +a O +SusD B-protein_type +- I-protein_type +like I-protein_type +SGBP I-protein_type +for O +glycan B-chemical +uptake O +in O +the O +Bacteroidetes B-taxonomy_domain +may O +be O +variable O +and O +substrate O +dependent O +. O + +Such O +is O +the O +case O +for O +XyGUL B-gene +from O +related O +Bacteroides B-taxonomy_domain +species O +, O +which O +may O +encode O +either O +one O +or O +two O +of O +these O +predicted O +SGBPs B-protein_type +, O +and O +these O +proteins O +vary O +considerably O +in O +length O +. O + +The O +extremely O +low O +similarity O +of O +these O +SGBPs B-protein_type +is O +striking O +in O +light O +of O +the O +moderate O +sequence O +conservation O +observed O +among O +homologous O +GHs B-protein_type +in O +syntenic O +PUL B-gene +. O + +This O +, O +together O +with O +the O +observation O +that O +these O +SGBPs B-protein_type +, O +as O +exemplified O +by O +BtSusE B-protein +and O +BtSusF B-protein +and O +the O +XyGUL B-gene +SGBP B-protein +- I-protein +B I-protein +of O +the O +present O +study O +, O +are O +expendable O +for O +polysaccharide B-chemical +growth O +, O +implies O +a O +high O +degree O +of O +evolutionary O +flexibility O +to O +enhance O +glycan B-chemical +capture O +at O +the O +cell O +surface O +. O + +However O +, O +the O +natural O +diversity O +of O +these O +proteins O +represents O +a O +rich O +source O +for O +the O +discovery O +of O +unique O +carbohydrate B-structure_element +- I-structure_element +binding I-structure_element +motifs I-structure_element +to O +both O +inform O +gut O +microbiology O +and O +generate O +new O +, O +specific O +carbohydrate B-chemical +analytical O +reagents O +. O + +In O +conclusion O +, O +the O +present O +study O +further O +illuminates O +the O +essential O +role O +that O +surface B-protein_type +- I-protein_type +glycan I-protein_type +binding I-protein_type +proteins I-protein_type +play O +in O +facilitating O +the O +catabolism O +of O +complex O +dietary O +carbohydrates B-chemical +by O +Bacteroidetes B-taxonomy_domain +. O + +The O +ability O +of O +our O +resident O +gut O +bacteria B-taxonomy_domain +to O +recognize O +polysaccharides B-chemical +is O +the O +first O +committed O +step O +of O +glycan B-chemical +consumption O +by O +these O +organisms O +, O +a O +critical O +process O +that O +influences O +the O +community O +structure O +and O +thus O +the O +metabolic O +output O +( O +i O +. O +e O +., O +short O +- O +chain O +fatty O +acid O +and O +metabolite O +profile O +) O +of O +these O +organisms O +. O + +Inhibiting O +complex O +IL B-protein +- I-protein +17A I-protein +and O +IL B-protein +- I-protein +17RA I-protein +interactions O +with O +a O +linear O +peptide B-chemical + +IL B-protein +- I-protein +17A I-protein +is O +a O +pro O +- O +inflammatory O +cytokine B-protein_type +that O +has O +been O +implicated O +in O +autoimmune O +and O +inflammatory O +diseases O +. O + +HAP B-chemical +binds O +specifically O +to O +IL B-protein +- I-protein +17A I-protein +and O +inhibits O +the O +interaction O +of O +the O +cytokine B-protein_type +with O +its O +receptor B-protein_type +, O +IL B-protein +- I-protein +17RA I-protein +. O + +Crystal B-experimental_method +structure I-experimental_method +studies I-experimental_method +revealed O +that O +two O +HAP B-chemical +molecules O +bind O +to O +one O +IL B-protein +- I-protein +17A I-protein +dimer B-oligomeric_state +symmetrically O +. O + +The O +N O +- O +terminal O +portions O +of O +HAP B-chemical +form O +a O +β B-structure_element +- I-structure_element +strand I-structure_element +that O +inserts O +between O +two O +IL B-protein +- I-protein +17A I-protein +monomers B-oligomeric_state +while O +the O +C O +- O +terminal O +section O +forms O +an O +α B-structure_element +helix I-structure_element +that O +directly O +blocks O +IL B-protein +- I-protein +17RA I-protein +from O +binding O +to O +the O +same O +region O +of O +IL B-protein +- I-protein +17A I-protein +. O + +IL B-protein +- I-protein +17A I-protein +signals O +through O +a O +specific O +cell O +surface O +receptor B-protein_type +complex O +which O +consists O +of O +IL B-protein +- I-protein +17RA I-protein +and O +IL B-protein +- I-protein +17RC I-protein +. O + +IL B-protein +- I-protein +17A I-protein +’ O +s O +downstream O +signaling O +leads O +to O +increased O +production O +of O +inflammatory O +cytokines B-protein_type +such O +as O +IL B-protein_type +- I-protein_type +6 I-protein_type +, O +IL B-protein_type +- I-protein_type +8 I-protein_type +, O +CCL B-protein_type +- I-protein_type +20 I-protein_type +and O +CXCL1 B-protein_type +by O +various O +mechanisms O +including O +stimulation O +of O +transcription O +and O +stabilization O +of O +mRNA B-chemical +. O + +Although O +various O +cell O +types O +have O +been O +reported O +to O +express O +IL B-protein +- I-protein +17RA I-protein +, O +the O +highest O +responses O +to O +IL B-protein +- I-protein +17A I-protein +come O +from O +epithelial O +cells O +, O +endothelial O +cells O +, O +keratinocytes O +and O +fibroblasts O +. O + +IL B-protein +- I-protein +17A I-protein +and O +its O +signaling O +is O +important O +in O +host O +defense O +against O +certain O +fungal O +and O +bacterial O +infections O +as O +demonstrated O +by O +patients O +with O +autoantibodies O +against O +IL B-protein +- I-protein +17A I-protein +and O +IL B-protein +- I-protein +17F I-protein +, O +or O +with O +inborn O +errors O +of O +IL B-protein_type +- I-protein_type +17 I-protein_type +immunity O +. O + +In O +addition O +to O +its O +physiological O +role O +, O +IL B-protein +- I-protein +17A I-protein +is O +a O +key O +pathogenic O +factor O +in O +inflammatory O +and O +autoimmune O +diseases O +. O + +In O +phase O +II O +and O +III O +clinical O +trials O +, O +neutralizing O +monoclonal O +antibodies B-protein_type +against O +IL B-protein +- I-protein +17A I-protein +( O +secukinumab B-chemical +and O +ixekizumab B-chemical +) O +or O +its O +receptor B-protein_type +IL B-protein +- I-protein +17RA I-protein +( O +brodalumab B-chemical +) O +are O +highly O +efficacious O +in O +treating O +moderate O +to O +severe O +plaque O +psoriasis O +and O +psoriatic O +arthritis O +. O + +Secukinumab B-chemical +has O +been O +approved O +recently O +as O +a O +new O +psoriasis O +drug O +by O +the O +US O +Food O +and O +Drug O +Administration O +( O +Cosentyx B-chemical +™). I-chemical + +In O +addition O +to O +psoriasis O +and O +psoriatic O +arthritis O +, O +IL B-protein +- I-protein +17A I-protein +blockade O +has O +also O +shown O +preclinical O +and O +clinical O +efficacies O +in O +ankylosing O +spondylitis O +and O +rheumatoid O +arthritis O +. O + +Among O +IL B-protein_type +- I-protein_type +17 I-protein_type +cytokines I-protein_type +, O +IL B-protein +- I-protein +17A I-protein +and O +IL B-protein +- I-protein +17F I-protein +share O +the O +highest O +homology O +. O + +Structures B-evidence +are O +known O +for O +apo B-protein_state +IL B-protein +- I-protein +17F I-protein +and O +its O +complex B-protein_state +with I-protein_state +IL B-protein +- I-protein +17RA I-protein +, O +for O +apo B-protein_state +IL B-protein +- I-protein +17A I-protein +, O +its O +complex B-protein_state +with I-protein_state +an O +antibody B-protein_type +Fab B-structure_element +, O +and O +its O +complex B-protein_state +with I-protein_state +IL B-protein +- I-protein +17RA I-protein +. O + +Developing O +small O +molecules O +targeting O +protein O +- O +protein O +interactions O +is O +difficult O +with O +particular O +challenges O +associated O +with O +the O +large O +, O +shallow O +IL B-site +- I-site +17A I-site +/ I-site +IL I-site +- I-site +17RA I-site +interfaces I-site +. O + +Our O +efforts O +resulted O +in O +discovery O +of O +a O +high B-chemical +affinity I-chemical +IL I-chemical +- I-chemical +17A I-chemical +peptide I-chemical +antagonist I-chemical +( O +HAP B-chemical +), O +which O +we O +attempted O +to O +increase O +the O +functional O +production O +and O +pharmacokinetics O +after O +fusing B-experimental_method +HAP B-chemical +to O +antibodies B-protein_type +for O +evaluation O +as O +a O +bispecific O +therapeutic O +in O +animal O +studies O +. O + +Unfortunately O +, O +this O +past O +work O +revealed O +stability O +issues O +of O +the O +uncapped B-protein_state +HAP B-chemical +in O +cell O +culture O +Here O +, O +we O +provide O +the O +details O +of O +the O +discovery O +and O +optimization O +that O +led O +to O +HAP B-chemical +and O +report O +the O +complex B-evidence +structure I-evidence +of O +IL B-protein +- I-protein +17A I-protein +with O +HAP B-chemical +, O +which O +provides O +structure O +based O +rationalization O +of O +peptide B-experimental_method +optimization I-experimental_method +and O +structure B-experimental_method +activity I-experimental_method +relationship I-experimental_method +( O +SAR B-experimental_method +). O + +Single O +clones O +were O +isolated O +and O +sub O +- O +cultured O +in O +growth O +medium O +, O +and O +culture O +supernatants O +were O +used O +in O +an O +enzyme B-experimental_method +- I-experimental_method +linked I-experimental_method +immunosorbent I-experimental_method +assay I-experimental_method +( O +ELISA B-experimental_method +) O +to O +identify O +specific O +IL B-protein +- I-protein +17A I-protein +- O +binding O +clones O +. O + +Approximately O +10 O +% O +of O +the O +clones O +that O +specifically O +bound O +to O +IL B-protein +- I-protein +17A I-protein +also O +prevented O +the O +cytokine B-protein_type +from O +binding O +to O +IL B-protein +- I-protein +17RA I-protein +. O + +A O +15 O +- O +mer O +linear O +peptide B-chemical +1 I-chemical +was O +shown O +to O +block O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +binding O +with O +an O +IC50 B-evidence +of O +80 O +nM O +in O +the O +competition B-experimental_method +ELISA I-experimental_method +assay I-experimental_method +( O +Table O +1 O +). O + +This O +peptide O +was O +then O +tested O +in O +a O +cell B-experimental_method +- I-experimental_method +based I-experimental_method +functional I-experimental_method +assay I-experimental_method +wherein O +production O +of O +GRO B-protein +- I-protein +α I-protein +in O +BJ O +human B-species +fibroblast O +cells O +was O +measured O +as O +a O +function O +of O +IL B-protein +- I-protein +17A I-protein +stimulation O +using O +1 O +ng O +/ O +ml O +IL B-protein +- I-protein +17A I-protein +. O + +Peptide B-chemical +1 I-chemical +was O +found O +to O +be O +active O +in O +this O +functional B-experimental_method +assay I-experimental_method +with O +an O +IC50 B-evidence +of O +370 O +nM O +. O + +Optimization O +of O +IL B-protein +- I-protein +17A I-protein +peptide O +inhibitors O + +A O +SAR B-experimental_method +campaign O +was O +undertaken O +to O +improve O +the O +potency O +of O +peptide B-chemical +1 I-chemical +. O + +When O +alanine B-residue_name +was O +already O +present O +( O +positions O +7 B-residue_number +and O +15 B-residue_number +), O +substitution B-experimental_method +was O +made O +with O +lysine B-residue_name +( O +Table O +1 O +, O +peptides B-chemical +3 I-chemical +– I-chemical +17 I-chemical +). O + +Positions O +1 B-residue_number +, O +2 B-residue_number +, O +4 B-residue_number +, O +5 B-residue_number +, O +7 B-residue_number +, O +14 B-residue_number +and O +15 B-residue_number +were O +shown O +to O +be O +amenable O +to O +substitution O +without O +significant O +loss O +( O +less O +than O +3 O +- O +fold O +) O +of O +binding B-evidence +affinity I-evidence +as O +measured O +by O +the O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +competition B-experimental_method +ELISA I-experimental_method +. O + +In O +order O +to O +rapidly O +evaluate O +the O +effects O +of O +substitution B-experimental_method +of O +natural O +amino O +acids O +at O +tolerant O +positions O +identified O +by O +the O +alanine B-experimental_method +scan I-experimental_method +, O +the O +lead O +sequence O +was O +subjected O +to O +site B-experimental_method +- I-experimental_method +specific I-experimental_method +saturation I-experimental_method +mutagenesis I-experimental_method +using O +MBP B-experimental_method +. O + +Each O +of O +the O +seven O +positions O +identified O +by O +the O +alanine B-experimental_method +scan I-experimental_method +was O +individually O +modified O +while O +keeping O +the O +rest O +of O +the O +sequence O +constant O +. O + +Peptides O +with O +beneficial O +point B-experimental_method +mutations I-experimental_method +at O +positions O +2 B-residue_number +, O +5 B-residue_number +, O +and O +14 B-residue_number +were O +synthesized B-experimental_method +and O +evaluated O +in O +the O +competition B-experimental_method +ELISA I-experimental_method +( O +Table O +1 O +). O + +Two O +of O +the O +changes O +, O +V2H B-mutant +( O +18 B-chemical +) O +or O +V2T B-mutant +( O +21 B-chemical +) O +displayed O +improved O +binding O +in O +the O +competition B-experimental_method +ELISA I-experimental_method +. O + +Introduction B-experimental_method +of O +a O +methionine B-residue_name +( O +27 B-chemical +) O +or O +a O +carboxamide B-chemical +( O +28 B-chemical +and O +29 B-chemical +) O +at O +position O +14 B-residue_number +was O +shown O +to O +improve O +the O +binding B-evidence +affinity I-evidence +of O +the O +lead O +peptide O +. O + +In O +general O +, O +there O +was O +good O +agreement O +between O +the O +respective O +binding B-evidence +affinities I-evidence +of O +the O +synthesized O +peptides O +and O +their O +MBP B-experimental_method +fusion I-experimental_method +counterparts O +, O +except O +for O +substitution B-experimental_method +of O +valine B-residue_name +at O +position O +2 B-residue_number +to O +a O +tryptophan B-residue_name +( O +22 B-chemical +), O +which O +resulted O +in O +a O +fivefold O +loss O +of O +affinity B-evidence +, O +for O +the O +free O +peptide O +when O +compared O +with O +the O +MBP B-experimental_method +fusion I-experimental_method +. O + +Combining O +the O +key O +amino O +- O +acid O +residues O +identified O +by O +SAR B-experimental_method +into O +a O +single O +peptide O +sequence O +resulted O +in O +peptide B-chemical +30 I-chemical +, O +named O +high B-chemical +affinity I-chemical +peptide I-chemical +( O +HAP B-chemical +), O +that O +was O +found O +to O +inhibit O +IL B-protein +- I-protein +17A I-protein +signaling O +in O +a O +BJ O +human B-species +fibroblast O +cell O +assay O +with O +an O +IC50 B-evidence +of O +17 O +nM O +, O +a O +more O +than O +20 O +- O +fold O +improvement O +over O +the O +phage B-experimental_method +peptide B-chemical +1 I-chemical +( O +Table O +2 O +and O +Supplementary O +Figure O +S2 O +). O + +We O +also O +examined O +the O +effect O +of O +removing O +the O +acetyl O +group O +at O +the O +N O +- O +terminus O +of O +HAP B-chemical +( O +which O +is O +present O +in O +all O +the O +peptides O +made O +, O +see O +Supplementary O +Material O +). O + +The O +un B-protein_state +- I-protein_state +capped I-protein_state +peptide B-chemical +( I-chemical +31 I-chemical +) I-chemical +had O +an O +IC50 B-evidence +of O +420 O +nM O +in O +the O +cell B-experimental_method +- I-experimental_method +based I-experimental_method +assay I-experimental_method +. O + +The O +loss O +of O +cellular O +activity O +of O +31 B-chemical +was O +most O +likely O +due O +to O +the O +degradation O +of O +the O +N O +- O +terminus O +of O +31 B-chemical +, O +since O +peptide O +31 B-chemical +was O +shown O +to O +be O +able O +to O +bind O +to O +IL B-protein +- I-protein +17A I-protein +with O +similar O +affinity O +as O +HAP B-chemical +itself O +. O + +Furthermore O +, O +our O +previous O +work O +had O +reported O +that O +in O +antibody B-experimental_method +fusions I-experimental_method +the O +uncapped B-protein_state +peptide B-chemical +was O +degraded O +under O +cell O +assay O +conditions O +with O +removal B-experimental_method +of I-experimental_method +the O +first B-residue_range +1 I-residue_range +- I-residue_range +3 I-residue_range +residues I-residue_range +to O +inactive O +products O +with O +the O +same O +N O +- O +terminal O +sequences O +as O +peptides B-chemical +32 I-chemical +– I-chemical +34 I-chemical +. O + +C O +- O +terminal O +truncations B-experimental_method +showed O +a O +more O +gradual O +reduction O +in O +activity O +( O +35 B-chemical +– I-chemical +37 I-chemical +; O +Table O +2 O +). O + +After O +deletion B-experimental_method +of I-experimental_method +three B-residue_range +amino I-residue_range +acids I-residue_range +from O +the O +C O +- O +terminal O +end O +( O +37 B-chemical +), O +the O +peptide O +is O +no O +longer O +active O +. O + +We O +reasoned O +that O +since O +the O +IL B-protein +- I-protein +17A I-protein +protein O +is O +almost O +exclusively O +present O +in O +a O +dimeric B-oligomeric_state +form O +, O +dimerizing B-oligomeric_state +the O +IL B-protein +- I-protein +17A I-protein +binding O +peptides O +could O +result O +in O +an O +improvement O +in O +binding B-evidence +affinity I-evidence +and O +inhibitory O +activity O +. O + +Homodimers B-oligomeric_state +of O +HAP B-chemical +were O +made O +through O +attachment O +of O +polyethylene B-chemical +glycol I-chemical +( O +PEG B-chemical +) O +spacers O +of O +different O +lengths O +at O +amino O +acids O +4 B-residue_number +, O +7 B-residue_number +and O +14 B-residue_number +, O +as O +these O +positions O +were O +identified O +in O +the O +alanine B-experimental_method +scan I-experimental_method +analysis I-experimental_method +as O +not O +contributing O +significantly O +to O +the O +activity O +, O +and O +at O +each O +N O +- O +terminus O +( O +Supplementary O +Table O +S2 O +). O + +Due O +to O +the O +high O +reactivity O +of O +the O +pentafluoroester B-chemical +( O +PFP B-chemical +) O +group O +used O +as O +the O +activating O +group O +in O +the O +PEG B-chemical +, O +the O +histidine B-residue_name +at O +position O +2 B-residue_number +and O +the O +lysine B-residue_name +at O +position O +15 B-residue_number +were O +replaced O +with O +threonine B-residue_name +and O +dimethyllysine B-residue_name +respectively O +to O +prevent O +formation O +of O +side O +products O +, O +which O +resulted O +in O +peptide B-chemical +38 I-chemical +that O +was O +comparable O +in O +activity O +with O +HAP B-chemical +. O + +This O +exercise O +revealed O +that O +several O +dimeric B-oligomeric_state +peptides B-chemical +with O +the O +longer O +PEG21 B-chemical +spacer O +were O +significantly O +more O +potent O +than O +the O +monomer B-oligomeric_state +peptide O +in O +the O +cell B-experimental_method +- I-experimental_method +based I-experimental_method +assay I-experimental_method +( O +Supplementary O +Table O +S2 O +). O + +The O +species O +cross O +- O +reactivity O +of O +the O +dimeric B-oligomeric_state +peptide B-chemical +45 I-chemical +and O +HAP B-chemical +were O +assessed O +in O +a O +murine B-experimental_method +functional I-experimental_method +cell I-experimental_method +assay I-experimental_method +using O +15 O +ng O +/ O +ml O +murine B-taxonomy_domain +IL B-protein +- I-protein +17A I-protein +. O + +Peptide B-chemical +45 I-chemical +blocked O +the O +receptor B-protein_type +binding O +of O +murine B-taxonomy_domain +IL B-protein +- I-protein +17A I-protein +although O +with O +potency O +two O +orders O +of O +magnitude O +weaker O +than O +that O +observed O +against O +human B-species +IL B-protein +- I-protein +17A I-protein +( O +IC50 B-evidence += O +41 O +nM O +vs O +IC50 B-evidence += O +0 O +. O +1 O +nM O +, O +respectively O +). O + +The O +monomer B-oligomeric_state +HAP B-chemical +was O +much O +weaker O +( O +IC50 B-evidence +> O +1 O +μM O +) O +in O +inhibiting O +murine B-taxonomy_domain +IL B-protein +- I-protein +17A I-protein +signaling O +( O +Supplementary O +Figure O +S4 O +). O + +Although O +the O +dimeric B-oligomeric_state +peptide B-chemical +45 I-chemical +is O +much O +more O +potent O +than O +HAP B-chemical +in O +the O +cell B-experimental_method +- I-experimental_method +based I-experimental_method +assay I-experimental_method +, O +in O +subsequent O +studies O +we O +decided O +to O +focus O +our O +efforts O +solely O +on O +characterizations O +of O +the O +monomeric B-oligomeric_state +peptide O +HAP B-chemical +in O +hopes O +to O +identify O +smaller O +peptide O +inhibitors O +containing O +the O +best O +minimal O +functional O +group O +. O + +HAP B-chemical +binds O +to O +immobilized O +human B-species +IL B-protein +- I-protein +17A I-protein +homodimer B-oligomeric_state +tightly O +( O +Table O +3 O +). O + +It O +has O +slightly O +weaker O +affinity B-evidence +for O +human B-species +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +F I-complex_assembly +heterodimer B-oligomeric_state +and O +> O +10 O +fold O +weaker O +affinity B-evidence +for O +mouse B-taxonomy_domain +IL B-protein +- I-protein +17A I-protein +( O +Table O +3 O +). O + +HAP B-chemical +does O +not O +show O +significant O +binding O +to O +immobilized O +human B-species +IL B-protein +- I-protein +17F I-protein +homodimer B-oligomeric_state +or O +IL B-protein +- I-protein +17RA I-protein +at O +concentrations O +up O +to O +100 O +nM O +. O + +Additionally O +, O +we O +investigated O +the O +antagonism O +of O +the O +human B-species +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +interaction O +by O +HAP B-chemical +using O +orthogonal O +methods O +including O +SPR B-experimental_method +and O +Förster B-experimental_method +resonance I-experimental_method +energy I-experimental_method +transfer I-experimental_method +( I-experimental_method +FRET I-experimental_method +) I-experimental_method +competition I-experimental_method +assays I-experimental_method +( O +Fig O +. O +1B O +, O +C O +). O + +In O +both O +assays O +, O +incubation O +of O +IL B-protein +- I-protein +17A I-protein +with O +HAP B-chemical +effectively O +blocks O +the O +binding O +of O +IL B-protein +- I-protein +17A I-protein +to O +immobilized B-protein_state +IL B-protein +- I-protein +17RA I-protein +with O +similar O +sub O +- O +nM O +IC50 B-evidence +( O +Table O +3 O +). O + +While O +either O +IL B-protein +- I-protein +17A I-protein +or O +TNF B-protein +- I-protein +α I-protein +alone O +can O +stimulate O +the O +release O +of O +multiple O +inflammatory O +cytokines B-protein_type +, O +when O +acting O +together O +they O +can O +synergistically O +enhance O +each O +other O +’ O +s O +effects O +( O +Supplementary O +Figure O +S5 O +). O + +These O +integrative O +responses O +to O +IL B-protein +- I-protein +17A I-protein +and O +TNF B-protein +- I-protein +α I-protein +in O +human B-species +keratinocytes O +have O +been O +reported O +to O +account O +for O +key O +inflammatory O +pathogenic O +circuits O +in O +psoriasis O +. O + +Thus O +, O +we O +chose O +to O +study O +HAP B-chemical +’ O +s O +efficacy O +in O +blocking O +the O +production O +of O +IL B-protein_type +- I-protein_type +8 I-protein_type +, O +IL B-protein_type +- I-protein_type +6 I-protein_type +and O +CCL B-protein_type +- I-protein_type +20 I-protein_type +by O +primary O +human B-species +keratinocytes O +stimulated O +by O +IL B-protein +- I-protein +17A I-protein +in O +the O +presence O +of O +TNF B-protein +- I-protein +α I-protein +, O +an O +assay O +which O +may O +be O +more O +disease O +- O +relevant O +. O + +HAP B-chemical +inhibits O +the O +production O +of O +all O +three O +cytokines B-protein_type +in O +a O +dose O +- O +dependent O +fashion O +( O +Fig O +. O +1D O +). O + +Significantly O +, O +the O +baseline O +levels O +of O +IL B-protein_type +- I-protein_type +8 I-protein_type +, O +IL B-protein_type +- I-protein_type +6 I-protein_type +and O +CCL B-protein_type +- I-protein_type +20 I-protein_type +stimulated O +by O +TNF B-protein +- I-protein +α I-protein +alone O +are O +not O +inhibited O +by O +HAP B-chemical +, O +further O +indicating O +the O +selectivity O +of O +HAP B-chemical +( O +Fig O +. O +1D O +). O + +Such O +pharmacological O +selectivity O +may O +be O +important O +to O +suppress O +inflammatory O +pathogenic O +circuits O +in O +psoriasis O +, O +while O +sparing O +the O +anti O +- O +infectious O +immune O +responses O +produced O +by O +TNF B-protein +- I-protein +α I-protein +. O + +As O +a O +reference O +, O +a O +commercial O +anti O +- O +IL B-protein +- I-protein +17A I-protein +antibody B-protein_type +( O +R O +& O +D O +Systems O +) O +inhibits O +the O +production O +of O +IL B-protein_type +- I-protein_type +8 I-protein_type +with O +an O +IC50 B-evidence +of O +13 O +(± O +6 O +) O +nM O +( O +N O += O +3 O +). O + +Indeed O +, O +the O +IC50 B-evidence +was O +14 O +(± O +9 O +) O +nM O +( O +N O += O +12 O +) O +for O +HAP B-chemical +inhibition O +of O +IL B-protein_type +- I-protein_type +8 I-protein_type +production O +when O +only O +5 O +ng O +/ O +ml O +IL B-protein +- I-protein +17A I-protein +was O +used O +in O +this O +assay O +. O + +Similar O +to O +keratinocytes B-experimental_method +assay I-experimental_method +results O +, O +while O +HAP B-chemical +inhibits O +IL B-protein +- I-protein +17A I-protein +stimulated O +IL B-protein_type +- I-protein_type +6 I-protein_type +production O +by O +BJ O +human B-species +fibroblast O +potently O +( O +IC50 B-evidence +of O +17 O +nM O +), O +it O +does O +not O +inhibit O +TNF B-protein +- I-protein +α I-protein +stimulated O +IL B-protein_type +- I-protein_type +6 I-protein_type +production O +at O +concentrations O +up O +to O +10 O +μM O +( O +Supplementary O +Figure O +S2 O +). O + +Extensive O +crystallization B-experimental_method +trials I-experimental_method +, O +either O +by O +co B-experimental_method +- I-experimental_method +crystallization I-experimental_method +or O +by O +soaking B-experimental_method +HAP B-chemical +into O +preformed O +apo B-protein_state +IL B-protein +- I-protein +17A I-protein +crystals B-evidence +, O +failed O +to O +lead O +to O +an O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O +crystals B-evidence +. O + +We O +theorized O +that O +HAP B-chemical +binding O +induced O +large O +conformational O +changes O +in O +IL B-protein +- I-protein +17A I-protein +that O +led O +to O +the O +difficulty O +of O +getting O +an O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +binary O +complex O +crystal B-evidence +. O + +We O +hypothesized O +that O +HAP B-chemical +may O +target O +the O +N O +- O +terminal O +of O +IL B-protein +- I-protein +17A I-protein +which O +is O +known O +to O +be O +more O +flexible O +than O +its O +C O +- O +terminal O +and O +conformational O +changes O +needed O +for O +HAP B-chemical +binding O +may O +be O +more O +likely O +there O +. O + +We O +designed O +an O +antibody B-protein_type +Fab B-structure_element +known O +to O +target O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +based O +on O +a O +published O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +Fab I-complex_assembly +complex O +crystal B-evidence +structure I-evidence +, O +and O +produced O +it O +in O +HEK293 O +cells O +. O + +In O +an O +SPR B-experimental_method +assay I-experimental_method +HAP B-chemical +and O +this O +Fab B-structure_element +were O +able O +to O +co O +- O +bind O +IL B-protein +- I-protein +17A I-protein +without O +large O +changes O +in O +their O +binding B-evidence +affinities I-evidence +and O +kinetics B-evidence +, O +confirming O +our O +hypothesis O +( O +Supplementary O +Figure O +S6 O +). O + +These O +were O +, O +respectively O +, O +a O +presumably O +more O +homogeneous O +form O +of O +IL B-protein +- I-protein +17A I-protein +that O +lacked B-protein_state +the O +disordered B-protein_state +N B-structure_element +- I-structure_element +terminal I-structure_element +peptide I-structure_element +and O +a O +full B-protein_state +- I-protein_state +length I-protein_state +form O +of O +the O +cytokine B-protein_type +with O +a O +full O +complement O +of O +disulfide B-ptm +bonds I-ptm +. O + +Both O +complexes O +crystallized B-experimental_method +in O +the O +space O +group O +of O +P321 O +, O +with O +half O +the O +complex O +( O +1 O +Fab B-structure_element +/ O +1 O +IL B-protein +- I-protein +17A I-protein +monomer B-oligomeric_state +/ O +1 O +HAP B-chemical +) O +in O +the O +asymmetric O +unit O +. O + +The O +intact B-protein_state +complex O +can O +be O +generated O +by O +applying O +crystallographic O +2 O +- O +fold O +symmetry O +. O + +Electron B-evidence +densities I-evidence +for O +HAP B-chemical +residues O +Ile1 B-residue_range +- I-residue_range +Asn14 I-residue_range +were O +readily O +interpretable O +with O +the O +exception O +of O +Lys15 B-residue_name_number +, O +which O +is O +disordered B-protein_state +. O + +When O +considering O +the O +protein O +, O +the O +complex B-evidence +structure I-evidence +containing O +the O +full B-protein_state +length I-protein_state +IL B-protein +- I-protein +17A I-protein +is O +identical O +to O +that O +of O +the O +truncated B-protein_state +IL B-protein +- I-protein +17A I-protein +, O +with O +the O +exception O +of O +Cys106 B-residue_name_number +( O +Ser106 B-residue_name_number +in O +the O +truncated B-protein_state +IL B-protein +- I-protein +17A I-protein +), O +which O +is O +disordered B-protein_state +. O + +Cys106 B-residue_name_number +is O +covalently O +linked O +to O +Cys10 B-residue_name_number +that O +resides O +in O +the O +disordered B-protein_state +N B-structure_element +- I-structure_element +terminal I-structure_element +peptide I-structure_element +in O +the O +full B-protein_state +length I-protein_state +IL B-protein +- I-protein +17A I-protein +. O + +Overall O +structure B-evidence +of O +Fab B-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O + +In O +a O +similar O +manner O +to O +the O +published O +structure B-evidence +of O +Fab B-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17A I-complex_assembly +complex O +, O +two O +Fab B-structure_element +molecules O +bind O +symmetrically O +to O +the O +C O +- O +terminal O +of O +the O +cytokine B-protein_type +dimer B-oligomeric_state +, O +interacting O +with O +epitopes O +from O +both O +monomers B-oligomeric_state +( O +Fig O +. O +2A O +). O + +Based O +on O +disclosed O +epitopes O +of O +Secukinumab B-chemical +and O +Ixekizumab B-chemical +, O +HAP B-chemical +binds O +to O +IL B-protein +- I-protein +17A I-protein +at O +an O +area O +that O +is O +also O +different O +from O +those O +of O +those O +two O +antibodies B-protein_type +. O + +The O +N O +- O +terminal O +5 B-residue_range +residues I-residue_range +of O +HAP B-chemical +, O +1IHVTI B-chemical +, O +form O +an O +amphipathic B-protein_state +β B-structure_element +- I-structure_element +strand I-structure_element +that O +inserts O +between O +β B-structure_element +- I-structure_element +strand I-structure_element +4 I-structure_element +of O +one O +IL B-protein +- I-protein +17A I-protein +monomer B-oligomeric_state +and O +β B-structure_element +- I-structure_element +strand I-structure_element +0 I-structure_element +( O +the O +first O +ordered O +peptide O +of O +IL B-protein +- I-protein +17A I-protein +) O +of O +the O +second O +monomer B-oligomeric_state +. O + +This O +β B-structure_element +- I-structure_element +strand I-structure_element +is O +parallel O +to O +both O +strands B-structure_element +0 I-structure_element +and I-structure_element +4 I-structure_element +( O +Fig O +. O +3B O +). O + +Strands B-structure_element +0 I-structure_element +of O +two O +IL B-protein +- I-protein +17A I-protein +monomer B-oligomeric_state +are O +antiparallel O +, O +as O +appeared O +in O +other O +IL B-protein +- I-protein +17A I-protein +structures B-evidence +. O + +As O +a O +comparison O +, O +an O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +complex B-evidence +structure I-evidence +( O +PDB O +code O +4HSA O +) O +is O +also O +shown O +with O +IL B-protein +- I-protein +17A I-protein +in O +the O +same O +orientation O +( O +Fig O +. O +2C O +). O + +IL B-protein +- I-protein +17RA I-protein +binds O +IL B-protein +- I-protein +17A I-protein +at O +three O +regions O +on O +the O +IL B-protein +- I-protein +17A I-protein +homodimer B-oligomeric_state +. O + +HAP B-chemical +binds O +IL B-protein +- I-protein +17A I-protein +at O +region B-structure_element +I I-structure_element +. O +Region B-structure_element +I I-structure_element +is O +formed O +by O +residues O +at O +the O +ends O +of O +β B-structure_element +strands I-structure_element +0 I-structure_element +and I-structure_element +4 I-structure_element +, O +and O +from O +loops B-structure_element +1 I-structure_element +– I-structure_element +2 I-structure_element +and O +3 B-structure_element +– I-structure_element +4 I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +( O +Fig O +. O +2 O +). O + +The O +most O +significant O +interactions O +between O +the O +α B-structure_element +helix I-structure_element +of O +HAP B-chemical +and O +IL B-protein +- I-protein +17A I-protein +involve O +Trp12 B-residue_name_number +of O +HAP B-chemical +, O +which O +binds O +in O +a O +hydrophobic B-site +pocket I-site +in O +IL B-protein +- I-protein +17A I-protein +formed O +by O +the O +side O +chains O +of O +Phe110 B-residue_name_number +, O +Tyr62 B-residue_name_number +, O +Pro59 B-residue_name_number +and O +the O +hydrophobic O +portion O +of O +the O +Arg101 B-residue_name_number +side O +chain O +( O +Fig O +. O +3A O +). O + +The O +Trp12 B-residue_name_number +side O +chain O +of O +HAP B-chemical +donates O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +to O +the O +main O +chain O +oxygen O +of O +Pro69 B-residue_name_number +of O +IL B-protein +- I-protein +17A I-protein +. O + +The O +positively O +charged O +Arg101 B-residue_name_number +side O +chain O +of O +the O +IL B-protein +- I-protein +17A I-protein +engages O +in O +a O +charge B-bond_interaction +- I-bond_interaction +helix I-bond_interaction +dipole I-bond_interaction +interaction I-bond_interaction +with O +the O +main O +chain O +oxygen O +of O +Trp12 B-residue_name_number +. O + +Additionally O +, O +Leu9 B-residue_name_number +and O +Ile13 B-residue_name_number +of O +the O +HAP B-chemical +have O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +IL B-protein +- I-protein +17A I-protein +, O +and O +the O +Asp8 B-residue_name_number +side O +chain O +has O +hydrogen B-bond_interaction +bond I-bond_interaction +and O +ion B-bond_interaction +pair I-bond_interaction +interactions I-bond_interaction +with O +Tyr62 B-residue_name_number +and O +Lys114 B-residue_name_number +of O +IL B-protein +- I-protein +17A I-protein +, O +respectively O +. O + +In O +region B-structure_element +I I-structure_element +, O +an O +IL B-protein +- I-protein +17RA I-protein +peptide O +interacts O +with O +IL B-protein +- I-protein +17A I-protein +in O +a O +very O +similar O +fashion O +to O +the O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +HAP B-chemical +. O + +The O +IL B-protein +- I-protein +17RA I-protein +peptide O +has O +sequences O +of O +27LDDSWI B-chemical +, O +and O +part O +of O +the O +peptide O +is O +also O +α B-structure_element +- I-structure_element +helical I-structure_element +( O +Fig O +. O +3B O +). O + +Leu7 B-residue_name_number +, O +Trp31 B-residue_name_number +and O +Ile32 B-residue_name_number +of O +IL B-protein +- I-protein +17RA I-protein +interact O +very O +similarly O +with O +the O +same O +residues O +of O +IL B-protein +- I-protein +17A I-protein +as O +Leu9 B-residue_name_number +, O +Trp12 B-residue_name_number +and O +Ile13 B-residue_name_number +of O +HAP B-chemical +( O +Fig O +. O +3B O +). O + +In O +this O +sense O +, O +the O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +HAP B-chemical +with O +a O +sequence O +of O +9LWDWI B-chemical +is O +a O +good O +mimetic O +of O +the O +27LDDSWI B-chemical +peptide O +of O +IL B-protein +- I-protein +17RA I-protein +. O + +The O +β B-structure_element +- I-structure_element +strand I-structure_element +of O +HAP B-chemical +has O +no O +equivalent O +in O +IL B-protein +- I-protein +17RA I-protein +. O + +The O +amphipathic B-protein_state +β B-structure_element +- I-structure_element +strand I-structure_element +of O +HAP B-chemical +orients O +the O +hydrophilic O +side O +chains O +of O +His2 B-residue_name_number +and O +Thr4 B-residue_name_number +outwards O +, O +and O +the O +hydrophobic O +side O +chains O +of O +Ile1 B-residue_name_number +, O +Val3 B-residue_name_number +and O +Ile5 B-residue_name_number +inward O +( O +Fig O +. O +3A O +). O + +β B-structure_element +- I-structure_element +strand I-structure_element +0 I-structure_element +in O +IL B-protein +- I-protein +17A I-protein +is O +also O +amphipathic B-protein_state +with O +the O +sequence O +of O +21TVMVNLNI B-chemical +. O + +In O +all O +IL B-protein +- I-protein +17A I-protein +structures B-evidence +obtained O +to O +date O +, O +β B-structure_element +- I-structure_element +strand I-structure_element +0 I-structure_element +orients O +the O +hydrophilic O +side O +chains O +of O +Thr21 B-residue_name_number +, O +Asn25 B-residue_name_number +and O +Asn27 B-residue_name_number +outward O +, O +and O +the O +hydrophobic O +side O +chains O +of O +Val22 B-residue_name_number +, O +Val24 B-residue_name_number +, O +Leu26 B-residue_name_number +and O +Ile28 B-residue_name_number +inward O +. O + +The O +binding B-site +pocket I-site +occupied O +by O +either O +Trp12 B-residue_name_number +of O +HAP B-chemical +or O +Trp31 B-residue_name_number +of O +IL B-protein +- I-protein +17RA I-protein +is O +not O +formed O +in O +the O +apo B-protein_state +IL B-protein +- I-protein +17A I-protein +structure B-evidence +( O +Fig O +. O +3C O +). O + +Particularly O +for O +HAP B-chemical +, O +β B-structure_element +- I-structure_element +strands I-structure_element +0 I-structure_element +have O +to O +shift O +out O +of O +the O +hydrophobic B-site +cleft I-site +formed O +by O +the O +main B-structure_element +body I-structure_element +of O +the O +IL B-protein +- I-protein +17A I-protein +by O +as O +much O +as O +10 O +Å O +between O +Cα O +atoms O +( O +Fig O +. O +3C O +). O + +Disruptions O +of O +the O +apo B-protein_state +IL B-protein +- I-protein +17A I-protein +structure B-evidence +by O +HAP B-chemical +binding O +are O +apparently O +compensated O +for O +by O +formation O +of O +the O +new O +interactions O +that O +involve O +almost O +the O +entire O +HAP B-chemical +molecule O +( O +Fig O +. O +3B O +). O + +The O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex B-evidence +structure I-evidence +obtained O +is O +very O +consistent O +with O +the O +observed O +SAR B-experimental_method +of O +our O +identified O +peptide O +inhibitors O +, O +explaining O +well O +how O +the O +evolution O +of O +the O +initial O +phage B-experimental_method +peptide B-chemical +1 I-chemical +to O +HAP B-chemical +and O +45 B-chemical +improved O +its O +potency O +( O +Supplementary O +Figure O +S7 O +). O + +The O +important O +interactions O +involving O +Trp12 B-residue_name_number +of O +HAP B-chemical +explain O +the O +> O +90 O +times O +drop O +in O +potency O +of O +the O +W12A B-mutant +variant O +( O +6 O +vs O +1 O +, O +Table O +1 O +). O + +The O +amphipathic B-protein_state +nature O +of O +the O +HAP B-chemical +β B-structure_element +- I-structure_element +strand I-structure_element +explains O +the O +preference O +of O +the O +hydrophilic O +residues O +at O +the O +2 B-residue_number +and O +4 B-residue_number +positions O +of O +peptides O +( O +14 B-chemical +, O +18 B-chemical +, O +19 B-chemical +, O +21 B-chemical +and O +23 B-chemical +vs O +1 B-chemical +and O +22 B-chemical +, O +Table O +1 O +). O + +All O +N O +- O +terminal O +residues O +of O +HAP B-chemical +are O +part O +of O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +with O +β B-structure_element +- I-structure_element +stands I-structure_element +0 I-structure_element +and I-structure_element +4 I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +, O +which O +explains O +why O +removal B-experimental_method +of I-experimental_method +the O +first B-residue_range +1 I-residue_range +– I-residue_range +3 I-residue_range +residues I-residue_range +completely O +abolishes O +the O +ability O +of O +HAP B-chemical +to O +block O +IL B-protein +- I-protein +17A I-protein +cell O +signaling O +( O +31 B-chemical +, O +32 B-chemical +and O +33 B-chemical +, O +Table O +2 O +). O + +Each O +peptide O +monomer B-oligomeric_state +in O +45 B-chemical +may O +not O +necessarily O +be O +more O +potent O +than O +HAP B-chemical +, O +but O +two O +monomer B-oligomeric_state +peptides O +within O +the O +same O +molecule O +that O +can O +simultaneously O +bind O +to O +IL B-protein +- I-protein +17A I-protein +can O +greatly O +improve O +its O +potency O +due O +to O +avidity O +effects O +. O + +HAP B-chemical +targets O +region B-structure_element +I I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +, O +an O +area O +that O +has O +the O +least O +sequence O +conservation O +in O +IL B-protein_type +- I-protein_type +17 I-protein_type +cytokines I-protein_type +. O + +This O +lack O +of O +sequence O +conservation O +in O +the O +HAP B-site +binding I-site +site I-site +explains O +the O +observed O +specificity O +of O +HAP B-chemical +binding O +to O +human B-species +IL B-protein +- I-protein +17A I-protein +. O + +This O +Phe B-structure_element +- I-structure_element +Phe I-structure_element +motif I-structure_element +is O +missing B-protein_state +in O +IL B-protein +- I-protein +17A I-protein +. O + +Sequence B-experimental_method +alignments I-experimental_method +between O +human B-species +and O +mouse B-taxonomy_domain +IL B-protein +- I-protein +17A I-protein +indicated O +that O +among O +IL B-protein +- I-protein +17A I-protein +residues O +that O +interacting O +with O +HAP B-chemical +, O +majority O +differences O +occur O +in O +strand B-structure_element +0 I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +which O +interacts O +with O +the O +N O +- O +terminal O +β B-structure_element +- I-structure_element +strand I-structure_element +of O +HAP B-chemical +. O + +In O +human B-species +IL B-protein +- I-protein +17A I-protein +the O +sequences O +are O +21TVMVNLNI B-chemical +, O +and O +in O +mouse B-taxonomy_domain +they O +are O +21NVKVNLKV B-chemical +. O + +Using O +a O +combination O +of O +phage B-experimental_method +display I-experimental_method +and O +SAR B-experimental_method +we O +have O +discovered O +novel O +peptides O +that O +are O +IL B-protein +- I-protein +17A I-protein +antagonists O +. O + +One O +of O +those O +peptides O +, O +HAP B-chemical +, O +also O +shows O +activity O +in O +inhibiting O +the O +production O +of O +multiple O +inflammatory O +cytokines B-protein_type +by O +primary O +human B-species +keratinocytes O +stimulated O +by O +IL B-protein +- I-protein +17A I-protein +and O +TNF B-protein +- I-protein +α I-protein +, O +a O +disease O +relevant O +- O +model O +. O + +With O +two O +HAP B-chemical +molecules O +covering O +both O +faces O +of O +the O +IL B-protein +- I-protein +17A I-protein +dimer B-oligomeric_state +, O +HAP B-chemical +can O +block O +IL B-protein +- I-protein +17RA I-protein +approaching O +from O +either O +face O +. O + +To O +form O +the O +1 O +: O +2 O +complex O +observed O +in O +crystal B-evidence +structure I-evidence +, O +it O +is O +important O +that O +there O +is O +no O +strong O +negative O +cooperativity O +in O +the O +binding O +of O +two O +HAP B-chemical +molecules O +. O + +In O +fact O +, O +in O +native B-experimental_method +electrospray I-experimental_method +ionization I-experimental_method +mass I-experimental_method +spectrometry I-experimental_method +analysis O +only O +1 O +: O +2 O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O +was O +observed O +even O +when O +IL B-protein +- I-protein +17A I-protein +was O +in O +excess O +( O +Supplementary O +Figure O +S8 O +), O +indicating O +a O +positive O +binding O +cooperativity O +that O +favors O +inhibition O +of O +IL B-protein +- I-protein +17RA I-protein +binding O +by O +HAP B-chemical +. O + +Due O +to O +the O +discontinuous O +nature O +of O +the O +IL B-site +- I-site +17A I-site +/ I-site +IL I-site +- I-site +17RA I-site +binding I-site +interface I-site +, O +it O +is O +classified O +as O +having O +tertiary O +structural O +epitopes O +on O +both O +binding O +partners O +, O +and O +is O +therefore O +hard O +to O +target O +using O +small O +molecules O +. O + +Our O +studies O +of O +HAP B-chemical +demonstrated O +an O +uncommon O +mode O +of O +action O +for O +a O +peptide O +in O +inhibiting O +such O +a O +difficult O +protein O +- O +protein O +interaction O +target O +, O +and O +suggest O +further O +possible O +improvements O +in O +its O +binding O +potency O +. O + +Homo O +- O +dimerization O +of O +HAP B-chemical +( O +45 B-chemical +) O +achieved O +sub O +- O +nanomolar O +potency O +against O +human B-species +IL B-protein +- I-protein +17A I-protein +in O +cell O +assay O +. O + +In O +the O +crystal B-evidence +structure I-evidence +, O +the O +distance O +between O +the O +carbonyl O +of O +Asn14 B-residue_name_number +of O +one O +HAP B-chemical +molecule O +and O +the O +N O +- O +terminus O +of O +the O +second O +is O +only O +15 O +. O +7 O +Å O +, O +suggesting O +the O +potential O +for O +more O +potent O +dimeric B-oligomeric_state +peptides B-chemical +to O +be O +designed O +by O +using O +linkers O +of O +different O +lengths O +at O +different O +positions O +. O + +Another O +direction O +of O +improving O +HAP B-chemical +is O +by O +reducing O +its O +size O +. O + +In O +summary O +, O +these O +peptide O +- O +based O +anti O +- O +IL B-protein +- I-protein +17A I-protein +modalities O +could O +be O +further O +developed O +as O +alternative O +therapeutic O +options O +to O +the O +reported O +monoclonal O +antibodies B-protein_type +. O + +We O +are O +also O +very O +interested O +in O +finding O +non O +- O +peptidic O +small O +molecule O +IL B-protein +- I-protein +17A I-protein +antagonists O +, O +and O +HAP B-chemical +can O +be O +used O +as O +an O +excellent O +tool O +peptide O +. O + +The O +strategy O +utilized O +in O +generating O +the O +complex O +structures B-evidence +of O +HAP B-chemical +may O +also O +be O +useful O +for O +enabling O +structure O +based O +design O +of O +some O +known O +small O +molecule O +IL O +- O +17A O +antagonists O +. O + +Binding O +of O +HAP B-chemical +to O +IL B-protein +- I-protein +17A I-protein +and O +inhibition O +of O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +are O +measured O +by O +SPR B-experimental_method +, O +FRET B-experimental_method +and O +cell B-experimental_method +- I-experimental_method +based I-experimental_method +assays I-experimental_method +. O + +( O +A O +) O +Typical O +SPR B-experimental_method +sensorgrams B-evidence +( O +black O +) O +of O +HAP B-chemical +at O +indicated O +concentrations O +binding O +to O +biotinylated B-protein_state +human B-species +IL B-protein +- I-protein +17A I-protein +immobilized O +on O +a O +streptavidin O +chip O +surface O +, O +fitted O +with O +single B-evidence +site I-evidence +binding I-evidence +model I-evidence +curves I-evidence +( O +red O +). O + +Kinetic O +parameters O +( O +ka B-evidence +, O +kd B-evidence +) O +were O +obtained O +by O +a O +global O +fit O +using O +three O +concentrations O +in O +triplicate O +. O + +Data O +are O +mean O +and O +error O +bars O +of O ++/− O +standard O +deviation O +of O +three O +measurements O +. O +( O +C O +) O +Inhibition O +of O +IL B-protein +- I-protein +17A I-protein +and O +IL B-protein +- I-protein +17RA I-protein +binding O +by O +HAP B-chemical +measured O +by O +FRET B-experimental_method +assay I-experimental_method +. O + +Data O +are O +mean O +and O +error O +bars O +of O ++/− O +standard O +deviation O +from O +299 O +experiments O +, O +each O +performed O +in O +duplicate O +. O +( O +D O +) O +Example O +of O +HAP B-chemical +selective O +inhibition O +of O +the O +production O +of O +IL B-protein_type +- I-protein_type +8 I-protein_type +( O +triangles O +), O +IL B-protein_type +- I-protein_type +6 I-protein_type +( O +squares O +) O +and O +CCL B-protein_type +- I-protein_type +20 I-protein_type +( O +circles O +) O +by O +primary O +human B-species +keratinocyte O +cells O +synergistically O +stimulated O +by O +100 O +ng O +/ O +ml O +IL B-protein +- I-protein +17A I-protein +and O +10 O +ng O +/ O +ml O +TNF B-protein +- I-protein +α I-protein +. O + +HAP B-chemical +does O +not O +inhibit O +the O +baseline O +production O +of O +IL B-protein_type +- I-protein_type +6 I-protein_type +, O +IL B-protein_type +- I-protein_type +8 I-protein_type +and O +CCL B-protein_type +- I-protein_type +20 I-protein_type +stimulated O +by O +10 O +ng O +/ O +ml O +TNF B-protein +- I-protein +α I-protein +alone O +( O +gray O +lines O +and O +symbols O +). O + +Two O +HAP B-chemical +molecules O +are O +colored O +blue O +and O +red O +, O +and O +IL B-protein +- I-protein +17A I-protein +monomers B-oligomeric_state +are O +colored O +ice O +blue O +and O +pink O +, O +respectively O +. O + +( O +A O +) O +Overview O +of O +the O +distinct O +binding B-site +sites I-site +of O +Fab B-structure_element +and O +HAP B-chemical +to O +IL B-protein +- I-protein +17A I-protein +. O + +( O +B O +) O +Close O +- O +in O +view O +of O +the O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +structure B-evidence +. O + +IL B-protein +- I-protein +17A I-protein +β B-structure_element +- I-structure_element +strands I-structure_element +are O +labelled O +. O + +Each O +of O +the O +two O +bound B-protein_state +HAP B-chemical +interacts O +with O +both O +monomers B-oligomeric_state +of O +the O +IL B-protein +- I-protein +17A I-protein +dimer B-oligomeric_state +. O + +Three O +distinct O +areas O +IL B-site +- I-site +17A I-site +/ I-site +IL I-site +- I-site +17RA I-site +interface I-site +are O +labeled O +. O + +Mechanism O +of O +the O +inhibition O +of O +the O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +IL I-complex_assembly +- I-complex_assembly +17RA I-complex_assembly +interaction O +by O +HAP B-chemical +. O + +IL B-protein +- I-protein +17A I-protein +dimer B-oligomeric_state +is O +in O +surface O +presentation O +( O +β B-structure_element +- I-structure_element +strands I-structure_element +0 I-structure_element +shown O +as O +ribbons O +for O +clarity O +). O + +HAP B-chemical +residues O +as O +well O +as O +key O +IL B-protein +- I-protein +17A I-protein +residues O +are O +labeled O +. O + +For O +clarity O +, O +a O +few O +HAP B-chemical +residues O +are O +also O +shown O +in O +stick O +model O +with O +carbon O +atoms O +colored O +green O +, O +oxygen O +in O +red O +and O +nitrogen O +in O +blue O +. O + +( O +B O +) O +I B-protein +- I-protein +17RA I-protein +( O +ribbon O +in O +gold O +) O +peptide O +Leu27 B-residue_range +- I-residue_range +Ile32 I-residue_range +binds O +to O +the O +same O +area O +as O +the O +HAP B-chemical +α B-structure_element +- I-structure_element +helix I-structure_element +. O + +Trp31 B-residue_name_number +of O +IL B-protein +- I-protein +17RA I-protein +binds O +to O +the O +same O +pocket B-site +in O +IL B-protein +- I-protein +17A I-protein +as O +Trp12 B-residue_name_number +of O +HAP B-chemical +. O +( O +C O +) O +As O +illustrated O +by O +overlay B-experimental_method +a O +single O +HAP B-chemical +molecule O +and O +β B-structure_element +- I-structure_element +strands I-structure_element +0 I-structure_element +( O +grey O +) O +of O +the O +IL B-complex_assembly +- I-complex_assembly +17A I-complex_assembly +/ I-complex_assembly +HAP I-complex_assembly +complex O +in O +the O +apo B-protein_state +IL B-protein +- I-protein +17A I-protein +structure B-evidence +, O +conformational O +changes O +in O +region B-structure_element +I I-structure_element +of O +IL B-protein +- I-protein +17A I-protein +are O +needed O +for O +binding O +of O +both O +the O +β B-structure_element +- I-structure_element +stand I-structure_element +and O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +the O +HAP B-chemical +. O + +Molecular O +Basis O +of O +Ligand O +- O +Dependent O +Regulation O +of O +NadR B-protein +, O +the O +Transcriptional B-protein_type +Repressor I-protein_type +of O +Meningococcal B-taxonomy_domain +Virulence O +Factor O +NadA B-protein + +Neisseria B-protein +adhesin I-protein +A I-protein +( O +NadA B-protein +) O +is O +present O +on O +the O +meningococcal B-taxonomy_domain +surface O +and O +contributes O +to O +adhesion O +to O +and O +invasion O +of O +human B-species +cells O +. O + +NadA B-protein +is O +also O +one O +of O +three O +recombinant O +antigens O +in O +the O +recently O +- O +approved O +Bexsero O +vaccine O +, O +which O +protects O +against O +serogroup B-taxonomy_domain +B I-taxonomy_domain +meningococcus I-taxonomy_domain +. O + +In O +the O +presence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +hydroxyphenylacetate I-chemical +( O +4 B-chemical +- I-chemical +HPA I-chemical +), O +a O +catabolite O +present O +in O +human B-species +saliva O +both O +under O +physiological O +conditions O +and O +during O +bacterial B-taxonomy_domain +infection O +, O +the O +binding O +of O +NadR B-protein +to O +the O +nadA B-gene +promoter O +is O +attenuated O +and O +nadA B-gene +expression O +is O +induced O +. O + +To O +gain O +insights O +into O +the O +regulation O +of O +NadR B-protein +mediated O +by O +4 B-chemical +- I-chemical +HPA I-chemical +, O +we O +combined O +structural B-experimental_method +, I-experimental_method +biochemical I-experimental_method +, I-experimental_method +and I-experimental_method +mutagenesis I-experimental_method +studies I-experimental_method +. O + +In O +particular O +, O +two O +new O +crystal B-evidence +structures I-evidence +of O +ligand B-protein_state +- I-protein_state +free I-protein_state +and O +ligand B-protein_state +- I-protein_state +bound I-protein_state +NadR B-protein +revealed O +( O +i O +) O +the O +molecular O +basis O +of O +‘ O +conformational O +selection O +’ O +by O +which O +a O +single O +molecule O +of O +4 B-chemical +- I-chemical +HPA I-chemical +binds O +and O +stabilizes O +dimeric B-oligomeric_state +NadR B-protein +in O +a O +conformation O +unsuitable O +for O +DNA O +- O +binding O +, O +( O +ii O +) O +molecular O +explanations O +for O +the O +binding O +specificities O +of O +different O +hydroxyphenylacetate B-chemical +ligands O +, O +including O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +which O +is O +produced O +during O +inflammation O +, O +( O +iii O +) O +the O +presence O +of O +a O +leucine B-residue_name +residue O +essential O +for O +dimerization O +and O +conserved B-protein_state +in O +many O +MarR B-protein_type +family O +proteins O +, O +and O +( O +iv O +) O +four O +residues O +( O +His7 B-residue_name_number +, O +Ser9 B-residue_name_number +, O +Asn11 B-residue_name_number +and O +Phe25 B-residue_name_number +), O +which O +are O +involved O +in O +binding O +4 B-chemical +- I-chemical +HPA I-chemical +, O +and O +were O +confirmed O +in O +vitro O +to O +have O +key O +roles O +in O +the O +regulatory O +mechanism O +in O +bacteria B-taxonomy_domain +. O + +Overall O +, O +this O +study O +deepens O +our O +molecular O +understanding O +of O +the O +sophisticated O +regulatory O +mechanisms O +of O +the O +expression O +of O +nadA B-gene +and O +other O +genes O +governed O +by O +NadR B-protein +, O +dependent O +on O +interactions O +with O +niche O +- O +specific O +signal O +molecules O +that O +may O +play O +important O +roles O +during O +meningococcal B-taxonomy_domain +pathogenesis O +. O + +The O +Bexsero O +vaccine O +protects O +against O +MenB B-species +and O +has O +recently O +been O +approved O +in O +> O +35 O +countries O +worldwide O +. O + +Neisseria B-protein +adhesin I-protein +A I-protein +( O +NadA B-protein +) O +present O +on O +the O +meningococcal B-taxonomy_domain +surface O +can O +mediate O +binding O +to O +human B-species +cells O +and O +is O +one O +of O +the O +three O +MenB B-species +vaccine O +protein O +antigens O +. O + +A O +deep O +understanding O +of O +nadA B-gene +expression O +is O +therefore O +important O +, O +otherwise O +the O +contribution O +of O +NadA B-protein +to O +vaccine O +- O +induced O +protection O +against O +meningococcal B-taxonomy_domain +meningitis O +may O +be O +underestimated O +. O + +These O +findings O +shed O +light O +on O +the O +regulation O +of O +NadR B-protein +, O +a O +key O +MarR B-protein_type +- O +family O +virulence O +factor O +of O +this O +important O +human B-species +pathogen O +. O + +The O +‘ O +Reverse B-experimental_method +Vaccinology I-experimental_method +’ O +approach O +was O +pioneered O +to O +identify O +antigens O +for O +a O +protein O +- O +based O +vaccine O +against O +serogroup B-species +B I-species +Neisseria I-species +meningitidis I-species +( O +MenB B-species +), O +a O +human B-species +pathogen O +causing O +potentially O +- O +fatal O +sepsis O +and O +invasive O +meningococcal B-taxonomy_domain +disease O +. O + +Indeed O +, O +Reverse B-experimental_method +Vaccinology I-experimental_method +identified O +Neisseria B-protein +adhesin I-protein +A I-protein +( O +NadA B-protein +), O +a O +surface O +- O +exposed O +protein O +involved O +in O +epithelial O +cell O +invasion O +and O +found O +in O +~ O +30 O +% O +of O +clinical O +isolates O +. O + +Recently O +, O +we O +reported O +the O +crystal B-evidence +structure I-evidence +of O +NadA B-protein +, O +providing O +insights O +into O +its O +biological O +and O +immunological O +functions O +. O + +Recombinant O +NadA B-protein +elicits O +a O +strong O +bactericidal O +immune O +response O +and O +is O +therefore O +included O +in O +the O +Bexsero O +vaccine O +that O +protects O +against O +MenB B-species +and O +which O +was O +recently O +approved O +in O +over O +35 O +countries O +worldwide O +. O + +Previous O +studies O +revealed O +that O +nadA B-gene +expression O +levels O +are O +mainly O +regulated O +by O +the O +Neisseria B-protein +adhesin I-protein +A I-protein +Regulator I-protein +( O +NadR B-protein +). O + +Studies O +of O +NadR B-protein +also O +have O +broader O +implications O +, O +since O +a O +genome O +- O +wide O +analysis O +of O +MenB B-species +wild B-protein_state +- I-protein_state +type I-protein_state +and O +nadR B-gene +knock B-protein_state +- I-protein_state +out I-protein_state +strains O +revealed O +that O +NadR B-protein +influences O +the O +regulation O +of O +> O +30 O +genes O +, O +including O +maf O +genes O +, O +from O +the O +multiple O +adhesin B-protein_type +family O +. O + +These O +genes O +encode O +a O +wide O +variety O +of O +proteins O +connected O +to O +many O +biological O +processes O +contributing O +to O +bacterial B-taxonomy_domain +survival O +, O +adaptation O +in O +the O +host O +niche O +, O +colonization O +and O +invasion O +. O + +NadR B-protein +belongs O +to O +the O +MarR B-protein_type +( O +Multiple B-protein_type +Antibiotic I-protein_type +Resistance I-protein_type +Regulator I-protein_type +) O +family O +, O +a O +group O +of O +ligand B-protein_type +- I-protein_type +responsive I-protein_type +transcriptional I-protein_type +regulators I-protein_type +ubiquitous O +in O +bacteria B-taxonomy_domain +and O +archaea B-taxonomy_domain +. O + +MarR B-protein_type +family O +proteins O +can O +promote O +bacterial B-taxonomy_domain +survival O +in O +the O +presence O +of O +antibiotics O +, O +toxic O +chemicals O +, O +organic O +solvents O +or O +reactive O +oxygen O +species O +and O +can O +regulate O +virulence O +factor O +expression O +. O + +MarR B-protein_type +homologues O +can O +act O +either O +as O +transcriptional O +repressors O +or O +as O +activators O +. O + +Although O +> O +50 O +MarR B-protein_type +family O +structures B-evidence +are O +known O +, O +a O +molecular O +understanding O +of O +their O +ligand O +- O +dependent O +regulatory O +mechanisms O +is O +still O +limited O +, O +often O +hampered O +by O +lack O +of O +identification O +of O +their O +ligands O +and O +/ O +or O +DNA O +targets O +. O + +A O +potentially O +interesting O +exception O +comes O +from O +the O +ligand B-protein_state +- I-protein_state +free I-protein_state +and O +salicylate B-protein_state +- I-protein_state +bound I-protein_state +forms O +of O +the O +Methanobacterium B-species +thermoautotrophicum I-species +protein O +MTH313 B-protein +which O +revealed O +that O +two O +salicylate B-chemical +molecules O +bind O +to O +one O +MTH313 B-protein +dimer B-oligomeric_state +and O +induce O +large O +conformational O +changes O +, O +apparently O +sufficient O +to O +prevent O +DNA O +binding O +. O + +However O +, O +it O +is O +unknown O +whether O +salicylate B-chemical +is O +a O +relevant O +in O +vivo O +ligand O +of O +either O +of O +these O +two O +proteins O +, O +which O +share O +~ O +20 O +% O +sequence O +identity O +with O +NadR B-protein +, O +rendering O +unclear O +the O +interpretation O +of O +these O +findings O +in O +relation O +to O +the O +regulatory O +mechanisms O +of O +NadR B-protein +or O +other O +MarR B-protein_type +family O +proteins O +. O + +NadR B-protein +binds O +nadA B-gene +on O +three O +different O +operators O +( O +OpI O +, O +OpII O +and O +OpIII O +). O + +4 B-chemical +- I-chemical +HPA I-chemical +is O +a O +small O +molecule O +derived O +from O +mammalian B-taxonomy_domain +aromatic O +amino O +acid O +catabolism O +and O +is O +released O +in O +human B-species +saliva O +, O +where O +it O +has O +been O +detected O +at O +micromolar O +concentration O +. O + +In O +the O +presence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +, O +NadR B-protein +is O +unable O +to O +bind O +the O +nadA B-gene +promoter O +and O +nadA B-gene +gene O +expression O +is O +induced O +. O + +In O +vivo O +, O +the O +presence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +in O +the O +host O +niche O +of O +N B-species +. I-species +meningitidis I-species +serves O +as O +an O +inducer O +of O +NadA B-protein +production O +, O +thereby O +promoting O +bacterial B-taxonomy_domain +adhesion O +to O +host O +cells O +. O + +Further O +, O +we O +recently O +reported O +that O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +, O +produced O +during O +inflammation O +, O +is O +another O +inducer O +of O +nadA B-gene +expression O +. O + +Extending O +our O +previous O +studies O +based O +on O +hydrogen B-experimental_method +- I-experimental_method +deuterium I-experimental_method +exchange I-experimental_method +mass I-experimental_method +spectrometry I-experimental_method +( O +HDX B-experimental_method +- I-experimental_method +MS I-experimental_method +), O +here O +we O +sought O +to O +reveal O +the O +molecular O +mechanisms O +and O +effects O +of O +NadR B-protein +/ O +HPA B-chemical +interactions O +via O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +, O +NMR B-experimental_method +spectroscopy I-experimental_method +and O +complementary O +biochemical B-experimental_method +and I-experimental_method +in I-experimental_method +vivo I-experimental_method +mutagenesis I-experimental_method +studies I-experimental_method +. O + +Standard O +chromatographic O +techniques O +were O +used O +to O +obtain O +a O +highly O +purified O +sample O +of O +NadR B-protein +( O +see O +Materials O +and O +Methods O +). O + +These O +data O +showed O +that O +NadR B-protein +was O +dimeric B-oligomeric_state +in O +solution O +, O +since O +the O +theoretical O +molecular O +mass O +of O +the O +NadR B-protein +dimer B-oligomeric_state +is O +33 O +. O +73 O +kDa O +; O +and O +, O +there O +was O +no O +change O +in O +oligomeric O +state O +on O +addition O +of O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +The O +thermal O +stability O +of O +NadR B-protein +was O +examined O +using O +differential B-experimental_method +scanning I-experimental_method +calorimetry I-experimental_method +( O +DSC B-experimental_method +). O + +The O +Tm B-evidence +of O +NadR B-protein +was O +67 O +. O +4 O +± O +0 O +. O +1 O +° O +C O +in O +the O +absence B-protein_state +of I-protein_state +ligand I-protein_state +, O +and O +was O +unaffected O +by O +salicylate B-chemical +. O + +However O +, O +an O +increased O +thermal O +stability O +was O +induced O +by O +4 B-chemical +- I-chemical +HPA I-chemical +and O +, O +to O +a O +lesser O +extent O +, O +by O +3 B-chemical +- I-chemical +HPA I-chemical +. O + +Interestingly O +, O +NadR B-protein +displayed O +the O +greatest O +Tm B-evidence +increase O +upon O +addition O +of O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +( O +Table O +1 O +and O +Fig O +1B O +). O + +Stability O +of O +NadR B-protein +is O +increased O +by O +small O +molecule O +ligands O +. O + +Melting B-evidence +- I-evidence +point I-evidence +( O +Tm B-evidence +) O +and O +its O +ligand O +- O +induced O +increase O +( O +ΔTm B-evidence +) O +derived O +from O +DSC B-experimental_method +thermostability B-experimental_method +experiments I-experimental_method +. O + +Dissociation B-evidence +constants I-evidence +( O +KD B-evidence +) O +of O +the O +NadR B-protein +/ O +ligand O +interactions O +from O +SPR B-experimental_method +steady I-experimental_method +- I-experimental_method +state I-experimental_method +binding I-experimental_method +experiments I-experimental_method +. O + +Ligand O +Tm B-evidence +(° O +C O +) O +ΔTm B-evidence +(° O +C O +) O +KD B-evidence +( O +mM O +) O +No O +ligand O +67 O +. O +4 O +± O +0 O +. O +1 O +n O +. O +a O +. O +n O +. O +a O +. O + +NadR B-protein +displays O +distinct O +binding B-evidence +affinities I-evidence +for O +hydroxyphenylacetate B-chemical +ligands O + +The O +SPR B-experimental_method +sensorgrams B-evidence +revealed O +very O +fast O +association O +and O +dissociation O +events O +, O +typical O +of O +small O +molecule O +ligands O +, O +thus O +prohibiting O +a O +detailed O +study O +of O +binding O +kinetics O +. O + +3 B-chemical +- I-chemical +HPA I-chemical +showed O +a O +weaker O +interaction O +, O +with O +a O +KD B-evidence +of O +2 O +. O +7 O +mM O +, O +while O +salicylate B-chemical +showed O +only O +a O +very O +weak O +response O +that O +did O +not O +reach O +saturation O +, O +indicating O +a O +non O +- O +specific O +interaction O +with O +NadR B-protein +. O +A O +ranking O +of O +these O +KD B-evidence +values O +showed O +that O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +was O +the O +tightest O +binder O +, O +and O +thus O +matched O +the O +ranking O +of O +ligand O +- O +induced O +Tm B-evidence +increases O +observed O +in O +the O +DSC B-experimental_method +experiments O +. O + +Although O +these O +KD B-evidence +values O +indicate O +rather O +weak O +interactions O +, O +they O +are O +similar O +to O +the O +values O +reported O +previously O +for O +the O +MarR B-protein_type +/ O +salicylate B-chemical +interaction O +( O +KD O +~ O +1 O +mM O +) O +and O +the O +MTH313 B-protein +/ O +salicylate B-chemical +interaction O +( O +KD O +2 O +– O +3 O +mM O +), O +and O +approximately O +20 O +- O +fold O +tighter O +than O +the O +ST1710 B-protein +/ O +salicylate B-chemical +interaction O +( O +KD O +~ O +20 O +mM O +). O + +Crystal B-evidence +structures I-evidence +of O +holo B-protein_state +- O +NadR B-protein +and O +apo B-protein_state +- O +NadR B-protein + +First O +, O +we O +crystallized B-experimental_method +NadR B-protein +( O +a O +selenomethionine B-experimental_method +- I-experimental_method +labelled I-experimental_method +derivative I-experimental_method +) O +in O +the O +presence O +of O +a O +200 O +- O +fold O +molar O +excess O +of O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +The O +structure B-evidence +of O +the O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +complex O +was O +determined O +at O +2 O +. O +3 O +Å O +resolution O +using O +a O +combination O +of O +the O +single B-experimental_method +- I-experimental_method +wavelength I-experimental_method +anomalous I-experimental_method +dispersion I-experimental_method +( O +SAD B-experimental_method +) O +and O +molecular B-experimental_method +replacement I-experimental_method +( O +MR B-experimental_method +) O +methods O +, O +and O +was O +refined O +to O +R B-evidence +work I-evidence +/ I-evidence +R I-evidence +free I-evidence +values O +of O +20 O +. O +9 O +/ O +26 O +. O +0 O +% O +( O +Table O +2 O +). O + +Despite O +numerous O +attempts O +, O +we O +were O +unable O +to O +obtain O +high O +- O +quality O +crystals B-evidence +of O +NadR B-protein +complexed B-protein_state +with I-protein_state +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +, O +3 B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +, O +3 B-chemical +- I-chemical +HPA I-chemical +or O +DNA O +targets O +. O + +However O +, O +it O +was O +eventually O +possible O +to O +crystallize B-experimental_method +apo B-protein_state +- O +NadR B-protein +, O +and O +the O +structure B-evidence +was O +determined O +at O +2 O +. O +7 O +Å O +resolution O +by O +MR B-experimental_method +methods O +using O +the O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +complex O +as O +the O +search O +model O +. O + +The O +apo B-protein_state +- O +NadR B-protein +structure B-evidence +was O +refined O +to O +R B-evidence +work I-evidence +/ I-evidence +R I-evidence +free I-evidence +values O +of O +19 O +. O +1 O +/ O +26 O +. O +8 O +% O +( O +Table O +2 O +). O + +The O +asymmetric O +unit O +of O +the O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +crystals B-evidence +( O +holo B-protein_state +- O +NadR B-protein +) O +contained O +one O +NadR B-protein +homodimer B-oligomeric_state +, O +while O +the O +apo B-protein_state +- O +NadR B-protein +crystals B-evidence +contained O +two O +homodimers B-oligomeric_state +. O + +Moreover O +, O +our O +SE B-experimental_method +- I-experimental_method +HPLC I-experimental_method +/ I-experimental_method +MALLS I-experimental_method +analyses O +( O +see O +above O +) O +revealed O +that O +in O +solution O +NadR B-protein +is O +dimeric B-oligomeric_state +, O +and O +previous O +studies O +using O +native B-experimental_method +mass I-experimental_method +spectrometry I-experimental_method +( O +MS B-experimental_method +) O +revealed O +dimers B-oligomeric_state +, O +not O +tetramers B-oligomeric_state +. O + +The O +NadR B-protein +homodimer B-oligomeric_state +bound B-protein_state +to I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +has O +a O +dimerization B-site +interface I-site +mostly O +involving O +the O +top O +of O +its O +‘ O +triangular B-protein_state +’ O +form O +, O +while O +the O +two O +DNA B-structure_element +- I-structure_element +binding I-structure_element +domains I-structure_element +are O +located O +at O +the O +base O +( O +Fig O +2A O +). O + +High O +- O +quality O +electron B-evidence +density I-evidence +maps I-evidence +allowed O +clear O +identification O +of O +the O +bound B-protein_state +ligand O +, O +4 B-chemical +- I-chemical +HPA I-chemical +( O +Fig O +2B O +). O + +The O +overall O +structure B-evidence +of O +NadR B-protein +shows O +dimensions O +of O +~ O +50 O +× O +65 O +× O +50 O +Å O +and O +a O +large O +homodimer B-site +interface I-site +that O +buries O +a O +total O +surface O +area O +of O +~ O +4800 O +Å2 O +. O + +Each O +NadR B-protein +monomer B-oligomeric_state +consists O +of O +six O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +two O +short B-structure_element +β I-structure_element +- I-structure_element +strands I-structure_element +, O +with O +helices B-structure_element +α1 B-structure_element +, O +α5 B-structure_element +, O +and O +α6 B-structure_element +forming O +the O +dimer B-site +interface I-site +. O + +Together O +, O +these O +structural O +elements O +constitute O +the O +winged B-structure_element +helix I-structure_element +- I-structure_element +turn I-structure_element +- I-structure_element +helix I-structure_element +( O +wHTH B-structure_element +) O +DNA B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +and O +, O +together O +with O +the O +dimeric B-oligomeric_state +organization O +, O +are O +the O +hallmarks O +of O +MarR B-protein_type +family O +structures B-evidence +. O + +The O +crystal B-evidence +structure I-evidence +of O +NadR B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +. O + +( O +A O +) O +The O +holo B-protein_state +- O +NadR B-protein +homodimer B-oligomeric_state +is O +depicted O +in O +green O +and O +blue O +for O +chains B-structure_element +A I-structure_element +and I-structure_element +B I-structure_element +respectively O +, O +while O +yellow O +sticks O +depict O +the O +4 B-chemical +- I-chemical +HPA I-chemical +ligand O +( O +labelled O +). O + +For O +simplicity O +, O +secondary O +structure O +elements O +are O +labelled O +for O +chain B-structure_element +B I-structure_element +only O +. O + +Red O +dashes O +show O +hypothetical O +positions O +of O +chain B-structure_element +B I-structure_element +residues O +88 B-residue_range +– I-residue_range +90 I-residue_range +that O +were O +not O +modeled O +due O +to O +lack O +of O +electron B-evidence +density I-evidence +. O + +( O +B O +) O +A O +zoom O +into O +the O +pocket B-site +occupied O +by O +4 B-chemical +- I-chemical +HPA I-chemical +shows O +that O +the O +ligand O +contacts O +both O +chains B-structure_element +A I-structure_element +and I-structure_element +B I-structure_element +; O +blue O +mesh O +shows O +electron B-evidence +density I-evidence +around O +4 B-chemical +- I-chemical +HPA I-chemical +calculated O +from O +a O +composite B-evidence +omit I-evidence +map I-evidence +( O +omitting O +4 B-chemical +- I-chemical +HPA I-chemical +), O +using O +phenix B-experimental_method +. O + +A O +single O +conserved B-protein_state +leucine B-residue_name +residue O +( O +L130 B-residue_name_number +) O +is O +crucial O +for O +dimerization O + +The O +NadR B-protein +dimer B-site +interface I-site +is O +formed O +by O +at O +least O +32 O +residues O +, O +which O +establish O +numerous O +inter O +- O +chain O +salt B-bond_interaction +bridges I-bond_interaction +or O +hydrogen B-bond_interaction +bonds I-bond_interaction +, O +and O +many O +hydrophobic B-bond_interaction +packing I-bond_interaction +interactions I-bond_interaction +( O +Fig O +3A O +and O +3B O +). O + +To O +determine O +which O +residues O +were O +most O +important O +for O +dimerization O +, O +we O +studied O +the O +interface B-site +in O +silico O +and O +identified O +several O +residues O +as O +potential O +mediators O +of O +key O +stabilizing O +interactions O +. O + +Each O +mutant B-protein_state +NadR B-protein +protein O +was O +purified O +, O +and O +then O +its O +oligomeric O +state O +was O +examined O +by O +analytical B-experimental_method +SE I-experimental_method +- I-experimental_method +HPLC I-experimental_method +. O + +Almost O +all O +the O +mutants O +showed O +the O +same O +elution O +profile O +as O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +NadR B-protein +protein O +. O + +Only O +the O +L130K B-mutant +mutation O +induced O +a O +notable O +change O +in O +the O +oligomeric O +state O +of O +NadR B-protein +( O +Fig O +3C O +). O + +Further O +, O +in O +SE B-experimental_method +- I-experimental_method +MALLS I-experimental_method +analyses O +, O +the O +L130K B-mutant +mutant B-protein_state +displayed O +two O +distinct O +species O +in O +solution O +, O +approximately O +80 O +% O +being O +monomeric B-oligomeric_state +( O +a O +19 O +kDa O +species O +), O +and O +only O +20 O +% O +retaining O +the O +typical O +native O +dimeric B-oligomeric_state +state O +( O +a O +35 O +kDa O +species O +) O +( O +Fig O +3D O +), O +demonstrating O +that O +Leu130 B-residue_name_number +is O +crucial O +for O +stable O +dimerization O +. O + +In O +contrast O +, O +most O +of O +the O +other O +residues O +identified O +in O +the O +NadR B-protein +dimer B-site +interface I-site +were O +poorly B-protein_state +conserved I-protein_state +in O +the O +MarR B-protein_type +family O +. O + +Analysis O +of O +the O +NadR B-protein +dimer B-site +interface I-site +. O + +( O +A O +) O +Both O +orientations O +show O +chain B-structure_element +A I-structure_element +, O +green O +backbone O +ribbon O +, O +colored O +red O +to O +highlight O +all O +locations O +involved O +in O +dimerization O +; O +namely O +, O +inter O +- O +chain O +salt B-bond_interaction +bridges I-bond_interaction +or O +hydrogen B-bond_interaction +bonds I-bond_interaction +involving O +Q4 B-residue_name_number +, O +S5 B-residue_name_number +, O +K6 B-residue_name_number +, O +H7 B-residue_name_number +, O +S9 B-residue_name_number +, O +I10 B-residue_name_number +, O +N11 B-residue_name_number +, O +I15 B-residue_name_number +, O +Q16 B-residue_name_number +, O +R18 B-residue_name_number +, O +D36 B-residue_name_number +, O +R43 B-residue_name_number +, O +A46 B-residue_name_number +, O +Q59 B-residue_name_number +, O +C61 B-residue_name_number +, O +Y104 B-residue_name_number +, O +D112 B-residue_name_number +, O +R114 B-residue_name_number +, O +Y115 B-residue_name_number +, O +D116 B-residue_name_number +, O +E119 B-residue_name_number +, O +K126 B-residue_name_number +, O +E136 B-residue_name_number +, O +E141 B-residue_name_number +, O +N145 B-residue_name_number +, O +and O +the O +hydrophobic B-bond_interaction +packing I-bond_interaction +interactions I-bond_interaction +involving O +I10 B-residue_name_number +, O +I12 B-residue_name_number +, O +L14 B-residue_name_number +, O +I15 B-residue_name_number +, O +R18 B-residue_name_number +, O +Y115 B-residue_name_number +, O +I118 B-residue_name_number +, O +L130 B-residue_name_number +, O +L133 B-residue_name_number +, O +L134 B-residue_name_number +and O +L137 B-residue_name_number +. O + +Chain B-structure_element +B I-structure_element +, O +grey O +surface O +, O +is O +marked O +blue O +to O +highlight O +residues O +probed O +by O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +( O +E136 B-residue_name_number +only O +makes O +a O +salt B-bond_interaction +bridge I-bond_interaction +with O +K126 B-residue_name_number +, O +therefore O +it O +was O +sufficient O +to O +make O +the O +K126A B-mutant +mutation O +to O +assess O +the O +importance O +of O +this O +ionic B-bond_interaction +interaction I-bond_interaction +; O +the O +H7 B-residue_name_number +position O +is O +labelled O +for O +monomer B-oligomeric_state +A B-structure_element +, O +since O +electron B-evidence +density I-evidence +was O +lacking O +for O +monomer B-oligomeric_state +B B-structure_element +). O +( O +B O +) O +A O +zoom O +into O +the O +environment O +of O +helix B-structure_element +α6 B-structure_element +to O +show O +how O +residue O +L130 B-residue_name_number +chain B-structure_element +B I-structure_element +( O +blue O +side O +chain O +) O +is O +a O +focus O +of O +hydrophobic B-bond_interaction +packing I-bond_interaction +interactions I-bond_interaction +with O +L130 B-residue_name_number +, O +L133 B-residue_name_number +, O +L134 B-residue_name_number +and O +L137 B-residue_name_number +of O +chain B-structure_element +A I-structure_element +( O +red O +side O +chains O +). O + +( O +C O +) O +SE B-experimental_method +- I-experimental_method +HPLC I-experimental_method +analyses O +of O +all O +mutant B-protein_state +forms O +of O +NadR B-protein +are O +compared O +with O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +protein O +. O + +The O +WT B-protein_state +and O +most O +of O +the O +mutants O +show O +a O +single O +elution O +peak O +with O +an O +absorbance O +maximum O +at O +17 O +. O +5 O +min O +. O + +The O +holo B-protein_state +- O +NadR B-protein +structure B-evidence +presents O +only O +one O +occupied O +ligand B-site +- I-site +binding I-site +pocket I-site + +The O +tunnel B-site +was O +lined O +with O +rather O +hydrophobic O +amino O +acids O +, O +and O +did O +not O +contain O +water B-chemical +molecules O +. O + +Unexpectedly O +, O +only O +one O +monomer B-oligomeric_state +of O +the O +holo B-protein_state +- O +NadR B-protein +homodimer B-oligomeric_state +contained O +4 B-chemical +- I-chemical +HPA I-chemical +in O +the O +binding B-site +pocket I-site +, O +whereas O +the O +corresponding O +pocket B-site +of O +the O +other O +monomer B-oligomeric_state +was O +unoccupied O +by O +ligand O +, O +despite O +the O +large O +excess O +of O +4 B-chemical +- I-chemical +HPA I-chemical +used O +in O +the O +crystallization O +conditions O +. O + +Inspection O +of O +the O +protein B-site +- I-site +ligand I-site +interaction I-site +network I-site +revealed O +no O +bonds O +from O +NadR B-protein +backbone O +groups O +to O +the O +ligand O +, O +but O +several O +key O +side O +chain O +mediated O +hydrogen B-bond_interaction +( I-bond_interaction +H I-bond_interaction +)- I-bond_interaction +bonds I-bond_interaction +and O +ionic B-bond_interaction +interactions I-bond_interaction +, O +most O +notably O +between O +the O +carboxylate O +group O +of O +4 B-chemical +- I-chemical +HPA I-chemical +and O +Ser9 B-residue_name_number +of O +chain B-structure_element +A I-structure_element +( O +SerA9 B-residue_name_number +), O +and O +chain B-structure_element +B I-structure_element +residues O +TrpB39 B-residue_name_number +, O +ArgB43 B-residue_name_number +and O +TyrB115 B-residue_name_number +( O +Fig O +4A O +). O + +Atomic O +details O +of O +NadR B-protein +/ O +HPA B-chemical +interactions O +. O + +A O +) O +A O +stereo O +- O +view O +zoom O +into O +the O +binding B-site +pocket I-site +showing O +side O +chain O +sticks O +for O +all O +interactions O +between O +NadR B-protein +and O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +4 B-chemical +- I-chemical +HPA I-chemical +is O +shown O +in O +yellow O +sticks O +, O +with O +oxygen O +atoms O +in O +red O +. O + +A O +water B-chemical +molecule O +is O +shown O +by O +the O +red O +sphere O +. O + +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +up O +to O +3 O +. O +6Å O +are O +shown O +as O +dashed O +lines O +. O + +The O +entire O +set O +of O +residues O +making O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +or O +non B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +contacts I-bond_interaction +with O +4 B-chemical +- I-chemical +HPA I-chemical +is O +as O +follows O +: O +SerA9 B-residue_name_number +, O +AsnA11 B-residue_name_number +, O +LeuB21 B-residue_name_number +, O +MetB22 B-residue_name_number +, O +PheB25 B-residue_name_number +, O +LeuB29 B-residue_name_number +, O +AspB36 B-residue_name_number +, O +TrpB39 B-residue_name_number +, O +ArgB43 B-residue_name_number +, O +ValB111 B-residue_name_number +and O +TyrB115 B-residue_name_number +( O +automated O +analysis O +performed O +using O +PDBsum B-experimental_method +and O +verified O +manually O +). O + +Side O +chains O +mediating O +hydrophobic B-bond_interaction +interactions I-bond_interaction +are O +shown O +in O +orange O +. O +( O +B O +) O +A O +model O +was O +prepared O +to O +visualize O +putative O +interactions O +of O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +( O +pink O +) O +with O +NadR B-protein +, O +revealing O +the O +potential O +for O +additional O +contacts O +( O +dashed O +lines O +) O +of O +the O +chloro O +moiety O +( O +green O +stick O +) O +with O +LeuB29 B-residue_name_number +and O +AspB36 B-residue_name_number +. O + +Notably O +, O +the O +phenyl O +ring O +of O +PheB25 B-residue_name_number +was O +positioned O +parallel O +to O +the O +phenyl O +ring O +of O +4 B-chemical +- I-chemical +HPA I-chemical +, O +potentially O +forming O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +parallel I-bond_interaction +- I-bond_interaction +displaced I-bond_interaction +stacking I-bond_interaction +interactions I-bond_interaction +. O + +Consequently O +, O +residues O +in O +the O +4 B-site +- I-site +HPA I-site +binding I-site +pocket I-site +are O +mostly O +contributed O +by O +NadR B-protein +chain B-structure_element +B I-structure_element +, O +and O +effectively O +created O +a O +polar O +‘ O +floor O +’ O +and O +a O +hydrophobic O +‘ O +ceiling O +’, O +which O +house O +the O +ligand O +. O + +Collectively O +, O +this O +mixed O +network O +of O +polar B-bond_interaction +and I-bond_interaction +hydrophobic I-bond_interaction +interactions I-bond_interaction +endows O +NadR B-protein +with O +a O +strong O +recognition O +pattern O +for O +HPAs B-chemical +, O +with O +additional O +medium O +- O +range O +interactions O +potentially O +established O +with O +the O +hydroxyl O +group O +at O +the O +4 O +- O +position O +. O + +Structure O +- O +activity O +relationships O +: O +molecular O +basis O +of O +enhanced O +stabilization O +by O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical + +We O +modelled B-experimental_method +the O +binding O +of O +other O +HPAs B-chemical +by O +in B-experimental_method +silico I-experimental_method +superposition I-experimental_method +onto O +4 B-chemical +- I-chemical +HPA I-chemical +in O +the O +holo B-protein_state +- O +NadR B-protein +structure B-evidence +, O +and O +thereby O +obtained O +molecular O +explanations O +for O +the O +binding O +specificities O +of O +diverse O +ligands O +. O + +For O +example O +, O +similar O +to O +4 B-chemical +- I-chemical +HPA I-chemical +, O +the O +binding O +of O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +could O +involve O +multiple O +bonds O +towards O +the O +carboxylate O +group O +of O +the O +ligand O +and O +some O +to O +the O +4 O +- O +hydroxyl O +group O +. O + +Additionally O +, O +the O +side O +chains O +of O +LeuB29 B-residue_name_number +and O +AspB36 B-residue_name_number +would O +be O +only O +2 O +. O +6 O +– O +3 O +. O +5 O +Å O +from O +the O +chlorine O +atom O +, O +thus O +providing O +van B-bond_interaction +der I-bond_interaction +Waals I-bond_interaction +’ I-bond_interaction +interactions I-bond_interaction +or O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +generate O +the O +additional O +binding B-evidence +affinity I-evidence +observed O +for O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +( O +Fig O +4B O +). O + +Finally O +, O +salicylate B-chemical +is O +presumably O +unable O +to O +specifically O +bind O +NadR B-protein +due O +to O +the O +2 O +- O +hydroxyl O +substitution O +and O +the O +shorter O +aliphatic O +chain O +connecting O +its O +carboxylate O +group O +( O +Fig O +1A O +): O +the O +compound O +simply O +seems O +too O +small O +to O +simultaneously O +establish O +the O +network O +of O +beneficial O +bonds O +observed O +in O +the O +NadR B-protein +/ O +HPA B-chemical +interactions O +. O + +Analysis O +of O +the O +pockets B-site +reveals O +the O +molecular O +basis O +for O +asymmetric O +binding O +and O +stoichiometry O + +However O +, O +studies O +based O +on O +tryptophan B-experimental_method +fluorescence I-experimental_method +were O +confounded O +by O +the O +fluorescence O +of O +the O +HPA B-chemical +ligands O +, O +and O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +( O +ITC B-experimental_method +) O +was O +unfeasible O +due O +to O +the O +need O +for O +very O +high O +concentrations O +of O +NadR B-protein +in O +the O +ITC B-experimental_method +chamber O +( O +due O +to O +the O +relatively O +low O +affinity O +), O +which O +exceeded O +the O +solubility O +limits O +of O +the O +protein O +. O + +However O +, O +it O +was O +possible O +to O +calculate O +the O +binding B-evidence +stoichiometry I-evidence +of O +the O +NadR B-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +interactions O +using O +an O +SPR B-experimental_method +- O +based O +approach O +. O + +This O +approach O +relies O +on O +the O +assumption O +that O +the O +captured O +protein O +(‘ O +the O +ligand O +’, O +according O +to O +SPR B-experimental_method +conventions O +) O +is O +100 O +% O +active O +and O +freely O +- O +accessible O +to O +potential O +interactors O +(‘ O +the O +analytes O +’). O + +Overall O +, O +the O +superposition B-experimental_method +revealed O +a O +high O +degree O +of O +structural O +similarity O +( O +Cα O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +rmsd B-evidence +) O +of O +1 O +. O +5Å O +), O +though O +on O +closer O +inspection O +a O +rotational O +difference O +of O +~ O +9 O +degrees O +along O +the O +long O +axis O +of O +helix B-structure_element +α6 B-structure_element +was O +observed O +, O +suggesting O +that O +4 B-chemical +- I-chemical +HPA I-chemical +induced O +a O +slight O +conformational O +change O +( O +Fig O +5A O +). O + +Most O +notably O +, O +atomic O +clashes O +between O +the O +ligand O +and O +the O +side O +chains O +of O +MetA22 B-residue_name_number +, O +PheA25 B-residue_name_number +and O +ArgA43 B-residue_name_number +would O +occur O +if O +4 B-chemical +- I-chemical +HPA I-chemical +were O +present O +in O +the O +monomer B-oligomeric_state +A B-structure_element +pocket B-site +( O +Fig O +5B O +). O + +Subsequently O +, O +analyses O +of O +the O +pockets B-site +in O +apo B-protein_state +- O +NadR B-protein +revealed O +that O +in O +the O +absence B-protein_state +of I-protein_state +ligand I-protein_state +the O +long O +Arg43 B-residue_name_number +side O +chain O +was O +always O +in O +the O +open O +‘ O +outward B-protein_state +’ O +position O +compatible O +with O +binding O +to O +the O +4 B-chemical +- I-chemical +HPA I-chemical +carboxylate O +group O +. O + +Structural O +differences O +of O +NadR B-protein +in O +ligand B-protein_state +- I-protein_state +bound I-protein_state +or O +free B-protein_state +forms O +. O + +( O +A O +) O +Aligned B-experimental_method +monomers B-oligomeric_state +of O +holo B-protein_state +- O +NadR B-protein +( O +chain B-structure_element +A I-structure_element +: O +green O +; O +chain B-structure_element +B I-structure_element +: O +blue O +), O +reveal O +major O +overall O +differences O +by O +the O +shift O +of O +helix B-structure_element +α6 B-structure_element +. O +( O +B O +) O +Comparison B-experimental_method +of O +the O +two O +binding B-site +pockets I-site +in O +holo B-protein_state +- O +NadR B-protein +shows O +that O +in O +the O +ligand B-protein_state +- I-protein_state +free I-protein_state +monomer B-oligomeric_state +A B-structure_element +( O +green O +) O +residues O +Met22 B-residue_name_number +, O +Phe25 B-residue_name_number +and O +Arg43 B-residue_name_number +adopt O +‘ O +inward B-protein_state +’ O +positions O +( O +highlighted O +by O +arrows O +) O +compared O +to O +the O +ligand B-protein_state +- I-protein_state +occupied I-protein_state +pocket B-site +( O +blue O +residues O +); O +these O +‘ O +inward B-protein_state +’ O +conformations O +appear O +unfavorable O +for O +binding O +of O +4 B-chemical +- I-chemical +HPA I-chemical +due O +to O +clashes O +with O +the O +4 O +- O +hydroxyl O +group O +, O +the O +phenyl O +ring O +and O +the O +carboxylate O +group O +, O +respectively O +. O + +In O +these O +crystals B-evidence +, O +the O +ArgA43 B-residue_name_number +side O +chain O +showed O +two O +alternate O +conformations O +, O +modelled O +with O +50 O +% O +occupancy O +in O +each O +state O +, O +as O +indicated O +by O +the O +two O +‘ O +mirrored O +’ O +arrows O +. O + +Finally O +, O +we O +applied O +15N B-experimental_method +heteronuclear I-experimental_method +solution I-experimental_method +NMR I-experimental_method +spectroscopy I-experimental_method +to O +examine O +the O +interaction O +of O +4 B-chemical +- I-chemical +HPA I-chemical +with O +apo B-protein_state +NadR B-protein +. O +We O +collected O +NMR B-experimental_method +spectra B-evidence +on O +NadR B-protein +in B-protein_state +the I-protein_state +presence I-protein_state +and O +absence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +( O +see O +Materials O +and O +Methods O +). O + +The O +1H B-experimental_method +- I-experimental_method +15N I-experimental_method +TROSY I-experimental_method +- I-experimental_method +HSQC I-experimental_method +spectrum B-evidence +of O +apo B-protein_state +- O +NadR B-protein +, O +acquired O +at O +25 O +° O +C O +, O +displayed O +approximately O +140 O +distinct O +peaks O +( O +Fig O +6A O +), O +most O +of O +which O +correspond O +to O +backbone O +amide O +N O +- O +H O +groups O +. O + +Upon O +the O +addition O +of O +4 B-chemical +- I-chemical +HPA I-chemical +, O +over O +45 O +peaks O +showed O +chemical O +shift O +perturbations O +, O +i O +. O +e O +. O +changed O +position O +in O +the O +spectrum O +or O +disappeared O +, O +while O +the O +remaining O +peaks O +remained O +unchanged O +. O + +This O +observation O +showed O +that O +4 B-chemical +- I-chemical +HPA I-chemical +was O +able O +to O +bind O +NadR B-protein +and O +induce O +notable O +changes O +in O +specific O +regions O +of O +the O +protein O +. O + +NMR B-experimental_method +spectra B-evidence +of O +NadR B-protein +in B-protein_state +the I-protein_state +presence I-protein_state +and O +absence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +. O + +( O +A O +) O +Superposition B-experimental_method +of O +two O +1H B-experimental_method +- I-experimental_method +15N I-experimental_method +TROSY I-experimental_method +- I-experimental_method +HSQC I-experimental_method +spectra B-evidence +recorded O +at O +25 O +° O +C O +on O +apo B-protein_state +- O +NadR B-protein +( O +cyan O +) O +and O +on O +NadR B-protein +in O +the O +presence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +( O +red O +). O + +The O +spectra B-evidence +acquired O +at O +10 O +° O +C O +are O +excluded O +from O +panel O +A O +for O +simplicity O +. O + +However O +, O +in O +the O +presence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +, O +the O +1H B-experimental_method +- I-experimental_method +15N I-experimental_method +TROSY I-experimental_method +- I-experimental_method +HSQC I-experimental_method +spectrum B-evidence +of O +NadR B-protein +displayed O +approximately O +140 O +peaks O +, O +as O +for O +apo B-protein_state +- O +NadR B-protein +, O +i O +. O +e O +. O +two O +distinct O +stable O +conformations O +( O +that O +might O +have O +potentially O +revealed O +the O +molecular O +asymmetry O +observed O +crystallographically B-experimental_method +) O +were O +not O +notable O +. O + +These O +doubled O +peaks O +may O +therefore O +reveal O +that O +the O +cooler O +temperature O +partially O +trapped O +the O +existence O +in O +solution O +of O +two O +distinct O +states O +, O +in O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +, O +with O +minor O +conformational O +differences O +occurring O +at O +least O +in O +proximity O +to O +the O +binding B-site +pocket I-site +. O + +Apo B-protein_state +- O +NadR B-protein +structures B-evidence +reveal O +intrinsic O +conformational O +flexibility O + +The O +apo B-protein_state +- O +NadR B-protein +crystal B-evidence +structure I-evidence +contained O +two O +homodimers B-oligomeric_state +in O +the O +asymmetric O +unit O +( O +chains B-structure_element +A I-structure_element ++ I-structure_element +B I-structure_element +and O +chains B-structure_element +C I-structure_element ++ I-structure_element +D I-structure_element +). O + +Upon O +overall O +structural B-experimental_method +superposition I-experimental_method +, O +these O +dimers B-oligomeric_state +revealed O +a O +few O +minor O +differences O +in O +the O +α6 B-structure_element +helix I-structure_element +( O +a O +major O +component O +of O +the O +dimer B-site +interface I-site +) O +and O +the O +helices B-structure_element +α4 B-structure_element +- I-structure_element +α5 I-structure_element +( O +the O +DNA B-site +binding I-site +region I-site +), O +and O +an O +rmsd B-evidence +of O +1 O +. O +55Å O +( O +Fig O +7A O +). O + +The O +slightly O +larger O +rmsd B-evidence +between O +the O +two O +apo B-protein_state +- O +homodimers B-oligomeric_state +, O +rather O +than O +between O +apo B-protein_state +- O +and O +holo B-protein_state +- O +homodimers B-oligomeric_state +, O +further O +indicate O +that O +apo B-protein_state +- O +NadR B-protein +possesses O +a O +notable O +degree O +of O +intrinsic O +conformational O +flexibility O +. O + +Overall O +apo B-protein_state +- O +and O +holo B-protein_state +- O +NadR B-protein +structures B-evidence +are O +similar O +. O + +( O +A O +) O +Pairwise B-experimental_method +alignment I-experimental_method +of O +the O +two O +distinct O +apo B-protein_state +- O +NadR B-protein +homodimers B-oligomeric_state +( O +AB B-structure_element +and O +CD B-structure_element +) O +present O +in O +the O +apo B-protein_state +- O +NadR B-protein +crystals B-evidence +. O +( O +B O +) O +Alignment B-experimental_method +of O +the O +holo B-protein_state +- O +NadR B-protein +homodimer B-oligomeric_state +( O +green O +and O +blue O +chains O +) O +onto O +the O +apo B-protein_state +- O +NadR B-protein +homodimers B-oligomeric_state +. O + +Here O +, O +larger O +differences O +are O +observed O +in O +the O +α6 B-structure_element +helices I-structure_element +( O +top O +). O + +4 B-chemical +- I-chemical +HPA I-chemical +stabilizes O +concerted O +conformational O +changes O +in O +NadR B-protein +that O +prevent O +DNA O +- O +binding O + +To O +further O +investigate O +the O +conformational O +rearrangements O +of O +NadR B-protein +, O +we O +performed O +local B-experimental_method +structural I-experimental_method +alignments I-experimental_method +using O +only O +a O +subset O +of O +residues O +in O +the O +DNA B-structure_element +- I-structure_element +binding I-structure_element +helix I-structure_element +( O +α4 B-structure_element +). O + +By O +selecting B-experimental_method +and O +aligning B-experimental_method +residues O +Arg64 B-residue_range +- I-residue_range +Ala77 I-residue_range +of O +one O +α4 B-structure_element +helix I-structure_element +per O +dimer B-oligomeric_state +, O +superposition B-experimental_method +of O +the O +holo B-protein_state +- O +homodimer B-oligomeric_state +onto O +the O +two O +apo B-protein_state +- O +homodimers B-oligomeric_state +revealed O +differences O +in O +the O +monomer B-oligomeric_state +conformations O +of O +each O +structure B-evidence +. O + +While O +one O +monomer B-oligomeric_state +from O +each O +structure B-evidence +was O +closely O +superimposable O +( O +Fig O +8A O +, O +left O +side O +), O +the O +second O +monomer B-oligomeric_state +displayed O +quite O +large O +differences O +( O +Fig O +8A O +, O +right O +side O +). O + +Most O +notably O +, O +the O +position O +of O +the O +DNA B-chemical +- O +binding O +helix B-structure_element +α4 B-structure_element +shifted O +by O +as O +much O +as O +6 O +Å O +( O +Fig O +8B O +). O + +Accordingly O +, O +helix B-structure_element +α4 B-structure_element +was O +also O +found O +to O +be O +one O +of O +the O +most O +dynamic O +regions O +in O +previous O +HDX B-experimental_method +- I-experimental_method +MS I-experimental_method +analyses O +of O +apo B-protein_state +- O +NadR B-protein +in O +solution O +. O + +The O +α4 B-structure_element +helices I-structure_element +aligned O +closely O +, O +Cα O +rmsd B-evidence +0 O +. O +2Å O +for O +14 O +residues O +. O + +( O +B O +) O +The O +relative O +positions O +of O +the O +α4 B-structure_element +helices I-structure_element +of O +the O +4 B-protein_state +- I-protein_state +HPA I-protein_state +- I-protein_state +bound I-protein_state +holo B-protein_state +homodimer B-oligomeric_state +chain B-structure_element +B I-structure_element +( O +blue O +), O +and O +of O +apo B-protein_state +homodimers B-oligomeric_state +AB B-structure_element +and O +CD B-structure_element +( O +showing O +chains B-structure_element +B I-structure_element +and I-structure_element +D I-structure_element +) O +in O +pale O +blue O +. O + +Dashes O +indicate O +the O +Ala77 B-residue_name_number +Cα O +atoms O +, O +in O +the O +most O +highly O +shifted O +region O +of O +the O +‘ O +non O +- O +fixed O +’ O +α4 B-structure_element +helix I-structure_element +. O + +( O +C O +) O +The O +double O +- O +stranded O +DNA B-chemical +molecule O +( O +grey O +cartoon O +) O +from O +the O +OhrR B-complex_assembly +- I-complex_assembly +ohrA I-complex_assembly +complex O +is O +shown O +after O +superposition B-experimental_method +with O +NadR B-protein +, O +to O +highlight O +the O +expected O +positions O +of O +the O +NadR B-protein +α4 B-structure_element +helices I-structure_element +in O +the O +DNA B-chemical +major O +grooves O +. O + +For O +clarity O +, O +only O +the O +α4 B-structure_element +helices I-structure_element +are O +shown O +in O +panels O +( O +B O +) O +and O +( O +C O +). O +( O +D O +) O +Upon O +comparison O +with O +the O +experimentally O +- O +determined O +OhrR B-complex_assembly +: I-complex_assembly +ohrA I-complex_assembly +structure B-evidence +( O +grey O +), O +the O +α4 B-structure_element +helix I-structure_element +of O +holo B-protein_state +- O +NadR B-protein +( O +blue O +) O +is O +shifted O +~ O +8Å O +out O +of O +the O +major O +groove O +. O + +In O +summary O +, O +compared O +to O +ligand B-protein_state +- I-protein_state +stabilized I-protein_state +holo B-protein_state +- O +NadR B-protein +, O +apo B-protein_state +- O +NadR B-protein +displayed O +an O +intrinsic O +flexibility O +focused O +in O +the O +DNA B-site +- I-site +binding I-site +region I-site +. O + +This O +was O +also O +evident O +in O +the O +greater O +disorder O +( O +i O +. O +e O +. O +less O +well O +- O +defined O +electron B-evidence +density I-evidence +) O +in O +the O +β1 B-structure_element +- I-structure_element +β2 I-structure_element +loops I-structure_element +of O +the O +apo B-protein_state +dimers B-oligomeric_state +( O +density B-evidence +for O +16 O +residues O +per O +dimer B-oligomeric_state +was O +missing O +) O +compared O +to O +the O +holo B-protein_state +dimer B-oligomeric_state +( O +density B-evidence +for O +only O +3 O +residues O +was O +missing O +). O + +In O +holo B-protein_state +- O +NadR B-protein +, O +the O +distance O +separating O +the O +two O +DNA O +- O +binding O +α4 B-structure_element +helices I-structure_element +was O +32 O +Å O +, O +while O +in O +apo B-protein_state +- O +NadR B-protein +it O +was O +29 O +Å O +for O +homodimer B-oligomeric_state +AB B-structure_element +, O +and O +34 O +Å O +for O +homodimer B-oligomeric_state +CD B-structure_element +( O +Fig O +8C O +). O + +Pairwise B-experimental_method +superpositions I-experimental_method +showed O +that O +the O +NadR B-protein +apo B-protein_state +- O +homodimer B-oligomeric_state +AB B-structure_element +was O +the O +most O +similar O +to O +OhrR B-protein +( O +rmsd B-evidence +2 O +. O +6 O +Å O +), O +while O +the O +holo B-protein_state +- O +homodimer B-oligomeric_state +was O +the O +most O +divergent O +( O +rmsd B-evidence +3 O +. O +3 O +Å O +) O +( O +Fig O +8C O +). O + +Assuming O +the O +same O +DNA B-chemical +- O +binding O +mechanism O +is O +used O +by O +OhrR B-protein +and O +NadR B-protein +, O +the O +apo B-protein_state +- O +homodimer B-oligomeric_state +AB B-structure_element +seems O +ideally O +pre O +- O +configured O +for O +DNA B-chemical +binding O +, O +while O +4 B-chemical +- I-chemical +HPA I-chemical +appeared O +to O +stabilize O +holo B-protein_state +- O +NadR B-protein +in O +a O +conformation O +poorly O +suited O +for O +DNA B-chemical +binding O +. O + +Specifically O +, O +in O +addition O +to O +the O +different O +inter B-evidence +- I-evidence +helical I-evidence +translational I-evidence +distances I-evidence +, O +the O +α4 B-structure_element +helices I-structure_element +in O +the O +holo B-protein_state +- O +NadR B-protein +homodimer B-oligomeric_state +were O +also O +reoriented O +, O +resulting O +in O +movement O +of O +α4 B-structure_element +out O +of O +the O +major O +groove O +, O +by O +up O +to O +8Å O +, O +and O +presumably O +preventing O +efficient O +DNA B-chemical +binding O +in O +the O +presence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +( O +Fig O +8D O +). O + +NadR B-protein +residues O +His7 B-residue_name_number +, O +Ser9 B-residue_name_number +, O +Asn11 B-residue_name_number +and O +Phe25 B-residue_name_number +are O +essential O +for O +regulation O +of O +NadA B-protein +expression O +in O +vivo O + +While O +previous O +studies O +had O +correctly O +suggested O +the O +involvement O +of O +several O +NadR B-protein +residues O +in O +ligand O +binding O +, O +the O +crystal B-evidence +structures I-evidence +presented O +here O +revealed O +additional O +residues O +with O +previously O +unknown O +roles O +in O +dimerization O +and O +/ O +or O +binding O +to O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +To O +explore O +the O +functional O +involvement O +of O +these O +residues O +, O +we O +characterized O +the O +behavior O +of O +four O +new O +NadR B-protein +mutants O +( O +H7A B-mutant +, O +S9A B-mutant +, O +N11A B-mutant +and O +F25A B-mutant +) O +in O +an O +in O +vivo O +assay O +using O +the O +previously O +described O +MC58 B-mutant +- I-mutant +Δ1843 I-mutant +nadR B-gene +- O +null O +mutant B-protein_state +strain O +, O +which O +was O +complemented O +either O +by O +wild B-protein_state +- I-protein_state +type I-protein_state +nadR B-gene +or O +by O +the O +nadR B-gene +mutants B-protein_state +. O + +NadA B-protein +protein O +abundance O +levels O +were O +assessed O +by O +Western B-experimental_method +blotting I-experimental_method +to O +evaluate O +the O +ability O +of O +the O +NadR B-protein +mutants B-protein_state +to O +repress O +the O +nadA B-gene +promoter O +, O +in O +the O +presence O +or O +absence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +The O +nadR B-gene +H7A B-mutant +, O +S9A B-mutant +and O +F25A B-mutant +complemented O +strains O +showed O +hyper O +- O +repression O +of O +nadA B-gene +expression O +in O +vivo O +, O +i O +. O +e O +. O +these O +mutants O +repressed O +nadA B-gene +more O +efficiently O +than O +the O +NadR B-protein +WT B-protein_state +protein O +, O +either O +in O +the O +presence O +or O +absence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +, O +while O +complementation O +with O +wild B-protein_state +- I-protein_state +type I-protein_state +nadR B-gene +resulted O +in O +high O +production O +of O +NadA B-protein +only O +in O +the O +presence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +( O +Fig O +9 O +). O + +Interestingly O +, O +and O +on O +the O +contrary O +, O +the O +nadR B-gene +N11A B-mutant +complemented O +strain O +showed O +hypo O +- O +repression O +( O +i O +. O +e O +. O +exhibited O +high O +expression O +of O +nadA B-gene +both O +in O +absence O +and O +presence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +). O + +Structure B-experimental_method +- I-experimental_method +based I-experimental_method +point I-experimental_method +mutations I-experimental_method +shed O +light O +on O +ligand O +- O +induced O +regulation O +of O +NadR B-protein +. O + +Complementation O +of O +ΔNadR B-mutant +with O +WT B-protein_state +NadR B-protein +enables O +induction O +of O +nadA B-gene +expression O +by O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +A O +detailed O +understanding O +of O +the O +in O +vitro O +repression O +of O +nadA B-gene +expression O +by O +the O +transcriptional B-protein_type +regulator I-protein_type +NadR B-protein +is O +important O +, O +both O +because O +it O +is O +a O +relevant O +disease O +- O +related O +model O +of O +how O +small O +- O +molecule O +ligands O +can O +regulate O +MarR B-protein_type +family O +proteins O +and O +thereby O +impact O +bacterial B-taxonomy_domain +virulence O +, O +and O +because O +nadA B-gene +expression O +levels O +are O +linked O +to O +the O +prediction O +of O +vaccine O +coverage O +. O + +The O +repressive O +activity O +of O +NadR B-protein +can O +be O +relieved O +by O +hydroxyphenylacetate B-chemical +( O +HPA B-chemical +) O +ligands O +, O +and O +HDX B-experimental_method +- I-experimental_method +MS I-experimental_method +studies O +previously O +indicated O +that O +4 B-chemical +- I-chemical +HPA I-chemical +stabilizes O +dimeric B-oligomeric_state +NadR B-protein +in O +a O +configuration O +incompatible O +with O +DNA O +binding O +. O + +Despite O +these O +and O +other O +studies O +, O +the O +molecular O +mechanisms O +by O +which O +ligands O +regulate O +MarR B-protein_type +family O +proteins O +are O +relatively O +poorly O +understood O +and O +likely O +differ O +depending O +on O +the O +specific O +ligand O +. O + +Firstly O +, O +we O +confirmed O +that O +NadR B-protein +is O +dimeric B-oligomeric_state +in O +solution O +and O +demonstrated O +that O +it O +retains O +its O +dimeric B-oligomeric_state +state O +in O +the O +presence B-protein_state +of I-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +, O +indicating O +that O +induction O +of O +a O +monomeric B-oligomeric_state +status O +is O +not O +the O +manner O +by O +which O +4 B-chemical +- I-chemical +HPA I-chemical +regulates O +NadR B-protein +. O +These O +observations O +were O +in O +agreement O +with O +( O +i O +) O +a O +previous O +study O +of O +NadR B-protein +performed O +using O +SEC B-experimental_method +and O +mass B-experimental_method +spectrometry I-experimental_method +, O +and O +( O +ii O +) O +crystallographic B-experimental_method +studies I-experimental_method +showing O +that O +several O +MarR B-protein_type +homologues O +are O +dimeric B-oligomeric_state +. O + +We O +also O +used O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +site I-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +to O +identify O +an O +important O +conserved B-protein_state +residue O +, O +Leu130 B-residue_name_number +, O +which O +stabilizes O +the O +NadR B-protein +dimer B-site +interface I-site +, O +knowledge O +of O +which O +may O +also O +inform O +future O +studies O +to O +explore O +the O +regulatory O +mechanisms O +of O +other O +MarR B-protein_type +family O +proteins O +. O + +Secondly O +, O +we O +assessed B-experimental_method +the I-experimental_method +thermal I-experimental_method +stability I-experimental_method +and O +unfolding O +of O +NadR B-protein +in B-protein_state +the I-protein_state +presence I-protein_state +or O +absence B-protein_state +of I-protein_state +ligands O +. O + +All O +DSC B-experimental_method +profiles B-evidence +showed O +a O +single O +peak O +, O +suggesting O +that O +a O +single O +unfolding O +event O +simultaneously O +disrupted O +the O +dimer B-oligomeric_state +and O +the O +monomer B-oligomeric_state +. O + +HPA O +ligands O +specifically O +increased O +the O +stability O +of O +NadR B-protein +. O +The O +largest O +effects O +were O +induced O +by O +the O +naturally O +- O +occurring O +compounds O +4 B-chemical +- I-chemical +HPA I-chemical +and O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +, O +which O +, O +in O +SPR B-experimental_method +assays I-experimental_method +, O +were O +found O +to O +bind O +NadR B-protein +with O +KD B-evidence +values O +of O +1 O +. O +5 O +mM O +and O +1 O +. O +1 O +mM O +, O +respectively O +. O + +Although O +these O +NadR B-protein +/ O +HPA B-chemical +interactions O +appeared O +rather O +weak O +, O +their O +distinct O +affinities O +and O +specificities O +matched O +their O +in O +vitro O +effects O +and O +their O +biological O +relevance O +appears O +similar O +to O +previous O +proposals O +that O +certain O +small O +molecules O +, O +including O +some O +antibiotics O +, O +in O +the O +millimolar O +concentration O +range O +may O +be O +broad O +inhibitors O +of O +MarR B-protein_type +family O +proteins O +. O + +Indeed O +, O +4 B-chemical +- I-chemical +HPA I-chemical +is O +found O +in O +human B-species +saliva O +and O +3Cl B-chemical +, I-chemical +4 I-chemical +- I-chemical +HPA I-chemical +is O +produced O +during O +inflammatory O +processes O +, O +suggesting O +that O +these O +natural O +ligands O +are O +encountered O +by O +N B-species +. I-species +meningitidis I-species +in O +the O +mucosa O +of O +the O +oropharynx O +during O +infections O +. O + +It O +is O +also O +possible O +that O +NadR B-protein +responds O +to O +currently O +unidentified O +HPA B-chemical +analogues O +. O + +Indeed O +, O +in O +the O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +complex O +there O +was O +a O +water B-chemical +molecule O +close O +to O +the O +carboxylate O +group O +and O +also O +a O +small O +unfilled O +tunnel B-site +~ O +5Å O +long O +, O +both O +factors O +suggesting O +that O +alternative O +larger O +ligands O +could O +occupy O +the O +pocket O +. O + +The O +ability O +to O +respond O +to O +various O +ligands O +might O +enable O +NadR B-protein +in O +vivo O +to O +orchestrate O +multiple O +response O +mechanisms O +and O +modulate O +expression O +of O +genes O +other O +than O +nadA B-gene +. O +Ultimately O +, O +confirmation O +of O +the O +relevance O +of O +each O +ligand O +will O +require O +a O +deeper O +understanding O +of O +the O +available O +concentration O +in O +vivo O +in O +the O +host O +niche O +during O +bacterial B-taxonomy_domain +colonization O +and O +inflammation O +. O + +Here O +, O +we O +determined O +the O +first O +crystal B-evidence +structures I-evidence +of O +apo B-protein_state +- O +NadR B-protein +and O +holo B-protein_state +- O +NadR B-protein +. O +These O +experimentally O +- O +determined O +structures B-evidence +enabled O +a O +new O +detailed O +characterization O +of O +the O +ligand B-site +- I-site +binding I-site +pocket I-site +. O + +In O +holo B-protein_state +- O +NadR B-protein +, O +4 B-chemical +- I-chemical +HPA I-chemical +interacted O +directly O +with O +at O +least O +11 O +polar O +and O +hydrophobic O +residues O +. O + +Several O +, O +but O +not O +all O +, O +of O +these O +interactions O +were O +predicted O +previously O +by O +homology B-experimental_method +modelling I-experimental_method +combined O +with O +ligand B-experimental_method +docking I-experimental_method +in O +silico O +. O + +Subsequently O +, O +we O +established O +the O +functional O +importance O +of O +His7 B-residue_name_number +, O +Ser9 B-residue_name_number +, O +Asn11 B-residue_name_number +and O +Phe25 B-residue_name_number +in O +the O +in O +vitro O +response O +of O +meningococcus B-taxonomy_domain +to O +4 B-chemical +- I-chemical +HPA I-chemical +, O +via O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +. O + +More O +unexpectedly O +, O +the O +crystal B-evidence +structure I-evidence +revealed O +that O +only O +one O +molecule O +of O +4 B-chemical +- I-chemical +HPA I-chemical +was O +bound B-protein_state +per O +NadR B-protein +dimer B-oligomeric_state +. O + +We O +also O +used O +heteronuclear B-experimental_method +NMR I-experimental_method +spectroscopy I-experimental_method +to O +detect O +substantial O +conformational O +changes O +of O +NadR B-protein +occurring O +in O +solution O +upon O +addition O +of O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +Moreover O +, O +NMR B-experimental_method +spectra B-evidence +at O +10 O +° O +C O +suggested O +the O +existence O +of O +two O +distinct O +conformations O +of O +NadR B-protein +in O +the O +vicinity O +of O +the O +ligand B-site +- I-site +binding I-site +pocket I-site +. O + +More O +powerfully O +, O +our O +unique O +crystallographic B-evidence +observation I-evidence +of O +this O +‘ O +occupied B-protein_state +vs O +unoccupied B-protein_state +site O +’ O +asymmetry O +in O +the O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +interaction O +is O +, O +to O +our O +knowledge O +, O +the O +first O +example O +reported O +for O +a O +MarR B-protein_type +family O +protein O +. O + +Such O +a O +mechanism O +indicates O +negative O +cooperativity O +, O +which O +may O +enhance O +the O +ligand O +- O +responsiveness O +of O +NadR B-protein +. O + +Comparisons O +of O +the O +NadR B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +- I-complex_assembly +HPA I-complex_assembly +complex O +with O +available O +MarR B-protein_type +family O +/ O +salicylate B-chemical +complexes O +revealed O +that O +4 B-chemical +- I-chemical +HPA I-chemical +has O +a O +previously O +unobserved O +binding O +mode O +. O + +Briefly O +, O +in O +the O +M B-species +. I-species +thermoautotrophicum I-species +MTH313 B-protein +dimer B-oligomeric_state +, O +one O +molecule O +of O +salicylate B-chemical +binds O +in O +the O +pocket B-site +of O +each O +monomer B-oligomeric_state +, O +though O +with O +two O +rather O +different O +positions O +and O +orientations O +, O +only O +one O +of O +which O +( O +site B-site +- I-site +1 I-site +) O +is O +thought O +to O +be O +biologically O +relevant O +( O +Fig O +10A O +). O + +In O +NadR B-protein +, O +the O +single O +molecule O +of O +4 B-chemical +- I-chemical +HPA I-chemical +binds O +in O +a O +position O +distinctly O +different O +from O +the O +salicylate B-site +binding I-site +site I-site +: O +translated O +by O +> O +10 O +Å O +and O +with O +a O +180 O +° O +inverted O +orientation O +( O +Fig O +10C O +). O + +( O +A O +) O +A O +structural B-experimental_method +alignment I-experimental_method +of O +MTH313 B-protein +chains B-structure_element +A I-structure_element +and I-structure_element +B I-structure_element +shows O +that O +salicylate B-chemical +is O +bound B-protein_state +in O +distinct O +locations O +in O +each O +monomer B-oligomeric_state +; O +site B-site +- I-site +1 I-site +( O +thought O +to O +be O +the O +biologically O +relevant O +site O +) O +and O +site B-site +- I-site +2 I-site +differ O +by O +~ O +7Å O +( O +indicated O +by O +black O +dotted O +line O +) O +and O +also O +by O +ligand O +orientation O +. O + +( O +C O +) O +Addition O +of O +holo B-protein_state +- O +NadR B-protein +( O +chain B-structure_element +B I-structure_element +, O +blue O +) O +to O +the O +alignment B-experimental_method +reveals O +that O +bound B-protein_state +4 B-chemical +- I-chemical +HPA I-chemical +differs O +in O +position O +by O +> O +10 O +Å O +compared O +to O +salicylate B-chemical +, O +and O +adopts O +a O +novel O +orientation O +. O + +Interestingly O +, O +a O +crystal B-evidence +structure I-evidence +was O +previously O +reported O +for O +a O +functionally O +- O +uncharacterized O +meningococcal B-taxonomy_domain +homologue O +of O +NadR B-protein +, O +termed O +NMB1585 B-protein +, O +which O +shares O +16 O +% O +sequence O +identity O +with O +NadR B-protein +. O +The O +two O +structures B-evidence +can O +be O +closely O +aligned O +( O +rmsd B-evidence +2 O +. O +3 O +Å O +), O +but O +NMB1585 B-protein +appears O +unsuited O +for O +binding O +HPAs B-chemical +, O +since O +its O +corresponding O +‘ B-site +pocket I-site +’ O +region O +is O +occupied O +by O +several O +bulky O +hydrophobic O +side O +chains O +. O + +It O +can O +be O +speculated O +that O +MarR B-protein_type +family O +members O +have O +evolved O +separately O +to O +engage O +distinct O +signaling O +molecules O +, O +thus O +enabling O +bacteria B-taxonomy_domain +to O +use O +the O +overall O +conserved O +MarR B-protein_type +scaffold O +to O +adapt O +and O +respond O +to O +diverse O +changing O +environmental O +stimuli O +experienced O +in O +their O +natural O +niches O +. O + +The O +apo B-protein_state +- O +NadR B-protein +crystal B-evidence +structures I-evidence +revealed O +two O +dimers B-oligomeric_state +with O +slightly O +different O +conformations O +, O +most O +divergent O +in O +the O +DNA B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +. O + +It O +is O +not O +unusual O +for O +a O +crystal B-evidence +structure I-evidence +to O +reveal O +multiple O +copies O +of O +the O +same O +protein O +in O +very O +slightly O +different O +conformations O +, O +which O +are O +likely O +representative O +of O +the O +lowest O +- O +energy O +conformations O +sampled O +by O +the O +dynamic O +ensemble O +of O +molecular O +states O +occurring O +in O +solution O +, O +and O +which O +likely O +have O +only O +small O +energetic O +differences O +, O +as O +described O +previously O +for O +MexR B-protein +( O +a O +MarR B-protein_type +protein O +) O +or O +more O +recently O +for O +the O +solute B-protein_type +- I-protein_type +binding I-protein_type +protein I-protein_type +FhuD2 B-protein +. O + +Further O +, O +the O +holo B-protein_state +- O +NadR B-protein +structure B-evidence +was O +overall O +more O +different O +from O +the O +two O +apo B-protein_state +- O +NadR B-protein +structures B-evidence +( O +rmsd B-evidence +values O +~ O +1 O +. O +3Å O +), O +suggesting O +that O +the O +ligand O +selected O +and O +stabilized O +yet O +another O +conformation O +of O +NadR B-protein +. O +These O +observations O +suggest O +that O +4 B-chemical +- I-chemical +HPA I-chemical +, O +and O +potentially O +other O +similar O +ligands O +, O +can O +shift O +the O +molecular O +equilibrium O +, O +changing O +the O +energy O +barriers O +that O +separate O +active B-protein_state +and O +inactive B-protein_state +states O +, O +and O +stabilizing O +the O +specific O +conformation O +of O +NadR B-protein +poorly O +suited O +to O +bind O +DNA B-chemical +. O + +Comparisons O +of O +the O +apo B-protein_state +- O +and O +holo B-protein_state +- O +NadR B-protein +structures B-evidence +revealed O +that O +the O +largest O +differences O +occurred O +in O +the O +DNA B-chemical +- O +binding O +helix B-structure_element +α4 B-structure_element +. O + +The O +shift O +of O +helix B-structure_element +α4 B-structure_element +in O +holo B-protein_state +- O +NadR B-protein +was O +also O +accompanied O +by O +rearrangements O +at O +the O +dimer B-site +interface I-site +, O +involving O +helices B-structure_element +α1 B-structure_element +, O +α5 B-structure_element +, O +and O +α6 B-structure_element +, O +and O +this O +holo B-protein_state +- O +form O +appeared O +poorly O +suited O +for O +DNA B-chemical +- O +binding O +when O +compared O +with O +the O +known O +OhrR B-complex_assembly +: I-complex_assembly +ohrA I-complex_assembly +complex O +. O + +One O +of O +the O +two O +conformations O +of O +apo B-protein_state +- O +NadR B-protein +appeared O +ideally O +suited O +for O +DNA B-chemical +- O +binding O +. O + +Overall O +, O +these O +analyses O +suggest O +that O +the O +apo B-protein_state +- O +NadR B-protein +dimer B-oligomeric_state +has O +a O +pre O +- O +existing O +equilibrium O +that O +samples O +a O +variety O +of O +conformations O +, O +some O +of O +which O +are O +compatible O +with O +DNA B-chemical +binding O +. O + +Subsequently O +, O +upon O +ligand O +binding O +, O +holo B-protein_state +- O +NadR B-protein +adopts O +a O +structure O +less O +suited O +for O +DNA B-chemical +- O +binding O +and O +this O +conformation O +is O +selected O +and O +stabilized O +by O +a O +network O +of O +protein O +- O +ligand O +interactions O +and O +concomitant O +rearrangements O +at O +the O +NadR B-protein +holo B-protein_state +dimer B-site +interface I-site +. O + +In O +an O +alternative O +and O +less O +extensive O +manner O +, O +the O +binding O +of O +two O +salicylate B-chemical +molecules O +to O +the O +M B-species +. I-species +thermoautotrophicum I-species +protein O +MTH313 B-protein +appeared O +to O +induce O +large O +changes O +in O +the O +wHTH B-structure_element +domain I-structure_element +, O +which O +was O +associated O +with O +reduced O +DNA O +- O +binding O +activity O +. O + +Here O +we O +have O +presented O +two O +new O +crystal B-evidence +structures I-evidence +of O +the O +transcription B-protein_type +factor I-protein_type +, O +NadR B-protein +, O +which O +regulates O +expression O +of O +the O +meningococcal B-taxonomy_domain +surface O +protein O +, O +virulence O +factor O +and O +vaccine O +antigen O +NadA B-protein +. O +Detailed O +structural B-experimental_method +analyses I-experimental_method +provided O +a O +molecular O +explanation O +for O +the O +ligand O +- O +responsive O +regulation O +by O +NadR B-protein +on O +the O +majority O +of O +the O +promoters O +of O +meningococcal B-taxonomy_domain +genes O +regulated O +by O +NadR B-protein +, O +including O +nadA B-gene +. O +Intriguingly O +, O +NadR B-protein +exhibits O +a O +reversed O +regulatory O +mechanism O +on O +a O +second O +class O +of O +promoters O +, O +including O +mafA B-gene +of O +the O +multiple O +adhesin O +family O +– O +i O +. O +e O +. O +NadR B-protein +represses O +these O +genes O +in O +the O +presence O +but O +not O +absence O +of O +4 B-chemical +- I-chemical +HPA I-chemical +. O + +Ultimately O +, O +knowledge O +of O +the O +ligand O +- O +dependent O +activity O +of O +NadR B-protein +will O +continue O +to O +deepen O +our O +understanding O +of O +nadA B-gene +expression O +levels O +, O +which O +influence O +meningococcal B-taxonomy_domain +pathogenesis O +. O + +The O +structure O +of O +NMB1585 B-protein +, O +a O +MarR O +- O +family O +regulator O +from O +Neisseria O +meningitidis O + +The O +nuclear B-protein_type +hormone I-protein_type +receptor I-protein_type +RORγ B-protein +regulates O +transcriptional O +genes O +involved O +in O +the O +production O +of O +the O +pro O +- O +inflammatory O +interleukin B-protein_type +IL B-protein_type +- I-protein_type +17 I-protein_type +which O +has O +been O +linked O +to O +autoimmune O +diseases O +such O +as O +rheumatoid O +arthritis O +, O +multiple O +sclerosis O +and O +inflammatory O +bowel O +disease O +. O + +This O +transcriptional O +activity O +of O +RORγ B-protein +is O +modulated O +through O +a O +protein O +- O +protein O +interaction O +involving O +the O +activation B-structure_element +function I-structure_element +2 I-structure_element +( I-structure_element +AF2 I-structure_element +) I-structure_element +helix I-structure_element +on O +the O +ligand B-structure_element +binding I-structure_element +domain I-structure_element +of O +RORγ B-protein +and O +a O +conserved B-protein_state +LXXLL B-structure_element +helix I-structure_element +motif I-structure_element +on O +coactivator O +proteins O +. O + +We O +identified O +a O +novel O +series O +of O +synthetic O +benzoxazinone B-chemical +ligands O +having O +an O +agonist B-protein_state +( O +BIO592 B-chemical +) O +and O +inverse B-protein_state +agonist I-protein_state +( O +BIO399 B-chemical +) O +mode O +of O +action O +in O +a O +FRET B-experimental_method +based I-experimental_method +assay I-experimental_method +. O + +We O +show O +that O +the O +AF2 B-structure_element +helix I-structure_element +of O +RORγ B-protein +is O +proteolytically B-protein_state +sensitive I-protein_state +when O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +binds O +. O + +Using O +x B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +we O +show O +how O +small O +modifications O +on O +the O +benzoxazinone B-chemical +agonist B-protein_state +BIO592 B-chemical +trigger O +inverse O +agonism O +of O +RORγ B-protein +. O + +The O +proteolytic O +sensitivity O +of O +the O +AF2 B-structure_element +helix I-structure_element +of O +RORγ B-protein +demonstrates O +that O +it O +destabilizes O +upon O +BIO399 B-chemical +inverse B-protein_state +agonist I-protein_state +binding O +perturbing O +the O +coactivator B-site +protein I-site +binding I-site +site I-site +. O + +Even O +though O +a O +high O +degree O +of O +sequence O +similarity O +exists O +between O +the O +RORs B-protein_type +, O +their O +functional O +roles O +in O +regulation O +for O +physiological O +processes O +involved O +in O +development O +and O +immunity O +are O +distinct O +. O + +During O +development O +, O +RORγ B-protein +regulates O +the O +transcriptional O +genes O +involved O +in O +the O +functioning O +of O +multiple O +pro O +- O +inflammatory O +lymphocyte O +lineages O +including O +T O +helper O +cells O +( O +TH17cells O +) O +which O +are O +necessary O +for O +IL B-protein_type +- I-protein_type +17 I-protein_type +production O +. O + +IL B-protein_type +- I-protein_type +17 I-protein_type +is O +a O +pro O +- O +inflammatory O +interleukin B-protein_type +linked O +to O +autoimmune O +diseases O +such O +as O +rheumatoid O +arthritis O +, O +multiple O +sclerosis O +and O +inflammatory O +bowel O +disease O +; O +making O +its O +transcriptional O +regulation O +through O +RORγ B-protein +an O +attractive O +therapeutic O +target O +. O + +RORγ B-protein +consists O +of O +an O +N O +- O +terminal O +DNA B-structure_element +binding I-structure_element +domain I-structure_element +( O +DBD B-structure_element +) O +connected O +to O +a O +C O +- O +terminal O +ligand B-structure_element +binding I-structure_element +domain I-structure_element +( O +LBD B-structure_element +) O +via O +a O +flexible O +hinge B-structure_element +region I-structure_element +. O + +The O +DBD B-structure_element +is O +composed O +of O +two O +zinc B-structure_element +fingers I-structure_element +that O +allow O +it O +to O +interact O +with O +specifically O +encoded O +regions O +on O +the O +DNA O +called O +the O +nuclear B-structure_element +receptor I-structure_element +response I-structure_element +elements I-structure_element +. O + +The O +LBD B-structure_element +consists O +of O +a O +coactivator B-site +protein I-site +binding I-site +pocket I-site +and O +a O +hydrophobic B-site +ligand I-site +binding I-site +site I-site +( O +LBS B-site +) O +which O +are O +responsible O +for O +regulating O +transcription O +. O + +The O +coactivator B-site +binding I-site +pocket I-site +of O +RORγ B-protein +recognizes O +a O +conserved B-protein_state +helix B-structure_element +motif I-structure_element +LXXLL I-structure_element +( O +where O +X O +can O +be O +any O +amino O +acid O +) O +on O +transcriptional O +coactivator O +complexes O +and O +recruits O +it O +to O +activate O +transcription O +. O + +In O +RORγ B-protein +, O +the O +conformation O +of O +the O +AF2 B-structure_element +helix I-structure_element +required O +to O +form O +the O +coactivator B-site +binding I-site +pocket I-site +is O +mediated O +by O +a O +salt B-bond_interaction +bridge I-bond_interaction +between O +His479 B-residue_name_number +and O +Tyr502 B-residue_name_number +in O +addition O +to O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +interactions I-bond_interaction +between O +Tyr502 B-residue_name_number +and O +Phe506 B-residue_name_number +. O + +The O +conformation O +of O +the O +AF2 B-structure_element +helix I-structure_element +can O +be O +modulated O +through O +targeted O +ligands O +which O +bind O +the O +LBS B-site +and O +increase O +the O +binding O +of O +the O +coactivator O +protein O +( O +agonists O +) O +or O +disrupt O +binding O +( O +inverse O +agonists O +) O +thereby O +enhancing O +or O +inhibiting O +transcription O +. O + +Since O +RORγ B-protein +has O +been O +demonstrated O +to O +play O +an O +important O +role O +in O +pro O +- O +inflammatory O +gene O +expression O +patterns O +implicated O +in O +several O +major O +autoimmune O +diseases O +, O +our O +aim O +was O +to O +develop O +RORγ B-protein +inverse O +agonists O +that O +would O +help O +down O +regulate O +pro O +- O +inflammatory O +gene O +transcription O +. O + +Finally O +, O +comparing O +binding B-evidence +modes I-evidence +of O +our O +benzoxazinone B-chemical +RORγ B-protein +crystal B-evidence +structures I-evidence +to O +other O +ROR B-protein_type +structures B-evidence +, O +we O +hypothesize O +a O +new O +mode O +of O +action O +for O +achieving O +inverse O +agonism O +and O +selectivity O +. O + +Interestingly O +, O +the O +structural O +difference O +between O +the O +agonist B-protein_state +BIO592 B-chemical +and O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +was O +minor O +; O +with O +the O +2 B-chemical +, I-chemical +3 I-chemical +- I-chemical +dihydrobenzo I-chemical +[ I-chemical +1 I-chemical +, I-chemical +4 I-chemical +] I-chemical +oxazepin I-chemical +- I-chemical +4 I-chemical +- I-chemical +one I-chemical +ring O +system O +of O +BIO399 B-chemical +being O +3 O +atoms O +larger O +than O +the O +benzo B-chemical +[ I-chemical +1 I-chemical +, I-chemical +4 I-chemical +] I-chemical +oxazine I-chemical +- I-chemical +3 I-chemical +- I-chemical +one I-chemical +ring O +system O +of O +BIO592 B-chemical +. O + +In O +order O +to O +understand O +how O +small O +changes O +in O +the O +core O +ring O +system O +leads O +to O +inverse O +agonism O +, O +we O +wanted O +to O +structurally O +determine O +the O +binding O +mode O +of O +both O +BIO592 B-chemical +and O +BIO399 B-chemical +in O +the O +LBS B-site +of O +RORγ B-protein +using O +x B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +Structure B-evidence +of O +the O +RORγ518 B-complex_assembly +- I-complex_assembly +BIO592 I-complex_assembly +- I-complex_assembly +EBI96 I-complex_assembly +ternary O +complex O +is O +in O +a O +transcriptionally O +active B-protein_state +conformation O + +RORγ518 B-protein +bound B-protein_state +to I-protein_state +agonist B-protein_state +BIO592 B-chemical +was O +crystallized B-experimental_method +with O +a O +truncated B-protein_state +form O +of O +the O +coactivator O +peptide O +EBI96 B-chemical +to O +a O +resolution O +of O +2 O +. O +6 O +Å O +( O +Fig O +. O +2a O +). O + +The O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +His479 B-residue_name_number +and O +Tyr502 B-residue_name_number +has O +been O +reported O +to O +be O +critical O +for O +RORγ B-protein +agonist B-protein_state +activity O +. O + +Disrupting O +this O +interaction O +through O +mutagenesis B-experimental_method +reduced O +transcriptional O +activity O +of O +RORγ B-protein +. O + +This O +reduced O +transcriptional O +activity O +has O +been O +attributed O +to O +the O +inability O +of O +the O +AF2 B-structure_element +helix I-structure_element +to O +complete O +the O +formation O +of O +the O +coactivator B-site +binding I-site +pocket I-site +necessary O +for O +coactivator O +proteins O +to O +bind O +. O + +This O +interaction O +is O +further O +stabilized O +through O +a O +conserved B-protein_state +charged B-structure_element +clamp I-structure_element +wherein O +the O +backbone O +amide O +of O +Tyr7 B-residue_name_number +and O +carbonyl O +of O +Leu11 B-residue_name_number +of O +EBI96 B-chemical +form O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +Glu504 B-residue_name_number +( O +helix12 B-structure_element +) O +and O +Lys336 B-residue_name_number +( O +helix3 B-structure_element +) O +of O +RORγ B-protein +. O + +Formation O +of O +this O +charged B-structure_element +clamp I-structure_element +is O +essential O +for O +RORγ B-protein +’ O +s O +function O +for O +playing O +a O +role O +in O +transcriptional O +activation O +and O +this O +has O +been O +corroborated O +through O +mutagenic B-experimental_method +studies I-experimental_method +in O +this O +region O +. O + +BIO592 B-chemical +binds O +in O +a O +collapsed B-protein_state +conformation O +stabilizing O +the O +agonist B-protein_state +conformation O +of O +RORγ B-protein + +a O +Collapsed O +binding O +mode O +of O +agonist B-protein_state +BIO592 B-chemical +in O +the O +hydrophobic O +LBS B-site +of O +RORγ B-protein +. O + +The O +sulfonyl B-chemical +group O +faces O +the O +entrance O +of O +the O +pocket B-site +, O +while O +the O +CF3 O +makes O +a O +hydrophobic B-bond_interaction +contact I-bond_interaction +with O +Ala327 B-residue_name_number +. O + +RORγ B-protein +AF2 B-structure_element +helix I-structure_element +is O +sensitive O +to O +proteolysis O +in O +the O +presence B-protein_state +of I-protein_state +Inverse B-protein_state +Agonist I-protein_state +BIO399 B-chemical + +Next O +, O +we O +attempted O +co B-experimental_method +- I-experimental_method +crystallization I-experimental_method +with O +the O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +. O + +However O +, O +extensive O +crystallization B-experimental_method +efforts O +with O +BIO399 B-chemical +and O +RORγ518 B-protein +or O +other O +AF2 B-structure_element +intact B-protein_state +constructs O +did O +not O +produce O +crystals B-evidence +. O + +We O +hypothesized O +that O +the O +RORγ518 B-protein +coactivator O +peptide O +interaction O +in O +the O +FRET B-experimental_method +assay I-experimental_method +was O +disrupted O +upon O +BIO399 B-chemical +binding O +and O +that O +a O +conformational O +rearrangement O +of O +the O +AF2 B-structure_element +helix I-structure_element +could O +have O +occurred O +, O +hindering O +crystallization B-experimental_method +. O + +The O +unfolding O +of O +the O +AF2 B-structure_element +helix I-structure_element +has O +been O +observed O +for O +other O +nuclear B-protein_type +hormone I-protein_type +receptors I-protein_type +when O +bound B-protein_state +to I-protein_state +an O +inverse B-protein_state +agonist I-protein_state +or O +antagonist O +. O + +We O +used O +partial B-experimental_method +proteolysis I-experimental_method +in O +combination O +with O +mass B-experimental_method +spectrometry I-experimental_method +to O +determine O +if O +BIO399 B-chemical +was O +causing O +the O +AF2 B-structure_element +helix I-structure_element +to O +unfold O +. O + +Results O +of O +the O +Actinase B-experimental_method +E I-experimental_method +proteolysis I-experimental_method +experiments O +on O +RORγ518 B-protein +, O +the O +ternary O +complex O +of O +RORγ518 B-protein +with O +agonist B-protein_state +BIO592 B-chemical +and O +coactivator O +EBI96 B-chemical +, O +or O +in O +the O +presence B-protein_state +of I-protein_state +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +supported O +our O +hypothesis O +. O + +Analysis O +of O +the O +fragmentation B-evidence +pattern I-evidence +showed O +minimal O +proteolytic O +removal O +of O +the O +AF2 B-structure_element +helix I-structure_element +by O +Actinase B-protein +E I-protein +on O +RORγ518 B-protein +alone O +( O +ending O +at O +504 B-residue_range +to I-residue_range +506 I-residue_range +) O +and O +the O +ternary B-protein_state +complex I-protein_state +remained O +primarily O +intact O +( O +ending O +at O +515 B-residue_number +/ O +518 B-residue_number +) O +( O +Additional O +file O +4 O +). O + +We O +attributed O +the O +inability O +to O +form O +crystals B-evidence +to O +the O +unfolding O +of O +the O +AF2 B-structure_element +helix I-structure_element +induced O +by O +BIO399 B-chemical +. O + +AF2 B-protein_state +truncated I-protein_state +RORγ B-complex_assembly +BIO399 I-complex_assembly +complex O +is O +more O +amenable O +to O +crystallization B-experimental_method + +a O +The O +binary O +structure B-evidence +of O +AF2 B-protein_state +- I-protein_state +truncated I-protein_state +RORγ B-protein +and O +BIO399 B-chemical +. O + +b O +The O +superposition B-experimental_method +of O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +( O +Cyan O +) O +and O +agonist B-protein_state +BIO592 B-chemical +( O +Green O +). O + +c O +Movement O +of O +Met358 B-residue_name_number +and O +His479 B-residue_name_number +in O +the O +BIO399 B-chemical +( O +Cyan O +) O +and O +BIO592 B-chemical +( O +Green O +) O +structures B-evidence + +The O +Actinase B-protein +E I-protein +treated O +RORγ518 B-complex_assembly +BIO399 I-complex_assembly +ternary O +complex O +( O +aeRORγ493 B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +) O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +readily O +in O +several O +PEG O +based O +conditions O +. O + +The O +structure B-evidence +of O +aeRORγ493 B-complex_assembly +/ I-complex_assembly +4 I-complex_assembly +BIO399 I-complex_assembly +complex O +was O +solved B-experimental_method +to O +2 O +. O +3 O +Å O +and O +adopted O +a O +similar O +core O +fold O +to O +the O +BIO592 B-chemical +agonist B-protein_state +crystal B-evidence +structure I-evidence +( O +Fig O +. O +5a O +, O +Additional O +file O +3 O +). O + +Inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +uses O +Met358 B-residue_name_number +as O +a O +trigger O +for O +inverse O +agonism O + +The O +majority O +of O +the O +side O +chains O +within O +4 O +Å O +of O +BIO399 B-chemical +and O +BIO592 B-chemical +adopt O +similar O +rotomer O +conformations O +with O +the O +exceptions O +of O +Met358 B-residue_name_number +and O +His479 B-residue_name_number +( O +Fig O +. O +5c O +). O + +The O +difference B-evidence +density I-evidence +map I-evidence +showed O +clear O +positive B-evidence +density I-evidence +for O +Met358 B-residue_name_number +in O +an O +alternate O +rotomer O +conformation O +compared O +to O +the O +one O +observed O +in O +the O +molecular B-experimental_method +replacement I-experimental_method +model I-experimental_method +or O +the O +other O +agonist B-protein_state +containing O +models O +( O +Additional O +file O +6 O +). O + +We O +tried O +to O +refine O +Met358 B-residue_name_number +in O +the O +same O +conformation O +as O +the O +molecular B-experimental_method +replacement I-experimental_method +model I-experimental_method +or O +the O +other O +agonist B-protein_state +containing O +models O +, O +but O +the O +results O +clearly O +indicated O +that O +this O +was O +not O +possible O +, O +thus O +confirming O +the O +new O +rotamer O +conformation O +for O +the O +Met358 B-residue_name_number +sidechain O +in O +the O +inverse B-protein_state +agonist I-protein_state +bound I-protein_state +structure B-evidence +. O + +The O +change O +in O +rotomer O +conformation O +of O +Met358 B-residue_name_number +between O +the O +agonist B-protein_state +and O +inverse B-protein_state +agonist I-protein_state +structures B-evidence +is O +attributed O +to O +the O +gem O +- O +dimethyl O +group O +on O +the O +larger O +7 O +membered O +benzoxazinone B-chemical +ring O +system O +of O +BIO399 B-chemical +. O + +The O +comparison B-experimental_method +of O +the O +two O +structures B-evidence +shows O +that O +the O +agonist B-protein_state +conformation O +observed O +in O +the O +BIO592 B-chemical +structure B-evidence +would O +be O +perturbed O +by O +BIO399 B-chemical +pushing O +Met358 B-residue_name_number +into O +Phe506 B-residue_name_number +of O +the O +AF2 B-structure_element +helix I-structure_element +indicating O +that O +Met358 B-residue_name_number +is O +a O +trigger O +for O +inducing O +inverse O +agonism O +in O +RORγ B-protein +( O +Fig O +. O +5c O +). O + +b O +Overlay B-experimental_method +of O +M358 B-residue_name_number +in O +RORγ B-protein +structure B-evidence +BIO596 B-chemical +( O +Green O +), O +BIO399 B-chemical +( O +Cyan O +), O +Digoxin B-chemical +( O +Yellow O +), O +Compound O +2 O +( O +Grey O +), O +Compound O +48 O +( O +Salmon O +) O +and O +Compound O +4j O +( O +Orange O +) O + +The O +co B-evidence +- I-evidence +crystal I-evidence +structure I-evidence +of O +RORγ B-protein +with O +T0901317 B-chemical +( O +PDB O +code O +: O +4NB6 O +), O +an O +inverse B-protein_state +agonist I-protein_state +of O +RORγ B-protein +( O +IC50 B-evidence +of O +54nM O +in O +an O +SRC1 B-experimental_method +displacement I-experimental_method +FRET I-experimental_method +assay I-experimental_method +and O +an O +IC50 B-evidence +of O +59nM O +in O +our O +FRET B-experimental_method +assay I-experimental_method +( O +Additional O +file O +7 O +)) O +shows O +that O +it O +adopts O +a O +collapsed B-protein_state +conformation O +similar O +to O +the O +structure B-evidence +of O +BIO399 B-chemical +described O +here O +. O + +The O +two O +compounds O +superimpose B-experimental_method +with O +an O +RMSD B-evidence +of O +0 O +. O +81 O +Å O +( O +Fig O +. O +6a O +). O + +The O +CF3 O +group O +on O +the O +hexafluoropropanol B-chemical +group O +of O +T0901317 B-chemical +was O +reported O +to O +fit O +the O +electron B-evidence +density I-evidence +in O +two O +conformations O +one O +of O +which O +pushes O +Met358 B-residue_name_number +into O +the O +vicinity O +of O +Phe506 B-residue_name_number +in O +the O +RORγ B-protein +BIO592 B-chemical +agonist B-protein_state +structure B-evidence +. O + +Co B-evidence +- I-evidence +crystal I-evidence +structures I-evidence +of O +RORγ B-protein +have O +been O +generated O +with O +several O +potent O +inverse O +agonists O +adopting O +a O +linear B-protein_state +conformation O +distinct O +from O +the O +collapsed B-protein_state +conformations O +seen O +for O +BIO399 B-chemical +and O +T090131718 B-chemical +. O + +BIO399 B-chemical +neither O +orients O +the O +sidechain O +of O +Trp317 B-residue_name_number +toward O +Tyr502 B-residue_name_number +nor O +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +His479 B-residue_name_number +suggesting O +its O +mode O +of O +action O +is O +distinct O +from O +linear O +inverse O +agonists O +( O +Additional O +file O +8 O +). O + +In O +the O +linear O +inverse B-protein_state +agonist I-protein_state +crystal B-evidence +structures I-evidence +the O +side O +chain O +of O +Met358 B-residue_name_number +resides O +in O +a O +similar O +position O +as O +the O +rotomer O +observed O +in O +RORγ B-protein +agonist B-protein_state +structures B-evidence +with O +BIO592 B-chemical +described O +here O +or O +as O +observed O +in O +the O +hydroxycholesterol B-chemical +derivatives O +and O +therefore O +would O +not O +trigger O +inverse O +agonism O +with O +these O +ligands O +( O +Fig O +. O +6b O +). O + +BIO399 B-chemical +shows O +selectivity O +for O +RORγ B-protein +over O +RORα B-protein +and O +RORβ B-protein +in O +a O +GAL4 B-experimental_method +Cellular I-experimental_method +Reporter I-experimental_method +Assay I-experimental_method + +a O +Overlay B-experimental_method +of O +RORα B-protein +( O +yellow O +), O +β B-protein +( O +pink O +) O +and O +γ B-protein +( O +cyan O +) O +showing O +side O +chain O +differences O +at O +Met358 B-residue_name_number +inverse O +agonism O +trigger O +position O +and O +( O +b O +) O +around O +the O +benzoxazinone B-chemical +ring O +system O +of O +BIO399 B-chemical + +In O +order O +to O +assess O +the O +in O +vivo O +selectivity O +profile O +of O +BIO399 B-chemical +a O +cellular B-experimental_method +reporter I-experimental_method +assay I-experimental_method +was O +implemented O +where O +the O +ligand B-structure_element +binding I-structure_element +domains I-structure_element +of O +ROR B-protein_type +α B-protein +, O +β B-protein +and O +γ B-protein +were O +fused B-experimental_method +to I-experimental_method +the O +DNA B-structure_element +binding I-structure_element +domain I-structure_element +of O +the O +transcriptional B-protein_type +factor I-protein_type +GAL4 B-protein +. O + +The O +ROR B-protein_type +- O +GAL4 B-protein +fusion O +proteins O +were O +expressed O +in O +cells O +with O +the O +luciferase O +reporter O +gene O +under O +the O +control O +of O +a O +GAL4 B-protein +promoter O +. O + +BIO399 B-chemical +inhibited O +the O +luciferase O +activity O +when O +added O +to O +the O +cells O +expressing O +the O +RORγ B-protein +- O +GAL4 B-protein +fusion O +with O +an O +in O +vivo O +IC50 B-evidence +of O +42 O +. O +5nM O +while O +showing O +> O +235 O +and O +28 O +fold O +selectivity O +over O +cells O +expressing O +GAL4 B-protein +fused O +to O +the O +LBD B-structure_element +of O +ROR B-protein_type +α B-protein +or O +β B-protein +, O +respectively O +( O +Table O +1 O +). O + +The O +LBS B-site +of O +RORs B-protein_type +share O +a O +high O +degree O +of O +similarity O +. O + +This O +selectivity O +profile O +for O +BIO399 B-chemical +is O +attributed O +to O +the O +shorter O +leucine B-residue_name +side O +chain O +in O +RORα B-protein +and O +β B-protein +which O +would O +not O +reach O +the O +phenylalanine B-residue_name +on O +the O +AF2 B-structure_element +helix I-structure_element +further O +underscoring O +the O +role O +of O +Met358 B-residue_name_number +as O +a O +trigger O +for O +RORγ B-protein +specific O +inverse O +agonism O +( O +Fig O +. O +7a O +). O + +We O +hypothesize O +that O +the O +two O +phenylalanine B-residue_name +residues O +in O +the O +LBS B-site +of O +RORα B-protein +occlude O +the O +dihydrobenzoxazepinone B-chemical +ring O +system O +of O +BIO399 B-chemical +from O +binding O +it O +and O +responsible O +for O +the O +increase O +in O +selectivity O +for O +RORα B-protein +over O +β B-protein +. O + +We O +have O +identified O +a O +novel O +series O +of O +synthetic O +benzoxazinone B-chemical +ligands O +which O +modulate O +the O +transcriptional O +activity O +of O +RORγ B-protein +in O +a O +FRET B-experimental_method +based I-experimental_method +assay I-experimental_method +. O + +Using O +partial B-experimental_method +proteolysis I-experimental_method +we O +show O +a O +conformational O +change O +which O +destabilizes O +the O +AF2 B-structure_element +helix I-structure_element +of O +RORγ B-protein +when O +the O +inverse B-protein_state +agonist I-protein_state +BIO399 B-chemical +binds O +. O + +The O +two O +RORγ B-protein +co B-evidence +- I-evidence +crystal I-evidence +structures I-evidence +reported O +here O +show O +how O +a O +small O +change O +to O +the O +core O +ring O +system O +can O +modulate O +the O +mode O +of O +action O +from O +agonist B-protein_state +( O +BIO592 B-chemical +) O +to O +inverse O +agonism O +( O +BIO399 B-chemical +). O + +Finally O +, O +we O +are O +reporting O +a O +newly O +identified O +trigger O +for O +achieving O +RORγ B-protein +specific O +inverse O +agonism O +in O +an O +in O +vivo O +setting O +through O +Met358 B-residue_name_number +which O +perturbs O +the O +agonist B-protein_state +conformation O +of O +the O +AF2 B-structure_element +helix I-structure_element +and O +prevents O +coactivator O +protein O +binding O +. O + +Bacterial B-taxonomy_domain +Microcompartments B-complex_assembly +( O +BMCs B-complex_assembly +) O +are O +proteinaceous O +organelles O +that O +encapsulate O +critical O +segments O +of O +autotrophic O +and O +heterotrophic O +metabolic O +pathways O +; O +they O +are O +functionally O +diverse O +and O +are O +found O +across O +23 O +different O +phyla O +. O + +The O +core O +enzyme O +phosphotransacylase B-protein_type +( O +PTAC B-protein_type +) O +recycles O +Coenzyme B-chemical +A I-chemical +and O +generates O +an O +acyl B-chemical +phosphate I-chemical +that O +can O +serve O +as O +an O +energy O +source O +. O + +The O +PTAC B-protein_type +predominantly O +associated O +with O +metabolosomes B-complex_assembly +( O +PduL B-protein_type +) O +has O +no O +sequence O +homology O +to O +the O +PTAC B-protein_type +ubiquitous O +among O +fermentative B-taxonomy_domain +bacteria I-taxonomy_domain +( O +Pta B-protein_type +). O + +Here O +, O +we O +report O +two O +high O +- O +resolution O +PduL B-protein_type +crystal B-evidence +structures I-evidence +with B-protein_state +bound I-protein_state +substrates I-protein_state +. O + +The O +PduL B-protein_type +fold B-structure_element +is O +unrelated O +to O +that B-structure_element +of O +Pta B-protein_type +; O +it O +contains O +a O +dimetal B-site +active I-site +site I-site +involved O +in O +a O +catalytic O +mechanism O +distinct O +from O +that O +of O +the O +housekeeping B-protein_state +PTAC B-protein_type +. O + +The O +PduL B-protein_type +structure B-evidence +, O +in O +the O +context O +of O +the O +catalytic O +core O +, O +completes O +our O +understanding O +of O +the O +structural O +basis O +of O +cofactor O +recycling O +in O +the O +metabolosome B-complex_assembly +lumen O +. O + +This O +study O +describes O +the O +structure B-evidence +of O +a O +novel O +phosphotransacylase B-protein_type +enzyme O +that O +facilitates O +the O +recycling O +of O +the O +essential O +cofactor O +acetyl B-chemical +- I-chemical +CoA I-chemical +within O +a O +bacterial B-taxonomy_domain +organelle O +and O +discusses O +the O +properties O +of O +the O +enzyme O +' O +s O +active B-site +site I-site +and O +how O +it O +is O +packaged O +into O +the O +organelle O +. O + +The O +phosphotransacylase B-protein_type +( O +Pta B-protein_type +) O +enzyme O +catalyzes O +the O +conversion O +between O +acyl B-chemical +- I-chemical +CoA I-chemical +and O +acyl B-chemical +- I-chemical +phosphate I-chemical +. O + +This O +reaction O +directly O +links O +an O +acyl B-chemical +- I-chemical +CoA I-chemical +with O +ATP B-chemical +generation O +via O +substrate O +- O +level O +phosphorylation O +, O +producing O +short B-chemical +- I-chemical +chain I-chemical +fatty I-chemical +acids I-chemical +( O +e O +. O +g O +., O +acetate B-chemical +), O +and O +also O +provides O +a O +path O +for O +short B-chemical +- I-chemical +chain I-chemical +fatty I-chemical +acids I-chemical +to O +enter O +central O +metabolism O +. O + +Due O +to O +this O +key O +function O +, O +Pta O +is O +conserved B-protein_state +across O +the O +bacterial B-taxonomy_domain +kingdom I-taxonomy_domain +. O + +Recently O +, O +a O +new O +type O +of O +phosphotransacylase B-protein_type +was O +described O +that O +shares O +no O +evolutionary O +relation O +to O +Pta B-protein_type +. O + +Not O +only O +does O +PduL B-protein_type +facilitate O +substrate O +level O +phosphorylation O +, O +but O +it O +also O +is O +critical O +for O +cofactor O +recycling O +within O +, O +and O +product O +efflux O +from O +, O +the O +organelle O +. O + +We O +solved B-experimental_method +the O +structure B-evidence +of O +this O +convergent B-protein_state +phosphotransacylase B-protein_type +and O +show O +that O +it O +is O +completely O +structurally O +different O +from O +Pta B-protein_type +, O +including O +its O +active B-site +site I-site +architecture O +. O + +Bacterial B-taxonomy_domain +Microcompartments B-complex_assembly +( O +BMCs B-complex_assembly +) O +are O +organelles O +that O +encapsulate O +enzymes O +for O +sequential O +biochemical O +reactions O +within O +a O +protein O +shell B-structure_element +. O + +The O +shell B-structure_element +is O +typically O +composed O +of O +three O +types O +of O +protein O +subunits O +, O +which O +form O +either O +hexagonal B-protein_state +( O +BMC B-complex_assembly +- I-complex_assembly +H I-complex_assembly +and O +BMC B-complex_assembly +- I-complex_assembly +T I-complex_assembly +) O +or O +pentagonal B-protein_state +( O +BMC B-complex_assembly +- I-complex_assembly +P I-complex_assembly +) O +tiles O +that O +assemble O +into O +a O +polyhedral B-protein_state +shell B-structure_element +. O + +The O +vitamin B-complex_assembly +B12 I-complex_assembly +- I-complex_assembly +dependent I-complex_assembly +propanediol I-complex_assembly +- I-complex_assembly +utilizing I-complex_assembly +( I-complex_assembly +PDU I-complex_assembly +) I-complex_assembly +BMC I-complex_assembly +was O +one O +of O +the O +first O +functionally O +characterized O +catabolic B-protein_state +BMCs B-complex_assembly +; O +subsequently O +, O +other O +types O +have O +been O +implicated O +in O +the O +degradation O +of O +ethanolamine B-chemical +, O +choline B-chemical +, O +fucose B-chemical +, O +rhamnose B-chemical +, O +and O +ethanol B-chemical +, O +all O +of O +which O +produce O +different O +aldehyde B-chemical +intermediates O +( O +Table O +1 O +). O + +More O +recently O +, O +bioinformatic B-experimental_method +studies I-experimental_method +have O +demonstrated O +the O +widespread O +distribution O +of O +BMCs B-complex_assembly +among O +diverse O +bacterial B-taxonomy_domain +phyla I-taxonomy_domain +and O +grouped O +them O +into O +23 O +different O +functional O +types O +. O + +The O +reactions O +carried O +out O +in O +the O +majority O +of O +catabolic B-protein_state +BMCs B-complex_assembly +( O +also O +known O +as O +metabolosomes B-complex_assembly +) O +fit O +a O +generalized O +biochemical O +paradigm O +for O +the O +oxidation O +of O +aldehydes B-chemical +( O +Fig O +1 O +). O + +This O +involves O +a O +BMC B-complex_assembly +- O +encapsulated O +signature O +enzyme O +that O +generates O +a O +toxic O +and O +/ O +or O +volatile O +aldehyde B-chemical +that O +the O +BMC B-complex_assembly +shell B-structure_element +sequesters O +from O +the O +cytosol O +. O + +These O +two O +cofactors O +are O +relatively O +large O +, O +and O +their O +diffusion O +across O +the O +protein B-structure_element +shell I-structure_element +is O +thought O +to O +be O +restricted O +, O +necessitating O +their O +regeneration O +within O +the O +BMC B-complex_assembly +lumen O +. O + +The O +final O +product O +of O +the O +BMC B-complex_assembly +, O +an O +acyl B-chemical +- I-chemical +phosphate I-chemical +, O +can O +then O +be O +used O +to O +generate O +ATP B-chemical +via O +acyl B-protein_type +kinase I-protein_type +, O +or O +revert O +back O +to O +acyl B-chemical +- I-chemical +CoA I-chemical +by O +Pta B-protein_type +for O +biosynthesis O +. O + +Collectively O +, O +the O +aldehyde B-protein_type +and I-protein_type +alcohol I-protein_type +dehydrogenases I-protein_type +, O +as O +well O +as O +the O +PTAC B-protein_type +, O +constitute O +the O +common O +metabolosome B-complex_assembly +core O +. O + +General O +biochemical O +model O +of O +aldehyde B-protein_state +- I-protein_state +degrading I-protein_state +BMCs B-complex_assembly +( O +metabolosomes B-complex_assembly +) O +illustrating O +the O +common O +metabolosome B-complex_assembly +core O +enzymes O +and O +reactions O +. O + +Characterized O +and O +predicted O +catabolic B-protein_state +BMC B-complex_assembly +( O +metabolosome B-complex_assembly +) O +types O +that O +represent O +the O +aldehyde B-chemical +- O +degrading O +paradigm O +( O +for O +definition O +of O +types O +see O +Kerfeld O +and O +Erbilgin O +). O + +Name O +PTAC B-protein_type +Type O +Sequestered O +Aldehyde B-chemical +PDU B-complex_assembly +* O +PduL B-protein_type +propionaldehyde B-chemical +EUT1 B-complex_assembly +PTA_PTB B-protein_type +acetaldehyde B-chemical +EUT2 B-complex_assembly +PduL B-protein_type +acetaldehyde B-chemical +ETU B-complex_assembly +None O +acetaldehyde B-chemical +GRM1 B-complex_assembly +/ I-complex_assembly +CUT I-complex_assembly +PduL B-protein_type +acetaldehyde B-chemical +GRM2 B-complex_assembly +PduL B-protein_type +acetaldehyde B-chemical +GRM3 B-complex_assembly +*, I-complex_assembly +4 I-complex_assembly +PduL B-protein_type +propionaldehyde B-chemical +GRM5 B-complex_assembly +/ I-complex_assembly +GRP I-complex_assembly +PduL B-protein_type +propionaldehyde B-chemical +PVM B-complex_assembly +* O +PduL B-protein_type +lactaldehyde B-chemical +RMM1 B-complex_assembly +, I-complex_assembly +2 I-complex_assembly +None O +unknown O +SPU B-complex_assembly +PduL B-protein_type +unknown O + +* O +PduL B-protein_type +from O +these O +functional O +types O +of O +metabolosomes B-complex_assembly +were O +purified O +in O +this O +study O +. O + +The O +concerted O +functioning O +of O +a O +PTAC B-protein_type +and O +an O +acetate B-protein_type +kinase I-protein_type +( O +Ack B-protein_type +) O +is O +crucial O +for O +ATP B-chemical +generation O +in O +the O +fermentation O +of O +pyruvate B-chemical +to O +acetate B-chemical +( O +see O +Reactions O +1 O +and O +2 O +). O + +Both O +enzymes O +are O +, O +however O +, O +not O +restricted O +to O +fermentative B-taxonomy_domain +organisms I-taxonomy_domain +. O + +Reaction O +1 O +: O +acetyl B-chemical +- I-chemical +S I-chemical +- I-chemical +CoA I-chemical ++ O +Pi B-chemical +←→ O +acetyl B-chemical +phosphate I-chemical ++ O +CoA B-chemical +- I-chemical +SH I-chemical +( O +PTAC B-protein_type +) O + +Reaction O +2 O +: O +acetyl B-chemical +phosphate I-chemical ++ O +ADP B-chemical +←→ O +acetate B-chemical ++ O +ATP B-chemical +( O +Ack B-protein_type +) O + +Pta B-protein_type +has O +been O +extensively O +characterized O +due O +to O +its O +key O +role O +in O +fermentation O +. O + +More O +recently O +, O +a O +second O +type O +of O +PTAC B-protein_type +without O +any O +sequence O +homology O +to O +Pta B-protein_type +was O +identified O +. O + +This O +protein O +, O +PduL B-protein_type +( O +Pfam O +domain O +PF06130 B-structure_element +), O +was O +shown O +to O +catalyze O +the O +conversion O +of O +propionyl B-chemical +- I-chemical +CoA I-chemical +to O +propionyl B-chemical +- I-chemical +phosphate I-chemical +and O +is O +associated O +with O +a O +BMC B-complex_assembly +involved O +in O +propanediol O +utilization O +, O +the O +PDU B-complex_assembly +BMC I-complex_assembly +. O + +Both O +pduL B-gene +and O +pta B-gene +genes O +can O +be O +found O +in O +genetic O +loci O +of O +functionally O +distinct O +BMCs B-complex_assembly +, O +although O +the O +PduL B-protein_type +type O +is O +much O +more O +prevalent O +, O +being O +found O +in O +all O +but O +one O +type O +of O +metabolosome B-gene +locus I-gene +: O +EUT1 B-gene +( O +Table O +1 O +). O + +Furthermore O +, O +in O +the O +Integrated O +Microbial O +Genomes O +Database O +, O +91 O +% O +of O +genomes O +that O +encode O +PF06130 B-structure_element +also O +encode O +genes O +for O +shell O +proteins O +. O + +As O +a O +member O +of O +the O +core O +biochemical O +machinery O +of O +functionally O +diverse O +aldehyde B-protein_state +- I-protein_state +oxidizing I-protein_state +metabolosomes B-complex_assembly +, O +PduL B-protein_type +must O +have O +a O +certain O +level O +of O +substrate O +plasticity O +( O +see O +Table O +1 O +) O +that O +is O +not O +required O +of O +Pta B-protein_type +, O +which O +has O +generally O +been O +observed O +to O +prefer O +acetyl B-chemical +- I-chemical +CoA I-chemical +. O +PduL B-protein_type +from O +the O +PDU B-complex_assembly +BMC I-complex_assembly +of O +Salmonella B-species +enterica I-species +favors O +propionyl B-chemical +- I-chemical +CoA I-chemical +over O +acetyl B-chemical +- I-chemical +CoA I-chemical +, O +and O +it O +is O +likely O +that O +PduL B-protein_type +orthologs O +in O +functionally O +diverse O +BMCs B-complex_assembly +would O +have O +substrate O +preferences O +for O +other O +CoA B-chemical +derivatives O +. O + +EPs B-structure_element +have O +also O +been O +observed O +to O +cause O +proteins O +to O +aggregate O +, O +and O +this O +has O +recently O +been O +suggested O +to O +be O +functionally O +relevant O +as O +an O +initial O +step O +in O +metabolosome B-complex_assembly +assembly O +, O +in O +which O +a O +multifunctional O +protein O +core O +is O +formed O +, O +around O +which O +the O +shell B-structure_element +assembles O +. O + +Of O +the O +three O +common O +metabolosome B-complex_assembly +core O +enzymes O +, O +crystal B-evidence +structures I-evidence +are O +available O +for O +both O +the O +alcohol B-protein_type +and I-protein_type +aldehyde I-protein_type +dehydrogenases I-protein_type +. O + +In O +contrast O +, O +the O +structure B-evidence +of O +PduL B-protein_type +, O +the O +PTAC B-protein_type +found O +in O +the O +vast O +majority O +of O +catabolic B-protein_state +BMCs B-complex_assembly +, O +has O +not O +been O +determined O +. O + +This O +is O +a O +major O +gap O +in O +our O +understanding O +of O +metabolosome B-complex_assembly +- O +encapsulated O +biochemistry O +and O +cofactor O +recycling O +. O + +Moreover O +, O +it O +will O +be O +useful O +for O +guiding O +efforts O +to O +engineer O +novel O +BMC B-complex_assembly +cores O +for O +biotechnological O +applications O +. O + +No O +available O +protein O +structures O +contain O +the O +PF06130 B-structure_element +domain O +, O +and O +homology B-experimental_method +searches I-experimental_method +using O +the O +primary O +structure O +of O +PduL B-protein_type +do O +not O +return O +any O +significant O +results O +that O +would O +allow O +prediction O +of O +the O +structure B-evidence +. O + +Moreover O +, O +the O +evident O +novelty O +of O +PduL B-protein_type +makes O +its O +structure B-evidence +interesting O +in O +the O +context O +of O +convergent O +evolution O +of O +PTAC B-protein_type +function O +; O +to O +- O +date O +, O +only O +the O +Pta B-protein_type +active B-site +site I-site +and O +catalytic O +mechanism O +is O +known O +. O + +We O +propose O +a O +catalytic O +mechanism O +analogous O +but O +yet O +distinct O +from O +the O +ubiquitous O +Pta B-protein_type +enzyme O +, O +highlighting O +the O +functional O +convergence O +of O +two O +enzymes O +with O +completely O +different O +structures O +and O +metal O +requirements O +. O + +We O +also O +investigate O +the O +quaternary O +structures O +of O +three O +different O +PduL B-protein_type +homologs O +and O +situate O +our O +findings O +in O +the O +context O +of O +organelle O +biogenesis O +in O +functionally O +diverse O +BMCs B-complex_assembly +. O + +We O +cloned B-experimental_method +, I-experimental_method +expressed I-experimental_method +, I-experimental_method +and I-experimental_method +purified I-experimental_method +three O +different O +PduL B-protein_type +homologs O +from O +functionally O +distinct O +BMCs B-complex_assembly +( O +Table O +1 O +): O +from O +the O +well O +- O +studied O +pdu B-gene +locus I-gene +in O +S B-species +. I-species +enterica I-species +Typhimurium I-species +LT2 I-species +( O +sPduL B-protein +), O +from O +the O +recently O +characterized O +pvm B-gene +locus I-gene +in O +Planctomyces B-species +limnophilus I-species +( O +pPduL B-protein +), O +and O +from O +the O +grm3 B-gene +locus I-gene +in O +Rhodopseudomonas B-species +palustris I-species +BisB18 I-species +( O +rPduL B-protein +). O + +While O +purifying O +full B-protein_state +- I-protein_state +length I-protein_state +sPduL B-protein +, O +we O +observed O +a O +tendency O +to O +aggregation O +as O +described O +previously O +, O +with O +a O +large O +fraction O +of O +the O +expressed O +protein O +found O +in O +the O +insoluble O +fraction O +in O +a O +white O +, O +cake O +- O +like O +pellet O +. O + +Similar O +differences O +in O +solubility O +were O +observed O +for O +pPduL B-protein +and O +rPduL B-protein +when O +comparing O +EP B-protein_state +- I-protein_state +truncated I-protein_state +forms O +to O +the O +full B-protein_state +- I-protein_state +length I-protein_state +protein O +, O +but O +none O +were O +quite O +as O +dramatic O +as O +for O +sPduL B-protein +. O +We O +confirmed O +that O +all O +homologs O +were O +active B-protein_state +( O +S1a O +and O +S1b O +Fig O +). O + +Among O +these O +, O +we O +were O +only O +able O +to O +obtain O +diffraction B-evidence +- I-evidence +quality I-evidence +crystals I-evidence +of O +rPduL B-protein +after O +removing B-experimental_method +the O +N O +- O +terminal O +putative O +EP B-structure_element +( O +33 B-residue_range +amino I-residue_range +acids I-residue_range +, O +also O +see O +Fig O +2a O +) O +( O +rPduLΔEP B-mutant +). O + +Truncated B-protein_state +rPduLΔEP B-mutant +had O +comparable O +enzymatic O +activity O +to O +the O +full B-protein_state +- I-protein_state +length I-protein_state +enzyme O +( O +S1a O +Fig O +). O + +Structural O +overview O +of O +R B-species +. I-species +palustris I-species +PduL B-protein_type +from O +the O +grm3 B-gene +locus I-gene +. O + +( O +a O +) O +Primary O +and O +secondary O +structure O +of O +rPduL B-protein +( O +tubes O +represent O +α B-structure_element +- I-structure_element +helices I-structure_element +, O +arrows O +β B-structure_element +- I-structure_element +sheets I-structure_element +and O +dashed O +line O +residues O +disordered O +in O +the O +structure B-evidence +. O + +The O +first B-residue_range +33 I-residue_range +amino I-residue_range +acids I-residue_range +are O +present O +only O +in O +the O +wildtype O +construct O +and O +contains O +the O +predicted O +EP B-structure_element +alpha B-structure_element +helix I-structure_element +, O +α0 B-structure_element +); O +the O +truncated B-protein_state +rPduLΔEP B-mutant +that O +was O +crystallized B-experimental_method +begins O +with O +M B-residue_name +- O +G B-residue_name +- O +V B-residue_name +. O +Coloring O +is O +according O +to O +structural O +domains O +( O +domain B-structure_element +1 I-structure_element +D36 B-residue_range +- I-residue_range +N46 I-residue_range +/ O +Q155 B-residue_range +- I-residue_range +C224 I-residue_range +, O +blue O +; O +loop B-structure_element +insertion I-structure_element +G61 B-residue_range +- I-residue_range +E81 I-residue_range +, O +grey O +; O +domain B-structure_element +2 I-structure_element +R47 B-residue_range +- I-residue_range +F60 I-residue_range +/ O +E82 B-residue_range +- I-residue_range +A154 I-residue_range +, O +red O +). O + +Metal B-site +coordination I-site +residues I-site +are O +highlighted O +in O +light O +blue O +and O +CoA B-site +contacting I-site +residues I-site +in O +magenta O +, O +residues O +contacting O +the O +CoA B-chemical +of O +the O +other O +chain O +are O +also O +outlined O +. O + +( O +b O +) O +Cartoon O +representation O +of O +the O +structure B-evidence +colored O +by O +domains O +and O +including O +secondary O +structure B-evidence +numbering O +. O + +Coenzyme B-chemical +A I-chemical +is O +shown O +in O +magenta O +sticks O +and O +Zinc B-chemical +( O +grey O +) O +as O +spheres O +. O + +We O +collected B-experimental_method +a I-experimental_method +native I-experimental_method +dataset I-experimental_method +from O +rPduLΔEP B-mutant +crystals B-evidence +diffracting O +to O +a O +resolution O +of O +1 O +. O +54 O +Å O +( O +Table O +2 O +). O + +Using O +a O +mercury B-experimental_method +- I-experimental_method +derivative I-experimental_method +crystal I-experimental_method +form O +diffracting O +to O +1 O +. O +99 O +Å O +( O +Table O +2 O +), O +we O +obtained O +high O +quality O +electron B-evidence +density I-evidence +for O +model O +building O +and O +used O +the O +initial O +model O +to O +refine O +against O +the O +native O +data O +to O +Rwork B-evidence +/ O +Rfree B-evidence +values O +of O +18 O +. O +9 O +/ O +22 O +. O +1 O +%. O + +There O +are O +two O +PduL B-protein_type +molecules O +in O +the O +asymmetric O +unit O +of O +the O +P212121 O +unit O +cell O +. O + +We O +were O +able O +to O +fit O +all O +of O +the O +primary O +structure O +of O +PduLΔEP B-mutant +into O +the O +electron B-evidence +density I-evidence +with O +the O +exception O +of O +three O +amino O +acids O +at O +the O +N O +- O +terminus O +and O +two O +amino O +acids O +at O +the O +C O +- O +terminus O +( O +Fig O +2a O +); O +the O +model O +is O +of O +excellent O +quality O +( O +Table O +2 O +). O + +Structurally O +, O +PduL B-protein_type +consists O +of O +two O +domains B-structure_element +( O +Fig O +2 O +, O +blue O +/ O +red O +), O +each O +a O +beta B-structure_element +- I-structure_element +barrel I-structure_element +that O +is O +capped O +on O +both O +ends O +by O +short O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +β B-structure_element +- I-structure_element +Barrel I-structure_element +2 I-structure_element +consists O +mainly O +of O +the O +central O +segment O +of O +primary O +structure O +( O +β2 B-structure_element +, O +β5 B-structure_element +– I-structure_element +β9 I-structure_element +; O +residues O +47 B-residue_range +– I-residue_range +60 I-residue_range +and O +82 B-residue_range +– I-residue_range +154 I-residue_range +) O +( O +Fig O +2 O +, O +red O +), O +but O +is O +interrupted O +by O +a O +short B-structure_element +two I-structure_element +- I-structure_element +strand I-structure_element +beta I-structure_element +sheet I-structure_element +( O +β3 B-structure_element +- I-structure_element +β4 I-structure_element +, O +residues O +61 B-residue_range +– I-residue_range +81 I-residue_range +). O + +This O +β B-structure_element +- I-structure_element +sheet I-structure_element +is O +involved O +in O +contacts O +between O +the O +two O +domains O +and O +forms O +a O +lid O +over O +the O +active B-site +site I-site +. O + +Residues O +in O +this O +region O +( O +Gln42 B-residue_name_number +, O +Pro43 B-residue_name_number +, O +Gly44 B-residue_name_number +), O +covering O +the O +active B-site +site I-site +, O +are O +strongly B-protein_state +conserved I-protein_state +( O +Fig O +3 O +). O + +This O +structural O +arrangement O +is O +completely O +different O +from O +the O +functionally O +related O +Pta B-protein_type +, O +which O +is O +composed O +of O +two O +domains B-structure_element +, O +each O +consisting O +of O +a O +central O +flat O +beta B-structure_element +sheet I-structure_element +with O +alpha B-structure_element +- I-structure_element +helices I-structure_element +on O +the O +top O +and O +bottom O +. O + +Residues O +100 O +% O +conserved O +across O +all O +PduL B-protein_type +homologs O +in O +our O +dataset O +are O +noted O +with O +an O +asterisk O +, O +and O +residues O +conserved O +in O +over O +90 O +% O +of O +sequences O +are O +noted O +with O +a O +colon O +. O + +There O +are O +two O +PduL B-protein_type +molecules O +in O +the O +asymmetric O +unit O +forming O +a O +butterfly B-protein_state +- I-protein_state +shaped I-protein_state +dimer B-oligomeric_state +( O +Fig O +4c O +). O + +Consistent O +with O +this O +, O +results O +from O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +of O +rPduLΔEP B-mutant +suggest O +that O +it O +is O +a O +dimer B-oligomeric_state +in O +solution O +( O +Fig O +5e O +). O + +The O +interface B-site +between O +the O +two O +chains O +buries O +882 O +Å2 O +per O +monomer B-oligomeric_state +and O +is O +mainly O +formed O +by O +α B-structure_element +- I-structure_element +helices I-structure_element +2 I-structure_element +and I-structure_element +4 I-structure_element +and O +parts O +of O +β B-structure_element +- I-structure_element +sheets I-structure_element +12 I-structure_element +and I-structure_element +14 I-structure_element +, O +as O +well O +as O +a O +π O +– O +π O +stacking O +of O +the O +adenine B-chemical +moiety O +of O +CoA B-chemical +with O +Phe116 B-residue_name_number +of O +the O +adjacent O +chain O +( O +Fig O +4c O +). O + +The O +folds O +of O +the O +two O +chains O +in O +the O +asymmetric O +unit O +are O +very O +similar O +, O +superimposing B-experimental_method +with O +a O +rmsd B-evidence +of O +0 O +. O +16 O +Å O +over O +2 O +, O +306 O +aligned O +atom O +pairs O +. O + +The O +peripheral O +helices B-structure_element +and O +the O +short B-structure_element +antiparallel I-structure_element +β3 I-structure_element +– I-structure_element +4 I-structure_element +sheet I-structure_element +mediate O +most O +of O +the O +crystal O +contacts O +. O + +Details O +of O +active B-site +site I-site +, O +dimeric B-oligomeric_state +assembly O +, O +and O +sequence O +conservation O +of O +PduL B-protein_type +. O + +( O +a O +, O +b O +) O +Proposed O +active B-site +site I-site +of O +PduL B-protein_type +with O +relevant O +residues O +shown O +as O +sticks O +in O +atom O +coloring O +( O +nitrogen B-chemical +blue O +, O +oxygen B-chemical +red O +, O +sulfur B-chemical +yellow O +), O +zinc B-chemical +as O +grey O +colored O +spheres O +and O +coordinating O +ordered O +water B-chemical +molecules O +in O +red O +. O + +( O +c O +) O +View O +of O +the O +dimer B-oligomeric_state +in O +the O +asymmetric O +unit O +from O +the O +side O +, O +domains B-structure_element +1 I-structure_element +and I-structure_element +2 I-structure_element +colored O +as O +in O +Fig O +2 O +and O +the O +two O +chains O +differentiated O +by O +blue O +/ O +red O +versus O +slate O +/ O +firebrick O +. O + +( O +d O +) O +Surface O +representation O +of O +the O +structure B-evidence +with O +indicated O +conservation O +( O +red O +: O +high O +, O +white O +: O +intermediate O +, O +yellow O +: O +low O +). O + +All O +chromatograms B-evidence +are O +cropped O +to O +show O +only O +the O +linear O +range O +of O +separation O +based O +on O +standard O +runs O +, O +shown O +in O +black O +squares O +with O +a O +dashed O +linear O +trend O +line O +. O + +Active B-site +Site I-site +Properties O + +CoA B-chemical +and O +the O +metal O +ions O +bind O +between O +the O +two O +domains O +, O +presumably O +in O +the O +active B-site +site I-site +( O +Figs O +2b O +and O +4a O +). O + +To O +identify O +the O +bound O +metals O +, O +we O +performed O +an O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +fluorescence I-experimental_method +scan I-experimental_method +on O +the O +crystals B-evidence +at O +various O +wavelengths O +( O +corresponding O +to O +the O +K O +- O +edges O +of O +Mn B-chemical +, O +Fe B-chemical +, O +Co B-chemical +, O +Ni B-chemical +, O +Cu B-chemical +, O +and O +Zn B-chemical +). O + +There O +was O +a O +large O +signal O +at O +the O +zinc O +edge O +, O +and O +we O +tested O +for O +the O +presence O +of O +zinc B-chemical +by O +collecting B-experimental_method +full I-experimental_method +data I-experimental_method +sets I-experimental_method +before I-experimental_method +and I-experimental_method +after I-experimental_method +the I-experimental_method +Zn I-experimental_method +K I-experimental_method +- I-experimental_method +edge I-experimental_method +( I-experimental_method +1 I-experimental_method +. I-experimental_method +2861 I-experimental_method +and I-experimental_method +1 I-experimental_method +. I-experimental_method +2822 I-experimental_method +Å I-experimental_method +, O +respectively O +). O + +The O +large O +differences O +between O +the O +anomalous O +signals O +confirm O +the O +presence O +of O +zinc B-chemical +at O +both O +metal O +sites O +( O +S3 O +Fig O +). O + +The O +first O +zinc B-chemical +ion O +( O +Zn1 B-chemical +) O +is O +in O +a O +tetrahedral O +coordination O +state O +with O +His48 B-residue_name_number +, O +His50 B-residue_name_number +, O +Glu109 B-residue_name_number +, O +and O +the O +CoA B-chemical +sulfur B-chemical +( O +Fig O +4a O +). O + +The O +nitrogen O +atom O +coordinating O +the O +zinc B-chemical +is O +the O +Nε O +in O +each O +histidine B-residue_name +residue O +, O +as O +is O +typical O +for O +this O +interaction O +. O + +The O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +structure B-evidence +aligns B-experimental_method +well O +with O +the O +CoA B-protein_state +- I-protein_state +bound I-protein_state +structure B-evidence +( O +0 O +. O +43 O +Å O +rmsd B-evidence +over O +2 O +, O +361 O +atoms O +for O +the O +monomer B-oligomeric_state +, O +0 O +. O +83 O +Å O +over O +5 O +, O +259 O +aligned O +atoms O +for O +the O +dimer B-oligomeric_state +). O + +Conserved B-protein_state +Arg103 B-residue_name_number +seems O +to O +be O +involved O +in O +maintaining O +the O +phosphate B-chemical +in O +that O +position O +. O + +An O +additional O +phosphate B-chemical +molecule O +is O +bound O +at O +a O +crystal O +contact O +interface O +, O +perhaps O +accounting O +for O +the O +14 O +Å O +shorter O +c O +- O +axis O +in O +the O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +crystal O +form O +( O +Table O +2 O +). O + +Interestingly O +, O +some O +of O +the O +residues O +important O +for O +dimerization O +of O +rPduL B-protein +, O +particularly O +Phe116 B-residue_name_number +, O +are O +poorly B-protein_state +conserved I-protein_state +across O +PduL B-protein_type +homologs O +associated O +with O +functionally O +diverse O +BMCs B-complex_assembly +( O +Figs O +4c O +and O +3 O +), O +suggesting O +that O +they O +may O +have O +alternative O +oligomeric O +states O +. O + +We O +tested O +this O +hypothesis O +by O +performing O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +on O +both O +full B-protein_state +- I-protein_state +length I-protein_state +and O +truncated O +variants O +( O +lacking B-protein_state +the O +EP B-structure_element +, O +ΔEP B-mutant +) O +of O +sPduL B-protein +, O +rPduL B-protein +, O +and O +pPduL B-protein +. O +These O +three O +homologs O +are O +found O +in O +functionally O +distinct O +BMCs B-complex_assembly +( O +Table O +1 O +). O + +It O +has O +been O +proposed O +that O +the O +catabolic B-protein_state +BMCs B-complex_assembly +may O +assemble O +in O +a O +core O +- O +first O +manner O +, O +with O +the O +luminal O +enzymes O +( O +signature O +enzyme O +, O +aldehyde B-protein_type +, I-protein_type +and I-protein_type +alcohol I-protein_type +dehydrogenases I-protein_type +and O +the O +BMC B-complex_assembly +PTAC B-protein_type +) O +forming O +an O +initial O +bolus O +, O +or O +prometabolosome O +, O +around O +which O +a O +shell B-structure_element +assembles O +. O + +We O +found O +that O +not O +only O +did O +the O +different O +orthologs O +appear O +to O +assemble O +into O +different O +oligomeric O +states O +, O +but O +that O +quaternary O +structure O +was O +dependent O +on O +whether O +or O +not O +the O +EP B-structure_element +was O +present O +. O + +Full B-protein_state +- I-protein_state +length I-protein_state +sPduL B-protein +was O +unstable O +in O +solution O +— O +precipitating O +over O +time O +— O +and O +eluted O +throughout O +the O +entire O +volume O +of O +a O +size O +exclusion O +column O +, O +indicating O +it O +was O +nonspecifically O +aggregating O +. O + +However O +, O +when O +the O +putative O +EP B-structure_element +( O +residues O +1 B-residue_range +– I-residue_range +27 I-residue_range +) O +was O +removed B-experimental_method +( O +sPduL B-mutant +ΔEP I-mutant +), O +the O +truncated B-protein_state +protein O +was O +stable O +and O +eluted O +as O +a O +single O +peak O +( O +Fig O +5a O +) O +consistent O +with O +the O +size O +of O +a O +monomer B-oligomeric_state +( O +Fig O +5d O +, O +blue O +curve O +). O + +In O +contrast O +, O +both O +full B-protein_state +- I-protein_state +length I-protein_state +rPduL B-protein +and O +pPduL B-protein +appeared O +to O +exist O +in O +two O +distinct O +oligomeric O +states O +( O +Fig O +5b O +and O +5c O +respectively O +, O +orange O +curves O +), O +one O +form O +of O +the O +approximate O +size O +of O +a O +dimer B-oligomeric_state +and O +the O +second O +, O +a O +higher O +molecular O +weight O +oligomer B-oligomeric_state +(~ O +150 O +kDa O +). O + +Upon O +deletion B-experimental_method +of O +the O +putative O +EP B-structure_element +( O +residues O +1 B-residue_range +– I-residue_range +47 I-residue_range +for O +rPduL B-protein +, O +and O +1 B-residue_range +– I-residue_range +20 I-residue_range +for O +pPduL B-protein +), O +there O +was O +a O +distinct O +change O +in O +the O +elution O +profiles O +( O +Fig O +5b O +and O +5c O +respectively O +, O +blue O +curves O +). O + +In O +contrast O +, O +rPduLΔEP B-mutant +eluted O +as O +one O +smaller O +oligomer O +, O +possibly O +a O +dimer B-oligomeric_state +. O + +We O +also O +analyzed O +purified O +rPduL B-protein +and O +rPduLΔEP B-mutant +by O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +coupled O +with O +multiangle B-experimental_method +light I-experimental_method +scattering I-experimental_method +( O +SEC B-experimental_method +- I-experimental_method +MALS I-experimental_method +) O +for O +a O +complementary O +approach O +to O +assessing O +oligomeric O +state O +. O + +SEC B-experimental_method +- I-experimental_method +MALS I-experimental_method +analysis O +of O +rPdulΔEP B-mutant +is O +consistent O +with O +a O +dimer B-oligomeric_state +( O +as O +observed O +in O +the O +crystal B-evidence +structure I-evidence +) O +with O +a O +weighted B-evidence +average I-evidence +( I-evidence +Mw I-evidence +) I-evidence +and I-evidence +number I-evidence +average I-evidence +( I-evidence +Mn I-evidence +) I-evidence +of I-evidence +the I-evidence +molar I-evidence +mass I-evidence +of O +58 O +. O +4 O +kDa O ++/− O +11 O +. O +2 O +% O +and O +58 O +. O +8 O +kDa O ++/− O +10 O +. O +9 O +%, O +respectively O +( O +S4a O +Fig O +). O + +This O +corresponds O +to O +an O +oligomeric O +state O +of O +six B-oligomeric_state +subunits I-oligomeric_state +( O +calculated O +molecular B-evidence +weight I-evidence +of O +144 O +kDa O +). O + +Collectively O +, O +these O +data O +strongly O +suggest O +that O +the O +N O +- O +terminal O +EP B-structure_element +of O +PduL B-protein_type +plays O +a O +role O +in O +defining O +the O +quaternary O +structure O +of O +the O +protein O +. O + +The O +BMC B-complex_assembly +shell B-structure_element +not O +only O +sequesters O +specific O +enzymes O +but O +also O +their O +cofactors O +, O +thereby O +establishing O +a O +private O +cofactor O +pool O +dedicated O +to O +the O +encapsulated O +reactions O +. O + +In O +catabolic B-protein_state +BMCs B-complex_assembly +, O +CoA B-chemical +and O +NAD B-chemical ++ I-chemical +must O +be O +continually O +recycled O +within O +the O +organelle O +( O +Fig O +1 O +). O + +Curiously O +, O +while O +the O +housekeeping B-protein_state +Pta B-protein_type +could O +provide O +this O +function O +, O +and O +indeed O +does O +so O +in O +the O +case O +of O +one O +type O +of O +ethanolamine B-complex_assembly +- I-complex_assembly +utilizing I-complex_assembly +( I-complex_assembly +EUT I-complex_assembly +) I-complex_assembly +BMC I-complex_assembly +, O +the O +evolutionarily O +unrelated O +PduL B-protein_type +fulfills O +this O +function O +for O +the O +majority O +of O +metabolosomes B-complex_assembly +using O +a O +novel O +structure B-evidence +and O +active B-site +site I-site +for O +convergent O +evolution O +of O +function O +. O + +The O +Tertiary O +Structure O +of O +PduL B-protein_type +Is O +Formed O +by O +Discontinuous O +Segments O +of O +Primary O +Structure O + +The O +structure B-evidence +of O +PduL B-protein_type +consists O +of O +two B-structure_element +β I-structure_element +- I-structure_element +barrel I-structure_element +domains I-structure_element +capped O +by O +short B-structure_element +alpha I-structure_element +helical I-structure_element +segments I-structure_element +( O +Fig O +2b O +). O + +The O +two O +domains O +are O +structurally O +very O +similar O +( O +superimposing B-experimental_method +with O +a O +rmsd B-evidence +of O +1 O +. O +34 O +Å O +( O +over O +123 O +out O +of O +320 O +/ O +348 O +aligned O +backbone O +atoms O +, O +S5a O +Fig O +). O + +However O +, O +the O +amino O +acid O +sequences O +of O +the O +two O +domains O +are O +only O +16 O +% O +identical O +( O +mainly O +the O +RHxH B-structure_element +motif I-structure_element +, O +β2 B-structure_element +and O +β10 B-structure_element +), O +and O +34 O +% O +similar O +. O + +Our O +structure B-evidence +reveals O +that O +the O +two O +assigned O +PF06130 B-structure_element +domains O +( O +Fig O +3 O +) O +do O +not O +form O +structurally O +discrete O +units O +; O +this O +reduces O +the O +apparent O +sequence O +conservation O +at O +the O +level O +of O +primary O +structure O +. O + +One O +strand B-structure_element +of O +the O +domain B-structure_element +1 I-structure_element +beta B-structure_element +barrel I-structure_element +( O +shown O +in O +blue O +in O +Fig O +2 O +) O +is O +contributed O +by O +the O +N O +- O +terminus O +, O +while O +the O +rest O +of O +the O +domain O +is O +formed O +by O +the O +residues O +from O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +the O +protein B-protein_type +. O + +When O +aligned B-experimental_method +by O +structure B-evidence +, O +the O +β1 B-structure_element +strand I-structure_element +of O +the O +first B-structure_element +domain I-structure_element +( O +Fig O +2a O +and O +2b O +, O +blue O +) O +corresponds O +to O +the O +final B-structure_element +strand I-structure_element +of O +the O +second B-structure_element +domain I-structure_element +( O +β9 B-structure_element +), O +effectively O +making O +the O +domains O +continuous O +if O +the O +first O +strand O +was O +transplanted O +to O +the O +C O +- O +terminus O +. O + +The O +closest O +structural O +homolog O +of O +the O +PduL B-protein_type +barrel B-structure_element +domain I-structure_element +is O +a O +subdomain O +of O +a O +multienzyme O +complex O +, O +the O +alpha B-structure_element +subunit I-structure_element +of O +ethylbenzene B-protein_type +dehydrogenase I-protein_type +( O +S5b O +Fig O +, O +rmsd B-evidence +of O +2 O +. O +26 O +Å O +over O +226 O +aligned O +atoms O +consisting O +of O +one O +beta B-structure_element +barrel I-structure_element +and O +one O +capping B-structure_element +helix I-structure_element +). O + +In O +contrast O +to O +PduL B-protein_type +, O +there O +is O +only O +one O +barrel B-structure_element +present O +in O +ethylbenzene B-protein_type +dehydrogenase I-protein_type +, O +and O +there O +is O +no O +comparable O +active B-site +site I-site +arrangement O +. O + +The O +PduL B-protein_type +signature O +primary O +structure O +, O +two O +PF06130 B-structure_element +domains O +, O +occurs O +in O +some O +multidomain O +proteins O +, O +most O +of O +them O +annotated O +as O +Acks B-protein_type +, O +suggesting O +that O +PduL B-protein_type +may O +also O +replace O +Pta B-protein_type +in O +variants O +of O +the O +phosphotransacetylase B-protein_type +- O +Ack B-protein_type +pathway O +. O + +These O +PduL B-protein_type +homologs O +lack B-protein_state +EPs B-structure_element +, O +and O +their B-protein_type +fusion O +to O +Ack B-protein_type +may O +have O +evolved O +as O +a O +way O +to O +facilitate O +substrate O +channeling O +between O +the O +two O +enzymes O +. O + +For O +BMC B-complex_assembly +- O +encapsulated O +proteins O +to O +properly O +function O +together O +, O +they O +must O +be O +targeted O +to O +the O +lumen O +and O +assemble O +into O +an O +organization O +that O +facilitates O +substrate O +/ O +product O +channeling O +among O +the O +different O +catalytic B-site +sites I-site +of O +the O +signature O +and O +core O +enzymes O +. O + +The O +N B-structure_element +- I-structure_element +terminal I-structure_element +extension I-structure_element +on O +PduL B-protein_type +homologs O +may O +serve O +both O +of O +these O +functions O +. O + +The B-structure_element +extension I-structure_element +shares O +many O +features O +with O +previously O +characterized O +EPs B-structure_element +: O +it O +is O +present O +only O +in O +homologs O +associated O +with O +BMC B-gene +loci I-gene +, O +and O +it O +is O +predicted O +to O +form O +an O +amphipathic B-protein_state +α B-structure_element +- I-structure_element +helix I-structure_element +. O + +Moreover O +, O +its O +removal B-experimental_method +affects O +the O +oligomeric O +state O +of O +the O +protein O +. O + +EP B-structure_element +- O +mediated O +oligomerization O +has O +been O +observed O +for O +the O +signature O +and O +core O +BMC B-complex_assembly +enzymes O +; O +for O +example O +, O +full B-protein_state +- I-protein_state +length I-protein_state +propanediol B-protein_type +dehydratase I-protein_type +and O +ethanolamine B-protein_type +ammonia I-protein_type +- I-protein_type +lyase I-protein_type +( O +signature O +enzymes O +for O +PDU B-complex_assembly +and O +EUT B-complex_assembly +BMCs I-complex_assembly +) O +subunits O +are O +also O +insoluble O +, O +but O +become O +soluble O +upon O +removal O +of O +the O +predicted O +EP B-structure_element +. O + +sPduL B-protein +has O +also O +previously O +been O +reported O +to O +localize O +to O +inclusion O +bodies O +when O +overexpressed B-experimental_method +; O +we O +show O +here O +that O +this O +is O +dependent O +on O +the O +presence O +of O +the O +EP B-structure_element +. O + +This O +propensity O +of O +the O +EP B-structure_element +to O +cause O +proteins O +to O +form O +complexes O +( O +Fig O +5 O +) O +might O +not O +be O +a O +coincidence O +, O +but O +could O +be O +a O +necessary O +step O +in O +the O +assembly O +of O +BMCs B-complex_assembly +. O + +Structured O +aggregation O +of O +the O +core O +enzymes O +has O +been O +proposed O +to O +be O +the O +initial O +step O +in O +metabolosome B-complex_assembly +assembly O +and O +is O +known O +to O +be O +the O +first O +step O +of O +β O +- O +carboxysome O +biogenesis O +, O +where O +the O +core O +enzyme O +Ribulose B-protein_type +Bisphosphate I-protein_type +Carboxylase I-protein_type +/ I-protein_type +Oxygenase I-protein_type +( O +RuBisCO B-protein_type +) O +is O +aggregated O +by O +the O +CcmM B-protein_type +protein O +. O + +Likewise O +, O +CsoS2 B-protein_type +, O +a O +protein O +in O +the O +α B-complex_assembly +- I-complex_assembly +carboxysome I-complex_assembly +core O +, O +also O +aggregates O +when O +purified O +and O +is O +proposed O +to O +facilitate O +the O +nucleation O +and O +encapsulation O +of O +RuBisCO B-protein_type +molecules O +in O +the O +lumen O +of O +the O +organelle O +. O + +This O +role O +for O +EPs B-structure_element +in O +BMC B-complex_assembly +assembly O +is O +in O +addition O +to O +their O +interaction O +with O +shell O +proteins O +. O + +Our O +PduL B-protein_type +crystals B-evidence +contained O +CoA B-chemical +that O +was O +captured O +from O +the O +Escherichia B-species +coli I-species +cytosol O +, O +indicating O +that O +the O +“ O +ground O +state O +” O +of O +PduL B-protein_type +is O +in O +the O +CoA B-protein_state +- I-protein_state +bound I-protein_state +form O +; O +this O +could O +provide O +an O +elegantly O +simple O +means O +of O +guaranteeing O +a O +1 O +: O +1 O +ratio O +of O +CoA B-complex_assembly +: I-complex_assembly +PduL I-complex_assembly +within O +the O +metabolosome B-complex_assembly +lumen O +. O + +Active B-site +Site I-site +Identification O +and O +Structural O +Insights O +into O +Catalysis O + +The O +active B-site +site I-site +of O +PduL B-protein_type +is O +formed O +at O +the O +interface B-site +of O +the O +two O +structural O +domains B-structure_element +( O +Fig O +2b O +). O + +As O +expected O +, O +the O +amino O +acid O +sequence O +conservation O +is O +highest O +in O +the O +region O +around O +the O +proposed O +active B-site +site I-site +( O +Fig O +4d O +); O +highly B-protein_state +conserved I-protein_state +residues O +are O +also O +involved O +in O +CoA B-chemical +binding O +( O +Figs O +2a O +and O +3 O +, O +residues O +Ser45 B-residue_name_number +, O +Lys70 B-residue_name_number +, O +Arg97 B-residue_name_number +, O +Leu99 B-residue_name_number +, O +His204 B-residue_name_number +, O +Asn211 B-residue_name_number +). O + +All O +of O +the O +metal B-site +- I-site +coordinating I-site +residues I-site +( O +Fig O +2a O +) O +are O +absolutely B-protein_state +conserved I-protein_state +, O +implicating O +them O +in O +catalysis O +or O +the O +correct O +spatial O +orientation O +of O +the O +substrates O +. O + +Arg103 B-residue_name_number +, O +which O +contacts O +the O +phosphate B-chemical +( O +Fig O +4b O +), O +is O +present O +in O +all O +PduL B-protein_type +homologs O +. O + +The O +close O +resemblance O +between O +the O +structures O +binding O +CoA B-chemical +and O +phosphate B-chemical +likely O +indicates O +that O +no O +large O +changes O +in O +protein O +conformation O +are O +involved O +in O +catalysis O +, O +and O +that O +our O +crystal B-evidence +structures I-evidence +are O +representative O +of O +the O +active B-protein_state +form O +. O + +There O +is O +a O +pocket B-site +nearby O +the O +active B-site +site I-site +between O +the O +well B-protein_state +- I-protein_state +conserved I-protein_state +residues O +Ser45 B-residue_name_number +and O +Ala154 B-residue_name_number +, O +which O +could O +accommodate O +the O +propionyl O +group O +( O +S6 O +Fig O +). O + +A O +homology B-experimental_method +model I-experimental_method +of O +sPduL B-protein +indicates O +that O +the O +residues O +making O +up O +this O +pocket B-site +and O +the O +surrounding O +active B-site +site I-site +region O +are O +identical O +to O +that O +of O +rPduL B-protein +, O +which O +is O +not O +surprising O +, O +because O +these O +two O +homologs O +presumably O +have O +the O +same O +propionyl B-chemical +- I-chemical +CoA I-chemical +substrate O +. O + +The O +homology B-experimental_method +model I-experimental_method +of O +pPduL B-protein +also O +has O +identical O +residues O +making O +up O +the O +pocket B-site +, O +but O +with O +a O +key O +difference O +in O +the O +vicinity O +of O +the O +active B-site +site I-site +: O +Gln77 B-residue_name_number +of O +rPduL B-protein +is O +replaced O +by O +a O +tyrosine B-residue_name +( O +Tyr77 B-residue_name_number +) O +in O +pPduL B-protein +. O +The O +physiological O +substrate O +of O +pPduL B-protein +( O +Table O +1 O +) O +is O +thought O +to O +be O +lactyl B-chemical +- I-chemical +CoA I-chemical +, O +which O +contains O +an O +additional O +hydroxyl O +group O +relative O +to O +propionyl B-chemical +- I-chemical +CoA I-chemical +. O +The O +presence O +of O +an O +aromatic B-protein_state +residue B-structure_element +at O +this O +position O +may O +underlie O +the O +substrate O +preference O +of O +the O +PduL B-protein_type +enzyme O +from O +the O +pvm B-gene +locus I-gene +. O + +The O +catalytic O +mechanism O +of O +Pta B-protein_type +involves O +the O +abstraction O +of O +a O +thiol O +hydrogen O +by O +an O +aspartate B-residue_name +residue O +, O +resulting O +in O +the O +nucleophilic O +attack O +of O +thiolate O +upon O +the O +carbonyl O +carbon O +of O +acetyl B-chemical +- I-chemical +phosphate I-chemical +, O +oriented O +by O +an O +arginine B-residue_name +and O +stabilized O +by O +a O +serine B-residue_name +— O +there O +are O +no O +metals O +involved O +. O + +In O +contrast O +, O +in O +the O +rPduL B-protein +structure B-evidence +, O +there O +are O +no O +conserved O +aspartate B-residue_name +residues O +in O +or O +around O +the O +active B-site +site I-site +, O +and O +the O +only O +well B-protein_state +- I-protein_state +conserved I-protein_state +glutamate B-residue_name +residue O +in O +the O +active B-site +site I-site +is O +involved O +in O +coordinating B-bond_interaction +one O +of O +the O +metal O +ions O +. O + +These O +observations O +strongly O +suggest O +that O +an O +acidic B-protein_state +residue B-structure_element +is O +not O +directly O +involved O +in O +catalysis O +by O +PduL B-protein_type +. O +Instead O +, O +the O +dimetal B-site +active I-site +site I-site +of O +PduL B-protein_type +may O +create O +a O +nucleophile O +from O +one O +of O +the O +hydroxyl O +groups O +on O +free O +phosphate B-chemical +to O +attack O +the O +carbonyl O +carbon O +of O +the O +thioester O +bond O +of O +an O +acyl B-chemical +- I-chemical +CoA I-chemical +. O +In O +the O +reverse O +direction O +, O +the O +metal O +ion O +( O +s O +) O +could O +stabilize O +the O +thiolate O +anion O +that O +would O +attack O +the O +carbonyl O +carbon O +of O +an O +acyl B-chemical +- I-chemical +phosphate I-chemical +; O +a O +similar O +mechanism O +has O +been O +described O +for O +phosphatases B-protein_type +where O +hydroxyl O +groups O +or O +hydroxide O +ions O +can O +act O +as O +a O +base O +when O +coordinated O +by O +a O +dimetal B-site +active I-site +site I-site +. O + +Our O +structures B-evidence +provide O +the O +foundation O +for O +studies O +to O +elucidate O +the O +details O +of O +the O +catalytic O +mechanism O +of O +PduL B-protein_type +. O +Conserved B-protein_state +residues O +in O +the O +active B-site +site I-site +that O +may O +contribute O +to O +substrate O +binding O +and O +/ O +or O +transition O +state O +stabilization O +include O +Ser127 B-residue_name_number +, O +Arg103 B-residue_name_number +, O +Arg194 B-residue_name_number +, O +Gln107 B-residue_name_number +, O +Gln74 B-residue_name_number +, O +and O +Gln B-residue_name_number +/ O +Glu77 B-residue_name_number +. O + +In O +the O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +crystal B-evidence +structure I-evidence +, O +Ser127 B-residue_name_number +and O +Arg103 B-residue_name_number +appear O +to O +position O +the O +phosphate B-chemical +( O +Fig O +4b O +). O + +Alternatively O +, O +Arg103 B-residue_name_number +might O +act O +as O +a O +base O +to O +render O +the O +phosphate B-chemical +more O +nucleophilic O +. O + +The O +functional O +groups O +of O +Gln74 B-residue_name_number +, O +Gln B-residue_name_number +/ O +Glu77 B-residue_name_number +, O +and O +Arg194 B-residue_name_number +are O +directed O +away O +from O +the O +active B-site +site I-site +in O +both O +CoA B-protein_state +and O +phosphate B-protein_state +- I-protein_state +bound I-protein_state +crystal B-evidence +structures I-evidence +and O +do O +not O +appear O +to O +be O +involved O +in O +hydrogen B-bond_interaction +bonding I-bond_interaction +with O +these O +substrates O +, O +although O +they O +could O +be O +important O +for O +positioning O +an O +acyl B-chemical +- I-chemical +phosphate I-chemical +. O + +This O +hypothesis O +is O +strengthened O +by O +the O +fact O +that O +the O +CoA B-protein_state +- I-protein_state +bound I-protein_state +crystals B-evidence +were O +obtained O +without O +added O +CoA B-chemical +, O +indicating O +that O +the O +protein O +bound B-protein_state +CoA B-chemical +from O +the O +E B-species +. I-species +coli I-species +expression O +strain O +and O +retained O +it O +throughout O +purification O +and O +crystallization O +. O + +Functional O +, O +but O +Not O +Structural O +, O +Convergence O +of O +PduL B-protein_type +and O +Pta B-protein_type + +PduL B-protein_type +and O +Pta B-protein_type +are O +mechanistically O +and O +structurally O +distinct O +enzymes O +that O +catalyze O +the O +same O +reaction O +, O +a O +prime O +example O +of O +evolutionary O +convergence O +upon O +a O +function O +. O + +There O +are O +several O +examples O +of O +such O +functional O +convergence O +of O +enzymes O +, O +although O +typically O +the O +enzymes O +have O +independently O +evolved O +similar O +, O +or O +even O +identical O +active B-site +sites I-site +; O +for O +example O +, O +the O +carbonic B-protein_type +anhydrase I-protein_type +family O +. O + +However O +, O +apparently O +less O +frequent O +is O +functional O +convergence O +that O +is O +supported O +by O +distinctly O +different O +active B-site +sites I-site +and O +accordingly O +catalytic O +mechanism O +, O +as O +revealed O +by O +comparison O +of O +the O +structures O +of O +Pta B-protein_type +and O +PduL B-protein_type +. O +One O +well O +- O +studied O +example O +of O +this O +is O +the O +β B-protein_type +- I-protein_type +lactamase I-protein_type +family O +of O +enzymes O +, O +in O +which O +the O +active B-site +site I-site +of O +Class O +A O +and O +Class O +C O +enzymes O +involve O +serine O +- O +based O +catalysis O +, O +but O +Class O +B O +enzymes O +are O +metalloproteins B-protein_type +. O + +This O +is O +not O +surprising O +, O +as O +β B-protein_type +- I-protein_type +lactamases I-protein_type +are O +not O +so O +widespread O +among O +bacteria B-taxonomy_domain +and O +therefore O +would O +be O +expected O +to O +have O +evolved O +independently O +several O +times O +as O +a O +defense O +mechanism O +against O +β O +- O +lactam O +antibiotics O +. O + +However O +, O +nearly O +all O +bacteria B-taxonomy_domain +encode O +Pta B-protein_type +, O +and O +it O +is O +not O +immediately O +clear O +why O +the O +Pta B-protein_type +/ O +PduL B-protein_type +functional O +convergence O +should O +have O +evolved O +: O +it O +would O +seem O +to O +be O +evolutionarily O +more O +resourceful O +for O +the O +Pta B-gene +- I-gene +encoding I-gene +gene I-gene +to O +be O +duplicated O +and O +repurposed O +for O +BMCs B-complex_assembly +, O +as O +is O +apparently O +the O +case O +in O +one O +type O +of O +BMC B-complex_assembly +— I-complex_assembly +EUT1 I-complex_assembly +( O +Table O +1 O +). O + +Further O +biochemical O +comparison O +between O +the O +two O +PTACs B-protein_type +will O +likely O +yield O +exciting O +results O +that O +could O +answer O +this O +evolutionary O +question O +. O + +BMCs B-complex_assembly +are O +now O +known O +to O +be O +widespread O +among O +the O +bacteria B-taxonomy_domain +and O +are O +involved O +in O +critical O +segments O +of O +both O +autotrophic O +and O +heterotrophic O +biochemical O +pathways O +that O +confer O +to O +the O +host O +organism O +a O +competitive O +( O +metabolic O +) O +advantage O +in O +select O +niches O +. O + +As O +one O +of O +the O +three O +common O +metabolosome B-complex_assembly +core O +enzymes O +, O +the O +structure B-evidence +of O +PduL B-protein_type +provides O +a O +key O +missing O +piece O +to O +our O +structural O +picture O +of O +the O +shared O +core O +biochemistry O +( O +Fig O +1 O +) O +of O +functionally O +diverse O +catabolic B-protein_state +BMCs B-complex_assembly +. O + +We O +have O +observed O +the O +oligomeric O +state O +differences O +of O +PduL B-protein_type +to O +correlate O +with O +the O +presence O +of O +an O +EP B-structure_element +, O +providing O +new O +insight O +into O +the O +function O +of O +this O +sequence O +extension O +in O +BMC B-complex_assembly +assembly O +. O + +Moreover O +, O +our O +results O +suggest O +a O +means O +for O +Coenzyme B-chemical +A I-chemical +incorporation O +during O +metabolosome B-complex_assembly +biogenesis O +. O + +The O +fact O +that O +PduL B-protein_type +is O +confined O +almost O +exclusively O +to O +metabolosomes B-complex_assembly +can O +be O +used O +to O +develop O +an O +inhibitor O +that O +blocks O +only O +PduL B-protein_type +and O +not O +Pta B-protein_type +as O +a O +way O +to O +selectively O +disrupt O +BMC B-complex_assembly +- O +based O +metabolism O +, O +while O +not O +affecting O +most O +commensal O +organisms O +that O +require O +PTAC B-protein_type +activity O +. O + +Biochemistry O +and O +Crystal B-evidence +Structure I-evidence +of O +Ectoine B-protein_type +Synthase I-protein_type +: O +A O +Metal B-protein_state +- I-protein_state +Containing I-protein_state +Member O +of O +the O +Cupin B-protein_type +Superfamily I-protein_type + +Ectoine B-chemical +is O +a O +compatible O +solute O +and O +chemical O +chaperone O +widely O +used O +by O +members O +of O +the O +Bacteria B-taxonomy_domain +and O +a O +few O +Archaea B-taxonomy_domain +to O +fend O +- O +off O +the O +detrimental O +effects O +of O +high O +external O +osmolarity O +on O +cellular O +physiology O +and O +growth O +. O + +Ectoine B-protein_type +synthase I-protein_type +( O +EctC B-protein_type +) O +catalyzes O +the O +last O +step O +in O +ectoine B-chemical +production O +and O +mediates O +the O +ring O +closure O +of O +the O +substrate O +N B-chemical +- I-chemical +gamma I-chemical +- I-chemical +acetyl I-chemical +- I-chemical +L I-chemical +- I-chemical +2 I-chemical +, I-chemical +4 I-chemical +- I-chemical +diaminobutyric I-chemical +acid I-chemical +through O +a O +water B-chemical +elimination O +reaction O +. O + +However O +, O +the O +crystal B-evidence +structure I-evidence +of O +ectoine B-protein_type +synthase I-protein_type +is O +not O +known O +and O +a O +clear O +understanding O +of O +how O +its O +fold O +contributes O +to O +enzyme O +activity O +is O +thus O +lacking O +. O + +Using O +the O +ectoine B-protein_type +synthase I-protein_type +from O +the O +cold O +- O +adapted O +marine B-taxonomy_domain +bacterium I-taxonomy_domain +Sphingopyxis B-species +alaskensis I-species +( O +Sa B-species +), O +we O +report O +here O +both O +a O +detailed O +biochemical O +characterization O +of O +the O +EctC B-protein +enzyme O +and O +the O +high O +- O +resolution O +crystal B-evidence +structure I-evidence +of O +its O +apo B-protein_state +- O +form O +. O + +Structural B-experimental_method +analysis I-experimental_method +classified O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +as O +a O +member O +of O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +The O +interface B-site +of O +the O +dimer B-oligomeric_state +assembly O +is O +shaped O +through O +backbone O +- O +contacts O +and O +weak O +hydrophobic B-bond_interaction +interactions I-bond_interaction +mediated O +by O +two O +beta B-structure_element +- I-structure_element +sheets I-structure_element +within O +each O +monomer B-oligomeric_state +. O + +We O +found O +that O +EctC B-protein +not O +only O +effectively O +converts O +its O +natural O +substrate O +N B-chemical +- I-chemical +gamma I-chemical +- I-chemical +acetyl I-chemical +- I-chemical +L I-chemical +- I-chemical +2 I-chemical +, I-chemical +4 I-chemical +- I-chemical +diaminobutyric I-chemical +acid I-chemical +into O +ectoine B-chemical +through O +a O +cyclocondensation O +reaction O +, O +but O +that O +it O +can O +also O +use O +the O +isomer O +N B-chemical +- I-chemical +alpha I-chemical +- I-chemical +acetyl I-chemical +- I-chemical +L I-chemical +- I-chemical +2 I-chemical +, I-chemical +4 I-chemical +- I-chemical +diaminobutyric I-chemical +acid I-chemical +as O +its O +substrate O +, O +albeit O +with O +substantially O +reduced O +catalytic B-evidence +efficiency I-evidence +. O + +An O +assessment O +of O +enzyme O +activity O +and O +iron B-chemical +content O +of O +these O +mutants O +give O +important O +clues O +for O +understanding O +the O +architecture O +of O +the O +active B-site +site I-site +positioned O +within O +the O +core O +of O +the O +EctC B-protein +cupin B-structure_element +barrel I-structure_element +. O + +Ectoine B-chemical +[( O +S B-chemical +)- I-chemical +2 I-chemical +- I-chemical +methyl I-chemical +- I-chemical +1 I-chemical +, I-chemical +4 I-chemical +, I-chemical +5 I-chemical +, I-chemical +6 I-chemical +- I-chemical +tetrahydropyrimidine I-chemical +- I-chemical +4 I-chemical +- I-chemical +carboxylic I-chemical +acid I-chemical +] O +and O +its O +derivative O +5 B-chemical +- I-chemical +hydroxyectoine I-chemical +[( O +4S B-chemical +, I-chemical +5S I-chemical +)- I-chemical +5 I-chemical +- I-chemical +hydroxy I-chemical +- I-chemical +2 I-chemical +- I-chemical +methyl I-chemical +- I-chemical +1 I-chemical +, I-chemical +4 I-chemical +, I-chemical +5 I-chemical +, I-chemical +6 I-chemical +- I-chemical +tetrahydropyrimidine I-chemical +- I-chemical +4 I-chemical +- I-chemical +carboxylic I-chemical +acid I-chemical +] O +are O +such O +compatible O +solutes O +. O + +Both O +marine B-taxonomy_domain +and I-taxonomy_domain +terrestrial I-taxonomy_domain +microorganisms I-taxonomy_domain +produce O +them O +widely O +in O +response O +to O +osmotic O +or O +temperature O +stress O +. O + +Synthesis O +of O +ectoine B-chemical +occurs O +from O +the O +intermediate O +metabolite O +L B-chemical +- I-chemical +aspartate I-chemical +- I-chemical +ß I-chemical +- I-chemical +semialdehyde I-chemical +and O +comprises O +the O +sequential O +activities O +of O +three O +enzymes O +: O +L B-protein_type +- I-protein_type +2 I-protein_type +, I-protein_type +4 I-protein_type +- I-protein_type +diaminobutyrate I-protein_type +transaminase I-protein_type +( O +EctB B-protein_type +; O +EC O +2 O +. O +6 O +. O +1 O +. O +76 O +), O +2 B-protein_type +, I-protein_type +4 I-protein_type +- I-protein_type +diaminobutyrate I-protein_type +acetyltransferase I-protein_type +( O +EctA B-protein_type +; O +EC O +2 O +. O +3 O +. O +1 O +. O +178 O +), O +and O +ectoine B-protein_type +synthase I-protein_type +( O +EctC B-protein_type +; O +EC O +4 O +. O +2 O +. O +1 O +. O +108 O +) O +( O +Fig O +1 O +). O + +The O +ectoine B-chemical +derivative O +5 B-chemical +- I-chemical +hydroxyectoine I-chemical +, O +a O +highly O +effective O +stress O +protectant O +in O +its O +own O +right O +, O +is O +synthesized O +by O +a O +substantial O +subgroup O +of O +the O +ectoine B-chemical +producers O +. O + +The O +remarkable O +function O +preserving O +effects O +of O +ectoines B-chemical +for O +macromolecules O +and O +cells O +, O +frequently O +also O +addressed O +as O +chemical O +chaperones O +, O +led O +to O +a O +substantial O +interest O +in O +exploiting O +these O +compounds O +for O +biotechnological O +purposes O +and O +medical O +applications O +. O + +Biosynthetic O +routes O +for O +ectoine B-chemical +and O +5 B-chemical +- I-chemical +hydroxyectoine I-chemical +. O + +Here O +we O +focus O +on O +ectoine B-protein_type +synthase I-protein_type +( O +EctC B-protein +), O +the O +key O +enzyme O +of O +the O +ectoine B-chemical +biosynthetic O +route O +( O +Fig O +1 O +). O + +Biochemical O +characterizations B-experimental_method +of O +ectoine B-protein_type +synthases I-protein_type +from O +the O +extremophiles B-taxonomy_domain +Halomonas B-species +elongata I-species +, O +Methylomicrobium B-species +alcaliphilum I-species +, O +and O +Acidiphilium B-species +cryptum I-species +, O +and O +from O +the O +nitrifying B-taxonomy_domain +archaeon I-taxonomy_domain +Nitrosopumilus B-species +maritimus I-species +have O +been O +carried O +out O +. O + +Each O +of O +these O +enzymes O +catalyzes O +as O +their O +main O +activity O +the O +cyclization O +of O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +acetyl I-chemical +- I-chemical +L I-chemical +- I-chemical +2 I-chemical +, I-chemical +4 I-chemical +- I-chemical +diaminobutyric I-chemical +acid I-chemical +( O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +), O +the O +reaction O +product O +of O +the O +2 B-protein_type +, I-protein_type +4 I-protein_type +- I-protein_type +diaminobutyrate I-protein_type +acetyltransferase I-protein_type +( O +EctA B-protein_type +), O +to O +ectoine B-chemical +with O +the O +concomitant O +release O +of O +a O +water B-chemical +molecule O +( O +Fig O +1 O +). O + +In O +side O +reactions O +, O +EctC B-protein +can O +promote O +the O +formation O +of O +the O +synthetic O +compatible O +solute O +5 B-chemical +- I-chemical +amino I-chemical +- I-chemical +3 I-chemical +, I-chemical +4 I-chemical +- I-chemical +dihydro I-chemical +- I-chemical +2H I-chemical +- I-chemical +pyrrole I-chemical +- I-chemical +2 I-chemical +- I-chemical +carboxylate I-chemical +( O +ADPC B-chemical +) O +through O +the O +cyclic O +condensation O +of O +two O +glutamine B-chemical +molecules O +and O +it O +also O +possesses O +a O +minor O +hydrolytic O +activity O +for O +ectoine B-chemical +and O +synthetic O +ectoine B-chemical +derivatives O +with O +either O +reduced O +or O +expanded O +ring O +sizes O +. O + +Although O +progress O +has O +been O +made O +with O +respect O +to O +the O +biochemical O +characterization O +of O +ectoine B-protein_type +synthase I-protein_type +, O +a O +clear O +understanding O +of O +how O +its O +structure B-evidence +contributes O +to O +its O +enzyme O +activity O +and O +reaction O +mechanism O +is O +still O +lacking O +. O +With O +this O +in O +mind O +, O +we O +have O +biochemically B-experimental_method +characterized I-experimental_method +the O +ectoine B-protein_type +synthase I-protein_type +from O +the O +cold O +- O +adapted O +marine B-taxonomy_domain +bacterium I-taxonomy_domain +Sphingopyxis B-species +alaskensis I-species +( O +Sa B-species +). O + +We O +demonstrate O +here O +for O +the O +first O +time O +that O +the O +ectoine B-protein_type +synthase I-protein_type +is O +a O +metal B-chemical +- O +dependent O +enzyme O +, O +with O +iron B-chemical +as O +the O +most O +likely O +physiologically O +relevant O +co O +- O +factor O +. O + +Overproduction B-experimental_method +, O +purification B-experimental_method +and O +oligomeric O +state O +of O +the O +ectoine B-protein_type +synthase I-protein_type +in O +solution O + +We O +focused O +our O +biochemical B-experimental_method +and I-experimental_method +structural I-experimental_method +studies I-experimental_method +on O +the O +ectoine B-protein_type +synthase I-protein_type +from O +S B-species +. I-species +alaskensis I-species +[( O +Sa B-species +) O +EctC B-protein +], O +a O +cold O +- O +adapted O +marine B-taxonomy_domain +ultra I-taxonomy_domain +- I-taxonomy_domain +microbacterium I-taxonomy_domain +, O +from O +which O +we O +recently O +also O +determined O +the O +crystal B-evidence +structure I-evidence +of O +the O +ectoine B-protein_type +hydroxylase I-protein_type +( O +EctD B-protein_type +) O +in B-protein_state +complex I-protein_state +with I-protein_state +either O +its O +substrate O +or O +its O +reaction O +product O +. O + +We O +expressed O +a O +codon O +- O +optimized O +version O +of O +the O +S B-species +. I-species +alaskensis I-species +ectC B-gene +gene O +in O +E B-species +. I-species +coli I-species +to O +produce O +a O +recombinant O +protein O +with O +a O +carboxy O +- O +terminally O +attached O +Strep B-experimental_method +- I-experimental_method +tag I-experimental_method +II I-experimental_method +affinity I-experimental_method +peptide I-experimental_method +to O +allow O +purification O +of O +the O +( O +Sa B-species +) O +EctC B-protein +- O +Strep B-experimental_method +- I-experimental_method +Tag I-experimental_method +- I-experimental_method +II I-experimental_method +protein O +by O +affinity B-experimental_method +chromatography I-experimental_method +. O + +Conventional O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +( O +SEC B-experimental_method +) O +has O +already O +shown O +that O +( O +Sa B-species +) O +EctC B-protein +preparations O +produced O +in O +this O +fashion O +are O +homogeneous O +and O +that O +the O +protein O +forms O +dimers B-oligomeric_state +in O +solution O +. O + +High B-experimental_method +performance I-experimental_method +liquid I-experimental_method +chromatography I-experimental_method +coupled O +with O +multi B-experimental_method +- I-experimental_method +angle I-experimental_method +light I-experimental_method +- I-experimental_method +scattering I-experimental_method +detection I-experimental_method +( O +HPLC B-experimental_method +- I-experimental_method +MALS I-experimental_method +) O +experiments O +carried O +out O +here O +confirmed O +that O +the O +purified O +( O +Sa B-species +) O +EctC B-protein +protein O +was O +mono O +- O +disperse O +and O +possessed O +a O +molecular O +mass O +of O +33 O +. O +0 O +± O +2 O +. O +3 O +kDa O +( O +S2b O +Fig O +). O + +This O +value O +corresponds O +very O +well O +with O +the O +theoretically O +calculated O +molecular O +mass O +of O +an O +( O +Sa B-species +) O +EctC B-protein +dimer B-oligomeric_state +( O +molecular O +mass O +of O +the O +monomer B-oligomeric_state +, O +including O +the O +Strep B-experimental_method +- I-experimental_method +tag I-experimental_method +II I-experimental_method +affinity I-experimental_method +peptide I-experimental_method +: O +16 O +. O +3 O +kDa O +). O + +Such O +a O +quaternary O +assembly O +as O +dimer B-oligomeric_state +has O +also O +been O +reported O +for O +the O +EctC B-protein_type +proteins I-protein_type +from O +H B-species +. I-species +elongata I-species +and O +N B-species +. I-species +maritimus I-species +. O + +The O +EctA B-protein +- O +produced O +substrate O +of O +the O +ectoine B-protein_type +synthase I-protein_type +, O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +acetyl I-chemical +- I-chemical +L I-chemical +- I-chemical +2 I-chemical +, I-chemical +4 I-chemical +- I-chemical +diaminobutyric I-chemical +acid I-chemical +( O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +) O +( O +Fig O +1 O +), O +is O +commercially O +not O +available O +. O + +We O +used O +alkaline O +hydrolysis O +of O +ectoine B-chemical +and O +subsequent O +chromatography O +on O +silica O +gel O +columns O +to O +obtain O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +in O +chemically O +highly O +purified O +form O +( O +S1a O +Fig O +). O + +This O +procedure O +also O +yielded O +the O +isomer O +of O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +, O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +acetyl I-chemical +- I-chemical +L I-chemical +- I-chemical +2 I-chemical +, I-chemical +4 I-chemical +- I-chemical +diaminobutyric I-chemical +acid I-chemical +( O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +) O +( O +S1b O +Fig O +). O + +Using O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +as O +the O +substrate O +, O +we O +initially O +evaluated O +a O +set O +of O +biochemical O +parameters O +of O +the O +recombinant O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +S B-species +. I-species +alaskensis I-species +, O +from O +which O +the O +studied O +ectoine B-protein_type +synthase I-protein_type +was O +originally O +derived O +, O +is O +a O +microorganism B-taxonomy_domain +that O +is O +well O +- O +adapted O +to O +a O +life O +in O +permanently O +cold O +ocean O +waters O +. O + +Consistent O +with O +the O +physicochemical O +attributes O +of O +this O +habitat O +, O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +was O +already O +enzymatically B-protein_state +active I-protein_state +at O +5 O +° O +C O +, O +had O +a O +temperature O +optimum O +of O +15 O +° O +C O +and O +was O +able O +to O +function O +over O +a O +broad O +range O +of O +temperatures O +( O +S3a O +Fig O +). O + +It O +possessed O +an O +alkaline B-protein_state +pH O +optimum O +of O +8 O +. O +5 O +( O +S3b O +Fig O +), O +a O +value O +similar O +to O +the O +ectoine B-protein_type +synthases I-protein_type +from O +the O +halo B-protein_state +- I-protein_state +tolerant I-protein_state +H B-species +. I-species +elongata I-species +( O +pH O +optimum O +of O +8 O +. O +5 O +to O +9 O +. O +0 O +), O +the O +alkaliphile B-taxonomy_domain +M B-species +. I-species +alcaliphilum I-species +( O +pH O +optimum O +of O +9 O +. O +0 O +), O +and O +the O +acidophile B-taxonomy_domain +Acidiphilium B-species +cryptum I-species +( O +pH O +optimum O +of O +8 O +. O +5 O +to O +9 O +. O +0 O +), O +whereas O +the O +EctC B-protein +protein O +from O +N B-species +. I-species +maritimus I-species +has O +a O +neutral B-protein_state +pH I-protein_state +optimum O +( O +pH O +7 O +. O +0 O +). O + +The O +salinity O +of O +the O +assay O +buffer O +had O +a O +significant O +influence O +on O +the O +maximal O +enzyme O +activity O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +An O +increase O +in O +either O +the O +NaCl B-chemical +or O +the O +KCl B-chemical +concentration O +led O +to O +an O +approximately O +5 O +- O +fold O +enhancement O +of O +the O +ectoine B-protein_type +synthase I-protein_type +activity O +. O + +The O +maximum O +enzyme O +activity O +of O +( O +Sa B-species +) O +EctC B-protein +occurred O +around O +250 O +mM O +NaCl B-chemical +or O +KCl B-chemical +, O +respectively O +. O + +Considerations O +based O +on O +bioinformatics O +suggests O +that O +EctC B-protein +belongs O +to O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +Most O +of O +these O +proteins O +contain O +catalytically O +important O +transition O +state O +metals O +such O +as O +iron B-chemical +, O +copper B-chemical +, O +zinc B-chemical +, O +manganese B-chemical +, O +cobalt B-chemical +, O +or O +nickel B-chemical +. O + +Cupins B-protein_type +contain O +two O +conserved B-protein_state +motifs O +: O +G B-structure_element +( I-structure_element +X I-structure_element +) I-structure_element +5HXH I-structure_element +( I-structure_element +X I-structure_element +) I-structure_element +3 I-structure_element +, I-structure_element +4E I-structure_element +( I-structure_element +X I-structure_element +) I-structure_element +6G I-structure_element +and O +G B-structure_element +( I-structure_element +X I-structure_element +) I-structure_element +5PXG I-structure_element +( I-structure_element +X I-structure_element +) I-structure_element +2H I-structure_element +( I-structure_element +X I-structure_element +) I-structure_element +3N I-structure_element +( O +the O +letters O +in O +bold O +represent O +those O +residues O +that O +often O +coordinate O +the O +metal B-chemical +). O + +Inspection O +of O +a O +previous O +alignment B-experimental_method +of I-experimental_method +the I-experimental_method +amino I-experimental_method +acid I-experimental_method +sequences I-experimental_method +of O +440 O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +revealed O +that O +the O +canonical O +metal B-structure_element +- I-structure_element +binding I-structure_element +motif I-structure_element +( O +s O +) O +of O +cupin B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +is O +not B-protein_state +conserved I-protein_state +among O +members O +of O +the O +extended O +ectoine B-protein_type +synthase I-protein_type +protein I-protein_type +family I-protein_type +. O + +An O +abbreviated O +alignment B-experimental_method +of I-experimental_method +the I-experimental_method +amino I-experimental_method +acid I-experimental_method +sequence I-experimental_method +of O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +is O +shown O +in O +Fig O +2 O +. O + +Abbreviated O +alignment B-experimental_method +of O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +. O + +Strictly B-protein_state +conserved I-protein_state +amino O +acid O +residues O +are O +shown O +in O +yellow O +. O + +Dots O +shown O +above O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +sequence O +indicate O +residues O +likely O +to O +be O +involved O +in O +iron B-chemical +- O +binding O +( O +red O +), O +ligand O +- O +binding O +( O +green O +) O +and O +stabilization O +of O +the O +loop O +- O +architecture O +( O +blue O +). O + +The O +conserved B-protein_state +residue O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +with O +so O +- O +far O +undefined O +functions O +is O +indicated O +by O +a O +green O +dot O +circled O +in O +red O +. O + +Secondary O +structural O +elements O +( O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +β B-structure_element +- I-structure_element +sheets I-structure_element +) O +found O +in O +the O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structure I-evidence +are O +projected O +onto O +the O +amino O +acid O +sequences O +of O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +. O + +For O +this O +analysis O +we O +used O +recombinant O +( O +Sa B-species +) O +EctC B-protein +preparations O +from O +three O +independent O +protein O +overproduction O +and O +purification O +experiments O +. O + +The O +ICP B-experimental_method +- I-experimental_method +MS I-experimental_method +analyses O +yielded O +an O +iron B-chemical +content O +of O +0 O +. O +66 O +± O +0 O +. O +06 O +mol O +iron B-chemical +per O +mol O +of O +protein O +and O +the O +used O +( O +Sa B-species +) O +EctC B-protein +protein O +preparations O +also O +contained O +a O +minor O +amount O +of O +zinc B-chemical +( O +0 O +. O +08 O +mol O +zinc B-chemical +per O +mol O +of O +protein O +). O + +All O +other O +assayed O +metals O +( O +copper B-chemical +and O +nickel B-chemical +) O +were O +only O +present O +in O +trace O +amounts O +( O +0 O +. O +01 O +mol O +metal B-chemical +per O +mol O +of O +protein O +, O +respectively O +). O + +The O +presence O +of O +iron B-chemical +in O +these O +( O +Sa B-species +) O +EctC B-protein +protein O +preparations O +was O +further O +confirmed O +by O +a O +colorimetric B-experimental_method +method I-experimental_method +that O +is O +based O +on O +an O +iron B-chemical +- O +complexing O +reagent O +; O +this O +procedure O +yielded O +an O +iron B-chemical +- O +content O +of O +0 O +. O +84 O +± O +0 O +. O +05 O +mol O +per O +mol O +of O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +Hence O +, O +both O +ICP B-experimental_method +- I-experimental_method +MS I-experimental_method +and O +the O +colorimetric B-experimental_method +method I-experimental_method +clearly O +established O +that O +the O +recombinantly O +produced O +ectoine B-protein_type +synthase I-protein_type +from O +S B-species +. I-species +alaskensis I-species +is O +an O +iron B-chemical +- O +containing O +protein O +. O + +The O +reason O +for O +this O +difference O +is O +not O +known O +, O +but O +indicates O +that O +the O +well O +established O +colorimetric B-experimental_method +assay I-experimental_method +probably O +overestimates O +the O +iron B-chemical +content O +of O +( O +Sa B-species +) O +EctC B-protein +protein O +preparations O +to O +a O +certain O +degree O +. O + +The O +iron B-chemical +detected O +in O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +preparations O +could O +serve O +a O +structural O +role O +, O +or O +most O +likely O +, O +could O +be O +critical O +for O +enzyme O +catalysis O +as O +is O +the O +case O +for O +many O +members O +of O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +The O +addition O +of O +very O +low O +concentrations O +of O +EDTA B-chemical +( O +0 O +. O +05 O +mM O +) O +to O +the O +EctC B-protein +enzyme O +already O +led O +to O +a O +noticeable O +inhibition O +of O +the O +ectoine B-protein_type +synthase I-protein_type +activity O +and O +the O +presence O +of O +1 O +mM O +EDTA B-chemical +completely O +inhibited O +the O +enzyme O +( O +Fig O +3a O +). O + +( O +a O +) O +Impact O +of O +the O +iron B-chemical +- O +chelator O +EDTA B-chemical +on O +the O +enzyme O +activity O +of O +the O +purified O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +Metal B-experimental_method +depletion I-experimental_method +and I-experimental_method +reconstitution I-experimental_method +experiments I-experimental_method +with O +( O +b O +) O +stoichiometric O +and O +( O +c O +) O +excess O +amounts O +of O +metals O +. O + +The O +( O +Sa B-species +) O +EctC B-protein +protein O +was O +present O +at O +a O +concentration O +of O +10 O +μM O +. O +The O +level O +of O +enzyme O +activity O +given O +in O +( O +b O +) O +is O +benchmarked O +relative O +to O +that O +of O +ectoine B-protein_type +synthase I-protein_type +enzyme B-experimental_method +assays I-experimental_method +in O +which O +1 O +mM O +FeCl2 B-chemical +was O +added O +. O + +The O +addition O +of O +FeCl2 B-chemical +to O +the O +enzyme B-experimental_method +assay I-experimental_method +restored O +enzyme O +activity O +to O +about O +38 O +%, O +whereas O +the O +addition O +of O +ZnCl2 B-chemical +or O +CoCl2 B-chemical +rescued O +( O +Sa B-species +) O +EctC B-protein +enzyme O +activity O +only O +to O +5 O +% O +and O +3 O +%, O +respectively O +. O + +All O +other O +tested O +metals O +, O +including O +Fe3 B-chemical ++, I-chemical +were O +unable O +to O +restore O +activity O +( O +Fig O +3b O +). O + +When O +the O +concentration O +of O +the O +various O +metals O +in O +the O +enzyme B-experimental_method +assay I-experimental_method +was O +increased O +100 O +- O +fold O +, O +Fe2 B-chemical ++ I-chemical +exhibited O +again O +the O +strongest O +stimulating O +effect O +on O +enzyme O +activity O +, O +and O +rescued O +enzyme O +activity O +to O +a O +degree O +similar O +to O +that O +exhibited O +by O +( O +Sa B-species +) O +EctC B-protein +protein O +preparations O +that O +had O +not O +been O +inactivated O +through O +EDTA B-chemical +treatment O +( O +Fig O +3c O +). O + +However O +, O +a O +large O +molar O +excess O +of O +other O +transition O +- O +state O +metals O +( O +zinc B-chemical +, O +cobalt B-chemical +, O +nickel B-chemical +, O +copper B-chemical +, O +and O +manganese B-chemical +) O +typically O +found O +in O +members O +of O +the O +cupin B-protein_type +superfamily I-protein_type +allowed O +the O +partial O +rescue O +of O +ectoine B-protein_type +synthase I-protein_type +activity O +as O +well O +( O +Fig O +3c O +). O + +This O +is O +in O +line O +with O +literature O +data O +showing O +that O +cupin B-protein_type +- I-protein_type +type I-protein_type +enzymes I-protein_type +are O +often O +promiscuous O +with O +respect O +to O +the O +use O +of O +the O +catalytically O +important O +metal B-chemical +. O + +Kinetic O +parameters O +of O +EctC B-protein +for O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +and O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical + +Based O +on O +the O +data O +presented O +in O +S3 O +Fig O +, O +we O +formulated O +an O +optimized O +activity B-experimental_method +assay I-experimental_method +for O +the O +ectoine B-protein_type +synthase I-protein_type +of O +S B-species +. I-species +alaskensis I-species +and O +used O +it O +to O +determined O +the O +kinetic O +parameters O +for O +the O +( O +Sa B-species +) O +EctC B-protein +enzyme O +for O +both O +its O +natural O +substrate O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +and O +the O +isomer O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +. O + +Given O +the O +chemical O +relatedness O +of O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +to O +the O +natural O +substrate O +( O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +) O +of O +the O +ectoine B-protein_type +synthase I-protein_type +( O +S1a O +and O +S1b O +Fig O +), O +we O +wondered O +whether O +( O +Sa B-species +) O +EctC B-protein +could O +also O +use O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +to O +produce O +ectoine B-chemical +. O + +However O +, O +both O +the O +affinity B-evidence +( O +Km B-evidence +) O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +and O +its O +catalytic B-evidence +efficiency I-evidence +( O +kcat B-evidence +/ I-evidence +Km I-evidence +) O +were O +strongly O +reduced O +in O +comparison O +with O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +. O + +Both O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +and O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +are O +concomitantly O +formed O +during O +the O +enzymatic O +hydrolysis O +of O +the O +ectoine B-chemical +ring O +during O +catabolism O +. O + +Our O +finding O +that O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +is O +a O +substrate O +for O +ectoine B-protein_type +synthase I-protein_type +has O +bearings O +for O +an O +understanding O +of O +the O +physiology O +of O +those O +microorganisms B-taxonomy_domain +that O +can O +both O +synthesize O +and O +catabolize O +ectoine B-chemical +. O + +However O +, O +these O +types O +of O +microorganisms B-taxonomy_domain +should O +still O +be O +able O +to O +largely O +avoid O +a O +futile O +cycle O +since O +the O +affinity B-evidence +of O +ectoine B-protein_type +synthase I-protein_type +for O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +and O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +, O +and O +its O +catalytic B-evidence +efficiency I-evidence +for O +the O +two O +compounds O +, O +differs O +substantially O +( O +S4a O +and O +S4b O +Fig O +). O + +Crystallization B-experimental_method +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O + +Since O +no O +crystal B-evidence +structure I-evidence +of O +ectoine B-protein_type +synthase I-protein_type +has O +been O +reported O +, O +we O +set O +out O +to O +crystallize B-experimental_method +the O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +Attempts O +to O +obtain O +crystals B-evidence +of O +( O +Sa B-species +) O +EctC B-protein +in B-protein_state +complex I-protein_state +either O +with O +its O +substrate O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +or O +its O +reaction O +product O +ectoine B-chemical +were O +not O +successful O +. O + +Attempts O +to O +solve O +the O +crystal B-evidence +structure I-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +by O +molecular B-experimental_method +replacement I-experimental_method +has O +previously O +failed O +. O + +However O +, O +we O +were O +able O +to O +obtain O +crystals B-evidence +of O +form O +B O +that O +were O +derivatized O +with O +mercury B-chemical +and O +these O +diffracted O +up O +to O +2 O +. O +8 O +Å O +( O +S1 O +Table O +). O + +This O +dataset O +was O +used O +to O +derive O +an O +initial O +structural B-evidence +model I-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +, O +which O +in O +turn O +was O +employed O +as O +a O +template O +for O +molecular B-experimental_method +replacement I-experimental_method +to O +phase O +the O +native O +dataset O +( O +2 O +. O +0 O +Å O +) O +of O +crystal O +form O +B O +. O +After O +several O +rounds O +of O +manual O +model O +building O +and O +refinement O +, O +four O +monomers B-oligomeric_state +of O +( O +Sa B-species +) O +EctC B-protein +were O +identified O +and O +the O +crystal B-evidence +structure I-evidence +was O +refined O +to O +a O +final O +Rcryst B-evidence +of O +21 O +. O +1 O +% O +and O +an O +Rfree B-evidence +of O +24 O +. O +8 O +% O +( O +S1 O +Table O +). O + +The O +two O +EctC B-protein +structures B-evidence +that O +we O +determined O +revealed O +that O +the O +ectoine B-protein_type +synthase I-protein_type +belongs O +to O +the O +cupin B-protein_type +superfamily I-protein_type +with O +respect O +to O +its O +overall O +fold O +( O +Fig O +4a O +– O +4c O +). O + +However O +, O +they O +represent O +two O +different O +states O +of O +the O +137 B-residue_range +amino I-residue_range +acids I-residue_range +comprising O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +2 O +). O + +First O +, O +the O +1 O +. O +2 O +Å O +structure B-evidence +reveals O +the O +spatial O +configuration O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +ranging O +from O +amino O +acid O +Met B-residue_range +- I-residue_range +1 I-residue_range +to I-residue_range +Glu I-residue_range +- I-residue_range +115 I-residue_range +; O +hence O +, O +it O +lacks B-protein_state +22 B-residue_range +amino I-residue_range +acids I-residue_range +at O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +of O +the O +authentic O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +In O +this O +structure B-evidence +no O +metal B-chemical +co O +- O +factor O +was O +identified O +. O + +The O +second O +crystal B-evidence +structure I-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +was O +solved B-experimental_method +at O +a O +resolution O +of O +2 O +. O +0 O +Å O +and O +contained O +four O +molecules O +of O +the O +protein O +in O +the O +asymmetric O +unit O +of O +which O +protomer B-oligomeric_state +A B-structure_element +comprised O +amino O +acid O +Met B-residue_range +- I-residue_range +1 I-residue_range +to I-residue_range +Gly I-residue_range +- I-residue_range +121 I-residue_range +and O +adopts O +a O +closed B-protein_state +conformation O +. O + +Hence O +, O +it O +still O +lacks B-protein_state +16 B-residue_range +amino I-residue_range +acid I-residue_range +residues O +of O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +of O +the O +authentic O +137 B-residue_range +amino I-residue_range +acids I-residue_range +comprising O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +2 O +). O + +We O +therefore O +cannot O +exclude O +that O +this O +crystal B-evidence +structure I-evidence +does O +not O +represent O +the O +fully B-protein_state +closed I-protein_state +state O +of O +the O +ectoine B-protein_type +synthase I-protein_type +; O +consequently O +, O +we O +tentatively O +termed O +it O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +. O + +Overall O +structure B-evidence +of O +the O +“ O +open B-protein_state +” O +and O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +crystal B-evidence +structures I-evidence +of O +( O +Sa B-species +) O +EctC B-protein +. O + +( O +a O +) O +The O +overall O +structure B-evidence +of O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +resolved O +at O +2 O +. O +0 O +Å O +is O +depicted O +in O +green O +in O +a O +cartoon O +( O +upper O +panel O +) O +and O +surface O +( O +lower O +panel O +) O +representation O +. O + +( O +b O +) O +The O +overall O +structure B-evidence +of O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +was O +resolved O +at O +1 O +. O +2 O +Å O +and O +is O +depicted O +in O +yellow O +in O +a O +cartoon O +( O +upper O +panel O +) O +and O +surface O +( O +lower O +panel O +) O +representation O +. O + +The O +overall O +structure B-evidence +of O +( O +Sa B-species +) O +EctC B-protein +is O +basically O +the O +same O +in O +both O +crystals B-evidence +except O +for O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +, O +which O +covers O +the O +entry O +of O +one O +side O +of O +the O +cupin B-structure_element +barrel I-structure_element +from O +the O +surroundings O +in O +monomer B-oligomeric_state +A B-structure_element +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +. O + +This O +is O +reflected O +by O +the O +calculated O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +RMSD B-evidence +) O +of O +the O +Cα O +atoms O +that O +was O +about O +0 O +. O +56 O +Å O +( O +over O +117 O +residues O +) O +when O +the O +four O +“ O +open B-protein_state +” O +monomers B-oligomeric_state +were O +compared O +with O +each O +other O +. O + +However O +, O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +monomer B-oligomeric_state +has O +a O +slightly O +higher O +RMSD B-evidence +of O +1 O +. O +4 O +Å O +( O +over O +117 O +residues O +) O +when O +compared O +with O +the O +“ O +open B-protein_state +” O +2 O +. O +0 O +Å O +structure B-evidence +. O + +Therefore O +, O +we O +describe O +in O +the O +following O +the O +overall O +structure B-evidence +for O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +form O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +and O +subsequently O +highlight O +the O +structural O +differences O +between O +the O +“ O +open B-protein_state +” O +and O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +forms O +in O +more O +detail O +. O + +The O +β B-structure_element +- I-structure_element +strands I-structure_element +form O +two O +anti B-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +: O +β2 B-structure_element +β3 B-structure_element +, O +β4 B-structure_element +, O +β11 B-structure_element +, O +β6 B-structure_element +, O +and O +β9 B-structure_element +, O +and O +a O +smaller O +three B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +( O +β7 B-structure_element +, O +β8 B-structure_element +, O +and O +β10 B-structure_element +), O +respectively O +. O + +These O +two O +β B-structure_element +- I-structure_element +sheets I-structure_element +pack O +against O +each O +other O +, O +forming O +a O +cup B-structure_element +- I-structure_element +shaped I-structure_element +β I-structure_element +- I-structure_element +sandwich I-structure_element +with O +a O +topology O +characteristic O +for O +the O +cupin B-structure_element +- I-structure_element +fold I-structure_element +. O + +In O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +, O +a O +longer O +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +tail I-structure_element +is O +visible O +in O +the O +electron B-evidence +density I-evidence +, O +folding O +into O +a O +small B-structure_element +helix I-structure_element +( O +α B-structure_element +- I-structure_element +II I-structure_element +) O +that O +closes O +the O +active B-site +site I-site +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +4a O +). O + +Structural B-experimental_method +comparison I-experimental_method +analyses I-experimental_method +using O +the O +DALI B-experimental_method +server I-experimental_method +revealed O +that O +( O +Sa B-species +) O +EctC B-protein +adopts O +a O +fold O +similar O +to O +other O +members O +of O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +The O +highest O +structural O +similarities O +are O +observed O +for O +the O +Cupin B-protein +2 I-protein +conserved I-protein +barrel I-protein +domain I-protein +protein I-protein +( O +YP_751781 B-protein +. I-protein +1 I-protein +) O +from O +Shewanella B-species +frigidimarina I-species +( O +PDB O +accession O +code O +: O +2PFW O +) O +with O +a O +Z B-evidence +- I-evidence +score I-evidence +of O +13 O +. O +1 O +and O +an O +RMSD B-evidence +of O +2 O +. O +2 O +Å O +over O +104 O +Cα O +- O +atoms O +( O +structural O +data O +for O +this O +protein O +have O +been O +deposited O +in O +the O +PDB O +but O +no O +publication O +connected O +to O +this O +structure B-evidence +is O +currently O +available O +), O +a O +manganese B-protein +- I-protein +containing I-protein +cupin I-protein +( O +TM1459 B-protein +) O +from O +Thermotoga B-species +maritima I-species +( O +PDB O +accession O +code O +: O +1VJ2 O +) O +with O +a O +Z B-evidence +- I-evidence +score I-evidence +of O +12 O +. O +8 O +and O +an O +RMSD B-evidence +of O +2 O +. O +0 O +Å O +over O +103 O +Cα O +- O +atoms O +, O +the O +cyclase B-protein_type +RemF B-protein +from O +Streptomyces B-species +resistomycificus I-species +( O +PDB O +accession O +code O +: O +3HT1 O +with O +a O +Z B-evidence +- I-evidence +score I-evidence +of O +11 O +. O +9 O +and O +an O +RMSD B-evidence +of O +1 O +. O +9 O +Å O +over O +102 O +Cα O +- O +atoms O +), O +and O +an O +auxin B-protein +- I-protein +binding I-protein +protein I-protein +1 I-protein +from O +Zea B-species +mays I-species +( O +PDB O +accession O +code O +: O +1LR5 O +) O +with O +an O +Z B-evidence +- I-evidence +score I-evidence +of O +11 O +. O +8 O +and O +an O +RMSD B-evidence +of O +2 O +. O +8 O +Å O +over O +104 O +Cα O +- O +atoms O +). O + +Next O +to O +RemF B-protein +and O +the O +aldos B-protein_type +- I-protein_type +2 I-protein_type +- I-protein_type +ulose I-protein_type +dehydratase I-protein_type +/ O +isomerase B-protein_type +, O +the O +ectoine B-protein_type +synthase I-protein_type +is O +only O +the O +third O +characterized O +dehydratase B-protein_type +within O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +In O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +crystal B-evidence +structure I-evidence +, O +( O +Sa B-species +) O +EctC B-protein +has O +crystallized B-experimental_method +as O +a O +dimer B-oligomeric_state +of O +dimers B-oligomeric_state +within O +the O +asymmetric O +unit O +. O + +This O +dimer B-oligomeric_state +( O +Fig O +5a O +and O +5b O +) O +is O +composed O +of O +two O +monomers B-oligomeric_state +arranged O +in O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +orientation O +and O +is O +stabilized O +via O +strong O +interactions O +mediated O +by O +two O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +strands I-structure_element +, O +β B-structure_element +- I-structure_element +strand I-structure_element +β1 B-structure_element +( O +sequence O +1MIVRN5 B-structure_element +) O +from O +monomer B-oligomeric_state +A B-structure_element +and O +β B-structure_element +- I-structure_element +strand I-structure_element +β8 B-structure_element +from O +monomer B-oligomeric_state +B B-structure_element +( O +sequence O +82GVMYAL87 B-structure_element +) O +( O +Fig O +5c O +). O + +The O +strong O +interactions O +between O +these O +β B-structure_element +- I-structure_element +strands I-structure_element +rely O +primarily O +on O +backbone O +contacts O +. O + +In O +addition O +to O +these O +interactions O +, O +some O +weaker O +hydrophobic B-bond_interaction +interactions I-bond_interaction +are O +also O +observed O +between O +the O +two O +monomers B-oligomeric_state +in O +some O +loops B-structure_element +connecting O +the O +β B-structure_element +- I-structure_element +strands I-structure_element +. O + +Both O +values O +fall O +within O +the O +range O +for O +known O +functional O +dimers B-oligomeric_state +. O + +Crystal B-evidence +structure I-evidence +of O +( O +Sa B-species +) O +EctC B-protein +. O + +( O +a O +) O +Top O +- O +view O +of O +the O +dimer B-oligomeric_state +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +The O +position O +of O +the O +water B-chemical +molecule O +, O +described O +in O +detail O +in O +the O +text O +, O +is O +shown O +in O +one O +of O +the O +monomers B-oligomeric_state +as O +an O +orange O +sphere O +. O +( O +b O +) O +Side O +- O +view O +of O +a O +( O +Sa B-species +) O +EctC B-protein +dimer B-oligomeric_state +allowing O +an O +assessment O +of O +the O +dimer B-site +interface I-site +formed O +by O +two O +β B-structure_element +- I-structure_element +strands I-structure_element +of O +each O +monomer B-oligomeric_state +. O + +( O +c O +) O +Close O +- O +up O +representation O +of O +the O +dimer B-site +interface I-site +mediated O +by O +beta B-structure_element +- I-structure_element +strand I-structure_element +β1 B-structure_element +and O +β6 B-structure_element +. O + +Indeed O +, O +a O +similar O +dimer B-oligomeric_state +configuration O +to O +the O +one O +described O +for O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +is O +observed O +with O +the O +same O +monomer B-oligomeric_state +- O +monomer B-oligomeric_state +interactions O +mediated O +by O +the O +two O +β B-structure_element +- I-structure_element +sheets I-structure_element +. O + +The O +crystallographic O +two O +- O +fold O +axis O +present O +within O +the O +crystal O +symmetry O +is O +located O +exactly O +in O +between O +the O +two O +monomers B-oligomeric_state +, O +resulting O +in O +a O +monomer B-oligomeric_state +within O +the O +asymmetric O +unit O +. O + +Hence O +, O +the O +same O +dimer B-oligomeric_state +observed O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +of O +( O +Sa B-species +) O +EctC B-protein +can O +also O +be O +observed O +in O +the O +“ O +open B-protein_state +” O +structure B-evidence +. O + +Interestingly O +, O +the O +proteins O +identified O +by O +the O +above O +- O +described O +DALI B-experimental_method +search I-experimental_method +not O +only O +have O +folds O +similar O +to O +EctC B-protein +, O +but O +are O +also O +functional O +dimers B-oligomeric_state +that O +adopt O +similar O +monomer B-oligomeric_state +- O +monomer B-oligomeric_state +interactions O +within O +the O +dimer B-oligomeric_state +assembly O +as O +deduced O +from O +the O +inspection O +of O +the O +corresponding O +PDB O +files O +( O +2PFW O +, O +3HT1 O +, O +1VJ2 O +, O +1LR5 O +). O + +Structural O +rearrangements O +of O +the O +flexible B-protein_state +( O +Sa B-species +) O +EctC B-protein +carboxy B-structure_element +- I-structure_element +terminus I-structure_element + +The O +cupin O +core O +represents O +the O +structural O +framework O +of O +ectoine B-protein_type +synthase I-protein_type +( O +Figs O +4 O +and O +5 O +). O + +The O +major O +difference O +in O +the O +two O +crystal B-evidence +structures I-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +reported O +here O +is O +the O +orientation O +of O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +. O + +Some O +amino O +acids O +located O +in O +the O +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +the O +137 B-residue_range +amino I-residue_range +acids I-residue_range +comprising O +( O +Sa B-species +) O +EctC B-protein +protein O +are O +highly B-protein_state +conserved I-protein_state +( O +Fig O +2 O +) O +within O +the O +extended B-protein_state +EctC B-protein_type +protein I-protein_type +family O +. O + +At O +the O +end O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β11 B-structure_element +, O +two O +consecutive O +conserved B-protein_state +proline B-residue_name +residues O +( O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +and O +Pro B-residue_name_number +- I-residue_name_number +110 I-residue_name_number +) O +are O +present O +that O +are O +responsible O +for O +a O +turn O +in O +the O +main O +chain O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +In O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +, O +the O +visible O +electron B-evidence +density I-evidence +of O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +is O +extended O +by O +7 B-residue_range +amino I-residue_range +acid I-residue_range +residues I-residue_range +and O +ends O +at O +position O +Gly B-residue_name_number +- I-residue_name_number +121 I-residue_name_number +. O + +Furthermore O +, O +this O +helix B-structure_element +is O +stabilized O +via O +interactions O +with O +the O +loop B-structure_element +region I-structure_element +between O +β B-structure_element +- I-structure_element +strands I-structure_element +β4 B-structure_element +and O +β6 B-structure_element +, O +thereby O +inducing O +a O +structural O +rearrangement O +. O + +This O +induces O +the O +formation O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +, O +which O +is O +not O +present O +when O +the O +small B-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +helix I-structure_element +is O +absent B-protein_state +as O +observed O +in O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +. O + +The O +position O +of O +this O +His B-residue_name +residue O +is O +slightly O +shifted O +in O +both O +( O +Sa B-species +) O +EctC B-protein +structures B-evidence +, O +likely O +the O +result O +of O +the O +formation O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +. O + +The O +consecutive O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +and O +Pro B-residue_name_number +- I-residue_name_number +110 I-residue_name_number +residues O +found O +at O +the O +end O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β11are B-structure_element +highly B-protein_state +conserved I-protein_state +in O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +( O +Fig O +2 O +). O + +They O +are O +responsible O +for O +redirecting O +the O +main O +chain O +of O +the O +remaining O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +( O +27 B-residue_range +amino I-residue_range +acid I-residue_range +residues I-residue_range +) O +of O +( O +Sa B-species +) O +EctC B-protein +to O +close O +the O +cupin B-structure_element +fold I-structure_element +. O + +In O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +this O +results O +in O +a O +complete O +closure O +of O +the O +entry O +of O +the O +cupin B-structure_element +barrel I-structure_element +( O +Fig O +4a O +to O +4c O +). O + +A O +search O +for O +partners O +interacting O +with O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +revealed O +that O +it O +interacts O +via O +its O +backbone O +oxygen O +with O +the O +side O +chain O +of O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +as O +visible O +in O +both O +the O +“ O +open B-protein_state +” O +and O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structures B-evidence +. O + +The O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +/ O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +interaction O +ensures O +the O +stable B-protein_state +orientation O +of O +both O +proline B-residue_name +residues O +at O +the O +end O +of O +β B-structure_element +- I-structure_element +strand I-structure_element +β11 B-structure_element +. O + +In O +addition O +to O +the O +interactions O +between O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +and O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +, O +the O +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +( O +Sa B-species +) O +EctC B-protein +is O +held O +in O +position O +via O +an O +interaction O +of O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +with O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +, O +which O +stabilizes O +the O +conformation O +of O +the O +small B-structure_element +helix I-structure_element +in O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +further O +. O + +Architecture O +of O +the O +presumed O +metal B-site +- I-site +binding I-site +site I-site +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +and O +its O +flexible B-protein_state +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +. O + +( O +a O +) O +The O +described O +water B-chemical +molecule O +( O +depicted O +as O +orange O +sphere O +) O +is O +bound O +via O +interactions O +with O +the O +side O +chains O +of O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +and O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +. O + +The O +position O +occupied O +by O +this O +water B-chemical +molecule O +represents O +probably O +the O +position O +of O +the O +Fe2 B-chemical ++ I-chemical +cofactor O +in O +the O +active B-site +side I-site +of O +the O +ectoine B-protein_type +synthase I-protein_type +. O + +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +interacts O +with O +the O +double B-structure_element +proline I-structure_element +motif I-structure_element +( O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +and O +Pro B-residue_name_number +- I-residue_name_number +110 I-residue_name_number +). O + +It O +is O +further O +stabilized O +via O +an O +interaction O +with O +the O +side O +chain O +of O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +which O +is O +localized O +in O +the O +flexible B-protein_state +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +( O +colored O +in O +orange O +) O +of O +( O +Sa B-species +) O +EctC B-protein +that O +is O +visible O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +. O + +Since O +( O +Sa B-species +) O +EctC B-protein +is O +a O +metal B-chemical +containing O +protein O +( O +Fig O +3 O +), O +we O +tried O +to O +fit O +either O +Fe2 B-chemical ++, I-chemical +or O +Zn2 B-chemical ++ I-chemical +ions O +into O +this O +density B-evidence +and O +also O +refined B-experimental_method +occupancy I-experimental_method +. O + +Only O +the O +refinement O +of O +Fe2 B-chemical ++ I-chemical +resulted O +in O +a O +visibly O +improved O +electron B-evidence +density I-evidence +, O +however O +with O +a O +low O +degree O +of O +occupancy O +. O + +This O +possible O +iron B-chemical +molecule O +is O +bound O +via O +interactions O +with O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +and O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +( O +Fig O +6a O +and O +6b O +). O + +The O +distance O +between O +the O +side O +chains O +of O +these O +residues O +and O +the O +( O +putative O +) O +iron B-chemical +co O +- O +factor O +is O +3 O +. O +1 O +Å O +for O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +2 O +. O +9 O +Å O +for O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +and O +2 O +. O +9 O +Å O +for O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +, O +respectively O +. O + +The O +position O +of O +this O +water B-chemical +molecule O +is O +described O +in O +more O +detail O +below O +and O +is O +highlighted O +in O +Figs O +5a O +and O +5b O +and O +6a O +and O +6b O +as O +a O +sphere O +. O + +Interestingly O +, O +all O +three O +amino O +acids O +coordinating O +this O +water B-chemical +molecule O +are O +strictly B-protein_state +conserved I-protein_state +within O +an O +alignment B-experimental_method +of O +440 O +members O +of O +the O +EctC B-protein_type +protein I-protein_type +family O +( O +for O +an O +abbreviated O +alignment O +of O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +see O +Fig O +2 O +). O + +In O +the O +“ O +open B-protein_state +” O +structure B-evidence +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +, O +electron B-evidence +density I-evidence +is O +visible O +where O +the O +presumptive O +iron B-chemical +is O +positioned O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +. O + +However O +, O +this O +electron B-evidence +density I-evidence +fits O +perfectly O +to O +a O +water B-chemical +molecule O +and O +not O +to O +an O +iron B-chemical +, O +and O +the O +water B-chemical +molecule O +was O +clearly O +visible O +after O +the O +refinement O +at O +this O +high O +resolution O +( O +1 O +. O +2 O +Å O +) O +of O +the O +“ O +open B-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +. O + +In O +a O +superimposition B-experimental_method +of O +both O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structures I-evidence +, O +the O +spatial O +arrangements O +of O +the O +side O +chains O +of O +the O +three O +amino O +acids O +( O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +and O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +) O +likely O +to O +contact O +the O +iron B-chemical +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +match O +nicely O +with O +those O +of O +the O +corresponding O +residues O +of O +the O +“ O +iron B-protein_state +- I-protein_state +free I-protein_state +” O +“ O +open B-protein_state +” O +structure B-evidence +( O +Fig O +6b O +). O + +In O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +, O +the O +hydroxyl O +- O +group O +of O +the O +side O +- O +chain O +of O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +points O +towards O +the O +iron B-chemical +( O +Fig O +6a O +and O +6b O +), O +but O +the O +corresponding O +distance O +( O +3 O +. O +9 O +Å O +) O +makes O +it O +highly O +unlikely O +that O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +is O +directly O +involved O +in O +metal B-chemical +binding O +. O + +Nevertheless O +, O +its O +substitution B-experimental_method +by O +an O +Ala B-residue_name +residue O +causes O +a O +strong O +decrease O +in O +iron B-chemical +- O +content O +and O +enzyme O +activity O +of O +the O +mutant B-protein_state +protein O +( O +Table O +1 O +). O + +Since O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +is O +strictly B-protein_state +conserved I-protein_state +in O +an O +alignment B-experimental_method +of O +440 O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +( O +Fig O +2 O +), O +we O +speculate O +that O +it O +might O +be O +involved O +in O +contacting O +the O +substrate O +of O +the O +ectoine B-protein_type +synthase I-protein_type +and O +that O +the O +absence B-protein_state +of I-protein_state +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +in O +our O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structures I-evidence +might O +endow O +the O +side O +chain O +of O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +with O +extra O +spatial O +flexibility O +. O + +To O +further O +analyze O +the O +putative O +iron B-site +binding I-site +site I-site +( O +Fig O +6a O +), O +we O +performed O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +site I-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +and O +assessed O +the O +resulting O +( O +Sa B-species +) O +EctC B-protein +variants O +for O +their O +iron B-chemical +content O +and O +studied O +their O +enzyme O +activity O +. O + +When O +those O +three O +residues O +( O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +) O +that O +likely O +form O +the O +mono B-site +- I-site +nuclear I-site +iron I-site +center I-site +in O +the O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structure I-evidence +were O +individually O +replaced B-experimental_method +by O +an O +Ala B-residue_name +residue O +, O +both O +the O +catalytic O +activity O +and O +the O +iron B-chemical +content O +of O +the O +mutant B-protein_state +proteins O +was O +strongly O +reduced O +( O +Table O +1 O +). O + +For O +some O +of O +the O +presumptive O +iron B-site +- I-site +coordinating I-site +residues I-site +, O +additional O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +experiments O +were O +carried O +out O +. O + +To O +verify O +the O +importance O +of O +the O +negative O +charge O +in O +the O +position O +of O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +we O +created O +an O +Asp B-residue_name +variant B-protein_state +. O + +This O +mutant B-protein_state +protein O +rescued O +the O +enzyme O +activity O +and O +iron B-chemical +content O +of O +the O +Ala B-residue_name +substitution B-experimental_method +substantially O +( O +Table O +1 O +). O + +Collectively O +, O +these O +data O +suggest O +that O +the O +hydroxyl O +group O +of O +the O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +side O +chain O +is O +needed O +for O +the O +binding O +of O +the O +iron B-chemical +( O +Fig O +6a O +). O + +We O +also O +replaced B-experimental_method +the O +presumptive O +iron B-site +- I-site +binding I-site +residue I-site +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +by O +an O +Asn B-residue_name +residue O +, O +yielding O +a O +( O +Sa B-species +) O +EctC B-protein +protein O +variant O +that O +possessed O +an O +enzyme O +activity O +of O +23 O +% O +and O +iron B-chemical +content O +of O +only O +14 O +% O +relative O +to O +that O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +protein O +( O +Table O +1 O +). O + +Collectively O +, O +the O +data O +addressing O +the O +functionality O +of O +the O +putative O +iron B-site +- I-site +coordinating I-site +residues I-site +( O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +) O +buttress O +our O +notion O +that O +the O +Fe2 B-chemical ++ I-chemical +present O +in O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +is O +of O +catalytic O +importance O +. O + +A O +chemically O +undefined O +ligand O +in O +the O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +provides O +clues O +for O +the O +binding O +of O +the O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +substrate O + +Despite O +considerable O +efforts O +, O +either O +by O +trying O +co B-experimental_method +- I-experimental_method +crystallization I-experimental_method +or O +soaking B-experimental_method +experiments I-experimental_method +, O +we O +were O +not O +able O +to O +obtain O +a O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structures I-evidence +that O +contained O +either O +the O +substrate O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +, O +or O +ectoine B-chemical +, O +the O +reaction O +product O +of O +ectoine B-protein_type +synthase I-protein_type +( O +Fig O +1 O +). O + +However O +, O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +where O +the O +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +loop I-structure_element +is O +largely O +resolved O +, O +a O +long O +stretched O +electron B-evidence +density I-evidence +feature O +was O +detected O +in O +the O +predicted O +active B-site +site I-site +of O +the O +enzyme O +; O +it O +remained O +visible O +after O +crystallographic B-experimental_method +refinement I-experimental_method +. O + +We O +tried O +to O +fit O +all O +compounds O +used O +in O +the O +buffers O +during O +purification B-experimental_method +and O +crystallization B-experimental_method +into O +the O +observed O +electron B-evidence +density I-evidence +, O +but O +none O +matched O +. O + +This O +observation O +indicates O +that O +the O +chemically O +undefined O +ligand O +was O +either O +trapped O +by O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +during O +its O +heterologous O +production O +in O +E B-species +. I-species +coli I-species +or O +during O +crystallization B-experimental_method +. O + +Estimating O +from O +the O +dimensions O +of O +the O +electron B-evidence +density I-evidence +feature I-evidence +, O +we O +modeled O +the O +chemically O +undefined O +compound O +trapped O +by O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +as O +a O +hexane B-chemical +- I-chemical +1 I-chemical +, I-chemical +6 I-chemical +- I-chemical +diol I-chemical +molecule O +( O +PDB O +identifier O +: O +HEZ O +) O +to O +best O +fit O +the O +observed O +electron B-evidence +density I-evidence +. O + +However O +, O +to O +the O +best O +of O +our O +knowledge O +, O +hexane B-chemical +- I-chemical +1 I-chemical +, I-chemical +6 I-chemical +- I-chemical +diol I-chemical +is O +not O +part O +of O +the O +E B-species +. I-species +coli I-species +metabolome O +. O + +We O +note O +that O +both O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +and O +hexane B-chemical +- I-chemical +1 I-chemical +, I-chemical +6 I-chemical +- I-chemical +diol I-chemical +are O +both O +C6 O +- O +compounds O +and O +display O +similar O +length O +( O +Fig O +7a O +). O + +A O +chemically O +undefined O +ligand O +is O +captured O +in O +the O +active B-site +site I-site +of O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structure I-evidence +. O + +( O +a O +) O +The O +observed O +electron B-evidence +density I-evidence +in O +the O +active B-site +site I-site +of O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +structure B-evidence +of O +( O +Sa B-species +) O +EctC B-protein +is O +modeled O +as O +a O +hexane B-chemical +- I-chemical +1 I-chemical +, I-chemical +6 I-chemical +- I-chemical +diol I-chemical +molecule O +and O +compared O +with O +the O +electron B-evidence +density I-evidence +of O +the O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +substrate O +of O +the O +ectoine B-protein_type +synthase I-protein_type +to O +emphasize O +the O +similarity O +in O +size O +of O +these O +compounds O +. O + +( O +b O +) O +The O +presumable O +binding B-site +site I-site +of O +the O +iron B-chemical +co O +- O +factor O +and O +of O +the O +modeled O +hexane B-chemical +- I-chemical +1 I-chemical +, I-chemical +6 I-chemical +- I-chemical +diol I-chemical +molecule O +is O +depicted O +. O + +The O +amino O +acid O +side O +chains O +involved O +in O +iron B-chemical +- O +ligand O +binding O +are O +colored O +in O +blue O +and O +those O +involved O +in O +the O +binding O +of O +the O +chemically O +undefined O +ligand O +are O +colored O +in O +green O +using O +a O +ball O +and O +stick O +representation O +. O + +The O +flexible B-protein_state +carboxy B-structure_element +- I-structure_element +terminal I-structure_element +loop I-structure_element +of O +( O +Sa B-species +) O +EctC B-protein +is O +highlighted O +in O +orange O +. O + +We O +refined B-experimental_method +the O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +with O +the O +trapped O +compound O +, O +and O +by O +doing O +so O +, O +the O +refinement O +parameters O +( O +especially O +R B-evidence +- I-evidence +and I-evidence +Rfree I-evidence +- I-evidence +factor I-evidence +) O +dropped O +by O +1 O +. O +5 O +%. O + +Remarkably O +, O +all O +of O +these O +residues O +are O +highly B-protein_state +conserved I-protein_state +throughout O +the O +extended O +EctC B-protein_type +protein I-protein_type +family O +( O +Fig O +2 O +). O + +Structure B-experimental_method +- I-experimental_method +guided I-experimental_method +site I-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +of O +the O +catalytic B-site +core I-site +of O +the O +ectoine B-protein_type +synthase I-protein_type + +In O +a O +previous O +alignment B-experimental_method +of I-experimental_method +the I-experimental_method +amino I-experimental_method +acid I-experimental_method +sequences I-experimental_method +of O +440 O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +, O +13 O +amino O +acids O +were O +identified O +as O +strictly B-protein_state +conserved I-protein_state +residues O +. O + +Amino O +acid O +residues O +Gly B-residue_name_number +- I-residue_name_number +64 I-residue_name_number +, O +Pro B-residue_name_number +- I-residue_name_number +109 I-residue_name_number +, O +and O +Gly B-residue_name_number +- I-residue_name_number +113 I-residue_name_number +likely O +fulfill O +structural O +roles O +since O +they O +are O +positioned O +either O +at O +the O +end O +or O +at O +the O +beginning O +of O +β B-structure_element +- I-structure_element +strands I-structure_element +and O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +We O +considered O +the O +remaining O +ten O +residues O +as O +important O +either O +for O +ligand O +binding O +, O +for O +catalysis O +, O +or O +for O +the O +structurally O +correct O +orientation O +of O +the O +flexible B-protein_state +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +. O + +In O +view O +of O +the O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +with O +the O +serendipitously O +trapped O +compound O +( O +Fig O +7b O +), O +we O +probed O +the O +functional O +importance O +of O +the O +seven O +residues O +that O +contact O +this O +ligand O +by O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +site I-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +( O +Table O +1 O +). O + +We O +benchmarked O +the O +activity O +of O +the O +( O +Sa B-species +) O +EctC B-protein +variants O +in O +a O +single B-experimental_method +time I-experimental_method +- I-experimental_method +point I-experimental_method +enzyme I-experimental_method +assay I-experimental_method +under O +conditions O +where O +10 O +μM O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +Sa B-species +) O +EctC B-protein +protein O +converted O +almost O +completely O +the O +supplied O +10 O +mM O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +substrate O +to O +9 O +. O +33 O +mM O +ectoine B-chemical +within O +a O +time O +frame O +of O +20 O +min O +. O + +In O +addition O +, O +we O +determined O +the O +iron B-chemical +content O +of O +each O +of O +the O +mutant B-protein_state +( O +Sa B-species +) O +EctC B-protein +protein O +by O +a O +colorimetric B-experimental_method +assay I-experimental_method +( O +Table O +1 O +). O + +The O +side O +chains O +of O +the O +evolutionarily B-protein_state +conserved I-protein_state +Trp B-residue_name_number +- I-residue_name_number +21 I-residue_name_number +, O +Ser B-residue_name_number +- I-residue_name_number +23 I-residue_name_number +, O +Thr B-residue_name_number +- I-residue_name_number +40 I-residue_name_number +, O +Cys B-residue_name_number +- I-residue_name_number +105 I-residue_name_number +, O +and O +Phe B-residue_name_number +- I-residue_name_number +107 I-residue_name_number +residues O +( O +Fig O +2 O +) O +make O +contacts O +with O +the O +chemically O +undefined O +ligand O +that O +we O +observed O +in O +the O +“ O +semi B-protein_state +- I-protein_state +closed I-protein_state +” O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +( O +Fig O +7b O +). O + +Thr B-residue_name_number +- I-residue_name_number +40 I-residue_name_number +is O +positioned O +on O +β B-structure_element +- I-structure_element +strand I-structure_element +β5 B-structure_element +and O +its O +side O +chain O +protrudes O +into O +the O +lumen O +of O +the O +cupin B-structure_element +barrel I-structure_element +formed O +by O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Fig O +7b O +). O + +We O +also O +replaced B-experimental_method +Phe B-residue_name_number +- I-residue_name_number +107 I-residue_name_number +with O +either O +an O +Tyr B-residue_name +or O +an O +Trp B-residue_name +residue O +: O +the O +Phe B-mutant +- I-mutant +107 I-mutant +/ I-mutant +Tyr I-mutant +substitution B-experimental_method +possessed O +near O +wild B-protein_state +- I-protein_state +type I-protein_state +enzyme O +activity O +( O +about O +95 O +%) O +and O +the O +full O +iron B-chemical +content O +, O +but O +the O +Phe B-mutant +- I-mutant +107 I-mutant +/ I-mutant +Trp I-mutant +substitution B-experimental_method +possessed O +only O +12 O +% O +enzyme O +activity O +and O +72 O +% O +iron B-chemical +content O +compared O +to O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +protein O +. O + +The O +properties O +of O +these O +mutant B-protein_state +proteins O +indicate O +that O +the O +aromatic O +side O +chain O +at O +position O +107 B-residue_number +of O +( O +Sa B-species +) O +EctC B-protein +is O +of O +importance O +but O +that O +a O +substitution B-experimental_method +with O +a O +bulky O +aromatic O +side O +chain O +is O +strongly O +detrimental O +to O +enzyme O +activity O +and O +concomitantly O +moderately O +impairs O +iron B-chemical +binding O +. O + +Replacement B-experimental_method +of O +the O +only O +Cys B-residue_name +residue O +in O +( O +Sa B-species +) O +EctC B-protein +( O +Cys B-residue_name_number +- I-residue_name_number +105 I-residue_name_number +; O +Fig O +2 O +) O +by O +a O +Ser B-residue_name +residue O +, O +a O +configuration O +that O +is O +naturally O +found O +in O +two O +EctC B-protein_type +proteins I-protein_type +among O +440 O +inspected O +amino O +acid O +sequences O +, O +yielded O +a O +( O +Sa B-species +) O +EctC B-protein +variant B-protein_state +with O +84 O +% O +wild B-protein_state +- I-protein_state +type I-protein_state +activity O +and O +an O +iron B-chemical +content O +similar O +to O +that O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +protein O +. O + +Since O +the O +side O +- O +chains O +of O +Cys B-residue_name +residues O +are O +chemically O +reactive O +and O +often O +participate O +in O +enzyme O +catalysis O +, O +Cys B-residue_name_number +- I-residue_name_number +105 I-residue_name_number +( O +or O +Ser B-residue_name_number +- I-residue_name_number +105 I-residue_name_number +) O +might O +serve O +such O +a O +role O +for O +ectoine B-protein_type +synthase I-protein_type +. O + +Based O +on O +the O +( O +Sa B-species +) O +EctC B-protein +crystal B-evidence +structures I-evidence +that O +we O +present O +here O +, O +we O +can O +currently O +not O +firmly O +understand O +why O +the O +replacement B-experimental_method +of O +Tyr B-residue_name_number +- I-residue_name_number +52 I-residue_name_number +by O +Ala B-residue_name +impairs O +enzyme O +function O +and O +iron B-chemical +content O +so O +drastically O +( O +Table O +1 O +). O + +This O +is O +different O +for O +the O +His B-mutant +- I-mutant +55 I-mutant +/ I-mutant +Ala I-mutant +substitution O +. O + +The O +individual O +substitution B-experimental_method +of O +either O +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +or O +His B-residue_name_number +- I-residue_name_number +55 I-residue_name_number +by O +an O +Ala B-residue_name +residue O +is O +predicted O +to O +disrupt O +this O +interactive B-site +network I-site +and O +therefore O +should O +affect O +enzyme O +activity O +. O + +Indeed O +, O +the O +Glu B-mutant +- I-mutant +115 I-mutant +/ I-mutant +Ala I-mutant +and O +the O +His B-mutant +- I-mutant +55 I-mutant +/ I-mutant +Ala I-mutant +substitutions O +possessed O +only O +21 O +% O +and O +16 O +% O +activity O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +protein O +, O +respectively O +( O +Table O +1 O +). O + +We O +also O +replaced B-experimental_method +Glu B-residue_name_number +- I-residue_name_number +115 I-residue_name_number +with O +a O +negatively O +charged O +residue O +( O +Asp B-residue_name +); O +this O +( O +Sa B-species +) O +EctC B-protein +variant O +possessed O +wild B-protein_state +- I-protein_state +type I-protein_state +levels O +of O +iron B-chemical +and O +still O +exhibited O +77 O +% O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +enzyme O +activity O +. O + +Collectively O +, O +these O +data O +suggest O +that O +the O +correct O +positioning O +of O +the O +carboxy B-structure_element +- I-structure_element +terminus I-structure_element +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +is O +of O +structural O +and O +functional O +importance O +for O +the O +activity O +of O +the O +ectoine B-protein_type +synthase I-protein_type +. O + +Residues O +Leu B-residue_name_number +- I-residue_name_number +87 I-residue_name_number +and O +Asp B-residue_name_number +- I-residue_name_number +91 I-residue_name_number +are O +highly B-protein_state +conserved I-protein_state +in O +the O +ectoine B-protein_type +synthase I-protein_type +protein O +family O +. O + +The O +replacement B-experimental_method +of O +Leu B-residue_name_number +- I-residue_name_number +87 I-residue_name_number +by O +Ala B-residue_name +led O +to O +a O +substantial O +drop O +in O +enzyme O +activity O +( O +Table O +1 O +). O + +Conversely O +, O +the O +replacement B-experimental_method +of O +Asp B-residue_name_number +- I-residue_name_number +91 I-residue_name_number +by O +Ala B-residue_name +and O +Glu B-residue_name +, O +resulted O +in O +( O +Sa B-species +) O +EctC B-protein +protein O +variants O +with O +80 O +% O +and O +98 O +% O +enzyme O +activity O +, O +respectively O +( O +Table O +1 O +). O + +We O +currently O +cannot O +comment O +on O +possible O +functional O +role O +Asp B-residue_name_number +- I-residue_name_number +91 I-residue_name_number +. O + +However O +, O +Leu B-residue_name_number +- I-residue_name_number +87 I-residue_name_number +is O +positioned O +at O +the O +end O +of O +one O +of O +the O +β B-structure_element +- I-structure_element +sheets I-structure_element +that O +form O +the O +dimer B-site +interface I-site +( O +Fig O +5c O +) O +and O +it O +might O +therefore O +possess O +a O +structural O +role O +. O + +It O +is O +also O +located O +near O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +one O +of O +the O +residues O +that O +probably O +coordinate O +the O +iron B-chemical +molecule O +with O +in O +the O +( O +Sa B-species +) O +EctC B-protein +active B-site +site I-site +( O +Fig O +6a O +) O +and O +therefore O +might O +exert O +indirect O +effects O +. O + +We O +note O +that O +His B-residue_name_number +- I-residue_name_number +117 I-residue_name_number +is O +located O +close O +to O +the O +chemically O +undefined O +ligand O +in O +the O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +( O +Fig O +7b O +) O +and O +might O +thus O +play O +a O +role O +in O +contacting O +the O +natural O +substrate O +of O +the O +ectoine B-protein_type +synthase I-protein_type +. O + +Both O +( O +Sa B-species +) O +EctC B-protein +protein O +variants O +exhibited O +wild B-protein_state +- I-protein_state +type I-protein_state +level O +enzyme O +activities O +and O +possessed O +a O +iron B-chemical +content O +matching O +that O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +Table O +1 O +). O + +This O +illustrates O +that O +not O +every O +amino O +acid O +substitution O +in O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +leads O +to O +an O +indiscriminate O +impairment O +of O +enzyme O +function O +and O +iron B-chemical +content O +. O + +The O +crystallographic B-evidence +data I-evidence +presented O +here O +firmly O +identify O +ectoine B-protein_type +synthase I-protein_type +( O +EctC B-protein +), O +an O +enzyme O +critical O +for O +the O +production O +of O +the O +microbial B-taxonomy_domain +cytoprotectant O +and O +chemical O +chaperone O +ectoine B-chemical +, O +as O +a O +new O +member O +of O +the O +cupin B-protein_type +superfamily I-protein_type +. O + +The O +overall O +fold O +and O +bowl O +shape O +of O +the O +( O +Sa B-species +) O +EctC B-protein +protein O +( O +Figs O +4 O +and O +5 O +) O +with O +its O +11 O +β B-structure_element +- I-structure_element +strands I-structure_element +( O +β1 B-structure_element +- I-structure_element +β11 I-structure_element +) O +and O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +α B-structure_element +- I-structure_element +I I-structure_element +and O +α B-structure_element +- I-structure_element +II I-structure_element +) O +closely O +adheres O +to O +the O +design O +principles O +typically O +found O +in O +crystal B-evidence +structures I-evidence +of O +cupins B-protein_type +. O + +In O +addition O +to O +the O +ectoine B-protein_type +synthase I-protein_type +, O +the O +polyketide B-protein_type +cyclase I-protein_type +RemF B-protein +is O +the O +only O +other O +currently O +known O +cupin B-protein_type +- I-protein_type +related I-protein_type +enzyme O +that O +catalyze O +a O +cyclocondensation O +reaction O +although O +the O +substrates O +of O +EctC B-protein +and O +RemF B-protein +are O +rather O +different O +. O + +The O +pro B-taxonomy_domain +- I-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +members O +of O +the O +cupin B-protein_type +superfamily I-protein_type +perform O +a O +variety O +of O +both O +enzymatic O +and O +non O +- O +enzymatic O +functions O +that O +are O +built O +upon O +a O +common O +structural O +scaffold O +. O + +Most O +cupins B-protein_type +contain O +transition O +state O +metals O +that O +can O +promote O +different O +types O +of O +chemical O +reactions O +. O + +We O +report O +here O +for O +the O +first O +time O +that O +the O +ectoine B-protein_type +synthase I-protein_type +is O +a O +metal B-chemical +- O +dependent O +enzyme O +. O + +ICP B-experimental_method +- I-experimental_method +MS I-experimental_method +, O +metal B-experimental_method +- I-experimental_method +depletion I-experimental_method +and I-experimental_method +reconstitution I-experimental_method +experiments I-experimental_method +( O +Fig O +3 O +) O +consistently O +identify O +iron B-chemical +as O +the O +biologically O +most O +relevant O +metal B-chemical +for O +the O +EctC B-protein +- O +catalyzed O +cyclocondensation O +reaction O +. O + +However O +, O +as O +observed O +with O +other O +cupins B-protein_type +, O +EctC B-protein +is O +a O +somewhat O +promiscuous O +enzyme O +as O +far O +as O +the O +catalytically O +important O +metal B-chemical +is O +concerned O +when O +they O +are O +provided O +in O +large O +molar O +excess O +( O +Fig O +3c O +). O + +Although O +some O +uncertainty O +remains O +with O +respect O +to O +the O +precise O +identity O +of O +amino O +acid O +residues O +that O +participate O +in O +metal B-chemical +binding O +by O +( O +Sa B-species +) O +EctC B-protein +, O +our O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +site I-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +experiments O +targeting O +the O +presumptive O +iron B-site +- I-site +binding I-site +residues I-site +( O +Fig O +6a O +and O +6b O +) O +demonstrate O +that O +none O +of O +them O +can O +be O +spared O +( O +Table O +1 O +). O + +The O +three O +residues O +( O +Glu B-residue_name_number +- I-residue_name_number +57 I-residue_name_number +, O +Tyr B-residue_name_number +- I-residue_name_number +85 I-residue_name_number +, O +His B-residue_name_number +- I-residue_name_number +93 I-residue_name_number +) O +that O +we O +deem O +to O +form O +it O +( O +Figs O +6 O +and O +7b O +) O +are O +strictly B-protein_state +conserved I-protein_state +in O +a O +large O +collection O +of O +EctC B-protein_type +- I-protein_type +type I-protein_type +proteins I-protein_type +originating O +from O +16 O +bacterial B-taxonomy_domain +and O +three O +archaeal B-taxonomy_domain +phyla O +( O +Fig O +2 O +). O + +We O +also O +show O +here O +for O +the O +first O +time O +that O +, O +in O +addition O +to O +its O +natural O +substrate O +N B-chemical +- I-chemical +γ I-chemical +- I-chemical +ADABA I-chemical +, O +EctC B-protein +also O +converts O +the O +isomer O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +into O +ectoine B-chemical +, O +albeit O +with O +a O +73 O +- O +fold O +reduced O +catalytic B-evidence +efficiency I-evidence +( O +S3a O +and O +S3b O +Fig O +). O + +Our O +finding O +that O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +serves O +as O +a O +substrate O +for O +ectoine B-protein_type +synthase I-protein_type +has O +physiologically O +relevant O +ramifications O +for O +those O +microorganisms B-taxonomy_domain +that O +can O +both O +synthesize O +and O +catabolize O +ectoine B-chemical +, O +since O +they O +need O +to O +prevent O +a O +futile O +cycle O +of O +synthesis O +and O +degradation O +when O +N B-chemical +- I-chemical +α I-chemical +- I-chemical +ADABA I-chemical +is O +produced O +as O +an O +intermediate O +in O +the O +catabolic O +route O +. O + +Although O +we O +cannot O +identify O +the O +true O +chemical O +nature O +of O +the O +C6 B-chemical +compound O +that O +was O +trapped O +in O +the O +( O +Sa B-species +) O +EctC B-protein +structure B-evidence +nor O +its O +precise O +origin O +, O +we O +treated O +this O +compound O +as O +a O +proxy O +for O +the O +natural O +substrate O +of O +ectoine B-protein_type +synthase I-protein_type +, O +which O +is O +a O +C6 O +compound O +as O +well O +( O +Fig O +7a O +). O + +Indeed O +, O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +of O +those O +five O +residues O +that O +contact O +the O +unknown O +C6 O +compound O +( O +Fig O +7b O +) O +yielded O +( O +Sa B-species +) O +EctC B-protein +variants O +with O +strongly O +impaired O +enzyme O +function O +but O +near O +wild B-protein_state +- I-protein_state +type I-protein_state +levels O +of O +iron B-chemical +( O +Table O +1 O +). O + +We O +therefore O +surmise O +that O +our O +crystallographic B-evidence +data I-evidence +and O +the O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +study I-experimental_method +reported O +here O +provide O +a O +structural O +and O +functional O +view O +into O +the O +architecture O +of O +the O +EctC B-protein +active B-site +site I-site +( O +Fig O +7b O +). O + +The O +ectoine B-protein_type +synthase I-protein_type +from O +the O +cold O +- O +adapted O +marine B-taxonomy_domain +bacterium I-taxonomy_domain +S B-species +. I-species +alaskensis I-species +can O +be O +considered O +as O +a O +psychrophilic O +enzyme O +( O +S3a O +Fig O +), O +types O +of O +proteins O +with O +a O +considerable O +structural O +flexibility O +. O + +It O +is O +hoped O +that O +these O +can O +be O +further O +employed O +to O +obtain O +EctC B-protein +crystal B-evidence +structures I-evidence +with O +either O +the O +substrate O +or O +the O +reaction O +product O +. O + +Together O +with O +our O +finding O +that O +ectoine B-protein_type +synthase I-protein_type +is O +metal B-protein_state +dependent I-protein_state +, O +these O +crystal B-evidence +structures I-evidence +should O +allow O +a O +more O +detailed O +understanding O +of O +the O +chemistry O +underlying O +the O +EctC B-protein +- O +catalyzed O +cyclocondensation O +reaction O +. O + +ADARs B-protein_type +( O +adenosine B-protein_type +deaminases I-protein_type +acting I-protein_type +on I-protein_type +RNA I-protein_type +) O +are O +editing B-protein_type +enzymes I-protein_type +that O +convert O +adenosine B-residue_name +( O +A B-residue_name +) O +to O +inosine B-residue_name +( O +I B-residue_name +) O +in O +duplex B-structure_element +RNA I-structure_element +, O +a O +modification O +reaction O +with O +wide O +- O +ranging O +consequences O +on O +RNA B-chemical +function O +. O + +Our O +understanding O +of O +the O +ADAR B-protein_type +reaction O +mechanism O +, O +origin O +of O +editing B-site +site I-site +selectivity O +and O +effect O +of O +mutations O +is O +limited O +by O +the O +lack O +of O +high O +- O +resolution O +structural B-evidence +data I-evidence +for O +complexes O +of O +ADARs B-protein_type +bound B-protein_state +to I-protein_state +substrate O +RNAs B-chemical +. O + +Here O +we O +describe O +four O +crystal B-evidence +structures I-evidence +of O +the O +deaminase B-structure_element +domain I-structure_element +of O +human B-species +ADAR2 B-protein +bound B-protein_state +to I-protein_state +RNA B-structure_element +duplexes I-structure_element +bearing O +a O +mimic O +of O +the O +deamination O +reaction O +intermediate O +. O + +These O +structures B-evidence +, O +together O +with O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +mutagenesis I-experimental_method +and O +RNA B-experimental_method +- I-experimental_method +modification I-experimental_method +experiments I-experimental_method +, O +explain O +the O +basis O +for O +ADAR B-protein_type +deaminase B-structure_element +domain I-structure_element +’ O +s O +dsRNA B-chemical +specificity O +, O +its O +base O +- O +flipping O +mechanism O +, O +and O +nearest O +neighbor O +preferences O +. O + +In O +addition O +, O +an O +ADAR2 B-protein +- O +specific O +RNA B-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +was O +identified O +near O +the O +enzyme O +active B-site +site I-site +rationalizing O +differences O +in O +selectivity O +observed O +between O +different O +ADARs B-protein_type +. O + +RNA B-chemical +editing O +reactions O +alter O +a O +transcript O +’ O +s O +genomically O +encoded O +sequence O +by O +inserting O +, O +deleting O +or O +modifying O +nucleotides O +. O + +Deamination O +of O +adenosine B-residue_name +( O +A B-residue_name +), O +the O +most O +common O +form O +of O +RNA B-chemical +editing O +in O +humans B-species +, O +generates O +inosine B-residue_name +( O +I B-residue_name +) O +at O +the O +corresponding O +nucleotide O +position O +. O + +Since O +I B-residue_name +base O +pairs O +with O +cytidine B-residue_name +( O +C B-residue_name +), O +it O +functions O +like O +guanosine B-residue_name +( O +G B-residue_name +) O +in O +cellular O +processes O +such O +as O +splicing O +, O +translation O +and O +reverse O +transcription O +. O + +A O +to O +I O +editing O +has O +wide O +- O +ranging O +consequences O +on O +RNA B-chemical +function O +including O +altering O +miRNA B-site +recognition I-site +sites I-site +, O +redirecting O +splicing O +and O +changing O +the O +meaning O +of O +specific O +codons O +. O + +ADAR B-protein_type +activity O +is O +required O +for O +nervous O +system O +function O +and O +altered O +editing O +has O +been O +linked O +to O +neurological O +disorders O +such O +as O +epilepsy O +and O +Prader O +Willi O +Syndrome O +. O + +In O +addition O +, O +mutations O +in O +the O +ADAR1 B-protein +gene O +are O +known O +to O +cause O +the O +autoimmune O +disease O +Aicardi O +- O +Goutieres O +Syndrome O +( O +AGS O +) O +and O +the O +skin O +disorder O +Dyschromatosis O +Symmetrica O +Hereditaria O +( O +DSH O +). O + +Hyper O +editing O +has O +been O +observed O +at O +certain O +sites O +in O +cancer O +cells O +, O +such O +as O +in O +the O +mRNA B-chemical +for O +AZIN1 B-protein +( O +antizyme B-protein +inhibitor I-protein +1 I-protein +). O + +However O +, O +hypo O +editing O +also O +occurs O +in O +cancer O +- O +derived O +cell O +lines O +exemplified O +by O +reduced O +editing O +observed O +in O +the O +message O +for O +glioma B-protein +- I-protein +associated I-protein +oncogene I-protein +1 I-protein +( O +Gli1 B-protein +). O + +The O +ADAR B-protein_type +proteins O +have O +a O +modular O +structure O +with O +double B-structure_element +stranded I-structure_element +RNA I-structure_element +binding I-structure_element +domains I-structure_element +( O +dsRBDs B-structure_element +) O +and O +a O +C O +- O +terminal O +deaminase B-structure_element +domain I-structure_element +( O +see O +Fig O +. O +1a O +for O +hADAR2 B-protein +domains O +). O + +ADARs B-protein_type +efficiently O +deaminate O +specific O +adenosines B-residue_name +in O +duplex B-structure_element +RNA I-structure_element +while O +leaving O +most O +adenosines B-residue_name +unmodified O +. O + +The O +mechanism O +of O +adenosine B-residue_name +deamination O +requires O +ADAR B-protein_type +to O +flip O +the O +reactive O +base O +out O +of O +an O +RNA B-chemical +double I-chemical +helix I-chemical +to O +access O +its O +active B-site +site I-site +. O + +How O +an O +enzyme O +could O +accomplish O +this O +task O +with O +a O +duplex B-structure_element +RNA I-structure_element +substrate O +is O +not O +known O +. O + +To O +address O +these O +knowledge O +gaps O +, O +we O +set O +out O +to O +trap O +the O +human B-species +ADAR2 B-protein +deaminase B-structure_element +domain I-structure_element +( O +aa299 O +– B-residue_range +701 I-residue_range +, O +hADAR2d B-mutant +) O +bound B-protein_state +to I-protein_state +different O +duplex B-structure_element +RNAs I-structure_element +and O +solve O +structures B-evidence +for O +the O +resulting O +complexes O +using O +x B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +Trapping O +the O +flipped B-protein_state +conformation O + +The O +ADAR B-protein_type +reaction O +involves O +the O +formation O +of O +a O +hydrated O +intermediate O +that O +loses O +ammonia O +to O +generate O +the O +inosine B-residue_name +- O +containing O +product O +RNA B-chemical +( O +for O +reaction O +scheme O +see O +Fig O +. O +1b O +). O + +The O +covalent O +hydrate O +of O +the O +nucleoside O +analog O +8 B-chemical +- I-chemical +azanebularine I-chemical +( O +N B-chemical +) O +mimics O +the O +proposed O +high O +- O +energy O +intermediate O +( O +for O +reaction O +scheme O +see O +Fig O +. O +1b O +). O + +In O +addition O +, O +for O +one O +of O +these O +duplexes O +( O +Bdf2 B-chemical +), O +we O +positioned O +the O +8 B-chemical +- I-chemical +azanebularine I-chemical +opposite O +either O +uridine B-residue_name +or O +cytidine B-residue_name +to O +mimic O +an O +A B-residue_name +- O +U B-residue_name +pair O +or O +A B-residue_name +- O +C B-residue_name +mismatch O +at O +the O +editing B-site +site I-site +creating O +a O +total O +of O +three O +different O +RNA B-chemical +substrates O +for O +structural O +studies O +( O +Fig O +. O +1c O +). O + +The O +hADAR2d B-mutant +protein O +( O +without B-protein_state +RNA I-protein_state +bound I-protein_state +) O +has O +been O +previously O +crystallized B-experimental_method +and O +structurally O +characterized O +revealing O +features O +of O +the O +active B-site +site I-site +including O +the O +presence O +of O +zinc B-chemical +. O + +For O +crystallization B-experimental_method +of O +hADAR2d B-complex_assembly +- I-complex_assembly +RNA I-complex_assembly +complexes O +, O +we O +used O +both O +the O +wild B-protein_state +type I-protein_state +( O +WT B-protein_state +) O +deaminase B-structure_element +domain I-structure_element +and O +a O +mutant B-protein_state +( O +E488Q B-mutant +) O +that O +has O +enhanced O +catalytic O +activity O +. O + +A O +description O +of O +the O +crystallization O +conditions O +, O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +diffraction I-experimental_method +data I-experimental_method +collection I-experimental_method +and I-experimental_method +solution I-experimental_method +of O +the O +structures B-evidence +can O +be O +found O +in O +Online O +Methods O +. O + +Four O +protein O +- O +RNA B-chemical +combinations O +generated O +diffracting O +crystals B-evidence +that O +resulted O +in O +high O +- O +resolution O +structures B-evidence +( O +hADAR2d B-complex_assembly +WT I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +U I-complex_assembly +, O +hADAR2d B-complex_assembly +WT I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +C I-complex_assembly +, O +hADAR2d B-complex_assembly +E488Q I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +C I-complex_assembly +, O +hADAR2d B-complex_assembly +E488Q I-complex_assembly +– I-complex_assembly +Gli1 I-complex_assembly +) O +( O +Table O +1 O +). O + +The O +large O +binding B-site +site I-site +( O +1493 O +Å2 O +RNA O +surface O +area O +and O +1277 O +Å2 O +protein O +surface O +area O +buried O +) O +observed O +for O +hADAR2d B-mutant +is O +consistent O +with O +recent O +footprinting B-experimental_method +studies I-experimental_method +. O + +Both O +strands O +of O +the O +RNA B-chemical +contact O +the O +protein O +with O +the O +majority O +of O +these O +interactions O +mediated O +through O +the O +phosphodiester O +- O +ribose O +backbone O +near O +the O +editing B-site +site I-site +( O +Fig O +. O +2c O +, O +Supplementary O +Fig O +. O +2 O +b O +– O +d O +). O + +The O +structures B-evidence +show O +a O +large O +deviation O +from O +A B-structure_element +- I-structure_element +form I-structure_element +RNA B-chemical +conformation O +at O +the O +editing B-site +site I-site +( O +Fig O +. O +2 O +, O +Fig O +. O +3 O +, O +Supplementary O +Video O +1 O +). O + +This O +interaction O +was O +expected O +given O +the O +proposed O +role O +of O +E396 B-residue_name_number +in O +mediating O +proton O +transfers O +to O +and O +from O +N1 O +of O +the O +substrate O +adenosine B-residue_name +. O + +The O +2 O +’- O +hydroxyl O +of O +8 B-chemical +- I-chemical +azanebularine I-chemical +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +the O +backbone O +carbonyl O +of O +T375 B-residue_name_number +while O +the O +T375 B-residue_name_number +side O +chain O +contacts O +its O +3 O +’- O +phosphodiester O +. O + +R455 B-residue_name_number +and O +K376 B-residue_name_number +help O +position O +the O +flipped B-protein_state +nucleotide B-chemical +in O +the O +active B-site +site I-site +by O +fastening O +the O +phosphate O +backbone O +flanking O +the O +editing B-site +site I-site +. O + +RNA B-chemical +binding O +does O +not O +alter O +IHP B-chemical +binding O +or O +the O +H B-site +- I-site +bonding I-site +network I-site +linking O +IHP B-chemical +to O +the O +active B-site +site I-site +. O + +The O +ADAR2 B-protein +base B-structure_element +- I-structure_element +flipping I-structure_element +loop I-structure_element +, O +bearing O +residue O +488 B-residue_number +, O +approaches O +the O +RNA B-structure_element +duplex I-structure_element +from O +the O +minor B-site +groove I-site +side O +at O +the O +editing B-site +site I-site +. O + +For O +instance O +, O +in O +the O +complex B-protein_state +with I-protein_state +hADAR2d B-mutant +E488Q B-mutant +and O +the O +Bdf2 B-chemical +- I-chemical +C I-chemical +duplex I-chemical +, O +the O +protein O +recognizes O +an O +orphaned B-protein_state +C B-residue_name +by O +donating O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +from O +Nε2 O +to O +cytosine B-residue_name +N3 O +and O +from O +its O +backbone O +NH O +to O +cytosine B-residue_name +O2 O +( O +Fig O +. O +3b O +). O + +Interestingly O +, O +the O +E488Q B-mutant +mutant B-protein_state +was O +discovered O +in O +a O +screen O +for O +highly B-protein_state +active I-protein_state +ADAR2 B-protein +mutants B-protein_state +and O +this O +residue O +was O +suggested O +to O +be O +involved O +in O +base O +flipping O +given O +its O +effect O +on O +editing O +substrates O +with O +a O +fluorescent O +nucleobase O +at O +the O +editing B-site +site I-site +. O + +ADARs B-protein_type +react O +preferentially O +with O +adenosines B-residue_name +in O +A B-structure_element +• I-structure_element +C I-structure_element +mismatches O +and O +A B-structure_element +- I-structure_element +U I-structure_element +pairs I-structure_element +over O +A B-structure_element +• I-structure_element +A I-structure_element +and O +A B-structure_element +• I-structure_element +G I-structure_element +mismatches O +. O + +A O +purine B-chemical +at O +the O +orphan B-protein_state +base B-chemical +position O +( O +in O +its O +anti O +conformation O +) O +would O +clash O +with O +the O +488 B-residue_number +residue O +explaining O +the O +preference O +for O +pyrimidines B-chemical +here O +. O + +The O +interaction O +of O +the O +488 B-residue_number +residue O +with O +the O +orphaned B-protein_state +base B-chemical +is O +reminiscent O +of O +an O +interaction O +observed O +for O +Hha B-protein_type +I I-protein_type +DNA I-protein_type +methyltransfersase I-protein_type +( O +MTase B-protein_type +), O +a O +duplex B-structure_element +DNA I-structure_element +modifying O +enzyme O +that O +also O +uses O +a O +base O +flipping O +mechanism O +to O +access O +2 B-residue_name +’- I-residue_name +deoxycytidine I-residue_name +( O +dC B-residue_name +) O +for O +methylation O +. O + +For O +that O +enzyme O +, O +Q237 B-residue_name_number +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +an O +orphaned B-protein_state +dG B-residue_name +while O +it O +fills O +the O +void O +left O +by O +the O +flipped B-protein_state +out I-protein_state +dC B-residue_name +( O +Supplementary O +Fig O +. O +4b O +). O + +In O +addition O +, O +two O +glycine B-residue_name +residues O +flank O +Q237 B-residue_name_number +allowing O +the O +loop B-structure_element +to O +adopt O +the O +conformation O +necessary O +for O +penetration O +into O +the O +helix B-structure_element +. O + +Such O +an O +approach O +requires O +deeper O +penetration O +of O +the O +intercalating B-site +residue I-site +to O +access O +the O +H B-site +- I-site +bonding I-site +sites I-site +on O +the O +orphaned B-protein_state +base B-chemical +, O +necessitating O +an O +additional O +conformational O +change O +in O +the O +RNA B-structure_element +duplex I-structure_element +. O + +This O +change O +includes O +shifting O +of O +the O +base O +pairs O +immediately O +5 O +’ O +to O +the O +editing B-site +site I-site +toward O +the O +helical O +axis O +and O +a O +widening O +of O +the O +major B-site +groove I-site +opposite O +the O +editing B-site +site I-site +( O +Figs O +. O +4a O +, O +4b O +, O +Supplementary O +Video O +1 O +). O + +In O +the O +case O +of O +the O +hADAR2d B-complex_assembly +WT I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +U I-complex_assembly +RNA B-chemical +, O +this O +shift O +is O +accompanied O +by O +a O +shearing O +of O +the O +U11 B-residue_name_number +- O +A13 B-residue_name_number +' O +base O +pair O +with O +U11 B-residue_name_number +shifted O +further O +in O +the O +direction O +of O +the O +major B-site +groove I-site +creating O +an O +unusual O +U B-structure_element +- I-structure_element +A I-structure_element +"""" I-structure_element +wobble I-structure_element +"""" I-structure_element +interaction O +with O +adenine B-residue_name +N6 O +and O +N1 O +within O +H B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +distance O +to O +uracil B-residue_name +N3H O +and O +O2 O +, O +respectively O +( O +Fig O +. O +4c O +, O +Supplementary O +Fig O +. O +3b O +). O + +This O +type O +of O +wobble O +pair O +has O +been O +observed O +before O +and O +requires O +either O +the O +imino O +tautomer O +of O +adenine B-residue_name +or O +the O +enol O +tautomer O +of O +uracil B-residue_name +. O + +This O +kink B-structure_element +is O +stabilized O +by O +interactions O +of O +the O +side O +chains O +of O +R510 B-residue_name_number +and O +S495 B-residue_name_number +with O +phosphodiesters O +in O +the O +RNA B-chemical +backbone O +of O +the O +unedited O +strand O +( O +Fig O +. O +4a O +). O + +Interestingly O +, O +ADAR2 B-protein +’ O +s O +flipping B-structure_element +loop I-structure_element +approach O +from O +the O +minor B-site +groove I-site +side O +is O +like O +that O +seen O +with O +certain O +DNA B-protein_type +repair I-protein_type +glycosylases I-protein_type +( O +e O +. O +g O +. O +UDG B-protein +, O +HOGG1 B-protein +, O +and O +AAG B-protein +) O +that O +also O +project O +intercalating O +residues O +from O +loops B-structure_element +bound B-protein_state +in I-protein_state +the O +minor B-site +groove I-site +( O +Supplementary O +Fig O +. O +5a O +). O + +However O +, O +these O +enzymes O +typically O +bend O +the O +DNA B-chemical +duplex I-chemical +at O +the O +site O +of O +modification O +to O +allow O +for O +penetration O +of O +intercalating O +residues O +and O +damage O +recognition O +. O + +While O +hADAR2d B-mutant +clearly O +alters O +the O +duplex O +conformation O +to O +gain O +access O +to O +the O +modification O +site O +from O +the O +minor B-site +groove I-site +, O +it O +does O +not O +bend O +the O +RNA B-structure_element +duplex I-structure_element +( O +Figs O +. O +2a O +, O +2b O +, O +4b O +). O + +Furthermore O +, O +ADARs B-protein_type +do O +not O +modify O +duplex B-structure_element +DNA I-structure_element +. O + +For O +instance O +, O +ADAR B-protein_type +can O +readily O +penetrate O +an O +A B-structure_element +- I-structure_element +form I-structure_element +helix I-structure_element +from O +the O +minor B-site +groove I-site +side O +and O +place O +the O +helix O +- O +penetrating O +residue O +in O +the O +space O +occupied O +by O +the O +editing B-site +site I-site +base O +( O +Supplementary O +Fig O +. O +6 O +). O + +However O +, O +this O +residue O +cannot O +penetrate O +the O +minor B-site +groove I-site +enough O +to O +occupy O +the O +base O +position O +in O +a O +B B-structure_element +- I-structure_element +form I-structure_element +helix I-structure_element +( O +Supplementary O +Fig O +. O +6 O +). O + +Furthermore O +, O +DNA B-chemical +lacks O +the O +2 O +’ O +hydroxyls O +that O +are O +used O +by O +ADAR B-protein_type +for O +substrate O +recognition O +( O +Fig O +. O +2c O +). O + +Thus O +, O +hADAR2d B-mutant +uses O +a O +substrate O +recognition O +and O +base O +flipping O +mechanism O +with O +similarities O +to O +other O +known O +nucleic B-protein_type +acid I-protein_type +- I-protein_type +modifying I-protein_type +enzymes I-protein_type +but O +uniquely O +suited O +for O +reaction O +with O +adenosine B-residue_name +in O +the O +context O +of O +duplex B-structure_element +RNA I-structure_element +. O + +Structures B-evidence +explain O +nearest O +neighbor O +preferences O + +Also O +, O +the O +minor B-site +groove I-site +edge O +of O +this O +pair O +is O +juxtaposed O +to O +the O +protein O +backbone O +at O +G489 B-residue_name_number +. O + +2AP B-structure_element +is O +an O +adenosine B-residue_name +analog O +that O +forms O +a O +base O +pair O +with O +uridine B-residue_name +of O +similar O +stability O +to O +a O +U B-structure_element +- I-structure_element +A I-structure_element +pair I-structure_element +, O +but O +places O +an O +amino O +group O +in O +the O +minor B-site +groove I-site +( O +Fig O +. O +5b O +). O + +Importantly O +, O +this O +substitution O +also O +resulted O +in O +an O +80 O +% O +reduction O +in O +rate O +, O +illustrating O +the O +detrimental O +effect O +of O +the O +amino O +group O +in O +the O +minor B-site +groove I-site +at O +this O +location O +. O + +In O +each O +of O +the O +hADAR2d B-complex_assembly +- I-complex_assembly +RNA I-complex_assembly +structures B-evidence +reported O +here O +, O +the O +backbone O +carbonyl O +oxygen O +at O +S486 B-residue_name_number +accepts O +an O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +from O +the O +2 O +- O +amino O +group O +of O +the O +G B-residue_name +on O +the O +3 O +’ O +side O +of O +the O +edited O +nucleotide O +( O +Fig O +. O +5d O +). O + +Guanine B-residue_name +is O +the O +only O +common O +nucleobase O +that O +presents O +an O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +donor O +in O +the O +RNA B-site +minor I-site +groove I-site +suggesting O +that O +other O +nucleotides O +in O +this O +position O +would O +reduce O +editing O +efficiency O +. O + +Indeed O +, O +mutating B-experimental_method +this O +base O +to O +A B-residue_name +, O +C B-residue_name +or O +U B-residue_name +, O +while O +maintaining O +base O +pairing O +at O +this O +position O +, O +reduced O +the O +rate O +of O +deamination O +by O +hADAR2d B-mutant +in O +Gli1 B-protein +mRNA B-chemical +model O +substrates O +( O +Supplementary O +Fig O +. O +7 O +a O +– O +b O +). O + +To O +test O +the O +importance O +of O +the O +amino O +group O +on O +the O +3 O +’ O +G B-residue_name +in O +the O +hADAR2d B-mutant +reaction O +, O +we O +prepared O +RNA B-structure_element +duplex I-structure_element +substrates O +with O +purine O +analogs O +on O +the O +3 O +’ O +side O +of O +the O +edited B-protein_state +A B-residue_name +( O +Fig O +. O +5e O +). O + +We O +tested O +a O +G B-residue_name +analog O +that O +lacks O +the O +2 O +- O +amino O +group O +( O +inosine B-residue_name +, O +I B-residue_name +) O +and O +one O +that O +blocks O +access O +to O +this O +amino O +group O +( O +N2 O +- O +methylguanosine O +( O +N2MeG O +). O + +In O +addition O +, O +we O +compared O +a O +3 O +’ O +A B-residue_name +to O +a O +3 O +’ O +2AP B-structure_element +since O +2AP B-structure_element +could O +form O +the O +H B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +interaction I-bond_interaction +observed O +with O +S486 B-residue_name_number +. O + +We O +found O +the O +substrate O +with O +a O +3 O +’ O +N2MeG O +to O +be O +unreactive O +to O +hADAR2d B-mutant +- O +catalyzed O +deamination O +confirming O +the O +importance O +of O +the O +observed O +close O +approach O +by O +the O +protein O +to O +the O +3 O +’ O +G B-residue_name +2 O +- O +amino O +group O +( O +Fig O +. O +5f O +). O + +This O +conclusion O +is O +further O +supported O +by O +the O +observation O +that O +deamination O +in O +the O +substrate O +with O +a O +3 O +’ O +2AP B-structure_element +is O +faster O +than O +in O +the O +substrate O +with O +a O +3 O +’ O +A B-residue_name +( O +Fig O +. O +5f O +). O + +The O +structures B-evidence +reported O +here O +identify O +RNA B-structure_element +- I-structure_element +binding I-structure_element +loops I-structure_element +of O +the O +ADAR B-protein_type +catalytic B-structure_element +domain I-structure_element +and O +suggest O +roles O +for O +several O +amino O +acids O +not O +previously O +known O +to O +be O +important O +for O +editing O +, O +either O +substrate O +binding O +or O +catalysis O +( O +Fig O +. O +6 O +). O + +The O +side O +chain O +for O +R510 B-residue_name_number +ion B-bond_interaction +- I-bond_interaction +pairs I-bond_interaction +with O +the O +3 O +’ O +phosphodiester O +of O +the O +orphaned B-protein_state +nucleotide B-chemical +( O +Figs O +. O +3a O +, O +3c O +). O + +This O +residue O +is O +conserved B-protein_state +in O +ADAR2s B-protein_type +and O +ADAR1s B-protein_type +, O +but O +is O +glutamine B-residue_name +in O +the O +editing B-protein_state +- I-protein_state +inactive I-protein_state +ADAR3s B-protein_type +( O +Supplementary O +Table O +1 O +). O + +Mutation B-experimental_method +of O +hADAR2d B-mutant +at O +this O +site O +to O +either O +glutamine B-residue_name +( O +R510Q B-mutant +) O +or O +to O +alanine B-residue_name +( O +R510A B-mutant +) O +reduced O +the O +measured O +deamination B-evidence +rate I-evidence +constant I-evidence +by O +approximately O +an O +order O +of O +magnitude O +( O +Fig O +. O +6c O +). O + +In O +addition O +, O +the O +contact O +point O +near O +the O +5 O +’ O +end O +of O +the O +unedited O +strand O +involves O +G593 B-residue_name_number +, O +K594 B-residue_name_number +and O +R348 B-residue_name_number +, O +residues O +completely B-protein_state +conserved I-protein_state +in O +the O +family O +of O +ADAR2s B-protein_type +( O +Fig O +. O +2c O +, O +Supplementary O +Table O +1 O +). O + +RNA B-chemical +binding O +leads O +to O +an O +ordering O +of O +the O +454 B-residue_range +– I-residue_range +477 I-residue_range +loop B-structure_element +, O +which O +was O +disordered B-protein_state +in O +the O +RNA B-protein_state +- I-protein_state +free I-protein_state +hADAR2d B-mutant +structure B-evidence +( O +Fig O +. O +1d O +, O +green O +) O +( O +Supplementary O +Video O +2 O +). O + +This O +loop B-structure_element +sequence O +is O +conserved B-protein_state +in O +ADAR2s B-protein_type +but O +different O +in O +the O +family O +of O +ADAR1s B-protein_type +( O +Fig O +. O +6d O +). O + +The O +substantial O +difference O +in O +sequence O +between O +the O +ADARs B-protein_type +in O +this O +RNA B-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +suggests O +differences O +in O +editing B-site +site I-site +selectivity O +between O +the O +two O +ADARs B-protein_type +arise O +, O +at O +least O +in O +part O +, O +from O +differences O +in O +how O +this O +loop B-structure_element +binds O +RNA B-chemical +substrates O +. O + +DNA B-protein_type +methylases I-protein_type +, O +DNA B-protein_type +repair I-protein_type +glycosylases I-protein_type +and O +RNA B-protein_type +loop I-protein_type +modifying I-protein_type +enzymes I-protein_type +are O +known O +that O +flip O +a O +nucleotide B-chemical +out O +of O +a O +base O +pair O +. O + +However O +, O +none O +of O +the O +structurally O +characterized O +base B-protein_type +- I-protein_type +flipping I-protein_type +enzymes I-protein_type +access O +their O +reactive B-site +sites I-site +from O +within O +a O +normal B-protein_state +base I-protein_state +- I-protein_state +paired I-protein_state +RNA B-structure_element +duplex I-structure_element +. O + +We O +are O +aware O +of O +one O +other O +protein O +- O +induced O +nucleotide O +flipping O +from O +an O +RNA B-structure_element +duplex I-structure_element +region O +. O + +Bacterial B-taxonomy_domain +initiation B-protein +factor I-protein +1 I-protein +( O +IF1 B-protein +) O +binds O +to O +the O +30S B-complex_assembly +ribosomal I-complex_assembly +subunit I-complex_assembly +at O +helix B-structure_element +44 I-structure_element +of O +16S B-chemical +RNA I-chemical +with O +A1492 B-residue_name_number +and O +A1493 B-residue_name_number +flipped B-protein_state +out I-protein_state +of O +the O +helix O +and O +bound B-protein_state +into I-protein_state +protein B-site +pockets I-site +( O +Supplementary O +Fig O +. O +5b O +). O + +Because O +the O +modification B-site +sites I-site +are O +not O +flanked O +on O +both O +sides O +by O +normal B-protein_state +duplex B-structure_element +, O +these O +enzymes O +do O +not O +experience O +the O +same O +limits O +in O +approach O +to O +the O +substrate O +that O +ADARs B-protein_type +do O +. O + +The O +fact O +that O +ADARs B-protein_type +must O +induce O +flipping O +from O +a O +normal B-protein_state +duplex B-structure_element +has O +implications O +on O +its O +preference O +for O +adenosines B-residue_name +flanked O +by O +certain O +base O +pairs O +, O +a O +phenomenon O +that O +was O +not O +well O +understood O +prior O +to O +this O +work O +. O + +In O +our O +structures B-evidence +, O +the O +flipped B-protein_state +out I-protein_state +8 B-chemical +- I-chemical +azanebularine I-chemical +is O +hydrated O +, O +mimicking O +the O +tetrahedral O +intermediate O +predicted O +for O +deamination O +of O +adenosine B-residue_name +( O +Figs O +. O +1b O +, O +3a O +, O +Supplementary O +Fig O +. O +3 O +a O +– O +b O +). O + +In O +addition O +, O +8 B-chemical +- I-chemical +azanebularine I-chemical +was O +found O +to O +adopt O +a O +2 O +’- O +endo O +sugar O +pucker O +with O +its O +2 O +’- O +hydroxyl O +H B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +protein O +backbone O +carbonyl O +at O +T375 B-residue_name_number +. O + +The O +2 O +’ O +endo O +conformation O +appears O +to O +facilitate O +access O +of O +the O +nucleobase O +to O +the O +zinc B-chemical +- O +bound O +water B-chemical +for O +nucleophilic O +attack O +at O +C6 O +. O + +For O +hADAR2 B-protein +, O +E488 B-residue_name_number +serves O +this O +role O +. O + +In O +the O +two O +structures B-evidence +with O +wild B-protein_state +type I-protein_state +hADAR2 B-protein +, O +the O +E488 B-residue_name_number +residue O +and O +orphan B-protein_state +base B-chemical +are O +in O +nearly O +identical O +positions O +( O +see O +Supplementary O +Fig O +. O +4a O +for O +overlay B-experimental_method +). O + +The O +pKa B-evidence +of O +E488 B-residue_name_number +in O +the O +ADAR B-complex_assembly +- I-complex_assembly +RNA I-complex_assembly +complex O +has O +not O +been O +measured O +, O +but O +proximity O +to O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +acceptors O +, O +such O +as O +cytidine B-residue_name +N3 O +, O +and O +insertion O +between O +stacked O +nucleobases O +, O +would O +undoubtedly O +elevate O +this O +value O +and O +could O +lead O +to O +a O +substantial O +fraction O +in O +the O +protonated B-protein_state +state O +at O +physiologically O +relevant O +pH O +. O +Since O +the O +glutamine B-residue_name +side O +chain O +is O +fully B-protein_state +protonated I-protein_state +under O +physiologically O +relevant O +conditions O +, O +a O +rate O +enhancement O +for O +the O +E488Q B-mutant +mutant B-protein_state +would O +be O +expected O +if O +the O +reaction O +requires O +E488 B-residue_name_number +protonation O +. O + +The O +interactions O +of O +hADAR2d B-mutant +with O +base O +pairs O +adjacent O +to O +the O +editing B-site +site I-site +adenosine B-residue_name +explain O +the O +known O +5 O +’ O +and O +3 O +’ O +nearest O +neighbor O +preferences O +( O +Fig O +. O +5 O +). O + +While O +these O +studies O +indicate O +the O +ADAR2 B-protein +catalytic B-structure_element +domain I-structure_element +makes O +an O +important O +contact O +to O +the O +3 O +’ O +nearest O +neighbor O +G B-residue_name +, O +Stefl O +et O +al O +. O +suggested O +the O +3 O +’ O +G B-residue_name +preference O +arises O +from O +dsRBD B-structure_element +binding O +selectivity O +for O +ADAR2 B-protein +. O + +These O +authors O +reported O +a O +model O +for O +ADAR2 B-protein +’ O +s O +dsRBDs B-structure_element +bound B-protein_state +to I-protein_state +an O +editing O +substrate O +based O +on O +NMR B-experimental_method +data O +from O +the O +isolated B-protein_state +dsRBDs B-structure_element +( O +lacking B-protein_state +the O +deaminase B-structure_element +domain I-structure_element +) O +and O +short O +RNA B-chemical +fragments O +derived O +from O +the O +GluR B-protein +- I-protein +B I-protein +R B-site +/ I-site +G I-site +site I-site +RNA B-chemical +. O + +They O +describe O +an O +interaction O +wherein O +the O +3 O +’ O +G B-residue_name +2 O +- O +amino O +group O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +the O +backbone O +carbonyl O +of O +S258 B-residue_name_number +found O +in O +the O +β1 B-structure_element +- I-structure_element +β2 I-structure_element +loop I-structure_element +of O +ADAR2 B-protein +’ O +s O +dsRBDII B-structure_element +. O + +It O +is O +not O +possible O +for O +the O +S486 B-residue_name_number +- O +3 O +’ O +G B-residue_name +interaction O +we O +describe O +here O +and O +the O +S258 B-residue_name_number +- O +3 O +’ O +G B-residue_name +interaction O +reported O +by O +Stefl O +et O +al O +. O +to O +exist O +in O +the O +same O +complex O +since O +both O +involve O +protein O +loops O +bound B-protein_state +in I-protein_state +the O +RNA B-chemical +minor B-site +groove I-site +at O +the O +same O +location O +. O + +Because O +our O +structures B-evidence +have O +captured O +the O +edited B-protein_state +nucleotide B-chemical +in O +the O +conformation O +required O +to O +access O +the O +active B-site +site I-site +, O +the O +interactions O +observed O +here O +are O +highly O +likely O +to O +occur O +during O +the O +deamination O +reaction O +at O +the O +editing B-site +site I-site +. O + +It O +is O +also O +possible O +that O +ADAR B-protein_type +dsRBD B-structure_element +and O +catalytic B-structure_element +domain I-structure_element +binding O +are O +sequential O +, O +with O +release O +of O +the O +dsRBD B-structure_element +from O +the O +RNA B-chemical +taking O +place O +prior O +to O +catalytic B-structure_element +domain I-structure_element +engagement O +and O +base O +flipping O +. O + +Given O +the O +conservation O +in O +RNA B-site +binding I-site +surface I-site +and O +active B-site +site I-site +residues O +, O +we O +expect O +the O +hADAR1 B-protein +catalytic B-structure_element +domain I-structure_element +to O +bind O +RNA B-chemical +with O +a O +similar O +orientation O +of O +the O +helix O +found O +in O +our O +hADAR2d B-complex_assembly +- I-complex_assembly +RNA I-complex_assembly +structures B-evidence +. O + +When O +one O +maps O +the O +locations O +of O +the O +AGS O +- O +associated O +mutations O +onto O +the O +hADAR2d B-complex_assembly +- I-complex_assembly +RNA I-complex_assembly +complex O +, O +two O +mutations O +involve O +residues O +in O +close O +proximity O +to O +the O +RNA B-chemical +(< O +4 O +Å O +) O +( O +Supplementary O +Fig O +. O +8a O +). O + +G487 B-residue_name_number +of O +hADAR2 B-protein +is O +found O +on O +the O +flipping B-structure_element +loop I-structure_element +near O +the O +RNA B-chemical +( O +Fig O +. O +3b O +). O + +Sequence O +in O +this O +loop B-structure_element +is O +highly B-protein_state +conserved I-protein_state +among O +ADARs B-protein_type +and O +corresponds O +to O +G1007 B-residue_name_number +in O +hADAR1 B-protein +( O +Supplementary O +Table O +2 O +). O + +Also O +, O +K376 B-residue_name_number +forms O +salt B-bond_interaction +bridges I-bond_interaction +with O +both O +the O +5 O +’ O +and O +3 O +’ O +phosphodiesters O +of O +the O +guanosine B-residue_name +on O +the O +3 O +’ O +side O +of O +the O +editing B-site +site I-site +( O +Fig O +. O +2 O +). O + +The O +corresponding O +residue O +in O +hADAR1 B-protein +( O +R892 B-residue_name_number +) O +could O +form O +similar O +contacts O +and O +the O +R892H B-mutant +mutation O +would O +likely O +alter O +this O +interaction O +. O + +a O +, O +Domain O +map O +for O +human B-species +ADAR2 B-protein +b O +, O +ADAR B-protein_type +reaction O +showing O +intermediate O +and O +8 B-chemical +- I-chemical +azanebularine I-chemical +( I-chemical +N I-chemical +) I-chemical +hydrate I-chemical +that O +mimics O +this O +structure B-evidence +c O +, O +Duplex B-structure_element +RNAs I-structure_element +used O +for O +crystallization B-experimental_method +. O + +Bdf2 B-chemical +duplex I-chemical +sequence O +is O +derived O +from O +an O +editing B-site +site I-site +found O +in O +S B-species +. I-species +cerevisiae I-species +Bdf2 B-chemical +mRNA I-chemical +and O +Gli1 B-protein +duplex O +has O +sequence O +surrounding O +the O +human B-species +Gli1 B-protein +mRNA B-chemical +editing B-site +site I-site +. O + +Structure B-evidence +of O +hADAR2d B-mutant +E488Q B-mutant +bound B-protein_state +to I-protein_state +the O +Bdf2 B-chemical +- I-chemical +C I-chemical +RNA I-chemical +duplex I-chemical +at O +2 O +. O +75 O +Å O +resolution O + +a O +, O +View O +of O +structure O +perpendicular O +to O +the O +dsRNA B-chemical +helical O +axis O +. O + +Colors O +correspond O +to O +those O +in O +Figs O +. O +1a O +and O +1c O +; O +flipped B-protein_state +out I-protein_state +base O +N O +is O +highlighted O +red O +, O +zinc B-chemical +in O +grey O +space O +- O +filling O +sphere O +, O +Q488 B-residue_name_number +in O +yellow O +, O +previously O +disordered B-protein_state +aa454 O +– B-residue_range +477 I-residue_range +loop B-structure_element +in O +green O +and O +inositol B-chemical +hexakisphosphate I-chemical +( O +IHP B-chemical +) O +in O +space O +filling O +. O + +ADAR B-protein_type +recognition O +of O +the O +flipped B-protein_state +out I-protein_state +and O +orphaned B-protein_state +nucleotides B-chemical + +a O +, O +Contacts O +to O +the O +editing B-site +site I-site +nucleotide B-chemical +( O +N O +) O +in O +the O +active B-site +site I-site +. O + +c O +, O +Orphan B-protein_state +nucleotide B-chemical +recognition O +in O +the O +hADAR2d B-complex_assembly +WT I-complex_assembly +– I-complex_assembly +Bdf2 I-complex_assembly +- I-complex_assembly +U I-complex_assembly +complex O +. O + +a O +, O +hADAR2d B-mutant +shifts O +the O +position O +of O +U11 B-residue_name_number +- O +A13 B-residue_name_number +’ O +base O +pair O +from O +ideal O +A B-structure_element +- I-structure_element +form I-structure_element +RNA I-structure_element +helix I-structure_element +( O +yellow O +). O + +b O +, O +Overlay B-experimental_method +of O +Bdf2 B-chemical +duplex I-chemical +RNA I-chemical +and O +idealized O +A B-structure_element +form I-structure_element +duplex I-structure_element +of O +same O +sequence O +( O +yellow O +) O +illustrating O +kink O +in O +strand O +and O +widening O +of O +major B-site +groove I-site +opposite O +editing B-site +site I-site +induced O +by O +hADAR2d B-mutant +. O + +Interactions O +with O +editing B-site +site I-site +nearest O +neighbor O +nucleotides B-chemical + +c O +, O +Comparison O +of O +deamination B-evidence +rate I-evidence +constants I-evidence +by O +hADAR2d B-mutant +at O +the O +editing B-site +site I-site +adenosine B-residue_name +( O +red O +) O +for O +duplexes O +bearing O +different O +5 O +’ O +nearest O +neighbors O +; O +krel B-evidence += O +kobs B-evidence +/( O +kobs B-evidence +for O +unmodified B-protein_state +RNA B-chemical +). O + +d O +, O +hADAR2 B-protein +S486 B-residue_name_number +backbone O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +with O +3 O +’ O +G B-residue_name +2 O +- O +amino O +group O +; O +e O +, O +RNA B-structure_element +duplex I-structure_element +substrates O +prepared O +with O +different O +3 O +’ O +nearest O +neighbor O +nucleotides O +adjacent O +to O +editing B-site +site I-site +indicated O +in O +red O +( O +I B-residue_name += O +inosine B-residue_name +, O +N2MeG O += O +N2 O +- O +methylguanosine O +, O +2AP B-structure_element += O +2 B-structure_element +- I-structure_element +aminopurine I-structure_element +). O + +krel B-evidence += O +kobs B-evidence +/( O +kobs B-evidence +for O +unmodified B-protein_state +RNA B-chemical +). O + +RNA B-structure_element +- I-structure_element +binding I-structure_element +loops I-structure_element +in O +the O +ADAR B-protein_type +catalytic B-structure_element +domain I-structure_element + +a O +, O +hADAR2 B-protein +residues O +that O +contact O +phosphodiester O +backbone O +near O +5 O +’ O +end O +of O +unedited O +strand O +. O + +b O +, O +Location O +of O +mutations O +introduced O +at O +protein B-site +- I-site +RNA I-site +interface I-site +. O + +c O +, O +Comparison O +of O +deamination B-evidence +rate I-evidence +constants I-evidence +of O +the O +different O +hADAR2d B-mutant +mutants O +( O +Log O +scale O +). O + +krel B-evidence += O +kobs B-evidence +for O +mutant B-protein_state +/ O +kobs B-evidence +for O +WT B-protein_state +. O + +d O +, O +Sequence B-experimental_method +alignment I-experimental_method +of O +ADAR2s B-protein_type +( O +A2 O +) O +and O +ADAR1s B-protein_type +( O +A1 O +) O +from O +different O +organisms O +with O +different O +levels O +of O +conservation O +colored O +( O +Yellow O +: O +conserved B-protein_state +in O +all O +ADAR1s B-protein_type +and O +ADAR2s B-protein_type +, O +red O +: O +conserved B-protein_state +in O +ADAR2s B-protein_type +, O +blue O +: O +conserved B-protein_state +in O +ADAR1s B-protein_type +. O + +e O +, O +Interaction O +of O +the O +ADAR B-structure_element +- I-structure_element +specific I-structure_element +RNA I-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +near O +the O +5 O +’ O +end O +of O +the O +edited O +strand O +. O + +Colors O +as O +in O +d O +, O +white O +: O +not B-protein_state +conserved I-protein_state +, O +flipped B-protein_state +out I-protein_state +base B-chemical +is O +shown O +in O +pink O +. O + +Structural O +basis O +for O +the O +regulation O +of O +enzymatic O +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +by O +domain O +- O +domain O +interactions O + +Here O +, O +we O +report O +the O +structures B-evidence +of O +four O +domains O +of O +Regnase B-protein +- I-protein +1 I-protein +from O +Mus B-species +musculus I-species +— O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +NTD B-structure_element +), O +PilT B-structure_element +N I-structure_element +- I-structure_element +terminus I-structure_element +like I-structure_element +( O +PIN B-structure_element +) O +domain O +, O +zinc B-structure_element +finger I-structure_element +( O +ZF B-structure_element +) O +domain O +and O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +CTD B-structure_element +). O + +The O +PIN B-structure_element +domain O +harbors O +the O +RNase B-protein_type +catalytic B-site +center I-site +; O +however O +, O +it O +is O +insufficient O +for O +enzymatic O +activity O +. O + +We O +found O +that O +the O +NTD B-structure_element +associates O +with O +the O +PIN B-structure_element +domain O +and O +significantly O +enhances O +its O +RNase B-protein_type +activity O +. O + +The O +PIN B-structure_element +domain O +forms O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomer B-oligomeric_state +and O +the O +dimer B-site +interface I-site +overlaps O +with O +the O +NTD B-site +binding I-site +site I-site +. O + +These O +results O +suggest O +that O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O +is O +tightly O +controlled O +by O +both O +intramolecular O +( O +NTD B-structure_element +- O +PIN B-structure_element +) O +and O +intermolecular O +( O +PIN B-structure_element +- O +PIN B-structure_element +) O +interactions O +. O + +The O +initial O +sensing O +of O +infection O +is O +mediated O +by O +a O +set O +of O +pattern B-protein_type +- I-protein_type +recognition I-protein_type +receptors I-protein_type +( O +PRRs B-protein_type +) O +such O +Toll B-protein_type +- I-protein_type +like I-protein_type +receptors I-protein_type +( O +TLRs B-protein_type +) O +and O +the O +intracellular O +signaling O +cascades O +triggered O +by O +TLRs B-protein_type +evoke O +transcriptional O +expression O +of O +inflammatory O +mediators O +that O +coordinate O +the O +elimination O +of O +pathogens O +and O +infected O +cells O +. O + +Regnase B-protein +- I-protein +1 I-protein +( O +also O +known O +as O +Zc3h12a B-protein +and O +MCPIP1 B-protein +) O +is O +an O +RNase B-protein_type +whose O +expression O +level O +is O +stimulated O +by O +lipopolysaccharides B-chemical +and O +prevents O +autoimmune O +diseases O +by O +directly O +controlling O +the O +stability O +of O +mRNAs B-chemical +of O +inflammatory O +genes O +such O +as O +interleukin O +( B-protein_type +IL I-protein_type +)- I-protein_type +6 I-protein_type +, O +IL B-protein_type +- I-protein_type +1β I-protein_type +, O +IL B-protein_type +- I-protein_type +2 I-protein_type +, O +and O +IL B-protein_type +- I-protein_type +12p40 I-protein_type +. O + +Regnase B-protein +- I-protein +1 I-protein +accelerates O +target O +mRNA B-chemical +degradation O +via O +their O +3 B-structure_element +′- I-structure_element +terminal I-structure_element +untranslated I-structure_element +region I-structure_element +( O +3 B-structure_element +′ I-structure_element +UTR I-structure_element +), O +and O +also O +degrades O +its O +own O +mRNA B-chemical +. O + +Several O +CCCH B-structure_element +- I-structure_element +type I-structure_element +ZF I-structure_element +motifs I-structure_element +in O +RNA B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +have O +been O +reported O +to O +directly O +bind O +RNA B-chemical +. O + +Here O +, O +we O +performed O +structural B-experimental_method +and I-experimental_method +functional I-experimental_method +analyses I-experimental_method +of O +individual O +domains O +of O +Regnase B-protein +- I-protein +1 I-protein +derived O +from O +Mus B-species +musculus I-species +in O +order O +to O +understand O +the O +catalytic O +activity O +in O +vitro O +. O + +The O +NTD B-structure_element +plays O +a O +crucial O +role O +in O +efficient O +cleavage O +of O +target O +mRNA B-chemical +, O +through O +intramolecular O +NTD B-structure_element +- O +PIN B-structure_element +interactions O +. O + +Moreover O +, O +Regnase B-protein +- I-protein +1 I-protein +functions O +as O +a O +dimer B-oligomeric_state +through O +intermolecular O +PIN B-structure_element +- O +PIN B-structure_element +interactions O +during O +cleavage O +of O +target O +mRNA B-chemical +. O + +Our O +findings O +suggest O +that O +Regnase B-protein +- I-protein +1 I-protein +cleaves O +its O +target O +mRNA B-chemical +by O +an O +NTD B-protein_state +- I-protein_state +activated I-protein_state +functional B-protein_state +PIN B-structure_element +dimer B-oligomeric_state +, O +while O +the O +ZF B-structure_element +increases O +RNA B-chemical +affinity O +in O +the O +vicinity O +of O +the O +PIN B-structure_element +dimer B-oligomeric_state +. O + +Domain O +structures B-evidence +of O +Regnase B-protein +- I-protein +1 I-protein + +We O +analyzed O +Rengase B-protein +- I-protein +1 I-protein +derived O +from O +Mus B-species +musculus I-species +and O +solved B-experimental_method +the O +structures B-evidence +of O +the O +four O +domains O +; O +NTD B-structure_element +, O +PIN B-structure_element +, O +ZF B-structure_element +, O +and O +CTD B-structure_element +individually O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +or O +NMR B-experimental_method +( O +Fig O +. O +1a O +– O +e O +). O + +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +was O +attempted O +for O +the O +fragment O +containing O +both O +the O +PIN B-structure_element +and O +ZF B-structure_element +domains O +, O +however O +, O +electron B-evidence +density I-evidence +was O +observed O +only O +for O +the O +PIN B-structure_element +domain O +( O +Fig O +. O +1c O +), O +consistent O +with O +a O +previous O +report O +on O +Regnase B-protein +- I-protein +1 I-protein +derived O +from O +Homo B-species +sapiens I-species +. O + +This O +suggests O +that O +the O +PIN B-structure_element +and O +ZF B-structure_element +domains O +exist O +independently O +without O +interacting O +with O +each O +other O +. O + +The O +domain O +structures B-evidence +of O +NTD B-structure_element +, O +ZF B-structure_element +, O +and O +CTD B-structure_element +were O +determined O +by O +NMR B-experimental_method +( O +Fig O +. O +1b O +, O +d O +, O +e O +). O + +The O +NTD B-structure_element +and O +CTD B-structure_element +are O +both O +composed O +of O +three O +α B-structure_element +helices I-structure_element +, O +and O +structurally O +resemble O +ubiquitin B-protein +conjugating I-protein +enzyme I-protein +E2 I-protein +K I-protein +( O +PDB O +ID O +: O +3K9O O +) O +and O +ubiquitin B-protein +associated I-protein +protein I-protein +1 I-protein +( O +PDB O +ID O +: O +4AE4 O +), O +respectively O +, O +according O +to O +the O +Dali B-experimental_method +server I-experimental_method +. O + +Although O +the O +PIN B-structure_element +domain O +is O +responsible O +for O +the O +catalytic O +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +, O +the O +roles O +of O +the O +other O +domains O +are O +largely O +unknown O +. O + +First O +, O +we O +evaluated O +a O +role O +of O +the O +NTD B-structure_element +and O +ZF B-structure_element +domains O +for O +mRNA B-chemical +binding O +by O +an O +in B-experimental_method +vitro I-experimental_method +gel I-experimental_method +shift I-experimental_method +assay I-experimental_method +( O +Fig O +. O +1f O +). O + +Fluorescently B-protein_state +5 I-protein_state +′- I-protein_state +labeled I-protein_state +RNA B-chemical +corresponding O +to O +nucleotides O +82 O +– O +106 O +of O +the O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +3 B-structure_element +′ I-structure_element +UTR I-structure_element +and O +the O +catalytically O +inactive B-protein_state +mutant B-protein_state +( O +D226N B-mutant +and O +D244N B-mutant +) O +of O +Regnase B-protein +- I-protein +1 I-protein +— O +hereafter O +referred O +to O +as O +the O +DDNN B-mutant +mutant B-protein_state +— O +were O +utilized O +. O + +Based O +on O +the O +decrease O +in O +the O +free O +RNA B-chemical +fluorescence O +band O +, O +we O +evaluated O +the O +contribution O +of O +each O +domain O +of O +Regnase B-protein +- I-protein +1 I-protein +to O +RNA B-chemical +binding O +. O + +While O +the O +RNA B-chemical +binding O +ability O +was O +not O +significantly O +changed O +in O +the O +presence B-protein_state +of I-protein_state +NTD B-structure_element +, O +it O +increased O +in O +the O +presence B-protein_state +of I-protein_state +the O +ZF B-structure_element +domain O +( O +Fig O +. O +1f O +, O +g O +and O +Supplementary O +Fig O +. O +1 O +). O + +Direct O +binding O +of O +the O +ZF B-structure_element +domain O +and O +RNA B-chemical +were O +confirmed O +by O +NMR B-experimental_method +spectral B-evidence +changes I-evidence +. O + +The O +fitting O +of O +the O +titration B-evidence +curve I-evidence +of O +Y314 B-residue_name_number +resulted O +in O +an O +apparent O +dissociation B-evidence +constant I-evidence +( O +Kd B-evidence +) O +of O +10 O +± O +1 O +. O +1 O +μM O +( O +Supplementary O +Fig O +. O +2 O +). O + +These O +results O +indicate O +that O +not O +only O +the O +PIN B-structure_element +but O +also O +the O +ZF B-structure_element +domain O +contribute O +to O +RNA B-chemical +binding O +, O +while O +the O +NTD B-structure_element +is O +not O +likely O +to O +be O +involved O +in O +direct O +interaction O +with O +RNA B-chemical +. O + +Contribution O +of O +each O +domain O +of O +Regnase B-protein +- I-protein +1 I-protein +to O +RNase B-protein_type +activity O + +The O +apparent O +half O +- O +life O +( O +T1 O +/ O +2 O +) O +of O +the O +RNase B-protein_type +activity O +was O +about O +20 O +minutes O +. O + +Regnase B-protein +- I-protein +1 I-protein +lacking B-protein_state +the O +ZF B-structure_element +domain O +generated O +a O +smaller O +but O +appreciable O +amount O +of O +cleaved O +product O +( O +T1 O +/ O +2 O +~ O +70 O +minutes O +), O +while O +those O +lacking B-protein_state +the O +NTD B-structure_element +did O +not O +generate O +cleaved O +products O +( O +T1 O +/ O +2 O +> O +90 O +minutes O +). O + +It O +should O +be O +noted O +that O +NTD B-mutant +- I-mutant +PIN I-mutant +( I-mutant +DDNN I-mutant +)- I-mutant +ZF I-mutant +, O +which O +possesses O +the O +NTD B-structure_element +but O +lacks B-protein_state +the O +catalytic B-site +residues I-site +in O +PIN B-structure_element +, O +completely O +lost O +all O +RNase B-protein_type +activity O +( O +Fig O +. O +1g O +, O +right O +panel O +), O +as O +expected O +, O +confirming O +that O +the O +RNase B-protein_type +catalytic B-site +center I-site +is O +located O +in O +the O +PIN B-structure_element +domain O +. O + +Taken O +together O +with O +the O +results O +in O +the O +previous O +section O +, O +we O +conclude O +that O +the O +NTD B-structure_element +is O +crucial O +for O +the O +RNase B-protein_type +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +in O +vitro O +, O +although O +it O +does O +not O +contribute O +to O +the O +direct O +mRNA B-chemical +binding O +. O + +Dimer B-oligomeric_state +formation O +of O +the O +PIN B-structure_element +domains O + +By O +comparison B-experimental_method +with I-experimental_method +the I-experimental_method +elution I-experimental_method +volume I-experimental_method +of I-experimental_method +standard I-experimental_method +marker I-experimental_method +proteins I-experimental_method +, O +the O +PIN B-structure_element +domain O +was O +assumed O +to O +be O +in O +equilibrium O +between O +a O +monomer B-oligomeric_state +and O +a O +dimer B-oligomeric_state +in O +solution O +at O +concentrations O +in O +the O +20 O +– O +200 O +μM O +range O +. O + +The O +crystal B-evidence +structure I-evidence +of O +the O +PIN B-structure_element +domain O +has O +been O +determined O +in O +three O +distinct O +crystal B-evidence +forms I-evidence +with O +a O +space O +group O +of O +P3121 O +( O +form O +I O +in O +this O +study O +and O +PDB O +ID O +3V33 O +), O +P3221 O +( O +form O +II O +in O +this O +study O +), O +and O +P41 O +( O +PDB O +ID O +3V32 O +and O +3V34 O +), O +respectively O +. O + +We O +found O +that O +the O +PIN B-structure_element +domain O +formed O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomer B-oligomeric_state +that O +was O +commonly O +observed O +in O +all O +three O +crystal B-evidence +forms I-evidence +in O +spite O +of O +the O +different O +crystallization O +conditions O +( O +Supplementary O +Fig O +. O +3 O +). O + +On O +the O +other O +hand O +, O +single B-experimental_method +mutations I-experimental_method +of O +side O +chains O +involved O +in O +the O +PIN B-structure_element +– O +PIN B-structure_element +oligomeric O +interaction O +resulted O +in O +monomer B-oligomeric_state +formation O +, O +judging O +from O +gel B-experimental_method +filtration I-experimental_method +( O +Fig O +. O +2a O +, O +b O +). O + +Wild B-protein_state +type I-protein_state +and O +monomeric B-oligomeric_state +PIN B-structure_element +mutants B-protein_state +( O +P212A B-mutant +and O +D278R B-mutant +) O +were O +also O +analyzed O +by O +NMR B-experimental_method +. O + +The O +spectra B-evidence +indicate O +that O +the O +dimer B-site +interface I-site +of O +the O +wild B-protein_state +type I-protein_state +PIN B-structure_element +domain O +were O +significantly O +broadened O +compared O +to O +the O +monomeric B-oligomeric_state +mutants B-protein_state +( O +Supplementary O +Fig O +. O +4 O +). O + +Interestingly O +, O +the O +monomeric B-oligomeric_state +PIN B-structure_element +mutants B-protein_state +P212A B-mutant +, O +R214A B-mutant +, O +and O +D278R B-mutant +had O +no O +significant O +RNase B-protein_type +activity O +for O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +in O +vitro O +( O +Fig O +. O +2c O +). O + +Domain O +- O +domain O +interaction O +between O +the O +NTD B-structure_element +and O +the O +PIN B-structure_element +domain O + +While O +the O +NTD B-structure_element +does O +not O +contribute O +to O +RNA B-chemical +binding O +( O +Fig O +. O +1f O +, O +g O +, O +and O +Supplementary O +Fig O +. O +1 O +), O +it O +increases O +the O +RNase B-protein_type +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +( O +Fig O +. O +1h O +). O + +In O +order O +to O +gain O +insight O +into O +the O +molecular O +mechanism O +of O +the O +NTD B-structure_element +- O +mediated O +enhancement O +of O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O +, O +we O +further O +investigated O +the O +domain O +- O +domain O +interaction O +between O +the O +NTD B-structure_element +and O +the O +PIN B-structure_element +domain O +using O +NMR B-experimental_method +. O + +We O +used O +the O +catalytically B-protein_state +inactive I-protein_state +monomeric B-oligomeric_state +PIN B-structure_element +mutant B-protein_state +possessing O +both O +the O +DDNN B-mutant +and O +D278R B-mutant +mutations O +to O +avoid O +dimer B-oligomeric_state +formation O +of O +the O +PIN B-structure_element +domain O +. O + +The O +NMR B-experimental_method +signals O +from O +the O +PIN B-structure_element +domain O +( O +residues O +V177 B-residue_name_number +, O +F210 B-residue_range +- I-residue_range +T211 I-residue_range +, O +R214 B-residue_name_number +, O +F228 B-residue_range +- I-residue_range +L232 I-residue_range +, O +and O +F234 B-residue_range +- I-residue_range +S236 I-residue_range +) O +exhibited O +significant O +chemical O +shift O +changes O +upon O +addition B-experimental_method +of I-experimental_method +the O +NTD B-structure_element +( O +Fig O +. O +3a O +). O + +These O +results O +clearly O +indicate O +a O +direct O +interaction O +between O +the O +PIN B-structure_element +domain O +and O +the O +NTD B-structure_element +. O + +Based O +on O +the O +titration B-evidence +curve I-evidence +for O +the O +chemical B-evidence +shift I-evidence +changes I-evidence +of O +L58 B-residue_name_number +, O +the O +apparent O +Kd B-evidence +between O +the O +isolated O +NTD B-structure_element +and O +PIN B-structure_element +was O +estimated O +to O +be O +110 O +± O +5 O +. O +8 O +μM O +. O +Considering O +the O +fact O +that O +the O +NTD B-structure_element +and O +PIN B-structure_element +domains O +are O +attached O +by O +a O +linker B-structure_element +, O +the O +actual O +binding B-evidence +affinity I-evidence +is O +expected O +much O +higher O +in O +the O +native B-protein_state +protein O +. O + +Mapping O +the O +residues O +with O +chemical O +shift O +changes O +reveals O +the O +putative O +PIN B-site +/ I-site +NTD I-site +interface I-site +, O +which O +includes O +a O +helix B-structure_element +that O +harbors O +catalytic O +residues O +D225 B-residue_name_number +and O +D226 B-residue_name_number +on O +the O +PIN B-structure_element +domain O +( O +Fig O +. O +3a O +). O + +Interestingly O +, O +the O +putative O +binding B-site +site I-site +for O +the O +NTD B-structure_element +overlaps O +with O +the O +PIN B-site +- I-site +PIN I-site +dimer I-site +interface I-site +, O +implying O +that O +NTD B-structure_element +binding O +can O +“ O +terminate O +” O +PIN B-structure_element +- O +PIN B-structure_element +oligomerization O +( O +Fig O +. O +2b O +). O + +An O +in B-experimental_method +silico I-experimental_method +docking I-experimental_method +of O +the O +NTD B-structure_element +and O +PIN B-structure_element +domains O +using O +chemical B-evidence +shift I-evidence +restraints I-evidence +provided O +a O +model O +consistent O +with O +the O +NMR B-experimental_method +experiments O +( O +Fig O +. O +3c O +). O + +Residues O +critical O +for O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O + +To O +gain O +insight O +into O +the O +residues O +critical O +for O +Regnase B-protein +- I-protein +1 I-protein +RNase B-protein_type +activity O +, O +each O +basic O +or O +aromatic O +residue O +located O +around O +the O +catalytic B-site +site I-site +of O +the O +PIN B-structure_element +oligomer B-oligomeric_state +was O +mutated B-experimental_method +to I-experimental_method +alanine B-residue_name +, O +and O +the O +oligomerization O +and O +RNase B-protein_type +activity O +were O +investigated O +( O +Fig O +. O +4 O +). O + +From O +the O +gel B-experimental_method +filtration I-experimental_method +assays I-experimental_method +, O +all O +mutants B-protein_state +except O +R214A B-mutant +formed O +dimers B-oligomeric_state +, O +suggesting O +that O +any O +lack O +of O +RNase B-protein_type +activity O +in O +the O +mutants B-protein_state +, O +except O +R214A B-mutant +, O +was O +directly O +due O +to O +mutational O +effects O +of O +the O +specific O +residues O +and O +not O +to O +abrogation O +of O +dimer B-oligomeric_state +formation O +. O + +The O +W182A B-mutant +, O +R183A B-mutant +, O +and O +R214A B-mutant +mutants B-protein_state +markedly O +lost O +cleavage O +activity O +for O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +as O +well O +as O +for O +Regnase B-protein +- I-protein +1 I-protein +mRNA B-chemical +. O + +The O +K184A B-mutant +, O +R215A B-mutant +, O +and O +R220A B-mutant +mutants B-protein_state +moderately O +but O +significantly O +decreased O +the O +cleavage O +activity O +for O +both O +target O +mRNAs B-chemical +. O + +The O +importance O +of O +K219 B-residue_name_number +and O +R247 B-residue_name_number +was O +slightly O +different O +for O +IL B-protein_type +- I-protein_type +6 I-protein_type +and O +Regnase B-protein +- I-protein +1 I-protein +mRNA B-chemical +; O +both O +K219 B-residue_name_number +and O +R247 B-residue_name_number +were O +more O +important O +in O +the O +cleavage O +of O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +than O +for O +Regnase B-protein +- I-protein +1 I-protein +mRNA B-chemical +. O + +The O +other O +mutated O +residues O +— O +K152 B-residue_name_number +, O +R158 B-residue_name_number +, O +R188 B-residue_name_number +, O +R200 B-residue_name_number +, O +K204 B-residue_name_number +, O +K206 B-residue_name_number +, O +K257 B-residue_name_number +, O +and O +R258 B-residue_name_number +— O +were O +not O +critical O +for O +RNase B-protein_type +activity O +. O + +The O +importance O +of O +residues O +W182 B-residue_name_number +and O +R183 B-residue_name_number +can O +readily O +be O +understood O +in O +terms O +of O +the O +monomeric B-oligomeric_state +PIN B-structure_element +structure B-evidence +as O +they O +are O +located O +near O +to O +the O +RNase B-protein_type +catalytic B-site +site I-site +; O +however O +, O +the O +importance O +of O +residue O +K184 B-residue_name_number +, O +which O +points O +away O +from O +the O +active B-site +site I-site +is O +more O +easily O +rationalized O +in O +terms O +of O +the O +oligomeric O +structure B-evidence +, O +in O +which O +the O +“ O +secondary O +” O +chain O +’ O +s O +residue O +K184 B-residue_name_number +is O +positioned O +near O +the O +“ O +primary B-protein_state +” I-protein_state +chain O +’ O +s O +catalytic B-site +site I-site +( O +Fig O +. O +4 O +). O + +In O +contrast O +, O +R214 B-residue_name_number +is O +important O +for O +oligomerization O +of O +the O +PIN B-structure_element +domain O +and O +the O +“ O +secondary O +” O +chain O +’ O +s O +residue O +R214 B-residue_name_number +is O +also O +positioned O +near O +the O +“ O +primary B-protein_state +” O +chain O +’ O +s O +active B-site +site I-site +within O +the O +dimer B-site +interface I-site +. O + +It O +should O +be O +noted O +that O +the O +putative B-site +- I-site +RNA I-site +binding I-site +residues I-site +K184 B-residue_name_number +and O +R214 B-residue_name_number +are O +unique O +to O +Regnase B-protein +- I-protein +1 I-protein +among O +PIN B-structure_element +domains O +. O + +Molecular O +mechanism O +of O +target O +mRNA B-chemical +cleavage O +by O +the O +PIN B-structure_element +dimer B-oligomeric_state + +One O +group O +consisted O +of O +catalytically B-protein_state +active I-protein_state +PIN B-structure_element +domains O +with O +mutation B-experimental_method +of I-experimental_method +basic O +residues O +found O +in O +the O +previous O +section O +to O +confer O +decreased O +RNase B-protein_type +activity O +( O +Fig O +. O +4 O +). O + +These O +were O +paired O +with O +a O +DDNN B-mutant +mutant B-protein_state +that O +had O +no O +RNase B-protein_type +activity O +by O +itself O +. O + +When O +any O +members O +of O +the O +two O +groups O +are O +mixed O +, O +two O +kinds O +of O +heterodimers B-oligomeric_state +can O +be O +formed O +: O +one O +is O +composed O +of O +a O +DDNN B-mutant +primary B-protein_state +PIN B-structure_element +and O +a O +basic O +residue O +mutant B-protein_state +secondary B-protein_state +PIN B-structure_element +and O +is O +expected O +to O +exhibit O +no O +RNase B-protein_type +activity O +; O +the O +other O +is O +composed O +of O +a O +basic O +residue O +mutant B-protein_state +primary B-protein_state +PIN B-structure_element +and O +a O +DDNN B-mutant +secondary B-protein_state +PIN B-structure_element +and O +is O +predicted O +to O +rescue O +RNase B-protein_type +activity O +( O +Fig O +. O +5a O +). O + +When O +we O +compared O +the O +fluorescence B-evidence +intensity I-evidence +of O +uncleaved B-protein_state +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +, O +basic O +residue O +mutants B-protein_state +W182A B-mutant +, O +K184A B-mutant +, O +R214A B-mutant +, O +and O +R220A B-mutant +were O +rescued O +upon O +addition O +of O +the O +DDNN B-mutant +mutant B-protein_state +( O +Fig O +. O +5b O +). O + +Rescue O +of O +K184A B-mutant +and O +R214A B-mutant +by O +the O +DDNN B-mutant +mutant B-protein_state +was O +also O +confirmed O +by O +a O +significant O +increase O +in O +the O +cleaved O +products O +. O + +This O +is O +particularly O +significant O +because O +the O +side O +chains O +of O +K184 B-residue_name_number +and O +R214 B-residue_name_number +in O +the O +primary B-protein_state +PIN B-structure_element +are O +oriented O +away O +from O +their O +own O +catalytic B-site +center I-site +, O +while O +those O +in O +the O +secondary B-protein_state +PIN B-structure_element +face O +toward O +the O +catalytic B-site +center I-site +of O +the O +primary B-protein_state +PIN B-structure_element +. O + +Taken O +together O +, O +the O +rescue O +experiments O +above O +support O +the O +proposed O +model O +in O +which O +the O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +dimer B-oligomeric_state +is O +functional O +in O +vitro O +. O + +We O +determined O +the O +individual O +domain O +structures B-evidence +of O +Regnase B-protein +- I-protein +1 I-protein +by O +NMR B-experimental_method +and O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +A O +Regnase B-protein +- I-protein +1 I-protein +construct O +consisting O +of O +PIN B-structure_element +and O +ZF B-structure_element +domains O +derived O +from O +Mus B-species +musculus I-species +was O +crystallized B-experimental_method +; O +however O +, O +the O +electron B-evidence +density I-evidence +of O +the O +ZF B-structure_element +domain O +was O +low O +, O +indicating O +that O +the O +ZF B-structure_element +domain O +is O +highly B-protein_state +mobile I-protein_state +in O +the O +absence B-protein_state +of I-protein_state +target O +mRNA B-chemical +or O +possibly O +other O +protein O +- O +protein O +interactions O +. O + +These O +results O +indicate O +that O +Regnase B-protein +- I-protein +1 I-protein +directly O +binds O +to O +RNA B-chemical +and O +precipitates O +under O +such O +experimental O +conditions O +. O + +The O +previously O +reported O +crystal B-evidence +structure I-evidence +of O +the O +Regnase B-protein +- I-protein +1 I-protein +PIN B-structure_element +domain O +derived O +from O +Homo B-species +sapiens I-species +is O +nearly O +identical O +to O +the O +one O +derived O +from O +Mus B-species +musculus I-species +in O +this O +study O +, O +with O +a O +backbone O +RMSD B-evidence +of O +0 O +. O +2 O +Å O +. O +The O +amino O +acid O +sequences O +corresponding O +to O +PIN B-structure_element +( O +residues O +134 B-residue_range +– I-residue_range +295 I-residue_range +) O +are O +the O +two O +non O +- O +identical O +residues O +are O +substituted O +with O +similar O +amino O +acids O +. O + +Both O +the O +mouse B-taxonomy_domain +and O +human B-species +PIN B-structure_element +domains O +form O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomers B-oligomeric_state +in O +three O +distinct O +crystal B-evidence +forms I-evidence +. O + +Rao O +and O +co O +- O +workers O +previously O +argued O +that O +PIN B-structure_element +dimerization O +is O +likely O +to O +be O +a O +crystallographic O +artifact O +with O +no O +physiological O +significance O +, O +since O +monomers B-oligomeric_state +were O +dominant O +in O +their O +analytical B-experimental_method +ultra I-experimental_method +- I-experimental_method +centrifugation I-experimental_method +experiments O +. O + +In O +contrast O +, O +our O +gel B-experimental_method +filtration I-experimental_method +data O +, O +mutational B-experimental_method +analyses I-experimental_method +, O +and O +NMR B-experimental_method +spectra B-evidence +all O +indicate O +that O +the O +PIN B-structure_element +domain O +forms O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +dimer B-oligomeric_state +in O +solution O +in O +a O +manner O +similar O +to O +the O +crystal B-evidence +structure I-evidence +. O + +Single B-experimental_method +mutations I-experimental_method +to O +residues O +involved O +in O +the O +putative O +oligomeric O +interaction O +of O +PIN B-structure_element +monomerized B-oligomeric_state +as O +expected O +and O +these O +mutants B-protein_state +lost O +their O +RNase B-protein_type +activity O +as O +well O +. O + +Moreover O +, O +our O +structure B-experimental_method +- I-experimental_method +based I-experimental_method +mutational I-experimental_method +analyses I-experimental_method +showed O +these O +two O +Regnase B-protein +- I-protein +1 I-protein +specific O +basic O +regions O +were O +essential O +for O +target O +mRNA B-chemical +cleavage O +in O +vitro O +. O + +The O +cleavage B-experimental_method +assay I-experimental_method +also O +showed O +that O +the O +NTD B-structure_element +is O +crucial O +for O +efficient O +mRNA B-chemical +cleavage O +. O + +Moreover O +, O +we O +found O +that O +the O +NTD B-structure_element +associates O +with O +the O +oligomeric B-site +surface I-site +of O +the O +primary B-protein_state +PIN B-structure_element +, O +docking O +to O +a O +helix B-structure_element +that O +harbors O +its O +catalytic B-site +residues I-site +( O +Figs O +2b O +and O +3a O +). O + +Taken O +together O +, O +this O +suggests O +that O +the O +NTD B-structure_element +and O +the O +PIN B-structure_element +domain O +compete O +for O +a O +common B-site +binding I-site +site I-site +. O + +The O +affinity B-evidence +of O +the O +domain O +- O +domain O +interaction O +between O +two O +PIN B-structure_element +domains O +( O +Kd B-evidence += O +~ O +10 O +− O +4 O +M O +) O +is O +similar O +to O +that O +of O +the O +NTD B-structure_element +- O +PIN B-structure_element +( O +Kd B-evidence += O +110 O +± O +5 O +. O +8 O +μM O +) O +interactions O +; O +however O +, O +the O +covalent O +connection O +corresponding O +to O +residues O +90 B-residue_range +– I-residue_range +133 I-residue_range +between O +the O +NTD B-structure_element +and O +the O +primary B-protein_state +PIN B-structure_element +will O +greatly O +enhance O +the O +intramolecular O +domain O +interaction O +in O +the O +case O +of O +full B-protein_state +- I-protein_state +length I-protein_state +Regnase B-protein +- I-protein +1 I-protein +. O + +Based O +on O +these O +structural B-experimental_method +and I-experimental_method +functional I-experimental_method +analyses I-experimental_method +of O +Regnase B-protein +- I-protein +1 I-protein +domain O +- O +domain O +interactions O +, O +we O +performed O +docking B-experimental_method +simulations I-experimental_method +of O +the O +NTD B-structure_element +, O +PIN B-structure_element +dimer B-oligomeric_state +, O +and O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +. O + +The O +docking B-experimental_method +result O +revealed O +multiple O +RNA B-chemical +binding O +modes O +that O +satisfied O +the O +experimental O +results O +in O +vitro O +( O +Supplementary O +Fig O +. O +7c O +, O +d O +), O +however O +, O +it O +should O +be O +noted O +that O +, O +in O +vivo O +, O +there O +would O +likely O +be O +many O +other O +RNA B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +that O +would O +protect O +loop B-structure_element +regions O +from O +cleavage O +by O +Regnase B-protein +- I-protein +1 I-protein +. O + +In O +the O +absence B-protein_state +of I-protein_state +target O +mRNA B-chemical +, O +the O +PIN B-structure_element +domain O +forms O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomers B-oligomeric_state +at O +high O +concentration O +. O + +While O +further O +investigations O +on O +the O +domain O +- O +domain O +interactions O +of O +Regnase B-protein +- I-protein +1 I-protein +in O +vivo O +are O +necessary O +, O +these O +intramolecular O +and O +intermolecular O +domain O +interactions O +of O +Regnase B-protein +- I-protein +1 I-protein +appear O +to O +structurally O +constrain O +Regnase B-protein +- I-protein +1activity I-protein +, O +which O +, O +in O +turn O +, O +enables O +tight O +regulation O +of O +immune O +responses O +. O + +Structural B-experimental_method +and I-experimental_method +functional I-experimental_method +analyses I-experimental_method +of O +Regnase B-protein +- I-protein +1 I-protein +. O + +Catalytic B-protein_state +Asp B-residue_name +residues O +were O +shown O +in O +sticks O +. O + +Three O +Cys B-residue_name +residues O +and O +one O +His B-residue_name +residue O +responsible O +for O +Zn2 O ++- O +binding O +were O +shown O +in O +sticks O +. O + +All O +the O +structures B-evidence +were O +colored O +in O +rainbow O +from O +N O +- O +terminus O +( O +blue O +) O +to O +C O +- O +terminus O +( O +red O +). O + +Fluorescence B-evidence +intensity I-evidence +of O +the O +free B-protein_state +IL B-protein_type +- I-protein_type +6 I-protein_type +in O +each O +sample O +was O +indicated O +as O +the O +percentage O +against O +that O +in O +the O +absence B-protein_state +of I-protein_state +Regnase B-protein +- I-protein +1 I-protein +. O + +The O +percentage O +of O +the O +bound O +IL B-protein_type +- I-protein_type +6 I-protein_type +was O +calculated O +based O +on O +the O +fluorescence B-evidence +intensities I-evidence +of O +the O +free O +IL B-protein_type +- I-protein_type +6 I-protein_type +quantified O +in O +( O +f O +). O + +Head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +oligomer B-oligomeric_state +formation O +of O +the O +PIN B-structure_element +domain O +is O +crucial O +for O +the O +RNase B-protein_type +activity O +of O +Regnase B-protein +- I-protein +1 I-protein +. O + +Two O +PIN B-structure_element +molecules O +in O +the O +crystal B-evidence +were O +colored O +white O +and O +green O +, O +respectively O +. O + +Catalytic B-site +residues I-site +and O +mutated O +residues O +were O +shown O +in O +sticks O +. O + +Residues O +important O +for O +the O +oligomeric O +interaction O +were O +colored O +red O +, O +while O +R215 B-residue_name_number +that O +was O +dispensable O +for O +the O +oligomeric O +interaction O +was O +colored O +blue O +. O +( O +c O +) O +RNase B-protein_type +activity O +of O +monomeric B-oligomeric_state +mutants B-protein_state +for O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +was O +analyzed O +. O + +Domain O +- O +domain O +interaction O +between O +the O +NTD B-structure_element +and O +the O +PIN B-structure_element +domain O +. O + +( O +a O +) O +NMR B-experimental_method +analyses I-experimental_method +of O +the O +NTD B-structure_element +- O +binding O +to O +the O +PIN B-structure_element +domain O +. O + +The O +residues O +with O +significant O +chemical O +shift O +changes O +were O +labeled O +in O +the O +overlaid B-experimental_method +spectra B-evidence +( O +left O +) O +and O +colored O +red O +on O +the O +surface O +and O +ribbon O +structure O +of O +the O +PIN B-structure_element +domain O +( O +right O +). O + +Pro B-residue_name +and O +the O +residues O +without O +analysis O +were O +colored O +black O +and O +gray O +, O +respectively O +. O + +( O +b O +) O +NMR B-experimental_method +analyses I-experimental_method +of O +the O +PIN B-structure_element +- O +binding O +to O +the O +NTD B-structure_element +. O + +The O +NTD B-structure_element +and O +the O +PIN B-structure_element +domain O +are O +shown O +in O +cyan O +and O +white O +, O +respectively O +. O + +Catalytic B-site +residues I-site +of O +the O +PIN B-structure_element +domain O +are O +shown O +in O +sticks O +, O +and O +the O +residues O +that O +exhibited O +significant B-evidence +chemical I-evidence +shift I-evidence +changes I-evidence +in O +( O +a O +, O +b O +) O +were O +labeled O +. O + +( O +a O +) O +In B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assay I-experimental_method +of O +basic O +residue O +mutants B-protein_state +for O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +. O + +( O +b O +) O +In B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assay I-experimental_method +of O +basic O +residue O +mutants B-protein_state +for O +Regnase B-protein +- I-protein +1 I-protein +mRNA B-chemical +. O + +The O +fluorescence B-evidence +intensity I-evidence +of O +the O +uncleaved B-protein_state +mRNA B-chemical +was O +quantified O +and O +the O +results O +were O +mapped O +on O +the O +PIN B-structure_element +dimer B-oligomeric_state +structure B-evidence +. O + +Mutated O +basic O +residues O +were O +shown O +in O +sticks O +and O +those O +with O +significantly O +reduced O +RNase B-protein_type +activities O +were O +colored O +red O +or O +yellow O +. O + +( O +a O +) O +Cartoon O +representation O +of O +the O +concept O +of O +the O +experiment O +. O +( O +b O +) O +In B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assay I-experimental_method +of O +Regnase B-protein +- I-protein +1 I-protein +for O +IL B-protein_type +- I-protein_type +6 I-protein_type +mRNA B-chemical +. O + +( O +c O +) O +In B-experimental_method +vitro I-experimental_method +cleavage I-experimental_method +assay I-experimental_method +of O +Regnase B-protein +- I-protein +1 I-protein +for O +Regnase B-protein +- I-protein +1 I-protein +mRNA B-chemical +. O + +The O +fluorescence B-evidence +intensity I-evidence +of O +the O +uncleaved B-protein_state +mRNA B-chemical +was O +quantified O +and O +the O +results O +were O +mapped O +on O +the O +PIN B-structure_element +dimer B-oligomeric_state +. O + +The O +mutations O +whose O +RNase B-protein_type +activities O +were O +not O +increased O +in O +the O +presence B-protein_state +of I-protein_state +DDNN B-mutant +mutant B-protein_state +were O +colored O +in O +blue O +on O +the O +primary O +PIN B-structure_element +. O + +Schematic O +representation O +of O +regulation O +of O +the O +Regnase B-protein +- I-protein +1 I-protein +catalytic O +activity O +through O +the O +domain O +- O +domain O +interactions O +. O + +RAD51 B-protein +forms O +oligomers B-oligomeric_state +by O +binding O +to O +another O +molecule O +of O +RAD51 B-protein +via O +an O +‘ O +FxxA B-structure_element +’ O +motif O +, O +and O +the O +same O +recognition O +sequence O +is O +similarly O +utilised O +to O +bind O +BRCA2 B-protein +. O + +We O +use O +mutants B-protein_state +of O +a O +tetrapeptide B-chemical +sequence O +to O +probe O +the O +binding O +interaction O +, O +using O +both O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +and O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +Where O +possible O +, O +comparison O +between O +our O +tetrapeptide B-experimental_method +mutational I-experimental_method +study I-experimental_method +and O +the O +previously O +reported O +mutations O +is O +made O +, O +discrepancies O +are O +discussed O +and O +the O +importance O +of O +secondary O +structure O +in O +interpreting O +alanine B-experimental_method +scanning I-experimental_method +and O +mutational O +data O +of O +this O +nature O +is O +considered O +. O + +Eukaryotic B-taxonomy_domain +RAD51 B-protein +, O +archeal B-taxonomy_domain +RadA B-protein +and O +prokaryotic B-taxonomy_domain +RecA B-protein +are O +a O +family O +of O +ATP B-protein_type +‐ I-protein_type +dependent I-protein_type +recombinases I-protein_type +involved O +in O +homologous O +recombination O +( O +HR O +) O +of O +double O +‐ O +strand O +breaks O +in O +DNA O +1 O +. O + +BRCA2 B-protein +especially O +has O +garnered O +much O +attention O +in O +a O +clinical O +context O +, O +as O +many O +mutations O +have O +been O +identified O +that O +drive O +an O +increased O +risk O +of O +cancer O +in O +individuals O +4 O +, O +5 O +. O + +Although O +the O +inactivation O +of O +the O +BRCA2 B-complex_assembly +: I-complex_assembly +RAD51 I-complex_assembly +DNA O +repair O +pathway O +can O +cause O +genomic O +instability O +and O +eventual O +tumour O +development O +, O +an O +inability O +to O +repair O +breaks O +in O +DNA O +may O +also O +engender O +a O +sensitivity O +to O +ionising O +radiation O +6 O +, O +7 O +. O + +For O +this O +reason O +it O +is O +hypothesised O +that O +in O +tumour O +cells O +with O +an O +intact O +BRCA2 B-complex_assembly +: I-complex_assembly +RAD51 I-complex_assembly +repair O +pathway O +, O +small O +molecules O +which O +prevent O +the O +interaction O +between O +RAD51 B-protein +and O +BRCA2 B-protein +may O +confer O +radiosensitivity O +by O +disabling O +the O +HR O +pathway O +8 O +. O + +The O +interaction O +between O +the O +two O +proteins O +is O +mediated O +by O +eight O +BRC B-structure_element +repeats I-structure_element +, O +which O +are O +characterised O +by O +a O +conserved B-protein_state +‘ B-structure_element +FxxA I-structure_element +’ I-structure_element +motif I-structure_element +9 I-structure_element +. O + +RAD51 B-protein +and O +RadA B-protein +proteins O +also O +contain O +an O +‘ O +FxxA B-structure_element +’ O +sequence O +( O +FTTA B-structure_element +for O +human B-species +RAD51 B-protein +) O +through O +which O +it O +can O +bind O +to O +other O +RAD51 B-protein +and O +RadA B-protein +molecules O +, O +and O +oligomerise O +to O +form O +higher O +order O +filament O +structures O +on O +DNA O +. O + +The O +common O +FxxA B-structure_element +motifs O +of O +both O +the O +BRC B-structure_element +repeats I-structure_element +and O +RAD51 B-protein +oligomerisation B-structure_element +sequence I-structure_element +are O +recognised O +by O +the O +same O +FxxA B-site +‐ I-site +binding I-site +site I-site +of O +RAD51 B-protein +. O + +In O +general O +, O +the O +dominant O +contribution O +of O +certain O +residues O +to O +the O +overall O +binding B-evidence +energy I-evidence +of O +a O +protein O +– O +protein O +interaction O +are O +known O +as O +‘ O +hot B-site +‐ I-site +spot I-site +’ O +residues O +. O + +Interestingly O +, O +small O +molecule O +inhibitors O +of O +PPIs O +are O +often O +found O +to O +occupy O +the O +same O +pockets B-site +which O +are O +otherwise O +occupied O +by O +hot B-site +‐ I-site +spot I-site +residues O +in O +the O +native B-protein_state +complex O +. O + +It O +is O +therefore O +of O +great O +interest O +to O +identify O +hot B-site +‐ I-site +spots I-site +in O +an O +effort O +to O +guide O +drug O +discovery O +efforts O +against O +a O +PPI O +. O + +Further O +, O +a O +correlation O +between O +residues O +that O +are O +strongly B-protein_state +conserved I-protein_state +and O +hot B-site +‐ I-site +spot I-site +residues O +has O +been O +reported O +10 O +. O + +Purely O +based O +on O +the O +amino O +acid O +consensus O +sequence O +reported O +by O +Pellegrini O +et O +al O +., O +11 O +phenylalanine B-residue_name +and O +alanine B-residue_name +would O +both O +be O +expected O +to O +be O +hot B-site +‐ I-site +spots I-site +and O +to O +a O +lesser O +extent O +, O +threonine B-residue_name +. O + +The O +importance O +of O +residues O +in O +the O +FxxA B-structure_element +motif O +has O +been O +probed O +by O +a O +variety O +of O +techniques O +, O +collated O +in O +Table O +1 O +. O + +Briefly O +, O +mutating B-experimental_method +phenylalanine B-residue_name +to O +glutamic B-residue_name +acid I-residue_name +inactivated B-protein_state +the O +BRC4 B-chemical +peptide O +and O +prevented O +RAD51 B-protein +oligomerisation O +11 O +, O +12 O +. O + +A O +phenylalanine B-protein_state +‐ I-protein_state +truncated I-protein_state +BRC4 B-chemical +is O +also O +found O +to O +be O +inactive B-protein_state +13 O +, O +however O +, O +introducing B-experimental_method +a O +tryptophan B-residue_name +for O +phenylalanine B-residue_name +was O +found O +to O +have O +no O +significant O +effect O +on O +BRC4 B-chemical +affinity B-evidence +12 O +. O + +A O +glutamine B-residue_name +replacing B-experimental_method +the O +histidine B-residue_name +in O +BRC4 B-chemical +maintains O +BRC4 B-chemical +activity O +13 O +. O + +Similarly O +, O +mutating B-experimental_method +alanine B-residue_name +to O +glutamic B-residue_name +acid I-residue_name +in O +the O +RAD51 B-protein +oligomerisation B-structure_element +sequence I-structure_element +11 O +or O +to O +serine B-residue_name +in O +BRC4 B-chemical +13 O +leads O +to O +loss O +of O +interaction O +in O +both O +cases O +. O + +Mutations O +identified O +in O +the O +clinic O +, O +in O +the O +FxxA B-structure_element +region O +of O +the O +BRC B-structure_element +repeats I-structure_element +of O +BRCA2 B-protein +are O +collated O +in O +Table O +1 O +14 O +. O + +For O +completeness O +, O +we O +present O +them O +here O +with O +this O +caveat O +, O +and O +to O +make O +the O +comment O +that O +these O +clinical O +mutations O +are O +consistent O +with O +abrogating O +the O +interaction O +with O +RAD51 B-protein +. O + +Summary O +of O +FxxA B-structure_element +‐ O +relevant O +mutations O +previously O +reported O +and O +degree O +of O +characterisation O +. O + +Mutation O +contexta O +Mutation O +FxxA B-structure_element +motif O +Technique O +used O +Effect O +RAD51 B-protein +( O +FTTA B-structure_element +) O +F86E B-mutant +ETTA B-structure_element +Immunoprecipitation B-experimental_method +11 O +No O +binding O +BRC4 B-chemical +( O +FHTA B-structure_element +) O +F1524E B-mutant +EHTA B-structure_element +Competitive B-experimental_method +ELISA I-experimental_method +12 O +Peptide O +inactive B-protein_state +BRC4 B-chemical +( O +FHTA B-structure_element +) O +F1524W B-mutant +WHTA B-structure_element +Competitive B-experimental_method +ELISA I-experimental_method +12 O +Comparable O +activity O +to O +WT B-protein_state +BRC4 B-chemical +( O +FHTA B-structure_element +) O +F1524V B-mutant +VHTA B-structure_element +BRCA2 B-protein +mutations O +database O +14 O +– O +BRC4 B-chemical +( O +FHTA B-structure_element +) O +ΔF1524 B-mutant +‐ O +HTA B-structure_element +Dissociation O +of O +RAD51 B-complex_assembly +‐ I-complex_assembly +DNA I-complex_assembly +complex O +13 O +Peptide O +inactive B-protein_state +BRC4 B-chemical +( O +FHTA B-structure_element +) O +H1525Q B-mutant +FQTA B-structure_element +Dissociation O +of O +RAD51 B-complex_assembly +‐ I-complex_assembly +DNA I-complex_assembly +complex O +13 O +Comparable O +activity O +BRC7 B-chemical +( O +FSTA B-structure_element +) O +S1979R B-mutant +FRTA B-structure_element +BRCA2 B-protein +mutations O +database O +14 O +– O +BRC3 B-chemical +( O +FQTA B-structure_element +) O +T1430A B-mutant +FQAA B-structure_element +RAD51 B-complex_assembly +: I-complex_assembly +DNA I-complex_assembly +bandshift B-experimental_method +assay I-experimental_method +3 O +Peptide O +inactive B-protein_state +BRC3 B-chemical +( O +FQTA B-structure_element +) O +T1430A B-mutant +FQAA B-structure_element +Electron B-experimental_method +microscopic I-experimental_method +visualisation O +of O +nucleoprotein O +filaments O +3 O +Peptide O +inactive B-protein_state +BRC1 B-chemical +( O +FRTA B-structure_element +) O +T1011R B-mutant +FRRA B-structure_element +BRCA2 B-protein +mutations O +database O +14 O +– O +BRC2 B-chemical +( O +FYSA B-structure_element +) O +S1221P B-mutant +FYPA B-structure_element +BRCA2 B-protein +mutations O +database O +14 O +– O +BRC2 B-chemical +( O +FYSA B-structure_element +) O +S1221Y B-mutant +FYYA B-structure_element +BRCA2 B-protein +mutations O +database O +14 O +– O +RAD51 B-protein +( O +FTTA B-structure_element +) O +A89E B-mutant +FTTE B-structure_element +Immunoprecipitation B-experimental_method +11 O +No O +binding O +BRC4 B-chemical +( O +FHTA B-structure_element +) O +A1527S B-mutant +FHTS B-structure_element +Dissociation O +of O +RAD51 B-complex_assembly +‐ I-complex_assembly +DNA I-complex_assembly +complex O +13 O +Peptide O +inactive B-protein_state + +In O +this O +work O +, O +we O +report O +the O +most O +detailed O +study O +of O +systematic B-experimental_method +mutations I-experimental_method +of O +peptides O +to O +probe O +the O +FxxA B-structure_element +‐ I-structure_element +binding I-structure_element +motif I-structure_element +to O +date O +. O + +We O +have O +chosen O +to O +focus O +on O +tetrapeptides B-chemical +, O +which O +allows O +us O +to O +examine O +the O +effect O +of O +mutation B-experimental_method +on O +the O +fundamental O +unit O +of O +binding O +, O +FxxA B-structure_element +, O +rather O +than O +in O +the O +context O +of O +either O +the O +BRC B-structure_element +repeat I-structure_element +or O +self B-structure_element +‐ I-structure_element +oligomerisation I-structure_element +sequence I-structure_element +. O + +The O +use O +of O +ITC B-experimental_method +is O +generally O +perceived O +as O +a O +gold O +‐ O +standard O +in O +protein O +– O +ligand O +characterisation O +, O +rather O +than O +a O +competitive B-experimental_method +assay I-experimental_method +which O +may O +be O +prone O +to O +aggregation O +artefacts O +. O + +Conservation O +of O +FxxA B-structure_element +motif O +( O +A O +) O +BRC4 B-chemical +peptide O +( O +green O +cartoon O +) O +bound B-protein_state +to I-protein_state +truncated B-protein_state +human B-species +RAD51 B-protein +( O +grey O +surface O +) O +( O +PDB O +: O +1n0w O +, O +11 O +). O + +The O +blue O +dashed O +box O +highlights O +the O +FxxA B-site +interaction I-site +pocket I-site +. O + +( O +B O +) O +Two O +interacting O +protein O +molecules O +of O +RAD51 B-protein +from O +Saccharomyces B-species +cerevisiae I-species +are O +shown O +. O + +The O +N O +‐ O +terminal O +domain O +of O +one O +RAD51 B-protein +protomer B-oligomeric_state +is O +highlighted O +in O +pink O +for O +clarity O +and O +the O +green O +arrow O +indicates O +the O +location O +of O +this O +protomer B-oligomeric_state +' O +s O +FxxA B-structure_element +oligomerisation I-structure_element +sequence I-structure_element +( O +PDB O +: O +1szp O +, O +29 O +). O +( O +C O +) O +Conservation O +of O +FxxA B-structure_element +motif O +across O +the O +human B-species +BRC B-structure_element +repeats I-structure_element +and O +( O +D O +) O +across O +21 O +eukaryotic B-taxonomy_domain +RAD51s B-protein_type +and O +24 O +RadAs B-protein_type +, O +with O +the O +size O +of O +the O +letters O +proportional O +to O +the O +degree O +of O +conservation O +. O + +We O +have O +mutated B-experimental_method +and I-experimental_method +truncated I-experimental_method +the O +tetrapeptide B-chemical +epitope O +FHTA B-structure_element +, O +and O +examined O +the O +effects O +both O +structurally O +and O +on O +the O +binding B-evidence +affinity I-evidence +with O +humanised B-protein_state +RadA B-protein +. O +As O +a O +comparative O +reference O +, O +we O +are O +using O +the O +FHTA B-structure_element +sequence O +derived O +from O +the O +most O +tightly O +binding O +BRC B-structure_element +repeat I-structure_element +, O +BRC4 B-chemical +22 O +. O + +The O +peptides O +used O +are O +N B-protein_state +‐ I-protein_state +acetylated I-protein_state +and O +C O +‐ O +amide O +terminated O +in O +order O +to O +provide O +the O +most O +relevant O +peptide O +in O +the O +context O +of O +a O +longer O +peptide O +chain O +. O + +Phe1524 B-residue_name_number +of O +BRC4 B-chemical +binds O +in O +a O +small O +surface B-site +pocket I-site +of O +human B-species +RAD51 B-protein +, O +defined O +by O +the O +hydrophobic O +side O +chains O +of O +residues O +Met158 B-residue_name_number +, O +Ile160 B-residue_name_number +, O +Ala192 B-residue_name_number +, O +Leu203 B-residue_name_number +and O +Met210 B-residue_name_number +. O + +The O +residue O +is O +highly B-protein_state +conserved I-protein_state +across O +BRC B-structure_element +repeats I-structure_element +and O +oligomerisation B-structure_element +sequences I-structure_element +. O + +Consistent O +with O +this O +, O +the O +truncated B-protein_state +HTA B-structure_element +tripeptide B-chemical +could O +not O +be O +detected O +to O +bind O +to O +humanised B-protein_state +, O +monomeric B-oligomeric_state +RadA B-protein +, O +HumRadA2 B-mutant +( O +Table O +2 O +, O +entry O +13 O +). O + +As O +previously O +discussed O +, O +there O +is O +some O +evidence O +that O +substituting B-experimental_method +a O +tryptophan B-residue_name +for O +the O +phenylalanine B-residue_name +at O +this O +position O +was O +tolerated O +in O +the O +context O +of O +BRC4 B-chemical +12 O +. O + +Therefore O +, O +the O +WHTA B-structure_element +peptide O +was O +tested O +and O +found O +to O +not O +only O +be O +tolerated O +, O +but O +to O +increase O +the O +binding B-evidence +affinity I-evidence +of O +the O +peptide O +approximately O +threefold O +. O + +The O +second O +position O +of O +the O +tetrapeptide B-chemical +was O +found O +to O +be O +largely O +invariant O +to O +changes O +in O +the O +side O +chains O +that O +were O +investigated O +. O + +Replacing B-experimental_method +the O +histidine B-residue_name +with O +an O +asparagine B-residue_name +, O +chosen O +to O +potentially O +mimic O +the O +hydrogen B-bond_interaction +bond I-bond_interaction +donor O +– O +acceptor O +nature O +of O +histidine B-residue_name +, O +resulted O +in O +a O +moderate O +, O +twofold O +decrease O +in O +potency O +( O +Table O +2 O +, O +entry O +4 O +). O + +Mutating B-experimental_method +to O +an O +alanine B-residue_name +, O +recapitulated O +the O +potency O +of O +FHTA B-structure_element +, O +implying O +that O +the O +interactions O +made O +by O +histidine B-residue_name +do O +not O +contribute O +overall O +to O +binding B-evidence +affinity I-evidence +( O +Table O +2 O +, O +entry O +3 O +). O + +Modelling O +suggests O +that O +a O +proline B-residue_name +in O +the O +second O +position O +would O +be O +expected O +to O +clash O +sterically O +with O +the O +surface O +of O +the O +protein O +, O +and O +provides O +a O +rationale O +for O +the O +lack O +of O +binding O +observed O +. O + +Threonine B-residue_name +was O +mutated B-experimental_method +to O +an O +alanine B-residue_name +, O +resulting O +in O +only O +a O +moderately O +weaker O +K B-evidence +D I-evidence +( O +twofold O +, O +Table O +2 O +, O +entry O +7 O +). O + +In O +the O +context O +of O +a O +tetrapeptide B-chemical +at O +least O +, O +this O +result O +implies O +a O +lack O +of O +importance O +of O +a O +threonine B-residue_name +at O +this O +position O +. O + +While O +the O +importance O +of O +the O +phenylalanine B-residue_name +may O +be O +possible O +to O +predict O +from O +examination O +of O +the O +crystal B-evidence +structure I-evidence +, O +the O +alanine B-residue_name +appears O +to O +be O +of O +much O +less O +importance O +in O +this O +regard O +. O + +It O +is O +, O +however O +, O +a O +highly B-protein_state +conserved I-protein_state +residue O +and O +clearly O +of O +interest O +for O +systematic O +mutation O +. O + +Removing B-experimental_method +the O +alanine B-residue_name +residue O +entirely O +produced O +the O +truncated B-protein_state +tripeptide B-chemical +FHT B-structure_element +, O +which O +did O +not O +bind O +( O +Table O +2 O +, O +entry O +12 O +). O + +The O +unnatural O +amino O +acid O +, O +α B-chemical +‐ I-chemical +amino I-chemical +butyric I-chemical +acid I-chemical +( O +U B-chemical +), O +was O +introduced O +at O +the O +fourth O +position O +, O +positioning O +an O +ethyl O +group O +into O +the O +alanine B-site +pocket I-site +( O +Table O +2 O +, O +entry O +9 O +). O + +Structural B-experimental_method +characterisation I-experimental_method +of O +peptide O +complexes O + +The O +corresponding O +PDB O +codes O +are O +indicated O +in O +Table O +2 O +and O +crystallographic B-evidence +data I-evidence +are O +found O +in O +the O +Supporting O +Information O +. O + +WHTA B-structure_element +peptide O +shows O +a O +relative O +dislocation O +when O +compared O +to O +FHTA B-structure_element +( O +Fig O +2A O +), O +with O +the O +entire O +ligand O +backbone O +of O +WHTA B-structure_element +shifted O +to O +accommodate O +the O +change O +in O +the O +position O +of O +the O +main O +chain O +carbon O +of O +the O +first O +residue O +, O +as O +the O +larger O +indole O +side O +chain O +fills O +the O +Phe B-site +pocket I-site +. O + +This O +shift O +is O +translated O +all O +the O +way O +to O +the O +alanine B-residue_name +side O +chain O +. O + +It O +is O +possible O +that O +this O +mutation B-experimental_method +is O +beneficial O +in O +the O +tetrapeptide B-chemical +context O +and O +neutral O +in O +the O +full B-protein_state +‐ I-protein_state +length I-protein_state +BRC4 B-chemical +context O +because O +the O +smaller O +peptide O +is O +less O +constrained O +and O +allowed O +to O +explore O +more O +conformations O +. O + +An O +attempt O +to O +combine O +both O +the O +tryptophan B-residue_name +and O +proline B-residue_name +mutations B-experimental_method +, O +however O +, O +led O +to O +no O +improvement O +for O +WHPA B-structure_element +peptide O +compared O +to O +FHTA B-structure_element +. O + +One O +possible O +explanation O +is O +that O +the O +‘ O +shifted O +’ O +binding O +mode O +observed O +in O +WHTA B-structure_element +was O +not O +compatible O +with O +the O +conformational O +restriction O +that O +the O +proline B-residue_name +of O +WHPA B-structure_element +introduced O +. O + +Comparison O +of O +different O +peptide O +complexes O +( O +A O +) O +Overlay B-experimental_method +with O +FHTA B-structure_element +( O +grey O +) O +and O +WHTA B-structure_element +( O +purple O +) O +showing O +a O +small O +relative O +displacement O +of O +the O +peptide O +backbone O +. O +( O +B O +) O +Superposition B-experimental_method +of O +FHTA B-structure_element +( O +grey O +) O +and O +FHPA B-structure_element +( O +yellow O +), O +showing O +conservation O +of O +backbone O +orientation O +( O +C O +) O +Overlay B-experimental_method +of O +FHTU B-structure_element +( O +green O +), O +FHTA B-structure_element +( O +grey O +) O +and O +FHTG B-structure_element +( O +cyan O +). O + +Although O +ΔH B-evidence +and O +ΔS B-evidence +are O +tabulated O +, O +the O +K B-evidence +Ds I-evidence +measured O +are O +relatively O +weak O +and O +necessarily O +performed O +under O +low O +c O +‐ O +value O +conditions O +. O + +In O +this O +experimental O +regime O +, O +nonsigmoidal O +curves O +are O +generated O +and O +therefore O +errors O +in O +ΔH B-evidence +are O +expected O +to O +be O +much O +higher O +than O +the O +errors O +from O +model O +fitting O +given O +in O +Table O +2 O +16 O +. O + +Such O +effects O +have O +been O +discussed O +by O +Klebe O +24 O +and O +Chodera O +and O +Mobley O +25 O +and O +will O +frustrate O +attempts O +to O +interpret O +the O +measured O +ΔΔH B-evidence +and O +ΔΔS B-evidence +. O + +The O +conserved B-protein_state +phenylalanine B-residue_name +and O +alanine B-residue_name +residues O +of O +the O +FHTA B-structure_element +sequence O +were O +both O +found O +to O +be O +essential O +for O +binding O +by O +ITC B-experimental_method +. O + +Conversely O +the O +second O +position O +histidine B-residue_name +residue O +, O +corresponding O +to O +the O +unconserved B-protein_state +His1525 B-residue_name_number +in O +the O +BRC4 B-chemical +sequence O +, O +could O +be O +mutated B-experimental_method +without O +significant O +effect O +on O +the O +peptide B-evidence +affinity I-evidence +. O + +The O +more O +general O +correlation O +between O +hot B-site +‐ I-site +spot I-site +residues O +in O +protein O +– O +protein O +interactions O +and O +the O +high B-protein_state +conservation I-protein_state +of O +such O +residues O +has O +been O +previously O +reported O +10 O +, O +26 O +. O + +Interestingly O +, O +however O +, O +the O +highly B-protein_state +conserved I-protein_state +threonine B-residue_name +residue O +could O +be O +mutated B-experimental_method +without O +affecting O +the O +peptide B-evidence +affinity I-evidence +. O + +This O +unexpected O +result O +, O +in O +the O +light O +of O +its O +very O +high B-protein_state +conservation I-protein_state +in O +the O +BRC B-structure_element +and O +oligomerisation B-structure_element +sequences I-structure_element +, O +begs O +the O +question O +of O +what O +the O +role O +of O +Thr1526 B-residue_name_number +is O +and O +highlights O +a O +potential O +pitfall O +and O +need O +for O +caution O +in O +the O +experimental O +design O +of O +alanine B-experimental_method +mutation I-experimental_method +studies I-experimental_method +. O + +As O +the O +FHTA B-structure_element +peptide B-chemical +is O +potentially O +a O +surrogate O +peptide O +for O +both O +the O +BRC B-structure_element +repeat I-structure_element +peptides O +and O +the O +RAD51 B-protein +self B-structure_element +‐ I-structure_element +oligomerisation I-structure_element +peptide I-structure_element +, O +it O +is O +useful O +to O +examine O +the O +role O +of O +Thr1526 B-residue_name_number +( O +BRC4 B-chemical +) O +and O +the O +analogous O +Thr87 B-residue_name_number +( O +RAD51 B-protein +) O +in O +both O +binding O +contexts O +in O +more O +detail O +. O + +Also O +, O +Thr1520 B-residue_name_number +is O +constrained O +by O +crystal O +contacts O +in O +this O +structure B-evidence +. O + +Lack B-protein_state +of I-protein_state +conservation I-protein_state +of O +this O +residue O +supports O +the O +idea O +that O +this O +interaction O +is O +not O +crucial O +for O +RAD51 B-complex_assembly +: I-complex_assembly +BRC I-complex_assembly +repeat I-complex_assembly +binding O +. O + +Residue O +numbering O +relates O +to O +the O +S B-species +. I-species +cerevisiae I-species +RAD51 B-protein +protein O +, O +the O +corresponding O +human B-species +residues O +are O +in O +parentheses O +. O + +With O +respect O +to O +understanding O +the O +RAD51 B-complex_assembly +: I-complex_assembly +RAD51 I-complex_assembly +interaction O +, O +no O +human B-species +crystal B-evidence +structure I-evidence +has O +been O +published O +, O +however O +, O +several O +oligomeric O +structures B-evidence +of O +archaeal B-taxonomy_domain +RadA B-protein +as O +well O +that O +of O +Saccharomyces B-species +cerevisiae I-species +RAD51 B-protein +have O +been O +reported O +27 O +, O +28 O +, O +29 O +. O + +Figure O +3B O +shows O +a O +highlight O +of O +the O +FxxA B-structure_element +portion O +of O +oligomerisation B-structure_element +peptide I-structure_element +from O +the O +S B-species +. I-species +cerevisiae I-species +RAD51 B-protein +structure B-evidence +, O +with O +residues O +in O +parentheses O +corresponding O +to O +the O +human B-species +RAD51 B-protein +protein O +. O + +In O +both O +structural O +contexts O +, O +the O +role O +of O +the O +third O +position O +threonine B-residue_name +in O +FxxA B-structure_element +seems O +to O +be O +in O +stabilising O +secondary O +structure O +; O +a O +β B-structure_element +‐ I-structure_element +turn I-structure_element +in O +the O +case O +of O +BRC B-structure_element +binding O +and O +an O +α B-structure_element +‐ I-structure_element +helix I-structure_element +in O +the O +case O +of O +RAD51 B-protein +oligomerisation O +. O + +In O +the O +tetrapeptide B-chemical +context O +these O +secondary O +interactions O +are O +not O +present O +and O +mutation B-experimental_method +of O +threonine B-residue_name +to O +alanine B-residue_name +would O +be O +expected O +to O +have O +little O +effect O +on O +affinity B-evidence +. O + +In O +line O +with O +this O +, O +although O +we O +observe O +a O +slight O +twofold O +weakening O +of O +peptide B-evidence +affinity I-evidence +, O +the O +effect O +is O +far O +from O +being O +as O +drastic O +or O +inactivating O +as O +reported O +in O +longer O +peptide O +backgrounds O +3 O +. O +It O +would O +be O +interesting O +to O +investigate O +the O +importance O +of O +this O +residue O +in O +the O +context O +of O +the O +BRC4 B-chemical +peptide O +, O +and O +the O +oligomerisation B-structure_element +peptide I-structure_element +. O + +Rather O +than O +indifference O +to O +alanine B-residue_name +mutation B-experimental_method +, O +a O +significant O +effect O +, O +via O +lack O +of O +secondary O +structure O +stabilisation O +, O +would O +be O +predicted O +, O +as O +indeed O +has O +been O +reported O +for O +BRC3 B-chemical +3 O +. O + +Proline B-residue_name +at O +the O +third O +position O +similarly O +improved O +potency O +. O + +Activity O +was O +lost O +by O +mutating B-experimental_method +the O +terminal O +alanine B-residue_name +to O +glycine B-residue_name +, O +but O +recovered O +somewhat O +with O +the O +novel O +α B-chemical +‐ I-chemical +amino I-chemical +butyric I-chemical +acid I-chemical +( O +U B-chemical +). O + +Threonine B-residue_name +was O +found O +to O +be O +relatively O +unimportant O +in O +the O +tetrapeptides B-chemical +but O +has O +been O +previously O +reported O +to O +be O +crucial O +in O +the O +context O +of O +BRC3 B-chemical +. O + +This O +may O +lead O +to O +a O +more O +general O +caution O +, O +that O +hot B-site +‐ I-site +spot I-site +data O +should O +be O +interpreted O +by O +considering O +the O +bound O +interaction O +with O +the O +protein O +, O +as O +well O +as O +the O +potential O +role O +in O +stabilising O +the O +bound O +peptide O +secondary O +structure O +. O + +In O +either O +case O +, O +the O +requirement O +for O +structural O +data O +in O +correctly O +interpreting O +alanine B-experimental_method +‐ I-experimental_method +scanning I-experimental_method +experiments I-experimental_method +is O +reinforced O +. O + +Summary O +of O +key O +observations O +( O +A O +) O +FxxA B-structure_element +motif O +sequence O +conservation O +of O +Rad51 B-protein +oligomerisation O +sequences O +and O +BRC B-structure_element +repeats I-structure_element +. O +( O +B O +) O +Highlight O +of O +SAR O +identified O +for O +the O +tetrapeptide B-chemical +. O + +Purple O +carbon O +is O +WHTA B-structure_element +, O +light O +blue O +is O +FATA B-structure_element +, O +yellow O +is O +FHPA B-structure_element +, O +cyan O +is O +FHTG B-structure_element +and O +grey O +carbon O +is O +FHTA B-structure_element +. O + +Note O +the O +C O +‐ O +terminal O +amide O +changes O +position O +in O +FHTG B-structure_element +without O +the O +anchoring O +methyl O +group O +. O + +Crystal B-evidence +Structure I-evidence +and O +Activity B-experimental_method +Studies I-experimental_method +of O +the O +C11 B-protein_type +Cysteine B-protein_type +Peptidase I-protein_type +from O +Parabacteroides B-species +merdae I-species +in O +the O +Human B-species +Gut O +Microbiome O +* O + +Clan B-protein_type +CD I-protein_type +cysteine I-protein_type +peptidases I-protein_type +, O +a O +structurally O +related O +group O +of O +peptidases B-protein_type +that O +include O +mammalian B-taxonomy_domain +caspases B-protein_type +, O +exhibit O +a O +wide O +range O +of O +important O +functions O +, O +along O +with O +a O +variety O +of O +specificities O +and O +activation O +mechanisms O +. O + +However O +, O +for O +the O +clostripain B-protein_type +family I-protein_type +( O +denoted O +C11 B-protein_type +), O +little O +is O +currently O +known O +. O + +PmC11 B-protein +is O +a O +monomeric B-oligomeric_state +cysteine B-protein_type +peptidase I-protein_type +that O +comprises O +an O +extended B-structure_element +caspase I-structure_element +- I-structure_element +like I-structure_element +α I-structure_element +/ I-structure_element +β I-structure_element +/ I-structure_element +α I-structure_element +sandwich I-structure_element +and O +an O +unusual O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +. O + +It O +shares O +core O +structural O +elements O +with O +clan B-protein_type +CD I-protein_type +cysteine I-protein_type +peptidases I-protein_type +but O +otherwise O +structurally O +differs O +from O +the O +other O +families O +in O +the O +clan O +. O + +Collectively O +, O +these O +data O +provide O +insights O +into O +the O +mechanism O +and O +activity O +of O +PmC11 B-protein +and O +a O +detailed O +framework O +for O +studies O +on O +C11 B-protein_type +peptidases I-protein_type +from O +other O +phylogenetic O +kingdoms O +. O + +In O +the O +MEROPS O +peptidase O +database O +, O +clan B-protein_type +CD I-protein_type +contains O +groups O +( O +or O +families O +) O +of O +cysteine B-protein_type +peptidases I-protein_type +that O +share O +some O +highly B-protein_state +conserved I-protein_state +structural O +elements O +. O + +Although O +seven O +families O +( O +C14 O +is O +additionally O +split O +into O +three O +subfamilies O +) O +have O +been O +described O +for O +this O +clan O +, O +crystal B-evidence +structures I-evidence +have O +only O +been O +determined O +from O +four O +: O +legumain B-protein +( O +C13 B-protein_type +), O +caspase B-protein +( O +C14a B-protein_type +), O +paracaspase B-protein +( O +C14b B-protein_type +( I-protein_type +P I-protein_type +), O +metacaspase B-protein +( O +C14b B-protein_type +( I-protein_type +M I-protein_type +), O +gingipain B-protein +( O +C25 B-protein_type +), O +and O +the O +cysteine B-structure_element +peptidase I-structure_element +domain I-structure_element +( O +CPD B-structure_element +) O +of O +various O +toxins O +( O +C80 B-protein_type +). O + +No O +structural O +information O +is O +available O +for O +clostripain B-protein +( O +C11 B-protein_type +), O +separase B-protein +( O +C50 B-protein_type +), O +or O +PrtH B-protein +- I-protein +peptidase I-protein +( O +C85 B-protein_type +). O + +Clan B-protein_type +CD I-protein_type +enzymes I-protein_type +have O +a O +highly B-protein_state +conserved I-protein_state +His B-site +/ I-site +Cys I-site +catalytic I-site +dyad I-site +and O +exhibit O +strict O +specificity O +for O +the O +P1 B-residue_number +residue O +of O +their O +substrates O +. O + +Interestingly O +, O +little O +is O +known O +about O +the O +structure O +or O +function O +of O +the O +C11 B-protein_type +proteins O +, O +despite O +their O +widespread O +distribution O +and O +its O +archetypal O +member O +, O +clostripain B-protein +from O +Clostridium B-species +histolyticum I-species +, O +first O +reported O +in O +the O +literature O +in O +1938 O +. O + +As O +part O +of O +an O +ongoing O +project O +to O +characterize O +commensal O +bacteria B-taxonomy_domain +in O +the O +microbiome O +that O +inhabit O +the O +human B-species +gut O +, O +the O +structure B-evidence +of O +C11 B-protein_type +peptidase I-protein_type +, O +PmC11 B-protein +, O +from O +Parabacteroides B-species +merdae I-species +was O +determined O +using O +the O +Joint O +Center O +for O +Structural O +Genomics O +( O +JCSG O +) O +4 O +HTP O +structural O +biology O +pipeline O +. O + +The O +structure B-experimental_method +was I-experimental_method +analyzed I-experimental_method +, O +and O +the O +enzyme O +was O +biochemically B-experimental_method +characterized I-experimental_method +to O +provide O +the O +first O +structure O +/ O +function O +correlation O +for O +a O +C11 B-protein_type +peptidase I-protein_type +. O + +Structure B-evidence +of O +PmC11 B-protein + +The O +crystal B-evidence +structure I-evidence +of O +the O +catalytically B-protein_state +active I-protein_state +form O +of O +PmC11 B-protein +revealed O +an O +extended B-structure_element +caspase I-structure_element +- I-structure_element +like I-structure_element +α I-structure_element +/ I-structure_element +β I-structure_element +/ I-structure_element +α I-structure_element +sandwich I-structure_element +architecture O +comprised O +of O +a O +central O +nine B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +, O +with O +an O +unusual O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +CTD B-structure_element +), O +starting O +at O +Lys250 B-residue_name_number +. O + +The O +central O +nine B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +( O +β1 B-structure_element +– I-structure_element +β9 I-structure_element +) O +of O +PmC11 B-protein +consists O +of O +six O +parallel B-structure_element +and O +three O +anti B-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +strands I-structure_element +with O +4 O +↑ O +3 O +↓ O +2 O +↑ O +1 O +↑ O +5 O +↑ O +6 O +↑ O +7 O +↓ O +8 O +↓ O +9 O +↑ O +topology O +( O +Fig O +. O +1A O +) O +and O +the O +overall O +structure B-evidence +includes O +14 O +α B-structure_element +- I-structure_element +helices I-structure_element +with O +six O +( O +α1 B-structure_element +– I-structure_element +α2 I-structure_element +and O +α4 B-structure_element +– I-structure_element +α7 I-structure_element +) O +closely O +surrounding O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +in O +an O +approximately O +parallel O +orientation O +. O + +Helices B-structure_element +α1 B-structure_element +, O +α7 B-structure_element +, O +and O +α6 B-structure_element +are O +located O +on O +one O +side O +of O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +with O +α2 B-structure_element +, O +α4 B-structure_element +, O +and O +α5 B-structure_element +on O +the O +opposite O +side O +( O +Fig O +. O +1A O +). O + +Helix B-structure_element +α3 B-structure_element +sits O +at O +the O +end O +of O +the O +loop B-structure_element +following O +β5 B-structure_element +( O +L5 B-structure_element +), O +just O +preceding O +the O +Lys147 B-residue_name_number +cleavage B-site +site I-site +, O +with O +both O +L5 B-structure_element +and O +α3 B-structure_element +pointing O +away O +from O +the O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +and O +toward O +the O +CTD B-structure_element +, O +which O +starts O +with O +α8 B-structure_element +. O + +A O +, O +primary B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +PmC11 B-protein +( O +Uniprot O +ID O +A7A9N3 O +) O +and O +clostripain B-protein +( O +Uniprot O +ID O +P09870 O +) O +from O +C B-species +. I-species +histolyticum I-species +with O +identical O +residues O +highlighted O +in O +gray O +shading O +. O + +Connecting O +loops B-structure_element +are O +colored O +gray O +, O +the O +main O +β B-structure_element +- I-structure_element +sheet I-structure_element +is O +in O +orange O +, O +with O +other O +strands O +in O +olive O +, O +α B-structure_element +- I-structure_element +helices I-structure_element +are O +in O +blue O +, O +and O +the O +nonapeptide B-structure_element +linker I-structure_element +of O +clostripain B-protein +that O +is O +excised O +upon O +autocleavage B-ptm +is O +underlined O +in O +red O +. O + +Sequences O +around O +the O +catalytic B-site +site I-site +of O +clostripain B-protein +and O +PmC11 B-protein +align O +well O +. O + +The O +position O +of O +the O +catalytic B-site +dyad I-site +( O +H B-residue_name +, O +C B-residue_name +) O +and O +the O +processing B-site +site I-site +( O +Lys147 B-residue_name_number +) O +are O +highlighted O +. O + +Helices O +( O +1 O +– O +14 O +) O +and O +β B-structure_element +- I-structure_element +strands I-structure_element +( O +1 O +– O +9 O +and O +A O +- O +F O +) O +are O +numbered O +from O +the O +N O +terminus O +. O + +The O +core B-structure_element +caspase I-structure_element +- I-structure_element +fold I-structure_element +is O +highlighted O +in O +a O +box O +. O + +C O +, O +tertiary O +structure O +of O +PmC11 B-protein +. O + +Loops O +are O +colored O +gray O +, O +the O +main O +β B-structure_element +- I-structure_element +sheet I-structure_element +is O +in O +orange O +, O +with O +other O +β B-structure_element +- I-structure_element +strands I-structure_element +in O +yellow O +, O +and O +α B-structure_element +- I-structure_element +helices I-structure_element +are O +in O +blue O +. O + +The O +CTD B-structure_element +of O +PmC11 B-protein +is O +composed O +of O +a O +tight B-structure_element +helical I-structure_element +bundle I-structure_element +formed O +from O +helices B-structure_element +α8 B-structure_element +– I-structure_element +α14 I-structure_element +and O +includes O +strands B-structure_element +βC B-structure_element +and O +βF B-structure_element +, O +and O +β B-structure_element +- I-structure_element +hairpin I-structure_element +βD B-structure_element +– I-structure_element +βE I-structure_element +. O +The O +CTD B-structure_element +sits O +entirely O +on O +one O +side O +of O +the O +enzyme O +interacting O +only O +with O +α3 B-structure_element +, O +α5 B-structure_element +, O +β9 B-structure_element +, O +and O +the O +loops B-structure_element +surrounding O +β8 B-structure_element +. O + +This B-structure_element +helix I-structure_element +makes O +a O +total O +of O +eight O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +CTD B-structure_element +, O +including O +one O +salt B-bond_interaction +bridge I-bond_interaction +( O +Arg191 B-residue_name_number +- O +Asp255 B-residue_name_number +) O +and O +is O +surrounded O +by O +the O +CTD B-structure_element +on O +one O +side O +and O +the O +main B-structure_element +core I-structure_element +of O +the O +enzyme O +on O +the O +other O +, O +acting O +like O +a O +linchpin O +holding O +both O +components O +together O +( O +Fig O +. O +1C O +). O + +The O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +is O +unique O +to O +PmC11 B-protein +within O +clan B-protein_type +CD I-protein_type +and O +structure B-experimental_method +comparisons I-experimental_method +for O +this B-structure_element +domain I-structure_element +alone I-structure_element +does O +not O +produce O +any O +hits O +in O +the O +PDB O +( O +DaliLite B-experimental_method +, O +PDBeFold B-experimental_method +), O +suggesting O +a O +completely O +novel O +fold O +. O + +As O +the O +archetypal O +and O +arguably O +most O +well O +studied O +member O +of O +clan B-protein_type +CD I-protein_type +, O +the O +caspases B-protein_type +were O +used O +as O +the O +basis O +to O +investigate O +the O +structure O +/ O +function O +relationships O +in O +PmC11 B-protein +, O +with O +caspase B-protein +- I-protein +7 I-protein +as O +the O +representative O +member O +. O + +Six O +of O +the O +central O +β B-structure_element +- I-structure_element +strands I-structure_element +in O +PmC11 B-protein +( O +β1 B-structure_element +– I-structure_element +β2 I-structure_element +and O +β5 B-structure_element +– I-structure_element +β8 I-structure_element +) O +share O +the O +same O +topology O +as O +the O +six B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +found O +in O +caspases B-protein_type +, O +with O +strands B-structure_element +β3 B-structure_element +, O +β4 B-structure_element +, O +and O +β9 B-structure_element +located O +on O +the O +outside O +of O +this O +core B-structure_element +structure I-structure_element +( O +Fig O +. O +1B O +, O +box O +). O + +A O +multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +C11 B-protein_type +proteins O +revealed O +that O +these O +residues O +are O +highly B-protein_state +conserved I-protein_state +( O +data O +not O +shown O +). O + +Biochemical B-experimental_method +and I-experimental_method +structural I-experimental_method +characterization I-experimental_method +of O +PmC11 B-protein +. O + +A O +, O +ribbon O +representation O +of O +the O +overall O +structure O +of O +PmC11 B-protein +illustrating O +the O +catalytic B-site +site I-site +, O +cleavage O +site O +displacement O +, O +and O +potential O +S1 B-site +binding I-site +site I-site +. O + +The O +two O +ends O +of O +the O +autolytic B-site +cleavage I-site +site I-site +( O +Lys147 B-residue_name_number +and O +Ala148 B-residue_name_number +, O +green O +) O +are O +displaced O +by O +19 O +. O +5 O +Å O +( O +thin O +black O +line O +) O +from O +one O +another O +and O +residues O +in O +the O +potential O +substrate B-site +binding I-site +pocket I-site +are O +highlighted O +in O +blue O +. O + +B O +, O +size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +of O +PmC11 B-protein +. O + +PmC11 O +migrates O +as O +a O +monomer B-oligomeric_state +with O +a O +molecular O +mass O +around O +41 O +kDa O +calculated O +from O +protein O +standards O +of O +known O +molecular O +weights O +. O + +Elution O +fractions O +across O +the O +major O +peak O +( O +1 O +– O +6 O +) O +were O +analyzed O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +on O +a O +4 O +– O +12 O +% O +gel O +in O +MES O +buffer O +. O + +C O +, O +the O +active B-protein_state +form O +of O +PmC11 B-protein +and O +two O +mutants O +, O +PmC11C179A B-mutant +( O +C O +) O +and O +PmC11K147A B-mutant +( O +K O +), O +were O +examined O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +( O +lane O +1 O +) O +and O +Western B-experimental_method +blot I-experimental_method +analysis O +using O +an O +anti O +- O +His O +antibody O +( O +lane O +2 O +) O +show O +that O +PmC11 B-protein +autoprocesses B-ptm +, O +whereas O +mutants O +, O +PmC11C179A B-mutant +and O +PmC11K147A B-mutant +, O +do O +not O +show O +autoprocessing B-ptm +in O +vitro O +. O + +D O +, O +cysteine O +peptidase O +activity O +of O +PmC11 B-protein +. O + +Km O +and O +Vmax B-evidence +of O +PmC11 B-protein +and O +K147A B-mutant +mutant O +were O +determined O +by O +monitoring O +change O +in O +the O +fluorescence O +corresponding O +to O +AMC O +release O +from O +Bz B-chemical +- I-chemical +R I-chemical +- I-chemical +AMC I-chemical +. O + +PmC11C179A O +( O +20 O +μg O +) O +was O +incubated O +overnight O +at O +37 O +° O +C O +with O +increasing O +amounts O +of O +processed O +PmC11 B-protein +and O +analyzed O +on O +a O +10 O +% O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +gel O +. O + +F O +, O +activity B-evidence +of O +PmC11 B-protein +against O +basic O +substrates O +. O + +G O +, O +electrostatic O +surface O +potential O +of O +PmC11 B-protein +shown O +in O +a O +similar O +orientation O +, O +where O +blue O +and O +red O +denote O +positively O +and O +negatively O +charged O +surface O +potential O +, O +respectively O +, O +contoured O +at O +± O +5 O +kT O +/ O +e O +. O + +Five O +of O +the O +α B-structure_element +- I-structure_element +helices I-structure_element +surrounding O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +PmC11 B-protein +( O +α1 B-structure_element +, O +α2 B-structure_element +, O +α4 B-structure_element +, O +α6 B-structure_element +, O +and O +α7 B-structure_element +) O +are O +found O +in O +similar O +positions O +to O +the O +five O +structurally B-protein_state +conserved I-protein_state +helices B-structure_element +in O +caspases B-protein_type +and O +other O +members O +of O +clan B-protein_type +CD I-protein_type +, O +apart O +from O +family O +C80 B-protein_type +. O + +Autoprocessing B-ptm +of O +PmC11 B-protein + +Purification B-experimental_method +of O +recombinant O +PmC11 B-protein +( O +molecular O +mass O += O +42 O +. O +6 O +kDa O +) O +revealed O +partial O +processing O +into O +two O +cleavage O +products O +of O +26 O +. O +4 O +and O +16 O +. O +2 O +kDa O +, O +related O +to O +the O +observed O +cleavage B-ptm +at O +Lys147 B-residue_name_number +in O +the O +crystal B-evidence +structure I-evidence +( O +Fig O +. O +2A O +). O + +Incubation B-experimental_method +of O +PmC11 B-protein +at O +37 O +° O +C O +for O +16 O +h O +, O +resulted O +in O +a O +fully B-protein_state +processed I-protein_state +enzyme O +that O +remained O +as O +an O +intact B-protein_state +monomer B-oligomeric_state +when O +applied O +to O +a O +size O +- O +exclusion O +column O +( O +Fig O +. O +2B O +). O + +The O +single O +cleavage B-site +site I-site +of O +PmC11 B-protein +at O +Lys147 B-residue_name_number +is O +found O +immediately O +after O +α3 B-structure_element +, O +in O +loop B-structure_element +L5 B-structure_element +within O +the O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +( O +Figs O +. O +1 O +, O +A O +and O +B O +, O +and O +2A O +). O + +Moreover O +, O +the O +C O +- O +terminal O +side O +of O +the O +cleavage B-site +site I-site +resides O +near O +the O +catalytic B-site +dyad I-site +with O +Ala148 B-residue_name_number +being O +4 O +. O +5 O +and O +5 O +. O +7 O +Å O +from O +His133 B-residue_name_number +and O +Cys179 B-residue_name_number +, O +respectively O +. O + +Thus O +, O +the O +cleavage B-ptm +would O +be O +required O +for O +full B-protein_state +activation I-protein_state +of O +PmC11 B-protein +. O + +To O +investigate O +this O +possibility O +, O +two O +mutant O +forms O +of O +the O +enzyme O +were O +created O +: O +PmC11C179A B-mutant +( O +a O +catalytically B-protein_state +inactive I-protein_state +mutant I-protein_state +) O +and O +PmC11K147A B-mutant +( O +a O +cleavage B-protein_state +- I-protein_state +site I-protein_state +mutant I-protein_state +). O + +Initial O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +and O +Western B-experimental_method +blot I-experimental_method +analysis O +of O +both O +mutants O +revealed O +no O +discernible O +processing O +occurred O +as O +compared O +with O +active B-protein_state +PmC11 B-protein +( O +Fig O +. O +2C O +). O + +The O +PmC11K147A B-mutant +mutant B-protein_state +enzyme O +had O +a O +markedly O +different O +reaction B-evidence +rate I-evidence +( O +Vmax B-evidence +) O +compared O +with O +WT B-protein_state +, O +where O +the O +reaction B-evidence +velocity I-evidence +of O +PmC11 B-protein +was O +10 O +times O +greater O +than O +that O +of O +PmC11K147A B-mutant +( O +Fig O +. O +2D O +). O + +Taken O +together O +, O +these O +data O +reveal O +that O +PmC11 B-protein +requires O +processing O +at O +Lys147 B-residue_name_number +for O +optimum O +activity O +. O + +This O +suggests O +that O +cleavage B-ptm +of O +PmC11C179A B-mutant +was O +most O +likely O +an O +effect O +of O +the O +increasing O +concentration O +of O +PmC11 B-protein +and O +intermolecular O +cleavage O +. O + +Substrate O +Specificity O +of O +PmC11 B-protein + +The O +autocatalytic B-ptm +cleavage I-ptm +of O +PmC11 B-protein +at O +Lys147 B-residue_name_number +( O +sequence O +KLK O +∧ O +A O +) O +demonstrates O +that O +the O +enzyme O +accepts O +substrates O +with O +Lys B-residue_name +in O +the O +P1 B-residue_number +position O +. O + +As O +expected O +, O +PmC11 B-protein +showed O +no O +activity O +against O +substrates O +with O +Pro B-residue_name +or O +Asp B-residue_name +in O +P1 B-residue_number +but O +was O +active B-protein_state +toward O +substrates O +with O +a O +basic O +residue O +in O +P1 B-residue_number +such O +as O +Bz B-chemical +- I-chemical +R I-chemical +- I-chemical +AMC I-chemical +, O +Z B-chemical +- I-chemical +GGR I-chemical +- I-chemical +AMC I-chemical +, O +and O +BOC B-chemical +- I-chemical +VLK I-chemical +- I-chemical +AMC I-chemical +. O + +The O +rate O +of O +cleavage O +was O +∼ O +3 O +- O +fold O +greater O +toward O +the O +single O +Arg B-residue_name +substrate O +Bz B-chemical +- I-chemical +R I-chemical +- I-chemical +AMC I-chemical +than O +for O +the O +other O +two O +( O +Fig O +. O +2F O +) O +and O +, O +unexpectedly O +, O +PmC11 B-protein +showed O +no O +activity O +toward O +BOC B-chemical +- I-chemical +K I-chemical +- I-chemical +AMC I-chemical +. O + +The O +catalytic B-site +dyad I-site +of O +PmC11 B-protein +sits O +near O +the O +bottom O +of O +an O +open B-protein_state +pocket B-site +on O +the O +surface O +of O +the O +enzyme O +at O +a O +conserved B-protein_state +location I-protein_state +in O +the O +clan O +CD B-protein_type +family I-protein_type +. O + +The O +PmC11 B-protein +structure B-evidence +reveals O +that O +the O +catalytic B-site +dyad I-site +forms O +part O +of O +a O +large O +acidic B-site +pocket I-site +( O +Fig O +. O +2G O +), O +consistent O +with O +a O +binding B-site +site I-site +for O +a O +basic O +substrate O +. O + +This O +pocket B-site +is O +lined O +with O +the O +potential O +functional O +side O +chains O +of O +Asn50 B-residue_name_number +, O +Asp177 B-residue_name_number +, O +and O +Thr204 B-residue_name_number +with O +Gly134 B-residue_name_number +, O +Asp207 B-residue_name_number +, O +and O +Met205 B-residue_name_number +also O +contributing O +to O +the O +pocket B-site +( O +Fig O +. O +2A O +). O + +Interestingly O +, O +these O +residues O +are O +in O +regions O +that O +are O +structurally B-protein_state +similar I-protein_state +to O +those O +involved O +in O +the O +S1 B-site +binding I-site +pockets I-site +of O +other O +clan B-protein_type +CD I-protein_type +members I-protein_type +( O +shown O +in O +Ref O +.). O + +A O +structure B-experimental_method +overlay I-experimental_method +of O +PmC11 B-protein +with O +the O +MALT1 B-protein +- I-protein +paracacaspase I-protein +( O +MALT1 B-protein +- I-protein +P I-protein +), O +in O +complex B-protein_state +with O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +, O +revealed O +that O +the O +PmC11 B-protein +dyad B-site +sits O +in O +a O +very O +similar O +position O +to O +that O +of O +active B-protein_state +MALT1 B-protein +- I-protein +P I-protein +and O +that O +Asn50 B-residue_name_number +, O +Asp177 B-residue_name_number +, O +and O +Asp207 B-residue_name_number +superimpose O +well O +with O +the O +principal O +MALT1 B-protein +- I-protein +P I-protein +inhibitor B-site +binding I-site +residues I-site +( O +Asp365 B-residue_name_number +, O +Asp462 B-residue_name_number +, O +and O +Glu500 B-residue_name_number +, O +respectively O +( O +VRPR B-chemical +- I-chemical +FMK I-chemical +from O +MALT1 B-protein +- I-protein +P I-protein +with O +the O +corresponding O +PmC11 B-protein +residues O +from O +the O +structural B-experimental_method +overlay I-experimental_method +is O +shown O +in O +Fig O +. O +1D O +), O +as O +described O +in O +Ref O +.). O + +Asp177 B-residue_name_number +is O +located O +near O +the O +catalytic B-protein_state +cysteine B-residue_name +and O +is O +conserved B-protein_state +throughout I-protein_state +the O +C11 B-protein_type +family I-protein_type +, O +suggesting O +it O +is O +the O +primary O +S1 B-site +binding I-site +site I-site +residue I-site +. O + +In O +the O +structure B-evidence +of O +PmC11 B-protein +, O +Asp207 B-residue_name_number +resides O +on O +a O +flexible O +loop B-structure_element +pointing O +away O +from O +the O +S1 B-site +binding I-site +pocket I-site +( O +Fig O +. O +3C O +). O + +However O +, O +this O +loop B-structure_element +has O +been O +shown O +to O +be O +important O +for O +substrate O +binding O +in O +clan B-protein_type +CD I-protein_type +and O +this O +residue O +could O +easily O +rotate O +and O +be O +involved O +in O +substrate O +binding O +in O +PmC11 B-protein +. O + +Thus O +, O +Asn50 B-residue_name_number +, O +Asp177 B-residue_name_number +, O +and O +Asp207 B-residue_name_number +are O +most O +likely O +responsible O +for O +the O +substrate O +specificity O +of O +PmC11 B-protein +. O + +PmC11 B-protein +binds O +and O +is O +inhibited O +by O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +and O +does O +not O +require O +Ca2 B-chemical ++ I-chemical +for O +activity O +. O + +A O +, O +PmC11 O +activity O +is O +inhibited O +by O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +. O + +PmC11 O +was O +incubated B-experimental_method +with O +(+) O +or O +without O +(−) O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +and O +the O +samples O +analyzed O +on O +a O +10 O +% O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +gel O +. O + +A O +size B-evidence +shift I-evidence +can O +be O +observed O +in O +the O +larger O +processed O +product O +of O +PmC11 B-protein +( O +26 O +. O +1 O +kDa O +). O + +C O +, O +PmC11 B-protein +with O +the O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +from O +the O +MALT1 B-protein +- I-protein +paracacaspase I-protein +( O +MALT1 B-protein +- I-protein +P I-protein +) O +superimposed B-experimental_method +. O + +A O +three B-experimental_method +- I-experimental_method +dimensional I-experimental_method +structural I-experimental_method +overlay I-experimental_method +of O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +from O +the O +MALT1 B-protein +- I-protein +P I-protein +complex O +onto O +PmC11 B-protein +. O + +The O +position O +and O +orientation O +of O +Z B-chemical +- I-chemical +VRPR I-chemical +- I-chemical +FMK I-chemical +was O +taken O +from O +superposition B-experimental_method +of O +the O +PmC11 B-protein +and O +MALTI_P B-protein +structures B-evidence +and O +indicates O +the O +presumed O +active B-site +site I-site +of O +PmC11 B-protein +. O + +Residues O +surrounding O +the O +inhibitor O +are O +labeled O +and O +represent O +potentially O +important O +binding B-site +site I-site +residues I-site +, O +labeled O +in O +black O +and O +shown O +in O +an O +atomic O +representation O +. O + +C O +, O +divalent O +cations O +do O +not O +increase O +the O +activity O +of O +PmC11 B-protein +. O + +Clostripain B-protein +from O +C B-species +. I-species +histolyticum I-species +is O +the O +founding O +member O +of O +the O +C11 B-protein_type +family I-protein_type +of O +peptidases B-protein_type +and O +contains O +an O +additional O +149 B-residue_range +residues I-residue_range +compared O +with O +PmC11 B-protein +. O + +Unlike O +PmC11 B-protein +, O +clostripain B-protein +has O +two O +cleavage B-site +sites I-site +( O +Arg181 B-residue_name_number +and O +Arg190 B-residue_name_number +), O +which O +results O +in O +the O +removal O +of O +a O +nonapeptide B-structure_element +, O +and O +is O +required O +for O +full B-protein_state +activation I-protein_state +of O +the O +enzyme O +( O +highlighted O +in O +Fig O +. O +1A O +). O + +As O +studies O +on O +clostripain B-protein +revealed O +addition O +of O +Ca2 B-chemical ++ I-chemical +ions O +are O +required O +for O +full B-protein_state +activation I-protein_state +, O +the O +Ca2 B-chemical ++ I-chemical +dependence O +of O +PmC11 B-protein +was O +examined O +. O + +Surprisingly O +, O +Ca2 B-chemical ++ I-chemical +did O +not O +enhance O +PmC11 B-protein +activity O +and O +, O +furthermore O +, O +other O +divalent O +cations O +, O +Mg2 B-chemical ++, I-chemical +Mn2 B-chemical ++, I-chemical +Co2 B-chemical ++, I-chemical +Fe2 B-chemical ++, I-chemical +Zn2 B-chemical ++, I-chemical +and O +Cu2 B-chemical ++, I-chemical +were O +not O +necessary O +for O +PmC11 B-protein +activity O +( O +Fig O +. O +3D O +). O + +In O +support O +of O +these O +findings O +, O +EGTA B-chemical +did O +not O +inhibit O +PmC11 B-protein +suggesting O +that O +, O +unlike O +clostripain B-protein +, O +PmC11 B-protein +does O +not O +require O +Ca2 B-chemical ++ I-chemical +or O +other O +divalent O +cations O +, O +for O +activity O +. O + +The O +crystal B-evidence +structure I-evidence +of O +PmC11 B-protein +now O +provides O +three O +- O +dimensional O +information O +for O +a O +member O +of O +the O +clostripain B-protein +C11 B-protein_type +family I-protein_type +of O +cysteine B-protein_type +peptidases I-protein_type +. O + +The O +enzyme O +exhibits O +all O +of O +the O +key O +structural O +elements O +of O +clan B-protein_type +CD I-protein_type +members I-protein_type +, O +but O +is O +unusual O +in O +that O +it O +has O +a O +nine O +- O +stranded O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +with O +a O +novel O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +. O + +The O +structural O +similarity O +of O +PmC11 B-protein +with O +its O +nearest O +structural O +neighbors O +in O +the O +PDB O +is O +decidedly O +low O +, O +overlaying O +better O +with O +six O +- O +stranded O +caspase B-protein +- I-protein +7 I-protein +than O +any O +of O +the O +other O +larger O +members O +of O +the O +clan O +( O +Table O +2 O +). O + +The O +substrate O +specificity O +of O +PmC11 B-protein +is O +Arg B-residue_name +/ O +Lys B-residue_name +and O +the O +crystal B-evidence +structure I-evidence +revealed O +an O +acidic B-site +pocket I-site +for O +specific O +binding O +of O +such O +basic O +substrates O +. O + +In O +addition O +, O +the O +structure B-evidence +suggested O +a O +mechanism O +of O +self O +- O +inhibition O +in O +both O +PmC11 B-protein +and O +clostripain B-protein +and O +an O +activation O +mechanism O +that O +requires O +autoprocessing B-ptm +. O + +PmC11 B-protein +differs O +from O +clostripain B-protein +in O +that O +is O +does O +not O +appear O +to O +require O +divalent O +cations O +for O +activation O +. O + +Several O +other O +members O +of O +clan B-protein_type +CD I-protein_type +require O +processing B-ptm +for O +full B-protein_state +activation I-protein_state +including O +legumain B-protein +, O +gingipain B-protein +- I-protein +R I-protein +, O +MARTX B-protein +- I-protein +CPD I-protein +, O +and O +the O +effector B-protein_type +caspases I-protein_type +, O +e O +. O +g O +. O +caspase B-protein +- I-protein +7 I-protein +. O + +To O +date O +, O +the O +effector B-protein_type +caspases I-protein_type +are O +the O +only O +group O +of O +enzymes O +that O +require O +cleavage B-ptm +of O +a O +loop B-structure_element +within O +the O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +. O + +All O +other O +clan B-protein_type +CD I-protein_type +members I-protein_type +requiring O +cleavage B-ptm +for O +full B-protein_state +activation I-protein_state +do O +so O +at O +sites B-site +external O +to O +their O +central O +sheets B-structure_element +. O + +In O +addition O +, O +several O +members O +of O +clan B-protein_type +CD I-protein_type +exhibit O +self O +- O +inhibition O +, O +whereby O +regions B-structure_element +of O +the O +enzyme O +block O +access O +to O +the O +active B-site +site I-site +. O + +Like O +PmC11 B-protein +, O +these O +structures O +show O +preformed O +catalytic O +machinery O +and O +, O +for O +a O +substrate O +to O +gain O +access O +, O +movement O +and O +/ O +or O +cleavage B-ptm +of O +the O +blocking B-structure_element +region I-structure_element +is O +required O +. O + +The O +structure B-evidence +of O +PmC11 B-protein +gives O +the O +first O +insight O +into O +this O +class O +of O +relatively O +unexplored O +family O +of O +proteins O +and O +should O +allow O +important O +catalytic O +and O +substrate O +binding O +residues O +to O +be O +identified O +in O +a O +variety O +of O +orthologues O +. O + +Indeed O +, O +insights O +gained O +from O +an O +analysis O +of O +the O +PmC11 B-protein +structure B-evidence +revealed O +the O +identity O +of O +the O +Trypanosoma B-species +brucei I-species +PNT1 B-protein +protein O +as O +a O +C11 B-protein_type +cysteine I-protein_type +peptidase I-protein_type +with O +an O +essential O +role O +in O +organelle O +replication O +. O + +The O +chemically O +most O +complex O +modification O +in O +eukaryotic B-taxonomy_domain +rRNA B-chemical +is O +the O +conserved B-protein_state +hypermodified B-protein_state +nucleotide B-chemical +N1 B-chemical +- I-chemical +methyl I-chemical +- I-chemical +N3 I-chemical +- I-chemical +aminocarboxypropyl I-chemical +- I-chemical +pseudouridine I-chemical +( O +m1acp3Ψ B-chemical +) O +located O +next O +to O +the O +P B-site +- I-site +site I-site +tRNA B-chemical +on O +the O +small O +subunit O +18S B-chemical +rRNA I-chemical +. O + +While O +S B-chemical +- I-chemical +adenosylmethionine I-chemical +was O +identified O +as O +the O +source O +of O +the O +aminocarboxypropyl B-chemical +( O +acp B-chemical +) O +group O +more O +than O +40 O +years O +ago O +the O +enzyme O +catalyzing O +the O +acp B-chemical +transfer O +remained O +elusive O +. O + +In O +functionally O +impaired O +Tsr3 B-protein +- O +mutants B-protein_state +, O +a O +reduced O +level O +of O +acp B-chemical +modification O +directly O +correlates O +with O +increased O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +accumulation O +. O + +The O +crystal B-evidence +structure I-evidence +of O +archaeal B-taxonomy_domain +Tsr3 B-protein +homologs O +revealed O +the O +same O +fold O +as O +in O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +- I-protein_type +methyltransferases I-protein_type +but O +a O +distinct O +SAM B-site +binding I-site +mode I-site +. O + +Structurally O +, O +Tsr3 B-protein +therefore O +represents O +a O +novel O +class O +of O +acp B-protein_type +transferase I-protein_type +enzymes O +. O + +During O +eukaryotic B-taxonomy_domain +ribosome O +biogenesis O +several O +dozens O +of O +rRNA B-chemical +nucleotides B-chemical +become O +chemically O +modified O +. O + +The O +most O +abundant O +rRNA B-chemical +modifications O +are O +methylations B-ptm +at O +the O +2 O +′- O +OH O +ribose B-chemical +moieties O +and O +isomerizations O +of O +uridine B-chemical +residues O +to O +pseudouridine B-chemical +, O +catalyzed O +by O +small B-complex_assembly +nucleolar I-complex_assembly +ribonucleoprotein I-complex_assembly +particles I-complex_assembly +( O +snoRNPs B-complex_assembly +). O + +In O +addition O +, O +18S B-chemical +and O +25S B-chemical +( O +yeast B-taxonomy_domain +)/ O +28S B-chemical +( O +humans B-species +) O +rRNAs B-chemical +contain O +several O +base O +modifications O +catalyzed O +by O +site O +- O +specific O +and O +snoRNA B-chemical +- O +independent O +enzymes O +. O + +In O +Saccharomyces B-species +cerevisiae I-species +18S B-chemical +rRNA I-chemical +contains O +four O +base O +methylations B-ptm +, O +two O +acetylations B-ptm +and O +a O +single O +3 B-chemical +- I-chemical +amino I-chemical +- I-chemical +3 I-chemical +- I-chemical +carboxypropyl I-chemical +( O +acp B-chemical +) O +modification O +, O +whereas O +six O +base O +methylations B-ptm +are O +present O +in O +the O +25S B-chemical +rRNA I-chemical +. O + +Ribosomal B-chemical +RNA I-chemical +modifications O +have O +been O +suggested O +to O +optimize O +ribosome O +function O +, O +although O +in O +most O +cases O +this O +remains O +to O +be O +clearly O +established O +. O + +They O +might O +contribute O +to O +increased O +RNA B-chemical +stability O +by O +providing O +additional O +hydrogen B-bond_interaction +bonds I-bond_interaction +( O +pseudouridines B-chemical +), O +improved O +base B-bond_interaction +stacking I-bond_interaction +( O +pseudouridines B-chemical +and O +base B-ptm +methylations I-ptm +) O +or O +an O +increased O +resistance O +against O +hydrolysis O +( O +ribose B-ptm +methylations I-ptm +). O + +Most O +modified O +rRNA B-chemical +nucleotides B-chemical +cluster O +in O +the O +vicinity O +of O +the O +decoding B-site +or O +the O +peptidyl B-site +transferase I-site +center I-site +, O +suggesting O +an O +influence O +on O +ribosome O +functionality O +and O +stability O +. O + +The O +chemically O +most O +complex O +modification O +is O +located O +in O +the O +loop B-structure_element +capping I-structure_element +helix I-structure_element +31 I-structure_element +of O +18S B-chemical +rRNA I-chemical +( O +Supplementary O +Figure O +S1B O +). O + +There O +a O +uridine B-residue_name +( O +U1191 B-residue_name_number +in O +yeast B-taxonomy_domain +) O +is O +modified O +to O +1 B-chemical +- I-chemical +methyl I-chemical +- I-chemical +3 I-chemical +-( I-chemical +3 I-chemical +- I-chemical +amino I-chemical +- I-chemical +3 I-chemical +- I-chemical +carboxypropyl I-chemical +)- I-chemical +pseudouridine I-chemical +( O +m1acp3Ψ B-chemical +, O +Figure O +1A O +). O + +This O +base O +modification O +was O +first O +described O +in O +1968 O +for O +hamster B-taxonomy_domain +cells O +and O +is O +conserved B-protein_state +in I-protein_state +eukaryotes B-taxonomy_domain +. O + +This O +hypermodified B-protein_state +nucleotide B-chemical +, O +which O +is O +located O +at O +the O +P B-site +- I-site +site I-site +tRNA B-chemical +, O +is O +synthesized O +in O +three O +steps O +beginning O +with O +the O +snR35 B-chemical +H B-structure_element +/ I-structure_element +ACA I-structure_element +snoRNP B-complex_assembly +guided O +conversion O +of O +uridine B-chemical +into O +pseudouridine B-chemical +. O + +In O +a O +second O +step O +, O +the O +essential O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +methyltransferase I-protein_type +Nep1 B-protein +/ O +Emg1 B-protein +modifies O +the O +pseudouridine B-chemical +to O +N1 B-chemical +- I-chemical +methylpseudouridine I-chemical +. O + +The O +final O +acp B-chemical +modification O +leading O +to O +N1 B-chemical +- I-chemical +methyl I-chemical +- I-chemical +N3 I-chemical +- I-chemical +aminocarboxypropyl I-chemical +- I-chemical +pseudouridine I-chemical +occurs O +late O +during O +40S B-complex_assembly +biogenesis O +in O +the O +cytoplasm O +, O +while O +the O +two O +former O +reactions O +are O +taking O +place O +in O +the O +nucleolus O +and O +nucleus O +, O +and O +is O +independent O +from O +pseudouridylation B-ptm +or O +methylation O +. O + +Both O +the O +methyl O +and O +the O +acp O +group O +are O +derived O +from O +S B-chemical +- I-chemical +adenosylmethionine I-chemical +( O +SAM B-chemical +), O +but O +the O +enzyme O +responsible O +for O +acp B-chemical +modification O +remained O +elusive O +for O +more O +than O +40 O +years O +. O + +The O +asterisk O +indicates O +the O +C1 O +- O +atom O +labeled O +in O +the O +14C B-experimental_method +- I-experimental_method +incorporation I-experimental_method +assay I-experimental_method +. O + +( O +B O +) O +RP B-experimental_method +- I-experimental_method +HPLC I-experimental_method +elution B-evidence +profile I-evidence +of O +yeast B-taxonomy_domain +18S B-chemical +rRNA I-chemical +nucleosides B-chemical +. O + +Wild B-protein_state +type I-protein_state +( O +WT B-protein_state +) O +and O +plasmid O +encoded O +18S B-chemical +rRNA I-chemical +( O +U1191U B-mutant +) O +show O +the O +14C B-chemical +- I-chemical +acp I-chemical +signal O +, O +whereas O +the O +14C B-chemical +- I-chemical +acp I-chemical +signal O +is O +missing O +in O +the O +U1191A B-mutant +mutant B-protein_state +plasmid O +encoded O +18S B-chemical +rRNA I-chemical +( O +U1191A B-mutant +) O +and O +Δtsr3 B-mutant +mutants O +( O +Δtsr3 B-mutant +). O + +All O +samples O +were O +loaded O +on O +the O +gel O +with O +two O +different O +amounts O +of O +5 O +and O +10 O +μl O +. O +( O +D O +) O +Primer B-experimental_method +extension I-experimental_method +analysis I-experimental_method +of O +acp B-chemical +modification O +in O +yeast B-taxonomy_domain +18S B-chemical +rRNA I-chemical +( O +right O +gel O +) O +including O +a O +sequencing O +ladder O +( O +left O +gel O +). O + +The O +primer O +extension O +stop O +at O +nucleotide O +1191 B-residue_number +is O +missing O +exclusively O +in O +Δtsr3 B-mutant +mutants O +and O +Δtsr3 B-mutant +Δsnr35 I-mutant +recombinants O +. O + +( O +E O +) O +Primer B-experimental_method +extension I-experimental_method +analysis I-experimental_method +of O +human B-species +18S B-chemical +rRNA I-chemical +after O +siRNA B-experimental_method +knockdown I-experimental_method +of O +HsNEP1 B-protein +/ O +EMG1 B-protein +( O +541 O +, O +542 O +and O +543 O +) O +and O +HsTSR3 B-protein +( O +544 O +and O +545 O +) O +( O +right O +gel O +), O +including O +a O +sequencing O +ladder O +( O +left O +gel O +). O + +The O +primer O +extension O +arrest O +is O +reduced O +in O +HTC116 O +cells O +transfected O +with O +siRNAs B-chemical +544 O +and O +545 O +. O + +The O +efficiency O +of O +siRNA B-chemical +mediated O +HsTSR3 B-protein +repression O +correlates O +with O +the O +primer B-evidence +extension I-evidence +signals I-evidence +( O +see O +Supplementary O +Figure O +S2A O +). O + +Only O +a O +few O +acp B-chemical +transferring O +enzymes O +have O +been O +characterized O +until O +now O +. O + +During O +the O +biosynthesis O +of O +wybutosine B-chemical +, O +a O +tricyclic O +nucleoside B-chemical +present O +in O +eukaryotic B-taxonomy_domain +and O +archaeal B-taxonomy_domain +phenylalanine B-chemical +tRNA B-chemical +, O +Tyw2 B-protein +( O +Trm12 B-protein +in O +yeast B-taxonomy_domain +) O +transfers O +an O +acp B-chemical +group O +from O +SAM B-chemical +to O +an O +acidic O +carbon O +atom O +. O + +Another O +acp B-chemical +modification O +has O +been O +described O +in O +the O +diphtamide B-chemical +biosynthesis O +pathway O +, O +where O +an O +acp B-chemical +group O +is O +transferred O +from O +SAM B-chemical +to O +the O +carbon O +atom O +of O +a O +histidine B-residue_name +residue O +of O +eukaryotic B-taxonomy_domain +translation B-protein_type +elongation I-protein_type +factor I-protein_type +2 I-protein_type +by O +use O +of O +a O +radical O +mechanism O +. O + +It O +is O +highly B-protein_state +conserved I-protein_state +among O +eukaryotes B-taxonomy_domain +and O +archaea B-taxonomy_domain +( O +Supplementary O +Figure O +S1A O +) O +and O +its O +deletion O +leads O +to O +an O +accumulation O +of O +the O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +precursor O +of O +18S B-chemical +rRNA I-chemical +, O +suggesting O +an O +influence O +on O +D B-site +- I-site +site I-site +cleavage O +during O +the O +maturation O +of O +the O +small O +ribosomal O +subunit O +. O + +However O +, O +its O +function O +remained O +unclear O +although O +recently O +a O +putative O +nuclease O +function O +during O +18S B-chemical +rRNA I-chemical +maturation O +was O +predicted O +. O + +Here O +, O +we O +identify O +Tsr3 B-protein +as O +the O +long O +- O +sought O +acp B-protein_type +transferase I-protein_type +that O +catalyzes O +the O +last O +step O +in O +the O +biosynthesis O +of O +the O +hypermodified B-protein_state +nucleotide B-chemical +m1acp3Ψ B-chemical +in O +yeast B-taxonomy_domain +and O +human B-species +cells O +. O + +Furthermore O +using O +catalytically B-protein_state +defective I-protein_state +mutants O +of O +yeast B-taxonomy_domain +Tsr3 B-protein +we O +demonstrated O +that O +the O +acp B-chemical +modification O +is O +required O +for O +18S B-chemical +rRNA I-chemical +maturation O +. O + +Surprisingly O +, O +the O +crystal B-evidence +structures I-evidence +of O +archaeal B-taxonomy_domain +homologs O +revealed O +that O +Tsr3 B-protein +is O +structurally O +similar O +to O +the O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +methyltransferases I-protein_type +. O + +Interestingly O +, O +the O +two O +structurally O +very O +different O +enzymes O +use O +similar O +strategies O +in O +binding O +the O +SAM B-chemical +- O +cofactor O +in O +order O +to O +ensure O +that O +in O +contrast O +to O +methyltransferases B-protein_type +the O +acp B-chemical +and O +not O +the O +methyl O +group O +of O +SAM B-chemical +is O +transferred O +to O +the O +substrate O +. O + +Tsr3 B-protein +is O +the O +enzyme O +responsible O +for O +18S B-chemical +rRNA I-chemical +acp B-chemical +modification O +in O +yeast B-taxonomy_domain +and O +humans B-species + +For O +the O +Δtsr3 B-mutant +deletion O +strain O +the O +HPLC B-evidence +elution I-evidence +profile I-evidence +of O +18S B-chemical +rRNA I-chemical +nucleosides B-chemical +( O +Figure O +1B O +) O +was O +very O +similar O +to O +that O +of O +the O +pseudouridine B-protein_type +- I-protein_type +N1 I-protein_type +methyltransferase I-protein_type +mutant B-protein_state +Δnep1 B-mutant +, O +where O +a O +shoulder O +at O +∼ O +7 O +. O +4 O +min O +elution O +time O +was O +missing O +in O +the O +elution O +profile O +. O + +As O +previously O +reported O +this O +shoulder O +was O +identified O +by O +ESI B-experimental_method +- I-experimental_method +MS I-experimental_method +as O +corresponding O +to O +m1acp3Ψ B-chemical +. O + +In O +order O +to O +directly O +analyze O +the O +presence O +of O +the O +acp B-chemical +modification O +of O +nucleotide B-chemical +1191 B-residue_number +we O +used O +an O +in B-experimental_method +vivo14C I-experimental_method +incorporation I-experimental_method +assay I-experimental_method +with O +1 B-chemical +- I-chemical +14C I-chemical +- I-chemical +methionine I-chemical +. O + +Whereas O +the O +acp B-chemical +labeling O +of O +18S B-chemical +rRNA I-chemical +was O +clearly O +present O +in O +the O +wild B-protein_state +type I-protein_state +strain O +no O +radioactive O +labeling O +could O +be O +observed O +in O +a O +Δtsr3 B-mutant +strain O +( O +Figure O +1C O +). O + +As O +previously O +shown O +, O +only O +the O +acp B-chemical +but O +none O +of O +the O +other O +modifications O +at O +U1191 B-residue_name_number +of O +yeast B-taxonomy_domain +18S B-chemical +rRNA I-chemical +blocks O +reverse O +transcriptase O +activity O +. O + +Therefore O +the O +presence O +of O +the O +acp B-chemical +modification O +can O +be O +directly O +assessed O +by O +primer B-experimental_method +extension I-experimental_method +. O + +Indeed O +, O +in O +wild B-protein_state +- I-protein_state +type I-protein_state +yeast B-taxonomy_domain +a O +strong O +primer B-evidence +extension I-evidence +stop I-evidence +signal I-evidence +occurred O +at O +position O +1192 B-residue_number +. O + +In O +contrast O +, O +in O +a O +Δtsr3 B-mutant +mutant B-protein_state +no O +primer O +extension O +stop O +signal O +was O +present O +at O +this O +position O +. O + +As O +expected O +, O +in O +a O +Δsnr35 B-mutant +deletion B-experimental_method +preventing O +pseudouridylation B-ptm +and O +N1 B-ptm +- I-ptm +methylation I-ptm +( O +resulting O +in O +acp3U B-chemical +) O +as O +well O +as O +in O +a O +Δnep1 B-mutant +deletion O +strain O +where O +pseudouridine B-chemical +is O +not B-protein_state +methylated I-protein_state +( O +resulting O +in O +acp3Ψ B-chemical +) O +a O +primer B-evidence +extension I-evidence +stop I-evidence +signal I-evidence +of O +similar O +intensity O +as O +in O +the O +wild B-protein_state +type I-protein_state +was O +observed O +. O + +In O +a O +Δtsr3 B-mutant +Δsnr35 I-mutant +double O +deletion O +strain O +the O +18S B-chemical +rRNA I-chemical +contains O +an O +unmodified B-protein_state +U B-chemical +and O +the O +primer O +extension O +stop O +signal O +was O +missing O +( O +Figure O +1D O +). O + +Human B-species +18S B-chemical +rRNA I-chemical +has O +also O +been O +shown O +to O +contain O +m1acp3Ψ B-ptm +in O +the O +18S B-chemical +rRNA I-chemical +at O +position O +1248 B-residue_number +. O + +After O +siRNA B-experimental_method +- I-experimental_method +mediated I-experimental_method +depletion I-experimental_method +of O +Tsr3 B-protein +in O +human B-species +colon O +carcinoma O +HCT116 O +(+/+) O +cells O +the O +acp B-evidence +primer I-evidence +extension I-evidence +arrest I-evidence +was O +reduced O +in O +comparison O +to O +cells O +transfected O +with O +a O +non O +- O +targeting O +scramble O +siRNA B-chemical +control O +( O +Figure O +1E O +, O +compare O +lanes O +544 O +and O +scramble O +). O + +The O +efficiency O +of O +siRNA B-chemical +- O +mediated O +depletion O +was O +established O +by O +RT B-experimental_method +- I-experimental_method +qPCR I-experimental_method +and O +found O +to O +be O +very O +high O +with O +siRNA B-chemical +544 O +( O +Supplementary O +Figure O +S2A O +, O +remaining O +TSR3 B-protein +mRNA O +level O +of O +2 O +%). O + +Thus O +, O +HsTsr3 B-protein +is O +also O +responsible O +for O +the O +acp B-chemical +modification O +of O +18S B-chemical +rRNA I-chemical +nucleotide B-chemical +Ψ1248 B-ptm +in O +helix B-structure_element +31 I-structure_element +. O + +Phenotypic O +characterization O +of O +Δtsr3 B-mutant +mutants O + +However O +, O +the O +Δtsr3 B-mutant +deletion O +was O +synthetic O +sick O +with O +a O +Δsnr35 B-mutant +deletion O +preventing O +pseudouridylation B-ptm +and O +Nep1 B-protein +- O +catalyzed O +methylation O +of O +nucleotide O +1191 B-residue_number +( O +Figure O +2A O +). O + +Interestingly O +, O +no O +increased O +growth O +defect O +could O +be O +observed O +for O +Δtsr3 B-mutant +Δnep1 I-mutant +recombinants O +containing O +the O +nep1 B-gene +suppressor O +mutation O +Δnop6 B-mutant +as O +well O +as O +for O +Δtsr3 B-mutant +Δsnr35 I-mutant +Δnep1 I-mutant +recombinants O +with O +unmodified B-protein_state +U1191 B-residue_name_number +( O +Supplementary O +Figure O +S2D O +and O +E O +). O + +Phenotypic O +characterization O +of O +yeast B-taxonomy_domain +TSR3 B-protein +deletion O +( O +Δtrs3 B-mutant +) O +and O +human B-species +TSR3 B-protein +depletion O +( O +siRNAs B-chemical +544 O +and O +545 O +) O +and O +cellular O +localization O +of O +yeast B-taxonomy_domain +Tsr3 B-protein +. O +( O +A O +) O +Growth O +of O +yeast B-taxonomy_domain +wild B-protein_state +type I-protein_state +, O +Δtsr3 B-mutant +, O +Δsnr35 B-mutant +and O +Δtsr3 B-mutant +Δsnr35 I-mutant +segregants O +after O +meiosis O +and O +tetrad O +dissection O +of O +Δtsr3 B-mutant +/ O +TSR3 B-protein +Δsnr35 B-mutant +/ O +SNR35 B-protein +heterozygous O +diploids O +. O + +The O +Δtsr3 B-mutant +deletion O +is O +synthetic O +sick O +with O +a O +Δsnr35 B-mutant +deletion O +preventing O +U1191 B-residue_name_number +pseudouridylation O +. O + +( O +B O +) O +In O +agar B-experimental_method +diffusion I-experimental_method +assays I-experimental_method +the O +yeast B-taxonomy_domain +Δtsr3 B-mutant +deletion B-protein_state +mutant I-protein_state +shows O +a O +hypersensitivity O +against O +paromomycin B-chemical +and O +hygromycin B-chemical +B I-chemical +which O +is O +further O +increased O +by O +recombination O +with O +Δsnr35 B-mutant +. O +( O +C O +) O +Northern B-experimental_method +blot I-experimental_method +analysis I-experimental_method +with O +an O +ITS1 O +hybridization O +probe O +after O +siRNA B-experimental_method +depletion I-experimental_method +of O +HsTSR3 B-protein +( O +siRNAs B-chemical +544 O +and O +545 O +) O +and O +a O +scrambled O +siRNA B-chemical +as O +control O +. O + +The O +accumulation O +of O +18SE B-chemical +and O +47S B-chemical +and O +/ O +or O +45S B-chemical +pre I-chemical +- I-chemical +RNAs I-chemical +is O +enforced O +upon O +HsTSR3 B-protein +depletion O +. O + +Right O +gel O +: O +Ethidium O +bromide O +staining O +showing O +18S B-chemical +and O +28S B-chemical +rRNAs I-chemical +. O + +( O +D O +) O +Cytoplasmic O +localization O +of O +yeast B-taxonomy_domain +Tsr3 B-protein +shown O +by O +fluorescence B-experimental_method +microscopy I-experimental_method +of O +GFP B-mutant +- I-mutant +fused I-mutant +Tsr3 I-mutant +. O + +From O +left O +to O +right O +: O +differential B-experimental_method +interference I-experimental_method +contrast I-experimental_method +( O +DIC B-experimental_method +), O +green O +fluorescence O +of O +GFP B-mutant +- I-mutant +Tsr3 I-mutant +, O +red O +fluorescence O +of O +Nop56 B-mutant +- I-mutant +mRFP I-mutant +as O +nucleolar O +marker O +, O +and O +merge O +of O +GFP B-mutant +- I-mutant +Tsr3 I-mutant +/ O +Nop56 B-mutant +- I-mutant +mRFP I-mutant +with O +DIC B-experimental_method +. O +( O +E O +) O +Elution B-evidence +profile I-evidence +( O +A254 O +) O +after O +sucrose B-experimental_method +gradient I-experimental_method +separation I-experimental_method +of O +yeast B-taxonomy_domain +ribosomal B-complex_assembly +subunits I-complex_assembly +and O +polysomes B-complex_assembly +( O +upper O +part O +) O +and O +western B-experimental_method +blot I-experimental_method +analysis O +of O +3xHA B-chemical +tagged O +Tsr3 B-protein +( O +Tsr3 B-mutant +- I-mutant +3xHA I-mutant +) O +after O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +separation O +of O +polysome O +profile O +fractions O +taken O +every O +20 O +s O +( O +lower O +part O +). O + +The O +TSR3 B-protein +gene O +was O +genetically O +modified O +at O +its O +native O +locus O +, O +resulting O +in O +a O +C O +- O +terminal O +fusion B-protein_state +of O +Tsr3 B-protein +with O +a O +3xHA B-chemical +epitope O +expressed O +by O +the O +native O +promotor O +in O +yeast B-taxonomy_domain +strain O +CEN O +. O +BM258 O +- O +5B O +. O + +The O +influence O +of O +the O +acp B-chemical +modification O +of O +nucleotide B-chemical +1191 B-residue_number +on O +ribosome O +function O +was O +analyzed O +by O +treating O +Δtsr3 B-mutant +mutants O +with O +protein O +synthesis O +inhibitors O +. O + +A O +minor O +effect O +on O +20S B-chemical +rRNA I-chemical +accumulation O +was O +also O +observed O +for O +Δsnr35 B-mutant +, O +but O +- O +probably O +due O +to O +different O +strain O +backgrounds O +– O +to O +a O +weaker O +extent O +than O +described O +earlier O +. O + +In O +human B-species +cells O +, O +the O +depletion B-experimental_method +of I-experimental_method +HsTsr3 B-protein +in O +HCT116 O +(+/+) O +cells O +caused O +an O +accumulation O +of O +the O +human B-species +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +equivalent O +18S B-chemical +- I-chemical +E I-chemical +suggesting O +an O +evolutionary O +conserved O +role O +of O +Tsr3 B-protein +in O +the O +late O +steps O +of O +18S B-chemical +rRNA I-chemical +processing O +( O +Figure O +2C O +and O +Supplementary O +Figure O +S2B O +). O + +Surprisingly O +, O +early O +nucleolar O +processing O +reactions O +were O +also O +inhibited O +, O +and O +this O +was O +observed O +in O +both O +yeast B-taxonomy_domain +Δtsr3 B-mutant +cells O +( O +see O +accumulation O +of O +35S B-complex_assembly +in O +Supplementary O +Figure O +S2C O +) O +and O +Tsr3 B-protein +depleted O +human B-species +cells O +( O +see O +47S B-complex_assembly +/ O +45S B-complex_assembly +accumulation O +in O +Figure O +2C O +and O +Northern B-experimental_method +blot I-experimental_method +quantification O +in O +Supplementary O +Figure O +S2B O +). O + +Consistent O +with O +its O +role O +in O +late O +18S B-chemical +rRNA I-chemical +processing O +, O +TSR3 B-protein +deletion O +leads O +to O +a O +ribosomal O +subunit O +imbalance O +with O +a O +reduced O +40S B-complex_assembly +to O +60S B-complex_assembly +ratio O +of O +0 O +. O +81 O +( O +σ O += O +0 O +. O +024 O +) O +which O +was O +further O +increased O +in O +a O +Δtsr3 B-mutant +Δsnr35 I-mutant +recombinant O +to O +0 O +. O +73 O +( O +σ O += O +0 O +. O +023 O +) O +( O +Supplementary O +Figure O +S2F O +). O + +Cellular O +localization O +of O +Tsr3 B-protein +in O +S B-species +. I-species +cerevisiae I-species + +Fluorescence B-experimental_method +microscopy I-experimental_method +of O +GFP B-protein_state +- I-protein_state +tagged I-protein_state +Tsr3 B-protein +localized O +the O +fusion O +protein O +in O +the O +cytoplasm O +of O +yeast B-taxonomy_domain +cells O +and O +no O +co O +- O +localization O +with O +the O +nucleolar O +marker O +protein O +Nop56 B-protein +could O +be O +observed O +( O +Figure O +2D O +). O + +This O +agrees O +with O +previous O +biochemical O +data O +suggesting O +that O +the O +acp B-chemical +modification O +of O +18S B-chemical +rRNA I-chemical +occurs O +late O +during O +40S B-complex_assembly +subunit O +biogenesis O +in O +the O +cytoplasm O +, O +and O +makes O +an O +additional O +nuclear O +localization O +as O +reported O +in O +a O +previous O +large O +- O +scale O +analysis O +unlikely O +. O + +After O +polysome B-experimental_method +gradient I-experimental_method +separation I-experimental_method +C O +- O +terminally O +epitope O +- O +labeled O +Tsr3 B-mutant +- I-mutant +3xHA I-mutant +was O +exclusively O +detectable O +in O +the O +low O +- O +density O +fraction O +( O +Figure O +2E O +). O + +Such O +distribution B-evidence +on I-evidence +a I-evidence +density I-evidence +gradient I-evidence +suggests O +that O +Tsr3 B-protein +only O +interacts O +transiently O +with O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +subunits I-complex_assembly +, O +which O +presumably O +explains O +why O +it O +was O +not O +characterized O +in O +pre B-experimental_method +- I-experimental_method +ribosome I-experimental_method +affinity I-experimental_method +purifications I-experimental_method +. O + +Searches O +for O +sequence O +homologs O +of O +S B-species +. I-species +cerevisiae I-species +Tsr3 B-protein +( O +ScTsr3 B-protein +) O +by O +us O +and O +others O +revealed O +that O +the O +genomes O +of O +many O +archaea B-taxonomy_domain +contain O +genes O +encoding O +Tsr3 B-protein_type +- I-protein_type +like I-protein_type +proteins I-protein_type +. O + +To O +locate O +the O +domains O +most O +important O +for O +Tsr3 B-protein +activity O +, O +ScTsr3 B-protein +fragments O +of O +different O +lengths O +containing O +the O +highly B-protein_state +conserved I-protein_state +central O +part O +were O +expressed B-experimental_method +in O +a O +Δtsr3 B-mutant +mutant B-protein_state +( O +Figure O +3A O +) O +and O +analyzed O +by O +primer B-experimental_method +extension I-experimental_method +( O +Figure O +3B O +) O +and O +Northern B-experimental_method +blotting I-experimental_method +( O +Figure O +3C O +). O + +Domain O +characterization O +of O +yeast B-taxonomy_domain +Tsr3 B-protein +and O +correlation O +of O +acp B-chemical +modification O +with O +late O +18S B-chemical +rRNA I-chemical +processing O +steps O +. O +( O +A O +) O +Scheme O +of O +the O +TSR3 B-protein +gene O +with O +truncation O +positions O +in O +the O +open O +reading O +frame O +. O + +TSR3 B-protein +fragments O +of O +different O +length O +were O +expressed O +under O +the O +native O +promotor O +from O +multicopy O +plasmids O +in O +a O +Δtsr3 B-mutant +deletion O +strain O +. O + +N O +- O +terminal O +deletions B-experimental_method +of O +36 B-residue_range +or O +45 B-residue_range +amino O +acids O +and O +C O +- O +terminal O +deletions B-experimental_method +of O +43 B-residue_range +or O +76 B-residue_range +residues O +show O +a O +primer B-evidence +extension I-evidence +stop I-evidence +comparable O +to O +the O +wild B-protein_state +type I-protein_state +. O + +Tsr3 B-protein +fragments O +37 B-residue_range +– I-residue_range +223 I-residue_range +or O +46 B-residue_range +– I-residue_range +223 I-residue_range +cause O +a O +nearly O +complete O +loss O +of O +the O +arrest O +signal O +. O + +The O +box O +highlights O +the O +shortest O +Tsr3 B-protein +fragment O +( O +aa O +46 B-residue_range +– I-residue_range +270 I-residue_range +) O +with O +wild B-protein_state +type I-protein_state +activity O +( O +strong O +primer B-evidence +extension I-evidence +block I-evidence +). O +( O +C O +) O +Northern B-experimental_method +blot I-experimental_method +analysis O +of O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +accumulation O +. O + +A O +weak O +20S B-chemical +rRNA I-chemical +signal O +, O +indicating O +normal O +processing O +, O +is O +observed O +for O +Tsr3 B-protein +fragment O +46 B-residue_range +– I-residue_range +270 I-residue_range +( O +highlighted O +in O +a O +box O +) O +showing O +its O +functionality O +. O + +Strong O +20S O +rRNA O +accumulation O +similar O +to O +that O +of O +the O +Δtsr3 B-mutant +deletion B-experimental_method +is O +observed O +for O +Tsr3 B-protein +fragments O +37 B-residue_range +– I-residue_range +223 I-residue_range +or O +46 B-residue_range +– I-residue_range +223 I-residue_range +. O + +Thus O +, O +the O +archaeal B-taxonomy_domain +homologs O +correspond O +to O +the O +functional O +core O +of O +Tsr3 B-protein +. O + +In O +order O +to O +define O +the O +structural O +basis O +for O +Tsr3 B-protein +function O +, O +homologs O +from O +thermophilic B-taxonomy_domain +archaea I-taxonomy_domain +were O +screened O +for O +crystallization B-experimental_method +. O + +We O +focused O +on O +archaeal B-taxonomy_domain +species O +containing O +a O +putative O +Nep1 B-protein +homolog O +suggesting O +that O +these O +species O +are O +in O +principle O +capable O +of O +synthesizing O +N1 B-chemical +- I-chemical +methyl I-chemical +- I-chemical +N3 I-chemical +- I-chemical +acp I-chemical +- I-chemical +pseudouridine I-chemical +. O + +Well O +diffracting O +crystals B-evidence +were O +obtained O +for O +Tsr3 B-protein +homologs O +from O +the O +two O +crenarchaeal B-taxonomy_domain +species O +Vulcanisaeta B-species +distributa I-species +( O +VdTsr3 B-protein +) O +and O +Sulfolobus B-species +solfataricus I-species +( O +SsTsr3 B-protein +) O +which O +share O +36 O +% O +( O +VdTsr3 B-protein +) O +and O +38 O +% O +( O +SsTsr3 B-protein +) O +identity O +with O +the O +ScTsr3 B-protein +core B-structure_element +region I-structure_element +( O +ScTsr3 B-protein +aa O +46 B-residue_range +– I-residue_range +223 I-residue_range +). O + +Crystals B-evidence +of O +VdTsr3 B-protein +diffracted O +to O +a O +resolution O +of O +1 O +. O +6 O +Å O +whereas O +crystals B-evidence +of O +SsTsr3 B-protein +diffracted O +to O +2 O +. O +25 O +Å O +. O +Serendipitously O +, O +VdTsr3 B-protein +was O +purified O +and O +crystallized B-experimental_method +in B-protein_state +complex I-protein_state +with I-protein_state +endogenous B-protein_state +( O +E B-species +. I-species +coli I-species +) O +SAM B-chemical +( O +Supplementary O +Figure O +S4 O +) O +while O +SsTsr3 B-protein +crystals B-evidence +contained O +the O +protein O +in O +the O +apo B-protein_state +state O +. O + +The O +structure B-evidence +of O +SsTsr3 B-protein +was O +solved O +by O +molecular B-experimental_method +replacement I-experimental_method +using O +VdTsr3 B-protein +as O +a O +search O +model O +( O +see O +Supplementary O +Table O +S1 O +for O +data O +collection O +and O +refinement O +statistics O +). O + +The O +structure B-evidence +of O +VdTsr3 B-protein +can O +be O +divided O +into O +two O +domains O +( O +Figure O +4A O +). O + +The O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +aa O +1 B-residue_range +– I-residue_range +92 I-residue_range +) O +has O +a O +mixed O +α B-structure_element +/ I-structure_element +β I-structure_element +- I-structure_element +structure I-structure_element +centered O +around O +a O +five B-structure_element +- I-structure_element +stranded I-structure_element +all I-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +( O +Figure O +4B O +) O +with O +the O +strand O +order O +β5 B-structure_element +↑- I-structure_element +β3 B-structure_element +↑- I-structure_element +β4 B-structure_element +↑- I-structure_element +β1 B-structure_element +↑- I-structure_element +β2 B-structure_element +↑. I-structure_element +The O +loops B-structure_element +connecting O +β1 B-structure_element +and O +β2 B-structure_element +, O +β3 B-structure_element +and O +β4 B-structure_element +and O +β4 B-structure_element +and O +β5 B-structure_element +include O +α B-structure_element +- I-structure_element +helices I-structure_element +α1 B-structure_element +, O +α2 B-structure_element +and O +α3 B-structure_element +, O +respectively O +. O + +The O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +aa O +93 B-residue_range +– I-residue_range +184 I-residue_range +) O +has O +a O +globular B-structure_element +all I-structure_element +α I-structure_element +- I-structure_element +helical I-structure_element +structure I-structure_element +comprising O +α B-structure_element +- I-structure_element +helices I-structure_element +α4 B-structure_element +to I-structure_element +α9 I-structure_element +. O + +Remarkably O +, O +the O +entire O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +92 B-residue_range +aa I-residue_range +) O +of O +the O +protein O +is O +threaded O +through O +the O +loop B-structure_element +which O +connects O +β B-structure_element +- I-structure_element +strand I-structure_element +β3 B-structure_element +and O +α B-structure_element +- I-structure_element +helix I-structure_element +α2 B-structure_element +of O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +. O + +The O +structure B-evidence +of O +SsTsr3 B-protein +in O +the O +apo B-protein_state +state O +is O +very O +similar O +to O +that O +of O +VdTsr3 B-protein +( O +Figure O +4C O +) O +with O +an O +RMSD B-evidence +for O +equivalent O +Cα O +atoms O +of O +1 O +. O +1 O +Å O +. O +The O +only O +significant O +difference O +in O +the O +global O +structure B-evidence +of O +the O +two O +proteins O +is O +the O +presence O +of O +an O +extended O +α B-structure_element +- I-structure_element +helix I-structure_element +α8 B-structure_element +and O +the O +absence B-protein_state +of I-protein_state +α B-structure_element +- I-structure_element +helix I-structure_element +α9 B-structure_element +in O +SsTsr3 B-protein +. O + +Tsr3 B-protein +has O +a O +fold O +similar O +to O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +methyltransferases I-protein_type +. O +( O +A O +) O +Cartoon O +representation O +of O +the O +X B-evidence +- I-evidence +ray I-evidence +structure I-evidence +of O +VdTsr3 B-protein +in O +two O +orientations O +. O + +The O +bound O +S B-chemical +- I-chemical +adenosylmethionine I-chemical +is O +shown O +in O +a O +stick O +representation O +and O +colored O +by O +atom O +type O +. O + +The O +color O +coding O +is O +the O +same O +as O +in O +( O +A O +). O +( O +C O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +X B-evidence +- I-evidence +ray I-evidence +structures I-evidence +of O +VdTsr3 B-protein +in O +the O +SAM B-protein_state +- I-protein_state +bound I-protein_state +state O +( O +red O +) O +and O +SsTsr3 B-protein +( O +blue O +) O +in O +the O +apo B-protein_state +state O +. O + +The O +locations O +of O +the O +α B-structure_element +- I-structure_element +helix I-structure_element +α8 B-structure_element +which O +is O +longer O +in O +SsTsr3 B-protein +and O +of O +α B-structure_element +- I-structure_element +helix I-structure_element +α9 B-structure_element +which O +is O +only O +present O +in O +VdTsr3 B-protein +are O +indicated O +. O +( O +D O +) O +Secondary O +structure O +cartoon O +( O +left O +) O +of O +S B-species +. I-species +pombe I-species +Trm10 B-protein +( O +pdb4jwf O +)— O +the O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +methyltransferase I-protein_type +structurally O +most O +similar O +to O +Tsr3 B-protein +and O +superposition B-experimental_method +of O +the O +VdTsr3 B-protein +and O +Trm10 B-protein +X B-evidence +- I-evidence +ray I-evidence +structures I-evidence +( O +right O +). O +( O +E O +) O +Analytical B-experimental_method +gel I-experimental_method +filtration I-experimental_method +profiles B-evidence +for O +VdTsr3 B-protein +( O +red O +) O +and O +SsTsr3 B-protein +( O +blue O +) O +show O +that O +both O +proteins O +are O +monomeric B-oligomeric_state +in O +solution O +. O + +Vd B-species +, O +Vulcanisaeta B-species +distributa I-species +; O +Ss B-species +, O +Sulfolobus B-species +solfataricus I-species +. O + +However O +, O +no O +structural O +similarity O +to O +an O +RLI B-structure_element +- I-structure_element +domain I-structure_element +was O +detectable O +. O + +This O +is O +in O +accordance O +with O +the O +functional O +analysis O +of O +alanine B-experimental_method +replacement I-experimental_method +mutations I-experimental_method +of O +cysteine B-residue_name +residues O +in O +ScTsr3 B-protein +( O +Supplementary O +Figure O +S3 O +). O + +The O +β B-structure_element +- I-structure_element +strand I-structure_element +topology I-structure_element +and O +the O +deep O +C O +- O +terminal O +trefoil B-structure_element +knot I-structure_element +of O +archaeal B-taxonomy_domain +Tsr3 B-protein +are O +the O +structural O +hallmarks O +of O +the O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +- I-protein_type +methyltransferase I-protein_type +fold O +. O + +In O +comparison O +to O +Tsr3 B-protein +the O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +element I-structure_element +of O +Trm10 B-protein +is O +extended O +by O +one O +additional O +β B-structure_element +- I-structure_element +strand I-structure_element +pairing O +to O +β2 B-structure_element +. O + +Furthermore O +, O +the O +trefoil B-structure_element +knot I-structure_element +of O +Trm10 B-protein +is O +not O +as O +deep O +as O +that O +of O +Tsr3 B-protein +( O +Figure O +4D O +). O + +Interestingly O +, O +Nep1 B-protein +— O +the O +enzyme O +preceding O +Tsr3 B-protein +in O +the O +biosynthetic O +pathway O +for O +the O +synthesis O +of O +m1acp3Ψ B-chemical +— O +also O +belongs O +to O +the O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +of I-protein_type +RNA I-protein_type +methyltransferases I-protein_type +. O + +However O +, O +the O +structural O +similarities O +between O +Nep1 B-protein +and O +Tsr3 B-protein +( O +DALI B-evidence +Z I-evidence +- I-evidence +score I-evidence +4 O +. O +4 O +) O +are O +less O +pronounced O +than O +between O +Tsr3 B-protein +and O +Trm10 B-protein +. O + +Most O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +- I-protein_type +methyltransferases I-protein_type +are O +homodimers B-oligomeric_state +. O + +So O +far O +, O +structural O +information O +is O +only O +available O +for O +one O +other O +enzyme O +that O +transfers O +the O +acp B-chemical +group O +from O +SAM B-chemical +to O +an O +RNA B-chemical +nucleotide B-chemical +. O + +Cofactor O +binding O +of O +Tsr3 B-protein + +The O +SAM B-site +- I-site +binding I-site +site I-site +of O +Tsr3 B-protein +is O +located O +in O +a O +deep O +crevice O +between O +the O +N B-structure_element +- I-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +domains I-structure_element +in O +the O +vicinity O +of O +the O +trefoil B-structure_element +knot I-structure_element +as O +typical O +for O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +- I-protein_type +methyltransferases I-protein_type +( O +Figure O +4A O +). O + +The O +adenine B-chemical +base O +of O +the O +cofactor O +is O +recognized O +by O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +its O +N1 O +nitrogen O +and O +the O +backbone O +amide O +of O +L93 B-residue_name_number +directly O +preceding O +β5 B-structure_element +as O +well O +as O +between O +its O +N6 O +- O +amino O +group O +and O +the O +backbone O +carbonyl O +group O +of O +Y108 B-residue_name_number +located O +in O +the O +loop B-structure_element +connecting O +β5 B-structure_element +in O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +and O +α4 B-structure_element +in O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +Figure O +5A O +). O + +Furthermore O +, O +the O +adenine B-chemical +base O +of O +SAM B-chemical +is O +involved O +in O +hydrophobic B-bond_interaction +packing I-bond_interaction +interactions I-bond_interaction +with O +the O +side O +chains O +of O +L45 B-residue_name_number +( O +β3 B-structure_element +), O +P47 B-residue_name_number +and O +W73 B-residue_name_number +( O +α3 B-structure_element +) O +in O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +as O +well O +as O +with O +L93 B-residue_name_number +, O +L110 B-residue_name_number +( O +both O +in O +the O +loop B-structure_element +connecting O +β5 B-structure_element +and O +α4 B-structure_element +) O +and O +A115 B-residue_name_number +( O +α5 B-structure_element +) O +in O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +. O + +The O +ribose B-chemical +2 O +′ O +and O +3 O +′ O +hydroxyl O +groups O +of O +SAM B-chemical +are O +hydrogen B-bond_interaction +bonded I-bond_interaction +to O +the O +backbone O +carbonyl O +group O +of O +I69 B-residue_name_number +. O + +The O +acp B-chemical +side O +chain O +of O +SAM B-chemical +is O +fixed O +in O +position O +by O +hydrogen B-bond_interaction +bonding I-bond_interaction +of O +its O +carboxylate O +group O +to O +the O +backbone O +amide O +and O +the O +side O +chain O +hydroxyl O +group O +of O +T19 B-residue_name_number +in O +α1 B-structure_element +as O +well O +as O +the O +backbone O +amide O +group O +of O +T112 B-residue_name_number +in O +α4 B-structure_element +( O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +). O + +Most O +importantly O +, O +the O +methyl O +group O +of O +SAM B-chemical +is O +buried O +in O +a O +hydrophobic B-site +pocket I-site +formed O +by O +the O +sidechains O +of O +W73 B-residue_name_number +and O +A76 B-residue_name_number +both O +located O +in O +α3 B-structure_element +( O +Figure O +5A O +and O +B O +). O + +W73 B-residue_name_number +is O +highly B-protein_state +conserved I-protein_state +in O +all O +known O +Tsr3 B-protein_type +proteins I-protein_type +, O +whereas O +A76 B-residue_name_number +can O +be O +replaced O +by O +other O +hydrophobic O +amino B-chemical +acids I-chemical +. O + +Consequently O +, O +the O +accessibility O +of O +this O +methyl O +group O +for O +a O +nucleophilic O +attack O +is O +strongly O +reduced O +in O +comparison O +with O +RNA B-protein_type +- I-protein_type +methyltransferases I-protein_type +such O +as O +Trm10 B-protein +( O +Figure O +5B O +, O +C O +). O + +In O +contrast O +, O +the O +acp B-chemical +side O +chain O +of O +SAM B-chemical +is O +accessible O +for O +reactions O +in O +the O +Tsr3 B-protein_state +- I-protein_state +bound I-protein_state +state O +( O +Figure O +5B O +). O + +( O +A O +) O +Close O +- O +up O +view O +of O +the O +SAM B-site +- I-site +binding I-site +pocket I-site +of O +VdTsr3 B-protein +. O + +Nitrogen O +atoms O +are O +dark O +blue O +, O +oxygen O +atoms O +red O +, O +sulfur B-chemical +atoms O +orange O +, O +carbon O +atoms O +of O +the O +protein O +light O +blue O +and O +carbon O +atoms O +of O +SAM B-chemical +yellow O +. O + +Hydrogen B-bond_interaction +bonds I-bond_interaction +are O +indicated O +by O +dashed O +lines O +. O + +( O +B O +) O +Solvent O +accessibility O +of O +the O +acp B-chemical +group O +of O +SAM B-chemical +bound B-protein_state +to I-protein_state +VdTsr3 B-protein +. O + +The O +solvent O +accessible O +surface O +of O +the O +protein O +is O +shown O +in O +semitransparent O +gray O +whereas O +SAM B-chemical +is O +show O +in O +a O +stick O +representation O +. O + +A O +red O +arrow O +indicates O +the O +reactive O +CH2 O +- O +moiety O +of O +the O +acp B-chemical +group O +. O +( O +C O +) O +Solvent O +accessibility O +of O +the O +SAM B-chemical +methyl O +group O +for O +SAM B-chemical +bound B-protein_state +to I-protein_state +the O +RNA B-protein_type +methyltransferase I-protein_type +Trm10 B-protein +. O + +A O +red O +arrow O +indicates O +the O +SAM B-chemical +methyl O +group O +. O +( O +D O +) O +Binding O +of O +SAM B-chemical +analogs O +to O +SsTsr3 B-protein +. O + +Tryptophan B-evidence +fluorescence I-evidence +quenching I-evidence +curves I-evidence +upon O +addition O +of O +SAM B-chemical +( O +blue O +), O +5 B-chemical +′- I-chemical +methyl I-chemical +- I-chemical +thioadenosine I-chemical +( O +red O +) O +and O +SAH B-chemical +( O +black O +). O + +( O +E O +) O +Binding O +of O +14C B-chemical +- I-chemical +labeled I-chemical +SAM I-chemical +to O +SsTsr3 B-protein +. O + +Radioactively O +labeled O +SAM B-chemical +is O +retained O +on O +a O +filter O +in O +the O +presence B-protein_state +of I-protein_state +SsTsr3 B-protein +. O + +Addition O +of O +unlabeled O +SAM B-chemical +competes O +with O +the O +binding O +of O +labeled O +SAM B-chemical +. O + +This O +correlates O +with O +a O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +accumulation O +comparable O +to O +the O +Δtsr3 B-mutant +deletion O +( O +right O +: O +northern B-experimental_method +blot I-experimental_method +). O + +Binding B-evidence +affinities I-evidence +for O +SAM B-chemical +and O +its O +analogs O +5 B-chemical +′- I-chemical +methylthioadenosin I-chemical +and O +SAH B-chemical +to O +SsTsr3 B-protein +were O +measured O +using O +tryptophan B-experimental_method +fluorescence I-experimental_method +quenching I-experimental_method +. O + +SsTsr3 B-protein +bound B-protein_state +SAM B-chemical +with O +a O +KD B-evidence +of O +6 O +. O +5 O +μM O +, O +which O +is O +similar O +to O +SAM B-evidence +- I-evidence +KD I-evidence +' I-evidence +s I-evidence +reported O +for O +several O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +methyltransferases I-protein_type +. O + +5 B-chemical +′- I-chemical +methylthioadenosin I-chemical +— O +the O +reaction O +product O +after O +the O +acp B-chemical +- O +transfer O +— O +binds O +only O +∼ O +2 O +. O +5 O +- O +fold O +weaker O +( O +KD O += O +16 O +. O +7 O +μM O +) O +compared O +to O +SAM B-chemical +. O + +This O +suggests O +that O +the O +hydrophobic B-bond_interaction +interaction I-bond_interaction +between O +SAM B-chemical +' O +s O +methyl O +group O +and O +the O +hydrophobic B-site +pocket I-site +of O +Tsr3 B-protein +is O +thermodynamically O +important O +for O +the O +interaction O +. O + +On O +the O +other O +hand O +, O +the O +loss O +of O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +the O +acp B-chemical +sidechain O +carboxylate O +group O +and O +the O +protein O +appears O +to O +be O +thermodynamically O +less O +important O +but O +these O +hydrogen B-bond_interaction +bonds I-bond_interaction +might O +play O +a O +crucial O +role O +for O +the O +proper O +orientation O +of O +the O +cofactor O +side O +chain O +in O +the O +substrate B-site +binding I-site +pocket I-site +. O + +Accordingly O +, O +a O +W66A B-mutant +- O +mutation B-experimental_method +( O +W73 B-residue_name_number +in O +VdTsr3 B-protein +) O +of O +SsTsr3 B-protein +significantly O +diminished O +SAM B-evidence +- I-evidence +binding I-evidence +in O +a O +filter B-experimental_method +binding I-experimental_method +assay I-experimental_method +compared O +to O +the O +wild B-protein_state +type I-protein_state +( O +Figure O +5E O +). O + +Furthermore O +, O +a O +W B-experimental_method +to I-experimental_method +A I-experimental_method +mutation I-experimental_method +at O +the O +equivalent O +position O +W114 B-residue_name_number +in O +ScTsr3 B-protein +strongly O +reduced O +the O +in O +vivo O +acp B-protein_type +transferase I-protein_type +activity O +( O +Figure O +5F O +). O + +The O +side O +chain O +hydroxyl O +group O +of O +T19 B-residue_name_number +seems O +of O +minor O +importance O +for O +SAM B-chemical +binding O +since O +mutations B-experimental_method +of O +T17 B-residue_name_number +( O +T19 B-residue_name_number +in O +VdTsr3 B-protein +) O +to O +either O +A B-residue_name +or O +D B-residue_name +did O +not O +significantly O +influence O +the O +SAM B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +SsTsr3 B-protein +( O +KD B-evidence +' O +s O += O +3 O +. O +9 O +or O +11 O +. O +2 O +mM O +, O +respectively O +). O + +Nevertheless O +, O +a O +mutation B-experimental_method +of O +the O +equivalent O +position O +S62 B-residue_name_number +of O +ScTsr3 B-protein +to O +D B-residue_name +, O +but O +not O +to O +A B-residue_name +, O +resulted O +in O +reduced O +acp B-chemical +modification O +in O +vivo O +, O +as O +shown O +by O +primer B-experimental_method +extension I-experimental_method +analysis I-experimental_method +( O +Figure O +5F O +). O + +The O +acp B-chemical +- O +transfer O +reaction O +catalyzed O +by O +Tsr3 B-protein +most O +likely O +requires O +the O +presence O +of O +a O +catalytic O +base O +in O +order O +to O +abstract O +a O +proton O +from O +the O +N3 O +imino O +group O +of O +the O +modified O +pseudouridine B-chemical +. O + +This O +residue O +is O +conserved B-protein_state +as I-protein_state +D B-residue_name +or O +E B-residue_name +both O +in O +archaeal B-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +Tsr3 B-protein +homologs O +. O + +Mutations B-experimental_method +of O +the O +corresponding O +residue O +in O +SsTsr3 B-protein +to O +A B-residue_name +( O +D63 B-residue_name_number +) O +does O +not O +significantly O +alter O +the O +SAM B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +protein O +( O +KD B-evidence += O +11 O +. O +0 O +μM O +). O + +RNA B-chemical +- O +binding O +of O +Tsr3 B-protein + +Analysis B-experimental_method +of I-experimental_method +the I-experimental_method +electrostatic I-experimental_method +surface I-experimental_method +properties I-experimental_method +of O +VdTsr3 B-protein +clearly O +identified O +positively B-site +charged I-site +surface I-site +patches I-site +in O +the O +vicinity O +of O +the O +SAM B-site +- I-site +binding I-site +site I-site +suggesting O +a O +putative O +RNA B-site +- I-site +binding I-site +site I-site +( O +Figure O +6A O +). O + +Furthermore O +, O +a O +negatively O +charged O +MES B-chemical +- O +ion O +is O +found O +in O +the O +crystal B-evidence +structure I-evidence +of O +VdTsr3 B-protein +complexed B-protein_state +to I-protein_state +the O +side O +chain O +of O +K22 B-residue_name_number +in O +helix B-structure_element +α1 B-structure_element +. O + +Its O +negatively O +charged O +sulfate B-chemical +group O +might O +mimic O +an O +RNA B-chemical +backbone O +phosphate O +. O + +Helix B-structure_element +α1 B-structure_element +contains O +two O +more O +positively O +charged O +amino O +acids O +K17 B-residue_name_number +and O +R25 B-residue_name_number +as O +does O +the O +loop B-structure_element +preceding O +it O +( O +R9 B-residue_name_number +). O + +Some O +of O +these O +amino O +acids O +are O +conserved B-protein_state +between O +archaeal B-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +Tsr3 B-protein +( O +Supplementary O +Figure O +S1A O +). O + +RNA O +- O +binding O +of O +Tsr3 B-protein +. O + +( O +A O +) O +Electrostatic O +charge O +distribution O +on O +the O +surface O +of O +VdTsr3 B-protein +. O + +SAM B-chemical +is O +shown O +in O +a O +stick O +representation O +. O + +Also O +shown O +in O +stick O +representation O +is O +a O +negatively O +charged O +MES B-chemical +ion O +. O + +Conserved B-protein_state +basic O +amino B-chemical +acids I-chemical +are O +labeled O +. O +( O +B O +) O +Comparison O +of O +the O +secondary O +structures O +of O +helix B-structure_element +31 I-structure_element +from O +the O +small O +ribosomal O +subunit O +rRNAs B-chemical +in O +S B-species +. I-species +cerevisiae I-species +and O +S B-species +. I-species +solfataricus I-species +with O +the O +location O +of O +the O +hypermodified B-protein_state +nucleotide B-chemical +indicated O +in O +red O +. O + +( O +C O +) O +Binding O +of O +SsTsr3 B-protein +to O +RNA B-chemical +. O + +5 O +′- O +fluoresceine B-chemical +labeled O +RNA B-chemical +oligonucleotides O +corresponding O +either O +to O +the O +native B-protein_state +( O +20mer B-oligomeric_state +– O +see O +inset O +) O +or O +a O +stabilized B-protein_state +( O +20mer_GC B-oligomeric_state +- O +inset O +) O +helix B-structure_element +31 I-structure_element +of O +the O +small O +ribosomal O +subunit O +rRNA B-chemical +from O +S B-species +. I-species +solfataricus I-species +were O +titrated B-experimental_method +with I-experimental_method +increasing I-experimental_method +amounts I-experimental_method +of O +SsTsr3 B-protein +and O +the O +changes O +in O +the O +fluoresceine B-chemical +fluorescence B-evidence +anisotropy I-evidence +were O +measured O +and O +fitted O +to O +a O +binding B-evidence +curve I-evidence +( O +20mer B-oligomeric_state +– O +red O +, O +20mer_GC B-oligomeric_state +– O +blue O +). O + +Oligo B-chemical +- I-chemical +U9 I-chemical +- I-chemical +RNA I-chemical +was O +used O +for O +comparison O +( O +black O +). O + +The O +20mer_GC B-oligomeric_state +RNA B-chemical +was O +also O +titrated B-experimental_method +with O +SsTsr3 B-protein +in O +the O +presence O +of O +2 O +mM O +SAM B-chemical +( O +purple O +). O +( O +D O +) O +Mutants B-protein_state +of O +ScTsr3 B-protein +R60 B-residue_name_number +, O +K65 B-residue_name_number +or O +R131 B-residue_name_number +( O +equivalent O +to O +K17 B-residue_name_number +, O +K22 B-residue_name_number +and O +R91 B-residue_name_number +in O +VdTsr3 B-protein +) O +expressed B-experimental_method +in O +Δtsr3 B-mutant +yeast B-taxonomy_domain +cells O +show O +a O +primer B-evidence +extension I-evidence +stop I-evidence +comparable O +to O +the O +wild B-protein_state +type I-protein_state +. O + +Combination B-experimental_method +of I-experimental_method +the I-experimental_method +three I-experimental_method +point I-experimental_method +mutations I-experimental_method +( O +R60A B-mutant +/ O +K65A B-mutant +/ O +R131A B-mutant +) O +leads O +to O +a O +strongly O +reduced O +acp B-chemical +modification O +of O +18S B-chemical +rRNA I-chemical +. O + +In O +order O +to O +explore O +the O +RNA O +- O +ligand O +specificity O +of O +Tsr3 B-protein +we O +titrated B-experimental_method +SsTsr3 B-protein +prepared O +in O +RNase B-protein_state +- I-protein_state +free I-protein_state +form O +with O +5 O +′- O +fluoresceine B-chemical +- O +labeled O +RNA B-chemical +and O +determined O +the O +affinity B-evidence +by O +fluorescence B-experimental_method +anisotropy I-experimental_method +measurements I-experimental_method +. O + +SsTsr3 B-protein +in O +the O +apo B-protein_state +state O +bound B-protein_state +a O +20mer B-oligomeric_state +RNA B-chemical +corresponding O +to O +helix B-structure_element +31 I-structure_element +of O +S B-species +. I-species +solfataricus I-species +16S B-chemical +rRNA I-chemical +( O +Figure O +6B O +) O +with O +a O +KD B-evidence +of O +1 O +. O +9 O +μM O +and O +to O +a O +version O +of O +this O +hairpin B-structure_element +stabilized O +by O +additional O +GC O +base O +pairs O +( O +20mer B-oligomeric_state +- I-oligomeric_state +GC I-oligomeric_state +) O +with O +a O +KD B-evidence +of O +0 O +. O +6 O +μM O +( O +Figure O +6C O +). O + +A O +single O +stranded O +oligoU B-chemical +- I-chemical +RNA I-chemical +bound B-protein_state +with O +a O +10 O +- O +fold O +- O +reduced O +affinity B-evidence +( O +6 O +. O +0 O +μM O +). O + +The O +presence O +of O +saturating O +amounts O +of O +SAM B-chemical +( O +2 O +mM O +) O +did O +not O +have O +a O +significant O +influence O +on O +the O +RNA B-evidence +- I-evidence +affinity I-evidence +of O +SsTsr3 B-protein +( O +KD B-evidence +of O +1 O +. O +7 O +μM O +for O +the O +20mer B-oligomeric_state +- I-oligomeric_state +GC I-oligomeric_state +- O +RNA B-chemical +) O +suggesting O +no O +cooperativity O +in O +substrate O +binding O +. O + +U1191 B-residue_name_number +is O +the O +only O +hypermodified B-protein_state +base O +in O +the O +yeast B-taxonomy_domain +18S B-chemical +rRNA I-chemical +and O +is O +strongly B-protein_state +conserved I-protein_state +in O +eukaryotes B-taxonomy_domain +. O + +Unexpectedly O +, O +archaeal B-taxonomy_domain +Tsr3 B-protein +has O +a O +structure B-evidence +similar O +to O +SPOUT B-protein_type +- I-protein_type +class I-protein_type +RNA I-protein_type +methyltransferases I-protein_type +, O +and O +it O +is O +the O +first O +example O +for O +an O +enzyme O +of O +this O +class O +transferring O +an O +acp B-chemical +group O +, O +due O +to O +a O +modified O +SAM B-site +- I-site +binding I-site +pocket I-site +that O +exposes O +the O +acp B-chemical +instead O +of O +the O +methyl O +group O +of O +SAM B-chemical +to O +its O +RNA B-chemical +substrate O +. O + +Similar O +to O +the O +structurally O +unrelated O +Rossmann B-protein_type +- I-protein_type +fold I-protein_type +Tyw2 I-protein_type +acp I-protein_type +transferase I-protein_type +, O +the O +SAM B-chemical +methyl O +group O +of O +Tsr3 B-protein +is O +bound O +in O +an O +inaccessible O +hydrophobic B-site +pocket I-site +whereas O +the O +acp B-chemical +side O +chain O +becomes O +accessible O +for O +a O +nucleophilic O +attack O +by O +the O +N3 O +of O +pseudouridine B-chemical +. O + +Thus O +, O +additional O +examples O +for O +acp B-protein_type +transferase I-protein_type +enzymes O +might O +be O +found O +with O +similarities O +to O +other O +structural O +classes O +of O +methyltransferases B-protein_type +. O + +In O +contrast O +to O +Nep1 B-protein +, O +the O +enzyme O +preceding O +Tsr3 B-protein +in O +the O +m1acp3Ψ B-chemical +biosynthesis O +pathway O +, O +Tsr3 B-protein +binds O +rather O +weakly O +and O +with O +little O +specificity O +to O +its O +isolated O +substrate O +RNA B-chemical +. O + +This O +suggests O +that O +Tsr3 B-protein +is O +not O +stably O +incorporated O +into O +pre B-complex_assembly +- I-complex_assembly +ribosomal I-complex_assembly +particles I-complex_assembly +and O +that O +its O +binding O +to O +the O +nascent O +ribosomal B-complex_assembly +subunit I-complex_assembly +possibly O +requires O +additional O +interactions O +with O +other O +pre O +- O +ribosomal O +components O +. O + +Consistently O +, O +in O +sucrose B-experimental_method +gradient I-experimental_method +analysis I-experimental_method +, O +Tsr3 B-protein +was O +found O +in O +low O +- O +molecular O +weight O +fractions O +rather O +than O +with O +pre B-complex_assembly +- I-complex_assembly +ribosome I-complex_assembly +containing O +high O +- O +molecular O +weight O +fractions O +. O + +In O +contrast O +to O +several O +enzymes O +that O +catalyze O +base O +specific O +modifications O +in O +rRNAs B-chemical +Tsr3 B-protein +is O +not O +an O +essential O +protein O +. O + +Typically O +, O +other O +small B-protein_type +subunit I-protein_type +rRNA I-protein_type +methyltransferases I-protein_type +as O +Dim1 B-protein +, O +Bud23 B-protein +and O +Nep1 B-protein +/ O +Emg1 B-protein +carry O +dual O +functions O +, O +in O +ribosome O +biogenesis O +and O +rRNA B-chemical +modification O +, O +and O +it O +is O +their O +involvement O +in O +pre B-chemical +- I-chemical +RNA I-chemical +processing O +that O +is O +essential O +rather O +than O +their O +RNA O +- O +methylating O +activity O +(, O +discussed O +in O +7 O +). O + +This O +demonstrates O +that O +, O +unlike O +the O +other O +small O +subunit O +rRNA B-chemical +base O +modifications O +, O +the O +acp B-chemical +modification O +is O +required O +for O +efficient O +pre B-chemical +- I-chemical +rRNA I-chemical +processing O +. O + +Recently O +, O +structural B-experimental_method +, I-experimental_method +functional I-experimental_method +, I-experimental_method +and I-experimental_method +CRAC I-experimental_method +( I-experimental_method +cross I-experimental_method +- I-experimental_method +linking I-experimental_method +and I-experimental_method +cDNA I-experimental_method +analysis I-experimental_method +) I-experimental_method +experiments I-experimental_method +of O +late O +assembly O +factors O +involved O +in O +cytoplasmic O +processing O +of O +40S B-complex_assembly +subunits I-complex_assembly +, O +along O +with O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +studies O +of O +the O +late B-protein_state +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +subunits I-complex_assembly +have O +provided O +important O +insights O +into O +late O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +processing O +. O + +Apart O +from O +most O +of O +the O +ribosomal O +proteins O +, O +cytoplasmic O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +particles I-complex_assembly +contain O +20S B-chemical +rRNA I-chemical +and O +at O +least O +seven O +non B-protein_type +- I-protein_type +ribosomal I-protein_type +proteins I-protein_type +including O +the O +D B-protein_type +- I-protein_type +site I-protein_type +endonuclease I-protein_type +Nob1 B-protein +as O +well O +as O +Tsr1 B-protein +, O +a O +putative O +GTPase B-protein_type +and O +Rio2 B-protein +which O +block O +the O +mRNA B-site +channel I-site +and O +the O +initiator B-site +tRNA I-site +binding I-site +site I-site +, O +respectively O +, O +thus O +preventing O +translation O +initiation O +. O + +The O +cleavage O +step O +most O +likely O +acts O +as O +a O +quality O +control O +check O +that O +ensures O +the O +proper O +40S B-complex_assembly +subunit I-complex_assembly +assembly O +with O +only O +completely O +processed O +precursors O +. O + +Interestingly O +, O +differences O +in O +the O +level O +of O +acp B-chemical +modification O +were O +demonstrated O +for O +different O +steps O +of O +the O +cytoplasmic O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +subunit I-complex_assembly +maturation O +after O +analyzing O +purified O +20S B-chemical +pre I-chemical +- I-chemical +rRNAs I-chemical +using O +different O +purification O +bait O +proteins O +. O + +In O +contrast O +, O +late O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +subunits I-complex_assembly +containing O +Nob1 B-protein +and O +Rio1 B-protein +or O +already O +associated O +with O +60S B-complex_assembly +subunits I-complex_assembly +in O +80S B-complex_assembly +- I-complex_assembly +like I-complex_assembly +particles I-complex_assembly +showed O +acp B-chemical +modification O +levels O +comparable O +to O +mature B-protein_state +40S B-complex_assembly +subunits I-complex_assembly +. O + +Thus O +, O +the O +acp B-chemical +transfer O +to O +m1Ψ1191 B-residue_name_number +occurs O +during O +the O +step O +at O +which O +Rio2 B-protein +leaves O +the O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +particle I-complex_assembly +. O + +These O +data O +and O +the O +finding O +that O +a O +missing O +acp B-chemical +modification O +hinders O +pre B-chemical +- I-chemical +20S I-chemical +rRNA I-chemical +processing O +, O +suggest O +that O +the O +acp B-chemical +modification O +together O +with O +the O +release O +of O +Rio2 B-protein +promotes O +the O +formation O +of O +the O +decoding B-site +site I-site +and O +thus O +D B-site +- I-site +site I-site +cleavage O +by O +Nob1 B-protein +. O + +The O +interrelation O +between O +acp B-chemical +modification O +and O +Rio2 B-protein +release O +is O +also O +supported O +by O +CRAC B-experimental_method +analysis I-experimental_method +showing O +that O +Rio2 B-protein +binds O +to O +helix B-structure_element +31 I-structure_element +next O +to O +the O +Ψ1191 B-residue_name_number +residue O +that O +receives O +the O +acp B-chemical +modification O +. O + +In O +summary O +, O +by O +identifying O +Tsr3 B-protein +as O +the O +enzyme O +responsible O +for O +introducing O +the O +acp B-chemical +group O +to O +the O +hypermodified B-protein_state +m1acp3Ψ B-chemical +nucleotide B-chemical +at O +position O +1191 B-residue_number +( O +yeast B-taxonomy_domain +)/ O +1248 B-residue_number +( O +humans B-species +) O +of O +18S B-chemical +rRNA I-chemical +we O +added O +one O +of O +the O +last O +remaining O +pieces O +to O +the O +puzzle O +of O +eukaryotic B-taxonomy_domain +small B-chemical +ribosomal I-chemical +subunit I-chemical +rRNA I-chemical +modifications O +. O + +The O +current O +data O +together O +with O +the O +finding O +that O +acp B-chemical +modification O +takes O +place O +at O +the O +very O +last O +step O +in O +pre B-complex_assembly +- I-complex_assembly +40S I-complex_assembly +subunit I-complex_assembly +maturation O +indicate O +that O +the O +acp B-chemical +modification O +probably O +supports O +the O +formation O +of O +the O +decoding B-site +site I-site +and O +efficient O +20S B-chemical +pre I-chemical +- I-chemical +rRNA I-chemical +D B-site +- I-site +site I-site +cleavage O +. O + +Furthermore O +, O +our O +structural B-evidence +data I-evidence +unravelled O +how O +the O +regioselectivity O +of O +SAM B-chemical +- O +dependent O +group O +transfer O +reactions O +can O +be O +tuned O +by O +distinct O +small O +evolutionary O +adaptions O +of O +the O +ligand B-site +binding I-site +pocket I-site +of O +SAM B-protein_type +- I-protein_type +binding I-protein_type +enzymes I-protein_type +. O + +Structural O +insights O +into O +the O +regulatory O +mechanism O +of O +the O +Pseudomonas B-species +aeruginosa I-species +YfiBNR B-complex_assembly +system O + +YfiBNR B-complex_assembly +is O +a O +recently O +identified O +bis B-chemical +-( I-chemical +3 I-chemical +’- I-chemical +5 I-chemical +’)- I-chemical +cyclic I-chemical +dimeric I-chemical +GMP I-chemical +( O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +) O +signaling O +system O +in O +opportunistic O +pathogens O +. O + +Here O +, O +we O +report O +the O +crystal B-evidence +structures I-evidence +of O +YfiB B-protein +alone B-protein_state +and O +of O +an O +active B-protein_state +mutant B-protein_state +YfiBL43P B-mutant +complexed B-protein_state +with I-protein_state +YfiR B-protein +with O +2 O +: O +2 O +stoichiometry O +. O + +In O +addition O +, O +our O +crystallographic B-experimental_method +analyses I-experimental_method +revealed O +that O +YfiR B-protein +binds O +Vitamin B-chemical +B6 I-chemical +( O +VB6 B-chemical +) O +or O +L B-chemical +- I-chemical +Trp I-chemical +at O +a O +YfiB B-site +- I-site +binding I-site +site I-site +and O +that O +both O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +are O +able O +to O +reduce O +YfiBL43P B-mutant +- O +induced O +biofilm O +formation O +. O + +An O +increase O +in O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +promotes O +biofilm O +formation O +, O +and O +a O +decrease O +results O +in O +biofilm O +degradation O +( O +Boehm O +et O +al O +.,; O +Duerig O +et O +al O +.,; O +Hickman O +et O +al O +.,; O +Jenal O +,; O +Romling O +et O +al O +.,). O + +The O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +level O +is O +regulated O +by O +two O +reciprocal O +enzyme O +systems O +, O +namely O +, O +diguanylate B-protein_type +cyclases I-protein_type +( O +DGCs B-protein_type +) O +that O +synthesize O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +and O +phosphodiesterases B-protein_type +( O +PDEs B-protein_type +) O +that O +hydrolyze O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +( O +Kulasakara O +et O +al O +.,; O +Ross O +et O +al O +.,; O +Ross O +et O +al O +.,). O +Many O +of O +these O +enzymes O +are O +multiple O +- O +domain O +proteins O +containing O +a O +variable O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +that O +commonly O +acts O +as O +a O +signal O +sensor O +or O +transduction O +module O +, O +followed O +by O +the O +relatively B-protein_state +conserved I-protein_state +GGDEF B-structure_element +motif I-structure_element +in O +DGCs B-protein_type +or O +EAL B-structure_element +/ I-structure_element +HD I-structure_element +- I-structure_element +GYP I-structure_element +domains I-structure_element +in O +PDEs B-protein_type +( O +Hengge O +,; O +Navarro O +et O +al O +.,; O +Schirmer O +and O +Jenal O +,). O + +In O +Pseudomonas B-species +aeruginosa I-species +in O +particular O +, O +42 O +genes O +containing O +putative O +DGCs B-protein_type +and O +/ O +or O +PDEs B-protein_type +were O +identified O +( O +Kulasakara O +et O +al O +.,). O + +However O +, O +due O +to O +the O +intricacy O +of O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +signaling O +networks O +and O +the O +diversity O +of O +experimental O +cues O +, O +the O +detailed O +mechanisms O +by O +which O +these O +signaling O +pathways O +specifically O +sense O +and O +integrate O +different O +inputs O +remain O +largely O +elusive O +. O + +Biofilm O +formation O +protects O +pathogenic O +bacteria B-taxonomy_domain +from O +antibiotic O +treatment O +, O +and O +c O +- O +di O +- O +GMP O +- O +regulated O +biofilm O +formation O +has O +been O +extensively O +studied O +in O +P B-species +. I-species +aeruginosa I-species +( O +Evans O +,; O +Kirisits O +et O +al O +.,; O +Malone O +,; O +Reinhardt O +et O +al O +.,). O + +In O +the O +lungs O +of O +cystic O +fibrosis O +( O +CF O +) O +patients O +, O +adherent O +biofilm O +formation O +and O +the O +appearance O +of O +small O +colony O +variant O +( O +SCV O +) O +morphologies O +of O +P B-species +. I-species +aeruginosa I-species +correlate O +with O +prolonged O +persistence O +of O +infection O +and O +poor O +lung O +function O +( O +Govan O +and O +Deretic O +,; O +Haussler O +et O +al O +.,; O +Haussler O +et O +al O +.,; O +Parsek O +and O +Singh O +,; O +Smith O +et O +al O +.,). O + +The O +YfiBNR B-complex_assembly +system O +contains O +three O +protein O +members O +and O +modulates O +intracellular O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +levels O +in O +response O +to O +signals O +received O +in O +the O +periplasm O +( O +Malone O +et O +al O +.,). O + +More O +recently O +, O +this O +system O +was O +also O +reported O +in O +other O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +bacteria I-taxonomy_domain +, O +such O +as O +Escherichia B-species +coli I-species +( O +Hufnagel O +et O +al O +.,; O +Raterman O +et O +al O +.,; O +Sanchez O +- O +Torres O +et O +al O +.,), O +Klebsiella B-species +pneumonia I-species +( O +Huertas O +et O +al O +.,) O +and O +Yersinia B-species +pestis I-species +( O +Ren O +et O +al O +.,). O + +YfiN B-protein +is O +an O +integral O +inner O +- O +membrane O +protein O +with O +two O +potential O +transmembrane B-structure_element +helices I-structure_element +, O +a O +periplasmic O +Per B-structure_element +- I-structure_element +Arnt I-structure_element +- I-structure_element +Sim I-structure_element +( O +PAS B-structure_element +) O +domain O +, O +and O +cytosolic O +domains O +containing O +a O +HAMP B-structure_element +domain I-structure_element +( O +mediate O +input O +- O +output O +signaling O +in O +histidine B-protein_type +kinases I-protein_type +, O +adenylyl B-protein_type +cyclases I-protein_type +, O +methyl B-protein_type +- I-protein_type +accepting I-protein_type +chemotaxis I-protein_type +proteins I-protein_type +, O +and O +phosphatases B-protein_type +) O +and O +a O +C O +- O +terminal O +GGDEF B-structure_element +domain I-structure_element +indicating O +a O +DGC B-protein_type +’ O +s O +function O +( O +Giardina O +et O +al O +.,; O +Malone O +et O +al O +.,). O + +YfiN B-protein +is O +repressed B-protein_state +by I-protein_state +specific O +interaction O +between O +its O +periplasmic O +PAS B-structure_element +domain I-structure_element +and O +the O +periplasmic O +protein O +YfiR B-protein +( O +Malone O +et O +al O +.,). O + +YfiB B-protein +is O +an O +OmpA B-protein_type +/ I-protein_type +Pal I-protein_type +- I-protein_type +like I-protein_type +outer O +- O +membrane O +lipoprotein B-protein_type +( O +Parsons O +et O +al O +.,) O +that O +can O +activate O +YfiN B-protein +by O +sequestering O +YfiR B-protein +( O +Malone O +et O +al O +.,) O +in O +an O +unknown O +manner O +. O + +Whether O +YfiB B-protein +directly O +recruits O +YfiR B-protein +or O +recruits O +YfiR B-protein +via O +a O +third O +partner O +is O +an O +open O +question O +. O + +It O +has O +been O +reported O +that O +the O +activation O +of O +YfiN B-protein +may O +be O +induced O +by O +redox O +- O +driven O +misfolding O +of O +YfiR B-protein +( O +Giardina O +et O +al O +.,; O +Malone O +et O +al O +.,; O +Malone O +et O +al O +.,). O + +It O +is O +also O +proposed O +that O +the O +sequestration O +of O +YfiR B-protein +by O +YfiB B-protein +can O +be O +induced O +by O +certain O +YfiB B-protein +- O +mediated O +cell O +wall O +stress O +, O +and O +mutagenesis B-experimental_method +studies I-experimental_method +revealed O +a O +number O +of O +activation B-structure_element +residues I-structure_element +of O +YfiB B-protein +that O +were O +located O +in O +close O +proximity O +to O +the O +predicted B-protein_state +first B-structure_element +helix I-structure_element +of O +the O +periplasmic B-structure_element +domain I-structure_element +( O +Malone O +et O +al O +.,). O + +In O +addition O +, O +quorum O +sensing O +- O +related O +dephosphorylation O +of O +the O +PAS B-structure_element +domain I-structure_element +of O +YfiN B-protein +may O +also O +be O +involved O +in O +the O +regulation O +( O +Ueda O +and O +Wood O +,; O +Xu O +et O +al O +.,). O + +Recently O +, O +we O +solved O +the O +crystal B-evidence +structure I-evidence +of O +YfiR B-protein +in O +both O +the O +non B-protein_state +- I-protein_state +oxidized I-protein_state +and O +the O +oxidized B-protein_state +states O +, O +revealing O +breakage O +/ O +formation O +of O +one O +disulfide B-ptm +bond I-ptm +( O +Cys71 B-residue_name_number +- O +Cys110 B-residue_name_number +) O +and O +local O +conformational O +change O +around O +the O +other O +one O +( O +Cys145 B-residue_name_number +- O +Cys152 B-residue_name_number +), O +indicating O +that O +Cys145 B-residue_name_number +- O +Cys152 B-residue_name_number +plays O +an O +important O +role O +in O +maintaining O +the O +correct O +folding O +of O +YfiR B-protein +( O +Yang O +et O +al O +.,). O + +Most O +recently O +, O +Li O +and O +coworkers O +reported O +the O +crystal B-evidence +structures I-evidence +of O +YfiB B-protein +( O +27 B-residue_range +– I-residue_range +168 I-residue_range +) O +alone B-protein_state +and O +YfiRC71S B-mutant +in B-protein_state +complex I-protein_state +with I-protein_state +YfiB B-protein +( O +59 B-residue_range +– I-residue_range +168 I-residue_range +) O +( O +Li O +et O +al O +.,). O + +Compared O +with O +the O +reported O +complex O +structure O +, O +YfiBL43P B-mutant +in O +our O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +structure B-evidence +has O +additional O +visible O +N O +- O +terminal O +residues O +44 B-residue_range +– I-residue_range +58 I-residue_range +that O +are O +shown O +to O +play O +essential O +roles O +in O +YfiB B-protein +activation O +and O +biofilm O +formation O +. O + +Therefore O +, O +we O +are O +able O +to O +visualize O +the O +detailed O +allosteric O +arrangement O +of O +the O +N O +- O +terminal O +structure O +of O +YfiB B-protein +and O +its O +important O +role O +in O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +interaction O +. O + +In O +addition O +, O +we O +found O +that O +the O +YfiBL43P B-mutant +shows O +a O +much O +higher O +PG B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +than O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +, O +most O +likely O +due O +to O +its O +more O +compact O +PG B-site +- I-site +binding I-site +pocket I-site +. O + +Overall O +structure B-evidence +of O +YfiB B-protein +. O +( O +A O +) O +The O +overall O +structure B-evidence +of O +the O +YfiB B-protein +monomer B-oligomeric_state +. O +( O +B O +) O +A O +topology O +diagram O +of O +the O +YfiB B-protein +monomer B-oligomeric_state +. O +( O +C O +and O +D O +) O +The O +analytical B-experimental_method +ultracentrifugation I-experimental_method +experiment O +results O +for O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +and O +YfiBL43P B-mutant + +The O +“ O +back B-protein_state +to I-protein_state +back I-protein_state +” O +dimer B-oligomeric_state +. O + +In O +addition O +, O +there O +is O +a O +short O +helix B-structure_element +turn I-structure_element +connecting O +the O +β4 B-structure_element +strand I-structure_element +and O +α4 B-structure_element +helix I-structure_element +( O +Fig O +. O +1A O +and O +1B O +). O + +Here O +, O +we O +refer O +to O +the O +two O +dimeric B-oligomeric_state +types O +as O +“ O +head B-protein_state +to I-protein_state +head I-protein_state +” O +and O +“ O +back B-protein_state +to I-protein_state +back I-protein_state +” O +according O +to O +the O +interacting O +mode O +( O +Fig O +. O +2A O +and O +2E O +), O +with O +the O +total O +buried O +surface O +areas O +being O +316 O +. O +8 O +Å2 O +and O +554 O +. O +3 O +Å2 O +, O +respectively O +. O + +The O +“ O +head B-protein_state +to I-protein_state +head I-protein_state +” O +dimer B-oligomeric_state +exhibits O +a O +clamp B-protein_state +shape I-protein_state +. O + +The O +“ O +back B-protein_state +to I-protein_state +back I-protein_state +” O +dimer B-oligomeric_state +presents O +a O +Y B-protein_state +shape I-protein_state +. O + +Overall O +structure B-evidence +of O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +and O +the O +conserved B-site +surface I-site +in O +YfiR B-protein +. O +( O +A O +) O +The O +overall O +structure B-evidence +of O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +. O + +The O +YfiBL43P B-mutant +molecules O +are O +shown O +in O +cyan O +and O +yellow O +. O + +To O +illustrate O +the O +differences O +between O +apo B-protein_state +YfiB B-protein +and O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +, O +the O +apo B-protein_state +YfiB B-protein +is O +shown O +in O +pink O +, O +except O +residues O +34 B-residue_range +– I-residue_range +70 I-residue_range +are O +shown O +in O +red O +, O +whereas O +the O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +is O +shown O +in O +cyan O +, O +except O +residues O +44 B-residue_range +– I-residue_range +70 I-residue_range +are O +shown O +in O +blue O +. O +( O +C O +) O +Close O +- O +up O +view O +of O +the O +differences O +between O +apo B-protein_state +YfiB B-protein +and O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +. O + +The O +residues O +proposed O +to O +contribute O +to O +YfiB B-protein +activation O +are O +illustrated O +in O +sticks O +. O + +The O +key O +residues O +in O +apo B-protein_state +YfiB B-protein +are O +shown O +in O +red O +and O +those O +in O +YfiBL43P B-mutant +are O +shown O +in O +blue O +. O +( O +D O +) O +Close O +- O +up O +views O +showing O +interactions O +in O +regions B-structure_element +I I-structure_element +and I-structure_element +II I-structure_element +. O + +YfiBL43P B-mutant +and O +YfiR B-protein +are O +shown O +in O +cyan O +and O +green O +, O +respectively O +. O +( O +E O +and O +F O +) O +The O +conserved B-site +surface I-site +in O +YfiR B-protein +contributes O +to O +the O +interaction O +with O +YfiB B-protein +. O +( O +G O +) O +The O +residues B-structure_element +of O +YfiR B-protein +responsible O +for O +interacting O +with O +YfiB B-protein +are O +shown O +in O +green O +sticks O +, O +and O +the O +proposed O +YfiN B-site +- I-site +interacting I-site +residues I-site +are O +shown O +in O +yellow O +sticks O +. O + +The O +red O +sticks O +, O +which O +represent O +the O +YfiB B-site +- I-site +interacting I-site +residues I-site +, O +are O +also O +responsible O +for O +the O +proposed O +interactions O +with O +YfiN B-protein + +It O +has O +been O +reported O +that O +single B-experimental_method +mutants I-experimental_method +of I-experimental_method +Q39 B-residue_name_number +, O +L43 B-residue_name_number +, O +F48 B-residue_name_number +and O +W55 B-residue_name_number +contribute O +to O +YfiB B-protein +activation O +leading O +to O +the O +induction O +of O +the O +SCV O +phenotype O +in O +P B-species +. I-species +aeruginosa I-species +PAO1 I-species +( O +Malone O +et O +al O +.,). O + +Therefore O +, O +we O +constructed B-experimental_method +two I-experimental_method +such I-experimental_method +single I-experimental_method +mutants I-experimental_method +of O +YfiB B-protein +( O +YfiBL43P B-mutant +and O +YfiBF48S B-mutant +). O + +As O +expected O +, O +both O +mutants O +form O +a O +stable B-protein_state +complex B-protein_state +with I-protein_state +YfiR B-protein +. O +Finally O +, O +we O +crystalized B-experimental_method +YfiR B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +YfiBL43P B-mutant +mutant B-protein_state +and O +solved O +the O +structure B-evidence +at O +1 O +. O +78 O +Å O +resolution O +by O +molecular B-experimental_method +replacement I-experimental_method +using O +YfiR B-protein +and O +YfiB B-protein +as O +models O +. O + +The O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +is O +a O +2 O +: O +2 O +heterotetramer B-oligomeric_state +( O +Fig O +. O +3A O +) O +in O +which O +the O +YfiR B-protein +dimer B-oligomeric_state +is O +clamped O +by O +two O +separated O +YfiBL43P B-mutant +molecules O +with O +a O +total O +buried O +surface O +area O +of O +3161 O +. O +2 O +Å2 O +. O + +The O +YfiR B-protein +dimer B-oligomeric_state +in O +the O +complex O +is O +identical O +to O +the O +non B-protein_state +- I-protein_state +oxidized I-protein_state +YfiR B-protein +dimer B-oligomeric_state +alone B-protein_state +( O +Yang O +et O +al O +.,), O +with O +only O +Cys145 B-residue_name_number +- O +Cys152 B-residue_name_number +of O +the O +two O +disulfide B-ptm +bonds I-ptm +well O +formed O +, O +suggesting O +Cys71 B-residue_name_number +- O +Cys110 B-residue_name_number +disulfide B-ptm +bond I-ptm +formation O +is O +not O +essential O +for O +forming O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +. O + +The O +observed O +changes O +in O +conformation O +of O +YfiB B-protein +and O +the O +results O +of O +mutagenesis B-experimental_method +suggest O +a O +mechanism O +by O +which O +YfiB B-protein +sequesters O +YfiR B-protein +. O + +The O +YfiB B-site +- I-site +YfiR I-site +interface I-site +can O +be O +divided O +into O +two O +regions O +( O +Fig O +. O +3A O +and O +3D O +). O + +Region B-structure_element +I I-structure_element +is O +formed O +by O +numerous O +main O +- O +chain O +and O +side O +- O +chain O +hydrophilic B-bond_interaction +interactions I-bond_interaction +between O +residues O +E45 B-residue_name_number +, O +G47 B-residue_name_number +and O +E53 B-residue_name_number +from O +the O +N O +- O +terminal O +extended O +loop B-structure_element +of O +YfiB B-protein +and O +residues O +S57 B-residue_name_number +, O +R60 B-residue_name_number +, O +A89 B-residue_name_number +and O +H177 B-residue_name_number +from O +YfiR B-protein +( O +Fig O +. O +3D O +- O +I O +( O +i O +)). O + +In O +region B-structure_element +II I-structure_element +, O +the O +side O +chains O +of O +R96 B-residue_name_number +, O +E98 B-residue_name_number +and O +E157 B-residue_name_number +from O +YfiB B-protein +interact O +with O +the O +side O +chains O +of O +E163 B-residue_name_number +, O +S146 B-residue_name_number +and O +R171 B-residue_name_number +from O +YfiR B-protein +, O +respectively O +. O + +Additionally O +, O +the O +main O +chains O +of O +I163 B-residue_name_number +and O +V165 B-residue_name_number +from O +YfiB B-protein +form O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +main O +chains O +of O +L166 B-residue_name_number +and O +A164 B-residue_name_number +from O +YfiR B-protein +, O +respectively O +, O +and O +the O +main O +chain O +of O +P166 B-residue_name_number +from O +YfiB B-protein +interacts O +with O +the O +side O +chain O +of O +R185 B-residue_name_number +from O +YfiR B-protein +( O +Fig O +. O +3D O +- O +II O +). O + +These O +two O +regions O +contribute O +a O +robust O +hydrogen B-site +- I-site +bonding I-site +network I-site +to O +the O +YfiB B-site +- I-site +YfiR I-site +interface I-site +, O +resulting O +in O +a O +tightly O +bound O +complex O +. O + +Based O +on O +the O +observations O +that O +two O +separated O +YfiBL43P B-mutant +molecules O +form O +a O +2 O +: O +2 O +complex O +structure B-evidence +with O +YfiR B-protein +dimer B-oligomeric_state +, O +we O +performed O +an O +analytical B-experimental_method +ultracentrifugation I-experimental_method +experiment O +to O +check O +the O +oligomeric O +states O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +and O +YfiBL43P B-mutant +. O + +The O +results O +showed O +that O +wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +exists O +in O +both O +monomeric B-oligomeric_state +and O +dimeric B-oligomeric_state +states O +in O +solution O +, O +while O +YfiBL43P B-mutant +primarily O +adopts O +the O +monomer B-oligomeric_state +state O +in O +solution O +( O +Fig O +. O +1C O +– O +D O +). O + +This O +suggests O +that O +the O +N O +- O +terminus O +of O +YfiB B-protein +plays O +an O +important O +role O +in O +forming O +the O +dimeric B-oligomeric_state +YfiB B-protein +in O +solution O +and O +that O +the O +conformational O +change O +of O +residue O +L43 B-residue_name_number +is O +associated O +with O +the O +stretch O +of O +the O +N O +- O +terminus O +and O +opening O +of O +the O +dimer B-oligomeric_state +. O + +The O +PG B-site +- I-site +binding I-site +site I-site +of O +YfiB B-protein + +The O +PG B-site +- I-site +binding I-site +site I-site +in O +YfiB B-protein +. O +( O +A O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +PG B-site +- I-site +binding I-site +sites I-site +of O +the O +H B-species +. I-species +influenzae I-species +Pal B-complex_assembly +/ I-complex_assembly +PG I-complex_assembly +- I-complex_assembly +P I-complex_assembly +complex O +and O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +complexed B-protein_state +with I-protein_state +sulfate B-chemical +ions O +. O + +( O +B O +) O +Close O +- O +up O +view O +showing O +the O +key O +residues O +of O +Pal B-protein_type +interacting O +with O +the O +m B-chemical +- I-chemical +Dap5 I-chemical +ε I-chemical +- I-chemical +carboxylate I-chemical +group O +of O +PG B-chemical +- I-chemical +P I-chemical +. O +Pal B-protein_type +is O +shown O +in O +wheat O +and O +PG B-chemical +- I-chemical +P I-chemical +is O +in O +magenta O +. O + +Apo B-protein_state +YfiB B-protein +is O +shown O +in O +yellow O +and O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +in O +cyan O +. O +( O +E O +and O +F O +) O +MST B-experimental_method +data O +and O +analysis O +for O +binding B-evidence +affinities I-evidence +of O +( O +E O +) O +YfiB B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +and O +( O +F O +) O +YfiBL43P B-mutant +with O +PG B-chemical +. O +( O +G O +) O +The O +sequence B-experimental_method +alignment I-experimental_method +of O +P B-species +. I-species +aeruginosa I-species +and O +E B-species +. I-species +coli I-species +sources O +of O +YfiB B-protein +, O +Pal B-protein_type +and O +the O +periplasmic B-structure_element +domain I-structure_element +of O +OmpA B-protein_type + +Previous O +homology B-experimental_method +modeling I-experimental_method +studies O +suggested O +that O +YfiB B-protein +contains O +a O +Pal B-site +- I-site +like I-site +PG I-site +- I-site +binding I-site +site I-site +( O +Parsons O +et O +al O +.,), O +and O +the O +mutation B-experimental_method +of I-experimental_method +two I-experimental_method +residues I-experimental_method +at O +this O +site O +, O +D102 B-residue_name_number +and O +G105 B-residue_name_number +, O +reduces O +the O +ability O +for O +biofilm O +formation O +and O +surface O +attachment O +( O +Malone O +et O +al O +.,). O + +In O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +, O +one O +sulfate B-chemical +ion O +is O +found O +at O +the O +bottom O +of O +each O +YfiBL43P B-mutant +molecule O +( O +Fig O +. O +3A O +) O +and O +forms O +a O +strong O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +D102 B-residue_name_number +of O +YfiBL43P B-mutant +( O +Fig O +. O +4A O +and O +4C O +). O + +Structural B-experimental_method +superposition I-experimental_method +between O +YfiBL43P B-mutant +and O +Haemophilus B-species +influenzae I-species +Pal B-protein_type +complexed B-protein_state +with I-protein_state +biosynthetic O +peptidoglycan B-chemical +precursor I-chemical +( O +PG B-chemical +- I-chemical +P I-chemical +), O +UDP B-chemical +- I-chemical +N I-chemical +- I-chemical +acetylmuramyl I-chemical +- I-chemical +L I-chemical +- I-chemical +Ala I-chemical +- I-chemical +α I-chemical +- I-chemical +D I-chemical +- I-chemical +Glu I-chemical +- I-chemical +m I-chemical +- I-chemical +Dap I-chemical +- I-chemical +D I-chemical +- I-chemical +Ala I-chemical +- I-chemical +D I-chemical +- I-chemical +Ala I-chemical +( O +m B-chemical +- I-chemical +Dap I-chemical +is O +meso B-chemical +- I-chemical +diaminopimelate I-chemical +) O +( O +PDB O +code O +: O +2aiz O +) O +( O +Parsons O +et O +al O +.,), O +revealed O +that O +the O +sulfate B-chemical +ion O +is O +located O +at O +the O +position O +of O +the O +m B-chemical +- I-chemical +Dap5 I-chemical +ϵ I-chemical +- I-chemical +carboxylate I-chemical +group O +in O +the O +Pal B-complex_assembly +/ I-complex_assembly +PG I-complex_assembly +- I-complex_assembly +P I-complex_assembly +complex O +( O +Fig O +. O +4A O +). O + +Similarly O +, O +in O +the O +YfiR B-protein_state +- I-protein_state +bound I-protein_state +YfiBL43P B-mutant +structure B-evidence +, O +the O +sulfate B-chemical +ion O +interacts O +with O +the O +side O +- O +chain O +atoms O +of O +D102 B-residue_name_number +( O +corresponding O +to O +D71 B-residue_name_number +in O +Pal B-protein_type +) O +and O +R117 B-residue_name_number +( O +corresponding O +to O +R86 B-residue_name_number +in O +Pal B-protein_type +) O +and O +the O +main O +- O +chain O +amide O +of O +N68 B-residue_name_number +( O +corresponding O +to O +D37 B-residue_name_number +in O +Pal B-protein_type +). O + +Moreover O +, O +a O +water B-chemical +molecule O +was O +found O +to O +bridge O +the O +sulfate B-chemical +ion O +and O +the O +side O +chains O +of O +N67 B-residue_name_number +and O +D102 B-residue_name_number +, O +strengthening O +the O +hydrogen B-site +bond I-site +network I-site +( O +Fig O +. O +4C O +). O + +Compared O +to O +YfiBL43P B-mutant +, O +the O +N68 B-residue_name_number +- O +containing O +loop B-structure_element +of O +the O +apo B-protein_state +YfiB B-protein +flips O +away O +about O +7 O +Å O +, O +and O +D102 B-residue_name_number +and O +R117 B-residue_name_number +swing O +slightly O +outward O +; O +thus O +, O +the O +PG B-site +- I-site +binding I-site +pocket I-site +is O +enlarged O +with O +no O +sulfate B-chemical +ion O +or O +water B-chemical +bound O +( O +Fig O +. O +4D O +). O + +As O +the O +experiment O +is O +performed O +in B-protein_state +the I-protein_state +absence I-protein_state +of I-protein_state +YfiR B-protein +, O +it O +suggests O +that O +an O +increase O +in O +the O +PG B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +YfiB B-protein +is O +not O +a O +result O +of O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +interaction O +and O +is O +highly O +coupled O +to O +the O +activation O +of O +YfiB B-protein +characterized O +by O +a O +stretched B-protein_state +N I-protein_state +- I-protein_state +terminal I-protein_state +conformation I-protein_state +. O + +The O +conserved B-site +surface I-site +in O +YfiR B-protein +is O +functional O +for O +binding O +YfiB B-protein +and O +YfiN B-protein + +Interestingly O +, O +the O +majority O +of O +this O +conserved B-site +surface I-site +contributes O +to O +the O +interaction O +with O +YfiB B-protein +( O +Fig O +. O +3E O +and O +3F O +). O + +Malone O +JG O +et O +al O +. O +have O +reported O +that O +F151 B-residue_name_number +, O +E163 B-residue_name_number +, O +I169 B-residue_name_number +and O +Q187 B-residue_name_number +, O +located O +near O +the O +C O +- O +terminus O +of O +YfiR B-protein +, O +comprise O +a O +putative O +YfiN B-site +binding I-site +site I-site +( O +Malone O +et O +al O +.,). O + +F151 B-residue_name_number +, O +E163 B-residue_name_number +and O +I169 B-residue_name_number +form O +a O +hydrophobic B-site +core I-site +while O +, O +Q187 B-residue_name_number +is O +located O +at O +the O +end O +of O +the O +α6 B-structure_element +helix I-structure_element +. O + +E163 B-residue_name_number +and O +I169 B-residue_name_number +are O +YfiB B-site +- I-site +interacting I-site +residues I-site +of O +YfiR B-protein +, O +in O +which O +E163 B-residue_name_number +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +R96 B-residue_name_number +of O +YfiB B-protein +( O +Fig O +. O +3D O +- O +II O +) O +and O +I169 B-residue_name_number +is O +involved O +in O +forming O +the O +L166 B-residue_name_number +/ O +I169 B-residue_name_number +/ O +V176 B-residue_name_number +/ O +P178 B-residue_name_number +/ O +L181 B-residue_name_number +hydrophobic B-site +core I-site +for O +anchoring O +F57 B-residue_name_number +of O +YfiB B-protein +( O +Fig O +. O +3D O +- O +I O +( O +ii O +)). O + +YfiR B-protein +binds O +small O +molecules O + +The O +electron B-evidence +densities I-evidence +of O +VB6 B-chemical +and O +Trp B-chemical +are O +countered O +at O +3 O +. O +0σ O +and O +2 O +. O +3σ O +, O +respectively O +, O +in O +| B-evidence +Fo I-evidence +|-| I-evidence +Fc I-evidence +| I-evidence +maps I-evidence +. O +( O +C O +) O +Superposition B-experimental_method +of O +the O +hydrophobic B-site +pocket I-site +of O +YfiR B-protein +with O +VB6 B-chemical +, O +L B-chemical +- I-chemical +Trp I-chemical +and O +F57 B-residue_name_number +of O +YfiB B-protein + +Intriguingly O +, O +a O +Dali B-experimental_method +search I-experimental_method +( O +Holm O +and O +Rosenstrom O +,) O +indicated O +that O +the O +closest O +homologs O +of O +YfiR B-protein +shared O +the O +characteristic O +of O +being O +able O +to O +bind O +several O +structurally O +similar O +small O +molecules O +, O +such O +as O +L B-chemical +- I-chemical +Trp I-chemical +, O +L B-chemical +- I-chemical +Phe I-chemical +, O +B O +- O +group O +vitamins O +and O +their O +analogs O +, O +encouraging O +us O +to O +test O +whether O +YfiR B-protein +can O +recognize O +these O +molecules O +. O + +For O +this O +purpose O +, O +we O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +YfiR B-protein +or O +soaked B-experimental_method +YfiR B-protein +crystals B-evidence +with O +different O +small O +molecules O +, O +including O +L B-chemical +- I-chemical +Trp I-chemical +and O +B O +- O +group O +vitamins O +. O + +Fortunately O +, O +we O +found O +obvious O +small B-evidence +- I-evidence +molecule I-evidence +density I-evidence +in O +the O +VB6 B-protein_state +- I-protein_state +bound I-protein_state +and O +Trp B-protein_state +- I-protein_state +bound I-protein_state +YfiR B-protein +crystal B-evidence +structures I-evidence +( O +Fig O +. O +5A O +and O +5B O +), O +and O +in O +both O +structures B-evidence +, O +the O +YfiR B-protein +dimers B-oligomeric_state +resemble O +the O +oxidized B-protein_state +YfiR B-protein +structure B-evidence +in O +which O +both O +two O +disulfide B-ptm +bonds I-ptm +are O +well O +formed O +( O +Yang O +et O +al O +.,). O + +Functional O +analysis O +of O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +. O +( O +A O +and O +B O +) O +The O +effect B-experimental_method +of I-experimental_method +increasing I-experimental_method +concentrations I-experimental_method +of O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +on O +YfiBL43P B-mutant +- O +induced O +attachment O +( O +bars O +). O + +The O +relative B-evidence +optical I-evidence +density I-evidence +is O +represented O +as O +curves O +. O + +Wild B-protein_state +- I-protein_state +type I-protein_state +YfiB B-protein +is O +used O +as O +negative O +control O +. O + +( O +C O +and O +D O +) O +BIAcore B-experimental_method +data O +and O +analysis O +for O +binding B-evidence +affinities I-evidence +of O +( O +C O +) O +VB6 B-chemical +and O +( O +D O +) O +L B-chemical +- I-chemical +Trp I-chemical +with O +YfiR B-protein +. O +( O +E O +– O +G O +) O +ITC B-experimental_method +data O +and O +analysis O +for O +titration B-experimental_method +of O +( O +E O +) O +YfiB B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +, O +( O +F O +) O +YfiBL43P O +, O +and O +( O +G O +) O +YfiBL43P B-mutant +/ O +F57A B-mutant +into O +YfiR B-protein + +Structural B-experimental_method +analyses I-experimental_method +revealed O +that O +the O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +molecules O +are O +bound B-protein_state +at I-protein_state +the O +periphery O +of O +the O +YfiR B-protein +dimer B-oligomeric_state +, O +but O +not O +at O +the O +dimer B-site +interface I-site +. O + +Interestingly O +, O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +were O +found O +to O +occupy O +the O +same O +hydrophobic B-site +pocket I-site +, O +formed O +by O +L166 B-residue_name_number +/ O +I169 B-residue_name_number +/ O +V176 B-residue_name_number +/ O +P178 B-residue_name_number +/ O +L181 B-residue_name_number +of O +YfiR B-protein +, O +which O +is O +also O +a O +binding B-site +pocket I-site +for O +F57 B-residue_name_number +of O +YfiB B-protein +, O +as O +observed O +in O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +( O +Fig O +. O +5C O +). O + +To O +evaluate O +the O +importance O +of O +F57 B-residue_name_number +in O +YfiBL43P B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +interaction O +, O +the O +binding B-evidence +affinities I-evidence +of O +YfiBL43P B-mutant +and O +YfiBL43P B-mutant +/ O +F57A B-mutant +for O +YfiR B-protein +were O +measured O +by O +isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +( O +ITC B-experimental_method +). O + +The O +results O +showed O +Kd B-evidence +values O +of O +1 O +. O +4 O +× O +10 O +− O +7 O +mol O +/ O +L O +and O +5 O +. O +3 O +× O +10 O +− O +7 O +mol O +/ O +L O +for O +YfiBL43P B-mutant +and O +YfiBL43P B-mutant +/ O +F57A B-mutant +, O +respectively O +, O +revealing O +that O +the O +YfiBL43P B-mutant +/ O +F57A B-mutant +mutant B-protein_state +caused O +a O +3 O +. O +8 O +- O +fold O +reduction O +in O +the O +binding B-evidence +affinity I-evidence +compared O +with O +the O +YfiBL43P B-mutant +mutant B-protein_state +( O +Fig O +. O +6F O +and O +6G O +). O + +Growth B-experimental_method +and I-experimental_method +surface I-experimental_method +attachment I-experimental_method +assays I-experimental_method +were O +carried O +out O +for O +the O +yfiB B-mutant +- I-mutant +L43P I-mutant +strain O +in O +the O +presence O +of O +increasing B-experimental_method +concentrations I-experimental_method +of O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +. O + +As O +shown O +in O +Fig O +. O +6A O +and O +6B O +, O +the O +over B-experimental_method +- I-experimental_method +expression I-experimental_method +of O +YfiBL43P B-mutant +induced O +strong O +surface O +attachment O +and O +much O +slower O +growth O +of O +the O +yfiB B-mutant +- I-mutant +L43P I-mutant +strain O +, O +and O +as O +expected O +, O +a O +certain O +amount O +of O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +( O +4 O +– O +6 O +mmol O +/ O +L O +for O +VB6 B-chemical +and O +6 O +– O +10 O +mmol O +/ O +L O +for O +L B-chemical +- I-chemical +Trp I-chemical +) O +could O +reduce O +the O +surface O +attachment O +. O + +In O +Helicobacter B-species +pylori I-species +in O +particular O +, O +VB6 B-chemical +biosynthetic O +enzymes O +act O +as O +novel O +virulence O +factors O +, O +and O +VB6 B-chemical +is O +required O +for O +full O +motility O +and O +virulence O +( O +Grubman O +et O +al O +.,). O + +In O +E B-species +. I-species +coli I-species +, O +mutants O +with O +decreased O +tryptophan B-chemical +synthesis O +show O +greater O +biofilm O +formation O +, O +and O +matured O +biofilm O +is O +degraded O +by O +L B-chemical +- I-chemical +tryptophan I-chemical +addition O +( O +Shimazaki O +et O +al O +.,). O + +To O +answer O +the O +question O +whether O +competition O +of O +VB6 B-chemical +or O +L B-chemical +- I-chemical +Trp I-chemical +for O +the O +YfiB B-protein +F57 B-site +- I-site +binding I-site +pocket I-site +of O +YfiR B-protein +plays O +an O +essential O +role O +in O +inhibiting O +biofilm O +formation O +, O +we O +measured O +the O +binding B-evidence +affinities I-evidence +of O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +for O +YfiR B-protein +via O +BIAcore B-experimental_method +experiments O +. O + +The O +results O +showed O +relatively O +weak O +Kd B-evidence +values O +of O +35 O +. O +2 O +mmol O +/ O +L O +and O +76 O +. O +9 O +mmol O +/ O +L O +for O +VB6 B-chemical +and O +L B-chemical +- I-chemical +Trp I-chemical +, O +respectively O +( O +Fig O +. O +6C O +and O +6D O +). O + +Previous O +studies O +suggested O +that O +in O +response O +to O +cell O +stress O +, O +YfiB B-protein +in O +the O +outer O +membrane O +sequesters O +the O +periplasmic O +protein O +YfiR B-protein +, O +releasing O +its O +inhibition O +of O +YfiN B-protein +on O +the O +inner O +membrane O +and O +thus O +inducing O +the O +diguanylate O +cyclase O +activity O +of O +YfiN B-protein +to O +allow O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +production O +( O +Giardina O +et O +al O +.,; O +Malone O +et O +al O +.,; O +Malone O +et O +al O +.,). O + +Here O +, O +we O +report O +the O +crystal B-evidence +structures I-evidence +of O +YfiB B-protein +alone B-protein_state +and O +an O +active B-protein_state +mutant B-protein_state +YfiBL43P B-mutant +in B-protein_state +complex I-protein_state +with I-protein_state +YfiR B-protein +, O +indicating O +that O +YfiR B-protein +forms O +a O +2 O +: O +2 O +complex B-protein_state +with I-protein_state +YfiB B-protein +via O +a O +region O +composed O +of O +conserved O +residues O +. O + +In O +addition O +to O +the O +preceding B-residue_range +8 I-residue_range +aa I-residue_range +loop B-structure_element +( O +from O +the O +lipid O +acceptor O +Cys26 B-residue_range +to I-residue_range +Gly34 I-residue_range +), O +the O +full B-protein_state +length I-protein_state +of O +the O +periplasmic O +portion O +of O +apo B-protein_state +YfiB B-protein +can O +reach O +approximately O +60 O +Å O +. O +It O +was O +reported O +that O +the O +distance O +between O +the O +outer O +membrane O +and O +the O +cell O +wall O +is O +approximately O +50 O +Å O +and O +that O +the O +thickness O +of O +the O +PG O +layer O +is O +approximately O +70 O +Å O +( O +Matias O +et O +al O +.,). O + +Thus O +, O +YfiB B-protein +alone B-protein_state +represents O +an O +inactive B-protein_state +form O +that O +may O +only O +partially O +insert O +into O +the O +PG O +matrix O +. O + +In O +addition O +to O +the O +17 B-residue_range +preceding I-residue_range +intracellular I-residue_range +residues I-residue_range +( O +from O +the O +lipid O +acceptor O +Cys26 B-residue_range +to I-residue_range +Leu43 I-residue_range +), O +the O +length O +of O +the O +intracellular O +portion O +of O +active B-protein_state +YfiB B-protein +may O +extend O +over O +100 O +Å O +, O +assuming O +a O +fully B-protein_state +stretched I-protein_state +conformation I-protein_state +. O + +The O +periplasmic B-structure_element +domain I-structure_element +of O +YfiB B-protein +and O +the O +YfiB B-complex_assembly +- I-complex_assembly +YfiR I-complex_assembly +complex O +are O +depicted O +according O +to O +the O +crystal B-evidence +structures I-evidence +. O + +The O +lipid O +acceptor O +Cys26 B-residue_name_number +is O +indicated O +as O +blue O +ball O +. O + +The O +PAS B-structure_element +domain I-structure_element +of O +YfiN B-protein +is O +shown O +as O +pink O +oval O +. O + +In O +this O +model O +, O +in O +response O +to O +a O +particular O +cell O +stress O +that O +is O +yet O +to O +be O +identified O +, O +the O +dimeric B-oligomeric_state +YfiB B-protein +is O +activated B-protein_state +from O +a O +compact B-protein_state +, O +inactive B-protein_state +conformation B-protein_state +to O +a O +stretched B-protein_state +conformation I-protein_state +, O +which O +possesses O +increased O +PG B-chemical +binding O +affinity O +. O + +The O +YfiBNR B-complex_assembly +system O +provides O +a O +good O +example O +of O +a O +delicate O +homeostatic O +system O +that O +integrates O +multiple O +signals O +to O +regulate O +the O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +level O +. O + +Homologs O +of O +the O +YfiBNR B-complex_assembly +system O +are O +functionally B-protein_state +conserved I-protein_state +in O +P B-species +. I-species +aeruginosa I-species +( O +Malone O +et O +al O +.,; O +Malone O +et O +al O +.,), O +E B-species +. I-species +coli I-species +( O +Hufnagel O +et O +al O +.,; O +Raterman O +et O +al O +.,; O +Sanchez O +- O +Torres O +et O +al O +.,), O +K B-species +. I-species +pneumonia I-species +( O +Huertas O +et O +al O +.,) O +and O +Y B-species +. I-species +pestis I-species +( O +Ren O +et O +al O +.,), O +where O +they O +affect O +c B-chemical +- I-chemical +di I-chemical +- I-chemical +GMP I-chemical +production O +and O +biofilm O +formation O +. O + +Hemi B-chemical +- I-chemical +methylated I-chemical +DNA I-chemical +opens O +a O +closed B-protein_state +conformation O +of O +UHRF1 B-protein +to O +facilitate O +its O +histone B-protein_type +recognition O + +UHRF1 B-protein +is O +an O +important O +epigenetic O +regulator O +for O +maintenance O +DNA O +methylation B-ptm +. O + +Here O +we O +show O +that O +UHRF1 B-protein +adopts O +a O +closed B-protein_state +conformation O +, O +in O +which O +a O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +( O +Spacer B-structure_element +) O +binds B-protein_state +to I-protein_state +the O +tandem B-structure_element +Tudor I-structure_element +domain I-structure_element +( O +TTD B-structure_element +) O +and O +inhibits O +H3K9me3 B-protein_type +recognition O +, O +whereas O +the O +SET B-structure_element +- I-structure_element +and I-structure_element +- I-structure_element +RING I-structure_element +- I-structure_element +associated I-structure_element +( O +SRA B-structure_element +) O +domain O +binds B-protein_state +to I-protein_state +the O +plant B-structure_element +homeodomain I-structure_element +( O +PHD B-structure_element +) O +and O +inhibits O +H3R2 B-site +recognition O +. O + +Hm B-chemical +- I-chemical +DNA I-chemical +impairs O +the O +intramolecular O +interactions O +and O +promotes O +H3K9me3 B-protein_type +recognition O +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +. O + +The O +Spacer B-structure_element +also O +facilitates O +UHRF1 B-complex_assembly +– I-complex_assembly +DNMT1 I-complex_assembly +interaction O +and O +enhances O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +SRA B-structure_element +. O + +When O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +binds B-protein_state +to I-protein_state +H3K9me3 B-protein_type +, O +SRA B-structure_element +- I-structure_element +Spacer I-structure_element +may O +exist O +in O +a O +dynamic O +equilibrium O +: O +either O +recognizes O +hm B-chemical +- I-chemical +DNA I-chemical +or O +recruits O +DNMT1 B-protein +to O +chromatin O +. O + +UHRF1 B-protein +is O +involved O +in O +the O +maintenance O +of O +DNA O +methylation B-ptm +, O +but O +the O +regulatory O +mechanism O +of O +this O +epigenetic O +regulator O +is O +unclear O +. O + +Here O +, O +the O +authors O +show O +that O +it O +has O +a O +closed B-protein_state +conformation O +and O +are O +able O +to O +make O +conclusions O +about O +the O +mechanism O +of O +recognition O +of O +epigenetic O +marks O +. O + +DNA O +methylation B-ptm +is O +an O +important O +epigenetic O +modification O +for O +gene O +repression O +, O +X O +- O +chromosome O +inactivation O +, O +genome O +imprinting O +and O +maintenance O +of O +genome O +stability O +. O + +Mammalian B-taxonomy_domain +DNA O +methylation B-ptm +is O +established O +by O +de O +novo O +DNA B-protein_type +methyltransferases I-protein_type +DNMT3A B-protein +/ I-protein +3B I-protein +, O +and O +DNA O +methylation B-ptm +patterns O +are O +maintained O +by O +maintenance O +DNA B-protein +methyltransferase I-protein +1 I-protein +( O +DNMT1 B-protein +) O +during O +DNA O +replication O +. O + +Ubiquitin B-protein +- I-protein +like I-protein +, I-protein +containing I-protein +PHD I-protein +and I-protein +RING I-protein +fingers I-protein +domains I-protein +, I-protein +1 I-protein +( O +UHRF1 B-protein +, O +also O +known O +as O +ICBP90 B-protein +and O +NP95 B-protein +in O +mouse B-taxonomy_domain +) O +was O +shown O +to O +be O +essential O +for O +maintenance O +DNA O +methylation B-ptm +through O +recruiting O +DNMT1 B-protein +to O +replication O +forks O +in O +S O +phase O +of O +the O +cell O +cycle O +. O + +UHRF1 B-protein +is O +essential O +for O +S O +phase O +entry O +and O +is O +involved O +in O +heterochromatin O +formation O +. O + +UHRF1 B-protein +is O +a O +multi O +- O +domain O +containing O +protein O +connecting O +histone B-protein_type +modification O +and O +DNA B-chemical +methylation B-ptm +. O + +As O +shown O +in O +Fig O +. O +1a O +, O +UHRF1 B-protein +is O +comprised O +of O +an O +N O +- O +terminal O +ubiquitin B-structure_element +- I-structure_element +like I-structure_element +domain I-structure_element +, O +followed O +by O +a O +tandem B-structure_element +Tudor I-structure_element +domain I-structure_element +( O +TTD B-structure_element +containing O +TTDN B-structure_element +and O +TTDC B-structure_element +sub O +- O +domains O +), O +a O +plant B-structure_element +homeodomain I-structure_element +( O +PHD B-structure_element +), O +a O +SET B-structure_element +- I-structure_element +and I-structure_element +- I-structure_element +RING I-structure_element +- I-structure_element +associated I-structure_element +( O +SRA B-structure_element +) O +domain O +, O +and O +a O +C O +- O +terminal O +really B-structure_element +interesting I-structure_element +new I-structure_element +gene I-structure_element +( O +RING B-structure_element +) O +domain O +. O + +We O +and O +other O +groups O +demonstrated O +that O +the O +TTD B-structure_element +and O +the O +PHD B-structure_element +coordinately O +recognize O +histone B-protein_type +H3K9me3 B-protein_type +, O +in O +which O +residue O +R2 B-residue_name_number +is O +recognized O +by O +the O +PHD B-structure_element +and O +tri B-ptm +- I-ptm +methylation I-ptm +of O +residue O +K9 B-residue_name_number +( O +K9me3 B-ptm +) O +is O +recognized O +by O +the O +TTD B-structure_element +. O + +The O +SRA B-structure_element +preferentially O +binds B-protein_state +to I-protein_state +hemi B-chemical +- I-chemical +methylated I-chemical +DNA I-chemical +( O +hm B-chemical +- I-chemical +DNA I-chemical +). O + +Recent O +studies O +show O +that O +the O +SRA B-structure_element +directly O +binds B-protein_state +to I-protein_state +replication B-structure_element +focus I-structure_element +targeting I-structure_element +sequence I-structure_element +( O +RFTS B-structure_element +) O +of O +DNMT1 B-protein +( O +RFTSDNMT1 B-protein +). O + +A O +spacer B-structure_element +region I-structure_element +( O +Fig O +. O +1a O +, O +designated O +Spacer B-structure_element +hereafter O +) O +connecting O +the O +SRA B-structure_element +and O +the O +RING B-structure_element +is O +rich O +in O +basic O +residues O +and O +predicted O +to O +be O +unstructured B-protein_state +for O +unknown O +function O +. O + +Recent O +study O +shows O +that O +phosphatidylinostiol B-chemical +phosphate I-chemical +PI5P B-chemical +binds B-protein_state +to I-protein_state +the O +Spacer B-structure_element +and O +induces O +a O +conformational O +change O +of O +UHRF1 B-protein +to O +allow O +the O +TTD B-structure_element +to O +recognize O +H3K9me3 B-protein_type +( O +ref O +.). O + +These O +studies O +indicate O +that O +UHRF1 B-protein +connects O +dynamic O +regulation O +of O +DNA B-chemical +methylation B-ptm +and O +H3K9me3 B-protein_type +, O +which O +are O +positively O +correlated O +in O +human B-species +genome O +. O + +Upon O +binding B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +, O +UHRF1 B-protein +impairs O +the O +intramolecular O +interactions O +and O +promotes O +the O +H3K9me3 B-protein_type +recognition O +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +, O +which O +may O +further O +enhance O +its O +genomic O +localization O +. O + +Our O +study O +reveals O +the O +mechanism O +for O +regulation O +of O +H3K9me3 B-protein_type +and O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +by O +UHRF1 B-protein +. O + +Hm B-chemical +- I-chemical +DNA I-chemical +facilitates O +histone B-protein_type +H3K9me3 B-protein_type +recognition O +by O +UHRF1 B-protein + +We O +first O +performed O +an O +in B-experimental_method +vitro I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +using O +biotinylated B-protein_state +histone B-protein_type +H3 B-protein_type +peptides O +and O +hm B-chemical +- I-chemical +DNA I-chemical +( O +Supplementary O +Table O +1 O +). O + +As O +shown O +in O +Fig O +. O +1b O +, O +hm B-chemical +- I-chemical +DNA I-chemical +largely O +enhanced O +the O +interaction O +between O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +unmethylated B-protein_state +histone B-protein_type +H3 B-protein_type +( O +H3K9me0 B-protein_type +) O +or O +H3K9me3 B-protein_type +peptide O +. O + +Compared O +with O +hm B-chemical +- I-chemical +DNA I-chemical +, O +um B-chemical +- I-chemical +DNA I-chemical +( O +unmethylated B-protein_state +DNA B-chemical +) O +or O +fm B-chemical +- I-chemical +DNA I-chemical +( O +fully B-protein_state +methylated I-protein_state +DNA B-chemical +) O +showed O +marginal O +effect O +on O +facilitating O +the O +interaction O +between O +UHRF1 B-protein +and O +histone B-protein_type +peptides O +, O +which O +is O +consistent O +with O +previous O +studies O +that O +UHRF1 B-protein +prefers O +hm B-chemical +- I-chemical +DNA I-chemical +for O +chromatin O +association O +( O +Supplementary O +Fig O +. O +1a O +). O + +In O +contrast O +, O +histone B-protein_type +peptides O +showed O +no O +enhancement O +on O +the O +interaction O +between O +hm B-chemical +- I-chemical +DNA I-chemical +and O +UHRF1 B-protein +( O +Fig O +. O +1c O +). O + +Our O +previous O +studies O +show O +that O +the O +PHD B-structure_element +recognizes O +H3K9me0 B-protein_type +and O +the O +TTD B-structure_element +and O +the O +PHD B-structure_element +together O +( O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +) O +coordinately O +recognize O +H3K9me3 B-protein_type +( O +refs O +.). O + +We O +noticed O +that O +the O +isolated B-protein_state +TTD B-structure_element +– I-structure_element +PHD I-structure_element +showed O +much O +higher O +(∼ O +31 O +- O +fold O +) O +binding B-evidence +affinity I-evidence +to O +H3K9me3 B-protein_type +peptide O +than O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +( O +Fig O +. O +1d O +and O +Supplementary O +Table O +2 O +), O +and O +the O +isolated O +PHD B-structure_element +showed O +much O +higher O +(∼ O +34 O +- O +fold O +) O +binding B-evidence +affinity I-evidence +to O +H3K9me0 B-protein_type +peptide O +than O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +( O +Fig O +. O +1e O +). O + +Intramolecular O +interaction O +within O +UHRF1 B-protein + +Interestingly O +, O +the O +TTD B-structure_element +directly O +bound B-protein_state +to I-protein_state +SRA B-structure_element +- I-structure_element +Spacer I-structure_element +but O +not O +the O +SRA B-structure_element +, O +suggesting O +that O +the O +Spacer B-structure_element +( O +residues O +587 B-residue_range +– I-residue_range +674 I-residue_range +) O +is O +important O +for O +the O +intramolecular O +interaction O +( O +Fig O +. O +2a O +). O + +The O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +also O +shows O +that O +the O +PHD B-structure_element +bound B-protein_state +to I-protein_state +the O +SRA B-structure_element +, O +which O +was O +further O +confirmed O +by O +the O +ITC B-experimental_method +measurements O +( O +KD B-evidence += O +26 O +. O +7 O +μM O +; O +Fig O +. O +2a O +, O +d O +). O + +Taken O +together O +, O +UHRF1 B-protein +seems O +to O +adopt O +a O +closed B-protein_state +form O +through O +intramolecular O +interactions O +( O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +and O +PHD B-structure_element +- I-structure_element +SRA I-structure_element +), O +which O +inhibit O +histone B-protein_type +H3 B-protein_type +tail O +recognition O +by O +UHRF1 B-protein +. O + +Overall O +structure B-evidence +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element + +To O +investigate O +the O +intramolecular O +interaction O +within O +UHRF1 B-protein +, O +we O +first O +mapped O +the O +minimal O +regions O +within O +the O +Spacer B-structure_element +for O +the O +interaction O +with O +the O +TTD B-structure_element +( O +Supplementary O +Fig O +. O +2a O +). O + +Internal O +deletions B-experimental_method +of O +the O +Spacer B-structure_element +, O +including O +SpacerΔ660 B-mutant +– I-mutant +664 I-mutant +, O +SpacerΔ665 B-mutant +– I-mutant +669 I-mutant +, O +SpacerΔ670 B-mutant +– I-mutant +674 I-mutant +and O +Spacer642 B-mutant +– I-mutant +674 I-mutant +, O +bound B-protein_state +to I-protein_state +the O +TTD B-structure_element +with O +comparable O +binding B-evidence +affinities I-evidence +to O +that O +of O +the O +Spacer B-structure_element +, O +whereas O +Spacer587 B-mutant +– I-mutant +641 I-mutant +showed O +no O +detectable O +interaction O +. O + +We O +next O +determined O +the O +solution B-evidence +structure I-evidence +of O +the O +TTD B-structure_element +( O +residues O +134 B-residue_range +– I-residue_range +285 I-residue_range +) O +bound B-protein_state +to I-protein_state +Spacer627 B-residue_range +– I-residue_range +674 I-residue_range +by O +conventional O +NMR B-experimental_method +techniques O +( O +Supplementary O +Table O +3 O +and O +Supplementary O +Fig O +. O +3a O +, O +b O +). O + +In O +the O +complex B-evidence +structure I-evidence +, O +each O +Tudor B-structure_element +domain I-structure_element +adopts O +a O +‘ B-structure_element +Royal I-structure_element +' I-structure_element +fold I-structure_element +containing O +a O +characteristic O +five B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +and O +the O +two O +Tudor B-structure_element +domains I-structure_element +tightly O +pack O +against O +each O +other O +with O +a O +buried O +area O +of O +573 O +Å2 O +( O +Fig O +. O +3a O +). O + +The O +TTD B-structure_element +adopts O +similar O +fold O +to O +that O +in O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +complex O +structure B-evidence +( O +PDB O +: O +4GY5 O +) O +with O +a O +root B-evidence +- I-evidence +mean I-evidence +- I-evidence +square I-evidence +deviation I-evidence +of O +1 O +. O +09 O +Å O +for O +128 O +Cα O +atoms O +, O +indicating O +that O +the O +Spacer B-structure_element +does O +not O +result O +in O +obvious O +conformational O +change O +of O +the O +TTD B-structure_element +( O +Fig O +. O +3b O +). O + +The O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +is O +mediated O +by O +a O +number O +of O +hydrogen B-bond_interaction +bonds I-bond_interaction +( O +Fig O +. O +3d O +). O + +The O +side O +chain O +of O +residue O +K648 B-residue_name_number +forms O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +carbonyl O +oxygen O +atom O +of O +D189 B-residue_name_number +and O +side O +chain O +of O +D190 B-residue_name_number +of O +the O +TTD B-structure_element +. O + +The O +side O +chain O +of O +residue O +R649 B-residue_name_number +packs B-bond_interaction +against I-bond_interaction +an O +acidic O +surface O +mainly O +formed O +by O +residues O +D142 B-residue_name_number +and O +E153 B-residue_name_number +. O + +Residue O +S651 B-residue_name_number +forms O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +main O +chain O +of O +residues O +G236 B-residue_name_number +and O +W238 B-residue_name_number +. O + +The O +interaction O +is O +further O +supported O +by O +hydrogen B-bond_interaction +bonds I-bond_interaction +formed O +between O +residues O +K650 B-residue_name_number +, O +A652 B-residue_name_number +, O +G653 B-residue_name_number +and O +G654 B-residue_name_number +of O +the O +Spacer B-structure_element +and O +residues O +N228 B-residue_name_number +, O +G236 B-residue_name_number +and O +W238 B-residue_name_number +of O +the O +TTD B-structure_element +, O +respectively O +. O + +In O +support O +of O +above O +structural B-experimental_method +analyses I-experimental_method +, O +mutation B-experimental_method +D142A B-mutant +/ O +E153A B-mutant +of O +the O +TTD B-structure_element +abolished O +its O +interaction O +with O +the O +Spacer B-structure_element +( O +Fig O +. O +3e O +). O + +As O +negative O +control O +, O +mutations B-experimental_method +S639D B-mutant +and O +S666D B-mutant +of O +the O +Spacer B-structure_element +showed O +little O +effect O +on O +the O +interaction O +. O + +Interestingly O +, O +phosphorylation B-ptm +at O +residue O +S651 B-residue_name_number +of O +UHRF1 B-protein +was O +observed O +in O +previous O +mass B-experimental_method +- I-experimental_method +spectrometry I-experimental_method +analyses O +. O + +Compared O +with O +the O +unmodified B-protein_state +peptide O +of O +Spacer642 B-mutant +– I-mutant +664 I-mutant +, O +a O +phosphorylation B-ptm +at O +S651 B-residue_name_number +markedly O +decreased O +the O +binding B-evidence +affinity I-evidence +to O +the O +TTD B-structure_element +( O +Supplementary O +Fig O +. O +2b O +), O +suggesting O +that O +the O +phosphorylation B-ptm +may O +regulate O +the O +intramolecular O +interaction O +within O +UHRF1 B-protein +. O + +The O +spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +by O +competing O +with O +the O +linker B-structure_element + +Previous O +studies O +indicate O +that O +the O +TTD B-structure_element +binds B-protein_state +to I-protein_state +a O +linker B-structure_element +region I-structure_element +connecting O +the O +TTD B-structure_element +and O +PHD B-structure_element +( O +residues O +286 B-residue_range +– I-residue_range +306 I-residue_range +, O +designated O +Linker B-structure_element +, O +Fig O +. O +1a O +), O +and O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +interaction O +is O +essential O +for O +H3K9me3 B-protein_type +recognition O +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +. O + +In O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +structure B-evidence +, O +residues O +R295 B-residue_name_number +, O +R296 B-residue_name_number +and O +S298 B-residue_name_number +of O +the O +Linker B-structure_element +adopt O +almost O +identical O +conformation O +to O +residues O +K648 B-residue_name_number +, O +R649 B-residue_name_number +and O +S651 B-residue_name_number +of O +the O +Spacer B-structure_element +in O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +structure B-evidence +, O +respectively O +. O + +Similar O +intramolecular O +contacts O +( O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +and O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +) O +were O +observed O +in O +the O +two O +structures B-evidence +( O +Fig O +. O +3b O +, O +d O +and O +Supplementary O +Fig O +. O +4a O +). O + +To O +test O +this O +hypothesis O +, O +we O +first O +investigated O +the O +potential O +competition O +between O +the O +Linker B-structure_element +and O +the O +Spacer B-structure_element +for O +their O +interaction O +with O +the O +TTD B-structure_element +. O + +The O +competitive B-experimental_method +ITC I-experimental_method +experiments O +show O +that O +TTD B-evidence +– I-evidence +Spacer I-evidence +binding I-evidence +affinity I-evidence +decreased O +by O +a O +factor O +of O +two O +in O +the O +presence B-protein_state +of I-protein_state +the O +Linker B-structure_element +, O +whereas O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +interaction O +was O +abolished O +in O +the O +presence B-protein_state +of I-protein_state +the O +Spacer B-structure_element +( O +Supplementary O +Fig O +. O +4c O +). O + +Compared O +with O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +( O +KD B-evidence += O +1 O +. O +48 O +μM O +), O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +decreased O +the O +binding B-evidence +affinity I-evidence +to O +the O +Spacer B-structure_element +( O +KD B-evidence += O +10 O +. O +68 O +μM O +), O +whereas O +mutation B-experimental_method +R295D B-mutant +/ O +R296D B-mutant +( O +within O +the O +Linker B-structure_element +and O +important O +for O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +interaction O +) O +of O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +showed O +minor O +decrease O +in O +the O +binding B-evidence +affinity I-evidence +( O +KD B-evidence += O +2 O +. O +69 O +μM O +; O +Fig O +. O +3g O +), O +indicating O +a O +competition O +between O +the O +Spacer B-structure_element +and O +the O +Linker B-structure_element +on O +the O +same O +binding B-site +site I-site +of O +the O +TTD B-structure_element +. O + +As O +indicated O +in O +Fig O +. O +3h O +, O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +UHRF1ΔSRA B-mutant +showed O +no O +interaction O +with O +GST B-protein_state +- I-protein_state +tagged I-protein_state +TTD B-structure_element +, O +Linker B-structure_element +or O +Spacer B-structure_element +, O +suggesting O +that O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +in B-protein_state +- I-protein_state +cis I-protein_state +within O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +or O +UHRF1ΔSRA B-mutant +prohibits O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +complex O +formation O +in B-protein_state +- I-protein_state +trans I-protein_state +. O + +In O +contrast O +, O +UHRF1ΔTTD B-mutant +bound B-protein_state +to I-protein_state +GST B-experimental_method +- O +TTD B-structure_element +, O +and O +UHRF1Δ627 B-mutant +– I-mutant +674 I-mutant +bound B-protein_state +to I-protein_state +GST B-experimental_method +- O +Spacer B-structure_element +, O +indicating O +that O +lack O +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +in B-protein_state +- I-protein_state +cis I-protein_state +, O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +complex O +could O +form O +in B-protein_state +- I-protein_state +trans I-protein_state +, O +supporting O +that O +the O +TTD B-structure_element +binds B-protein_state +to I-protein_state +the O +Spacer B-structure_element +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +. O + +Moreover O +, O +GST B-experimental_method +- O +Linker B-structure_element +showed O +very O +weak O +if O +not O +undetectable O +interaction O +with O +wild B-protein_state +- I-protein_state +type I-protein_state +or O +deletions O +of O +UHRF1 B-protein +, O +suggesting O +that O +TTD B-structure_element +– I-structure_element +Linker I-structure_element +interaction O +is O +much O +weaker O +than O +that O +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +. O + +The O +spacer B-structure_element +inhibits O +H3K9me3 B-protein_type +recognition O +by O +the O +isolated O +TTD B-structure_element + +Our O +previous O +study O +indicates O +that O +H3K9me3 B-protein_type +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +in O +different O +manner O +in O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +( O +ref O +.) O +and O +TTD B-complex_assembly +- I-complex_assembly +H3K9me3 I-complex_assembly +( O +PDB O +: O +2L3R O +) O +structures B-evidence +. O + +As O +shown O +in O +Supplementary O +Fig O +. O +4e O +, O +the O +Spacer B-structure_element +inhibited O +TTD B-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +interaction O +, O +whereas O +its O +TTD B-protein_state +- I-protein_state +binding I-protein_state +defective I-protein_state +mutants B-protein_state +of O +the O +Spacer B-structure_element +or O +the O +SRA B-structure_element +( O +a O +negative O +control O +) O +markedly O +decreased O +the O +inhibition O +. O + +Compared O +with O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +, O +UHRF1Δ627 B-mutant +– I-mutant +674 I-mutant +enhanced O +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +by O +a O +factor O +of O +four O +( O +Supplementary O +Fig O +. O +4f O +). O + +The O +restoration O +of O +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +is O +not O +dramatic O +because O +the O +PHD B-structure_element +still O +binds B-protein_state +to I-protein_state +histone B-protein_type +H3 B-protein_type +in O +both O +proteins O +. O + +To O +exclude O +this O +effect O +, O +we O +performed O +the O +assay O +using O +UHRF1D334A B-mutant +, O +which O +abolishes B-protein_state +H3R2 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +PHD B-structure_element +. O + +UHRF1D334A B-mutant +showed O +undetectable O +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +, O +whereas O +UHRF1D334A B-mutant +& O +Δ627 B-mutant +– I-mutant +674 I-mutant +dramatically O +restored O +its O +H3K9me3 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +( O +KD B-evidence += O +8 O +. O +69 O +μM O +; O +Supplementary O +Fig O +. O +4f O +), O +indicating O +that O +H3K9me3 B-protein_type +recognition O +by O +the O +TTD B-structure_element +is O +blocked O +by O +the O +Spacer B-structure_element +through O +competitive O +interaction O +with O +the O +TTD B-structure_element +. O + +Moreover O +, O +the O +R295D B-mutant +/ O +R296D B-mutant +mutant B-protein_state +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +showed O +decreased O +binding B-evidence +affinity I-evidence +to O +H3K9me3 B-protein_type +( O +eightfold O +lower O +than O +wild B-protein_state +type I-protein_state +), O +suggesting O +that O +mutation B-experimental_method +of O +R295D B-mutant +/ O +R296D B-mutant +favours O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +and O +therefore O +promotes O +UHRF1 B-protein +to O +exhibit O +a O +more O +stable O +closed B-protein_state +conformation O +( O +Supplementary O +Fig O +. O +4g O +). O + +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +complex O +inhibits O +TTD B-structure_element +– I-structure_element +spacer I-structure_element +interaction O + +Interestingly O +, O +pre B-experimental_method +- I-experimental_method +incubation I-experimental_method +of O +H3K9me3 B-protein_type +peptide O +completely O +blocked O +the O +interaction O +between O +the O +Spacer B-structure_element +and O +the O +TTD B-structure_element +alone B-protein_state +or O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +( O +Supplementary O +Fig O +. O +4h O +), O +whereas O +the O +presence B-protein_state +of I-protein_state +the O +Spacer B-structure_element +partially O +impaired O +the O +interaction O +between O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +and O +H3K9me3 B-protein_type +( O +Fig O +. O +2c O +). O + +The O +results O +are O +also O +consistent O +with O +the O +previous O +observation O +that O +the O +interaction O +between O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +and O +the O +Spacer B-structure_element +is O +much O +weaker O +( O +KD B-evidence += O +10 O +. O +68 O +μM O +, O +Fig O +. O +3g O +) O +than O +that O +between O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +and O +H3K9me3 B-protein_type +( O +KD B-evidence += O +0 O +. O +15 O +μM O +, O +Fig O +. O +1d O +). O + +These O +results O +suggest O +that O +once O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +binds B-protein_state +to I-protein_state +H3K9me3 B-protein_type +, O +UHRF1 B-protein +will O +be O +locked O +by O +H3K9me3 B-protein_type +and O +the O +Spacer B-structure_element +is O +unlikely O +to O +fold O +back O +for O +the O +intramolecular O +interaction O +. O + +Hm B-chemical +- I-chemical +DNA I-chemical +disrupts O +intramolecular O +interaction O +within O +UHRF1 B-protein + +To O +investigate O +whether O +hm B-chemical +- I-chemical +DNA I-chemical +could O +open B-protein_state +the O +closed B-protein_state +conformation O +of O +UHRF1 B-protein +, O +we O +first O +measured O +the O +intramolecular O +interaction O +using O +UHRF1 B-protein +truncations B-experimental_method +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +. O + +The O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +show O +that O +the O +PHD B-structure_element +bound B-protein_state +to I-protein_state +the O +SRA B-structure_element +and O +such O +interaction O +was O +impaired O +by O +the O +addition O +of O +hm B-chemical +- I-chemical +DNA I-chemical +( O +Fig O +. O +4a O +). O + +H3 B-experimental_method +peptide I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +show O +that O +hm B-chemical +- I-chemical +DNA I-chemical +only O +enhanced O +the O +H3K9me0 B-evidence +- I-evidence +binding I-evidence +affinities I-evidence +of O +UHRF1 B-protein +truncations B-experimental_method +containing O +PHD B-structure_element +- I-structure_element +SRA I-structure_element +, O +such O +as O +PHD B-structure_element +- I-structure_element +SRA I-structure_element +, O +TTD B-structure_element +- I-structure_element +PHD I-structure_element +- I-structure_element +SRA I-structure_element +, O +TTD B-structure_element +- I-structure_element +PHD I-structure_element +- I-structure_element +SRA I-structure_element +- I-structure_element +Spacer I-structure_element +, O +UHRF1ΔTTD B-mutant +and O +UHRF1ΔSpacer B-mutant +( O +Fig O +. O +4b O +). O + +The O +result O +indicates O +that O +hm B-chemical +- I-chemical +DNA I-chemical +disrupts O +PHD B-structure_element +– I-structure_element +SRA I-structure_element +interaction O +and O +facilitates O +H3K9me0 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +PHD B-structure_element +in O +a O +manner O +independent O +on O +the O +TTD B-structure_element +or O +the O +Spacer B-structure_element +. O + +Moreover O +, O +the O +TTD B-structure_element +or O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +bound B-protein_state +to I-protein_state +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +and O +the O +interaction O +was O +impaired O +by O +the O +addition O +of O +hm B-chemical +- I-chemical +DNA I-chemical +( O +Fig O +. O +4c O +). O + +The O +ITC B-experimental_method +measurements O +show O +that O +the O +presence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +markedly O +impaired O +the O +interaction O +between O +the O +TTD B-structure_element +and O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +( O +Supplementary O +Fig O +. O +5a O +). O + +However O +, O +the O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +was O +not O +affected O +by O +the O +presence B-protein_state +of I-protein_state +the O +hm B-chemical +- I-chemical +DNA I-chemical +, O +indicating O +that O +hm B-chemical +- I-chemical +DNA I-chemical +displaces O +the O +Spacer B-structure_element +from O +the O +TTD B-structure_element +in O +a O +SRA B-structure_element +- O +dependent O +manner O +( O +Supplementary O +Fig O +. O +5b O +). O + +To O +investigate O +whether O +hm B-chemical +- I-chemical +DNA I-chemical +disrupts O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +, O +we O +monitored O +the O +conformational O +changes O +of O +UHRF1 B-protein +using O +its O +histone B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +as O +read O +- O +out O +. O + +UHRF1D334A B-mutant +was O +used O +to O +exclude O +the O +effect O +of O +H3K9me0 B-protein_type +recognition O +by O +the O +PHD B-structure_element +. O + +As O +expected O +, O +all O +D334A B-mutant +- O +containing O +mutants B-protein_state +showed O +undetectable O +interaction O +with O +H3K9me0 B-protein_type +( O +Fig O +. O +4d O +). O + +UHRF1D334A B-mutant +bound B-protein_state +to I-protein_state +H3K9me3 B-protein_type +peptide O +in O +the O +presence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +, O +but O +showed O +no O +interaction O +in O +the O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +, O +which O +is O +consistent O +with O +the O +ITC B-experimental_method +experiments O +( O +Supplementary O +Fig O +. O +4f O +). O + +In O +contrast O +, O +UHRF1D334A B-mutant +& O +Δ627 B-mutant +– I-mutant +674 I-mutant +strongly O +bound B-protein_state +to I-protein_state +H3K9me3 B-protein_type +even O +in O +the O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +( O +Fig O +. O +4d O +), O +indicating O +that O +the O +deletion B-experimental_method +of O +the O +Spacer B-structure_element +releases O +otherwise O +blocked O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +for O +H3K9me3 B-protein_type +recognition O +. O + +The O +results O +further O +support O +the O +conclusion O +that O +the O +Spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +the O +intramolecular O +interactions O +are O +disrupted O +by O +hm B-chemical +- I-chemical +DNA I-chemical +. O + +We O +next O +performed O +similar O +peptide B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +using O +two O +mutants B-protein_state +( O +N228C B-mutant +/ O +G653C B-mutant +and O +R235C B-mutant +/ O +G654C B-mutant +) O +generated O +on O +UHRF1D334A B-mutant +. O + +As O +negative O +controls O +, O +H3K9me3 B-protein_type +recognition O +by O +UHRF1D334A B-mutant +or O +UHRF1D334A B-mutant +& O +Δ627 B-mutant +– I-mutant +674 I-mutant +is O +not O +affected O +by O +DTT B-chemical +. O + +We O +have O +previously O +demonstrated O +that O +hm B-chemical +- I-chemical +DNA I-chemical +also O +disrupts O +PHD B-structure_element +– I-structure_element +SRA I-structure_element +interaction O +and O +facilitates O +H3K9me0 B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +PHD B-structure_element +in O +a O +manner O +independent O +on O +the O +TTD B-structure_element +or O +the O +Spacer B-structure_element +. O + +Taken O +together O +, O +hm B-chemical +- I-chemical +DNA I-chemical +disrupts O +the O +intramolecular O +interactions O +within O +UHRF1 B-protein +, O +and O +therefore O +facilitates O +the O +coordinate O +recognition O +of O +H3K9me3 B-protein_type +by O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +. O + +The O +spacer B-structure_element +enhances O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +SRA B-structure_element + +To O +investigate O +how O +hm B-chemical +- I-chemical +DNA I-chemical +impairs O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +, O +we O +tested O +whether O +the O +Spacer B-structure_element +is O +involved O +in O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +by O +the O +SRA B-structure_element +, O +which O +is O +the O +only O +known O +domain O +for O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +within O +UHRF1 B-protein +. O + +In O +the O +electrophoretic B-experimental_method +mobility I-experimental_method +- I-experimental_method +shift I-experimental_method +assay I-experimental_method +, O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +showed O +higher O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +than O +the O +SRA B-structure_element +alone B-protein_state +( O +Supplementary O +Fig O +. O +6a O +). O + +ITC B-experimental_method +measurements O +show O +that O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +bound B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +with O +a O +much O +higher O +binding B-evidence +affinity I-evidence +( O +KD B-evidence += O +1 O +. O +75 O +μM O +) O +than O +the O +SRA B-structure_element +( O +KD B-evidence += O +25 O +. O +12 O +μM O +), O +whereas O +the O +Spacer B-structure_element +alone B-protein_state +showed O +no O +interaction O +with O +hm B-chemical +- I-chemical +DNA I-chemical +( O +Fig O +. O +5a O +). O + +In O +the O +fluorescence B-experimental_method +polarization I-experimental_method +( I-experimental_method +FP I-experimental_method +) O +measurements O +, O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +, O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +UHRF1ΔTTD B-mutant +showed O +comparable O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinities I-evidence +( O +Fig O +. O +5b O +and O +Supplementary O +Table O +4 O +), O +suggesting O +that O +UHRF1 B-protein +binds B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +no O +matter O +UHRF1 B-protein +adopts O +a O +closed B-protein_state +form O +or O +not O +. O + +In O +contrast O +, O +UHRF1ΔSRA B-mutant +abolished O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +, O +indicating O +that O +the O +SRA B-structure_element +is O +essential O +for O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +. O + +Compared O +with O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +, O +UHRF1Δ627 B-mutant +– I-mutant +674 I-mutant +decreased O +the O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +by O +a O +factor O +of O +14 O +( O +Fig O +. O +5b O +), O +further O +supporting O +that O +the O +Spacer B-structure_element +plays O +an O +important O +role O +in O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +. O + +In O +addition O +, O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinities I-evidence +of O +SRA B-structure_element +or O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +did O +not O +obviously O +vary O +upon O +the O +change O +of O +DNA O +lengths O +but O +did O +decrease O +with O +the O +increasing O +salt O +concentrations O +( O +Supplementary O +Fig O +. O +6b O +, O +c O +and O +Supplementary O +Table O +5 O +). O + +We O +next O +mapped O +the O +minimal O +region O +of O +the O +Spacer B-structure_element +for O +the O +enhancement O +of O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +. O + +SRA B-mutant +– I-mutant +Spacer I-mutant +- I-mutant +661 I-mutant +( O +residues O +414 B-residue_range +– I-residue_range +661 I-residue_range +) O +still O +maintained O +strong O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +comparable O +to O +that O +of O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +( O +residues O +414 B-residue_range +– I-residue_range +674 I-residue_range +), O +whereas O +SRA B-mutant +– I-mutant +Spacer I-mutant +- I-mutant +652 I-mutant +and O +SRA B-mutant +– I-mutant +Spacer I-mutant +- I-mutant +642 I-mutant +markedly O +decreased O +their O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinities I-evidence +( O +Fig O +. O +5c O +), O +indicating O +that O +residues O +642 B-residue_range +– I-residue_range +661 I-residue_range +are O +important O +for O +enhancing O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +SRA B-structure_element +. O + +This O +minimal O +region O +largely O +overlaps O +with O +the O +Spacer B-structure_element +region O +( O +643 B-residue_range +– I-residue_range +655 I-residue_range +) O +essential O +for O +TTD B-structure_element +interaction O +. O + +We O +also O +determined O +the O +crystal B-evidence +structure I-evidence +of O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +bound B-protein_state +to I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +at O +3 O +. O +15 O +Å O +resolution O +( O +Supplementary O +Table O +6 O +and O +Supplementary O +Fig O +. O +7a O +). O + +Intriguingly O +, O +no O +electron B-evidence +density I-evidence +was O +observed O +for O +the O +Spacer B-structure_element +. O + +A O +possible O +explanation O +is O +that O +the O +Spacer B-structure_element +facilitates O +SRA B-complex_assembly +– I-complex_assembly +hm I-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +interaction O +through O +nonspecific O +salt B-bond_interaction +bridge I-bond_interaction +contacts O +because O +DNA B-chemical +is O +rich O +in O +acidic O +groups O +and O +the O +Spacer B-structure_element +is O +rich O +in O +basic O +residues O +( O +Supplementary O +Fig O +. O +7b O +). O + +The O +nonspecific O +interaction O +is O +consistent O +with O +the O +previous O +observation O +that O +UHRF1 B-protein +has O +no O +DNA B-chemical +sequence O +selectivity O +besides O +hm B-chemical +- I-chemical +CpG I-chemical +dinucleotide I-chemical +. O + +The O +spacer B-structure_element +is O +important O +for O +PCH O +localization O +of O +UHRF1 B-protein + +For O +the O +NIH3T3 O +cells O +expressing O +wild B-protein_state +- I-protein_state +type I-protein_state +UHRF1 B-protein +, O +most O +cells O +(∼ O +74 O +. O +6 O +%) O +showed O +a O +focal O +pattern O +of O +protein O +that O +is O +co O +- O +localized O +with O +4 B-chemical +, I-chemical +6 I-chemical +- I-chemical +diamidino I-chemical +- I-chemical +2 I-chemical +- I-chemical +phenylindole I-chemical +( O +DAPI B-chemical +) O +foci O +( O +Fig O +. O +5d O +), O +whereas O +the O +rest O +cells O +showed O +a O +diffuse O +nuclear O +staining O +pattern O +. O + +The O +result O +is O +consistent O +with O +the O +previous O +studies O +that O +UHRF1 B-protein +is O +mainly O +localized O +to O +highly B-protein_state +methylated I-protein_state +pericentromeric O +heterochromatin O +( O +PCH O +). O + +In O +contrast O +, O +for O +the O +cells O +expressing O +UHRF1Δ627 B-mutant +– I-mutant +674 I-mutant +, O +a O +spacer B-protein_state +deletion I-protein_state +mutant I-protein_state +with O +decreased O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +( O +Fig O +. O +5b O +), O +only O +∼ O +22 O +. O +1 O +% O +cells O +showed O +co O +- O +localization O +with O +DAPI B-chemical +. O + +Previous O +reports O +have O +shown O +that O +the O +H3K9me3 B-protein_type +recognition O +of O +UHRF1 B-protein +also O +plays O +an O +important O +role O +in O +its O +heterochromatin O +localization O +. O + +Because O +manipulation O +of O +endogenous O +hm B-chemical +- I-chemical +DNA I-chemical +in O +cells O +is O +technically O +challenging O +, O +we O +used O +UHRF1ΔSRA B-mutant +( O +lacks B-protein_state +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +but O +maintains O +closed B-protein_state +conformation O +, O +Figs O +3h O +and O +5b O +) O +to O +test O +whether O +closed B-protein_state +conformation O +of O +UHRF1 B-protein +exists O +in O +vivo O +. O + +In O +NIH3T3 O +cells O +, O +UHRF1ΔSRA B-mutant +largely O +decreased O +chromatin O +association O +( O +Fig O +. O +5d O +). O + +Only O +∼ O +4 O +. O +8 O +% O +cells O +expressing O +UHRF1ΔSRA B-mutant +showed O +an O +intermediate O +enrichment O +, O +but O +not O +characteristic O +focal O +pattern O +, O +at O +DAPI B-chemical +foci O +, O +whereas O +the O +majority O +of O +the O +cells O +showed O +a O +diffuse O +nuclear O +staining O +pattern O +. O + +The O +spacer B-structure_element +facilitates O +UHRF1 B-complex_assembly +– I-complex_assembly +DNMT1 I-complex_assembly +interaction O + +Previous O +studies O +show O +that O +UHRF1 B-protein +recruits O +DNMT1 B-protein +to O +hm B-chemical +- I-chemical +DNA I-chemical +for O +maintenance O +DNA B-chemical +methylation B-ptm +through O +the O +interaction O +between O +the O +SRA B-structure_element +and O +RFTSDNMT1 B-protein +( O +refs O +). O + +We O +confirmed O +the O +direct O +interaction O +between O +RFTSDNMT1 B-protein +and O +the O +SRA B-structure_element +in O +a O +solution O +with O +low O +salt O +concentration O +( O +50 O +mM O +NaCl B-chemical +), O +but O +observed O +weak O +or O +undetectable O +interaction O +in O +a O +solution O +with O +higher O +salt O +concentrations O +( O +100 O +or O +150 O +mM O +NaCl B-chemical +) O +( O +Supplementary O +Fig O +. O +8a O +). O + +Compared O +with O +the O +SRA B-structure_element +, O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +exhibited O +stronger O +interaction O +with O +RFTSDNMT1 B-protein +. O + +In O +addition O +, O +RFTSDNMT1 B-protein +bound B-protein_state +to I-protein_state +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +with O +a O +binding B-evidence +affinity I-evidence +of O +7 O +. O +09 O +μM O +, O +but O +showed O +no O +detectable O +interaction O +with O +the O +SRA B-structure_element +( O +Supplementary O +Fig O +. O +8b O +). O + +Interestingly O +, O +the O +addition O +of O +hm B-chemical +- I-chemical +DNA I-chemical +abolished O +the O +interaction O +between O +RFTSDNMT1 B-protein +and O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +, O +suggesting O +that O +hm B-chemical +- I-chemical +DNA I-chemical +also O +regulates O +UHRF1 B-complex_assembly +– I-complex_assembly +DNMT1 I-complex_assembly +interaction O +( O +Supplementary O +Fig O +. O +8c O +). O + +These O +results O +indicate O +that O +the O +Spacer B-structure_element +facilitates O +the O +interaction O +between O +RFTSDNMT1 B-protein +and O +the O +SRA B-structure_element +, O +and O +the O +interaction O +is O +impaired O +by O +the O +presence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +. O + +We O +next O +tested O +whether O +the O +UHRF1 B-complex_assembly +– I-complex_assembly +DNMT1 I-complex_assembly +interaction O +is O +regulated O +by O +the O +conformational O +change O +of O +UHRF1 B-protein +. O + +Because O +the O +addition O +of O +hm B-chemical +- I-chemical +DNA I-chemical +disrupts O +the O +interaction O +between O +the O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +and O +RFTSDNMT1 B-protein +, O +we O +used O +various O +truncations B-experimental_method +to O +mimic O +open B-protein_state +and O +closed B-protein_state +forms O +of O +UHRF1 B-protein +. O + +As O +the O +deletion B-experimental_method +of I-experimental_method +the O +TTD B-structure_element +allows O +UHRF1 B-protein +to O +adopt O +an O +open B-protein_state +conformation O +, O +the O +results O +suggest O +that O +RFTSDNMT1 B-protein +binds B-protein_state +to I-protein_state +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +when O +UHRF1 B-protein +adopts O +an O +open B-protein_state +conformation O +in O +the O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +. O + +In O +support O +of O +above O +observations O +, O +the O +addition B-experimental_method +of O +large O +amount O +of O +RFTSDNMT1 B-protein +impaired O +the O +interaction O +between O +UHRF1 B-protein +and O +hm B-chemical +- I-chemical +DNA I-chemical +( O +Supplementary O +Fig O +. O +8d O +), O +suggesting O +an O +existence O +of O +dynamic O +equilibrium O +between O +UHRF1 B-complex_assembly +– I-complex_assembly +hm I-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +and O +UHRF1 B-complex_assembly +– I-complex_assembly +DNMT1 I-complex_assembly +complexes O +. O + +According O +to O +the O +above O +results O +, O +we O +here O +proposed O +a O +working O +model O +for O +hm B-chemical +- I-chemical +DNA I-chemical +- O +mediated O +regulation O +of O +UHRF1 B-protein +conformation O +( O +Fig O +. O +5f O +). O + +In O +the O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +( O +A O +), O +UHRF1 B-protein +prefers O +a O +closed B-protein_state +conformation O +, O +in O +which O +the O +Spacer B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +by O +competing O +with O +the O +Linker B-structure_element +and O +the O +SRA B-structure_element +binds B-protein_state +to I-protein_state +the O +PHD B-structure_element +. O + +As O +a O +result O +, O +the O +recognition O +of O +histone B-protein_type +H3K9me3 B-protein_type +by O +the O +TTD B-structure_element +is O +blocked O +by O +the O +Spacer B-structure_element +, O +and O +recognition O +of O +unmodified B-protein_state +histone B-protein_type +H3 B-protein_type +( O +H3R2 B-site +) O +by O +the O +PHD B-structure_element +is O +inhibited O +by O +the O +SRA B-structure_element +. O + +The O +interaction O +between O +UHRF1 B-protein +and O +DNMT1 B-protein +is O +also O +weak O +because O +the O +Spacer B-structure_element +is O +unable O +to O +facilitate O +the O +intermolecular O +interaction O +. O + +In O +the O +presence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +( O +B O +), O +UHRF1 B-protein +prefers O +an O +open B-protein_state +conformation O +, O +in O +which O +the O +SRA B-structure_element +binds B-protein_state +to I-protein_state +the O +hm B-chemical +- I-chemical +DNA I-chemical +; O +the O +Spacer B-structure_element +dissociates O +from O +the O +TTD B-structure_element +and O +facilitates O +the O +interaction O +between O +the O +SRA B-structure_element +and O +hm B-chemical +- I-chemical +DNA I-chemical +; O +the O +Linker B-structure_element +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +and O +allows O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +to O +recognize O +histone B-protein_type +H3K9me3 B-protein_type +. O + +When O +UHRF1 B-protein +adopts O +an O +open B-protein_state +conformation O +and O +has O +already O +bound B-protein_state +to I-protein_state +H3K9me3 B-protein_type +( O +B O +), O +the O +interaction O +between O +H3K9me3 B-protein_type +and O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +further O +prevents O +the O +Spacer B-structure_element +from O +folding O +back O +to O +interact O +with O +the O +TTD B-structure_element +, O +and O +therefore O +locks O +UHRF1 B-protein +in O +an O +open B-protein_state +conformation O +. O + +The O +association O +of O +UHRF1 B-protein +to O +the O +histone B-protein_type +may O +facilitate O +the O +ubiquitination B-ptm +of O +histone B-protein_type +tail O +( O +mediated O +by O +RING B-structure_element +domain O +) O +for O +DNMT1 B-protein +targeting O +. O + +Moreover O +, O +through O +a O +mechanism O +yet O +to O +be O +fully O +elucidated O +, O +DNMT1 B-protein +targets O +hm B-chemical +- I-chemical +DNA I-chemical +for O +maintenance O +DNA O +methylation B-ptm +, O +probably O +through O +interaction O +with O +the O +histone B-protein_type +ubiquitylation B-ptm +and O +/ O +or O +SRA B-structure_element +- I-structure_element +Spacer I-structure_element +. O + +The O +P B-evidence +( I-evidence +r I-evidence +) I-evidence +function I-evidence +obtained O +from O +small B-experimental_method +- I-experimental_method +angle I-experimental_method +X I-experimental_method +- I-experimental_method +ray I-experimental_method +scattering I-experimental_method +( O +SAXS B-experimental_method +) O +measurements O +of O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +SRA I-complex_assembly +– I-complex_assembly +Spacer I-complex_assembly +– I-complex_assembly +hm I-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +complex O +showed O +a O +broader O +distribution O +than O +that O +of O +the O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +SRA I-complex_assembly +– I-complex_assembly +Spacer I-complex_assembly +alone O +, O +supporting O +the O +proposed O +model O +that O +UHRF1 B-protein +adopts O +an O +open B-protein_state +conformation O +in O +the O +presence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +( O +Supplementary O +Fig O +. O +8e O +). O + +We O +have O +tried O +crystallizing B-experimental_method +more O +than O +three O +sub O +- O +constructs O +with B-protein_state +and O +without B-protein_state +DNA B-chemical +across O +over O +1 O +, O +200 O +crystallization O +conditions O +but O +failed O +to O +determine O +the O +structure B-evidence +of O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +SRA I-complex_assembly +– I-complex_assembly +Spacer I-complex_assembly +in O +the O +absence B-protein_state +or O +presence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +. O + +Our O +previous O +studies O +show O +that O +phosphorylation B-ptm +at O +S639 B-residue_name_number +within O +the O +Spacer B-structure_element +disrupts O +interaction O +between O +UHRF1 B-protein +and O +deubiquitylase B-protein_type +USP7 B-protein +and O +decreases O +UHRF1 B-protein +stability O +in O +the O +M O +phase O +of O +the O +cell O +cycle O +. O + +The O +Spacer B-structure_element +was O +predicted O +to O +contain O +two O +nuclear B-structure_element +localization I-structure_element +signals I-structure_element +, O +residues O +581 B-residue_range +– I-residue_range +600 I-residue_range +and O +648 B-residue_range +- I-residue_range +670 I-residue_range +( O +ref O +.). O + +In O +this O +report O +, O +we O +found O +that O +the O +Spacer B-structure_element +( O +i O +) O +binds B-protein_state +to I-protein_state +the O +TTD B-structure_element +in O +the O +closed B-protein_state +form O +of O +UHRF1 B-protein +and O +inhibits O +its O +interaction O +with O +H3K9me3 B-protein_type +; O +( O +ii O +) O +facilitates O +hm B-chemical +- I-chemical +DNA I-chemical +recognition O +by O +the O +SRA B-structure_element +and O +( O +iii O +) O +facilitates O +the O +interaction O +between O +the O +SRA B-structure_element +and O +RFTSDNMT1 B-protein +. O + +The O +result O +suggests O +that O +PI5P B-chemical +may O +facilitate O +the O +conformational O +change O +of O +UHRF1 B-protein +induced O +by O +hm B-chemical +- I-chemical +DNA I-chemical +when O +UHRF1 B-protein +is O +recruited O +to O +chromatin O +. O + +In O +addition O +, O +mass B-experimental_method +- I-experimental_method +spectrometry I-experimental_method +analyses O +have O +identified O +several O +phosphorylation B-site +sites I-site +( O +S639 B-residue_name_number +, O +S651 B-residue_name_number +, O +S661 B-residue_name_number +) O +within O +the O +Spacer B-structure_element +, O +suggesting O +that O +post O +- O +translational O +modification O +may O +add O +another O +layer O +of O +regulation O +of O +UHRF1 B-protein +( O +refs O +). O + +It O +has O +been O +well O +characterized O +that O +the O +SRA B-structure_element +of O +UHRF1 B-protein +preferentially O +recognizes O +hm B-chemical +- I-chemical +DNA I-chemical +through O +a O +base O +- O +flipping O +mechanism O +. O + +Our O +study O +demonstrates O +that O +the O +Spacer B-structure_element +markedly O +enhances O +the O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +SRA B-structure_element +and O +the O +deletion B-experimental_method +of I-experimental_method +the O +Spacer B-structure_element +impairs O +heterochromatin O +localization O +of O +UHRF1 B-protein +, O +indicating O +that O +the O +Spacer B-structure_element +is O +essential O +for O +recognition O +of O +hm B-chemical +- I-chemical +DNA I-chemical +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +. O + +Thus O +, O +although O +UHRF2 B-protein +exhibits O +the O +histone O +- O +and O +hm O +- O +DNA O +- O +binding O +activities O +, O +the O +difference O +in O +the O +Spacer B-structure_element +region O +may O +contribute O +to O +the O +functional O +differences O +between O +UHRF1 B-protein +and O +UHRF2 B-protein +. O + +This O +is O +also O +consistent O +with O +previous O +finding O +that O +UHRF2 B-protein +is O +unable O +to O +replace O +UHRF1 B-protein +to O +maintain O +the O +DNA B-chemical +methylation B-ptm +. O + +However O +, O +little O +is O +known O +about O +the O +crosstalk O +between O +these O +two O +epigenetic O +marks O +within O +UHRF1 B-protein +. O + +We O +have O +shown O +that O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +and O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +strongly O +bind O +to O +hm B-chemical +- I-chemical +DNA I-chemical +( O +0 O +. O +35 O +and O +0 O +. O +49 O +μM O +, O +respectively O +) O +and O +the O +Spacer B-structure_element +plays O +an O +important O +role O +in O +PCH O +localization O +( O +Fig O +. O +5d O +). O + +As O +a O +result O +, O +when O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +dissociates O +from O +hm B-chemical +- I-chemical +DNA I-chemical +and O +binds B-protein_state +to I-protein_state +DNMT1 B-protein +with O +a O +currently O +unknown O +mechanism O +, O +UHRF1 B-protein +may O +keep O +the O +complex O +associated O +with O +chromatin O +through O +the O +interaction O +between O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +and O +H3K9me3 B-protein_type +( O +or O +PHD B-structure_element +- O +H3 B-protein_type +), O +and O +make O +it O +possible O +for O +DNMT1 B-protein +to O +target O +proper O +DNA B-chemical +substrate O +for O +methylation B-ptm +. O + +This O +explanation O +agrees O +nicely O +with O +previous O +observations O +and O +clarifies O +the O +importance O +of O +coordinate O +recognition O +of O +H3K9me3 B-protein_type +and O +hm B-chemical +- I-chemical +DNA I-chemical +by O +UHRF1 B-protein +for O +maintenance O +DNA O +methylation B-ptm +. O + +UHRF1 B-protein +is O +essential O +for O +maintenance O +DNA B-chemical +methylation B-ptm +through O +recruiting O +DNMT1 B-protein +to O +DNA B-chemical +replication O +forks O +during O +S O +phase O +. O + +DNMT1 B-protein +binds B-protein_state +to I-protein_state +ubiquitylated B-protein_state +histone B-protein_type +H3 B-protein_type +and O +ubiquitylation B-ptm +is O +required O +for O +maintenance O +of O +DNA B-chemical +methylation B-ptm +in O +vivo O +. O + +In O +this O +study O +, O +we O +found O +that O +both O +TTD B-structure_element +and O +PHD B-structure_element +are O +regulated O +by O +hm B-chemical +- I-chemical +DNA I-chemical +to O +recognize O +histone B-protein_type +tail O +. O + +Thus O +, O +the O +closed B-protein_state +form O +UHRF1 B-protein +may O +prevent O +miss O +localization O +of O +URHF1 B-protein +, O +whereas O +only O +the O +UHRF1 B-protein +in O +open B-protein_state +conformation O +( O +induced O +by O +hm B-chemical +- I-chemical +DNA I-chemical +) O +could O +properly O +binds B-protein_state +to I-protein_state +histone B-protein_type +tail O +for O +ubiquitylation B-ptm +and O +subsequent O +DNA B-chemical +methylation B-ptm +. O + +Binding O +of O +UHRF1 B-protein +to O +hm B-chemical +- I-chemical +DNA I-chemical +may O +serve O +as O +a O +switch O +for O +its O +recruitment O +of O +DNMT1 B-protein +. O + +Hm B-chemical +- I-chemical +DNA I-chemical +facilities O +histione B-protein_type +tails O +recognition O +by O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +. O + +( O +a O +) O +Colour O +- O +coded O +domain O +structure O +of O +human B-species +UHRF1 B-protein +. O + +Note O +that O +the O +conserved B-protein_state +motif O +( O +green O +background O +) O +of O +the O +Linker B-structure_element +( O +residues O +286 B-residue_range +– I-residue_range +306 I-residue_range +) O +and O +the O +Spacer B-structure_element +( O +residues O +587 B-residue_range +– I-residue_range +674 I-residue_range +) O +bind O +to O +the O +TTD B-structure_element +in O +a O +similar O +manner O +( O +Fig O +. O +3b O +). O +( O +b O +) O +Hm B-chemical +- I-chemical +DNA I-chemical +facilities O +histone B-protein_type +H3 B-protein_type +and O +H3K9me3 B-protein_type +recognition O +by O +UHRF1 B-protein +. O + +Purified O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +was O +incubated O +with O +biotinylated B-protein_state +H3 B-protein_type +( O +1 B-residue_range +– I-residue_range +21 I-residue_range +) O +or O +H3K9me3 B-protein_type +( O +1 B-residue_range +– I-residue_range +21 I-residue_range +) O +peptides O +in O +the O +presence O +or O +absence B-protein_state +of I-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +( O +molar O +ratio O +UHRF1 B-protein +/ O +hm B-chemical +- I-chemical +DNA I-chemical += O +1 O +: O +2 O +). O + +Full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +was O +incubated B-experimental_method +with I-experimental_method +biotinylated B-protein_state +hm B-chemical +- I-chemical +DNA I-chemical +in O +the O +presence O +or O +absence B-protein_state +of I-protein_state +H3 B-protein_type +( O +1 B-residue_range +– I-residue_range +17 I-residue_range +) O +or O +H3K9me3 B-protein_type +( O +1 B-residue_range +– I-residue_range +17 I-residue_range +) O +peptides O +and O +analysed O +as O +in O +b O +. O +( O +d O +, O +e O +) O +Superimposed O +ITC B-experimental_method +enthalpy B-evidence +plots I-evidence +for O +binding O +of O +H3K9me3 B-protein_type +peptide O +( O +1 B-residue_range +– I-residue_range +17 I-residue_range +) O +to O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +and O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +( O +d O +), O +and O +H3 B-protein_type +peptide O +( O +1 B-residue_range +– I-residue_range +17 I-residue_range +) O +to O +the O +PHD B-structure_element +and O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +( O +e O +). O + +The O +estimated O +binding B-evidence +affinities I-evidence +( O +KD B-evidence +) O +are O +listed O +. O + +( O +a O +) O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +for O +the O +intramolecular O +interactions O +. O + +The O +isolated O +domains O +of O +UHRF1 B-protein +were O +incubated B-experimental_method +with O +GST B-protein_state +- I-protein_state +tagged I-protein_state +TTD B-structure_element +or O +PHD B-structure_element +immobilized O +on O +glutathione O +resin O +. O + +The O +bound O +proteins O +were O +analysed O +by O +SDS B-experimental_method +– I-experimental_method +PAGE I-experimental_method +and O +Coomassie O +blue O +staining O +. O + +( O +b O +, O +d O +) O +Superimposed O +ITC B-experimental_method +enthalpy B-evidence +plots I-evidence +for O +the O +intramolecular O +interactions O +of O +isolated O +UHRF1 B-protein +domains O +. O + +ND O +, O +not O +detectable O +. O +( O +c O +) O +Superimposed O +ITC B-experimental_method +enthalpy B-evidence +plots I-evidence +for O +the O +binding O +of O +H3K9me3 B-protein_type +to O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +in O +the O +absence B-protein_state +or O +presence B-protein_state +of I-protein_state +the O +Spacer B-structure_element +( O +molar O +ratio O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +/ O +Spacer B-structure_element += O +1 O +: O +2 O +). O +( O +e O +) O +Superimposed O +ITC B-experimental_method +enthalpy B-evidence +plots I-evidence +for O +the O +binding O +of O +H3 B-protein_type +to O +PHD B-structure_element +– I-structure_element +SRA I-structure_element +or O +PHD B-structure_element +in O +the O +absence B-protein_state +or O +presence B-protein_state +of I-protein_state +the O +SRA B-structure_element +( O +molar O +ratio O +PHD B-structure_element +/ O +SRA B-structure_element += O +1 O +: O +1 O +or O +1 O +: O +2 O +). O + +NMR B-experimental_method +structure B-evidence +of O +the O +TTD B-structure_element +bound B-protein_state +to I-protein_state +the O +Spacer B-structure_element +. O + +( O +a O +) O +Ribbon O +representation O +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +structure B-evidence +. O + +N O +- O +and O +C O +- O +termini O +of O +the O +Spacer B-structure_element +are O +indicated O +. O + +The O +TTD B-structure_element +is O +coloured O +in O +green O +, O +and O +the O +Spacer B-structure_element +is O +coloured O +in O +yellow O +. O + +( O +b O +) O +Superimposition B-experimental_method +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +and O +TTD B-complex_assembly +– I-complex_assembly +PHD I-complex_assembly +– I-complex_assembly +H3K9me3 I-complex_assembly +( O +4GY5 O +. O +PDB O +) O +structures B-evidence +shown O +in O +ribbon O +representations O +. O + +The O +TTD B-structure_element +is O +coloured O +in O +green O +and O +the O +Spacer B-structure_element +in O +yellow O +in O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +structure B-evidence +. O + +( O +c O +) O +Electrostatic O +potential O +surface O +representation O +of O +the O +TTD B-structure_element +with O +the O +Spacer B-structure_element +shown O +in O +ribbon O +representation O +. O + +The O +critical O +residues O +on O +the O +Spacer B-structure_element +for O +the O +interaction O +are O +shown O +in O +stick O +representation O +. O + +( O +d O +) O +Close O +- O +up O +view O +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +interaction O +. O + +Hydrogen B-bond_interaction +bonds I-bond_interaction +are O +indicated O +as O +dashed O +lines O +. O + +( O +e O +– O +g O +) O +Superimposed O +ITC B-experimental_method +enthalpy B-evidence +plots I-evidence +for O +the O +interaction O +between O +the O +Spacer B-structure_element +and O +the O +TTD B-structure_element +( O +or O +TTD B-structure_element +– I-structure_element +PHD I-structure_element +) O +with O +the O +estimated O +binding B-evidence +affinity I-evidence +( O +KD B-evidence +) O +indicated O +. O + +Wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +proteins O +for O +the O +measurements O +are O +indicated O +. O + +( O +h O +) O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +for O +the O +intramolecular O +interactions O +. O + +The O +wild B-protein_state +- I-protein_state +type I-protein_state +or O +indicated O +truncations O +of O +UHRF1 B-protein +were O +incubated O +with O +GST B-protein_state +- I-protein_state +tagged I-protein_state +TTD B-structure_element +, O +Linker B-structure_element +or O +Spacer B-structure_element +. O + +Hm B-chemical +- I-chemical +DNA I-chemical +impairs O +the O +intramolecular O +interaction O +of O +UHRF1 B-protein +and O +facilitates O +its O +histone B-protein_type +recognition O +. O + +( O +a O +) O +Hm B-chemical +- I-chemical +DNA I-chemical +impairs O +the O +intramolecular O +interaction O +of O +PHD B-structure_element +– I-structure_element +SRA I-structure_element +. O + +The O +SRA B-structure_element +was O +incubated B-experimental_method +with O +GST B-protein_state +- I-protein_state +tagged I-protein_state +PHD B-structure_element +in O +the O +presence B-protein_state +of I-protein_state +increasing O +concentrations O +of O +hm B-chemical +- I-chemical +DNA I-chemical +and O +immobilized O +on O +glutathione O +resin O +. O + +( O +b O +) O +Purified O +fragments O +of O +UHRF1 B-protein +were O +analysed O +by O +histone B-experimental_method +peptide I-experimental_method +( O +H3K9me0 B-protein_type +) O +pull B-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +as O +described O +in O +Fig O +. O +1b O +. O +( O +c O +) O +Hm B-chemical +- I-chemical +DNA I-chemical +impairs O +the O +intramolecular O +interaction O +of O +TTD B-structure_element +– I-structure_element +Spacer I-structure_element +. O + +The O +assays O +were O +performed O +in O +the O +presence O +(+ O +DTT B-chemical +) O +or O +absence O +(− O +DTT B-chemical +) O +of O +15 O +mM O +DTT B-chemical +. O + +The O +Spacer B-structure_element +facilitates O +hm B-chemical +- I-chemical +DNA I-chemical +– O +SRA B-structure_element +interaction O +and O +DNMT1 B-complex_assembly +– I-complex_assembly +UHRF1 I-complex_assembly +interaction O +. O + +( O +a O +) O +Superimposed O +ITC B-experimental_method +enthalpy B-evidence +plots I-evidence +for O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinities I-evidence +of O +the O +SRA B-structure_element +, O +the O +Spacer B-structure_element +and O +SRA B-structure_element +– I-structure_element +Spacer I-structure_element +. O +( O +b O +, O +c O +) O +Superimposed O +fluorescence B-evidence +polarization I-evidence +( I-evidence +FP I-evidence +) I-evidence +plots I-evidence +for O +hm B-evidence +- I-evidence +DNA I-evidence +- I-evidence +binding I-evidence +affinities I-evidence +of O +truncations O +or O +full B-protein_state +- I-protein_state +length I-protein_state +UHRF1 B-protein +. O + +Scale O +bar O +, O +5 O +μm O +. O +( O +e O +) O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +experiment I-experimental_method +for O +the O +interactions O +between O +wild B-protein_state +- I-protein_state +type I-protein_state +or O +truncations B-experimental_method +of O +UHRF1 B-protein +and O +RFTSDNMT1 B-protein +as O +described O +in O +Fig O +. O +2a O +. O +( O +f O +) O +Working O +model O +for O +hm B-chemical +- I-chemical +DNA I-chemical +- O +mediated O +conformational O +changes O +of O +UHRF1 B-protein +, O +as O +described O +in O +the O +Discussion O +. O + +X B-evidence +- I-evidence +ray I-evidence +Crystallographic I-evidence +Structures I-evidence +of O +a O +Trimer B-oligomeric_state +, O +Dodecamer B-oligomeric_state +, O +and O +Annular B-site +Pore I-site +Formed O +by O +an O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +β B-structure_element +- I-structure_element +Hairpin I-structure_element + +High O +- O +resolution O +structures B-evidence +of O +oligomers B-oligomeric_state +formed O +by O +the O +β B-protein +- I-protein +amyloid I-protein +peptide I-protein +Aβ B-protein +are O +needed O +to O +understand O +the O +molecular O +basis O +of O +Alzheimer O +’ O +s O +disease O +and O +develop O +therapies O +. O + +Two O +covalent O +constraints O +act O +in O +tandem O +to O +stabilize O +the O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +peptide O +in O +a O +hairpin B-structure_element +conformation O +: O +a O +δ B-protein_state +- I-protein_state +linked I-protein_state +ornithine B-residue_name +turn B-structure_element +connecting O +positions O +17 B-residue_number +and O +36 B-residue_number +to O +create O +a O +macrocycle O +and O +an O +intramolecular O +disulfide B-ptm +linkage I-ptm +between O +positions O +24 B-residue_number +and O +29 B-residue_number +. O + +Three O +β B-structure_element +- I-structure_element +hairpin I-structure_element +monomers B-oligomeric_state +assemble O +to O +form O +a O +triangular B-protein_state +trimer B-oligomeric_state +, O +four O +trimers B-oligomeric_state +assemble O +in O +a O +tetrahedral O +arrangement O +to O +form O +a O +dodecamer B-oligomeric_state +, O +and O +five O +dodecamers B-oligomeric_state +pack O +together O +to O +form O +an O +annular B-site +pore I-site +. O + +High O +- O +resolution O +structures B-evidence +of O +oligomers B-oligomeric_state +formed O +by O +the O +β B-protein +- I-protein +amyloid I-protein +peptide I-protein +Aβ B-protein +are O +desperately O +needed O +to O +understand O +the O +molecular O +basis O +of O +Alzheimer O +’ O +s O +disease O +and O +ultimately O +develop O +preventions O +or O +treatments O +. O + +Over O +the O +last O +two O +decades O +the O +role O +of O +Aβ B-protein +oligomers B-oligomeric_state +in O +the O +pathophysiology O +of O +Alzheimer O +’ O +s O +disease O +has O +begun O +to O +unfold O +. O + +Aβ B-protein +isolated O +from O +the O +brains O +of O +young O +plaque O +- O +free O +Tg2576 O +mice B-taxonomy_domain +forms O +a O +mixture O +of O +low O +molecular O +weight O +oligomers B-oligomeric_state +. O + +A O +56 O +kDa O +soluble O +oligomer B-oligomeric_state +identified O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +was O +found O +to O +be O +especially O +important O +within O +this O +mixture O +. O + +This O +oligomer B-oligomeric_state +was O +termed O +Aβ B-complex_assembly +* I-complex_assembly +56 I-complex_assembly +and O +appears O +to O +be O +a O +dodecamer B-oligomeric_state +of O +Aβ B-protein +. O + +Smaller O +oligomers B-oligomeric_state +with O +molecular O +weights O +consistent O +with O +trimers B-oligomeric_state +, O +hexamers B-oligomeric_state +, O +and O +nonamers B-oligomeric_state +were O +also O +identified O +within O +the O +mixture O +of O +low O +molecular O +weight O +oligomers B-oligomeric_state +. O + +Recently O +, O +Aβ B-protein +trimers B-oligomeric_state +and O +Aβ B-complex_assembly +* I-complex_assembly +56 I-complex_assembly +were O +identified O +in O +the O +brains O +of O +cognitively O +normal O +humans B-species +and O +were O +found O +to O +increase O +with O +age O +. O + +A O +type O +of O +large O +oligomers B-oligomeric_state +called O +annular B-complex_assembly +protofibrils I-complex_assembly +( O +APFs B-complex_assembly +) O +have O +also O +been O +observed O +in O +the O +brains O +of O +transgenic O +mice B-taxonomy_domain +and O +isolated O +from O +the O +brains O +of O +Alzheimer O +’ O +s O +patients O +. O + +APFs B-complex_assembly +were O +first O +discovered O +in O +vitro O +using O +chemically B-protein_state +synthesized I-protein_state +Aβ B-protein +that O +aggregated O +into O +porelike B-structure_element +structures I-structure_element +that O +could O +be O +observed O +by O +atomic B-experimental_method +force I-experimental_method +microscopy I-experimental_method +( O +AFM B-experimental_method +) O +and O +transmission B-experimental_method +electron I-experimental_method +microscopy I-experimental_method +( O +TEM B-experimental_method +). O + +Lashuel O +et O +al O +. O +observed O +APFs B-complex_assembly +with O +an O +outer O +diameter O +that O +ranged O +from O +7 O +– O +10 O +nm O +and O +an O +inner O +diameter O +that O +ranged O +from O +1 O +. O +5 O +– O +2 O +nm O +, O +consistent O +with O +molecular O +weights O +of O +150 O +– O +250 O +kDa O +. O + +Kayed O +et O +al O +. O +observed O +APFs B-complex_assembly +with O +an O +outer O +diameter O +that O +ranged O +from O +8 O +– O +25 O +nm O +, O +which O +were O +composed O +of O +small B-protein_state +spherical I-protein_state +Aβ B-protein +oligomers B-oligomeric_state +, O +3 O +– O +5 O +nm O +in O +diameter O +. O + +Although O +the O +APFs B-complex_assembly +in O +these O +studies O +differ O +in O +size O +, O +they O +share O +a O +similar O +annular O +morphology O +and O +appear O +to O +be O +composed O +of O +smaller O +oligomers B-oligomeric_state +. O + +APFs B-complex_assembly +have O +also O +been O +observed O +in O +the O +brains O +of O +APP23 O +transgenic O +mice B-taxonomy_domain +by O +immunofluorescence B-experimental_method +with O +an O +anti O +- O +APF B-complex_assembly +antibody O +and O +were O +found O +to O +accumulate O +in O +neuronal O +processes O +and O +synapses O +. O + +In O +a O +subsequent O +study O +, O +APFs B-complex_assembly +were O +isolated O +from O +the O +brains O +of O +Alzheimer O +’ O +s O +patients O +by O +immunoprecipitation B-experimental_method +with O +an O +anti O +- O +APF B-complex_assembly +antibody O +. O + +These O +APFs B-complex_assembly +had O +an O +outer O +diameter O +that O +ranged O +from O +11 O +– O +14 O +nm O +and O +an O +inner O +diameter O +that O +ranged O +from O +2 O +. O +5 O +– O +4 O +nm O +. O + +Dimers B-oligomeric_state +of O +Aβ B-protein +have O +also O +been O +isolated O +from O +the O +brains O +of O +Alzheimer O +’ O +s O +patients O +.− O +Aβ B-protein +dimers B-oligomeric_state +inhibit O +long O +- O +term O +potentiation O +in O +mice B-taxonomy_domain +and O +promote O +hyperphosphorylation B-ptm +of O +the O +microtubule B-protein +- I-protein +associated I-protein +protein I-protein +tau I-protein +, O +leading O +to O +neuritic O +damage O +. O + +Aβ B-protein +dimers B-oligomeric_state +have O +only O +been O +isolated O +from O +human B-species +or O +transgenic O +mouse B-taxonomy_domain +brains O +that O +contain O +the O +pathognomonic O +fibrillar B-protein_state +Aβ B-protein +plaques O +associated O +with O +Alzheimer O +’ O +s O +disease O +. O + +Furthermore O +, O +the O +endogenous O +rise O +of O +Aβ B-protein +dimers B-oligomeric_state +in O +the O +brains O +of O +Tg2576 O +and O +J20 O +transgenic O +mice B-taxonomy_domain +coincides O +with O +the O +deposition O +of O +Aβ B-protein +plaques O +. O + +These O +observations O +suggest O +that O +the O +Aβ B-protein +trimers B-oligomeric_state +, O +hexamers B-oligomeric_state +, O +dodecamers B-oligomeric_state +, O +and O +related O +assemblies O +may O +be O +associated O +with O +presymptomatic O +neurodegeneration O +, O +while O +Aβ B-protein +dimers B-oligomeric_state +are O +more O +closely O +associated O +with O +fibril O +formation O +and O +plaque O +deposition O +during O +symptomatic O +Alzheimer O +’ O +s O +disease O +.− O + +Techniques O +such O +as O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +, O +TEM B-experimental_method +, O +and O +AFM B-experimental_method +have O +only O +provided O +information O +about O +the O +molecular O +weights O +, O +sizes O +, O +morphologies O +, O +and O +stoichiometry O +of O +Aβ B-protein +oligomers B-oligomeric_state +. O + +High O +- O +resolution O +structural B-experimental_method +studies I-experimental_method +of O +Aβ B-protein +have O +primarily O +focused O +on O +Aβ B-protein +fibrils B-oligomeric_state +and O +Aβ B-protein +monomers B-oligomeric_state +. O + +Solid B-experimental_method +- I-experimental_method +state I-experimental_method +NMR I-experimental_method +spectroscopy I-experimental_method +studies O +of O +Aβ B-protein +fibrils B-oligomeric_state +revealed O +that O +Aβ B-protein +fibrils B-oligomeric_state +are O +generally O +composed O +of O +extended O +networks O +of O +in B-structure_element +- I-structure_element +register I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +.− O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallographic I-experimental_method +studies I-experimental_method +using O +fragments O +of O +Aβ B-protein +have O +provided O +additional O +information O +about O +how O +Aβ B-protein +fibrils B-oligomeric_state +pack O +. O + +Solution B-experimental_method +- I-experimental_method +phase I-experimental_method +NMR I-experimental_method +and O +solid B-experimental_method +- I-experimental_method +state I-experimental_method +NMR I-experimental_method +have O +been O +used O +to O +study O +the O +structures B-evidence +of O +the O +Aβ B-protein +monomers B-oligomeric_state +within O +oligomeric O +assemblies O +.− O +A O +major O +finding O +from O +these O +studies O +is O +that O +oligomeric O +assemblies O +of O +Aβ B-protein +are O +primarily O +composed O +of O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +. O + +Many O +of O +these O +studies O +have O +reported O +the O +monomer B-oligomeric_state +subunit B-structure_element +as O +adopting O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +conformation O +, O +in O +which O +the O +hydrophobic O +central B-structure_element +and O +C B-structure_element +- I-structure_element +terminal I-structure_element +regions I-structure_element +form O +an O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +. O + +In O +2008 O +, O +Hoyer O +et O +al O +. O +reported O +the O +NMR B-experimental_method +structure B-evidence +of O +an O +Aβ B-protein +monomer B-oligomeric_state +bound B-protein_state +to I-protein_state +an O +artificial B-chemical +binding I-chemical +protein I-chemical +called O +an O +affibody B-chemical +( O +PDB O +2OTK O +). O + +Sequestering O +Aβ B-protein +within O +the O +affibody B-chemical +prevents O +its O +fibrilization O +and O +reduces O +its O +neurotoxicity O +, O +providing O +evidence O +that O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +structure O +may O +contribute O +to O +the O +ability O +of O +Aβ B-protein +to O +form O +neurotoxic O +oligomers B-oligomeric_state +. O + +In O +a O +related O +study O +, O +Sandberg O +et O +al O +. O +constrained O +Aβ B-protein +in O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +conformation O +by O +mutating B-experimental_method +residues O +A21 B-residue_name_number +and O +A30 B-residue_name_number +to O +cysteine B-residue_name +and O +forming O +an O +intramolecular O +disulfide B-ptm +bond I-ptm +. O + +The O +oligomers B-oligomeric_state +with O +a O +molecular O +weight O +of O +∼ O +100 O +kDa O +that O +were O +isolated O +by O +SEC B-experimental_method +were O +toxic O +toward O +neuronally O +derived O +SH O +- O +SY5Y O +cells O +. O + +This O +study O +provides O +evidence O +for O +the O +role O +of O +β B-structure_element +- I-structure_element +hairpin I-structure_element +structure O +in O +Aβ B-protein +oligomerization O +and O +neurotoxicity O +. O + +Inspired O +by O +these O +β B-structure_element +- I-structure_element +hairpin I-structure_element +structures B-evidence +, O +our O +laboratory O +developed O +a O +macrocyclic O +β B-structure_element +- I-structure_element +sheet I-structure_element +peptide O +derived O +from O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +designed O +to O +mimic O +an O +Aβ B-protein +β B-structure_element +- I-structure_element +hairpin I-structure_element +and O +reported O +its O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +. O + +This O +peptide O +( O +peptide B-mutant +1 I-mutant +) O +consists O +of O +two O +β B-structure_element +- I-structure_element +strands I-structure_element +comprising O +Aβ17 B-protein +– B-residue_range +23 I-residue_range +and O +Aβ30 B-protein +– B-residue_range +36 I-residue_range +covalently O +linked O +by O +two O +δ B-protein_state +- I-protein_state +linked I-protein_state +ornithine B-residue_name +( O +δOrn B-structure_element +) O +β B-structure_element +- I-structure_element +turn I-structure_element +mimics O +. O + +The O +δOrn B-structure_element +that O +connects O +residues O +D23 B-residue_name_number +and O +A30 B-residue_name_number +replaces O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +. O + +The O +δOrn B-structure_element +that O +connects O +residues O +L17 B-residue_name_number +and O +V36 B-residue_name_number +enforces O +β B-structure_element +- I-structure_element +hairpin I-structure_element +structure O +. O + +We O +incorporated O +an O +N O +- O +methyl O +group O +at O +position O +G33 B-residue_name_number +to O +prevent O +uncontrolled O +aggregation O +and O +precipitation O +of O +the O +peptide O +. O + +To O +improve O +the O +solubility O +of O +the O +peptide O +we O +replaced B-experimental_method +M35 B-residue_name_number +with O +the O +hydrophilic O +isostere O +of O +methionine B-residue_name +, O +ornithine B-residue_name +( O +α B-protein_state +- I-protein_state +linked I-protein_state +) O +( O +Figure O +1B O +). O + +The O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +1 I-mutant +reveals O +that O +it O +folds O +to O +form O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +that O +assembles O +to O +form O +trimers B-oligomeric_state +and O +that O +the O +trimers B-oligomeric_state +further O +assemble O +to O +form O +hexamers B-oligomeric_state +and O +dodecamers B-oligomeric_state +. O + +( O +A O +) O +Cartoon O +illustrating O +the O +design O +of O +peptides B-chemical +1 I-chemical +and I-chemical +2 I-chemical +and O +their O +relationship O +to O +an O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +β B-structure_element +- I-structure_element +hairpin I-structure_element +. O + +( O +B O +) O +Chemical O +structure O +of O +peptide B-mutant +1 I-mutant +illustrating O +Aβ17 B-protein +– O +23 O +and O +Aβ30 B-protein +– O +36 O +, O +M35Orn O +, O +the O +N O +- O +methyl O +group O +, O +and O +the O +δ B-protein_state +- I-protein_state +linked I-protein_state +ornithine B-residue_name +turns B-structure_element +. O +( O +C O +) O +Chemical O +structure O +of O +peptide B-mutant +2 I-mutant +illustrating O +Aβ17 B-protein +– O +36 O +, O +the O +N O +- O +methyl O +group O +, O +the O +disulfide B-ptm +bond I-ptm +across O +positions O +24 B-residue_number +and O +29 B-residue_number +, O +and O +the O +δ B-protein_state +- I-protein_state +linked I-protein_state +ornithine B-residue_name +turn B-structure_element +. O + +Our O +design O +of O +peptide B-mutant +1 I-mutant +omitted O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +. O + +To O +visualize O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +, O +we O +performed O +replica B-experimental_method +- I-experimental_method +exchange I-experimental_method +molecular I-experimental_method +dynamics I-experimental_method +( O +REMD B-experimental_method +) O +simulations B-experimental_method +on O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +using O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +coordinates I-evidence +of O +Aβ17 B-protein +– B-residue_range +23 I-residue_range +and O +Aβ30 B-protein +– B-residue_range +36 I-residue_range +from O +peptide B-mutant +1 I-mutant +. O + +These O +studies O +provided O +a O +working O +model O +for O +a O +trimer B-oligomeric_state +of O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +β B-structure_element +- I-structure_element +hairpins I-structure_element +and O +demonstrated O +that O +the O +trimer B-oligomeric_state +should O +be O +capable O +of O +accommodating O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +. O + +We O +designed O +peptide B-mutant +2 I-mutant +as O +a O +homologue O +of O +peptide B-mutant +1 I-mutant +that O +embodies O +these O +ideas O +. O + +Peptide B-mutant +2 I-mutant +contains O +a O +methionine B-residue_name +residue O +at O +position O +35 B-residue_number +and O +an O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +with O +residues O +24 B-residue_number +and O +29 B-residue_number +( O +Val B-residue_name +and O +Gly B-residue_name +) O +mutated B-experimental_method +to O +cysteine B-residue_name +and O +linked O +by O +a O +disulfide B-ptm +bond I-ptm +( O +Figure O +1C O +). O + +Here O +, O +we O +describe O +the O +development O +of O +peptide B-mutant +2 I-mutant +and O +report O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structures I-evidence +of O +the O +trimer B-oligomeric_state +, O +dodecamer B-oligomeric_state +, O +and O +annular B-site +pore I-site +observed O +within O +the O +crystal B-evidence +structure I-evidence +. O + +Development O +of O +Peptide B-mutant +2 I-mutant + +We O +developed O +peptide B-mutant +2 I-mutant +from O +peptide B-mutant +1 I-mutant +by O +an O +iterative O +process O +, O +in O +which O +we O +first O +attempted O +to O +restore O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +without O +a O +disulfide B-ptm +linkage I-ptm +. O + +We O +envisioned O +peptide B-mutant +3 I-mutant +as O +a O +homologue O +of O +peptide B-mutant +1 I-mutant +with O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +in O +place O +of O +the O +δOrn B-structure_element +that O +connects O +D23 B-residue_name_number +and O +A30 B-residue_name_number +and O +p B-chemical +- I-chemical +iodophenylalanine I-chemical +( O +FI B-chemical +) O +in O +place O +of O +F19 B-residue_name_number +. O + +After O +determining O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +the O +p B-chemical +- I-chemical +iodophenylalanine I-chemical +variant O +we O +attempt O +to O +determine O +the O +structure B-evidence +of O +the O +native O +phenylalanine B-residue_name +compound O +by O +isomorphous B-experimental_method +replacement I-experimental_method +. O + +We O +postulate O +that O +the O +loss O +of O +the O +δOrn B-structure_element +constraint O +leads O +to O +conformational O +heterogeneity O +that O +prevents O +peptide B-mutant +3 I-mutant +from O +crystallizing O +. O + +We O +designed O +peptide B-mutant +4 I-mutant +to O +embody O +this O +idea O +, O +mutating B-experimental_method +Val24 B-residue_name_number +and O +Gly29 B-residue_name_number +to O +cysteine B-residue_name +and O +forming O +an O +interstrand O +disulfide B-ptm +linkage I-ptm +. O + +Residues O +V24 B-residue_name_number +and O +G29 B-residue_name_number +form O +a O +non B-bond_interaction +- I-bond_interaction +hydrogen I-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +pair I-bond_interaction +, O +which O +can O +readily O +accommodate O +disulfide B-ptm +linkages I-ptm +in O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +. O + +Disulfide B-ptm +bonds I-ptm +across O +non B-bond_interaction +- I-bond_interaction +hydrogen I-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +pairs I-bond_interaction +stabilize O +β B-structure_element +- I-structure_element +hairpins I-structure_element +, O +while O +disulfide B-ptm +bonds I-ptm +across O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +pairs I-bond_interaction +do O +not O +. O + +Although O +the O +disulfide B-ptm +bond I-ptm +between O +positions O +24 B-residue_number +and O +29 B-residue_number +helps O +stabilize O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +, O +it O +does O +not O +alter O +the O +charge O +or O +substantially O +change O +the O +hydrophobicity O +of O +the O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +β B-structure_element +- I-structure_element +hairpin I-structure_element +. O + +We O +were O +gratified O +to O +find O +that O +peptide B-mutant +4 I-mutant +afforded O +crystals B-evidence +suitable O +for O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +As O +the O +next O +step O +in O +the O +iterative O +process O +, O +we O +determined B-experimental_method +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +this O +peptide O +( O +PDB O +5HOW O +). O + +We O +completed O +the O +iterative O +process O +— O +from O +1 O +to O +3 O +to O +4 O +to O +2 O +— O +by O +successfully O +determining O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +2 I-mutant +( O +PDB O +5HOX O +and O +5HOY O +). O + +The O +following O +sections O +describe O +the O +synthesis O +of O +peptides B-mutant +2 I-mutant +– I-mutant +4 I-mutant +and O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +2 I-mutant +. O + +Synthesis O +of O +Peptides B-mutant +2 I-mutant +– I-mutant +4 I-mutant + +We O +synthesized O +peptides B-mutant +2 I-mutant +– I-mutant +4 I-mutant +by O +similar O +procedures O +to O +those O +we O +have O +developed O +for O +other O +macrocyclic O +peptides O +. O + +In O +synthesizing O +peptides B-mutant +2 I-mutant +and I-mutant +4 I-mutant +we O +formed O +the O +disulfide B-ptm +linkage I-ptm +after O +macrolactamization O +and O +deprotection O +of O +the O +acid O +- O +labile O +side O +chain O +protecting O +groups O +. O + +Crystallization B-experimental_method +, O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +Crystallographic I-experimental_method +Data I-experimental_method +Collection I-experimental_method +, O +Data O +Processing O +, O +and O +Structure B-experimental_method +Determination I-experimental_method +of O +Peptides B-mutant +2 I-mutant +and I-mutant +4 I-mutant + +We O +screened B-experimental_method +crystallization I-experimental_method +conditions I-experimental_method +for O +peptide B-mutant +4 I-mutant +in O +a O +96 O +- O +well O +- O +plate O +format O +using O +three O +different O +Hampton O +Research O +crystallization O +kits O +( O +Crystal O +Screen O +, O +Index O +, O +and O +PEG O +/ O +Ion O +) O +with O +three O +ratios O +of O +peptide O +and O +mother O +liquor O +per O +condition O +( O +864 O +experiments O +). O + +Peptide B-mutant +4 I-mutant +afforded O +crystals B-evidence +in O +a O +single O +set O +of O +conditions O +containing O +HEPES O +buffer O +and O +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +— O +the O +same O +crystallization O +conditions O +that O +afforded O +crystals B-evidence +of O +peptide B-mutant +1 I-mutant +. O + +We O +further O +optimized O +these O +conditions O +to O +rapidly O +(∼ O +72 O +h O +) O +yield O +crystals B-evidence +suitable O +for O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +Crystal B-evidence +diffraction I-evidence +data I-evidence +for O +peptides B-mutant +4 I-mutant +and I-mutant +2 I-mutant +were O +collected O +in O +- O +house O +with O +a O +Rigaku O +MicroMax O +007HF O +X O +- O +ray O +diffractometer O +at O +1 O +. O +54 O +Å O +wavelength O +. O + +Data O +from O +peptides B-mutant +4 I-mutant +and I-mutant +2 I-mutant +suitable O +for O +refinement O +at O +2 O +. O +30 O +Å O +were O +obtained O +from O +the O +diffractometer O +; O +data O +from O +peptide B-mutant +2 I-mutant +suitable O +for O +refinement O +at O +1 O +. O +90 O +Å O +were O +obtained O +from O +the O +synchrotron O +. O + +Phases B-evidence +for O +peptide B-mutant +4 I-mutant +were O +determined O +by O +single B-experimental_method +- I-experimental_method +wavelength I-experimental_method +anomalous I-experimental_method +diffraction I-experimental_method +( O +SAD B-experimental_method +) O +phasing B-experimental_method +by O +using O +the O +coordinates O +of O +the O +iodine B-evidence +anomalous I-evidence +signal I-evidence +from O +p B-chemical +- I-chemical +iodophenylalanine I-chemical +. O + +Phases B-evidence +for O +peptide B-mutant +2 I-mutant +were O +determined O +by O +isomorphous B-experimental_method +replacement I-experimental_method +of O +peptide B-mutant +4 I-mutant +. O + +The O +structures B-evidence +of O +peptides B-mutant +2 I-mutant +and I-mutant +4 I-mutant +were O +solved B-experimental_method +and O +refined O +in O +the O +P6122 O +space O +group O +. O + +The O +asymmetric O +unit O +of O +each O +peptide B-chemical +consists O +of O +six O +monomers B-oligomeric_state +, O +arranged O +as O +two O +trimers B-oligomeric_state +. O + +Peptides B-mutant +2 I-mutant +and I-mutant +4 I-mutant +form O +morphologically O +identical O +structures O +and O +assemblies O +in O +the O +crystal B-evidence +lattice I-evidence +. O + +X B-evidence +- I-evidence +ray I-evidence +Crystallographic I-evidence +Structure I-evidence +of O +Peptide B-mutant +2 I-mutant +and O +the O +Oligomers B-oligomeric_state +It O +Forms O + +Eight O +residues O +make O +up O +each O +surface O +of O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +: O +L17 B-residue_name_number +, O +F19 B-residue_name_number +, O +A21 B-residue_name_number +, O +D23 B-residue_name_number +, O +A30 B-residue_name_number +, O +I32 B-residue_name_number +, O +L34 B-residue_name_number +, O +and O +V36 B-residue_name_number +make O +up O +one O +surface O +; O +V18 B-residue_name_number +, O +F20 B-residue_name_number +, O +E22 B-residue_name_number +, O +C24 B-residue_name_number +, O +C29 B-residue_name_number +, O +I31 B-residue_name_number +, O +G33 B-residue_name_number +, O +and O +M35 B-residue_name_number +make O +up O +the O +other O +surface O +. O + +The O +β B-structure_element +- I-structure_element +strands I-structure_element +of O +the O +monomers B-oligomeric_state +in O +the O +asymmetric O +unit O +are O +virtually O +identical O +, O +differing O +primarily O +in O +rotamers O +of O +F20 B-residue_name_number +, O +E22 B-residue_name_number +, O +C24 B-residue_name_number +, O +C29 B-residue_name_number +, O +I31 B-residue_name_number +, O +and O +M35 B-residue_name_number +( O +Figure O +S1 O +). O + +We O +refined B-experimental_method +three O +of O +the O +β B-structure_element +- I-structure_element +hairpins I-structure_element +with O +intact B-protein_state +disulfide B-ptm +linkages I-ptm +and O +three O +with O +thiols O +to O +represent O +cleaved B-protein_state +disulfide B-ptm +linkages I-ptm +in O +the O +synchrotron O +data O +set O +( O +PDB O +5HOX O +). O + +X O +- O +ray O +crystallographic O +structure O +of O +peptide B-mutant +2 I-mutant +( O +PDB O +5HOX O +, O +synchrotron O +data O +set O +). O +( O +A O +) O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +a O +representative O +β B-structure_element +- I-structure_element +hairpin I-structure_element +monomer B-oligomeric_state +formed O +by O +peptide B-mutant +2 I-mutant +. O +( O +B O +) O +Overlay B-experimental_method +of O +the O +six O +β B-structure_element +- I-structure_element +hairpin I-structure_element +monomers B-oligomeric_state +in O +the O +asymmetric O +unit O +. O + +The O +β B-structure_element +- I-structure_element +hairpins I-structure_element +are O +shown O +as O +cartoons O +to O +illustrate O +the O +differences O +in O +the O +Aβ25 B-protein +– B-residue_range +28 I-residue_range +loops B-structure_element +. O + +The O +Aβ25 B-protein +– B-residue_range +28 I-residue_range +loops B-structure_element +of O +the O +six O +monomers B-oligomeric_state +within O +the O +asymmetric O +unit O +vary O +substantially O +in O +backbone O +geometry O +and O +side O +chain O +rotamers O +( O +Figures O +2B O +and O +S1 O +). O + +The O +electron B-evidence +density I-evidence +for O +the O +loops B-structure_element +is O +weak O +and O +diffuse O +compared O +to O +the O +electron B-evidence +density I-evidence +for O +the O +β B-structure_element +- I-structure_element +strands I-structure_element +. O + +The O +B B-evidence +values I-evidence +for O +the O +loops B-structure_element +are O +large O +, O +indicating O +that O +the O +loops B-structure_element +are O +dynamic O +and O +not O +well O +ordered O +. O + +Thus O +, O +the O +differences O +in O +backbone O +geometry O +and O +side O +chain O +rotamers O +among O +the O +loops B-structure_element +are O +likely O +of O +little O +significance O +and O +should O +be O +interpreted O +with O +caution O +. O + +Peptide B-mutant +2 I-mutant +assembles O +into O +oligomers B-oligomeric_state +similar O +in O +morphology O +to O +those O +formed O +by O +peptide B-mutant +1 I-mutant +. O + +Like O +peptide B-mutant +1 I-mutant +, O +peptide B-mutant +2 I-mutant +forms O +a O +triangular B-protein_state +trimer B-oligomeric_state +, O +and O +four O +trimers B-oligomeric_state +assemble O +to O +form O +a O +dodecamer B-oligomeric_state +. O + +In O +the O +higher O +- O +order O +assembly O +of O +the O +dodecamers B-oligomeric_state +formed O +by O +peptide B-mutant +2 I-mutant +a O +new O +structure B-evidence +emerges O +, O +not O +seen O +in O +peptide B-mutant +1 I-mutant +, O +an O +annular B-site +pore I-site +consisting O +of O +five O +dodecamers B-oligomeric_state +. O + +Trimer B-oligomeric_state + +Peptide B-mutant +2 I-mutant +forms O +a O +trimer B-oligomeric_state +, O +much O +like O +that O +which O +we O +observed O +previously O +for O +peptide B-mutant +1 I-mutant +, O +in O +which O +three O +β B-structure_element +- I-structure_element +hairpins I-structure_element +assemble O +to O +form O +an O +equilateral B-structure_element +triangle I-structure_element +( O +Figure O +3A O +). O + +The O +two O +trimers B-oligomeric_state +are O +almost O +identical O +in O +structure O +, O +differing O +slightly O +among O +side O +chain O +rotamers O +and O +loop B-structure_element +conformations O +. O + +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +the O +trimer B-oligomeric_state +formed O +by O +peptide B-mutant +2 I-mutant +. O +( O +A O +) O +Triangular B-protein_state +trimer B-oligomeric_state +. O + +The O +three O +water B-chemical +molecules O +in O +the O +center O +hole O +of O +the O +trimer B-oligomeric_state +are O +shown O +as O +spheres O +. O +( O +B O +) O +Detailed O +view O +of O +the O +intermolecular O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +the O +main O +chains O +of O +V18 B-residue_name_number +and O +E22 B-residue_name_number +and O +δOrn B-structure_element +and O +C24 B-residue_name_number +, O +at O +the O +three O +corners O +of O +the O +triangular B-protein_state +trimer B-oligomeric_state +. O +( O +C O +) O +The O +F19 B-residue_name_number +face O +of O +the O +trimer B-oligomeric_state +, O +with O +key O +side O +chains O +shown O +as O +spheres O +. O +( O +D O +) O +The O +F20 B-residue_name_number +face O +of O +the O +trimer B-oligomeric_state +, O +with O +key O +side O +chains O +as O +spheres O +. O + +At O +the O +corners O +of O +the O +trimer B-oligomeric_state +, O +the O +pairs O +of O +β B-structure_element +- I-structure_element +hairpin I-structure_element +monomers B-oligomeric_state +form O +four O +hydrogen B-bond_interaction +bonds I-bond_interaction +: O +two O +between O +the O +main O +chains O +of O +V18 B-residue_name_number +and O +E22 B-residue_name_number +and O +two O +between O +δOrn B-structure_element +and O +the O +main O +chain O +of O +C24 B-residue_name_number +( O +Figure O +3B O +). O + +At O +each O +corner O +, O +the O +side O +chains O +of O +residues O +L17 B-residue_name_number +, O +F19 B-residue_name_number +, O +and O +V36 B-residue_name_number +of O +one O +β B-structure_element +- I-structure_element +hairpin I-structure_element +pack O +against O +the O +side O +chains O +of O +residues O +A21 B-residue_name_number +, O +I32 B-residue_name_number +, O +L34 B-residue_name_number +, O +and O +also O +D23 B-residue_name_number +of O +the O +adjacent O +β B-structure_element +- I-structure_element +hairpin I-structure_element +to O +create O +a O +hydrophobic B-site +cluster I-site +( O +Figure O +3C O +). O +The O +three O +hydrophobic B-site +clusters I-site +create O +a O +large O +hydrophobic B-site +surface I-site +on O +one O +face O +of O +the O +trimer B-oligomeric_state +. O + +The O +other O +face O +of O +the O +trimer B-oligomeric_state +displays O +a O +smaller O +hydrophobic B-site +surface I-site +, O +which O +includes O +the O +side O +chains O +of O +residues O +V18 B-residue_name_number +, O +F20 B-residue_name_number +, O +and O +I31 B-residue_name_number +of O +the O +three O +β B-structure_element +- I-structure_element +hairpins I-structure_element +( O +Figure O +3D O +). O + +In O +subsequent O +discussion O +, O +we O +designate O +the O +former O +surface O +the O +“ O +F19 B-residue_name_number +face O +” O +and O +the O +latter O +surface O +the O +“ O +F20 B-residue_name_number +face O +”. O + +Dodecamer B-oligomeric_state + +Four O +trimers B-oligomeric_state +assemble O +to O +form O +a O +dodecamer B-oligomeric_state +. O + +The O +four O +trimers B-oligomeric_state +arrange O +in O +a O +tetrahedral B-protein_state +fashion O +, O +creating O +a O +central B-site +cavity I-site +inside O +the O +dodecamer B-oligomeric_state +. O +Because O +each O +trimer B-oligomeric_state +is O +triangular B-protein_state +, O +the O +resulting O +arrangement O +resembles O +an O +octahedron B-protein_state +. O + +Figure O +4B O +illustrates O +the O +tetrahedral B-protein_state +arrangement O +of O +the O +four O +trimers B-oligomeric_state +. O + +X O +- O +ray O +crystallographic O +structure O +of O +the O +dodecamer B-oligomeric_state +formed O +by O +peptide B-mutant +2 I-mutant +. O +( O +A O +) O +View O +of O +the O +dodecamer B-oligomeric_state +that O +illustrates O +the O +octahedral B-protein_state +shape O +. O +( O +B O +) O +View O +of O +the O +dodecamer B-oligomeric_state +that O +illustrates O +the O +tetrahedral B-protein_state +arrangement O +of O +the O +four O +trimers B-oligomeric_state +that O +comprise O +the O +dodecamer B-oligomeric_state +. O +( O +C O +) O +View O +of O +two O +trimer B-oligomeric_state +subunits B-structure_element +from O +inside O +the O +cavity B-site +of O +the O +dodecamer B-oligomeric_state +. O + +The O +F19 B-residue_name_number +faces O +of O +the O +trimers B-oligomeric_state +line O +the O +interior O +of O +the O +dodecamer B-oligomeric_state +. O + +At O +the O +six O +vertices O +, O +hydrophobic B-bond_interaction +packing I-bond_interaction +between O +the O +side O +chains O +of O +L17 B-residue_name_number +, O +L34 B-residue_name_number +, O +and O +V36 B-residue_name_number +helps O +stabilize O +the O +dodecamer B-oligomeric_state +( O +Figures O +4C O +and O +D O +). O + +Salt O +bridges O +between O +the O +side O +chains O +of O +D23 B-residue_name_number +and O +δOrn B-structure_element +at O +the O +vertices O +further O +stabilize O +the O +dodecamer B-oligomeric_state +. O + +Each O +of O +the O +six O +vertices O +includes O +two O +Aβ25 B-protein +– B-residue_range +28 I-residue_range +loops B-structure_element +that O +extend O +past O +the O +core B-structure_element +of O +the O +dodecamer B-oligomeric_state +without O +making O +any O +substantial O +intermolecular O +contacts O +. O + +In O +the O +crystal B-evidence +lattice I-evidence +, O +each O +F20 B-residue_name_number +face O +of O +one O +dodecamer B-oligomeric_state +packs O +against O +an O +F20 B-residue_name_number +face O +of O +another O +dodecamer B-oligomeric_state +. O + +Although O +the O +asymmetric O +unit O +comprises O +half O +a O +dodecamer B-oligomeric_state +, O +the O +crystal B-evidence +lattice I-evidence +may O +be O +thought O +of O +as O +being O +built O +of O +dodecamers B-oligomeric_state +. O + +The O +shape O +and O +length O +of O +the O +electron B-evidence +density I-evidence +is O +consistent O +with O +the O +structure B-evidence +of O +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +, O +which O +is O +an O +essential O +component O +of O +the O +crystallization O +conditions O +. O + +Although O +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +is O +a O +heterogeneous O +mixture O +with O +varying O +chain O +lengths O +and O +stereochemistry O +, O +we O +modeled O +a O +single O +stereoisomer O +with O +nine O +propylene O +glycol O +units O +( O +n O += O +9 O +) O +to O +fit O +the O +electron B-evidence +density I-evidence +. O + +The O +Jeffamine B-chemical +M I-chemical +- I-chemical +600 I-chemical +appears O +to O +stabilize O +the O +dodecamer B-oligomeric_state +by O +occupying O +the O +central B-site +cavity I-site +and O +making O +hydrophobic B-bond_interaction +contacts I-bond_interaction +with O +residues O +lining O +the O +cavity B-site +( O +Figure O +S3 O +). O + +In O +a O +dodecamer B-oligomeric_state +formed O +by O +full B-protein_state +- I-protein_state +length I-protein_state +Aβ B-protein +, O +the O +hydrophobic O +C O +- O +terminal O +residues O +( O +Aβ37 B-protein +– B-residue_range +40 I-residue_range +or O +Aβ37 B-protein +– B-residue_range +42 I-residue_range +) O +might O +play O +a O +similar O +role O +in O +filling O +the O +dodecamer B-oligomeric_state +and O +thus O +create O +a O +packed O +hydrophobic B-site +core I-site +within O +the O +central B-site +cavity I-site +of O +the O +dodecamer B-oligomeric_state +. O + +Five O +dodecamers B-oligomeric_state +assemble O +to O +form O +an O +annular O +porelike B-structure_element +structure O +( O +Figure O +5A O +). O + +Hydrophobic B-bond_interaction +packing I-bond_interaction +between O +the O +F20 B-residue_name_number +faces O +of O +trimers B-oligomeric_state +displayed O +on O +the O +outer O +surface O +of O +each O +dodecamer B-oligomeric_state +stabilizes O +the O +porelike O +assembly O +. O + +Hydrophobic B-bond_interaction +packing I-bond_interaction +between O +the O +side O +chains O +of O +F20 B-residue_name_number +, O +I31 B-residue_name_number +, O +and O +E22 B-residue_name_number +stabilizes O +these O +interfaces B-site +( O +Figure O +5D O +and O +E O +). O + +The O +eclipsed B-protein_state +interfaces B-site +occur O +between O +dodecamers B-structure_element +1 I-structure_element +and I-structure_element +2 I-structure_element +, O +1 B-structure_element +and I-structure_element +5 I-structure_element +, O +and O +3 B-structure_element +and I-structure_element +4 I-structure_element +, O +as O +shown O +in O +Figure O +5A O +. O + +The O +annular B-site +pore I-site +is O +not O +completely O +flat O +, O +instead O +, O +adopting O +a O +slightly O +puckered O +shape O +, O +which O +accommodates O +the O +eclipsed B-protein_state +and O +staggered B-protein_state +interfaces B-site +. O + +The O +hydrophilic O +side O +chains O +of O +S26 B-residue_name_number +, O +N27 B-residue_name_number +, O +and O +K28 B-residue_name_number +decorate O +the O +hole O +. O + +( O +B O +) O +Eclipsed B-site +interface I-site +between O +dodecamers B-structure_element +1 I-structure_element +and I-structure_element +2 I-structure_element +( O +side O +view O +). O + +The O +diameter O +of O +the O +hole O +in O +the O +center O +of O +the O +pore B-site +is O +∼ O +2 O +nm O +. O + +The O +thickness O +of O +the O +pore B-site +is O +∼ O +5 O +nm O +, O +which O +is O +comparable O +to O +that O +of O +a O +lipid O +bilayer O +membrane O +. O + +It O +is O +important O +to O +note O +that O +the O +annular B-site +pore I-site +formed O +by O +peptide B-mutant +2 I-mutant +is O +not O +a O +discrete O +unit O +in O +the O +crystal B-evidence +lattice I-evidence +. O + +The O +crystal B-evidence +lattice I-evidence +shows O +how O +the O +dodecamers B-oligomeric_state +can O +further O +assemble O +to O +form O +larger O +structures O +. O + +Each O +dodecamer B-oligomeric_state +may O +be O +thought O +of O +as O +a O +tetravalent O +building O +block O +with O +the O +potential O +to O +assemble O +on O +all O +four O +faces O +to O +form O +higher O +- O +order O +supramolecular O +assemblies O +. O + +The O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallographic I-experimental_method +study I-experimental_method +of O +peptide B-mutant +2 I-mutant +described O +here O +provides O +high O +- O +resolution O +structures B-evidence +of O +oligomers B-oligomeric_state +formed O +by O +an O +Aβ17 B-protein +– B-residue_range +36 I-residue_range +β B-structure_element +- I-structure_element +hairpin I-structure_element +. 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O + +Monomeric B-oligomeric_state +Aβ B-protein +folds O +to O +form O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +in O +which O +the O +hydrophobic O +central B-structure_element +and O +C B-structure_element +- I-structure_element +terminal I-structure_element +regions I-structure_element +form O +an O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +. O + +Five O +dodecamers B-oligomeric_state +assemble O +to O +form O +an O +annular B-site +pore I-site +. O + +The O +model O +put O +forth O +in O +Figure O +6 O +is O +consistent O +with O +the O +current O +understanding O +of O +endogenous O +Aβ B-protein +oligomerization O +and O +explains O +at O +atomic O +resolution O +many O +key O +observations O +about O +Aβ B-protein +oligomers B-oligomeric_state +. O + +Two O +general O +types O +of O +endogenous O +Aβ B-protein +oligomers B-oligomeric_state +have O +been O +observed O +: O +Aβ B-protein +oligomers B-oligomeric_state +that O +occur O +on O +a O +pathway O +to O +fibrils B-oligomeric_state +, O +or O +“ O +fibrillar B-protein_state +oligomers B-oligomeric_state +”, O +and O +Aβ B-protein +oligomers B-oligomeric_state +that O +evade O +a O +fibrillar B-protein_state +fate O +, O +or O +“ O +nonfibrillar B-protein_state +oligomers B-oligomeric_state +”.− O +Fibrillar B-protein_state +oligomers B-oligomeric_state +accumulate O +in O +Alzheimer O +’ O +s O +disease O +later O +than O +nonfibrillar B-protein_state +oligomers B-oligomeric_state +and O +coincide O +with O +the O +deposition O +of O +plaques O +. O + +Nonfibrillar B-protein_state +oligomers B-oligomeric_state +accumulate O +early O +in O +Alzheimer O +’ O +s O +disease O +before O +plaque O +deposition O +. O + +Fibrillar B-protein_state +oligomers B-oligomeric_state +are O +recognized O +by O +the O +OC O +antibody O +but O +not O +the O +A11 O +antibody O +, O +whereas O +nonfibrillar B-protein_state +oligomers B-oligomeric_state +are O +recognized O +by O +the O +A11 O +antibody O +but O +not O +the O +OC O +antibody O +. O + +These O +criteria O +have O +been O +used O +to O +classify O +the O +Aβ B-protein +oligomers B-oligomeric_state +that O +accumulate O +in O +vivo O +. O + +Aβ B-protein +dimers B-oligomeric_state +have O +been O +classified O +as O +fibrillar B-protein_state +oligomers B-oligomeric_state +, O +whereas O +Aβ B-protein +trimers B-oligomeric_state +, O +Aβ B-complex_assembly +* I-complex_assembly +56 I-complex_assembly +, O +and O +APFs B-complex_assembly +have O +been O +classified O +as O +nonfibrillar B-protein_state +oligomers B-oligomeric_state +. O + +Larson O +and O +Lesné O +proposed O +a O +model O +for O +the O +endogenous O +production O +of O +nonfibrillar B-protein_state +oligomers B-oligomeric_state +that O +explains O +these O +observations O +. O + +In O +this O +model O +, O +folded B-protein_state +Aβ B-protein +monomer B-oligomeric_state +assembles O +into O +a O +trimer B-oligomeric_state +, O +the O +trimer B-oligomeric_state +further O +assembles O +into O +hexamers B-oligomeric_state +and O +dodecamers B-oligomeric_state +, O +and O +the O +dodecamers B-oligomeric_state +further O +assemble O +to O +form O +annular B-complex_assembly +protofibrils I-complex_assembly +. O + +The O +hierarchical O +assembly O +of O +peptide B-mutant +2 I-mutant +is O +consistent O +with O +this O +model O +; O +and O +the O +trimer B-oligomeric_state +, O +dodecamer B-oligomeric_state +, O +and O +annular B-site +pore I-site +formed O +by O +peptide B-mutant +2 I-mutant +may O +share O +similarities O +to O +the O +trimers B-oligomeric_state +, O +Aβ B-complex_assembly +* I-complex_assembly +56 I-complex_assembly +, O +and O +APFs B-complex_assembly +observed O +in O +vivo O +. O + +The O +crystallographically B-evidence +observed I-evidence +annular B-site +pore I-site +formed O +by O +peptide B-mutant +2 I-mutant +is O +morphologically O +similar O +to O +the O +APFs B-complex_assembly +formed O +by O +full B-protein_state +- I-protein_state +length I-protein_state +Aβ B-protein +. O + +The O +annular B-site +pore I-site +formed O +by O +peptide B-mutant +2 I-mutant +is O +comparable O +in O +size O +to O +the O +APFs B-complex_assembly +prepared O +in O +vitro O +or O +isolated O +from O +Alzheimer O +’ O +s O +brains O +( O +Figure O +7 O +and O +Table O +1 O +). O + +The O +dodecamers B-oligomeric_state +that O +comprise O +the O +annular B-site +pore I-site +exhibit O +two O +modes O +of O +assembly O +— O +eclipsed B-protein_state +interactions O +and O +staggered B-protein_state +interactions O +between O +the O +F20 B-residue_name_number +faces O +of O +trimers B-oligomeric_state +within O +dodecamers B-oligomeric_state +. O + +These O +two O +modes O +of O +assembly O +might O +reflect O +a O +dynamic O +interaction O +between O +dodecamers B-oligomeric_state +, O +which O +could O +permit O +assemblies O +of O +more O +dodecamers B-oligomeric_state +into O +larger O +annular B-site +pores I-site +. O + +Annular B-site +Pores I-site +Formed O +by O +Aβ B-protein +and O +Peptide B-mutant +2 I-mutant + +annular B-site +pore I-site +source O +outer O +diameter O +inner O +diameter O +observation O +method O +peptide B-chemical +2 O +∼ O +11 O +– O +12 O +nm O +∼ O +2 O +nm O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +synthetic B-protein_state +Aβ B-protein +7 O +– O +10 O +nm O +1 O +. O +5 O +– O +2 O +nm O +TEM B-experimental_method +synthetic O +Aβ B-protein +16 O +nm O +not O +reported O +AFM B-experimental_method +synthetic O +Aβ B-protein +8 O +– O +25 O +nm O +not O +reported O +TEM B-experimental_method +Alzheimer O +’ O +s O +brain O +11 O +– O +14 O +nm O +2 O +. O +5 O +– O +4 O +nm O +TEM B-experimental_method + +Dot B-experimental_method +blot I-experimental_method +analysis O +shows O +that O +peptide B-mutant +2 I-mutant +is O +reactive O +toward O +the O +A11 O +antibody O +( O +Figure O +S5 O +). O + +Further O +studies O +are O +needed O +to O +elucidate O +the O +species O +that O +peptide B-mutant +2 I-mutant +forms O +in O +solution O +and O +to O +study O +their O +biological O +properties O +. O + +Preliminary O +attempts O +to O +study O +these O +species O +by O +SEC B-experimental_method +and O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +have O +not O +provided O +a O +clear O +measure O +of O +the O +structures B-evidence +formed O +in O +solution O +. 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O + +This O +mode O +of O +assembly O +is O +not O +unique O +to O +Aβ B-protein +. O + +The O +foldon B-structure_element +domain I-structure_element +of O +bacteriophage B-species +T4 I-species +fibritin B-protein +is O +composed O +of O +three O +β B-structure_element +- I-structure_element +hairpins I-structure_element +that O +assemble O +into O +a O +triangular B-protein_state +trimer B-oligomeric_state +similar O +to O +the O +triangular B-protein_state +trimer B-oligomeric_state +formed O +by O +peptide B-mutant +2 I-mutant +. O + +Additionally O +, O +our O +research O +group O +has O +observed O +a O +similar O +assembly O +of O +a O +β B-structure_element +- I-structure_element +hairpin I-structure_element +peptide O +derived O +from O +β2 B-protein +- I-protein +microglobulin I-protein +. O + +Although O +we O +began O +these O +studies O +with O +a O +relatively O +simple O +hypothesis O +— O +that O +the O +trimers B-oligomeric_state +and O +dodecamers B-oligomeric_state +formed O +by O +peptide B-mutant +1 I-mutant +could O +accommodate O +the O +Aβ24 B-protein +– B-residue_range +29 I-residue_range +loop B-structure_element +— O +an O +even O +more O +exciting O +finding O +has O +emerged O +— O +that O +the O +dodecamers B-oligomeric_state +can O +assemble O +to O +form O +annular B-site +pores I-site +. O + +This O +finding O +could O +not O +have O +been O +anticipated O +from O +the O +X B-evidence +- I-evidence +ray I-evidence +crystallographic I-evidence +structure I-evidence +of O +peptide B-mutant +1 I-mutant +and O +reveals O +a O +new O +level O +of O +hierarchical O +assembly O +that O +recapitulates O +micrographic O +observations O +of O +annular B-complex_assembly +protofibrils I-complex_assembly +. O + +The O +crystallographically B-evidence +observed I-evidence +dodecamer B-oligomeric_state +, O +in O +turn O +, O +recapitulates O +the O +observation O +of O +Aβ B-complex_assembly +* I-complex_assembly +56 I-complex_assembly +, O +which O +appears O +to O +be O +a O +dodecamer B-oligomeric_state +of O +Aβ B-protein +. O + +We O +believe O +this O +iterative O +, O +“ O +bottom O +up O +” O +approach O +of O +identifying O +the O +minimal O +modification O +required O +to O +crystallize B-experimental_method +Aβ B-protein +peptides O +will O +ultimately O +allow O +larger O +fragments O +of O +Aβ B-protein +to O +be O +crystallized B-experimental_method +, O +thus O +providing O +greater O +insights O +into O +the O +structures B-evidence +of O +Aβ B-protein +oligomers B-oligomeric_state +. O + +Some O +estrogen B-protein +receptor I-protein +‐ I-protein +α I-protein +( O +ERα B-protein +)‐ O +targeted O +breast O +cancer O +therapies O +such O +as O +tamoxifen B-chemical +have O +tissue O +‐ O +selective O +or O +cell O +‐ O +specific O +activities O +, O +while O +others O +have O +similar O +activities O +in O +different O +cell O +types O +. O + +Ligands O +that O +regulate O +the O +dynamics O +and O +stability O +of O +the O +coactivator B-site +‐ I-site +binding I-site +site I-site +in O +the O +C O +‐ O +terminal O +ligand B-structure_element +‐ I-structure_element +binding I-structure_element +domain I-structure_element +, O +called O +activation B-structure_element +function I-structure_element +‐ I-structure_element +2 I-structure_element +( O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +), O +showed O +similar O +activity O +profiles O +in O +different O +cell O +types O +. O + +Such O +ligands O +induced O +breast O +cancer O +cell O +proliferation O +in O +a O +manner O +that O +was O +predicted O +by O +the O +canonical O +recruitment O +of O +the O +coactivators O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +and O +induction O +of O +the O +GREB1 B-protein +proliferative O +gene O +. O + +Small O +‐ O +molecule O +ligands O +control O +receptor O +activity O +by O +modulating O +recruitment O +of O +effector O +enzymes O +to O +distal O +regions O +of O +the O +receptor O +, O +relative O +to O +the O +ligand B-site +‐ I-site +binding I-site +site I-site +. O + +For O +example O +, O +selective O +estrogen B-protein_type +receptor I-protein_type +modulators I-protein_type +( O +SERMs B-protein_type +) O +such O +as O +tamoxifen B-chemical +( O +Nolvadex B-chemical +®; I-chemical +AstraZeneca O +) O +or O +raloxifene B-chemical +( O +Evista B-chemical +®; I-chemical +Eli O +Lilly O +) O +( O +Fig O +1A O +) O +block O +the O +ERα B-protein +‐ O +mediated O +proliferative O +effects O +of O +the O +native O +estrogen B-chemical +, O +17β B-chemical +‐ I-chemical +estradiol I-chemical +( O +E2 B-chemical +), O +on O +breast O +cancer O +cells O +, O +but O +promote O +beneficial O +estrogenic O +effects O +on O +bone O +mineral O +density O +and O +adverse O +estrogenic O +effects O +such O +as O +uterine O +proliferation O +, O +fatty O +liver O +, O +or O +stroke O +( O +Frolik O +et O +al O +, O +1996 O +; O +Fisher O +et O +al O +, O +1998 O +; O +McDonnell O +et O +al O +, O +2002 O +; O +Jordan O +, O +2003 O +). O + +Allosteric O +control O +of O +ERα B-protein +activity O + +E2 B-chemical +‐ O +rings O +are O +numbered O +A O +‐ O +D O +. O +The O +E O +‐ O +ring O +is O +the O +common O +site O +of O +attachment O +for O +BSC O +found O +in O +many O +SERMS B-protein_type +. O + +Schematic O +illustration O +of O +the O +canonical O +ERα B-protein +signaling O +pathway O +. O + +Branched O +causality O +model O +for O +ERα B-protein +‐ O +mediated O +cell O +proliferation O +. O + +ERα B-protein +contains O +structurally B-protein_state +conserved I-protein_state +globular B-structure_element +domains I-structure_element +of O +the O +nuclear B-protein_type +receptor I-protein_type +superfamily I-protein_type +, O +including O +a O +DNA B-structure_element +‐ I-structure_element +binding I-structure_element +domain I-structure_element +( O +DBD B-structure_element +) O +that O +is O +connected O +by O +a O +flexible B-protein_state +hinge B-structure_element +region I-structure_element +to O +the O +ligand B-structure_element +‐ I-structure_element +binding I-structure_element +domain I-structure_element +( O +LBD B-structure_element +), O +as O +well O +as O +unstructured B-protein_state +AB B-structure_element +and O +F B-structure_element +domains O +at O +its O +amino O +and O +carboxyl O +termini O +, O +respectively O +( O +Fig O +1B O +). O + +The O +LBD B-structure_element +contains O +a O +ligand O +‐ O +dependent O +coactivator B-site +‐ I-site +binding I-site +site I-site +called O +activation B-structure_element +function I-structure_element +‐ I-structure_element +2 I-structure_element +( O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +). O + +However O +, O +the O +agonist O +activity O +of O +SERMs B-protein_type +derives O +from O +activation B-structure_element +function I-structure_element +‐ I-structure_element +1 I-structure_element +( O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +)— O +a O +coactivator B-site +recruitment I-site +site I-site +located O +in O +the O +AB B-structure_element +domain O +( O +Berry O +et O +al O +, O +1990 O +; O +Shang O +& O +Brown O +, O +2002 O +; O +Abot O +et O +al O +, O +2013 O +). O + +AF B-structure_element +‐ I-structure_element +1 I-structure_element +and O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +bind O +distinct O +but O +overlapping O +sets O +of O +coregulators O +( O +Webb O +et O +al O +, O +1998 O +; O +Endoh O +et O +al O +, O +1999 O +; O +Delage O +‐ O +Mourroux O +et O +al O +, O +2000 O +; O +Yi O +et O +al O +, O +2015 O +). O + +AF B-structure_element +‐ I-structure_element +2 I-structure_element +binds O +the O +signature O +LxxLL B-structure_element +motif I-structure_element +peptides O +of O +coactivators O +such O +as O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +( O +also O +known O +as O +SRC B-protein +‐ I-protein +1 I-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +). O + +In O +the O +canonical O +model O +of O +the O +ERα B-protein +signaling O +pathway O +( O +Fig O +1C O +), O +E2 B-protein_state +‐ I-protein_state +bound I-protein_state +ERα B-protein +forms O +a O +homodimer B-oligomeric_state +that O +binds O +DNA O +at O +estrogen B-site +‐ I-site +response I-site +elements I-site +( O +EREs B-site +), O +recruits O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +( O +Metivier O +et O +al O +, O +2003 O +; O +Johnson O +& O +O O +' O +Malley O +, O +2012 O +), O +and O +activates O +the O +GREB1 B-protein +gene O +, O +which O +is O +required O +for O +proliferation O +of O +ERα B-protein +‐ O +positive O +breast O +cancer O +cells O +( O +Ghosh O +et O +al O +, O +2000 O +; O +Rae O +et O +al O +, O +2005 O +; O +Deschenes O +et O +al O +, O +2007 O +; O +Liu O +et O +al O +, O +2012 O +; O +Srinivasan O +et O +al O +, O +2013 O +). O + +However O +, O +ERα B-protein +‐ O +mediated O +proliferative O +responses O +vary O +in O +a O +ligand O +‐ O +dependent O +manner O +( O +Srinivasan O +et O +al O +, O +2013 O +); O +thus O +, O +it O +is O +not O +known O +whether O +this O +canonical O +model O +is O +widely O +applicable O +across O +diverse O +ERα B-protein +ligands O +. O + +The O +simplest O +response O +model O +for O +ligand O +‐ O +specific O +proliferative O +effects O +is O +a O +linear O +causality O +model O +, O +where O +the O +degree O +of O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +determines O +GREB1 B-protein +expression O +, O +which O +in O +turn O +drives O +ligand O +‐ O +specific O +cell O +proliferation O +( O +Fig O +1D O +). O + +To O +test O +these O +signaling O +models O +, O +we O +profiled O +a O +diverse O +library O +of O +ERα B-protein +ligands O +using O +systems O +biology O +approaches O +to O +X B-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +and O +chemical B-experimental_method +biology I-experimental_method +( O +Srinivasan O +et O +al O +, O +2013 O +), O +including O +a O +series O +of O +quantitative O +bioassays O +for O +ERα B-protein +function O +that O +were O +statistically O +robust O +and O +reproducible O +, O +based O +on O +the O +Z B-evidence +’‐ I-evidence +statistic I-evidence +( O +Fig O +EV1A O +and O +B O +; O +see O +Materials O +and O +Methods O +). O + +Our O +findings O +here O +indicate O +that O +specific O +structural O +perturbations O +can O +be O +tied O +to O +ligand O +‐ O +selective O +domain O +usage O +and O +signaling O +patterns O +, O +thus O +providing O +a O +framework O +for O +structure O +‐ O +based O +design O +of O +improved O +breast O +cancer O +therapeutics O +, O +and O +understanding O +the O +different O +phenotypic O +effects O +of O +environmental O +estrogens B-chemical +. O + +High O +‐ O +throughput O +screens O +for O +ERα B-protein +ligand O +profiling O + +Summary O +of O +ligand B-experimental_method +screening I-experimental_method +assays I-experimental_method +used O +to O +measure O +ER O +‐ O +mediated O +activities O +. O + +ERE B-structure_element +, O +estrogen B-structure_element +‐ I-structure_element +response I-structure_element +element I-structure_element +; O +Luc B-experimental_method +, O +luciferase B-experimental_method +reporter I-experimental_method +gene I-experimental_method +; O +M2H B-experimental_method +, O +mammalian B-experimental_method +2 I-experimental_method +‐ I-experimental_method +hybrid I-experimental_method +; O +UAS B-structure_element +, O +upstream B-structure_element +‐ I-structure_element +activating I-structure_element +sequence I-structure_element +. O + +Strength O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +signaling O +does O +not O +determine O +cell O +‐ O +specific O +signaling O + +These O +include O +15 O +indirect O +modulator O +series O +, O +which O +lack B-protein_state +a O +SERM B-protein_type +‐ I-protein_type +like I-protein_type +side O +chain O +and O +modulate O +coactivator O +binding O +indirectly O +from O +the O +ligand B-site +‐ I-site +binding I-site +pocket I-site +( O +Fig O +2A O +– O +E O +; O +Dataset O +EV1 O +) O +( O +Zheng O +et O +al O +, O +2012 O +) O +( O +Zhu O +et O +al O +, O +2012 O +) O +( O +Muthyala O +et O +al O +, O +2003 O +; O +Seo O +et O +al O +, O +2006 O +) O +( O +Srinivasan O +et O +al O +, O +2013 O +) O +( O +Wang O +et O +al O +, O +2012 O +) O +( O +Liao O +et O +al O +, O +2014 O +) O +( O +Min O +et O +al O +, O +2013 O +). O + +Ligand B-experimental_method +profiling I-experimental_method +using O +our O +quantitative B-experimental_method +bioassays I-experimental_method +revealed O +a O +wide O +range O +of O +ligand O +‐ O +induced O +GREB1 B-protein +expression O +, O +reporter O +gene O +activities O +, O +ERα B-protein +‐ O +coactivator O +interactions O +, O +and O +proliferative O +effects O +on O +MCF O +‐ O +7 O +breast O +cancer O +cells O +( O +Figs O +EV1 O +and O +EV2A O +– O +J O +). O + +This O +wide O +variance O +enabled O +us O +to O +probe O +specific O +features O +of O +ERα B-protein +signaling O +using O +ligand B-experimental_method +class I-experimental_method +analyses I-experimental_method +, O +and O +identify O +signaling O +patterns O +shared O +by O +specific O +ligand O +series O +or O +scaffolds O +. O + +Classes O +of O +compounds O +in O +the O +ERα B-protein +ligand O +library O + +Structural O +details O +of O +the O +ERα B-protein +LBD B-structure_element +bound B-protein_state +to I-protein_state +the O +indicated O +ligands O +. O + +Unlike O +E2 B-chemical +( O +PDB O +1GWR O +), O +TAM B-chemical +is O +a O +direct O +modulator O +with O +a O +BSC O +that O +dislocates O +h12 B-structure_element +to O +block O +the O +NCOA2 B-site +‐ I-site +binding I-site +site I-site +( O +PDB O +3ERT O +). O + +OBHS B-chemical +is O +an O +indirect O +modulator O +that O +dislocates O +the O +h11 B-structure_element +C O +‐ O +terminus O +to O +destabilize O +the O +h11 B-site +– I-site +h12 I-site +interface I-site +( O +PDB O +4ZN9 O +). O + +To O +this O +end O +, O +we O +compared O +the O +average O +ligand O +‐ O +induced O +GREB1 B-protein +mRNA O +levels O +in O +MCF O +‐ O +7 O +cells O +and O +3 B-experimental_method +× I-experimental_method +ERE I-experimental_method +‐ I-experimental_method +Luc I-experimental_method +reporter O +gene O +activity O +in O +Ishikawa O +endometrial O +cancer O +cells O +( O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +) O +or O +in O +HepG2 O +cells O +transfected O +with O +wild B-protein_state +‐ I-protein_state +type I-protein_state +ERα B-protein +( O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +) O +( O +Figs O +3A O +and O +EV2A O +– O +C O +). O + +Direct O +modulators O +showed O +significant O +differences O +in O +average O +activity O +between O +cell O +types O +except O +OBHS B-chemical +‐ I-chemical +ASC I-chemical +analogs O +, O +which O +had O +similar O +low O +agonist O +activities O +in O +the O +three O +cell O +types O +. O + +While O +it O +was O +known O +that O +direct O +modulators O +such O +as O +tamoxifen B-chemical +drive O +cell O +‐ O +specific O +signaling O +, O +these O +experiments O +reveal O +that O +indirect O +modulators O +also O +drive O +cell O +‐ O +specific O +signaling O +, O +since O +eight O +of O +fourteen O +classes O +showed O +significant O +differences O +in O +average O +activity O +( O +Figs O +3A O +and O +EV2A O +– O +C O +). O + +Ligand O +‐ O +specific O +signaling O +underlies O +ERα B-protein +‐ O +mediated O +cell O +proliferation O + +( O +A O +) O +Ligand O +‐ O +specific O +ERα B-protein +activities O +in O +HepG2 O +, O +Ishikawa O +and O +MCF O +‐ O +7 O +cells O +. O + +The O +ligand O +‐ O +induced O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +and O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activities O +and O +GREB1 B-protein +mRNA O +levels O +are O +shown O +by O +scaffold O +( O +mean O ++ O +SD O +). O + +( O +B O +) O +Ligand O +class B-experimental_method +analysis I-experimental_method +of O +the O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +and O +ERα B-mutant +‐ I-mutant +ΔAB I-mutant +activities O +in O +HepG2 O +cells O +. O + +Significant O +sensitivity O +to O +AB B-structure_element +domain O +deletion O +was O +determined O +by O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +‐ I-experimental_method +test I-experimental_method +( O +n O += O +number O +of O +ligands O +per O +scaffold O +in O +Fig O +2 O +). O + +Correlation B-experimental_method +and I-experimental_method +regression I-experimental_method +analyses I-experimental_method +in O +a O +large O +test O +set O +. O + +In O +cluster O +1 O +, O +the O +first O +three O +comparisons O +( O +rows O +) O +showed O +significant O +positive O +correlations O +( O +F B-experimental_method +‐ I-experimental_method +test I-experimental_method +for O +nonzero O +slope O +, O +P B-evidence +≤ O +0 O +. O +05 O +). O + +In O +cluster O +2 O +, O +only O +one O +of O +these O +comparisons O +revealed O +a O +significant O +positive O +correlation O +, O +while O +none O +was O +significant O +in O +cluster O +3 O +. O ++, O +statistically O +significant O +correlations O +gained O +by O +deletion B-experimental_method +of O +the O +AB B-structure_element +or O +F B-structure_element +domains O +. O + +−, O +significant O +correlations O +lost O +upon O +deletion O +of O +AB B-structure_element +or O +F B-structure_element +domains O +. O + +Tamoxifen B-chemical +depends O +on O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +for O +its O +cell O +‐ O +specific O +activity O +( O +Sakamoto O +et O +al O +, O +2002 O +); O +therefore O +, O +we O +asked O +whether O +cell O +‐ O +specific O +signaling O +observed O +here O +is O +due O +to O +a O +similar O +dependence O +on O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +for O +activity O +( O +Fig O +EV1 O +). O + +While O +E2 B-chemical +showed O +similar O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +and O +ERα B-mutant +‐ I-mutant +ΔAB I-mutant +activities O +, O +tamoxifen B-chemical +showed O +complete O +loss O +of O +activity O +without B-protein_state +the O +AB B-structure_element +domain O +( O +Fig O +EV1B O +). O + +These O +“ O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +‐ O +sensitive O +” O +activities O +were O +exhibited O +by O +both O +direct O +and O +indirect O +modulators O +, O +and O +were O +not O +limited O +to O +scaffolds O +that O +showed O +cell O +‐ O +specific O +signaling O +( O +Fig O +3A O +and O +B O +). O + +Thus O +, O +the O +strength O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +signaling O +does O +not O +determine O +cell O +‐ O +specific O +signaling O +. O + +Identifying O +cell O +‐ O +specific O +signaling O +clusters O +in O +ERα B-protein +ligand O +classes O + +For O +each O +ligand O +class O +or O +scaffold O +, O +we O +calculated O +the O +Pearson B-evidence +' I-evidence +s I-evidence +correlation I-evidence +coefficient I-evidence +, O +r B-evidence +, O +for O +pairwise O +comparison O +of O +activity O +profiles O +in O +breast O +( O +GREB1 B-protein +), O +liver O +( O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +), O +and O +endometrial O +cells O +( O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +). O + +The O +value O +of O +r B-evidence +ranges O +from O +− O +1 O +to O +1 O +, O +and O +it O +defines O +the O +extent O +to O +which O +the O +data O +fit O +a O +straight O +line O +when O +compounds O +show O +similar O +agonist O +/ O +antagonist O +activity O +profiles O +between O +cell O +types O +( O +Fig O +EV3A O +). O + +We O +also O +calculated O +the O +coefficient B-evidence +of I-evidence +determination I-evidence +, O +r B-evidence +2 I-evidence +, O +which O +describes O +the O +percentage O +of O +variance O +in O +a O +dependent O +variable O +such O +as O +proliferation O +that O +can O +be O +predicted O +by O +an O +independent O +variable O +such O +as O +GREB1 B-protein +expression O +. O + +We O +present O +both O +calculations O +as O +r B-evidence +2 I-evidence +to O +readily O +compare O +signaling O +specificities O +using O +a O +heat O +map O +on O +which O +the O +red O +– O +yellow O +palette O +indicates O +significant O +positive O +correlations O +( O +P B-evidence +≤ O +0 O +. O +05 O +, O +F B-experimental_method +‐ I-experimental_method +test I-experimental_method +for O +nonzero O +slope O +), O +while O +the O +blue O +palette O +denotes O +negative O +correlations O +( O +Fig O +3C O +– O +F O +). O + +The O +side O +chain O +of O +OBHS B-chemical +‐ I-chemical +BSC I-chemical +analogs O +induces O +cell O +‐ O +specific O +signaling O + +Correlation O +analysis O +of O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-mutant +‐ I-mutant +ΔAB I-mutant +activity O +versus O +endogenous O +ERα B-protein +activity O +of O +OBHS B-chemical +analogs O +. O + +In O +panel O +( O +D O +), O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +activity O +from O +panel O +( O +B O +) O +is O +shown O +for O +comparison O +. O + +Correlation O +analysis O +of O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-mutant +‐ I-mutant +ΔF I-mutant +activity O +versus O +endogenous O +ERα B-protein +activities O +of O +OBHS B-chemical +analogs O +. O + +Correlation O +analysis O +of O +MCF O +‐ O +7 O +cell O +proliferation O +versus O +NCOA2 B-protein +/ I-protein +3 I-protein +recruitment O +or O +GREB1 B-protein +levels O +observed O +in O +response O +to O +( O +G O +) O +OBHS B-chemical +‐ I-chemical +N I-chemical +and O +( O +H O +) O +OBHS B-chemical +‐ I-chemical +BSC I-chemical +analogs O +. O + +Scaffolds O +in O +cluster O +1 O +exhibited O +strongly O +correlated O +GREB1 B-protein +levels O +, O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +and O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O +profiles O +across O +the O +three O +cell O +types O +( O +Fig O +3C O +lanes O +1 O +– O +4 O +), O +suggesting O +these O +ligands O +use O +similar O +ERα B-protein +signaling O +pathways O +in O +the O +breast O +, O +endometrial O +, O +and O +liver O +cell O +types O +. O + +This O +cluster O +includes O +WAY B-chemical +‐ I-chemical +C I-chemical +, O +OBHS B-chemical +, O +OBHS B-chemical +‐ I-chemical +N I-chemical +, O +and O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +, O +all O +of O +which O +are O +indirect O +modulators O +. O + +This O +cluster O +includes O +two O +classes O +of O +direct O +modulators O +( O +cyclofenil B-chemical +‐ I-chemical +ASC I-chemical +and O +WAY B-chemical +dimer I-chemical +), O +and O +six O +classes O +of O +indirect O +modulators O +( O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +, O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +, O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +2 I-chemical +and O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +, O +furan B-chemical +, O +and O +WAY B-chemical +‐ I-chemical +D I-chemical +). O + +For O +example O +, O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +, O +furan B-chemical +, O +and O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +2 I-chemical +drove O +positively O +correlated O +GREB1 B-protein +levels O +and O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +but O +not O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +activity O +( O +Fig O +3C O +lanes O +5 O +– O +7 O +). O + +In O +contrast O +, O +WAY B-chemical +dimer I-chemical +and O +WAY B-chemical +‐ I-chemical +D I-chemical +analogs O +drove O +positively O +correlated O +GREB1 B-protein +levels O +and O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +but O +not O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O +( O +Fig O +3C O +lanes O +8 O +and O +9 O +). O + +This O +is O +demonstrated O +by O +directly O +comparing O +the O +signaling O +specificities O +of O +matched O +OBHS B-chemical +( O +indirect O +modulator O +, O +cluster O +1 O +) O +and O +OBHS B-chemical +‐ I-chemical +BSC I-chemical +analogs O +( O +direct O +modulator O +, O +cluster O +3 O +), O +which O +differ O +only O +in O +the O +basic O +side O +chain O +( O +Fig O +2E O +). O + +The O +activities O +of O +OBHS B-chemical +analogs O +were O +positively O +correlated O +across O +the O +three O +cell O +types O +, O +but O +the O +side O +chain O +of O +OBHS B-chemical +‐ I-chemical +BSC I-chemical +analogs O +was O +sufficient O +to O +abolish O +these O +correlations O +( O +Figs O +3C O +lanes O +1 O +and O +19 O +, O +and O +EV3A O +– O +C O +). O + +This O +demonstrates O +that O +the O +signaling O +specificities O +underlying O +these O +positive O +correlations O +are O +not O +modified O +by O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +. O + +Despite O +this O +nearly O +complete O +lack O +of O +activity O +, O +the O +pattern O +of O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-mutant +‐ I-mutant +ΔAB I-mutant +activity O +was O +still O +highly O +correlated O +with O +the O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O +and O +GREB1 B-protein +expression O +( O +Fig O +EV3D O +and O +E O +), O +demonstrating O +that O +very O +small O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +activities O +can O +be O +amplified O +by O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +to O +produce O +robust O +signals O +. O + +Similarly O +, O +deletion B-experimental_method +of I-experimental_method +the O +F B-structure_element +domain O +did O +not O +abolish O +correlations O +between O +the O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +and O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +or O +GREB1 B-protein +levels O +induced O +by O +OBHS B-chemical +analogs O +( O +Fig O +EV3F O +). O + +Deletion B-experimental_method +of I-experimental_method +the O +AB B-structure_element +or O +F B-structure_element +domain O +altered O +correlations O +for O +six O +of O +the O +eight O +scaffolds O +in O +this O +cluster O +( O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +, O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +, O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +, O +WAY B-chemical +‐ I-chemical +D I-chemical +, O +WAY B-chemical +dimer I-chemical +, O +and O +cyclofenil B-chemical +‐ I-chemical +ASC I-chemical +) O +( O +Fig O +3D O +lanes O +5 O +– O +12 O +). O + +For O +ligands O +in O +cluster O +3 O +, O +we O +could O +not O +eliminate O +a O +role O +for O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +in O +determining O +signaling O +specificity O +, O +since O +this O +cluster O +lacked O +positively O +correlated O +activity O +profiles O +( O +Fig O +3C O +), O +and O +deletion B-experimental_method +of I-experimental_method +the O +AB B-structure_element +or O +F B-structure_element +domain O +rarely O +induced O +such O +correlations O +( O +Fig O +3D O +), O +except O +for O +A B-chemical +‐ I-chemical +CD I-chemical +and O +OBHS B-chemical +‐ I-chemical +ASC I-chemical +analogs O +, O +where O +deletion B-experimental_method +of I-experimental_method +the O +AB B-structure_element +domain O +or O +F B-structure_element +domain O +led O +to O +positive O +correlations O +with O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O +and O +/ O +or O +GREB1 B-protein +levels O +( O +Fig O +3D O +lanes O +13 O +and O +18 O +). O + +Ligand O +‐ O +specific O +control O +of O +GREB1 B-protein +expression O + +To O +determine O +whether O +ligand O +classes O +control O +expression O +of O +native O +ERα B-protein +target O +genes O +through O +the O +canonical O +linear O +signaling O +pathway O +, O +we O +performed O +pairwise B-experimental_method +linear I-experimental_method +regression I-experimental_method +analyses I-experimental_method +using O +ERα B-complex_assembly +– I-complex_assembly +NCOA1 I-complex_assembly +/ I-complex_assembly +2 I-complex_assembly +/ I-complex_assembly +3 I-complex_assembly +interactions O +in O +M2H B-experimental_method +assay I-experimental_method +as O +independent O +predictors O +of O +GREB1 B-protein +expression O +( O +the O +dependent O +variable O +) O +( O +Figs O +EV1 O +and O +EV2A O +, O +F O +– O +H O +). O + +In O +cluster O +1 O +, O +the O +recruitment O +of O +NCOA1 B-protein +and O +NCOA2 B-protein +was O +highest O +for O +WAY B-chemical +‐ I-chemical +C I-chemical +, O +followed O +by O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +, O +OBHS B-chemical +‐ I-chemical +N I-chemical +, O +and O +OBHS B-chemical +series O +, O +while O +for O +NCOA3 B-protein +, O +OBHS B-chemical +‐ I-chemical +N I-chemical +compounds O +induced O +the O +most O +recruitment O +and O +OBHS B-chemical +ligands O +were O +inverse O +agonists O +( O +Fig O +EV2F O +– O +H O +). O + +GREB1 B-protein +levels O +induced O +by O +OBHS B-chemical +analogs O +were O +determined O +by O +recruitment O +of O +NCOA1 B-protein +but O +not O +NCOA2 B-protein +/ I-protein +3 I-protein +( O +Fig O +3E O +lane O +1 O +), O +suggesting O +that O +there O +may O +be O +alternate O +or O +preferential O +use O +of O +these O +coactivators O +by O +different O +classes O +. O + +However O +, O +in O +cluster O +1 O +, O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +generally O +predicted O +GREB1 B-protein +levels O +( O +Fig O +3E O +lanes O +1 O +– O +4 O +), O +consistent O +with O +the O +canonical O +signaling O +model O +( O +Fig O +1D O +). O + +For O +clusters O +2 O +and O +3 O +, O +GREB1 B-protein +activity O +was O +generally O +not O +predicted O +by O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +. O + +Direct O +modulators O +showed O +low O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +( O +Fig O +EV2F O +– O +H O +), O +but O +only O +OBHS B-chemical +‐ I-chemical +ASC I-chemical +analogs O +had O +NCOA2 B-protein +recruitment O +profiles O +that O +predicted O +a O +full O +range O +of O +effects O +on O +GREB1 B-protein +levels O +( O +Figs O +3E O +lanes O +9 O +, O +11 O +, O +18 O +– O +19 O +, O +and O +EV2A O +). O + +The O +indirect O +modulators O +in O +clusters O +2 O +and O +3 O +stimulated O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +and O +GREB1 B-protein +expression O +with O +substantial O +variance O +( O +Figs O +3A O +and O +EV2F O +– O +H O +). O + +However O +, O +ligand O +‐ O +induced O +GREB1 B-protein +levels O +were O +generally O +not O +determined O +by O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +( O +Fig O +3E O +lanes O +5 O +– O +19 O +), O +consistent O +with O +an O +alternate O +causality O +model O +( O +Fig O +1E O +). O + +Out O +of O +11 O +indirect O +modulator O +series O +in O +cluster O +2 O +or O +3 O +, O +only O +the O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +class O +had O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +profiles O +that O +predicted O +GREB1 B-protein +levels O +( O +Fig O +3E O +lane O +12 O +). O + +To O +determine O +mechanisms O +for O +ligand O +‐ O +dependent O +control O +of O +breast O +cancer O +cell O +proliferation O +, O +we O +performed O +linear B-experimental_method +regression I-experimental_method +analyses I-experimental_method +across O +the O +19 O +scaffolds O +using O +MCF O +‐ O +7 O +cell O +proliferation O +as O +the O +dependent O +variable O +, O +and O +the O +other O +activities O +as O +independent O +variables O +( O +Fig O +3F O +). O + +In O +cluster O +1 O +, O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +and O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activities O +, O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +, O +and O +GREB1 B-protein +levels O +generally O +predicted O +the O +proliferative O +response O +( O +Fig O +3F O +lanes O +2 O +– O +4 O +). O + +With O +the O +OBHS B-chemical +‐ I-chemical +N I-chemical +compounds O +, O +NCOA3 B-protein +and O +GREB1 B-protein +showed O +near O +perfect O +prediction O +of O +proliferation O +( O +Fig O +EV3G O +), O +with O +unexplained O +variance O +similar O +to O +the O +noise O +in O +the O +assays O +. O + +The O +lack O +of O +significant O +predictors O +for O +OBHS B-chemical +analogs O +( O +Fig O +3F O +lane O +1 O +) O +reflects O +their O +small O +range O +of O +proliferative O +effects O +on O +MCF O +‐ O +7 O +cells O +( O +Fig O +EV2I O +). O + +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +, O +cyclofenil B-chemical +, O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +, O +and O +imidazopyridine B-chemical +analogs O +had O +NCOA1 B-protein +/ I-protein +3 I-protein +recruitment O +profiles O +that O +predicted O +their O +proliferative O +effects O +, O +without O +determining O +GREB1 B-protein +levels O +( O +Fig O +3E O +and O +F O +, O +lanes O +5 O +and O +14 O +– O +16 O +). O + +Similarly O +, O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +, O +cyclofenil B-chemical +‐ I-chemical +ASC I-chemical +, O +and O +OBHS B-chemical +‐ I-chemical +ASC I-chemical +had O +positively O +correlated O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +recruitment O +and O +GREB1 B-protein +levels O +, O +but O +none O +of O +these O +activities O +determined O +their O +proliferative O +effects O +( O +Fig O +3E O +and O +F O +lanes O +11 O +– O +12 O +and O +18 O +). O + +For O +ligands O +that O +show O +cell O +‐ O +specific O +signaling O +, O +ERα B-protein +‐ O +mediated O +recruitment O +of O +other O +coregulators O +and O +activation O +of O +other O +target O +genes O +likely O +determine O +their O +proliferative O +effects O +on O +MCF O +‐ O +7 O +cells O +. O + +We O +also O +questioned O +whether O +promoter O +occupancy O +by O +coactivators O +is O +statistically O +robust O +and O +reproducible O +for O +ligand O +class O +analysis O +using O +a O +chromatin B-experimental_method +immunoprecipitation I-experimental_method +( I-experimental_method +ChIP I-experimental_method +)‐ I-experimental_method +based I-experimental_method +quantitative I-experimental_method +assay I-experimental_method +, I-experimental_method +and O +whether O +it O +has O +a O +better O +predictive O +power O +than O +the O +M2H B-experimental_method +assay I-experimental_method +. O + +ERα B-protein +and O +NCOA3 B-protein +cycle O +on O +and O +off O +the O +GREB1 B-protein +promoter O +( O +Nwachukwu O +et O +al O +, O +2014 O +). O + +Therefore O +, O +we O +first O +performed O +a O +time B-experimental_method +‐ I-experimental_method +course I-experimental_method +study I-experimental_method +, O +and O +found O +that O +E2 B-chemical +and O +the O +WAY B-chemical +‐ I-chemical +C I-chemical +analog O +, O +AAPII B-chemical +‐ I-chemical +151 I-chemical +‐ I-chemical +4 I-chemical +, O +induced O +recruitment O +of O +NCOA3 B-protein +to O +the O +GREB1 B-protein +promoter O +in O +a O +temporal O +cycle O +that O +peaked O +after O +45 O +min O +in O +MCF O +‐ O +7 O +cells O +( O +Fig O +4A O +). O + +At O +this O +time O +point O +, O +other O +WAY B-chemical +‐ I-chemical +C I-chemical +analogs O +also O +induced O +recruitment O +of O +NCOA3 B-protein +at O +this O +site O +to O +varying O +degrees O +( O +Fig O +4B O +). O + +The O +Z B-evidence +’ I-evidence +for O +this O +assay O +was O +0 O +. O +6 O +, O +showing O +statistical O +robustness O +( O +see O +Materials O +and O +Methods O +). O + +We O +prepared O +biological O +replicates O +with O +different O +cell O +passage O +numbers O +and O +separately O +prepared O +samples O +, O +which O +showed O +r B-evidence +2 I-evidence +of O +0 O +. O +81 O +, O +demonstrating O +high O +reproducibility O +( O +Fig O +4C O +). O + +Kinetic B-experimental_method +ChIP I-experimental_method +assay I-experimental_method +examining O +recruitment O +of O +NCOA3 B-protein +to O +the O +GREB1 B-protein +gene O +in O +MCF O +‐ O +7 O +cells O +stimulated O +with O +E2 B-chemical +or O +the O +indicated O +WAY B-chemical +‐ I-chemical +C I-chemical +analog O +. O + +NCOA3 B-protein +occupancy O +at O +GREB1 B-protein +was O +compared O +by O +ChIP B-experimental_method +assay I-experimental_method +45 O +min O +after O +stimulation O +with O +vehicle O +, O +E2 B-chemical +, O +or O +the O +WAY B-chemical +‐ I-chemical +C I-chemical +analogs O +. O + +In O +panel O +( O +B O +), O +the O +average O +recruitment O +of O +two O +biological O +replicates O +are O +shown O +as O +mean O ++ O +SEM O +, O +and O +the O +Z B-evidence +‐ I-evidence +score I-evidence +is O +indicated O +. O + +Linear B-experimental_method +regression I-experimental_method +analyses I-experimental_method +comparing O +the O +ability O +of O +NCOA3 B-protein +recruitment O +, O +measured O +by O +ChIP B-experimental_method +or O +M2H B-experimental_method +, O +to O +predict O +other O +agonist O +activities O +of O +WAY B-chemical +‐ I-chemical +C I-chemical +analogs O +. O +* O +Significant O +positive O +correlation O +( O +F B-experimental_method +‐ I-experimental_method +test I-experimental_method +for O +nonzero O +slope O +, O +P B-evidence +‐ I-evidence +value I-evidence +). O + +Thus O +, O +the O +simplified O +coactivator B-experimental_method +‐ I-experimental_method +binding I-experimental_method +assay I-experimental_method +showed O +much O +greater O +predictive O +power O +than O +the O +ChIP B-experimental_method +assay I-experimental_method +for O +ligand O +‐ O +specific O +effects O +on O +GREB1 B-protein +expression O +and O +cell O +proliferation O +. O + +One O +difference O +between O +MCF O +‐ O +7 O +breast O +cancer O +cells O +and O +Ishikawa O +endometrial O +cancer O +cells O +is O +the O +contribution O +of O +ERβ B-protein +to O +estrogenic O +response O +, O +as O +Ishikawa O +cells O +may O +express O +ERβ B-protein +( O +Bhat O +& O +Pezzuto O +, O +2001 O +). O + +When O +overexpressed B-experimental_method +in O +MCF O +‐ O +7 O +cells O +, O +ERβ B-protein +alters O +E2 B-chemical +‐ O +induced O +expression O +of O +only O +a O +subset O +of O +ERα B-protein +‐ O +target O +genes O +( O +Wu O +et O +al O +, O +2011 O +), O +raising O +the O +possibility O +that O +ligand O +‐ O +induced O +ERβ B-protein +activity O +may O +contribute O +to O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activities O +, O +and O +thus O +underlie O +the O +lack O +of O +correlation O +between O +the O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +and O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +activities O +or O +GREB1 B-protein +levels O +induced O +by O +cell O +‐ O +specific O +modulators O +in O +cluster O +2 O +and O +cluster O +3 O +( O +Fig O +3C O +). O + +To O +test O +this O +idea O +, O +we O +determined O +the O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERβ O +activity O +profiles O +of O +the O +ligands O +( O +Fig O +EV1 O +). O + +All O +direct O +modulator O +and O +two O +indirect O +modulator O +scaffolds O +( O +OBHS B-chemical +and O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +) O +lacked O +ERβ O +agonist O +activity O +. O + +Nevertheless O +, O +the O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activities O +of O +both O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +and O +cyclofenil B-chemical +analogs O +were O +better O +predicted O +by O +their O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERα B-protein +‐ O +WT B-protein_state +than O +L B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +ERβ B-protein +activities O +( O +Fig O +EV4A O +and O +B O +). O + +ERβ B-protein +activity O +is O +not O +an O +independent O +predictor O +of O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O + +ERβ B-protein +activity O +in O +HepG2 O +cells O +rarely O +correlates O +with O +E B-experimental_method +‐ I-experimental_method +Luc I-experimental_method +activity O +. O + +Data O +information O +: O +The O +r O +2 O +and O +P B-evidence +values I-evidence +for O +the O +indicated O +correlations O +are O +shown O +in O +both O +panels O +. O +* O +Significant O +positive O +correlation O +( O +F B-experimental_method +‐ I-experimental_method +test I-experimental_method +for O +nonzero O +slope O +, O +P B-evidence +‐ I-evidence +value I-evidence +) O + +To O +overcome O +barriers O +to O +crystallization B-experimental_method +of O +ERα B-protein +LBD B-structure_element +complexes O +, O +we O +developed O +a O +conformation B-experimental_method +‐ I-experimental_method +trapping I-experimental_method +X I-experimental_method +‐ I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +approach O +using O +the O +ERα B-mutant +‐ I-mutant +Y537S I-mutant +mutation O +( O +Nettles O +et O +al O +, O +2008 O +; O +Bruning O +et O +al O +, O +2010 O +; O +Srinivasan O +et O +al O +, O +2013 O +). O + +To O +further O +validate O +this O +approach O +, O +we O +solved B-experimental_method +the O +structure B-evidence +of O +the O +ERα B-mutant +‐ I-mutant +Y537S I-mutant +LBD B-structure_element +in B-protein_state +complex I-protein_state +with I-protein_state +diethylstilbestrol B-chemical +( O +DES B-chemical +), O +which O +bound O +identically O +in O +the O +wild B-protein_state +‐ I-protein_state +type I-protein_state +and O +ERα B-mutant +‐ I-mutant +Y537S I-mutant +LBDs B-structure_element +, O +demonstrating O +again O +that O +this O +surface O +mutation O +stabilizes O +h12 B-structure_element +dynamics O +to O +facilitate O +crystallization O +without O +changing O +ligand O +binding O +( O +Appendix O +Fig O +S1A O +and O +B O +) O +( O +Nettles O +et O +al O +, O +2008 O +; O +Bruning O +et O +al O +, O +2010 O +; O +Delfosse O +et O +al O +, O +2012 O +). O + +Using O +this O +approach O +, O +we O +solved B-experimental_method +76 O +ERα B-protein +LBD B-structure_element +structures B-evidence +in O +the O +active B-protein_state +conformation I-protein_state +and O +bound B-protein_state +to I-protein_state +ligands I-protein_state +studied O +here O +( O +Appendix O +Fig O +S1C O +). O + +Eleven O +of O +these O +structures B-evidence +have O +been O +published O +, O +while O +65 O +are O +new O +, O +including O +the O +DES B-protein_state +‐ I-protein_state +bound I-protein_state +ERα B-mutant +‐ I-mutant +Y537S I-mutant +LBD B-structure_element +. O + +We O +present O +57 O +of O +these O +new O +structures B-evidence +here O +( O +Dataset O +EV2 O +), O +while O +the O +remaining O +eight O +new O +structures B-evidence +bound B-protein_state +to I-protein_state +OBHS B-chemical +‐ I-chemical +N I-chemical +analogs O +will O +be O +published O +elsewhere O +( O +S O +. O +Srinivasan O +et O +al O +, O +in O +preparation O +). O + +The O +indirect O +modulator O +scaffolds O +in O +cluster O +1 O +did O +not O +show O +cell O +‐ O +specific O +signaling O +( O +Fig O +3C O +), O +but O +shared O +common O +structural O +perturbations O +that O +we O +designed O +to O +modulate O +h12 B-structure_element +dynamics O +. O + +Based O +on O +our O +original O +OBHS B-chemical +structure B-evidence +, O +the O +OBHS B-chemical +, O +OBHS B-chemical +‐ I-chemical +N I-chemical +, O +and O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +compounds O +were O +modified O +with O +h11 B-structure_element +‐ O +directed O +pendant O +groups O +( O +Zheng O +et O +al O +, O +2012 O +; O +Zhu O +et O +al O +, O +2012 O +; O +Liao O +et O +al O +, O +2014 O +). O + +Superposing B-experimental_method +the O +LBDs B-structure_element +based O +on O +the O +class O +of O +bound O +ligands O +provides O +an O +ensemble O +view O +of O +the O +structural O +variance O +and O +clarifies O +what O +part O +of O +the O +ligand B-site +‐ I-site +binding I-site +pocket I-site +is O +differentially O +perturbed O +or O +targeted O +. O + +We O +observed O +that O +the O +OBHS B-chemical +‐ I-chemical +N I-chemical +analogs O +displaced O +h11 B-structure_element +along O +a O +vector O +away O +from O +Leu354 B-residue_name_number +in O +a O +region O +of O +h3 B-structure_element +that O +is O +unaffected O +by O +the O +ligands O +, O +and O +toward O +the O +dimer B-site +interface I-site +. O + +For O +the O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +, O +the O +displacement O +of O +h11 B-structure_element +was O +in O +a O +perpendicular O +direction O +, O +away O +from O +Ile424 B-residue_name_number +in O +h8 B-structure_element +and O +toward O +h12 B-structure_element +. O + +Remarkably O +, O +these O +individual O +inter B-evidence +‐ I-evidence +atomic I-evidence +distances I-evidence +showed O +a O +ligand O +class O +‐ O +specific O +ability O +to O +significantly O +predict O +proliferative O +effects O +( O +Fig O +5E O +and O +F O +), O +demonstrating O +the O +feasibility O +of O +developing O +a O +minimal O +set O +of O +activity O +predictors O +from O +crystal B-evidence +structures I-evidence +. O + +Structure B-experimental_method +‐ I-experimental_method +class I-experimental_method +analysis I-experimental_method +of O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +. O + +Arrows O +indicate O +chemical O +variance O +in O +the O +orientation O +of O +the O +different O +h11 B-structure_element +‐ O +directed O +ligand O +side O +groups O +( O +PDB O +5DK9 O +, O +5DKB O +, O +5DKE O +, O +5DKG O +, O +5DKS O +, O +5DL4 O +, O +5DLR O +, O +5DMC O +, O +5DMF O +and O +5DP0 O +). O + +Panel O +( O +B O +) O +shows O +the O +crystal B-evidence +structure I-evidence +of O +a O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analog O +‐ O +bound O +ERα B-protein +LBD B-structure_element +( O +PDB O +5DLR O +). O + +The O +h11 B-site +– I-site +h12 I-site +interface I-site +( O +circled O +) O +includes O +the O +C O +‐ O +terminal O +part O +of O +h11 B-structure_element +. O + +This O +region O +was O +expanded O +in O +panel O +( O +C O +), O +where O +the O +10 O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analog O +‐ O +bound O +ERα B-protein +LBD B-structure_element +structures B-evidence +( O +see O +Datasets O +EV1 O +and O +EV2 O +) O +were O +superposed B-experimental_method +to O +show O +variations O +in O +the O +h11 B-structure_element +C O +‐ O +terminus O +( O +PDB O +5DK9 O +, O +5DKB O +, O +5DKE O +, O +5DKG O +, O +5DKS O +, O +5DL4 O +, O +5DLR O +, O +5DMC O +, O +5DMF O +, O +and O +5DP0 O +). O + +Inter B-evidence +‐ I-evidence +atomic I-evidence +distances I-evidence +predict O +the O +proliferative O +effects O +of O +specific O +ligand O +series O +. O + +Ile424 B-residue_name_number +– O +His524 B-residue_name_number +distance B-evidence +measured O +in O +the O +crystal B-evidence +structures I-evidence +correlates O +with O +the O +proliferative O +effect O +of O +triaryl B-chemical +‐ I-chemical +ethylene I-chemical +analogs O +in O +MCF O +‐ O +7 O +cells O +. O + +In O +contrast O +, O +the O +Leu354 B-residue_name_number +– O +Leu525 B-residue_name_number +distance B-evidence +correlates O +with O +the O +proliferative O +effects O +of O +OBHS B-chemical +‐ I-chemical +N I-chemical +analogs O +in O +MCF O +‐ O +7 O +cells O +. O + +Structure B-experimental_method +‐ I-experimental_method +class I-experimental_method +analysis I-experimental_method +of O +WAY B-chemical +‐ I-chemical +C I-chemical +analogs O +. O + +WAY B-chemical +‐ I-chemical +C I-chemical +side O +groups O +subtly O +nudge O +h12 B-structure_element +Leu540 B-residue_name_number +. O + +ERα B-protein +LBD B-structure_element +structures B-evidence +bound B-protein_state +to I-protein_state +4 O +distinct O +WAY B-chemical +‐ I-chemical +C I-chemical +analogs O +were O +superposed B-experimental_method +( O +PDB O +4 O +IU7 O +, O +4IV4 O +, O +4IVW O +, O +4IW6 O +) O +( O +see O +Datasets O +EV1 O +and O +EV2 O +). O + +Structure B-experimental_method +‐ I-experimental_method +class I-experimental_method +analysis I-experimental_method +of O +indirect O +modulators O + +Structure B-experimental_method +‐ I-experimental_method +class I-experimental_method +analysis I-experimental_method +of O +indirect O +modulators O +in O +cluster O +1 O +. O + +Crystal B-evidence +structures I-evidence +of O +the O +ERα B-protein +LBD B-structure_element +bound B-protein_state +to I-protein_state +OBHS B-chemical +and O +OBHS B-chemical +‐ I-chemical +N I-chemical +analogs O +were O +superposed B-experimental_method +. O + +Arrows O +indicate O +chemical O +variance O +in O +the O +orientation O +of O +the O +different O +h11 B-structure_element +‐ O +directed O +ligand O +side O +groups O +. O + +Panel O +( O +B O +) O +shows O +the O +ligand O +‐ O +induced O +conformational O +variation O +at O +the O +C O +‐ O +terminal O +region O +of O +h11 B-structure_element +( O +OBHS B-chemical +: O +PDB O +4ZN9 O +, O +4ZNH O +, O +4ZNS O +, O +4ZNT O +, O +4ZNU O +, O +4ZNV O +, O +and O +4ZNW O +; O +OBHS B-chemical +‐ I-chemical +N I-chemical +: O +PDB O +4ZUB O +, O +4ZUC O +, O +4ZWH O +, O +4ZWK O +, O +5BNU O +, O +5BP6 O +, O +5BPR O +, O +and O +5BQ4 O +). O + +Structure B-experimental_method +‐ I-experimental_method +class I-experimental_method +analysis I-experimental_method +of O +indirect O +modulators O +in O +clusters O +2 O +and O +3 O +. O + +Crystal B-evidence +structures I-evidence +of O +the O +ERα B-protein +LBD B-structure_element +bound B-protein_state +to I-protein_state +ligands O +with O +cell O +‐ O +specific O +activities O +were O +superposed B-experimental_method +. O + +As O +visualized O +in O +four O +LBD B-structure_element +structures B-evidence +( O +Srinivasan O +et O +al O +, O +2013 O +), O +WAY B-chemical +‐ I-chemical +C I-chemical +analogs O +were O +designed O +with O +small O +substitutions O +that O +slightly O +nudge O +h12 B-structure_element +Leu540 B-residue_name_number +, O +without O +exiting O +the O +ligand B-site +‐ I-site +binding I-site +pocket I-site +( O +Fig O +5G O +and O +H O +). O + +Therefore O +, O +changing O +h12 B-structure_element +dynamics O +maintains O +the O +canonical O +signaling O +pathway O +defined O +by O +E2 B-chemical +( O +Fig O +1D O +) O +to O +support O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +‐ O +driven O +signaling O +and O +recruit O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +for O +GREB1 B-protein +‐ O +stimulated O +proliferation O +. O + +Therefore O +, O +we O +examined O +another O +50 O +LBD B-structure_element +structures B-evidence +containing O +ligands O +in O +clusters O +2 O +and O +3 O +. O + +These O +structures B-evidence +demonstrated O +that O +cell O +‐ O +specific O +activity O +derived O +from O +altering O +the O +shape O +of O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +without O +an O +extended O +side O +chain O +. O + +Ligands O +in O +cluster O +2 O +and O +cluster O +3 O +showed O +conformational O +heterogeneity O +in O +parts O +of O +the O +scaffold O +that O +were O +directed O +toward O +multiple O +regions O +of O +the O +receptor O +including O +h3 B-structure_element +, O +h8 B-structure_element +, O +h11 B-structure_element +, O +h12 B-structure_element +, O +and O +/ O +or O +the O +β B-structure_element +‐ I-structure_element +sheets I-structure_element +( O +Fig O +EV5C O +– O +G O +). O + +This O +difference O +in O +ligand O +positioning O +altered O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +via O +a O +shift O +in O +the O +N O +‐ O +terminus O +of O +h12 B-structure_element +, O +which O +directly O +contacts O +the O +coactivator O +. O + +This O +effect O +is O +evident O +in O +a O +single O +structure B-evidence +due O +to O +its O +1 O +Å O +magnitude O +( O +Fig O +6A O +and O +B O +). O + +The O +shifts O +in O +h12 B-structure_element +residues O +Asp538 B-residue_name_number +and O +Leu539 B-residue_name_number +led O +to O +rotation O +of O +the O +coactivator O +peptide O +( O +Fig O +6C O +). O + +Thus O +, O +cell O +‐ O +specific O +activity O +can O +stem O +from O +perturbation O +of O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +without O +an O +extended O +side O +chain O +, O +which O +presumably O +alters O +the O +receptor O +– O +coregulator O +interaction O +profile O +. O + +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +2 I-chemical +/ I-chemical +3 I-chemical +analogs O +subtly O +distort O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +. O + +The O +h3 B-site +– I-site +h12 I-site +interface I-site +( O +circled O +) O +at O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +( O +pink O +) O +was O +expanded O +in O +panels O +( O +B O +, O +C O +). O + +The O +S B-protein_state +‐ I-protein_state +OBHS I-protein_state +‐ I-protein_state +2 I-protein_state +/ I-protein_state +3 I-protein_state +‐ I-protein_state +bound I-protein_state +ERα B-protein +LBDs B-structure_element +were O +superposed B-experimental_method +to O +show O +shifts O +in O +h3 B-structure_element +( O +panel O +B O +) O +and O +the O +NCOA2 B-protein +peptide O +docked O +at O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +( O +panel O +C O +). O + +Crystal B-evidence +structures I-evidence +show O +that O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +shift O +h3 B-structure_element +and O +h11 B-structure_element +further O +apart O +compared O +to O +an O +A O +‐ O +CD O +‐ O +ring O +estrogen B-chemical +( O +PDB O +4PPS O +, O +5DRM O +, O +5DRJ O +). O + +The O +2F O +o O +‐ O +F O +c O +electron O +density O +map O +and O +F O +o O +‐ O +F O +c O +difference O +map O +of O +a O +2 B-protein_state +, I-protein_state +5 I-protein_state +‐ I-protein_state +DTP I-protein_state +‐ I-protein_state +bound I-protein_state +structure B-evidence +( O +PDB O +5DRJ O +) O +were O +contoured O +at O +1 O +. O +0 O +sigma O +and O +± O +3 O +. O +0 O +sigma O +, O +respectively O +. O + +Average O +( O +mean O ++ O +SEM O +) O +α B-evidence +‐ I-evidence +carbon I-evidence +distance I-evidence +measured O +from O +h3 B-structure_element +Thr347 B-residue_name_number +to O +h11 B-structure_element +Leu525 B-residue_name_number +of O +A B-protein_state +‐ I-protein_state +CD I-protein_state +‐, I-protein_state +2 I-protein_state +, I-protein_state +5 I-protein_state +‐ I-protein_state +DTP I-protein_state +‐, I-protein_state +and I-protein_state +3 I-protein_state +, I-protein_state +4 I-protein_state +‐ I-protein_state +DTPD I-protein_state +‐ I-protein_state +bound I-protein_state +ERα B-protein +LBDs B-structure_element +. O + +* O +Two O +‐ O +tailed O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +‐ I-experimental_method +test I-experimental_method +, O +P B-evidence += O +0 O +. O +002 O +( O +PDB O +A B-chemical +‐ I-chemical +CD I-chemical +: O +5DI7 O +, O +5DID O +, O +5DIE O +, O +5DIG O +, O +and O +4PPS O +; O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +: O +4IWC O +, O +5DRM O +, O +and O +5DRJ O +; O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +: O +5DTV O +and O +5DU5 O +). O + +Crystal B-evidence +structures I-evidence +show O +that O +a O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +analog O +shifts O +h3 B-structure_element +( O +F B-structure_element +) O +and O +the O +NCOA2 B-protein +( O +G O +) O +peptide O +compared O +to O +an O +A B-chemical +‐ I-chemical +CD I-chemical +‐ O +ring O +estrogen B-chemical +( O +PDB O +4PPS O +, O +5DTV O +). O + +The O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +showed O +perturbation O +of O +h11 B-structure_element +, O +as O +well O +as O +h3 B-structure_element +, O +which O +forms O +part O +of O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +. O + +These O +compounds O +bind O +the O +LBD B-structure_element +in O +an O +unusual O +fashion O +because O +they O +have O +a O +phenol O +‐ O +to O +‐ O +phenol O +length O +of O +~ O +12 O +Å O +, O +which O +is O +longer O +than O +steroids O +and O +other O +prototypical O +ERα B-protein +agonists O +that O +are O +~ O +10 O +Å O +in O +length O +. O + +To O +quantify O +this O +difference O +, O +we O +compared O +the O +distance B-evidence +between O +α O +‐ O +carbons O +at O +h3 B-structure_element +Thr347 B-residue_name_number +and O +h11 B-structure_element +Leu525 B-residue_name_number +in O +the O +set O +of O +structures B-evidence +containing O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +( O +n O += O +3 O +) O +or O +A B-chemical +‐ I-chemical +CD I-chemical +‐ O +ring O +analogs O +( O +n O += O +5 O +) O +( O +Fig O +6E O +). O + +We O +observed O +a O +difference O +of O +0 O +. O +4 O +Å O +that O +was O +significant O +( O +two O +‐ O +tailed O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +‐ I-experimental_method +test I-experimental_method +, O +P B-evidence += O +0 O +. O +002 O +) O +due O +to O +the O +very O +tight O +clustering O +of O +the O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +‐ O +induced O +LBD B-structure_element +conformation O +. O + +The O +shifts O +in O +h3 B-structure_element +suggest O +these O +compounds O +are O +positioned O +to O +alter O +coregulator O +preferences O +. O + +The O +crystal B-evidence +structure I-evidence +of O +ERα B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +a O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +is O +unknown O +; O +however O +, O +we O +solved B-experimental_method +two O +crystal B-evidence +structures I-evidence +of O +ERα B-protein +bound B-protein_state +to I-protein_state +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +analogs O +and O +one O +structure B-evidence +containing O +a O +furan B-chemical +ligand O +— O +all O +of O +which O +have O +a O +3 O +, O +4 O +‐ O +diaryl O +configuration O +( O +Fig O +2 O +; O +Datasets O +EV1 O +and O +EV2 O +). O + +In O +these O +structures B-evidence +, O +the O +A O +‐ O +ring O +mimetic O +of O +the O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +scaffold O +bound O +h3 B-structure_element +Glu353 B-residue_name_number +as O +expected O +, O +but O +the O +other O +phenol O +wrapped O +around O +h3 B-structure_element +to O +form O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +Thr347 B-residue_name_number +, O +indicating O +a O +change O +in O +binding O +epitopes O +in O +the O +ERα B-protein +ligand B-site +‐ I-site +binding I-site +pocket I-site +( O +Fig O +6F O +). O + +The O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +analogs O +also O +induced O +a O +shift O +in O +h3 B-structure_element +positioning O +, O +which O +translated O +again O +into O +a O +shift O +in O +the O +bound O +coactivator O +peptide O +( O +Fig O +6F O +). O + +Therefore O +, O +these O +indirect O +modulators O +, O +including O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +2 I-chemical +, O +S B-chemical +‐ I-chemical +OBHS I-chemical +‐ I-chemical +3 I-chemical +, O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +, O +and O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTPD I-chemical +analogs O +— O +all O +of O +which O +show O +cell O +‐ O +specific O +activity O +profiles O +— O +induced O +shifts O +in O +h3 B-structure_element +and O +h12 B-structure_element +that O +were O +transmitted O +to O +the O +coactivator O +peptide O +via O +an O +altered O +AF B-site +‐ I-site +2 I-site +surface I-site +. O + +To O +test O +whether O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +shows O +changes O +in O +shape O +in O +solution O +, O +we O +used O +the O +microarray B-experimental_method +assay I-experimental_method +for I-experimental_method +real I-experimental_method +‐ I-experimental_method +time I-experimental_method +coregulator I-experimental_method +– I-experimental_method +nuclear I-experimental_method +receptor I-experimental_method +interaction I-experimental_method +( O +MARCoNI B-experimental_method +) O +analysis O +( O +Aarts O +et O +al O +, O +2013 O +). O + +Here O +, O +the O +ligand O +‐ O +dependent O +interactions O +of O +the O +ERα B-protein +LBD B-structure_element +with O +over O +150 O +distinct O +LxxLL B-structure_element +motif I-structure_element +peptides O +were O +assayed O +to O +define O +structural O +fingerprints O +for O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +, O +in O +a O +manner O +similar O +to O +the O +use O +of O +phage B-experimental_method +display I-experimental_method +peptides I-experimental_method +as O +structural O +probes O +( O +Connor O +et O +al O +, O +2001 O +). O + +However O +, O +there O +was O +a O +unique O +cluster O +of O +peptides O +that O +were O +recruited O +by O +E2 B-chemical +but O +not O +the O +2 B-chemical +, I-chemical +5 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +. O + +In O +contrast O +, O +3 B-chemical +, I-chemical +4 I-chemical +‐ I-chemical +DTP I-chemical +analogs O +dismissed O +most O +of O +the O +peptides O +from O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +( O +Fig O +6H O +). O + +Indirect O +modulators O +in O +cluster O +1 O +avoid O +this O +by O +perturbing O +the O +h11 B-site +– I-site +h12 I-site +interface I-site +, O +and O +modulating O +the O +dynamics O +of O +h12 B-structure_element +without O +changing O +the O +shape O +of O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +when O +stabilized O +. O + +We O +found O +a O +very O +strong O +set O +of O +predictors O +, O +where O +ligands O +in O +cluster O +1 O +, O +defined O +by O +similar O +signaling O +across O +cell O +types O +, O +showed O +indirect O +modulation O +of O +h12 B-structure_element +dynamics O +via O +the O +h11 B-site +– I-site +12 I-site +interface I-site +or O +slight O +contact O +with O +h12 B-structure_element +. O + +For O +ligands O +in O +cluster O +1 O +, O +deletion B-experimental_method +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +reduced O +activity O +to O +varying O +degrees O +, O +but O +did O +not O +change O +the O +underlying O +signaling O +patterns O +established O +through O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +. O + +In O +contrast O +, O +an O +extended O +side O +chain O +designed O +to O +directly O +reposition O +h12 B-structure_element +and O +completely O +disrupt O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +results O +in O +cell O +‐ O +specific O +signaling O +. O + +Compared O +to O +cluster O +1 O +, O +the O +structural O +rules O +are O +less O +clear O +in O +clusters O +2 O +and O +3 O +, O +but O +a O +number O +of O +indirect O +modulator O +classes O +perturbed O +the O +LBD B-structure_element +conformation O +at O +the O +intersection O +of O +h3 B-structure_element +, O +the O +h12 B-structure_element +N O +‐ O +terminus O +, O +and O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +. O + +For O +direct O +and O +indirect O +modulators O +in O +cluster O +2 O +or O +3 O +, O +the O +canonical O +ERα B-protein +signaling O +pathway O +involving O +recruitment O +of O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +and O +induction O +of O +GREB1 B-protein +did O +not O +generally O +predict O +their O +proliferative O +effects O +, O +indicating O +an O +alternate O +causal O +model O +( O +Fig O +1E O +). O + +These O +principles O +outlined O +above O +provide O +a O +structural O +basis O +for O +how O +the O +ligand B-site +– I-site +receptor I-site +interface I-site +leads O +to O +different O +signaling O +specificities O +through O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +and O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +. O + +Completely O +blocking O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +with O +an O +extended O +side O +chain O +or O +altering O +the O +shape O +of O +AF B-structure_element +‐ I-structure_element +2 I-structure_element +changes O +the O +preference O +away O +from O +NCOA1 B-protein +/ I-protein +2 I-protein +/ I-protein +3 I-protein +for O +determining O +GREB1 B-protein +levels O +and O +proliferation O +of O +breast O +cancer O +cells O +. O + +AF B-structure_element +‐ I-structure_element +2 I-structure_element +blockade O +also O +allows O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +to O +function O +independently O +, O +which O +is O +important O +since O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +drives O +tissue O +‐ O +selective O +effects O +in O +vivo O +. O + +This O +was O +demonstrated O +with O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +knockout O +mice O +that O +show O +E2 B-chemical +‐ O +dependent O +vascular O +protection O +, O +but O +not O +uterine O +proliferation O +, O +thus O +highlighting O +the O +role O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +in O +tissue O +‐ O +selective O +or O +cell O +‐ O +specific O +signaling O +( O +Billon O +‐ O +Gales O +et O +al O +, O +2009 O +; O +Abot O +et O +al O +, O +2013 O +). O + +Here O +, O +we O +examined O +many O +LBD B-structure_element +structures B-evidence +and O +tested O +several O +variables O +that O +were O +not O +predictive O +, O +including O +ERβ B-protein +activity O +, O +the O +strength O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +signaling O +, O +and O +NCOA3 B-protein +occupancy O +at O +the O +GREB1 B-protein +gene O +. O + +Similarly O +, O +we O +visualized O +structures B-evidence +to O +identify O +patterns O +. O + +For O +example O +, O +phage B-experimental_method +display I-experimental_method +was O +used O +to O +identify O +the O +androgen O +receptor O +interactome O +, O +which O +was O +cloned O +into O +an O +M2H B-experimental_method +library O +and O +used O +to O +identify O +clusters O +of O +ligand O +‐ O +selective O +interactions O +( O +Norris O +et O +al O +, O +2009 O +). O + +Indeed O +, O +the O +most O +anti O +‐ O +proliferative O +compound O +in O +the O +OBHS B-chemical +‐ I-chemical +N I-chemical +series O +had O +a O +fulvestrant O +‐ O +like O +profile O +across O +a O +battery O +of O +assays O +( O +S O +. O +Srinivasan O +et O +al O +, O +in O +preparation O +). O + +Secondly O +, O +our O +finding O +that O +WAY B-chemical +‐ I-chemical +C I-chemical +compounds O +do O +not O +rely O +of O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +for O +signaling O +efficacy O +may O +derive O +from O +the O +slight O +contacts O +with O +h12 B-structure_element +observed O +in O +crystal B-evidence +structures I-evidence +( O +Figs O +3B O +and O +5H O +), O +unlike O +other O +compounds O +in O +cluster O +1 O +that O +dislocate O +h11 B-structure_element +and O +rely O +on O +AF B-structure_element +‐ I-structure_element +1 I-structure_element +for O +signaling O +efficacy O +( O +Figs O +3B O +and O +5C O +, O +and O +EV5B O +). O + +Some O +of O +these O +ligands O +altered O +the O +shape O +of O +the O +AF B-site +‐ I-site +2 I-site +surface I-site +by O +perturbing O +the O +h3 B-site +– I-site +h12 I-site +interface I-site +, O +thus O +providing O +a O +route O +to O +new O +SERM O +‐ O +like O +activity O +profiles O +by O +combining O +indirect O +and O +direct O +modulation O +of O +receptor O +structure O +. O + +Incorporation O +of O +statistical O +approaches O +to O +understand O +relationships O +between O +structure O +and O +signaling O +variables O +moves O +us O +toward O +predictive O +models O +for O +complex O +ERα B-protein +‐ O +mediated O +responses O +such O +as O +in O +vivo O +uterine O +proliferation O +or O +tumor O +growth O +, O +and O +more O +generally O +toward O +structure O +‐ O +based O +design O +for O +other O +allosteric O +drug O +targets O +including O +GPCRs B-protein_type +and O +other O +nuclear B-protein_type +receptors I-protein_type +. O + +Structure B-evidence +of O +a O +quinolone O +- O +stabilized O +cleavage O +complex O +of O +topoisomerase B-complex_assembly +IV I-complex_assembly +from O +Klebsiella B-species +pneumoniae I-species +and O +comparison O +with O +a O +related O +Streptococcus B-species +pneumoniae I-species +complex O + +Crystal B-evidence +structures I-evidence +of O +the O +cleavage O +complexes O +of O +topoisomerase B-complex_assembly +IV I-complex_assembly +from O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +( O +K B-species +. I-species +pneumoniae I-species +) O +and O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +( O +S B-species +. I-species +pneumoniae I-species +) O +bacterial B-taxonomy_domain +pathogens O +stabilized O +by O +the O +clinically O +important O +antibacterial O +drug O +levofloxacin B-chemical +are O +presented O +, O +analysed O +and O +compared O +. O + +Klebsiella B-species +pneumoniae I-species +is O +a O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +bacterium I-taxonomy_domain +that O +is O +responsible O +for O +a O +range O +of O +common O +infections O +, O +including O +pulmonary O +pneumonia O +, O +bloodstream O +infections O +and O +meningitis O +. O + +Certain O +strains O +of O +Klebsiella B-taxonomy_domain +have O +become O +highly O +resistant O +to O +antibiotics O +. O + +Despite O +the O +vast O +amount O +of O +research O +carried O +out O +on O +this O +class O +of O +bacteria B-taxonomy_domain +, O +the O +molecular O +structure B-evidence +of O +its O +topoisomerase B-complex_assembly +IV I-complex_assembly +, O +a O +type B-protein_type +II I-protein_type +topoisomerase I-protein_type +essential O +for O +catalysing O +chromosomal O +segregation O +, O +had O +remained O +unknown O +. O + +In O +this O +paper O +, O +the O +structure B-evidence +of O +its O +DNA B-chemical +- O +cleavage O +complex O +is O +reported O +at O +3 O +. O +35 O +Å O +resolution O +. O + +The O +complex O +is O +comprised O +of O +ParC B-protein +breakage B-structure_element +- I-structure_element +reunion I-structure_element +and O +ParE B-protein +TOPRIM B-structure_element +domains O +of O +K B-species +. I-species +pneumoniae I-species +topoisomerase B-complex_assembly +IV I-complex_assembly +with O +DNA B-chemical +stabilized O +by O +levofloxacin B-chemical +, O +a O +broad O +- O +spectrum O +fluoroquinolone B-chemical +antimicrobial O +agent O +. O + +Klebsiella B-taxonomy_domain +is O +a O +genus O +belonging O +to O +the O +Enterobacteriaceae B-taxonomy_domain +family O +of O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +bacilli I-taxonomy_domain +, O +which O +is O +divided O +into O +seven O +species O +with O +demonstrated O +similarities O +in O +DNA B-chemical +homology O +: O +K B-species +. I-species +pneumoniae I-species +, O +K B-species +. I-species +ozaenae I-species +, O +K B-species +. I-species +rhinoscleromatis I-species +, O +K B-species +. I-species +oxytoca I-species +, O +K B-species +. I-species +planticola I-species +, O +K B-species +. I-species +terrigena I-species +and O +K B-species +. I-species +ornithinolytica I-species +. O + +K B-species +. I-species +pneumoniae I-species +is O +the O +most O +medically O +important O +species O +of O +the O +genus O +owing O +to O +its O +high O +resistance O +to O +antibiotics O +. O + +However O +, O +common O +treatments O +( O +based O +on O +in B-experimental_method +vitro I-experimental_method +susceptibility I-experimental_method +testing I-experimental_method +) O +are O +the O +polymyxins B-chemical +, O +tigecycline B-chemical +and O +, O +less O +frequently O +, O +aminoglycoside B-chemical +antibiotics O +( O +Arnold O +et O +al O +., O +2011 O +). O + +Another O +effective O +strategy O +involves O +the O +limited O +use O +of O +certain O +antimicrobials O +, O +specifically O +fluoroquinolones B-chemical +and O +cephalo B-chemical +­ I-chemical +sporins I-chemical +( O +Gasink O +et O +al O +., O +2009 O +). O + +These O +include O +combinations O +of O +existing O +β O +- O +lactam O +antibiotics O +with O +new O +β B-protein_type +- I-protein_type +lactamase I-protein_type +inhibitors O +able O +to O +circumvent O +KPC O +resistance O +. O + +Neoglycosides B-chemical +are O +novel O +aminoglycosides B-chemical +that O +have O +activity O +against O +KPC O +- O +producing O +bacteria B-taxonomy_domain +that O +are O +also O +being O +developed O +( O +Chen O +et O +al O +., O +2012 O +). O + +Type B-protein_type +II I-protein_type +topoisomerase I-protein_type +enzymes I-protein_type +play O +important O +roles O +in O +prokaryotic B-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +DNA B-chemical +replication O +, O +recombination O +and O +transcription O +( O +Drlica O +et O +al O +., O +2008 O +; O +Laponogov O +et O +al O +., O +2013 O +; O +Lee O +et O +al O +., O +2013 O +; O +Nitiss O +, O +2009a O +, O +b O +; O +Schoeffler O +& O +Berger O +, O +2008 O +; O +Sissi O +& O +Palumbo O +, O +2009 O +; O +Vos O +et O +al O +., O +2011 O +; O +Wendorff O +et O +al O +., O +2012 O +; O +Wu O +et O +al O +., O +2011 O +, O +2013 O +). O + +Both O +enzymes O +act O +via O +a O +double O +- O +strand O +DNA B-chemical +break O +involving O +a O +cleavage O +complex O +and O +are O +targets O +for O +quinolone O +antimicrobials O +that O +act O +by O +trapping O +these O +enzymes O +at O +the O +DNA B-chemical +- O +cleavage O +stage O +and O +preventing O +strand O +re O +- O +joining O +( O +Drlica O +et O +al O +., O +2008 O +). O + +It O +is O +active O +against O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +and O +Gram B-taxonomy_domain +- I-taxonomy_domain +negative I-taxonomy_domain +bacteria I-taxonomy_domain +and O +functions O +by O +inhibiting O +gyrase B-protein_type +and O +topoisomerase B-complex_assembly +IV I-complex_assembly +( O +Drlica O +& O +Zhao O +, O +1997 O +; O +Laponogov O +et O +al O +., O +2010 O +). O + +Here O +, O +we O +report O +the O +first O +three O +- O +dimensional O +X B-evidence +- I-evidence +ray I-evidence +structure I-evidence +of O +a O +K B-species +. I-species +pneumoniae I-species +topoisomerase B-complex_assembly +IV I-complex_assembly +ParC B-complex_assembly +/ I-complex_assembly +ParE I-complex_assembly +cleavage O +complex O +with O +DNA B-chemical +stabilized O +by O +levofloxacin B-chemical +. O + +The O +crystal B-evidence +structure I-evidence +provides O +structural O +information O +on O +topoisomerase B-complex_assembly +IV I-complex_assembly +from O +K B-species +. I-species +pneumoniae I-species +, O +a O +pathogen O +for O +which O +drug O +resistance O +is O +a O +serious O +concern O +. O + +The O +structure B-evidence +of O +the O +ParC B-complex_assembly +/ I-complex_assembly +ParE I-complex_assembly +– O +DNA B-site +– I-site +levofloxacin I-site +binding I-site +site I-site +highlights O +the O +details O +of O +the O +cleavage O +- O +complex O +assembly O +that O +are O +essential O +for O +the O +rational O +design O +of O +Klebsiella B-taxonomy_domain +topoisomerase B-protein_type +inhibitors O +. O + +We O +have O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +the O +K B-species +. I-species +pneumoniae I-species +topoisomerase B-complex_assembly +IV I-complex_assembly +ParC B-complex_assembly +/ I-complex_assembly +ParE I-complex_assembly +breakage B-structure_element +- I-structure_element +reunion I-structure_element +domain O +( O +ParC55 B-protein +; O +residues O +1 B-residue_range +– I-residue_range +490 I-residue_range +) O +and O +ParE B-protein +TOPRIM B-structure_element +domain O +( O +ParE30 B-protein +; O +residues O +390 B-residue_range +– I-residue_range +631 I-residue_range +) O +with O +a O +precut O +34 O +bp O +DNA B-chemical +duplex O +( O +the O +E B-site +- I-site +site I-site +), O +stabilized O +by O +levofloxacin B-chemical +. O + +The O +X B-evidence +- I-evidence +ray I-evidence +crystal I-evidence +structure I-evidence +of O +the O +complex O +was O +determined O +to O +3 O +. O +35 O +Å O +resolution O +, O +revealing O +a O +closed B-protein_state +ParC55 B-protein +dimer B-oligomeric_state +flanked O +by O +two O +ParE30 B-protein +monomers B-oligomeric_state +( O +Figs O +. O +1 O +▸, O +2 O +▸ O +and O +3 O +▸). O + +The O +overall O +architecture O +of O +this O +complex O +is O +similar O +to O +that O +found O +for O +S B-species +. I-species +pneumoniae I-species +topoisomerase O +– O +DNA O +– O +drug O +complexes O +( O +Laponogov O +et O +al O +., O +2009 O +, O +2010 O +). O + +Residues O +6 B-residue_range +– I-residue_range +30 I-residue_range +of O +the O +N O +- O +terminal O +α B-structure_element +- I-structure_element +helix I-structure_element +α1 B-structure_element +of O +the O +ParC B-protein +subunit O +again O +embrace O +the O +ParE B-protein +subunit O +, O +‘ O +hugging O +’ O +the O +ParE B-protein +subunits O +close O +to O +either O +side O +of O +the O +ParC B-protein +dimer B-oligomeric_state +( O +Laponogov O +et O +al O +., O +2010 O +). O + +Deletion B-experimental_method +of I-experimental_method +this O +‘ O +arm B-structure_element +’ O +α1 B-structure_element +results O +in O +loss B-protein_state +of I-protein_state +DNA I-protein_state +- I-protein_state +cleavage I-protein_state +activity I-protein_state +( O +Laponogov O +et O +al O +., O +2007 O +) O +and O +is O +clearly O +very O +important O +in O +complex O +stability O +( O +Fig O +. O +3 O +▸). O + +This O +structural O +feature O +was O +absent O +in O +our O +original O +ParC55 B-protein +structure B-evidence +( O +Laponogov O +et O +al O +., O +2007 O +; O +Sohi O +et O +al O +., O +2008 O +). O + +The O +C B-protein +- I-protein +subunit I-protein +provides O +the O +WHD B-structure_element +( O +winged B-structure_element +- I-structure_element +helix I-structure_element +domain I-structure_element +; O +a O +CAP B-structure_element +- I-structure_element +like I-structure_element +structure I-structure_element +; O +McKay O +& O +Steitz O +, O +1981 O +) O +and O +the O +‘ O +tower B-structure_element +’ O +which O +form O +the O +U B-structure_element +groove I-structure_element +- O +shaped O +protein O +region O +into O +which O +the O +G B-structure_element +- I-structure_element +gate I-structure_element +DNA B-chemical +binds O +with O +an O +induced O +U O +- O +shaped O +bend O +. O + +The O +lower O +C B-structure_element +- I-structure_element +gate I-structure_element +region O +( O +Fig O +. O +3 O +▸) O +consists O +of O +the O +same O +disposition O +of O +pairs O +of O +two O +long B-structure_element +α I-structure_element +- I-structure_element +helices I-structure_element +terminated O +by O +a O +spanning O +short B-structure_element +α I-structure_element +- I-structure_element +helix I-structure_element +forming O +a O +30 O +Å O +wide O +DNA B-site +- I-site +accommodating I-site +cavity I-site +through O +which O +the O +T B-structure_element +- I-structure_element +gate I-structure_element +DNA B-chemical +passes O +as O +found O +in O +the O +S B-species +. I-species +pneumoniae I-species +complex O +. O + +Owing O +to O +the O +structural O +similarity O +, O +it O +appears O +that O +the O +topo B-complex_assembly +­ I-complex_assembly +isomerases I-complex_assembly +IV I-complex_assembly +from O +K B-species +. I-species +pneumoniae I-species +and O +S B-species +. I-species +pneumoniae I-species +are O +likely O +to O +follow O +a O +similar O +overall O +topoisomerase B-protein_type +catalytic O +cycle O +as O +shown O +in O +Fig O +. O +4 O +▸; O +we O +have O +confirmation O +of O +one O +intermediate O +from O +our O +recent O +structure B-evidence +of O +the O +full B-protein_state +complex I-protein_state +( O +the O +holoenzyme B-protein_state +less O +the O +CTD B-structure_element +β I-structure_element +- I-structure_element +pinwheel I-structure_element +domain O +) O +with O +the O +ATPase B-structure_element +domain I-structure_element +in O +the O +open B-protein_state +conformation O +( O +Laponogov O +et O +al O +., O +2013 O +). O + +The O +G B-structure_element +- I-structure_element +gate I-structure_element +DNA B-chemical +for O +the O +S B-species +. I-species +pneumoniae I-species +complex O +consists O +of O +an O +18 O +- O +base O +- O +pair O +E B-site +- I-site +site I-site +sequence O +( O +our O +designation O +for O +a O +DNA B-site +site I-site +which O +we O +first O +found O +from O +DNA B-experimental_method +- I-experimental_method +mapping I-experimental_method +studies I-experimental_method +; O +Leo O +et O +al O +., O +2005 O +; O +Arnoldi O +et O +al O +., O +2013 O +; O +Fig O +. O +1 O +▸). O + +The O +crystallized B-experimental_method +complex O +was O +formed O +by O +turning O +over O +the O +topoisomerase B-protein_type +tetramer B-oligomeric_state +in O +the O +presence B-protein_state +of I-protein_state +DNA B-chemical +and O +levofloxacin B-chemical +and O +crystallizing B-experimental_method +the O +product O +. O + +In O +contrast O +, O +the O +K B-species +. I-species +pneumoniae I-species +complex O +was O +formed O +by O +co B-experimental_method +- I-experimental_method +crystallizing I-experimental_method +the O +topoisomerase B-protein_type +tetramer B-oligomeric_state +complex O +in O +the O +presence B-protein_state +of I-protein_state +a O +34 O +- O +base O +- O +pair O +pre B-protein_state +- I-protein_state +cleaved I-protein_state +DNA B-chemical +in O +the O +presence B-protein_state +of I-protein_state +levofloxacin B-chemical +. O + +We O +have O +been O +able O +to O +unambiguously O +read O +off O +the O +DNA B-chemical +sequences O +in O +the O +electron B-evidence +- I-evidence +density I-evidence +maps I-evidence +. O + +For O +the O +ParC B-protein +subunits O +, O +the O +figures O +are O +40 O +. O +8 O +identity O +and O +55 O +. O +6 O +% O +homology O +between O +the O +two O +organisms O +. O + +The O +binding O +of O +levofloxacin B-chemical +in O +the O +K B-species +. I-species +pneumoniae I-species +complex O +is O +shown O +in O +Figs O +. O +2 O +▸, O +3 O +▸ O +and O +5 O +▸ O +and O +is O +hemi O +- O +intercalated O +into O +the O +DNA B-chemical +and O +stacked O +against O +the O +DNA B-chemical +bases O +at O +the O +cleavage B-site +site I-site +( O +positions O +− B-residue_number +1 I-residue_number +and O ++ B-residue_number +1 I-residue_number +of O +the O +four O +- O +base O +- O +pair O +staggered O +cut O +in O +the O +34 O +- O +mer O +DNA B-chemical +) O +which O +is O +similar O +to O +that O +found O +for O +the O +S B-species +. I-species +pneumoniae I-species +complex O +. O + +Fig O +. O +5 O +▸ O +presents O +side O +- O +by O +- O +side O +views O +of O +the O +K B-species +. I-species +pneumoniae I-species +and O +S B-species +. I-species +pneumoniae I-species +active B-site +sites I-site +which O +shows O +that O +levofloxacin B-chemical +binds O +in O +a O +very O +similar O +manner O +in O +these O +two O +complexes O +with O +extensive O +π B-bond_interaction +– I-bond_interaction +π I-bond_interaction +stacking I-bond_interaction +interaction I-bond_interaction +between O +the O +bases O +and O +the O +drug O +. O + +The O +methylpiperazine B-chemical +at O +C7 O +( O +using O +the O +conventional O +quinolone B-chemical +numbering O +; O +C9 O +in O +the O +IUPAC O +numbering O +) O +on O +the O +drug O +extends O +towards O +residues O +Glu474 B-residue_name_number +and O +Glu475 B-residue_name_number +for O +S B-species +. I-species +pneumoniae I-species +and O +towards O +Gln460 B-residue_name_number +and O +Glu461 B-residue_name_number +for O +K B-species +. I-species +pneumoniae I-species +, O +where O +the O +glutamate B-residue_name_number +at I-residue_name_number +474 I-residue_name_number +is O +substituted O +by O +a O +glutamine B-residue_name_number +at I-residue_name_number +460 I-residue_name_number +in O +the O +Klebsiella B-taxonomy_domain +strain O +. O + +Obviously O +, O +the O +drug O +– O +ParE B-protein +interaction O +in O +this O +region O +is O +less O +strong O +compared O +with O +PD B-chemical +0305970 I-chemical +binding O +to O +the O +S B-species +. I-species +pneumoniae I-species +DNA B-chemical +complex O +, O +where O +PD B-chemical +0305970 I-chemical +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +to O +ParE B-protein +residue O +Asp475 B-residue_name_number +and O +can O +form O +one O +to O +Asp474 B-residue_name_number +if O +the O +bond O +rotates O +( O +Laponogov O +et O +al O +., O +2010 O +). O + +This O +may O +explain O +why O +drug O +- O +resistance O +mutations O +for O +levofloxacin B-chemical +are O +more O +likely O +to O +form O +in O +the O +ParC B-protein +subunits O +rather O +than O +in O +the O +ParE B-protein +subunits O +. O + +For O +both O +complexes O +there O +is O +a O +Mg2 B-chemical ++ I-chemical +ion O +bound B-protein_state +to I-protein_state +levofloxacin B-chemical +between O +the O +carbonyl O +group O +at O +position O +4 O +of O +the O +quinolone B-chemical +and O +the O +carboxyl O +at O +position O +6 O +( O +Figs O +. O +2 O +▸ O +and O +5 O +▸ O +and O +Supplementary O +Fig O +. O +2 O +▸). O + +For O +S B-species +. I-species +pneumoniae I-species +topoisomerase B-complex_assembly +IV I-complex_assembly +, O +one O +of O +the O +O O +atoms O +of O +the O +carboxyl O +of O +Asp83 B-residue_name_number +points O +towards O +the O +Mg2 B-chemical ++ I-chemical +ion O +and O +is O +within O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +distance O +( O +5 O +. O +04 O +Å O +) O +through O +an O +Mg2 B-chemical ++- I-chemical +coordinated O +water B-chemical +. O + +For O +K B-species +. I-species +pneumoniae I-species +both O +of O +the O +carboxyl O +O O +atoms O +are O +pointing O +towards O +the O +Mg2 B-chemical ++ I-chemical +ion O +at O +distances O +of O +4 O +. O +86 O +and O +4 O +. O +23 O +Å O +. O +These O +residues O +are O +ordered O +in O +only O +one O +of O +the O +two O +dimers B-oligomeric_state +in O +the O +K B-species +. I-species +pneumoniae I-species +crystal B-evidence +( O +the O +one O +in O +which O +the O +C7 O +group O +is O +pointing O +towards O +the O +DNA B-chemical +away O +from O +ParE B-protein +, O +although O +the O +conformations O +of O +these O +two O +groups O +on O +the O +drug O +are O +probably O +not O +correlated O +). O + +The O +topoisomerase B-complex_assembly +IV I-complex_assembly +ParE27 B-complex_assembly +- I-complex_assembly +ParC55 I-complex_assembly +fusion O +protein O +from O +K B-species +. I-species +pneumoniae I-species +was O +fully O +active O +in O +promoting O +levofloxacin B-chemical +- O +mediated O +cleavage O +of O +DNA B-chemical +( O +Fig O +. O +6 O +▸). O + +In O +the O +absence B-protein_state +of I-protein_state +the O +drug B-chemical +and O +ATP B-chemical +, O +the O +protein O +converted O +supercoiled O +pBR322 O +into O +a O +ladder O +of O +bands O +corresponding O +to O +relaxed O +DNA B-chemical +. O + +The O +inclusion O +of O +levofloxacin B-chemical +produced O +linear O +DNA B-chemical +in O +a O +dose O +- O +dependent O +and O +ATP B-chemical +- O +independent O +fashion O +. O + +Interestingly O +, O +K B-species +. I-species +pneumoniae I-species +strains O +are O +much O +more O +susceptible O +to O +levofloxacin B-chemical +than O +S B-species +. I-species +pneumoniae I-species +, O +with O +typical O +MIC O +values O +of O +0 O +. O +016 O +and O +1 O +mg O +l O +− O +1 O +, O +respectively O +( O +Odenholt O +& O +Cars O +, O +2006 O +), O +reflecting O +differences O +in O +multiple O +factors O +( O +in O +addition O +to O +binding B-evidence +affinity I-evidence +) O +that O +influence O +drug O +responses O +, O +including O +membrane O +, O +peptidoglycan O +structure O +, O +drug O +- O +uptake O +and O +efflux O +mechanisms O +. O + +In O +summary O +, O +we O +have O +determined O +the O +first O +structure B-evidence +of O +a O +quinolone B-chemical +– O +DNA B-chemical +cleavage O +complex O +involving O +a O +type B-protein_type +II I-protein_type +topo I-protein_type +­ I-protein_type +isomerase I-protein_type +from O +K B-species +. I-species +pneumoniae I-species +. O + +Protein O +and O +DNA B-chemical +used O +in O +the O +co B-experimental_method +- I-experimental_method +crystallization I-experimental_method +experiment O +. O + +( O +a O +) O +Coloured O +diagram O +of O +the O +protein O +constructs O +used O +in O +crystallization B-experimental_method +. O + +( O +b O +) O +DNA B-chemical +sequences O +used O +in O +crystallization B-experimental_method +. O + +Chemical O +structure O +of O +levofloxacin B-chemical +( O +a O +) O +and O +its O +conformations O +observed O +within O +the O +active B-site +sites I-site +of O +S B-species +. I-species +pneumoniae I-species +topoisomerase B-complex_assembly +IV I-complex_assembly +( O +b O +) O +and O +K B-species +. I-species +pneumoniae I-species +topoisomerase B-complex_assembly +IV I-complex_assembly +( O +c O +, O +d O +). O + +Electron B-evidence +- I-evidence +density I-evidence +maps I-evidence +( O +2F O +obs O +− O +F O +calc O +) O +are O +shown O +as O +meshes O +for O +the O +drug O +molecules O +contoured O +at O +1 O +. O +5σ O +and O +are O +limited O +to O +a O +distance O +of O +2 O +. O +3 O +Å O +from O +the O +drug O +atoms O +. O + +Overall O +orthogonal O +views O +of O +the O +cleavage O +complex O +of O +topoisomerase B-complex_assembly +IV I-complex_assembly +from O +K B-species +. I-species +pneumoniae I-species +in O +surface O +( O +left O +) O +and O +cartoon O +( O +right O +) O +representations O +. O + +The O +ParC B-protein +subunit O +is O +in O +blue O +, O +ParE B-protein +is O +in O +yellow O +and O +DNA B-chemical +is O +in O +cyan O +. O + +The O +bound B-protein_state +quinolone B-chemical +molecules O +( O +levofloxacin B-chemical +) O +are O +in O +red O +and O +are O +shown O +using O +van O +der O +Waals O +representation O +. O + +Schematic O +representation O +of O +the O +catalytic O +cycle O +of O +type B-protein_type +II I-protein_type +topoisomerases I-protein_type +. O + +The O +ParC B-protein +N O +- O +terminal O +domain O +( O +ParC55 B-protein +) O +is O +in O +grey O +, O +the O +ParC B-protein +C O +- O +terminal O +β B-structure_element +-­ I-structure_element +pinwheel I-structure_element +domain I-structure_element +is O +in O +silver O +, O +the O +ParE B-protein +N O +- O +terminal O +ATPase B-structure_element +domain I-structure_element +is O +in O +red O +, O +the O +ParE B-protein +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +( O +ParE30 B-protein +) O +is O +in O +yellow O +, O +the O +G B-structure_element +- I-structure_element +gate I-structure_element +DNA B-chemical +is O +in O +green O +and O +the O +T B-structure_element +- I-structure_element +segment I-structure_element +DNA B-chemical +is O +in O +purple O +. O + +Detailed O +views O +of O +the O +active B-site +sites I-site +of O +topoisomerase B-complex_assembly +IV I-complex_assembly +from O +S B-species +. I-species +pneumoniae I-species +and O +K B-species +. I-species +pneumoniae I-species +with O +quinolone B-chemical +molecules O +bound B-protein_state +. O + +The O +DNA B-chemical +is O +shown O +in O +silver O +/ O +cyan O +. O + +Supercoiled O +plasmid O +pBR322 O +( O +400 O +ng O +) O +was O +incubated O +with O +topoisomerase B-complex_assembly +IV I-complex_assembly +proteins O +( O +400 O +ng O +) O +in O +the O +absence O +or O +presence B-protein_state +of I-protein_state +levofloxacin B-chemical +at O +the O +indicated O +concentrations O +. O + +Cryo B-experimental_method +- I-experimental_method +electron I-experimental_method +microscopy I-experimental_method +( O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +) O +methods O +are O +now O +being O +used O +to O +determine O +structures B-evidence +at O +near O +- O +atomic O +resolution O +and O +have O +great O +promise O +in O +molecular O +pharmacology O +, O +especially O +in O +the O +context O +of O +mapping O +the O +binding O +of O +small O +- O +molecule O +ligands O +to O +protein O +complexes O +that O +display O +conformational O +flexibility O +. O + +Dysregulation O +of O +GDH B-protein_type +leads O +to O +a O +variety O +of O +metabolic O +and O +neurologic O +disorders O +. O + +We O +show O +that O +the O +binding O +of O +the O +coenzyme O +NADH B-chemical +alone O +or O +in O +concert O +with O +GTP B-chemical +results O +in O +a O +binary O +mixture O +in O +which O +the O +enzyme O +is O +in O +either O +an O +“ O +open B-protein_state +” O +or O +“ O +closed B-protein_state +” O +state O +. O + +Whereas O +the O +structure B-evidence +of O +NADH B-chemical +in O +the O +active B-site +site I-site +is O +similar O +between O +the O +open B-protein_state +and O +closed B-protein_state +states O +, O +it O +is O +unexpectedly O +different O +at O +the O +regulatory B-site +site I-site +. O + +Our O +studies O +thus O +demonstrate O +that O +even O +in O +instances O +when O +there O +is O +considerable O +structural O +information O +available O +from O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +, O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +methods O +can O +provide O +useful O +complementary O +insights O +into O +regulatory O +mechanisms O +for O +dynamic O +protein O +complexes O +. O + +Recent O +advances O +in O +cryo B-experimental_method +- I-experimental_method +electron I-experimental_method +microscopy I-experimental_method +( O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +) O +allow O +determination O +of O +structures B-evidence +of O +small O +protein O +complexes O +and O +membrane O +proteins O +at O +near O +- O +atomic O +resolution O +, O +marking O +a O +critical O +shift O +in O +the O +structural O +biology O +field O +. O + +One O +specific O +area O +of O +broad O +general O +interest O +in O +drug O +discovery O +is O +the O +localization O +of O +bound O +ligands O +and O +cofactors O +under O +conditions O +in O +which O +efforts O +at O +crystallization B-experimental_method +have O +not O +been O +successful O +because O +of O +structural O +heterogeneity O +. O + +Recent O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +analyses O +have O +already O +demonstrated O +that O +it O +is O +now O +possible O +to O +use O +single B-experimental_method +- I-experimental_method +particle I-experimental_method +cryo I-experimental_method +- I-experimental_method +EM I-experimental_method +methods O +to O +localize O +small O +bound O +ligands O +or O +inhibitors O +on O +target O +proteins O +. O + +Here O +, O +we O +address O +this O +question O +using O +mammalian B-taxonomy_domain +glutamate B-protein_type +dehydrogenase I-protein_type +as O +an O +example O +. O + +Glutamate B-protein_type +dehydrogenase I-protein_type +( O +GDH B-protein_type +) O +is O +a O +highly B-protein_state +conserved I-protein_state +enzyme O +expressed O +in O +most O +organisms O +. O + +GDH B-protein_type +plays O +a O +central O +role O +in O +glutamate B-chemical +metabolism O +by O +catalyzing O +the O +reversible O +oxidative O +deamination O +of O +glutamate B-chemical +to O +generate O +α B-chemical +- I-chemical +ketoglutarate I-chemical +and O +ammonia B-chemical +, O +with O +the O +concomitant O +transfer O +of O +a O +pair O +of O +electrons O +to O +either O +NAD B-chemical ++ I-chemical +or O +NADP B-chemical ++. I-chemical + +Regulation O +of O +GDH B-protein_type +is O +tightly O +controlled O +through O +multiple O +allosteric O +mechanisms O +. O + +Extensive O +biochemical B-experimental_method +and I-experimental_method +crystallographic I-experimental_method +studies I-experimental_method +have O +characterized O +the O +enzymatic O +activity O +of O +GDH B-protein_type +and O +its O +modulation O +by O +a O +chemically O +diverse O +group O +of O +compounds O +such O +as O +nucleotides O +, O +amino O +acids O +, O +steroid O +hormones O +, O +antipsychotic O +drugs O +, O +and O +natural O +products O +. O + +The O +second O +, O +a O +nucleotide B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +( O +NBD B-structure_element +) O +with O +a O +Rossmann B-structure_element +fold I-structure_element +, O +defines O +one O +face O +of O +the O +catalytic B-site +cleft I-site +bounded O +by O +the O +core O +domain O +. O + +During O +the O +catalytic O +cycle O +, O +the O +NBD B-structure_element +executes O +a O +large O +movement O +, O +hinged O +around O +a O +“ B-structure_element +pivot I-structure_element +” I-structure_element +helix I-structure_element +, O +that O +closes O +the O +catalytic B-site +cleft I-site +, O +and O +drives O +a O +large O +conformational O +change O +in O +the O +hexamer B-oligomeric_state +from O +open B-protein_state +to O +closed B-protein_state +states O +( O +Fig O +. O +1B O +). O + +The O +third O +domain O +, O +dubbed O +the O +“ O +antenna B-structure_element +,” O +is O +an O +evolutionary O +acquisition O +in O +protista B-taxonomy_domain +and O +animals B-taxonomy_domain +. O + +Antennae B-structure_element +of O +adjacent O +protomers B-oligomeric_state +in O +each O +trimer B-oligomeric_state +intercalate O +to O +form O +a O +bundle O +, O +perpendicular O +to O +the O +pivot B-structure_element +helices I-structure_element +, O +that O +protrudes O +along O +the O +distal O +extremes O +of O +the O +3 O +- O +fold O +axis O +. O + +When O +a O +protomer B-oligomeric_state +undergoes O +a O +conformational O +change O +, O +the O +rotation O +of O +its O +pivot B-structure_element +helix I-structure_element +is O +transferred O +through O +the O +antenna B-structure_element +to O +the O +adjacent O +subunit B-structure_element +. O + +The O +influence O +of O +the O +antenna B-structure_element +, O +present O +only O +in O +protozoan B-taxonomy_domain +and O +metazoan B-taxonomy_domain +enzymes O +, O +has O +been O +proposed O +to O +explain O +its O +cooperative O +behavior O +, O +which O +is O +absent O +in O +bacterial B-taxonomy_domain +homologs O +. O + +Only O +three O +protomers B-oligomeric_state +are O +shown O +in O +the O +top O +view O +for O +purposes O +of O +visual O +clarity O +. O + +The O +transition O +between O +“ O +closed B-protein_state +” O +and O +“ O +open B-protein_state +” O +states O +of O +GDH B-protein_type +is O +modulated O +by O +two O +allosteric B-site +sites I-site +in O +each O +protomer B-oligomeric_state +( O +Fig O +. O +1A O +), O +which O +are O +differentially O +bound B-protein_state +by I-protein_state +GTP B-chemical +( O +an O +inhibitor O +) O +and O +ADP B-chemical +( O +an O +activator O +). O + +In O +the O +first O +site O +, O +which O +sits O +next O +to O +the O +pivot B-structure_element +helix I-structure_element +at O +the O +base O +of O +the O +antenna B-structure_element +( O +the O +“ O +GTP B-site +binding I-site +site I-site +”), O +GTP B-chemical +binding O +is O +known O +to O +act O +as O +an O +inhibitor O +, O +preventing O +release O +of O +the O +reaction O +product O +from O +the O +catalytic B-site +site I-site +by O +stabilizing O +the O +closed B-protein_state +conformation O +of O +the O +catalytic B-site +cleft I-site +. O + +In O +the O +second O +“ O +regulatory B-site +site I-site +”, O +which O +is O +situated O +near O +the O +pivot B-structure_element +helix I-structure_element +between O +adjacent O +protomers B-oligomeric_state +, O +ADP B-chemical +acts O +as O +an O +activator O +of O +enzymatic O +activity O +, O +presumably O +by O +hastening O +the O +opening O +of O +the O +catalytic B-site +cleft I-site +that O +leads O +to O +the O +release O +of O +the O +reaction O +product O +. O + +Interestingly O +, O +it O +has O +also O +been O +shown O +that O +the O +coenzyme O +NADH B-chemical +can O +bind O +to O +the O +regulatory B-site +site I-site +( O +also O +bound B-protein_state +by I-protein_state +the O +activator O +ADP B-chemical +), O +exerting O +a O +converse O +, O +inhibitory O +effect O +on O +GDH B-protein_type +product O +release O +, O +although O +the O +role O +this O +may O +play O +in O +vivo O +is O +not O +entirely O +clear O +. O + +Nearly O +all O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +structures B-evidence +of O +mammalian B-taxonomy_domain +GDH B-protein_type +are O +in O +the O +closed B-protein_state +conformation O +, O +and O +the O +few O +structures B-evidence +that O +are O +in O +the O +open B-protein_state +conformation O +are O +at O +lower O +resolution O +( O +Table O +1 O +). O + +Of O +those O +structures B-evidence +in O +the O +closed B-protein_state +conformation O +, O +most O +include O +NAD B-chemical +[ I-chemical +P I-chemical +] I-chemical +H I-chemical +, O +GTP B-chemical +, O +and O +glutamate B-chemical +( O +or O +, O +alternately O +, O +NAD B-chemical ++, I-chemical +GTP B-chemical +, O +and O +α B-chemical +- I-chemical +ketoglutarate I-chemical +). O + +However O +, O +the O +effects O +of O +coenzyme O +and O +GTP B-chemical +, O +bound B-protein_state +alone I-protein_state +or O +in O +concert O +in O +the O +absence B-protein_state +of I-protein_state +glutamate B-chemical +, O +have O +not O +been O +analyzed O +by O +crystallographic O +methods O +. O + +Here O +, O +we O +report O +single B-experimental_method +- I-experimental_method +particle I-experimental_method +cryo I-experimental_method +- I-experimental_method +electron I-experimental_method +microscopy I-experimental_method +( O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +) O +studies O +that O +show O +that O +under O +these O +conditions O +enzyme O +complexes O +coexist O +in O +both O +closed B-protein_state +and O +open B-protein_state +conformations O +. O + +We O +show O +that O +the O +structures B-evidence +in O +both O +states O +can O +be O +resolved O +at O +near O +- O +atomic O +resolution O +, O +suggesting O +a O +molecular O +mechanism O +for O +synergistic O +inhibition O +of O +GDH B-protein_type +by O +NADH B-chemical +and O +GTP B-chemical +( O +see O +Table O +2 O +for O +detailed O +information O +on O +all O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +- O +derived O +structures B-evidence +that O +we O +report O +in O +this O +work O +). O + +X B-experimental_method +- I-experimental_method +ray I-experimental_method +structures B-evidence +of O +mammalian B-taxonomy_domain +GDH B-protein_type +reported O +in O +both O +the O +open B-protein_state +and O +closed B-protein_state +conformations O + +GDH B-protein +Ligands O +PDB O +ID O +Conformation O +Resolution O +WT B-protein_state +NADH B-chemical ++ O +GLU B-chemical ++ O +GTP B-chemical +3MW9 O +Closed B-protein_state +2 O +. O +4 O +WT B-protein_state +Glu B-chemical +, O +GTP B-chemical +, O +NADPH B-chemical +, O +and O +Bithionol O +3ETD O +Closed B-protein_state +2 O +. O +5 O +WT B-protein_state +Glu B-chemical +, O +NADPH B-chemical +, O +GTP B-chemical ++ O +GW5074 O +3ETG O +Closed B-protein_state +2 O +. O +5 O +WT B-protein_state +apo B-protein_state +1L1F O +Open B-protein_state +2 O +. O +7 O +WT B-protein_state +NADPH B-chemical +, O +glutamate B-chemical +, O +and O +GTP B-chemical +1HWZ O +Closed B-protein_state +2 O +. O +8 O +WT B-protein_state +NADPH B-chemical ++ O +GLU B-chemical ++ O +GTP B-chemical ++ O +Zinc B-chemical +3MVQ O +Closed B-protein_state +2 O +. O +94 O +WT B-protein_state +NADPH B-chemical +, O +Glu B-chemical +, O +GTP B-chemical +, O +Hexachlorophene O +3ETE O +Closed B-protein_state +3 O +WT B-protein_state +NAD B-chemical +, O +PO4 B-chemical +, O +and O +2 B-chemical +- I-chemical +oxoglutarate I-chemical +1HWY O +Closed B-protein_state +3 O +. O +2 O +WT B-protein_state +NADPH B-chemical ++ O +GLU B-chemical ++ O +Eu O +3MVO O +Closed B-protein_state +3 O +. O +23 O +R463A O +mutant B-protein_state +apo B-protein_state +1NR1 O +Open B-protein_state +3 O +. O +3 O +WT B-protein_state +apo B-protein_state +1NR7 O +Open B-protein_state +3 O +. O +3 O +WT B-protein_state +ADP B-chemical +1NQT O +Open B-protein_state +3 O +. O +5 O +WT B-protein_state +NADPH B-chemical +and O +Epicatechin O +- O +3 O +- O +gallate O +( O +Ecg O +) O +3QMU O +Open B-protein_state +3 O +. O +62 O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +of O +mammalian B-taxonomy_domain +GDH B-protein_type +determined O +for O +this O +study O + +GDH B-protein_type +Ligands O +EMDB O +ID O +PDB O +ID O +Conformation O +Resolution O +Particles O +WT B-protein_state +apo B-protein_state +EMD O +- O +6630 O +3JCZ O +Open B-protein_state +3 O +. O +26 O +22462 O +WT B-protein_state +GTP B-chemical +EMD O +- O +6631 O +3JD0 O +Open B-protein_state +3 O +. O +47 O +39439 O +WT B-protein_state +NADH B-chemical +EMD O +- O +6635 O +3JD2 O +Open B-protein_state +3 O +. O +27 O +34716 O +WT B-protein_state +NADH B-chemical +EMD O +- O +6634 O +3JD1 O +Closed B-protein_state +3 O +. O +27 O +34926 O +WT B-protein_state +NADH B-chemical ++ O +GTP B-chemical +EMD O +- O +6632 O +3JD3 O +Open B-protein_state +3 O +. O +55 O +14793 O +WT B-protein_state +NADH B-chemical ++ O +GTP B-chemical +EMD O +- O +6633 O +3JD4 O +Closed B-protein_state +3 O +. O +40 O +20429 O + +To O +explore O +the O +conformational O +landscape O +of O +apo B-protein_state +- O +GDH B-protein +, O +we O +first O +determined O +its O +structure B-evidence +in O +the O +absence B-protein_state +of I-protein_state +any O +added O +ligands O +( O +Supplemental O +Fig O +. O +1 O +, O +Fig O +. O +2 O +, O +A O +– O +C O +). O + +The O +density B-evidence +map I-evidence +, O +refined O +to O +an O +average O +resolution O +of O +∼ O +3 O +. O +0 O +Å O +( O +Supplemental O +Fig O +. O +2 O +), O +is O +in O +the O +open B-protein_state +conformation O +and O +closely O +matches O +the O +model O +of O +unliganded B-protein_state +GDH B-protein +derived O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +at O +3 O +. O +3 O +Å O +resolution O +( O +PDB O +ID O +1NR7 O +). O + +The O +variation O +in O +local O +resolution O +from O +the O +core O +to O +the O +periphery O +, O +as O +reported O +by O +ResMap B-experimental_method +( O +Supplemental O +Fig O +. O +3D O +), O +is O +consistent O +with O +the O +B B-evidence +- I-evidence +factor I-evidence +gradient I-evidence +observed O +in O +the O +crystal B-evidence +structure I-evidence +( O +Supplemental O +Fig O +. O +3A O +). O + +Extensive O +classification O +without O +imposing O +symmetry O +yielded O +only O +open B-protein_state +structures B-evidence +and O +failed O +to O +detect O +any O +closed B-protein_state +catalytic B-site +cleft I-site +in O +the O +unliganded B-protein_state +enzyme O +, O +suggesting O +that O +all O +six O +protomers B-oligomeric_state +are O +in O +the O +open B-protein_state +conformation O +. O + +Consistent O +with O +this O +conclusion O +, O +the O +loops B-structure_element +connecting O +the O +β B-structure_element +- I-structure_element +strands I-structure_element +of O +the O +Rossmann B-structure_element +fold I-structure_element +are O +well O +- O +defined O +( O +Fig O +. O +2B O +), O +implying O +that O +there O +is O +little O +movement O +at O +the O +NBD B-structure_element +, O +as O +the O +transition O +between O +closed B-protein_state +and O +open B-protein_state +states O +is O +associated O +with O +NBD B-structure_element +movement O +( O +Fig O +. O +1B O +). O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +of O +GDH B-protein +in O +unliganded B-protein_state +and O +NADH B-protein_state +- I-protein_state +bound I-protein_state +states O +. O +( O +A O +) O +Refined O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +map B-evidence +of O +unliganded B-protein_state +GDH B-protein +at O +∼ O +3 O +Å O +resolution O +. O + +( O +B O +, O +C O +) O +Illustration O +of O +density B-evidence +map I-evidence +in O +the O +regions O +that O +contain O +the O +Rossmann B-structure_element +nucleotide I-structure_element +binding I-structure_element +fold I-structure_element +( O +B O +), O +pivot B-structure_element +and I-structure_element +antenna I-structure_element +helices I-structure_element +( O +C O +) O +in O +the O +unliganded B-protein_state +GDH B-protein +map B-evidence +. O +( O +D O +) O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +- O +derived O +density B-evidence +maps I-evidence +for O +two O +coexisting O +conformations O +that O +are O +present O +when O +GDH B-protein +is O +bound B-protein_state +to I-protein_state +the O +cofactor O +NADH B-chemical +. O + +Each O +protomer B-oligomeric_state +is O +shown O +in O +a O +different O +color O +and O +densities B-evidence +for O +NADH B-chemical +bound B-protein_state +in I-protein_state +both O +regulatory B-site +( O +red O +) O +and O +catalytic B-site +( O +purple O +) O +sites B-site +on O +one O +protomer B-oligomeric_state +are O +indicated O +. O + +The O +overall O +quaternary O +structures O +of O +the O +two O +conformations O +are O +essentially O +the O +same O +as O +that O +of O +the O +open B-protein_state +and O +closed B-protein_state +states O +observed O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +To O +test O +the O +effect O +of O +NADH B-chemical +binding O +on O +GDH B-protein +conformation O +in O +solution O +, O +we O +determined O +the O +structure B-evidence +of O +this O +binary O +complex O +using O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +methods O +combined O +with O +three B-experimental_method +- I-experimental_method +dimensional I-experimental_method +classification I-experimental_method +. O + +Two O +dominant O +conformational O +states O +, O +in O +an O +all O +open B-protein_state +or O +all O +closed B-protein_state +conformation O +were O +detected O +, O +segregated O +( O +Fig O +. O +2D O +), O +and O +further O +refined O +to O +near O +- O +atomic O +resolution O +(∼ O +3 O +. O +3 O +Å O +; O +Supplemental O +Fig O +. O +2 O +). O + +Densities B-evidence +for O +12 O +molecules O +of O +bound B-protein_state +NADH B-chemical +were O +identified O +in O +maps B-evidence +of O +both O +open B-protein_state +and O +closed B-protein_state +states O +( O +Supplemental O +Fig O +. O +4 O +). O + +The O +NADH B-protein_state +- I-protein_state +bound I-protein_state +closed B-protein_state +conformation O +matches O +the O +structure B-evidence +of O +the O +quaternary O +complex O +observed O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +, O +with O +the O +exception O +that O +density B-evidence +corresponding O +to O +GTP B-chemical +and O +glutamate B-chemical +was O +absent O +in O +the O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +- O +derived O +map B-evidence +. O + +Thus O +, O +closure O +of O +the O +catalytic B-site +cleft I-site +is O +accompanied O +by O +a O +quaternary O +structural O +change O +that O +can O +be O +described O +as O +a O +global O +bending O +of O +the O +structure B-evidence +about O +an O +axis O +that O +runs O +parallel O +to O +the O +pivot B-structure_element +helix I-structure_element +, O +accompanied O +by O +an O +expansion O +of O +the O +core O +( O +Figs O +. O +1A O +and O +2D O +). O + +Detailed O +analysis O +of O +the O +GDH B-complex_assembly +/ I-complex_assembly +NADH I-complex_assembly +structures B-evidence +shows O +that O +both O +the O +adenosine O +and O +nicotinamide O +moieties O +of O +NADH B-chemical +bind O +to O +the O +catalytic B-site +site I-site +within O +the O +NBD B-structure_element +in O +nearly O +the O +same O +orientation O +in O +both O +the O +open B-protein_state +and O +the O +closed B-protein_state +states O +, O +and O +display O +closely O +comparable O +interactions O +with O +the O +Rossmann B-structure_element +fold I-structure_element +( O +Fig O +. O +3 O +, O +A O +and O +B O +). O + +At O +the O +regulatory B-site +site I-site +, O +where O +either O +ADP B-chemical +can O +bind O +as O +an O +activator O +or O +NADH B-chemical +can O +bind O +as O +an O +inhibitor O +, O +the O +binding O +of O +the O +adenine O +moiety O +of O +NADH B-chemical +is O +nearly O +identical O +between O +the O +two O +conformers O +. O + +In O +the O +closed B-protein_state +state O +, O +the O +nicotinamide O +group O +is O +oriented O +toward O +the O +center O +of O +the O +hexamer B-oligomeric_state +, O +inserted O +into O +a O +narrow O +cavity B-site +between O +two O +adjacent O +subunits B-structure_element +in O +the O +trimer B-oligomeric_state +. O + +There O +are O +extensive O +interactions O +between O +NADH B-chemical +and O +the O +residues O +lining O +this O +cavity B-site +, O +which O +may O +explain O +the O +well O +- O +defined O +density B-evidence +of O +this O +portion O +of O +NADH B-chemical +in O +the O +closed B-protein_state +state O +. O + +Detailed O +view O +of O +NADH B-chemical +conformation O +in O +catalytic B-site +and I-site +regulatory I-site +sites I-site +. O +( O +A O +, O +B O +) O +NADH B-chemical +density B-evidence +( O +purple O +) O +and O +interactions O +in O +the O +catalytic B-site +sites I-site +of O +closed B-protein_state +( O +A O +) O +and O +open B-protein_state +( O +B O +) O +states O +. O +( O +C O +, O +D O +) O +NADH B-chemical +density B-evidence +( O +red O +) O +and O +interactions O +in O +the O +regulatory B-site +sites I-site +of O +closed B-protein_state +( O +C O +) O +and O +open B-protein_state +( O +D O +) O +states O +. O + +In O +the O +open B-protein_state +state O +, O +the O +binding O +of O +ADP B-chemical +or O +NADH B-chemical +is O +further O +stabilized O +by O +His209 B-residue_name_number +, O +a O +residue O +that O +undergoes O +a O +large O +movement O +during O +the O +transition O +from O +open B-protein_state +to O +closed B-protein_state +conformation O +( O +Fig O +. O +3 O +, O +C O +and O +D O +). O + +In O +the O +closed B-protein_state +conformation O +, O +however O +, O +this O +key O +histidine B-residue_name +residue O +is O +> O +10 O +. O +5 O +Å O +away O +from O +the O +nearest O +phosphate O +group O +on O +NADH B-chemical +, O +altering O +a O +critical O +stabilization O +point O +within O +the O +regulatory B-site +site I-site +. O + +In O +the O +absence B-protein_state +of I-protein_state +NADH B-chemical +, O +GTP B-chemical +binds O +weakly O +to O +GDH B-protein +with O +a O +dissociation B-evidence +constant I-evidence +of O +∼ O +20 O +μM O +. O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +analysis O +of O +GDH B-protein +incubated B-protein_state +with I-protein_state +GTP B-chemical +resulted O +in O +a O +structure B-evidence +at O +an O +overall O +resolution O +of O +3 O +. O +5 O +Å O +, O +showing O +that O +it O +is O +in O +an O +open B-protein_state +conformation O +( O +Supplemental O +Fig O +. O +6 O +), O +with O +all O +NBDs B-structure_element +in O +the O +open B-protein_state +state O +. O + +The O +density B-evidence +for O +GTP B-chemical +is O +not O +very O +well O +defined O +, O +suggesting O +considerable O +wobble O +in O +the O +binding B-site +site I-site +. O + +Subtraction B-experimental_method +of O +the O +GTP B-protein_state +- I-protein_state +bound I-protein_state +map B-evidence +with O +that O +of O +the O +apo B-protein_state +state O +shows O +that O +GTP B-chemical +binding O +can O +nevertheless O +be O +visualized O +specifically O +in O +the O +GTP B-site +binding I-site +site I-site +( O +Supplemental O +Fig O +. O +6 O +). O + +To O +further O +dissect O +the O +roles O +of O +NADH B-chemical +and O +GTP B-chemical +in O +the O +transition O +from O +the O +open B-protein_state +to O +closed B-protein_state +conformations O +, O +we O +next O +determined B-experimental_method +structures B-evidence +of O +GDH B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +both O +NADH B-chemical +and O +GTP B-chemical +, O +but O +without B-protein_state +glutamate B-chemical +. O + +Reconstruction B-experimental_method +without I-experimental_method +classification I-experimental_method +, O +however O +, O +yields O +a O +structure B-evidence +clearly O +in O +the O +closed B-protein_state +conformation O +, O +suggesting O +that O +, O +in O +coordination O +with O +NADH B-chemical +, O +GTP B-chemical +may O +further O +stabilize O +the O +closed B-protein_state +conformation O +. O + +The O +location O +of O +GTP B-chemical +in O +the O +open B-protein_state +and O +closed B-protein_state +states O +of O +the O +GDH B-complex_assembly +/ I-complex_assembly +NADH I-complex_assembly +/ I-complex_assembly +GTP I-complex_assembly +complex O +is O +similar O +to O +that O +in O +the O +crystal B-evidence +structure I-evidence +observed O +in O +the O +presence B-protein_state +of I-protein_state +NADH B-chemical +, O +GTP B-chemical +, O +and O +glutamate B-chemical +. O + +Likewise O +, O +the O +position O +of O +NADH B-chemical +in O +the O +open B-protein_state +and O +closed B-protein_state +states O +closely O +resembles O +the O +position O +of O +NADH B-chemical +in O +the O +GDH B-complex_assembly +/ I-complex_assembly +NADH I-complex_assembly +open B-protein_state +and O +closed B-protein_state +structures B-evidence +. O + +One O +key O +difference O +between O +the O +open B-protein_state +and O +closed B-protein_state +states O +of O +these O +structures B-evidence +is O +the O +position O +of O +the O +His209 B-residue_name_number +residue O +: O +As O +mentioned O +above O +, O +His209 B-residue_name_number +swings O +away O +from O +the O +adenine O +moiety O +of O +NADH B-chemical +in O +the O +closed B-protein_state +state O +. O + +Thus O +, O +GTP B-chemical +binding O +to O +GDH B-protein +appears O +synergistic O +with O +NADH B-chemical +and O +displaces O +the O +conformational O +landscape O +toward O +the O +closed B-protein_state +state O +. O + +( O +A O +, O +B O +) O +Observation O +of O +co O +- O +existing O +open B-protein_state +( O +A O +) O +and O +closed B-protein_state +( O +B O +) O +conformations O +in O +the O +GDH B-complex_assembly +- I-complex_assembly +NADH I-complex_assembly +- I-complex_assembly +GTP I-complex_assembly +ternary O +complex O +. O + +Densities B-evidence +for O +GTP B-chemical +( O +yellow O +) O +as O +well O +as O +NADH B-chemical +bound B-protein_state +to I-protein_state +both O +catalytic B-site +( O +purple O +) O +and O +regulatory B-site +( O +red O +) O +sites B-site +in O +each O +protomer B-oligomeric_state +are O +shown O +. O + +Whereas O +the O +orientation O +in O +which O +NADH B-chemical +binds O +at O +the O +catalytic B-site +site I-site +is O +similar O +for O +both O +conformations O +, O +the O +orientation O +of O +the O +nicotinamide O +portion O +of O +NADH B-chemical +in O +the O +regulatory B-site +site I-site +is O +different O +between O +the O +open B-protein_state +and O +closed B-protein_state +conformations O +( O +Figs O +. O +3 O +and O +4 O +). O + +In O +the O +closed B-protein_state +state O +, O +the O +nicotinamide O +moiety O +is O +inserted O +into O +a O +well O +- O +defined O +cavity B-site +at O +the O +interface B-site +between O +two O +adjacent O +protomers B-oligomeric_state +in O +the O +trimer B-oligomeric_state +. O + +As O +mentioned O +above O +, O +this O +cavity B-site +is O +much O +narrower O +in O +the O +open B-protein_state +state O +, O +suggesting O +that O +this O +cavity B-site +may O +be O +unavailable O +to O +the O +NADH B-chemical +nicotinamide O +moiety O +when O +the O +enzyme O +is O +in O +the O +open B-protein_state +conformation O +. O + +The O +rapid O +emergence O +of O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +as O +a O +tool O +for O +near O +- O +atomic O +resolution O +structure B-experimental_method +determination I-experimental_method +provides O +new O +opportunities O +for O +complementing O +atomic O +resolution O +information O +from O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +, O +as O +illustrated O +here O +with O +GDH B-protein +. O + +Perhaps O +the O +most O +important O +contribution O +of O +these O +methods O +is O +the O +prospect O +that O +when O +there O +are O +discrete O +subpopulations O +present O +, O +the O +structure B-evidence +of O +each O +state O +can O +be O +determined O +at O +near O +- O +atomic O +resolution O +. O +What O +we O +demonstrate O +here O +with O +GDH B-protein +is O +that O +by O +employing O +three B-experimental_method +- I-experimental_method +dimensional I-experimental_method +image I-experimental_method +classification I-experimental_method +approaches I-experimental_method +, O +we O +not O +only O +can O +isolate O +distinct O +, O +coexisting O +conformations O +, O +but O +we O +can O +also O +localize O +small O +molecule O +ligands O +in O +each O +of O +these O +conformations O +. O + +Investigation O +of O +the O +Interaction O +between O +Cdc42 B-protein +and O +Its O +Effector O +TOCA1 B-protein + +Transducer B-protein +of I-protein +Cdc42 I-protein +- I-protein +dependent I-protein +actin I-protein +assembly I-protein +protein I-protein +1 I-protein +( O +TOCA1 B-protein +) O +is O +an O +effector O +of O +the O +Rho B-protein_type +family I-protein_type +small I-protein_type +G I-protein_type +protein I-protein_type +Cdc42 B-protein +. O + +We O +have O +found O +that O +the O +TOCA1 B-protein +HR1 B-structure_element +, O +like O +the O +closely O +related O +CIP4 B-protein +HR1 B-structure_element +, O +has O +interesting O +structural O +features O +that O +are O +not O +observed O +in O +other O +HR1 B-structure_element +domains O +. O + +We O +have O +also O +investigated O +the O +binding O +of O +the O +TOCA B-protein +HR1 B-structure_element +domain O +to O +Cdc42 B-protein +and O +the O +potential O +ternary O +complex O +between O +Cdc42 B-protein +and O +the O +G B-site +protein I-site +- I-site +binding I-site +regions I-site +of O +TOCA1 B-protein +and O +a O +member O +of O +the O +Wiskott B-protein_type +- I-protein_type +Aldrich I-protein_type +syndrome I-protein_type +protein I-protein_type +family I-protein_type +, O +N B-protein +- I-protein +WASP I-protein +. O + +TOCA1 B-protein +binds O +Cdc42 B-protein +with O +micromolar O +affinity O +, O +in O +contrast O +to O +the O +nanomolar O +affinity O +of O +the O +N B-protein +- I-protein +WASP I-protein +G B-site +protein I-site +- I-site +binding I-site +region I-site +for O +Cdc42 B-protein +. O + +NMR B-experimental_method +experiments O +show O +that O +the O +Cdc42 B-site +- I-site +binding I-site +domain I-site +from O +N B-protein +- I-protein +WASP I-protein +is O +able O +to O +displace O +TOCA1 B-protein +HR1 B-structure_element +from O +Cdc42 B-protein +, O +whereas O +the O +N B-protein +- I-protein +WASP I-protein +domain O +but O +not O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +inhibits O +actin O +polymerization O +. O + +This O +suggests O +that O +TOCA1 B-protein +binding O +to O +Cdc42 B-protein +is O +an O +early O +step O +in O +the O +Cdc42 B-protein +- O +dependent O +pathways O +that O +govern O +actin O +dynamics O +, O +and O +the O +differential O +binding B-evidence +affinities I-evidence +of O +the O +effectors O +facilitate O +a O +handover O +from O +TOCA1 B-protein +to O +N B-protein +- I-protein +WASP I-protein +, O +which O +can O +then O +drive O +recruitment O +of O +the O +actin O +- O +modifying O +machinery O +. O + +The O +Ras B-protein_type +superfamily I-protein_type +of O +small B-protein_type +GTPases I-protein_type +comprises O +over O +150 O +members O +that O +regulate O +a O +multitude O +of O +cellular O +processes O +in O +eukaryotes B-taxonomy_domain +. O + +The O +superfamily O +can O +be O +divided O +into O +five O +families O +based O +on O +structural O +and O +functional O +similarities O +: O +Ras B-protein_type +, O +Rho B-protein_type +, O +Rab B-protein_type +, O +Arf B-protein_type +, O +and O +Ran B-protein_type +. O + +These O +molecular O +switches O +cycle O +between O +active B-protein_state +, O +GTP B-protein_state +- I-protein_state +bound I-protein_state +, O +and O +inactive B-protein_state +, O +GDP B-protein_state +- I-protein_state +bound I-protein_state +, O +states O +with O +the O +help O +of O +auxiliary O +proteins O +. O + +The O +guanine B-protein_type +nucleotide I-protein_type +exchange I-protein_type +factors I-protein_type +mediate O +formation O +of O +the O +active B-protein_state +state O +by O +promoting O +the O +dissociation O +of O +GDP B-chemical +, O +allowing O +GTP B-chemical +to O +bind O +. O + +The O +GTPase B-protein_type +- I-protein_type +activating I-protein_type +proteins I-protein_type +stimulate O +the O +rate O +of O +intrinsic O +GTP B-chemical +hydrolysis O +, O +mediating O +the O +return O +to O +the O +inactive B-protein_state +state O +( O +reviewed O +in O +Ref O +.). O + +These O +regions O +are O +responsible O +for O +“ O +sensing O +” O +the O +nucleotide O +state O +, O +with O +the O +GTP B-protein_state +- I-protein_state +bound I-protein_state +state O +showing O +greater O +rigidity O +and O +the O +GDP B-protein_state +- I-protein_state +bound I-protein_state +state O +adopting O +a O +more O +relaxed O +conformation O +( O +reviewed O +in O +Ref O +.). O + +In O +the O +active B-protein_state +state O +, O +G B-protein_type +proteins I-protein_type +bind O +to O +an O +array O +of O +downstream O +effectors O +, O +through O +which O +they O +exert O +their O +extensive O +roles O +within O +the O +cell O +. O + +The O +Rho B-protein_type +family I-protein_type +comprises O +20 O +members O +, O +of O +which O +three O +, O +RhoA B-protein +, O +Rac1 B-protein +, O +and O +Cdc42 B-protein +, O +have O +been O +relatively O +well O +studied O +. O + +RhoA B-protein +acts O +to O +rearrange O +existing O +actin O +structures O +to O +form O +stress O +fibers O +, O +whereas O +Rac1 B-protein +and O +Cdc42 B-protein +promote O +de O +novo O +actin O +polymerization O +to O +form O +lamellipodia O +and O +filopodia O +, O +respectively O +. O + +A O +number O +of O +RhoA B-protein +and O +Rac1 B-protein +effector O +proteins O +, O +including O +the O +formins O +and O +members O +of O +the O +protein B-protein_type +kinase I-protein_type +C I-protein_type +- I-protein_type +related I-protein_type +kinase I-protein_type +( O +PRK B-protein_type +) O +6 B-protein_type +family O +, O +along O +with O +Cdc42 B-protein +effectors O +, O +including O +the O +Wiskott B-protein_type +- I-protein_type +Aldrich I-protein_type +syndrome I-protein_type +( O +WASP B-protein_type +) O +family O +and O +the O +transducer O +of O +Cdc42 B-protein_type +- I-protein_type +dependent I-protein_type +actin I-protein_type +assembly I-protein_type +( O +TOCA B-protein_type +) O +family O +, O +have O +also O +been O +linked O +to O +the O +pathways O +that O +govern O +cytoskeletal O +dynamics O +. O + +Cdc42 B-protein +effectors O +, O +TOCA1 B-protein +and O +the O +ubiquitously O +expressed O +member O +of O +the O +WASP B-protein_type +family I-protein_type +, O +N B-protein +- I-protein +WASP I-protein +, O +have O +been O +implicated O +in O +the O +regulation O +of O +actin O +polymerization O +downstream O +of O +Cdc42 B-protein +and O +phosphatidylinositol B-chemical +4 I-chemical +, I-chemical +5 I-chemical +- I-chemical +bisphosphate I-chemical +( O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +). O + +N B-protein +- I-protein +WASP I-protein +exists O +in O +an O +autoinhibited B-protein_state +conformation I-protein_state +, O +which O +is O +released O +upon O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +and O +Cdc42 B-protein +binding O +or O +by O +other O +factors O +, O +such O +as O +phosphorylation O +. O + +Following O +their O +release O +, O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +regions I-structure_element +of O +N B-protein +- I-protein +WASP I-protein +are O +free O +to O +interact O +with O +G B-protein_type +- I-protein_type +actin I-protein_type +and O +a O +known O +nucleator O +of O +actin O +assembly O +, O +the O +Arp2 B-complex_assembly +/ I-complex_assembly +3 I-complex_assembly +complex O +. O + +The O +importance O +of O +TOCA1 B-protein +in O +actin O +polymerization O +has O +been O +demonstrated O +in O +a O +range O +of O +in O +vitro O +and O +in O +vivo O +studies O +, O +but O +the O +exact O +role O +of O +TOCA1 B-protein +in O +the O +many O +pathways O +involving O +actin O +assembly O +remains O +unclear O +. O + +TOCA1 B-protein +comprises O +an O +N O +- O +terminal O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +, O +a O +central B-structure_element +homology I-structure_element +region I-structure_element +1 I-structure_element +( O +HR1 B-structure_element +) O +domain O +, O +and O +a O +C O +- O +terminal O +SH3 B-structure_element +domain O +. O + +The O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +is O +a O +known O +dimerization O +, O +membrane O +- O +binding O +, O +and O +membrane O +- O +deforming O +module O +found O +in O +a O +number O +of O +cell O +signaling O +proteins O +. O + +The O +HR1 B-structure_element +domain O +has O +been O +directly O +implicated O +in O +the O +interaction O +between O +TOCA1 B-protein +and O +Cdc42 B-protein +, O +representing O +the O +first O +Cdc42 B-protein +- O +HR1 B-structure_element +domain O +interaction O +to O +be O +identified O +. O + +Other O +HR1 B-structure_element +domains O +studied O +so O +far O +, O +including O +those O +from O +the O +PRK B-protein_type +family I-protein_type +, O +have O +been O +found O +to O +bind O +their O +cognate O +Rho O +family O +G B-protein_type +protein I-protein_type +- O +binding O +partner O +with O +high O +specificity O +and O +affinities B-evidence +in O +the O +nanomolar O +range O +. O + +These O +HR1 B-structure_element +domains O +, O +however O +, O +show O +specificity O +for O +Cdc42 B-protein +, O +rather O +than O +RhoA B-protein +or O +Rac1 B-protein +. O + +Furthermore O +, O +the O +biological O +function O +of O +the O +interaction O +between O +TOCA1 B-protein +and O +Cdc42 B-protein +remains O +poorly O +understood O +, O +and O +so O +far O +there O +has O +been O +no O +biophysical O +or O +structural O +insight O +. O + +The O +interactions O +of O +TOCA1 B-protein +and O +N B-protein +- I-protein +WASP I-protein +with O +Cdc42 B-protein +as O +well O +as O +with O +each O +other O +have O +raised O +questions O +as O +to O +whether O +the O +two O +Cdc42 B-protein +effectors O +can O +interact O +with O +a O +single O +molecule O +of O +Cdc42 B-protein +simultaneously O +. O + +There O +is O +some O +evidence O +for O +a O +ternary O +complex O +between O +Cdc42 B-protein +, O +N B-protein +- I-protein +WASP I-protein +, O +and O +TOCA1 B-protein +, O +but O +there O +was O +no O +direct O +demonstration O +of O +simultaneous O +contacts O +between O +the O +two O +effectors O +and O +a O +single O +molecule O +of O +Cdc42 B-protein +. O + +Nonetheless O +, O +the O +substantial O +difference O +between O +the O +structures B-evidence +of O +the O +G B-site +protein I-site +- I-site +binding I-site +regions I-site +of O +the O +two O +effectors O +is O +intriguing O +and O +implies O +that O +they O +bind O +to O +Cdc42 B-protein +quite O +differently O +, O +providing O +motivation O +for O +investigating O +the O +possibility O +that O +Cdc42 B-protein +can O +bind O +both O +effectors O +concurrently O +. O + +WASP B-protein_type +interacts O +with O +Cdc42 B-protein +via O +a O +conserved B-protein_state +, O +unstructured B-structure_element +binding I-structure_element +motif I-structure_element +known O +as O +the O +Cdc42 B-structure_element +- I-structure_element +and I-structure_element +Rac I-structure_element +- I-structure_element +interactive I-structure_element +binding I-structure_element +region I-structure_element +( O +CRIB B-structure_element +), O +which O +forms O +an O +intermolecular B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +, O +expanding O +the O +anti O +- O +parallel O +β2 B-structure_element +and I-structure_element +β3 I-structure_element +strands I-structure_element +of O +Cdc42 B-protein +. O + +In O +contrast O +, O +the O +TOCA B-protein_type +family I-protein_type +proteins I-protein_type +are O +thought O +to O +interact O +via O +the O +HR1 B-structure_element +domain O +, O +which O +may O +form O +a O +triple B-structure_element +coiled I-structure_element +- I-structure_element +coil I-structure_element +with O +switch B-site +II I-site +of O +Rac1 B-protein +, O +like O +the O +HR1b B-structure_element +domain O +of O +PRK1 B-protein +. O + +Here O +, O +we O +present O +the O +solution B-experimental_method +NMR I-experimental_method +structure B-evidence +of O +the O +HR1 B-structure_element +domain O +of O +TOCA1 B-protein +, O +providing O +the O +first O +structural B-evidence +data I-evidence +for O +this O +protein O +. O + +Finally O +, O +we O +investigate O +the O +potential O +ternary O +complex O +between O +Cdc42 B-protein +and O +the O +G B-site +protein I-site +- I-site +binding I-site +regions I-site +of O +TOCA1 B-protein +and O +N B-protein +- I-protein +WASP I-protein +, O +contributing O +to O +our O +understanding O +of O +G B-protein_type +protein I-protein_type +- O +effector O +interactions O +as O +well O +as O +the O +roles O +of O +Cdc42 B-protein +, O +N B-protein +- I-protein +WASP I-protein +, O +and O +TOCA1 B-protein +in O +the O +pathways O +that O +govern O +actin B-protein_type +dynamics O +. O + +Cdc42 B-protein +- O +TOCA1 B-protein +Binding O + +TOCA1 B-protein +was O +identified O +in O +Xenopus B-taxonomy_domain +extracts O +as O +a O +protein O +necessary O +for O +Cdc42 B-protein +- O +dependent O +actin O +assembly O +and O +was O +shown O +to O +bind O +to O +Cdc42 B-complex_assembly +· I-complex_assembly +GTPγS I-complex_assembly +but O +not O +to O +Cdc42 B-complex_assembly +· I-complex_assembly +GDP I-complex_assembly +or O +to O +Rac1 B-protein +and O +RhoA B-protein +. O +Given O +its O +homology O +to O +other O +Rho B-site +family I-site +binding I-site +modules I-site +, O +it O +is O +likely O +that O +the O +HR1 B-structure_element +domain O +of O +TOCA1 B-protein +is O +sufficient O +to O +bind O +Cdc42 B-protein +. O + +The O +C B-species +. I-species +elegans I-species +TOCA1 B-protein +orthologues O +also O +bind O +to O +Cdc42 B-protein +via O +their O +consensus O +HR1 B-structure_element +domain O +. O + +The O +HR1 B-structure_element +domains O +from O +the O +PRK B-protein_type +family I-protein_type +bind O +their O +G B-protein_type +protein I-protein_type +partners O +with O +a O +high O +affinity O +, O +exhibiting O +a O +range O +of O +submicromolar O +dissociation B-evidence +constants I-evidence +( O +Kd B-evidence +) O +as O +low O +as O +26 O +nm O +. O + +A O +Kd B-evidence +in O +the O +nanomolar O +range O +was O +therefore O +expected O +for O +the O +interaction O +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +with O +Cdc42 B-protein +. O + +We O +generated O +an O +X B-species +. I-species +tropicalis I-species +TOCA1 B-protein +HR1 B-structure_element +domain O +construct O +encompassing O +residues O +330 B-residue_range +– I-residue_range +426 I-residue_range +. O + +This O +region O +comprises O +the O +complete O +HR1 B-structure_element +domain O +based O +on O +secondary O +structure O +predictions O +and O +sequence B-experimental_method +alignments I-experimental_method +with O +another O +TOCA B-protein_type +family I-protein_type +member O +, O +CIP4 B-protein +, O +whose O +structure B-evidence +has O +been O +determined O +. O + +The O +interaction O +between O +[ B-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +· I-complex_assembly +Cdc42 I-complex_assembly +and O +a O +C O +- O +terminally O +His B-protein_state +- I-protein_state +tagged I-protein_state +TOCA1 B-protein +HR1 B-structure_element +domain O +construct O +was O +investigated O +using O +SPA B-experimental_method +. O + +The O +binding B-evidence +isotherm I-evidence +for O +the O +interaction O +is O +shown O +in O +Fig O +. O +1A O +, O +together O +with O +the O +Cdc42 B-protein +- O +PAK B-protein +interaction O +as O +a O +positive O +control O +. O + +The O +binding O +of O +TOCA1 B-protein +HR1 B-structure_element +to O +Cdc42 B-protein +was O +unexpectedly O +weak O +, O +with O +a O +Kd B-evidence +of O +> O +1 O +μm O +. O + +It O +was O +not O +possible O +to O +estimate O +the O +Kd B-evidence +more O +accurately O +using O +direct O +SPA B-experimental_method +experiments O +, O +because O +saturation O +could O +not O +be O +reached O +due O +to O +nonspecific O +signal O +at O +higher O +protein O +concentrations O +. O + +The O +TOCA1 B-protein +HR1 B-structure_element +- O +Cdc42 B-protein +interaction O +is O +low O +affinity O +. O + +A O +, O +curves O +derived O +from O +direct B-experimental_method +binding I-experimental_method +assays I-experimental_method +in O +which O +the O +indicated O +concentrations O +of O +Cdc42Δ7Q61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +were O +incubated B-experimental_method +with O +30 O +nm O +GST B-mutant +- I-mutant +PAK I-mutant +or O +HR1 B-mutant +- I-mutant +His6 I-mutant +in O +SPAs B-experimental_method +. O + +The O +SPA B-experimental_method +signal O +was O +corrected O +by O +subtraction O +of O +control O +data O +with O +no O +GST B-mutant +- I-mutant +PAK I-mutant +or O +HR1 B-mutant +- I-mutant +His6 I-mutant +. O + +The O +Kd B-evidence +values O +derived O +for O +the O +ACK B-protein +GBD B-structure_element +with O +Cdc42Δ7 B-mutant +and O +full B-protein_state +- I-protein_state +length I-protein_state +Cdc42 B-protein +were O +0 O +. O +032 O +± O +0 O +. O +01 O +and O +0 O +. O +011 O +± O +0 O +. O +01 O +μm O +, O +respectively O +. O + +Isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +was O +carried O +out O +, O +but O +no O +heat O +changes O +were O +observed O +at O +a O +range O +of O +concentrations O +and O +temperatures O +( O +data O +not O +shown O +), O +suggesting O +that O +the O +interaction O +is O +predominantly O +entropically O +driven O +. O + +The O +affinity B-evidence +was O +therefore O +determined O +using O +competition B-experimental_method +SPAs I-experimental_method +. O + +A O +complex O +of O +a O +GST B-experimental_method +fusion I-experimental_method +of O +the O +GBD B-structure_element +of O +ACK B-protein +, O +which O +binds O +with O +a O +high O +affinity O +to O +Cdc42 B-protein +, O +with O +radiolabeled O +[ B-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +· I-complex_assembly +Cdc42 I-complex_assembly +was O +preformed O +, O +and O +the O +effect O +of O +increasing B-experimental_method +concentrations I-experimental_method +of O +untagged B-protein_state +TOCA1 B-protein +HR1 B-structure_element +domain O +was O +examined O +. O + +Competition O +of O +GST B-mutant +- I-mutant +ACK I-mutant +GBD B-structure_element +bound B-protein_state +to I-protein_state +[ B-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +· I-complex_assembly +Cdc42 I-complex_assembly +by O +free B-protein_state +ACK B-protein +GBD B-structure_element +was O +used O +as O +a O +control O +and O +to O +establish O +the O +value O +of O +background O +counts O +when O +Cdc42 B-protein +is O +fully O +displaced O +. O + +Free B-protein_state +ACK B-protein +competed O +with O +itself O +with O +an O +affinity B-evidence +of O +32 O +nm O +, O +similar O +to O +the O +value O +obtained O +by O +direct B-experimental_method +binding I-experimental_method +of O +23 O +nm O +. O + +The O +TOCA1 B-protein +HR1 B-structure_element +domain O +also O +fully O +competed O +with O +the O +GST B-mutant +- I-mutant +ACK I-mutant +but O +bound B-protein_state +with O +an O +affinity B-evidence +of O +6 O +μm O +( O +Fig O +. O +1 O +, O +B O +and O +C O +), O +in O +agreement O +with O +the O +low O +affinity B-evidence +observed O +in O +the O +direct B-experimental_method +binding I-experimental_method +experiments I-experimental_method +. O + +The O +Cdc42 B-protein +construct O +used O +in O +the O +binding B-experimental_method +assays I-experimental_method +has O +seven B-residue_range +residues I-residue_range +deleted B-experimental_method +from O +the O +C O +terminus O +to O +facilitate O +purification O +. O + +These O +residues O +are O +not O +generally O +required O +for O +G B-protein_type +protein I-protein_type +- O +effector O +interactions O +, O +including O +the O +interaction O +between O +RhoA B-protein +and O +the O +PRK1 B-protein +HR1a B-structure_element +domain O +. O + +In O +contrast O +, O +the O +C O +terminus O +of O +Rac1 B-protein +contains O +a O +polybasic O +sequence O +, O +which O +is O +crucial O +for O +Rac1 B-protein +binding O +to O +the O +HR1b B-structure_element +domain O +from O +PRK1 B-protein +. O + +The O +binding B-experimental_method +experiments I-experimental_method +were O +repeated O +with O +full B-protein_state +- I-protein_state +length I-protein_state +[ B-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +· I-complex_assembly +Cdc42 I-complex_assembly +, O +but O +the O +affinity B-evidence +of O +the O +HR1 B-structure_element +domain O +for O +full B-protein_state +- I-protein_state +length I-protein_state +Cdc42 B-protein +was O +similar O +to O +its O +affinity B-evidence +for O +truncated B-protein_state +Cdc42 B-protein +( O +Kd B-evidence +≈ O +5 O +μm O +; O +Fig O +. O +1C O +). O + +Another O +possible O +explanation O +for O +the O +low O +affinities B-evidence +observed O +was O +that O +the O +HR1 B-structure_element +domain O +alone B-protein_state +is O +not O +sufficient O +for O +maximal O +binding O +of O +the O +TOCA B-protein_type +proteins I-protein_type +to O +Cdc42 B-protein +and O +that O +the O +other O +domains O +are O +required O +. O + +Indeed O +, O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +downs I-experimental_method +performed O +with O +in O +vitro O +translated O +human B-species +TOCA1 B-protein +fragments O +had O +suggested O +that O +residues O +N O +- O +terminal O +to O +the O +HR1 B-structure_element +domain O +may O +be O +required O +to O +stabilize O +the O +HR1 B-structure_element +domain O +structure O +. O + +Furthermore O +, O +both O +BAR B-structure_element +and O +SH3 B-structure_element +domains O +have O +been O +implicated O +in O +interactions O +with O +small O +G B-protein_type +proteins I-protein_type +( O +e O +. O +g O +. O +the O +BAR B-structure_element +domain O +of O +Arfaptin2 B-protein +binds O +to O +Rac1 B-protein +and O +Arl1 B-protein +), O +while O +an O +SH3 B-structure_element +domain O +mediates O +the O +interaction O +between O +Rac1 B-protein +and O +the O +guanine B-protein +nucleotide I-protein +exchange I-protein +factor I-protein +, O +β B-protein +- I-protein +PIX I-protein +. O + +TOCA1 B-protein +dimerizes B-oligomeric_state +via O +its O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +, O +which O +could O +also O +affect O +Cdc42 B-protein +binding O +, O +for O +example O +by O +presenting O +two O +HR1 B-structure_element +domains O +for O +Cdc42 B-protein +interactions O +. O + +Various O +TOCA1 B-protein +fragments O +( O +Fig O +. O +2A O +) O +were O +therefore O +assessed O +for O +binding O +to O +full B-protein_state +- I-protein_state +length I-protein_state +Cdc42 B-protein +by O +direct O +SPA B-experimental_method +. O + +The O +isolated O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +showed O +no O +binding O +to O +full B-protein_state +- I-protein_state +length I-protein_state +Cdc42 B-protein +( O +Fig O +. O +2B O +). O + +The O +HR1 B-mutant +- I-mutant +SH3 I-mutant +protein O +could O +not O +be O +purified O +to O +homogeneity O +as O +a O +fusion O +protein O +, O +so O +it O +was O +assayed O +in O +competition B-experimental_method +assays I-experimental_method +after O +cleavage O +of O +the O +His O +tag O +. O + +This O +construct O +competed O +with O +GST B-mutant +- I-mutant +ACK I-mutant +GBD B-structure_element +to O +give O +a O +similar O +affinity O +to O +the O +HR1 B-structure_element +domain O +alone B-protein_state +( O +Kd B-evidence += O +4 O +. O +6 O +± O +4 O +μm O +; O +Fig O +. O +2C O +). O + +Taken O +together O +, O +these O +data O +suggest O +that O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +sufficient O +for O +maximal O +binding O +and O +that O +this O +binding O +is O +of O +a O +relatively O +low O +affinity O +compared O +with O +many O +other O +Cdc42 B-protein +· O +effector O +complexes O +. O + +The O +Cdc42 B-complex_assembly +- I-complex_assembly +HR1 I-complex_assembly +interaction O +is O +of O +low O +affinity O +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +protein O +and O +in O +TOCA1 B-protein +paralogues O +. O + +A O +, O +diagram O +illustrating O +the O +TOCA1 B-protein +constructs O +assayed O +for O +Cdc42 B-protein +binding O +. O + +Domain O +boundaries O +are O +derived O +from O +secondary O +structure O +predictions O +; O +B O +, O +binding B-evidence +curves I-evidence +derived O +from O +direct B-experimental_method +binding I-experimental_method +assays I-experimental_method +, O +in O +which O +the O +indicated O +concentrations O +of O +Cdc42Δ7Q61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +were O +incubated B-experimental_method +with O +30 O +nm O +GST B-mutant +- I-mutant +ACK I-mutant +or O +His B-protein_state +- I-protein_state +tagged I-protein_state +TOCA1 B-protein +constructs O +, O +as O +indicated O +, O +in O +SPAs B-experimental_method +. O + +C O +– O +E O +, O +representative O +examples O +of O +competition B-experimental_method +SPA I-experimental_method +experiments O +carried O +out O +with O +the O +indicated O +concentrations O +of O +the O +TOCA1 B-protein +HR1 B-mutant +- I-mutant +SH3 I-mutant +construct O +titrated B-experimental_method +into O +30 O +nm O +GST B-mutant +- I-mutant +ACK I-mutant +and O +30 O +nm O +Cdc42Δ7Q61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +( O +C O +) O +or O +HR1CIP4 B-structure_element +( O +D O +) O +or O +HR1FBP17 B-structure_element +( O +E O +) O +titrated B-experimental_method +into O +30 O +nm O +GST B-mutant +- I-mutant +ACK I-mutant +and O +30 O +nm O +Cdc42FLQ61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +. O + +The O +HR1 B-structure_element +domains O +from O +FBP17 B-protein +and O +CIP4 B-protein +were O +purified B-experimental_method +and O +assayed O +for O +Cdc42 B-protein +binding O +in O +competition B-experimental_method +SPAs I-experimental_method +, O +analogous O +to O +those O +carried O +out O +with O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +. O + +Structure B-evidence +of O +the O +TOCA1 B-protein +HR1 B-structure_element +Domain O + +Because O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +was O +sufficient O +for O +maximal O +Cdc42 B-protein +- O +binding O +, O +we O +used O +this O +construct O +for O +structural O +studies O +. O + +2 O +, O +778 O +non O +- O +degenerate O +NOE B-evidence +restraints I-evidence +were O +used O +in O +initial O +structure B-experimental_method +calculations I-experimental_method +( O +1 O +, O +791 O +unambiguous O +and O +987 O +ambiguous O +), O +derived O +from O +three O +- O +dimensional O +15N B-experimental_method +- I-experimental_method +separated I-experimental_method +NOESY I-experimental_method +and O +13C B-experimental_method +- I-experimental_method +separated I-experimental_method +NOESY I-experimental_method +experiments O +. O + +There O +were O +1 O +, O +845 O +unambiguous O +NOEs B-evidence +and O +757 O +ambiguous O +NOEs B-evidence +after O +eight O +iterations O +. O + +Table O +1 O +indicates O +that O +the O +HR1 B-structure_element +domain O +structure B-evidence +is O +well O +defined O +by O +the O +NMR B-experimental_method +data O +. O + +a O +< O +SA O +>, O +the O +average B-evidence +root I-evidence +mean I-evidence +square I-evidence +deviations I-evidence +for O +the O +ensemble O +± O +S O +. O +D O +. O + +b O +< O +SA O +> O +c O +, O +values O +for O +the O +structure B-evidence +that O +is O +closest O +to O +the O +mean O +. O + +The O +structure B-evidence +closest O +to O +the O +mean O +is O +shown O +in O +Fig O +. O +3A O +. O + +A O +sequence B-experimental_method +alignment I-experimental_method +illustrating O +the O +secondary O +structure O +elements O +of O +the O +TOCA1 B-protein +and O +CIP4 B-protein +HR1 B-structure_element +domains O +and O +the O +HR1a B-structure_element +and O +HR1b B-structure_element +domains O +from O +PRK1 B-protein +is O +shown O +in O +Fig O +. O +3B O +. O + +Flexible O +regions O +at O +the O +N O +and O +C O +termini O +( O +residues O +330 B-residue_range +– I-residue_range +333 I-residue_range +and O +421 B-residue_range +– I-residue_range +426 I-residue_range +) O +are O +omitted O +for O +clarity O +. O + +B O +, O +a O +sequence B-experimental_method +alignment I-experimental_method +of O +the O +HR1 B-structure_element +domains O +from O +TOCA1 B-protein +, O +CIP4 B-protein +, O +and O +PRK1 B-protein +. O + +The O +secondary O +structure O +was O +deduced O +using O +Stride B-experimental_method +, O +based O +on O +the O +Ramachandran B-evidence +angles I-evidence +, O +and O +is O +indicated O +as O +follows O +: O +gray O +, O +turn O +; O +yellow O +, O +α B-structure_element +- I-structure_element +helix I-structure_element +; O +blue O +, O +310 B-structure_element +helix I-structure_element +; O +white O +, O +coil O +. O + +C O +, O +a O +close O +- O +up O +of O +the O +N O +- O +terminal O +region O +of O +TOCA1 B-protein +HR1 B-structure_element +, O +indicating O +some O +of O +the O +NOEs B-evidence +defining O +its O +position O +with O +respect O +to O +the O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +Dotted O +lines O +, O +NOE B-evidence +restraints I-evidence +. O + +In O +the O +HR1a B-structure_element +domain O +of O +PRK1 B-protein +, O +a O +region O +N O +- O +terminal O +to O +helix B-structure_element +1 I-structure_element +forms O +a O +short B-structure_element +α I-structure_element +- I-structure_element +helix I-structure_element +, O +which O +packs O +against O +both O +helices O +of O +the O +HR1 B-structure_element +domain O +. O + +This O +region O +of O +TOCA1 B-protein +HR1 B-structure_element +( O +residues O +334 B-residue_range +– I-residue_range +340 I-residue_range +) O +is O +well O +defined O +in O +the O +family O +of O +structures B-evidence +( O +Fig O +. O +3A O +) O +but O +does O +not O +form O +an O +α B-structure_element +- I-structure_element +helix I-structure_element +. O + +It O +instead O +forms O +a O +series O +of O +turns O +, O +defined O +by O +NOE B-evidence +restraints I-evidence +observed O +between O +residues O +separated O +by O +one O +( O +residues O +332 B-residue_range +– I-residue_range +334 I-residue_range +, O +333 B-residue_range +– I-residue_range +335 I-residue_range +, O +etc O +.) O +or O +two O +( O +residues O +337 B-residue_range +– I-residue_range +340 I-residue_range +) O +residues O +in O +the O +sequence O +and O +the O +φ B-evidence +and I-evidence +ψ I-evidence +angles I-evidence +, O +assessed O +using O +Stride B-experimental_method +. O + +These O +turns O +cause O +the O +chain O +to O +reverse O +direction O +, O +allowing O +the O +N O +- O +terminal O +segment O +( O +residues O +334 B-residue_range +– I-residue_range +340 I-residue_range +) O +to O +contact O +both O +helices O +of O +the O +HR1 B-structure_element +domain O +. O + +The O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +of O +TOCA1 B-protein +HR1 B-structure_element +are O +separated O +by O +a O +long O +loop B-structure_element +of O +10 O +residues O +( O +residues O +380 B-residue_range +– I-residue_range +389 I-residue_range +) O +that O +contains O +two O +short B-structure_element +310 I-structure_element +helices I-structure_element +( O +residues O +381 B-residue_range +– I-residue_range +383 I-residue_range +and O +386 B-residue_range +– I-residue_range +389 I-residue_range +). O + +Interestingly O +, O +side O +chains O +of O +residues O +within O +the O +loop B-structure_element +region I-structure_element +point O +back O +toward O +helix B-structure_element +1 I-structure_element +; O +for O +example O +, O +there O +are O +numerous O +distinct O +NOEs O +between O +the O +side O +chains O +of O +Asn B-residue_name_number +- I-residue_name_number +380 I-residue_name_number +and O +Met B-residue_name_number +- I-residue_name_number +383 I-residue_name_number +of O +the O +loop B-structure_element +region I-structure_element +and O +Tyr B-residue_name_number +- I-residue_name_number +377 I-residue_name_number +and O +Val B-residue_name_number +- I-residue_name_number +376 I-residue_name_number +of O +helix B-structure_element +1 I-structure_element +( O +Fig O +. O +3D O +). O + +The O +backbone O +NH O +and O +CHα O +groups O +of O +Gly B-residue_name_number +- I-residue_name_number +384 I-residue_name_number +and O +Asp B-residue_name_number +- I-residue_name_number +385 I-residue_name_number +also O +show O +NOEs O +with O +the O +side O +chain O +of O +Tyr B-residue_name_number +- I-residue_name_number +377 I-residue_name_number +. O + +Mapping O +the O +TOCA1 B-protein +and O +Cdc42 B-site +Binding I-site +Interfaces I-site + +The O +HR1TOCA1 B-site +- I-site +Cdc42 I-site +interface I-site +was O +investigated O +using O +NMR B-experimental_method +spectroscopy I-experimental_method +. O + +A O +series O +of O +15N B-experimental_method +HSQC I-experimental_method +experiments O +was O +recorded O +on O +15N B-chemical +- O +labeled B-protein_state +TOCA1 B-protein +HR1 B-structure_element +domain O +in O +the O +presence B-protein_state +of I-protein_state +increasing B-experimental_method +concentrations I-experimental_method +of O +unlabeled B-protein_state +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +to O +map O +the O +Cdc42 B-site +- I-site +binding I-site +surface I-site +. O + +A O +comparison O +of O +the O +15N B-experimental_method +HSQC I-experimental_method +spectra B-evidence +of O +free B-protein_state +HR1 B-structure_element +and O +HR1 B-structure_element +in O +the O +presence B-protein_state +of I-protein_state +excess O +Cdc42 B-protein +shows O +that O +although O +some O +peaks O +were O +shifted O +, O +several O +were O +much O +broader O +in O +the O +complex O +, O +and O +a O +considerable O +subset O +had O +disappeared O +( O +Fig O +. O +4A O +). O + +This O +behavior O +cannot O +be O +explained O +by O +the O +increase O +in O +molecular O +mass O +( O +from O +12 O +to O +33 O +kDa O +) O +when O +Cdc42 B-protein +binds O +and O +is O +more O +likely O +to O +be O +due O +to O +conformational O +exchange O +. O + +A O +peak O +that O +disappeared O +or O +had O +a O +CSP B-experimental_method +above O +the O +mean O +CSP B-experimental_method +for O +the O +spectrum O +was O +considered O +to O +be O +significantly O +affected O +. O + +A O +, O +the O +15N B-experimental_method +HSQC I-experimental_method +of O +200 O +μm O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +shown O +in O +the O +free B-protein_state +form I-protein_state +( O +black O +) O +and O +in O +the O +presence B-protein_state +of I-protein_state +a O +4 O +- O +fold O +molar O +excess O +of O +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +( O +red O +). O + +B O +, O +CSPs B-experimental_method +were O +calculated O +as O +described O +under O +“ O +Experimental O +Procedures O +” O +and O +are O +shown O +for O +backbone O +and O +side O +chain O +NH O +groups O +. O + +The O +mean O +CSP B-experimental_method +is O +marked O +with O +a O +red O +line O +. O + +Those O +that O +were O +not O +traceable O +due O +to O +spectral O +overlap O +were O +assigned O +a O +CSP B-experimental_method +of O +zero O +and O +are O +marked O +with O +an O +asterisk O +below O +the O +bar O +. O + +Residues O +with O +affected O +side O +chain O +CSPs B-experimental_method +derived O +from O +13C B-experimental_method +HSQCs I-experimental_method +are O +marked O +with O +green O +asterisks O +above O +the O +bars O +. O + +C O +, O +a O +schematic O +representation O +of O +the O +HR1 B-structure_element +domain O +. O + +Residues O +with O +significantly O +affected O +backbone O +or O +side O +chain O +chemical O +shifts O +when O +Cdc42 B-protein_state +bound I-protein_state +and O +that O +are O +buried O +are O +colored O +dark O +blue O +, O +whereas O +those O +that O +are O +solvent B-protein_state +- I-protein_state +accessible I-protein_state +are O +colored O +yellow O +. O + +Residues O +with O +significantly O +affected O +backbone O +and O +side O +chain O +groups O +that O +are O +solvent B-protein_state +- I-protein_state +accessible I-protein_state +are O +colored O +red O +. O + +A O +close O +- O +up O +of O +the O +binding B-site +region I-site +is O +shown O +, O +with O +affected O +side O +chain O +heavy O +atoms O +shown O +as O +sticks O +. O + +D O +, O +the O +G B-site +protein I-site +- I-site +binding I-site +region I-site +is O +marked O +in O +red O +onto O +structures B-evidence +of O +the O +HR1 B-structure_element +domains O +as O +indicated O +. O + +15N B-experimental_method +HSQC I-experimental_method +shift I-experimental_method +mapping I-experimental_method +experiments O +report O +on O +changes O +to O +amide O +groups O +, O +which O +are O +mainly O +inaccessible O +because O +they O +are O +buried O +inside O +the O +helices B-structure_element +and O +are O +involved O +in O +hydrogen B-bond_interaction +bonds I-bond_interaction +. O + +Therefore O +, O +13C B-experimental_method +HSQC I-experimental_method +and O +methyl B-experimental_method +- I-experimental_method +selective I-experimental_method +SOFAST I-experimental_method +- I-experimental_method +HMQC I-experimental_method +experiments O +were O +also O +recorded O +on O +15N B-chemical +, O +13C B-chemical +- O +labeled B-protein_state +TOCA1 B-protein +HR1 B-structure_element +to O +yield O +more O +information O +on O +side O +chain O +involvement O +. O + +The O +changes O +were O +localized O +to O +one O +end O +of O +the O +coiled B-structure_element +- I-structure_element +coil I-structure_element +, O +and O +the O +binding B-site +site I-site +appeared O +to O +include O +residues O +from O +both O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +the O +loop B-structure_element +region I-structure_element +that O +joins O +them O +. O + +The O +residues O +in O +the O +interhelical B-structure_element +loop I-structure_element +and O +helix B-structure_element +1 I-structure_element +that O +contact O +each O +other O +( O +Fig O +. O +3D O +) O +show O +shift O +changes O +in O +their O +backbone O +NH O +and O +side O +chains O +in O +the O +presence B-protein_state +of I-protein_state +Cdc42 B-protein +. O + +For O +example O +, O +the O +side O +chain O +of O +Asn B-residue_name_number +- I-residue_name_number +380 I-residue_name_number +and O +the O +backbones O +of O +Val B-residue_name_number +- I-residue_name_number +376 I-residue_name_number +and O +Tyr B-residue_name_number +- I-residue_name_number +377 I-residue_name_number +were O +significantly O +affected O +but O +are O +all O +buried O +in O +the O +free B-protein_state +TOCA1 B-protein +HR1 B-structure_element +structure B-evidence +, O +indicating O +that O +local O +conformational O +changes O +in O +the O +loop B-structure_element +may O +facilitate O +complex O +formation O +. O + +The O +chemical B-experimental_method +shift I-experimental_method +mapping I-experimental_method +data O +indicate O +that O +the O +G B-site +protein I-site +- I-site +binding I-site +region I-site +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +broadly O +similar O +to O +that O +of O +the O +CIP4 B-protein +and O +PRK1 B-protein +HR1 B-structure_element +domains O +( O +Figs O +. O +3B O +and O +4D O +). O + +The O +overall O +CSP B-experimental_method +was O +calculated O +for O +each O +residue O +. O + +Detailed O +side O +chain O +data O +could O +not O +be O +obtained O +for O +all O +residues O +due O +to O +spectral O +overlap O +, O +but O +constant B-experimental_method +time I-experimental_method +13C I-experimental_method +HSQC I-experimental_method +and O +methyl B-experimental_method +- I-experimental_method +selective I-experimental_method +SOFAST I-experimental_method +- I-experimental_method +HMQC I-experimental_method +experiments O +provided O +further O +information O +on O +certain O +well O +resolved O +side O +chains O +( O +marked O +with O +green O +asterisks O +in O +Fig O +. O +5B O +). O + +B O +, O +CSPs B-experimental_method +are O +shown O +for O +backbone O +NH O +groups O +. O + +The O +red O +line O +indicates O +the O +mean O +CSP B-experimental_method +, O +plus O +one O +S O +. O +D O +. O +Residues O +that O +disappeared O +in O +the O +presence B-protein_state +of I-protein_state +Cdc42 B-protein +were O +assigned O +a O +CSP B-experimental_method +of O +0 O +. O +1 O +and O +are O +indicated O +with O +open O +bars O +. O + +C O +, O +the O +residues O +with O +significantly O +affected O +backbone O +and O +side O +chain O +groups O +are O +highlighted O +on O +an O +NMR B-experimental_method +structure B-evidence +of O +free B-protein_state +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +; O +those O +that O +are O +buried O +are O +colored O +dark O +blue O +, O +whereas O +those O +that O +are O +solvent B-protein_state +- I-protein_state +accessible I-protein_state +are O +colored O +red O +. O + +Residues O +without O +information O +from O +shift B-experimental_method +mapping I-experimental_method +are O +colored O +gray O +. O + +As O +many O +of O +the O +peaks O +disappeared O +, O +the O +mean B-evidence +chemical I-evidence +shift I-evidence +change I-evidence +was O +relatively O +low O +, O +so O +a O +threshold O +of O +the O +mean O +plus O +one O +S O +. O +D O +. O +value O +was O +used O +to O +define O +a O +significant O +CSP B-experimental_method +. O + +Parts O +of O +the O +switch B-site +regions I-site +( O +Fig O +. O +5 O +, O +B O +and O +C O +) O +are O +invisible O +in O +NMR B-experimental_method +spectra B-evidence +recorded O +on O +free B-protein_state +Cdc42 B-protein +due O +to O +conformational O +exchange O +. O + +These O +switch B-site +regions I-site +become O +visible O +in O +Cdc42 B-protein +and O +other O +small O +G B-protein_type +protein I-protein_type +· O +effector O +complexes O +due O +to O +a O +decrease O +in O +conformational O +freedom O +upon O +complex O +formation O +. O + +Indeed O +, O +Ser B-residue_name_number +- I-residue_name_number +30 I-residue_name_number +of O +switch B-site +I I-site +and O +Arg B-residue_name_number +- I-residue_name_number +66 I-residue_name_number +, O +Arg B-residue_name_number +- I-residue_name_number +68 I-residue_name_number +, O +Leu B-residue_name_number +- I-residue_name_number +70 I-residue_name_number +, O +and O +Ser B-residue_name_number +- I-residue_name_number +71 I-residue_name_number +of O +switch B-site +II I-site +are O +visible O +in O +free B-protein_state +Cdc42 B-protein +but O +disappear O +in O +the O +presence B-protein_state +of I-protein_state +the O +HR1 B-structure_element +domain O +. O + +This O +suggests O +that O +the O +switch B-site +regions I-site +are O +not O +rigidified O +in O +the O +HR1 B-structure_element +complex O +and O +are O +still O +in O +conformational O +exchange O +. O + +Nevertheless O +, O +mapping O +of O +the O +affected O +residues O +onto O +the O +NMR B-experimental_method +structure B-evidence +of O +free B-protein_state +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +( O +Fig O +. O +5C O +) O +8 O +shows O +that O +, O +although O +they O +are O +relatively O +widespread O +compared O +with O +changes O +in O +the O +HR1 B-structure_element +domain O +, O +in O +general O +, O +they O +are O +on O +the O +face O +of O +the O +protein O +that O +includes O +the O +switches B-site +. O + +Modeling O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +HR1 I-complex_assembly +Complex O + +HADDOCK B-experimental_method +was O +therefore O +used O +to O +perform O +rigid B-experimental_method +body I-experimental_method +docking I-experimental_method +based O +on O +the O +structures B-evidence +of O +free B-protein_state +HR1 B-structure_element +domain O +and O +Cdc42 B-protein +and O +ambiguous O +interaction O +restraints O +derived O +from O +the O +titration B-experimental_method +experiments I-experimental_method +described O +above O +. O + +The O +orientation O +of O +the O +HR1 B-structure_element +domain O +with O +respect O +to O +Cdc42 B-protein +cannot O +be O +definitively O +concluded O +in O +the O +absence O +of O +unambiguous O +distance O +restraints O +; O +hence O +, O +HADDOCK B-experimental_method +produced O +a O +set O +of O +models O +in O +which O +the O +HR1 B-structure_element +domain O +contacts O +the O +same O +surface O +on O +Cdc42 B-protein +but O +is O +in O +various O +orientations O +with O +respect O +to O +Cdc42 B-protein +. O + +A O +representative O +model O +from O +this O +cluster O +is O +shown O +in O +Fig O +. O +6A O +alongside O +the O +Rac1 B-complex_assembly +- I-complex_assembly +HR1b I-complex_assembly +structure B-evidence +( O +PDB O +code O +2RMK O +) O +in O +Fig O +. O +6B O +. O + +A O +, O +a O +representative O +model O +of O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1 I-complex_assembly +complex O +from O +the O +cluster O +closest O +to O +the O +lowest O +energy O +model O +produced O +using O +HADDOCK B-experimental_method +. O + +Residues O +of O +Cdc42 B-protein +that O +are O +affected O +in O +the O +presence B-protein_state +of I-protein_state +the O +HR1 B-structure_element +domain O +but O +are O +not O +in O +close O +proximity O +to O +it O +are O +colored O +in O +red O +and O +labeled O +. O + +B O +, O +structure B-evidence +of O +Rac1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +HR1b B-structure_element +domain O +of O +PRK1 B-protein +( O +PDB O +code O +2RMK O +). O + +Contact O +residues O +of O +RhoA B-protein +and O +Rac1 B-protein +to O +PRK1 B-protein +HR1a B-structure_element +and O +HR1b B-structure_element +, O +respectively O +, O +are O +colored O +cyan O +. O + +Residues O +equivalent O +to O +Rac1 B-protein +and O +RhoA B-protein +contact B-site +sites I-site +but O +that O +are O +invisible O +in O +free B-protein_state +Cdc42 B-protein +are O +gray O +. O + +The O +four O +lowest O +energy O +structures B-evidence +in O +the O +chosen O +HADDOCK B-experimental_method +cluster O +are O +shown O +overlaid O +, O +with O +the O +residues O +of O +interest O +shown O +as O +sticks O +and O +labeled O +. O + +Cdc42 O +is O +shown O +in O +cyan O +, O +and O +TOCA1 B-protein +is O +shown O +in O +purple O +. O + +A O +sequence B-experimental_method +alignment I-experimental_method +of O +RhoA B-protein +, O +Cdc42 B-protein +, O +and O +Rac1 B-protein +is O +shown O +in O +Fig O +. O +6C O +. O + +The O +RhoA B-protein +and O +Rac1 B-protein +contact O +residues O +in O +the O +switch B-site +regions I-site +are O +invisible O +in O +the O +spectra B-evidence +of O +Cdc42 B-protein +, O +but O +they O +are O +generally O +conserved B-protein_state +between O +all O +three O +G B-protein_type +proteins I-protein_type +. O + +Several O +Cdc42 B-protein +residues O +identified O +by O +chemical B-experimental_method +shift I-experimental_method +mapping I-experimental_method +are O +not O +in O +close O +contact O +in O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +model O +( O +Fig O +. O +6A O +). O + +Other O +residues O +that O +are O +affected O +in O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +complex O +but O +that O +do O +not O +correspond O +to O +contact O +residues O +of O +RhoA B-protein +or O +Rac1 B-protein +( O +Fig O +. O +6C O +) O +include O +Gln B-residue_name_number +- I-residue_name_number +2Cdc42 I-residue_name_number +, O +Lys B-residue_name_number +- I-residue_name_number +16Cdc42 I-residue_name_number +, O +Thr B-residue_name_number +- I-residue_name_number +52Cdc42 I-residue_name_number +, O +and O +Arg B-residue_name_number +- I-residue_name_number +68Cdc42 I-residue_name_number +. O + +Competition O +between O +N B-protein +- I-protein +WASP I-protein +and O +TOCA1 B-protein + +From O +the O +known O +interactions O +and O +effects O +of O +the O +proteins O +in O +biological O +systems O +, O +it O +has O +been O +suggested O +that O +TOCA1 B-protein +and O +N B-protein +- I-protein +WASP I-protein +could O +bind O +Cdc42 B-protein +simultaneously O +. O + +An O +overlay B-experimental_method +of O +the O +HADDOCK B-experimental_method +model B-evidence +of O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1TOCA1 I-complex_assembly +complex O +and O +the O +structure B-evidence +of O +Cdc42 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +GBD B-structure_element +of O +the O +N B-protein +- I-protein +WASP I-protein +homologue O +, O +WASP B-protein +( O +PDB O +code O +1CEE O +), O +shows O +that O +the O +HR1 B-structure_element +and O +GBD B-site +binding I-site +sites I-site +only O +partly O +overlap O +, O +and O +, O +therefore O +, O +a O +ternary O +complex O +remained O +possible O +( O +Fig O +. O +7A O +). O + +Interestingly O +, O +the O +presence B-protein_state +of I-protein_state +the O +TOCA1 B-protein +HR1 B-structure_element +would O +not O +prevent O +the O +core O +CRIB B-structure_element +of O +WASP B-protein +from O +binding O +to O +Cdc42 B-protein +, O +although O +the O +regions O +C O +- O +terminal O +to O +the O +CRIB B-structure_element +that O +are O +required O +for O +high O +affinity O +binding O +of O +WASP B-protein +would O +interfere O +sterically O +with O +the O +TOCA1 B-protein +HR1 B-structure_element +. O + +The O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +displaces O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +. O + +A O +, O +the O +model O +of O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +HR1 B-structure_element +domain O +complex O +overlaid O +with O +the O +Cdc42 B-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +structure B-evidence +. O + +Cdc42 O +is O +shown O +in O +green O +, O +and O +TOCA1 B-protein +is O +shown O +in O +purple O +. O + +A O +semitransparent O +surface O +representation O +of O +Cdc42 B-protein +and O +WASP B-protein +is O +shown O +overlaid O +with O +the O +schematic O +. O + +B O +, O +competition B-experimental_method +SPA I-experimental_method +experiments O +carried O +out O +with O +indicated O +concentrations O +of O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +construct O +titrated B-experimental_method +into O +30 O +nm O +GST B-mutant +- I-mutant +ACK I-mutant +or O +GST B-mutant +- I-mutant +WASP I-mutant +GBD B-structure_element +and O +30 O +nm O +Cdc42Δ7Q61L B-complex_assembly +·[ I-complex_assembly +3H I-complex_assembly +] I-complex_assembly +GTP I-complex_assembly +. O + +C O +, O +Selected O +regions O +of O +the O +15N B-experimental_method +HSQC I-experimental_method +of O +145 O +μm O +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +with O +the O +indicated O +ratios O +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +, O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +, O +or O +both O +, O +showing O +that O +the O +TOCA B-protein +HR1 B-structure_element +domain O +does O +not O +displace O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +. O + +The O +Kd B-evidence +that O +was O +determined O +( O +37 O +nm O +) O +is O +consistent O +with O +the O +previously O +reported O +affinity B-evidence +. O + +Unlabeled B-protein_state +HR1TOCA1 B-structure_element +was O +then O +added O +to O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +N I-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +complex O +, O +and O +no O +changes O +were O +seen O +, O +suggesting O +that O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +was O +not O +displaced O +even O +in O +the O +presence B-protein_state +of I-protein_state +a O +5 O +- O +fold O +excess O +of O +HR1TOCA1 B-structure_element +. O + +Furthermore O +, O +15N B-chemical +- O +TOCA1 B-protein +HR1 B-structure_element +was O +monitored O +in O +the O +presence B-protein_state +of I-protein_state +unlabeled B-protein_state +Cdc42Δ7Q61L B-complex_assembly +· I-complex_assembly +GMPPNP I-complex_assembly +( O +1 O +: O +1 O +) O +before O +and O +after O +the O +addition O +of O +0 O +. O +25 O +and O +1 O +. O +0 O +eq O +of O +unlabeled B-protein_state +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +. O + +When O +in O +fast O +exchange O +, O +the O +NMR B-experimental_method +signal O +represents O +a O +population O +- O +weighted O +average O +between O +free B-protein_state +and O +bound B-protein_state +states O +, O +so O +the O +intermediate O +spectrum B-evidence +indicates O +that O +the O +population O +comprises O +a O +mixture O +of O +free B-protein_state +and O +bound B-protein_state +HR1 B-structure_element +domain O +. O + +Again O +, O +the O +experiments O +were O +recorded O +on O +protein O +samples O +far O +in O +excess O +of O +the O +individual O +Kd B-evidence +values O +( O +600 O +μm O +each O +protein O +). O + +These O +data O +indicate O +that O +the O +HR1 B-structure_element +domain O +is O +displaced O +from O +Cdc42 B-protein +by O +N B-protein +- I-protein +WASP I-protein +and O +that O +a O +ternary O +complex O +comprising O +TOCA1 B-protein +HR1 B-structure_element +, O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +, O +and O +Cdc42 B-protein +is O +not O +formed O +. O + +Taken O +together O +, O +the O +data O +in O +Fig O +. O +7 O +, O +C O +and O +D O +, O +indicate O +unidirectional O +competition O +for O +Cdc42 B-protein +binding O +in O +which O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +displaces O +TOCA1 B-protein +HR1 B-structure_element +but O +not O +vice O +versa O +. O + +To O +extend O +these O +studies O +to O +a O +more O +complex O +system O +and O +to O +assess O +the O +ability O +of O +TOCA1 B-protein +HR1 B-structure_element +to O +compete O +with O +full B-protein_state +- I-protein_state +length I-protein_state +N B-protein +- I-protein +WASP I-protein +, O +pyrene B-experimental_method +actin I-experimental_method +assays I-experimental_method +were O +employed O +. O + +These O +assays O +, O +described O +in O +detail O +elsewhere O +, O +were O +carried O +out O +using O +pyrene B-chemical +actin I-chemical +- O +supplemented O +Xenopus B-taxonomy_domain +extracts O +into O +which O +exogenous O +TOCA1 B-protein +HR1 B-structure_element +domain O +or O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +was O +added O +, O +to O +assess O +their O +effects O +on O +actin B-protein_type +polymerization O +. O + +Endogenous O +N B-protein +- I-protein +WASP I-protein +is O +present O +at O +∼ O +100 O +nm O +in O +Xenopus B-taxonomy_domain +extracts O +, O +whereas O +TOCA1 B-protein +is O +present O +at O +a O +10 O +- O +fold O +lower O +concentration O +than O +N B-protein +- I-protein +WASP I-protein +. O + +The O +addition B-experimental_method +of O +the O +isolated O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +significantly O +inhibited O +the O +polymerization O +of O +actin B-protein_type +at O +concentrations O +as O +low O +as O +100 O +nm O +and O +completely O +abolished O +polymerization O +at O +higher O +concentrations O +( O +Fig O +. O +8 O +). O + +The O +addition B-experimental_method +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +to O +100 O +μm O +had O +no O +significant O +effect O +on O +the O +rate O +of O +actin B-protein_type +polymerization O +or O +maximum B-evidence +fluorescence I-evidence +. O + +This O +is O +consistent O +with O +endogenous B-protein_state +N B-protein +- I-protein +WASP I-protein +, O +activated O +by O +other O +components O +of O +the O +assay O +, O +outcompeting O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +for O +Cdc42 B-protein +binding O +. O + +Actin O +polymerization O +downstream O +of O +Cdc42 B-complex_assembly +· I-complex_assembly +N I-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +is O +inhibited B-protein_state +by O +excess O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +but O +not O +by O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +. O + +Fluorescence B-evidence +curves I-evidence +show O +actin O +polymerization O +in O +the O +presence B-protein_state +of I-protein_state +increasing B-experimental_method +concentrations I-experimental_method +of O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +or O +TOCA1 B-protein +HR1 B-structure_element +domain O +as O +indicated O +. O + +The O +Cdc42 B-protein +- O +TOCA1 B-protein +Interaction O + +The O +TOCA1 B-protein +HR1 B-structure_element +domain O +alone B-protein_state +is O +sufficient O +for O +Cdc42 B-protein +binding O +in O +vitro O +, O +yet O +the O +affinity B-evidence +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +for O +Cdc42 B-protein +is O +remarkably O +low O +( O +Kd B-evidence +≈ O +5 O +μm O +). O + +A O +single O +binding B-site +interface I-site +on O +both O +the O +HR1 B-structure_element +domain O +and O +Cdc42 B-protein +can O +be O +concluded O +from O +the O +data O +presented O +here O +. O + +Furthermore O +, O +the O +interfaces B-site +are O +comparable O +with O +those O +of O +other O +G B-protein_type +protein I-protein_type +- O +HR1 B-structure_element +interactions O +( O +Fig O +. O +4 O +), O +and O +the O +lowest O +energy O +model B-evidence +produced O +in O +rigid B-experimental_method +body I-experimental_method +docking I-experimental_method +resembles O +previously O +studied O +G B-complex_assembly +protein I-complex_assembly +· I-complex_assembly +HR1 I-complex_assembly +complexes O +( O +Fig O +. O +6 O +). O + +It O +seems O +, O +therefore O +, O +that O +the O +interaction O +, O +despite O +its O +relatively O +low O +affinity O +, O +is O +specific O +and O +sterically O +similar O +to O +other O +HR1 B-structure_element +domain O +- O +G B-protein_type +protein I-protein_type +interactions O +. O + +A O +short O +region O +N O +- O +terminal O +to O +the O +coiled B-structure_element +- I-structure_element +coil I-structure_element +exhibits O +a O +series O +of O +turns O +and O +contacts O +residues O +of O +both O +helices O +of O +the O +coiled B-structure_element +- I-structure_element +coil I-structure_element +( O +Fig O +. O +3 O +). O + +The O +corresponding O +sequence O +in O +CIP4 B-protein +also O +includes O +a O +series O +of O +turns O +but O +is O +flexible O +, O +whereas O +in O +the O +HR1a B-structure_element +domain O +of O +PRK1 B-protein +, O +the O +equivalent O +region O +adopts O +an O +α B-structure_element +- I-structure_element +helical I-structure_element +structure I-structure_element +that O +packs O +against O +the O +coiled B-structure_element +- I-structure_element +coil I-structure_element +. O + +The O +interhelical B-structure_element +loops I-structure_element +of O +TOCA1 B-protein +and O +CIP4 B-protein +differ O +from O +the O +same O +region O +in O +the O +HR1 B-structure_element +domains O +of O +PRK1 B-protein +in O +that O +they O +are O +longer O +and O +contain O +two O +short O +stretches O +of O +310 B-structure_element +- I-structure_element +helix I-structure_element +. O + +This O +region O +lies O +within O +the O +G B-site +protein I-site +- I-site +binding I-site +surface I-site +of O +all O +of O +the O +HR1 B-structure_element +domains O +( O +Fig O +. O +4D O +). O + +TOCA1 B-protein +and O +CIP4 B-protein +both O +bind O +weakly O +to O +Cdc42 B-protein +, O +whereas O +the O +HR1a B-structure_element +domain O +of O +PRK1 B-protein +binds O +tightly O +to O +RhoA B-protein +and O +Rac1 B-protein +, O +and O +the O +HR1b B-structure_element +domain O +binds O +to O +Rac1 B-protein +. O + +The O +structural O +features O +shared O +by O +TOCA1 B-protein +and O +CIP4 B-protein +may O +therefore O +be O +related O +to O +Cdc42 B-protein +binding O +specificity O +and O +the O +low O +affinities O +. O + +In O +free B-protein_state +TOCA1 B-protein +, O +the O +side O +chains O +of O +the O +interhelical B-structure_element +region I-structure_element +make O +extensive O +contacts O +with O +residues O +in O +helix B-structure_element +1 I-structure_element +. O + +Many O +of O +these O +residues O +are O +significantly O +affected O +in O +the O +presence B-protein_state +of I-protein_state +Cdc42 B-protein +, O +so O +it O +is O +likely O +that O +the O +conformation O +of O +this O +loop B-structure_element +is O +altered O +in O +the O +Cdc42 B-protein +complex O +. O + +These O +observations O +therefore O +provide O +a O +molecular O +mechanism O +whereby O +mutation B-experimental_method +of O +Met383 B-residue_name_number +- O +Gly384 B-residue_name_number +- O +Asp385 B-residue_name_number +to O +Ile383 B-residue_name_number +- O +Ser384 B-residue_name_number +- O +Thr385 B-residue_name_number +abolishes O +TOCA1 B-protein +binding O +to O +Cdc42 B-protein +. O + +The O +lowest O +energy O +model B-evidence +produced O +by O +HADDOCK B-experimental_method +using O +ambiguous O +interaction O +restraints O +from O +the O +titration B-evidence +data O +resembled O +the O +NMR B-experimental_method +structures B-evidence +of O +RhoA B-protein +and O +Rac1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +their O +HR1 B-structure_element +domain O +partners O +. O + +For O +example O +, O +Phe B-residue_name_number +- I-residue_name_number +56Cdc42 I-residue_name_number +, O +which O +is O +not O +visible O +in O +free B-protein_state +Cdc42 B-protein +or O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1TOCA1 I-complex_assembly +, O +is O +close O +to O +the O +TOCA1 B-protein +HR1 B-structure_element +( O +Fig O +. O +6A O +). O + +Phe B-residue_name_number +- I-residue_name_number +56Cdc42 I-residue_name_number +, O +which O +is O +a O +Trp B-residue_name +in O +both O +Rac1 B-protein +and O +RhoA B-protein +( O +Fig O +. O +6C O +), O +is O +thought O +to O +pack O +behind O +switch B-site +I I-site +when O +Cdc42 B-protein +interacts O +with O +ACK B-protein +, O +maintaining O +the O +switch O +in O +a O +binding O +- O +competent O +orientation O +. O + +This O +residue O +has O +also O +been O +identified O +as O +important O +for O +Cdc42 B-protein +- O +WASP B-protein +binding O +. O + +Phe B-residue_name_number +- I-residue_name_number +56Cdc42 I-residue_name_number +is O +therefore O +likely O +to O +be O +involved O +in O +the O +Cdc42 B-protein +- O +TOCA1 B-protein +interaction O +, O +probably O +by O +stabilizing O +the O +position O +of O +switch B-site +I I-site +. O + +Gln B-residue_name_number +- I-residue_name_number +2Cdc42 I-residue_name_number +, O +which O +has O +also O +been O +identified O +as O +a O +contact O +residue O +in O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +ACK I-complex_assembly +complex O +, O +contacts O +Val B-residue_name_number +- I-residue_name_number +376TOCA1 I-residue_name_number +and O +Asn B-residue_name_number +- I-residue_name_number +380TOCA1 I-residue_name_number +in O +the O +model O +and O +disrupts O +the O +contacts O +between O +the O +interhelical B-structure_element +loop I-structure_element +and O +the O +first B-structure_element +helix I-structure_element +of O +the O +TOCA1 B-protein +coiled B-structure_element +- I-structure_element +coil I-structure_element +. O + +The O +position O +equivalent O +to O +Lys B-residue_name_number +- I-residue_name_number +372TOCA1 I-residue_name_number +in O +PRK1 B-protein +is O +Glu B-residue_name_number +- I-residue_name_number +58HR1a I-residue_name_number +or O +Gln B-residue_name_number +- I-residue_name_number +151HR1b I-residue_name_number +. O + +Thr B-residue_name_number +- I-residue_name_number +52Cdc42 I-residue_name_number +- O +Lys B-residue_name_number +- I-residue_name_number +372TOCA1 I-residue_name_number +may O +therefore O +represent O +a O +specific O +Cdc42 B-protein +- O +HR1TOCA1 B-structure_element +contact O +. O + +Arg B-residue_name_number +- I-residue_name_number +68Cdc42 I-residue_name_number +of O +switch B-site +II I-site +is O +positioned O +close O +to O +Glu B-residue_name_number +- I-residue_name_number +395TOCA1 I-residue_name_number +( O +Fig O +. O +6D O +), O +suggesting O +a O +direct O +electrostatic O +contact O +between O +switch B-site +II I-site +of O +Cdc42 B-protein +and O +helix B-structure_element +2 I-structure_element +of O +the O +HR1 B-structure_element +domain O +. O + +The O +equivalent O +Arg B-residue_name +in O +Rac1 B-protein +and O +RhoA B-protein +is O +pointing O +away O +from O +the O +HR1 B-structure_element +domains O +of O +PRK1 B-protein +. O + +The O +solution B-evidence +structure I-evidence +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +presented O +here O +, O +along O +with O +the O +model O +of O +the O +HR1TOCA1 B-complex_assembly +· I-complex_assembly +Cdc42 I-complex_assembly +complex O +is O +consistent O +with O +a O +conserved O +mode O +of O +binding O +across O +the O +known O +HR1 B-structure_element +domain O +- O +Rho O +family O +interactions O +, O +despite O +their O +differing O +affinities O +. O + +The O +weak O +binding O +prevented O +detailed O +structural B-experimental_method +and I-experimental_method +thermodynamic I-experimental_method +studies I-experimental_method +of O +the O +complex O +. O + +We O +have O +previously O +postulated O +that O +the O +inherent O +flexibility O +of O +HR1 B-structure_element +domains O +contributes O +to O +their O +ability O +to O +bind O +to O +different O +Rho B-protein_type +family I-protein_type +G I-protein_type +proteins I-protein_type +, O +with O +Rho O +- O +binding O +HR1 B-structure_element +domains O +displaying O +increased O +flexibility O +, O +reflected O +in O +their O +lower O +melting B-evidence +temperatures I-evidence +( O +Tm B-evidence +) O +and O +Rac B-protein_type +binders O +being O +more O +rigid O +. O + +The O +Tm B-evidence +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +61 O +. O +9 O +° O +C O +( O +data O +not O +shown O +), O +which O +is O +the O +highest O +Tm B-evidence +that O +we O +have O +measured O +for O +an O +HR1 B-structure_element +domain O +thus O +far O +. O + +As O +such O +, O +the O +ability O +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +to O +bind O +to O +Cdc42 B-protein +( O +a O +close O +relative O +of O +Rac1 B-protein +rather O +than O +RhoA B-protein +) O +fits O +this O +trend O +. O + +The O +low O +affinity O +of O +the O +Cdc42 B-protein +- O +HR1TOCA1 B-structure_element +interaction O +is O +consistent O +with O +a O +tightly O +spatially O +and O +temporally O +regulated O +pathway O +, O +requiring O +combinatorial O +signals O +leading O +to O +a O +series O +of O +coincident O +weak O +interactions O +that O +elicit O +full O +activation O +. O + +The O +HR1 B-structure_element +domains O +from O +other O +TOCA B-protein_type +family I-protein_type +members I-protein_type +, O +CIP4 B-protein +and O +FBP17 B-protein +, O +also O +bind O +at O +low O +micromolar O +affinities O +to O +Cdc42 B-protein +, O +so O +the O +low O +affinity O +interaction O +appears O +to O +be O +commonplace O +among O +this O +family O +of O +HR1 B-protein_type +domain I-protein_type +proteins I-protein_type +, O +in O +contrast O +to O +the O +PRK B-protein_type +family I-protein_type +. O + +The O +low O +affinity O +of O +the O +HR1TOCA1 B-structure_element +- O +Cdc42 B-protein +interaction O +in O +the O +context O +of O +the O +physiological O +concentration O +of O +TOCA1 B-protein +in O +Xenopus B-taxonomy_domain +extracts O +(∼ O +10 O +nm O +) O +suggests O +that O +binding O +between O +TOCA1 B-protein +and O +Cdc42 B-protein +is O +likely O +to O +occur O +in O +vivo O +only O +when O +TOCA1 B-protein +is O +at O +high O +local O +concentrations O +and O +membrane O +- O +localized O +and O +therefore O +in O +close O +proximity O +to O +activated B-protein_state +Cdc42 B-protein +. O + +For O +example O +, O +electrostatic B-bond_interaction +interactions I-bond_interaction +between O +the O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +and O +the O +membrane O +are O +required O +for O +TOCA1 B-protein +recruitment O +to O +membrane O +vesicles O +and O +tubules O +, O +and O +TOCA1 B-protein +- O +dependent O +actin O +polymerization O +is O +known O +to O +depend O +specifically O +on O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +. O + +Furthermore O +, O +the O +isolated B-experimental_method +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +of O +FBP17 B-protein +has O +been O +shown O +to O +induce O +membrane O +tubulation O +of O +brain O +liposomes O +and O +BAR B-structure_element +domain O +proteins O +that O +promote O +tubulation O +cluster O +on O +membranes O +at O +high O +densities O +. O + +Once O +at O +the O +membrane O +, O +high O +local O +concentrations O +of O +TOCA1 B-protein +could O +exceed O +the O +Kd B-evidence +of O +F B-structure_element +- I-structure_element +BAR I-structure_element +dimerization B-oligomeric_state +( O +likely O +to O +be O +comparable O +with O +that O +of O +the O +FCHo2 B-protein +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +( O +2 O +. O +5 O +μm O +)) O +and O +that O +of O +the O +Cdc42 B-protein +- O +HR1TOCA1 B-structure_element +interaction O +. O + +Cdc42 B-protein +- O +HR1TOCA1 B-structure_element +binding O +would O +then O +be O +favorable O +, O +as O +long O +as O +coincident O +activation O +of O +Cdc42 B-protein +had O +occurred O +, O +leading O +to O +stabilization O +of O +TOCA1 B-protein +at O +the O +membrane O +and O +downstream O +activation O +of O +N B-protein +- I-protein +WASP I-protein +. O + +It O +has O +been O +postulated O +that O +WASP B-protein_type +and O +N B-protein +- I-protein +WASP I-protein +exist O +in O +equilibrium O +between O +folded B-protein_state +( O +inactive B-protein_state +) O +and O +unfolded B-protein_state +( O +active B-protein_state +) O +forms O +, O +and O +the O +affinity B-evidence +of O +Cdc42 B-protein +for O +the O +unfolded B-protein_state +WASP B-protein_type +proteins O +is O +significantly O +enhanced O +. O + +The O +unfolded B-protein_state +, O +high O +affinity O +state O +of O +WASP B-protein_type +is O +represented O +by O +a O +short O +peptide B-chemical +, O +the O +GBD B-structure_element +, O +which O +binds O +with O +a O +low O +nanomolar O +affinity O +to O +Cdc42 B-protein +. O + +In O +contrast O +, O +the O +best O +estimate O +of O +the O +affinity B-evidence +of O +full B-protein_state +- I-protein_state +length I-protein_state +WASP B-protein_type +for O +Cdc42 B-protein +is O +low O +micromolar O +. O + +In O +the O +inactive B-protein_state +state O +of O +WASP B-protein_type +, O +the O +actin O +- O +and O +Arp2 B-complex_assembly +/ I-complex_assembly +3 I-complex_assembly +- O +binding O +VCA B-structure_element +domain O +contacts O +the O +GBD B-structure_element +, O +competing O +for O +Cdc42 B-protein +binding O +. O + +The O +high O +affinity O +of O +Cdc42 B-protein +for O +the O +unfolded B-protein_state +, O +active B-protein_state +form O +pushes O +the O +equilibrium O +in O +favor O +of O +( B-protein +N I-protein +-) I-protein +WASP I-protein +activation O +. O + +Binding O +of O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +to O +the O +basic O +region O +just O +N O +- O +terminal O +to O +the O +GBD B-structure_element +further O +favors O +the O +active B-protein_state +conformation O +. O + +A O +substantial O +body O +of O +data O +has O +illuminated O +the O +complex O +regulation O +of O +WASP B-protein_type +/ I-protein_type +N I-protein_type +- I-protein_type +WASP I-protein_type +proteins I-protein_type +, O +and O +current O +evidence O +suggests O +that O +these O +allosteric O +activation O +mechanisms O +and O +oligomerization O +combine O +to O +regulate O +WASP B-protein_type +activity O +, O +allowing O +the O +synchronization O +and O +integration O +of O +multiple O +potential O +activation O +signals O +( O +reviewed O +in O +Ref O +.). O + +We O +envisage O +that O +TOCA1 B-protein +is O +first O +recruited O +to O +the O +appropriate O +membrane O +in O +response O +to O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +via O +its O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +, O +where O +the O +local O +increase O +in O +concentration O +favors O +F B-structure_element +- I-structure_element +BAR I-structure_element +- O +mediated O +dimerization B-oligomeric_state +of O +TOCA1 B-protein +. O + +Cdc42 B-protein +is O +activated O +in O +response O +to O +co O +- O +incident O +signals O +and O +can O +then O +bind O +to O +TOCA1 B-protein +, O +further O +stabilizing O +TOCA1 B-protein +at O +the O +membrane O +. O + +TOCA1 B-protein +can O +then O +recruit O +N B-protein +- I-protein +WASP I-protein +via O +an O +interaction O +between O +its O +SH3 B-structure_element +domain O +and O +the O +N B-protein +- I-protein +WASP I-protein +proline B-structure_element +- I-structure_element +rich I-structure_element +region I-structure_element +. O + +The O +recruitment O +of O +N B-protein +- I-protein +WASP I-protein +alone B-protein_state +and O +of O +the O +N B-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +WIP I-complex_assembly +complex O +by O +TOCA1 B-protein +and O +FBP17 B-protein +has O +been O +demonstrated O +. O + +It O +may O +therefore O +be O +envisaged O +that O +WIP B-protein +and O +TOCA1 B-protein +exert O +opposing O +allosteric O +effects O +on O +N B-protein +- I-protein +WASP I-protein +, O +with O +TOCA1 B-protein +favoring O +the O +unfolded B-protein_state +, O +active B-protein_state +conformation O +of O +N B-protein +- I-protein +WASP I-protein +and O +increasing O +its O +affinity O +for O +Cdc42 B-protein +. O + +TOCA1 B-protein +may O +also O +activate O +N B-protein +- I-protein +WASP I-protein +by O +effective O +oligomerization O +because O +clustering O +of O +TOCA1 B-protein +at O +the O +membrane O +following O +coincident O +interactions O +with O +PI B-chemical +( I-chemical +4 I-chemical +, I-chemical +5 I-chemical +) I-chemical +P2 I-chemical +and O +Cdc42 B-protein +would O +in O +turn O +lead O +to O +clustering O +of O +N B-protein +- I-protein +WASP I-protein +, O +in O +addition O +to O +pushing O +the O +equilibrium O +toward O +the O +unfolded B-protein_state +, O +active B-protein_state +state O +. O + +In O +a O +cellular O +context O +, O +full B-protein_state +- I-protein_state +length I-protein_state +TOCA1 B-protein +and O +N B-protein +- I-protein +WASP I-protein +are O +likely O +to O +have O +similar O +affinities B-evidence +for O +active B-protein_state +Cdc42 B-protein +, O +but O +in O +the O +unfolded B-protein_state +, O +active B-protein_state +conformation O +, O +the O +affinity B-evidence +of O +N B-protein +- I-protein +WASP I-protein +for O +Cdc42 B-protein +dramatically O +increases O +. O + +Our O +binding B-evidence +data I-evidence +suggest O +that O +TOCA1 B-protein +HR1 B-structure_element +binding O +is O +not O +allosterically O +regulated O +, O +and O +our O +NMR B-experimental_method +data O +, O +along O +with O +the O +high O +stability B-protein_state +of O +TOCA1 B-protein +HR1 B-structure_element +, O +suggest O +that O +there O +is O +no O +widespread O +conformational O +change O +in O +the O +presence B-protein_state +of I-protein_state +Cdc42 B-protein +. O + +As O +full B-protein_state +- I-protein_state +length I-protein_state +TOCA1 B-protein +and O +the O +isolated B-protein_state +HR1 B-structure_element +domain O +bind O +Cdc42 B-protein +with O +similar O +affinities O +, O +the O +N B-protein +- I-protein +WASP I-protein +- O +Cdc42 B-protein +interaction O +will O +be O +favored O +because O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +can O +easily O +outcompete O +the O +TOCA1 B-protein +HR1 B-structure_element +for O +Cdc42 B-protein +. O + +In O +such O +an O +array O +of O +molecules O +localized O +to O +a O +discrete O +region O +of O +the O +membrane O +, O +it O +is O +plausible O +that O +WASP B-protein +could O +bind O +to O +a O +second O +Cdc42 B-protein +molecule O +rather O +than O +displacing O +TOCA1 B-protein +from O +its O +cognate O +Cdc42 B-protein +. O + +Our O +NMR B-experimental_method +and O +affinity B-evidence +data I-evidence +, O +however O +, O +are O +consistent O +with O +displacement O +of O +the O +TOCA1 B-protein +HR1 B-structure_element +by O +the O +N B-protein +- I-protein +WASP I-protein +GBD B-structure_element +. O + +The O +commonly O +used O +MGD B-mutant +→ I-mutant +IST I-mutant +( O +Cdc42 B-protein_state +- I-protein_state +binding I-protein_state +deficient I-protein_state +) O +mutant O +of O +TOCA1 B-protein +has O +a O +reduced O +ability O +to O +activate O +the O +N B-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +WIP I-complex_assembly +complex O +, O +further O +indicating O +the O +importance O +of O +the O +Cdc42 B-protein +- O +HR1TOCA1 B-structure_element +interaction O +prior O +to O +downstream O +activation O +of O +N B-protein +- I-protein +WASP I-protein +. O + +In O +light O +of O +this O +, O +we O +favor O +an O +“ O +effector O +handover O +” O +scheme O +whereby O +TOCA1 B-protein +interacts O +with O +Cdc42 B-protein +prior O +to O +N B-protein +- I-protein +WASP I-protein +activation O +, O +after O +which O +N B-protein +- I-protein +WASP I-protein +displaces O +TOCA1 B-protein +from O +its O +bound B-protein_state +Cdc42 B-protein +in O +order O +to O +be O +fully B-protein_state +activated I-protein_state +rather O +than O +binding O +a O +second O +Cdc42 B-protein +molecule O +. O + +Potentially O +, O +the O +TOCA1 B-protein +- O +Cdc42 B-protein +interaction O +functions O +to O +position O +N B-protein +- I-protein +WASP I-protein +and O +Cdc42 B-protein +such O +that O +they O +are O +poised O +to O +interact O +with O +high O +affinity O +. O + +The O +concomitant O +release O +of O +TOCA1 B-protein +from O +Cdc42 B-protein +while O +still O +bound B-protein_state +to I-protein_state +N B-protein +- I-protein +WASP I-protein +presumably O +enhances O +the O +ability O +of O +TOCA1 B-protein +to O +further O +activate O +N B-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +WIP I-complex_assembly +- O +induced O +actin O +polymerization O +. O + +Hence O +, O +actin O +polymerization O +cannot O +occur O +until O +F B-structure_element +- I-structure_element +BAR I-structure_element +domains O +are O +poised O +for O +membrane O +distortion O +. O + +Our O +model O +of O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1TOCA1 I-complex_assembly +complex O +indicates O +a O +mechanism O +by O +which O +such O +a O +handover O +could O +take O +place O +( O +Fig O +. O +9 O +) O +because O +it O +shows O +that O +the O +effector B-site +binding I-site +sites I-site +only O +partially O +overlap O +on O +Cdc42 B-protein +. O + +The O +lysine B-residue_name +residues O +thought O +to O +be O +involved O +in O +an O +electrostatic O +steering O +mechanism O +in O +WASP B-protein +- O +Cdc42 B-protein +binding O +are O +conserved O +in O +N B-protein +- I-protein +WASP I-protein +and O +would O +be O +able O +to O +interact O +with O +Cdc42 B-protein +even O +when O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +is O +already O +bound B-protein_state +. O + +It O +has O +been O +postulated O +that O +the O +initial O +interactions O +between O +this O +basic O +region O +and O +Cdc42 B-protein +could O +stabilize O +the O +active B-protein_state +conformation O +of O +WASP B-protein +, O +leading O +to O +high O +affinity O +binding O +between O +the O +core O +CRIB B-structure_element +and O +Cdc42 B-protein +. O + +Step O +1 O +, O +TOCA1 B-protein +is O +recruited O +to O +the O +membrane O +via O +its O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +and O +/ O +or O +Cdc42 B-protein +interactions O +. O + +F O +- O +BAR O +oligomerization O +is O +expected O +to O +occur O +following O +membrane O +binding O +, O +but O +a O +single O +monomer B-oligomeric_state +is O +shown O +for O +clarity O +. O + +Step O +2 O +, O +N B-protein +- I-protein +WASP I-protein +exists O +in O +an O +inactive B-protein_state +, O +folded B-protein_state +conformation O +. O + +The O +TOCA1 B-protein +SH3 B-structure_element +domain O +interacts O +with O +N B-protein +- I-protein +WASP I-protein +, O +causing O +an O +activatory O +allosteric O +effect O +. O + +Step O +3 O +, O +electrostatic B-bond_interaction +interactions I-bond_interaction +between O +Cdc42 B-protein +and O +the O +basic O +region O +upstream O +of O +the O +CRIB B-structure_element +initiate O +Cdc42 B-complex_assembly +· I-complex_assembly +N I-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +binding O +. O + +Step O +4 O +, O +the O +core O +CRIB B-structure_element +binds O +with O +high O +affinity O +while O +the O +region O +C O +- O +terminal O +to O +the O +CRIB B-structure_element +displaces O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +and O +increases O +the O +affinity O +of O +the O +N B-protein +- I-protein +WASP I-protein +- O +Cdc42 O +interaction O +further O +. O + +The O +VCA B-structure_element +domain O +is O +released O +for O +downstream O +interactions O +, O +and O +actin O +polymerization O +proceeds O +. O + +WH1 O +, O +WASP B-structure_element +homology I-structure_element +1 I-structure_element +domain I-structure_element +; O +PP B-structure_element +, O +proline B-structure_element +- I-structure_element +rich I-structure_element +region I-structure_element +; O +VCA B-structure_element +, O +verprolin B-structure_element +homology I-structure_element +, I-structure_element +cofilin I-structure_element +homology I-structure_element +, I-structure_element +acidic I-structure_element +region I-structure_element +. O + +The O +analogous O +HR1 B-structure_element +domains O +from O +other O +TOCA1 B-protein_type +family I-protein_type +members O +, O +FBP17 B-protein +and O +CIP4 B-protein +, O +also O +exhibit O +micromolar O +affinity O +for O +Cdc42 B-protein +. O + +A O +role O +for O +the O +TOCA1 B-protein +-, O +FBP17 B-protein +-, O +and O +CIP4 B-protein +- O +Cdc42 B-protein +interactions O +in O +the O +recruitment O +of O +these O +proteins O +to O +the O +membrane O +therefore O +appears O +unlikely O +. O + +Instead O +, O +our O +findings O +agree O +with O +earlier O +suggestions O +that O +the O +F B-structure_element +- I-structure_element +BAR I-structure_element +domain O +is O +responsible O +for O +membrane O +recruitment O +. O + +The O +role O +of O +the O +Cdc42 B-protein +- O +TOCA1 B-protein +interaction O +remains O +somewhat O +elusive O +, O +but O +it O +may O +serve O +to O +position O +activated B-protein_state +Cdc42 B-protein +and O +N B-protein +- I-protein +WASP I-protein +to O +allow O +full B-protein_state +activation I-protein_state +of O +N B-protein +- I-protein +WASP I-protein +and O +as O +such O +serve O +to O +couple O +F B-structure_element +- I-structure_element +BAR I-structure_element +- O +mediated O +membrane O +deformation O +with O +N B-protein +- I-protein +WASP I-protein +activation O +. O + +It O +is O +clear O +from O +the O +data O +presented O +here O +that O +TOCA1 B-protein +and O +N B-protein +- I-protein +WASP I-protein +do O +not O +bind O +Cdc42 B-protein +simultaneously O +and O +that O +N B-protein +- I-protein +WASP I-protein +is O +likely O +to O +outcompete O +TOCA1 B-protein +for O +Cdc42 B-protein +binding O +. O + +We O +therefore O +postulate O +an O +effector O +handover O +mechanism O +based O +on O +current O +evidence O +surrounding O +WASP B-protein +/ O +N B-protein +- I-protein +WASP I-protein +activation O +and O +our O +model O +of O +the O +Cdc42 B-complex_assembly +· I-complex_assembly +HR1TOCA1 I-complex_assembly +complex O +. O + +The O +displacement O +of O +the O +TOCA1 B-protein +HR1 B-structure_element +domain O +from O +Cdc42 B-protein +by O +N B-protein +- I-protein +WASP I-protein +may O +represent O +a O +unidirectional O +step O +in O +the O +pathway O +of O +Cdc42 B-complex_assembly +· I-complex_assembly +N I-complex_assembly +- I-complex_assembly +WASP I-complex_assembly +· I-complex_assembly +TOCA1 I-complex_assembly +- O +dependent O +actin O +assembly O +. O + +The O +dynamic B-protein_state +organization O +of O +fungal B-taxonomy_domain +acetyl B-protein_type +- I-protein_type +CoA I-protein_type +carboxylase I-protein_type + +Here O +we O +report O +the O +crystal B-evidence +structure I-evidence +of O +the O +yeast B-taxonomy_domain +ACC B-protein_type +CD B-structure_element +, O +revealing O +a O +unique O +four O +- O +domain O +organization O +. O + +A O +regulatory B-structure_element +loop I-structure_element +, O +which O +is O +phosphorylated B-protein_state +at O +the O +key O +functional O +phosphorylation B-site +site I-site +of O +fungal B-taxonomy_domain +ACC B-protein_type +, O +wedges O +into O +a O +crevice O +between O +two O +domains O +of O +CD B-structure_element +. O + +In O +contrast O +to O +related O +carboxylases B-protein_type +, O +large O +- O +scale O +conformational O +changes O +are O +required O +for O +substrate O +turnover O +, O +and O +are O +mediated O +by O +the O +CD B-structure_element +under O +phosphorylation B-ptm +control O +. O + +Acetyl B-protein_type +- I-protein_type +CoA I-protein_type +carboxylases I-protein_type +are O +central O +regulatory O +hubs O +of O +fatty O +acid O +metabolism O +and O +are O +important O +targets O +for O +drug O +development O +in O +obesity O +and O +cancer O +. O + +Here O +, O +the O +authors O +demonstrate O +that O +the O +regulation O +of O +these O +highly B-protein_state +dynamic I-protein_state +enzymes B-protein_type +in O +fungi B-taxonomy_domain +is O +governed O +by O +a O +mechanism O +based O +on O +phosphorylation B-ptm +- O +dependent O +conformational O +variability O +. O + +By O +catalysing O +this O +rate O +- O +limiting O +step O +in O +fatty O +- O +acid O +biosynthesis O +, O +ACC B-protein_type +plays O +a O +key O +role O +in O +anabolic O +metabolism O +. O + +Furthermore O +, O +elevated O +ACC B-protein_type +activity O +is O +observed O +in O +malignant O +tumours O +. O + +A O +direct O +link O +between O +ACC B-protein_type +and O +cancer O +is O +provided O +by O +cancer O +- O +associated O +mutations B-mutant +in O +the O +breast B-protein +cancer I-protein +susceptibility I-protein +gene I-protein +1 I-protein +( O +BRCA1 B-protein +), O +which O +relieve O +inhibitory O +interactions O +of O +BRCA1 B-protein +with O +ACC B-protein_type +. O + +Thus O +, O +ACC B-protein_type +is O +a O +relevant O +drug O +target O +for O +type O +2 O +diabetes O +and O +cancer O +. O + +Microbial B-taxonomy_domain +ACCs B-protein_type +are O +also O +the O +principal O +target O +of O +antifungal O +and O +antibiotic O +compounds O +, O +such O +as O +Soraphen B-chemical +A I-chemical +. O + +Carboxyltransferase B-protein_type +( O +CT B-protein_type +) O +transfers O +the O +activated O +carboxyl B-chemical +group O +from O +carboxybiotin B-chemical +to O +acetyl B-chemical +- I-chemical +CoA I-chemical +to O +yield O +malonyl B-chemical +- I-chemical +CoA I-chemical +. O +Prokaryotic B-taxonomy_domain +ACCs B-protein_type +are O +transient B-protein_state +assemblies O +of O +individual O +BC B-protein_type +, O +CT B-protein_type +and O +BCCP B-protein_type +subunits O +. O + +Eukaryotic B-taxonomy_domain +ACCs B-protein_type +, O +instead O +, O +are O +multienzymes B-protein_type +, O +which O +integrate O +all O +functional O +components O +into O +a O +single O +polypeptide O +chain O +of O +∼ O +2 O +, O +300 O +amino O +acids O +. O + +Human B-species +ACC B-protein_type +occurs O +in O +two O +closely O +related O +isoforms B-protein_state +, O +ACC1 B-protein +and O +2 B-protein +, O +located O +in O +the O +cytosol O +and O +at O +the O +outer O +mitochondrial O +membrane O +, O +respectively O +. O + +In O +addition O +to O +the O +canonical O +ACC B-structure_element +components I-structure_element +, O +eukaryotic B-taxonomy_domain +ACCs B-protein_type +contain O +two O +non B-protein_state +- I-protein_state +catalytic I-protein_state +regions B-structure_element +, O +the O +large O +central B-structure_element +domain I-structure_element +( O +CD B-structure_element +) O +and O +the O +BC B-structure_element +– I-structure_element +CT I-structure_element +interaction I-structure_element +domain I-structure_element +( O +BT B-structure_element +). O + +The O +BT B-structure_element +domain O +has O +been O +visualized O +in O +bacterial B-taxonomy_domain +carboxylases B-protein_type +, O +where O +it O +mediates O +contacts O +between O +α B-structure_element +- I-structure_element +and O +β B-structure_element +- I-structure_element +subunits I-structure_element +. O + +Structural B-experimental_method +studies I-experimental_method +on O +the O +functional O +architecture O +of O +intact B-protein_state +ACCs B-protein_type +have O +been O +hindered O +by O +their O +huge O +size O +and O +pronounced O +dynamics O +, O +as O +well O +as O +the O +transient B-protein_state +assembly O +mode O +of O +bacterial B-taxonomy_domain +ACCs B-protein_type +. O + +However O +, O +crystal B-evidence +structures I-evidence +of O +individual O +components O +or O +domains O +from O +prokaryotic B-taxonomy_domain +and O +eukaryotic B-taxonomy_domain +ACCs B-protein_type +, O +respectively O +, O +have O +been O +solved O +. O + +The O +structure B-experimental_method +determination I-experimental_method +of O +the O +holoenzymes B-protein_state +of O +bacterial B-taxonomy_domain +biotin B-protein_type +- I-protein_type +dependent I-protein_type +carboxylases I-protein_type +, O +which O +lack B-protein_state +the O +characteristic O +CD B-structure_element +, O +such O +as O +the O +pyruvate B-protein_type +carboxylase I-protein_type +( O +PC B-protein_type +), O +propionyl B-protein_type +- I-protein_type +CoA I-protein_type +carboxylase I-protein_type +, O +3 B-protein_type +- I-protein_type +methyl I-protein_type +- I-protein_type +crotonyl I-protein_type +- I-protein_type +CoA I-protein_type +carboxylase I-protein_type +and O +a O +long B-protein_type +- I-protein_type +chain I-protein_type +acyl I-protein_type +- I-protein_type +CoA I-protein_type +carboxylase I-protein_type +revealed O +strikingly O +divergent O +architectures O +despite O +a O +general O +conservation O +of O +all O +functional O +components O +. O + +Human B-species +ACC1 B-protein +is O +regulated B-protein_state +allosterically I-protein_state +, O +via O +specific O +protein O +– O +protein O +interactions O +, O +and O +by O +reversible O +phosphorylation B-ptm +. O + +Dynamic O +polymerization O +of O +human B-species +ACC1 B-protein +is O +linked O +to O +increased O +activity O +and O +is O +regulated B-protein_state +allosterically I-protein_state +by O +the O +activator O +citrate B-chemical +and O +the O +inhibitor O +palmitate B-chemical +, O +or O +by O +binding O +of O +the O +small O +protein O +MIG B-protein +- I-protein +12 I-protein +( O +ref O +.). O + +Human B-species +ACC1 B-protein +is O +further O +regulated O +by O +specific O +phosphorylation B-ptm +- O +dependent O +binding O +of O +BRCA1 B-protein +to O +Ser1263 B-residue_name_number +in O +the O +CD B-structure_element +. O + +Furthermore O +, O +phosphorylation B-ptm +by O +AMP B-protein +- I-protein +activated I-protein +protein I-protein +kinase I-protein +( O +AMPK B-protein +) O +and O +cAMP B-protein +- I-protein +dependent I-protein +protein I-protein +kinase I-protein +( O +PKA B-protein +) O +leads O +to O +a O +decrease O +in O +ACC1 B-protein +activity O +. O + +AMPK B-protein +phosphorylates O +ACC1 B-protein +in O +vitro O +at O +Ser80 B-residue_name_number +, O +Ser1201 B-residue_name_number +and O +Ser1216 B-residue_name_number +and O +PKA B-protein +at O +Ser78 B-residue_name_number +and O +Ser1201 B-residue_name_number +. O + +However O +, O +regulatory O +effects O +on O +ACC1 B-protein +activity O +are O +mainly O +mediated O +by O +phosphorylation B-ptm +of O +Ser80 B-residue_name_number +and O +Ser1201 B-residue_name_number +( O +refs O +). O + +Phosphorylated B-protein_state +Ser80 B-residue_name_number +, O +which O +is O +highly B-protein_state +conserved I-protein_state +only O +in O +higher B-taxonomy_domain +eukaryotes I-taxonomy_domain +, O +presumably O +binds O +into O +the O +Soraphen B-site +A I-site +- I-site +binding I-site +pocket I-site +. O + +The O +regulatory O +Ser1201 B-residue_name_number +shows O +only O +moderate B-protein_state +conservation I-protein_state +across O +higher B-taxonomy_domain +eukaryotes I-taxonomy_domain +, O +while O +the O +phosphorylated B-protein_state +Ser1216 B-residue_name_number +is O +highly B-protein_state +conserved I-protein_state +across O +all O +eukaryotes B-taxonomy_domain +. O + +However O +, O +no O +effect O +of O +Ser1216 B-residue_name_number +phosphorylation B-ptm +on O +ACC B-protein_type +activity O +has O +been O +reported O +in O +higher B-taxonomy_domain +eukaryotes I-taxonomy_domain +. O + +The O +BRCA1 B-protein +- O +interacting O +phosphoserine B-residue_name +position O +is O +not B-protein_state +conserved I-protein_state +in O +fungal B-taxonomy_domain +ACC B-protein_type +, O +and O +no O +other O +phospho O +- O +dependent O +protein O +– O +protein O +interactions O +of O +fungal B-taxonomy_domain +ACC B-protein_type +have O +been O +described O +. O + +In O +yeast B-taxonomy_domain +ACC B-protein_type +, O +phosphorylation B-site +sites I-site +have O +been O +identified O +at O +Ser2 B-residue_name_number +, O +Ser735 B-residue_name_number +, O +Ser1148 B-residue_name_number +, O +Ser1157 B-residue_name_number +and O +Ser1162 B-residue_name_number +( O +ref O +.). O + +Of O +these O +, O +only O +Ser1157 B-residue_name_number +is O +highly B-protein_state +conserved I-protein_state +in O +fungal B-taxonomy_domain +ACC B-protein_type +and O +aligns B-experimental_method +to I-experimental_method +Ser1216 B-residue_name_number +in O +human B-species +ACC1 B-protein +. O + +Its O +phosphorylation B-ptm +by O +the O +AMPK B-protein +homologue O +SNF1 B-protein +results O +in O +strongly O +reduced O +ACC B-protein_type +activity O +. O + +Here O +we O +provide O +the O +structure B-evidence +of O +Saccharomyces B-species +cerevisiae I-species +( O +Sce B-species +) O +ACC B-protein_type +CD B-structure_element +, O +intermediate O +- O +and O +low O +- O +resolution O +structures B-evidence +of O +human B-species +( O +Hsa B-species +) O +ACC B-protein_type +CD B-structure_element +and O +larger B-mutant +fragments I-mutant +of O +fungal B-taxonomy_domain +ACC B-protein_type +from O +Chaetomium B-species +thermophilum I-species +( O +Cth B-species +; O +Fig O +. O +1a O +). O + +The O +organization O +of O +the O +yeast B-taxonomy_domain +ACC B-protein_type +CD B-structure_element + +First O +, O +we O +focused O +on O +structure B-experimental_method +determination I-experimental_method +of O +the O +82 O +- O +kDa O +CD B-structure_element +. O + +The O +overall O +extent O +of O +the O +SceCD B-species +is O +70 O +by O +75 O +Å O +( O +Fig O +. O +1b O +and O +Supplementary O +Fig O +. O +1a O +, O +b O +), O +and O +the O +attachment O +points O +of O +the O +N O +- O +terminal O +26 B-structure_element +- I-structure_element +residue I-structure_element +linker I-structure_element +to O +the O +BCCP B-structure_element +domain O +and O +the O +C O +- O +terminal O +CT B-structure_element +domain O +are O +separated O +by O +46 O +Å O +( O +the O +N O +- O +and O +C O +termini O +are O +indicated O +with O +spheres O +in O +Fig O +. O +1b O +). O + +SceCD B-species +comprises O +four O +distinct O +domains O +, O +an O +N O +- O +terminal O +α B-structure_element +- I-structure_element +helical I-structure_element +domain I-structure_element +( O +CDN B-structure_element +), O +and O +a O +central O +four B-structure_element +- I-structure_element +helix I-structure_element +bundle I-structure_element +linker I-structure_element +domain I-structure_element +( O +CDL B-structure_element +), O +followed O +by O +two O +α B-structure_element +– I-structure_element +β I-structure_element +- I-structure_element +fold I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +domains I-structure_element +( O +CDC1 B-structure_element +/ O +CDC2 B-structure_element +). O + +CDN B-structure_element +adopts O +a O +letter O +C B-protein_state +shape I-protein_state +, O +where O +one O +of O +the O +ends O +is O +a O +regular B-structure_element +four I-structure_element +- I-structure_element +helix I-structure_element +bundle I-structure_element +( O +Nα3 B-structure_element +- I-structure_element +6 I-structure_element +), O +the O +other O +end O +is O +a O +helical B-structure_element +hairpin I-structure_element +( O +Nα8 B-structure_element +, I-structure_element +9 I-structure_element +) O +and O +the O +bridging B-structure_element +region I-structure_element +comprises O +six O +helices B-structure_element +( O +Nα1 B-structure_element +, I-structure_element +2 I-structure_element +, I-structure_element +7 I-structure_element +, I-structure_element +10 I-structure_element +– I-structure_element +12 I-structure_element +). O + +CDL B-structure_element +does O +not O +interact O +with O +CDN B-structure_element +apart O +from O +the O +covalent O +linkage O +and O +forms O +only O +a O +small O +contact O +to O +CDC2 B-structure_element +via O +a O +loop B-structure_element +between O +Lα2 B-structure_element +/ I-structure_element +α3 I-structure_element +and O +the O +N O +- O +terminal O +end O +of O +Lα1 B-structure_element +, O +with O +an O +interface B-site +area O +of O +400 O +Å2 O +. O + +CDC1 B-structure_element +/ O +CDC2 B-structure_element +share O +a O +common O +fold O +; O +they O +are O +composed O +of O +six B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheets I-structure_element +flanked O +on O +one O +side O +by O +two O +long B-structure_element +, I-structure_element +bent I-structure_element +helices I-structure_element +inserted O +between O +strands B-structure_element +β3 B-structure_element +/ I-structure_element +β4 I-structure_element +and O +β4 B-structure_element +/ I-structure_element +β5 I-structure_element +. O + +CDC2 B-structure_element +is O +extended B-protein_state +at O +its O +C O +terminus O +by O +an O +additional O +β B-structure_element +- I-structure_element +strand I-structure_element +and O +an O +irregular B-structure_element +β I-structure_element +- I-structure_element +hairpin I-structure_element +. O + +On O +the O +basis O +of O +a O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +of O +main O +chain O +atom O +positions O +of O +2 O +. O +2 O +Å O +, O +CDC1 B-structure_element +/ O +CDC2 B-structure_element +are O +structurally O +more O +closely O +related O +to O +each O +other O +than O +to O +any O +other O +protein O +( O +Fig O +. O +1c O +); O +they O +may O +thus O +have O +evolved O +by O +duplication O +. O + +Close O +structural O +homologues O +could O +not O +be O +found O +for O +the O +CDN B-structure_element +or O +the O +CDC B-structure_element +domains O +. O + +MS B-experimental_method +analysis O +of O +dissolved B-experimental_method +crystals I-experimental_method +confirmed O +the O +phosphorylated B-protein_state +state O +of O +Ser1157 B-residue_name_number +also O +in O +SceCD B-species +crystals B-evidence +. O + +In O +the O +SceCD B-species +crystal B-evidence +structure I-evidence +, O +the O +phosphorylated B-protein_state +Ser1157 B-residue_name_number +resides O +in O +a O +regulatory B-structure_element +36 I-structure_element +- I-structure_element +amino I-structure_element +- I-structure_element +acid I-structure_element +loop I-structure_element +between O +strands B-structure_element +β2 B-structure_element +and O +β3 B-structure_element +of O +CDC1 B-structure_element +( O +Fig O +. O +1b O +, O +d O +), O +which O +contains O +two O +additional O +less B-protein_state +- I-protein_state +conserved I-protein_state +phosphorylation B-site +sites I-site +( O +Ser1148 B-residue_name_number +and O +Ser1162 B-residue_name_number +) O +confirmed O +in O +yeast B-taxonomy_domain +, O +but O +not O +occupied O +here O +. O + +This O +regulatory B-structure_element +loop I-structure_element +wedges O +between O +the O +CDC1 B-structure_element +and O +CDC2 B-structure_element +domains O +and O +provides O +the O +largest O +contribution O +to O +the O +interdomain B-site +interface I-site +. O + +The O +N O +- O +terminal O +region O +of O +the O +regulatory B-structure_element +loop I-structure_element +also O +directly O +contacts O +the O +C O +- O +terminal O +region O +of O +CDC2 B-structure_element +leading O +into O +CT B-structure_element +. O + +Phosphoserine B-residue_name_number +1157 I-residue_name_number +is O +tightly O +bound O +by O +two O +highly B-protein_state +conserved I-protein_state +arginines B-residue_name +( O +Arg1173 B-residue_name_number +and O +Arg1260 B-residue_name_number +) O +of O +CDC1 B-structure_element +( O +Fig O +. O +1d O +). O + +Phosphorylation B-ptm +of O +the O +regulatory B-structure_element +loop I-structure_element +thus O +determines O +interdomain O +interactions O +of O +CDC1 B-structure_element +and O +CDC2 B-structure_element +, O +suggesting O +that O +it O +may O +exert O +its O +regulatory O +function O +by O +modifying O +the O +overall O +structure O +and O +dynamics O +of O +the O +CD B-structure_element +. O + +The O +functional O +role O +of O +Ser1157 B-residue_name_number +was O +confirmed O +by O +an O +activity B-experimental_method +assay I-experimental_method +based O +on O +the O +incorporation O +of O +radioactive O +carbonate O +into O +acid O +non O +- O +volatile O +material O +. O + +Phosphorylated B-protein_state +SceACC B-protein +shows O +only O +residual O +activity O +( O +kcat B-evidence += O +0 O +. O +4 O +± O +0 O +. O +2 O +s O +− O +1 O +, O +s O +. O +d O +. O +based O +on O +five O +replicate O +measurements O +), O +which O +increases O +16 O +- O +fold O +( O +kcat B-evidence += O +6 O +. O +5 O +± O +0 O +. O +3 O +s O +− O +1 O +) O +after O +dephosphorylation O +with O +λ B-protein_type +protein I-protein_type +phosphatase I-protein_type +. O + +The O +values O +obtained O +for O +dephosphorylated B-protein_state +SceACC B-protein +are O +comparable O +to O +earlier O +measurements O +of O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +yeast B-taxonomy_domain +ACC B-protein_type +expressed B-experimental_method +in I-experimental_method +E B-species +. I-species +coli I-species +. O + +The O +variable O +CD B-structure_element +is O +conserved B-protein_state +between O +yeast B-taxonomy_domain +and O +human B-species + +To O +compare O +the O +organization O +of O +fungal B-taxonomy_domain +and O +human B-species +ACC B-protein_type +CD B-structure_element +, O +we O +determined B-experimental_method +the I-experimental_method +structure I-experimental_method +of O +a O +human B-species +ACC1 B-mutant +fragment I-mutant +that O +comprises O +the O +BT B-structure_element +and O +CD B-structure_element +domains O +( O +HsaBT B-mutant +- I-mutant +CD I-mutant +), O +but O +lacks B-protein_state +the O +mobile O +BCCP B-structure_element +in O +between O +( O +Fig O +. O +1a O +). O + +An O +experimentally B-evidence +phased I-evidence +map I-evidence +was O +obtained O +at O +3 O +. O +7 O +Å O +resolution O +for O +a O +cadmium B-chemical +- O +derivatized O +crystal O +and O +was O +interpreted O +by O +a O +poly O +- O +alanine O +model O +( O +Fig O +. O +1e O +and O +Table O +1 O +). O + +Each O +of O +the O +four O +CD B-structure_element +domains O +in O +HsaBT B-mutant +- I-mutant +CD I-mutant +individually O +resembles O +the O +corresponding O +SceCD B-species +domain O +; O +however O +, O +human B-species +and O +yeast B-taxonomy_domain +CDs B-structure_element +exhibit O +distinct O +overall O +structures B-evidence +. O + +In O +agreement O +with O +their O +tight O +interaction O +in O +SceCD B-species +, O +the O +relative O +spatial O +arrangement O +of O +CDL B-structure_element +and O +CDC1 B-structure_element +is O +preserved O +in O +HsaBT B-mutant +- I-mutant +CD I-mutant +, O +but O +the O +human B-species +CDL B-structure_element +/ O +CDC1 B-structure_element +didomain O +is O +tilted O +by O +30 O +° O +based O +on O +a O +superposition B-experimental_method +of O +human B-species +and O +yeast B-taxonomy_domain +CDC2 B-structure_element +( O +Supplementary O +Fig O +. O +1c O +). O + +As O +a O +result O +, O +the O +N O +terminus O +of O +CDL B-structure_element +at O +helix B-structure_element +Lα1 B-structure_element +, O +which O +connects O +to O +CDN B-structure_element +, O +is O +shifted O +by O +12 O +Å O +. O +Remarkably O +, O +CDN B-structure_element +of O +HsaBT B-mutant +- I-mutant +CD I-mutant +adopts O +a O +completely O +different O +orientation O +compared O +with O +SceCD B-species +. O + +With O +CDL B-structure_element +/ O +CDC1 B-structure_element +superposed B-experimental_method +, O +CDN B-structure_element +in O +HsaBT B-mutant +- I-mutant +CD I-mutant +is O +rotated O +by O +160 O +° O +around O +a O +hinge B-structure_element +at O +the O +connection O +of O +CDN B-structure_element +/ O +CDL B-structure_element +( O +Supplementary O +Fig O +. O +1d O +). O + +This O +rotation O +displaces O +the O +N O +terminus O +of O +CDN B-structure_element +in O +HsaBT B-mutant +- I-mutant +CD I-mutant +by O +51 O +Å O +compared O +with O +SceCD B-species +, O +resulting O +in O +a O +separation O +of O +the O +attachment O +points O +of O +the O +N O +- O +terminal O +linker B-structure_element +to O +the O +BCCP B-structure_element +domain I-structure_element +and O +the O +C O +- O +terminal O +CT B-structure_element +domain O +by O +67 O +Å O +( O +the O +attachment O +points O +are O +indicated O +with O +spheres O +in O +Fig O +. O +1e O +). O + +The O +BT B-structure_element +domain O +of O +HsaBT B-mutant +- I-mutant +CD I-mutant +consists O +of O +a O +helix B-structure_element +that O +is O +surrounded O +at O +its O +N O +terminus O +by O +an O +antiparallel B-structure_element +eight I-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +barrel I-structure_element +. O + +It O +resembles O +the O +BT B-structure_element +of O +propionyl B-protein_type +- I-protein_type +CoA I-protein_type +carboxylase I-protein_type +; O +only O +the O +four O +C O +- O +terminal O +strands B-structure_element +of I-structure_element +the I-structure_element +β I-structure_element +- I-structure_element +barrel I-structure_element +are O +slightly O +tilted O +. O + +The O +highly B-protein_state +conserved I-protein_state +Ser1216 B-residue_name_number +( O +corresponding O +to O +S B-species +. I-species +cerevisiae I-species +Ser1157 B-residue_name_number +), O +as O +well O +as O +Ser1201 B-residue_name_number +, O +both O +in O +the O +regulatory B-structure_element +loop I-structure_element +discussed O +above O +, O +are O +not B-protein_state +phosphorylated I-protein_state +. O + +MS B-experimental_method +analysis O +of O +the O +HsaBT B-mutant +- I-mutant +CD I-mutant +crystallization B-evidence +sample I-evidence +reveals O +partial O +proteolytic O +digestion O +of O +the O +regulatory B-structure_element +loop I-structure_element +. O + +The O +absence B-protein_state +of I-protein_state +the O +regulatory B-structure_element +loop I-structure_element +might O +be O +linked O +to O +the O +less B-protein_state +- I-protein_state +restrained I-protein_state +interface B-site +of O +CDL B-structure_element +/ O +CDC1 B-structure_element +and O +CDC2 B-structure_element +and O +altered O +relative O +orientations O +of O +these O +domains B-structure_element +. O + +At O +the O +level O +of O +isolated B-experimental_method +yeast B-taxonomy_domain +and O +human B-species +CD B-structure_element +, O +the O +structural B-experimental_method +analysis I-experimental_method +indicates O +the O +presence O +of O +at O +least O +two O +hinges B-structure_element +, O +one O +with O +large O +- O +scale O +flexibility O +at O +the O +CDN B-structure_element +/ I-structure_element +CDL I-structure_element +connection I-structure_element +, O +and O +one O +with O +tunable O +plasticity O +between O +CDL B-structure_element +/ O +CDC1 B-structure_element +and O +CDC2 B-structure_element +, O +plausibly O +affected O +by O +phosphorylation B-ptm +in O +the O +regulatory B-structure_element +loop I-structure_element +region O +. O + +The O +integration O +of O +CD B-structure_element +into O +the O +fungal B-taxonomy_domain +ACC B-protein_type +multienzyme I-protein_type + +No O +crystals O +diffracting O +to O +sufficient O +resolution O +were O +obtained O +for O +larger B-mutant +BC I-mutant +- I-mutant +containing I-mutant +fragments I-mutant +, O +or O +for O +full B-protein_state +- I-protein_state +length I-protein_state +Cth B-species +or O +SceACC B-protein +. O + +To O +improve B-experimental_method +crystallizability I-experimental_method +, O +we O +generated B-experimental_method +ΔBCCP B-mutant +variants I-mutant +of O +full B-protein_state +- I-protein_state +length I-protein_state +ACC B-protein_type +, O +which O +, O +based O +on O +SAXS B-experimental_method +analysis I-experimental_method +, O +preserve O +properties O +of O +intact B-protein_state +ACC B-protein_type +( O +Supplementary O +Table O +1 O +and O +Supplementary O +Fig O +. O +2a O +– O +c O +). O + +For O +CthΔBCCP B-mutant +, O +crystals B-evidence +diffracting O +to O +8 O +. O +4 O +Å O +resolution O +were O +obtained O +. O + +However O +, O +molecular B-experimental_method +replacement I-experimental_method +did O +not O +reveal O +a O +unique O +positioning O +of O +the O +BC B-structure_element +domain O +. O + +Still O +, O +these B-evidence +structures I-evidence +contribute O +considerably O +to O +the O +visualization O +of O +an O +intrinsically O +dynamic B-protein_state +fungal B-taxonomy_domain +ACC B-protein_type +. O + +In O +all O +these O +crystal B-evidence +structures I-evidence +, O +the O +CT B-structure_element +domains O +build O +a O +canonical O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +dimer B-oligomeric_state +, O +with O +active B-site +sites I-site +formed O +by O +contributions O +from O +both O +protomers B-oligomeric_state +( O +Fig O +. O +2 O +and O +Supplementary O +Fig O +. O +3a O +). O + +The O +connection B-structure_element +of O +CD B-structure_element +and O +CT B-structure_element +is O +provided O +by O +a O +10 B-residue_range +- I-residue_range +residue I-residue_range +peptide I-residue_range +stretch I-residue_range +, O +which O +links O +the O +N O +terminus O +of O +CT B-structure_element +to O +the O +irregular B-structure_element +β I-structure_element +- I-structure_element +hairpin I-structure_element +/ I-structure_element +β I-structure_element +- I-structure_element +strand I-structure_element +extension I-structure_element +of O +CDC2 B-structure_element +( O +Supplementary O +Fig O +. O +3b O +). O + +The O +connecting B-structure_element +region I-structure_element +is O +remarkably O +similar O +in O +isolated B-protein_state +CD B-structure_element +and O +CthCD B-mutant +- I-mutant +CTCter I-mutant +structures B-evidence +, O +indicating O +inherent O +conformational O +stability O +. O + +CD B-structure_element +/ O +CT B-structure_element +contacts O +are O +only O +formed O +in O +direct O +vicinity O +of O +the O +covalent O +linkage O +and O +involve O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +extension I-structure_element +of O +CDC2 B-structure_element +as O +well O +as O +the O +loop B-structure_element +between O +strands B-structure_element +β2 I-structure_element +/ I-structure_element +β3 I-structure_element +of O +the O +CT B-structure_element +N I-structure_element +- I-structure_element +lobe I-structure_element +, O +which O +contains O +a O +conserved B-protein_state +RxxGxN B-structure_element +motif I-structure_element +. O + +The O +neighbouring O +loop B-structure_element +on O +the O +CT B-structure_element +side O +( O +between O +CT B-structure_element +β1 B-structure_element +/ O +β2 B-structure_element +) O +is O +displaced O +by O +2 O +. O +5 O +Å O +compared O +to O +isolated B-protein_state +CT B-structure_element +structures B-evidence +( O +Supplementary O +Fig O +. O +3c O +). O + +Indeed O +, O +the O +comparison O +of O +the O +positioning O +of O +eight O +instances O +of O +the O +C O +- O +terminal O +part O +of O +CD B-structure_element +relative O +to O +CT B-structure_element +in O +crystal B-evidence +structures I-evidence +determined B-experimental_method +here O +, O +reveals O +flexible O +interdomain O +linking O +( O +Fig O +. O +3a O +). O + +The O +CDC2 B-site +/ I-site +CT I-site +interface I-site +acts O +as O +a O +true B-structure_element +hinge I-structure_element +with O +observed O +rotation O +up O +to O +16 O +°, O +which O +results O +in O +a O +translocation O +of O +the O +distal O +end O +of O +CDC2 B-structure_element +by O +8 O +Å O +. O + +The O +interface B-site +between O +CDC2 B-structure_element +and O +CDL B-structure_element +/ O +CDC1 B-structure_element +, O +which O +is O +mediated O +by O +the O +phosphorylated B-protein_state +regulatory B-structure_element +loop I-structure_element +in O +the O +SceCD B-species +structure B-evidence +, O +is O +less O +variable O +than O +the O +CD B-structure_element +– I-structure_element +CT I-structure_element +junction I-structure_element +, O +and O +permits O +only O +limited O +rotation O +and O +tilting O +( O +Fig O +. O +3b O +). O + +Analysis O +of O +the O +impact O +of O +phosphorylation B-ptm +on O +the O +interface B-site +between O +CDC2 B-structure_element +and O +CDL B-structure_element +/ O +CDC1 B-structure_element +in O +CthACC B-mutant +variant I-mutant +structures B-evidence +is O +precluded O +by O +the O +limited O +crystallographic O +resolution O +. O + +The O +CDN B-structure_element +domain O +positioning O +relative O +to O +CDL B-structure_element +/ O +CDC1 B-structure_element +is O +highly O +variable O +with O +three O +main O +orientations O +observed O +in O +the O +structures B-evidence +of O +SceCD B-species +and O +the O +larger B-mutant +CthACC I-mutant +fragments I-mutant +: O +CDN B-structure_element +tilts O +, O +resulting O +in O +a O +displacement O +of O +its O +N O +terminus O +by O +23 O +Å O +( O +Fig O +. O +4a O +, O +observed O +in O +both O +protomers B-oligomeric_state +of O +CthCD B-mutant +- I-mutant +CT I-mutant +and O +one O +protomer B-oligomeric_state +of O +CthΔBCCP B-mutant +, O +denoted O +as O +CthCD B-mutant +- I-mutant +CT1 I-mutant +/ I-mutant +2 I-mutant +and O +CthΔBCCP1 B-mutant +, O +respectively O +). O + +In O +addition O +, O +CDN B-structure_element +can O +rotate O +around O +hinges B-structure_element +in O +the O +connection O +between O +CDN B-structure_element +/ O +CDL B-structure_element +by O +70 O +° O +( O +Fig O +. O +4b O +, O +observed O +in O +the O +second O +protomer B-oligomeric_state +of O +CthΔBCCP B-mutant +, O +denoted O +as O +CthΔBCCP2 B-mutant +) O +and O +160 O +° O +( O +Fig O +. O +4c O +, O +observed O +in O +SceCD B-species +) O +leading O +to O +displacement O +of O +the O +anchor B-site +site I-site +for O +the O +BCCP B-structure_element +linker I-structure_element +by O +up O +to O +33 O +and O +40 O +Å O +, O +respectively O +. O + +Conformational O +variability O +in O +the O +CD B-structure_element +thus O +contributes O +considerably O +to O +variations O +in O +the O +spacing O +between O +the O +BC B-structure_element +and O +CT B-structure_element +domains O +, O +and O +may O +extend O +to O +distance O +variations O +beyond O +the O +mobility O +range O +of O +the O +flexibly B-protein_state +tethered I-protein_state +BCCP B-structure_element +. O + +On O +the O +basis O +of O +the O +occurrence O +of O +related O +conformational O +changes O +between O +fungal B-taxonomy_domain +and O +human B-species +ACC B-mutant +fragments I-mutant +, O +the O +observed O +set O +of O +conformations O +may O +well O +represent O +general O +states O +present O +in O +all O +eukaryotic B-taxonomy_domain +ACCs B-protein_type +. O + +Large O +- O +scale O +conformational O +variability O +of O +fungal B-taxonomy_domain +ACC B-protein_type + +To O +obtain O +a O +comprehensive O +view O +of O +fungal B-taxonomy_domain +ACC B-protein_type +dynamics O +in B-protein_state +solution I-protein_state +, O +we O +employed O +SAXS B-experimental_method +and O +EM B-experimental_method +. O + +The O +smooth O +appearance O +of O +scattering B-evidence +curves I-evidence +and O +derived B-evidence +distance I-evidence +distributions I-evidence +might O +indicate O +substantial O +interdomain O +flexibility O +( O +Supplementary O +Fig O +. O +2a O +– O +c O +). O + +Direct O +observation O +of O +individual O +full B-protein_state +- I-protein_state +length I-protein_state +CthACC B-protein +particles B-evidence +, O +according O +to O +MS B-experimental_method +results O +predominantly O +in O +a O +phosphorylated B-protein_state +low B-protein_state +- I-protein_state +activity I-protein_state +state I-protein_state +, O +in O +negative B-experimental_method +stain I-experimental_method +EM I-experimental_method +reveals O +a O +large O +set O +of O +conformations O +from O +rod B-protein_state +- I-protein_state +like I-protein_state +extended I-protein_state +to O +U B-protein_state +- I-protein_state +shaped I-protein_state +particles B-evidence +. O + +The O +flexibility O +in O +the O +CDC2 B-structure_element +/ I-structure_element +CT I-structure_element +hinge I-structure_element +appears O +substantially O +larger O +than O +the O +variations O +observed O +in O +the O +set O +of O +crystal B-evidence +structures I-evidence +. O + +The O +BC B-structure_element +domain O +is O +not O +completely O +disordered O +, O +but O +laterally O +attached O +to O +BT B-structure_element +/ O +CDN B-structure_element +in O +a O +generally B-protein_state +conserved I-protein_state +position I-protein_state +, O +albeit O +with O +increased O +flexibility O +. O + +Surprisingly O +, O +in O +both O +the O +linear B-protein_state +and I-protein_state +U I-protein_state +- I-protein_state +shaped I-protein_state +conformations I-protein_state +, O +the O +approximate O +distances O +between O +the O +BC B-structure_element +and O +CT B-structure_element +active B-site +sites I-site +would O +remain O +larger O +than O +110 O +Å O +. O +These O +observed O +distances O +are O +considerably O +larger O +than O +in O +static B-protein_state +structures B-evidence +of O +any O +other O +related O +biotin B-protein_type +- I-protein_type +dependent I-protein_type +carboxylase I-protein_type +. O + +Furthermore O +, O +based O +on O +an O +average O +length O +of O +the O +BCCP B-structure_element +– I-structure_element +CD I-structure_element +linker I-structure_element +in O +fungal B-taxonomy_domain +ACC B-protein_type +of O +26 B-residue_range +amino I-residue_range +acids I-residue_range +, O +mobility O +of O +the O +BCCP B-structure_element +alone O +would O +not O +be O +sufficient O +to O +bridge O +the O +active B-site +sites I-site +of O +BC B-structure_element +and O +CT B-structure_element +. O + +Altogether O +, O +the O +architecture O +of O +fungal B-taxonomy_domain +ACC B-protein_type +is O +based O +on O +the O +central O +dimeric B-oligomeric_state +CT B-structure_element +domain O +( O +Fig O +. O +4d O +). O + +In O +fungal B-taxonomy_domain +ACC B-protein_type +, O +however O +, O +Ser1157 B-residue_name_number +in O +the O +regulatory B-structure_element +loop I-structure_element +of O +the O +CD B-structure_element +is O +the O +only O +phosphorylation B-site +site I-site +that O +has O +been O +demonstrated O +to O +be O +both O +phosphorylated B-protein_state +in O +vivo O +and O +involved O +in O +the O +regulation O +of O +ACC B-protein_type +activity O +. O + +In O +its O +phosphorylated B-protein_state +state O +, O +the O +regulatory B-structure_element +loop I-structure_element +containing O +Ser1157 B-residue_name_number +wedges O +between O +CDC1 B-structure_element +/ O +CDC2 B-structure_element +and O +presumably O +limits O +the O +conformational B-protein_state +freedom I-protein_state +at O +this O +interdomain B-site +interface I-site +. O + +The O +current O +data O +thus O +suggest O +that O +regulation O +of O +fungal B-taxonomy_domain +ACC B-protein_type +is O +mediated O +by O +controlling O +the O +dynamics O +of O +the O +unique B-protein_state +CD B-structure_element +, O +rather O +than O +directly O +affecting O +catalytic O +turnover O +at O +the O +active B-site +sites I-site +of O +BC B-structure_element +and O +CT B-structure_element +. O + +Most O +recently O +, O +a O +crystal B-evidence +structure I-evidence +of O +near B-protein_state +full I-protein_state +- I-protein_state +length I-protein_state +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +ACC B-protein_type +from O +S B-species +. I-species +cerevisae I-species +( O +lacking B-protein_state +only I-protein_state +21 B-residue_range +N O +- O +terminal O +amino O +acids O +, O +here O +denoted O +as O +flACC B-protein +) O +was O +published O +by O +Wei O +and O +Tong O +. O + +In O +flACC B-protein +, O +the O +ACC B-protein_type +dimer B-oligomeric_state +obeys O +twofold O +symmetry O +and O +assembles O +in O +a O +triangular B-protein_state +architecture I-protein_state +with O +dimeric B-oligomeric_state +BC B-structure_element +domains O +( O +Supplementary O +Fig O +. O +5a O +). O + +In O +their O +study O +, O +mutational B-experimental_method +data I-experimental_method +indicate O +a O +requirement O +for O +BC O +dimerization O +for O +catalytic O +activity O +. O + +The O +transition O +from O +the O +elongated B-protein_state +open I-protein_state +shape I-protein_state +, O +observed O +in O +our O +experiments O +, O +towards O +a O +compact B-protein_state +triangular I-protein_state +shape I-protein_state +is O +based O +on O +an O +intricate O +interplay O +of O +several O +hinge O +- O +bending O +motions O +in O +the O +CD B-structure_element +( O +Fig O +. O +4d O +). O + +Comparison B-experimental_method +of O +flACC B-protein +with O +our O +CthΔBCCP B-mutant +structure B-evidence +reveals O +the O +CDC2 B-structure_element +/ I-structure_element +CT I-structure_element +hinge I-structure_element +as O +a O +major O +contributor O +to O +conformational O +flexibility O +( O +Supplementary O +Fig O +. O +5b O +, O +c O +). O + +The O +only O +bona O +fide O +regulatory B-protein_state +phophorylation B-site +site I-site +of O +fungal B-taxonomy_domain +ACC B-protein_type +in O +the O +regulatory B-structure_element +loop I-structure_element +is O +directly O +participating O +in O +CDC1 B-structure_element +/ O +CDC2 B-structure_element +domain O +interactions O +and O +thus O +stabilizes O +the O +hinge B-structure_element +conformation I-structure_element +. O + +In O +flACC B-protein +, O +the O +regulatory B-structure_element +loop I-structure_element +is O +mostly B-protein_state +disordered I-protein_state +, O +illustrating O +the O +increased O +flexibility O +due O +to O +the O +absence O +of O +the O +phosphoryl B-chemical +group O +. O + +Only O +in O +three O +out O +of O +eight O +observed O +protomers B-oligomeric_state +a O +short B-structure_element +peptide I-structure_element +stretch O +( O +including O +Ser1157 B-residue_name_number +) O +was O +modelled B-evidence +. O + +In O +those O +instances O +the O +Ser1157 B-residue_name_number +residue O +is O +located O +at O +a O +distance O +of O +14 O +– O +20 O +Å O +away O +from O +the O +location O +of O +the O +phosphorylated B-protein_state +serine B-residue_name +observed O +here O +, O +based O +on O +superposition B-experimental_method +of O +either O +CDC1 B-structure_element +or O +CDC2 B-structure_element +. O + +Applying B-experimental_method +the O +conformation O +of O +the O +CDC1 B-structure_element +/ I-structure_element +CDC2 I-structure_element +hinge I-structure_element +observed O +in O +SceCD B-species +on O +flACC B-protein +leads O +to O +CDN B-structure_element +sterically O +clashing O +with O +CDC2 B-structure_element +and O +BT B-structure_element +/ O +CDN B-structure_element +clashing O +with O +CT B-structure_element +( O +Supplementary O +Fig O +. O +6a O +, O +b O +). O + +Thus O +, O +in O +accordance O +with O +the O +results O +presented O +here O +, O +phosphorylation B-ptm +of O +Ser1157 B-residue_name_number +in O +SceACC B-protein +most O +likely O +limits O +flexibility O +in O +the O +CDC1 B-structure_element +/ I-structure_element +CDC2 I-structure_element +hinge I-structure_element +such O +that O +activation O +through O +BC B-structure_element +dimerization O +is O +not O +possible O +( O +Fig O +. O +4d O +), O +which O +however O +does O +not O +exclude O +intermolecular O +dimerization O +. O + +In O +addition O +, O +EM B-experimental_method +micrographs B-evidence +of O +phosphorylated B-protein_state +and O +dephosphorylated B-protein_state +SceACC B-protein +display O +for O +both O +samples O +mainly O +elongated B-protein_state +and I-protein_state +U I-protein_state +- I-protein_state +shaped I-protein_state +conformations I-protein_state +and O +reveal O +no O +apparent O +differences O +in O +particle B-evidence +shape I-evidence +distributions I-evidence +( O +Supplementary O +Fig O +. O +7 O +). O + +This O +implicates O +that O +the O +triangular B-protein_state +shape I-protein_state +with O +dimeric B-oligomeric_state +BC B-structure_element +domains O +has O +a O +low O +population O +also O +in O +the O +active B-protein_state +form I-protein_state +, O +even O +though O +a O +biasing O +influence O +of O +grid O +preparation O +cannot O +be O +excluded O +completely O +. O + +Together O +, O +this O +structural B-evidence +information I-evidence +suggests O +that O +variable O +carrier O +protein O +tethering O +is O +not O +sufficient O +for O +efficient O +substrate O +transfer O +and O +catalysis O +in O +any O +of O +these O +systems O +. O + +The O +determination B-experimental_method +of I-experimental_method +a I-experimental_method +set I-experimental_method +of I-experimental_method +crystal B-evidence +structures I-evidence +of O +SceACC B-protein +in O +two O +states O +, O +unphosphorylated B-protein_state +and O +phosphorylated B-protein_state +at O +the O +major B-site +regulatory I-site +site I-site +Ser1157 B-residue_name_number +, O +provides O +a O +unique O +depiction O +of O +multienzyme O +regulation O +by O +post O +- O +translational O +modification O +( O +Fig O +. O +4d O +). O + +The O +regulation O +of O +activity O +thus O +results O +from O +restrained O +large O +- O +scale O +conformational O +dynamics O +rather O +than O +a O +direct O +or O +indirect O +influence O +on O +active B-site +site I-site +structure I-site +. O + +To O +our O +best O +knowledge O +, O +ACC B-protein_type +is O +the O +first O +multienzyme B-protein_type +for O +which O +such O +a O +phosphorylation B-ptm +- O +dependent O +mechanical O +control O +mechanism O +has O +been O +visualized O +. O + +However O +, O +the O +example O +of O +ACC B-protein_type +now O +demonstrates O +the O +possibility O +of O +regulating O +activity O +by O +controlled O +dynamics O +of O +non B-structure_element +- I-structure_element +enzymatic I-structure_element +linker I-structure_element +regions I-structure_element +also O +in O +other O +families O +of O +carrier B-protein_type +- I-protein_type +dependent I-protein_type +multienzymes I-protein_type +. O + +The O +phosphorylated B-protein_state +central B-structure_element +domain I-structure_element +of O +yeast B-taxonomy_domain +ACC B-protein_type +. O + +( O +a O +) O +Schematic O +overview O +of O +the O +domain O +organization O +of O +eukaryotic B-taxonomy_domain +ACCs B-protein_type +. O + +Crystallized B-evidence +constructs I-evidence +are O +indicated O +. O + +( O +b O +) O +Cartoon O +representation O +of O +the O +SceCD B-species +crystal B-evidence +structure I-evidence +. O + +The O +regulatory B-structure_element +loop I-structure_element +is O +shown O +as O +bold O +cartoon O +, O +and O +the O +phosphorylated B-protein_state +Ser1157 B-residue_name_number +is O +marked O +by O +a O +red O +triangle O +. O + +The O +attachment O +points O +to O +the O +N O +- O +terminal O +BCCP B-structure_element +domain O +and O +the O +C O +- O +terminal O +CT B-structure_element +domain O +are O +indicated O +with O +spheres O +. O + +Architecture O +of O +the O +CD B-structure_element +– O +CT B-structure_element +core O +of O +fungal B-taxonomy_domain +ACC B-protein_type +. O + +One O +protomer B-oligomeric_state +is O +shown O +in O +colour O +and O +one O +in O +grey O +. O + +Individual O +domains O +are O +labelled O +; O +the O +active B-site +site I-site +of O +CT B-structure_element +and O +the O +position O +of O +the O +conserved B-protein_state +regulatory B-protein_state +phosphoserine B-site +site I-site +based O +on O +SceCD B-species +are O +indicated O +by O +an O +asterisk O +and O +a O +triangle O +, O +respectively O +. O + +( O +a O +) O +Hinge B-structure_element +properties O +of O +the O +CDC2 B-structure_element +– I-structure_element +CT I-structure_element +connection I-structure_element +analysed O +by O +a O +CT B-experimental_method +- I-experimental_method +based I-experimental_method +superposition I-experimental_method +of O +eight O +instances O +of O +the O +CDC2 B-mutant +- I-mutant +CT I-mutant +segment I-mutant +. O + +For O +clarity O +, O +only O +one O +protomer B-oligomeric_state +of O +CthCD B-mutant +- I-mutant +CTCter1 I-mutant +is O +shown O +in O +full O +colour O +as O +reference O +. O + +The O +range O +of O +hinge O +bending O +is O +indicated O +and O +the O +connection O +points O +between O +CDC2 B-structure_element +and O +CT B-structure_element +( O +blue O +) O +as O +well O +as O +between O +CDC1 B-structure_element +and O +CDC2 B-structure_element +( O +green O +and O +grey O +) O +are O +marked O +as O +spheres O +. O + +One O +protomer B-oligomeric_state +of O +CthΔBCCP B-mutant +is O +shown O +in O +colour O +, O +the O +CDL B-structure_element +domains O +are O +omitted O +for O +clarity O +and O +the O +position O +of O +the O +phosphorylated B-protein_state +serine B-residue_name +based O +on O +SceCD B-species +is O +indicated O +with O +a O +red O +triangle O +. O + +The O +connection O +points O +from O +CDC1 B-structure_element +to O +CDC2 B-structure_element +and O +to O +CDL B-structure_element +are O +represented O +by O +green O +spheres O +. O + +The O +conformational O +dynamics O +of O +fungal B-taxonomy_domain +ACC B-protein_type +. O + +Domains O +other O +than O +CDN B-structure_element +and O +CDL B-structure_element +/ O +CDC1 B-structure_element +are O +omitted O +for O +clarity O +. O + +The O +domains O +are O +labelled O +and O +the O +distances O +between O +the O +N O +termini O +of O +CDN B-structure_element +( O +spheres O +) O +in O +the O +compared O +structures O +are O +indicated O +. O + +( O +d O +) O +Schematic O +model O +of O +fungal B-taxonomy_domain +ACC B-protein_type +showing O +the O +intrinsic O +, O +regulated O +flexibility O +of O +CD B-structure_element +in O +the O +phosphorylated B-protein_state +inhibited B-protein_state +or O +the O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +activated B-protein_state +state O +. O + +Flexibility O +of O +the O +CDC2 B-structure_element +/ O +CT B-structure_element +and O +CDN B-structure_element +/ O +CDL B-structure_element +hinges B-structure_element +is O +illustrated O +by O +arrows O +. O + +The O +Ser1157 B-residue_name_number +phosphorylation B-ptm +site O +and O +the O +regulatory B-structure_element +loop I-structure_element +are O +schematically O +indicated O +in O +magenta O +. O + +Crystal B-evidence +structure I-evidence +of O +SEL1L B-protein +: O +Insight O +into O +the O +roles O +of O +SLR B-structure_element +motifs O +in O +ERAD O +pathway O + +SEL1L B-protein +, O +a O +component O +of O +the O +ERAD O +machinery O +, O +plays O +an O +important O +role O +in O +selecting O +and O +transporting O +ERAD O +substrates O +for O +degradation O +. O + +We O +have O +determined O +the O +crystal B-evidence +structure I-evidence +of O +the O +mouse B-taxonomy_domain +SEL1L B-protein +central B-structure_element +domain I-structure_element +comprising O +five O +Sel1 B-structure_element +- I-structure_element +Like I-structure_element +Repeats I-structure_element +( O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +to I-structure_element +9 I-structure_element +; O +hereafter O +called O +SEL1Lcent B-structure_element +). O + +Furthermore O +, O +we O +discovered O +that O +the O +SLR B-structure_element +- I-structure_element +C I-structure_element +, O +comprising O +SLR B-structure_element +motifs I-structure_element +10 I-structure_element +and I-structure_element +11 I-structure_element +, O +of O +SEL1L B-protein +directly O +interacts O +with O +the O +N O +- O +terminus O +luminal B-structure_element +loops I-structure_element +of O +HRD1 B-protein +. O + +Therefore O +, O +we O +propose O +that O +certain O +SLR B-structure_element +motifs O +of O +SEL1L B-protein +play O +a O +unique O +role O +in O +membrane O +bound O +ERAD O +machinery O +. O + +Protein O +quality O +control O +in O +the O +endoplasmic O +reticulum O +( O +ER O +) O +is O +essential O +for O +maintenance O +of O +cellular O +homeostasis O +in O +eukaryotes B-taxonomy_domain +and O +is O +implicated O +in O +many O +severe O +diseases O +. O + +Terminally O +misfolded O +proteins O +in O +the O +lumen O +or O +membrane O +of O +the O +ER O +are O +retrotranslocated O +into O +the O +cytosol O +, O +polyubiquitinated B-protein_state +, O +and O +degraded O +by O +the O +proteasome B-complex_assembly +. O + +Accumulating O +studies O +have O +identified O +key O +components O +for O +ERAD O +, O +including O +HRD1 B-protein +, O +SEL1L B-protein +( O +Hrd3p B-protein +), O +Derlin B-protein +- I-protein +1 I-protein +, I-protein +- I-protein +2 I-protein +, I-protein +- I-protein +3 I-protein +( O +Der1p B-protein +), O +HERP B-protein +- I-protein +1 I-protein +, I-protein +- I-protein +2 I-protein +( O +Usa1p B-protein +), O +OS9 B-protein +( O +Yos9 B-protein +), O +XTP B-protein +- I-protein +B I-protein +, O +and O +Grp94 B-protein +, O +all O +of O +which O +are O +involved O +in O +the O +recognition O +and O +translocation O +of O +the O +ERAD O +substrates O +in O +yeast B-taxonomy_domain +and O +metazoans B-taxonomy_domain +. O + +Yeast B-taxonomy_domain +ERAD O +components O +, O +which O +have O +been O +extensively O +characterized O +through O +genetic B-experimental_method +and I-experimental_method +biochemical I-experimental_method +studies I-experimental_method +, O +are O +comparable O +with O +mammalian B-taxonomy_domain +ERAD O +components O +, O +sharing O +similar O +molecular O +functions O +and O +structural O +composition O +. O + +The O +HRD1 B-protein +E3 B-protein_type +ubiquitin I-protein_type +ligase I-protein_type +, O +which O +is O +embedded O +in O +the O +ER O +membrane O +, O +is O +involved O +in O +translocating O +ERAD O +substrates O +across O +the O +ER O +membrane O +and O +catalyzing O +substrate O +ubiquitination O +via O +its O +cytosolic O +RING B-structure_element +finger I-structure_element +domain I-structure_element +. O + +SEL1L B-protein +, O +the O +mammalian B-taxonomy_domain +homolog O +of O +Hrd3p B-protein +, O +associates O +with O +HRD1 B-protein +, O +mediates O +HRD1 B-protein +interactions O +with O +the O +ER O +luminal O +lectin B-protein_type +OS9 B-protein +, O +and O +recognizes O +substrates O +to O +be O +degraded O +. O + +Recent O +research O +based O +on O +the O +inducible O +Sel1l B-gene +knockout B-experimental_method +mouse I-experimental_method +model O +highlights O +the O +physiological O +functions O +of O +SEL1L B-protein +. O + +However O +, O +despite O +the O +functional O +importance O +of O +SEL1L B-protein +, O +the O +molecular O +structure B-evidence +of O +SEL1L B-protein +has O +not O +been O +solved O +. O + +Previous O +biochemical B-experimental_method +studies I-experimental_method +reveal O +that O +SEL1L B-protein +is O +a O +type B-protein_type +I I-protein_type +transmembrane I-protein_type +protein I-protein_type +and O +has O +a O +large O +luminal B-structure_element +domain I-structure_element +comprising O +sets O +of O +repeated B-structure_element +Sel1 I-structure_element +- I-structure_element +like I-structure_element +( O +SLR B-structure_element +) O +motifs O +. O + +The O +SLR B-structure_element +motif O +is O +a O +structural O +motif O +that O +closely O +resembles O +the O +tetratricopeptide B-structure_element +- I-structure_element +repeat I-structure_element +( O +TPR B-structure_element +) O +motif O +, O +which O +is O +a O +protein O +- O +protein O +interaction O +module O +. O + +Furthermore O +, O +while O +repeated O +SLR B-structure_element +motifs O +are O +commonly O +found O +in O +tandem O +arrays O +, O +the O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +are O +, O +according O +to O +the O +primary O +structure O +prediction O +of O +SEL1L B-protein +, O +interspersed O +among O +other O +sequences O +in O +the O +luminal B-structure_element +domain I-structure_element +and O +form O +three O +SLR B-structure_element +domain O +clusters O +. O + +Therefore O +, O +the O +way O +in O +which O +these O +unique O +structural O +features O +of O +SEL1L B-protein +are O +related O +to O +its O +critical O +function O +in O +ERAD O +remains O +to O +be O +elucidated O +. O + +To O +clearly O +understand O +the O +biochemical O +role O +of O +the O +SLR B-structure_element +domains O +of O +SEL1L B-protein +in O +ERAD O +, O +we O +determined O +the O +crystal B-evidence +structure I-evidence +of O +the O +central O +SLR B-structure_element +domain O +of O +SEL1L B-protein +. O + +We O +found O +that O +the O +central B-structure_element +domain I-structure_element +of O +SEL1L B-protein +, O +comprising O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +through I-structure_element +9 I-structure_element +( O +SEL1Lcent B-structure_element +), O +forms O +a O +tight O +dimer B-oligomeric_state +with O +two O +- O +fold O +symmetry O +due O +to O +domain O +swapping O +of O +the O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +. O + +We O +also O +found O +that O +SLR B-structure_element +- I-structure_element +C I-structure_element +, O +consisting O +of O +SLR B-structure_element +motifs I-structure_element +10 I-structure_element +and I-structure_element +11 I-structure_element +, O +directly O +interacts O +with O +the O +N O +- O +terminus O +luminal B-structure_element +loop I-structure_element +of O +HRD1 B-protein +. O + +Structure B-experimental_method +Determination I-experimental_method +of O +SEL1Lcent B-structure_element + +The O +Mus B-species +musculus I-species +SEL1L B-protein +protein O +contains O +790 O +amino O +acids O +and O +has O +17 O +% O +sequence O +identity O +to O +its O +yeast B-taxonomy_domain +homolog O +, O +Hrd3p B-protein +. O + +Mouse B-taxonomy_domain +SEL1L B-protein +contains O +a O +fibronectin B-structure_element +type I-structure_element +II I-structure_element +domain I-structure_element +at O +the O +N O +- O +terminus O +, O +followed O +by O +11 O +SLR B-structure_element +motifs O +and O +a O +single O +transmembrane B-structure_element +domain I-structure_element +at O +the O +C O +- O +terminus O +( O +Fig O +. O +1A O +). O + +However O +, O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +protein O +aggregated O +in O +solution O +and O +produced O +no O +soluble O +protein O +. O + +However O +, O +the O +central B-structure_element +region I-structure_element +of O +SEL1L B-protein +, O +comprising O +residues O +337 B-residue_range +– I-residue_range +554 I-residue_range +, O +was O +soluble O +and O +homogenous O +in O +size O +, O +as O +determined O +by O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +. O + +To O +define O +compact O +domain O +boundaries O +for O +the O +central B-structure_element +region I-structure_element +of O +SEL1L B-protein +, O +we O +digested B-experimental_method +the I-experimental_method +protein I-experimental_method +with I-experimental_method +trypsin I-experimental_method +and O +analyzed O +the O +proteolysis O +products O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +and O +N B-experimental_method +- I-experimental_method +terminal I-experimental_method +sequencing I-experimental_method +. O + +The O +results O +of O +this O +preliminary O +biochemical O +analysis O +suggested O +that O +SEL1L B-protein +residues O +348 B-residue_range +– I-residue_range +533 I-residue_range +( O +SEL1Lcent B-structure_element +) O +would O +be O +amenable O +to O +structural B-experimental_method +analysis I-experimental_method +( O +Fig O +. O +1A O +). O + +Crystals B-evidence +of O +SEL1Lcent B-structure_element +grew O +in O +space O +group O +P21 O +with O +four O +copies O +of O +SEL1Lcent B-structure_element +( O +a O +total O +of O +82 O +kDa O +) O +in O +the O +asymmetric O +unit O +. O + +The O +structure B-evidence +was O +determined O +by O +the O +single B-experimental_method +- I-experimental_method +wavelength I-experimental_method +anomalous I-experimental_method +diffraction I-experimental_method +( O +SAD B-experimental_method +) O +method O +using O +selenium B-chemical +as O +the O +anomalous O +scatterer O +( O +Table O +1 O +and O +Methods O +). O + +The O +assignment O +of O +residues O +during O +model O +building O +was O +aided O +by O +the O +selenium B-chemical +atom O +positions O +, O +and O +the O +structure B-evidence +was O +refined O +with O +native O +data O +to O +2 O +. O +6 O +Å O +resolution O +with O +Rwork B-evidence +/ I-evidence +Rfree I-evidence +values O +of O +20 O +. O +7 O +/ O +27 O +. O +7 O +%. O + +Overall O +Structure B-evidence +of O +SEL1Lcent B-structure_element + +The O +mouse B-taxonomy_domain +SEL1Lcent B-structure_element +crystallized B-experimental_method +as O +a O +homodimer B-oligomeric_state +, O +and O +there O +were O +two O +homodimers B-oligomeric_state +in O +the O +crystal O +asymmetric O +unit O +( O +Fig O +. O +1B O +, O +C O +, O +Supplementary O +Fig O +. O +1 O +). O + +The O +two O +SEL1Lcent B-structure_element +molecules O +dimerize B-oligomeric_state +in O +a O +head B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +tail I-protein_state +manner O +through O +a O +two B-site +- I-site +fold I-site +symmetry I-site +interface I-site +resulting O +in O +a O +cosmos O +- O +like O +shaped O +structure B-evidence +( O +Fig O +. O +1B O +). O + +The O +resulting O +structure B-evidence +resembles O +the O +yin O +- O +yang O +symbol O +with O +overall O +dimensions O +of O +60 O +× O +60 O +× O +25 O +Å O +, O +where O +a O +SEL1Lcent B-structure_element +monomer B-oligomeric_state +corresponds O +to O +half O +the O +symbol O +. O + +The O +dimer B-oligomeric_state +formation O +buries O +a O +surface O +area O +of O +1670 O +Å2 O +for O +each O +monomer B-oligomeric_state +, O +and O +no O +significant O +differences O +between O +the O +protomers B-oligomeric_state +were O +displayed O +( O +final O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +RMSD B-evidence +) O +of O +0 O +. O +6 O +Å O +for O +all O +Cα O +atoms O +). O + +Each O +protomer B-oligomeric_state +is O +composed O +of O +ten O +α B-structure_element +- I-structure_element +helices I-structure_element +, O +which O +form O +the O +five O +SLRs B-structure_element +, O +resulting O +in O +an O +elongated O +curved O +structure O +, O +confirming O +the O +primary O +structure O +prediction O +( O +Fig O +. O +1D O +). O + +The O +α B-structure_element +- I-structure_element +helices I-structure_element +subdivide O +the O +structure B-evidence +into O +five O +pairs O +( O +A B-structure_element +and O +B B-structure_element +) O +as O +shown O +in O +a O +number O +of O +TPRs B-structure_element +and O +SLRs B-structure_element +. O + +In O +addition O +, O +a O +longer O +loop B-structure_element +, O +consisting O +of O +approximately O +eight O +amino O +acids O +, O +is O +inserted O +between O +helix B-structure_element +B I-structure_element +of O +one O +SLR B-structure_element +and O +helix B-structure_element +A I-structure_element +of O +the O +next O +SLR B-structure_element +. O + +This O +arrangement O +is O +a O +unique O +feature O +for O +SLRs B-structure_element +among O +the O +major O +classes O +of O +repeats O +containing O +an O +α B-structure_element +- I-structure_element +solenoid I-structure_element +. O + +This O +unique O +conformation O +of O +helix B-structure_element +9B I-structure_element +most O +likely O +contributes O +to O +formation O +of O +the O +dimer B-oligomeric_state +structure O +of O +SEL1Lcent B-structure_element +, O +as O +detailed O +below O +. O + +With O +the O +exception O +of O +the O +last O +SLR B-structure_element +, O +the O +four O +α B-structure_element +- I-structure_element +helix I-structure_element +pairs O +possess O +similar O +conformations O +, O +with O +RMSD B-evidence +values O +of O +0 O +. O +7 O +Å O +for O +all O +Cα O +atoms O +. O + +Although O +the O +sequence O +similarity O +for O +the O +pairwise B-experimental_method +alignments I-experimental_method +varies O +between O +25 O +% O +and O +35 O +%, O +all O +the O +residues O +present O +in O +the O +SLR B-structure_element +motifs O +are O +conserved B-protein_state +among O +the O +five O +pairs O +. O + +The O +SLR B-structure_element +domain O +of O +SLR B-structure_element +- I-structure_element +M I-structure_element +ends O +at O +residue O +524 B-residue_number +, O +and O +C O +- O +terminal O +amino O +acids O +525 B-residue_range +– I-residue_range +533 I-residue_range +of O +the O +protein O +are O +not O +visible O +in O +the O +electron B-evidence +density I-evidence +map I-evidence +, O +suggesting O +that O +this O +region O +is O +highly B-protein_state +flexible I-protein_state +. O + +Since O +no O +information O +regarding O +dimer B-oligomeric_state +formation O +by O +SEL1L B-protein +through O +its O +SLR B-structure_element +motifs O +is O +available O +, O +we O +tested O +whether O +the O +SEL1Lcent B-structure_element +dimer B-oligomeric_state +shown O +in O +our O +crystal B-evidence +structure I-evidence +is O +a O +biological O +unit O +. O + +First O +, O +we O +cross B-experimental_method +- I-experimental_method +linked I-experimental_method +SEL1Lcent B-structure_element +or O +a O +longer O +construct O +of O +SEL1L B-protein +( O +SEL1Llong B-mutant +, O +residues O +337 B-residue_range +– I-residue_range +554 I-residue_range +) O +using O +various O +concentrations O +of O +glutaraldehyde B-chemical +( O +GA B-chemical +) O +or O +dimethyl B-chemical +suberimidate I-chemical +( O +DMS B-chemical +) O +and O +analyzed O +the O +products O +by O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +. O + +We O +detected O +bands O +at O +the O +mass O +of O +a O +dimer B-oligomeric_state +for O +both O +SEL1Lcent B-structure_element +and O +SEL1Llong B-mutant +when O +cross B-experimental_method +- I-experimental_method +linked I-experimental_method +with O +low O +concentrations O +of O +GA B-chemical +( O +0 O +. O +005 O +%) O +or O +DMS B-chemical +( O +0 O +. O +3 O +mM O +) O +( O +Supplementary O +Fig O +. O +2A O +, O +B O +). O + +Next O +, O +we O +conducted O +analytical B-experimental_method +ultracentrifugation I-experimental_method +of O +SEL1Lcent B-structure_element +. O + +Consistent O +with O +the O +cross B-experimental_method +- I-experimental_method +linking I-experimental_method +data O +, O +analytical B-experimental_method +ultracentrifugation I-experimental_method +revealed O +that O +the O +molecular B-evidence +weight I-evidence +of O +SEL1Lcent B-structure_element +corresponds O +to O +a O +dimer B-oligomeric_state +( O +Supplementary O +Fig O +. O +2C O +). O + +Taken O +together O +, O +these O +data O +indicate O +that O +some O +kind O +of O +dimer B-oligomeric_state +is O +formed O +in O +solution O +. O + +Dimer B-site +Interface I-site +of O +SEL1Lcent B-structure_element + +In O +contrast O +to O +a O +previously O +described O +SLR B-protein_type +motif I-protein_type +containing I-protein_type +proteins I-protein_type +that O +exist O +as O +monomers B-oligomeric_state +in O +solution O +, O +SEL1Lcent B-structure_element +forms O +an O +intimate O +two B-site +- I-site +fold I-site +homotypic I-site +dimer I-site +interface I-site +( O +Figs O +1B O +and O +2A O +). O + +However O +, O +no O +interactions O +were O +seen O +between O +the O +two O +- O +fold O +- O +related O +protomers B-oligomeric_state +through O +the O +concave B-site +inner I-site +surfaces I-site +themselves O +. O + +Rather O +, O +the O +unique O +structure O +of O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +, O +consisting O +of O +two O +parallel O +helices O +( O +9A B-structure_element +and O +9B B-structure_element +), O +is O +located O +in O +the O +space O +generated O +by O +the O +concave B-site +surface I-site +and O +provides O +an O +extensive O +dimerization B-site +interface I-site +between O +the O +two O +- O +fold O +- O +related O +molecules O +( O +Fig O +. O +2A O +). O + +Three O +major O +contact B-site +interfaces I-site +are O +involved O +in O +the O +interactions O +, O +and O +all O +interfaces B-site +are O +symmetrically O +related O +between O +the O +dimer B-oligomeric_state +subunits O +( O +Fig O +. O +2A O +). O + +Structure B-experimental_method +- I-experimental_method +based I-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +135 O +SEL1L B-protein +phylogenetic O +sequences O +using O +a O +ConSurf B-experimental_method +server I-experimental_method +revealed O +that O +the O +surface O +residues O +in O +the O +dimer B-site +interfaces I-site +were O +highly B-protein_state +conserved I-protein_state +among O +the O +SEL1L B-protein +orthologs O +( O +Fig O +. O +1E O +). O + +First O +, O +helix B-structure_element +9B I-structure_element +of O +each O +SEL1Lcent B-structure_element +subunit O +interacts O +with O +residues O +lining O +the O +inner B-site +groove I-site +from O +the O +SLR B-structure_element +α B-structure_element +- I-structure_element +helices I-structure_element +( O +5B B-structure_element +, O +6B B-structure_element +, O +7B B-structure_element +, O +and O +8B B-structure_element +) O +from O +its O +counterpart O +. O + +In O +addition O +to O +hydrophobic B-bond_interaction +interactions I-bond_interaction +, O +the O +side O +chain O +hydroxyl O +group O +of O +Tyr B-residue_name_number +519 I-residue_name_number +and O +the O +main O +- O +chain O +oxygen O +of O +Ile B-residue_name_number +515 I-residue_name_number +form O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +to O +the O +side O +chain O +of O +the O +conserved B-protein_state +Gln B-residue_name_number +377 I-residue_name_number +and O +His B-residue_name_number +381 I-residue_name_number +on O +helix B-structure_element +5B I-structure_element +of O +the O +two O +- O +fold O +- O +related O +protomer B-oligomeric_state +. O + +The O +side O +chain O +of O +Gln B-residue_name_number +523 I-residue_name_number +forms O +an O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +to O +the O +side O +chain O +of O +Asp B-residue_name_number +480 I-residue_name_number +on O +the O +two O +- O +fold O +- O +related O +protomer B-oligomeric_state +( O +Fig O +. O +2A O +, O +Interface B-site +1 I-site +detail O +). O + +In O +this O +interface B-site +, O +the O +interacting O +residues O +on O +helix B-structure_element +9A I-structure_element +, O +including O +Leu B-residue_name_number +503 I-residue_name_number +, O +Tyr B-residue_name_number +499 I-residue_name_number +, O +and O +the O +aliphatic O +side O +chain O +of O +Lys B-residue_name_number +500 I-residue_name_number +, O +form O +an O +extensive O +network O +of O +van B-bond_interaction +der I-bond_interaction +Waals I-bond_interaction +contacts I-bond_interaction +with O +the O +hydrophobic O +residues O +of O +the O +counterpart O +helix B-structure_element +5A I-structure_element +, O +including O +Tyr B-residue_name_number +360 I-residue_name_number +, O +Leu B-residue_name_number +356 I-residue_name_number +, O +Tyr B-residue_name_number +359 I-residue_name_number +, O +and O +Leu B-residue_name_number +363 I-residue_name_number +. O + +In O +addition O +to O +hydrophobic B-bond_interaction +interactions I-bond_interaction +, O +the O +side O +chains O +of O +Asn B-residue_name_number +507 I-residue_name_number +and O +Ser B-residue_name_number +510 I-residue_name_number +on O +helix B-structure_element +9A I-structure_element +make O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +with O +highly B-protein_state +conserved I-protein_state +Arg B-residue_name_number +384 I-residue_name_number +in O +the O +loop B-structure_element +between O +helix B-structure_element +5B I-structure_element +and O +6A B-structure_element +from O +the O +two O +- O +fold O +- O +related O +protomer B-oligomeric_state +( O +Fig O +. O +2A O +, O +Interface O +2 O +detail O +). O + +Third O +, O +the O +helix B-structure_element +9B I-structure_element +from O +each O +protomer B-oligomeric_state +is O +involved O +in O +the O +dimer B-oligomeric_state +interaction O +by O +forming O +a O +two O +- O +fold O +antiparallel O +symmetry O +. O + +In O +particular O +, O +the O +side O +chains O +of O +hydrophobic O +residues O +, O +including O +Phe B-residue_name_number +518 I-residue_name_number +, O +Leu B-residue_name_number +521 I-residue_name_number +, O +and O +Met B-residue_name_number +524 I-residue_name_number +, O +are O +directed O +toward O +each O +other O +, O +where O +they O +make O +both O +inter O +- O +and O +intramolecular O +contacts O +( O +Fig O +. O +2A O +, O +Interface B-site +3 I-site +detail O +). O + +To O +further O +investigate O +the O +interactions O +observed O +in O +our O +crystal B-evidence +structure I-evidence +, O +we O +generated O +a O +C O +- O +terminal O +deletion B-protein_state +mutant I-protein_state +( O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +) O +lacking B-protein_state +SLR B-structure_element +motif I-structure_element +9 I-structure_element +( O +helix B-structure_element +9A I-structure_element +and O +9B B-structure_element +) O +from O +SEL1Lcent B-structure_element +for O +comparative O +analysis O +. O + +The O +deletion B-protein_state +mutant I-protein_state +and O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +SEL1Lcent B-structure_element +showed O +no O +difference O +in O +spectra B-evidence +by O +CD B-experimental_method +spectroscopy I-experimental_method +, O +indicating O +that O +the O +deletion B-experimental_method +of O +the O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +did O +not O +affect O +the O +secondary O +structure O +of O +SEL1Lcent B-structure_element +( O +Supplementary O +Fig O +. O +3 O +). O + +Additionally O +, O +to O +further O +validate O +the O +key O +residues O +involved O +in O +dimer B-oligomeric_state +formation O +, O +we O +generated O +a O +triple B-protein_state +point I-protein_state +mutant I-protein_state +( O +Interface B-site +1 I-site +, O +I515A B-mutant +, O +L516A B-mutant +, O +and O +Y519A B-mutant +) O +of O +the O +hydrophobic O +residues O +that O +are O +involved O +in O +dimerization B-oligomeric_state +. O + +The O +triple B-protein_state +point I-protein_state +mutant I-protein_state +eluted O +at O +the O +monomer B-oligomeric_state +position O +upon O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +, O +while O +the O +negative O +control O +point B-protein_state +mutant I-protein_state +( O +Q460A B-mutant +) O +eluted O +at O +the O +same O +position O +as O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +. O + +Notably O +, O +a O +single B-experimental_method +- I-experimental_method +residue I-experimental_method +mutation I-experimental_method +( O +L521A B-mutant +in O +interface B-site +3 I-site +) O +abolished B-protein_state +the I-protein_state +dimerization I-protein_state +of O +SEL1Lcent B-structure_element +( O +Fig O +. O +2B O +). O + +Taken O +together O +, O +these O +structural B-evidence +and I-evidence +biochemical I-evidence +data I-evidence +demonstrate O +that O +SEL1Lcent B-structure_element +exists O +as O +a O +dimer B-oligomeric_state +in O +solution O +and O +that O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +in O +SEL1Lcent B-structure_element +plays O +an O +important O +role O +in O +generating O +a O +two O +- O +fold O +dimerization B-site +interface I-site +. O + +The O +Two O +Glycine B-residue_name +Residues O +( O +G512 B-residue_name_number +and O +G513 B-residue_name_number +) O +Create O +a O +Hinge B-structure_element +for O +Domain O +Swapping O +of O +SLR B-structure_element +Motif I-structure_element +9 I-structure_element + +Based O +on O +the O +prediction O +, O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +contains O +a O +total O +of O +11 O +SLR B-structure_element +motifs O +, O +and O +our O +construct O +corresponds O +to O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +through I-structure_element +9 I-structure_element +. O + +According O +to O +our O +crystal B-evidence +structure I-evidence +, O +the O +central O +axis O +of O +helix B-structure_element +9B I-structure_element +is O +almost O +parallel O +to O +that O +of O +helix B-structure_element +9A I-structure_element +( O +Fig O +. O +3B O +). O + +However O +, O +this O +unusual O +conformation O +of O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +seems O +to O +be O +essential O +for O +dimer B-oligomeric_state +formation O +, O +as O +described O +earlier O +. O + +The O +phi O +and O +psi O +dihedrals O +are O +100 O +° O +and O +20 O +° O +for O +Gly B-residue_name_number +512 I-residue_name_number +, O +and O +110 O +° O +and O +− O +20 O +° O +for O +Gly B-residue_name_number +513 I-residue_name_number +, O +respectively O +( O +Fig O +. O +3C O +). O + +The O +involvement O +of O +a O +glycine B-residue_name +residue O +in O +forming O +a O +hinge B-structure_element +for O +domain O +swapping O +has O +been O +reported O +previously O +. O + +The O +significance O +of O +Gly B-residue_name_number +513 I-residue_name_number +is O +further O +highlighted O +by O +its O +absolute B-protein_state +conservation I-protein_state +among O +different O +species O +, O +including O +the O +budding B-taxonomy_domain +yeast I-taxonomy_domain +homolog O +Hrd3p B-protein +. O + +To O +further O +investigate O +the O +importance O +of O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +in O +the O +unusual O +SLR B-structure_element +motif O +geometry O +, O +we O +generated O +a O +point B-experimental_method +mutation I-experimental_method +( O +Gly B-mutant +to I-mutant +Ala I-mutant +), O +which O +restricts O +the O +flexibility O +. O +Although O +the O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +residues O +are O +closely O +surrounded O +by O +helix B-structure_element +9B I-structure_element +from O +the O +counter O +protomer B-oligomeric_state +, O +there O +is O +enough O +space O +for O +the O +side O +chain O +of O +alanine B-residue_name +, O +suggesting O +that O +no O +steric O +hindrance O +would O +be O +caused O +by O +the O +mutation B-experimental_method +( O +Fig O +. O +3C O +). O + +However O +, O +the O +double B-protein_state +mutant I-protein_state +( O +G512A B-mutant +/ O +G513A B-mutant +) O +eluted O +over O +a O +broad O +range O +and O +much O +earlier O +than O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +, O +suggesting O +that O +mutation B-experimental_method +of O +the O +residues O +involved O +in O +the O +hinge B-structure_element +linking O +helix B-structure_element +9A I-structure_element +and O +9B B-structure_element +significantly O +affected O +the O +geometry O +of O +helix B-structure_element +9B I-structure_element +in O +generating O +domain O +swapping O +, O +and O +eventually O +altered O +the O +overall O +oligomeric O +state O +of O +SEL1Lcent B-structure_element +into O +a O +polydisperse O +pattern O +( O +Fig O +. O +3D O +, O +Supplementary O +Fig O +. O +6 O +). O + +When O +the O +residues O +were O +mutated B-experimental_method +to I-experimental_method +lysine B-residue_name +( O +G512K B-mutant +/ O +G513K B-mutant +), O +the O +mutant B-protein_state +not O +only O +restricted O +the O +geometry O +of O +residues O +at O +the O +hinge B-structure_element +but O +also O +generated O +steric O +hindrance O +during O +interaction O +with O +the O +counter O +protomer B-oligomeric_state +of O +SEL1Lcent B-structure_element +, O +thereby O +inhibiting O +self O +- O +association O +of O +SEL1Lcent B-structure_element +completely O +. O + +A O +previous O +study O +shows O +that O +induction O +of O +steric O +hindrance O +by O +mutation B-experimental_method +destabilizes O +the O +dimerization B-site +interface I-site +of O +a O +different O +protein O +, O +ClC B-protein_type +transporter I-protein_type +. O + +Collectively O +, O +these O +data O +suggest O +that O +the O +Gly B-residue_name_number +512 I-residue_name_number +and O +Gly B-residue_name_number +513 I-residue_name_number +at O +the O +connection O +between O +helix B-structure_element +9A I-structure_element +and O +9B B-structure_element +play O +a O +crucial O +role O +in O +forming O +the O +domain B-protein_state +- I-protein_state +swapped I-protein_state +conformation O +that O +enables O +dimer B-oligomeric_state +formation O +. O + +SEL1L B-protein +Forms O +Self B-oligomeric_state +- I-oligomeric_state +oligomers I-oligomeric_state +through O +SEL1Lcent B-structure_element +domain O +in O +vivo O + +Next O +, O +we O +examined O +if O +SEL1L B-protein +also O +forms O +self B-oligomeric_state +- I-oligomeric_state +oligomers I-oligomeric_state +in O +vivo O +using O +HEK293T O +cells O +. O + +A O +co B-experimental_method +- I-experimental_method +immunoprecipitation I-experimental_method +assay I-experimental_method +using O +an O +anti O +- O +FLAG B-experimental_method +antibody O +followed O +by O +Western B-experimental_method +blot I-experimental_method +analysis O +using O +an O +anti O +- O +HA B-experimental_method +antibody O +showed O +that O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +forms O +self B-oligomeric_state +- I-oligomeric_state +oligomers I-oligomeric_state +in O +vivo O +( O +Fig O +. O +4A O +). O + +Co B-experimental_method +- I-experimental_method +immunoprecipitation I-experimental_method +analysis I-experimental_method +showed O +that O +the O +SEL1Lcent B-structure_element +was O +sufficient O +to O +physically O +interact O +with O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +, O +while O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +failed O +to O +do O +so O +( O +Fig O +. O +4A O +). O + +Interestingly O +, O +however O +, O +the O +expression O +level O +of O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +was O +consistently O +lower O +than O +that O +of O +SEL1Lcent B-structure_element +( O +Fig O +. O +4A O +, O +B O +). O + +Semi B-experimental_method +- I-experimental_method +quantitative I-experimental_method +RT I-experimental_method +- I-experimental_method +PCR I-experimental_method +revealed O +no O +significant O +difference O +in O +transcriptional O +levels O +of O +the O +two O +constructs O +( O +data O +not O +shown O +). O + +We O +speculated O +that O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +could O +be O +secreted O +while O +the O +SEL1Lcent B-structure_element +is O +retained O +in O +the O +ER O +by O +association O +with O +the O +endogenous O +ERAD O +complex O +. O + +We O +next O +examined O +if O +the O +reason O +why O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +failed O +to O +bind O +to O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +may O +be O +because O +of O +the O +lower O +level O +of O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +in O +the O +ER O +lumen O +compared O +to O +SEL1Lcent B-structure_element +fragment O +. O + +In O +order O +to O +retain O +two O +SEL1L B-protein +fragments O +in O +the O +ER O +lumen O +, O +we O +added O +KDEL B-structure_element +ER B-structure_element +retention I-structure_element +sequence I-structure_element +to O +the O +C O +- O +terminus O +of O +both O +fragments O +. O + +Indeed O +, O +the O +addition O +of O +KDEL B-structure_element +peptide O +increased O +the O +level O +of O +SEL1L348 B-mutant +– I-mutant +497 I-mutant +in O +the O +ER O +lumen O +( O +Fig O +. O +4D O +, O +E O +) O +and O +the O +immunostaining B-experimental_method +analysis O +showed O +both O +constructs O +were O +well O +localized O +to O +the O +ER O +( O +Fig O +. O +4C O +). O + +We O +further O +analyzed O +whether O +SEL1Lcent B-structure_element +may O +competitively O +inhibit O +the O +self O +- O +oligomerization O +of O +SEL1L B-protein +in O +vivo O +. O + +These O +data O +suggest O +that O +the O +SEL1L B-protein +forms O +self B-oligomeric_state +- I-oligomeric_state +oligomers I-oligomeric_state +and O +the O +oligomerization O +is O +mediated O +by O +the O +SEL1Lcent B-structure_element +domain O +in O +vivo O +. O + +Structural B-experimental_method +Comparison I-experimental_method +of O +SEL1L B-protein +SLRs B-structure_element +with O +TPRs B-structure_element +or O +SLRs B-structure_element +of O +Other O +Proteins O + +Previous O +studies O +reveal O +that O +TPRs B-structure_element +and O +SLRs B-structure_element +have O +similar O +consensus O +sequences O +, O +suggesting O +that O +their O +three O +- O +dimensional O +structures O +are O +also O +similar O +. O + +The O +superposition B-experimental_method +of O +isolated O +TPRs B-structure_element +from O +Cdc23 B-protein +( O +S B-species +. I-species +pombe I-species +, O +cell B-protein +division I-protein +cycle I-protein +23 I-protein +homolog O +, O +PDB O +code O +3ZN3 O +) O +and O +SLRs B-structure_element +from O +HcpC B-protein +( O +Helicobacter B-protein +Cysteine I-protein +- I-protein +rich I-protein +Protein I-protein +C I-protein +, O +PDB O +code O +1OUV O +) O +yields O +RMSDs B-evidence +below O +1 O +Å O +, O +confirming O +that O +the O +isolated O +repeats O +are O +indeed O +similar O +. O + +This O +is O +relevant O +to O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +, O +as O +isolated O +SLR B-structure_element +motifs O +from O +SEL1Lcent B-structure_element +showed O +good O +structural B-experimental_method +alignment I-experimental_method +with O +isolated O +TPRs B-structure_element +( O +RMSD B-evidence +1 O +. O +6 O +Å O +for O +all O +Cα O +chains O +) O +from O +Cdc23N B-protein +- O +term O +and O +SLRs B-structure_element +( O +RMSD B-evidence +0 O +. O +6 O +Å O +for O +all O +Cα O +chains O +) O +from O +HcpC B-protein +( O +Fig O +. O +5A O +). O + +However O +, O +superimposing B-experimental_method +the O +structure B-evidence +of O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +to I-structure_element +9 I-structure_element +from O +SEL1Lcent B-structure_element +onto O +the O +overall O +Cdc23N B-protein +- O +term O +or O +full B-protein_state +- I-protein_state +length I-protein_state +HcpC B-protein +structures B-evidence +revealed O +that O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +to I-structure_element +9 I-structure_element +in O +SEL1Lcent B-structure_element +have O +a O +different O +superhelical O +structure O +than O +either O +Cdc23 B-protein +or O +HcpC B-protein +( O +RMSD B-evidence +values O +of O +> O +2 O +. O +5 O +Å O +for O +Cα O +atoms O +) O +( O +Fig O +. O +5B O +). O + +The O +differences O +may O +result O +from O +the O +differing O +numbers O +of O +residues O +in O +the O +loops B-structure_element +and O +differences O +in O +antiparallel B-structure_element +helix I-structure_element +packing O +. O + +Moreover O +, O +there O +are O +conserved B-protein_state +disulfide B-ptm +bonds I-ptm +in O +the O +SLR B-structure_element +motifs O +of O +HcpC B-protein +and O +HcpB B-protein +, O +but O +no O +such O +bonds O +are O +observed O +in O +SEL1Lcent B-structure_element +. O + +These O +factors O +contribute O +to O +the O +differences O +in O +the O +overall O +conformation O +of O +the O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +and O +other O +SLR B-protein_type +or I-protein_type +TPR I-protein_type +motif I-protein_type +- I-protein_type +containing I-protein_type +proteins I-protein_type +. O + +Another O +major O +difference O +in O +the O +structure B-evidence +of O +SLR B-structure_element +motifs O +between O +SEL1L B-protein +and O +HcpC B-protein +is O +the O +oligomeric O +state O +of O +proteins O +. O + +The O +TPR B-structure_element +motif O +is O +involved O +in O +the O +dimerization B-oligomeric_state +of O +proteins O +such O +as O +Cdc23 B-protein +, O +Cdc16 B-protein +, O +and O +Cdc27 B-protein +. O + +In O +particular O +, O +the O +N O +- O +terminal O +domain O +of O +Cdc23 B-protein +( O +Cdc23N B-protein +- O +term O +) O +has O +a O +TPR B-structure_element +- O +motif O +organization O +similar O +to O +that O +of O +the O +SLR B-structure_element +motif O +in O +SEL1Lcent B-structure_element +. O + +The O +seven O +TPR B-structure_element +motifs O +of O +Cdc23N B-protein +- O +term O +are O +assembled O +into O +a O +superhelical B-structure_element +structure I-structure_element +, O +generating O +a O +hollow O +surface O +and O +encircling O +its O +dimer B-oligomeric_state +counterpart O +in O +an O +interlocking O +clasp O +- O +like O +arrangement O +( O +Fig O +. O +5C O +). O + +The O +TPR B-structure_element +motif I-structure_element +1 I-structure_element +( O +TPR1 B-structure_element +) O +of O +each O +Cdc23N B-protein +- O +term O +subunit O +is O +located O +in O +the O +hollow O +surface O +of O +the O +counter O +subunit O +and O +interacts O +with O +residues O +lining O +the O +inner B-site +groove I-site +TPR B-structure_element +α B-structure_element +- I-structure_element +helices I-structure_element +, O +generating O +two O +- O +fold O +symmetry O +homotype O +interactions O +. O + +However O +, O +in O +this O +structure B-evidence +, O +a O +conformational O +change O +in O +the O +TPR B-structure_element +motif O +itself O +is O +not O +observed O +. O + +Self O +- O +association O +of O +HcpC B-protein +has O +not O +been O +reported O +, O +and O +there O +is O +no O +domain B-protein_state +- I-protein_state +swapped I-protein_state +structure O +in O +the O +SLR B-structure_element +motifs O +of O +HcpC B-protein +, O +in O +contrast O +to O +that O +observed O +in O +SEL1Lcent B-structure_element +. O + +Although O +SEL1L B-protein +contains O +a O +number O +of O +SLR B-structure_element +motifs O +comparable O +to O +HcpC B-protein +, O +the O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +are O +interrupted O +by O +other O +sequences O +, O +making O +three O +SLR B-structure_element +motif O +clusters O +( O +Fig O +. O +1A O +). O + +The O +interrupted O +SLR B-structure_element +motifs O +may O +be O +required O +for O +dimerization B-oligomeric_state +of O +SEL1Lcent B-structure_element +, O +as O +five O +SLR B-structure_element +motifs O +are O +more O +than O +enough O +to O +form O +the O +semicircle B-structure_element +of I-structure_element +the I-structure_element +yin I-structure_element +- I-structure_element +yang I-structure_element +symbol O +( O +Fig O +. O +1B O +). O + +Helix B-structure_element +5A I-structure_element +from O +SLR B-structure_element +motif I-structure_element +5 I-structure_element +meets O +helix B-structure_element +9A I-structure_element +from O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +of O +the O +counterpart O +SEL1L B-protein +. O + +If O +the O +SLR B-structure_element +motifs I-structure_element +5 I-structure_element +to I-structure_element +9 I-structure_element +were O +not O +isolated O +from O +other O +SLR B-structure_element +motifs O +, O +steric O +hindrance O +could O +interfere O +with O +dimerization B-oligomeric_state +of O +SEL1L B-protein +. O + +TPR B-structure_element +and O +SLR B-structure_element +motifs O +are O +generally O +involved O +in O +protein O +- O +protein O +interaction O +modules O +, O +and O +the O +sequences O +between O +the O +SLR B-structure_element +motifs O +of O +SEL1L B-protein +might O +actually O +facilitate O +the O +self O +- O +association O +of O +this O +protein O +. O + +SLR B-structure_element +- I-structure_element +C I-structure_element +of O +SEL1L B-protein +Binds O +HRD1 B-protein +N O +- O +terminus O +Luminal B-structure_element +Loop I-structure_element + +Based O +on O +the O +structural B-evidence +data I-evidence +presented O +herein O +, O +a O +possible O +arrangement O +of O +membrane O +- O +associated O +ERAD O +components O +in O +mammals B-taxonomy_domain +, O +highlighting O +the O +molecular O +functions O +of O +SLR B-structure_element +domains O +in O +SEL1L B-protein +, O +is O +shown O +in O +Fig O +. O +6C O +. O + +We O +suggest O +that O +the O +middle O +SLR B-structure_element +domains O +are O +involved O +in O +the O +dimerization B-oligomeric_state +of O +SEL1L B-protein +based O +on O +the O +crystal B-evidence +structure I-evidence +and O +biochemical O +data O +. O + +SLR B-structure_element +- I-structure_element +C I-structure_element +, O +which O +contains O +SLR B-structure_element +motifs I-structure_element +10 I-structure_element +to I-structure_element +11 I-structure_element +, O +might O +be O +involved O +in O +the O +interaction O +with O +HRD1 B-protein +. O + +The O +Hrd3p B-protein +residues O +664 B-residue_range +– I-residue_range +695 I-residue_range +correspond O +to O +mouse B-taxonomy_domain +SEL1L B-protein +residues O +696 B-residue_range +– I-residue_range +727 I-residue_range +, O +which O +include O +the O +entire O +helix B-structure_element +11B I-structure_element +( O +residue O +697 B-residue_range +– I-residue_range +709 I-residue_range +) O +of O +SLR B-structure_element +motif I-structure_element +11 I-structure_element +and O +a O +well B-protein_state +- I-protein_state +conserved I-protein_state +adjacent O +region O +( O +Supplementary O +Fig O +. O +4 O +). O + +This O +observation O +is O +supported O +by O +the O +following O +: O +( O +1 O +) O +the O +meticulous O +range O +of O +SLR B-structure_element +motif I-structure_element +10 I-structure_element +to I-structure_element +11 I-structure_element +is O +newly O +established O +from O +a O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +SLR I-experimental_method +motif I-experimental_method +alignment I-experimental_method +, O +based O +on O +the O +present O +structure B-experimental_method +study I-experimental_method +, O +and O +( O +2 O +) O +the O +relatively O +high O +sequence B-protein_state +conservation I-protein_state +between O +mammalian B-taxonomy_domain +SEL1L B-protein +and O +yeast B-taxonomy_domain +Hrd3p B-protein +around O +SLR B-structure_element +motifs I-structure_element +10 I-structure_element +to I-structure_element +11 I-structure_element +, O +which O +contain O +contact O +regions O +with O +HRD1 B-protein +( O +Hrd1p B-protein +) O +( O +Supplementary O +Figs O +. O +4 O +and O +5 O +). O + +To O +address O +this O +hypothesis O +, O +we O +prepared O +constructs O +encoding O +mouse B-taxonomy_domain +HRD1 B-protein +luminal O +fragments O +fused B-experimental_method +to I-experimental_method +GST I-experimental_method +as O +shown O +in O +Fig O +. O +6A O +, O +and O +tested O +their O +ability O +to O +bind O +certain O +SLR B-structure_element +motifs O +in O +SEL1L B-protein +. O + +Figure O +6B O +shows O +that O +the O +SLR B-structure_element +- I-structure_element +C I-structure_element +, O +consisting O +of O +SLR B-structure_element +motifs I-structure_element +10 I-structure_element +and I-structure_element +11 I-structure_element +, O +exclusively O +interacts O +with O +N O +- O +terminal O +luminal B-structure_element +loop I-structure_element +( O +residues O +21 B-residue_range +– I-residue_range +42 I-residue_range +) O +of O +HRD1 B-protein +. O + +The O +molecular O +functions O +of O +SLR B-structure_element +- I-structure_element +N I-structure_element +are O +unclear O +. O + +SEL1Lcent B-structure_element +contains O +a O +putative O +N B-site +- I-site +glycosylation I-site +site I-site +, O +Asn B-residue_name_number +427 I-residue_name_number +, O +which O +is O +highly B-protein_state +conserved I-protein_state +among O +different O +species O +and O +structurally O +exposed O +to O +the O +surface O +of O +the O +SEL1L B-protein +dimer B-oligomeric_state +according O +to O +the O +crystal B-evidence +structure I-evidence +( O +Fig O +. O +6C O +). O + +Many O +reports O +demonstrate O +that O +membrane O +- O +bound O +ERAD O +machinery O +proteins O +in O +yeast B-taxonomy_domain +, O +such O +as O +Hrd1p B-protein +, O +Der1p B-protein +, O +and O +Usa1p B-protein +, O +are O +involved O +in O +oligomerization O +of O +ERAD O +components O +. O + +The O +Hrd1p B-protein +complex O +forms O +dimers B-oligomeric_state +upon O +sucrose B-experimental_method +gradient I-experimental_method +sedimentation I-experimental_method +and O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +. O + +Previous O +data O +show O +that O +HA B-protein_state +- I-protein_state +epitope I-protein_state +- I-protein_state +tagged I-protein_state +Hrd3p B-protein +or O +Hrd1p B-protein +efficiently O +co O +- O +precipitate O +with O +unmodified B-protein_state +Hrd3p B-protein +and O +Hrd1p B-protein +, O +respectively O +, O +suggesting O +that O +both O +Hrd1p B-protein +and O +Hrd3p B-protein +homodimers B-oligomeric_state +are O +involved O +in O +self O +- O +association O +of O +the O +Hrd B-complex_assembly +complex O +. O + +Considering O +that O +the O +functional O +and O +structural O +composition O +of O +ERAD O +components O +are O +conserved O +in O +both O +yeast B-taxonomy_domain +and O +mammals B-taxonomy_domain +, O +we O +propose O +that O +the O +mammalian B-taxonomy_domain +ERAD O +components O +also O +form O +self O +- O +associating O +oligomers B-oligomeric_state +. O + +We O +need O +to O +further O +test O +whether O +there O +are O +contacts O +involved O +in O +dimer B-oligomeric_state +formation O +in O +SEL1L B-protein +in O +addition O +to O +those O +in O +the O +SLR B-structure_element +- I-structure_element +M I-structure_element +region O +. O + +In O +yeast B-taxonomy_domain +, O +Usa1p B-protein +acts O +as O +a O +scaffold O +for O +Hrd1p B-protein +and O +Der1p B-protein +, O +in O +which O +the O +N O +- O +terminus O +of O +Usa1p B-protein +interacts O +with O +the O +C O +- O +terminal O +34 O +amino O +acids O +of O +Hrd1p B-protein +in O +the O +cytosol O +to O +induce O +oligomerization O +of O +Hrd1p B-protein +, O +which O +is O +essential O +for O +its O +activity O +. O + +Although O +mammalian B-taxonomy_domain +HERP B-protein_type +has O +sequences O +and O +domains O +that O +are O +conserved B-protein_state +in I-protein_state +Usa1p B-protein +, O +the O +molecular O +function O +of O +HERP B-protein_type +is O +not O +clearly O +related O +to O +that O +of O +Usa1p B-protein +. O + +This O +is O +further O +supported O +by O +our O +data O +showing O +that O +the O +SLR B-structure_element +- I-structure_element +C I-structure_element +of O +SEL1L B-protein +directly O +interacts O +with O +the O +luminal O +fragment O +of O +HRD1 B-protein +in O +the O +ER O +lumen O +. O + +Although O +the O +organization O +of O +membrane O +- O +bound O +HRD B-complex_assembly +complex O +components O +may O +be O +very O +similar O +between O +metazoans B-taxonomy_domain +and O +yeast B-taxonomy_domain +, O +the O +molecular O +details O +of O +interactions O +between O +the O +components O +may O +not O +necessarily O +be O +conserved O +. O + +Likewise O +, O +the O +dimerization B-oligomeric_state +of O +SEL1L B-protein +might O +provide O +stability O +for O +the O +mammalian B-taxonomy_domain +HRD B-complex_assembly +oligomer B-oligomeric_state +complex O +. O + +Further O +cell O +biological O +studies O +are O +required O +to O +clarify O +whether O +SEL1L B-protein +( O +Hrd3p B-protein +) O +dimerization B-oligomeric_state +could O +be O +cooperative O +with O +the O +oligomerization O +of O +the O +HRD B-complex_assembly +complex O +. O + +Considering O +that O +it O +is O +very O +important O +for O +the O +function O +of O +the O +HRD B-complex_assembly +complex O +that O +the O +components O +assemble O +as O +oligomers B-oligomeric_state +, O +we O +believe O +that O +the O +self O +- O +association O +of O +SEL1L B-protein +strongly O +contributes O +to O +generating O +active B-protein_state +forms O +of O +the O +HRD B-complex_assembly +complex O +, O +even O +in O +the O +absence B-protein_state +of I-protein_state +Usa1p B-protein +, O +in O +metazoans B-taxonomy_domain +. O + +These O +findings O +should O +provide O +a O +foundation O +for O +molecular O +- O +level O +studies O +to O +understand O +the O +membrane O +- O +associated O +HRD B-complex_assembly +complex O +assembly O +in O +ERAD O +. O + +( O +A O +) O +The O +diagram O +shows O +the O +domain O +structure O +of O +Mus B-species +musculus I-species +SEL1L B-protein +, O +as O +defined O +by O +proteolytic B-experimental_method +mapping I-experimental_method +and O +sequence B-experimental_method +/ I-experimental_method +structure I-experimental_method +analysis I-experimental_method +. O + +We O +determined O +the O +crystal B-evidence +structure I-evidence +of O +the O +SLR B-structure_element +- I-structure_element +M I-structure_element +, O +residues O +348 B-residue_range +– I-residue_range +533 I-residue_range +. O +( O +B O +) O +Ribbon O +diagram O +of O +the O +biological O +unit O +of O +the O +SEL1Lcent B-structure_element +, O +viewed O +along O +the O +two O +- O +fold O +NCS O +axis O +. O + +The O +crystal B-evidence +structure I-evidence +was O +determined O +by O +SAD B-experimental_method +phasing I-experimental_method +using O +selenium B-chemical +as O +the O +anomalous O +scatterer O +and O +refined O +to O +2 O +. O +6 O +Å O +resolution O +( O +Table O +1 O +). O + +( O +C O +) O +SEL1Lcent B-structure_element +ribbon O +diagram O +rotated O +90 O +° O +around O +a O +horizontal O +axis O +relative O +to O +( O +B O +). O + +( O +D O +) O +One O +protomer B-oligomeric_state +of O +the O +SEL1Lcent B-structure_element +dimer B-oligomeric_state +. O + +Starting O +from O +the O +N O +- O +terminus O +, O +SEL1Lcent B-structure_element +has O +five O +SLR B-structure_element +motifs O +comprising O +ten O +α B-structure_element +helices I-structure_element +. O + +Each O +SLR B-structure_element +motif O +( O +from O +5 O +to O +9 O +) O +is O +indicated O +in O +a O +different O +color O +. O +( O +E O +) O +Evolutionary O +conservation O +of O +surface O +residues O +in O +SEL1Lcent B-structure_element +, O +calculated O +using O +ConSurf B-experimental_method +, O +from O +a O +structure B-experimental_method +- I-experimental_method +based I-experimental_method +alignment I-experimental_method +of O +135 O +SEL1L B-protein +sequences O +. O + +The O +surface O +is O +colored O +from O +red O +( O +high O +) O +to O +white O +( O +poor O +) O +according O +to O +the O +degree O +of O +conservation O +in O +the O +SEL1L B-protein +phylogenetic O +orthologs O +. O + +The O +ribbon O +diagram O +of O +the O +counterpart O +protomer B-oligomeric_state +is O +drawn O +to O +show O +the O +orientation O +of O +the O +SEL1Lcent B-structure_element +dimer B-oligomeric_state +. O + +Dimer B-site +Interface I-site +of O +SEL1Lcent B-structure_element +. O + +Three O +distinct O +contact B-site +regions I-site +are O +indicated O +with O +labeled O +boxes O +. O + +The O +close O +- O +up O +view O +on O +the O +right O +shows O +the O +residues O +of O +SEL1Lcent B-structure_element +that O +contribute O +to O +dimer B-oligomeric_state +formation O +via O +the O +three O +contact B-site +interfaces I-site +. O + +The O +yellow O +dotted O +lines O +indicate O +intermolecular O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +two O +protomers B-oligomeric_state +of O +SEL1Lcent B-structure_element +. O +( O +B O +) O +Size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +( O +SEC B-experimental_method +) O +analysis O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +dimeric B-site +interface I-site +SEL1Lcent B-structure_element +mutants B-protein_state +to O +compare O +the O +oligomeric O +states O +of O +the O +proteins O +. O + +The O +standard O +molecular O +masses O +for O +the O +SEC B-experimental_method +experiments O +( O +top O +) O +were O +obtained O +from O +the O +following O +proteins O +: O +aldolase O +, O +158 O +kDa O +; O +cobalbumin O +, O +75 O +kDa O +; O +ovalbumin O +, O +44 O +kDa O +; O +and O +carbonic O +anhydrase O +, O +29 O +kDa O +. O + +Domain O +Swapping O +for O +Dimerization B-oligomeric_state +of O +SEL1Lcent B-structure_element +. O + +The O +11 O +SLR B-structure_element +motifs O +were O +aligned B-experimental_method +based O +on O +the O +present O +crystal B-evidence +structure I-evidence +of O +SEL1Lcent B-structure_element +. O + +The O +secondary O +structure O +elements O +are O +indicated O +above O +the O +sequences O +, O +with O +helices B-structure_element +depicted O +as O +cylinders O +. O + +The O +GG B-structure_element +sequence O +in O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +, O +which O +creates O +the O +hinge B-structure_element +for O +domain O +swapping O +( O +see O +text O +), O +is O +shaded O +yellow O +. O + +Stars O +below O +the O +sequences O +indicate O +the O +specific O +residues O +that O +commonly O +appear O +in O +SLRs B-structure_element +. O + +( O +B O +) O +Structure B-experimental_method +alignment I-experimental_method +of O +five O +SLR B-structure_element +motifs O +in O +SEL1Lcent B-structure_element +is O +shown O +to O +highlight O +the O +unusual O +geometry O +of O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +. O + +Each O +SLR B-structure_element +motif O +is O +shown O +in O +a O +different O +color O +. O + +Size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +was O +conducted O +as O +described O +in O +Fig O +. O +2B O +. O + +SEL1L B-protein +forms O +self B-oligomeric_state +- I-oligomeric_state +oligomer I-oligomeric_state +mediated O +by O +the O +SEL1Lcent B-structure_element +domain O +in O +vivo O +. O + +( O +A O +) O +HEK293T O +cells O +were O +transfected O +with O +the O +indicated O +plasmid O +constructs O +and O +the O +lysates O +were O +immunoprecipitated B-experimental_method +with O +an O +anti O +- O +FLAG B-experimental_method +antibody O +followed O +by O +western B-experimental_method +blot I-experimental_method +analysis O +using O +an O +anti O +- O +HA B-experimental_method +antibody O +. O + +The O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +- O +FLAG B-experimental_method +was O +co B-experimental_method +- I-experimental_method +immunoprecipitated I-experimental_method +with O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +- O +HA B-experimental_method +. O + +Also O +, O +SEL1Lcent B-structure_element +was O +co B-experimental_method +- I-experimental_method +immunoprecipitated I-experimental_method +with O +the O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +while O +the O +SLR B-structure_element +motif I-structure_element +9 I-structure_element +deletion B-experimental_method +failed O +to O +do O +so O +. O +( O +B O +) O +The O +HEK293T O +cells O +were O +transfected O +with O +the O +indicated O +plasmid O +constructs O +and O +the O +cell O +lysate O +and O +culture O +media O +were O +analyzed O +by O +western B-experimental_method +blot I-experimental_method +analysis O +and O +immunoprecipitation B-experimental_method +respectively O +. O + +The O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +forms O +self B-oligomeric_state +- I-oligomeric_state +oligomers I-oligomeric_state +and O +the O +SEL1Lcent B-mutant +- I-mutant +FLAG I-mutant +- I-mutant +KDEL I-mutant +was O +co B-experimental_method +- I-experimental_method +immunoprecipitated I-experimental_method +with O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +- O +HA B-experimental_method +. O + +SEL1L348 B-mutant +– I-mutant +497 I-mutant +- I-mutant +FLAG I-mutant +- I-mutant +KDEL I-mutant +did O +not O +co O +- O +immunoprecipitate O +with O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +- O +HA B-experimental_method +. O + +The O +white O +asterisks O +indicate O +non O +- O +specific O +bands O +. O +( O +E O +) O +SEL1Lcent B-mutant +- I-mutant +HA I-mutant +- I-mutant +KDEL I-mutant +competitively O +inhibited O +self O +- O +oligomerization O +of O +full B-protein_state +- I-protein_state +length I-protein_state +SEL1L B-protein +. O + +The O +indicated O +plasmid O +constructs O +were O +transfected O +and O +immunoprecipitation B-experimental_method +assay I-experimental_method +was O +performed O +using O +an O +anti O +- O +FLAG B-experimental_method +antibody O +followed O +by O +western B-experimental_method +blot I-experimental_method +analysis O +using O +an O +anti O +- O +HA B-experimental_method +antibody O +. O + +The O +red O +rectangle O +indicates O +competitively O +inhibited O +SEL1L B-protein +self O +- O +oligomer B-oligomeric_state +formation O +by O +the O +increasing O +doses O +of O +SEL1Lcent B-mutant +- I-mutant +HA I-mutant +- I-mutant +KDEL I-mutant +. O +( O +F O +) O +L521A B-mutant +point B-protein_state +mutant I-protein_state +in O +SEL1Lcent B-structure_element +did O +not O +inhibit O +the O +self O +- O +association O +of O +SEL1L B-protein +. O + +Comparison O +of O +SLR B-structure_element +in O +SEL1L B-protein +with O +TPR B-structure_element +or O +Other O +SLR B-protein_type +- I-protein_type +Containing I-protein_type +Proteins I-protein_type +. O + +The O +SEL1L B-protein +, O +Cdc23 B-protein +, O +and O +HcpC B-protein +are O +colored O +magenta O +, O +green O +and O +cyan O +, O +respectively O +. O + +Both O +SEL1Lcent B-structure_element +schematics O +are O +identically O +oriented O +for O +comparison O +. O + +The O +Cα O +atoms O +of O +the O +residues O +in O +each O +α B-structure_element +- I-structure_element +solenoid I-structure_element +domain I-structure_element +are O +superimposed B-experimental_method +with O +a O +root B-evidence +- I-evidence +mean I-evidence +- I-evidence +squared I-evidence +deviation I-evidence +of O +3 O +. O +3 O +Å O +for O +Cdc23 B-protein +and O +SEL1Lcent B-structure_element +( O +left O +), O +and O +2 O +. O +5 O +Å O +for O +HcpC B-protein +and O +SEL1Lcent B-structure_element +( O +right O +). O + +SEL1Lcent B-structure_element +, O +Cdc23 B-protein +, O +and O +HcpC B-protein +are O +colored O +as O +in O +( O +A O +). O +( O +C O +) O +Ribbon O +diagram O +showing O +the O +overall O +structure B-evidence +of O +Cdc23N B-protein +- O +term O +( O +left O +) O +and O +SEL1Lcent B-structure_element +( O +right O +) O +to O +compare O +their O +similarities O +regarding O +dimer B-oligomeric_state +formation O +through O +domain O +swapping O +. O + +The O +Role O +of O +SLR B-structure_element +- I-structure_element +C I-structure_element +in O +ERAD O +machinery O +and O +Model O +for O +the O +Organization O +of O +Proteins O +in O +Membrane O +- O +Associated O +ERAD O +Components O +. O + +( O +A O +) O +Schematic O +diagram O +shows O +three O +HRD1 B-protein +fragment O +constructs O +used O +in O +the O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +experiment O +. O +( O +B O +) O +Pull B-experimental_method +- I-experimental_method +down I-experimental_method +experiments I-experimental_method +to O +examine O +the O +interactions O +between O +HRD B-complex_assembly +luminal B-structure_element +loops I-structure_element +and O +certain O +SLR B-structure_element +motifs O +of O +SEL1L B-protein +. O + +( O +C O +) O +Schematic O +representation O +of O +the O +organization O +of O +metazoan B-taxonomy_domain +ERAD O +components O +in O +the O +ER O +membrane O +. O + +We O +hypothesized O +that O +the O +interrupted O +SLR B-structure_element +motifs O +of O +SEL1L B-protein +have O +distinct O +functions O +such O +that O +the O +SLR B-structure_element +- I-structure_element +M I-structure_element +is O +important O +for O +dimer B-oligomeric_state +formation O +of O +the O +protein O +, O +and O +SLR B-structure_element +- I-structure_element +C I-structure_element +is O +involved O +in O +the O +interaction O +with O +HRD1 B-protein +in O +the O +ER O +lumen O +. O + +The O +surface O +representation O +of O +SEL1Lcent B-structure_element +is O +placed O +in O +the O +same O +orientation O +as O +that O +shown O +in O +the O +schematic O +model O +to O +show O +that O +the O +putative O +N B-site +- I-site +glycosylation I-site +site I-site +, O +residue O +N427 B-residue_name_number +( O +indicated O +in O +yellow O +), O +is O +exposed O +on O +the O +surface O +of O +the O +protein O +. O + +Sequence B-experimental_method +alignment I-experimental_method +suggests O +that O +both O +kinases B-protein_type +belong O +to O +the O +ribulokinase B-protein_type +- I-protein_type +like I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +, O +a O +sub O +- O +family O +of O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +. O + +Here O +we O +solved B-experimental_method +the O +structures B-evidence +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +in O +both O +their O +apo B-protein_state +forms O +and O +in B-protein_state +complex I-protein_state +with I-protein_state +nucleotide B-chemical +substrates O +. O + +The O +two O +kinases O +exhibit O +nearly O +identical O +overall O +architecture O +, O +with O +both O +kinases B-protein_type +possessing O +ATP B-chemical +hydrolysis O +activity O +in O +the O +absence B-protein_state +of I-protein_state +substrates I-protein_state +. O + +In O +addition O +, O +our O +enzymatic B-experimental_method +assays I-experimental_method +suggested O +that O +SePSK B-protein +has O +the O +capability O +to O +phosphorylate O +D B-chemical +- I-chemical +ribulose I-chemical +. O + +Moreover O +, O +our O +structural B-experimental_method +comparison I-experimental_method +with O +other O +family O +members O +suggests O +that O +there O +are O +major O +conformational O +changes O +in O +SePSK B-protein +upon O +substrate O +binding O +, O +facilitating O +the O +catalytic O +process O +. O + +Phosphorylation B-ptm +is O +one O +of O +the O +various O +pivotal O +modifications O +of O +carbohydrates B-chemical +, O +and O +is O +catalyzed O +by O +specific O +sugar B-protein_type +kinases I-protein_type +. O + +These O +kinases B-protein_type +exhibit O +considerable O +differences O +in O +their O +folding O +pattern O +and O +substrate O +specificity O +. O + +Based O +on O +sequence B-experimental_method +analysis I-experimental_method +, O +they O +can O +be O +divided O +into O +four O +families O +, O +namely O +HSP B-protein_type +70_NBD I-protein_type +family I-protein_type +, O +FGGY B-protein_type +family I-protein_type +, O +Mer_B B-protein_type +like I-protein_type +family I-protein_type +and O +Parm_like B-protein_type +family I-protein_type +. O + +These O +sugar B-chemical +substrates O +include O +L B-chemical +- I-chemical +ribulose I-chemical +, O +erythritol B-chemical +, O +L B-chemical +- I-chemical +fuculose I-chemical +, O +D B-chemical +- I-chemical +glycerol I-chemical +, O +D B-chemical +- I-chemical +gluconate I-chemical +, O +L B-chemical +- I-chemical +xylulose I-chemical +, O +D B-chemical +- I-chemical +ribulose I-chemical +, O +L B-chemical +- I-chemical +rhamnulose I-chemical +and O +D B-chemical +- I-chemical +xylulose I-chemical +. O + +Structures B-evidence +reported O +in O +the O +Protein O +Data O +Bank O +of O +the O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +exhibit O +a O +similar O +overall O +architecture O +containing O +two O +protein O +domains O +, O +one O +of O +which O +is O +responsible O +for O +the O +binding O +of O +substrate O +, O +while O +the O +second O +is O +used O +for O +binding O +cofactor O +ATP B-chemical +. O + +While O +the O +binding B-site +pockets I-site +for O +substrates O +are O +at O +the O +same O +position O +, O +each O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +uses O +different O +substrate B-site +- I-site +binding I-site +residues I-site +, O +resulting O +in O +high O +substrate O +specificity O +. O + +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +display O +a O +sequence O +identity O +of O +44 O +. O +9 O +%, O +and O +belong O +to O +the O +ribulokinase B-protein_type +- I-protein_type +like I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +, O +a O +sub O +- O +family O +of O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +. O + +Members O +of O +this O +sub O +- O +family O +are O +responsible O +for O +the O +phosphorylation B-ptm +of O +sugars B-chemical +similar O +to O +L B-chemical +- I-chemical +ribulose I-chemical +and O +D B-chemical +- I-chemical +ribulose I-chemical +. O + +The O +sequence O +and O +the O +substrate O +specificity O +of O +ribulokinase B-protein_type +- I-protein_type +like I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +are O +different O +, O +but O +they O +share O +the O +common O +folding O +feature O +with O +two O +domains O +. O + +Domain B-structure_element +I I-structure_element +exhibits O +a O +ribonuclease B-structure_element +H I-structure_element +- I-structure_element +like I-structure_element +folding I-structure_element +pattern I-structure_element +, O +and O +is O +responsible O +for O +the O +substrate O +binding O +, O +while O +domain B-structure_element +II I-structure_element +possesses O +an O +actin B-structure_element +- I-structure_element +like I-structure_element +ATPase I-structure_element +domain I-structure_element +that O +binds O +cofactor O +ATP B-chemical +. O + +It O +was O +shown O +that O +XK B-protein +- I-protein +2 I-protein +( O +At5g49650 B-gene +) O +located O +in O +the O +cytosol O +is O +indeed O +xylulose B-protein_type +kinase I-protein_type +. O + +However O +, O +the O +function O +of O +XK B-protein +- I-protein +1 I-protein +( O +At2g21370 B-gene +) O +inside O +the O +chloroplast O +stroma O +has O +remained O +unknown O +. O + +SePSK B-protein +from O +Synechococcus B-species +elongatus I-species +strain I-species +PCC I-species +7942 I-species +is O +the O +homolog O +of O +AtXK B-protein +- I-protein +1 I-protein +, O +though O +its O +physiological O +function O +and O +substrates O +remain O +unclear O +. O + +In O +order O +to O +obtain O +functional O +and O +structural O +information O +about O +these O +two O +proteins O +, O +here O +we O +reported O +the O +crystal B-evidence +structures I-evidence +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +. O + +Our O +findings O +provide O +new O +details O +of O +the O +catalytic O +mechanism O +of O +SePSK B-protein +and O +lay O +the O +foundation O +for O +future O +studies O +into O +its O +homologs O +in O +eukaryotes B-taxonomy_domain +. O + +Overall O +structures B-evidence +of O +apo B-protein_state +- O +SePSK B-protein +and O +apo B-protein_state +- O +AtXK B-protein +- I-protein +1 I-protein + +We O +therefore O +used O +single B-experimental_method +isomorphous I-experimental_method +replacement I-experimental_method +anomalous I-experimental_method +scattering I-experimental_method +method I-experimental_method +( O +SIRAS B-experimental_method +) O +for O +successful O +solution O +of O +the O +apo B-protein_state +- O +SePSK B-protein +structure B-evidence +at O +a O +resolution O +of O +2 O +. O +3 O +Å O +. O +Subsequently O +, O +the O +apo B-protein_state +- O +SePSK B-protein +structure B-evidence +was O +used O +as O +molecular B-experimental_method +replacement I-experimental_method +model I-experimental_method +to O +solve O +all O +other O +structures B-evidence +identified O +in O +this O +study O +. O + +The O +amino O +- O +acid O +residues O +were O +traced O +from O +Val2 B-residue_name_number +to O +His419 B-residue_name_number +, O +except O +for O +the O +Met1 B-residue_name_number +residue O +and O +the O +seven O +residues O +at O +the O +C O +- O +termini O +. O + +Apo B-protein_state +- O +SePSK B-protein +contains O +two O +domains O +referred O +to O +further O +on O +as O +domain B-structure_element +I I-structure_element +and O +domain B-structure_element +II I-structure_element +( O +Fig O +1A O +). O + +Domain B-structure_element +I I-structure_element +consists O +of O +non O +- O +contiguous O +portions O +of O +the O +polypeptide O +chains O +( O +aa O +. O + +2 B-residue_range +– I-residue_range +228 I-residue_range +and O +aa O +. O + +402 B-residue_range +– I-residue_range +419 I-residue_range +), O +exhibiting O +11 O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +11 O +β B-structure_element +- I-structure_element +sheets I-structure_element +. O + +In O +addition O +, O +four O +β B-structure_element +- I-structure_element +sheets I-structure_element +( O +β7 B-structure_element +, O +β10 B-structure_element +, O +β12 B-structure_element +and O +β16 B-structure_element +) O +and O +five O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +α8 B-structure_element +, O +α9 B-structure_element +, O +α13 B-structure_element +, O +α14 B-structure_element +and O +α15 B-structure_element +) O +flank O +the O +left O +side O +of O +the O +core B-structure_element +region I-structure_element +. O + +229 B-residue_range +– I-residue_range +401 I-residue_range +and O +classified O +into O +B2 B-structure_element +( O +β31 B-structure_element +/ O +β29 B-structure_element +/ O +β22 B-structure_element +/ O +β23 B-structure_element +/ O +β25 B-structure_element +/ O +β24 B-structure_element +) O +and O +A3 B-structure_element +( O +α26 B-structure_element +/ O +α27 B-structure_element +/ O +α28 B-structure_element +/ O +α30 B-structure_element +) O +( O +Fig O +1A O +and O +S1 O +Fig O +). O + +In O +the O +SePSK B-protein +structure B-evidence +, O +B1 B-structure_element +and O +B2 B-structure_element +are O +sandwiched O +by O +A1 B-structure_element +, O +A2 B-structure_element +and O +A3 B-structure_element +, O +and O +the O +whole O +structure B-evidence +shows O +the O +A1 B-structure_element +/ O +B1 B-structure_element +/ O +A2 B-structure_element +/ O +B2 B-structure_element +/ O +A3 B-structure_element +( O +α B-structure_element +/ O +β B-structure_element +/ O +α B-structure_element +/ O +β B-structure_element +/ O +α B-structure_element +) O +folding O +pattern O +, O +which O +is O +in O +common O +with O +other O +members O +of O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +( O +S2 O +Fig O +). O + +The O +overall O +folding O +of O +SePSK B-protein +resembles O +a O +clip O +, O +with O +A2 B-structure_element +of O +domain B-structure_element +I I-structure_element +acting O +as O +a O +hinge B-structure_element +region I-structure_element +. O + +Overall O +structures B-evidence +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +. O + +The O +secondary O +structural O +elements O +are O +indicated O +( O +α B-structure_element +- I-structure_element +helix I-structure_element +: O +cyan O +, O +β B-structure_element +- I-structure_element +sheet I-structure_element +: O +yellow O +). O + +The O +secondary O +structural O +elements O +are O +indicated O +( O +α B-structure_element +- I-structure_element +helix I-structure_element +: O +green O +, O +β B-structure_element +- I-structure_element +sheet I-structure_element +: O +wheat O +). O + +Apo B-protein_state +- O +AtXK B-protein +- I-protein +1 I-protein +exhibits O +a O +folding O +pattern O +similar O +to O +that O +of O +SePSK B-protein +in O +line O +with O +their O +high O +sequence O +identity O +( O +Fig O +1B O +and O +S1 O +Fig O +). O + +However O +, O +superposition B-experimental_method +of O +structures B-evidence +of O +AtXK B-protein +- I-protein +1 I-protein +and O +SePSK B-protein +shows O +some O +differences O +, O +especially O +at O +the O +loop B-structure_element +regions I-structure_element +. O + +A O +considerable O +difference O +is O +found O +in O +the O +loop3 B-structure_element +linking O +β3 B-structure_element +and O +α4 B-structure_element +, O +which O +is O +stretched O +out O +in O +the O +AtXK B-protein +- I-protein +1 I-protein +structure B-evidence +, O +while O +in O +the O +SePSK B-protein +structure B-evidence +, O +it O +is O +bent O +back O +towards O +the O +inner O +part O +. O + +The O +corresponding O +residues O +between O +these O +two O +structures B-evidence +( O +SePSK B-protein +- O +Lys35 B-residue_name_number +and O +AtXK B-protein +- I-protein +1 I-protein +- O +Lys48 B-residue_name_number +) O +have O +a O +distance O +of O +15 O +. O +4 O +Å O +( O +S3 O +Fig O +). O + +Activity B-experimental_method +assays I-experimental_method +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein + +In O +order O +to O +understand O +the O +function O +of O +these O +two O +kinases O +, O +we O +performed O +structural B-experimental_method +comparison I-experimental_method +using O +Dali B-experimental_method +server I-experimental_method +. O + +The O +structures B-evidence +most O +closely O +related O +to O +SePSK B-protein +are O +xylulose B-protein_type +kinase I-protein_type +, O +glycerol B-protein_type +kinase I-protein_type +and O +ribulose B-protein_type +kinase I-protein_type +, O +implying O +that O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +might O +function O +similarly O +to O +these O +kinases B-protein_type +. O + +We O +first O +tested O +whether O +both O +enzymes O +possessed O +ATP B-chemical +hydrolysis O +activity O +in O +the O +absence B-protein_state +of I-protein_state +substrates O +. O + +As O +shown O +in O +Fig O +2A O +, O +both O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +exhibited O +ATP B-chemical +hydrolysis O +activity O +. O + +To O +further O +identify O +the O +actual O +substrate O +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +, O +five O +different O +sugar O +molecules O +, O +including O +D B-chemical +- I-chemical +ribulose I-chemical +, O +L B-chemical +- I-chemical +ribulose I-chemical +, O +D B-chemical +- I-chemical +xylulose I-chemical +, O +L B-chemical +- I-chemical +xylulose I-chemical +and O +Glycerol B-chemical +, O +were O +used O +in O +enzymatic B-experimental_method +activity I-experimental_method +assays I-experimental_method +. O + +As O +shown O +in O +Fig O +2B O +, O +the O +ATP B-chemical +hydrolysis O +activity O +of O +SePSK B-protein +greatly O +increased O +upon O +adding O +D B-chemical +- I-chemical +ribulose I-chemical +than O +adding O +other O +potential O +substrates O +, O +suggesting O +that O +it O +has O +D B-protein_type +- I-protein_type +ribulose I-protein_type +kinase I-protein_type +activity O +. O + +The O +enzymatic B-experimental_method +activity I-experimental_method +assays I-experimental_method +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +. O + +( O +A O +) O +The O +ATP B-chemical +hydrolysis O +activity O +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +. O + +( O +B O +) O +The O +ATP B-chemical +hydrolysis O +activity O +of O +SePSK B-protein +with O +addition O +of O +five O +different O +substrates O +. O + +The O +substrates O +are O +DR B-chemical +( O +D B-chemical +- I-chemical +ribulose I-chemical +), O +LR B-chemical +( O +L B-chemical +- I-chemical +ribulose I-chemical +), O +DX B-chemical +( O +D B-chemical +- I-chemical +xylulose I-chemical +), O +LX B-chemical +( O +L B-chemical +- I-chemical +xylulose I-chemical +) O +and O +GLY B-chemical +( O +Glycerol B-chemical +). O +( O +C O +) O +The O +ATP B-chemical +hydrolysis O +activity O +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +with O +or O +without O +D B-chemical +- I-chemical +ribulose I-chemical +. O +( O +D O +) O +The O +ATP B-chemical +hydrolysis O +activity O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +and O +single O +- O +site O +mutants O +of O +SePSK B-protein +. O + +Three O +single O +- O +site O +mutants O +of O +SePSK B-protein +are O +D8A B-mutant +- O +SePSK B-protein +, O +T11A B-mutant +- O +SePSK B-protein +and O +D221A B-mutant +- O +SePSK B-protein +. O + +Mutations B-experimental_method +of O +the O +corresponding O +residue O +in O +xylulose B-protein_type +kinase I-protein_type +and O +glycerol B-protein_type +kinase I-protein_type +from O +Escherichia B-species +coli I-species +greatly O +reduced O +their O +activity O +. O + +To O +identify O +the O +function O +of O +these O +three O +residues O +of O +SePSK B-protein +, O +we O +constructed O +D8A B-mutant +, O +T11A B-mutant +and O +D221A B-mutant +mutants B-protein_state +. O + +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +possess O +a O +similar O +ATP B-site +binding I-site +site I-site + +To O +obtain O +more O +detailed O +information O +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +in B-protein_state +complex I-protein_state +with I-protein_state +ATP B-chemical +, O +we O +soaked B-experimental_method +the O +apo B-protein_state +- O +crystals B-evidence +in O +the O +reservoir O +adding O +cofactor O +ATP B-chemical +, O +and O +obtained O +the O +structures B-evidence +of O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +bound B-protein_state +with I-protein_state +ATP B-chemical +at O +the O +resolution O +of O +2 O +. O +3 O +Å O +and O +1 O +. O +8 O +Å O +, O +respectively O +. O + +In O +both O +structures B-evidence +, O +a O +strong O +electron B-evidence +density I-evidence +was O +found O +in O +the O +conserved B-protein_state +ATP B-site +binding I-site +pocket I-site +, O +but O +can O +only O +be O +fitted O +with O +an O +ADP B-chemical +molecule O +( O +S4 O +Fig O +). O + +This O +result O +was O +consistent O +with O +our O +enzymatic B-experimental_method +activity I-experimental_method +assays I-experimental_method +where O +SePSK B-protein +and O +AtXK B-protein +- I-protein +1 I-protein +showed O +ATP B-chemical +hydrolysis O +activity O +without O +adding O +any O +substrates O +( O +Fig O +2A O +and O +2C O +). O + +To O +avoid O +hydrolysis O +of O +ATP B-chemical +, O +we O +soaked B-experimental_method +the O +crystals B-evidence +of O +apo B-protein_state +- O +SePSK B-protein +and O +apo B-protein_state +- O +AtXK B-protein +- I-protein +1 I-protein +into O +the O +reservoir O +adding O +AMP B-chemical +- I-chemical +PNP I-chemical +. O + +However O +, O +we O +found O +that O +the O +electron B-evidence +densities I-evidence +of O +γ O +- O +phosphate B-chemical +group O +of O +AMP B-chemical +- I-chemical +PNP I-chemical +( O +AMP B-chemical +- I-chemical +PNP I-chemical +γ O +- O +phosphate B-chemical +) O +are O +still O +weak O +in O +the O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +and O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +AtXK I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +structures B-evidence +, O +suggesting O +high O +flexibility O +of O +ATP B-chemical +- O +γ O +- O +phosphate B-chemical +. O + +The O +γ O +- O +phosphate B-chemical +group O +of O +ATP B-chemical +is O +transferred O +to O +the O +sugar B-chemical +substrate O +during O +the O +reaction O +process O +, O +so O +this O +flexibility O +might O +be O +important O +for O +the O +ability O +of O +these O +kinases B-protein_type +. O + +The O +overall O +structures B-evidence +as O +well O +as O +the O +coordination O +modes O +of O +ADP B-chemical +and O +AMP B-chemical +- I-chemical +PNP I-chemical +in O +the O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +AtXK I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +, O +ADP B-complex_assembly +- I-complex_assembly +AtXK I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +, O +ADP B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +and O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +structures B-evidence +are O +nearly O +identical O +( O +S5 O +Fig O +), O +therefore O +the O +structure B-evidence +of O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +is O +used O +here O +to O +describe O +the O +structural O +details O +and O +to O +compare O +with O +those O +of O +other O +family O +members O +. O + +The O +AMP B-site +- I-site +PNP I-site +binding I-site +pocket I-site +consists O +of O +four B-structure_element +α I-structure_element +- I-structure_element +helices I-structure_element +( O +α26 B-structure_element +, O +α28 B-structure_element +, O +α27 B-structure_element +and O +α30 B-structure_element +) O +and O +forms O +a O +shape B-protein_state +resembling I-protein_state +a I-protein_state +half I-protein_state +- I-protein_state +fist I-protein_state +( O +Fig O +3A O +and O +3B O +). O + +The O +head O +group O +of O +the O +AMP B-chemical +- I-chemical +PNP I-chemical +is O +embedded O +in O +a O +pocket B-site +surrounded O +by O +Trp383 B-residue_name_number +, O +Asn380 B-residue_name_number +, O +Gly376 B-residue_name_number +and O +Gly377 B-residue_name_number +. O + +The O +purine O +ring O +of O +AMP B-chemical +- I-chemical +PNP I-chemical +is O +positioned O +in O +parallel O +to O +the O +indole O +ring O +of O +Trp383 B-residue_name_number +. O + +In O +addition O +, O +it O +is O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +with O +the O +side O +chain O +amide O +of O +Asn380 B-residue_name_number +( O +Fig O +3B O +). O + +The O +tail O +of O +AMP B-chemical +- I-chemical +PNP I-chemical +points O +to O +the O +hinge B-structure_element +region I-structure_element +of O +SePSK B-protein +, O +and O +its O +α O +- O +phosphate B-chemical +and O +β O +- O +phosphate B-chemical +groups O +are O +stabilized O +by O +Gly376 B-residue_name_number +and O +Ser243 B-residue_name_number +, O +respectively O +. O + +Together O +, O +this O +structure B-evidence +clearly O +shows O +that O +the O +AMP B-chemical +- I-chemical +PNP I-chemical +- O +β O +- O +phosphate B-chemical +is O +sticking O +out O +of O +the O +ATP B-site +binding I-site +pocket I-site +, O +thus O +the O +γ O +- O +phosphate B-chemical +group O +is O +at O +the O +empty O +space O +between O +domain B-structure_element +I I-structure_element +and O +domain B-structure_element +II I-structure_element +and O +is O +unconstrained O +in O +its O +movement O +by O +the O +protein O +. O + +Structure B-evidence +of O +SePSK B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +AMP B-chemical +- I-chemical +PNP I-chemical +. O + +( O +A O +) O +The O +electron B-evidence +density I-evidence +of O +AMP B-chemical +- I-chemical +PNP I-chemical +. O + +The O +AMP B-chemical +- I-chemical +PNP I-chemical +is O +depicted O +as O +sticks O +with O +its O +ǀFoǀ B-evidence +- I-evidence +ǀFcǀ I-evidence +map I-evidence +contoured O +at O +3 O +σ O +shown O +as O +cyan O +mesh O +. O + +The O +head O +of O +AMP B-chemical +- I-chemical +PNP I-chemical +is O +sandwiched B-bond_interaction +by I-bond_interaction +four O +residues O +( O +Leu293 B-residue_name_number +, O +Gly376 B-residue_name_number +, O +Gly377 B-residue_name_number +and O +Trp383 B-residue_name_number +). O + +The O +four O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +α26 B-structure_element +, O +α28 B-structure_element +, O +α27 B-structure_element +and O +α30 B-structure_element +) O +are O +labeled O +in O +red O +. O + +The O +AMP B-chemical +- I-chemical +PNP I-chemical +and O +coordinated O +residues O +are O +shown O +as O +sticks O +. O + +The O +potential O +substrate B-site +binding I-site +site I-site +in O +SePSK B-protein + +The O +results O +from O +our O +activity B-experimental_method +assays I-experimental_method +suggested O +that O +SePSK B-protein +has O +D B-protein_type +- I-protein_type +ribulose I-protein_type +kinase I-protein_type +activity O +. O + +To O +better O +understand O +the O +interaction O +pattern O +between O +SePSK B-protein +and O +D B-chemical +- I-chemical +ribulose I-chemical +, O +the O +apo B-protein_state +- O +SePSK B-protein +crystals B-experimental_method +were I-experimental_method +soaked I-experimental_method +into I-experimental_method +the O +reservoir B-experimental_method +with O +10 O +mM O +D B-chemical +- I-chemical +ribulose I-chemical +( O +RBL B-chemical +) O +and O +the O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +structure B-evidence +was O +solved B-experimental_method +. O + +As O +shown O +in O +S6 O +Fig O +, O +two O +residual O +electron B-evidence +densities I-evidence +are O +visible O +in O +domain B-structure_element +I I-structure_element +, O +which O +can O +be O +interpreted O +as O +two O +D B-chemical +- I-chemical +ribulose I-chemical +molecules O +with O +reasonable O +fit O +. O + +As O +shown O +in O +Fig O +4A O +, O +the O +nearest O +distance O +between O +the O +carbon O +skeleton O +of O +two O +D B-chemical +- I-chemical +ribulose I-chemical +molecules O +are O +approx O +. O + +RBL1 B-residue_name_number +is O +located O +in O +the O +pocket B-site +consisting O +of O +α21 B-structure_element +and O +the O +loop B-structure_element +between O +β6 B-structure_element +and I-structure_element +β7 I-structure_element +. O + +The O +O4 O +and O +O5 O +of O +RBL1 B-residue_name_number +are O +coordinated B-bond_interaction +with I-bond_interaction +the O +side O +chain O +carboxyl O +group O +of O +Asp221 B-residue_name_number +. O + +This O +pocket B-site +is O +at O +a O +similar O +position O +of O +substrate B-site +binding I-site +site I-site +of O +other O +sugar B-protein_type +kinase I-protein_type +, O +such O +as O +L B-protein +- I-protein +ribulokinase I-protein +( O +PDB O +code O +: O +3QDK O +) O +( O +S7 O +Fig O +). O + +Glu329 B-residue_name_number +in O +3QDK O +has O +no O +counterpart O +in O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +structure B-evidence +. O + +In O +addition O +, O +although O +Lys208 B-residue_name_number +of O +L B-protein +- I-protein +ribulokinase I-protein +has O +the O +corresponding O +residue O +( O +Lys163 B-residue_name_number +) O +in O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +structure B-evidence +, O +the O +hydrogen B-bond_interaction +bond I-bond_interaction +of O +Lys163 B-residue_name_number +is O +broken O +because O +of O +the O +conformational O +change O +of O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +α9 B-structure_element +and O +α13 B-structure_element +) O +of O +SePSK B-protein +. O + +The O +binding O +of O +D B-chemical +- I-chemical +ribulose I-chemical +( O +RBL B-chemical +) O +with O +SePSK B-protein +. O + +( O +A O +) O +The O +electrostatic B-evidence +potential I-evidence +surface I-evidence +map I-evidence +of O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +and O +a O +zoom O +- O +in O +view O +of O +RBL B-site +binding I-site +site I-site +. O + +The O +RBL1 B-residue_name_number +and O +RBL2 B-residue_name_number +are O +depicted O +as O +sticks O +. O +( O +B O +) O +Interaction O +of O +two O +D B-chemical +- I-chemical +ribulose I-chemical +molecules O +( O +RBL1 B-residue_name_number +and O +RBL2 B-residue_name_number +) O +with O +SePSK B-protein +. O + +The O +hydrogen B-bond_interaction +bonds I-bond_interaction +are O +indicated O +by O +the O +black O +dashed O +lines O +and O +the O +numbers O +near O +the O +dashed O +lines O +are O +the O +distances O +( O +Å O +). O +( O +C O +) O +The O +binding B-experimental_method +affinity I-experimental_method +assays I-experimental_method +of O +SePSK B-protein +with O +D B-chemical +- I-chemical +ribulose I-chemical +. O + +Single B-experimental_method +- I-experimental_method +cycle I-experimental_method +kinetic I-experimental_method +data I-experimental_method +are O +reflecting O +the O +interaction O +of O +SePSK B-protein +and O +D8A B-mutant +- O +SePSK B-protein +with O +D B-chemical +- I-chemical +ribulose I-chemical +. O + +It O +shows O +two O +experimental O +sensorgrams B-evidence +after O +minus O +the O +empty O +sensorgrams B-evidence +. O + +Dissociation B-evidence +rate I-evidence +constant I-evidence +of O +wild B-protein_state +type I-protein_state +and O +D8A B-mutant +- O +SePSK B-protein +are O +3 O +ms O +- O +1 O +and O +9 O +ms O +- O +1 O +, O +respectively O +. O + +The O +binding B-site +pocket I-site +of O +RBL2 B-residue_name_number +with O +relatively O +weak O +electron B-evidence +density I-evidence +is O +near O +the O +N O +- O +terminal O +region O +of O +SePSK B-protein +and O +is O +negatively O +charged O +. O + +The O +hydroxyl O +group O +of O +Ser12 B-residue_name_number +coordinates B-bond_interaction +with I-bond_interaction +O2 O +of O +RBL2 B-residue_name_number +. O + +The O +backbone O +amide O +nitrogens O +of O +Gly13 B-residue_name_number +and O +Arg15 B-residue_name_number +also O +keep O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +RBL2 B-residue_name_number +( O +Fig O +4B O +). O + +In O +the O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +structure B-evidence +, O +a O +2 O +. O +6 O +Å O +hydrogen B-bond_interaction +bond I-bond_interaction +is O +present O +between O +RBL2 B-residue_name_number +and O +Ser12 B-residue_name_number +( O +Fig O +4B O +), O +while O +in O +the O +AtXK B-protein +- I-protein +1 I-protein +structure B-evidence +this O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +corresponding O +residue O +( O +Ser22 B-residue_name_number +) O +is O +broken O +. O + +In O +addition O +, O +our O +enzymatic B-experimental_method +assays I-experimental_method +indicated O +that O +Asp8 B-residue_name_number +is O +important O +for O +the O +activity O +of O +SePSK B-protein +( O +Fig O +2D O +). O + +To O +further O +verified O +this O +result O +, O +we O +measured O +the O +binding B-evidence +affinity I-evidence +for O +D B-chemical +- I-chemical +ribulose I-chemical +of O +both O +wild B-protein_state +type I-protein_state +( O +WT B-protein_state +) O +and O +D8A B-mutant +mutant B-protein_state +of O +SePSK B-protein +using O +a O +surface B-experimental_method +plasmon I-experimental_method +resonance I-experimental_method +method I-experimental_method +. O + +The O +results O +showed O +that O +the O +affinity B-evidence +of O +D8A B-mutant +- O +SePSK B-protein +with O +D B-chemical +- I-chemical +ribulose I-chemical +is O +weaker O +than O +that O +of O +WT B-protein_state +with O +a O +reduction O +of O +approx O +. O + +Dissociation B-evidence +rate I-evidence +constant I-evidence +( O +Kd B-evidence +) O +of O +wild B-protein_state +type I-protein_state +and O +D8A B-mutant +- O +SePSK B-protein +are O +3 O +ms O +- O +1 O +and O +9 O +ms O +- O +1 O +, O +respectively O +. O + +The O +results O +implied O +that O +the O +second B-site +RBL I-site +binding I-site +site I-site +plays O +a O +role O +in O +the O +D B-protein_type +- I-protein_type +ribulose I-protein_type +kinase I-protein_type +function O +of O +SePSK B-protein +. O + +Simulated O +conformational O +change O +of O +SePSK B-protein +during O +the O +catalytic O +process O + +It O +was O +reported O +earlier O +that O +the O +crossing O +angle O +between O +the O +domain B-structure_element +I I-structure_element +and O +domain B-structure_element +II I-structure_element +in O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +is O +different O +. O + +As O +reported O +previously O +, O +members O +of O +the O +sugar B-protein_type +kinase I-protein_type +family O +undergo O +a O +conformational O +change O +to O +narrow O +the O +crossing O +angle O +between O +two O +domains O +and O +reduce O +the O +distance O +between O +substrate O +and O +ATP B-chemical +in O +order O +to O +facilitate O +the O +catalytic O +reaction O +of O +phosphorylation B-ptm +of O +sugar O +substrates O +. O + +After O +comparing O +structures B-evidence +of O +apo B-protein_state +- O +SePSK B-protein +, O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +and O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +, O +we O +noticed O +that O +these O +structures B-evidence +presented O +here O +are O +similar O +. O + +Since O +the O +two O +domains O +of O +SePSK B-protein +are O +widely O +separated O +in O +this O +structure B-evidence +, O +we O +hypothesize O +that O +our O +structures B-evidence +of O +SePSK B-protein +represent O +its O +open B-protein_state +form O +, O +and O +that O +a O +conformational O +rearrangement O +must O +occur O +to O +switch O +to O +the O +closed B-protein_state +state O +in O +order O +to O +facilitate O +the O +catalytic O +process O +of O +phosphorylation B-ptm +of O +sugar O +substrates O +. O + +For O +studying O +such O +potential O +conformational O +change O +, O +a O +simulation B-experimental_method +on O +the O +Hingeprot B-experimental_method +Server I-experimental_method +was O +performed O +to O +predict O +the O +movement O +of O +different O +SePSK B-protein +domains O +. O + +Based O +on O +the O +above O +results O +, O +SePSK B-protein +is O +divided O +into O +two O +rigid O +parts O +. O + +The O +domain B-structure_element +I I-structure_element +of O +RBL B-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +( O +aa O +. O +1 B-residue_range +– I-residue_range +228 I-residue_range +, O +aa O +. O +402 B-residue_range +– I-residue_range +421 I-residue_range +) O +and O +the O +domain B-structure_element +II I-structure_element +of O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +( O +aa O +. O +229 B-residue_range +– I-residue_range +401 I-residue_range +) O +were O +superposed B-experimental_method +with O +structures B-evidence +, O +including O +apo B-protein_state +- O +AtXK B-protein +- I-protein +1 I-protein +, O +apo B-protein_state +- O +SePSK B-protein +, O +xylulose B-protein_type +kinase I-protein_type +from O +Lactobacillus B-species +acidophilus I-species +( O +PDB O +code O +: O +3LL3 O +) O +and O +the O +S58W B-mutant +mutant B-protein_state +form O +of O +glycerol B-protein_type +kinase I-protein_type +from O +Escherichia B-species +coli I-species +( O +PDB O +code O +: O +1GLJ O +). O + +After O +superposition B-experimental_method +, O +the O +distances O +of O +AMP B-chemical +- I-chemical +PNP I-chemical +γ O +- O +phosphate B-chemical +and O +the O +fifth O +hydroxyl O +group O +of O +RBL1 B-residue_name_number +are O +7 O +. O +9 O +Å O +( O +superposed B-experimental_method +with O +AtXK B-protein +- I-protein +1 I-protein +), O +7 O +. O +4 O +Å O +( O +superposed B-experimental_method +with O +SePSK B-protein +), O +6 O +. O +6 O +Å O +( O +superposed B-experimental_method +with O +3LL3 O +) O +and O +6 O +. O +1 O +Å O +( O +superposed B-experimental_method +with O +1GLJ O +). O + +This O +distance O +between O +RBL2 B-residue_name_number +and O +AMP B-chemical +- I-chemical +PNP I-chemical +- O +γ O +- O +phosphate B-chemical +is O +close O +enough O +to O +facilitate O +phosphate B-chemical +transferring O +. O + +Simulated O +conformational O +change O +of O +SePSK B-protein +during O +the O +catalytic O +process O +. O + +The O +structures B-evidence +are O +shown O +as O +cartoon O +and O +the O +ligands O +are O +shown O +as O +sticks O +. O + +Domain B-structure_element +I I-structure_element +from O +D B-complex_assembly +- I-complex_assembly +ribulose I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +( O +green O +) O +and O +Domain B-structure_element +II I-structure_element +from O +AMP B-complex_assembly +- I-complex_assembly +PNP I-complex_assembly +- I-complex_assembly +SePSK I-complex_assembly +( O +cyan O +) O +are O +superposed B-experimental_method +with O +apo B-protein_state +- O +AtXK B-protein +- I-protein +1 I-protein +( O +1st O +), O +apo B-protein_state +- O +SePSK B-protein +( O +2nd O +), O +3LL3 O +( O +3rd O +) O +and O +1GLJ O +( O +4th O +), O +respectively O +. O + +The O +numbers O +near O +the O +black O +dashed O +lines O +show O +the O +distances O +( O +Å O +) O +between O +two O +nearest O +atoms O +of O +RBL B-chemical +and O +AMP B-chemical +- I-chemical +PNP I-chemical +. O + +Our O +results O +provide O +the O +detailed O +information O +about O +the O +interaction O +of O +SePSK B-protein +with O +ATP B-chemical +and O +substrates O +. O + +Moreover O +, O +structural B-experimental_method +superposition I-experimental_method +results O +enable O +us O +to O +visualize O +the O +conformational O +change O +of O +SePSK B-protein +during O +the O +catalytic O +process O +. O + +In O +conclusion O +, O +our O +results O +provide O +important O +information O +for O +a O +more O +detailed O +understanding O +of O +the O +mechanisms O +of O +SePSK B-protein +and O +other O +members O +of O +FGGY B-protein_type +family I-protein_type +carbohydrate I-protein_type +kinases I-protein_type +. O + +Structural O +insights O +into O +the O +Escherichia B-species +coli I-species +lysine B-protein_type +decarboxylases I-protein_type +and O +molecular O +determinants O +of O +interaction O +with O +the O +AAA B-protein_type ++ I-protein_type +ATPase I-protein_type +RavA B-protein + +A O +unique O +macromolecular O +cage O +formed O +by O +two O +decamers B-oligomeric_state +of O +the O +Escherichia B-species +coli I-species +LdcI B-protein +and O +five O +hexamers B-oligomeric_state +of O +the O +AAA B-protein_type ++ I-protein_type +ATPase I-protein_type +RavA B-protein +was O +shown O +to O +counteract O +acid O +stress O +under O +starvation O +. O + +Comparison B-experimental_method +with O +each O +other O +and O +with O +available O +structures B-evidence +uncovers O +differences O +between O +LdcI B-protein +and O +LdcC B-protein +explaining O +why O +only O +the O +acid B-protein_type +stress I-protein_type +response I-protein_type +enzyme I-protein_type +is O +capable O +of O +binding O +RavA B-protein +. O +We O +identify O +interdomain O +movements O +associated O +with O +the O +pH B-protein_state +- I-protein_state +dependent I-protein_state +enzyme O +activation O +and O +with O +the O +RavA B-protein +binding O +. O + +Multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +coupled O +to O +a O +phylogenetic B-experimental_method +analysis I-experimental_method +reveals O +that O +certain O +enterobacteria B-taxonomy_domain +exert O +evolutionary O +pressure O +on O +the O +lysine B-protein_type +decarboxylase I-protein_type +towards O +the O +cage O +- O +like O +assembly O +with O +RavA B-protein +, O +implying O +that O +this O +complex O +may O +have O +an O +important O +function O +under O +particular O +stress O +conditions O +. O + +Enterobacterial B-taxonomy_domain +inducible B-protein_state +decarboxylases B-protein_type +of O +basic B-protein_state +amino B-chemical +acids I-chemical +lysine B-residue_name +, O +arginine B-residue_name +and O +ornithine B-residue_name +have O +a O +common O +evolutionary O +origin O +and O +belong O +to O +the O +α B-protein_type +- I-protein_type +family I-protein_type +of O +pyridoxal B-chemical +- I-chemical +5 I-chemical +′- I-chemical +phosphate I-chemical +( O +PLP B-chemical +)- O +dependent O +enzymes O +. O + +Each O +decarboxylase B-protein_type +is O +induced O +by O +an O +excess O +of O +the O +target O +amino B-chemical +acid I-chemical +and O +a O +specific O +range O +of O +extracellular O +pH O +, O +and O +works O +in O +conjunction O +with O +a O +cognate O +inner B-protein_type +membrane I-protein_type +antiporter I-protein_type +. O + +Consequently O +, O +these O +enzymes O +buffer O +both O +the O +bacterial B-taxonomy_domain +cytoplasm O +and O +the O +local O +extracellular O +environment O +. O + +These O +amino B-protein_type +acid I-protein_type +decarboxylases I-protein_type +are O +therefore O +called O +acid O +stress O +inducible B-protein_state +or O +biodegradative B-protein_state +to O +distinguish O +them O +from O +their O +biosynthetic B-protein_state +lysine B-protein_type +and I-protein_type +ornithine I-protein_type +decarboxylase I-protein_type +paralogs O +catalysing O +the O +same O +reaction O +but O +responsible O +for O +the O +polyamine B-chemical +production O +at O +neutral B-protein_state +pH I-protein_state +. O + +Inducible B-protein_state +enterobacterial B-taxonomy_domain +amino B-protein_type +acid I-protein_type +decarboxylases I-protein_type +have O +been O +intensively O +studied O +since O +the O +early O +1940 O +because O +the O +ability O +of O +bacteria B-taxonomy_domain +to O +withstand O +acid O +stress O +can O +be O +linked O +to O +their O +pathogenicity O +in O +humans B-species +. O + +In O +particular O +, O +the O +inducible B-protein_state +lysine B-protein_type +decarboxylase I-protein_type +LdcI B-protein +( O +or O +CadA B-protein +) O +attracts O +attention O +due O +to O +its O +broad B-protein_state +pH I-protein_state +range I-protein_state +of O +activity O +and O +its O +capacity O +to O +promote O +survival O +and O +growth O +of O +pathogenic O +enterobacteria B-taxonomy_domain +such O +as O +Salmonella B-species +enterica I-species +serovar I-species +Typhimurium I-species +, O +Vibrio B-species +cholerae I-species +and O +Vibrio B-species +vulnificus I-species +under O +acidic O +conditions O +. O + +Furthermore O +, O +both O +LdcI B-protein +and O +the O +biosynthetic B-protein_state +lysine B-protein_type +decarboxylase I-protein_type +LdcC B-protein +of O +uropathogenic B-species +Escherichia I-species +coli I-species +( O +UPEC B-species +) O +appear O +to O +play O +an O +important O +role O +in O +increased O +resistance O +of O +this O +pathogen O +to O +nitrosative O +stress O +produced O +by O +nitric B-chemical +oxide I-chemical +and O +other O +damaging O +reactive O +nitrogen O +intermediates O +accumulating O +during O +the O +course O +of O +urinary O +tract O +infections O +( O +UTI O +). O + +This O +effect O +is O +attributed O +to O +cadaverine B-chemical +, O +the O +diamine O +produced O +by O +decarboxylation O +of O +lysine B-residue_name +by O +LdcI B-protein +and O +LdcC B-protein +, O +that O +was O +shown O +to O +enhance O +UPEC B-species +colonisation O +of O +the O +bladder O +. O + +Both O +acid B-protein_state +pH I-protein_state +and O +cadaverine B-chemical +induce O +closure O +of O +outer O +membrane O +porins B-protein_type +thereby O +contributing O +to O +bacterial B-taxonomy_domain +protection O +from O +acid O +stress O +, O +but O +also O +from O +certain O +antibiotics O +, O +by O +reduction O +in O +membrane O +permeability O +. O + +The O +crystal B-evidence +structure I-evidence +of O +the O +E B-species +. I-species +coli I-species +LdcI B-protein +as O +well O +as O +its O +low O +resolution O +characterisation O +by O +electron B-experimental_method +microscopy I-experimental_method +( O +EM B-experimental_method +) O +showed O +that O +it O +is O +a O +decamer B-oligomeric_state +made O +of O +two O +pentameric B-oligomeric_state +rings B-structure_element +. O + +Ten O +years O +ago O +we O +showed O +that O +the O +E B-species +. I-species +coli I-species +AAA B-protein_type ++ I-protein_type +ATPase I-protein_type +RavA B-protein +, O +involved O +in O +multiple O +stress O +response O +pathways O +, O +tightly O +interacted O +with O +LdcI B-protein +but O +was O +not O +capable O +of O +binding O +to O +LdcC B-protein +. O +We O +described O +how O +two O +double O +pentameric B-oligomeric_state +rings B-structure_element +of O +the O +LdcI B-protein +tightly O +associate O +with O +five O +hexameric B-oligomeric_state +rings B-structure_element +of O +RavA B-protein +to O +form O +a O +unique O +cage O +- O +like O +architecture O +that O +enables O +the O +bacterium B-taxonomy_domain +to O +withstand O +acid O +stress O +even O +under O +conditions O +of O +nutrient O +deprivation O +eliciting O +stringent O +response O +. O + +The O +main O +determinants O +of O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +assembly O +appeared O +to O +be O +the O +N O +- O +terminal O +loop B-structure_element +of O +the O +LARA B-structure_element +domain I-structure_element +of O +RavA B-protein +and O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +LdcI B-protein +. O + +In O +spite O +of O +this O +wealth O +of O +structural B-evidence +information I-evidence +, O +the O +fact O +that O +LdcC B-protein +does O +not O +interact O +with O +RavA B-protein +, O +although O +the O +two O +lysine B-protein_type +decarboxylases I-protein_type +are O +69 O +% O +identical O +and O +84 O +% O +similar O +, O +and O +the O +physiological O +significance O +of O +the O +absence O +of O +this O +interaction O +remained O +unexplored O +. O + +Finally O +, O +we O +performed O +multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +22 O +lysine B-protein_type +decarboxylases I-protein_type +from O +Enterobacteriaceae B-taxonomy_domain +containing O +the O +ravA B-gene +- I-gene +viaA I-gene +operon I-gene +in O +their O +genome O +. O + +This O +fascinating O +parallelism O +between O +the O +propensity O +for O +RavA B-protein +binding O +and O +the O +genetic O +environment O +of O +an O +enterobacterial B-taxonomy_domain +lysine B-protein_type +decarboxylase I-protein_type +, O +as O +well O +as O +the O +high B-protein_state +degree I-protein_state +of I-protein_state +conservation I-protein_state +of O +this O +small B-structure_element +structural I-structure_element +motif I-structure_element +, O +emphasize O +the O +functional O +importance O +of O +the O +interaction O +between O +biodegradative B-protein_state +enterobacterial B-taxonomy_domain +lysine B-protein_type +decarboxylases I-protein_type +and O +the O +AAA B-protein_type ++ I-protein_type +ATPase I-protein_type +RavA B-protein +. O + +CryoEM B-experimental_method +3D B-evidence +reconstructions I-evidence +of O +LdcC B-protein +, O +LdcIa B-protein +and O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly + +In O +the O +frame O +of O +this O +work O +, O +we O +produced O +two O +novel O +subnanometer O +resolution O +cryoEM B-experimental_method +reconstructions B-evidence +of O +the O +E B-species +. I-species +coli I-species +lysine B-protein_type +decarboxylases I-protein_type +at O +pH B-protein_state +optimal I-protein_state +for O +their O +enzymatic O +activity O +– O +a O +5 O +. O +5 O +Å O +resolution O +cryoEM B-experimental_method +map B-evidence +of O +the O +LdcC B-protein +( O +pH B-protein_state +7 I-protein_state +. I-protein_state +5 I-protein_state +) O +for O +which O +no O +3D O +structural O +information O +has O +been O +previously O +available O +( O +Figs O +1A O +, O +B O +and O +S1 O +), O +and O +a O +6 O +. O +1 O +Å O +resolution O +cryoEM B-experimental_method +map B-evidence +of O +the O +LdcIa B-protein +, O +( O +pH B-protein_state +6 I-protein_state +. I-protein_state +2 I-protein_state +) O +( O +Figs O +1C O +, O +D O +and O +S2 O +). O + +In O +addition O +, O +we O +improved O +our O +earlier O +cryoEM B-experimental_method +map B-evidence +of O +the O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +complex O +from O +7 O +. O +5 O +Å O +to O +6 O +. O +2 O +Å O +resolution O +( O +Figs O +1E O +, O +F O +and O +S3 O +). O + +Based O +on O +these O +reconstructions B-evidence +, O +reliable O +pseudoatomic B-evidence +models I-evidence +of O +the O +three O +assemblies O +were O +obtained O +by O +flexible B-experimental_method +fitting I-experimental_method +of I-experimental_method +either O +the O +crystal B-evidence +structure I-evidence +of O +LdcIi B-protein +or O +a O +derived O +structural B-experimental_method +homology I-experimental_method +model I-experimental_method +of O +LdcC B-protein +( O +Table O +S1 O +). O + +Significant O +differences O +between O +these O +pseudoatomic B-evidence +models I-evidence +can O +be O +interpreted O +as O +movements O +between O +specific O +biological O +states O +of O +the O +proteins O +as O +described O +below O +. O + +The O +wing B-structure_element +domains I-structure_element +as O +a O +stable O +anchor O +at O +the O +center O +of O +the O +double B-structure_element +- I-structure_element +ring I-structure_element + +As O +a O +first O +step O +of O +a O +comparative O +analysis O +, O +we O +superimposed B-experimental_method +the O +three O +cryoEM B-experimental_method +reconstructions B-evidence +( O +LdcIa B-protein +, O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +and O +LdcC B-protein +) O +and O +the O +crystal B-evidence +structure I-evidence +of O +the O +LdcIi B-protein +decamer B-oligomeric_state +( O +Fig O +. O +2 O +and O +Movie O +S1 O +). O + +This O +superposition B-experimental_method +reveals O +that O +the O +densities B-evidence +lining O +the O +central B-structure_element +hole I-structure_element +of O +the O +toroid B-structure_element +are O +roughly O +at O +the O +same O +location O +, O +while O +the O +rest O +of O +the O +structure B-evidence +exhibits O +noticeable O +changes O +. O + +Specifically O +, O +at O +the O +center O +of O +the O +double B-structure_element +- I-structure_element +ring I-structure_element +the O +wing B-structure_element +domains I-structure_element +of O +the O +subunits O +provide O +the O +conserved B-protein_state +basis O +for O +the O +assembly O +with O +the O +lowest B-evidence +root I-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +RMSD B-evidence +) O +( O +between O +1 O +. O +4 O +and O +2 O +Å O +for O +the O +Cα O +atoms O +only O +), O +whereas O +the O +peripheral O +CTDs B-structure_element +containing O +the O +RavA B-site +binding I-site +interface I-site +manifest O +the O +highest O +RMSD B-evidence +( O +up O +to O +4 O +. O +2 O +Å O +) O +( O +Table O +S2 O +). O + +This O +preservation O +of O +the O +central B-structure_element +part I-structure_element +of O +the O +double O +- O +ring O +assembly O +may O +help O +the O +enzymes O +to O +maintain O +their O +decameric B-oligomeric_state +state O +upon O +activation O +and O +incorporation O +into O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +. O + +The O +core B-structure_element +domain I-structure_element +and O +the O +active B-site +site I-site +rearrangements O +upon O +pH B-protein_state +- I-protein_state +dependent I-protein_state +enzyme O +activation O +and O +LARA O +binding O + +Both O +visual B-experimental_method +inspection I-experimental_method +( O +Fig O +. O +2 O +) O +and O +RMSD B-experimental_method +calculations I-experimental_method +( O +Table O +S2 O +) O +show O +that O +globally O +the O +three O +structures B-evidence +at O +active B-protein_state +pH I-protein_state +( O +LdcIa B-protein +, O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +and O +LdcC B-protein +) O +are O +more O +similar O +to O +each O +other O +than O +to O +the O +structure O +determined O +at O +high B-protein_state +pH I-protein_state +conditions O +( O +LdcIi B-protein +). O + +The O +decameric B-oligomeric_state +enzyme O +is O +built O +of O +five O +dimers B-oligomeric_state +associating O +into O +a O +5 B-structure_element +- I-structure_element +fold I-structure_element +symmetrical I-structure_element +double I-structure_element +- I-structure_element +ring I-structure_element +( O +two O +monomers B-oligomeric_state +making O +a O +dimer B-oligomeric_state +are O +delineated O +in O +Fig O +. O +1 O +). O + +In O +addition O +, O +our O +earlier O +biochemical B-experimental_method +observation I-experimental_method +that O +the O +enzymatic O +activity O +of O +LdcIa B-protein +is O +unaffected O +by O +RavA B-protein +binding O +is O +consistent O +with O +the O +relatively O +small O +changes O +undergone O +by O +the O +active B-site +site I-site +upon O +transition O +from O +LdcIa B-protein +to O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +. O + +Worthy O +of O +note O +, O +our O +previous O +comparison O +of O +the O +crystal B-evidence +structure I-evidence +of O +LdcIi B-protein +with O +that O +of O +the O +inducible B-protein_state +arginine B-protein_type +decarboxylase I-protein_type +AdiA B-protein +revealed O +high B-protein_state +conservation I-protein_state +of O +the O +PLP B-site +- I-site +coordinating I-site +residues I-site +and O +identified O +a O +patch B-site +of I-site +negatively I-site +charged I-site +residues I-site +lining O +the O +active B-site +site I-site +channel I-site +as O +a O +potential O +binding B-site +site I-site +for O +the O +target O +amino B-chemical +acid I-chemical +substrate O +( O +Figs O +S3 O +and O +S4 O +in O +ref O +.). O + +Rearrangements O +of O +the O +ppGpp B-site +binding I-site +pocket I-site +upon O +pH B-protein_state +- I-protein_state +dependent I-protein_state +enzyme O +activation O +and O +LARA B-structure_element +binding O + +Whereas O +the O +crystal B-evidence +structure I-evidence +of O +the O +ppGpp B-complex_assembly +- I-complex_assembly +LdcIi I-complex_assembly +was O +solved B-experimental_method +to O +2 O +Å O +resolution O +, O +only O +a O +4 O +. O +1 O +Å O +resolution O +structure B-evidence +of O +the O +ppGpp B-protein_state +- I-protein_state +free I-protein_state +LdcIi B-protein +could O +be O +obtained O +. O + +At O +this O +resolution O +, O +the O +apo B-protein_state +- O +LdcIi B-protein +and O +ppGpp B-complex_assembly +- I-complex_assembly +LdcIi I-complex_assembly +structures B-evidence +( O +both O +solved O +at O +pH B-protein_state +8 I-protein_state +. I-protein_state +5 I-protein_state +) O +appeared O +indistinguishable O +except O +for O +the O +presence O +of O +ppGpp B-chemical +( O +Fig O +. O +S11 O +in O +ref O +. O +). O + +Thus O +, O +we O +speculated O +that O +inhibition O +of O +LdcI B-protein +by O +ppGpp B-chemical +would O +be O +accompanied O +by O +a O +transduction O +of O +subtle O +structural O +changes O +at O +the O +level O +of O +individual O +amino B-chemical +acid I-chemical +side O +chains O +between O +the O +ppGpp B-site +binding I-site +pocket I-site +and O +the O +active B-site +site I-site +of O +the O +enzyme O +. O + +While O +differences O +in O +the O +ppGpp B-site +binding I-site +site I-site +could O +indeed O +be O +visualized O +( O +Fig O +. O +S4 O +), O +the O +level O +of O +resolution O +warns O +against O +speculations O +about O +their O +significance O +. O + +The O +fact O +that O +interaction O +with O +RavA B-protein +reduces O +the O +ppGpp B-chemical +affinity O +for O +LdcI B-protein +despite O +the O +long O +distance O +of O +~ O +30 O +Å O +between O +the O +LARA B-site +domain I-site +binding I-site +site I-site +and O +the O +closest O +ppGpp B-site +binding I-site +pocket I-site +( O +Fig O +. O +S5 O +) O +seems O +to O +favor O +an O +allosteric O +regulation O +mechanism O +. O + +Swinging O +and O +stretching O +of O +the O +CTDs B-structure_element +upon O +pH B-protein_state +- I-protein_state +dependent I-protein_state +LdcI B-protein +activation O +and O +LARA B-structure_element +binding O + +Inspection O +of O +the O +superimposed B-experimental_method +decameric B-oligomeric_state +structures B-evidence +( O +Figs O +2 O +and O +S6 O +) O +suggests O +a O +depiction O +of O +the O +wing B-structure_element +domains I-structure_element +as O +an O +anchor O +around O +which O +the O +peripheral O +CTDs B-structure_element +swing O +. O + +Indeed O +, O +all O +CTDs B-structure_element +have O +very O +similar O +structures O +( O +RMSDmin B-evidence +< O +1 O +Å O +). O + +The O +LdcIi B-protein +monomer B-oligomeric_state +is O +the O +most B-protein_state +compact I-protein_state +, O +whereas O +LdcIa B-protein +and O +especially O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +gradually B-protein_state +extend I-protein_state +their O +CTDs B-structure_element +towards O +the O +LARA B-structure_element +domain I-structure_element +of O +RavA B-protein +( O +Figs O +2 O +and O +4 O +). O + +In O +our O +previous O +contribution O +, O +based O +on O +the O +fit O +of O +the O +LdcIi B-protein +and O +the O +LARA B-structure_element +crystal B-evidence +structures I-evidence +into O +the O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +cryoEM B-experimental_method +density B-evidence +, O +we O +predicted O +that O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +interaction O +should O +involve O +the O +C O +- O +terminal O +two B-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +of O +the O +LdcI B-protein +. O +Our O +present O +cryoEM B-experimental_method +maps B-evidence +and O +pseudoatomic B-evidence +models I-evidence +provide O +first O +structure O +- O +based O +insights O +into O +the O +differences O +between O +the O +inducible B-protein_state +and O +the O +constitutive B-protein_state +lysine B-protein_type +decarboxylases I-protein_type +. O + +Therefore O +, O +we O +wanted O +to O +check O +the O +influence O +of O +the O +primary O +sequence O +of O +the O +two O +proteins O +in O +this O +region O +on O +their O +ability O +to O +interact O +with O +RavA B-protein +. O +To O +this O +end O +, O +we O +swapped B-experimental_method +the O +relevant O +β B-structure_element +- I-structure_element +sheets I-structure_element +of O +the O +two O +proteins O +and O +produced O +their O +chimeras B-mutant +, O +namely O +LdcIC B-mutant +( O +i O +. O +e O +. O +LdcI B-protein +with O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +LdcC B-protein +) O +and O +LdcCI B-mutant +( O +i O +. O +e O +. O +LdcC B-protein +with O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +LdcI B-protein +) O +( O +Fig O +. O +5A O +– O +C O +). O + +Both B-mutant +constructs I-mutant +could O +be O +purified O +and O +could O +form O +decamers B-oligomeric_state +visually O +indistinguishable O +from O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +proteins O +. O + +As O +expected O +, O +binding O +of O +LdcI B-protein +to O +RavA B-protein +was O +completely O +abolished O +by O +this O +procedure O +and O +no O +LdcIC B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +complex O +could O +be O +detected O +. O + +On O +the O +contrary O +, O +introduction B-experimental_method +of O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +LdcI B-protein +into O +LdcC B-protein +led O +to O +an O +assembly O +of O +the O +LdcCI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +complex O +. O + +On O +the O +negative B-experimental_method +stain I-experimental_method +EM I-experimental_method +grid I-experimental_method +, O +the O +chimeric B-protein_state +cages O +appeared O +less O +rigid O +than O +the O +native B-protein_state +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +, O +which O +probably O +means O +that O +the O +environment O +of O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +contributes O +to O +the O +efficiency O +of O +the O +interaction O +and O +the O +stability O +of O +the O +entire O +architecture O +( O +Fig O +. O +5D O +– O +F O +). O + +The O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +of O +a O +lysine B-protein_type +decarboxylase I-protein_type +is O +a O +highly B-protein_state +conserved I-protein_state +signature O +allowing O +to O +distinguish O +between O +LdcI B-protein +and O +LdcC B-protein + +Alignment B-experimental_method +of I-experimental_method +the I-experimental_method +primary I-experimental_method +sequences I-experimental_method +of O +the O +E B-species +. I-species +coli I-species +LdcI B-protein +and O +LdcC B-protein +shows O +that O +some O +amino O +acid O +residues O +of O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +are O +the O +same O +in O +the O +two O +proteins O +, O +whereas O +others O +are O +notably O +different O +in O +chemical O +nature O +. O + +Importantly O +, O +most O +of O +the O +amino O +acid O +differences O +between O +the O +two O +enzymes O +are O +located O +in O +this O +very B-structure_element +region I-structure_element +. O + +First O +of O +all O +, O +consensus B-evidence +sequence I-evidence +for O +the O +entire O +lysine B-protein_type +decarboxylase I-protein_type +family O +was O +derived O +. O + +Second O +, O +the O +phylogenetic B-experimental_method +analysis I-experimental_method +clearly O +split O +the O +lysine B-protein_type +decarboxylases I-protein_type +into O +two O +groups O +( O +Fig O +. O +6A O +). O + +Thus O +, O +consensus B-evidence +sequences I-evidence +could O +also O +be O +determined O +for O +each O +of O +the O +two O +groups O +( O +Figs O +6B O +, O +C O +and O +S7 O +). O + +Inspection O +of O +these O +consensus B-evidence +sequences I-evidence +revealed O +important O +differences O +between O +the O +groups O +regarding O +charge O +, O +size O +and O +hydrophobicity O +of O +several O +residues O +precisely O +at O +the O +level O +of O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +that O +is O +responsible O +for O +the O +interaction O +with O +RavA B-protein +( O +Fig O +. O +6B O +– O +D O +). O + +For O +example O +, O +in O +our O +previous O +study O +, O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutations I-experimental_method +identified O +Y697 B-residue_name_number +as O +critically O +required O +for O +the O +RavA B-protein +binding O +. O + +Our O +current O +analysis O +shows O +that O +Y697 B-residue_name_number +is O +strictly B-protein_state +conserved I-protein_state +in O +the O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +group O +whereas O +the O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +enzymes O +always B-protein_state +have I-protein_state +a O +lysine B-residue_name +in O +this O +position O +; O +it O +also O +uncovers O +several O +other O +residues O +potentially O +essential O +for O +the O +interaction O +with O +RavA B-protein +which O +can O +now O +be O +addressed O +by O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +. O + +The O +third O +and O +most O +remarkable O +finding O +was O +that O +exactly O +the O +same O +separation O +into O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +and O +“ O +LdcC B-protein_type +”- I-protein_type +like I-protein_type +groups O +can O +be O +obtained O +based O +on O +a O +comparison O +of O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheets I-structure_element +only O +, O +without O +taking O +the O +rest O +of O +the O +primary O +sequence O +into O +account O +. O + +Therefore O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +emerges O +as O +being O +a O +highly B-protein_state +conserved I-protein_state +signature B-structure_element +sequence I-structure_element +, O +sufficient O +to O +unambiguously O +discriminate O +between O +the O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +and O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +enterobacterial B-taxonomy_domain +lysine B-protein_type +decarboxylases I-protein_type +independently O +of O +any O +other O +information O +( O +Figs O +6 O +and O +S7 O +). O + +Thus O +, O +enterobacteria B-taxonomy_domain +identified O +here O +( O +Fig O +. O +6 O +, O +Table O +S4 O +) O +appear O +to O +exert O +evolutionary O +pressure O +on O +the O +biodegradative B-protein_state +lysine B-protein_type +decarboxylase I-protein_type +towards O +the O +RavA B-protein +binding O +. O + +One O +of O +the O +elucidated O +roles O +of O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +is O +to O +maintain O +LdcI B-protein +activity O +under O +conditions O +of O +enterobacterial B-taxonomy_domain +starvation O +by O +preventing O +LdcI B-protein +inhibition O +by O +the O +stringent B-chemical +response I-chemical +alarmone I-chemical +ppGpp B-chemical +. O + +Furthermore O +, O +the O +recently O +documented O +interaction O +of O +both O +LdcI B-protein +and O +RavA B-protein +with O +specific O +subunits B-structure_element +of O +the O +respiratory B-protein_type +complex I-protein_type +I I-protein_type +, O +together O +with O +the O +unanticipated O +link O +between O +RavA B-protein +and O +maturation O +of O +numerous O +iron B-protein_type +- I-protein_type +sulfur I-protein_type +proteins I-protein_type +, O +tend O +to O +suggest O +an O +additional O +intriguing O +function O +for O +this O +3 O +. O +5 O +MDa O +assembly O +. O + +The O +conformational O +rearrangements O +of O +LdcI B-protein +upon O +enzyme O +activation O +and O +RavA B-protein +binding O +revealed O +in O +this O +work O +, O +and O +our O +amazing O +finding O +that O +the O +molecular O +determinant O +of O +the O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +interaction O +is O +the O +one O +that O +straightforwardly O +determines O +if O +a O +particular O +enterobacterial B-taxonomy_domain +lysine B-protein_type +decarboxylase I-protein_type +belongs O +to O +“ O +LdcI B-protein_type +- I-protein_type +like I-protein_type +” O +or O +“ O +LdcC B-protein_type +- I-protein_type +like I-protein_type +” O +proteins O +, O +should O +give O +a O +new O +impetus O +to O +functional O +studies O +of O +the O +unique O +LdcI B-complex_assembly +- I-complex_assembly +RavA I-complex_assembly +cage O +. O + +Besides O +, O +the O +structures B-evidence +and O +the O +pseudoatomic B-evidence +models I-evidence +of O +the O +active B-protein_state +ppGpp B-protein_state +- I-protein_state +free I-protein_state +states O +of O +both O +the O +biodegradative B-protein_state +and O +the O +biosynthetic B-protein_state +E B-species +. I-species +coli I-species +lysine B-protein_type +decarboxylases I-protein_type +offer O +an O +additional O +tool O +for O +analysis O +of O +their O +role O +in O +UPEC B-species +infectivity O +. O + +Together O +with O +the O +apo B-protein_state +- O +LdcI B-protein +and O +ppGpp B-complex_assembly +- I-complex_assembly +LdcIi I-complex_assembly +crystal B-evidence +structures I-evidence +, O +our O +cryoEM B-experimental_method +reconstructions B-evidence +provide O +a O +structural O +framework O +for O +future O +studies O +of O +structure O +- O +function O +relationships O +of O +lysine B-protein_type +decarboxylases I-protein_type +from O +other O +enterobacteria B-taxonomy_domain +and O +even O +of O +their O +homologues O +outside O +Enterobacteriaceae B-taxonomy_domain +. O +For O +example O +, O +the O +lysine B-protein_type +decarboxylase I-protein_type +of O +Eikenella B-species +corrodens I-species +is O +thought O +to O +play O +a O +major O +role O +in O +the O +periodontal O +disease O +and O +its O +inhibitors O +were O +shown O +to O +retard O +gingivitis O +development O +. O + +3D O +cryoEM B-experimental_method +reconstructions B-evidence +of O +LdcC B-protein +, O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +and O +LdcIa B-protein +. O + +In O +the O +rest O +of O +the O +protomers B-oligomeric_state +, O +the O +wing B-structure_element +, O +core B-structure_element +and O +C B-structure_element +- I-structure_element +terminal I-structure_element +domains I-structure_element +are O +colored O +from O +light O +to O +dark O +in O +shades O +of O +green O +for O +LdcC B-protein +( O +A O +), O +pink O +for O +LdcIa B-protein +( O +C O +) O +and O +blue O +for O +LdcI B-protein +in O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +( O +E O +). O + +In O +( O +E O +), O +the O +LARA B-structure_element +domain I-structure_element +density O +is O +shown O +in O +dark O +grey O +. O + +Two O +monomers B-oligomeric_state +making O +a O +dimer B-oligomeric_state +are O +delineated O +. O + +Scale O +bar O +50 O +Å O +. O +( O +B O +, O +D O +, O +F O +) O +One O +protomer B-oligomeric_state +from O +the O +cryoEM B-experimental_method +map B-evidence +of O +the O +LdcC B-protein +( O +B O +), O +LdcIa B-protein +( O +D O +) O +and O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +( O +F O +) O +in O +light O +grey O +with O +the O +pseudoatomic B-evidence +model I-evidence +represented O +as O +cartoons O +and O +colored O +as O +the O +densities O +in O +( O +A O +, O +C O +, O +E O +). O + +Superposition B-experimental_method +of O +the O +pseudoatomic B-evidence +models I-evidence +of O +LdcC B-protein +, O +LdcI B-protein +from O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +and O +LdcIa B-protein +colored O +as O +in O +Fig O +. O +1 O +, O +and O +the O +crystal B-evidence +structure I-evidence +of O +LdcIi B-protein +in O +shades O +of O +yellow O +. O + +Conformational O +rearrangements O +in O +the O +enzyme O +active B-site +site I-site +. O + +( O +A O +) O +LdcIi B-protein +crystal B-evidence +structure I-evidence +, O +with O +one O +ring B-structure_element +represented O +as O +a O +grey O +surface O +and O +the O +second O +as O +a O +cartoon O +. O + +A O +monomer B-oligomeric_state +with O +its O +PLP B-chemical +cofactor O +is O +delineated O +. O + +The O +PLP B-chemical +moieties O +of O +the O +cartoon O +ring B-structure_element +are O +shown O +in O +red O +. O + +One O +monomer B-oligomeric_state +is O +colored O +in O +shades O +of O +yellow O +as O +in O +Figs O +1 O +and O +2 O +, O +while O +the O +monomer B-oligomeric_state +related O +by O +C2 O +symmetry O +is O +grey O +. O + +The O +active B-site +site I-site +is O +boxed O +. O + +Stretching O +of O +the O +LdcI B-protein +monomer B-oligomeric_state +upon O +pH B-protein_state +- I-protein_state +dependent I-protein_state +enzyme O +activation O +and O +LARA B-structure_element +binding O +. O + +( O +A O +– O +C O +) O +A O +slice O +through O +the O +pseudoatomic B-evidence +models I-evidence +of O +the O +LdcI B-protein +monomers B-oligomeric_state +extracted O +from O +the O +superimposed B-experimental_method +decamers B-oligomeric_state +( O +Fig O +. O +2 O +) O +The O +rectangle O +indicates O +the O +regions O +enlarged O +in O +( O +D O +– O +F O +). O + +( O +A O +) O +compares O +LdcIi B-protein +( O +yellow O +) O +and O +LdcIa B-protein +( O +pink O +), O +( O +B O +) O +compares O +LdcIa B-protein +( O +pink O +) O +and O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +( O +blue O +), O +and O +( O +C O +) O +compares O +LdcIi B-protein +( O +yellow O +), O +LdcIa B-protein +( O +pink O +) O +and O +LdcI B-complex_assembly +- I-complex_assembly +LARA I-complex_assembly +( O +blue O +) O +simultaneously O +in O +order O +to O +show O +the O +progressive O +stretching O +described O +in O +the O +text O +. O + +The O +cryoEM B-experimental_method +density B-evidence +of O +the O +LARA B-structure_element +domain I-structure_element +is O +represented O +as O +a O +grey O +surface O +to O +show O +the O +position O +of O +the O +binding B-site +site I-site +and O +the O +direction O +of O +the O +movement O +. O + +Analysis O +of O +the O +LdcIC B-mutant +and O +LdcCI B-mutant +chimeras B-mutant +. O + +( O +A O +) O +A O +slice O +through O +the O +pseudoatomic B-evidence +models I-evidence +of O +the O +LdcIa B-protein +( O +purple O +) O +and O +LdcC B-protein +( O +green O +) O +monomers B-oligomeric_state +extracted O +from O +the O +superimposed B-experimental_method +decamers B-oligomeric_state +( O +Fig O +. O +2 O +). O +( O +B O +) O +The O +C O +- O +terminal O +β B-structure_element +- I-structure_element +sheet I-structure_element +in O +LdcIa B-protein +and O +LdcC B-protein +enlarged O +from O +( O +A O +, O +C O +) O +Exchanged O +primary O +sequences O +( O +capital O +letters O +) O +and O +their O +immediate O +vicinity O +( O +lower O +case O +letters O +) O +colored O +as O +in O +( O +A O +, O +B O +), O +with O +the O +corresponding O +secondary O +structure O +elements O +and O +the O +amino O +acid O +numbering O +shown O +. O + +Sequence B-experimental_method +analysis I-experimental_method +of O +enterobacterial B-taxonomy_domain +lysine B-protein_type +decarboxylases I-protein_type +. O + +Numbering O +as O +in O +E B-species +. I-species +coli I-species +. O + +Structural O +basis O +for O +Mep2 B-protein_type +ammonium B-protein_type +transceptor I-protein_type +activation O +by O +phosphorylation B-ptm + +Here O +we O +report O +X B-evidence +- I-evidence +ray I-evidence +crystal I-evidence +structures I-evidence +of O +the O +Mep2 B-protein_type +orthologues O +from O +Saccharomyces B-species +cerevisiae I-species +and O +Candida B-species +albicans I-species +and O +show O +that O +under O +nitrogen O +- O +sufficient O +conditions O +the O +transporters B-protein_type +are O +not B-protein_state +phosphorylated I-protein_state +and O +present O +in O +closed B-protein_state +, O +inactive B-protein_state +conformations O +. O + +The O +crystal B-evidence +structure I-evidence +of O +phosphorylation B-protein_state +- I-protein_state +mimicking I-protein_state +Mep2 B-mutant +variants I-mutant +from O +C B-species +. I-species +albicans I-species +show O +large O +conformational O +changes O +in O +a O +conserved B-protein_state +and O +functionally O +important O +region O +of O +the O +CTR B-structure_element +. O + +The O +results O +allow O +us O +to O +propose O +a O +model O +for O +regulation O +of O +eukaryotic B-taxonomy_domain +ammonium B-chemical +transport O +by O +phosphorylation B-ptm +. O + +Mep2 B-protein_type +proteins I-protein_type +are O +tightly O +regulated O +fungal B-taxonomy_domain +ammonium B-protein_type +transporters I-protein_type +. O + +Here O +, O +the O +authors O +report O +the O +crystal B-evidence +structures I-evidence +of O +closed B-protein_state +states O +of O +Mep2 B-protein_type +proteins I-protein_type +and O +propose O +a O +model O +for O +their O +regulation O +by B-experimental_method +comparing I-experimental_method +them I-experimental_method +with I-experimental_method +the O +open B-protein_state +ammonium B-protein_type +transporters I-protein_type +of O +bacteria B-taxonomy_domain +. O + +Transceptors B-protein_type +are O +membrane B-protein_type +proteins I-protein_type +that O +function O +not O +only O +as O +transporters O +but O +also O +as O +receptors O +/ O +sensors O +during O +nutrient O +sensing O +to O +activate O +downstream O +signalling O +pathways O +. O + +While O +most O +studies O +have O +focused O +on O +the O +Saccharomyces B-species +cerevisiae I-species +transceptors B-protein_type +for O +phosphate B-chemical +( O +Pho84 B-protein +), O +amino B-chemical +acids I-chemical +( O +Gap1 B-protein +) O +and O +ammonium B-chemical +( O +Mep2 B-protein +), O +transceptors B-protein_type +are O +found O +in O +higher B-taxonomy_domain +eukaryotes I-taxonomy_domain +as O +well O +( O +for O +example O +, O +the O +mammalian B-taxonomy_domain +SNAT2 B-protein +amino B-protein_type +- I-protein_type +acid I-protein_type +transporter I-protein_type +and O +the O +GLUT2 B-protein +glucose B-protein_type +transporter I-protein_type +). O + +One O +hypothesis O +is O +that O +downstream O +signalling O +is O +dependent O +on O +a O +specific O +conformation O +of O +the O +transporter B-protein_type +. O + +They O +belong O +to O +the O +Amt B-protein_type +/ I-protein_type +Mep I-protein_type +/ I-protein_type +Rh I-protein_type +family I-protein_type +of I-protein_type +transporters I-protein_type +that O +are O +present O +in O +all B-taxonomy_domain +kingdoms I-taxonomy_domain +of I-taxonomy_domain +life I-taxonomy_domain +and O +they O +take O +up O +ammonium B-chemical +from O +the O +extracellular O +environment O +. O + +Of O +these O +, O +only O +Mep2 B-protein_type +proteins I-protein_type +function O +as O +ammonium B-chemical +receptors O +/ O +sensors O +in O +fungal B-taxonomy_domain +development O +. O + +Under O +conditions O +of O +nitrogen O +limitation O +, O +Mep2 B-protein +initiates O +a O +signalling O +cascade O +that O +results O +in O +a O +switch O +from O +the O +yeast O +form O +to O +filamentous O +( O +pseudohyphal O +) O +growth O +that O +may O +be O +required O +for O +fungal B-taxonomy_domain +pathogenicity O +. O + +In O +addition O +, O +Mep2 B-protein +is O +also O +important O +for O +uptake O +of O +ammonium B-chemical +produced O +by O +growth O +on O +other O +nitrogen B-chemical +sources O +. O + +With O +the O +exception O +of O +the O +human B-species +RhCG B-protein +structure B-evidence +, O +no O +structural O +information O +is O +available O +for O +eukaryotic B-taxonomy_domain +ammonium B-protein_type +transporters I-protein_type +. O + +The O +proteins O +form O +stable B-protein_state +trimers B-oligomeric_state +, O +with O +each O +monomer B-oligomeric_state +having O +11 O +transmembrane B-structure_element +( O +TM B-structure_element +) O +helices B-structure_element +and O +a O +central B-site +channel I-site +for O +the O +transport O +of O +ammonium B-chemical +. O + +All O +structures B-evidence +show O +the O +transporters B-protein_type +in O +open B-protein_state +conformations O +. O + +In O +animals B-taxonomy_domain +, O +this O +is O +due O +to O +toxicity O +of O +elevated O +intracellular O +ammonium B-chemical +levels O +, O +whereas O +for O +microorganisms B-taxonomy_domain +ammonium B-chemical +is O +a O +preferred O +nitrogen O +source O +. O + +In O +bacteria B-taxonomy_domain +, O +amt B-gene +genes O +are O +present O +in O +an O +operon O +with O +glnK B-gene +, O +encoding O +a O +PII B-protein_type +- I-protein_type +like I-protein_type +signal I-protein_type +transduction I-protein_type +class I-protein_type +protein I-protein_type +. O + +By O +binding O +tightly O +to O +Amt B-protein_type +proteins I-protein_type +without O +inducing O +a O +conformational O +change O +in O +the O +transporter B-protein_type +, O +GlnK B-protein_type +sterically O +blocks O +ammonium B-chemical +conductance O +when O +nitrogen O +levels O +are O +sufficient O +. O + +Under O +conditions O +of O +nitrogen B-chemical +limitation O +, O +GlnK B-protein_type +becomes O +uridylated B-protein_state +, O +blocking O +its O +ability O +to O +bind O +and O +inhibit O +Amt B-protein_type +proteins I-protein_type +. O + +Importantly O +, O +eukaryotes B-taxonomy_domain +do O +not O +have O +GlnK B-protein_type +orthologues O +and O +have O +a O +different O +mechanism O +for O +regulation O +of O +ammonium B-chemical +transport O +activity O +. O + +In O +plants B-taxonomy_domain +, O +transporter B-protein_type +phosphorylation B-ptm +and O +dephosphorylation B-ptm +are O +known O +to O +regulate O +activity O +. O + +In O +S B-species +. I-species +cerevisiae I-species +, O +phosphorylation B-ptm +of O +Ser457 B-residue_name_number +within O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +( O +CTR B-structure_element +) O +in O +the O +cytoplasm O +was O +recently O +proposed O +to O +cause O +Mep2 B-protein_type +opening O +, O +possibly O +via O +inducing O +a O +conformational O +change O +. O + +To O +elucidate O +the O +mechanism O +of O +Mep2 B-protein_type +transport O +regulation O +, O +we O +present O +here O +X B-evidence +- I-evidence +ray I-evidence +crystal I-evidence +structures I-evidence +of O +the O +Mep2 B-protein_type +transceptors I-protein_type +from O +S B-species +. I-species +cerevisiae I-species +and O +C B-species +. I-species +albicans I-species +. O + +The O +most O +striking O +difference O +is O +the O +fact O +that O +the O +Mep2 B-protein_type +proteins I-protein_type +have O +closed B-protein_state +conformations O +. O + +The O +putative O +phosphorylation B-site +site I-site +is O +solvent B-protein_state +accessible I-protein_state +and O +located O +in O +a O +negatively B-site +charged I-site +pocket I-site +∼ O +30 O +Å O +away O +from O +the O +channel B-site +exit I-site +. O + +The O +channels B-site +of O +phosphorylation B-protein_state +- I-protein_state +mimicking I-protein_state +mutants I-protein_state +of O +C B-species +. I-species +albicans I-species +Mep2 B-protein +are O +still O +closed B-protein_state +but O +show O +large O +conformational O +changes O +within O +a O +conserved B-protein_state +part O +of O +the O +CTR B-structure_element +. O + +General O +architecture O +of O +Mep2 B-protein_type +ammonium B-protein_type +transceptors I-protein_type + +Of O +these O +, O +Mep2 B-protein +from O +C B-species +. I-species +albicans I-species +( O +CaMep2 B-protein +) O +showed O +superior O +stability O +in O +relatively O +harsh O +detergents O +such O +as O +nonyl O +- O +glucoside O +, O +allowing O +structure B-experimental_method +determination I-experimental_method +in O +two O +different O +crystal B-evidence +forms I-evidence +to O +high O +resolution O +( O +up O +to O +1 O +. O +5 O +Å O +). O + +Despite O +different O +crystal O +packing O +( O +Supplementary O +Table O +1 O +), O +the O +two O +CaMep2 B-protein +structures B-evidence +are O +identical O +to O +each O +other O +and O +very O +similar O +to O +ScMep2 B-protein +( O +Cα O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +. I-evidence + +( O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +)= O +0 O +. O +7 O +Å O +for O +434 O +residues O +), O +with O +the O +main O +differences O +confined O +to O +the O +N O +terminus O +and O +the O +CTR B-structure_element +( O +Fig O +. O +1 O +). O + +Electron B-evidence +density I-evidence +is O +visible O +for O +the O +entire O +polypeptide O +chains O +, O +with O +the O +exception O +of O +the O +C O +- O +terminal O +43 B-residue_range +( O +ScMep2 B-protein +) O +and O +25 B-residue_range +residues O +( O +CaMep2 B-protein +), O +which O +are O +poorly B-protein_state +conserved I-protein_state +and O +presumably O +disordered B-protein_state +. O + +Both O +Mep2 B-protein_type +proteins I-protein_type +show O +the O +archetypal O +trimeric B-oligomeric_state +assemblies O +in O +which O +each O +monomer B-oligomeric_state +consists O +of O +11 O +TM B-structure_element +helices I-structure_element +surrounding O +a O +central B-structure_element +pore I-structure_element +. O + +Important O +functional O +features O +such O +as O +the O +extracellular O +ammonium B-site +binding I-site +site I-site +, O +the O +Phe B-site +gate I-site +and O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +within O +the O +hydrophobic B-site +channel I-site +are O +all O +very O +similar O +to O +those O +present O +in O +the O +bacterial B-taxonomy_domain +transporters B-protein_type +and O +RhCG B-protein +. O + +In O +the O +remainder O +of O +the O +manuscript O +, O +we O +will O +specifically O +discuss O +CaMep2 B-protein +due O +to O +the O +superior O +resolution O +of O +the O +structure B-evidence +. O + +Unless O +specifically O +stated O +, O +the O +drawn O +conclusions O +also O +apply O +to O +ScMep2 B-protein +. O + +While O +the O +overall O +architecture O +of O +Mep2 B-protein +is O +similar O +to O +that O +of O +the O +prokaryotic B-taxonomy_domain +transporters B-protein_type +( O +Cα O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +. I-evidence +with O +Amt B-protein +- I-protein +1 I-protein += O +1 O +. O +4 O +Å O +for O +361 O +residues O +), O +there O +are O +large O +differences O +within O +the O +N O +terminus O +, O +intracellular B-structure_element +loops I-structure_element +( O +ICLs B-structure_element +) O +ICL1 B-structure_element +and O +ICL3 B-structure_element +, O +and O +the O +CTR B-structure_element +. O + +Moreover O +, O +the O +N O +terminus O +of O +one O +monomer B-oligomeric_state +interacts O +with O +the O +extended O +extracellular B-structure_element +loop I-structure_element +ECL5 B-structure_element +of O +a O +neighbouring O +monomer B-oligomeric_state +. O + +Together O +with O +additional O +, O +smaller O +differences O +in O +other O +extracellular B-structure_element +loops I-structure_element +, O +these O +changes O +generate O +a O +distinct O +vestibule B-structure_element +leading O +to O +the O +ammonium B-site +binding I-site +site I-site +that O +is O +much O +more O +pronounced O +than O +in O +the O +bacterial B-taxonomy_domain +proteins O +. O + +The O +N O +- O +terminal O +vestibule B-structure_element +and O +the O +resulting O +inter O +- O +monomer B-oligomeric_state +interactions O +likely O +increase O +the O +stability O +of O +the O +Mep2 B-protein +trimer B-oligomeric_state +, O +in O +support O +of O +data O +for O +plant B-taxonomy_domain +AMT B-protein_type +proteins I-protein_type +. O + +However O +, O +given O +that O +an O +N O +- O +terminal O +deletion B-protein_state +mutant I-protein_state +( O +2 B-mutant +- I-mutant +27Δ I-mutant +) O +grows O +as O +well O +as O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +Mep2 B-protein +on O +minimal O +ammonium B-chemical +medium O +( O +Fig O +. O +3 O +and O +Supplementary O +Fig O +. O +1 O +), O +the O +importance O +of O +the O +N O +terminus O +for O +Mep2 B-protein +activity O +is O +not O +clear O +. O + +In O +the O +vicinity O +of O +the O +Mep2 B-protein +channel B-site +exit I-site +, O +the O +cytoplasmic O +end O +of O +TM2 B-structure_element +has O +unwound O +, O +generating O +a O +longer O +ICL1 B-structure_element +even O +though O +there O +are O +no O +insertions O +in O +this O +region O +compared O +to O +the O +bacterial B-taxonomy_domain +proteins O +( O +Figs O +2 O +and O +4 O +). O + +The O +largest O +backbone O +movements O +of O +equivalent O +residues O +within O +ICL1 B-structure_element +are O +∼ O +10 O +Å O +, O +markedly O +affecting O +the O +conserved B-protein_state +basic B-protein_state +RxK B-structure_element +motif I-structure_element +( O +Fig O +. O +4 O +). O + +In O +addition O +to O +changing O +the O +RxK B-structure_element +motif I-structure_element +, O +the O +movement O +of O +ICL1 B-structure_element +has O +another O +, O +crucial O +functional O +consequence O +. O + +At O +the O +C O +- O +terminal O +end O +of O +TM1 B-structure_element +, O +the O +side O +- O +chain O +hydroxyl O +group O +of O +the O +relatively B-protein_state +conserved I-protein_state +Tyr49 B-residue_name_number +( O +Tyr53 B-residue_name_number +in O +ScMep2 B-protein +) O +makes O +a O +strong O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +ɛ2 O +nitrogen O +atom O +of O +the O +absolutely B-protein_state +conserved I-protein_state +His342 B-residue_name_number +of O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +( O +His348 B-residue_name_number +in O +ScMep2 B-protein +), O +closing O +the O +channel B-site +( O +Figs O +4 O +and O +5 O +). O + +In O +bacterial B-taxonomy_domain +Amt B-protein_type +proteins I-protein_type +, O +this O +Tyr B-residue_name +side O +chain O +is O +rotated O +∼ O +4 O +Å O +away O +as O +a O +result O +of O +the O +different O +conformation O +of O +TM1 B-structure_element +, O +leaving O +the O +channel B-site +open B-protein_state +and O +the O +histidine B-residue_name +available O +for O +its O +putative O +role O +in O +substrate O +transport O +( O +Supplementary O +Fig O +. O +2 O +). O + +Compared O +with O +ICL1 B-structure_element +, O +the O +backbone O +conformational O +changes O +observed O +for O +the O +neighbouring O +ICL2 B-structure_element +are O +smaller O +, O +but O +large O +shifts O +are O +nevertheless O +observed O +for O +the O +conserved B-protein_state +residues O +Glu140 B-residue_name_number +and O +Arg141 B-residue_name_number +( O +Fig O +. O +4 O +). O + +Finally O +, O +the O +important O +ICL3 B-structure_element +linking O +the O +pseudo B-structure_element +- I-structure_element +symmetrical I-structure_element +halves I-structure_element +( O +TM1 B-structure_element +- I-structure_element +5 I-structure_element +and O +TM6 B-structure_element +- I-structure_element +10 I-structure_element +) O +of O +the O +transporter B-protein_type +is O +also O +shifted O +up O +to O +∼ O +10 O +Å O +and O +forms O +an O +additional O +barrier O +that O +closes O +the O +channel B-site +on O +the O +cytoplasmic O +side O +( O +Fig O +. O +5 O +). O + +This O +two O +- O +tier O +channel B-structure_element +block I-structure_element +likely O +ensures O +that O +very O +little O +ammonium B-chemical +transport O +will O +take O +place O +under O +nitrogen B-chemical +- O +sufficient O +conditions O +. O + +The O +closed B-protein_state +state O +of O +the O +channel B-site +might O +also O +explain O +why O +no B-evidence +density I-evidence +, O +which O +could O +correspond O +to O +ammonium B-chemical +( O +or O +water B-chemical +), O +is O +observed O +in O +the O +hydrophobic O +part O +of O +the O +Mep2 B-protein +channel B-site +close O +to O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +. O + +Significantly O +, O +this O +is O +also O +true O +for O +ScMep2 B-protein +, O +which O +was O +crystallized B-experimental_method +in O +the O +presence O +of O +0 O +. O +2 O +M O +ammonium B-chemical +ions O +( O +see O +Methods O +section O +). O + +The O +final O +region O +in O +Mep2 B-protein +that O +shows O +large O +differences O +compared O +with O +the O +bacterial B-taxonomy_domain +transporters B-protein_type +is O +the O +CTR B-structure_element +. O + +By O +contrast O +, O +in O +the O +structures B-evidence +of O +bacterial B-taxonomy_domain +proteins O +, O +the O +CTR B-structure_element +is O +docked O +tightly O +onto O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +the O +transporters B-protein_type +( O +corresponding O +to O +TM1 B-structure_element +- I-structure_element +5 I-structure_element +), O +resulting O +in O +a O +more O +compact B-protein_state +structure B-evidence +. O + +In O +Amt B-protein +- I-protein +1 I-protein +and O +other O +bacterial B-taxonomy_domain +ammonium B-protein_type +transporters I-protein_type +, O +these O +CTR B-structure_element +residues O +interact O +with O +residues O +within O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +the O +protein O +. O + +Similar O +interactions O +were O +also O +modelled B-experimental_method +in O +the O +active B-protein_state +, O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +plant B-taxonomy_domain +AtAmt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +structure B-evidence +( O +for O +example O +, O +Y467 B-residue_name_number +- O +H239 B-residue_name_number +and O +D458 B-residue_name_number +- O +K71 B-residue_name_number +). O + +Also O +noteworthy O +is O +Asp381 B-residue_name_number +, O +the O +side O +chain O +of O +which O +interacts O +strongly O +with O +the O +positive O +dipole O +on O +the O +N O +- O +terminal O +end O +of O +TM2 B-structure_element +. O + +This O +interaction O +in O +the O +centre O +of O +the O +protein O +may O +be O +particularly O +important O +to O +stabilize O +the O +open B-protein_state +conformations O +of O +ammonium B-protein_type +transporters I-protein_type +. O + +In O +the O +Mep2 B-protein +structures B-evidence +, O +none O +of O +the O +interactions O +mentioned O +above O +are O +present O +. O + +Phosphorylation B-site +target I-site +site I-site +is O +at O +the O +periphery O +of O +Mep2 B-protein + +Recently O +Boeckstaens O +et O +al O +. O +provided O +evidence O +that O +Ser457 B-residue_name_number +in O +ScMep2 B-protein +( O +corresponding O +to O +Ser453 B-residue_name_number +in O +CaMep2 B-protein +) O +is O +phosphorylated B-protein_state +by O +the O +TORC1 B-protein_type +effector I-protein_type +kinase I-protein_type +Npr1 B-protein +under O +nitrogen B-chemical +- O +limiting O +conditions O +. O + +In O +the O +absence B-protein_state +of I-protein_state +Npr1 B-protein +, O +plasmid B-experimental_method +- I-experimental_method +encoded I-experimental_method +WT B-protein_state +Mep2 B-protein +in O +a O +S B-species +. I-species +cerevisiae I-species +mep1 B-mutant +- I-mutant +3Δ I-mutant +strain O +( O +triple B-mutant +mepΔ I-mutant +) O +does O +not O +allow O +growth O +on O +low O +concentrations O +of O +ammonium B-chemical +, O +suggesting O +that O +the O +transporter B-protein_type +is O +inactive B-protein_state +( O +Fig O +. O +3 O +and O +Supplementary O +Fig O +. O +1 O +). O + +Collectively O +, O +these O +data O +suggest O +that O +phosphorylation B-ptm +of O +Ser457 B-residue_name_number +opens O +the O +Mep2 B-protein +channel B-site +to O +allow O +ammonium B-chemical +uptake O +. O + +Ser457 B-residue_name_number +is O +located O +in O +a O +part O +of O +the O +CTR B-structure_element +that O +is O +conserved B-protein_state +in O +a O +subgroup O +of O +Mep2 B-protein_type +proteins I-protein_type +, O +but O +which O +is O +not O +present O +in O +bacterial B-taxonomy_domain +proteins O +( O +Fig O +. O +2 O +). O + +Where O +is O +the O +AI B-structure_element +region I-structure_element +and O +the O +Npr1 B-protein +phosphorylation B-site +site I-site +located O +? O +Our O +structures B-evidence +reveal O +that O +surprisingly O +, O +the O +AI B-structure_element +region I-structure_element +is O +folded O +back O +onto O +the O +CTR B-structure_element +and O +is O +not O +located O +near O +the O +centre O +of O +the O +trimer B-oligomeric_state +as O +expected O +from O +the O +bacterial B-taxonomy_domain +structures B-evidence +( O +Fig O +. O +4 O +). O + +The O +AI B-structure_element +regions I-structure_element +have O +very O +similar O +conformations O +in O +CaMep2 B-protein +and O +ScMep2 B-protein +, O +despite O +considerable O +differences O +in O +the O +rest O +of O +the O +CTR B-structure_element +( O +Fig O +. O +6 O +). O + +Strikingly O +, O +the O +Npr1 B-site +target I-site +serine I-site +residue O +is O +located O +at O +the O +periphery O +of O +the O +trimer B-oligomeric_state +, O +far O +away O +(∼ O +30 O +Å O +) O +from O +any O +channel B-site +exit I-site +( O +Fig O +. O +6 O +). O + +This O +makes O +sense O +since O +the O +proteins O +were O +expressed O +in O +rich O +medium O +and O +confirms O +the O +recent O +suggestion O +by O +Boeckstaens O +et O +al O +. O +that O +the O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +form O +of O +Mep2 B-protein +corresponds O +to O +the O +inactive B-protein_state +state O +. O + +For O +ScMep2 B-protein +, O +Ser457 B-residue_name_number +is O +the O +most O +C O +- O +terminal O +residue O +for O +which O +electron B-evidence +density I-evidence +is O +visible O +, O +indicating O +that O +the O +region O +beyond O +Ser457 B-residue_name_number +is O +disordered B-protein_state +. O + +In O +CaMep2 B-protein +, O +the O +visible O +part O +of O +the O +sequence O +extends O +for O +two O +residues O +beyond O +Ser453 B-residue_name_number +( O +Fig O +. O +6 O +). O + +Interestingly O +, O +a O +ScMep2 B-protein +457Δ B-mutant +truncation B-protein_state +mutant I-protein_state +in O +which O +a O +His O +- O +tag O +directly O +follows O +Ser457 B-residue_name_number +is O +highly O +expressed O +but O +has O +low B-protein_state +activity I-protein_state +( O +Fig O +. O +3 O +and O +Supplementary O +Fig O +. O +1b O +), O +suggesting O +that O +the O +His O +- O +tag O +interferes O +with O +phosphorylation B-ptm +by O +Npr1 B-protein +. O + +Given O +that O +Ser457 B-residue_name_number +/ O +453 B-residue_number +is O +far O +from O +any O +channel B-site +exit I-site +( O +Fig O +. O +6 O +), O +the O +crucial O +question O +is O +how O +phosphorylation B-ptm +opens O +the O +Mep2 B-protein +channel B-site +to O +generate O +an O +active B-protein_state +transporter B-protein_type +. O + +Boeckstaens O +et O +al O +. O +proposed O +that O +phosphorylation B-ptm +does O +not O +affect O +channel O +activity O +directly O +, O +but O +instead O +relieves O +inhibition O +by O +the O +AI B-structure_element +region I-structure_element +. O + +We O +obtained O +a O +similar O +result O +for O +ammonium O +uptake O +by O +the O +446Δ B-mutant +mutant B-protein_state +( O +Fig O +. O +3 O +), O +supporting O +the O +data O +from O +Marini O +et O +al O +. O +We O +then O +constructed B-experimental_method +and I-experimental_method +purified I-experimental_method +the O +analogous O +CaMep2 B-protein +442Δ B-mutant +truncation B-protein_state +mutant I-protein_state +and O +determined B-experimental_method +the O +crystal B-evidence +structure I-evidence +using O +data O +to O +3 O +. O +4 O +Å O +resolution O +. O + +The O +structure B-evidence +shows O +that O +removal B-experimental_method +of I-experimental_method +the O +AI B-structure_element +region I-structure_element +markedly O +increases O +the O +dynamics O +of O +the O +cytoplasmic B-structure_element +parts I-structure_element +of O +the O +transporter B-protein_type +. O + +Density B-evidence +for O +ICL3 B-structure_element +and O +the O +CTR B-structure_element +beyond O +residue O +Arg415 B-residue_name_number +is O +missing O +in O +the O +442Δ B-mutant +mutant B-protein_state +, O +and O +the O +density B-evidence +for O +the O +other O +ICLs B-structure_element +including O +ICL1 B-structure_element +is O +generally O +poor O +with O +visible O +parts O +of O +the O +structure B-evidence +having O +high O +B O +- O +factors O +( O +Fig O +. O +7 O +). O + +Why O +then O +does O +this O +mutant O +appear O +to O +be O +constitutively O +active B-protein_state +? O +We O +propose O +two O +possibilities O +. O + +The O +first O +one O +is O +that O +the O +open B-protein_state +state O +is O +disfavoured O +by O +crystallization B-experimental_method +because O +of O +lower O +stability O +or O +due O +to O +crystal O +packing O +constraints O +. O + +The O +second O +possibility O +is O +that O +the O +Tyr B-site +– I-site +His I-site +hydrogen I-site +bond I-site +has O +to O +be O +disrupted O +by O +the O +incoming O +substrate O +to O +open B-protein_state +the O +channel O +. O + +The O +latter O +model O +would O +fit O +well O +with O +the O +NH3 B-chemical +/ O +H B-chemical ++ I-chemical +symport O +model O +in O +which O +the O +proton O +is O +relayed O +by O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +. O + +The O +importance O +of O +the O +Tyr B-site +– I-site +His I-site +hydrogen I-site +bond I-site +is O +underscored O +by O +the O +fact O +that O +its O +removal B-experimental_method +in O +the O +ScMep2 B-protein +Y53A B-mutant +mutant B-protein_state +results O +in O +a O +constitutively B-protein_state +active I-protein_state +transporter B-protein_type +( O +Fig O +. O +3 O +). O + +Phosphorylation B-ptm +causes O +a O +conformational O +change O +in O +the O +CTR B-structure_element + +Do O +the O +Mep2 B-protein +structures B-evidence +provide O +any O +clues O +regarding O +the O +potential O +effect O +of O +phosphorylation B-ptm +? O + +The O +side O +- O +chain O +hydroxyl O +of O +Ser457 B-residue_name_number +/ O +453 B-residue_number +is O +located O +in O +a O +well O +- O +defined O +electronegative B-site +pocket I-site +that O +is O +solvent B-protein_state +accessible I-protein_state +( O +Fig O +. O +6 O +). O + +The O +closest O +atoms O +to O +the O +serine B-residue_name +hydroxyl O +group O +are O +the O +backbone O +carbonyl O +atoms O +of O +Asp419 B-residue_name_number +, O +Glu420 B-residue_name_number +and O +Glu421 B-residue_name_number +, O +which O +are O +3 O +– O +4 O +Å O +away O +. O + +To O +test O +this O +hypothesis O +, O +we O +determined B-experimental_method +the O +structure B-evidence +of O +the O +phosphorylation B-protein_state +- I-protein_state +mimicking I-protein_state +R452D B-mutant +/ I-mutant +S453D I-mutant +protein O +( O +hereafter O +termed O +‘ O +DD B-mutant +mutant I-mutant +'), O +using O +data O +to O +a O +resolution O +of O +2 O +. O +4 O +Å O +. O +The O +additional B-experimental_method +mutation I-experimental_method +of I-experimental_method +the O +arginine B-residue_name +preceding O +the O +phosphorylation B-site +site I-site +was O +introduced O +( O +i O +) O +to O +increase O +the O +negative O +charge O +density O +and O +make O +it O +more O +comparable O +to O +a O +phosphate B-chemical +at O +neutral O +pH O +, O +and O +( O +ii O +) O +to O +further O +destabilize O +the O +interactions O +of O +the O +AI B-structure_element +region I-structure_element +with O +the O +main B-structure_element +body I-structure_element +of O +the O +transporter B-protein_type +( O +Fig O +. O +6 O +). O + +The O +ammonium B-chemical +uptake O +activity O +of O +the O +S B-species +. I-species +cerevisiae I-species +version O +of O +the O +DD B-mutant +mutant I-mutant +is O +the O +same O +as O +that O +of O +WT B-protein_state +Mep2 B-protein +and O +the O +S453D B-mutant +mutant B-protein_state +, O +indicating O +that O +the O +mutations O +do O +not O +affect O +transporter O +functionality O +in O +the O +triple B-mutant +mepΔ I-mutant +background O +( O +Fig O +. O +3 O +). O + +Unexpectedly O +, O +the O +AI B-structure_element +segment I-structure_element +containing O +the O +mutated O +residues O +has O +only O +undergone O +a O +slight O +shift O +compared O +with O +the O +WT B-protein_state +protein O +( O +Fig O +. O +8 O +and O +Supplementary O +Fig O +. O +3 O +). O + +In O +addition O +, O +residues O +Glu420 B-residue_range +- I-residue_range +Leu423 I-residue_range +including O +Glu421 B-residue_name_number +of O +the O +ExxGxD B-structure_element +motif I-structure_element +are O +now O +disordered B-protein_state +( O +Fig O +. O +8 O +and O +Supplementary O +Fig O +. O +3 O +). O + +To O +exclude O +the O +possibility O +that O +the O +additional O +R452D B-mutant +mutation O +is O +responsible O +for O +the O +observed O +changes O +, O +we O +also O +determined B-experimental_method +the O +structure B-evidence +of O +the O +‘ O +single B-mutant +D I-mutant +' O +S453D B-mutant +mutant B-protein_state +. O + +To O +supplement O +the O +crystal B-evidence +structures I-evidence +, O +we O +also O +performed O +modelling B-experimental_method +and O +MD B-experimental_method +studies O +of O +WT B-protein_state +CaMep2 B-protein +, O +the O +DD B-mutant +mutant I-mutant +and O +phosphorylated B-protein_state +protein O +( O +S453J B-mutant +). O + +In O +the O +WT B-protein_state +structure B-evidence +, O +the O +acidic O +residues O +Asp419 B-residue_name_number +, O +Glu420 B-residue_name_number +and O +Glu421 B-residue_name_number +are O +within O +hydrogen B-bond_interaction +bonding I-bond_interaction +distance O +of O +Ser453 B-residue_name_number +. O + +The O +protein O +backbone O +has O +an O +average O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +. I-evidence +of O +only O +∼ O +3 O +Å O +during O +the O +200 O +- O +ns O +simulation B-experimental_method +, O +indicating O +that O +the O +protein O +is O +stable B-protein_state +. O + +There O +is O +flexibility O +in O +the O +side O +chains O +of O +the O +acidic O +residues O +so O +that O +they O +are O +able O +to O +form O +stable B-protein_state +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +Ser453 B-residue_name_number +. O + +In O +particular O +, O +persistent O +hydrogen B-bond_interaction +bonds I-bond_interaction +are O +observed O +between O +the O +Ser453 B-residue_name_number +hydroxyl O +group O +and O +the O +acidic O +group O +of O +Glu420 B-residue_name_number +, O +and O +also O +between O +the O +amine O +group O +of O +Ser453 B-residue_name_number +and O +the O +backbone O +carbonyl O +of O +Glu420 B-residue_name_number +( O +Supplementary O +Fig O +. O +5 O +). O + +The O +DD B-mutant +mutant I-mutant +is O +also O +stable B-protein_state +during O +the O +simulations B-experimental_method +, O +but O +the O +average O +backbone O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +of O +∼ O +3 O +. O +6 O +Å O +suggests O +slightly O +more O +conformational O +flexibility O +than O +WT B-protein_state +. O + +For O +example O +, O +the O +distance B-evidence +between O +the O +Asp453 B-residue_name_number +acidic O +oxygens O +and O +the O +Glu420 B-residue_name_number +acidic O +oxygens O +increases O +from O +∼ O +7 O +to O +> O +22 O +Å O +after O +200 O +ns O +simulations B-experimental_method +, O +and O +thus O +these O +residues O +are O +not O +interacting O +. O + +The O +protein O +is O +structurally B-protein_state +stable I-protein_state +throughout O +the O +simulation B-experimental_method +with O +little O +deviation O +in O +the O +other O +parts O +of O +the O +protein O +. O + +The O +movement O +of O +the O +acidic O +residues O +away O +from O +Arg452 B-residue_name_number +and O +Sep453 B-residue_name_number +is O +more O +pronounced O +in O +this O +simulation B-experimental_method +in O +comparison O +with O +the O +movement O +away O +from O +Asp452 B-residue_name_number +and O +Asp453 B-residue_name_number +in O +the O +DD B-mutant +mutant I-mutant +. O + +The O +short B-structure_element +helix I-structure_element +formed O +by O +residues O +Leu427 B-residue_range +to I-residue_range +Asp438 I-residue_range +unravels O +during O +the O +simulations B-experimental_method +to O +a O +disordered B-protein_state +state O +. O + +However O +, O +the O +conformational O +changes O +for O +the O +phosphomimetic B-mutant +mutants I-mutant +in O +the O +crystals B-evidence +are O +confined O +to O +the O +CTR B-structure_element +( O +Fig O +. O +8 O +), O +and O +the O +channels B-site +are O +still O +closed B-protein_state +( O +Supplementary O +Fig O +. O +2 O +). O + +The O +fact O +that O +the O +S453D B-mutant +structure B-evidence +was O +obtained O +in O +the O +presence O +of O +10 O +mM O +ammonium B-chemical +ions O +suggests O +that O +the O +crystallization B-experimental_method +process O +favours O +closed B-protein_state +states O +of O +the O +Mep2 B-protein +channels B-site +. O + +Knowledge O +about O +ammonium B-protein_type +transporter I-protein_type +structure B-evidence +has O +been O +obtained O +from O +experimental O +and O +theoretical O +studies O +on O +bacterial B-taxonomy_domain +family O +members O +. O + +These O +efforts O +have O +advanced O +our O +knowledge O +considerably O +but O +have O +not O +yet O +yielded O +atomic O +- O +level O +answers O +to O +several O +important O +mechanistic O +questions O +, O +including O +how O +ammonium B-chemical +transport O +is O +regulated O +in O +eukaryotes B-taxonomy_domain +and O +the O +mechanism O +of O +ammonium B-chemical +signalling O +. O + +Interestingly O +, O +phosphomimetic B-mutant +mutations I-mutant +introduced O +into O +one O +monomer B-oligomeric_state +inactivate O +the O +entire O +trimer B-oligomeric_state +, O +indicating O +that O +( O +i O +) O +heterotrimerization O +occurs O +and O +( O +ii O +) O +the O +CTR B-structure_element +mediates O +allosteric O +regulation O +of O +ammonium B-chemical +transport O +activity O +via O +phosphorylation B-ptm +. O + +Contrasting O +with O +the O +plant B-taxonomy_domain +transporters B-protein_type +, O +the O +inactive B-protein_state +states O +of O +Mep2 B-protein_type +proteins I-protein_type +under O +conditions O +of O +high O +ammonium B-chemical +are O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +, O +with O +channels B-site +that O +are O +closed B-protein_state +on O +the O +cytoplasmic O +side O +. O + +The O +reason O +why O +similar O +transporters B-protein_type +such O +as O +A B-species +. I-species +thaliana I-species +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +and O +Mep2 B-protein +are O +regulated O +in O +opposite O +ways O +by O +phosphorylation B-ptm +( O +inactivation B-protein_state +in O +plants B-taxonomy_domain +and O +activation B-protein_state +in O +fungi B-taxonomy_domain +) O +is O +not O +known O +. O + +In O +fungi B-taxonomy_domain +, O +preventing O +ammonium B-chemical +entry O +via O +channel O +closure O +in O +ammonium B-protein_type +transporters I-protein_type +would O +be O +one O +way O +to O +alleviate O +ammonium B-chemical +toxicity O +, O +in O +addition O +to O +ammonium B-chemical +excretion O +via O +Ato B-protein_type +transporters B-protein_type +and O +amino O +- O +acid O +secretion O +. O + +By O +determining O +the O +first O +structures B-evidence +of O +closed B-protein_state +ammonium B-protein_type +transporters I-protein_type +and O +comparing B-experimental_method +those O +structures B-evidence +with O +the O +permanently B-protein_state +open I-protein_state +bacterial B-taxonomy_domain +proteins O +, O +we O +demonstrate O +that O +Mep2 B-protein_type +channel B-site +closure O +is O +likely O +due O +to O +movements O +of O +the O +CTR B-structure_element +and O +ICL1 B-structure_element +and O +ICL3 B-structure_element +. O + +More O +specifically O +, O +the O +close O +interactions O +between O +the O +CTR B-structure_element +and O +ICL1 B-structure_element +/ O +ICL3 B-structure_element +present O +in O +open B-protein_state +transporters B-protein_type +are O +disrupted O +, O +causing O +ICL3 B-structure_element +to O +move O +outwards O +and O +block O +the O +channel B-site +( O +Figs O +4 O +and O +9a O +). O + +In O +addition O +, O +ICL1 B-structure_element +has O +shifted O +inwards O +to O +contribute O +to O +the O +channel B-site +closure O +by O +engaging O +His2 B-residue_name_number +from O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +via O +hydrogen B-bond_interaction +bonding I-bond_interaction +with O +a O +highly B-protein_state +conserved I-protein_state +tyrosine B-residue_name +hydroxyl O +group O +. O + +Importantly O +, O +the O +structural B-evidence +similarities I-evidence +in O +the O +TM B-structure_element +parts I-structure_element +of O +Mep2 B-protein +and O +AfAmt B-protein +- I-protein +1 I-protein +( O +Fig O +. O +5a O +) O +suggest O +that O +channel B-site +opening O +/ O +closure O +does O +not O +require O +substantial O +changes O +in O +the O +residues O +lining O +the O +channel B-site +. O + +How O +exactly O +the O +channel B-site +opens O +and O +whether O +opening O +is O +intra O +- O +monomeric O +are O +still O +open B-protein_state +questions O +; O +it O +is O +possible O +that O +the O +change O +in O +the O +CTR B-structure_element +may O +disrupt O +its O +interactions O +with O +ICL3 B-structure_element +of O +the O +neighbouring O +monomer B-oligomeric_state +( O +Fig O +. O +9b O +), O +which O +could O +result O +in O +opening O +of O +the O +neighbouring O +channel B-site +via O +inward O +movement O +of O +its O +ICL3 B-structure_element +. O + +Owing O +to O +the O +crosstalk O +between O +monomers B-oligomeric_state +, O +a O +single O +phosphorylation B-ptm +event O +might O +lead O +to O +opening O +of O +the O +entire O +trimer B-oligomeric_state +, O +although O +this O +has O +not O +yet O +been O +tested O +( O +Fig O +. O +9b O +). O + +Whether O +or O +not O +Mep2 B-protein_type +channel B-site +opening O +requires O +, O +in O +addition O +to O +phosphorylation B-ptm +, O +disruption O +of O +the O +Tyr B-site +– I-site +His2 I-site +interaction I-site +by O +the O +ammonium B-chemical +substrate O +is O +not O +yet O +clear O +. O + +Is O +our O +model O +for O +opening O +and O +closing O +of O +Mep2 B-protein +channels B-site +valid O +for O +other O +eukaryotic B-taxonomy_domain +ammonium B-protein_type +transporters I-protein_type +? O +Our O +structural B-evidence +data I-evidence +support O +previous O +studies O +and O +clarify O +the O +central O +role O +of O +the O +CTR B-structure_element +and O +cytoplasmic B-structure_element +loops I-structure_element +in O +the O +transition O +between O +closed B-protein_state +and O +open B-protein_state +states O +. O + +Nevertheless O +, O +given O +the O +central O +role O +of O +absolutely B-protein_state +conserved I-protein_state +residues O +within O +the O +ICL1 B-site +- I-site +ICL3 I-site +- I-site +CTR I-site +interaction I-site +network I-site +( O +Fig O +. O +4 O +), O +we O +propose O +that O +the O +structural O +basics O +of O +fungal B-taxonomy_domain +ammonium B-chemical +transporter O +activation O +are O +conserved B-protein_state +. O + +The O +fact O +that O +Mep2 B-protein_type +orthologues O +of O +distantly O +related O +fungi B-taxonomy_domain +are O +fully O +functional O +in O +ammonium B-chemical +transport O +and O +signalling O +in O +S B-species +. I-species +cerevisiae I-species +supports O +this O +notion O +. O + +It O +should O +also O +be O +noted O +that O +the O +tyrosine B-residue_name +residue O +interacting O +with O +His2 B-residue_name_number +is O +highly B-protein_state +conserved I-protein_state +in O +fungal B-taxonomy_domain +Mep2 B-protein_type +orthologues O +, O +suggesting O +that O +the O +Tyr B-site +– I-site +His2 I-site +hydrogen I-site +bond I-site +might O +be O +a O +general O +way O +to O +close B-protein_state +Mep2 B-protein_type +proteins I-protein_type +. O + +With O +regards O +to O +plant B-taxonomy_domain +AMTs B-protein_type +, O +it O +has O +been O +proposed O +that O +phosphorylation B-ptm +at O +T460 B-residue_name_number +generates O +conformational O +changes O +that O +would O +close O +the O +neighbouring O +pore B-site +via O +the O +C B-structure_element +terminus I-structure_element +. O + +This O +assumption O +was O +based O +partly O +on O +a O +homology B-experimental_method +model I-experimental_method +for O +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +based O +on O +the O +( O +open B-protein_state +) O +archaebacterial B-taxonomy_domain +AfAmt B-protein +- I-protein +1 I-protein +structure B-evidence +, O +which O +suggested O +that O +the O +C B-structure_element +terminus I-structure_element +of O +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +would O +extend O +further O +to O +the O +neighbouring O +monomer B-oligomeric_state +. O + +Based O +on O +the O +available O +structural B-evidence +information I-evidence +, O +we O +consider O +it O +more O +likely O +that O +phosphorylation O +- O +mediated O +pore O +closure O +in O +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +is O +intra O +- O +monomeric O +, O +via O +disruption O +of O +the O +interactions O +between O +the O +CTR B-structure_element +and O +ICL1 B-structure_element +/ O +ICL3 B-structure_element +( O +for O +example O +, O +Y467 B-residue_name_number +- O +H239 B-residue_name_number +and O +D458 B-residue_name_number +- O +K71 B-residue_name_number +). O + +There O +is O +generally O +no O +equivalent O +for O +CaMep2 B-protein +Tyr49 B-residue_name_number +in O +plant B-taxonomy_domain +AMTs B-protein_type +, O +indicating O +that O +a O +Tyr B-site +– I-site +His2 I-site +hydrogen I-site +bond I-site +as O +observed O +in O +Mep2 B-protein +may O +not O +contribute O +to O +the O +closed B-protein_state +state O +in O +plant B-taxonomy_domain +transporters B-protein_type +. O + +We O +propose O +that O +intra B-site +- I-site +monomeric I-site +CTR I-site +- I-site +ICL1 I-site +/ I-site +ICL3 I-site +interactions I-site +lie O +at O +the O +basis O +of O +regulation O +of O +both O +fungal B-taxonomy_domain +and O +plant B-taxonomy_domain +ammonium B-protein_type +transporters I-protein_type +; O +close O +interactions O +generate O +open B-protein_state +channels B-site +, O +whereas O +the O +lack B-protein_state +of I-protein_state +‘ O +intra O +-' O +interactions O +leads O +to O +inactive B-protein_state +states O +. O + +The O +need O +to O +regulate O +in O +opposite O +ways O +may O +be O +the O +reason O +why O +the O +phosphorylation B-site +sites I-site +are O +in O +different O +parts O +of O +the O +CTR B-structure_element +, O +that O +is O +, O +centrally O +located O +close O +to O +the O +ExxGxD B-structure_element +motif I-structure_element +in O +AMTs B-protein_type +and O +peripherally O +in O +Mep2 B-protein +. O + +Our O +model O +also O +provides O +an O +explanation O +for O +the O +observation O +that O +certain B-mutant +mutations I-mutant +within O +the O +CTR B-structure_element +completely O +abolish O +transport O +activity O +. O + +An O +example O +of O +an O +inactivating O +residue O +is O +the O +glycine B-residue_name +of O +the O +ExxGxD B-structure_element +motif I-structure_element +of O +the O +CTR B-structure_element +. O + +Mutation B-experimental_method +of O +this O +residue O +( O +G393 B-residue_name_number +in O +EcAmtB B-protein +; O +G456 B-residue_name_number +in O +AtAmt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +) O +inactivates O +transporters B-protein_type +as O +diverse O +as O +Escherichia B-species +coli I-species +AmtB B-protein +and O +A B-species +. I-species +thaliana I-species +Amt B-protein +- I-protein +1 I-protein +; I-protein +1 I-protein +( O +refs O +). O + +Regulation O +and O +modulation O +of O +membrane O +transport O +by O +phosphorylation B-ptm +is O +known O +to O +occur O +in O +, O +for O +example O +, O +aquaporins B-protein_type +and O +urea B-protein_type +transporters I-protein_type +, O +and O +is O +likely O +to O +be O +a O +common O +theme O +for O +eukaryotic B-taxonomy_domain +channels B-protein_type +and O +transporters B-protein_type +. O + +With O +respect O +to O +ammonium B-chemical +transport O +, O +phosphorylation B-ptm +has O +thus O +far O +only O +been O +shown O +for O +A B-species +. I-species +thaliana I-species +AMTs B-protein_type +and O +for O +S B-species +. I-species +cerevisiae I-species +Mep2 B-protein +( O +refs O +). O + +However O +, O +the O +absence B-protein_state +of I-protein_state +GlnK B-protein_type +proteins I-protein_type +in O +eukaryotes B-taxonomy_domain +suggests O +that O +phosphorylation B-ptm +- O +based O +regulation O +of O +ammonium B-chemical +transport O +may O +be O +widespread O +. O + +With O +respect O +to O +Mep2 B-protein_type +- O +mediated O +signalling O +to O +induce O +pseudohyphal O +growth O +, O +two O +models O +have O +been O +put O +forward O +as O +to O +how O +this O +occurs O +and O +why O +it O +is O +specific O +to O +Mep2 B-protein_type +proteins I-protein_type +. O + +In O +one O +model O +, O +signalling O +is O +proposed O +to O +depend O +on O +the O +nature O +of O +the O +transported O +substrate O +, O +which O +might O +be O +different O +in O +certain O +subfamilies O +of O +ammonium B-protein_type +transporters I-protein_type +( O +for O +example O +, O +Mep1 B-protein +/ O +Mep3 B-protein +versus O +Mep2 B-protein +). O + +For O +example O +, O +NH3 B-chemical +uniport O +or O +symport O +of O +NH3 B-chemical +/ O +H B-chemical ++ I-chemical +might O +result O +in O +changes O +in O +local O +pH O +, O +but O +NH4 B-chemical ++ I-chemical +uniport O +might O +not O +, O +and O +this O +difference O +might O +determine O +signalling O +. O + +In O +the O +other O +model O +, O +signalling O +is O +thought O +to O +require O +a O +distinct O +conformation O +of O +the O +Mep2 B-protein +transporter B-protein_type +occurring O +during O +the O +transport O +cycle O +. O + +While O +the O +current O +study O +does O +not O +specifically O +address O +the O +mechanism O +of O +signalling O +underlying O +pseudohyphal O +growth O +, O +our O +structures B-evidence +do O +show O +that O +Mep2 B-protein_type +proteins I-protein_type +can O +assume O +different O +conformations O +. O + +It O +is O +clear O +that O +ammonium B-chemical +transport O +across O +biomembranes O +remains O +a O +fascinating O +and O +challenging O +field O +in O +large O +part O +due O +to O +the O +unique O +properties O +of O +the O +substrate O +. O + +Our O +Mep2 B-protein +structural O +work O +now O +provides O +a O +foundation O +for O +future O +studies O +to O +uncover O +the O +details O +of O +the O +structural O +changes O +that O +occur O +during O +eukaryotic B-taxonomy_domain +ammonium B-chemical +transport O +and O +signaling O +, O +and O +to O +assess O +the O +possibility O +to O +utilize O +small O +molecules O +to O +shut O +down O +ammonium B-chemical +sensing O +and O +downstream O +signalling O +pathways O +in O +pathogenic O +fungi B-taxonomy_domain +. O + +The O +region O +showing O +ICL1 B-structure_element +( O +blue O +), O +ICL3 B-structure_element +( O +green O +) O +and O +the O +CTR B-structure_element +( O +red O +) O +is O +boxed O +for O +comparison O +. O + +( O +b O +) O +CaMep2 B-protein +trimer B-oligomeric_state +viewed O +from O +the O +intracellular O +side O +( O +right O +). O + +The O +CTR B-structure_element +is O +boxed O +. O + +( O +c O +) O +Overlay B-experimental_method +of O +ScMep2 B-protein +( O +grey O +) O +and O +CaMep2 B-protein +( O +rainbow O +), O +illustrating O +the O +differences O +in O +the O +CTRs B-structure_element +. O + +Sequence B-evidence +conservation I-evidence +in O +ammonium B-protein_type +transporters I-protein_type +. O + +The O +secondary O +structure O +elements O +observed O +for O +CaMep2 B-protein +are O +indicated O +, O +with O +the O +numbers O +corresponding O +to O +the O +centre O +of O +the O +TM B-structure_element +segment I-structure_element +. O + +Coloured O +residues O +are O +functionally O +important O +and O +correspond O +to O +those O +of O +the O +Phe B-site +gate I-site +( O +blue O +), O +the O +binding B-site +site I-site +Trp B-residue_name +residue O +( O +magenta O +) O +and O +the O +twin O +- O +His O +motif O +( O +red O +). O + +Growth B-experimental_method +of O +ScMep2 B-mutant +variants I-mutant +on O +low O +ammonium O +medium O +. O + +( O +a O +) O +The O +triple B-mutant +mepΔ I-mutant +strain O +( O +black O +) O +and O +triple O +mepΔ O +npr1Δ O +strain O +( O +grey O +) O +containing O +plasmids O +expressing O +WT B-protein_state +and O +variant B-mutant +ScMep2 I-mutant +were O +grown B-experimental_method +on I-experimental_method +minimal I-experimental_method +medium I-experimental_method +containing O +1 O +mM O +ammonium B-chemical +sulphate I-chemical +. O + +The O +quantified O +cell B-evidence +density I-evidence +reflects O +logarithmic O +growth O +after O +24 O +h O +. O +Error O +bars O +are O +the O +s O +. O +d O +. O +for O +three O +replicates O +of O +each O +strain O +( O +b O +) O +The O +strains O +used O +in O +a O +were O +also O +serially O +diluted O +and O +spotted O +onto O +minimal O +agar O +plates O +containing O +glutamate B-chemical +( O +0 O +. O +1 O +%) O +or O +ammonium B-chemical +sulphate I-chemical +( O +1 O +mM O +), O +and O +grown O +for O +3 O +days O +at O +30 O +° O +C O +. O + +Structural O +differences O +between O +Mep2 B-protein +and O +bacterial B-taxonomy_domain +ammonium O +transporters O +. O + +The O +numbering O +is O +for O +CaMep2 B-protein +. O + +( O +c O +) O +Conserved B-protein_state +residues O +in O +ICL1 B-structure_element +- I-structure_element +3 I-structure_element +and O +the O +CTR B-structure_element +. O + +Views O +from O +the O +cytosol O +for O +CaMep2 B-protein +( O +left O +) O +and O +AfAmt B-protein +- I-protein +1 I-protein +, O +highlighting O +the O +large O +differences O +in O +conformation O +of O +the O +conserved B-protein_state +residues O +in O +ICL1 B-structure_element +( O +RxK O +motif O +; O +blue O +), O +ICL2 B-structure_element +( O +ER B-structure_element +motif I-structure_element +; O +cyan O +), O +ICL3 B-structure_element +( O +green O +) O +and O +the O +CTR B-structure_element +( O +red O +). O + +The O +labelled O +residues O +are O +analogous O +within O +both O +structures B-evidence +. O + +Channel O +closures O +in O +Mep2 B-protein +. O + +( O +a O +) O +Stereo O +superposition B-experimental_method +of O +AfAmt B-protein +- I-protein +1 I-protein +and O +CaMep2 B-protein +showing O +the O +residues O +of O +the O +Phe B-site +gate I-site +, O +His2 B-residue_name_number +of O +the O +twin B-structure_element +- I-structure_element +His I-structure_element +motif I-structure_element +and O +the O +tyrosine B-residue_name +residue O +Y49 B-residue_name_number +in O +TM1 B-structure_element +that O +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +His2 B-residue_name_number +in O +CaMep2 B-protein +. O +( O +b O +) O +Surface O +views O +from O +the O +side O +in O +rainbow O +colouring O +, O +showing O +the O +two O +- O +tier O +channel B-structure_element +block I-structure_element +( O +indicated O +by O +the O +arrows O +) O +in O +CaMep2 B-protein +. O + +The O +Npr1 B-protein +kinase B-protein_type +target O +Ser453 B-residue_name_number +is O +dephosphorylated B-protein_state +and O +located O +in O +an O +electronegative B-site +pocket I-site +. O + +The O +phosphorylation B-ptm +target O +residue O +Ser453 B-residue_name_number +is O +labelled O +in O +bold O +. O + +( O +b O +) O +Overlay B-experimental_method +of O +the O +CTRs B-structure_element +of O +ScMep2 B-protein +( O +grey O +) O +and O +CaMep2 B-protein +( O +green O +), O +showing O +the O +similar O +electronegative O +environment O +surrounding O +the O +phosphorylation B-site +site I-site +( O +P O +). O + +The O +AI B-structure_element +regions I-structure_element +are O +coloured O +magenta O +. O + +( O +c O +) O +Cytoplasmic O +view O +of O +the O +Mep2 B-protein +trimer B-oligomeric_state +indicating O +the O +large O +distance O +between O +Ser453 B-residue_name_number +and O +the O +channel B-site +exits I-site +( O +circles O +; O +Ile52 B-residue_name_number +lining O +the O +channel B-site +exit I-site +is O +shown O +). O + +Effect O +of O +removal B-experimental_method +of O +the O +AI B-structure_element +region I-structure_element +on O +Mep2 B-protein +structure B-evidence +. O + +( O +a O +) O +Side O +views O +for O +WT B-protein_state +CaMep2 B-protein +( O +left O +) O +and O +the O +truncation B-protein_state +mutant I-protein_state +442Δ B-mutant +( O +right O +). O + +The O +latter O +is O +shown O +as O +a O +putty O +model O +according O +to O +B O +- O +factors O +to O +illustrate O +the O +disorder B-protein_state +in O +the O +protein O +on O +the O +cytoplasmic O +side O +. O + +Missing O +regions O +are O +labelled O +. O +( O +b O +) O +Stereo O +superpositions B-experimental_method +of O +WT B-protein_state +CaMep2 B-protein +and O +the O +truncation B-protein_state +mutant I-protein_state +. O + +2Fo O +– O +Fc O +electron O +density O +( O +contoured O +at O +1 O +. O +0 O +σ O +) O +for O +residues O +Tyr49 B-residue_name_number +and O +His342 B-residue_name_number +is O +shown O +for O +the O +truncation B-protein_state +mutant I-protein_state +. O + +( O +a O +) O +Cytoplasmic O +view O +of O +the O +DD B-mutant +mutant I-mutant +trimer B-oligomeric_state +, O +with O +WT B-protein_state +CaMep2 B-protein +superposed B-experimental_method +in O +grey O +for O +one O +of O +the O +monomers B-oligomeric_state +. O + +The O +AI B-structure_element +region I-structure_element +is O +coloured O +magenta O +. O + +( O +b O +) O +Monomer B-oligomeric_state +side O +- O +view O +superposition B-experimental_method +of O +WT B-protein_state +CaMep2 B-protein +and O +the O +DD B-mutant +mutant I-mutant +, O +showing O +the O +conformational O +change O +and O +disorder O +around O +the O +ExxGxD B-structure_element +motif I-structure_element +. O + +Side O +chains O +for O +residues O +452 B-residue_number +and O +453 B-residue_number +are O +shown O +as O +stick O +models O +. O + +Schematic O +model O +for O +phosphorylation O +- O +based O +regulation O +of O +Mep2 B-protein +ammonium O +transporters O +. O + +( O +a O +) O +In O +the O +closed B-protein_state +, O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +state O +( O +i O +), O +the O +CTR B-structure_element +( O +magenta O +) O +and O +ICL3 B-structure_element +( O +green O +) O +are O +far O +apart O +with O +the O +latter O +blocking O +the O +intracellular O +channel B-site +exit I-site +( O +indicated O +with O +a O +hatched O +circle O +). O + +Upon O +phosphorylation B-ptm +and O +mimicked B-protein_state +by O +the O +CaMep2 B-protein +S453D B-mutant +and O +DD B-mutant +mutants I-mutant +( O +ii O +), O +the O +region O +around O +the O +ExxGxD B-structure_element +motif I-structure_element +undergoes O +a O +conformational O +change O +that O +results O +in O +the O +CTR B-structure_element +interacting O +with O +the O +inward O +- O +moving O +ICL3 B-structure_element +, O +opening O +the O +channel B-site +( O +full O +circle O +) O +( O +iii O +). O + +The O +open B-protein_state +- O +channel B-site +Mep2 B-protein +structure B-evidence +is O +represented O +by O +archaebacterial B-taxonomy_domain +Amt B-protein +- I-protein +1 I-protein +and O +shown O +in O +lighter O +colours O +consistent O +with O +Fig O +. O +4 O +. O + +As O +discussed O +in O +the O +text O +, O +similar O +structural O +arrangements O +may O +occur O +in O +plant B-taxonomy_domain +AMTs B-protein_type +. O + +Template O +- O +dependent O +nucleotide O +addition O +in O +the O +reverse O +( O +3 O +′- O +5 O +′) O +direction O +by O +Thg1 B-protein_type +- I-protein_type +like I-protein_type +protein I-protein_type + +Structures B-evidence +of O +Thg1 B-protein_type +- I-protein_type +like I-protein_type +proteins I-protein_type +provide O +insight O +into O +the O +template O +- O +dependent O +nucleotide O +addition O +in O +the O +reverse O +( O +3 O +′- O +5 O +′) O +direction O +. O + +Thg1 B-protein_type +- I-protein_type +like I-protein_type +protein I-protein_type +( O +TLP B-protein_type +) O +catalyzes O +the O +addition O +of O +a O +nucleotide O +to O +the O +5 O +′- O +end O +of O +truncated O +transfer B-chemical +RNA I-chemical +( O +tRNA B-chemical +) O +species O +in O +a O +Watson O +- O +Crick O +template O +– O +dependent O +manner O +. O + +The O +reaction O +proceeds O +in O +two O +steps O +: O +the O +activation O +of O +the O +5 O +′- O +end O +by O +adenosine B-chemical +5 I-chemical +′- I-chemical +triphosphate I-chemical +( O +ATP B-chemical +)/ O +guanosine B-chemical +5 I-chemical +′- I-chemical +triphosphate I-chemical +( O +GTP B-chemical +), O +followed O +by O +nucleotide O +addition O +. O + +Structural B-experimental_method +analyses I-experimental_method +of O +the O +TLP B-protein_type +and O +its O +reaction O +intermediates O +have O +revealed O +the O +atomic O +detail O +of O +the O +template O +- O +dependent O +elongation O +reaction O +in O +the O +3 O +′- O +5 O +′ O +direction O +. O + +The O +enzyme O +creates O +two O +substrate B-site +binding I-site +sites I-site +for O +the O +first O +- O +and O +second O +- O +step O +reactions O +in O +the O +vicinity O +of O +one O +reaction B-site +center I-site +consisting O +of O +two O +Mg2 B-chemical ++ I-chemical +ions O +, O +and O +the O +two O +reactions O +are O +executed O +at O +the O +same O +reaction B-site +center I-site +in O +a O +stepwise O +fashion O +. O + +When O +the O +incoming O +nucleotide B-chemical +is O +bound B-protein_state +to I-protein_state +the O +second B-site +binding I-site +site I-site +with O +Watson B-bond_interaction +- I-bond_interaction +Crick I-bond_interaction +hydrogen I-bond_interaction +bonds I-bond_interaction +, O +the O +3 O +′- O +OH O +of O +the O +incoming O +nucleotide O +and O +the O +5 B-chemical +′- I-chemical +triphosphate I-chemical +of O +the O +tRNA B-chemical +are O +moved O +to O +the O +reaction B-site +center I-site +where O +the O +first O +reaction O +has O +occurred O +. O + +Although O +TLP B-protein_type +and O +Thg1 B-protein +have O +similar O +tetrameric B-oligomeric_state +organization O +, O +the O +tRNA B-chemical +binding O +mode O +of O +TLP B-protein_type +is O +different O +from O +that O +of O +Thg1 B-protein +, O +a O +tRNAHis B-protein_type +- I-protein_type +specific I-protein_type +G I-protein_type +− I-protein_type +1 I-protein_type +addition I-protein_type +enzyme I-protein_type +. O + +Each O +tRNAHis B-chemical +binds O +to O +three O +of O +the O +four O +Thg1 B-protein +tetramer B-oligomeric_state +subunits B-structure_element +, O +whereas O +in O +TLP B-protein_type +, O +tRNA B-chemical +only O +binds O +to O +a O +dimer B-site +interface I-site +and O +the O +elongation O +reaction O +is O +terminated O +by O +measuring O +the O +accepter B-structure_element +stem I-structure_element +length O +through O +the O +flexible B-protein_state +β B-structure_element +- I-structure_element +hairpin I-structure_element +. O + +All O +polynucleotide O +chain O +elongation O +reactions O +, O +whether O +with O +DNA B-chemical +or O +RNA B-chemical +, O +proceed O +in O +the O +5 O +′- O +3 O +′ O +direction O +. O + +This O +reaction O +involves O +the O +nucleophilic O +attack O +of O +a O +3 O +′- O +OH O +of O +the O +terminal O +nucleotide O +in O +the O +elongating O +chain O +on O +the O +α O +- O +phosphate B-chemical +of O +an O +incoming O +nucleotide O +. O + +This O +elongation O +reaction O +of O +DNA B-chemical +/ O +RNA B-chemical +chains O +is O +in O +clear O +contrast O +to O +the O +elongation O +of O +protein O +chains O +in O +which O +the O +high O +energy O +of O +the O +incoming O +aminoacyl B-chemical +- I-chemical +tRNA I-chemical +is O +not O +used O +for O +its O +own O +addition O +but O +for O +the O +addition O +of O +the O +next O +monomer B-oligomeric_state +( O +termed O +head O +polymerization O +). O + +In O +this O +case O +, O +the O +5 O +′- O +end O +of O +tRNA B-chemical +is O +first O +activated O +using O +adenosine B-chemical +5 I-chemical +′- I-chemical +triphosphate I-chemical +( O +ATP B-chemical +)/ O +guanosine B-chemical +5 I-chemical +′- I-chemical +triphosphate I-chemical +( O +GTP B-chemical +), O +followed O +by O +nucleophilic O +attack O +of O +a O +3 O +′- O +OH O +on O +the O +incoming O +nucleotide O +[ O +nucleoside B-chemical +5 I-chemical +′- I-chemical +triphosphate I-chemical +( O +NTP B-chemical +)] O +to O +yield O +pppN B-chemical +- I-chemical +tRNA I-chemical +. O + +Thus O +, O +the O +energy O +in O +the O +triphosphate B-chemical +bond O +of O +the O +incoming O +nucleotide O +is O +not O +used O +for O +its O +own O +addition O +but O +is O +reserved O +for O +subsequent O +polymerization O +( O +that O +is O +, O +head O +polymerization O +) O +( O +Fig O +. O +1 O +). O + +Top O +: O +Reaction O +scheme O +of O +3 O +′- O +5 O +′ O +elongation O +by O +Thg1 B-protein +/ O +TLP B-protein_type +family O +proteins O +. O + +Bottom O +: O +Reaction O +scheme O +of O +5 O +′- O +3 O +′ O +elongation O +by O +DNA B-protein_type +/ I-protein_type +RNA I-protein_type +polymerases I-protein_type +. O + +The O +best O +- O +characterized O +member O +of O +this O +family O +of O +proteins O +is O +eukaryotic B-taxonomy_domain +Thg1 B-protein +( O +tRNAHis B-protein_type +guanylyltransferase I-protein_type +), O +which O +catalyzes O +the O +nontemplated O +addition O +of O +a O +guanylate O +to O +the O +5 O +′- O +end O +of O +immature O +tRNAHis B-chemical +. O + +Therefore O +, O +Thg1 B-protein +is O +essential O +to O +the O +fidelity O +of O +protein O +synthesis O +in O +eukaryotes B-taxonomy_domain +. O + +However O +, O +Thg1 B-protein +homologs O +or O +TLPs B-protein_type +are O +found O +in O +organisms O +in O +which O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +is O +genetically O +encoded O +, O +and O +thus O +, O +posttranscriptional O +modification O +is O +not O +required O +. O + +TLPs B-protein_type +have O +been O +shown O +to O +catalyze O +5 O +′- O +end O +nucleotide O +addition O +to O +truncated O +tRNA B-chemical +species O +in O +vitro O +in O +a O +Watson O +- O +Crick O +template O +– O +dependent O +manner O +. O + +This O +function O +of O +TLPs B-protein_type +is O +not O +limited O +to O +tRNAHis B-chemical +but O +occurs O +efficiently O +with O +other O +tRNAs B-chemical +. O + +Furthermore O +, O +the O +yeast B-taxonomy_domain +homolog O +( O +Thg1p B-protein +) O +has O +been O +shown O +to O +interact O +with O +the O +replication O +origin O +recognition O +complex O +for O +DNA B-chemical +replication O +, O +and O +the O +plant B-taxonomy_domain +homolog O +( O +ICA1 B-protein +) O +was O +identified O +as O +a O +protein O +affecting O +the O +capacity O +to O +repair O +DNA B-chemical +damage O +. O + +These O +observations O +suggest O +that O +TLPs B-protein_type +may O +have O +more O +general O +functions O +in O +DNA B-chemical +/ O +RNA B-chemical +repair O +. O + +The O +3 O +′- O +5 O +′ O +addition O +reaction O +catalyzed O +by O +Thg1 B-protein +occurs O +through O +three O +reaction O +steps O +. O + +In O +the O +first O +step O +, O +the O +5 O +′- O +monophosphorylated O +tRNAHis B-chemical +, O +which O +is O +cleaved O +by O +ribonuclease B-protein_type +P I-protein_type +from O +pre B-chemical +- I-chemical +tRNAHis I-chemical +, O +is O +activated O +by O +ATP B-chemical +, O +creating O +a O +5 O +′- O +adenylylated O +tRNAHis B-chemical +intermediate O +. O + +In O +the O +second O +step O +, O +the O +3 O +′- O +OH O +of O +the O +incoming O +GTP B-chemical +attacks O +the O +activated O +intermediate O +, O +yielding O +pppG B-chemical +− I-chemical +1 I-chemical +- I-chemical +tRNAHis I-chemical +. O + +Finally O +, O +the O +pyrophosphate B-chemical +is O +removed O +, O +and O +mature O +pG B-chemical +− I-chemical +1 I-chemical +- I-chemical +tRNAHis I-chemical +is O +created O +. O + +The O +crystal B-evidence +structure I-evidence +of O +human B-species +Thg1 B-protein +( O +HsThg1 B-protein +) O +shows O +that O +its O +catalytic B-site +core I-site +shares O +structural O +homology O +with O +canonical O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +nucleotide I-protein_type +polymerases I-protein_type +, O +such O +as O +T7 B-protein_type +DNA I-protein_type +/ I-protein_type +RNA I-protein_type +polymerases I-protein_type +. O + +The O +crystal B-evidence +structure I-evidence +of O +TLP B-protein_type +from O +Bacillus B-species +thuringiensis I-species +shows O +that O +it O +shares O +a O +similar O +tetrameric B-oligomeric_state +assembly O +and O +active B-site +- I-site +site I-site +architecture O +with O +HsThg1 B-protein +. O + +However O +, O +in O +this O +structural B-experimental_method +analysis I-experimental_method +, O +the O +5 O +′- O +end O +of O +tRNAHis B-chemical +was O +not O +activated O +and O +the O +second O +substrate O +( O +GTP B-chemical +) O +was O +not B-protein_state +bound I-protein_state +. O + +Here O +, O +we O +successfully O +solved B-experimental_method +the O +structure B-evidence +of O +TLP B-protein_type +from O +the O +methanogenic B-taxonomy_domain +archaeon I-taxonomy_domain +Methanosarcina B-species +acetivorans I-species +( O +MaTLP B-protein +) O +in B-protein_state +complex I-protein_state +with I-protein_state +ppptRNAPheΔ1 B-chemical +, O +which O +mimics O +the O +activated O +intermediate O +of O +the O +repair O +substrate O +. O + +Although O +TLP B-protein_type +and O +Thg1 B-protein +have O +similar O +tetrameric B-oligomeric_state +organization O +, O +the O +mode O +of O +tRNA B-chemical +binding O +is O +different O +in O +TLP B-protein_type +. O + +Furthermore O +, O +we O +obtained O +the O +structure B-evidence +in O +which O +the O +GTP B-chemical +analog O +( O +GDPNP B-chemical +) O +was O +inserted O +into O +this O +complex O +to O +form O +a O +Watson B-bond_interaction +- I-bond_interaction +Crick I-bond_interaction +base I-bond_interaction +pair I-bond_interaction +with O +C72 B-residue_name_number +at O +the O +3 O +′- O +end O +region O +of O +the O +tRNA B-chemical +. O + +On O +the O +basis O +of O +these O +structures B-evidence +, O +we O +discuss O +the O +reaction O +mechanism O +of O +template O +- O +dependent O +reverse O +( O +3 O +′- O +5 O +′) O +polymerization O +in O +comparison O +with O +canonical O +5 O +′- O +3 O +′ O +polymerization O +. O + +Anticodon O +- O +independent O +binding O +of O +ppptRNAPheΔ1 B-chemical +to O +MaTLP B-protein + +Previous O +biochemical B-experimental_method +experiments I-experimental_method +have O +suggested O +that O +ppptRNAPheΔ1 B-chemical +, O +in O +which O +the O +5 O +′- O +end O +of O +tRNAPhe B-chemical +was O +triphosphorylated O +and O +G1 B-residue_name_number +was O +deleted B-experimental_method +, O +can O +be O +an O +efficient O +substrate O +for O +the O +repair O +reaction O +( O +guanylyl O +transfer O +) O +of O +Thg1 B-protein +/ O +TLP B-protein_type +. O + +The O +crystal B-evidence +contained O +a O +dimer B-oligomeric_state +of O +TLP B-protein_type +( O +A B-structure_element +and O +B B-structure_element +molecules O +) O +and O +one O +tRNA B-chemical +in O +an O +asymmetric O +unit O +. O + +Two O +dimers B-oligomeric_state +in O +the O +crystal B-evidence +further O +assembled O +as O +a O +dimer B-oligomeric_state +of O +dimers B-oligomeric_state +by O +the O +crystallographic O +twofold O +axis O +( O +Fig O +. O +2 O +). O + +This O +tetrameric B-oligomeric_state +structure B-evidence +and O +4 O +: O +2 O +stoichiometry O +of O +the O +TLP B-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +complex O +are O +the O +same O +as O +those O +of O +the O +CaThg1 B-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +complex O +. O + +However O +, O +whereas O +the O +AB B-structure_element +and O +CD B-structure_element +dimers B-oligomeric_state +of O +tetrameric B-oligomeric_state +CaThg1 B-protein +play O +different O +roles O +, O +respectively O +recognizing O +the O +accepter B-structure_element +stem I-structure_element +and O +anticodon O +of O +tRNAHis B-chemical +, O +the O +AB B-structure_element +dimer B-oligomeric_state +and O +its O +symmetry O +mate O +( O +CD B-structure_element +dimer B-oligomeric_state +) O +on O +tetrameric B-oligomeric_state +MaTLP B-protein +independently O +bind O +one O +molecule O +of O +tRNA B-chemical +( O +fig O +. O +S2 O +), O +recognizing O +the O +tRNA B-chemical +accepter B-structure_element +stem I-structure_element +and O +elbow B-structure_element +region I-structure_element +. O + +Thus O +, O +consistent O +with O +the O +notion O +that O +MaTLP B-protein +is O +an O +anticodon B-protein_type +- I-protein_type +independent I-protein_type +repair I-protein_type +enzyme I-protein_type +, O +the O +anticodon O +was O +not O +recognized O +in O +the O +MaTLP B-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +complex O +, O +whereas O +the O +binding O +mode O +of O +CaThg1 B-protein +is O +for O +the O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +addition O +reaction O +, O +therefore O +the O +His B-residue_name +anticodon O +has O +to O +be O +recognized O +( O +see O +“ O +Dual O +binding O +mode O +for O +tRNA B-chemical +repair O +”). O + +Structure B-evidence +of O +the O +MaTLP B-protein +complex B-protein_state +with I-protein_state +ppptRNAPheΔ1 B-chemical +. O + +Left O +: O +One O +molecule O +of O +the O +tRNA B-chemical +substrate O +( O +ppptRNAPheΔ1 B-chemical +) O +is O +bound B-protein_state +to I-protein_state +the O +MaTLP B-protein +dimer B-oligomeric_state +. O + +The O +AB B-structure_element +and O +CD B-structure_element +dimers B-oligomeric_state +are O +further O +dimerized B-oligomeric_state +by O +the O +crystallographic O +twofold O +axis O +to O +form O +a O +tetrameric B-oligomeric_state +structure B-evidence +( O +dimer B-oligomeric_state +of O +dimers B-oligomeric_state +). O + +The O +CD B-structure_element +dimer B-oligomeric_state +is O +omitted O +for O +clarity O +. O + +The O +accepter B-structure_element +stem I-structure_element +of O +the O +tRNA B-chemical +is O +recognized O +by O +molecule O +A O +( O +yellow O +), O +and O +the O +elbow B-structure_element +region I-structure_element +by O +molecule O +B O +( O +blue O +). O + +The O +β B-structure_element +- I-structure_element +hairpin I-structure_element +region O +of O +molecule O +B O +is O +shown O +in O +red O +. O + +The O +elbow B-structure_element +region I-structure_element +of O +the O +tRNA B-chemical +substrate O +was O +recognized O +by O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +of O +molecule O +B O +of O +MaTLP B-protein +. O + +The O +N O +atoms O +in O +the O +side O +chain O +of O +R215 B-residue_name_number +in O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +region O +of O +MaTLP B-protein +were O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +phosphate B-chemical +groups O +of O +U55 B-residue_name_number +and O +G57 B-residue_name_number +. O + +This O +β B-structure_element +- I-structure_element +hairpin I-structure_element +region O +was O +disordered B-protein_state +in O +the O +crystal B-evidence +structure I-evidence +of O +the O +CaThg1 B-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +complex O +. O + +The O +accepter B-structure_element +stem I-structure_element +of O +the O +tRNA B-chemical +substrate O +was O +recognized O +by O +molecule O +A O +of O +MaTLP B-protein +. O + +R136 B-residue_name_number +was O +also O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +base O +of O +C72 B-residue_name_number +( O +the O +Watson O +- O +Crick O +bond O +partner O +of O +ΔG1 O +). O + +The O +triphosphate B-chemical +moiety O +at O +the O +5 O +′- O +end O +of O +the O +tRNA B-chemical +was O +bonded O +to O +the O +D21 B-residue_range +- I-residue_range +K26 I-residue_range +region O +. O + +These O +phosphates B-chemical +were O +also O +coordinated B-bond_interaction +to I-bond_interaction +two O +metal O +ions O +, O +presumably O +Mg2 B-chemical ++ I-chemical +( O +Mg2 B-chemical ++ I-chemical +A O +and O +Mg2 B-chemical ++ I-chemical +B O +) O +because O +they O +were O +observed O +at O +the O +same O +positions O +( O +figs O +. O +S3 O +and O +S4 O +) O +previously O +identified O +by O +CaThg1 B-protein +and O +HsThg1 B-protein +structures B-evidence +. O + +These O +ions O +were O +in O +turn O +coordinated B-bond_interaction +by I-bond_interaction +the O +O O +atoms O +of O +the O +side O +chains O +of O +D21 B-residue_name_number +and O +D69 B-residue_name_number +and O +the O +main O +- O +chain O +O O +of O +G22 B-residue_name_number +( O +fig O +. O +S3A O +). O + +Mutation B-experimental_method +of O +D29 B-residue_name_number +and O +D76 B-residue_name_number +in O +HsThg1 B-protein +( O +corresponding O +to O +D21 B-residue_name_number +and O +D69 B-residue_name_number +of O +MaTLP B-protein +) O +has O +been O +shown O +to O +markedly O +decrease O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +addition O +activity O +. O + +Template O +- O +dependent O +binding O +of O +the O +GTP B-chemical +analog O +to O +the O +MaTLP B-complex_assembly +- I-complex_assembly +ppptRNAPheΔ1 I-complex_assembly +complex O + +Here O +, O +we O +successfully O +obtained O +the O +structure B-evidence +of O +the O +ternary O +complex O +of O +MaTLP B-protein +, O +5 O +′- O +activated O +tRNA B-chemical +( O +ppptRNAPheΔ1 B-chemical +), O +and O +the O +GTP B-chemical +analog O +( O +GDPNP B-chemical +) O +( O +Fig O +. O +3 O +and O +fig O +. O +S4 O +) O +by O +soaking B-experimental_method +the O +MaTLP B-complex_assembly +- I-complex_assembly +ppptRNAPheΔ1 I-complex_assembly +complex O +crystal B-evidence +in O +a O +solution O +containing O +GDPNP B-chemical +. O + +The O +5 O +′- O +end O +( O +position O +2 O +) O +of O +the O +tRNA B-chemical +moved O +significantly O +( O +Fig O +. O +3C O +) O +due O +to O +the O +insertion O +of O +GDPNP B-chemical +. O + +Together O +with O +the O +observation O +that O +the O +3 O +′- O +OH O +of O +the O +incoming O +GTP B-chemical +analog O +was O +within O +coordination O +distance O +( O +2 O +. O +8 O +Å O +) O +to O +Mg2 B-chemical ++ I-chemical +A O +( O +fig O +. O +S3B O +) O +and O +was O +able O +to O +execute O +a O +nucleophilic O +attack O +on O +the O +α O +- O +phosphate B-chemical +of O +the O +5 O +′- O +end O +, O +this O +structure B-evidence +indicates O +that O +the O +elongation O +reaction O +( O +second O +reaction O +) O +takes O +place O +at O +the O +same O +reaction B-site +center I-site +where O +the O +activation O +reaction O +( O +first O +reaction O +) O +occurs O +. O + +Structural O +change O +of O +the O +tRNA B-chemical +( O +ppptRNAPheΔ1 B-chemical +). O + +Structural O +change O +of O +the O +tRNA B-chemical +( O +ppptRNAPheΔ1 B-chemical +) O +accepter B-structure_element +stem I-structure_element +in O +MaTLP B-protein +caused O +by O +insertion O +of O +GDPNP B-chemical +. O +( O +A O +) O +Structure B-evidence +before O +GDPNP B-chemical +binding O +. O + +The O +triphosphate B-chemical +of O +the O +GDPNP B-chemical +was O +also O +bonded O +to O +the O +third O +Mg2 B-chemical ++ I-chemical +( O +Mg2 B-chemical ++ I-chemical +C O +), O +which O +, O +unlike O +Mg2 B-chemical ++ I-chemical +A O +and O +Mg2 B-chemical ++ I-chemical +B O +, O +is O +not O +coordinated B-bond_interaction +by I-bond_interaction +the O +TLP B-protein_type +molecule O +( O +fig O +. O +S3B O +). O + +This O +triphosphate B-chemical +binding O +mode O +is O +the O +same O +as O +that O +for O +the O +second B-site +nucleotide I-site +binding I-site +site I-site +in O +Thg1 B-protein +. O + +However O +, O +in O +previous O +analyses O +, O +the O +base O +moiety O +at O +the O +second B-site +site I-site +was O +either O +invisible O +or O +far O +beyond O +the O +reaction O +distance O +of O +the O +phosphate B-chemical +, O +and O +therefore O +, O +flipping O +of O +the O +base O +was O +expected O +to O +occur O +. O + +tRNA B-experimental_method +binding I-experimental_method +and I-experimental_method +repair I-experimental_method +experiments I-experimental_method +of O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +mutants B-protein_state + +To O +confirm O +tRNA B-chemical +recognition O +by O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +, O +we O +created B-experimental_method +mutation I-experimental_method +variants I-experimental_method +with O +altered O +residues O +in O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +region O +. O + +Then O +, O +tRNA B-experimental_method +binding I-experimental_method +and I-experimental_method +enzymatic I-experimental_method +activities I-experimental_method +were I-experimental_method +measured I-experimental_method +. O + +β B-structure_element +- I-structure_element +Hairpin I-structure_element +deletion B-protein_state +variant I-protein_state +delR198 B-mutant +- I-mutant +R215 I-mutant +almost O +completely O +abolished O +the O +binding O +of O +tRNAPheΔ1 B-chemical +( O +fig O +. O +S6 O +). O + +Furthermore O +, O +the O +enzymatic O +activities O +of O +delR198 B-mutant +- I-mutant +R215 I-mutant +and O +delG202 B-mutant +- I-mutant +E210 I-mutant +were O +very O +weak O +( O +5 O +. O +2 O +and O +13 O +. O +5 O +%, O +respectively O +) O +compared O +with O +wild B-protein_state +type I-protein_state +, O +whereas O +mutations B-experimental_method +( O +N179A B-mutant +and O +F174A B-mutant +/ O +N179A B-mutant +/ O +R188A B-mutant +) O +on O +the O +anticodon B-site +recognition I-site +site I-site +[ O +deduced O +from O +the O +Thg1 B-complex_assembly +- I-complex_assembly +tRNAHis I-complex_assembly +complex O +structure B-evidence +] O +had O +no O +effect O +on O +the O +catalytic O +activity O +( O +Fig O +. O +4A O +). O + +Experiments O +using O +the O +tRNAHisΔ1 B-chemical +substrate O +gave O +similar O +results O +( O +Fig O +. O +4A O +). O + +All O +these O +results O +are O +consistent O +with O +the O +crystal B-evidence +structure I-evidence +and O +suggest O +that O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +plays O +an O +important O +role O +in O +anticodon O +- O +independent O +binding O +of O +the O +tRNA B-chemical +substrate O +. O + +Residues O +in O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +are O +not B-protein_state +well I-protein_state +conserved I-protein_state +, O +except O +for O +R215 B-residue_name_number +( O +fig O +. O +S5 O +). O + +Mutants B-protein_state +R215A B-mutant +and O +R215A B-mutant +/ O +S213A B-mutant +, O +in O +which O +the O +completely B-protein_state +conserved I-protein_state +R215 B-residue_name_number +was O +changed B-experimental_method +to O +alanine B-residue_name +, O +showed O +a O +moderate O +effect O +on O +the O +activity O +( O +27 O +. O +3 O +and O +16 O +. O +3 O +%, O +respectively O +). O + +Thus O +, O +specific O +interactions O +with O +the O +conserved B-protein_state +R215 B-residue_name_number +and O +van B-bond_interaction +der I-bond_interaction +Waals I-bond_interaction +contacts I-bond_interaction +to O +residues O +in O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +would O +be O +important O +for O +tRNA B-chemical +recognition O +. O + +Mutational B-experimental_method +analysis I-experimental_method +of O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +and O +anticodon B-site +binding I-site +region I-site +. O + +The O +activity O +to O +tRNAPheΔ1 B-chemical +is O +about O +10 O +% O +of O +ppptRNAPheΔ1 B-chemical +. O + +Termination O +of O +the O +elongation O +reaction O +by O +measuring O +the O +accepter B-structure_element +stem I-structure_element + +TLPs B-protein_type +catalyze O +the O +Watson O +- O +Crick O +template O +– O +dependent O +elongation O +or O +repair O +reaction O +for O +5 O +′- O +end O +truncated O +tRNAPhe B-chemical +substrates O +lacking O +G1 B-residue_name_number +only O +( O +tRNAPheΔ1 B-chemical +), O +or O +lacking O +both O +G1 B-residue_name_number +and O +G2 B-residue_name_number +( O +tRNAPheΔ1 B-chemical +, I-chemical +2 I-chemical +), O +whereas O +they O +do O +not O +show O +any O +activity O +with O +intact O +tRNAPhe B-chemical +( O +thus O +, O +repair O +is O +unnecessary O +). O + +How O +TLP B-protein_type +distinguishes O +between O +tRNAs B-chemical +that O +need O +5 O +′- O +end O +repair O +from O +ones O +that O +do O +not O +, O +or O +in O +other O +words O +, O +how O +the O +elongation O +reaction O +is O +properly O +terminated O +, O +remains O +unknown O +. O + +The O +present O +structure B-evidence +of O +the O +MaTLP B-complex_assembly +- I-complex_assembly +ppptRNAPheΔ1 I-complex_assembly +complex O +shows O +that O +, O +unlike O +Thg1 B-protein +, O +the O +TLP B-protein_type +dimer B-oligomeric_state +binds O +one O +molecule O +of O +tRNA B-chemical +by O +recognizing O +the O +elbow B-structure_element +region I-structure_element +by O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +of O +molecule O +B B-structure_element +and O +the O +5 O +′- O +end O +by O +molecule O +A B-structure_element +. O +Therefore O +, O +we O +speculated O +that O +the O +flexible B-protein_state +nature O +of O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +enables O +the O +recognition O +of O +tRNA B-chemical +substrates O +with O +different O +accepter B-structure_element +stem I-structure_element +lengths O +. O + +To O +confirm O +this O +speculation O +, O +we O +used B-experimental_method +computer I-experimental_method +graphics I-experimental_method +to I-experimental_method +examine I-experimental_method +whether O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +region O +was O +able O +to O +bind O +tRNA B-chemical +substrates O +with O +different O +accepter B-structure_element +stem I-structure_element +lengths O +when O +the O +5 O +′- O +end O +was O +properly O +placed O +in O +the O +reaction B-site +site I-site +. O + +When O +the O +5 O +′- O +end O +was O +placed O +in O +the O +reaction O +site O +, O +the O +body O +of O +the O +tRNA B-chemical +molecule O +shifted O +in O +a O +manner O +dependent O +on O +the O +accepter B-structure_element +stem I-structure_element +length O +. O + +The O +tRNA B-chemical +body O +also O +rotated O +because O +of O +the O +helical O +nature O +of O +the O +accepter B-structure_element +stem I-structure_element +( O +fig O +. O +S7 O +). O + +This O +model O +structure B-evidence +showed O +that O +the O +accepter B-structure_element +stem I-structure_element +of O +intact O +tRNAPhe B-chemical +was O +too O +long O +for O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +to O +recognize O +its O +elbow B-structure_element +region I-structure_element +, O +whereas O +tRNAPheΔ1 B-chemical +and O +tRNAPheΔ1 B-chemical +, I-chemical +2 I-chemical +were O +recognized O +by O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +region O +( O +fig O +. O +S7 O +), O +which O +is O +consistent O +with O +previous O +experiments O +. O + +On O +the O +basis O +of O +these O +model O +structures B-evidence +, O +we O +concluded O +that O +the O +TLP B-protein_type +molecule O +can O +properly O +terminate O +elongation O +by O +measuring O +the O +accepter B-structure_element +stem I-structure_element +length O +of O +tRNA B-chemical +substrates O +. O + +Dual O +binding O +mode O +for O +tRNA B-chemical +repair O + +The O +present O +structural B-experimental_method +analysis I-experimental_method +revealed O +that O +although O +TLP B-protein_type +and O +Thg1 B-protein +have O +a O +similar O +tetrameric B-oligomeric_state +architecture O +, O +they O +have O +different O +binding O +modes O +for O +tRNAs B-chemical +: O +Thg1 B-protein +is O +bound B-protein_state +to I-protein_state +tRNAHis B-chemical +as O +a O +tetramer B-oligomeric_state +, O +whereas O +TLP B-protein_type +is O +bound B-protein_state +to I-protein_state +tRNAPhe B-chemical +as O +a O +dimer B-oligomeric_state +. O + +This O +difference O +in O +the O +tRNA B-chemical +binding O +modes O +is O +closely O +related O +to O +their O +enzymatic O +functions O +. O + +The O +tetrameric B-oligomeric_state +architecture O +of O +the O +Thg1 B-protein +molecule O +allows O +it O +to O +access O +both O +regions O +located O +at O +the O +opposite O +side O +of O +the O +tRNA B-chemical +molecule O +[ O +the O +AB B-structure_element +dimer B-oligomeric_state +recognizes O +the O +accepter B-structure_element +stem I-structure_element +and O +CD B-structure_element +dimer B-oligomeric_state +anticodon O +]. O + +In O +contrast O +, O +the O +binding O +mode O +of O +TLP B-protein_type +corresponds O +to O +the O +anticodon O +- O +independent O +repair O +reactions O +of O +5 O +′- O +truncated O +general O +tRNAs B-chemical +. O + +This O +binding O +mode O +is O +also O +suitable O +for O +the O +correct O +termination O +of O +the O +elongation O +or O +repair O +reaction O +by O +measuring O +the O +length O +of O +the O +accepter B-structure_element +stem I-structure_element +by O +the O +flexible B-protein_state +β B-structure_element +- I-structure_element +hairpin I-structure_element +. O + +Because O +tRNAHis B-chemical +requires O +an O +extra O +guanosine B-chemical +( O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +) O +at O +the O +5 O +′- O +end O +, O +the O +repair O +enzyme O +has O +to O +extend O +the O +5 O +′- O +end O +by O +one O +more O +nucleotide O +than O +other O +tRNAs B-chemical +. O + +Here O +, O +we O +showed O +that O +the O +TLP B-protein_type +mutants B-protein_state +, O +wherein O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +is O +truncated B-protein_state +and O +tRNAPheΔ1 B-chemical +binding O +ability O +is O +lost O +, O +can O +still O +bind O +to O +tRNAPhe B-chemical +( O +GUG B-chemical +) O +whose O +anticodon O +is O +changed O +to O +that O +for O +His B-residue_name +( O +fig O +. O +S6 O +, O +C O +, O +H O +, O +and O +I O +). O + +Also O +, O +the O +intact O +tRNAPhe B-chemical +, O +which O +is O +not O +recognized O +by O +TLP B-protein_type +( O +Fig O +. O +4B O +and O +fig O +. O +S6E O +), O +can O +be O +recognized O +when O +its O +anticodon O +is O +changed O +to O +that O +for O +His B-residue_name +( O +fig O +. O +S6D O +). O + +Furthermore O +, O +the O +TLP B-protein_type +variant B-protein_state +( O +F174A B-mutant +/ O +N179A B-mutant +/ O +R188A B-mutant +) O +whose O +anticodon B-site +recognition I-site +site I-site +[ O +deduced O +from O +the O +Thg1 B-complex_assembly +- I-complex_assembly +tRNAHis I-complex_assembly +complex O +structure B-evidence +] O +is O +disrupted O +has O +been O +shown O +to O +have O +a O +reduced O +catalytic O +activity O +to O +tRNAHisΔ B-chemical +− I-chemical +1 I-chemical +( O +Fig O +. O +4B O +). O + +All O +these O +experimental O +results O +indicate O +that O +TLP B-protein_type +recognizes O +and O +binds O +tRNAs B-chemical +carrying O +the O +His B-residue_name +anticodon O +in O +the O +same O +way O +that O +Thg1 B-protein +recognizes O +tRNAHis B-chemical +. O + +The O +elongation O +or O +repair O +reaction O +normally O +terminates O +when O +the O +5 O +′- O +end O +reaches O +position O +1 O +, O +but O +when O +the O +His B-residue_name +anticodon O +is O +present O +, O +TLP B-protein_type +binds O +the O +tRNA B-chemical +in O +the O +second O +mode O +by O +recognizing O +the O +anticodon O +to O +execute O +the O +G B-residue_name_number +− I-residue_name_number +1 I-residue_name_number +addition O +reaction O +. O + +By O +having O +two O +different O +binding O +modes O +, O +TLP B-protein_type +can O +manage O +this O +special O +feature O +of O +tRNAHis B-chemical +. O + +The O +Thg1 B-protein +/ O +TLP B-protein_type +family O +of O +proteins O +extends O +tRNA B-chemical +chains O +in O +the O +3 O +′- O +5 O +′ O +direction O +. O + +First O +, O +the O +5 B-chemical +′- I-chemical +phosphate I-chemical +is O +activated O +by O +GTP B-chemical +/ O +ATP B-chemical +. O + +Then O +, O +the O +activated O +phosphate B-chemical +is O +attacked O +by O +the O +incoming O +nucleotide O +, O +resulting O +in O +an O +extension O +by O +one O +nucleotide O +at O +the O +5 O +′- O +end O +. O + +Here O +, O +we O +successfully O +solved B-experimental_method +for O +the O +first O +time O +the O +intermediate O +structures B-evidence +of O +the O +template O +- O +dependent O +3 O +′- O +5 O +′ O +elongation O +complex O +of O +MaTLP B-protein +. O + +Figure O +5 O +is O +a O +schematic O +diagram O +of O +the O +3 O +′- O +5 O +′ O +addition O +reaction O +of O +TLP B-protein_type +. O + +The O +structure B-evidence +of O +the O +MaTLP B-complex_assembly +- I-complex_assembly +ppptRNAPheΔ1 I-complex_assembly +complex O +, O +wherein O +β O +- O +and O +γ O +- O +phosphates B-chemical +coordinate B-bond_interaction +with I-bond_interaction +Mg2 B-chemical ++ I-chemical +A O +and O +Mg2 B-chemical ++ I-chemical +B O +, O +respectively O +( O +Figs O +. O +3A O +and O +5C O +′), O +may O +represent O +this O +activated O +intermediate O +. O + +Subsequent O +binding O +of O +an O +incoming O +nucleotide B-chemical +to O +site B-site +2 I-site +followed O +by O +formation O +of O +the O +Watson B-bond_interaction +- I-bond_interaction +Crick I-bond_interaction +base I-bond_interaction +pair I-bond_interaction +with O +a O +nucleotide B-chemical +in O +the O +template O +strand O +conveys O +the O +3 O +′- O +OH O +of O +the O +incoming O +nucleotide B-chemical +to O +the O +position O +of O +deprotonation O +by O +Mg2 B-chemical ++ I-chemical +A O +and O +the O +5 B-chemical +′- I-chemical +triphosphate I-chemical +of O +the O +tRNA B-chemical +to O +the O +reaction B-site +center I-site +( O +Figs O +. O +3B O +and O +5D O +). O + +Thus O +, O +the O +present O +structure B-evidence +shows O +that O +this O +3 B-protein_type +′- I-protein_type +5 I-protein_type +′ I-protein_type +elongation I-protein_type +enzyme I-protein_type +utilizes O +a O +reaction B-site +center I-site +homologous O +to O +that O +of O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +elongation I-protein_type +enzymes I-protein_type +for O +both O +activation O +and O +elongation O +in O +a O +stepwise O +fashion O +. O + +It O +should O +be O +noted O +that O +TLP B-protein_type +has O +evolved O +to O +allow O +the O +occurrence O +of O +these O +two O +elaborate O +reaction O +steps O +within O +one O +reaction B-site +center I-site +. O + +A O +, O +B O +, O +and O +C O +( O +in O +green O +) O +represent O +binding B-site +sites I-site +for O +Mg2 B-chemical ++ I-chemical +A O +, O +Mg2 B-chemical ++ I-chemical +B O +, O +and O +Mg2 B-chemical ++ I-chemical +C O +. O +P B-site +( O +in O +blue O +) O +represents O +the O +phosphate B-site +binding I-site +sites I-site +; O +O O +− O +( O +in O +red O +) O +is O +the O +binding B-site +site I-site +for O +the O +deprotonated O +OH O +group O +. O + +Important O +TLP B-protein_type +residues O +for O +tRNA B-chemical +and O +Mg2 B-chemical ++ I-chemical +binding O +are O +also O +shown O +. O +( O +B O +) O +Structure B-evidence +of O +the O +activation O +complex O +( O +corresponding O +to O +fig O +. O +S8 O +). O + +GTP B-chemical +/ O +ATP B-chemical +binds O +to O +triphosphate B-site +binding I-site +site I-site +1 I-site +; O +the O +deprotonated O +OH O +group O +of O +the O +5 B-chemical +′- I-chemical +phosphate I-chemical +attacks O +the O +α O +- O +phosphate B-chemical +of O +GTP B-chemical +/ O +ATP B-chemical +, O +and O +PPi B-chemical +( O +inorganic B-chemical +pyrophosphate I-chemical +) O +is O +released O +. O +( O +C O +) O +Possible O +structure B-evidence +after O +the O +activation O +step O +as O +suggested O +from O +the O +structure B-evidence +of O +( O +C O +′). O + +( O +C O +′) O +Structure B-evidence +before O +the O +elongation O +reaction O +( O +corresponding O +to O +Fig O +. O +3A O +). O + +The O +5 B-chemical +′- I-chemical +triphosphate I-chemical +of O +the O +tRNA B-chemical +binds O +to O +the O +same O +site O +as O +for O +activation O +of O +the O +5 O +′- O +terminus O +of O +the O +tRNA B-chemical +in O +( O +B O +). O + +The O +base O +of O +the O +incoming O +GTP B-chemical +forms O +a O +Watson B-bond_interaction +- I-bond_interaction +Crick I-bond_interaction +hydrogen I-bond_interaction +bond I-bond_interaction +with O +the O +nucleotide B-chemical +at O +position O +72 B-residue_number +in O +the O +template O +chain O +and O +a O +base B-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +interaction I-bond_interaction +with O +a O +neighboring O +base O +( O +G2 B-residue_name_number +). O + +The O +3 O +′- O +OH O +of O +the O +incoming O +GTP B-chemical +is O +deprotonated O +by O +Mg2 B-chemical ++ I-chemical +A O +and O +attacks O +the O +α O +- O +phosphate B-chemical +to O +form O +a O +covalent O +bond O +. O +( O +E O +) O +After O +the O +elongation O +reaction O +, O +the O +triphosphate B-chemical +of O +the O +new O +nucleotide B-chemical +is O +placed O +on O +site B-site +1 I-site +, O +as O +in O +( O +C O +′), O +and O +is O +ready O +for O +the O +next O +reaction O +. O + +Figure O +6 O +compares O +the O +3 O +′- O +5 O +′ O +and O +5 O +′- O +3 O +′ O +elongation O +mechanisms O +, O +showing O +the O +symmetrical O +nature O +of O +both O +elongation O +reactions O +using O +a O +similar O +reaction B-site +center I-site +composed O +of O +Mg2 B-chemical ++ I-chemical +A O +and O +Mg2 B-chemical ++ I-chemical +B O +in O +the O +conserved B-protein_state +catalytic B-site +core I-site +. O + +In O +TLP B-protein_type +, O +which O +carries O +out O +3 O +′- O +5 O +′ O +elongation O +, O +the O +3 O +′- O +OH O +of O +the O +incoming O +nucleotide O +attacks O +the O +5 O +′- O +activated O +phosphate B-chemical +of O +the O +tRNA B-chemical +to O +form O +a O +phosphodiester O +bond O +, O +whereas O +in O +the O +T7 B-protein +RNA I-protein +polymerase I-protein +, O +a O +representative O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +DNA I-protein_type +/ I-protein_type +RNA I-protein_type +polymerase I-protein_type +, O +the O +3 O +′- O +OH O +of O +the O +3 O +′- O +terminal O +nucleotide O +of O +the O +RNA B-chemical +attacks O +the O +activated O +phosphate B-chemical +of O +the O +incoming O +nucleotide O +to O +form O +a O +phosphodiester O +bond O +. O + +Mg2 B-chemical ++ I-chemical +A O +activates O +the O +3 O +′- O +OH O +of O +the O +incoming O +nucleotide O +in O +TLP B-protein_type +and O +the O +3 O +′- O +OH O +of O +the O +3 O +′- O +end O +of O +the O +RNA B-chemical +chain O +in O +T7 B-protein +RNA I-protein +polymerase I-protein +. O + +Symmetrical O +relationship O +between O +3 O +′- O +5 O +′ O +elongation O +by O +TLP B-protein_type +( O +this O +study O +) O +( O +left O +) O +and O +5 O +′- O +3 O +′ O +elongation O +by O +T7 B-protein +RNA I-protein +polymerase I-protein +[ O +Protein O +Data O +Bank O +( O +PDB O +) O +ID O +: O +1S76 O +] O +( O +right O +). O + +In O +the O +3 O +′- O +5 O +′ O +elongation O +reaction O +, O +the O +3 O +′- O +OH O +of O +the O +incoming O +nucleotide O +attacks O +the O +5 O +′- O +activated O +phosphate B-chemical +of O +the O +tRNA B-chemical +to O +form O +a O +phosphodiester O +bond O +, O +whereas O +in O +the O +5 O +′- O +3 O +′ O +elongation O +reaction O +, O +the O +3 O +′- O +OH O +of O +the O +3 O +′- O +terminal O +nucleotide O +of O +the O +RNA B-chemical +attacks O +the O +activated O +phosphate B-chemical +of O +the O +incoming O +nucleotide O +to O +form O +a O +phosphodiester O +bond O +. O + +Green O +spheres O +represent O +Mg2 B-chemical ++ I-chemical +ions O +. O + +Because O +the O +chemical O +roles O +of O +tRNA B-chemical +and O +the O +incoming O +nucleotide O +are O +reversed O +in O +these O +two O +reactions O +, O +these O +two O +substrates O +are O +inserted O +into O +a O +similar O +reaction B-site +center I-site +from O +opposite O +directions O +( O +Fig O +. O +6 O +). O + +For O +this O +reason O +, O +TLP B-protein_type +requires O +a O +mechanism O +that O +activates O +the O +5 O +′- O +terminus O +of O +the O +tRNA B-chemical +during O +the O +initial O +step O +of O +the O +reaction O +. O + +Our O +analysis O +showed O +that O +the O +initial O +activation O +and O +subsequent O +elongation O +reactions O +occur O +sequentially O +at O +one O +reaction B-site +center I-site +. O + +In O +this O +case O +, O +the O +enzyme O +needs O +to O +create O +two O +substrate B-site +binding I-site +sites I-site +for O +two O +different O +reactions O +in O +the O +vicinities O +of O +one O +reaction B-site +center I-site +. O + +These O +structural O +features O +of O +the O +TLP B-protein_type +molecule O +suggest O +that O +development O +of O +an O +activation B-site +reaction I-site +site I-site +is O +a O +prerequisite O +for O +developing O +the O +3 B-protein_type +′- I-protein_type +5 I-protein_type +′ I-protein_type +elongation I-protein_type +enzyme I-protein_type +. O + +This O +is O +clearly O +more O +difficult O +than O +developing O +the O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +elongation I-protein_type +enzyme I-protein_type +, O +wherein O +the O +activation B-site +reaction I-site +site I-site +is O +not O +necessary O +, O +and O +which O +may O +be O +the O +primary O +reason O +why O +the O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +elongation I-protein_type +enzyme I-protein_type +has O +been O +exclusively O +developed O +. O + +Here O +, O +we O +established O +a O +structural O +basis O +for O +3 O +′- O +5 O +′ O +nucleotide O +elongation O +and O +showed O +that O +TLP B-protein_type +has O +evolved O +to O +acquire O +a O +two O +- O +step O +Watson O +- O +Crick O +template O +– O +dependent O +3 O +′- O +5 O +′ O +elongation O +reaction O +using O +the O +catalytic B-site +center I-site +homologous O +to O +5 B-protein_type +′- I-protein_type +3 I-protein_type +′ I-protein_type +elongation I-protein_type +enzymes I-protein_type +. O + +The O +active B-site +site I-site +of O +this O +enzyme O +is O +created O +at O +the O +dimerization B-site +interface I-site +. O + +The O +dimerization O +also O +endows O +this O +protein O +with O +the O +ability O +to O +measure O +the O +length O +of O +the O +accepter B-structure_element +stem I-structure_element +of O +the O +tRNA B-chemical +substrate O +, O +so O +that O +the O +enzyme O +can O +properly O +terminate O +the O +elongation O +reaction O +. O + +The O +detailed O +molecular O +mechanism O +of O +the O +Thg1 B-protein +/ O +TLP B-protein_type +family O +established O +by O +our O +analysis O +will O +open O +up O +new O +perspectives O +in O +our O +understanding O +of O +3 O +′- O +5 O +′ O +versus O +5 O +′- O +3 O +′ O +polymerization O +and O +the O +molecular O +evolution O +of O +template B-protein_type +- I-protein_type +dependent I-protein_type +polymerases I-protein_type +. O + +Transcribed O +tRNAs B-chemical +were O +purified O +by O +a O +HiTrap O +DEAE O +FF O +column O +( O +GE O +Healthcare O +) O +as O +previously O +described O +. O + +The O +highly O +conserved O +tRNAHis O +guanylyltransferase O +Thg1p B-protein +interacts O +with O +the O +origin O +recognition O +complex O +and O +is O +required O +for O +the O +G2 O +/ O +M O +phase O +transition O +in O +the O +yeast O +Saccharomyces O +cerevisiae O + +To O +support O +antibody B-protein_type +therapeutic O +development O +, O +the O +crystal B-evidence +structures I-evidence +of O +a O +set O +of O +16 O +germline O +variants O +composed O +of O +4 O +different O +kappa B-structure_element +light I-structure_element +chains I-structure_element +paired O +with O +4 O +different O +heavy B-structure_element +chains I-structure_element +have O +been O +determined O +. O + +All O +four O +heavy B-structure_element +chains I-structure_element +of O +the O +antigen B-structure_element +- I-structure_element +binding I-structure_element +fragments I-structure_element +( O +Fabs B-structure_element +) O +have O +the O +same O +complementarity B-structure_element +- I-structure_element +determining I-structure_element +region I-structure_element +( O +CDR B-structure_element +) O +H3 B-structure_element +that O +was O +reported O +in O +an O +earlier O +Fab B-structure_element +structure B-evidence +. O + +The O +structure B-experimental_method +analyses I-experimental_method +include O +comparisons O +of O +the O +overall O +structures B-evidence +, O +canonical O +structures B-evidence +of O +the O +CDRs B-structure_element +and O +the O +VH B-complex_assembly +: I-complex_assembly +VL I-complex_assembly +packing B-bond_interaction +interactions I-bond_interaction +. O + +The O +CDR B-structure_element +conformations O +for O +the O +most O +part O +are O +tightly O +clustered O +, O +especially O +for O +the O +ones O +with O +shorter O +lengths O +. O + +CDR B-structure_element +H3 B-structure_element +, O +despite O +having O +the O +same O +amino O +acid O +sequence O +, O +exhibits O +the O +largest O +conformational O +diversity O +. O + +About O +half O +of O +the O +structures B-evidence +have O +CDR B-structure_element +H3 B-structure_element +conformations O +similar O +to O +that O +of O +the O +parent O +; O +the O +others O +diverge O +significantly O +. O + +The O +stem B-structure_element +regions I-structure_element +of O +14 O +of O +the O +variant O +pairs O +are O +in O +the O +‘ O +kinked B-protein_state +’ O +conformation O +, O +and O +only O +2 O +are O +in O +the O +extended B-protein_state +conformation O +. O + +The O +packing O +of O +the O +VH B-structure_element +and O +VL B-structure_element +domains O +is O +consistent O +with O +our O +knowledge O +of O +antibody B-protein_type +structure B-evidence +, O +and O +the O +tilt B-evidence +angles I-evidence +between O +these O +domains O +cover O +a O +range O +of O +11 O +degrees O +. O + +Two O +of O +16 O +structures B-evidence +showed O +particularly O +large O +variations O +in O +the O +tilt B-evidence +angles I-evidence +when O +compared O +with O +the O +other O +pairings O +. O + +At O +present O +, O +therapeutic O +antibodies B-protein_type +are O +the O +largest O +class O +of O +biotherapeutic O +proteins O +that O +are O +in O +clinical O +trials O +. O + +The O +use O +of O +monoclonal O +antibodies B-protein_type +as O +therapeutics O +began O +in O +the O +early O +1980s O +, O +and O +their O +composition O +has O +transitioned O +from O +murine B-taxonomy_domain +antibodies B-protein_type +to O +generally O +less O +immunogenic O +humanized O +and O +human B-species +antibodies B-protein_type +. O + +The O +technologies O +currently O +used O +to O +obtain O +human B-species +antibodies B-protein_type +include O +transgenic O +mice B-taxonomy_domain +containing O +human B-species +antibody B-protein_type +repertoires O +, O +cloning O +directly O +from O +human B-species +B O +cells O +, O +and O +in B-experimental_method +vitro I-experimental_method +selection I-experimental_method +from O +antibody B-experimental_method +libraries I-experimental_method +using O +various O +display O +technologies O +. O + +Once O +a O +candidate O +antibody B-protein_type +is O +identified O +, O +protein B-experimental_method +engineering I-experimental_method +is O +usually O +required O +to O +produce O +a O +molecule O +with O +the O +right O +biophysical O +and O +functional O +properties O +. O + +All O +engineering O +efforts O +are O +guided O +by O +our O +understanding O +of O +the O +atomic B-evidence +structures I-evidence +of O +antibodies B-protein_type +. O + +Today O +' O +s O +antibody B-protein_type +modeling O +approaches O +, O +which O +normally O +focus O +on O +the O +variable B-structure_element +region I-structure_element +, O +are O +being O +developed O +by O +the O +application O +of O +structural O +principles O +and O +insights O +that O +are O +evolving O +as O +our O +knowledge O +of O +antibody B-protein_type +structures B-evidence +continues O +to O +expand O +. O + +Five O +different O +antibody B-protein_type +isotypes O +occur O +, O +IgG B-protein +, O +IgD B-protein +, O +IgE B-protein +, O +IgA B-protein +and O +IgM B-protein +, O +and O +each O +isotype O +has O +a O +unique O +role O +in O +the O +adaptive O +immune O +system O +. O + +Isotypes O +IgG B-protein +, O +IgD B-protein +and O +IgA B-protein +each O +have O +4 O +domains O +, O +one O +variable B-structure_element +( O +V B-structure_element +) O +and O +3 O +constant B-structure_element +( O +C B-structure_element +) O +domains O +, O +while O +IgE B-protein +and O +IgM B-protein +each O +have O +the O +same O +4 O +domains O +along O +with O +an O +additional O +C B-structure_element +domain I-structure_element +. O + +Both O +κ B-structure_element +and O +λ B-structure_element +polypeptide O +chains O +are O +composed O +of O +a O +single O +V B-structure_element +domain I-structure_element +and O +a O +single O +C B-structure_element +domain I-structure_element +. O + +All O +immunoglobulin B-protein_type +chains I-protein_type +have O +an O +N O +- O +terminal O +V B-structure_element +domain I-structure_element +followed O +by O +1 O +to O +4 O +C B-structure_element +domains I-structure_element +, O +depending O +upon O +the O +chain O +type O +. O + +In O +antibodies B-protein_type +, O +the O +heavy B-structure_element +and I-structure_element +light I-structure_element +chain I-structure_element +V B-structure_element +domains I-structure_element +pack O +together O +forming O +the O +antigen B-site +combining I-site +site I-site +. O + +The O +sequence O +diversity O +of O +the O +CDR B-structure_element +regions I-structure_element +presents O +a O +substantial O +challenge O +to O +antibody B-protein_type +modeling O +. O + +However O +, O +an O +initial O +structural B-experimental_method +analysis I-experimental_method +of O +the O +combining B-site +sites I-site +of O +the O +small O +set O +of O +structures B-evidence +of O +immunoglobulin O +fragments O +available O +in O +the O +1980s O +found O +that O +5 O +of O +the O +6 O +hypervariable B-structure_element +loops I-structure_element +or O +CDRs B-structure_element +had O +canonical O +structures O +( O +a O +limited O +set O +of O +main O +- O +chain O +conformations O +). O + +Furthermore O +, O +studies O +of O +antibody B-protein_type +sequences O +revealed O +that O +the O +total O +number O +of O +canonical O +structures O +are O +limited O +for O +each O +CDR B-structure_element +, O +indicating O +possibly O +that O +antigen O +recognition O +may O +be O +affected O +by O +structural O +restrictions O +at O +the O +antigen B-site +- I-site +binding I-site +site I-site +. O + +Later O +studies O +found O +that O +the O +CDR B-structure_element +loop I-structure_element +length O +is O +the O +primary O +determining O +factor O +of O +antigen B-site +- I-site +binding I-site +site I-site +topography O +because O +it O +is O +the O +primary O +factor O +for O +determining O +a O +canonical O +structure O +. O + +Additional O +efforts O +have O +led O +to O +our O +current O +understanding O +that O +the O +LC B-structure_element +CDRs B-structure_element +L1 B-structure_element +, O +L2 B-structure_element +, O +and O +L3 B-structure_element +have O +preferred O +sets O +of O +canonical O +structures O +based O +on O +length O +and O +amino O +acid O +sequence O +composition O +. O + +This O +was O +also O +found O +to O +be O +the O +case O +for O +the O +H1 B-structure_element +and O +H2 B-structure_element +CDRs B-structure_element +. O + +Recently O +, O +a O +comprehensive O +CDR B-structure_element +classification O +scheme O +was O +reported O +identifying O +72 O +clusters O +of O +conformations O +observed O +in O +antibody B-protein_type +structures B-evidence +. O + +The O +knowledge O +and O +predictability O +of O +these O +CDR B-structure_element +canonical O +structures B-evidence +have O +greatly O +advanced O +antibody B-protein_type +modeling O +efforts O +. O + +In O +contrast O +to O +CDRs B-structure_element +L1 B-structure_element +, O +L2 B-structure_element +, O +L3 B-structure_element +, O +H1 B-structure_element +and O +H2 B-structure_element +, O +no O +canonical O +structures B-evidence +have O +been O +observed O +for O +CDR B-structure_element +H3 B-structure_element +, O +which O +is O +the O +most O +variable O +in O +length O +and O +amino O +acid O +sequence O +. O + +In O +the O +torso B-structure_element +region I-structure_element +, O +2 O +primary O +groups O +could O +be O +identified O +, O +which O +led O +to O +sequence O +- O +based O +rules O +that O +can O +predict O +with O +some O +degree O +of O +reliability O +the O +conformation O +of O +the O +stem B-structure_element +region I-structure_element +. O + +The O +cataloging O +and O +development O +of O +the O +rules O +for O +predicting O +the O +conformation O +of O +the O +anchor B-structure_element +region I-structure_element +of O +CDR B-structure_element +H3 B-structure_element +continue O +to O +be O +refined O +, O +producing O +new O +insight O +into O +the O +CDR B-structure_element +H3 B-structure_element +conformations O +and O +new O +tools O +for O +antibody B-protein_type +engineering O +. O + +Although O +antibody B-protein_type +modeling O +is O +improving O +, O +the O +latest O +assessment O +revealed O +a O +number O +of O +challenges O +that O +need O +to O +be O +overcome O +to O +provide O +accurate O +3 O +- O +dimensional O +models O +of O +antibody B-protein_type +V B-structure_element +regions I-structure_element +, O +including O +accuracies O +in O +the O +modeling O +of O +CDR B-structure_element +H3 B-structure_element +. O + +To O +support O +antibody B-protein_type +engineering O +and O +therapeutic O +development O +efforts O +, O +a O +phage B-experimental_method +library I-experimental_method +was O +designed O +and O +constructed O +based O +on O +a O +limited O +number O +of O +scaffolds O +built O +with O +frequently O +used O +human B-species +germ O +- O +line O +IGV B-structure_element +and O +IGJ B-structure_element +gene O +segments O +that O +encode O +antigen B-site +combining I-site +sites I-site +suitable O +for O +recognition O +of O +peptides O +and O +proteins O +. O + +Selection O +of O +these O +genes O +was O +based O +on O +the O +high O +frequency O +of O +their O +use O +and O +their O +cognate O +canonical O +structures B-evidence +that O +were O +found O +binding O +to O +peptides O +and O +proteins O +, O +as O +well O +as O +their O +ability O +to O +be O +expressed B-experimental_method +in I-experimental_method +bacteria I-experimental_method +and O +displayed B-experimental_method +on I-experimental_method +filamentous I-experimental_method +phage I-experimental_method +. O + +The O +implementation O +of O +the O +library O +involves O +the O +diversification O +of O +the O +human B-species +germline O +genes O +to O +mimic O +that O +found O +in O +natural O +human B-species +libraries O +. O + +The O +crystal B-experimental_method +structure I-experimental_method +determinations I-experimental_method +and O +structural B-experimental_method +analyses I-experimental_method +of O +all O +germline O +Fabs B-structure_element +in O +the O +library O +described O +above O +along O +with O +the O +structures B-evidence +of O +a O +fourth O +HC B-structure_element +germline O +, O +IGHV3 B-mutant +- I-mutant +53 I-mutant +( O +H3 B-mutant +- I-mutant +53 I-mutant +), O +paired O +with O +the O +4 O +LCs B-structure_element +of O +the O +library O +have O +been O +carried O +out O +to O +support O +antibody B-protein_type +therapeutic O +development O +. O + +The O +structures B-evidence +and O +their O +analyses O +provide O +a O +foundation O +for O +future O +antibody B-protein_type +engineering O +and O +structure O +determination O +efforts O +. O + +Crystal B-evidence +structures I-evidence + +Crystal B-evidence +data I-evidence +, O +X B-evidence +- I-evidence +ray I-evidence +data I-evidence +, O +and O +refinement B-evidence +statistics I-evidence +. O + +( O +Continued O +) O +Crystal B-evidence +data I-evidence +, O +X B-evidence +- I-evidence +ray I-evidence +data I-evidence +, O +and O +refinement B-evidence +statistics I-evidence +. O + +The O +crystal B-evidence +structures I-evidence +of O +a O +germline B-experimental_method +library I-experimental_method +composed O +of O +16 O +Fabs B-structure_element +generated O +by O +combining O +4 O +HCs B-structure_element +( O +H1 B-mutant +- I-mutant +69 I-mutant +, O +H3 B-mutant +- I-mutant +23 I-mutant +, O +H3 B-mutant +- I-mutant +53 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +) O +and O +4 O +LCs B-structure_element +( O +L1 B-mutant +- I-mutant +39 I-mutant +, O +L3 B-mutant +- I-mutant +11 I-mutant +, O +L3 B-mutant +- I-mutant +20 I-mutant +and O +L4 B-mutant +- I-mutant +1 I-mutant +) O +have O +been O +determined O +. O + +The O +Fab B-structure_element +heavy O +and O +light B-structure_element +chain I-structure_element +sequences O +for O +the O +variants O +numbered O +according O +to O +Chothia O +are O +shown O +in O +Fig O +. O +S1 O +. O + +The O +four O +different O +HCs B-structure_element +all O +have O +the O +same O +CDR B-structure_element +H3 B-structure_element +sequence O +, O +ARYDGIYGELDF B-structure_element +. O + +Variations O +occur O +in O +the O +pH O +( O +buffer O +) O +and O +the O +additives O +, O +and O +, O +in O +group O +3 O +, O +PEG B-chemical +3350 I-chemical +is O +the O +precipitant O +for O +one O +variants O +while O +ammonium B-chemical +sulfate I-chemical +is O +the O +precipitant O +for O +the O +other O +two O +. O + +The O +similarity O +in O +the O +crystal B-evidence +forms I-evidence +is O +attributed O +in O +part O +to O +cross O +- O +seeding O +using O +the O +microseed B-experimental_method +matrix I-experimental_method +screening I-experimental_method +for O +groups O +2 O +and O +3 O +. O + +The O +crystal B-evidence +structures I-evidence +of O +the O +16 O +Fabs B-structure_element +have O +been O +determined O +at O +resolutions O +ranging O +from O +3 O +. O +3 O +Å O +to O +1 O +. O +65 O +Å O +( O +Table O +1 O +). O + +The O +number O +of O +Fab B-structure_element +molecules O +in O +the O +crystallographic O +asymmetric O +unit O +varies O +from O +1 O +( O +for O +12 O +Fabs B-structure_element +) O +to O +2 O +( O +for O +4 O +Fabs B-structure_element +). O + +Overall O +the O +structures B-evidence +are O +fairly O +complete O +, O +and O +, O +as O +can O +be O +expected O +, O +the O +models O +for O +the O +higher O +resolution O +structures B-evidence +are O +more O +complete O +than O +those O +for O +the O +lower O +resolution O +structures B-evidence +( O +Table O +S1 O +). O + +Invariably O +, O +the O +HCs B-structure_element +have O +more O +disorder B-protein_state +than O +the O +LCs B-structure_element +. O + +For O +the O +LC B-structure_element +, O +the O +disorder B-protein_state +is O +observed O +at O +2 O +of O +the O +C O +- O +terminal O +residues O +with O +few O +exceptions O +. O + +Apart O +from O +the O +C O +- O +terminus O +, O +only O +a O +few O +surface O +residues O +in O +LC B-structure_element +are O +disordered B-protein_state +. O + +The O +HCs B-structure_element +feature O +the O +largest O +number O +of O +disordered B-protein_state +residues O +, O +with O +the O +lower O +resolution O +structures B-evidence +having O +the O +most O +. O + +The O +C O +- O +terminal O +residues O +including O +the O +6xHis O +tags O +are O +disordered B-protein_state +in O +all O +16 O +structures B-evidence +. O + +One O +involves O +the O +loop B-structure_element +connecting O +the O +first O +2 O +β B-structure_element +- I-structure_element +strands I-structure_element +of O +the O +constant B-structure_element +domain I-structure_element +( O +in O +all O +Fabs B-structure_element +except O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +, O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +). O + +CDR B-structure_element +H1 B-structure_element +and O +CDR B-structure_element +H2 B-structure_element +also O +show O +some O +degree O +of O +disorder B-protein_state +, O +but O +to O +a O +lesser O +extent O +. O + +CDR B-structure_element +canonical O +structures B-evidence + +Several O +CDR B-structure_element +definitions O +have O +evolved O +over O +decades O +of O +antibody B-protein_type +research O +. O + +Depending O +on O +the O +focus O +of O +the O +study O +, O +the O +CDR B-structure_element +boundaries O +differ O +slightly O +between O +various O +definitions O +. O + +In O +this O +work O +, O +we O +use O +the O +CDR B-structure_element +definition O +of O +North O +et O +al O +., O +which O +is O +similar O +to O +that O +of O +Martin O +with O +the O +following O +exceptions O +: O +1 O +) O +CDRs B-structure_element +H1 B-structure_element +and O +H3 B-structure_element +begin O +immediately O +after O +the O +Cys B-residue_name +; O +and O +2 O +) O +CDR B-structure_element +L2 B-structure_element +includes O +an O +additional O +residue O +at O +the O +N O +- O +terminal O +side O +, O +typically O +Tyr B-residue_name +. O + +CDR B-structure_element +H1 B-structure_element + +The O +superposition B-experimental_method +of O +CDR B-structure_element +H1 B-structure_element +backbones O +for O +all O +HC B-complex_assembly +: I-complex_assembly +LC I-complex_assembly +pairs O +with O +heavy B-structure_element +chains I-structure_element +: O +( O +A O +) O +H1 B-mutant +- I-mutant +69 I-mutant +, O +( O +B O +) O +H3 B-mutant +- I-mutant +23 I-mutant +, O +( O +C O +) O +H3 B-mutant +- I-mutant +53 I-mutant +and O +( O +D O +) O +H5 B-mutant +- I-mutant +51 I-mutant +. O + +CDRs B-structure_element +are O +defined O +using O +the O +Dunbrack O +convention O +[ O +12 O +]. O + +Assignments O +for O +2 O +copies O +of O +the O +Fab B-structure_element +in O +the O +asymmetric O +unit O +are O +given O +for O +5 O +structures B-evidence +. O + +The O +four O +HCs B-structure_element +feature O +CDR B-structure_element +H1 B-structure_element +of O +the O +same O +length O +, O +and O +their O +sequences O +are O +highly O +similar O +( O +Table O +2 O +). O + +Three O +of O +the O +HCs B-structure_element +, O +H3 B-mutant +- I-mutant +23 I-mutant +, O +H3 B-mutant +- I-mutant +53 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +, O +have O +the O +same O +canonical O +structure O +, O +H1 B-mutant +- I-mutant +13 I-mutant +- I-mutant +1 I-mutant +, O +and O +the O +backbone O +conformations O +are O +tightly O +clustered O +for O +each O +set O +of O +Fab B-structure_element +structures B-evidence +as O +reflected O +in O +the O +rmsd B-evidence +values I-evidence +( O +Fig O +. O +1B O +- O +D O +). O + +Some O +deviation O +is O +observed O +for O +H3 B-mutant +- I-mutant +53 I-mutant +, O +mostly O +due O +to O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +, O +which O +exhibits O +a O +significant O +degree O +of O +disorder O +in O +CDR B-structure_element +H1 B-structure_element +. O + +The O +electron B-evidence +density I-evidence +for O +the O +backbone O +is O +weak O +and O +discontinuous O +, O +and O +completely O +missing O +for O +several O +side O +chains O +. O + +The O +CDR B-structure_element +H1 B-structure_element +structures B-evidence +with O +H1 B-mutant +- I-mutant +69 I-mutant +shown O +in O +Fig O +. O +1A O +are O +quite O +variable O +, O +both O +for O +the O +structures B-evidence +with O +different O +LCs B-structure_element +and O +for O +the O +copies O +of O +the O +same O +Fab B-structure_element +in O +the O +asymmetric O +unit O +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +and O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +. O + +In O +total O +, O +6 O +independent O +Fab B-structure_element +structures B-evidence +produce O +5 O +different O +canonical O +structures B-evidence +, O +namely O +H1 B-mutant +- I-mutant +13 I-mutant +- I-mutant +1 I-mutant +, O +H1 B-mutant +- I-mutant +13 I-mutant +- I-mutant +3 I-mutant +, O +H1 B-mutant +- I-mutant +13 I-mutant +- I-mutant +4 I-mutant +, O +H1 B-mutant +- I-mutant +13 I-mutant +- I-mutant +6 I-mutant +and O +H1 B-mutant +- I-mutant +13 I-mutant +- I-mutant +10 I-mutant +. O + +Glycine B-residue_name +introduces O +the O +possibility O +of O +a O +higher O +degree O +of O +conformational O +flexibility O +that O +undoubtedly O +translates O +to O +the O +differences O +observed O +, O +and O +contributes O +to O +the O +elevated O +thermal O +parameters O +for O +the O +atoms O +in O +the O +amino O +acid O +residues O +in O +this O +region O +. O + +CDR B-structure_element +H2 B-structure_element + +The O +canonical O +structures O +of O +CDR B-structure_element +H2 B-structure_element +have O +fairly O +consistent O +conformations O +( O +Table O +2 O +, O +Fig O +. O +2 O +). O + +Each O +of O +the O +4 O +HCs B-structure_element +adopts O +only O +one O +canonical O +structure O +regardless O +of O +the O +pairing O +LC B-structure_element +. O + +Germlines O +H1 B-mutant +- I-mutant +69 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +have O +the O +same O +canonical O +structure O +assignment O +H2 B-mutant +- I-mutant +10 I-mutant +- I-mutant +1 I-mutant +, O +H3 B-mutant +- I-mutant +23 I-mutant +has O +H2 B-mutant +- I-mutant +10 I-mutant +- I-mutant +2 I-mutant +, O +and O +H3 B-mutant +- I-mutant +53 I-mutant +has O +H2 B-mutant +- I-mutant +9 I-mutant +- I-mutant +3 I-mutant +. O + +The O +conformations O +for O +all O +of O +these O +CDR B-structure_element +H2s B-structure_element +are O +tightly O +clustered O +( O +Fig O +. O +2 O +). O + +In O +one O +case O +, O +in O +the O +second O +Fab B-structure_element +of O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +CDR B-structure_element +H2 B-structure_element +is O +partially B-protein_state +disordered I-protein_state +( O +Δ55 B-mutant +- I-mutant +60 I-mutant +). O + +Although O +three O +of O +the O +germlines O +have O +CDR B-structure_element +H2 B-structure_element +of O +the O +same O +length O +, O +10 B-residue_range +residues I-residue_range +, O +they O +adopt O +2 O +distinctively O +different O +conformations O +depending O +mostly O +on O +the O +residue O +at O +position O +71 B-residue_number +from O +the O +so O +- O +called O +CDR B-structure_element +H4 B-structure_element +. O + +Germlines O +H1 B-mutant +- I-mutant +69 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +are O +unique O +in O +the O +human B-species +repertoire O +in O +having O +an O +Ala B-residue_name +at O +position O +71 B-residue_number +that O +leaves O +enough O +space O +for O +H B-structure_element +- O +Pro52a B-residue_name_number +to O +pack O +deeper O +against O +CDR B-structure_element +H4 B-structure_element +so O +that O +the O +following O +residues O +53 B-residue_number +and O +54 B-residue_number +point O +toward O +the O +putative O +antigen O +. O + +Conformations O +of O +CDR B-structure_element +H2 B-structure_element +in O +H1 B-mutant +- I-mutant +69 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +, O +both O +of O +which O +have O +canonical O +structure O +H2 B-mutant +- I-mutant +10 I-mutant +- I-mutant +1 I-mutant +, O +show O +little O +deviation O +within O +each O +set O +of O +4 O +structures B-evidence +. O + +However O +, O +there O +is O +a O +significant O +shift O +of O +the O +CDR B-structure_element +as O +a O +rigid O +body O +when O +the O +2 O +sets O +are O +superimposed B-experimental_method +. O + +Most O +likely O +this O +is O +the O +result O +of O +interaction O +of O +CDR B-structure_element +H2 B-structure_element +with O +CDR B-structure_element +H1 B-structure_element +, O +namely O +with O +the O +residue O +at O +position O +33 B-residue_number +( O +residue O +11 O +of O +13 O +in O +CDR B-structure_element +H1 B-structure_element +). O + +Germline O +H1 B-mutant +- I-mutant +69 I-mutant +has O +Ala B-residue_name +at O +position O +33 B-residue_number +whereas O +in O +H5 B-mutant +- I-mutant +51 I-mutant +position O +33 B-residue_number +is O +occupied O +by O +a O +bulky O +Trp B-residue_name +, O +which O +stacks O +against O +H B-structure_element +- O +Tyr52 B-residue_name_number +and O +drives O +CDR B-structure_element +H2 B-structure_element +away O +from O +the O +center O +. O + +CDR B-structure_element +L1 B-structure_element + +The O +four O +LC B-structure_element +CDRs B-structure_element +L1 B-structure_element +feature O +3 O +different O +lengths O +( O +11 B-residue_range +, O +12 B-residue_range +and O +17 B-residue_range +residues O +) O +having O +a O +total O +of O +4 O +different O +canonical O +structure O +assignments O +. O + +Of O +these O +LCs B-structure_element +, O +L1 B-mutant +- I-mutant +39 I-mutant +and O +L3 B-mutant +- I-mutant +11 I-mutant +have O +the O +same O +canonical O +structure O +, O +L1 B-mutant +- I-mutant +11 I-mutant +- I-mutant +1 I-mutant +, O +and O +superimpose B-experimental_method +very O +well O +( O +Fig O +. O +3A O +, O +B O +). O + +L4 B-mutant +- I-mutant +1 I-mutant +has O +the O +longest O +CDR B-structure_element +L1 B-structure_element +, O +composed O +of O +17 B-residue_range +amino I-residue_range +acid I-residue_range +residues I-residue_range +( O +Fig O +. O +3D O +). O + +Despite O +this O +, O +the O +conformations O +are O +tightly O +clustered O +( O +rmsd B-evidence +is O +0 O +. O +20 O +Å O +). O + +The O +backbone O +conformations O +of O +the O +stem B-structure_element +regions I-structure_element +superimpose O +well O +. O + +This O +is O +the O +tip O +of O +the O +loop B-structure_element +region I-structure_element +, O +which O +appears O +to O +have O +similar O +conformations O +that O +fan O +out O +the O +structures B-evidence +because O +of O +the O +slight O +differences O +in O +torsion O +angles O +in O +the O +backbone O +near O +Tyr30a B-residue_name_number +and O +Lys30f B-residue_name_number +. O + +L3 B-mutant +- I-mutant +20 I-mutant +is O +the O +most O +variable O +in O +CDR B-structure_element +L1 B-structure_element +among O +the O +4 O +germlines O +as O +indicated O +by O +an O +rmsd B-evidence +of O +0 O +. O +54 O +Å O +( O +Fig O +. O +3C O +). O + +The O +fourth O +member O +of O +the O +set O +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +was O +crystallized B-experimental_method +with O +2 O +Fabs B-structure_element +in O +the O +asymmetric O +unit O +. O + +This O +reflects O +the O +lack O +of O +accuracy O +in O +the O +structure B-evidence +due O +to O +low O +resolution O +of O +the O +X B-evidence +- I-evidence +ray I-evidence +data I-evidence +( O +3 O +. O +3 O +Å O +). O + +CDR B-structure_element +L2 B-structure_element + +The O +superposition B-experimental_method +of O +CDR B-structure_element +L2 B-structure_element +backbones O +for O +all O +HC B-complex_assembly +: I-complex_assembly +LC I-complex_assembly +pairs O +with O +light B-structure_element +chains I-structure_element +: O +( O +A O +) O +L1 B-mutant +- I-mutant +39 I-mutant +, O +( O +B O +) O +L3 B-mutant +- I-mutant +11 I-mutant +, O +( O +C O +) O +L3 B-mutant +- I-mutant +20 I-mutant +and O +( O +D O +) O +L4 B-mutant +- I-mutant +1 I-mutant +. O + +All O +four O +LCs B-structure_element +have O +CDR B-structure_element +L2 B-structure_element +of O +the O +same O +length O +and O +canonical O +structure O +, O +L2 B-mutant +- I-mutant +8 I-mutant +- I-mutant +1 I-mutant +( O +Table O +2 O +). O + +The O +CDR B-structure_element +L2 B-structure_element +conformations O +for O +each O +of O +the O +LCs B-structure_element +paired O +with O +the O +4 O +HCs B-structure_element +are O +clustered O +more O +tightly O +than O +any O +of O +the O +other O +CDRs B-structure_element +( O +rmsd B-evidence +values O +are O +in O +the O +range O +0 O +. O +09 O +- O +0 O +. O +16 O +Å O +), O +and O +all O +4 O +sets O +have O +virtually O +the O +same O +conformation O +despite O +the O +sequence O +diversity O +of O +the O +loop B-structure_element +. O + +CDR B-structure_element +L3 B-structure_element + +The O +superposition B-experimental_method +of O +CDR B-structure_element +L3 B-structure_element +backbones O +for O +all O +HC B-complex_assembly +: I-complex_assembly +LC I-complex_assembly +pairs O +with O +light B-structure_element +chains I-structure_element +: O +( O +A O +) O +L1 B-mutant +- I-mutant +39 I-mutant +, O +( O +B O +) O +L3 B-mutant +- I-mutant +11 I-mutant +, O +( O +C O +) O +L3 B-mutant +- I-mutant +20 I-mutant +and O +( O +D O +) O +L4 B-mutant +- I-mutant +1 I-mutant +. O + +The O +conformations O +of O +CDR B-structure_element +L3 B-structure_element +for O +L1 B-mutant +- I-mutant +39 I-mutant +, O +L3 B-mutant +- I-mutant +11 I-mutant +, O +and O +particularly O +for O +L320 O +, O +are O +not O +as O +tightly O +clustered O +as O +those O +of O +L4 B-mutant +- I-mutant +1 I-mutant +( O +Fig O +. O +5 O +). O + +The O +slight O +conformational O +variability O +occurs O +in O +the O +region O +of O +amino O +acid O +residues O +90 B-residue_range +- I-residue_range +92 I-residue_range +, O +which O +is O +in O +contact O +with O +CDR B-structure_element +H3 B-structure_element +. O + +CDR B-structure_element +H3 B-structure_element +conformational O +diversity O + +The O +loop B-structure_element +and O +the O +2 O +β B-structure_element +- I-structure_element +strands I-structure_element +of O +the O +CDR B-structure_element +H3 B-structure_element +in O +this O +‘ O +parent O +’ O +structure B-evidence +are O +stabilized O +by O +H B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +between O +the O +carbonyl O +oxygen O +and O +peptide O +nitrogen O +atoms O +in O +the O +2 O +strands O +. 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O + +Another O +four O +of O +the O +Fabs B-structure_element +, O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +, O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +, O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +have O +missing O +side O +- O +chain O +atoms O +. O + +The O +variations O +in O +CDR B-structure_element +H3 B-structure_element +conformation O +are O +illustrated O +in O +Fig O +. 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O + +Melting B-evidence +temperatures I-evidence +( O +Tm B-evidence +) O +were O +measured O +for O +all O +Fabs B-structure_element +using O +differential B-experimental_method +scanning I-experimental_method +calorimetry I-experimental_method +( O +Table O +5 O +). O + +In O +addition O +, O +L1 B-mutant +- I-mutant +39 I-mutant +provides O +a O +much O +higher O +degree O +of O +stabilization O +than O +the O +other O +3 O +LC B-structure_element +germlines O +when O +combined O +with O +any O +of O +the O +HCs B-structure_element +. O + +As O +a O +result O +, O +the O +Tm B-evidence +for O +pairs O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L1 I-complex_assembly +- I-complex_assembly +39 I-complex_assembly +is O +12 O +- O +13 O +° O +higher O +than O +for O +pairs O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +H3 B-complex_assembly +- I-complex_assembly +53 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +, O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L4 I-complex_assembly +- I-complex_assembly +1 I-complex_assembly +. O + +These O +findings O +correlate O +well O +with O +the O +degree O +of O +conformational O +disorder O +observed O +in O +the O +crystal B-evidence +structures I-evidence +. O + +Parts O +of O +CDR B-structure_element +H3 B-structure_element +main O +chain O +are O +completely O +disordered B-protein_state +, O +and O +were O +not O +modeled O +in O +Fabs B-structure_element +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +H5 B-complex_assembly +- I-complex_assembly +51 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +11 I-complex_assembly +that O +have O +the O +lowest O +Tms B-evidence +in O +the O +set O +. O + +All O +those O +molecules O +are O +relatively O +unstable O +, O +as O +is O +reflected O +in O +their O +low O +Tms B-evidence +. O + +This O +is O +the O +first O +report O +of O +a O +systematic B-experimental_method +structural I-experimental_method +investigation I-experimental_method +of O +a O +phage B-experimental_method +germline I-experimental_method +library I-experimental_method +. O + +The O +16 O +Fab B-structure_element +structures B-evidence +offer O +a O +unique O +look O +at O +all O +pairings O +of O +4 O +different O +HCs B-structure_element +( O +H1 B-mutant +- I-mutant +69 I-mutant +, O +H3 B-mutant +- I-mutant +23 I-mutant +, O +H3 B-mutant +- I-mutant +53 I-mutant +, O +and O +H5 B-mutant +- I-mutant +51 I-mutant +) O +and O +4 O +different O +LCs B-structure_element +( O +L1 B-mutant +- I-mutant +39 I-mutant +, O +L3 B-mutant +- I-mutant +11 I-mutant +, O +L3 B-mutant +- I-mutant +20 I-mutant +and O +L4 B-mutant +- I-mutant +1 I-mutant +), O +all O +with O +the O +same O +CDR B-structure_element +H3 B-structure_element +. O + +The O +structural B-evidence +data I-evidence +set O +taken O +as O +a O +whole O +provides O +insight O +into O +how O +the O +backbone O +conformations O +of O +the O +CDRs B-structure_element +of O +a O +specific O +heavy O +or O +light B-structure_element +chain I-structure_element +vary O +when O +it O +is O +paired O +with O +4 O +different O +light O +or O +heavy B-structure_element +chains I-structure_element +, O +respectively O +. O + +A O +large O +variability O +in O +the O +CDR B-structure_element +conformations O +for O +the O +sets O +of O +HCs B-structure_element +and O +LCs B-structure_element +is O +observed O +. O + +In O +some O +cases O +the O +CDR B-structure_element +conformations O +for O +all O +members O +of O +a O +set O +are O +virtually O +identical O +, O +for O +others O +subtle O +changes O +occur O +in O +a O +few O +members O +of O +a O +set O +, O +and O +in O +some O +cases O +larger O +deviations O +are O +observed O +within O +a O +set O +. O + +The O +five O +variants O +that O +crystallized B-experimental_method +with O +2 O +copies O +of O +the O +Fab B-structure_element +in O +the O +asymmetric O +unit O +serve O +somewhat O +as O +controls O +for O +the O +influence O +of O +crystal O +packing O +on O +the O +conformations O +of O +the O +CDRs B-structure_element +. O + +In O +four O +of O +the O +5 O +structures B-evidence +the O +CDR B-structure_element +conformations O +are O +consistent O +. O + +In O +only O +one O +case O +, O +that O +of O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +( O +the O +lowest O +resolution O +structure B-evidence +), O +do O +we O +see O +differences O +in O +the O +conformations O +of O +the O +2 O +copies O +of O +CDRs B-structure_element +H1 B-structure_element +and O +L1 B-structure_element +. O + +This O +variability O +is O +likely O +a O +result O +of O +2 O +factors O +, O +crystal O +packing O +interactions O +and O +internal O +instability O +of O +the O +variable B-structure_element +domain I-structure_element +. O + +For O +the O +CDRs B-structure_element +with O +canonical O +structures O +, O +the O +largest O +changes O +in O +conformation O +occur O +for O +CDR B-structure_element +H1 B-structure_element +of O +H1 B-mutant +- I-mutant +69 I-mutant +and O +H3 B-mutant +- I-mutant +53 I-mutant +. O + +The O +other O +2 O +HCs B-structure_element +, O +H3 B-mutant +- I-mutant +23 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +, O +have O +canonical O +structures O +that O +are O +remarkably B-protein_state +well I-protein_state +conserved I-protein_state +( O +Fig O +. O +1 O +). O + +H1 B-mutant +- I-mutant +69 I-mutant +is O +unique O +in O +having O +a O +pair O +of O +glycine B-residue_name +residues O +at O +positions O +26 B-residue_number +and O +27 B-residue_number +, O +which O +provide O +more O +conformational B-protein_state +freedom I-protein_state +in O +CDR B-structure_element +H1 B-structure_element +. O + +Besides O +IGHV1 B-mutant +- I-mutant +69 I-mutant +, O +only O +the O +germlines O +of O +the O +VH4 B-structure_element +family O +possess O +double O +glycines B-residue_name +in O +CDR B-structure_element +H1 B-structure_element +, O +and O +it O +will O +be O +interesting O +to O +see O +if O +they O +are O +also O +conformationally B-protein_state +unstable I-protein_state +. O + +More O +than O +half O +of O +the O +variants O +retain O +the O +conformation O +of O +the O +parent O +despite O +having O +differences O +in O +the O +VH B-complex_assembly +: I-complex_assembly +VL I-complex_assembly +pairing O +. O + +This O +subset O +includes O +2 O +structures B-evidence +with O +2 O +copies O +of O +the O +Fab B-structure_element +in O +the O +asymmetric O +unit O +, O +all O +of O +which O +are O +nearly O +identical O +in O +conformation O +. O + +The O +remaining O +8 O +structures B-evidence +exhibit O +“ O +non O +- O +parental O +” O +conformations O +, O +indicating O +that O +the O +VH B-structure_element +and O +VL B-structure_element +context O +can O +also O +be O +a O +dominating O +factor O +influencing O +CDR B-structure_element +H3 B-structure_element +. O + +In O +looking O +at O +a O +possible O +correlation O +between O +the O +tilt B-evidence +angle I-evidence +and O +the O +conformation O +of O +CDR B-structure_element +H3 B-structure_element +, O +no O +clear O +trends O +are O +observed O +. O + +The O +absolute O +VH B-complex_assembly +: I-complex_assembly +VL I-complex_assembly +orientation B-evidence +parameters I-evidence +for O +the O +2 O +Fabs B-structure_element +( O +Table O +S2 O +) O +show O +significant O +deviation B-evidence +in O +HL B-structure_element +, O +LC1 B-structure_element +and O +HC2 B-structure_element +values O +( O +2 O +- O +3 O +standard O +deviations O +from O +the O +mean O +). O + +As O +noted O +in O +the O +Results O +section O +, O +the O +2 O +variants O +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +are O +outliers O +in O +terms O +of O +the O +tilt B-evidence +angle I-evidence +; O +at O +the O +same O +time O +, O +both O +have O +the O +smallest O +VH B-site +: I-site +VL I-site +interface I-site +. O + +These O +smaller O +interfaces B-site +may O +perhaps O +translate O +to O +a O +significant O +deviation O +in O +how O +VH B-structure_element +is O +oriented O +relative O +to O +VL B-structure_element +than O +the O +other O +variants O +. O + +These O +deviations O +from O +the O +other O +variants O +can O +also O +be O +seen O +to O +some O +extent O +in O +VH B-complex_assembly +: I-complex_assembly +VL I-complex_assembly +orientation O +parameters O +in O +Table O +S2 O +, O +as O +well O +as O +in O +the O +smaller O +number O +of O +residues O +involved O +in O +the O +VH B-site +: I-site +VL I-site +interfaces I-site +of O +these O +2 O +variants O +( O +Fig O +. O +S5 O +). O + +These O +differences O +undoubtedly O +influence O +the O +conformation O +of O +the O +CDRs B-structure_element +, O +in O +particular O +CDR B-structure_element +H1 B-structure_element +( O +Fig O +. O +1A O +) O +and O +CDR B-structure_element +L1 B-structure_element +( O +Fig O +. O +3C O +), O +especially O +with O +the O +tandem O +glycines B-residue_name +and O +multiple O +serines B-residue_name +present O +, O +respectively O +. O + +As O +indicated O +by O +the O +melting B-evidence +temperatures I-evidence +, O +germlines O +H1 B-mutant +- I-mutant +69 I-mutant +and O +H3 B-mutant +- I-mutant +23 I-mutant +for O +HC B-structure_element +and O +germline O +L1 B-mutant +- I-mutant +39 I-mutant +for O +LC B-structure_element +produce O +more O +stable B-protein_state +Fabs B-structure_element +compared O +to O +the O +other O +germlines O +in O +the O +experimental O +set O +. O + +One O +possible O +explanation O +of O +the O +clear O +preference O +of O +LC B-structure_element +germline O +L1 B-mutant +- I-mutant +39 I-mutant +is O +that O +CDR B-structure_element +L3 B-structure_element +has O +smaller O +residues O +at O +positions O +91 B-residue_number +and O +94 B-residue_number +, O +allowing O +for O +more O +room O +to O +accommodate O +CDR B-structure_element +H3 B-structure_element +. O + +Other O +germlines O +have O +bulky O +residues O +, O +Tyr B-residue_name +, O +Arg B-residue_name +and O +Trp B-residue_name +, O +at O +these O +positions O +, O +whereas O +L1 B-mutant +- I-mutant +39 I-mutant +has O +Ser B-residue_name +and O +Thr B-residue_name +. O + +Various O +combinations O +of O +germline O +sequences O +for O +VL B-structure_element +and O +VH B-structure_element +impose O +certain O +constraints O +on O +CDR B-structure_element +H3 B-structure_element +, O +which O +has O +to O +adapt O +to O +the O +environment O +. O + +A O +more O +compact B-protein_state +CDR B-structure_element +L3 B-structure_element +may O +be O +beneficial O +in O +this O +situation O +. O + +While O +pairings O +with O +H3 B-mutant +- I-mutant +53 I-mutant +and O +H5 B-mutant +- I-mutant +51 I-mutant +may O +be O +safely O +called O +a O +mismatch O +, O +those O +with O +H1 B-mutant +- I-mutant +69 I-mutant +and O +H3 B-mutant +- I-mutant +23 I-mutant +have O +Tms B-evidence +about O +5 O +- O +6 O +° O +higher O +. O + +Curiously O +, O +the O +2 O +Fabs B-structure_element +, O +H1 B-complex_assembly +- I-complex_assembly +69 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +and O +H3 B-complex_assembly +- I-complex_assembly +23 I-complex_assembly +: I-complex_assembly +L3 I-complex_assembly +- I-complex_assembly +20 I-complex_assembly +, O +deviate O +markedly O +in O +their O +tilt B-evidence +angles I-evidence +from O +the O +rest O +of O +the O +panel O +. O + +It O +is O +possible O +that O +by O +adopting O +extreme O +tilt B-evidence +angles I-evidence +the O +structure B-evidence +modulates O +CDR B-structure_element +H3 B-structure_element +and O +its O +environment O +, O +which O +apparently O +cannot O +be O +achieved O +solely O +by O +conformational O +rearrangement O +of O +the O +CDR B-structure_element +. O + +Overall O +, O +the O +stability O +of O +the O +Fab B-structure_element +, O +as O +measured O +by O +Tm B-evidence +, O +is O +a O +result O +of O +the O +mutual O +adjustment O +of O +the O +HC B-structure_element +and O +LC B-structure_element +variable B-structure_element +domains I-structure_element +and O +adjustment O +of O +CDR B-structure_element +H3 B-structure_element +to O +the O +VH B-site +: I-site +VL I-site +cleft I-site +. O + +In O +summary O +, O +the O +analysis O +of O +this O +structural B-experimental_method +library I-experimental_method +of O +germline O +variants O +composed O +of O +all O +pairs O +of O +4 O +HCs B-structure_element +and O +4LCs O +, O +all O +with O +the O +same O +CDR B-structure_element +H3 B-structure_element +, O +offers O +some O +unique O +insights O +into O +antibody B-protein_type +structure B-evidence +and O +how O +pairing O +and O +sequence O +may O +influence O +, O +or O +not O +, O +the O +canonical O +structures O +of O +the O +L1 B-structure_element +, O +L2 B-structure_element +, O +L3 B-structure_element +, O +H1 B-structure_element +and O +H2 B-structure_element +CDRs B-structure_element +. O + +Comparison O +of O +the O +CDR B-structure_element +H3s B-structure_element +reveals O +a O +large O +set O +of O +variants O +with O +conformations O +similar O +to O +the O +parent O +, O +while O +a O +second O +set O +has O +significant O +conformational O +variability O +, O +indicating O +that O +both O +the O +sequence O +and O +the O +structural O +context O +define O +the O +CDR B-structure_element +H3 B-structure_element +conformation O +. O + +Furthermore O +, O +antibody B-protein_type +CDRs B-structure_element +, O +H3 B-structure_element +in O +particular O +, O +may O +go O +through O +conformational O +changes O +upon O +binding O +their O +targets O +, O +making O +structural O +prediction O +for O +docking O +purposes O +an O +even O +more O +difficult O +task O +. O + +Fortunately O +, O +for O +most O +applications O +of O +antibody B-protein_type +modeling O +, O +such O +as O +engineering O +affinity O +and O +biophysical O +properties O +, O +an O +accurate O +CDR B-structure_element +H3 B-structure_element +structure B-evidence +is O +not O +always O +necessary O +. O + +The O +set O +of O +16 O +germline O +Fab B-structure_element +structures B-evidence +offers O +a O +unique O +dataset O +to O +facilitate O +software O +development O +for O +antibody B-protein_type +modeling O +. O + +The O +results O +essentially O +support O +the O +underlying O +idea O +of O +canonical O +structures B-evidence +, O +indicating O +that O +most O +CDRs B-structure_element +with O +germline O +sequences O +tend O +to O +adopt O +predefined O +conformations O +. O + +From O +this O +point O +of O +view O +, O +a O +novel O +approach O +to O +design O +combinatorial O +antibody B-protein_type +libraries O +would O +be O +to O +cover O +the O +range O +of O +CDR B-structure_element +conformations O +that O +may O +not O +necessarily O +coincide O +with O +the O +germline O +usage O +in O +the O +human B-species +repertoire O +. O + +This O +would O +insure O +more O +structural O +diversity O +, O +leading O +to O +a O +more O +diverse O +panel O +of O +antibodies B-protein_type +that O +would O +bind O +to O +a O +broad O +spectrum O +of O +targets O +. O + +Structure B-evidence +of O +the O +Response B-protein_type +Regulator I-protein_type +NsrR B-protein +from O +Streptococcus B-species +agalactiae I-species +, O +Which O +Is O +Involved O +in O +Lantibiotic B-chemical +Resistance O + +Lantibiotics B-chemical +are O +antimicrobial B-chemical +peptides I-chemical +produced O +by O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +bacteria I-taxonomy_domain +. O + +The O +expression O +of O +the O +genes O +responsible O +for O +lantibiotic B-chemical +resistance O +is O +regulated O +by O +a O +specific O +two B-complex_assembly +- I-complex_assembly +component I-complex_assembly +system I-complex_assembly +consisting O +of O +a O +histidine B-protein_type +kinase I-protein_type +and O +a O +response B-protein_type +regulator I-protein_type +. O + +Here O +, O +we O +focused O +on O +a O +response B-protein_type +regulator I-protein_type +involved O +in O +lantibiotic B-chemical +resistance O +, O +NsrR B-protein +from O +Streptococcus B-species +agalactiae I-species +, O +and O +determined O +the O +crystal B-evidence +structures I-evidence +of O +its O +N O +- O +terminal O +receiver B-structure_element +domain I-structure_element +and O +C O +- O +terminal O +DNA B-structure_element +- I-structure_element +binding I-structure_element +effector I-structure_element +domain I-structure_element +. O + +Amino O +acids O +involved O +in O +phosphorylation B-ptm +, O +dimerization O +, O +and O +DNA B-chemical +- O +binding O +were O +identified O +and O +demonstrated O +to O +be O +conserved B-protein_state +in O +lantibiotic B-protein_type +resistance I-protein_type +regulators I-protein_type +. O + +This O +has O +led O +to O +the O +search O +for O +novel O +antibiotics O +that O +can O +be O +used O +as O +pharmaceuticals O +against O +human B-species +pathogenic O +bacteria B-taxonomy_domain +. O + +Lantibiotics B-chemical +are O +small O +antimicrobial B-chemical +peptides I-chemical +( O +30 O +– O +50 O +amino O +acids O +in O +length O +), O +which O +are O +produced O +by O +several O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +bacterial I-taxonomy_domain +strains O +. O + +Lantibiotics B-chemical +are O +for O +example O +highly O +effective O +against O +various O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +, O +human B-species +pathogenic O +bacteria B-taxonomy_domain +including O +Streptococcus B-species +pneumoniae I-species +and O +several O +methicillin B-species +- I-species +resistant I-species +Staphylococcus I-species +aureus I-species +( O +MRSA B-species +) O +strains O +. O + +The O +high O +potency O +of O +lantibiotics B-chemical +for O +medical O +usage O +has O +already O +been O +noticed O +, O +and O +several O +lantibiotics B-chemical +are O +already O +included O +in O +clinical O +trials O +. O + +Nisin B-chemical +is O +the O +most O +prominent O +member O +of O +the O +lantibiotic B-chemical +family O +and O +is O +able O +to O +inhibit O +cell O +growth O +, O +penetrates O +the O +membranes O +of O +various O +Gram B-taxonomy_domain +- I-taxonomy_domain +positive I-taxonomy_domain +bacteria I-taxonomy_domain +, O +and O +is O +characterized O +by O +five O +specific O +( O +methyl O +-) O +lanthionine O +rings O +, O +which O +are O +crucial O +for O +stability O +and O +activity O +in O +the O +nanomolar O +range O +. O + +This O +immunity O +system O +consists O +of O +a O +membrane B-protein_type +– I-protein_type +associated I-protein_type +lipoprotein I-protein_type +( O +usually O +referred O +to O +as O +LanI B-protein_type +) O +and O +/ O +or O +an O +ABC B-protein_type +transporter I-protein_type +( O +termed O +as O +LanFEG B-protein_type +and O +comprising O +three O +subunits O +). O + +Although O +some O +lantibiotics B-chemical +such O +as O +Pep5 B-chemical +, O +epicidin B-chemical +, O +epilancin B-chemical +, O +and O +lactocin B-chemical +S I-chemical +only O +require O +LanI B-protein_type +for O +immunity O +, O +other O +lantibiotics B-chemical +with O +a O +dual O +mode O +of O +action O +involving O +pore O +formation O +and O +lipid O +II O +binding O +such O +as O +nisin B-chemical +, O +subtilin B-chemical +, O +epidermin B-chemical +, O +gallidermin B-chemical +, O +and O +lacticin B-chemical +3147 I-chemical +require O +additionally O +the O +presence O +of O +LanFEG B-protein_type +. O + +Structural B-evidence +data I-evidence +are O +reported O +for O +the O +immunity B-protein_type +proteins I-protein_type +NisI B-protein +from O +Lactococcus B-species +lactis I-species +, O +SpaI B-protein +from O +Bacillus B-species +subtilis I-species +and O +MlbQ B-protein +from O +the O +lantibiotic B-chemical +NAI B-chemical +- I-chemical +107 I-chemical +producer O +strain O +Microbispora B-species +ATCC I-species +PTA I-species +- I-species +5024 I-species +. O + +Recently O +, O +gene O +clusters O +were O +identified O +in O +certain O +clinically O +relevant O +human B-species +pathogenic O +strains O +such O +as O +Streptococcus B-species +agalactiae I-species +, O +S B-species +. I-species +aureus I-species +, O +and O +others O +that O +confer O +inherent O +resistance O +against O +specific O +lantibiotics B-chemical +such O +as O +nisin B-chemical +and O +resemble O +the O +genetic O +architecture O +of O +the O +lantibiotic O +immunity O +genes O +found O +in O +the O +producing O +strains O +. O + +Within O +these O +resistance O +operons O +, O +genes O +encoding O +for O +a O +membrane B-protein_type +- I-protein_type +associated I-protein_type +protease I-protein_type +and O +an O +ABC B-protein_type +transporter I-protein_type +were O +identified O +. O + +Expression O +of O +these O +proteins O +provides O +resistance O +against O +lantibiotics B-chemical +. O + +Bacteria B-taxonomy_domain +have O +the O +ability O +to O +sense O +and O +survive O +various O +environmental O +stimuli O +through O +adaptive O +responses O +, O +which O +are O +regulated O +by O +TCSs B-complex_assembly +. O + +The O +absence B-protein_state +of I-protein_state +TCSs B-complex_assembly +within O +mammals B-taxonomy_domain +makes O +them O +unique O +targets O +for O +novel O +antimicrobial O +drugs O +. O + +Some O +examples O +of O +TCS B-complex_assembly +are O +: O +BraRS B-protein +in O +S B-species +. I-species +aureus I-species +which O +is O +induced O +by O +bacitracin B-chemical +, O +nisin B-chemical +and O +nukacin B-chemical +- I-chemical +ISK I-chemical +- I-chemical +1 I-chemical +resistance O +, O +BceRS B-protein +in O +Bacillus B-taxonomy_domain +spp I-taxonomy_domain +. O +that O +is O +induced O +by O +actagardine B-chemical +and O +mersacidin B-chemical +resistance O +, O +LcrRS B-protein +in O +Streptococcus B-species +mutans I-species +induced O +by O +nukacin B-chemical +- I-chemical +ISK I-chemical +- I-chemical +1 I-chemical +and O +lacticin B-chemical +481 I-chemical +and O +LisRK B-protein +of O +Listeria B-species +monocytogenes I-species +induced O +by O +nisin B-chemical +resistance O +. O + +Furthermore O +, O +multiple O +lantibiotics B-chemical +can O +induce O +the O +TCS B-complex_assembly +CprRK B-protein +from O +Clostridium B-species +difficile I-species +, O +leading O +to O +the O +expression O +of O +the O +genes O +localized O +on O +the O +cpr B-gene +operon O +, O +resulting O +in O +resistance O +against O +several O +lantibiotics B-chemical +of O +which O +nisin B-chemical +, O +gallidermin B-chemical +, O +subtilin B-chemical +, O +and O +mutacin B-chemical +1140 I-chemical +are O +some O +examples O +. O + +It O +has O +been O +suggested O +that O +in O +addition O +to O +conferring O +general O +resistance O +against O +lantibiotics B-chemical +, O +the O +BceAB B-protein_type +- I-protein_type +type I-protein_type +transporters I-protein_type +assist O +in O +signalling O +as O +via O +the O +presence O +of O +a O +large O +extracellular B-structure_element +domain I-structure_element +within O +the O +transmembrane B-structure_element +segment I-structure_element +indicated O +by O +experimental O +evidence O +from O +various O +systems O +. O + +Homologous O +operons O +have O +been O +identified O +in O +various O +human B-species +pathogenic O +strains O +such O +as O +Staphylococcus B-species +epidermis I-species +and O +Streptococcus B-species +ictaluri I-species +based O +on O +the O +high O +sequence O +identity O +of O +NSR B-protein +and O +NsrFP B-protein +. O + +In O +this O +gene O +cluster O +, O +the O +TCS B-complex_assembly +NsrRK B-protein +is O +responsible O +for O +the O +expression O +of O +the O +nsr B-gene +and O +nsrFP B-gene +genes O +. O + +The O +similarity O +of O +the O +TCS B-complex_assembly +within O +all O +the O +described O +nisin B-chemical +resistance O +operons O +suggests O +an O +expression O +specifically O +induced O +by O +nisin B-chemical +. O + +Thus O +, O +NsrRK B-protein +might O +be O +a O +useful O +target O +to O +combat O +inherently O +pathogenic O +lantibiotic B-chemical +- O +resistant O +strains O +. O + +Generally O +, O +RRs B-protein_type +consist O +of O +two O +distinct O +structural O +domains O +, O +a O +receiver B-structure_element +domain I-structure_element +( O +RD B-structure_element +) O +and O +an O +effector B-structure_element +domain I-structure_element +( O +ED B-structure_element +), O +that O +are O +separated O +from O +each O +other O +by O +a O +flexible B-protein_state +linker B-structure_element +. O + +RDs B-structure_element +contain O +a O +highly B-protein_state +conserved I-protein_state +aspartate B-residue_name +residue O +, O +which O +acts O +as O +a O +phosphoryl O +acceptor O +that O +becomes O +phosphorylated B-protein_state +by O +the O +kinase B-structure_element +domain I-structure_element +of O +the O +histidine B-protein_type +kinase I-protein_type +upon O +reception O +of O +an O +external O +signal O +. O + +The O +RRs B-protein_type +are O +classified O +into O +different O +subfamilies O +depending O +on O +the O +three O +- O +dimensional O +structure O +of O +their O +EDs B-structure_element +. O + +The O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +is O +the O +largest O +subgroup O +of O +RRs B-protein_type +and O +comprises O +approximately O +40 O +% O +of O +all O +response B-protein_type +regulators I-protein_type +in O +bacteria B-taxonomy_domain +. O + +The O +various O +structures B-evidence +of O +RRs B-protein_type +reveal O +that O +in O +addition O +to O +being O +in O +either O +“ O +inactive B-protein_state +” O +or O +“ O +active B-protein_state +” O +state O +, O +the O +RRs B-protein_type +can O +also O +exist O +in O +two O +distinct O +conformations O +: O +“ O +open B-protein_state +” O +and O +“ O +closed B-protein_state +”. O + +MtrA B-protein +and O +PrrA B-protein +exhibit O +a O +very B-protein_state +compact I-protein_state +, O +closed B-protein_state +structure B-evidence +with O +the O +DNA B-structure_element +- I-structure_element +binding I-structure_element +sequence I-structure_element +, O +called O +recognition B-structure_element +helix I-structure_element +, O +of O +the O +ED B-structure_element +being O +inaccessible O +to O +DNA B-chemical +. O + +Here O +, O +we O +describe O +the O +crystal B-evidence +structures I-evidence +of O +the O +N O +- O +terminal O +RD B-structure_element +and O +the O +C O +- O +terminal O +ED B-structure_element +of O +the O +lantibiotic B-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +RR I-protein_type +NsrR B-protein +from O +S B-species +. I-species +agalactiae I-species +. O + +NsrR B-protein +is O +part O +of O +the O +nisin B-chemical +resistance O +operon O +. O + +The O +expression O +of O +the O +genes O +of O +this O +operon O +is O +induced O +by O +a O +TCS B-complex_assembly +consisting O +of O +the O +HK B-protein_type +NsrK B-protein +and O +the O +RR B-protein_type +NsrR B-protein +. O +Based O +on O +the O +crystal B-evidence +structures I-evidence +of O +both O +the O +domains O +, O +modeling O +was O +employed O +to O +shed O +light O +on O +the O +putative O +DNA B-protein_state +- I-protein_state +bound I-protein_state +state O +of O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +. O + +Both O +bands O +were O +subjected O +to O +mass B-experimental_method +spectrometry I-experimental_method +analysis I-experimental_method +. O + +The O +analysis O +revealed O +that O +the O +larger O +fragment O +(**) O +represents O +the O +N O +- O +terminal O +receiver B-structure_element +domain I-structure_element +( O +residues O +1 B-residue_range +– I-residue_range +119 I-residue_range +; O +referred O +to O +as O +NsrR B-protein +- O +RD B-structure_element +) O +whereas O +the O +smaller O +fragment O +(***) O +contained O +the O +C O +- O +terminal O +DNA B-structure_element +- I-structure_element +binding I-structure_element +effector I-structure_element +domain I-structure_element +of O +NsrR B-protein +( O +residues O +129 B-residue_range +– I-residue_range +243 I-residue_range +including O +21 O +amino O +acids O +derived O +from O +the O +expression O +tag O +; O +referred O +to O +as O +NsrR B-protein +- O +ED B-structure_element +) O +( O +Fig O +1C O +). O + +Residues O +120 B-residue_range +– I-residue_range +128 I-residue_range +form O +the O +linker B-structure_element +connecting O +the O +RD B-structure_element +and O +ED B-structure_element +. O + +Mass B-experimental_method +spectrometry I-experimental_method +analysis I-experimental_method +did O +not O +reveal O +the O +presence O +of O +any O +specific O +protease O +in O +the O +purified O +NsrR B-protein +sample O +. O + +Furthermore O +, O +addition O +of O +a O +protease O +inhibitor O +, O +such O +as O +PMSF B-chemical +( O +Phenylmethylsulfonyl B-chemical +fluoride I-chemical +) O +and O +AEBSF B-chemical +{ O +4 B-chemical +-( I-chemical +2 I-chemical +- I-chemical +Aminoethyl I-chemical +) I-chemical +benzenesulfonyl I-chemical +fluoride I-chemical +hydrochloride I-chemical +}, O +even O +at O +high O +concentrations O +, O +did O +not O +inhibit O +proteolysis O +( O +data O +not O +shown O +). O + +Purification B-experimental_method +of O +NsrR B-protein +and O +SDS B-experimental_method +PAGE I-experimental_method +analysis O +of O +purified O +NsrR B-protein +directly O +and O +one O +week O +after O +purification O +. O + +( O +a O +) O +Elution B-evidence +profile I-evidence +of O +size B-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +step O +of O +NsrR B-protein +. O +The O +y O +- O +axis O +represents O +the O +UV O +absorption O +of O +the O +protein O +at O +280 O +nm O +, O +while O +the O +x O +- O +axis O +represents O +the O +elution O +volume O +. O + +( O +b O +) O +Freshly O +purified O +NsrR B-protein +protein O +, O +and O +( O +c O +) O +NsrR B-protein +protein O +after O +one O +week O +. O + +Lanes O +: O +M O +represents O +the O +PAGE O +Ruler O +Unstained O +Ladder O +; O +1 O +: O +NsrR B-protein +after O +a O +two O +- O +step O +purification O +; O +2 O +: O +NsrR B-protein +one O +week O +after O +purification O +. O + +* O +corresponds O +to O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +protein O +at O +27 O +kDa O +, O +while O +** O +and O +*** O +correspond O +to O +the O +NsrR B-protein +- O +RD B-structure_element +and O +NsrR B-protein +- O +ED B-structure_element +domain O +at O +around O +13 O +kDa O +, O +respectively O +. O + +Since O +formation O +of O +the O +crystals B-evidence +took O +around O +one O +month O +, O +it O +is O +not O +surprising O +that O +this O +cleavage O +also O +occurred O +in O +the O +crystallization O +drop O +. O + +Initially O +, O +we O +tried O +to O +solve O +the O +structure B-evidence +of O +NsrR B-protein +by O +molecular B-experimental_method +replacement I-experimental_method +, O +which O +was O +not O +successful O +. O + +Therefore O +, O +we O +tried O +heavy B-experimental_method +atom I-experimental_method +phasing I-experimental_method +using O +a O +platinum B-chemical +compound O +. O + +This O +succeeded O +for O +the O +rectangular O +plate O +- O +shaped O +crystals B-evidence +. O + +Therefore O +, O +we O +thought O +that O +these O +crystals B-evidence +contained O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +of O +NsrR B-protein +and O +successfully O +phased O +this O +dataset O +using O +molecular B-experimental_method +replacement I-experimental_method +with O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +of O +PhoB B-protein +( O +PDB O +code O +: O +1B00 O +; O +as O +a O +template O +. O + +This O +approach O +revealed O +that O +this O +crystal O +form O +indeed O +contained O +two O +monomers B-oligomeric_state +of O +the O +RD B-structure_element +of O +NsrR B-protein +in O +the O +asymmetric O +unit O +. O + +Since O +both O +crystals O +forms O +were O +obtained O +in O +the O +same O +drop O +it O +is O +not O +surprising O +that O +, O +when O +dissolving O +several O +crystals B-evidence +and O +performing O +subsequent O +mass B-experimental_method +- I-experimental_method +spectrometry I-experimental_method +to O +identify O +the O +protein O +in O +the O +crystals B-evidence +, O +it O +yielded O +peptide O +fragments O +throughout O +the O +NsrR B-protein +sequence O +. O + +In O +summary O +, O +the O +two O +crystal B-evidence +forms I-evidence +contained O +one O +of O +the O +two O +domains O +, O +respectively O +, O +such O +that O +both O +domains O +were O +successfully O +crystallized B-experimental_method +. O + +However O +, O +a O +part O +of O +the O +linker B-structure_element +region I-structure_element +( O +residues O +120 B-residue_range +– I-residue_range +128 I-residue_range +; O +120RRSQQFIQQ128 B-structure_element +; O +underlined O +are O +the O +amino O +acid O +residues O +not O +visible O +in O +either O +domain O +) O +could O +not O +be O +traced O +in O +the O +electron B-evidence +density I-evidence +. O + +Overall O +structure B-evidence +of O +the O +N O +- O +terminal O +NsrR B-protein +receiver B-structure_element +domain I-structure_element +( O +NsrR B-protein +- O +RD B-structure_element +) O + +The O +structure B-evidence +of O +the O +NsrR B-protein +- O +RD B-structure_element +was O +determined O +at O +a O +resolution O +of O +1 O +. O +8 O +Å O +( O +Table O +1 O +). O + +The O +structure B-evidence +contained O +many O +ethylene B-chemical +glycol I-chemical +molecules O +arising O +from O +the O +cryo O +- O +protecting O +procedure O +. O + +The O +asymmetric O +unit O +contains O +two O +copies O +of O +NsrR B-protein +- O +RD B-structure_element +. O + +For O +Asn85 B-residue_name_number +, O +Asp86 B-residue_name_number +, O +and O +Glu87 B-residue_name_number +of O +chain B-structure_element +A I-structure_element +, O +poor O +electron B-evidence +density I-evidence +was O +observed O +for O +the O +side O +chains O +and O +, O +thus O +, O +these O +side O +chains O +were O +deleted O +during O +refinement O +and O +are O +not O +present O +in O +the O +final O +structure B-evidence +. O + +Since O +the O +two O +monomers B-oligomeric_state +of O +NsrR B-protein +- O +RD B-structure_element +were O +virtually O +identical O +( O +rmsd B-evidence +of O +0 O +. O +6 O +Å O +over O +116 O +Cα O +atoms O +for O +the O +two O +monomers B-oligomeric_state +). O + +Therefore O +, O +the O +overall O +structure B-evidence +is O +described O +for O +monomer B-oligomeric_state +A B-structure_element +only O +. O + +NsrR B-protein +- O +RD B-structure_element +structurally O +adopts O +a O +αβ B-structure_element +doubly I-structure_element +- I-structure_element +wound I-structure_element +fold I-structure_element +previously O +observed O +in O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +type I-protein_type +regulators I-protein_type +. O + +Five O +β B-structure_element +- I-structure_element +strands I-structure_element +( O +β1 B-structure_element +- I-structure_element +β5 I-structure_element +) O +are O +arranged O +in O +a O +parallel O +fashion O +constituting O +the O +central O +core O +of O +the O +structure B-evidence +, O +which O +is O +surrounded O +by O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +α1 B-structure_element +and O +α5 B-structure_element +) O +on O +one O +and O +three O +helices B-structure_element +( O +α2 B-structure_element +, O +α3 B-structure_element +, O +α4 B-structure_element +) O +on O +the O +other O +side O +( O +Fig O +2 O +). O + +Structure B-evidence +of O +NsrR B-protein +- O +RD B-structure_element +. O + +Comparison B-experimental_method +with O +structures B-evidence +of O +other O +receiver B-structure_element +domains I-structure_element + +NsrR B-protein +belongs O +to O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +family I-protein_type +of O +RRs B-protein_type +. O + +Superimposition B-experimental_method +of O +the O +structures B-evidence +revealed O +that O +helix B-structure_element +α4 B-structure_element +is O +slightly O +rotated O +outward O +in O +NsrR B-protein +- O +RD B-structure_element +( O +Fig O +2 O +). O + +In O +receiver B-structure_element +domains I-structure_element +of O +response B-protein_type +regulators I-protein_type +, O +helix B-structure_element +α4 B-structure_element +has O +been O +shown O +to O +be O +a O +crucial O +part O +of O +the O +dimerization B-site +interface I-site +. O + +Furthermore O +, O +helix B-structure_element +α4 B-structure_element +in O +NsrR B-protein +is O +shorter O +than O +in O +other O +RRs B-protein_type +. O + +The O +first B-structure_element +helical I-structure_element +turn I-structure_element +is O +unwound B-protein_state +and O +adopts O +an O +unstructured B-protein_state +region O +( O +see O +Fig O +2 O +). O + +A O +slightly O +outward O +rotation O +or O +unwinding O +of O +helix B-structure_element +α4 B-structure_element +has O +been O +observed O +in O +the O +structures B-evidence +of O +other O +RD B-structure_element +of O +regulators O +. O + +For O +example O +, O +the O +structure B-evidence +of O +BaeR B-protein +and O +RegX3 B-protein +displayed O +a O +completely O +unwound B-protein_state +helix B-structure_element +α4 B-structure_element +. O + +In O +the O +structure B-evidence +of O +DrrD B-protein +, O +helix B-structure_element +α4 B-structure_element +is O +only O +partially O +displaced O +. O + +In O +the O +receiver B-structure_element +domain I-structure_element +of O +NsrR B-protein +, O +helix B-structure_element +α4 B-structure_element +is O +also O +partially O +displaced O +but O +in O +a O +different O +direction O +( O +S1 O +Fig O +). O + +Inspection O +of O +the O +crystal O +contacts O +revealed O +no O +major O +interactions O +in O +this O +region O +that O +could O +have O +influenced O +the O +orientation O +of O +helix B-structure_element +α4 B-structure_element +. O + +The O +structures B-evidence +of O +the O +RD B-structure_element +and O +ED B-structure_element +domains O +of O +NsrR B-protein +aligned B-experimental_method +to O +other O +response B-protein_type +regulators I-protein_type +. O + +This O +structural O +homology O +is O +also O +reflected O +by O +the O +low O +rmsd B-evidence +of O +1 O +. O +9 O +Å O +over O +117 O +Cα O +atoms O +after O +superimposition B-experimental_method +of O +the O +receiver B-structure_element +domains I-structure_element +of O +NsrR B-protein +and O +KdpE B-protein +( O +Table O +2 O +). O + +Furthermore O +, O +the O +orientation O +of O +the O +helix B-structure_element +α4 B-structure_element +in O +NsrR B-protein +is O +close O +to O +that O +present O +in O +KdpE B-protein +( O +S1 O +Fig O +). O + +Active B-site +site I-site +residues O +and O +dimerization O + +All O +RRs B-protein_type +contain O +a O +highly B-protein_state +conserved I-protein_state +aspartate B-residue_name +residue O +in O +the O +active B-site +site I-site +( O +Fig O +3 O +; O +shown O +in O +red O +). O + +Phosphorylation B-ptm +of O +this O +aspartate B-residue_name +residue O +induces O +a O +conformational O +change O +leading O +to O +the O +activation O +of O +the O +effector B-structure_element +domain I-structure_element +that O +binds O +DNA B-chemical +and O +regulates O +the O +transcription O +of O +target O +genes O +. O + +Sequence B-experimental_method +alignment I-experimental_method +of O +NsrR B-protein +protein O +with O +other O +response B-protein_type +regulators I-protein_type +. O + +A O +sequence B-experimental_method +alignment I-experimental_method +of O +NsrR B-protein +with O +RRs B-protein_type +belonging O +to O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +( O +marked O +in O +grey O +) O +and O +RRs B-protein_type +involved O +in O +lantibiotic B-chemical +resistance O +( O +black O +) O +is O +shown O +. O + +The O +active B-site +site I-site +aspartate B-residue_name +residue O +( O +highlighted O +in O +red O +), O +the O +residues O +forming O +the O +acidic B-site +pocket I-site +surrounding O +it O +( O +highlighted O +in O +pink O +), O +the O +switch B-site +residues I-site +( O +highlighted O +in O +blue O +), O +the O +conserved B-protein_state +lysine B-residue_name +residue O +( O +highlighted O +in O +green O +), O +the O +highly B-protein_state +conserved I-protein_state +residues O +of O +the O +linker B-structure_element +region I-structure_element +( O +colored O +in O +purple O +), O +the O +residues O +involved O +in O +dimer B-site +interface I-site +of O +receiver B-structure_element +domain I-structure_element +( O +highlighted O +in O +yellow O +), O +residues O +involved O +in O +interdomain O +interactions O +( O +shown O +in O +orange O +boxes O +and O +in O +cyan O +) O +and O +the O +residues O +involved O +in O +interaction O +with O +DNA B-chemical +( O +colored O +in O +blue O +) O +are O +shown O +. O + +In O +NsrR B-protein +, O +Glu12 B-residue_name_number +, O +Asp13 B-residue_name_number +, O +and O +Asp55 B-residue_name_number +are O +in O +close O +proximity O +of O +a O +highly B-protein_state +conserved I-protein_state +Lys104 B-residue_name_number +residue O +( O +highlighted O +in O +green O +in O +Fig O +3 O +). O + +Location O +of O +the O +highly B-protein_state +conserved I-protein_state +Asp55 B-residue_name_number +and O +inactive B-protein_state +state O +conformation O +of O +the O +key O +switch B-site +residues I-site +, O +Ser82 B-residue_name_number +and O +Phe101 B-residue_name_number +in O +NsrR B-protein +- O +RD B-structure_element +. O + +NsrR B-protein +( O +represented O +in O +yellow O +) O +displays O +a O +geometry O +representing O +the O +inactive B-protein_state +state O +as O +deduced O +from O +the O +inactive B-protein_state +state O +structure B-evidence +of O +PhoB B-protein +( O +shown O +in O +brown O +, O +PDB O +code O +1B00 O +) O +( O +a O +). O + +However O +, O +the O +structure B-evidence +of O +NsrR B-protein +- O +RD B-structure_element +did O +not O +contain O +any O +divalent O +ion O +. O + +Instead O +, O +a O +water B-chemical +molecule O +is O +present O +, O +which O +interacts O +with O +Glu12 B-residue_name_number +of O +the O +acidic B-site +pocket I-site +, O +Lys104 B-residue_name_number +, O +and O +another O +water B-chemical +molecule O +in O +the O +vicinity O +. O + +As O +seen O +in O +the O +alignment B-experimental_method +( O +Fig O +3 O +, O +highlighted O +in O +blue O +), O +these O +signature O +residues O +( O +Ser B-residue_name +/ O +Thr B-residue_name +and O +Phe B-residue_name +/ O +Tyr B-residue_name +) O +are O +highly B-protein_state +conserved I-protein_state +in O +the O +lantibiotic B-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +RRs I-protein_type +. O + +The O +orientation O +of O +the O +side O +chains O +of O +these O +residues O +determines O +whether O +the O +RD B-structure_element +is O +in O +an O +active B-protein_state +or O +inactive B-protein_state +state O +. O + +In O +the O +inactive B-protein_state +state O +, O +the O +phenylalanine B-residue_name +or O +tyrosine B-residue_name +residue O +faces O +away O +from O +the O +active B-site +site I-site +, O +and O +the O +corresponding O +serine B-residue_name +or O +threonine B-residue_name +residue O +adopts O +an O +outward B-protein_state +- I-protein_state +facing I-protein_state +conformation O +as O +well O +( O +Fig O +4A O +). O + +By O +sequence B-experimental_method +alignment I-experimental_method +with O +other O +lantibiotic B-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +RRs I-protein_type +, O +these O +“ O +signature B-site +switch I-site +residues I-site +” O +are O +identified O +as O +Ser82 B-residue_name_number +and O +Phe101 B-residue_name_number +in O +NsrR B-protein +( O +see O +above O +). O + +Furthermore O +, O +the O +second B-site +switch I-site +residue I-site +is O +mostly O +a O +tyrosine B-residue_name +, O +with O +NsrR B-protein +, O +BraR B-protein +, O +and O +BceR B-protein +being O +the O +only O +exceptions O +containing O +a O +phenylalanine B-residue_name +at O +that O +position O +. O + +As O +mentioned O +above O +, O +RRs B-protein_type +contain O +a O +phosphorylation B-protein_state +- I-protein_state +activated I-protein_state +switch B-site +and O +normally O +exist O +in O +equilibrium O +between O +the O +active B-protein_state +and O +inactive B-protein_state +conformations O +. O + +Therefore O +, O +we O +performed O +a O +DALI B-experimental_method +search I-experimental_method +and O +focused O +on O +RD B-structure_element +domains O +that O +were O +structurally O +determined O +as O +functional B-protein_state +dimers B-oligomeric_state +. O + +In O +this O +context O +, O +the O +dimer B-oligomeric_state +of O +full B-protein_state +- I-protein_state +length I-protein_state +KdpE B-protein +from O +E B-species +. I-species +coli I-species +( O +Z B-evidence +- I-evidence +score I-evidence +18 O +. O +8 O +, O +rmsd B-evidence +1 O +. O +9 O +Å O +over O +117 O +Cα O +atoms O +) O +( O +PDB O +code O +: O +4KNY O +) O +and O +the O +structure B-evidence +of O +the O +functional B-protein_state +dimer B-oligomeric_state +of O +the O +RD B-structure_element +of O +KdpE B-protein +from O +E B-species +. I-species +coli I-species +( O +PDB O +code O +: O +1ZH2 O +) O +represent O +the O +most O +structurally O +related O +structures B-evidence +. O + +We O +aligned B-experimental_method +NsrR B-protein +- O +RD B-structure_element +on O +both O +monomers B-oligomeric_state +of O +the O +RD B-structure_element +of O +KdpE B-protein +. O +Since O +helix B-structure_element +α4 B-structure_element +of O +NsrR B-protein +- O +RD B-structure_element +is O +orientated O +slightly O +different O +when O +compared O +with O +other O +structures B-evidence +of O +RDs B-structure_element +( O +Fig O +2 O +), O +helix B-structure_element +α4 B-structure_element +and O +the O +N O +- O +terminal O +loop B-structure_element +of O +one O +monomer B-oligomeric_state +were O +clashing O +with O +the O +second O +monomer B-oligomeric_state +( O +S2A O +Fig O +). O + +Therefore O +, O +helix B-structure_element +α4 B-structure_element +and O +the O +N O +- O +terminal O +loop B-structure_element +were O +shifted O +to O +the O +position O +of O +KdpE B-protein +by O +primarily O +modifying O +backbone O +torsion O +angles O +in O +the O +region O +immediately O +C O +- O +terminal O +to O +helix B-structure_element +α4 B-structure_element +. O + +The O +dimeric B-site +interface I-site +is O +formed O +by O +α4 B-structure_element +- I-structure_element +β5 I-structure_element +- I-structure_element +α5 I-structure_element +of O +RD B-structure_element +( O +Fig O +5A O +), O +as O +previously O +observed O +in O +other O +RRs B-protein_type +. O + +In O +KdpE B-protein +, O +a O +network O +of O +salt B-bond_interaction +bridges I-bond_interaction +and O +other O +electrostatic B-bond_interaction +interactions I-bond_interaction +stabilize O +the O +interface B-site +within O +a O +single O +monomer B-oligomeric_state +as O +well O +as O +between O +the O +monomers B-oligomeric_state +. O + +Functional O +dimer B-oligomeric_state +orientation O +of O +the O +RDs B-structure_element +of O +NsrR B-protein +. O + +( O +b O +) O +Zoom O +- O +in O +of O +the O +dimeric B-site +interface I-site +mediated O +by O +α4 B-structure_element +- I-structure_element +β5 I-structure_element +- I-structure_element +α5 I-structure_element +. O + +The O +monomer B-oligomeric_state +- O +monomer B-oligomeric_state +interactions O +are O +facilitated O +by O +hydrophobic O +residues O +( O +displayed O +as O +spheres O +), O +inter O +- O +and O +intra O +- O +domain O +interactions O +( O +displayed O +as O +sticks O +). O + +Conserved O +intermolecular O +electrostatic B-bond_interaction +interactions I-bond_interaction +further O +stabilize O +the O +monomer B-oligomeric_state +- O +monomer B-oligomeric_state +interaction O +of O +KdpE B-protein +and O +are O +formed O +between O +Asp97 B-residue_name_number +( O +β5 B-structure_element +) O +and O +Arg111 B-residue_name_number +( O +α5 B-structure_element +), O +Asp96 B-residue_name_number +( O +α4 B-structure_element +– I-structure_element +β5 I-structure_element +loop I-structure_element +) O +and O +Arg118 B-residue_name_number +( O +α5 B-structure_element +), O +and O +Asp92 B-residue_name_number +( O +α4 B-structure_element +) O +and O +Arg113 B-residue_name_number +( O +α5 B-structure_element +). O + +Some O +of O +these O +interactions O +can O +also O +be O +identified O +in O +the O +dimeric B-oligomeric_state +model O +of O +NsrR B-protein +- O +RD B-structure_element +. O + +Asp99 B-residue_name_number +( O +α4 B-structure_element +– I-structure_element +β5 I-structure_element +loop I-structure_element +; O +Fig O +3 O +, O +shown O +in O +cyan O +) O +points O +toward O +the O +side O +chain O +of O +Arg121 B-residue_name_number +. O + +This O +interaction O +is O +also O +observed O +in O +KdpE B-protein +( O +Asp96 B-residue_name_number +( O +α4 B-structure_element +– I-structure_element +β5 I-structure_element +loop I-structure_element +) O +and O +Arg118 B-residue_name_number +( O +α5 B-structure_element +)). O + +In O +KdpE B-protein +, O +Arg111 B-residue_name_number +is O +additionally O +stabilized O +by O +another O +intra O +- O +molecular O +salt B-bond_interaction +bridge I-bond_interaction +with O +Glu107 B-residue_name_number +( O +α5 B-structure_element +). O + +Interestingly O +, O +in O +NsrR B-protein +- O +RD B-structure_element +this O +amino O +acid O +corresponds O +to O +Val110 B-residue_name_number +( O +highlighted O +in O +yellow O +in O +Fig O +3 O +). O + +As O +observed O +in O +this O +alignment B-experimental_method +, O +the O +above O +- O +mentioned O +arginine B-residue_name +residue O +( O +Arg111 B-residue_name_number +in O +KdpE B-protein +) O +is O +either O +an O +arginine B-residue_name +or O +a O +lysine B-residue_name +residue O +( O +Lys114 B-residue_name_number +in O +NsrR B-protein +) O +in O +all O +RRs B-protein_type +used O +in O +the O +alignment B-experimental_method +( O +Fig O +3 O +, O +shown O +in O +cyan O +). O + +Interestingly O +, O +whenever O +an O +arginine B-residue_name +is O +present O +at O +this O +position O +( O +Arg111 B-residue_name_number +in O +KdpE B-protein +), O +a O +glutamate B-residue_name +( O +Glu107 B-residue_name_number +in O +KdpE B-protein +) O +is O +present O +as O +well O +, O +presumably O +stabilizing O +the O +arginine B-residue_name +side O +chain O +. O + +However O +, O +when O +a O +lysine B-residue_name +is O +present O +at O +this O +position O +, O +the O +glutamate B-residue_name +is O +exchanged O +to O +a O +hydrophobic O +residue O +contributing O +to O +the O +hydrophobic B-site +patch I-site +described O +above O +. O + +Additionally O +, O +it O +has O +been O +shown O +for O +PhoB B-protein +from O +E B-species +. I-species +coli I-species +and O +PhoP B-protein +from O +B B-species +. I-species +subtilis I-species +that O +mutating B-experimental_method +the O +corresponding O +residues O +involved O +in O +dimerisation O +( O +residues O +Asp100 B-residue_name_number +, O +Val110 B-residue_name_number +and O +Lys114 B-residue_name_number +in O +NsrR B-protein +) O +results O +in O +monomeric B-oligomeric_state +form O +of O +response B-protein_type +regulator I-protein_type +which O +has O +lost B-protein_state +the I-protein_state +ability I-protein_state +to I-protein_state +dimerize I-protein_state +as O +well O +as O +display O +reduced O +DNA B-chemical +binding O +capabilities O +. O + +The O +structure B-evidence +of O +NsrR B-protein +- O +ED B-structure_element +from O +S B-species +. I-species +agalactiae I-species +was O +determined O +using O +experimental O +phases O +from O +a O +single B-experimental_method +- I-experimental_method +wavelength I-experimental_method +anomalous I-experimental_method +dispersion I-experimental_method +dataset I-experimental_method +from O +the O +rectangular O +plate O +- O +shaped O +crystal O +derivatized O +with O +platinum B-chemical +at O +a O +resolution O +of O +1 O +. O +6 O +Å O +in O +space O +group O +P21212 O +. O + +The O +Rwork B-evidence +and O +Rfree B-evidence +values O +after O +refinement O +were O +0 O +. O +18 O +and O +0 O +. O +22 O +, O +respectively O +. O + +The O +latter O +is O +Glu128 B-residue_name_number +( O +last O +residue O +of O +the O +linker B-structure_element +region I-structure_element +) O +of O +chain B-structure_element +B I-structure_element +that O +is O +involved O +in O +crystal O +contacts O +and O +, O +therefore O +, O +likely O +adopts O +an O +unfavorable O +conformation O +. O + +The O +structure B-evidence +contained O +a O +few O +ethylene B-chemical +glycol I-chemical +molecules O +introduced O +by O +the O +cryo O +- O +protecting O +procedure O +. O + +The O +C O +- O +terminal O +effector B-structure_element +DNA I-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +of O +NsrR B-protein +is O +about O +13 O +kDa O +in O +size O +and O +consists O +of O +residues O +129 B-residue_range +– I-residue_range +243 I-residue_range +( O +including O +21 O +amino O +acid O +residues O +of O +the O +expression O +tag O +). O + +For O +Asp147 B-residue_name_number +of O +chain B-structure_element +A I-structure_element +and O +Glu174 B-residue_name_number +of O +chain B-structure_element +B I-structure_element +, O +poor O +electron B-evidence +density I-evidence +was O +observed O +for O +the O +side O +chains O +and O +, O +thus O +, O +these O +side O +chains O +were O +removed O +during O +refinement O +. O + +An O +overlay B-experimental_method +revealed O +that O +both O +monomers B-oligomeric_state +display O +high O +similarity O +in O +their O +overall O +structure B-evidence +with O +an O +rmsd B-evidence +of O +0 O +. O +5 O +Å O +over O +95 O +Cα O +atoms O +. O + +We O +therefore O +describe O +for O +the O +overall O +structure B-evidence +only O +monomer B-oligomeric_state +A B-structure_element +. O + +The O +ED B-structure_element +domain O +of O +NsrR B-protein +consists O +of O +six O +β B-structure_element +- I-structure_element +strands I-structure_element +and O +three O +α B-structure_element +- I-structure_element +helices I-structure_element +in O +a O +β6 B-structure_element +- I-structure_element +β7 I-structure_element +- I-structure_element +β8 I-structure_element +- I-structure_element +β9 I-structure_element +- I-structure_element +α6 I-structure_element +- I-structure_element +α7 I-structure_element +- I-structure_element +α8 I-structure_element +- I-structure_element +β10 I-structure_element +- I-structure_element +β11 I-structure_element +topology O +( O +the O +secondary O +structure O +elements O +are O +counted O +in O +continuation O +of O +those O +of O +the O +RD B-structure_element +). O + +The O +effector B-structure_element +domain I-structure_element +starts O +with O +a O +4 B-structure_element +- I-structure_element +stranded I-structure_element +antiparallel I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +, O +followed O +by O +three O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +eventually O +ends O +in O +a O +C O +- O +terminal O +β B-structure_element +- I-structure_element +hairpin I-structure_element +( O +Fig O +6 O +). O + +Cartoon O +representation O +of O +the O +C O +- O +terminal O +effector B-structure_element +domain I-structure_element +of O +NsrR B-protein +( O +green O +; O +recognition B-structure_element +helix I-structure_element +in O +cyan O +). O + +The O +structural O +areas O +with O +the O +highest O +variations O +compared O +to O +the O +effector B-structure_element +domains I-structure_element +of O +DrrB B-protein +( O +pink O +, O +1P2F O +), O +MtrA B-protein +( O +grey O +, O +2GWR O +), O +and O +PhoB B-protein +( O +blue O +, O +1GXQ O +) O +are O +marked O +. O + +The O +transactivation B-structure_element +loop I-structure_element +of O +MtrA B-protein +is O +missing B-protein_state +in O +the O +structure B-evidence +, O +therefore O +, O +the O +two O +termini O +are O +connected O +by O +a O +dashed O +line O +. O + +The O +characteristic O +feature O +of O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +of O +RRs B-protein_type +is O +a O +winged B-structure_element +helix I-structure_element +- I-structure_element +turn I-structure_element +- I-structure_element +helix I-structure_element +( O +wHTH B-structure_element +) O +fold O +that O +is O +adopted O +by O +the O +α7 B-structure_element +- I-structure_element +loop I-structure_element +- I-structure_element +α8 I-structure_element +segment I-structure_element +in O +full B-protein_state +- I-protein_state +length I-protein_state +and O +single O +effector B-structure_element +domain I-structure_element +structures B-evidence +of O +RRs B-protein_type +of O +this O +subfamily O +. O + +The O +second O +helix B-structure_element +of O +the O +wHTH B-structure_element +motif O +is O +important O +for O +DNA B-chemical +- O +binding O +and O +, O +therefore O +, O +is O +termed O +“ O +recognition B-structure_element +helix I-structure_element +” O +( O +shown O +in O +cyan O +in O +Fig O +6 O +). O + +Furthermore O +, O +a O +helix B-structure_element +within O +the O +HTH B-structure_element +motif O +, O +named O +“ O +positioning B-structure_element +helix I-structure_element +”, O +is O +important O +for O +proper O +orientation O +and O +positioning O +of O +the O +loop B-structure_element +between O +these O +two O +helices O +and O +is O +referred O +to O +as O +“ O +transactivation B-structure_element +loop I-structure_element +” O +( O +also O +called O +α B-structure_element +- I-structure_element +loop I-structure_element +; O +Fig O +6 O +). O + +The O +16 O +- O +residue O +long O +, O +solvent B-protein_state +- I-protein_state +exposed I-protein_state +recognition B-structure_element +helix I-structure_element +α8 B-structure_element +of O +NsrR B-protein +- O +ED B-structure_element +contains O +four O +positively O +charged O +residues O +that O +can O +potentially O +interact O +with O +DNA B-chemical +. O + +These O +are O +Arg198 B-residue_name_number +, O +Arg200 B-residue_name_number +, O +Lys201 B-residue_name_number +, O +and O +Lys202 B-residue_name_number +. O + +When O +comparing O +the O +sequence O +of O +NsrR B-protein +with O +PhoB B-protein +, O +KdpE B-protein +, O +and O +MtrA B-protein +, O +the O +alignment B-experimental_method +( O +Fig O +3 O +, O +colored O +in O +blue O +) O +emphasizes O +the O +variations O +at O +these O +positions O +, O +except O +for O +Arg200 B-residue_name_number +, O +which O +is O +conserved B-protein_state +throughout O +the O +lantibiotic B-protein_type +resistance I-protein_type +RRs I-protein_type +. O + +Additionally O +, O +Lys202 B-residue_name_number +is O +also O +highly B-protein_state +conserved I-protein_state +throughout O +the O +family O +of O +RRs B-protein_type +except O +PhoB B-protein +, O +clearly O +reflecting O +differences O +in O +the O +sequences O +of O +DNA B-chemical +to O +be O +bound O +. O + +Comparison O +with O +structures B-evidence +of O +other O +effector B-structure_element +domains I-structure_element + +We O +performed O +a O +DALI B-experimental_method +search I-experimental_method +to O +identify O +structurally O +related O +proteins O +to O +NsrR B-protein +- O +ED B-structure_element +. O + +Here O +the O +structure B-evidence +of O +the O +effector B-structure_element +domain I-structure_element +of O +PhoB B-protein +from O +E B-species +. I-species +coli I-species +( O +PDB O +code O +: O +1GXQ O +) O +( O +Z B-evidence +- I-evidence +score I-evidence +of O +13 O +. O +7 O +) O +is O +structurally O +the O +most O +closely O +related O +. O + +Similar O +to O +the O +PhoB B-protein +effector B-structure_element +domain I-structure_element +, O +a O +9 O +- O +residues O +long O +loop B-structure_element +( O +amino O +acid O +182 B-residue_range +– I-residue_range +189 I-residue_range +) O +is O +also O +present O +in O +the O +structure B-evidence +of O +NsrR B-protein +- O +ED B-structure_element +that O +connects O +helices B-structure_element +α7 B-structure_element +and O +α8 B-structure_element +. O + +Therefore O +, O +we O +superimposed B-experimental_method +the O +Cα O +traces O +of O +the O +effector B-structure_element +domain I-structure_element +of O +NsrR B-protein +( O +NsrR B-protein +- O +ED B-structure_element +) O +with O +other O +previously O +determined O +effector B-structure_element +domains I-structure_element +from O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +family I-protein_type +such O +as O +DrrB B-protein +, O +MtrA B-protein +and O +of O +only O +the O +effector B-structure_element +domain I-structure_element +structure B-evidence +of O +PhoB B-protein +from O +E B-species +. I-species +coli I-species +. O + +The O +highest O +variations O +( O +Fig O +6 O +) O +are O +visible O +in O +in O +the O +loop B-structure_element +regions O +α7 B-structure_element +- I-structure_element +α8 I-structure_element +, O +which O +corresponds O +to O +the O +transactivation B-structure_element +loop I-structure_element +. O + +In O +many O +RRs B-protein_type +this O +transactivation B-structure_element +loop I-structure_element +along O +with O +the O +recognition B-structure_element +helix I-structure_element +α8 B-structure_element +, O +form O +inter O +- O +domain O +contacts O +in O +the O +inactive B-protein_state +state O +and O +are O +only O +exposed O +upon O +activation O +of O +the O +RRs B-protein_type +via O +a O +conformational O +change O +where O +the O +N O +- O +and O +C O +- O +terminal O +domains O +move O +away O +from O +each O +other O +. O + +Linker B-structure_element +region I-structure_element + +The O +linkers B-structure_element +that O +connect O +the O +RDs B-structure_element +and O +EDs B-structure_element +in O +response B-protein_type +regulators I-protein_type +are O +highly B-protein_state +variable I-protein_state +with O +respect O +to O +both O +length O +and O +sequence O +. O + +Similar O +to O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +family I-protein_type +, O +the O +lantibiotic B-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +family I-protein_type +of I-protein_type +response I-protein_type +regulators I-protein_type +also O +displays O +diverse O +linker B-structure_element +regions I-structure_element +, O +which O +are O +recognized O +in O +sequence B-experimental_method +alignments I-experimental_method +by O +the O +introduction O +of O +gaps O +( O +Fig O +3 O +). O + +Interestingly O +, O +two O +arginine B-residue_name +residues O +( O +Arg120 B-residue_name_number +and O +Arg121 B-residue_name_number +in O +NsrR B-protein +; O +Fig O +3 O +, O +shown O +in O +purple O +) O +at O +the O +end O +of O +the O +RDs B-structure_element +seem O +to O +be O +strictly B-protein_state +conserved I-protein_state +throughout O +the O +family O +of O +response B-protein_type +regulators I-protein_type +in O +both O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +and I-protein_type +lantibiotic I-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +RRs I-protein_type +, O +indicating O +a O +conserved B-protein_state +similarity O +. O + +This O +aspartate B-residue_name +residue O +is O +identified O +in O +NsrR B-protein +as O +Asp99 B-residue_name_number +( O +see O +above O +). O + +Arginine B-residue_name_number +121 I-residue_name_number +of O +NsrR B-protein +points O +towards O +this O +Asp99 B-residue_name_number +residue O +however O +, O +the O +distance O +for O +a O +salt B-bond_interaction +bridge I-bond_interaction +interaction O +is O +too O +large O +. O + +Nonetheless O +, O +the O +structures B-evidence +of O +NsrR B-protein +- O +RD B-structure_element +and O +NsrR B-protein +- O +ED B-structure_element +together O +provide O +the O +required O +structural O +knowledge O +to O +predict O +the O +linker B-structure_element +region I-structure_element +that O +joins O +the O +receiver B-structure_element +and I-structure_element +effector I-structure_element +domains I-structure_element +. O + +The O +linker B-structure_element +region I-structure_element +of O +NsrR B-protein +consists O +of O +approximately O +nine O +residues O +( O +Fig O +3 O +), O +comprising O +120RRSQQFIQQ128 B-structure_element +( O +underlined O +residues O +are O +neither O +present O +in O +the O +structure B-evidence +of O +RD B-structure_element +nor O +in O +ED B-structure_element +of O +NsrR B-protein +) O +and O +contains O +two O +positively O +charged O +amino O +acids O +. O + +To O +achieve O +this O +, O +we O +first O +carefully O +analyzed O +the O +outcome O +of O +the O +Dali B-experimental_method +search I-experimental_method +for O +each O +domain O +and O +identified O +structurally O +highly O +similar O +proteins O +( O +based O +on O +Z B-evidence +- I-evidence +scores I-evidence +and O +rmsd B-evidence +values O +) O +and O +choose O +the O +full B-protein_state +- I-protein_state +length I-protein_state +structures B-evidence +previously O +reported O +. O + +In O +solution O +, O +RRs B-protein_type +exist O +in O +equilibrium O +between O +the O +active B-protein_state +and O +inactive B-protein_state +state O +, O +which O +is O +shifted O +towards O +the O +active B-protein_state +state O +upon O +phosphorylation B-ptm +of O +the O +ED B-structure_element +. O + +This O +results O +in O +oligomerization O +of O +the O +RR B-protein_type +and O +a O +higher O +affinity O +towards O +DNA B-chemical +. O + +Based O +on O +the O +above O +- O +mentioned O +criteria O +, O +the O +structure B-evidence +of O +MtrA B-protein +from O +M B-species +. I-species +tuberculosis I-species +, O +crystallized B-experimental_method +in O +an O +inactive B-protein_state +and O +non B-protein_state +- I-protein_state +phosphorylated I-protein_state +state O +, O +seemed O +best O +suited O +for O +modeling O +purposes O +. O + +Furthermore O +, O +the O +linker B-structure_element +between O +the O +two O +domains O +of O +MtrA B-protein +contains O +nine O +amino O +acids O +and O +is O +of O +similar O +length O +as O +the O +linker B-structure_element +of O +NsrR B-protein +. O +We O +aligned B-experimental_method +the O +NsrR B-protein +- O +RD B-structure_element +and O +- O +ED B-structure_element +to O +the O +corresponding O +MtrA B-protein +domains O +and O +evaluated O +the O +structure B-evidence +. O + +The O +missing B-protein_state +linker B-structure_element +is O +represented O +by O +a O +dotted O +line O +. O + +( O +b O +) O +Active B-protein_state +state O +conformation O +: O +A O +model O +of O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +in O +active B-protein_state +conformation O +based O +on O +the O +alignment B-experimental_method +of O +both O +the O +domains O +of O +NsrR B-protein +to O +the O +structure B-evidence +of O +DNA B-protein_state +bound I-protein_state +structure B-evidence +of O +KdpE B-protein +( O +PDB O +code O +: O +4KNY O +), O +adopting O +an O +active B-protein_state +open B-protein_state +conformation O +, O +where O +the O +other O +molecule O +of O +NsrR B-protein +is O +shown O +in O +shades O +of O +blue O +with O +the O +recognition B-structure_element +helix I-structure_element +colored O +in O +green O +. O + +In O +MtrA B-protein +, O +the O +two O +domains O +interact O +via O +the O +α4 B-site +- I-site +β5 I-site +- I-site +α5 I-site +interface I-site +of O +the O +receiver B-structure_element +domain I-structure_element +and O +the O +end O +of O +α7 B-structure_element +, O +α7 B-structure_element +- I-structure_element +α8 I-structure_element +loop I-structure_element +and O +α8 B-structure_element +of O +the O +effector B-structure_element +domain I-structure_element +. O + +Both O +interfaces B-site +have O +been O +shown O +to O +form O +functionally O +important O +contact O +areas O +in O +the O +active B-protein_state +state O +within O +members O +of O +the O +OmpR B-protein_type +/ I-protein_type +PhoB I-protein_type +subfamily I-protein_type +. O + +In O +our O +model O +of O +full B-protein_state +- I-protein_state +length I-protein_state +NsrR B-protein +, O +a O +similar O +orientation O +between O +the O +domains O +is O +observed O +, O +contributing O +to O +the O +inter O +- O +domain O +interactions O +. O + +The O +inactive B-protein_state +conformation O +of O +MtrA B-protein +is O +supported O +by O +the O +orientation O +of O +the O +side O +chain O +of O +Tyr102 B-residue_name_number +, O +which O +points O +away O +from O +the O +active B-protein_state +Asp56 B-residue_name_number +residue O +, O +while O +the O +side O +chain O +of O +Tyr102 B-residue_name_number +interacts O +with O +Asp190 B-residue_name_number +of O +the O +RD B-structure_element +of O +MtrA B-protein +, O +thereby O +stabilizing O +its O +closed B-protein_state +conformation O +. O + +In O +the O +model O +of O +NsrR B-protein +, O +similar O +amino O +acids O +are O +present O +, O +Phe101 B-residue_name_number +( O +switch B-site +residue I-site +) O +and O +Asp188 B-residue_name_number +( O +Fig O +3 O +, O +represented O +by O +orange O +boxes O +) O +forming O +a O +likewise O +similar O +network O +of O +interaction O +. O + +Next O +, O +we O +were O +interested O +in O +the O +active B-protein_state +conformation O +of O +the O +NsrR B-protein +protein O +adopting O +an O +active B-protein_state +“ O +open B-protein_state +” O +conformation O +in O +the O +dimeric B-oligomeric_state +state O +. O + +We O +compared B-experimental_method +and I-experimental_method +aligned I-experimental_method +the O +NsrR B-protein +- O +RD B-structure_element +and O +ED B-structure_element +on O +the O +dimeric B-oligomeric_state +structure B-evidence +of O +KdpE B-protein +that O +was O +solved B-experimental_method +in O +the O +DNA B-protein_state +- I-protein_state +bound I-protein_state +state O +( O +Fig O +7B O +). O + +Also O +the O +linker B-structure_element +region I-structure_element +of O +KdpE B-protein +is O +of O +similar O +length O +as O +of O +NsrR B-protein +, O +which O +suggests O +that O +the O +distance O +in O +the O +DNA B-protein_state +- I-protein_state +bound I-protein_state +state O +between O +the O +RD B-structure_element +and O +ED B-structure_element +of O +NsrR B-protein +will O +be O +similar O +to O +that O +in O +the O +KdpE B-protein +active B-protein_state +dimer B-oligomeric_state +. O + +We O +superimposed B-experimental_method +the O +ED B-structure_element +of O +NsrR B-protein +with O +the O +DNA B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +of O +KdpE B-protein +resulting O +in O +a O +reasonably O +well O +- O +aligned O +structure B-evidence +( O +rmsd B-evidence +of O +2 O +. O +6Å O +over O +86 O +Cα O +atoms O +; O +Table O +2 O +). O + +As O +a O +result O +, O +a O +highly B-site +positive I-site +groove I-site +is O +created O +by O +the O +two O +ED B-structure_element +domains O +of O +NsrR B-protein +which O +likely O +represents O +the O +DNA B-site +binding I-site +site I-site +as O +observed O +in O +KdpE B-protein +. O +A O +prediction O +of O +the O +putative O +promoter O +sequence O +that O +NsrR B-protein +binds O +via O +the O +BPROM O +online O +server O +was O +performed O +( O +S3 O +Fig O +). O + +A O +promoter O +region O +was O +identified O +upstream O +of O +the O +nsr B-gene +operon O +. O + +However O +, O +the O +regulation O +of O +the O +predicted O +promoter O +and O +the O +DNA B-chemical +binding O +by O +NsrR B-protein +has O +to O +be O +confirmed O +. O + +The O +structure B-evidence +of O +the O +response B-protein_type +regulator I-protein_type +NsrR B-protein +from O +S B-species +. I-species +agalactiae I-species +presented O +in O +this O +study O +is O +the O +first O +structural O +information O +available O +for O +the O +subgroup O +of O +lantibiotic B-protein_type +resistance I-protein_type +- I-protein_type +associated I-protein_type +RRs I-protein_type +. O + +Visualizing O +chaperone B-protein_type +- O +assisted O +protein O +folding O + +Challenges O +in O +determining O +the O +structures B-evidence +of O +heterogeneous O +and O +dynamic O +protein O +complexes O +have O +greatly O +hampered O +past O +efforts O +to O +obtain O +a O +mechanistic O +understanding O +of O +many O +important O +biological O +processes O +. O + +One O +such O +process O +is O +chaperone B-protein_type +- O +assisted O +protein O +folding O +, O +where O +obtaining O +structural O +ensembles O +of O +chaperone B-protein_type +: O +substrate O +complexes O +would O +ultimately O +reveal O +how O +chaperones B-protein_type +help O +proteins O +fold O +into O +their O +native O +state O +. O + +To O +address O +this O +problem O +, O +we O +devised O +a O +novel O +structural O +biology O +approach O +based O +on O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +, O +termed O +Residual B-experimental_method +Electron I-experimental_method +and I-experimental_method +Anomalous I-experimental_method +Density I-experimental_method +( O +READ B-experimental_method +). O + +The O +ensemble O +shows O +that O +Spy B-protein_state +- I-protein_state +associated I-protein_state +Im7 B-protein +samples O +conformations O +ranging O +from O +unfolded B-protein_state +to O +partially O +folded B-protein_state +and O +native B-protein_state +- O +like O +states O +, O +and O +reveals O +how O +a O +substrate O +can O +explore O +its O +folding O +landscape O +while O +bound B-protein_state +to I-protein_state +a O +chaperone B-protein_type +. O + +High O +- O +resolution O +structural B-evidence +models I-evidence +of O +protein O +- O +protein O +interactions O +are O +critical O +for O +obtaining O +mechanistic O +insights O +into O +biological O +processes O +. O + +However O +, O +many O +protein O +- O +protein O +interactions O +are O +highly B-protein_state +dynamic I-protein_state +, O +making O +it O +difficult O +to O +obtain O +high O +- O +resolution O +data O +. O + +Recent O +advances O +in O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +and O +NMR B-experimental_method +spectroscopy I-experimental_method +continue O +to O +improve O +our O +ability O +to O +analyze O +biomolecules O +that O +exist O +in O +multiple O +conformations O +. O + +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +has O +historically O +provided O +valuable O +information O +on O +small O +- O +scale O +conformational O +changes O +, O +but O +observing O +large O +- O +amplitude O +heterogeneous O +conformational O +changes O +often O +falls O +beyond O +the O +reach O +of O +current O +crystallographic O +techniques O +. O + +It O +is O +clear O +that O +molecular O +chaperones B-protein_type +aid O +in O +protein O +folding O +. O + +Structural B-evidence +models I-evidence +of O +chaperone B-protein_type +- O +substrate O +complexes O +have O +recently O +begun O +to O +provide O +information O +as O +to O +how O +a O +chaperone B-protein_type +can O +recognize O +its O +substrate O +. O + +For O +most O +chaperones B-protein_type +, O +it O +is O +still O +unclear O +whether O +the O +chaperone B-protein_type +actively O +participates O +in O +and O +affects O +the O +folding O +of O +the O +substrate O +proteins O +, O +or O +merely O +provides O +a O +suitable O +microenvironment O +enabling O +the O +substrate O +to O +fold O +on O +its O +own O +. O + +This O +is O +a O +truly O +fundamental O +question O +in O +the O +chaperone B-protein_type +field O +, O +and O +one O +that O +has O +eluded O +the O +community O +largely O +because O +of O +the O +highly B-protein_state +dynamic I-protein_state +nature O +of O +the O +chaperone B-protein_type +- O +substrate O +complexes O +. O + +The O +crystal B-evidence +structure I-evidence +of O +Spy B-protein +revealed O +that O +it O +forms O +a O +thin O +α O +- O +helical O +homodimeric B-oligomeric_state +cradle B-site +. O + +Crosslinking B-experimental_method +and I-experimental_method +genetic I-experimental_method +experiments I-experimental_method +suggested O +that O +Spy B-protein +interacts O +with O +substrates O +somewhere O +on O +its O +concave O +side O +. O + +By O +using O +a O +novel O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +- O +based O +approach O +to O +model O +disorder O +in O +crystal B-evidence +structures I-evidence +, O +we O +have O +now O +determined O +the O +high O +- O +resolution O +ensemble B-evidence +of O +the O +dynamic B-protein_state +Spy B-complex_assembly +: I-complex_assembly +Im7 I-complex_assembly +complex O +. O + +This O +work O +provides O +a O +detailed O +view O +of O +chaperone B-protein_type +- O +mediated O +protein O +folding O +and O +shows O +how O +substrates O +like O +Im7 B-protein +find O +their O +native O +fold O +while O +bound B-protein_state +to I-protein_state +their O +chaperones B-protein_type +. O + +Crystallizing B-experimental_method +the O +Spy B-complex_assembly +: I-complex_assembly +Im7 I-complex_assembly +complex O + +We O +reasoned O +that O +to O +obtain O +crystals B-evidence +of O +complexes O +between O +Spy B-protein +( O +domain O +boundaries O +in O +Supplementary O +Fig O +. O +1 O +) O +and O +its O +substrate O +proteins O +, O +our O +best O +approach O +was O +to O +identify O +crystallization B-experimental_method +conditions I-experimental_method +that O +yielded O +Spy B-protein +crystals B-evidence +in O +the O +presence B-protein_state +of I-protein_state +protein O +substrates O +but O +not O +in O +their O +absence B-protein_state +. O + +We O +therefore O +screened B-experimental_method +crystallization B-experimental_method +conditions I-experimental_method +for O +Spy B-protein +with O +four O +different O +substrate O +proteins O +: O +a O +fragment O +of O +the O +largely O +unfolded B-protein_state +bovine B-taxonomy_domain +α B-chemical +- I-chemical +casein I-chemical +protein O +, O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +E B-species +. I-species +coli I-species +Im7 B-protein +, O +an O +unfolded B-protein_state +variant O +of O +Im7 B-protein +( O +L18A B-mutant +L19A B-mutant +L37A B-mutant +), O +and O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +Im7 B-protein +( O +Im76 B-mutant +- I-mutant +45 I-mutant +), O +which O +encompasses O +the O +entire O +Spy B-structure_element +- I-structure_element +binding I-structure_element +portion I-structure_element +of O +Im7 B-protein +. O + +We O +found O +conditions O +in O +which O +all O +four O +substrates O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +with B-protein_state +Spy B-protein +, O +but O +in O +which O +Spy B-protein +alone B-protein_state +did O +not O +yield O +crystals B-evidence +. O + +Subsequent O +crystal B-experimental_method +washing I-experimental_method +and I-experimental_method +dissolution I-experimental_method +experiments O +confirmed O +the O +presence O +of O +the O +substrates O +in O +the O +co B-experimental_method +- I-experimental_method +crystals I-experimental_method +( O +Supplementary O +Fig O +. O +2 O +). O + +The O +crystals B-evidence +diffracted O +to O +~ O +1 O +. O +8 O +Å O +resolution O +. O + +However O +, O +modeling O +of O +the O +substrate O +in O +the O +complex O +proved O +to O +be O +a O +substantial O +challenge O +, O +as O +the O +electron B-evidence +density I-evidence +of O +the O +substrate O +was O +discontinuous O +and O +fragmented O +. O + +Even O +the O +minimal B-structure_element +binding I-structure_element +portion I-structure_element +of O +Im7 B-protein +( O +Im76 B-mutant +- I-mutant +45 I-mutant +) O +showed O +highly O +dispersed O +electron B-evidence +density I-evidence +( O +Fig O +. O +1a O +). O + +We O +hypothesized O +that O +the O +fragmented O +density B-evidence +was O +due O +to O +multiple O +, O +partially O +occupied O +conformations O +of O +the O +substrate O +bound O +within O +the O +crystal B-evidence +. O + +Thus O +, O +we O +developed O +a O +new O +approach O +to O +interpret O +the O +chaperone B-protein_state +- I-protein_state +bound I-protein_state +substrate O +in O +multiple O +conformations O +. O + +READ B-experimental_method +: O +a O +strategy O +to O +visualize O +heterogeneous O +and O +dynamic O +biomolecules O + +( O +2 O +) O +We O +then O +labeled O +individual O +residues O +in O +the O +flexible B-protein_state +regions O +of O +the O +substrate O +with O +the O +strong O +anomalous O +scatterer O +iodine B-chemical +, O +which O +serves O +to O +locate O +these O +residues O +in O +three O +- O +dimensional O +space O +using O +their O +anomalous B-evidence +density I-evidence +. O + +( O +3 O +) O +We O +performed O +molecular B-experimental_method +dynamics I-experimental_method +( O +MD B-experimental_method +) O +simulations B-experimental_method +to O +generate O +a O +pool O +of O +energetically O +reasonable O +conformations O +of O +the O +dynamic B-protein_state +complex O +and O +( O +4 O +) O +applied O +a O +sample B-experimental_method +- I-experimental_method +and I-experimental_method +- I-experimental_method +select I-experimental_method +algorithm I-experimental_method +to O +determine O +the O +minimal O +set O +of O +substrate O +conformations O +that O +fit O +both O +the O +residual B-evidence +and I-evidence +anomalous I-evidence +density I-evidence +. O + +Importantly O +, O +even O +though O +we O +only O +labeled O +a O +subset O +of O +the O +residues O +in O +the O +flexible B-protein_state +regions O +of O +the O +substrate O +with O +iodine B-chemical +, O +the O +residual B-evidence +electron I-evidence +density I-evidence +can O +provide O +spatial O +information O +on O +many O +of O +the O +other O +flexible B-protein_state +residues O +. O + +The O +electron B-evidence +density I-evidence +then O +allowed O +us O +to O +connect O +the O +labeled O +residues O +of O +the O +substrate O +by O +confining O +the O +protein O +chain O +within O +regions O +of O +detectable O +density B-evidence +. O + +In O +this O +way O +, O +the O +two O +forms O +of O +data O +together O +were O +able O +to O +describe O +multiple O +conformations O +of O +the O +substrate O +within O +the O +crystal B-evidence +. O + +As O +described O +in O +detail O +below O +, O +we O +developed O +the O +READ B-experimental_method +method O +to O +uncover O +the O +ensemble O +of O +conformations O +that O +the O +Spy B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +of O +Im7 B-protein +( O +i O +. O +e O +., O +Im76 B-mutant +- I-mutant +45 I-mutant +) O +adopts O +while O +bound B-protein_state +to I-protein_state +Spy B-protein +. O + +However O +, O +we O +believe O +that O +READ B-experimental_method +will O +prove O +generally O +applicable O +to O +visualizing O +heterogeneous O +and O +dynamic O +complexes O +that O +have O +previously O +escaped O +detailed O +structural O +analysis O +. O + +Collecting O +READ B-experimental_method +data O +for O +the O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +complex O + +To O +apply O +the O +READ B-experimental_method +technique I-experimental_method +to O +the O +folding O +mechanism O +employed O +by O +the O +chaperone B-protein_type +Spy B-protein +, O +we O +selected O +Im76 B-mutant +- I-mutant +45 I-mutant +for O +further O +investigation O +because O +NMR B-experimental_method +data O +suggested O +that O +Im76 B-mutant +- I-mutant +45 I-mutant +could O +recapitulate O +unfolded B-protein_state +, O +partially O +folded B-protein_state +, O +and O +native O +- O +like O +states O +of O +Im7 B-protein +( O +Supplementary O +Fig O +. O +3 O +). O + +To O +introduce O +the O +anomalous O +scatterer O +iodine B-chemical +, O +we O +replaced B-experimental_method +eight O +Im76 B-mutant +- I-mutant +45 I-mutant +residues O +with O +the O +non O +- O +canonical O +amino O +acid O +4 B-chemical +- I-chemical +iodophenylalanine I-chemical +( O +pI B-chemical +- I-chemical +Phe I-chemical +). O + +Its O +strong O +anomalous B-evidence +scattering I-evidence +allowed O +us O +to O +track O +the O +positions O +of O +these O +individual O +Im76 B-mutant +- I-mutant +45 I-mutant +residues O +one O +at O +a O +time O +, O +potentially O +even O +if O +the O +residue O +was O +found O +in O +several O +locations O +in O +the O +same O +crystal B-evidence +. O + +Consistent O +with O +our O +electron B-evidence +density I-evidence +map I-evidence +, O +we O +found O +that O +the O +majority O +of O +anomalous B-evidence +signals I-evidence +emerged O +in O +the O +cradle B-site +of O +Spy B-protein +, O +implying O +that O +this O +is O +the O +likely O +Im7 B-protein +substrate B-site +binding I-site +site I-site +. O + +Together O +, O +these O +results O +indicated O +that O +the O +Im7 B-protein +substrate O +binds O +Spy B-protein +in O +multiple O +conformations O +. O + +READ B-experimental_method +sample B-experimental_method +- I-experimental_method +and I-experimental_method +- I-experimental_method +select I-experimental_method +procedure O + +To O +determine O +the O +structural O +ensemble O +that O +Im76 B-mutant +- I-mutant +45 I-mutant +adopts O +while O +bound B-protein_state +to I-protein_state +Spy B-protein +, O +we O +combined O +the O +residual B-evidence +electron I-evidence +density I-evidence +and O +the O +anomalous B-evidence +signals I-evidence +from O +our O +pI B-chemical +- I-chemical +Phe I-chemical +substituted O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +complexes O +. O + +To O +generate O +an O +accurate O +depiction O +of O +the O +chaperone B-protein_type +- O +substrate O +interactions O +, O +we O +devised O +a O +selection O +protocol O +based O +on O +a O +sample B-experimental_method +- I-experimental_method +and I-experimental_method +- I-experimental_method +select I-experimental_method +procedure O +employed O +in O +NMR B-experimental_method +spectroscopy I-experimental_method +. O + +If O +successful O +, O +the O +selection O +identifies O +the O +smallest O +group O +of O +specific O +conformations O +that O +best O +fits O +the O +residual B-evidence +electron I-evidence +density I-evidence +and O +anomalous B-evidence +signals I-evidence +. O + +The O +READ B-experimental_method +sample B-experimental_method +- I-experimental_method +and I-experimental_method +- I-experimental_method +select I-experimental_method +algorithm I-experimental_method +is O +diagrammed O +in O +Fig O +. O +2 O +. O + +Prior O +to O +performing O +the O +selection O +, O +we O +generated O +a O +large O +and O +diverse O +pool O +of O +chaperone B-protein_type +- O +substrate O +complexes O +using O +coarse B-experimental_method +- I-experimental_method +grained I-experimental_method +MD I-experimental_method +simulations I-experimental_method +in O +a O +pseudo B-experimental_method +- I-experimental_method +crystal I-experimental_method +environment I-experimental_method +( O +Fig O +. O +2 O +and O +Supplementary O +Fig O +. O +4 O +). O + +The O +coarse B-experimental_method +- I-experimental_method +grained I-experimental_method +simulations I-experimental_method +are O +based O +on O +a O +single O +- O +residue O +resolution O +model O +for O +protein O +folding O +and O +were O +extended O +here O +to O +describe O +Spy B-complex_assembly +- I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +binding O +events O +( O +Online O +Methods O +). O + +The O +initial O +conditions O +of O +the O +binding B-experimental_method +simulations I-experimental_method +are O +not O +biased O +toward O +a O +particular O +conformation O +of O +the O +substrate O +or O +any O +specific O +chaperone B-protein_type +- O +substrate O +interaction O +( O +Online O +Methods O +). O + +Im76 B-mutant +- I-mutant +45 I-mutant +binds O +and O +unbinds O +to O +Spy B-protein +throughout O +the O +simulations B-experimental_method +. O + +From O +the O +MD B-experimental_method +simulations B-experimental_method +, O +we O +extracted O +~ O +10 O +, O +000 O +diverse O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +complexes O +to O +be O +used O +by O +the O +ensuing O +selection O +. O + +Each O +complex O +within O +this O +pool O +comprises O +one O +Spy B-protein +dimer B-oligomeric_state +bound B-protein_state +to I-protein_state +a O +single O +Im76 B-mutant +- I-mutant +45 I-mutant +substrate O +. O + +This O +pool O +was O +then O +used O +by O +the O +selection O +algorithm O +to O +identify O +the O +minimal O +ensemble O +that O +best O +satisfies O +both O +the O +residual B-evidence +electron I-evidence +and I-evidence +anomalous I-evidence +crystallographic I-evidence +data I-evidence +. O + +The O +anomalous B-evidence +scattering I-evidence +portion O +of O +the O +selection O +uses O +our O +basic O +knowledge O +of O +pI B-chemical +- I-chemical +Phe I-chemical +geometry O +: O +the O +iodine B-chemical +is O +separated O +from O +its O +respective O +Cα O +atom O +in O +each O +coarse O +- O +grained O +conformer O +by O +6 O +. O +5 O +Å O +. O +The O +selection O +then O +picks O +ensembles O +that O +best O +reproduce O +the O +collection O +of O +iodine B-chemical +anomalous B-evidence +signals I-evidence +. O + +To O +make O +the O +electron B-experimental_method +density I-experimental_method +selection I-experimental_method +practical O +, O +we O +needed O +to O +develop O +a O +method O +to O +rapidly O +evaluate O +the O +agreement O +between O +the O +selected O +sub O +- O +ensembles O +and O +the O +experimental O +electron B-evidence +density I-evidence +on O +- O +the O +- O +fly O +during O +the O +selection O +procedure O +. O + +To O +accomplish O +this O +task O +, O +we O +generated O +a O +compressed O +version O +of O +the O +experimental O +2mFo B-evidence +− I-evidence +DFc I-evidence +electron I-evidence +density I-evidence +map I-evidence +for O +use O +in O +the O +selection O +. O + +This O +process O +provided O +us O +with O +a O +target O +map B-evidence +that O +the O +ensuing O +selection O +tried O +to O +recapitulate O +. O + +To O +reduce O +the O +extent O +of O +3D O +space O +to O +be O +explored O +, O +this O +compressed O +map B-evidence +was O +created O +by O +only O +using O +density B-evidence +from O +regions O +of O +space O +significantly O +sampled O +by O +Im76 B-mutant +- I-mutant +45 I-mutant +in O +the O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +MD B-experimental_method +simulations B-experimental_method +. O + +For O +each O +of O +the O +~ O +10 O +, O +000 O +complexes O +in O +the O +coarse B-experimental_method +- I-experimental_method +grained I-experimental_method +MD B-experimental_method +pool O +, O +the O +electron B-evidence +density I-evidence +at O +the O +Cα O +positions O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +was O +extracted O +and O +used O +to O +construct O +an O +electron B-evidence +density I-evidence +map I-evidence +( O +Online O +Methods O +). O + +These O +individual O +electron B-evidence +density I-evidence +maps I-evidence +from O +the O +separate O +conformers O +could O +then O +be O +combined O +( O +Fig O +. O +2 O +) O +and O +compared O +to O +the O +averaged O +experimental O +electron B-evidence +density I-evidence +map I-evidence +as O +part O +of O +the O +selection O +algorithm O +. O + +This O +approach O +allowed O +us O +to O +simultaneously O +use O +both O +the O +iodine B-chemical +anomalous B-evidence +signals I-evidence +and O +the O +residual B-evidence +electron I-evidence +density I-evidence +in O +the O +selection O +procedure O +. O + +The O +selection O +resulted O +in O +small O +ensembles O +from O +the O +MD B-experimental_method +pool O +that O +best O +fit O +the O +READ B-experimental_method +data O +( O +Fig O +. O +1c O +, O +d O +). O + +Before O +analyzing O +the O +details O +of O +the O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +complex O +, O +we O +first O +engaged O +in O +a O +series O +of O +validation O +tests O +to O +verify O +the O +ensemble O +and O +selection O +procedure O +( O +Supplementary O +Note O +1 O +, O +Figures O +1c O +, O +d O +, O +Supplemental O +Figures O +5 O +- O +7 O +). O + +Of O +note O +, O +the O +final O +six O +- O +membered O +ensemble O +was O +the O +largest O +ensemble O +that O +could O +simultaneously O +decrease O +the O +RFree B-evidence +and O +pass O +the O +10 B-experimental_method +- I-experimental_method +fold I-experimental_method +cross I-experimental_method +- I-experimental_method +validation I-experimental_method +test I-experimental_method +. O + +This O +ensemble O +depicts O +the O +conformations O +that O +the O +substrate O +Im76 B-mutant +- I-mutant +45 I-mutant +adopts O +while O +bound B-protein_state +to I-protein_state +the O +chaperone B-protein_type +Spy B-protein +( O +Fig O +. O +3 O +Supplementary O +Movie O +1 O +, O +and O +Table O +1 O +). O + +Folding O +and O +interactions O +of O +Im7 B-protein +while O +bound B-protein_state +to I-protein_state +Spy B-protein + +Our O +results O +showed O +that O +by O +using O +this O +novel O +READ B-experimental_method +approach O +, O +we O +were O +able O +to O +obtain O +structural O +information O +about O +the O +dynamic O +interaction O +of O +a O +chaperone B-protein_type +with O +its O +substrate O +protein O +. O + +We O +were O +particularly O +interested O +in O +finding O +answers O +to O +one O +of O +the O +most O +fundamental O +questions O +in O +chaperone B-protein_type +biology O +— O +how O +does O +chaperone B-protein_type +binding O +affect O +substrate O +structure O +and O +vice O +versa O +. O + +We O +found O +these O +conformations O +to O +be O +highly O +heterogeneous O +and O +to O +include O +unfolded B-protein_state +, O +partially B-protein_state +folded I-protein_state +, O +and O +native B-protein_state +- I-protein_state +like I-protein_state +states O +( O +Fig O +. O +3 O +). O + +We O +constructed O +a O +contact B-evidence +map I-evidence +of O +the O +complex O +, O +which O +shows O +the O +frequency O +of O +interactions O +for O +chaperone B-protein_type +- O +substrate O +residue O +pairs O +( O +Fig O +. O +4 O +). O + +We O +found O +that O +the O +primary O +interaction B-site +sites I-site +on O +Spy B-protein +reside O +at O +the O +N O +and O +C O +termini O +( O +Arg122 B-residue_name_number +, O +Thr124 B-residue_name_number +, O +and O +Phe29 B-residue_name_number +) O +as O +well O +as O +on O +the O +concave O +face O +of O +the O +chaperone B-protein_type +( O +Arg61 B-residue_name_number +, O +Arg43 B-residue_name_number +, O +Lys47 B-residue_name_number +, O +His96 B-residue_name_number +, O +and O +Met46 B-residue_name_number +). O + +With O +respect O +to O +the O +substrate O +, O +we O +observed O +that O +nearly O +every O +residue O +in O +Im76 B-mutant +- I-mutant +45 I-mutant +is O +in O +contact O +with O +Spy B-protein +( O +Fig O +. O +4a O +). O + +However O +, O +we O +did O +notice O +that O +despite O +this O +uniformity O +, O +regions O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +preferentially O +interact O +with O +different O +regions O +in O +Spy B-protein +( O +Fig O +. O +4b O +). O + +Not O +unexpectedly O +, O +we O +found O +that O +as O +Im76 B-mutant +- I-mutant +45 I-mutant +progresses O +from O +the O +unfolded B-protein_state +to O +the O +native B-protein_state +state O +, O +its O +interactions O +with O +Spy B-protein +shift O +accordingly O +. O + +This O +shift O +in O +contacts O +is O +likely O +due O +to O +hydrophobic O +residues O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +preferentially O +forming O +intra O +- O +molecular O +contacts O +upon O +folding O +( O +i O +. O +e O +., O +hydrophobic O +collapse O +), O +effectively O +removing O +themselves O +from O +the O +interaction B-site +sites I-site +. O + +The O +diversity O +of O +conformations O +and O +binding B-site +sites I-site +observed O +here O +emphasizes O +the O +dynamic O +and O +heterogeneous O +nature O +of O +the O +chaperone B-protein_type +- O +substrate O +ensemble O +. O + +Comparing O +the O +structure B-evidence +of O +Spy B-protein +in O +its O +substrate B-protein_state +- I-protein_state +bound I-protein_state +and O +apo B-protein_state +states O +revealed O +that O +the O +Spy B-protein +dimer B-oligomeric_state +also O +undergoes O +significant O +conformational O +changes O +upon O +substrate O +binding O +( O +Fig O +. O +5a O +and O +Supplementary O +Movie O +2 O +). O + +Upon O +substrate O +binding O +, O +the O +Spy B-protein +dimer B-oligomeric_state +twists O +9 O +° O +about O +its O +center O +relative O +to O +its O +apo B-protein_state +form O +. O + +This O +twist O +yields O +asymmetry O +and O +results O +in O +substantially O +different O +interaction O +patterns O +in O +the O +two O +Spy B-protein +monomers B-oligomeric_state +( O +Fig O +. O +4b O +). O + +It O +is O +possible O +that O +this O +twist O +serves O +to O +increase O +heterogeneity O +in O +Spy B-protein +by O +providing O +more O +binding O +poses O +. O + +This O +increased O +disorder O +might O +explain O +how O +Spy B-protein +is O +able O +to O +recognize O +and O +bind O +different O +substrates O +and O +/ O +or O +differing O +conformations O +of O +the O +same O +substrate O +. O + +Importantly O +, O +we O +observed O +the O +same O +structural O +changes O +in O +Spy B-protein +regardless O +of O +which O +of O +the O +four O +substrates O +was O +bound O +( O +Fig O +. O +5b O +, O +Table O +1 O +). O + +The O +RMSD B-evidence +between O +the O +well B-protein_state +- I-protein_state +folded I-protein_state +sections O +of O +Spy B-protein +in O +the O +four O +chaperone B-protein_type +- O +substrate O +complexes O +was O +very O +small O +, O +less O +than O +0 O +. O +3 O +Å O +. O +Combined O +with O +competition B-experimental_method +experiments I-experimental_method +showing O +that O +the O +substrates O +compete O +in O +solution O +for O +Spy B-protein +binding O +( O +Fig O +. O +5c O +and O +Supplementary O +Fig O +. O +8 O +), O +we O +conclude O +that O +all O +the O +tested O +substrates O +share O +the O +same O +overall O +Spy B-site +binding I-site +site I-site +. O + +To O +shed O +light O +on O +how O +chaperones B-protein_type +interact O +with O +their O +substrates O +, O +we O +developed O +a O +novel O +structural O +biology O +method O +( O +READ B-experimental_method +) O +and O +applied O +it O +to O +determine O +a O +conformational B-evidence +ensemble I-evidence +of O +the O +chaperone B-protein_type +Spy B-protein +bound B-protein_state +to I-protein_state +substrate I-protein_state +. O + +As O +a O +substrate O +, O +we O +used O +Im76 B-mutant +- I-mutant +45 I-mutant +, O +the O +chaperone B-structure_element +- I-structure_element +interacting I-structure_element +portion I-structure_element +of O +the O +protein O +- O +folding O +model O +protein O +Im7 B-protein +. O + +This O +substrate O +- O +chaperone B-protein_type +ensemble O +helps O +accomplish O +the O +longstanding O +goal O +of O +obtaining O +a O +detailed O +view O +of O +how O +a O +chaperone B-protein_type +aids O +protein O +folding O +. O + +We O +recently O +showed O +that O +Im7 B-protein +can O +fold O +while O +remaining O +continuously B-protein_state +bound I-protein_state +to I-protein_state +Spy B-protein +. O + +The O +structures B-evidence +of O +our O +ensemble B-evidence +agree O +well O +with O +lower O +- O +resolution O +crosslinking O +data O +, O +which O +indicate O +that O +chaperone B-protein_type +- O +substrate O +interactions O +primarily O +occur O +on O +the O +concave B-site +surface I-site +of O +Spy B-protein +. O + +This O +model O +is O +consistent O +with O +previous O +studies O +postulating O +that O +the O +flexible O +binding O +of O +chaperones B-protein_type +allows O +for O +substrate O +protein O +folding O +. O + +The O +amphipathic O +concave B-site +surface I-site +of O +Spy B-protein +likely O +facilitates O +this O +flexible O +binding O +and O +may O +be O +a O +crucial O +feature O +for O +Spy B-protein +and O +potentially O +other O +chaperones B-protein_type +, O +allowing O +them O +to O +bind O +multiple O +conformations O +of O +many O +different O +substrates O +. O + +In O +contrast O +to O +Spy B-protein +’ O +s O +binding B-site +hotspots I-site +, O +Im76 B-mutant +- I-mutant +45 I-mutant +displays O +substantially O +less O +specificity O +in O +its O +binding B-site +sites I-site +. O + +Nearly O +all O +Im76 B-mutant +- I-mutant +45 I-mutant +residues O +come O +in O +contact O +with O +Spy B-protein +. O + +Unfolded B-protein_state +substrate O +conformers O +interact O +with O +Spy B-protein +through O +both O +hydrophobic B-bond_interaction +and I-bond_interaction +hydrophilic I-bond_interaction +interactions I-bond_interaction +, O +whereas O +the O +binding O +of O +native B-protein_state +- I-protein_state +like I-protein_state +states O +is O +mainly O +hydrophilic O +. O + +This O +trend O +suggests O +that O +complex O +formation O +between O +an O +ATP B-protein_state +- I-protein_state +independent I-protein_state +chaperone B-protein_type +and O +its O +unfolded B-protein_state +substrate O +may O +initially O +involve O +hydrophobic B-bond_interaction +interactions I-bond_interaction +, O +effectively O +shielding O +the O +exposed O +aggregation O +- O +sensitive O +hydrophobic B-site +regions I-site +in O +the O +substrate O +. O + +Once O +the O +substrate O +begins O +to O +fold O +within O +this O +protected O +environment O +, O +it O +progressively O +buries O +its O +own O +hydrophobic O +residues O +, O +and O +its O +interactions O +with O +the O +chaperone B-protein_type +shift O +towards O +becoming O +more O +electrostatic O +. O + +Notably O +, O +the O +most O +frequent O +contacts O +between O +Spy B-protein +and O +Im76 B-mutant +- I-mutant +45 I-mutant +are O +charge B-bond_interaction +- I-bond_interaction +charge I-bond_interaction +interactions I-bond_interaction +. O + +The O +negatively O +charged O +Im7 B-protein +residues O +Glu21 B-residue_name_number +, O +Asp32 B-residue_name_number +, O +and O +Asp35 B-residue_name_number +reside O +on O +the O +surface O +of O +Im7 B-protein +and O +form O +interactions O +with O +Spy B-protein +’ O +s O +positively O +charged O +cradle B-site +in O +both O +the O +unfolded B-protein_state +and O +native B-protein_state +- I-protein_state +like I-protein_state +states O +. O + +This O +proximity O +likely O +causes O +electrostatic O +repulsion O +that O +destabilizes O +Im7 B-protein +’ O +s O +native B-protein_state +state O +. O + +Interaction O +with O +Spy B-protein +’ O +s O +positively O +- O +charged O +residues O +likely O +relieves O +the O +charge O +repulsion O +between O +Asp32 B-residue_name_number +and O +Asp35 B-residue_name_number +, O +promoting O +their O +compaction O +into O +a O +helical B-protein_state +conformation I-protein_state +. O + +Recently O +, O +we O +employed O +a O +genetic B-experimental_method +selection I-experimental_method +system I-experimental_method +to O +improve O +the O +chaperone B-protein_type +activity O +of O +Spy B-protein +. O + +This O +selection O +resulted O +in O +“ O +Super O +Spy B-protein +” O +variants B-protein_state +that O +were O +more O +effective O +at O +both O +preventing O +aggregation O +and O +promoting O +protein O +folding O +. O + +In O +conjunction O +with O +our O +bound B-protein_state +Im76 B-mutant +- I-mutant +45 I-mutant +ensemble B-evidence +, O +these O +mutants O +now O +allowed O +us O +to O +investigate O +structural O +features O +important O +to O +chaperone B-protein_type +function O +. O + +Previous O +analysis O +revealed O +that O +the O +Super O +Spy B-protein +variants B-protein_state +either O +bound B-protein_state +Im7 B-protein +tighter O +than O +WT B-protein_state +Spy B-protein +, O +increased O +chaperone B-protein_type +flexibility O +as O +measured O +via O +H B-experimental_method +/ I-experimental_method +D I-experimental_method +exchange I-experimental_method +, O +or O +both O +. O + +Our O +ensemble B-evidence +revealed O +that O +two O +of O +the O +Super O +Spy B-protein +mutations B-protein_state +( O +H96L B-mutant +and O +Q100L B-mutant +) O +form O +part O +of O +the O +chaperone B-site +contact I-site +surface I-site +that O +binds O +to O +Im76 B-mutant +- I-mutant +45 I-mutant +( O +Fig O +. O +4a O +). O + +Moreover O +, O +our O +co B-evidence +- I-evidence +structure I-evidence +suggests O +that O +the O +L32P B-mutant +substitution O +, O +which O +increases O +Spy B-protein +’ O +s O +flexibility O +, O +could O +operate O +by O +unhinging O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +helix I-structure_element +and O +effectively O +expanding O +the O +size O +of O +the O +disordered B-protein_state +linker B-structure_element +. O + +This O +possibility O +is O +supported O +by O +the O +Spy B-protein +: O +substrate O +structures B-evidence +, O +in O +which O +the O +linker B-structure_element +region I-structure_element +becomes O +more O +flexible O +compared O +to O +the O +apo B-protein_state +state O +( O +Fig O +. O +6a O +). O + +By O +sampling O +multiple O +conformations O +, O +this O +linker B-structure_element +region I-structure_element +may O +allow O +diverse O +substrate O +conformations O +to O +be O +accommodated O +. O + +Other O +Super O +Spy B-protein +mutations B-protein_state +( O +F115I B-mutant +and O +F115L B-mutant +) O +caused O +increased O +flexibility O +but O +not O +tighter O +substrate O +binding O +. O + +This O +residue O +does O +not O +directly O +contact O +Im76 B-mutant +- I-mutant +45 I-mutant +in O +our O +READ B-experimental_method +- O +derived O +ensemble B-evidence +. O + +Instead O +, O +when O +Spy B-protein +is O +bound B-protein_state +to I-protein_state +substrate O +, O +F115 B-residue_name_number +engages O +in O +close O +CH O +⋯ O +π O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +Tyr104 B-residue_name_number +( O +Fig O +. O +6b O +). O + +Overall O +, O +comparison O +of O +our O +ensemble B-evidence +to O +the O +Super O +Spy B-protein +variants B-protein_state +provides O +specific O +examples O +to O +corroborate O +the O +importance O +of O +conformational O +flexibility O +in O +chaperone B-protein_type +- O +substrate O +interactions O +. O + +Despite O +extensive O +studies O +, O +exactly O +how O +complex O +chaperone B-protein_type +machines O +help O +proteins O +fold O +remains O +controversial O +. O + +We O +speculate O +that O +many O +other O +chaperones B-protein_type +could O +utilize O +a O +similar O +strategy O +. O + +ATP B-chemical +and O +co O +- O +chaperone B-protein_type +dependencies O +may O +have O +emerged O +later O +through O +evolution O +to O +better O +modulate O +and O +control O +chaperone B-protein_type +action O +. O + +In O +addition O +to O +insights O +into O +chaperone B-protein_type +function O +, O +this O +work O +presents O +a O +new O +method O +for O +determining O +heterogeneous O +structural O +ensembles O +via O +a O +hybrid O +methodology O +of O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +and O +computational B-experimental_method +modeling I-experimental_method +. O + +Heterogeneous O +dynamic O +complexes O +or O +disordered B-protein_state +regions O +of O +single O +proteins O +, O +once O +considered O +solely O +approachable O +by O +NMR B-experimental_method +spectroscopy I-experimental_method +, O +can O +now O +be O +visualized O +through O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +. O + +This O +is O +the O +residual O +density B-evidence +that O +is O +used O +in O +the O +READ B-experimental_method +selection O +. O + +Multiple O +iodine B-chemical +positions O +were O +detected O +for O +most O +residues O +. O + +Agreement O +to O +the O +residual O +Im76 B-mutant +- I-mutant +45 I-mutant +electron B-evidence +density I-evidence +( O +c O +) O +and O +anomalous B-evidence +iodine I-evidence +signals I-evidence +( O +d O +) O +for O +ensembles O +of O +varying O +size O +generated O +by O +randomly O +choosing O +from O +the O +MD B-experimental_method +pool O +( O +blue O +) O +and O +from O +the O +selection O +procedure O +( O +black O +). O + +The O +cost B-evidence +function I-evidence +, O +χ2 B-evidence +, O +decreases O +as O +the O +agreement O +to O +the O +experimental O +data O +increases O +and O +is O +defined O +in O +the O +Online O +Methods O +. O + +Flowchart O +of O +the O +READ B-experimental_method +sample B-experimental_method +- I-experimental_method +and I-experimental_method +- I-experimental_method +select I-experimental_method +process O +. O + +Spy B-protein +is O +depicted O +as O +a O +gray O +surface O +and O +the O +Im76 B-mutant +- I-mutant +45 I-mutant +conformer O +is O +shown O +as O +orange O +balls O +. O + +Atoms O +that O +were O +either O +not O +directly O +selected O +in O +the O +READ B-experimental_method +procedure O +, O +or O +whose O +position O +could O +not O +be O +justified O +based O +on O +agreement O +with O +the O +residual B-evidence +electron I-evidence +density I-evidence +were O +removed O +, O +leading O +to O +non O +- O +contiguous O +sections O +. O + +Residues O +of O +the O +Spy B-protein +flexible O +linker B-structure_element +region I-structure_element +that O +fit O +the O +residual B-evidence +electron I-evidence +density I-evidence +are O +shown O +as O +larger O +gray O +spheres O +. O + +Shown O +below O +each O +ensemble O +member O +is O +the O +RMSD B-evidence +of O +each O +conformer O +to O +the O +native B-protein_state +state O +of O +Im76 B-mutant +- I-mutant +45 I-mutant +, O +as O +well O +as O +the O +percentage O +of O +contacts O +between O +Im76 B-mutant +- I-mutant +45 I-mutant +and O +Spy B-protein +that O +are O +hydrophobic O +. O + +Contact B-evidence +maps I-evidence +of O +Spy B-complex_assembly +: I-complex_assembly +Im76 I-complex_assembly +- I-complex_assembly +45 I-complex_assembly +complex O +. O + +Contacts O +to O +the O +two O +Spy B-protein +monomers B-oligomeric_state +are O +depicted O +separately O +. O + +Note O +that O +the O +flexible B-protein_state +linker B-structure_element +region I-structure_element +of O +Spy B-protein +( O +residues O +47 B-residue_range +– I-residue_range +57 I-residue_range +) O +is O +not O +represented O +in O +the O +2D O +contact B-evidence +maps I-evidence +. O + +Spy B-protein +conformation O +changes O +upon O +substrate O +binding O +. O + +The O +Super O +Spy B-protein +mutants O +F115L B-mutant +, O +F115I B-mutant +, O +and O +L32P B-mutant +are O +proposed O +to O +gain O +activity O +by O +increasing O +the O +flexibility O +or O +size O +of O +this O +linker B-structure_element +region I-structure_element +. O + +Tdp2 B-protein +deficiencies O +are O +linked O +to O +neurological O +disease O +and O +cellular O +sensitivity O +to O +Top2 B-protein_type +poisons O +. O + +Modeling O +of O +a O +proposed O +Tdp2 B-protein +reaction O +coordinate O +, O +combined O +with O +mutagenesis B-experimental_method +and O +biochemical B-experimental_method +studies I-experimental_method +support O +a O +single O +Mg2 B-chemical ++- I-chemical +ion O +mechanism O +assisted O +by O +a O +phosphotyrosyl B-site +- I-site +arginine I-site +cation I-site +- I-site +π I-site +interface I-site +. O + +We O +further O +identify O +a O +Tdp2 B-protein +active B-site +site I-site +SNP O +that O +ablates B-protein_state +Tdp2 B-protein +Mg2 B-chemical ++ I-chemical +binding O +and O +catalytic O +activity O +, O +impairs O +Tdp2 B-protein +mediated O +NHEJ O +of O +tyrosine B-residue_name +blocked O +termini O +, O +and O +renders O +cells O +sensitive O +to O +the O +anticancer O +agent O +etoposide B-chemical +. O + +Collectively O +, O +our O +results O +provide O +a O +structural O +mechanism O +for O +Tdp2 B-protein +engagement O +of O +heterogeneous O +DNA B-chemical +damage O +that O +causes O +Top2 B-protein_type +poisoning O +, O +and O +indicate O +that O +evaluation O +of O +Tdp2 B-protein +status O +may O +be O +an O +important O +personalized O +medicine O +biomarker O +informing O +on O +individual O +sensitivities O +to O +chemotherapeutic O +Top2 B-protein_type +poisons O +. O + +Topoisomerases B-protein_type +relieve O +topological O +DNA B-chemical +strain O +and O +entanglement O +to O +facilitate O +critical O +nuclear O +DNA B-chemical +transactions O +including O +DNA B-chemical +replication O +, O +transcription O +and O +cell O +division O +. O + +The O +mammalian B-taxonomy_domain +type B-protein_type +II I-protein_type +topoisomerases I-protein_type +Top2α B-protein +and O +Top2β B-protein +enzymes O +generate O +transient O +, O +reversible O +DNA B-chemical +double O +strand O +breaks O +( O +DSBs O +) O +to O +drive O +topological O +transactions O +. O + +Reversibility O +of O +Top2 B-protein_type +DNA B-chemical +cleavage O +reactions O +is O +facilitated O +by O +formation O +of O +covalent O +enzyme O +phosphotyrosyl B-ptm +linkages I-ptm +between O +the O +5 B-chemical +′- I-chemical +phosphate I-chemical +ends O +of O +the O +incised O +duplex O +and O +an O +active B-site +site I-site +Top2 B-protein_type +tyrosine B-residue_name +, O +resulting O +in O +Top2 B-protein_type +cleavage O +complexes O +( O +Top2cc B-complex_assembly +). O + +The O +Top2cc B-complex_assembly +protein O +– O +DNA B-chemical +adduct O +is O +a O +unique O +threat O +to O +genomic O +integrity O +which O +must O +be O +resolved O +to O +prevent O +catastrophic O +Top2cc B-complex_assembly +collisions O +with O +the O +cellular O +replication O +and O +transcription O +machineries O +. O + +Importantly O +, O +Top2 B-protein_type +is O +also O +poisoned O +when O +it O +engages O +abundant O +endogenous O +DNA B-chemical +damage O +not O +limited O +to O +but O +including O +ribonucleotides O +, O +abasic O +sites O +and O +alkylation O +damage O +such O +as O +exocyclic O +DNA B-chemical +adducts O +arising O +from O +bioactivation O +of O +the O +vinyl O +chloride O +carcinogen O +( O +Figure O +1A O +). O + +In O +the O +case O +of O +DNA B-chemical +damage O +- O +triggered O +Top2cc B-complex_assembly +, O +compound O +DNA B-chemical +lesions O +arise O +that O +consist O +of O +the O +instigating O +lesion O +, O +and O +a O +DNA B-chemical +DSB O +bearing O +a O +bulky O +terminal O +5 O +′- O +linked O +Top2 B-protein_type +DNA B-chemical +– O +protein O +crosslink O +. O + +Precisely O +how O +the O +cellular O +DNA B-chemical +repair O +machinery O +navigates O +these O +complex O +lesions O +is O +an O +important O +aspect O +of O +Top2cc B-complex_assembly +repair O +that O +has O +not O +yet O +been O +explored O +. O + +( O +A O +) O +Unrepaired O +DNA B-chemical +damage O +and O +repair O +intermediates O +such O +as O +bulky O +DNA B-chemical +adducts O +, O +ribonucleotides O +or O +abasic O +sites O +can O +poison O +Top2 B-protein_type +and O +trap O +Top2 B-protein_type +cleavage O +complex O +( O +Top2cc B-complex_assembly +), O +resulting O +in O +a O +DSB O +with O +a O +5 O +′– O +Top2 B-protein_type +protein O +adduct O +linked O +by O +a O +phosphotyrosine B-residue_name +bond O +. O + +PDB O +entry O +5HT2 O +is O +displayed O +, O +also O +see O +Table O +1 O +. O +( O +E O +) O +Structure B-evidence +of O +mTdp2cat B-structure_element +bound B-protein_state +to I-protein_state +5 B-chemical +′- I-chemical +phosphate I-chemical +DNA I-chemical +( O +product O +complex O +) O +containing O +THF B-chemical +( O +yellow O +). O + +PDB O +entry O +5INK O +is O +displayed O +, O +also O +see O +Table O +1 O +. O +( O +F O +) O +Structure B-evidence +of O +mTdp2cat B-structure_element +in O +the O +absence B-protein_state +of I-protein_state +DNA B-chemical +showing O +the O +extended B-protein_state +3 B-structure_element +- I-structure_element +helix I-structure_element +loop I-structure_element +( O +tan O +) O +open B-protein_state +- O +conformation O +of O +the O +DNA B-site +- I-site +binding I-site +grasp I-site +as O +seen O +in O +monomer B-oligomeric_state +E B-structure_element +of O +the O +apo B-protein_state +structure B-evidence +. O + +Tyrosyl B-protein +DNA I-protein +phosphodiesterase I-protein +2 I-protein +( O +Tdp2 B-protein +) O +directly O +hydrolyzes O +5 B-ptm +′- I-ptm +phosphotyrosyl I-ptm +( O +5 B-ptm +′- I-ptm +Y I-ptm +) O +linkages B-ptm +, O +and O +is O +a O +key O +modulator O +of O +cellular O +resistance O +to O +chemotherapeutic O +Top2 B-protein_type +poisons O +. O + +Tdp2 B-protein +knockdown B-experimental_method +sensitizes O +A549 O +lung O +cancer O +cells O +to O +etoposide B-chemical +, O +and O +increases O +formation O +of O +nuclear O +γH2AX O +foci O +, O +a O +marker O +of O +DSBs O +, O +underlining O +the O +importance O +of O +Tdp2 B-protein +in O +cellular O +Top2cc B-complex_assembly +repair O +. O + +Tdp2 B-protein +is O +overexpressed O +in O +lung O +cancers O +, O +is O +transcriptionally O +up O +- O +regulated O +in O +mutant B-protein_state +p53 B-protein +cells O +and O +mediates O +mutant B-protein_state +p53 B-protein +gain O +of O +function O +phenotypes O +, O +which O +can O +lead O +to O +acquisition O +of O +therapy O +resistance O +during O +cancer O +progression O +. O + +The O +importance O +of O +Tdp2 B-protein +in O +mediating O +topoisomerase B-protein_type +biology O +is O +further O +underlined O +by O +the O +facts O +that O +human B-species +TDP2 B-protein +inactivating O +mutations O +are O +found O +in O +individuals O +with O +intellectual O +disabilities O +, O +seizures O +and O +ataxia O +, O +and O +at O +the O +cellular O +level O +, O +loss B-protein_state +of I-protein_state +Tdp2 B-protein +inhibits O +Top2β B-protein +- O +dependent O +transcription O +. O + +It O +is O +possible O +that O +TDP2 B-protein +single O +nucleotide O +polymorphisms O +( O +SNPs O +) O +encode O +mutations O +that O +impact O +Tdp2 B-protein +function O +, O +but O +the O +molecular O +underpinnings O +for O +such O +Tdp2 B-protein +deficiencies O +are O +not O +understood O +. O + +Previously O +we O +reported O +high O +- O +resolution O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystal B-evidence +structures I-evidence +of O +the O +minimal B-protein_state +catalytically I-protein_state +active I-protein_state +endonuclease B-structure_element +/ I-structure_element +exonuclease I-structure_element +/ I-structure_element +phosphatase I-structure_element +( O +EEP B-structure_element +) O +domain O +of O +mouse B-taxonomy_domain +Tdp2 B-protein +( O +mTdp2cat B-structure_element +) O +bound B-protein_state +to I-protein_state +a O +DNA B-chemical +substrate O +mimic O +, O +and O +a O +5 B-protein_state +′- I-protein_state +phosphorylated I-protein_state +reaction O +product O +. O + +First O +, O +it O +is O +unclear O +if O +Tdp2 B-protein +processes O +phosphotyrosyl B-ptm +linkages I-ptm +in O +the O +context O +of O +DNA B-chemical +damage O +that O +triggers O +Top2cc B-complex_assembly +, O +and O +if O +so O +, O +how O +the O +enzyme O +can O +accommodate O +such O +complex O +DNA B-chemical +damage O +within O +its O +active B-site +site I-site +. O + +Based O +on O +metal B-protein_state +- I-protein_state +bound I-protein_state +Tdp2 B-protein +structures B-evidence +, O +we O +also O +proposed O +a O +single O +Mg2 B-chemical ++ I-chemical +mediated O +catalytic O +mechanism O +, O +but O +this O +mechanism O +requires O +further O +scrutiny O +and O +characterization O +. O + +Herein O +, O +we O +report O +an O +integrated O +structure B-experimental_method +- I-experimental_method +function I-experimental_method +study I-experimental_method +of O +the O +Tdp2 B-protein +reaction O +mechanism O +, O +including O +a O +description O +of O +new O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +structures B-evidence +of O +ligand B-protein_state +- I-protein_state +free I-protein_state +Tdp2 B-protein +, O +and O +Tdp2 B-protein +bound B-protein_state +to I-protein_state +abasic O +and O +alkylated O +( O +1 B-chemical +- I-chemical +N6 I-chemical +- I-chemical +etheno I-chemical +- I-chemical +adenine I-chemical +) O +DNA B-chemical +damage O +. O + +We O +further O +establish O +that O +DNA B-chemical +damage O +binding O +in O +the O +Tdp2 B-protein +active B-site +site I-site +is O +linked O +to O +conformational O +change O +and O +binding O +of O +metal O +cofactor O +. O + +Tdp2 B-protein +processing O +of O +compound O +DNA B-chemical +damage O + +Two O +potent O +Top2 B-protein_type +poisons O +include O +bulky O +alkylated O +DNA B-chemical +helix O +- O +distorting O +DNA B-chemical +base O +adducts O +( O +e O +. O +g O +. O +1 B-chemical +- I-chemical +N6 I-chemical +- I-chemical +ethenoadenine I-chemical +, O +ϵA B-chemical +) O +and O +abundant O +abasic O +sites O +( O +Figure O +1A O +). O + +Whether O +Tdp2 B-protein +processes O +phosphotyrosyl B-ptm +linkages I-ptm +within O +these O +diverse O +structural O +contexts O +is O +not O +known O +. O + +We O +then O +evaluated O +the O +ability O +of O +a O +recombinant O +purified O +mouse B-taxonomy_domain +Tdp2 B-protein +catalytic B-structure_element +domain I-structure_element +( O +mTdp2cat B-structure_element +) O +to O +release O +PNP B-chemical +( O +a O +structural O +mimic O +of O +a O +topoisomerase B-protein_type +tyrosine B-residue_name +) O +from O +the O +5 O +′- O +terminus O +of O +compound O +damaged O +DNA B-chemical +substrates O +using O +a O +colorimetric B-experimental_method +assay I-experimental_method +( O +Figure O +1B O +). O + +We O +observe O +robust O +Tdp2 B-protein +- O +dependent O +release O +of O +PNP B-chemical +from O +5 O +′- O +modified O +oligonucleotides O +in O +the O +context O +of O +dA B-chemical +- I-chemical +PNP I-chemical +, O +ϵA B-chemical +- I-chemical +PNP I-chemical +or O +the O +abasic O +- O +site O +analog O +tetrahydrofuran B-chemical +spacer I-chemical +( O +THF B-chemical +) O +( O +Figure O +1C O +). O + +Thus O +, O +Tdp2 B-protein +efficiently O +cleaves O +phosphotyrosyl B-ptm +linkages I-ptm +in O +the O +context O +of O +a O +compound O +5 O +′ O +lesions O +composed O +of O +abasic O +or O +bulky O +DNA B-chemical +base O +adduct O +DNA B-chemical +damage O +. O + +In O +these O +Tdp2 B-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +complex O +structures B-evidence +, O +mTdp2cat B-structure_element +adopts O +a O +mixed B-structure_element +α I-structure_element +- I-structure_element +β I-structure_element +fold I-structure_element +typified O +by O +a O +central O +12 B-structure_element +- I-structure_element +stranded I-structure_element +anti I-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +- I-structure_element +sandwich I-structure_element +enveloped O +by O +several O +helical O +elements O +that O +mold O +the O +Tdp2 B-protein +active B-site +site I-site +. O + +One O +half O +of O +the O +molecule O +contributes O +to O +formation O +of O +the O +walls O +of O +the O +DNA B-site +- I-site +binding I-site +cleft I-site +that O +embraces O +the O +terminal O +position O +of O +the O +damaged O +DNA B-chemical +substrate O +. O + +In O +the O +DNA B-protein_state +lesion I-protein_state +- I-protein_state +bound I-protein_state +state O +, O +two O +key O +DNA B-chemical +binding O +elements O +, O +the O +β B-structure_element +- I-structure_element +2 I-structure_element +- I-structure_element +helix I-structure_element +- I-structure_element +β I-structure_element +( O +β2Hβ B-structure_element +) O +‘ O +grasp B-structure_element +’, O +and O +‘ O +helical B-structure_element +cap I-structure_element +’ O +mold O +the O +substrate B-site +binding I-site +trench I-site +and O +direct O +the O +ssDNA B-chemical +of O +a O +5 O +′- O +overhang O +substrate O +into O +the O +active B-site +site I-site +. O + +A O +comparison O +to O +an O +additional O +new O +structure B-evidence +of O +DNA B-protein_state +- I-protein_state +free I-protein_state +Tdp2 B-protein +( O +apo B-protein_state +state O +, O +Figure O +1F O +) O +shows O +that O +this O +loop B-structure_element +is O +conformationally B-protein_state +mobile I-protein_state +and O +important O +for O +engaging O +DNA B-chemical +substrates O +. O + +The O +mode O +of O +engagement O +of O +the O +5 O +′- O +nucleobase O +of O +the O +bulky O +ϵA B-chemical +adduct O +describes O +a O +mechanism O +for O +Tdp2 B-protein +to O +bind O +5 B-protein_state +′- I-protein_state +tyrosylated I-protein_state +substrates O +that O +contain O +diverse O +forms O +of O +DNA B-chemical +damage O +. O + +The O +5 B-chemical +′- I-chemical +ϵA I-chemical +nucleobase O +is O +recognized O +by O +an O +extended O +Tdp2 B-protein +van B-site +Der I-site +Waals I-site +interaction I-site +surface I-site +, O +referred O +to O +here O +as O +the O +‘ O +hydrophobic B-site +wall I-site +’ O +that O +is O +assembled O +with O +the O +sidechains O +of O +residues O +Leu315 B-residue_name_number +and O +Ile317 B-residue_name_number +( O +Figure O +2A O +and O +B O +). O + +Structures B-evidence +of O +mTdp2cat B-structure_element +bound B-protein_state +to I-protein_state +DNA B-chemical +damage O +that O +triggers O +Top2 B-protein_type +poisoning O +. O + +mTdp2cat B-structure_element +is O +colored O +by O +electrostatic O +surface O +potential O +( O +red O += O +negative O +, O +blue O += O +positive O +, O +gray O += O +neutral O +/ O +hydrophobic O +). O + +( O +B O +) O +σ B-evidence +- I-evidence +A I-evidence +weighted I-evidence +2Fo I-evidence +- I-evidence +Fc I-evidence +electron I-evidence +density I-evidence +map I-evidence +( O +at O +1 O +. O +43 O +Å O +resolution O +, O +contoured O +at O +2 O +. O +0 O +σ O +) O +for O +the O +ϵA B-chemical +DNA I-chemical +complex O +. O + +The O +ϵA B-chemical +nucleotide O +is O +shown O +in O +yellow O +and O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +from O +the O +ϵA B-chemical +O4 O +′ O +to O +inner O +- O +sphere O +water B-chemical +is O +shown O +as O +gray O +dashes O +. O +( O +C O +) O +Structure B-evidence +of O +mTdp2cat B-structure_element +bound B-protein_state +to I-protein_state +5 B-chemical +′- I-chemical +phosphate I-chemical +DNA I-chemical +( O +product O +complex O +) O +containing O +THF B-chemical +( O +yellow O +), O +Mg2 B-chemical ++ I-chemical +( O +magenta O +) O +and O +its O +inner O +- O +sphere O +waters B-chemical +( O +gray O +). O + +PDB O +entry O +5INK O +is O +displayed O +. O +( O +D O +) O +σ B-evidence +- I-evidence +A I-evidence +weighted I-evidence +2Fo I-evidence +- I-evidence +Fc I-evidence +electron I-evidence +density I-evidence +map I-evidence +( O +at O +2 O +. O +15 O +Å O +resolution O +, O +contoured O +at O +2 O +. O +0 O +σ O +) O +for O +THF B-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +complex O +. O + +For O +comparison O +, O +we O +also O +determined B-experimental_method +a O +structure B-evidence +of O +an O +undamaged O +5 B-chemical +′- I-chemical +adenine I-chemical +( O +5 B-chemical +′- I-chemical +dA I-chemical +) O +bound B-protein_state +to I-protein_state +Tdp2 B-protein +at O +1 O +. O +55 O +Å O +( O +PDB O +entry O +5INL O +). O + +Therefore O +, O +structurally O +diverse O +undamaged O +or O +alkylated O +bases O +( O +e O +. O +g O +. O +ϵG B-chemical +, O +ϵT B-chemical +) O +could O +likely O +be O +accommodated O +in O +the O +Tdp2 B-protein +active B-site +site I-site +via O +planar B-bond_interaction +base I-bond_interaction +stacking I-bond_interaction +with O +the O +active B-site +site I-site +facing O +hydrophobic B-site +wall I-site +of O +the O +β2Hβ B-structure_element +motif O +. O + +Interestingly O +, O +in O +the O +absence B-protein_state +of I-protein_state +a O +nucleobase O +, O +O4 O +′ O +of O +the O +THF B-chemical +ring O +adopts O +a O +close O +approach O +( O +2 O +. O +8 O +Å O +) O +to O +a O +water B-chemical +molecule O +that O +directly O +participates O +in O +the O +outer O +sphere O +single O +Mg2 B-chemical ++ I-chemical +ion B-bond_interaction +coordination I-bond_interaction +shell I-bond_interaction +( O +Figure O +2D O +). O + +These O +collective O +differences O +may O +explain O +the O +slight O +, O +but O +statistically O +significant O +elevated O +activity O +on O +the O +THF B-chemical +substrate O +( O +Figure O +1C O +). O + +In O +this O +arrangement O +, O +six O +solvent O +molecules O +form O +a O +channel O +under O +the O +β2Hβ B-site +- I-site +grasp I-site +, O +ending O +with O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +peptide O +backbone O +of O +the O +Mg2 B-chemical ++ I-chemical +ligand O +Asp358 B-residue_name_number +. O + +To O +test O +this O +hypothesis O +, O +we O +crystallized B-experimental_method +Tdp2 B-protein +in O +the O +absence B-protein_state +of I-protein_state +DNA B-chemical +and O +determined O +a O +DNA B-protein_state +free I-protein_state +Tdp2 B-protein +structure B-evidence +to O +2 O +. O +4 O +Å O +resolution O +( O +PDB O +entry O +5INM O +; O +Figures O +1F O +and O +3A O +). O + +( O +A O +) O +The O +open B-protein_state +, O +3 B-structure_element +- I-structure_element +helix I-structure_element +conformation O +( O +tan O +) O +of O +flexible B-protein_state +active B-structure_element +- I-structure_element +site I-structure_element +loop I-structure_element +observed O +in O +monomer B-oligomeric_state +E B-structure_element +of O +the O +DNA B-protein_state +- I-protein_state +free I-protein_state +mTdp2cat B-structure_element +structure B-evidence +( O +PDB O +entry O +5INM O +) O +is O +supported O +by O +T309 B-residue_name_number +( O +green O +), O +which O +packs O +against O +the O +EEP B-structure_element +core O +. O + +mTdp2cat B-structure_element +WT B-protein_state +( O +lanes O +1 O +– O +13 O +) O +or O +mTdp2cat B-structure_element +D358N B-mutant +( O +lanes O +14 O +– O +26 O +) O +were O +incubated O +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +Mg2 B-chemical ++ I-chemical +and O +/ O +or O +a O +12 O +nt O +self O +annealing O +, O +5 O +′- O +phosphorylated O +DNA B-chemical +( O +substrate O +‘ O +12 O +nt O +’ O +in O +Supplementary O +Table O +S1 O +), O +then O +reacted O +with O +0 O +. O +6 O +, O +1 O +. O +7 O +or O +5 O +ng O +μl O +− O +1 O +of O +trypsin O +. O + +This O +crystal O +form O +contains O +5 O +Tdp2 B-protein +protein O +molecules O +in O +the O +asymmetric O +unit O +, O +with O +variations O +in O +active B-site +site I-site +Mg2 B-chemical ++ I-chemical +occupancy O +and O +substrate B-structure_element +binding I-structure_element +loops I-structure_element +observed O +for O +the O +individual O +protomers B-oligomeric_state +. O + +The O +most O +striking O +feature O +of O +the O +DNA B-protein_state +ligand I-protein_state +- I-protein_state +free I-protein_state +state O +is O +that O +the O +active B-site +site I-site +β2Hβ I-site +- I-site +grasp I-site +can O +adopt O +alternative O +structures O +that O +are O +distinct O +from O +the O +DNA B-protein_state +- I-protein_state +bound I-protein_state +, O +closed B-protein_state +β2Hβ B-site +DNA I-site +binding I-site +grasp I-site +( O +Figure O +3A O +and O +B O +). O + +In O +one O +monomer B-oligomeric_state +( O +chain B-structure_element +‘ I-structure_element +E I-structure_element +’), I-structure_element +the O +grasp B-structure_element +adopts O +an O +‘ O +open B-protein_state +’ O +3 B-structure_element +- I-structure_element +helix I-structure_element +loop I-structure_element +conformation O +that O +projects O +away O +from O +the O +EEP B-structure_element +catalytic B-site +core I-site +. O + +Two O +monomers B-oligomeric_state +have O +variable O +disordered B-protein_state +states O +for O +which O +much O +of O +the O +DNA B-structure_element +binding I-structure_element +loop I-structure_element +is O +not O +visible O +in O +the O +electron B-evidence +density I-evidence +. O + +The O +remaining O +two O +molecules O +in O +the O +DNA B-protein_state +- I-protein_state +free I-protein_state +crystal B-evidence +form I-evidence +are O +closed B-protein_state +β2Hβ B-structure_element +conformers O +similar O +to O +the O +DNA B-protein_state +bound I-protein_state +structures B-evidence +( O +Figure O +3C O +). O + +Thus O +, O +we O +posit O +that O +Tdp2 B-protein +DNA B-chemical +binding O +conformationally O +selects O +the O +closed B-protein_state +form O +of O +the O +β2Hβ B-site +grasp I-site +, O +rather O +than O +inducing O +closure O +upon O +binding O +. O + +A O +detailed O +analysis O +of O +the O +extended B-protein_state +3 B-structure_element +- I-structure_element +helix I-structure_element +conformation O +shows O +that O +the O +substrate B-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +is O +able O +to O +undergo O +metamorphic O +structural O +changes O +. O + +In O +this O +open B-protein_state +form O +, O +residues O +Asn312 B-residue_range +- I-residue_range +Leu315 I-residue_range +are O +distal O +from O +the O +active B-site +site I-site +and O +solvent B-protein_state +- I-protein_state +exposed I-protein_state +( O +orange O +sticks O +, O +Figure O +3A O +), O +while O +Thr309 B-residue_name_number +( O +green O +surface O +, O +Figure O +3A O +) O +packs O +into O +a O +shallow O +pocket B-site +of O +the O +EEP B-structure_element +core O +to O +anchor O +the O +loop B-structure_element +. O + +By O +comparison O +, O +the O +closed B-protein_state +β2Hβ B-site +grasp I-site +conformer O +is O +stabilized O +by O +Asn312 B-residue_name_number +and O +Asn314 B-residue_name_number +binding O +into O +two O +β2Hβ B-site +docking I-site +pockets I-site +, O +and O +Leu315 B-residue_name_number +engagement O +of O +the O +5 O +′- O +terminal O +nucleobase O +( O +Figure O +3B O +). O + +Stabilization O +of O +the O +closed B-protein_state +β2Hβ B-site +- I-site +grasp I-site +conformation O +is O +linked O +to O +the O +active B-site +site I-site +through O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +Trp307 B-residue_name_number +and O +the O +Mg2 B-site ++ I-site +coordinating I-site +residue I-site +Asp358 B-residue_name_number +. O + +To O +evaluate O +Mg2 B-chemical ++ I-chemical +and O +DNA B-chemical +- O +dependent O +Tdp2 B-protein +structural O +states O +in O +solution O +, O +we O +probed O +mTdp2cat B-structure_element +conformations O +using O +limited B-experimental_method +trypsin I-experimental_method +and I-experimental_method +chymotrypsin I-experimental_method +proteolysis I-experimental_method +( O +Figure O +3C O +– O +E O +). O + +In B-protein_state +the I-protein_state +absence I-protein_state +of I-protein_state +DNA B-chemical +or O +Mg2 B-chemical ++, I-chemical +mTdp2cat B-structure_element +is O +efficiently O +cleaved O +in O +the O +metamorphic O +DNA B-site +binding I-site +grasp I-site +at O +one O +site O +by O +trypsin B-experimental_method +( O +Arg316 B-residue_name_number +), O +or O +at O +two O +positions O +by O +chymotrypsin B-experimental_method +( O +Trp307 B-residue_name_number +and O +Leu315 B-residue_name_number +). O + +By O +comparison O +, O +Mg2 B-chemical ++, I-chemical +and O +to O +a O +greater O +extent O +Mg2 B-chemical ++/ I-chemical +DNA B-chemical +mixtures O +( O +compare O +Figure O +3 O +, O +lanes O +4 O +, O +7 O +and O +13 O +) O +protect O +mTdp2cat B-structure_element +from O +proteolytic O +cleavage O +. O + +Interestingly O +, O +addition O +of O +Mg2 B-chemical ++ I-chemical +alone O +protects O +against O +proteolysis O +as O +well O +. O + +This O +is O +consistent O +with O +Mg2 B-chemical ++ I-chemical +stabilizing O +the O +closed B-protein_state +conformation O +of O +the O +β2Hβ B-site +- I-site +grasp I-site +through O +an O +extended O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +network I-bond_interaction +with O +Asp358 B-residue_name_number +and O +the O +indole O +ring O +of O +the O +β2Hβ B-site +- I-site +grasp I-site +residue O +Trp307 B-residue_name_number +( O +also O +discussion O +below O +on O +Tdp2 B-protein +active B-site +site I-site +SNPs O +). O + +To O +assess O +structural O +conservation O +of O +Tdp2 B-protein +conformational O +changes O +between O +human B-species +and O +mouse B-taxonomy_domain +Tdp2 B-protein +, O +we O +also O +determined B-experimental_method +a O +3 O +. O +2 O +Å O +resolution O +structure B-evidence +of O +the O +human B-species +Tdp2cat B-structure_element +domain O +bound B-protein_state +to I-protein_state +a O +DNA B-chemical +5 I-chemical +′- I-chemical +PO4 I-chemical +terminus O +product O +complex O +( O +PDB O +entry O +5INO O +). O + +Comparisons O +of O +the O +human B-species +hTdp2cat B-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +complex O +structure B-evidence +to O +the O +mTdp2cat B-structure_element +DNA B-protein_state +bound I-protein_state +state O +show O +a O +high O +level O +of O +conservation O +of O +the O +DNA B-protein_state +- I-protein_state +bound I-protein_state +conformations O +( O +Supplementary O +Figure O +S3A O +). O + +Moreover O +, O +similar O +to O +mTdp2cat B-structure_element +, O +proteolytic O +protection O +of O +the O +hTdp2cat B-structure_element +substrate B-structure_element +binding I-structure_element +loop I-structure_element +occurs O +with O +addition O +of O +Mg2 B-chemical ++ I-chemical +and O +DNA B-chemical +( O +Supplementary O +Figure O +S3B O +). O + +Tdp2 B-protein +metal O +ion O +dependence O + +Consistently O +in O +high O +- O +resolution O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +structural I-experimental_method +analyses I-experimental_method +we O +, O +and O +others O +observe O +a O +single O +Mg2 B-chemical ++ I-chemical +metal O +bound B-protein_state +in I-protein_state +the O +Tdp2 B-protein +active B-site +site I-site +. O + +This O +includes O +the O +DNA B-protein_state +- I-protein_state +free I-protein_state +( O +Figure O +3A O +), O +DNA B-protein_state +damage I-protein_state +bound I-protein_state +( O +Figure O +3B O +) O +and O +reaction B-protein_state +product I-protein_state +- I-protein_state +bound I-protein_state +crystal B-evidence +forms I-evidence +of O +mouse B-taxonomy_domain +, O +( O +PDB O +entry O +4GZ1 O +), O +D B-species +. I-species +rerio I-species +( O +PDB O +entry O +4FPV O +) O +and O +C B-species +. I-species +elegans I-species +Tdp2 B-protein +( O +PDB O +entry O +4FVA O +). O + +In O +these O +experiments O +, O +at O +limiting O +Mg2 B-chemical ++ I-chemical +concentrations O +, O +Ca2 B-chemical ++ I-chemical +addition O +to O +Tdp2 B-protein +reactions O +stimulated O +activity O +. O + +In O +fact O +, O +divalent B-chemical +metals I-chemical +have O +been O +observed O +in O +the O +Tdp2 B-protein +protein O +– O +DNA B-chemical +complexes O +( O +PDB O +entry O +4GZ2 O +) O +distal O +to O +the O +active B-site +center I-site +, O +and O +we O +propose O +this O +might O +account O +for O +varied O +results O +in O +different O +studies O +. O + +To O +further O +probe O +the O +metal O +ion O +dependence O +of O +the O +Tdp2 B-protein +phosphodiesterase B-protein_type +reaction O +, O +we O +performed O +metal B-experimental_method +ion I-experimental_method +binding I-experimental_method +assays I-experimental_method +, O +determined O +crystal B-evidence +structures I-evidence +in O +the O +presence B-protein_state +of I-protein_state +varied O +divalent B-chemical +metals I-chemical +( O +Mn2 B-chemical ++ I-chemical +and O +Ca2 B-chemical ++), I-chemical +and O +analyzed O +metal O +ion O +dependence O +of O +the O +Tdp2 B-protein +phosphotyrosyl B-protein_type +phosphodiesterase I-protein_type +reaction O +( O +Figure O +4 O +). O + +Metal O +cofactor O +interactions O +with O +Tdp2 B-protein +. O +( O +A O +) O +Intrinsic B-evidence +tryptophan I-evidence +fluorescence I-evidence +of O +mTdp2cat B-structure_element +was O +used O +to O +monitor O +a O +conformational O +response O +to O +divalent O +metal O +ion O +binding O +. O + +Both O +Mg2 B-chemical ++ I-chemical +and O +Ca2 B-chemical ++ I-chemical +induce O +a O +conformational O +change O +which O +elicits O +an O +increase O +in O +tryptophan B-evidence +fluorescence I-evidence +of O +mTdp2cat B-structure_element +in O +the O +presence B-protein_state +and O +absence B-protein_state +of I-protein_state +DNA B-chemical +, O +while O +D358N B-mutant +active B-site +site I-site +mutant B-protein_state +of O +mTdp2cat B-structure_element +is O +unresponsive B-protein_state +to O +Mg2 B-chemical ++. I-chemical +( O +B O +) O +mTdp2cat B-structure_element +activity O +assayed O +on O +a O +T5PNP B-chemical +substrate O +as O +a O +function O +of O +Mg2 B-chemical ++ I-chemical +and O +Ca2 B-chemical ++ I-chemical +concentration O +. O + +A O +53σ O +peak O +in O +the O +anomalous B-evidence +difference I-evidence +Fourier I-evidence +map I-evidence +( O +data O +collected O +at O +λ O += O +1 O +. O +5418 O +Å O +) O +supports O +Mn2 B-chemical ++ I-chemical +as O +the O +identity O +of O +this O +atom O +. O + +( O +D O +) O +Comparison O +of O +Ca2 B-chemical ++ I-chemical +( O +green O +Ca2 B-chemical ++ I-chemical +ion O +, O +orange O +DNA B-chemical +) O +( O +PDB O +entry O +5INQ O +), O +and O +Mg2 B-chemical ++ I-chemical +( O +magenta O +Mg2 B-chemical ++ I-chemical +ion O +, O +yellow O +DNA B-chemical +) O +( O +PDB O +entry O +4GZ1 O +) O +mTdp2cat B-complex_assembly +– I-complex_assembly +DNA I-complex_assembly +structures B-evidence +shows O +that O +Ca2 B-chemical ++ I-chemical +distorts O +the O +5 B-site +′- I-site +phosphate I-site +binding I-site +mode I-site +. O + +Our O +proteolysis B-experimental_method +results O +indicate O +a O +Mg2 B-chemical ++- I-chemical +dependent O +Tdp2 B-protein +conformational O +response O +to O +metal O +binding O +. O + +The O +Tdp2 B-protein +active B-site +site I-site +has O +three O +tryptophan B-residue_name +residues O +within O +10 O +Å O +of O +the O +metal B-site +binding I-site +center I-site +, O +so O +we O +assayed O +intrinsic B-evidence +tryptophan I-evidence +fluorescence I-evidence +to O +detect O +metal O +- O +induced O +conformational O +changes O +in O +mTdp2cat B-structure_element +. O + +We O +then O +measured O +effects O +of O +metal O +ion O +concentrations O +on O +Tdp2 B-protein +cleavage O +of O +p B-chemical +- I-chemical +nitrophenyl I-chemical +- I-chemical +thymidine I-chemical +- I-chemical +5 I-chemical +′- I-chemical +phosphate I-chemical +by O +mTdp2cat B-structure_element +. O + +This O +small O +molecule O +substrate O +is O +not O +expected O +to O +be O +influenced O +by O +metal O +– O +DNA B-chemical +coordination O +outside O +of O +the O +active B-site +site I-site +. O + +Inclusion O +of O +ultrapure O +Ca2 B-chemical ++ I-chemical +( O +1 O +mM O +or O +10 O +mM O +) O +results O +in O +a O +dose O +- O +dependent O +inhibition O +but O +not O +stimulation O +Tdp2 B-protein +activity O +, O +even O +in O +conditions O +of O +limiting O +Mg2 B-chemical ++ I-chemical +( O +Figure O +4B O +). O + +We O +performed O +the O +same O +titrations B-experimental_method +with O +human B-species +hTdp2FL B-protein +and O +hTdp2cat B-structure_element +( O +Supplementary O +Figure O +S4 O +), O +and O +find O +similar O +stimulation O +of O +activity O +by O +Mg2 B-chemical ++ I-chemical +and O +inhibition O +by O +Ca2 B-chemical ++. I-chemical + +To O +further O +evaluate O +the O +structural O +influence O +of O +divalent O +cations O +on O +the O +Tdp2 B-protein +active B-site +site I-site +, O +we O +determined O +crystal B-evidence +structures I-evidence +by O +soaking B-experimental_method +crystals I-experimental_method +with O +metal O +cofactors O +that O +either O +support B-protein_state +( O +Mn2 B-chemical ++) I-chemical +or O +inhibit B-protein_state +( O +Ca2 B-chemical ++, I-chemical +Figure O +4B O +) O +the O +Tdp2 B-protein +reaction O +( O +PDB O +entries O +5INP O +and O +5INQ O +). O + +Anomalous B-evidence +difference I-evidence +Fourier I-evidence +maps I-evidence +of O +the O +Tdp2 B-complex_assembly +– I-complex_assembly +DNA I-complex_assembly +– I-complex_assembly +Mn2 I-complex_assembly ++ I-complex_assembly +complex O +show O +a O +single O +binding B-site +site I-site +for O +Mn2 B-chemical ++ I-chemical +in O +each O +Tdp2 B-protein +active B-site +site I-site +( O +Figure O +4C O +), O +with O +octahedral B-bond_interaction +coordination I-bond_interaction +and O +bond O +lengths O +typical O +for O +Mn2 B-chemical ++ I-chemical +ligands O +( O +Supplementary O +Table O +S3 O +). O + +The O +Mn2 B-chemical ++ I-chemical +ion O +is O +positioned O +in O +the O +Tdp2 B-protein +active B-site +site I-site +similar O +to O +the O +Mg2 B-protein_state ++- I-protein_state +bound I-protein_state +complex O +( O +Figure O +2C O +), O +which O +is O +consistent O +with O +the O +ability O +of O +Mn2 B-chemical ++ I-chemical +to O +support O +robust O +Tdp2 B-protein +catalytic O +activity O +. O + +Although O +Ca2 B-chemical ++ I-chemical +is O +also O +octahedrally B-bond_interaction +coordinated I-bond_interaction +, O +longer O +bond O +lengths O +for O +the O +Ca2 B-chemical ++ I-chemical +ligands O +( O +Supplementary O +Table O +S3 O +) O +shift O +the O +Ca2 B-chemical ++ I-chemical +ion O +relative O +to O +the O +Mg2 B-site ++ I-site +ion I-site +site I-site +. O + +Interestingly O +, O +bi B-bond_interaction +- I-bond_interaction +dentate I-bond_interaction +inner I-bond_interaction +sphere I-bond_interaction +metal I-bond_interaction +contacts I-bond_interaction +from O +the O +Ca2 B-chemical ++ I-chemical +ion O +to O +Glu162 B-residue_name_number +distort O +the O +active B-site +site I-site +phosphate I-site +- I-site +binding I-site +mode I-site +, O +and O +dislodge O +the O +5 B-chemical +′- I-chemical +PO4 I-chemical +out O +of O +the O +Tdp2 B-protein +active B-site +site I-site +( O +Figure O +4D O +). O + +Together O +with O +results O +showing O +that O +under O +the O +conditions O +examined O +here O +, O +Ca2 B-chemical ++ I-chemical +inhibits O +rather O +than O +stimulates O +the O +Tdp2 B-protein +reaction O +, O +the O +divalent B-protein_state +metal I-protein_state +bound I-protein_state +Tdp2 B-protein +structures B-evidence +provide O +a O +mechanism O +for O +Ca2 B-chemical ++- I-chemical +mediated O +inhibition O +of O +the O +Tdp2 B-protein +reaction O +. O + +Modeling O +the O +Tdp2 B-protein +reaction O +coordinate O + +Next O +, O +to O +examine O +the O +feasibility O +of O +our O +proposed O +single O +Mg2 B-chemical ++ I-chemical +mechanism O +, O +we O +simulated B-experimental_method +the O +Tdp2 B-protein +reaction O +coordinate O +with O +hybrid B-experimental_method +QM I-experimental_method +/ I-experimental_method +MM I-experimental_method +modeling I-experimental_method +using O +Tdp2 B-protein +substrate B-protein_state +analog I-protein_state +- I-protein_state +and O +product B-protein_state +- I-protein_state +bound I-protein_state +structures B-evidence +as O +guides O +. O + +Previous O +structural B-experimental_method +analyses I-experimental_method +showed O +that O +the O +superposition B-experimental_method +of O +a O +DNA B-chemical +substrate O +mimic O +( O +5 B-chemical +′- I-chemical +aminohexanol I-chemical +) O +and O +product O +( O +5 B-chemical +′- I-chemical +PO4 I-chemical +) O +complexes O +delineates O +a O +probable O +Tdp2 B-protein +reaction O +trajectory O +characterized O +by O +inversion O +of O +stereochemistry O +about O +the O +adducted O +5 O +′- O +phosphorus O +. O + +In O +this O +scheme O +( O +Figure O +5A O +), O +a O +candidate O +nucleophilic O +water B-chemical +that O +is O +strongly O +hydrogen B-bond_interaction +bonded I-bond_interaction +to O +Asp272 B-residue_name_number +and O +Asn274 B-residue_name_number +, O +is O +well O +positioned O +for O +the O +in O +- O +line O +nucleophilic O +attack O +∼ O +180 O +° O +opposite O +of O +the O +P O +– O +O O +bond O +of O +the O +5 O +′- O +Tyr O +adduct O +. O + +Structure O +- O +function O +analysis O +of O +the O +Tdp2 B-protein +reaction O +mechanism O +. O + +( O +A O +) O +Proposed O +mechanism O +for O +hydrolysis O +of O +phosphotyrosine B-residue_name +bond O +by O +Tdp2 B-protein +. O + +Residues O +in O +green O +form O +the O +binding B-site +- I-site +site I-site +for O +the O +5 B-residue_name +′- I-residue_name +tyrosine I-residue_name +( O +red O +) O +and O +phosphate B-chemical +, O +yellow O +bind O +the O +5 O +′ O +nucleotide O +and O +blue O +bind O +nucleotides O +2 O +– O +3 O +. O + +Residue O +numbers O +shown O +are O +for O +the O +mTdp2 B-protein +homolog O +. O +( O +B O +) O +Free B-evidence +energy I-evidence +during O +the O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +simulation I-experimental_method +as O +a O +function O +of O +distance O +between O +the O +nucleophilic O +water B-chemical +and O +5 O +′- O +phosphorus O +atom O +. O + +Reaction O +proceeds O +from O +right O +to O +left O +. O +( O +C O +) O +Models O +for O +the O +mTdp2cat B-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +complex O +during O +the O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +reaction I-experimental_method +path I-experimental_method +simulation I-experimental_method +showing O +the O +substrate O +( O +left O +, O +tan O +), O +transition O +state O +intermediate O +( O +center O +, O +cyan O +) O +and O +product O +( O +right O +, O +pink O +) O +states O +. O + +Electrostatic B-evidence +potential I-evidence +color O +gradient O +extends O +from O +positive O +( O +red O +) O +through O +neutral O +( O +gray O +), O +to O +negative O +( O +blue O +). O +( O +E O +) O +Bar O +graph O +displaying O +the O +relative O +activity O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +human B-species +MBP B-experimental_method +- O +hTdp2cat B-structure_element +fusion B-experimental_method +proteins I-experimental_method +on O +the O +three O +substrates O +. O + +Release O +of O +PNP B-chemical +from O +PNP B-chemical +phosphate B-chemical +and O +T5PNP B-chemical +was O +detected O +as O +an O +increase O +in O +absorbance O +at O +415 O +nm O +. O + +Reaction B-evidence +rates I-evidence +are O +expressed O +as O +the O +percent O +of O +activity O +relative O +to O +wildtype B-protein_state +MBP B-experimental_method +- O +hTdp2cat B-structure_element +; O +error O +bars O +, O +s O +. O +d O +. O + +Mutants O +of O +hTdp2 B-protein +( O +black O +) O +and O +the O +equivalent O +residue O +in O +mTdp2 B-protein +( O +tan O +) O +are O +indicated O +. O + +We O +examined O +the O +energy O +profile O +of O +the O +nucleophilic O +attack O +of O +the O +water B-chemical +molecule O +by O +using O +the O +distance O +between O +the O +water B-chemical +oxygen O +and O +the O +P O +atom O +on O +the O +phosphate O +moiety O +as O +the O +sole O +reaction O +coordinate O +in O +the O +present O +calculation O +( O +Figure O +5B O +and O +C O +). O + +A O +starting O +model O +was O +generated O +from O +atomic O +coordinates O +of O +the O +mTdp2cat B-structure_element +5 B-chemical +′– I-chemical +aminohexanol I-chemical +substrate O +analog O +structure B-evidence +( O +PDB O +4GZ0 O +) O +with O +a O +tyrosine B-residue_name +replacing O +the O +5 B-chemical +′- I-chemical +aminohexanol I-chemical +then O +adding O +the O +Mg2 B-chemical ++ I-chemical +and O +inner O +- O +sphere O +waters B-chemical +from O +the O +mTdp2 B-complex_assembly +- I-complex_assembly +DNA I-complex_assembly +product O +structure B-evidence +( O +PDB O +, O +4GZ1 O +), O +and O +running O +an O +initial O +round O +of O +molecular B-experimental_method +dynamics I-experimental_method +simulation I-experimental_method +( O +10 O +ns O +) O +to O +allow O +the O +system O +to O +reach O +an O +equilibrium O +. O + +After O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +optimization I-experimental_method +of O +this O +model O +( O +Figure O +5C O +, O +‘ O +i O +- O +substrate O +’), O +the O +O O +– O +P O +distance O +is O +3 O +. O +4 O +Å O +, O +which O +is O +in O +agreement O +with O +the O +range O +of O +distances O +observed O +in O +the O +mTdp2cat B-structure_element +5 B-chemical +′- I-chemical +aminohexanol I-chemical +substrate O +analog O +structure B-evidence +( O +3 O +. O +2 O +– O +3 O +. O +4 O +Å O +). O + +In O +the O +subsequent O +two O +steps O +of O +the O +simulation B-experimental_method +, O +as O +the O +water B-chemical +- O +phosphate O +O O +– O +P O +distance O +reduces O +to O +1 O +. O +98 O +Å O +, O +a O +key O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +the O +nucleophilic O +water B-chemical +and O +Asp272 B-residue_name_number +shortens O +to O +1 O +. O +38 O +Å O +as O +the O +water B-chemical +H O +– O +O O +bond O +approaches O +the O +point O +of O +dissociation O +. O + +The O +second O +proton O +on O +the O +water B-chemical +nucleophile O +maintains O +a O +strong O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +Asn274 B-residue_name_number +throughout O +the O +reaction O +, O +implicating O +this O +residue O +in O +orienting O +the O +water B-chemical +nucleophile O +during O +the O +reaction O +. O + +Concomitant O +with O +this O +, O +the O +phosphotyrosyl B-ptm +O O +– O +P O +bond O +weakens O +( O +d O += O +1 O +. O +89 O +Å O +), O +and O +the O +formation O +of O +the O +penta O +- O +covalent O +transition O +state O +( O +Figure O +5C O +‘ O +ii O +- O +transition O +state O +’) O +is O +observed O +. O + +The O +final O +steps O +show O +inversion O +of O +stereochemistry O +at O +the O +phosphate B-chemical +, O +along O +with O +lengthening O +and O +breaking O +of O +the O +phosphotyrosyl B-ptm +O O +– O +P O +bond O +. O + +Of O +note O +, O +both O +nitrogens O +of O +the O +imidazole O +side O +chain O +of O +His B-residue_name_number +359 I-residue_name_number +require O +protonation O +for O +stability O +of O +the O +simulation B-experimental_method +. O + +Asp B-residue_name_number +326 I-residue_name_number +makes O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +to O +N O +∂ O +1 O +of O +His359 B-residue_name_number +, O +suggesting O +that O +this O +salt B-bond_interaction +bridge I-bond_interaction +could O +stabilize O +the O +protonated B-protein_state +form O +of O +His359 B-residue_name_number +as O +has O +been O +demonstrated O +for O +the O +analogous O +Asp B-residue_name +- O +His B-residue_name +pair O +in O +the O +EEP B-structure_element +domain O +of O +APE1 B-protein +, O +which O +elevates O +the O +pKa B-evidence +of O +this O +His B-residue_name +above O +8 O +. O +0 O +. O +In O +our O +model O +, O +the O +transition O +state O +contains O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +the O +doubly B-protein_state +protonated I-protein_state +His359 B-residue_name_number +and O +the O +phosphate B-chemical +oxygen O +that O +also O +coordinates O +with O +the O +single O +catalytic O +Mg2 B-chemical ++, I-chemical +while O +the O +second O +His359 B-residue_name_number +imidazole O +proton O +maintains O +a O +H B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +with O +the O +Asp326 B-residue_name_number +residue O +throughout O +the O +reaction O +. O + +In O +the O +final O +optimized O +structure B-evidence +, O +the O +observed O +product O +state O +( O +Figure O +5C O +, O +‘ O +iii O +- O +product O +’) O +is O +found O +in O +a O +conformation O +that O +is O +7 O +. O +4 O +kcal O +mol O +− O +1 O +more O +stable O +than O +the O +initial O +reactive O +state O +( O +Figure O +5B O +). O + +The O +tyrosine B-residue_name +oxy O +- O +anion O +product O +is O +coordinated B-bond_interaction +to I-bond_interaction +the O +Mg2 B-chemical ++ I-chemical +ion O +with O +a O +2 O +. O +0 O +Å O +distance O +, O +which O +is O +the O +shortest O +of O +the O +six O +Mg2 B-chemical ++ I-chemical +ligands O +( O +including O +three O +water B-chemical +molecules O +, O +one O +of O +the O +free O +oxygens O +on O +the O +phosphate B-chemical +group O +and O +the O +Glu162 B-residue_name_number +residue O +), O +indicating O +the O +single O +Mg2 B-chemical ++ I-chemical +greatly O +stabilizes O +the O +product O +oxy O +- O +anion O +. O + +An O +additional O +striking O +feature O +gleaned O +from O +the O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +modeling I-experimental_method +is O +the O +putative O +binding O +mode O +of O +the O +Top2 B-protein_type +tyrosine B-residue_name +- O +leaving O +group O +. O + +A O +trio O +of O +conserved B-protein_state +residues O +( O +Tyr B-residue_name_number +188 I-residue_name_number +, O +Arg B-residue_name_number +216 I-residue_name_number +and O +Ser B-residue_name_number +239 I-residue_name_number +) O +forms O +the O +walls O +of O +a O +conserved B-protein_state +Top2 B-protein_type +tyrosine B-site +binding I-site +pocket I-site +. O + +We O +propose O +this O +cation B-bond_interaction +– I-bond_interaction +π I-bond_interaction +interaction I-bond_interaction +further O +contributes O +to O +tuned O +stabilization O +of O +the O +negatively O +charged O +phenolate O +reaction O +product O +. O + +Consistent O +with O +this O +, O +analysis O +of O +electrostatic B-evidence +potential I-evidence +of O +the O +phosphotyrosyl B-ptm +moiety O +using O +Gaussian O +09 O +. O +D01 O +in O +the O +presence B-protein_state +and O +absence B-protein_state +of I-protein_state +the O +Arg216 B-residue_name_number +guanidinium O +reveals O +Arg216 B-residue_name_number +is O +strongly O +electron O +withdrawing O +( O +Figure O +5D O +). O + +We O +further O +examined O +the O +contribution O +of O +this O +cation B-bond_interaction +– I-bond_interaction +π I-bond_interaction +interaction I-bond_interaction +to O +the O +reaction O +chemistry O +by O +moving O +the O +guanidinium O +group O +of O +Arg216 B-residue_name_number +from O +the O +QM B-experimental_method +system O +to O +the O +MM B-experimental_method +system O +as O +either O +a O ++ O +1 O +or O +∼ O +0 O +charge O +species O +, O +and O +re O +- O +computed O +energy B-evidence +penalties I-evidence +for O +each O +step O +in O +the O +reaction O +coordinate O +( O +Supplementary O +Figure O +S5A O +). O + +Removing O +Arg216 B-residue_name_number +from O +the O +quantum O +subsystem O +incurs O +an O +∼ O +2 O +kcal O +mol O +− O +1 O +penalty O +in O +the O +transition O +state O +and O +product O +complex O +. O + +Removing O +the O ++ O +1 O +charge O +on O +the O +Arg216 B-residue_name_number +has O +a O +minimal O +impact O +on O +the O +transition O +state O +, O +but O +incurs O +an O +additional O +∼ O +2 O +kcal O +mol O +− O +1 O +penalty O +in O +the O +product O +complex O +. O + +Tdp2 B-protein +mutational B-experimental_method +analysis I-experimental_method + +For O +example O +, O +mutations B-experimental_method +impacting O +Tdp2 B-protein +active B-site +site I-site +chemistry O +and O +phosphotyrosyl B-ptm +bond O +cleavage O +should O +similarly O +affect O +catalysis O +on O +all O +three O +substrates O +, O +but O +mutants O +impacting O +DNA B-chemical +damage O +binding O +might O +only O +impair O +catalysis O +on O +5 B-ptm +′- I-ptm +Y I-ptm +and O +T5PNP B-chemical +but O +not O +PNPP B-chemical +that O +lacks O +a O +nucleobase O +. O + +To O +test O +if O +this O +proposed O +Lewis O +base O +is O +critical O +for O +reaction O +chemistry O +we O +mutated B-experimental_method +it O +to B-experimental_method +a O +His B-residue_name +, O +which O +could O +alternatively O +support O +metal O +binding O +, O +as O +well O +as O +bulky O +hydrophobic O +residues O +( O +Leu B-residue_name +and O +Met B-residue_name +) O +that O +we O +predict O +would O +block O +the O +water B-site +- I-site +binding I-site +site I-site +. O + +Similar O +to O +a O +previously O +characterized O +hD262N B-mutant +mutation O +, O +all O +three O +substitutions B-experimental_method +ablate O +activity O +, O +supporting O +essential O +roles O +for O +hAsp262 B-residue_name_number +( O +mAsp272 B-residue_name_number +) O +in O +catalysis O +. O + +Next O +, O +we O +mutated B-experimental_method +key O +elements O +of O +the O +mobile O +loop O +( O +β2Hβ B-site +hydrophobic I-site +wall I-site +, O +Figure O +2A O +and O +C O +). O + +The O +hL305W B-mutant +substitution O +that O +we O +expect O +to O +have O +the O +most O +distorting O +impact O +on O +conformation O +of O +the O +β2Hβ B-site +hydrophobic I-site +wall I-site +also O +has O +the O +largest O +impact O +on O +catalysis O +of O +the O +DNA B-chemical +substrate O +5 B-ptm +′- I-ptm +Y I-ptm +. O +By O +comparison O +, O +as O +predicted O +by O +our O +model O +where O +β2Hβ B-structure_element +dictates O +key O +interactions O +with O +undamaged O +and O +damaged O +nucleobases O +, O +all O +of O +these O +substitutions B-experimental_method +have O +little O +impact O +on O +PNPP B-chemical +(> O +90 O +% O +activity O +). O + +Third O +, O +we O +altered O +properties O +of O +the O +proposed O +enzyme B-site +substrate I-site +cation I-site +– I-site +π I-site +interface I-site +. O + +No O +activity O +was O +detected O +for O +a O +mutant B-protein_state +that O +removes O +the O +positive O +charge O +at O +this O +position O +( O +hR206A B-mutant +). O + +The O +precise O +geometry O +of O +this O +pocket B-site +is O +also O +critical O +for O +catalysis O +as O +replacement B-experimental_method +of O +hArg206 B-residue_name_number +( O +mArg216 B-residue_name_number +) O +with O +a O +lysine B-residue_name +also O +results O +in O +a O +profound O +decrease O +in O +catalysis O +(< O +5 O +% O +activity O +on O +5 B-ptm +′- I-ptm +Y I-ptm +, O +no O +detectable O +activity O +on O +T5PNP B-chemical +or O +PNPP B-chemical +). O + +Similarly O +, O +mutation B-experimental_method +of O +hTyr178 B-residue_name_number +that O +structurally O +scaffolds O +the O +hArg206 B-residue_name_number +( O +mArg216 B-residue_name_number +) O +guanidinium O +also O +significantly O +impacts O +activity O +, O +with O +Y178F B-mutant +and O +Y178W B-mutant +having O +< O +25 O +% O +activity O +on O +all O +substrates O +. O + +Fourth O +, O +we O +evaluated O +roles O +for O +the O +hHis351 B-residue_name_number +– O +hAsp316 B-residue_name_number +( O +mAsp326 B-residue_name_number +– O +mHis359 B-residue_name_number +) O +transition O +state O +stabilization O +charge O +pair O +. O + +Thus O +altogether O +, O +our O +mutational O +data O +support O +key O +roles O +for O +the O +active B-site +site I-site +Lewis O +base O +aspartate B-residue_name +, O +mobile B-protein_state +substrate B-structure_element +engagement I-structure_element +loops I-structure_element +, O +enzyme O +– O +substrate O +cation B-bond_interaction +– I-bond_interaction +π I-bond_interaction +interactions I-bond_interaction +, O +and O +active B-site +site I-site +transition O +state O +stabilizing O +charge B-bond_interaction +interaction I-bond_interaction +in O +supporting O +Tdp2 B-protein +catalysis O +. O + +A O +Tdp2 B-protein +active B-site +site I-site +single O +nucleotide O +polymorphism O +impairs O +Tdp2 B-protein +function O + +Recently O +, O +it O +was O +found O +that O +inactivation O +of O +TDP2 B-protein +by O +a O +splice O +- O +site O +mutation O +is O +associated O +with O +neurological O +disease O +and O +confers O +hypersensitivity O +to O +Top2 B-protein_type +poisons O +. O + +We O +considered O +whether O +human B-species +SNPs O +causing O +missense O +mutations O +might O +also O +impact O +Tdp2 B-protein +DNA B-chemical +– O +protein O +crosslink O +repair O +functions O +established O +here O +as O +well O +as O +Tdp2 B-protein +- O +mediated O +NHEJ O +of O +blocked O +DNA B-chemical +termini O +. O + +We O +identified O +two O +SNPs O +in O +human B-species +TDP2 B-protein +curated O +in O +the O +NCBI O +SNP O +database O +that O +result O +in O +missense O +mutations O +within O +the O +DNA B-site +processing I-site +active I-site +site I-site +: O +rs199602263 B-gene +( O +minor O +allele O +frequency O +0 O +. O +0002 O +), O +which O +substitutes O +hAsp350 B-residue_name_number +for O +Asn B-residue_name +, O +and O +rs77273535 B-gene +( O +minor O +allele O +frequency O +0 O +. O +004 O +, O +which O +substitutes O +hIle307 B-residue_name_number +for O +Val B-residue_name +) O +( O +Figure O +6A O +). O + +We O +show O +the O +hD350N B-mutant +substitution B-experimental_method +severely O +impairs O +activity O +on O +all O +substrates O +tested O +in O +vitro O +, O +whereas O +hI307V B-mutant +only O +has O +a O +mild O +impact O +on O +catalysis O +( O +Figure O +6B O +– O +D O +). O + +To O +better O +understand O +the O +basis O +for O +the O +D350N B-mutant +catalytic O +defect O +, O +we O +analyzed O +the O +structural O +environment O +of O +this O +substitution O +based O +on O +the O +high O +- O +resolution O +structures B-evidence +of O +mTdp2cat B-structure_element +( O +Figure O +6A O +). O + +Here O +, O +hAsp350 B-residue_name_number +( O +mAsp358 B-residue_name_number +) O +serves O +as O +a O +structural O +nexus O +linking O +active B-site +site I-site +metal O +binding O +to O +substrate B-structure_element +binding I-structure_element +loop I-structure_element +conformations O +. O + +Tdp2 B-protein +SNPs O +impair O +function O +. O +( O +A O +) O +Active B-site +site I-site +residues O +mutated O +by O +TDP2 B-protein +SNPs O +. O + +D350N B-mutant +( O +mTdp2 B-protein +D358N B-mutant +) O +and O +I307V B-mutant +( O +mTdp2 B-protein +I317V B-mutant +) O +substitutions B-experimental_method +are O +mapped O +onto O +the O +Tdp2 B-protein +active B-site +site I-site +of O +the O +high O +- O +resolution O +mTdp2cat B-structure_element +structure B-evidence +( O +4GZ1 O +). O + +( O +B O +) O +Coomassie O +blue O +stained O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +gel O +of O +purified O +WT B-protein_state +and O +mutant B-protein_state +MBP B-experimental_method +- O +hTdp2cat B-structure_element +proteins O +used O +for O +assays O +in O +panels O +C O +and O +D O +. O +( O +C O +) O +Activity O +of O +WT B-protein_state +and O +mutant B-protein_state +MBP B-experimental_method +- O +hTdp2cat B-structure_element +proteins O +on O +a O +5 B-chemical +′– I-chemical +phosphotyrosyl I-chemical +– I-chemical +DNA I-chemical +oligonucleotides I-chemical +with O +3 O +′- O +fluorescein B-chemical +label O +. O + +Release O +of O +PNP B-chemical +from O +PNP B-chemical +phosphate I-chemical +( O +PNPP B-chemical +) O +and O +was O +detected O +as O +an O +increase O +in O +absorbance O +at O +415 O +nm O +, O +whereas O +the O +5 B-ptm +′- I-ptm +Y I-ptm +substrate O +is O +quantification O +of O +activity O +in O +a O +gel B-experimental_method +based I-experimental_method +assay I-experimental_method +shown O +in O +Figure O +6C O +. O + +To O +define O +the O +molecular O +basis O +for O +the O +hD350N B-mutant +( O +mD358N B-mutant +) O +defect O +, O +we O +crystallized B-experimental_method +and I-experimental_method +determined I-experimental_method +the O +structure B-evidence +of O +the O +DNA B-protein_state +- I-protein_state +free I-protein_state +form O +of O +the O +mD358N B-mutant +protein O +to O +2 O +. O +8Å O +resolution O +( O +PDB O +entry O +5INN O +). O + +This O +structure B-evidence +shows O +the O +D358N B-mutant +mutation B-experimental_method +disrupts O +the O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +Asp358 B-residue_name_number +and O +Trp307 B-residue_name_number +, O +shifts O +the O +position O +of O +Asn358 B-residue_name_number +and O +destabilizes O +Trp307 B-residue_name_number +. O + +Consequently O +, O +poor O +electron B-evidence +density I-evidence +is O +visible O +for O +the O +β2Hβ B-structure_element +loop I-structure_element +which O +is O +mostly O +disordered B-protein_state +( O +Supplementary O +Figure O +S6 O +). O + +Tdp2 B-protein +facilitates O +NHEJ O +repair O +of O +5 B-residue_name +′- I-residue_name +phosphotyrosine I-residue_name +adducted O +DSBs O + +Overall O +, O +our O +Tdp2 B-protein +structure B-experimental_method +/ I-experimental_method +activity I-experimental_method +studies I-experimental_method +reveal O +a O +tuned O +, O +5 B-ptm +′- I-ptm +detyrosylation I-ptm +DNA B-chemical +end O +processing O +activity O +and O +it O +has O +been O +demonstrated O +that O +Tdp2 B-protein +could O +enable O +repair O +of O +Top2 B-protein_type +damage O +by O +the O +non O +- O +homologous O +end O +- O +joining O +( O +NHEJ O +) O +pathway O +. O + +Interestingly O +, O +hTdp2cat B-structure_element +is O +slightly O +more O +effective O +than O +hTdp2FL B-protein +in O +promoting O +NHEJ O +of O +adducted O +ends O +, O +while O +a O +catalytically B-protein_state +deficient I-protein_state +E152Q B-mutant +mutant B-protein_state +was O +inactive B-protein_state +in O +this O +assay O +, O +supporting O +the O +notion O +that O +Tdp2 B-protein +catalytic O +activity O +is O +required O +to O +support O +NHEJ O +of O +phosphotyrosyl B-ptm +blocked O +DSBs O +( O +Supplementary O +Figure O +S7A O +). O + +We O +confirmed O +that O +efficient O +joining O +of O +the O +same O +tyrosine B-residue_name +- O +adducted O +substrate O +in O +cells O +( O +Figure O +7B O +) O +was O +dependent O +on O +both O +NHEJ O +( O +reduced O +over O +10 O +- O +fold O +in O +ligase O +IV O +deficient O +HCT O +116 O +cells O +; O +Supplementary O +Figure O +S7B O +), O +and O +Tdp2 B-protein +( O +reduced O +5 O +- O +fold O +in O +Tdp2 B-protein +deficient O +MEFs O +; O +Figure O +7C O +). O + +Moreover O +, O +products O +with O +error O +( O +i O +. O +e O +. O +junctions O +have O +missing O +sequence O +flanking O +the O +adducted O +terminus O +) O +are O +twice O +as O +frequent O +in O +cells O +deficient O +in O +Tdp2 B-protein +( O +Figure O +7D O +). O + +Therefore O +, O +in O +accord O +with O +previous O +work O +, O +joining O +of O +tyrosine B-residue_name +adducted O +ends O +after O +Tdp2 B-protein +- O +mediated O +detyrosylation B-ptm +is O +both O +more O +efficient O +and O +more O +accurate O +than O +joining O +after O +endonucleolytic O +excision O +( O +e O +. O +g O +. O +mediated O +by O +Artemis B-protein +or O +the O +Mre11 B-complex_assembly +/ I-complex_assembly +Rad50 I-complex_assembly +/ I-complex_assembly +Nbs1 I-complex_assembly +complex O +). O + +Effects O +of O +Tdp2 B-protein +active B-site +site I-site +SNP O +- O +encoded O +mutants O +on O +cellular O +Tdp2 B-protein +functions O +. O +( O +A O +) O +Cy5 B-chemical +labeled O +substrates O +with O +5 B-chemical +′- I-chemical +phosphate I-chemical +termini O +( O +Lanes O +1 O +– O +4 O +) O +or O +5 B-protein_state +′- I-protein_state +tyrosylated I-protein_state +termini O +( O +Lanes O +5 O +– O +9 O +) O +were O +incubated O +with O +Ku B-protein +, O +the O +NHEJ B-protein_type +ligase I-protein_type +( O +XRCC4 B-protein +, O +ligase B-protein +IV I-protein +and O +XLF B-protein +; O +X O +- O +L O +- O +X O +) O +and O +1 O +nM O +hTdp2FL B-protein +as O +indicated O +(+) O +for O +5 O +min O +at O +37 O +° O +C O +. O + +Concatemer O +ligation O +products O +were O +detected O +by O +5 O +% O +native B-experimental_method +PAGE I-experimental_method +. O + +( O +B O +) O +Workflow O +diagram O +of O +cellular B-experimental_method +end I-experimental_method +joining I-experimental_method +assays I-experimental_method +. O + +After O +1 O +h O +, O +DNA B-chemical +was O +recovered O +from O +cells O +and O +repair O +efficiency O +by O +qPCR B-experimental_method +or O +sequencing B-experimental_method +as O +indicated O +. O +( O +C O +) O +qPCR B-experimental_method +assessment O +of O +cellular O +end O +joining O +efficiency O +of O +the O +tyrosylated B-protein_state +substrate O +comparing O +results O +from O +wildtype B-protein_state +MEF O +cells O +to O +Tdp2 B-protein +−/− O +cells O +and O +Tdp2 B-protein +−/− O +cells O +complemented O +with O +wildtype B-protein_state +or O +the O +noted O +hTDP2FL B-protein +variants O +; O +Joining O +efficiency O +shown O +is O +the O +ratio O +of O +junctions O +recovered O +relative O +to O +WT B-protein_state +cells O +. O + +We O +next O +compared O +the O +ability O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +hTdp2FL B-protein +variants O +to O +complement O +Tdp2 B-protein +deficient O +mouse B-taxonomy_domain +embryonic O +fibroblasts O +( O +Supplementary O +Figure O +S7C O +). O + +Joining O +of O +extrachromosomal O +DNA B-chemical +with O +phosphotyrosine B-residue_name +blocked O +ends O +, O +both O +in O +terms O +of O +efficiency O +( O +Figure O +7C O +) O +and O +fidelity O +( O +Figure O +7D O +), O +was O +indistinguishable O +comparing O +MEFs O +from O +a O +wild B-protein_state +- I-protein_state +type I-protein_state +mouse B-taxonomy_domain +, O +MEFs O +from O +a O +Tdp2 B-protein +-/- O +mouse B-taxonomy_domain +overexpressing O +wild B-protein_state +- I-protein_state +type I-protein_state +human B-species +Tdp2 B-protein +, O +and O +Tdp2 B-protein +-/- O +MEFs O +overexpressing O +the O +I307V B-mutant +variant B-protein_state +human B-species +Tdp2 B-protein +. O + +In O +contrast O +, O +joining O +of O +5 O +′ O +phosphotyrosine O +- O +blocked O +ends O +was O +reduced O +5 O +- O +fold O +in O +Tdp2 B-protein +-/- O +MEFs O +, O +and O +an O +equivalent O +defect O +was O +observed O +in O +Tdp2 B-protein +-/- O +MEFs O +overexpressing O +Tdp2 B-protein +D350N B-mutant +. O + +Moreover O +, O +the O +frequency O +of O +inaccurate O +repair O +was O +2 O +- O +fold O +higher O +in O +both O +Tdp2 B-protein +deficient O +cells O +and O +Tdp2 B-protein +deficient O +cells O +overexpressing O +D350N B-mutant +, O +relative O +to O +cells O +expressing O +wild B-protein_state +type I-protein_state +Tdp2 B-protein +or O +hTdp2 B-protein +I307V B-mutant +( O +Figure O +7D O +). O + +Expression O +of O +wild B-protein_state +type I-protein_state +or O +I307V B-mutant +human B-species +Tdp2 B-protein +in O +Tdp2 B-protein +-/- O +MEFs O +was O +also O +sufficient O +to O +confer O +levels O +of O +resistance O +to O +etoposide B-chemical +comparable O +to O +the O +matched O +wild B-protein_state +- I-protein_state +type I-protein_state +MEF O +line O +, O +while O +overexpression B-experimental_method +of O +human B-species +D350N B-mutant +Tdp2 B-protein +had O +no O +apparent O +complementation O +activity O +( O +Figure O +7E O +). O + +The O +rare O +D350N B-mutant +variant B-protein_state +is O +thus O +inactive B-protein_state +by O +all O +metrics O +analyzed O +. O + +Understanding O +how O +cells O +cope O +with O +complex O +DNA B-chemical +breaks O +bearing O +topoisomerase O +– O +DNA B-chemical +protein O +crosslinks O +is O +key O +to O +deciphering O +individual O +responses O +to O +chemotherapeutic O +outcomes O +and O +genotoxic O +agents O +that O +poison O +Top2 B-protein_type +. O + +This O +mechanistic O +dissection O +of O +Tdp2 B-protein +interactions O +with O +damaged O +DNA B-chemical +and O +metal O +cofactor O +provides O +a O +detailed O +molecular O +understanding O +of O +the O +mechanism O +of O +Tdp2 B-protein +DNA B-chemical +protein O +crosslink O +processing O +. O + +The O +properties O +of O +complex O +DNA B-chemical +strand O +breaks O +bearing O +Top2 B-protein_type +- O +DNA B-chemical +protein O +crosslinks O +necessitate O +that O +Tdp2 B-protein +accommodates O +both O +damaged O +nucleic O +acid O +as O +well O +as O +the O +topoisomerase B-protein_type +protein O +in O +its O +active B-site +site I-site +for O +catalysis O +. O + +The O +Tdp2 B-protein +substrate B-site +interaction I-site +groove I-site +facilitates O +DNA B-chemical +- O +protein O +conjugate O +recognition O +in O +two O +important O +ways O +. O + +First O +, O +the O +nucleic B-site +acid I-site +binding I-site +trench I-site +is O +assembled O +by O +a O +dynamic B-protein_state +β2Hβ B-structure_element +DNA I-structure_element +damage I-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +that O +is O +capable O +of O +recognizing O +and O +processing O +diverse O +phosphotyrosyl B-ptm +linkages I-ptm +even O +in O +the O +context O +of O +bulky O +adducts O +such O +as O +ϵA B-chemical +. O +This O +is O +achieved O +by O +binding O +of O +nucleic O +acid O +‘ O +bases O +out O +’ O +by O +an O +extended O +base B-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +hydrophobic B-site +wall I-site +of O +the O +β2Hβ B-structure_element +- I-structure_element +loop I-structure_element +. O + +Secondly O +, O +our O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +analysis O +further O +highlights O +an O +enzyme O +– O +substrate O +cation B-bond_interaction +– I-bond_interaction +π I-bond_interaction +interaction I-bond_interaction +as O +an O +additional O +key O +feature O +of O +the O +Tdp2 B-protein +protein O +– O +DNA B-chemical +crosslink O +binding O +and O +reversal O +. O + +The O +strictly B-protein_state +conserved I-protein_state +active B-site +site I-site +Arg216 B-residue_name_number +appears O +optimally O +positioned O +to O +stabilize O +a O +delocalized O +charge O +on O +the O +phenolate O +product O +of O +the O +phosphotyrosyl B-ptm +cleavage O +reaction O +through O +molecular O +orbital O +overlap O +and O +polarization O +of O +the O +leaving O +group O +. O + +To O +our O +knowledge O +, O +this O +is O +the O +first O +proposed O +example O +of O +a O +substrate B-site +cation I-site +– I-site +π I-site +interface I-site +exploited O +to O +promote O +a O +phosphoryl O +- O +transfer O +reaction O +. O + +This O +unique O +feature O +likely O +provides O +an O +additional O +level O +of O +substrate O +- O +specificity O +for O +Tdp2 B-protein +by O +restricting O +activity O +to O +hydrolysis O +of O +aromatic O +adducts O +characteristic O +of O +Top2cc B-complex_assembly +, O +picornaviral B-taxonomy_domain +protein O +– O +RNA B-chemical +and O +Hepatitis B-taxonomy_domain +B I-taxonomy_domain +Virus I-taxonomy_domain +( O +HBV B-taxonomy_domain +) O +protein O +– O +DNA B-chemical +processing O +intermediates O +. O + +By O +comparison O +, O +other O +EEP B-structure_element +nucleases B-protein_type +such O +as O +Ape1 B-protein +and O +Ape2 B-protein +have O +evolved O +robust O +DNA B-chemical +damage O +specific O +endonucleolytic O +and O +exonucleolytic O +activities O +not O +shared O +with O +Tdp2 B-protein +. O + +The O +dynamic O +nature O +of O +the O +Tdp2 B-protein +active B-site +site I-site +presents O +opportunities O +for O +enzyme O +regulation O +. O + +However O +, O +whether O +additional O +protein O +factors O +can O +bind O +to O +Tdp2 B-protein +and O +modulate O +assembly O +/ O +disassembly O +of O +the O +Tdp2 B-protein +β2Hβ B-structure_element +- I-structure_element +loop I-structure_element +is O +unknown O +. O + +Furthermore O +, O +high O +- O +resolution O +structures B-evidence +of O +mouse B-taxonomy_domain +( O +Figures O +3 O +and O +4 O +) O +and O +C B-species +. I-species +elegans I-species +Tdp2 B-protein +show O +that O +a O +single O +metal O +ion O +typifies O +the O +Tdp2 B-protein +active B-site +site I-site +from O +worms B-taxonomy_domain +to O +man B-taxonomy_domain +. O + +Herein O +, O +we O +report O +five O +additional O +lines O +of O +evidence O +from O +metal O +binding O +detected O +by O +intrinsic B-experimental_method +tryptophan I-experimental_method +fluorescence I-experimental_method +, O +crystallographic B-experimental_method +analysis I-experimental_method +of O +varied O +metal O +cofactor O +complexes O +, O +mutagenesis B-experimental_method +, O +Ca2 B-experimental_method ++ I-experimental_method +inhibition I-experimental_method +studies I-experimental_method +and O +QM B-experimental_method +/ I-experimental_method +MM I-experimental_method +analysis I-experimental_method +that O +all O +support O +a O +feasible O +single O +Mg2 B-chemical ++ I-chemical +mediated O +Tdp2 B-protein +catalytic O +mechanism O +. O + +Given O +Tdp2 B-protein +variation O +in O +the O +human B-species +population O +, O +links O +to O +neurological O +disease O +and O +viral O +pathogenesis O +, O +our O +finding O +that O +TDP2 B-protein +SNPs O +ablate O +catalytic O +activity O +has O +probable O +implications O +for O +modulation O +of O +cancer O +chemotherapy O +, O +susceptibility O +to O +environmentally O +linked O +Top2 B-protein_type +poisons O +, O +and O +viral B-taxonomy_domain +infection O +. O + +Lastly O +, O +Tdp2 B-protein +inhibitors O +may O +synergize O +or O +potentiate O +cytotoxic O +effects O +of O +current O +anticancer O +treatments O +that O +target O +Tdp2 B-protein +. O + +Thus O +, O +we O +anticipate O +this O +atomic O +- O +level O +and O +mechanistic O +definition O +of O +the O +molecular O +determinants O +of O +Tdp2 B-protein +catalysis O +and O +conformational O +changes O +driven O +by O +DNA B-chemical +– O +protein O +and O +protein O +– O +protein O +interactions O +will O +foster O +unique O +strategies O +for O +the O +development O +of O +Tdp2 B-protein +targeted O +small O +molecule O +interventions O +. O + +Mechanism O +of O +extracellular O +ion O +exchange O +and O +binding B-site +- I-site +site I-site +occlusion O +in O +the O +sodium B-protein_type +- I-protein_type +calcium I-protein_type +exchanger I-protein_type + +Na B-protein_type ++/ I-protein_type +Ca2 I-protein_type ++ I-protein_type +exchangers I-protein_type +utilize O +the O +Na B-chemical ++ I-chemical +electrochemical O +gradient O +across O +the O +plasma O +membrane O +to O +extrude O +intracellular O +Ca2 B-chemical ++, I-chemical +and O +play O +a O +central O +role O +in O +Ca2 B-chemical ++ I-chemical +homeostasis O +. O + +This O +analysis O +defines O +the O +binding O +mode O +and O +relative O +affinity O +of O +these O +ions O +, O +establishes O +the O +structural O +basis O +for O +the O +anticipated O +3Na O ++: B-chemical +1Ca2 O ++ B-chemical +exchange O +stoichiometry O +, O +and O +reveals O +the O +conformational O +changes O +at O +the O +onset O +of O +the O +alternating O +- O +access O +transport O +mechanism O +. O + +An O +independent O +analysis O +of O +the O +dynamics O +and O +conformational B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscape I-evidence +of O +NCX_Mj B-protein +in O +different O +ion B-protein_state +- I-protein_state +occupancy I-protein_state +states O +, O +based O +on O +enhanced B-experimental_method +- I-experimental_method +sampling I-experimental_method +molecular I-experimental_method +- I-experimental_method +dynamics I-experimental_method +simulations I-experimental_method +, O +demonstrates O +that O +the O +crystal B-evidence +structures I-evidence +reflect O +mechanistically O +relevant O +, O +interconverting O +conformations O +. O + +These O +calculations B-experimental_method +also O +reveal O +the O +mechanism O +by O +which O +the O +outward B-protein_state +- O +to O +- O +inward B-protein_state +transition O +is O +controlled O +by O +the O +ion O +- O +occupancy O +state O +, O +thereby O +explaining O +the O +emergence O +of O +strictly O +- O +coupled O +Na B-chemical ++/ I-chemical +Ca2 B-chemical ++ I-chemical +antiport O +. O + +Na B-protein_type ++/ I-protein_type +Ca2 I-protein_type ++ I-protein_type +exchangers I-protein_type +( O +NCX B-protein_type +) O +play O +physiologically O +essential O +roles O +in O +Ca2 B-chemical ++ I-chemical +signaling O +and O +homeostasis O +. O + +NCX B-protein_type +catalyzes O +the O +uphill O +extrusion O +of O +intracellular O +Ca2 B-chemical ++ I-chemical +across O +the O +cell O +membrane O +, O +by O +coupling O +this O +process O +to O +the O +downhill O +permeation O +of O +Na B-chemical ++ I-chemical +into O +the O +cell O +, O +with O +a O +3 O +Na B-chemical ++ I-chemical +to O +1 O +Ca2 B-chemical ++ I-chemical +stoichiometry O +. O + +Each O +of O +these O +halves B-structure_element +contains O +a O +highly B-protein_state +conserved I-protein_state +region O +, O +referred O +to O +as O +α B-structure_element +- I-structure_element +repeat I-structure_element +, O +known O +to O +be O +important O +for O +ion O +binding O +and O +translocation O +; O +in O +eukaryotic B-taxonomy_domain +NCX B-protein_type +, O +the O +two O +halves B-structure_element +are O +connected O +by O +a O +large O +intracellular B-structure_element +regulatory I-structure_element +domain I-structure_element +, O +which O +is O +absent B-protein_state +in O +microbial B-taxonomy_domain +NCX B-protein_type +( O +Supplementary O +Fig O +. O +1 O +). O + +Despite O +a O +long O +history O +of O +physiological O +and O +functional O +studies O +, O +the O +molecular O +mechanism O +of O +NCX B-protein_type +has O +been O +elusive O +, O +owing O +to O +the O +lack O +of O +structural O +information O +. O + +This O +structure B-evidence +shows O +the O +exchanger B-protein_type +in O +an O +outward B-protein_state +- I-protein_state +facing I-protein_state +conformation O +and O +reveals O +four O +putative O +ion B-site +- I-site +binding I-site +sites I-site +, O +denominated O +internal B-site +( O +Sint B-site +), O +external B-site +( O +Sext B-site +), O +Ca2 B-site ++- I-site +binding I-site +( O +SCa B-site +) O +and O +middle B-site +( O +Smid B-site +), O +clustered O +in O +the O +center O +of O +the O +protein O +and O +occluded B-protein_state +from I-protein_state +the O +solvent O +( O +Fig O +. O +1a O +- O +b O +). O + +In O +this O +study O +, O +we O +set O +out O +to O +determine O +the O +structures B-evidence +of O +outward B-protein_state +- I-protein_state +facing I-protein_state +wild B-protein_state +- I-protein_state +type I-protein_state +NCX_Mj B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +Na B-chemical ++, I-chemical +Ca2 B-chemical ++ I-chemical +and O +Sr2 B-chemical ++, I-chemical +at O +various O +concentrations O +. O + +These O +calculations B-experimental_method +also O +reveal O +how O +the O +ion O +occupancy O +state O +of O +the O +outward B-protein_state +- I-protein_state +facing I-protein_state +exchanger B-protein_type +determines O +the O +feasibility O +of O +the O +transition O +to O +the O +inward B-protein_state +- I-protein_state +facing I-protein_state +conformation O +, O +thereby O +addressing O +a O +key O +outstanding O +question O +in O +secondary O +- O +active O +transport O +, O +namely O +how O +the O +transported O +substrates O +control O +the O +alternating O +- O +access O +mechanism O +. O + +The O +assignment O +of O +the O +four O +central B-site +binding I-site +sites I-site +identified O +in O +the O +previously O +reported O +NCX_Mj B-protein +structure B-evidence +was O +hampered O +by O +the O +presence O +of O +both O +Na B-chemical ++ I-chemical +and O +Ca2 B-chemical ++ I-chemical +in O +the O +protein O +crystals B-evidence +. O + +To O +conclusively O +clarify O +this O +assignment O +, O +we O +first O +set O +out O +to O +examine O +the O +Na B-chemical ++ I-chemical +occupancy O +of O +these O +sites O +without O +Ca2 B-chemical ++. I-chemical + +Crystals B-evidence +were O +grown O +in O +150 O +mM O +NaCl B-chemical +using O +the O +lipidic B-experimental_method +cubic I-experimental_method +phase I-experimental_method +( O +LCP B-experimental_method +) O +technique O +. O + +The O +crystallization O +solutions O +around O +the O +LCP B-experimental_method +droplets O +were O +then O +slowly O +replaced O +by O +solutions O +containing O +different O +concentrations O +of O +NaCl B-chemical +and O +EGTA B-chemical +( O +Methods O +). O + +X B-experimental_method +- I-experimental_method +ray I-experimental_method +diffraction I-experimental_method +of O +these O +soaked O +crystals B-evidence +revealed O +a O +Na B-chemical ++- I-chemical +dependent O +variation O +in O +the O +electron B-evidence +- I-evidence +density I-evidence +distribution I-evidence +at O +sites O +Sext B-site +, O +SCa B-site +and O +Sint B-site +, O +indicating O +a O +Na B-chemical ++ I-chemical +occupancy O +change O +( O +Fig O +. O +1c O +). O + +Occupancy B-experimental_method +refinement I-experimental_method +indicated O +two O +Na B-chemical ++ I-chemical +ions O +bind O +to O +Sint B-site +and O +SCa B-site +at O +low O +Na B-chemical ++ I-chemical +concentrations O +( O +Fig O +. O +1c O +), O +with O +a O +slight O +preference O +for O +Sint B-site +( O +Table O +1 O +). O + +Binding O +of O +a O +third O +Na B-chemical ++ I-chemical +to O +Sext B-site +occurs O +at O +higher O +concentrations O +, O +as O +no O +density B-evidence +was O +observed O +there O +at O +10 O +mM O +Na B-chemical ++ I-chemical +or O +lower O +( O +Fig O +. O +1c O +); O +Sext B-site +is O +however O +partially O +occupied O +at O +20 O +mM O +Na B-chemical ++, I-chemical +and O +fully O +occupied O +at O +150 O +mM O +( O +Fig O +. O +1c O +). O + +Indeed O +, O +two O +observations O +indicate O +that O +a O +water B-chemical +molecule O +rather O +than O +a O +Na B-chemical ++ I-chemical +ion O +occupies O +Smid B-site +, O +as O +was O +predicted O +in O +a O +recent O +simulation B-experimental_method +study O +. O + +First O +, O +the O +electron B-evidence +density I-evidence +at O +Smid B-site +does O +not O +depend O +significantly O +on O +the O +Na B-chemical ++ I-chemical +concentration O +. O + +Second O +, O +the O +protein O +coordination O +geometry O +at O +Smid B-site +is O +clearly O +suboptimal O +for O +Na B-chemical ++ I-chemical +( O +Supplementary O +Fig O +. O +1d O +). O + +It O +can O +be O +inferred O +from O +this O +assignment O +that O +Glu54 B-residue_name_number +and O +Glu213 B-residue_name_number +are O +ionized O +, O +while O +Asp240 B-residue_name_number +, O +which O +flanks O +Smid B-site +( O +and O +is O +replaced O +by O +Asn B-residue_name +in O +eukaryotic B-taxonomy_domain +NCX B-protein_type +) O +would O +be O +protonated B-protein_state +, O +as O +indicated O +by O +the O +abovementioned O +simulation B-experimental_method +study O +. O + +Na B-chemical ++- I-chemical +dependent O +conformational O +change O + +The O +NCX_Mj B-protein +structures B-evidence +in O +various O +Na B-chemical ++ I-chemical +concentrations O +also O +reveal O +that O +Na B-chemical ++ I-chemical +binding O +to O +Sext B-site +is O +coupled O +to O +a O +subtle O +but O +important O +conformational O +change O +( O +Fig O +. O +2 O +). O + +TM7b B-structure_element +occludes O +the O +four O +central B-site +binding I-site +sites I-site +from O +the O +external O +solution O +, O +with O +the O +backbone O +carbonyl O +of O +Ala206 B-residue_name_number +coordinating B-bond_interaction +the O +Na B-chemical ++ I-chemical +ion O +( O +Fig O +. O +2b O +- O +d O +). O + +TM7a B-structure_element +also O +forms O +hydrophobic B-bond_interaction +contacts I-bond_interaction +with O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +TM6 B-structure_element +. O + +The O +straightening O +of O +TM7ab B-structure_element +also O +opens O +up O +a O +passageway O +from O +the O +external O +solution O +to O +Sext B-site +and O +Smid B-site +, O +while O +SCa B-site +and O +Sint B-site +remain O +occluded B-protein_state +( O +Fig O +. O +2d O +). O + +Thus O +, O +the O +structures B-evidence +at O +high B-protein_state +and O +low B-protein_state +Na B-chemical ++ I-chemical +concentrations O +represent O +the O +outward B-protein_state +- I-protein_state +facing I-protein_state +occluded B-protein_state +and O +partially B-protein_state +open I-protein_state +states O +, O +respectively O +. O + +This O +conformational O +change O +is O +dependent O +on O +the O +Na B-chemical ++ I-chemical +occupancy O +of O +Sext B-site +and O +occurs O +when O +Na B-chemical ++ I-chemical +already O +occupies O +Sint B-site +and O +SCa B-site +. O + +Our O +crystallographic B-experimental_method +titration I-experimental_method +experiment I-experimental_method +indicates O +that O +the O +K1 B-evidence +/ I-evidence +2 I-evidence +of O +this O +Na B-chemical ++- I-chemical +driven O +conformational O +transition O +is O +~ O +20 O +mM O +. O +At O +this O +concentration O +, O +Sext B-site +is O +partially B-protein_state +occupied I-protein_state +and O +the O +NCX_Mj B-protein +crystal B-evidence +is O +a O +mixture O +of O +both O +the O +occluded B-protein_state +and O +partially B-protein_state +open I-protein_state +conformations O +. O + +The O +finding O +that O +the O +Na B-chemical ++ I-chemical +occupancy O +change O +from O +2 O +to O +3 O +ions O +coincides O +with O +a O +conformational O +change O +of O +the O +transporter B-protein_type +also O +provides O +a O +rationale O +to O +the O +Hill B-evidence +coefficient I-evidence +of O +the O +Na B-chemical ++- I-chemical +dependent O +activation O +process O +in O +eukaryotic B-taxonomy_domain +NCX B-protein_type +. O + +Extracellular O +Ca2 B-chemical ++ I-chemical +and O +Sr2 B-chemical ++ I-chemical +binding O +and O +their O +competition O +with O +Na B-chemical ++ I-chemical + +To O +determine O +how O +Ca2 B-chemical ++ I-chemical +binds O +to O +NCX_Mj B-protein +and O +competes O +with O +Na B-chemical ++, I-chemical +we O +first O +titrated B-experimental_method +the I-experimental_method +crystals I-experimental_method +with O +Sr2 B-chemical ++ I-chemical +( O +Methods O +). O + +Protein B-experimental_method +crystals I-experimental_method +soaked I-experimental_method +with O +10 O +mM O +Sr2 B-chemical ++ I-chemical +and O +2 O +. O +5 O +mM O +Na B-chemical ++ I-chemical +revealed O +a O +strong O +electron B-evidence +- I-evidence +density I-evidence +peak I-evidence +at O +site O +SCa B-site +, O +indicating O +binding O +of O +a O +single O +Sr2 B-chemical ++ I-chemical +ion O +( O +Fig O +. O +3a O +). O + +Binding O +of O +Sr2 B-chemical ++, I-chemical +however O +, O +excludes O +Na B-chemical ++ I-chemical +entirely O +. O + +Crystal B-experimental_method +titrations I-experimental_method +with O +decreasing B-experimental_method +Sr2 B-chemical ++ I-chemical +or O +increasing B-experimental_method +Na B-chemical ++ I-chemical +demonstrated O +that O +Sr2 B-chemical ++ I-chemical +binds O +to O +the O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +with O +low O +affinity O +, O +and O +that O +it O +can O +be O +out O +- O +competed O +by O +Na B-chemical ++ I-chemical +even O +at O +low O +concentrations O +( O +Supplementary O +Note O +1 O +and O +Supplementary O +Fig O +. O +2a O +- O +b O +). O + +Binding O +of O +Ca2 B-chemical ++ I-chemical +to O +both O +sites O +simultaneously O +is O +highly O +improbable O +due O +to O +their O +close O +proximity O +, O +and O +at O +least O +one O +water B-chemical +molecule O +can O +be O +discerned O +coordinating B-bond_interaction +the O +ion O +( O +Fig O +. O +3b O +). O + +The O +partial B-protein_state +Ca2 B-chemical ++ I-chemical +occupancy B-protein_state +at O +Smid B-site +is O +likely O +caused O +by O +Asp240 B-residue_name_number +, O +which O +flanks O +this O +site O +and O +can O +in O +principle O +coordinate B-bond_interaction +Ca2 B-chemical ++. I-chemical + +Previous O +functional B-experimental_method +and I-experimental_method +computational I-experimental_method +studies I-experimental_method +, O +however O +, O +indicate O +Asp240 B-residue_name_number +becomes O +protonated B-protein_state +during O +transport O +. O + +Indeed O +, O +in O +most O +NCX B-protein_type +proteins O +Asp240 B-residue_name_number +is O +substituted B-experimental_method +by O +Asn B-residue_name +, O +which O +would O +likely O +weaken O +or O +abrogate O +Ca2 B-chemical ++ I-chemical +binding O +to O +Smid B-site +. O + +SCa B-site +is O +therefore O +the O +functional O +Ca2 B-site ++ I-site +site I-site +. O + +Similarly O +to O +Sr2 B-chemical ++, I-chemical +Ca2 B-chemical ++ I-chemical +binds O +with O +low O +affinity B-evidence +to O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +and O +can O +be O +readily O +displaced O +by O +Na B-chemical ++ I-chemical +( O +Supplementary O +Note O +1 O +and O +Supplementary O +Fig O +. O +2c O +). O + +Specifically O +, O +our O +crystallographic B-experimental_method +titration I-experimental_method +assay I-experimental_method +indicates O +Ca2 B-chemical ++ I-chemical +binds O +with O +sub O +- O +millimolar O +affinity B-evidence +, O +in O +good O +agreement O +with O +the O +external O +apparent O +Ca2 B-evidence ++ I-evidence +affinities I-evidence +deduced O +functionally O +for O +cardiac O +NCX B-protein_type +( O +Km B-evidence +~ O +0 O +. O +32 O +mM O +) O +and O +NCX_Mj B-protein +( O +Km B-evidence +~ O +0 O +. O +175 O +mM O +). O + +Taken O +together O +, O +these O +crystal B-experimental_method +titration I-experimental_method +experiments I-experimental_method +demonstrate O +that O +the O +four O +binding B-site +sites I-site +in O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +exhibit O +different O +specificity O +: O +Sint B-site +and O +Sext B-site +are O +Na B-chemical ++ I-chemical +specific O +whereas O +SCa B-site +, O +previously O +hypothesized O +to O +be O +Ca2 B-chemical ++ I-chemical +specific O +, O +can O +also O +bind O +Na B-chemical ++, I-chemical +confirming O +our O +earlier O +simulation B-experimental_method +study O +, O +as O +well O +as O +Sr2 B-chemical ++; I-chemical +Smid B-site +can O +also O +transiently O +accommodate O +Ca2 B-chemical ++ I-chemical +but O +during O +transport O +Smid B-site +is O +most O +likely O +occupied O +by O +water B-chemical +. O + +The O +ion B-site +- I-site +binding I-site +sites I-site +in O +NCX_Mj B-protein +can O +therefore O +accommodate O +up O +to O +three O +Na B-chemical ++ I-chemical +ions O +or O +a O +single O +divalent O +ion O +, O +and O +occupancy O +by O +Na B-chemical ++ I-chemical +and O +Ca2 B-chemical ++ I-chemical +( O +or O +Sr2 B-chemical ++) I-chemical +are O +mutually O +exclusive O +, O +as O +was O +deduced O +for O +eukaryotic B-taxonomy_domain +exchangers B-protein_type +. O + +A O +structure B-evidence +of O +NCX_Mj B-protein +without B-protein_state +Na B-chemical ++ I-chemical +or O +Ca2 B-chemical ++ I-chemical +bound B-protein_state + +We O +were O +able O +to O +determine O +an O +apo B-protein_state +- O +state O +structure B-evidence +of O +NCX_Mj B-protein +, O +by O +crystallizing B-experimental_method +the O +protein O +at O +lower B-protein_state +pH I-protein_state +and O +in O +the O +absence B-protein_state +of I-protein_state +Na B-chemical ++ I-chemical +( O +Methods O +). O + +This O +structure B-evidence +is O +similar O +to O +the O +partially B-protein_state +open I-protein_state +structure B-evidence +with O +two O +Na B-chemical ++ I-chemical +or O +either O +one O +Ca2 B-chemical ++ I-chemical +or O +one O +Sr2 B-chemical ++ I-chemical +ion O +, O +with O +two O +noticeable O +differences O +. O + +First O +, O +TM7ab B-structure_element +along O +with O +the O +extracellular B-structure_element +half I-structure_element +of O +the O +TM6 B-structure_element +and O +TM1 B-structure_element +swing O +further O +away O +from O +the O +protein O +core O +( O +Fig O +. O +3c O +), O +resulting O +in O +a O +slightly O +wider O +passageway O +into O +the O +binding B-site +sites I-site +. O + +Second O +, O +Glu54 B-residue_name_number +and O +Glu213 B-residue_name_number +side O +chains O +rotate O +away O +from O +the O +binding B-site +sites I-site +and O +appear O +to O +form O +hydrogen B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +with O +residues O +involved O +in O +ion B-bond_interaction +coordination I-bond_interaction +in O +the O +fully B-protein_state +Na I-protein_state ++- I-protein_state +loaded I-protein_state +structure B-evidence +( O +Fig O +. O +3d O +). O + +This O +apo B-protein_state +structure B-evidence +might O +therefore O +represent O +the O +unloaded B-protein_state +, O +open B-protein_state +state O +of O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +. O + +Such O +interpretation O +would O +be O +consistent O +with O +the O +computer B-experimental_method +simulations I-experimental_method +reported O +below O +. O + +Indeed O +, O +transport B-experimental_method +assays I-experimental_method +of O +NCX_Mj B-protein +have O +shown O +that O +even O +in O +the O +presence B-protein_state +of I-protein_state +Na B-chemical ++ I-chemical +or O +Ca2 B-chemical ++, I-chemical +low B-protein_state +pH I-protein_state +inactivates B-protein_state +the O +transport O +cycle O +. O + +NCX B-protein_type +must O +be O +loaded O +either O +with O +3 O +Na B-chemical ++ I-chemical +or O +1 O +Ca2 B-chemical ++, I-chemical +and O +therefore O +functions O +as O +an O +antiporter B-protein_type +; O +symporters B-protein_type +, O +by O +contrast O +, O +undergo O +the O +alternating O +- O +access O +transition O +only O +when O +all O +substrates O +and O +coupling O +ions O +are O +concurrently O +bound B-protein_state +, O +or O +in O +the O +apo B-protein_state +state O +. O + +This O +computational O +analysis O +was O +based O +solely O +on O +the O +published O +structure B-evidence +of O +NCX_Mj B-protein +, O +independently O +of O +the O +crystallographic B-experimental_method +studies I-experimental_method +described O +above O +. O + +A O +series O +of O +exploratory O +MD B-experimental_method +simulations I-experimental_method +was O +initially O +carried O +out O +to O +examine O +what O +features O +of O +the O +NCX_Mj B-protein +structure B-evidence +might O +depend O +on O +the O +ion B-site +- I-site +binding I-site +sites I-site +occupancy O +. O + +The O +Na B-chemical ++ I-chemical +ions O +at O +SCa B-site +and O +Sint B-site +were O +displaced O +subsequently O +, O +and O +an O +analogous O +simulation B-experimental_method +was O +then O +carried O +out O +. O + +These O +initial O +simulations B-experimental_method +revealed O +noticeable O +changes O +in O +the O +transporter B-protein_type +, O +consistent O +with O +those O +observed O +in O +the O +new O +crystal B-evidence +structures I-evidence +. O + +The O +most O +notable O +change O +upon O +displacement O +of O +Na B-chemical ++ I-chemical +from O +Sext B-site +was O +the O +straightening O +of O +TM7ab B-structure_element +( O +Fig O +. O +4a O +). O + +When O +3 O +Na B-chemical ++ I-chemical +ions O +are O +bound B-protein_state +, O +TM7ab B-structure_element +primarily O +folds O +as O +two O +distinct O +, O +non O +- O +collinear O +α B-structure_element +- I-structure_element +helical I-structure_element +fragments I-structure_element +, O +owing O +to O +the O +loss O +of O +the O +backbone O +carbonyl O +- O +amide O +hydrogen B-bond_interaction +- I-bond_interaction +bonds I-bond_interaction +between O +F202 B-residue_name_number +and O +A206 B-residue_name_number +, O +and O +T203 B-residue_name_number +and O +F207 B-residue_name_number +( O +Fig O +. O +4b O +). O + +This O +distortion O +occludes O +Sext B-site +from O +the O +exterior O +( O +Fig O +. O +4d O +, O +4h O +- O +i O +) O +and O +appears O +to O +be O +induced O +by O +the O +Na B-chemical ++ I-chemical +ion O +itself O +, O +which O +pulls O +the O +carbonyl O +group O +of O +A206 B-residue_name_number +into O +its O +coordination O +sphere O +( O +Fig O +. O +4g O +). O + +Together O +, O +these O +changes O +open O +a O +second O +aqueous B-site +channel I-site +leading O +directly O +into O +SCa B-site +and O +Sint B-site +( O +Fig O +. O +4f O +, O +Fig O +. O +4h O +- O +i O +). O + +The O +transporter B-protein_type +thus O +becomes O +fully B-protein_state +outward I-protein_state +- I-protein_state +open I-protein_state +. O + +To O +more O +rigorously O +characterize O +the O +influence O +of O +the O +ion O +- O +occupancy O +state O +on O +the O +conformational O +dynamics O +of O +the O +exchanger B-protein_type +, O +we O +carried O +out O +a O +series O +of O +enhanced O +- O +sampling O +MD B-experimental_method +calculations I-experimental_method +designed O +to O +reversibly O +simulate O +the O +transition O +between O +the O +outward B-protein_state +- I-protein_state +occluded I-protein_state +and O +fully B-protein_state +outward I-protein_state +- I-protein_state +open I-protein_state +states O +, O +and O +thus O +quantify O +the O +free B-evidence +- I-evidence +energy I-evidence +landscape I-evidence +encompassing O +these O +states O +( O +Methods O +). O + +As O +above O +, O +we O +initially O +examined O +three O +occupancy O +states O +, O +namely O +with O +Na B-chemical ++ I-chemical +in O +Sext B-site +, O +SCa B-site +and O +Sint B-site +, O +with O +Na B-chemical ++ I-chemical +only O +at O +SCa B-site +and O +Sint B-site +, O +and O +without B-protein_state +Na B-chemical ++. I-chemical + +When O +all O +Na B-site ++ I-site +sites I-site +are O +occupied O +, O +the O +global O +free B-evidence +- I-evidence +energy I-evidence +minimum I-evidence +corresponds O +to O +a O +conformation O +in O +which O +the O +ions O +are O +maximally O +coordinated O +by O +the O +protein O +( O +Fig O +. O +5a O +, O +5c O +); O +TM7ab B-structure_element +is O +bent O +and O +packs O +closely O +with O +TM2 B-structure_element +and O +TM3 B-structure_element +, O +and O +so O +the O +binding B-site +sites I-site +are O +occluded O +from O +the O +solvent O +( O +Fig O +. O +5b O +). O + +The O +Na B-chemical ++ I-chemical +ion O +at O +Sext B-site +remains O +fully B-protein_state +coordinated I-protein_state +, O +but O +an O +ordered O +water B-chemical +molecule O +now O +mediates O +its O +interaction O +with O +A206 B-residue_name_number +: O +O O +, O +relieving O +the O +strain O +on O +the O +F202 B-residue_name_number +: O +O O +– O +A206 B-residue_name_number +: O +N O +hydrogen B-bond_interaction +- I-bond_interaction +bond I-bond_interaction +( O +Fig O +. O +5c O +). O + +Interestingly O +, O +this O +doubly O +occupied O +state O +can O +also O +access O +conformations O +in O +which O +the O +second O +aqueous B-site +channel I-site +mentioned O +above O +, O +i O +. O +e O +. O +leading O +to O +SCa B-site +between O +TM7 B-structure_element +and O +TM2 B-structure_element +and O +over O +the O +gating B-structure_element +helices I-structure_element +TM1 B-structure_element +and O +TM6 B-structure_element +, O +also O +becomes O +open B-protein_state +( O +Fig O +. O +5b O +- O +c O +). O + +Crucially O +, O +though O +, O +the O +free B-evidence +- I-evidence +energy I-evidence +landscape I-evidence +for O +this O +partially B-protein_state +occupied I-protein_state +state O +demonstrates O +that O +the O +occluded B-protein_state +conformation O +is O +no O +longer O +energetically O +feasible O +( O +Fig O +. O +5a O +). O + +Displacement O +of O +the O +two O +remaining O +Na B-chemical ++ I-chemical +ions O +from O +SCa B-site +and O +Sint B-site +further O +reshapes O +the O +free B-evidence +- I-evidence +energy I-evidence +landscape I-evidence +of O +the O +transporter B-protein_type +( O +No O +ions O +, O +Fig O +. O +5a O +), O +which O +now O +can O +only O +adopt O +a O +fully B-protein_state +open I-protein_state +state O +featuring O +the O +two O +aqueous B-site +channels I-site +( O +Fig O +. O +5b O +- O +c O +). O + +The O +transition O +to O +the O +occluded B-protein_state +state O +in O +this O +apo B-protein_state +state O +is O +again O +energetically O +unfeasible O +. O + +From O +a O +mechanistic O +standpoint O +, O +it O +is O +satisfying O +to O +observe O +how O +the O +open B-protein_state +and O +semi B-protein_state +- I-protein_state +open I-protein_state +states O +are O +each O +compatible O +with O +two O +different O +Na B-chemical ++ I-chemical +occupancies O +, O +explaining O +how O +sequential O +Na B-chemical ++ I-chemical +binding O +to O +energetically O +accessible O +conformations O +( O +prior O +to O +those O +binding O +events O +) O +progressively O +reshape O +the O +free B-evidence +- I-evidence +energy I-evidence +landscape I-evidence +of O +the O +transporter B-protein_type +; O +by O +contrast O +, O +the O +occluded B-protein_state +conformation O +is O +forbidden O +unless O +the O +Na B-protein_state ++ I-protein_state +occupancy I-protein_state +is I-protein_state +complete I-protein_state +. O + +This O +processivity O +is O +logical O +since O +three O +Na B-chemical ++ I-chemical +ions O +are O +involved O +, O +but O +also O +implies O +that O +in O +the O +Ca2 B-protein_state ++- I-protein_state +bound I-protein_state +state O +, O +which O +includes O +a O +single O +ion O +, O +the O +transporter B-protein_type +ought O +to O +be O +able O +to O +access O +all O +three O +major O +conformations O +, O +i O +. O +e O +. O +the O +outward B-protein_state +- I-protein_state +open I-protein_state +state O +, O +in O +order O +to O +release O +( O +or O +re O +- O +bind O +) O +Ca2 B-chemical ++, I-chemical +but O +also O +the O +occluded B-protein_state +conformation O +, O +and O +thus O +the O +semi B-protein_state +- I-protein_state +open I-protein_state +intermediate O +, O +in O +order O +to O +transition O +to O +the O +inward B-protein_state +- I-protein_state +open I-protein_state +state O +. O + +By O +contrast O +, O +occupancy O +by O +H B-chemical ++, I-chemical +which O +as O +mentioned O +are O +not O +transported O +, O +might O +be O +compatible O +with O +a O +semi B-protein_state +- I-protein_state +open I-protein_state +state O +as O +well O +as O +with O +the O +fully B-protein_state +open I-protein_state +conformation O +, O +but O +should O +not O +be O +conducive O +to O +occlusion O +. O + +To O +assess O +this O +hypothesis O +, O +we O +carried O +out O +enhanced B-experimental_method +- I-experimental_method +sampling I-experimental_method +simulations I-experimental_method +for O +the O +Ca2 B-protein_state ++ I-protein_state +and O +H B-protein_state ++- I-protein_state +bound I-protein_state +states O +of O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +analogous O +to O +those O +described O +above O +for O +Na B-chemical ++ I-chemical +( O +see O +Supplementary O +Note O +2 O +and O +Supplementary O +Fig O +. O +3 O +- O +4 O +for O +details O +on O +how O +the O +structures B-evidence +of O +the O +Ca2 B-protein_state ++- I-protein_state +bound I-protein_state +state O +was O +predicted O +). O + +The O +calculated B-experimental_method +free B-evidence +- I-evidence +energy I-evidence +landscape I-evidence +for O +Ca2 B-protein_state ++- I-protein_state +bound I-protein_state +NCX_Mj B-protein +confirms O +the O +hypothesis O +outlined O +above O +( O +1 O +× O +Ca2 B-chemical ++, I-chemical +Fig O +. O +6a O +): O +consistent O +with O +the O +fact O +that O +NCX_Mj B-protein +transports O +a O +single O +Ca2 B-chemical ++, I-chemical +the O +occluded B-protein_state +, O +dehydrated B-protein_state +conformation O +is O +one O +of O +the O +major O +energetic O +minima O +, O +but O +clearly O +the O +exchanger B-protein_type +can O +also O +adopt O +the O +semi B-protein_state +- I-protein_state +open I-protein_state +and O +open B-protein_state +states O +that O +would O +be O +required O +for O +Ca2 B-chemical ++ I-chemical +release O +and O +Na B-chemical ++ I-chemical +entry O +, O +via O +either O +of O +the O +aqueous B-site +access I-site +channels I-site +that O +lead O +to O +Sext B-site +and O +SCa B-site +( O +Fig O +. O +6b O +- O +c O +). O + +By O +contrast O +, O +protonation B-protein_state +of O +Glu54 B-residue_name_number +and O +Glu213 B-residue_name_number +makes O +the O +occluded B-protein_state +conformation O +energetically O +unfeasible O +, O +consistent O +with O +the O +fact O +that O +NCX_Mj B-protein +does O +not O +transport O +protons B-chemical +; O +in O +this O +H B-protein_state ++- I-protein_state +bound I-protein_state +state O +, O +though O +, O +the O +exchanger B-protein_type +can O +adopt O +the O +semi B-protein_state +- I-protein_state +open I-protein_state +conformation O +captured O +in O +the O +low B-protein_state +pH I-protein_state +, O +apo B-protein_state +crystal B-evidence +structure I-evidence +( O +2 O +× O +H B-chemical ++, I-chemical +Fig O +. O +6a O +- O +c O +). O + +Taken O +together O +, O +this O +systematic B-experimental_method +computational I-experimental_method +analysis I-experimental_method +of O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +clearly O +demonstrates O +that O +the O +alternating O +- O +access O +and O +ion O +- O +recognition O +mechanisms O +in O +this O +Na B-protein_type ++/ I-protein_type +Ca2 I-protein_type ++ I-protein_type +exchanger I-protein_type +are O +coupled O +through O +the O +influence O +that O +the O +bound O +ions O +have O +on O +the O +free B-evidence +- I-evidence +energy I-evidence +landscape I-evidence +of O +the O +protein O +, O +which O +in O +turn O +determines O +whether O +or O +not O +the O +occluded B-protein_state +conformation O +is O +energetically O +feasible O +. O + +The O +alternating O +- O +access O +hypothesis O +implicitly O +dictates O +that O +the O +switch O +between O +outward B-protein_state +- O +and O +inward B-protein_state +- I-protein_state +open I-protein_state +conformations O +of O +a O +given O +secondary B-protein_state +- I-protein_state +active I-protein_state +transporter B-protein_type +must O +not O +occur O +unless O +the O +appropriate O +type O +and O +number O +of O +substrates O +are O +recognized O +. O + +It O +is O +however O +also O +non O +- O +trivial O +: O +antiporters B-protein_type +, O +for O +example O +, O +do O +not O +undergo O +the O +alternating O +- O +access O +transition O +without O +a O +cargo O +, O +but O +this O +is O +precisely O +how O +membrane B-protein_type +symporters I-protein_type +reset O +their O +transport O +cycles O +. O + +Here O +, O +we O +have O +provided O +novel O +insights O +into O +this O +intriguing O +mechanism O +of O +conformational O +control O +through O +structural B-experimental_method +studies I-experimental_method +and O +quantitative B-experimental_method +molecular I-experimental_method +simulations I-experimental_method +of O +a O +Na B-protein_type ++/ I-protein_type +Ca2 I-protein_type ++ I-protein_type +exchanger I-protein_type +. O + +Specifically O +, O +our O +studies O +of O +NCX_Mj B-protein +reveal O +the O +mechanism O +of O +forward O +ion O +exchange O +( O +Fig O +. O +7 O +). O + +The O +internal O +symmetry O +of O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +and O +the O +inward B-protein_state +- I-protein_state +facing I-protein_state +crystal B-evidence +structures I-evidence +of O +several O +Ca2 B-protein_type ++/ I-protein_type +H I-protein_type ++ I-protein_type +exchangers I-protein_type +indicate O +that O +the O +alternating O +- O +access O +mechanism O +of O +NCX B-protein_type +proteins O +entails O +a O +sliding O +motion O +of O +TM1 B-structure_element +and O +TM6 B-structure_element +relative O +to O +the O +rest O +of O +the O +transporter B-protein_type +. O + +Here O +, O +we O +demonstrate O +that O +conformational O +changes O +in O +the O +extracellular B-structure_element +region I-structure_element +of O +the O +TM2 B-structure_element +- I-structure_element +TM3 I-structure_element +and O +TM7 B-structure_element +- I-structure_element +TM8 I-structure_element +bundle I-structure_element +precede O +and O +are O +necessary O +for O +the O +transition O +, O +and O +are O +associated O +with O +ion O +recognition O +and O +/ O +or O +release O +. O + +The O +most O +apparent O +of O +these O +changes O +involves O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +TM7 B-structure_element +( O +TM7ab B-structure_element +); O +together O +with O +more O +subtle O +displacements O +in O +TM2 B-structure_element +and O +TM3 B-structure_element +, O +this O +change O +in O +TM7ab B-structure_element +correlates O +with O +the O +opening O +and O +closing O +of O +two O +distinct O +aqueous B-site +channels I-site +leading O +into O +the O +ion B-site +- I-site +binding I-site +sites I-site +from O +the O +extracellular O +solution O +. O + +Interestingly O +, O +the O +bending O +of O +TM7 B-structure_element +associated O +with O +the O +occlusion O +of O +the O +ion B-site +- I-site +binding I-site +sites I-site +also O +unlocks O +its O +interaction O +with O +TM6 B-structure_element +, O +and O +thus O +enables O +TM6 B-structure_element +and O +TM1 B-structure_element +to O +freely O +slide O +to O +the O +inward B-protein_state +- I-protein_state +facing I-protein_state +conformation O +. O + +The O +crystal B-evidence +structures I-evidence +of O +NCX_Mj B-protein +reported O +here O +, O +with O +either O +Na B-chemical ++, I-chemical +Ca2 B-chemical ++, I-chemical +Sr2 B-chemical ++ I-chemical +or O +H B-chemical ++ I-chemical +bound B-protein_state +, O +capture O +the O +exchanger B-protein_type +in O +different O +conformational O +states O +. O + +These O +states O +can O +only O +represent O +a O +subset O +among O +all O +possible O +, O +but O +they O +ought O +to O +reflect O +inherent O +preferences O +of O +the O +transporter B-protein_type +, O +modulated O +by O +the O +experimental O +conditions O +. O + +Indeed O +, O +we O +show O +that O +it O +is O +the O +presence O +or O +absence O +of O +the O +occluded B-protein_state +state O +in O +this O +landscape O +that O +explains O +the O +antiport O +function O +of O +NCX_Mj B-protein +and O +its O +3Na O ++: B-chemical +1Ca2 O ++ B-chemical +stoichiometry O +. O + +In O +multiple O +ways O +, O +our O +findings O +provide O +an O +explanation O +for O +, O +existing O +functional O +, O +biochemical O +and O +biophysical O +data O +for O +both O +NCX_Mj B-protein +and O +its O +eukaryotic B-taxonomy_domain +homologues O +. O + +The O +striking O +quantitative O +agreement O +between O +the O +ion B-evidence +- I-evidence +binding I-evidence +affinities I-evidence +inferred O +from O +our O +crystallographic B-experimental_method +titrations I-experimental_method +and O +the O +Km B-evidence +and O +K1 B-evidence +/ I-evidence +2 I-evidence +values I-evidence +previously O +deduced O +from O +functional B-experimental_method +assays I-experimental_method +has O +been O +discussed O +above O +. O + +Consistent O +with O +that O +finding O +, O +mutations O +that O +have O +been O +shown O +to O +inactivate O +or O +diminish O +the O +transport O +activity O +of O +NCX_Mj B-protein +and O +cardiac O +NCX B-protein_type +perfectly O +map O +to O +the O +first O +ion O +- O +coordination O +shell O +in O +our O +NCX_Mj B-protein +structures B-evidence +( O +Supplementary O +Fig O +. O +4c O +- O +d O +). O + +The O +crystallographic B-evidence +data I-evidence +also O +provides O +the O +long O +- O +sought O +structural O +basis O +for O +the O +‘ O +two O +- O +site O +’ O +model O +proposed O +to O +describe O +competitive O +cation O +binding O +in O +eukaryotic B-taxonomy_domain +NCX B-protein_type +, O +underscoring O +the O +relevance O +of O +these O +studies O +of O +NCX_Mj B-protein +as O +a O +prototypical O +Na B-protein_type ++/ I-protein_type +Ca2 I-protein_type ++ I-protein_type +exchanger I-protein_type +. O + +The O +Sext B-site +site O +, O +by O +contrast O +, O +might O +be O +thought O +as O +an O +activation B-site +site I-site +for O +inward O +Na B-chemical ++ I-chemical +translocation O +, O +since O +this O +is O +where O +the O +third O +Na B-chemical ++ I-chemical +ion O +binds O +at O +high O +Na B-chemical ++ I-chemical +concentration O +, O +enabling O +the O +transition O +to O +the O +occluded B-protein_state +state O +. O + +Indeed O +, O +structures B-evidence +of O +NCX_Mj B-protein +bound B-protein_state +to I-protein_state +Cd2 B-chemical ++ I-chemical +or O +Mn2 B-chemical ++, I-chemical +both O +of O +which O +inhibit O +transport O +, O +show O +these O +ions O +at O +Smid B-site +; O +by O +contrast O +, O +Sr2 B-chemical ++ I-chemical +binds O +only O +to O +SCa B-site +, O +and O +accordingly O +, O +is O +transported O +by O +NCX B-protein_type +similarly O +to O +calcium B-chemical +. O + +Lastly O +, O +our O +theory O +that O +occlusion O +of O +NCX_Mj B-protein +is O +selectively O +induced O +upon O +Ca2 B-chemical ++ I-chemical +or O +Na B-chemical ++ I-chemical +recognition O +is O +consonant O +with O +a O +recent O +analysis O +of O +the O +rate O +of O +hydrogen B-experimental_method +- I-experimental_method +deuterium I-experimental_method +exchange I-experimental_method +( O +HDX B-experimental_method +) O +in O +NCX_Mj B-protein +, O +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +these O +ions O +, O +in O +conditions O +that O +favor O +outward B-protein_state +- I-protein_state +facing I-protein_state +conformations O +. O + +We O +interpret O +these O +observations O +as O +reflecting O +that O +the O +solvent O +accessibility O +of O +the O +protein O +interior O +is O +diminished O +upon O +ion O +recognition O +, O +consistent O +with O +our O +finding O +that O +opening O +and O +closing O +of O +extracellular O +aqueous O +pathways O +to O +the O +ion B-site +- I-site +binding I-site +sites I-site +depend O +on O +ion O +occupancy O +state O +. O + +In O +addition O +, O +the O +increased O +compactness O +of O +the O +protein O +tertiary O +structure O +in O +the O +occluded B-protein_state +state O +would O +also O +slow O +down O +the O +dynamics O +of O +the O +secondary O +- O +structure O +elements O +, O +and O +thus O +further O +reduce O +the O +HDX B-evidence +rate I-evidence +. O + +Our O +data O +would O +also O +explain O +the O +observation O +that O +the O +reduction O +in O +the O +HDX B-evidence +rate I-evidence +is O +comparable O +for O +Na B-chemical ++ I-chemical +and O +Ca2 B-chemical ++, I-chemical +as O +well O +as O +the O +finding O +that O +the O +degree O +of O +deuterium O +incorporation O +remains O +non O +- O +negligible O +even O +under O +saturating O +ion O +concentrations O +. O + +As O +the O +calculated B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscapes I-evidence +show O +, O +Na B-chemical ++ I-chemical +and O +Ca2 B-chemical ++ I-chemical +induce O +the O +occlusion O +of O +the O +transporter B-protein_type +in O +a O +comparable O +manner O +, O +and O +yet O +the O +ion B-protein_state +- I-protein_state +bound I-protein_state +states O +retain O +the O +ability O +to O +explore O +conformations O +that O +are O +partially O +or O +fully B-protein_state +open I-protein_state +to O +the O +extracellular O +solution O +, O +precisely O +so O +as O +to O +be O +able O +to O +unload O +and O +re O +- O +load O +the O +substrates O +. O + +Colored O +spheres O +represent O +the O +bound O +Na B-chemical ++ I-chemical +( O +green O +) O +and O +water B-chemical +( O +red O +). O + +( O +b O +) O +Structural O +details O +and O +definition O +of O +the O +four O +central B-site +binding I-site +sites I-site +. O + +Further O +details O +are O +shown O +in O +Supplementary O +Fig O +. O +1 O +. O +( O +c O +) O +Concentration O +- O +dependent O +change O +in O +Na B-chemical ++ I-chemical +occupancy O +( O +see O +also O +Table O +1 O +). O + +All O +Fo B-evidence +– I-evidence +Fc I-evidence +ion I-evidence +- I-evidence +omit I-evidence +maps I-evidence +are O +calculated O +to O +2 O +. O +4 O +Å O +and O +contoured O +at O +3σ O +for O +comparison O +. O + +At O +20 O +mM O +Na B-chemical ++, I-chemical +both O +conformations O +co O +- O +exist O +. O + +Na B-chemical ++- I-chemical +occupancy O +dependent O +conformational O +change O +in O +NCX_Mj B-protein +. O + +( O +a O +) O +Superimposition B-experimental_method +of O +the O +NCX_Mj B-protein +crystal B-evidence +structures I-evidence +obtained O +in O +high O +( O +100 O +mM O +, O +cyan O +cylinders O +) O +and O +low O +( O +10 O +mM O +, O +brown O +cylinders O +) O +Na B-chemical ++ I-chemical +concentrations O +. O + +( O +b O +) O +Close O +- O +up O +view O +of O +the O +interface B-site +between O +TM6 B-structure_element +and O +TM7ab B-structure_element +in O +the O +NCX_Mj B-protein +structures B-evidence +obtained O +at O +high O +and O +low O +Na B-chemical ++ I-chemical +concentrations O +( O +top O +and O +lower O +panels O +, O +respectively O +). O + +( O +c O +) O +Close O +- O +up O +view O +of O +the O +Na B-site ++- I-site +binding I-site +sites I-site +. O + +Residues O +surrounding O +this O +site O +are O +also O +indicated O +; O +note O +A206 B-residue_name_number +( O +labeled O +in O +red O +) O +coordinates B-bond_interaction +Na B-chemical ++ I-chemical +at O +Sext B-site +via O +its O +backbone O +carbonyl O +oxygen O +. O + +( O +d O +) O +Extracellular O +solvent O +accessibility O +of O +the O +ion B-site +binding I-site +sites I-site +in O +the O +structures B-evidence +at O +high B-protein_state +and O +low B-protein_state +[ O +Na B-chemical ++]. I-chemical + +Putative O +solvent B-site +channels I-site +are O +represented O +as O +light O +- O +purple O +surfaces O +. O + +Divalent O +cation O +binding O +and O +apo B-protein_state +structure B-evidence +of O +NCX_Mj B-protein +. O +( O +a O +) O +A O +single O +Sr2 B-chemical ++ I-chemical +( O +dark O +blue O +sphere O +) O +binds O +at O +SCa B-site +in O +crystals B-experimental_method +titrated I-experimental_method +with O +10 O +mM O +Sr2 B-chemical ++ I-chemical +and O +2 O +. O +5 O +mM O +Na B-chemical ++ I-chemical +( O +see O +also O +Supplementary O +Fig O +. O +2 O +). O + +There O +are O +no O +significant O +changes O +in O +the O +side O +- O +chains O +involved O +in O +ion O +coordination O +, O +relative O +to O +the O +Na B-protein_state ++- I-protein_state +bound I-protein_state +state O +. O + +T50 B-residue_name_number +and O +T209 B-residue_name_number +( O +labeled O +in O +red O +) O +coordinate B-bond_interaction +Sr2 B-chemical ++ I-chemical +through O +their O +backbone O +carbonyl O +- O +oxygen O +atoms O +. O + +High O +Na B-chemical ++ I-chemical +concentration O +( O +100 O +mM O +) O +completely O +displaces O +Sr2 B-chemical ++ I-chemical +and O +reverts O +NCX_Mj B-protein +to O +the O +occluded B-protein_state +state O +( O +right O +panel O +). O + +The O +contour O +level O +of O +the O +Fo B-evidence +– I-evidence +Fc I-evidence +omit I-evidence +map I-evidence +of O +the O +structure B-evidence +at O +high O +Na B-chemical ++ I-chemical +concentration O +was O +lowered O +( O +to O +4σ O +) O +so O +as O +to O +visualize O +the O +density B-evidence +from O +Na B-chemical ++ I-chemical +ions O +and O +H2O B-chemical +. O + +( O +b O +) O +Ca2 B-chemical ++ I-chemical +( O +tanned O +spheres O +) O +binds O +either O +to O +SCa B-site +or O +Smid B-site +in O +crystals B-experimental_method +titrated I-experimental_method +with O +10 O +mM O +Ca2 B-chemical ++ I-chemical +and O +2 O +. O +5 O +mM O +Na B-chemical ++ I-chemical +( O +see O +also O +Supplementary O +Fig O +. O +2 O +). O + +The O +relative O +occupancies O +are O +55 O +% O +and O +45 O +%, O +respectively O +. O +( O +c O +) O +Superimposition B-experimental_method +of O +NCX_Mj B-protein +structures B-evidence +obtained O +at O +low O +Na B-chemical ++ I-chemical +concentration O +( O +10 O +mM O +) O +and O +pH O +6 O +. O +5 O +( O +brown O +) O +and O +in O +the O +absence B-protein_state +of I-protein_state +Na B-chemical ++ I-chemical +and O +pH B-protein_state +4 I-protein_state +( O +light O +green O +), O +referred O +to O +as O +apo B-protein_state +state O +. O +( O +d O +) O +Close O +- O +up O +view O +of O +the O +ion B-site +- I-site +binding I-site +sites I-site +in O +the O +apo B-protein_state +( O +or O +high B-protein_state +H I-protein_state ++) I-protein_state +state O +. O + +The O +side O +chains O +of O +E54 B-residue_name_number +and O +E213 B-residue_name_number +from O +the O +low B-protein_state +Na I-protein_state ++ I-protein_state +structure B-evidence +are O +also O +shown O +( O +light O +brown O +) O +for O +comparison O +. O + +White O +spheres O +indicate O +the O +location O +Sint B-site +, O +Smid B-site +SCa B-site +. O +( O +e O +) O +Extracellular O +solvent O +accessibility O +of O +the O +ion B-site +- I-site +binding I-site +sites I-site +in O +apo B-protein_state +NCX_Mj B-protein +. O + +Spontaneous O +changes O +in O +the O +structure B-evidence +of O +outward B-protein_state +- I-protein_state +occluded I-protein_state +, O +fully B-protein_state +Na I-protein_state ++- I-protein_state +occupied I-protein_state +NCX_Mj B-protein +( O +PDB O +code O +3V5U O +) O +upon O +sequential O +displacement O +of O +Na B-chemical ++. I-chemical + +( O +a O +) O +Representative O +simulation B-experimental_method +snapshots O +of O +NCX_Mj B-protein +( O +Methods O +) O +with O +Na B-chemical ++ I-chemical +bound B-protein_state +at I-protein_state +Sext B-site +, O +SCa B-site +and O +Sint B-site +( O +orange O +cartoons O +, O +green O +spheres O +) O +and O +with O +Na B-chemical ++ I-chemical +bound B-protein_state +only I-protein_state +at I-protein_state +SCa B-site +and O +Sint B-site +( O +marine O +cartoons O +, O +yellow O +spheres O +) O +( O +b O +) O +Close O +- O +up O +of O +the O +backbone O +of O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +half I-structure_element +of O +TM7 B-structure_element +( O +TM7ab B-structure_element +), O +in O +the O +same O +Na B-chemical ++ I-chemical +occupancy O +states O +depicted O +in O +( O +a O +). O + +( O +c O +) O +Representative O +simulation B-evidence +snapshots I-evidence +( O +same O +as O +above O +) O +with O +Na B-chemical ++ I-chemical +bound B-protein_state +at I-protein_state +SCa B-site +and O +Sint B-site +( O +marine O +cartoons O +, O +yellow O +spheres O +) O +and O +without B-protein_state +any O +Na B-chemical ++ I-chemical +bound B-protein_state +( O +grey O +cartoons O +). O + +Approximate O +distances O +between O +TM2 B-structure_element +, O +TM3 B-structure_element +and O +TM7 B-structure_element +are O +indicated O +in O +Å O +. O +( O +e O +) O +Close O +- O +up O +of O +the O +ion B-site +- I-site +binding I-site +region I-site +in O +the O +partially B-protein_state +Na I-protein_state ++- I-protein_state +occupied I-protein_state +state O +. O + +( O +f O +) O +Close O +- O +up O +of O +the O +ion B-site +- I-site +binding I-site +region I-site +in O +the O +Na B-protein_state ++- I-protein_state +free I-protein_state +state O +. O +( O +g O +- O +i O +) O +Analytical O +descriptors O +of O +the O +changes O +just O +described O +, O +calculated O +from O +the O +simulations B-experimental_method +of O +each O +Na B-protein_state ++- I-protein_state +occupancy I-protein_state +state O +depicted O +in O +panels O +( O +a O +- O +f O +). O + +( O +g O +) O +Probability B-evidence +distributions I-evidence +of O +an O +analytical O +descriptor O +of O +the O +backbone O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +pattern O +in O +TM7ab B-structure_element +( O +Eq O +. O +2 O +). O +( O +h O +) O +Mean O +value O +( O +with O +standard O +deviation O +) O +of O +a O +quantitative O +descriptor O +of O +the O +solvent O +accessibility O +of O +the O +Sext B-site +site O +( O +Eq O +. O +1 O +). O +( O +i O +) O +Mean O +value O +( O +with O +standard O +deviation O +) O +of O +a O +quantitative O +descriptor O +of O +the O +solvent O +accessibility O +of O +the O +SCa B-site +site O +( O +Eq O +. O +1 O +). O + +The O +free B-evidence +energy I-evidence +is O +plotted O +as O +a O +function O +of O +two O +coordinates O +, O +each O +describing O +the O +degree O +of O +opening O +of O +the O +aqueous B-site +channels I-site +leading O +to O +the O +Sext B-site +and O +SCa B-site +sites O +, O +respectively O +( O +see O +Methods O +). O + +Black O +circles O +map O +the O +X B-evidence +- I-evidence +ray I-evidence +structures I-evidence +of O +NCX_Mj B-protein +obtained O +at O +high B-protein_state +and O +low B-protein_state +Na B-chemical ++ I-chemical +concentration O +, O +as O +well O +as O +that O +at O +low B-protein_state +pH I-protein_state +, O +reported O +in O +this O +study O +. O + +( O +b O +) O +Density B-evidence +isosurfaces I-evidence +for O +water B-chemical +molecules O +within O +12 O +Å O +of O +the O +ion B-site +- I-site +binding I-site +region I-site +( O +grey O +volumes O +), O +for O +each O +of O +the O +major O +conformational B-evidence +free I-evidence +- I-evidence +energy I-evidence +minima I-evidence +in O +each O +ion O +- O +occupancy O +state O +. O + +Na B-chemical ++ I-chemical +ions O +are O +shown O +as O +green O +spheres O +. O + +The O +two O +inverted B-structure_element +- I-structure_element +topology I-structure_element +repeats I-structure_element +in O +the O +transporter B-protein_type +structure B-evidence +( O +transparent O +cartoons O +) O +are O +colored O +differently O +( O +TM1 B-structure_element +- I-structure_element +5 I-structure_element +, O +orange O +; O +TM6 B-structure_element +- I-structure_element +10 I-structure_element +, O +marine O +). O + +( O +c O +) O +Close O +- O +up O +views O +of O +the O +ion B-site +- I-site +binding I-site +region I-site +in O +the O +same O +conformational B-evidence +free I-evidence +- I-evidence +energy I-evidence +minima I-evidence +. O + +Key O +residues O +involved O +in O +Na B-chemical ++ I-chemical +and O +water B-chemical +coordination O +( O +W O +) O +are O +highlighted O +( O +sticks O +, O +black O +lines O +). O + +The O +water B-evidence +- I-evidence +density I-evidence +maps I-evidence +in O +( O +b O +) O +is O +shown O +here O +as O +a O +grey O +mesh O +. O + +Note O +D240 B-residue_name_number +is O +protonated O +, O +while O +E54 B-residue_name_number +and O +E213 B-residue_name_number +are O +ionized O +. O + +Thermodynamic O +basis O +for O +the O +proposed O +mechanism O +of O +substrate O +control O +of O +the O +alternating O +- O +access O +transition O +of O +NCX B-protein_type +. O +( O +a O +) O +Calculated B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscapes I-evidence +for O +outward B-protein_state +- I-protein_state +facing I-protein_state +NCX_Mj B-protein +, O +for O +the O +Ca2 B-chemical ++ I-chemical +and O +the O +fully B-protein_state +protonated I-protein_state +state O +. O + +The O +free B-evidence +energy I-evidence +is O +plotted O +as O +in O +Fig O +. O +5 O +. O + +Black O +circles O +map O +the O +crystal B-evidence +structures I-evidence +obtained O +at O +high O +Ca2 B-chemical ++ I-chemical +concentration O +and O +at O +low B-protein_state +pH I-protein_state +( O +or O +high B-protein_state +H I-protein_state ++) I-protein_state +reported O +in O +this O +study O +. O + +( O +b O +) O +Water B-evidence +- I-evidence +density I-evidence +isosurfaces I-evidence +analogous O +to O +those O +in O +Fig O +. O +5 O +are O +shown O +for O +each O +of O +the O +major O +conformational O +free B-evidence +- I-evidence +energy I-evidence +minima I-evidence +in O +the O +free B-evidence +- I-evidence +energy I-evidence +maps I-evidence +. O + +The O +Ca2 B-chemical ++ I-chemical +ion O +is O +shown O +as O +a O +red O +sphere O +; O +the O +protein O +is O +shown O +as O +in O +Fig O +. O +5 O +. O +( O +c O +) O +Close O +- O +up O +views O +of O +the O +ion B-site +- I-site +binding I-site +region I-site +in O +the O +same O +conformational B-evidence +free I-evidence +- I-evidence +energy I-evidence +minima I-evidence +. O + +Key O +residues O +involved O +in O +Ca2 B-chemical ++ I-chemical +and O +water B-chemical +coordination O +( O +W O +) O +are O +highlighted O +( O +sticks O +, O +black O +lines O +). O + +The O +water B-evidence +- I-evidence +density I-evidence +maps I-evidence +in O +( O +b O +) O +are O +shown O +here O +as O +a O +grey O +mesh O +. O + +In O +the O +occluded B-protein_state +state O +with O +Ca2 B-chemical ++ I-chemical +bound B-protein_state +, O +helix B-structure_element +TM7ab B-structure_element +bends O +in O +the O +same O +way O +as O +in O +the O +fully B-protein_state +occupied I-protein_state +Na B-chemical ++ I-chemical +state O +, O +as O +the O +carbonyl O +of O +Ala206 B-residue_name_number +forms O +a O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +interaction I-bond_interaction +with O +Ser210 B-residue_name_number +. O + +Structural O +mechanism O +of O +extracellular O +forward O +ion O +exchange O +in O +NCX B-protein_type +. O + +The O +carbonyl O +groups O +of O +Ala47 B-residue_name_number +( O +on O +TM2b B-structure_element +) O +and O +Ala206 B-residue_name_number +( O +on O +TM7b B-structure_element +), O +and O +the O +side O +chains O +of O +Glu54 B-residue_name_number +( O +on O +TM2c B-structure_element +) O +and O +Glu213 B-residue_name_number +( O +on O +TM7c B-structure_element +) O +are O +highlighted O +; O +these O +are O +four O +of O +the O +key O +residues O +for O +ion O +chelation O +and O +conformational O +changes O +. O + +The O +green O +open O +cylinders O +represent O +the O +gating B-structure_element +helices I-structure_element +TM1 B-structure_element +and O +TM6 B-structure_element +. O + +These O +states O +and O +their O +connectivity O +can O +also O +be O +deduced O +from O +the O +calculated B-evidence +free I-evidence +- I-evidence +energy I-evidence +landscapes I-evidence +, O +which O +also O +reveal O +a O +Ca2 B-protein_state ++- I-protein_state +loaded I-protein_state +outward B-protein_state +- I-protein_state +facing I-protein_state +occluded B-protein_state +state O +, O +and O +an O +unloaded B-protein_state +, O +fully B-protein_state +open I-protein_state +state O +. 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O + +Altogether O +, O +these O +results O +advance O +the O +mechanistic O +understanding O +of O +molecular O +recognition O +for O +a O +major O +class O +of O +splice B-site +site I-site +signals O +. O + +Here O +, O +the O +authors O +report O +U2AF65 B-protein +structures B-evidence +and O +single B-experimental_method +molecule I-experimental_method +FRET I-experimental_method +that O +reveal O +mechanistic O +insights O +into O +splice B-site +site I-site +recognition O +. O + +The O +differential O +skipping O +or O +inclusion O +of O +alternatively O +spliced O +pre B-structure_element +- I-structure_element +mRNA I-structure_element +regions I-structure_element +is O +a O +major O +source O +of O +diversity O +for O +nearly O +all O +human B-species +gene O +transcripts O +. O + +The O +splice B-site +sites I-site +are O +marked O +by O +relatively O +short B-structure_element +consensus I-structure_element +sequences I-structure_element +and O +are O +regulated O +by O +additional O +pre B-structure_element +- I-structure_element +mRNA I-structure_element +motifs I-structure_element +( O +reviewed O +in O +ref O +.). O + +At O +the O +3 B-site +′ I-site +splice I-site +site I-site +of O +the O +major O +intron O +class O +, O +these O +include O +a O +polypyrimidine B-chemical +( I-chemical +Py I-chemical +) I-chemical +tract I-chemical +comprising O +primarily O +Us B-residue_name +or O +Cs B-residue_name +, O +which O +is O +preceded O +by O +a O +branch B-site +point I-site +sequence I-site +( O +BPS B-site +) O +that O +ultimately O +serves O +as O +the O +nucleophile O +in O +the O +splicing O +reaction O +and O +an O +AG B-chemical +- I-chemical +dinucleotide I-chemical +at O +the O +3 B-site +′ I-site +splice I-site +site I-site +junction O +. O + +Disease O +- O +causing O +mutations O +often O +compromise O +pre B-chemical +- I-chemical +mRNA I-chemical +splicing O +( O +reviewed O +in O +refs O +), O +yet O +a O +priori O +predictions O +of O +splice B-site +sites I-site +and O +the O +consequences O +of O +their O +mutations O +are O +challenged O +by O +the O +brevity O +and O +degeneracy O +of O +known O +splice B-site +site I-site +sequences O +. O + +High O +- O +resolution O +structures B-evidence +of O +intact B-protein_state +splicing B-complex_assembly +factor I-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complexes O +would O +offer O +key O +insights O +regarding O +the O +juxtaposition O +of O +the O +distinct O +splice B-site +site I-site +consensus O +sequences O +and O +their O +relationship O +to O +disease O +- O +causing O +point O +mutations O +. O + +The O +early O +- O +stage O +pre B-protein_type +- I-protein_type +mRNA I-protein_type +splicing I-protein_type +factor I-protein_type +U2AF65 B-protein +is O +essential O +for O +viability O +in O +vertebrates B-taxonomy_domain +and O +other O +model O +organisms O +( O +for O +example O +, O +ref O +.). O + +A O +tightly O +controlled O +assembly B-complex_assembly +among O +U2AF65 B-protein +, O +the O +pre B-chemical +- I-chemical +mRNA I-chemical +, O +and O +partner O +proteins O +sequentially O +identifies O +the O +3 B-site +′ I-site +splice I-site +site I-site +and O +promotes O +association O +of O +the O +spliceosome B-complex_assembly +, O +which O +ultimately O +accomplishes O +the O +task O +of O +splicing O +. O + +Initially O +U2AF65 B-protein +recognizes O +the O +Py B-chemical +- I-chemical +tract I-chemical +splice B-site +site I-site +signal O +. O + +In O +turn O +, O +the O +ternary B-complex_assembly +complex I-complex_assembly +of O +U2AF65 B-protein +with O +SF1 B-protein +and O +U2AF35 B-protein +identifies O +the O +surrounding O +BPS B-site +and O +3 B-site +′ I-site +splice I-site +site I-site +junctions O +. O + +Subsequently O +U2AF65 B-protein +recruits O +the O +U2 B-complex_assembly +small I-complex_assembly +nuclear I-complex_assembly +ribonucleoprotein I-complex_assembly +particle I-complex_assembly +( O +snRNP B-complex_assembly +) O +and O +ultimately O +dissociates O +from O +the O +active B-protein_state +spliceosome B-complex_assembly +. O + +Biochemical B-experimental_method +characterizations I-experimental_method +of O +U2AF65 B-protein +demonstrated O +that O +tandem O +RNA B-structure_element +recognition I-structure_element +motifs I-structure_element +( O +RRM1 B-structure_element +and O +RRM2 B-structure_element +) O +recognize O +the O +Py B-chemical +tract I-chemical +( O +Fig O +. O +1a O +). O + +A O +subsequent O +NMR B-experimental_method +structure B-evidence +characterized O +the O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +arrangement O +of O +the O +minimal B-protein_state +U2AF65 B-protein +RRM1 B-structure_element +and O +RRM2 B-structure_element +connected O +by O +a O +linker B-structure_element +of O +natural B-protein_state +length I-protein_state +( O +U2AF651 B-mutant +, I-mutant +2 I-mutant +), O +yet O +depended O +on O +the O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +crystal B-evidence +structures I-evidence +for O +RNA B-chemical +interactions O +and O +an O +ab O +initio O +model O +for O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +conformation O +. O + +As O +such O +, O +the O +molecular O +mechanisms O +for O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +by O +the O +intact B-protein_state +U2AF65 B-structure_element +– I-structure_element +RNA I-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +remained O +unknown O +. O + +Here O +, O +we O +use O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +and O +biochemical B-experimental_method +studies I-experimental_method +to O +reveal O +new O +roles O +in O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +for O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +and O +key O +residues O +surrounding O +the O +core B-protein_state +U2AF65 B-protein +RRMs B-structure_element +. O + +Cognate O +U2AF65 B-protein +– O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +requires O +RRM B-structure_element +extensions I-structure_element + +The O +RNA B-evidence +affinity I-evidence +of O +the O +minimal B-protein_state +U2AF651 B-mutant +, I-mutant +2 I-mutant +domain O +comprising O +the O +core B-protein_state +RRM1 B-structure_element +– O +RRM2 B-structure_element +folds B-structure_element +( O +U2AF651 B-mutant +, I-mutant +2 I-mutant +, O +residues O +148 B-residue_range +– I-residue_range +336 I-residue_range +) O +is O +relatively O +weak O +compared O +with O +full B-protein_state +- I-protein_state +length I-protein_state +U2AF65 B-protein +( O +Fig O +. O +1a O +, O +b O +; O +Supplementary O +Fig O +. O +1 O +). O + +Historically O +, O +this O +difference O +was O +attributed O +to O +the O +U2AF65 B-protein +arginine B-structure_element +– I-structure_element +serine I-structure_element +rich I-structure_element +domain I-structure_element +, O +which O +contacts O +pre B-complex_assembly +- I-complex_assembly +mRNA I-complex_assembly +– I-complex_assembly +U2 I-complex_assembly +snRNA I-complex_assembly +duplexes I-complex_assembly +outside O +of O +the O +Py B-chemical +tract I-chemical +. O + +We O +noticed O +that O +the O +RNA B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +the O +U2AF651 B-mutant +, I-mutant +2 I-mutant +domain O +was O +greatly O +enhanced O +by O +the O +addition B-experimental_method +of I-experimental_method +seven I-experimental_method +and I-experimental_method +six I-experimental_method +residues I-experimental_method +at O +the O +respective O +N O +and O +C O +termini O +of O +the O +minimal B-protein_state +RRM1 B-structure_element +and O +RRM2 B-structure_element +( O +U2AF651 B-mutant +, I-mutant +2L I-mutant +, O +residues O +141 B-residue_range +– I-residue_range +342 I-residue_range +; O +Fig O +. O +1a O +). O + +Likewise O +, O +both O +U2AF651 B-mutant +, I-mutant +2L I-mutant +and O +full B-protein_state +- I-protein_state +length I-protein_state +U2AF65 B-protein +showed O +similar O +sequence B-evidence +specificity I-evidence +for O +U B-structure_element +- I-structure_element +rich I-structure_element +stretches I-structure_element +in O +the O +5 B-site +′- I-site +region I-site +of O +the O +Py B-chemical +tract I-chemical +and O +promiscuity O +for O +C B-structure_element +- I-structure_element +rich I-structure_element +regions I-structure_element +in O +the O +3 B-site +′- I-site +region I-site +( O +Fig O +. O +1c O +, O +Supplementary O +Fig O +. O +1e O +– O +h O +). O + +U2AF65 B-protein_state +- I-protein_state +bound I-protein_state +Py B-chemical +tract I-chemical +comprises O +nine O +contiguous B-structure_element +nucleotides B-chemical + +To O +investigate O +the O +structural O +basis O +for O +cognate O +U2AF65 B-protein +recognition O +of O +a O +contiguous B-structure_element +Py B-chemical +tract I-chemical +, O +we O +determined B-experimental_method +four O +crystal B-evidence +structures I-evidence +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +bound B-protein_state +to I-protein_state +Py B-chemical +- I-chemical +tract I-chemical +oligonucleotides I-chemical +( O +Fig O +. O +2a O +; O +Table O +1 O +). O + +By O +sequential B-experimental_method +boot I-experimental_method +strapping I-experimental_method +( O +Methods O +), O +we O +optimized O +the O +oligonucleotide B-chemical +length O +, O +the O +position O +of O +a O +Br B-chemical +- I-chemical +dU I-chemical +, O +and O +the O +identity O +of O +the O +terminal O +nucleotide B-chemical +( O +rU B-residue_name +, O +dU B-residue_name +and O +rC B-residue_name +) O +to O +achieve O +full O +views O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +bound B-protein_state +to I-protein_state +contiguous B-structure_element +Py B-chemical +tracts I-chemical +at O +up O +to O +1 O +. O +5 O +Å O +resolution O +. O + +The O +protein O +and O +oligonucleotide B-chemical +conformations O +are O +nearly O +identical O +among O +the O +four O +new O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +( O +Supplementary O +Fig O +. O +2a O +). O + +The O +U2AF651 B-mutant +, I-mutant +2L I-mutant +RRM1 B-structure_element +and O +RRM2 B-structure_element +associate O +with O +the O +Py B-chemical +tract I-chemical +in O +a O +parallel B-protein_state +, O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +arrangement O +( O +shown O +for O +representative O +structure O +iv O +in O +Fig O +. O +2b O +, O +c O +; O +Supplementary O +Movie O +1 O +). O + +An O +extended B-protein_state +conformation I-protein_state +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +traverses O +across O +the O +α B-structure_element +- I-structure_element +helical I-structure_element +surface I-structure_element +of O +RRM1 B-structure_element +and O +the O +central O +β B-structure_element +- I-structure_element +strands I-structure_element +of O +RRM2 B-structure_element +and O +is O +well O +defined O +in O +the O +electron B-evidence +density I-evidence +( O +Fig O +. O +2b O +). O + +The O +extensions B-structure_element +at O +the O +N O +terminus O +of O +RRM1 B-structure_element +and O +C O +terminus O +of O +RRM2 B-structure_element +adopt O +well O +- O +ordered O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +We O +compare O +the O +global O +conformation O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +with O +the O +prior O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +crystal B-evidence +structure I-evidence +and O +U2AF651 B-mutant +, I-mutant +2 I-mutant +NMR B-experimental_method +structure B-evidence +in O +the O +Supplementary O +Discussion O +and O +Supplementary O +Fig O +. O +2 O +. O + +Based O +on O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +structures B-evidence +, O +we O +originally O +hypothesized O +that O +the O +U2AF65 B-protein +RRMs B-structure_element +would O +bind O +the O +minimal B-protein_state +seven O +nucleotides B-chemical +observed O +in O +these O +structures B-evidence +. O + +Qualitatively O +, O +a O +subset O +of O +the O +U2AF651 B-site +, I-site +2L I-site +- I-site +nucleotide I-site +- I-site +binding I-site +sites I-site +( O +sites B-site +1 I-site +– I-site +3 I-site +and O +7 B-site +– I-site +9 I-site +) O +share O +similar O +locations O +to O +those O +of O +the O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +structures B-evidence +( O +Supplementary O +Figs O +2c O +, O +d O +and O +3 O +). O + +Yet O +, O +only O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +interactions O +at O +sites B-site +1 I-site +and I-site +7 I-site +are O +nearly O +identical O +to O +those O +of O +the O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +structures B-evidence +( O +Supplementary O +Fig O +. O +3a O +, O +f O +). O + +In O +striking O +departures O +from O +prior O +partial O +views O +, O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +reveal O +three O +unanticipated O +nucleotide B-site +- I-site +binding I-site +sites I-site +at O +the O +centre O +of O +the O +Py B-chemical +tract I-chemical +, O +as O +well O +as O +numerous O +new O +interactions O +that O +underlie O +cognate O +recognition O +of O +the O +Py B-chemical +tract I-chemical +( O +Fig O +. O +3a O +– O +h O +). O + +The O +U2AF651 B-mutant +, I-mutant +2L I-mutant +RRM2 B-structure_element +, O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +and O +RRM1 B-structure_element +concomitantly O +recognize O +the O +three O +central O +nucleotides B-chemical +of O +the O +Py B-chemical +tract I-chemical +, O +which O +are O +likely O +to O +coordinate O +the O +conformational O +arrangement O +of O +these O +disparate O +portions O +of O +the O +protein O +. O + +Residues O +in O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +comprise O +a O +centrally O +located O +binding B-site +site I-site +for O +the O +fifth B-residue_number +nucleotide B-chemical +on O +the O +RRM2 B-site +surface I-site +and O +abutting O +the O +RRM1 B-site +/ I-site +RRM2 I-site +interface I-site +( O +Fig O +. O +3d O +). O + +The O +backbone O +amide O +of O +the O +linker B-structure_element +V254 B-residue_name_number +and O +the O +carbonyl O +of O +T252 B-residue_name_number +engage O +in O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +rU5 B-residue_name_number +- O +O4 O +and O +- O +N3H O +atoms O +. O + +In O +the O +C O +- O +terminal O +β B-structure_element +- I-structure_element +strand I-structure_element +of O +RRM1 B-structure_element +, O +the O +side O +chains O +of O +K225 B-residue_name_number +and O +R227 B-residue_name_number +donate O +additional O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +rU5 B-residue_name_number +- O +O2 O +lone O +pair O +electrons O +. O + +The O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +also O +participates O +in O +the O +preceding O +rU4 B-site +- I-site +binding I-site +site I-site +, O +where O +the O +V254 B-residue_name_number +backbone O +carbonyl O +and O +D256 B-residue_name_number +carboxylate O +position O +the O +K260 B-residue_name_number +side O +chain O +to O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +rU4 B-residue_name_number +- O +O4 O +( O +Fig O +. O +3c O +). O + +This O +nucleotide B-chemical +twists O +to O +face O +away O +from O +the O +U2AF65 B-protein +linker B-structure_element +and O +instead O +inserts O +the O +rU6 B-residue_name_number +- O +uracil B-residue_name +into O +a O +sandwich O +between O +the O +β2 B-structure_element +/ I-structure_element +β3 I-structure_element +loops I-structure_element +of O +RRM1 B-structure_element +and O +RRM2 B-structure_element +. O + +The O +rU6 B-residue_name_number +base O +edge O +is O +relatively O +solvent B-protein_state +exposed I-protein_state +; O +accordingly O +, O +the O +rU6 B-residue_name_number +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +U2AF65 B-protein +are O +water B-chemical +mediated O +apart O +from O +a O +single O +direct O +interaction O +by O +the O +RRM1 B-structure_element +- O +N196 B-residue_name_number +side O +chain O +. O + +Mutagenesis B-experimental_method +of O +either O +V254 B-residue_name_number +in O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +to O +proline B-residue_name +or O +RRM1 B-structure_element +– O +R227 B-residue_name_number +to O +alanine B-residue_name +, O +which O +remove O +the O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +fifth B-residue_number +uracil B-residue_name +- O +O4 O +or O +- O +O2 O +, O +reduced O +the O +affinities B-evidence +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +for O +the O +representative O +AdML B-gene +Py B-chemical +tract I-chemical +by O +four O +- O +or O +five O +- O +fold O +, O +respectively O +. O + +The O +energetic O +penalties O +due O +to O +these O +mutations O +( O +ΔΔG B-evidence +0 O +. O +8 O +– O +0 O +. O +9 O +kcal O +mol O +− O +1 O +) O +are O +consistent O +with O +the O +loss O +of O +each O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +rU5 B-residue_name_number +base O +and O +support O +the O +relevance O +of O +the O +central O +nucleotide O +interactions O +observed O +in O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +. O + +The O +N B-structure_element +- I-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +extensions I-structure_element +of O +the O +U2AF65 B-protein +RRM1 B-structure_element +and O +RRM2 B-structure_element +directly O +contact O +the O +bound B-protein_state +Py B-chemical +tract I-chemical +. O + +Rather O +than O +interacting O +with O +a O +new O +5 O +′- O +terminal O +nucleotide B-chemical +as O +we O +had O +hypothesized O +, O +the O +C O +- O +terminal O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +RRM2 B-structure_element +instead O +folds O +across O +one O +surface O +of O +rU3 B-residue_name_number +in O +the O +third B-site +binding I-site +site I-site +( O +Fig O +. O +3b O +). O + +There O +, O +a O +salt B-bond_interaction +bridge I-bond_interaction +between O +the O +K340 B-residue_name_number +side O +chain O +and O +nucleotide B-chemical +phosphate O +, O +as O +well O +as O +G338 B-residue_name_number +- O +base O +stacking B-bond_interaction +and O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +the O +backbone O +amide O +of O +G338 B-residue_name_number +and O +the O +rU3 B-residue_name_number +- O +O4 O +, O +secure O +the O +RRM2 B-structure_element +extension I-structure_element +. O + +Consequently O +, O +the O +U2AF651 B-protein_state +, I-protein_state +2L I-protein_state +- I-protein_state +bound I-protein_state +rU2 B-residue_name_number +- O +O4 O +and O +- O +N3H O +form O +dual O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +K329 B-residue_name_number +backbone O +atoms O +( O +Fig O +. O +3a O +), O +rather O +than O +a O +single O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +K329 B-residue_name_number +side O +chain O +as O +in O +the O +prior O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +structure B-evidence +( O +Supplementary O +Fig O +. O +3b O +). O + +At O +the O +N O +terminus O +, O +the O +α B-structure_element +- I-structure_element +helical I-structure_element +extension I-structure_element +of O +U2AF65 B-protein +RRM1 B-structure_element +positions O +the O +Q147 B-residue_name_number +side O +chain O +to O +bridge O +the O +eighth B-residue_number +and O +ninth B-residue_number +nucleotides B-chemical +at O +the O +3 B-site +′ I-site +terminus I-site +of O +the O +Py B-chemical +tract I-chemical +( O +Fig O +. O +3f O +– O +h O +). O + +The O +Q147 B-residue_name_number +residue O +participates O +in O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +- O +N3H O +of O +the O +eighth B-residue_number +uracil B-residue_name +and O +- O +O2 O +of O +the O +ninth B-residue_number +pyrimidine B-chemical +. O + +The O +adjacent O +R146 B-residue_name_number +guanidinium O +group O +donates O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +3 O +′- O +terminal O +ribose B-chemical +- O +O2 O +′ O +and O +O3 O +′ O +atoms O +, O +where O +it O +could O +form O +a O +salt B-bond_interaction +bridge I-bond_interaction +with O +a O +phospho O +- O +diester O +group O +in O +the O +context O +of O +a O +longer O +pre B-chemical +- I-chemical +mRNA I-chemical +. O + +Consistent O +with O +loss O +of O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +ninth B-residue_number +pyrimidine B-chemical +- O +O2 O +( O +ΔΔG B-evidence +1 O +. O +0 O +kcal O +mol O +− O +1 O +), O +mutation B-experimental_method +of O +the O +Q147 B-residue_name_number +to O +an O +alanine B-residue_name +reduced O +U2AF651 B-evidence +, I-evidence +2L I-evidence +affinity I-evidence +for O +the O +AdML B-gene +Py B-chemical +tract I-chemical +by O +five O +- O +fold O +( O +Fig O +. O +3i O +; O +Supplementary O +Fig O +. O +4c O +). O + +We O +compare B-experimental_method +U2AF65 B-protein +interactions O +with O +uracil B-residue_name +relative O +to O +cytosine B-residue_name +pyrimidines B-chemical +at O +the O +ninth B-site +binding I-site +site I-site +in O +Fig O +. O +3g O +, O +h O +and O +the O +Supplementary O +Discussion O +. O + +The O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +reveal O +that O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +mediates O +an O +extensive B-site +interface I-site +with O +the O +second O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +RRM1 B-structure_element +, O +the O +β2 B-structure_element +/ I-structure_element +β3 I-structure_element +strands I-structure_element +of O +RRM2 B-structure_element +and O +the O +N O +- O +terminal O +α B-structure_element +- I-structure_element +helical I-structure_element +extension I-structure_element +of O +RRM1 B-structure_element +. O + +Altogether O +, O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +residues O +( O +R228 B-residue_range +– I-residue_range +K260 I-residue_range +) O +bury O +2 O +, O +800 O +Å2 O +of O +surface O +area O +in O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +holo B-protein_state +- I-protein_state +protein I-protein_state +, O +suggestive O +of O +a O +cognate B-site +interface I-site +compared O +with O +1 O +, O +900 O +Å2 O +for O +a O +typical O +protein O +– O +protein O +complex O +. O + +The O +path O +of O +the O +linker B-structure_element +initiates O +at O +P229 B-residue_name_number +following O +the O +core B-protein_state +RRM1 B-structure_element +β B-structure_element +- I-structure_element +strand I-structure_element +, O +in O +a O +kink B-structure_element +that O +is O +positioned O +by O +intra B-bond_interaction +- I-bond_interaction +molecular I-bond_interaction +stacking I-bond_interaction +among O +the O +consecutive O +R228 B-residue_name_number +, O +Y232 B-residue_name_number +and O +P234 B-residue_name_number +side O +chains O +( O +Fig O +. O +4a O +, O +lower O +right O +). O + +In O +the O +neighbouring O +apical O +region O +of O +the O +linker B-structure_element +, O +the O +V244 B-residue_name_number +and O +V246 B-residue_name_number +side O +chains O +pack O +in O +a O +hydrophobic B-site +pocket I-site +between O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +of O +the O +core B-protein_state +RRM1 B-structure_element +. O + +The O +adjacent O +V249 B-residue_name_number +and O +V250 B-residue_name_number +are O +notable O +for O +their O +respective O +interactions O +that O +connect O +RRM1 B-structure_element +and O +RRM2 B-structure_element +at O +this O +distal O +interface B-site +from O +the O +RNA B-site +- I-site +binding I-site +site I-site +( O +Fig O +. O +4a O +, O +top O +). O + +A O +third B-structure_element +kink I-structure_element +stacks B-bond_interaction +P247 B-residue_name_number +and O +G248 B-residue_name_number +with O +Y245 B-residue_name_number +and O +re O +- O +orients O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +of O +the O +linker B-structure_element +towards O +the O +RRM2 B-structure_element +and O +bound B-protein_state +RNA B-chemical +. O + +Few O +direct O +contacts O +are O +made O +between O +the O +remaining O +residues O +of O +the O +linker B-structure_element +and O +the O +U2AF65 B-protein +RRM2 B-structure_element +; O +instead O +, O +the O +C O +- O +terminal O +conformation O +of O +the O +linker B-structure_element +appears O +primarily O +RNA B-chemical +mediated O +( O +Fig O +. O +3c O +, O +d O +). O + +We O +investigated O +whether O +the O +observed O +contacts O +between O +the O +RRMs B-structure_element +and O +linker B-structure_element +were O +critical O +for O +RNA O +binding O +by O +structure B-experimental_method +- I-experimental_method +guided I-experimental_method +mutagenesis I-experimental_method +( O +Fig O +. O +4b O +). O + +We O +titrated B-experimental_method +these O +mutant B-protein_state +U2AF651 B-mutant +, I-mutant +2L I-mutant +proteins O +into O +fluorescein B-chemical +- O +labelled O +AdML B-gene +Py B-chemical +- I-chemical +tract I-chemical +RNA I-chemical +and O +fit O +the O +fluorescence B-evidence +anisotropy I-evidence +changes I-evidence +to O +obtain O +the O +apparent O +equilibrium B-evidence +affinities I-evidence +( O +Supplementary O +Fig O +. O +4d O +– O +h O +). O + +We O +introduced O +glycine B-residue_name +substitutions B-experimental_method +to O +maximally O +reduce O +the O +buried O +surface O +area O +without O +directly O +interfering O +with O +its O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +backbone O +atoms O +and O +the O +base O +. O + +First O +, O +we O +replaced B-experimental_method +V249 B-residue_name_number +and O +V250 B-residue_name_number +at O +the O +RRM1 B-site +/ I-site +RRM2 I-site +interface I-site +and O +V254 B-residue_name_number +at O +the O +bound B-protein_state +RNA B-chemical +site O +with O +glycine B-residue_name +( O +3Gly B-mutant +). O + +However O +, O +the O +resulting O +decrease O +in O +the O +AdML B-gene +RNA B-evidence +affinity I-evidence +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +- I-mutant +3Gly I-mutant +mutant B-protein_state +relative O +to O +wild B-protein_state +- I-protein_state +type I-protein_state +protein B-protein +was O +not O +significant O +( O +Fig O +. O +4b O +). O + +In O +parallel O +, O +we O +replaced B-experimental_method +five O +linker B-structure_element +residues I-structure_element +( O +S251 B-residue_name_number +, O +T252 B-residue_name_number +, O +V253 B-residue_name_number +, O +V254 B-residue_name_number +and O +P255 B-residue_name_number +) O +at O +the O +fifth B-site +nucleotide I-site +- I-site +binding I-site +site I-site +with O +glycines B-residue_name +( O +5Gly B-mutant +) O +and O +also O +found O +that O +the O +RNA B-evidence +affinity I-evidence +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +- I-mutant +5Gly I-mutant +mutant B-protein_state +likewise O +decreased O +only O +slightly O +relative O +to O +wild B-protein_state +- I-protein_state +type I-protein_state +protein B-protein +. O + +A O +more O +conservative B-experimental_method +substitution I-experimental_method +of O +these O +five O +residues O +( O +251 B-residue_range +– I-residue_range +255 I-residue_range +) O +with O +an O +unrelated O +sequence O +capable O +of O +backbone O +- O +mediated O +hydrogen B-bond_interaction +bonds I-bond_interaction +( O +STVVP B-mutant +> I-mutant +NLALA I-mutant +) O +confirmed O +the O +subtle O +impact O +of O +this O +versatile O +inter B-structure_element +- I-structure_element +RRM I-structure_element +sequence I-structure_element +on O +affinity B-evidence +for O +the O +AdML B-gene +Py B-chemical +tract I-chemical +. O + +Finally O +, O +to O +ensure O +that O +these O +selective O +mutations O +were O +sufficient O +to O +disrupt O +the O +linker B-structure_element +/ O +RRM B-structure_element +contacts O +, O +we O +substituted B-experimental_method +glycine B-residue_name +for O +the O +majority O +of O +buried O +hydrophobic O +residues O +in O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +( O +including O +M144 B-residue_name_number +, O +L235 B-residue_name_number +, O +M238 B-residue_name_number +, O +V244 B-residue_name_number +, O +V246 B-residue_name_number +, O +V249 B-residue_name_number +, O +V250 B-residue_name_number +, O +S251 B-residue_name_number +, O +T252 B-residue_name_number +, O +V253 B-residue_name_number +, O +V254 B-residue_name_number +, O +P255 B-residue_name_number +; O +called O +12Gly B-mutant +). O + +To O +test O +the O +interplay O +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +with O +its O +N O +- O +and O +C O +- O +terminal O +RRM B-structure_element +extensions I-structure_element +, O +we O +constructed B-experimental_method +an O +internal O +linker B-experimental_method +deletion I-experimental_method +of O +20 B-residue_range +- I-residue_range +residues I-residue_range +within O +the O +extended B-protein_state +RNA B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +( O +dU2AF651 B-mutant +, I-mutant +2L I-mutant +). O + +We O +found O +that O +the O +affinity B-evidence +of O +dU2AF651 B-mutant +, I-mutant +2L I-mutant +for O +the O +AdML B-gene +RNA B-chemical +was O +significantly O +reduced O +relative O +to O +U2AF651 B-mutant +, I-mutant +2L I-mutant +( O +four O +- O +fold O +, O +Figs O +1b O +and O +4b O +; O +Supplementary O +Fig O +. O +4i O +). O + +Yet O +, O +it O +is O +well O +known O +that O +the O +linker B-experimental_method +deletion I-experimental_method +in O +the O +context O +of O +the O +minimal B-protein_state +RRM1 B-structure_element +– O +RRM2 B-structure_element +boundaries O +has O +no O +detectable O +effect O +on O +the O +RNA B-evidence +affinities I-evidence +of O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +compared O +with O +U2AF651 B-mutant +, I-mutant +2 I-mutant +( O +refs O +; O +Figs O +1b O +and O +4b O +; O +Supplementary O +Fig O +. O +4j O +). O + +The O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +suggest O +that O +an O +extended B-protein_state +conformation I-protein_state +of O +the O +truncated B-protein_state +dU2AF651 B-mutant +, I-mutant +2 I-mutant +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +would O +suffice O +to O +connect O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +RRM1 B-structure_element +C O +terminus O +to O +the O +N O +terminus O +of O +RRM2 B-structure_element +( O +24 O +Å O +distance O +between O +U2AF651 B-mutant +, I-mutant +2L I-mutant +R227 B-residue_name_number +- O +Cα O +– O +H259 B-residue_name_number +- O +Cα O +atoms O +), O +which O +agrees O +with O +the O +greater O +RNA B-evidence +affinities I-evidence +of O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +and O +U2AF651 B-mutant +, I-mutant +2 I-mutant +dual B-protein_state +RRMs B-structure_element +compared O +with O +the O +individual B-protein_state +U2AF65 B-protein +RRMs B-structure_element +. O + +This O +difference O +indicates O +that O +the O +linearly B-protein_state +distant I-protein_state +regions B-structure_element +of O +the O +U2AF65 B-protein +primary O +sequence O +, O +including O +Q147 B-residue_name_number +in O +the O +N O +- O +terminal O +RRM1 B-structure_element +extension I-structure_element +and O +R227 B-residue_name_number +/ O +V254 B-residue_name_number +in O +the O +N O +-/ O +C O +- O +terminal O +linker B-structure_element +regions I-structure_element +at O +the O +fifth B-site +nucleotide I-site +site I-site +, O +cooperatively O +recognize O +the O +Py B-chemical +tract I-chemical +. O + +Altogether O +, O +we O +conclude O +that O +the O +conformation O +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +is O +key O +for O +recognizing O +RNA B-chemical +and O +is O +positioned O +by O +the O +RRM B-structure_element +extension I-structure_element +but O +otherwise O +relatively O +independent O +of O +the O +side O +chain O +composition O +. O + +Importance O +of O +U2AF65 B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +contacts O +for O +pre B-chemical +- I-chemical +mRNA I-chemical +splicing O + +We O +proceeded O +to O +test O +the O +importance O +of O +new O +U2AF65 B-complex_assembly +– I-complex_assembly +Py I-complex_assembly +- I-complex_assembly +tract I-complex_assembly +interactions O +for O +splicing O +of O +a O +model O +pre B-chemical +- I-chemical +mRNA I-chemical +substrate O +in O +a O +human B-species +cell O +line O +( O +Fig O +. O +5 O +; O +Supplementary O +Fig O +. O +5 O +). O + +When O +transfected B-experimental_method +into O +HEK293T O +cells O +containing O +only O +endogenous B-protein_state +U2AF65 B-protein +, O +the O +PY B-site +splice I-site +site I-site +is O +used O +and O +the O +remaining O +transcript O +remains O +unspliced O +. O + +When O +co B-experimental_method +- I-experimental_method +transfected I-experimental_method +with O +an O +expression B-experimental_method +plasmid I-experimental_method +for O +wild B-protein_state +- I-protein_state +type I-protein_state +U2AF65 B-protein +, O +use O +of O +the O +py B-site +splice I-site +site I-site +significantly O +increases O +( O +by O +more O +than O +five O +- O +fold O +) O +and O +as O +documented O +converts O +a O +fraction O +of O +the O +unspliced O +to O +spliced O +transcript O +. O + +The O +strong O +PY B-site +splice I-site +site I-site +is O +insensitive O +to O +added O +U2AF65 B-protein +, O +suggesting O +that O +endogenous B-protein_state +U2AF65 B-protein +levels O +are O +sufficient O +to O +saturate O +this O +site O +( O +Supplementary O +Fig O +. O +5b O +). O + +We O +introduced O +the O +triple B-experimental_method +mutation I-experimental_method +( O +V254P B-mutant +/ O +R227A B-mutant +/ O +Q147A B-mutant +) O +that O +significantly O +reduced O +U2AF651 B-mutant +, I-mutant +2L I-mutant +association O +with O +the O +Py B-chemical +tract I-chemical +( O +Fig O +. O +4b O +) O +in O +the O +context O +of O +full B-protein_state +- I-protein_state +length I-protein_state +U2AF65 B-protein +( O +U2AF65 B-mutant +- I-mutant +3Mut I-mutant +). O + +Co B-experimental_method +- I-experimental_method +transfection I-experimental_method +of O +the O +U2AF65 B-mutant +- I-mutant +3Mut I-mutant +with O +the O +pyPY B-chemical +splicing O +substrate O +significantly O +reduced O +splicing O +of O +the O +weak O +‘ B-site +py I-site +' I-site +splice I-site +site I-site +relative O +to O +wild B-protein_state +- I-protein_state +type I-protein_state +U2AF65 B-protein +( O +Fig O +. O +5b O +, O +c O +). O + +We O +conclude O +that O +the O +Py B-chemical +- I-chemical +tract I-chemical +interactions O +with O +these O +residues O +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +and O +RRM B-structure_element +extensions I-structure_element +are O +important O +for O +splicing O +as O +well O +as O +for O +binding O +a O +representative O +of O +the O +major B-structure_element +U2 I-structure_element +- I-structure_element +class I-structure_element +of I-structure_element +splice I-structure_element +sites I-structure_element +. O + +This O +minor O +U2AF65 B-protein +RRM1 B-site +/ I-site +RRM2 I-site +interface I-site +, O +coupled O +with O +the O +versatile O +sequence O +of O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +, O +highlighted O +the O +potential O +role O +for O +inter B-structure_element +- I-structure_element +RRM I-structure_element +conformational O +dynamics O +in O +U2AF65 B-protein +- O +splice O +site O +recognition O +. O + +Yet O +, O +small B-experimental_method +- I-experimental_method +angle I-experimental_method +X I-experimental_method +- I-experimental_method +ray I-experimental_method +scattering I-experimental_method +( O +SAXS B-experimental_method +) O +data O +indicated O +that O +both O +the O +minimal B-protein_state +U2AF651 B-mutant +, I-mutant +2 I-mutant +and O +longer O +constructs O +comprise O +a O +highly B-protein_state +diverse I-protein_state +continuum I-protein_state +of I-protein_state +conformations I-protein_state +in O +the O +absence B-protein_state +of I-protein_state +RNA B-chemical +that O +includes O +the O +‘ O +closed B-protein_state +' O +and O +‘ O +open B-protein_state +' O +conformations O +. O + +To O +complement O +the O +static O +portraits O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structure B-evidence +that O +we O +had O +determined O +by O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +, O +we O +used O +smFRET B-experimental_method +to O +characterize O +the O +probability B-evidence +distribution I-evidence +functions I-evidence +and O +time O +dependence O +of O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +conformational O +dynamics O +in O +solution O +. O + +The O +inter B-structure_element +- I-structure_element +RRM I-structure_element +dynamics O +of O +U2AF65 B-protein +were O +followed O +using O +FRET B-experimental_method +between O +fluorophores B-chemical +attached O +to O +RRM1 B-structure_element +and O +RRM2 B-structure_element +( O +Fig O +. O +6a O +, O +b O +, O +Methods O +). O + +The O +positions O +of O +single O +cysteine B-residue_name +mutations B-experimental_method +for O +fluorophore B-chemical +attachment O +( O +A181C B-mutant +in O +RRM1 B-structure_element +and O +Q324C B-mutant +in O +RRM2 B-structure_element +) O +were O +chosen O +based O +on O +inspection O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +and O +the O +‘ O +closed B-protein_state +' O +model O +of O +apo B-protein_state +- O +U2AF651 B-mutant +, I-mutant +2 I-mutant +. O + +Criteria O +included O +( O +i O +) O +residue O +locations O +that O +are O +distant O +from O +and O +hence O +not O +expected O +to O +interfere O +with O +the O +RRM B-complex_assembly +/ I-complex_assembly +RNA I-complex_assembly +or O +inter B-site +- I-site +RRM I-site +interfaces I-site +, O +( O +ii O +) O +inter O +- O +dye O +distances O +( O +50 O +Å O +for O +U2AF651 B-complex_assembly +, I-complex_assembly +2L I-complex_assembly +– I-complex_assembly +Py I-complex_assembly +tract I-complex_assembly +and O +30 O +Å O +for O +the O +closed B-protein_state +apo B-protein_state +- O +model O +) O +that O +are O +expected O +to O +be O +near O +the O +Förster B-experimental_method +radius I-experimental_method +( I-experimental_method +Ro I-experimental_method +) I-experimental_method +for O +the O +Cy3 B-chemical +/ O +Cy5 B-chemical +pair O +( O +56 O +Å O +), O +where O +changes O +in O +the O +efficiency O +of O +energy O +transfer O +are O +most O +sensitive O +to O +distance O +, O +and O +( O +iii O +) O +FRET B-evidence +efficiencies I-evidence +that O +are O +calculated O +to O +be O +significantly O +greater O +for O +the O +‘ O +closed B-protein_state +' O +apo B-protein_state +- O +model O +as O +opposed O +to O +the O +‘ O +open B-protein_state +' O +RNA B-protein_state +- I-protein_state +bound I-protein_state +structures B-evidence +( O +by O +∼ O +30 O +%). O + +The O +FRET B-evidence +efficiencies I-evidence +of O +either O +of O +these O +structurally O +characterized O +conformations O +also O +are O +expected O +to O +be O +significantly O +greater O +than O +elongated B-protein_state +U2AF65 B-protein +conformations O +that O +lack B-protein_state +inter O +- O +RRM B-structure_element +contacts O +. O + +Double O +- O +cysteine B-residue_name +variant B-protein_state +of O +U2AF651 B-mutant +, I-mutant +2 I-mutant +was O +modified B-experimental_method +with O +equimolar O +amount O +of O +Cy3 B-chemical +and O +Cy5 B-chemical +. O + +Only O +traces B-evidence +that O +showed O +single O +photobleaching O +events O +for O +both O +donor O +and O +acceptor O +dyes O +and O +anti O +- O +correlated O +changes O +in O +acceptor O +and O +donor O +fluorescence O +were O +included O +in O +smFRET B-experimental_method +data O +analysis O +. O + +The O +double O +- O +labelled O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +protein O +was O +tethered B-protein_state +to O +a O +slide O +via O +biotin B-chemical +- I-chemical +NTA I-chemical +/ I-chemical +Ni I-chemical ++ I-chemical +2 I-chemical +resin I-chemical +. O + +Virtually O +no O +fluorescent O +molecules O +were O +detected O +in O +the O +absence B-protein_state +of I-protein_state +biotin B-chemical +- I-chemical +NTA I-chemical +/ I-chemical +Ni I-chemical ++ I-chemical +2 I-chemical +, O +which O +demonstrates O +the O +absence B-protein_state +of I-protein_state +detectable O +non O +- O +specific O +binding O +of O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +to O +the O +slide O +. O + +The O +FRET B-evidence +distribution I-evidence +histogram I-evidence +built O +from O +more O +than O +a O +thousand O +traces B-evidence +of O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +in O +the O +absence B-protein_state +of I-protein_state +ligand B-chemical +showed O +an O +extremely O +broad O +distribution O +centred O +at O +a O +FRET B-evidence +efficiency I-evidence +of O +∼ O +0 O +. O +4 O +( O +Fig O +. O +6d O +). O + +Despite O +the O +large O +width O +of O +the O +FRET B-evidence +- I-evidence +distribution I-evidence +histogram I-evidence +, O +the O +majority O +( O +80 O +%) O +of O +traces B-evidence +that O +showed O +fluctuations O +sampled O +only O +two O +distinct O +FRET B-evidence +states I-evidence +( O +for O +example O +, O +Supplementary O +Fig O +. O +7a O +). O + +Approximately O +70 O +% O +of O +observed O +fluctuations O +were O +interchanges O +between O +the O +∼ O +0 O +. O +65 O +and O +∼ O +0 O +. O +45 O +FRET B-evidence +values I-evidence +( O +Supplementary O +Fig O +. O +7b O +). O + +We O +cannot O +exclude O +a O +possibility O +that O +tethering O +of O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +to O +the O +microscope O +slide O +introduces O +structural O +heterogeneity O +into O +the O +protein O +and O +, O +thus O +, O +contributes O +to O +the O +breadth O +of O +the O +FRET B-evidence +distribution I-evidence +histogram I-evidence +. O + +However O +, O +the O +presence O +of O +repetitive O +fluctuations O +between O +particular O +FRET B-evidence +values I-evidence +supports O +the O +hypothesis O +that O +RNA B-protein_state +- I-protein_state +free I-protein_state +U2AF65 B-protein +samples O +several O +distinct O +conformations O +. O + +This O +result O +is O +consistent O +with O +the O +broad O +ensembles O +of O +extended B-protein_state +solution O +conformations O +that O +best O +fit O +the O +SAXS B-experimental_method +data O +collected O +for O +U2AF651 B-mutant +, I-mutant +2 I-mutant +as O +well O +as O +for O +a O +longer O +construct O +( O +residues O +136 B-residue_range +– I-residue_range +347 I-residue_range +). O + +We O +conclude O +that O +weak O +contacts O +between O +the O +U2AF65 B-protein +RRM1 B-structure_element +and O +RRM2 B-structure_element +permit O +dissociation O +of O +these O +RRMs B-structure_element +in O +the O +absence B-protein_state +of I-protein_state +RNA B-chemical +. O + +U2AF65 B-protein +conformational O +selection O +and O +induced O +fit O +by O +bound B-protein_state +RNA B-chemical + +We O +next O +used O +smFRET B-experimental_method +to O +probe O +the O +conformational O +selection O +of O +distinct O +inter B-structure_element +- I-structure_element +RRM I-structure_element +arrangements O +following O +association O +of O +U2AF65 B-protein +with O +the O +AdML B-gene +Py B-chemical +- I-chemical +tract I-chemical +prototype O +. O + +Addition O +of O +the O +AdML B-gene +RNA B-chemical +to O +tethered B-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +selectively O +increases O +a O +fraction O +of O +molecules O +showing O +an O +∼ O +0 O +. O +45 O +apparent O +FRET B-evidence +efficiency I-evidence +, O +suggesting O +that O +RNA O +binding O +stabilizes O +a O +single O +conformation O +, O +which O +corresponds O +to O +the O +0 O +. O +45 O +FRET B-evidence +state I-evidence +( O +Fig O +. O +6e O +, O +f O +). O + +To O +assess O +the O +possible O +contributions O +of O +RNA B-protein_state +- I-protein_state +free I-protein_state +conformations O +of O +U2AF65 B-protein +and O +/ O +or O +structural O +heterogeneity O +introduced O +by O +tethering B-experimental_method +of O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +to O +the O +slide O +to O +the O +observed O +distribution B-evidence +of I-evidence +FRET I-evidence +values I-evidence +, O +we O +reversed B-experimental_method +the I-experimental_method +immobilization I-experimental_method +scheme I-experimental_method +. O + +We O +tethered B-protein_state +the O +AdML B-gene +RNA B-chemical +to O +the O +slide O +via O +a O +biotinylated B-chemical +oligonucleotide I-chemical +DNA I-chemical +handle O +and O +added B-experimental_method +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +in O +the O +absence B-protein_state +of I-protein_state +biotin B-chemical +- I-chemical +NTA I-chemical +resin I-chemical +( O +Fig O +. O +6g O +, O +h O +; O +Supplementary O +Fig O +. O +7c O +– O +g O +). O + +We O +examined O +the O +effect O +on O +U2AF651 B-mutant +, I-mutant +2L I-mutant +conformations O +of O +purine B-experimental_method +interruptions I-experimental_method +that O +often O +occur O +in O +relatively O +degenerate O +human B-species +Py B-chemical +tracts I-chemical +. O + +We O +introduced B-experimental_method +an O +rArA B-chemical +purine B-chemical +dinucleotide I-chemical +within O +a O +variant O +of O +the O +AdML B-gene +Py B-chemical +tract I-chemical +( O +detailed O +in O +Methods O +). O + +Insertion B-experimental_method +of O +adenine B-chemical +nucleotides I-chemical +decreased O +binding B-evidence +affinity I-evidence +of O +U2AF65 B-protein +to O +RNA B-chemical +by O +approximately O +five O +- O +fold O +. O + +Nevertheless O +, O +in O +the O +presence O +of O +saturating O +concentrations O +of O +rArA B-chemical +- O +interrupted O +RNA B-chemical +slide B-protein_state +- I-protein_state +tethered I-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +showed O +a O +prevalent O +∼ O +0 O +. O +45 O +apparent O +FRET B-evidence +value I-evidence +( O +Fig O +. O +6i O +, O +j O +), O +which O +was O +also O +predominant O +in O +the O +presence O +of O +continuous O +Py B-chemical +tract I-chemical +. O + +It O +should O +be O +noted O +that O +inferring O +distances O +from O +FRET B-evidence +values I-evidence +is O +prone O +to O +significant O +error O +because O +of O +uncertainties O +in O +the O +determination O +of O +fluorophore O +orientation O +factor O +κ2 O +and O +Förster O +radius O +R0 O +, O +the O +parameters O +used O +in O +distance O +calculations O +. O + +Importantly O +, O +the O +majority O +of O +traces B-evidence +(∼ O +70 O +%) O +of O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +bound B-protein_state +to I-protein_state +the O +slide O +- O +tethered O +RNA B-chemical +lacked O +FRET O +fluctuations O +and O +predominately O +exhibited O +a O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +( O +for O +example O +, O +Fig O +. O +6g O +). O + +The O +remaining O +∼ O +30 O +% O +of O +traces B-evidence +for O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +bound B-protein_state +to I-protein_state +the O +slide O +- O +tethered O +RNA B-chemical +showed O +fluctuations O +between O +distinct O +FRET B-evidence +values I-evidence +. O + +Hidden B-experimental_method +Markov I-experimental_method +modelling I-experimental_method +analysis I-experimental_method +of O +smFRET B-experimental_method +traces B-evidence +suggests O +that O +RNA B-protein_state +- I-protein_state +bound I-protein_state +U2AF651 B-mutant +, I-mutant +2L I-mutant +can O +sample O +at O +least O +two O +other O +conformations O +corresponding O +to O +∼ O +0 O +. O +7 O +– O +0 O +. O +8 O +and O +∼ O +0 O +. O +3 O +FRET B-evidence +values I-evidence +in O +addition O +to O +the O +predominant O +conformation O +corresponding O +to O +the O +0 O +. O +45 O +FRET B-evidence +state I-evidence +. O + +Although O +a O +compact O +conformation O +( O +or O +multiple O +conformations O +) O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +corresponding O +to O +∼ O +0 O +. O +7 O +– O +0 O +. O +8 O +FRET B-evidence +values I-evidence +can O +bind O +RNA B-chemical +, O +on O +RNA B-chemical +binding O +, O +these O +compact B-protein_state +conformations O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +transition O +into O +a O +more O +stable O +structural O +state O +that O +corresponds O +to O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +and O +is O +likely O +similar O +to O +the O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +inter B-structure_element +- I-structure_element +RRM I-structure_element +- O +arrangement O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +crystal B-evidence +structures I-evidence +. O + +The O +U2AF65 B-protein +structures B-evidence +and O +analyses B-evidence +presented O +here O +represent O +a O +successful O +step O +towards O +defining O +a O +molecular O +map O +of O +the O +3 B-site +′ I-site +splice I-site +site I-site +. O + +Truncation B-experimental_method +of O +U2AF65 B-protein +to O +the O +core B-protein_state +RRM1 B-structure_element +– I-structure_element +RRM2 I-structure_element +region I-structure_element +reduces O +its O +RNA B-evidence +affinity I-evidence +by O +100 O +- O +fold O +. O + +Likewise O +, O +deletion B-experimental_method +of O +20 B-residue_range +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +residues I-structure_element +significantly O +reduces O +U2AF65 B-protein +– O +RNA B-chemical +binding O +only O +when O +introduced O +in O +the O +context O +of O +the O +longer B-protein_state +U2AF651 B-mutant +, I-mutant +2L I-mutant +construct O +comprising O +the O +RRM B-structure_element +extensions I-structure_element +, O +which O +in O +turn O +position O +the O +linker B-structure_element +for O +RNA B-chemical +interactions O +. O + +Notably O +, O +a O +triple B-protein_state +mutation I-protein_state +of O +three O +residues O +( O +V254P B-mutant +, O +Q147A B-mutant +and O +R227A B-mutant +) O +in O +the O +respective O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +, O +N B-structure_element +- I-structure_element +and I-structure_element +C I-structure_element +- I-structure_element +terminal I-structure_element +extensions I-structure_element +non O +- O +additively O +reduce O +RNA B-evidence +binding I-evidence +by O +150 O +- O +fold O +. O + +The O +implications O +of O +this O +finding O +for O +U2AF65 B-protein +conservation O +and O +Py B-chemical +- I-chemical +tract I-chemical +recognition O +are O +detailed O +in O +the O +Supplementary O +Discussion O +. O + +Recently O +, O +high B-experimental_method +- I-experimental_method +throughput I-experimental_method +sequencing I-experimental_method +studies I-experimental_method +have O +shown O +that O +somatic O +mutations O +in O +pre B-protein_type +- I-protein_type +mRNA I-protein_type +splicing I-protein_type +factors I-protein_type +occur O +in O +the O +majority O +of O +patients O +with O +myelodysplastic O +syndrome O +( O +MDS O +). O + +MDS O +- O +relevant O +mutations O +are O +common O +in O +the O +small B-protein_state +U2AF B-protein_type +subunit I-protein_type +( O +U2AF35 B-protein +, O +or O +U2AF1 B-protein +), O +yet O +such O +mutations O +are O +rare O +in O +the O +large B-protein_state +U2AF65 B-protein +subunit O +( O +also O +called O +U2AF2 B-protein +)— O +possibly O +due O +to O +the O +selective O +versus O +nearly O +universal O +requirements O +of O +these O +factors O +for O +splicing O +. O + +A O +confirmed O +somatic O +mutation O +of O +U2AF65 B-protein +in O +patients O +with O +MDS O +, O +L187V B-mutant +, O +is O +located O +on O +a O +solvent B-site +- I-site +exposed I-site +surface I-site +of O +RRM1 B-structure_element +that O +is O +distinct O +from O +the O +RNA B-site +interface I-site +( O +Fig O +. O +7a O +). O + +This O +L187 B-residue_name_number +surface O +is O +oriented O +towards O +the O +N O +terminus O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +construct O +, O +where O +it O +is O +expected O +to O +abut O +the O +U2AF35 B-site +- I-site +binding I-site +site I-site +in O +the O +context O +of O +the O +full B-protein_state +- I-protein_state +length I-protein_state +U2AF B-protein +heterodimer B-oligomeric_state +. O + +Likewise O +, O +an O +unconfirmed O +M144I B-mutant +mutation O +reported O +by O +the O +same O +group O +corresponds O +to O +the O +N O +- O +terminal O +residue O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +, O +which O +is O +separated O +by O +only O +∼ O +20 O +residues O +from O +the O +U2AF35 B-site +- I-site +binding I-site +site I-site +. O + +As O +such O +, O +we O +suggest O +that O +the O +MDS O +- O +relevant O +U2AF65 B-protein +mutations O +contribute O +to O +MDS O +progression O +indirectly O +, O +by O +destabilizing O +a O +relevant O +conformation O +of O +the O +conjoined O +U2AF35 B-protein +subunit O +rather O +than O +affecting O +U2AF65 B-protein +functions O +in O +RNA B-chemical +binding O +or O +spliceosome B-complex_assembly +recruitment O +per O +se O +. O + +An O +increased O +prevalence O +of O +the O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +following O +U2AF65 B-protein +– O +RNA B-chemical +binding O +, O +coupled O +with O +the O +apparent O +absence B-protein_state +of I-protein_state +transitions O +in O +many O +∼ O +0 O +. O +45 O +- O +value O +single O +molecule O +traces B-evidence +( O +for O +example O +, O +Fig O +. O +6e O +), O +suggests O +a O +population O +shift O +in O +which O +RNA B-chemical +binds O +to O +( O +and O +draws O +the O +equilibrium O +towards O +) O +a O +pre B-protein_state +- I-protein_state +configured I-protein_state +inter B-structure_element +- I-structure_element +RRM I-structure_element +proximity O +that O +most O +often O +corresponds O +to O +the O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +. O + +Examples O +of O +‘ O +extended B-protein_state +conformational O +selection O +' O +during O +ligand O +binding O +have O +been O +characterized O +for O +a O +growing O +number O +of O +macromolecules O +( O +for O +example O +, O +adenylate B-protein_type +kinase I-protein_type +, O +LAO B-protein_type +- I-protein_type +binding I-protein_type +protein I-protein_type +, O +poly B-protein_type +- I-protein_type +ubiquitin I-protein_type +, O +maltose B-protein_type +- I-protein_type +binding I-protein_type +protein I-protein_type +and O +the O +preQ1 B-protein_type +riboswitch I-protein_type +, O +among O +others O +). O + +Here O +, O +the O +majority O +of O +changes O +in O +smFRET B-experimental_method +traces B-evidence +for O +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +bound B-protein_state +to I-protein_state +slide O +- O +tethered O +RNA B-chemical +began O +at O +high O +( O +0 O +. O +65 O +– O +0 O +. O +8 O +) O +FRET B-evidence +value I-evidence +and O +transition O +to O +the O +predominant O +0 O +. O +45 O +FRET B-evidence +value I-evidence +( O +Supplementary O +Fig O +. O +7c O +– O +g O +). O + +These O +transitions O +could O +correspond O +to O +rearrangement O +from O +the O +‘ O +closed B-protein_state +' O +NMR B-experimental_method +/ O +PRE B-experimental_method +- O +based O +U2AF65 B-protein +conformation O +in O +which O +the O +RNA B-site +- I-site +binding I-site +surface I-site +of O +only O +a O +single B-protein_state +RRM B-structure_element +is O +exposed O +and O +available O +for O +RNA O +binding O +, O +to O +the O +structural O +state O +seen O +in O +the O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +, O +RNA B-protein_state +- I-protein_state +bound I-protein_state +crystal B-evidence +structure I-evidence +. O + +The O +finding O +that O +U2AF65 B-protein +recognizes O +a O +nine O +base O +pair O +Py B-chemical +tract I-chemical +contributes O +to O +an O +elusive O +‘ O +code O +' O +for O +predicting O +splicing O +patterns O +from O +primary O +sequences O +in O +the O +post O +- O +genomic O +era O +( O +reviewed O +in O +ref O +.). O + +Further O +research O +will O +be O +needed O +to O +understand O +the O +roles O +of O +SF1 B-protein +and O +U2AF35 B-protein +subunits O +in O +the O +conformational O +equilibria O +underlying O +U2AF65 B-protein +association O +with O +Py B-chemical +tracts I-chemical +. O + +Moreover O +, O +structural O +differences O +among O +U2AF65 B-protein +homologues O +and O +paralogues O +may O +regulate O +splice B-site +site I-site +selection O +. O + +Ultimately O +, O +these O +guidelines O +will O +assist O +the O +identification O +of O +3 B-site +′ I-site +splice I-site +sites I-site +and O +the O +relationship O +of O +disease O +- O +causing O +mutations O +to O +penalties O +for O +U2AF65 B-protein +association O +. O + +The O +intact B-protein_state +U2AF65 B-protein +RRM1 B-structure_element +/ O +RRM2 B-structure_element +- O +containing O +domain O +and O +flanking O +residues O +are O +required O +for O +binding O +contiguous B-structure_element +Py B-chemical +tracts I-chemical +. O + +( O +a O +) O +Domain O +organization O +of O +full B-protein_state +- I-protein_state +length I-protein_state +( O +fl B-protein_state +) O +U2AF65 B-protein +and O +constructs O +used O +for O +RNA B-chemical +binding O +and O +structural O +experiments O +. O + +An O +internal O +deletion O +( O +d B-mutant +, O +Δ B-mutant +) O +of O +residues O +238 B-residue_range +– I-residue_range +257 I-residue_range +removes O +a O +portion O +of O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +from O +the O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +and O +dU2AF651 B-mutant +, I-mutant +2L I-mutant +constructs O +. O + +( O +b O +) O +Comparison O +of O +the O +apparent O +equilibrium B-evidence +affinities I-evidence +of O +various O +U2AF65 B-protein +constructs O +for O +binding O +the O +prototypical O +AdML B-gene +Py B-chemical +tract I-chemical +( O +5 B-chemical +′- I-chemical +CCCUUUUUUUUCC I-chemical +- I-chemical +3 I-chemical +′). I-chemical + +The O +flU2AF65 B-protein +protein O +includes O +a O +heterodimerization B-structure_element +domain I-structure_element +of O +the O +U2AF35 B-protein +subunit O +to O +promote O +solubility O +and O +folding O +. O + +The O +apparent O +equilibrium B-evidence +dissociation I-evidence +constants I-evidence +( O +KD B-evidence +) O +for O +binding O +the O +AdML B-gene +13mer O +are O +as O +follows O +: O +flU2AF65 B-protein +, O +30 O +± O +3 O +nM O +; O +U2AF651 B-mutant +, I-mutant +2L I-mutant +, O +35 O +± O +6 O +nM O +; O +U2AF651 B-mutant +, I-mutant +2 I-mutant +, O +3 O +, O +600 O +± O +300 O +nM O +. O +( O +c O +) O +Comparison O +of O +the O +RNA B-evidence +sequence I-evidence +specificities I-evidence +of O +flU2AF65 B-protein +and O +U2AF651 B-mutant +, I-mutant +2L I-mutant +constructs O +binding O +C B-structure_element +- I-structure_element +rich I-structure_element +Py B-chemical +tracts I-chemical +with O +4U O +' O +s O +embedded O +in O +either O +the O +5 O +′- O +( O +light O +grey O +fill O +) O +or O +3 O +′- O +( O +dark O +grey O +fill O +) O +regions O +. O + +The O +KD B-evidence +' O +s O +for O +binding O +5 B-chemical +′- I-chemical +CCUUUUCCCCCCC I-chemical +- I-chemical +3 I-chemical +′ I-chemical +are O +: O +flU2AF65 B-protein +, O +41 O +± O +2 O +nM O +; O +U2AF651 B-mutant +, I-mutant +2L I-mutant +, O +31 O +± O +3 O +nM O +. O +The O +KD B-evidence +' O +s O +for O +binding O +5 B-chemical +′- I-chemical +CCCCCCCUUUUCC I-chemical +- I-chemical +3 I-chemical +′ I-chemical +are O +: O +flU2AF65 B-protein +, O +414 O +± O +12 O +nM O +; O +U2AF651 B-mutant +, I-mutant +2L I-mutant +, O +417 O +± O +10 O +nM O +. O +Bar O +graphs O +are O +hatched O +to O +match O +the O +constructs O +shown O +in O +a O +. O +The O +average B-evidence +apparent I-evidence +equilibrium I-evidence +affinity I-evidence +( O +KA B-evidence +) O +and O +s O +. O +e O +. O +m O +. O +for O +three O +independent O +titrations O +are O +plotted O +. O + +The O +purified O +protein O +and O +average B-evidence +fitted I-evidence +fluorescence I-evidence +anisotropy I-evidence +RNA I-evidence +- I-evidence +binding I-evidence +curves I-evidence +are O +shown O +in O +Supplementary O +Fig O +. O +1 O +. O + +Structures B-evidence +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +recognizing O +a O +contiguous B-structure_element +Py B-chemical +tract I-chemical +. O + +( O +a O +) O +Alignment B-experimental_method +of O +oligonucleotide B-chemical +sequences O +that O +were O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +in O +the O +indicated O +U2AF651 B-mutant +, I-mutant +2L I-mutant +structures B-evidence +. O + +The O +regions O +of O +RRM1 B-structure_element +, O +RRM2 B-structure_element +and O +linker B-structure_element +contacts O +are O +indicated O +above O +by O +respective O +black O +and O +blue O +arrows O +from O +N O +- O +to O +C O +- O +terminus O +. O + +For O +clarity O +, O +we O +consistently O +number O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +nucleotide B-site +- I-site +binding I-site +sites I-site +from O +one O +to O +nine O +, O +although O +in O +some O +cases O +the O +co B-experimental_method +- I-experimental_method +crystallized I-experimental_method +oligonucleotide B-chemical +comprises O +eight O +nucleotides B-chemical +and O +as O +such O +leaves O +the O +first B-site +binding I-site +site I-site +empty O +. O + +Crystallographic O +statistics O +are O +given O +in O +Table O +1 O +and O +the O +overall O +conformations O +of O +U2AF651 B-mutant +, I-mutant +2L I-mutant +and O +prior O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +/ O +U2AF651 B-mutant +, I-mutant +2 I-mutant +structures B-evidence +are O +compared O +in O +Supplementary O +Fig O +. O +2 O +. O + +BrdU B-chemical +, O +5 B-chemical +- I-chemical +bromo I-chemical +- I-chemical +deoxy I-chemical +- I-chemical +uridine I-chemical +; O +d B-chemical +, O +deoxy B-chemical +- I-chemical +ribose I-chemical +; O +P B-chemical +-, I-chemical +5 B-chemical +′- I-chemical +phosphorylation I-chemical +; O +r B-chemical +, O +ribose B-chemical +. O + +Representative O +views O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +interactions O +with O +each O +new O +nucleotide B-chemical +of O +the O +bound B-protein_state +Py B-chemical +tract I-chemical +. O + +The O +apparent O +equilibrium B-evidence +dissociation I-evidence +constants I-evidence +( O +KD B-evidence +) O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +mutant B-protein_state +proteins O +are O +: O +wild B-protein_state +type I-protein_state +( O +WT B-protein_state +), O +35 O +± O +6 O +nM O +; O +R227A B-mutant +, O +166 O +± O +2 O +nM O +; O +V254P B-mutant +, O +137 O +± O +10 O +nM O +; O +Q147A B-mutant +, O +171 O +± O +21 O +nM O +. O +The O +average O +KA B-evidence +and O +s O +. O +e O +. O +m O +. O +for O +three O +independent O +titrations O +are O +plotted O +. O + +The O +U2AF65 B-protein +linker B-structure_element +/ O +RRM B-structure_element +and O +inter O +- O +RRM B-structure_element +interactions O +. O + +( O +a O +) O +Contacts O +of O +the O +U2AF65 B-protein +inter B-structure_element +- I-structure_element +RRM I-structure_element +linker I-structure_element +with O +the O +RRMs B-structure_element +. O + +A O +semi O +- O +transparent O +space O +- O +filling O +surface O +is O +shown O +for O +the O +RRM1 B-structure_element +( O +green O +) O +and O +RRM2 B-structure_element +( O +light O +blue O +). O + +Residues O +V249 B-residue_name_number +, O +V250 B-residue_name_number +, O +V254 B-residue_name_number +( O +yellow O +) O +are O +mutated B-experimental_method +to O +V249G B-mutant +/ O +V250G B-mutant +/ O +V254G B-mutant +in O +the O +3Gly B-mutant +mutant I-mutant +; O +residues O +S251 B-residue_name_number +, O +T252 B-residue_name_number +, O +V253 B-residue_name_number +, O +P255 B-residue_name_number +( O +red O +) O +along O +with O +V254 B-residue_name_number +are O +mutated B-experimental_method +to O +S251G B-mutant +/ O +T252G B-mutant +/ O +V253G B-mutant +/ O +V254G B-mutant +/ O +P255G B-mutant +in O +the O +5Gly B-mutant +mutant I-mutant +or O +to O +S251N B-mutant +/ O +T252L B-mutant +/ O +V253A B-mutant +/ O +V254L B-mutant +/ O +P255A B-mutant +in O +the O +NLALA B-mutant +mutant I-mutant +; O +residues O +M144 B-residue_name_number +, O +L235 B-residue_name_number +, O +M238 B-residue_name_number +, O +V244 B-residue_name_number +, O +V246 B-residue_name_number +( O +orange O +) O +along O +with O +V249 B-residue_name_number +, O +V250 B-residue_name_number +, O +S251 B-residue_name_number +, O +T252 B-residue_name_number +, O +V253 B-residue_name_number +, O +V254 B-residue_name_number +, O +P255 B-residue_name_number +are O +mutated B-experimental_method +to O +M144G B-mutant +/ O +L235G B-mutant +/ O +M238G B-mutant +/ O +V244G B-mutant +/ O +V246G B-mutant +/ O +V249G B-mutant +/ O +V250G B-mutant +/ O +S251G B-mutant +/ O +T252G B-mutant +/ O +V253G B-mutant +/ O +V254G B-mutant +/ O +P255G B-mutant +in O +the O +12Gly B-mutant +mutant I-mutant +. O + +The O +central O +panel O +shows O +an O +overall O +view O +with O +stick O +diagrams O +for O +mutated O +residues O +; O +boxed O +regions O +are O +expanded O +to O +show O +the O +C O +- O +terminal O +( O +bottom O +left O +) O +and O +central B-structure_element +linker I-structure_element +regions I-structure_element +( O +top O +) O +at O +the O +inter B-structure_element +- I-structure_element +RRM I-structure_element +interfaces I-structure_element +, O +and O +N O +- O +terminal O +linker O +region O +contacts O +with O +RRM1 B-structure_element +( O +bottom O +right O +). O + +( O +b O +) O +Bar O +graph O +of O +apparent O +equilibrium B-evidence +affinities I-evidence +( O +KA B-evidence +) O +for O +the O +AdML B-gene +Py B-chemical +tract I-chemical +( O +5 B-chemical +′- I-chemical +CCCUUUUUUUUCC I-chemical +- I-chemical +3 I-chemical +′) I-chemical +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +blue O +) O +U2AF651 B-mutant +, I-mutant +2L I-mutant +protein O +compared O +with O +mutations O +of O +the O +residues O +shown O +in O +a O +: O +3Gly B-mutant +( O +yellow O +), O +5Gly B-mutant +( O +red O +), O +NLALA B-mutant +( O +hatched O +red O +), O +12Gly B-mutant +( O +orange O +) O +and O +the O +linker B-experimental_method +deletions I-experimental_method +dU2AF651 B-mutant +, I-mutant +2 I-mutant +in O +the O +minimal B-protein_state +RRM1 B-structure_element +– I-structure_element +RRM2 I-structure_element +region I-structure_element +( O +residues O +148 B-residue_range +– I-residue_range +237 I-residue_range +, O +258 B-residue_range +– I-residue_range +336 I-residue_range +) O +or O +dU2AF651 B-mutant +, I-mutant +2L I-mutant +( O +residues O +141 B-residue_range +– I-residue_range +237 I-residue_range +, O +258 B-residue_range +– I-residue_range +342 I-residue_range +). O + +The O +apparent O +equilibrium B-evidence +dissociation I-evidence +constants I-evidence +( O +KD B-evidence +) O +of O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +mutant B-protein_state +proteins O +are O +: O +wild B-protein_state +type I-protein_state +( O +WT B-protein_state +), O +35 O +± O +6 O +nM O +; O +3Gly B-mutant +, O +47 O +± O +4 O +nM O +; O +5Gly B-mutant +, O +61 O +± O +3 O +nM O +; O +12Gly B-mutant +, O +88 O +± O +21 O +nM O +; O +NLALA B-mutant +, O +45 O +± O +3 O +nM O +; O +dU2AF651 B-mutant +, I-mutant +2L I-mutant +, O +123 O +± O +5 O +nM O +; O +dU2AF651 B-mutant +, I-mutant +2 I-mutant +, O +5000 O +± O +100 O +nM O +; O +3Mut B-mutant +, O +5630 O +± O +70 O +nM O +. O +The O +average O +KA B-evidence +and O +s O +. O +e O +. O +m O +. O +for O +three O +independent O +titrations O +are O +plotted O +. O + +The O +fitted O +fluorescence O +anisotropy O +RNA B-evidence +- I-evidence +binding I-evidence +curves I-evidence +are O +shown O +in O +Supplementary O +Fig O +. O +4d O +– O +j O +. O +( O +c O +) O +Close O +view O +of O +the O +U2AF65 B-protein +RRM1 B-site +/ I-site +RRM2 I-site +interface I-site +following O +a O +two O +- O +fold O +rotation O +about O +the O +x O +- O +axis O +relative O +to O +a O +. O + +U2AF65 B-protein +inter O +- O +domain O +residues O +are O +important O +for O +splicing O +a O +representative O +pre B-chemical +- I-chemical +mRNA I-chemical +substrate O +in O +human B-species +cells O +. O + +( O +a O +) O +Schematic O +diagram O +of O +the O +pyPY B-chemical +reporter O +minigene O +construct O +comprising O +two O +alternative O +splice B-site +sites I-site +preceded O +by O +either O +the O +weak O +IgM O +Py B-chemical +tract I-chemical +( O +py B-chemical +) O +or O +the O +strong O +AdML B-gene +Py B-chemical +tract I-chemical +( O +PY B-chemical +) O +( O +sequences O +inset O +). O + +RNA O +binding O +stabilizes O +the O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +conformation O +of O +U2AF65 B-protein +RRMs B-structure_element +. O + +( O +a O +, O +b O +) O +Views O +of O +FRET B-experimental_method +pairs O +chosen O +to O +follow O +the O +relative O +movement O +of O +RRM1 B-structure_element +and O +RRM2 B-structure_element +on O +the O +crystal B-evidence +structure I-evidence +of O +‘ O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +' O +U2AF651 B-mutant +, I-mutant +2L I-mutant +RRMs B-structure_element +bound B-protein_state +to I-protein_state +a O +Py B-chemical +- I-chemical +tract I-chemical +oligonucleotide I-chemical +( O +a O +, O +representative O +structure O +iv O +) O +or O +‘ O +closed B-protein_state +' O +NMR B-experimental_method +/ O +PRE B-experimental_method +- O +based O +model O +of O +U2AF651 B-mutant +, I-mutant +2 I-mutant +( O +b O +, O +PDB O +ID O +2YH0 O +) O +in O +identical O +orientations O +of O +RRM2 B-structure_element +. O + +The O +untethered B-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +protein O +( O +1 O +nM O +) O +was O +added O +to O +AdML B-gene +RNA B-chemical +– I-chemical +polyethylene I-chemical +- I-chemical +glycol I-chemical +- I-chemical +linker I-chemical +– I-chemical +DNA I-chemical +oligonucleotide I-chemical +( O +10 O +nM O +), O +which O +was O +immobilized O +on O +the O +microscope O +slide O +by O +annealing O +with O +a O +complementary O +biotinyl B-chemical +- I-chemical +DNA I-chemical +oligonucleotide I-chemical +( O +black O +vertical O +line O +). O + +Additional O +traces B-evidence +for O +untethered B-protein_state +, O +RNA B-protein_state +- I-protein_state +bound I-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +are O +shown O +in O +Supplementary O +Fig O +. O +7c O +, O +d O +. O +Histograms B-evidence +( O +d O +, O +f O +, O +h O +, O +j O +) O +show O +the O +distribution B-evidence +of I-evidence +FRET I-evidence +values I-evidence +in O +RNA B-protein_state +- I-protein_state +free I-protein_state +, O +slide B-protein_state +- I-protein_state +tethered I-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +( O +d O +); O +AdML B-gene +RNA B-protein_state +- I-protein_state +bound I-protein_state +, O +slide B-protein_state +- I-protein_state +tethered I-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +( O +f O +); O +AdML B-gene +RNA B-protein_state +- I-protein_state +bound I-protein_state +, O +untethered B-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +( O +h O +) O +and O +adenosine O +- O +interrupted O +RNA B-protein_state +- I-protein_state +bound I-protein_state +, O +slide B-protein_state +- I-protein_state +tethered I-protein_state +U2AF651 B-mutant +, I-mutant +2LFRET I-mutant +( O +Cy3 B-chemical +/ O +Cy5 B-chemical +) O +( O +j O +). O + +MDS O +- O +relevant O +mutated O +residues O +of O +U2AF65 B-protein +are O +shown O +as O +yellow O +spheres O +( O +L187 B-residue_name_number +and O +M144 B-residue_name_number +). O + +( O +b O +) O +Following O +binding O +to O +the O +Py B-chemical +- I-chemical +tract I-chemical +RNA I-chemical +, O +a O +conformation O +corresponding O +to O +high B-evidence +FRET I-evidence +and O +consistent O +with O +the O +‘ O +closed B-protein_state +', O +back B-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +back I-protein_state +apo B-protein_state +- O +U2AF65 B-protein +model O +resulting O +from O +PRE B-experimental_method +/ O +NMR B-experimental_method +characterization O +( O +PDB O +ID O +2YH0 O +) O +often O +transitions O +to O +a O +conformation O +corresponding O +to O +∼ O +0 O +. O +45 O +FRET B-evidence +value I-evidence +, O +which O +is O +consistent O +with O +‘ O +open B-protein_state +', O +side B-protein_state +- I-protein_state +by I-protein_state +- I-protein_state +side I-protein_state +RRMs B-structure_element +such O +as O +the O +U2AF651 B-mutant +, I-mutant +2L I-mutant +crystal B-evidence +structures I-evidence +. O + +RRM1 B-structure_element +, O +green O +; O +RRM2 B-structure_element +, O +pale O +blue O +; O +RRM B-structure_element +extensions I-structure_element +/ O +linker B-structure_element +, O +blue O +. O + +RNA B-chemical +protects O +a O +nucleoprotein B-complex_assembly +complex O +against O +radiation O +damage O + +Systematic O +analysis O +of O +radiation O +damage O +within O +a O +protein B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complex O +over O +a O +large O +dose O +range O +( O +1 O +. O +3 O +– O +25 O +MGy O +) O +reveals O +significant O +differential O +susceptibility O +of O +RNA B-chemical +and O +protein O +. O + +A O +new O +method O +of O +difference B-experimental_method +electron I-experimental_method +- I-experimental_method +density I-experimental_method +quantification I-experimental_method +is O +presented O +. O + +Radiation O +damage O +during O +macromolecular B-experimental_method +X I-experimental_method +- I-experimental_method +ray I-experimental_method +crystallographic I-experimental_method +data I-experimental_method +collection I-experimental_method +is O +still O +the O +main O +impediment O +for O +many O +macromolecular B-experimental_method +structure I-experimental_method +determinations I-experimental_method +. O + +Although O +this O +has O +been O +well O +characterized O +within O +protein O +crystals B-evidence +, O +far O +less O +is O +known O +about O +specific O +damage O +effects O +within O +the O +larger O +class O +of O +nucleoprotein O +complexes O +. O + +Here O +, O +a O +methodology O +has O +been O +developed O +whereby O +per B-evidence +- I-evidence +atom I-evidence +density I-evidence +changes I-evidence +could O +be O +quantified O +with O +increasing O +dose O +over O +a O +wide O +( O +1 O +. O +3 O +– O +25 O +. O +0 O +MGy O +) O +range O +and O +at O +higher O +resolution O +( O +1 O +. O +98 O +Å O +) O +than O +the O +previous O +systematic O +specific O +damage O +study O +on O +a O +protein O +– O +DNA B-chemical +complex O +. O + +Additionally O +, O +the O +method O +enabled O +a O +quantification O +of O +the O +reduction O +in O +radiation O +- O +induced O +Lys B-residue_name +and O +Phe B-residue_name +disordering O +upon O +RNA B-chemical +binding O +directly O +from O +the O +electron B-evidence +density I-evidence +. O + +With O +the O +wide O +use O +of O +high O +- O +flux O +third O +- O +generation O +synchrotron O +sources O +, O +radiation O +damage O +( O +RD O +) O +has O +once O +again O +become O +a O +dominant O +reason O +for O +the O +failure O +of O +structure B-experimental_method +determination I-experimental_method +using O +macromolecular B-experimental_method +crystallography I-experimental_method +( O +MX B-experimental_method +) O +in O +experiments O +conducted O +both O +at O +room O +temperature O +and O +under O +cryocooled O +conditions O +( O +100 O +K O +). O + +Significant O +progress O +has O +been O +made O +in O +recent O +years O +in O +understanding O +the O +inevitable O +manifestations O +of O +X O +- O +ray O +- O +induced O +RD O +within O +protein O +crystals B-evidence +, O +and O +there O +is O +now O +a O +body O +of O +literature O +on O +possible O +strategies O +to O +mitigate O +the O +effects O +of O +RD O +( O +e O +. O +g O +. O +Zeldin O +, O +Brockhauser O +et O +al O +., O +2013 O +; O +Bourenkov O +& O +Popov O +, O +2010 O +). O + +Dose O +is O +defined O +as O +the O +absorbed O +energy O +per O +unit O +mass O +of O +crystal O +in O +grays O +( O +Gy O +; O +1 O +Gy O += O +1 O +J O +kg O +− O +1 O +), O +and O +is O +the O +metric O +against O +which O +damage O +progression O +should O +be O +monitored O +during O +MX B-experimental_method +data O +collection O +, O +as O +opposed O +to O +time O +. O + +SRD O +has O +been O +well O +characterized O +in O +a O +large O +range O +of O +proteins O +, O +and O +is O +seen O +to O +follow O +a O +reproducible O +order O +: O +metallo O +- O +centre O +reduction O +, O +disulfide B-ptm +- I-ptm +bond I-ptm +cleavage O +, O +acidic O +residue O +decarboxylation O +and O +methionine O +methylthio O +cleavage O +( O +Ravelli O +& O +McSweeney O +, O +2000 O +; O +Burmeister O +, O +2000 O +; O +Weik O +et O +al O +., O +2000 O +; O +Yano O +et O +al O +., O +2005 O +). O + +There O +are O +a O +number O +of O +cases O +where O +SRD O +manifestations O +have O +compromised O +the O +biological O +information O +extracted O +from O +MX B-experimental_method +- I-experimental_method +determined I-experimental_method +structures B-evidence +at O +much O +lower O +doses O +than O +the O +recommended O +30 O +MGy O +limit O +, O +leading O +to O +false O +structural O +interpretations O +of O +protein O +mechanisms O +. O + +Active B-site +- I-site +site I-site +residues I-site +appear O +to O +be O +particularly O +susceptible O +, O +particularly O +for O +photosensitive O +proteins O +and O +in O +instances O +where O +chemical O +strain O +is O +an O +intrinsic O +feature O +of O +the O +reaction O +mechanism O +. O + +Since O +the O +majority O +of O +SRD B-experimental_method +studies I-experimental_method +to O +date O +have O +focused O +on O +proteins O +, O +much O +less O +is O +known O +about O +the O +effects O +of O +X O +- O +ray O +irradiation O +on O +the O +wider O +class O +of O +crystalline O +nucleoprotein B-complex_assembly +complexes O +or O +how O +to O +correct O +for O +such O +radiation O +- O +induced O +structural O +changes O +. O + +Understanding O +RD O +to O +such O +complexes O +is O +crucial O +, O +since O +DNA B-chemical +is O +rarely O +naked O +within O +a O +cell O +, O +instead O +dynamically O +interacting O +with O +proteins O +, O +facilitating O +replication O +, O +transcription O +, O +modification O +and O +DNA B-chemical +repair O +. O + +It O +is O +essential O +to O +understand O +how O +these O +increasingly O +complex O +macromolecular O +structures B-evidence +are O +affected O +by O +the O +radiation O +used O +to O +solve O +them O +. O + +Investigations O +on O +sub O +- O +ionization O +- O +level O +LEEs O +( O +0 O +– O +15 O +eV O +) O +interacting O +with O +both O +dried O +and O +aqueous O +oligonucleotides O +( O +Alizadeh O +& O +Sanche O +, O +2014 O +; O +Simons O +, O +2006 O +) O +concluded O +that O +resonant O +electron O +attachment O +to O +DNA B-chemical +bases O +and O +the O +sugar O +- O +phosphate O +backbone O +could O +lead O +to O +the O +preferential O +cleavage O +of O +strong O +(∼ O +4 O +eV O +, O +385 O +kJ O +mol O +− O +1 O +) O +sugar O +- O +phosphate O +C O +— O +O O +covalent O +bonds O +within O +the O +DNA B-chemical +backbone O +and O +then O +base O +- O +sugar O +N1 O +— O +C O +bonds O +, O +eventually O +leading O +to O +single O +- O +strand O +breakages O +( O +SSBs O +; O +Ptasińska O +& O +Sanche O +, O +2007 O +). O + +Electrons O +have O +been O +shown O +to O +be O +mobile O +at O +77 O +K O +by O +electron B-experimental_method +spin I-experimental_method +resonance I-experimental_method +spectroscopy I-experimental_method +studies O +( O +Symons O +, O +1997 O +; O +Jones O +et O +al O +., O +1987 O +), O +with O +rapid O +electron O +quantum O +tunnelling O +and O +positive O +hole O +migration O +along O +the O +protein O +backbone O +and O +through O +stacked O +DNA B-chemical +bases O +indicated O +as O +a O +dominant O +mechanism O +by O +which O +oxidative O +and O +reductive O +damage O +localizes O +at O +distances O +from O +initial O +ionization B-site +sites I-site +at O +100 O +K O +( O +O O +’ O +Neill O +et O +al O +., O +2002 O +). O + +The O +investigation O +of O +naturally O +forming O +nucleoprotein O +complexes O +circumvents O +the O +inherent O +challenges O +in O +making O +controlled O +comparisons O +of O +damage O +mechanisms O +between O +protein O +and O +nucleic O +acids O +crystallized B-experimental_method +separately O +. O +Recently O +, O +for O +a O +well O +characterized O +bacterial B-taxonomy_domain +protein O +– O +DNA B-chemical +complex O +( O +C B-complex_assembly +. I-complex_assembly +Esp1396I I-complex_assembly +; O +PDB O +entry O +3clc O +; O +resolution O +2 O +. O +8 O +Å O +; O +McGeehan O +et O +al O +., O +2008 O +) O +it O +was O +concluded O +that O +over O +a O +wide O +dose O +range O +( O +2 O +. O +1 O +– O +44 O +. O +6 O +MGy O +) O +the O +protein O +was O +far O +more O +susceptible O +to O +SRD O +than O +the O +DNA B-chemical +within O +the O +crystal B-evidence +( O +Bury O +et O +al O +., O +2015 O +). O + +Only O +at O +doses O +above O +20 O +MGy O +were O +precursors O +of O +phosphodiester O +- O +bond O +cleavage O +observed O +within O +AT B-structure_element +- I-structure_element +rich I-structure_element +regions I-structure_element +of O +the O +35 O +- O +mer O +DNA B-chemical +. O + +For O +crystalline O +complexes O +such O +as O +C B-complex_assembly +. I-complex_assembly +Esp1396I I-complex_assembly +, O +whether O +the O +protein O +is O +intrinsically O +more O +susceptible O +to O +X O +- O +ray O +- O +induced O +damage O +or O +whether O +the O +protein O +scavenges O +electrons O +to O +protect O +the O +DNA B-chemical +remains O +unclear O +in O +the O +absence O +of O +a O +non O +- O +nucleic O +acid O +- O +bound O +protein O +control O +obtained O +under O +exactly O +the O +same O +crystallization O +and O +data O +- O +collection O +conditions O +. O + +To O +monitor O +the O +effects O +of O +nucleic O +acid O +binding O +on O +protein O +damage O +susceptibility O +, O +a O +crystal B-evidence +containing O +two O +protein O +molecules O +per O +asymmetric O +unit O +, O +only O +one O +of O +which O +was O +bound B-protein_state +to I-protein_state +RNA B-chemical +, O +is O +reported O +here O +( O +Fig O +. O +1 O +▸). O + +Using O +newly O +developed O +methodology O +, O +we O +present O +a O +controlled B-experimental_method +SRD I-experimental_method +investigation O +at O +1 O +. O +98 O +Å O +resolution O +using O +a O +large O +(∼ O +91 O +kDa O +) O +crystalline O +protein B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complex O +: O +trp B-protein_type +RNA I-protein_type +- I-protein_type +binding I-protein_type +attenuation I-protein_type +protein I-protein_type +( O +TRAP B-complex_assembly +) O +bound B-protein_state +to I-protein_state +a O +53 O +bp O +RNA B-chemical +sequence O +( B-chemical +GAGUU I-chemical +) I-chemical +10GAG I-chemical +( O +PDB O +entry O +1gtf O +; O +Hopcroft O +et O +al O +., O +2002 O +). O + +It O +binds O +with O +high O +affinity O +( O +K B-evidence +d I-evidence +≃ O +1 O +. O +0 O +nM O +) O +to O +RNA B-chemical +segments O +containing O +11 O +GAG B-structure_element +/ I-structure_element +UAG I-structure_element +triplets I-structure_element +separated O +by O +two O +or O +three O +spacer B-structure_element +nucleotides I-structure_element +( O +Elliott O +et O +al O +., O +2001 O +) O +to O +regulate O +the O +transcription O +of O +tryptophan B-chemical +biosynthetic O +genes O +in O +Bacillus B-species +subtilis I-species +( O +Antson O +et O +al O +., O +1999 O +). O + +In O +this O +structure B-evidence +, O +the O +bases O +of O +the O +G1 B-chemical +- I-chemical +A2 I-chemical +- I-chemical +G3 I-chemical +nucleotides O +form O +direct O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +protein O +, O +unlike O +the O +U4 B-chemical +- I-chemical +U5 I-chemical +nucleotides O +, O +which O +appear O +to O +be O +more O +flexible O +. O + +Ten O +successive O +1 O +. O +98 O +Å O +resolution O +MX B-experimental_method +data O +sets O +were O +collected O +from O +the O +same O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +crystal B-evidence +to O +analyse O +X O +- O +ray O +- O +induced O +structural O +changes O +over O +a O +large O +dose O +range O +( O +d O +1 O += O +1 O +. O +3 O +MGy O +to O +d O +10 O += O +25 O +. O +0 O +MGy O +). O + +To O +avoid O +the O +previous O +necessity O +for O +visual O +inspection O +of O +electron B-evidence +- I-evidence +density I-evidence +maps I-evidence +to O +detect O +SRD B-site +sites I-site +, O +a O +computational O +approach O +was O +designed O +to O +quantify O +the O +electron B-evidence +- I-evidence +density I-evidence +change I-evidence +for O +each O +refined O +atom O +with O +increasing O +dose O +, O +thus O +providing O +a O +rapid O +systematic O +method O +for O +SRD O +study O +on O +such O +large O +multimeric O +complexes O +. O + +By O +employing O +the O +high O +11 O +- O +fold O +structural O +symmetry O +within O +each O +TRAP B-complex_assembly +macromolecule O +, O +this O +approach O +permitted O +a O +thorough O +statistical O +quantification O +of O +the O +RD O +effects O +of O +RNA B-chemical +binding O +to O +TRAP B-complex_assembly +. O + +Per B-experimental_method +- I-experimental_method +atom I-experimental_method +quantification I-experimental_method +of I-experimental_method +electron I-experimental_method +density I-experimental_method + +To O +quantify O +the O +exact O +effects O +of O +nucleic O +acid O +binding O +to O +a O +protein O +on O +SRD O +susceptibility O +, O +a O +high O +- O +throughput O +and O +automated O +pipeline O +was O +created O +to O +systematically O +calculate O +the O +electron B-evidence +- I-evidence +density I-evidence +change I-evidence +for O +every O +refined O +atom O +within O +the O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +structure B-evidence +as O +a O +function O +of O +dose O +. O + +This O +provides O +an O +atom O +- O +specific O +quantification O +of O +density B-evidence +– I-evidence +dose I-evidence +dynamics I-evidence +, O +which O +was O +previously O +lacking O +within O +the O +field O +. O + +Previous O +studies O +have O +characterized O +SRD B-site +sites I-site +by O +reporting O +magnitudes O +of O +F B-evidence +obs I-evidence +( I-evidence +d I-evidence +n I-evidence +) I-evidence +− I-evidence +F I-evidence +obs I-evidence +( I-evidence +d I-evidence +1 I-evidence +) I-evidence +Fourier I-evidence +difference I-evidence +map I-evidence +peaks I-evidence +in O +terms O +of O +the O +sigma B-evidence +( O +σ B-evidence +) O +contour O +level O +( O +the O +number O +of O +standard B-evidence +deviations I-evidence +from O +the O +mean B-evidence +map I-evidence +electron I-evidence +- I-evidence +density I-evidence +value I-evidence +) O +at O +which O +peaks O +become O +visible O +. O + +However O +, O +these O +σ B-evidence +levels O +depend O +on O +the O +standard B-evidence +deviation I-evidence +values O +of O +the O +map B-evidence +, O +which O +can O +deviate O +between O +data O +sets O +, O +and O +are O +thus O +unsuitable O +for O +quantitative O +comparison O +of O +density B-evidence +between O +different O +dose O +data O +sets O +. O + +Large O +positive O +D B-evidence +loss I-evidence +values O +indicate O +radiation O +- O +induced O +atomic O +disordering O +reproducibly O +throughout O +the O +unit O +cells O +with O +respect O +to O +the O +initial O +low O +- O +dose O +data O +set O +. O + +For O +each O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +data O +set O +, O +the O +D B-evidence +loss I-evidence +metric I-evidence +successfully O +identified O +the O +recognized O +forms O +of O +protein O +SRD B-experimental_method +( O +Fig O +. O +2 O +▸ O +a O +), O +with O +clear O +Glu B-residue_name +and O +Asp B-residue_name +side O +- O +chain O +decarboxylation O +even O +in O +the O +first O +difference B-evidence +map I-evidence +calculated O +( O +3 O +. O +9 O +MGy O +; O +Fig O +. O +3 O +▸ O +a O +). O + +The O +main O +sequence O +of O +TRAP B-complex_assembly +does O +not O +contain O +any O +Trp B-residue_name +and O +Cys B-residue_name +residues O +( O +and O +thus O +contains O +no O +disulfide O +bonds O +). O + +Even O +for O +radiation O +- O +insensitive O +residues O +( O +e O +. O +g O +. O +Gly B-residue_name +) O +the O +average O +D B-evidence +loss I-evidence +increases O +with O +dose O +: O +this O +is O +the O +effect O +of O +global O +radiation O +damage O +, O +since O +as O +dose O +increases O +the O +electron B-evidence +density I-evidence +associated O +with O +each O +refined O +atom O +becomes O +weaker O +as O +the O +atomic O +occupancy O +decreases O +( O +Fig O +. O +2 O +▸ O +b O +). O + +Only O +Glu B-residue_name +and O +Asp B-residue_name +residues O +exhibit O +a O +rate O +of O +D B-evidence +loss I-evidence +increase O +that O +consistently O +exceeds O +the O +average O +decay O +( O +Fig O +. O +2 O +▸ O +b O +, O +dashed O +line O +) O +at O +each O +dose O +. O + +RNA B-chemical +is O +less O +susceptible O +to O +electron B-evidence +- I-evidence +density I-evidence +loss O +than O +protein O +within O +the O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complex O + +Visual B-experimental_method +inspection I-experimental_method +of I-experimental_method +Fourier B-evidence +difference I-evidence +maps I-evidence +illustrated O +the O +clear O +lack O +of O +RNA B-chemical +electron B-evidence +- I-evidence +density I-evidence +degradation I-evidence +with O +increasing O +dose O +compared O +with O +the O +obvious O +protein O +damage O +manifestations O +( O +Figs O +. O +3 O +▸ O +b O +and O +3 O +▸ O +c O +). O + +Only O +at O +the O +highest O +doses O +investigated O +(> O +20 O +MGy O +) O +was O +density O +loss O +observed O +at O +the O +RNA B-chemical +phosphate O +and O +C O +— O +O O +bonds O +of O +the O +phosphodiester O +backbone O +. O + +However O +, O +the O +median O +D B-evidence +loss I-evidence +was O +lower O +by O +a O +factor O +of O +> O +2 O +for O +RNA B-chemical +P O +atoms O +than O +for O +Glu B-residue_name +and O +Asp B-residue_name +side O +- O +chain O +groups O +at O +25 O +. O +0 O +MGy O +( O +Supplementary O +Fig O +. O +S4 O +), O +and O +furthermore O +could O +not O +be O +numerically O +distinguished O +from O +Gly B-residue_name +Cα O +atoms O +within O +TRAP B-complex_assembly +, O +which O +are O +not O +radiation O +- O +sensitive O +at O +the O +doses O +tested O +here O +( O +Supplementary O +Fig O +. O +S3 O +). O + +RNA B-chemical +binding O +protects O +radiation O +- O +sensitive O +residues O + +Hotelling B-experimental_method +’ I-experimental_method +s I-experimental_method +T I-experimental_method +- I-experimental_method +squared I-experimental_method +test I-experimental_method +( O +the O +multivariate O +counterpart O +of O +Student B-experimental_method +’ I-experimental_method +s I-experimental_method +t I-experimental_method +- I-experimental_method +test I-experimental_method +) O +was O +used O +to O +reject O +the O +null O +hypothesis O +that O +the O +means O +of O +the O +D B-evidence +loss I-evidence +metric I-evidence +were O +equal O +for O +the O +bound B-protein_state +and O +nonbound B-protein_state +groups O +in O +Fig O +. O +5 O +▸. O + +A O +significant O +reduction O +in O +D B-evidence +loss I-evidence +is O +seen O +for O +Glu36 B-residue_name_number +in O +RNA B-protein_state +- I-protein_state +bound I-protein_state +compared O +with O +nonbound B-protein_state +TRAP B-complex_assembly +, O +indicative O +of O +a O +lower O +rate O +of O +side O +- O +chain O +decarboxylation O +( O +Fig O +. O +5 O +▸ O +a O +; O +p O += O +6 O +. O +06 O +× O +10 O +− O +5 O +). O + +For O +each O +TRAP B-complex_assembly +ring B-structure_element +subunit B-structure_element +, O +the O +Glu36 B-residue_name_number +side O +- O +chain O +carboxyl O +group O +accepts O +a O +pair O +of O +hydrogen B-bond_interaction +bonds I-bond_interaction +from O +the O +two O +N O +atoms O +of O +the O +G3 B-residue_name_number +RNA B-chemical +base O +. O + +In O +our O +analysis O +, O +Asp39 B-residue_name_number +in O +the O +TRAP B-complex_assembly +–( I-complex_assembly +GAGUU I-complex_assembly +) I-complex_assembly +10GAG I-complex_assembly +structure B-evidence +appears O +to O +exhibit O +two O +distinct O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +G1 B-residue_name_number +base O +within O +each O +of O +the O +11 O +TRAP B-site +– I-site +RNA I-site +interfaces I-site +, O +as O +does O +Glu36 B-residue_name_number +to O +G3 B-residue_name_number +; O +however O +, O +the O +reduction O +in O +density B-evidence +disordering O +upon O +RNA B-chemical +binding O +is O +far O +less O +significant O +for O +Asp39 B-residue_name_number +than O +for O +Glu36 B-residue_name_number +( O +Fig O +. O +5 O +▸ O +b O +, O +p O += O +0 O +. O +0925 O +). O + +RNA B-chemical +binding O +reduces O +radiation O +- O +induced O +disorder O +on O +the O +atomic O +scale O + +One O +oxygen O +( O +O O +∊ O +1 O +) O +of O +Glu42 B-residue_name_number +appears O +to O +form O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +to O +a O +nearby O +water B-chemical +within O +each O +TRAP B-site +RNA I-site +- I-site +binding I-site +pocket I-site +, O +with O +the O +other O +( O +O O +∊ O +2 O +) O +being O +involved O +in O +a O +salt B-bond_interaction +- I-bond_interaction +bridge I-bond_interaction +interaction O +with O +Arg58 B-residue_name_number +( O +Hopcroft O +et O +al O +., O +2002 O +; O +Antson O +et O +al O +., O +1999 O +). O + +The O +density B-evidence +- I-evidence +change I-evidence +dynamics I-evidence +were O +statistically O +indistinguishable O +between O +bound B-protein_state +and O +nonbound B-protein_state +TRAP B-complex_assembly +for O +each O +Glu42 B-residue_name_number +carboxyl O +group O +Cδ O +atom O +( O +p O += O +0 O +. O +435 O +), O +indicating O +that O +upon O +RNA B-chemical +binding O +the O +conserved O +salt B-bond_interaction +- I-bond_interaction +bridge I-bond_interaction +interaction O +ultimately O +dictated O +the O +overall O +Glu42 B-residue_name_number +decarboxylation O +rate O +. O + +With O +increasing O +dose O +, O +the O +D B-evidence +loss I-evidence +associated O +with O +the O +Phe32 B-residue_name_number +side O +chain O +was O +significantly O +reduced O +upon O +RNA B-chemical +binding O +( O +Fig O +. O +5 O +▸ O +e O +; O +Phe32 B-residue_name_number +Cζ O +; O +p O += O +0 O +. O +0014 O +), O +an O +indication O +that O +radiation O +- O +induced O +conformation O +disordering O +of O +Phe32 B-residue_name_number +had O +been O +reduced O +. O + +The O +extended O +aliphatic O +Lys37 B-residue_name_number +side O +chain O +stacks O +against O +the O +nearby O +G1 B-residue_name_number +base O +, O +making O +a O +series O +of O +nonpolar B-bond_interaction +contacts I-bond_interaction +within O +each O +RNA B-site +- I-site +binding I-site +interface I-site +. O + +The O +D B-evidence +loss I-evidence +for O +Lys37 B-residue_name_number +side O +- O +chain O +atoms O +was O +also O +reduced O +when O +stacked B-bond_interaction +against O +the O +G1 B-residue_name_number +base O +( O +Fig O +. O +5 O +▸ O +f O +; O +p O += O +0 O +. O +0243 O +for O +Lys37 B-residue_name_number +C O +∊ O +atoms O +). O + +Representative O +Phe32 B-residue_name_number +and O +Lys37 B-residue_name_number +atoms O +were O +selected O +to O +illustrate O +these O +trends O +. O + +Compared O +with O +previous O +studies O +, O +the O +results O +provide O +a O +further O +step O +in O +the O +detailed O +characterization O +of O +SRD O +effects O +in O +MX B-experimental_method +. O + +Our O +method O +­ O +ology O +, O +which O +eliminated O +tedious O +and O +error O +- O +prone O +visual O +inspection O +, O +permitted O +the O +determination O +on O +a O +per O +- O +atom O +basis O +of O +the O +most O +damaged O +sites O +, O +as O +characterized O +by O +F B-evidence +obs I-evidence +( I-evidence +d I-evidence +n I-evidence +) I-evidence +− I-evidence +F I-evidence +obs I-evidence +( I-evidence +d I-evidence +1 I-evidence +) I-evidence +Fourier I-evidence +difference I-evidence +map I-evidence +peaks I-evidence +between O +successive O +data O +sets O +collected O +from O +the O +same O +crystal B-evidence +. O + +The O +RNA B-chemical +was O +found O +to O +be O +substantially O +more O +radiation B-protein_state +- I-protein_state +resistant I-protein_state +than O +the O +protein O +, O +even O +at O +the O +highest O +doses O +investigated O +(∼ O +25 O +. O +0 O +MGy O +), O +which O +is O +in O +strong O +concurrence O +with O +our O +previous O +SRD B-experimental_method +investigation I-experimental_method +of O +the O +C B-complex_assembly +. I-complex_assembly +Esp1396I I-complex_assembly +protein O +– O +DNA B-chemical +complex O +( O +Bury O +et O +al O +., O +2015 O +). O + +RNA B-chemical +backbone O +disordering O +thus O +appears O +to O +be O +the O +main O +radiation O +- O +induced O +effect O +in O +RNA B-chemical +, O +with O +the O +protein O +– O +base O +interactions O +maintained O +even O +at O +high O +doses O +(> O +20 O +MGy O +). O + +The O +U4 B-residue_name_number +phosphate B-chemical +exhibited O +marginally O +larger O +D B-evidence +loss I-evidence +values O +above O +20 O +MGy O +than O +G1 B-residue_name_number +, O +A2 B-residue_name_number +and O +G3 B-residue_name_number +( O +Supplementary O +Fig O +. O +S4 O +). O + +Consequently O +, O +no O +clear O +single O +- O +strand O +breaks O +could O +be O +located O +, O +and O +since O +RNA B-chemical +- O +binding O +within O +the O +current O +TRAP B-complex_assembly +–( I-complex_assembly +GAGUU I-complex_assembly +) I-complex_assembly +10GAG I-complex_assembly +complex O +is O +mediated O +predominantly O +through O +base O +– O +protein O +interactions O +, O +the O +biological O +integrity O +of O +the O +RNA B-chemical +complex O +was O +dictated O +by O +the O +rate O +at O +which O +protein O +decarboxylation O +occurred O +. O + +RNA B-chemical +interacting O +with O +TRAP B-complex_assembly +was O +shown O +to O +offer O +significant O +protection O +against O +radiation O +- O +induced O +structural O +changes O +. O + +Both O +Glu36 B-residue_name_number +and O +Asp39 B-residue_name_number +bind O +directly O +to O +RNA B-chemical +, O +each O +through O +two O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +guanine B-chemical +bases O +( O +G3 B-residue_name_number +and O +G1 B-residue_name_number +, O +respectively O +). O + +However O +, O +compared O +with O +Asp39 B-residue_name_number +, O +Glu36 B-residue_name_number +is O +strikingly O +less O +decarboxylated O +when O +bound B-protein_state +to I-protein_state +RNA B-chemical +( O +Fig O +. O +4 O +▸). O + +This O +is O +in O +good O +agreement O +with O +previous O +mutagenesis B-experimental_method +and I-experimental_method +nucleoside I-experimental_method +analogue I-experimental_method +studies I-experimental_method +( O +Elliott O +et O +al O +., O +2001 O +), O +which O +indicated O +that O +the O +G1 B-residue_name_number +nucleotide O +does O +not O +bind O +to O +TRAP B-complex_assembly +as O +strongly O +as O +do O +A2 B-residue_name_number +and O +G3 B-residue_name_number +, O +and O +plays O +little O +role O +in O +the O +high O +RNA B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +TRAP B-complex_assembly +( O +K B-evidence +d I-evidence +≃ O +1 O +. O +1 O +± O +0 O +. O +4 O +nM O +). O + +Thus O +, O +another O +factor O +must O +be O +responsible O +for O +this O +clear O +reduction O +in O +Glu36 B-residue_name_number +CO2 O +decarboxyl O +­ O +ation O +in O +RNA B-protein_state +- I-protein_state +bound I-protein_state +TRAP B-complex_assembly +. O + +When O +bound B-protein_state +to I-protein_state +RNA B-chemical +, O +the O +average O +solvent O +- O +accessible O +area O +of O +the O +Glu36 B-residue_name_number +side O +- O +chain O +O O +atoms O +is O +reduced O +from O +∼ O +15 O +to O +0 O +Å2 O +. O + +The O +electron B-evidence +- I-evidence +recombination I-evidence +rate I-evidence +K I-evidence +− I-evidence +1 I-evidence +remains O +high O +, O +however O +, O +owing O +to O +rapid O +electron O +migration O +through O +the O +protein B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complex O +to O +refill O +the O +Glu36 B-residue_name_number +positive B-site +hole I-site +( O +the O +precursor O +for O +Glu B-residue_name +decarboxylation O +). O + +The O +prevalence O +of O +radical O +attack O +from O +solvent O +channels O +surrounding O +the O +protein O +in O +the O +crystal B-evidence +is O +a O +questionable O +cause O +, O +considering O +previous O +observations O +indicating O +that O +the O +strongly O +oxidizing O +hydroxyl O +radical O +is O +immobile O +at O +100 O +K O +( O +Allan O +et O +al O +., O +2013 O +; O +Owen O +et O +al O +., O +2012 O +). O + +By O +comparing O +equivalent O +acidic O +residues O +with B-protein_state +and O +without B-protein_state +RNA B-chemical +, O +we O +have O +now O +deconvoluted O +the O +role O +of O +solvent O +accessibility O +from O +other O +local O +protein O +environment O +factors O +, O +and O +thus O +propose O +a O +suitable O +mechanism O +by O +which O +exceptionally O +low O +solvent O +accessibility O +can O +reduce O +the O +rate O +of O +decarboxylation O +. O + +Apart O +from O +these O +RNA B-site +- I-site +binding I-site +interfaces I-site +, O +RNA B-chemical +binding O +was O +seen O +to O +enhance O +decarboxylation O +for O +residues O +Glu50 B-residue_name_number +, O +Glu71 B-residue_name_number +and O +Glu73 B-residue_name_number +, O +all O +of O +which O +are O +involved O +in O +crystal O +contacts O +between O +TRAP B-complex_assembly +rings B-structure_element +( O +Fig O +. O +4 O +▸ O +c O +). O + +Radiation O +- O +induced O +side O +- O +chain O +conformational O +changes O +have O +been O +poorly O +characterized O +in O +previous O +SRD B-experimental_method +investigations I-experimental_method +owing O +to O +their O +strong O +dependence O +on O +packing O +density O +and O +geometric O +strain O +. O + +Such O +structural O +changes O +are O +known O +to O +have O +significant O +roles O +within O +enzymatic O +pathways O +, O +and O +experimenters O +must O +be O +aware O +of O +these O +possible O +confounding O +factors O +when O +assigning O +true O +functional O +mechanisms O +using O +MX B-experimental_method +. O + +Our O +results O +show O +that O +RNA B-chemical +binding O +to O +TRAP B-complex_assembly +physically O +stabilizes O +non O +- O +acidic O +residues O +within O +the O +TRAP B-complex_assembly +macromolecule O +, O +most O +notably O +Lys37 B-residue_name_number +and O +Phe32 B-residue_name_number +, O +which O +stack O +against O +the O +G1 B-residue_name_number +and O +G3 B-residue_name_number +bases O +, O +respectively O +. O + +In O +TRAP B-complex_assembly +, O +D B-evidence +loss I-evidence +increased O +at O +a O +similar O +rate O +for O +both O +the O +Tyr B-residue_name +O O +atoms O +and O +aromatic O +ring B-structure_element +atoms O +, O +suggesting O +that O +full O +ring B-structure_element +conformational O +disordering O +is O +more O +likely O +. O + +The O +RNA B-chemical +- O +stabilization O +effects O +on O +protein O +are O +observed O +at O +short O +ranges O +and O +are O +restricted O +to O +within O +the O +RNA B-site +- I-site +binding I-site +interfaces I-site +around O +the O +TRAP B-complex_assembly +ring B-structure_element +. O + +For O +example O +, O +Asp17 B-residue_name_number +is O +located O +∼ O +6 O +. O +8 O +Å O +from O +the O +G1 B-residue_name_number +base O +, O +outside O +the O +RNA B-site +- I-site +binding I-site +interfaces I-site +, O +and O +has O +indistinguishable O +Cγ O +atom O +D O +loss B-evidence +dose I-evidence +- I-evidence +dynamics I-evidence +between O +RNA B-protein_state +- I-protein_state +bound I-protein_state +and O +nonbound B-protein_state +TRAP B-complex_assembly +( O +p O +> O +0 O +. O +9 O +). O + +An O +increase O +in O +the O +dose O +at O +which O +functionally O +important O +residues O +remain O +intact O +has O +biological O +ramifications O +for O +understanding O +the O +mechanisms O +at O +which O +ionizing O +radiation O +damage O +is O +mitigated O +within O +naturally O +forming O +DNA B-complex_assembly +– I-complex_assembly +protein I-complex_assembly +and O +RNA B-complex_assembly +– I-complex_assembly +protein I-complex_assembly +complexes O +. O + +Observations O +of O +lower O +protein O +radiation O +- O +sensitivity O +in O +DNA B-protein_state +- I-protein_state +bound I-protein_state +forms O +have O +been O +recorded O +in O +solution O +at O +RT O +at O +much O +lower O +doses O +(∼ O +1 O +kGy O +) O +than O +those O +used O +for O +typical O +MX B-experimental_method +experiments O +[ O +e O +. O +g O +. O +an O +oestrogen O +response O +element O +– O +receptor O +complex O +( O +Stísová O +et O +al O +., O +2006 O +) O +and O +a O +DNA B-protein_type +glycosylase I-protein_type +and O +its O +abasic B-site +DNA I-site +target I-site +site I-site +( O +Gillard O +et O +al O +., O +2004 O +)]. O + +In O +these O +studies O +, O +the O +main O +damaging O +species O +is O +predicted O +to O +be O +the O +oxidizing O +hydroxyl O +radical O +produced O +through O +solvent O +irradiation O +, O +which O +is O +known O +to O +add O +to O +double O +covalent O +bonds O +within O +both O +DNA B-chemical +and O +RNA B-chemical +bases O +to O +induce O +strand O +breaks O +and O +base O +modification O +( O +Spotheim O +- O +Maurizot O +& O +Davídková O +, O +2011 O +; O +Chance O +et O +al O +., O +1997 O +). O + +It O +was O +suggested O +that O +physical O +screening O +of O +DNA B-chemical +by O +protein O +shielded O +the O +DNA B-site +– I-site +protein I-site +interaction I-site +sites I-site +from O +radical O +damage O +, O +yielding O +an O +extended O +life O +- O +dose O +for O +the O +nucleoprotein O +complex O +compared O +with O +separate O +protein O +and O +DNA B-chemical +constituents O +at O +RT O +. O + +However O +, O +in O +the O +current O +MX B-experimental_method +study O +at O +100 O +K O +, O +the O +main O +damaging O +species O +are O +believed O +to O +be O +migrating O +LEEs O +and O +holes O +produced O +directly O +within O +the O +protein B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +components O +or O +in O +closely O +associated O +solvent O +. O + +The O +results O +presented O +here O +suggest O +that O +biologically O +relevant O +nucleoprotein B-complex_assembly +complexes O +also O +exhibit O +prolonged O +life O +- O +doses O +under O +the O +effect O +of O +LEE O +- O +induced O +structural O +changes O +, O +involving O +direct O +physical O +protection O +of O +key O +RNA B-site +- I-site +binding I-site +residues I-site +. O + +When O +exposed O +to O +X O +- O +rays O +, O +these O +residues O +will O +be O +preferentially O +damaged O +by O +X O +- O +rays O +and O +subsequently O +reduce O +the O +affinity O +with O +which O +TRAP B-complex_assembly +binds O +to O +RNA B-chemical +. O + +Within O +the O +cellular O +environment O +, O +this O +mechanism O +could O +reduce O +the O +risk O +that O +radiation O +- O +damaged O +proteins O +might O +bind O +to O +RNA B-chemical +, O +thus O +avoiding O +the O +detrimental O +introduction O +of O +incorrect O +DNA B-chemical +- O +repair O +, O +transcriptional O +and O +base O +- O +modification O +pathways O +. O + +The O +TRAP B-complex_assembly +–( I-complex_assembly +GAGUU I-complex_assembly +) I-complex_assembly +10GAG I-complex_assembly +complex O +asymmetric O +unit O +( O +PDB O +entry O +1gtf O +; O +Hopcroft O +et O +al O +., O +2002 O +). O + +Bound B-protein_state +tryptophan B-chemical +ligands O +are O +represented O +as O +coloured O +spheres O +. O + +( O +a O +) O +Electron O +- O +density O +loss O +sites O +as O +indicated O +by O +D O +loss O +in O +the O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complex O +crystal B-evidence +by O +residue O +/ O +nucleotide O +type O +for O +five O +doses O +[ O +sites O +determined O +above O +the O +4 O +× O +average O +D O +loss O +threshold O +, O +calculated O +over O +the O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +structure B-evidence +for O +the O +first O +difference B-evidence +map I-evidence +: O +F O +obs O +( O +d O +2 O +) O +− O +F O +obs O +( O +d O +1 O +)]. O + +The O +average O +D O +loss O +( O +calculated O +over O +the O +whole O +TRAP B-complex_assembly +asymmetric O +unit O +) O +is O +shown O +at O +each O +dose O +( O +dashed O +line O +). O + +Radiation O +- O +induced O +protein O +disordering O +is O +evident O +across O +the O +large O +dose O +range O +( O +b O +, O +c O +); O +in O +comparison O +, O +no O +clear O +deterioration O +of O +the O +RNA B-chemical +density B-evidence +was O +observed O +. O + +D O +loss O +calculated O +for O +all O +side O +- O +chain O +carboxyl O +group O +Glu B-residue_name +Cδ O +and O +Asp B-residue_name +Cγ O +atoms O +within O +the O +TRAP B-complex_assembly +– I-complex_assembly +RNA I-complex_assembly +complex O +for O +a O +dose O +of O +19 O +. O +3 O +MGy O +( O +d O +8 O +). O + +Residues O +have O +been O +grouped O +by O +amino O +- O +acid O +number O +, O +and O +split O +into O +bound B-protein_state +and O +nonbound B-protein_state +groupings O +, O +with O +each O +bar O +representing O +the O +mean O +calculated O +over O +11 O +equivalent O +atoms O +around O +a O +TRAP B-complex_assembly +ring B-structure_element +. O + +D O +loss O +against O +dose O +for O +( O +a O +) O +Glu36 B-residue_name_number +Cδ O +, O +( O +b O +) O +Asp39 B-residue_name_number +Cγ O +, O +( O +c O +) O +Glu42 B-residue_name_number +O O +∊ O +1 O +, O +( O +d O +) O +Glu42 B-residue_name_number +O O +∊ O +2 O +, O +( O +e O +) O +Phe32 B-residue_name_number +Cζ O +and O +( O +f O +) O +Lys37 B-residue_name_number +C O +∊ O +atoms O +. O + +95 O +% O +CI O +are O +included O +for O +each O +set O +of O +11 O +equivalent O +atoms O +grouped O +as O +bound B-protein_state +/ O +nonbound B-protein_state +. O + +RNA B-site +- I-site +binding I-site +interface I-site +interactions O +are O +shown O +for O +TRAP B-complex_assembly +chain O +N O +, O +with O +the O +F O +obs O +( O +d O +7 O +) O +− O +F O +obs O +( O +d O +1 O +) O +Fourier O +difference O +map O +( O +dose O +16 O +. O +7 O +MGy O +) O +overlaid O +and O +contoured O +at O +a O +± O +4σ O +level O +. O + +A O +conserved O +motif O +in O +JNK B-protein_type +/ I-protein_type +p38 I-protein_type +- I-protein_type +specific I-protein_type +MAPK I-protein_type +phosphatases I-protein_type +as O +a O +determinant O +for O +JNK1 B-protein +recognition O +and O +inactivation O + +MAPK B-protein_type +signalling O +efficiency O +and O +specificity O +is O +modulated O +by O +protein O +– O +protein O +interactions O +between O +individual O +MAPKs B-protein_type +and O +the O +docking B-structure_element +motifs I-structure_element +in O +cognate O +binding O +partners O +. O + +Here O +we O +report O +the O +crystal B-evidence +structure I-evidence +of O +JNK1 B-protein +bound B-protein_state +to I-protein_state +the O +catalytic B-structure_element +domain I-structure_element +of O +MKP7 B-protein +at O +2 O +. O +4 O +- O +Å O +resolution O +, O +providing O +high O +- O +resolution O +structural O +insight O +into O +the O +FXF B-site +- I-site +docking I-site +interaction I-site +. O + +Here O +, O +the O +authors O +report O +the O +structure B-evidence +of O +MKP7 B-protein +bound B-protein_state +to I-protein_state +JNK1 B-protein +and O +characterise O +the O +conserved B-protein_state +MKP B-protein_type +- O +MAPK B-protein_type +interaction O +. O + +The O +mitogen B-protein_type +- I-protein_type +activated I-protein_type +protein I-protein_type +kinases I-protein_type +( O +MAPKs B-protein_type +) O +are O +central O +components O +of O +the O +signal O +- O +transduction O +pathways O +, O +which O +mediate O +the O +cellular O +response O +to O +a O +variety O +of O +extracellular O +stimuli O +, O +ranging O +from O +growth O +factors O +to O +environmental O +stresses O +. O + +The O +MAPK B-protein_type +signalling O +pathways O +are O +evolutionally O +highly O +conserved O +. O + +The O +basic O +assembly O +of O +MAPK B-protein_type +pathways O +is O +a O +three O +- O +tier O +kinase B-protein_type +module O +that O +establishes O +a O +sequential O +activation O +cascade O +: O +a O +MAPK B-protein_type +kinase I-protein_type +kinase I-protein_type +activates O +a O +MAPK B-protein_type +kinase I-protein_type +, O +which O +in O +turn O +activates O +a O +MAPK B-protein_type +. O + +The O +three O +best O +- O +characterized O +MAPK B-protein_type +signalling O +pathways O +are O +mediated O +by O +the O +kinases B-protein_type +extracellular B-protein_type +signal I-protein_type +- I-protein_type +regulated I-protein_type +kinase I-protein_type +( O +ERK B-protein_type +), O +c B-protein_type +- I-protein_type +Jun I-protein_type +N I-protein_type +- I-protein_type +terminal I-protein_type +kinase I-protein_type +( O +JNK B-protein_type +) O +and O +p38 B-protein_type +. O + +The O +ERK B-protein_type +pathway O +is O +activated O +by O +various O +mitogens O +and O +phorbol O +esters O +, O +whereas O +the O +JNK B-protein_type +and O +p38 B-protein_type +pathways O +are O +stimulated O +mainly O +by O +environmental O +stress O +and O +inflammatory O +cytokines B-protein_type +. O + +After O +activation O +, O +each O +MAPK B-protein_type +phosphorylates O +a O +distinct O +set O +of O +protein O +substrates O +, O +which O +act O +as O +the O +critical O +effectors O +that O +enable O +cells O +to O +mount O +the O +appropriate O +responses O +to O +varied O +stimuli O +. O + +Two O +types O +of O +docking B-structure_element +motifs I-structure_element +have O +been O +identified O +in O +MAPK B-protein_type +substrates O +and O +cognate O +proteins O +: O +kinase B-structure_element +- I-structure_element +interacting I-structure_element +motif I-structure_element +( O +D B-structure_element +- I-structure_element +motif I-structure_element +) O +and O +FXF B-structure_element +- I-structure_element +motif I-structure_element +( O +also O +called O +DEF B-structure_element +motif I-structure_element +, O +docking B-site +site I-site +for O +ERK B-protein_type +FXF B-structure_element +). O + +The O +D B-site +- I-site +motif I-site +- I-site +docking I-site +site I-site +( O +D B-site +- I-site +site I-site +) O +in O +MAPKs B-protein_type +is O +situated O +in O +a O +noncatalytic B-site +region I-site +opposite O +of O +the O +kinase B-protein_type +catalytic B-site +pocket I-site +and O +is O +comprised O +of O +a O +highly B-site +acidic I-site +patch I-site +and O +a O +hydrophobic B-site +groove I-site +. O + +D B-structure_element +- I-structure_element +motifs I-structure_element +are O +found O +in O +many O +MAPK B-protein_type +- I-protein_type +interacting I-protein_type +proteins I-protein_type +, O +including O +substrates O +, O +activating O +kinases B-protein_type +and O +inactivating O +phosphatases B-protein_type +, O +as O +well O +as O +scaffolding O +proteins O +. O + +A O +second B-structure_element +docking I-structure_element +motif I-structure_element +for O +MAPKs B-protein_type +consists O +of O +two O +Phe B-residue_name +residues O +separated O +by O +one O +residue O +( O +FXF B-structure_element +- I-structure_element +motif I-structure_element +). O + +This O +motif O +has O +been O +observed O +in O +several O +MAPK B-protein_type +substrates O +. O + +The O +FXF B-site +- I-site +motif I-site +- I-site +binding I-site +site I-site +of O +ERK2 B-protein +has O +been O +mapped O +to O +a O +hydrophobic B-site +pocket I-site +formed O +between O +the O +P B-site ++ I-site +1 I-site +site I-site +, O +αG B-structure_element +helix I-structure_element +and O +the O +MAPK B-structure_element +insert I-structure_element +. O + +However O +, O +the O +generality O +and O +mechanism O +of O +the O +FXF B-structure_element +- O +mediated O +interaction O +is O +unclear O +. O + +The O +physiological O +outcome O +of O +MAPK B-protein_type +signalling O +depends O +on O +both O +the O +magnitude O +and O +the O +duration O +of O +kinase O +activation O +. O + +MKPs B-protein_type +constitute O +a O +group O +of O +DUSPs B-protein_type +that O +are O +characterized O +by O +their O +ability O +to O +dephosphorylate O +both O +phosphotyrosine B-residue_name +and O +phosphoserine B-residue_name +/ O +phospho B-residue_name +- I-residue_name +threonine I-residue_name +residues O +within O +a O +substrate O +. O + +Dysregulated O +expression O +of O +MKPs B-protein_type +has O +been O +associated O +with O +pathogenesis O +of O +various O +diseases O +, O +and O +understanding O +their O +precise O +recognition O +mechanism O +presents O +an O +important O +challenge O +and O +opportunity O +for O +drug O +development O +. O + +This O +structure B-evidence +reveals O +the O +molecular O +mechanism O +underlying O +the O +docking O +interaction O +between O +MKP7 B-protein +and O +JNK1 B-protein +. O + +In O +the O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +complex O +, O +a O +hydrophobic B-structure_element +motif I-structure_element +( O +285FNFL288 B-structure_element +) O +that O +initiates O +the O +helix B-structure_element +α5 B-structure_element +in O +the O +MKP7 B-protein +catalytic B-structure_element +domain I-structure_element +directly O +binds O +to O +the O +FXF B-site +- I-site +motif I-site +- I-site +binding I-site +site I-site +on O +JNK1 B-protein +, O +providing O +the O +structural O +insight O +into O +the O +classic O +FXF B-site +- I-site +type I-site +docking I-site +interaction I-site +. O + +Biochemical B-experimental_method +and I-experimental_method +modelling I-experimental_method +studies I-experimental_method +further O +demonstrate O +that O +the O +molecular O +interactions O +mediate O +this O +key O +element O +for O +substrate O +recognition O +are O +highly O +conserved O +among O +all O +MKP B-protein_type +- I-protein_type +family I-protein_type +members I-protein_type +. O + +Thus O +, O +our O +study O +reveals O +a O +hitherto O +unrecognized O +interaction O +mode O +for O +encoding O +complex O +target O +specificity O +among O +MAPK B-protein_type +isoforms I-protein_type +. O + +Interaction O +of O +JNK1 B-protein +with O +the O +MKP7 B-protein +catalytic B-structure_element +domain I-structure_element + +MKPs B-protein_type +represent O +a O +distinct O +subfamily O +within O +a O +larger O +group O +of O +DUSPs B-protein_type +. O + +In O +mammalian B-taxonomy_domain +cells O +, O +the O +MKP B-protein_type +subfamily I-protein_type +includes O +10 O +distinct O +catalytically B-protein_state +active I-protein_state +MKPs B-protein_type +. O + +All O +MKPs B-protein_type +contain O +a O +highly B-protein_state +conserved I-protein_state +C O +- O +terminal O +catalytic B-structure_element +domain I-structure_element +( O +CD B-structure_element +) O +and O +an O +N O +- O +terminal O +kinase B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +( O +KBD B-structure_element +). O + +On O +the O +basis O +of O +sequence O +similarity O +, O +substrate O +specificity O +and O +predominant O +subcellular O +localization O +, O +the O +MKP B-protein_type +family I-protein_type +can O +be O +further O +divided O +into O +three O +groups O +( O +Fig O +. O +1 O +). O + +Biochemical B-experimental_method +and I-experimental_method +structural I-experimental_method +studies I-experimental_method +have O +revealed O +that O +the O +KBD B-structure_element +of O +MKPs B-protein_type +is O +critical O +for O +MKP3 B-protein +docking O +to O +ERK2 B-protein +, O +and O +MKP5 B-protein +binding O +to O +p38α B-protein +, O +although O +their O +binding O +mechanisms O +are O +completely O +different O +. O + +However O +, O +it O +is O +unknown O +if O +other O +MAPKs B-protein_type +can O +interact O +with O +the O +KBD B-structure_element +of O +their O +cognate O +phosphatases B-protein_type +in O +the O +same O +manner O +as O +observed O +for O +recognition O +of O +ERK2 B-protein +and O +p38α B-protein +by O +their O +MKPs B-protein_type +, O +or O +whether O +they O +recognize O +distinct O +docking B-structure_element +motifs I-structure_element +of O +MKPs B-protein_type +. O + +MKP7 B-protein +, O +the O +biggest O +molecule O +in O +the O +MKP B-protein_type +family I-protein_type +, O +selectively O +inactivates O +JNK B-protein_type +and O +p38 B-protein_type +following O +stress O +activation O +. O + +In O +addition O +to O +the O +CD B-structure_element +and O +KBD B-structure_element +, O +MKP7 B-protein +has O +a O +long O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +that O +contains O +both O +nuclear O +localization O +and O +export O +sequences O +by O +which O +MKP7 B-protein +shuttles O +between O +the O +nucleus O +and O +the O +cytoplasm O +( O +Fig O +. O +2a O +). O + +To O +quantitatively O +assess O +the O +contribution O +of O +the O +N B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +to O +the O +MKP7 B-protein +- O +catalysed O +JNK1 B-protein +dephosphorylation B-ptm +, O +we O +first O +measured O +the O +kinetic B-evidence +parameters O +of O +the O +C O +- O +terminal O +truncation B-experimental_method +of O +MKP7 B-protein +( O +MKP7ΔC304 B-mutant +, O +residues O +5 B-residue_range +– I-residue_range +303 I-residue_range +) O +and O +MKP7 B-protein +- O +CD B-structure_element +( O +residues O +156 B-residue_range +– I-residue_range +301 I-residue_range +) O +towards O +phosphorylated B-protein_state +JNK1 B-protein +( O +pJNK1 B-protein_state +). O + +Figure O +2b O +shows O +the O +variation B-evidence +of I-evidence +initial I-evidence +rates I-evidence +of O +the O +MKP7ΔC304 B-mutant +and O +MKP7 B-protein +- O +CD B-structure_element +- O +catalysed O +reaction O +with O +the O +concentration O +of O +phospho B-protein_state +- O +JNK1 B-protein +. O +Because O +the O +concentrations O +of O +MKP7 B-protein +and O +pJNK1 B-protein_state +were O +comparable O +in O +the O +reaction O +, O +the O +assumption O +that O +the O +free O +- O +substrate O +concentration O +is O +equal O +to O +the O +total O +substrate O +concentration O +is O +not O +valid O +. O + +The O +kcat B-evidence +and O +Km B-evidence +of O +the O +MKP7 B-protein +- O +CD B-structure_element +( O +0 O +. O +028 O +s O +− O +1 O +and O +0 O +. O +26 O +μM O +) O +so O +determined O +were O +nearly O +identical O +to O +those O +of O +MKP7ΔC304 B-mutant +( O +0 O +. O +029 O +s O +− O +1 O +and O +0 O +. O +27 O +μM O +), O +indicating O +that O +the O +MKP7 B-protein +- O +KBD B-structure_element +has O +no O +effect O +on O +enzyme O +catalysis O +. O + +We O +next O +examined O +the O +interaction O +of O +JNK1 B-protein +with O +the O +CD B-structure_element +and O +KBD B-structure_element +of O +MKP7 B-protein +by O +gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +. O + +When O +3 O +molar O +equivalents O +of O +CD B-structure_element +were O +mixed O +with O +1 O +molar O +equivalent O +of O +JNK1 B-protein +, O +a O +significant O +amount O +of O +CD B-structure_element +co O +- O +migrated O +with O +JNK1 B-protein +to O +earlier O +fractions O +, O +and O +the O +excess O +amount O +of O +CD B-structure_element +was O +eluted O +from O +the O +size O +exclusion O +column O +as O +a O +monomer B-oligomeric_state +, O +indicating O +stable O +complex O +formation O +( O +Fig O +. O +2c O +). O + +In O +contrast O +, O +no O +KBD B-complex_assembly +– I-complex_assembly +JNK1 I-complex_assembly +complex O +was O +detected O +when O +3 O +molar O +equivalents O +of O +KBD B-structure_element +were O +mixed O +with O +1 O +molar O +equivalent O +of O +JNK1 B-protein +. O + +Taken O +together O +, O +our O +data O +indicate O +that O +the O +CD B-structure_element +of O +MKP7 B-protein +, O +but O +not O +the O +KBD B-structure_element +domain O +, O +is O +responsible O +for O +JNK B-protein_type +substrate O +- O +binding O +and O +enzymatic O +specificity O +. O + +Crystal B-evidence +structure I-evidence +of O +JNK1 B-protein +in B-protein_state +complex I-protein_state +with I-protein_state +the O +MKP7 B-protein +- O +CD B-structure_element + +The O +overall O +folding O +of O +MKP7 B-protein +- O +CD B-structure_element +is O +typical O +of O +DUSPs B-protein_type +, O +with O +a O +central O +twisted B-structure_element +five I-structure_element +- I-structure_element +stranded I-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +surrounded O +by O +six O +α B-structure_element +- I-structure_element +helices I-structure_element +. O + +One O +side O +of O +the O +β B-structure_element +- I-structure_element +sheet I-structure_element +is O +covered O +with O +two O +α B-structure_element +- I-structure_element +helices I-structure_element +and O +the O +other O +is O +covered O +with O +four O +α B-structure_element +- I-structure_element +helices I-structure_element +( O +Fig O +. O +3b O +). O + +The O +catalytic B-structure_element +domain I-structure_element +of O +MKP7 B-protein +interacts O +with O +JNK1 B-protein +through O +a O +contiguous O +surface O +area O +that O +is O +remote O +from O +the O +active B-site +site I-site +. O + +MKP7 B-protein +- O +CD B-structure_element +is O +positioned O +onto O +the O +JNK1 B-protein +molecule O +so O +that O +the O +active B-site +site I-site +of O +the O +phosphatase B-protein_type +faces O +towards O +the O +activation B-structure_element +segment I-structure_element +. O + +The O +most O +striking O +difference O +is O +that O +helix B-structure_element +α0 B-structure_element +and O +loop B-structure_element +α0 B-structure_element +– I-structure_element +β1 I-structure_element +of O +VHR B-protein +are O +absent O +in O +MKP7 B-protein +- O +CD B-structure_element +. O + +Another O +region O +that O +cannot O +be O +aligned O +with O +VHR B-protein +is O +found O +in O +loop B-structure_element +β3 B-structure_element +– I-structure_element +β4 I-structure_element +. O + +The O +active B-site +site I-site +of O +MKP7 B-protein +consists O +of O +the O +phosphate B-structure_element +- I-structure_element +binding I-structure_element +loop I-structure_element +( O +P B-structure_element +- I-structure_element +loop I-structure_element +, O +Cys244 B-residue_name_number +- O +Leu245 B-residue_name_number +- O +Ala246 B-residue_name_number +- O +Gly247 B-residue_name_number +- O +Ile248 B-residue_name_number +- O +Ser249 B-residue_name_number +- O +Arg250 B-residue_name_number +), O +and O +Asp213 B-residue_name_number +in O +the O +general B-structure_element +acid I-structure_element +loop I-structure_element +( O +Fig O +. O +3b O +and O +Supplementary O +Fig O +. O +1b O +). O + +The O +MKP7 B-protein +- O +CD B-structure_element +structure B-evidence +near O +the O +active B-site +site I-site +exhibits O +a O +typical O +active B-protein_state +conformation I-protein_state +as O +found O +in O +VHR B-protein +and O +other O +PTPs B-protein_type +. O + +The O +catalytic B-site +residue I-site +, O +Cys244 B-residue_name_number +, O +is O +located O +just O +after O +strand B-structure_element +β5 B-structure_element +and O +optimally O +positioned O +for O +nucleophilic O +attack O +. O + +Asp213 B-residue_name_number +in O +MKP7 B-protein +also O +adopts O +a O +position O +similar O +to O +that O +of O +Asp92 B-residue_name_number +in O +VHR B-protein +( O +Supplementary O +Fig O +. O +1c O +), O +indicating O +that O +Asp213 B-residue_name_number +is O +likely O +to O +function O +as O +the O +general O +acid O +in O +MKP7 B-protein +. O + +It O +is O +located O +3 O +. O +36 O +Å O +from O +the O +Cys244 B-residue_name_number +side O +chain O +and O +makes O +electrostatic B-bond_interaction +interactions I-bond_interaction +with O +the O +dipole O +moment O +of O +helix B-structure_element +α3 B-structure_element +and O +with O +several O +main O +- O +chain O +amide O +groups O +. O + +The O +side O +chain O +of O +strictly B-protein_state +conserved I-protein_state +Arg250 B-residue_name_number +is O +oriented O +towards O +the O +negatively O +charged O +chloride B-chemical +, O +similar O +to O +the O +canonical O +phosphate B-structure_element +- I-structure_element +coordinating I-structure_element +conformation I-structure_element +. O + +The O +difference O +in O +the O +polarity O +/ O +hydrophobicity O +of O +the O +surface O +may O +also O +point O +to O +the O +origin O +of O +the O +differences O +in O +the O +substrate O +- O +recognition O +mechanism O +for O +these O +two O +phosphatases B-protein_type +( O +Supplementary O +Fig O +. O +1e O +, O +f O +). O + +As O +a O +result O +, O +the O +buried O +solvent O +- O +accessible O +surface O +area O +is O +∼ O +1 O +, O +315 O +Å O +. O +In O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +domain I-structure_element +, O +JNK1 B-protein +has O +an O +insertion O +after O +the O +helix B-structure_element +αG B-structure_element +. O +This O +insertion O +consists O +of O +two O +helices B-structure_element +( O +α1L14 B-structure_element +and O +α2L14 B-structure_element +) O +that O +are O +common O +to O +all O +members O +of O +the O +MAPK B-protein_type +family I-protein_type +. O + +The O +interactive B-site +surface I-site +in O +JNK1 B-protein +, O +formed O +by O +the O +helices B-structure_element +αG B-structure_element +and O +α2L14 B-structure_element +, O +displays O +a O +hydrophobic B-site +region I-site +, O +centred O +at O +Trp234 B-residue_name_number +( O +Fig O +. O +3d O +). O + +The O +MKP7 B-site +- I-site +docking I-site +region I-site +includes O +two O +helices B-structure_element +, O +α4 B-structure_element +and O +α5 B-structure_element +, O +and O +the O +general B-structure_element +acid I-structure_element +loop I-structure_element +. O + +In O +addition O +, O +there O +are O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +Ser282 B-residue_name_number +and O +Asn286 B-residue_name_number +of O +MKP7 B-protein +and O +His230 B-residue_name_number +and O +Thr255 B-residue_name_number +of O +JNK1 B-protein +, O +and O +the O +main O +chain O +of O +Phe215 B-residue_name_number +in O +the O +general B-structure_element +acid I-structure_element +loop I-structure_element +of O +MKP7 B-protein +is O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +the O +side O +chain O +of O +Gln253 B-residue_name_number +in O +JNK1 B-protein +. O + +The O +second B-site +interactive I-site +area I-site +involves O +the O +α4 B-structure_element +helix I-structure_element +of O +MKP7 B-protein +and O +charged O +/ O +polar O +residues O +of O +JNK1 B-protein +( O +Fig O +. O +3e O +). O + +The O +carboxylate O +of O +Asp268 B-residue_name_number +in O +MKP7 B-protein +forms O +a O +salt B-bond_interaction +bridge I-bond_interaction +with O +side O +chain O +of O +Arg263 B-residue_name_number +in O +JNK1 B-protein +, O +and O +Lys275 B-residue_name_number +of O +MKP7 B-protein +forms O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +and O +a O +salt B-bond_interaction +bridge I-bond_interaction +with O +Thr228 B-residue_name_number +and O +Asp229 B-residue_name_number +of O +JNK1 B-protein +, O +respectively O +. O + +Mutational B-experimental_method +analysis I-experimental_method +of O +the O +JNK1 B-site +– I-site +MKP7 I-site +docking I-site +interface I-site + +To O +assess O +the O +importance O +of O +the O +aforementioned O +interactions O +, O +we O +generated O +a O +series O +of O +point B-experimental_method +mutations I-experimental_method +on O +the O +MKP7 B-protein +- O +CD B-structure_element +and O +examined O +their O +effect O +on O +the O +MKP7 B-protein +- O +catalysed O +JNK1 B-protein +dephosphorylation B-ptm +( O +Fig O +. O +4a O +). O + +When O +the O +hydrophobic O +residues O +Phe285 B-residue_name_number +and O +Phe287 B-residue_name_number +on O +the O +α5 B-structure_element +of O +MKP7 B-protein +- O +CD B-structure_element +were O +replaced B-experimental_method +by O +Asp B-residue_name +or O +Ala B-residue_name +, O +their O +phosphatase O +activities O +for O +JNK1 B-protein +dephosphorylation B-ptm +decreased O +∼ O +10 O +- O +fold O +. O + +In O +comparison O +, O +replacement B-experimental_method +of O +the O +other O +residues O +( O +Phe215 B-residue_name_number +, O +Asp268 B-residue_name_number +, O +Lys275 B-residue_name_number +, O +Ser282 B-residue_name_number +, O +Asn286 B-residue_name_number +and O +Leu292 B-residue_name_number +) O +with O +an O +Ala B-residue_name +or O +Asp B-residue_name +individually O +led O +to O +a O +modest O +decrease O +in O +catalytic O +efficiencies O +, O +suggesting O +that O +this O +position O +may O +only O +affect O +some O +selectivity O +of O +MKP B-protein_type +. O + +Mutation B-experimental_method +of O +Leu288 B-residue_name_number +markedly O +reduced O +its O +solubility O +when O +expressed O +in O +Escherichia B-species +coli I-species +, O +resulting O +in O +the O +insoluble O +aggregation O +of O +the O +mutant B-protein_state +protein O +. O + +Gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +further O +confirmed O +the O +key O +role O +of O +Phe285 B-residue_name_number +in O +the O +MKP7 B-protein +– O +JNK1 B-protein +interaction O +: O +no O +F285D B-complex_assembly +– I-complex_assembly +JNK1 I-complex_assembly +complex O +was O +detected O +when O +3 O +molar O +equivalents O +of O +MKP7 B-protein +- O +CD B-structure_element +( O +F285D B-mutant +) O +were O +mixed O +with O +1 O +molar O +equivalent O +of O +JNK1 B-protein +( O +Fig O +. O +4b O +). O + +We O +also O +generated O +a O +series O +of O +point B-experimental_method +mutations I-experimental_method +in O +the O +JNK1 B-protein +and O +assessed O +the O +effect O +on O +JNK1 B-protein +binding O +using O +the O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assay I-experimental_method +( O +Fig O +. O +4c O +). O + +Substitution B-experimental_method +at O +Asp229 B-residue_name_number +, O +Trp234 B-residue_name_number +, O +Thr255 B-residue_name_number +, O +Val256 B-residue_name_number +, O +Tyr259 B-residue_name_number +and O +Val260 B-residue_name_number +significantly O +reduced O +the O +binding B-evidence +affinity I-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +for O +JNK B-protein_type +. O + +To O +determine O +whether O +the O +deficiencies O +in O +their O +abilities O +to O +bind O +partner O +proteins O +or O +carry O +out O +catalytic O +function O +are O +owing O +to O +misfolding O +of O +the O +purified O +mutant B-protein_state +proteins O +, O +we O +also O +examined O +the O +folding O +properties O +of O +the O +JNK1 B-protein +and O +MKP7 B-protein +mutants B-protein_state +with O +circular B-experimental_method +dichroism I-experimental_method +. O + +The O +spectra B-evidence +of O +these O +mutants B-protein_state +are O +similar O +to O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +proteins O +, O +indicating O +that O +these O +mutants B-protein_state +fold O +as O +well O +as O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +proteins O +( O +Fig O +. O +4d O +, O +e O +). O + +Taken O +together O +, O +these O +results O +are O +consistent O +with O +the O +present O +crystallographic B-evidence +model I-evidence +, O +which O +reveal O +the O +hydrophobic B-bond_interaction +contacts I-bond_interaction +between O +the O +MKP7 B-protein +catalytic B-structure_element +domain I-structure_element +and O +JNK1 B-protein +have O +a O +predominant O +role O +in O +the O +enzyme O +– O +substrate O +interaction O +, O +and O +hydrophobic O +residue O +Phe285 B-residue_name_number +in O +the O +MKP7 B-protein +- O +CD B-structure_element +is O +a O +key O +residue O +for O +its O +high O +- O +affinity O +binding O +to O +JNK1 B-protein +. O + +It O +has O +previously O +been O +reported O +that O +several O +cytosolic O +and O +inducible O +nuclear O +MKPs B-protein_type +undergo O +catalytic O +activation O +upon O +interaction O +with O +the O +MAPK B-protein_type +substrates O +. O + +This O +allosteric O +activation O +of O +MKP3 B-protein +has O +been O +well O +- O +documented O +in O +vitro O +using O +pNPP B-chemical +, O +a O +small O +- O +molecule O +phosphotyrosine B-residue_name +analogue O +of O +its O +normal O +substrate O +. O + +We O +then O +assayed O +pNPPase B-protein_type +activities O +of O +MKP7ΔC304 B-mutant +and O +MKP7 B-protein +- O +CD B-structure_element +in O +the O +presence B-protein_state +of I-protein_state +JNK1 B-protein +. O + +The O +small O +pNPP B-chemical +molecule O +binds O +directly O +at O +the O +enzyme O +active B-site +site I-site +and O +can O +be O +used O +to O +probe O +the O +reaction O +mechanism O +of O +protein B-protein_type +phosphatases I-protein_type +. O + +We O +therefore O +examined O +the O +effects O +of O +the O +MKP7 B-protein +- O +CD B-structure_element +mutants B-protein_state +on O +their O +pNPPase B-protein_type +activities O +. O + +In O +the O +JNK1 B-complex_assembly +/ I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +complex O +structure B-evidence +, O +Phe287 B-residue_name_number +of O +MKP7 B-protein +does O +not O +make O +contacts O +with O +JNK1 B-protein +substrate O +. O + +It O +penetrates O +into O +a O +pocket B-site +formed O +by O +residues O +from O +the O +P B-structure_element +- I-structure_element +loop I-structure_element +and O +general B-structure_element +acid I-structure_element +loop I-structure_element +and O +forms O +hydrophobic B-bond_interaction +contacts I-bond_interaction +with O +the O +aliphatic O +portions O +of O +side O +chains O +of O +Arg250 B-residue_name_number +, O +Glu217 B-residue_name_number +and O +Ile219 B-residue_name_number +, O +suggesting O +that O +Phe287 B-residue_name_number +in O +MKP7 B-protein +would O +play O +a O +similar O +role O +to O +that O +of O +its O +structural O +counterpart O +in O +the O +PTPs B-protein_type +( O +Gln266 B-residue_name_number +in O +PTP1B B-protein +) O +and O +VHR B-protein +( O +Phe166 B-residue_name_number +in O +VHR B-protein +) O +in O +the O +precise O +alignment O +of O +active B-site +- I-site +site I-site +residues I-site +in O +MKP7 B-protein +with O +respect O +to O +the O +substrate O +for O +efficient O +catalysis O +( O +Supplementary O +Fig O +. O +2c O +). O + +Kinase B-protein +- I-protein +associated I-protein +phosphatase I-protein +( O +KAP B-protein +), O +a O +member O +of O +the O +DUSP B-protein_type +family I-protein_type +, O +plays O +a O +crucial O +role O +in O +cell O +cycle O +regulation O +by O +dephosphorylating O +the O +pThr160 B-ptm +residue O +of O +CDK2 B-protein +( O +cyclin B-protein +- I-protein +dependent I-protein +kinase I-protein +2 I-protein +). O + +The O +crystal B-evidence +structure I-evidence +of O +the O +CDK2 B-complex_assembly +/ I-complex_assembly +KAP I-complex_assembly +complex O +has O +been O +determined O +at O +3 O +. O +0 O +Å O +( O +Fig O +. O +5a O +). O + +The O +interface B-site +between O +these O +two O +proteins O +consists O +of O +three O +discontinuous O +contact O +regions O +. O + +Structural B-experimental_method +analysis I-experimental_method +and O +sequence B-experimental_method +alignment I-experimental_method +reveal O +that O +one O +of O +the O +few O +differences O +between O +MKP7 B-protein +- O +CD B-structure_element +and O +KAP B-protein +in O +the O +substrate B-site +- I-site +binding I-site +region I-site +is O +the O +presence O +of O +the O +motif O +FNFL B-structure_element +in O +MKP7 B-protein +- O +CD B-structure_element +, O +which O +corresponds O +to O +IKQY B-structure_element +in O +KAP B-protein +( O +Fig O +. O +5c O +). O + +The O +substitution B-experimental_method +of O +the O +two O +hydrophobic O +residues O +with O +charged O +/ O +polar O +residues O +( O +F285I B-mutant +/ O +N286K B-mutant +) O +seriously O +disrupts O +the O +hydrophobic B-bond_interaction +interaction I-bond_interaction +required O +for O +MKP7 B-protein +binding O +on O +JNK1 B-protein +( O +Fig O +. O +4a O +). O + +These O +data O +indicated O +that O +a O +unique O +hydrophobic B-site +pocket I-site +formed O +between O +the O +MAPK B-structure_element +insert I-structure_element +and O +αG B-structure_element +helix I-structure_element +plays O +a O +major O +role O +in O +the O +substrate O +recognition O +by O +MKPs B-protein_type +. O + +F B-site +- I-site +site I-site +interaction O +is O +crucial O +for O +JNK1 B-protein +inactivation O +in O +vivo O + +To O +assess O +the O +effects O +of O +MKP7 B-protein +and O +its O +mutants B-protein_state +on O +the O +activation O +of O +endogenous O +JNK B-protein_type +in O +vivo O +, O +HEK293T O +cells O +were O +transfected O +with O +blank O +vector O +or O +with O +HA B-protein_state +- I-protein_state +tagged I-protein_state +constructs O +for O +full B-protein_state +- I-protein_state +length I-protein_state +MKP7 B-protein +, O +MKP7ΔC304 B-mutant +and O +MKP7 B-protein +- O +CD B-structure_element +or O +MKP7 B-protein +mutants B-protein_state +, O +and O +stimulated O +with O +ultraviolet O +or O +etoposide B-chemical +treatment O +. O + +As O +shown O +in O +Fig O +. O +6a O +– O +c O +, O +immunobloting B-experimental_method +showed O +similar O +expression O +levels O +for O +the O +different O +MKP7 B-protein +constructs O +in O +all O +the O +cells O +. O + +Overexpressed B-experimental_method +full B-protein_state +- I-protein_state +length I-protein_state +MKP7 B-protein +, O +MKP7ΔC304 B-mutant +and O +MKP7 B-protein +- O +CD B-structure_element +significantly O +reduced O +the O +endogenous O +level O +of O +phosphorylated B-protein_state +JNK B-protein_type +compared O +with O +vector O +- O +transfected O +cells O +. O + +In O +agreement O +with O +the O +in B-experimental_method +vitro I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +results O +, O +the O +mutants B-protein_state +D229A B-mutant +, O +W234D B-mutant +and O +Y259D B-mutant +were O +not O +co O +- O +precipitated O +with O +MKP7 B-protein +, O +and O +the O +I231D B-mutant +mutant B-protein_state +had O +only O +little O +effect O +on O +the O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +interaction O +( O +Fig O +. O +6d O +and O +Supplementary O +Fig O +. O +3a O +). O + +Activation O +of O +the O +JNK B-protein_type +signalling O +pathway O +is O +frequently O +associated O +with O +apoptotic O +cell O +death O +, O +and O +inhibition O +of O +JNK B-protein_type +can O +prevent O +apoptotic O +death O +of O +multiple O +cells O +. O + +To O +examine O +whether O +the O +inhibition O +of O +JNK B-protein_type +activity O +by O +MKP7 B-protein +would O +provide O +protections O +against O +the O +apoptosis O +, O +we O +analysed O +the O +rate O +of O +apoptosis O +in O +ultraviolet O +- O +irradiated O +cells O +transfected O +with O +MKP7 B-protein +( O +wild B-protein_state +type I-protein_state +or O +mutants B-protein_state +) O +by O +flow B-experimental_method +cytometry I-experimental_method +. O + +The O +results O +showed O +similar O +apoptotic O +rates O +between O +cells O +transfected O +with O +blank O +vector O +or O +with O +MKP7 B-protein +( O +wild B-protein_state +type I-protein_state +or O +mutants B-protein_state +) O +under O +unstimulated O +conditions O +( O +Supplementary O +Fig O +. O +3b O +), O +while O +ultraviolet O +- O +irradiation O +significantly O +increased O +apoptotic O +rate O +in O +cells O +transfected O +with O +blank O +vector O +( O +Fig O +. O +6e O +). O + +Moreover O +, O +treatment O +of O +cells O +expressing O +MKP7 B-protein +- O +KBD B-structure_element +mutants B-protein_state +( O +R56A B-mutant +/ O +R57A B-mutant +and O +V63A B-mutant +/ O +I65A B-mutant +) O +decreased O +the O +apoptosis O +rates O +to O +a O +similar O +extent O +as O +MKP7 B-protein +wild B-protein_state +type I-protein_state +did O +. O + +Taken O +together O +, O +our O +results O +suggested O +that O +FXF B-structure_element +- I-structure_element +motif I-structure_element +- O +mediated O +, O +rather O +than O +KBD B-structure_element +- O +mediated O +, O +interaction O +is O +essential O +for O +MKP7 B-protein +to O +block O +ultraviolet O +- O +induced O +apoptosis O +. O + +A O +similar O +docking O +mechanism O +for O +JNK1 B-protein +recognition O +by O +MKP5 B-protein + +MKP5 B-protein +belongs O +to O +the O +same O +subfamily O +as O +MKP7 B-protein +. O + +The O +KBD B-structure_element +of O +MKP5 B-protein +interacts O +with O +the O +D B-site +- I-site +site I-site +of O +p38α B-protein +to O +mediate O +the O +enzyme O +– O +substrate O +interaction O +. O + +In O +contrast O +to O +p38α B-protein +substrate O +, O +deletion B-experimental_method +of I-experimental_method +the O +MKP5 B-protein +- O +KBD B-structure_element +had O +little O +effects O +on O +the O +kinetic O +parameters O +for O +the O +JNK1 B-protein +dephosphorylation O +, O +indicating O +that O +the O +KBD B-structure_element +of O +MKP5 B-protein +is O +not O +required O +for O +the O +JNK1 B-protein +dephosphorylation O +( O +Fig O +. O +7b O +). O + +The O +substrate B-evidence +specificity I-evidence +constant I-evidence +kcat B-evidence +/ I-evidence +Km I-evidence +value O +for O +MKP5 B-protein +- O +CD B-structure_element +was O +calculated O +as O +1 O +. O +0 O +× O +105 O +M O +− O +1 O +s O +− O +1 O +, O +which O +is O +very O +close O +to O +that O +of O +MKP7 B-protein +- O +CD B-structure_element +( O +1 O +. O +07 O +× O +105 O +M O +− O +1 O +s O +− O +1 O +). O + +The O +crystal B-evidence +structure I-evidence +of O +human B-species +MKP5 B-protein +- O +CD B-structure_element +has O +been O +determined O +. O + +Given O +the O +distinct O +interaction O +mode O +revealed O +by O +the O +crystal B-evidence +structure I-evidence +of O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +, O +one O +obvious O +question O +is O +whether O +this O +is O +a O +general O +mechanism O +used O +by O +all O +members O +of O +the O +JNK B-protein_type +- I-protein_type +specific I-protein_type +MKPs I-protein_type +. O + +To O +address O +this O +issue O +, O +we O +first O +examined O +the O +docking O +ability O +of O +JNK1 B-protein +to O +the O +KBD B-structure_element +and O +CD B-structure_element +of O +MKP5 B-protein +using O +gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +and O +pull B-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +. O + +It O +can O +be O +seen O +from O +gel B-experimental_method +filtration I-experimental_method +experiments I-experimental_method +that O +JNK1 B-protein +can O +forms O +a O +stable B-protein_state +heterodimer B-oligomeric_state +with O +MKP5 B-protein +- O +CD B-structure_element +in O +solution O +, O +but O +no O +detectable O +interaction O +was O +found O +with O +the O +KBD B-structure_element +domain O +( O +Fig O +. O +7d O +). O + +The O +catalytic B-structure_element +domain I-structure_element +of O +MKP5 B-protein +, O +but O +not O +its O +KBD B-structure_element +, O +was O +able O +to O +pull O +- O +down O +a O +detectable O +amount O +of O +JNK1 B-protein +( O +Fig O +. O +7e O +), O +implicating O +a O +different O +substrate O +- O +recognition O +mechanisms O +for O +p38 B-protein_type +and O +JNK B-protein_type +MAPKs B-protein_type +. O + +To O +further O +test O +our O +hypothesis O +, O +we O +generated O +forms O +of O +MKP5 B-protein +- O +CD B-structure_element +bearing O +mutations B-experimental_method +corresponding O +to O +the O +changes O +we O +made O +on O +MKP7 B-protein +- O +CD B-structure_element +on O +the O +basis O +of O +sequence B-experimental_method +and I-experimental_method +structural I-experimental_method +alignment I-experimental_method +and O +examined O +their O +effects O +on O +the O +phosphatase B-protein_type +activity O +. O + +In O +addition O +, O +there O +were O +no O +significant O +differences O +in O +the O +CD B-evidence +spectra I-evidence +between O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +proteins O +, O +indicating O +that O +the O +overall O +structures B-evidence +of O +these O +mutants B-protein_state +did O +not O +change O +significantly O +from O +that O +of O +wild B-protein_state +- I-protein_state +type I-protein_state +MKP5 B-protein +protein O +( O +Supplementary O +Fig O +. O +4a O +). O + +In O +particular O +, O +Leu449 B-residue_name_number +of O +MKP5 B-protein +, O +which O +is O +equivalent O +to O +the O +key O +residue O +Phe285 B-residue_name_number +of O +MKP7 B-protein +, O +buried O +deeply O +within O +the O +hydrophobic B-site +pocket I-site +of O +JNK1 B-protein +in O +the O +same O +way O +as O +Phe285 B-residue_name_number +in O +the O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +complex O +( O +Supplementary O +Fig O +. O +4d O +). O + +Despite O +the O +strong O +similarities O +between O +JNK1 B-protein +– O +MKP5 B-protein +- O +CD B-structure_element +and O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +, O +however O +, O +there O +are O +differences O +. O + +Asp268 B-residue_name_number +of O +MKP7 B-protein +- O +CD B-structure_element +forms O +salt B-bond_interaction +bridge I-bond_interaction +with O +JNK1 B-protein +Arg263 B-residue_name_number +, O +whereas O +the O +corresponding O +residue O +Thr432 B-residue_name_number +in O +MKP5 B-protein +- O +CD B-structure_element +may O +not O +interact O +with O +JNK1 B-protein +. O + +In O +addition O +, O +the O +key O +interacting O +residues O +of O +MKP7 B-protein +- O +CD B-structure_element +, O +Phe215 B-residue_name_number +, O +Leu267 B-residue_name_number +and O +Leu288 B-residue_name_number +, O +are O +replaced O +by O +less O +hydrophobic O +residues O +, O +Asn379 B-residue_name_number +, O +Met431 B-residue_name_number +and O +Met452 B-residue_name_number +in O +MKP5 B-protein +- O +CD B-structure_element +( O +Fig O +. O +5c O +), O +respectively O +, O +which O +may O +result O +in O +weaker O +hydrophobic B-bond_interaction +interactions I-bond_interaction +between O +MKP5 B-protein +- O +CD B-structure_element +and O +JNK1 B-protein +. O + +This O +is O +consistent O +with O +the O +experimental O +observation O +showing O +that O +JNK1 B-protein +binds O +to O +MKP7 B-protein +- O +CD B-structure_element +much O +more O +tightly O +than O +MKP5 B-protein +- O +CD B-structure_element +( O +Km O +value O +of O +MKP5 B-protein +- O +CD B-structure_element +for O +pJNK1 B-protein_state +substrate O +is O +∼ O +20 O +- O +fold O +higher O +than O +that O +of O +MKP7 B-protein +- O +CD B-structure_element +). O + +The O +MAPKs B-protein_type +p38 B-protein_type +, O +ERK B-protein_type +and O +JNK B-protein_type +, O +are O +central O +to O +evolutionarily O +conserved O +signalling O +pathways O +that O +are O +present O +in O +all O +eukaryotic B-taxonomy_domain +cells O +. O + +Each O +MAPK B-protein_type +cascade O +is O +activated O +in O +response O +to O +a O +diverse O +array O +of O +extracellular O +signals O +and O +culminates O +in O +the O +dual B-ptm +- I-ptm +phosphorylation I-ptm +of O +a O +threonine B-residue_name +and O +a O +tyrosine B-residue_name +residue O +in O +the O +MAPK B-structure_element +- I-structure_element +activation I-structure_element +loop I-structure_element +. O + +The O +propagation O +of O +MAPK B-protein_type +signals O +is O +attenuated O +through O +the O +actions O +of O +the O +MKPs B-protein_type +. O + +Most O +studies O +have O +focused O +on O +the O +dephosphorylation O +of O +MAPKs B-protein_type +by O +phosphatases B-protein_type +containing O +the O +‘ O +kinase B-structure_element +- I-structure_element +interaction I-structure_element +motif I-structure_element +' O +( O +D B-structure_element +- I-structure_element +motif I-structure_element +), O +including O +a O +group O +of O +DUSPs B-protein_type +( O +MKPs B-protein_type +) O +and O +a O +distinct O +subfamily O +of O +tyrosine B-protein_type +phosphatases I-protein_type +( O +HePTP B-protein +, O +STEP B-protein +and O +PTP B-protein +- I-protein +SL I-protein +). O + +Crystal B-evidence +structures I-evidence +of O +ERK2 B-protein +bound B-protein_state +with I-protein_state +the O +D B-structure_element +- I-structure_element +motif I-structure_element +sequences O +derived O +from O +MKP3 B-protein +and O +HePTP B-protein +have O +been O +reported O +. O + +The O +particular O +amino O +acids O +and O +their O +spacing O +within O +D B-structure_element +- I-structure_element +motif I-structure_element +sequences O +and O +amino O +acid O +composition O +of O +the O +docking B-site +sites I-site +on O +MAPKs B-protein_type +appear O +to O +determine O +the O +specificity O +of O +D B-structure_element +- I-structure_element +motifs I-structure_element +for O +individual O +MAPKs B-protein_type +. O + +Recently O +, O +the O +crystal B-evidence +structure I-evidence +of O +a O +complex O +between O +the O +KBD B-structure_element +of O +MKP5 B-protein +and O +p38α B-protein +has O +been O +obtained O +. O + +This O +complex O +has O +revealed O +a O +distinct O +interaction O +mode O +for O +MKP5 B-protein +. O + +The O +KBD B-structure_element +of O +MKP5 B-protein +binds O +to O +p38α B-protein +in O +the O +opposite O +polypeptide O +direction O +compared O +with O +how O +the O +D B-structure_element +- I-structure_element +motif I-structure_element +of O +MKP3 B-protein +binds O +to O +ERK2 B-protein +. O + +Further O +structural B-experimental_method +and I-experimental_method +biochemical I-experimental_method +studies I-experimental_method +indicate O +that O +KBD B-structure_element +of O +MKP7 B-protein +may O +interact O +with O +p38α B-protein +in O +a O +similar O +manner O +to O +that O +of O +MKP5 B-protein +. O + +In O +contrast O +to O +MKP5 B-protein +, O +removal B-experimental_method +of I-experimental_method +the O +KBD B-structure_element +domain O +from O +MKP7 B-protein +does O +not O +drastically O +affect O +enzyme O +catalysis O +, O +and O +the O +kinetic O +parameters O +of O +MKP7 B-protein +- O +CD B-structure_element +for O +p38α B-protein +substrate O +are O +very O +similar O +to O +those O +for O +JNK1 B-protein +substrate O +. O + +Taken O +together O +, O +these O +results O +suggest O +that O +MKP7 B-protein +utilizes O +a O +bipartite O +recognition O +mechanism O +to O +achieve O +the O +efficiency O +and O +fidelity O +of O +p38α B-protein +signalling O +. O + +This O +hydrophobic B-site +site I-site +was O +first O +identified O +by O +changes B-evidence +in I-evidence +deuterium I-evidence +exchange I-evidence +profiles I-evidence +, O +and O +is O +near O +the O +MAPK B-structure_element +insertion I-structure_element +and O +helix B-structure_element +αG B-structure_element +. O +Interestingly O +, O +many O +of O +the O +equivalent O +residues O +in O +JNK1 B-protein +, O +important O +for O +MKP7 B-protein +- O +CD B-structure_element +recognition O +, O +are O +also O +used O +for O +substrate O +binding O +by O +ERK2 B-protein +( O +ref O +.), O +indicating O +that O +this O +site O +is O +overlapped O +with O +the O +DEF B-site +- I-site +site I-site +previously O +identified O +in O +ERK2 B-protein +( O +Fig O +. O +5d O +). O + +MKP3 B-protein +is O +highly O +specific O +in O +dephosphorylating O +and O +inactivating O +ERK2 B-protein +, O +and O +the O +phosphatase O +activity O +of O +the O +MKP3 B-protein +- O +catalysed O +pNPP B-chemical +reaction O +can O +be O +markedly O +increased O +in O +the O +presence B-protein_state +of I-protein_state +ERK2 B-protein +( O +refs O +). O + +Therefore O +, O +it O +is O +tempting O +to O +speculate O +that O +the O +catalytic B-structure_element +domain I-structure_element +of O +MKP3 B-protein +may O +bind O +to O +ERK2 B-protein +in O +a O +manner O +analogous O +to O +the O +way O +by O +which O +MKP7 B-protein +- O +CD B-structure_element +binds O +to O +JNK1 B-protein +. O + +A O +comprehensive O +examination O +of O +the O +molecular O +basis O +of O +the O +specific O +ERK2 B-protein +recognition O +by O +MKP3 B-protein +is O +underway O +. O + +The O +ongoing O +work O +demonstrates O +that O +although O +the O +overall O +interaction O +modes O +are O +similar O +between O +the O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +and O +ERK2 B-complex_assembly +– I-complex_assembly +MKP3 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +complexes O +, O +the O +ERK2 B-complex_assembly +– I-complex_assembly +MKP3 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +interaction O +is O +less O +extensive O +and O +helix B-structure_element +α4 B-structure_element +from O +MKP3 B-protein +- O +CD B-structure_element +does O +not O +interact O +directly O +with O +ERK2 B-protein +. O + +The O +FXF B-structure_element +- I-structure_element +motif I-structure_element +- O +mediated O +interaction O +is O +critical O +for O +both O +pERK2 B-protein_state +inactivation O +and O +ERK2 B-protein +- O +induced O +MKP3 B-protein +activation O +( O +manuscript O +in O +preparation O +). O + +This O +structure B-evidence +reveals O +an O +FXF B-site +- I-site +docking I-site +interaction I-site +mode I-site +between O +MAPK B-protein_type +and O +MKP B-protein_type +. O + +Results O +from O +biochemical B-experimental_method +characterization I-experimental_method +of O +the O +Phe285 B-residue_name_number +and O +Phe287 B-residue_name_number +MKP7 B-protein +mutants B-protein_state +combined O +with O +structural B-evidence +information I-evidence +support O +the O +conclusion O +that O +the O +two O +Phe B-residue_name +residues O +serve O +different O +roles O +in O +the O +catalytic O +reaction O +. O + +Phe285 B-residue_name_number +is O +essential O +for O +JNK1 B-protein +substrate O +binding O +, O +whereas O +Phe287 B-residue_name_number +plays O +a O +role O +for O +the O +precise O +alignment O +of O +active B-site +- I-site +site I-site +residues I-site +, O +which O +are O +important O +for O +transition O +- O +state O +stabilization O +. O + +This O +newly O +identified O +FXF B-structure_element +- I-structure_element +type I-structure_element +motif I-structure_element +is O +present O +in O +all O +MKPs B-protein_type +, O +except O +that O +the O +residue O +at O +the O +first O +position O +in O +MKP5 B-protein +is O +an O +equivalent O +hydrophobic O +leucine B-residue_name +residue O +( O +see O +also O +Fig O +. O +7f O +, O +g O +), O +suggesting O +that O +these O +two O +Phe B-residue_name +residues O +would O +play O +a O +similar O +role O +in O +facilitating O +substrate O +recognition O +and O +catalysis O +, O +respectively O +. O + +An O +important O +feature O +of O +MKP B-protein_type +– O +JNK1 B-protein +interactions O +is O +that O +MKP7 B-protein +or O +MKP5 B-protein +only O +interact O +with O +the O +F B-site +- I-site +site I-site +of O +JNK1 B-protein +. O + +One O +possible O +explanation O +is O +that O +JNK1 B-protein +needs O +to O +use O +the O +D B-site +- I-site +site I-site +to O +interact O +with O +JIP B-protein +- I-protein +1 I-protein +, O +a O +scaffold O +protein O +for O +JNK B-protein_type +signalling O +. O + +The O +N O +- O +terminal O +JNK B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +of O +JIP B-protein +- I-protein +1 I-protein +interacts O +with O +the O +D B-site +- I-site +site I-site +on O +JNK B-protein_type +and O +this O +interaction O +is O +required O +for O +JIP B-protein +- I-protein +1 I-protein +- O +mediated O +enhancement O +of O +JNK B-protein_type +activation O +. O + +Domain O +structures B-evidence +of O +ten O +human B-species +MKPs B-protein_type +and O +the O +atypical O +VHR B-protein +. O + +On O +the O +basis O +of O +sequence O +similarity O +, O +protein O +structure B-evidence +, O +substrate O +specificity O +and O +subcellular O +localization O +, O +the O +ten O +members O +of O +MKP B-protein_type +family I-protein_type +can O +be O +divided O +into O +three O +groups O +. O + +The O +first O +subfamily O +comprises O +MKP1 B-protein +, O +MKP2 B-protein +, O +PAC1 B-protein +and O +hVH3 B-protein +, O +which O +are O +inducible B-protein_state +nuclear B-protein_type +phosphatases I-protein_type +and O +can O +dephosphorylate O +ERK B-protein_type +( O +and O +JNK B-protein_type +, O +p38 B-protein_type +) O +MAPKs B-protein_type +. O + +The O +second O +subfamily O +contains O +MKP3 B-protein +, O +MKP4 B-protein +and O +MKPX B-protein +, O +which O +are O +cytoplasmic O +ERK B-protein_type +- I-protein_type +specific I-protein_type +MKPs I-protein_type +. O + +The O +third O +subfamily O +comprises O +MKP5 B-protein +, O +MKP7 B-protein +and O +hVH5 B-protein +, O +which O +were O +located O +in O +both O +nucleus O +and O +cytoplasm O +, O +and O +selectively O +inactivate O +JNK B-protein_type +and O +p38 B-protein_type +. O + +All O +MKPs B-protein_type +contain O +both O +the O +CD B-structure_element +and O +KBD B-structure_element +domains O +, O +whereas O +VHR B-protein +, O +an O +atypical O +MKP B-protein_type +, O +only O +contains O +a O +highly B-protein_state +conserved I-protein_state +catalytic B-structure_element +domain I-structure_element +. O + +In O +addition O +to O +the O +CD B-structure_element +and O +KBD B-structure_element +, O +MKP7 B-protein +contains O +a O +unique O +long O +C B-structure_element +- I-structure_element +terminal I-structure_element +region I-structure_element +that O +contains O +NES B-structure_element +, O +NLS B-structure_element +and O +PEST B-structure_element +motifs I-structure_element +, O +which O +has O +no O +effect O +on O +the O +binding O +ability O +and O +phosphatase O +activity O +of O +MKP7 B-protein +toward O +MAPKs B-protein_type +. O + +NES B-structure_element +, O +nuclear B-structure_element +export I-structure_element +signal I-structure_element +; O +NLS B-structure_element +, O +nuclear B-structure_element +localization I-structure_element +signal I-structure_element +; O +PEST B-structure_element +, O +C B-structure_element +- I-structure_element +terminal I-structure_element +sequence I-structure_element +rich I-structure_element +in O +prolines B-residue_name +, O +glutamates B-residue_name +, O +serines B-residue_name +and O +threonines B-residue_name +. O + +MKP7 B-protein +- O +CD B-structure_element +is O +crucial O +for O +JNK1 B-protein +binding O +and O +enzyme O +catalysis O +. O + +( O +a O +) O +Domain O +organization O +of O +human B-species +MKP7 B-protein +and O +JNK1 B-protein +. O + +The O +KBD B-structure_element +and O +CD B-structure_element +of O +MKP7 B-protein +are O +shown O +in O +green O +and O +blue O +, O +and O +the O +N B-structure_element +- I-structure_element +lobe I-structure_element +and O +C B-structure_element +- I-structure_element +lobe I-structure_element +of O +JNK1 B-protein +are O +coloured O +in O +lemon O +and O +yellow O +, O +respectively O +. O + +The O +error O +bars O +represent O +s O +. O +e O +. O +m O +. O +( O +c O +) O +Gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +for O +interaction O +of O +JNK1 B-protein +with O +MKP7 B-protein +- O +CD B-structure_element +and O +MKP7 B-protein +- O +KBD B-structure_element +. O + +The O +top O +panel O +shows O +the O +relative O +affinities B-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +and O +MKP7 B-protein +- O +KBD B-structure_element +to O +JNK1 B-protein +, O +with O +the O +affinity B-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +defined O +as O +100 O +%; O +the O +middle O +panel O +is O +the O +electrophoretic O +pattern O +of O +MKP7 B-protein +and O +JNK1 B-protein +after O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +. O + +The O +protein O +amounts O +of O +MKP7 B-protein +used O +are O +shown O +at O +the O +bottom O +. O + +The O +JNK1 B-protein +is O +shown O +in O +surface O +representation O +coloured O +according O +to O +electrostatic O +potential O +( O +positive O +, O +blue O +; O +negative O +, O +red O +). O + +( O +e O +) O +Interaction B-site +networks I-site +mainly O +involving O +helices B-structure_element +α4 B-structure_element +and O +α5 B-structure_element +from O +MKP7 B-protein +- O +CD B-structure_element +, O +and O +αG B-structure_element +and O +α2L14 B-structure_element +of O +JNK1 B-protein +. O + +Blue O +dashed O +lines O +represent O +polar B-bond_interaction +interactions I-bond_interaction +. O + +( O +f O +) O +The O +2Fo B-evidence +− I-evidence +Fc I-evidence +omit I-evidence +map I-evidence +( O +contoured O +at O +1 O +. O +5σ O +) O +clearly O +shows O +electron B-evidence +density I-evidence +for O +the O +285FNFL288 B-structure_element +segment I-structure_element +of O +MKP7 B-protein +- O +CD B-structure_element +. O + +Mutational B-experimental_method +analysis I-experimental_method +on O +interactions O +between O +MKP7 B-protein +- O +CD B-structure_element +and O +JNK1 B-protein +. O + +Residues O +involved O +in O +hydrophobic B-bond_interaction +and I-bond_interaction +hydrophilic I-bond_interaction +contacts I-bond_interaction +are O +coloured O +in O +red O +and O +blue O +, O +respectively O +. O +( O +b O +) O +Gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +for O +interaction O +of O +JNK1 B-protein +with O +MKP7 B-protein +- O +CD B-structure_element +mutant B-protein_state +F285D B-mutant +. O + +( O +c O +) O +Pull B-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +of O +MKP7 B-protein +- O +CD B-structure_element +by O +GST B-protein_state +- I-protein_state +tagged I-protein_state +JNK1 B-protein +mutants B-protein_state +. O + +The O +top O +panel O +shows O +the O +relative O +affinities B-evidence +of O +MKP7 B-protein +- O +CD B-structure_element +to O +JNK1 B-protein +mutants B-protein_state +, O +with O +the O +affinity B-evidence +of O +wild B-protein_state +- I-protein_state +type I-protein_state +JNK1 B-protein +defined O +as O +100 O +%, O +the O +middle O +panel O +is O +the O +electrophoretic O +pattern O +of O +MKP7 B-protein +- O +CD B-structure_element +and O +JNK1 B-protein +mutants B-protein_state +after O +GST B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +. O + +The O +protein O +amounts O +of O +MKP7 B-protein +- O +CD B-structure_element +used O +are O +shown O +at O +the O +bottom O +. O +( O +d O +) O +Circular B-experimental_method +dichroism I-experimental_method +spectra B-evidence +for O +MKP7 B-protein +- O +CD B-structure_element +wild B-protein_state +type I-protein_state +and O +mutants B-protein_state +. O + +Measurements O +were O +averaged O +for O +three O +scans O +. O +( O +e O +) O +Circular B-experimental_method +dichroism I-experimental_method +spectra B-evidence +for O +JNK1 B-protein +wild B-protein_state +type I-protein_state +and O +mutants B-protein_state +. O + +Comparison O +of O +CDK2 B-complex_assembly +- I-complex_assembly +KAP I-complex_assembly +and O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +. O + +( O +a O +) O +Superposition B-experimental_method +of O +the O +complex O +structures B-evidence +of O +CDK2 B-complex_assembly +- I-complex_assembly +KAP I-complex_assembly +( O +PDB O +1FQ1 O +) O +and O +JNK1 B-complex_assembly +– I-complex_assembly +MKP7 I-complex_assembly +- I-complex_assembly +CD I-complex_assembly +. O + +The O +N B-structure_element +- I-structure_element +lobe I-structure_element +and O +C B-structure_element +- I-structure_element +lobe I-structure_element +of O +CDK2 B-protein +are O +coloured O +in O +grey O +and O +pink O +, O +respectively O +, O +and O +KAP B-protein +is O +coloured O +in O +green O +. O + +The O +interactions O +between O +these O +two O +proteins O +consist O +of O +three O +discontinuous O +contact B-site +regions I-site +, O +centred O +at O +the O +multiple O +hydrogen B-bond_interaction +bonds I-bond_interaction +between O +the O +pThr160 B-ptm +of O +CDK2 B-protein +and O +the O +active B-site +site I-site +of O +KAP B-protein +( O +region B-structure_element +I I-structure_element +). O + +Interestingly O +, O +the O +recognition O +of O +CDK2 B-protein +by O +KAP B-protein +is O +augmented O +by O +a O +similar O +interface B-site +as O +that O +observed O +in O +the O +complex O +of O +JNK1 B-protein +and O +MKP7 B-protein +- O +CD B-structure_element +( O +region B-structure_element +II I-structure_element +). O + +( O +b O +) O +Interactions O +networks O +at O +the O +auxiliary B-structure_element +region I-structure_element +II I-structure_element +mainly O +involving O +helix B-structure_element +α7 B-structure_element +from O +KAP B-protein +and O +the O +αG B-structure_element +helix I-structure_element +and O +following O +L14 B-structure_element +loop I-structure_element +of O +CDK2 B-protein +. O + +The O +CDK2 B-protein +is O +shown O +in O +surface O +representation O +coloured O +according O +to O +the O +electrostatic O +potential O +( O +positive O +, O +blue O +; O +negative O +, O +red O +). O + +Residues O +of O +KAP B-protein +and O +CDK2 B-protein +are O +highlighted O +as O +green O +and O +red O +sticks O +, O +respectively O +. O + +Residues O +of O +MKP7 B-protein +- O +CD B-structure_element +involved O +in O +JNK1 B-protein +recognition O +are O +indicated O +by O +cyan O +asterisks O +, O +and O +the O +conserved B-protein_state +FXF B-structure_element +- I-structure_element +motif I-structure_element +is O +highlighted O +in O +cyan O +. O + +The O +secondary O +structure O +assignments O +of O +MKP7 B-protein +- O +CD B-structure_element +and O +KAP B-protein +are O +shown O +above O +and O +below O +each O +sequence O +. O + +Residues O +of O +JNK1 B-protein +involved O +in O +recognition O +of O +MKP7 B-protein +are O +indicated O +by O +orange O +asterisks O +, O +and O +those O +forming O +the O +F B-site +- I-site +site I-site +are O +highlighted O +in O +yellow O +. O + +( O +a O +– O +c O +) O +FXF B-structure_element +- I-structure_element +motif I-structure_element +is O +essential O +for O +the O +dephosphorylation O +of O +JNK B-protein_type +by O +MKP7 B-protein +. O + +Shown O +is O +a O +typical O +immunoblot O +for O +phosphorylated B-protein_state +JNK B-protein_type +from O +three O +independent O +experiments O +. O + +( O +d O +) O +F B-site +- I-site +site I-site +is O +required O +for O +JNK1 B-protein +to O +interact O +with O +MKP7 B-protein +. O + +Whole O +- O +cell O +extracts O +were O +then O +immunoprecipitated B-experimental_method +with O +antibody O +against O +Myc O +for O +MKP7 B-protein +; O +immunobloting O +was O +carried O +out O +with O +antibodies O +indicated O +. O + +IP B-experimental_method +, O +immunoprecipitation B-experimental_method +; O +TCL O +, O +total O +cell O +lysate O +. O + +( O +e O +) O +Effect O +of O +MKP7 B-protein +( O +wild B-protein_state +type I-protein_state +or O +mutants B-protein_state +) O +expression O +on O +ultraviolet O +- O +induced O +apoptosis O +. O + +HeLa O +cells O +were O +infected O +with O +lentiviruses B-taxonomy_domain +expressing O +MKP7 B-protein +full B-protein_state +- I-protein_state +length I-protein_state +and O +its O +mutants B-protein_state +. O + +Cells O +were O +then O +subjected O +to O +flow B-experimental_method +cytometry I-experimental_method +analysis O +. O + +The O +results O +using O +Annexin B-chemical +- I-chemical +V I-chemical +stain O +for O +membrane O +phosphatidylserine O +eversion O +, O +combined O +with O +propidium B-chemical +iodide I-chemical +( O +PI B-chemical +) O +uptake O +to O +evaluate O +cells O +whose O +membranes O +had O +been O +compromised O +. O + +Staining O +with O +both O +Annexin B-chemical +- I-chemical +V I-chemical +and O +PI B-chemical +indicate O +apoptosis O +( O +upper O +right O +quadrant O +). O + +( O +f O +) O +Statistical O +analysis O +of O +apoptotic O +cells O +( O +mean O +± O +s O +. O +e O +. O +m O +., O +n O += O +3 O +), O +* B-evidence +P I-evidence +< O +0 O +. O +05 O +, O +*** B-evidence +P I-evidence +< O +0 O +. O +001 O +( O +ANOVA B-experimental_method +followed O +by O +Tukey B-experimental_method +' I-experimental_method +s I-experimental_method +test I-experimental_method +). O + +( O +a O +) O +Domain O +organization O +of O +human B-species +MKP5 B-protein +. O + +The O +KBD B-structure_element +and O +CD B-structure_element +of O +MKP5 B-protein +are O +shown O +in O +brown O +and O +grey O +, O +respectively O +. O +( O +b O +) O +Plots B-evidence +of I-evidence +initial I-evidence +velocity I-evidence +of O +the O +MKP5 B-protein +- O +catalysed O +reaction O +versus O +phospho B-protein_state +- O +JNK1 B-protein +concentration O +. O + +The O +error O +bars O +represent O +s O +. O +e O +. O +m O +. O +( O +c O +) O +Structural B-experimental_method +comparison I-experimental_method +of O +the O +JNK B-site +- I-site +interacting I-site +residues I-site +on O +MKP5 B-protein +- O +CD B-structure_element +( O +PDB O +1ZZW O +) O +and O +MKP7 B-protein +- O +CD B-structure_element +. O + +The O +corresponding O +residues O +on O +MKP5 B-protein +are O +depicted O +as O +orange O +sticks O +, O +and O +MKP5 B-protein +residues O +numbers O +are O +in O +parentheses O +. O + +( O +d O +) O +Gel B-experimental_method +filtration I-experimental_method +analysis I-experimental_method +for O +interaction O +of O +JNK1 B-protein +with O +MKP5 B-protein +- O +CD B-structure_element +and O +MKP5 B-protein +- O +KBD B-structure_element +. O +( O +e O +) O +GST B-experimental_method +- I-experimental_method +mediated I-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +for O +interaction O +of O +JNK1 B-protein +with O +MKP5 B-protein +- O +CD B-structure_element +and O +MKP5 B-protein +- O +KBD B-structure_element +. O + +The O +panels O +are O +arranged O +the O +same O +as O +in O +Fig O +. O +2d O +. O +( O +f O +) O +Effects O +of O +mutations B-experimental_method +in O +MKP5 B-protein +- O +CD B-structure_element +on O +the O +JNK1 B-protein +dephosphorylation O +( O +mean O +± O +s O +. O +e O +. O +m O +., O +n O += O +3 O +). O + +( O +g O +) O +Effects O +of O +mutations B-experimental_method +in O +MKP5 B-protein +- O +CD B-structure_element +on O +the O +pNPP B-chemical +hydrolysis O +reaction O +( O +mean O +± O +s O +. O +e O +. O +m O +., O +n O += O +3 O +). O + +Mechanistic O +insight O +into O +a O +peptide B-protein_type +hormone I-protein_type +signaling O +complex O +mediating O +floral O +organ O +abscission O + +Plants B-taxonomy_domain +constantly O +renew O +during O +their O +life O +cycle O +and O +thus O +require O +to O +shed O +senescent O +and O +damaged O +organs O +. O + +It O +is O +unknown O +how O +expression O +of O +IDA B-protein +in O +the O +abscission O +zone O +leads O +to O +HAESA B-protein +activation O +. O + +Here O +we O +show O +that O +IDA B-protein +is O +sensed O +directly O +by O +the O +HAESA B-protein +ectodomain B-structure_element +. O + +A O +central O +hydroxyproline B-residue_name +residue O +anchors O +IDA B-protein +to O +the O +receptor O +. O + +Plants B-taxonomy_domain +can O +shed O +their O +leaves O +, O +flowers O +or O +other O +organs O +when O +they O +no O +longer O +need O +them O +. O +But O +how O +does O +a O +leaf O +or O +a O +flower O +know O +when O +to O +let O +go O +? O +A O +receptor B-protein_type +protein I-protein_type +called O +HAESA B-protein +is O +found O +on O +the O +surface O +of O +the O +cells O +that O +surround O +a O +future O +break O +point O +on O +the O +plant O +. O +When O +its O +time O +to O +shed O +an O +organ O +, O +a O +hormone B-chemical +called O +IDA B-protein +instructs O +HAESA B-protein +to O +trigger O +the O +shedding O +process O +. O + +However O +, O +the O +molecular O +details O +of O +how O +IDA B-protein +triggers O +organ O +shedding O +are O +not O +clear O +. O + +The O +shedding O +of O +floral O +organs O +( O +or O +leaves O +) O +can O +be O +easily O +studied O +in O +a O +model O +plant B-taxonomy_domain +called O +Arabidopsis B-taxonomy_domain +. O + +Santiago O +et O +al O +. O +used O +protein B-experimental_method +biochemistry I-experimental_method +, O +structural B-experimental_method +biology I-experimental_method +and O +genetics B-experimental_method +to O +uncover O +how O +the O +IDA B-protein +hormone B-chemical +activates O +HAESA B-protein +. O + +The O +experiments O +show O +that O +IDA B-protein +binds B-protein_state +directly I-protein_state +to I-protein_state +a O +canyon B-protein_state +shaped I-protein_state +pocket B-site +in O +HAESA B-protein +that O +extends O +out O +from O +the O +surface O +of O +the O +cell O +. O + +IDA B-protein +binding O +to O +HAESA B-protein +allows O +another O +receptor B-protein_type +protein I-protein_type +called O +SERK1 B-protein +to B-protein_state +bind I-protein_state +to I-protein_state +HAESA B-protein +, O +which O +results O +in O +the O +release O +of O +signals O +inside O +the O +cell O +that O +trigger O +the O +shedding O +of O +organs O +. O + +The O +HAESA B-protein +ectodomain B-structure_element +folds O +into O +a O +superhelical B-structure_element +assembly I-structure_element +of O +21 O +leucine B-structure_element +- I-structure_element +rich I-structure_element +repeats I-structure_element +. O + +The O +calculated O +molecular O +mass O +is O +65 O +. O +7 O +kDa O +, O +the O +actual O +molecular O +mass O +obtained O +by O +mass B-experimental_method +spectrometry I-experimental_method +is O +74 O +, O +896 O +Da O +, O +accounting O +for O +the O +N B-chemical +- I-chemical +glycans I-chemical +. O +( O +B O +) O +Ribbon O +diagrams O +showing O +front O +( O +left O +panel O +) O +and O +side O +views O +( O +right O +panel O +) O +of O +the O +isolated O +HAESA B-protein +LRR B-structure_element +domain I-structure_element +. O + +The O +N O +- O +( O +residues O +20 B-residue_range +– I-residue_range +88 I-residue_range +) O +and O +C O +- O +terminal O +( O +residues O +593 B-residue_range +– I-residue_range +615 I-residue_range +) O +capping B-structure_element +domains I-structure_element +are O +shown O +in O +yellow O +, O +the O +central O +21 O +LRR B-structure_element +motifs I-structure_element +are O +in O +blue O +and O +disulphide B-ptm +bonds I-ptm +are O +highlighted O +in O +green O +( O +in O +bonds O +representation O +). O +( O +C O +) O +Structure B-experimental_method +based I-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +the O +21 O +leucine B-structure_element +- I-structure_element +rich I-structure_element +repeats I-structure_element +in O +HAESA B-protein +with O +the O +plant B-taxonomy_domain +LRR B-structure_element +consensus O +sequence O +shown O +for O +comparison O +. O + +Conserved B-protein_state +hydrophobic B-protein_state +residues B-structure_element +are O +shaded O +in O +gray O +, O +N B-site +- I-site +glycosylation I-site +sites I-site +visible O +in O +our O +structures B-evidence +are O +highlighted O +in O +blue O +, O +cysteine B-residue_name +residues O +involved O +in O +disulphide B-ptm +bridge I-ptm +formation O +in O +green O +. O +( O +D O +) O +Asn B-ptm +- I-ptm +linked I-ptm +glycans I-ptm +mask O +the O +N O +- O +terminal O +portion O +of O +the O +HAESA B-protein +ectodomain B-structure_element +. O + +Oligomannose B-chemical +core O +structures O +( O +containing O +two O +N B-chemical +- I-chemical +actylglucosamines I-chemical +and O +three O +terminal O +mannose B-chemical +units O +) O +as O +found O +in O +Trichoplusia B-species +ni I-species +cells O +and O +in O +plants B-taxonomy_domain +were O +modeled O +onto O +the O +seven O +glycosylation B-site +sites I-site +observed O +in O +our O +HAESA B-protein +structures B-evidence +, O +to O +visualize O +the O +surface O +areas O +potentially O +not O +masked O +by O +carbohydrate B-chemical +. O + +The O +HAESA B-protein +ectodomain B-structure_element +is O +shown O +in O +blue O +( O +in O +surface O +representation O +), O +the O +glycan B-chemical +structures O +are O +shown O +in O +yellow O +. O + +Hydrophobic B-bond_interaction +contacts I-bond_interaction +and O +a O +hydrogen B-site +- I-site +bond I-site +network I-site +mediate O +the O +interaction O +between O +HAESA B-protein +and O +the O +peptide B-protein_type +hormone I-protein_type +IDA B-protein +. O + +( O +A O +) O +Details O +of O +the O +IDA B-site +binding I-site +pocket I-site +. O + +HAESA B-protein +is O +shown O +in O +blue O +( O +ribbon O +diagram O +), O +the O +C O +- O +terminal O +Arg B-structure_element +- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +( O +left O +panel O +), O +the O +central O +Hyp B-structure_element +anchor I-structure_element +( O +center O +) O +and O +the O +N O +- O +terminal O +Pro B-structure_element +- I-structure_element +rich I-structure_element +motif I-structure_element +in O +IDA B-protein +( O +right O +panel O +) O +are O +shown O +in O +yellow O +( O +in O +bonds O +representation O +). O + +HAESA B-site +interface I-site +residues I-site +are O +shown O +as O +sticks O +, O +selected O +hydrogen B-bond_interaction +bond I-bond_interaction +interactions I-bond_interaction +are O +denoted O +as O +dotted O +lines O +( O +in O +magenta O +). O +( O +B O +) O +View O +of O +the O +complete O +IDA B-protein +( O +in O +bonds O +representation O +, O +in O +yellow O +) O +binding B-site +pocket I-site +in O +HAESA B-protein +( O +surface O +view O +, O +in O +blue O +). O + +Orientation O +as O +in O +( O +A O +). O +( O +C O +) O +Structure B-experimental_method +based I-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +leucine B-structure_element +- I-structure_element +rich I-structure_element +repeats I-structure_element +in O +HAESA B-protein +with O +the O +plant B-taxonomy_domain +LRR B-structure_element +consensus B-evidence +sequence I-evidence +shown O +for O +comparison O +. O + +The O +IDA B-complex_assembly +- I-complex_assembly +HAESA I-complex_assembly +and O +SERK1 B-complex_assembly +- I-complex_assembly +HAESA I-complex_assembly +complex O +interfaces B-site +are O +conserved B-protein_state +among O +HAESA B-protein +and O +HAESA B-protein_type +- I-protein_type +like I-protein_type +proteins I-protein_type +from O +different O +plant B-taxonomy_domain +species O +. O + +Structure B-experimental_method +- I-experimental_method +based I-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +the O +HAESA B-protein_type +family I-protein_type +members I-protein_type +: O +Arabidopsis B-species +thaliana I-species +HAESA B-protein +( O +Uniprot O +( O +http O +:// O +www O +. O +uniprot O +. O +org O +) O +ID O +P47735 O +), O +Arabidopsis B-species +thaliana I-species +HSL2 B-protein +( O +Uniprot O +ID O +C0LGX3 O +), O +Capsella B-species +rubella I-species +HAESA B-protein +( O +Uniprot O +ID O +R0F2U6 O +), O +Citrus B-species +clementina I-species +HSL2 B-protein +( O +Uniprot O +ID O +V4U227 O +), O +Vitis B-species +vinifera I-species +HAESA B-protein +( O +Uniprot O +ID O +F6HM39 O +). O + +The O +peptide B-protein_type +hormone I-protein_type +IDA B-protein +binds O +to O +the O +HAESA B-protein +LRR B-structure_element +ectodomain I-structure_element +. O + +( O +A O +) O +Multiple B-experimental_method +sequence I-experimental_method +alignment I-experimental_method +of O +selected O +IDA B-protein_type +family I-protein_type +members I-protein_type +. O + +Details O +of O +the O +interactions O +between O +the O +central O +Hyp B-structure_element +anchor I-structure_element +in O +IDA B-protein +and O +the O +C O +- O +terminal O +Arg B-structure_element +- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +with O +HAESA B-protein +are O +highlighted O +in O +( O +E O +) O +and O +( O +F O +), O +respectively O +. O + +Hydrogren O +bonds O +are O +depicted O +as O +dotted O +lines O +( O +in O +magenta O +), O +a O +water B-chemical +molecule O +is O +shown O +as O +a O +red O +sphere O +. O + +During O +their O +growth O +, O +development O +and O +reproduction O +plants B-taxonomy_domain +use O +cell O +separation O +processes O +to O +detach O +no O +- O +longer O +required O +, O +damaged O +or O +senescent O +organs O +. O + +Abscission O +of O +floral O +organs O +in O +Arabidopsis B-taxonomy_domain +is O +a O +model O +system O +to O +study O +these O +cell O +separation O +processes O +in O +molecular O +detail O +. O + +The O +LRR B-structure_element +- I-structure_element +RKs I-structure_element +HAESA B-protein +( O +greek O +: O +to O +adhere O +to O +) O +and O +HAESA B-protein +- I-protein +LIKE I-protein +2 I-protein +( O +HSL2 B-protein +) O +redundantly O +control O +floral O +abscission O +. O + +Loss O +- O +of O +- O +function O +of O +the O +secreted O +small O +protein O +INFLORESCENCE B-protein +DEFICIENT I-protein +IN I-protein +ABSCISSION I-protein +( O +IDA B-protein +) O +causes O +floral O +organs O +to O +remain O +attached O +while O +its O +over O +- O +expression O +leads O +to O +premature O +shedding O +. O + +It O +has O +been O +demonstrated O +that O +a O +dodecamer B-structure_element +peptide B-chemical +within O +EPIP B-structure_element +is O +able O +to O +activate O +HAESA B-protein +and O +HSL2 B-protein +in O +transient B-experimental_method +assays I-experimental_method +in O +tobacco B-taxonomy_domain +cells O +. O + +This B-structure_element +sequence I-structure_element +motif I-structure_element +is O +highly B-protein_state +conserved I-protein_state +among O +IDA B-protein_type +family I-protein_type +members I-protein_type +( O +IDA B-protein_type +- I-protein_type +LIKE I-protein_type +PROTEINS I-protein_type +, O +IDLs B-protein_type +) O +and O +contains O +a O +central O +Pro B-residue_name +residue O +, O +presumed O +to O +be O +post B-protein_state +- I-protein_state +translationally I-protein_state +modified I-protein_state +to O +hydroxyproline B-residue_name +( O +Hyp B-residue_name +; O +Figure O +1A O +). O + +The O +available O +genetic O +and O +biochemical O +evidence O +suggests O +that O +IDA B-protein +and O +HAESA B-protein +together O +control O +floral O +abscission O +, O +but O +it O +is O +poorly O +understood O +if O +IDA B-protein +is O +directly O +sensed O +by O +the O +receptor B-protein_type +kinase I-protein_type +HAESA B-protein +and O +how O +IDA B-protein +binding O +at O +the O +cell O +surface O +would O +activate O +the O +receptor O +. O + +IDA B-protein +directly O +binds O +to O +the O +LRR B-structure_element +domain I-structure_element +of O +HAESA B-protein + +Active B-protein_state +IDA B-protein_type +- I-protein_type +family I-protein_type +peptide I-protein_type +hormones I-protein_type +are O +hydroxyprolinated B-protein_state +dodecamers B-structure_element +. O + +Note O +that O +Pro58IDA B-residue_name_number +and O +Leu67IDA B-residue_name_number +are O +the O +first O +residues O +defined O +by O +electron B-evidence +density I-evidence +when O +bound B-protein_state +to I-protein_state +the O +HAESA B-protein +ectodomain B-structure_element +. O +( O +D O +) O +Table O +summaries O +for O +equilibrium B-evidence +dissociation I-evidence +constants I-evidence +( O +Kd B-evidence +), O +binding B-evidence +enthalpies I-evidence +( O +ΔH B-evidence +), O +binding B-evidence +entropies I-evidence +( O +ΔS B-evidence +) O +and O +stoichoimetries O +( O +N O +) O +for O +different O +IDA B-chemical +peptides I-chemical +binding O +to O +the O +HAESA B-protein +ectodomain B-structure_element +( O +± O +fitting O +errors O +; O +n O +. O +d O +. O + +Root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +( O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +.) I-evidence +is O +1 O +. O +0 O +Å O +comparing O +100 O +corresponding O +atoms O +. O + +The O +receptor B-protein_type +kinase I-protein_type +SERK1 B-protein +acts O +as O +a O +HAESA B-protein_type +co I-protein_type +- I-protein_type +receptor I-protein_type +and O +promotes O +high O +- O +affinity O +IDA B-protein +sensing O +. O + +( O +A O +) O +Petal B-experimental_method +break I-experimental_method +- I-experimental_method +strength I-experimental_method +assays I-experimental_method +measure O +the O +force O +( O +expressed O +in O +gram O +equivalents O +) O +required O +to O +remove O +the O +petals O +from O +the O +flower O +of O +serk B-gene +mutant B-protein_state +plants B-taxonomy_domain +compared O +to O +haesa B-gene +/ O +hsl2 B-gene +mutant B-protein_state +and O +Col O +- O +0 O +wild B-protein_state +- I-protein_state +type I-protein_state +flowers O +. O + +Petal O +break O +- O +strength O +was O +found O +significantly O +increased O +in O +almost O +all O +positions O +( O +indicated O +with O +a O +*) O +for O +haesa B-gene +/ O +hsl2 B-gene +and O +serk1 B-gene +- I-gene +1 I-gene +mutant B-protein_state +plants B-taxonomy_domain +with O +respect O +to O +the O +Col O +- O +0 O +control O +. O + +( O +B O +) O +Analytical B-experimental_method +size I-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +. O + +The O +HAESA B-protein +LRR B-structure_element +domain I-structure_element +elutes O +as O +a O +monomer B-oligomeric_state +( O +black O +dotted O +line O +), O +as O +does O +the O +isolated O +SERK1 B-protein +ectodomain B-structure_element +( O +blue O +dotted O +line O +). O + +A O +HAESA B-complex_assembly +– I-complex_assembly +IDA I-complex_assembly +– I-complex_assembly +SERK1 I-complex_assembly +complex O +elutes O +as O +an O +apparent O +heterodimer B-oligomeric_state +( O +red O +line O +), O +while O +a O +mixture O +of O +HAESA B-protein +and O +SERK1 B-protein +yields O +two O +isolated O +peaks O +that O +correspond O +to O +monomeric B-oligomeric_state +HAESA B-protein +and O +SERK1 B-protein +, O +respectively O +( O +black O +line O +). O + +Void O +( O +V0 O +) O +volume O +and O +total O +volume O +( O +Vt O +) O +are O +shown O +, O +together O +with O +elution O +volumes O +for O +molecular O +mass O +standards O +( O +A O +, O +Thyroglobulin B-protein +, O +669 O +, O +000 O +Da O +; O +B O +, O +Ferritin B-protein +, O +440 O +, O +00 O +Da O +, O +C O +, O +Aldolase B-protein +, O +158 O +, O +000 O +Da O +; O +D O +, O +Conalbumin B-protein +, O +75 O +, O +000 O +Da O +; O +E O +, O +Ovalbumin B-protein +, O +44 O +, O +000 O +Da O +; O +F O +, O +Carbonic B-protein +anhydrase I-protein +, O +29 O +, O +000 O +Da O +). O + +A O +SDS B-experimental_method +PAGE I-experimental_method +of O +the O +peak O +fractions O +is O +shown O +alongside O +. O + +Purified O +HAESA B-protein +and O +SERK1 B-protein +are O +~ O +75 O +and O +~ O +28 O +kDa O +, O +respectively O +. O +( O +C O +) O +Isothermal B-experimental_method +titration I-experimental_method +calorimetry I-experimental_method +of O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +Hyp64 B-ptm +→ I-ptm +Pro I-ptm +IDA B-protein +versus O +the O +HAESA B-protein +and O +SERK1 B-protein +ectodomains B-structure_element +. O + +no O +detectable O +binding O +) O +( O +D O +) O +Analytical B-experimental_method +size I-experimental_method +- I-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +in O +the O +presence B-protein_state +of I-protein_state +the O +IDA B-protein +Hyp64 B-ptm +→ I-ptm +Pro I-ptm +mutant B-protein_state +peptide B-chemical +reveals O +no O +complex O +formation O +between O +HAESA B-protein +and O +SERK1 B-protein +ectodomains B-structure_element +. O + +( O +E O +) O +In B-experimental_method +vitro I-experimental_method +kinase I-experimental_method +assays I-experimental_method +of O +the O +HAESA B-protein +and O +SERK1 B-protein +kinase B-structure_element +domains I-structure_element +. O + +Wild B-protein_state +- I-protein_state +type I-protein_state +HAESA B-protein +and O +SERK1 B-protein +kinase B-structure_element +domains I-structure_element +( O +KDs B-structure_element +) O +exhibit O +auto O +- O +phosphorylation O +activities O +( O +lanes O +1 O ++ O +3 O +). O + +Transphosphorylation O +activity O +from O +the O +active B-protein_state +kinase O +to O +the O +mutated B-protein_state +form O +can O +be O +observed O +in O +both O +directions O +( O +lanes O +5 O ++ O +6 O +). O + +A O +Hyp B-protein_state +- I-protein_state +modified I-protein_state +dodecamer B-structure_element +comprising O +the O +highly B-protein_state +conserved I-protein_state +PIP B-structure_element +motif I-structure_element +in O +IDA B-protein +( O +Figure O +1A O +) O +interacts O +with O +HAESA B-protein +with O +1 O +: O +1 O +stoichiometry O +( O +N O +) O +and O +with O +a O +dissociation B-evidence +constant I-evidence +( O +Kd B-evidence +) O +of O +~ O +20 O +μM O +( O +Figure O +1B O +). O + +We O +next O +determined O +crystal B-evidence +structures I-evidence +of O +the O +apo B-protein_state +HAESA B-protein +ectodomain B-structure_element +and O +of O +a O +HAESA B-complex_assembly +- I-complex_assembly +IDA I-complex_assembly +complex O +, O +at O +1 O +. O +74 O +and O +1 O +. O +86 O +Å O +resolution O +, O +respectively O +( O +Figure O +1C O +; O +Figure O +1 O +— O +figure O +supplement O +1B O +– O +D O +; O +Tables O +1 O +, O +2 O +). O + +IDA B-protein +binds O +in O +a O +completely B-protein_state +extended I-protein_state +conformation I-protein_state +along O +the O +inner O +surface O +of O +the O +HAESA B-protein +ectodomain B-structure_element +, O +covering O +LRRs B-structure_element +2 I-structure_element +– I-structure_element +14 I-structure_element +( O +Figure O +1C O +, O +D O +, O +Figure O +1 O +— O +figure O +supplement O +2 O +). O + +The O +central O +Hyp64IDA B-ptm +is O +buried O +in O +a O +specific O +pocket B-site +formed O +by O +HAESA B-protein +LRRs B-structure_element +8 I-structure_element +– I-structure_element +10 I-structure_element +, O +with O +its O +hydroxyl O +group O +establishing O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +the O +strictly B-protein_state +conserved I-protein_state +Glu266HAESA B-residue_name_number +and O +with O +a O +water B-chemical +molecule O +, O +which O +in O +turn O +is O +coordinated O +by O +the O +main O +chain O +oxygens O +of O +Phe289HAESA B-residue_name_number +and O +Ser311HAESA B-residue_name_number +( O +Figure O +1E O +; O +Figure O +1 O +— O +figure O +supplement O +3 O +). O + +The O +restricted O +size O +of O +the O +Hyp B-site +pocket I-site +suggests O +that O +IDA B-protein +does O +not O +require O +arabinosylation B-ptm +of O +Hyp64IDA B-ptm +for O +activity O +in O +vivo O +, O +a O +modification O +that O +has O +been O +reported O +for O +Hyp B-residue_name +residues O +in O +plant B-taxonomy_domain +CLE B-protein_type +peptide I-protein_type +hormones I-protein_type +. O + +The O +C O +- O +terminal O +Arg B-structure_element +- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +in O +IDA B-protein +maps O +to O +a O +cavity B-site +formed O +by O +HAESA B-protein +LRRs B-structure_element +11 I-structure_element +– I-structure_element +14 I-structure_element +( O +Figure O +1D O +, O +F O +). O + +Mutation B-experimental_method +of O +Arg417HSL2 B-residue_name_number +( O +which O +corresponds O +to O +Arg409HAESA B-residue_name_number +) O +causes O +a O +loss O +- O +of O +- O +function O +phenotype O +in O +HSL2 B-protein +, O +which O +indicates O +that O +the O +peptide B-site +binding I-site +pockets I-site +in O +different O +HAESA B-protein_type +receptors I-protein_type +have O +common O +structural O +and O +sequence O +features O +. O + +Indeed O +, O +we O +find O +many O +of O +the O +residues O +contributing O +to O +the O +formation O +of O +the O +IDA B-site +binding I-site +surface I-site +in O +HAESA B-protein +to O +be O +conserved B-protein_state +in O +HSL2 B-protein +and O +in O +other O +HAESA B-protein_type +- I-protein_type +type I-protein_type +receptors I-protein_type +in O +different O +plant B-taxonomy_domain +species O +( O +Figure O +1 O +— O +figure O +supplement O +3 O +). O + +A O +N O +- O +terminal O +Pro B-structure_element +- I-structure_element +rich I-structure_element +motif I-structure_element +in O +IDA B-protein +makes O +contacts O +with O +LRRs B-structure_element +2 I-structure_element +– I-structure_element +6 I-structure_element +of O +the O +receptor O +( O +Figure O +1D O +, O +Figure O +1 O +— O +figure O +supplement O +2A O +– O +C O +). O + +Other O +hydrophobic B-bond_interaction +and I-bond_interaction +polar I-bond_interaction +interactions I-bond_interaction +are O +mediated O +by O +Ser62IDA B-residue_name_number +, O +Ser65IDA B-residue_name_number +and O +by O +backbone O +atoms O +along O +the O +IDA B-chemical +peptide I-chemical +( O +Figure O +1D O +, O +Figure O +1 O +— O +figure O +supplement O +2A O +– O +C O +). O + +HAESA B-protein +specifically O +senses O +IDA B-protein_type +- I-protein_type +family I-protein_type +dodecamer B-structure_element +peptides B-chemical + +We O +next O +investigated O +whether O +HAESA B-protein +binds O +N B-protein_state +- I-protein_state +terminally I-protein_state +extended I-protein_state +versions O +of O +IDA B-protein +. O + +In O +this O +structure B-evidence +, O +no O +additional O +electron B-evidence +density I-evidence +accounts O +for O +the O +PKGV B-structure_element +motif I-structure_element +at O +the O +IDA B-protein +N O +- O +terminus O +( O +Figure O +2A O +, O +B O +). O + +Consistently O +, O +PKGV B-mutant +- I-mutant +IDA I-mutant +and O +IDA B-protein +have O +similar O +binding B-evidence +affinities I-evidence +in O +our O +ITC B-experimental_method +assays I-experimental_method +, O +further O +indicating O +that O +HAESA B-protein +senses O +a O +dodecamer B-structure_element +peptide B-chemical +comprising O +residues O +58 B-residue_range +- I-residue_range +69IDA I-residue_range +( O +Figure O +2D O +). O + +IDL1 B-protein +, O +which O +can O +rescue O +IDA B-protein +loss O +- O +of O +- O +function O +mutants O +when O +introduced O +in O +abscission O +zone O +cells O +, O +can O +also O +be O +sensed O +by O +HAESA B-protein +, O +albeit O +with O +lower O +affinity B-evidence +( O +Figure O +2D O +). O + +A O +2 O +. O +56 O +Å O +co B-evidence +- I-evidence +crystal I-evidence +structure I-evidence +with O +IDL1 B-protein +reveals O +that O +different O +IDA B-protein_type +family I-protein_type +members I-protein_type +use O +a O +common O +binding O +mode O +to O +interact O +with O +HAESA B-protein_type +- I-protein_type +type I-protein_type +receptors I-protein_type +( O +Figure O +2A O +– O +C O +, O +E O +, O +Table O +2 O +). O + +Notably O +, O +HAESA B-protein +can O +discriminate O +between O +IDLs B-protein_type +and O +functionally B-protein_state +unrelated I-protein_state +dodecamer B-structure_element +peptides B-chemical +with O +Hyp B-ptm +modifications I-ptm +, O +such O +as O +CLV3 B-protein +( O +Figures O +2D O +, O +7 O +). O + +The O +co B-protein_type +- I-protein_type +receptor I-protein_type +kinase I-protein_type +SERK1 B-protein +allows O +for O +high O +- O +affinity O +IDA O +sensing O + +As O +all O +five O +SERK B-protein_type +family I-protein_type +members I-protein_type +appear O +to O +be O +expressed O +in O +the O +Arabidopsis B-taxonomy_domain +abscission O +zone O +, O +we O +quantified O +their O +relative O +contribution O +to O +floral O +abscission O +in O +Arabidopsis B-taxonomy_domain +using O +a O +petal B-experimental_method +break I-experimental_method +- I-experimental_method +strength I-experimental_method +assay I-experimental_method +. O + +Our O +experiments O +suggest O +that O +among O +the O +SERK B-protein_type +family I-protein_type +members I-protein_type +, O +SERK1 B-protein +is O +a O +positive O +regulator O +of O +floral O +abscission O +. O + +Possibly O +because O +SERKs B-protein_type +have O +additional O +roles O +in O +plant O +development O +such O +as O +in O +pollen O +formation O +and O +brassinosteroid O +signaling O +, O +we O +found O +that O +higher O +- O +order O +SERK O +mutants O +exhibit O +pleiotropic O +phenotypes O +in O +the O +flower O +, O +rendering O +their O +analysis O +and O +comparison O +by O +quantitative B-experimental_method +petal I-experimental_method +break I-experimental_method +- I-experimental_method +strength I-experimental_method +assays I-experimental_method +difficult O +. O + +We O +thus O +focused O +on O +analyzing O +the O +contribution O +of O +SERK1 B-protein +to O +HAESA B-protein +ligand O +sensing O +and O +receptor O +activation O +. O + +We O +next O +quantified O +the O +contribution O +of O +SERK1 B-protein +to O +IDA B-protein +recognition O +by O +HAESA B-protein +. O + +We O +found O +that O +HAESA B-protein +senses O +IDA B-protein +with O +a O +~ O +60 O +fold O +higher O +binding B-evidence +affinity I-evidence +in O +the O +presence B-protein_state +of I-protein_state +SERK1 B-protein +, O +suggesting O +that O +SERK1 B-protein +is O +involved O +in O +the O +specific O +recognition O +of O +the O +peptide B-protein_type +hormone I-protein_type +( O +Figure O +3C O +). O + +This O +suggests O +that O +IDA B-protein +itself O +promotes O +receptor O +– O +co O +- O +receptor O +association O +, O +as O +previously O +described O +for O +the O +steroid B-chemical +hormone I-chemical +brassinolide B-chemical +and O +for O +other O +LRR B-complex_assembly +- I-complex_assembly +RK I-complex_assembly +complexes O +. O + +Importantly O +, O +hydroxyprolination B-ptm +of O +IDA B-protein +is O +critical O +for O +HAESA B-complex_assembly +- I-complex_assembly +IDA I-complex_assembly +- I-complex_assembly +SERK1 I-complex_assembly +complex O +formation O +( O +Figure O +3C O +, O +D O +). O + +Upon O +IDA B-protein +binding O +at O +the O +cell O +surface O +, O +the O +kinase B-structure_element +domains I-structure_element +of O +HAESA B-protein +and O +SERK1 B-protein +, O +which O +have O +been O +shown O +to O +be O +active B-protein_state +protein B-protein_type +kinases I-protein_type +, O +may O +interact O +in O +the O +cytoplasm O +to O +activate O +each O +other O +. O + +Consistently O +, O +the O +HAESA B-protein +kinase B-structure_element +domain I-structure_element +can O +transphosphorylate O +SERK1 B-protein +and O +vice O +versa O +in O +in O +vitro O +transphosphorylation B-experimental_method +assays I-experimental_method +( O +Figure O +3E O +). O + +Crystal B-evidence +structure I-evidence +of O +a O +HAESA B-complex_assembly +– I-complex_assembly +IDA I-complex_assembly +– I-complex_assembly +SERK1 I-complex_assembly +signaling O +complex O +. O + +( O +A O +) O +Overview O +of O +the O +ternary O +complex O +with O +HAESA B-protein +in O +blue O +( O +surface O +representation O +), O +IDA B-protein +in O +yellow O +( O +bonds O +representation O +) O +and O +SERK1 B-protein +in O +orange O +( O +surface O +view O +). O +( O +B O +) O +The O +HAESA B-protein +ectodomain B-structure_element +undergoes O +a O +conformational O +change O +upon O +SERK1 B-protein +co O +- O +receptor O +binding O +. O + +Shown O +are O +Cα O +traces O +of O +a O +structural B-experimental_method +superposition I-experimental_method +of O +the O +unbound B-protein_state +( O +yellow O +) O +and O +SERK1 B-protein_state +- I-protein_state +bound I-protein_state +( O +blue O +) O +HAESA B-protein +ectodomains B-structure_element +( O +r B-evidence +. I-evidence +m I-evidence +. I-evidence +s I-evidence +. I-evidence +d I-evidence +. I-evidence +is O +1 O +. O +5 O +Å O +between O +572 O +corresponding O +Cα O +atoms O +). O + +SERK1 B-protein +( O +in O +orange O +) O +and O +IDA B-protein +( O +in O +red O +) O +are O +shown O +alongside O +. O + +The O +N O +- O +terminal O +capping B-structure_element +domain I-structure_element +of O +SERK1 B-protein +( O +in O +orange O +) O +directly O +contacts O +the O +C O +- O +terminal O +part O +of O +IDA B-protein +( O +in O +yellow O +, O +in O +bonds O +representation O +) O +and O +the O +receptor B-protein_type +HAESA B-protein +( O +in O +blue O +). O + +Polar B-bond_interaction +contacts I-bond_interaction +of O +SERK1 B-protein +with O +IDA B-protein +are O +shown O +in O +magenta O +, O +with O +the O +HAESA B-protein +LRR B-structure_element +domain I-structure_element +in O +gray O +. O +( O +D O +) O +Details O +of O +the O +zipper B-structure_element +- I-structure_element +like I-structure_element +SERK1 B-site +- I-site +HAESA I-site +interface I-site +. O + +Polar B-bond_interaction +interactions I-bond_interaction +are O +highlighted O +as O +dotted O +lines O +( O +in O +magenta O +). O + +HAESA B-protein +LRRs B-structure_element +16 I-structure_element +– I-structure_element +21 I-structure_element +and O +its O +C O +- O +terminal O +capping B-structure_element +domain I-structure_element +undergo O +a O +conformational O +change O +upon O +SERK1 B-protein +binding O +( O +Figure O +4B O +). O + +The O +SERK1 B-protein +ectodomain B-structure_element +interacts O +with O +the O +IDA B-site +peptide I-site +binding I-site +site I-site +using O +a O +loop B-structure_element +region I-structure_element +( O +residues O +51 B-residue_range +- I-residue_range +59SERK1 I-residue_range +) O +from O +its O +N O +- O +terminal O +cap B-structure_element +( O +Figure O +4A O +, O +C O +). O + +SERK1 B-protein +binds O +HAESA B-protein +using O +these O +two O +distinct O +interaction B-site +surfaces I-site +( O +Figure O +1 O +— O +figure O +supplement O +3 O +), O +with O +the O +N B-structure_element +- I-structure_element +cap I-structure_element +of O +the O +SERK1 B-protein +LRR B-structure_element +domain I-structure_element +partially O +covering O +the O +IDA B-site +peptide I-site +binding I-site +cleft I-site +. O + +The O +IDA B-protein +C B-structure_element +- I-structure_element +terminal I-structure_element +motif I-structure_element +is O +required O +for O +HAESA B-complex_assembly +- I-complex_assembly +SERK1 I-complex_assembly +complex O +formation O +and O +for O +IDA O +bioactivity O +. O + +( O +A O +) O +Size B-experimental_method +exclusion I-experimental_method +chromatography I-experimental_method +experiments O +similar O +to O +Figure O +3B O +, O +D O +reveal O +that O +IDA B-protein +mutant B-protein_state +peptides B-chemical +targeting O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +motif I-structure_element +do O +not O +form O +biochemically B-protein_state +stable I-protein_state +HAESA B-complex_assembly +- I-complex_assembly +IDA I-complex_assembly +- I-complex_assembly +SERK1 I-complex_assembly +complexes O +. O + +Purified B-experimental_method +HAESA B-protein +and O +SERK1 B-protein +are O +~ O +75 O +and O +~ O +28 O +kDa O +, O +respectively O +. O + +Left O +panel O +: O +IDA B-mutant +K66A I-mutant +/ I-mutant +R67A I-mutant +; O +center O +: O +IDA B-mutant +ΔN69 I-mutant +, O +right O +panel O +: O +SDS B-experimental_method +- I-experimental_method +PAGE I-experimental_method +of O +peak O +fractions O +. O + +Note O +that O +the O +HAESA B-protein +and O +SERK1 B-protein +input O +lanes O +have O +already O +been O +shown O +in O +Figure O +3D O +. O +( O +B O +) O +Isothermal B-evidence +titration I-evidence +thermographs I-evidence +of O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +IDA B-chemical +peptides I-chemical +titrated B-experimental_method +into O +a O +HAESA B-protein +- O +SERK1 B-protein +mixture O +in O +the O +cell O +. O + +Table O +summaries O +for O +calorimetric B-evidence +binding I-evidence +constants I-evidence +and O +stoichoimetries O +for O +different O +IDA B-chemical +peptides I-chemical +binding O +to O +the O +HAESA B-protein +– O +SERK1 B-protein +ectodomain B-structure_element +mixture O +( O +± O +fitting O +errors O +; O +n O +. O +d O +. O + +( O +C O +) O +Quantitative O +petal B-experimental_method +break I-experimental_method +- I-experimental_method +strength I-experimental_method +assay I-experimental_method +for O +Col O +- O +0 O +wild B-protein_state +- I-protein_state +type I-protein_state +flowers O +and O +35S B-gene +:: O +IDA B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +and O +35S B-gene +:: O +IDA B-mutant +K66A I-mutant +/ I-mutant +R67A I-mutant +mutant B-protein_state +flowers O +. O + +Up O +to O +inflorescence O +position O +4 O +, O +petal O +break O +in O +35S B-gene +:: O +IDA B-mutant +K66A I-mutant +/ I-mutant +R67A I-mutant +mutant B-protein_state +plants B-taxonomy_domain +was O +significantly O +increased O +compared O +to O +both O +Col O +- O +0 O +control O +plants B-taxonomy_domain +( O +b O +) O +and O +35S B-gene +:: O +IDA B-protein +plants B-taxonomy_domain +( O +c O +) O +( O +D O +) O +Normalized O +expression O +levels O +( O +relative O +expression O +± O +standard O +error O +; O +ida O +: O +- O +0 O +. O +02 O +± O +0 O +. O +001 O +; O +Col O +- O +0 O +: O +1 O +± O +0 O +. O +11 O +; O +35S B-gene +:: O +IDA B-protein +124 O +± O +0 O +. O +75 O +; O +35S B-gene +:: O +IDA B-mutant +K66A I-mutant +/ I-mutant +R67A I-mutant +: O +159 O +± O +0 O +. O +58 O +) O +of O +IDA B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutant B-protein_state +transcripts O +in O +the O +35S B-experimental_method +promoter I-experimental_method +over I-experimental_method +- I-experimental_method +expression I-experimental_method +lines I-experimental_method +analyzed O +in O +( O +C O +). O +( O +E O +) O +Magnified O +view O +of O +representative O +abscission O +zones O +from O +35S B-gene +:: O +IDA B-protein +, O +Col O +- O +0 O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +35S B-gene +:: O +IDA B-mutant +K66A I-mutant +/ I-mutant +R67A I-mutant +double B-protein_state +- I-protein_state +mutant I-protein_state +T3 B-experimental_method +transgenic I-experimental_method +lines I-experimental_method +. O + +15 O +out O +of O +15 O +35S B-gene +:: O +IDA B-protein +plants B-taxonomy_domain +, O +0 O +out O +of O +15 O +Col O +- O +0 O +plants B-taxonomy_domain +and O +0 O +out O +of O +15 O +35S B-gene +:: O +IDA B-mutant +K66A I-mutant +/ I-mutant +R67A I-mutant +double B-protein_state +- I-protein_state +mutant I-protein_state +plants B-taxonomy_domain +, O +showed O +an O +enlarged O +abscission O +zone O +, O +respectively O +( O +3 O +independent O +lines O +were O +analyzed O +). O + +The O +four O +C O +- O +terminal O +residues O +in O +IDA B-protein +( O +Lys66IDA B-residue_range +- I-residue_range +Asn69IDA I-residue_range +) O +are O +conserved B-protein_state +among O +IDA B-protein_type +family I-protein_type +members I-protein_type +and O +are O +in O +direct O +contact O +with O +SERK1 B-protein +( O +Figures O +1A O +, O +4C O +). O + +We O +thus O +assessed O +their O +contribution O +to O +HAESA B-complex_assembly +– I-complex_assembly +SERK1 I-complex_assembly +complex O +formation O +. O + +A O +synthetic B-protein_state +Lys66IDA B-mutant +/ I-mutant +Arg67IDA I-mutant +→ I-mutant +Ala I-mutant +mutant B-protein_state +peptide B-chemical +( O +IDA B-mutant +K66A I-mutant +/ I-mutant +R66A I-mutant +) O +showed O +a O +10 O +fold O +reduced O +binding B-evidence +affinity I-evidence +when O +titrated B-experimental_method +in O +a O +HAESA B-protein +/ O +SERK1 B-protein +protein O +solution O +( O +Figures O +5A O +, O +B O +, O +2D O +). O + +We O +over B-experimental_method +- I-experimental_method +expressed I-experimental_method +full B-protein_state +- I-protein_state +length I-protein_state +wild B-protein_state +- I-protein_state +type I-protein_state +IDA B-protein +or O +this O +Lys66IDA B-mutant +/ I-mutant +Arg67IDA I-mutant +→ I-mutant +Ala I-mutant +double B-protein_state +- I-protein_state +mutant I-protein_state +to O +similar O +levels O +in O +Col O +- O +0 O +Arabidopsis B-taxonomy_domain +plants B-taxonomy_domain +( O +Figure O +5D O +). O + +We O +found O +that O +over B-experimental_method +- I-experimental_method +expression I-experimental_method +of O +wild B-protein_state +- I-protein_state +type I-protein_state +IDA B-protein +leads O +to O +early O +floral O +abscission O +and O +an O +enlargement O +of O +the O +abscission O +zone O +( O +Figure O +5C O +– O +E O +). O + +In O +contrast O +, O +over B-experimental_method +- I-experimental_method +expression I-experimental_method +of O +the O +IDA B-mutant +Lys66IDA I-mutant +/ I-mutant +Arg67IDA I-mutant +→ I-mutant +Ala I-mutant +double B-protein_state +mutant I-protein_state +significantly O +delays O +floral O +abscission O +when O +compared O +to O +wild B-protein_state +- I-protein_state +type I-protein_state +control O +plants B-taxonomy_domain +, O +suggesting O +that O +the O +mutant B-protein_state +IDA B-chemical +peptide I-chemical +has O +reduced O +activity O +in O +planta B-taxonomy_domain +( O +Figure O +5C O +– O +E O +). O + +In O +agreement O +with O +our O +structures B-evidence +and O +biochemical B-experimental_method +assays I-experimental_method +, O +this O +experiment O +suggests O +a O +role O +of O +the O +conserved B-protein_state +IDA B-protein +C O +- O +terminus O +in O +the O +control O +of O +floral O +abscission O +. O + +For O +a O +rapidly O +growing O +number O +of O +plant B-taxonomy_domain +signaling O +pathways O +, O +SERK B-protein_type +proteins I-protein_type +act O +as O +these O +essential O +co B-protein_type +- I-protein_type +receptors I-protein_type +(; O +). O + +SERK1 O +has O +been O +previously O +reported O +as O +a O +positive O +regulator O +in O +plant B-taxonomy_domain +embryogenesis O +, O +male O +sporogenesis O +, O +brassinosteroid O +signaling O +and O +in O +phytosulfokine O +perception O +. O + +Recent O +findings O +by O +and O +our O +mechanistic O +studies O +now O +also O +support O +a O +positive O +role O +for O +SERK1 B-protein +in O +floral O +abscission O +. O + +It O +has O +been O +previously O +suggested O +that O +SERK1 B-protein +can O +inhibit O +cell O +separation O +. O + +However O +our O +results O +show O +that O +SERK1 B-protein +also O +can O +activate O +this O +process O +upon O +IDA B-protein +sensing O +, O +indicating O +that O +SERKs B-protein_type +may O +fulfill O +several O +different O +functions O +in O +the O +course O +of O +the O +abscission O +process O +. O + +While O +the O +sequence O +of O +the O +mature B-protein_state +IDA B-chemical +peptide I-chemical +has O +not O +been O +experimentally O +determined O +in O +planta B-taxonomy_domain +, O +our O +HAESA B-complex_assembly +- I-complex_assembly +IDA I-complex_assembly +complex O +structures B-evidence +and O +calorimetry B-evidence +assays I-evidence +suggest O +that O +active B-protein_state +IDLs B-protein_type +are O +hydroxyprolinated B-protein_state +dodecamers B-structure_element +. O + +It O +will O +be O +thus O +interesting O +to O +see O +if O +proteolytic O +processing O +of O +full B-protein_state +- I-protein_state +length I-protein_state +IDA B-protein +in O +vivo O +is O +regulated O +in O +a O +cell O +- O +type O +or O +tissue O +- O +specific O +manner O +. O + +The O +central O +Hyp B-residue_name +residue O +in O +IDA B-protein +is O +found O +buried O +in O +the O +HAESA B-protein +peptide B-site +binding I-site +surface I-site +and O +thus O +this O +post O +- O +translational O +modification O +may O +regulate O +IDA B-protein +bioactivity O +. O + +In O +our O +quantitative B-experimental_method +biochemical I-experimental_method +assays I-experimental_method +, O +the O +presence B-protein_state +of I-protein_state +SERK1 B-protein +dramatically O +increases O +the O +HAESA B-protein +binding O +specificity O +and O +affinity O +for O +IDA B-protein +. O + +This O +observation O +is O +consistent O +with O +our O +complex O +structure B-evidence +in O +which O +receptor O +and O +co O +- O +receptor O +together O +form O +the O +IDA B-site +binding I-site +pocket I-site +. O + +The O +fact O +that O +SERK1 B-protein +specifically O +interacts O +with O +the O +very O +C O +- O +terminus O +of O +IDLs B-protein_type +may O +allow O +for O +the O +rational O +design O +of O +peptide B-chemical +hormone I-chemical +antagonists I-chemical +, O +as O +previously O +demonstrated O +for O +the O +brassinosteroid O +pathway O +. O + +Importantly O +, O +our O +calorimetry B-experimental_method +assays I-experimental_method +reveal O +that O +the O +SERK1 B-protein +ectodomain B-structure_element +binds B-protein_state +HAESA B-protein +with O +nanomolar O +affinity O +, O +but O +only O +in O +the O +presence B-protein_state +of I-protein_state +IDA B-protein +( O +Figure O +3C O +). O + +This O +ligand O +- O +induced O +formation O +of O +a O +receptor O +– O +co O +- O +receptor O +complex O +may O +allow O +the O +HAESA B-protein +and O +SERK1 B-protein +kinase B-structure_element +domains I-structure_element +to O +efficiently O +trans O +- O +phosphorylate O +and O +activate O +each O +other O +in O +the O +cytoplasm O +. O + +SERK1 B-protein +uses O +partially O +overlapping O +surface O +areas O +to O +activate O +different O +plant B-taxonomy_domain +signaling B-protein_type +receptors I-protein_type +. O + +Left O +panel O +: O +Ribbon O +diagram O +of O +HAESA B-protein +( O +in O +blue O +), O +SERK1 B-protein +( O +in O +orange O +) O +and O +IDA B-protein +( O +in O +bonds O +and O +surface O +represention O +). O + +Right O +panel O +: O +Ribbon O +diagram O +of O +the O +plant B-taxonomy_domain +steroid B-protein_type +receptor I-protein_type +BRI1 B-protein +( O +in O +blue O +) O +bound B-protein_state +to I-protein_state +brassinolide B-chemical +( O +in O +gray O +, O +in O +bonds O +representation O +) O +and O +to O +SERK1 B-protein +, O +shown O +in O +the O +same O +orientation O +( O +PDB O +- O +ID O +. O +4lsx O +). O + +( O +B O +) O +View O +of O +the O +inner O +surface O +of O +the O +SERK1 B-protein +LRR B-structure_element +domain I-structure_element +( O +PDB O +- O +ID O +4lsc O +, O +surface O +representation O +, O +in O +gray O +). O + +A O +ribbon O +diagram O +of O +SERK1 B-protein +in O +the O +same O +orientation O +is O +shown O +alongside O +. O + +Residues O +interacting O +with O +the O +HAESA B-protein +or O +BRI1 B-protein +LRR B-structure_element +domains I-structure_element +are O +shown O +in O +orange O +or O +magenta O +, O +respectively O +. O + +Comparison B-experimental_method +of O +our O +HAESA B-complex_assembly +– I-complex_assembly +IDA I-complex_assembly +– I-complex_assembly +SERK1 I-complex_assembly +structure B-evidence +with O +the O +brassinosteroid O +receptor O +signaling O +complex O +, O +where O +SERK1 B-protein +also O +acts O +as O +co B-protein_type +- I-protein_type +receptor I-protein_type +, O +reveals O +an O +overall O +conserved B-protein_state +mode O +of O +SERK1 B-protein +binding O +, O +while O +the O +ligand B-site +binding I-site +pockets I-site +map O +to O +very O +different O +areas O +in O +the O +corresponding O +receptors O +( O +LRRs B-structure_element +2 I-structure_element +– I-structure_element +14 I-structure_element +; O +HAESA B-protein +; O +LRRs B-structure_element +21 I-structure_element +– I-structure_element +25 I-structure_element +, O +BRI1 B-protein +) O +and O +may O +involve O +an O +island O +domain O +( O +BRI1 B-protein +) O +or O +not O +( O +HAESA B-protein +) O +( O +Figure O +6A O +). O + +These O +residues O +are O +not O +involved O +in O +the O +sensing O +of O +the O +steroid B-chemical +hormone I-chemical +brassinolide B-chemical +. O + +In O +both O +cases O +however O +, O +the O +co O +- O +receptor O +completes O +the O +hormone B-site +binding I-site +pocket I-site +. O + +Different O +plant B-taxonomy_domain +peptide B-protein_type +hormone I-protein_type +families I-protein_type +contain O +a O +C O +- O +terminal O +( B-structure_element +Arg I-structure_element +)- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +, O +which O +in O +IDA B-protein +represents O +the O +co B-site +- I-site +receptor I-site +recognition I-site +site I-site +. O + +Our O +experiments O +reveal O +that O +SERK1 B-protein +recognizes O +a O +C O +- O +terminal O +Arg B-structure_element +- I-structure_element +His I-structure_element +- I-structure_element +Asn I-structure_element +motif I-structure_element +in O +IDA B-protein +. O + +Importantly O +, O +this B-structure_element +motif I-structure_element +can O +also O +be O +found O +in O +other O +peptide B-protein_type +hormone I-protein_type +families I-protein_type +( O +Figure O +7 O +). O + +Internal B-site +ribosome I-site +entry I-site +sites I-site +( O +IRESs B-site +) O +mediate O +cap O +- O +independent O +translation O +of O +viral B-taxonomy_domain +mRNAs B-chemical +. O + +Using O +electron B-experimental_method +cryo I-experimental_method +- I-experimental_method +microscopy I-experimental_method +of O +a O +single O +specimen O +, O +we O +present O +five O +ribosome B-complex_assembly +structures B-evidence +formed O +with O +the O +Taura B-species +syndrome I-species +virus I-species +IRES B-site +and O +translocase B-protein_type +eEF2 B-complex_assembly +• I-complex_assembly +GTP I-complex_assembly +bound B-protein_state +with I-protein_state +sordarin B-chemical +. O + +The O +structures B-evidence +suggest O +a O +trajectory O +of O +IRES B-site +translocation O +, O +required O +for O +translation O +initiation B-protein_state +, O +and O +provide O +an O +unprecedented O +view O +of O +eEF2 B-protein +dynamics O +. O + +The O +IRES B-site +rearranges O +from O +extended B-protein_state +to O +bent B-protein_state +to O +extended B-protein_state +conformations O +. O + +This O +inchworm B-protein_state +- O +like O +movement O +is O +coupled O +with O +ribosomal O +inter O +- O +subunit O +rotation O +and O +40S B-complex_assembly +head B-structure_element +swivel O +. O + +eEF2 B-protein +, O +attached O +to O +the O +60S B-complex_assembly +subunit B-structure_element +, O +slides O +along O +the O +rotating O +40S B-complex_assembly +subunit B-structure_element +to O +enter O +the O +A B-site +site I-site +. O + +Its O +diphthamide B-ptm +- O +bearing O +tip O +at O +domain O +IV B-structure_element +separates O +the O +tRNA B-structure_element +- I-structure_element +mRNA I-structure_element +- I-structure_element +like I-structure_element +pseudoknot I-structure_element +I I-structure_element +( O +PKI B-structure_element +) O +of O +the O +IRES B-site +from O +the O +decoding B-site +center I-site +. O + +To O +efficiently O +compete O +with O +host O +mRNAs B-chemical +and O +engage O +in O +translation O +under O +stress O +, O +some O +viral B-taxonomy_domain +mRNAs B-chemical +undergo O +cap O +- O +independent O +translation O +. O + +To O +this O +end O +, O +internal B-site +ribosome I-site +entry I-site +site I-site +( O +IRES B-site +) O +RNAs B-chemical +are O +employed O +( O +reviewed O +in O +. O + +An O +IRES B-site +is O +located O +at O +the O +5 B-structure_element +’ I-structure_element +untranslated I-structure_element +region I-structure_element +of O +the O +viral B-taxonomy_domain +mRNA B-chemical +, O +preceding O +an O +open B-structure_element +reading I-structure_element +frame I-structure_element +( O +ORF B-structure_element +). O + +To O +initiate O +translation O +, O +a O +structured B-protein_state +IRES B-site +RNA B-chemical +interacts O +with O +the O +40S B-complex_assembly +subunit B-structure_element +or O +the O +80S B-complex_assembly +ribosome I-complex_assembly +, O +resulting O +in O +precise O +positioning O +of O +the O +downstream O +start O +codon O +in O +the O +small B-protein_state +40S B-complex_assembly +subunit B-structure_element +. O + +Upon O +peptide O +bond O +formation O +, O +the O +two O +tRNAs B-chemical +and O +their O +respective O +mRNA B-chemical +codons O +translocate O +from O +the O +A B-site +and I-site +P I-site +to O +P B-site +and I-site +E I-site +( I-site +exit I-site +) I-site +sites I-site +, O +freeing O +the O +A B-site +site I-site +for O +the O +next O +elongator O +tRNA B-chemical +. O + +An O +unusual O +strategy O +of O +initiation B-protein_state +is O +used O +by O +intergenic B-structure_element +- I-structure_element +region I-structure_element +( O +IGR B-structure_element +) O +IRESs B-site +found O +in O +Dicistroviridae B-species +arthropod I-species +- O +infecting O +viruses B-taxonomy_domain +. O + +These O +include O +shrimp B-taxonomy_domain +- O +infecting O +Taura B-species +syndrome I-species +virus I-species +( O +TSV B-species +), O +and O +insect B-taxonomy_domain +viruses O +Plautia B-species +stali I-species +intestine I-species +virus I-species +( O +PSIV B-species +) O +and O +Cricket B-species +paralysis I-species +virus I-species +( O +CrPV B-species +). O + +The O +IGR B-structure_element +IRES B-site +mRNAs B-chemical +do O +not O +contain O +an O +AUG O +start O +codon O +. O + +As O +such O +, O +this O +group O +of O +IRESs B-site +represents O +the O +most O +streamlined O +mechanism O +of O +eukaryotic B-taxonomy_domain +translation O +initiation B-protein_state +. O + +Early O +electron B-experimental_method +cryo I-experimental_method +- I-experimental_method +microscopy I-experimental_method +( O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +) O +studies O +have O +found O +that O +the O +CrPV B-species +IRES B-site +packs O +in O +the O +ribosome B-complex_assembly +intersubunit B-site +space I-site +. O + +Recent O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +of O +ribosome B-protein_state +- I-protein_state +bound I-protein_state +TSV B-species +IRES B-site +and O +CrPV B-species +IRES B-site +revealed O +that O +IGR B-structure_element +IRESs B-site +position O +the O +ORF B-structure_element +by O +mimicking O +a O +translating O +ribosome B-complex_assembly +bound B-protein_state +with I-protein_state +tRNA B-chemical +and O +mRNA B-chemical +. O + +The O +~ O +200 O +- O +nt O +IRES B-site +RNAs B-chemical +span O +from O +the O +A B-site +site I-site +beyond O +the O +E B-site +site I-site +. O + +A O +conserved B-protein_state +tRNA B-structure_element +- I-structure_element +mRNA I-structure_element +– I-structure_element +like I-structure_element +structural I-structure_element +element I-structure_element +of O +pseudoknot B-structure_element +I I-structure_element +( O +PKI B-structure_element +) O +interacts O +with O +the O +decoding B-site +center I-site +in O +the O +A B-site +site I-site +of O +the O +40S B-complex_assembly +subunit B-structure_element +. O + +The O +downstream O +initiation O +codon O +— O +coding O +for O +alanine B-residue_name +— O +is O +placed O +in O +the O +mRNA B-site +tunnel I-site +, O +preceding O +the O +decoding B-site +center I-site +. O + +PKI B-structure_element +of O +IGR B-structure_element +IRESs B-site +therefore O +mimics O +an O +A B-site +- I-site +site I-site +elongator O +tRNA B-chemical +interacting O +with O +an O +mRNA B-chemical +sense O +codon O +, O +but O +not O +a O +P B-site +- I-site +site I-site +initiator O +tRNAMet B-chemical +and O +the O +AUG O +start O +codon O +. O + +How O +this O +non O +- O +canonical O +initiation B-protein_state +complex O +transitions O +to O +the O +elongation O +step O +is O +not O +fully O +understood O +. O + +A O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structure B-evidence +of O +the O +ribosome B-complex_assembly +bound B-protein_state +with I-protein_state +a O +CrPV B-species +IRES B-site +and O +release B-protein_type +factor I-protein_type +eRF1 B-protein +occupying O +the O +A B-site +site I-site +provided O +insight O +into O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +state O +. O + +In O +this O +structure B-evidence +, O +PKI B-structure_element +is O +positioned O +in O +the O +P B-site +site I-site +and O +the O +first O +mRNA B-chemical +codon O +is O +located O +in O +the O +A B-site +site I-site +. O + +How O +the O +large B-protein_state +IRES B-site +RNA B-chemical +translocates O +within O +the O +ribosome B-complex_assembly +, O +allowing O +PKI B-structure_element +translocation O +from O +the O +A B-site +to I-site +P I-site +site I-site +is O +not O +known O +. O + +The O +structural O +similarity O +of O +PKI B-structure_element +and O +the O +tRNA B-chemical +anticodon B-structure_element +stem I-structure_element +loop I-structure_element +( O +ASL B-structure_element +) O +bound B-protein_state +to I-protein_state +a O +codon O +suggests O +that O +their O +mechanisms O +of O +translocation O +are O +similar O +to O +some O +extent O +. O + +Translocation O +of O +the O +IRES B-site +or O +tRNA B-complex_assembly +- I-complex_assembly +mRNA I-complex_assembly +requires O +eukaryotic B-taxonomy_domain +elongation B-protein +factor I-protein +2 I-protein +( O +eEF2 B-protein +), O +a O +structural O +and O +functional O +homolog O +of O +the O +well O +- O +studied O +bacterial B-taxonomy_domain +EF B-protein +- I-protein +G I-protein +. O +Pre B-protein_state +- I-protein_state +translocation I-protein_state +tRNA B-protein_state +- I-protein_state +bound I-protein_state +ribosomes B-complex_assembly +contain O +a O +peptidyl B-chemical +- I-chemical +and I-chemical +deacyl I-chemical +- I-chemical +tRNA I-chemical +, O +both O +base O +- O +paired O +to O +mRNA B-chemical +codons O +in O +the O +A B-site +and I-site +P I-site +sites I-site +( O +termed O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +). O + +Translocation O +of O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +involves O +two O +major O +large O +- O +scale O +ribosome B-complex_assembly +rearrangements O +( O +Figure O +1 O +— O +figure O +supplement O +1 O +) O +( O +reviewed O +in O +). O + +First O +, O +studies O +of O +bacterial B-taxonomy_domain +ribosomes B-complex_assembly +showed O +that O +a O +~ O +10 O +° O +rotation O +of O +the O +small B-structure_element +subunit I-structure_element +relative O +to O +the O +large B-structure_element +subunit I-structure_element +, O +known O +as O +intersubunit O +rotation O +, O +or O +ratcheting O +, O +is O +required O +for O +translocation O +. O + +In O +the O +rotated B-protein_state +pre B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +, O +the O +peptidyl B-chemical +- I-chemical +tRNA I-chemical +binds O +the O +A B-site +site I-site +of O +the O +small B-structure_element +subunit I-structure_element +with O +its O +ASL B-structure_element +and O +the O +P B-site +site I-site +of O +the O +large B-structure_element +subunit I-structure_element +with O +the O +CCA O +3 O +’ O +end O +( O +A B-protein_state +/ I-protein_state +P I-protein_state +hybrid I-protein_state +state O +). O + +Concurrently O +, O +the O +deacyl B-chemical +- I-chemical +tRNA I-chemical +interacts O +with O +the O +P B-site +site I-site +of O +the O +small B-structure_element +subunit I-structure_element +and O +the O +E B-site +site I-site +of O +the O +large B-structure_element +subunit I-structure_element +( O +P B-protein_state +/ I-protein_state +E I-protein_state +hybrid I-protein_state +state O +). O + +The O +ribosome B-complex_assembly +can O +undergo O +spontaneous O +, O +thermally O +- O +driven O +forward O +- O +reverse O +rotation O +that O +shifts O +the O +two O +tRNAs B-chemical +between O +the O +hybrid B-protein_state +and O +' O +classical B-protein_state +' O +states O +while O +the O +anticodon B-structure_element +stem I-structure_element +loops I-structure_element +remain O +non B-protein_state +- I-protein_state +translocated I-protein_state +. O + +Binding O +of O +EF B-protein +- I-protein +G I-protein +next O +to O +the O +A B-site +site I-site +and O +reverse O +rotation O +of O +the O +small B-structure_element +subunit I-structure_element +results O +in O +translocation O +of O +both O +ASLs B-structure_element +on O +the O +small B-structure_element +subunit I-structure_element +. O + +EF B-protein +- I-protein +G I-protein +is O +thought O +to O +' O +unlock O +' O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +, O +allowing O +movement O +of O +the O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +, O +however O +the O +structural O +details O +of O +this O +unlocking O +are O +not O +known O +. O + +The O +second O +large O +- O +scale O +rearrangement O +involves O +rotation O +, O +or O +swiveling O +, O +of O +the O +head B-structure_element +of O +the O +small B-structure_element +subunit I-structure_element +relative O +to O +the O +body B-structure_element +. O + +The O +head B-structure_element +can O +rotate O +by O +up O +to O +~ O +20 O +° O +around O +the O +axis O +nearly O +orthogonal O +to O +that O +of O +intersubunit O +rotation O +, O +in O +the O +absence B-protein_state +of I-protein_state +tRNA B-chemical +or O +in O +the O +presence B-protein_state +of I-protein_state +a O +single O +P B-site +/ O +E B-site +tRNA B-chemical +and O +eEF2 B-protein +or O +EF B-protein +- I-protein +G I-protein +. O +Förster B-experimental_method +resonance I-experimental_method +energy I-experimental_method +transfer I-experimental_method +( O +FRET B-experimental_method +) O +data O +suggest O +that O +head B-structure_element +swivel O +of O +the O +rotated B-protein_state +small B-structure_element +subunit I-structure_element +facilitates O +EF B-protein +- I-protein +G I-protein +- O +mediated O +movement O +of O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +. O + +Structures B-evidence +of O +the O +70S B-complex_assembly +• I-complex_assembly +EF I-complex_assembly +- I-complex_assembly +G I-complex_assembly +complex O +bound B-protein_state +with I-protein_state +two O +nearly B-protein_state +translocated I-protein_state +tRNAs B-chemical +, O +exhibit O +a O +large O +18 O +° O +to O +21 O +° O +head B-structure_element +swivel O +in O +a O +mid B-protein_state +- I-protein_state +rotated I-protein_state +subunit B-structure_element +, O +whereas O +no O +head B-structure_element +swivel O +is O +observed O +in O +the O +fully B-protein_state +rotated I-protein_state +pre B-protein_state +- I-protein_state +translocation I-protein_state +or O +in O +the O +non B-protein_state +- I-protein_state +rotated I-protein_state +post B-protein_state +- I-protein_state +translocation I-protein_state +70S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +EF I-complex_assembly +- I-complex_assembly +G I-complex_assembly +structures B-evidence +. O + +The O +structural O +role O +of O +head B-structure_element +swivel O +is O +not O +fully O +understood O +. O + +The O +head B-structure_element +swivel O +was O +proposed O +to O +facilitate O +transition O +of O +the O +tRNA B-chemical +from O +the O +P B-site +to I-site +E I-site +site I-site +by O +widening O +a O +constriction B-site +between O +these O +sites O +on O +the O +30S B-complex_assembly +subunit B-structure_element +. O + +This O +widening O +allows O +the O +ASL B-structure_element +to O +sample O +positions O +between O +the O +P B-site +and I-site +E I-site +sites I-site +. O + +Whether O +and O +how O +the O +head B-structure_element +swivel O +mediates O +tRNA B-chemical +transition O +from O +the O +A B-site +to I-site +P I-site +site I-site +remains O +unknown O +. O + +Comparison O +of O +70S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +and O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +translocation O +complexes O +. O + +The O +large O +ribosomal O +subunit B-structure_element +is O +shown O +in O +cyan O +; O +the O +small B-structure_element +subunit I-structure_element +in O +light O +yellow O +( O +head B-structure_element +) O +and O +wheat O +- O +yellow O +( O +body B-structure_element +), O +elongation B-protein +factor I-protein +G I-protein +( O +EF B-protein +- I-protein +G I-protein +) O +is O +shown O +in O +green O +. O + +Nucleotides O +C1054 B-residue_name_number +, O +G966 B-residue_name_number +and O +G693 B-residue_name_number +of O +16S B-chemical +rRNA I-chemical +are O +shown O +in O +black O +to O +denote O +the O +A B-site +, I-site +P I-site +and I-site +E I-site +sites I-site +, O +respectively O +. O + +The O +extents O +of O +the O +30S B-complex_assembly +subunit B-structure_element +rotation O +and O +head B-structure_element +swivel O +relative O +to O +their O +positions O +in O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +structure B-evidence +are O +shown O +with O +arrows O +. O + +References O +and O +PDB O +codes O +of O +the O +structures B-evidence +are O +shown O +. O + +( O +b O +) O +Structures B-evidence +of O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +complexes O +in O +the O +absence B-protein_state +and O +presence B-protein_state +of I-protein_state +eEF2 B-protein +( O +this O +work O +). O + +Unresolved O +regions O +of O +the O +IRES B-site +in O +densities B-evidence +for O +Structures B-evidence +III I-evidence +and I-evidence +V I-evidence +are O +shown O +in O +gray O +. O + +Schematic O +of O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +refinement O +and O +classification O +procedures O +. O + +All O +particles B-experimental_method +were O +initially O +aligned O +to O +a O +single O +model O +. O + +3D B-experimental_method +classification I-experimental_method +using O +a O +3D B-evidence +mask I-evidence +around O +the O +40S B-complex_assembly +head B-structure_element +, O +TSV B-species +IRES B-site +and O +eEF2 B-protein +, O +of O +the O +4x O +binned O +stack B-bond_interaction +was O +used O +to O +identify O +particles B-experimental_method +containing O +both O +the O +IRES B-site +and O +eEF2 B-protein +. O + +Subsequent O +3D B-experimental_method +classification I-experimental_method +using O +a O +2D B-evidence +mask I-evidence +comprising O +PKI B-structure_element +and O +domain O +IV B-structure_element +of O +eEF2 B-protein +yielded O +5 O +' O +purified O +' O +classes O +representing O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +. O +Sub B-experimental_method +- I-experimental_method +classification I-experimental_method +of O +each O +class O +did O +not O +yield O +additional O +classes O +, O +but O +helped O +improve O +density B-evidence +in O +the O +PKI B-structure_element +region O +of O +class O +III O +( O +estimated O +resolution O +and O +percentage O +of O +particles B-experimental_method +in O +the O +sub B-experimental_method +- I-experimental_method +classified I-experimental_method +reconstruction B-evidence +are O +shown O +in O +parentheses O +). O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +density B-evidence +of O +Structures B-evidence +I I-evidence +- I-evidence +V I-evidence +. O + +The O +maps B-evidence +in O +all O +panels O +were O +B O +- O +softened O +by O +applying O +a O +B O +- O +factor O +of O +30 O +Å2 O +. O + +( O +a O +- O +e O +) O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +map B-evidence +of O +Structures B-evidence +I I-evidence +, I-evidence +II I-evidence +, I-evidence +III I-evidence +, I-evidence +IV I-evidence +and I-evidence +V I-evidence +. O +( O +f O +- O +j O +) O +Local O +resolution O +of O +unfiltered O +and O +unmasked O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +reconstructions B-evidence +, O +assessed O +using O +Blocres B-experimental_method +from O +the O +BSoft O +package O +, O +for O +Structures B-evidence +I I-evidence +, I-evidence +II I-evidence +, I-evidence +III I-evidence +, I-evidence +IV I-evidence +and I-evidence +V I-evidence +. O +( O +k O +- O +o O +) O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +density B-evidence +for O +the O +TSV B-species +IRES B-site +( O +red O +model O +) O +and O +eEF2 B-protein +( O +green O +model O +) O +in O +Structures B-evidence +I I-evidence +, I-evidence +II I-evidence +, I-evidence +III I-evidence +, I-evidence +IV I-evidence +and I-evidence +V I-evidence +. O +( O +p O +) O +Fourier B-evidence +shell I-evidence +correlation I-evidence +( O +FSC B-evidence +) O +curves B-evidence +for O +Structures B-evidence +I I-evidence +- I-evidence +V I-evidence +. O +The O +horizontal O +axis O +is O +labeled O +with O +spatial O +frequency O +Å O +- O +1 O +and O +with O +Å O +. O +The O +resolutions O +stated O +in O +the O +text O +correspond O +to O +an O +FSC B-evidence +threshold O +value O +of O +0 O +. O +143 O +, O +shown O +as O +a O +dotted O +line O +, O +for O +the O +FREALIGN B-experimental_method +- O +derived O +FSC B-evidence +(' O +Part_FSC O +'). O + +Nucleotides O +C1274 B-residue_name_number +, O +U1191 B-residue_name_number +of O +the O +40S B-complex_assembly +head B-structure_element +and O +G904 B-residue_name_number +of O +the O +platform B-site +( O +C1054 B-residue_name_number +, O +G966 B-residue_name_number +and O +G693 B-residue_name_number +in O +E B-species +. I-species +coli I-species +16S B-chemical +rRNA I-chemical +) O +are O +shown O +in O +black O +to O +denote O +the O +A B-site +, I-site +P I-site +and I-site +E I-site +sites I-site +, O +respectively O +. O + +( O +b O +) O +Schematic O +representation O +of O +the O +structures B-evidence +shown O +in O +panel O +a O +, O +denoting O +the O +conformations O +of O +the O +small B-structure_element +subunit I-structure_element +relative O +to O +the O +large B-structure_element +subunit I-structure_element +. O + +A B-site +, I-site +P I-site +and I-site +E I-site +sites I-site +are O +shown O +as O +rectangles O +. O + +All O +measurements O +are O +relative O +to O +the O +non B-protein_state +- I-protein_state +rotated I-protein_state +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +structure B-evidence +. O + +We O +sought O +to O +address O +the O +following O +questions O +by O +structural B-experimental_method +visualization I-experimental_method +of O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +translocation O +complexes O +: O +( O +1 O +) O +How O +does O +a O +large O +IRES B-site +RNA B-chemical +move O +through O +the O +restricted O +intersubunit O +space O +, O +bringing O +PKI B-structure_element +from O +the O +A B-site +to I-site +P I-site +site I-site +of O +the O +small B-structure_element +subunit I-structure_element +? O +( O +2 O +) O +How O +does O +eEF2 B-protein +mediate O +IRES B-site +translocation O +? O +( O +3 O +) O +Does O +IRES B-site +translocation O +involve O +large O +rearrangements O +in O +the O +ribosome B-complex_assembly +, O +similar O +to O +tRNA B-chemical +translocation O +? O +( O +4 O +) O +What O +, O +if O +any O +, O +is O +the O +mechanistic O +role O +of O +40S B-complex_assembly +head B-structure_element +rotation O +in O +IRES B-site +translocation O +? O + +We O +used O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +to O +visualize O +80S B-complex_assembly +• I-complex_assembly +TSV I-complex_assembly +IRES I-complex_assembly +complexes O +formed O +in O +the O +presence B-protein_state +of I-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +GTP I-complex_assembly +and O +the O +translation O +inhibitor O +sordarin B-chemical +, O +which O +stabilizes O +eEF2 B-protein +on O +the O +ribosome B-complex_assembly +. O + +Maximum B-experimental_method +- I-experimental_method +likelihood I-experimental_method +classification I-experimental_method +using O +FREALIGN B-experimental_method +identified O +five O +IRES B-protein_state +- I-protein_state +eEF2 I-protein_state +- I-protein_state +bound I-protein_state +ribosome B-complex_assembly +structures B-evidence +within O +a O +single O +sample O +( O +Figures O +1 O +and O +2 O +). O + +The O +structures B-evidence +differ O +in O +the O +positions O +and O +conformations O +of O +ribosomal O +subunits O +( O +Figures O +1b O +and O +2 O +), O +IRES B-site +RNA B-chemical +( O +Figures O +3 O +and O +4 O +) O +and O +eEF2 B-protein +( O +Figures O +5 O +and O +6 O +). O + +This O +ensemble O +of O +structures B-evidence +allowed O +us O +to O +reconstruct O +a O +sequence O +of O +steps O +in O +IRES B-site +translocation O +induced O +by O +eEF2 B-protein +. O + +We O +used O +single B-experimental_method +- I-experimental_method +particle I-experimental_method +cryo I-experimental_method +- I-experimental_method +EM I-experimental_method +and O +maximum B-experimental_method +- I-experimental_method +likelihood I-experimental_method +image I-experimental_method +classification I-experimental_method +in O +FREALIGN B-experimental_method +to O +obtain O +three O +- O +dimensional O +density B-evidence +maps I-evidence +from O +a O +single O +specimen O +. O + +The O +translocation O +complex O +was O +formed O +using O +S B-species +. I-species +cerevisiae I-species +80S B-complex_assembly +ribosomes I-complex_assembly +, O +Taura B-species +syndrome I-species +virus I-species +IRES B-site +, O +and O +S B-species +. I-species +cerevisiae I-species +eEF2 B-protein +in O +the O +presence B-protein_state +of I-protein_state +GTP B-chemical +and O +the O +eEF2 B-protein +- O +binding O +translation O +inhibitor O +sordarin B-chemical +. O + +This O +approach O +revealed O +five O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +structures B-evidence +at O +average O +resolutions O +of O +3 O +. O +5 O +to O +4 O +. O +2 O +Å O +, O +sufficient O +to O +locate O +IRES B-site +domains O +and O +to O +resolve O +individual O +residues O +in O +the O +core O +regions O +of O +the O +ribosome B-complex_assembly +and O +eEF2 B-protein +( O +Figures O +3c O +, O +d O +, O +and O +5f O +, O +h O +; O +see O +also O +Figure O +1 O +— O +figure O +supplement O +2 O +and O +Figure O +5 O +— O +figure O +supplement O +2 O +), O +including O +the O +post O +- O +translational O +modification O +diphthamide B-ptm +699 I-ptm +( O +Figure O +3c O +). 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O + +( O +b O +) O +Solvent O +view O +( O +opposite O +from O +that O +shown O +in O +( O +a O +)) O +of O +the O +40S B-complex_assembly +subunit B-structure_element +in O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +structure B-evidence +( O +INIT B-complex_assembly +; O +PDB O +3J6Y O +) O +and O +in O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +Structures B-evidence +I I-evidence +, I-evidence +II I-evidence +, I-evidence +III I-evidence +, I-evidence +IV I-evidence +and I-evidence +V I-evidence +( O +this O +work O +). O + +The O +structures B-evidence +are O +colored O +as O +in O +Figure O +1 O +. 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O + +( O +d O +) O +Comparison O +of O +conformations O +of O +the O +L1 B-structure_element +and O +P B-structure_element +stalks I-structure_element +of O +the O +large B-structure_element +subunit I-structure_element +in O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +with O +those O +in O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +and O +tRNA B-protein_state +- I-protein_state +bound I-protein_state +80S B-complex_assembly +structures B-evidence +. O + +Superpositions B-experimental_method +were O +performed O +by O +structural B-experimental_method +alignments I-experimental_method +of O +25S B-chemical +ribosomal I-chemical +RNAs I-chemical +. O + +The O +central B-structure_element +protuberance I-structure_element +( O +CP B-structure_element +) O +is O +labeled O +. 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O + +The O +maximum O +head B-structure_element +swivel O +is O +observed O +in O +the O +mid B-protein_state +- I-protein_state +rotated I-protein_state +complexes O +II B-evidence +and I-evidence +III I-evidence +, O +in O +which O +PKI B-structure_element +transitions O +from O +the O +A B-site +to I-site +P I-site +site I-site +, O +while O +eEF2 B-protein +occupies O +the O +A B-site +site I-site +partially O +. O + +These O +observations O +suggest O +that O +eEF2 B-protein +is O +necessary O +for O +inducing O +or O +stabilizing O +the O +large O +head B-structure_element +swivel O +of O +the O +40S B-complex_assembly +subunit B-structure_element +characteristic O +for O +IRES B-site +translocation O +intermediates O +. O + +IRES B-site +rearrangements O + +Comparison O +of O +the O +TSV B-species +IRES B-site +and O +eEF2 B-protein +positions O +in O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +. 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O + +Positions O +of O +the O +IRES B-site +relative O +to O +proteins O +uS7 B-protein +, O +uS11 B-protein +and O +eS25 B-protein +. O + +( O +a O +) O +Intra O +- O +IRES B-site +rearrangements O +from O +the O +80S B-complex_assembly +* I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +structure B-evidence +( O +INIT B-complex_assembly +; O +PDB O +3J6Y O +,) O +to O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +. O +For O +each O +structure B-evidence +( O +shown O +in O +red O +), O +the O +conformation O +from O +a O +preceding O +structure B-evidence +is O +shown O +in O +light O +red O +for O +comparison O +. O + +Superpositions B-experimental_method +were O +obtained O +by O +structural B-experimental_method +alignments I-experimental_method +of O +18S B-chemical +rRNA I-chemical +. O + +( O +b O +) O +Positions O +of O +the O +IRES B-site +and O +eEF2 B-protein +relative O +to O +those O +of O +classical O +P B-site +- I-site +and I-site +E I-site +- I-site +site I-site +tRNAs B-chemical +in O +the O +80S B-complex_assembly +• I-complex_assembly +tRNA I-complex_assembly +complex O +. O +( O +c O +) O +Positions O +of O +the O +IRES B-site +relative O +to O +proteins O +uS11 B-protein +( O +40S B-site +platform I-site +) O +and O +uS7 B-protein +and O +eS25 B-protein +( O +40S B-complex_assembly +head B-structure_element +), O +which O +interact O +with O +the O +5 B-structure_element +′ I-structure_element +domain I-structure_element +of O +the O +IRES B-site +in O +the O +initiation B-protein_state +state O +( O +left O +panel O +). O + +In O +all O +panels O +, O +superpositions B-experimental_method +were O +obtained O +by O +structural B-experimental_method +alignments I-experimental_method +of O +the O +18S B-chemical +rRNAs I-chemical +. 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O + +2668 B-residue_range +– I-residue_range +2687 I-residue_range +) O +and O +protein O +uL5 B-protein +( O +collectively O +labeled O +as O +central B-structure_element +protuberance I-structure_element +, O +CP B-structure_element +, O +in O +the O +upper O +- O +row O +first O +figure O +, O +and O +individually O +labeled O +in O +the O +lower O +- O +row O +first O +figure O +). O + +Structures B-evidence +of O +translocation O +complexes O +of O +the O +bacterial B-taxonomy_domain +70S B-complex_assembly +ribosome I-complex_assembly +bound B-protein_state +with I-protein_state +two O +tRNAs B-chemical +and O +yeast B-taxonomy_domain +80S B-complex_assembly +complexes B-protein_state +with I-protein_state +tRNAs B-chemical +are O +shown O +in O +the O +upper O +row O +and O +labeled O +. O + +Structures B-evidence +of O +the O +80S B-complex_assembly +complexes B-protein_state +with I-protein_state +tRNAs B-chemical +are O +shown O +in O +the O +lower O +row O +in O +a O +view O +similar O +to O +that O +for O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +complex O +. O + +( O +a O +) O +Secondary O +structure B-evidence +of O +the O +TSV B-species +IRES B-site +. O + +The O +TSV B-species +IRES B-site +comprises O +two O +domains O +: O +the O +5 B-structure_element +' I-structure_element +domain I-structure_element +( O +blue O +) O +and O +the O +PKI B-structure_element +domain O +( O +red O +). O + +The O +open B-structure_element +reading I-structure_element +frame I-structure_element +( O +gray O +) O +is O +immediately O +following O +pseudoknot B-structure_element +I I-structure_element +( O +PKI B-structure_element +). O + +( O +b O +) O +Three O +- O +dimensional O +structure B-evidence +of O +the O +TSV B-species +IRES B-site +( O +Structure B-evidence +II I-evidence +). O + +( O +c O +) O +Positions O +of O +the O +IRES B-site +and O +eEF2 B-protein +on O +the O +small B-structure_element +subunit I-structure_element +in O +Structures B-evidence +I I-evidence +to I-evidence +V I-evidence +. O +The O +initiation B-protein_state +- O +state O +IRES B-site +is O +shown O +in O +gray O +. O + +The O +insert O +shows O +density O +for O +interaction O +of O +diphthamide O +699 O +( B-protein +eEF2 O +; O +green O +) O +with O +the O +codon O +- O +anticodon O +- O +like O +helix O +( B-structure_element +PKI O +; O +red O +) O +in O +Structure O +V O +. O +( O +d O +and O +e O +) O +Density O +of O +the O +P O +site O +in O +Structure O +V O +shows O +that O +interactions O +of O +PKI O +with O +the O +18S O +rRNA O +nucleotides O +( O +c O +) O +are O +nearly O +identical O +to O +those O +in O +the O +P O +site O +of O +the O +2tRNA O +• O +mRNA O +- O +bound O +70S O +ribosome O +( O +d O +). O + +In O +each O +structure B-evidence +, O +the O +TSV B-species +IRES B-site +adopts O +a O +distinct O +conformation O +in O +the O +intersubunit O +space O +of O +the O +ribosome B-complex_assembly +( O +Figures O +3 O +and O +4 O +). O + +The O +IRES B-site +( O +nt O +6758 B-residue_range +– I-residue_range +6952 I-residue_range +) O +consists O +of O +two O +globular O +parts O +( O +Figure O +3a O +): O +the O +5 B-structure_element +’- I-structure_element +region I-structure_element +( O +domains O +I B-structure_element +and O +II B-structure_element +, O +nt O +6758 B-residue_range +– I-residue_range +6888 I-residue_range +) O +and O +the O +PKI B-structure_element +domain O +( O +domain O +III B-structure_element +, O +nt O +6889 B-residue_range +– I-residue_range +6952 I-residue_range +). O + +The O +PKI B-structure_element +domain O +comprises O +PKI B-structure_element +and O +stem B-structure_element +loop I-structure_element +3 I-structure_element +( O +SL3 B-structure_element +), O +which O +stacks O +on O +top O +of O +the O +stem O +of O +PKI B-structure_element +. O + +The O +6953GCU O +triplet O +immediately O +following O +the O +PKI B-structure_element +domain O +is O +the O +first O +codon O +of O +the O +open B-structure_element +reading I-structure_element +frame I-structure_element +. O + +In O +the O +eEF2 B-protein_state +- I-protein_state +free I-protein_state +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +complex O +( O +INIT B-complex_assembly +), O +the O +bulk O +of O +the O +5 B-structure_element +’- I-structure_element +domain I-structure_element +( O +nt O +. O + +In O +Structures B-evidence +I I-evidence +to I-evidence +IV I-evidence +, O +these O +contacts O +remain O +as O +in O +the O +initiation B-complex_assembly +complex I-complex_assembly +( O +Figure O +1a O +). O + +Specifically O +, O +the O +L1 B-structure_element +. I-structure_element +1 I-structure_element +region I-structure_element +interacts O +with O +the O +L1 B-structure_element +stalk I-structure_element +of O +the O +large B-structure_element +subunit I-structure_element +, O +while O +SL4 B-structure_element +and O +SL5 B-structure_element +bind O +at O +the O +side O +of O +the O +40S B-complex_assembly +head B-structure_element +and O +interact O +with O +proteins O +uS7 B-protein +, O +uS11 B-protein +and O +eS25 B-protein +( O +Figure O +3 O +— O +figure O +supplement O +2 O +and O +Figure O +3 O +— O +figure O +supplement O +3 O +; O +ribosomal O +proteins O +are O +termed O +according O +to O +). O + +In O +Structures B-evidence +I I-evidence +- I-evidence +IV I-evidence +, O +the O +minor B-site +groove I-site +of O +SL4 B-structure_element +( O +at O +nt O +6840 B-residue_range +– I-residue_range +6846 I-residue_range +) O +binds O +next O +to O +an O +α B-structure_element +- I-structure_element +helix I-structure_element +of O +uS7 B-protein +, O +which O +is O +rich O +in O +positively O +charged O +residues O +( O +K212 B-residue_name_number +, O +K213 B-residue_name_number +, O +R219 B-residue_name_number +and O +K222 B-residue_name_number +). O + +The O +tip O +of O +SL4 B-structure_element +binds O +in O +the O +vicinity O +of O +R157 B-residue_name_number +in O +the O +β B-structure_element +- I-structure_element +hairpin I-structure_element +of O +uS7 B-protein +and O +of O +Y58 B-residue_name_number +in O +uS11 B-protein +. O + +In O +Structure B-evidence +V I-evidence +, O +however O +, O +the O +density B-evidence +for O +SL5 B-structure_element +is O +missing O +suggesting O +that O +SL5 B-structure_element +is O +mobile B-protein_state +, O +while O +weak O +SL4 B-structure_element +density B-evidence +suggests O +that O +SL4 B-structure_element +is O +shifted O +along O +the O +surface O +of O +uS7 B-protein +, O +~ O +20 O +Å O +away O +from O +its O +initial O +position O +( O +Figure O +3 O +— O +figure O +supplement O +2c O +). O + +The O +L1 B-structure_element +. I-structure_element +1 I-structure_element +region I-structure_element +remains O +in O +contact O +with O +the O +L1 B-structure_element +stalk I-structure_element +( O +Figure O +3 O +— O +figure O +supplement O +3 O +). O + +Inchworm B-protein_state +- O +like O +translocation O +of O +the O +TSV B-species +IRES B-site +. O + +The O +shape O +of O +the O +IRES B-site +changes O +considerably O +from O +the O +initiation B-protein_state +state O +to O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +, O +from O +an O +extended B-protein_state +to O +compact B-protein_state +to O +extended B-protein_state +conformation O +( O +Figure O +4 O +; O +see O +also O +Figure O +3 O +— O +figure O +supplement O +2a O +). O + +Because O +in O +Structures B-evidence +I I-evidence +to I-evidence +IV I-evidence +the O +PKI B-structure_element +domain O +shifts O +toward O +the O +P B-site +site I-site +, O +while O +the O +5 O +’ O +remains O +unchanged O +near O +the O +E B-site +site I-site +, O +the O +distance O +between O +the O +domains O +shortens O +( O +Figure O +4 O +). O + +In O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +state O +, O +the O +A B-protein_state +- I-protein_state +site I-protein_state +- I-protein_state +bound I-protein_state +PKI B-structure_element +is O +separated O +from O +SL4 B-structure_element +by O +almost O +50 O +Å O +( O +Figure O +4 O +). O + +In O +Structures B-evidence +I I-evidence +and I-evidence +II I-evidence +, O +the O +PKI B-structure_element +is O +partially O +retracted O +from O +the O +A B-site +site I-site +and O +the O +distance O +from O +SL4 B-structure_element +shortens O +to O +~ O +35 O +Å O +. O +As O +PKI B-structure_element +moves O +toward O +the O +P B-site +site I-site +in O +Structures B-evidence +III I-evidence +and I-evidence +IV I-evidence +, O +the O +PKI B-structure_element +domain O +approaches O +to O +within O +~ O +25 O +Å O +of O +SL4 B-structure_element +. O + +Because O +the O +5 B-structure_element +’- I-structure_element +domain I-structure_element +in O +the O +following O +structure B-evidence +( I-evidence +V I-evidence +) I-evidence +moves O +by O +~ O +20 O +Å O +along O +the O +40S B-complex_assembly +head B-structure_element +, O +the O +IRES B-site +returns O +to O +an O +extended B-protein_state +conformation O +(~ O +45 O +Å O +) O +that O +is O +similar O +to O +that O +in O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +complex O +. O + +2668 B-residue_range +– I-residue_range +2687 I-residue_range +) O +and O +protein O +uL5 B-protein +( O +Figure O +3 O +— O +figure O +supplement O +6 O +). O + +This O +position O +of O +SL3 B-structure_element +is O +~ O +25 O +Å O +away O +from O +that O +in O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +state O +, O +in O +which O +PKI B-structure_element +and O +SL3 B-structure_element +closely O +mimic O +the O +ASL B-structure_element +and O +elbow B-structure_element +of O +the O +A B-site +- I-site +site I-site +tRNA B-chemical +, O +respectively O +. O + +As O +such O +, O +the O +transition O +from O +the O +initiation B-protein_state +state O +to O +Structure B-evidence +I I-evidence +involves O +repositioning O +of O +SL3 B-structure_element +around O +the O +A B-structure_element +- I-structure_element +site I-structure_element +finger I-structure_element +, O +resembling O +the O +transition O +between O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +A B-site +/ I-site +P I-site +and O +A B-site +/ I-site +P I-site +* I-site +tRNA B-chemical +. O + +The O +second O +set O +of O +major O +structural O +changes O +involves O +interaction O +of O +the O +P B-site +site I-site +region I-site +of O +the O +large B-structure_element +subunit I-structure_element +with O +the O +hinge B-structure_element +point I-structure_element +of O +the O +IRES B-site +bending O +between O +the O +5 B-structure_element +´ I-structure_element +domain I-structure_element +and O +the O +PKI B-structure_element +domain O +( O +nt O +. O +6886 B-residue_range +– I-residue_range +6890 I-residue_range +). O + +In O +the O +highly B-protein_state +bent I-protein_state +Structures B-evidence +III I-evidence +and I-evidence +IV I-evidence +, O +the O +hinge B-structure_element +region I-structure_element +interacts O +with O +the O +universally B-protein_state +conserved I-protein_state +uL5 B-protein +and O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +tail I-structure_element +of O +eL42 B-protein +( O +Figure O +3 O +— O +figure O +supplement O +7 O +). O + +Another O +local O +rearrangement O +concerns O +loop B-structure_element +3 I-structure_element +, O +also O +known O +as O +the O +variable B-structure_element +loop I-structure_element +region I-structure_element +, O +which O +connects O +the O +ASL B-structure_element +- I-structure_element +and I-structure_element +mRNA I-structure_element +- I-structure_element +like I-structure_element +parts I-structure_element +of O +PKI B-structure_element +. O + +This O +loop B-structure_element +is O +poorly O +resolved O +in O +Structures B-evidence +I I-evidence +through I-evidence +IV I-evidence +, O +suggesting O +conformational O +flexibility O +in O +agreement O +with O +structural B-experimental_method +studies I-experimental_method +of O +the O +isolated B-protein_state +PKI B-structure_element +and O +biochemical B-experimental_method +studies I-experimental_method +of O +unbound B-protein_state +IRESs B-site +. O + +6945 B-residue_range +– I-residue_range +6946 I-residue_range +) O +interacts O +with O +R148 B-residue_name_number +and O +R157 B-residue_name_number +in O +β B-structure_element +- I-structure_element +hairpin I-structure_element +of O +uS7 B-protein +. O + +This O +interpretation O +is O +consistent O +with O +the O +recent O +observation O +that O +alterations O +in O +loop B-structure_element +3 I-structure_element +of O +the O +CrPV B-species +IRES B-site +result O +in O +decreased O +efficiency O +of O +translocation O +. O + +eEF2 B-protein +structures B-evidence + +Elements O +of O +the O +80S B-complex_assembly +ribosome I-complex_assembly +that O +contact O +eEF2 B-protein +in O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +. O + +The O +view O +and O +colors O +are O +as O +in O +Figure O +5b O +: O +eEF2 B-protein +is O +shown O +in O +green O +, O +IRES B-site +RNA B-chemical +in O +red O +, O +40S B-complex_assembly +subunit B-structure_element +elements O +in O +orange O +, O +60S B-complex_assembly +in O +cyan O +/ O +teal O +. O + +The O +switch B-structure_element +loop I-structure_element +I I-structure_element +in O +Structure B-evidence +I I-evidence +is O +shown O +in O +blue O +. O + +The O +putative O +position O +of O +the O +switch B-structure_element +loop I-structure_element +I I-structure_element +, O +unresolved O +in O +the O +density B-evidence +of O +Structure B-evidence +II I-evidence +, O +is O +shown O +with O +a O +dashed O +line O +. O + +Conformations O +and O +interactions O +of O +eEF2 B-protein +. O + +( O +a O +) O +Conformations O +of O +eEF2 B-protein +in O +Structures B-evidence +I I-evidence +- I-evidence +V I-evidence +and O +domain O +organization O +of O +eEF2 B-protein +are O +shown O +. O + +Roman O +numerals O +denote O +eEF2 B-protein +domains O +. O + +Superposition B-experimental_method +was O +obtained O +by O +structural B-experimental_method +alignment I-experimental_method +of O +domains O +I B-structure_element +and O +II B-structure_element +. O + +( O +b O +) O +Elements O +of O +the O +80S B-complex_assembly +ribosome I-complex_assembly +in O +Structures B-evidence +I I-evidence +and I-evidence +V I-evidence +that O +contact O +eEF2 B-protein +. O + +eEF2 B-protein +is O +shown O +in O +green O +, O +IRES B-site +RNA B-chemical +in O +red O +, O +40S B-complex_assembly +subunit B-structure_element +elements O +in O +orange O +, O +60S B-complex_assembly +in O +cyan O +/ O +teal O +. O + +( O +c O +) O +Comparison O +of O +conformations O +of O +eEF2 B-complex_assembly +• I-complex_assembly +sordarin I-complex_assembly +in O +Structure B-evidence +I I-evidence +( O +light O +green O +) O +with O +those O +of O +free B-protein_state +apo B-protein_state +- O +eEF2 B-protein +( O +magenta O +) O +and O +eEF2 B-complex_assembly +• I-complex_assembly +sordarin I-complex_assembly +( O +teal O +). O + +( O +d O +) O +Interactions O +of O +the O +GTPase B-structure_element +domains I-structure_element +with O +the O +40S B-complex_assembly +and O +60S B-complex_assembly +subunits B-structure_element +in O +Structure B-evidence +I I-evidence +( O +colored O +in O +green O +/ O +blue O +, O +eEF2 B-protein +; O +orange O +, O +40S B-complex_assembly +; O +cyan O +/ O +teal O +, O +60S B-complex_assembly +) O +and O +in O +Structure B-evidence +II I-evidence +( O +gray O +). O + +( O +e O +) O +Comparison O +of O +the O +GTP B-protein_state +- I-protein_state +like I-protein_state +conformation O +of O +eEF2 B-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +in O +Structure B-evidence +I I-evidence +( O +light O +green O +) O +with O +those O +of O +70S B-protein_state +- I-protein_state +bound I-protein_state +elongation B-protein_type +factors I-protein_type +EF B-complex_assembly +- I-complex_assembly +Tu I-complex_assembly +• I-complex_assembly +GDPCP I-complex_assembly +( O +teal O +) O +and O +EF B-complex_assembly +- I-complex_assembly +G I-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +• I-complex_assembly +fusidic I-complex_assembly +acid I-complex_assembly +( O +magenta O +; O +fusidic O +acid O +not O +shown O +). O +( O +f O +) O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +density B-evidence +showing O +guanosine B-chemical +diphosphate I-chemical +bound B-protein_state +in I-protein_state +the O +GTPase B-site +center I-site +( O +green O +) O +next O +to O +the O +sarcin B-structure_element +- I-structure_element +ricin I-structure_element +loop I-structure_element +of O +25S B-chemical +rRNA I-chemical +( O +cyan O +) O +of O +Structure B-evidence +II I-evidence +. O +( O +g O +) O +Comparison O +of O +the O +sordarin B-site +- I-site +binding I-site +sites I-site +in O +the O +ribosome B-protein_state +- I-protein_state +bound I-protein_state +( O +light O +green O +; O +Structure B-evidence +II I-evidence +) O +and O +isolated O +eEF2 B-protein +( O +teal O +). O + +( O +h O +) O +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +density B-evidence +showing O +the O +sordarin B-site +- I-site +binding I-site +pocket I-site +of O +eEF2 B-protein +( O +Structure B-evidence +II I-evidence +). O + +The O +elongation B-protein_type +factor I-protein_type +consists O +of O +three O +dynamic O +superdomains B-structure_element +: O +an O +N O +- O +terminal O +globular O +superdomain B-structure_element +formed O +by O +the O +G B-structure_element +( I-structure_element +GTPase I-structure_element +) I-structure_element +domain I-structure_element +( O +domain O +I B-structure_element +) O +and O +domain O +II B-structure_element +; O +a O +linker B-structure_element +domain I-structure_element +III I-structure_element +; O +and O +a O +C O +- O +terminal O +superdomain B-structure_element +comprising O +domains O +IV B-structure_element +and O +V B-structure_element +( O +Figure O +5a O +). O + +Domain O +IV B-structure_element +extends O +from O +the O +main O +body B-structure_element +and O +is O +critical O +for O +translocation O +catalyzed O +by O +eEF2 B-protein +or O +EF B-protein +- I-protein +G I-protein +. O +ADP B-ptm +- I-ptm +ribosylation I-ptm +of O +eEF2 B-protein +at O +the O +tip O +of O +domain O +IV B-structure_element +or O +deletion B-experimental_method +of O +domain O +IV B-structure_element +from O +EF B-protein +- I-protein +G I-protein +abrogate O +translocation O +. O + +In O +post B-protein_state +- I-protein_state +translocation I-protein_state +- O +like O +80S B-complex_assembly +• I-complex_assembly +tRNA I-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +complexes O +, O +domain O +IV B-structure_element +binds O +in O +the O +40S B-complex_assembly +A B-site +site I-site +, O +suggesting O +direct O +involvement O +of O +domain O +IV B-structure_element +in O +translocation O +of O +tRNA B-chemical +from O +the O +A B-site +to I-site +P I-site +site I-site +. O + +The O +global O +conformations O +of O +eEF2 B-protein +( O +Figure O +5a O +) O +are O +similar O +in O +these O +structures B-evidence +( O +all O +- O +atom O +RMSD B-evidence +≤ O +2 O +Å O +), O +but O +the O +positions O +of O +eEF2 B-protein +relative O +to O +the O +40S B-complex_assembly +subunit B-structure_element +differ O +substantially O +as O +a O +result O +of O +40S B-complex_assembly +subunit B-structure_element +rotation O +( O +Figure O +2 O +— O +source O +data O +1 O +). O + +Although O +the O +P B-structure_element +/ I-structure_element +L11 I-structure_element +stalk I-structure_element +is O +known O +to O +be O +dynamic O +, O +its O +position O +remains O +unchanged O +from O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +: O +all O +- O +atom O +root B-evidence +- I-evidence +mean I-evidence +- I-evidence +square I-evidence +differences I-evidence +for O +the O +25S B-chemical +rRNA I-chemical +of O +the O +P B-structure_element +stalk I-structure_element +( O +nt O +1223 B-residue_range +– I-residue_range +1286 I-residue_range +) O +are O +within O +2 O +. O +5 O +Å O +. O +However O +, O +with O +respect O +to O +its O +position O +in O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +complex O +in O +the O +absence B-protein_state +of I-protein_state +eEF2 B-protein +and O +in O +the O +80S B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +, O +the O +P B-structure_element +stalk I-structure_element +is O +shifted O +by O +~ O +13 O +Å O +toward O +the O +A B-site +site I-site +( O +Figure O +2d O +). O + +The O +sarcin B-structure_element +- I-structure_element +ricin I-structure_element +loop I-structure_element +interacts O +with O +the O +GTP B-site +- I-site +binding I-site +site I-site +of O +eEF2 B-protein +( O +Figures O +5d O +and O +f O +). O + +Repositioning O +( O +sliding O +) O +of O +the O +positively B-site +- I-site +charged I-site +cluster I-site +of O +domain O +IV B-structure_element +of O +eEF2 B-protein +over O +the O +phosphate O +backbone O +( O +red O +) O +of O +the O +18S B-structure_element +helices I-structure_element +33 I-structure_element +and I-structure_element +34 I-structure_element +. O + +Electrostatic O +surface O +of O +eEF2 B-protein +is O +shown O +; O +negatively O +and O +positively O +charged O +regions O +are O +shown O +in O +red O +and O +blue O +, O +respectively O +. O + +Interactions O +of O +eEF2 B-protein +with O +the O +40S B-complex_assembly +subunit B-structure_element +. O + +( O +c O +) O +Rearrangements O +, O +from O +Structure B-evidence +I I-evidence +through I-evidence +V I-evidence +, O +of O +a O +positively O +charged O +cluster O +of O +eEF2 B-protein +( O +K613 B-residue_name_number +, O +R617 B-residue_name_number +and O +R631 B-residue_name_number +) O +positioned O +over O +the O +phosphate O +backbone O +of O +18S B-structure_element +helices I-structure_element +33 I-structure_element +and I-structure_element +34 I-structure_element +, O +suggesting O +a O +role O +of O +electrostatic B-bond_interaction +interactions I-bond_interaction +in O +eEF2 B-protein +diffusion O +over O +the O +40S B-complex_assembly +surface O +. O + +( O +d O +) O +Shift O +of O +the O +tip O +of O +domain O +III B-structure_element +of O +eEF2 B-protein +, O +interacting O +with O +uS12 B-protein +upon O +reverse O +subunit B-structure_element +rotation O +from O +Structure B-evidence +I I-evidence +to I-evidence +Structure I-evidence +V I-evidence +. O +Structure B-evidence +I I-evidence +colored O +as O +in O +Figure O +1 O +, O +except O +uS12 B-protein +, O +which O +is O +in O +purple O +; O +Structure B-evidence +V I-evidence +is O +in O +gray O +. O + +This O +limited O +flexibility O +of O +the O +ribosome B-protein_state +- I-protein_state +bound I-protein_state +eEF2 B-protein +is O +likely O +the O +result O +of O +simultaneous O +fixation O +of O +eEF2 B-protein +superdomains B-structure_element +, O +via O +domains O +I B-structure_element +and O +V B-structure_element +, O +by O +the O +GTPase B-site +- I-site +associated I-site +center I-site +of O +the O +large B-structure_element +subunit I-structure_element +. O + +eEF2 B-protein +settles O +into O +the O +A B-site +site I-site +from O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +, O +as O +the O +tip O +of O +domain O +IV B-structure_element +shifts O +by O +~ O +10 O +Å O +relative O +to O +the O +body B-structure_element +and O +by O +~ O +20 O +Å O +relative O +to O +the O +swiveling O +head B-structure_element +. O + +At O +the O +central O +region O +of O +eEF2 B-protein +, O +domains O +II B-structure_element +and O +III B-structure_element +contact O +the O +40S B-complex_assembly +body B-structure_element +( O +mainly O +at O +nucleotides O +48 B-residue_range +– I-residue_range +52 I-residue_range +and O +429 B-residue_range +– I-residue_range +432 I-residue_range +of O +18S B-chemical +rRNA I-chemical +helix B-structure_element +5 I-structure_element +and O +uS12 B-protein +). O + +From O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +, O +these O +central O +domains O +migrate O +by O +~ O +10 O +Å O +along O +the O +40S B-complex_assembly +surface O +( O +Figure O +6c O +). O + +Comparison O +of O +eEF2 B-protein +conformations O +reveals O +that O +in O +Structure B-evidence +V I-evidence +, O +domain O +III B-structure_element +is O +displaced O +as O +a O +result O +of O +interaction O +with O +uS12 B-protein +, O +as O +discussed O +below O +. O + +In O +summary O +, O +between O +Structures B-evidence +I I-evidence +and I-evidence +V I-evidence +, O +a O +step O +- O +wise O +translocation O +of O +PKI B-structure_element +by O +~ O +15 O +Å O +from O +the O +A B-site +to I-site +P I-site +site I-site +- O +within O +the O +40S B-complex_assembly +subunit B-structure_element +– O +occurs O +simultaneously O +with O +the O +~ O +11 O +Å O +side O +- O +way O +entry O +of O +domain O +IV B-structure_element +into O +the O +A B-site +site I-site +coupled O +with O +~ O +3 O +to O +5 O +Å O +inter O +- O +domain O +rearrangements O +in O +eEF2 B-protein +. O + +These O +shifts O +occur O +during O +the O +reverse O +rotation O +of O +the O +40S B-complex_assembly +body B-structure_element +coupled O +with O +the O +forward O +- O +then O +- O +reverse O +head B-structure_element +swivel O +. O + +To O +elucidate O +the O +detailed O +structural O +mechanism O +of O +IRES B-site +translocation O +and O +the O +roles O +of O +eEF2 B-protein +and O +ribosome B-complex_assembly +rearrangements O +, O +we O +describe O +in O +the O +following O +sections O +the O +interactions O +of O +PKI B-structure_element +and O +eEF2 B-protein +with O +the O +ribosomal O +A B-site +and I-site +P I-site +sites I-site +in O +Structures B-evidence +I I-evidence +through I-evidence +V I-evidence +( O +Figure O +2g O +; O +see O +also O +Figure O +1 O +— O +figure O +supplement O +1 O +). O + +Structure B-evidence +I I-evidence +represents O +a O +pre B-protein_state +- I-protein_state +translocation I-protein_state +IRES B-site +and O +initial O +entry O +of O +eEF2 B-protein +in O +a O +GTP B-chemical +- O +like O +state O + +In O +the O +fully B-protein_state +rotated I-protein_state +Structure B-evidence +I I-evidence +, O +PKI B-structure_element +is O +shifted O +toward O +the O +P B-site +site I-site +by O +~ O +3 O +Å O +relative O +to O +its O +position O +in O +the O +initiation B-complex_assembly +complex I-complex_assembly +but O +maintains O +interactions O +with O +the O +partially B-protein_state +swiveled I-protein_state +head B-structure_element +. O + +The O +C1274 B-residue_name_number +: O +G6953 B-residue_name_number +base O +pair O +provides O +a O +stacking B-site +platform I-site +for O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +– I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +. O + +Interactions O +of O +the O +residues O +at O +the O +eEF2 B-protein +tip O +with O +the O +decoding B-site +center I-site +of O +the O +IRES B-protein_state +- I-protein_state +bound I-protein_state +ribosome B-complex_assembly +. O + +The O +histidine B-site +- I-site +diphthamide I-site +tip I-site +of O +eEF2 B-protein +is O +shown O +in O +green O +. O + +A B-site +and I-site +P I-site +sites I-site +are O +schematically O +demarcated O +by O +dotted O +lines O +. O + +In O +Structure B-evidence +I I-evidence +, O +PKI B-structure_element +does O +not O +contact O +these O +nucleotides O +( O +Figures O +2g O +and O +7 O +). O + +The O +position O +of O +eEF2 B-protein +on O +the O +40S B-complex_assembly +subunit B-structure_element +of O +Structure B-evidence +I I-evidence +is O +markedly O +distinct O +from O +those O +in O +Structures B-evidence +II I-evidence +to I-evidence +V I-evidence +. O +The O +translocase B-protein_type +interacts O +with O +the O +40S B-complex_assembly +body B-structure_element +but O +does O +not O +contact O +the O +head B-structure_element +( O +Figures O +5b O +and O +6a O +; O +Figure O +5 O +— O +figure O +supplement O +1 O +). O + +Domain O +IV B-structure_element +is O +partially O +engaged O +with O +the O +body B-structure_element +A B-site +site I-site +. O + +The O +tip O +of O +domain O +IV B-structure_element +is O +wedged O +between O +PKI B-structure_element +and O +decoding B-site +- I-site +center I-site +nucleotides O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +, O +which O +are O +bulged O +out O +of O +h44 O +. O + +This O +tip O +contains O +the O +histidine B-site +- I-site +diphthamide I-site +triad I-site +( O +H583 B-residue_name_number +, O +H694 B-residue_name_number +and O +Diph699 B-ptm +), O +which O +interacts O +with O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +- I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +and O +A1756 B-residue_name_number +( O +Figure O +7 O +). O + +The O +trimethylamino O +end O +of O +Diph699 B-ptm +packs O +over O +A1756 B-residue_name_number +( O +Figure O +7 O +). O + +The O +opposite O +surface O +of O +the O +tail O +is O +oriented O +toward O +the O +minor B-site +- I-site +groove I-site +side O +of O +the O +second O +base O +pair O +of O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +helix I-structure_element +( O +G6906 B-residue_name_number +: O +C6951 B-residue_name_number +). O + +Thus O +, O +in O +comparison O +with O +the O +initiation B-protein_state +state O +, O +the O +histidine B-site +- I-site +diphthamide I-site +tip I-site +of O +eEF2 B-protein +replaces O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +– I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +. O + +The O +splitting O +of O +the O +interaction O +of O +A1755 B-residue_name_number +- O +A1756 B-residue_name_number +and O +PKI B-structure_element +is O +achieved O +by O +providing O +the O +histidine B-site +- I-site +diphthamine I-site +tip I-site +as O +a O +binding O +partner O +for O +both O +A1756 B-residue_name_number +and O +the O +minor B-site +groove I-site +of O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +helix I-structure_element +( O +Figure O +7 O +). O + +Unlike O +in O +Structures B-evidence +II I-evidence +to I-evidence +V I-evidence +, O +the O +conformation O +of O +the O +eEF2 B-protein +GTPase B-site +center I-site +in O +Structure B-evidence +I I-evidence +resembles O +that O +of O +a O +GTP B-protein_state +- I-protein_state +bound I-protein_state +translocase B-protein_type +( O +Figure O +5e O +). O + +In O +translational B-protein_type +GTPases I-protein_type +, O +switch B-structure_element +loops I-structure_element +I I-structure_element +and I-structure_element +II I-structure_element +are O +involved O +in O +the O +GTPase B-protein_type +activity O +( O +reviewed O +in O +). O + +The O +histidine B-residue_name +resides O +next O +to O +the O +backbone O +of O +G3028 B-residue_name_number +of O +the O +sarcin B-structure_element +- I-structure_element +ricin I-structure_element +loop I-structure_element +and O +near O +the O +diphosphate O +of O +GDP B-chemical +( O +Figure O +5e O +). O + +By O +contrast O +, O +switch B-structure_element +loop I-structure_element +I I-structure_element +( O +aa O +50 B-residue_range +– I-residue_range +70 I-residue_range +in O +S B-species +. I-species +cerevisiae I-species +eEF2 B-protein +) O +is O +resolved O +only O +in O +Structure B-evidence +I I-evidence +( O +Figure O +5 O +— O +figure O +supplement O +2 O +). O + +Structure B-evidence +II I-evidence +reveals O +PKI B-structure_element +between O +the O +body B-structure_element +A B-site +and I-site +P I-site +sites I-site +and O +eEF2 B-protein +partially O +advanced O +into O +the O +A B-site +site I-site + +In O +Structure B-evidence +II I-evidence +, O +relative O +to O +Structure B-evidence +I I-evidence +, O +PKI B-structure_element +is O +further O +shifted O +along O +the O +40S B-complex_assembly +body B-structure_element +, O +traversing O +~ O +4 O +Å O +toward O +the O +P B-site +site I-site +( O +Figures O +2e O +, O +f O +, O +and O +g O +), O +while O +stacking B-bond_interaction +on O +C1274 B-residue_name_number +at O +the O +head B-structure_element +A B-site +site I-site +. O + +Thus O +, O +the O +intermediate O +position O +of O +PKI B-structure_element +is O +possible O +due O +to O +a O +large O +swivel O +of O +the O +head B-structure_element +relative O +to O +the O +body B-structure_element +, O +which O +brings O +the O +head B-structure_element +A B-site +site I-site +close O +to O +the O +body B-structure_element +P B-site +site I-site +. O + +Domain O +IV B-structure_element +of O +eEF2 B-protein +is O +further O +entrenched O +in O +the O +A B-site +site I-site +by O +~ O +3 O +Å O +relative O +to O +the O +body B-structure_element +and O +~ O +8 O +Å O +relative O +to O +the O +head B-structure_element +, O +preserving O +its O +interactions O +with O +PKI B-structure_element +. O + +The O +decoding B-site +center I-site +residues O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +are O +rearranged O +to O +pack O +inside O +helix B-structure_element +44 I-structure_element +, O +making O +room O +for O +eEF2 B-protein +. O + +This O +conformation O +of O +decoding B-site +center I-site +residues O +is O +also O +observed O +in O +the O +absence B-protein_state +of I-protein_state +A B-site +- I-site +site I-site +ligands O +. O + +Here O +, O +a O +positively B-site +charged I-site +surface I-site +of O +eEF2 B-protein +, O +formed O +by O +K613 B-residue_name_number +, O +R617 B-residue_name_number +and O +R631 B-residue_name_number +contacts O +the O +phosphate O +backbone O +of O +helix B-structure_element +33 I-structure_element +( O +Figures O +6c O +; O +see O +also O +Figure O +6 O +— O +figure O +supplement O +1 O +). O + +Consistent O +with O +the O +similar O +head B-structure_element +swivels O +in O +Structure B-evidence +III I-evidence +and O +Structure B-evidence +II I-evidence +, O +relative O +positions O +of O +the O +40S B-complex_assembly +head B-structure_element +A B-site +site I-site +and O +body B-structure_element +P B-site +site I-site +remain O +as O +in O +Structure B-evidence +II I-evidence +. O + +The O +map B-evidence +allows O +placement O +of O +PKI B-structure_element +at O +the O +body B-structure_element +P B-site +site I-site +( O +Figure O +1 O +— O +figure O +supplement O +3 O +). O + +Lower O +resolution O +of O +the O +map B-evidence +in O +this O +region O +suggests O +that O +PKI B-structure_element +is O +somewhat O +destabilized O +in O +the O +vicinity O +of O +the O +body B-structure_element +P B-site +site I-site +in O +the O +absence B-protein_state +of I-protein_state +stacking B-bond_interaction +with O +the O +foundations O +of O +the O +head B-structure_element +A B-site +site I-site +( O +C1274 B-residue_name_number +) O +or O +P B-site +site I-site +( O +U1191 B-residue_name_number +). O + +Structure B-evidence +IV I-evidence +represents O +a O +highly B-protein_state +bent I-protein_state +IRES B-site +with O +PKI B-structure_element +partially O +accommodated O +in O +the O +P B-site +site I-site + +In O +Structure B-evidence +IV I-evidence +, O +the O +40S B-complex_assembly +subunit B-structure_element +is O +almost O +non B-protein_state +- I-protein_state +rotated I-protein_state +relative O +to O +the O +60S B-complex_assembly +subunit B-structure_element +, O +and O +the O +40S B-complex_assembly +head B-structure_element +is O +mid B-protein_state +- I-protein_state +swiveled I-protein_state +. O + +Unwinding O +of O +the O +head B-structure_element +moves O +the O +head B-structure_element +P B-site +- I-site +site I-site +residue O +U1191 B-residue_name_number +and O +body B-structure_element +P B-site +- I-site +site I-site +residue O +C1637 B-residue_name_number +closer O +together O +, O +resulting O +in O +a O +partially O +restored O +40S B-complex_assembly +P B-site +site I-site +. O + +Whereas O +C1637 B-residue_name_number +forms O +a O +stacking B-site +platform I-site +for O +the O +last O +base O +pair O +of O +PKI B-structure_element +, O +U1191 B-residue_name_number +does O +not O +yet O +stack B-bond_interaction +on O +PKI B-structure_element +because O +the O +head B-structure_element +remains O +partially O +swiveled O +. O + +This O +renders O +PKI B-structure_element +partially O +accommodated O +in O +the O +P B-site +site I-site +( O +Figure O +2g O +). O + +Unwinding O +of O +the O +40S B-complex_assembly +head B-structure_element +also O +positions O +the O +head B-structure_element +A B-site +site I-site +closer O +to O +the O +body B-structure_element +A B-site +site I-site +. O + +This O +results O +in O +rearrangements O +of O +eEF2 B-protein +interactions O +with O +the O +head B-structure_element +, O +allowing O +eEF2 B-protein +to O +advance O +further O +into O +the O +A B-site +site I-site +. O + +Structure B-evidence +V I-evidence +represents O +an O +extended B-protein_state +IRES B-site +with O +PKI B-structure_element +fully O +accommodated O +in O +the O +P B-site +site I-site +and O +domain O +IV B-structure_element +of O +eEF2 B-protein +in O +the O +A B-site +site I-site + +A O +notable O +conformational O +change O +in O +eEF2 B-protein +from O +that O +in O +the O +preceding O +Structures B-evidence +is O +visible O +in O +the O +position O +of O +domain O +III B-structure_element +, O +which O +contacts O +uS12 B-protein +( O +Figure O +6d O +). O + +In O +Structure B-evidence +V I-evidence +, O +protein O +uS12 B-protein +is O +shifted O +along O +with O +the O +40S B-complex_assembly +body B-structure_element +as O +a O +result O +of O +intersubunit O +rotation O +. O + +Specifically O +, O +the O +C B-structure_element +- I-structure_element +terminal I-structure_element +tail I-structure_element +of O +uS12 B-protein +packs O +against O +the O +β B-structure_element +- I-structure_element +barrel I-structure_element +of O +domain O +II B-structure_element +, O +while O +the O +β B-structure_element +- I-structure_element +barrel I-structure_element +of O +uS12 B-protein +packs O +against O +helix B-structure_element +A I-structure_element +of O +domain O +III B-structure_element +. O + +This O +shifts O +the O +tip O +of O +helix B-structure_element +A I-structure_element +of O +domain O +III B-structure_element +( O +at O +aa O +500 B-residue_number +) O +by O +~ O +5 O +Å O +( O +relative O +to O +that O +in O +Structure B-evidence +I I-evidence +) O +toward O +domain O +I B-structure_element +. O +Although O +domain O +III B-structure_element +remains O +in O +contact O +with O +domain O +V B-structure_element +, O +the O +shift O +occurs O +in O +the O +direction O +that O +could O +eventually O +disconnect O +the O +β B-structure_element +- I-structure_element +platforms I-structure_element +of O +these O +domains O +. O + +The O +amide O +at O +the O +diphthamide B-ptm +end O +interacts O +with O +N2 O +of O +G6906 B-residue_name_number +and O +O2 O +and O +O2 O +’ O +of O +C6951 B-residue_name_number +( O +corresponding O +to O +nt O +2 O +of O +the O +codon O +). O + +The O +trimethylamino O +- O +group O +is O +positioned O +over O +the O +ribose O +of O +C6952 B-residue_name_number +( O +codon O +nt O +3 O +). O + +IRES B-site +translocation O +mechanism O + +Four O +views O +( O +scenes O +) O +are O +shown O +: O +( O +1 O +) O +A O +view O +down O +the O +intersubunit O +space O +, O +with O +the O +head B-structure_element +of O +the O +40S B-complex_assembly +subunit B-structure_element +oriented O +toward O +a O +viewer O +, O +as O +in O +Figure O +1a O +; O +( O +2 O +) O +A O +view O +at O +the O +solvent O +side O +of O +the O +40S B-complex_assembly +subunit B-structure_element +, O +with O +the O +40S B-complex_assembly +head B-structure_element +shown O +at O +the O +top O +, O +as O +in O +Figure O +2 O +— O +figure O +supplement O +1 O +; O +( O +3 O +) O +A O +view O +down O +at O +the O +subunit O +interface O +of O +the O +40S B-complex_assembly +subunit B-structure_element +; O +( O +4 O +) O +A O +close O +- O +up O +view O +of O +the O +decoding B-site +center I-site +( O +A B-site +site I-site +) O +and O +the O +P B-site +site I-site +, O +as O +in O +Figure O +2g O +. O +Each O +scene O +is O +shown O +twice O +. O + +We O +propose O +that O +together O +with O +the O +previously O +reported O +initiation B-protein_state +state O +, O +these O +structures B-evidence +represent O +the O +trajectory O +of O +eEF2 B-protein +- O +induced O +IRES B-site +translocation O +( O +shown O +as O +an O +animation O +in O +http O +:// O +labs O +. O +umassmed O +. O +edu O +/ O +korostelevlab O +/ O +msc O +/ O +iresmovie O +. O +gif O +and O +Video O +1 O +). O + +Our O +structures B-evidence +reveal O +previously O +unseen O +intermediate O +states O +of O +eEF2 B-protein +or O +EF B-protein +- I-protein +G I-protein +engagement O +with O +the O +A B-site +site I-site +, O +providing O +the O +structural O +basis O +for O +the O +mechanism O +of O +translocase B-protein_type +action O +. O + +Furthermore O +, O +they O +provide O +insight O +into O +the O +mechanism O +of O +eEF2 B-complex_assembly +• I-complex_assembly +GTP I-complex_assembly +association O +with O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +and O +eEF2 B-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +dissociation O +from O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +, O +also O +delineating O +the O +mechanism O +of O +translation O +inhibition O +by O +the O +antifungal O +drug O +sordarin B-chemical +. O + +At O +the O +start O +( O +initiation B-protein_state +state O +), O +the O +IRES B-site +adopts O +an O +extended B-protein_state +conformation O +( O +extended B-protein_state +inchworm I-protein_state +). O + +The O +front B-structure_element +' I-structure_element +legs I-structure_element +' O +( O +SL4 B-structure_element +and O +SL5 B-structure_element +) O +of O +the O +5 B-structure_element +’- I-structure_element +domain I-structure_element +( O +front B-structure_element +end I-structure_element +) O +are O +attached O +to O +the O +40S B-complex_assembly +head B-structure_element +proteins O +uS7 B-protein +, O +uS11 B-protein +and O +eS25 B-protein +( O +Figure O +3 O +— O +figure O +supplement O +2 O +). O + +PKI B-structure_element +, O +representing O +the O +hind B-structure_element +end I-structure_element +, O +is O +bound B-protein_state +in I-protein_state +the O +A B-site +site I-site +. O + +This O +shortens O +the O +distance O +between O +PKI B-structure_element +and O +SL4 B-structure_element +by O +up O +to O +20 O +Å O +relative O +to O +the O +initiating O +IRES B-site +structure B-evidence +, O +resulting O +in O +a O +bent B-protein_state +IRES B-site +conformation O +( O +bent B-protein_state +inchworm I-protein_state +). O + +In O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +CrPV B-species +IRES B-site +structure B-evidence +, O +the O +5 B-structure_element +’- I-structure_element +domain I-structure_element +similarly O +protrudes O +between O +the O +subunits O +and O +interacts O +with O +the O +L1 B-structure_element +stalk I-structure_element +, O +as O +in O +the O +initiation B-protein_state +state O +for O +this O +IRES B-site +. O + +This O +underlines O +structural O +similarity O +for O +the O +TSV B-species +and O +CrPV B-species +IRES B-site +translocation O +mechanisms O +. O + +One O +of O +the O +mechanistic O +scenarios O +( O +discussed O +in O +) O +involves O +binding O +of O +the O +first O +aminoacyl B-chemical +- I-chemical +tRNA I-chemical +to O +the O +post B-protein_state +- I-protein_state +translocated I-protein_state +IRES B-site +mRNA B-chemical +frame O +shifted O +by O +one O +nucleotide O +( O +predominantly O +a O ++ O +1 O +frame O +shift O +). O + +It O +is O +likely O +that O +alternative O +frame O +setting O +occurs O +following O +eEF2 B-protein +release O +and O +that O +this O +depends O +on O +transient O +displacement O +of O +the O +start O +codon O +in O +the O +decoding B-site +center I-site +, O +allowing O +binding O +of O +the O +corresponding O +amino B-chemical +acyl I-chemical +- I-chemical +tRNA I-chemical +to O +an O +off O +- O +frame O +codon O +. O + +Further O +structural B-experimental_method +studies I-experimental_method +involving O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +• I-complex_assembly +tRNA I-complex_assembly +complexes O +are O +necessary O +to O +understand O +the O +mechanisms O +underlying O +alternative O +reading O +frame O +selection O +. O + +This O +is O +consistent O +with O +the O +observations O +that O +the O +intergenic O +IRESs B-site +are O +prone O +to O +reverse O +translocation O +. O + +This O +contrasts O +with O +the O +post B-protein_state +- I-protein_state +translocated I-protein_state +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +, O +in O +which O +the O +classical O +P B-site +and I-site +E I-site +- I-site +site I-site +tRNAs B-chemical +are O +stabilized O +in O +the O +non B-protein_state +- I-protein_state +rotated I-protein_state +ribosome B-complex_assembly +after O +translocase B-protein_type +release O +. O + +In O +the O +initiation B-protein_state +state O +, O +the O +IRES B-site +resembles O +a O +pre B-protein_state +- I-protein_state +translocation I-protein_state +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +reduced O +to O +the O +A B-site +/ I-site +P I-site +- O +tRNA B-chemical +anticodon B-structure_element +- I-structure_element +stem I-structure_element +loop I-structure_element +and O +elbow B-structure_element +in O +the O +A B-site +site I-site +and O +the O +P B-site +/ I-site +E I-site +- O +tRNA B-chemical +elbow B-structure_element +contacting O +the O +L1 B-structure_element +stalk I-structure_element +. O + +Because O +the O +anticodon B-structure_element +- I-structure_element +stem I-structure_element +loop I-structure_element +of O +the O +A B-site +- O +tRNA B-chemical +is O +sufficient O +for O +translocation O +completion O +, O +we O +ascribe O +the O +meta O +- O +stability O +of O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +IRES B-site +to O +the O +absence B-protein_state +of I-protein_state +the O +P B-site +/ I-site +E I-site +- O +tRNA B-chemical +elements O +, O +either O +the O +ASL B-structure_element +or O +the O +acceptor O +arm O +, O +or O +both O +. O + +Furthermore O +, O +interactions O +of O +SL4 B-structure_element +and O +SL5 B-structure_element +with O +the O +40S B-complex_assembly +subunit B-structure_element +likely O +contribute O +to O +stabilization O +of O +pre B-protein_state +- I-protein_state +translocation I-protein_state +structures B-evidence +. O + +Partitioned O +roles O +of O +40S B-complex_assembly +subunit B-structure_element +rearrangements O + +Our O +structures B-evidence +delineate O +the O +mechanistic O +functions O +for O +intersubunit O +rotation O +and O +head B-structure_element +swivel O +in O +translocation O +. O + +Specifically O +, O +intersubunit O +rotation O +allows O +eEF2 B-protein +entry O +into O +the O +A B-site +site I-site +, O +while O +the O +head B-structure_element +swivel O +mediates O +PKI B-structure_element +translocation O +. O + +This O +suggests O +that O +the O +subunits B-structure_element +are O +capable O +of O +spontaneous O +rotation O +, O +as O +is O +the O +case O +for O +tRNA B-protein_state +- I-protein_state +bound I-protein_state +pre B-protein_state +- I-protein_state +translocation I-protein_state +complexes O +. O + +This O +allows O +eEF2 B-protein +to O +move O +into O +the O +A B-site +site I-site +. O + +As O +such O +, O +reverse O +intersubunit O +rotation O +facilitates O +full O +docking O +of O +eEF2 B-protein +in O +the O +A B-site +site I-site +. O + +Because O +the O +histidine B-site +- I-site +diphthamide I-site +tip I-site +of O +eEF2 B-protein +( O +H583 B-residue_name_number +, O +H694 B-residue_name_number +and O +Diph699 B-ptm +) O +attaches O +to O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +- I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +, O +eEF2 B-protein +appears O +to O +directly O +force O +PKI B-structure_element +out O +of O +the O +A B-site +site I-site +. O + +The O +fully B-protein_state +swiveled I-protein_state +conformations O +of O +Structures B-evidence +II I-evidence +and I-evidence +III I-evidence +represent O +the O +mid O +- O +point O +of O +translocation O +, O +in O +which O +PKI B-structure_element +relocates O +between O +the O +head B-structure_element +A B-site +site I-site +and O +body B-structure_element +P B-site +site I-site +. O + +We O +note O +that O +such O +mid O +- O +states O +have O +not O +been O +observed O +for O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +, O +but O +their O +formation O +can O +explain O +the O +formation O +of O +subsequent O +pe B-protein_state +/ I-protein_state +E I-protein_state +hybrid I-protein_state +and O +ap B-protein_state +/ I-protein_state +P I-protein_state +chimeric I-protein_state +structures B-evidence +( O +Figure O +1 O +— O +figure O +supplement O +1 O +). O + +Reverse O +swivel O +from O +Structure B-evidence +III I-evidence +to I-evidence +V I-evidence +brings O +the O +head B-structure_element +to O +the O +non B-protein_state +- I-protein_state +swiveled I-protein_state +position O +, O +restoring O +the O +A B-site +and I-site +P I-site +sites I-site +on O +the O +small B-structure_element +subunit I-structure_element +. O + +The O +functions O +of O +eEF2 B-protein +in O +translocation O + +To O +our O +knowledge O +, O +our O +work O +provides O +the O +first O +high O +- O +resolution O +view O +of O +the O +dynamics O +of O +a O +ribosomal B-protein_type +translocase I-protein_type +that O +is O +inferred O +from O +an O +ensemble O +of O +structures B-evidence +sampled O +under O +uniform O +conditions O +. O + +The O +structures B-evidence +, O +therefore O +, O +offer O +a O +unique O +opportunity O +to O +address O +the O +role O +of O +the O +elongation B-protein_type +factors I-protein_type +during O +translocation O +. O + +While O +the O +ribosome B-complex_assembly +itself O +has O +the O +capacity O +to O +translocate O +in O +the O +absence B-protein_state +of I-protein_state +the O +translocase B-protein_type +, O +spontaneous O +translocation O +is O +slow O +. O + +EF B-protein +- I-protein +G I-protein +enhances O +the O +translocation O +rate O +by O +several O +orders O +of O +magnitude O +, O +aided O +by O +an O +additional O +2 O +- O +to O +50 O +- O +fold O +boost O +from O +GTP B-chemical +hydrolysis O +. O + +Due O +to O +the O +lack O +of O +structures B-evidence +of O +translocation O +intermediates O +, O +the O +mechanistic O +role O +of O +eEF2 B-protein +/ O +EF B-protein +- I-protein +G I-protein +is O +not O +fully O +understood O +. O + +The O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +structures B-evidence +reported O +here O +suggest O +two O +main O +roles O +for O +eEF2 B-protein +in O +translocation O +. O + +As O +discussed O +above O +, O +the O +first O +role O +is O +to O +directly O +shift O +PKI B-structure_element +out O +of O +the O +A B-site +site I-site +upon O +spontaneous O +reverse O +intersubunit O +rotation O +. O + +In O +our O +structures B-evidence +, O +the O +tip O +of O +domain O +IV B-structure_element +docks O +next O +to O +PKI B-structure_element +, O +with O +diphthamide B-ptm +699 I-ptm +fit O +into O +the O +minor B-site +groove I-site +of O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +- I-structure_element +like I-structure_element +helix I-structure_element +of O +PKI B-structure_element +( O +Figure O +7 O +). O + +This O +arrangement O +rationalizes O +inactivation O +of O +eEF2 B-protein +by O +diphtheria B-protein_type +toxin I-protein_type +, O +which O +catalyzes O +ADP B-ptm +- I-ptm +ribosylation I-ptm +of O +the O +diphthamide B-ptm +( O +reviewed O +in O +). O + +The O +enzyme O +ADP B-ptm +- I-ptm +ribosylates I-ptm +the O +NE2 O +atom O +of O +the O +imidazole O +ring O +, O +which O +in O +our O +structures B-evidence +interacts O +with O +the O +first O +two O +residues O +of O +the O +anticodon B-structure_element +- I-structure_element +like I-structure_element +strand I-structure_element +of O +PKI B-structure_element +. O + +As O +eEF2 B-protein +shifts O +PKI B-structure_element +toward O +the O +P B-site +site I-site +in O +the O +course O +of O +reverse O +intersubunit O +rotation O +, O +the O +60S B-protein_state +- I-protein_state +attached I-protein_state +translocase B-protein_type +migrates O +along O +the O +surface O +of O +the O +40S B-complex_assembly +subunit B-structure_element +, O +guided O +by O +electrostatic B-bond_interaction +interactions I-bond_interaction +. O + +Positively B-site +- I-site +charged I-site +patches I-site +of O +domains O +II B-structure_element +and O +III B-structure_element +( O +R391 B-residue_name_number +, O +K394 B-residue_name_number +, O +R433 B-residue_name_number +, O +R510 B-residue_name_number +) O +and O +IV B-structure_element +( O +K613 B-residue_name_number +, O +R617 B-residue_name_number +, O +R609 B-residue_name_number +, O +R631 B-residue_name_number +, O +K651 B-residue_name_number +) O +slide O +over O +rRNA B-chemical +of O +the O +40S B-complex_assembly +body B-structure_element +( O +h5 B-structure_element +) O +and O +head B-structure_element +( O +h18 B-structure_element +and O +h33 B-structure_element +/ O +h34 B-structure_element +), O +respectively O +. O + +For O +example O +, O +between O +Structures B-evidence +II I-evidence +and I-evidence +V I-evidence +, O +the O +K613 B-residue_name_number +/ O +R617 B-residue_name_number +/ O +R631 B-residue_name_number +cluster O +of O +domain O +IV B-structure_element +hops O +by O +~ O +19 O +Å O +( O +for O +Cα O +of O +R617 B-residue_name_number +) O +from O +the O +phosphate O +backbone O +of O +h33 B-structure_element +( O +at O +nt O +1261 B-residue_range +– I-residue_range +1264 I-residue_range +) O +to O +that O +of O +the O +neighboring O +h34 B-structure_element +( O +at O +nt O +1442 B-residue_range +– I-residue_range +1445 I-residue_range +). O + +Thus O +, O +sliding O +of O +eEF2 B-protein +involves O +reorganization O +of O +electrostatic B-bond_interaction +, I-bond_interaction +perhaps I-bond_interaction +isoenergetic I-bond_interaction +interactions I-bond_interaction +, O +echoing O +those O +implied O +in O +extraordinarily O +fast O +ribosome B-complex_assembly +inactivation O +rates O +by O +the O +small O +- O +protein O +ribotoxins O +and O +in O +fast O +protein O +association O +and O +diffusion O +along O +DNA O +. O + +Comparison B-experimental_method +of O +our O +structures B-evidence +with O +the O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +initiation B-protein_state +structure B-evidence +reveals O +the O +structural O +basis O +for O +the O +second O +key O +function O +of O +the O +translocase B-protein_type +: O +' O +unlocking O +' O +of O +intrasubunit O +rearrangements O +that O +are O +required O +for O +step O +- O +wise O +translocation O +of O +PKI B-structure_element +on O +the O +small B-structure_element +subunit I-structure_element +. O + +The O +unlocking O +model O +of O +the O +ribosome B-complex_assembly +• I-complex_assembly +2tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +pre B-protein_state +- I-protein_state +translocation I-protein_state +complex O +has O +been O +proposed O +decades O +ago O +and O +functional O +requirement O +of O +the O +translocase B-protein_type +in O +this O +process O +has O +been O +implicated O +. O + +FRET B-evidence +data I-evidence +indicate O +that O +translocation O +of O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +on O +the O +70S B-complex_assembly +ribosome I-complex_assembly +requires O +a O +forward O +- O +and O +- O +reverse O +head B-structure_element +swivel O +, O +which O +may O +be O +related O +to O +the O +unlocking O +phenomenon O +. O + +Our O +structures B-evidence +suggest O +that O +eEF2 B-protein +induces O +head B-structure_element +swivel O +by O +' O +unlocking O +' O +the O +head B-structure_element +- O +body B-structure_element +interactions O +( O +Figure O +7 O +). O + +Binding O +of O +the O +ASL B-structure_element +to O +the O +A B-site +site I-site +is O +known O +from O +structural B-experimental_method +studies I-experimental_method +of O +bacterial B-taxonomy_domain +ribosomes B-complex_assembly +to O +result O +in O +' O +domain B-protein_state +closure I-protein_state +' O +of O +the O +small B-structure_element +subunit I-structure_element +, O +i O +. O +e O +. O +closer O +association O +of O +the O +head B-structure_element +, O +shoulder B-structure_element +and O +body B-structure_element +domains O +. O + +The O +domain O +closure O +' O +locks O +' O +cognate O +tRNA B-chemical +in O +the O +A B-site +site I-site +via O +stacking B-bond_interaction +on O +the O +head B-structure_element +A B-site +site I-site +( O +C1274 B-residue_name_number +in O +S B-species +. I-species +cerevisiae I-species +or O +C1054 B-residue_name_number +in O +E B-species +. I-species +coli I-species +) O +and O +interactions O +with O +the O +body B-structure_element +A B-site +- I-site +site I-site +nucleotides O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +( O +A1492 B-residue_name_number +and O +A1493 B-residue_name_number +in O +E B-species +. I-species +coli I-species +). O + +The O +histidine B-ptm +- I-ptm +diphthamide I-ptm +- O +induced O +disengagement O +of O +PKI B-structure_element +from O +A1755 B-residue_name_number +and O +A1756 B-residue_name_number +therefore O +provides O +the O +structural O +definition O +for O +the O +' O +unlocking O +' O +mode O +of O +eEF2 B-protein +action O +. O + +In O +summary O +, O +our O +structures B-evidence +are O +consistent O +with O +a O +model O +of O +eEF2 B-protein +- O +induced O +translocation O +in O +which O +both O +PKI B-structure_element +and O +eEF2 B-protein +passively O +migrate O +into O +the O +P B-site +and I-site +A I-site +site I-site +, O +respectively O +, O +during O +spontaneous O +40S B-complex_assembly +body B-structure_element +rotation O +and O +head B-structure_element +swivel O +, O +the O +latter O +being O +allowed O +by O +' O +unlocking O +' O +of O +the O +A B-site +site I-site +by O +eEF2 B-protein +. O + +Insights O +into O +eEF2 B-protein +association O +with O +and O +dissociation O +from O +the O +ribosome B-complex_assembly + +The O +conformational O +rearrangements O +in O +eEF2 B-protein +from O +Structure B-evidence +I I-evidence +through O +Structure B-evidence +V I-evidence +provide O +insights O +into O +the O +mechanisms O +of O +eEF2 B-protein +association O +with O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +and O +dissociation O +from O +the O +post B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +. O + +In O +the O +fully B-protein_state +- I-protein_state +rotated I-protein_state +pre B-protein_state +- I-protein_state +translocation I-protein_state +- O +like O +Structure B-evidence +I I-evidence +, O +an O +additional O +interaction O +exists O +. O + +The O +interaction O +between O +h14 B-structure_element +and O +switch B-structure_element +loop I-structure_element +I I-structure_element +is O +not O +resolved O +in O +Structures B-evidence +II I-evidence +to I-evidence +V I-evidence +, O +in O +all O +of O +which O +the O +small B-structure_element +subunit I-structure_element +is O +partially B-protein_state +rotated I-protein_state +or O +non B-protein_state +- I-protein_state +rotated I-protein_state +, O +so O +that O +helix B-structure_element +14 I-structure_element +is O +placed O +at O +least O +6 O +Å O +farther O +from O +eEF2 B-protein +( O +Figure O +5d O +). O + +We O +conclude O +that O +unlike O +other O +conformations O +of O +the O +ribosome B-complex_assembly +, O +the O +fully B-protein_state +rotated I-protein_state +40S B-complex_assembly +subunit B-structure_element +of O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +ribosome B-complex_assembly +provides O +an O +interaction B-site +surface I-site +, O +complementing O +the O +P B-structure_element +stalk I-structure_element +and O +SRL B-structure_element +, O +for O +binding O +of O +the O +GTP B-protein_state +- I-protein_state +bound I-protein_state +translocase B-protein_type +. O + +This O +structural O +basis O +rationalizes O +the O +observation O +of O +transient O +stabilization O +of O +the O +rotated B-protein_state +70S B-complex_assembly +ribosome I-complex_assembly +upon O +EF B-complex_assembly +- I-complex_assembly +G I-complex_assembly +• I-complex_assembly +GTP I-complex_assembly +binding O +and O +prior O +to O +translocation O +. O + +The O +most O +pronounced O +inter O +- O +domain O +rearrangement O +in O +eEF2 B-protein +involves O +movement O +of O +domain O +III B-structure_element +. O + +In O +the O +rotated B-protein_state +or O +mid B-protein_state +- I-protein_state +rotated I-protein_state +Structures B-evidence +I I-evidence +through I-evidence +III I-evidence +, O +this O +domain O +remains O +rigidly O +associated O +with O +domain O +V B-structure_element +and O +the O +N O +- O +terminal O +superdomain B-structure_element +and O +does O +not O +undergo O +noticeable O +rearrangements O +. O + +In O +Structure B-evidence +V I-evidence +, O +however O +, O +the O +tip O +of O +helix B-structure_element +A I-structure_element +of O +domain O +III B-structure_element +is O +displaced O +toward O +domain O +I B-structure_element +by O +~ O +5 O +Å O +relative O +to O +that O +in O +mid B-protein_state +- I-protein_state +rotated I-protein_state +or O +fully B-protein_state +rotated I-protein_state +structures B-evidence +. O + +This O +displacement O +is O +caused O +by O +the O +8 O +Å O +movement O +of O +the O +40S B-complex_assembly +body B-structure_element +protein O +uS12 B-protein +upon O +reverse O +intersubunit O +rotation O +from O +Structure B-evidence +I I-evidence +to I-evidence +V I-evidence +( O +Figure O +6d O +). O + +We O +propose O +that O +the O +shift O +of O +domain O +III B-structure_element +by O +uS12 B-protein +initiates O +intra O +- O +domain O +rearrangements O +in O +eEF2 B-protein +, O +which O +unstack O +the O +β B-structure_element +- I-structure_element +platform I-structure_element +of O +domain O +III B-structure_element +from O +that O +of O +domain O +V B-structure_element +. O +This O +would O +result O +in O +a O +conformation O +characteristic O +of O +free B-protein_state +eEF2 B-protein +and O +EF B-protein +- I-protein +G I-protein +in O +which O +the O +β B-structure_element +- I-structure_element +platforms I-structure_element +are O +nearly O +perpendicular O +. O + +As O +we O +discuss O +below O +, O +Structure B-evidence +V I-evidence +captures O +a O +' O +pre B-protein_state +- I-protein_state +unstacking I-protein_state +' O +state O +due O +to O +stabilization O +of O +the O +interface B-site +between O +domains O +III B-structure_element +and O +V B-structure_element +by O +sordarin B-chemical +. O + +Sordarin B-chemical +stabilizes O +GDP B-protein_state +- I-protein_state +bound I-protein_state +eEF2 B-protein +on O +the O +ribosome B-complex_assembly + +Our O +structures B-evidence +therefore O +indicate O +that O +sordarin B-chemical +stalls O +eEF2 B-protein +on O +the O +ribosome B-complex_assembly +in O +the O +GDP B-protein_state +- I-protein_state +bound I-protein_state +form O +, O +i O +. O +e O +. O +following O +GTP B-chemical +hydrolysis O +and O +phosphate O +release O +. O + +In O +all O +five O +structures B-evidence +, O +sordarin B-chemical +is O +bound B-protein_state +between O +domains O +III B-structure_element +and O +V B-structure_element +of O +eEF2 B-protein +, O +stabilized O +by O +hydrophobic B-bond_interaction +interactions I-bond_interaction +identical O +to O +those O +in O +the O +isolated B-protein_state +eEF2 B-complex_assembly +• I-complex_assembly +sordarin I-complex_assembly +complex O +( O +Figures O +5g O +and O +h O +). O + +In O +the O +nearly B-protein_state +non I-protein_state +- I-protein_state +rotated I-protein_state +post B-protein_state +- I-protein_state +translocation I-protein_state +Structure B-evidence +V I-evidence +, O +the O +tip O +of O +domain O +III B-structure_element +is O +shifted O +, O +however O +the O +interface B-site +between O +domains O +III B-structure_element +and O +V B-structure_element +remains O +unchanged O +, O +suggesting O +strong O +stabilization O +of O +this O +interface B-site +by O +sordarin B-chemical +. O + +We O +propose O +that O +sordarin B-chemical +acts O +to O +prevent O +full O +reverse O +rotation O +and O +release O +of O +eEF2 B-complex_assembly +• I-complex_assembly +GDP I-complex_assembly +by O +stabilizing O +the O +interdomain B-site +interface I-site +and O +thus O +blocking O +uS12 B-protein +- O +induced O +disengagement O +of O +domain O +III B-structure_element +from O +domain O +V B-structure_element +. O + +Implications O +for O +tRNA B-chemical +and O +mRNA B-chemical +translocation O +during O +translation O + +Despite O +an O +impressive O +body B-structure_element +of O +biochemical B-evidence +, I-evidence +fluorescence I-evidence +and I-evidence +structural I-evidence +data I-evidence +accumulated O +since O +then O +, O +translocation O +remains O +the O +least O +understood O +step O +of O +elongation O +. O + +However O +, O +visualization O +of O +the O +eEF2 B-protein +/ O +EF B-protein +- I-protein +G I-protein +- O +induced O +translocation O +is O +confined O +to O +very O +early O +pre B-protein_state +- I-protein_state +EF I-protein_state +- I-protein_state +G I-protein_state +- I-protein_state +entry I-protein_state +states O +and O +late O +( O +almost B-protein_state +translocated I-protein_state +or O +fully B-protein_state +translocated I-protein_state +) O +states O +, O +leaving O +most O +of O +the O +path O +from O +the O +A B-site +to I-site +the I-site +P I-site +site I-site +uncharacterized O +( O +Figure O +1 O +— O +figure O +supplement O +1 O +). O + +First O +, O +we O +propose O +that O +tRNA B-chemical +and O +IRES B-site +translocations O +occur O +via O +the O +same O +general O +trajectory O +. O + +This O +is O +evident O +from O +the O +fact O +that O +ribosome B-complex_assembly +rearrangements O +in O +translocation O +are O +inherent O +to O +the O +ribosome B-complex_assembly +and O +likely O +occur O +in O +similar O +ways O +in O +both O +cases O +. O + +Furthermore O +, O +the O +step O +- O +wise O +coupling O +of O +ribosome B-complex_assembly +dynamics O +with O +IRES B-site +translocation O +is O +overall O +consistent O +with O +that O +observed O +for O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +translocation O +in O +solution O +. O + +For O +example O +, O +fluorescence B-experimental_method +and I-experimental_method +biochemical I-experimental_method +studies I-experimental_method +revealed O +that O +the O +early O +pre B-protein_state +- I-protein_state +translocation I-protein_state +EF B-protein_state +- I-protein_state +G I-protein_state +- I-protein_state +bound I-protein_state +ribosomes B-complex_assembly +are O +fully B-protein_state +rotated I-protein_state +and O +translocation O +of O +the O +tRNA B-complex_assembly +- I-complex_assembly +mRNA I-complex_assembly +complex O +occurs O +during O +reverse O +rotation O +of O +the O +small B-structure_element +subunit I-structure_element +, O +coupled O +with O +head B-structure_element +swivel O +. O + +The O +sequence O +of O +ribosome B-complex_assembly +rearrangements O +during O +IRES B-site +translocation O +also O +agrees O +with O +that O +inferred O +from O +70S B-complex_assembly +• I-complex_assembly +EF I-complex_assembly +- I-complex_assembly +G I-complex_assembly +structures B-evidence +, O +including O +those O +in O +which O +the O +A B-site +- I-site +to I-site +- I-site +P I-site +- I-site +site I-site +translocating O +tRNA B-chemical +was O +not O +present O +. O + +Specifically O +, O +an O +earlier O +translocation O +intermediate O +ribosome B-complex_assembly +( O +TIpre O +) O +was O +proposed O +to O +adopt O +a O +rotated B-protein_state +( O +7 O +– O +9 O +°) O +body B-structure_element +and O +a O +partly B-protein_state +rotated I-protein_state +head B-structure_element +( O +5 O +– O +7 O +. O +5 O +°), O +in O +agreement O +with O +the O +conformation O +of O +our O +Structure B-evidence +I I-evidence +. O +The O +most B-protein_state +swiveled I-protein_state +head B-structure_element +( O +18 O +– O +21 O +°) O +was O +observed O +in O +a O +mid B-protein_state +- I-protein_state +rotated I-protein_state +ribosome B-complex_assembly +( O +3 O +– O +5 O +°) O +of O +a O +later O +translocation O +intermediate O +TIpost O +, O +similar O +to O +the O +conformation O +of O +our O +Structure B-evidence +III I-evidence +. O + +Overall O +, O +these O +correlations O +suggest O +that O +the O +intermediate O +locations O +of O +the O +elusive O +A B-site +- I-site +to I-site +- I-site +P I-site +- I-site +site I-site +translocating O +tRNA B-chemical +are O +similar O +to O +those O +of O +PKI B-structure_element +in O +our O +structures B-evidence +. O + +We O +deem O +the O +pre B-protein_state +- I-protein_state +translocation I-protein_state +complex O +locked B-protein_state +, O +because O +the O +A B-protein_state +- I-protein_state +site I-protein_state +bound I-protein_state +ASL O +- O +mRNA B-chemical +is O +stabilized O +by O +interactions O +with O +the O +decoding B-site +center I-site +. O + +Unlocking O +involves O +separation O +of O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +helix I-structure_element +from O +the O +decoding B-site +center I-site +residues O +by O +the O +protruding O +tip O +of O +eEF2 B-protein +/ O +EF B-protein +- I-protein +G I-protein +( O +Figure O +7 O +), O +occurring O +in O +the O +fully B-protein_state +rotated I-protein_state +ribosome B-complex_assembly +at O +an O +early O +pre B-protein_state +- I-protein_state +translocation I-protein_state +step O +. O + +Third O +, O +our O +findings O +uncover O +a O +new O +role O +of O +the O +head B-structure_element +swivel O +. O + +Finally O +, O +the O +similar O +populations O +of O +particles B-experimental_method +( O +within O +a O +2X O +range O +) O +in O +our O +80S B-complex_assembly +• I-complex_assembly +IRES I-complex_assembly +• I-complex_assembly +eEF2 I-complex_assembly +reconstructions B-evidence +( O +Figure O +1 O +— O +figure O +supplement O +2 O +) O +suggest O +that O +the O +intermediate O +translocation O +states O +sample O +several O +energetically O +similar O +and O +interconverting O +conformations O +. O + +This O +is O +consistent O +with O +the O +idea O +of O +a O +rather O +flat O +energy O +landscape O +of O +translocation O +, O +suggested O +by O +recent O +work O +that O +measured O +mechanical O +work O +produced O +by O +the O +ribosome B-complex_assembly +during O +translocation O +. O + +Our O +findings O +implicate O +, O +however O +, O +that O +the O +energy O +landscape O +is O +not O +completely O +flat O +and O +contains O +local O +minima O +for O +transient O +positions O +of O +the O +codon B-structure_element +- I-structure_element +anticodon I-structure_element +helix I-structure_element +between O +the O +A B-site +and I-site +P I-site +sites I-site +. O + +The O +shift O +of O +the O +PKI B-structure_element +with O +respect O +to O +the O +body B-structure_element +occurs O +during O +forward O +head B-structure_element +swivel O +in O +two O +major O +sub O +- O +steps O +of O +~ O +4 O +Å O +each O +( O +initiation B-complex_assembly +complex I-complex_assembly +to O +I B-evidence +, O +and O +I B-evidence +to O +II B-evidence +), O +after O +which O +PKI B-structure_element +undergoes O +small O +shifts O +to O +settle O +in O +the O +body B-structure_element +P B-site +site I-site +in O +Structures B-evidence +III I-evidence +, I-evidence +IV I-evidence +and I-evidence +V I-evidence +( O +Figure O +2 O +— O +source O +data O +1 O +). O + +We O +note O +that O +four O +of O +our O +near O +- O +atomic O +resolution O +maps B-evidence +comprised O +~ O +30 O +, O +000 O +particles B-experimental_method +each O +, O +the O +minimum O +number O +required O +for O +a O +near B-evidence +- I-evidence +atomic I-evidence +- I-evidence +resolution I-evidence +reconstruction I-evidence +of O +the O +ribosome B-complex_assembly +. O + +Translation O +of O +viral B-taxonomy_domain +mRNA B-chemical + +The O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +structures B-evidence +demonstrate O +that O +the O +TSV B-species +IRES B-site +structurally O +and O +dynamically O +represents O +a O +chimera O +of O +the O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +translocating O +complex O +( O +A B-complex_assembly +/ I-complex_assembly +P I-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +• I-complex_assembly +P I-complex_assembly +/ I-complex_assembly +E I-complex_assembly +- I-complex_assembly +tRNA I-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +). O + +Like O +in O +the O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +translocating O +complex O +in O +which O +the O +two O +tRNAs B-chemical +move O +independently O +of O +each O +other O +, O +the O +PKI B-structure_element +domain O +moves O +relative O +to O +the O +5 B-structure_element +´- I-structure_element +domain I-structure_element +, O +causing O +the O +IRES B-site +to O +undergo O +an O +inchworm B-protein_state +- O +walk O +translocation O +. O + +A O +large O +structural O +difference O +between O +the O +IRES B-site +and O +the O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +complex O +exists O +, O +however O +, O +in O +that O +the O +IRES B-site +lacks B-protein_state +three O +out O +of O +six O +tRNA B-structure_element +- I-structure_element +like I-structure_element +domains I-structure_element +involved O +in O +tRNA B-chemical +translocation O +. O + +Although O +structurally O +handicapped O +, O +the O +TSV B-species +IRES B-site +manages O +to O +translocate O +by O +employing O +ribosome B-complex_assembly +dynamics O +that O +are O +remarkably O +similar O +to O +that O +in O +2tRNA B-complex_assembly +• I-complex_assembly +mRNA I-complex_assembly +translocation O +. O + +The O +uniformity O +of O +ribosome B-complex_assembly +dynamics O +underscores O +the O +idea O +that O +translocation O +is O +an O +inherent O +and O +structurally O +- O +optimized O +property O +of O +the O +ribosome B-complex_assembly +, O +supported O +also O +by O +translocation O +activity O +in O +the O +absence B-protein_state +of I-protein_state +the O +elongation B-protein_type +factor I-protein_type +. O + +This O +property O +is O +rendered O +by O +the O +relative O +mobility O +of O +the O +three O +major O +building O +blocks O +, O +the O +60S B-complex_assembly +subunit B-structure_element +and O +the O +40S B-complex_assembly +head B-structure_element +and O +body B-structure_element +, O +assisted O +by O +ligand B-structure_element +- I-structure_element +interacting I-structure_element +extensions I-structure_element +including O +the O +L1 B-structure_element +stalk I-structure_element +and O +the O +P B-structure_element +stalk I-structure_element +. O + +Viral B-taxonomy_domain +mRNAs B-chemical +have O +evolved O +to O +adopt O +an O +atypical O +structure B-evidence +to O +employ O +the O +inherent O +ribosome B-complex_assembly +dynamics O +, O +to O +be O +able O +to O +hijack O +the O +host O +translational O +machinery O +in O +a O +simple O +fashion O +. O + +Our O +current O +understanding O +of O +macromolecular O +machines O +, O +such O +as O +the O +ribosome B-complex_assembly +, O +is O +often O +limited O +by O +a O +gap O +between O +biophysical B-experimental_method +/ I-experimental_method +biochemical I-experimental_method +studies I-experimental_method +and O +structural B-experimental_method +studies I-experimental_method +. O + +High O +- O +resolution O +crystal B-evidence +structures I-evidence +, O +on O +the O +other O +hand O +, O +can O +provide O +static O +images O +of O +an O +assembly O +, O +and O +the O +structural O +dynamics O +can O +only O +be O +inferred O +by O +comparing O +structures B-evidence +that O +are O +usually O +obtained O +in O +different O +experiments O +and O +under O +different O +, O +often O +non O +- O +native O +, O +conditions O +. O + +Cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +offers O +the O +possibility O +of O +obtaining O +integrated O +information O +of O +both O +structure B-evidence +and O +dynamics O +as O +demonstrated O +in O +lower O +- O +resolution O +studies O +of O +bacterial B-taxonomy_domain +ribosome B-complex_assembly +complexes O +. O + +This O +is O +presumably O +one O +of O +the O +reasons O +why O +most O +recent O +studies O +of O +ribosome B-complex_assembly +complexes O +have O +focused O +on O +a O +single O +high O +- O +resolution O +structure B-evidence +despite O +the O +non O +- O +uniform O +local O +resolution O +of O +the O +maps B-evidence +that O +likely O +reflects O +structural O +heterogeneity O +. O + +The O +computational O +efficiency O +of O +FREALIGN B-experimental_method +has O +allowed O +us O +to O +classify O +a O +relatively O +large O +dataset O +( O +1 O +. O +1 O +million O +particles B-experimental_method +) O +into O +15 O +classes O +( O +Figure O +1 O +— O +figure O +supplement O +2 O +) O +and O +obtain O +eight O +near O +- O +atomic O +- O +resolution O +structures B-evidence +from O +it O +. O + +The O +classification O +, O +which O +followed O +an O +initial O +alignment O +of O +all O +particles B-experimental_method +to O +a O +single O +reference O +, O +required O +about O +130 O +, O +000 O +CPU O +hours O +or O +about O +five O +to O +six O +full O +days O +on O +a O +1000 O +- O +CPU O +cluster O +. O + +Therefore O +, O +cryo B-experimental_method +- I-experimental_method +EM I-experimental_method +has O +the O +potential O +to O +become O +a O +standard O +tool O +for O +uncovering O +detailed O +dynamic O +pathways O +of O +complex O +macromolecular O +machines O +. O + +The O +N O +- O +terminal O +propeptides B-structure_element +protecting O +the O +active B-site +- I-site +site I-site +threonines B-residue_name +are O +autocatalytically B-ptm +released O +only O +on O +completion O +of O +assembly O +. O + +However O +, O +the O +trigger O +for O +the O +self O +- O +activation O +and O +the O +reason O +for O +the O +strict B-protein_state +conservation I-protein_state +of O +threonine B-residue_name +as O +the O +active O +site O +nucleophile O +remain O +enigmatic O +. O + +Here O +we O +use O +mutagenesis B-experimental_method +, O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +and O +biochemical B-experimental_method +assays I-experimental_method +to O +suggest O +that O +Lys33 B-residue_name_number +initiates O +nucleophilic O +attack O +of O +the O +propeptide B-structure_element +by O +deprotonating O +the O +Thr1 B-residue_name_number +hydroxyl O +group O +and O +that O +both O +residues O +together O +with O +Asp17 B-residue_name_number +are O +part O +of O +a O +catalytic B-site +triad I-site +. O + +Substitution B-experimental_method +of O +Thr1 B-residue_name_number +by O +Cys B-residue_name +disrupts O +the O +interaction O +with O +Lys33 B-residue_name_number +and O +inactivates B-protein_state +the O +proteasome B-complex_assembly +. O + +Although O +a O +Thr1Ser B-mutant +mutant B-protein_state +is O +active B-protein_state +, O +it O +is O +less O +efficient O +compared O +with O +wild B-protein_state +type I-protein_state +because O +of O +the O +unfavourable O +orientation O +of O +Ser1 B-residue_name_number +towards O +incoming O +substrates O +. O + +The O +proteasome B-complex_assembly +, O +an O +essential O +molecular O +machine O +, O +is O +a O +threonine B-protein_type +protease I-protein_type +, O +but O +the O +evolution O +and O +the O +components O +of O +its O +proteolytic O +centre O +are O +unclear O +. O + +Here O +, O +the O +authors O +use O +structural O +biology O +and O +biochemistry O +to O +investigate O +the O +role O +of O +proteasome B-complex_assembly +active B-site +site I-site +residues O +on O +maturation O +and O +activity O +. O + +The O +20S B-complex_assembly +proteasome I-complex_assembly +core I-complex_assembly +particle I-complex_assembly +( O +CP B-complex_assembly +) O +is O +the O +key O +non B-protein_type +- I-protein_type +lysosomal I-protein_type +protease I-protein_type +of O +eukaryotic B-taxonomy_domain +cells O +. O + +Its O +seven O +different O +α B-protein +and O +seven O +different O +β B-protein +subunits I-protein +assemble O +into O +four O +heptameric B-oligomeric_state +rings B-structure_element +that O +are O +stacked O +on O +each O +other O +to O +form O +a O +hollow B-structure_element +cylinder I-structure_element +. O + +Release O +of O +the O +propeptides B-structure_element +creates O +a O +functionally O +active B-protein_state +CP B-complex_assembly +that O +cleaves O +proteins O +into O +short O +peptides O +. O + +Nucleophilic O +attack O +of O +Thr1Oγ B-residue_name_number +on O +the O +carbonyl O +carbon O +atom O +of O +the O +scissile O +peptide O +bond O +creates O +a O +first O +cleavage O +product O +and O +a O +covalent O +acyl O +- O +enzyme O +intermediate O +. O + +Hydrolysis O +of O +this O +complex B-complex_assembly +by O +the O +addition O +of O +a O +nucleophilic O +water B-chemical +molecule O +regenerates O +the O +enzyme B-complex_assembly +and O +releases O +the O +second O +peptide B-chemical +fragment O +. O + +The O +proteasome B-complex_assembly +belongs O +to O +the O +family O +of O +N B-protein_type +- I-protein_type +terminal I-protein_type +nucleophilic I-protein_type +( I-protein_type +Ntn I-protein_type +) I-protein_type +hydrolases I-protein_type +, O +and O +the O +free B-protein_state +N O +- O +terminal O +amine O +group O +of O +Thr1 B-residue_name_number +was O +proposed O +to O +deprotonate O +the O +Thr1 B-residue_name_number +hydroxyl O +group O +to O +generate O +a O +nucleophilic O +Thr1Oγ B-residue_name_number +for O +peptide O +- O +bond O +cleavage O +. O + +An O +alternative O +candidate O +for O +deprotonating O +the O +Thr1 B-residue_name_number +hydroxyl O +group O +is O +the O +side O +chain O +of O +Lys33 B-residue_name_number +as O +it O +is O +within O +hydrogen B-bond_interaction +- I-bond_interaction +bonding I-bond_interaction +distance O +to O +Thr1OH B-residue_name_number +( O +2 O +. O +7 O +Å O +). O + +In O +principle O +it O +could O +function O +as O +the O +general O +base O +during O +both O +autocatalytic B-ptm +removal I-ptm +of O +the O +propeptide B-structure_element +and O +protein O +substrate O +cleavage O +. O + +Here O +we O +provide O +experimental O +evidences O +for O +this O +distinct O +view O +of O +the O +proteasome B-complex_assembly +active B-site +- I-site +site I-site +mechanism O +. O + +Furthermore O +, O +we O +determine O +the O +advantages O +of O +Thr B-residue_name +over O +Cys B-residue_name +or O +Ser B-residue_name +as O +the O +active O +- O +site O +nucleophile O +using O +X B-experimental_method +- I-experimental_method +ray I-experimental_method +crystallography I-experimental_method +together O +with O +activity B-experimental_method +and I-experimental_method +inhibition I-experimental_method +assays I-experimental_method +. O + +Proteasome B-complex_assembly +- O +mediated O +degradation O +of O +cell O +- O +cycle O +regulators O +and O +potentially O +toxic O +misfolded O +proteins O +is O +required O +for O +the O +viability O +of O +eukaryotic B-taxonomy_domain +cells O +. O + +Viability O +is O +restored O +if O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +subunit O +has O +its O +propeptide B-structure_element +( O +pp B-chemical +) O +deleted B-experimental_method +but I-experimental_method +expressed I-experimental_method +separately I-experimental_method +in O +trans B-protein_state +( O +β5 B-mutant +- I-mutant +T1A I-mutant +pp B-chemical +trans B-protein_state +), O +although O +substantial O +phenotypic O +impairment O +remains O +( O +Table O +1 O +). O + +Sequencing B-experimental_method +of I-experimental_method +the I-experimental_method +plasmids I-experimental_method +, O +testing O +them O +in O +both O +published O +yeast B-taxonomy_domain +strain O +backgrounds O +and O +site B-experimental_method +- I-experimental_method +directed I-experimental_method +mutagenesis I-experimental_method +revealed O +that O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +mutant B-protein_state +pp B-chemical +cis B-protein_state +is O +viable O +, O +but O +suffers O +from O +a O +marked O +growth O +defect O +that O +requires O +extended O +incubation O +of O +4 O +– O +5 O +days O +for O +initial O +colony O +formation O +( O +Table O +1 O +and O +Supplementary O +Methods O +). O + +We O +also O +identified O +an O +additional O +point O +mutation O +K81R B-mutant +in O +subunit O +β5 B-protein +that O +was O +present O +in O +the O +allele O +used O +in O +ref O +.. O +This B-experimental_method +single I-experimental_method +amino I-experimental_method +- I-experimental_method +acid I-experimental_method +exchange I-experimental_method +is O +located O +at O +the O +interface B-site +of O +the O +subunits O +α4 B-protein +, O +β4 B-protein +and O +β5 B-protein +( O +Supplementary O +Fig O +. O +1b O +) O +and O +might O +weakly O +promote O +CP B-complex_assembly +assembly O +by O +enhancing O +inter O +- O +subunit O +contacts O +. O + +The O +slightly O +better O +growth O +of O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +mutant B-protein_state +allowed O +us O +to O +solve O +the O +crystal B-evidence +structure I-evidence +of O +a O +yeast B-taxonomy_domain +proteasome B-complex_assembly +( O +yCP B-complex_assembly +) O +with O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +mutation O +, O +which O +is O +discussed O +in O +the O +following O +section O +( O +for O +details O +see O +Supplementary O +Note O +1 O +). O + +Propeptide B-structure_element +conformation O +and O +triggering O +of O +autolysis B-ptm + +In O +the O +final O +steps O +of O +proteasome B-complex_assembly +biogenesis O +, O +the O +propeptides B-structure_element +are O +autocatalytically B-ptm +cleaved I-ptm +from O +the O +mature B-protein_state +β B-protein +- I-protein +subunit I-protein +domains I-protein +. O + +For O +subunit O +β1 B-protein +, O +this O +process O +was O +previously O +inferred O +to O +require O +that O +the O +propeptide B-structure_element +residue O +at O +position O +(- B-residue_number +2 I-residue_number +) I-residue_number +of O +the O +subunit O +precursor O +occupies O +the O +S1 B-site +specificity I-site +pocket I-site +of O +the O +substrate B-site +- I-site +binding I-site +channel I-site +formed O +by O +amino O +acid O +45 B-residue_number +( O +for O +details O +see O +Supplementary O +Note O +2 O +). O + +Furthermore O +, O +it O +was O +observed O +that O +the O +prosegment B-structure_element +forms O +an O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +in O +the O +active B-site +site I-site +, O +and O +that O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +adopts O +a O +γ B-structure_element +- I-structure_element +turn I-structure_element +conformation I-structure_element +, O +which O +by O +definition O +is O +characterized O +by O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +Leu B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +O O +and O +Thr1NH B-residue_name_number +( O +ref O +.). O + +Here O +we O +again O +analysed O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +mutant B-protein_state +crystallographically B-experimental_method +but O +in O +addition O +determined O +the O +structures B-evidence +of O +the O +β2 B-mutant +- I-mutant +T1A I-mutant +single O +and O +β1 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +β2 I-mutant +- I-mutant +T1A I-mutant +double O +mutants O +( O +Protein O +Data O +Bank O +( O +PDB O +) O +entry O +codes O +are O +provided O +in O +Supplementary O +Table O +1 O +). O + +However O +, O +the O +γ B-structure_element +- I-structure_element +turn I-structure_element +conformation I-structure_element +and O +the O +associated O +hydrogen B-bond_interaction +bond I-bond_interaction +initially O +proposed O +is O +for O +geometric O +and O +chemical O +reasons O +inappropriate O +and O +would O +not O +perfectly O +position O +the O +carbonyl O +carbon O +atom O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +for O +nucleophilic O +attack O +by O +Thr1 B-residue_name_number +. O + +Surprisingly O +, O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +is O +completely O +extended O +and O +forces O +the O +histidine B-residue_name +side O +chain O +at O +position O +(- B-residue_number +2 I-residue_number +) I-residue_number +to O +occupy O +the O +S2 B-site +instead O +of O +the O +S1 B-site +pocket I-site +, O +thereby O +disrupting O +the O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +. O + +Nonetheless O +, O +the O +carbonyl O +carbon O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +would O +be O +ideally O +placed O +for O +nucleophilic O +attack O +by O +Thr1Oγ B-residue_name_number +( O +Fig O +. O +1c O +and O +Supplementary O +Fig O +. O +2c O +, O +d O +). O + +As O +the O +K81R B-mutant +mutation O +is O +located O +far O +from O +the O +active B-site +site I-site +( O +Thr1Cα B-residue_name_number +– O +Arg81Cα B-residue_name_number +: O +24 O +Å O +), O +any O +influence O +on O +propeptide B-structure_element +conformation O +can O +be O +excluded O +. O + +Instead O +, O +the O +plasticity O +of O +the O +β5 B-protein +S1 B-site +pocket I-site +caused O +by O +the O +rotational O +flexibility O +of O +Met45 B-residue_name_number +might O +prevent O +stable O +accommodation O +of O +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +in O +the O +S1 B-site +site I-site +and O +thus O +also O +promote O +its O +immediate O +release O +after O +autolysis B-ptm +. O + +Remarkably O +, O +eukaryotic B-taxonomy_domain +proteasomal O +β5 B-protein +subunits O +bear O +a O +His B-residue_name +residue O +in O +position O +(- B-residue_number +2 I-residue_number +) I-residue_number +of O +the O +propeptide B-structure_element +( O +Supplementary O +Fig O +. O +3a O +). O + +In O +agreement O +, O +the O +chymotrypsin O +- O +like O +( O +ChT O +- O +L O +) O +activity O +of O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +N I-mutant +and O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +F I-mutant +mutant B-protein_state +yCPs B-complex_assembly +was O +impaired O +in O +situ O +and O +in O +vitro O +( O +Supplementary O +Fig O +. O +3c O +). O + +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +and O +Phe B-residue_name +/ O +Lys B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +were O +visualized O +at O +low O +occupancy O +, O +while O +Ala B-residue_name +/ O +Asn B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +could O +not O +be O +assigned O +. O + +This O +observation O +indicates O +a O +mixture O +of O +processed B-protein_state +and O +unprocessed B-protein_state +β5 B-protein +subunits O +and O +partially O +impaired O +autolysis B-ptm +, O +thereby O +excluding O +any O +essential O +role O +of O +residue O +(- B-residue_number +2 I-residue_number +) I-residue_number +as O +the O +general O +base O +. O + +Next O +, O +we O +examined O +the O +effect O +of O +residue O +(- B-residue_number +2 I-residue_number +) I-residue_number +on O +the O +orientation O +of O +the O +propeptide B-structure_element +by O +creating B-experimental_method +mutants I-experimental_method +that I-experimental_method +combine I-experimental_method +the O +T1A B-mutant +( O +K81R B-mutant +) O +mutation B-experimental_method +( I-experimental_method +s I-experimental_method +) I-experimental_method +with O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +L I-mutant +, O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +T I-mutant +or O +H B-mutant +(- I-mutant +2 I-mutant +) I-mutant +A I-mutant +substitutions B-experimental_method +. O + +Leu B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +is O +encoded O +in O +the O +yeast B-taxonomy_domain +β1 B-protein +subunit O +precursor O +( O +Supplementary O +Fig O +. O +3a O +); O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +is O +generally O +part O +of O +β2 B-protein +- O +propeptides B-structure_element +( O +Supplementary O +Fig O +. O +3a O +); O +and O +Ala B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +was O +expected O +to O +fit O +the O +β5 B-protein +- O +S1 B-site +pocket I-site +without O +inducing O +conformational O +changes O +of O +Met45 B-residue_name_number +, O +allowing O +it O +to O +accommodate O +‘ O +β1 O +- O +like O +' O +propeptide O +positioning O +. O + +As O +expected O +from O +β5 B-mutant +- I-mutant +T1A I-mutant +mutants O +, O +the O +yeasts B-taxonomy_domain +show O +severe O +growth O +phenotypes O +, O +with O +minor O +variations O +( O +Supplementary O +Fig O +. O +4a O +and O +Table O +1 O +). O + +We O +determined O +crystal B-evidence +structures I-evidence +of O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +L I-mutant +- I-mutant +T1A I-mutant +, O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +T I-mutant +- I-mutant +T1A I-mutant +and O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +A I-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +mutants O +( O +Supplementary O +Table O +1 O +). O + +For O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +A I-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +variant O +, O +only O +the O +residues O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +and O +Ala B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +could O +be O +visualized O +, O +indicating O +that O +Ala B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +leads O +to O +insufficient O +stabilization O +of O +the O +propeptide B-structure_element +in O +the O +substrate B-site +- I-site +binding I-site +channel I-site +( O +Supplementary O +Fig O +. O +4d O +). O + +By O +contrast O +, O +the O +prosegments B-structure_element +of O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +L I-mutant +- I-mutant +T1A I-mutant +and O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +T I-mutant +- I-mutant +T1A I-mutant +mutants O +were O +significantly O +better O +resolved O +in O +the O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +- I-evidence +density I-evidence +maps I-evidence +yet O +not O +at O +full O +occupancy O +( O +Supplementary O +Fig O +. O +4b O +, O +c O +and O +Supplementary O +Table O +1 O +), O +suggesting O +that O +the O +natural O +propeptide B-structure_element +bearing O +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +is O +most O +favourable O +. O + +This O +result O +proves O +that O +the O +naturally O +occurring O +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +of O +the O +β5 B-protein +propeptide B-structure_element +does O +not O +stably O +fit O +into O +the O +S1 B-site +site I-site +. O + +Since O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +adopts O +the O +same O +position O +in O +both O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +WT B-protein_state +) O +and O +mutant B-protein_state +β5 B-protein +propeptides B-structure_element +, O +and O +since O +in O +all O +cases O +its O +carbonyl O +carbon O +is O +perfectly O +placed O +for O +nucleophilic O +attack O +by O +Thr1Oγ B-residue_name_number +( O +Fig O +. O +2b O +), O +we O +propose O +that O +neither O +binding O +of O +residue O +(- B-residue_number +2 I-residue_number +) I-residue_number +to O +the O +S1 B-site +pocket I-site +nor O +formation O +of O +the O +antiparallel B-structure_element +β I-structure_element +- I-structure_element +sheet I-structure_element +is O +essential O +for O +autolysis B-ptm +of O +the O +propeptide B-structure_element +. O + +Next O +, O +we O +determined O +the O +crystal B-evidence +structure I-evidence +of O +a O +chimeric B-protein_state +yCP B-complex_assembly +having O +the O +yeast B-taxonomy_domain +β1 B-protein +- O +propeptide B-structure_element +replaced B-experimental_method +by I-experimental_method +its O +β5 B-protein +counterpart B-structure_element +. O + +As O +proven O +by O +the O +β2 B-mutant +- I-mutant +T1A I-mutant +crystal B-evidence +structures I-evidence +, O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +O O +. O +Although O +this O +interaction O +was O +not O +observed O +for O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +T I-mutant +- I-mutant +T1A I-mutant +mutant B-protein_state +( O +Fig O +. O +2c O +and O +Supplementary O +Fig O +. O +4c O +, O +i O +), O +exchange B-experimental_method +of O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +by O +Val B-residue_name +in O +β2 B-protein +, O +a O +conservative O +mutation O +regarding O +size O +but O +drastic O +with O +respect O +to O +polarity O +, O +was O +found O +to O +inhibit O +maturation O +of O +this O +subunit O +( O +Fig O +. O +2d O +and O +Supplementary O +Fig O +. O +4e O +, O +j O +). O + +In O +particular O +, O +Val B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +is O +displaced O +from O +the O +S1 B-site +site I-site +and O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +is O +severely O +shifted O +( O +movement O +of O +the O +carbonyl O +oxygen O +atom O +of O +3 O +. O +8 O +Å O +), O +thereby O +preventing O +nucleophilic O +attack O +of O +Thr1 B-residue_name_number +( O +Fig O +. O +2d O +and O +Supplementary O +Fig O +. O +4j O +, O +k O +). O + +These O +results O +further O +confirm O +that O +correct O +positioning O +of O +the O +active B-site +- I-site +site I-site +residues I-site +and O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +is O +decisive O +for O +the O +maturation O +of O +the O +proteasome B-complex_assembly +. O + +Proton O +shuttling O +from O +the O +proteasomal O +active B-site +site I-site +Thr1OH B-residue_name_number +to O +Thr1NH2 B-residue_name_number +via O +a O +nucleophilic O +water B-chemical +molecule O +was O +suggested O +to O +initiate O +peptide O +- O +bond O +hydrolysis O +. O + +However O +, O +in O +the O +immature B-protein_state +particle B-complex_assembly +Thr1NH2 B-residue_name_number +is O +blocked O +by O +the O +propeptide B-structure_element +and O +cannot O +activate O +Thr1Oγ B-residue_name_number +. O + +A O +proposed O +catalytic B-site +tetrad I-site +model O +involving O +Thr1OH B-residue_name_number +, O +Thr1NH2 B-residue_name_number +, O +Lys33NH2 B-residue_name_number +and O +Asp17Oδ B-residue_name_number +, O +as O +well O +as O +a O +nucleophilic O +water B-chemical +molecule O +as O +the O +proton O +shuttle O +appeared O +to O +accommodate O +all O +possible O +views O +of O +the O +proteasomal O +active B-site +site I-site +. O + +Twenty O +years O +later O +, O +with O +a O +plethora O +of O +yCP B-complex_assembly +X B-evidence +- I-evidence +ray I-evidence +structures I-evidence +in O +hand O +, O +we O +decided O +to O +re O +- O +analyse O +the O +active B-site +site I-site +of O +the O +proteasome B-complex_assembly +and O +to O +resolve O +the O +uncertainty O +regarding O +the O +nature O +of O +the O +general O +base O +. O + +This O +discrepancy O +in O +growth O +was O +traced O +to O +an O +additional O +point O +mutation O +L B-mutant +(- I-mutant +49 I-mutant +) I-mutant +S I-mutant +in O +the O +β5 B-protein +- O +propeptide B-structure_element +of O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +mutant B-protein_state +( O +see O +also O +Supplementary O +Note O +1 O +). O + +This O +structural O +alteration O +destroys O +active B-site +- I-site +site I-site +integrity O +and O +abolishes O +catalytic O +activity O +of O +the O +β5 B-protein +active B-site +site I-site +( O +Supplementary O +Fig O +. O +5a O +). O + +Additional O +proof O +for O +the O +key O +function O +of O +Lys33 B-residue_name_number +was O +obtained O +from O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +mutant B-protein_state +, O +with O +the O +propeptide B-structure_element +expressed B-experimental_method +separately I-experimental_method +from O +the O +main O +subunit O +( O +pp B-chemical +trans B-protein_state +). O + +The O +Thr1 B-residue_name_number +N O +terminus O +of O +this O +mutant B-protein_state +is O +not O +blocked O +by O +the O +propeptide B-structure_element +, O +yet O +its O +catalytic O +activity O +is O +reduced O +by O +∼ O +83 O +% O +( O +Supplementary O +Fig O +. O +6b O +). O + +Consistent O +with O +this O +, O +the O +crystal B-evidence +structure I-evidence +of O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +pp B-chemical +trans B-protein_state +mutant B-protein_state +in B-protein_state +complex I-protein_state +with I-protein_state +carfilzomib B-chemical +only O +showed O +partial O +occupancy O +of O +the O +ligand O +at O +the O +β5 B-protein +active B-site +sites I-site +( O +Supplementary O +Fig O +. O +5b O +and O +Supplementary O +Table O +1 O +). O + +Since O +no O +acetylation B-ptm +of O +the O +Thr1 B-residue_name_number +N O +terminus O +was O +observed O +for O +the O +β5 B-mutant +- I-mutant +K33A I-mutant +pp B-chemical +trans B-protein_state +apo B-protein_state +crystal B-evidence +structure I-evidence +, O +the O +reduced O +reactivity O +towards O +substrates O +and O +inhibitors O +indicates O +that O +Lys33NH2 B-residue_name_number +, O +rather O +than O +Thr1NH2 B-residue_name_number +, O +deprotonates O +and O +activates O +Thr1OH B-residue_name_number +. O + +Remarkably O +, O +the O +solvent O +molecule O +occupies O +the O +position O +normally O +taken O +by O +Lys33NH2 B-residue_name_number +in O +the O +WT B-protein_state +proteasome B-complex_assembly +structure B-evidence +( O +Fig O +. O +3c O +), O +further O +corroborating O +the O +essential O +role O +of O +Lys33 B-residue_name_number +as O +the O +general O +base O +for O +autolysis B-ptm +and O +proteolysis O +. O + +Conservative B-experimental_method +substitution I-experimental_method +of O +Lys33 B-residue_name_number +by O +Arg B-residue_name +delays O +autolysis B-ptm +of O +the O +β5 B-protein +precursor O +and O +impairs O +yeast B-taxonomy_domain +growth O +( O +for O +details O +see O +Supplementary O +Note O +1 O +). O + +While O +Thr1 B-residue_name_number +occupies O +the O +same O +position O +as O +in O +WT B-protein_state +yCPs B-complex_assembly +, O +Arg33 B-residue_name_number +is O +unable O +to O +hydrogen B-bond_interaction +bond I-bond_interaction +to O +Asp17 B-residue_name_number +, O +thereby O +inactivating O +the O +β5 B-protein +active B-site +site I-site +( O +Supplementary O +Fig O +. O +5e O +). O + +The O +conservative B-experimental_method +mutation I-experimental_method +of O +Asp17 B-residue_name_number +to O +Asn B-residue_name +in O +subunit O +β5 B-protein +of O +the O +yCP B-complex_assembly +also O +provokes O +a O +severe O +growth O +defect O +( O +Supplementary O +Note O +1 O +, O +Supplementary O +Fig O +. O +6a O +and O +Table O +1 O +). O + +Notably O +, O +only O +with O +the O +additional O +point O +mutation O +L B-mutant +(- I-mutant +49 I-mutant +) I-mutant +S I-mutant +present O +in O +the O +β5 B-protein +propeptide B-structure_element +could O +we O +purify O +a O +small O +amount O +of O +the O +β5 B-mutant +- I-mutant +D17N I-mutant +mutant B-protein_state +yCP B-complex_assembly +. O + +As O +determined O +by O +crystallographic B-experimental_method +analysis I-experimental_method +, O +this O +mutant B-protein_state +β5 B-protein +subunit O +was O +partially B-protein_state +processed I-protein_state +( O +Table O +1 O +) O +but O +displayed O +impaired O +reactivity O +towards O +the O +proteasome B-complex_assembly +inhibitor O +carfilzomib B-chemical +compared O +with O +the O +subunits O +β1 B-protein +and O +β2 B-protein +, O +and O +with O +WT B-protein_state +β5 B-protein +( O +Supplementary O +Fig O +. O +7a O +). O + +Autolysis B-ptm +and O +residual O +catalytic O +activity O +of O +the O +β5 B-mutant +- I-mutant +D17N I-mutant +mutants O +may O +originate O +from O +the O +carbonyl O +group O +of O +Asn17 B-residue_name_number +, O +which O +albeit O +to O +a O +lower O +degree O +still O +can O +polarize O +Lys33 B-residue_name_number +for O +the O +activation O +of O +Thr1 B-residue_name_number +. O + +Strikingly O +, O +although O +the O +X B-evidence +- I-evidence +ray I-evidence +data I-evidence +on O +the O +β5 B-mutant +- I-mutant +D17N I-mutant +mutant B-protein_state +with O +the O +propeptide B-structure_element +expressed B-experimental_method +in O +cis B-protein_state +and O +in O +trans B-protein_state +looked O +similar O +, O +there O +was O +a O +pronounced O +difference O +in O +their O +growth O +phenotypes O +observed O +( O +Supplementary O +Fig O +. O +6a O +and O +Supplementary O +Fig O +. O +7b O +). O + +This O +model O +is O +also O +consistent O +with O +the O +fact O +that O +no O +defined O +water B-chemical +molecule O +is O +observed O +in O +the O +mature B-protein_state +WT B-protein_state +proteasomal O +active B-site +site I-site +that O +could O +shuttle O +the O +proton O +from O +Thr1Oγ B-residue_name_number +to O +Thr1NH2 B-residue_name_number +. O + +To O +explore O +this O +active B-site +- I-site +site I-site +model O +further O +, O +we O +exchanged B-experimental_method +the I-experimental_method +conserved I-experimental_method +Asp166 B-residue_name_number +residue O +for O +Asn B-residue_name +in O +the O +yeast B-taxonomy_domain +β5 B-protein +subunit O +. O + +Asp166Oδ B-residue_name_number +is O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +Thr1NH2 B-residue_name_number +via O +Ser129OH B-residue_name_number +and O +Ser169OH B-residue_name_number +, O +and O +therefore O +was O +proposed O +to O +be O +involved O +in O +catalysis O +. O + +Instead O +, O +a O +water B-chemical +molecule O +is O +bound B-protein_state +to I-protein_state +Ser129OH B-residue_name_number +and O +Thr1NH2 B-residue_name_number +( O +Supplementary O +Fig O +. O +8b O +), O +which O +may O +enable O +precursor B-ptm +processing I-ptm +. O + +The O +hydrogen B-bond_interaction +bonds I-bond_interaction +involving O +Ser169OH B-residue_name_number +are O +intact O +and O +may O +account O +for O +residual O +substrate O +turnover O +. O + +In O +the O +carfilzomib B-complex_assembly +complex I-complex_assembly +structure B-evidence +, O +Thr1Oγ B-residue_name_number +and O +Thr1N B-residue_name_number +incorporate O +into O +a O +morpholine O +ring O +structure O +and O +Ser129 B-residue_name_number +adopts O +its O +WT B-protein_state +- O +like O +orientation O +. O + +In O +the O +MG132 B-protein_state +- I-protein_state +bound I-protein_state +state I-protein_state +, O +Thr1N B-residue_name_number +is O +unmodified B-protein_state +, O +and O +we O +again O +observe O +that O +Ser129 B-residue_name_number +is O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +a O +water B-chemical +molecule O +instead O +of O +Asn166 B-residue_name_number +. O + +Whereas O +Asn B-residue_name +can O +to O +some O +degree O +replace O +Asp166 B-residue_name_number +due O +to O +its O +carbonyl O +group O +in O +the O +side O +chain O +, O +Ala B-residue_name +at O +this O +position O +was O +found O +to O +prevent O +both O +autolysis B-ptm +and O +catalysis O +. O + +Substitution B-experimental_method +of O +the O +active B-site +- I-site +site I-site +Thr1 B-residue_name_number +by O +Cys B-residue_name + +Mutation B-experimental_method +of O +Thr1 B-residue_name_number +to O +Cys B-residue_name +inactivates O +the O +20S B-complex_assembly +proteasome I-complex_assembly +from O +the O +archaeon B-taxonomy_domain +T B-species +. I-species +acidophilum I-species +. O + +In O +yeast B-taxonomy_domain +, O +this O +mutation B-experimental_method +causes O +a O +strong O +growth O +defect O +( O +Fig O +. O +4a O +and O +Table O +1 O +), O +although O +the O +propeptide B-structure_element +is O +hydrolysed O +, O +as O +shown O +here O +by O +its O +X B-evidence +- I-evidence +ray I-evidence +structure I-evidence +. O + +In O +one O +of O +the O +two O +β5 B-protein +subunits O +, O +however O +, O +we O +found O +the O +cleaved B-protein_state +propeptide B-structure_element +still B-protein_state +bound I-protein_state +in O +the O +substrate B-site +- I-site +binding I-site +channel I-site +( O +Fig O +. O +4c O +). O + +On O +the O +basis O +of O +the O +phenotype O +of O +the O +T1C B-mutant +mutant B-protein_state +and O +the O +propeptide B-structure_element +remnant O +identified O +in O +its O +active B-site +site I-site +, O +we O +suppose O +that O +autolysis B-ptm +is O +retarded O +and O +may O +not O +have O +been O +completed O +before O +crystallization B-experimental_method +. O + +In O +agreement O +, O +soaking B-experimental_method +crystals I-experimental_method +with O +the O +CP B-complex_assembly +inhibitors O +bortezomib B-chemical +or O +carfilzomib B-chemical +modifies O +only O +the O +β1 B-protein +and O +β2 B-protein +active B-site +sites I-site +, O +while O +leaving O +the O +β5 B-mutant +- I-mutant +T1C I-mutant +proteolytic B-site +centres I-site +unmodified B-protein_state +even O +though O +they O +are O +only O +partially O +occupied O +by O +the O +cleaved B-protein_state +propeptide B-structure_element +remnant O +. O + +Consequently O +, O +the O +hydrogen B-bond_interaction +bond I-bond_interaction +bridging O +the O +active O +- O +site O +nucleophile O +and O +Lys33 B-residue_name_number +in O +WT B-protein_state +CPs B-complex_assembly +is O +broken O +with O +Cys1 B-residue_name_number +. O + +Together O +, O +these O +observations O +suggest O +that O +efficient O +peptide O +- O +bond O +hydrolysis O +requires O +that O +Lys33NH2 B-residue_name_number +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +the O +active O +site O +nucleophile O +. O + +The O +benefit O +of O +Thr B-residue_name +over O +Ser B-residue_name +as O +the O +active O +- O +site O +nucleophile O + +All O +proteasomes B-complex_assembly +strictly B-protein_state +employ I-protein_state +threonine B-residue_name +as O +the O +active B-site +- I-site +site I-site +residue I-site +instead O +of O +serine B-residue_name +. O + +To O +investigate O +the O +reason O +for O +this O +singularity O +, O +we O +analysed O +a O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +, O +which O +is O +viable O +but O +suffers O +from O +growth O +defects O +( O +Fig O +. O +4a O +and O +Table O +1 O +). O + +Activity B-experimental_method +assays I-experimental_method +with O +the O +β5 B-protein +- O +specific O +substrate O +Suc B-chemical +- I-chemical +LLVY I-chemical +- I-chemical +AMC I-chemical +demonstrated O +that O +the O +ChT O +- O +L O +activity O +of O +the O +T1S B-mutant +mutant B-protein_state +is O +reduced O +by O +40 O +– O +45 O +% O +compared O +with O +WT B-protein_state +proteasomes B-complex_assembly +depending O +on O +the O +incubation O +temperature O +( O +Fig O +. O +4b O +and O +Supplementary O +Fig O +. O +9c O +). O + +By O +contrast O +, O +turnover O +of O +the O +substrate O +Z B-chemical +- I-chemical +GGL I-chemical +- I-chemical +pNA I-chemical +, O +used O +to O +monitor O +ChT O +- O +L O +activity O +in O +situ O +but O +in O +a O +less O +quantitative O +fashion O +, O +is O +not O +detectably O +impaired O +( O +Supplementary O +Fig O +. O +9a O +). O + +Compared O +with O +Thr1Oγ B-residue_name_number +in O +WT B-protein_state +CP B-complex_assembly +structures B-evidence +, O +Ser1Oγ B-residue_name_number +is O +rotated O +by O +60 O +°. O + +The O +active B-site +- I-site +site I-site +residue I-site +Thr1 B-residue_name_number +is O +fixed O +in O +its O +position O +, O +as O +its O +methyl O +group O +is O +engaged O +in O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +Thr3 B-residue_name_number +and O +Ala46 B-residue_name_number +( O +Fig O +. O +4h O +). O + +Consequently O +, O +the O +hydroxyl O +group O +of O +Thr1 B-residue_name_number +requires O +no O +reorientation O +before O +substrate O +cleavage O +and O +is O +thus O +more O +catalytically O +efficient O +than O +Ser1 B-residue_name_number +. O + +In O +vitro O +, O +the O +mutant B-protein_state +proteasome B-complex_assembly +is O +less O +susceptible O +to O +proteasome B-complex_assembly +inhibition O +by O +bortezomib B-chemical +( O +3 O +. O +7 O +- O +fold O +) O +and O +carfilzomib B-chemical +( O +1 O +. O +8 O +- O +fold O +; O +Fig O +. O +5 O +). O + +Nevertheless O +, O +inhibitor B-complex_assembly +complex I-complex_assembly +structures B-evidence +indicate O +identical O +binding O +modes O +compared O +with O +the O +WT B-protein_state +yCP B-complex_assembly +structures B-evidence +, O +with B-protein_state +the I-protein_state +same I-protein_state +inhibitors I-protein_state +. O + +Notably O +, O +the O +affinity B-evidence +of O +the O +tetrapeptide O +carfilzomib B-chemical +is O +less O +impaired O +, O +as O +it O +is O +better O +stabilized O +in O +the O +substrate B-site +- I-site +binding I-site +channel I-site +than O +the O +dipeptide O +bortezomib B-chemical +, O +which O +lacks O +a O +defined O +P3 O +site O +and O +has O +only O +a O +few O +interactions O +with O +the O +surrounding O +protein O +. O + +Hence O +, O +the O +mean B-evidence +residence I-evidence +time I-evidence +of O +carfilzomib B-chemical +at O +the O +active B-site +site I-site +is O +prolonged O +and O +the O +probability O +to O +covalently O +react O +with O +Ser1 B-residue_name_number +is O +increased O +. O + +Considered O +together O +, O +these O +results O +provide O +a O +plausible O +explanation O +for O +the O +invariance O +of O +threonine B-residue_name +as O +the O +active O +- O +site O +nucleophile O +in O +proteasomes B-complex_assembly +in O +all O +three O +domains O +of O +life O +, O +as O +well O +as O +in O +proteasome B-protein_type +- I-protein_type +like I-protein_type +proteases I-protein_type +such O +as O +HslV B-protein +( O +ref O +.). O + +The O +β B-protein +- I-protein +subunit I-protein +propeptides B-structure_element +, O +particularly O +that O +of O +β5 B-protein +, O +are O +key O +factors O +that O +help O +drive O +proper O +assembly O +of O +the O +CP B-complex_assembly +complex O +. O + +In O +addition O +, O +they O +prevent O +irreversible O +inactivation O +of O +the O +Thr1 B-residue_name_number +N O +terminus O +by O +N B-ptm +- I-ptm +acetylation I-ptm +. O + +In O +eukaryotes B-taxonomy_domain +, O +deletion O +of O +or O +failure O +to O +cleave O +the O +β1 B-protein +and O +β2 B-protein +propeptides B-structure_element +is O +well O +tolerated O +. O + +However O +, O +removal B-experimental_method +of I-experimental_method +the O +β5 B-protein +prosegment B-structure_element +or O +any O +interference O +with O +its O +cleavage O +causes O +severe O +phenotypic O +defects O +. O + +Depending O +on O +the O +(- B-residue_number +2 I-residue_number +) I-residue_number +residue O +we O +observed O +various O +propeptide B-structure_element +conformations O +, O +but O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +is O +in O +all O +structures B-evidence +perfectly O +located O +for O +the O +nucleophilic O +attack O +by O +Thr1Oγ B-residue_name_number +, O +although O +it O +does O +not O +adopt O +the O +tight B-structure_element +turn I-structure_element +observed O +for O +the O +prosegment B-structure_element +of O +subunit O +β1 B-protein +. O + +In O +this O +regard O +, O +inappropriate O +N B-ptm +- I-ptm +acetylation I-ptm +of O +the O +Thr1 B-residue_name_number +N O +terminus O +cannot O +be O +removed O +by O +Thr1Oγ B-residue_name_number +due O +to O +the O +rotational O +freedom O +and O +flexibility O +of O +the O +acetyl O +group O +. O + +Autolytic O +activation O +of O +the O +CP B-complex_assembly +constitutes O +one O +of O +the O +final O +steps O +of O +proteasome O +biogenesis O +, O +but O +the O +trigger O +for O +propeptide B-ptm +cleavage I-ptm +had O +remained O +enigmatic O +. O + +We O +propose O +a O +catalytic B-site +triad I-site +for O +the O +active B-site +site I-site +of O +the O +CP B-complex_assembly +consisting O +of O +residues O +Thr1 B-residue_name_number +, O +Lys33 B-residue_name_number +and O +Asp B-residue_name +/ O +Glu17 B-residue_name_number +, O +which O +are O +conserved O +among O +all O +proteolytically O +active O +eukaryotic B-taxonomy_domain +, O +bacterial B-taxonomy_domain +and O +archaeal B-taxonomy_domain +proteasome B-complex_assembly +subunits O +. O + +Analogously O +to O +the O +proteasome B-complex_assembly +, O +a O +Thr B-site +– I-site +Lys I-site +– I-site +Asp I-site +triad I-site +is O +also O +found O +in O +L B-protein_type +- I-protein_type +asparaginase I-protein_type +. O + +Thus O +, O +specific O +protein O +surroundings O +can O +significantly O +alter O +the O +chemical O +properties O +of O +amino O +acids O +such O +as O +Lys B-residue_name +to O +function O +as O +an O +acid O +– O +base O +catalyst O +. O + +The O +resulting O +uncharged O +Thr1NH2 B-residue_name_number +is O +hydrogen B-bond_interaction +- I-bond_interaction +bridged I-bond_interaction +to O +the O +C3 O +- O +OH O +group O +. O + +Cleavage O +of O +the O +scissile O +peptide O +bond O +requires O +protonation O +of O +the O +emerging O +free O +amine O +, O +and O +in O +the O +proteasome B-complex_assembly +, O +the O +Thr1 B-residue_name_number +amine O +group O +is O +likely O +to O +assume O +this O +function O +. O + +Analogously O +, O +Thr1NH3 B-residue_name_number ++ O +might O +promote O +the O +bivalent O +reaction O +mode O +of O +epoxyketone O +inhibitors O +by O +protonating O +the O +epoxide O +moiety O +to O +create O +a O +positively O +charged O +trivalent O +oxygen O +atom O +that O +is O +subsequently O +nucleophilically O +attacked O +by O +Thr1NH2 B-residue_name_number +. O + +Breakdown O +of O +this O +tetrahedral O +transition O +state O +releases O +the O +Thr1 B-residue_name_number +N O +terminus O +that O +is O +protonated O +by O +aspartic B-residue_name_number +acid I-residue_name_number +166 I-residue_name_number +via O +Ser129OH B-residue_name_number +to O +yield O +Thr1NH3 B-residue_name_number ++. O + +The O +residues O +Ser129 B-residue_name_number +and O +Asp166 B-residue_name_number +are O +expected O +to O +increase O +the O +pKa O +value O +of O +Thr1N B-residue_name_number +, O +thereby O +favouring O +its O +charged O +state O +. O + +Consistent O +with O +playing O +an O +essential O +role O +in O +proton O +shuttling O +, O +the O +mutation B-experimental_method +D166A B-mutant +prevents O +autolysis B-ptm +of O +the O +archaeal B-taxonomy_domain +CP B-complex_assembly +and O +the O +exchange B-experimental_method +D166N B-mutant +impairs O +catalytic O +activity O +of O +the O +yeast B-taxonomy_domain +CP B-complex_assembly +about O +60 O +%. O + +This O +interpretation O +agrees O +with O +the O +strongly O +reduced O +catalytic O +activity O +of O +the O +β5 B-mutant +- I-mutant +D166N I-mutant +mutant B-protein_state +on O +the O +one O +hand O +, O +and O +the O +ability O +to O +react O +readily O +with O +carfilzomib B-chemical +on O +the O +other O +. O + +Hence O +, O +the O +proteasome B-complex_assembly +can O +be O +viewed O +as O +having O +a O +second B-site +triad I-site +that O +is O +essential O +for O +efficient O +proteolysis O +. O + +While O +Lys33NH2 B-residue_name_number +and O +Asp17Oδ B-residue_name_number +are O +required O +to O +deprotonate O +the O +Thr1 B-residue_name_number +hydroxyl O +side O +chain O +, O +Ser129OH B-residue_name_number +and O +Asp166OH B-residue_name_number +serve O +to O +protonate O +the O +N O +- O +terminal O +amine O +group O +of O +Thr1 B-residue_name_number +. O + +In O +accord O +with O +the O +proposed O +Thr1 B-residue_name_number +– O +Lys33 B-residue_name_number +– O +Asp17 B-residue_name_number +catalytic B-site +triad I-site +, O +crystallographic B-evidence +data I-evidence +on O +the O +proteolytically B-protein_state +inactive I-protein_state +β5 B-mutant +- I-mutant +T1C I-mutant +mutant B-protein_state +demonstrate O +that O +the O +interaction O +of O +Lys33NH2 B-residue_name_number +and O +Cys1 B-residue_name_number +is O +broken O +. O + +However O +, O +owing O +to O +Cys B-residue_name +being O +a O +strong O +nucleophile O +, O +the O +propeptide B-structure_element +can O +still O +be O +cleaved B-protein_state +off O +over O +time O +. O + +While O +only O +one O +single O +turnover O +is O +necessary O +for O +autolysis B-ptm +, O +continuous O +enzymatic O +activity O +is O +required O +for O +significant O +and O +detectable O +substrate O +hydrolysis O +. O + +Notably O +, O +in O +the O +Ntn B-protein_type +hydrolase I-protein_type +penicillin B-protein_type +acylase I-protein_type +, O +substitution B-experimental_method +of O +the O +catalytic B-protein_state +N O +- O +terminal O +Ser B-residue_name +residue O +by O +Cys B-residue_name +also O +inactivates B-protein_state +the O +enzyme B-protein_type +but O +still O +enables O +precursor B-ptm +processing I-ptm +. O + +To O +investigate O +why O +the O +CP B-complex_assembly +specifically O +employs O +threonine B-residue_name +as O +its O +active B-site +- I-site +site I-site +residue I-site +, O +we O +used O +a O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +of O +the O +yCP B-complex_assembly +and O +characterized O +it O +biochemically B-experimental_method +and I-experimental_method +structurally I-experimental_method +. O + +Structural B-evidence +analyses I-evidence +support O +these O +findings O +with O +the O +T1S B-mutant +mutant B-protein_state +and O +provide O +an O +explanation O +for O +the O +strict B-protein_state +use I-protein_state +of I-protein_state +Thr B-residue_name +residues O +in O +proteasomes B-complex_assembly +. O + +Notably O +, O +proteolytically B-protein_state +active I-protein_state +proteasome B-complex_assembly +subunits O +from O +archaea B-taxonomy_domain +, O +yeast B-taxonomy_domain +and O +mammals B-taxonomy_domain +, O +including O +constitutive O +, O +immuno O +- O +and O +thymoproteasome O +subunits O +, O +either O +encode O +Thr B-residue_name +or O +Ile B-residue_name +at O +position O +3 B-residue_number +, O +indicating O +the O +importance O +of O +the O +Cγ O +for O +fixing O +the O +position O +of O +the O +nucleophilic O +Thr1 B-residue_name_number +. O + +Similarly O +, O +although O +the O +serine B-residue_name +mutant B-protein_state +is O +active B-protein_state +, O +threonine B-residue_name +is O +more O +efficient O +in O +the O +context O +of O +the O +proteasome B-complex_assembly +active B-site +site I-site +. O + +( O +a O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +and O +the O +matured B-protein_state +WT B-protein_state +β1 B-protein +active B-site +- I-site +site I-site +Thr1 B-residue_name_number +. O + +The O +major O +determinant O +of O +the O +S1 B-site +specificity I-site +pocket I-site +, O +residue O +45 B-residue_number +, O +is O +depicted O +. O + +The O +black O +arrow O +indicates O +the O +attack O +of O +Thr1Oγ B-residue_name_number +onto O +the O +carbonyl O +carbon O +atom O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +). I-residue_name_number + +( O +b O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +and O +the O +β2 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +highlights O +subtle O +differences O +in O +their O +conformations O +, O +but O +illustrates O +that O +Ala1 B-residue_name_number +and O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +match O +well O +. O + +( O +c O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +, O +the O +β2 B-mutant +- I-mutant +T1A I-mutant +and O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +propeptide B-structure_element +remnants O +depict O +their O +differences O +in O +conformation O +. O + +While O +residue O +(- B-residue_number +2 I-residue_number +) I-residue_number +of O +the O +β1 B-protein +and O +β2 B-protein +prosegments B-structure_element +fit O +the O +S1 B-site +pocket I-site +, O +His B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +of O +the O +β5 B-protein +propeptide B-structure_element +occupies O +the O +S2 B-site +pocket I-site +. O + +Nonetheless O +, O +in O +all O +mutants O +the O +carbonyl O +carbon O +atom O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +is O +ideally O +placed O +for O +the O +nucleophilic O +attack O +by O +Thr1Oγ B-residue_name_number +. O + +The O +hydrogen B-bond_interaction +bond I-bond_interaction +between O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +OH O +and O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +O O +(∼ O +2 O +. O +8 O +Å O +) O +is O +indicated O +by O +a O +black O +dashed O +line O +. O + +( O +a O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +and O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +L I-mutant +- I-mutant +T1A I-mutant +mutant B-protein_state +propeptide B-structure_element +. O + +The O +(- B-residue_number +2 I-residue_number +) I-residue_number +residues O +of O +both O +prosegments B-structure_element +point O +into O +the O +S1 B-site +pocket I-site +. O + +While O +the O +residues O +(- B-residue_range +2 I-residue_range +) I-residue_range +to I-residue_range +(- I-residue_range +4 I-residue_range +) I-residue_range +vary O +in O +their O +conformation O +, O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +and O +Ala1 B-residue_name_number +are O +located O +in O +all O +structures B-evidence +at O +the O +same O +positions O +. O + +( O +c O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β2 B-mutant +- I-mutant +T1A I-mutant +propeptide B-structure_element +and O +the O +β5 B-mutant +- I-mutant +H I-mutant +(- I-mutant +2 I-mutant +) I-mutant +T I-mutant +- I-mutant +T1A I-mutant +mutant B-protein_state +propeptide B-structure_element +. O + +The O +(- B-residue_number +2 I-residue_number +) I-residue_number +residues O +of O +both O +prosegments B-structure_element +point O +into O +the O +S1 B-site +pocket I-site +, O +but O +only O +Thr B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +OH O +of O +β2 B-protein +forms O +a O +hydrogen B-bond_interaction +bridge I-bond_interaction +to O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +O O +( O +black O +dashed O +line O +). O + +Notably O +, O +Val B-residue_name_number +(- I-residue_name_number +2 I-residue_name_number +) I-residue_name_number +of O +the O +latter O +does O +not O +occupy O +the O +S1 B-site +pocket I-site +, O +thereby O +changing O +the O +orientation O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +) I-residue_name_number +and O +preventing O +nucleophilic O +attack O +of O +Thr1Oγ B-residue_name_number +on O +the O +carbonyl O +carbon O +atom O +of O +Gly B-residue_name_number +(- I-residue_name_number +1 I-residue_name_number +). I-residue_name_number + +Architecture O +and O +proposed O +reaction O +mechanism O +of O +the O +proteasomal O +active B-site +site I-site +. O + +Thr1OH B-residue_name_number +is O +hydrogen B-bond_interaction +- I-bond_interaction +bonded I-bond_interaction +to O +Lys33NH2 B-residue_name_number +( O +2 O +. O +7 O +Å O +), O +which O +in O +turn O +interacts O +with O +Asp17Oδ B-residue_name_number +. O + +The O +Thr1 B-residue_name_number +N O +terminus O +is O +engaged O +in O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +Ser129Oγ B-residue_name_number +, O +the O +carbonyl O +oxygen O +of O +residue O +168 B-residue_number +, O +Ser169Oγ B-residue_name_number +and O +Asp166Oδ B-residue_name_number +. O +( O +b O +) O +The O +orientations O +of O +the O +active B-site +- I-site +site I-site +residues I-site +involved O +in O +hydrogen B-bond_interaction +bonding I-bond_interaction +are O +strictly B-protein_state +conserved I-protein_state +in O +each O +proteolytic B-site +centre I-site +, O +as O +shown O +by O +superposition B-experimental_method +of O +the O +β B-protein +subunits I-protein +. O + +In O +the O +latter O +, O +a O +water B-chemical +molecule O +( O +red O +sphere O +) O +is O +found O +at O +the O +position O +where O +in O +the O +WT B-protein_state +structure O +the O +side O +chain O +amine O +group O +of O +Lys33 B-residue_name_number +is O +located O +. O + +Similarly O +to O +Lys33 B-residue_name_number +, O +the O +water B-chemical +molecule O +hydrogen B-bond_interaction +bonds I-bond_interaction +to O +Arg19O B-residue_name_number +, O +Asp17Oδ B-residue_name_number +and O +Thr1OH B-residue_name_number +. O + +( O +d O +) O +Proposed O +chemical O +reaction O +mechanism O +for O +autocatalytic B-ptm +precursor I-ptm +processing I-ptm +and O +proteolysis O +in O +the O +proteasome B-complex_assembly +. O + +The O +active B-site +- I-site +site I-site +Thr1 B-residue_name_number +is O +depicted O +in O +blue O +, O +the O +propeptide B-structure_element +segment O +and O +the O +peptide O +substrate O +are O +coloured O +in O +green O +, O +whereas O +the O +scissile O +peptide O +bond O +is O +highlighted O +in O +red O +. O + +Autolysis B-ptm +( O +left O +set O +of O +structures O +) O +is O +initiated O +by O +deprotonation O +of O +Thr1OH B-residue_name_number +via O +Lys33NH2 B-residue_name_number +and O +the O +formation O +of O +a O +tetrahedral O +transition O +state O +. O + +The O +strictly B-protein_state +conserved I-protein_state +oxyanion O +hole O +Gly47NH B-residue_name_number +stabilizing O +the O +negatively O +charged O +intermediate O +is O +illustrated O +as O +a O +semicircle O +. O + +Collapse O +of O +the O +transition O +state O +frees O +the O +Thr1 B-residue_name_number +N O +terminus O +( O +by O +completing O +an O +N O +- O +to O +- O +O O +acyl O +shift O +of O +the O +propeptide B-structure_element +), O +which O +is O +subsequently O +protonated O +by O +Asp166OH B-residue_name_number +via O +Ser129OH B-residue_name_number +. O + +Next O +, O +Thr1NH2 B-residue_name_number +polarizes O +a O +water B-chemical +molecule O +for O +the O +nucleophilic O +attack O +of O +the O +acyl O +- O +enzyme O +intermediate O +. O + +On O +hydrolysis O +of O +the O +latter O +, O +the O +active B-site +- I-site +site I-site +Thr1 B-residue_name_number +is O +ready O +for O +catalysis O +( O +right O +set O +of O +structures O +). O + +The O +charged O +Thr1 B-residue_name_number +N O +terminus O +may O +engage O +in O +the O +orientation O +of O +the O +amide O +moiety O +and O +donate O +a O +proton O +to O +the O +emerging O +N O +terminus O +of O +the O +C O +- O +terminal O +cleavage O +product O +. O + +The O +resulting O +deprotonated O +Thr1NH2 B-residue_name_number +finally O +activates O +a O +water B-chemical +molecule O +for O +hydrolysis O +of O +the O +acyl O +- O +enzyme O +. O + +The O +proteasome B-complex_assembly +favours O +threonine B-residue_name +as O +the O +active O +- O +site O +nucleophile O +. O + +( O +a O +) O +Growth B-experimental_method +tests I-experimental_method +by I-experimental_method +serial I-experimental_method +dilution I-experimental_method +of O +WT B-protein_state +and O +pre2 O +( O +β5 B-protein +) O +mutant B-protein_state +yeast B-taxonomy_domain +cultures O +reveal O +growth O +defects O +of O +the O +active B-site +- I-site +site I-site +mutants B-experimental_method +under O +the O +indicated O +conditions O +after O +2 O +days O +( O +2 O +d O +) O +of O +incubation O +. O + +( O +b O +) O +Purified O +WT B-protein_state +and O +mutant B-protein_state +proteasomes B-complex_assembly +were O +tested O +for O +their O +chymotrypsin O +- O +like O +activity O +( O +β5 B-protein +) O +using O +the O +substrate O +Suc B-chemical +- I-chemical +LLVY I-chemical +- I-chemical +AMC I-chemical +. O + +The O +prosegment B-structure_element +is O +cleaved B-protein_state +but O +still B-protein_state +bound I-protein_state +in O +the O +substrate B-site +- I-site +binding I-site +channel I-site +. O + +( O +d O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β5 B-mutant +- I-mutant +T1A I-mutant +- I-mutant +K81R I-mutant +and O +the O +β5 B-mutant +- I-mutant +T1C I-mutant +mutant B-protein_state +subunits O +onto O +the O +WT B-protein_state +β5 B-protein +subunit O +. O +( O +e O +) O +Structural B-experimental_method +superposition I-experimental_method +of O +the O +β5 B-mutant +- I-mutant +T1C I-mutant +propeptide B-structure_element +onto O +the O +β1 B-mutant +- I-mutant +T1A I-mutant +active B-site +site I-site +( O +blue O +) O +and O +the O +WT B-protein_state +β5 B-protein +active B-site +site I-site +in B-protein_state +complex I-protein_state +with I-protein_state +the O +proteasome B-complex_assembly +inhibitor O +MG132 B-chemical +( O +ref O +.). O + +The O +inhibitor B-chemical +as O +well O +as O +the O +propeptides B-structure_element +adopt O +similar O +conformations O +in O +the O +substrate B-site +- I-site +binding I-site +channel I-site +. O + +The O +2FO B-evidence +– I-evidence +FC I-evidence +electron I-evidence +- I-evidence +density I-evidence +map I-evidence +for O +Ser1 B-residue_name_number +( O +blue O +mesh O +contoured O +at O +1σ O +) O +is O +illustrated O +. O + +( O +h O +) O +The O +methyl O +group O +of O +Thr1 B-residue_name_number +is O +anchored O +by O +hydrophobic B-bond_interaction +interactions I-bond_interaction +with O +Ala46Cβ B-residue_name_number +and O +Thr3Cγ B-residue_name_number +. O + +Inhibition O +of O +WT B-protein_state +and O +mutant B-protein_state +β5 B-mutant +- I-mutant +T1S I-mutant +proteasomes B-complex_assembly +by O +bortezomib B-chemical +and O +carfilzomib B-chemical +. O + +Inhibition B-experimental_method +assays I-experimental_method +( O +left O +panel O +). O + +Purified O +yeast B-taxonomy_domain +proteasomes B-complex_assembly +were O +tested O +for O +the O +susceptibility O +of O +their O +ChT O +- O +L O +( O +β5 B-protein +) O +activity O +to O +inhibition O +by O +bortezomib B-chemical +and O +carfilzomib B-chemical +using O +the O +substrate O +Suc B-chemical +- I-chemical +LLVY I-chemical +- I-chemical +AMC I-chemical +. O + +IC50 B-evidence +values I-evidence +were O +determined O +in O +triplicate O +; O +s O +. O +d O +.' O +s O +are O +indicated O +by O +error O +bars O +. O + +Note O +that O +IC50 B-evidence +values I-evidence +depend O +on O +time O +and O +enzyme O +concentration O +. O + +Proteasomes B-complex_assembly +( O +final O +concentration O +: O +66 O +nM O +) O +were O +incubated O +with O +inhibitor O +for O +45 O +min O +before O +substrate O +addition O +( O +final O +concentration O +: O +200 O +μM O +). O + +Structures B-evidence +of O +the O +β5 B-mutant +- I-mutant +T1S I-mutant +mutant B-protein_state +in O +complex B-complex_assembly +with I-complex_assembly +both I-complex_assembly +ligands I-complex_assembly +( O +green O +) O +prove O +the O +reactivity O +of O +Ser1 B-residue_name_number +( O +right O +panel O +). O + +The O +WT B-protein_state +proteasome B-complex_assembly +: I-complex_assembly +inhibitor I-complex_assembly +complex I-complex_assembly +structures B-evidence +( O +inhibitor O +in O +grey O +; O +Thr1 B-residue_name_number +in O +black O +) O +are O +superimposed B-experimental_method +and O +demonstrate O +that O +mutation B-experimental_method +of O +Thr1 B-residue_name_number +to O +Ser B-residue_name +does O +not O +affect O +the O +binding O +mode O +of O +bortezomib B-chemical +or O +carfilzomib B-chemical +. O + +The O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +is O +a O +reader O +of O +histone B-protein_type +crotonylation B-ptm + +The O +discovery O +of O +new O +histone B-protein_type +modifications O +is O +unfolding O +at O +startling O +rates O +, O +however O +, O +the O +identification O +of O +effectors O +capable O +of O +interpreting O +these O +modifications O +has O +lagged O +behind O +. O + +Here O +we O +report O +the O +YEATS B-structure_element +domain I-structure_element +as O +an O +effective O +reader O +of O +histone B-protein_type +lysine B-residue_name +crotonylation B-ptm +– O +an O +epigenetic O +signature O +associated O +with O +active O +transcription O +. O + +We O +show O +that O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +engages O +crotonyllysine B-residue_name +via O +a O +unique O +π B-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +π I-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +mechanism O +and O +that O +other O +YEATS B-structure_element +domains I-structure_element +have O +crotonyllysine B-residue_name +binding O +activity O +. O + +The O +crotonyllysine B-residue_name +mark O +on O +histone B-protein_type +H3K18 B-protein_type +is O +produced O +by O +p300 B-protein +, O +a O +histone B-protein_type +acetyltransferase I-protein_type +also O +responsible O +for O +acetylation B-ptm +of O +histones O +. O + +Owing O +to O +some O +differences O +in O +their O +genomic O +distribution O +, O +the O +crotonyllysine B-residue_name +and O +acetyllysine B-residue_name +( O +Kac B-residue_name +) O +modifications O +have O +been O +linked O +to O +distinct O +functional O +outcomes O +. O + +p300 B-protein +- O +catalyzed O +histone B-protein_type +crotonylation B-ptm +, O +which O +is O +likely O +metabolically O +regulated O +, O +stimulates O +transcription O +to O +a O +greater O +degree O +than O +p300 B-protein +- O +catalyzed O +acetylation B-ptm +. O + +The O +discovery O +of O +individual O +biological O +roles O +for O +the O +crotonyllysine B-residue_name +and O +acetyllysine B-residue_name +marks O +suggests O +that O +these O +PTMs O +can O +be O +read O +by O +distinct O +readers O +. O + +The O +family O +of O +acetyllysine B-residue_name +readers O +has O +been O +expanded O +with O +the O +discovery O +that O +the O +YEATS B-structure_element +( O +Yaf9 B-protein +, O +ENL B-protein +, O +AF9 B-protein +, O +Taf14 B-protein +, O +Sas5 B-protein +) O +domains O +of O +human B-species +AF9 B-protein +and O +yeast B-taxonomy_domain +Taf14 B-protein +are O +capable O +of O +recognizing O +the O +histone B-protein_type +mark O +H3K9ac B-protein_type +. O + +Similarly O +, O +activation O +of O +a O +subset O +of O +genes O +and O +DNA O +damage O +repair O +in O +yeast B-taxonomy_domain +require O +the O +acetyllysine B-residue_name +binding O +activity O +of O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +. O + +Consistent O +with O +its O +role O +in O +gene O +regulation O +, O +Taf14 B-protein +was O +identified O +as O +a O +core O +component O +of O +the O +transcription O +factor O +complexes O +TFIID B-complex_assembly +and O +TFIIF B-complex_assembly +. O + +However O +, O +Taf14 B-protein +is O +also O +found O +in O +a O +number O +of O +chromatin O +- O +remodeling O +complexes O +( O +i O +. O +e O +., O +INO80 B-complex_assembly +, O +SWI B-complex_assembly +/ I-complex_assembly +SNF I-complex_assembly +and O +RSC B-complex_assembly +) O +and O +the O +histone B-protein_type +acetyltransferase I-protein_type +complex O +NuA3 B-complex_assembly +, O +indicating O +a O +multifaceted O +role O +of O +Taf14 B-protein +in O +transcriptional O +regulation O +and O +chromatin O +biology O +. O + +We O +found O +that O +H3K9cr B-protein_type +is O +present O +in O +yeast B-taxonomy_domain +and O +is O +dynamically O +regulated O +. O + +The O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +adopts O +an O +immunoglobin B-structure_element +- I-structure_element +like I-structure_element +β I-structure_element +sandwich I-structure_element +fold I-structure_element +containing O +eight O +anti B-structure_element +- I-structure_element +parallel I-structure_element +β I-structure_element +strands I-structure_element +linked O +by O +short O +loops B-structure_element +that O +form O +a O +binding B-site +site I-site +for O +H3K9cr B-protein_type +( O +Fig O +. O +1b O +). O + +The O +most O +striking O +feature O +of O +the O +crotonyllysine B-residue_name +recognition O +mechanism O +is O +the O +unique O +coordination O +of O +crotonylated B-protein_state +lysine B-residue_name +residue O +. O + +The O +fully O +extended O +side O +chain O +of O +K9cr B-ptm +transverses O +the O +narrow O +tunnel O +, O +crossing O +the O +β B-structure_element +sandwich I-structure_element +at O +right O +angle O +in O +a O +corkscrew O +- O +like O +manner O +( O +Fig O +. O +1b O +and O +Supplementary O +Figure O +1b O +). O + +The O +side O +chain O +of O +Trp81 B-residue_name_number +appears O +to O +adopt O +two O +conformations O +, O +one O +of O +which O +provides O +maximum O +π B-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +with O +the O +alkene O +functional O +group O +while O +the O +other O +rotamer O +affords O +maximum O +π B-bond_interaction +- I-bond_interaction +stacking I-bond_interaction +with O +the O +amide O +π O +electrons O +( O +Supplementary O +Fig O +. O +1c O +). O + +The O +dual O +conformation O +of O +Trp81 B-residue_name_number +is O +likely O +due O +to O +the O +conjugated O +nature O +of O +the O +C O += O +C O +and O +C O += O +O O +π O +- O +orbitals O +within O +the O +crotonyl B-chemical +functional O +group O +. O + +The O +π B-bond_interaction +bond I-bond_interaction +conjugation O +of O +the O +crotonyl B-chemical +group O +gives O +rise O +to O +a O +dipole O +moment O +of O +the O +alkene O +moiety O +, O +resulting O +in O +a O +partial O +positive O +charge O +on O +the O +β O +- O +carbon O +( O +Cβ O +) O +and O +a O +partial O +negative O +charge O +on O +the O +α O +- O +carbon O +( O +Cα O +). O + +This O +provides O +the O +capability O +for O +the O +alkene O +moiety O +to O +form O +electrostatic B-bond_interaction +contacts I-bond_interaction +, O +as O +Cα O +and O +Cβ O +lay O +within O +electrostatic B-bond_interaction +interaction I-bond_interaction +distances O +of O +the O +carbonyl O +oxygen O +of O +Gln79 B-residue_name_number +and O +of O +the O +hydroxyl O +group O +of O +Thr61 B-residue_name_number +, O +respectively O +. O + +Extra O +stabilization O +of O +K9cr B-ptm +is O +attained O +by O +a O +hydrogen B-bond_interaction +bond I-bond_interaction +formed O +between O +its O +carbonyl O +oxygen O +and O +the O +backbone O +nitrogen O +of O +Trp81 B-residue_name_number +, O +as O +well O +as O +a O +water B-chemical +- O +mediated O +hydrogen B-bond_interaction +bond I-bond_interaction +with O +the O +backbone O +carbonyl O +group O +of O +Gly82 B-residue_name_number +( O +Fig O +1d O +). O + +This O +distinctive O +mechanism O +was O +corroborated O +through O +mapping O +the O +Taf14 B-protein +YEATS B-site +- I-site +H3K9cr I-site +binding I-site +interface I-site +in O +solution O +using O +NMR B-experimental_method +chemical I-experimental_method +shift I-experimental_method +perturbation I-experimental_method +analysis I-experimental_method +( O +Supplementary O +Fig O +. O +2a O +, O +b O +). O + +The O +dissociation B-evidence +constant I-evidence +( O +Kd B-evidence +) O +for O +the O +Taf14 B-complex_assembly +YEATS I-complex_assembly +- I-complex_assembly +H3K9cr5 I-complex_assembly +- I-complex_assembly +13 I-complex_assembly +complex O +was O +found O +to O +be O +9 O +. O +5 O +μM O +, O +as O +measured O +by O +fluorescence B-experimental_method +spectroscopy I-experimental_method +( O +Supplementary O +Fig O +. O +2c O +). O + +This O +value O +is O +in O +the O +range O +of O +binding B-evidence +affinities I-evidence +exhibited O +by O +the O +majority O +of O +histone O +readers O +, O +thus O +attesting O +to O +the O +physiological O +relevance O +of O +the O +H3K9cr B-protein_type +recognition O +by O +Taf14 B-protein +. O + +Both O +H3K9cr B-protein_type +and O +H3K9ac B-protein_type +were O +detected O +in O +yeast B-taxonomy_domain +histones B-protein_type +; O +to O +our O +knowledge O +, O +this O +is O +the O +first O +report O +of O +H3K9cr B-protein_type +occurring O +in O +yeast B-taxonomy_domain +. O + +Towards O +this O +end O +, O +we O +probed O +extracts O +derived O +from O +yeast B-taxonomy_domain +cells O +in O +which O +major O +yeast B-taxonomy_domain +HATs B-protein_type +( O +HAT1 B-protein +, O +Gcn5 B-protein +, O +and O +Rtt109 B-protein +) O +or O +HDACs B-protein_type +( O +Rpd3 B-protein +, O +Hos1 B-protein +, O +and O +Hos2 B-protein +) O +were O +deleted B-experimental_method +. O + +As O +shown O +in O +Figure O +2a O +, O +b O +and O +Supplementary O +Fig O +. O +3e O +, O +H3K9cr B-protein_type +levels O +were O +abolished O +or O +reduced O +considerably O +in O +the O +HAT B-protein_type +deletion B-experimental_method +strains O +, O +whereas O +they O +were O +dramatically O +increased O +in O +the O +HDAC B-protein_type +deletion B-experimental_method +strains O +. O + +Furthermore O +, O +fluctuations O +in O +the O +H3K9cr B-protein_type +levels O +were O +more O +substantial O +than O +fluctuations O +in O +the O +corresponding O +H3K9ac B-protein_type +levels O +. O + +Together O +, O +these O +results O +reveal O +that O +H3K9cr B-protein_type +is O +a O +dynamic O +mark O +of O +chromatin O +in O +yeast B-taxonomy_domain +and O +suggest O +an O +important O +role O +for O +this O +modification O +in O +transcription O +as O +it O +is O +regulated O +by O +HATs B-protein_type +and O +HDACs B-protein_type +. O + +We O +have O +previously O +shown O +that O +among O +acetylated B-protein_state +histone B-protein_type +marks O +, O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +prefers O +acetylated B-protein_state +H3K9 B-protein_type +( O +also O +see O +Supplementary O +Fig O +. O +3b O +), O +however O +it O +binds O +to O +H3K9cr B-protein_type +tighter O +. O + +The O +selectivity O +of O +Taf14 B-protein +towards O +crotonyllysine B-residue_name +was O +substantiated O +by O +1H B-experimental_method +, I-experimental_method +15N I-experimental_method +HSQC I-experimental_method +experiments O +, O +in O +which O +either O +H3K9cr5 B-chemical +- I-chemical +13 I-chemical +or O +H3K9ac5 B-chemical +- I-chemical +13 I-chemical +peptide O +was O +titrated B-experimental_method +into O +the O +15N B-protein_state +- I-protein_state +labeled I-protein_state +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +( O +Fig O +. O +2c O +and O +Supplementary O +Fig O +. O +4a O +, O +b O +). O + +In O +contrast O +, O +binding O +of O +H3K9ac B-protein_type +resulted O +in O +an O +intermediate O +exchange O +, O +which O +is O +characteristic O +of O +a O +weaker O +association O +. O + +Furthermore O +, O +crosspeaks B-evidence +of O +Gly80 B-residue_name_number +and O +Trp81 B-residue_name_number +of O +the O +YEATS B-structure_element +domain I-structure_element +were O +uniquely O +perturbed O +by O +H3K9cr B-protein_type +and O +H3K9ac B-protein_type +, O +indicating O +a O +different O +chemical O +environment O +in O +the O +respective O +crotonyllysine B-site +and I-site +acetyllysine I-site +binding I-site +pockets I-site +( O +Supplementary O +Fig O +. O +4a O +). O + +These O +differences O +support O +our O +model O +that O +Trp81 B-residue_name_number +adopts O +two O +conformations O +upon O +complex O +formation O +with O +the O +H3K9cr B-protein_type +mark O +as O +compared O +to O +H3K9ac B-protein_type +( O +Supplementary O +Figs O +. O +1c O +, O +d O +and O +4c O +). O + +One O +of O +the O +conformations O +, O +characterized O +by O +the O +π O +stacking O +involving O +two O +aromatic O +residues O +and O +the O +alkene O +group O +, O +is O +observed O +only O +in O +the O +YEATS B-complex_assembly +- I-complex_assembly +H3K9cr I-complex_assembly +complex O +. O + +As O +shown O +in O +Figure O +2d O +and O +Supplementary O +Fig O +. O +5a O +, O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +binds O +more O +strongly O +to O +H3K9cr1 B-chemical +- I-chemical +20 I-chemical +, O +as O +compared O +to O +other O +acylated B-protein_state +histone O +peptides O +. O + +The O +preference O +for O +H3K9cr B-protein_type +over O +H3K9ac B-protein_type +, O +H3K9pr B-protein_type +and O +H3K9bu B-protein_type +was O +supported O +by O +1H B-experimental_method +, I-experimental_method +15N I-experimental_method +HSQC I-experimental_method +titration I-experimental_method +experiments I-experimental_method +. O + +Addition O +of O +H3K9ac1 B-chemical +- I-chemical +20 I-chemical +, O +H3K9pr1 B-chemical +- I-chemical +20 I-chemical +, O +and O +H3K9bu1 B-chemical +- I-chemical +20 I-chemical +peptides O +caused O +chemical B-evidence +shift I-evidence +perturbations I-evidence +in O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +in O +intermediate O +exchange O +regime O +, O +implying O +that O +these O +interactions O +are O +weaker O +compared O +to O +the O +interaction O +with O +the O +H3K9cr1 B-chemical +- I-chemical +20 I-chemical +peptide O +( O +Supplementary O +Fig O +. O +5b O +). O + +From O +comparative B-experimental_method +structural I-experimental_method +analysis I-experimental_method +of O +the O +YEATS O +complexes O +, O +Gly80 B-residue_name_number +emerged O +as O +candidate O +residue O +potentially O +responsible O +for O +the O +preference O +for O +crotonyllysine B-residue_name +. O + +In O +attempt O +to O +generate O +a O +mutant O +capable O +of O +accommodating O +a O +short O +acetyl O +moiety O +but O +discriminating O +against O +a O +longer O +, O +planar O +crotonyl B-chemical +moiety O +, O +we O +mutated B-protein_state +Gly80 B-residue_name_number +to O +more O +bulky O +residues O +, O +however O +all O +mutants B-protein_state +of I-protein_state +Gly80 B-residue_name_number +lost O +their O +binding O +activities O +towards O +either O +acylated B-protein_state +peptide O +, O +suggesting O +that O +Gly80 B-residue_name_number +is O +absolutely O +required O +for O +the O +interaction O +. O + +To O +determine O +if O +the O +binding O +to O +crotonyllysine B-residue_name +is O +conserved B-protein_state +, O +we O +tested O +human B-species +YEATS B-structure_element +domains I-structure_element +by O +pull B-experimental_method +- I-experimental_method +down I-experimental_method +experiments I-experimental_method +using O +singly O +and O +multiply O +acetylated B-protein_state +, O +propionylated B-protein_state +, O +butyrylated B-protein_state +, O +and O +crotonylated B-protein_state +histone B-protein_type +peptides O +( O +Supplementary O +Fig O +. O +6 O +). O + +We O +found O +that O +all O +YEATS B-structure_element +domains I-structure_element +tested O +are O +capable O +of O +binding O +to O +crotonyllysine B-residue_name +peptides O +, O +though O +they O +display O +variable O +preferences O +for O +the O +acyl O +moieties O +. O + +While O +YEATS2 B-protein +and O +ENL B-protein +showed O +selectivity O +for O +the O +crotonylated B-protein_state +peptides O +, O +GAS41 B-protein +and O +AF9 B-protein +bound O +acylated B-protein_state +peptides O +almost O +equally O +well O +. O + +We O +assayed O +a O +large O +set O +of O +BDs B-structure_element +in O +pull B-experimental_method +- I-experimental_method +down I-experimental_method +experiments I-experimental_method +and O +found O +that O +this O +module O +is O +highly O +specific O +for O +acetyllysine B-residue_name +and O +propionyllysine B-residue_name +containing O +peptides O +( O +Supplementary O +Fig O +. O +7 O +). O + +However O +, O +bromodomains B-structure_element +did O +not O +interact O +( O +or O +associated O +very O +weakly O +) O +with O +longer O +acyl O +modifications O +, O +including O +crotonyllysine B-residue_name +, O +as O +in O +the O +case O +of O +BDs B-structure_element +of O +TAF1 B-protein +and O +BRD2 B-protein +, O +supporting O +recent O +reports O +. O + +These O +results O +demonstrate O +that O +the O +YEATS B-structure_element +domain I-structure_element +is O +currently O +the O +sole O +reader O +of O +crotonyllysine B-residue_name +. O + +In O +conclusion O +, O +we O +have O +identified O +the O +YEATS B-structure_element +domain I-structure_element +of O +Taf14 B-protein +as O +the O +first O +reader O +of O +histone B-protein_type +crotonylation B-ptm +. O + +We O +further O +demonstrate O +that O +H3K9cr B-protein_type +exists O +in O +yeast B-taxonomy_domain +and O +is O +dynamically O +regulated O +by O +HATs B-protein_type +and O +HDACs B-protein_type +. O + +Furthermore O +, O +the O +functional O +significance O +of O +crotonyllysine B-residue_name +recognition O +by O +other O +YEATS B-protein_type +proteins O +will O +be O +of O +great O +importance O +to O +elucidate O +and O +compare O +. O + +The O +structural O +mechanism O +for O +the O +recognition O +of O +H3K9cr B-protein_type + +( O +a O +) O +Chemical O +structure O +of O +crotonyllysine B-residue_name +. O +( O +b O +) O +The O +crystal B-evidence +structure I-evidence +of O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +( O +wheat O +) O +in B-protein_state +complex I-protein_state +with I-protein_state +the O +H3K9cr5 B-chemical +- I-chemical +13 I-chemical +peptide O +( O +green O +). O +( O +c O +) O +H3K9cr B-protein_type +is O +stabilized O +via O +an O +extensive O +network O +of O +intermolecular O +electrostatic B-bond_interaction +and I-bond_interaction +polar I-bond_interaction +interactions I-bond_interaction +with O +the O +Taf14 B-protein +YEATS B-structure_element +domain I-structure_element +. O + +Total O +H3 B-protein_type +was O +used O +as O +a O +loading O +control O +. O + +( O +c O +) O +Superimposed O +1H B-experimental_method +, I-experimental_method +15N I-experimental_method +HSQC I-experimental_method +spectra B-evidence +of O +Taf14 B-protein +YEATS B-structure_element +recorded O +as O +H3K9cr5 B-chemical +- I-chemical +13 I-chemical +and O +H3K9ac5 B-chemical +- I-chemical +13 I-chemical +peptides O +were O +titrated B-experimental_method +in O +. O + +Spectra B-evidence +are O +color O +coded O +according O +to O +the O +protein O +: O +peptide O +molar O +ratio O +. O + +( O +d O +) O +Western B-experimental_method +blot I-experimental_method +analyses O +of O +peptide B-experimental_method +pull I-experimental_method +- I-experimental_method +down I-experimental_method +assays I-experimental_method +using O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +mutated B-protein_state +Taf14 B-protein +YEATS B-structure_element +domains I-structure_element +and O +indicated O +peptides O +. O + +Cellular O +homeostasis O +requires O +correct O +delivery O +of O +cell B-protein_type +- I-protein_type +surface I-protein_type +receptor I-protein_type +proteins O +( O +cargo O +) O +to O +their O +target O +subcellular O +compartments O +. O + +The O +adapter B-protein_type +proteins I-protein_type +Tom1 B-protein +and O +Tollip B-protein +are O +involved O +in O +sorting O +of O +ubiquitinated B-ptm +cargo O +in O +endosomal O +compartments O +. O + +Recruitment O +of O +Tom1 B-protein +to O +the O +endosomal O +compartments O +is O +mediated O +by O +its O +GAT B-structure_element +domain O +’ O +s O +association O +to O +Tollip B-protein +’ O +s O +Tom1 B-structure_element +- I-structure_element +binding I-structure_element +domain I-structure_element +( O +TBD B-structure_element +). O + +In O +this O +data O +article O +, O +we O +report O +the O +solution B-experimental_method +NMR I-experimental_method +- O +derived O +structure B-evidence +of O +the O +Tom1 B-protein +GAT B-structure_element +domain O +. O + +The O +estimated O +protein O +structure B-evidence +exhibits O +a O +bundle O +of O +three O +helical O +elements O +. O + +We O +compare B-experimental_method +the O +Tom1 B-protein +GAT B-structure_element +structure B-evidence +with O +those O +structures B-evidence +corresponding O +to O +the O +Tollip B-protein +TBD B-protein_state +- I-protein_state +and O +ubiquitin B-protein_state +- I-protein_state +bound I-protein_state +states O +. O + +Subject O +area O +Biology O +More O +specific O +subject O +area O +Structural O +biology O +Type O +of O +data O +Table O +, O +text O +file O +, O +graph O +, O +figures O +How O +data O +was O +acquired O +Circular B-experimental_method +dichroism I-experimental_method +and O +NMR B-experimental_method +. O + +The O +Tom1 B-protein +GAT B-structure_element +domain O +solution B-evidence +structure I-evidence +will O +provide O +additional O +tools O +for O +modulating O +its O +biological O +function O +. O + +Tom1 B-protein +GAT B-structure_element +can O +adopt O +distinct O +conformations O +upon O +ligand O +binding O +. O + +A O +conformational O +response O +of O +the O +Tom1 B-protein +GAT B-structure_element +domain O +upon O +Tollip B-protein +TBD B-structure_element +binding O +can O +serve O +as O +an O +example O +to O +explain O +mutually O +exclusive O +ligand O +binding O +events O +. O + +Analysis O +of O +the O +far B-experimental_method +- I-experimental_method +UV I-experimental_method +circular I-experimental_method +dichroism I-experimental_method +( O +CD B-experimental_method +) O +spectrum B-evidence +of O +the O +Tom B-protein +1 I-protein +GAT B-structure_element +domain O +( O +Fig O +. O +1 O +) O +predicts O +58 O +. O +7 O +% O +α B-structure_element +- I-structure_element +helix I-structure_element +, O +3 O +% O +β B-structure_element +- I-structure_element +strand I-structure_element +, O +15 O +. O +5 O +% O +turn O +, O +and O +22 O +. O +8 O +% O +disordered O +regions O +. O + +Representative O +far B-experimental_method +- I-experimental_method +UV I-experimental_method +CD I-experimental_method +spectrum B-evidence +of O +the O +His B-experimental_method +- I-experimental_method +Tom1 B-protein +GAT B-structure_element +domain O +. O + +( O +A O +) O +Stereo O +view O +displaying O +the O +best O +- O +fit O +backbone B-experimental_method +superposition I-experimental_method +of O +the O +refined O +structures B-evidence +for O +the O +Tom1 B-protein +GAT B-structure_element +domain O +. O + +Helices O +are O +shown O +in O +orange O +, O +whereas O +loops O +are O +colored O +in O +green O +. O +( O +B O +) O +Ribbon O +illustration O +of O +the O +Tom1 B-protein +GAT B-structure_element +domain O +. O + +( O +A O +) O +Two O +views O +of O +the O +superimposed B-experimental_method +structures I-experimental_method +of O +the O +Tom1 B-protein +GAT B-structure_element +domain O +in O +the O +free B-protein_state +state O +( O +gray O +) O +with O +that O +in O +the O +Tollip B-protein +TBD B-protein_state +- I-protein_state +bound I-protein_state +state O +( O +red O +). O +( O +B O +) O +Two O +views O +of O +the O +superimposed B-experimental_method +structures I-experimental_method +of O +the O +Tom1 B-protein +GAT B-structure_element +domain O +( O +gray O +) O +with O +that O +in O +the O +Ub B-protein_state +- I-protein_state +bound I-protein_state +state O +( O +green O +). O + +deviations O +were O +obtained O +by O +superimposing B-experimental_method +residues O +215 B-residue_range +– I-residue_range +309 I-residue_range +of O +Tom1 B-protein +GAT B-structure_element +among O +10 O +lowest O +energy O +refined O +structures B-evidence +. O + +Haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +/ O +Sigma B-protein +- I-protein +2 I-protein +receptor O +facilitates O +cancer O +proliferation O +and O +chemoresistance O + +Progesterone B-protein +- I-protein +receptor I-protein +membrane I-protein +component I-protein +1 I-protein +( O +PGRMC1 B-protein +/ O +Sigma B-protein +- I-protein +2 I-protein +receptor I-protein +) O +is O +a O +haem B-protein_type +- I-protein_type +containing I-protein_type +protein I-protein_type +that O +interacts O +with O +epidermal B-protein_type +growth I-protein_type +factor I-protein_type +receptor I-protein_type +( O +EGFR B-protein_type +) O +and O +cytochromes B-protein_type +P450 I-protein_type +to O +regulate O +cancer O +proliferation O +and O +chemoresistance O +; O +its O +structural O +basis O +remains O +unknown O +. O + +Here O +crystallographic B-experimental_method +analyses I-experimental_method +of O +the O +PGRMC1 B-protein +cytosolic B-structure_element +domain I-structure_element +at O +1 O +. O +95 O +Å O +resolution O +reveal O +that O +it O +forms O +a O +stable B-protein_state +dimer B-oligomeric_state +through O +stacking B-bond_interaction +interactions I-bond_interaction +of O +two O +protruding O +haem B-chemical +molecules O +. O + +The O +haem B-chemical +iron B-chemical +is O +five B-bond_interaction +- I-bond_interaction +coordinated I-bond_interaction +by I-bond_interaction +Tyr113 B-residue_name_number +, O +and O +the O +open O +surface B-site +of O +the O +haem B-chemical +mediates O +dimerization B-oligomeric_state +. O + +Carbon B-chemical +monoxide I-chemical +( O +CO B-chemical +) O +interferes O +with O +PGRMC1 B-protein +dimerization B-oligomeric_state +by O +binding O +to O +the O +sixth B-site +coordination I-site +site I-site +of O +the O +haem B-chemical +. O + +Haem B-chemical +- O +mediated O +PGRMC1 B-protein +dimerization B-oligomeric_state +is O +required O +for O +interactions O +with O +EGFR B-protein_type +and O +cytochromes B-protein_type +P450 I-protein_type +, O +cancer O +proliferation O +and O +chemoresistance O +against O +anti O +- O +cancer O +drugs O +; O +these O +events O +are O +attenuated O +by O +either O +CO B-chemical +or O +haem B-chemical +deprivation O +in O +cancer O +cells O +. O + +This O +study O +demonstrates O +protein O +dimerization B-oligomeric_state +via O +haem B-bond_interaction +– I-bond_interaction +haem I-bond_interaction +stacking I-bond_interaction +, O +which O +has O +not O +been O +seen O +in O +eukaryotes B-taxonomy_domain +, O +and O +provides O +insights O +into O +its O +functional O +significance O +in O +cancer O +. O + +Here O +, O +the O +authors O +determine O +the O +structure B-evidence +of O +the O +cytosolic B-structure_element +domain I-structure_element +of O +PGRMC1 B-protein +, O +which O +forms O +a O +dimer B-oligomeric_state +via O +haem B-bond_interaction +– I-bond_interaction +haem I-bond_interaction +stacking I-bond_interaction +, O +and O +propose O +how O +this O +interaction O +could O +be O +involved O +in O +its O +function O +. O + +Much O +attention O +has O +been O +paid O +to O +the O +roles O +of O +haem B-chemical +- O +iron B-chemical +in O +cancer O +development O +. O + +Increased O +dietary O +intake O +of O +haem B-chemical +is O +a O +risk O +factor O +for O +several O +types O +of O +cancer O +. O + +On O +the O +other O +hand O +, O +carbon B-chemical +monoxide I-chemical +( O +CO B-chemical +), O +the O +gaseous O +mediator O +generated O +by O +oxidative O +degradation O +of O +haem B-chemical +via O +haem B-protein_type +oxygenase I-protein_type +( O +HO B-protein_type +), O +inhibits O +tumour O +growth O +. O + +Thus O +, O +a O +tenuous O +balance O +between O +free O +haem B-chemical +and O +CO B-chemical +plays O +key O +roles O +in O +cancer O +development O +and O +chemoresistance O +, O +although O +the O +underlying O +mechanisms O +are O +not O +fully O +understood O +. O + +To O +gain O +insight O +into O +the O +underlying O +mechanisms O +, O +we O +took O +chemical O +biological O +approaches O +using O +affinity B-experimental_method +nanobeads I-experimental_method +carrying O +haem B-chemical +and O +identified O +progesterone B-protein +- I-protein +receptor I-protein +membrane I-protein +component I-protein +1 I-protein +( O +PGRMC1 B-protein +) O +as O +a O +haem B-chemical +- O +binding O +protein O +from O +mouse B-taxonomy_domain +liver O +extracts O +( O +Supplementary O +Fig O +. O +1 O +). O + +PGRMC1 B-protein +is O +a O +member O +of O +the O +membrane B-protein_type +- I-protein_type +associated I-protein_type +progesterone I-protein_type +receptor I-protein_type +( O +MAPR B-protein_type +) O +family O +with O +a O +cytochrome B-structure_element +b5 I-structure_element +- I-structure_element +like I-structure_element +haem B-site +- I-site +binding I-site +region I-site +, O +and O +is O +known O +to O +be O +highly B-protein_state +expressed I-protein_state +in O +various O +types O +of O +cancers O +. O + +PGRMC1 B-protein +is O +anchored O +to O +the O +cell O +membrane O +through O +the O +N O +- O +terminal O +transmembrane B-structure_element +helix I-structure_element +and O +interacts O +with O +epidermal B-protein_type +growth I-protein_type +factor I-protein_type +receptor I-protein_type +( O +EGFR B-protein_type +) O +and O +cytochromes B-protein_type +P450 I-protein_type +( O +ref O +). O + +While O +PGRMC1 B-protein +is O +implicated O +in O +cell O +proliferation O +and O +cholesterol O +biosynthesis O +, O +the O +structural O +basis O +on O +which O +PGRMC1 B-protein +exerts O +its O +function O +remains O +largely O +unknown O +. O + +Here O +we O +show O +that O +PGRMC1 B-protein +exhibits O +a O +unique O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +. O + +X B-evidence +- I-evidence +ray I-evidence +crystal I-evidence +structure I-evidence +of O +PGRMC1 B-protein + +We O +solved B-experimental_method +the O +crystal B-evidence +structure I-evidence +of O +the O +haem B-protein_state +- I-protein_state +bound I-protein_state +PGRMC1 B-protein +cytosolic B-structure_element +domain I-structure_element +( O +a O +. O +a O +. O +72 B-residue_range +– I-residue_range +195 I-residue_range +) O +at O +1 O +. O +95 O +Å O +resolution O +( O +Supplementary O +Fig O +. O +2 O +). O + +In O +the O +presence B-protein_state +of I-protein_state +haem B-chemical +, O +PGRMC1 B-protein +forms O +a O +dimeric B-oligomeric_state +structure O +largely O +through O +hydrophobic B-bond_interaction +interactions I-bond_interaction +between O +the O +haem B-chemical +moieties O +of O +two O +monomers B-oligomeric_state +( O +Fig O +. O +1a O +, O +Table O +1 O +and O +Supplementary O +Fig O +. O +3 O +; O +a O +stereo O +- O +structural O +image O +is O +shown O +in O +Supplementary O +Fig O +4 O +). O + +In O +cytochrome B-protein_type +b5 I-protein_type +, O +the O +haem B-chemical +iron B-chemical +is O +six B-bond_interaction +- I-bond_interaction +coordinated I-bond_interaction +by I-bond_interaction +two O +axial O +histidine B-residue_name +residues O +. O + +These O +histidines B-residue_name +are O +missing B-protein_state +in O +PGRMC1 B-protein +, O +and O +the O +haem B-chemical +iron B-chemical +is O +five B-bond_interaction +- I-bond_interaction +coordinated I-bond_interaction +by I-bond_interaction +Tyr113 B-residue_name_number +( O +Y113 B-residue_name_number +) O +alone B-protein_state +( O +Fig O +. O +1b O +and O +Supplementary O +Fig O +. O +3 O +). O + +A O +homologous B-structure_element +helix I-structure_element +that O +holds O +haem B-chemical +in O +cytochrome B-protein_type +b5 I-protein_type +is O +longer O +, O +shifts O +away O +from O +haem B-chemical +, O +and O +does O +not O +form O +a O +coordinate O +bond O +in O +PGRMC1 B-protein +( O +Fig O +. O +1c O +). O + +Consequently O +, O +the O +five O +- O +coordinated O +haem B-chemical +of O +PGRMC1 B-protein +has O +an O +open O +surface B-site +that O +allows O +its O +dimerization B-oligomeric_state +through O +hydrophobic B-bond_interaction +haem I-bond_interaction +– I-bond_interaction +haem I-bond_interaction +stacking I-bond_interaction +. O + +Contrary O +to O +our O +finding O +, O +Kaluka O +et O +al O +. O +recently O +reported O +that O +Tyr164 B-residue_name_number +of O +PGRMC1 B-protein +is O +the O +axial O +ligand O +of O +haem B-chemical +because O +mutation B-experimental_method +of O +this O +residue O +impairs O +haem B-chemical +binding O +. O + +This O +is O +consistent O +with O +observations O +by O +Min O +et O +al O +. O +that O +Tyr B-residue_name_number +107 I-residue_name_number +and O +Tyr113 B-residue_name_number +of O +PGRMC1 B-protein +are O +involved O +in O +binding O +with O +haem B-chemical +. O + +These O +amino O +acid O +residues O +are O +conserved B-protein_state +among O +MAPR B-protein_type +family O +members O +( O +Supplementary O +Fig O +. O +5a O +), O +suggesting O +that O +these O +proteins O +share O +the O +ability O +to O +exhibit O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +. O + +PGRMC1 B-protein +exhibits O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +in O +solution O + +In O +the O +PGRMC1 B-protein +crystal B-evidence +, O +two O +different O +types O +of O +crystal O +contacts O +( O +chain O +A O +– O +A O +″ O +and O +A O +– O +B O +) O +were O +observed O +in O +addition O +to O +the O +haem B-chemical +- O +mediated O +dimer B-oligomeric_state +( O +chain O +A O +– O +A O +′) O +( O +Supplementary O +Figs O +3 O +and O +6a O +). O + +To O +confirm O +that O +haem B-chemical +- O +assisted O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +occurs O +in O +solution O +, O +we O +analysed O +the O +structure B-evidence +of O +apo B-protein_state +- O +and O +haem B-protein_state +- I-protein_state +bound I-protein_state +PGMRC1 B-protein +by O +two B-experimental_method +- I-experimental_method +dimensional I-experimental_method +nuclear I-experimental_method +magnetic I-experimental_method +resonance I-experimental_method +( O +NMR B-experimental_method +) O +using O +heteronuclear B-experimental_method +single I-experimental_method +- I-experimental_method +quantum I-experimental_method +coherence I-experimental_method +and I-experimental_method +transverse I-experimental_method +relaxation I-experimental_method +- I-experimental_method +optimized I-experimental_method +spectroscopy I-experimental_method +( O +Supplementary O +Figs O +6b O +and O +7 O +). O + +NMR B-experimental_method +signals O +from O +some O +amino O +acid O +residues O +of O +PGRMC1 B-protein +disappeared O +due O +to O +the O +paramagnetic O +relaxation O +effect O +of O +haem B-chemical +( O +Supplementary O +Figs O +6b O +); O +these O +residues O +were O +located O +in O +the O +haem B-site +- I-site +binding I-site +region I-site +. O + +Furthermore O +, O +free B-evidence +energy I-evidence +of I-evidence +dissociation I-evidence +predicted O +by O +PISA B-experimental_method +suggested O +that O +the O +haem B-chemical +- O +mediated O +dimer B-oligomeric_state +is O +stable B-protein_state +in O +solution O +while O +the O +other O +potential O +interactions O +are O +not O +. O + +We O +also O +attempted O +to O +predict O +the O +secondary O +structure O +of O +PGRMC1 B-protein +through O +NMR B-experimental_method +data O +by O +calculating O +with O +TALOS B-experimental_method ++ I-experimental_method +program I-experimental_method +( O +Supplementary O +Fig O +. O +8 O +); O +the O +prediction O +suggested O +that O +the O +overall O +secondary O +structure O +is O +comparable O +between O +apo B-protein_state +- O +and O +haem B-protein_state +- I-protein_state +bound I-protein_state +forms O +of O +PGRMC1 B-protein +in O +solution O +. O + +Mass B-experimental_method +spectrometry I-experimental_method +( O +MS B-experimental_method +) O +analyses O +under O +non B-experimental_method +- I-experimental_method +denaturing I-experimental_method +condition I-experimental_method +demonstrated O +that O +the O +apo B-protein_state +- O +monomer B-oligomeric_state +PGRMC1 B-protein +resulted O +in O +dimerization B-oligomeric_state +by O +binding O +with O +haem B-chemical +( O +Fig O +. O +2a O +). O + +It O +should O +be O +noted O +that O +a O +disulfide B-ptm +bond I-ptm +between O +two O +Cys129 B-residue_name_number +residues O +is O +observed O +in O +the O +crystal B-evidence +of O +PGRMC1 B-protein +( O +Fig O +. O +1a O +), O +while O +Cys129 B-residue_name_number +is O +not B-protein_state +conserved I-protein_state +among O +the O +MAPR B-protein_type +family O +proteins O +( O +Supplementary O +Fig O +. O +5a O +). O + +This O +observation O +led O +us O +to O +examine O +whether O +or O +not O +the O +disulfide B-ptm +bond I-ptm +contributes O +to O +PGRMC1 B-protein +dimerization B-oligomeric_state +. O + +MS B-experimental_method +analyses O +under O +non B-experimental_method +- I-experimental_method +denaturing I-experimental_method +conditions I-experimental_method +clearly O +showed O +that O +the O +Cys129Ser B-mutant +( O +C129S B-mutant +) O +mutant B-protein_state +is O +dimerized B-protein_state +in O +the O +presence B-protein_state +of I-protein_state +haem B-chemical +, O +indicating O +that O +the O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +occurs O +independently O +of O +the O +disulfide B-ptm +bond I-ptm +formation O +via O +Cys129 B-residue_name_number +( O +Fig O +. O +2a O +). O + +Supporting O +this O +, O +MS B-experimental_method +analyses O +under O +denaturing B-experimental_method +conditions I-experimental_method +showed O +that O +haem B-chemical +- O +mediated O +PGRMC1 B-protein +dimer B-oligomeric_state +is O +completely O +dissociated O +into O +monomer B-oligomeric_state +, O +indicating O +that O +dimerization B-oligomeric_state +of O +this O +kind O +is O +not O +mediated O +by O +any O +covalent O +bond O +such O +as O +disulfide B-ptm +bond I-ptm +( O +Supplementary O +Fig O +. O +9 O +). O + +We O +also O +analysed O +the O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +by O +diffusion B-experimental_method +- I-experimental_method +ordered I-experimental_method +NMR I-experimental_method +spectroscopy I-experimental_method +( O +DOSY B-experimental_method +) O +analyses O +( O +Table O +2 O +, O +Supplementary O +Fig O +. O +10 O +). O + +The O +results O +suggested O +that O +the O +hydrodynamic B-evidence +radius I-evidence +of O +haem B-protein_state +- I-protein_state +bound I-protein_state +PGRMC1 B-protein +is O +larger O +than O +that O +of O +apo B-protein_state +- O +PGRMC1 B-protein +. O + +To O +further O +evaluate O +changes O +in O +molecular O +weights O +in O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +, O +sedimentation B-experimental_method +velocity I-experimental_method +analytical I-experimental_method +ultracentrifugation I-experimental_method +( O +SV B-experimental_method +- I-experimental_method +AUC I-experimental_method +) O +analysis O +was O +carried O +out O +. O + +Whereas O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +wt B-protein_state +) O +apo B-protein_state +- O +PGRMC1 B-protein +appeared O +at O +a O +1 O +. O +9 O +S O +peak O +as O +monomer B-oligomeric_state +, O +the O +haem B-chemical +- O +binding O +PGRMC1 B-protein +was O +converted O +into O +dimer B-oligomeric_state +at O +a O +3 O +. O +1 O +S O +peak O +( O +Fig O +. O +2b O +). O + +The O +sedimentation B-evidence +coefficients I-evidence +calculated O +on O +the O +basis O +of O +the O +crystal B-evidence +structure I-evidence +were O +1 O +. O +71 O +S O +for O +monomer B-oligomeric_state +and O +2 O +. O +56 O +S O +for O +dimer B-oligomeric_state +( O +Supplementary O +Fig O +. O +11 O +, O +upper O +panel O +). O + +The O +results O +showed O +that O +the O +PGRMC1 B-protein +dimer B-oligomeric_state +is O +not O +dissociated O +into O +monomer B-oligomeric_state +at O +all O +concentrations O +examined O +( O +Supplementary O +Fig O +. O +11 O +, O +lower O +panel O +), O +suggesting O +that O +the O +Kd B-evidence +value O +of O +haem B-chemical +- O +mediated O +dimer B-oligomeric_state +of O +PGRMC1 B-protein +is O +under O +3 O +. O +5 O +μmol O +l O +− O +1 O +. O + +A O +value O +of O +this O +kind O +implies O +that O +the O +PGRMC1 B-protein +dimer B-oligomeric_state +is O +more O +stable O +than O +other O +dimers B-oligomeric_state +of O +extracellular B-structure_element +domain I-structure_element +of O +membrane B-protein_type +proteins I-protein_type +such O +as O +Toll B-protein +like I-protein +receptor I-protein +9 I-protein +( O +dimerization B-oligomeric_state +Kd B-evidence +of O +20 O +μmol O +l O +− O +1 O +) O +( O +ref O +.) O +and O +plexin B-protein +A2 I-protein +receptor I-protein +( O +dimerization B-oligomeric_state +Kd B-evidence +higher O +than O +300 O +μmol O +l O +− O +1 O +) O +( O +ref O +.). O + +The O +current O +analytical O +data O +confirmed O +that O +apo B-protein_state +- O +PGRMC1 B-protein +monomer B-oligomeric_state +converts O +into O +dimer B-oligomeric_state +by O +binding O +to O +haem B-chemical +in O +solution O +( O +Table O +2 O +). O + +These O +results O +raised O +the O +possibility O +that O +the O +function O +of O +PGRMC1 B-protein +is O +regulated O +by O +intracellular O +haem B-chemical +concentrations O +. O + +CO B-chemical +inhibits O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein + +Crystallographic B-experimental_method +analyses I-experimental_method +revealed O +that O +Tyr113 B-residue_name_number +of O +PGRMC1 B-protein +is O +an O +axial O +ligand O +for O +haem B-chemical +and O +contributes O +to O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +( O +Fig O +. O +1a O +). O + +Analysis O +of O +UV B-evidence +- I-evidence +visible I-evidence +spectra I-evidence +revealed O +that O +the O +heme B-chemical +of O +PGRMC1 B-protein +is O +reducible O +from O +ferric B-protein_state +to O +ferrous B-protein_state +state O +, O +thus O +allowing O +CO B-chemical +binding O +( O +Fig O +. O +3a O +). O + +Furthermore O +, O +the O +UV B-evidence +- I-evidence +visible I-evidence +spectrum I-evidence +of O +the O +wild B-protein_state +type I-protein_state +PGRMC1 B-protein +was O +the O +same O +as O +that O +of O +the O +C129S B-mutant +mutant B-protein_state +of O +PGRMC1 B-protein +, O +and O +the O +R B-evidence +/ I-evidence +Z I-evidence +ratio I-evidence +determined O +by O +the O +intensities O +between O +the O +Soret O +band O +( O +394 O +nm O +) O +peak O +and O +the O +274 O +- O +nm O +peak O +showed O +that O +these O +proteins O +were O +fully B-protein_state +loaded I-protein_state +with I-protein_state +haem B-chemical +( O +Supplementary O +Fig O +. O +12 O +). O + +Analysis O +of O +the O +ferric B-protein_state +form O +of O +PGRMC1 B-protein +using O +resonance B-experimental_method +Raman I-experimental_method +spectroscopy I-experimental_method +( O +Supplementary O +Fig O +. O +13 O +) O +showed O +that O +the O +relative O +intensity O +of O +oxidation O +and O +spin O +state O +marker O +bands O +( O +ν4 O +and O +ν3 O +) O +is O +close O +to O +1 O +. O +0 O +, O +which O +is O +consistent O +with O +it O +being O +a O +haem B-chemical +protein O +with O +a O +proximal O +Tyr B-residue_name +coordination O +. O + +A O +specific O +Raman B-evidence +shift I-evidence +peaking O +at O +vFe O +– O +CO O += O +500 O +cm O +− O +1 O +demonstrated O +that O +the O +CO B-protein_state +- I-protein_state +bound I-protein_state +haem B-chemical +of O +PGRMC1 B-protein +is O +six O +- O +coordinated O +( O +Supplementary O +Fig O +. O +13 O +). O + +Since O +PGRMC1 B-protein +dimerization B-oligomeric_state +involves O +the O +open O +surface B-site +of O +haem B-chemical +on O +the O +opposite O +side O +of O +the O +axial O +Tyr113 B-residue_name_number +, O +no O +space O +for O +CO B-chemical +binding O +is O +available O +in O +the O +dimeric B-oligomeric_state +structure B-evidence +( O +Fig O +. O +3b O +). O + +This O +prompted O +us O +to O +ask O +if O +CO B-chemical +binding O +to O +haem B-chemical +causes O +dissociation O +of O +the O +PGRMC1 B-protein +dimer B-oligomeric_state +. O + +Analysis O +by O +gel B-experimental_method +filtration I-experimental_method +chromatography I-experimental_method +revealed O +that O +the O +relative O +molecular O +sizes O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +and O +the O +C129S B-mutant +mutant B-protein_state +of O +PGRMC1 B-protein +are O +increased O +by O +adding O +haem B-chemical +to O +apo B-protein_state +- O +PGRMC1 B-protein +regardless O +of O +the O +oxidation O +state O +of O +the O +iron B-chemical +( O +Fig O +. O +3c O +), O +which O +is O +in O +agreement O +with O +the O +results O +in O +Table O +1 O +. O + +CO B-chemical +application O +to O +ferrous B-protein_state +PGRMC1 B-protein +abolished O +the O +haem B-chemical +- O +dependent O +increase O +in O +its O +molecular O +size O +. O + +Under O +this O +reducing O +condition O +in O +the O +presence B-protein_state +of I-protein_state +dithionite B-chemical +, O +analyses O +of O +UV B-evidence +- I-evidence +visible I-evidence +spectra I-evidence +indicated O +that O +CO B-chemical +- O +binding O +with O +haem B-complex_assembly +- I-complex_assembly +PGRMC1 I-complex_assembly +is O +stable B-protein_state +, O +showing O +only O +20 O +% O +reduction O +of O +the O +absorbance O +at O +412 O +nm O +within O +2 O +h O +( O +Supplementary O +Fig O +. O +14 O +). O + +Furthermore O +, O +the O +Tyr113Phe B-mutant +( O +Y113F B-mutant +) O +mutant B-protein_state +of O +PGRMC1 B-protein +was O +not O +responsive O +to O +haem B-chemical +. O + +These O +results O +suggest O +that O +CO B-chemical +favours O +the O +six O +- O +coordinate O +form O +of O +haem B-chemical +and O +interferes O +with O +the O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +. O + +To O +examine O +the O +inhibitory O +effects O +of O +CO B-chemical +on O +haem B-chemical +- O +mediated O +PGRMC1 B-protein +dimerization B-oligomeric_state +, O +SV B-experimental_method +- I-experimental_method +AUC I-experimental_method +analysis O +was O +carried O +out O +. O + +The O +peak O +corresponding O +to O +the O +haem B-chemical +/ O +PGRMC1 B-protein +dimer B-oligomeric_state +was O +detected O +under O +reducing O +conditions O +in O +the O +presence B-protein_state +of I-protein_state +dithionite B-chemical +( O +Supplementary O +Fig O +. O +15 O +, O +middle O +panel O +). O + +These O +observations O +raised O +the O +transition O +model O +for O +structural O +regulation O +of O +PGRMC1 B-protein +in O +response O +to O +haem B-chemical +( O +Fig O +. O +3d O +). O + +As O +mentioned O +above O +, O +apo B-protein_state +- O +PGRMC1 B-protein +exists O +as O +monomer B-oligomeric_state +. O + +By O +binding O +with O +haem B-chemical +( O +binding O +Kd B-evidence += O +50 O +nmol O +l O +− O +1 O +), O +PGRMC1 B-protein +forms O +a O +stable B-protein_state +dimer B-oligomeric_state +( O +dimerization B-oligomeric_state +Kd B-evidence +<< O +3 O +. O +5 O +μmol O +l O +− O +1 O +) O +through O +stacking B-bond_interaction +of O +the O +two O +open O +surfaces B-site +of O +the O +five O +- O +coordinated O +haem B-chemical +molecules O +in O +each O +monomer B-oligomeric_state +. O + +CO B-chemical +induces O +the O +dissociation O +of O +the O +haem B-chemical +- O +mediated O +dimer B-oligomeric_state +of O +PGRMC1 B-protein +by O +interfering O +with O +the O +haem B-site +- I-site +stacking I-site +interface I-site +via O +formation O +of O +the O +six O +- O +coordinated O +CO B-complex_assembly +- I-complex_assembly +haem I-complex_assembly +- I-complex_assembly +PGRMC1 I-complex_assembly +complex O +. O + +Such O +a O +dynamic O +structural O +regulation O +led O +us O +to O +further O +examine O +the O +regulation O +of O +PGRMC1 B-protein +functions O +in O +cancer O +cells O +. O + +PGRMC1 B-protein +dimerization B-oligomeric_state +is O +required O +for O +binding O +to O +EGFR B-protein_type + +Because O +PGRMC1 B-protein +is O +known O +to O +interact O +with O +EGFR B-protein_type +and O +to O +accelerate O +tumour O +progression O +, O +we O +examined O +the O +effect O +of O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +on O +its O +interaction O +with O +EGFR B-protein_type +by O +using O +purified O +proteins O +. O + +As O +shown O +in O +Fig O +. O +4a O +, O +the O +cytosolic B-structure_element +domain I-structure_element +of O +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +, O +but O +not O +the O +Y113F B-mutant +mutant B-protein_state +, O +interacted O +with O +purified O +EGFR B-protein_type +in O +a O +haem B-chemical +- O +dependent O +manner O +. O + +We O +further O +analysed O +the O +intracellular O +interaction O +between O +PGRMC1 B-protein +and O +EGFR B-protein_type +. O + +FLAG B-protein_state +- I-protein_state +tagged I-protein_state +PGRMC1 B-protein +ectopically B-experimental_method +expressed I-experimental_method +in O +human B-species +colon O +cancer O +HCT116 O +cells O +was O +immunoprecipitated B-experimental_method +with O +anti O +- O +FLAG O +antibody O +, O +and O +co B-experimental_method +- I-experimental_method +immunoprecipitated I-experimental_method +EGFR B-protein_type +and O +endogenous B-protein_state +PGRMC1 B-protein +binding O +to O +FLAG O +- O +PGRMC1 B-protein +were O +detected O +by O +Western B-experimental_method +blotting I-experimental_method +( O +Fig O +. O +4c O +). O + +Whereas O +FLAG B-protein_state +- I-protein_state +tagged I-protein_state +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +interacted O +with O +endogenous B-protein_state +PGRMC1 B-protein +and O +EGFR B-protein_type +, O +the O +Y113F B-mutant +mutant B-protein_state +did O +not O +. O + +We O +also O +examined O +the O +effect O +of O +succinylacetone B-chemical +( O +SA B-chemical +), O +an O +inhibitor O +of O +haem B-chemical +biosynthesis O +( O +Fig O +. O +4d O +). O + +As O +expected O +, O +SA B-chemical +significantly O +reduced B-protein_state +PGRMC1 B-protein +dimerization B-oligomeric_state +and O +its O +interaction O +with O +EGFR B-protein_type +( O +Fig O +. O +4e O +), O +indicating O +that O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGMRC1 B-protein +is O +critical O +for O +its O +binding O +to O +EGFR B-protein_type +. O + +PGRMC1 B-protein +dimer B-oligomeric_state +facilitates O +EGFR B-protein_type +- O +mediated O +cancer O +growth O + +Next O +, O +we O +investigated O +the O +functional O +significance O +of O +PGRMC1 B-protein +dimerization B-oligomeric_state +in O +EGFR B-protein_type +signaling O +. O + +EGF B-protein_type +- O +induced O +phosphorylations B-ptm +of O +EGFR B-protein_type +and O +its O +downstream O +targets O +AKT B-protein_type +and O +ERK B-protein_type +were O +decreased O +by O +PGRMC1 B-protein +knockdown B-protein_state +( O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +) O +( O +Fig O +. O +4f O +). O + +These O +results O +suggested O +that O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +is O +critical O +for O +EGFR B-protein_type +signaling O +. O + +Chemosensitivity O +enhancement O +by O +two O +different O +shRNAs B-chemical +to O +PGRMC1 B-protein +was O +seen O +also O +in O +HCT116 O +cells O +and O +human B-species +hepatoma O +HuH7 O +cells O +( O +Supplementary O +Fig O +. O +17 O +). O + +Furthermore O +, O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +inhibited O +spheroid O +formation O +of O +HCT116 O +cells O +in O +culture O +, O +and O +this O +inhibition O +was O +reversed O +by O +co B-experimental_method +- I-experimental_method +expression I-experimental_method +of O +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +but O +not O +of O +the O +Y113F B-mutant +mutant B-protein_state +( O +Fig O +. O +5c O +and O +Supplementary O +Fig O +. O +18 O +). O + +Thus O +, O +PGRMC1 B-protein +dimerization B-oligomeric_state +is O +important O +for O +cancer O +cell O +proliferation O +and O +chemoresistance O +. O + +Ten O +days O +after O +intra B-experimental_method +- I-experimental_method +splenic I-experimental_method +implantation I-experimental_method +of O +HCT116 O +cells O +that O +were O +genetically O +tagged O +with O +a O +fluorescent O +protein O +Venus O +, O +the O +group O +implanted O +with O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +showed O +a O +significant O +decrease O +of O +liver O +metastasis O +in O +comparison O +with O +the O +control O +group O +( O +Fig O +. O +5d O +). O + +Since O +PGRMC1 B-protein +has O +been O +shown O +to O +interact O +with O +cytochromes B-protein_type +P450 I-protein_type +( O +ref O +), O +we O +investigated O +whether O +the O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +is O +necessary O +for O +their O +interactions O +. O + +Recombinant O +CYP1A2 B-protein +and O +CYP3A4 B-protein +including O +a O +microsomal O +formulation O +containing O +cytochrome B-protein_type +b5 I-protein_type +and O +cytochrome B-protein +P450 I-protein +reductase I-protein +, O +drug O +- O +metabolizing O +cytochromes B-protein_type +P450 I-protein_type +, O +interacted O +with O +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +, O +but O +not O +with O +the O +Y113F B-mutant +mutant B-protein_state +, O +in O +a O +haem B-chemical +- O +dependent O +manner O +( O +Fig O +. O +6a O +, O +b O +). O + +Moreover O +, O +the O +interaction O +of O +PGRMC1 B-protein +with O +CYP1A2 B-protein +was O +blocked O +by O +CORM3 B-chemical +under O +reducing O +conditions O +( O +Fig O +. O +6c O +), O +indicating O +that O +PGRMC1 B-protein +dimerization B-oligomeric_state +is O +necessary O +for O +its O +interaction O +with O +cytochromes B-protein_type +P450 I-protein_type +. O + +Doxorubicin B-chemical +is O +an O +anti O +- O +cancer O +reagent O +that O +is O +metabolized O +into O +inactive O +doxorubicinol B-chemical +by O +CYP2D6 B-protein +and O +CYP3A4 B-protein +( O +Fig O +. O +6d O +). O + +PGRMC1 B-mutant +- I-mutant +KD I-mutant +significantly O +suppressed O +the O +conversion O +of O +doxorubicin B-chemical +to O +doxorubicinol B-chemical +( O +Fig O +. O +6d O +) O +and O +increased O +sensitivity O +to O +doxorubicin B-chemical +( O +Fig O +. O +6e O +). O + +Enhanced O +doxorubicin B-chemical +sensitivity O +was O +modestly O +but O +significantly O +induced O +by O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +. O + +To O +gain O +further O +insight O +into O +the O +interaction O +between O +PGRMC1 B-protein +and O +cytochromes B-protein_type +P450 I-protein_type +, O +surface B-experimental_method +plasmon I-experimental_method +resonance I-experimental_method +analyses I-experimental_method +were O +conducted O +using O +recombinant O +CYP51 B-protein +and O +PGRMC1 B-protein +. O + +This O +was O +based O +on O +a O +previous O +study O +showing O +that O +PGRMC1 B-protein +binds O +to O +CYP51 B-protein +and O +enhances O +cholesterol O +biosynthesis O +by O +CYP51 B-protein +( O +refs O +). O + +CYP51 B-protein +interacted O +with O +PGRMC1 B-protein +in O +a O +concentration O +- O +dependent O +manner O +in O +the O +presence B-protein_state +of I-protein_state +haem B-chemical +, O +but O +not O +in O +its O +absence B-protein_state +( O +Supplementary O +Fig O +. O +19 O +), O +suggesting O +the O +requirement O +for O +the O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +. O + +The O +Kd B-evidence +value O +of O +PGRMC1 B-protein +binding O +to O +CYP51 B-protein +was O +in O +a O +micromolar O +range O +and O +comparable O +with O +those O +of O +other O +haem B-chemical +proteins O +, O +such O +as O +cytochrome B-protein +P450 I-protein +reductase I-protein +and O +neuroglobin B-protein +/ O +Gαi1 B-protein +( O +ref O +.), O +suggesting O +that O +haem B-chemical +- O +dependent O +PGRMC1 B-protein +interaction O +with O +CYP51 B-protein +is O +biologically O +relevant O +. O + +In O +this O +study O +, O +we O +showed O +that O +PGRMC1 B-protein +dimerizes B-oligomeric_state +by O +stacking B-bond_interaction +interactions I-bond_interaction +of O +haem B-chemical +molecules O +from O +each O +monomer B-oligomeric_state +. O + +Recently O +, O +Lucas O +et O +al O +. O +reported O +that O +translationally B-protein_type +- I-protein_type +controlled I-protein_type +tumour I-protein_type +protein I-protein_type +was O +dimerized B-protein_state +by O +binding O +with O +haem B-chemical +, O +but O +its O +structural O +basis O +remains O +unclear O +. O + +This O +is O +the O +report O +showing O +crystallographic O +evidence O +that O +indicates O +roles O +of O +the O +direct O +haem B-bond_interaction +– I-bond_interaction +haem I-bond_interaction +stacking I-bond_interaction +in O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +in O +eukaryotes B-taxonomy_domain +, O +although O +a O +few O +examples O +are O +known O +in O +bacteria B-taxonomy_domain +. O + +Sequence B-experimental_method +alignments I-experimental_method +show O +that O +haem B-site +- I-site +binding I-site +residues I-site +( O +Tyr113 B-residue_name_number +, O +Tyr107 B-residue_name_number +, O +Lys163 B-residue_name_number +and O +Tyr164 B-residue_name_number +) O +in O +PGRMC1 B-protein +are O +conserved B-protein_state +among O +MAPR B-protein_type +proteins O +( O +Supplementary O +Fig O +. O +5 O +). O + +Since O +the O +Y113 B-residue_name_number +residue O +is O +involved O +in O +the O +putative O +consensus B-structure_element +motif I-structure_element +of O +phosphorylation B-ptm +by O +tyrosine B-protein_type +kinases I-protein_type +such O +as O +Abl B-protein_type +and O +Lck B-protein_type +, O +we O +investigated O +whether O +phosphorylated B-protein_state +Y113 B-residue_name_number +is O +present O +in O +HCT116 O +cells O +by O +ESI B-experimental_method +- I-experimental_method +MS I-experimental_method +analysis O +. O + +While O +the O +effects O +of O +PGRMC1 B-protein +on O +cholesterol O +synthesis O +mediated O +by O +CYP51 B-protein +have O +been O +well O +documented O +in O +yeast B-taxonomy_domain +and O +human B-species +cells O +, O +it O +has O +not O +been O +clear O +whether O +drug O +- O +metabolizing O +CYP B-protein_type +activities O +are O +regulated O +by O +PGRMC1 B-protein +. O + +Szczesna O +- O +Skorupa O +and O +Kemper O +reported O +that O +PGRMC1 B-protein +exhibited O +an O +inhibitory O +effect O +on O +CYP3A4 B-protein +drug O +metabolizing O +activity O +by O +competitively O +binding O +with O +cytochrome B-protein +P450 I-protein +reductase I-protein +( O +CPR B-protein +) O +in O +HEK293 O +or O +HepG2 O +cells O +. O + +On O +the O +other O +hand O +, O +Oda O +et O +al O +. O +reported O +that O +PGRMC1 B-protein +had O +no O +effect O +to O +CYP2E1 B-protein +and O +CYP3A4 B-protein +activities O +in O +HepG2 O +cell O +. O + +Several O +other O +groups O +showed O +that O +PGRMC1 B-protein +enhanced O +chemoresistance O +in O +several O +cancer O +cells O +such O +as O +uterine O +sarcoma O +, O +breast O +cancer O +, O +endometrial O +tumour O +and O +ovarian O +cancer O +; O +however O +, O +no O +evidence O +of O +PGRMC1 B-protein +- O +dependent O +regulation O +of O +CYP B-protein_type +activity O +was O +provided O +. O + +Our O +results O +showed O +that O +PGRMC1 B-protein +contributes O +to O +enhancement O +of O +the O +doxorubicin B-chemical +metabolism O +, O +which O +is O +mediated O +by O +CYP2D6 B-protein +or O +CYP3A4 B-protein +in O +human B-species +colon O +cancer O +HCT116 O +cells O +( O +Fig O +. O +6d O +). O + +We O +showed O +that O +haem B-chemical +- O +mediated O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +enhances O +proliferation O +and O +chemoresistance O +of O +cancer O +cells O +through O +binding O +to O +and O +regulating O +EGFR B-protein_type +and O +cytochromes B-protein_type +P450 I-protein_type +( O +illustrated O +in O +Fig O +. O +7 O +). O + +Since O +the O +haem B-evidence +- I-evidence +binding I-evidence +affinity I-evidence +of O +PGRMC1 B-protein +is O +lower O +than O +those O +of O +constitutive B-protein_state +haem B-protein_type +- I-protein_type +binding I-protein_type +proteins I-protein_type +such O +as O +myoglobin B-protein +, O +PGMRC1 B-protein +is O +probably O +interconverted O +between O +apo B-protein_state +- O +monomer B-oligomeric_state +and O +haem B-protein_state +- I-protein_state +bound I-protein_state +dimer B-oligomeric_state +forms O +in O +response O +to O +changes O +in O +the O +intracellular O +haem B-chemical +concentration O +. O + +Moreover O +, O +exposure O +of O +cancer O +cells O +to O +stimuli O +such O +as O +hypoxia O +, O +radiation O +and O +chemotherapy O +causes O +cell O +damages O +and O +leads O +to O +protein O +degradation O +, O +resulting O +in O +increased O +levels O +of O +TCA O +cycle O +intermediates O +and O +in O +an O +enhanced O +haem B-chemical +biosynthesis O +. O + +On O +the O +other O +hand O +, O +excessive O +haem B-chemical +induces O +HO B-protein +- I-protein +1 I-protein +, O +the O +enzyme O +that O +oxidatively O +degrades O +haem B-chemical +and O +generates O +CO B-chemical +. O + +This O +idea O +is O +consistent O +with O +the O +observation O +that O +HO B-protein +- I-protein +1 I-protein +induction O +or O +CO B-chemical +inhibits O +tumour O +growth O +. O + +Besides O +the O +regulatory O +roles O +of O +PGRMC1 B-protein +/ O +Sigma B-protein +- I-protein +2 I-protein +receptor O +in O +proliferation O +and O +chemoresistance O +in O +cancer O +cells O +( O +ref O +.), O +recent O +reports O +show O +that O +PGRMC1 B-protein +is O +able O +to O +bind O +to O +amyloid B-protein +beta I-protein +oligomer B-oligomeric_state +to O +enhance O +its O +neurotoxicity O +. O + +The O +roles O +of O +haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +in O +the O +functional O +regulation O +of O +its O +target O +proteins O +deserve O +further O +studies O +to O +find O +evidence O +that O +therapeutic O +interventions O +to O +interfere O +with O +the O +function O +of O +the O +dimer B-oligomeric_state +may O +control O +varied O +disease O +conditions O +. O + +Alzheimer O +' O +s O +therapeutics O +targeting O +amyloid O +beta O +1 O +- O +42 O +oligomers O +II O +: O +Sigma O +- O +2 O +/ O +PGRMC1 O +receptors O +mediate O +Abeta O +42 O +oligomer B-oligomeric_state +binding O +and O +synaptotoxicity O + +X B-evidence +- I-evidence +ray I-evidence +crystal I-evidence +structure I-evidence +of O +PGRMC1 B-protein +. O + +( O +a O +) O +Structure O +of O +the O +PGRMC1 B-protein +dimer B-oligomeric_state +formed O +through O +stacked O +haems B-chemical +. O + +Two O +PGRMC1 B-protein +subunits B-structure_element +( O +blue O +and O +green O +ribbons O +) O +dimerize B-oligomeric_state +via O +stacking B-bond_interaction +of O +the O +haem B-chemical +molecules O +. O + +PGRCM1 B-protein +is O +dimerized B-protein_state +by O +binding O +with O +haem B-chemical +. O + +( O +a O +) O +Mass B-experimental_method +spectrometric I-experimental_method +analyses O +of O +the O +wild B-protein_state +- I-protein_state +type I-protein_state +( O +wt B-protein_state +) O +PGRMC1 B-protein +or O +the O +C129S B-mutant +mutant B-protein_state +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +haem B-chemical +under O +non O +- O +denaturing O +condition O +. O + +Both O +proteins O +had O +identical O +lengths O +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +). O + +( O +b O +) O +SV B-experimental_method +- I-experimental_method +AUC I-experimental_method +analyses O +of O +the O +wt B-protein_state +- O +PGRMC1 B-protein +and O +the O +C129S B-mutant +mutant B-protein_state +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +) O +in O +the O +presence B-protein_state +or O +absence B-protein_state +of I-protein_state +haem B-chemical +. O + +( O +c O +) O +Difference B-evidence +absorption I-evidence +spectra I-evidence +of O +PGRMC1 B-protein +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +) O +titrated B-experimental_method +with I-experimental_method +haem B-chemical +( O +left O +panel O +). O + +The O +titration B-evidence +curve I-evidence +of O +haem B-chemical +to O +PGRMC1 B-protein +( O +right O +panel O +). O + +The O +absorbance B-evidence +difference I-evidence +at O +400 O +nm O +is O +plotted O +against O +the O +haem B-chemical +concentration O +. O + +Carbon B-chemical +monoxide I-chemical +inhibits O +haem B-chemical +- O +dependent O +PGRMC1 B-protein +dimerization B-oligomeric_state +. O + +Haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +is O +necessary O +for O +tumour O +proliferation O +mediated O +by O +EGFR B-protein_type +signalling O +. O + +( O +c O +) O +FLAG O +- O +PGRMC1 B-protein +wt B-protein_state +or O +Y113F B-mutant +( O +full B-protein_state +length I-protein_state +) O +was O +over B-experimental_method +- I-experimental_method +expressed I-experimental_method +in O +HCT116 O +cells O +and O +immunoprecipitated B-experimental_method +with O +anti O +- O +FLAG O +antibody O +- O +conjugated O +beads O +. O + +( O +d O +) O +HCT116 O +cells O +were O +treated O +with O +or O +without O +250 O +μmol O +l O +− O +1 O +of O +succinylacetone B-chemical +( O +SA B-chemical +) O +for O +48 O +h O +. O +The O +intracellular O +haem B-chemical +was O +extracted O +and O +quantified O +by O +reverse B-experimental_method +- I-experimental_method +phase I-experimental_method +HPLC I-experimental_method +. O + +of O +four O +separate O +experiments O +. O +** O +P O +< O +0 O +. O +01 O +using O +unpaired O +Student B-experimental_method +' I-experimental_method +s I-experimental_method +t I-experimental_method +- I-experimental_method +test I-experimental_method +. O +( O +e O +) O +Co B-experimental_method +- I-experimental_method +immunoprecipitation I-experimental_method +assay I-experimental_method +was O +performed O +as O +in O +( O +c O +) O +with O +or O +without O +SA B-chemical +treatment O +in O +HCT116 O +cells O +. O + +( O +g O +, O +h O +) O +HCT116 O +cells O +were O +treated O +with O +or O +without O +EGF B-protein_type +, O +SA B-chemical +, O +RuCl3 B-chemical +and O +CORM3 B-chemical +as O +indicated O +, O +and O +components O +of O +the O +EGFR B-protein_type +signaling O +pathway O +were O +detected O +by O +Western B-experimental_method +blotting I-experimental_method +. O + +Haem B-chemical +- O +dependent O +dimerization B-oligomeric_state +of O +PGRMC1 B-protein +accelerates O +tumour O +growth O +through O +the O +EGFR B-protein_type +signaling O +pathway O +. O + +( O +a O +) O +Nucleotide O +sequences O +of O +PGRMC1 B-protein +targeted O +by O +shRNA B-chemical +and O +of O +the O +shRNA B-protein_state +- I-protein_state +resistant I-protein_state +full B-protein_state +length I-protein_state +PGRMC1 B-protein +expression O +vector O +. O + +Stable O +PGRMC1 B-mutant +- I-mutant +knockdown I-mutant +( O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +) O +HCT116 O +cells O +were O +transiently B-experimental_method +transfected I-experimental_method +with O +the O +shRNA B-protein_state +- I-protein_state +resistant I-protein_state +expression B-experimental_method +vector I-experimental_method +of O +wild B-protein_state +- I-protein_state +type I-protein_state +PGRMC1 B-protein +( O +wt B-protein_state +) O +or O +the O +Y113F B-mutant +mutant B-protein_state +( O +Y113F B-mutant +). O + +( O +b O +) O +Erlotinib B-chemical +was O +added O +to O +HCT116 O +( O +control O +) O +cells O +, O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +or O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +expressing O +shRNA B-protein_state +- I-protein_state +resistant I-protein_state +PGRMC1 B-protein +wt B-protein_state +or O +Y113F B-mutant +, O +and O +cell O +viability O +was O +examined O +by O +MTT B-experimental_method +assay I-experimental_method +. O + +of O +four O +separate O +experiments O +. O +* B-evidence +P I-evidence +< O +0 O +. O +01 O +using O +ANOVA B-experimental_method +with O +Fischer B-experimental_method +' I-experimental_method +s I-experimental_method +LSD I-experimental_method +test I-experimental_method +. O + +( O +c O +) O +Spheroid O +formation O +in O +control O +and O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +HCT116 O +cells O +. O + +The O +graph O +represents O +mean O +± O +s O +. O +e O +. O +of O +each O +spheroid O +size O +. O +* B-evidence +P I-evidence +< O +0 O +. O +01 O +using O +ANOVA B-experimental_method +with O +Fischer B-experimental_method +' I-experimental_method +s I-experimental_method +LSD I-experimental_method +test I-experimental_method +. O + +Scale O +bar O +: O +0 O +. O +1 O +mm O +. O +( O +d O +) O +Tumour O +- O +bearing O +livers O +of O +NOG O +mice O +at O +10 O +days O +after O +intrasplenic B-experimental_method +injection I-experimental_method +of O +HCT116 O +( O +control O +) O +or O +PGRMC1 B-mutant +- I-mutant +KD I-mutant +cells O +. O + +( O +a O +, O +b O +) O +FLAG O +- O +PGRMC1 B-protein +wild B-protein_state +- I-protein_state +type I-protein_state +( O +wt B-protein_state +) O +and O +Y113F B-mutant +mutant B-protein_state +proteins O +( O +a O +. O +a O +. O +44 B-residue_range +– I-residue_range +195 I-residue_range +), O +in O +either O +apo B-protein_state +or O +haem B-protein_state +- I-protein_state +bound I-protein_state +form O +, O +were O +incubated B-experimental_method +with O +CYP1A2 B-protein +( O +a O +) O +or O +CYP3A4 B-protein +( O +b O +) O +and O +immunoprecipitated B-experimental_method +with O +anti O +- O +FLAG O +antibody O +- O +conjugated O +beads O +. O + +( O +c O +) O +Binding B-experimental_method +assay I-experimental_method +was O +performed O +as O +in O +( O +a O +) O +using O +haem B-protein_state +- I-protein_state +bound I-protein_state +FLAG O +- O +PGRMC1 B-protein +wt B-protein_state +and O +CYP1A2 B-protein +with O +or O +without O +RuCl3 B-chemical +and O +CORM3 B-chemical +. O + +( O +d O +) O +Schematic O +illustration O +of O +doxorubicin B-chemical +metabolism O +is O +shown O +on O +the O +left O +. O + +Schematic O +diagram O +for O +the O +regulation O +of O +PGRMC1 B-protein +functions O +. O + +Apo B-protein_state +- O +PGRMC1 B-protein +exists O +as O +an O +inactive B-protein_state +monomer B-oligomeric_state +. O + +On O +binding B-protein_state +to I-protein_state +haem B-chemical +, O +PGRMC1 B-protein +forms O +a O +dimer B-oligomeric_state +through O +stacking B-bond_interaction +interactions I-bond_interaction +between O +the O +haem B-chemical +moieties O +, O +which O +enables O +PGRMC1 B-protein +to O +interact O +with O +EGFR B-protein_type +and O +cytochromes B-protein_type +P450 I-protein_type +, O +leading O +to O +an O +enhanced O +proliferation O +and O +chemoresistance O +of O +cancer O +cells O +. O + +Apo O +form O +Haem O +- O +bound O +form O +Mass O +( O +Da O +) O +Mass O +( O +Da O +) O +aPGRMC1 O +wt O +( O +a O +. O +a O +. O +44 O +– O +195 O +) O +ESI O +- O +MS O +— O +17 O +, O +844 O +. O +14 O +— O +36 O +, O +920 O +. O +19 O +Theoretical O +17 O +, O +843 O +. O +65 O +36 O +, O +918 O +. O +06 O +Hydrodynamic O +radius O +10 O +− O +9 O +( O +m O +) O +MW O +( O +kDa O +) O +Hydrodynamic O +radius O +10 O +− O +9 O +( O +m O +) O +MW O +( O +kDa O +) O +DOSY O +2 O +. O +04 O +– O +2 O +. O +15 O +20 O +2 O +. O +94 O +– O +3 O +. O +02 O +42 O +S20 O +, O +w O +( O +S O +) O +MW O +( O +kDa O +) O +S20 O +, O +w O +( O +S O +) O +MW O +( O +kDa O +) O +SV O +- O +AUC O +1 O +. O +9 O +17 O +. O +6 O +3 O +. O +1 O +35 O +. O +5 O +bPGRMC1 O +C129S B-mutant +( O +a O +. O +a O +. O +44 O +– O +195 O +) O +ESI O +- O +MS O +— O +17 O +, O +827 O +. O +91 O +— O +36 O +, O +887 O +. O +07 O +Theoretical O +17 O +, O +827 O +. O +59 O +36 O +, O +885 O +. O +6 O +S20 O +, O +w O +( O +S O +) O +MW O +( O +kDa O +) O +S20 O +, O +w O +( O +S O +) O +MW O +( O +kDa O +) O +SV O +- O +AUC O +2 O +. O +0 O +18 O +. O +1 O +3 O +. O +1 O +35 O +. O +8 O + +Differences O +in O +molecular O +weights O +of O +the O +wild O +- O +type O +( O +wt O +; O +a O +) O +and O +the O +C129S B-mutant +mutant O +( O +b O +) O +PGRMC1 O +proteins O +in O +the O +absence O +( O +apo O +form O +) O +or O +the O +presence O +of O +haem O +( O +haem O +- O +bound O +form O +). O + +The O +protein O +sizes O +of O +the O +wt O +and O +C129S B-mutant +PGRMC1 O +cytosolic O +domains O +( O +a O +. O +a O +. O +44 O +– O +195 O +) O +in O +the O +presence O +or O +absence O +of O +haem O +were O +estimated O +by O +ESI O +- O +MS O +, O +DOSY O +and O +SV O +- O +AUC O +. O + +Hotspot O +autoimmune O +T B-protein_type +cell I-protein_type +receptor I-protein_type +binding O +underlies O +pathogen O +and O +insulin B-chemical +peptide O +cross O +- O +reactivity O + +However O +, O +the O +mechanisms O +that O +allow O +the O +clonal O +T B-complex_assembly +cell I-complex_assembly +antigen I-complex_assembly +receptor I-complex_assembly +( O +TCR B-complex_assembly +) O +to O +functionally O +engage O +multiple O +peptide B-complex_assembly +– I-complex_assembly +major I-complex_assembly +histocompatibility I-complex_assembly +complexes I-complex_assembly +( O +pMHC B-complex_assembly +) O +are O +unclear O +. O + +Here O +, O +we O +studied O +multiligand O +discrimination O +by O +a O +human B-species +, O +preproinsulin B-protein +reactive O +, O +MHC B-complex_assembly +class O +- O +I O +– O +restricted O +CD8 O ++ O +T O +cell O +clone O +( O +1E6 O +) O +that O +can O +recognize O +over O +1 O +million O +different O +peptides O +. O + +We O +generated O +high O +- O +resolution O +structures B-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +bound B-protein_state +to I-protein_state +7 O +altered B-chemical +peptide I-chemical +ligands I-chemical +, O +including O +a O +pathogen O +- O +derived O +peptide O +that O +was O +an O +order O +of O +magnitude O +more O +potent O +than O +the O +natural O +self O +- O +peptide O +. O + +Evaluation O +of O +these O +structures B-evidence +demonstrated O +that O +binding O +was O +stabilized O +through O +a O +conserved O +lock O +- O +and O +- O +key O +– O +like O +minimal O +binding O +footprint O +that O +enables O +1E6 B-complex_assembly +TCR I-complex_assembly +to O +tolerate O +vast O +numbers O +of O +substitutions O +outside O +of O +this O +so O +- O +called O +hotspot O +. O + +Highly O +potent O +antigens O +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +engaged O +with O +a O +strong O +antipathogen B-evidence +- I-evidence +like I-evidence +binding I-evidence +affinity I-evidence +; O +this O +engagement O +was O +governed O +though O +an O +energetic O +switch O +from O +an O +enthalpically O +to O +entropically O +driven O +interaction O +compared O +with O +the O +natural O +autoimmune O +ligand O +. O + +This O +ability O +is O +required O +to O +enable O +the O +estimated O +25 O +million O +distinct O +TCRs B-complex_assembly +expressed O +in O +humans B-species +to O +provide O +effective O +immune O +coverage O +against O +all O +possible O +foreign O +peptide O +antigens O +. O + +Several O +mechanisms O +, O +by O +which O +TCRs B-complex_assembly +could O +bind O +to O +a O +large O +number O +of O +different O +peptide B-complex_assembly +- I-complex_assembly +MHC I-complex_assembly +( O +pMHC B-complex_assembly +), O +have O +been O +proposed O +. O + +Both O +MHC B-complex_assembly +and O +peptide B-chemical +have O +also O +been O +shown O +to O +undergo O +structural O +changes O +upon O +TCR B-complex_assembly +binding O +, O +mediating O +an O +induced O +fit O +between O +the O +TCR B-complex_assembly +and O +pMHC B-complex_assembly +. O + +Other O +studies O +, O +mainly O +in O +the O +murine B-taxonomy_domain +system O +, O +have O +demonstrated O +that O +the O +same O +TCR B-complex_assembly +can O +interact O +with O +different O +pMHCs B-complex_assembly +using O +a O +common O +or O +divergent O +modality O +. O + +Recent O +studies O +in O +model O +murine B-taxonomy_domain +systems O +demonstrate O +that O +TCR B-complex_assembly +cross O +- O +reactivity O +can O +be O +governed O +by O +recognition O +of O +a O +conserved O +region O +in O +the O +peptide O +that O +allows O +tolerance O +of O +peptide O +sequence O +variation O +outside O +of O +this O +hotspot O +. O + +CD8 O ++ O +T O +cells O +that O +recognize O +HLA B-complex_assembly +- I-complex_assembly +A I-complex_assembly +* I-complex_assembly +0201 I-complex_assembly +– I-complex_assembly +ALWGPDPAAA I-complex_assembly +have O +been O +shown O +to O +populate O +insulitic O +lesions O +in O +patients O +with O +type O +1 O +diabetes O +( O +T1D O +). O + +We O +demonstrated O +that O +the O +TCR B-complex_assembly +from O +the O +1E6 O +T O +cell O +clone O +bound B-protein_state +to I-protein_state +HLA B-complex_assembly +- I-complex_assembly +A I-complex_assembly +* I-complex_assembly +0201 I-complex_assembly +– I-complex_assembly +ALWGPDPAAA I-complex_assembly +using O +a O +limited O +footprint O +and O +very O +weak O +binding B-evidence +affinity I-evidence +. O + +This O +first O +experimental O +evidence O +of O +a O +high O +level O +of O +CD8 O ++ O +T O +cell O +cross O +- O +reactivity O +in O +a O +human B-species +autoimmune O +disease O +system O +hinted O +toward O +molecular O +mimicry O +by O +a O +more O +potent O +pathogenic O +peptide O +as O +a O +potential O +mechanism O +leading O +to O +β O +cell O +destruction O +. O + +Here O +, O +we O +solved B-experimental_method +the O +structure B-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +with O +7 O +altered B-chemical +peptide I-chemical +ligands I-chemical +( O +APLs B-chemical +) O +determined O +by O +our O +previously O +published O +combinatorial B-experimental_method +peptide I-experimental_method +library I-experimental_method +( I-experimental_method +CPL I-experimental_method +) I-experimental_method +screening I-experimental_method +, O +2 O +of O +which O +mapped O +within O +human B-species +pathogens O +. O + +These O +APLs B-chemical +differed O +from O +the O +natural O +preproinsulin B-protein +peptide O +by O +up O +to O +7 O +of O +10 O +residues O +. O + +We O +also O +solved B-experimental_method +the O +structure B-evidence +of O +each O +unligated B-protein_state +APL B-chemical +to O +investigate O +whether O +structural O +changes O +occurred O +before O +or O +after O +binding O +— O +which O +, O +combined O +with O +an O +in O +- O +depth O +cellular B-experimental_method +and I-experimental_method +biophysical I-experimental_method +analysis I-experimental_method +of O +the O +1E6 O +interaction O +with O +each O +APL B-chemical +, O +demonstrated O +the O +molecular O +mechanism O +mediating O +the O +high O +level O +of O +cross O +- O +reactivity O +exhibited O +by O +this O +preproinsulin B-protein +- O +reactive O +human B-species +CD8 O ++ O +T O +cell O +clone O +. O + +The O +1E6 O +T O +cell O +clone O +recognizes O +APLs B-chemical +across O +a O +large O +dynamic O +range O +. O + +We O +have O +previously O +demonstrated O +that O +the O +1E6 O +T O +cell O +clone O +can O +recognize O +over O +1 O +million O +different O +peptides O +with O +a O +potency O +comparable O +with O +, O +or O +better O +than O +, O +the O +cognate O +preproinsulin B-protein +peptide O +ALWGPDPAAA B-chemical +. O + +Two O +of O +these O +peptides O +, O +MVWGPDPLYV B-chemical +and O +RQFGPDWIVA B-chemical +( O +bold O +text O +signifies O +amino O +acids O +that O +are O +different O +from O +the O +index O +preproinsulin B-protein +– O +derived O +sequence O +), O +are O +contained O +within O +the O +proteomes O +of O +the O +human B-species +pathogens O +Bacteroides B-species +fragilis I-species +/ I-species +thetaiotaomicron I-species +and O +Clostridium B-species +asparagiforme I-species +, O +respectively O +. O + +Competitive B-experimental_method +functional I-experimental_method +testing I-experimental_method +revealed O +that O +the O +preproinsulin B-protein +- O +derived O +sequence O +ALWGPDPAAA B-chemical +was O +one O +of O +the O +least O +potent O +targets O +for O +1E6 O +, O +with O +only O +the O +MVWGPDPLYV B-chemical +and O +YLGGPDFPTI B-chemical +demonstrating O +a O +similar O +low O +- O +activity O +profile O +in O +MIP B-protein +- I-protein +1β I-protein +secretion O +and O +target O +killing O +assays O +( O +Figure O +1 O +, O +A O +and O +B O +). O + +The O +RQFGPDWIVA B-chemical +sequence O +( O +present O +in O +C B-species +. I-species +asparagiforme I-species +) O +activated O +the O +1E6 O +T O +cell O +with O +around O +1 O +log O +– O +greater O +potency O +compared O +with O +ALWGPDPAAA B-chemical +. O + +At O +the O +other O +end O +of O +the O +spectrum O +, O +the O +RQFGPDFPTI B-chemical +peptide O +stimulated O +MIP B-protein +- I-protein +1β I-protein +release O +and O +killing O +by O +1E6 O +at O +an O +exogenous O +peptide O +concentration O +2 O +– O +3 O +logs O +lower O +compared O +with O +ALWGPDPAAA B-chemical +. O + +The O +pattern O +of O +peptide O +potency O +was O +closely O +mirrored O +by O +pMHC B-complex_assembly +tetramer B-experimental_method +staining I-experimental_method +experiments O +( O +Figure O +1C O +and O +plots O +shown O +in O +Supplemental O +Figure O +1 O +; O +supplemental O +material O +available O +online O +with O +this O +article O +; O +doi O +: O +10 O +. O +1172 O +/ O +JCI85679DS1 O +). O + +To O +parallel O +the O +functional O +analysis O +, O +we O +also O +performed O +thermal B-experimental_method +melt I-experimental_method +( O +Tm B-evidence +) O +experiments O +using O +synchrotron B-experimental_method +radiation I-experimental_method +circular I-experimental_method +dichroism I-experimental_method +( O +SRCD B-experimental_method +) O +to O +investigate O +the O +stability O +of O +each O +APL B-chemical +( O +Figure O +1D O +). O + +This O +pattern O +of O +stability O +did O +not O +correlate O +with O +the O +T O +cell O +activation O +or O +tetramer B-experimental_method +staining I-experimental_method +experiments O +, O +indicating O +that O +peptide O +binding O +to O +the O +MHC B-complex_assembly +do O +not O +explain O +ligand O +potency O +. O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +can O +bind O +peptides O +with O +strong O +antipathogen O +- O +like O +affinities O +. O + +We O +, O +and O +others O +, O +have O +previously O +demonstrated O +that O +antipathogenic O +TCRs B-complex_assembly +tend O +to O +bind O +with O +stronger O +affinity B-evidence +compared O +with O +self O +- O +reactive O +TCRs B-complex_assembly +, O +likely O +a O +consequence O +of O +the O +deletion O +of O +T O +cells O +with O +high O +- O +affinity B-evidence +self O +- O +reactive O +TCR B-complex_assembly +during O +thymic O +selection O +. O + +First O +, O +the O +1E6 O +T O +cell O +could O +still O +functionally O +respond O +to O +peptide O +when O +the O +TCR B-evidence +binding I-evidence +affinity I-evidence +was O +extremely O +weak O +, O +e O +. O +g O +., O +the O +1E6 B-evidence +TCR I-evidence +binding I-evidence +affinity I-evidence +for O +the O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +peptide O +was O +KD B-evidence += O +~ O +600 O +μM O +. O +Second O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +bound B-protein_state +to I-protein_state +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +with O +KD B-evidence += O +0 O +. O +5 O +μM O +, O +equivalent O +to O +the O +binding B-evidence +affinity I-evidence +of O +the O +very O +strongest O +antipathogen O +TCRs B-complex_assembly +. O + +Third O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +bound B-protein_state +to I-protein_state +A2 B-chemical +- I-chemical +RQFGPDWIVA I-chemical +peptide O +, O +within O +the O +C B-species +. I-species +asparagiforme I-species +proteome O +, O +with O +approximately O +4 O +- O +fold O +stronger O +affinity B-evidence +than O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +, O +demonstrating O +the O +potential O +for O +a O +pathogen O +- O +derived O +antigen O +to O +initiate O +a O +response O +to O +the O +self O +- O +derived O +sequence O +. O + +To O +confirm O +the O +affinity B-evidence +spread O +detected O +by O +SPR B-experimental_method +, O +and O +to O +evaluate O +whether O +experiments O +performed O +using O +soluble O +molecules O +were O +biologically O +relevant O +to O +events O +at O +the O +T O +cell O +surface O +, O +we O +determined O +the O +effective O +2D B-evidence +affinity I-evidence +of O +each O +APL B-chemical +using O +an O +adhesion B-experimental_method +frequency I-experimental_method +assay I-experimental_method +in O +which O +a O +human B-species +rbc O +coated O +in O +pMHC B-complex_assembly +acted O +as O +an O +adhesion O +sensor O +. O + +In O +agreement O +with O +SPR B-experimental_method +experiments O +, O +the O +range O +of O +2D B-evidence +affinities I-evidence +we O +detected O +differed O +by O +around O +3 O +logs O +, O +with O +the O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +generating O +the O +weakest O +2D B-evidence +affinity I-evidence +( O +2 O +. O +6 O +× O +10 O +– O +5 O +AcKa B-evidence +μm4 O +) O +and O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +the O +strongest O +( O +4 O +. O +5 O +× O +10 O +– O +2 O +AcKa B-evidence +μm4 O +) O +( O +Figure O +2J O +). O + +Of O +note O +, O +these O +data O +demonstrate O +a O +close O +agreement O +between O +the O +3D B-evidence +affinity I-evidence +values O +generated O +using O +SPR B-experimental_method +and O +2D B-evidence +affinity I-evidence +values O +generated O +using O +adhesion O +frequency O +assays O +. O + +All O +structures B-evidence +were O +solved B-experimental_method +in O +space O +group O +P1 O +to O +2 O +– O +3 O +Å O +resolution O +with O +crystallographic O +Rwork B-evidence +/ I-evidence +Rfree I-evidence +ratios I-evidence +within O +accepted O +limits O +as O +shown O +in O +the O +theoretically O +expected O +distribution O +( O +ref O +. O +and O +Supplemental O +Table O +1 O +). O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +used O +a O +very O +similar O +overall O +binding O +modality O +to O +engage O +all O +of O +the O +APLs B-chemical +, O +with O +root B-evidence +mean I-evidence +square I-evidence +deviation I-evidence +ranging O +between O +0 O +. O +81 O +and O +1 O +. O +12 O +Å2 O +( O +compared O +with O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +ALWGPDPAAA I-complex_assembly +). O + +Overall O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +used O +a O +canonical O +binding O +mode O +to O +engage O +each O +APL B-chemical +with O +the O +TCR B-complex_assembly +α B-structure_element +- I-structure_element +chain I-structure_element +positioned O +over O +the O +MHC B-complex_assembly +class I-complex_assembly +I I-complex_assembly +( O +MHCI B-complex_assembly +) O +α2 B-structure_element +- I-structure_element +helix I-structure_element +and O +the O +TCR B-complex_assembly +β B-structure_element +- I-structure_element +chain I-structure_element +over O +the O +MHCI B-complex_assembly +α B-structure_element +- I-structure_element +1 I-structure_element +helix I-structure_element +, O +straddling O +the O +peptide O +cargo O +. O + +However O +, O +subtle O +differences O +in O +the O +respective O +interfaces B-site +were O +apparent O +( O +discussed O +below O +) O +and O +resulted O +in O +altered O +binding B-evidence +affinities I-evidence +of O +the O +respective O +complexes O +. O + +Interactions O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +different O +APLs B-chemical +are O +focused O +around O +a O +conserved B-protein_state +GPD B-structure_element +peptide I-structure_element +motif I-structure_element +. O + +We O +next O +performed O +an O +in O +- O +depth O +atomic B-experimental_method +analysis I-experimental_method +of O +the O +contacts O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +each O +APL B-chemical +to O +determine O +the O +structural O +basis O +for O +the O +altered O +T O +cell O +peptide O +sensitivities O +and O +TCR B-evidence +binding I-evidence +affinities I-evidence +( O +Table O +2 O +). O + +Concomitant O +with O +our O +global O +analysis O +of O +1E6 B-complex_assembly +TCR I-complex_assembly +binding O +to O +the O +APLs B-chemical +, O +we O +observed O +a O +common O +interaction O +element O +, O +consistent O +with O +our O +previous O +findings O +, O +that O +utilized O +TCR B-complex_assembly +residues O +Tyr97α B-residue_name_number +and O +Trp97β B-residue_name_number +, O +forming O +an O +aromatic B-structure_element +cap I-structure_element +over O +a O +central O +GPD B-structure_element +motif I-structure_element +that O +was O +present O +in O +all O +of O +the O +APLs B-chemical +( O +Figure O +4 O +). O + +Interactions O +between O +these O +2 O +TCR B-complex_assembly +and O +3 O +peptide O +residues O +accounted O +for O +41 O +%– O +50 O +% O +of O +the O +total O +contacts O +across O +all O +complexes O +( O +Table O +2 O +), O +demonstrating O +the O +conserved B-protein_state +peptide O +centric O +binding O +mode O +utilized O +by O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +. O + +This O +fixed O +anchoring O +between O +the O +2 O +molecules O +was O +important O +for O +stabilization O +of O +the O +TCR B-complex_assembly +- I-complex_assembly +pMHC I-complex_assembly +complex O +, O +as O +— O +although O +other O +peptides O +without O +the O +‘ B-structure_element +GDP I-structure_element +’ I-structure_element +motif I-structure_element +were O +tested O +and O +shown O +to O +activate O +the O +1E6 O +T O +cell O +clone O +— O +we O +were O +unable O +to O +measure O +robust O +affinities B-evidence +using O +SPR B-experimental_method +( O +data O +not O +shown O +). O + +These O +data O +support O +the O +requirement O +for O +a O +conserved B-protein_state +interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +the O +GPD B-structure_element +motif I-structure_element +, O +as O +we O +observed O +in O +our O +previously O +published O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +ALWGPDPAAA I-complex_assembly +structure B-evidence +. O + +Although O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +formed O +a O +similar O +overall O +interaction O +with O +each O +APL B-chemical +, O +the O +stabilization O +between O +the O +TCR B-complex_assembly +and O +the O +GPD B-structure_element +motif I-structure_element +enabled O +fine O +differences O +in O +the O +contact B-site +network I-site +with O +both O +the O +peptide B-chemical +and O +MHC B-site +surface I-site +that O +allowed O +discrimination O +between O +each O +ligand O +( O +Figure O +5 O +). O + +For O +example O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +made O +only O +47 O +peptide O +contacts O +with O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +( O +KD B-evidence += O +~ O +600 O +μM O +) O +compared O +with O +63 O +and O +57 O +contacts O +with O +A2 B-chemical +- I-chemical +YQFGPDFPIA I-chemical +( O +KD B-evidence += O +7 O +. O +4 O +μM O +) O +and O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +( O +KD B-evidence += O +0 O +. O +5 O +μM O +), O +respectively O +. O + +For O +example O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +made O +64 O +peptide O +contacts O +with O +A2 B-chemical +- I-chemical +YLGGPDFPTI I-chemical +( O +KD B-evidence += O +~ O +400 O +μM O +) O +compared O +with O +43 O +contacts O +with O +A2 B-chemical +- I-chemical +RQWGPDPAAV I-chemical +( O +KD B-evidence += O +7 O +. O +8 O +μM O +). O + +The O +most O +important O +peptide O +modification O +in O +terms O +of O +generating O +new O +contacts O +was O +peptide O +position O +1 B-residue_number +. O + +We O +have O +previously O +shown O +that O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +uses O +a O +rigid O +lock O +- O +and O +- O +key O +mechanism O +during O +binding O +to O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +. O + +In O +order O +to O +determine O +whether O +any O +of O +the O +APLs B-chemical +required O +an O +induced O +fit O +mechanism O +during O +binding O +that O +could O +explain O +the O +difference O +in O +free B-evidence +binding I-evidence +energy I-evidence +( O +ΔG B-evidence +) O +between O +each O +complex O +( O +Table O +2 O +), O +we O +solved B-experimental_method +the O +unligated B-protein_state +structures B-evidence +of O +all O +7 O +APLs B-chemical +( O +the O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +structure B-evidence +has O +been O +previously O +published O +and O +was O +used O +in O +this O +comparison O +, O +ref O +.) O +( O +Figure O +6 O +and O +Supplemental O +Table O +2 O +). O + +The O +unligated B-protein_state +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +( O +KD B-evidence += O +~ O +600 O +μM O +) O +structure B-evidence +revealed O +that O +the O +side O +chain O +Tyr9 B-residue_name_number +swung O +around O +8 O +Å O +in O +the O +complex O +structure B-evidence +, O +subsequently O +making O +contacts O +with O +TCR B-complex_assembly +residues O +Asp30β B-residue_name_number +and O +Asn51β B-residue_name_number +( O +Figure O +6A O +and O +Figure O +5A O +, O +respectively O +). O + +This O +movement O +could O +result O +in O +an O +entropic O +penalty O +contributing O +to O +the O +weak O +TCR B-evidence +binding I-evidence +affinity I-evidence +we O +observed O +for O +this O +ligand O +. O + +Additional O +small O +movements O +in O +the O +Cα O +backbone O +of O +the O +peptide O +around O +peptide O +residue O +Asp6 B-residue_name_number +were O +apparent O +in O +the O +A2 B-chemical +- I-chemical +YLGGPDFPTI I-chemical +( O +KD B-evidence += O +~ O +400 O +μM O +), O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +( O +KD B-evidence += O +~ O +208 O +μM O +), O +and O +A2 B-chemical +- I-chemical +RQFGPDWIVA I-chemical +( O +KD B-evidence += O +44 O +. O +4 O +μM O +) O +structures B-evidence +( O +Figure O +6 O +, O +B O +, O +C O +, O +and O +E O +). O + +Apart O +from O +the O +case O +of O +A2 B-chemical +- I-chemical +AQWGPDAAA I-chemical +( O +KD B-evidence += O +61 O +. O +9 O +μM O +), O +these O +observations O +support O +the O +conclusion O +that O +the O +higher O +- O +affinity B-evidence +ligands O +required O +less O +conformational O +melding O +during O +binding O +, O +which O +could O +be O +energetically O +beneficial O +( O +lower O +entopic O +cost O +) O +during O +ligation O +with O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +. O + +MHC B-complex_assembly +residue O +Arg65 B-residue_name_number +that O +forms O +part O +of O +the O +MHC B-site +restriction I-site +triad I-site +( O +Arg65 B-residue_name_number +, O +Ala69 B-residue_name_number +, O +and O +Gln155 B-residue_name_number +) O +played O +a O +central O +role O +in O +TCR B-complex_assembly +- O +MHC B-complex_assembly +contacts O +, O +with O +Gln155 B-residue_name_number +playing O +a O +less O +important O +role O +and O +Ala69 B-residue_name_number +playing O +no O +role O +in O +binding O +at O +the O +interface B-site +( O +Figure O +7 O +). O + +For O +instance O +, O +contacts O +were O +made O +between O +TCR B-complex_assembly +residue O +Val53β B-residue_name_number +and O +MHC B-complex_assembly +residue O +Gln72 B-residue_name_number +in O +all O +APLs B-chemical +except O +for O +in O +the O +weakest O +affinity B-evidence +ligand O +pair O +, O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +MVWGPDPLYV I-complex_assembly +, O +in O +which O +a O +subtle O +change O +in O +TCR B-complex_assembly +conformation O +— O +probably O +mediated O +by O +different O +peptide O +contacts O +— O +abrogated O +this O +interaction O +( O +Figure O +7A O +). O + +Our O +analysis O +of O +the O +contact B-site +network I-site +provided O +some O +clues O +that O +could O +explain O +the O +different O +antigen O +potencies O +and O +binding B-evidence +affinities I-evidence +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +the O +different O +APLs B-chemical +. O + +The O +overall O +free B-evidence +binding I-evidence +energies I-evidence +( O +ΔG B-evidence +°) I-evidence +were O +between O +– O +4 O +. O +4 O +and O +– O +8 O +. O +6 O +kcal O +/ O +mol O +, O +reflecting O +the O +wide O +range O +of O +TCR B-evidence +binding I-evidence +affinities I-evidence +we O +observed O +for O +the O +different O +APLs B-chemical +. O + +The O +enthalpic O +contribution O +in O +each O +complex O +did O +not O +follow O +a O +clear O +trend O +with O +affinity B-evidence +, O +with O +all O +but O +the O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQFGPDFPTI I-complex_assembly +interaction O +( O +ΔH B-evidence +° I-evidence += O +6 O +. O +3 O +kcal O +/ O +mol O +) O +generating O +an O +energetically O +favorable O +enthalpy B-evidence +value O +( O +ΔH B-evidence +° I-evidence += O +– O +3 O +. O +7 O +to O +– O +11 O +. O +4 O +kcal O +/ O +mol O +); O +this O +indicated O +a O +net O +gain O +in O +electrostatic O +interactions O +during O +complex O +formation O +. O + +For O +instance O +, O +the O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +, O +A2 B-chemical +- I-chemical +AQWGPDAAA I-chemical +, O +and O +A2 B-chemical +- I-chemical +RQFGPDWIVA I-chemical +( O +KD B-evidence += O +~ O +208 O +μM O +, O +KD B-evidence += O +61 O +. O +9 O +μM O +, O +and O +KD B-evidence += O +44 O +. O +4 O +μM O +, O +respectively O +) O +were O +all O +entropically O +unfavorable O +( O +TΔS B-evidence +° I-evidence += O +– O +2 O +. O +9 O +to O +– O +5 O +. O +6 O +kcal O +/ O +mol O +), O +indicating O +a O +net O +change O +from O +disorder O +to O +order O +. O + +Furthermore O +, O +the O +structures O +of O +the O +unligated B-protein_state +pMHCs B-complex_assembly +demonstrated O +that O +, O +for O +these O +stronger O +- O +affinity B-evidence +ligands O +, O +there O +was O +less O +conformational O +difference O +between O +the O +TCR B-complex_assembly +ligated B-protein_state +pMHCs B-complex_assembly +compared O +with O +the O +weaker O +- O +affinity B-evidence +ligands O +( O +Figure O +6 O +). O + +The O +potential O +requirement O +for O +a O +larger O +degree O +of O +induced O +fit O +during O +binding O +to O +these O +weaker O +- O +affinity B-evidence +ligands O +is O +consistent O +with O +the O +larger O +entropic O +penalties O +observed O +for O +these O +interactions O +. O + +Potential O +epitopes O +for O +1E6 B-complex_assembly +TCR I-complex_assembly +occur O +commonly O +in O +the O +viral B-taxonomy_domain +proteome O +. O + +We O +searched O +a O +database O +of O +over O +1 O +, O +924 O +, O +572 O +unique O +decamer O +peptides B-chemical +from O +the O +proteome O +of O +viral B-taxonomy_domain +pathogens O +that O +are O +known O +, O +or O +strongly O +suspected O +, O +to O +infect O +humans B-species +. O + +Three O +hundred O +forty O +- O +two O +of O +these O +decamers O +conformed O +to O +the O +motif O +xxxGPDxxxx B-structure_element +. O + +Of O +these O +, O +53 O +peptides O +contained O +the O +motif O +xOxGPDxxxO B-structure_element +, O +where O +O O +is O +one O +of O +the O +hydrophobic O +amino O +acid O +residues O +A B-residue_name +, O +V B-residue_name +, O +I B-residue_name +, O +L B-residue_name +, O +M B-residue_name +, O +Y B-residue_name +, O +F B-residue_name +, O +and O +W B-residue_name +that O +might O +allow O +binding O +to O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +( O +Supplemental O +Table O +4 O +). O + +Thus O +, O +there O +are O +many O +pathogen O +- O +encoded O +peptides O +that O +could O +act O +as O +agonists O +for O +the O +1E6 O +T O +cell O +beyond O +the O +MVWGPDPLYV B-chemical +and O +RQFGPDWIVA B-chemical +sequences O +studied O +here O +. O + +Extension O +of O +these O +analyses O +to O +include O +the O +larger O +genomes O +of O +bacterial B-taxonomy_domain +pathogens O +would O +be O +expected O +to O +considerably O +increase O +these O +numbers O +. O + +T O +cell O +antigen O +discrimination O +is O +governed O +by O +an O +interaction O +between O +the O +clonally O +expressed O +TCR B-complex_assembly +and O +pMHC B-complex_assembly +, O +mediated O +by O +the O +chemical O +characteristics O +of O +the O +interacting O +molecules O +. O + +It O +has O +recently O +become O +clear O +that O +TCR B-complex_assembly +cross O +- O +reactivity O +with O +large O +numbers O +of O +different O +pMHC B-complex_assembly +ligands O +is O +essential O +to O +plug O +holes O +in O +T O +cell O +immune O +coverage O +that O +pathogens O +could O +exploit O +. O + +This O +notion O +is O +attractive O +because O +the O +CDR B-structure_element +loops I-structure_element +, O +which O +form O +the O +TCR B-site +antigen I-site +- I-site +binding I-site +site I-site +, O +are O +usually O +the O +most O +flexible O +part O +of O +the O +TCR B-complex_assembly +and O +have O +the O +ability O +to O +mold O +around O +differently O +shaped O +ligands O +. O + +Notably O +among O +these O +studies O +, O +Garcia O +and O +colleagues O +recently O +used O +the O +alloreactive B-protein_state +murine B-taxonomy_domain +TCR B-complex_assembly +- O +MHC B-complex_assembly +pair O +of O +the O +42F3 B-protein +TCR B-complex_assembly +and O +H2 B-protein +- I-protein +Ld I-protein +to O +demonstrate O +recognition O +of O +a O +large O +number O +of O +different O +peptides O +via O +conserved B-protein_state +hotspot B-site +contacts O +with O +prominent O +up O +- O +facing O +peptide O +residues O +. O + +Sethi O +and O +colleagues O +recently O +demonstrated O +that O +the O +MHCII B-protein_type +- O +restricted O +Hy B-protein +. I-protein +1B11 I-protein +TCR B-complex_assembly +, O +which O +was O +isolated O +from O +a O +patient O +with O +multiple O +sclerosis O +, O +could O +anchor O +into O +a O +deep B-site +pocket I-site +formed O +from O +peptide O +residues O +2 B-residue_number +, O +3 B-residue_number +, O +and O +5 B-residue_number +( O +from O +MBP85 B-protein +– I-protein +99 I-protein +bound B-protein_state +to I-protein_state +HLA B-protein +- I-protein +DQ1 I-protein +). O + +First O +, O +we O +currently O +know O +nothing O +about O +how O +human B-species +MHCI B-complex_assembly +– O +restricted O +TCRs B-complex_assembly +mediate O +cross O +- O +reactivity O +in O +the O +context O +of O +a O +clinically O +relevant O +model O +of O +autoimmunity O +, O +thought O +to O +be O +a O +major O +pathway O +of O +disease O +initiation O +in O +several O +autoimmune O +diseases O +. O + +Here O +, O +we O +investigated O +a O +highly O +cross O +- O +reactive O +MHCI B-complex_assembly +- O +restricted O +TCR B-complex_assembly +isolated O +from O +a O +patient O +with O +T1D O +that O +recognizes O +an O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +– O +restricted O +preproinsulin B-protein +signal B-structure_element +peptide I-structure_element +( O +ALWGPDPAAA15 B-chemical +– I-chemical +24 I-chemical +). O + +Human B-species +CD8 O ++ O +T O +cell O +clones O +expressing O +TCRs B-complex_assembly +with O +this O +specificity O +mediate O +the O +destruction O +of O +β O +cells O +, O +have O +been O +found O +in O +islets O +early O +in O +infection O +, O +and O +are O +proposed O +to O +be O +a O +major O +driver O +of O +disease O +. O + +We O +solved B-experimental_method +the O +structure B-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +with O +7 O +APLs B-chemical +to O +enable O +a O +comprehensive O +analysis O +of O +the O +molecular O +basis O +of O +TCR B-complex_assembly +degeneracy O +. O + +Overall O +, O +the O +difference O +in O +antigen O +potency O +correlated O +well O +with O +the O +binding B-evidence +energy I-evidence +( O +ΔG B-evidence +° I-evidence +kcal O +/ O +mol O +) O +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +for O +the O +different O +epitopes O +, O +which O +ranged O +from O +values O +of O +ΔG B-evidence +° I-evidence += O +~– O +4 O +. O +4 O +to O +– O +8 O +. O +6 O +kcal O +/ O +mol O +( O +calculated O +from O +3D B-evidence +affinity I-evidence +data O +) O +or O +2D B-evidence +affinity I-evidence +values O +of O +AcKa B-evidence += O +2 O +. O +5 O +× O +10 O +– O +5 O +to O +4 O +. O +4 O +× O +10 O +– O +2 O +μm4 O +. O + +The O +weaker O +end O +of O +this O +spectrum O +extends O +our O +understanding O +of O +the O +limits O +in O +which O +T O +cells O +can O +functionally O +operate O +in O +terms O +of O +TCR B-evidence +3D I-evidence +binding I-evidence +affinity I-evidence +and O +is O +in O +line O +with O +the O +types O +of O +very O +low O +affinity B-evidence +, O +yet O +fully O +functional O +self O +- O +reactive O +CD8 O ++ O +T O +cells O +we O +have O +observed O +in O +tumor O +- O +infiltrating O +lymphocytes O +. O + +Previous O +studies O +of O +autoreactive O +TCRs B-complex_assembly +have O +shown O +that O +their O +binding O +mode O +is O +generally O +atypical O +, O +either O +due O +to O +an O +unusual O +binding O +manner O +, O +weak O +TCR B-evidence +binding I-evidence +affinity I-evidence +, O +an O +unstable B-protein_state +pMHC B-complex_assembly +, O +or O +a O +combination O +of O +these O +factors O +. O + +Our O +data O +demonstrate O +the O +potential O +for O +an O +autoreactive O +TCR B-complex_assembly +to O +bind O +with O +a O +conventional O +binding O +mode O +to O +a O +stable B-protein_state +pMHC B-complex_assembly +with O +antipathogen O +- O +like O +affinity B-evidence +( O +KD B-evidence += O +0 O +. O +5 O +μM O +) O +depending O +on O +the O +peptide O +sequence O +. O + +Our O +structural B-experimental_method +analysis I-experimental_method +revealed O +that O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +bound B-protein_state +with O +a O +conserved B-protein_state +conformation I-protein_state +across O +all O +APLs B-chemical +investigated O +. O + +This O +binding O +orientation O +was O +mediated O +through O +a O +focused O +interaction O +with O +TCR B-complex_assembly +residues O +Tyr97α B-residue_name_number +and O +Trp97β B-residue_name_number +that O +formed O +an O +aromatic B-structure_element +cap I-structure_element +over O +a O +central O +‘ B-structure_element +GDP I-structure_element +’ I-structure_element +motif I-structure_element +that O +was O +common O +to O +all O +APLs B-chemical +. O + +We O +have O +previously O +demonstrated O +the O +importance O +of O +the O +GPD B-structure_element +motif I-structure_element +using O +a O +peptide B-experimental_method +library I-experimental_method +scan I-experimental_method +, O +as O +well O +as O +a O +CPL B-experimental_method +scan I-experimental_method +approach O +. O + +This O +hotspot O +binding O +, O +defined O +as O +a O +localized O +cluster O +of O +interactions O +that O +dominate O +binding O +energy O +during O +protein O +- O +protein O +interactions O +, O +has O +been O +previously O +shown O +to O +contribute O +to O +TCR B-complex_assembly +recognition O +of O +MHC B-complex_assembly +as O +a O +mechanism O +that O +tunes O +T O +cell O +cross O +- O +reactivity O +by O +providing O +fixed O +anchor O +points O +that O +enable O +TCRs B-complex_assembly +to O +tolerate O +a O +variable O +peptide O +cargo O +. O + +Alternatively O +, O +interactions O +between O +the O +TCR B-complex_assembly +and O +peptide O +have O +been O +shown O +to O +dominate O +the O +energetic O +landscape O +during O +ligand O +engagement O +, O +ensuring O +that O +T O +cells O +retain O +peptide O +specificity O +. O + +These O +findings O +are O +also O +analogous O +to O +the O +observed O +binding O +mode O +of O +the O +Hy B-protein +. I-protein +1B11 I-protein +TCR B-complex_assembly +, O +in O +which O +one O +aromatic O +residue O +of O +the O +TCR B-complex_assembly +CDR3α B-structure_element +loop I-structure_element +anchored O +into O +a O +pocket O +created O +by O +a O +conserved O +peptide O +motif O +. O + +In O +both O +of O +these O +examples O +, O +self O +- O +recognition O +is O +mediated O +by O +TCR B-complex_assembly +residues O +with O +aromatic O +side O +chains O +. O + +Despite O +some O +weak O +statistical O +correlation O +between O +the O +surface B-evidence +complementarity I-evidence +( O +SC B-evidence +) O +and O +affinity B-evidence +, O +closer O +inspection O +of O +the O +interface B-site +revealed O +no O +obvious O +structural O +signature O +that O +could O +definitively O +explain O +the O +differences O +in O +antigen B-evidence +potency I-evidence +and O +TCR B-evidence +binding I-evidence +strength I-evidence +between O +the O +different O +ligands O +. O + +However O +, O +similar O +to O +our O +findings O +in O +other O +systems O +, O +modifications O +to O +residues O +outside O +of O +the O +canonical O +central B-structure_element +peptide I-structure_element +bulge I-structure_element +were O +important O +for O +generating O +new O +interactions O +. O + +For O +example O +, O +all O +of O +the O +stronger O +ligands O +encoded O +larger O +side O +chains O +( O +Arg B-residue_name +or O +Tyr B-residue_name +) O +at O +peptide O +position O +1 B-residue_number +that O +enabled O +new O +interactions O +with O +1E6 O +not O +present O +with O +the O +Ala B-residue_name +at O +this O +position O +in O +the O +natural O +preproinsulin B-protein +peptide B-chemical +. O + +We O +have O +recently O +demonstrated O +how O +a O +suboptimal O +position O +2 B-residue_number +anchor B-structure_element +in O +a O +melanoma O +- O +derived O +antigen O +can O +improve O +TCR B-complex_assembly +binding O +through O +a O +similar O +mechanism O +. O + +These O +results O +challenge O +the O +notion O +that O +the O +most O +potent O +peptide O +antigens O +exhibit O +the O +greatest O +pMHC B-complex_assembly +stability O +and O +have O +implications O +for O +the O +design O +of O +anchor O +residue O +– O +modified O +heteroclitic O +peptides O +for O +vaccination O +. O + +These O +parameters O +aligned O +well O +with O +structural B-evidence +data I-evidence +, O +demonstrating O +that O +TCRs B-complex_assembly +engaged O +pMHC B-complex_assembly +using O +an O +induced O +fit O +binding O +mode O +. O + +However O +, O +more O +recent O +data O +have O +shown O +that O +TCRs B-complex_assembly +can O +utilize O +a O +range O +of O +energetic O +strategies O +during O +pMHC B-complex_assembly +binding O +, O +currently O +with O +no O +obvious O +pattern O +in O +terms O +of O +TCR B-evidence +affinity I-evidence +, O +binding O +mechanism O +, O +or O +specificity O +( O +pathogen O +, O +cancer O +, O +or O +self O +- O +ligands O +). O + +Although O +no O +energetic O +signature O +appears O +to O +exist O +for O +different O +TCRs B-complex_assembly +, O +we O +used O +thermodynamic B-experimental_method +analysis I-experimental_method +here O +to O +explore O +whether O +changes O +in O +energetics O +could O +help O +explain O +ligand O +discrimination O +by O +a O +single O +TCR B-complex_assembly +. O + +The O +weaker O +APL B-chemical +ligands O +were O +characterized O +by O +favorable O +enthalpy B-evidence +and O +unfavorable O +entropy B-evidence +, O +whereas O +the O +stronger O +ligands O +progressively O +shifted O +to O +favorable O +entropy B-evidence +. O + +Thus O +, O +the O +enhanced O +antigen O +potency O +was O +probably O +mediated O +through O +a O +shift O +from O +an O +induced O +fit O +to O +a O +lock O +- O +and O +- O +key O +interaction O +between O +the O +stronger O +ligands O +( O +less O +requirement O +for O +energetically O +unfavorable O +disorder O +- O +to O +- O +order O +changes O +), O +resulting O +in O +a O +more O +energetically O +favorable O +ΔG B-evidence +value I-evidence +. O + +The O +RQFGPDWIVA B-chemical +peptide O +, O +which O +was O +substantially O +more O +potent O +than O +the O +preproinsulin B-protein +peptide O +, O +is O +within O +the O +proteome O +of O +a O +common O +human B-species +pathogen O +( O +C B-species +. I-species +asparagiforme I-species +), O +demonstrating O +the O +potential O +for O +an O +encounter O +between O +a O +naive O +1E6 O +- O +like O +T O +cell O +and O +a O +foreign O +peptide O +with O +a O +more O +potent O +ligand O +that O +might O +then O +break O +self O +- O +tolerance O +. O + +Indeed O +, O +we O +found O +over O +50 O +decamer O +peptides O +from O +the O +proteome O +of O +likely O +, O +or O +known O +, O +human B-species +viral B-taxonomy_domain +pathogens O +alone O +that O +contained O +both O +the O +conserved B-protein_state +central O +GPD B-structure_element +motif I-structure_element +and O +anchor B-structure_element +residues I-structure_element +at O +positions O +2 B-residue_number +and O +10 B-residue_number +that O +would O +enable O +binding O +to O +HLA B-protein +- I-protein +A I-protein +* I-protein +02 I-protein +: I-protein +01 I-protein +. O + +In O +summary O +, O +this O +investigation O +into O +the O +molecular O +basis O +of O +T O +cell O +cross O +- O +reactivity O +using O +a O +clinically O +relevant O +cytotoxic O +CD8 O ++ O +T O +cell O +clone O +that O +kills O +human B-species +pancreatic O +β O +cells O +provides O +answers O +to O +a O +number O +of O +previously O +outstanding O +questions O +. O + +First O +, O +our O +data O +shows O +that O +a O +single O +TCR B-complex_assembly +has O +the O +potential O +to O +functionally O +( O +assessed O +through O +T O +cell O +activation O +) O +bind O +to O +different O +ligands O +with O +affinities B-evidence +ranging O +across O +3 O +orders O +of O +magnitude O +. O + +Second O +, O +this O +is O +the O +first O +example O +in O +which O +ligands O +have O +been O +identified O +and O +characterized O +for O +a O +human B-species +autoreactive O +TCR B-complex_assembly +that O +are O +substantially O +more O +potent O +than O +the O +natural O +self O +- O +ligand O +, O +demonstrating O +the O +potential O +for O +a O +pathogenic O +ligand O +to O +break O +self O +- O +tolerance O +and O +prime O +self O +- O +reactive O +T O +cells O +. O + +Third O +, O +this O +first O +structural B-experimental_method +analysis I-experimental_method +of O +a O +cross O +- O +reactive O +human B-species +MHCI B-complex_assembly +– O +restricted O +autoimmune O +TCR B-complex_assembly +showed O +that O +degeneracy O +was O +mediated O +through O +TCR B-complex_assembly +- I-complex_assembly +pMHC I-complex_assembly +anchoring O +by O +a O +conserved B-protein_state +minimal B-structure_element +binding I-structure_element +peptide I-structure_element +motif I-structure_element +. O + +Our O +demonstration O +of O +the O +molecular O +mechanism O +governing O +cross O +- O +reactivity O +by O +this O +preproinsulin B-protein +reactive O +human B-species +CD8 O ++ O +T O +cell O +clone O +supports O +the O +notion O +first O +put O +forward O +by O +Wucherpfennig O +and O +Strominger O +that O +molecular O +mimicry O +could O +mediate O +autoimmunity O +and O +has O +far O +- O +reaching O +implications O +for O +the O +complex O +nature O +of O +T O +cell O +antigen O +discrimination O +. O + +The O +1E6 O +T O +cell O +clone O +reacts O +with O +a O +broad O +sensitivity O +range O +to O +APLs B-chemical +. O + +( O +A O +and O +B O +) O +The O +1E6 O +T O +cell O +clone O +was O +tested O +in O +a O +peptide B-experimental_method +dilution I-experimental_method +assay I-experimental_method +, O +in O +triplicate O +, O +with O +MVWGPDPLYV B-chemical +( O +gray O +), O +YLGGPDFPTI B-chemical +( O +red O +), O +ALWGPDPAAA B-chemical +( O +blue O +), O +AQWGPDPAAA B-chemical +( O +green O +), O +RQFGPDWIVA B-chemical +( O +dark O +blue O +), O +RQWGPDPAAV B-chemical +( O +purple O +), O +YQFGPDFPTA B-chemical +( O +yellow O +), O +and O +RQFGPDFPTI B-chemical +( O +cyan O +) O +peptides O +presented O +by O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +– O +expressing O +C1R O +cells O +for O +release O +of O +MIP B-protein +- I-protein +1β I-protein +( O +A O +) O +and O +killing O +( O +B O +). O + +Tm B-evidence +values O +were O +calculated O +using O +a O +Boltzmann B-experimental_method +fit I-experimental_method +to I-experimental_method +each I-experimental_method +set I-experimental_method +of I-experimental_method +data I-experimental_method +. O + +( O +A O +– O +H O +) O +Binding B-evidence +affinity I-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +interaction O +at O +25 O +° O +C O +using O +SPR B-experimental_method +. O + +Eight O +serial O +dilutions O +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +were O +measured O +( O +shown O +in O +the O +inset O +); O +representative O +data O +from O +3 O +independent O +experiments O +are O +plotted O +. O + +In O +order O +to O +calculate O +each O +response O +, O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +was O +also O +injected O +over O +a O +control O +sample O +( O +HLA B-complex_assembly +- I-complex_assembly +A I-complex_assembly +* I-complex_assembly +0201 I-complex_assembly +– I-complex_assembly +ILAKFLHWL I-complex_assembly +) O +that O +was O +deducted O +from O +the O +experimental O +data O +. O + +( O +J O +) O +Effective B-evidence +2D I-evidence +affinity I-evidence +( O +AcKa B-evidence +) O +calculated O +using O +adhesion B-experimental_method +frequency I-experimental_method +assays I-experimental_method +, O +using O +at O +least O +5 O +cell O +pairs O +, O +and O +calculated O +as O +an O +average O +of O +100 O +cell O +cell O +contacts O +. O + +( O +K O +) O +Effective B-evidence +2D I-evidence +affinity I-evidence +plotted O +against O +1 O +/ O +EC50 B-evidence +showing O +Pearson B-experimental_method +’ I-experimental_method +s I-experimental_method +coefficient I-experimental_method +analysis I-experimental_method +( O +r B-evidence +) O +and O +P B-evidence +value I-evidence +. O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +uses O +a O +conserved O +binding O +mode O +to O +engage O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +and O +the O +APLs B-chemical +. O + +( O +A O +) O +Superposition B-experimental_method +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +multicolored O +illustration O +) O +in B-protein_state +complex I-protein_state +with I-protein_state +all O +7 O +APLs B-chemical +( O +multicolored O +sticks O +) O +and O +the O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +ligand O +using O +the O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +( O +gray O +illustration O +) O +molecule O +to O +align B-experimental_method +all O +of O +the O +structures B-evidence +. O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +each O +peptide O +are O +colored O +according O +to O +the O +APL B-chemical +used O +in O +the O +complex O +as O +in O +Figure O +1 O +. O +( O +B O +) O +Position O +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +CDR B-structure_element +loops I-structure_element +( O +multicolored O +lines O +) O +in O +each O +complex O +. O + +The O +ALWGPDPAAA B-chemical +peptide O +( O +green O +sticks O +) O +is O +shown O +in O +the O +HLA B-site +- I-site +A I-site +* I-site +0201 I-site +binding I-site +groove I-site +( O +gray O +surface O +). O +( O +C O +) O +The O +Cα O +backbone O +conformation O +of O +each O +APL B-chemical +( O +multicolored O +illustration O +) O +in O +the O +context O +of O +the O +HLA B-protein +- I-protein +A I-protein +* I-protein +0201 I-protein +α1 B-structure_element +helices I-structure_element +( O +gray O +illustration O +). O +( O +D O +) O +Crossing B-evidence +angle I-evidence +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +multicolored O +lines O +) O +calculated O +using O +previously O +published O +parameters O +in O +the O +context O +of O +the O +ALWGPDPAAA B-chemical +peptide O +( O +green O +sticks O +) O +bound B-protein_state +in I-protein_state +the O +HLA B-site +- I-site +A I-site +* I-site +0201 I-site +binding I-site +groove I-site +( O +gray O +surface O +). O + +A O +conserved O +interaction O +with O +a O +GPD B-structure_element +motif I-structure_element +underpins O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +interaction O +with O +the O +APLs B-chemical +. O + +Interaction O +between O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +gray O +illustration O +) O +residues O +Tyr97α B-residue_name_number +and O +Tyr97β B-residue_name_number +( O +the O +position O +of O +these O +side O +chains O +in O +the O +TCR B-complex_assembly +in B-protein_state +complex I-protein_state +with I-protein_state +all O +7 O +APLs B-chemical +, O +and O +the O +previously O +reported O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +epitope O +, O +is O +shown O +in O +multicolored O +sticks O +; O +ref O +.) O +and O +the O +GPD B-structure_element +peptide I-structure_element +motif I-structure_element +( O +the O +position O +of O +these O +side O +chains O +in O +all O +7 O +APLs B-chemical +and O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +in B-protein_state +complex I-protein_state +with I-protein_state +the O +1E6 B-complex_assembly +TCR I-complex_assembly +is O +shown O +in O +multicolored O +sticks O +). O + +The O +rest O +of O +the O +peptide O +, O +and O +the O +MHCα1 B-complex_assembly +helix B-structure_element +, O +are O +shown O +as O +a O +gray O +illustration O +. O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +makes O +distinct O +peptide O +contacts O +with O +peripheral O +APL B-chemical +residues O +. O + +Interactions O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +peptide O +residues O +outside O +of O +the O +conserved B-protein_state +GPD B-structure_element +motif I-structure_element +. O + +The O +MHCα1 B-complex_assembly +helix B-structure_element +is O +shown O +in O +gray O +illustrations O +. O + +Hydrogen B-bond_interaction +bonds I-bond_interaction +are O +shown O +as O +red O +dotted O +lines O +; O +van B-bond_interaction +der I-bond_interaction +Waals I-bond_interaction +( I-bond_interaction +vdW I-bond_interaction +) I-bond_interaction +contacts I-bond_interaction +are O +shown O +as O +black O +dotted O +lines O +. O + +Boxes O +show O +total O +contacts O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +and O +each O +peptide O +ligand O +. O + +( O +D O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +green O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +AQWGPDPAAA I-chemical +( O +green O +illustration O +and O +sticks O +). O +( O +E O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +dark O +blue O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +RQFGPDWIVA I-chemical +( O +dark O +blue O +illustration O +and O +sticks O +). O +( O +F O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +purple O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +RQWGPDPAAV I-chemical +( O +purple O +illustration O +and O +sticks O +). O +( O +G O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +yellow O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +YQFGPDFPTA I-chemical +( O +yellow O +illustration O +and O +sticks O +). O +( O +H O +) O +Interaction O +between O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +( O +cyan O +illustration O +and O +sticks O +) O +and O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +( O +cyan O +illustration O +and O +sticks O +). O + +Comparison O +of O +ligated B-protein_state +and O +unligated B-protein_state +APLs B-chemical +. O + +Peptide O +sequences O +are O +shown O +underneath O +each O +structure B-evidence +aligned O +with O +the O +peptide O +structure B-evidence +. O + +( O +A O +) O +A2 B-chemical +- I-chemical +MVWGPDPLYV I-chemical +( O +black O +sticks O +). O + +A O +large O +conformational O +shift O +was O +observed O +for O +Tyr8 B-residue_name_number +in O +the O +ligated B-protein_state +versus O +unligated B-protein_state +states O +( O +black O +circle O +). O +( O +B O +) O +A2 B-chemical +- I-chemical +YLGGPDFPTI I-chemical +( O +red O +sticks O +). O +( O +C O +) O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +( O +blue O +sticks O +) O +reproduced O +from O +previous O +published O +data O +. O +( O +D O +) O +A2 B-chemical +- I-chemical +AQWGPDPAAA I-chemical +( O +green O +sticks O +). O +( O +E O +) O +A2 B-chemical +- I-chemical +RQFGPDWIVA I-chemical +( O +dark O +blue O +sticks O +). O +( O +F O +) O +A2 B-chemical +- I-chemical +RQWGPDPAAV I-chemical +( O +purple O +sticks O +). O +( O +G O +) O +A2 B-chemical +- I-chemical +YQFGPDFPTA I-chemical +( O +yellow O +sticks O +). O +( O +H O +) O +A2 B-chemical +- I-chemical +RQFGPDFPTI I-chemical +( O +cyan O +sticks O +). O + +The O +1E6 B-complex_assembly +TCR I-complex_assembly +makes O +distinct O +peptide O +contacts O +with O +the O +MHC B-site +surface I-site +depending O +on O +the O +peptide O +cargo O +. O + +MHCα1 B-complex_assembly +helix B-structure_element +are O +shown O +in O +gray O +illustrations O +. O + +Thermodynamic B-experimental_method +analysis I-experimental_method +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +with O +A2 B-chemical +- I-chemical +ALWGPDPAAA I-chemical +and O +the O +APLs B-chemical +. O + +Eight O +serial O +dilutions O +of O +the O +1E6 B-complex_assembly +TCR I-complex_assembly +were O +injected O +, O +in O +duplicate O +, O +over O +each O +immobilized O +APL B-chemical +and O +A2 B-chemical +- I-chemical +ALW I-chemical +at O +5 O +° O +C O +, O +13 O +° O +C O +, O +18 O +° O +C O +, O +25 O +° O +C O +, O +30 O +° O +C O +, O +and O +37 O +° O +C O +. O + +The O +equilibrium B-evidence +binding I-evidence +constant I-evidence +( O +KD B-evidence +) O +values O +were O +calculated O +using O +a O +nonlinear B-experimental_method +curve I-experimental_method +fit I-experimental_method +( O +y O += O +[ O +P1x O +]/[ O +P2 O ++ O +X O +]), O +and O +thermodynamic O +parameters O +were O +calculated O +according O +to O +the O +Gibbs B-experimental_method +- I-experimental_method +Helmholtz I-experimental_method +equation I-experimental_method +( O +ΔG B-evidence +° I-evidence += O +ΔH B-evidence +− O +TΔS B-evidence +°). I-evidence + +The O +binding B-evidence +free I-evidence +energies I-evidence +, O +ΔG B-evidence +° I-evidence +( O +ΔG B-evidence +° I-evidence += O +RTlnKD O +), O +were O +plotted O +against O +temperature O +( O +K O +) O +using O +nonlinear B-experimental_method +regression I-experimental_method +to O +fit O +the O +3 O +- O +parameters O +van B-experimental_method +’ I-experimental_method +t I-experimental_method +Hoff I-experimental_method +equation I-experimental_method +( O +RT B-evidence +ln I-evidence +KD I-evidence += O +ΔH B-evidence +° I-evidence +– O +TΔS B-evidence +° I-evidence ++ O +ΔCp B-evidence +°[ I-evidence +T O +- O +T0 O +] O +– O +TΔCp B-evidence +° I-evidence +ln O +[ O +T O +/ O +T0 O +] O +with O +T0 O += O +298 O +K O +). O + +( O +A O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +ALWGPDPAAA I-complex_assembly +; O +( O +B O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +AQWGPDPAAA I-complex_assembly +; O +( O +C O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQFGPDWIVA I-complex_assembly +; O +( O +D O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQWGPDPAAV I-complex_assembly +, O +( O +E O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +YQFGPDFPTA I-complex_assembly +; O +and O +( O +F O +) O +1E6 B-complex_assembly +- I-complex_assembly +A2 I-complex_assembly +- I-complex_assembly +RQFGPDFPTI I-complex_assembly +. O + +1E6 B-complex_assembly +TCR I-complex_assembly +- I-complex_assembly +pMHC I-complex_assembly +contacts O +, O +affinity B-experimental_method +measurements I-experimental_method +and O +thermodynamics B-experimental_method + +The O +immunity B-protein_type +- I-protein_type +related I-protein_type +GTPase I-protein_type +Irga6 B-protein +dimerizes B-oligomeric_state +in O +a O +parallel B-protein_state +head I-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +head I-protein_state +fashion O + +The O +immunity B-protein_type +- I-protein_type +related I-protein_type +GTPases I-protein_type +( O +IRGs B-protein_type +) O +constitute O +a O +powerful O +cell O +- O +autonomous O +resistance O +system O +against O +several O +intracellular O +pathogens O +. O + +Irga6 B-protein +is O +a O +dynamin B-protein_type +- I-protein_type +like I-protein_type +protein I-protein_type +that O +oligomerizes O +at O +the O +parasitophorous O +vacuolar O +membrane O +( O +PVM O +) O +of O +Toxoplasma B-species +gondii I-species +leading O +to O +its O +vesiculation O +. O + +Based O +on O +a O +previous O +biochemical B-experimental_method +analysis I-experimental_method +, O +it O +has O +been O +proposed O +that O +the O +GTPase B-structure_element +domains I-structure_element +of O +Irga6 B-protein +dimerize B-oligomeric_state +in O +an O +antiparallel B-protein_state +fashion O +during O +oligomerization O +. O + +Contrary O +to O +the O +previous O +model O +, O +the O +structure B-evidence +shows O +that O +the O +GTPase B-structure_element +domains I-structure_element +dimerize B-oligomeric_state +in O +a O +parallel B-protein_state +fashion O +. O + +The O +nucleotides B-chemical +in O +the O +center O +of O +the O +interface B-site +participate O +in O +dimerization O +by O +forming O +symmetric O +contacts O +with O +each O +other O +and O +with O +the O +switch B-site +I I-site +region O +of O +the O +opposing O +Irga6 B-protein +molecule O +. O + +The O +latter O +contact O +appears O +to O +activate O +GTP B-chemical +hydrolysis O +by O +stabilizing O +the O +position O +of O +the O +catalytic B-protein_state +glutamate B-residue_name_number +106 I-residue_name_number +in O +switch B-site +I I-site +close O +to O +the O +active B-site +site I-site +. O + +The O +Irga6 B-protein +structure B-evidence +features O +a O +parallel B-protein_state +GTPase B-structure_element +domain I-structure_element +dimer B-oligomeric_state +, O +which O +appears O +to O +be O +a O +unifying O +feature O +of O +all O +dynamin B-protein_type +and O +septin B-protein_type +superfamily O +members O +. O + +Immunity B-protein_type +- I-protein_type +related I-protein_type +GTPases I-protein_type +( O +IRGs B-protein_type +) O +comprise O +a O +family O +of O +dynamin B-protein_type +- I-protein_type +related I-protein_type +cell I-protein_type +- I-protein_type +autonomous I-protein_type +resistance I-protein_type +proteins I-protein_type +targeting O +intracellular O +pathogens O +, O +such O +as O +Mycobacterium B-species +tuberculosis I-species +, O +Mycobacterium B-species +avium I-species +, O +Listeria B-species +monocytogenes I-species +, O +Trypanosoma B-species +cruzi I-species +, O +and O +Toxoplasma B-species +gondii I-species +. O + +In O +mice B-taxonomy_domain +, O +the O +23 O +IRG B-protein_type +members O +are O +induced O +by O +interferons B-protein_type +, O +whereas O +the O +single O +human B-species +homologue O +is O +constitutively O +expressed O +in O +some O +tissues O +, O +especially O +in O +testis O +. O + +In O +non O +- O +infected O +cells O +, O +most O +IRGs B-protein_type +are O +largely O +cytosolic O +. O + +In O +this O +way O +, O +IRGs B-protein_type +contribute O +to O +the O +release O +of O +the O +pathogen O +into O +the O +cytoplasm O +and O +its O +subsequent O +destruction O +. O + +Irga6 B-protein +, O +one O +of O +the O +effector O +IRG B-protein_type +proteins O +, O +localizes O +to O +the O +intact O +parasitophorous O +vacuole O +membrane O +( O +PVM O +) O +and O +, O +after O +disruption O +of O +the O +PVM O +, O +is O +found O +associated O +with O +vesicular O +accumulations O +, O +presumably O +derived O +from O +the O +PVM O +. O + +A O +myristoylation B-site +site I-site +at O +Gly2 B-residue_name_number +is O +necessary O +for O +the O +recruitment O +to O +the O +PVM O +but O +not O +for O +the O +weak O +constitutive O +binding O +to O +the O +ER O +membrane O +. O + +An O +internally O +oriented O +antibody O +epitope O +on O +helix B-structure_element +A I-structure_element +between O +positions O +20 B-residue_range +and I-residue_range +24 I-residue_range +was O +demonstrated O +to O +be O +accessible O +in O +the O +GTP B-protein_state +-, I-protein_state +but O +not O +in O +the O +GDP B-protein_state +- I-protein_state +bound I-protein_state +state O +. O + +This O +indicates O +large O +- O +scale O +structural O +changes O +upon O +GTP B-chemical +binding O +that O +probably O +include O +exposure O +of O +the O +myristoyl O +group O +, O +enhancing O +binding O +to O +the O +PVM O +. O + +Biochemical B-experimental_method +studies I-experimental_method +indicated O +that O +Irga6 B-protein +hydrolyses O +GTP B-chemical +in O +a O +cooperative O +manner O +and O +forms O +GTP B-protein_state +- I-protein_state +dependent I-protein_state +oligomers B-oligomeric_state +in O +vitro O +and O +in O +vivo O +. O + +Crystal B-evidence +structures I-evidence +of O +Irga6 B-protein +in O +various O +nucleotide B-protein_state +- I-protein_state +loaded I-protein_state +states O +revealed O +the O +basic O +architecture O +of O +IRG B-protein_type +proteins O +, O +including O +a O +GTPase B-structure_element +domain I-structure_element +and O +a O +composite B-structure_element +helical I-structure_element +domain I-structure_element +. O + +These O +studies O +additionally O +showed O +a O +dimerization B-site +interface I-site +in O +the O +nucleotide B-protein_state +- I-protein_state +free I-protein_state +protein O +as O +well O +as O +in O +all O +nucleotide B-protein_state +- I-protein_state +bound I-protein_state +states O +. O + +It O +involves O +a O +GTPase B-site +domain I-site +surface I-site +, O +which O +is O +located O +at O +the O +opposite O +side O +of O +the O +nucleotide O +, O +and O +an O +interface B-site +in O +the O +helical B-structure_element +domain I-structure_element +, O +with O +a O +water B-chemical +- O +filled O +gap O +between O +the O +two O +contact B-site +surfaces I-site +. O + +Recent O +structural B-experimental_method +studies I-experimental_method +indicated O +that O +a O +' O +G B-site +interface I-site +' O +is O +typical O +of O +dynamin B-protein_type +superfamily O +members O +, O +such O +as O +dynamin B-protein_type +, O +MxA B-protein +, O +the O +guanylate B-protein +binding I-protein +protein I-protein +- I-protein +1 I-protein +( O +GBP B-protein +- I-protein +1 I-protein +), O +atlastin B-protein_type +and O +the O +bacterial B-taxonomy_domain +dynamin B-protein_type +- I-protein_type +like I-protein_type +proteins I-protein_type +( O +BDLP B-protein_type +). O + +In O +dynamin B-protein_type +, O +the O +G B-site +interface I-site +includes O +residues O +in O +the O +phosphate B-structure_element +binding I-structure_element +loop I-structure_element +, O +the O +two O +switch B-site +regions I-site +, O +the O +' O +trans B-structure_element +stabilizing I-structure_element +loop I-structure_element +' O +and O +the O +' O +G4 B-structure_element +loop I-structure_element +'. O + +For O +Irga6 B-protein +, O +it O +was O +demonstrated O +that O +besides O +residues O +in O +the O +switch B-site +I I-site +and O +switch B-site +II I-site +regions O +, O +the O +3 O +'- O +OH O +group O +of O +the O +ribose O +participates O +in O +this O +interface B-site +. O + +This O +structure B-evidence +was O +obtained O +by O +soaking B-experimental_method +GMPPNP B-chemical +in O +nucleotide B-protein_state +- I-protein_state +free I-protein_state +crystals B-evidence +of O +Irga6 B-protein +, O +an O +approach O +which O +may O +have O +interfered O +with O +nucleotide O +- O +induced O +domain O +rearrangements O +. O + +To O +clarify O +the O +dimerization O +mode O +via O +the O +G B-site +interface I-site +, O +we O +determined B-experimental_method +the O +GMPPNP B-protein_state +- I-protein_state +bound I-protein_state +crystal B-evidence +structure I-evidence +of O +a O +non B-protein_state +- I-protein_state +oligomerizing I-protein_state +Irga6 B-protein +variant B-protein_state +. O + +Previous O +results O +indicated O +that O +Irga6 B-protein +mutations B-experimental_method +in O +a O +loosely O +defined O +surface B-site +region I-site +( O +the O +"""" O +secondary B-site +patch I-site +""")," O +which O +is O +distant O +from O +the O +G B-site +- I-site +interface I-site +and O +only O +slightly O +overlapping O +with O +the O +backside B-site +interface I-site +( O +see O +below O +), O +individually O +reduced O +GTP B-chemical +- O +dependent O +oligomerization O +. O + +A O +combination B-experimental_method +of I-experimental_method +four I-experimental_method +of I-experimental_method +these I-experimental_method +mutations I-experimental_method +( O +R31E B-mutant +, O +K32E B-mutant +, O +K176E B-mutant +, O +and O +K246E B-mutant +) O +essentially O +eliminated B-protein_state +GTP B-chemical +- O +dependent O +assembly O +( O +Additional O +file O +1 O +: O +Figure O +S1 O +) O +and O +allowed O +crystallization B-experimental_method +of O +Irga6 B-protein +in O +the O +presence B-protein_state +of I-protein_state +GMPPNP B-chemical +. O + +The O +asymmetric O +unit O +contained O +seven O +Irga6 B-protein +molecules O +that O +were O +arranged O +in O +a O +helical O +pattern O +along O +the O +long O +cell O +axis O +( O +Additional O +file O +1 O +: O +Figure O +S2 O +). O + +a O +Schematic O +view O +of O +the O +domain O +architecture O +of O +mouse B-taxonomy_domain +Irga6 B-protein +. O + +The O +nucleotide O +and O +Mg2 B-chemical ++ I-chemical +ion O +( O +green O +) O +are O +shown O +in O +sphere O +representation O +. O + +The O +GTPase B-structure_element +domain I-structure_element +dimer B-oligomeric_state +is O +boxed O +. O + +d O +Magnification O +of O +the O +contact B-site +sites I-site +. O + +e O +Superposition B-experimental_method +of O +different O +switch B-site +I I-site +conformations O +in O +the O +asymmetric O +unit O +; O +the O +same O +colors O +as O +in O +Additional O +file O +1 O +: O +Figure O +S2 O +are O +used O +for O +the O +switch B-site +I I-site +regions O +of O +the O +individual O +subunits O +. O + +Switch B-site +I I-site +residues O +of O +subunit O +A B-structure_element +( O +yellow O +) O +involved O +in O +ribose O +binding O +are O +labelled O +and O +shown O +in O +stick O +representation O +. O + +Like O +other O +dynamin B-protein_type +superfamily O +members O +, O +the O +GTPase B-structure_element +domain I-structure_element +of O +Irga6 B-protein +comprises O +a O +canonical O +GTPase B-structure_element +domain I-structure_element +fold O +, O +with O +a O +central O +β B-structure_element +- I-structure_element +sheet I-structure_element +surrounded O +by O +helices B-structure_element +on O +both O +sides O +( O +Fig O +. O +1a O +- O +c O +). O + +The O +helical B-structure_element +domain I-structure_element +is O +a O +bipartite O +structure O +composed O +of O +helices B-structure_element +αA B-structure_element +- I-structure_element +C I-structure_element +at O +the O +N O +- O +terminus O +and O +helix B-structure_element +αF B-structure_element +- I-structure_element +L I-structure_element +at O +the O +C O +- O +terminus O +of O +the O +GTPase B-structure_element +domain I-structure_element +. O + +Overall O +, O +the O +seven O +molecules O +in O +the O +asymmetric O +unit O +are O +very O +similar O +to O +each O +other O +, O +with O +root B-evidence +mean I-evidence +square I-evidence +deviations I-evidence +( O +rmsd B-evidence +) O +ranging O +from O +0 O +. O +32 O +– O +0 O +. O +45 O +Å O +over O +all O +Cα O +atoms O +. O + +The O +seven O +Irga6 B-protein +molecules O +in O +the O +asymmetric O +unit O +form O +various O +higher O +order O +contacts O +in O +the O +crystals B-evidence +. O + +Another O +assembly B-site +interface I-site +with O +a O +buried O +surface O +area O +of O +450 O +Å2 O +, O +which O +we O +call O +the O +“ O +tertiary B-site +patch I-site +”, O +was O +formed O +via O +two O +interaction B-site +sites I-site +in O +the O +helical B-structure_element +domains I-structure_element +( O +Additional O +file O +1 O +: O +Figure O +S2c O +, O +d O +, O +S3 O +). O + +In O +this O +interface B-site +, O +helices B-structure_element +αK B-structure_element +from O +two O +adjacent O +molecules O +form O +a O +hydrogen B-bond_interaction +bonding I-bond_interaction +network I-bond_interaction +involving O +residues O +373 B-residue_range +- I-residue_range +376 I-residue_range +. O + +Furthermore O +, O +two O +adjacent O +helices B-structure_element +αA B-structure_element +form O +hydrophobic B-bond_interaction +contacts I-bond_interaction +. O + +It O +was O +previously O +shown O +that O +the O +double B-protein_state +mutation I-protein_state +L372R B-mutant +/ O +A373R B-mutant +did O +not O +prevent O +GTP B-chemical +- O +induced O +assembly O +, O +so O +there O +is O +currently O +no O +evidence O +supporting O +an O +involvement O +of O +this O +interface B-site +in O +higher O +- O +order O +oligomerization O +. O + +This O +assembly O +results O +in O +a O +butterfly B-protein_state +- I-protein_state +shaped I-protein_state +Irga6 B-protein +dimer B-oligomeric_state +in O +which O +the O +helical B-structure_element +domains I-structure_element +protrude O +in O +parallel B-protein_state +orientations O +( O +Fig O +. O +1b O +, O +Additional O +file O +1 O +: O +Figure O +S3 O +). O + +In O +contrast O +, O +the O +other O +six O +molecules O +in O +the O +asymmetric O +unit O +do O +not O +assemble O +via O +the O +G B-site +interface I-site +. O + +The O +G B-site +interface I-site +in O +molecule O +A O +can O +be O +subdivided O +into O +three O +distinct O +contact B-site +sites I-site +( O +Fig O +. O +1c O +, O +d O +). O + +Contact B-site +site I-site +I I-site +is O +formed O +between O +R159 B-residue_name_number +and O +K161 B-residue_name_number +in O +the O +trans B-structure_element +stabilizing I-structure_element +loops I-structure_element +, O +and O +S132 B-residue_name_number +in O +the O +switch B-site +II I-site +regions O +of O +the O +opposing O +molecules O +. O + +In O +contact B-site +site I-site +III I-site +, O +G103 B-residue_name_number +of O +switch B-site +I I-site +interacts O +via O +its O +main O +chain O +nitrogen O +with O +the O +exocyclic O +2 O +’- O +OH O +and O +3 O +’- O +OH O +groups O +of O +the O +opposing O +ribose B-chemical +in O +trans O +, O +whereas O +the O +two O +opposing O +exocyclic O +3 O +’- O +OH O +group O +of O +the O +ribose B-chemical +form O +hydrogen B-bond_interaction +bonds I-bond_interaction +with O +each O +other O +. O + +Via O +the O +ribose B-chemical +contact O +, O +switch B-site +I I-site +is O +pulled O +towards O +the O +opposing O +nucleotide B-chemical +( O +Fig O +. O +1e O +). O + +E106 B-residue_name_number +was O +previously O +shown O +to O +be O +essential O +for O +catalysis O +, O +and O +the O +observed O +interactions O +in O +contact B-site +site I-site +III I-site +explain O +how O +dimerization O +via O +the O +ribose B-chemical +is O +directly O +coupled O +to O +the O +activation O +of O +GTP B-chemical +hydrolysis O +. O + +The O +G B-site +interface I-site +is O +in O +full O +agreement O +with O +previously O +published O +biochemical O +data O +that O +indicate O +crucial O +roles O +of O +E77 B-residue_name_number +, O +G103 B-residue_name_number +, O +E106 B-residue_name_number +, O +S132 B-residue_name_number +, O +R159 B-residue_name_number +, O +K161 B-residue_name_number +, O +K162 B-residue_name_number +, O +D164 B-residue_name_number +, O +N191 B-residue_name_number +, O +and O +K196 B-residue_name_number +for O +oligomerization O +and O +oligomerization O +- O +induced O +GTP B-chemical +hydrolysis O +. O + +All O +of O +these O +residues O +directly O +participate O +in O +contacts O +( O +G103 B-residue_name_number +, O +S132 B-residue_name_number +, O +R159 B-residue_name_number +, O +and O +K161 B-residue_name_number +) O +or O +are O +in O +direct O +vicinity O +to O +the O +interface B-site +( O +E77 B-residue_name_number +, O +E106 B-residue_name_number +, O +K162 B-residue_name_number +, O +D164 B-residue_name_number +, O +and O +N191 B-residue_name_number +). O + +Residues O +E77 B-residue_name_number +, O +K162 B-residue_name_number +, O +and O +D164 B-residue_name_number +appear O +to O +orient O +the O +trans B-structure_element +stabilizing I-structure_element +loop I-structure_element +which O +is O +involved O +in O +interface B-site +formation O +in O +contact B-site +site I-site +II I-site +. O + +In O +the O +earlier O +model O +of O +an O +anti B-protein_state +- I-protein_state +parallel I-protein_state +G B-site +interface I-site +, O +it O +was O +not O +possible O +to O +position O +the O +side O +chain O +of O +R159 B-residue_name_number +to O +avoid O +steric O +conflict O +. O + +In O +the O +present O +structure B-evidence +, O +the O +side O +- O +chain O +of O +R159 B-residue_name_number +projects O +laterally O +along O +the O +G B-site +interface I-site +and O +, O +therefore O +, O +does O +not O +cause O +a O +steric O +conflict O +. O + +A O +conserved O +dimerization O +mode O +via O +the O +G B-site +interface I-site +in O +dynamin B-protein_type +and O +septin B-protein_type +GTPases I-protein_type +. O + +The O +overall O +architecture O +of O +the O +parallel B-protein_state +GTPase B-structure_element +domain I-structure_element +dimer B-oligomeric_state +of O +Irga6 B-protein +is O +related O +to O +that O +of O +other O +dynamin B-protein_type +and O +septin B-protein_type +superfamily O +proteins O +. O + +Irga6 B-protein +immunity B-protein +- I-protein +related I-protein +GTPase I-protein +6 I-protein +, O +GMPPNP B-chemical +5 B-chemical +'- I-chemical +guanylyl I-chemical +imidodiphosphate I-chemical +, O +GTP B-chemical +guanosine B-chemical +- I-chemical +triphosphate I-chemical +, O +BDLP B-protein_type +bacterial B-taxonomy_domain +dynamin B-protein_type +like I-protein_type +protein I-protein_type +, O +GIMAP2 B-protein +, O +GTPase B-protein +of I-protein +immunity I-protein +associated I-protein +protein I-protein +2 I-protein + +The O +buried O +surface O +area O +per O +molecule O +( O +BSA O +) O +of O +the O +G B-site +interface I-site +in O +Irga6 B-protein +is O +relatively O +small O +( O +470 O +Å2 O +) O +compared O +to O +that O +of O +other O +dynamin B-protein_type +superfamily O +members O +, O +such O +as O +dynamin B-protein_type +( O +BSA O +: O +1400 O +Å2 O +), O +atlastin B-protein_type +( O +BSA O +: O +820 O +Å2 O +), O +GBP B-protein +- I-protein +1 I-protein +( O +BSA O +: O +2060 O +Å2 O +), O +BDLP B-protein_type +( O +BSA O +: O +2300 O +Å2 O +) O +or O +the O +septin B-protein +- I-protein +related I-protein +GTPase I-protein +of I-protein +immunity I-protein +associated I-protein +protein I-protein +2 I-protein +( O +GIMAP2 B-protein +) O +( O +BSA O +: O +590 O +Å2 O +) O +( O +Fig O +. O +2 O +). O + +However O +, O +the O +relative O +orientations O +of O +the O +GTPase B-structure_element +domains I-structure_element +in O +these O +dimers B-oligomeric_state +are O +strikingly O +similar O +, O +and O +the O +same O +elements O +, O +such O +as O +switch B-site +I I-site +, O +switch B-site +II I-site +, O +the O +trans B-structure_element +activating I-structure_element +and I-structure_element +G4 I-structure_element +loops I-structure_element +are O +involved O +in O +the O +parallel B-protein_state +dimerization O +mode O +in O +all O +of O +these O +GTPase B-protein_type +families O +. O + +IRG B-protein_type +proteins O +are O +crucial O +mediators O +of O +the O +innate O +immune O +response O +in O +mice B-taxonomy_domain +against O +a O +specific O +subset O +of O +intracellular O +pathogens O +, O +all O +of O +which O +enter O +the O +cell O +to O +form O +a O +membrane O +- O +bounded O +vacuole O +without O +engagement O +of O +the O +phagocytic O +machinery O +. O + +As O +members O +of O +the O +dynamin B-protein_type +superfamily O +, O +IRGs B-protein_type +oligomerize O +at O +cellular O +membranes O +in O +response O +to O +GTP B-chemical +binding O +. O + +Oligomerization O +and O +oligomerization O +- O +induced O +GTP B-chemical +hydrolysis O +are O +thought O +to O +induce O +membrane O +remodeling O +events O +ultimately O +leading O +to O +disruption O +of O +the O +PVM O +. O + +Recent O +structural B-experimental_method +and I-experimental_method +mechanistic I-experimental_method +analyses I-experimental_method +have O +begun O +to O +unravel O +the O +molecular O +basis O +for O +the O +membrane O +- O +remodeling O +activity O +and O +mechano O +- O +chemical O +function O +of O +some O +members O +( O +reviewed O +in O +). O + +However O +, O +for O +other O +dynamin B-protein_type +superfamily O +members O +such O +as O +IRGs B-protein_type +, O +the O +molecular O +basis O +for O +GTP B-chemical +hydrolysis O +and O +the O +exact O +role O +of O +the O +mechano O +- O +chemical O +function O +are O +still O +unclear O +. O + +Such O +a O +low O +affinity O +interaction O +mode O +may O +allow O +reversibility O +of O +oligomerization O +following O +GTP B-chemical +hydrolysis O +. O + +Similar O +low O +affinity O +G B-site +interface I-site +interactions O +were O +reported O +for O +dynamin B-protein_type +and O +MxA B-protein +. O + +The O +dimerization O +mode O +is O +strikingly O +different O +from O +the O +previously O +proposed O +anti B-protein_state +- I-protein_state +parallel I-protein_state +model O +that O +was O +based O +on O +the O +crystal B-evidence +structure I-evidence +of O +the O +signal B-protein_type +recognition I-protein_type +particle I-protein_type +GTPase I-protein_type +, O +SRP54 B-protein +and O +its O +homologous O +receptor O +. O + +However O +, O +the O +G B-site +dimer I-site +interface I-site +is O +reminiscent O +of O +the O +GTPase B-structure_element +domain I-structure_element +dimers B-oligomeric_state +observed O +for O +several O +other O +dynamin B-protein_type +superfamily O +members O +, O +such O +as O +dynamin B-protein_type +, O +GBP1 B-protein +, O +atlastin B-protein_type +, O +and O +BDLP B-protein_type +. O + +It O +was O +recently O +shown O +that O +septin B-protein_type +and O +septin B-protein_type +- I-protein_type +related I-protein_type +GTPases I-protein_type +, O +such O +as O +the O +Tocs B-protein_type +GTPases I-protein_type +or O +GTPases B-protein_type +of I-protein_type +immunity I-protein_type +related I-protein_type +proteins I-protein_type +( O +GIMAPs B-protein_type +), O +also O +employ O +a O +GTP B-chemical +- O +dependent O +parallel B-protein_state +dimerization O +mode O +. O + +Importantly O +, O +our O +analysis O +indicates O +that O +IRGs B-protein_type +are O +not O +outliers O +, O +but O +bona O +- O +fide O +representatives O +of O +the O +dynamin B-protein_type +superfamily O +. O + +Whereas O +the O +overall O +dimerization O +mode O +is O +similar O +in O +septin B-protein_type +and O +dynamin B-protein_type +GTPases I-protein_type +, O +family O +- O +specific O +differences O +in O +the O +G B-site +interface I-site +and O +the O +oligomerization B-site +interfaces I-site +exist O +. O + +For O +example O +, O +the O +involvement O +of O +the O +2 O +’ O +and O +3 O +’- O +OH O +groups O +of O +the O +ribose B-chemical +in O +the O +dimerization B-site +interface I-site +of O +Irga6 B-protein +has O +not O +been O +observed O +for O +other O +dynamin B-protein_type +and O +septin B-protein_type +superfamily O +members O +. O + +The O +surface O +- O +exposed O +location O +of O +the O +ribose O +in O +the O +Irga6 B-protein +structure B-evidence +, O +with O +a O +wide B-protein_state +- I-protein_state +open I-protein_state +nucleotide B-site +- I-site +binding I-site +pocket I-site +, O +facilitates O +its O +engagement O +in O +the O +dimerization B-site +interface I-site +. O + +This O +contact O +, O +in O +turn O +, O +appears O +to O +activate O +GTP B-chemical +hydrolysis O +by O +inducing O +rearrangements O +in O +switch B-site +I I-site +and O +the O +positioning O +of O +the O +catalytic B-protein_state +E106 B-residue_name_number +. O + +In O +dynamin B-protein_type +, O +the O +corresponding O +serine B-residue_name +residue O +coordinates O +a O +sodium B-chemical +ion O +that O +is O +crucial O +for O +GTP B-chemical +hydrolysis O +. O + +Higher O +resolution O +structures B-evidence +of O +the O +Irga6 B-protein +dimer B-oligomeric_state +in O +the O +presence B-protein_state +of I-protein_state +a O +transition O +state O +analogue O +are O +required O +to O +show O +whether O +Gly79 B-residue_name_number +directly O +participates O +in O +GTP B-chemical +hydrolysis O +or O +whether O +it O +may O +also O +position O +a O +catalytic O +cation O +. O + +In O +dynamin B-protein_type +, O +further O +assembly B-site +sites I-site +are O +provided O +by O +the O +helical B-structure_element +domains I-structure_element +which O +assemble O +in O +a O +criss O +- O +cross O +fashion O +to O +form O +a O +helical B-structure_element +filament I-structure_element +. O + +In O +dynamin B-protein_type +- I-protein_type +related I-protein_type +Eps15 I-protein_type +homology I-protein_type +domain I-protein_type +containing I-protein_type +proteins I-protein_type +( O +EHDs B-protein_type +), O +a O +second B-site +assembly I-site +interface I-site +is O +present O +in O +the O +GTPase B-structure_element +domain I-structure_element +. O + +Further O +structural B-experimental_method +studies I-experimental_method +, O +especially O +electron B-experimental_method +microscopy I-experimental_method +analysis I-experimental_method +of O +the O +Irga6 B-protein +oligomers B-oligomeric_state +, O +are O +required O +to O +clarify O +the O +assembly O +mode O +via O +the O +helical B-structure_element +domains I-structure_element +and O +to O +show O +how O +these O +interfaces B-site +cooperate O +with O +the O +G B-site +interface I-site +to O +mediate O +the O +regulated O +assembly O +on O +a O +membrane O +surface O +. O + +Notably O +, O +we O +did O +not O +observe O +major O +rearrangements O +of O +the O +helical B-structure_element +domain I-structure_element +versus O +the O +GTPase B-structure_element +domain I-structure_element +in O +the O +Irga6 B-protein +molecules O +that O +dimerized B-protein_state +via O +the O +G B-site +interface I-site +. O + +In O +a O +manner O +similar O +to O +BDLP B-protein_type +, O +such O +large O +- O +scale O +conformational O +changes O +may O +be O +induced O +by O +membrane O +binding O +. O + +Our O +structural B-experimental_method +analysis I-experimental_method +and O +the O +identification O +of O +the O +G B-site +- I-site +interface I-site +paves O +the O +way O +for O +determining O +the O +specific O +assembly O +of O +Irga6 B-protein +into O +a O +membrane O +- O +associated O +scaffold O +as O +the O +prerequisite O +to O +understand O +its O +action O +as O +an O +anti O +- O +parasitic O +machine O +. O + +Our O +study O +indicates O +that O +Irg B-protein_type +proteins O +dimerize B-oligomeric_state +via O +the O +G B-site +interface I-site +in O +a O +parallel B-protein_state +head I-protein_state +- I-protein_state +to I-protein_state +- I-protein_state +head I-protein_state +fashion O +thereby O +facilitating O +GTPase B-protein_type +activation O +. O + +These O +findings O +contribute O +to O +a O +molecular O +understanding O +of O +the O +anti O +- O +parasitic O +action O +of O +the O +Irg B-protein_type +protein O +family O +and O +suggest O +that O +Irgs B-protein_type +are O +bona O +- O +fide O +members O +of O +the O +dynamin B-protein_type +superfamily O +. O + diff --git a/annotation_JSON/annotations.json b/annotation_JSON/annotations.json new file mode 100644 index 0000000000000000000000000000000000000000..b9ace15b24eac23b6f92e43ad8b1cf60c82732d7 --- /dev/null +++ b/annotation_JSON/annotations.json @@ -0,0 +1,297350 @@ +{ + "PMC4850273": { + "annotations": [ + { + "sid": 0, + "sent": "Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont", + "section": "TITLE", + "ner": [ + [ + 24, + 34, + "Xyloglucan", + "chemical" + ], + [ + 62, + 67, + "Human", + "species" + ] + ] + }, + { + "sid": 1, + "sent": "Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as \u201cdietary fiber.\u201d However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 31, + "Polysaccharide utilization loci", + "gene" + ], + [ + 33, + 36, + "PUL", + "gene" + ], + [ + 69, + 74, + "human", + "species" + ], + [ + 79, + 92, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 161, + 174, + "carbohydrates", + "chemical" + ], + [ + 241, + 244, + "PUL", + "gene" + ], + [ + 339, + 344, + "human", + "species" + ], + [ + 345, + 354, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 2, + "sent": "In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood.", + "section": "ABSTRACT", + "ner": [ + [ + 38, + 60, + "complex polysaccharide", + "chemical" + ], + [ + 130, + 142, + "glycosidases", + "protein_type" + ] + ] + }, + { + "sid": 3, + "sent": "Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide.", + "section": "ABSTRACT", + "ner": [ + [ + 21, + 82, + "biochemical, structural, and reverse genetic characterization", + "experimental_method" + ], + [ + 97, + 133, + "cell surface glycan-binding proteins", + "protein_type" + ], + [ + 135, + 140, + "SGBPs", + "protein_type" + ], + [ + 155, + 183, + "xyloglucan utilization locus", + "gene" + ], + [ + 185, + 190, + "XyGUL", + "gene" + ], + [ + 197, + 215, + "Bacteroides ovatus", + "species" + ], + [ + 266, + 275, + "vegetable", + "taxonomy_domain" + ], + [ + 276, + 290, + "polysaccharide", + "chemical" + ] + ] + }, + { + "sid": 4, + "sent": "Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 20, + "Biochemical analysis", + "experimental_method" + ], + [ + 40, + 72, + "outer membrane-anchored proteins", + "protein_type" + ], + [ + 130, + 140, + "xyloglucan", + "chemical" + ], + [ + 142, + 145, + "XyG", + "chemical" + ], + [ + 147, + 161, + "polysaccharide", + "chemical" + ] + ] + }, + { + "sid": 5, + "sent": "The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the \u03b2-glucan backbone.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 25, + 31, + "SGBP-A", + "protein" + ], + [ + 35, + 39, + "SusD", + "protein" + ], + [ + 56, + 61, + "bound", + "protein_state" + ], + [ + 62, + 65, + "XyG", + "chemical" + ], + [ + 66, + 85, + "tetradecasaccharide", + "chemical" + ], + [ + 106, + 135, + "carbohydrate-binding platform", + "site" + ], + [ + 180, + 188, + "\u03b2-glucan", + "chemical" + ] + ] + }, + { + "sid": 6, + "sent": "The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain.", + "section": "ABSTRACT", + "ner": [ + [ + 12, + 25, + "tetra-modular", + "structure_element" + ], + [ + 26, + 35, + "structure", + "evidence" + ], + [ + 39, + 45, + "SGBP-B", + "protein" + ], + [ + 62, + 82, + "tandem Ig-like folds", + "structure_element" + ], + [ + 89, + 92, + "XyG", + "chemical" + ], + [ + 124, + 141, + "C-terminal domain", + "structure_element" + ] + ] + }, + { + "sid": 7, + "sent": "Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B.\u00a0ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes.", + "section": "ABSTRACT", + "ner": [ + [ + 27, + 37, + "affinities", + "evidence" + ], + [ + 42, + 45, + "XyG", + "chemical" + ], + [ + 47, + 71, + "reverse-genetic analysis", + "experimental_method" + ], + [ + 85, + 91, + "SGBP-B", + "protein" + ], + [ + 146, + 162, + "oligosaccharides", + "chemical" + ], + [ + 188, + 194, + "SGBP-A", + "protein" + ], + [ + 221, + 233, + "carbohydrate", + "chemical" + ], + [ + 264, + 267, + "XyG", + "chemical" + ], + [ + 307, + 313, + "SGBP-A", + "protein" + ], + [ + 318, + 324, + "SGBP-B", + "protein" + ], + [ + 366, + 378, + "carbohydrate", + "chemical" + ], + [ + 390, + 399, + "B.\u00a0ovatus", + "species" + ], + [ + 429, + 438, + "vegetable", + "taxonomy_domain" + ], + [ + 439, + 450, + "xyloglucans", + "chemical" + ], + [ + 471, + 484, + "Bacteroidetes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 8, + "sent": "The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute \u201cdietary fiber.\u201d Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 17, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 31, + 39, + "bacteria", + "taxonomy_domain" + ], + [ + 47, + 52, + "human", + "species" + ], + [ + 103, + 126, + "complex polysaccharides", + "chemical" + ], + [ + 267, + 280, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 299, + 304, + "plant", + "taxonomy_domain" + ], + [ + 315, + 330, + "polysaccharides", + "chemical" + ] + ] + }, + { + "sid": 9, + "sent": "Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide.", + "section": "ABSTRACT", + "ner": [ + [ + 27, + 77, + "biochemical, crystallographic, and genetic insight", + "experimental_method" + ], + [ + 91, + 122, + "surface glycan-binding proteins", + "protein_type" + ], + [ + 140, + 158, + "Bacteroides ovatus", + "species" + ], + [ + 159, + 187, + "xyloglucan utilization locus", + "gene" + ], + [ + 189, + 194, + "XyGUL", + "gene" + ], + [ + 245, + 254, + "vegetable", + "taxonomy_domain" + ], + [ + 255, + 269, + "polysaccharide", + "chemical" + ] + ] + }, + { + "sid": 10, + "sent": "Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria.", + "section": "ABSTRACT", + "ner": [ + [ + 61, + 83, + "complex polysaccharide", + "chemical" + ], + [ + 125, + 138, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 231, + 239, + "bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 11, + "sent": "The human gut microbiota influences the course of human development and health, playing key roles in immune stimulation, intestinal cell proliferation, and metabolic balance.", + "section": "INTRO", + "ner": [ + [ + 4, + 9, + "human", + "species" + ], + [ + 14, + 24, + "microbiota", + "taxonomy_domain" + ], + [ + 50, + 55, + "human", + "species" + ] + ] + }, + { + "sid": 12, + "sent": "This microbial community is largely bacterial, with the Bacteroidetes, Firmicutes, and Actinobacteria comprising the dominant phyla.", + "section": "INTRO", + "ner": [ + [ + 5, + 14, + "microbial", + "taxonomy_domain" + ], + [ + 36, + 45, + "bacterial", + "taxonomy_domain" + ], + [ + 56, + 69, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 71, + 81, + "Firmicutes", + "taxonomy_domain" + ], + [ + 87, + 101, + "Actinobacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 13, + "sent": "The ability to acquire energy from carbohydrates of dietary or host origin is central to the adaptation of human gut bacterial species to their niche.", + "section": "INTRO", + "ner": [ + [ + 35, + 48, + "carbohydrates", + "chemical" + ], + [ + 107, + 112, + "human", + "species" + ], + [ + 117, + 126, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 14, + "sent": "More importantly, this makes diet a tractable way to manipulate the abundance and metabolic output of the microbiota toward improved human health.", + "section": "INTRO", + "ner": [ + [ + 106, + 116, + "microbiota", + "taxonomy_domain" + ], + [ + 133, + 138, + "human", + "species" + ] + ] + }, + { + "sid": 15, + "sent": "However, there is a paucity of data regarding how the vast array of complex carbohydrate structures are selectively recognized and imported by members of the microbiota, a critical process that enables these organisms to thrive in the competitive gut environment.", + "section": "INTRO", + "ner": [ + [ + 68, + 88, + "complex carbohydrate", + "chemical" + ], + [ + 158, + 168, + "microbiota", + "taxonomy_domain" + ] + ] + }, + { + "sid": 16, + "sent": "The human gut bacteria Bacteroidetes share a profound capacity for dietary glycan degradation, with many species containing >250 predicted carbohydrate-active enzymes (CAZymes), compared to 50 to 100 within many Firmicutes and only 17 in the human genome devoted toward carbohydrate utilization.", + "section": "INTRO", + "ner": [ + [ + 4, + 9, + "human", + "species" + ], + [ + 14, + 22, + "bacteria", + "taxonomy_domain" + ], + [ + 23, + 36, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 75, + 81, + "glycan", + "chemical" + ], + [ + 212, + 222, + "Firmicutes", + "taxonomy_domain" + ], + [ + 242, + 247, + "human", + "species" + ] + ] + }, + { + "sid": 17, + "sent": "A remarkable feature of the Bacteroidetes is the packaging of genes for carbohydrate catabolism into discrete polysaccharide utilization loci (PUL), which are transcriptionally regulated by specific substrate signatures.", + "section": "INTRO", + "ner": [ + [ + 28, + 41, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 110, + 141, + "polysaccharide utilization loci", + "gene" + ], + [ + 143, + 146, + "PUL", + "gene" + ] + ] + }, + { + "sid": 18, + "sent": "The archetypal PUL-encoded system is the starch utilization system (Sus) (Fig.\u00a01B) of Bacteroides thetaiotaomicron.", + "section": "INTRO", + "ner": [ + [ + 15, + 18, + "PUL", + "gene" + ], + [ + 41, + 66, + "starch utilization system", + "complex_assembly" + ], + [ + 68, + 71, + "Sus", + "complex_assembly" + ], + [ + 86, + 114, + "Bacteroides thetaiotaomicron", + "species" + ] + ] + }, + { + "sid": 19, + "sent": "The Sus includes a lipid-anchored, outer membrane endo-amylase, SusG; a TonB-dependent transporter (TBDT), SusC, which imports oligosaccharides with the help of an associated starch-binding protein, SusD; two additional carbohydrate-binding lipoproteins, SusE and SusF; and two periplasmic exo-glucosidases, SusA and SusB, which generate glucose for transport into the cytoplasm.", + "section": "INTRO", + "ner": [ + [ + 4, + 7, + "Sus", + "complex_assembly" + ], + [ + 19, + 33, + "lipid-anchored", + "protein_state" + ], + [ + 50, + 62, + "endo-amylase", + "protein_type" + ], + [ + 64, + 68, + "SusG", + "protein" + ], + [ + 72, + 98, + "TonB-dependent transporter", + "protein_type" + ], + [ + 100, + 104, + "TBDT", + "protein_type" + ], + [ + 107, + 111, + "SusC", + "protein" + ], + [ + 127, + 143, + "oligosaccharides", + "chemical" + ], + [ + 175, + 197, + "starch-binding protein", + "protein_type" + ], + [ + 199, + 203, + "SusD", + "protein" + ], + [ + 220, + 253, + "carbohydrate-binding lipoproteins", + "protein_type" + ], + [ + 255, + 259, + "SusE", + "protein" + ], + [ + 264, + 268, + "SusF", + "protein" + ], + [ + 290, + 306, + "exo-glucosidases", + "protein_type" + ], + [ + 308, + 312, + "SusA", + "protein" + ], + [ + 317, + 321, + "SusB", + "protein" + ], + [ + 338, + 345, + "glucose", + "chemical" + ] + ] + }, + { + "sid": 20, + "sent": "The importance of PUL as a successful evolutionary strategy is underscored by the observation that Bacteroidetes such as B.\u00a0thetaiotaomicron and Bacteroides ovatus devote ~18% of their genomes to these systems.", + "section": "INTRO", + "ner": [ + [ + 18, + 21, + "PUL", + "gene" + ], + [ + 99, + 112, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 121, + 140, + "B.\u00a0thetaiotaomicron", + "species" + ], + [ + 145, + 163, + "Bacteroides ovatus", + "species" + ] + ] + }, + { + "sid": 21, + "sent": "Moving beyond seminal genomic and transcriptomic analyses, the current state-of-the-art PUL characterization involves combined reverse-genetic, biochemical, and structural studies to illuminate the molecular details of PUL function.", + "section": "INTRO", + "ner": [ + [ + 88, + 91, + "PUL", + "gene" + ], + [ + 127, + 179, + "reverse-genetic, biochemical, and structural studies", + "experimental_method" + ], + [ + 219, + 222, + "PUL", + "gene" + ] + ] + }, + { + "sid": 22, + "sent": "Xyloglucan and the Bacteroides ovatus xyloglucan utilization locus (XyGUL). (A) Representative structures of common xyloglucans using the Consortium for Functional Glycomics Symbol Nomenclature (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml).", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "Xyloglucan", + "chemical" + ], + [ + 19, + 37, + "Bacteroides ovatus", + "species" + ], + [ + 38, + 66, + "xyloglucan utilization locus", + "gene" + ], + [ + 68, + 73, + "XyGUL", + "gene" + ], + [ + 95, + 105, + "structures", + "evidence" + ], + [ + 116, + 127, + "xyloglucans", + "chemical" + ] + ] + }, + { + "sid": 23, + "sent": "Cleavage sites for BoXyGUL glycosidases (GHs) are indicated for solanaceous xyloglucan. (B) BtSus and BoXyGUL. (C) Localization of BoXyGUL-encoded proteins in cellular membranes and concerted modes of action in the degradation of xyloglucans to monosaccharides.", + "section": "FIG", + "ner": [ + [ + 19, + 26, + "BoXyGUL", + "gene" + ], + [ + 27, + 39, + "glycosidases", + "protein_type" + ], + [ + 41, + 44, + "GHs", + "protein_type" + ], + [ + 64, + 75, + "solanaceous", + "taxonomy_domain" + ], + [ + 76, + 86, + "xyloglucan", + "chemical" + ], + [ + 92, + 97, + "BtSus", + "gene" + ], + [ + 102, + 109, + "BoXyGUL", + "gene" + ], + [ + 131, + 138, + "BoXyGUL", + "gene" + ], + [ + 230, + 241, + "xyloglucans", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "The location of SGBP-A/B is presented in this work; the location of GH5 has been empirically determined, and the enzymes have been placed based upon their predicted cellular location.", + "section": "FIG", + "ner": [ + [ + 16, + 22, + "SGBP-A", + "protein" + ], + [ + 23, + 24, + "B", + "protein" + ], + [ + 68, + 71, + "GH5", + "protein" + ] + ] + }, + { + "sid": 25, + "sent": "We recently reported the detailed molecular characterization of a PUL that confers the ability of the human gut commensal B.\u00a0ovatus ATCC 8483 to grow on a prominent family of plant cell wall glycans, the xyloglucans (XyG).", + "section": "INTRO", + "ner": [ + [ + 66, + 69, + "PUL", + "gene" + ], + [ + 102, + 107, + "human", + "species" + ], + [ + 122, + 141, + "B.\u00a0ovatus ATCC 8483", + "species" + ], + [ + 175, + 180, + "plant", + "taxonomy_domain" + ], + [ + 191, + 198, + "glycans", + "chemical" + ], + [ + 204, + 215, + "xyloglucans", + "chemical" + ], + [ + 217, + 220, + "XyG", + "chemical" + ] + ] + }, + { + "sid": 26, + "sent": "XyG variants (Fig.\u00a01A) constitute up to 25% of the dry weight of common vegetables.", + "section": "INTRO", + "ner": [ + [ + 0, + 3, + "XyG", + "chemical" + ], + [ + 72, + 82, + "vegetables", + "taxonomy_domain" + ] + ] + }, + { + "sid": 27, + "sent": "Analogous to the Sus locus, the xyloglucan utilization locus (XyGUL) encodes a cohort of carbohydrate-binding, -hydrolyzing, and -importing proteins (Fig.\u00a01B and C).", + "section": "INTRO", + "ner": [ + [ + 17, + 26, + "Sus locus", + "gene" + ], + [ + 32, + 60, + "xyloglucan utilization locus", + "gene" + ], + [ + 62, + 67, + "XyGUL", + "gene" + ], + [ + 89, + 148, + "carbohydrate-binding, -hydrolyzing, and -importing proteins", + "protein_type" + ] + ] + }, + { + "sid": 28, + "sent": "The number of glycoside hydrolases (GHs) encoded by the XyGUL is, however, more expansive than that by the Sus locus (Fig.\u00a01B), which reflects the greater complexity of glycosidic linkages found in XyG vis-\u00e0-vis starch.", + "section": "INTRO", + "ner": [ + [ + 14, + 34, + "glycoside hydrolases", + "protein_type" + ], + [ + 36, + 39, + "GHs", + "protein_type" + ], + [ + 56, + 61, + "XyGUL", + "gene" + ], + [ + 107, + 116, + "Sus locus", + "gene" + ], + [ + 198, + 201, + "XyG", + "chemical" + ], + [ + 212, + 218, + "starch", + "chemical" + ] + ] + }, + { + "sid": 29, + "sent": "Whereas our previous study focused on the characterization of the linkage specificity of these GHs, a key outstanding question regarding this locus is how XyG recognition is mediated at the cell surface.", + "section": "INTRO", + "ner": [ + [ + 95, + 98, + "GHs", + "protein_type" + ], + [ + 155, + 158, + "XyG", + "chemical" + ] + ] + }, + { + "sid": 30, + "sent": "In the archetypal starch utilization system of B.\u00a0thetaiotaomicron, starch binding to the cell surface is mediated at eight distinct starch-binding sites distributed among four surface glycan-binding proteins (SGBPs): two within the amylase SusG, one within SusD, two within SusE, and three within SusF. The functional redundancy of many of these sites is high: whereas SusD is essential for growth on starch, combined mutations of the SusE, SusF, and SusG binding sites are required to impair growth on the polysaccharide.", + "section": "INTRO", + "ner": [ + [ + 18, + 43, + "starch utilization system", + "complex_assembly" + ], + [ + 47, + 66, + "B.\u00a0thetaiotaomicron", + "species" + ], + [ + 133, + 153, + "starch-binding sites", + "site" + ], + [ + 177, + 208, + "surface glycan-binding proteins", + "protein_type" + ], + [ + 210, + 215, + "SGBPs", + "protein_type" + ], + [ + 233, + 240, + "amylase", + "protein_type" + ], + [ + 241, + 245, + "SusG", + "protein" + ], + [ + 258, + 262, + "SusD", + "protein" + ], + [ + 275, + 279, + "SusE", + "protein" + ], + [ + 298, + 302, + "SusF", + "protein" + ], + [ + 370, + 374, + "SusD", + "protein" + ], + [ + 402, + 408, + "starch", + "chemical" + ], + [ + 436, + 440, + "SusE", + "protein" + ], + [ + 442, + 446, + "SusF", + "protein" + ], + [ + 452, + 456, + "SusG", + "protein" + ], + [ + 457, + 470, + "binding sites", + "site" + ], + [ + 508, + 522, + "polysaccharide", + "chemical" + ] + ] + }, + { + "sid": 31, + "sent": "Bacteroidetes PUL ubiquitously encode homologs of SusC and SusD, as well as proteins whose genes are immediately downstream of susD, akin to susE/F, and these are typically annotated as \u201cputative lipoproteins\u201d.", + "section": "INTRO", + "ner": [ + [ + 0, + 13, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 14, + 17, + "PUL", + "gene" + ], + [ + 50, + 54, + "SusC", + "protein" + ], + [ + 59, + 63, + "SusD", + "protein" + ], + [ + 127, + 131, + "susD", + "gene" + ], + [ + 141, + 147, + "susE/F", + "gene" + ], + [ + 187, + 195, + "putative", + "protein_state" + ], + [ + 196, + 208, + "lipoproteins", + "protein_type" + ] + ] + }, + { + "sid": 32, + "sent": "The genes coding for these proteins, sometimes referred to as \u201csusE/F positioned,\u201d display products with a wide variation in amino acid sequence and which have little or no homology to other PUL-encoded proteins or known carbohydrate-binding proteins.", + "section": "INTRO", + "ner": [ + [ + 63, + 69, + "susE/F", + "gene" + ], + [ + 191, + 194, + "PUL", + "gene" + ], + [ + 221, + 250, + "carbohydrate-binding proteins", + "protein_type" + ] + ] + }, + { + "sid": 33, + "sent": "As the Sus SGBPs remain the only structurally characterized cohort to date, we therefore wondered whether such glycan binding and function are extended to other PUL that target more complex and heterogeneous polysaccharides, such as XyG.", + "section": "INTRO", + "ner": [ + [ + 7, + 10, + "Sus", + "complex_assembly" + ], + [ + 11, + 16, + "SGBPs", + "protein_type" + ], + [ + 111, + 117, + "glycan", + "chemical" + ], + [ + 161, + 164, + "PUL", + "gene" + ], + [ + 208, + 223, + "polysaccharides", + "chemical" + ], + [ + 233, + 236, + "XyG", + "chemical" + ] + ] + }, + { + "sid": 34, + "sent": "We describe here the detailed functional and structural characterization of the noncatalytic SGBPs encoded by Bacova_02651 and Bacova_02650 of the XyGUL, here referred to as SGBP-A and SGBP-B, to elucidate their molecular roles in carbohydrate acquisition in vivo.", + "section": "INTRO", + "ner": [ + [ + 30, + 72, + "functional and structural characterization", + "experimental_method" + ], + [ + 80, + 92, + "noncatalytic", + "protein_state" + ], + [ + 93, + 98, + "SGBPs", + "protein_type" + ], + [ + 110, + 122, + "Bacova_02651", + "gene" + ], + [ + 127, + 139, + "Bacova_02650", + "gene" + ], + [ + 147, + 152, + "XyGUL", + "gene" + ], + [ + 174, + 180, + "SGBP-A", + "protein" + ], + [ + 185, + 191, + "SGBP-B", + "protein" + ] + ] + }, + { + "sid": 35, + "sent": "Combined biochemical, structural, and reverse-genetic approaches clearly illuminate the distinct, yet complementary, functions that these two proteins play in XyG recognition as it impacts the physiology of B.\u00a0ovatus.", + "section": "INTRO", + "ner": [ + [ + 9, + 64, + "biochemical, structural, and reverse-genetic approaches", + "experimental_method" + ], + [ + 159, + 162, + "XyG", + "chemical" + ], + [ + 207, + 216, + "B.\u00a0ovatus", + "species" + ] + ] + }, + { + "sid": 36, + "sent": "These data extend our current understanding of the Sus-like glycan uptake paradigm within the Bacteroidetes and reveals how the complex dietary polysaccharide xyloglucan is recognized at the cell surface.", + "section": "INTRO", + "ner": [ + [ + 60, + 66, + "glycan", + "chemical" + ], + [ + 94, + 107, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 144, + 158, + "polysaccharide", + "chemical" + ], + [ + 159, + 169, + "xyloglucan", + "chemical" + ] + ] + }, + { + "sid": 37, + "sent": "SGBP-A and SGBP-B are cell-surface-localized, xyloglucan-specific binding proteins.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "SGBP-A", + "protein" + ], + [ + 11, + 17, + "SGBP-B", + "protein" + ], + [ + 22, + 82, + "cell-surface-localized, xyloglucan-specific binding proteins", + "protein_type" + ] + ] + }, + { + "sid": 38, + "sent": "SGBP-A, encoded by the XyGUL locus tag Bacova_02651 (Fig.\u00a01B), shares 26% amino acid sequence identity (40% similarity) with its homolog, B.\u00a0thetaiotaomicron SusD, and similar homology with the SusD-like proteins encoded within syntenic XyGUL identified in our earlier work.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "SGBP-A", + "protein" + ], + [ + 23, + 28, + "XyGUL", + "gene" + ], + [ + 39, + 51, + "Bacova_02651", + "gene" + ], + [ + 138, + 157, + "B.\u00a0thetaiotaomicron", + "species" + ], + [ + 158, + 162, + "SusD", + "protein" + ], + [ + 194, + 212, + "SusD-like proteins", + "protein_type" + ], + [ + 237, + 242, + "XyGUL", + "gene" + ] + ] + }, + { + "sid": 39, + "sent": "In contrast, SGBP-B, encoded by locus tag Bacova_02650, displays little sequence similarity to the products of similarly positioned genes in syntenic XyGUL nor to any other gene product among the diversity of Bacteroidetes PUL.", + "section": "RESULTS", + "ner": [ + [ + 13, + 19, + "SGBP-B", + "protein" + ], + [ + 42, + 54, + "Bacova_02650", + "gene" + ], + [ + 150, + 155, + "XyGUL", + "gene" + ], + [ + 209, + 222, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 223, + 226, + "PUL", + "gene" + ] + ] + }, + { + "sid": 40, + "sent": "Whereas sequence similarity among SusC/SusD homolog pairs often serves as a hallmark for PUL identification, the sequence similarities of downstream genes encoding SGBPs are generally too low to allow reliable bioinformatic classification of their products into protein families, let alone prediction of function.", + "section": "RESULTS", + "ner": [ + [ + 34, + 38, + "SusC", + "protein" + ], + [ + 39, + 43, + "SusD", + "protein" + ], + [ + 89, + 92, + "PUL", + "gene" + ], + [ + 164, + 169, + "SGBPs", + "protein_type" + ] + ] + }, + { + "sid": 41, + "sent": "Hence, there is a critical need for the elucidation of detailed structure-function relationships among PUL SGBPs, in light of the manifold glycan structures in nature.", + "section": "RESULTS", + "ner": [ + [ + 103, + 106, + "PUL", + "gene" + ], + [ + 107, + 112, + "SGBPs", + "protein_type" + ], + [ + 139, + 145, + "glycan", + "chemical" + ] + ] + }, + { + "sid": 42, + "sent": "Immunofluorescence of formaldehyde-fixed, nonpermeabilized cells grown in minimal medium with XyG as the sole carbon source to induce XyGUL expression, reveals that both SGBP-A and SGBP-B are presented on the cell surface by N-terminal lipidation, as predicted by signal peptide analysis with SignalP (Fig.\u00a02).", + "section": "RESULTS", + "ner": [ + [ + 0, + 18, + "Immunofluorescence", + "experimental_method" + ], + [ + 94, + 97, + "XyG", + "chemical" + ], + [ + 134, + 139, + "XyGUL", + "gene" + ], + [ + 170, + 176, + "SGBP-A", + "protein" + ], + [ + 181, + 187, + "SGBP-B", + "protein" + ], + [ + 236, + 246, + "lipidation", + "ptm" + ] + ] + }, + { + "sid": 43, + "sent": "Here, the SGBPs very likely work in concert with the cell-surface-localized endo-xyloglucanase B.\u00a0ovatus GH5 (BoGH5) to recruit and cleave XyG for subsequent periplasmic import via the SusC-like TBDT of the XyGUL (Fig.\u00a01B and C).", + "section": "RESULTS", + "ner": [ + [ + 10, + 15, + "SGBPs", + "protein_type" + ], + [ + 53, + 94, + "cell-surface-localized endo-xyloglucanase", + "protein_type" + ], + [ + 95, + 104, + "B.\u00a0ovatus", + "species" + ], + [ + 105, + 108, + "GH5", + "protein" + ], + [ + 110, + 115, + "BoGH5", + "protein" + ], + [ + 139, + 142, + "XyG", + "chemical" + ], + [ + 185, + 199, + "SusC-like TBDT", + "protein_type" + ], + [ + 207, + 212, + "XyGUL", + "gene" + ] + ] + }, + { + "sid": 44, + "sent": "SGBP-A and SGBP-B visualized by immunofluorescence.", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "SGBP-A", + "protein" + ], + [ + 11, + 17, + "SGBP-B", + "protein" + ], + [ + 32, + 50, + "immunofluorescence", + "experimental_method" + ] + ] + }, + { + "sid": 45, + "sent": "Formalin-fixed, nonpermeabilized B.\u00a0ovatus cells were grown in minimal medium plus XyG, probed with custom rabbit antibodies to SGBP-A or SGBP-B, and then stained with Alexa Fluor 488 goat anti-rabbit IgG. (A) Overlay of bright-field and FITC images of B.\u00a0ovatus cells labeled with anti-SGBP-A. (B) Overlay of bright-field and FITC images of B.\u00a0ovatus cells labeled with anti-SGBP-B. (C) Bright-field image of \u0394SGBP-B cells labeled with anti-SGBP-B antibodies.", + "section": "FIG", + "ner": [ + [ + 33, + 42, + "B.\u00a0ovatus", + "species" + ], + [ + 83, + 86, + "XyG", + "chemical" + ], + [ + 128, + 134, + "SGBP-A", + "protein" + ], + [ + 138, + 144, + "SGBP-B", + "protein" + ], + [ + 210, + 217, + "Overlay", + "experimental_method" + ], + [ + 221, + 249, + "bright-field and FITC images", + "evidence" + ], + [ + 253, + 262, + "B.\u00a0ovatus", + "species" + ], + [ + 299, + 306, + "Overlay", + "experimental_method" + ], + [ + 310, + 338, + "bright-field and FITC images", + "evidence" + ], + [ + 342, + 351, + "B.\u00a0ovatus", + "species" + ], + [ + 388, + 406, + "Bright-field image", + "evidence" + ], + [ + 410, + 417, + "\u0394SGBP-B", + "mutant" + ] + ] + }, + { + "sid": 46, + "sent": "(D) FITC images of \u0394SGBP-B cells labeled with anti-SGBP-B antibodies.", + "section": "FIG", + "ner": [ + [ + 4, + 15, + "FITC images", + "evidence" + ], + [ + 19, + 26, + "\u0394SGBP-B", + "mutant" + ] + ] + }, + { + "sid": 47, + "sent": "Cells lacking SGBP-A (\u0394SGBP-A) do not grow on XyG and therefore could not be tested in parallel.", + "section": "FIG", + "ner": [ + [ + 6, + 13, + "lacking", + "protein_state" + ], + [ + 14, + 20, + "SGBP-A", + "protein" + ], + [ + 22, + 29, + "\u0394SGBP-A", + "mutant" + ], + [ + 46, + 49, + "XyG", + "chemical" + ] + ] + }, + { + "sid": 48, + "sent": "In our initial study focused on the functional characterization of the glycoside hydrolases of the XyGUL, we reported preliminary affinity PAGE and isothermal titration calorimetry (ITC) data indicating that both SGBP-A and SGBP-B are competent xyloglucan-binding proteins (affinity constant [Ka] values of 3.74 \u00d7 105\u00a0M\u22121 and 4.98 \u00d7 104\u00a0M\u22121, respectively [23]).", + "section": "RESULTS", + "ner": [ + [ + 71, + 91, + "glycoside hydrolases", + "protein_type" + ], + [ + 99, + 104, + "XyGUL", + "gene" + ], + [ + 130, + 143, + "affinity PAGE", + "experimental_method" + ], + [ + 148, + 180, + "isothermal titration calorimetry", + "experimental_method" + ], + [ + 182, + 185, + "ITC", + "experimental_method" + ], + [ + 213, + 219, + "SGBP-A", + "protein" + ], + [ + 224, + 230, + "SGBP-B", + "protein" + ], + [ + 245, + 272, + "xyloglucan-binding proteins", + "protein_type" + ], + [ + 274, + 291, + "affinity constant", + "evidence" + ], + [ + 293, + 295, + "Ka", + "evidence" + ] + ] + }, + { + "sid": 49, + "sent": "Additional affinity PAGE analysis (Fig.\u00a03) demonstrates that SGBP-A also has moderate affinity for the artificial soluble cellulose derivative hydroxyethyl cellulose [HEC; a \u03b2(1 \u2192 4)-glucan] and limited affinity for mixed-linkage \u03b2(1\u21923)/\u03b2(1\u21924)-glucan (MLG) and glucomannan (GM; mixed glucosyl and mannosyl backbone), which together indicate general binding to polysaccharide backbone residues and major contributions from side-chain recognition.", + "section": "RESULTS", + "ner": [ + [ + 11, + 24, + "affinity PAGE", + "experimental_method" + ], + [ + 61, + 67, + "SGBP-A", + "protein" + ], + [ + 143, + 165, + "hydroxyethyl cellulose", + "chemical" + ], + [ + 167, + 170, + "HEC", + "chemical" + ], + [ + 174, + 189, + "\u03b2(1 \u2192 4)-glucan", + "chemical" + ], + [ + 216, + 250, + "mixed-linkage \u03b2(1\u21923)/\u03b2(1\u21924)-glucan", + "chemical" + ], + [ + 252, + 255, + "MLG", + "chemical" + ], + [ + 261, + 272, + "glucomannan", + "chemical" + ], + [ + 274, + 276, + "GM", + "chemical" + ], + [ + 284, + 292, + "glucosyl", + "chemical" + ], + [ + 297, + 305, + "mannosyl", + "chemical" + ], + [ + 360, + 374, + "polysaccharide", + "chemical" + ] + ] + }, + { + "sid": 50, + "sent": "In contrast, SGBP-B bound to HEC more weakly than SGBP-A and did not bind to MLG or GM.", + "section": "RESULTS", + "ner": [ + [ + 13, + 19, + "SGBP-B", + "protein" + ], + [ + 29, + 32, + "HEC", + "chemical" + ], + [ + 50, + 56, + "SGBP-A", + "protein" + ], + [ + 77, + 80, + "MLG", + "chemical" + ], + [ + 84, + 86, + "GM", + "chemical" + ] + ] + }, + { + "sid": 51, + "sent": "Neither SGBP recognized galactomannan (GGM), starch, carboxymethylcellulose, or mucin (see Fig.\u00a0S1 in the supplemental material).", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "SGBP", + "protein_type" + ], + [ + 24, + 37, + "galactomannan", + "chemical" + ], + [ + 39, + 42, + "GGM", + "chemical" + ], + [ + 45, + 51, + "starch", + "chemical" + ], + [ + 53, + 75, + "carboxymethylcellulose", + "chemical" + ], + [ + 80, + 85, + "mucin", + "chemical" + ] + ] + }, + { + "sid": 52, + "sent": "Together, these results highlight the high specificities of SGBP-A and SGBP-B for XyG, which is concordant with their association with XyG-specific GHs in the XyGUL, as well as transcriptomic analysis indicating that B.\u00a0ovatus has discrete PUL for MLG, GM, and GGM (11).", + "section": "RESULTS", + "ner": [ + [ + 60, + 66, + "SGBP-A", + "protein" + ], + [ + 71, + 77, + "SGBP-B", + "protein" + ], + [ + 82, + 85, + "XyG", + "chemical" + ], + [ + 135, + 151, + "XyG-specific GHs", + "protein_type" + ], + [ + 159, + 164, + "XyGUL", + "gene" + ], + [ + 217, + 226, + "B.\u00a0ovatus", + "species" + ], + [ + 240, + 243, + "PUL", + "gene" + ], + [ + 248, + 251, + "MLG", + "chemical" + ], + [ + 253, + 255, + "GM", + "chemical" + ], + [ + 261, + 264, + "GGM", + "chemical" + ] + ] + }, + { + "sid": 53, + "sent": "Notably, the absence of carbohydrate-binding modules in the GHs encoded by the XyGUL implies that noncatalytic recognition of xyloglucan is mediated entirely by SGBP-A and -B.", + "section": "RESULTS", + "ner": [ + [ + 24, + 52, + "carbohydrate-binding modules", + "site" + ], + [ + 60, + 63, + "GHs", + "protein_type" + ], + [ + 79, + 84, + "XyGUL", + "gene" + ], + [ + 126, + 136, + "xyloglucan", + "chemical" + ], + [ + 161, + 167, + "SGBP-A", + "protein" + ], + [ + 172, + 174, + "-B", + "protein" + ] + ] + }, + { + "sid": 54, + "sent": "SGBP-A and SGBP-B preferentially bind xyloglucan.", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "SGBP-A", + "protein" + ], + [ + 11, + 17, + "SGBP-B", + "protein" + ], + [ + 38, + 48, + "xyloglucan", + "chemical" + ] + ] + }, + { + "sid": 55, + "sent": "Affinity electrophoresis (10% acrylamide) of SGBP-A and SGBP-B with BSA as a control protein.", + "section": "FIG", + "ner": [ + [ + 0, + 24, + "Affinity electrophoresis", + "experimental_method" + ], + [ + 45, + 51, + "SGBP-A", + "protein" + ], + [ + 56, + 62, + "SGBP-B", + "protein" + ], + [ + 68, + 71, + "BSA", + "protein" + ] + ] + }, + { + "sid": 56, + "sent": "All samples were loaded on the same gel next to the BSA controls; thin black lines indicate where intervening lanes were removed from the final image for both space and clarity.", + "section": "FIG", + "ner": [ + [ + 52, + 55, + "BSA", + "protein" + ] + ] + }, + { + "sid": 57, + "sent": "The percentage of polysaccharide incorporated into each native gel is displayed.", + "section": "FIG", + "ner": [ + [ + 18, + 32, + "polysaccharide", + "chemical" + ] + ] + }, + { + "sid": 58, + "sent": "The vanguard endo-xyloglucanase of the XyGUL, BoGH5, preferentially cleaves the polysaccharide at unbranched glucosyl residues to generate xylogluco-oligosaccharides (XyGOs) comprising a Glc4 backbone with variable side-chain galactosylation (XyGO1) (Fig.\u00a01A; n = 1) as the limit of digestion products in vitro; controlled digestion and fractionation by size exclusion chromatography allow the production of higher-order oligosaccharides (e.g., XyGO2) (Fig.\u00a01A; n = 2).", + "section": "RESULTS", + "ner": [ + [ + 13, + 31, + "endo-xyloglucanase", + "protein_type" + ], + [ + 39, + 44, + "XyGUL", + "gene" + ], + [ + 46, + 51, + "BoGH5", + "protein" + ], + [ + 80, + 94, + "polysaccharide", + "chemical" + ], + [ + 109, + 117, + "glucosyl", + "chemical" + ], + [ + 139, + 165, + "xylogluco-oligosaccharides", + "chemical" + ], + [ + 167, + 172, + "XyGOs", + "chemical" + ], + [ + 187, + 200, + "Glc4 backbone", + "structure_element" + ], + [ + 206, + 241, + "variable side-chain galactosylation", + "structure_element" + ], + [ + 243, + 248, + "XyGO1", + "chemical" + ], + [ + 312, + 350, + "controlled digestion and fractionation", + "experimental_method" + ], + [ + 354, + 383, + "size exclusion chromatography", + "experimental_method" + ], + [ + 421, + 437, + "oligosaccharides", + "chemical" + ], + [ + 445, + 450, + "XyGO2", + "chemical" + ] + ] + }, + { + "sid": 59, + "sent": "ITC demonstrates that SGBP-A binds to XyG polysaccharide and XyGO2 (based on a Glc8 backbone) with essentially equal affinities, while no binding of XyGO1 (Glc4 backbone) was detectable (Table\u00a01; see Fig.\u00a0S2 and S3 in the supplemental material).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "ITC", + "experimental_method" + ], + [ + 22, + 28, + "SGBP-A", + "protein" + ], + [ + 38, + 41, + "XyG", + "chemical" + ], + [ + 42, + 56, + "polysaccharide", + "chemical" + ], + [ + 61, + 66, + "XyGO2", + "chemical" + ], + [ + 79, + 92, + "Glc8 backbone", + "structure_element" + ], + [ + 117, + 127, + "affinities", + "evidence" + ], + [ + 149, + 154, + "XyGO1", + "chemical" + ], + [ + 156, + 169, + "Glc4 backbone", + "structure_element" + ] + ] + }, + { + "sid": 60, + "sent": "Similarly, SGBP-B also bound to XyG and XyGO2 with approximately equal affinities, although in both cases, Ka values were nearly 10-fold lower than those for SGBP-A. Also in contrast to SGBP-A, SGBP-B also bound to XyGO1, yet the affinity for this minimal repeating unit was poor, with a Ka value of ca. 1 order of magnitude lower than for XyG and XyGO2.", + "section": "RESULTS", + "ner": [ + [ + 11, + 17, + "SGBP-B", + "protein" + ], + [ + 23, + 31, + "bound to", + "protein_state" + ], + [ + 32, + 35, + "XyG", + "chemical" + ], + [ + 40, + 45, + "XyGO2", + "chemical" + ], + [ + 71, + 81, + "affinities", + "evidence" + ], + [ + 107, + 109, + "Ka", + "evidence" + ], + [ + 158, + 164, + "SGBP-A", + "protein" + ], + [ + 186, + 192, + "SGBP-A", + "protein" + ], + [ + 194, + 200, + "SGBP-B", + "protein" + ], + [ + 206, + 214, + "bound to", + "protein_state" + ], + [ + 215, + 220, + "XyGO1", + "chemical" + ], + [ + 230, + 238, + "affinity", + "evidence" + ], + [ + 248, + 270, + "minimal repeating unit", + "structure_element" + ], + [ + 288, + 290, + "Ka", + "evidence" + ], + [ + 340, + 343, + "XyG", + "chemical" + ], + [ + 348, + 353, + "XyGO2", + "chemical" + ] + ] + }, + { + "sid": 61, + "sent": "Together, these data clearly suggest that polysaccharide binding of both SGBPs is fulfilled by a dimer of the minimal repeat, corresponding to XyGO2 (cf.", + "section": "RESULTS", + "ner": [ + [ + 42, + 56, + "polysaccharide", + "chemical" + ], + [ + 73, + 78, + "SGBPs", + "protein_type" + ], + [ + 97, + 102, + "dimer", + "oligomeric_state" + ], + [ + 110, + 124, + "minimal repeat", + "structure_element" + ], + [ + 143, + 148, + "XyGO2", + "chemical" + ] + ] + }, + { + "sid": 62, + "sent": "The observation by affinity PAGE that these proteins specifically recognize XyG is further substantiated by their lack of binding for the undecorated oligosaccharide cellotetraose (Table\u00a01; see Fig.\u00a0S3).", + "section": "RESULTS", + "ner": [ + [ + 19, + 32, + "affinity PAGE", + "experimental_method" + ], + [ + 76, + 79, + "XyG", + "chemical" + ], + [ + 150, + 165, + "oligosaccharide", + "chemical" + ], + [ + 166, + 179, + "cellotetraose", + "chemical" + ] + ] + }, + { + "sid": 63, + "sent": "Furthermore, SGBP-A binds cellohexaose with ~770-fold weaker affinity than XyG, while SGBP-B displays no detectable binding to this linear hexasaccharide.", + "section": "RESULTS", + "ner": [ + [ + 13, + 19, + "SGBP-A", + "protein" + ], + [ + 26, + 38, + "cellohexaose", + "chemical" + ], + [ + 61, + 69, + "affinity", + "evidence" + ], + [ + 75, + 78, + "XyG", + "chemical" + ], + [ + 86, + 92, + "SGBP-B", + "protein" + ], + [ + 139, + 153, + "hexasaccharide", + "chemical" + ] + ] + }, + { + "sid": 64, + "sent": "To provide molecular-level insight into how the XyGUL SGBPs equip B.\u00a0ovatus to specifically harvest XyG from the gut environment, we performed X-ray crystallography analysis of both SGBP-A and SGPB-B in oligosaccharide-complex forms.", + "section": "RESULTS", + "ner": [ + [ + 48, + 53, + "XyGUL", + "gene" + ], + [ + 54, + 59, + "SGBPs", + "protein_type" + ], + [ + 66, + 75, + "B.\u00a0ovatus", + "species" + ], + [ + 100, + 103, + "XyG", + "chemical" + ], + [ + 143, + 164, + "X-ray crystallography", + "experimental_method" + ], + [ + 182, + 188, + "SGBP-A", + "protein" + ], + [ + 193, + 199, + "SGPB-B", + "protein" + ], + [ + 203, + 232, + "oligosaccharide-complex forms", + "complex_assembly" + ] + ] + }, + { + "sid": 65, + "sent": "Summary of thermodynamic parameters for wild-type SGBP-A and SGBP-B obtained by isothermal titration calorimetry at 25\u00b0Ca", + "section": "TABLE", + "ner": [ + [ + 40, + 49, + "wild-type", + "protein_state" + ], + [ + 50, + 56, + "SGBP-A", + "protein" + ], + [ + 61, + 67, + "SGBP-B", + "protein" + ], + [ + 80, + 112, + "isothermal titration calorimetry", + "experimental_method" + ] + ] + }, + { + "sid": 66, + "sent": "Carbohydrate\tKa (M\u22121)\t\u0394G (kcal\u00a0\u22c5\u00a0mol\u22121)\t\u0394H (kcal\u00a0\u22c5\u00a0mol\u22121)\tT\u0394S (kcal\u00a0\u22c5\u00a0mol\u22121)\t \tSGBP-A\tSGBP-B\tSGBP-A\tSGBP-B\tSGBP-A\tSGBP-B\tSGBP-A\tSGBP-B\t \tXyGb\t(4.4 \u00b1 0.1) \u00d7 105\t(5.7 \u00b1 0.2) \u00d7 104\t\u22127.7\t\u22126.5\t\u221214 \u00b1 3\t\u221214 \u00b1 2\t\u22126.5\t\u22127.6\t \tXyGO2c\t3.0 \u00d7 105\t2.0 \u00d7 104\t\u22127.5\t\u22125.9\t\u221217.2\t\u221217.6\t\u22129.7\t\u221211.7\t \tXyGO1\tNBd\t(2.4 \u00b1 0.1) \u00d7 103\tNB\t\u22124.6\tNB\t\u22124.4 \u00b1 0.2\tNB\t0.2\t \tCellohexaose\t568.0 \u00b1 291.0\tNB\t\u22123.8\tNB\t\u221216 \u00b1 8\tNB\t\u221212.7\tNB\t \tCellotetraose\tNB\tNB\tNB\tNB\tNB\tNB\tNB\tNB\t \t", + "section": "TABLE", + "ner": [ + [ + 22, + 24, + "\u0394G", + "evidence" + ] + ] + }, + { + "sid": 67, + "sent": "SGBP-A is a SusD homolog with an extensive glycan-binding platform.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "SGBP-A", + "protein" + ], + [ + 12, + 16, + "SusD", + "protein" + ], + [ + 43, + 66, + "glycan-binding platform", + "site" + ] + ] + }, + { + "sid": 68, + "sent": "As anticipated by sequence similarity, the high-resolution tertiary structure of apo-SGBP-A (1.36\u00a0\u00c5, Rwork = 14.7%, Rfree = 17.4%, residues 28 to 546) (Table\u00a02) displays the canonical \u201cSusD-like\u201d protein fold dominated by four tetratrico-peptide repeat (TPR) motifs that cradle the rest of the structure (Fig.\u00a04A).", + "section": "RESULTS", + "ner": [ + [ + 68, + 77, + "structure", + "evidence" + ], + [ + 81, + 84, + "apo", + "protein_state" + ], + [ + 85, + 91, + "SGBP-A", + "protein" + ], + [ + 101, + 106, + "Rwork", + "evidence" + ], + [ + 116, + 121, + "Rfree", + "evidence" + ], + [ + 140, + 149, + "28 to 546", + "residue_range" + ], + [ + 184, + 208, + "\u201cSusD-like\u201d protein fold", + "structure_element" + ], + [ + 227, + 252, + "tetratrico-peptide repeat", + "structure_element" + ], + [ + 254, + 257, + "TPR", + "structure_element" + ], + [ + 294, + 303, + "structure", + "evidence" + ] + ] + }, + { + "sid": 69, + "sent": "Specifically, SGBP-A overlays B.\u00a0thetaiotaomicron SusD (BtSusD) with a root mean square deviation (RMSD) value of 2.2\u00a0\u00c5 for 363 C\u03b1 pairs, which is notable given the 26% amino acid identity (40% similarity) between these homologs (Fig.\u00a04C).", + "section": "RESULTS", + "ner": [ + [ + 14, + 20, + "SGBP-A", + "protein" + ], + [ + 21, + 29, + "overlays", + "experimental_method" + ], + [ + 30, + 49, + "B.\u00a0thetaiotaomicron", + "species" + ], + [ + 50, + 54, + "SusD", + "protein" + ], + [ + 56, + 62, + "BtSusD", + "protein" + ], + [ + 71, + 97, + "root mean square deviation", + "evidence" + ], + [ + 99, + 103, + "RMSD", + "evidence" + ] + ] + }, + { + "sid": 70, + "sent": "Cocrystallization of SGBP-A with XyGO2 generated a substrate complex structure (2.3\u00a0\u00c5, Rwork = 21.8%, Rfree = 24.8%, residues 36 to 546) (Fig.\u00a04A and B; Table\u00a02) that revealed the distinct binding-site architecture of the XyG binding protein.", + "section": "RESULTS", + "ner": [ + [ + 0, + 17, + "Cocrystallization", + "experimental_method" + ], + [ + 21, + 27, + "SGBP-A", + "protein" + ], + [ + 33, + 38, + "XyGO2", + "chemical" + ], + [ + 51, + 68, + "substrate complex", + "complex_assembly" + ], + [ + 69, + 78, + "structure", + "evidence" + ], + [ + 87, + 92, + "Rwork", + "evidence" + ], + [ + 102, + 107, + "Rfree", + "evidence" + ], + [ + 126, + 135, + "36 to 546", + "residue_range" + ], + [ + 189, + 201, + "binding-site", + "site" + ], + [ + 222, + 241, + "XyG binding protein", + "protein_type" + ] + ] + }, + { + "sid": 71, + "sent": "The SGBP-A:XyGO2 complex superimposes closely with the apo structure (RMSD of 0.6\u00a0\u00c5) and demonstrates that no major conformational change occurs upon substrate binding; small deviations in the orientation of several surface loops are likely the result of differential crystal packing.", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "SGBP-A:XyGO2", + "complex_assembly" + ], + [ + 25, + 37, + "superimposes", + "experimental_method" + ], + [ + 55, + 58, + "apo", + "protein_state" + ], + [ + 59, + 68, + "structure", + "evidence" + ], + [ + 70, + 74, + "RMSD", + "evidence" + ] + ] + }, + { + "sid": 72, + "sent": "It is particularly notable that although the location of the ligand-binding site is conserved between SGBP-A and SusD, that of SGBP-A displays an ~29-\u00c5-long aromatic platform to accommodate the extended, linear XyG chain (see reference for a review of XyG secondary structure), versus the shorter, ~18-\u00c5-long, site within SusD that complements the helical conformation of amylose (Fig.\u00a04C and D).", + "section": "RESULTS", + "ner": [ + [ + 61, + 80, + "ligand-binding site", + "site" + ], + [ + 84, + 93, + "conserved", + "protein_state" + ], + [ + 102, + 108, + "SGBP-A", + "protein" + ], + [ + 113, + 117, + "SusD", + "protein" + ], + [ + 127, + 133, + "SGBP-A", + "protein" + ], + [ + 157, + 174, + "aromatic platform", + "site" + ], + [ + 211, + 214, + "XyG", + "chemical" + ], + [ + 252, + 255, + "XyG", + "chemical" + ], + [ + 310, + 314, + "site", + "site" + ], + [ + 322, + 326, + "SusD", + "protein" + ], + [ + 372, + 379, + "amylose", + "chemical" + ] + ] + }, + { + "sid": 73, + "sent": "Molecular structure of SGBP-A (Bacova_02651). (A) Overlay of SGBP-A from the apo (rainbow) and XyGO2 (gray) structures.", + "section": "FIG", + "ner": [ + [ + 10, + 19, + "structure", + "evidence" + ], + [ + 23, + 29, + "SGBP-A", + "protein" + ], + [ + 31, + 43, + "Bacova_02651", + "gene" + ], + [ + 50, + 57, + "Overlay", + "experimental_method" + ], + [ + 61, + 67, + "SGBP-A", + "protein" + ], + [ + 77, + 80, + "apo", + "protein_state" + ], + [ + 95, + 100, + "XyGO2", + "chemical" + ], + [ + 108, + 118, + "structures", + "evidence" + ] + ] + }, + { + "sid": 74, + "sent": "The apo structure is color ramped from blue to red.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "apo", + "protein_state" + ], + [ + 8, + 17, + "structure", + "evidence" + ] + ] + }, + { + "sid": 75, + "sent": "An omit map (2\u03c3) for XyGO2 (orange and red sticks) is displayed.", + "section": "FIG", + "ner": [ + [ + 3, + 11, + "omit map", + "evidence" + ], + [ + 21, + 26, + "XyGO2", + "chemical" + ] + ] + }, + { + "sid": 76, + "sent": "(B) Close-up view of the omit map as in panel A, rotated 90\u00b0 clockwise.", + "section": "FIG", + "ner": [ + [ + 25, + 33, + "omit map", + "evidence" + ] + ] + }, + { + "sid": 77, + "sent": "(C) Overlay of the C\u03b1 backbones of SGBP-A (black) with XyGO2 (orange and red spheres) and BtSusD (blue) with maltoheptaose (pink and red spheres), highlighting the conservation of the glycan-binding site location.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "Overlay", + "experimental_method" + ], + [ + 35, + 41, + "SGBP-A", + "protein" + ], + [ + 55, + 60, + "XyGO2", + "chemical" + ], + [ + 90, + 96, + "BtSusD", + "protein" + ], + [ + 109, + 122, + "maltoheptaose", + "chemical" + ], + [ + 184, + 203, + "glycan-binding site", + "site" + ] + ] + }, + { + "sid": 78, + "sent": "(D) Close-up of the SGBP-A (black and orange) and SusD (blue and pink) glycan-binding sites.", + "section": "FIG", + "ner": [ + [ + 20, + 26, + "SGBP-A", + "protein" + ], + [ + 50, + 54, + "SusD", + "protein" + ], + [ + 71, + 91, + "glycan-binding sites", + "site" + ] + ] + }, + { + "sid": 79, + "sent": "The approximate length of each glycan-binding site is displayed, colored to match the protein structures. (E) Stereo view of the xyloglucan-binding site of SGBP-A, displaying all residues within 4 \u00c5 of the ligand.", + "section": "FIG", + "ner": [ + [ + 31, + 50, + "glycan-binding site", + "site" + ], + [ + 86, + 104, + "protein structures", + "evidence" + ], + [ + 129, + 152, + "xyloglucan-binding site", + "site" + ], + [ + 156, + 162, + "SGBP-A", + "protein" + ] + ] + }, + { + "sid": 80, + "sent": "The backbone glucose residues are numbered from the nonreducing end; xylose residues are labeled X1 and X2.", + "section": "FIG", + "ner": [ + [ + 13, + 20, + "glucose", + "chemical" + ], + [ + 69, + 75, + "xylose", + "chemical" + ], + [ + 97, + 99, + "X1", + "residue_name_number" + ], + [ + 104, + 106, + "X2", + "residue_name_number" + ] + ] + }, + { + "sid": 81, + "sent": "Potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms.", + "section": "FIG", + "ner": [ + [ + 10, + 39, + "hydrogen-bonding interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 82, + "sent": "Seven of the eight backbone glucosyl residues of XyGO2 could be convincingly modeled in the ligand electron density, and only two \u03b1(1\u21926)-linked xylosyl residues were observed (Fig.\u00a04B; cf.", + "section": "RESULTS", + "ner": [ + [ + 28, + 36, + "glucosyl", + "chemical" + ], + [ + 49, + 54, + "XyGO2", + "chemical" + ], + [ + 92, + 115, + "ligand electron density", + "evidence" + ], + [ + 130, + 151, + "\u03b1(1\u21926)-linked xylosyl", + "chemical" + ] + ] + }, + { + "sid": 83, + "sent": "Indeed, the electron density for the ligand suggests some disorder, which may arise from multiple oligosaccharide orientations along the binding site.", + "section": "RESULTS", + "ner": [ + [ + 12, + 28, + "electron density", + "evidence" + ], + [ + 98, + 113, + "oligosaccharide", + "chemical" + ], + [ + 137, + 149, + "binding site", + "site" + ] + ] + }, + { + "sid": 84, + "sent": "Three aromatic residues\u2014W82, W283, W306\u2014comprise the flat platform that stacks along the naturally twisted \u03b2-glucan backbone (Fig.\u00a04E).", + "section": "RESULTS", + "ner": [ + [ + 24, + 27, + "W82", + "residue_name_number" + ], + [ + 29, + 33, + "W283", + "residue_name_number" + ], + [ + 35, + 39, + "W306", + "residue_name_number" + ], + [ + 53, + 66, + "flat platform", + "site" + ], + [ + 72, + 78, + "stacks", + "bond_interaction" + ], + [ + 107, + 115, + "\u03b2-glucan", + "chemical" + ] + ] + }, + { + "sid": 85, + "sent": "The functional importance of this platform is underscored by the observation that the W82A W283A W306A mutant of SGBP-A, designated SGBP-A*, is completely devoid of XyG affinity (Table\u00a03; see Fig.\u00a0S4 in the supplemental material).", + "section": "RESULTS", + "ner": [ + [ + 34, + 42, + "platform", + "site" + ], + [ + 86, + 90, + "W82A", + "mutant" + ], + [ + 91, + 96, + "W283A", + "mutant" + ], + [ + 97, + 102, + "W306A", + "mutant" + ], + [ + 103, + 109, + "mutant", + "protein_state" + ], + [ + 113, + 119, + "SGBP-A", + "protein" + ], + [ + 132, + 139, + "SGBP-A*", + "mutant" + ], + [ + 144, + 177, + "completely devoid of XyG affinity", + "protein_state" + ] + ] + }, + { + "sid": 86, + "sent": "Dissection of the individual contribution of these residues reveals that the W82A mutant displays a significant 4.9-fold decrease in the Ka value for XyG, while the W306A substitution completely abolishes XyG binding.", + "section": "RESULTS", + "ner": [ + [ + 77, + 81, + "W82A", + "mutant" + ], + [ + 82, + 88, + "mutant", + "protein_state" + ], + [ + 137, + 139, + "Ka", + "evidence" + ], + [ + 150, + 153, + "XyG", + "chemical" + ], + [ + 165, + 170, + "W306A", + "mutant" + ], + [ + 171, + 183, + "substitution", + "experimental_method" + ], + [ + 195, + 216, + "abolishes XyG binding", + "protein_state" + ] + ] + }, + { + "sid": 87, + "sent": "Contrasting with the clear importance of these hydrophobic interactions, there are remarkably few hydrogen-bonding interactions with the ligand, which are provided by R65, N83, and S308, which are proximal to Glc5 and Glc3.", + "section": "RESULTS", + "ner": [ + [ + 47, + 71, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 98, + 127, + "hydrogen-bonding interactions", + "bond_interaction" + ], + [ + 137, + 143, + "ligand", + "chemical" + ], + [ + 167, + 170, + "R65", + "residue_name_number" + ], + [ + 172, + 175, + "N83", + "residue_name_number" + ], + [ + 181, + 185, + "S308", + "residue_name_number" + ], + [ + 209, + 213, + "Glc5", + "residue_name_number" + ], + [ + 218, + 222, + "Glc3", + "residue_name_number" + ] + ] + }, + { + "sid": 88, + "sent": "Most surprising in light of the saccharide-binding data, however, was a lack of extensive recognition of the XyG side chains; only Y84 appeared to provide a hydrophobic interface for a xylosyl residue (Xyl1).", + "section": "RESULTS", + "ner": [ + [ + 32, + 55, + "saccharide-binding data", + "evidence" + ], + [ + 109, + 112, + "XyG", + "chemical" + ], + [ + 131, + 134, + "Y84", + "residue_name_number" + ], + [ + 157, + 178, + "hydrophobic interface", + "site" + ], + [ + 185, + 192, + "xylosyl", + "chemical" + ], + [ + 202, + 206, + "Xyl1", + "residue_name_number" + ] + ] + }, + { + "sid": 89, + "sent": "Summary of thermodynamic parameters for site-directed mutants of SGBP-A and SGBP-B obtained by ITC with XyG at 25\u00b0Ca", + "section": "TABLE", + "ner": [ + [ + 65, + 71, + "SGBP-A", + "protein" + ], + [ + 76, + 82, + "SGBP-B", + "protein" + ], + [ + 95, + 98, + "ITC", + "experimental_method" + ], + [ + 104, + 107, + "XyG", + "chemical" + ] + ] + }, + { + "sid": 90, + "sent": "Protein name\tKa\t\u0394G (kcal\u00a0\u22c5\u00a0mol\u22121)\t\u0394H (kcal\u00a0\u22c5\u00a0mol\u22121)\tT\u0394S (kcal\u00a0\u22c5\u00a0mol\u22121)\t \tFold changeb\tM\u22121\t \tSGBP-A(W82A W283A W306A)\tND\tNB\tNB\tNB\tNB\t \tSGBP-A(W82A)c\t4.9\t9.1 \u00d7 104\t\u22126.8\t\u22126.3\t0.5\t \tSGBP-A(W306)\tND\tNB\tNB\tNB\tNB\t \tSGBP-B(230\u2013489)\t0.7\t(8.6 \u00b1 0.20) \u00d7 104\t\u22126.7\t\u221214.9 \u00b1 0.1\t\u22128.2\t \tSGBP-B(Y363A)\t19.7\t(2.9 \u00b1 0.10) \u00d7 103\t\u22124.7\t\u221218.1 \u00b1 0.1\t\u221213.3\t \tSGBP-B(W364A)\tND\tWeak\tWeak\tWeak\tWeak\t \tSGBP-B(F414A)\t3.2\t(1.80 \u00b1 0.03) \u00d7 104\t\u22125.8\t\u221211.4 \u00b1 0.1\t\u22125.6\t \t", + "section": "TABLE", + "ner": [ + [ + 13, + 15, + "Ka", + "evidence" + ], + [ + 16, + 18, + "\u0394G", + "evidence" + ], + [ + 34, + 36, + "\u0394H", + "evidence" + ], + [ + 52, + 55, + "T\u0394S", + "evidence" + ], + [ + 92, + 98, + "SGBP-A", + "protein" + ], + [ + 99, + 103, + "W82A", + "mutant" + ], + [ + 104, + 109, + "W283A", + "mutant" + ], + [ + 110, + 115, + "W306A", + "mutant" + ], + [ + 134, + 140, + "SGBP-A", + "protein" + ], + [ + 141, + 145, + "W82A", + "mutant" + ], + [ + 178, + 184, + "SGBP-A", + "protein" + ], + [ + 185, + 189, + "W306", + "residue_name_number" + ], + [ + 208, + 214, + "SGBP-B", + "protein" + ], + [ + 215, + 222, + "230\u2013489", + "residue_range" + ], + [ + 271, + 277, + "SGBP-B", + "protein" + ], + [ + 278, + 283, + "Y363A", + "mutant" + ], + [ + 334, + 340, + "SGBP-B", + "protein" + ], + [ + 341, + 346, + "W364A", + "mutant" + ], + [ + 373, + 379, + "SGBP-B", + "protein" + ], + [ + 380, + 385, + "F414A", + "mutant" + ] + ] + }, + { + "sid": 91, + "sent": "Binding thermodynamics are based on the concentration of the binding unit, XyGO2.", + "section": "TABLE", + "ner": [ + [ + 75, + 80, + "XyGO2", + "chemical" + ] + ] + }, + { + "sid": 92, + "sent": "Weak binding represents a Ka of <500 M\u22121.", + "section": "TABLE", + "ner": [ + [ + 26, + 28, + "Ka", + "evidence" + ] + ] + }, + { + "sid": 93, + "sent": "Ka fold change = Ka of wild-type protein/Ka of mutant protein for xyloglucan binding.", + "section": "TABLE", + "ner": [ + [ + 0, + 2, + "Ka", + "evidence" + ], + [ + 17, + 19, + "Ka", + "evidence" + ], + [ + 23, + 32, + "wild-type", + "protein_state" + ], + [ + 41, + 43, + "Ka", + "evidence" + ], + [ + 66, + 76, + "xyloglucan", + "chemical" + ] + ] + }, + { + "sid": 94, + "sent": "SGBP-B has a multimodular structure with a single, C-terminal glycan-binding domain.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "SGBP-B", + "protein" + ], + [ + 62, + 83, + "glycan-binding domain", + "structure_element" + ] + ] + }, + { + "sid": 95, + "sent": "The crystal structure of full-length SGBP-B in complex with XyGO2 (2.37\u00a0\u00c5, Rwork = 19.9%, Rfree = 23.9%, residues 34 to 489) (Table\u00a02) revealed an extended structure composed of three tandem immunoglobulin (Ig)-like domains (domains A, B, and C) followed at the C terminus by a novel xyloglucan-binding domain (domain D) (Fig.\u00a05A).", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 25, + 36, + "full-length", + "protein_state" + ], + [ + 37, + 43, + "SGBP-B", + "protein" + ], + [ + 44, + 59, + "in complex with", + "protein_state" + ], + [ + 60, + 65, + "XyGO2", + "chemical" + ], + [ + 75, + 80, + "Rwork", + "evidence" + ], + [ + 90, + 95, + "Rfree", + "evidence" + ], + [ + 114, + 123, + "34 to 489", + "residue_range" + ], + [ + 156, + 165, + "structure", + "evidence" + ], + [ + 184, + 223, + "tandem immunoglobulin (Ig)-like domains", + "structure_element" + ], + [ + 233, + 234, + "A", + "structure_element" + ], + [ + 236, + 237, + "B", + "structure_element" + ], + [ + 243, + 244, + "C", + "structure_element" + ], + [ + 284, + 309, + "xyloglucan-binding domain", + "structure_element" + ], + [ + 318, + 319, + "D", + "structure_element" + ] + ] + }, + { + "sid": 96, + "sent": "Domains A, B, and C display similar \u03b2-sandwich folds; domains B (residues 134 to 230) and C (residues 231 to 313) can be superimposed onto domain A (residues 34 to 133) with RMSDs of 1.1 and 1.2\u00a0\u00c5, respectively, for 47 atom pairs (23% and 16% sequence identity, respectively).", + "section": "RESULTS", + "ner": [ + [ + 8, + 9, + "A", + "structure_element" + ], + [ + 11, + 12, + "B", + "structure_element" + ], + [ + 18, + 19, + "C", + "structure_element" + ], + [ + 36, + 52, + "\u03b2-sandwich folds", + "structure_element" + ], + [ + 62, + 63, + "B", + "structure_element" + ], + [ + 74, + 84, + "134 to 230", + "residue_range" + ], + [ + 90, + 91, + "C", + "structure_element" + ], + [ + 102, + 112, + "231 to 313", + "residue_range" + ], + [ + 121, + 133, + "superimposed", + "experimental_method" + ], + [ + 146, + 147, + "A", + "structure_element" + ], + [ + 158, + 167, + "34 to 133", + "residue_range" + ], + [ + 174, + 179, + "RMSDs", + "evidence" + ] + ] + }, + { + "sid": 97, + "sent": "These domains also display similarity to the C-terminal \u03b2-sandwich domains of many GH13 enzymes, including the cyclodextrin glucanotransferase of Geobacillus stearothermophilus (Fig.\u00a05B).", + "section": "RESULTS", + "ner": [ + [ + 0, + 13, + "These domains", + "structure_element" + ], + [ + 56, + 74, + "\u03b2-sandwich domains", + "structure_element" + ], + [ + 83, + 95, + "GH13 enzymes", + "protein_type" + ], + [ + 111, + 142, + "cyclodextrin glucanotransferase", + "protein_type" + ], + [ + 146, + 176, + "Geobacillus stearothermophilus", + "species" + ] + ] + }, + { + "sid": 98, + "sent": "Such domains are not typically involved in carbohydrate binding.", + "section": "RESULTS", + "ner": [ + [ + 0, + 12, + "Such domains", + "structure_element" + ], + [ + 43, + 55, + "carbohydrate", + "chemical" + ] + ] + }, + { + "sid": 99, + "sent": "Indeed, visual inspection of the SGBP-B structure, as well as individual production of the A and B domains and affinity PAGE analysis (see Fig.\u00a0S5 in the supplemental material), indicates that these domains do not contribute to XyG capture.", + "section": "RESULTS", + "ner": [ + [ + 8, + 25, + "visual inspection", + "experimental_method" + ], + [ + 33, + 39, + "SGBP-B", + "protein" + ], + [ + 40, + 49, + "structure", + "evidence" + ], + [ + 91, + 92, + "A", + "structure_element" + ], + [ + 97, + 98, + "B", + "structure_element" + ], + [ + 111, + 124, + "affinity PAGE", + "experimental_method" + ], + [ + 228, + 231, + "XyG", + "chemical" + ] + ] + }, + { + "sid": 100, + "sent": "On the other hand, production of the fused domains C and D in tandem (SGBP-B residues 230 to 489) retains complete binding of xyloglucan in vitro, with the observed slight increase in affinity likely arising from a reduced potential for steric hindrance of the smaller protein construct during polysaccharide interactions (Table\u00a03).", + "section": "RESULTS", + "ner": [ + [ + 19, + 29, + "production", + "experimental_method" + ], + [ + 37, + 58, + "fused domains C and D", + "mutant" + ], + [ + 70, + 76, + "SGBP-B", + "protein" + ], + [ + 86, + 96, + "230 to 489", + "residue_range" + ], + [ + 126, + 136, + "xyloglucan", + "chemical" + ], + [ + 294, + 308, + "polysaccharide", + "chemical" + ] + ] + }, + { + "sid": 101, + "sent": "While neither the full-length protein nor domain D displays structural homology to known XyG-binding proteins, the topology of SGBP-B resembles the xylan-binding protein Bacova_04391 (PDB 3ORJ) encoded within a xylan-targeting PUL of B.\u00a0ovatus (Fig.\u00a05C).", + "section": "RESULTS", + "ner": [ + [ + 18, + 29, + "full-length", + "protein_state" + ], + [ + 49, + 50, + "D", + "structure_element" + ], + [ + 89, + 109, + "XyG-binding proteins", + "protein_type" + ], + [ + 127, + 133, + "SGBP-B", + "protein" + ], + [ + 148, + 169, + "xylan-binding protein", + "protein_type" + ], + [ + 170, + 182, + "Bacova_04391", + "protein" + ], + [ + 211, + 216, + "xylan", + "chemical" + ], + [ + 227, + 230, + "PUL", + "gene" + ], + [ + 234, + 243, + "B.\u00a0ovatus", + "species" + ] + ] + }, + { + "sid": 102, + "sent": "The structure-based alignment of these proteins reveals 17% sequence identity, with a core RMSD of 3.6\u00a0\u00c5 for 253 aligned residues.", + "section": "RESULTS", + "ner": [ + [ + 4, + 29, + "structure-based alignment", + "experimental_method" + ], + [ + 91, + 95, + "RMSD", + "evidence" + ] + ] + }, + { + "sid": 103, + "sent": "While there is no substrate-complexed structure of Bacova_04391 available, the binding site is predicted to include W241 and Y404, which are proximal to the XyGO binding site in SGBP-B. However, the opposing, clamp-like arrangement of these residues in Bacova_04391 is clearly distinct from the planar surface arrangement of the residues that interact with XyG in SGBP-B (described below).", + "section": "RESULTS", + "ner": [ + [ + 51, + 63, + "Bacova_04391", + "protein" + ], + [ + 79, + 91, + "binding site", + "site" + ], + [ + 116, + 120, + "W241", + "residue_name_number" + ], + [ + 125, + 129, + "Y404", + "residue_name_number" + ], + [ + 157, + 174, + "XyGO binding site", + "site" + ], + [ + 178, + 184, + "SGBP-B", + "protein" + ], + [ + 199, + 231, + "opposing, clamp-like arrangement", + "protein_state" + ], + [ + 235, + 249, + "these residues", + "structure_element" + ], + [ + 253, + 265, + "Bacova_04391", + "protein" + ], + [ + 295, + 321, + "planar surface arrangement", + "site" + ], + [ + 329, + 337, + "residues", + "structure_element" + ], + [ + 357, + 360, + "XyG", + "chemical" + ], + [ + 364, + 370, + "SGBP-B", + "protein" + ] + ] + }, + { + "sid": 104, + "sent": "Multimodular structure of SGBP-B (Bacova_02650). (A) Full-length structure of SGBP-B, color coded by domain as indicated.", + "section": "FIG", + "ner": [ + [ + 26, + 32, + "SGBP-B", + "protein" + ], + [ + 34, + 46, + "Bacova_02650", + "gene" + ], + [ + 53, + 64, + "Full-length", + "protein_state" + ], + [ + 65, + 74, + "structure", + "evidence" + ], + [ + 78, + 84, + "SGBP-B", + "protein" + ] + ] + }, + { + "sid": 105, + "sent": "Prolines between domains are indicated as spheres.", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "Prolines", + "residue_name" + ] + ] + }, + { + "sid": 106, + "sent": "An omit map (2\u03c3) for XyGO2 is displayed to highlight the location of the glycan-binding site.", + "section": "FIG", + "ner": [ + [ + 3, + 11, + "omit map", + "evidence" + ], + [ + 21, + 26, + "XyGO2", + "chemical" + ], + [ + 73, + 92, + "glycan-binding site", + "site" + ] + ] + }, + { + "sid": 107, + "sent": "(B) Overlay of SGBP-B domains A, B, and C (colored as in panel A), with a C-terminal Ig-like domain of the G. stearothermophilus cyclodextrin glucanotransferase (PDB 1CYG [residues 375 to 493]) in green. (C) C\u03b1 overlay of SGBP-B (gray) and Bacova_04391 (PDB 3ORJ) (pink).", + "section": "FIG", + "ner": [ + [ + 15, + 21, + "SGBP-B", + "protein" + ], + [ + 30, + 31, + "A", + "structure_element" + ], + [ + 33, + 34, + "B", + "structure_element" + ], + [ + 40, + 41, + "C", + "structure_element" + ], + [ + 85, + 99, + "Ig-like domain", + "structure_element" + ], + [ + 107, + 128, + "G. stearothermophilus", + "species" + ], + [ + 129, + 160, + "cyclodextrin glucanotransferase", + "protein_type" + ], + [ + 181, + 191, + "375 to 493", + "residue_range" + ], + [ + 211, + 218, + "overlay", + "experimental_method" + ], + [ + 222, + 228, + "SGBP-B", + "protein" + ], + [ + 240, + 252, + "Bacova_04391", + "protein" + ] + ] + }, + { + "sid": 108, + "sent": "(D) Close-up omit map for the XyGO2 ligand, contoured at 2\u03c3. (E) Stereo view of the xyloglucan-binding site of SGBP-B, displaying all residues within 4 \u00c5 of the ligand.", + "section": "FIG", + "ner": [ + [ + 13, + 21, + "omit map", + "evidence" + ], + [ + 30, + 35, + "XyGO2", + "chemical" + ], + [ + 84, + 107, + "xyloglucan-binding site", + "site" + ], + [ + 111, + 117, + "SGBP-B", + "protein" + ] + ] + }, + { + "sid": 109, + "sent": "The backbone glucose residues are numbered from the nonreducing end, xylose residues are shown as X1, X2, and X3, potential hydrogen-bonding interactions are shown as dashed lines, and the distance is shown in angstroms.", + "section": "FIG", + "ner": [ + [ + 13, + 20, + "glucose", + "chemical" + ], + [ + 69, + 75, + "xylose", + "chemical" + ], + [ + 98, + 100, + "X1", + "residue_name_number" + ], + [ + 102, + 104, + "X2", + "residue_name_number" + ], + [ + 110, + 112, + "X3", + "residue_name_number" + ], + [ + 124, + 153, + "hydrogen-bonding interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 110, + "sent": "Inspection of the tertiary structure indicates that domains C and D are effectively inseparable, with a contact interface of 396\u00a0\u00c52.", + "section": "RESULTS", + "ner": [ + [ + 27, + 36, + "structure", + "evidence" + ], + [ + 60, + 61, + "C", + "structure_element" + ], + [ + 66, + 67, + "D", + "structure_element" + ] + ] + }, + { + "sid": 111, + "sent": "Domains A, B, and C do not pack against each other.", + "section": "RESULTS", + "ner": [ + [ + 8, + 9, + "A", + "structure_element" + ], + [ + 11, + 12, + "B", + "structure_element" + ], + [ + 18, + 19, + "C", + "structure_element" + ] + ] + }, + { + "sid": 112, + "sent": "Moreover, the five-residue linkers between these first three domains all feature a proline as the middle residue, suggesting significant conformational rigidity (Fig.\u00a05A).", + "section": "RESULTS", + "ner": [ + [ + 14, + 34, + "five-residue linkers", + "structure_element" + ], + [ + 83, + 90, + "proline", + "residue_name" + ], + [ + 98, + 112, + "middle residue", + "structure_element" + ] + ] + }, + { + "sid": 113, + "sent": "Despite the lack of sequence and structural conservation, a similarly positioned proline joins the Ig-like domains of the xylan-binding Bacova_04391 and the starch-binding proteins SusE and SusF. We speculate that this is a biologically important adaptation that serves to project the glycan binding site of these proteins far from the membrane surface.", + "section": "RESULTS", + "ner": [ + [ + 81, + 88, + "proline", + "residue_name" + ], + [ + 99, + 114, + "Ig-like domains", + "structure_element" + ], + [ + 136, + 148, + "Bacova_04391", + "protein" + ], + [ + 157, + 180, + "starch-binding proteins", + "protein_type" + ], + [ + 181, + 185, + "SusE", + "protein" + ], + [ + 190, + 194, + "SusF", + "protein" + ], + [ + 285, + 304, + "glycan binding site", + "site" + ] + ] + }, + { + "sid": 114, + "sent": "Any mobility of SGBP-B on the surface of the cell (beyond lateral diffusion within the membrane) is likely imparted by the eight-residue linker that spans the predicted lipidated Cys (C28) and the first \u03b2-strand of domain A. Other outer membrane proteins from various Sus-like systems possess a similar 10- to 20-amino-acid flexible linker between the lipidated Cys that tethers the protein to the outside the cell and the first secondary structure element.", + "section": "RESULTS", + "ner": [ + [ + 16, + 22, + "SGBP-B", + "protein" + ], + [ + 123, + 143, + "eight-residue linker", + "structure_element" + ], + [ + 169, + 178, + "lipidated", + "protein_state" + ], + [ + 179, + 182, + "Cys", + "residue_name" + ], + [ + 184, + 187, + "C28", + "residue_name_number" + ], + [ + 197, + 211, + "first \u03b2-strand", + "structure_element" + ], + [ + 222, + 223, + "A", + "structure_element" + ], + [ + 231, + 254, + "outer membrane proteins", + "protein_type" + ], + [ + 268, + 284, + "Sus-like systems", + "complex_assembly" + ], + [ + 303, + 339, + "10- to 20-amino-acid flexible linker", + "structure_element" + ], + [ + 352, + 361, + "lipidated", + "protein_state" + ], + [ + 362, + 365, + "Cys", + "residue_name" + ] + ] + }, + { + "sid": 115, + "sent": "Analogously, the outer membrane-anchored endo-xyloglucanase BoGH5 of the XyGUL contains a 100-amino-acid, all-\u03b2-strand, N-terminal module and flexible linker that imparts conformational flexibility and distances the catalytic module from the cell surface.", + "section": "RESULTS", + "ner": [ + [ + 17, + 40, + "outer membrane-anchored", + "protein_state" + ], + [ + 41, + 59, + "endo-xyloglucanase", + "protein_type" + ], + [ + 60, + 65, + "BoGH5", + "protein" + ], + [ + 73, + 78, + "XyGUL", + "gene" + ], + [ + 90, + 118, + "100-amino-acid, all-\u03b2-strand", + "structure_element" + ], + [ + 120, + 137, + "N-terminal module", + "structure_element" + ], + [ + 142, + 157, + "flexible linker", + "structure_element" + ], + [ + 216, + 232, + "catalytic module", + "structure_element" + ] + ] + }, + { + "sid": 116, + "sent": "XyG binds to domain D of SGBP-B at the concave interface of the top \u03b2-sheet, with binding mediated by loops connecting the \u03b2-strands.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "XyG", + "chemical" + ], + [ + 4, + 12, + "binds to", + "protein_state" + ], + [ + 20, + 21, + "D", + "structure_element" + ], + [ + 25, + 31, + "SGBP-B", + "protein" + ], + [ + 39, + 56, + "concave interface", + "site" + ], + [ + 68, + 75, + "\u03b2-sheet", + "structure_element" + ], + [ + 102, + 107, + "loops", + "structure_element" + ], + [ + 123, + 132, + "\u03b2-strands", + "structure_element" + ] + ] + }, + { + "sid": 117, + "sent": "Six glucosyl residues, comprising the main chain, and three branching xylosyl residues of XyGO2 can be modeled in the density (Fig.\u00a05D; cf.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "glucosyl", + "chemical" + ], + [ + 70, + 77, + "xylosyl", + "chemical" + ], + [ + 90, + 95, + "XyGO2", + "chemical" + ], + [ + 118, + 125, + "density", + "evidence" + ] + ] + }, + { + "sid": 118, + "sent": "The backbone is flat, with less of the \u201ctwisted-ribbon\u201d geometry observed in some cello- and xylogluco-oligosaccharides.", + "section": "RESULTS", + "ner": [ + [ + 82, + 119, + "cello- and xylogluco-oligosaccharides", + "chemical" + ] + ] + }, + { + "sid": 119, + "sent": "The aromatic platform created by W330, W364, and Y363 spans four glucosyl residues, compared to the longer platform of SGBP-A, which supports six glucosyl residues (Fig.\u00a05E).", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "aromatic platform", + "site" + ], + [ + 33, + 37, + "W330", + "residue_name_number" + ], + [ + 39, + 43, + "W364", + "residue_name_number" + ], + [ + 49, + 53, + "Y363", + "residue_name_number" + ], + [ + 65, + 73, + "glucosyl", + "chemical" + ], + [ + 100, + 106, + "longer", + "protein_state" + ], + [ + 107, + 115, + "platform", + "site" + ], + [ + 119, + 125, + "SGBP-A", + "protein" + ], + [ + 146, + 154, + "glucosyl", + "chemical" + ] + ] + }, + { + "sid": 120, + "sent": "The Y363A site-directed mutant of SGBP-B displays a 20-fold decrease in the Ka for XyG, while the W364A mutant lacks XyG binding (Table\u00a03; see Fig.\u00a0S6 in the supplemental material).", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "Y363A", + "mutant" + ], + [ + 10, + 30, + "site-directed mutant", + "experimental_method" + ], + [ + 34, + 40, + "SGBP-B", + "protein" + ], + [ + 76, + 78, + "Ka", + "evidence" + ], + [ + 83, + 86, + "XyG", + "chemical" + ], + [ + 98, + 103, + "W364A", + "mutant" + ], + [ + 104, + 110, + "mutant", + "protein_state" + ], + [ + 111, + 128, + "lacks XyG binding", + "protein_state" + ] + ] + }, + { + "sid": 121, + "sent": "There are no additional contacts between the protein and the \u03b2-glucan backbone and surprisingly few interactions with the side-chain xylosyl residues, despite that fact that ITC data demonstrate that SGBP-B does not measurably bind the cellohexaose (Table\u00a01).", + "section": "RESULTS", + "ner": [ + [ + 61, + 69, + "\u03b2-glucan", + "chemical" + ], + [ + 133, + 140, + "xylosyl", + "chemical" + ], + [ + 174, + 177, + "ITC", + "experimental_method" + ], + [ + 200, + 206, + "SGBP-B", + "protein" + ], + [ + 236, + 248, + "cellohexaose", + "chemical" + ] + ] + }, + { + "sid": 122, + "sent": "F414 stacks with the xylosyl residue of Glc3, while Q407 is positioned for hydrogen bonding with the O4 of xylosyl residue Xyl1.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "F414", + "residue_name_number" + ], + [ + 5, + 11, + "stacks", + "bond_interaction" + ], + [ + 21, + 28, + "xylosyl", + "chemical" + ], + [ + 40, + 44, + "Glc3", + "residue_name_number" + ], + [ + 52, + 56, + "Q407", + "residue_name_number" + ], + [ + 75, + 91, + "hydrogen bonding", + "bond_interaction" + ], + [ + 107, + 114, + "xylosyl", + "chemical" + ], + [ + 123, + 127, + "Xyl1", + "residue_name_number" + ] + ] + }, + { + "sid": 123, + "sent": "Surprisingly, an F414A mutant of SGBP-B displays only a mild 3-fold decrease in the Ka value for XyG, again suggesting that glycan recognition is primarily mediated via contact with the \u03b2-glucan backbone (Table\u00a03; see Fig.\u00a0S6).", + "section": "RESULTS", + "ner": [ + [ + 17, + 22, + "F414A", + "mutant" + ], + [ + 23, + 29, + "mutant", + "protein_state" + ], + [ + 33, + 39, + "SGBP-B", + "protein" + ], + [ + 84, + 86, + "Ka", + "evidence" + ], + [ + 97, + 100, + "XyG", + "chemical" + ], + [ + 124, + 130, + "glycan", + "chemical" + ] + ] + }, + { + "sid": 124, + "sent": "Additional residues surrounding the binding site, including Y369 and E412, may contribute to the recognition of more highly decorated XyG, but precisely how this is mediated is presently unclear.", + "section": "RESULTS", + "ner": [ + [ + 11, + 19, + "residues", + "structure_element" + ], + [ + 36, + 48, + "binding site", + "site" + ], + [ + 60, + 64, + "Y369", + "residue_name_number" + ], + [ + 69, + 73, + "E412", + "residue_name_number" + ], + [ + 134, + 137, + "XyG", + "chemical" + ] + ] + }, + { + "sid": 125, + "sent": "Hoping to achieve a higher-resolution view of the SGBP-B\u2013xyloglucan interaction, we solved the crystal structure of the fused CD domains in complex with XyGO2 (1.57\u00a0\u00c5, Rwork = 15.6%, Rfree = 17.1%, residues 230 to 489) (Table\u00a02).", + "section": "RESULTS", + "ner": [ + [ + 50, + 56, + "SGBP-B", + "protein" + ], + [ + 57, + 67, + "xyloglucan", + "chemical" + ], + [ + 84, + 90, + "solved", + "experimental_method" + ], + [ + 95, + 112, + "crystal structure", + "evidence" + ], + [ + 120, + 136, + "fused CD domains", + "mutant" + ], + [ + 137, + 152, + "in complex with", + "protein_state" + ], + [ + 153, + 158, + "XyGO2", + "chemical" + ], + [ + 168, + 173, + "Rwork", + "evidence" + ], + [ + 183, + 188, + "Rfree", + "evidence" + ], + [ + 207, + 217, + "230 to 489", + "residue_range" + ] + ] + }, + { + "sid": 126, + "sent": "The CD domains of the truncated and full-length proteins superimpose with a 0.4-\u00c5 RMSD of the C\u03b1 backbone, with no differences in the position of any of the glycan-binding residues (see Fig.\u00a0S7A in the supplemental material).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "CD domains", + "structure_element" + ], + [ + 22, + 31, + "truncated", + "protein_state" + ], + [ + 36, + 47, + "full-length", + "protein_state" + ], + [ + 57, + 68, + "superimpose", + "experimental_method" + ], + [ + 82, + 86, + "RMSD", + "evidence" + ], + [ + 157, + 180, + "glycan-binding residues", + "site" + ] + ] + }, + { + "sid": 127, + "sent": "While density is observed for XyGO2, the ligand could not be unambiguously modeled into this density to achieve a reasonable fit between the X-ray data and the known stereochemistry of the sugar (see Fig.\u00a0S7B and C).", + "section": "RESULTS", + "ner": [ + [ + 6, + 13, + "density", + "evidence" + ], + [ + 30, + 35, + "XyGO2", + "chemical" + ], + [ + 93, + 100, + "density", + "evidence" + ], + [ + 141, + 151, + "X-ray data", + "evidence" + ] + ] + }, + { + "sid": 128, + "sent": "While this may occur for a number of reasons in crystal structures, it is likely that the poor ligand density even at higher resolution is due to movement or multiple orientations of the sugar averaged throughout the lattice.", + "section": "RESULTS", + "ner": [ + [ + 48, + 66, + "crystal structures", + "evidence" + ], + [ + 187, + 192, + "sugar", + "chemical" + ] + ] + }, + { + "sid": 129, + "sent": "SGBP-A and SGBP-B have distinct, coordinated functions in vivo.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "SGBP-A", + "protein" + ], + [ + 11, + 17, + "SGBP-B", + "protein" + ] + ] + }, + { + "sid": 130, + "sent": "The similarity of the glycan specificity of SGBP-A and SGBP-B presents an intriguing conundrum regarding their individual roles in XyG utilization by B.\u00a0ovatus.", + "section": "RESULTS", + "ner": [ + [ + 22, + 28, + "glycan", + "chemical" + ], + [ + 44, + 50, + "SGBP-A", + "protein" + ], + [ + 55, + 61, + "SGBP-B", + "protein" + ], + [ + 131, + 134, + "XyG", + "chemical" + ], + [ + 150, + 159, + "B.\u00a0ovatus", + "species" + ] + ] + }, + { + "sid": 131, + "sent": "To disentangle the functions of SGBP-A and SGBP-B in XyG recognition and uptake, we created individual in-frame deletion and complementation mutant strains of B.\u00a0ovatus.", + "section": "RESULTS", + "ner": [ + [ + 32, + 38, + "SGBP-A", + "protein" + ], + [ + 43, + 49, + "SGBP-B", + "protein" + ], + [ + 53, + 56, + "XyG", + "chemical" + ], + [ + 103, + 147, + "in-frame deletion and complementation mutant", + "experimental_method" + ], + [ + 159, + 168, + "B.\u00a0ovatus", + "species" + ] + ] + }, + { + "sid": 132, + "sent": "In these growth experiments, overnight cultures of strains grown on minimal medium plus glucose were back-diluted 1:100-fold into minimal medium containing 5\u00a0mg/ml of the reported carbohydrate.", + "section": "RESULTS", + "ner": [ + [ + 9, + 27, + "growth experiments", + "experimental_method" + ], + [ + 88, + 95, + "glucose", + "chemical" + ], + [ + 180, + 192, + "carbohydrate", + "chemical" + ] + ] + }, + { + "sid": 133, + "sent": "Growth on glucose displayed the shortest lag time for each strain, and so lag times were normalized for each carbohydrate by subtracting the lag time of that strain in glucose (Fig.\u00a06; see Fig.\u00a0S8 in the supplemental material).", + "section": "RESULTS", + "ner": [ + [ + 10, + 17, + "glucose", + "chemical" + ], + [ + 41, + 49, + "lag time", + "evidence" + ], + [ + 74, + 83, + "lag times", + "evidence" + ], + [ + 109, + 121, + "carbohydrate", + "chemical" + ], + [ + 141, + 149, + "lag time", + "evidence" + ], + [ + 168, + 175, + "glucose", + "chemical" + ] + ] + }, + { + "sid": 134, + "sent": "A strain in which the entire XyGUL is deleted displays a lag of 24.5\u00a0h during growth on glucose compared to the isogenic parental wild-type (WT) \u0394tdk strain, for which exponential growth lags for 19.8\u00a0h (see Fig.\u00a0S8D).", + "section": "RESULTS", + "ner": [ + [ + 29, + 34, + "XyGUL", + "gene" + ], + [ + 38, + 45, + "deleted", + "experimental_method" + ], + [ + 57, + 60, + "lag", + "evidence" + ], + [ + 88, + 95, + "glucose", + "chemical" + ], + [ + 130, + 139, + "wild-type", + "protein_state" + ], + [ + 141, + 143, + "WT", + "protein_state" + ], + [ + 145, + 149, + "\u0394tdk", + "mutant" + ], + [ + 187, + 191, + "lags", + "evidence" + ] + ] + }, + { + "sid": 135, + "sent": "It is unknown whether this is because cultures were not normalized by the starting optical density (OD) or viable cells or reflects a minor defect for glucose utilization.", + "section": "RESULTS", + "ner": [ + [ + 151, + 158, + "glucose", + "chemical" + ] + ] + }, + { + "sid": 136, + "sent": "The former seems more likely as the growth rates are nearly identical for these strains on glucose and xylose.", + "section": "RESULTS", + "ner": [ + [ + 91, + 98, + "glucose", + "chemical" + ], + [ + 103, + 109, + "xylose", + "chemical" + ] + ] + }, + { + "sid": 137, + "sent": "The \u0394XyGUL and WT \u0394tdk strains display normalized lag times on xylose within experimental error, and curiously some of the mutant and complemented strains display a nominally shorter lag time on xylose than the WT \u0394tdk strain.", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "\u0394XyGUL", + "mutant" + ], + [ + 15, + 17, + "WT", + "protein_state" + ], + [ + 18, + 22, + "\u0394tdk", + "mutant" + ], + [ + 50, + 59, + "lag times", + "evidence" + ], + [ + 63, + 69, + "xylose", + "chemical" + ], + [ + 183, + 191, + "lag time", + "evidence" + ], + [ + 195, + 201, + "xylose", + "chemical" + ], + [ + 211, + 213, + "WT", + "protein_state" + ], + [ + 214, + 218, + "\u0394tdk", + "mutant" + ] + ] + }, + { + "sid": 138, + "sent": "Complementation of the \u0394SGBP-A strain (\u0394SGBP-A::SGBP-A) restores growth to wild-type rates on xyloglucan and XyGO1, yet the calculated rate of the complemented strain is ~72% that of the WT \u0394tdk strain on XyGO2; similar results were obtained for the SGBP-B complemented strain despite the fact that the growth curves do not appear much different (see Fig.\u00a0S8C and F).", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Complementation", + "experimental_method" + ], + [ + 23, + 30, + "\u0394SGBP-A", + "mutant" + ], + [ + 39, + 46, + "\u0394SGBP-A", + "mutant" + ], + [ + 48, + 54, + "SGBP-A", + "protein" + ], + [ + 75, + 84, + "wild-type", + "protein_state" + ], + [ + 94, + 104, + "xyloglucan", + "chemical" + ], + [ + 109, + 114, + "XyGO1", + "chemical" + ], + [ + 187, + 189, + "WT", + "protein_state" + ], + [ + 190, + 194, + "\u0394tdk", + "mutant" + ], + [ + 205, + 210, + "XyGO2", + "chemical" + ], + [ + 250, + 256, + "SGBP-B", + "protein" + ] + ] + }, + { + "sid": 139, + "sent": "The reason for this observation on XyGO2 is unclear, as the \u0394SGBP-B mutant does not have a significantly different growth rate from the WT on XyGO2.", + "section": "RESULTS", + "ner": [ + [ + 35, + 40, + "XyGO2", + "chemical" + ], + [ + 60, + 67, + "\u0394SGBP-B", + "mutant" + ], + [ + 68, + 74, + "mutant", + "protein_state" + ], + [ + 136, + 138, + "WT", + "protein_state" + ], + [ + 142, + 147, + "XyGO2", + "chemical" + ] + ] + }, + { + "sid": 140, + "sent": "Growth of select XyGUL mutants on xyloglucan and oligosaccharides.", + "section": "FIG", + "ner": [ + [ + 17, + 22, + "XyGUL", + "gene" + ], + [ + 34, + 44, + "xyloglucan", + "chemical" + ], + [ + 49, + 65, + "oligosaccharides", + "chemical" + ] + ] + }, + { + "sid": 141, + "sent": "B. ovatus mutants were created in a thymidine kinase deletion (\u0394tdk) mutant as described previously.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "B. ovatus", + "species" + ], + [ + 36, + 61, + "thymidine kinase deletion", + "mutant" + ], + [ + 63, + 67, + "\u0394tdk", + "mutant" + ] + ] + }, + { + "sid": 142, + "sent": "SGBP-A* denotes the Bacova_02651 (W82A W283A W306A) allele, and the GH9 gene is Bacova_02649.", + "section": "FIG", + "ner": [ + [ + 0, + 7, + "SGBP-A*", + "mutant" + ], + [ + 20, + 32, + "Bacova_02651", + "gene" + ], + [ + 34, + 38, + "W82A", + "mutant" + ], + [ + 39, + 44, + "W283A", + "mutant" + ], + [ + 45, + 50, + "W306A", + "mutant" + ], + [ + 68, + 71, + "GH9", + "protein" + ], + [ + 80, + 92, + "Bacova_02649", + "gene" + ] + ] + }, + { + "sid": 143, + "sent": "Growth was measured over time in minimal medium containing (A) XyG, (B) XyGO2, (C) XyGO1, (D) glucose, and (E) xylose.", + "section": "FIG", + "ner": [ + [ + 63, + 66, + "XyG", + "chemical" + ], + [ + 72, + 77, + "XyGO2", + "chemical" + ], + [ + 83, + 88, + "XyGO1", + "chemical" + ], + [ + 94, + 101, + "glucose", + "chemical" + ], + [ + 111, + 117, + "xylose", + "chemical" + ] + ] + }, + { + "sid": 144, + "sent": "In panel F, the growth rate of each strain on the five carbon sources is displayed, and in panel G, the normalized lag time of each culture, relative to its growth on glucose, is displayed.", + "section": "FIG", + "ner": [ + [ + 115, + 123, + "lag time", + "evidence" + ], + [ + 167, + 174, + "glucose", + "chemical" + ] + ] + }, + { + "sid": 145, + "sent": "Solid bars indicate conditions that are not statistically significant from the WT \u0394tdk cultures grown on the indicated carbohydrate, while open bars indicate a P value of <0.005 compared to the WT \u0394tdk strain.", + "section": "FIG", + "ner": [ + [ + 79, + 81, + "WT", + "protein_state" + ], + [ + 82, + 86, + "\u0394tdk", + "mutant" + ], + [ + 119, + 131, + "carbohydrate", + "chemical" + ], + [ + 194, + 196, + "WT", + "protein_state" + ], + [ + 197, + 201, + "\u0394tdk", + "mutant" + ] + ] + }, + { + "sid": 146, + "sent": "Conditions denoted by the same letter (b, c, or d) are not statistically significant from each other but are significantly different from the condition labeled \u201ca.\u201d Complementation of \u0394SGBP-A and \u0394SBGP-B was performed by allelic exchange of the wild-type genes back into the genome for expression via the native promoter: these growth curves, quantified rates and lag times are displayed in Fig.\u00a0S8 in the supplemental material.", + "section": "FIG", + "ner": [ + [ + 184, + 191, + "\u0394SGBP-A", + "mutant" + ], + [ + 196, + 203, + "\u0394SBGP-B", + "mutant" + ], + [ + 245, + 254, + "wild-type", + "protein_state" + ], + [ + 364, + 373, + "lag times", + "evidence" + ] + ] + }, + { + "sid": 147, + "sent": "The \u0394SGBP-A (\u0394Bacova_02651) strain (cf.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "\u0394SGBP-A", + "mutant" + ], + [ + 13, + 26, + "\u0394Bacova_02651", + "mutant" + ] + ] + }, + { + "sid": 148, + "sent": "Fig.\u00a01B) was completely incapable of growth on XyG, XyGO1, and XyGO2, indicating that SGBP-A is essential for XyG utilization (Fig.\u00a06).", + "section": "RESULTS", + "ner": [ + [ + 47, + 50, + "XyG", + "chemical" + ], + [ + 52, + 57, + "XyGO1", + "chemical" + ], + [ + 63, + 68, + "XyGO2", + "chemical" + ], + [ + 86, + 92, + "SGBP-A", + "protein" + ], + [ + 110, + 113, + "XyG", + "chemical" + ] + ] + }, + { + "sid": 149, + "sent": "This result mirrors our previous data for the canonical Sus of B.\u00a0thetaiotaomicron, which revealed that a homologous \u0394susD mutant is unable to grow on starch or malto-oligosaccharides, despite normal cell surface expression of all other PUL-encoded proteins.", + "section": "RESULTS", + "ner": [ + [ + 56, + 59, + "Sus", + "complex_assembly" + ], + [ + 63, + 82, + "B.\u00a0thetaiotaomicron", + "species" + ], + [ + 117, + 122, + "\u0394susD", + "mutant" + ], + [ + 123, + 129, + "mutant", + "protein_state" + ], + [ + 151, + 157, + "starch", + "chemical" + ], + [ + 161, + 183, + "malto-oligosaccharides", + "chemical" + ], + [ + 237, + 240, + "PUL", + "gene" + ] + ] + }, + { + "sid": 150, + "sent": "More recently, we demonstrated that this phenotype is due to the loss of the physical presence of SusD; complementation of \u0394susD with SusD*, a triple site-directed mutant (W96A W320A Y296A) that ablates glycan binding, restores B.\u00a0thetaiotaomicron growth on malto-oligosaccharides and starch when sus transcription is induced by maltose addition.", + "section": "RESULTS", + "ner": [ + [ + 98, + 102, + "SusD", + "protein" + ], + [ + 104, + 119, + "complementation", + "experimental_method" + ], + [ + 123, + 128, + "\u0394susD", + "mutant" + ], + [ + 134, + 139, + "SusD*", + "mutant" + ], + [ + 143, + 170, + "triple site-directed mutant", + "protein_state" + ], + [ + 172, + 176, + "W96A", + "mutant" + ], + [ + 177, + 182, + "W320A", + "mutant" + ], + [ + 183, + 188, + "Y296A", + "mutant" + ], + [ + 195, + 217, + "ablates glycan binding", + "protein_state" + ], + [ + 228, + 247, + "B.\u00a0thetaiotaomicron", + "species" + ], + [ + 258, + 280, + "malto-oligosaccharides", + "chemical" + ], + [ + 285, + 291, + "starch", + "chemical" + ], + [ + 297, + 300, + "sus", + "gene" + ], + [ + 329, + 336, + "maltose", + "chemical" + ] + ] + }, + { + "sid": 151, + "sent": "Similarly, the function of SGBP-A extends beyond glycan binding.", + "section": "RESULTS", + "ner": [ + [ + 27, + 33, + "SGBP-A", + "protein" + ], + [ + 49, + 55, + "glycan", + "chemical" + ] + ] + }, + { + "sid": 152, + "sent": "Complementation of \u0394SGBP-A with the SGBP-A* (W82A W283A W306A) variant, which does not bind XyG, supports growth on XyG and XyGOs (Fig.\u00a06; \u0394SGBP-A::SGBP-A*), with growth rates that are ~70% that of the WT.", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Complementation", + "experimental_method" + ], + [ + 19, + 26, + "\u0394SGBP-A", + "mutant" + ], + [ + 36, + 43, + "SGBP-A*", + "mutant" + ], + [ + 45, + 49, + "W82A", + "mutant" + ], + [ + 50, + 55, + "W283A", + "mutant" + ], + [ + 56, + 61, + "W306A", + "mutant" + ], + [ + 83, + 91, + "not bind", + "protein_state" + ], + [ + 92, + 95, + "XyG", + "chemical" + ], + [ + 116, + 119, + "XyG", + "chemical" + ], + [ + 124, + 129, + "XyGOs", + "chemical" + ], + [ + 139, + 146, + "\u0394SGBP-A", + "mutant" + ], + [ + 148, + 155, + "SGBP-A*", + "mutant" + ], + [ + 202, + 204, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 153, + "sent": "In previous studies, we observed that carbohydrate binding by SusD enhanced the sensitivity of the cells to limiting concentrations of malto-oligosaccharides by several orders of magnitude, such that the addition of 0.5\u00a0g/liter maltose was required to restore growth of the \u0394susD::SusD* strain on starch, which nonetheless occurred following an extended lag phase.", + "section": "RESULTS", + "ner": [ + [ + 38, + 50, + "carbohydrate", + "chemical" + ], + [ + 62, + 66, + "SusD", + "protein" + ], + [ + 228, + 235, + "maltose", + "chemical" + ], + [ + 274, + 279, + "\u0394susD", + "mutant" + ], + [ + 281, + 286, + "SusD*", + "mutant" + ], + [ + 297, + 303, + "starch", + "chemical" + ], + [ + 354, + 363, + "lag phase", + "evidence" + ] + ] + }, + { + "sid": 154, + "sent": "In contrast, the \u0394SGBP-A::SGBP-A* strain does not display an extended lag time on any of the xyloglucan substrates compared to the WT (Fig.\u00a06).", + "section": "RESULTS", + "ner": [ + [ + 17, + 24, + "\u0394SGBP-A", + "mutant" + ], + [ + 26, + 33, + "SGBP-A*", + "mutant" + ], + [ + 70, + 78, + "lag time", + "evidence" + ], + [ + 93, + 103, + "xyloglucan", + "chemical" + ], + [ + 131, + 133, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 155, + "sent": "The specific glycan signal that upregulates BoXyGUL is currently unknown.", + "section": "RESULTS", + "ner": [ + [ + 13, + 19, + "glycan", + "chemical" + ], + [ + 44, + 51, + "BoXyGUL", + "gene" + ] + ] + }, + { + "sid": 156, + "sent": "From our present data, we cannot eliminate the possibility that the glycan binding by SGBP-A enhances transcriptional activation of the XyGUL.", + "section": "RESULTS", + "ner": [ + [ + 68, + 74, + "glycan", + "chemical" + ], + [ + 86, + 92, + "SGBP-A", + "protein" + ], + [ + 136, + 141, + "XyGUL", + "gene" + ] + ] + }, + { + "sid": 157, + "sent": "However, the modest rate defect displayed by the SGBP-A::SGBP-A* strain suggests that recognition of XyG and product import is somewhat less efficient in these cells.", + "section": "RESULTS", + "ner": [ + [ + 49, + 55, + "SGBP-A", + "protein" + ], + [ + 57, + 64, + "SGBP-A*", + "mutant" + ], + [ + 101, + 104, + "XyG", + "chemical" + ] + ] + }, + { + "sid": 158, + "sent": "Intriguingly, the \u0394SGBP-B strain (\u0394Bacova_02650) (cf.", + "section": "RESULTS", + "ner": [ + [ + 18, + 25, + "\u0394SGBP-B", + "mutant" + ], + [ + 34, + 47, + "\u0394Bacova_02650", + "mutant" + ] + ] + }, + { + "sid": 159, + "sent": "Fig.\u00a01B) exhibited a minor growth defect on both XyG and XyGO2, with rates 84.6% and 93.9% that of the WT \u0394tdk strain.", + "section": "RESULTS", + "ner": [ + [ + 49, + 52, + "XyG", + "chemical" + ], + [ + 57, + 62, + "XyGO2", + "chemical" + ], + [ + 103, + 105, + "WT", + "protein_state" + ], + [ + 106, + 110, + "\u0394tdk", + "mutant" + ] + ] + }, + { + "sid": 160, + "sent": "However, growth of the \u0394SGBP-B strain on XyGO1 was 54.2% the rate of the parental strain, despite the fact that SGBP-B binds this substrate ca.", + "section": "RESULTS", + "ner": [ + [ + 23, + 30, + "\u0394SGBP-B", + "mutant" + ], + [ + 41, + 46, + "XyGO1", + "chemical" + ], + [ + 112, + 118, + "SGBP-B", + "protein" + ] + ] + }, + { + "sid": 161, + "sent": "10-fold more weakly than XyGO2 and XyG (Fig.\u00a06; Table\u00a01).", + "section": "RESULTS", + "ner": [ + [ + 25, + 30, + "XyGO2", + "chemical" + ], + [ + 35, + 38, + "XyG", + "chemical" + ] + ] + }, + { + "sid": 162, + "sent": "As such, the data suggest that SGBP-A can compensate for the loss of function of SGBP-B on longer oligo- and polysaccharides, while SGBP-B may adapt the cell to recognize smaller oligosaccharides efficiently.", + "section": "RESULTS", + "ner": [ + [ + 31, + 37, + "SGBP-A", + "protein" + ], + [ + 81, + 87, + "SGBP-B", + "protein" + ], + [ + 98, + 124, + "oligo- and polysaccharides", + "chemical" + ], + [ + 132, + 138, + "SGBP-B", + "protein" + ], + [ + 179, + 195, + "oligosaccharides", + "chemical" + ] + ] + }, + { + "sid": 163, + "sent": "Indeed, a double mutant, consisting of a crippled SGBP-A and a deletion of SGBP-B (\u0394SGBP-A::SGBP-A*/\u0394SGBP-B), exhibits an extended lag time on both XyG and XyGO2, as well as XyGO1.", + "section": "RESULTS", + "ner": [ + [ + 10, + 23, + "double mutant", + "protein_state" + ], + [ + 41, + 49, + "crippled", + "protein_state" + ], + [ + 50, + 56, + "SGBP-A", + "protein" + ], + [ + 63, + 74, + "deletion of", + "experimental_method" + ], + [ + 75, + 81, + "SGBP-B", + "protein" + ], + [ + 83, + 90, + "\u0394SGBP-A", + "mutant" + ], + [ + 92, + 99, + "SGBP-A*", + "mutant" + ], + [ + 100, + 107, + "\u0394SGBP-B", + "mutant" + ], + [ + 131, + 139, + "lag time", + "evidence" + ], + [ + 148, + 151, + "XyG", + "chemical" + ], + [ + 156, + 161, + "XyGO2", + "chemical" + ], + [ + 174, + 179, + "XyGO1", + "chemical" + ] + ] + }, + { + "sid": 164, + "sent": "Taken together, the data indicate that SGBP-A and SGBP-B functionally complement each other in the capture of XyG polysaccharide, while SGBP-B may allow B.\u00a0ovatus to scavenge smaller XyGOs liberated by other gut commensals.", + "section": "RESULTS", + "ner": [ + [ + 39, + 45, + "SGBP-A", + "protein" + ], + [ + 50, + 56, + "SGBP-B", + "protein" + ], + [ + 110, + 113, + "XyG", + "chemical" + ], + [ + 114, + 128, + "polysaccharide", + "chemical" + ], + [ + 136, + 142, + "SGBP-B", + "protein" + ], + [ + 153, + 162, + "B.\u00a0ovatus", + "species" + ], + [ + 183, + 188, + "XyGOs", + "chemical" + ] + ] + }, + { + "sid": 165, + "sent": "This additional role of SGBP-B is especially notable in the context of studies on BtSusE and BtSusF (positioned similarly in the archetypal Sus locus) (Fig.\u00a01B), for which growth defects on starch or malto-oligosaccharides have never been observed.", + "section": "RESULTS", + "ner": [ + [ + 24, + 30, + "SGBP-B", + "protein" + ], + [ + 82, + 88, + "BtSusE", + "protein" + ], + [ + 93, + 99, + "BtSusF", + "protein" + ], + [ + 140, + 149, + "Sus locus", + "gene" + ], + [ + 190, + 196, + "starch", + "chemical" + ], + [ + 200, + 222, + "malto-oligosaccharides", + "chemical" + ] + ] + }, + { + "sid": 166, + "sent": "Beyond SGBP-A and SGBP-B, we speculated that the catalytically feeble endo-xyloglucanase GH9, which is expendable for growth in the presence of GH5, might also play a role in glycan binding to the cell surface.", + "section": "RESULTS", + "ner": [ + [ + 7, + 13, + "SGBP-A", + "protein" + ], + [ + 18, + 24, + "SGBP-B", + "protein" + ], + [ + 49, + 69, + "catalytically feeble", + "protein_state" + ], + [ + 70, + 88, + "endo-xyloglucanase", + "protein_type" + ], + [ + 89, + 92, + "GH9", + "protein" + ], + [ + 144, + 147, + "GH5", + "protein" + ], + [ + 175, + 181, + "glycan", + "chemical" + ] + ] + }, + { + "sid": 167, + "sent": "However, combined deletion of the genes encoding GH9 (encoded by Bacova_02649) and SGBP-B does not exacerbate the growth defect on XyGO1 (Fig.\u00a06; \u0394SGBP-B/\u0394GH9).", + "section": "RESULTS", + "ner": [ + [ + 9, + 48, + "combined deletion of the genes encoding", + "experimental_method" + ], + [ + 49, + 52, + "GH9", + "protein" + ], + [ + 65, + 77, + "Bacova_02649", + "gene" + ], + [ + 83, + 89, + "SGBP-B", + "protein" + ], + [ + 131, + 136, + "XyGO1", + "chemical" + ], + [ + 146, + 153, + "\u0394SGBP-B", + "mutant" + ], + [ + 154, + 158, + "\u0394GH9", + "mutant" + ] + ] + }, + { + "sid": 168, + "sent": "The necessity of SGBP-B is elevated in the SGBP-A* strain, as the \u0394SGBP-A::SGBP-A*/ \u0394SGBP-B mutant displays an extended lag during growth on XyG and xylogluco-oligosaccharides, while growth rate differences are more subtle.", + "section": "RESULTS", + "ner": [ + [ + 17, + 23, + "SGBP-B", + "protein" + ], + [ + 43, + 50, + "SGBP-A*", + "mutant" + ], + [ + 66, + 73, + "\u0394SGBP-A", + "mutant" + ], + [ + 75, + 82, + "SGBP-A*", + "mutant" + ], + [ + 84, + 91, + "\u0394SGBP-B", + "mutant" + ], + [ + 92, + 98, + "mutant", + "protein_state" + ], + [ + 120, + 123, + "lag", + "evidence" + ], + [ + 141, + 144, + "XyG", + "chemical" + ], + [ + 149, + 175, + "xylogluco-oligosaccharides", + "chemical" + ] + ] + }, + { + "sid": 169, + "sent": "The precise reason for this lag is unclear, but recapitulating our findings on the role of SusD in malto-oligosaccharide sensing in B.\u00a0thetaiotaomicron, this extended lag may be due to inefficient import and thus sensing of xyloglucan in the environment in the absence of glycan binding by essential SGBPs.", + "section": "RESULTS", + "ner": [ + [ + 28, + 31, + "lag", + "evidence" + ], + [ + 91, + 95, + "SusD", + "protein" + ], + [ + 99, + 120, + "malto-oligosaccharide", + "chemical" + ], + [ + 132, + 151, + "B.\u00a0thetaiotaomicron", + "species" + ], + [ + 167, + 170, + "lag", + "evidence" + ], + [ + 224, + 234, + "xyloglucan", + "chemical" + ], + [ + 272, + 278, + "glycan", + "chemical" + ], + [ + 300, + 305, + "SGBPs", + "protein_type" + ] + ] + }, + { + "sid": 170, + "sent": "Our previous work demonstrates that B.\u00a0ovatus cells grown in minimal medium plus glucose express low levels of the XyGUL transcript.", + "section": "RESULTS", + "ner": [ + [ + 36, + 45, + "B.\u00a0ovatus", + "species" + ], + [ + 81, + 88, + "glucose", + "chemical" + ], + [ + 115, + 120, + "XyGUL", + "gene" + ] + ] + }, + { + "sid": 171, + "sent": "Thus, in our experiments, we presume that each strain, initially grown in glucose, expresses low levels of the XyGUL transcript and thus low levels of the XyGUL-encoded surface proteins, including the vanguard GH5.", + "section": "RESULTS", + "ner": [ + [ + 74, + 81, + "glucose", + "chemical" + ], + [ + 111, + 116, + "XyGUL", + "gene" + ], + [ + 155, + 160, + "XyGUL", + "gene" + ], + [ + 210, + 213, + "GH5", + "protein" + ] + ] + }, + { + "sid": 172, + "sent": "Presumably without glycan binding by the SGBPs, the GH5 protein cannot efficiently process xyloglucan, and/or the lack of SGBP function prevents efficient capture and import of the processed oligosaccharides.", + "section": "RESULTS", + "ner": [ + [ + 19, + 25, + "glycan", + "chemical" + ], + [ + 41, + 46, + "SGBPs", + "protein_type" + ], + [ + 52, + 55, + "GH5", + "protein" + ], + [ + 91, + 101, + "xyloglucan", + "chemical" + ], + [ + 122, + 126, + "SGBP", + "protein_type" + ], + [ + 191, + 207, + "oligosaccharides", + "chemical" + ] + ] + }, + { + "sid": 173, + "sent": "It may then be that only after a sufficient amount of glycan is processed and imported by the cell is XyGUL upregulated and exponential growth on the glycan can begin.", + "section": "RESULTS", + "ner": [ + [ + 54, + 60, + "glycan", + "chemical" + ], + [ + 102, + 107, + "XyGUL", + "gene" + ], + [ + 150, + 156, + "glycan", + "chemical" + ] + ] + }, + { + "sid": 174, + "sent": "We hypothesize that during exponential growth the essential role of SGBP-A extends beyond glycan recognition, perhaps due to a critical interaction with the TBDT.", + "section": "RESULTS", + "ner": [ + [ + 68, + 74, + "SGBP-A", + "protein" + ], + [ + 90, + 96, + "glycan", + "chemical" + ], + [ + 157, + 161, + "TBDT", + "protein_type" + ] + ] + }, + { + "sid": 175, + "sent": "In the BtSus, SusD and the TBDT SusC interact, and we speculate that this interaction is necessary for glycan uptake, as suggested by the fact that a \u0394susD mutant cannot grow on starch, but a \u0394susD::SusD* strain regains this ability if a transcriptional activator of the sus operon is supplied.", + "section": "RESULTS", + "ner": [ + [ + 7, + 12, + "BtSus", + "gene" + ], + [ + 14, + 18, + "SusD", + "protein" + ], + [ + 27, + 31, + "TBDT", + "protein_type" + ], + [ + 32, + 36, + "SusC", + "protein" + ], + [ + 103, + 109, + "glycan", + "chemical" + ], + [ + 150, + 155, + "\u0394susD", + "mutant" + ], + [ + 156, + 162, + "mutant", + "protein_state" + ], + [ + 178, + 184, + "starch", + "chemical" + ], + [ + 192, + 197, + "\u0394susD", + "mutant" + ], + [ + 199, + 204, + "SusD*", + "mutant" + ], + [ + 238, + 263, + "transcriptional activator", + "protein_type" + ], + [ + 271, + 281, + "sus operon", + "gene" + ] + ] + }, + { + "sid": 176, + "sent": "Likewise, such cognate interactions between homologous protein pairs such as SGBP-A and its TBDT may underlie our observation that a \u0394SGBP-A mutant cannot grow on xyloglucan.", + "section": "RESULTS", + "ner": [ + [ + 77, + 83, + "SGBP-A", + "protein" + ], + [ + 92, + 96, + "TBDT", + "protein_type" + ], + [ + 133, + 140, + "\u0394SGBP-A", + "mutant" + ], + [ + 141, + 147, + "mutant", + "protein_state" + ], + [ + 163, + 173, + "xyloglucan", + "chemical" + ] + ] + }, + { + "sid": 177, + "sent": "However, unlike the Sus, in which elimination of SusE and SusF does not affect growth on starch, SGBP-B appears to have a dedicated role in growth on small xylogluco-oligosaccharides.", + "section": "RESULTS", + "ner": [ + [ + 20, + 23, + "Sus", + "complex_assembly" + ], + [ + 34, + 48, + "elimination of", + "experimental_method" + ], + [ + 49, + 53, + "SusE", + "protein" + ], + [ + 58, + 62, + "SusF", + "protein" + ], + [ + 89, + 95, + "starch", + "chemical" + ], + [ + 97, + 103, + "SGBP-B", + "protein" + ], + [ + 156, + 182, + "xylogluco-oligosaccharides", + "chemical" + ] + ] + }, + { + "sid": 178, + "sent": "The ability of gut-adapted microorganisms to thrive in the gastrointestinal tract is critically dependent upon their ability to efficiently recognize, cleave, and import glycans.", + "section": "RESULTS", + "ner": [ + [ + 27, + 41, + "microorganisms", + "taxonomy_domain" + ], + [ + 170, + 177, + "glycans", + "chemical" + ] + ] + }, + { + "sid": 179, + "sent": "The human gut, in particular, is a densely packed ecosystem with hundreds of species, in which there is potential for both competition and synergy in the utilization of different substrates.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "human", + "species" + ] + ] + }, + { + "sid": 180, + "sent": "Recent work has elucidated that Bacteroidetes cross-feed during growth on many glycans; the glycoside hydrolases expressed by one species liberate oligosaccharides for consumption by other members of the community.", + "section": "RESULTS", + "ner": [ + [ + 32, + 45, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 79, + 86, + "glycans", + "chemical" + ], + [ + 92, + 112, + "glycoside hydrolases", + "protein_type" + ], + [ + 147, + 163, + "oligosaccharides", + "chemical" + ] + ] + }, + { + "sid": 181, + "sent": "Thus, understanding glycan capture at the cell surface is fundamental to explaining, and eventually predicting, how the carbohydrate content of the diet shapes the gut community structure as well as its causative health effects.", + "section": "RESULTS", + "ner": [ + [ + 20, + 26, + "glycan", + "chemical" + ] + ] + }, + { + "sid": 182, + "sent": "Here, we demonstrate that the surface glycan binding proteins encoded within the BoXyGUL play unique and essential roles in the acquisition of the ubiquitous and abundant vegetable polysaccharide xyloglucan.", + "section": "RESULTS", + "ner": [ + [ + 30, + 61, + "surface glycan binding proteins", + "protein_type" + ], + [ + 81, + 88, + "BoXyGUL", + "gene" + ], + [ + 171, + 180, + "vegetable", + "taxonomy_domain" + ], + [ + 181, + 195, + "polysaccharide", + "chemical" + ], + [ + 196, + 206, + "xyloglucan", + "chemical" + ] + ] + }, + { + "sid": 183, + "sent": "Yet, a number of questions remain regarding the molecular interplay of SGBPs with their cotranscribed cohort of glycoside hydrolases and TonB-dependent transporters.", + "section": "RESULTS", + "ner": [ + [ + 71, + 76, + "SGBPs", + "protein_type" + ], + [ + 112, + 132, + "glycoside hydrolases", + "protein_type" + ], + [ + 137, + 164, + "TonB-dependent transporters", + "protein_type" + ] + ] + }, + { + "sid": 184, + "sent": "A particularly understudied aspect of glycan utilization is the mechanism of import via TBDTs (SusC homologs) (Fig.\u00a01), which are ubiquitous and defining components of all PUL.", + "section": "RESULTS", + "ner": [ + [ + 38, + 44, + "glycan", + "chemical" + ], + [ + 88, + 93, + "TBDTs", + "protein_type" + ], + [ + 95, + 99, + "SusC", + "protein" + ], + [ + 172, + 175, + "PUL", + "gene" + ] + ] + }, + { + "sid": 185, + "sent": "PUL-encoded TBDTs in Bacteroidetes are larger than the well-characterized iron-targeting TBDTs from many Proteobacteria and are further distinguished as the only known glycan-importing TBDTs coexpressed with an SGBP.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "PUL", + "gene" + ], + [ + 12, + 17, + "TBDTs", + "protein_type" + ], + [ + 21, + 34, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 74, + 94, + "iron-targeting TBDTs", + "protein_type" + ], + [ + 105, + 119, + "Proteobacteria", + "taxonomy_domain" + ], + [ + 168, + 190, + "glycan-importing TBDTs", + "protein_type" + ], + [ + 211, + 215, + "SGBP", + "protein_type" + ] + ] + }, + { + "sid": 186, + "sent": "A direct interaction between the BtSusC TBDT and the SusD SGBP has been previously demonstrated, as has an interaction between the homologous components encoded by an N-glycan-scavenging PUL of Capnocytophaga canimorsus.", + "section": "RESULTS", + "ner": [ + [ + 33, + 39, + "BtSusC", + "protein" + ], + [ + 40, + 44, + "TBDT", + "protein_type" + ], + [ + 53, + 57, + "SusD", + "protein" + ], + [ + 58, + 62, + "SGBP", + "protein_type" + ], + [ + 169, + 175, + "glycan", + "chemical" + ], + [ + 187, + 190, + "PUL", + "gene" + ], + [ + 194, + 219, + "Capnocytophaga canimorsus", + "species" + ] + ] + }, + { + "sid": 187, + "sent": "Our observation here that the physical presence of the SusD homolog SGBP-A, independent of XyG-binding ability, is both necessary and sufficient for XyG utilization further supports a model of glycan import whereby the SusC-like TBDTs and the SusD-like SGBPs must be intimately associated to support glycan uptake (Fig.\u00a01C).", + "section": "RESULTS", + "ner": [ + [ + 55, + 59, + "SusD", + "protein" + ], + [ + 68, + 74, + "SGBP-A", + "protein" + ], + [ + 91, + 94, + "XyG", + "chemical" + ], + [ + 149, + 152, + "XyG", + "chemical" + ], + [ + 193, + 199, + "glycan", + "chemical" + ], + [ + 219, + 234, + "SusC-like TBDTs", + "protein_type" + ], + [ + 243, + 258, + "SusD-like SGBPs", + "protein_type" + ], + [ + 300, + 306, + "glycan", + "chemical" + ] + ] + }, + { + "sid": 188, + "sent": "It is yet presently unclear whether this interaction is static or dynamic and to what extent the association of cognate TBDT/SGBPs is dependent upon the structure of the carbohydrate to be imported.", + "section": "RESULTS", + "ner": [ + [ + 120, + 124, + "TBDT", + "protein_type" + ], + [ + 125, + 130, + "SGBPs", + "protein_type" + ], + [ + 170, + 182, + "carbohydrate", + "chemical" + ] + ] + }, + { + "sid": 189, + "sent": "On the other hand, there is clear evidence for independent TBDTs in Bacteroidetes that do not require SGBP association for activity.", + "section": "RESULTS", + "ner": [ + [ + 59, + 64, + "TBDTs", + "protein_type" + ], + [ + 68, + 81, + "Bacteroidetes", + "taxonomy_domain" + ], + [ + 102, + 106, + "SGBP", + "protein_type" + ] + ] + }, + { + "sid": 190, + "sent": "For example, it was recently demonstrated that expression of nanO, which encodes a SusC-like TBDT as part of a sialic-acid-targeting PUL from B.\u00a0fragilis, restored growth on this monosaccharide in a mutant strain of E.\u00a0coli.", + "section": "RESULTS", + "ner": [ + [ + 61, + 65, + "nanO", + "gene" + ], + [ + 83, + 97, + "SusC-like TBDT", + "protein_type" + ], + [ + 133, + 136, + "PUL", + "gene" + ], + [ + 142, + 153, + "B.\u00a0fragilis", + "species" + ], + [ + 179, + 193, + "monosaccharide", + "chemical" + ], + [ + 216, + 223, + "E.\u00a0coli", + "species" + ] + ] + }, + { + "sid": 191, + "sent": "In this instance, coexpression of the susD-like gene nanU was not required, nor did the expression of the nanU gene enhance growth kinetics.", + "section": "RESULTS", + "ner": [ + [ + 38, + 42, + "susD", + "gene" + ], + [ + 53, + 57, + "nanU", + "gene" + ], + [ + 106, + 110, + "nanU", + "gene" + ] + ] + }, + { + "sid": 192, + "sent": "Similarly, the deletion of BT1762 encoding a fructan-targeting SusD-like protein in B.\u00a0thetaiotaomicron did not result in a dramatic loss of growth on fructans.", + "section": "RESULTS", + "ner": [ + [ + 27, + 33, + "BT1762", + "gene" + ], + [ + 45, + 80, + "fructan-targeting SusD-like protein", + "protein_type" + ], + [ + 84, + 103, + "B.\u00a0thetaiotaomicron", + "species" + ], + [ + 151, + 159, + "fructans", + "chemical" + ] + ] + }, + { + "sid": 193, + "sent": "Thus, the strict dependence on a SusD-like SGBP for glycan uptake in the Bacteroidetes may be variable and substrate dependent.", + "section": "RESULTS", + "ner": [ + [ + 33, + 47, + "SusD-like SGBP", + "protein_type" + ], + [ + 52, + 58, + "glycan", + "chemical" + ], + [ + 73, + 86, + "Bacteroidetes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 194, + "sent": "Furthermore, considering the broader distribution of TBDTs in PUL lacking SGBPs (sometimes known as carbohydrate utilization containing TBDT [CUT] loci; see reference and reviewed in reference) across bacterial phyla, it appears that the intimate biophysical association of these substrate-transport and -binding proteins is the result of specific evolution within the Bacteroidetes.", + "section": "RESULTS", + "ner": [ + [ + 53, + 58, + "TBDTs", + "protein_type" + ], + [ + 62, + 65, + "PUL", + "gene" + ], + [ + 74, + 79, + "SGBPs", + "protein_type" + ], + [ + 100, + 151, + "carbohydrate utilization containing TBDT [CUT] loci", + "gene" + ], + [ + 201, + 210, + "bacterial", + "taxonomy_domain" + ], + [ + 369, + 382, + "Bacteroidetes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 195, + "sent": "Equally intriguing is the observation that while SusD-like proteins such as SGBP-A share moderate primary and high tertiary structural conservation, the genes for the SGBPs encoded immediately downstream (Fig.\u00a01B [sometimes referred to as \u201csusE positioned\u201d]) encode glycan-binding lipoproteins with little or no sequence or structural conservation, even among syntenic PUL that target the same polysaccharide.", + "section": "RESULTS", + "ner": [ + [ + 49, + 67, + "SusD-like proteins", + "protein_type" + ], + [ + 76, + 82, + "SGBP-A", + "protein" + ], + [ + 167, + 172, + "SGBPs", + "protein_type" + ], + [ + 266, + 293, + "glycan-binding lipoproteins", + "protein_type" + ], + [ + 369, + 372, + "PUL", + "gene" + ], + [ + 394, + 408, + "polysaccharide", + "chemical" + ] + ] + }, + { + "sid": 196, + "sent": "Such is the case for XyGUL from related Bacteroides species, which may encode either one or two of these predicted SGBPs, and these proteins vary considerably in length.", + "section": "RESULTS", + "ner": [ + [ + 21, + 26, + "XyGUL", + "gene" + ], + [ + 40, + 51, + "Bacteroides", + "taxonomy_domain" + ], + [ + 115, + 120, + "SGBPs", + "protein_type" + ] + ] + }, + { + "sid": 197, + "sent": "The extremely low similarity of these SGBPs is striking in light of the moderate sequence conservation observed among homologous GHs in syntenic PUL.", + "section": "RESULTS", + "ner": [ + [ + 38, + 43, + "SGBPs", + "protein_type" + ], + [ + 129, + 132, + "GHs", + "protein_type" + ], + [ + 145, + 148, + "PUL", + "gene" + ] + ] + }, + { + "sid": 198, + "sent": "This, together with the observation that these SGBPs, as exemplified by BtSusE and BtSusF and the XyGUL SGBP-B of the present study, are expendable for polysaccharide growth, implies a high degree of evolutionary flexibility to enhance glycan capture at the cell surface.", + "section": "RESULTS", + "ner": [ + [ + 47, + 52, + "SGBPs", + "protein_type" + ], + [ + 72, + 78, + "BtSusE", + "protein" + ], + [ + 83, + 89, + "BtSusF", + "protein" + ], + [ + 98, + 103, + "XyGUL", + "gene" + ], + [ + 104, + 110, + "SGBP-B", + "protein" + ], + [ + 152, + 166, + "polysaccharide", + "chemical" + ], + [ + 236, + 242, + "glycan", + "chemical" + ] + ] + }, + { + "sid": 199, + "sent": "Because the intestinal ecosystem is a dense consortium of bacteria that must compete for their nutrients, these multimodular SGBPs may reflect ongoing evolutionary experiments to enhance glycan uptake efficiency.", + "section": "RESULTS", + "ner": [ + [ + 58, + 66, + "bacteria", + "taxonomy_domain" + ], + [ + 125, + 130, + "SGBPs", + "protein_type" + ], + [ + 187, + 193, + "glycan", + "chemical" + ] + ] + }, + { + "sid": 200, + "sent": "Whether organisms that express longer SGBPs, extending further above the cell surface toward the extracellular environment, are better equipped to compete for available carbohydrates is presently unknown.", + "section": "RESULTS", + "ner": [ + [ + 38, + 43, + "SGBPs", + "protein_type" + ], + [ + 169, + 182, + "carbohydrates", + "chemical" + ] + ] + }, + { + "sid": 201, + "sent": "However, the natural diversity of these proteins represents a rich source for the discovery of unique carbohydrate-binding motifs to both inform gut microbiology and generate new, specific carbohydrate analytical reagents.", + "section": "RESULTS", + "ner": [ + [ + 102, + 129, + "carbohydrate-binding motifs", + "structure_element" + ], + [ + 189, + 201, + "carbohydrate", + "chemical" + ] + ] + }, + { + "sid": 202, + "sent": "In conclusion, the present study further illuminates the essential role that surface-glycan binding proteins play in facilitating the catabolism of complex dietary carbohydrates by Bacteroidetes.", + "section": "RESULTS", + "ner": [ + [ + 77, + 108, + "surface-glycan binding proteins", + "protein_type" + ], + [ + 164, + 177, + "carbohydrates", + "chemical" + ], + [ + 181, + 194, + "Bacteroidetes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 203, + "sent": "The ability of our resident gut bacteria to recognize polysaccharides is the first committed step of glycan consumption by these organisms, a critical process that influences the community structure and thus the metabolic output (i.e., short-chain fatty acid and metabolite profile) of these organisms.", + "section": "RESULTS", + "ner": [ + [ + 32, + 40, + "bacteria", + "taxonomy_domain" + ], + [ + 54, + 69, + "polysaccharides", + "chemical" + ], + [ + 101, + 107, + "glycan", + "chemical" + ] + ] + }, + { + "sid": 204, + "sent": "A molecular understanding of glycan uptake by human gut bacteria is therefore central to the development of strategies to improve human health through manipulation of the microbiota.", + "section": "RESULTS", + "ner": [ + [ + 29, + 35, + "glycan", + "chemical" + ], + [ + 46, + 51, + "human", + "species" + ], + [ + 56, + 64, + "bacteria", + "taxonomy_domain" + ], + [ + 130, + 135, + "human", + "species" + ], + [ + 171, + 181, + "microbiota", + "taxonomy_domain" + ] + ] + } + ] + }, + "PMC4869123": { + "annotations": [ + { + "sid": 0, + "sent": "Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide", + "section": "TITLE", + "ner": [ + [ + 19, + 25, + "IL-17A", + "protein" + ], + [ + 30, + 37, + "IL-17RA", + "protein" + ], + [ + 65, + 72, + "peptide", + "chemical" + ] + ] + }, + { + "sid": 1, + "sent": "IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 6, + "IL-17A", + "protein" + ], + [ + 29, + 37, + "cytokine", + "protein_type" + ] + ] + }, + { + "sid": 2, + "sent": "Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking.", + "section": "ABSTRACT", + "ner": [ + [ + 11, + 21, + "antibodies", + "protein_type" + ], + [ + 33, + 39, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 3, + "sent": "A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions.", + "section": "ABSTRACT", + "ner": [ + [ + 2, + 41, + "high affinity IL-17A peptide antagonist", + "chemical" + ], + [ + 43, + 46, + "HAP", + "chemical" + ], + [ + 51, + 62, + "15 residues", + "residue_range" + ], + [ + 86, + 109, + "phage-display screening", + "experimental_method" + ], + [ + 122, + 157, + "saturation mutagenesis optimization", + "experimental_method" + ], + [ + 162, + 186, + "amino acid substitutions", + "experimental_method" + ] + ] + }, + { + "sid": 4, + "sent": "HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 3, + "HAP", + "chemical" + ], + [ + 26, + 32, + "IL-17A", + "protein" + ], + [ + 69, + 77, + "cytokine", + "protein_type" + ], + [ + 87, + 95, + "receptor", + "protein_type" + ], + [ + 97, + 104, + "IL-17RA", + "protein" + ] + ] + }, + { + "sid": 5, + "sent": "Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines.", + "section": "ABSTRACT", + "ner": [ + [ + 18, + 23, + "human", + "species" + ], + [ + 31, + 34, + "HAP", + "chemical" + ], + [ + 83, + 92, + "cytokines", + "protein_type" + ] + ] + }, + { + "sid": 6, + "sent": "Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 25, + "Crystal structure studies", + "experimental_method" + ], + [ + 44, + 47, + "HAP", + "chemical" + ], + [ + 70, + 76, + "IL-17A", + "protein" + ], + [ + 77, + 82, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 7, + "sent": "The N-terminal portions of HAP form a \u03b2-strand that inserts between two IL-17A monomers while the C-terminal section forms an \u03b1 helix that directly blocks IL-17RA from binding to the same region of IL-17A.", + "section": "ABSTRACT", + "ner": [ + [ + 27, + 30, + "HAP", + "chemical" + ], + [ + 38, + 46, + "\u03b2-strand", + "structure_element" + ], + [ + 72, + 78, + "IL-17A", + "protein" + ], + [ + 79, + 87, + "monomers", + "oligomeric_state" + ], + [ + 126, + 133, + "\u03b1 helix", + "structure_element" + ], + [ + 155, + 162, + "IL-17RA", + "protein" + ], + [ + 198, + 204, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 8, + "sent": "The family of IL-17 cytokines and receptors consists of six polypeptides, IL-17A-F, and five receptors, IL-17RA-E. IL-17A is secreted from activated Th17 cells, and several innate immune T cell types including macrophages, neutrophils, natural killer cells, and dendritic cells.", + "section": "INTRO", + "ner": [ + [ + 14, + 29, + "IL-17 cytokines", + "protein_type" + ], + [ + 74, + 82, + "IL-17A-F", + "protein" + ], + [ + 104, + 113, + "IL-17RA-E", + "protein" + ], + [ + 115, + 121, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 9, + "sent": "IL-17A signals through a specific cell surface receptor complex which consists of IL-17RA and IL-17RC.", + "section": "INTRO", + "ner": [ + [ + 0, + 6, + "IL-17A", + "protein" + ], + [ + 47, + 55, + "receptor", + "protein_type" + ], + [ + 82, + 89, + "IL-17RA", + "protein" + ], + [ + 94, + 101, + "IL-17RC", + "protein" + ] + ] + }, + { + "sid": 10, + "sent": "IL-17A\u2019s downstream signaling leads to increased production of inflammatory cytokines such as IL-6, IL-8, CCL-20 and CXCL1 by various mechanisms including stimulation of transcription and stabilization of mRNA.", + "section": "INTRO", + "ner": [ + [ + 0, + 6, + "IL-17A", + "protein" + ], + [ + 76, + 85, + "cytokines", + "protein_type" + ], + [ + 94, + 98, + "IL-6", + "protein_type" + ], + [ + 100, + 104, + "IL-8", + "protein_type" + ], + [ + 106, + 112, + "CCL-20", + "protein_type" + ], + [ + 117, + 122, + "CXCL1", + "protein_type" + ], + [ + 205, + 209, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 11, + "sent": "Although various cell types have been reported to express IL-17RA, the highest responses to IL-17A come from epithelial cells, endothelial cells, keratinocytes and fibroblasts.", + "section": "INTRO", + "ner": [ + [ + 58, + 65, + "IL-17RA", + "protein" + ], + [ + 92, + 98, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 12, + "sent": "IL-17A and its signaling is important in host defense against certain fungal and bacterial infections as demonstrated by patients with autoantibodies against IL-17A and IL-17F, or with inborn errors of IL-17 immunity.", + "section": "INTRO", + "ner": [ + [ + 0, + 6, + "IL-17A", + "protein" + ], + [ + 158, + 164, + "IL-17A", + "protein" + ], + [ + 169, + 175, + "IL-17F", + "protein" + ], + [ + 202, + 207, + "IL-17", + "protein_type" + ] + ] + }, + { + "sid": 13, + "sent": "In addition to its physiological role, IL-17A is a key pathogenic factor in inflammatory and autoimmune diseases.", + "section": "INTRO", + "ner": [ + [ + 39, + 45, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 14, + "sent": "In phase II and III clinical trials, neutralizing monoclonal antibodies against IL-17A (secukinumab and ixekizumab) or its receptor IL-17RA (brodalumab) are highly efficacious in treating moderate to severe plaque psoriasis and psoriatic arthritis.", + "section": "INTRO", + "ner": [ + [ + 61, + 71, + "antibodies", + "protein_type" + ], + [ + 80, + 86, + "IL-17A", + "protein" + ], + [ + 88, + 99, + "secukinumab", + "chemical" + ], + [ + 104, + 114, + "ixekizumab", + "chemical" + ], + [ + 123, + 131, + "receptor", + "protein_type" + ], + [ + 132, + 139, + "IL-17RA", + "protein" + ], + [ + 141, + 151, + "brodalumab", + "chemical" + ] + ] + }, + { + "sid": 15, + "sent": "Secukinumab has been approved recently as a new psoriasis drug by the US Food and Drug Administration (Cosentyx\u2122).", + "section": "INTRO", + "ner": [ + [ + 0, + 11, + "Secukinumab", + "chemical" + ], + [ + 103, + 112, + "Cosentyx\u2122", + "chemical" + ] + ] + }, + { + "sid": 16, + "sent": "In addition to psoriasis and psoriatic arthritis, IL-17A blockade has also shown preclinical and clinical efficacies in ankylosing spondylitis and rheumatoid arthritis.", + "section": "INTRO", + "ner": [ + [ + 50, + 56, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 17, + "sent": "Among IL-17 cytokines, IL-17A and IL-17F share the highest homology.", + "section": "INTRO", + "ner": [ + [ + 6, + 21, + "IL-17 cytokines", + "protein_type" + ], + [ + 23, + 29, + "IL-17A", + "protein" + ], + [ + 34, + 40, + "IL-17F", + "protein" + ] + ] + }, + { + "sid": 18, + "sent": "These polypeptides form covalent homodimers, and IL-17A and IL-17F also form an IL-17A/IL-17F hetereodimer.", + "section": "INTRO", + "ner": [ + [ + 24, + 32, + "covalent", + "protein_state" + ], + [ + 33, + 43, + "homodimers", + "oligomeric_state" + ], + [ + 49, + 55, + "IL-17A", + "protein" + ], + [ + 60, + 66, + "IL-17F", + "protein" + ], + [ + 80, + 93, + "IL-17A/IL-17F", + "complex_assembly" + ], + [ + 94, + 106, + "hetereodimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 19, + "sent": "Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA.", + "section": "INTRO", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 25, + 28, + "apo", + "protein_state" + ], + [ + 29, + 35, + "IL-17F", + "protein" + ], + [ + 44, + 56, + "complex with", + "protein_state" + ], + [ + 57, + 64, + "IL-17RA", + "protein" + ], + [ + 70, + 73, + "apo", + "protein_state" + ], + [ + 74, + 80, + "IL-17A", + "protein" + ], + [ + 86, + 98, + "complex with", + "protein_state" + ], + [ + 102, + 110, + "antibody", + "protein_type" + ], + [ + 111, + 114, + "Fab", + "structure_element" + ], + [ + 124, + 136, + "complex with", + "protein_state" + ], + [ + 137, + 144, + "IL-17RA", + "protein" + ] + ] + }, + { + "sid": 20, + "sent": "In these structures, both IL-17A and IL-17F adopt a cysteine-knot fold with two intramolecular disulfides and two interchain disulfide bonds that covalently link two monomers.", + "section": "INTRO", + "ner": [ + [ + 9, + 19, + "structures", + "evidence" + ], + [ + 26, + 32, + "IL-17A", + "protein" + ], + [ + 37, + 43, + "IL-17F", + "protein" + ], + [ + 52, + 65, + "cysteine-knot", + "structure_element" + ], + [ + 95, + 105, + "disulfides", + "ptm" + ], + [ + 125, + 140, + "disulfide bonds", + "ptm" + ], + [ + 166, + 174, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 21, + "sent": "There has been active research in identifying orally available chemical entities that would functionally antagonize IL-17A-mediated signaling.", + "section": "INTRO", + "ner": [ + [ + 116, + 122, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 22, + "sent": "Developing small molecules targeting protein-protein interactions is difficult with particular challenges associated with the large, shallow IL-17A/IL-17RA interfaces.", + "section": "INTRO", + "ner": [ + [ + 141, + 166, + "IL-17A/IL-17RA interfaces", + "site" + ] + ] + }, + { + "sid": 23, + "sent": "Since IL-17RA is a shared receptor for at least IL-17A, IL-17F, IL-17A/IL-17F and IL-17E, we chose to seek IL-17A-specific inhibitors that may have more defined pharmacological responses than IL-17RA inhibitors.", + "section": "INTRO", + "ner": [ + [ + 6, + 13, + "IL-17RA", + "protein" + ], + [ + 26, + 34, + "receptor", + "protein_type" + ], + [ + 48, + 54, + "IL-17A", + "protein" + ], + [ + 56, + 62, + "IL-17F", + "protein" + ], + [ + 64, + 77, + "IL-17A/IL-17F", + "complex_assembly" + ], + [ + 82, + 88, + "IL-17E", + "protein" + ], + [ + 107, + 113, + "IL-17A", + "protein" + ], + [ + 192, + 199, + "IL-17RA", + "protein" + ] + ] + }, + { + "sid": 24, + "sent": "Our efforts resulted in discovery of a high affinity IL-17A peptide antagonist (HAP), which we attempted to increase the functional production and pharmacokinetics after fusing HAP to antibodies for evaluation as a bispecific therapeutic in animal studies.", + "section": "INTRO", + "ner": [ + [ + 39, + 78, + "high affinity IL-17A peptide antagonist", + "chemical" + ], + [ + 80, + 83, + "HAP", + "chemical" + ], + [ + 170, + 176, + "fusing", + "experimental_method" + ], + [ + 177, + 180, + "HAP", + "chemical" + ], + [ + 184, + 194, + "antibodies", + "protein_type" + ] + ] + }, + { + "sid": 25, + "sent": "Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR).", + "section": "INTRO", + "ner": [ + [ + 63, + 71, + "uncapped", + "protein_state" + ], + [ + 72, + 75, + "HAP", + "chemical" + ], + [ + 167, + 170, + "HAP", + "chemical" + ], + [ + 186, + 203, + "complex structure", + "evidence" + ], + [ + 207, + 213, + "IL-17A", + "protein" + ], + [ + 219, + 222, + "HAP", + "chemical" + ], + [ + 274, + 294, + "peptide optimization", + "experimental_method" + ], + [ + 299, + 330, + "structure activity relationship", + "experimental_method" + ], + [ + 332, + 335, + "SAR", + "experimental_method" + ] + ] + }, + { + "sid": 26, + "sent": "Identification of IL-17A peptide inhibitors", + "section": "RESULTS", + "ner": [ + [ + 18, + 24, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 27, + "sent": "Peptides specifically binding to human IL-17A were identified from phage panning using cyclic and linear peptide libraries (Supplementary Figure S1).", + "section": "RESULTS", + "ner": [ + [ + 33, + 38, + "human", + "species" + ], + [ + 39, + 45, + "IL-17A", + "protein" + ], + [ + 67, + 80, + "phage panning", + "experimental_method" + ], + [ + 87, + 122, + "cyclic and linear peptide libraries", + "experimental_method" + ] + ] + }, + { + "sid": 28, + "sent": "Positive phage pools were then sub-cloned into a maltose-binding protein (MBP) fusion system.", + "section": "RESULTS", + "ner": [ + [ + 0, + 20, + "Positive phage pools", + "experimental_method" + ], + [ + 31, + 41, + "sub-cloned", + "experimental_method" + ], + [ + 49, + 92, + "maltose-binding protein (MBP) fusion system", + "experimental_method" + ] + ] + }, + { + "sid": 29, + "sent": "Single clones were isolated and sub-cultured in growth medium, and culture supernatants were used in an enzyme-linked immunosorbent assay (ELISA) to identify specific IL-17A-binding clones.", + "section": "RESULTS", + "ner": [ + [ + 104, + 137, + "enzyme-linked immunosorbent assay", + "experimental_method" + ], + [ + 139, + 144, + "ELISA", + "experimental_method" + ], + [ + 167, + 173, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 30, + "sent": "The positive binding supernatants were tested for the ability to block biotinylated IL-17A signaling through IL-17RA in an IL-17A/IL-17RA competition ELISA assay where unlabeled IL-17A was used as positive control to inhibit biotinylated IL-17A binding.", + "section": "RESULTS", + "ner": [ + [ + 71, + 83, + "biotinylated", + "protein_state" + ], + [ + 84, + 90, + "IL-17A", + "protein" + ], + [ + 109, + 116, + "IL-17RA", + "protein" + ], + [ + 123, + 137, + "IL-17A/IL-17RA", + "complex_assembly" + ], + [ + 138, + 161, + "competition ELISA assay", + "experimental_method" + ], + [ + 178, + 184, + "IL-17A", + "protein" + ], + [ + 225, + 237, + "biotinylated", + "protein_state" + ], + [ + 238, + 244, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 31, + "sent": "Approximately 10% of the clones that specifically bound to IL-17A also prevented the cytokine from binding to IL-17RA.", + "section": "RESULTS", + "ner": [ + [ + 59, + 65, + "IL-17A", + "protein" + ], + [ + 85, + 93, + "cytokine", + "protein_type" + ], + [ + 110, + 117, + "IL-17RA", + "protein" + ] + ] + }, + { + "sid": 32, + "sent": "Sequences identified from phage clones were chemically synthesized (Supplementary Table 1) and tested for inhibition of IL-17A binding to IL-17RA (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 26, + 38, + "phage clones", + "experimental_method" + ], + [ + 44, + 66, + "chemically synthesized", + "experimental_method" + ], + [ + 120, + 126, + "IL-17A", + "protein" + ], + [ + 138, + 145, + "IL-17RA", + "protein" + ] + ] + }, + { + "sid": 33, + "sent": "A 15-mer linear peptide 1 was shown to block IL-17A/IL-17RA binding with an IC50 of 80\u2009nM in the competition ELISA assay (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 16, + 25, + "peptide 1", + "chemical" + ], + [ + 45, + 59, + "IL-17A/IL-17RA", + "complex_assembly" + ], + [ + 76, + 80, + "IC50", + "evidence" + ], + [ + 97, + 120, + "competition ELISA assay", + "experimental_method" + ] + ] + }, + { + "sid": 34, + "sent": "This peptide was then tested in a cell-based functional assay wherein production of GRO-\u03b1 in BJ human fibroblast cells was measured as a function of IL-17A stimulation using 1\u2009ng/ml IL-17A.", + "section": "RESULTS", + "ner": [ + [ + 34, + 61, + "cell-based functional assay", + "experimental_method" + ], + [ + 84, + 89, + "GRO-\u03b1", + "protein" + ], + [ + 96, + 101, + "human", + "species" + ], + [ + 149, + 155, + "IL-17A", + "protein" + ], + [ + 182, + 188, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 35, + "sent": "Peptide 1 was found to be active in this functional assay with an IC50 of 370\u2009nM.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Peptide 1", + "chemical" + ], + [ + 41, + 57, + "functional assay", + "experimental_method" + ], + [ + 66, + 70, + "IC50", + "evidence" + ] + ] + }, + { + "sid": 36, + "sent": "Optimization of IL-17A peptide inhibitors", + "section": "RESULTS", + "ner": [ + [ + 16, + 22, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 37, + "sent": "A SAR campaign was undertaken to improve the potency of peptide 1.", + "section": "RESULTS", + "ner": [ + [ + 2, + 5, + "SAR", + "experimental_method" + ], + [ + 56, + 65, + "peptide 1", + "chemical" + ] + ] + }, + { + "sid": 38, + "sent": "An alanine scan of peptide 2, an analogue of 1 with a lysine to arginine substitution at position 14, was initiated.", + "section": "RESULTS", + "ner": [ + [ + 3, + 15, + "alanine scan", + "experimental_method" + ], + [ + 19, + 28, + "peptide 2", + "chemical" + ], + [ + 45, + 46, + "1", + "chemical" + ], + [ + 54, + 60, + "lysine", + "residue_name" + ], + [ + 64, + 72, + "arginine", + "residue_name" + ], + [ + 73, + 85, + "substitution", + "experimental_method" + ], + [ + 98, + 100, + "14", + "residue_number" + ] + ] + }, + { + "sid": 39, + "sent": "When alanine was already present (positions 7 and 15), substitution was made with lysine (Table 1, peptides 3\u201317).", + "section": "RESULTS", + "ner": [ + [ + 5, + 12, + "alanine", + "residue_name" + ], + [ + 44, + 45, + "7", + "residue_number" + ], + [ + 50, + 52, + "15", + "residue_number" + ], + [ + 55, + 67, + "substitution", + "experimental_method" + ], + [ + 82, + 88, + "lysine", + "residue_name" + ], + [ + 99, + 112, + "peptides 3\u201317", + "chemical" + ] + ] + }, + { + "sid": 40, + "sent": "Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA.", + "section": "RESULTS", + "ner": [ + [ + 10, + 11, + "1", + "residue_number" + ], + [ + 13, + 14, + "2", + "residue_number" + ], + [ + 16, + 17, + "4", + "residue_number" + ], + [ + 19, + 20, + "5", + "residue_number" + ], + [ + 22, + 23, + "7", + "residue_number" + ], + [ + 25, + 27, + "14", + "residue_number" + ], + [ + 32, + 34, + "15", + "residue_number" + ], + [ + 124, + 140, + "binding affinity", + "evidence" + ], + [ + 160, + 174, + "IL-17A/IL-17RA", + "complex_assembly" + ], + [ + 175, + 192, + "competition ELISA", + "experimental_method" + ] + ] + }, + { + "sid": 41, + "sent": "In particular, at position 5 (13), substitution of methionine with alanine resulted in a seven fold improvement in potency (80\u2009nM versus 11\u2009nM respectively).", + "section": "RESULTS", + "ner": [ + [ + 27, + 28, + "5", + "residue_number" + ], + [ + 30, + 32, + "13", + "chemical" + ], + [ + 35, + 47, + "substitution", + "experimental_method" + ], + [ + 51, + 61, + "methionine", + "residue_name" + ], + [ + 67, + 74, + "alanine", + "residue_name" + ] + ] + }, + { + "sid": 42, + "sent": "In order to rapidly evaluate the effects of substitution of natural amino acids at tolerant positions identified by the alanine scan, the lead sequence was subjected to site-specific saturation mutagenesis using MBP.", + "section": "RESULTS", + "ner": [ + [ + 44, + 56, + "substitution", + "experimental_method" + ], + [ + 120, + 132, + "alanine scan", + "experimental_method" + ], + [ + 169, + 205, + "site-specific saturation mutagenesis", + "experimental_method" + ], + [ + 212, + 215, + "MBP", + "experimental_method" + ] + ] + }, + { + "sid": 43, + "sent": "Each of the seven positions identified by the alanine scan was individually modified while keeping the rest of the sequence constant.", + "section": "RESULTS", + "ner": [ + [ + 46, + 58, + "alanine scan", + "experimental_method" + ] + ] + }, + { + "sid": 44, + "sent": "Modifications at positions 2 and 14 were shown to display improvement in binding affinity (data not shown).", + "section": "RESULTS", + "ner": [ + [ + 27, + 28, + "2", + "residue_number" + ], + [ + 33, + 35, + "14", + "residue_number" + ], + [ + 73, + 89, + "binding affinity", + "evidence" + ] + ] + }, + { + "sid": 45, + "sent": "Peptides with beneficial point mutations at positions 2, 5, and 14 were synthesized and evaluated in the competition ELISA (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 25, + 40, + "point mutations", + "experimental_method" + ], + [ + 54, + 55, + "2", + "residue_number" + ], + [ + 57, + 58, + "5", + "residue_number" + ], + [ + 64, + 66, + "14", + "residue_number" + ], + [ + 72, + 83, + "synthesized", + "experimental_method" + ], + [ + 105, + 122, + "competition ELISA", + "experimental_method" + ] + ] + }, + { + "sid": 46, + "sent": "Two of the changes, V2H (18) or V2T (21) displayed improved binding in the competition ELISA.", + "section": "RESULTS", + "ner": [ + [ + 20, + 23, + "V2H", + "mutant" + ], + [ + 25, + 27, + "18", + "chemical" + ], + [ + 32, + 35, + "V2T", + "mutant" + ], + [ + 37, + 39, + "21", + "chemical" + ], + [ + 75, + 92, + "competition ELISA", + "experimental_method" + ] + ] + }, + { + "sid": 47, + "sent": "Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine.", + "section": "RESULTS", + "ner": [ + [ + 10, + 21, + "replacement", + "experimental_method" + ], + [ + 25, + 35, + "methionine", + "residue_name" + ], + [ + 48, + 49, + "5", + "residue_number" + ], + [ + 55, + 62, + "alanine", + "residue_name" + ], + [ + 118, + 128, + "isoleucine", + "residue_name" + ], + [ + 130, + 132, + "24", + "chemical" + ], + [ + 135, + 142, + "leucine", + "residue_name" + ], + [ + 144, + 146, + "25", + "chemical" + ], + [ + 152, + 158, + "valine", + "residue_name" + ], + [ + 160, + 162, + "26", + "chemical" + ], + [ + 256, + 262, + "valine", + "residue_name" + ], + [ + 267, + 277, + "isoleucine", + "residue_name" + ], + [ + 278, + 290, + "replacements", + "experimental_method" + ], + [ + 310, + 317, + "alanine", + "residue_name" + ] + ] + }, + { + "sid": 48, + "sent": "Introduction of a methionine (27) or a carboxamide (28 and 29) at position 14 was shown to improve the binding affinity of the lead peptide.", + "section": "RESULTS", + "ner": [ + [ + 0, + 12, + "Introduction", + "experimental_method" + ], + [ + 18, + 28, + "methionine", + "residue_name" + ], + [ + 30, + 32, + "27", + "chemical" + ], + [ + 39, + 50, + "carboxamide", + "chemical" + ], + [ + 52, + 54, + "28", + "chemical" + ], + [ + 59, + 61, + "29", + "chemical" + ], + [ + 75, + 77, + "14", + "residue_number" + ], + [ + 103, + 119, + "binding affinity", + "evidence" + ] + ] + }, + { + "sid": 49, + "sent": "In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion.", + "section": "RESULTS", + "ner": [ + [ + 60, + 78, + "binding affinities", + "evidence" + ], + [ + 117, + 127, + "MBP fusion", + "experimental_method" + ], + [ + 153, + 165, + "substitution", + "experimental_method" + ], + [ + 169, + 175, + "valine", + "residue_name" + ], + [ + 188, + 189, + "2", + "residue_number" + ], + [ + 195, + 205, + "tryptophan", + "residue_name" + ], + [ + 207, + 209, + "22", + "chemical" + ], + [ + 249, + 257, + "affinity", + "evidence" + ], + [ + 303, + 313, + "MBP fusion", + "experimental_method" + ] + ] + }, + { + "sid": 50, + "sent": "Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17\u2009nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2).", + "section": "RESULTS", + "ner": [ + [ + 52, + 55, + "SAR", + "experimental_method" + ], + [ + 99, + 109, + "peptide 30", + "chemical" + ], + [ + 117, + 138, + "high affinity peptide", + "chemical" + ], + [ + 140, + 143, + "HAP", + "chemical" + ], + [ + 172, + 178, + "IL-17A", + "protein" + ], + [ + 197, + 202, + "human", + "species" + ], + [ + 233, + 237, + "IC50", + "evidence" + ], + [ + 289, + 294, + "phage", + "experimental_method" + ], + [ + 295, + 304, + "peptide 1", + "chemical" + ] + ] + }, + { + "sid": 51, + "sent": "We also examined the effect of removing the acetyl group at the N-terminus of HAP (which is present in all the peptides made, see Supplementary Material).", + "section": "RESULTS", + "ner": [ + [ + 78, + 81, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 52, + "sent": "The un-capped peptide (31) had an IC50 of 420\u2009nM in the cell-based assay.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "un-capped", + "protein_state" + ], + [ + 14, + 26, + "peptide (31)", + "chemical" + ], + [ + 34, + 38, + "IC50", + "evidence" + ], + [ + 56, + 72, + "cell-based assay", + "experimental_method" + ] + ] + }, + { + "sid": 53, + "sent": "The loss of cellular activity of 31 was most likely due to the degradation of the N-terminus of 31, since peptide 31 was shown to be able to bind to IL-17A with similar affinity as HAP itself.", + "section": "RESULTS", + "ner": [ + [ + 33, + 35, + "31", + "chemical" + ], + [ + 96, + 98, + "31", + "chemical" + ], + [ + 114, + 116, + "31", + "chemical" + ], + [ + 149, + 155, + "IL-17A", + "protein" + ], + [ + 181, + 184, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 54, + "sent": "Furthermore, our previous work had reported that in antibody fusions the uncapped peptide was degraded under cell assay conditions with removal of the first 1-3 residues to inactive products with the same N-terminal sequences as peptides 32\u201334.", + "section": "RESULTS", + "ner": [ + [ + 52, + 68, + "antibody fusions", + "experimental_method" + ], + [ + 73, + 81, + "uncapped", + "protein_state" + ], + [ + 82, + 89, + "peptide", + "chemical" + ], + [ + 136, + 146, + "removal of", + "experimental_method" + ], + [ + 151, + 169, + "first 1-3 residues", + "residue_range" + ], + [ + 229, + 243, + "peptides 32\u201334", + "chemical" + ] + ] + }, + { + "sid": 55, + "sent": "In this work, 32\u201334 are capped by protective acetyl group and reflect the same inactivity as reported.", + "section": "RESULTS", + "ner": [ + [ + 14, + 19, + "32\u201334", + "chemical" + ], + [ + 24, + 30, + "capped", + "protein_state" + ] + ] + }, + { + "sid": 56, + "sent": "C-terminal truncations showed a more gradual reduction in activity (35\u201337; Table 2).", + "section": "RESULTS", + "ner": [ + [ + 11, + 22, + "truncations", + "experimental_method" + ], + [ + 68, + 73, + "35\u201337", + "chemical" + ] + ] + }, + { + "sid": 57, + "sent": "After deletion of three amino acids from the C-terminal end (37), the peptide is no longer active.", + "section": "RESULTS", + "ner": [ + [ + 6, + 17, + "deletion of", + "experimental_method" + ], + [ + 18, + 35, + "three amino acids", + "residue_range" + ], + [ + 61, + 63, + "37", + "chemical" + ] + ] + }, + { + "sid": 58, + "sent": "Dimerization of HAP can further increase its potency", + "section": "RESULTS", + "ner": [ + [ + 16, + 19, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 59, + "sent": "We reasoned that since the IL-17A protein is almost exclusively present in a dimeric form, dimerizing the IL-17A binding peptides could result in an improvement in binding affinity and inhibitory activity.", + "section": "RESULTS", + "ner": [ + [ + 27, + 33, + "IL-17A", + "protein" + ], + [ + 77, + 84, + "dimeric", + "oligomeric_state" + ], + [ + 91, + 101, + "dimerizing", + "oligomeric_state" + ], + [ + 106, + 112, + "IL-17A", + "protein" + ], + [ + 164, + 180, + "binding affinity", + "evidence" + ] + ] + }, + { + "sid": 60, + "sent": "Homodimers of HAP were made through attachment of polyethylene glycol (PEG) spacers of different lengths at amino acids 4, 7 and 14, as these positions were identified in the alanine scan analysis as not contributing significantly to the activity, and at each N-terminus (Supplementary Table S2).", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "Homodimers", + "oligomeric_state" + ], + [ + 14, + 17, + "HAP", + "chemical" + ], + [ + 50, + 69, + "polyethylene glycol", + "chemical" + ], + [ + 71, + 74, + "PEG", + "chemical" + ], + [ + 120, + 121, + "4", + "residue_number" + ], + [ + 123, + 124, + "7", + "residue_number" + ], + [ + 129, + 131, + "14", + "residue_number" + ], + [ + 175, + 196, + "alanine scan analysis", + "experimental_method" + ] + ] + }, + { + "sid": 61, + "sent": "Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP.", + "section": "RESULTS", + "ner": [ + [ + 34, + 50, + "pentafluoroester", + "chemical" + ], + [ + 52, + 55, + "PFP", + "chemical" + ], + [ + 99, + 102, + "PEG", + "chemical" + ], + [ + 108, + 117, + "histidine", + "residue_name" + ], + [ + 130, + 131, + "2", + "residue_number" + ], + [ + 140, + 146, + "lysine", + "residue_name" + ], + [ + 159, + 161, + "15", + "residue_number" + ], + [ + 181, + 190, + "threonine", + "residue_name" + ], + [ + 195, + 209, + "dimethyllysine", + "residue_name" + ], + [ + 280, + 290, + "peptide 38", + "chemical" + ], + [ + 328, + 331, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 62, + "sent": "This exercise revealed that several dimeric peptides with the longer PEG21 spacer were significantly more potent than the monomer peptide in the cell-based assay (Supplementary Table S2).", + "section": "RESULTS", + "ner": [ + [ + 36, + 43, + "dimeric", + "oligomeric_state" + ], + [ + 44, + 52, + "peptides", + "chemical" + ], + [ + 69, + 74, + "PEG21", + "chemical" + ], + [ + 122, + 129, + "monomer", + "oligomeric_state" + ], + [ + 145, + 161, + "cell-based assay", + "experimental_method" + ] + ] + }, + { + "sid": 63, + "sent": "Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1\u2009nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21\u2009nM (Supplementary Scheme S2).", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "Peptide 45", + "chemical" + ], + [ + 12, + 21, + "dimerized", + "oligomeric_state" + ], + [ + 42, + 47, + "PEG21", + "chemical" + ], + [ + 67, + 69, + "14", + "residue_number" + ], + [ + 145, + 149, + "IC50", + "evidence" + ], + [ + 232, + 239, + "monomer", + "oligomeric_state" + ], + [ + 262, + 267, + "PEG21", + "chemical" + ], + [ + 286, + 288, + "14", + "residue_number" + ], + [ + 292, + 302, + "peptide 48", + "chemical" + ], + [ + 310, + 314, + "IC50", + "evidence" + ] + ] + }, + { + "sid": 64, + "sent": "The species cross-reactivity of the dimeric peptide 45 and HAP were assessed in a murine functional cell assay using 15\u2009ng/ml murine IL-17A.", + "section": "RESULTS", + "ner": [ + [ + 36, + 43, + "dimeric", + "oligomeric_state" + ], + [ + 44, + 54, + "peptide 45", + "chemical" + ], + [ + 59, + 62, + "HAP", + "chemical" + ], + [ + 82, + 110, + "murine functional cell assay", + "experimental_method" + ], + [ + 126, + 132, + "murine", + "taxonomy_domain" + ], + [ + 133, + 139, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 65, + "sent": "Peptide 45 blocked the receptor binding of murine IL-17A although with potency two orders of magnitude weaker than that observed against human IL-17A (IC50\u2009=\u200941\u2009nM vs IC50\u2009=\u20090.1\u2009nM, respectively).", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "Peptide 45", + "chemical" + ], + [ + 23, + 31, + "receptor", + "protein_type" + ], + [ + 43, + 49, + "murine", + "taxonomy_domain" + ], + [ + 50, + 56, + "IL-17A", + "protein" + ], + [ + 137, + 142, + "human", + "species" + ], + [ + 143, + 149, + "IL-17A", + "protein" + ], + [ + 151, + 155, + "IC50", + "evidence" + ], + [ + 167, + 171, + "IC50", + "evidence" + ] + ] + }, + { + "sid": 66, + "sent": "The monomer HAP was much weaker (IC50 >1\u2009\u03bcM) in inhibiting murine IL-17A signaling (Supplementary Figure S4).", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "monomer", + "oligomeric_state" + ], + [ + 12, + 15, + "HAP", + "chemical" + ], + [ + 33, + 37, + "IC50", + "evidence" + ], + [ + 59, + 65, + "murine", + "taxonomy_domain" + ], + [ + 66, + 72, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 67, + "sent": "Although the dimeric peptide 45 is much more potent than HAP in the cell-based assay, in subsequent studies we decided to focus our efforts solely on characterizations of the monomeric peptide HAP in hopes to identify smaller peptide inhibitors containing the best minimal functional group.", + "section": "RESULTS", + "ner": [ + [ + 13, + 20, + "dimeric", + "oligomeric_state" + ], + [ + 21, + 31, + "peptide 45", + "chemical" + ], + [ + 57, + 60, + "HAP", + "chemical" + ], + [ + 68, + 84, + "cell-based assay", + "experimental_method" + ], + [ + 175, + 184, + "monomeric", + "oligomeric_state" + ], + [ + 193, + 196, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 68, + "sent": "Orthogonal assays to confirm HAP antagonism", + "section": "RESULTS", + "ner": [ + [ + 29, + 32, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 69, + "sent": "To further characterize the interaction of HAP with IL-17A, we set out to determine its in vitro binding affinity, specificity and kinetic profile using Surface Plasmon Resonance (SPR) methods (Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 43, + 46, + "HAP", + "chemical" + ], + [ + 52, + 58, + "IL-17A", + "protein" + ], + [ + 97, + 113, + "binding affinity", + "evidence" + ], + [ + 131, + 146, + "kinetic profile", + "evidence" + ], + [ + 153, + 178, + "Surface Plasmon Resonance", + "experimental_method" + ], + [ + 180, + 183, + "SPR", + "experimental_method" + ] + ] + }, + { + "sid": 70, + "sent": "HAP binds to immobilized human IL-17A homodimer tightly (Table 3).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "HAP", + "chemical" + ], + [ + 25, + 30, + "human", + "species" + ], + [ + 31, + 37, + "IL-17A", + "protein" + ], + [ + 38, + 47, + "homodimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 71, + "sent": "It has slightly weaker affinity for human IL-17A/F heterodimer and >10 fold weaker affinity for mouse IL-17A (Table 3).", + "section": "RESULTS", + "ner": [ + [ + 23, + 31, + "affinity", + "evidence" + ], + [ + 36, + 41, + "human", + "species" + ], + [ + 42, + 50, + "IL-17A/F", + "complex_assembly" + ], + [ + 51, + 62, + "heterodimer", + "oligomeric_state" + ], + [ + 83, + 91, + "affinity", + "evidence" + ], + [ + 96, + 101, + "mouse", + "taxonomy_domain" + ], + [ + 102, + 108, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 72, + "sent": "HAP does not show significant binding to immobilized human IL-17F homodimer or IL-17RA at concentrations up to 100\u2009nM.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "HAP", + "chemical" + ], + [ + 53, + 58, + "human", + "species" + ], + [ + 59, + 65, + "IL-17F", + "protein" + ], + [ + 66, + 75, + "homodimer", + "oligomeric_state" + ], + [ + 79, + 86, + "IL-17RA", + "protein" + ] + ] + }, + { + "sid": 73, + "sent": "Additionally, we investigated the antagonism of the human IL-17A/IL-17RA interaction by HAP using orthogonal methods including SPR and F\u00f6rster resonance energy transfer (FRET) competition assays (Fig. 1B,C).", + "section": "RESULTS", + "ner": [ + [ + 52, + 57, + "human", + "species" + ], + [ + 58, + 72, + "IL-17A/IL-17RA", + "complex_assembly" + ], + [ + 88, + 91, + "HAP", + "chemical" + ], + [ + 127, + 130, + "SPR", + "experimental_method" + ], + [ + 135, + 194, + "F\u00f6rster resonance energy transfer (FRET) competition assays", + "experimental_method" + ] + ] + }, + { + "sid": 74, + "sent": "In both assays, incubation of IL-17A with HAP effectively blocks the binding of IL-17A to immobilized IL-17RA with similar sub-nM IC50 (Table 3).", + "section": "RESULTS", + "ner": [ + [ + 30, + 36, + "IL-17A", + "protein" + ], + [ + 42, + 45, + "HAP", + "chemical" + ], + [ + 80, + 86, + "IL-17A", + "protein" + ], + [ + 90, + 101, + "immobilized", + "protein_state" + ], + [ + 102, + 109, + "IL-17RA", + "protein" + ], + [ + 130, + 134, + "IC50", + "evidence" + ] + ] + }, + { + "sid": 75, + "sent": "HAP blocks IL-17A signaling in a human primary cell assay", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "HAP", + "chemical" + ], + [ + 11, + 17, + "IL-17A", + "protein" + ], + [ + 33, + 38, + "human", + "species" + ] + ] + }, + { + "sid": 76, + "sent": "While either IL-17A or TNF-\u03b1 alone can stimulate the release of multiple inflammatory cytokines, when acting together they can synergistically enhance each other\u2019s effects (Supplementary Figure S5).", + "section": "RESULTS", + "ner": [ + [ + 13, + 19, + "IL-17A", + "protein" + ], + [ + 23, + 28, + "TNF-\u03b1", + "protein" + ], + [ + 86, + 95, + "cytokines", + "protein_type" + ] + ] + }, + { + "sid": 77, + "sent": "These integrative responses to IL-17A and TNF-\u03b1 in human keratinocytes have been reported to account for key inflammatory pathogenic circuits in psoriasis.", + "section": "RESULTS", + "ner": [ + [ + 31, + 37, + "IL-17A", + "protein" + ], + [ + 42, + 47, + "TNF-\u03b1", + "protein" + ], + [ + 51, + 56, + "human", + "species" + ] + ] + }, + { + "sid": 78, + "sent": "Thus, we chose to study HAP\u2019s efficacy in blocking the production of IL-8, IL-6 and CCL-20 by primary human keratinocytes stimulated by IL-17A in the presence of TNF-\u03b1, an assay which may be more disease-relevant.", + "section": "RESULTS", + "ner": [ + [ + 24, + 27, + "HAP", + "chemical" + ], + [ + 69, + 73, + "IL-8", + "protein_type" + ], + [ + 75, + 79, + "IL-6", + "protein_type" + ], + [ + 84, + 90, + "CCL-20", + "protein_type" + ], + [ + 102, + 107, + "human", + "species" + ], + [ + 136, + 142, + "IL-17A", + "protein" + ], + [ + 162, + 167, + "TNF-\u03b1", + "protein" + ] + ] + }, + { + "sid": 79, + "sent": "HAP inhibits the production of all three cytokines in a dose-dependent fashion (Fig. 1D).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "HAP", + "chemical" + ], + [ + 41, + 50, + "cytokines", + "protein_type" + ] + ] + }, + { + "sid": 80, + "sent": "Significantly, the baseline levels of IL-8, IL-6 and CCL-20 stimulated by TNF-\u03b1 alone are not inhibited by HAP, further indicating the selectivity of HAP (Fig. 1D).", + "section": "RESULTS", + "ner": [ + [ + 38, + 42, + "IL-8", + "protein_type" + ], + [ + 44, + 48, + "IL-6", + "protein_type" + ], + [ + 53, + 59, + "CCL-20", + "protein_type" + ], + [ + 74, + 79, + "TNF-\u03b1", + "protein" + ], + [ + 107, + 110, + "HAP", + "chemical" + ], + [ + 150, + 153, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 81, + "sent": "Such pharmacological selectivity may be important to suppress inflammatory pathogenic circuits in psoriasis, while sparing the anti-infectious immune responses produced by TNF-\u03b1.", + "section": "RESULTS", + "ner": [ + [ + 172, + 177, + "TNF-\u03b1", + "protein" + ] + ] + }, + { + "sid": 82, + "sent": "The relatively high IC50 values in this assay (Table 3) are probably due to the high IL-17A concentration (100\u2009ng/ml) needed for detection of IL-6.", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "IC50", + "evidence" + ], + [ + 85, + 91, + "IL-17A", + "protein" + ], + [ + 142, + 146, + "IL-6", + "protein_type" + ] + ] + }, + { + "sid": 83, + "sent": "As a reference, a commercial anti-IL-17A antibody (R&D Systems) inhibits the production of IL-8 with an IC50 of 13(\u00b16) nM (N\u2009=\u20093).", + "section": "RESULTS", + "ner": [ + [ + 34, + 40, + "IL-17A", + "protein" + ], + [ + 41, + 49, + "antibody", + "protein_type" + ], + [ + 91, + 95, + "IL-8", + "protein_type" + ], + [ + 104, + 108, + "IC50", + "evidence" + ] + ] + }, + { + "sid": 84, + "sent": "Indeed, the IC50 was 14(\u00b19) nM (N\u2009=\u200912) for HAP inhibition of IL-8 production when only 5\u2009ng/ml IL-17A was used in this assay.", + "section": "RESULTS", + "ner": [ + [ + 12, + 16, + "IC50", + "evidence" + ], + [ + 44, + 47, + "HAP", + "chemical" + ], + [ + 62, + 66, + "IL-8", + "protein_type" + ], + [ + 96, + 102, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 85, + "sent": "In patients, the concentration of IL-17A in psoriatic lesions is reported to be 0.01\u2009ng/ml, well below the EC50 (5\u201310ng/ml) of IL-17A induced IL-8 production in vitro.", + "section": "RESULTS", + "ner": [ + [ + 34, + 40, + "IL-17A", + "protein" + ], + [ + 127, + 133, + "IL-17A", + "protein" + ], + [ + 142, + 146, + "IL-8", + "protein_type" + ] + ] + }, + { + "sid": 86, + "sent": "Similar to keratinocytes assay results, while HAP inhibits IL-17A stimulated IL-6 production by BJ human fibroblast potently (IC50 of 17\u2009nM), it does not inhibit TNF-\u03b1 stimulated IL-6 production at concentrations up to 10\u2009\u03bcM (Supplementary Figure S2).", + "section": "RESULTS", + "ner": [ + [ + 11, + 30, + "keratinocytes assay", + "experimental_method" + ], + [ + 46, + 49, + "HAP", + "chemical" + ], + [ + 59, + 65, + "IL-17A", + "protein" + ], + [ + 77, + 81, + "IL-6", + "protein_type" + ], + [ + 99, + 104, + "human", + "species" + ], + [ + 126, + 130, + "IC50", + "evidence" + ], + [ + 162, + 167, + "TNF-\u03b1", + "protein" + ], + [ + 179, + 183, + "IL-6", + "protein_type" + ] + ] + }, + { + "sid": 87, + "sent": "Crystallization and structure determination", + "section": "RESULTS", + "ner": [ + [ + 0, + 43, + "Crystallization and structure determination", + "experimental_method" + ] + ] + }, + { + "sid": 88, + "sent": "Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals.", + "section": "RESULTS", + "ner": [ + [ + 10, + 32, + "crystallization trials", + "experimental_method" + ], + [ + 44, + 62, + "co-crystallization", + "experimental_method" + ], + [ + 69, + 76, + "soaking", + "experimental_method" + ], + [ + 77, + 80, + "HAP", + "chemical" + ], + [ + 96, + 99, + "apo", + "protein_state" + ], + [ + 100, + 106, + "IL-17A", + "protein" + ], + [ + 107, + 115, + "crystals", + "evidence" + ], + [ + 138, + 148, + "IL-17A/HAP", + "complex_assembly" + ], + [ + 157, + 165, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 89, + "sent": "We theorized that HAP binding induced large conformational changes in IL-17A that led to the difficulty of getting an IL-17A/HAP binary complex crystal.", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "HAP", + "chemical" + ], + [ + 70, + 76, + "IL-17A", + "protein" + ], + [ + 118, + 128, + "IL-17A/HAP", + "complex_assembly" + ], + [ + 144, + 151, + "crystal", + "evidence" + ] + ] + }, + { + "sid": 90, + "sent": "It is known that an antibody antigen-binding fragment (Fab) can be used as crystallization chaperones in crystallizing difficult targets.", + "section": "RESULTS", + "ner": [ + [ + 20, + 28, + "antibody", + "protein_type" + ], + [ + 29, + 53, + "antigen-binding fragment", + "structure_element" + ], + [ + 55, + 58, + "Fab", + "structure_element" + ] + ] + }, + { + "sid": 91, + "sent": "We hypothesized that HAP may target the N-terminal of IL-17A which is known to be more flexible than its C-terminal and conformational changes needed for HAP binding may be more likely there.", + "section": "RESULTS", + "ner": [ + [ + 21, + 24, + "HAP", + "chemical" + ], + [ + 54, + 60, + "IL-17A", + "protein" + ], + [ + 154, + 157, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 92, + "sent": "We designed an antibody Fab known to target the C-terminal half of IL-17A based on a published IL-17A/Fab complex crystal structure, and produced it in HEK293 cells.", + "section": "RESULTS", + "ner": [ + [ + 15, + 23, + "antibody", + "protein_type" + ], + [ + 24, + 27, + "Fab", + "structure_element" + ], + [ + 48, + 63, + "C-terminal half", + "structure_element" + ], + [ + 67, + 73, + "IL-17A", + "protein" + ], + [ + 95, + 105, + "IL-17A/Fab", + "complex_assembly" + ], + [ + 114, + 131, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 93, + "sent": "In an SPR assay HAP and this Fab were able to co-bind IL-17A without large changes in their binding affinities and kinetics, confirming our hypothesis (Supplementary Figure S6).", + "section": "RESULTS", + "ner": [ + [ + 6, + 15, + "SPR assay", + "experimental_method" + ], + [ + 16, + 19, + "HAP", + "chemical" + ], + [ + 29, + 32, + "Fab", + "structure_element" + ], + [ + 54, + 60, + "IL-17A", + "protein" + ], + [ + 92, + 110, + "binding affinities", + "evidence" + ], + [ + 115, + 123, + "kinetics", + "evidence" + ] + ] + }, + { + "sid": 94, + "sent": "Furthermore, since it binds to an area far away from that of HAP (see below), this Fab should have minimum effects on HAP binding conformation.", + "section": "RESULTS", + "ner": [ + [ + 61, + 64, + "HAP", + "chemical" + ], + [ + 83, + 86, + "Fab", + "structure_element" + ], + [ + 118, + 121, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 95, + "sent": "Crystals of Fab/IL-17A/HAP ternary complex were obtained readily in crystallization screens.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Crystals", + "evidence" + ], + [ + 12, + 26, + "Fab/IL-17A/HAP", + "complex_assembly" + ], + [ + 68, + 91, + "crystallization screens", + "experimental_method" + ] + ] + }, + { + "sid": 96, + "sent": "Crystallization of IL-17A and its binding partners was accomplished using two forms of IL-17A.", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Crystallization", + "experimental_method" + ], + [ + 19, + 25, + "IL-17A", + "protein" + ], + [ + 87, + 93, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 97, + "sent": "These were, respectively, a presumably more homogeneous form of IL-17A that lacked the disordered N-terminal peptide and a full-length form of the cytokine with a full complement of disulfide bonds.", + "section": "RESULTS", + "ner": [ + [ + 64, + 70, + "IL-17A", + "protein" + ], + [ + 76, + 82, + "lacked", + "protein_state" + ], + [ + 87, + 97, + "disordered", + "protein_state" + ], + [ + 98, + 116, + "N-terminal peptide", + "structure_element" + ], + [ + 123, + 134, + "full-length", + "protein_state" + ], + [ + 147, + 155, + "cytokine", + "protein_type" + ], + [ + 182, + 197, + "disulfide bonds", + "ptm" + ] + ] + }, + { + "sid": 98, + "sent": "Crystals of the Fab/truncated IL-17A/HAP complex diffracted to 2.2\u2009\u00c5, and the Fab/full length IL-17A/HAP complex diffracted to 3.0\u2009\u00c5 (Supplementary Table S3).", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Crystals", + "evidence" + ], + [ + 16, + 40, + "Fab/truncated IL-17A/HAP", + "complex_assembly" + ], + [ + 78, + 104, + "Fab/full length IL-17A/HAP", + "complex_assembly" + ] + ] + }, + { + "sid": 99, + "sent": "Both structures were solved by molecular replacement.", + "section": "RESULTS", + "ner": [ + [ + 5, + 15, + "structures", + "evidence" + ], + [ + 31, + 52, + "molecular replacement", + "experimental_method" + ] + ] + }, + { + "sid": 100, + "sent": "Both complexes crystallized in the space group of P321, with half the complex (1 Fab/1 IL-17A monomer/1 HAP) in the asymmetric unit.", + "section": "RESULTS", + "ner": [ + [ + 15, + 27, + "crystallized", + "experimental_method" + ], + [ + 81, + 84, + "Fab", + "structure_element" + ], + [ + 87, + 93, + "IL-17A", + "protein" + ], + [ + 94, + 101, + "monomer", + "oligomeric_state" + ], + [ + 104, + 107, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 101, + "sent": "The intact complex can be generated by applying crystallographic 2-fold symmetry.", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "intact", + "protein_state" + ] + ] + }, + { + "sid": 102, + "sent": "Electron densities for HAP residues Ile1-Asn14 were readily interpretable with the exception of Lys15, which is disordered.", + "section": "RESULTS", + "ner": [ + [ + 0, + 18, + "Electron densities", + "evidence" + ], + [ + 23, + 26, + "HAP", + "chemical" + ], + [ + 36, + 46, + "Ile1-Asn14", + "residue_range" + ], + [ + 96, + 101, + "Lys15", + "residue_name_number" + ], + [ + 112, + 122, + "disordered", + "protein_state" + ] + ] + }, + { + "sid": 103, + "sent": "When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered.", + "section": "RESULTS", + "ner": [ + [ + 34, + 51, + "complex structure", + "evidence" + ], + [ + 67, + 78, + "full length", + "protein_state" + ], + [ + 79, + 85, + "IL-17A", + "protein" + ], + [ + 114, + 123, + "truncated", + "protein_state" + ], + [ + 124, + 130, + "IL-17A", + "protein" + ], + [ + 154, + 160, + "Cys106", + "residue_name_number" + ], + [ + 162, + 168, + "Ser106", + "residue_name_number" + ], + [ + 176, + 185, + "truncated", + "protein_state" + ], + [ + 186, + 192, + "IL-17A", + "protein" + ], + [ + 204, + 214, + "disordered", + "protein_state" + ] + ] + }, + { + "sid": 104, + "sent": "Cys106 is covalently linked to Cys10 that resides in the disordered N-terminal peptide in the full length IL-17A.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Cys106", + "residue_name_number" + ], + [ + 31, + 36, + "Cys10", + "residue_name_number" + ], + [ + 57, + 67, + "disordered", + "protein_state" + ], + [ + 68, + 86, + "N-terminal peptide", + "structure_element" + ], + [ + 94, + 105, + "full length", + "protein_state" + ], + [ + 106, + 112, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 105, + "sent": "Overall structure of Fab/IL-17A/HAP complex", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 21, + 35, + "Fab/IL-17A/HAP", + "complex_assembly" + ] + ] + }, + { + "sid": 106, + "sent": "In a similar manner to the published structure of Fab/IL-17A complex, two Fab molecules bind symmetrically to the C-terminal of the cytokine dimer, interacting with epitopes from both monomers (Fig. 2A).", + "section": "RESULTS", + "ner": [ + [ + 37, + 46, + "structure", + "evidence" + ], + [ + 50, + 60, + "Fab/IL-17A", + "complex_assembly" + ], + [ + 74, + 77, + "Fab", + "structure_element" + ], + [ + 132, + 140, + "cytokine", + "protein_type" + ], + [ + 141, + 146, + "dimer", + "oligomeric_state" + ], + [ + 184, + 192, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 107, + "sent": "Two copies of HAP bind to the N-terminal of the cytokine dimer, also symmetrically, and each HAP molecule also interacts with both IL-17A monomers (Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "HAP", + "chemical" + ], + [ + 48, + 56, + "cytokine", + "protein_type" + ], + [ + 57, + 62, + "dimer", + "oligomeric_state" + ], + [ + 93, + 96, + "HAP", + "chemical" + ], + [ + 131, + 137, + "IL-17A", + "protein" + ], + [ + 138, + 146, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 108, + "sent": "Based on disclosed epitopes of Secukinumab and Ixekizumab, HAP binds to IL-17A at an area that is also different from those of those two antibodies.", + "section": "RESULTS", + "ner": [ + [ + 31, + 42, + "Secukinumab", + "chemical" + ], + [ + 47, + 57, + "Ixekizumab", + "chemical" + ], + [ + 59, + 62, + "HAP", + "chemical" + ], + [ + 72, + 78, + "IL-17A", + "protein" + ], + [ + 137, + 147, + "antibodies", + "protein_type" + ] + ] + }, + { + "sid": 109, + "sent": "The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic \u03b2-strand that inserts between \u03b2-strand 4 of one IL-17A monomer and \u03b2-strand 0 (the first ordered peptide of IL-17A) of the second monomer.", + "section": "RESULTS", + "ner": [ + [ + 15, + 25, + "5 residues", + "residue_range" + ], + [ + 29, + 32, + "HAP", + "chemical" + ], + [ + 34, + 40, + "1IHVTI", + "chemical" + ], + [ + 50, + 61, + "amphipathic", + "protein_state" + ], + [ + 62, + 70, + "\u03b2-strand", + "structure_element" + ], + [ + 92, + 102, + "\u03b2-strand 4", + "structure_element" 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C-terminal 8 residues of the HAP that are ordered in the structure, 7ADLWDWIN, form an amphipathic \u03b1-helix interacting with the second IL-17A monomer.", + "section": "RESULTS", + "ner": [ + [ + 15, + 25, + "8 residues", + "residue_range" + ], + [ + 33, + 36, + "HAP", + "chemical" + ], + [ + 61, + 70, + "structure", + "evidence" + ], + [ + 72, + 81, + "7ADLWDWIN", + "chemical" + ], + [ + 91, + 102, + "amphipathic", + "protein_state" + ], + [ + 103, + 110, + "\u03b1-helix", + "structure_element" + ], + [ + 139, + 145, + "IL-17A", + "protein" + ], + [ + 146, + 153, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 113, + "sent": "Pro6 of HAP makes a transition between the N-terminal \u03b2-strand and the C-terminal \u03b1-helix of HAP.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "Pro6", + "residue_name_number" + ], + [ + 8, + 11, + "HAP", + "chemical" + ], + [ + 54, + 62, + "\u03b2-strand", + "structure_element" + ], + [ + 82, + 89, + "\u03b1-helix", + "structure_element" + ], + [ + 93, + 96, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 114, + "sent": "As a comparison, an IL-17A/IL-17RA complex structure (PDB code 4HSA) is also shown with IL-17A in the same orientation (Fig. 2C).", + "section": "RESULTS", + "ner": [ + [ + 20, + 34, + "IL-17A/IL-17RA", + "complex_assembly" + ], + [ + 35, + 52, + "complex structure", + "evidence" + ], + [ + 88, + 94, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 115, + "sent": "Inhibition mechanism of IL-17A signaling by HAP", + "section": "RESULTS", + "ner": [ + [ + 24, + 30, + "IL-17A", + "protein" + ], + [ + 44, + 47, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 116, + "sent": "IL-17RA binds IL-17A at three regions on the IL-17A homodimer.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "IL-17RA", + "protein" + ], + [ + 14, + 20, + "IL-17A", + "protein" + ], + [ + 45, + 51, + "IL-17A", + "protein" + ], + [ + 52, + 61, + "homodimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 117, + "sent": "HAP binds IL-17A at region I. Region I is formed by residues at the ends of \u03b2 strands 0 and 4, and from loops 1\u20132 and 3\u20134 of IL-17A (Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "HAP", + "chemical" + ], + [ + 10, + 16, + "IL-17A", + "protein" + ], + [ + 20, + 28, + "region I", + "structure_element" + ], + [ + 30, + 38, + "Region I", + "structure_element" + ], + [ + 76, + 93, + "\u03b2 strands 0 and 4", + "structure_element" + ], + [ + 104, + 113, + "loops 1\u20132", + "structure_element" + ], + [ + 118, + 121, + "3\u20134", + "structure_element" + ], + [ + 125, + 131, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 118, + "sent": "Conformational changes in region I induced by HAP binding alone may allosterically affect IL-17RA binding, but more importantly, the \u03b1-helix of HAP directly competes with IL-17RA for binding to IL-17A (Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 26, + 34, + "region I", + "structure_element" + ], + [ + 46, + 49, + "HAP", + "chemical" + ], + [ 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135, + "Trp12", + "residue_name_number" + ] + ] + }, + { + "sid": 122, + "sent": "Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively.", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "Leu9", + "residue_name_number" + ], + [ + 23, + 28, + "Ile13", + "residue_name_number" + ], + [ + 36, + 39, + "HAP", + "chemical" + ], + [ + 45, + 69, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 75, + 81, + "IL-17A", + "protein" + ], + [ + 91, + 95, + "Asp8", + "residue_name_number" + ], + [ + 111, + 124, + "hydrogen bond", + "bond_interaction" + ], + [ + 129, + 150, + "ion pair interactions", + "bond_interaction" + ], + [ + 156, + 161, + "Tyr62", + "residue_name_number" + ], + [ + 166, + 172, + "Lys114", + "residue_name_number" + ], + [ + 176, + 182, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 123, + "sent": "In region I, an IL-17RA peptide interacts with IL-17A in a very similar fashion to the \u03b1-helix of HAP.", + "section": "RESULTS", + "ner": [ + [ + 3, + 11, + "region I", + "structure_element" + ], + [ + 16, + 23, + "IL-17RA", + "protein" + ], + [ + 47, + 53, + "IL-17A", + "protein" + ], + [ + 87, + 94, + "\u03b1-helix", + "structure_element" + ], + [ + 98, + 101, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 124, + "sent": "The IL-17RA peptide has sequences of 27LDDSWI, and part of the peptide is also \u03b1-helical (Fig. 3B).", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "IL-17RA", + "protein" + ], + [ + 37, + 45, + "27LDDSWI", + "chemical" + ], + [ + 79, + 88, + "\u03b1-helical", + "structure_element" + ] + ] + }, + { + "sid": 125, + "sent": "Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B).", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "Leu7", + "residue_name_number" + ], + [ + 6, + 11, + "Trp31", + "residue_name_number" + ], + [ + 16, + 21, + "Ile32", + "residue_name_number" + ], + [ + 25, + 32, + "IL-17RA", + "protein" + ], + [ + 83, + 89, + "IL-17A", + "protein" + ], + [ + 93, + 97, + "Leu9", + "residue_name_number" + ], + [ + 99, + 104, + "Trp12", + "residue_name_number" + ], + [ + 109, + 114, + "Ile13", + "residue_name_number" + ], + [ + 118, + 121, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 126, + "sent": "In this sense, the \u03b1-helix of HAP with a sequence of 9LWDWI is a good mimetic of the 27LDDSWI peptide of IL-17RA.", + "section": "RESULTS", + "ner": [ + [ + 19, + 26, + "\u03b1-helix", + "structure_element" + ], + [ + 30, + 33, + "HAP", + "chemical" + ], + [ + 53, + 59, + "9LWDWI", + "chemical" + ], + [ + 85, + 93, + "27LDDSWI", + "chemical" + ], + [ + 105, + 112, + "IL-17RA", + "protein" + ] + ] + }, + { + "sid": 127, + "sent": "The \u03b2-strand of HAP has no equivalent in IL-17RA.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "\u03b2-strand", + "structure_element" + ], + [ + 16, + 19, + "HAP", + "chemical" + ], + [ + 41, + 48, + "IL-17RA", + "protein" + ] + ] + }, + { + "sid": 128, + "sent": "However, it mimics the \u03b2-strand 0 of IL-17A.", + "section": "RESULTS", + "ner": [ + [ + 23, + 33, + "\u03b2-strand 0", + "structure_element" + ], + [ + 37, + 43, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 129, + "sent": "The amphipathic \u03b2-strand of HAP orients the hydrophilic side chains of His2 and Thr4 outwards, and the hydrophobic side chains of Ile1, Val3 and Ile5 inward (Fig. 3A).", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "amphipathic", + "protein_state" + ], + [ + 16, + 24, + "\u03b2-strand", + "structure_element" + ], + [ + 28, + 31, + "HAP", + "chemical" + ], + [ + 71, + 75, + "His2", + "residue_name_number" + ], + [ + 80, + 84, + "Thr4", + "residue_name_number" + ], + [ + 130, + 134, + "Ile1", + "residue_name_number" + ], + [ + 136, + 140, + "Val3", + "residue_name_number" + ], + [ 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"RESULTS", + "ner": [ + [ + 26, + 32, + "IL-17A", + "protein" + ], + [ + 53, + 56, + "HAP", + "chemical" + ], + [ + 61, + 68, + "IL-17RA", + "protein" + ] + ] + }, + { + "sid": 134, + "sent": "Particularly for HAP, \u03b2-strands 0 have to shift out of the hydrophobic cleft formed by the main body of the IL-17A by as much as 10\u2009\u00c5 between C\u03b1 atoms (Fig. 3C).", + "section": "RESULTS", + "ner": [ + [ + 17, + 20, + "HAP", + "chemical" + ], + [ + 22, + 33, + "\u03b2-strands 0", + "structure_element" + ], + [ + 59, + 76, + "hydrophobic cleft", + "site" + ], + [ + 91, + 100, + "main body", + "structure_element" + ], + [ + 108, + 114, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 135, + "sent": "Disruptions of the apo IL-17A structure by HAP binding are apparently compensated for by formation of the new interactions that involve almost the entire HAP molecule (Fig. 3B).", + "section": "RESULTS", + "ner": [ + [ + 19, + 22, + "apo", + "protein_state" + ], + [ + 23, + 29, + "IL-17A", + "protein" + ], + [ + 30, + 39, + "structure", + "evidence" + ], + [ + 43, + 46, + "HAP", + "chemical" + ], + [ + 154, + 157, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 136, + "sent": "Structure basis for the observed SAR of peptides", + "section": "RESULTS", + "ner": [ + [ + 33, + 36, + "SAR", + "experimental_method" + ] + ] + }, + { + "sid": 137, + "sent": "The IL-17A/HAP complex structure obtained is very consistent with the observed SAR of our identified peptide inhibitors, explaining well how the evolution of the initial phage peptide 1 to HAP and 45 improved its potency (Supplementary Figure S7).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "IL-17A/HAP", + "complex_assembly" + ], + [ + 15, + 32, + "complex structure", + "evidence" + ], + [ + 79, + 82, + "SAR", + "experimental_method" + ], + [ + 170, + 175, + "phage", + "experimental_method" + ], + [ + 176, + 185, + "peptide 1", + "chemical" + ], + [ + 189, + 192, + "HAP", + "chemical" + ], + [ + 197, + 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"chemical" + ], + [ + 216, + 218, + "32", + "chemical" + ], + [ + 223, + 225, + "33", + "chemical" + ] + ] + }, + { + "sid": 141, + "sent": "The C-terminal Asn14 and Lys15 of HAP are not directly involved in interactions with IL-17A, and this is reflected in the gradual reduction in activity caused by C-terminal truncations (35 and 36, Table 2).", + "section": "RESULTS", + "ner": [ + [ + 15, + 20, + "Asn14", + "residue_name_number" + ], + [ + 25, + 30, + "Lys15", + "residue_name_number" + ], + [ + 34, + 37, + "HAP", + "chemical" + ], + [ + 85, + 91, + "IL-17A", + "protein" + ], + [ + 173, + 184, + "truncations", + "experimental_method" + ], + [ + 186, + 188, + "35", + "chemical" + ], + [ + 193, + 195, + "36", + "chemical" + ] + ] + }, + { + "sid": 142, + "sent": "Each peptide monomer in 45 may not necessarily be more potent than HAP, but two monomer peptides within the same molecule that can simultaneously bind to IL-17A can greatly improve its potency due to avidity effects.", + "section": "RESULTS", + "ner": [ + [ + 13, + 20, + "monomer", + "oligomeric_state" + ], + [ + 24, + 26, + "45", + "chemical" + ], + [ + 67, + 70, + "HAP", + "chemical" + ], + [ + 80, + 87, + "monomer", + "oligomeric_state" + ], + [ + 154, + 160, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 143, + "sent": "HAP targets region I of IL-17A, an area that has the least sequence conservation in IL-17 cytokines.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "HAP", + "chemical" + ], + [ + 12, + 20, + "region I", + "structure_element" + ], + [ + 24, + 30, + "IL-17A", + "protein" + ], + [ + 84, + 99, + "IL-17 cytokines", + "protein_type" + ] + ] + }, + { + "sid": 144, + "sent": "This lack of sequence conservation in the HAP binding site explains the observed specificity of HAP binding to human IL-17A.", + "section": "RESULTS", + "ner": [ + [ + 42, + 58, + "HAP binding site", + "site" + ], + [ + 96, + 99, + "HAP", + "chemical" + ], + [ + 111, + 116, + "human", + "species" + ], + [ + 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"missing", + "protein_state" + ], + [ + 33, + 39, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 147, + "sent": "Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal \u03b2-strand of HAP.", + "section": "RESULTS", + "ner": [ + [ + 0, + 19, + "Sequence alignments", + "experimental_method" + ], + [ + 28, + 33, + "human", + "species" + ], + [ + 38, + 43, + "mouse", + "taxonomy_domain" + ], + [ + 44, + 50, + "IL-17A", + "protein" + ], + [ + 72, + 78, + "IL-17A", + "protein" + ], + [ + 110, + 113, + "HAP", + "chemical" + ], + [ + 145, + 153, + "strand 0", + "structure_element" + ], + [ + 157, + 163, + "IL-17A", + "protein" + ], + [ + 200, + 208, + "\u03b2-strand", + "structure_element" + ], + [ + 212, + 215, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 148, + "sent": "In human IL-17A the sequences are 21TVMVNLNI, and in mouse they are 21NVKVNLKV.", + "section": "RESULTS", + "ner": [ + [ + 3, + 8, + "human", + "species" + ], + [ + 9, + 15, + "IL-17A", + "protein" + ], + [ + 34, + 44, + "21TVMVNLNI", + "chemical" + ], + [ + 53, + 58, + "mouse", + "taxonomy_domain" + ], + [ + 68, + 78, + "21NVKVNLKV", + "chemical" + ] + ] + }, + { + "sid": 149, + "sent": "Using a combination of phage display and SAR we have discovered novel peptides that are IL-17A antagonists.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 36, + "phage display", + "experimental_method" + ], + [ + 41, + 44, + "SAR", + "experimental_method" + ], + [ + 88, + 94, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 150, + "sent": "One of those peptides, HAP, also shows activity in inhibiting the production of multiple inflammatory cytokines by primary human keratinocytes stimulated by IL-17A and TNF-\u03b1, a disease relevant-model.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 26, + "HAP", + "chemical" + ], + [ + 102, + 111, + "cytokines", + "protein_type" + ], + [ + 123, + 128, + "human", + "species" + ], + [ + 157, + 163, + "IL-17A", + "protein" + ], + [ + 168, + 173, + "TNF-\u03b1", + "protein" + ] + ] + }, + { + "sid": 151, + "sent": "We have also determined the complex structure of IL-17A/HAP, which provides the structural basis for HAP\u2019s antagonism to IL-17A signaling.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 23, + "determined", + "experimental_method" + ], + [ + 28, + 45, + "complex structure", + "evidence" + ], + [ + 49, + 59, + "IL-17A/HAP", + "complex_assembly" + ], + [ + 101, + 104, + "HAP", + "chemical" + ], + [ + 121, + 127, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 152, + "sent": "During IL-17A signaling, IL-17A binds to one copy of IL-17RA and one copy of IL-17RC.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 13, + "IL-17A", + "protein" + ], + [ + 25, + 31, + "IL-17A", + "protein" + ], + [ + 53, + 60, + "IL-17RA", + "protein" + ], + [ + 77, + 84, + "IL-17RC", + "protein" + ] + ] + }, + { + "sid": 153, + "sent": "Since apo IL-17A is a homodimer with 2 fold symmetry, IL-17RA potentially can bind to either face of the IL-17A dimer.", + "section": "DISCUSS", + "ner": [ + [ + 6, + 9, + "apo", + "protein_state" + ], + [ + 10, + 16, + "IL-17A", + "protein" + ], + [ + 22, + 31, + "homodimer", + "oligomeric_state" + ], + [ + 54, + 61, + "IL-17RA", + "protein" + ], + [ + 105, + 111, + "IL-17A", + "protein" + ], + [ + 112, + 117, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 154, + "sent": "With two HAP molecules covering both faces of the IL-17A dimer, HAP can block IL-17RA approaching from either face.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 12, + "HAP", + "chemical" + ], + [ + 50, + 56, + "IL-17A", + "protein" + ], + [ + 57, + 62, + "dimer", + "oligomeric_state" + ], + [ + 64, + 67, + "HAP", + "chemical" + ], + [ + 78, + 85, + "IL-17RA", + "protein" + ] + ] + }, + { + "sid": 155, + "sent": "To form the 1:2 complex observed in crystal structure, it is important that there is no strong negative cooperativity in the binding of two HAP molecules.", + "section": "DISCUSS", + "ner": [ + [ + 36, + 53, + "crystal structure", + "evidence" + ], + [ + 140, + 143, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 156, + "sent": "In fact, in native electrospray ionization mass spectrometry analysis only 1:2 IL-17A/HAP complex was observed even when IL-17A was in excess (Supplementary Figure S8), indicating a positive binding cooperativity that favors inhibition of IL-17RA binding by HAP.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 60, + "native electrospray ionization mass spectrometry", + "experimental_method" + ], + [ + 79, + 89, + "IL-17A/HAP", + "complex_assembly" + ], + [ + 121, + 127, + "IL-17A", + "protein" + ], + [ + 239, + 246, + "IL-17RA", + "protein" + ], + [ + 258, + 261, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 157, + "sent": "HAP, with only 15 residues, can achieve almost the same binding affinity as the much larger IL-17RA molecule, indicating a more efficient way of binding to IL-17A.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 3, + "HAP", + "chemical" + ], + [ + 15, + 26, + "15 residues", + "residue_range" + ], + [ + 56, + 72, + "binding affinity", + "evidence" + ], + [ + 92, + 99, + "IL-17RA", + "protein" + ], + [ + 156, + 162, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 158, + "sent": "The interaction of IL-17A with IL-17RA has an extensive interface, covering ~2,200\u2009\u00c52 surface area of IL-17A.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 25, + "IL-17A", + "protein" + ], + [ + 31, + 38, + "IL-17RA", + "protein" + ], + [ + 56, + 65, + "interface", + "site" + ], + [ + 102, + 108, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 159, + "sent": "Due to the discontinuous nature of the IL-17A/IL-17RA binding interface, it is classified as having tertiary structural epitopes on both binding partners, and is therefore hard to target using small molecules.", + "section": "DISCUSS", + "ner": [ + [ + 39, + 71, + "IL-17A/IL-17RA binding interface", + "site" + ] + ] + }, + { + "sid": 160, + "sent": "Our studies of HAP demonstrated an uncommon mode of action for a peptide in inhibiting such a difficult protein-protein interaction target, and suggest further possible improvements in its binding potency.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 18, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 161, + "sent": "One way of further improving HAP\u2019s potency is by dimerization.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 32, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 162, + "sent": "Homo-dimerization of HAP (45) achieved sub-nanomolar potency against human IL-17A in cell assay.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 24, + "HAP", + "chemical" + ], + [ + 26, + 28, + "45", + "chemical" + ], + [ + 69, + 74, + "human", + "species" + ], + [ + 75, + 81, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 163, + "sent": "In the crystal structure, the distance between the carbonyl of Asn14 of one HAP molecule and the N-terminus of the second is only 15.7\u2009\u00c5, suggesting the potential for more potent dimeric peptides to be designed by using linkers of different lengths at different positions.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 24, + "crystal structure", + "evidence" + ], + [ + 63, + 68, + "Asn14", + "residue_name_number" + ], + [ + 76, + 79, + "HAP", + "chemical" + ], + [ + 179, + 186, + "dimeric", + "oligomeric_state" + ], + [ + 187, + 195, + "peptides", + "chemical" + ] + ] + }, + { + "sid": 164, + "sent": "Another direction of improving HAP is by reducing its size.", + "section": "DISCUSS", + "ner": [ + [ + 31, + 34, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 165, + "sent": "As demonstrated by the crystal structure, binding of the \u03b1-helix of HAP should be sufficient for preventing IL-17RA binding to IL-17A.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 40, + "crystal structure", + "evidence" + ], + [ + 57, + 64, + "\u03b1-helix", + "structure_element" + ], + [ + 68, + 71, + "HAP", + "chemical" + ], + [ + 108, + 115, + "IL-17RA", + "protein" + ], + [ + 127, + 133, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 166, + "sent": "Theoretically, it is possible to design chemicals such as stapled \u03b1-helical peptides to block \u03b1-helix-mediated IL-17A/IL-17RA interactions.", + "section": "DISCUSS", + "ner": [ + [ + 94, + 101, + "\u03b1-helix", + "structure_element" + ], + [ + 111, + 125, + "IL-17A/IL-17RA", + "complex_assembly" + ] + ] + }, + { + "sid": 167, + "sent": "In summary, these peptide-based anti-IL-17A modalities could be further developed as alternative therapeutic options to the reported monoclonal antibodies.", + "section": "DISCUSS", + "ner": [ + [ + 37, + 43, + "IL-17A", + "protein" + ], + [ + 144, + 154, + "antibodies", + "protein_type" + ] + ] + }, + { + "sid": 168, + "sent": "We are also very interested in finding non-peptidic small molecule IL-17A antagonists, and HAP can be used as an excellent tool peptide.", + "section": "DISCUSS", + "ner": [ + [ + 67, + 73, + "IL-17A", + "protein" + ], + [ + 91, + 94, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 169, + "sent": "The strategy utilized in generating the complex structures of HAP may also be useful for enabling structure based design of some known small molecule IL-17A antagonists.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 58, + "structures", + "evidence" + ], + [ + 62, + 65, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 170, + "sent": "Binding of HAP to IL-17A and inhibition of IL-17A/IL-17RA are measured by SPR, FRET and cell-based assays.", + "section": "FIG", + "ner": [ + [ + 11, + 14, + "HAP", + "chemical" + ], + [ + 18, + 24, + "IL-17A", + "protein" + ], + [ + 43, + 57, + "IL-17A/IL-17RA", + "complex_assembly" + ], + [ + 74, + 77, + "SPR", + "experimental_method" + ], + [ + 79, + 83, + "FRET", + "experimental_method" + ], + [ + 88, + 105, + "cell-based assays", + "experimental_method" + ] + ] + }, + { + "sid": 171, + "sent": "(A) Typical SPR sensorgrams (black) of HAP at indicated concentrations binding to biotinylated human IL-17A immobilized on a streptavidin chip surface, fitted with single site binding model curves (red).", + "section": "FIG", + "ner": [ + [ + 12, + 15, + "SPR", + "experimental_method" + ], + [ + 16, + 27, + "sensorgrams", + "evidence" + ], + [ + 39, + 42, + "HAP", + "chemical" + ], + [ + 82, + 94, + "biotinylated", + "protein_state" + ], + [ + 95, + 100, + "human", + "species" + ], + [ + 101, + 107, + "IL-17A", + "protein" + ], + [ + 164, + 196, + "single site binding model curves", + "evidence" + ] + ] + }, + { + "sid": 172, + "sent": "Kinetic parameters (ka, kd) were obtained by a global fit using three concentrations in triplicate.", + "section": "FIG", + "ner": [ + [ + 20, + 22, + "ka", + "evidence" + ], + [ + 24, + 26, + "kd", + "evidence" + ] + ] + }, + { + "sid": 173, + "sent": "KD determined by the standard equation, KD\u2009=\u2009kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA.", + "section": "FIG", + "ner": [ + [ + 0, + 2, + "KD", + "evidence" + ], + [ + 40, + 42, + "KD", + "evidence" + ], + [ + 45, + 47, + "kd", + "evidence" + ], + [ + 48, + 50, + "ka", + "evidence" + ], + [ + 56, + 59, + "HAP", + "chemical" + ], + [ + 69, + 72, + "SPR", + "experimental_method" + ], + [ + 86, + 92, + "IL-17A", + "protein" + ], + [ + 104, + 115, + "immobilized", + "protein_state" + ], + [ + 116, + 123, + "IL-17RA", + "protein" + ] + ] + }, + { + "sid": 174, + "sent": "Data are mean and error bars of +/\u2212 standard deviation of three measurements. (C) Inhibition of IL-17A and IL-17RA binding by HAP measured by FRET assay.", + "section": "FIG", + "ner": [ + [ + 96, + 102, + "IL-17A", + "protein" + ], + [ + 107, + 114, + "IL-17RA", + "protein" + ], + [ + 126, + 129, + "HAP", + "chemical" + ], + [ + 142, + 152, + "FRET assay", + "experimental_method" + ] + ] + }, + { + "sid": 175, + "sent": "Data are mean and error bars of +/\u2212 standard deviation from 299 experiments, each performed in duplicate. (D) Example of HAP selective inhibition of the production of IL-8 (triangles), IL-6 (squares) and CCL-20 (circles) by primary human keratinocyte cells synergistically stimulated by 100\u2009ng/ml IL-17A and 10\u2009ng/ml TNF-\u03b1.", + "section": "FIG", + "ner": [ + [ + 121, + 124, + "HAP", + "chemical" + ], + [ + 167, + 171, + "IL-8", + "protein_type" + ], + [ + 185, + 189, + "IL-6", + "protein_type" + ], + [ + 204, + 210, + "CCL-20", + "protein_type" + ], + [ + 232, + 237, + "human", + "species" + ], + [ + 297, + 303, + "IL-17A", + "protein" + ], + [ + 317, + 322, + "TNF-\u03b1", + "protein" + ] + ] + }, + { + "sid": 176, + "sent": "HAP does not inhibit the baseline production of IL-6, IL-8 and CCL-20 stimulated by 10\u2009ng/ml TNF-\u03b1 alone (gray lines and symbols).", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "HAP", + "chemical" + ], + [ + 48, + 52, + "IL-6", + "protein_type" + ], + [ + 54, + 58, + "IL-8", + "protein_type" + ], + [ + 63, + 69, + "CCL-20", + "protein_type" + ], + [ + 93, + 98, + "TNF-\u03b1", + "protein" + ] + ] + }, + { + "sid": 177, + "sent": "Overall structure of the Fab/IL-17A/HAP complex in ribbon presentation.", + "section": "FIG", + "ner": [ + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 25, + 39, + "Fab/IL-17A/HAP", + "complex_assembly" + ] + ] + }, + { + "sid": 178, + "sent": "Two HAP molecules are colored blue and red, and IL-17A monomers are colored ice blue and pink, respectively.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "HAP", + "chemical" + ], + [ + 48, + 54, + "IL-17A", + "protein" + ], + [ + 55, + 63, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 179, + "sent": "(A) Overview of the distinct binding sites of Fab and HAP to IL-17A.", + "section": "FIG", + "ner": [ + [ + 29, + 42, + "binding sites", + "site" + ], + [ + 46, + 49, + "Fab", + "structure_element" + ], + [ + 54, + 57, + "HAP", + "chemical" + ], + [ + 61, + 67, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 180, + "sent": "(B) Close-in view of the IL-17A/HAP structure.", + "section": "FIG", + "ner": [ + [ + 25, + 35, + "IL-17A/HAP", + "complex_assembly" + ], + [ + 36, + 45, + "structure", + "evidence" + ] + ] + }, + { + "sid": 181, + "sent": "IL-17A \u03b2-strands are labelled.", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "IL-17A", + "protein" + ], + [ + 7, + 16, + "\u03b2-strands", + "structure_element" + ] + ] + }, + { + "sid": 182, + "sent": "Each of the two bound HAP interacts with both monomers of the IL-17A dimer.", + "section": "FIG", + "ner": [ + [ + 16, + 21, + "bound", + "protein_state" + ], + [ + 22, + 25, + "HAP", + "chemical" + ], + [ + 46, + 54, + "monomers", + "oligomeric_state" + ], + [ + 62, + 68, + "IL-17A", + "protein" + ], + [ + 69, + 74, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 183, + "sent": "(C) As a comparison, the IL-17A/IL-17RA complex was shown with IL-17A in the same orientation.", + "section": "FIG", + "ner": [ + [ + 25, + 39, + "IL-17A/IL-17RA", + "complex_assembly" + ], + [ + 63, + 69, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 184, + "sent": "Three distinct areas IL-17A/IL-17RA interface are labeled.", + "section": "FIG", + "ner": [ + [ + 21, + 45, + "IL-17A/IL-17RA interface", + "site" + ] + ] + }, + { + "sid": 185, + "sent": "Mechanism of the inhibition of the IL-17A/IL-17RA interaction by HAP.", + "section": "FIG", + "ner": [ + [ + 35, + 49, + "IL-17A/IL-17RA", + "complex_assembly" + ], + [ + 65, + 68, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 186, + "sent": "(A) HAP binds at region I of IL-17A.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "HAP", + "chemical" + ], + [ + 17, + 25, + "region I", + "structure_element" + ], + [ + 29, + 35, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 187, + "sent": "IL-17A dimer is in surface presentation (\u03b2-strands 0 shown as ribbons for clarity).", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "IL-17A", + "protein" + ], + [ + 7, + 12, + "dimer", + "oligomeric_state" + ], + [ + 41, + 52, + "\u03b2-strands 0", + "structure_element" + ] + ] + }, + { + "sid": 188, + "sent": "Polar interactions are shown in dashes.", + "section": "FIG", + "ner": [ + [ + 0, + 18, + "Polar interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 189, + "sent": "HAP residues as well as key IL-17A residues are labeled.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "HAP", + "chemical" + ], + [ + 28, + 34, + "IL-17A", + "protein" + ] + ] + }, + { + "sid": 190, + "sent": "For clarity, a few HAP residues are also shown in stick model with carbon atoms colored green, oxygen in red and nitrogen in blue.", + "section": "FIG", + "ner": [ + [ + 19, + 22, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 191, + "sent": "(B) I-17RA (ribbon in gold) peptide Leu27-Ile32 binds to the same area as the HAP \u03b1-helix.", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "I-17RA", + "protein" + ], + [ + 36, + 47, + "Leu27-Ile32", + "residue_range" + ], + [ + 78, + 81, + "HAP", + "chemical" + ], + [ + 82, + 89, + "\u03b1-helix", + "structure_element" + ] + ] + }, + { + "sid": 192, + "sent": "Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and \u03b2-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the \u03b2-stand and \u03b1-helix of the HAP.", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "Trp31", + "residue_name_number" + ], + [ + 9, + 16, + "IL-17RA", + "protein" + ], + [ + 35, + 41, + "pocket", + "site" + ], + [ + 45, + 51, + "IL-17A", + "protein" + ], + [ + 55, + 60, + "Trp12", + "residue_name_number" + ], + [ + 64, + 67, + "HAP", + "chemical" + ], + [ + 91, + 98, + "overlay", + "experimental_method" + ], + [ + 108, + 111, + "HAP", + "chemical" + ], + [ + 125, + 136, + "\u03b2-strands 0", + "structure_element" + ], + [ + 151, + 161, + "IL-17A/HAP", + "complex_assembly" + ], + [ + 177, + 180, + "apo", + "protein_state" + ], + [ + 181, + 187, + "IL-17A", + "protein" + ], + [ + 188, + 197, + "structure", + "evidence" + ], + [ + 225, + 233, + "region I", + "structure_element" + ], + [ + 237, + 243, + "IL-17A", + "protein" + ], + [ + 279, + 286, + "\u03b2-stand", + "structure_element" + ], + [ + 291, + 298, + "\u03b1-helix", + "structure_element" + ], + [ + 306, + 309, + "HAP", + "chemical" + ] + ] + }, + { + "sid": 193, + "sent": "Notice that the Trp binding pocket for W12 of HAP or W31 of IL-17RA is missing in the apo structure.", + "section": "FIG", + "ner": [ + [ + 16, + 34, + "Trp binding pocket", + "site" + ], + [ + 39, + 42, + "W12", + "residue_name_number" + ], + [ + 46, + 49, + "HAP", + "chemical" + ], + [ + 53, + 56, + "W31", + "residue_name_number" + ], + [ + 60, + 67, + "IL-17RA", + "protein" + ], + [ + 86, + 89, + "apo", + "protein_state" + ], + [ + 90, + 99, + "structure", + "evidence" + ] + ] + }, + { + "sid": 194, + "sent": "ELISA competition activity of peptide analogues of 1.", + "section": "TABLE", + "ner": [ + [ + 0, + 26, + "ELISA competition activity", + "experimental_method" + ] + ] + } + ] + }, + "PMC4841544": { + "annotations": [ + { + "sid": 0, + "sent": "Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA", + "section": "TITLE", + "ner": [ + [ + 50, + 54, + "NadR", + "protein" + ], + [ + 60, + 85, + "Transcriptional Repressor", + "protein_type" + ], + [ + 89, + 102, + "Meningococcal", + "taxonomy_domain" + ], + [ + 120, + 124, + "NadA", + "protein" + ] + ] + }, + { + "sid": 1, + "sent": " Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells.", + "section": "ABSTRACT", + "ner": [ + [ + 1, + 20, + "Neisseria adhesin A", + "protein" + ], + [ + 22, + 26, + "NadA", + "protein" + ], + [ + 46, + 59, + "meningococcal", + "taxonomy_domain" + ], + [ + 115, + 120, + "human", + "species" + ] + ] + }, + { + "sid": 2, + "sent": "NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 4, + "NadA", + "protein" + ], + [ + 112, + 137, + "serogroup B meningococcus", + "taxonomy_domain" + ] + ] + }, + { + "sid": 3, + "sent": "The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response.", + "section": "ABSTRACT", + "ner": [ + [ + 14, + 18, + "NadA", + "protein" + ], + [ + 26, + 35, + "bacterial", + "taxonomy_domain" + ], + [ + 173, + 178, + "human", + "species" + ], + [ + 231, + 240, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 4, + "sent": "It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo.", + "section": "ABSTRACT", + "ner": [ + [ + 70, + 74, + "nadA", + "gene" + ], + [ + 136, + 161, + "transcriptional regulator", + "protein_type" + ], + [ + 162, + 166, + "NadR", + "protein" + ], + [ + 168, + 197, + "Neisseria adhesin A Regulator", + "protein" + ] + ] + }, + { + "sid": 5, + "sent": "NadR binds the nadA promoter and represses gene transcription.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 4, + "NadR", + "protein" + ], + [ + 15, + 19, + "nadA", + "gene" + ], + [ + 0, + 4, + "NadR", + "protein" + ], + [ + 15, + 19, + "nadA", + "gene" + ] + ] + }, + { + "sid": 6, + "sent": "In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced.", + "section": "ABSTRACT", + "ner": [ + [ + 7, + 18, + "presence of", + "protein_state" + ], + [ + 19, + 41, + "4-hydroxyphenylacetate", + "chemical" + ], + [ + 43, + 48, + "4-HPA", + "chemical" + ], + [ + 75, + 80, + "human", + "species" + ], + [ + 135, + 144, + "bacterial", + "taxonomy_domain" + ], + [ + 171, + 175, + "NadR", + "protein" + ], + [ + 183, + 187, + "nadA", + "gene" + ], + [ + 215, + 219, + "nadA", + "gene" + ] + ] + }, + { + "sid": 7, + "sent": "NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 4, + "NadR", + "protein" + ], + [ + 61, + 74, + "meningococcal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 8, + "sent": "To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies.", + "section": "ABSTRACT", + "ner": [ + [ + 40, + 44, + "NadR", + "protein" + ], + [ + 57, + 62, + "4-HPA", + "chemical" + ], + [ + 76, + 124, + "structural, biochemical, and mutagenesis studies", + "experimental_method" + ] + ] + }, + { + "sid": 9, + "sent": "In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of \u2018conformational selection\u2019 by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria.", + "section": "ABSTRACT", + "ner": [ + [ + 23, + 41, + "crystal structures", + "evidence" + ], + [ + 45, + 56, + "ligand-free", + "protein_state" + ], + [ + 61, + 73, + "ligand-bound", + "protein_state" + ], + [ + 74, + 78, + "NadR", + "protein" + ], + [ + 172, + 177, + "4-HPA", + "chemical" + ], + [ + 199, + 206, + "dimeric", + "oligomeric_state" + ], + [ + 207, + 211, + "NadR", + "protein" + ], + [ + 329, + 349, + "hydroxyphenylacetate", + "chemical" + ], + [ + 369, + 378, + "3Cl,4-HPA", + "chemical" + ], + [ + 442, + 449, + "leucine", + "residue_name" + ], + [ + 489, + 498, + "conserved", + "protein_state" + ], + [ + 507, + 511, + "MarR", + "protein_type" + ], + [ + 553, + 557, + "His7", + "residue_name_number" + ], + [ + 559, + 563, + "Ser9", + "residue_name_number" + ], + [ + 565, + 570, + "Asn11", + "residue_name_number" + ], + [ + 575, + 580, + "Phe25", + "residue_name_number" + ], + [ + 613, + 618, + "4-HPA", + "chemical" + ], + [ + 697, + 705, + "bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 10, + "sent": "Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis.", + "section": "ABSTRACT", + "ner": [ + [ + 120, + 124, + "nadA", + "gene" + ], + [ + 153, + 157, + "NadR", + "protein" + ], + [ + 259, + 272, + "meningococcal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 11, + "sent": "Serogroup B meningococcus (MenB) causes fatal sepsis and invasive meningococcal disease, particularly in young children and adolescents, as highlighted by recent MenB outbreaks in universities of the United States and Canada.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 25, + "Serogroup B meningococcus", + "taxonomy_domain" + ], + [ + 27, + 31, + "MenB", + "species" + ], + [ + 66, + 79, + "meningococcal", + "taxonomy_domain" + ], + [ + 162, + 166, + "MenB", + "species" + ] + ] + }, + { + "sid": 12, + "sent": "The Bexsero vaccine protects against MenB and has recently been approved in > 35 countries worldwide.", + "section": "ABSTRACT", + "ner": [ + [ + 37, + 41, + "MenB", + "species" + ] + ] + }, + { + "sid": 13, + "sent": "Neisseria adhesin A (NadA) present on the meningococcal surface can mediate binding to human cells and is one of the three MenB vaccine protein antigens.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 19, + "Neisseria adhesin A", + "protein" + ], + [ + 21, + 25, + "NadA", + "protein" + ], + [ + 42, + 55, + "meningococcal", + "taxonomy_domain" + ], + [ + 87, + 92, + "human", + "species" + ], + [ + 123, + 127, + "MenB", + "species" + ] + ] + }, + { + "sid": 14, + "sent": "The amount of NadA exposed on the meningococcal surface also influences the antibody-mediated serum bactericidal response measured in vitro.", + "section": "ABSTRACT", + "ner": [ + [ + 14, + 18, + "NadA", + "protein" + ], + [ + 34, + 47, + "meningococcal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 15, + "sent": "A deep understanding of nadA expression is therefore important, otherwise the contribution of NadA to vaccine-induced protection against meningococcal meningitis may be underestimated.", + "section": "ABSTRACT", + "ner": [ + [ + 24, + 28, + "nadA", + "gene" + ], + [ + 94, + 98, + "NadA", + "protein" + ], + [ + 137, + 150, + "meningococcal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 16, + "sent": "The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. Here, we present functional, biochemical and high-resolution structural data on NadR. Our studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by \u2018conformational selection\u2019 of inactive forms.", + "section": "ABSTRACT", + "ner": [ + [ + 33, + 37, + "NadA", + "protein" + ], + [ + 58, + 101, + "ligand-responsive transcriptional repressor", + "protein_type" + ], + [ + 102, + 106, + "NadR", + "protein" + ], + [ + 125, + 184, + "functional, biochemical and high-resolution structural data", + "evidence" + ], + [ + 188, + 192, + "NadR", + "protein" + ], + [ + 273, + 293, + "hydroxyphenylacetate", + "chemical" + ], + [ + 377, + 381, + "NadR", + "protein" + ], + [ + 416, + 424, + "inactive", + "protein_state" + ] + ] + }, + { + "sid": 17, + "sent": "These findings shed light on the regulation of NadR, a key MarR-family virulence factor of this important human pathogen.", + "section": "ABSTRACT", + "ner": [ + [ + 47, + 51, + "NadR", + "protein" + ], + [ + 59, + 63, + "MarR", + "protein_type" + ], + [ + 106, + 111, + "human", + "species" + ] + ] + }, + { + "sid": 18, + "sent": "The \u2018Reverse Vaccinology\u2019 approach was pioneered to identify antigens for a protein-based vaccine against serogroup B Neisseria meningitidis (MenB), a human pathogen causing potentially-fatal sepsis and invasive meningococcal disease.", + "section": "INTRO", + "ner": [ + [ + 5, + 24, + "Reverse Vaccinology", + "experimental_method" + ], + [ + 106, + 140, + "serogroup B Neisseria meningitidis", + "species" + ], + [ + 142, + 146, + "MenB", + "species" + ], + [ + 151, + 156, + "human", + "species" + ], + [ + 212, + 225, + "meningococcal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 19, + "sent": "Indeed, Reverse Vaccinology identified Neisseria adhesin A (NadA), a surface-exposed protein involved in epithelial cell invasion and found in ~30% of clinical isolates.", + "section": "INTRO", + "ner": [ + [ + 8, + 27, + "Reverse Vaccinology", + "experimental_method" + ], + [ + 39, + 58, + "Neisseria adhesin A", + "protein" + ], + [ + 60, + 64, + "NadA", + "protein" + ] + ] + }, + { + "sid": 20, + "sent": "Recently, we reported the crystal structure of NadA, providing insights into its biological and immunological functions.", + "section": "INTRO", + "ner": [ + [ + 26, + 43, + "crystal structure", + "evidence" + ], + [ + 47, + 51, + "NadA", + "protein" + ] + ] + }, + { + "sid": 21, + "sent": "Recombinant NadA elicits a strong bactericidal immune response and is therefore included in the Bexsero vaccine that protects against MenB and which was recently approved in over 35 countries worldwide.", + "section": "INTRO", + "ner": [ + [ + 12, + 16, + "NadA", + "protein" + ], + [ + 134, + 138, + "MenB", + "species" + ] + ] + }, + { + "sid": 22, + "sent": "Previous studies revealed that nadA expression levels are mainly regulated by the Neisseria adhesin A Regulator (NadR).", + "section": "INTRO", + "ner": [ + [ + 31, + 35, + "nadA", + "gene" + ], + [ + 82, + 111, + "Neisseria adhesin A Regulator", + "protein" + ], + [ + 113, + 117, + "NadR", + "protein" + ] + ] + }, + { + "sid": 23, + "sent": "Although additional factors influence nadA expression, we focused on its regulation by NadR, the major mediator of NadA phase variable expression.", + "section": "INTRO", + "ner": [ + [ + 38, + 42, + "nadA", + "gene" + ], + [ + 87, + 91, + "NadR", + "protein" + ], + [ + 115, + 119, + "NadA", + "protein" + ] + ] + }, + { + "sid": 24, + "sent": "Studies of NadR also have broader implications, since a genome-wide analysis of MenB wild-type and nadR knock-out strains revealed that NadR influences the regulation of > 30 genes, including maf genes, from the multiple adhesin family.", + "section": "INTRO", + "ner": [ + [ + 11, + 15, + "NadR", + "protein" + ], + [ + 80, + 84, + "MenB", + "species" + ], + [ + 85, + 94, + "wild-type", + "protein_state" + ], + [ + 99, + 103, + "nadR", + "gene" + ], + [ + 104, + 113, + "knock-out", + "protein_state" + ], + [ + 136, + 140, + "NadR", + "protein" + ], + [ + 221, + 228, + "adhesin", + "protein_type" + ] + ] + }, + { + "sid": 25, + "sent": "These genes encode a wide variety of proteins connected to many biological processes contributing to bacterial survival, adaptation in the host niche, colonization and invasion.", + "section": "INTRO", + "ner": [ + [ + 101, + 110, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 26, + "sent": "NadR belongs to the MarR (Multiple Antibiotic Resistance Regulator) family, a group of ligand-responsive transcriptional regulators ubiquitous in bacteria and archaea.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "NadR", + "protein" + ], + [ + 20, + 24, + "MarR", + "protein_type" + ], + [ + 26, + 66, + "Multiple Antibiotic Resistance Regulator", + "protein_type" + ], + [ + 87, + 131, + "ligand-responsive transcriptional regulators", + "protein_type" + ], + [ + 146, + 154, + "bacteria", + "taxonomy_domain" + ], + [ + 159, + 166, + "archaea", + "taxonomy_domain" + ] + ] + }, + { + "sid": 27, + "sent": "MarR family proteins can promote bacterial survival in the presence of antibiotics, toxic chemicals, organic solvents or reactive oxygen species and can regulate virulence factor expression.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "MarR", + "protein_type" + ], + [ + 33, + 42, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 28, + "sent": "MarR homologues can act either as transcriptional repressors or as activators.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "MarR", + "protein_type" + ] + ] + }, + { + "sid": 29, + "sent": "Although > 50 MarR family structures are known, a molecular understanding of their ligand-dependent regulatory mechanisms is still limited, often hampered by lack of identification of their ligands and/or DNA targets.", + "section": "INTRO", + "ner": [ + [ + 14, + 18, + "MarR", + "protein_type" + ], + [ + 26, + 36, + "structures", + "evidence" + ] + ] + }, + { + "sid": 30, + "sent": "A potentially interesting exception comes from the ligand-free and salicylate-bound forms of the Methanobacterium thermoautotrophicum protein MTH313 which revealed that two salicylate molecules bind to one MTH313 dimer and induce large conformational changes, apparently sufficient to prevent DNA binding.", + "section": "INTRO", + "ner": [ + [ + 51, + 62, + "ligand-free", + "protein_state" + ], + [ + 67, + 83, + "salicylate-bound", + "protein_state" + ], + [ + 97, + 133, + "Methanobacterium thermoautotrophicum", + "species" + ], + [ + 142, + 148, + "MTH313", + "protein" + ], + [ + 173, + 183, + "salicylate", + "chemical" + ], + [ + 206, + 212, + "MTH313", + "protein" + ], + [ + 213, + 218, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 31, + "sent": "However, the homologous archeal Sulfolobus tokodaii protein ST1710 presented essentially the same structure in ligand-free and salicylate-bound forms, apparently contrasting the mechanism proposed for MTH313.", + "section": "INTRO", + "ner": [ + [ + 24, + 31, + "archeal", + "taxonomy_domain" + ], + [ + 32, + 51, + "Sulfolobus tokodaii", + "species" + ], + [ + 60, + 66, + "ST1710", + "protein" + ], + [ + 98, + 107, + "structure", + "evidence" + ], + [ + 111, + 122, + "ligand-free", + "protein_state" + ], + [ + 127, + 143, + "salicylate-bound", + "protein_state" + ], + [ + 201, + 207, + "MTH313", + "protein" + ] + ] + }, + { + "sid": 32, + "sent": "Despite these apparent differences, MTH313 and ST1710 bind salicylate in approximately the same site, between their dimerization and DNA-binding domains.", + "section": "INTRO", + "ner": [ + [ + 36, + 42, + "MTH313", + "protein" + ], + [ + 47, + 53, + "ST1710", + "protein" + ], + [ + 59, + 69, + "salicylate", + "chemical" + ], + [ + 116, + 152, + "dimerization and DNA-binding domains", + "structure_element" + ] + ] + }, + { + "sid": 33, + "sent": "However, it is unknown whether salicylate is a relevant in vivo ligand of either of these two proteins, which share ~20% sequence identity with NadR, rendering unclear the interpretation of these findings in relation to the regulatory mechanisms of NadR or other MarR family proteins.", + "section": "INTRO", + "ner": [ + [ + 31, + 41, + "salicylate", + "chemical" + ], + [ + 144, + 148, + "NadR", + "protein" + ], + [ + 249, + 253, + "NadR", + "protein" + ], + [ + 263, + 267, + "MarR", + "protein_type" + ] + ] + }, + { + "sid": 34, + "sent": "NadR binds nadA on three different operators (OpI, OpII and OpIII).", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "NadR", + "protein" + ], + [ + 11, + 15, + "nadA", + "gene" + ] + ] + }, + { + "sid": 35, + "sent": "The DNA-binding activity of NadR is attenuated in vitro upon addition of various hydroxyphenylacetate (HPA) derivatives, including 4-HPA.", + "section": "INTRO", + "ner": [ + [ + 28, + 32, + "NadR", + "protein" + ], + [ + 81, + 101, + "hydroxyphenylacetate", + "chemical" + ], + [ + 103, + 106, + "HPA", + "chemical" + ], + [ + 131, + 136, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 36, + "sent": "4-HPA is a small molecule derived from mammalian aromatic amino acid catabolism and is released in human saliva, where it has been detected at micromolar concentration.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "4-HPA", + "chemical" + ], + [ + 39, + 48, + "mammalian", + "taxonomy_domain" + ], + [ + 99, + 104, + "human", + "species" + ] + ] + }, + { + "sid": 37, + "sent": "In the presence of 4-HPA, NadR is unable to bind the nadA promoter and nadA gene expression is induced.", + "section": "INTRO", + "ner": [ + [ + 19, + 24, + "4-HPA", + "chemical" + ], + [ + 26, + 30, + "NadR", + "protein" + ], + [ + 53, + 57, + "nadA", + "gene" + ], + [ + 71, + 75, + "nadA", + "gene" + ] + ] + }, + { + "sid": 38, + "sent": "In vivo, the presence of 4-HPA in the host niche of N. meningitidis serves as an inducer of NadA production, thereby promoting bacterial adhesion to host cells.", + "section": "INTRO", + "ner": [ + [ + 25, + 30, + "4-HPA", + "chemical" + ], + [ + 52, + 67, + "N. meningitidis", + "species" + ], + [ + 92, + 96, + "NadA", + "protein" + ], + [ + 127, + 136, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 39, + "sent": "Further, we recently reported that 3Cl,4-HPA, produced during inflammation, is another inducer of nadA expression.", + "section": "INTRO", + "ner": [ + [ + 35, + 44, + "3Cl,4-HPA", + "chemical" + ], + [ + 98, + 102, + "nadA", + "gene" + ] + ] + }, + { + "sid": 40, + "sent": "Extending our previous studies based on hydrogen-deuterium exchange mass spectrometry (HDX-MS), here we sought to reveal the molecular mechanisms and effects of NadR/HPA interactions via X-ray crystallography, NMR spectroscopy and complementary biochemical and in vivo mutagenesis studies.", + "section": "INTRO", + "ner": [ + [ + 40, + 85, + "hydrogen-deuterium exchange mass spectrometry", + "experimental_method" + ], + [ + 87, + 93, + "HDX-MS", + "experimental_method" + ], + [ + 161, + 165, + "NadR", + "protein" + ], + [ + 166, + 169, + "HPA", + "chemical" + ], + [ + 187, + 208, + "X-ray crystallography", + "experimental_method" + ], + [ + 210, + 226, + "NMR spectroscopy", + "experimental_method" + ], + [ + 245, + 288, + "biochemical and in vivo mutagenesis studies", + "experimental_method" + ] + ] + }, + { + "sid": 41, + "sent": "We obtained detailed new insights into ligand specificity, how the ligand allosterically influences the DNA-binding ability of NadR, and the regulation of nadA expression, thus also providing a deeper structural understanding of the ligand-responsive MarR super-family.", + "section": "INTRO", + "ner": [ + [ + 127, + 131, + "NadR", + "protein" + ], + [ + 155, + 159, + "nadA", + "gene" + ], + [ + 251, + 255, + "MarR", + "protein_type" + ] + ] + }, + { + "sid": 42, + "sent": "Moreover, these findings are important because the activity of NadR impacts the potential coverage provided by anti-NadA antibodies elicited by the Bexsero vaccine and influences host-bacteria interactions that contribute to meningococcal pathogenesis.", + "section": "INTRO", + "ner": [ + [ + 63, + 67, + "NadR", + "protein" + ], + [ + 116, + 120, + "NadA", + "protein" + ], + [ + 184, + 192, + "bacteria", + "taxonomy_domain" + ], + [ + 225, + 238, + "meningococcal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 43, + "sent": "NadR is dimeric and is stabilized by specific hydroxyphenylacetate ligands", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "NadR", + "protein" + ], + [ + 8, + 15, + "dimeric", + "oligomeric_state" + ], + [ + 46, + 66, + "hydroxyphenylacetate", + "chemical" + ] + ] + }, + { + "sid": 44, + "sent": "Recombinant NadR was produced in E. coli using an expression construct prepared from N. meningitidis serogroup B strain MC58.", + "section": "RESULTS", + "ner": [ + [ + 12, + 16, + "NadR", + "protein" + ], + [ + 33, + 40, + "E. coli", + "species" + ], + [ + 50, + 70, + "expression construct", + "experimental_method" + ], + [ + 85, + 124, + "N. meningitidis serogroup B strain MC58", + "species" + ] + ] + }, + { + "sid": 45, + "sent": "Standard chromatographic techniques were used to obtain a highly purified sample of NadR (see Materials and Methods).", + "section": "RESULTS", + "ner": [ + [ + 84, + 88, + "NadR", + "protein" + ] + ] + }, + { + "sid": 46, + "sent": "In analytical size-exclusion high-performance liquid chromatography (SE-HPLC) experiments coupled with multi-angle laser light scattering (MALLS), NadR presented a single species with an absolute molecular mass of 35 kDa (S1 Fig).", + "section": "RESULTS", + "ner": [ + [ + 3, + 67, + "analytical size-exclusion high-performance liquid chromatography", + "experimental_method" + ], + [ + 69, + 76, + "SE-HPLC", + "experimental_method" + ], + [ + 103, + 137, + "multi-angle laser light scattering", + "experimental_method" + ], + [ + 139, + 144, + "MALLS", + "experimental_method" + ], + [ + 147, + 151, + "NadR", + "protein" + ] + ] + }, + { + "sid": 47, + "sent": "These data showed that NadR was dimeric in solution, since the theoretical molecular mass of the NadR dimer is 33.73 kDa; and, there was no change in oligomeric state on addition of 4-HPA.", + "section": "RESULTS", + "ner": [ + [ + 23, + 27, + "NadR", + "protein" + ], + [ + 32, + 39, + "dimeric", + "oligomeric_state" + ], + [ + 97, + 101, + "NadR", + "protein" + ], + [ + 102, + 107, + "dimer", + "oligomeric_state" + ], + [ + 182, + 187, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 48, + "sent": "The thermal stability of NadR was examined using differential scanning calorimetry (DSC).", + "section": "RESULTS", + "ner": [ + [ + 25, + 29, + "NadR", + "protein" + ], + [ + 49, + 82, + "differential scanning calorimetry", + "experimental_method" + ], + [ + 84, + 87, + "DSC", + "experimental_method" + ] + ] + }, + { + "sid": 49, + "sent": "Since ligand-binding often increases protein stability, we also investigated the effect of various HPAs (Fig 1A) on the melting temperature (Tm) of NadR. As a control of specificity, we also tested salicylate, a known ligand of some MarR proteins previously reported to increase the Tm of ST1710 and MTH313.", + "section": "RESULTS", + "ner": [ + [ + 99, + 103, + "HPAs", + "chemical" + ], + [ + 120, + 139, + "melting temperature", + "evidence" + ], + [ + 141, + 143, + "Tm", + "evidence" + ], + [ + 148, + 152, + "NadR", + "protein" + ], + [ + 198, + 208, + "salicylate", + "chemical" + ], + [ + 233, + 237, + "MarR", + "protein_type" + ], + [ + 283, + 285, + "Tm", + "evidence" + ], + [ + 289, + 295, + "ST1710", + "protein" + ], + [ + 300, + 306, + "MTH313", + "protein" + ] + ] + }, + { + "sid": 50, + "sent": "The Tm of NadR was 67.4 \u00b1 0.1\u00b0C in the absence of ligand, and was unaffected by salicylate.", + "section": "RESULTS", + "ner": [ + [ + 4, + 6, + "Tm", + "evidence" + ], + [ + 10, + 14, + "NadR", + "protein" + ], + [ + 39, + 56, + "absence of ligand", + "protein_state" + ], + [ + 80, + 90, + "salicylate", + "chemical" + ] + ] + }, + { + "sid": 51, + "sent": "However, an increased thermal stability was induced by 4-HPA and, to a lesser extent, by 3-HPA.", + "section": "RESULTS", + "ner": [ + [ + 55, + 60, + "4-HPA", + "chemical" + ], + [ + 89, + 94, + "3-HPA", + "chemical" + ] + ] + }, + { + "sid": 52, + "sent": "Interestingly, NadR displayed the greatest Tm increase upon addition of 3Cl,4-HPA (Table 1 and Fig 1B).", + "section": "RESULTS", + "ner": [ + [ + 15, + 19, + "NadR", + "protein" + ], + [ + 43, + 45, + "Tm", + "evidence" + ], + [ + 72, + 81, + "3Cl,4-HPA", + "chemical" + ] + ] + }, + { + "sid": 53, + "sent": "Stability of NadR is increased by small molecule ligands.", + "section": "FIG", + "ner": [ + [ + 13, + 17, + "NadR", + "protein" + ] + ] + }, + { + "sid": 54, + "sent": " (A) Molecular structures of 3-HPA (MW 152.2), 4-HPA (MW 152.2), 3Cl,4-HPA (MW 186.6) and salicylic acid (MW 160.1). (B) DSC profiles, colored as follows: apo-NadR (violet), NadR+salicylate (red), NadR+3-HPA (green), NadR+4-HPA (blue), NadR+3Cl,4-HPA (pink).", + "section": "FIG", + "ner": [ + [ + 29, + 34, + "3-HPA", + "chemical" + ], + [ + 47, + 52, + "4-HPA", + "chemical" + ], + [ + 65, + 74, + "3Cl,4-HPA", + "chemical" + ], + [ + 90, + 104, + "salicylic acid", + "chemical" + ], + [ + 121, + 124, + "DSC", + "experimental_method" + ], + [ + 125, + 133, + "profiles", + "evidence" + ], + [ + 155, + 158, + "apo", + "protein_state" + ], + [ + 159, + 163, + "NadR", + "protein" + ], + [ + 174, + 189, + "NadR+salicylate", + "complex_assembly" + ], + [ + 197, + 207, + "NadR+3-HPA", + "complex_assembly" + ], + [ + 217, + 227, + "NadR+4-HPA", + "complex_assembly" + ], + [ + 236, + 250, + "NadR+3Cl,4-HPA", + "complex_assembly" + ] + ] + }, + { + "sid": 55, + "sent": "All DSC profiles are representative of triplicate experiments.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "DSC", + "experimental_method" + ], + [ + 8, + 16, + "profiles", + "evidence" + ] + ] + }, + { + "sid": 56, + "sent": "Melting-point (Tm) and its ligand-induced increase (\u0394Tm) derived from DSC thermostability experiments.", + "section": "TABLE", + "ner": [ + [ + 0, + 13, + "Melting-point", + "evidence" + ], + [ + 15, + 17, + "Tm", + "evidence" + ], + [ + 52, + 55, + "\u0394Tm", + "evidence" + ], + [ + 70, + 73, + "DSC", + "experimental_method" + ], + [ + 74, + 101, + "thermostability experiments", + "experimental_method" + ] + ] + }, + { + "sid": 57, + "sent": "Dissociation constants (KD) of the NadR/ligand interactions from SPR steady-state binding experiments.", + "section": "TABLE", + "ner": [ + [ + 0, + 22, + "Dissociation constants", + "evidence" + ], + [ + 24, + 26, + "KD", + "evidence" + ], + [ + 35, + 39, + "NadR", + "protein" + ], + [ + 65, + 101, + "SPR steady-state binding experiments", + "experimental_method" + ] + ] + }, + { + "sid": 58, + "sent": "Ligand\tTm (\u00b0C)\t\u0394Tm (\u00b0C)\tKD (mM)\t \tNo ligand\t67.4 \u00b1 0.1\tn.a.\tn.a.", + "section": "TABLE", + "ner": [ + [ + 7, + 9, + "Tm", + "evidence" + ], + [ + 15, + 18, + "\u0394Tm", + "evidence" + ], + [ + 24, + 26, + "KD", + "evidence" + ] + ] + }, + { + "sid": 59, + "sent": "\t \t3-HPA\t70.0 \u00b1 0.1\t2.7\t2.7 \u00b1 0.1\t \t4-HPA\t70.7 \u00b1 0.1\t3.3\t1.5 \u00b1 0.1\t \t3Cl,4-HPA\t71.3 \u00b1 0.2\t3.9\t1.1 \u00b1 0.1\t \t", + "section": "TABLE", + "ner": [ + [ + 2, + 7, + "3-HPA", + "chemical" + ], + [ + 35, + 40, + "4-HPA", + "chemical" + ], + [ + 68, + 77, + "3Cl,4-HPA", + "chemical" + ] + ] + }, + { + "sid": 60, + "sent": "NadR displays distinct binding affinities for hydroxyphenylacetate ligands", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "NadR", + "protein" + ], + [ + 23, + 41, + "binding affinities", + "evidence" + ], + [ + 46, + 66, + "hydroxyphenylacetate", + "chemical" + ] + ] + }, + { + "sid": 61, + "sent": "To further investigate the binding of HPAs to NadR, we used surface plasmon resonance (SPR).", + "section": "RESULTS", + "ner": [ + [ + 38, + 42, + "HPAs", + "chemical" + ], + [ + 46, + 50, + "NadR", + "protein" + ], + [ + 60, + 85, + "surface plasmon resonance", + "experimental_method" + ], + [ + 87, + 90, + "SPR", + "experimental_method" + ] + ] + }, + { + "sid": 62, + "sent": "The SPR sensorgrams revealed very fast association and dissociation events, typical of small molecule ligands, thus prohibiting a detailed study of binding kinetics.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "SPR", + "experimental_method" + ], + [ + 8, + 19, + "sensorgrams", + "evidence" + ] + ] + }, + { + "sid": 63, + "sent": "However, steady-state SPR analyses of the NadR-HPA interactions allowed determination of the equilibrium dissociation constants (KD) (Table 1 and S2 Fig).", + "section": "RESULTS", + "ner": [ + [ + 9, + 25, + "steady-state SPR", + "experimental_method" + ], + [ + 42, + 50, + "NadR-HPA", + "complex_assembly" + ], + [ + 93, + 127, + "equilibrium dissociation constants", + "evidence" + ], + [ + 129, + 131, + "KD", + "evidence" + ] + ] + }, + { + "sid": 64, + "sent": "The interactions of 4-HPA and 3Cl,4-HPA with NadR exhibited KD values of 1.5 mM and 1.1 mM, respectively.", + "section": "RESULTS", + "ner": [ + [ + 20, + 25, + "4-HPA", + "chemical" + ], + [ + 30, + 39, + "3Cl,4-HPA", + "chemical" + ], + [ + 45, + 49, + "NadR", + "protein" + ], + [ + 60, + 62, + "KD", + "evidence" + ] + ] + }, + { + "sid": 65, + "sent": "3-HPA showed a weaker interaction, with a KD of 2.7 mM, while salicylate showed only a very weak response that did not reach saturation, indicating a non-specific interaction with NadR. A ranking of these KD values showed that 3Cl,4-HPA was the tightest binder, and thus matched the ranking of ligand-induced Tm increases observed in the DSC experiments.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "3-HPA", + "chemical" + ], + [ + 42, + 44, + "KD", + "evidence" + ], + [ + 62, + 72, + "salicylate", + "chemical" + ], + [ + 180, + 184, + "NadR", + "protein" + ], + [ + 205, + 207, + "KD", + "evidence" + ], + [ + 227, + 236, + "3Cl,4-HPA", + "chemical" + ], + [ + 309, + 311, + "Tm", + "evidence" + ], + [ + 338, + 341, + "DSC", + "experimental_method" + ] + ] + }, + { + "sid": 66, + "sent": "Although these KD values indicate rather weak interactions, they are similar to the values reported previously for the MarR/salicylate interaction (KD ~1 mM) and the MTH313/salicylate interaction (KD 2\u20133 mM), and approximately 20-fold tighter than the ST1710/salicylate interaction (KD ~20 mM).", + "section": "RESULTS", + "ner": [ + [ + 15, + 17, + "KD", + "evidence" + ], + [ + 119, + 123, + "MarR", + "protein_type" + ], + [ + 124, + 134, + "salicylate", + "chemical" + ], + [ + 166, + 172, + "MTH313", + "protein" + ], + [ + 173, + 183, + "salicylate", + "chemical" + ], + [ + 252, + 258, + "ST1710", + "protein" + ], + [ + 259, + 269, + "salicylate", + "chemical" + ] + ] + }, + { + "sid": 67, + "sent": "Crystal structures of holo-NadR and apo-NadR", + "section": "RESULTS", + "ner": [ + [ + 0, + 18, + "Crystal structures", + "evidence" + ], + [ + 22, + 26, + "holo", + "protein_state" + ], + [ + 27, + 31, + "NadR", + "protein" + ], + [ + 36, + 39, + "apo", + "protein_state" + ], + [ + 40, + 44, + "NadR", + "protein" + ] + ] + }, + { + "sid": 68, + "sent": "To fully characterize the NadR/HPA interactions, we sought to determine crystal structures of NadR in ligand-bound (holo) and ligand-free (apo) forms.", + "section": "RESULTS", + "ner": [ + [ + 26, + 30, + "NadR", + "protein" + ], + [ + 31, + 34, + "HPA", + "chemical" + ], + [ + 72, + 90, + "crystal structures", + "evidence" + ], + [ + 94, + 98, + "NadR", + "protein" + ], + [ + 102, + 114, + "ligand-bound", + "protein_state" + ], + [ + 116, + 120, + "holo", + "protein_state" + ], + [ + 126, + 137, + "ligand-free", + "protein_state" + ], + [ + 139, + 142, + "apo", + "protein_state" + ] + ] + }, + { + "sid": 69, + "sent": "First, we crystallized NadR (a selenomethionine-labelled derivative) in the presence of a 200-fold molar excess of 4-HPA.", + "section": "RESULTS", + "ner": [ + [ + 10, + 22, + "crystallized", + "experimental_method" + ], + [ + 23, + 27, + "NadR", + "protein" + ], + [ + 31, + 67, + "selenomethionine-labelled derivative", + "experimental_method" + ], + [ + 115, + 120, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 70, + "sent": "The structure of the NadR/4-HPA complex was determined at 2.3 \u00c5 resolution using a combination of the single-wavelength anomalous dispersion (SAD) and molecular replacement (MR) methods, and was refined to R work/R free values of 20.9/26.0% (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 21, + 31, + "NadR/4-HPA", + "complex_assembly" + ], + [ + 102, + 140, + "single-wavelength anomalous dispersion", + "experimental_method" + ], + [ + 142, + 145, + "SAD", + "experimental_method" + ], + [ + 151, + 172, + "molecular replacement", + "experimental_method" + ], + [ + 174, + 176, + "MR", + "experimental_method" + ], + [ + 206, + 219, + "R work/R free", + "evidence" + ] + ] + }, + { + "sid": 71, + "sent": "Despite numerous attempts, we were unable to obtain high-quality crystals of NadR complexed with 3Cl,4-HPA, 3,4-HPA, 3-HPA or DNA targets.", + "section": "RESULTS", + "ner": [ + [ + 65, + 73, + "crystals", + "evidence" + ], + [ + 77, + 81, + "NadR", + "protein" + ], + [ + 82, + 96, + "complexed with", + "protein_state" + ], + [ + 97, + 106, + "3Cl,4-HPA", + "chemical" + ], + [ + 108, + 115, + "3,4-HPA", + "chemical" + ], + [ + 117, + 122, + "3-HPA", + "chemical" + ] + ] + }, + { + "sid": 72, + "sent": "However, it was eventually possible to crystallize apo-NadR, and the structure was determined at 2.7 \u00c5 resolution by MR methods using the NadR/4-HPA complex as the search model.", + "section": "RESULTS", + "ner": [ + [ + 39, + 50, + "crystallize", + "experimental_method" + ], + [ + 51, + 54, + "apo", + "protein_state" + ], + [ + 55, + 59, + "NadR", + "protein" + ], + [ + 69, + 78, + "structure", + "evidence" + ], + [ + 117, + 119, + "MR", + "experimental_method" + ], + [ + 138, + 148, + "NadR/4-HPA", + "complex_assembly" + ] + ] + }, + { + "sid": 73, + "sent": "The apo-NadR structure was refined to R work/R free values of 19.1/26.8% (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "apo", + "protein_state" + ], + [ + 8, + 12, + "NadR", + "protein" + ], + [ + 13, + 22, + "structure", + "evidence" + ], + [ + 38, + 51, + "R work/R free", + "evidence" + ] + ] + }, + { + "sid": 74, + "sent": "Data collection and refinement statistics for NadR structures.", + "section": "TABLE", + "ner": [ + [ + 46, + 50, + "NadR", + "protein" + ], + [ + 51, + 61, + "structures", + "evidence" + ] + ] + }, + { + "sid": 75, + "sent": "The asymmetric unit of the NadR/4-HPA crystals (holo-NadR) contained one NadR homodimer, while the apo-NadR crystals contained two homodimers.", + "section": "RESULTS", + "ner": [ + [ + 27, + 37, + "NadR/4-HPA", + "complex_assembly" + ], + [ + 38, + 46, + "crystals", + "evidence" + ], + [ + 48, + 52, + "holo", + "protein_state" + ], + [ + 53, + 57, + "NadR", + "protein" + ], + [ + 73, + 77, + "NadR", + "protein" + ], + [ + 78, + 87, + "homodimer", + "oligomeric_state" + ], + [ + 99, + 102, + "apo", + "protein_state" + ], + [ + 103, + 107, + "NadR", + "protein" + ], + [ + 108, + 116, + "crystals", + "evidence" + ], + [ + 131, + 141, + "homodimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 76, + "sent": "In the apo-NadR crystals, the two homodimers were related by a rotation of ~90\u00b0; the observed association of the two dimers was presumably merely an effect of crystal packing, since the interface between the two homodimers is small (< 550 \u00c52 of buried surface area), and is not predicted to be physiologically relevant by the PISA software.", + "section": "RESULTS", + "ner": [ + [ + 7, + 10, + "apo", + "protein_state" + ], + [ + 11, + 15, + "NadR", + "protein" + ], + [ + 16, + 24, + "crystals", + "evidence" + ], + [ + 34, + 44, + "homodimers", + "oligomeric_state" + ], + [ + 117, + 123, + "dimers", + "oligomeric_state" + ], + [ + 186, + 195, + "interface", + "site" + ], + [ + 212, + 222, + "homodimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 77, + "sent": "Moreover, our SE-HPLC/MALLS analyses (see above) revealed that in solution NadR is dimeric, and previous studies using native mass spectrometry (MS) revealed dimers, not tetramers.", + "section": "RESULTS", + "ner": [ + [ + 14, + 27, + "SE-HPLC/MALLS", + "experimental_method" + ], + [ + 75, + 79, + "NadR", + "protein" + ], + [ + 83, + 90, + "dimeric", + "oligomeric_state" + ], + [ + 119, + 143, + "native mass spectrometry", + "experimental_method" + ], + [ + 145, + 147, + "MS", + "experimental_method" + ], + [ + 158, + 164, + "dimers", + "oligomeric_state" + ], + [ + 170, + 179, + "tetramers", + "oligomeric_state" + ] + ] + }, + { + "sid": 78, + "sent": "The NadR homodimer bound to 4-HPA has a dimerization interface mostly involving the top of its \u2018triangular\u2019 form, while the two DNA-binding domains are located at the base (Fig 2A).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "NadR", + "protein" + ], + [ + 9, + 18, + "homodimer", + "oligomeric_state" + ], + [ + 19, + 27, + "bound to", + "protein_state" + ], + [ + 28, + 33, + "4-HPA", + "chemical" + ], + [ + 40, + 62, + "dimerization interface", + "site" + ], + [ + 96, + 106, + "triangular", + "protein_state" + ], + [ + 128, + 147, + "DNA-binding domains", + "structure_element" + ] + ] + }, + { + "sid": 79, + "sent": "High-quality electron density maps allowed clear identification of the bound ligand, 4-HPA (Fig 2B).", + "section": "RESULTS", + "ner": [ + [ + 13, + 34, + "electron density maps", + "evidence" + ], + [ + 71, + 76, + "bound", + "protein_state" + ], + [ + 85, + 90, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 80, + "sent": "The overall structure of NadR shows dimensions of ~50 \u00d7 65 \u00d7 50 \u00c5 and a large homodimer interface that buries a total surface area of ~ 4800 \u00c52.", + "section": "RESULTS", + "ner": [ + [ + 12, + 21, + "structure", + "evidence" + ], + [ + 25, + 29, + "NadR", + "protein" + ], + [ + 78, + 97, + "homodimer interface", + "site" + ] + ] + }, + { + "sid": 81, + "sent": "Each NadR monomer consists of six \u03b1-helices and two short \u03b2-strands, with helices \u03b11, \u03b15, and \u03b16 forming the dimer interface.", + "section": "RESULTS", + "ner": [ + [ + 5, + 9, + "NadR", + "protein" + ], + [ + 10, + 17, + "monomer", + "oligomeric_state" + ], + [ + 34, + 43, + "\u03b1-helices", + "structure_element" + ], + [ + 52, + 67, + "short \u03b2-strands", + "structure_element" + ], + [ + 74, + 81, + "helices", + "structure_element" + ], + [ + 82, + 84, + "\u03b11", + "structure_element" + ], + [ + 86, + 88, + "\u03b15", + "structure_element" + ], + [ + 94, + 96, + "\u03b16", + "structure_element" + ], + [ + 109, + 124, + "dimer interface", + "site" + ] + ] + }, + { + "sid": 82, + "sent": "Helices \u03b13 and \u03b14 form a helix-turn-helix motif, followed by the \u201cwing motif\u201d comprised of two short antiparallel \u03b2-strands (\u03b21-\u03b22) linked by a relatively long and flexible loop.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Helices", + "structure_element" + ], + [ + 8, + 10, + "\u03b13", + "structure_element" + ], + [ + 15, + 17, + "\u03b14", + "structure_element" + ], + [ + 25, + 47, + "helix-turn-helix motif", + "structure_element" + ], + [ + 66, + 76, + "wing motif", + "structure_element" + ], + [ + 95, + 123, + "short antiparallel \u03b2-strands", + "structure_element" + ], + [ + 125, + 130, + "\u03b21-\u03b22", + "structure_element" + ], + [ + 173, + 177, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 83, + "sent": "Interestingly, in the \u03b14-\u03b22 region, the stretch of residues from R64-R91 presents seven positively-charged side chains, all available for potential interactions with DNA.", + "section": "RESULTS", + "ner": [ + [ + 22, + 34, + "\u03b14-\u03b22 region", + "structure_element" + ], + [ + 65, + 72, + "R64-R91", + "residue_range" + ], + [ + 166, + 169, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 84, + "sent": "Together, these structural elements constitute the winged helix-turn-helix (wHTH) DNA-binding domain and, together with the dimeric organization, are the hallmarks of MarR family structures.", + "section": "RESULTS", + "ner": [ + [ + 51, + 74, + "winged helix-turn-helix", + "structure_element" + ], + [ + 76, + 80, + "wHTH", + "structure_element" + ], + [ + 82, + 100, + "DNA-binding domain", + "structure_element" + ], + [ + 124, + 131, + "dimeric", + "oligomeric_state" + ], + [ + 167, + 171, + "MarR", + "protein_type" + ], + [ + 179, + 189, + "structures", + "evidence" + ] + ] + }, + { + "sid": 85, + "sent": "The crystal structure of NadR in complex with 4-HPA.", + "section": "FIG", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 25, + 29, + "NadR", + "protein" + ], + [ + 30, + 45, + "in complex with", + "protein_state" + ], + [ + 46, + 51, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 86, + "sent": " (A) The holo-NadR homodimer is depicted in green and blue for chains A and B respectively, while yellow sticks depict the 4-HPA ligand (labelled).", + "section": "FIG", + "ner": [ + [ + 9, + 13, + "holo", + "protein_state" + ], + [ + 14, + 18, + "NadR", + "protein" + ], + [ + 19, + 28, + "homodimer", + "oligomeric_state" + ], + [ + 63, + 77, + "chains A and B", + "structure_element" + ], + [ + 123, + 128, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 87, + "sent": "For simplicity, secondary structure elements are labelled for chain B only.", + "section": "FIG", + "ner": [ + [ + 62, + 69, + "chain B", + "structure_element" + ] + ] + }, + { + "sid": 88, + "sent": "Red dashes show hypothetical positions of chain B residues 88\u201390 that were not modeled due to lack of electron density.", + "section": "FIG", + "ner": [ + [ + 42, + 49, + "chain B", + "structure_element" + ], + [ + 59, + 64, + "88\u201390", + "residue_range" + ], + [ + 102, + 118, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 89, + "sent": "(B) A zoom into the pocket occupied by 4-HPA shows that the ligand contacts both chains A and B; blue mesh shows electron density around 4-HPA calculated from a composite omit map (omitting 4-HPA), using phenix.", + "section": "FIG", + "ner": [ + [ + 20, + 26, + "pocket", + "site" + ], + [ + 39, + 44, + "4-HPA", + "chemical" + ], + [ + 81, + 95, + "chains A and B", + "structure_element" + ], + [ + 113, + 129, + "electron density", + "evidence" + ], + [ + 137, + 142, + "4-HPA", + "chemical" + ], + [ + 161, + 179, + "composite omit map", + "evidence" + ], + [ + 190, + 195, + "4-HPA", + "chemical" + ], + [ + 204, + 210, + "phenix", + "experimental_method" + ] + ] + }, + { + "sid": 90, + "sent": "The map is contoured at 1\u03c3 and the figure was prepared with a density mesh carve factor of 1.7, using Pymol (www.pymol.org).", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "map", + "evidence" + ], + [ + 62, + 74, + "density mesh", + "evidence" + ] + ] + }, + { + "sid": 91, + "sent": "A single conserved leucine residue (L130) is crucial for dimerization", + "section": "RESULTS", + "ner": [ + [ + 9, + 18, + "conserved", + "protein_state" + ], + [ + 19, + 26, + "leucine", + "residue_name" + ], + [ + 36, + 40, + "L130", + "residue_name_number" + ] + ] + }, + { + "sid": 92, + "sent": "The NadR dimer interface is formed by at least 32 residues, which establish numerous inter-chain salt bridges or hydrogen bonds, and many hydrophobic packing interactions (Fig 3A and 3B).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "NadR", + "protein" + ], + [ + 9, + 24, + "dimer interface", + "site" + ], + [ + 97, + 109, + "salt bridges", + "bond_interaction" + ], + [ + 113, + 127, + "hydrogen bonds", + "bond_interaction" + ], + [ + 138, + 170, + "hydrophobic packing interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 93, + "sent": "To determine which residues were most important for dimerization, we studied the interface in silico and identified several residues as potential mediators of key stabilizing interactions.", + "section": "RESULTS", + "ner": [ + [ + 81, + 90, + "interface", + "site" + ] + ] + }, + { + "sid": 94, + "sent": "Using site-directed mutagenesis, a panel of eight mutant NadR proteins was prepared (including mutations H7A, S9A, N11A, D112A, R114A, Y115A, K126A, L130K and L133K), sufficient to explore the entire dimer interface.", + "section": "RESULTS", + "ner": [ + [ + 6, + 31, + "site-directed mutagenesis", + "experimental_method" + ], + [ + 50, + 56, + "mutant", + "protein_state" + ], + [ + 57, + 61, + "NadR", + "protein" + ], + [ + 105, + 108, + "H7A", + "mutant" + ], + [ + 110, + 113, + "S9A", + "mutant" + ], + [ + 115, + 119, + "N11A", + "mutant" + ], + [ + 121, + 126, + "D112A", + "mutant" + ], + [ + 128, + 133, + "R114A", + "mutant" + ], + [ + 135, + 140, + "Y115A", + "mutant" + ], + [ + 142, + 147, + "K126A", + "mutant" + ], + [ + 149, + 154, + "L130K", + "mutant" + ], + [ + 159, + 164, + "L133K", + "mutant" + ], + [ + 200, + 215, + "dimer interface", + "site" + ] + ] + }, + { + "sid": 95, + "sent": "Each mutant NadR protein was purified, and then its oligomeric state was examined by analytical SE-HPLC.", + "section": "RESULTS", + "ner": [ + [ + 5, + 11, + "mutant", + "protein_state" + ], + [ + 12, + 16, + "NadR", + "protein" + ], + [ + 85, + 103, + "analytical SE-HPLC", + "experimental_method" + ] + ] + }, + { + "sid": 96, + "sent": "Almost all the mutants showed the same elution profile as the wild-type (WT) NadR protein.", + "section": "RESULTS", + "ner": [ + [ + 62, + 71, + "wild-type", + "protein_state" + ], + [ + 73, + 75, + "WT", + "protein_state" + ], + [ + 77, + 81, + "NadR", + "protein" + ] + ] + }, + { + "sid": 97, + "sent": "Only the L130K mutation induced a notable change in the oligomeric state of NadR (Fig 3C).", + "section": "RESULTS", + "ner": [ + [ + 9, + 14, + "L130K", + "mutant" + ], + [ + 76, + 80, + "NadR", + "protein" + ] + ] + }, + { + "sid": 98, + "sent": "Further, in SE-MALLS analyses, the L130K mutant displayed two distinct species in solution, approximately 80% being monomeric (a 19 kDa species), and only 20% retaining the typical native dimeric state (a 35 kDa species) (Fig 3D), demonstrating that Leu130 is crucial for stable dimerization.", + "section": "RESULTS", + "ner": [ + [ + 12, + 20, + "SE-MALLS", + "experimental_method" + ], + [ + 35, + 40, + "L130K", + "mutant" + ], + [ + 41, + 47, + "mutant", + "protein_state" + ], + [ + 116, + 125, + "monomeric", + "oligomeric_state" + ], + [ + 188, + 195, + "dimeric", + "oligomeric_state" + ], + [ + 250, + 256, + "Leu130", + "residue_name_number" + ] + ] + }, + { + "sid": 99, + "sent": "It is notable that L130 is usually present as Leu, or an alternative bulky hydrophobic amino acid (e.g. Phe, Val), in many MarR family proteins, suggesting a conserved role in stabilizing the dimer interface.", + "section": "RESULTS", + "ner": [ + [ + 19, + 23, + "L130", + "residue_name_number" + ], + [ + 46, + 49, + "Leu", + "residue_name" + ], + [ + 104, + 107, + "Phe", + "residue_name" + ], + [ + 109, + 112, + "Val", + "residue_name" + ], + [ + 123, + 127, + "MarR", + "protein_type" + ], + [ + 158, + 167, + "conserved", + "protein_state" + ], + [ + 192, + 207, + "dimer interface", + "site" + ] + ] + }, + { + "sid": 100, + "sent": "In contrast, most of the other residues identified in the NadR dimer interface were poorly conserved in the MarR family.", + "section": "RESULTS", + "ner": [ + [ + 58, + 62, + "NadR", + "protein" + ], + [ + 63, + 78, + "dimer interface", + "site" + ], + [ + 84, + 100, + "poorly conserved", + "protein_state" + ], + [ + 108, + 112, + "MarR", + "protein_type" + ] + ] + }, + { + "sid": 101, + "sent": "Analysis of the NadR dimer interface.", + "section": "FIG", + "ner": [ + [ + 16, + 20, + "NadR", + "protein" + ], + [ + 21, + 36, + "dimer interface", + "site" + ] + ] + }, + { + "sid": 102, + "sent": " (A) Both orientations show chain A, green backbone ribbon, colored red to highlight all locations involved in dimerization; namely, inter-chain salt bridges or hydrogen bonds involving Q4, S5, K6, H7, S9, I10, N11, I15, Q16, R18, D36, R43, A46, Q59, C61, Y104, D112, R114, Y115, D116, E119, K126, E136, E141, N145, and the hydrophobic packing interactions involving I10, I12, L14, I15, R18, Y115, I118, L130, L133, L134 and L137.", + "section": "FIG", + "ner": [ + [ + 28, + 35, + "chain A", + "structure_element" + ], + [ + 145, + 157, + "salt bridges", + "bond_interaction" + ], + [ + 161, + 175, + "hydrogen bonds", + "bond_interaction" + ], + [ + 186, + 188, + "Q4", + "residue_name_number" + ], + [ + 190, + 192, + "S5", + "residue_name_number" + ], + [ + 194, + 196, + "K6", + "residue_name_number" + ], + [ + 198, + 200, + "H7", + "residue_name_number" + ], + [ + 202, + 204, + "S9", + "residue_name_number" + ], + [ + 206, + 209, + "I10", + "residue_name_number" + ], + [ + 211, + 214, + "N11", + "residue_name_number" + ], + [ + 216, + 219, + "I15", + "residue_name_number" + ], + [ + 221, + 224, + "Q16", + "residue_name_number" + ], + [ + 226, + 229, + "R18", + "residue_name_number" + ], + [ + 231, + 234, + "D36", + "residue_name_number" + ], + [ + 236, + 239, + "R43", + "residue_name_number" + ], + [ + 241, + 244, + "A46", + "residue_name_number" + ], + [ + 246, + 249, + "Q59", + "residue_name_number" + ], + [ + 251, + 254, + "C61", + "residue_name_number" + ], + [ + 256, + 260, + "Y104", + "residue_name_number" + ], + [ + 262, + 266, + "D112", + "residue_name_number" + ], + [ + 268, + 272, + "R114", + "residue_name_number" + ], + [ + 274, + 278, + "Y115", + "residue_name_number" + ], + [ + 280, + 284, + "D116", + "residue_name_number" + ], + [ + 286, + 290, + "E119", + "residue_name_number" + ], + [ + 292, + 296, + "K126", + "residue_name_number" + ], + [ + 298, + 302, + "E136", + "residue_name_number" + ], + [ + 304, + 308, + "E141", + "residue_name_number" + ], + [ + 310, + 314, + "N145", + "residue_name_number" + ], + [ + 324, + 356, + "hydrophobic packing interactions", + "bond_interaction" + ], + [ + 367, + 370, + "I10", + "residue_name_number" + ], + [ + 372, + 375, + "I12", + "residue_name_number" + ], + [ + 377, + 380, + "L14", + "residue_name_number" + ], + [ + 382, + 385, + "I15", + "residue_name_number" + ], + [ + 387, + 390, + "R18", + "residue_name_number" + ], + [ + 392, + 396, + "Y115", + "residue_name_number" + ], + [ + 398, + 402, + "I118", + "residue_name_number" + ], + [ + 404, + 408, + "L130", + "residue_name_number" + ], + [ + 410, + 414, + "L133", + "residue_name_number" + ], + [ + 416, + 420, + "L134", + "residue_name_number" + ], + [ + 425, + 429, + "L137", + "residue_name_number" + ] + ] + }, + { + "sid": 103, + "sent": "Chain B, grey surface, is marked blue to highlight residues probed by site-directed mutagenesis (E136 only makes a salt bridge with K126, therefore it was sufficient to make the K126A mutation to assess the importance of this ionic interaction; the H7 position is labelled for monomer A, since electron density was lacking for monomer B). (B) A zoom into the environment of helix \u03b16 to show how residue L130 chain B (blue side chain) is a focus of hydrophobic packing interactions with L130, L133, L134 and L137 of chain A (red side chains).", + "section": "FIG", + "ner": [ + [ + 0, + 7, + "Chain B", + "structure_element" + ], + [ + 70, + 95, + "site-directed mutagenesis", + "experimental_method" + ], + [ + 97, + 101, + "E136", + "residue_name_number" + ], + [ + 115, + 126, + "salt bridge", + "bond_interaction" + ], + [ + 132, + 136, + "K126", + "residue_name_number" + ], + [ + 178, + 183, + "K126A", + "mutant" + ], + [ + 226, + 243, + "ionic interaction", + "bond_interaction" + ], + [ + 249, + 251, + "H7", + "residue_name_number" + ], + [ + 277, + 284, + "monomer", + "oligomeric_state" + ], + [ + 285, + 286, + "A", + "structure_element" + ], + [ + 294, + 310, + "electron density", + "evidence" + ], + [ + 327, + 334, + "monomer", + "oligomeric_state" + ], + [ + 335, + 336, + "B", + "structure_element" + ], + [ + 374, + 379, + "helix", + "structure_element" + ], + [ + 380, + 382, + "\u03b16", + "structure_element" + ], + [ + 403, + 407, + "L130", + "residue_name_number" + ], + [ + 408, + 415, + "chain B", + "structure_element" + ], + [ + 448, + 480, + "hydrophobic packing interactions", + "bond_interaction" + ], + [ + 486, + 490, + "L130", + "residue_name_number" + ], + [ + 492, + 496, + "L133", + "residue_name_number" + ], + [ + 498, + 502, + "L134", + "residue_name_number" + ], + [ + 507, + 511, + "L137", + "residue_name_number" + ], + [ + 515, + 522, + "chain A", + "structure_element" + ] + ] + }, + { + "sid": 104, + "sent": "(C) SE-HPLC analyses of all mutant forms of NadR are compared with the wild-type (WT) protein.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "SE-HPLC", + "experimental_method" + ], + [ + 28, + 34, + "mutant", + "protein_state" + ], + [ + 44, + 48, + "NadR", + "protein" + ], + [ + 71, + 80, + "wild-type", + "protein_state" + ], + [ + 82, + 84, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 105, + "sent": "The WT and most of the mutants show a single elution peak with an absorbance maximum at 17.5 min.", + "section": "FIG", + "ner": [ + [ + 4, + 6, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 106, + "sent": "Only the mutation L130K has a noteworthy effect on the oligomeric state, inducing a second peak with a longer retention time and a second peak maximum at 18.6 min.", + "section": "FIG", + "ner": [ + [ + 18, + 23, + "L130K", + "mutant" + ] + ] + }, + { + "sid": 107, + "sent": "To a much lesser extent, the L133K mutation also appears to induce a \u2018shoulder\u2019 to the main peak, suggesting very weak ability to disrupt the dimer. (D) SE-HPLC/MALLS analyses of the L130K mutant, shows 20% dimer and 80% monomer.", + "section": "FIG", + "ner": [ + [ + 29, + 34, + "L133K", + "mutant" + ], + [ + 142, + 147, + "dimer", + "oligomeric_state" + ], + [ + 153, + 166, + "SE-HPLC/MALLS", + "experimental_method" + ], + [ + 183, + 188, + "L130K", + "mutant" + ], + [ + 189, + 195, + "mutant", + "protein_state" + ], + [ + 207, + 212, + "dimer", + "oligomeric_state" + ], + [ + 221, + 228, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 108, + "sent": "The holo-NadR structure presents only one occupied ligand-binding pocket", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "holo", + "protein_state" + ], + [ + 9, + 13, + "NadR", + "protein" + ], + [ + 14, + 23, + "structure", + "evidence" + ], + [ + 51, + 72, + "ligand-binding pocket", + "site" + ] + ] + }, + { + "sid": 109, + "sent": "The NadR/4-HPA structure revealed the ligand-binding site nestled between the dimerization and DNA-binding domains (Fig 2).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "NadR/4-HPA", + "complex_assembly" + ], + [ + 15, + 24, + "structure", + "evidence" + ], + [ + 38, + 57, + "ligand-binding site", + "site" + ], + [ + 78, + 114, + "dimerization and DNA-binding domains", + "structure_element" + ] + ] + }, + { + "sid": 110, + "sent": "The ligand showed a different position and orientation compared to salicylate complexed with MTH313 and ST1710 (see Discussion).", + "section": "RESULTS", + "ner": [ + [ + 67, + 77, + "salicylate", + "chemical" + ], + [ + 78, + 92, + "complexed with", + "protein_state" + ], + [ + 93, + 99, + "MTH313", + "protein" + ], + [ + 104, + 110, + "ST1710", + "protein" + ] + ] + }, + { + "sid": 111, + "sent": "The binding pocket was almost entirely filled by 4-HPA and one water molecule, although there also remained a small tunnel 2-4\u00c5 in diameter and 5-6\u00c5 long leading from the pocket (proximal to the 4-hydroxyl position) to the protein surface.", + "section": "RESULTS", + "ner": [ + [ + 4, + 18, + "binding pocket", + "site" + ], + [ + 49, + 54, + "4-HPA", + "chemical" + ], + [ + 63, + 68, + "water", + "chemical" + ], + [ + 116, + 122, + "tunnel", + "site" + ], + [ + 171, + 177, + "pocket", + "site" + ] + ] + }, + { + "sid": 112, + "sent": "The tunnel was lined with rather hydrophobic amino acids, and did not contain water molecules.", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "tunnel", + "site" + ], + [ + 78, + 83, + "water", + "chemical" + ] + ] + }, + { + "sid": 113, + "sent": "Unexpectedly, only one monomer of the holo-NadR homodimer contained 4-HPA in the binding pocket, whereas the corresponding pocket of the other monomer was unoccupied by ligand, despite the large excess of 4-HPA used in the crystallization conditions.", + "section": "RESULTS", + "ner": [ + [ + 23, + 30, + "monomer", + "oligomeric_state" + ], + [ + 38, + 42, + "holo", + "protein_state" + ], + [ + 43, + 47, + "NadR", + "protein" + ], + [ + 48, + 57, + "homodimer", + "oligomeric_state" + ], + [ + 68, + 73, + "4-HPA", + "chemical" + ], + [ + 81, + 95, + "binding pocket", + "site" + ], + [ + 123, + 129, + "pocket", + "site" + ], + [ + 143, + 150, + "monomer", + "oligomeric_state" + ], + [ + 205, + 210, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 114, + "sent": "Inspection of the protein-ligand interaction network revealed no bonds from NadR backbone groups to the ligand, but several key side chain mediated hydrogen (H)-bonds and ionic interactions, most notably between the carboxylate group of 4-HPA and Ser9 of chain A (SerA9), and chain B residues TrpB39, ArgB43 and TyrB115 (Fig 4A).", + "section": "RESULTS", + "ner": [ + [ + 18, + 52, + "protein-ligand interaction network", + "site" + ], + [ + 76, + 80, + "NadR", + "protein" + ], + [ + 148, + 166, + "hydrogen (H)-bonds", + "bond_interaction" + ], + [ + 171, + 189, + "ionic interactions", + "bond_interaction" + ], + [ + 237, + 242, + "4-HPA", + "chemical" + ], + [ + 247, + 251, + "Ser9", + "residue_name_number" + ], + [ + 255, + 262, + "chain A", + "structure_element" + ], + [ + 264, + 269, + "SerA9", + "residue_name_number" + ], + [ + 276, + 283, + "chain B", + "structure_element" + ], + [ + 293, + 299, + "TrpB39", + "residue_name_number" + ], + [ + 301, + 307, + "ArgB43", + "residue_name_number" + ], + [ + 312, + 319, + "TyrB115", + "residue_name_number" + ] + ] + }, + { + "sid": 115, + "sent": "At the other \u2018end\u2019 of the ligand, the 4-hydroxyl group was proximal to AspB36, with which it may establish an H-bond (see bond distances in Table 3).", + "section": "RESULTS", + "ner": [ + [ + 71, + 77, + "AspB36", + "residue_name_number" + ], + [ + 110, + 116, + "H-bond", + "bond_interaction" + ] + ] + }, + { + "sid": 116, + "sent": "The water molecule observed in the pocket was bound by the carboxylate group and the side chains of SerA9 and AsnA11.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "water", + "chemical" + ], + [ + 100, + 105, + "SerA9", + "residue_name_number" + ], + [ + 110, + 116, + "AsnA11", + "residue_name_number" + ] + ] + }, + { + "sid": 117, + "sent": "Atomic details of NadR/HPA interactions.", + "section": "FIG", + "ner": [ + [ + 18, + 22, + "NadR", + "protein" + ], + [ + 23, + 26, + "HPA", + "chemical" + ] + ] + }, + { + "sid": 118, + "sent": " A) A stereo-view zoom into the binding pocket showing side chain sticks for all interactions between NadR and 4-HPA.", + "section": "FIG", + "ner": [ + [ + 32, + 46, + "binding pocket", + "site" + ], + [ + 102, + 106, + "NadR", + "protein" + ], + [ + 111, + 116, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 119, + "sent": "Green and blue ribbons depict NadR chains A and B, respectively.", + "section": "FIG", + "ner": [ + [ + 30, + 34, + "NadR", + "protein" + ], + [ + 35, + 49, + "chains A and B", + "structure_element" + ] + ] + }, + { + "sid": 120, + "sent": "4-HPA is shown in yellow sticks, with oxygen atoms in red.", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 121, + "sent": "A water molecule is shown by the red sphere.", + "section": "FIG", + "ner": [ + [ + 2, + 7, + "water", + "chemical" + ] + ] + }, + { + "sid": 122, + "sent": "H-bonds up to 3.6\u00c5 are shown as dashed lines.", + "section": "FIG", + "ner": [ + [ + 0, + 7, + "H-bonds", + "bond_interaction" + ] + ] + }, + { + "sid": 123, + "sent": "The entire set of residues making H-bonds or non-bonded contacts with 4-HPA is as follows: SerA9, AsnA11, LeuB21, MetB22, PheB25, LeuB29, AspB36, TrpB39, ArgB43, ValB111 and TyrB115 (automated analysis performed using PDBsum and verified manually).", + "section": "FIG", + "ner": [ + [ + 34, + 41, + "H-bonds", + "bond_interaction" + ], + [ + 45, + 64, + "non-bonded contacts", + "bond_interaction" + ], + [ + 70, + 75, + "4-HPA", + "chemical" + ], + [ + 91, + 96, + "SerA9", + "residue_name_number" + ], + [ + 98, + 104, + "AsnA11", + "residue_name_number" + ], + [ + 106, + 112, + "LeuB21", + "residue_name_number" + ], + [ + 114, + 120, + "MetB22", + "residue_name_number" + ], + [ + 122, + 128, + "PheB25", + "residue_name_number" + ], + [ + 130, + 136, + "LeuB29", + "residue_name_number" + ], + [ + 138, + 144, + "AspB36", + "residue_name_number" + ], + [ + 146, + 152, + "TrpB39", + "residue_name_number" + ], + [ + 154, + 160, + "ArgB43", + "residue_name_number" + ], + [ + 162, + 169, + "ValB111", + "residue_name_number" + ], + [ + 174, + 181, + "TyrB115", + "residue_name_number" + ], + [ + 218, + 224, + "PDBsum", + "experimental_method" + ] + ] + }, + { + "sid": 124, + "sent": "Residues AsnA11 and ArgB18 likely make indirect yet local contributions to ligand binding, mainly by stabilizing the position of AspB36.", + "section": "FIG", + "ner": [ + [ + 9, + 15, + "AsnA11", + "residue_name_number" + ], + [ + 20, + 26, + "ArgB18", + "residue_name_number" + ], + [ + 129, + 135, + "AspB36", + "residue_name_number" + ] + ] + }, + { + "sid": 125, + "sent": "Side chains mediating hydrophobic interactions are shown in orange. (B) A model was prepared to visualize putative interactions of 3Cl,4-HPA (pink) with NadR, revealing the potential for additional contacts (dashed lines) of the chloro moiety (green stick) with LeuB29 and AspB36.", + "section": "FIG", + "ner": [ + [ + 22, + 46, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 131, + 140, + "3Cl,4-HPA", + "chemical" + ], + [ + 153, + 157, + "NadR", + "protein" + ], + [ + 262, + 268, + "LeuB29", + "residue_name_number" + ], + [ + 273, + 279, + "AspB36", + "residue_name_number" + ] + ] + }, + { + "sid": 126, + "sent": "List of 4-HPA atoms bound to NadR via ionic interactions and/or H-bonds.", + "section": "TABLE", + "ner": [ + [ + 8, + 13, + "4-HPA", + "chemical" + ], + [ + 29, + 33, + "NadR", + "protein" + ], + [ + 38, + 56, + "ionic interactions", + "bond_interaction" + ], + [ + 64, + 71, + "H-bonds", + "bond_interaction" + ] + ] + }, + { + "sid": 127, + "sent": "4-HPA atom\tNadR residue/atom\tDistance (\u00c5)\t \tO2\tTrpB39/NE1\t2.83\t \tO2\tArgB43/NH1\t2.76\t \tO1\tArgB43/NH1\t3.84\t \tO1\tSerA9/OG\t2.75\t \tO1\tTyrB115/OH\t2.50\t \tO2\tWater (*Ser9/Asn11)\t2.88\t \tOH\tAspB36/OD1/OD2\t3.6/3.7\t \t", + "section": "TABLE", + "ner": [ + [ + 0, + 5, + "4-HPA", + "chemical" + ], + [ + 11, + 15, + "NadR", + "protein" + ], + [ + 47, + 53, + "TrpB39", + "residue_name_number" + ], + [ + 68, + 74, + "ArgB43", + "residue_name_number" + ], + [ + 89, + 95, + "ArgB43", + "residue_name_number" + ], + [ + 110, + 115, + "SerA9", + "residue_name_number" + ], + [ + 129, + 136, + "TyrB115", + "residue_name_number" + ], + [ + 150, + 155, + "Water", + "chemical" + ], + [ + 158, + 162, + "Ser9", + "residue_name_number" + ], + [ + 163, + 168, + "Asn11", + "residue_name_number" + ], + [ + 180, + 186, + "AspB36", + "residue_name_number" + ] + ] + }, + { + "sid": 128, + "sent": "* Bond distance between the ligand carboxylate group and the water molecule, which in turn makes H-bond to the SerA9 and AsnA11 side chains.", + "section": "TABLE", + "ner": [ + [ + 61, + 66, + "water", + "chemical" + ], + [ + 97, + 103, + "H-bond", + "bond_interaction" + ], + [ + 111, + 116, + "SerA9", + "residue_name_number" + ], + [ + 121, + 127, + "AsnA11", + "residue_name_number" + ] + ] + }, + { + "sid": 129, + "sent": "In addition to the H-bonds involving the carboxylate and hydroxyl groups of 4-HPA, binding of the phenyl moiety appeared to be stabilized by several van der Waals\u2019 contacts, particularly those involving the hydrophobic side chain atoms of LeuB21, MetB22, PheB25, LeuB29 and ValB111 (Fig 4A).", + "section": "RESULTS", + "ner": [ + [ + 19, + 26, + "H-bonds", + "bond_interaction" + ], + [ + 76, + 81, + "4-HPA", + "chemical" + ], + [ + 149, + 172, + "van der Waals\u2019 contacts", + "bond_interaction" + ], + [ + 239, + 245, + "LeuB21", + "residue_name_number" + ], + [ + 247, + 253, + "MetB22", + "residue_name_number" + ], + [ + 255, + 261, + "PheB25", + "residue_name_number" + ], + [ + 263, + 269, + "LeuB29", + "residue_name_number" + ], + [ + 274, + 281, + "ValB111", + "residue_name_number" + ] + ] + }, + { + "sid": 130, + "sent": "Notably, the phenyl ring of PheB25 was positioned parallel to the phenyl ring of 4-HPA, potentially forming \u03c0-\u03c0 parallel-displaced stacking interactions.", + "section": "RESULTS", + "ner": [ + [ + 28, + 34, + "PheB25", + "residue_name_number" + ], + [ + 81, + 86, + "4-HPA", + "chemical" + ], + [ + 108, + 152, + "\u03c0-\u03c0 parallel-displaced stacking interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 131, + "sent": "Consequently, residues in the 4-HPA binding pocket are mostly contributed by NadR chain B, and effectively created a polar \u2018floor\u2019 and a hydrophobic \u2018ceiling\u2019, which house the ligand.", + "section": "RESULTS", + "ner": [ + [ + 30, + 50, + "4-HPA binding pocket", + "site" + ], + [ + 77, + 81, + "NadR", + "protein" + ], + [ + 82, + 89, + "chain B", + "structure_element" + ] + ] + }, + { + "sid": 132, + "sent": "Collectively, this mixed network of polar and hydrophobic interactions endows NadR with a strong recognition pattern for HPAs, with additional medium-range interactions potentially established with the hydroxyl group at the 4-position.", + "section": "RESULTS", + "ner": [ + [ + 36, + 70, + "polar and hydrophobic interactions", + "bond_interaction" + ], + [ + 78, + 82, + "NadR", + "protein" + ], + [ + 121, + 125, + "HPAs", + "chemical" + ] + ] + }, + { + "sid": 133, + "sent": "Structure-activity relationships: molecular basis of enhanced stabilization by 3Cl,4-HPA", + "section": "RESULTS", + "ner": [ + [ + 79, + 88, + "3Cl,4-HPA", + "chemical" + ] + ] + }, + { + "sid": 134, + "sent": "We modelled the binding of other HPAs by in silico superposition onto 4-HPA in the holo-NadR structure, and thereby obtained molecular explanations for the binding specificities of diverse ligands.", + "section": "RESULTS", + "ner": [ + [ + 3, + 11, + "modelled", + "experimental_method" + ], + [ + 33, + 37, + "HPAs", + "chemical" + ], + [ + 41, + 64, + "in silico superposition", + "experimental_method" + ], + [ + 70, + 75, + "4-HPA", + "chemical" + ], + [ + 83, + 87, + "holo", + "protein_state" + ], + [ + 88, + 92, + "NadR", + "protein" + ], + [ + 93, + 102, + "structure", + "evidence" + ] + ] + }, + { + "sid": 135, + "sent": "For example, similar to 4-HPA, the binding of 3Cl,4-HPA could involve multiple bonds towards the carboxylate group of the ligand and some to the 4-hydroxyl group.", + "section": "RESULTS", + "ner": [ + [ + 24, + 29, + "4-HPA", + "chemical" + ], + [ + 46, + 55, + "3Cl,4-HPA", + "chemical" + ] + ] + }, + { + "sid": 136, + "sent": "Additionally, the side chains of LeuB29 and AspB36 would be only 2.6\u20133.5 \u00c5 from the chlorine atom, thus providing van der Waals\u2019 interactions or H-bonds to generate the additional binding affinity observed for 3Cl,4-HPA (Fig 4B).", + "section": "RESULTS", + "ner": [ + [ + 33, + 39, + "LeuB29", + "residue_name_number" + ], + [ + 44, + 50, + "AspB36", + "residue_name_number" + ], + [ + 114, + 141, + "van der Waals\u2019 interactions", + "bond_interaction" + ], + [ + 145, + 152, + "H-bonds", + "bond_interaction" + ], + [ + 180, + 196, + "binding affinity", + "evidence" + ], + [ + 210, + 219, + "3Cl,4-HPA", + "chemical" + ] + ] + }, + { + "sid": 137, + "sent": "The presence of a single hydroxyl group at position 2, as in 2-HPA, rather than at position 4, would eliminate the possibility of favorable interactions with AspB36, resulting in the lack of NadR regulation by 2-HPA described previously.", + "section": "RESULTS", + "ner": [ + [ + 61, + 66, + "2-HPA", + "chemical" + ], + [ + 158, + 164, + "AspB36", + "residue_name_number" + ], + [ + 191, + 195, + "NadR", + "protein" + ], + [ + 210, + 215, + "2-HPA", + "chemical" + ] + ] + }, + { + "sid": 138, + "sent": "Finally, salicylate is presumably unable to specifically bind NadR due to the 2-hydroxyl substitution and the shorter aliphatic chain connecting its carboxylate group (Fig 1A): the compound simply seems too small to simultaneously establish the network of beneficial bonds observed in the NadR/HPA interactions.", + "section": "RESULTS", + "ner": [ + [ + 9, + 19, + "salicylate", + "chemical" + ], + [ + 62, + 66, + "NadR", + "protein" + ], + [ + 289, + 293, + "NadR", + "protein" + ], + [ + 294, + 297, + "HPA", + "chemical" + ] + ] + }, + { + "sid": 139, + "sent": "Analysis of the pockets reveals the molecular basis for asymmetric binding and stoichiometry", + "section": "RESULTS", + "ner": [ + [ + 16, + 23, + "pockets", + "site" + ] + ] + }, + { + "sid": 140, + "sent": "However, studies based on tryptophan fluorescence were confounded by the fluorescence of the HPA ligands, and isothermal titration calorimetry (ITC) was unfeasible due to the need for very high concentrations of NadR in the ITC chamber (due to the relatively low affinity), which exceeded the solubility limits of the protein.", + "section": "RESULTS", + "ner": [ + [ + 26, + 49, + "tryptophan fluorescence", + "experimental_method" + ], + [ + 93, + 96, + "HPA", + "chemical" + ], + [ + 110, + 142, + "isothermal titration calorimetry", + "experimental_method" + ], + [ + 144, + 147, + "ITC", + "experimental_method" + ], + [ + 212, + 216, + "NadR", + "protein" + ], + [ + 224, + 227, + "ITC", + "experimental_method" + ] + ] + }, + { + "sid": 141, + "sent": "However, it was possible to calculate the binding stoichiometry of the NadR-HPA interactions using an SPR-based approach.", + "section": "RESULTS", + "ner": [ + [ + 42, + 63, + "binding stoichiometry", + "evidence" + ], + [ + 71, + 79, + "NadR-HPA", + "complex_assembly" + ], + [ + 102, + 105, + "SPR", + "experimental_method" + ] + ] + }, + { + "sid": 142, + "sent": "In SPR, the signal measured is proportional to the total molecular mass proximal to the sensor surface; consequently, if the molecular weights of the interactors are known, then the stoichiometry of the resulting complex can be determined.", + "section": "RESULTS", + "ner": [ + [ + 3, + 6, + "SPR", + "experimental_method" + ] + ] + }, + { + "sid": 143, + "sent": "This approach relies on the assumption that the captured protein (\u2018the ligand\u2019, according to SPR conventions) is 100% active and freely-accessible to potential interactors (\u2018the analytes\u2019).", + "section": "RESULTS", + "ner": [ + [ + 93, + 96, + "SPR", + "experimental_method" + ] + ] + }, + { + "sid": 144, + "sent": "Firstly, NadR is expected to be covalently immobilized on the sensor chip as a dimer in random orientations, since it is a stable dimer in solution and has sixteen lysines well-distributed around its surface, all able to act as potential sites for amine coupling to the chip, and none of which are close to the ligand-binding pocket.", + "section": "RESULTS", + "ner": [ + [ + 9, + 13, + "NadR", + "protein" + ], + [ + 79, + 84, + "dimer", + "oligomeric_state" + ], + [ + 123, + 129, + "stable", + "protein_state" + ], + [ + 130, + 135, + "dimer", + "oligomeric_state" + ], + [ + 164, + 171, + "lysines", + "residue_name" + ], + [ + 311, + 332, + "ligand-binding pocket", + "site" + ] + ] + }, + { + "sid": 145, + "sent": "Secondly, the HPA analytes are all very small (MW 150\u2013170, Fig 1A) and therefore are expected to be able to diffuse readily into all potential binding sites, irrespective of the random orientations of the immobilized NadR dimers on the chip.", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "HPA", + "chemical" + ], + [ + 143, + 156, + "binding sites", + "site" + ], + [ + 217, + 221, + "NadR", + "protein" + ], + [ + 222, + 228, + "dimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 146, + "sent": "The stoichiometry of the NadR-HPA interactions was determined using Eq 1 (see Materials and Methods), and revealed stoichiometries of 1.13 for 4-HPA, 1.02 for 3-HPA, and 1.21 for 3Cl,4-HPA, strongly suggesting that one NadR dimer bound to 1 HPA analyte molecule.", + "section": "RESULTS", + "ner": [ + [ + 25, + 33, + "NadR-HPA", + "complex_assembly" + ], + [ + 115, + 130, + "stoichiometries", + "evidence" + ], + [ + 143, + 148, + "4-HPA", + "chemical" + ], + [ + 159, + 164, + "3-HPA", + "chemical" + ], + [ + 179, + 188, + "3Cl,4-HPA", + "chemical" + ], + [ + 219, + 223, + "NadR", + "protein" + ], + [ + 224, + 229, + "dimer", + "oligomeric_state" + ], + [ + 230, + 238, + "bound to", + "protein_state" + ], + [ + 241, + 244, + "HPA", + "chemical" + ] + ] + }, + { + "sid": 147, + "sent": "The crystallographic data, supported by the SPR studies of binding stoichiometry, revealed the lack of a second 4-HPA molecule in the homodimer, suggesting negative co-operativity, a phenomenon previously described for the MTH313/salicylate interaction and for other MarR family proteins.", + "section": "RESULTS", + "ner": [ + [ + 4, + 25, + "crystallographic data", + "evidence" + ], + [ + 44, + 47, + "SPR", + "experimental_method" + ], + [ + 59, + 80, + "binding stoichiometry", + "evidence" + ], + [ + 112, + 117, + "4-HPA", + "chemical" + ], + [ + 134, + 143, + "homodimer", + "oligomeric_state" + ], + [ + 223, + 229, + "MTH313", + "protein" + ], + [ + 230, + 240, + "salicylate", + "chemical" + ], + [ + 267, + 271, + "MarR", + "protein_type" + ] + ] + }, + { + "sid": 148, + "sent": "To explore the molecular basis of asymmetry in holo-NadR, we superposed its ligand-free monomer (chain A) onto the ligand-occupied monomer (chain B).", + "section": "RESULTS", + "ner": [ + [ + 47, + 51, + "holo", + "protein_state" + ], + [ + 52, + 56, + "NadR", + "protein" + ], + [ + 61, + 71, + "superposed", + "experimental_method" + ], + [ + 76, + 87, + "ligand-free", + "protein_state" + ], + [ + 88, + 95, + "monomer", + "oligomeric_state" + ], + [ + 97, + 104, + "chain A", + "structure_element" + ], + [ + 115, + 130, + "ligand-occupied", + "protein_state" + ], + [ + 131, + 138, + "monomer", + "oligomeric_state" + ], + [ + 140, + 147, + "chain B", + "structure_element" + ] + ] + }, + { + "sid": 149, + "sent": "Overall, the superposition revealed a high degree of structural similarity (C\u03b1 root mean square deviation (rmsd) of 1.5\u00c5), though on closer inspection a rotational difference of ~9 degrees along the long axis of helix \u03b16 was observed, suggesting that 4-HPA induced a slight conformational change (Fig 5A).", + "section": "RESULTS", + "ner": [ + [ + 13, + 26, + "superposition", + "experimental_method" + ], + [ + 79, + 105, + "root mean square deviation", + "evidence" + ], + [ + 107, + 111, + "rmsd", + "evidence" + ], + [ + 212, + 217, + "helix", + "structure_element" + ], + [ + 218, + 220, + "\u03b16", + "structure_element" + ], + [ + 251, + 256, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 150, + "sent": "However, since residues of helix \u03b16 were not directly involved in ligand binding, an explanation for the lack of 4-HPA in monomer A did not emerge by analyzing only these backbone atom positions, suggesting that a more complex series of allosteric events may occur.", + "section": "RESULTS", + "ner": [ + [ + 27, + 32, + "helix", + "structure_element" + ], + [ + 33, + 35, + "\u03b16", + "structure_element" + ], + [ + 113, + 118, + "4-HPA", + "chemical" + ], + [ + 122, + 129, + "monomer", + "oligomeric_state" + ], + [ + 130, + 131, + "A", + "structure_element" + ] + ] + }, + { + "sid": 151, + "sent": "Indeed, we noted interesting differences in the side chains of Met22, Phe25 and Arg43, which in monomer B are used to contact the ligand while in monomer A they partially occupied the pocket and collectively reduced its volume significantly.", + "section": "RESULTS", + "ner": [ + [ + 63, + 68, + "Met22", + "residue_name_number" + ], + [ + 70, + 75, + "Phe25", + "residue_name_number" + ], + [ + 80, + 85, + "Arg43", + "residue_name_number" + ], + [ + 96, + 103, + "monomer", + "oligomeric_state" + ], + [ + 104, + 105, + "B", + "structure_element" + ], + [ + 146, + 153, + "monomer", + "oligomeric_state" + ], + [ + 154, + 155, + "A", + "structure_element" + ], + [ + 184, + 190, + "pocket", + "site" + ] + ] + }, + { + "sid": 152, + "sent": "Specifically, upon analysis with the CASTp software, the pocket in chain B containing the 4-HPA exhibited a total volume of approximately 370 \u00c53, while the pocket in chain A was occupied by these three side chains that adopted \u2018inward\u2019 positions and thereby divided the space into a few much smaller pockets, each with volume < 50 \u00c53, evidently rendering chain A unfavorable for ligand binding.", + "section": "RESULTS", + "ner": [ + [ + 37, + 42, + "CASTp", + "experimental_method" + ], + [ + 57, + 63, + "pocket", + "site" + ], + [ + 67, + 74, + "chain B", + "structure_element" + ], + [ + 90, + 95, + "4-HPA", + "chemical" + ], + [ + 156, + 162, + "pocket", + "site" + ], + [ + 166, + 173, + "chain A", + "structure_element" + ], + [ + 228, + 234, + "inward", + "protein_state" + ], + [ + 355, + 362, + "chain A", + "structure_element" + ] + ] + }, + { + "sid": 153, + "sent": "Most notably, atomic clashes between the ligand and the side chains of MetA22, PheA25 and ArgA43 would occur if 4-HPA were present in the monomer A pocket (Fig 5B).", + "section": "RESULTS", + "ner": [ + [ + 71, + 77, + "MetA22", + "residue_name_number" + ], + [ + 79, + 85, + "PheA25", + "residue_name_number" + ], + [ + 90, + 96, + "ArgA43", + "residue_name_number" + ], + [ + 112, + 117, + "4-HPA", + "chemical" + ], + [ + 138, + 145, + "monomer", + "oligomeric_state" + ], + [ + 146, + 147, + "A", + "structure_element" + ], + [ + 148, + 154, + "pocket", + "site" + ] + ] + }, + { + "sid": 154, + "sent": "Subsequently, analyses of the pockets in apo-NadR revealed that in the absence of ligand the long Arg43 side chain was always in the open \u2018outward\u2019 position compatible with binding to the 4-HPA carboxylate group.", + "section": "RESULTS", + "ner": [ + [ + 30, + 37, + "pockets", + "site" + ], + [ + 41, + 44, + "apo", + "protein_state" + ], + [ + 45, + 49, + "NadR", + "protein" + ], + [ + 71, + 88, + "absence of ligand", + "protein_state" + ], + [ + 98, + 103, + "Arg43", + "residue_name_number" + ], + [ + 139, + 146, + "outward", + "protein_state" + ], + [ + 188, + 193, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 155, + "sent": "In contrast, the apo-form Met22 and Phe25 residues were still encroaching the spaces of the 4-hydroxyl group and the phenyl ring of the ligand, respectively (Fig 5C).", + "section": "RESULTS", + "ner": [ + [ + 17, + 20, + "apo", + "protein_state" + ], + [ + 26, + 31, + "Met22", + "residue_name_number" + ], + [ + 36, + 41, + "Phe25", + "residue_name_number" + ] + ] + }, + { + "sid": 156, + "sent": "The \u2018outward\u2019 position of Arg43 generated an open apo-form pocket with volume approximately 380\u00c53.", + "section": "RESULTS", + "ner": [ + [ + 5, + 12, + "outward", + "protein_state" + ], + [ + 26, + 31, + "Arg43", + "residue_name_number" + ], + [ + 45, + 49, + "open", + "protein_state" + ], + [ + 50, + 53, + "apo", + "protein_state" + ], + [ + 59, + 65, + "pocket", + "site" + ] + ] + }, + { + "sid": 157, + "sent": "Taken together, these observations suggest that Arg43 is a major determinant of ligand binding, and that its \u2018inward\u2019 position inhibits the binding of 4-HPA to the empty pocket of holo-NadR.", + "section": "RESULTS", + "ner": [ + [ + 48, + 53, + "Arg43", + "residue_name_number" + ], + [ + 110, + 116, + "inward", + "protein_state" + ], + [ + 151, + 156, + "4-HPA", + "chemical" + ], + [ + 170, + 176, + "pocket", + "site" + ], + [ + 180, + 184, + "holo", + "protein_state" + ], + [ + 185, + 189, + "NadR", + "protein" + ] + ] + }, + { + "sid": 158, + "sent": "Structural differences of NadR in ligand-bound or free forms.", + "section": "FIG", + "ner": [ + [ + 26, + 30, + "NadR", + "protein" + ], + [ + 34, + 46, + "ligand-bound", + "protein_state" + ], + [ + 50, + 54, + "free", + "protein_state" + ] + ] + }, + { + "sid": 159, + "sent": " (A) Aligned monomers of holo-NadR (chain A: green; chain B: blue), reveal major overall differences by the shift of helix \u03b16. (B) Comparison of the two binding pockets in holo-NadR shows that in the ligand-free monomer A (green) residues Met22, Phe25 and Arg43 adopt \u2018inward\u2019 positions (highlighted by arrows) compared to the ligand-occupied pocket (blue residues); these \u2018inward\u2019 conformations appear unfavorable for binding of 4-HPA due to clashes with the 4-hydroxyl group, the phenyl ring and the carboxylate group, respectively.", + "section": "FIG", + "ner": [ + [ + 5, + 12, + "Aligned", + "experimental_method" + ], + [ + 13, + 21, + "monomers", + "oligomeric_state" + ], + [ + 25, + 29, + "holo", + "protein_state" + ], + [ + 30, + 34, + "NadR", + "protein" + ], + [ + 36, + 43, + "chain A", + "structure_element" + ], + [ + 52, + 59, + "chain B", + "structure_element" + ], + [ + 117, + 122, + "helix", + "structure_element" + ], + [ + 123, + 125, + "\u03b16", + "structure_element" + ], + [ + 131, + 141, + "Comparison", + "experimental_method" + ], + [ + 153, + 168, + "binding pockets", + "site" + ], + [ + 172, + 176, + "holo", + "protein_state" + ], + [ + 177, + 181, + "NadR", + "protein" + ], + [ + 200, + 211, + "ligand-free", + "protein_state" + ], + [ + 212, + 219, + "monomer", + "oligomeric_state" + ], + [ + 220, + 221, + "A", + "structure_element" + ], + [ + 239, + 244, + "Met22", + "residue_name_number" + ], + [ + 246, + 251, + "Phe25", + "residue_name_number" + ], + [ + 256, + 261, + "Arg43", + "residue_name_number" + ], + [ + 269, + 275, + "inward", + "protein_state" + ], + [ + 327, + 342, + "ligand-occupied", + "protein_state" + ], + [ + 343, + 349, + "pocket", + "site" + ], + [ + 374, + 380, + "inward", + "protein_state" + ], + [ + 430, + 435, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 160, + "sent": "In these crystals, the ArgA43 side chain showed two alternate conformations, modelled with 50% occupancy in each state, as indicated by the two \u2018mirrored\u2019 arrows.", + "section": "FIG", + "ner": [ + [ + 9, + 17, + "crystals", + "evidence" + ], + [ + 23, + 29, + "ArgA43", + "residue_name_number" + ] + ] + }, + { + "sid": 161, + "sent": "The inner conformer is the one that would display major clashes if 4-HPA were present. (C) Comparison of the empty pocket from holo-NadR (green residues) with the four empty pockets of apo-NadR (grey residues), shows that in the absence of 4-HPA the Arg43 side chain is always observed in the \u2018outward\u2019 conformation.", + "section": "FIG", + "ner": [ + [ + 67, + 72, + "4-HPA", + "chemical" + ], + [ + 115, + 121, + "pocket", + "site" + ], + [ + 127, + 131, + "holo", + "protein_state" + ], + [ + 132, + 136, + "NadR", + "protein" + ], + [ + 174, + 181, + "pockets", + "site" + ], + [ + 185, + 188, + "apo", + "protein_state" + ], + [ + 189, + 193, + "NadR", + "protein" + ], + [ + 229, + 239, + "absence of", + "protein_state" + ], + [ + 240, + 245, + "4-HPA", + "chemical" + ], + [ + 250, + 255, + "Arg43", + "residue_name_number" + ], + [ + 294, + 301, + "outward", + "protein_state" + ] + ] + }, + { + "sid": 162, + "sent": "Finally, we applied 15N heteronuclear solution NMR spectroscopy to examine the interaction of 4-HPA with apo NadR. We collected NMR spectra on NadR in the presence and absence of 4-HPA (see Materials and Methods).", + "section": "RESULTS", + "ner": [ + [ + 20, + 63, + "15N heteronuclear solution NMR spectroscopy", + "experimental_method" + ], + [ + 94, + 99, + "4-HPA", + "chemical" + ], + [ + 105, + 108, + "apo", + "protein_state" + ], + [ + 109, + 113, + "NadR", + "protein" + ], + [ + 128, + 131, + "NMR", + "experimental_method" + ], + [ + 132, + 139, + "spectra", + "evidence" + ], + [ + 143, + 147, + "NadR", + "protein" + ], + [ + 148, + 163, + "in the presence", + "protein_state" + ], + [ + 168, + 178, + "absence of", + "protein_state" + ], + [ + 179, + 184, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 163, + "sent": "The 1H-15N TROSY-HSQC spectrum of apo-NadR, acquired at 25\u00b0C, displayed approximately 140 distinct peaks (Fig 6A), most of which correspond to backbone amide N-H groups.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "1H-15N TROSY-HSQC", + "experimental_method" + ], + [ + 22, + 30, + "spectrum", + "evidence" + ], + [ + 34, + 37, + "apo", + "protein_state" + ], + [ + 38, + 42, + "NadR", + "protein" + ] + ] + }, + { + "sid": 164, + "sent": "The broad spectral dispersion and the number of peaks observed, which is close to the number of expected backbone amide N-H groups for this polypeptide, confirmed that apo-NadR is well-folded under these conditions and exhibits one conformation appreciable on the NMR timescale, i.e. in the NMR experiments at 25\u00b0C, two or more distinct conformations of apo-NadR monomers were not readily apparent.", + "section": "RESULTS", + "ner": [ + [ + 168, + 171, + "apo", + "protein_state" + ], + [ + 172, + 176, + "NadR", + "protein" + ], + [ + 180, + 191, + "well-folded", + "protein_state" + ], + [ + 264, + 267, + "NMR", + "experimental_method" + ], + [ + 291, + 294, + "NMR", + "experimental_method" + ], + [ + 354, + 357, + "apo", + "protein_state" + ], + [ + 358, + 362, + "NadR", + "protein" + ], + [ + 363, + 371, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 165, + "sent": "Upon the addition of 4-HPA, over 45 peaks showed chemical shift perturbations, i.e. changed position in the spectrum or disappeared, while the remaining peaks remained unchanged.", + "section": "RESULTS", + "ner": [ + [ + 21, + 26, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 166, + "sent": "This observation showed that 4-HPA was able to bind NadR and induce notable changes in specific regions of the protein.", + "section": "RESULTS", + "ner": [ + [ + 29, + 34, + "4-HPA", + "chemical" + ], + [ + 52, + 56, + "NadR", + "protein" + ] + ] + }, + { + "sid": 167, + "sent": "NMR spectra of NadR in the presence and absence of 4-HPA.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "NMR", + "experimental_method" + ], + [ + 4, + 11, + "spectra", + "evidence" + ], + [ + 15, + 19, + "NadR", + "protein" + ], + [ + 20, + 35, + "in the presence", + "protein_state" + ], + [ + 40, + 50, + "absence of", + "protein_state" + ], + [ + 51, + 56, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 168, + "sent": " (A) Superposition of two 1H-15N TROSY-HSQC spectra recorded at 25\u00b0C on apo-NadR (cyan) and on NadR in the presence of 4-HPA (red).", + "section": "FIG", + "ner": [ + [ + 5, + 18, + "Superposition", + "experimental_method" + ], + [ + 26, + 43, + "1H-15N TROSY-HSQC", + "experimental_method" + ], + [ + 44, + 51, + "spectra", + "evidence" + ], + [ + 72, + 75, + "apo", + "protein_state" + ], + [ + 76, + 80, + "NadR", + "protein" + ], + [ + 95, + 99, + "NadR", + "protein" + ], + [ + 107, + 118, + "presence of", + "protein_state" + ], + [ + 119, + 124, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 169, + "sent": "(B,C) Overlay of selected regions of the 1H-15N TROSY-HSQC spectra acquired at 25\u00b0C of apo-NadR (cyan) and NadR/4-HPA (red) superimposed with the spectra acquired at 10\u00b0C of apo-NadR (blue) and NadR/4-HPA (green).", + "section": "FIG", + "ner": [ + [ + 6, + 13, + "Overlay", + "experimental_method" + ], + [ + 41, + 58, + "1H-15N TROSY-HSQC", + "experimental_method" + ], + [ + 59, + 66, + "spectra", + "evidence" + ], + [ + 87, + 90, + "apo", + "protein_state" + ], + [ + 91, + 95, + "NadR", + "protein" + ], + [ + 107, + 117, + "NadR/4-HPA", + "complex_assembly" + ], + [ + 124, + 136, + "superimposed", + "experimental_method" + ], + [ + 146, + 153, + "spectra", + "evidence" + ], + [ + 174, + 177, + "apo", + "protein_state" + ], + [ + 178, + 182, + "NadR", + "protein" + ], + [ + 194, + 204, + "NadR/4-HPA", + "complex_assembly" + ] + ] + }, + { + "sid": 170, + "sent": "The spectra acquired at 10\u00b0C are excluded from panel A for simplicity.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "spectra", + "evidence" + ] + ] + }, + { + "sid": 171, + "sent": "However, in the presence of 4-HPA, the 1H-15N TROSY-HSQC spectrum of NadR displayed approximately 140 peaks, as for apo-NadR, i.e. two distinct stable conformations (that might have potentially revealed the molecular asymmetry observed crystallographically) were not notable.", + "section": "RESULTS", + "ner": [ + [ + 16, + 27, + "presence of", + "protein_state" + ], + [ + 28, + 33, + "4-HPA", + "chemical" + ], + [ + 39, + 56, + "1H-15N TROSY-HSQC", + "experimental_method" + ], + [ + 57, + 65, + "spectrum", + "evidence" + ], + [ + 69, + 73, + "NadR", + "protein" + ], + [ + 116, + 119, + "apo", + "protein_state" + ], + [ + 120, + 124, + "NadR", + "protein" + ], + [ + 236, + 256, + "crystallographically", + "experimental_method" + ] + ] + }, + { + "sid": 172, + "sent": "Considering the small size, fast diffusion and relatively low binding affinity of 4-HPA, it would not be surprising if the ligand associates and dissociates rapidly on the NMR time scale, resulting in only one set of peaks whose chemical shifts represent the average environment of the bound and unbound states.", + "section": "RESULTS", + "ner": [ + [ + 62, + 78, + "binding affinity", + "evidence" + ], + [ + 82, + 87, + "4-HPA", + "chemical" + ], + [ + 172, + 175, + "NMR", + "experimental_method" + ], + [ + 286, + 291, + "bound", + "protein_state" + ], + [ + 296, + 303, + "unbound", + "protein_state" + ] + ] + }, + { + "sid": 173, + "sent": "Interestingly, by cooling the samples to 10\u00b0C, we observed that a number of those peaks strongly affected by 4-HPA (and therefore likely to be in the ligand-binding site) demonstrated evidence of peak splitting, i.e. a tendency to become two distinct peaks rather than one single peak (Fig 6B and 6C).", + "section": "RESULTS", + "ner": [ + [ + 109, + 114, + "4-HPA", + "chemical" + ], + [ + 150, + 169, + "ligand-binding site", + "site" + ] + ] + }, + { + "sid": 174, + "sent": "These doubled peaks may therefore reveal that the cooler temperature partially trapped the existence in solution of two distinct states, in presence or absence of 4-HPA, with minor conformational differences occurring at least in proximity to the binding pocket.", + "section": "RESULTS", + "ner": [ + [ + 140, + 148, + "presence", + "protein_state" + ], + [ + 152, + 162, + "absence of", + "protein_state" + ], + [ + 163, + 168, + "4-HPA", + "chemical" + ], + [ + 247, + 261, + "binding pocket", + "site" + ] + ] + }, + { + "sid": 175, + "sent": "Although more comprehensive NMR experiments and full chemical shift assignment of the spectra would be required to precisely define this multi-state behavior, the NMR data clearly demonstrate that NadR exhibits conformational flexibility which is modulated by 4-HPA in solution.", + "section": "RESULTS", + "ner": [ + [ + 28, + 31, + "NMR", + "experimental_method" + ], + [ + 86, + 93, + "spectra", + "evidence" + ], + [ + 163, + 166, + "NMR", + "experimental_method" + ], + [ + 197, + 201, + "NadR", + "protein" + ], + [ + 260, + 265, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 176, + "sent": "Apo-NadR structures reveal intrinsic conformational flexibility", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "Apo", + "protein_state" + ], + [ + 4, + 8, + "NadR", + "protein" + ], + [ + 9, + 19, + "structures", + "evidence" + ] + ] + }, + { + "sid": 177, + "sent": "The apo-NadR crystal structure contained two homodimers in the asymmetric unit (chains A+B and chains C+D).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "apo", + "protein_state" + ], + [ + 8, + 12, + "NadR", + "protein" + ], + [ + 13, + 30, + "crystal structure", + "evidence" + ], + [ + 45, + 55, + "homodimers", + "oligomeric_state" + ], + [ + 80, + 90, + "chains A+B", + "structure_element" + ], + [ + 95, + 105, + "chains C+D", + "structure_element" + ] + ] + }, + { + "sid": 178, + "sent": "Upon overall structural superposition, these dimers revealed a few minor differences in the \u03b16 helix (a major component of the dimer interface) and the helices \u03b14-\u03b15 (the DNA binding region), and an rmsd of 1.55\u00c5 (Fig 7A).", + "section": "RESULTS", + "ner": [ + [ + 13, + 37, + "structural superposition", + "experimental_method" + ], + [ + 45, + 51, + "dimers", + "oligomeric_state" + ], + [ + 92, + 100, + "\u03b16 helix", + "structure_element" + ], + [ + 127, + 142, + "dimer interface", + "site" + ], + [ + 152, + 159, + "helices", + "structure_element" + ], + [ + 160, + 165, + "\u03b14-\u03b15", + "structure_element" + ], + [ + 171, + 189, + "DNA binding region", + "site" + ], + [ + 199, + 203, + "rmsd", + "evidence" + ] + ] + }, + { + "sid": 179, + "sent": "Similarly, the entire holo-homodimer could be closely superposed onto each of the apo-homodimers, showing rmsd values of 1.29\u00c5 and 1.31\u00c5, and with more notable differences in the \u03b16 helix positions (Fig 7B).", + "section": "RESULTS", + "ner": [ + [ + 22, + 26, + "holo", + "protein_state" + ], + [ + 27, + 36, + "homodimer", + "oligomeric_state" + ], + [ + 46, + 64, + "closely superposed", + "experimental_method" + ], + [ + 82, + 85, + "apo", + "protein_state" + ], + [ + 86, + 96, + "homodimers", + "oligomeric_state" + ], + [ + 106, + 110, + "rmsd", + "evidence" + ], + [ + 179, + 187, + "\u03b16 helix", + "structure_element" + ] + ] + }, + { + "sid": 180, + "sent": "The slightly larger rmsd between the two apo-homodimers, rather than between apo- and holo-homodimers, further indicate that apo-NadR possesses a notable degree of intrinsic conformational flexibility.", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "rmsd", + "evidence" + ], + [ + 41, + 44, + "apo", + "protein_state" + ], + [ + 45, + 55, + "homodimers", + "oligomeric_state" + ], + [ + 77, + 80, + "apo", + "protein_state" + ], + [ + 86, + 90, + "holo", + "protein_state" + ], + [ + 91, + 101, + "homodimers", + "oligomeric_state" + ], + [ + 125, + 128, + "apo", + "protein_state" + ], + [ + 129, + 133, + "NadR", + "protein" + ] + ] + }, + { + "sid": 181, + "sent": "Overall apo- and holo-NadR structures are similar.", + "section": "FIG", + "ner": [ + [ + 8, + 11, + "apo", + "protein_state" + ], + [ + 17, + 21, + "holo", + "protein_state" + ], + [ + 22, + 26, + "NadR", + "protein" + ], + [ + 27, + 37, + "structures", + "evidence" + ] + ] + }, + { + "sid": 182, + "sent": " (A) Pairwise alignment of the two distinct apo-NadR homodimers (AB and CD) present in the apo-NadR crystals. (B) Alignment of the holo-NadR homodimer (green and blue chains) onto the apo-NadR homodimers.", + "section": "FIG", + "ner": [ + [ + 5, + 23, + "Pairwise alignment", + "experimental_method" + ], + [ + 44, + 47, + "apo", + "protein_state" + ], + [ + 48, + 52, + "NadR", + "protein" + ], + [ + 53, + 63, + "homodimers", + "oligomeric_state" + ], + [ + 65, + 67, + "AB", + "structure_element" + ], + [ + 72, + 74, + "CD", + "structure_element" + ], + [ + 91, + 94, + "apo", + "protein_state" + ], + [ + 95, + 99, + "NadR", + "protein" + ], + [ + 100, + 108, + "crystals", + "evidence" + ], + [ + 114, + 123, + "Alignment", + "experimental_method" + ], + [ + 131, + 135, + "holo", + "protein_state" + ], + [ + 136, + 140, + "NadR", + "protein" + ], + [ + 141, + 150, + "homodimer", + "oligomeric_state" + ], + [ + 184, + 187, + "apo", + "protein_state" + ], + [ + 188, + 192, + "NadR", + "protein" + ], + [ + 193, + 203, + "homodimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 183, + "sent": "Here, larger differences are observed in the \u03b16 helices (top).", + "section": "FIG", + "ner": [ + [ + 45, + 55, + "\u03b16 helices", + "structure_element" + ] + ] + }, + { + "sid": 184, + "sent": "4-HPA stabilizes concerted conformational changes in NadR that prevent DNA-binding", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "4-HPA", + "chemical" + ], + [ + 53, + 57, + "NadR", + "protein" + ] + ] + }, + { + "sid": 185, + "sent": "To further investigate the conformational rearrangements of NadR, we performed local structural alignments using only a subset of residues in the DNA-binding helix (\u03b14).", + "section": "RESULTS", + "ner": [ + [ + 60, + 64, + "NadR", + "protein" + ], + [ + 79, + 106, + "local structural alignments", + "experimental_method" + ], + [ + 146, + 163, + "DNA-binding helix", + "structure_element" + ], + [ + 165, + 167, + "\u03b14", + "structure_element" + ] + ] + }, + { + "sid": 186, + "sent": "By selecting and aligning residues Arg64-Ala77 of one \u03b14 helix per dimer, superposition of the holo-homodimer onto the two apo-homodimers revealed differences in the monomer conformations of each structure.", + "section": "RESULTS", + "ner": [ + [ + 3, + 12, + "selecting", + "experimental_method" + ], + [ + 17, + 25, + "aligning", + "experimental_method" + ], + [ + 35, + 46, + "Arg64-Ala77", + "residue_range" + ], + [ + 54, + 62, + "\u03b14 helix", + "structure_element" + ], + [ + 67, + 72, + "dimer", + "oligomeric_state" + ], + [ + 74, + 87, + "superposition", + "experimental_method" + ], + [ + 95, + 99, + "holo", + "protein_state" + ], + [ + 100, + 109, + "homodimer", + "oligomeric_state" + ], + [ + 123, + 126, + "apo", + "protein_state" + ], + [ + 127, + 137, + "homodimers", + "oligomeric_state" + ], + [ + 166, + 173, + "monomer", + "oligomeric_state" + ], + [ + 196, + 205, + "structure", + "evidence" + ] + ] + }, + { + "sid": 187, + "sent": "While one monomer from each structure was closely superimposable (Fig 8A, left side), the second monomer displayed quite large differences (Fig 8A, right side).", + "section": "RESULTS", + "ner": [ + [ + 10, + 17, + "monomer", + "oligomeric_state" + ], + [ + 28, + 37, + "structure", + "evidence" + ], + [ + 97, + 104, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 188, + "sent": "Most notably, the position of the DNA-binding helix \u03b14 shifted by as much as 6 \u00c5 (Fig 8B).", + "section": "RESULTS", + "ner": [ + [ + 34, + 37, + "DNA", + "chemical" + ], + [ + 46, + 51, + "helix", + "structure_element" + ], + [ + 52, + 54, + "\u03b14", + "structure_element" + ] + ] + }, + { + "sid": 189, + "sent": "Accordingly, helix \u03b14 was also found to be one of the most dynamic regions in previous HDX-MS analyses of apo-NadR in solution.", + "section": "RESULTS", + "ner": [ + [ + 13, + 18, + "helix", + "structure_element" + ], + [ + 19, + 21, + "\u03b14", + "structure_element" + ], + [ + 87, + 93, + "HDX-MS", + "experimental_method" + ], + [ + 106, + 109, + "apo", + "protein_state" + ], + [ + 110, + 114, + "NadR", + "protein" + ] + ] + }, + { + "sid": 190, + "sent": "Structural comparisons of NadR and modelling of interactions with DNA.", + "section": "FIG", + "ner": [ + [ + 0, + 22, + "Structural comparisons", + "experimental_method" + ], + [ + 26, + 30, + "NadR", + "protein" + ], + [ + 66, + 69, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 191, + "sent": " (A) The holo-homodimer structure is shown as green and blue cartoons, for chain A and B, respectively, while the two homodimers of apo-NadR are both cyan and pale blue for chains A/C and B/D, respectively.", + "section": "FIG", + "ner": [ + [ + 9, + 13, + "holo", + "protein_state" + ], + [ + 14, + 23, + "homodimer", + "oligomeric_state" + ], + [ + 24, + 33, + "structure", + "evidence" + ], + [ + 75, + 88, + "chain A and B", + "structure_element" + ], + [ + 118, + 128, + "homodimers", + "oligomeric_state" + ], + [ + 132, + 135, + "apo", + "protein_state" + ], + [ + 136, + 140, + "NadR", + "protein" + ], + [ + 180, + 183, + "A/C", + "structure_element" + ], + [ + 188, + 191, + "B/D", + "structure_element" + ] + ] + }, + { + "sid": 192, + "sent": "The three homodimers (chains AB holo, AB apo, and CD apo) were overlaid by structural alignment exclusively of all heavy atoms in residues R64-A77 (shown in red, with side chain sticks) of chains A holo, A apo, and C apo, belonging to helix \u03b14 (left).", + "section": "FIG", + "ner": [ + [ + 10, + 20, + "homodimers", + "oligomeric_state" + ], + [ + 29, + 31, + "AB", + "structure_element" + ], + [ + 32, + 36, + "holo", + "protein_state" + ], + [ + 38, + 40, + "AB", + "structure_element" + ], + [ + 41, + 44, + "apo", + "protein_state" + ], + [ + 50, + 52, + "CD", + "structure_element" + ], + [ + 53, + 56, + "apo", + "protein_state" + ], + [ + 63, + 71, + "overlaid", + "experimental_method" + ], + [ + 75, + 95, + "structural alignment", + "experimental_method" + ], + [ + 139, + 146, + "R64-A77", + "residue_range" + ], + [ + 196, + 197, + "A", + "structure_element" + ], + [ + 198, + 202, + "holo", + "protein_state" + ], + [ + 204, + 205, + "A", + "structure_element" + ], + [ + 206, + 209, + "apo", + "protein_state" + ], + [ + 215, + 216, + "C", + "structure_element" + ], + [ + 217, + 220, + "apo", + "protein_state" + ], + [ + 235, + 240, + "helix", + "structure_element" + ], + [ + 241, + 243, + "\u03b14", + "structure_element" + ] + ] + }, + { + "sid": 193, + "sent": "The \u03b14 helices aligned closely, C\u03b1 rmsd 0.2\u00c5 for 14 residues.", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "\u03b14 helices", + "structure_element" + ], + [ + 35, + 39, + "rmsd", + "evidence" + ] + ] + }, + { + "sid": 194, + "sent": "(B) The relative positions of the \u03b14 helices of the 4-HPA-bound holo homodimer chain B (blue), and of apo homodimers AB and CD (showing chains B and D) in pale blue.", + "section": "FIG", + "ner": [ + [ + 34, + 44, + "\u03b14 helices", + "structure_element" + ], + [ + 52, + 63, + "4-HPA-bound", + "protein_state" + ], + [ + 64, + 68, + "holo", + "protein_state" + ], + [ + 69, + 78, + "homodimer", + "oligomeric_state" + ], + [ + 79, + 86, + "chain B", + "structure_element" + ], + [ + 102, + 105, + "apo", + "protein_state" + ], + [ + 106, + 116, + "homodimers", + "oligomeric_state" + ], + [ + 117, + 119, + "AB", + "structure_element" + ], + [ + 124, + 126, + "CD", + "structure_element" + ], + [ + 136, + 150, + "chains B and D", + "structure_element" + ] + ] + }, + { + "sid": 195, + "sent": "Dashes indicate the Ala77 C\u03b1 atoms, in the most highly shifted region of the \u2018non-fixed\u2019 \u03b14 helix.", + "section": "FIG", + "ner": [ + [ + 20, + 25, + "Ala77", + "residue_name_number" + ], + [ + 89, + 97, + "\u03b14 helix", + "structure_element" + ] + ] + }, + { + "sid": 196, + "sent": "(C) The double-stranded DNA molecule (grey cartoon) from the OhrR-ohrA complex is shown after superposition with NadR, to highlight the expected positions of the NadR \u03b14 helices in the DNA major grooves.", + "section": "FIG", + "ner": [ + [ + 24, + 27, + "DNA", + "chemical" + ], + [ + 61, + 70, + "OhrR-ohrA", + "complex_assembly" + ], + [ + 94, + 107, + "superposition", + "experimental_method" + ], + [ + 113, + 117, + "NadR", + "protein" + ], + [ + 162, + 166, + "NadR", + "protein" + ], + [ + 167, + 177, + "\u03b14 helices", + "structure_element" + ], + [ + 185, + 188, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 197, + "sent": "For clarity, only the \u03b14 helices are shown in panels (B) and (C). (D) Upon comparison with the experimentally-determined OhrR:ohrA structure (grey), the \u03b14 helix of holo-NadR (blue) is shifted ~8\u00c5 out of the major groove.", + "section": "FIG", + "ner": [ + [ + 22, + 32, + "\u03b14 helices", + "structure_element" + ], + [ + 121, + 130, + "OhrR:ohrA", + "complex_assembly" + ], + [ + 131, + 140, + "structure", + "evidence" + ], + [ + 153, + 161, + "\u03b14 helix", + "structure_element" + ], + [ + 165, + 169, + "holo", + "protein_state" + ], + [ + 170, + 174, + "NadR", + "protein" + ] + ] + }, + { + "sid": 198, + "sent": "However, structural comparisons revealed that the shift of holo-NadR helix \u03b14 induced by the presence of 4-HPA was also accompanied by several changes at the holo dimer interface, while such extensive structural differences were not observed in the apo dimer interfaces, particularly notable when comparing the \u03b16 helices (S3 Fig).", + "section": "RESULTS", + "ner": [ + [ + 9, + 31, + "structural comparisons", + "experimental_method" + ], + [ + 59, + 63, + "holo", + "protein_state" + ], + [ + 64, + 68, + "NadR", + "protein" + ], + [ + 69, + 74, + "helix", + "structure_element" + ], + [ + 75, + 77, + "\u03b14", + "structure_element" + ], + [ + 93, + 104, + "presence of", + "protein_state" + ], + [ + 105, + 110, + "4-HPA", + "chemical" + ], + [ + 158, + 162, + "holo", + "protein_state" + ], + [ + 163, + 178, + "dimer interface", + "site" + ], + [ + 249, + 252, + "apo", + "protein_state" + ], + [ + 253, + 269, + "dimer interfaces", + "site" + ], + [ + 311, + 321, + "\u03b16 helices", + "structure_element" + ] + ] + }, + { + "sid": 199, + "sent": "In summary, compared to ligand-stabilized holo-NadR, apo-NadR displayed an intrinsic flexibility focused in the DNA-binding region.", + "section": "RESULTS", + "ner": [ + [ + 24, + 41, + "ligand-stabilized", + "protein_state" + ], + [ + 42, + 46, + "holo", + "protein_state" + ], + [ + 47, + 51, + "NadR", + "protein" + ], + [ + 53, + 56, + "apo", + "protein_state" + ], + [ + 57, + 61, + "NadR", + "protein" + ], + [ + 112, + 130, + "DNA-binding region", + "site" + ] + ] + }, + { + "sid": 200, + "sent": "This was also evident in the greater disorder (i.e. less well-defined electron density) in the \u03b21-\u03b22 loops of the apo dimers (density for 16 residues per dimer was missing) compared to the holo dimer (density for only 3 residues was missing).", + "section": "RESULTS", + "ner": [ + [ + 70, + 86, + "electron density", + "evidence" + ], + [ + 95, + 106, + "\u03b21-\u03b22 loops", + "structure_element" + ], + [ + 114, + 117, + "apo", + "protein_state" + ], + [ + 118, + 124, + "dimers", + "oligomeric_state" + ], + [ + 126, + 133, + "density", + "evidence" + ], + [ + 154, + 159, + "dimer", + "oligomeric_state" + ], + [ + 189, + 193, + "holo", + "protein_state" + ], + [ + 194, + 199, + "dimer", + "oligomeric_state" + ], + [ + 201, + 208, + "density", + "evidence" + ] + ] + }, + { + "sid": 201, + "sent": "In holo-NadR, the distance separating the two DNA-binding \u03b14 helices was 32 \u00c5, while in apo-NadR it was 29 \u00c5 for homodimer AB, and 34 \u00c5 for homodimer CD (Fig 8C).", + "section": "RESULTS", + "ner": [ + [ + 3, + 7, + "holo", + "protein_state" + ], + [ + 8, + 12, + "NadR", + "protein" + ], + [ + 58, + 68, + "\u03b14 helices", + "structure_element" + ], + [ + 88, + 91, + "apo", + "protein_state" + ], + [ + 92, + 96, + "NadR", + "protein" + ], + [ + 113, + 122, + "homodimer", + "oligomeric_state" + ], + [ + 123, + 125, + "AB", + "structure_element" + ], + [ + 140, + 149, + "homodimer", + "oligomeric_state" + ], + [ + 150, + 152, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 202, + "sent": "Thus, the apo-homodimer AB presented the DNA-binding helices in a conformation similar to that observed in the protein:DNA complex of OhrR:ohrA from Bacillus subtilis (Fig 8C).", + "section": "RESULTS", + "ner": [ + [ + 10, + 13, + "apo", + "protein_state" + ], + [ + 14, + 23, + "homodimer", + "oligomeric_state" + ], + [ + 24, + 26, + "AB", + "structure_element" + ], + [ + 41, + 60, + "DNA-binding helices", + "structure_element" + ], + [ + 134, + 143, + "OhrR:ohrA", + "complex_assembly" + ], + [ + 149, + 166, + "Bacillus subtilis", + "species" + ] + ] + }, + { + "sid": 203, + "sent": "Interestingly, OhrR contacts ohrA across 22 base pairs (bp), and similarly the main NadR target sites identified in the nadA promoter (the operators Op I and Op II) both span 22 bp.", + "section": "RESULTS", + "ner": [ + [ + 15, + 19, + "OhrR", + "protein" + ], + [ + 29, + 33, + "ohrA", + "gene" + ], + [ + 84, + 88, + "NadR", + "protein" + ], + [ + 89, + 101, + "target sites", + "site" + ], + [ + 120, + 124, + "nadA", + "gene" + ] + ] + }, + { + "sid": 204, + "sent": "Pairwise superpositions showed that the NadR apo-homodimer AB was the most similar to OhrR (rmsd 2.6 \u00c5), while the holo-homodimer was the most divergent (rmsd 3.3 \u00c5) (Fig 8C).", + "section": "RESULTS", + "ner": [ + [ + 0, + 23, + "Pairwise superpositions", + "experimental_method" + ], + [ + 40, + 44, + "NadR", + "protein" + ], + [ + 45, + 48, + "apo", + "protein_state" + ], + [ + 49, + 58, + "homodimer", + "oligomeric_state" + ], + [ + 59, + 61, + "AB", + "structure_element" + ], + [ + 86, + 90, + "OhrR", + "protein" + ], + [ + 92, + 96, + "rmsd", + "evidence" + ], + [ + 115, + 119, + "holo", + "protein_state" + ], + [ + 120, + 129, + "homodimer", + "oligomeric_state" + ], + [ + 154, + 158, + "rmsd", + "evidence" + ] + ] + }, + { + "sid": 205, + "sent": "Assuming the same DNA-binding mechanism is used by OhrR and NadR, the apo-homodimer AB seems ideally pre-configured for DNA binding, while 4-HPA appeared to stabilize holo-NadR in a conformation poorly suited for DNA binding.", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "DNA", + "chemical" + ], + [ + 51, + 55, + "OhrR", + "protein" + ], + [ + 60, + 64, + "NadR", + "protein" + ], + [ + 70, + 73, + "apo", + "protein_state" + ], + [ + 74, + 83, + "homodimer", + "oligomeric_state" + ], + [ + 84, + 86, + "AB", + "structure_element" + ], + [ + 120, + 123, + "DNA", + "chemical" + ], + [ + 139, + 144, + "4-HPA", + "chemical" + ], + [ + 167, + 171, + "holo", + "protein_state" + ], + [ + 172, + 176, + "NadR", + "protein" + ], + [ + 213, + 216, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 206, + "sent": "Specifically, in addition to the different inter-helical translational distances, the \u03b14 helices in the holo-NadR homodimer were also reoriented, resulting in movement of \u03b14 out of the major groove, by up to 8\u00c5, and presumably preventing efficient DNA binding in the presence of 4-HPA (Fig 8D).", + "section": "RESULTS", + "ner": [ + [ + 43, + 80, + "inter-helical translational distances", + "evidence" + ], + [ + 86, + 96, + "\u03b14 helices", + "structure_element" + ], + [ + 104, + 108, + "holo", + "protein_state" + ], + [ + 109, + 113, + "NadR", + "protein" + ], + [ + 114, + 123, + "homodimer", + "oligomeric_state" + ], + [ + 171, + 173, + "\u03b14", + "structure_element" + ], + [ + 248, + 251, + "DNA", + "chemical" + ], + [ + 279, + 284, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 207, + "sent": "When aligned with OhrR, the apo-homodimer CD presented yet another different intermediate conformation (rmsd 2.9\u00c5), apparently not ideally pre-configured for DNA binding, but which in solution can presumably readily adopt the AB conformation due to the intrinsic flexibility described above.", + "section": "RESULTS", + "ner": [ + [ + 5, + 12, + "aligned", + "experimental_method" + ], + [ + 18, + 22, + "OhrR", + "protein" + ], + [ + 28, + 31, + "apo", + "protein_state" + ], + [ + 32, + 41, + "homodimer", + "oligomeric_state" + ], + [ + 42, + 44, + "CD", + "structure_element" + ], + [ + 104, + 108, + "rmsd", + "evidence" + ], + [ + 158, + 161, + "DNA", + "chemical" + ], + [ + 226, + 228, + "AB", + "structure_element" + ] + ] + }, + { + "sid": 208, + "sent": "NadR residues His7, Ser9, Asn11 and Phe25 are essential for regulation of NadA expression in vivo", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "NadR", + "protein" + ], + [ + 14, + 18, + "His7", + "residue_name_number" + ], + [ + 20, + 24, + "Ser9", + "residue_name_number" + ], + [ + 26, + 31, + "Asn11", + "residue_name_number" + ], + [ + 36, + 41, + "Phe25", + "residue_name_number" + ], + [ + 74, + 78, + "NadA", + "protein" + ] + ] + }, + { + "sid": 209, + "sent": "While previous studies had correctly suggested the involvement of several NadR residues in ligand binding, the crystal structures presented here revealed additional residues with previously unknown roles in dimerization and/or binding to 4-HPA.", + "section": "RESULTS", + "ner": [ + [ + 74, + 78, + "NadR", + "protein" + ], + [ + 111, + 129, + "crystal structures", + "evidence" + ], + [ + 238, + 243, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 210, + "sent": "To explore the functional involvement of these residues, we characterized the behavior of four new NadR mutants (H7A, S9A, N11A and F25A) in an in vivo assay using the previously described MC58-\u03941843 nadR-null mutant strain, which was complemented either by wild-type nadR or by the nadR mutants.", + "section": "RESULTS", + "ner": [ + [ + 99, + 103, + "NadR", + "protein" + ], + [ + 113, + 116, + "H7A", + "mutant" + ], + [ + 118, + 121, + "S9A", + "mutant" + ], + [ + 123, + 127, + "N11A", + "mutant" + ], + [ + 132, + 136, + "F25A", + "mutant" + ], + [ + 189, + 199, + "MC58-\u03941843", + "mutant" + ], + [ + 200, + 204, + "nadR", + "gene" + ], + [ + 210, + 216, + "mutant", + "protein_state" + ], + [ + 258, + 267, + "wild-type", + "protein_state" + ], + [ + 268, + 272, + "nadR", + "gene" + ], + [ + 283, + 287, + "nadR", + "gene" + ], + [ + 288, + 295, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 211, + "sent": "NadA protein abundance levels were assessed by Western blotting to evaluate the ability of the NadR mutants to repress the nadA promoter, in the presence or absence of 4-HPA.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "NadA", + "protein" + ], + [ + 47, + 63, + "Western blotting", + "experimental_method" + ], + [ + 95, + 99, + "NadR", + "protein" + ], + [ + 100, + 107, + "mutants", + "protein_state" + ], + [ + 123, + 127, + "nadA", + "gene" + ], + [ + 168, + 173, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 212, + "sent": "The nadR H7A, S9A and F25A complemented strains showed hyper-repression of nadA expression in vivo, i.e. these mutants repressed nadA more efficiently than the NadR WT protein, either in the presence or absence of 4-HPA, while complementation with wild-type nadR resulted in high production of NadA only in the presence of 4-HPA (Fig 9).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "nadR", + "gene" + ], + [ + 9, + 12, + "H7A", + "mutant" + ], + [ + 14, + 17, + "S9A", + "mutant" + ], + [ + 22, + 26, + "F25A", + "mutant" + ], + [ + 75, + 79, + "nadA", + "gene" + ], + [ + 129, + 133, + "nadA", + "gene" + ], + [ + 160, + 164, + "NadR", + "protein" + ], + [ + 165, + 167, + "WT", + "protein_state" + ], + [ + 214, + 219, + "4-HPA", + "chemical" + ], + [ + 248, + 257, + "wild-type", + "protein_state" + ], + [ + 258, + 262, + "nadR", + "gene" + ], + [ + 294, + 298, + "NadA", + "protein" + ], + [ + 323, + 328, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 213, + "sent": "Interestingly, and on the contrary, the nadR N11A complemented strain showed hypo-repression (i.e. exhibited high expression of nadA both in absence and presence of 4-HPA).", + "section": "RESULTS", + "ner": [ + [ + 40, + 44, + "nadR", + "gene" + ], + [ + 45, + 49, + "N11A", + "mutant" + ], + [ + 128, + 132, + "nadA", + "gene" + ], + [ + 165, + 170, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 214, + "sent": "This mutagenesis data revealed that NadR residues His7, Ser9, Asn11 and Phe25 play key roles in the ligand-mediated regulation of NadR; they are each involved in the controlled de-repression of the nadA promoter and synthesis of NadA in response to 4-HPA in vivo.", + "section": "RESULTS", + "ner": [ + [ + 5, + 16, + "mutagenesis", + "experimental_method" + ], + [ + 36, + 40, + "NadR", + "protein" + ], + [ + 50, + 54, + "His7", + "residue_name_number" + ], + [ + 56, + 60, + "Ser9", + "residue_name_number" + ], + [ + 62, + 67, + "Asn11", + "residue_name_number" + ], + [ + 72, + 77, + "Phe25", + "residue_name_number" + ], + [ + 130, + 134, + "NadR", + "protein" + ], + [ + 198, + 202, + "nadA", + "gene" + ], + [ + 229, + 233, + "NadA", + "protein" + ], + [ + 249, + 254, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 215, + "sent": "Structure-based point mutations shed light on ligand-induced regulation of NadR.", + "section": "FIG", + "ner": [ + [ + 0, + 31, + "Structure-based point mutations", + "experimental_method" + ], + [ + 75, + 79, + "NadR", + "protein" + ] + ] + }, + { + "sid": 216, + "sent": "Western blot analyses of wild-type (WT) strain (lanes 1\u20132) or isogenic nadR knockout strains (\u0394NadR) complemented to express the indicated NadR WT or mutant proteins (lanes 3\u201312) or not complemented (lanes 13\u201314), grown in the presence (even lanes) or absence (odd lanes) of 5mM 4-HPA, showing NadA and NadR expression.", + "section": "FIG", + "ner": [ + [ + 0, + 12, + "Western blot", + "experimental_method" + ], + [ + 25, + 34, + "wild-type", + "protein_state" + ], + [ + 36, + 38, + "WT", + "protein_state" + ], + [ + 71, + 75, + "nadR", + "gene" + ], + [ + 94, + 99, + "\u0394NadR", + "mutant" + ], + [ + 139, + 143, + "NadR", + "protein" + ], + [ + 144, + 146, + "WT", + "protein_state" + ], + [ + 150, + 156, + "mutant", + "protein_state" + ], + [ + 279, + 284, + "4-HPA", + "chemical" + ], + [ + 294, + 298, + "NadA", + "protein" + ], + [ + 303, + 307, + "NadR", + "protein" + ] + ] + }, + { + "sid": 217, + "sent": "Complementation of \u0394NadR with WT NadR enables induction of nadA expression by 4-HPA.", + "section": "FIG", + "ner": [ + [ + 19, + 24, + "\u0394NadR", + "mutant" + ], + [ + 30, + 32, + "WT", + "protein_state" + ], + [ + 33, + 37, + "NadR", + "protein" + ], + [ + 59, + 63, + "nadA", + "gene" + ], + [ + 78, + 83, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 218, + "sent": "The H7A, S9A and F25A mutants efficiently repress nadA expression but are less ligand-responsive than WT NadR. The N11A mutant does not efficiently repress nadA expression either in presence or absence of 4-HPA. (The protein abundance levels of the meningococcal factor H binding protein (fHbp) were used as a gel loading control).", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "H7A", + "mutant" + ], + [ + 9, + 12, + "S9A", + "mutant" + ], + [ + 17, + 21, + "F25A", + "mutant" + ], + [ + 50, + 54, + "nadA", + "gene" + ], + [ + 102, + 104, + "WT", + "protein_state" + ], + [ + 105, + 109, + "NadR", + "protein" + ], + [ + 115, + 119, + "N11A", + "mutant" + ], + [ + 120, + 126, + "mutant", + "protein_state" + ], + [ + 156, + 160, + "nadA", + "gene" + ], + [ + 205, + 210, + "4-HPA", + "chemical" + ], + [ + 249, + 262, + "meningococcal", + "taxonomy_domain" + ], + [ + 263, + 287, + "factor H binding protein", + "protein" + ], + [ + 289, + 293, + "fHbp", + "protein" + ] + ] + }, + { + "sid": 219, + "sent": "NadA is a surface-exposed meningococcal protein contributing to pathogenesis, and is one of three main antigens present in the vaccine Bexsero.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "NadA", + "protein" + ], + [ + 26, + 39, + "meningococcal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 220, + "sent": "A detailed understanding of the in vitro repression of nadA expression by the transcriptional regulator NadR is important, both because it is a relevant disease-related model of how small-molecule ligands can regulate MarR family proteins and thereby impact bacterial virulence, and because nadA expression levels are linked to the prediction of vaccine coverage.", + "section": "DISCUSS", + "ner": [ + [ + 55, + 59, + "nadA", + "gene" + ], + [ + 78, + 103, + "transcriptional regulator", + "protein_type" + ], + [ + 104, + 108, + "NadR", + "protein" + ], + [ + 218, + 222, + "MarR", + "protein_type" + ], + [ + 258, + 267, + "bacterial", + "taxonomy_domain" + ], + [ + 291, + 295, + "nadA", + "gene" + ] + ] + }, + { + "sid": 221, + "sent": "The repressive activity of NadR can be relieved by hydroxyphenylacetate (HPA) ligands, and HDX-MS studies previously indicated that 4-HPA stabilizes dimeric NadR in a configuration incompatible with DNA binding.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 31, + "NadR", + "protein" + ], + [ + 51, + 71, + "hydroxyphenylacetate", + "chemical" + ], + [ + 73, + 76, + "HPA", + "chemical" + ], + [ + 91, + 97, + "HDX-MS", + "experimental_method" + ], + [ + 132, + 137, + "4-HPA", + "chemical" + ], + [ + 149, + 156, + "dimeric", + "oligomeric_state" + ], + [ + 157, + 161, + "NadR", + "protein" + ] + ] + }, + { + "sid": 222, + "sent": "Despite these and other studies, the molecular mechanisms by which ligands regulate MarR family proteins are relatively poorly understood and likely differ depending on the specific ligand.", + "section": "DISCUSS", + "ner": [ + [ + 84, + 88, + "MarR", + "protein_type" + ] + ] + }, + { + "sid": 223, + "sent": "Given the importance of NadR-mediated regulation of NadA levels in the contexts of meningococcal pathogenesis, we sought to characterize NadR, and its interaction with ligands, at atomic resolution.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 28, + "NadR", + "protein" + ], + [ + 52, + 56, + "NadA", + "protein" + ], + [ + 83, + 96, + "meningococcal", + "taxonomy_domain" + ], + [ + 137, + 141, + "NadR", + "protein" + ] + ] + }, + { + "sid": 224, + "sent": "Firstly, we confirmed that NadR is dimeric in solution and demonstrated that it retains its dimeric state in the presence of 4-HPA, indicating that induction of a monomeric status is not the manner by which 4-HPA regulates NadR. These observations were in agreement with (i) a previous study of NadR performed using SEC and mass spectrometry, and (ii) crystallographic studies showing that several MarR homologues are dimeric.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 31, + "NadR", + "protein" + ], + [ + 35, + 42, + "dimeric", + "oligomeric_state" + ], + [ + 92, + 99, + "dimeric", + "oligomeric_state" + ], + [ + 113, + 124, + "presence of", + "protein_state" + ], + [ + 125, + 130, + "4-HPA", + "chemical" + ], + [ + 163, + 172, + "monomeric", + "oligomeric_state" + ], + [ + 207, + 212, + "4-HPA", + "chemical" + ], + [ + 223, + 227, + "NadR", + "protein" + ], + [ + 295, + 299, + "NadR", + "protein" + ], + [ + 316, + 319, + "SEC", + "experimental_method" + ], + [ + 324, + 341, + "mass spectrometry", + "experimental_method" + ], + [ + 352, + 376, + "crystallographic studies", + "experimental_method" + ], + [ + 398, + 402, + "MarR", + "protein_type" + ], + [ + 418, + 425, + "dimeric", + "oligomeric_state" + ] + ] + }, + { + "sid": 225, + "sent": "We also used structure-guided site-directed mutagenesis to identify an important conserved residue, Leu130, which stabilizes the NadR dimer interface, knowledge of which may also inform future studies to explore the regulatory mechanisms of other MarR family proteins.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 55, + "structure-guided site-directed mutagenesis", + "experimental_method" + ], + [ + 81, + 90, + "conserved", + "protein_state" + ], + [ + 100, + 106, + "Leu130", + "residue_name_number" + ], + [ + 129, + 133, + "NadR", + "protein" + ], + [ + 134, + 149, + "dimer interface", + "site" + ], + [ + 247, + 251, + "MarR", + "protein_type" + ] + ] + }, + { + "sid": 226, + "sent": "Secondly, we assessed the thermal stability and unfolding of NadR in the presence or absence of ligands.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 43, + "assessed the thermal stability", + "experimental_method" + ], + [ + 61, + 65, + "NadR", + "protein" + ], + [ + 66, + 81, + "in the presence", + "protein_state" + ], + [ + 85, + 95, + "absence of", + "protein_state" + ] + ] + }, + { + "sid": 227, + "sent": "All DSC profiles showed a single peak, suggesting that a single unfolding event simultaneously disrupted the dimer and the monomer.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 7, + "DSC", + "experimental_method" + ], + [ + 8, + 16, + "profiles", + "evidence" + ], + [ + 109, + 114, + "dimer", + "oligomeric_state" + ], + [ + 123, + 130, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 228, + "sent": "HPA ligands specifically increased the stability of NadR. The largest effects were induced by the naturally-occurring compounds 4-HPA and 3Cl,4-HPA, which, in SPR assays, were found to bind NadR with KD values of 1.5 mM and 1.1 mM, respectively.", + "section": "DISCUSS", + "ner": [ + [ + 52, + 56, + "NadR", + "protein" + ], + [ + 128, + 133, + "4-HPA", + "chemical" + ], + [ + 138, + 147, + "3Cl,4-HPA", + "chemical" + ], + [ + 159, + 169, + "SPR assays", + "experimental_method" + ], + [ + 190, + 194, + "NadR", + "protein" + ], + [ + 200, + 202, + "KD", + "evidence" + ] + ] + }, + { + "sid": 229, + "sent": "Although these NadR/HPA interactions appeared rather weak, their distinct affinities and specificities matched their in vitro effects and their biological relevance appears similar to previous proposals that certain small molecules, including some antibiotics, in the millimolar concentration range may be broad inhibitors of MarR family proteins.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 19, + "NadR", + "protein" + ], + [ + 20, + 23, + "HPA", + "chemical" + ], + [ + 326, + 330, + "MarR", + "protein_type" + ] + ] + }, + { + "sid": 230, + "sent": "Indeed, 4-HPA is found in human saliva and 3Cl,4-HPA is produced during inflammatory processes, suggesting that these natural ligands are encountered by N. meningitidis in the mucosa of the oropharynx during infections.", + "section": "DISCUSS", + "ner": [ + [ + 8, + 13, + "4-HPA", + "chemical" + ], + [ + 26, + 31, + "human", + "species" + ], + [ + 43, + 52, + "3Cl,4-HPA", + "chemical" + ], + [ + 153, + 168, + "N. meningitidis", + "species" + ] + ] + }, + { + "sid": 231, + "sent": "It is also possible that NadR responds to currently unidentified HPA analogues.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 29, + "NadR", + "protein" + ], + [ + 65, + 68, + "HPA", + "chemical" + ] + ] + }, + { + "sid": 232, + "sent": "Indeed, in the NadR/4-HPA complex there was a water molecule close to the carboxylate group and also a small unfilled tunnel ~5\u00c5 long, both factors suggesting that alternative larger ligands could occupy the pocket.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 25, + "NadR/4-HPA", + "complex_assembly" + ], + [ + 46, + 51, + "water", + "chemical" + ], + [ + 118, + 124, + "tunnel", + "site" + ] + ] + }, + { + "sid": 233, + "sent": "The ability to respond to various ligands might enable NadR in vivo to orchestrate multiple response mechanisms and modulate expression of genes other than nadA. Ultimately, confirmation of the relevance of each ligand will require a deeper understanding of the available concentration in vivo in the host niche during bacterial colonization and inflammation.", + "section": "DISCUSS", + "ner": [ + [ + 55, + 59, + "NadR", + "protein" + ], + [ + 156, + 160, + "nadA", + "gene" + ], + [ + 319, + 328, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 234, + "sent": "Here, we determined the first crystal structures of apo-NadR and holo-NadR. These experimentally-determined structures enabled a new detailed characterization of the ligand-binding pocket.", + "section": "DISCUSS", + "ner": [ + [ + 30, + 48, + "crystal structures", + "evidence" + ], + [ + 52, + 55, + "apo", + "protein_state" + ], + [ + 56, + 60, + "NadR", + "protein" + ], + [ + 65, + 69, + "holo", + "protein_state" + ], + [ + 70, + 74, + "NadR", + "protein" + ], + [ + 108, + 118, + "structures", + "evidence" + ], + [ + 166, + 187, + "ligand-binding pocket", + "site" + ] + ] + }, + { + "sid": 235, + "sent": "In holo-NadR, 4-HPA interacted directly with at least 11 polar and hydrophobic residues.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 7, + "holo", + "protein_state" + ], + [ + 8, + 12, + "NadR", + "protein" + ], + [ + 14, + 19, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 236, + "sent": "Several, but not all, of these interactions were predicted previously by homology modelling combined with ligand docking in silico.", + "section": "DISCUSS", + "ner": [ + [ + 73, + 91, + "homology modelling", + "experimental_method" + ], + [ + 106, + 120, + "ligand docking", + "experimental_method" + ] + ] + }, + { + "sid": 237, + "sent": "Subsequently, we established the functional importance of His7, Ser9, Asn11 and Phe25 in the in vitro response of meningococcus to 4-HPA, via site-directed mutagenesis.", + "section": "DISCUSS", + "ner": [ + [ + 58, + 62, + "His7", + "residue_name_number" + ], + [ + 64, + 68, + "Ser9", + "residue_name_number" + ], + [ + 70, + 75, + "Asn11", + "residue_name_number" + ], + [ + 80, + 85, + "Phe25", + "residue_name_number" + ], + [ + 114, + 127, + "meningococcus", + "taxonomy_domain" + ], + [ + 131, + 136, + "4-HPA", + "chemical" + ], + [ + 142, + 167, + "site-directed mutagenesis", + "experimental_method" + ] + ] + }, + { + "sid": 238, + "sent": "More unexpectedly, the crystal structure revealed that only one molecule of 4-HPA was bound per NadR dimer.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 40, + "crystal structure", + "evidence" + ], + [ + 76, + 81, + "4-HPA", + "chemical" + ], + [ + 86, + 91, + "bound", + "protein_state" + ], + [ + 96, + 100, + "NadR", + "protein" + ], + [ + 101, + 106, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 239, + "sent": "We confirmed this stoichiometry in solution using SPR methods.", + "section": "DISCUSS", + "ner": [ + [ + 50, + 53, + "SPR", + "experimental_method" + ] + ] + }, + { + "sid": 240, + "sent": "We also used heteronuclear NMR spectroscopy to detect substantial conformational changes of NadR occurring in solution upon addition of 4-HPA.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 43, + "heteronuclear NMR spectroscopy", + "experimental_method" + ], + [ + 92, + 96, + "NadR", + "protein" + ], + [ + 136, + 141, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 241, + "sent": "Moreover, NMR spectra at 10\u00b0C suggested the existence of two distinct conformations of NadR in the vicinity of the ligand-binding pocket.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 13, + "NMR", + "experimental_method" + ], + [ + 14, + 21, + "spectra", + "evidence" + ], + [ + 87, + 91, + "NadR", + "protein" + ], + [ + 115, + 136, + "ligand-binding pocket", + "site" + ] + ] + }, + { + "sid": 242, + "sent": "More powerfully, our unique crystallographic observation of this \u2018occupied vs unoccupied site\u2019 asymmetry in the NadR/4-HPA interaction is, to our knowledge, the first example reported for a MarR family protein.", + "section": "DISCUSS", + "ner": [ + [ + 28, + 56, + "crystallographic observation", + "evidence" + ], + [ + 66, + 74, + "occupied", + "protein_state" + ], + [ + 78, + 88, + "unoccupied", + "protein_state" + ], + [ + 112, + 122, + "NadR/4-HPA", + "complex_assembly" + ], + [ + 190, + 194, + "MarR", + "protein_type" + ] + ] + }, + { + "sid": 243, + "sent": "Structural analyses suggested that \u2018inward\u2019 side chain positions of Met22, Phe25 and especially Arg43 precluded binding of a second ligand molecule.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 19, + "Structural analyses", + "experimental_method" + ], + [ + 36, + 42, + "inward", + "protein_state" + ], + [ + 68, + 73, + "Met22", + "residue_name_number" + ], + [ + 75, + 80, + "Phe25", + "residue_name_number" + ], + [ + 96, + 101, + "Arg43", + "residue_name_number" + ] + ] + }, + { + "sid": 244, + "sent": "Such a mechanism indicates negative cooperativity, which may enhance the ligand-responsiveness of NadR.", + "section": "DISCUSS", + "ner": [ + [ + 98, + 102, + "NadR", + "protein" + ] + ] + }, + { + "sid": 245, + "sent": "Comparisons of the NadR/4-HPA complex with available MarR family/salicylate complexes revealed that 4-HPA has a previously unobserved binding mode.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 29, + "NadR/4-HPA", + "complex_assembly" + ], + [ + 53, + 57, + "MarR", + "protein_type" + ], + [ + 65, + 75, + "salicylate", + "chemical" + ], + [ + 100, + 105, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 246, + "sent": "Briefly, in the M. thermoautotrophicum MTH313 dimer, one molecule of salicylate binds in the pocket of each monomer, though with two rather different positions and orientations, only one of which (site-1) is thought to be biologically relevant (Fig 10A).", + "section": "DISCUSS", + "ner": [ + [ + 16, + 38, + "M. thermoautotrophicum", + "species" + ], + [ + 39, + 45, + "MTH313", + "protein" + ], + [ + 46, + 51, + "dimer", + "oligomeric_state" + ], + [ + 69, + 79, + "salicylate", + "chemical" + ], + [ + 93, + 99, + "pocket", + "site" + ], + [ + 108, + 115, + "monomer", + "oligomeric_state" + ], + [ + 197, + 203, + "site-1", + "site" + ] + ] + }, + { + "sid": 247, + "sent": "In the S. tokodaii protein ST1710, salicylate binds to the same position in each monomer of the dimer, in a site equivalent to the putative biologically relevant site of MTH313 (Fig 10B).", + "section": "DISCUSS", + "ner": [ + [ + 7, + 18, + "S. tokodaii", + "species" + ], + [ + 27, + 33, + "ST1710", + "protein" + ], + [ + 35, + 45, + "salicylate", + "chemical" + ], + [ + 81, + 88, + "monomer", + "oligomeric_state" + ], + [ + 96, + 101, + "dimer", + "oligomeric_state" + ], + [ + 170, + 176, + "MTH313", + "protein" + ] + ] + }, + { + "sid": 248, + "sent": "Unlike other MarR family proteins which revealed multiple ligand binding interactions, we observed only 1 molecule of 4-HPA bound to NadR, suggesting a more specific and less promiscuous interaction.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 17, + "MarR", + "protein_type" + ], + [ + 118, + 123, + "4-HPA", + "chemical" + ], + [ + 124, + 132, + "bound to", + "protein_state" + ], + [ + 133, + 137, + "NadR", + "protein" + ] + ] + }, + { + "sid": 249, + "sent": "In NadR, the single molecule of 4-HPA binds in a position distinctly different from the salicylate binding site: translated by > 10 \u00c5 and with a 180\u00b0 inverted orientation (Fig 10C).", + "section": "DISCUSS", + "ner": [ + [ + 3, + 7, + "NadR", + "protein" + ], + [ + 32, + 37, + "4-HPA", + "chemical" + ], + [ + 88, + 111, + "salicylate binding site", + "site" + ] + ] + }, + { + "sid": 250, + "sent": "NadR shows a ligand binding site distinct from other MarR homologues.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "NadR", + "protein" + ], + [ + 13, + 32, + "ligand binding site", + "site" + ], + [ + 53, + 57, + "MarR", + "protein_type" + ] + ] + }, + { + "sid": 251, + "sent": " (A) A structural alignment of MTH313 chains A and B shows that salicylate is bound in distinct locations in each monomer; site-1 (thought to be the biologically relevant site) and site-2 differ by ~7\u00c5 (indicated by black dotted line) and also by ligand orientation.", + "section": "FIG", + "ner": [ + [ + 7, + 27, + "structural alignment", + "experimental_method" + ], + [ + 31, + 37, + "MTH313", + "protein" + ], + [ + 38, + 52, + "chains A and B", + "structure_element" + ], + [ + 64, + 74, + "salicylate", + "chemical" + ], + [ + 78, + 83, + "bound", + "protein_state" + ], + [ + 114, + 121, + "monomer", + "oligomeric_state" + ], + [ + 123, + 129, + "site-1", + "site" + ], + [ + 181, + 187, + "site-2", + "site" + ] + ] + }, + { + "sid": 252, + "sent": "(B) A structural alignment of MTH313 chain A and ST1710 (pink) (C\u03b1 rmsd 2.3\u00c5), shows that they bind salicylate in equivalent sites (differing by only ~3\u00c5) and with the same orientation.", + "section": "FIG", + "ner": [ + [ + 6, + 26, + "structural alignment", + "experimental_method" + ], + [ + 30, + 36, + "MTH313", + "protein" + ], + [ + 37, + 44, + "chain A", + "structure_element" + ], + [ + 49, + 55, + "ST1710", + "protein" + ], + [ + 67, + 71, + "rmsd", + "evidence" + ], + [ + 100, + 110, + "salicylate", + "chemical" + ] + ] + }, + { + "sid": 253, + "sent": "(C) Addition of holo-NadR (chain B, blue) to the alignment reveals that bound 4-HPA differs in position by > 10 \u00c5 compared to salicylate, and adopts a novel orientation.", + "section": "FIG", + "ner": [ + [ + 16, + 20, + "holo", + "protein_state" + ], + [ + 21, + 25, + "NadR", + "protein" + ], + [ + 27, + 34, + "chain B", + "structure_element" + ], + [ + 49, + 58, + "alignment", + "experimental_method" + ], + [ + 72, + 77, + "bound", + "protein_state" + ], + [ + 78, + 83, + "4-HPA", + "chemical" + ], + [ + 126, + 136, + "salicylate", + "chemical" + ] + ] + }, + { + "sid": 254, + "sent": "Interestingly, a crystal structure was previously reported for a functionally-uncharacterized meningococcal homologue of NadR, termed NMB1585, which shares 16% sequence identity with NadR. The two structures can be closely aligned (rmsd 2.3 \u00c5), but NMB1585 appears unsuited for binding HPAs, since its corresponding \u2018pocket\u2019 region is occupied by several bulky hydrophobic side chains.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 34, + "crystal structure", + "evidence" + ], + [ + 94, + 107, + "meningococcal", + "taxonomy_domain" + ], + [ + 121, + 125, + "NadR", + "protein" + ], + [ + 134, + 141, + "NMB1585", + "protein" + ], + [ + 183, + 187, + "NadR", + "protein" + ], + [ + 197, + 207, + "structures", + "evidence" + ], + [ + 232, + 236, + "rmsd", + "evidence" + ], + [ + 249, + 256, + "NMB1585", + "protein" + ], + [ + 286, + 290, + "HPAs", + "chemical" + ], + [ + 316, + 323, + "\u2018pocket", + "site" + ] + ] + }, + { + "sid": 255, + "sent": "It can be speculated that MarR family members have evolved separately to engage distinct signaling molecules, thus enabling bacteria to use the overall conserved MarR scaffold to adapt and respond to diverse changing environmental stimuli experienced in their natural niches.", + "section": "DISCUSS", + "ner": [ + [ + 26, + 30, + "MarR", + "protein_type" + ], + [ + 124, + 132, + "bacteria", + "taxonomy_domain" + ], + [ + 162, + 166, + "MarR", + "protein_type" + ] + ] + }, + { + "sid": 256, + "sent": "Alternatively, it is possible that other MarR homologues (e.g. NMB1585) may have no extant functional binding pocket and thus may have lost the ability to respond to a ligand, acting instead as constitutive DNA-binding regulatory proteins.", + "section": "DISCUSS", + "ner": [ + [ + 41, + 45, + "MarR", + "protein_type" + ], + [ + 63, + 70, + "NMB1585", + "protein" + ], + [ + 102, + 116, + "binding pocket", + "site" + ], + [ + 207, + 210, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 257, + "sent": "The apo-NadR crystal structures revealed two dimers with slightly different conformations, most divergent in the DNA-binding domain.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 7, + "apo", + "protein_state" + ], + [ + 8, + 12, + "NadR", + "protein" + ], + [ + 13, + 31, + "crystal structures", + "evidence" + ], + [ + 45, + 51, + "dimers", + "oligomeric_state" + ], + [ + 113, + 131, + "DNA-binding domain", + "structure_element" + ] + ] + }, + { + "sid": 258, + "sent": "It is not unusual for a crystal structure to reveal multiple copies of the same protein in very slightly different conformations, which are likely representative of the lowest-energy conformations sampled by the dynamic ensemble of molecular states occurring in solution, and which likely have only small energetic differences, as described previously for MexR (a MarR protein) or more recently for the solute-binding protein FhuD2.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 41, + "crystal structure", + "evidence" + ], + [ + 356, + 360, + "MexR", + "protein" + ], + [ + 364, + 368, + "MarR", + "protein_type" + ], + [ + 403, + 425, + "solute-binding protein", + "protein_type" + ], + [ + 426, + 431, + "FhuD2", + "protein" + ] + ] + }, + { + "sid": 259, + "sent": "Further, the holo-NadR structure was overall more different from the two apo-NadR structures (rmsd values ~1.3\u00c5), suggesting that the ligand selected and stabilized yet another conformation of NadR. These observations suggest that 4-HPA, and potentially other similar ligands, can shift the molecular equilibrium, changing the energy barriers that separate active and inactive states, and stabilizing the specific conformation of NadR poorly suited to bind DNA.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 17, + "holo", + "protein_state" + ], + [ + 18, + 22, + "NadR", + "protein" + ], + [ + 23, + 32, + "structure", + "evidence" + ], + [ + 73, + 76, + "apo", + "protein_state" + ], + [ + 77, + 81, + "NadR", + "protein" + ], + [ + 82, + 92, + "structures", + "evidence" + ], + [ + 94, + 98, + "rmsd", + "evidence" + ], + [ + 193, + 197, + "NadR", + "protein" + ], + [ + 231, + 236, + "4-HPA", + "chemical" + ], + [ + 357, + 363, + "active", + "protein_state" + ], + [ + 368, + 376, + "inactive", + "protein_state" + ], + [ + 430, + 434, + "NadR", + "protein" + ], + [ + 457, + 460, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 260, + "sent": "Comparisons of the apo- and holo-NadR structures revealed that the largest differences occurred in the DNA-binding helix \u03b14.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 22, + "apo", + "protein_state" + ], + [ + 28, + 32, + "holo", + "protein_state" + ], + [ + 33, + 37, + "NadR", + "protein" + ], + [ + 38, + 48, + "structures", + "evidence" + ], + [ + 103, + 106, + "DNA", + "chemical" + ], + [ + 115, + 120, + "helix", + "structure_element" + ], + [ + 121, + 123, + "\u03b14", + "structure_element" + ] + ] + }, + { + "sid": 261, + "sent": "The shift of helix \u03b14 in holo-NadR was also accompanied by rearrangements at the dimer interface, involving helices \u03b11, \u03b15, and \u03b16, and this holo-form appeared poorly suited for DNA-binding when compared with the known OhrR:ohrA complex.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 18, + "helix", + "structure_element" + ], + [ + 19, + 21, + "\u03b14", + "structure_element" + ], + [ + 25, + 29, + "holo", + "protein_state" + ], + [ + 30, + 34, + "NadR", + "protein" + ], + [ + 81, + 96, + "dimer interface", + "site" + ], + [ + 108, + 115, + "helices", + "structure_element" + ], + [ + 116, + 118, + "\u03b11", + "structure_element" + ], + [ + 120, + 122, + "\u03b15", + "structure_element" + ], + [ + 128, + 130, + "\u03b16", + "structure_element" + ], + [ + 141, + 145, + "holo", + "protein_state" + ], + [ + 178, + 181, + "DNA", + "chemical" + ], + [ + 219, + 228, + "OhrR:ohrA", + "complex_assembly" + ] + ] + }, + { + "sid": 262, + "sent": "While some flexibility of helix \u03b14 was also observed in the two apo-structures, concomitant changes in the dimer interfaces were not observed, possibly due to the absence of ligand.", + "section": "DISCUSS", + "ner": [ + [ + 26, + 31, + "helix", + "structure_element" + ], + [ + 32, + 34, + "\u03b14", + "structure_element" + ], + [ + 64, + 67, + "apo", + "protein_state" + ], + [ + 68, + 78, + "structures", + "evidence" + ], + [ + 107, + 123, + "dimer interfaces", + "site" + ], + [ + 163, + 180, + "absence of ligand", + "protein_state" + ] + ] + }, + { + "sid": 263, + "sent": "One of the two conformations of apo-NadR appeared ideally suited for DNA-binding.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 35, + "apo", + "protein_state" + ], + [ + 36, + 40, + "NadR", + "protein" + ], + [ + 69, + 72, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 264, + "sent": "Overall, these analyses suggest that the apo-NadR dimer has a pre-existing equilibrium that samples a variety of conformations, some of which are compatible with DNA binding.", + "section": "DISCUSS", + "ner": [ + [ + 41, + 44, + "apo", + "protein_state" + ], + [ + 45, + 49, + "NadR", + "protein" + ], + [ + 50, + 55, + "dimer", + "oligomeric_state" + ], + [ + 162, + 165, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 265, + "sent": "The noted flexibility may also explain how NadR can adapt to bind various DNA target sequences with slightly different structural features.", + "section": "DISCUSS", + "ner": [ + [ + 43, + 47, + "NadR", + "protein" + ], + [ + 74, + 77, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 266, + "sent": "Subsequently, upon ligand binding, holo-NadR adopts a structure less suited for DNA-binding and this conformation is selected and stabilized by a network of protein-ligand interactions and concomitant rearrangements at the NadR holo dimer interface.", + "section": "DISCUSS", + "ner": [ + [ + 35, + 39, + "holo", + "protein_state" + ], + [ + 40, + 44, + "NadR", + "protein" + ], + [ + 80, + 83, + "DNA", + "chemical" + ], + [ + 223, + 227, + "NadR", + "protein" + ], + [ + 228, + 232, + "holo", + "protein_state" + ], + [ + 233, + 248, + "dimer interface", + "site" + ] + ] + }, + { + "sid": 267, + "sent": "In an alternative and less extensive manner, the binding of two salicylate molecules to the M. thermoautotrophicum protein MTH313 appeared to induce large changes in the wHTH domain, which was associated with reduced DNA-binding activity.", + "section": "DISCUSS", + "ner": [ + [ + 64, + 74, + "salicylate", + "chemical" + ], + [ + 92, + 114, + "M. thermoautotrophicum", + "species" + ], + [ + 123, + 129, + "MTH313", + "protein" + ], + [ + 170, + 181, + "wHTH domain", + "structure_element" + ] + ] + }, + { + "sid": 268, + "sent": "Here we have presented two new crystal structures of the transcription factor, NadR, which regulates expression of the meningococcal surface protein, virulence factor and vaccine antigen NadA. Detailed structural analyses provided a molecular explanation for the ligand-responsive regulation by NadR on the majority of the promoters of meningococcal genes regulated by NadR, including nadA. Intriguingly, NadR exhibits a reversed regulatory mechanism on a second class of promoters, including mafA of the multiple adhesin family\u2013i.e. NadR represses these genes in the presence but not absence of 4-HPA.", + "section": "DISCUSS", + "ner": [ + [ + 31, + 49, + "crystal structures", + "evidence" + ], + [ + 57, + 77, + "transcription factor", + "protein_type" + ], + [ + 79, + 83, + "NadR", + "protein" + ], + [ + 119, + 132, + "meningococcal", + "taxonomy_domain" + ], + [ + 187, + 191, + "NadA", + "protein" + ], + [ + 202, + 221, + "structural analyses", + "experimental_method" + ], + [ + 295, + 299, + "NadR", + "protein" + ], + [ + 336, + 349, + "meningococcal", + "taxonomy_domain" + ], + [ + 369, + 373, + "NadR", + "protein" + ], + [ + 385, + 389, + "nadA", + "gene" + ], + [ + 405, + 409, + "NadR", + "protein" + ], + [ + 493, + 497, + "mafA", + "gene" + ], + [ + 534, + 538, + "NadR", + "protein" + ], + [ + 596, + 601, + "4-HPA", + "chemical" + ] + ] + }, + { + "sid": 269, + "sent": "The latter may influence the surface abundance or secretion of maf proteins, an emerging class of highly conserved meningococcal putative adhesins and toxins with many important roles.", + "section": "DISCUSS", + "ner": [ + [ + 98, + 114, + "highly conserved", + "protein_state" + ], + [ + 115, + 128, + "meningococcal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 270, + "sent": "Further work is required to investigate how the two different promoter types influence the ligand-responsiveness of NadR during bacterial infection and may provide insights into the regulatory mechanisms occurring during these host-pathogen interactions.", + "section": "DISCUSS", + "ner": [ + [ + 116, + 120, + "NadR", + "protein" + ], + [ + 128, + 137, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 271, + "sent": "Ultimately, knowledge of the ligand-dependent activity of NadR will continue to deepen our understanding of nadA expression levels, which influence meningococcal pathogenesis.", + "section": "DISCUSS", + "ner": [ + [ + 58, + 62, + "NadR", + "protein" + ], + [ + 108, + 112, + "nadA", + "gene" + ], + [ + 148, + 161, + "meningococcal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 272, + "sent": "Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of the MarR family", + "section": "REF", + "ner": [ + [ + 21, + 25, + "ohrA", + "gene" + ] + ] + }, + { + "sid": 273, + "sent": "The structure of NMB1585, a MarR-family regulator from Neisseria meningitidis", + "section": "REF", + "ner": [ + [ + 17, + 24, + "NMB1585", + "protein" + ] + ] + } + ] + }, + "PMC4888278": { + "annotations": [ + { + "sid": 0, + "sent": "Structural determinant for inducing RORgamma specific inverse agonism triggered by a synthetic benzoxazinone ligand", + "section": "TITLE", + "ner": [ + [ + 36, + 44, + "RORgamma", + "protein" + ], + [ + 95, + 108, + "benzoxazinone", + "chemical" + ] + ] + }, + { + "sid": 1, + "sent": "The nuclear hormone receptor ROR\u03b3 regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 28, + "nuclear hormone receptor", + "protein_type" + ], + [ + 29, + 33, + "ROR\u03b3", + "protein" + ], + [ + 117, + 128, + "interleukin", + "protein_type" + ], + [ + 129, + 134, + "IL-17", + "protein_type" + ] + ] + }, + { + "sid": 2, + "sent": "This transcriptional activity of ROR\u03b3 is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of ROR\u03b3 and a conserved LXXLL helix motif on coactivator proteins.", + "section": "ABSTRACT", + "ner": [ + [ + 33, + 37, + "ROR\u03b3", + "protein" + ], + [ + 103, + 136, + "activation function 2 (AF2) helix", + "structure_element" + ], + [ + 144, + 165, + "ligand binding domain", + "structure_element" + ], + [ + 169, + 173, + "ROR\u03b3", + "protein" + ], + [ + 180, + 189, + "conserved", + "protein_state" + ], + [ + 190, + 207, + "LXXLL helix motif", + "structure_element" + ] + ] + }, + { + "sid": 3, + "sent": "Our goal was to develop a ROR\u03b3 specific inverse agonist that would help down regulate pro-inflammatory gene transcription by disrupting the protein protein interaction with coactivator proteins as a therapeutic agent.", + "section": "ABSTRACT", + "ner": [ + [ + 26, + 30, + "ROR\u03b3", + "protein" + ], + [ + 40, + 55, + "inverse agonist", + "protein_state" + ] + ] + }, + { + "sid": 4, + "sent": "We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay.", + "section": "ABSTRACT", + "ner": [ + [ + 42, + 55, + "benzoxazinone", + "chemical" + ], + [ + 74, + 81, + "agonist", + "protein_state" + ], + [ + 83, + 89, + "BIO592", + "chemical" + ], + [ + 95, + 110, + "inverse agonist", + "protein_state" + ], + [ + 112, + 118, + "BIO399", + "chemical" + ], + [ + 140, + 156, + "FRET based assay", + "experimental_method" + ] + ] + }, + { + "sid": 5, + "sent": "We show that the AF2 helix of ROR\u03b3 is proteolytically sensitive when inverse agonist BIO399 binds.", + "section": "ABSTRACT", + "ner": [ + [ + 17, + 26, + "AF2 helix", + "structure_element" + ], + [ + 30, + 34, + "ROR\u03b3", + "protein" + ], + [ + 38, + 63, + "proteolytically sensitive", + "protein_state" + ], + [ + 69, + 84, + "inverse agonist", + "protein_state" + ], + [ + 85, + 91, + "BIO399", + "chemical" + ] + ] + }, + { + "sid": 6, + "sent": "Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of ROR\u03b3.", + "section": "ABSTRACT", + "ner": [ + [ + 6, + 27, + "x-ray crystallography", + "experimental_method" + ], + [ + 67, + 80, + "benzoxazinone", + "chemical" + ], + [ + 81, + 88, + "agonist", + "protein_state" + ], + [ + 89, + 95, + "BIO592", + "chemical" + ], + [ + 123, + 127, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 7, + "sent": "Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for ROR\u03b3 over ROR sub-family members \u03b1 and \u03b2.", + "section": "ABSTRACT", + "ner": [ + [ + 9, + 31, + "in vivo reporter assay", + "experimental_method" + ], + [ + 50, + 65, + "inverse agonist", + "protein_state" + ], + [ + 66, + 72, + "BIO399", + "chemical" + ], + [ + 99, + 103, + "ROR\u03b3", + "protein" + ], + [ + 109, + 112, + "ROR", + "protein_type" + ], + [ + 132, + 133, + "\u03b1", + "protein" + ], + [ + 138, + 139, + "\u03b2", + "protein" + ] + ] + }, + { + "sid": 8, + "sent": "The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of ROR\u03b3.", + "section": "ABSTRACT", + "ner": [ + [ + 14, + 27, + "benzoxazinone", + "chemical" + ], + [ + 54, + 64, + "FRET assay", + "experimental_method" + ], + [ + 73, + 80, + "agonist", + "protein_state" + ], + [ + 82, + 88, + "BIO592", + "chemical" + ], + [ + 93, + 108, + "inverse agonist", + "protein_state" + ], + [ + 110, + 116, + "BIO399", + "chemical" + ], + [ + 161, + 168, + "agonist", + "protein_state" + ], + [ + 185, + 189, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 9, + "sent": "The proteolytic sensitivity of the AF2 helix of ROR\u03b3 demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site.", + "section": "ABSTRACT", + "ner": [ + [ + 35, + 44, + "AF2 helix", + "structure_element" + ], + [ + 48, + 52, + "ROR\u03b3", + "protein" + ], + [ + 92, + 98, + "BIO399", + "chemical" + ], + [ + 99, + 114, + "inverse agonist", + "protein_state" + ], + [ + 138, + 170, + "coactivator protein binding site", + "site" + ] + ] + }, + { + "sid": 10, + "sent": "Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on ROR\u03b3 as the trigger for ROR\u03b3 specific inverse agonism.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 28, + "structural investigation", + "experimental_method" + ], + [ + 36, + 42, + "BIO592", + "chemical" + ], + [ + 43, + 50, + "agonist", + "protein_state" + ], + [ + 55, + 61, + "BIO399", + "chemical" + ], + [ + 62, + 77, + "inverse agonist", + "protein_state" + ], + [ + 78, + 88, + "structures", + "evidence" + ], + [ + 108, + 114, + "Met358", + "residue_name_number" + ], + [ + 118, + 122, + "ROR\u03b3", + "protein" + ], + [ + 142, + 146, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 11, + "sent": "Retinoid-related orphan receptor gamma (ROR\u03b3) is a transcription factor belonging to a sub-family of nuclear receptors that includes two closely related members ROR\u03b1 and ROR\u03b2.", + "section": "INTRO", + "ner": [ + [ + 0, + 38, + "Retinoid-related orphan receptor gamma", + "protein" + ], + [ + 40, + 44, + "ROR\u03b3", + "protein" + ], + [ + 51, + 71, + "transcription factor", + "protein_type" + ], + [ + 101, + 118, + "nuclear receptors", + "protein_type" + ], + [ + 161, + 165, + "ROR\u03b1", + "protein" + ], + [ + 170, + 174, + "ROR\u03b2", + "protein" + ] + ] + }, + { + "sid": 12, + "sent": "Even though a high degree of sequence similarity exists between the RORs, their functional roles in regulation for physiological processes involved in development and immunity are distinct.", + "section": "INTRO", + "ner": [ + [ + 68, + 72, + "RORs", + "protein_type" + ] + ] + }, + { + "sid": 13, + "sent": "During development, ROR\u03b3 regulates the transcriptional genes involved in the functioning of multiple pro-inflammatory lymphocyte lineages including T helper cells (TH17cells) which are necessary for IL-17 production.", + "section": "INTRO", + "ner": [ + [ + 20, + 24, + "ROR\u03b3", + "protein" + ], + [ + 199, + 204, + "IL-17", + "protein_type" + ] + ] + }, + { + "sid": 14, + "sent": "IL-17 is a pro-inflammatory interleukin linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease; making its transcriptional regulation through ROR\u03b3 an attractive therapeutic target.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "IL-17", + "protein_type" + ], + [ + 28, + 39, + "interleukin", + "protein_type" + ], + [ + 197, + 201, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 15, + "sent": "ROR\u03b3 consists of an N-terminal DNA binding domain (DBD) connected to a C-terminal ligand binding domain (LBD) via a flexible hinge region.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "ROR\u03b3", + "protein" + ], + [ + 31, + 49, + "DNA binding domain", + "structure_element" + ], + [ + 51, + 54, + "DBD", + "structure_element" + ], + [ + 82, + 103, + "ligand binding domain", + "structure_element" + ], + [ + 105, + 108, + "LBD", + "structure_element" + ], + [ + 125, + 137, + "hinge region", + "structure_element" + ] + ] + }, + { + "sid": 16, + "sent": "The DBD is composed of two zinc fingers that allow it to interact with specifically encoded regions on the DNA called the nuclear receptor response elements.", + "section": "INTRO", + "ner": [ + [ + 4, + 7, + "DBD", + "structure_element" + ], + [ + 27, + 39, + "zinc fingers", + "structure_element" + ], + [ + 122, + 156, + "nuclear receptor response elements", + "structure_element" + ] + ] + }, + { + "sid": 17, + "sent": "The LBD consists of a coactivator protein binding pocket and a hydrophobic ligand binding site (LBS) which are responsible for regulating transcription.", + "section": "INTRO", + "ner": [ + [ + 4, + 7, + "LBD", + "structure_element" + ], + [ + 22, + 56, + "coactivator protein binding pocket", + "site" + ], + [ + 63, + 94, + "hydrophobic ligand binding site", + "site" + ], + [ + 96, + 99, + "LBS", + "site" + ] + ] + }, + { + "sid": 18, + "sent": "The coactivator binding pocket of ROR\u03b3 recognizes a conserved helix motif LXXLL (where X can be any amino acid) on transcriptional coactivator complexes and recruits it to activate transcription.", + "section": "INTRO", + "ner": [ + [ + 4, + 30, + "coactivator binding pocket", + "site" + ], + [ + 34, + 38, + "ROR\u03b3", + "protein" + ], + [ + 52, + 61, + "conserved", + "protein_state" + ], + [ + 62, + 79, + "helix motif LXXLL", + "structure_element" + ] + ] + }, + { + "sid": 19, + "sent": "Like other nuclear hormone receptors, ROR\u03b3\u2019s helix12 which makes up the C-termini of the LBD is an essential part of the coactivator binding pocket and is commonly referred to as the activation function helix 2 (AF2).", + "section": "INTRO", + "ner": [ + [ + 11, + 36, + "nuclear hormone receptors", + "protein_type" + ], + [ + 38, + 42, + "ROR\u03b3", + "protein" + ], + [ + 45, + 52, + "helix12", + "structure_element" + ], + [ + 89, + 92, + "LBD", + "structure_element" + ], + [ + 121, + 147, + "coactivator binding pocket", + "site" + ], + [ + 183, + 210, + "activation function helix 2", + "structure_element" + ], + [ + 212, + 215, + "AF2", + "structure_element" + ] + ] + }, + { + "sid": 20, + "sent": "In ROR\u03b3, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to \u03c0- \u03c0 interactions between Tyr502 and Phe506.", + "section": "INTRO", + "ner": [ + [ + 3, + 7, + "ROR\u03b3", + "protein" + ], + [ + 33, + 42, + "AF2 helix", + "structure_element" + ], + [ + 64, + 90, + "coactivator binding pocket", + "site" + ], + [ + 108, + 119, + "salt bridge", + "bond_interaction" + ], + [ + 128, + 134, + "His479", + "residue_name_number" + ], + [ + 139, + 145, + "Tyr502", + "residue_name_number" + ], + [ + 161, + 178, + "\u03c0- \u03c0 interactions", + "bond_interaction" + ], + [ + 187, + 193, + "Tyr502", + "residue_name_number" + ], + [ + 198, + 204, + "Phe506", + "residue_name_number" + ] + ] + }, + { + "sid": 21, + "sent": "The conformation of the AF2 helix can be modulated through targeted ligands which bind the LBS and increase the binding of the coactivator protein (agonists) or disrupt binding (inverse agonists) thereby enhancing or inhibiting transcription.", + "section": "INTRO", + "ner": [ + [ + 24, + 33, + "AF2 helix", + "structure_element" + ], + [ + 91, + 94, + "LBS", + "site" + ] + ] + }, + { + "sid": 22, + "sent": "Since ROR\u03b3 has been demonstrated to play an important role in pro-inflammatory gene expression patterns implicated in several major autoimmune diseases, our aim was to develop ROR\u03b3 inverse agonists that would help down regulate pro-inflammatory gene transcription.", + "section": "INTRO", + "ner": [ + [ + 6, + 10, + "ROR\u03b3", + "protein" + ], + [ + 176, + 180, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 23, + "sent": "FRET results for agonist BIO592 (a) and Inverse Agonist BIO399 (b)", + "section": "FIG", + "ner": [ + [ + 0, + 12, + "FRET results", + "evidence" + ], + [ + 17, + 24, + "agonist", + "protein_state" + ], + [ + 25, + 31, + "BIO592", + "chemical" + ], + [ + 40, + 55, + "Inverse Agonist", + "protein_state" + ], + [ + 56, + 62, + "BIO399", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "Here we present the identification of two synthetic benzoxazinone ROR\u03b3 ligands, a weak agonist BIO592 (Fig.\u00a01a) and an inverse agonist BIO399 (Fig.\u00a01b) which were identified using a Fluorescence Resonance Energy transfer (FRET) based assay that monitored coactivator peptide recruitment.", + "section": "INTRO", + "ner": [ + [ + 52, + 65, + "benzoxazinone", + "chemical" + ], + [ + 66, + 70, + "ROR\u03b3", + "protein" + ], + [ + 87, + 94, + "agonist", + "protein_state" + ], + [ + 95, + 101, + "BIO592", + "chemical" + ], + [ + 119, + 134, + "inverse agonist", + "protein_state" + ], + [ + 135, + 141, + "BIO399", + "chemical" + ], + [ + 182, + 239, + "Fluorescence Resonance Energy transfer (FRET) based assay", + "experimental_method" + ] + ] + }, + { + "sid": 25, + "sent": "Using partial proteolysis in combination with mass spectrometry analysis we demonstrate that the AF2 helix of ROR\u03b3 destabilizes upon BIO399 (inverse agonist) binding.", + "section": "INTRO", + "ner": [ + [ + 6, + 25, + "partial proteolysis", + "experimental_method" + ], + [ + 46, + 63, + "mass spectrometry", + "experimental_method" + ], + [ + 97, + 106, + "AF2 helix", + "structure_element" + ], + [ + 110, + 114, + "ROR\u03b3", + "protein" + ], + [ + 133, + 139, + "BIO399", + "chemical" + ], + [ + 141, + 156, + "inverse agonist", + "protein_state" + ] + ] + }, + { + "sid": 26, + "sent": "Finally, comparing binding modes of our benzoxazinone ROR\u03b3 crystal structures to other ROR structures, we hypothesize a new mode of action for achieving inverse agonism and selectivity.", + "section": "INTRO", + "ner": [ + [ + 19, + 32, + "binding modes", + "evidence" + ], + [ + 40, + 53, + "benzoxazinone", + "chemical" + ], + [ + 54, + 58, + "ROR\u03b3", + "protein" + ], + [ + 59, + 77, + "crystal structures", + "evidence" + ], + [ + 87, + 90, + "ROR", + "protein_type" + ], + [ + 91, + 101, + "structures", + "evidence" + ] + ] + }, + { + "sid": 27, + "sent": "Using a FRET based assay we discovered agonist BIO592 (Fig.\u00a01a) which increased the coactivator peptide TRAP220 recruitment to ROR\u03b3 (EC50 0f 58nM and Emax of 130\u00a0%) and a potent inverse agonist BIO399 (Fig.\u00a01b) which inhibited coactivator recruitment (IC50: 4.7nM).", + "section": "RESULTS", + "ner": [ + [ + 8, + 24, + "FRET based assay", + "experimental_method" + ], + [ + 39, + 46, + "agonist", + "protein_state" + ], + [ + 47, + 53, + "BIO592", + "chemical" + ], + [ + 104, + 111, + "TRAP220", + "chemical" + ], + [ + 127, + 131, + "ROR\u03b3", + "protein" + ], + [ + 133, + 137, + "EC50", + "evidence" + ], + [ + 150, + 154, + "Emax", + "evidence" + ], + [ + 178, + 193, + "inverse agonist", + "protein_state" + ], + [ + 194, + 200, + "BIO399", + "chemical" + ], + [ + 252, + 256, + "IC50", + "evidence" + ] + ] + }, + { + "sid": 28, + "sent": "Interestingly, the structural difference between the agonist BIO592 and inverse agonist BIO399 was minor; with the 2,3-dihydrobenzo[1,4]oxazepin-4-one ring system of BIO399 being 3 atoms larger than the benzo[1,4]oxazine-3-one ring system of BIO592.", + "section": "RESULTS", + "ner": [ + [ + 53, + 60, + "agonist", + "protein_state" + ], + [ + 61, + 67, + "BIO592", + "chemical" + ], + [ + 72, + 87, + "inverse agonist", + "protein_state" + ], + [ + 88, + 94, + "BIO399", + "chemical" + ], + [ + 115, + 150, + "2,3-dihydrobenzo[1,4]oxazepin-4-one", + "chemical" + ], + [ + 166, + 172, + "BIO399", + "chemical" + ], + [ + 203, + 226, + "benzo[1,4]oxazine-3-one", + "chemical" + ], + [ + 242, + 248, + "BIO592", + "chemical" + ] + ] + }, + { + "sid": 29, + "sent": "In order to understand how small changes in the core ring system leads to inverse agonism, we wanted to structurally determine the binding mode of both BIO592 and BIO399 in the LBS of ROR\u03b3 using x-ray crystallography.", + "section": "RESULTS", + "ner": [ + [ + 152, + 158, + "BIO592", + "chemical" + ], + [ + 163, + 169, + "BIO399", + "chemical" + ], + [ + 177, + 180, + "LBS", + "site" + ], + [ + 184, + 188, + "ROR\u03b3", + "protein" + ], + [ + 195, + 216, + "x-ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 30, + "sent": "Structure of the ROR\u03b3518-BIO592-EBI96 ternary complex is in a transcriptionally active conformation", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 17, + 37, + "ROR\u03b3518-BIO592-EBI96", + "complex_assembly" + ], + [ + 80, + 86, + "active", + "protein_state" + ] + ] + }, + { + "sid": 31, + "sent": " a The ternary structure of ROR\u03b3518 BIO592 and EBI96.", + "section": "FIG", + "ner": [ + [ + 7, + 24, + "ternary structure", + "evidence" + ], + [ + 28, + 35, + "ROR\u03b3518", + "protein" + ], + [ + 36, + 42, + "BIO592", + "chemical" + ], + [ + 47, + 52, + "EBI96", + "chemical" + ] + ] + }, + { + "sid": 32, + "sent": "b ROR\u03b3 AF2 helix in the agonist conformation.", + "section": "FIG", + "ner": [ + [ + 2, + 6, + "ROR\u03b3", + "protein" + ], + [ + 7, + 16, + "AF2 helix", + "structure_element" + ], + [ + 24, + 31, + "agonist", + "protein_state" + ] + ] + }, + { + "sid": 33, + "sent": "c EBI96 coactivator peptide bound in the coactivator pocket of ROR\u03b3", + "section": "FIG", + "ner": [ + [ + 2, + 7, + "EBI96", + "chemical" + ], + [ + 28, + 36, + "bound in", + "protein_state" + ], + [ + 41, + 59, + "coactivator pocket", + "site" + ], + [ + 63, + 67, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 34, + "sent": "ROR\u03b3518 bound to agonist BIO592 was crystallized with a truncated form of the coactivator peptide EBI96 to a resolution of 2.6\u00a0\u00c5 (Fig.\u00a02a).", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "ROR\u03b3518", + "protein" + ], + [ + 8, + 16, + "bound to", + "protein_state" + ], + [ + 17, + 24, + "agonist", + "protein_state" + ], + [ + 25, + 31, + "BIO592", + "chemical" + ], + [ + 36, + 48, + "crystallized", + "experimental_method" + ], + [ + 56, + 65, + "truncated", + "protein_state" + ], + [ + 98, + 103, + "EBI96", + "chemical" + ] + ] + }, + { + "sid": 35, + "sent": "The structure of the ternary complex had features similar to other ROR agonist coactivator structures in a transcriptionally active canonical three layer helix fold with the AF2 helix in the agonist conformation.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 67, + 70, + "ROR", + "protein_type" + ], + [ + 71, + 78, + "agonist", + "protein_state" + ], + [ + 91, + 101, + "structures", + "evidence" + ], + [ + 107, + 131, + "transcriptionally active", + "protein_state" + ], + [ + 132, + 164, + "canonical three layer helix fold", + "protein_state" + ], + [ + 174, + 183, + "AF2 helix", + "structure_element" + ], + [ + 191, + 198, + "agonist", + "protein_state" + ] + ] + }, + { + "sid": 36, + "sent": "The agonist conformation is stabilized by a hydrogen bond between His479 and Tyr502, in addition to \u03c0-\u03c0 interactions between His479, Tyr502 and Phe506 (Fig.\u00a02b).", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "agonist", + "protein_state" + ], + [ + 44, + 57, + "hydrogen bond", + "bond_interaction" + ], + [ + 66, + 72, + "His479", + "residue_name_number" + ], + [ + 77, + 83, + "Tyr502", + "residue_name_number" + ], + [ + 100, + 116, + "\u03c0-\u03c0 interactions", + "bond_interaction" + ], + [ + 125, + 131, + "His479", + "residue_name_number" + ], + [ + 133, + 139, + "Tyr502", + "residue_name_number" + ], + [ + 144, + 150, + "Phe506", + "residue_name_number" + ] + ] + }, + { + "sid": 37, + "sent": "The hydrogen bond between His479 and Tyr502 has been reported to be critical for ROR\u03b3 agonist activity.", + "section": "RESULTS", + "ner": [ + [ + 4, + 17, + "hydrogen bond", + "bond_interaction" + ], + [ + 26, + 32, + "His479", + "residue_name_number" + ], + [ + 37, + 43, + "Tyr502", + "residue_name_number" + ], + [ + 81, + 85, + "ROR\u03b3", + "protein" + ], + [ + 86, + 93, + "agonist", + "protein_state" + ] + ] + }, + { + "sid": 38, + "sent": "Disrupting this interaction through mutagenesis reduced transcriptional activity of ROR\u03b3.", + "section": "RESULTS", + "ner": [ + [ + 36, + 47, + "mutagenesis", + "experimental_method" + ], + [ + 84, + 88, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 39, + "sent": "This reduced transcriptional activity has been attributed to the inability of the AF2 helix to complete the formation of the coactivator binding pocket necessary for coactivator proteins to bind.", + "section": "RESULTS", + "ner": [ + [ + 82, + 91, + "AF2 helix", + "structure_element" + ], + [ + 125, + 151, + "coactivator binding pocket", + "site" + ] + ] + }, + { + "sid": 40, + "sent": "Electron density for the coactivator peptide EBI96 was observed for residues EFPYLLSLLG which formed a \u03b1-helix stabilized through hydrophobic interactions with the coactivator binding pocket on ROR\u03b3 (Fig.\u00a02c).", + "section": "RESULTS", + "ner": [ + [ + 0, + 16, + "Electron density", + "evidence" + ], + [ + 45, + 50, + "EBI96", + "chemical" + ], + [ + 77, + 87, + "EFPYLLSLLG", + "structure_element" + ], + [ + 103, + 110, + "\u03b1-helix", + "structure_element" + ], + [ + 130, + 154, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 164, + 190, + "coactivator binding pocket", + "site" + ], + [ + 194, + 198, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 41, + "sent": "This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of ROR\u03b3.", + "section": "RESULTS", + "ner": [ + [ + 49, + 58, + "conserved", + "protein_state" + ], + [ + 59, + 72, + "charged clamp", + "structure_element" + ], + [ + 103, + 107, + "Tyr7", + "residue_name_number" + ], + [ + 124, + 129, + "Leu11", + "residue_name_number" + ], + [ + 133, + 138, + "EBI96", + "chemical" + ], + [ + 144, + 158, + "hydrogen bonds", + "bond_interaction" + ], + [ + 164, + 170, + "Glu504", + "residue_name_number" + ], + [ + 172, + 179, + "helix12", + "structure_element" + ], + [ + 185, + 191, + "Lys336", + "residue_name_number" + ], + [ + 193, + 199, + "helix3", + "structure_element" + ], + [ + 204, + 208, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 42, + "sent": "Formation of this charged clamp is essential for ROR\u03b3\u2019s function for playing a role in transcriptional activation and this has been corroborated through mutagenic studies in this region.", + "section": "RESULTS", + "ner": [ + [ + 18, + 31, + "charged clamp", + "structure_element" + ], + [ + 49, + 53, + "ROR\u03b3", + "protein" + ], + [ + 153, + 170, + "mutagenic studies", + "experimental_method" + ] + ] + }, + { + "sid": 43, + "sent": "BIO592 binds in a collapsed conformation stabilizing the agonist conformation of ROR\u03b3", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "BIO592", + "chemical" + ], + [ + 18, + 27, + "collapsed", + "protein_state" + ], + [ + 57, + 64, + "agonist", + "protein_state" + ], + [ + 81, + 85, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 44, + "sent": " a Collapsed binding mode of agonist BIO592 in the hydrophobic LBS of ROR\u03b3.", + "section": "FIG", + "ner": [ + [ + 29, + 36, + "agonist", + "protein_state" + ], + [ + 37, + 43, + "BIO592", + "chemical" + ], + [ + 63, + 66, + "LBS", + "site" + ], + [ + 70, + 74, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 45, + "sent": "b Benzoxazinone ring system of agonist BIO592 packing against His479 of ROR\u03b3 stabilizing agonist conformation of the AF2 helix", + "section": "FIG", + "ner": [ + [ + 2, + 15, + "Benzoxazinone", + "chemical" + ], + [ + 31, + 38, + "agonist", + "protein_state" + ], + [ + 39, + 45, + "BIO592", + "chemical" + ], + [ + 62, + 68, + "His479", + "residue_name_number" + ], + [ + 72, + 76, + "ROR\u03b3", + "protein" + ], + [ + 89, + 96, + "agonist", + "protein_state" + ], + [ + 117, + 126, + "AF2 helix", + "structure_element" + ] + ] + }, + { + "sid": 46, + "sent": "BIO592 bound in a collapsed conformational state in the LBS of ROR\u03b3 with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig.\u00a03a, Additional file 3).", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "BIO592", + "chemical" + ], + [ + 7, + 15, + "bound in", + "protein_state" + ], + [ + 18, + 27, + "collapsed", + "protein_state" + ], + [ + 56, + 59, + "LBS", + "site" + ], + [ + 63, + 67, + "ROR\u03b3", + "protein" + ], + [ + 77, + 83, + "xylene", + "chemical" + ], + [ + 121, + 127, + "pocket", + "site" + ], + [ + 135, + 159, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 165, + 171, + "Val376", + "residue_name_number" + ], + [ + 173, + 179, + "Phe378", + "residue_name_number" + ], + [ + 181, + 187, + "Phe388", + "residue_name_number" + ], + [ + 192, + 198, + "Phe401", + "residue_name_number" + ], + [ + 209, + 228, + "ethyl-benzoxazinone", + "chemical" + ], + [ + 249, + 273, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 279, + 285, + "Trp317", + "residue_name_number" + ], + [ + 287, + 293, + "Leu324", + "residue_name_number" + ], + [ + 295, + 301, + "Met358", + "residue_name_number" + ], + [ + 303, + 309, + "Leu391", + "residue_name_number" + ], + [ + 311, + 318, + "Ile 400", + "residue_name_number" + ], + [ + 323, + 329, + "His479", + "residue_name_number" + ] + ] + }, + { + "sid": 47, + "sent": "The sulfonyl group faces the entrance of the pocket, while the CF3 makes a hydrophobic contact with Ala327.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "sulfonyl", + "chemical" + ], + [ + 45, + 51, + "pocket", + "site" + ], + [ + 75, + 94, + "hydrophobic contact", + "bond_interaction" + ], + [ + 100, + 106, + "Ala327", + "residue_name_number" + ] + ] + }, + { + "sid": 48, + "sent": "Hydrophobic interaction between the ethyl group of the benzoxazinone and His479 reinforce the His479 sidechain position for making the hydrogen bond with Tyr502 thereby stabilizing the agonist conformation (Fig.\u00a03b).", + "section": "RESULTS", + "ner": [ + [ + 0, + 23, + "Hydrophobic interaction", + "bond_interaction" + ], + [ + 55, + 68, + "benzoxazinone", + "chemical" + ], + [ + 73, + 79, + "His479", + "residue_name_number" + ], + [ + 94, + 100, + "His479", + "residue_name_number" + ], + [ + 135, + 148, + "hydrogen bond", + "bond_interaction" + ], + [ + 154, + 160, + "Tyr502", + "residue_name_number" + ], + [ + 185, + 192, + "agonist", + "protein_state" + ] + ] + }, + { + "sid": 49, + "sent": "ROR\u03b3 AF2 helix is sensitive to proteolysis in the presence of Inverse Agonist BIO399", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "ROR\u03b3", + "protein" + ], + [ + 5, + 14, + "AF2 helix", + "structure_element" + ], + [ + 50, + 61, + "presence of", + "protein_state" + ], + [ + 62, + 77, + "Inverse Agonist", + "protein_state" + ], + [ + 78, + 84, + "BIO399", + "chemical" + ] + ] + }, + { + "sid": 50, + "sent": "Next, we attempted co-crystallization with the inverse agonist BIO399.", + "section": "RESULTS", + "ner": [ + [ + 19, + 37, + "co-crystallization", + "experimental_method" + ], + [ + 47, + 62, + "inverse agonist", + "protein_state" + ], + [ + 63, + 69, + "BIO399", + "chemical" + ] + ] + }, + { + "sid": 51, + "sent": "However, extensive crystallization efforts with BIO399 and ROR\u03b3518 or other AF2 intact constructs did not produce crystals.", + "section": "RESULTS", + "ner": [ + [ + 19, + 34, + "crystallization", + "experimental_method" + ], + [ + 48, + 54, + "BIO399", + "chemical" + ], + [ + 59, + 66, + "ROR\u03b3518", + "protein" + ], + [ + 76, + 79, + "AF2", + "structure_element" + ], + [ + 80, + 86, + "intact", + "protein_state" + ], + [ + 114, + 122, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 52, + "sent": "We hypothesized that the ROR\u03b3518 coactivator peptide interaction in the FRET assay was disrupted upon BIO399 binding and that a conformational rearrangement of the AF2 helix could have occurred, hindering crystallization.", + "section": "RESULTS", + "ner": [ + [ + 25, + 32, + "ROR\u03b3518", + "protein" + ], + [ + 72, + 82, + "FRET assay", + "experimental_method" + ], + [ + 102, + 108, + "BIO399", + "chemical" + ], + [ + 164, + 173, + "AF2 helix", + "structure_element" + ], + [ + 205, + 220, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 53, + "sent": "Specific proteolytic positions on ROR\u03b3518 when treated with Actinase E alone (Green) or in the presence of BIO399 (Red) and shared proteolytic sites (Yellow)", + "section": "FIG", + "ner": [ + [ + 34, + 41, + "ROR\u03b3518", + "protein" + ], + [ + 47, + 59, + "treated with", + "experimental_method" + ], + [ + 60, + 70, + "Actinase E", + "protein" + ], + [ + 95, + 106, + "presence of", + "protein_state" + ], + [ + 107, + 113, + "BIO399", + "chemical" + ], + [ + 131, + 148, + "proteolytic sites", + "site" + ] + ] + }, + { + "sid": 54, + "sent": "The unfolding of the AF2 helix has been observed for other nuclear hormone receptors when bound to an inverse agonist or antagonist.", + "section": "RESULTS", + "ner": [ + [ + 21, + 30, + "AF2 helix", + "structure_element" + ], + [ + 59, + 84, + "nuclear hormone receptors", + "protein_type" + ], + [ + 90, + 98, + "bound to", + "protein_state" + ], + [ + 102, + 117, + "inverse agonist", + "protein_state" + ] + ] + }, + { + "sid": 55, + "sent": "We used partial proteolysis in combination with mass spectrometry to determine if BIO399 was causing the AF2 helix to unfold.", + "section": "RESULTS", + "ner": [ + [ + 8, + 27, + "partial proteolysis", + "experimental_method" + ], + [ + 48, + 65, + "mass spectrometry", + "experimental_method" + ], + [ + 82, + 88, + "BIO399", + "chemical" + ], + [ + 105, + 114, + "AF2 helix", + "structure_element" + ] + ] + }, + { + "sid": 56, + "sent": "Results of the Actinase E proteolysis experiments on ROR\u03b3518, the ternary complex of ROR\u03b3518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis.", + "section": "RESULTS", + "ner": [ + [ + 15, + 37, + "Actinase E proteolysis", + "experimental_method" + ], + [ + 53, + 60, + "ROR\u03b3518", + "protein" + ], + [ + 85, + 92, + "ROR\u03b3518", + "protein" + ], + [ + 98, + 105, + "agonist", + "protein_state" + ], + [ + 106, + 112, + "BIO592", + "chemical" + ], + [ + 129, + 134, + "EBI96", + "chemical" + ], + [ + 146, + 157, + "presence of", + "protein_state" + ], + [ + 158, + 173, + "inverse agonist", + "protein_state" + ], + [ + 174, + 180, + "BIO399", + "chemical" + ] + ] + }, + { + "sid": 57, + "sent": "Analysis of the fragmentation pattern showed minimal proteolytic removal of the AF2 helix by Actinase E on ROR\u03b3518 alone (ending at 504 to 506) and the ternary complex remained primarily intact (ending at 515/518) (Additional file 4).", + "section": "RESULTS", + "ner": [ + [ + 16, + 37, + "fragmentation pattern", + "evidence" + ], + [ + 80, + 89, + "AF2 helix", + "structure_element" + ], + [ + 93, + 103, + "Actinase E", + "protein" + ], + [ + 107, + 114, + "ROR\u03b3518", + "protein" + ], + [ + 132, + 142, + "504 to 506", + "residue_range" + ], + [ + 152, + 167, + "ternary complex", + "protein_state" + ], + [ + 205, + 208, + "515", + "residue_number" + ], + [ + 209, + 212, + "518", + "residue_number" + ] + ] + }, + { + "sid": 58, + "sent": "However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig.\u00a04, Additional file 5).", + "section": "RESULTS", + "ner": [ + [ + 16, + 27, + "presence of", + "protein_state" + ], + [ + 28, + 43, + "inverse agonist", + "protein_state" + ], + [ + 44, + 50, + "BIO399", + "chemical" + ], + [ + 56, + 75, + "proteolytic pattern", + "evidence" + ], + [ + 178, + 181, + "494", + "residue_number" + ], + [ + 182, + 185, + "495", + "residue_number" + ], + [ + 226, + 235, + "AF2 helix", + "structure_element" + ], + [ + 259, + 262, + "APO", + "protein_state" + ], + [ + 266, + 289, + "ternary agonist complex", + "protein_state" + ] + ] + }, + { + "sid": 59, + "sent": "Several rounds of cocrystallization attempts with ROR\u03b3518 or other ROR\u03b3 AF2 helix containing constructs complexed with BIO399 had not produced crystals.", + "section": "RESULTS", + "ner": [ + [ + 18, + 35, + "cocrystallization", + "experimental_method" + ], + [ + 50, + 57, + "ROR\u03b3518", + "protein" + ], + [ + 67, + 71, + "ROR\u03b3", + "protein" + ], + [ + 72, + 81, + "AF2 helix", + "structure_element" + ], + [ + 104, + 118, + "complexed with", + "protein_state" + ], + [ + 119, + 125, + "BIO399", + "chemical" + ], + [ + 143, + 151, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 60, + "sent": "We attributed the inability to form crystals to the unfolding of the AF2 helix induced by BIO399.", + "section": "RESULTS", + "ner": [ + [ + 36, + 44, + "crystals", + "evidence" + ], + [ + 69, + 78, + "AF2 helix", + "structure_element" + ], + [ + 90, + 96, + "BIO399", + "chemical" + ] + ] + }, + { + "sid": 61, + "sent": "We reasoned that if we could remove the unfolded AF2 helix using proteolysis we could produce a binary complex more amenable to crystallization.", + "section": "RESULTS", + "ner": [ + [ + 40, + 48, + "unfolded", + "protein_state" + ], + [ + 49, + 58, + "AF2 helix", + "structure_element" + ], + [ + 65, + 76, + "proteolysis", + "experimental_method" + ], + [ + 128, + 143, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 62, + "sent": "AF2 truncated ROR\u03b3 BIO399 complex is more amenable to crystallization", + "section": "RESULTS", + "ner": [ + [ + 0, + 13, + "AF2 truncated", + "protein_state" + ], + [ + 14, + 25, + "ROR\u03b3 BIO399", + "complex_assembly" + ], + [ + 54, + 69, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 63, + "sent": " a The binary structure of AF2-truncated ROR\u03b3 and BIO399.", + "section": "FIG", + "ner": [ + [ + 14, + 23, + "structure", + "evidence" + ], + [ + 27, + 40, + "AF2-truncated", + "protein_state" + ], + [ + 41, + 45, + "ROR\u03b3", + "protein" + ], + [ + 50, + 56, + "BIO399", + "chemical" + ] + ] + }, + { + "sid": 64, + "sent": "b The superposition of inverse agonist BIO399 (Cyan) and agonist BIO592 (Green).", + "section": "FIG", + "ner": [ + [ + 6, + 19, + "superposition", + "experimental_method" + ], + [ + 23, + 38, + "inverse agonist", + "protein_state" + ], + [ + 39, + 45, + "BIO399", + "chemical" + ], + [ + 57, + 64, + "agonist", + "protein_state" + ], + [ + 65, + 71, + "BIO592", + "chemical" + ] + ] + }, + { + "sid": 65, + "sent": "c Movement of Met358 and His479 in the BIO399 (Cyan) and BIO592 (Green) structures", + "section": "FIG", + "ner": [ + [ + 14, + 20, + "Met358", + "residue_name_number" + ], + [ + 25, + 31, + "His479", + "residue_name_number" + ], + [ + 39, + 45, + "BIO399", + "chemical" + ], + [ + 57, + 63, + "BIO592", + "chemical" + ], + [ + 72, + 82, + "structures", + "evidence" + ] + ] + }, + { + "sid": 66, + "sent": "The Actinase E treated ROR\u03b3518 BIO399 ternary complex (aeROR\u03b3493/4) co-crystallized readily in several PEG based conditions.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "Actinase E", + "protein" + ], + [ + 23, + 37, + "ROR\u03b3518 BIO399", + "complex_assembly" + ], + [ + 55, + 66, + "aeROR\u03b3493/4", + "complex_assembly" + ], + [ + 68, + 83, + "co-crystallized", + "experimental_method" + ] + ] + }, + { + "sid": 67, + "sent": "The structure of aeROR\u03b3493/4 BIO399 complex was solved to 2.3\u00a0\u00c5 and adopted a similar core fold to the BIO592 agonist crystal structure (Fig.\u00a05a, Additional file 3).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 17, + 35, + "aeROR\u03b3493/4 BIO399", + "complex_assembly" + ], + [ + 48, + 54, + "solved", + "experimental_method" + ], + [ + 103, + 109, + "BIO592", + "chemical" + ], + [ + 110, + 117, + "agonist", + "protein_state" + ], + [ + 118, + 135, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 68, + "sent": "The aeROR\u03b3493/4 BIO399 structure diverged at the c-terminal end of Helix 11 from the ROR\u03b3518 BIO592 EBI96 structure, where helix 11 unwinds into a random coil after residue L475.", + "section": "RESULTS", + "ner": [ + [ + 4, + 22, + "aeROR\u03b3493/4 BIO399", + "complex_assembly" + ], + [ + 23, + 32, + "structure", + "evidence" + ], + [ + 67, + 75, + "Helix 11", + "structure_element" + ], + [ + 85, + 105, + "ROR\u03b3518 BIO592 EBI96", + "complex_assembly" + ], + [ + 106, + 115, + "structure", + "evidence" + ], + [ + 123, + 131, + "helix 11", + "structure_element" + ], + [ + 173, + 177, + "L475", + "residue_name_number" + ] + ] + }, + { + "sid": 69, + "sent": "Inverse agonist BIO399 uses Met358 as a trigger for inverse agonism", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Inverse agonist", + "protein_state" + ], + [ + 16, + 22, + "BIO399", + "chemical" + ], + [ + 28, + 34, + "Met358", + "residue_name_number" + ] + ] + }, + { + "sid": 70, + "sent": "BIO399 binds to the ligand binding site of ROR\u03b3 adopting a collapsed conformation as seen with BIO592 where the two compounds superimpose with an RMSD of 0.72\u00a0\u00c5 (Fig.\u00a05b).", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "BIO399", + "chemical" + ], + [ + 20, + 39, + "ligand binding site", + "site" + ], + [ + 43, + 47, + "ROR\u03b3", + "protein" + ], + [ + 59, + 68, + "collapsed", + "protein_state" + ], + [ + 95, + 101, + "BIO592", + "chemical" + ], + [ + 126, + 137, + "superimpose", + "experimental_method" + ], + [ + 146, + 150, + "RMSD", + "evidence" + ] + ] + }, + { + "sid": 71, + "sent": "The majority of the side chains within 4\u00a0\u00c5 of BIO399 and BIO592 adopt similar rotomer conformations with the exceptions of Met358 and His479 (Fig.\u00a05c).", + "section": "RESULTS", + "ner": [ + [ + 46, + 52, + "BIO399", + "chemical" + ], + [ + 57, + 63, + "BIO592", + "chemical" + ], + [ + 123, + 129, + "Met358", + "residue_name_number" + ], + [ + 134, + 140, + "His479", + "residue_name_number" + ] + ] + }, + { + "sid": 72, + "sent": "The difference density map showed clear positive density for Met358 in an alternate rotomer conformation compared to the one observed in the molecular replacement model or the other agonist containing models (Additional file 6).", + "section": "RESULTS", + "ner": [ + [ + 4, + 26, + "difference density map", + "evidence" + ], + [ + 40, + 56, + "positive density", + "evidence" + ], + [ + 61, + 67, + "Met358", + "residue_name_number" + ], + [ + 141, + 168, + "molecular replacement model", + "experimental_method" + ], + [ + 182, + 189, + "agonist", + "protein_state" + ] + ] + }, + { + "sid": 73, + "sent": "We tried to refine Met358 in the same conformation as the molecular replacement model or the other agonist containing models, but the results clearly indicated that this was not possible, thus confirming the new rotamer conformation for the Met358 sidechain in the inverse agonist bound structure.", + "section": "RESULTS", + "ner": [ + [ + 19, + 25, + "Met358", + "residue_name_number" + ], + [ + 58, + 85, + "molecular replacement model", + "experimental_method" + ], + [ + 99, + 106, + "agonist", + "protein_state" + ], + [ + 241, + 247, + "Met358", + "residue_name_number" + ], + [ + 265, + 286, + "inverse agonist bound", + "protein_state" + ], + [ + 287, + 296, + "structure", + "evidence" + ] + ] + }, + { + "sid": 74, + "sent": "The change in rotomer conformation of Met358 between the agonist and inverse agonist structures is attributed to the gem-dimethyl group on the larger 7 membered benzoxazinone ring system of BIO399.", + "section": "RESULTS", + "ner": [ + [ + 38, + 44, + "Met358", + "residue_name_number" + ], + [ + 57, + 64, + "agonist", + "protein_state" + ], + [ + 69, + 84, + "inverse agonist", + "protein_state" + ], + [ + 85, + 95, + "structures", + "evidence" + ], + [ + 161, + 174, + "benzoxazinone", + "chemical" + ], + [ + 190, + 196, + "BIO399", + "chemical" + ] + ] + }, + { + "sid": 75, + "sent": "The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in ROR\u03b3 (Fig.\u00a05c).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "comparison", + "experimental_method" + ], + [ + 26, + 36, + "structures", + "evidence" + ], + [ + 52, + 59, + "agonist", + "protein_state" + ], + [ + 89, + 95, + "BIO592", + "chemical" + ], + [ + 96, + 105, + "structure", + "evidence" + ], + [ + 128, + 134, + "BIO399", + "chemical" + ], + [ + 143, + 149, + "Met358", + "residue_name_number" + ], + [ + 155, + 161, + "Phe506", + "residue_name_number" + ], + [ + 169, + 178, + "AF2 helix", + "structure_element" + ], + [ + 195, + 201, + "Met358", + "residue_name_number" + ], + [ + 247, + 251, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 76, + "sent": "BIO399 and Inverse agonist T0901317 bind in a collapsed conformation distinct from other ROR\u03b3 Inverse Agonists Cocrystal structures", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "BIO399", + "chemical" + ], + [ + 11, + 26, + "Inverse agonist", + "protein_state" + ], + [ + 27, + 35, + "T0901317", + "chemical" + ], + [ + 46, + 55, + "collapsed", + "protein_state" + ], + [ + 89, + 93, + "ROR\u03b3", + "protein" + ], + [ + 111, + 131, + "Cocrystal structures", + "evidence" + ] + ] + }, + { + "sid": 77, + "sent": " a Overlay of ROR\u03b3 structures bound to BIO596 (Green), BIO399 (Cyan) and T0901317 (Pink).", + "section": "FIG", + "ner": [ + [ + 3, + 10, + "Overlay", + "experimental_method" + ], + [ + 14, + 18, + "ROR\u03b3", + "protein" + ], + [ + 19, + 29, + "structures", + "evidence" + ], + [ + 30, + 38, + "bound to", + "protein_state" + ], + [ + 39, + 45, + "BIO596", + "chemical" + ], + [ + 55, + 61, + "BIO399", + "chemical" + ], + [ + 73, + 81, + "T0901317", + "chemical" + ] + ] + }, + { + "sid": 78, + "sent": "b Overlay of M358 in ROR\u03b3 structure BIO596 (Green), BIO399 (Cyan), Digoxin (Yellow), Compound 2 (Grey), Compound 48 (Salmon) and Compound 4j (Orange)", + "section": "FIG", + "ner": [ + [ + 2, + 9, + "Overlay", + "experimental_method" + ], + [ + 13, + 17, + "M358", + "residue_name_number" + ], + [ + 21, + 25, + "ROR\u03b3", + "protein" + ], + [ + 26, + 35, + "structure", + "evidence" + ], + [ + 36, + 42, + "BIO596", + "chemical" + ], + [ + 52, + 58, + "BIO399", + "chemical" + ], + [ + 67, + 74, + "Digoxin", + "chemical" + ] + ] + }, + { + "sid": 79, + "sent": "The co-crystal structure of ROR\u03b3 with T0901317 (PDB code: 4NB6), an inverse agonist of ROR\u03b3 (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here.", + "section": "RESULTS", + "ner": [ + [ + 4, + 24, + "co-crystal structure", + "evidence" + ], + [ + 28, + 32, + "ROR\u03b3", + "protein" + ], + [ + 38, + 46, + "T0901317", + "chemical" + ], + [ + 68, + 83, + "inverse agonist", + "protein_state" + ], + [ + 87, + 91, + "ROR\u03b3", + "protein" + ], + [ + 93, + 97, + "IC50", + "evidence" + ], + [ + 112, + 140, + "SRC1 displacement FRET assay", + "experimental_method" + ], + [ + 148, + 152, + "IC50", + "evidence" + ], + [ + 168, + 178, + "FRET assay", + "experimental_method" + ], + [ + 223, + 232, + "collapsed", + "protein_state" + ], + [ + 261, + 270, + "structure", + "evidence" + ], + [ + 274, + 280, + "BIO399", + "chemical" + ] + ] + }, + { + "sid": 80, + "sent": "The two compounds superimpose with an RMSD of 0.81\u00a0\u00c5 (Fig.\u00a06a).", + "section": "RESULTS", + "ner": [ + [ + 18, + 29, + "superimpose", + "experimental_method" + ], + [ + 38, + 42, + "RMSD", + "evidence" + ] + ] + }, + { + "sid": 81, + "sent": "The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the ROR\u03b3 BIO592 agonist structure.", + "section": "RESULTS", + "ner": [ + [ + 21, + 39, + "hexafluoropropanol", + "chemical" + ], + [ + 49, + 57, + "T0901317", + "chemical" + ], + [ + 82, + 98, + "electron density", + "evidence" + ], + [ + 140, + 146, + "Met358", + "residue_name_number" + ], + [ + 168, + 174, + "Phe506", + "residue_name_number" + ], + [ + 182, + 186, + "ROR\u03b3", + "protein" + ], + [ + 187, + 193, + "BIO592", + "chemical" + ], + [ + 194, + 201, + "agonist", + "protein_state" + ], + [ + 202, + 211, + "structure", + "evidence" + ] + ] + }, + { + "sid": 82, + "sent": "We hypothesize that since the Met358 sidechain conformation in the T0901317 ROR\u03b3 structure is not in the BIO399 conformation, this difference could account for the 10-fold reduction in the inverse agonism for T0901317 compared to BIO399 in the FRET assay.", + "section": "RESULTS", + "ner": [ + [ + 30, + 36, + "Met358", + "residue_name_number" + ], + [ + 67, + 75, + "T0901317", + "chemical" + ], + [ + 76, + 80, + "ROR\u03b3", + "protein" + ], + [ + 81, + 90, + "structure", + "evidence" + ], + [ + 105, + 111, + "BIO399", + "chemical" + ], + [ + 209, + 217, + "T0901317", + "chemical" + ], + [ + 230, + 236, + "BIO399", + "chemical" + ], + [ + 244, + 254, + "FRET assay", + "experimental_method" + ] + ] + }, + { + "sid": 83, + "sent": "Co-crystal structures of ROR\u03b3 have been generated with several potent inverse agonists adopting a linear conformation distinct from the collapsed conformations seen for BIO399 and T090131718.", + "section": "RESULTS", + "ner": [ + [ + 0, + 21, + "Co-crystal structures", + "evidence" + ], + [ + 25, + 29, + "ROR\u03b3", + "protein" + ], + [ + 98, + 104, + "linear", + "protein_state" + ], + [ + 136, + 145, + "collapsed", + "protein_state" + ], + [ + 169, + 175, + "BIO399", + "chemical" + ], + [ + 180, + 190, + "T090131718", + "chemical" + ] + ] + }, + { + "sid": 84, + "sent": "The inverse agonist activity for these compounds has been attributed to orientating Trp317 to clash with Tyr502 or a direct inverse agonist hydrogen bonding event with His479, both of which would perturb the agonist conformation of ROR\u03b3.", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "inverse agonist", + "protein_state" + ], + [ + 84, + 90, + "Trp317", + "residue_name_number" + ], + [ + 105, + 111, + "Tyr502", + "residue_name_number" + ], + [ + 124, + 139, + "inverse agonist", + "protein_state" + ], + [ + 140, + 156, + "hydrogen bonding", + "bond_interaction" + ], + [ + 168, + 174, + "His479", + "residue_name_number" + ], + [ + 208, + 215, + "agonist", + "protein_state" + ], + [ + 232, + 236, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 85, + "sent": "BIO399 neither orients the sidechain of Trp317 toward Tyr502 nor forms a hydrogen bond with His479 suggesting its mode of action is distinct from linear inverse agonists (Additional file 8).", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "BIO399", + "chemical" + ], + [ + 40, + 46, + "Trp317", + "residue_name_number" + ], + [ + 54, + 60, + "Tyr502", + "residue_name_number" + ], + [ + 73, + 86, + "hydrogen bond", + "bond_interaction" + ], + [ + 92, + 98, + "His479", + "residue_name_number" + ] + ] + }, + { + "sid": 86, + "sent": "In the linear inverse agonist crystal structures the side chain of Met358 resides in a similar position as the rotomer observed in ROR\u03b3 agonist structures with BIO592 described here or as observed in the hydroxycholesterol derivatives and therefore would not trigger inverse agonism with these ligands (Fig.\u00a06b).", + "section": "RESULTS", + "ner": [ + [ + 14, + 29, + "inverse agonist", + "protein_state" + ], + [ + 30, + 48, + "crystal structures", + "evidence" + ], + [ + 67, + 73, + "Met358", + "residue_name_number" + ], + [ + 131, + 135, + "ROR\u03b3", + "protein" + ], + [ + 136, + 143, + "agonist", + "protein_state" + ], + [ + 144, + 154, + "structures", + "evidence" + ], + [ + 160, + 166, + "BIO592", + "chemical" + ], + [ + 204, + 222, + "hydroxycholesterol", + "chemical" + ] + ] + }, + { + "sid": 87, + "sent": "BIO399 shows selectivity for ROR\u03b3 over ROR\u03b1 and ROR\u03b2 in a GAL4 Cellular Reporter Assay", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "BIO399", + "chemical" + ], + [ + 29, + 33, + "ROR\u03b3", + "protein" + ], + [ + 39, + 43, + "ROR\u03b1", + "protein" + ], + [ + 48, + 52, + "ROR\u03b2", + "protein" + ], + [ + 58, + 86, + "GAL4 Cellular Reporter Assay", + "experimental_method" + ] + ] + }, + { + "sid": 88, + "sent": "GAL4 cell assay selectivity profile for BIO399 toward ROR\u03b1 and ROR\u03b2 in GAL4", + "section": "TABLE", + "ner": [ + [ + 0, + 15, + "GAL4 cell assay", + "experimental_method" + ], + [ + 40, + 46, + "BIO399", + "chemical" + ], + [ + 54, + 58, + "ROR\u03b1", + "protein" + ], + [ + 63, + 67, + "ROR\u03b2", + "protein" + ], + [ + 71, + 75, + "GAL4", + "protein" + ] + ] + }, + { + "sid": 89, + "sent": " a Overlay of ROR\u03b1 (yellow), \u03b2 (pink) and \u03b3 (cyan) showing side chain differences at Met358 inverse agonism trigger position and (b) around the benzoxazinone ring system of BIO399", + "section": "FIG", + "ner": [ + [ + 3, + 10, + "Overlay", + "experimental_method" + ], + [ + 14, + 18, + "ROR\u03b1", + "protein" + ], + [ + 29, + 30, + "\u03b2", + "protein" + ], + [ + 42, + 43, + "\u03b3", + "protein" + ], + [ + 85, + 91, + "Met358", + "residue_name_number" + ], + [ + 144, + 157, + "benzoxazinone", + "chemical" + ], + [ + 173, + 179, + "BIO399", + "chemical" + ] + ] + }, + { + "sid": 90, + "sent": "In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR \u03b1, \u03b2 and \u03b3 were fused to the DNA binding domain of the transcriptional factor GAL4.", + "section": "RESULTS", + "ner": [ + [ + 54, + 60, + "BIO399", + "chemical" + ], + [ + 63, + 86, + "cellular reporter assay", + "experimental_method" + ], + [ + 113, + 135, + "ligand binding domains", + "structure_element" + ], + [ + 139, + 142, + "ROR", + "protein_type" + ], + [ + 143, + 144, + "\u03b1", + "protein" + ], + [ + 146, + 147, + "\u03b2", + "protein" + ], + [ + 152, + 153, + "\u03b3", + "protein" + ], + [ + 159, + 167, + "fused to", + "experimental_method" + ], + [ + 172, + 190, + "DNA binding domain", + "structure_element" + ], + [ + 198, + 220, + "transcriptional factor", + "protein_type" + ], + [ + 221, + 225, + "GAL4", + "protein" + ] + ] + }, + { + "sid": 91, + "sent": "The ROR-GAL4 fusion proteins were expressed in cells with the luciferase reporter gene under the control of a GAL4 promoter.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "ROR", + "protein_type" + ], + [ + 8, + 12, + "GAL4", + "protein" + ], + [ + 110, + 114, + "GAL4", + "protein" + ] + ] + }, + { + "sid": 92, + "sent": "BIO399 inhibited the luciferase activity when added to the cells expressing the ROR\u03b3-GAL4 fusion with an in vivo IC50 of 42.5nM while showing >235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR \u03b1 or \u03b2, respectively (Table\u00a01).", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "BIO399", + "chemical" + ], + [ + 80, + 84, + "ROR\u03b3", + "protein" + ], + [ + 85, + 89, + "GAL4", + "protein" + ], + [ + 113, + 117, + "IC50", + "evidence" + ], + [ + 193, + 197, + "GAL4", + "protein" + ], + [ + 211, + 214, + "LBD", + "structure_element" + ], + [ + 218, + 221, + "ROR", + "protein_type" + ], + [ + 222, + 223, + "\u03b1", + "protein" + ], + [ + 227, + 228, + "\u03b2", + "protein" + ] + ] + }, + { + "sid": 93, + "sent": "The LBS of RORs share a high degree of similarity.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "LBS", + "site" + ], + [ + 11, + 15, + "RORs", + "protein_type" + ] + ] + }, + { + "sid": 94, + "sent": "However, the inverse agonism trigger of BIO399, residue Met358, is a leucine in both ROR\u03b1 and \u03b2.", + "section": "RESULTS", + "ner": [ + [ + 40, + 46, + "BIO399", + "chemical" + ], + [ + 56, + 62, + "Met358", + "residue_name_number" + ], + [ + 69, + 76, + "leucine", + "residue_name" + ], + [ + 85, + 89, + "ROR\u03b1", + "protein" + ], + [ + 94, + 95, + "\u03b2", + "protein" + ] + ] + }, + { + "sid": 95, + "sent": "This selectivity profile for BIO399 is attributed to the shorter leucine side chain in ROR\u03b1 and \u03b2 which would not reach the phenylalanine on the AF2 helix further underscoring the role of Met358 as a trigger for ROR\u03b3 specific inverse agonism (Fig.\u00a07a).", + "section": "RESULTS", + "ner": [ + [ + 29, + 35, + "BIO399", + "chemical" + ], + [ + 65, + 72, + "leucine", + "residue_name" + ], + [ + 87, + 91, + "ROR\u03b1", + "protein" + ], + [ + 96, + 97, + "\u03b2", + "protein" + ], + [ + 124, + 137, + "phenylalanine", + "residue_name" + ], + [ + 145, + 154, + "AF2 helix", + "structure_element" + ], + [ + 188, + 194, + "Met358", + "residue_name_number" + ], + [ + 212, + 216, + "ROR\u03b3", + "protein" + ] + ] + }, + { + "sid": 96, + "sent": "Furthermore, ROR\u03b1 contains two phenylalanine residues in its LBS whereas ROR\u03b2 and \u03b3 have a leucine in the same position (Fig.\u00a06b).", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "ROR\u03b1", + "protein" + ], + [ + 31, + 44, + "phenylalanine", + "residue_name" + ], + [ + 61, + 64, + "LBS", + "site" + ], + [ + 73, + 77, + "ROR\u03b2", + "protein" + ], + [ + 82, + 83, + "\u03b3", + "protein" + ], + [ + 91, + 98, + "leucine", + "residue_name" + ] + ] + }, + { + "sid": 97, + "sent": "We hypothesize that the two phenylalanine residues in the LBS of ROR\u03b1 occlude the dihydrobenzoxazepinone ring system of BIO399 from binding it and responsible for the increase in selectivity for ROR\u03b1 over \u03b2.", + "section": "RESULTS", + "ner": [ + [ + 28, + 41, + "phenylalanine", + "residue_name" + ], + [ + 58, + 61, + "LBS", + "site" + ], + [ + 65, + 69, + "ROR\u03b1", + "protein" + ], + [ + 82, + 104, + "dihydrobenzoxazepinone", + "chemical" + ], + [ + 120, + 126, + "BIO399", + "chemical" + ], + [ + 195, + 199, + "ROR\u03b1", + "protein" + ], + [ + 205, + 206, + "\u03b2", + "protein" + ] + ] + }, + { + "sid": 98, + "sent": "We have identified a novel series of synthetic benzoxazinone ligands which modulate the transcriptional activity of ROR\u03b3 in a FRET based assay.", + "section": "CONCL", + "ner": [ + [ + 47, + 60, + "benzoxazinone", + "chemical" + ], + [ + 116, + 120, + "ROR\u03b3", + "protein" + ], + [ + 126, + 142, + "FRET based assay", + "experimental_method" + ] + ] + }, + { + "sid": 99, + "sent": "Using partial proteolysis we show a conformational change which destabilizes the AF2 helix of ROR\u03b3 when the inverse agonist BIO399 binds.", + "section": "CONCL", + "ner": [ + [ + 6, + 25, + "partial proteolysis", + "experimental_method" + ], + [ + 81, + 90, + "AF2 helix", + "structure_element" + ], + [ + 94, + 98, + "ROR\u03b3", + "protein" + ], + [ + 108, + 123, + "inverse agonist", + "protein_state" + ], + [ + 124, + 130, + "BIO399", + "chemical" + ] + ] + }, + { + "sid": 100, + "sent": "The two ROR\u03b3 co-crystal structures reported here show how a small change to the core ring system can modulate the mode of action from agonist (BIO592) to inverse agonism (BIO399).", + "section": "CONCL", + "ner": [ + [ + 8, + 12, + "ROR\u03b3", + "protein" + ], + [ + 13, + 34, + "co-crystal structures", + "evidence" + ], + [ + 134, + 141, + "agonist", + "protein_state" + ], + [ + 143, + 149, + "BIO592", + "chemical" + ], + [ + 171, + 177, + "BIO399", + "chemical" + ] + ] + }, + { + "sid": 101, + "sent": "Finally, we are reporting a newly identified trigger for achieving ROR\u03b3 specific inverse agonism in an in vivo setting through Met358 which perturbs the agonist conformation of the AF2 helix and prevents coactivator protein binding.", + "section": "CONCL", + "ner": [ + [ + 67, + 71, + "ROR\u03b3", + "protein" + ], + [ + 127, + 133, + "Met358", + "residue_name_number" + ], + [ + 153, + 160, + "agonist", + "protein_state" + ], + [ + 181, + 190, + "AF2 helix", + "structure_element" + ] + ] + } + ] + }, + "PMC4784909": { + "annotations": [ + { + "sid": 0, + "sent": "The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle", + "section": "TITLE", + "ner": [ + [ + 24, + 34, + "Coenzyme A", + "chemical" + ], + [ + 50, + 59, + "Bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 1, + "sent": "Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 9, + "Bacterial", + "taxonomy_domain" + ], + [ + 10, + 27, + "Microcompartments", + "complex_assembly" + ], + [ + 29, + 33, + "BMCs", + "complex_assembly" + ] + ] + }, + { + "sid": 2, + "sent": "The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate.", + "section": "ABSTRACT", + "ner": [ + [ + 16, + 25, + "catabolic", + "protein_state" + ], + [ + 26, + 30, + "BMCs", + "complex_assembly" + ], + [ + 32, + 45, + "metabolosomes", + "complex_assembly" + ], + [ + 141, + 149, + "aldehyde", + "chemical" + ] + ] + }, + { + "sid": 3, + "sent": "The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source.", + "section": "ABSTRACT", + "ner": [ + [ + 16, + 35, + "phosphotransacylase", + "protein_type" + ], + [ + 37, + 41, + "PTAC", + "protein_type" + ], + [ + 52, + 62, + "Coenzyme A", + "chemical" + ], + [ + 80, + 94, + "acyl phosphate", + "chemical" + ] + ] + }, + { + "sid": 4, + "sent": "The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta).", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 8, + "PTAC", + "protein_type" + ], + [ + 39, + 52, + "metabolosomes", + "complex_assembly" + ], + [ + 54, + 58, + "PduL", + "protein_type" + ], + [ + 92, + 96, + "PTAC", + "protein_type" + ], + [ + 114, + 135, + "fermentative bacteria", + "taxonomy_domain" + ], + [ + 137, + 140, + "Pta", + "protein_type" + ] + ] + }, + { + "sid": 5, + "sent": "Here, we report two high-resolution PduL crystal structures with bound substrates.", + "section": "ABSTRACT", + "ner": [ + [ + 36, + 40, + "PduL", + "protein_type" + ], + [ + 41, + 59, + "crystal structures", + "evidence" + ], + [ + 60, + 81, + "with bound substrates", + "protein_state" + ] + ] + }, + { + "sid": 6, + "sent": "The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 8, + "PduL", + "protein_type" + ], + [ + 9, + 13, + "fold", + "structure_element" + ], + [ + 30, + 34, + "that", + "structure_element" + ], + [ + 38, + 41, + "Pta", + "protein_type" + ], + [ + 57, + 76, + "dimetal active site", + "site" + ], + [ + 137, + 149, + "housekeeping", + "protein_state" + ], + [ + 150, + 154, + "PTAC", + "protein_type" + ] + ] + }, + { + "sid": 7, + "sent": "Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution.", + "section": "ABSTRACT", + "ner": [ + [ + 13, + 17, + "PduL", + "protein_type" + ], + [ + 22, + 25, + "Pta", + "protein_type" + ] + ] + }, + { + "sid": 8, + "sent": "The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 8, + "PduL", + "protein_type" + ], + [ + 9, + 18, + "structure", + "evidence" + ], + [ + 139, + 151, + "metabolosome", + "complex_assembly" + ] + ] + }, + { + "sid": 9, + "sent": "This study describes the structure of a novel phosphotransacylase enzyme that facilitates the recycling of the essential cofactor acetyl-CoA within a bacterial organelle and discusses the properties of the enzyme's active site and how it is packaged into the organelle.", + "section": "ABSTRACT", + "ner": [ + [ + 25, + 34, + "structure", + "evidence" + ], + [ + 46, + 65, + "phosphotransacylase", + "protein_type" + ], + [ + 130, + 140, + "acetyl-CoA", + "chemical" + ], + [ + 150, + 159, + "bacterial", + "taxonomy_domain" + ], + [ + 215, + 226, + "active site", + "site" + ] + ] + }, + { + "sid": 10, + "sent": "In metabolism, molecules with \u201chigh-energy\u201d bonds (e.g., ATP and Acetyl~CoA) are critical for both catabolic and anabolic processes.", + "section": "ABSTRACT", + "ner": [ + [ + 57, + 60, + "ATP", + "chemical" + ], + [ + 65, + 75, + "Acetyl~CoA", + "chemical" + ] + ] + }, + { + "sid": 11, + "sent": "The phosphotransacylase (Pta) enzyme catalyzes the conversion between acyl-CoA and acyl-phosphate.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 23, + "phosphotransacylase", + "protein_type" + ], + [ + 25, + 28, + "Pta", + "protein_type" + ], + [ + 70, + 78, + "acyl-CoA", + "chemical" + ], + [ + 83, + 97, + "acyl-phosphate", + "chemical" + ] + ] + }, + { + "sid": 12, + "sent": "This reaction directly links an acyl-CoA with ATP generation via substrate-level phosphorylation, producing short-chain fatty acids (e.g., acetate), and also provides a path for short-chain fatty acids to enter central metabolism.", + "section": "ABSTRACT", + "ner": [ + [ + 32, + 40, + "acyl-CoA", + "chemical" + ], + [ + 46, + 49, + "ATP", + "chemical" + ], + [ + 108, + 131, + "short-chain fatty acids", + "chemical" + ], + [ + 139, + 146, + "acetate", + "chemical" + ], + [ + 178, + 201, + "short-chain fatty acids", + "chemical" + ] + ] + }, + { + "sid": 13, + "sent": "Due to this key function, Pta is conserved across the bacterial kingdom.", + "section": "ABSTRACT", + "ner": [ + [ + 33, + 42, + "conserved", + "protein_state" + ], + [ + 54, + 71, + "bacterial kingdom", + "taxonomy_domain" + ] + ] + }, + { + "sid": 14, + "sent": "Recently, a new type of phosphotransacylase was described that shares no evolutionary relation to Pta.", + "section": "ABSTRACT", + "ner": [ + [ + 24, + 43, + "phosphotransacylase", + "protein_type" + ], + [ + 98, + 101, + "Pta", + "protein_type" + ] + ] + }, + { + "sid": 15, + "sent": "This enzyme, PduL, is exclusively associated with organelles called bacterial microcompartments, which are used to catabolize various compounds.", + "section": "ABSTRACT", + "ner": [ + [ + 13, + 17, + "PduL", + "protein_type" + ], + [ + 22, + 33, + "exclusively", + "protein_state" + ], + [ + 68, + 77, + "bacterial", + "taxonomy_domain" + ], + [ + 78, + 95, + "microcompartments", + "complex_assembly" + ] + ] + }, + { + "sid": 16, + "sent": "Not only does PduL facilitate substrate level phosphorylation, but it also is critical for cofactor recycling within, and product efflux from, the organelle.", + "section": "ABSTRACT", + "ner": [ + [ + 14, + 18, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 17, + "sent": "We solved the structure of this convergent phosphotransacylase and show that it is completely structurally different from Pta, including its active site architecture.", + "section": "ABSTRACT", + "ner": [ + [ + 3, + 9, + "solved", + "experimental_method" + ], + [ + 14, + 23, + "structure", + "evidence" + ], + [ + 32, + 42, + "convergent", + "protein_state" + ], + [ + 43, + 62, + "phosphotransacylase", + "protein_type" + ], + [ + 122, + 125, + "Pta", + "protein_type" + ], + [ + 141, + 152, + "active site", + "site" + ] + ] + }, + { + "sid": 18, + "sent": "Bacterial Microcompartments (BMCs) are organelles that encapsulate enzymes for sequential biochemical reactions within a protein shell.", + "section": "INTRO", + "ner": [ + [ + 0, + 9, + "Bacterial", + "taxonomy_domain" + ], + [ + 10, + 27, + "Microcompartments", + "complex_assembly" + ], + [ + 29, + 33, + "BMCs", + "complex_assembly" + ], + [ + 129, + 134, + "shell", + "structure_element" + ] + ] + }, + { + "sid": 19, + "sent": "The shell is typically composed of three types of protein subunits, which form either hexagonal (BMC-H and BMC-T) or pentagonal (BMC-P) tiles that assemble into a polyhedral shell.", + "section": "INTRO", + "ner": [ + [ + 4, + 9, + "shell", + "structure_element" + ], + [ + 86, + 95, + "hexagonal", + "protein_state" + ], + [ + 97, + 102, + "BMC-H", + "complex_assembly" + ], + [ + 107, + 112, + "BMC-T", + "complex_assembly" + ], + [ + 117, + 127, + "pentagonal", + "protein_state" + ], + [ + 129, + 134, + "BMC-P", + "complex_assembly" + ], + [ + 163, + 173, + "polyhedral", + "protein_state" + ], + [ + 174, + 179, + "shell", + "structure_element" + ] + ] + }, + { + "sid": 20, + "sent": "The facets of the shell are composed primarily of hexamers that are typically perforated by pores lined with highly conserved, polar residues that presumably function as the conduits for metabolites into and out of the shell.", + "section": "INTRO", + "ner": [ + [ + 18, + 23, + "shell", + "structure_element" + ], + [ + 50, + 58, + "hexamers", + "oligomeric_state" + ], + [ + 92, + 97, + "pores", + "site" + ], + [ + 109, + 125, + "highly conserved", + "protein_state" + ], + [ + 127, + 132, + "polar", + "protein_state" + ], + [ + 133, + 141, + "residues", + "structure_element" + ], + [ + 219, + 224, + "shell", + "structure_element" + ] + ] + }, + { + "sid": 21, + "sent": "The vitamin B12-dependent propanediol-utilizing (PDU) BMC was one of the first functionally characterized catabolic BMCs; subsequently, other types have been implicated in the degradation of ethanolamine, choline, fucose, rhamnose, and ethanol, all of which produce different aldehyde intermediates (Table 1).", + "section": "INTRO", + "ner": [ + [ + 4, + 57, + "vitamin B12-dependent propanediol-utilizing (PDU) BMC", + "complex_assembly" + ], + [ + 106, + 115, + "catabolic", + "protein_state" + ], + [ + 116, + 120, + "BMCs", + "complex_assembly" + ], + [ + 191, + 203, + "ethanolamine", + "chemical" + ], + [ + 205, + 212, + "choline", + "chemical" + ], + [ + 214, + 220, + "fucose", + "chemical" + ], + [ + 222, + 230, + "rhamnose", + "chemical" + ], + [ + 236, + 243, + "ethanol", + "chemical" + ], + [ + 276, + 284, + "aldehyde", + "chemical" + ] + ] + }, + { + "sid": 22, + "sent": "More recently, bioinformatic studies have demonstrated the widespread distribution of BMCs among diverse bacterial phyla and grouped them into 23 different functional types.", + "section": "INTRO", + "ner": [ + [ + 15, + 36, + "bioinformatic studies", + "experimental_method" + ], + [ + 86, + 90, + "BMCs", + "complex_assembly" + ], + [ + 105, + 120, + "bacterial phyla", + "taxonomy_domain" + ] + ] + }, + { + "sid": 23, + "sent": "The reactions carried out in the majority of catabolic BMCs (also known as metabolosomes) fit a generalized biochemical paradigm for the oxidation of aldehydes (Fig 1).", + "section": "INTRO", + "ner": [ + [ + 45, + 54, + "catabolic", + "protein_state" + ], + [ + 55, + 59, + "BMCs", + "complex_assembly" + ], + [ + 75, + 88, + "metabolosomes", + "complex_assembly" + ], + [ + 150, + 159, + "aldehydes", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "This involves a BMC-encapsulated signature enzyme that generates a toxic and/or volatile aldehyde that the BMC shell sequesters from the cytosol.", + "section": "INTRO", + "ner": [ + [ + 16, + 19, + "BMC", + "complex_assembly" + ], + [ + 89, + 97, + "aldehyde", + "chemical" + ], + [ + 107, + 110, + "BMC", + "complex_assembly" + ], + [ + 111, + 116, + "shell", + "structure_element" + ] + ] + }, + { + "sid": 25, + "sent": "The aldehyde is subsequently converted into an acyl-CoA by aldehyde dehydrogenase, which uses NAD+ and CoA as cofactors.", + "section": "INTRO", + "ner": [ + [ + 4, + 12, + "aldehyde", + "chemical" + ], + [ + 47, + 55, + "acyl-CoA", + "chemical" + ], + [ + 59, + 81, + "aldehyde dehydrogenase", + "protein_type" + ], + [ + 94, + 98, + "NAD+", + "chemical" + ], + [ + 103, + 106, + "CoA", + "chemical" + ] + ] + }, + { + "sid": 26, + "sent": "These two cofactors are relatively large, and their diffusion across the protein shell is thought to be restricted, necessitating their regeneration within the BMC lumen.", + "section": "INTRO", + "ner": [ + [ + 73, + 86, + "protein shell", + "structure_element" + ], + [ + 160, + 163, + "BMC", + "complex_assembly" + ] + ] + }, + { + "sid": 27, + "sent": "NAD+ is recycled via alcohol dehydrogenase, and CoA is recycled via phosphotransacetylase (PTAC) (Fig 1).", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "NAD+", + "chemical" + ], + [ + 21, + 42, + "alcohol dehydrogenase", + "protein_type" + ], + [ + 48, + 51, + "CoA", + "chemical" + ], + [ + 68, + 89, + "phosphotransacetylase", + "protein_type" + ], + [ + 91, + 95, + "PTAC", + "protein_type" + ] + ] + }, + { + "sid": 28, + "sent": "The final product of the BMC, an acyl-phosphate, can then be used to generate ATP via acyl kinase, or revert back to acyl-CoA by Pta for biosynthesis.", + "section": "INTRO", + "ner": [ + [ + 25, + 28, + "BMC", + "complex_assembly" + ], + [ + 33, + 47, + "acyl-phosphate", + "chemical" + ], + [ + 78, + 81, + "ATP", + "chemical" + ], + [ + 86, + 97, + "acyl kinase", + "protein_type" + ], + [ + 117, + 125, + "acyl-CoA", + "chemical" + ], + [ + 129, + 132, + "Pta", + "protein_type" + ] + ] + }, + { + "sid": 29, + "sent": "Collectively, the aldehyde and alcohol dehydrogenases, as well as the PTAC, constitute the common metabolosome core.", + "section": "INTRO", + "ner": [ + [ + 18, + 53, + "aldehyde and alcohol dehydrogenases", + "protein_type" + ], + [ + 70, + 74, + "PTAC", + "protein_type" + ], + [ + 98, + 110, + "metabolosome", + "complex_assembly" + ] + ] + }, + { + "sid": 30, + "sent": "General biochemical model of aldehyde-degrading BMCs (metabolosomes) illustrating the common metabolosome core enzymes and reactions.", + "section": "FIG", + "ner": [ + [ + 29, + 47, + "aldehyde-degrading", + "protein_state" + ], + [ + 48, + 52, + "BMCs", + "complex_assembly" + ], + [ + 54, + 67, + "metabolosomes", + "complex_assembly" + ], + [ + 93, + 105, + "metabolosome", + "complex_assembly" + ] + ] + }, + { + "sid": 31, + "sent": "Substrates and cofactors involving the PTAC reaction are shown in red; other substrates and enzymes are shown in black, and other cofactors are shown in gray.", + "section": "FIG", + "ner": [ + [ + 39, + 43, + "PTAC", + "protein_type" + ] + ] + }, + { + "sid": 32, + "sent": "Characterized and predicted catabolic BMC (metabolosome) types that represent the aldehyde-degrading paradigm (for definition of types see Kerfeld and Erbilgin).", + "section": "TABLE", + "ner": [ + [ + 28, + 37, + "catabolic", + "protein_state" + ], + [ + 38, + 41, + "BMC", + "complex_assembly" + ], + [ + 43, + 55, + "metabolosome", + "complex_assembly" + ], + [ + 82, + 90, + "aldehyde", + "chemical" + ] + ] + }, + { + "sid": 33, + "sent": "Name\tPTAC Type\tSequestered Aldehyde\t \tPDU*\tPduL\tpropionaldehyde\t \tEUT1\tPTA_PTB\tacetaldehyde\t \tEUT2\tPduL\tacetaldehyde\t \tETU\tNone\tacetaldehyde\t \tGRM1/CUT\tPduL\tacetaldehyde\t \tGRM2\tPduL\tacetaldehyde\t \tGRM3*,4\tPduL\tpropionaldehyde\t \tGRM5/GRP\tPduL\tpropionaldehyde\t \tPVM*\tPduL\tlactaldehyde\t \tRMM1,2\tNone\tunknown\t \tSPU\tPduL\tunknown\t \t", + "section": "TABLE", + "ner": [ + [ + 5, + 9, + "PTAC", + "protein_type" + ], + [ + 27, + 35, + "Aldehyde", + "chemical" + ], + [ + 38, + 41, + "PDU", + "complex_assembly" + ], + [ + 43, + 47, + "PduL", + "protein_type" + ], + [ + 48, + 63, + "propionaldehyde", + "chemical" + ], + [ + 66, + 70, + "EUT1", + "complex_assembly" + ], + [ + 71, + 78, + "PTA_PTB", + "protein_type" + ], + [ + 79, + 91, + "acetaldehyde", + "chemical" + ], + [ + 94, + 98, + "EUT2", + "complex_assembly" + ], + [ + 99, + 103, + "PduL", + "protein_type" + ], + [ + 104, + 116, + "acetaldehyde", + "chemical" + ], + [ + 119, + 122, + "ETU", + "complex_assembly" + ], + [ + 128, + 140, + "acetaldehyde", + "chemical" + ], + [ + 143, + 151, + "GRM1/CUT", + "complex_assembly" + ], + [ + 152, + 156, + "PduL", + "protein_type" + ], + [ + 157, + 169, + "acetaldehyde", + "chemical" + ], + [ + 172, + 176, + "GRM2", + "complex_assembly" + ], + [ + 177, + 181, + "PduL", + "protein_type" + ], + [ + 182, + 194, + "acetaldehyde", + "chemical" + ], + [ + 197, + 204, + "GRM3*,4", + "complex_assembly" + ], + [ + 205, + 209, + "PduL", + "protein_type" + ], + [ + 210, + 225, + "propionaldehyde", + "chemical" + ], + [ + 228, + 236, + "GRM5/GRP", + "complex_assembly" + ], + [ + 237, + 241, + "PduL", + "protein_type" + ], + [ + 242, + 257, + "propionaldehyde", + "chemical" + ], + [ + 260, + 263, + "PVM", + "complex_assembly" + ], + [ + 265, + 269, + "PduL", + "protein_type" + ], + [ + 270, + 282, + "lactaldehyde", + "chemical" + ], + [ + 285, + 291, + "RMM1,2", + "complex_assembly" + ], + [ + 307, + 310, + "SPU", + "complex_assembly" + ], + [ + 311, + 315, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 34, + "sent": "* PduL from these functional types of metabolosomes were purified in this study.", + "section": "TABLE", + "ner": [ + [ + 2, + 6, + "PduL", + "protein_type" + ], + [ + 38, + 51, + "metabolosomes", + "complex_assembly" + ] + ] + }, + { + "sid": 35, + "sent": "The activities of core enzymes are not confined to BMC-associated functions: aldehyde and alcohol dehydrogenases are utilized in diverse metabolic reactions, and PTAC catalyzes a key biochemical reaction in the process of obtaining energy during fermentation.", + "section": "INTRO", + "ner": [ + [ + 51, + 54, + "BMC", + "complex_assembly" + ], + [ + 77, + 112, + "aldehyde and alcohol dehydrogenases", + "protein_type" + ], + [ + 162, + 166, + "PTAC", + "protein_type" + ] + ] + }, + { + "sid": 36, + "sent": "The concerted functioning of a PTAC and an acetate kinase (Ack) is crucial for ATP generation in the fermentation of pyruvate to acetate (see Reactions 1 and 2).", + "section": "INTRO", + "ner": [ + [ + 31, + 35, + "PTAC", + "protein_type" + ], + [ + 43, + 57, + "acetate kinase", + "protein_type" + ], + [ + 59, + 62, + "Ack", + "protein_type" + ], + [ + 79, + 82, + "ATP", + "chemical" + ], + [ + 117, + 125, + "pyruvate", + "chemical" + ], + [ + 129, + 136, + "acetate", + "chemical" + ] + ] + }, + { + "sid": 37, + "sent": "Both enzymes are, however, not restricted to fermentative organisms.", + "section": "INTRO", + "ner": [ + [ + 45, + 67, + "fermentative organisms", + "taxonomy_domain" + ] + ] + }, + { + "sid": 38, + "sent": "They can also work in the reverse direction to activate acetate to the CoA-thioester.", + "section": "INTRO", + "ner": [ + [ + 56, + 63, + "acetate", + "chemical" + ], + [ + 71, + 84, + "CoA-thioester", + "chemical" + ] + ] + }, + { + "sid": 39, + "sent": "This occurs, for example, during acetoclastic methanogenesis in the archaeal Methanosarcina species.", + "section": "INTRO", + "ner": [ + [ + 68, + 76, + "archaeal", + "taxonomy_domain" + ], + [ + 77, + 99, + "Methanosarcina species", + "taxonomy_domain" + ] + ] + }, + { + "sid": 40, + "sent": " Reaction 1: acetyl-S-CoA + Pi \u2190\u2192 acetyl phosphate + CoA-SH (PTAC)", + "section": "INTRO", + "ner": [ + [ + 13, + 25, + "acetyl-S-CoA", + "chemical" + ], + [ + 28, + 30, + "Pi", + "chemical" + ], + [ + 34, + 50, + "acetyl phosphate", + "chemical" + ], + [ + 53, + 59, + "CoA-SH", + "chemical" + ], + [ + 61, + 65, + "PTAC", + "protein_type" + ] + ] + }, + { + "sid": 41, + "sent": " Reaction 2: acetyl phosphate + ADP \u2190\u2192 acetate + ATP (Ack)", + "section": "INTRO", + "ner": [ + [ + 13, + 29, + "acetyl phosphate", + "chemical" + ], + [ + 32, + 35, + "ADP", + "chemical" + ], + [ + 39, + 46, + "acetate", + "chemical" + ], + [ + 49, + 52, + "ATP", + "chemical" + ], + [ + 54, + 57, + "Ack", + "protein_type" + ] + ] + }, + { + "sid": 42, + "sent": "The canonical PTAC, Pta, is an ancient enzyme found in some eukaryotes and archaea, and widespread among the bacteria; 90% of the bacterial genomes in the Integrated Microbial Genomes database contain a gene encoding the PTA_PTB phosphotransacylase (Pfam domain PF01515).", + "section": "INTRO", + "ner": [ + [ + 14, + 18, + "PTAC", + "protein_type" + ], + [ + 20, + 23, + "Pta", + "protein_type" + ], + [ + 60, + 70, + "eukaryotes", + "taxonomy_domain" + ], + [ + 75, + 82, + "archaea", + "taxonomy_domain" + ], + [ + 109, + 117, + "bacteria", + "taxonomy_domain" + ], + [ + 130, + 139, + "bacterial", + "taxonomy_domain" + ], + [ + 221, + 248, + "PTA_PTB phosphotransacylase", + "protein_type" + ], + [ + 262, + 269, + "PF01515", + "structure_element" + ] + ] + }, + { + "sid": 43, + "sent": "Pta has been extensively characterized due to its key role in fermentation.", + "section": "INTRO", + "ner": [ + [ + 0, + 3, + "Pta", + "protein_type" + ] + ] + }, + { + "sid": 44, + "sent": "More recently, a second type of PTAC without any sequence homology to Pta was identified.", + "section": "INTRO", + "ner": [ + [ + 32, + 36, + "PTAC", + "protein_type" + ], + [ + 70, + 73, + "Pta", + "protein_type" + ] + ] + }, + { + "sid": 45, + "sent": "This protein, PduL (Pfam domain PF06130), was shown to catalyze the conversion of propionyl-CoA to propionyl-phosphate and is associated with a BMC involved in propanediol utilization, the PDU BMC.", + "section": "INTRO", + "ner": [ + [ + 14, + 18, + "PduL", + "protein_type" + ], + [ + 32, + 39, + "PF06130", + "structure_element" + ], + [ + 82, + 95, + "propionyl-CoA", + "chemical" + ], + [ + 99, + 118, + "propionyl-phosphate", + "chemical" + ], + [ + 144, + 147, + "BMC", + "complex_assembly" + ], + [ + 189, + 196, + "PDU BMC", + "complex_assembly" + ] + ] + }, + { + "sid": 46, + "sent": "Both pduL and pta genes can be found in genetic loci of functionally distinct BMCs, although the PduL type is much more prevalent, being found in all but one type of metabolosome locus: EUT1 (Table 1).", + "section": "INTRO", + "ner": [ + [ + 5, + 9, + "pduL", + "gene" + ], + [ + 14, + 17, + "pta", + "gene" + ], + [ + 78, + 82, + "BMCs", + "complex_assembly" + ], + [ + 97, + 101, + "PduL", + "protein_type" + ], + [ + 166, + 184, + "metabolosome locus", + "gene" + ], + [ + 186, + 190, + "EUT1", + "gene" + ] + ] + }, + { + "sid": 47, + "sent": "Furthermore, in the Integrated Microbial Genomes Database, 91% of genomes that encode PF06130 also encode genes for shell proteins.", + "section": "INTRO", + "ner": [ + [ + 86, + 93, + "PF06130", + "structure_element" + ] + ] + }, + { + "sid": 48, + "sent": "As a member of the core biochemical machinery of functionally diverse aldehyde-oxidizing metabolosomes, PduL must have a certain level of substrate plasticity (see Table 1) that is not required of Pta, which has generally been observed to prefer acetyl-CoA. PduL from the PDU BMC of Salmonella enterica favors propionyl-CoA over acetyl-CoA, and it is likely that PduL orthologs in functionally diverse BMCs would have substrate preferences for other CoA derivatives.", + "section": "INTRO", + "ner": [ + [ + 70, + 88, + "aldehyde-oxidizing", + "protein_state" + ], + [ + 89, + 102, + "metabolosomes", + "complex_assembly" + ], + [ + 104, + 108, + "PduL", + "protein_type" + ], + [ + 197, + 200, + "Pta", + "protein_type" + ], + [ + 246, + 256, + "acetyl-CoA", + "chemical" + ], + [ + 258, + 262, + "PduL", + "protein_type" + ], + [ + 272, + 279, + "PDU BMC", + "complex_assembly" + ], + [ + 283, + 302, + "Salmonella enterica", + "species" + ], + [ + 310, + 323, + "propionyl-CoA", + "chemical" + ], + [ + 329, + 339, + "acetyl-CoA", + "chemical" + ], + [ + 363, + 367, + "PduL", + "protein_type" + ], + [ + 402, + 406, + "BMCs", + "complex_assembly" + ], + [ + 450, + 453, + "CoA", + "chemical" + ] + ] + }, + { + "sid": 49, + "sent": "Another distinctive feature of BMC-associated PduL homologs is an N-terminal encapsulation peptide (EP) that is thought to \u201ctarget\u201d proteins for encapsulation by the BMC shell.", + "section": "INTRO", + "ner": [ + [ + 31, + 45, + "BMC-associated", + "protein_state" + ], + [ + 46, + 50, + "PduL", + "protein_type" + ], + [ + 77, + 98, + "encapsulation peptide", + "structure_element" + ], + [ + 100, + 102, + "EP", + "structure_element" + ], + [ + 166, + 169, + "BMC", + "complex_assembly" + ], + [ + 170, + 175, + "shell", + "structure_element" + ] + ] + }, + { + "sid": 50, + "sent": "EPs are frequently found on BMC-associated proteins and have been shown to interact with shell proteins.", + "section": "INTRO", + "ner": [ + [ + 0, + 3, + "EPs", + "structure_element" + ], + [ + 28, + 51, + "BMC-associated proteins", + "protein_type" + ] + ] + }, + { + "sid": 51, + "sent": "EPs have also been observed to cause proteins to aggregate, and this has recently been suggested to be functionally relevant as an initial step in metabolosome assembly, in which a multifunctional protein core is formed, around which the shell assembles.", + "section": "INTRO", + "ner": [ + [ + 0, + 3, + "EPs", + "structure_element" + ], + [ + 147, + 159, + "metabolosome", + "complex_assembly" + ], + [ + 238, + 243, + "shell", + "structure_element" + ] + ] + }, + { + "sid": 52, + "sent": "Of the three common metabolosome core enzymes, crystal structures are available for both the alcohol and aldehyde dehydrogenases.", + "section": "INTRO", + "ner": [ + [ + 20, + 32, + "metabolosome", + "complex_assembly" + ], + [ + 47, + 65, + "crystal structures", + "evidence" + ], + [ + 93, + 128, + "alcohol and aldehyde dehydrogenases", + "protein_type" + ] + ] + }, + { + "sid": 53, + "sent": "In contrast, the structure of PduL, the PTAC found in the vast majority of catabolic BMCs, has not been determined.", + "section": "INTRO", + "ner": [ + [ + 17, + 26, + "structure", + "evidence" + ], + [ + 30, + 34, + "PduL", + "protein_type" + ], + [ + 40, + 44, + "PTAC", + "protein_type" + ], + [ + 75, + 84, + "catabolic", + "protein_state" + ], + [ + 85, + 89, + "BMCs", + "complex_assembly" + ] + ] + }, + { + "sid": 54, + "sent": "This is a major gap in our understanding of metabolosome-encapsulated biochemistry and cofactor recycling.", + "section": "INTRO", + "ner": [ + [ + 44, + 56, + "metabolosome", + "complex_assembly" + ] + ] + }, + { + "sid": 55, + "sent": "Moreover, it will be useful for guiding efforts to engineer novel BMC cores for biotechnological applications.", + "section": "INTRO", + "ner": [ + [ + 66, + 69, + "BMC", + "complex_assembly" + ] + ] + }, + { + "sid": 56, + "sent": "The primary structure of PduL homologs is subdivided into two PF06130 domains, each roughly 80 residues in length.", + "section": "INTRO", + "ner": [ + [ + 25, + 29, + "PduL", + "protein_type" + ], + [ + 62, + 69, + "PF06130", + "structure_element" + ], + [ + 92, + 113, + "80 residues in length", + "residue_range" + ] + ] + }, + { + "sid": 57, + "sent": "No available protein structures contain the PF06130 domain, and homology searches using the primary structure of PduL do not return any significant results that would allow prediction of the structure.", + "section": "INTRO", + "ner": [ + [ + 44, + 51, + "PF06130", + "structure_element" + ], + [ + 64, + 81, + "homology searches", + "experimental_method" + ], + [ + 113, + 117, + "PduL", + "protein_type" + ], + [ + 191, + 200, + "structure", + "evidence" + ] + ] + }, + { + "sid": 58, + "sent": "Moreover, the evident novelty of PduL makes its structure interesting in the context of convergent evolution of PTAC function; to-date, only the Pta active site and catalytic mechanism is known.", + "section": "INTRO", + "ner": [ + [ + 33, + 37, + "PduL", + "protein_type" + ], + [ + 48, + 57, + "structure", + "evidence" + ], + [ + 112, + 116, + "PTAC", + "protein_type" + ], + [ + 145, + 148, + "Pta", + "protein_type" + ], + [ + 149, + 160, + "active site", + "site" + ] + ] + }, + { + "sid": 59, + "sent": "Here we report high-resolution crystal structures of a PduL-type PTAC in both CoA- and phosphate-bound forms, completing our understanding of the structural basis of catalysis by the metabolosome common core enzymes.", + "section": "INTRO", + "ner": [ + [ + 31, + 49, + "crystal structures", + "evidence" + ], + [ + 55, + 69, + "PduL-type PTAC", + "protein_type" + ], + [ + 78, + 82, + "CoA-", + "protein_state" + ], + [ + 87, + 102, + "phosphate-bound", + "protein_state" + ], + [ + 183, + 195, + "metabolosome", + "complex_assembly" + ] + ] + }, + { + "sid": 60, + "sent": "We propose a catalytic mechanism analogous but yet distinct from the ubiquitous Pta enzyme, highlighting the functional convergence of two enzymes with completely different structures and metal requirements.", + "section": "INTRO", + "ner": [ + [ + 80, + 83, + "Pta", + "protein_type" + ] + ] + }, + { + "sid": 61, + "sent": "We also investigate the quaternary structures of three different PduL homologs and situate our findings in the context of organelle biogenesis in functionally diverse BMCs.", + "section": "INTRO", + "ner": [ + [ + 65, + 69, + "PduL", + "protein_type" + ], + [ + 167, + 171, + "BMCs", + "complex_assembly" + ] + ] + }, + { + "sid": 62, + "sent": "Structure Determination of PduL", + "section": "RESULTS", + "ner": [ + [ + 0, + 23, + "Structure Determination", + "experimental_method" + ], + [ + 27, + 31, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 63, + "sent": "We cloned, expressed, and purified three different PduL homologs from functionally distinct BMCs (Table 1): from the well-studied pdu locus in S. enterica Typhimurium LT2 (sPduL), from the recently characterized pvm locus in Planctomyces limnophilus (pPduL), and from the grm3 locus in Rhodopseudomonas palustris BisB18 (rPduL).", + "section": "RESULTS", + "ner": [ + [ + 3, + 34, + "cloned, expressed, and purified", + "experimental_method" + ], + [ + 51, + 55, + "PduL", + "protein_type" + ], + [ + 92, + 96, + "BMCs", + "complex_assembly" + ], + [ + 130, + 139, + "pdu locus", + "gene" + ], + [ + 143, + 170, + "S. enterica Typhimurium LT2", + "species" + ], + [ + 172, + 177, + "sPduL", + "protein" + ], + [ + 212, + 221, + "pvm locus", + "gene" + ], + [ + 225, + 249, + "Planctomyces limnophilus", + "species" + ], + [ + 251, + 256, + "pPduL", + "protein" + ], + [ + 272, + 282, + "grm3 locus", + "gene" + ], + [ + 286, + 319, + "Rhodopseudomonas palustris BisB18", + "species" + ], + [ + 321, + 326, + "rPduL", + "protein" + ] + ] + }, + { + "sid": 64, + "sent": "While purifying full-length sPduL, we observed a tendency to aggregation as described previously, with a large fraction of the expressed protein found in the insoluble fraction in a white, cake-like pellet.", + "section": "RESULTS", + "ner": [ + [ + 16, + 27, + "full-length", + "protein_state" + ], + [ + 28, + 33, + "sPduL", + "protein" + ] + ] + }, + { + "sid": 65, + "sent": "Remarkably, after removing the N-terminal putative EP (27 amino acids), most of the sPduL\u0394EP protein was in the soluble fraction upon cell lysis.", + "section": "RESULTS", + "ner": [ + [ + 18, + 26, + "removing", + "experimental_method" + ], + [ + 51, + 53, + "EP", + "structure_element" + ], + [ + 55, + 69, + "27 amino acids", + "residue_range" + ], + [ + 84, + 92, + "sPduL\u0394EP", + "mutant" + ] + ] + }, + { + "sid": 66, + "sent": "Similar differences in solubility were observed for pPduL and rPduL when comparing EP-truncated forms to the full-length protein, but none were quite as dramatic as for sPduL. We confirmed that all homologs were active (S1a and S1b Fig).", + "section": "RESULTS", + "ner": [ + [ + 52, + 57, + "pPduL", + "protein" + ], + [ + 62, + 67, + "rPduL", + "protein" + ], + [ + 83, + 95, + "EP-truncated", + "protein_state" + ], + [ + 109, + 120, + "full-length", + "protein_state" + ], + [ + 169, + 174, + "sPduL", + "protein" + ], + [ + 212, + 218, + "active", + "protein_state" + ] + ] + }, + { + "sid": 67, + "sent": "Among these, we were only able to obtain diffraction-quality crystals of rPduL after removing the N-terminal putative EP (33 amino acids, also see Fig 2a) (rPduL\u0394EP).", + "section": "RESULTS", + "ner": [ + [ + 41, + 69, + "diffraction-quality crystals", + "evidence" + ], + [ + 73, + 78, + "rPduL", + "protein" + ], + [ + 85, + 93, + "removing", + "experimental_method" + ], + [ + 118, + 120, + "EP", + "structure_element" + ], + [ + 122, + 136, + "33 amino acids", + "residue_range" + ], + [ + 156, + 164, + "rPduL\u0394EP", + "mutant" + ] + ] + }, + { + "sid": 68, + "sent": "Truncated rPduL\u0394EP had comparable enzymatic activity to the full-length enzyme (S1a Fig).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Truncated", + "protein_state" + ], + [ + 10, + 18, + "rPduL\u0394EP", + "mutant" + ], + [ + 60, + 71, + "full-length", + "protein_state" + ] + ] + }, + { + "sid": 69, + "sent": "Structural overview of R. palustris PduL from the grm3 locus.", + "section": "FIG", + "ner": [ + [ + 23, + 35, + "R. palustris", + "species" + ], + [ + 36, + 40, + "PduL", + "protein_type" + ], + [ + 50, + 60, + "grm3 locus", + "gene" + ] + ] + }, + { + "sid": 70, + "sent": "(a) Primary and secondary structure of rPduL (tubes represent \u03b1-helices, arrows \u03b2-sheets and dashed line residues disordered in the structure.", + "section": "FIG", + "ner": [ + [ + 39, + 44, + "rPduL", + "protein" + ], + [ + 62, + 71, + "\u03b1-helices", + "structure_element" + ], + [ + 80, + 88, + "\u03b2-sheets", + "structure_element" + ], + [ + 132, + 141, + "structure", + "evidence" + ] + ] + }, + { + "sid": 71, + "sent": "The first 33 amino acids are present only in the wildtype construct and contains the predicted EP alpha helix, \u03b10); the truncated rPduL\u0394EP that was crystallized begins with M-G-V. Coloring is according to structural domains (domain 1 D36-N46/Q155-C224, blue; loop insertion G61-E81, grey; domain 2 R47-F60/E82-A154, red).", + "section": "FIG", + "ner": [ + [ + 4, + 24, + "first 33 amino acids", + "residue_range" + ], + [ + 95, + 97, + "EP", + "structure_element" + ], + [ + 98, + 109, + "alpha helix", + "structure_element" + ], + [ + 111, + 113, + "\u03b10", + "structure_element" + ], + [ + 120, + 129, + "truncated", + "protein_state" + ], + [ + 130, + 138, + "rPduL\u0394EP", + "mutant" + ], + [ + 148, + 160, + "crystallized", + "experimental_method" + ], + [ + 173, + 174, + "M", + "residue_name" + ], + [ + 175, + 176, + "G", + "residue_name" + ], + [ + 177, + 178, + "V", + "residue_name" + ], + [ + 225, + 233, + "domain 1", + "structure_element" + ], + [ + 234, + 241, + "D36-N46", + "residue_range" + ], + [ + 242, + 251, + "Q155-C224", + "residue_range" + ], + [ + 259, + 273, + "loop insertion", + "structure_element" + ], + [ + 274, + 281, + "G61-E81", + "residue_range" + ], + [ + 289, + 297, + "domain 2", + "structure_element" + ], + [ + 298, + 305, + "R47-F60", + "residue_range" + ], + [ + 306, + 314, + "E82-A154", + "residue_range" + ] + ] + }, + { + "sid": 72, + "sent": "Metal coordination residues are highlighted in light blue and CoA contacting residues in magenta, residues contacting the CoA of the other chain are also outlined.", + "section": "FIG", + "ner": [ + [ + 0, + 27, + "Metal coordination residues", + "site" + ], + [ + 62, + 85, + "CoA contacting residues", + "site" + ], + [ + 122, + 125, + "CoA", + "chemical" + ] + ] + }, + { + "sid": 73, + "sent": "(b) Cartoon representation of the structure colored by domains and including secondary structure numbering.", + "section": "FIG", + "ner": [ + [ + 34, + 43, + "structure", + "evidence" + ], + [ + 87, + 96, + "structure", + "evidence" + ] + ] + }, + { + "sid": 74, + "sent": "Coenzyme A is shown in magenta sticks and Zinc (grey) as spheres.", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "Coenzyme A", + "chemical" + ], + [ + 42, + 46, + "Zinc", + "chemical" + ] + ] + }, + { + "sid": 75, + "sent": "We collected a native dataset from rPduL\u0394EP crystals diffracting to a resolution of 1.54 \u00c5 (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 3, + 29, + "collected a native dataset", + "experimental_method" + ], + [ + 35, + 43, + "rPduL\u0394EP", + "mutant" + ], + [ + 44, + 52, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 76, + "sent": "Using a mercury-derivative crystal form diffracting to 1.99 \u00c5 (Table 2), we obtained high quality electron density for model building and used the initial model to refine against the native data to Rwork/Rfree values of 18.9/22.1%.", + "section": "RESULTS", + "ner": [ + [ + 8, + 34, + "mercury-derivative crystal", + "experimental_method" + ], + [ + 98, + 114, + "electron density", + "evidence" + ], + [ + 198, + 203, + "Rwork", + "evidence" + ], + [ + 204, + 209, + "Rfree", + "evidence" + ] + ] + }, + { + "sid": 77, + "sent": "There are two PduL molecules in the asymmetric unit of the P212121 unit cell.", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 78, + "sent": "We were able to fit all of the primary structure of PduL\u0394EP into the electron density with the exception of three amino acids at the N-terminus and two amino acids at the C-terminus (Fig 2a); the model is of excellent quality (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 52, + 59, + "PduL\u0394EP", + "mutant" + ], + [ + 69, + 85, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 79, + "sent": "A CoA cofactor as well as two metal ions are clearly resolved in the density (for omit maps of CoA see S2 Fig).", + "section": "RESULTS", + "ner": [ + [ + 2, + 5, + "CoA", + "chemical" + ], + [ + 69, + 76, + "density", + "evidence" + ], + [ + 82, + 91, + "omit maps", + "evidence" + ], + [ + 95, + 98, + "CoA", + "chemical" + ] + ] + }, + { + "sid": 80, + "sent": "Structurally, PduL consists of two domains (Fig 2, blue/red), each a beta-barrel that is capped on both ends by short \u03b1-helices.", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "PduL", + "protein_type" + ], + [ + 35, + 42, + "domains", + "structure_element" + ], + [ + 69, + 80, + "beta-barrel", + "structure_element" + ], + [ + 118, + 127, + "\u03b1-helices", + "structure_element" + ] + ] + }, + { + "sid": 81, + "sent": "\u03b2-Barrel 1 consists of the N-terminal \u03b2 strand and \u03b2 strands from the C-terminal half of the polypeptide chain (\u03b21, \u03b210-\u03b214; residues 37\u201346 and 155\u2013224).", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "\u03b2-Barrel 1", + "structure_element" + ], + [ + 38, + 46, + "\u03b2 strand", + "structure_element" + ], + [ + 51, + 60, + "\u03b2 strands", + "structure_element" + ], + [ + 70, + 85, + "C-terminal half", + "structure_element" + ], + [ + 112, + 114, + "\u03b21", + "structure_element" + ], + [ + 116, + 123, + "\u03b210-\u03b214", + "structure_element" + ], + [ + 134, + 139, + "37\u201346", + "residue_range" + ], + [ + 144, + 151, + "155\u2013224", + "residue_range" + ] + ] + }, + { + "sid": 82, + "sent": "\u03b2-Barrel 2 consists mainly of the central segment of primary structure (\u03b22, \u03b25\u2013\u03b29; residues 47\u201360 and 82\u2013154) (Fig 2, red), but is interrupted by a short two-strand beta sheet (\u03b23-\u03b24, residues 61\u201381).", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "\u03b2-Barrel 2", + "structure_element" + ], + [ + 72, + 74, + "\u03b22", + "structure_element" + ], + [ + 76, + 81, + "\u03b25\u2013\u03b29", + "structure_element" + ], + [ + 92, + 97, + "47\u201360", + "residue_range" + ], + [ + 102, + 108, + "82\u2013154", + "residue_range" + ], + [ + 148, + 175, + "short two-strand beta sheet", + "structure_element" + ], + [ + 177, + 182, + "\u03b23-\u03b24", + "structure_element" + ], + [ + 193, + 198, + "61\u201381", + "residue_range" + ] + ] + }, + { + "sid": 83, + "sent": "This \u03b2-sheet is involved in contacts between the two domains and forms a lid over the active site.", + "section": "RESULTS", + "ner": [ + [ + 5, + 12, + "\u03b2-sheet", + "structure_element" + ], + [ + 86, + 97, + "active site", + "site" + ] + ] + }, + { + "sid": 84, + "sent": "Residues in this region (Gln42, Pro43, Gly44), covering the active site, are strongly conserved (Fig 3).", + "section": "RESULTS", + "ner": [ + [ + 25, + 30, + "Gln42", + "residue_name_number" + ], + [ + 32, + 37, + "Pro43", + "residue_name_number" + ], + [ + 39, + 44, + "Gly44", + "residue_name_number" + ], + [ + 60, + 71, + "active site", + "site" + ], + [ + 77, + 95, + "strongly conserved", + "protein_state" + ] + ] + }, + { + "sid": 85, + "sent": "This structural arrangement is completely different from the functionally related Pta, which is composed of two domains, each consisting of a central flat beta sheet with alpha-helices on the top and bottom.", + "section": "RESULTS", + "ner": [ + [ + 82, + 85, + "Pta", + "protein_type" + ], + [ + 112, + 119, + "domains", + "structure_element" + ], + [ + 155, + 165, + "beta sheet", + "structure_element" + ], + [ + 171, + 184, + "alpha-helices", + "structure_element" + ] + ] + }, + { + "sid": 86, + "sent": "Primary structure conservation of the PduL protein family.", + "section": "FIG", + "ner": [ + [ + 38, + 42, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 87, + "sent": "Sequence logo calculated from the multiple sequence alignment of PduL homologs (see Materials and Methods), but not including putative EP sequences.", + "section": "FIG", + "ner": [ + [ + 34, + 61, + "multiple sequence alignment", + "experimental_method" + ], + [ + 65, + 69, + "PduL", + "protein_type" + ], + [ + 112, + 125, + "not including", + "protein_state" + ], + [ + 135, + 137, + "EP", + "structure_element" + ] + ] + }, + { + "sid": 88, + "sent": "Residues 100% conserved across all PduL homologs in our dataset are noted with an asterisk, and residues conserved in over 90% of sequences are noted with a colon.", + "section": "FIG", + "ner": [ + [ + 35, + 39, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 89, + "sent": "The sequences aligning to the PF06130 domain (determined by BLAST) are highlighted in red and blue.", + "section": "FIG", + "ner": [ + [ + 30, + 37, + "PF06130", + "structure_element" + ] + ] + }, + { + "sid": 90, + "sent": "The position numbers shown correspond to the residue numbering of rPduL; note that some positions in the logo represent gaps in the rPduL sequence.", + "section": "FIG", + "ner": [ + [ + 66, + 71, + "rPduL", + "protein" + ], + [ + 132, + 137, + "rPduL", + "protein" + ] + ] + }, + { + "sid": 91, + "sent": "There are two PduL molecules in the asymmetric unit forming a butterfly-shaped dimer (Fig 4c).", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "PduL", + "protein_type" + ], + [ + 62, + 78, + "butterfly-shaped", + "protein_state" + ], + [ + 79, + 84, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 92, + "sent": "Consistent with this, results from size exclusion chromatography of rPduL\u0394EP suggest that it is a dimer in solution (Fig 5e).", + "section": "RESULTS", + "ner": [ + [ + 35, + 64, + "size exclusion chromatography", + "experimental_method" + ], + [ + 68, + 76, + "rPduL\u0394EP", + "mutant" + ], + [ + 98, + 103, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 93, + "sent": "The interface between the two chains buries 882 \u00c52 per monomer and is mainly formed by \u03b1-helices 2 and 4 and parts of \u03b2-sheets 12 and 14, as well as a \u03c0\u2013\u03c0 stacking of the adenine moiety of CoA with Phe116 of the adjacent chain (Fig 4c).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "interface", + "site" + ], + [ + 55, + 62, + "monomer", + "oligomeric_state" + ], + [ + 87, + 104, + "\u03b1-helices 2 and 4", + "structure_element" + ], + [ + 118, + 136, + "\u03b2-sheets 12 and 14", + "structure_element" + ], + [ + 171, + 178, + "adenine", + "chemical" + ], + [ + 189, + 192, + "CoA", + "chemical" + ], + [ + 198, + 204, + "Phe116", + "residue_name_number" + ] + ] + }, + { + "sid": 94, + "sent": "The folds of the two chains in the asymmetric unit are very similar, superimposing with a rmsd of 0.16 \u00c5 over 2,306 aligned atom pairs.", + "section": "RESULTS", + "ner": [ + [ + 69, + 82, + "superimposing", + "experimental_method" + ], + [ + 90, + 94, + "rmsd", + "evidence" + ] + ] + }, + { + "sid": 95, + "sent": "The peripheral helices and the short antiparallel \u03b23\u20134 sheet mediate most of the crystal contacts.", + "section": "RESULTS", + "ner": [ + [ + 15, + 22, + "helices", + "structure_element" + ], + [ + 31, + 60, + "short antiparallel \u03b23\u20134 sheet", + "structure_element" + ] + ] + }, + { + "sid": 96, + "sent": "Details of active site, dimeric assembly, and sequence conservation of PduL.", + "section": "FIG", + "ner": [ + [ + 11, + 22, + "active site", + "site" + ], + [ + 24, + 31, + "dimeric", + "oligomeric_state" + ], + [ + 71, + 75, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 97, + "sent": "(a,b) Proposed active site of PduL with relevant residues shown as sticks in atom coloring (nitrogen blue, oxygen red, sulfur yellow), zinc as grey colored spheres and coordinating ordered water molecules in red.", + "section": "FIG", + "ner": [ + [ + 15, + 26, + "active site", + "site" + ], + [ + 30, + 34, + "PduL", + "protein_type" + ], + [ + 92, + 100, + "nitrogen", + "chemical" + ], + [ + 107, + 113, + "oxygen", + "chemical" + ], + [ + 119, + 125, + "sulfur", + "chemical" + ], + [ + 135, + 139, + "zinc", + "chemical" + ], + [ + 189, + 194, + "water", + "chemical" + ] + ] + }, + { + "sid": 98, + "sent": "Distances between atom centers are indicated in \u00c5. (a) Coenzyme A containing, (b) phosphate-bound structure.", + "section": "FIG", + "ner": [ + [ + 55, + 65, + "Coenzyme A", + "chemical" + ], + [ + 82, + 97, + "phosphate-bound", + "protein_state" + ], + [ + 98, + 107, + "structure", + "evidence" + ] + ] + }, + { + "sid": 99, + "sent": "(c) View of the dimer in the asymmetric unit from the side, domains 1 and 2 colored as in Fig 2 and the two chains differentiated by blue/red versus slate/firebrick.", + "section": "FIG", + "ner": [ + [ + 16, + 21, + "dimer", + "oligomeric_state" + ], + [ + 60, + 75, + "domains 1 and 2", + "structure_element" + ] + ] + }, + { + "sid": 100, + "sent": "The asterisk and double arrow marks the location of the \u03c0\u2013\u03c0 interaction between F116 and the CoA base of the other dimer chain.", + "section": "FIG", + "ner": [ + [ + 56, + 71, + "\u03c0\u2013\u03c0 interaction", + "bond_interaction" + ], + [ + 80, + 84, + "F116", + "residue_name_number" + ], + [ + 93, + 96, + "CoA", + "chemical" + ], + [ + 115, + 120, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 101, + "sent": "(d) Surface representation of the structure with indicated conservation (red: high, white: intermediate, yellow: low).", + "section": "FIG", + "ner": [ + [ + 34, + 43, + "structure", + "evidence" + ] + ] + }, + { + "sid": 102, + "sent": "Size exclusion chromatography of PduL homologs.", + "section": "FIG", + "ner": [ + [ + 0, + 29, + "Size exclusion chromatography", + "experimental_method" + ], + [ + 33, + 37, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 103, + "sent": "(a)\u2013(c): Chromatograms of sPduL (a), rPduL (b), and pPduL (c) with (orange) or without (blue) the predicted EP, post-nickel affinity purification, applied over a preparative size exclusion column (see Materials and Methods).", + "section": "FIG", + "ner": [ + [ + 9, + 22, + "Chromatograms", + "evidence" + ], + [ + 26, + 31, + "sPduL", + "protein" + ], + [ + 37, + 42, + "rPduL", + "protein" + ], + [ + 52, + 57, + "pPduL", + "protein" + ], + [ + 108, + 110, + "EP", + "structure_element" + ], + [ + 117, + 145, + "nickel affinity purification", + "experimental_method" + ] + ] + }, + { + "sid": 104, + "sent": "(d)\u2013(f): Chromatograms of sPduL (d), rPduL (e), and pPduL (f) post-preparative size exclusion chromatography with different size fractions separated, applied over an analytical size exclusion column (see Materials and Methods).", + "section": "FIG", + "ner": [ + [ + 9, + 22, + "Chromatograms", + "evidence" + ], + [ + 26, + 31, + "sPduL", + "protein" + ], + [ + 37, + 42, + "rPduL", + "protein" + ], + [ + 52, + 57, + "pPduL", + "protein" + ], + [ + 79, + 108, + "size exclusion chromatography", + "experimental_method" + ] + ] + }, + { + "sid": 105, + "sent": "All chromatograms are cropped to show only the linear range of separation based on standard runs, shown in black squares with a dashed linear trend line.", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "chromatograms", + "evidence" + ] + ] + }, + { + "sid": 106, + "sent": "Active Site Properties", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Active Site", + "site" + ] + ] + }, + { + "sid": 107, + "sent": "CoA and the metal ions bind between the two domains, presumably in the active site (Figs 2b and 4a).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "CoA", + "chemical" + ], + [ + 71, + 82, + "active site", + "site" + ] + ] + }, + { + "sid": 108, + "sent": "To identify the bound metals, we performed an X-ray fluorescence scan on the crystals at various wavelengths (corresponding to the K-edges of Mn, Fe, Co, Ni, Cu, and Zn).", + "section": "RESULTS", + "ner": [ + [ + 46, + 69, + "X-ray fluorescence scan", + "experimental_method" + ], + [ + 77, + 85, + "crystals", + "evidence" + ], + [ + 142, + 144, + "Mn", + "chemical" + ], + [ + 146, + 148, + "Fe", + "chemical" + ], + [ + 150, + 152, + "Co", + "chemical" + ], + [ + 154, + 156, + "Ni", + "chemical" + ], + [ + 158, + 160, + "Cu", + "chemical" + ], + [ + 166, + 168, + "Zn", + "chemical" + ] + ] + }, + { + "sid": 109, + "sent": "There was a large signal at the zinc edge, and we tested for the presence of zinc by collecting full data sets before and after the Zn K-edge (1.2861 and 1.2822 \u00c5, respectively).", + "section": "RESULTS", + "ner": [ + [ + 77, + 81, + "zinc", + "chemical" + ], + [ + 85, + 162, + "collecting full data sets before and after the Zn K-edge (1.2861 and 1.2822 \u00c5", + "experimental_method" + ] + ] + }, + { + "sid": 110, + "sent": "The large differences between the anomalous signals confirm the presence of zinc at both metal sites (S3 Fig).", + "section": "RESULTS", + "ner": [ + [ + 76, + 80, + "zinc", + "chemical" + ] + ] + }, + { + "sid": 111, + "sent": "The first zinc ion (Zn1) is in a tetrahedral coordination state with His48, His50, Glu109, and the CoA sulfur (Fig 4a).", + "section": "RESULTS", + "ner": [ + [ + 10, + 14, + "zinc", + "chemical" + ], + [ + 20, + 23, + "Zn1", + "chemical" + ], + [ + 69, + 74, + "His48", + "residue_name_number" + ], + [ + 76, + 81, + "His50", + "residue_name_number" + ], + [ + 83, + 89, + "Glu109", + "residue_name_number" + ], + [ + 99, + 102, + "CoA", + "chemical" + ], + [ + 103, + 109, + "sulfur", + "chemical" + ] + ] + }, + { + "sid": 112, + "sent": "The second (Zn2) is in octahedral coordination by three conserved histidine residues (His157, His159 and His204) as well as three water molecules (Fig 4a).", + "section": "RESULTS", + "ner": [ + [ + 12, + 15, + "Zn2", + "chemical" + ], + [ + 56, + 65, + "conserved", + "protein_state" + ], + [ + 66, + 75, + "histidine", + "residue_name" + ], + [ + 86, + 92, + "His157", + "residue_name_number" + ], + [ + 94, + 100, + "His159", + "residue_name_number" + ], + [ + 105, + 111, + "His204", + "residue_name_number" + ], + [ + 130, + 135, + "water", + "chemical" + ] + ] + }, + { + "sid": 113, + "sent": "The nitrogen atom coordinating the zinc is the N\u03b5 in each histidine residue, as is typical for this interaction.", + "section": "RESULTS", + "ner": [ + [ + 35, + 39, + "zinc", + "chemical" + ], + [ + 58, + 67, + "histidine", + "residue_name" + ] + ] + }, + { + "sid": 114, + "sent": "When the crystals were soaked in a sodium phosphate solution for 2 d prior to data collection, the CoA dissociates, and density for a phosphate molecule is visible at the active site (Table 2, Fig 4b).", + "section": "RESULTS", + "ner": [ + [ + 9, + 29, + "crystals were soaked", + "experimental_method" + ], + [ + 35, + 51, + "sodium phosphate", + "chemical" + ], + [ + 99, + 102, + "CoA", + "chemical" + ], + [ + 120, + 127, + "density", + "evidence" + ], + [ + 134, + 143, + "phosphate", + "chemical" + ], + [ + 171, + 182, + "active site", + "site" + ] + ] + }, + { + "sid": 115, + "sent": "The phosphate-bound structure aligns well with the CoA-bound structure (0.43 \u00c5 rmsd over 2,361 atoms for the monomer, 0.83 \u00c5 over 5,259 aligned atoms for the dimer).", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "phosphate-bound", + "protein_state" + ], + [ + 20, + 29, + "structure", + "evidence" + ], + [ + 30, + 36, + "aligns", + "experimental_method" + ], + [ + 51, + 60, + "CoA-bound", + "protein_state" + ], + [ + 61, + 70, + "structure", + "evidence" + ], + [ + 79, + 83, + "rmsd", + "evidence" + ], + [ + 109, + 116, + "monomer", + "oligomeric_state" + ], + [ + 158, + 163, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 116, + "sent": "The phosphate contacts both zinc atoms (Fig 4b) and replaces the coordination by CoA at Zn1; the coordination for Zn2 changes from octahedral with three bound waters to tetrahedral with a phosphate ion as one of the ligands (Fig 4b).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "phosphate", + "chemical" + ], + [ + 28, + 32, + "zinc", + "chemical" + ], + [ + 81, + 84, + "CoA", + "chemical" + ], + [ + 88, + 91, + "Zn1", + "chemical" + ], + [ + 114, + 117, + "Zn2", + "chemical" + ], + [ + 159, + 165, + "waters", + "chemical" + ], + [ + 188, + 197, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 117, + "sent": "Conserved Arg103 seems to be involved in maintaining the phosphate in that position.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Conserved", + "protein_state" + ], + [ + 10, + 16, + "Arg103", + "residue_name_number" + ], + [ + 57, + 66, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 118, + "sent": "The two zinc atoms are slightly closer together in the phosphate-bound form (5.8 \u00c5 vs 6.3 \u00c5), possibly due to the bridging effect of the phosphate.", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "zinc", + "chemical" + ], + [ + 55, + 70, + "phosphate-bound", + "protein_state" + ], + [ + 137, + 146, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 119, + "sent": "An additional phosphate molecule is bound at a crystal contact interface, perhaps accounting for the 14 \u00c5 shorter c-axis in the phosphate-bound crystal form (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 14, + 23, + "phosphate", + "chemical" + ], + [ + 128, + 143, + "phosphate-bound", + "protein_state" + ] + ] + }, + { + "sid": 120, + "sent": "Oligomeric States of PduL Orthologs Are Influenced by the EP", + "section": "RESULTS", + "ner": [ + [ + 21, + 25, + "PduL", + "protein_type" + ], + [ + 58, + 60, + "EP", + "structure_element" + ] + ] + }, + { + "sid": 121, + "sent": "Interestingly, some of the residues important for dimerization of rPduL, particularly Phe116, are poorly conserved across PduL homologs associated with functionally diverse BMCs (Figs 4c and 3), suggesting that they may have alternative oligomeric states.", + "section": "RESULTS", + "ner": [ + [ + 66, + 71, + "rPduL", + "protein" + ], + [ + 86, + 92, + "Phe116", + "residue_name_number" + ], + [ + 98, + 114, + "poorly conserved", + "protein_state" + ], + [ + 122, + 126, + "PduL", + "protein_type" + ], + [ + 173, + 177, + "BMCs", + "complex_assembly" + ] + ] + }, + { + "sid": 122, + "sent": "We tested this hypothesis by performing size exclusion chromatography on both full-length and truncated variants (lacking the EP, \u0394EP) of sPduL, rPduL, and pPduL. These three homologs are found in functionally distinct BMCs (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 40, + 69, + "size exclusion chromatography", + "experimental_method" + ], + [ + 78, + 89, + "full-length", + "protein_state" + ], + [ + 114, + 121, + "lacking", + "protein_state" + ], + [ + 126, + 128, + "EP", + "structure_element" + ], + [ + 130, + 133, + "\u0394EP", + "mutant" + ], + [ + 138, + 143, + "sPduL", + "protein" + ], + [ + 145, + 150, + "rPduL", + "protein" + ], + [ + 156, + 161, + "pPduL", + "protein" + ], + [ + 219, + 223, + "BMCs", + "complex_assembly" + ] + ] + }, + { + "sid": 123, + "sent": "It has been proposed that the catabolic BMCs may assemble in a core-first manner, with the luminal enzymes (signature enzyme, aldehyde, and alcohol dehydrogenases and the BMC PTAC) forming an initial bolus, or prometabolosome, around which a shell assembles.", + "section": "RESULTS", + "ner": [ + [ + 30, + 39, + "catabolic", + "protein_state" + ], + [ + 40, + 44, + "BMCs", + "complex_assembly" + ], + [ + 126, + 162, + "aldehyde, and alcohol dehydrogenases", + "protein_type" + ], + [ + 171, + 174, + "BMC", + "complex_assembly" + ], + [ + 175, + 179, + "PTAC", + "protein_type" + ], + [ + 242, + 247, + "shell", + "structure_element" + ] + ] + }, + { + "sid": 124, + "sent": "Given the diversity of signature enzymes (Table 1), it is plausible that PduL orthologs may adopt different oligomeric states that reflect the differences in the proteins being packaged with them in the organelle lumen.", + "section": "RESULTS", + "ner": [ + [ + 73, + 77, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 125, + "sent": "We found that not only did the different orthologs appear to assemble into different oligomeric states, but that quaternary structure was dependent on whether or not the EP was present.", + "section": "RESULTS", + "ner": [ + [ + 170, + 172, + "EP", + "structure_element" + ] + ] + }, + { + "sid": 126, + "sent": "Full-length sPduL was unstable in solution\u2014precipitating over time\u2014and eluted throughout the entire volume of a size exclusion column, indicating it was nonspecifically aggregating.", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Full-length", + "protein_state" + ], + [ + 12, + 17, + "sPduL", + "protein" + ] + ] + }, + { + "sid": 127, + "sent": "However, when the putative EP (residues 1\u201327) was removed (sPduL \u0394EP), the truncated protein was stable and eluted as a single peak (Fig 5a) consistent with the size of a monomer (Fig 5d, blue curve).", + "section": "RESULTS", + "ner": [ + [ + 27, + 29, + "EP", + "structure_element" + ], + [ + 40, + 44, + "1\u201327", + "residue_range" + ], + [ + 50, + 57, + "removed", + "experimental_method" + ], + [ + 59, + 68, + "sPduL \u0394EP", + "mutant" + ], + [ + 75, + 84, + "truncated", + "protein_state" + ], + [ + 171, + 178, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 128, + "sent": "In contrast, both full-length rPduL and pPduL appeared to exist in two distinct oligomeric states (Fig 5b and 5c respectively, orange curves), one form of the approximate size of a dimer and the second, a higher molecular weight oligomer (~150 kDa).", + "section": "RESULTS", + "ner": [ + [ + 18, + 29, + "full-length", + "protein_state" + ], + [ + 30, + 35, + "rPduL", + "protein" + ], + [ + 40, + 45, + "pPduL", + "protein" + ], + [ + 181, + 186, + "dimer", + "oligomeric_state" + ], + [ + 229, + 237, + "oligomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 129, + "sent": "Upon deletion of the putative EP (residues 1\u201347 for rPduL, and 1\u201320 for pPduL), there was a distinct change in the elution profiles (Fig 5b and 5c respectively, blue curves).", + "section": "RESULTS", + "ner": [ + [ + 5, + 13, + "deletion", + "experimental_method" + ], + [ + 30, + 32, + "EP", + "structure_element" + ], + [ + 43, + 47, + "1\u201347", + "residue_range" + ], + [ + 52, + 57, + "rPduL", + "protein" + ], + [ + 63, + 67, + "1\u201320", + "residue_range" + ], + [ + 72, + 77, + "pPduL", + "protein" + ] + ] + }, + { + "sid": 130, + "sent": "pPduL\u0394EP eluted as two smaller forms, possibly corresponding to a trimer and a monomer.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "pPduL\u0394EP", + "mutant" + ], + [ + 66, + 72, + "trimer", + "oligomeric_state" + ], + [ + 79, + 86, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 131, + "sent": "In contrast, rPduL\u0394EP eluted as one smaller oligomer, possibly a dimer.", + "section": "RESULTS", + "ner": [ + [ + 13, + 21, + "rPduL\u0394EP", + "mutant" + ], + [ + 65, + 70, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 132, + "sent": "We also analyzed purified rPduL and rPduL\u0394EP by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) for a complementary approach to assessing oligomeric state.", + "section": "RESULTS", + "ner": [ + [ + 26, + 31, + "rPduL", + "protein" + ], + [ + 36, + 44, + "rPduL\u0394EP", + "mutant" + ], + [ + 48, + 77, + "size exclusion chromatography", + "experimental_method" + ], + [ + 91, + 118, + "multiangle light scattering", + "experimental_method" + ], + [ + 120, + 128, + "SEC-MALS", + "experimental_method" + ] + ] + }, + { + "sid": 133, + "sent": "SEC-MALS analysis of rPdul\u0394EP is consistent with a dimer (as observed in the crystal structure) with a weighted average (Mw) and number average (Mn) of the molar mass of 58.4 kDa +/\u2212 11.2% and 58.8 kDa +/\u2212 10.9%, respectively (S4a Fig).", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "SEC-MALS", + "experimental_method" + ], + [ + 21, + 29, + "rPdul\u0394EP", + "mutant" + ], + [ + 51, + 56, + "dimer", + "oligomeric_state" + ], + [ + 77, + 94, + "crystal structure", + "evidence" + ], + [ + 103, + 166, + "weighted average (Mw) and number average (Mn) of the molar mass", + "evidence" + ] + ] + }, + { + "sid": 134, + "sent": "rPduL full length runs as Mw = 140.3 kDa +/\u2212 1.2% and Mn = 140.5 kDa +/\u2212 1.2%.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "rPduL", + "protein" + ], + [ + 6, + 17, + "full length", + "protein_state" + ], + [ + 26, + 28, + "Mw", + "evidence" + ], + [ + 54, + 56, + "Mn", + "evidence" + ] + ] + }, + { + "sid": 135, + "sent": "This corresponds to an oligomeric state of six subunits (calculated molecular weight of 144 kDa).", + "section": "RESULTS", + "ner": [ + [ + 43, + 55, + "six subunits", + "oligomeric_state" + ], + [ + 68, + 84, + "molecular weight", + "evidence" + ] + ] + }, + { + "sid": 136, + "sent": "Collectively, these data strongly suggest that the N-terminal EP of PduL plays a role in defining the quaternary structure of the protein.", + "section": "RESULTS", + "ner": [ + [ + 62, + 64, + "EP", + "structure_element" + ], + [ + 68, + 72, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 137, + "sent": "The BMC shell not only sequesters specific enzymes but also their cofactors, thereby establishing a private cofactor pool dedicated to the encapsulated reactions.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 7, + "BMC", + "complex_assembly" + ], + [ + 8, + 13, + "shell", + "structure_element" + ] + ] + }, + { + "sid": 138, + "sent": "In catabolic BMCs, CoA and NAD+ must be continually recycled within the organelle (Fig 1).", + "section": "DISCUSS", + "ner": [ + [ + 3, + 12, + "catabolic", + "protein_state" + ], + [ + 13, + 17, + "BMCs", + "complex_assembly" + ], + [ + 19, + 22, + "CoA", + "chemical" + ], + [ + 27, + 31, + "NAD+", + "chemical" + ] + ] + }, + { + "sid": 139, + "sent": "Homologs of the predominant cofactor utilizer (aldehyde dehydrogenase) and NAD+ regenerator (alcohol dehydrogenase) have been structurally characterized, but until now structural information was lacking for PduL, which recycles CoA in the organelle lumen.", + "section": "DISCUSS", + "ner": [ + [ + 47, + 69, + "aldehyde dehydrogenase", + "protein_type" + ], + [ + 75, + 79, + "NAD+", + "chemical" + ], + [ + 93, + 114, + "alcohol dehydrogenase", + "protein_type" + ], + [ + 207, + 211, + "PduL", + "protein_type" + ], + [ + 228, + 231, + "CoA", + "chemical" + ] + ] + }, + { + "sid": 140, + "sent": "Curiously, while the housekeeping Pta could provide this function, and indeed does so in the case of one type of ethanolamine-utilizing (EUT) BMC, the evolutionarily unrelated PduL fulfills this function for the majority of metabolosomes using a novel structure and active site for convergent evolution of function.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 33, + "housekeeping", + "protein_state" + ], + [ + 34, + 37, + "Pta", + "protein_type" + ], + [ + 113, + 145, + "ethanolamine-utilizing (EUT) BMC", + "complex_assembly" + ], + [ + 176, + 180, + "PduL", + "protein_type" + ], + [ + 224, + 237, + "metabolosomes", + "complex_assembly" + ], + [ + 252, + 261, + "structure", + "evidence" + ], + [ + 266, + 277, + "active site", + "site" + ] + ] + }, + { + "sid": 141, + "sent": "The Tertiary Structure of PduL Is Formed by Discontinuous Segments of Primary Structure", + "section": "DISCUSS", + "ner": [ + [ + 26, + 30, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 142, + "sent": "The structure of PduL consists of two \u03b2-barrel domains capped by short alpha helical segments (Fig 2b).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 17, + 21, + "PduL", + "protein_type" + ], + [ + 34, + 54, + "two \u03b2-barrel domains", + "structure_element" + ], + [ + 65, + 93, + "short alpha helical segments", + "structure_element" + ] + ] + }, + { + "sid": 143, + "sent": "The two domains are structurally very similar (superimposing with a rmsd of 1.34 \u00c5 (over 123 out of 320/348 aligned backbone atoms, S5a Fig).", + "section": "DISCUSS", + "ner": [ + [ + 47, + 60, + "superimposing", + "experimental_method" + ], + [ + 68, + 72, + "rmsd", + "evidence" + ] + ] + }, + { + "sid": 144, + "sent": "However, the amino acid sequences of the two domains are only 16% identical (mainly the RHxH motif, \u03b22 and \u03b210), and 34% similar.", + "section": "DISCUSS", + "ner": [ + [ + 88, + 98, + "RHxH motif", + "structure_element" + ], + [ + 100, + 102, + "\u03b22", + "structure_element" + ], + [ + 107, + 110, + "\u03b210", + "structure_element" + ] + ] + }, + { + "sid": 145, + "sent": "Our structure reveals that the two assigned PF06130 domains (Fig 3) do not form structurally discrete units; this reduces the apparent sequence conservation at the level of primary structure.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 44, + 51, + "PF06130", + "structure_element" + ] + ] + }, + { + "sid": 146, + "sent": "One strand of the domain 1 beta barrel (shown in blue in Fig 2) is contributed by the N-terminus, while the rest of the domain is formed by the residues from the C-terminal half of the protein.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 10, + "strand", + "structure_element" + ], + [ + 18, + 26, + "domain 1", + "structure_element" + ], + [ + 27, + 38, + "beta barrel", + "structure_element" + ], + [ + 162, + 177, + "C-terminal half", + "structure_element" + ], + [ + 185, + 192, + "protein", + "protein_type" + ] + ] + }, + { + "sid": 147, + "sent": "When aligned by structure, the \u03b21 strand of the first domain (Fig 2a and 2b, blue) corresponds to the final strand of the second domain (\u03b29), effectively making the domains continuous if the first strand was transplanted to the C-terminus.", + "section": "DISCUSS", + "ner": [ + [ + 5, + 12, + "aligned", + "experimental_method" + ], + [ + 16, + 25, + "structure", + "evidence" + ], + [ + 31, + 40, + "\u03b21 strand", + "structure_element" + ], + [ + 48, + 60, + "first domain", + "structure_element" + ], + [ + 102, + 114, + "final strand", + "structure_element" + ], + [ + 122, + 135, + "second domain", + "structure_element" + ], + [ + 137, + 139, + "\u03b29", + "structure_element" + ] + ] + }, + { + "sid": 148, + "sent": "Refined domain assignment based on our structure should be able to predict domains of PF06130 homologs much more accurately.", + "section": "DISCUSS", + "ner": [ + [ + 39, + 48, + "structure", + "evidence" + ], + [ + 86, + 93, + "PF06130", + "structure_element" + ] + ] + }, + { + "sid": 149, + "sent": "The closest structural homolog of the PduL barrel domain is a subdomain of a multienzyme complex, the alpha subunit of ethylbenzene dehydrogenase (S5b Fig, rmsd of 2.26 \u00c5 over 226 aligned atoms consisting of one beta barrel and one capping helix).", + "section": "DISCUSS", + "ner": [ + [ + 38, + 42, + "PduL", + "protein_type" + ], + [ + 43, + 56, + "barrel domain", + "structure_element" + ], + [ + 102, + 115, + "alpha subunit", + "structure_element" + ], + [ + 119, + 145, + "ethylbenzene dehydrogenase", + "protein_type" + ], + [ + 156, + 160, + "rmsd", + "evidence" + ], + [ + 212, + 223, + "beta barrel", + "structure_element" + ], + [ + 232, + 245, + "capping helix", + "structure_element" + ] + ] + }, + { + "sid": 150, + "sent": "In contrast to PduL, there is only one barrel present in ethylbenzene dehydrogenase, and there is no comparable active site arrangement.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 19, + "PduL", + "protein_type" + ], + [ + 39, + 45, + "barrel", + "structure_element" + ], + [ + 57, + 83, + "ethylbenzene dehydrogenase", + "protein_type" + ], + [ + 112, + 123, + "active site", + "site" + ] + ] + }, + { + "sid": 151, + "sent": "The PduL signature primary structure, two PF06130 domains, occurs in some multidomain proteins, most of them annotated as Acks, suggesting that PduL may also replace Pta in variants of the phosphotransacetylase-Ack pathway.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 8, + "PduL", + "protein_type" + ], + [ + 42, + 49, + "PF06130", + "structure_element" + ], + [ + 122, + 126, + "Acks", + "protein_type" + ], + [ + 144, + 148, + "PduL", + "protein_type" + ], + [ + 166, + 169, + "Pta", + "protein_type" + ], + [ + 189, + 210, + "phosphotransacetylase", + "protein_type" + ], + [ + 211, + 214, + "Ack", + "protein_type" + ] + ] + }, + { + "sid": 152, + "sent": "These PduL homologs lack EPs, and their fusion to Ack may have evolved as a way to facilitate substrate channeling between the two enzymes.", + "section": "DISCUSS", + "ner": [ + [ + 6, + 10, + "PduL", + "protein_type" + ], + [ + 20, + 24, + "lack", + "protein_state" + ], + [ + 25, + 28, + "EPs", + "structure_element" + ], + [ + 34, + 39, + "their", + "protein_type" + ], + [ + 50, + 53, + "Ack", + "protein_type" + ] + ] + }, + { + "sid": 153, + "sent": "Implications for Metabolosome Core Assembly", + "section": "DISCUSS", + "ner": [ + [ + 17, + 29, + "Metabolosome", + "complex_assembly" + ] + ] + }, + { + "sid": 154, + "sent": "For BMC-encapsulated proteins to properly function together, they must be targeted to the lumen and assemble into an organization that facilitates substrate/product channeling among the different catalytic sites of the signature and core enzymes.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 7, + "BMC", + "complex_assembly" + ], + [ + 196, + 211, + "catalytic sites", + "site" + ] + ] + }, + { + "sid": 155, + "sent": "The N-terminal extension on PduL homologs may serve both of these functions.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 24, + "N-terminal extension", + "structure_element" + ], + [ + 28, + 32, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 156, + "sent": "The extension shares many features with previously characterized EPs: it is present only in homologs associated with BMC loci, and it is predicted to form an amphipathic \u03b1-helix.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 13, + "The extension", + "structure_element" + ], + [ + 65, + 68, + "EPs", + "structure_element" + ], + [ + 117, + 125, + "BMC loci", + "gene" + ], + [ + 158, + 169, + "amphipathic", + "protein_state" + ], + [ + 170, + 177, + "\u03b1-helix", + "structure_element" + ] + ] + }, + { + "sid": 157, + "sent": "Moreover, its removal affects the oligomeric state of the protein.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 21, + "removal", + "experimental_method" + ] + ] + }, + { + "sid": 158, + "sent": "EP-mediated oligomerization has been observed for the signature and core BMC enzymes; for example, full-length propanediol dehydratase and ethanolamine ammonia-lyase (signature enzymes for PDU and EUT BMCs) subunits are also insoluble, but become soluble upon removal of the predicted EP.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 2, + "EP", + "structure_element" + ], + [ + 73, + 76, + "BMC", + "complex_assembly" + ], + [ + 99, + 110, + "full-length", + "protein_state" + ], + [ + 111, + 134, + "propanediol dehydratase", + "protein_type" + ], + [ + 139, + 165, + "ethanolamine ammonia-lyase", + "protein_type" + ], + [ + 189, + 192, + "PDU", + "complex_assembly" + ], + [ + 197, + 205, + "EUT BMCs", + "complex_assembly" + ], + [ + 285, + 287, + "EP", + "structure_element" + ] + ] + }, + { + "sid": 159, + "sent": "sPduL has also previously been reported to localize to inclusion bodies when overexpressed; we show here that this is dependent on the presence of the EP.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "sPduL", + "protein" + ], + [ + 77, + 90, + "overexpressed", + "experimental_method" + ], + [ + 151, + 153, + "EP", + "structure_element" + ] + ] + }, + { + "sid": 160, + "sent": "This propensity of the EP to cause proteins to form complexes (Fig 5) might not be a coincidence, but could be a necessary step in the assembly of BMCs.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 25, + "EP", + "structure_element" + ], + [ + 147, + 151, + "BMCs", + "complex_assembly" + ] + ] + }, + { + "sid": 161, + "sent": "Structured aggregation of the core enzymes has been proposed to be the initial step in metabolosome assembly and is known to be the first step of \u03b2-carboxysome biogenesis, where the core enzyme Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) is aggregated by the CcmM protein.", + "section": "DISCUSS", + "ner": [ + [ + 87, + 99, + "metabolosome", + "complex_assembly" + ], + [ + 194, + 237, + "Ribulose Bisphosphate Carboxylase/Oxygenase", + "protein_type" + ], + [ + 239, + 246, + "RuBisCO", + "protein_type" + ], + [ + 269, + 273, + "CcmM", + "protein_type" + ] + ] + }, + { + "sid": 162, + "sent": "Likewise, CsoS2, a protein in the \u03b1-carboxysome core, also aggregates when purified and is proposed to facilitate the nucleation and encapsulation of RuBisCO molecules in the lumen of the organelle.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 15, + "CsoS2", + "protein_type" + ], + [ + 34, + 47, + "\u03b1-carboxysome", + "complex_assembly" + ], + [ + 150, + 157, + "RuBisCO", + "protein_type" + ] + ] + }, + { + "sid": 163, + "sent": "This role for EPs in BMC assembly is in addition to their interaction with shell proteins.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 17, + "EPs", + "structure_element" + ], + [ + 21, + 24, + "BMC", + "complex_assembly" + ] + ] + }, + { + "sid": 164, + "sent": "Moreover, the PduL crystal structures offer a clue as to how required cofactors enter the BMC lumen during assembly.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 18, + "PduL", + "protein_type" + ], + [ + 19, + 37, + "crystal structures", + "evidence" + ], + [ + 90, + 93, + "BMC", + "complex_assembly" + ] + ] + }, + { + "sid": 165, + "sent": "Free CoA and NAD+/H could potentially be bound to the enzymes as the core assembles and is encapsulated.", + "section": "DISCUSS", + "ner": [ + [ + 5, + 8, + "CoA", + "chemical" + ], + [ + 13, + 17, + "NAD+", + "chemical" + ], + [ + 18, + 19, + "H", + "chemical" + ] + ] + }, + { + "sid": 166, + "sent": "Our PduL crystals contained CoA that was captured from the Escherichia coli cytosol, indicating that the \u201cground state\u201d of PduL is in the CoA-bound form; this could provide an elegantly simple means of guaranteeing a 1:1 ratio of CoA:PduL within the metabolosome lumen.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 8, + "PduL", + "protein_type" + ], + [ + 9, + 17, + "crystals", + "evidence" + ], + [ + 28, + 31, + "CoA", + "chemical" + ], + [ + 59, + 75, + "Escherichia coli", + "species" + ], + [ + 123, + 127, + "PduL", + "protein_type" + ], + [ + 138, + 147, + "CoA-bound", + "protein_state" + ], + [ + 230, + 238, + "CoA:PduL", + "complex_assembly" + ], + [ + 250, + 262, + "metabolosome", + "complex_assembly" + ] + ] + }, + { + "sid": 167, + "sent": "Active Site Identification and Structural Insights into Catalysis", + "section": "DISCUSS", + "ner": [ + [ + 0, + 11, + "Active Site", + "site" + ] + ] + }, + { + "sid": 168, + "sent": "The active site of PduL is formed at the interface of the two structural domains (Fig 2b).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 15, + "active site", + "site" + ], + [ + 19, + 23, + "PduL", + "protein_type" + ], + [ + 41, + 50, + "interface", + "site" + ], + [ + 73, + 80, + "domains", + "structure_element" + ] + ] + }, + { + "sid": 169, + "sent": "As expected, the amino acid sequence conservation is highest in the region around the proposed active site (Fig 4d); highly conserved residues are also involved in CoA binding (Figs 2a and 3, residues Ser45, Lys70, Arg97, Leu99, His204, Asn211).", + "section": "DISCUSS", + "ner": [ + [ + 95, + 106, + "active site", + "site" + ], + [ + 117, + 133, + "highly conserved", + "protein_state" + ], + [ + 164, + 167, + "CoA", + "chemical" + ], + [ + 201, + 206, + "Ser45", + "residue_name_number" + ], + [ + 208, + 213, + "Lys70", + "residue_name_number" + ], + [ + 215, + 220, + "Arg97", + "residue_name_number" + ], + [ + 222, + 227, + "Leu99", + "residue_name_number" + ], + [ + 229, + 235, + "His204", + "residue_name_number" + ], + [ + 237, + 243, + "Asn211", + "residue_name_number" + ] + ] + }, + { + "sid": 170, + "sent": "All of the metal-coordinating residues (Fig 2a) are absolutely conserved, implicating them in catalysis or the correct spatial orientation of the substrates.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 38, + "metal-coordinating residues", + "site" + ], + [ + 52, + 72, + "absolutely conserved", + "protein_state" + ] + ] + }, + { + "sid": 171, + "sent": "Arg103, which contacts the phosphate (Fig 4b), is present in all PduL homologs.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 6, + "Arg103", + "residue_name_number" + ], + [ + 27, + 36, + "phosphate", + "chemical" + ], + [ + 65, + 69, + "PduL", + "protein_type" + ] + ] + }, + { + "sid": 172, + "sent": "The close resemblance between the structures binding CoA and phosphate likely indicates that no large changes in protein conformation are involved in catalysis, and that our crystal structures are representative of the active form.", + "section": "DISCUSS", + "ner": [ + [ + 53, + 56, + "CoA", + "chemical" + ], + [ + 61, + 70, + "phosphate", + "chemical" + ], + [ + 174, + 192, + "crystal structures", + "evidence" + ], + [ + 219, + 225, + "active", + "protein_state" + ] + ] + }, + { + "sid": 173, + "sent": "The native substrate for the forward reaction of rPduL and pPduL, propionyl-CoA, most likely binds to the enzyme in the same way at the observed nucleotide and pantothenic acid moiety, but the propionyl group in the CoA-thioester might point in a different direction.", + "section": "DISCUSS", + "ner": [ + [ + 49, + 54, + "rPduL", + "protein" + ], + [ + 59, + 64, + "pPduL", + "protein" + ], + [ + 66, + 79, + "propionyl-CoA", + "chemical" + ], + [ + 145, + 155, + "nucleotide", + "chemical" + ], + [ + 160, + 176, + "pantothenic acid", + "chemical" + ], + [ + 216, + 229, + "CoA-thioester", + "chemical" + ] + ] + }, + { + "sid": 174, + "sent": "There is a pocket nearby the active site between the well-conserved residues Ser45 and Ala154, which could accommodate the propionyl group (S6 Fig).", + "section": "DISCUSS", + "ner": [ + [ + 11, + 17, + "pocket", + "site" + ], + [ + 29, + 40, + "active site", + "site" + ], + [ + 53, + 67, + "well-conserved", + "protein_state" + ], + [ + 77, + 82, + "Ser45", + "residue_name_number" + ], + [ + 87, + 93, + "Ala154", + "residue_name_number" + ] + ] + }, + { + "sid": 175, + "sent": "A homology model of sPduL indicates that the residues making up this pocket and the surrounding active site region are identical to that of rPduL, which is not surprising, because these two homologs presumably have the same propionyl-CoA substrate.", + "section": "DISCUSS", + "ner": [ + [ + 2, + 16, + "homology model", + "experimental_method" + ], + [ + 20, + 25, + "sPduL", + "protein" + ], + [ + 69, + 75, + "pocket", + "site" + ], + [ + 96, + 107, + "active site", + "site" + ], + [ + 140, + 145, + "rPduL", + "protein" + ], + [ + 224, + 237, + "propionyl-CoA", + "chemical" + ] + ] + }, + { + "sid": 176, + "sent": "The homology model of pPduL also has identical residues making up the pocket, but with a key difference in the vicinity of the active site: Gln77 of rPduL is replaced by a tyrosine (Tyr77) in pPduL. The physiological substrate of pPduL (Table 1) is thought to be lactyl-CoA, which contains an additional hydroxyl group relative to propionyl-CoA. The presence of an aromatic residue at this position may underlie the substrate preference of the PduL enzyme from the pvm locus.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 18, + "homology model", + "experimental_method" + ], + [ + 22, + 27, + "pPduL", + "protein" + ], + [ + 70, + 76, + "pocket", + "site" + ], + [ + 127, + 138, + "active site", + "site" + ], + [ + 140, + 145, + "Gln77", + "residue_name_number" + ], + [ + 149, + 154, + "rPduL", + "protein" + ], + [ + 172, + 180, + "tyrosine", + "residue_name" + ], + [ + 182, + 187, + "Tyr77", + "residue_name_number" + ], + [ + 192, + 197, + "pPduL", + "protein" + ], + [ + 230, + 235, + "pPduL", + "protein" + ], + [ + 263, + 273, + "lactyl-CoA", + "chemical" + ], + [ + 331, + 344, + "propionyl-CoA", + "chemical" + ], + [ + 365, + 373, + "aromatic", + "protein_state" + ], + [ + 374, + 381, + "residue", + "structure_element" + ], + [ + 444, + 448, + "PduL", + "protein_type" + ], + [ + 465, + 474, + "pvm locus", + "gene" + ] + ] + }, + { + "sid": 177, + "sent": "Indeed, in the majority of PduLs encoded in pvm loci, Gln77 is substituted by either a Tyr or Phe, whereas it is typically a Gln or Glu in PduLs in all other BMC types that degrade acetyl- or propionyl-CoA. A comparison of the PduL active site to that of the functionally identical Pta suggests that the two enzymes have distinctly different mechanisms.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 32, + "PduLs", + "protein_type" + ], + [ + 44, + 52, + "pvm loci", + "gene" + ], + [ + 54, + 59, + "Gln77", + "residue_name_number" + ], + [ + 87, + 90, + "Tyr", + "residue_name" + ], + [ + 94, + 97, + "Phe", + "residue_name" + ], + [ + 125, + 128, + "Gln", + "residue_name" + ], + [ + 132, + 135, + "Glu", + "residue_name" + ], + [ + 139, + 144, + "PduLs", + "protein_type" + ], + [ + 158, + 161, + "BMC", + "complex_assembly" + ], + [ + 181, + 188, + "acetyl-", + "chemical" + ], + [ + 192, + 205, + "propionyl-CoA", + "chemical" + ], + [ + 209, + 219, + "comparison", + "experimental_method" + ], + [ + 227, + 231, + "PduL", + "protein_type" + ], + [ + 232, + 243, + "active site", + "site" + ], + [ + 282, + 285, + "Pta", + "protein_type" + ] + ] + }, + { + "sid": 178, + "sent": "The catalytic mechanism of Pta involves the abstraction of a thiol hydrogen by an aspartate residue, resulting in the nucleophilic attack of thiolate upon the carbonyl carbon of acetyl-phosphate, oriented by an arginine and stabilized by a serine \u2014there are no metals involved.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 30, + "Pta", + "protein_type" + ], + [ + 82, + 91, + "aspartate", + "residue_name" + ], + [ + 178, + 194, + "acetyl-phosphate", + "chemical" + ], + [ + 211, + 219, + "arginine", + "residue_name" + ], + [ + 240, + 246, + "serine", + "residue_name" + ] + ] + }, + { + "sid": 179, + "sent": "In contrast, in the rPduL structure, there are no conserved aspartate residues in or around the active site, and the only well-conserved glutamate residue in the active site is involved in coordinating one of the metal ions.", + "section": "DISCUSS", + "ner": [ + [ + 20, + 25, + "rPduL", + "protein" + ], + [ + 26, + 35, + "structure", + "evidence" + ], + [ + 60, + 69, + "aspartate", + "residue_name" + ], + [ + 96, + 107, + "active site", + "site" + ], + [ + 122, + 136, + "well-conserved", + "protein_state" + ], + [ + 137, + 146, + "glutamate", + "residue_name" + ], + [ + 162, + 173, + "active site", + "site" + ], + [ + 189, + 201, + "coordinating", + "bond_interaction" + ] + ] + }, + { + "sid": 180, + "sent": "These observations strongly suggest that an acidic residue is not directly involved in catalysis by PduL. Instead, the dimetal active site of PduL may create a nucleophile from one of the hydroxyl groups on free phosphate to attack the carbonyl carbon of the thioester bond of an acyl-CoA. In the reverse direction, the metal ion(s) could stabilize the thiolate anion that would attack the carbonyl carbon of an acyl-phosphate; a similar mechanism has been described for phosphatases where hydroxyl groups or hydroxide ions can act as a base when coordinated by a dimetal active site.", + "section": "DISCUSS", + "ner": [ + [ + 44, + 50, + "acidic", + "protein_state" + ], + [ + 51, + 58, + "residue", + "structure_element" + ], + [ + 100, + 104, + "PduL", + "protein_type" + ], + [ + 119, + 138, + "dimetal active site", + "site" + ], + [ + 142, + 146, + "PduL", + "protein_type" + ], + [ + 212, + 221, + "phosphate", + "chemical" + ], + [ + 280, + 288, + "acyl-CoA", + "chemical" + ], + [ + 412, + 426, + "acyl-phosphate", + "chemical" + ], + [ + 471, + 483, + "phosphatases", + "protein_type" + ], + [ + 564, + 583, + "dimetal active site", + "site" + ] + ] + }, + { + "sid": 181, + "sent": "Our structures provide the foundation for studies to elucidate the details of the catalytic mechanism of PduL. Conserved residues in the active site that may contribute to substrate binding and/or transition state stabilization include Ser127, Arg103, Arg194, Gln107, Gln74, and Gln/Glu77.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 105, + 109, + "PduL", + "protein_type" + ], + [ + 111, + 120, + "Conserved", + "protein_state" + ], + [ + 137, + 148, + "active site", + "site" + ], + [ + 236, + 242, + "Ser127", + "residue_name_number" + ], + [ + 244, + 250, + "Arg103", + "residue_name_number" + ], + [ + 252, + 258, + "Arg194", + "residue_name_number" + ], + [ + 260, + 266, + "Gln107", + "residue_name_number" + ], + [ + 268, + 273, + "Gln74", + "residue_name_number" + ], + [ + 279, + 282, + "Gln", + "residue_name_number" + ], + [ + 283, + 288, + "Glu77", + "residue_name_number" + ] + ] + }, + { + "sid": 182, + "sent": "In the phosphate-bound crystal structure, Ser127 and Arg103 appear to position the phosphate (Fig 4b).", + "section": "DISCUSS", + "ner": [ + [ + 7, + 22, + "phosphate-bound", + "protein_state" + ], + [ + 23, + 40, + "crystal structure", + "evidence" + ], + [ + 42, + 48, + "Ser127", + "residue_name_number" + ], + [ + 53, + 59, + "Arg103", + "residue_name_number" + ], + [ + 83, + 92, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 183, + "sent": "Alternatively, Arg103 might act as a base to render the phosphate more nucleophilic.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 21, + "Arg103", + "residue_name_number" + ], + [ + 56, + 65, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 184, + "sent": "The functional groups of Gln74, Gln/Glu77, and Arg194 are directed away from the active site in both CoA and phosphate-bound crystal structures and do not appear to be involved in hydrogen bonding with these substrates, although they could be important for positioning an acyl-phosphate.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 30, + "Gln74", + "residue_name_number" + ], + [ + 32, + 35, + "Gln", + "residue_name_number" + ], + [ + 36, + 41, + "Glu77", + "residue_name_number" + ], + [ + 47, + 53, + "Arg194", + "residue_name_number" + ], + [ + 81, + 92, + "active site", + "site" + ], + [ + 101, + 104, + "CoA", + "protein_state" + ], + [ + 109, + 124, + "phosphate-bound", + "protein_state" + ], + [ + 125, + 143, + "crystal structures", + "evidence" + ], + [ + 180, + 196, + "hydrogen bonding", + "bond_interaction" + ], + [ + 272, + 286, + "acyl-phosphate", + "chemical" + ] + ] + }, + { + "sid": 185, + "sent": "The free CoA-bound form is presumably poised for attack upon an acyl-phosphate, indicating that the enzyme initially binds CoA as opposed to acyl-phosphate.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 18, + "CoA-bound", + "protein_state" + ], + [ + 64, + 78, + "acyl-phosphate", + "chemical" + ], + [ + 123, + 126, + "CoA", + "chemical" + ], + [ + 141, + 155, + "acyl-phosphate", + "chemical" + ] + ] + }, + { + "sid": 186, + "sent": "This hypothesis is strengthened by the fact that the CoA-bound crystals were obtained without added CoA, indicating that the protein bound CoA from the E. coli expression strain and retained it throughout purification and crystallization.", + "section": "DISCUSS", + "ner": [ + [ + 53, + 62, + "CoA-bound", + "protein_state" + ], + [ + 63, + 71, + "crystals", + "evidence" + ], + [ + 100, + 103, + "CoA", + "chemical" + ], + [ + 133, + 138, + "bound", + "protein_state" + ], + [ + 139, + 142, + "CoA", + "chemical" + ], + [ + 152, + 159, + "E. coli", + "species" + ] + ] + }, + { + "sid": 187, + "sent": "The phosphate-bound structure indicates that in the opposite reaction direction phosphate is bound first, and then an acyl-CoA enters.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 19, + "phosphate-bound", + "protein_state" + ], + [ + 20, + 29, + "structure", + "evidence" + ], + [ + 80, + 89, + "phosphate", + "chemical" + ], + [ + 118, + 126, + "acyl-CoA", + "chemical" + ] + ] + }, + { + "sid": 188, + "sent": "The two high-resolution crystal structures presented here will serve as the foundation for mechanistic studies on this noncanonical PTAC enzyme to determine how the dimetal active site functions to catalyze both forward and reverse reactions.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 42, + "crystal structures", + "evidence" + ], + [ + 132, + 136, + "PTAC", + "protein_type" + ], + [ + 165, + 184, + "dimetal active site", + "site" + ] + ] + }, + { + "sid": 189, + "sent": "Functional, but Not Structural, Convergence of PduL and Pta", + "section": "DISCUSS", + "ner": [ + [ + 47, + 51, + "PduL", + "protein_type" + ], + [ + 56, + 59, + "Pta", + "protein_type" + ] + ] + }, + { + "sid": 190, + "sent": "PduL and Pta are mechanistically and structurally distinct enzymes that catalyze the same reaction, a prime example of evolutionary convergence upon a function.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "PduL", + "protein_type" + ], + [ + 9, + 12, + "Pta", + "protein_type" + ] + ] + }, + { + "sid": 191, + "sent": "There are several examples of such functional convergence of enzymes, although typically the enzymes have independently evolved similar, or even identical active sites; for example, the carbonic anhydrase family.", + "section": "DISCUSS", + "ner": [ + [ + 155, + 167, + "active sites", + "site" + ], + [ + 186, + 204, + "carbonic anhydrase", + "protein_type" + ] + ] + }, + { + "sid": 192, + "sent": "However, apparently less frequent is functional convergence that is supported by distinctly different active sites and accordingly catalytic mechanism, as revealed by comparison of the structures of Pta and PduL. One well-studied example of this is the \u03b2-lactamase family of enzymes, in which the active site of Class A and Class C enzymes involve serine-based catalysis, but Class B enzymes are metalloproteins.", + "section": "DISCUSS", + "ner": [ + [ + 102, + 114, + "active sites", + "site" + ], + [ + 199, + 202, + "Pta", + "protein_type" + ], + [ + 207, + 211, + "PduL", + "protein_type" + ], + [ + 253, + 264, + "\u03b2-lactamase", + "protein_type" + ], + [ + 297, + 308, + "active site", + "site" + ], + [ + 396, + 411, + "metalloproteins", + "protein_type" + ] + ] + }, + { + "sid": 193, + "sent": "This is not surprising, as \u03b2-lactamases are not so widespread among bacteria and therefore would be expected to have evolved independently several times as a defense mechanism against \u03b2-lactam antibiotics.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 39, + "\u03b2-lactamases", + "protein_type" + ], + [ + 68, + 76, + "bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 194, + "sent": "However, nearly all bacteria encode Pta, and it is not immediately clear why the Pta/PduL functional convergence should have evolved: it would seem to be evolutionarily more resourceful for the Pta-encoding gene to be duplicated and repurposed for BMCs, as is apparently the case in one type of BMC\u2014EUT1 (Table 1).", + "section": "DISCUSS", + "ner": [ + [ + 20, + 28, + "bacteria", + "taxonomy_domain" + ], + [ + 36, + 39, + "Pta", + "protein_type" + ], + [ + 81, + 84, + "Pta", + "protein_type" + ], + [ + 85, + 89, + "PduL", + "protein_type" + ], + [ + 194, + 211, + "Pta-encoding gene", + "gene" + ], + [ + 248, + 252, + "BMCs", + "complex_assembly" + ], + [ + 295, + 303, + "BMC\u2014EUT1", + "complex_assembly" + ] + ] + }, + { + "sid": 195, + "sent": "There could be some intrinsic biochemical difference between the two enzymes that renders PduL a more attractive candidate for encapsulation in a BMC\u2014for example, PduL might be more amenable to tight packaging, or is better suited for the chemical microenvironment formed within the lumen of the BMC, which can be quite different from the cytosol.", + "section": "DISCUSS", + "ner": [ + [ + 90, + 94, + "PduL", + "protein_type" + ], + [ + 146, + 149, + "BMC", + "complex_assembly" + ], + [ + 163, + 167, + "PduL", + "protein_type" + ], + [ + 296, + 299, + "BMC", + "complex_assembly" + ] + ] + }, + { + "sid": 196, + "sent": "Further biochemical comparison between the two PTACs will likely yield exciting results that could answer this evolutionary question.", + "section": "DISCUSS", + "ner": [ + [ + 47, + 52, + "PTACs", + "protein_type" + ] + ] + }, + { + "sid": 197, + "sent": "BMCs are now known to be widespread among the bacteria and are involved in critical segments of both autotrophic and heterotrophic biochemical pathways that confer to the host organism a competitive (metabolic) advantage in select niches.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "BMCs", + "complex_assembly" + ], + [ + 46, + 54, + "bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 198, + "sent": "As one of the three common metabolosome core enzymes, the structure of PduL provides a key missing piece to our structural picture of the shared core biochemistry (Fig 1) of functionally diverse catabolic BMCs.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 39, + "metabolosome", + "complex_assembly" + ], + [ + 58, + 67, + "structure", + "evidence" + ], + [ + 71, + 75, + "PduL", + "protein_type" + ], + [ + 195, + 204, + "catabolic", + "protein_state" + ], + [ + 205, + 209, + "BMCs", + "complex_assembly" + ] + ] + }, + { + "sid": 199, + "sent": "We have observed the oligomeric state differences of PduL to correlate with the presence of an EP, providing new insight into the function of this sequence extension in BMC assembly.", + "section": "DISCUSS", + "ner": [ + [ + 53, + 57, + "PduL", + "protein_type" + ], + [ + 95, + 97, + "EP", + "structure_element" + ], + [ + 169, + 172, + "BMC", + "complex_assembly" + ] + ] + }, + { + "sid": 200, + "sent": "Moreover, our results suggest a means for Coenzyme A incorporation during metabolosome biogenesis.", + "section": "DISCUSS", + "ner": [ + [ + 42, + 52, + "Coenzyme A", + "chemical" + ], + [ + 74, + 86, + "metabolosome", + "complex_assembly" + ] + ] + }, + { + "sid": 201, + "sent": "A detailed understanding of the underlying principles governing the assembly and internal structural organization of BMCs is a requisite for synthetic biologists to design custom nanoreactors that use BMC architectures as a template.", + "section": "DISCUSS", + "ner": [ + [ + 117, + 121, + "BMCs", + "complex_assembly" + ], + [ + 201, + 204, + "BMC", + "complex_assembly" + ] + ] + }, + { + "sid": 202, + "sent": "Furthermore, given the growing number of metabolosomes implicated in pathogenesis, the PduL structure will be useful in the development of therapeutics.", + "section": "DISCUSS", + "ner": [ + [ + 41, + 54, + "metabolosomes", + "complex_assembly" + ], + [ + 87, + 91, + "PduL", + "protein_type" + ], + [ + 92, + 101, + "structure", + "evidence" + ] + ] + }, + { + "sid": 203, + "sent": "The fact that PduL is confined almost exclusively to metabolosomes can be used to develop an inhibitor that blocks only PduL and not Pta as a way to selectively disrupt BMC-based metabolism, while not affecting most commensal organisms that require PTAC activity.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 18, + "PduL", + "protein_type" + ], + [ + 53, + 66, + "metabolosomes", + "complex_assembly" + ], + [ + 120, + 124, + "PduL", + "protein_type" + ], + [ + 133, + 136, + "Pta", + "protein_type" + ], + [ + 169, + 172, + "BMC", + "complex_assembly" + ], + [ + 249, + 253, + "PTAC", + "protein_type" + ] + ] + } + ] + }, + "PMC4795551": { + "annotations": [ + { + "sid": 0, + "sent": "Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily", + "section": "TITLE", + "ner": [ + [ + 17, + 34, + "Crystal Structure", + "evidence" + ], + [ + 38, + 54, + "Ectoine Synthase", + "protein_type" + ], + [ + 58, + 74, + "Metal-Containing", + "protein_state" + ], + [ + 89, + 106, + "Cupin Superfamily", + "protein_type" + ] + ] + }, + { + "sid": 1, + "sent": "Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 7, + "Ectoine", + "chemical" + ], + [ + 84, + 92, + "Bacteria", + "taxonomy_domain" + ], + [ + 103, + 110, + "Archaea", + "taxonomy_domain" + ] + ] + }, + { + "sid": 2, + "sent": "Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 16, + "Ectoine synthase", + "protein_type" + ], + [ + 18, + 22, + "EctC", + "protein_type" + ], + [ + 51, + 58, + "ectoine", + "chemical" + ], + [ + 117, + 157, + "N-gamma-acetyl-L-2,4-diaminobutyric acid", + "chemical" + ], + [ + 168, + 173, + "water", + "chemical" + ] + ] + }, + { + "sid": 3, + "sent": "However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking.", + "section": "ABSTRACT", + "ner": [ + [ + 13, + 30, + "crystal structure", + "evidence" + ], + [ + 34, + 50, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 4, + "sent": "Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.", + "section": "ABSTRACT", + "ner": [ + [ + 10, + 26, + "ectoine synthase", + "protein_type" + ], + [ + 49, + 65, + "marine bacterium", + "taxonomy_domain" + ], + [ + 66, + 89, + "Sphingopyxis alaskensis", + "species" + ], + [ + 91, + 93, + "Sa", + "species" + ], + [ + 163, + 167, + "EctC", + "protein" + ], + [ + 199, + 216, + "crystal structure", + "evidence" + ], + [ + 224, + 227, + "apo", + "protein_state" + ] + ] + }, + { + "sid": 5, + "sent": "Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 19, + "Structural analysis", + "experimental_method" + ], + [ + 36, + 38, + "Sa", + "species" + ], + [ + 39, + 43, + "EctC", + "protein" + ], + [ + 71, + 88, + "cupin superfamily", + "protein_type" + ] + ] + }, + { + "sid": 6, + "sent": "EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 4, + "EctC", + "protein" + ], + [ + 13, + 18, + "dimer", + "oligomeric_state" + ], + [ + 26, + 38, + "head-to-tail", + "protein_state" + ], + [ + 80, + 97, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 7, + "sent": "The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 13, + "interface", + "site" + ], + [ + 21, + 26, + "dimer", + "oligomeric_state" + ], + [ + 81, + 105, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 122, + 133, + "beta-sheets", + "structure_element" + ], + [ + 146, + 153, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 8, + "sent": "We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme.", + "section": "ABSTRACT", + "ner": [ + [ + 32, + 48, + "ectoine synthase", + "protein_type" + ], + [ + 83, + 88, + "metal", + "chemical" + ], + [ + 100, + 146, + "metal depletion and reconstitution experiments", + "experimental_method" + ], + [ + 160, + 164, + "EctC", + "protein" + ], + [ + 180, + 194, + "iron-dependent", + "protein_state" + ] + ] + }, + { + "sid": 9, + "sent": "We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency.", + "section": "ABSTRACT", + "ner": [ + [ + 14, + 18, + "EctC", + "protein" + ], + [ + 71, + 111, + "N-gamma-acetyl-L-2,4-diaminobutyric acid", + "chemical" + ], + [ + 117, + 124, + "ectoine", + "chemical" + ], + [ + 199, + 239, + "N-alpha-acetyl-L-2,4-diaminobutyric acid", + "chemical" + ], + [ + 292, + 312, + "catalytic efficiency", + "evidence" + ] + ] + }, + { + "sid": 10, + "sent": "Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 42, + "Structure-guided site-directed mutagenesis", + "experimental_method" + ], + [ + 94, + 125, + "evolutionarily highly conserved", + "protein_state" + ], + [ + 145, + 164, + "EctC protein family", + "protein_type" + ], + [ + 206, + 223, + "iron-binding site", + "site" + ], + [ + 296, + 300, + "EctC", + "protein" + ] + ] + }, + { + "sid": 11, + "sent": "An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.", + "section": "ABSTRACT", + "ner": [ + [ + 37, + 41, + "iron", + "chemical" + ], + [ + 130, + 141, + "active site", + "site" + ], + [ + 176, + 180, + "EctC", + "protein" + ], + [ + 181, + 193, + "cupin barrel", + "structure_element" + ] + ] + }, + { + "sid": 12, + "sent": "Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes.", + "section": "INTRO", + "ner": [ + [ + 0, + 7, + "Ectoine", + "chemical" + ], + [ + 9, + 68, + "(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid", + "chemical" + ], + [ + 89, + 105, + "5-hydroxyectoine", + "chemical" + ], + [ + 107, + 180, + "(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid", + "chemical" + ] + ] + }, + { + "sid": 13, + "sent": "Both marine and terrestrial microorganisms produce them widely in response to osmotic or temperature stress.", + "section": "INTRO", + "ner": [ + [ + 5, + 42, + "marine and terrestrial microorganisms", + "taxonomy_domain" + ] + ] + }, + { + "sid": 14, + "sent": "Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-\u00df-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).", + "section": "INTRO", + "ner": [ + [ + 13, + 20, + "ectoine", + "chemical" + ], + [ + 61, + 87, + "L-aspartate-\u00df-semialdehyde", + "chemical" + ], + [ + 146, + 180, + "L-2,4-diaminobutyrate transaminase", + "protein_type" + ], + [ + 182, + 186, + "EctB", + "protein_type" + ], + [ + 202, + 239, + "2,4-diaminobutyrate acetyltransferase", + "protein_type" + ], + [ + 241, + 245, + "EctA", + "protein_type" + ], + [ + 266, + 282, + "ectoine synthase", + "protein_type" + ], + [ + 284, + 288, + "EctC", + "protein_type" + ] + ] + }, + { + "sid": 15, + "sent": "The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers.", + "section": "INTRO", + "ner": [ + [ + 4, + 11, + "ectoine", + "chemical" + ], + [ + 23, + 39, + "5-hydroxyectoine", + "chemical" + ], + [ + 144, + 151, + "ectoine", + "chemical" + ] + ] + }, + { + "sid": 16, + "sent": "This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily.", + "section": "INTRO", + "ner": [ + [ + 45, + 52, + "ectoine", + "chemical" + ], + [ + 81, + 100, + "ectoine hydroxylase", + "protein_type" + ], + [ + 102, + 106, + "EctD", + "protein_type" + ], + [ + 138, + 219, + "non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily", + "protein_type" + ] + ] + }, + { + "sid": 17, + "sent": "The remarkable function preserving effects of ectoines for macromolecules and cells, frequently also addressed as chemical chaperones, led to a substantial interest in exploiting these compounds for biotechnological purposes and medical applications.", + "section": "INTRO", + "ner": [ + [ + 46, + 54, + "ectoines", + "chemical" + ] + ] + }, + { + "sid": 18, + "sent": "Biosynthetic routes for ectoine and 5-hydroxyectoine.", + "section": "FIG", + "ner": [ + [ + 24, + 31, + "ectoine", + "chemical" + ], + [ + 36, + 52, + "5-hydroxyectoine", + "chemical" + ] + ] + }, + { + "sid": 19, + "sent": "Scheme of the ectoine and 5-hydroxyectoine biosynthetic pathway.", + "section": "FIG", + "ner": [ + [ + 14, + 21, + "ectoine", + "chemical" + ], + [ + 26, + 42, + "5-hydroxyectoine", + "chemical" + ] + ] + }, + { + "sid": 20, + "sent": "Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1).", + "section": "INTRO", + "ner": [ + [ + 17, + 33, + "ectoine synthase", + "protein_type" + ], + [ + 35, + 39, + "EctC", + "protein" + ], + [ + 64, + 71, + "ectoine", + "chemical" + ] + ] + }, + { + "sid": 21, + "sent": "Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.", + "section": "INTRO", + "ner": [ + [ + 12, + 29, + "characterizations", + "experimental_method" + ], + [ + 33, + 50, + "ectoine synthases", + "protein_type" + ], + [ + 60, + 73, + "extremophiles", + "taxonomy_domain" + ], + [ + 74, + 92, + "Halomonas elongata", + "species" + ], + [ + 94, + 123, + "Methylomicrobium alcaliphilum", + "species" + ], + [ + 129, + 149, + "Acidiphilium cryptum", + "species" + ], + [ + 164, + 183, + "nitrifying archaeon", + "taxonomy_domain" + ], + [ + 184, + 208, + "Nitrosopumilus maritimus", + "species" + ] + ] + }, + { + "sid": 22, + "sent": "Each of these enzymes catalyzes as their main activity the cyclization of N-\u03b3-acetyl-L-2,4-diaminobutyric acid (N-\u03b3-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).", + "section": "INTRO", + "ner": [ + [ + 74, + 110, + "N-\u03b3-acetyl-L-2,4-diaminobutyric acid", + "chemical" + ], + [ + 112, + 121, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 152, + 189, + "2,4-diaminobutyrate acetyltransferase", + "protein_type" + ], + [ + 191, + 195, + "EctA", + "protein_type" + ], + [ + 201, + 208, + "ectoine", + "chemical" + ], + [ + 243, + 248, + "water", + "chemical" + ] + ] + }, + { + "sid": 23, + "sent": "In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.", + "section": "INTRO", + "ner": [ + [ + 19, + 23, + "EctC", + "protein" + ], + [ + 85, + 129, + "5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate", + "chemical" + ], + [ + 131, + 135, + "ADPC", + "chemical" + ], + [ + 176, + 185, + "glutamine", + "chemical" + ], + [ + 250, + 257, + "ectoine", + "chemical" + ], + [ + 272, + 279, + "ectoine", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).", + "section": "INTRO", + "ner": [ + [ + 84, + 100, + "ectoine synthase", + "protein_type" + ], + [ + 135, + 144, + "structure", + "evidence" + ], + [ + 248, + 275, + "biochemically characterized", + "experimental_method" + ], + [ + 280, + 296, + "ectoine synthase", + "protein_type" + ], + [ + 319, + 335, + "marine bacterium", + "taxonomy_domain" + ], + [ + 336, + 359, + "Sphingopyxis alaskensis", + "species" + ], + [ + 361, + 363, + "Sa", + "species" + ] + ] + }, + { + "sid": 25, + "sent": "We demonstrate here for the first time that the ectoine synthase is a metal-dependent enzyme, with iron as the most likely physiologically relevant co-factor.", + "section": "INTRO", + "ner": [ + [ + 48, + 64, + "ectoine synthase", + "protein_type" + ], + [ + 70, + 75, + "metal", + "chemical" + ], + [ + 99, + 103, + "iron", + "chemical" + ] + ] + }, + { + "sid": 26, + "sent": "The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily.", + "section": "INTRO", + "ner": [ + [ + 4, + 8, + "EctC", + "protein" + ], + [ + 25, + 30, + "dimer", + "oligomeric_state" + ], + [ + 51, + 70, + "structural analysis", + "experimental_method" + ], + [ + 104, + 121, + "cupin superfamily", + "protein_type" + ] + ] + }, + { + "sid": 27, + "sent": "The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 \u00c5 and 2.0 \u00c5, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.", + "section": "INTRO", + "ner": [ + [ + 8, + 26, + "crystal structures", + "evidence" + ], + [ + 56, + 58, + "Sa", + "species" + ], + [ + 59, + 63, + "EctC", + "protein" + ], + [ + 157, + 182, + "site-directed mutagenesis", + "experimental_method" + ], + [ + 205, + 236, + "evolutionarily highly conserved", + "protein_state" + ], + [ + 266, + 278, + "EctC protein", + "protein_type" + ], + [ + 337, + 351, + "catalytic core", + "site" + ], + [ + 359, + 375, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 28, + "sent": "Overproduction, purification and oligomeric state of the ectoine synthase in solution", + "section": "RESULTS", + "ner": [ + [ + 0, + 14, + "Overproduction", + "experimental_method" + ], + [ + 16, + 28, + "purification", + "experimental_method" + ], + [ + 57, + 73, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 29, + "sent": "We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.", + "section": "RESULTS", + "ner": [ + [ + 15, + 49, + "biochemical and structural studies", + "experimental_method" + ], + [ + 57, + 73, + "ectoine synthase", + "protein_type" + ], + [ + 79, + 92, + "S. alaskensis", + "species" + ], + [ + 95, + 97, + "Sa", + "species" + ], + [ + 98, + 102, + "EctC", + "protein" + ], + [ + 120, + 147, + "marine ultra-microbacterium", + "taxonomy_domain" + ], + [ + 192, + 209, + "crystal structure", + "evidence" + ], + [ + 217, + 236, + "ectoine hydroxylase", + "protein_type" + ], + [ + 238, + 242, + "EctD", + "protein_type" + ], + [ + 244, + 259, + "in complex with", + "protein_state" + ] + ] + }, + { + "sid": 30, + "sent": "We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.", + "section": "RESULTS", + "ner": [ + [ + 46, + 59, + "S. alaskensis", + "species" + ], + [ + 60, + 64, + "ectC", + "gene" + ], + [ + 73, + 80, + "E. coli", + "species" + ], + [ + 149, + 178, + "Strep-tag II affinity peptide", + "experimental_method" + ], + [ + 209, + 211, + "Sa", + "species" + ], + [ + 212, + 216, + "EctC", + "protein" + ], + [ + 217, + 229, + "Strep-Tag-II", + "experimental_method" + ], + [ + 241, + 264, + "affinity chromatography", + "experimental_method" + ] + ] + }, + { + "sid": 31, + "sent": "The (Sa)EctC protein was overproduced and isolated with good yields (30\u201340 mg L-1 of culture) and purity (S2a Fig).", + "section": "RESULTS", + "ner": [ + [ + 5, + 7, + "Sa", + "species" + ], + [ + 8, + 12, + "EctC", + "protein" + ] + ] + }, + { + "sid": 32, + "sent": "Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution.", + "section": "RESULTS", + "ner": [ + [ + 13, + 42, + "size-exclusion chromatography", + "experimental_method" + ], + [ + 44, + 47, + "SEC", + "experimental_method" + ], + [ + 73, + 75, + "Sa", + "species" + ], + [ + 76, + 80, + "EctC", + "protein" + ], + [ + 162, + 168, + "dimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 33, + "sent": "High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 \u00b1 2.3 kDa (S2b Fig).", + "section": "RESULTS", + "ner": [ + [ + 0, + 38, + "High performance liquid chromatography", + "experimental_method" + ], + [ + 52, + 90, + "multi-angle light-scattering detection", + "experimental_method" + ], + [ + 92, + 101, + "HPLC-MALS", + "experimental_method" + ], + [ + 161, + 163, + "Sa", + "species" + ], + [ + 164, + 168, + "EctC", + "protein" + ] + ] + }, + { + "sid": 34, + "sent": "This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa).", + "section": "RESULTS", + "ner": [ + [ + 89, + 91, + "Sa", + "species" + ], + [ + 92, + 96, + "EctC", + "protein" + ], + [ + 97, + 102, + "dimer", + "oligomeric_state" + ], + [ + 126, + 133, + "monomer", + "oligomeric_state" + ], + [ + 149, + 178, + "Strep-tag II affinity peptide", + "experimental_method" + ] + ] + }, + { + "sid": 35, + "sent": "Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus.", + "section": "RESULTS", + "ner": [ + [ + 30, + 35, + "dimer", + "oligomeric_state" + ], + [ + 67, + 80, + "EctC proteins", + "protein_type" + ], + [ + 86, + 97, + "H. elongata", + "species" + ], + [ + 102, + 114, + "N. maritimus", + "species" + ] + ] + }, + { + "sid": 36, + "sent": "Biochemical properties of the ectoine synthase", + "section": "RESULTS", + "ner": [ + [ + 30, + 46, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 37, + "sent": "The EctA-produced substrate of the ectoine synthase, N-\u03b3-acetyl-L-2,4-diaminobutyric acid (N-\u03b3-ADABA) (Fig 1), is commercially not available.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "EctA", + "protein" + ], + [ + 35, + 51, + "ectoine synthase", + "protein_type" + ], + [ + 53, + 89, + "N-\u03b3-acetyl-L-2,4-diaminobutyric acid", + "chemical" + ], + [ + 91, + 100, + "N-\u03b3-ADABA", + "chemical" + ] + ] + }, + { + "sid": 38, + "sent": "We used alkaline hydrolysis of ectoine and subsequent chromatography on silica gel columns to obtain N-\u03b3-ADABA in chemically highly purified form (S1a Fig).", + "section": "RESULTS", + "ner": [ + [ + 31, + 38, + "ectoine", + "chemical" + ], + [ + 101, + 110, + "N-\u03b3-ADABA", + "chemical" + ] + ] + }, + { + "sid": 39, + "sent": "This procedure also yielded the isomer of N-\u03b3-ADABA, N-\u03b1-acetyl-L-2,4-diaminobutyric acid (N-\u03b1-ADABA) (S1b Fig).", + "section": "RESULTS", + "ner": [ + [ + 42, + 51, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 53, + 89, + "N-\u03b1-acetyl-L-2,4-diaminobutyric acid", + "chemical" + ], + [ + 91, + 100, + "N-\u03b1-ADABA", + "chemical" + ] + ] + }, + { + "sid": 40, + "sent": "N-\u03b1-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "N-\u03b1-ADABA", + "chemical" + ], + [ + 60, + 64, + "EctC", + "protein" + ], + [ + 70, + 84, + "microorganisms", + "taxonomy_domain" + ], + [ + 94, + 101, + "ectoine", + "chemical" + ] + ] + }, + { + "sid": 41, + "sent": "Using N-\u03b3-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein.", + "section": "RESULTS", + "ner": [ + [ + 6, + 15, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 109, + 111, + "Sa", + "species" + ], + [ + 112, + 116, + "EctC", + "protein" + ] + ] + }, + { + "sid": 42, + "sent": "S. alaskensis, from which the studied ectoine synthase was originally derived, is a microorganism that is well-adapted to a life in permanently cold ocean waters.", + "section": "RESULTS", + "ner": [ + [ + 0, + 13, + "S. alaskensis", + "species" + ], + [ + 38, + 54, + "ectoine synthase", + "protein_type" + ], + [ + 84, + 97, + "microorganism", + "taxonomy_domain" + ] + ] + }, + { + "sid": 43, + "sent": "Consistent with the physicochemical attributes of this habitat, the (Sa)EctC protein was already enzymatically active at 5\u00b0C, had a temperature optimum of 15\u00b0C and was able to function over a broad range of temperatures (S3a Fig).", + "section": "RESULTS", + "ner": [ + [ + 69, + 71, + "Sa", + "species" + ], + [ + 72, + 76, + "EctC", + "protein" + ], + [ + 97, + 117, + "enzymatically active", + "protein_state" + ] + ] + }, + { + "sid": 44, + "sent": "It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).", + "section": "RESULTS", + "ner": [ + [ + 16, + 24, + "alkaline", + "protein_state" + ], + [ + 77, + 94, + "ectoine synthases", + "protein_type" + ], + [ + 104, + 117, + "halo-tolerant", + "protein_state" + ], + [ + 118, + 129, + "H. elongata", + "species" + ], + [ + 162, + 173, + "alkaliphile", + "taxonomy_domain" + ], + [ + 174, + 189, + "M. alcaliphilum", + "species" + ], + [ + 219, + 229, + "acidophile", + "taxonomy_domain" + ], + [ + 230, + 250, + "Acidiphilium cryptum", + "species" + ], + [ + 291, + 295, + "EctC", + "protein" + ], + [ + 309, + 321, + "N. maritimus", + "species" + ], + [ + 328, + 338, + "neutral pH", + "protein_state" + ] + ] + }, + { + "sid": 45, + "sent": "The salinity of the assay buffer had a significant influence on the maximal enzyme activity of the (Sa)EctC protein.", + "section": "RESULTS", + "ner": [ + [ + 100, + 102, + "Sa", + "species" + ], + [ + 103, + 107, + "EctC", + "protein" + ] + ] + }, + { + "sid": 46, + "sent": "An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity.", + "section": "RESULTS", + "ner": [ + [ + 26, + 30, + "NaCl", + "chemical" + ], + [ + 38, + 41, + "KCl", + "chemical" + ], + [ + 106, + 122, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 47, + "sent": "The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively.", + "section": "RESULTS", + "ner": [ + [ + 32, + 34, + "Sa", + "species" + ], + [ + 35, + 39, + "EctC", + "protein" + ], + [ + 63, + 67, + "NaCl", + "chemical" + ], + [ + 71, + 74, + "KCl", + "chemical" + ] + ] + }, + { + "sid": 48, + "sent": "(Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig).", + "section": "RESULTS", + "ner": [ + [ + 1, + 3, + "Sa", + "species" + ], + [ + 4, + 8, + "EctC", + "protein" + ], + [ + 97, + 101, + "NaCl", + "chemical" + ], + [ + 106, + 109, + "KCl", + "chemical" + ] + ] + }, + { + "sid": 49, + "sent": "The stimulation of EctC enzyme activity by salts has previously also been observed for other ectoine synthases.", + "section": "RESULTS", + "ner": [ + [ + 19, + 23, + "EctC", + "protein" + ], + [ + 93, + 110, + "ectoine synthases", + "protein_type" + ] + ] + }, + { + "sid": 50, + "sent": "The ectoine synthase is a metal-containing protein", + "section": "RESULTS", + "ner": [ + [ + 4, + 20, + "ectoine synthase", + "protein_type" + ], + [ + 26, + 50, + "metal-containing protein", + "protein_type" + ] + ] + }, + { + "sid": 51, + "sent": "Considerations based on bioinformatics suggests that EctC belongs to the cupin superfamily.", + "section": "RESULTS", + "ner": [ + [ + 53, + 57, + "EctC", + "protein" + ], + [ + 73, + 90, + "cupin superfamily", + "protein_type" + ] + ] + }, + { + "sid": 52, + "sent": "Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.", + "section": "RESULTS", + "ner": [ + [ + 87, + 91, + "iron", + "chemical" + ], + [ + 93, + 99, + "copper", + "chemical" + ], + [ + 101, + 105, + "zinc", + "chemical" + ], + [ + 107, + 116, + "manganese", + "chemical" + ], + [ + 118, + 124, + "cobalt", + "chemical" + ], + [ + 129, + 135, + "nickel", + "chemical" + ] + ] + }, + { + "sid": 53, + "sent": "Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal).", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Cupins", + "protein_type" + ], + [ + 19, + 28, + "conserved", + "protein_state" + ], + [ + 37, + 57, + "G(X)5HXH(X)3,4E(X)6G", + "structure_element" + ], + [ + 62, + 80, + "G(X)5PXG(X)2H(X)3N", + "structure_element" + ], + [ + 153, + 158, + "metal", + "chemical" + ] + ] + }, + { + "sid": 54, + "sent": "Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.", + "section": "RESULTS", + "ner": [ + [ + 25, + 62, + "alignment of the amino acid sequences", + "experimental_method" + ], + [ + 70, + 88, + "EctC-type proteins", + "protein_type" + ], + [ + 117, + 136, + "metal-binding motif", + "structure_element" + ], + [ + 143, + 162, + "cupin-type proteins", + "protein_type" + ], + [ + 166, + 179, + "not conserved", + "protein_state" + ], + [ + 210, + 241, + "ectoine synthase protein family", + "protein_type" + ] + ] + }, + { + "sid": 55, + "sent": "An abbreviated alignment of the amino acid sequence of EctC-type proteins is shown in Fig 2.", + "section": "RESULTS", + "ner": [ + [ + 15, + 51, + "alignment of the amino acid sequence", + "experimental_method" + ], + [ + 55, + 73, + "EctC-type proteins", + "protein_type" + ] + ] + }, + { + "sid": 56, + "sent": "Abbreviated alignment of EctC-type proteins.", + "section": "FIG", + "ner": [ + [ + 12, + 21, + "alignment", + "experimental_method" + ], + [ + 25, + 43, + "EctC-type proteins", + "protein_type" + ] + ] + }, + { + "sid": 57, + "sent": "The amino acid sequences of 20 selected EctC-type proteins are compared.", + "section": "FIG", + "ner": [ + [ + 40, + 58, + "EctC-type proteins", + "protein_type" + ] + ] + }, + { + "sid": 58, + "sent": "Strictly conserved amino acid residues are shown in yellow.", + "section": "FIG", + "ner": [ + [ + 0, + 18, + "Strictly conserved", + "protein_state" + ] + ] + }, + { + "sid": 59, + "sent": "Dots shown above the (Sa)EctC protein sequence indicate residues likely to be involved in iron-binding (red), ligand-binding (green) and stabilization of the loop-architecture (blue).", + "section": "FIG", + "ner": [ + [ + 22, + 24, + "Sa", + "species" + ], + [ + 25, + 29, + "EctC", + "protein" + ], + [ + 90, + 94, + "iron", + "chemical" + ] + ] + }, + { + "sid": 60, + "sent": "The conserved residue Tyr-52 with so-far undefined functions is indicated by a green dot circled in red.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "conserved", + "protein_state" + ], + [ + 22, + 28, + "Tyr-52", + "residue_name_number" + ] + ] + }, + { + "sid": 61, + "sent": "Secondary structural elements (\u03b1-helices and \u03b2-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.", + "section": "FIG", + "ner": [ + [ + 31, + 40, + "\u03b1-helices", + "structure_element" + ], + [ + 45, + 53, + "\u03b2-sheets", + "structure_element" + ], + [ + 69, + 71, + "Sa", + "species" + ], + [ + 72, + 76, + "EctC", + "protein" + ], + [ + 77, + 94, + "crystal structure", + "evidence" + ], + [ + 142, + 160, + "EctC-type proteins", + "protein_type" + ] + ] + }, + { + "sid": 62, + "sent": "Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).", + "section": "RESULTS", + "ner": [ + [ + 40, + 59, + "metal-binding motif", + "structure_element" + ], + [ + 140, + 145, + "metal", + "chemical" + ], + [ + 178, + 180, + "Sa", + "species" + ], + [ + 181, + 185, + "EctC", + "protein" + ], + [ + 197, + 239, + "inductive-coupled plasma mass spectrometry", + "experimental_method" + ], + [ + 241, + 247, + "ICP-MS", + "experimental_method" + ] + ] + }, + { + "sid": 63, + "sent": "For this analysis we used recombinant (Sa)EctC preparations from three independent protein overproduction and purification experiments.", + "section": "RESULTS", + "ner": [ + [ + 39, + 41, + "Sa", + "species" + ], + [ + 42, + 46, + "EctC", + "protein" + ] + ] + }, + { + "sid": 64, + "sent": "The ICP-MS analyses yielded an iron content of 0.66 \u00b1 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "ICP-MS", + "experimental_method" + ], + [ + 31, + 35, + "iron", + "chemical" + ], + [ + 63, + 67, + "iron", + "chemical" + ], + [ + 101, + 103, + "Sa", + "species" + ], + [ + 104, + 108, + "EctC", + "protein" + ], + [ + 163, + 167, + "zinc", + "chemical" + ], + [ + 178, + 182, + "zinc", + "chemical" + ] + ] + }, + { + "sid": 65, + "sent": "All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively).", + "section": "RESULTS", + "ner": [ + [ + 26, + 32, + "copper", + "chemical" + ], + [ + 37, + 43, + "nickel", + "chemical" + ], + [ + 90, + 95, + "metal", + "chemical" + ] + ] + }, + { + "sid": 66, + "sent": "The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 \u00b1 0.05 mol per mol of (Sa)EctC protein.", + "section": "RESULTS", + "ner": [ + [ + 16, + 20, + "iron", + "chemical" + ], + [ + 31, + 33, + "Sa", + "species" + ], + [ + 34, + 38, + "EctC", + "protein" + ], + [ + 87, + 106, + "colorimetric method", + "experimental_method" + ], + [ + 127, + 131, + "iron", + "chemical" + ], + [ + 178, + 182, + "iron", + "chemical" + ], + [ + 222, + 224, + "Sa", + "species" + ], + [ + 225, + 229, + "EctC", + "protein" + ] + ] + }, + { + "sid": 67, + "sent": "Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein.", + "section": "RESULTS", + "ner": [ + [ + 12, + 18, + "ICP-MS", + "experimental_method" + ], + [ + 27, + 46, + "colorimetric method", + "experimental_method" + ], + [ + 99, + 115, + "ectoine synthase", + "protein_type" + ], + [ + 121, + 134, + "S. alaskensis", + "species" + ], + [ + 141, + 145, + "iron", + "chemical" + ] + ] + }, + { + "sid": 68, + "sent": "We note in this context, that the values obtained for the iron content of the (Sa)EctC proteins varied by approximately 10 to 20% between the two methods.", + "section": "RESULTS", + "ner": [ + [ + 58, + 62, + "iron", + "chemical" + ], + [ + 79, + 81, + "Sa", + "species" + ], + [ + 82, + 86, + "EctC", + "protein" + ] + ] + }, + { + "sid": 69, + "sent": "The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree.", + "section": "RESULTS", + "ner": [ + [ + 85, + 103, + "colorimetric assay", + "experimental_method" + ], + [ + 131, + 135, + "iron", + "chemical" + ], + [ + 148, + 150, + "Sa", + "species" + ], + [ + 151, + 155, + "EctC", + "protein" + ] + ] + }, + { + "sid": 70, + "sent": "A metal cofactor is important for the catalytic activity of EctC", + "section": "RESULTS", + "ner": [ + [ + 2, + 7, + "metal", + "chemical" + ], + [ + 60, + 64, + "EctC", + "protein" + ] + ] + }, + { + "sid": 71, + "sent": "The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "iron", + "chemical" + ], + [ + 26, + 28, + "Sa", + "species" + ], + [ + 29, + 33, + "EctC", + "protein" + ], + [ + 180, + 197, + "cupin superfamily", + "protein_type" + ] + ] + }, + { + "sid": 72, + "sent": "To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.", + "section": "RESULTS", + "ner": [ + [ + 31, + 40, + "incubated", + "experimental_method" + ], + [ + 46, + 48, + "Sa", + "species" + ], + [ + 49, + 53, + "EctC", + "protein" + ], + [ + 61, + 91, + "with increasing concentrations", + "experimental_method" + ], + [ + 99, + 104, + "metal", + "chemical" + ], + [ + 114, + 147, + "ethylene-diamine-tetraacetic-acid", + "chemical" + ], + [ + 149, + 153, + "EDTA", + "chemical" + ], + [ + 180, + 196, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 73, + "sent": "The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a).", + "section": "RESULTS", + "ner": [ + [ + 43, + 47, + "EDTA", + "chemical" + ], + [ + 65, + 69, + "EctC", + "protein" + ], + [ + 123, + 139, + "ectoine synthase", + "protein_type" + ], + [ + 174, + 178, + "EDTA", + "chemical" + ] + ] + }, + { + "sid": 74, + "sent": "Dependency of the ectoine synthase activity on metals.", + "section": "FIG", + "ner": [ + [ + 18, + 34, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 75, + "sent": "(a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein.", + "section": "FIG", + "ner": [ + [ + 18, + 22, + "iron", + "chemical" + ], + [ + 32, + 36, + "EDTA", + "chemical" + ], + [ + 77, + 79, + "Sa", + "species" + ], + [ + 80, + 84, + "EctC", + "protein" + ] + ] + }, + { + "sid": 76, + "sent": "Metal depletion and reconstitution experiments with (b) stoichiometric and (c) excess amounts of metals.", + "section": "FIG", + "ner": [ + [ + 0, + 46, + "Metal depletion and reconstitution experiments", + "experimental_method" + ] + ] + }, + { + "sid": 77, + "sent": "The (Sa)EctC protein was present at a concentration of 10 \u03bcM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added.", + "section": "FIG", + "ner": [ + [ + 5, + 7, + "Sa", + "species" + ], + [ + 8, + 12, + "EctC", + "protein" + ], + [ + 139, + 155, + "ectoine synthase", + "protein_type" + ], + [ + 156, + 169, + "enzyme assays", + "experimental_method" + ], + [ + 184, + 189, + "FeCl2", + "chemical" + ] + ] + }, + { + "sid": 78, + "sent": "We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 \u03bcM) of various metals to the (Sa)EctC enzyme.", + "section": "RESULTS", + "ner": [ + [ + 21, + 32, + "inactivated", + "protein_state" + ], + [ + 65, + 69, + "EDTA", + "chemical" + ], + [ + 73, + 81, + "dialysis", + "experimental_method" + ], + [ + 150, + 152, + "Sa", + "species" + ], + [ + 153, + 157, + "EctC", + "protein" + ] + ] + }, + { + "sid": 79, + "sent": "The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.", + "section": "RESULTS", + "ner": [ + [ + 16, + 21, + "FeCl2", + "chemical" + ], + [ + 29, + 41, + "enzyme assay", + "experimental_method" + ], + [ + 105, + 110, + "ZnCl2", + "chemical" + ], + [ + 114, + 119, + "CoCl2", + "chemical" + ], + [ + 129, + 131, + "Sa", + "species" + ], + [ + 132, + 136, + "EctC", + "protein" + ] + ] + }, + { + "sid": 80, + "sent": "All other tested metals, including Fe3+, were unable to restore activity (Fig 3b).", + "section": "RESULTS", + "ner": [ + [ + 35, + 39, + "Fe3+", + "chemical" + ] + ] + }, + { + "sid": 81, + "sent": "When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c).", + "section": "RESULTS", + "ner": [ + [ + 52, + 64, + "enzyme assay", + "experimental_method" + ], + [ + 89, + 93, + "Fe2+", + "chemical" + ], + [ + 233, + 235, + "Sa", + "species" + ], + [ + 236, + 240, + "EctC", + "protein" + ], + [ + 300, + 304, + "EDTA", + "chemical" + ] + ] + }, + { + "sid": 82, + "sent": "However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).", + "section": "RESULTS", + "ner": [ + [ + 64, + 68, + "zinc", + "chemical" + ], + [ + 70, + 76, + "cobalt", + "chemical" + ], + [ + 78, + 84, + "nickel", + "chemical" + ], + [ + 86, + 92, + "copper", + "chemical" + ], + [ + 98, + 107, + "manganese", + "chemical" + ], + [ + 143, + 160, + "cupin superfamily", + "protein_type" + ], + [ + 191, + 207, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 83, + "sent": "This is in line with literature data showing that cupin-type enzymes are often promiscuous with respect to the use of the catalytically important metal.", + "section": "RESULTS", + "ner": [ + [ + 50, + 68, + "cupin-type enzymes", + "protein_type" + ], + [ + 146, + 151, + "metal", + "chemical" + ] + ] + }, + { + "sid": 84, + "sent": "Kinetic parameters of EctC for N-\u03b3-ADABA and N-\u03b1-ADABA", + "section": "RESULTS", + "ner": [ + [ + 22, + 26, + "EctC", + "protein" + ], + [ + 31, + 40, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 45, + 54, + "N-\u03b1-ADABA", + "chemical" + ] + ] + }, + { + "sid": 85, + "sent": "Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-\u03b3-ADABA and the isomer N-\u03b1-ADABA.", + "section": "RESULTS", + "ner": [ + [ + 66, + 80, + "activity assay", + "experimental_method" + ], + [ + 89, + 105, + "ectoine synthase", + "protein_type" + ], + [ + 109, + 122, + "S. alaskensis", + "species" + ], + [ + 181, + 183, + "Sa", + "species" + ], + [ + 184, + 188, + "EctC", + "protein" + ], + [ + 227, + 236, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 252, + 261, + "N-\u03b1-ADABA", + "chemical" + ] + ] + }, + { + "sid": 86, + "sent": "The EctC-catalyzed ring-closure of N-\u03b3-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 \u00b1 0.5 mM, a vmax of 25.0 \u00b1 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "EctC", + "protein" + ], + [ + 35, + 44, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 53, + 60, + "ectoine", + "chemical" + ], + [ + 71, + 96, + "Michaelis-Menten-kinetics", + "experimental_method" + ], + [ + 114, + 116, + "Km", + "evidence" + ], + [ + 136, + 140, + "vmax", + "evidence" + ], + [ + 166, + 170, + "kcat", + "evidence" + ] + ] + }, + { + "sid": 87, + "sent": "Given the chemical relatedness of N-\u03b1-ADABA to the natural substrate (N-\u03b3-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-\u03b1-ADABA to produce ectoine.", + "section": "RESULTS", + "ner": [ + [ + 34, + 43, + "N-\u03b1-ADABA", + "chemical" + ], + [ + 70, + 79, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 88, + 104, + "ectoine synthase", + "protein_type" + ], + [ + 145, + 147, + "Sa", + "species" + ], + [ + 148, + 152, + "EctC", + "protein" + ], + [ + 168, + 177, + "N-\u03b1-ADABA", + "chemical" + ], + [ + 189, + 196, + "ectoine", + "chemical" + ] + ] + }, + { + "sid": 88, + "sent": "(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 \u00b1 2.9 mM, a vmax of 24.6 \u00b1 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).", + "section": "RESULTS", + "ner": [ + [ + 1, + 3, + "Sa", + "species" + ], + [ + 4, + 8, + "EctC", + "protein" + ], + [ + 38, + 63, + "Michaelis-Menten-kinetics", + "experimental_method" + ], + [ + 87, + 89, + "Km", + "evidence" + ], + [ + 110, + 114, + "vmax", + "evidence" + ], + [ + 140, + 144, + "kcat", + "evidence" + ] + ] + }, + { + "sid": 89, + "sent": "Hence, N-\u03b1-ADABA is a newly recognized substrate for ectoine synthase.", + "section": "RESULTS", + "ner": [ + [ + 7, + 16, + "N-\u03b1-ADABA", + "chemical" + ], + [ + 53, + 69, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 90, + "sent": "However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-\u03b3-ADABA.", + "section": "RESULTS", + "ner": [ + [ + 18, + 26, + "affinity", + "evidence" + ], + [ + 28, + 30, + "Km", + "evidence" + ], + [ + 40, + 42, + "Sa", + "species" + ], + [ + 43, + 47, + "EctC", + "protein" + ], + [ + 64, + 84, + "catalytic efficiency", + "evidence" + ], + [ + 86, + 93, + "kcat/Km", + "evidence" + ], + [ + 136, + 145, + "N-\u03b3-ADABA", + "chemical" + ] + ] + }, + { + "sid": 91, + "sent": "The Km dropped fife-fold from 4.9 \u00b1 0.5 mM to 25.4 \u00b1 2.9 mM, and the catalytic efficiency was reduced from 1.47 mM-1 s-1 to 0.02 mM-1 s-1, a 73-fold decrease.", + "section": "RESULTS", + "ner": [ + [ + 4, + 6, + "Km", + "evidence" + ], + [ + 69, + 89, + "catalytic efficiency", + "evidence" + ] + ] + }, + { + "sid": 92, + "sent": "Both N-\u03b3-ADABA and N-\u03b1-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism.", + "section": "RESULTS", + "ner": [ + [ + 5, + 14, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 19, + 28, + "N-\u03b1-ADABA", + "chemical" + ], + [ + 93, + 100, + "ectoine", + "chemical" + ] + ] + }, + { + "sid": 93, + "sent": "Our finding that N-\u03b1-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine.", + "section": "RESULTS", + "ner": [ + [ + 17, + 26, + "N-\u03b1-ADABA", + "chemical" + ], + [ + 46, + 62, + "ectoine synthase", + "protein_type" + ], + [ + 124, + 138, + "microorganisms", + "taxonomy_domain" + ], + [ + 179, + 186, + "ectoine", + "chemical" + ] + ] + }, + { + "sid": 94, + "sent": "However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-\u03b3-ADABA and N-\u03b1-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).", + "section": "RESULTS", + "ner": [ + [ + 24, + 38, + "microorganisms", + "taxonomy_domain" + ], + [ + 102, + 110, + "affinity", + "evidence" + ], + [ + 114, + 130, + "ectoine synthase", + "protein_type" + ], + [ + 135, + 144, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 149, + 158, + "N-\u03b1-ADABA", + "chemical" + ], + [ + 168, + 188, + "catalytic efficiency", + "evidence" + ] + ] + }, + { + "sid": 95, + "sent": "Crystallization of the (Sa)EctC protein", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Crystallization", + "experimental_method" + ], + [ + 24, + 26, + "Sa", + "species" + ], + [ + 27, + 31, + "EctC", + "protein" + ] + ] + }, + { + "sid": 96, + "sent": "Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein.", + "section": "RESULTS", + "ner": [ + [ + 9, + 26, + "crystal structure", + "evidence" + ], + [ + 30, + 46, + "ectoine synthase", + "protein_type" + ], + [ + 80, + 91, + "crystallize", + "experimental_method" + ], + [ + 97, + 99, + "Sa", + "species" + ], + [ + 100, + 104, + "EctC", + "protein" + ] + ] + }, + { + "sid": 97, + "sent": "Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-\u03b3-ADABA or its reaction product ectoine were not successful.", + "section": "RESULTS", + "ner": [ + [ + 19, + 27, + "crystals", + "evidence" + ], + [ + 32, + 34, + "Sa", + "species" + ], + [ + 35, + 39, + "EctC", + "protein" + ], + [ + 40, + 50, + "in complex", + "protein_state" + ], + [ + 77, + 86, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 111, + 118, + "ectoine", + "chemical" + ] + ] + }, + { + "sid": 98, + "sent": "However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained.", + "section": "RESULTS", + "ner": [ + [ + 13, + 26, + "crystal forms", + "evidence" + ], + [ + 35, + 37, + "Sa", + "species" + ], + [ + 38, + 42, + "EctC", + "protein" + ], + [ + 58, + 68, + "absence of", + "protein_state" + ] + ] + }, + { + "sid": 99, + "sent": "Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed.", + "section": "RESULTS", + "ner": [ + [ + 22, + 39, + "crystal structure", + "evidence" + ], + [ + 48, + 50, + "Sa", + "species" + ], + [ + 51, + 55, + "EctC", + "protein" + ], + [ + 67, + 88, + "molecular replacement", + "experimental_method" + ] + ] + }, + { + "sid": 100, + "sent": "However, we were able to obtain crystals of form B that were derivatized with mercury and these diffracted up to 2.8 \u00c5 (S1 Table).", + "section": "RESULTS", + "ner": [ + [ + 32, + 40, + "crystals", + "evidence" + ], + [ + 78, + 85, + "mercury", + "chemical" + ] + ] + }, + { + "sid": 101, + "sent": "This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 \u00c5) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).", + "section": "RESULTS", + "ner": [ + [ + 43, + 59, + "structural model", + "evidence" + ], + [ + 68, + 70, + "Sa", + "species" + ], + [ + 71, + 75, + "EctC", + "protein" + ], + [ + 130, + 151, + "molecular replacement", + "experimental_method" + ], + [ + 274, + 282, + "monomers", + "oligomeric_state" + ], + [ + 287, + 289, + "Sa", + "species" + ], + [ + 290, + 294, + "EctC", + "protein" + ], + [ + 319, + 336, + "crystal structure", + "evidence" + ], + [ + 360, + 366, + "Rcryst", + "evidence" + ], + [ + 383, + 388, + "Rfree", + "evidence" + ] + ] + }, + { + "sid": 102, + "sent": "Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 \u00c5) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table).", + "section": "RESULTS", + "ner": [ + [ + 11, + 18, + "monomer", + "oligomeric_state" + ], + [ + 27, + 36, + "structure", + "evidence" + ], + [ + 64, + 85, + "molecular replacement", + "experimental_method" + ], + [ + 192, + 198, + "Rcryst", + "evidence" + ], + [ + 215, + 220, + "Rfree", + "evidence" + ] + ] + }, + { + "sid": 103, + "sent": "Overall fold of the (Sa)EctC protein", + "section": "RESULTS", + "ner": [ + [ + 21, + 23, + "Sa", + "species" + ], + [ + 24, + 28, + "EctC", + "protein" + ] + ] + }, + { + "sid": 104, + "sent": "The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a\u20134c).", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "EctC", + "protein" + ], + [ + 13, + 23, + "structures", + "evidence" + ], + [ + 61, + 77, + "ectoine synthase", + "protein_type" + ], + [ + 93, + 110, + "cupin superfamily", + "protein_type" + ] + ] + }, + { + "sid": 105, + "sent": "However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2).", + "section": "RESULTS", + "ner": [ + [ + 52, + 67, + "137 amino acids", + "residue_range" + ], + [ + 80, + 82, + "Sa", + "species" + ], + [ + 83, + 87, + "EctC", + "protein" + ] + ] + }, + { + "sid": 106, + "sent": "First, the 1.2 \u00c5 structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.", + "section": "RESULTS", + "ner": [ + [ + 17, + 26, + "structure", + "evidence" + ], + [ + 69, + 71, + "Sa", + "species" + ], + [ + 72, + 76, + "EctC", + "protein" + ], + [ + 109, + 125, + "Met-1 to Glu-115", + "residue_range" + ], + [ + 137, + 142, + "lacks", + "protein_state" + ], + [ + 143, + 157, + "22 amino acids", + "residue_range" + ], + [ + 165, + 181, + "carboxy-terminus", + "structure_element" + ], + [ + 200, + 202, + "Sa", + "species" + ], + [ + 203, + 207, + "EctC", + "protein" + ] + ] + }, + { + "sid": 107, + "sent": "This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the \u201copen\u201d (Sa)EctC structure (Fig 4b).", + "section": "RESULTS", + "ner": [ + [ + 5, + 14, + "structure", + "evidence" + ], + [ + 25, + 29, + "open", + "protein_state" + ], + [ + 79, + 92, + "cupin barrels", + "structure_element" + ], + [ + 139, + 143, + "open", + "protein_state" + ], + [ + 146, + 148, + "Sa", + "species" + ], + [ + 149, + 153, + "EctC", + "protein" + ], + [ + 154, + 163, + "structure", + "evidence" + ] + ] + }, + { + "sid": 108, + "sent": "In this structure no metal co-factor was identified.", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 21, + 26, + "metal", + "chemical" + ] + ] + }, + { + "sid": 109, + "sent": "The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 \u00c5 and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.", + "section": "RESULTS", + "ner": [ + [ + 11, + 28, + "crystal structure", + "evidence" + ], + [ + 37, + 39, + "Sa", + "species" + ], + [ + 40, + 44, + "EctC", + "protein" + ], + [ + 57, + 63, + "solved", + "experimental_method" + ], + [ + 165, + 173, + "protomer", + "oligomeric_state" + ], + [ + 174, + 175, + "A", + "structure_element" + ], + [ + 197, + 213, + "Met-1 to Gly-121", + "residue_range" + ], + [ + 227, + 233, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 110, + "sent": "Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).", + "section": "RESULTS", + "ner": [ + [ + 16, + 21, + "lacks", + "protein_state" + ], + [ + 22, + 35, + "16 amino acid", + "residue_range" + ], + [ + 52, + 68, + "carboxy-terminus", + "structure_element" + ], + [ + 86, + 101, + "137 amino acids", + "residue_range" + ], + [ + 114, + 116, + "Sa", + "species" + ], + [ + 117, + 121, + "EctC", + "protein" + ] + ] + }, + { + "sid": 111, + "sent": "We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the \u201csemi-closed\u201d (Sa)EctC structure.", + "section": "RESULTS", + "ner": [ + [ + 38, + 55, + "crystal structure", + "evidence" + ], + [ + 79, + 91, + "fully closed", + "protein_state" + ], + [ + 105, + 121, + "ectoine synthase", + "protein_type" + ], + [ + 167, + 178, + "semi-closed", + "protein_state" + ], + [ + 181, + 183, + "Sa", + "species" + ], + [ + 184, + 188, + "EctC", + "protein" + ], + [ + 189, + 198, + "structure", + "evidence" + ] + ] + }, + { + "sid": 112, + "sent": "Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the \u201copen\u201d EctC structure.", + "section": "RESULTS", + "ner": [ + [ + 31, + 39, + "monomers", + "oligomeric_state" + ], + [ + 86, + 102, + "Met-1 to Glu-115", + "residue_range" + ], + [ + 144, + 148, + "open", + "protein_state" + ], + [ + 150, + 154, + "EctC", + "protein" + ], + [ + 155, + 164, + "structure", + "evidence" + ] + ] + }, + { + "sid": 113, + "sent": "Overall structure of the \u201copen\u201d and \u201csemi-closed\u201d crystal structures of (Sa)EctC.", + "section": "FIG", + "ner": [ + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 26, + 30, + "open", + "protein_state" + ], + [ + 37, + 48, + "semi-closed", + "protein_state" + ], + [ + 50, + 68, + "crystal structures", + "evidence" + ], + [ + 73, + 75, + "Sa", + "species" + ], + [ + 76, + 80, + "EctC", + "protein" + ] + ] + }, + { + "sid": 114, + "sent": "(a) The overall structure of the \u201csemi-closed\u201d (Sa)EctC resolved at 2.0 \u00c5 is depicted in green in a cartoon (upper panel) and surface (lower panel) representation.", + "section": "FIG", + "ner": [ + [ + 16, + 25, + "structure", + "evidence" + ], + [ + 34, + 45, + "semi-closed", + "protein_state" + ], + [ + 48, + 50, + "Sa", + "species" + ], + [ + 51, + 55, + "EctC", + "protein" + ] + ] + }, + { + "sid": 115, + "sent": "The \u03b2-strands are numbered \u03b21-\u03b211 and the helices \u03b1-I to \u03b1-II.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "\u03b2-strands", + "structure_element" + ], + [ + 27, + 33, + "\u03b21-\u03b211", + "structure_element" + ], + [ + 42, + 49, + "helices", + "structure_element" + ], + [ + 50, + 61, + "\u03b1-I to \u03b1-II", + "structure_element" + ] + ] + }, + { + "sid": 116, + "sent": "(b) The overall structure of the \u201copen\u201d (Sa)EctC was resolved at 1.2 \u00c5 and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation.", + "section": "FIG", + "ner": [ + [ + 16, + 25, + "structure", + "evidence" + ], + [ + 34, + 38, + "open", + "protein_state" + ], + [ + 41, + 43, + "Sa", + "species" + ], + [ + 44, + 48, + "EctC", + "protein" + ] + ] + }, + { + "sid": 117, + "sent": "The entrance to the active site of the ectoine synthase is marked.", + "section": "FIG", + "ner": [ + [ + 20, + 31, + "active site", + "site" + ], + [ + 39, + 55, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 118, + "sent": "(c) Overlay of the \u201csemi-closed\u201d and \u201copen\u201d (Sa)EctC structures.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "Overlay", + "experimental_method" + ], + [ + 20, + 31, + "semi-closed", + "protein_state" + ], + [ + 38, + 42, + "open", + "protein_state" + ], + [ + 45, + 47, + "Sa", + "species" + ], + [ + 48, + 52, + "EctC", + "protein" + ], + [ + 53, + 63, + "structures", + "evidence" + ] + ] + }, + { + "sid": 119, + "sent": "The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the \u201csemi-closed\u201d structure.", + "section": "RESULTS", + "ner": [ + [ + 12, + 21, + "structure", + "evidence" + ], + [ + 26, + 28, + "Sa", + "species" + ], + [ + 29, + 33, + "EctC", + "protein" + ], + [ + 64, + 72, + "crystals", + "evidence" + ], + [ + 88, + 104, + "carboxy-terminus", + "structure_element" + ], + [ + 148, + 160, + "cupin barrel", + "structure_element" + ], + [ + 186, + 193, + "monomer", + "oligomeric_state" + ], + [ + 194, + 195, + "A", + "structure_element" + ], + [ + 204, + 215, + "semi-closed", + "protein_state" + ], + [ + 217, + 226, + "structure", + "evidence" + ] + ] + }, + { + "sid": 120, + "sent": "This is reflected by the calculated root mean square deviation (RMSD) of the C\u03b1 atoms that was about 0.56 \u00c5 (over 117 residues) when the four \u201copen\u201d monomers were compared with each other.", + "section": "RESULTS", + "ner": [ + [ + 36, + 62, + "root mean square deviation", + "evidence" + ], + [ + 64, + 68, + "RMSD", + "evidence" + ], + [ + 143, + 147, + "open", + "protein_state" + ], + [ + 149, + 157, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 121, + "sent": "However, the \u201csemi-closed\u201d monomer has a slightly higher RMSD of 1.4 \u00c5 (over 117 residues) when compared with the \u201copen\u201d 2.0 \u00c5 structure.", + "section": "RESULTS", + "ner": [ + [ + 14, + 25, + "semi-closed", + "protein_state" + ], + [ + 27, + 34, + "monomer", + "oligomeric_state" + ], + [ + 57, + 61, + "RMSD", + "evidence" + ], + [ + 115, + 119, + "open", + "protein_state" + ], + [ + 127, + 136, + "structure", + "evidence" + ] + ] + }, + { + "sid": 122, + "sent": "Therefore, we describe in the following the overall structure for the \u201csemi-closed\u201d form of the (Sa)EctC protein and subsequently highlight the structural differences between the \u201copen\u201d and \u201csemi-closed\u201d forms in more detail.", + "section": "RESULTS", + "ner": [ + [ + 52, + 61, + "structure", + "evidence" + ], + [ + 71, + 82, + "semi-closed", + "protein_state" + ], + [ + 97, + 99, + "Sa", + "species" + ], + [ + 100, + 104, + "EctC", + "protein" + ], + [ + 180, + 184, + "open", + "protein_state" + ], + [ + 191, + 202, + "semi-closed", + "protein_state" + ] + ] + }, + { + "sid": 123, + "sent": "The structure of the \u201csemi-closed\u201d (Sa)EctC protein consists of 11 \u03b2-strands (\u03b21-\u03b211) and two \u03b1-helices (\u03b1-I and \u03b1-II) (Fig 4a).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 22, + 33, + "semi-closed", + "protein_state" + ], + [ + 36, + 38, + "Sa", + "species" + ], + [ + 39, + 43, + "EctC", + "protein" + ], + [ + 67, + 76, + "\u03b2-strands", + "structure_element" + ], + [ + 78, + 84, + "\u03b21-\u03b211", + "structure_element" + ], + [ + 94, + 103, + "\u03b1-helices", + "structure_element" + ], + [ + 105, + 108, + "\u03b1-I", + "structure_element" + ], + [ + 113, + 117, + "\u03b1-II", + "structure_element" + ] + ] + }, + { + "sid": 124, + "sent": "The \u03b2-strands form two anti-parallel \u03b2-sheets: \u03b22 \u03b23, \u03b24, \u03b211, \u03b26, and \u03b29, and a smaller three-stranded \u03b2-sheet (\u03b27, \u03b28, and \u03b210), respectively.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "\u03b2-strands", + "structure_element" + ], + [ + 23, + 45, + "anti-parallel \u03b2-sheets", + "structure_element" + ], + [ + 47, + 49, + "\u03b22", + "structure_element" + ], + [ + 50, + 52, + "\u03b23", + "structure_element" + ], + [ + 54, + 56, + "\u03b24", + "structure_element" + ], + [ + 58, + 61, + "\u03b211", + "structure_element" + ], + [ + 63, + 65, + "\u03b26", + "structure_element" + ], + [ + 71, + 73, + "\u03b29", + "structure_element" + ], + [ + 89, + 111, + "three-stranded \u03b2-sheet", + "structure_element" + ], + [ + 113, + 115, + "\u03b27", + "structure_element" + ], + [ + 117, + 119, + "\u03b28", + "structure_element" + ], + [ + 125, + 128, + "\u03b210", + "structure_element" + ] + ] + }, + { + "sid": 125, + "sent": "These two \u03b2-sheets pack against each other, forming a cup-shaped \u03b2-sandwich with a topology characteristic for the cupin-fold.", + "section": "RESULTS", + "ner": [ + [ + 10, + 18, + "\u03b2-sheets", + "structure_element" + ], + [ + 54, + 75, + "cup-shaped \u03b2-sandwich", + "structure_element" + ], + [ + 115, + 125, + "cupin-fold", + "structure_element" + ] + ] + }, + { + "sid": 126, + "sent": "Hence, (Sa)EctC adopts an overall bowl shape in which one side is opened towards the solvent (Fig 4a to 4c).", + "section": "RESULTS", + "ner": [ + [ + 8, + 10, + "Sa", + "species" + ], + [ + 11, + 15, + "EctC", + "protein" + ] + ] + }, + { + "sid": 127, + "sent": "In the \u201csemi-closed\u201d structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (\u03b1-II) that closes the active site of the (Sa)EctC protein (Fig 4a).", + "section": "RESULTS", + "ner": [ + [ + 8, + 19, + "semi-closed", + "protein_state" + ], + [ + 21, + 30, + "structure", + "evidence" + ], + [ + 41, + 62, + "carboxy-terminal tail", + "structure_element" + ], + [ + 81, + 97, + "electron density", + "evidence" + ], + [ + 114, + 125, + "small helix", + "structure_element" + ], + [ + 127, + 131, + "\u03b1-II", + "structure_element" + ], + [ + 149, + 160, + "active site", + "site" + ], + [ + 169, + 171, + "Sa", + "species" + ], + [ + 172, + 176, + "EctC", + "protein" + ] + ] + }, + { + "sid": 128, + "sent": "The formation of this \u03b1-II helix induces a reorientation and shift of a long unstructured loop (as observed in the \u201copen\u201d structure) connecting \u03b24 and \u03b26, resulting in the formation of the stable \u03b2-strand \u03b25 as observed in the \u201csemi-closed\u201dstate of the (Sa)EctC protein (Fig 4a).", + "section": "RESULTS", + "ner": [ + [ + 22, + 32, + "\u03b1-II helix", + "structure_element" + ], + [ + 77, + 89, + "unstructured", + "protein_state" + ], + [ + 90, + 94, + "loop", + "structure_element" + ], + [ + 116, + 120, + "open", + "protein_state" + ], + [ + 122, + 131, + "structure", + "evidence" + ], + [ + 144, + 146, + "\u03b24", + "structure_element" + ], + [ + 151, + 153, + "\u03b26", + "structure_element" + ], + [ + 189, + 195, + "stable", + "protein_state" + ], + [ + 196, + 204, + "\u03b2-strand", + "structure_element" + ], + [ + 205, + 207, + "\u03b25", + "structure_element" + ], + [ + 228, + 239, + "semi-closed", + "protein_state" + ], + [ + 254, + 256, + "Sa", + "species" + ], + [ + 257, + 261, + "EctC", + "protein" + ] + ] + }, + { + "sid": 129, + "sent": "Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily.", + "section": "RESULTS", + "ner": [ + [ + 0, + 30, + "Structural comparison analyses", + "experimental_method" + ], + [ + 41, + 52, + "DALI server", + "experimental_method" + ], + [ + 68, + 70, + "Sa", + "species" + ], + [ + 71, + 75, + "EctC", + "protein" + ], + [ + 122, + 139, + "cupin superfamily", + "protein_type" + ] + ] + }, + { + "sid": 130, + "sent": "The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 \u00c5 over 104 C\u03b1-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 \u00c5 over 103 C\u03b1-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 \u00c5 over 102 C\u03b1-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 \u00c5 over 104 C\u03b1-atoms).", + "section": "RESULTS", + "ner": [ + [ + 57, + 96, + "Cupin 2 conserved barrel domain protein", + "protein" + ], + [ + 98, + 109, + "YP_751781.1", + "protein" + ], + [ + 116, + 140, + "Shewanella frigidimarina", + "species" + ], + [ + 175, + 182, + "Z-score", + "evidence" + ], + [ + 198, + 202, + "RMSD", + "evidence" + ], + [ + 332, + 341, + "structure", + "evidence" + ], + [ + 369, + 395, + "manganese-containing cupin", + "protein" + ], + [ + 397, + 403, + "TM1459", + "protein" + ], + [ + 410, + 429, + "Thermotoga maritima", + "species" + ], + [ + 464, + 471, + "Z-score", + "evidence" + ], + [ + 487, + 491, + "RMSD", + "evidence" + ], + [ + 524, + 531, + "cyclase", + "protein_type" + ], + [ + 532, + 536, + "RemF", + "protein" + ], + [ + 542, + 571, + "Streptomyces resistomycificus", + "species" + ], + [ + 605, + 612, + "Z-score", + "evidence" + ], + [ + 628, + 632, + "RMSD", + "evidence" + ], + [ + 669, + 692, + "auxin-binding protein 1", + "protein" + ], + [ + 698, + 706, + "Zea mays", + "species" + ], + [ + 742, + 749, + "Z-score", + "evidence" + ], + [ + 765, + 769, + "RMSD", + "evidence" + ] + ] + }, + { + "sid": 131, + "sent": "Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction.", + "section": "RESULTS", + "ner": [ + [ + 18, + 22, + "EctC", + "protein" + ], + [ + 43, + 61, + "polyketide cyclase", + "protein_type" + ], + [ + 62, + 66, + "RemF", + "protein" + ], + [ + 88, + 101, + "cupin-related", + "protein_type" + ] + ] + }, + { + "sid": 132, + "sent": "Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "RemF", + "protein" + ], + [ + 21, + 46, + "aldos-2-ulose dehydratase", + "protein_type" + ], + [ + 47, + 56, + "isomerase", + "protein_type" + ], + [ + 62, + 78, + "ectoine synthase", + "protein_type" + ], + [ + 111, + 122, + "dehydratase", + "protein_type" + ], + [ + 134, + 151, + "cupin superfamily", + "protein_type" + ] + ] + }, + { + "sid": 133, + "sent": "Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure", + "section": "RESULTS", + "ner": [ + [ + 16, + 20, + "EctC", + "protein" + ], + [ + 21, + 36, + "dimer interface", + "site" + ], + [ + 57, + 59, + "Sa", + "species" + ], + [ + 60, + 64, + "EctC", + "protein" + ], + [ + 65, + 82, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 134, + "sent": "Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution.", + "section": "RESULTS", + "ner": [ + [ + 9, + 12, + "SEC", + "experimental_method" + ], + [ + 30, + 39, + "HPLC-MALS", + "experimental_method" + ], + [ + 82, + 98, + "ectoine synthase", + "protein_type" + ], + [ + 104, + 117, + "S. alaskensis", + "species" + ], + [ + 123, + 128, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 135, + "sent": "The crystal structure of this protein reflects this quaternary arrangement.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 136, + "sent": "In the \u201csemi-closed\u201d crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.", + "section": "RESULTS", + "ner": [ + [ + 8, + 19, + "semi-closed", + "protein_state" + ], + [ + 21, + 38, + "crystal structure", + "evidence" + ], + [ + 41, + 43, + "Sa", + "species" + ], + [ + 44, + 48, + "EctC", + "protein" + ], + [ + 53, + 65, + "crystallized", + "experimental_method" + ], + [ + 71, + 76, + "dimer", + "oligomeric_state" + ], + [ + 80, + 86, + "dimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 137, + "sent": "This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel \u03b2-strands, \u03b2-strand \u03b21 (sequence 1MIVRN5) from monomer A and \u03b2-strand \u03b28 from monomer B (sequence 82GVMYAL87) (Fig 5c).", + "section": "RESULTS", + "ner": [ + [ + 5, + 10, + "dimer", + "oligomeric_state" + ], + [ + 46, + 54, + "monomers", + "oligomeric_state" + ], + [ + 69, + 81, + "head-to-tail", + "protein_state" + ], + [ + 152, + 174, + "antiparallel \u03b2-strands", + "structure_element" + ], + [ + 176, + 184, + "\u03b2-strand", + "structure_element" + ], + [ + 185, + 187, + "\u03b21", + "structure_element" + ], + [ + 198, + 205, + "1MIVRN5", + "structure_element" + ], + [ + 212, + 219, + "monomer", + "oligomeric_state" + ], + [ + 220, + 221, + "A", + "structure_element" + ], + [ + 226, + 234, + "\u03b2-strand", + "structure_element" + ], + [ + 235, + 237, + "\u03b28", + "structure_element" + ], + [ + 243, + 250, + "monomer", + "oligomeric_state" + ], + [ + 251, + 252, + "B", + "structure_element" + ], + [ + 263, + 273, + "82GVMYAL87", + "structure_element" + ] + ] + }, + { + "sid": 138, + "sent": "The strong interactions between these \u03b2-strands rely primarily on backbone contacts.", + "section": "RESULTS", + "ner": [ + [ + 38, + 47, + "\u03b2-strands", + "structure_element" + ] + ] + }, + { + "sid": 139, + "sent": "In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the \u03b2-strands.", + "section": "RESULTS", + "ner": [ + [ + 47, + 71, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 106, + 114, + "monomers", + "oligomeric_state" + ], + [ + 123, + 128, + "loops", + "structure_element" + ], + [ + 144, + 153, + "\u03b2-strands", + "structure_element" + ] + ] + }, + { + "sid": 140, + "sent": "As calculated with PDBePISA, the surface area buried upon dimer formation is 1462 \u00c52, which is 20.5% of the total accessible surface of a monomer of this protein.", + "section": "RESULTS", + "ner": [ + [ + 19, + 27, + "PDBePISA", + "experimental_method" + ], + [ + 58, + 63, + "dimer", + "oligomeric_state" + ], + [ + 138, + 145, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 141, + "sent": "Both values fall within the range for known functional dimers.", + "section": "RESULTS", + "ner": [ + [ + 55, + 61, + "dimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 142, + "sent": "Crystal structure of (Sa)EctC.", + "section": "FIG", + "ner": [ + [ + 0, + 17, + "Crystal structure", + "evidence" + ], + [ + 22, + 24, + "Sa", + "species" + ], + [ + 25, + 29, + "EctC", + "protein" + ] + ] + }, + { + "sid": 143, + "sent": "(a) Top-view of the dimer of the (Sa)EctC protein.", + "section": "FIG", + "ner": [ + [ + 20, + 25, + "dimer", + "oligomeric_state" + ], + [ + 34, + 36, + "Sa", + "species" + ], + [ + 37, + 41, + "EctC", + "protein" + ] + ] + }, + { + "sid": 144, + "sent": "The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two \u03b2-strands of each monomer.", + "section": "FIG", + "ner": [ + [ + 20, + 25, + "water", + "chemical" + ], + [ + 92, + 100, + "monomers", + "oligomeric_state" + ], + [ + 142, + 144, + "Sa", + "species" + ], + [ + 145, + 149, + "EctC", + "protein" + ], + [ + 150, + 155, + "dimer", + "oligomeric_state" + ], + [ + 186, + 201, + "dimer interface", + "site" + ], + [ + 216, + 225, + "\u03b2-strands", + "structure_element" + ], + [ + 234, + 241, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 145, + "sent": "(c) Close-up representation of the dimer interface mediated by beta-strand \u03b21 and \u03b26.", + "section": "FIG", + "ner": [ + [ + 35, + 50, + "dimer interface", + "site" + ], + [ + 63, + 74, + "beta-strand", + "structure_element" + ], + [ + 75, + 77, + "\u03b21", + "structure_element" + ], + [ + 82, + 84, + "\u03b26", + "structure_element" + ] + ] + }, + { + "sid": 146, + "sent": "In the \u201copen\u201d (Sa)EctC structure, one monomer is present in the asymmetric unit.", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "open", + "protein_state" + ], + [ + 15, + 17, + "Sa", + "species" + ], + [ + 18, + 22, + "EctC", + "protein" + ], + [ + 23, + 32, + "structure", + "evidence" + ], + [ + 38, + 45, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 147, + "sent": "We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well.", + "section": "RESULTS", + "ner": [ + [ + 35, + 42, + "packing", + "experimental_method" + ], + [ + 60, + 67, + "monomer", + "oligomeric_state" + ], + [ + 68, + 75, + "monomer", + "oligomeric_state" + ], + [ + 169, + 174, + "dimer", + "oligomeric_state" + ], + [ + 202, + 214, + "crystal form", + "evidence" + ] + ] + }, + { + "sid": 148, + "sent": "Indeed, a similar dimer configuration to the one described for the \u201csemi-closed\u201d (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two \u03b2-sheets.", + "section": "RESULTS", + "ner": [ + [ + 18, + 23, + "dimer", + "oligomeric_state" + ], + [ + 68, + 79, + "semi-closed", + "protein_state" + ], + [ + 82, + 84, + "Sa", + "species" + ], + [ + 85, + 89, + "EctC", + "protein" + ], + [ + 90, + 99, + "structure", + "evidence" + ], + [ + 126, + 133, + "monomer", + "oligomeric_state" + ], + [ + 134, + 141, + "monomer", + "oligomeric_state" + ], + [ + 175, + 183, + "\u03b2-sheets", + "structure_element" + ] + ] + }, + { + "sid": 149, + "sent": "The crystallographic two-fold axis present within the crystal symmetry is located exactly in between the two monomers, resulting in a monomer within the asymmetric unit.", + "section": "RESULTS", + "ner": [ + [ + 109, + 117, + "monomers", + "oligomeric_state" + ], + [ + 134, + 141, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 150, + "sent": "Hence, the same dimer observed in the \u201csemi-closed\u201d structure of (Sa)EctC can also be observed in the \u201copen\u201d structure.", + "section": "RESULTS", + "ner": [ + [ + 16, + 21, + "dimer", + "oligomeric_state" + ], + [ + 39, + 50, + "semi-closed", + "protein_state" + ], + [ + 52, + 61, + "structure", + "evidence" + ], + [ + 66, + 68, + "Sa", + "species" + ], + [ + 69, + 73, + "EctC", + "protein" + ], + [ + 103, + 107, + "open", + "protein_state" + ], + [ + 109, + 118, + "structure", + "evidence" + ] + ] + }, + { + "sid": 151, + "sent": "Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).", + "section": "RESULTS", + "ner": [ + [ + 62, + 73, + "DALI search", + "experimental_method" + ], + [ + 105, + 109, + "EctC", + "protein" + ], + [ + 135, + 141, + "dimers", + "oligomeric_state" + ], + [ + 161, + 168, + "monomer", + "oligomeric_state" + ], + [ + 169, + 176, + "monomer", + "oligomeric_state" + ], + [ + 201, + 206, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 152, + "sent": "Structural rearrangements of the flexible (Sa)EctC carboxy-terminus", + "section": "RESULTS", + "ner": [ + [ + 33, + 41, + "flexible", + "protein_state" + ], + [ + 43, + 45, + "Sa", + "species" + ], + [ + 46, + 50, + "EctC", + "protein" + ], + [ + 51, + 67, + "carboxy-terminus", + "structure_element" + ] + ] + }, + { + "sid": 153, + "sent": "The cupin core represents the structural framework of ectoine synthase (Figs 4 and 5).", + "section": "RESULTS", + "ner": [ + [ + 54, + 70, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 154, + "sent": "The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus.", + "section": "RESULTS", + "ner": [ + [ + 32, + 50, + "crystal structures", + "evidence" + ], + [ + 59, + 61, + "Sa", + "species" + ], + [ + 62, + 66, + "EctC", + "protein" + ], + [ + 115, + 131, + "carboxy-terminus", + "structure_element" + ] + ] + }, + { + "sid": 155, + "sent": "Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.", + "section": "RESULTS", + "ner": [ + [ + 32, + 55, + "carboxy-terminal region", + "structure_element" + ], + [ + 63, + 78, + "137 amino acids", + "residue_range" + ], + [ + 91, + 93, + "Sa", + "species" + ], + [ + 94, + 98, + "EctC", + "protein" + ], + [ + 111, + 127, + "highly conserved", + "protein_state" + ], + [ + 147, + 155, + "extended", + "protein_state" + ], + [ + 156, + 168, + "EctC protein", + "protein_type" + ] + ] + }, + { + "sid": 156, + "sent": "At the end of \u03b2-strand \u03b211, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.", + "section": "RESULTS", + "ner": [ + [ + 14, + 22, + "\u03b2-strand", + "structure_element" + ], + [ + 23, + 26, + "\u03b211", + "structure_element" + ], + [ + 44, + 53, + "conserved", + "protein_state" + ], + [ + 54, + 61, + "proline", + "residue_name" + ], + [ + 72, + 79, + "Pro-109", + "residue_name_number" + ], + [ + 84, + 91, + "Pro-110", + "residue_name_number" + ], + [ + 163, + 165, + "Sa", + "species" + ], + [ + 166, + 170, + "EctC", + "protein" + ] + ] + }, + { + "sid": 157, + "sent": "In the \u201csemi-closed\u201d (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.", + "section": "RESULTS", + "ner": [ + [ + 8, + 19, + "semi-closed", + "protein_state" + ], + [ + 22, + 24, + "Sa", + "species" + ], + [ + 25, + 29, + "EctC", + "protein" + ], + [ + 30, + 39, + "structure", + "evidence" + ], + [ + 53, + 69, + "electron density", + "evidence" + ], + [ + 77, + 93, + "carboxy-terminus", + "structure_element" + ], + [ + 109, + 130, + "7 amino acid residues", + "residue_range" + ], + [ + 152, + 159, + "Gly-121", + "residue_name_number" + ] + ] + }, + { + "sid": 158, + "sent": "These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).", + "section": "RESULTS", + "ner": [ + [ + 41, + 52, + "small helix", + "structure_element" + ], + [ + 70, + 74, + "open", + "protein_state" + ], + [ + 75, + 81, + "cavity", + "site" + ], + [ + 89, + 99, + "cupin-fold", + "structure_element" + ], + [ + 108, + 110, + "Sa", + "species" + ], + [ + 111, + 115, + "EctC", + "protein" + ] + ] + }, + { + "sid": 159, + "sent": "Furthermore, this helix is stabilized via interactions with the loop region between \u03b2-strands \u03b24 and \u03b26, thereby inducing a structural rearrangement.", + "section": "RESULTS", + "ner": [ + [ + 18, + 23, + "helix", + "structure_element" + ], + [ + 64, + 75, + "loop region", + "structure_element" + ], + [ + 84, + 93, + "\u03b2-strands", + "structure_element" + ], + [ + 94, + 96, + "\u03b24", + "structure_element" + ], + [ + 101, + 103, + "\u03b26", + "structure_element" + ] + ] + }, + { + "sid": 160, + "sent": "This induces the formation of \u03b2-strand \u03b25, which is not present when the small C-terminal helix is absent as observed in the \u201copen\u201d (Sa)EctC structure.", + "section": "RESULTS", + "ner": [ + [ + 30, + 38, + "\u03b2-strand", + "structure_element" + ], + [ + 39, + 41, + "\u03b25", + "structure_element" + ], + [ + 73, + 95, + "small C-terminal helix", + "structure_element" + ], + [ + 99, + 105, + "absent", + "protein_state" + ], + [ + 126, + 130, + "open", + "protein_state" + ], + [ + 133, + 135, + "Sa", + "species" + ], + [ + 136, + 140, + "EctC", + "protein" + ], + [ + 141, + 150, + "structure", + "evidence" + ] + ] + }, + { + "sid": 161, + "sent": "As a result, the newly formed \u03b2-strand \u03b25 is reoriented and moved by 2.4 \u00c5 within the \u201csemi-closed\u201d (Sa)EctC structure (Fig 4a to 4c).", + "section": "RESULTS", + "ner": [ + [ + 30, + 38, + "\u03b2-strand", + "structure_element" + ], + [ + 39, + 41, + "\u03b25", + "structure_element" + ], + [ + 87, + 98, + "semi-closed", + "protein_state" + ], + [ + 101, + 103, + "Sa", + "species" + ], + [ + 104, + 108, + "EctC", + "protein" + ], + [ + 109, + 118, + "structure", + "evidence" + ] + ] + }, + { + "sid": 162, + "sent": "It is worth mentioning that \u03b2-strand \u03b25 is located next to His-93, which in all likelihood involved in metal binding (see below).", + "section": "RESULTS", + "ner": [ + [ + 28, + 36, + "\u03b2-strand", + "structure_element" + ], + [ + 37, + 39, + "\u03b25", + "structure_element" + ], + [ + 59, + 65, + "His-93", + "residue_name_number" + ], + [ + 103, + 108, + "metal", + "chemical" + ] + ] + }, + { + "sid": 163, + "sent": "The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of \u03b2-strand \u03b25.", + "section": "RESULTS", + "ner": [ + [ + 21, + 24, + "His", + "residue_name" + ], + [ + 62, + 64, + "Sa", + "species" + ], + [ + 65, + 69, + "EctC", + "protein" + ], + [ + 70, + 80, + "structures", + "evidence" + ], + [ + 120, + 128, + "\u03b2-strand", + "structure_element" + ], + [ + 129, + 131, + "\u03b25", + "structure_element" + ] + ] + }, + { + "sid": 164, + "sent": "Therefore the sealing of the cupin fold, as described above, seem to have an indirect influence on the architecture of the postulated iron-binding site.", + "section": "RESULTS", + "ner": [ + [ + 29, + 39, + "cupin fold", + "structure_element" + ], + [ + 134, + 151, + "iron-binding site", + "site" + ] + ] + }, + { + "sid": 165, + "sent": "The consecutive Pro-109 and Pro-110 residues found at the end of \u03b2-strand \u03b211are highly conserved in EctC-type proteins (Fig 2).", + "section": "RESULTS", + "ner": [ + [ + 16, + 23, + "Pro-109", + "residue_name_number" + ], + [ + 28, + 35, + "Pro-110", + "residue_name_number" + ], + [ + 65, + 73, + "\u03b2-strand", + "structure_element" + ], + [ + 74, + 77, + "\u03b211", + "structure_element" + ], + [ + 81, + 97, + "highly conserved", + "protein_state" + ], + [ + 101, + 119, + "EctC-type proteins", + "protein_type" + ] + ] + }, + { + "sid": 166, + "sent": "They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold.", + "section": "RESULTS", + "ner": [ + [ + 69, + 85, + "carboxy-terminus", + "structure_element" + ], + [ + 87, + 109, + "27 amino acid residues", + "residue_range" + ], + [ + 115, + 117, + "Sa", + "species" + ], + [ + 118, + 122, + "EctC", + "protein" + ], + [ + 136, + 146, + "cupin fold", + "structure_element" + ] + ] + }, + { + "sid": 167, + "sent": "In the \u201csemi-closed\u201d structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c).", + "section": "RESULTS", + "ner": [ + [ + 8, + 19, + "semi-closed", + "protein_state" + ], + [ + 21, + 30, + "structure", + "evidence" + ], + [ + 86, + 98, + "cupin barrel", + "structure_element" + ] + ] + }, + { + "sid": 168, + "sent": "In the \u201copen\u201d (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "open", + "protein_state" + ], + [ + 15, + 17, + "Sa", + "species" + ], + [ + 18, + 22, + "EctC", + "protein" + ], + [ + 23, + 32, + "structure", + "evidence" + ], + [ + 39, + 46, + "proline", + "residue_name" + ], + [ + 75, + 91, + "electron density", + "evidence" + ], + [ + 124, + 131, + "Pro-110", + "residue_name_number" + ], + [ + 137, + 153, + "electron density", + "evidence" + ], + [ + 213, + 229, + "carboxy-terminus", + "structure_element" + ] + ] + }, + { + "sid": 169, + "sent": "A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the \u201copen\u201d and \u201csemi-closed\u201d (Sa)EctC structures.", + "section": "RESULTS", + "ner": [ + [ + 39, + 46, + "Pro-109", + "residue_name_number" + ], + [ + 121, + 127, + "His-55", + "residue_name_number" + ], + [ + 152, + 156, + "open", + "protein_state" + ], + [ + 163, + 174, + "semi-closed", + "protein_state" + ], + [ + 177, + 179, + "Sa", + "species" + ], + [ + 180, + 184, + "EctC", + "protein" + ], + [ + 185, + 195, + "structures", + "evidence" + ] + ] + }, + { + "sid": 170, + "sent": "The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of \u03b2-strand \u03b211.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "Pro-109", + "residue_name_number" + ], + [ + 12, + 18, + "His-55", + "residue_name_number" + ], + [ + 43, + 49, + "stable", + "protein_state" + ], + [ + 70, + 77, + "proline", + "residue_name" + ], + [ + 101, + 109, + "\u03b2-strand", + "structure_element" + ], + [ + 110, + 113, + "\u03b211", + "structure_element" + ] + ] + }, + { + "sid": 171, + "sent": "Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.", + "section": "RESULTS", + "ner": [ + [ + 12, + 19, + "proline", + "residue_name" + ], + [ + 49, + 72, + "carboxy-terminal region", + "structure_element" + ], + [ + 81, + 83, + "Sa", + "species" + ], + [ + 84, + 88, + "EctC", + "protein" + ], + [ + 117, + 123, + "His-55", + "residue_name_number" + ], + [ + 129, + 136, + "Pro-109", + "residue_name_number" + ] + ] + }, + { + "sid": 172, + "sent": "In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.", + "section": "RESULTS", + "ner": [ + [ + 40, + 47, + "Pro-109", + "residue_name_number" + ], + [ + 52, + 58, + "His-55", + "residue_name_number" + ], + [ + 64, + 87, + "carboxy-terminal region", + "structure_element" + ], + [ + 92, + 94, + "Sa", + "species" + ], + [ + 95, + 99, + "EctC", + "protein" + ], + [ + 142, + 149, + "Glu-115", + "residue_name_number" + ], + [ + 155, + 161, + "His-55", + "residue_name_number" + ], + [ + 204, + 215, + "small helix", + "structure_element" + ], + [ + 223, + 239, + "carboxy-terminus", + "structure_element" + ] + ] + }, + { + "sid": 173, + "sent": "The interaction between Glu-115 and His-55 is only visible in the \u201csemi-closed\u201d structure where the partially extended carboxy-terminus is resolved in the electron density.", + "section": "RESULTS", + "ner": [ + [ + 24, + 31, + "Glu-115", + "residue_name_number" + ], + [ + 36, + 42, + "His-55", + "residue_name_number" + ], + [ + 67, + 78, + "semi-closed", + "protein_state" + ], + [ + 80, + 89, + "structure", + "evidence" + ], + [ + 100, + 118, + "partially extended", + "protein_state" + ], + [ + 119, + 135, + "carboxy-terminus", + "structure_element" + ], + [ + 155, + 171, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 174, + "sent": "In the \u201copen\u201d structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b).", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "open", + "protein_state" + ], + [ + 14, + 23, + "structure", + "evidence" + ], + [ + 32, + 34, + "Sa", + "species" + ], + [ + 35, + 39, + "EctC", + "protein" + ], + [ + 87, + 94, + "Glu-115", + "residue_name_number" + ] + ] + }, + { + "sid": 175, + "sent": "Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the \u201copen\u201d (Sa)EctC structure and consequently results in less well defined electron density in this region.", + "section": "RESULTS", + "ner": [ + [ + 105, + 121, + "carboxy-terminus", + "structure_element" + ], + [ + 130, + 134, + "open", + "protein_state" + ], + [ + 137, + 139, + "Sa", + "species" + ], + [ + 140, + 144, + "EctC", + "protein" + ], + [ + 145, + 154, + "structure", + "evidence" + ], + [ + 201, + 217, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 176, + "sent": "Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus.", + "section": "FIG", + "ner": [ + [ + 29, + 47, + "metal-binding site", + "site" + ], + [ + 56, + 58, + "Sa", + "species" + ], + [ + 59, + 63, + "EctC", + "protein" + ], + [ + 80, + 88, + "flexible", + "protein_state" + ], + [ + 89, + 105, + "carboxy-terminus", + "structure_element" + ] + ] + }, + { + "sid": 177, + "sent": "(a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93.", + "section": "FIG", + "ner": [ + [ + 18, + 23, + "water", + "chemical" + ], + [ + 111, + 117, + "Glu-57", + "residue_name_number" + ], + [ + 119, + 125, + "Tyr-85", + "residue_name_number" + ], + [ + 131, + 137, + "His-93", + "residue_name_number" + ] + ] + }, + { + "sid": 178, + "sent": "The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase.", + "section": "FIG", + "ner": [ + [ + 30, + 35, + "water", + "chemical" + ], + [ + 85, + 89, + "Fe2+", + "chemical" + ], + [ + 106, + 117, + "active side", + "site" + ], + [ + 125, + 141, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 179, + "sent": "His-55 interacts with the double proline motif (Pro-109 and Pro-110).", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "His-55", + "residue_name_number" + ], + [ + 26, + 46, + "double proline motif", + "structure_element" + ], + [ + 48, + 55, + "Pro-109", + "residue_name_number" + ], + [ + 60, + 67, + "Pro-110", + "residue_name_number" + ] + ] + }, + { + "sid": 180, + "sent": "It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the \u201csemi-closed\u201d (Sa)EctC structure.", + "section": "FIG", + "ner": [ + [ + 67, + 74, + "Glu-115", + "residue_name_number" + ], + [ + 101, + 109, + "flexible", + "protein_state" + ], + [ + 110, + 126, + "carboxy-terminus", + "structure_element" + ], + [ + 151, + 153, + "Sa", + "species" + ], + [ + 154, + 158, + "EctC", + "protein" + ], + [ + 183, + 194, + "semi-closed", + "protein_state" + ], + [ + 197, + 199, + "Sa", + "species" + ], + [ + 200, + 204, + "EctC", + "protein" + ], + [ + 205, + 214, + "structure", + "evidence" + ] + ] + }, + { + "sid": 181, + "sent": "(b) An overlay of the \u201copen\u201d (colored in light blue) and the \u201csemi-closed\u201d (colored in green) structure of the (Sa)EctC protein.", + "section": "FIG", + "ner": [ + [ + 7, + 14, + "overlay", + "experimental_method" + ], + [ + 23, + 27, + "open", + "protein_state" + ], + [ + 62, + 73, + "semi-closed", + "protein_state" + ], + [ + 94, + 103, + "structure", + "evidence" + ], + [ + 112, + 114, + "Sa", + "species" + ], + [ + 115, + 119, + "EctC", + "protein" + ] + ] + }, + { + "sid": 182, + "sent": "The putative iron binding site of (Sa)EctC", + "section": "RESULTS", + "ner": [ + [ + 13, + 30, + "iron binding site", + "site" + ], + [ + 35, + 37, + "Sa", + "species" + ], + [ + 38, + 42, + "EctC", + "protein" + ] + ] + }, + { + "sid": 183, + "sent": "In the \u201csemi-closed\u201d structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.", + "section": "RESULTS", + "ner": [ + [ + 8, + 19, + "semi-closed", + "protein_state" + ], + [ + 21, + 30, + "structure", + "evidence" + ], + [ + 35, + 37, + "Sa", + "species" + ], + [ + 38, + 42, + "EctC", + "protein" + ], + [ + 61, + 69, + "monomers", + "oligomeric_state" + ], + [ + 120, + 136, + "electron density", + "evidence" + ], + [ + 159, + 171, + "cupin barrel", + "structure_element" + ] + ] + }, + { + "sid": 184, + "sent": "Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.", + "section": "RESULTS", + "ner": [ + [ + 7, + 9, + "Sa", + "species" + ], + [ + 10, + 14, + "EctC", + "protein" + ], + [ + 20, + 25, + "metal", + "chemical" + ], + [ + 77, + 81, + "Fe2+", + "chemical" + ], + [ + 86, + 90, + "Zn2+", + "chemical" + ], + [ + 106, + 113, + "density", + "evidence" + ], + [ + 123, + 140, + "refined occupancy", + "experimental_method" + ] + ] + }, + { + "sid": 185, + "sent": "Only the refinement of Fe2+ resulted in a visibly improved electron density, however with a low degree of occupancy.", + "section": "RESULTS", + "ner": [ + [ + 23, + 27, + "Fe2+", + "chemical" + ], + [ + 59, + 75, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 186, + "sent": "This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b).", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "iron", + "chemical" + ], + [ + 59, + 65, + "Glu-57", + "residue_name_number" + ], + [ + 67, + 73, + "Tyr-85", + "residue_name_number" + ], + [ + 78, + 84, + "His-93", + "residue_name_number" + ] + ] + }, + { + "sid": 187, + "sent": "The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 \u00c5 for Glu-57, 2.9 \u00c5 for Tyr-85, and 2.9 \u00c5 for His-93, respectively.", + "section": "RESULTS", + "ner": [ + [ + 74, + 78, + "iron", + "chemical" + ], + [ + 102, + 108, + "Glu-57", + "residue_name_number" + ], + [ + 120, + 126, + "Tyr-85", + "residue_name_number" + ], + [ + 142, + 148, + "His-93", + "residue_name_number" + ] + ] + }, + { + "sid": 188, + "sent": "These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure.", + "section": "RESULTS", + "ner": [ + [ + 51, + 69, + "iron binding sites", + "site" + ], + [ + 106, + 116, + "absence of", + "protein_state" + ], + [ + 146, + 148, + "Sa", + "species" + ], + [ + 149, + 153, + "EctC", + "protein" + ], + [ + 154, + 171, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 189, + "sent": "Since both the refinement and the distance did not clearly identify an iron molecule, we decided to conservatively place a water molecule at this position.", + "section": "RESULTS", + "ner": [ + [ + 71, + 75, + "iron", + "chemical" + ], + [ + 123, + 128, + "water", + "chemical" + ] + ] + }, + { + "sid": 190, + "sent": "The position of this water molecule is described in more detail below and is highlighted in Figs 5a and 5b and 6a and 6b as a sphere.", + "section": "RESULTS", + "ner": [ + [ + 21, + 26, + "water", + "chemical" + ] + ] + }, + { + "sid": 191, + "sent": "Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2).", + "section": "RESULTS", + "ner": [ + [ + 55, + 60, + "water", + "chemical" + ], + [ + 74, + 92, + "strictly conserved", + "protein_state" + ], + [ + 103, + 112, + "alignment", + "experimental_method" + ], + [ + 135, + 147, + "EctC protein", + "protein_type" + ], + [ + 188, + 206, + "EctC-type proteins", + "protein_type" + ] + ] + }, + { + "sid": 192, + "sent": "In the \u201copen\u201d structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the \u201csemi-closed\u201d structure.", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "open", + "protein_state" + ], + [ + 14, + 23, + "structure", + "evidence" + ], + [ + 32, + 34, + "Sa", + "species" + ], + [ + 35, + 39, + "EctC", + "protein" + ], + [ + 49, + 65, + "electron density", + "evidence" + ], + [ + 99, + 103, + "iron", + "chemical" + ], + [ + 126, + 137, + "semi-closed", + "protein_state" + ], + [ + 139, + 148, + "structure", + "evidence" + ] + ] + }, + { + "sid": 193, + "sent": "However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 \u00c5) of the \u201copen\u201d (Sa)EctC structure.", + "section": "RESULTS", + "ner": [ + [ + 14, + 30, + "electron density", + "evidence" + ], + [ + 51, + 56, + "water", + "chemical" + ], + [ + 80, + 84, + "iron", + "chemical" + ], + [ + 94, + 99, + "water", + "chemical" + ], + [ + 190, + 194, + "open", + "protein_state" + ], + [ + 197, + 199, + "Sa", + "species" + ], + [ + 200, + 204, + "EctC", + "protein" + ], + [ + 205, + 214, + "structure", + "evidence" + ] + ] + }, + { + "sid": 194, + "sent": "In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the \u201csemi-closed\u201d structure match nicely with those of the corresponding residues of the \u201ciron-free\u201d \u201copen\u201d structure (Fig 6b).", + "section": "RESULTS", + "ner": [ + [ + 5, + 20, + "superimposition", + "experimental_method" + ], + [ + 30, + 32, + "Sa", + "species" + ], + [ + 33, + 37, + "EctC", + "protein" + ], + [ + 38, + 56, + "crystal structures", + "evidence" + ], + [ + 128, + 134, + "Glu-57", + "residue_name_number" + ], + [ + 136, + 142, + "Tyr-85", + "residue_name_number" + ], + [ + 148, + 154, + "His-93", + "residue_name_number" + ], + [ + 178, + 182, + "iron", + "chemical" + ], + [ + 191, + 202, + "semi-closed", + "protein_state" + ], + [ + 204, + 213, + "structure", + "evidence" + ], + [ + 276, + 285, + "iron-free", + "protein_state" + ], + [ + 288, + 292, + "open", + "protein_state" + ], + [ + 294, + 303, + "structure", + "evidence" + ] + ] + }, + { + "sid": 195, + "sent": "Only His-93 is slightly rotated inwards in the \u201csemi-closed\u201d structure, most likely due to formation of \u03b2-strand \u03b25 as described above.", + "section": "RESULTS", + "ner": [ + [ + 5, + 11, + "His-93", + "residue_name_number" + ], + [ + 48, + 59, + "semi-closed", + "protein_state" + ], + [ + 61, + 70, + "structure", + "evidence" + ], + [ + 104, + 112, + "\u03b2-strand", + "structure_element" + ], + [ + 113, + 115, + "\u03b25", + "structure_element" + ] + ] + }, + { + "sid": 196, + "sent": "Taken together, this observations indicate, that the architecture of the presumptive iron-binding site is pre-set for the binding of the catalytically important metal by the ectoine synthase.", + "section": "RESULTS", + "ner": [ + [ + 85, + 102, + "iron-binding site", + "site" + ], + [ + 161, + 166, + "metal", + "chemical" + ], + [ + 174, + 190, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 197, + "sent": "Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of \u03b2-strand \u03b25) in the \u201copen\u201d and \u201csemi-closed\u201d (Sa)EctC structures.", + "section": "RESULTS", + "ner": [ + [ + 66, + 72, + "Tyr-52", + "residue_name_number" + ], + [ + 87, + 91, + "loop", + "structure_element" + ], + [ + 109, + 117, + "\u03b2-strand", + "structure_element" + ], + [ + 118, + 120, + "\u03b25", + "structure_element" + ], + [ + 130, + 134, + "open", + "protein_state" + ], + [ + 141, + 152, + "semi-closed", + "protein_state" + ], + [ + 155, + 157, + "Sa", + "species" + ], + [ + 158, + 162, + "EctC", + "protein" + ], + [ + 163, + 173, + "structures", + "evidence" + ] + ] + }, + { + "sid": 198, + "sent": "In the \u201csemi-closed\u201d structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 \u00c5) makes it highly unlikely that Tyr-52 is directly involved in metal binding.", + "section": "RESULTS", + "ner": [ + [ + 8, + 19, + "semi-closed", + "protein_state" + ], + [ + 21, + 30, + "structure", + "evidence" + ], + [ + 72, + 78, + "Tyr-52", + "residue_name_number" + ], + [ + 98, + 102, + "iron", + "chemical" + ], + [ + 189, + 195, + "Tyr-52", + "residue_name_number" + ], + [ + 220, + 225, + "metal", + "chemical" + ] + ] + }, + { + "sid": 199, + "sent": "Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 18, + 30, + "substitution", + "experimental_method" + ], + [ + 37, + 40, + "Ala", + "residue_name" + ], + [ + 77, + 81, + "iron", + "chemical" + ], + [ + 117, + 123, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 200, + "sent": "It becomes apparent from an overlay of the \u201copen\u201d and \u201csemi-closed\u201d (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).", + "section": "RESULTS", + "ner": [ + [ + 28, + 35, + "overlay", + "experimental_method" + ], + [ + 44, + 48, + "open", + "protein_state" + ], + [ + 55, + 66, + "semi-closed", + "protein_state" + ], + [ + 69, + 71, + "Sa", + "species" + ], + [ + 72, + 76, + "EctC", + "protein" + ], + [ + 77, + 95, + "crystal structures", + "evidence" + ], + [ + 119, + 125, + "Tyr-52", + "residue_name_number" + ], + [ + 176, + 180, + "iron", + "chemical" + ], + [ + 253, + 258, + "metal", + "chemical" + ], + [ + 269, + 275, + "Glu-57", + "residue_name_number" + ], + [ + 277, + 283, + "Tyr-85", + "residue_name_number" + ], + [ + 289, + 295, + "His-93", + "residue_name_number" + ] + ] + }, + { + "sid": 201, + "sent": "Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-\u03b3-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.", + "section": "RESULTS", + "ner": [ + [ + 6, + 12, + "Tyr-52", + "residue_name_number" + ], + [ + 16, + 34, + "strictly conserved", + "protein_state" + ], + [ + 41, + 50, + "alignment", + "experimental_method" + ], + [ + 58, + 76, + "EctC-type proteins", + "protein_type" + ], + [ + 160, + 176, + "ectoine synthase", + "protein_type" + ], + [ + 190, + 200, + "absence of", + "protein_state" + ], + [ + 201, + 210, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 219, + 221, + "Sa", + "species" + ], + [ + 222, + 226, + "EctC", + "protein" + ], + [ + 227, + 245, + "crystal structures", + "evidence" + ], + [ + 276, + 282, + "Tyr-52", + "residue_name_number" + ] + ] + }, + { + "sid": 202, + "sent": "To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity.", + "section": "RESULTS", + "ner": [ + [ + 32, + 49, + "iron binding site", + "site" + ], + [ + 73, + 115, + "structure-guided site-directed mutagenesis", + "experimental_method" + ], + [ + 144, + 146, + "Sa", + "species" + ], + [ + 147, + 151, + "EctC", + "protein" + ], + [ + 171, + 175, + "iron", + "chemical" + ] + ] + }, + { + "sid": 203, + "sent": "When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 27, + 33, + "Glu-57", + "residue_name_number" + ], + [ + 35, + 41, + "Tyr-85", + "residue_name_number" + ], + [ + 43, + 49, + "His-93", + "residue_name_number" + ], + [ + 72, + 96, + "mono-nuclear iron center", + "site" + ], + [ + 105, + 107, + "Sa", + "species" + ], + [ + 108, + 112, + "EctC", + "protein" + ], + [ + 113, + 130, + "crystal structure", + "evidence" + ], + [ + 149, + 157, + "replaced", + "experimental_method" + ], + [ + 164, + 167, + "Ala", + "residue_name" + ], + [ + 213, + 217, + "iron", + "chemical" + ], + [ + 233, + 239, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 204, + "sent": "For some of the presumptive iron-coordinating residues, additional site-directed mutagenesis experiments were carried out.", + "section": "RESULTS", + "ner": [ + [ + 28, + 54, + "iron-coordinating residues", + "site" + ], + [ + 67, + 92, + "site-directed mutagenesis", + "experimental_method" + ] + ] + }, + { + "sid": 205, + "sent": "To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant.", + "section": "RESULTS", + "ner": [ + [ + 67, + 73, + "Glu-57", + "residue_name_number" + ], + [ + 89, + 92, + "Asp", + "residue_name" + ], + [ + 93, + 100, + "variant", + "protein_state" + ] + ] + }, + { + "sid": 206, + "sent": "This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 5, + 11, + "mutant", + "protein_state" + ], + [ + 52, + 56, + "iron", + "chemical" + ], + [ + 72, + 75, + "Ala", + "residue_name" + ], + [ + 76, + 88, + "substitution", + "experimental_method" + ] + ] + }, + { + "sid": 207, + "sent": "We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.", + "section": "RESULTS", + "ner": [ + [ + 8, + 16, + "replaced", + "experimental_method" + ], + [ + 17, + 23, + "Tyr-85", + "residue_name_number" + ], + [ + 38, + 41, + "Phe", + "residue_name" + ], + [ + 47, + 50, + "Trp", + "residue_name" + ], + [ + 68, + 74, + "mutant", + "protein_state" + ], + [ + 126, + 130, + "iron", + "chemical" + ] + ] + }, + { + "sid": 208, + "sent": "Collectively, these data suggest that the hydroxyl group of the Tyr-85 side chain is needed for the binding of the iron (Fig 6a).", + "section": "RESULTS", + "ner": [ + [ + 64, + 70, + "Tyr-85", + "residue_name_number" + ], + [ + 115, + 119, + "iron", + "chemical" + ] + ] + }, + { + "sid": 209, + "sent": "We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 8, + 16, + "replaced", + "experimental_method" + ], + [ + 33, + 53, + "iron-binding residue", + "site" + ], + [ + 54, + 60, + "His-93", + "residue_name_number" + ], + [ + 67, + 70, + "Asn", + "residue_name" + ], + [ + 92, + 94, + "Sa", + "species" + ], + [ + 95, + 99, + "EctC", + "protein" + ], + [ + 161, + 165, + "iron", + "chemical" + ], + [ + 210, + 219, + "wild-type", + "protein_state" + ] + ] + }, + { + "sid": 210, + "sent": "Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.", + "section": "RESULTS", + "ner": [ + [ + 68, + 94, + "iron-coordinating residues", + "site" + ], + [ + 96, + 102, + "Glu-57", + "residue_name_number" + ], + [ + 104, + 110, + "Tyr-85", + "residue_name_number" + ], + [ + 112, + 118, + "His-93", + "residue_name_number" + ], + [ + 149, + 153, + "Fe2+", + "chemical" + ], + [ + 170, + 172, + "Sa", + "species" + ], + [ + 173, + 177, + "EctC", + "protein" + ] + ] + }, + { + "sid": 211, + "sent": "A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-\u03b3-ADABA substrate", + "section": "RESULTS", + "ner": [ + [ + 38, + 40, + "Sa", + "species" + ], + [ + 41, + 45, + "EctC", + "protein" + ], + [ + 46, + 55, + "structure", + "evidence" + ], + [ + 94, + 103, + "N-\u03b3-ADABA", + "chemical" + ] + ] + }, + { + "sid": 212, + "sent": "Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-\u03b3-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).", + "section": "RESULTS", + "ner": [ + [ + 47, + 65, + "co-crystallization", + "experimental_method" + ], + [ + 69, + 88, + "soaking experiments", + "experimental_method" + ], + [ + 120, + 122, + "Sa", + "species" + ], + [ + 123, + 127, + "EctC", + "protein" + ], + [ + 128, + 146, + "crystal structures", + "evidence" + ], + [ + 183, + 192, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 197, + 204, + "ectoine", + "chemical" + ], + [ + 230, + 246, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 213, + "sent": "However, in the \u201csemi-closed\u201d (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.", + "section": "RESULTS", + "ner": [ + [ + 17, + 28, + "semi-closed", + "protein_state" + ], + [ + 31, + 33, + "Sa", + "species" + ], + [ + 34, + 38, + "EctC", + "protein" + ], + [ + 39, + 48, + "structure", + "evidence" + ], + [ + 59, + 80, + "carboxy-terminal loop", + "structure_element" + ], + [ + 119, + 135, + "electron density", + "evidence" + ], + [ + 174, + 185, + "active site", + "site" + ], + [ + 227, + 254, + "crystallographic refinement", + "experimental_method" + ] + ] + }, + { + "sid": 214, + "sent": "This is in contrast to the high-resolution \u201copen\u201d structure of the (Sa)EctC protein where no additional electron density was observed after refinement.", + "section": "RESULTS", + "ner": [ + [ + 44, + 48, + "open", + "protein_state" + ], + [ + 50, + 59, + "structure", + "evidence" + ], + [ + 68, + 70, + "Sa", + "species" + ], + [ + 71, + 75, + "EctC", + "protein" + ], + [ + 104, + 120, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 215, + "sent": "We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched.", + "section": "RESULTS", + "ner": [ + [ + 57, + 69, + "purification", + "experimental_method" + ], + [ + 74, + 89, + "crystallization", + "experimental_method" + ], + [ + 108, + 124, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 216, + "sent": "This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization.", + "section": "RESULTS", + "ner": [ + [ + 91, + 93, + "Sa", + "species" + ], + [ + 94, + 98, + "EctC", + "protein" + ], + [ + 145, + 152, + "E. coli", + "species" + ], + [ + 163, + 178, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 217, + "sent": "Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments.", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "PEG", + "chemical" + ], + [ + 76, + 83, + "density", + "evidence" + ], + [ + 121, + 124, + "PEG", + "chemical" + ], + [ + 159, + 162, + "PEG", + "chemical" + ], + [ + 207, + 210, + "PEG", + "chemical" + ] + ] + }, + { + "sid": 218, + "sent": "Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density.", + "section": "RESULTS", + "ner": [ + [ + 38, + 62, + "electron density feature", + "evidence" + ], + [ + 125, + 127, + "Sa", + "species" + ], + [ + 128, + 132, + "EctC", + "protein" + ], + [ + 146, + 161, + "hexane-1,6-diol", + "chemical" + ], + [ + 218, + 234, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 219, + "sent": "However, to the best of our knowledge, hexane-1,6-diol is not part of the E. coli metabolome.", + "section": "RESULTS", + "ner": [ + [ + 39, + 54, + "hexane-1,6-diol", + "chemical" + ], + [ + 74, + 81, + "E. coli", + "species" + ] + ] + }, + { + "sid": 220, + "sent": "Despite these notable limitations, we considered the serendipitously trapped compound as a mock ligand that might provide useful insights into the spatial positioning of the true EctC substrate and those residues that coordinate it within the ectoine synthase active site.", + "section": "RESULTS", + "ner": [ + [ + 179, + 183, + "EctC", + "protein" + ], + [ + 243, + 259, + "ectoine synthase", + "protein_type" + ], + [ + 260, + 271, + "active site", + "site" + ] + ] + }, + { + "sid": 221, + "sent": "We note that both N-\u03b3-ADABA and hexane-1,6-diol are both C6-compounds and display similar length (Fig 7a).", + "section": "RESULTS", + "ner": [ + [ + 18, + 27, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 32, + 47, + "hexane-1,6-diol", + "chemical" + ] + ] + }, + { + "sid": 222, + "sent": "A chemically undefined ligand is captured in the active site of the \u201csemi-closed\u201d (Sa)EctC crystal structure.", + "section": "FIG", + "ner": [ + [ + 49, + 60, + "active site", + "site" + ], + [ + 69, + 80, + "semi-closed", + "protein_state" + ], + [ + 83, + 85, + "Sa", + "species" + ], + [ + 86, + 90, + "EctC", + "protein" + ], + [ + 91, + 108, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 223, + "sent": "(a) The observed electron density in the active site of the \u201csemi-closed\u201d structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-\u03b3-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.", + "section": "FIG", + "ner": [ + [ + 17, + 33, + "electron density", + "evidence" + ], + [ + 41, + 52, + "active site", + "site" + ], + [ + 61, + 72, + "semi-closed", + "protein_state" + ], + [ + 74, + 83, + "structure", + "evidence" + ], + [ + 88, + 90, + "Sa", + "species" + ], + [ + 91, + 95, + "EctC", + "protein" + ], + [ + 112, + 127, + "hexane-1,6-diol", + "chemical" + ], + [ + 159, + 175, + "electron density", + "evidence" + ], + [ + 183, + 192, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 210, + 226, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 224, + "sent": "(b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted.", + "section": "FIG", + "ner": [ + [ + 19, + 31, + "binding site", + "site" + ], + [ + 39, + 43, + "iron", + "chemical" + ], + [ + 73, + 88, + "hexane-1,6-diol", + "chemical" + ] + ] + }, + { + "sid": 225, + "sent": "The amino acid side chains involved in iron-ligand binding are colored in blue and those involved in the binding of the chemically undefined ligand are colored in green using a ball and stick representation.", + "section": "FIG", + "ner": [ + [ + 39, + 43, + "iron", + "chemical" + ] + ] + }, + { + "sid": 226, + "sent": "The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange.", + "section": "FIG", + "ner": [ + [ + 4, + 12, + "flexible", + "protein_state" + ], + [ + 13, + 34, + "carboxy-terminal loop", + "structure_element" + ], + [ + 39, + 41, + "Sa", + "species" + ], + [ + 42, + 46, + "EctC", + "protein" + ] + ] + }, + { + "sid": 227, + "sent": "The electron density was calculated as an omit map and contoured at 1.0 \u03c3.", + "section": "FIG", + "ner": [ + [ + 4, + 20, + "electron density", + "evidence" + ], + [ + 42, + 50, + "omit map", + "evidence" + ] + ] + }, + { + "sid": 228, + "sent": "We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%.", + "section": "RESULTS", + "ner": [ + [ + 3, + 10, + "refined", + "experimental_method" + ], + [ + 16, + 18, + "Sa", + "species" + ], + [ + 19, + 23, + "EctC", + "protein" + ], + [ + 24, + 33, + "structure", + "evidence" + ], + [ + 116, + 135, + "R- and Rfree-factor", + "evidence" + ] + ] + }, + { + "sid": 229, + "sent": "We also calculated an omit map and the electron density reappeared (Fig 7b).", + "section": "RESULTS", + "ner": [ + [ + 22, + 30, + "omit map", + "evidence" + ], + [ + 39, + 55, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 230, + "sent": "When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of \u03b2-sheet \u03b23, Thr-40 located in \u03b2-sheet \u03b24, and Cys-105 and Phe-107, which are both part of \u03b2-sheet \u03b211.", + "section": "RESULTS", + "ner": [ + [ + 61, + 63, + "Sa", + "species" + ], + [ + 64, + 68, + "EctC", + "protein" + ], + [ + 98, + 103, + "bound", + "protein_state" + ], + [ + 126, + 132, + "Trp-21", + "residue_name_number" + ], + [ + 137, + 143, + "Ser-23", + "residue_name_number" + ], + [ + 147, + 154, + "\u03b2-sheet", + "structure_element" + ], + [ + 155, + 157, + "\u03b23", + "structure_element" + ], + [ + 159, + 165, + "Thr-40", + "residue_name_number" + ], + [ + 177, + 184, + "\u03b2-sheet", + "structure_element" + ], + [ + 185, + 187, + "\u03b24", + "structure_element" + ], + [ + 193, + 200, + "Cys-105", + "residue_name_number" + ], + [ + 205, + 212, + "Phe-107", + "residue_name_number" + ], + [ + 237, + 244, + "\u03b2-sheet", + "structure_element" + ], + [ + 245, + 248, + "\u03b211", + "structure_element" + ] + ] + }, + { + "sid": 231, + "sent": "Remarkably, all of these residues are highly conserved throughout the extended EctC protein family (Fig 2).", + "section": "RESULTS", + "ner": [ + [ + 38, + 54, + "highly conserved", + "protein_state" + ], + [ + 79, + 91, + "EctC protein", + "protein_type" + ] + ] + }, + { + "sid": 232, + "sent": "Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase", + "section": "RESULTS", + "ner": [ + [ + 0, + 42, + "Structure-guided site-directed mutagenesis", + "experimental_method" + ], + [ + 50, + 64, + "catalytic core", + "site" + ], + [ + 72, + 88, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 233, + "sent": "In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues.", + "section": "RESULTS", + "ner": [ + [ + 14, + 51, + "alignment of the amino acid sequences", + "experimental_method" + ], + [ + 59, + 77, + "EctC-type proteins", + "protein_type" + ], + [ + 113, + 131, + "strictly conserved", + "protein_state" + ] + ] + }, + { + "sid": 234, + "sent": "These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).", + "section": "RESULTS", + "ner": [ + [ + 32, + 38, + "Thr-40", + "residue_name_number" + ], + [ + 40, + 46, + "Tyr-52", + "residue_name_number" + ], + [ + 48, + 54, + "His-55", + "residue_name_number" + ], + [ + 56, + 62, + "Glu-57", + "residue_name_number" + ], + [ + 64, + 70, + "Gly-64", + "residue_name_number" + ], + [ + 72, + 78, + "Tyr-85", + "residue_name_number" + ], + [ + 80, + 86, + "Leu-87", + "residue_name_number" + ], + [ + 88, + 94, + "His-93", + "residue_name_number" + ], + [ + 96, + 103, + "Phe-107", + "residue_name_number" + ], + [ + 105, + 112, + "Pro-109", + "residue_name_number" + ], + [ + 114, + 121, + "Gly-113", + "residue_name_number" + ], + [ + 123, + 130, + "Glu-115", + "residue_name_number" + ], + [ + 136, + 143, + "His-117", + "residue_name_number" + ], + [ + 152, + 154, + "Sa", + "species" + ], + [ + 155, + 159, + "EctC", + "protein" + ] + ] + }, + { + "sid": 235, + "sent": "Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of \u03b2-strands and \u03b1-helices.", + "section": "RESULTS", + "ner": [ + [ + 20, + 26, + "Gly-64", + "residue_name_number" + ], + [ + 28, + 35, + "Pro-109", + "residue_name_number" + ], + [ + 41, + 48, + "Gly-113", + "residue_name_number" + ], + [ + 148, + 157, + "\u03b2-strands", + "structure_element" + ], + [ + 162, + 171, + "\u03b1-helices", + "structure_element" + ] + ] + }, + { + "sid": 236, + "sent": "We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein.", + "section": "RESULTS", + "ner": [ + [ + 147, + 155, + "flexible", + "protein_state" + ], + [ + 156, + 172, + "carboxy-terminus", + "structure_element" + ], + [ + 181, + 183, + "Sa", + "species" + ], + [ + 184, + 188, + "EctC", + "protein" + ] + ] + }, + { + "sid": 237, + "sent": "As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a).", + "section": "RESULTS", + "ner": [ + [ + 39, + 45, + "Glu-57", + "residue_name_number" + ], + [ + 47, + 53, + "Tyr-85", + "residue_name_number" + ], + [ + 59, + 65, + "His-93", + "residue_name_number" + ], + [ + 91, + 95, + "iron", + "chemical" + ] + ] + }, + { + "sid": 238, + "sent": "In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 16, + 18, + "Sa", + "species" + ], + [ + 19, + 23, + "EctC", + "protein" + ], + [ + 24, + 33, + "structure", + "evidence" + ], + [ + 172, + 214, + "structure-guided site-directed mutagenesis", + "experimental_method" + ] + ] + }, + { + "sid": 239, + "sent": "Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations.", + "section": "RESULTS", + "ner": [ + [ + 14, + 20, + "mutant", + "protein_state" + ], + [ + 22, + 24, + "Sa", + "species" + ], + [ + 25, + 29, + "EctC", + "protein" + ], + [ + 59, + 66, + "E. coli", + "species" + ], + [ + 83, + 106, + "affinity chromatography", + "experimental_method" + ] + ] + }, + { + "sid": 240, + "sent": "We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 \u03bcM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-\u03b3-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.", + "section": "RESULTS", + "ner": [ + [ + 36, + 38, + "Sa", + "species" + ], + [ + 39, + 43, + "EctC", + "protein" + ], + [ + 58, + 88, + "single time-point enzyme assay", + "experimental_method" + ], + [ + 125, + 134, + "wild-type", + "protein_state" + ], + [ + 136, + 138, + "Sa", + "species" + ], + [ + 139, + 143, + "EctC", + "protein" + ], + [ + 199, + 208, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 230, + 237, + "ectoine", + "chemical" + ] + ] + }, + { + "sid": 241, + "sent": "In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 31, + 35, + "iron", + "chemical" + ], + [ + 59, + 65, + "mutant", + "protein_state" + ], + [ + 67, + 69, + "Sa", + "species" + ], + [ + 70, + 74, + "EctC", + "protein" + ], + [ + 88, + 106, + "colorimetric assay", + "experimental_method" + ] + ] + }, + { + "sid": 242, + "sent": "The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the \u201csemi-closed\u201d (Sa)EctC structure (Fig 7b).", + "section": "RESULTS", + "ner": [ + [ + 23, + 47, + "evolutionarily conserved", + "protein_state" + ], + [ + 48, + 54, + "Trp-21", + "residue_name_number" + ], + [ + 56, + 62, + "Ser-23", + "residue_name_number" + ], + [ + 64, + 70, + "Thr-40", + "residue_name_number" + ], + [ + 72, + 79, + "Cys-105", + "residue_name_number" + ], + [ + 85, + 92, + "Phe-107", + "residue_name_number" + ], + [ + 186, + 197, + "semi-closed", + "protein_state" + ], + [ + 200, + 202, + "Sa", + "species" + ], + [ + 203, + 207, + "EctC", + "protein" + ], + [ + 208, + 217, + "structure", + "evidence" + ] + ] + }, + { + "sid": 243, + "sent": "We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins.", + "section": "RESULTS", + "ner": [ + [ + 3, + 11, + "replaced", + "experimental_method" + ], + [ + 43, + 46, + "Ala", + "residue_name" + ], + [ + 107, + 111, + "iron", + "chemical" + ], + [ + 127, + 133, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 244, + "sent": "Thr-40 is positioned on \u03b2-strand \u03b25 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Thr-40", + "residue_name_number" + ], + [ + 24, + 32, + "\u03b2-strand", + "structure_element" + ], + [ + 33, + 35, + "\u03b25", + "structure_element" + ], + [ + 87, + 99, + "cupin barrel", + "structure_element" + ], + [ + 115, + 117, + "Sa", + "species" + ], + [ + 118, + 122, + "EctC", + "protein" + ] + ] + }, + { + "sid": 245, + "sent": "We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.", + "section": "RESULTS", + "ner": [ + [ + 8, + 16, + "replaced", + "experimental_method" + ], + [ + 17, + 24, + "Phe-107", + "residue_name_number" + ], + [ + 40, + 43, + "Tyr", + "residue_name" + ], + [ + 50, + 53, + "Trp", + "residue_name" + ], + [ + 67, + 78, + "Phe-107/Tyr", + "mutant" + ], + [ + 79, + 91, + "substitution", + "experimental_method" + ], + [ + 107, + 116, + "wild-type", + "protein_state" + ], + [ + 158, + 162, + "iron", + "chemical" + ], + [ + 180, + 191, + "Phe-107/Trp", + "mutant" + ], + [ + 192, + 204, + "substitution", + "experimental_method" + ], + [ + 248, + 252, + "iron", + "chemical" + ], + [ + 277, + 286, + "wild-type", + "protein_state" + ] + ] + }, + { + "sid": 246, + "sent": "The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.", + "section": "RESULTS", + "ner": [ + [ + 24, + 30, + "mutant", + "protein_state" + ], + [ + 90, + 93, + "107", + "residue_number" + ], + [ + 98, + 100, + "Sa", + "species" + ], + [ + 101, + 105, + "EctC", + "protein" + ], + [ + 134, + 146, + "substitution", + "experimental_method" + ], + [ + 260, + 264, + "iron", + "chemical" + ] + ] + }, + { + "sid": 247, + "sent": "Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Replacement", + "experimental_method" + ], + [ + 24, + 27, + "Cys", + "residue_name" + ], + [ + 40, + 42, + "Sa", + "species" + ], + [ + 43, + 47, + "EctC", + "protein" + ], + [ + 49, + 56, + "Cys-105", + "residue_name_number" + ], + [ + 70, + 73, + "Ser", + "residue_name" + ], + [ + 130, + 143, + "EctC proteins", + "protein_type" + ], + [ + 197, + 199, + "Sa", + "species" + ], + [ + 200, + 204, + "EctC", + "protein" + ], + [ + 205, + 212, + "variant", + "protein_state" + ], + [ + 222, + 231, + "wild-type", + "protein_state" + ], + [ + 248, + 252, + "iron", + "chemical" + ], + [ + 284, + 293, + "wild-type", + "protein_state" + ] + ] + }, + { + "sid": 248, + "sent": "However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 13, + 24, + "Cys-105/Ala", + "mutant" + ], + [ + 25, + 32, + "variant", + "protein_state" + ], + [ + 49, + 71, + "catalytically inactive", + "protein_state" + ], + [ + 102, + 106, + "iron", + "chemical" + ] + ] + }, + { + "sid": 249, + "sent": "Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase.", + "section": "RESULTS", + "ner": [ + [ + 25, + 28, + "Cys", + "residue_name" + ], + [ + 105, + 112, + "Cys-105", + "residue_name_number" + ], + [ + 117, + 124, + "Ser-105", + "residue_name_number" + ], + [ + 154, + 170, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 250, + "sent": "We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 16, + 40, + "amino acid substitutions", + "experimental_method" + ], + [ + 99, + 103, + "iron", + "chemical" + ], + [ + 128, + 138, + "Tyr-52/Ala", + "mutant" + ], + [ + 147, + 157, + "His-55/Ala", + "mutant" + ], + [ + 159, + 161, + "Sa", + "species" + ], + [ + 162, + 166, + "EctC", + "protein" + ] + ] + }, + { + "sid": 251, + "sent": "Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 14, + 16, + "Sa", + "species" + ], + [ + 17, + 21, + "EctC", + "protein" + ], + [ + 22, + 40, + "crystal structures", + "evidence" + ], + [ + 110, + 121, + "replacement", + "experimental_method" + ], + [ + 125, + 131, + "Tyr-52", + "residue_name_number" + ], + [ + 135, + 138, + "Ala", + "residue_name" + ], + [ + 167, + 171, + "iron", + "chemical" + ] + ] + }, + { + "sid": 252, + "sent": "This is different for the His-55/Ala substitution.", + "section": "RESULTS", + "ner": [ + [ + 26, + 36, + "His-55/Ala", + "mutant" + ] + ] + }, + { + "sid": 253, + "sent": "The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).", + "section": "RESULTS", + "ner": [ + [ + 4, + 27, + "carboxy-terminal region", + "structure_element" + ], + [ + 36, + 38, + "Sa", + "species" + ], + [ + 39, + 43, + "EctC", + "protein" + ], + [ + 98, + 105, + "Glu-115", + "residue_name_number" + ], + [ + 111, + 117, + "His-55", + "residue_name_number" + ], + [ + 125, + 131, + "His-55", + "residue_name_number" + ], + [ + 155, + 162, + "Pro-110", + "residue_name_number" + ] + ] + }, + { + "sid": 254, + "sent": "Each of these residues is evolutionarily highly conserved.", + "section": "RESULTS", + "ner": [ + [ + 26, + 57, + "evolutionarily highly conserved", + "protein_state" + ] + ] + }, + { + "sid": 255, + "sent": "The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity.", + "section": "RESULTS", + "ner": [ + [ + 15, + 27, + "substitution", + "experimental_method" + ], + [ + 38, + 45, + "Glu-115", + "residue_name_number" + ], + [ + 49, + 55, + "His-55", + "residue_name_number" + ], + [ + 62, + 65, + "Ala", + "residue_name" + ], + [ + 103, + 122, + "interactive network", + "site" + ] + ] + }, + { + "sid": 256, + "sent": "Indeed, the Glu-115/Ala and the His-55/Ala substitutions possessed only 21% and 16% activity of the wild-type protein, respectively (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 12, + 23, + "Glu-115/Ala", + "mutant" + ], + [ + 32, + 42, + "His-55/Ala", + "mutant" + ], + [ + 100, + 109, + "wild-type", + "protein_state" + ] + ] + }, + { + "sid": 257, + "sent": "The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "Glu-115/Ala", + "mutant" + ], + [ + 16, + 22, + "mutant", + "protein_state" + ], + [ + 33, + 42, + "wild-type", + "protein_state" + ], + [ + 53, + 57, + "iron", + "chemical" + ], + [ + 71, + 75, + "iron", + "chemical" + ], + [ + 91, + 101, + "His-55/Ala", + "mutant" + ], + [ + 138, + 147, + "wild-type", + "protein_state" + ] + ] + }, + { + "sid": 258, + "sent": "We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.", + "section": "RESULTS", + "ner": [ + [ + 8, + 16, + "replaced", + "experimental_method" + ], + [ + 17, + 24, + "Glu-115", + "residue_name_number" + ], + [ + 60, + 63, + "Asp", + "residue_name" + ], + [ + 72, + 74, + "Sa", + "species" + ], + [ + 75, + 79, + "EctC", + "protein" + ], + [ + 98, + 107, + "wild-type", + "protein_state" + ], + [ + 118, + 122, + "iron", + "chemical" + ], + [ + 150, + 159, + "wild-type", + "protein_state" + ] + ] + }, + { + "sid": 259, + "sent": "Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase.", + "section": "RESULTS", + "ner": [ + [ + 69, + 85, + "carboxy-terminus", + "structure_element" + ], + [ + 94, + 96, + "Sa", + "species" + ], + [ + 97, + 101, + "EctC", + "protein" + ], + [ + 177, + 193, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 260, + "sent": "Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family.", + "section": "RESULTS", + "ner": [ + [ + 9, + 15, + "Leu-87", + "residue_name_number" + ], + [ + 20, + 26, + "Asp-91", + "residue_name_number" + ], + [ + 31, + 47, + "highly conserved", + "protein_state" + ], + [ + 55, + 71, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 261, + "sent": "The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "replacement", + "experimental_method" + ], + [ + 19, + 25, + "Leu-87", + "residue_name_number" + ], + [ + 29, + 32, + "Ala", + "residue_name" + ] + ] + }, + { + "sid": 262, + "sent": "Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 16, + 27, + "replacement", + "experimental_method" + ], + [ + 31, + 37, + "Asp-91", + "residue_name_number" + ], + [ + 41, + 44, + "Ala", + "residue_name" + ], + [ + 49, + 52, + "Glu", + "residue_name" + ], + [ + 67, + 69, + "Sa", + "species" + ], + [ + 70, + 74, + "EctC", + "protein" + ] + ] + }, + { + "sid": 263, + "sent": "We currently cannot comment on possible functional role Asp-91.", + "section": "RESULTS", + "ner": [ + [ + 56, + 62, + "Asp-91", + "residue_name_number" + ] + ] + }, + { + "sid": 264, + "sent": "However, Leu-87 is positioned at the end of one of the \u03b2-sheets that form the dimer interface (Fig 5c) and it might therefore possess a structural role.", + "section": "RESULTS", + "ner": [ + [ + 9, + 15, + "Leu-87", + "residue_name_number" + ], + [ + 55, + 63, + "\u03b2-sheets", + "structure_element" + ], + [ + 78, + 93, + "dimer interface", + "site" + ] + ] + }, + { + "sid": 265, + "sent": "It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects.", + "section": "RESULTS", + "ner": [ + [ + 24, + 30, + "Tyr-85", + "residue_name_number" + ], + [ + 81, + 85, + "iron", + "chemical" + ], + [ + 108, + 110, + "Sa", + "species" + ], + [ + 111, + 115, + "EctC", + "protein" + ], + [ + 116, + 127, + "active site", + "site" + ] + ] + }, + { + "sid": 266, + "sent": "His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "His-117", + "residue_name_number" + ], + [ + 13, + 31, + "strictly conserved", + "protein_state" + ], + [ + 48, + 60, + "substitution", + "experimental_method" + ], + [ + 67, + 70, + "Ala", + "residue_name" + ], + [ + 137, + 141, + "iron", + "chemical" + ] + ] + }, + { + "sid": 267, + "sent": "We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase.", + "section": "RESULTS", + "ner": [ + [ + 13, + 20, + "His-117", + "residue_name_number" + ], + [ + 81, + 83, + "Sa", + "species" + ], + [ + 84, + 88, + "EctC", + "protein" + ], + [ + 89, + 98, + "structure", + "evidence" + ], + [ + 178, + 194, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 268, + "sent": "As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.", + "section": "RESULTS", + "ner": [ + [ + 31, + 54, + "mutagenesis experiments", + "experimental_method" + ], + [ + 64, + 75, + "substituted", + "experimental_method" + ], + [ + 76, + 82, + "Thr-41", + "residue_name_number" + ], + [ + 87, + 93, + "His-51", + "residue_name_number" + ], + [ + 117, + 145, + "not evolutionarily conserved", + "protein_state" + ], + [ + 149, + 167, + "EctC-type proteins", + "protein_type" + ], + [ + 173, + 176, + "Ala", + "residue_name" + ] + ] + }, + { + "sid": 269, + "sent": "Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 6, + 8, + "Sa", + "species" + ], + [ + 9, + 13, + "EctC", + "protein" + ], + [ + 41, + 50, + "wild-type", + "protein_state" + ], + [ + 91, + 95, + "iron", + "chemical" + ], + [ + 125, + 134, + "wild-type", + "protein_state" + ] + ] + }, + { + "sid": 270, + "sent": "This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content.", + "section": "RESULTS", + "ner": [ + [ + 64, + 66, + "Sa", + "species" + ], + [ + 67, + 71, + "EctC", + "protein" + ], + [ + 141, + 145, + "iron", + "chemical" + ] + ] + }, + { + "sid": 271, + "sent": "The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 25, + "crystallographic data", + "evidence" + ], + [ + 57, + 73, + "ectoine synthase", + "protein_type" + ], + [ + 75, + 79, + "EctC", + "protein" + ], + [ + 127, + 136, + "microbial", + "taxonomy_domain" + ], + [ + 175, + 182, + "ectoine", + "chemical" + ], + [ + 207, + 224, + "cupin superfamily", + "protein_type" + ] + ] + }, + { + "sid": 272, + "sent": "The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 \u03b2-strands (\u03b21-\u03b211) and two \u03b1-helices (\u03b1-I and \u03b1-II) closely adheres to the design principles typically found in crystal structures of cupins.", + "section": "DISCUSS", + "ner": [ + [ + 40, + 42, + "Sa", + "species" + ], + [ + 43, + 47, + "EctC", + "protein" + ], + [ + 83, + 92, + "\u03b2-strands", + "structure_element" + ], + [ + 94, + 100, + "\u03b21-\u03b211", + "structure_element" + ], + [ + 110, + 119, + "\u03b1-helices", + "structure_element" + ], + [ + 121, + 124, + "\u03b1-I", + "structure_element" + ], + [ + 129, + 133, + "\u03b1-II", + "structure_element" + ], + [ + 195, + 213, + "crystal structures", + "evidence" + ], + [ + 217, + 223, + "cupins", + "protein_type" + ] + ] + }, + { + "sid": 273, + "sent": "In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 35, + "ectoine synthase", + "protein_type" + ], + [ + 41, + 59, + "polyketide cyclase", + "protein_type" + ], + [ + 60, + 64, + "RemF", + "protein" + ], + [ + 99, + 112, + "cupin-related", + "protein_type" + ], + [ + 190, + 194, + "EctC", + "protein" + ], + [ + 199, + 203, + "RemF", + "protein" + ] + ] + }, + { + "sid": 274, + "sent": "As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as \u201cRemF-like\u201d proteins.", + "section": "DISCUSS", + "ner": [ + [ + 50, + 54, + "EctC", + "protein" + ], + [ + 59, + 63, + "RemF", + "protein" + ], + [ + 162, + 180, + "EctC-type proteins", + "protein_type" + ], + [ + 215, + 224, + "microbial", + "taxonomy_domain" + ], + [ + 246, + 255, + "RemF-like", + "protein_type" + ] + ] + }, + { + "sid": 275, + "sent": "The pro- and eukaryotic members of the cupin superfamily perform a variety of both enzymatic and non-enzymatic functions that are built upon a common structural scaffold.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 8, + "pro-", + "taxonomy_domain" + ], + [ + 13, + 23, + "eukaryotic", + "taxonomy_domain" + ], + [ + 39, + 56, + "cupin superfamily", + "protein_type" + ] + ] + }, + { + "sid": 276, + "sent": "Most cupins contain transition state metals that can promote different types of chemical reactions.", + "section": "DISCUSS", + "ner": [ + [ + 5, + 11, + "cupins", + "protein_type" + ] + ] + }, + { + "sid": 277, + "sent": "Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 38, + "cupin-related proteins", + "protein_type" + ], + [ + 64, + 82, + "metallo-chaperones", + "protein_type" + ], + [ + 88, + 93, + "bound", + "protein_state" + ], + [ + 94, + 99, + "metal", + "chemical" + ], + [ + 138, + 150, + "active sites", + "site" + ] + ] + }, + { + "sid": 278, + "sent": "We report here for the first time that the ectoine synthase is a metal-dependent enzyme.", + "section": "DISCUSS", + "ner": [ + [ + 43, + 59, + "ectoine synthase", + "protein_type" + ], + [ + 65, + 70, + "metal", + "chemical" + ] + ] + }, + { + "sid": 279, + "sent": "ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 6, + "ICP-MS", + "experimental_method" + ], + [ + 8, + 54, + "metal-depletion and reconstitution experiments", + "experimental_method" + ], + [ + 85, + 89, + "iron", + "chemical" + ], + [ + 124, + 129, + "metal", + "chemical" + ], + [ + 138, + 142, + "EctC", + "protein" + ] + ] + }, + { + "sid": 280, + "sent": "However, as observed with other cupins, EctC is a somewhat promiscuous enzyme as far as the catalytically important metal is concerned when they are provided in large molar excess (Fig 3c).", + "section": "DISCUSS", + "ner": [ + [ + 32, + 38, + "cupins", + "protein_type" + ], + [ + 40, + 44, + "EctC", + "protein" + ], + [ + 116, + 121, + "metal", + "chemical" + ] + ] + }, + { + "sid": 281, + "sent": "Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1).", + "section": "DISCUSS", + "ner": [ + [ + 114, + 119, + "metal", + "chemical" + ], + [ + 132, + 134, + "Sa", + "species" + ], + [ + 135, + 139, + "EctC", + "protein" + ], + [ + 145, + 187, + "structure-guided site-directed mutagenesis", + "experimental_method" + ], + [ + 226, + 247, + "iron-binding residues", + "site" + ] + ] + }, + { + "sid": 282, + "sent": "The architecture of the metal center of ectoine synthase seems to be subjected to considerable evolutionary constraints.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 36, + "metal center", + "site" + ], + [ + 40, + 56, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 283, + "sent": "The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).", + "section": "DISCUSS", + "ner": [ + [ + 20, + 26, + "Glu-57", + "residue_name_number" + ], + [ + 28, + 34, + "Tyr-85", + "residue_name_number" + ], + [ + 36, + 42, + "His-93", + "residue_name_number" + ], + [ + 88, + 106, + "strictly conserved", + "protein_state" + ], + [ + 132, + 150, + "EctC-type proteins", + "protein_type" + ], + [ + 171, + 180, + "bacterial", + "taxonomy_domain" + ], + [ + 191, + 199, + "archaeal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 284, + "sent": "We also show here for the first time that, in addition to its natural substrate N-\u03b3-ADABA, EctC also converts the isomer N-\u03b1-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig).", + "section": "DISCUSS", + "ner": [ + [ + 80, + 89, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 91, + 95, + "EctC", + "protein" + ], + [ + 121, + 130, + "N-\u03b1-ADABA", + "chemical" + ], + [ + 136, + 143, + "ectoine", + "chemical" + ], + [ + 175, + 195, + "catalytic efficiency", + "evidence" + ] + ] + }, + { + "sid": 285, + "sent": "Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 22, + "active site", + "site" + ], + [ + 26, + 42, + "ectoine synthase", + "protein_type" + ], + [ + 147, + 151, + "EctC", + "protein" + ], + [ + 207, + 211, + "ADPC", + "chemical" + ], + [ + 251, + 260, + "glutamine", + "chemical" + ] + ] + }, + { + "sid": 286, + "sent": "Our finding that N-\u03b1-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-\u03b1-ADABA is produced as an intermediate in the catabolic route.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 26, + "N-\u03b1-ADABA", + "chemical" + ], + [ + 53, + 69, + "ectoine synthase", + "protein_type" + ], + [ + 123, + 137, + "microorganisms", + "taxonomy_domain" + ], + [ + 178, + 185, + "ectoine", + "chemical" + ], + [ + 263, + 272, + "N-\u03b1-ADABA", + "chemical" + ] + ] + }, + { + "sid": 287, + "sent": "Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a).", + "section": "DISCUSS", + "ner": [ + [ + 60, + 62, + "C6", + "chemical" + ], + [ + 97, + 99, + "Sa", + "species" + ], + [ + 100, + 104, + "EctC", + "protein" + ], + [ + 105, + 114, + "structure", + "evidence" + ], + [ + 204, + 220, + "ectoine synthase", + "protein_type" + ] + ] + }, + { + "sid": 288, + "sent": "We assumed that its location and mode of binding gives, in all likelihood, clues as to the position of the true substrate N-\u03b3-ADABA within the EctC active site.", + "section": "DISCUSS", + "ner": [ + [ + 122, + 131, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 143, + 147, + "EctC", + "protein" + ], + [ + 148, + 159, + "active site", + "site" + ] + ] + }, + { + "sid": 289, + "sent": "Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1).", + "section": "DISCUSS", + "ner": [ + [ + 8, + 33, + "site-directed mutagenesis", + "experimental_method" + ], + [ + 112, + 114, + "Sa", + "species" + ], + [ + 115, + 119, + "EctC", + "protein" + ], + [ + 177, + 186, + "wild-type", + "protein_state" + ], + [ + 197, + 201, + "iron", + "chemical" + ] + ] + }, + { + "sid": 290, + "sent": "This set of data and the fact that the targeted residues are strongly conserved among EctC-type proteins (Fig 2) is consistent with their potential role in N-\u03b3-ADABA binding or enzyme catalysis.", + "section": "DISCUSS", + "ner": [ + [ + 61, + 79, + "strongly conserved", + "protein_state" + ], + [ + 86, + 104, + "EctC-type proteins", + "protein_type" + ], + [ + 156, + 165, + "N-\u03b3-ADABA", + "chemical" + ] + ] + }, + { + "sid": 291, + "sent": "We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b).", + "section": "DISCUSS", + "ner": [ + [ + 30, + 51, + "crystallographic data", + "evidence" + ], + [ + 60, + 91, + "site-directed mutagenesis study", + "experimental_method" + ], + [ + 176, + 180, + "EctC", + "protein" + ], + [ + 181, + 192, + "active site", + "site" + ] + ] + }, + { + "sid": 292, + "sent": "The ectoine synthase from the cold-adapted marine bacterium S. alaskensis can be considered as a psychrophilic enzyme (S3a Fig), types of proteins with a considerable structural flexibility.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 20, + "ectoine synthase", + "protein_type" + ], + [ + 43, + 59, + "marine bacterium", + "taxonomy_domain" + ], + [ + 60, + 73, + "S. alaskensis", + "species" + ] + ] + }, + { + "sid": 293, + "sent": "This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-\u03b3-ADABA or ectoine.", + "section": "DISCUSS", + "ner": [ + [ + 64, + 82, + "crystal structures", + "evidence" + ], + [ + 90, + 101, + "full-length", + "protein_state" + ], + [ + 103, + 105, + "Sa", + "species" + ], + [ + 106, + 110, + "EctC", + "protein" + ], + [ + 119, + 134, + "in complex with", + "protein_state" + ], + [ + 142, + 151, + "N-\u03b3-ADABA", + "chemical" + ], + [ + 155, + 162, + "ectoine", + "chemical" + ] + ] + }, + { + "sid": 294, + "sent": "Because microbial ectoine producers can colonize ecological niches with rather different physicochemical attributes, it seems promising to exploit this considerable biodiversity to identify EctC proteins with enhanced protein stability.", + "section": "DISCUSS", + "ner": [ + [ + 8, + 17, + "microbial", + "taxonomy_domain" + ], + [ + 18, + 25, + "ectoine", + "chemical" + ], + [ + 190, + 203, + "EctC proteins", + "protein_type" + ] + ] + }, + { + "sid": 295, + "sent": "It is hoped that these can be further employed to obtain EctC crystal structures with either the substrate or the reaction product.", + "section": "DISCUSS", + "ner": [ + [ + 57, + 61, + "EctC", + "protein" + ], + [ + 62, + 80, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 296, + "sent": "Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction.", + "section": "DISCUSS", + "ner": [ + [ + 31, + 47, + "ectoine synthase", + "protein_type" + ], + [ + 51, + 66, + "metal dependent", + "protein_state" + ], + [ + 74, + 92, + "crystal structures", + "evidence" + ], + [ + 168, + 172, + "EctC", + "protein" + ] + ] + } + ] + }, + "PMC4918759": { + "annotations": [ + { + "sid": 0, + "sent": "Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity", + "section": "TITLE", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 14, + 19, + "human", + "species" + ], + [ + 20, + 25, + "ADAR2", + "protein" + ], + [ + 26, + 34, + "bound to", + "protein_state" + ], + [ + 35, + 40, + "dsRNA", + "chemical" + ] + ] + }, + { + "sid": 1, + "sent": "ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 5, + "ADARs", + "protein_type" + ], + [ + 7, + 41, + "adenosine deaminases acting on RNA", + "protein_type" + ], + [ + 47, + 62, + "editing enzymes", + "protein_type" + ], + [ + 76, + 85, + "adenosine", + "residue_name" + ], + [ + 87, + 88, + "A", + "residue_name" + ], + [ + 93, + 100, + "inosine", + "residue_name" + ], + [ + 102, + 103, + "I", + "residue_name" + ], + [ + 108, + 118, + "duplex RNA", + "structure_element" + ], + [ + 178, + 181, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 2, + "sent": "Our understanding of the ADAR reaction mechanism, origin of editing site selectivity and effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs.", + "section": "ABSTRACT", + "ner": [ + [ + 25, + 29, + "ADAR", + "protein_type" + ], + [ + 60, + 72, + "editing site", + "site" + ], + [ + 151, + 166, + "structural data", + "evidence" + ], + [ + 184, + 189, + "ADARs", + "protein_type" + ], + [ + 190, + 198, + "bound to", + "protein_state" + ], + [ + 209, + 213, + "RNAs", + "chemical" + ] + ] + }, + { + "sid": 3, + "sent": "Here we describe four crystal structures of the deaminase domain of human ADAR2 bound to RNA duplexes bearing a mimic of the deamination reaction intermediate.", + "section": "ABSTRACT", + "ner": [ + [ + 22, + 40, + "crystal structures", + "evidence" + ], + [ + 48, + 64, + "deaminase domain", + "structure_element" + ], + [ + 68, + 73, + "human", + "species" + ], + [ + 74, + 79, + "ADAR2", + "protein" + ], + [ + 80, + 88, + "bound to", + "protein_state" + ], + [ + 89, + 101, + "RNA duplexes", + "structure_element" + ] + ] + }, + { + "sid": 4, + "sent": "These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis for ADAR deaminase domain\u2019s dsRNA specificity, its base-flipping mechanism, and nearest neighbor preferences.", + "section": "ABSTRACT", + "ner": [ + [ + 6, + 16, + "structures", + "evidence" + ], + [ + 32, + 60, + "structure-guided mutagenesis", + "experimental_method" + ], + [ + 65, + 93, + "RNA-modification experiments", + "experimental_method" + ], + [ + 117, + 121, + "ADAR", + "protein_type" + ], + [ + 122, + 138, + "deaminase domain", + "structure_element" + ], + [ + 141, + 146, + "dsRNA", + "chemical" + ] + ] + }, + { + "sid": 5, + "sent": "In addition, an ADAR2-specific RNA-binding loop was identified near the enzyme active site rationalizing differences in selectivity observed between different ADARs.", + "section": "ABSTRACT", + "ner": [ + [ + 16, + 21, + "ADAR2", + "protein" + ], + [ + 31, + 47, + "RNA-binding loop", + "structure_element" + ], + [ + 79, + 90, + "active site", + "site" + ], + [ + 159, + 164, + "ADARs", + "protein_type" + ] + ] + }, + { + "sid": 6, + "sent": "Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.", + "section": "ABSTRACT", + "ner": [ + [ + 85, + 89, + "ADAR", + "protein_type" + ], + [ + 116, + 121, + "human", + "species" + ] + ] + }, + { + "sid": 7, + "sent": "RNA editing reactions alter a transcript\u2019s genomically encoded sequence by inserting, deleting or modifying nucleotides.", + "section": "INTRO", + "ner": [ + [ + 0, + 3, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 8, + "sent": "Deamination of adenosine (A), the most common form of RNA editing in humans, generates inosine (I) at the corresponding nucleotide position.", + "section": "INTRO", + "ner": [ + [ + 15, + 24, + "adenosine", + "residue_name" + ], + [ + 26, + 27, + "A", + "residue_name" + ], + [ + 54, + 57, + "RNA", + "chemical" + ], + [ + 69, + 75, + "humans", + "species" + ], + [ + 87, + 94, + "inosine", + "residue_name" + ], + [ + 96, + 97, + "I", + "residue_name" + ] + ] + }, + { + "sid": 9, + "sent": "Since I base pairs with cytidine (C), it functions like guanosine (G) in cellular processes such as splicing, translation and reverse transcription.", + "section": "INTRO", + "ner": [ + [ + 6, + 7, + "I", + "residue_name" + ], + [ + 24, + 32, + "cytidine", + "residue_name" + ], + [ + 34, + 35, + "C", + "residue_name" + ], + [ + 56, + 65, + "guanosine", + "residue_name" + ], + [ + 67, + 68, + "G", + "residue_name" + ] + ] + }, + { + "sid": 10, + "sent": "A to I editing has wide-ranging consequences on RNA function including altering miRNA recognition sites, redirecting splicing and changing the meaning of specific codons.", + "section": "INTRO", + "ner": [ + [ + 48, + 51, + "RNA", + "chemical" + ], + [ + 80, + 103, + "miRNA recognition sites", + "site" + ] + ] + }, + { + "sid": 11, + "sent": "Two different enzymes carry out A to I editing in humans; ADAR1 and ADAR2.", + "section": "INTRO", + "ner": [ + [ + 50, + 56, + "humans", + "species" + ], + [ + 58, + 63, + "ADAR1", + "protein" + ], + [ + 68, + 73, + "ADAR2", + "protein" + ] + ] + }, + { + "sid": 12, + "sent": "ADAR activity is required for nervous system function and altered editing has been linked to neurological disorders such as epilepsy and Prader Willi Syndrome.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "ADAR", + "protein_type" + ] + ] + }, + { + "sid": 13, + "sent": "In addition, mutations in the ADAR1 gene are known to cause the autoimmune disease Aicardi-Goutieres Syndrome (AGS) and the skin disorder Dyschromatosis Symmetrica Hereditaria (DSH).", + "section": "INTRO", + "ner": [ + [ + 30, + 35, + "ADAR1", + "protein" + ] + ] + }, + { + "sid": 14, + "sent": "Hyper editing has been observed at certain sites in cancer cells, such as in the mRNA for AZIN1 (antizyme inhibitor 1).", + "section": "INTRO", + "ner": [ + [ + 81, + 85, + "mRNA", + "chemical" + ], + [ + 90, + 95, + "AZIN1", + "protein" + ], + [ + 97, + 117, + "antizyme inhibitor 1", + "protein" + ] + ] + }, + { + "sid": 15, + "sent": "However, hypo editing also occurs in cancer-derived cell lines exemplified by reduced editing observed in the message for glioma-associated oncogene 1 (Gli1).", + "section": "INTRO", + "ner": [ + [ + 122, + 150, + "glioma-associated oncogene 1", + "protein" + ], + [ + 152, + 156, + "Gli1", + "protein" + ] + ] + }, + { + "sid": 16, + "sent": "The ADAR proteins have a modular structure with double stranded RNA binding domains (dsRBDs) and a C-terminal deaminase domain (see Fig. 1a for hADAR2 domains).", + "section": "INTRO", + "ner": [ + [ + 4, + 8, + "ADAR", + "protein_type" + ], + [ + 48, + 83, + "double stranded RNA binding domains", + "structure_element" + ], + [ + 85, + 91, + "dsRBDs", + "structure_element" + ], + [ + 110, + 126, + "deaminase domain", + "structure_element" + ], + [ + 144, + 150, + "hADAR2", + "protein" + ] + ] + }, + { + "sid": 17, + "sent": "ADARs efficiently deaminate specific adenosines in duplex RNA while leaving most adenosines unmodified.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "ADARs", + "protein_type" + ], + [ + 37, + 47, + "adenosines", + "residue_name" + ], + [ + 51, + 61, + "duplex RNA", + "structure_element" + ], + [ + 81, + 91, + "adenosines", + "residue_name" + ] + ] + }, + { + "sid": 18, + "sent": "The mechanism of adenosine deamination requires ADAR to flip the reactive base out of an RNA double helix to access its active site.", + "section": "INTRO", + "ner": [ + [ + 17, + 26, + "adenosine", + "residue_name" + ], + [ + 48, + 52, + "ADAR", + "protein_type" + ], + [ + 89, + 105, + "RNA double helix", + "chemical" + ], + [ + 120, + 131, + "active site", + "site" + ] + ] + }, + { + "sid": 19, + "sent": "How an enzyme could accomplish this task with a duplex RNA substrate is not known.", + "section": "INTRO", + "ner": [ + [ + 48, + 58, + "duplex RNA", + "structure_element" + ] + ] + }, + { + "sid": 20, + "sent": "Furthermore, how an ADAR deaminase domain contributes to editing site selectivity is also unknown, since no structures of ADAR deaminase domain-RNA complexes have been reported.", + "section": "INTRO", + "ner": [ + [ + 20, + 24, + "ADAR", + "protein_type" + ], + [ + 25, + 41, + "deaminase domain", + "structure_element" + ], + [ + 57, + 69, + "editing site", + "site" + ], + [ + 108, + 118, + "structures", + "evidence" + ], + [ + 122, + 147, + "ADAR deaminase domain-RNA", + "complex_assembly" + ] + ] + }, + { + "sid": 21, + "sent": "To address these knowledge gaps, we set out to trap the human ADAR2 deaminase domain (aa299\u2013701, hADAR2d) bound to different duplex RNAs and solve structures for the resulting complexes using x-ray crystallography.", + "section": "INTRO", + "ner": [ + [ + 56, + 61, + "human", + "species" + ], + [ + 62, + 67, + "ADAR2", + "protein" + ], + [ + 68, + 84, + "deaminase domain", + "structure_element" + ], + [ + 88, + 95, + "299\u2013701", + "residue_range" + ], + [ + 97, + 104, + "hADAR2d", + "mutant" + ], + [ + 106, + 114, + "bound to", + "protein_state" + ], + [ + 125, + 136, + "duplex RNAs", + "structure_element" + ], + [ + 147, + 157, + "structures", + "evidence" + ], + [ + 192, + 213, + "x-ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 22, + "sent": "We then evaluated the importance of protein-RNA contacts using structure-guided mutagenesis and RNA-modification experiments coupled with adenosine deamination kinetics.", + "section": "INTRO", + "ner": [ + [ + 44, + 47, + "RNA", + "chemical" + ], + [ + 63, + 91, + "structure-guided mutagenesis", + "experimental_method" + ], + [ + 96, + 124, + "RNA-modification experiments", + "experimental_method" + ], + [ + 138, + 168, + "adenosine deamination kinetics", + "experimental_method" + ] + ] + }, + { + "sid": 23, + "sent": "Trapping the flipped conformation", + "section": "RESULTS", + "ner": [ + [ + 13, + 20, + "flipped", + "protein_state" + ] + ] + }, + { + "sid": 24, + "sent": "The ADAR reaction involves the formation of a hydrated intermediate that loses ammonia to generate the inosine-containing product RNA (for reaction scheme see Fig. 1b).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "ADAR", + "protein_type" + ], + [ + 103, + 110, + "inosine", + "residue_name" + ], + [ + 130, + 133, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 25, + "sent": "The covalent hydrate of the nucleoside analog 8-azanebularine (N) mimics the proposed high-energy intermediate (for reaction scheme see Fig. 1b).", + "section": "RESULTS", + "ner": [ + [ + 46, + 61, + "8-azanebularine", + "chemical" + ], + [ + 63, + 64, + "N", + "chemical" + ] + ] + }, + { + "sid": 26, + "sent": "For trapping hADAR2d bound to RNA for crystallography, we incorporated 8-azanebularine into duplex RNAs shown recently to be excellent substrates for deamination by hADAR2d (for duplex sequence see Fig. 1c) (for characterization of protein\u2013RNA complex see Supplementary Fig. 1).", + "section": "RESULTS", + "ner": [ + [ + 13, + 20, + "hADAR2d", + "mutant" + ], + [ + 21, + 29, + "bound to", + "protein_state" + ], + [ + 30, + 33, + "RNA", + "chemical" + ], + [ + 38, + 53, + "crystallography", + "experimental_method" + ], + [ + 71, + 86, + "8-azanebularine", + "chemical" + ], + [ + 92, + 103, + "duplex RNAs", + "structure_element" + ], + [ + 165, + 172, + "hADAR2d", + "mutant" + ], + [ + 240, + 243, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 27, + "sent": "In addition, for one of these duplexes (Bdf2), we positioned the 8-azanebularine opposite either uridine or cytidine to mimic an A-U pair or A-C mismatch at the editing site creating a total of three different RNA substrates for structural studies (Fig. 1c).", + "section": "RESULTS", + "ner": [ + [ + 40, + 44, + "Bdf2", + "chemical" + ], + [ + 65, + 80, + "8-azanebularine", + "chemical" + ], + [ + 97, + 104, + "uridine", + "residue_name" + ], + [ + 108, + 116, + "cytidine", + "residue_name" + ], + [ + 129, + 130, + "A", + "residue_name" + ], + [ + 131, + 132, + "U", + "residue_name" + ], + [ + 141, + 142, + "A", + "residue_name" + ], + [ + 143, + 144, + "C", + "residue_name" + ], + [ + 161, + 173, + "editing site", + "site" + ], + [ + 210, + 213, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 28, + "sent": "The hADAR2d protein (without RNA bound) has been previously crystallized and structurally characterized revealing features of the active site including the presence of zinc.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "hADAR2d", + "mutant" + ], + [ + 21, + 38, + "without RNA bound", + "protein_state" + ], + [ + 60, + 72, + "crystallized", + "experimental_method" + ], + [ + 130, + 141, + "active site", + "site" + ], + [ + 168, + 172, + "zinc", + "chemical" + ] + ] + }, + { + "sid": 29, + "sent": "In addition, an inositol hexakisphosphate (IHP) molecule was found buried in the core of the protein hydrogen bonded to numerous conserved polar residues.", + "section": "RESULTS", + "ner": [ + [ + 16, + 41, + "inositol hexakisphosphate", + "chemical" + ], + [ + 43, + 46, + "IHP", + "chemical" + ], + [ + 101, + 116, + "hydrogen bonded", + "bond_interaction" + ] + ] + }, + { + "sid": 30, + "sent": "For crystallization of hADAR2d-RNA complexes, we used both the wild type (WT) deaminase domain and a mutant (E488Q) that has enhanced catalytic activity.", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "crystallization", + "experimental_method" + ], + [ + 23, + 34, + "hADAR2d-RNA", + "complex_assembly" + ], + [ + 63, + 72, + "wild type", + "protein_state" + ], + [ + 74, + 76, + "WT", + "protein_state" + ], + [ + 78, + 94, + "deaminase domain", + "structure_element" + ], + [ + 101, + 107, + "mutant", + "protein_state" + ], + [ + 109, + 114, + "E488Q", + "mutant" + ] + ] + }, + { + "sid": 31, + "sent": "A description of the crystallization conditions, X-ray diffraction data collection and solution of the structures can be found in Online Methods.", + "section": "RESULTS", + "ner": [ + [ + 49, + 95, + "X-ray diffraction data collection and solution", + "experimental_method" + ], + [ + 103, + 113, + "structures", + "evidence" + ] + ] + }, + { + "sid": 32, + "sent": "Four protein-RNA combinations generated diffracting crystals that resulted in high-resolution structures (hADAR2d WT\u2013Bdf2-U, hADAR2d WT\u2013Bdf2-C, hADAR2d E488Q\u2013Bdf2-C, hADAR2d E488Q\u2013Gli1) (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 13, + 16, + "RNA", + "chemical" + ], + [ + 52, + 60, + "crystals", + "evidence" + ], + [ + 94, + 104, + "structures", + "evidence" + ], + [ + 106, + 123, + "hADAR2d WT\u2013Bdf2-U", + "complex_assembly" + ], + [ + 125, + 142, + "hADAR2d WT\u2013Bdf2-C", + "complex_assembly" + ], + [ + 144, + 164, + "hADAR2d E488Q\u2013Bdf2-C", + "complex_assembly" + ], + [ + 166, + 184, + "hADAR2d E488Q\u2013Gli1", + "complex_assembly" + ] + ] + }, + { + "sid": 33, + "sent": "In each of these complexes, the protein binds the RNA on one face of the duplex over ~ 20 bp using a positively charged surface near the zinc-containing active site (Fig. 2, Supplementary Fig. 2a).", + "section": "RESULTS", + "ner": [ + [ + 50, + 53, + "RNA", + "chemical" + ], + [ + 137, + 164, + "zinc-containing active site", + "site" + ] + ] + }, + { + "sid": 34, + "sent": "The large binding site (1493 \u00c52 RNA surface area and 1277 \u00c52 protein surface area buried) observed for hADAR2d is consistent with recent footprinting studies.", + "section": "RESULTS", + "ner": [ + [ + 10, + 22, + "binding site", + "site" + ], + [ + 103, + 110, + "hADAR2d", + "mutant" + ], + [ + 137, + 157, + "footprinting studies", + "experimental_method" + ] + ] + }, + { + "sid": 35, + "sent": "Both strands of the RNA contact the protein with the majority of these interactions mediated through the phosphodiester-ribose backbone near the editing site (Fig. 2c, Supplementary Fig. 2 b\u2013d).", + "section": "RESULTS", + "ner": [ + [ + 20, + 23, + "RNA", + "chemical" + ], + [ + 145, + 157, + "editing site", + "site" + ] + ] + }, + { + "sid": 36, + "sent": "The structures show a large deviation from A-form RNA conformation at the editing site (Fig. 2, Fig. 3, Supplementary Video 1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 43, + 49, + "A-form", + "structure_element" + ], + [ + 50, + 53, + "RNA", + "chemical" + ], + [ + 74, + 86, + "editing site", + "site" + ] + ] + }, + { + "sid": 37, + "sent": "The 8-azanebularine is flipped out of the helix and bound into the active site as its covalent hydrate where it interacts with several amino acids including V351, T375, K376, E396 and R455 (Fig. 3a, Supplementary Fig. 3a).", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "8-azanebularine", + "chemical" + ], + [ + 23, + 34, + "flipped out", + "protein_state" + ], + [ + 42, + 47, + "helix", + "structure_element" + ], + [ + 52, + 62, + "bound into", + "protein_state" + ], + [ + 67, + 78, + "active site", + "site" + ], + [ + 157, + 161, + "V351", + "residue_name_number" + ], + [ + 163, + 167, + "T375", + "residue_name_number" + ], + [ + 169, + 173, + "K376", + "residue_name_number" + ], + [ + 175, + 179, + "E396", + "residue_name_number" + ], + [ + 184, + 188, + "R455", + "residue_name_number" + ] + ] + }, + { + "sid": 38, + "sent": "The side chain of E396 H-bonds to purine N1 and O6.", + "section": "RESULTS", + "ner": [ + [ + 18, + 22, + "E396", + "residue_name_number" + ], + [ + 23, + 30, + "H-bonds", + "bond_interaction" + ], + [ + 34, + 40, + "purine", + "chemical" + ] + ] + }, + { + "sid": 39, + "sent": "This interaction was expected given the proposed role of E396 in mediating proton transfers to and from N1 of the substrate adenosine.", + "section": "RESULTS", + "ner": [ + [ + 57, + 61, + "E396", + "residue_name_number" + ], + [ + 124, + 133, + "adenosine", + "residue_name" + ] + ] + }, + { + "sid": 40, + "sent": "The 2\u2019-hydroxyl of 8-azanebularine H-bonds to the backbone carbonyl of T375 while the T375 side chain contacts its 3\u2019-phosphodiester.", + "section": "RESULTS", + "ner": [ + [ + 19, + 34, + "8-azanebularine", + "chemical" + ], + [ + 35, + 42, + "H-bonds", + "bond_interaction" + ], + [ + 71, + 75, + "T375", + "residue_name_number" + ], + [ + 86, + 90, + "T375", + "residue_name_number" + ] + ] + }, + { + "sid": 41, + "sent": "R455 and K376 help position the flipped nucleotide in the active site by fastening the phosphate backbone flanking the editing site.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "R455", + "residue_name_number" + ], + [ + 9, + 13, + "K376", + "residue_name_number" + ], + [ + 32, + 39, + "flipped", + "protein_state" + ], + [ + 40, + 50, + "nucleotide", + "chemical" + ], + [ + 58, + 69, + "active site", + "site" + ], + [ + 119, + 131, + "editing site", + "site" + ] + ] + }, + { + "sid": 42, + "sent": "The R455 side chain ion pairs with the 5\u2019-phosphodiester of 8-azanebularine while the K376 side chain contacts its 3\u2019-phosphodiester.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "R455", + "residue_name_number" + ], + [ + 20, + 29, + "ion pairs", + "bond_interaction" + ], + [ + 60, + 75, + "8-azanebularine", + "chemical" + ], + [ + 86, + 90, + "K376", + "residue_name_number" + ] + ] + }, + { + "sid": 43, + "sent": "Lastly, the side chain of V351 provides a hydrophobic surface for interaction with the nucleobase of the edited nucleotide.", + "section": "RESULTS", + "ner": [ + [ + 26, + 30, + "V351", + "residue_name_number" + ], + [ + 42, + 61, + "hydrophobic surface", + "site" + ], + [ + 105, + 111, + "edited", + "protein_state" + ], + [ + 112, + 122, + "nucleotide", + "chemical" + ] + ] + }, + { + "sid": 44, + "sent": "RNA binding does not alter IHP binding or the H-bonding network linking IHP to the active site.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "RNA", + "chemical" + ], + [ + 27, + 30, + "IHP", + "chemical" + ], + [ + 46, + 63, + "H-bonding network", + "site" + ], + [ + 72, + 75, + "IHP", + "chemical" + ], + [ + 83, + 94, + "active site", + "site" + ] + ] + }, + { + "sid": 45, + "sent": "ADARs use a unique mechanism to modify duplex RNA", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "ADARs", + "protein_type" + ], + [ + 39, + 49, + "duplex RNA", + "structure_element" + ] + ] + }, + { + "sid": 46, + "sent": "The ADAR2 base-flipping loop, bearing residue 488, approaches the RNA duplex from the minor groove side at the editing site.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "ADAR2", + "protein" + ], + [ + 10, + 28, + "base-flipping loop", + "structure_element" + ], + [ + 46, + 49, + "488", + "residue_number" + ], + [ + 66, + 76, + "RNA duplex", + "structure_element" + ], + [ + 86, + 98, + "minor groove", + "site" + ], + [ + 111, + 123, + "editing site", + "site" + ] + ] + }, + { + "sid": 47, + "sent": "The side chain of this amino acid penetrates the helix where it occupies the space vacated by the flipped out base and H-bonds to the complementary strand orphaned base and to the 2\u2019 hydroxyl of the nucleotide immediately 5\u2019 to the editing site (Figs. 3b, 3c).", + "section": "RESULTS", + "ner": [ + [ + 98, + 109, + "flipped out", + "protein_state" + ], + [ + 110, + 114, + "base", + "chemical" + ], + [ + 119, + 126, + "H-bonds", + "bond_interaction" + ], + [ + 155, + 163, + "orphaned", + "protein_state" + ], + [ + 164, + 168, + "base", + "chemical" + ], + [ + 232, + 244, + "editing site", + "site" + ] + ] + }, + { + "sid": 48, + "sent": "In the four structures reported here, three different combinations of helix-penetrating residue and orphan base are observed (i.e. E488 + U, E488 + C and Q488 + C) and all three combinations show the same side chain and base positions (Figs. 3b, 3c, Supplementary Fig. 4a for overlay of all three).", + "section": "RESULTS", + "ner": [ + [ + 12, + 22, + "structures", + "evidence" + ], + [ + 100, + 106, + "orphan", + "protein_state" + ], + [ + 107, + 111, + "base", + "chemical" + ], + [ + 131, + 135, + "E488", + "residue_name_number" + ], + [ + 138, + 139, + "U", + "residue_name" + ], + [ + 141, + 145, + "E488", + "residue_name_number" + ], + [ + 148, + 149, + "C", + "residue_name" + ], + [ + 154, + 158, + "Q488", + "residue_name_number" + ], + [ + 161, + 162, + "C", + "residue_name" + ], + [ + 276, + 283, + "overlay", + "experimental_method" + ] + ] + }, + { + "sid": 49, + "sent": "For instance, in the complex with hADAR2d E488Q and the Bdf2-C duplex, the protein recognizes an orphaned C by donating H-bonds from N\u03b52 to cytosine N3 and from its backbone NH to cytosine O2 (Fig. 3b).", + "section": "RESULTS", + "ner": [ + [ + 21, + 33, + "complex with", + "protein_state" + ], + [ + 34, + 41, + "hADAR2d", + "mutant" + ], + [ + 42, + 47, + "E488Q", + "mutant" + ], + [ + 56, + 69, + "Bdf2-C duplex", + "chemical" + ], + [ + 97, + 105, + "orphaned", + "protein_state" + ], + [ + 106, + 107, + "C", + "residue_name" + ], + [ + 120, + 127, + "H-bonds", + "bond_interaction" + ], + [ + 140, + 148, + "cytosine", + "residue_name" + ], + [ + 180, + 188, + "cytosine", + "residue_name" + ] + ] + }, + { + "sid": 50, + "sent": "In the complex with hADAR2d WT and the Bdf2-U duplex, a similar interaction is observed with the E488 backbone NH hydrogen bonded to the uracil O2 and the E488 side chain H-bonded to the uracil N3H (Fig. 3c).", + "section": "RESULTS", + "ner": [ + [ + 7, + 19, + "complex with", + "protein_state" + ], + [ + 20, + 27, + "hADAR2d", + "mutant" + ], + [ + 28, + 30, + "WT", + "protein_state" + ], + [ + 39, + 52, + "Bdf2-U duplex", + "chemical" + ], + [ + 97, + 101, + "E488", + "residue_name_number" + ], + [ + 114, + 129, + "hydrogen bonded", + "bond_interaction" + ], + [ + 137, + 143, + "uracil", + "residue_name" + ], + [ + 155, + 159, + "E488", + "residue_name_number" + ], + [ + 171, + 179, + "H-bonded", + "bond_interaction" + ], + [ + 187, + 193, + "uracil", + "residue_name" + ] + ] + }, + { + "sid": 51, + "sent": "Interestingly, the E488Q mutant was discovered in a screen for highly active ADAR2 mutants and this residue was suggested to be involved in base flipping given its effect on editing substrates with a fluorescent nucleobase at the editing site.", + "section": "RESULTS", + "ner": [ + [ + 19, + 24, + "E488Q", + "mutant" + ], + [ + 25, + 31, + "mutant", + "protein_state" + ], + [ + 63, + 76, + "highly active", + "protein_state" + ], + [ + 77, + 82, + "ADAR2", + "protein" + ], + [ + 83, + 90, + "mutants", + "protein_state" + ], + [ + 230, + 242, + "editing site", + "site" + ] + ] + }, + { + "sid": 52, + "sent": "ADARs react preferentially with adenosines in A\u2022C mismatches and A-U pairs over A\u2022A and A\u2022G mismatches.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "ADARs", + "protein_type" + ], + [ + 32, + 42, + "adenosines", + "residue_name" + ], + [ + 46, + 49, + "A\u2022C", + "structure_element" + ], + [ + 65, + 74, + "A-U pairs", + "structure_element" + ], + [ + 80, + 83, + "A\u2022A", + "structure_element" + ], + [ + 88, + 91, + "A\u2022G", + "structure_element" + ] + ] + }, + { + "sid": 53, + "sent": "A purine at the orphan base position (in its anti conformation) would clash with the 488 residue explaining the preference for pyrimidines here.", + "section": "RESULTS", + "ner": [ + [ + 2, + 8, + "purine", + "chemical" + ], + [ + 16, + 22, + "orphan", + "protein_state" + ], + [ + 23, + 27, + "base", + "chemical" + ], + [ + 85, + 88, + "488", + "residue_number" + ], + [ + 127, + 138, + "pyrimidines", + "chemical" + ] + ] + }, + { + "sid": 54, + "sent": "The interaction of the 488 residue with the orphaned base is reminiscent of an interaction observed for Hha I DNA methyltransfersase (MTase), a duplex DNA modifying enzyme that also uses a base flipping mechanism to access 2\u2019-deoxycytidine (dC) for methylation.", + "section": "RESULTS", + "ner": [ + [ + 23, + 26, + "488", + "residue_number" + ], + [ + 44, + 52, + "orphaned", + "protein_state" + ], + [ + 53, + 57, + "base", + "chemical" + ], + [ + 104, + 132, + "Hha I DNA methyltransfersase", + "protein_type" + ], + [ + 134, + 139, + "MTase", + "protein_type" + ], + [ + 144, + 154, + "duplex DNA", + "structure_element" + ], + [ + 223, + 239, + "2\u2019-deoxycytidine", + "residue_name" + ], + [ + 241, + 243, + "dC", + "residue_name" + ] + ] + }, + { + "sid": 55, + "sent": "For that enzyme, Q237 H-bonds to an orphaned dG while it fills the void left by the flipped out dC (Supplementary Fig. 4b).", + "section": "RESULTS", + "ner": [ + [ + 17, + 21, + "Q237", + "residue_name_number" + ], + [ + 22, + 29, + "H-bonds", + "bond_interaction" + ], + [ + 36, + 44, + "orphaned", + "protein_state" + ], + [ + 45, + 47, + "dG", + "residue_name" + ], + [ + 84, + 95, + "flipped out", + "protein_state" + ], + [ + 96, + 98, + "dC", + "residue_name" + ] + ] + }, + { + "sid": 56, + "sent": "In addition, two glycine residues flank Q237 allowing the loop to adopt the conformation necessary for penetration into the helix.", + "section": "RESULTS", + "ner": [ + [ + 17, + 24, + "glycine", + "residue_name" + ], + [ + 40, + 44, + "Q237", + "residue_name_number" + ], + [ + 58, + 62, + "loop", + "structure_element" + ], + [ + 124, + 129, + "helix", + "structure_element" + ] + ] + }, + { + "sid": 57, + "sent": "The flipping loop in ADAR2 (i.e. aa487\u2013489) also has the helix-penetrating residue flanked by glycines.", + "section": "RESULTS", + "ner": [ + [ + 4, + 17, + "flipping loop", + "structure_element" + ], + [ + 21, + 26, + "ADAR2", + "protein" + ], + [ + 35, + 42, + "487\u2013489", + "residue_range" + ], + [ + 94, + 102, + "glycines", + "residue_name" + ] + ] + }, + { + "sid": 58, + "sent": "However, unlike the case of the DNA MTase that approaches the DNA from the major groove, the ADAR2 loop approaches the duplex from the minor groove side.", + "section": "RESULTS", + "ner": [ + [ + 32, + 41, + "DNA MTase", + "protein_type" + ], + [ + 62, + 65, + "DNA", + "chemical" + ], + [ + 75, + 87, + "major groove", + "site" + ], + [ + 93, + 98, + "ADAR2", + "protein" + ], + [ + 99, + 103, + "loop", + "structure_element" + ], + [ + 119, + 125, + "duplex", + "structure_element" + ], + [ + 135, + 147, + "minor groove", + "site" + ] + ] + }, + { + "sid": 59, + "sent": "Such an approach requires deeper penetration of the intercalating residue to access the H-bonding sites on the orphaned base, necessitating an additional conformational change in the RNA duplex.", + "section": "RESULTS", + "ner": [ + [ + 52, + 73, + "intercalating residue", + "site" + ], + [ + 88, + 103, + "H-bonding sites", + "site" + ], + [ + 111, + 119, + "orphaned", + "protein_state" + ], + [ + 120, + 124, + "base", + "chemical" + ], + [ + 183, + 193, + "RNA duplex", + "structure_element" + ] + ] + }, + { + "sid": 60, + "sent": "This change includes shifting of the base pairs immediately 5\u2019 to the editing site toward the helical axis and a widening of the major groove opposite the editing site (Figs. 4a, 4b, Supplementary Video 1).", + "section": "RESULTS", + "ner": [ + [ + 70, + 82, + "editing site", + "site" + ], + [ + 129, + 141, + "major groove", + "site" + ], + [ + 155, + 167, + "editing site", + "site" + ] + ] + }, + { + "sid": 61, + "sent": "In the case of the hADAR2d WT\u2013Bdf2-U RNA, this shift is accompanied by a shearing of the U11-A13' base pair with U11 shifted further in the direction of the major groove creating an unusual U-A \"wobble\" interaction with adenine N6 and N1 within H-bonding distance to uracil N3H and O2, respectively (Fig. 4c, Supplementary Fig. 3b).", + "section": "RESULTS", + "ner": [ + [ + 19, + 36, + "hADAR2d WT\u2013Bdf2-U", + "complex_assembly" + ], + [ + 37, + 40, + "RNA", + "chemical" + ], + [ + 89, + 92, + "U11", + "residue_name_number" + ], + [ + 93, + 96, + "A13", + "residue_name_number" + ], + [ + 113, + 116, + "U11", + "residue_name_number" + ], + [ + 157, + 169, + "major groove", + "site" + ], + [ + 190, + 202, + "U-A \"wobble\"", + "structure_element" + ], + [ + 220, + 227, + "adenine", + "residue_name" + ], + [ + 245, + 254, + "H-bonding", + "bond_interaction" + ], + [ + 267, + 273, + "uracil", + "residue_name" + ] + ] + }, + { + "sid": 62, + "sent": "This type of wobble pair has been observed before and requires either the imino tautomer of adenine or the enol tautomer of uracil.", + "section": "RESULTS", + "ner": [ + [ + 92, + 99, + "adenine", + "residue_name" + ], + [ + 124, + 130, + "uracil", + "residue_name" + ] + ] + }, + { + "sid": 63, + "sent": "The ADAR-induced distortion in RNA conformation results in a kink in the RNA strand opposite the editing site (Fig. 4b).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "ADAR", + "protein_type" + ], + [ + 31, + 34, + "RNA", + "chemical" + ], + [ + 61, + 65, + "kink", + "structure_element" + ], + [ + 73, + 76, + "RNA", + "chemical" + ], + [ + 97, + 109, + "editing site", + "site" + ] + ] + }, + { + "sid": 64, + "sent": "This kink is stabilized by interactions of the side chains of R510 and S495 with phosphodiesters in the RNA backbone of the unedited strand (Fig. 4a).", + "section": "RESULTS", + "ner": [ + [ + 5, + 9, + "kink", + "structure_element" + ], + [ + 62, + 66, + "R510", + "residue_name_number" + ], + [ + 71, + 75, + "S495", + "residue_name_number" + ], + [ + 104, + 107, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 65, + "sent": "Interestingly, ADAR2\u2019s flipping loop approach from the minor groove side is like that seen with certain DNA repair glycosylases (e.g. UDG, HOGG1, and AAG) that also project intercalating residues from loops bound in the minor groove (Supplementary Fig. 5a).", + "section": "RESULTS", + "ner": [ + [ + 15, + 20, + "ADAR2", + "protein" + ], + [ + 23, + 36, + "flipping loop", + "structure_element" + ], + [ + 55, + 67, + "minor groove", + "site" + ], + [ + 104, + 127, + "DNA repair glycosylases", + "protein_type" + ], + [ + 134, + 137, + "UDG", + "protein" + ], + [ + 139, + 144, + "HOGG1", + "protein" + ], + [ + 150, + 153, + "AAG", + "protein" + ], + [ + 201, + 206, + "loops", + "structure_element" + ], + [ + 207, + 215, + "bound in", + "protein_state" + ], + [ + 220, + 232, + "minor groove", + "site" + ] + ] + }, + { + "sid": 66, + "sent": "However, these enzymes typically bend the DNA duplex at the site of modification to allow for penetration of intercalating residues and damage recognition.", + "section": "RESULTS", + "ner": [ + [ + 42, + 52, + "DNA duplex", + "chemical" + ] + ] + }, + { + "sid": 67, + "sent": "While hADAR2d clearly alters the duplex conformation to gain access to the modification site from the minor groove, it does not bend the RNA duplex (Figs. 2a, 2b, 4b).", + "section": "RESULTS", + "ner": [ + [ + 6, + 13, + "hADAR2d", + "mutant" + ], + [ + 102, + 114, + "minor groove", + "site" + ], + [ + 137, + 147, + "RNA duplex", + "structure_element" + ] + ] + }, + { + "sid": 68, + "sent": "Furthermore, ADARs do not modify duplex DNA.", + "section": "RESULTS", + "ner": [ + [ + 13, + 18, + "ADARs", + "protein_type" + ], + [ + 33, + 43, + "duplex DNA", + "structure_element" + ] + ] + }, + { + "sid": 69, + "sent": "The DNA B-form helix has groove widths and depths that would prevent productive interactions with ADAR.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "DNA", + "chemical" + ], + [ + 8, + 20, + "B-form helix", + "structure_element" + ], + [ + 98, + 102, + "ADAR", + "protein_type" + ] + ] + }, + { + "sid": 70, + "sent": "For instance, ADAR can readily penetrate an A-form helix from the minor groove side and place the helix-penetrating residue in the space occupied by the editing site base (Supplementary Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "ADAR", + "protein_type" + ], + [ + 44, + 56, + "A-form helix", + "structure_element" + ], + [ + 66, + 78, + "minor groove", + "site" + ], + [ + 153, + 165, + "editing site", + "site" + ] + ] + }, + { + "sid": 71, + "sent": "However, this residue cannot penetrate the minor groove enough to occupy the base position in a B-form helix (Supplementary Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 43, + 55, + "minor groove", + "site" + ], + [ + 96, + 108, + "B-form helix", + "structure_element" + ] + ] + }, + { + "sid": 72, + "sent": "Furthermore, DNA lacks the 2\u2019 hydroxyls that are used by ADAR for substrate recognition (Fig. 2c).", + "section": "RESULTS", + "ner": [ + [ + 13, + 16, + "DNA", + "chemical" + ], + [ + 57, + 61, + "ADAR", + "protein_type" + ] + ] + }, + { + "sid": 73, + "sent": "Thus, hADAR2d uses a substrate recognition and base flipping mechanism with similarities to other known nucleic acid-modifying enzymes but uniquely suited for reaction with adenosine in the context of duplex RNA.", + "section": "RESULTS", + "ner": [ + [ + 6, + 13, + "hADAR2d", + "mutant" + ], + [ + 104, + 134, + "nucleic acid-modifying enzymes", + "protein_type" + ], + [ + 173, + 182, + "adenosine", + "residue_name" + ], + [ + 201, + 211, + "duplex RNA", + "structure_element" + ] + ] + }, + { + "sid": 74, + "sent": "Structures explain nearest neighbor preferences", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ] + ] + }, + { + "sid": 75, + "sent": "ADARs have a preference for editing adenosines with 5\u2019 nearest neighbor U (or A) and 3\u2019 nearest neighbor G. The ADAR2 flipping loop occupies the minor groove spanning the three base pairs that include the nearest neighbor nucleotides flanking the edited base (Figs. 3b, 3c).", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "ADARs", + "protein_type" + ], + [ + 36, + 46, + "adenosines", + "residue_name" + ], + [ + 72, + 73, + "U", + "residue_name" + ], + [ + 78, + 79, + "A", + "residue_name" + ], + [ + 105, + 106, + "G", + "residue_name" + ], + [ + 112, + 117, + "ADAR2", + "protein" + ], + [ + 118, + 131, + "flipping loop", + "structure_element" + ], + [ + 145, + 157, + "minor groove", + "site" + ] + ] + }, + { + "sid": 76, + "sent": "As described above, the base pair including the 5\u2019 nearest neighbor U (U11-A13\u2019 in the Bdf2 duplex) is shifted from the position it would occupy in a typical A-form helix to accommodate the loop (Fig. 4a).", + "section": "RESULTS", + "ner": [ + [ + 68, + 69, + "U", + "residue_name" + ], + [ + 71, + 74, + "U11", + "residue_name_number" + ], + [ + 75, + 78, + "A13", + "residue_name_number" + ], + [ + 87, + 91, + "Bdf2", + "chemical" + ], + [ + 158, + 170, + "A-form helix", + "structure_element" + ], + [ + 190, + 194, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 77, + "sent": "Also, the minor groove edge of this pair is juxtaposed to the protein backbone at G489.", + "section": "RESULTS", + "ner": [ + [ + 10, + 22, + "minor groove", + "site" + ], + [ + 82, + 86, + "G489", + "residue_name_number" + ] + ] + }, + { + "sid": 78, + "sent": "Modeling a G-C or C-G pair at this position (i.e. 5\u2019 G or 5\u2019 C) suggests a 2-amino group in the minor groove would clash with the protein at G489 (Fig. 5a, Supplementary Fig. 7c).", + "section": "RESULTS", + "ner": [ + [ + 11, + 26, + "G-C or C-G pair", + "structure_element" + ], + [ + 53, + 54, + "G", + "residue_name" + ], + [ + 61, + 62, + "C", + "residue_name" + ], + [ + 96, + 108, + "minor groove", + "site" + ], + [ + 141, + 145, + "G489", + "residue_name_number" + ] + ] + }, + { + "sid": 79, + "sent": "Indeed, replacing the U-A pair adjacent to the editing site with a C-G pair in the Gli1 duplex substrate resulted in an 80% reduction in the rate of hADAR2d-catalyzed deamination (Figs. 5b, 5c).", + "section": "RESULTS", + "ner": [ + [ + 22, + 30, + "U-A pair", + "structure_element" + ], + [ + 47, + 59, + "editing site", + "site" + ], + [ + 67, + 75, + "C-G pair", + "structure_element" + ], + [ + 83, + 87, + "Gli1", + "protein" + ], + [ + 149, + 156, + "hADAR2d", + "mutant" + ] + ] + }, + { + "sid": 80, + "sent": "To determine whether this effect arises from an increase in local duplex stability from the C-G for U-A substitution or from the presence of the 2-amino group, we replaced the U-A pair with a U-2-aminopurine (2AP) pair.", + "section": "RESULTS", + "ner": [ + [ + 176, + 184, + "U-A pair", + "structure_element" + ], + [ + 192, + 218, + "U-2-aminopurine (2AP) pair", + "structure_element" + ] + ] + }, + { + "sid": 81, + "sent": "2AP is an adenosine analog that forms a base pair with uridine of similar stability to a U-A pair, but places an amino group in the minor groove (Fig. 5b).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "2AP", + "structure_element" + ], + [ + 10, + 19, + "adenosine", + "residue_name" + ], + [ + 55, + 62, + "uridine", + "residue_name" + ], + [ + 89, + 97, + "U-A pair", + "structure_element" + ], + [ + 132, + 144, + "minor groove", + "site" + ] + ] + }, + { + "sid": 82, + "sent": "Importantly, this substitution also resulted in an 80% reduction in rate, illustrating the detrimental effect of the amino group in the minor groove at this location.", + "section": "RESULTS", + "ner": [ + [ + 136, + 148, + "minor groove", + "site" + ] + ] + }, + { + "sid": 83, + "sent": "These observations suggest that hADAR2\u2019s 5\u2019 nearest neighbor preference for U (or A) is due to a destabilizing clash with the protein backbone at G489 that results from the presence of an amino group in the minor groove at this location for sequences with 5\u2019 nearest neighbor G or C. However, the observed clash is not severe and the enzyme would be able to accommodate G or C 5\u2019 nearest neighbors by slight structural perturbations, explaining why this sequence preference is not an absolute requirement.", + "section": "RESULTS", + "ner": [ + [ + 32, + 38, + "hADAR2", + "protein" + ], + [ + 76, + 77, + "U", + "residue_name" + ], + [ + 82, + 83, + "A", + "residue_name" + ], + [ + 146, + 150, + "G489", + "residue_name_number" + ], + [ + 207, + 219, + "minor groove", + "site" + ], + [ + 276, + 277, + "G", + "residue_name" + ], + [ + 281, + 282, + "C", + "residue_name" + ], + [ + 370, + 371, + "G", + "residue_name" + ], + [ + 375, + 376, + "C", + "residue_name" + ] + ] + }, + { + "sid": 84, + "sent": "In each of the hADAR2d-RNA structures reported here, the backbone carbonyl oxygen at S486 accepts an H-bond from the 2-amino group of the G on the 3\u2019 side of the edited nucleotide (Fig. 5d).", + "section": "RESULTS", + "ner": [ + [ + 15, + 26, + "hADAR2d-RNA", + "complex_assembly" + ], + [ + 27, + 37, + "structures", + "evidence" + ], + [ + 85, + 89, + "S486", + "residue_name_number" + ], + [ + 101, + 107, + "H-bond", + "bond_interaction" + ], + [ + 138, + 139, + "G", + "residue_name" + ] + ] + }, + { + "sid": 85, + "sent": "Guanine is the only common nucleobase that presents an H-bond donor in the RNA minor groove suggesting that other nucleotides in this position would reduce editing efficiency.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Guanine", + "residue_name" + ], + [ + 55, + 61, + "H-bond", + "bond_interaction" + ], + [ + 75, + 91, + "RNA minor groove", + "site" + ] + ] + }, + { + "sid": 86, + "sent": "Indeed, mutating this base to A, C or U, while maintaining base pairing at this position, reduced the rate of deamination by hADAR2d in Gli1 mRNA model substrates (Supplementary Fig. 7 a\u2013b).", + "section": "RESULTS", + "ner": [ + [ + 8, + 16, + "mutating", + "experimental_method" + ], + [ + 30, + 31, + "A", + "residue_name" + ], + [ + 33, + 34, + "C", + "residue_name" + ], + [ + 38, + 39, + "U", + "residue_name" + ], + [ + 125, + 132, + "hADAR2d", + "mutant" + ], + [ + 136, + 140, + "Gli1", + "protein" + ], + [ + 141, + 145, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 87, + "sent": "To test the importance of the amino group on the 3\u2019 G in the hADAR2d reaction, we prepared RNA duplex substrates with purine analogs on the 3\u2019 side of the edited A (Fig. 5e).", + "section": "RESULTS", + "ner": [ + [ + 52, + 53, + "G", + "residue_name" + ], + [ + 61, + 68, + "hADAR2d", + "mutant" + ], + [ + 91, + 101, + "RNA duplex", + "structure_element" + ], + [ + 155, + 161, + "edited", + "protein_state" + ], + [ + 162, + 163, + "A", + "residue_name" + ] + ] + }, + { + "sid": 88, + "sent": "We tested a G analog that lacks the 2-amino group (inosine, I) and one that blocks access to this amino group (N2-methylguanosine (N2MeG).", + "section": "RESULTS", + "ner": [ + [ + 12, + 13, + "G", + "residue_name" + ], + [ + 51, + 58, + "inosine", + "residue_name" + ], + [ + 60, + 61, + "I", + "residue_name" + ] + ] + }, + { + "sid": 89, + "sent": "In addition, we compared a 3\u2019 A to a 3\u2019 2AP since 2AP could form the H-bonding interaction observed with S486.", + "section": "RESULTS", + "ner": [ + [ + 30, + 31, + "A", + "residue_name" + ], + [ + 40, + 43, + "2AP", + "structure_element" + ], + [ + 50, + 53, + "2AP", + "structure_element" + ], + [ + 69, + 90, + "H-bonding interaction", + "bond_interaction" + ], + [ + 105, + 109, + "S486", + "residue_name_number" + ] + ] + }, + { + "sid": 90, + "sent": "We found the substrate with a 3\u2019 N2MeG to be unreactive to hADAR2d-catalyzed deamination confirming the importance of the observed close approach by the protein to the 3\u2019 G 2-amino group (Fig. 5f).", + "section": "RESULTS", + "ner": [ + [ + 59, + 66, + "hADAR2d", + "mutant" + ], + [ + 171, + 172, + "G", + "residue_name" + ] + ] + }, + { + "sid": 91, + "sent": "In addition, the substrate with a 3\u2019 I displayed a reduced deamination rate compared to the substrate with a 3\u2019 G suggesting the observed H-bond to the 2-amino group contributes to the 3\u2019 nearest neighbor selectivity (Fig. 5f).", + "section": "RESULTS", + "ner": [ + [ + 37, + 38, + "I", + "residue_name" + ], + [ + 51, + 75, + "reduced deamination rate", + "evidence" + ], + [ + 112, + 113, + "G", + "residue_name" + ], + [ + 138, + 144, + "H-bond", + "bond_interaction" + ] + ] + }, + { + "sid": 92, + "sent": "This conclusion is further supported by the observation that deamination in the substrate with a 3\u2019 2AP is faster than in the substrate with a 3\u2019 A (Fig. 5f).", + "section": "RESULTS", + "ner": [ + [ + 100, + 103, + "2AP", + "structure_element" + ], + [ + 146, + 147, + "A", + "residue_name" + ] + ] + }, + { + "sid": 93, + "sent": "RNA-binding loops of the ADAR catalytic domain", + "section": "RESULTS", + "ner": [ + [ + 0, + 17, + "RNA-binding loops", + "structure_element" + ], + [ + 25, + 29, + "ADAR", + "protein_type" + ], + [ + 30, + 46, + "catalytic domain", + "structure_element" + ] + ] + }, + { + "sid": 94, + "sent": "The structures reported here identify RNA-binding loops of the ADAR catalytic domain and suggest roles for several amino acids not previously known to be important for editing, either substrate binding or catalysis (Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 38, + 55, + "RNA-binding loops", + "structure_element" + ], + [ + 63, + 67, + "ADAR", + "protein_type" + ], + [ + 68, + 84, + "catalytic domain", + "structure_element" + ] + ] + }, + { + "sid": 95, + "sent": "The side chain for R510 ion-pairs with the 3\u2019 phosphodiester of the orphaned nucleotide (Figs. 3a, 3c).", + "section": "RESULTS", + "ner": [ + [ + 19, + 23, + "R510", + "residue_name_number" + ], + [ + 24, + 33, + "ion-pairs", + "bond_interaction" + ], + [ + 68, + 76, + "orphaned", + "protein_state" + ], + [ + 77, + 87, + "nucleotide", + "chemical" + ] + ] + }, + { + "sid": 96, + "sent": "This residue is conserved in ADAR2s and ADAR1s, but is glutamine in the editing-inactive ADAR3s (Supplementary Table 1).", + "section": "RESULTS", + "ner": [ + [ + 16, + 25, + "conserved", + "protein_state" + ], + [ + 29, + 35, + "ADAR2s", + "protein_type" + ], + [ + 40, + 46, + "ADAR1s", + "protein_type" + ], + [ + 55, + 64, + "glutamine", + "residue_name" + ], + [ + 72, + 88, + "editing-inactive", + "protein_state" + ], + [ + 89, + 95, + "ADAR3s", + "protein_type" + ] + ] + }, + { + "sid": 97, + "sent": "Mutation of hADAR2d at this site to either glutamine (R510Q) or to alanine (R510A) reduced the measured deamination rate constant by approximately an order of magnitude (Fig. 6c).", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Mutation", + "experimental_method" + ], + [ + 12, + 19, + "hADAR2d", + "mutant" + ], + [ + 43, + 52, + "glutamine", + "residue_name" + ], + [ + 54, + 59, + "R510Q", + "mutant" + ], + [ + 67, + 74, + "alanine", + "residue_name" + ], + [ + 76, + 81, + "R510A", + "mutant" + ], + [ + 104, + 129, + "deamination rate constant", + "evidence" + ] + ] + }, + { + "sid": 98, + "sent": "In addition, the contact point near the 5\u2019 end of the unedited strand involves G593, K594 and R348, residues completely conserved in the family of ADAR2s (Fig. 2c, Supplementary Table 1).", + "section": "RESULTS", + "ner": [ + [ + 79, + 83, + "G593", + "residue_name_number" + ], + [ + 85, + 89, + "K594", + "residue_name_number" + ], + [ + 94, + 98, + "R348", + "residue_name_number" + ], + [ + 109, + 129, + "completely conserved", + "protein_state" + ], + [ + 147, + 153, + "ADAR2s", + "protein_type" + ] + ] + }, + { + "sid": 99, + "sent": "Mutation of any of these residues to alanine (G593A, K594A, R348A) substantially reduces editing activity (Fig. 6c).", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Mutation", + "experimental_method" + ], + [ + 37, + 44, + "alanine", + "residue_name" + ], + [ + 46, + 51, + "G593A", + "mutant" + ], + [ + 53, + 58, + "K594A", + "mutant" + ], + [ + 60, + 65, + "R348A", + "mutant" + ] + ] + }, + { + "sid": 100, + "sent": "In addition, mutation of G593 to glutamic acid (G593E) resulted in a nearly two orders of magnitude reduction in rate, consistent with proximity of this residue to the negatively charged phosphodiester backbone of the RNA (Fig. 6c).", + "section": "RESULTS", + "ner": [ + [ + 13, + 21, + "mutation", + "experimental_method" + ], + [ + 25, + 29, + "G593", + "residue_name_number" + ], + [ + 33, + 46, + "glutamic acid", + "residue_name" + ], + [ + 48, + 53, + "G593E", + "mutant" + ], + [ + 218, + 221, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 101, + "sent": "RNA binding leads to an ordering of the 454\u2013477 loop, which was disordered in the RNA-free hADAR2d structure (Fig. 1d, green) (Supplementary Video 2).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "RNA", + "chemical" + ], + [ + 40, + 47, + "454\u2013477", + "residue_range" + ], + [ + 48, + 52, + "loop", + "structure_element" + ], + [ + 64, + 74, + "disordered", + "protein_state" + ], + [ + 82, + 90, + "RNA-free", + "protein_state" + ], + [ + 91, + 98, + "hADAR2d", + "mutant" + ], + [ + 99, + 108, + "structure", + "evidence" + ] + ] + }, + { + "sid": 102, + "sent": "This loop binds the RNA duplex contacting the minor groove near the editing site and inserting into the adjacent major groove (Fig. 6e).", + "section": "RESULTS", + "ner": [ + [ + 5, + 9, + "loop", + "structure_element" + ], + [ + 20, + 30, + "RNA duplex", + "structure_element" + ], + [ + 46, + 58, + "minor groove", + "site" + ], + [ + 68, + 80, + "editing site", + "site" + ], + [ + 113, + 125, + "major groove", + "site" + ] + ] + }, + { + "sid": 103, + "sent": "This loop sequence is conserved in ADAR2s but different in the family of ADAR1s (Fig. 6d).", + "section": "RESULTS", + "ner": [ + [ + 5, + 9, + "loop", + "structure_element" + ], + [ + 22, + 31, + "conserved", + "protein_state" + ], + [ + 35, + 41, + "ADAR2s", + "protein_type" + ], + [ + 73, + 79, + "ADAR1s", + "protein_type" + ] + ] + }, + { + "sid": 104, + "sent": "The substantial difference in sequence between the ADARs in this RNA-binding loop suggests differences in editing site selectivity between the two ADARs arise, at least in part, from differences in how this loop binds RNA substrates.", + "section": "RESULTS", + "ner": [ + [ + 51, + 56, + "ADARs", + "protein_type" + ], + [ + 65, + 81, + "RNA-binding loop", + "structure_element" + ], + [ + 106, + 118, + "editing site", + "site" + ], + [ + 147, + 152, + "ADARs", + "protein_type" + ], + [ + 207, + 211, + "loop", + "structure_element" + ], + [ + 218, + 221, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 105, + "sent": "Base flipping is a well-characterized mechanism by which nucleic acid modifying enzymes gain access to sites of reaction that are otherwise buried in base-paired structures.", + "section": "DISCUSS", + "ner": [ + [ + 57, + 87, + "nucleic acid modifying enzymes", + "protein_type" + ], + [ + 162, + 172, + "structures", + "evidence" + ] + ] + }, + { + "sid": 106, + "sent": "DNA methylases, DNA repair glycosylases and RNA loop modifying enzymes are known that flip a nucleotide out of a base pair.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 14, + "DNA methylases", + "protein_type" + ], + [ + 16, + 39, + "DNA repair glycosylases", + "protein_type" + ], + [ + 44, + 70, + "RNA loop modifying enzymes", + "protein_type" + ], + [ + 93, + 103, + "nucleotide", + "chemical" + ] + ] + }, + { + "sid": 107, + "sent": "However, none of the structurally characterized base-flipping enzymes access their reactive sites from within a normal base-paired RNA duplex.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 69, + "base-flipping enzymes", + "protein_type" + ], + [ + 83, + 97, + "reactive sites", + "site" + ], + [ + 112, + 130, + "normal base-paired", + "protein_state" + ], + [ + 131, + 141, + "RNA duplex", + "structure_element" + ] + ] + }, + { + "sid": 108, + "sent": "We are aware of one other protein-induced nucleotide flipping from an RNA duplex region.", + "section": "DISCUSS", + "ner": [ + [ + 70, + 80, + "RNA duplex", + "structure_element" + ] + ] + }, + { + "sid": 109, + "sent": "Bacterial initiation factor 1 (IF1) binds to the 30S ribosomal subunit at helix 44 of 16S RNA with A1492 and A1493 flipped out of the helix and bound into protein pockets (Supplementary Fig. 5b).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 9, + "Bacterial", + "taxonomy_domain" + ], + [ + 10, + 29, + "initiation factor 1", + "protein" + ], + [ + 31, + 34, + "IF1", + "protein" + ], + [ + 49, + 70, + "30S ribosomal subunit", + "complex_assembly" + ], + [ + 74, + 82, + "helix 44", + "structure_element" + ], + [ + 86, + 93, + "16S RNA", + "chemical" + ], + [ + 99, + 104, + "A1492", + "residue_name_number" + ], + [ + 109, + 114, + "A1493", + "residue_name_number" + ], + [ + 115, + 126, + "flipped out", + "protein_state" + ], + [ + 144, + 154, + "bound into", + "protein_state" + ], + [ + 155, + 170, + "protein pockets", + "site" + ] + ] + }, + { + "sid": 110, + "sent": "However, these nucleotides are located in a highly distorted and dynamic duplex region containing several mismatches and are predisposed to undergo this conformational change.", + "section": "DISCUSS", + "ner": [ + [ + 44, + 60, + "highly distorted", + "protein_state" + ], + [ + 65, + 72, + "dynamic", + "protein_state" + ], + [ + 73, + 86, + "duplex region", + "structure_element" + ] + ] + }, + { + "sid": 111, + "sent": "Thus, this system is not illustrative of base flipping from a normal duplex and does not involve an enzyme that must carryout a chemical reaction on the flipped out nucleotide.", + "section": "DISCUSS", + "ner": [ + [ + 62, + 68, + "normal", + "protein_state" + ], + [ + 153, + 164, + "flipped out", + "protein_state" + ], + [ + 165, + 175, + "nucleotide", + "chemical" + ] + ] + }, + { + "sid": 112, + "sent": "Other RNA modification enzymes are known that flip nucleotides out of loops, even from base pairs in loop regions (pseudoU synthetase, tRNA transglycosylase, and restrictocin bound to sarcin/ricin loop of 28S rRNA) (Supplementary Fig. 5b).", + "section": "DISCUSS", + "ner": [ + [ + 6, + 30, + "RNA modification enzymes", + "protein_type" + ], + [ + 115, + 133, + "pseudoU synthetase", + "protein_type" + ], + [ + 135, + 156, + "tRNA transglycosylase", + "protein_type" + ], + [ + 162, + 174, + "restrictocin", + "protein" + ], + [ + 175, + 183, + "bound to", + "protein_state" + ], + [ + 184, + 201, + "sarcin/ricin loop", + "structure_element" + ], + [ + 205, + 213, + "28S rRNA", + "chemical" + ] + ] + }, + { + "sid": 113, + "sent": "Because the modification sites are not flanked on both sides by normal duplex, these enzymes do not experience the same limits in approach to the substrate that ADARs do.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 30, + "modification sites", + "site" + ], + [ + 64, + 70, + "normal", + "protein_state" + ], + [ + 71, + 77, + "duplex", + "structure_element" + ], + [ + 161, + 166, + "ADARs", + "protein_type" + ] + ] + }, + { + "sid": 114, + "sent": "The fact that ADARs must induce flipping from a normal duplex has implications on its preference for adenosines flanked by certain base pairs, a phenomenon that was not well understood prior to this work.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 19, + "ADARs", + "protein_type" + ], + [ + 48, + 54, + "normal", + "protein_state" + ], + [ + 55, + 61, + "duplex", + "structure_element" + ], + [ + 101, + 111, + "adenosines", + "residue_name" + ] + ] + }, + { + "sid": 115, + "sent": "In our structures, the flipped out 8-azanebularine is hydrated, mimicking the tetrahedral intermediate predicted for deamination of adenosine (Figs. 1b, 3a, Supplementary Fig. 3 a\u2013b).", + "section": "DISCUSS", + "ner": [ + [ + 7, + 17, + "structures", + "evidence" + ], + [ + 23, + 34, + "flipped out", + "protein_state" + ], + [ + 35, + 50, + "8-azanebularine", + "chemical" + ], + [ + 132, + 141, + "adenosine", + "residue_name" + ] + ] + }, + { + "sid": 116, + "sent": "Our use of 8-azanebularine, with its high propensity to form a covalent hydrate, allowed us to capture a true mimic of the tetrahedral intermediate and reveal the interactions between the deaminase active site and the reactive nucleotide.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 26, + "8-azanebularine", + "chemical" + ], + [ + 188, + 197, + "deaminase", + "protein_type" + ], + [ + 198, + 209, + "active site", + "site" + ] + ] + }, + { + "sid": 117, + "sent": "In addition, 8-azanebularine was found to adopt a 2\u2019-endo sugar pucker with its 2\u2019-hydroxyl H-bonded to the protein backbone carbonyl at T375.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 28, + "8-azanebularine", + "chemical" + ], + [ + 92, + 100, + "H-bonded", + "bond_interaction" + ], + [ + 137, + 141, + "T375", + "residue_name_number" + ] + ] + }, + { + "sid": 118, + "sent": "The 2\u2019 endo conformation appears to facilitate access of the nucleobase to the zinc-bound water for nucleophilic attack at C6.", + "section": "DISCUSS", + "ner": [ + [ + 79, + 83, + "zinc", + "chemical" + ], + [ + 90, + 95, + "water", + "chemical" + ] + ] + }, + { + "sid": 119, + "sent": "Several other base-flipping enzymes stabilize the altered nucleic acid conformation by intercalation of an amino acid side chain into the space vacated by the flipped out base.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 35, + "base-flipping enzymes", + "protein_type" + ], + [ + 159, + 170, + "flipped out", + "protein_state" + ], + [ + 171, + 175, + "base", + "chemical" + ] + ] + }, + { + "sid": 120, + "sent": "For hADAR2, E488 serves this role.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 10, + "hADAR2", + "protein" + ], + [ + 12, + 16, + "E488", + "residue_name_number" + ] + ] + }, + { + "sid": 121, + "sent": "In the two structures with wild type hADAR2, the E488 residue and orphan base are in nearly identical positions (see Supplementary Fig. 4a for overlay).", + "section": "DISCUSS", + "ner": [ + [ + 11, + 21, + "structures", + "evidence" + ], + [ + 27, + 36, + "wild type", + "protein_state" + ], + [ + 37, + 43, + "hADAR2", + "protein" + ], + [ + 49, + 53, + "E488", + "residue_name_number" + ], + [ + 66, + 72, + "orphan", + "protein_state" + ], + [ + 73, + 77, + "base", + "chemical" + ], + [ + 143, + 150, + "overlay", + "experimental_method" + ] + ] + }, + { + "sid": 122, + "sent": "Thus, the E488 side chain directly contacts each orphan base, likely by accepting an H-bond from uracil N3H or by donating an H-bond to cytidine N3.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 14, + "E488", + "residue_name_number" + ], + [ + 49, + 55, + "orphan", + "protein_state" + ], + [ + 56, + 60, + "base", + "chemical" + ], + [ + 85, + 91, + "H-bond", + "bond_interaction" + ], + [ + 97, + 103, + "uracil", + "residue_name" + ], + [ + 126, + 132, + "H-bond", + "bond_interaction" + ], + [ + 136, + 144, + "cytidine", + "residue_name" + ] + ] + }, + { + "sid": 123, + "sent": "The latter interaction requires E488 to be protonated.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 36, + "E488", + "residue_name_number" + ], + [ + 43, + 53, + "protonated", + "protein_state" + ] + ] + }, + { + "sid": 124, + "sent": "The pKa of E488 in the ADAR-RNA complex has not been measured, but proximity to H-bond acceptors, such as cytidine N3, and insertion between stacked nucleobases, would undoubtedly elevate this value and could lead to a substantial fraction in the protonated state at physiologically relevant pH. Since the glutamine side chain is fully protonated under physiologically relevant conditions, a rate enhancement for the E488Q mutant would be expected if the reaction requires E488 protonation.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 7, + "pKa", + "evidence" + ], + [ + 11, + 15, + "E488", + "residue_name_number" + ], + [ + 23, + 31, + "ADAR-RNA", + "complex_assembly" + ], + [ + 80, + 86, + "H-bond", + "bond_interaction" + ], + [ + 106, + 114, + "cytidine", + "residue_name" + ], + [ + 247, + 257, + "protonated", + "protein_state" + ], + [ + 306, + 315, + "glutamine", + "residue_name" + ], + [ + 330, + 346, + "fully protonated", + "protein_state" + ], + [ + 417, + 422, + "E488Q", + "mutant" + ], + [ + 423, + 429, + "mutant", + "protein_state" + ], + [ + 473, + 477, + "E488", + "residue_name_number" + ] + ] + }, + { + "sid": 125, + "sent": "The interactions of hADAR2d with base pairs adjacent to the editing site adenosine explain the known 5\u2019 and 3\u2019 nearest neighbor preferences (Fig. 5).", + "section": "DISCUSS", + "ner": [ + [ + 20, + 27, + "hADAR2d", + "mutant" + ], + [ + 60, + 72, + "editing site", + "site" + ], + [ + 73, + 82, + "adenosine", + "residue_name" + ] + ] + }, + { + "sid": 126, + "sent": "While these studies indicate the ADAR2 catalytic domain makes an important contact to the 3\u2019 nearest neighbor G, Stefl et al. suggested the 3\u2019 G preference arises from dsRBD binding selectivity for ADAR2.", + "section": "DISCUSS", + "ner": [ + [ + 33, + 38, + "ADAR2", + "protein" + ], + [ + 39, + 55, + "catalytic domain", + "structure_element" + ], + [ + 110, + 111, + "G", + "residue_name" + ], + [ + 143, + 144, + "G", + "residue_name" + ], + [ + 168, + 173, + "dsRBD", + "structure_element" + ], + [ + 198, + 203, + "ADAR2", + "protein" + ] + ] + }, + { + "sid": 127, + "sent": "These authors reported a model for ADAR2\u2019s dsRBDs bound to an editing substrate based on NMR data from the isolated dsRBDs (lacking the deaminase domain) and short RNA fragments derived from the GluR-B R/G site RNA.", + "section": "DISCUSS", + "ner": [ + [ + 35, + 40, + "ADAR2", + "protein" + ], + [ + 43, + 49, + "dsRBDs", + "structure_element" + ], + [ + 50, + 58, + "bound to", + "protein_state" + ], + [ + 89, + 92, + "NMR", + "experimental_method" + ], + [ + 107, + 115, + "isolated", + "protein_state" + ], + [ + 116, + 122, + "dsRBDs", + "structure_element" + ], + [ + 124, + 131, + "lacking", + "protein_state" + ], + [ + 136, + 152, + "deaminase domain", + "structure_element" + ], + [ + 164, + 167, + "RNA", + "chemical" + ], + [ + 195, + 201, + "GluR-B", + "protein" + ], + [ + 202, + 210, + "R/G site", + "site" + ], + [ + 211, + 214, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 128, + "sent": "They describe an interaction wherein the 3\u2019 G 2-amino group H-bonds to the backbone carbonyl of S258 found in the \u03b21-\u03b22 loop of ADAR2\u2019s dsRBDII.", + "section": "DISCUSS", + "ner": [ + [ + 44, + 45, + "G", + "residue_name" + ], + [ + 60, + 67, + "H-bonds", + "bond_interaction" + ], + [ + 96, + 100, + "S258", + "residue_name_number" + ], + [ + 114, + 124, + "\u03b21-\u03b22 loop", + "structure_element" + ], + [ + 128, + 133, + "ADAR2", + "protein" + ], + [ + 136, + 143, + "dsRBDII", + "structure_element" + ] + ] + }, + { + "sid": 129, + "sent": "It is not possible for the S486-3\u2019G interaction we describe here and the S258-3\u2019G interaction reported by Stefl et al. to exist in the same complex since both involve protein loops bound in the RNA minor groove at the same location.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 31, + "S486", + "residue_name_number" + ], + [ + 34, + 35, + "G", + "residue_name" + ], + [ + 73, + 77, + "S258", + "residue_name_number" + ], + [ + 80, + 81, + "G", + "residue_name" + ], + [ + 181, + 189, + "bound in", + "protein_state" + ], + [ + 194, + 197, + "RNA", + "chemical" + ], + [ + 198, + 210, + "minor groove", + "site" + ] + ] + }, + { + "sid": 130, + "sent": "Because our structures have captured the edited nucleotide in the conformation required to access the active site, the interactions observed here are highly likely to occur during the deamination reaction at the editing site.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 22, + "structures", + "evidence" + ], + [ + 41, + 47, + "edited", + "protein_state" + ], + [ + 48, + 58, + "nucleotide", + "chemical" + ], + [ + 102, + 113, + "active site", + "site" + ], + [ + 212, + 224, + "editing site", + "site" + ] + ] + }, + { + "sid": 131, + "sent": "However, since dsRBDs are known to bind promiscuously with duplex RNA, it is possible that the S258-3\u2019G interaction found in a complex lacking the deaminase domain is not relevant to catalysis at the editing site.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 21, + "dsRBDs", + "structure_element" + ], + [ + 59, + 69, + "duplex RNA", + "structure_element" + ], + [ + 95, + 99, + "S258", + "residue_name_number" + ], + [ + 102, + 103, + "G", + "residue_name" + ], + [ + 135, + 146, + "lacking the", + "protein_state" + ], + [ + 147, + 163, + "deaminase domain", + "structure_element" + ], + [ + 200, + 212, + "editing site", + "site" + ] + ] + }, + { + "sid": 132, + "sent": "It is also possible that ADAR dsRBD and catalytic domain binding are sequential, with release of the dsRBD from the RNA taking place prior to catalytic domain engagement and base flipping.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 29, + "ADAR", + "protein_type" + ], + [ + 30, + 35, + "dsRBD", + "structure_element" + ], + [ + 40, + 56, + "catalytic domain", + "structure_element" + ], + [ + 101, + 106, + "dsRBD", + "structure_element" + ], + [ + 116, + 119, + "RNA", + "chemical" + ], + [ + 142, + 158, + "catalytic domain", + "structure_element" + ] + ] + }, + { + "sid": 133, + "sent": "Aicardi-Goutieres Syndrome (AGS) and Dyschromatosis Symmetrica Hereditaria (DSH) are human diseases caused by mutations in the human ADAR1 gene and several of the disease-associated mutations are found in the deaminase domain.", + "section": "DISCUSS", + "ner": [ + [ + 85, + 90, + "human", + "species" + ], + [ + 127, + 132, + "human", + "species" + ], + [ + 133, + 138, + "ADAR1", + "protein" + ], + [ + 209, + 225, + "deaminase domain", + "structure_element" + ] + ] + }, + { + "sid": 134, + "sent": "Given the conservation in RNA binding surface and active site residues, we expect the hADAR1 catalytic domain to bind RNA with a similar orientation of the helix found in our hADAR2d-RNA structures.", + "section": "DISCUSS", + "ner": [ + [ + 26, + 45, + "RNA binding surface", + "site" + ], + [ + 50, + 61, + "active site", + "site" + ], + [ + 86, + 92, + "hADAR1", + "protein" + ], + [ + 93, + 109, + "catalytic domain", + "structure_element" + ], + [ + 118, + 121, + "RNA", + "chemical" + ], + [ + 175, + 186, + "hADAR2d-RNA", + "complex_assembly" + ], + [ + 187, + 197, + "structures", + "evidence" + ] + ] + }, + { + "sid": 135, + "sent": "When one maps the locations of the AGS-associated mutations onto the hADAR2d-RNA complex, two mutations involve residues in close proximity to the RNA (< 4 \u00c5) (Supplementary Fig. 8a).", + "section": "DISCUSS", + "ner": [ + [ + 69, + 80, + "hADAR2d-RNA", + "complex_assembly" + ], + [ + 147, + 150, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 136, + "sent": "G487 of hADAR2 is found on the flipping loop near the RNA (Fig. 3b).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "G487", + "residue_name_number" + ], + [ + 8, + 14, + "hADAR2", + "protein" + ], + [ + 31, + 44, + "flipping loop", + "structure_element" + ], + [ + 54, + 57, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 137, + "sent": "Sequence in this loop is highly conserved among ADARs and corresponds to G1007 in hADAR1 (Supplementary Table 2).", + "section": "DISCUSS", + "ner": [ + [ + 17, + 21, + "loop", + "structure_element" + ], + [ + 25, + 41, + "highly conserved", + "protein_state" + ], + [ + 48, + 53, + "ADARs", + "protein_type" + ], + [ + 73, + 78, + "G1007", + "residue_name_number" + ], + [ + 82, + 88, + "hADAR1", + "protein" + ] + ] + }, + { + "sid": 138, + "sent": "An arginine at this position would preclude close approach of the flipping loop to the RNA, preventing E1008 insertion and base flipping into the active site (Supplementary Fig. 8b).", + "section": "DISCUSS", + "ner": [ + [ + 3, + 11, + "arginine", + "residue_name" + ], + [ + 66, + 79, + "flipping loop", + "structure_element" + ], + [ + 87, + 90, + "RNA", + "chemical" + ], + [ + 103, + 108, + "E1008", + "residue_name_number" + ], + [ + 146, + 157, + "active site", + "site" + ] + ] + }, + { + "sid": 139, + "sent": "This is consistent with the observation that the G1007R mutation in hADAR1 inhibits RNA editing activity.", + "section": "DISCUSS", + "ner": [ + [ + 49, + 55, + "G1007R", + "mutant" + ], + [ + 68, + 74, + "hADAR1", + "protein" + ], + [ + 84, + 87, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 140, + "sent": "Also, K376 forms salt bridges with both the 5\u2019 and 3\u2019 phosphodiesters of the guanosine on the 3\u2019 side of the editing site (Fig. 2).", + "section": "DISCUSS", + "ner": [ + [ + 6, + 10, + "K376", + "residue_name_number" + ], + [ + 17, + 29, + "salt bridges", + "bond_interaction" + ], + [ + 77, + 86, + "guanosine", + "residue_name" + ], + [ + 109, + 121, + "editing site", + "site" + ] + ] + }, + { + "sid": 141, + "sent": "The corresponding residue in hADAR1 (R892) could form similar contacts and the R892H mutation would likely alter this interaction.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 35, + "hADAR1", + "protein" + ], + [ + 37, + 41, + "R892", + "residue_name_number" + ], + [ + 79, + 84, + "R892H", + "mutant" + ] + ] + }, + { + "sid": 142, + "sent": "In summary, the structures described here establish human ADAR2 as a base-flipping enzyme that uses a unique mechanism well suited for modifying duplex RNA.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 26, + "structures", + "evidence" + ], + [ + 52, + 57, + "human", + "species" + ], + [ + 58, + 63, + "ADAR2", + "protein" + ], + [ + 145, + 155, + "duplex RNA", + "structure_element" + ] + ] + }, + { + "sid": 143, + "sent": "In addition, this work provides a basis for understanding the role of the ADAR catalytic domain in determining editing site selectivity and additional structural context to evaluate the impact of ADAR mutations associated with human disease.", + "section": "DISCUSS", + "ner": [ + [ + 74, + 78, + "ADAR", + "protein_type" + ], + [ + 79, + 95, + "catalytic domain", + "structure_element" + ], + [ + 111, + 123, + "editing site", + "site" + ], + [ + 196, + 200, + "ADAR", + "protein_type" + ], + [ + 227, + 232, + "human", + "species" + ] + ] + }, + { + "sid": 144, + "sent": "Human ADAR2 and modified RNAs for crystallography", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "Human", + "species" + ], + [ + 6, + 11, + "ADAR2", + "protein" + ], + [ + 25, + 29, + "RNAs", + "chemical" + ], + [ + 34, + 49, + "crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 145, + "sent": "a, Domain map for human ADAR2 b, ADAR reaction showing intermediate and 8-azanebularine (N) hydrate that mimics this structure c, Duplex RNAs used for crystallization.", + "section": "FIG", + "ner": [ + [ + 18, + 23, + "human", + "species" + ], + [ + 24, + 29, + "ADAR2", + "protein" + ], + [ + 33, + 37, + "ADAR", + "protein_type" + ], + [ + 72, + 99, + "8-azanebularine (N) hydrate", + "chemical" + ], + [ + 117, + 126, + "structure", + "evidence" + ], + [ + 130, + 141, + "Duplex RNAs", + "structure_element" + ], + [ + 151, + 166, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 146, + "sent": "Bdf2 duplex sequence is derived from an editing site found in S. cerevisiae Bdf2 mRNA and Gli1 duplex has sequence surrounding the human Gli1 mRNA editing site.", + "section": "FIG", + "ner": [ + [ + 0, + 11, + "Bdf2 duplex", + "chemical" + ], + [ + 40, + 52, + "editing site", + "site" + ], + [ + 62, + 75, + "S. cerevisiae", + "species" + ], + [ + 76, + 85, + "Bdf2 mRNA", + "chemical" + ], + [ + 90, + 94, + "Gli1", + "protein" + ], + [ + 131, + 136, + "human", + "species" + ], + [ + 137, + 141, + "Gli1", + "protein" + ], + [ + 142, + 146, + "mRNA", + "chemical" + ], + [ + 147, + 159, + "editing site", + "site" + ] + ] + }, + { + "sid": 147, + "sent": "Structure of hADAR2d E488Q bound to the Bdf2-C RNA duplex at 2.75 \u00c5 resolution", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 13, + 20, + "hADAR2d", + "mutant" + ], + [ + 21, + 26, + "E488Q", + "mutant" + ], + [ + 27, + 35, + "bound to", + "protein_state" + ], + [ + 40, + 57, + "Bdf2-C RNA duplex", + "chemical" + ] + ] + }, + { + "sid": 148, + "sent": "a, View of structure perpendicular to the dsRNA helical axis.", + "section": "FIG", + "ner": [ + [ + 42, + 47, + "dsRNA", + "chemical" + ] + ] + }, + { + "sid": 149, + "sent": "Colors correspond to those in Figs. 1a and 1c; flipped out base N is highlighted red, zinc in grey space-filling sphere, Q488 in yellow, previously disordered aa454\u2013477 loop in green and inositol hexakisphosphate (IHP) in space filling.", + "section": "FIG", + "ner": [ + [ + 47, + 58, + "flipped out", + "protein_state" + ], + [ + 86, + 90, + "zinc", + "chemical" + ], + [ + 121, + 125, + "Q488", + "residue_name_number" + ], + [ + 148, + 158, + "disordered", + "protein_state" + ], + [ + 161, + 168, + "454\u2013477", + "residue_range" + ], + [ + 169, + 173, + "loop", + "structure_element" + ], + [ + 187, + 212, + "inositol hexakisphosphate", + "chemical" + ], + [ + 214, + 217, + "IHP", + "chemical" + ] + ] + }, + { + "sid": 150, + "sent": "A transparent surface is shown for the hADAR2d protein.", + "section": "FIG", + "ner": [ + [ + 39, + 46, + "hADAR2d", + "mutant" + ] + ] + }, + { + "sid": 151, + "sent": "b, View of structure along the dsRNA helical axis.", + "section": "FIG", + "ner": [ + [ + 31, + 36, + "dsRNA", + "chemical" + ] + ] + }, + { + "sid": 152, + "sent": "c, Summary of the contacts between hADAR2d E488Q and the Bdf2-C RNA duplex.", + "section": "FIG", + "ner": [ + [ + 35, + 42, + "hADAR2d", + "mutant" + ], + [ + 43, + 48, + "E488Q", + "mutant" + ], + [ + 57, + 74, + "Bdf2-C RNA duplex", + "chemical" + ] + ] + }, + { + "sid": 153, + "sent": "ADAR recognition of the flipped out and orphaned nucleotides", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "ADAR", + "protein_type" + ], + [ + 24, + 35, + "flipped out", + "protein_state" + ], + [ + 40, + 48, + "orphaned", + "protein_state" + ], + [ + 49, + 60, + "nucleotides", + "chemical" + ] + ] + }, + { + "sid": 154, + "sent": "a, Contacts to the editing site nucleotide (N) in the active site.", + "section": "FIG", + "ner": [ + [ + 19, + 31, + "editing site", + "site" + ], + [ + 32, + 42, + "nucleotide", + "chemical" + ], + [ + 54, + 65, + "active site", + "site" + ] + ] + }, + { + "sid": 155, + "sent": "b, Orphan nucleotide recognition in the hADAR2d E488Q\u2013Bdf2-C complex.", + "section": "FIG", + "ner": [ + [ + 3, + 9, + "Orphan", + "protein_state" + ], + [ + 10, + 20, + "nucleotide", + "chemical" + ], + [ + 40, + 60, + "hADAR2d E488Q\u2013Bdf2-C", + "complex_assembly" + ] + ] + }, + { + "sid": 156, + "sent": "c, Orphan nucleotide recognition in the hADAR2d WT\u2013Bdf2-U complex.", + "section": "FIG", + "ner": [ + [ + 3, + 9, + "Orphan", + "protein_state" + ], + [ + 10, + 20, + "nucleotide", + "chemical" + ], + [ + 40, + 57, + "hADAR2d WT\u2013Bdf2-U", + "complex_assembly" + ] + ] + }, + { + "sid": 157, + "sent": "Other ADAR-induced changes in RNA conformation", + "section": "FIG", + "ner": [ + [ + 6, + 10, + "ADAR", + "protein_type" + ], + [ + 30, + 33, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 158, + "sent": "a, hADAR2d shifts the position of U11-A13\u2019 base pair from ideal A-form RNA helix (yellow).", + "section": "FIG", + "ner": [ + [ + 3, + 10, + "hADAR2d", + "mutant" + ], + [ + 34, + 37, + "U11", + "residue_name_number" + ], + [ + 38, + 41, + "A13", + "residue_name_number" + ], + [ + 64, + 80, + "A-form RNA helix", + "structure_element" + ] + ] + }, + { + "sid": 159, + "sent": "b, Overlay of Bdf2 duplex RNA and idealized A form duplex of same sequence (yellow) illustrating kink in strand and widening of major groove opposite editing site induced by hADAR2d.", + "section": "FIG", + "ner": [ + [ + 3, + 10, + "Overlay", + "experimental_method" + ], + [ + 14, + 29, + "Bdf2 duplex RNA", + "chemical" + ], + [ + 44, + 57, + "A form duplex", + "structure_element" + ], + [ + 128, + 140, + "major groove", + "site" + ], + [ + 150, + 162, + "editing site", + "site" + ], + [ + 174, + 181, + "hADAR2d", + "mutant" + ] + ] + }, + { + "sid": 160, + "sent": "c, Unusual \u201cwobble\u201d A13\u2019-U11 interaction in the hADAR2d WT\u2013Bdf2-U complex shown in stick with H-bond indicated with yellow dashes and distances shown in \u00c5. The position of this base pair in the hADAR2d E488Q\u2013Bdf2-C duplex is shown in wire with H-bonds shown with gray dashes.", + "section": "FIG", + "ner": [ + [ + 20, + 23, + "A13", + "residue_name_number" + ], + [ + 25, + 28, + "U11", + "residue_name_number" + ], + [ + 48, + 65, + "hADAR2d WT\u2013Bdf2-U", + "complex_assembly" + ], + [ + 94, + 100, + "H-bond", + "bond_interaction" + ], + [ + 194, + 214, + "hADAR2d E488Q\u2013Bdf2-C", + "complex_assembly" + ], + [ + 244, + 251, + "H-bonds", + "bond_interaction" + ] + ] + }, + { + "sid": 161, + "sent": "Interactions with editing site nearest neighbor nucleotides", + "section": "FIG", + "ner": [ + [ + 18, + 30, + "editing site", + "site" + ], + [ + 48, + 59, + "nucleotides", + "chemical" + ] + ] + }, + { + "sid": 162, + "sent": "a, The minor groove edge of the U11-A13\u2019 base pair from the Bdf2 duplex approaches G489; model with a C-G pair at this position suggests a clash with the G 2-amino group b, RNA duplex substrates prepared with different 5\u2019 nearest neighbor nucleotides adjacent to editing site indicated in red (2AP = 2-aminopurine).", + "section": "FIG", + "ner": [ + [ + 7, + 19, + "minor groove", + "site" + ], + [ + 32, + 35, + "U11", + "residue_name_number" + ], + [ + 36, + 39, + "A13", + "residue_name_number" + ], + [ + 60, + 71, + "Bdf2 duplex", + "chemical" + ], + [ + 83, + 87, + "G489", + "residue_name_number" + ], + [ + 102, + 110, + "C-G pair", + "structure_element" + ], + [ + 154, + 155, + "G", + "residue_name" + ], + [ + 173, + 183, + "RNA duplex", + "structure_element" + ], + [ + 263, + 275, + "editing site", + "site" + ], + [ + 294, + 297, + "2AP", + "structure_element" + ], + [ + 300, + 313, + "2-aminopurine", + "structure_element" + ] + ] + }, + { + "sid": 163, + "sent": "c, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 5\u2019 nearest neighbors; krel = kobs/(kobs for unmodified RNA).", + "section": "FIG", + "ner": [ + [ + 17, + 43, + "deamination rate constants", + "evidence" + ], + [ + 47, + 54, + "hADAR2d", + "mutant" + ], + [ + 62, + 74, + "editing site", + "site" + ], + [ + 75, + 84, + "adenosine", + "residue_name" + ], + [ + 144, + 148, + "krel", + "evidence" + ], + [ + 151, + 155, + "kobs", + "evidence" + ], + [ + 157, + 161, + "kobs", + "evidence" + ], + [ + 166, + 176, + "unmodified", + "protein_state" + ], + [ + 177, + 180, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 164, + "sent": "d, hADAR2 S486 backbone H-bond with 3\u2019 G 2-amino group; e, RNA duplex substrates prepared with different 3\u2019 nearest neighbor nucleotides adjacent to editing site indicated in red (I = inosine, N2MeG = N2-methylguanosine, 2AP = 2-aminopurine).", + "section": "FIG", + "ner": [ + [ + 3, + 9, + "hADAR2", + "protein" + ], + [ + 10, + 14, + "S486", + "residue_name_number" + ], + [ + 24, + 30, + "H-bond", + "bond_interaction" + ], + [ + 39, + 40, + "G", + "residue_name" + ], + [ + 59, + 69, + "RNA duplex", + "structure_element" + ], + [ + 149, + 161, + "editing site", + "site" + ], + [ + 180, + 181, + "I", + "residue_name" + ], + [ + 184, + 191, + "inosine", + "residue_name" + ], + [ + 221, + 224, + "2AP", + "structure_element" + ], + [ + 227, + 240, + "2-aminopurine", + "structure_element" + ] + ] + }, + { + "sid": 165, + "sent": "f, Comparison of deamination rate constants by hADAR2d at the editing site adenosine (red) for duplexes bearing different 3\u2019 nearest neighbors.", + "section": "FIG", + "ner": [ + [ + 17, + 43, + "deamination rate constants", + "evidence" + ], + [ + 47, + 54, + "hADAR2d", + "mutant" + ], + [ + 62, + 74, + "editing site", + "site" + ], + [ + 75, + 84, + "adenosine", + "residue_name" + ] + ] + }, + { + "sid": 166, + "sent": "krel = kobs/(kobs for unmodified RNA).", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "krel", + "evidence" + ], + [ + 7, + 11, + "kobs", + "evidence" + ], + [ + 13, + 17, + "kobs", + "evidence" + ], + [ + 22, + 32, + "unmodified", + "protein_state" + ], + [ + 33, + 36, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 167, + "sent": "RNA-binding loops in the ADAR catalytic domain", + "section": "FIG", + "ner": [ + [ + 0, + 17, + "RNA-binding loops", + "structure_element" + ], + [ + 25, + 29, + "ADAR", + "protein_type" + ], + [ + 30, + 46, + "catalytic domain", + "structure_element" + ] + ] + }, + { + "sid": 168, + "sent": "a, hADAR2 residues that contact phosphodiester backbone near 5\u2019 end of unedited strand.", + "section": "FIG", + "ner": [ + [ + 3, + 9, + "hADAR2", + "protein" + ] + ] + }, + { + "sid": 169, + "sent": "b, Location of mutations introduced at protein-RNA interface.", + "section": "FIG", + "ner": [ + [ + 39, + 60, + "protein-RNA interface", + "site" + ] + ] + }, + { + "sid": 170, + "sent": "c, Comparison of deamination rate constants of the different hADAR2d mutants (Log scale).", + "section": "FIG", + "ner": [ + [ + 17, + 43, + "deamination rate constants", + "evidence" + ], + [ + 61, + 68, + "hADAR2d", + "mutant" + ] + ] + }, + { + "sid": 171, + "sent": "krel = kobs for mutant/kobs for WT.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "krel", + "evidence" + ], + [ + 7, + 11, + "kobs", + "evidence" + ], + [ + 16, + 22, + "mutant", + "protein_state" + ], + [ + 23, + 27, + "kobs", + "evidence" + ], + [ + 32, + 34, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 172, + "sent": "d, Sequence alignment of ADAR2s (A2) and ADAR1s (A1) from different organisms with different levels of conservation colored (Yellow: conserved in all ADAR1s and ADAR2s, red: conserved in ADAR2s, blue: conserved in ADAR1s.", + "section": "FIG", + "ner": [ + [ + 3, + 21, + "Sequence alignment", + "experimental_method" + ], + [ + 25, + 31, + "ADAR2s", + "protein_type" + ], + [ + 41, + 47, + "ADAR1s", + "protein_type" + ], + [ + 133, + 142, + "conserved", + "protein_state" + ], + [ + 150, + 156, + "ADAR1s", + "protein_type" + ], + [ + 161, + 167, + "ADAR2s", + "protein_type" + ], + [ + 174, + 183, + "conserved", + "protein_state" + ], + [ + 187, + 193, + "ADAR2s", + "protein_type" + ], + [ + 201, + 210, + "conserved", + "protein_state" + ], + [ + 214, + 220, + "ADAR1s", + "protein_type" + ] + ] + }, + { + "sid": 173, + "sent": "e, Interaction of the ADAR-specific RNA-binding loop near the 5\u2019 end of the edited strand.", + "section": "FIG", + "ner": [ + [ + 22, + 52, + "ADAR-specific RNA-binding loop", + "structure_element" + ] + ] + }, + { + "sid": 174, + "sent": "Colors as in d, white: not conserved, flipped out base is shown in pink.", + "section": "FIG", + "ner": [ + [ + 23, + 36, + "not conserved", + "protein_state" + ], + [ + 38, + 49, + "flipped out", + "protein_state" + ], + [ + 50, + 54, + "base", + "chemical" + ] + ] + } + ] + }, + "PMC4772114": { + "annotations": [ + { + "sid": 0, + "sent": "Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions", + "section": "TITLE", + "ner": [ + [ + 61, + 70, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 1, + "sent": "Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 9, + "Regnase-1", + "protein" + ], + [ + 16, + 21, + "RNase", + "protein_type" + ], + [ + 44, + 49, + "mRNAs", + "chemical" + ], + [ + 80, + 84, + "IL-6", + "protein_type" + ], + [ + 89, + 97, + "IL-12p40", + "protein_type" + ] + ] + }, + { + "sid": 2, + "sent": "Here, we report the structures of four domains of Regnase-1 from Mus musculus\u2014the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD).", + "section": "ABSTRACT", + "ner": [ + [ + 20, + 30, + "structures", + "evidence" + ], + [ + 50, + 59, + "Regnase-1", + "protein" + ], + [ + 65, + 77, + "Mus musculus", + "species" + ], + [ + 82, + 99, + "N-terminal domain", + "structure_element" + ], + [ + 101, + 104, + "NTD", + "structure_element" + ], + [ + 107, + 127, + "PilT N-terminus like", + "structure_element" + ], + [ + 129, + 132, + "PIN", + "structure_element" + ], + [ + 142, + 153, + "zinc finger", + "structure_element" + ], + [ + 155, + 157, + "ZF", + "structure_element" + ], + [ + 170, + 187, + "C-terminal domain", + "structure_element" + ], + [ + 189, + 192, + "CTD", + "structure_element" + ] + ] + }, + { + "sid": 3, + "sent": "The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 7, + "PIN", + "structure_element" + ], + [ + 27, + 32, + "RNase", + "protein_type" + ], + [ + 33, + 49, + "catalytic center", + "site" + ] + ] + }, + { + "sid": 4, + "sent": "We found that the NTD associates with the PIN domain and significantly enhances its RNase activity.", + "section": "ABSTRACT", + "ner": [ + [ + 18, + 21, + "NTD", + "structure_element" + ], + [ + 42, + 45, + "PIN", + "structure_element" + ], + [ + 84, + 89, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 5, + "sent": "The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 7, + "PIN", + "structure_element" + ], + [ + 23, + 35, + "head-to-tail", + "protein_state" + ], + [ + 36, + 44, + "oligomer", + "oligomeric_state" + ], + [ + 53, + 68, + "dimer interface", + "site" + ], + [ + 87, + 103, + "NTD binding site", + "site" + ] + ] + }, + { + "sid": 6, + "sent": "Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro.", + "section": "ABSTRACT", + "ner": [ + [ + 15, + 24, + "mutations", + "experimental_method" + ], + [ + 34, + 37, + "PIN", + "structure_element" + ], + [ + 61, + 66, + "RNase", + "protein_type" + ], + [ + 118, + 121, + "NTD", + "structure_element" + ], + [ + 146, + 151, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 7, + "sent": "These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions.", + "section": "ABSTRACT", + "ner": [ + [ + 27, + 36, + "Regnase-1", + "protein" + ], + [ + 37, + 42, + "RNase", + "protein_type" + ], + [ + 98, + 101, + "NTD", + "structure_element" + ], + [ + 102, + 105, + "PIN", + "structure_element" + ], + [ + 127, + 130, + "PIN", + "structure_element" + ], + [ + 131, + 134, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 8, + "sent": "The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells.", + "section": "INTRO", + "ner": [ + [ + 57, + 86, + "pattern-recognition receptors", + "protein_type" + ], + [ + 88, + 92, + "PRRs", + "protein_type" + ], + [ + 99, + 118, + "Toll-like receptors", + "protein_type" + ], + [ + 120, + 124, + "TLRs", + "protein_type" + ], + [ + 180, + 184, + "TLRs", + "protein_type" + ] + ] + }, + { + "sid": 9, + "sent": "Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1\u03b2, IL-2, and IL-12p40.", + "section": "INTRO", + "ner": [ + [ + 0, + 9, + "Regnase-1", + "protein" + ], + [ + 25, + 32, + "Zc3h12a", + "protein" + ], + [ + 37, + 43, + "MCPIP1", + "protein" + ], + [ + 51, + 56, + "RNase", + "protein_type" + ], + [ + 97, + 116, + "lipopolysaccharides", + "chemical" + ], + [ + 191, + 196, + "mRNAs", + "chemical" + ], + [ + 239, + 245, + "(IL)-6", + "protein_type" + ], + [ + 247, + 252, + "IL-1\u03b2", + "protein_type" + ], + [ + 254, + 258, + "IL-2", + "protein_type" + ], + [ + 264, + 272, + "IL-12p40", + "protein_type" + ] + ] + }, + { + "sid": 10, + "sent": "Regnase-1 accelerates target mRNA degradation via their 3\u2032-terminal untranslated region (3\u2032UTR), and also degrades its own mRNA.", + "section": "INTRO", + "ner": [ + [ + 0, + 9, + "Regnase-1", + "protein" + ], + [ + 29, + 33, + "mRNA", + "chemical" + ], + [ + 56, + 87, + "3\u2032-terminal untranslated region", + "structure_element" + ], + [ + 89, + 94, + "3\u2032UTR", + "structure_element" + ], + [ + 123, + 127, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 11, + "sent": "Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc\u2013finger (ZF) domain, which are conserved among Regnase family members.", + "section": "INTRO", + "ner": [ + [ + 0, + 9, + "Regnase-1", + "protein" + ], + [ + 25, + 39, + "Regnase family", + "protein_type" + ], + [ + 61, + 81, + "PilT N-terminus like", + "structure_element" + ], + [ + 83, + 86, + "PIN", + "structure_element" + ], + [ + 109, + 130, + "CCCH-type zinc\u2013finger", + "structure_element" + ], + [ + 132, + 134, + "ZF", + "structure_element" + ], + [ + 154, + 163, + "conserved", + "protein_state" + ], + [ + 170, + 192, + "Regnase family members", + "protein_type" + ] + ] + }, + { + "sid": 12, + "sent": "Recently, the crystal structure of the Regnase-1 PIN domain derived from Homo sapiens was reported.", + "section": "INTRO", + "ner": [ + [ + 14, + 31, + "crystal structure", + "evidence" + ], + [ + 39, + 48, + "Regnase-1", + "protein" + ], + [ + 49, + 52, + "PIN", + "structure_element" + ], + [ + 73, + 85, + "Homo sapiens", + "species" + ] + ] + }, + { + "sid": 13, + "sent": "The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity.", + "section": "INTRO", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 91, + 94, + "Asp", + "residue_name" + ], + [ + 113, + 129, + "catalytic center", + "site" + ], + [ + 144, + 148, + "Mg2+", + "chemical" + ], + [ + 177, + 182, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 14, + "sent": "Several CCCH-type ZF motifs in RNA-binding proteins have been reported to directly bind RNA.", + "section": "INTRO", + "ner": [ + [ + 8, + 27, + "CCCH-type ZF motifs", + "structure_element" + ], + [ + 31, + 51, + "RNA-binding proteins", + "protein_type" + ], + [ + 88, + 91, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 15, + "sent": "In addition, Regnase-1 has been predicted to possess other domains in the N- and C- terminal regions.", + "section": "INTRO", + "ner": [ + [ + 13, + 22, + "Regnase-1", + "protein" + ], + [ + 74, + 100, + "N- and C- terminal regions", + "structure_element" + ] + ] + }, + { + "sid": 16, + "sent": "However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved.", + "section": "INTRO", + "ner": [ + [ + 13, + 22, + "structure", + "evidence" + ], + [ + 43, + 45, + "ZF", + "structure_element" + ], + [ + 54, + 71, + "N-terminal domain", + "structure_element" + ], + [ + 73, + 76, + "NTD", + "structure_element" + ], + [ + 82, + 99, + "C-terminal domain", + "structure_element" + ], + [ + 101, + 104, + "CTD", + "structure_element" + ], + [ + 109, + 118, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 17, + "sent": "Here, we performed structural and functional analyses of individual domains of Regnase-1 derived from Mus musculus in order to understand the catalytic activity in vitro.", + "section": "INTRO", + "ner": [ + [ + 19, + 53, + "structural and functional analyses", + "experimental_method" + ], + [ + 79, + 88, + "Regnase-1", + "protein" + ], + [ + 102, + 114, + "Mus musculus", + "species" + ] + ] + }, + { + "sid": 18, + "sent": "Our data revealed that the catalytic activity of Regnase-1 is regulated through both intra and intermolecular domain interactions in vitro.", + "section": "INTRO", + "ner": [ + [ + 49, + 58, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 19, + "sent": "The NTD plays a crucial role in efficient cleavage of target mRNA, through intramolecular NTD-PIN interactions.", + "section": "INTRO", + "ner": [ + [ + 4, + 7, + "NTD", + "structure_element" + ], + [ + 61, + 65, + "mRNA", + "chemical" + ], + [ + 90, + 93, + "NTD", + "structure_element" + ], + [ + 94, + 97, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 20, + "sent": "Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA.", + "section": "INTRO", + "ner": [ + [ + 10, + 19, + "Regnase-1", + "protein" + ], + [ + 35, + 40, + "dimer", + "oligomeric_state" + ], + [ + 64, + 67, + "PIN", + "structure_element" + ], + [ + 68, + 71, + "PIN", + "structure_element" + ], + [ + 111, + 115, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 21, + "sent": "Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer.", + "section": "INTRO", + "ner": [ + [ + 26, + 35, + "Regnase-1", + "protein" + ], + [ + 55, + 59, + "mRNA", + "chemical" + ], + [ + 66, + 79, + "NTD-activated", + "protein_state" + ], + [ + 80, + 90, + "functional", + "protein_state" + ], + [ + 91, + 94, + "PIN", + "structure_element" + ], + [ + 95, + 100, + "dimer", + "oligomeric_state" + ], + [ + 112, + 114, + "ZF", + "structure_element" + ], + [ + 125, + 128, + "RNA", + "chemical" + ], + [ + 161, + 164, + "PIN", + "structure_element" + ], + [ + 165, + 170, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 22, + "sent": "Domain structures of Regnase-1", + "section": "RESULTS", + "ner": [ + [ + 7, + 17, + "structures", + "evidence" + ], + [ + 21, + 30, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 23, + "sent": "We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a\u2013e).", + "section": "RESULTS", + "ner": [ + [ + 12, + 21, + "Rengase-1", + "protein" + ], + [ + 35, + 47, + "Mus musculus", + "species" + ], + [ + 52, + 58, + "solved", + "experimental_method" + ], + [ + 63, + 73, + "structures", + "evidence" + ], + [ + 95, + 98, + "NTD", + "structure_element" + ], + [ + 100, + 103, + "PIN", + "structure_element" + ], + [ + 105, + 107, + "ZF", + "structure_element" + ], + [ + 113, + 116, + "CTD", + "structure_element" + ], + [ + 133, + 154, + "X-ray crystallography", + "experimental_method" + ], + [ + 158, + 161, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 24, + "sent": "X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens.", + "section": "RESULTS", + "ner": [ + [ + 0, + 21, + "X-ray crystallography", + "experimental_method" + ], + [ + 73, + 76, + "PIN", + "structure_element" + ], + [ + 81, + 83, + "ZF", + "structure_element" + ], + [ + 102, + 118, + "electron density", + "evidence" + ], + [ + 145, + 148, + "PIN", + "structure_element" + ], + [ + 204, + 213, + "Regnase-1", + "protein" + ], + [ + 227, + 239, + "Homo sapiens", + "species" + ] + ] + }, + { + "sid": 25, + "sent": "This suggests that the PIN and ZF domains exist independently without interacting with each other.", + "section": "RESULTS", + "ner": [ + [ + 23, + 26, + "PIN", + "structure_element" + ], + [ + 31, + 33, + "ZF", + "structure_element" + ] + ] + }, + { + "sid": 26, + "sent": "The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e).", + "section": "RESULTS", + "ner": [ + [ + 11, + 21, + "structures", + "evidence" + ], + [ + 25, + 28, + "NTD", + "structure_element" + ], + [ + 30, + 32, + "ZF", + "structure_element" + ], + [ + 38, + 41, + "CTD", + "structure_element" + ], + [ + 61, + 64, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 27, + "sent": "The NTD and CTD are both composed of three \u03b1 helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "NTD", + "structure_element" + ], + [ + 12, + 15, + "CTD", + "structure_element" + ], + [ + 43, + 52, + "\u03b1 helices", + "structure_element" + ], + [ + 80, + 113, + "ubiquitin conjugating enzyme E2 K", + "protein" + ], + [ + 133, + 163, + "ubiquitin associated protein 1", + "protein" + ], + [ + 211, + 222, + "Dali server", + "experimental_method" + ] + ] + }, + { + "sid": 28, + "sent": "Contribution of each domain of Regnase-1 to the mRNA binding activity", + "section": "RESULTS", + "ner": [ + [ + 31, + 40, + "Regnase-1", + "protein" + ], + [ + 48, + 52, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 29, + "sent": "Although the PIN domain is responsible for the catalytic activity of Regnase-1, the roles of the other domains are largely unknown.", + "section": "RESULTS", + "ner": [ + [ + 13, + 16, + "PIN", + "structure_element" + ], + [ + 69, + 78, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 30, + "sent": "First, we evaluated a role of the NTD and ZF domains for mRNA binding by an in vitro gel shift assay (Fig. 1f).", + "section": "RESULTS", + "ner": [ + [ + 34, + 37, + "NTD", + "structure_element" + ], + [ + 42, + 44, + "ZF", + "structure_element" + ], + [ + 57, + 61, + "mRNA", + "chemical" + ], + [ + 76, + 100, + "in vitro gel shift assay", + "experimental_method" + ] + ] + }, + { + "sid": 31, + "sent": "Fluorescently 5\u2032-labeled RNA corresponding to nucleotides 82\u2013106 of the IL-6 mRNA 3\u2032UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1\u2014hereafter referred to as the DDNN mutant\u2014were utilized.", + "section": "RESULTS", + "ner": [ + [ + 0, + 24, + "Fluorescently 5\u2032-labeled", + "protein_state" + ], + [ + 25, + 28, + "RNA", + "chemical" + ], + [ + 72, + 76, + "IL-6", + "protein_type" + ], + [ + 77, + 81, + "mRNA", + "chemical" + ], + [ + 82, + 87, + "3\u2032UTR", + "structure_element" + ], + [ + 110, + 118, + "inactive", + "protein_state" + ], + [ + 119, + 125, + "mutant", + "protein_state" + ], + [ + 127, + 132, + "D226N", + "mutant" + ], + [ + 137, + 142, + "D244N", + "mutant" + ], + [ + 147, + 156, + "Regnase-1", + "protein" + ], + [ + 186, + 190, + "DDNN", + "mutant" + ], + [ + 191, + 197, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 32, + "sent": "Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA.", + "section": "RESULTS", + "ner": [ + [ + 36, + 45, + "Regnase-1", + "protein" + ], + [ + 51, + 63, + "fluorescence", + "evidence" + ], + [ + 67, + 71, + "free", + "protein_state" + ], + [ + 72, + 75, + "RNA", + "chemical" + ], + [ + 103, + 112, + "Regnase-1", + "protein" + ], + [ + 113, + 121, + "bound to", + "protein_state" + ], + [ + 126, + 129, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 33, + "sent": "Based on the decrease in the free RNA fluorescence band, we evaluated the contribution of each domain of Regnase-1 to RNA binding.", + "section": "RESULTS", + "ner": [ + [ + 34, + 37, + "RNA", + "chemical" + ], + [ + 105, + 114, + "Regnase-1", + "protein" + ], + [ + 118, + 121, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 34, + "sent": "While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1).", + "section": "RESULTS", + "ner": [ + [ + 10, + 13, + "RNA", + "chemical" + ], + [ + 67, + 78, + "presence of", + "protein_state" + ], + [ + 79, + 82, + "NTD", + "structure_element" + ], + [ + 104, + 115, + "presence of", + "protein_state" + ], + [ + 120, + 122, + "ZF", + "structure_element" + ] + ] + }, + { + "sid": 35, + "sent": "Direct binding of the ZF domain and RNA were confirmed by NMR spectral changes.", + "section": "RESULTS", + "ner": [ + [ + 22, + 24, + "ZF", + "structure_element" + ], + [ + 36, + 39, + "RNA", + "chemical" + ], + [ + 58, + 61, + "NMR", + "experimental_method" + ], + [ + 62, + 78, + "spectral changes", + "evidence" + ] + ] + }, + { + "sid": 36, + "sent": "The fitting of the titration curve of Y314 resulted in an apparent dissociation constant (Kd) of 10\u2009\u00b1\u20091.1\u2009\u03bcM (Supplementary Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 19, + 34, + "titration curve", + "evidence" + ], + [ + 38, + 42, + "Y314", + "residue_name_number" + ], + [ + 67, + 88, + "dissociation constant", + "evidence" + ], + [ + 90, + 92, + "Kd", + "evidence" + ] + ] + }, + { + "sid": 37, + "sent": "These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA.", + "section": "RESULTS", + "ner": [ + [ + 41, + 44, + "PIN", + "structure_element" + ], + [ + 58, + 60, + "ZF", + "structure_element" + ], + [ + 82, + 85, + "RNA", + "chemical" + ], + [ + 105, + 108, + "NTD", + "structure_element" + ], + [ + 165, + 168, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 38, + "sent": "Contribution of each domain of Regnase-1 to RNase activity", + "section": "RESULTS", + "ner": [ + [ + 31, + 40, + "Regnase-1", + "protein" + ], + [ + 44, + 49, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 39, + "sent": "In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5\u2032-labeled RNA corresponding to nucleotides 82\u2013106 of the IL-6 mRNA 3\u2032UTR (Fig. 1g).", + "section": "RESULTS", + "ner": [ + [ + 56, + 61, + "RNase", + "protein_type" + ], + [ + 74, + 83, + "Regnase-1", + "protein" + ], + [ + 101, + 124, + "in vitro cleavage assay", + "experimental_method" + ], + [ + 131, + 155, + "fluorescently 5\u2032-labeled", + "protein_state" + ], + [ + 156, + 159, + "RNA", + "chemical" + ], + [ + 203, + 207, + "IL-6", + "protein_type" + ], + [ + 208, + 212, + "mRNA", + "chemical" + ], + [ + 213, + 218, + "3\u2032UTR", + "structure_element" + ] + ] + }, + { + "sid": 40, + "sent": "Regnase-1 constructs consisting of NTD-PIN-ZF completely cleaved the target mRNA and generated the cleaved products.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Regnase-1", + "protein" + ], + [ + 35, + 45, + "NTD-PIN-ZF", + "mutant" + ], + [ + 76, + 80, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 41, + "sent": "The apparent half-life (T1/2) of the RNase activity was about 20\u2009minutes.", + "section": "RESULTS", + "ner": [ + [ + 37, + 42, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 42, + "sent": "Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2\u2009~\u200970\u2009minutes), while those lacking the NTD did not generate cleaved products (T1/2\u2009>\u200990\u2009minutes).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Regnase-1", + "protein" + ], + [ + 10, + 17, + "lacking", + "protein_state" + ], + [ + 22, + 24, + "ZF", + "structure_element" + ], + [ + 127, + 134, + "lacking", + "protein_state" + ], + [ + 139, + 142, + "NTD", + "structure_element" + ] + ] + }, + { + "sid": 43, + "sent": "It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain.", + "section": "RESULTS", + "ner": [ + [ + 24, + 40, + "NTD-PIN(DDNN)-ZF", + "mutant" + ], + [ + 62, + 65, + "NTD", + "structure_element" + ], + [ + 70, + 75, + "lacks", + "protein_state" + ], + [ + 80, + 98, + "catalytic residues", + "site" + ], + [ + 102, + 105, + "PIN", + "structure_element" + ], + [ + 127, + 132, + "RNase", + "protein_type" + ], + [ + 199, + 204, + "RNase", + "protein_type" + ], + [ + 205, + 221, + "catalytic center", + "site" + ], + [ + 240, + 243, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 44, + "sent": "Taken together with the results in the previous section, we conclude that the NTD is crucial for the RNase activity of Regnase-1 in vitro, although it does not contribute to the direct mRNA binding.", + "section": "RESULTS", + "ner": [ + [ + 78, + 81, + "NTD", + "structure_element" + ], + [ + 101, + 106, + "RNase", + "protein_type" + ], + [ + 119, + 128, + "Regnase-1", + "protein" + ], + [ + 185, + 189, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 45, + "sent": "Dimer formation of the PIN domains", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "Dimer", + "oligomeric_state" + ], + [ + 23, + 26, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 46, + "sent": "During purification by gel filtration, the PIN domain exhibited extremely asymmetric elution peaks in a concentration dependent manner (Fig. 2a).", + "section": "RESULTS", + "ner": [ + [ + 7, + 19, + "purification", + "experimental_method" + ], + [ + 23, + 37, + "gel filtration", + "experimental_method" + ], + [ + 43, + 46, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 47, + "sent": "By comparison with the elution volume of standard marker proteins, the PIN domain was assumed to be in equilibrium between a monomer and a dimer in solution at concentrations in the 20\u2013200\u2009\u03bcM range.", + "section": "RESULTS", + "ner": [ + [ + 3, + 65, + "comparison with the elution volume of standard marker proteins", + "experimental_method" + ], + [ + 71, + 74, + "PIN", + "structure_element" + ], + [ + 125, + 132, + "monomer", + "oligomeric_state" + ], + [ + 139, + 144, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 48, + "sent": "The crystal structure of the PIN domain has been determined in three distinct crystal forms with a space group of P3121 (form I in this study and PDB ID 3V33), P3221 (form II in this study), and P41 (PDB ID 3V32 and 3V34), respectively.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 29, + 32, + "PIN", + "structure_element" + ], + [ + 78, + 91, + "crystal forms", + "evidence" + ] + ] + }, + { + "sid": 49, + "sent": "We found that the PIN domain formed a head-to-tail oligomer that was commonly observed in all three crystal forms in spite of the different crystallization conditions (Supplementary Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "PIN", + "structure_element" + ], + [ + 38, + 50, + "head-to-tail", + "protein_state" + ], + [ + 51, + 59, + "oligomer", + "oligomeric_state" + ], + [ + 100, + 113, + "crystal forms", + "evidence" + ] + ] + }, + { + "sid": 50, + "sent": "Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Mutation", + "experimental_method" + ], + [ + 12, + 18, + "Arg215", + "residue_name_number" + ], + [ + 71, + 89, + "oligomeric surface", + "site" + ], + [ + 94, + 97, + "Glu", + "residue_name" + ], + [ + 112, + 119, + "monomer", + "oligomeric_state" + ], + [ + 120, + 125, + "dimer", + "oligomeric_state" + ], + [ + 154, + 163, + "wild type", + "protein_state" + ] + ] + }, + { + "sid": 51, + "sent": "On the other hand, single mutations of side chains involved in the PIN\u2013PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b).", + "section": "RESULTS", + "ner": [ + [ + 19, + 35, + "single mutations", + "experimental_method" + ], + [ + 67, + 70, + "PIN", + "structure_element" + ], + [ + 71, + 74, + "PIN", + "structure_element" + ], + [ + 110, + 117, + "monomer", + "oligomeric_state" + ], + [ + 142, + 156, + "gel filtration", + "experimental_method" + ] + ] + }, + { + "sid": 52, + "sent": "Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Wild type", + "protein_state" + ], + [ + 14, + 23, + "monomeric", + "oligomeric_state" + ], + [ + 24, + 27, + "PIN", + "structure_element" + ], + [ + 28, + 35, + "mutants", + "protein_state" + ], + [ + 37, + 42, + "P212A", + "mutant" + ], + [ + 47, + 52, + "D278R", + "mutant" + ], + [ + 76, + 79, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 53, + "sent": "The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "spectra", + "evidence" + ], + [ + 30, + 45, + "dimer interface", + "site" + ], + [ + 53, + 62, + "wild type", + "protein_state" + ], + [ + 63, + 66, + "PIN", + "structure_element" + ], + [ + 119, + 128, + "monomeric", + "oligomeric_state" + ], + [ + 129, + 136, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 54, + "sent": "These results indicate that the PIN domain forms a head-to-tail oligomer in solution similar to the crystal structure.", + "section": "RESULTS", + "ner": [ + [ + 32, + 35, + "PIN", + "structure_element" + ], + [ + 51, + 63, + "head-to-tail", + "protein_state" + ], + [ + 64, + 72, + "oligomer", + "oligomeric_state" + ], + [ + 100, + 117, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 55, + "sent": "Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c).", + "section": "RESULTS", + "ner": [ + [ + 19, + 28, + "monomeric", + "oligomeric_state" + ], + [ + 29, + 32, + "PIN", + "structure_element" + ], + [ + 33, + 40, + "mutants", + "protein_state" + ], + [ + 41, + 46, + "P212A", + "mutant" + ], + [ + 48, + 53, + "R214A", + "mutant" + ], + [ + 59, + 64, + "D278R", + "mutant" + ], + [ + 84, + 89, + "RNase", + "protein_type" + ], + [ + 103, + 107, + "IL-6", + "protein_type" + ], + [ + 108, + 112, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 56, + "sent": "The side chains of these residues point away from the catalytic center on the same molecule (Fig. 2b).", + "section": "RESULTS", + "ner": [ + [ + 54, + 70, + "catalytic center", + "site" + ] + ] + }, + { + "sid": 57, + "sent": "Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro.", + "section": "RESULTS", + "ner": [ + [ + 29, + 41, + "head-to-tail", + "protein_state" + ], + [ + 42, + 45, + "PIN", + "structure_element" + ], + [ + 78, + 81, + "NTD", + "structure_element" + ], + [ + 100, + 109, + "Regnase-1", + "protein" + ], + [ + 110, + 115, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 58, + "sent": "Domain-domain interaction between the NTD and the PIN domain", + "section": "RESULTS", + "ner": [ + [ + 38, + 41, + "NTD", + "structure_element" + ], + [ + 50, + 53, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 59, + "sent": "While the NTD does not contribute to RNA binding (Fig. 1f,g, and Supplementary Fig. 1), it increases the RNase activity of Regnase-1 (Fig. 1h).", + "section": "RESULTS", + "ner": [ + [ + 10, + 13, + "NTD", + "structure_element" + ], + [ + 37, + 40, + "RNA", + "chemical" + ], + [ + 105, + 110, + "RNase", + "protein_type" + ], + [ + 123, + 132, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 60, + "sent": "In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR.", + "section": "RESULTS", + "ner": [ + [ + 61, + 64, + "NTD", + "structure_element" + ], + [ + 89, + 98, + "Regnase-1", + "protein" + ], + [ + 99, + 104, + "RNase", + "protein_type" + ], + [ + 181, + 184, + "NTD", + "structure_element" + ], + [ + 193, + 196, + "PIN", + "structure_element" + ], + [ + 210, + 213, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 61, + "sent": "We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain.", + "section": "RESULTS", + "ner": [ + [ + 12, + 34, + "catalytically inactive", + "protein_state" + ], + [ + 35, + 44, + "monomeric", + "oligomeric_state" + ], + [ + 45, + 48, + "PIN", + "structure_element" + ], + [ + 49, + 55, + "mutant", + "protein_state" + ], + [ + 76, + 80, + "DDNN", + "mutant" + ], + [ + 85, + 90, + "D278R", + "mutant" + ], + [ + 110, + 115, + "dimer", + "oligomeric_state" + ], + [ + 133, + 136, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 62, + "sent": "The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "NMR", + "experimental_method" + ], + [ + 25, + 28, + "PIN", + "structure_element" + ], + [ + 46, + 50, + "V177", + "residue_name_number" + ], + [ + 52, + 61, + "F210-T211", + "residue_range" + ], + [ + 63, + 67, + "R214", + "residue_name_number" + ], + [ + 69, + 78, + "F228-L232", + "residue_range" + ], + [ + 84, + 93, + "F234-S236", + "residue_range" + ], + [ + 145, + 156, + "addition of", + "experimental_method" + ], + [ + 161, + 164, + "NTD", + "structure_element" + ] + ] + }, + { + "sid": 63, + "sent": "Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5).", + "section": "RESULTS", + "ner": [ + [ + 15, + 26, + "addition of", + "experimental_method" + ], + [ + 31, + 34, + "PIN", + "structure_element" + ], + [ + 43, + 46, + "NMR", + "experimental_method" + ], + [ + 68, + 71, + "R56", + "residue_name_number" + ], + [ + 73, + 80, + "L58-G59", + "residue_range" + ], + [ + 86, + 93, + "V86-H88", + "residue_range" + ], + [ + 101, + 104, + "NTD", + "structure_element" + ], + [ + 157, + 160, + "D53", + "residue_name_number" + ], + [ + 162, + 165, + "F55", + "residue_name_number" + ], + [ + 167, + 170, + "K57", + "residue_name_number" + ], + [ + 172, + 179, + "Y60-S61", + "residue_range" + ], + [ + 181, + 184, + "V68", + "residue_name_number" + ], + [ + 186, + 193, + "T80-G83", + "residue_range" + ], + [ + 195, + 198, + "L85", + "residue_name_number" + ], + [ + 204, + 207, + "G89", + "residue_name_number" + ], + [ + 215, + 218, + "NTD", + "structure_element" + ], + [ + 258, + 261, + "N79", + "residue_name_number" + ] + ] + }, + { + "sid": 64, + "sent": "These results clearly indicate a direct interaction between the PIN domain and the NTD.", + "section": "RESULTS", + "ner": [ + [ + 64, + 67, + "PIN", + "structure_element" + ], + [ + 83, + 86, + "NTD", + "structure_element" + ] + ] + }, + { + "sid": 65, + "sent": "Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110\u2009\u00b1\u20095.8\u2009\u03bcM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein.", + "section": "RESULTS", + "ner": [ + [ + 13, + 28, + "titration curve", + "evidence" + ], + [ + 37, + 59, + "chemical shift changes", + "evidence" + ], + [ + 63, + 66, + "L58", + "residue_name_number" + ], + [ + 81, + 83, + "Kd", + "evidence" + ], + [ + 105, + 108, + "NTD", + "structure_element" + ], + [ + 113, + 116, + "PIN", + "structure_element" + ], + [ + 181, + 184, + "NTD", + "structure_element" + ], + [ + 189, + 192, + "PIN", + "structure_element" + ], + [ + 219, + 225, + "linker", + "structure_element" + ], + [ + 238, + 254, + "binding affinity", + "evidence" + ], + [ + 286, + 292, + "native", + "protein_state" + ] + ] + }, + { + "sid": 66, + "sent": "Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a).", + "section": "RESULTS", + "ner": [ + [ + 70, + 87, + "PIN/NTD interface", + "site" + ], + [ + 106, + 111, + "helix", + "structure_element" + ], + [ + 144, + 148, + "D225", + "residue_name_number" + ], + [ + 153, + 157, + "D226", + "residue_name_number" + ], + [ + 165, + 168, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 67, + "sent": "Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can \u201cterminate\u201d PIN-PIN oligomerization (Fig. 2b).", + "section": "RESULTS", + "ner": [ + [ + 28, + 40, + "binding site", + "site" + ], + [ + 49, + 52, + "NTD", + "structure_element" + ], + [ + 71, + 94, + "PIN-PIN dimer interface", + "site" + ], + [ + 110, + 113, + "NTD", + "structure_element" + ], + [ + 138, + 141, + "PIN", + "structure_element" + ], + [ + 142, + 145, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 68, + "sent": "An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c).", + "section": "RESULTS", + "ner": [ + [ + 3, + 20, + "in silico docking", + "experimental_method" + ], + [ + 28, + 31, + "NTD", + "structure_element" + ], + [ + 36, + 39, + "PIN", + "structure_element" + ], + [ + 54, + 79, + "chemical shift restraints", + "evidence" + ], + [ + 117, + 120, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 69, + "sent": "Residues critical for Regnase-1 RNase activity", + "section": "RESULTS", + "ner": [ + [ + 22, + 31, + "Regnase-1", + "protein" + ], + [ + 32, + 37, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 70, + "sent": "To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 47, + 56, + "Regnase-1", + "protein" + ], + [ + 57, + 62, + "RNase", + "protein_type" + ], + [ + 123, + 137, + "catalytic site", + "site" + ], + [ + 145, + 148, + "PIN", + "structure_element" + ], + [ + 149, + 157, + "oligomer", + "oligomeric_state" + ], + [ + 162, + 172, + "mutated to", + "experimental_method" + ], + [ + 173, + 180, + "alanine", + "residue_name" + ], + [ + 210, + 215, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 71, + "sent": "From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation.", + "section": "RESULTS", + "ner": [ + [ + 9, + 30, + "gel filtration assays", + "experimental_method" + ], + [ + 36, + 43, + "mutants", + "protein_state" + ], + [ + 51, + 56, + "R214A", + "mutant" + ], + [ + 64, + 70, + "dimers", + "oligomeric_state" + ], + [ + 100, + 105, + "RNase", + "protein_type" + ], + [ + 122, + 129, + "mutants", + "protein_state" + ], + [ + 138, + 143, + "R214A", + "mutant" + ], + [ + 234, + 239, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 72, + "sent": "The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "W182A", + "mutant" + ], + [ + 11, + 16, + "R183A", + "mutant" + ], + [ + 22, + 27, + "R214A", + "mutant" + ], + [ + 28, + 35, + "mutants", + "protein_state" + ], + [ + 72, + 76, + "IL-6", + "protein_type" + ], + [ + 77, + 81, + "mRNA", + "chemical" + ], + [ + 97, + 106, + "Regnase-1", + "protein" + ], + [ + 107, + 111, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 73, + "sent": "The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "K184A", + "mutant" + ], + [ + 11, + 16, + "R215A", + "mutant" + ], + [ + 22, + 27, + "R220A", + "mutant" + ], + [ + 28, + 35, + "mutants", + "protein_state" + ], + [ + 113, + 118, + "mRNAs", + "chemical" + ] + ] + }, + { + "sid": 74, + "sent": "The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA.", + "section": "RESULTS", + "ner": [ + [ + 18, + 22, + "K219", + "residue_name_number" + ], + [ + 27, + 31, + "R247", + "residue_name_number" + ], + [ + 59, + 63, + "IL-6", + "protein_type" + ], + [ + 68, + 77, + "Regnase-1", + "protein" + ], + [ + 78, + 82, + "mRNA", + "chemical" + ], + [ + 89, + 93, + "K219", + "residue_name_number" + ], + [ + 98, + 102, + "R247", + "residue_name_number" + ], + [ + 142, + 146, + "IL-6", + "protein_type" + ], + [ + 147, + 151, + "mRNA", + "chemical" + ], + [ + 161, + 170, + "Regnase-1", + "protein" + ], + [ + 171, + 175, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 75, + "sent": "The other mutated residues\u2014K152, R158, R188, R200, K204, K206, K257, and R258\u2014were not critical for RNase activity.", + "section": "RESULTS", + "ner": [ + [ + 27, + 31, + "K152", + "residue_name_number" + ], + [ + 33, + 37, + "R158", + "residue_name_number" + ], + [ + 39, + 43, + "R188", + "residue_name_number" + ], + [ + 45, + 49, + "R200", + "residue_name_number" + ], + [ + 51, + 55, + "K204", + "residue_name_number" + ], + [ + 57, + 61, + "K206", + "residue_name_number" + ], + [ + 63, + 67, + "K257", + "residue_name_number" + ], + [ + 73, + 77, + "R258", + "residue_name_number" + ], + [ + 100, + 105, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 76, + "sent": "The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the \u201csecondary\u201d chain\u2019s residue K184 is positioned near the \u201cprimary\u201d chain\u2019s catalytic site (Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 27, + 31, + "W182", + "residue_name_number" + ], + [ + 36, + 40, + "R183", + "residue_name_number" + ], + [ + 83, + 92, + "monomeric", + "oligomeric_state" + ], + [ + 93, + 96, + "PIN", + "structure_element" + ], + [ + 97, + 106, + "structure", + "evidence" + ], + [ + 139, + 144, + "RNase", + "protein_type" + ], + [ + 145, + 159, + "catalytic site", + "site" + ], + [ + 196, + 200, + "K184", + "residue_name_number" + ], + [ + 229, + 240, + "active site", + "site" + ], + [ + 296, + 305, + "structure", + "evidence" + ], + [ + 348, + 352, + "K184", + "residue_name_number" + ], + [ + 377, + 385, + "primary\u201d", + "protein_state" + ], + [ + 394, + 408, + "catalytic site", + "site" + ] + ] + }, + { + "sid": 77, + "sent": "In contrast, R214 is important for oligomerization of the PIN domain and the \u201csecondary\u201d chain\u2019s residue R214 is also positioned near the \u201cprimary\u201d chain\u2019s active site within the dimer interface.", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "R214", + "residue_name_number" + ], + [ + 58, + 61, + "PIN", + "structure_element" + ], + [ + 105, + 109, + "R214", + "residue_name_number" + ], + [ + 139, + 146, + "primary", + "protein_state" + ], + [ + 156, + 167, + "active site", + "site" + ], + [ + 179, + 194, + "dimer interface", + "site" + ] + ] + }, + { + "sid": 78, + "sent": "It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains.", + "section": "RESULTS", + "ner": [ + [ + 28, + 57, + "putative-RNA binding residues", + "site" + ], + [ + 58, + 62, + "K184", + "residue_name_number" + ], + [ + 67, + 71, + "R214", + "residue_name_number" + ], + [ + 86, + 95, + "Regnase-1", + "protein" + ], + [ + 102, + 105, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 79, + "sent": "Molecular mechanism of target mRNA cleavage by the PIN dimer", + "section": "RESULTS", + "ner": [ + [ + 30, + 34, + "mRNA", + "chemical" + ], + [ + 51, + 54, + "PIN", + "structure_element" + ], + [ + 55, + 60, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 80, + "sent": "Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain.", + "section": "RESULTS", + "ner": [ + [ + 4, + 26, + "mutational experiments", + "experimental_method" + ], + [ + 55, + 60, + "dimer", + "oligomeric_state" + ], + [ + 100, + 109, + "secondary", + "protein_state" + ], + [ + 110, + 113, + "PIN", + "structure_element" + ], + [ + 136, + 145, + "Regnase-1", + "protein" + ], + [ + 153, + 173, + "RNA binding residues", + "site" + ], + [ + 183, + 194, + "active site", + "site" + ], + [ + 202, + 209, + "primary", + "protein_state" + ], + [ + 210, + 213, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 81, + "sent": "If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together.", + "section": "RESULTS", + "ner": [ + [ + 50, + 72, + "catalytically inactive", + "protein_state" + ], + [ + 73, + 76, + "PIN", + "structure_element" + ], + [ + 83, + 86, + "PIN", + "structure_element" + ], + [ + 87, + 94, + "lacking", + "protein_state" + ], + [ + 108, + 128, + "RNA-binding residues", + "site" + ], + [ + 141, + 149, + "inactive", + "protein_state" + ], + [ + 174, + 180, + "active", + "protein_state" + ] + ] + }, + { + "sid": 82, + "sent": "In order to test this hypothesis, we performed in vitro cleavage assays using combinations of Regnase-1 mutants that had no or decreased RNase activities by themselves (Fig. 5).", + "section": "RESULTS", + "ner": [ + [ + 47, + 71, + "in vitro cleavage assays", + "experimental_method" + ], + [ + 94, + 103, + "Regnase-1", + "protein" + ], + [ + 104, + 111, + "mutants", + "protein_state" + ], + [ + 137, + 142, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 83, + "sent": "One group consisted of catalytically active PIN domains with mutation of basic residues found in the previous section to confer decreased RNase activity (Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 23, + 43, + "catalytically active", + "protein_state" + ], + [ + 44, + 47, + "PIN", + "structure_element" + ], + [ + 61, + 72, + "mutation of", + "experimental_method" + ], + [ + 138, + 143, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 84, + "sent": "These were paired with a DDNN mutant that had no RNase activity by itself.", + "section": "RESULTS", + "ner": [ + [ + 25, + 29, + "DDNN", + "mutant" + ], + [ + 30, + 36, + "mutant", + "protein_state" + ], + [ + 49, + 54, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 85, + "sent": "When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a).", + "section": "RESULTS", + "ner": [ + [ + 59, + 71, + "heterodimers", + "oligomeric_state" + ], + [ + 108, + 112, + "DDNN", + "mutant" + ], + [ + 113, + 120, + "primary", + "protein_state" + ], + [ + 121, + 124, + "PIN", + "structure_element" + ], + [ + 145, + 151, + "mutant", + "protein_state" + ], + [ + 152, + 161, + "secondary", + "protein_state" + ], + [ + 162, + 165, + "PIN", + "structure_element" + ], + [ + 196, + 201, + "RNase", + "protein_type" + ], + [ + 253, + 259, + "mutant", + "protein_state" + ], + [ + 260, + 267, + "primary", + "protein_state" + ], + [ + 268, + 271, + "PIN", + "structure_element" + ], + [ + 278, + 282, + "DDNN", + "mutant" + ], + [ + 283, + 292, + "secondary", + "protein_state" + ], + [ + 293, + 296, + "PIN", + "structure_element" + ], + [ + 324, + 329, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 86, + "sent": "When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b).", + "section": "RESULTS", + "ner": [ + [ + 21, + 43, + "fluorescence intensity", + "evidence" + ], + [ + 47, + 56, + "uncleaved", + "protein_state" + ], + [ + 57, + 61, + "IL-6", + "protein_type" + ], + [ + 62, + 66, + "mRNA", + "chemical" + ], + [ + 82, + 89, + "mutants", + "protein_state" + ], + [ + 90, + 95, + "W182A", + "mutant" + ], + [ + 97, + 102, + "K184A", + "mutant" + ], + [ + 104, + 109, + "R214A", + "mutant" + ], + [ + 115, + 120, + "R220A", + "mutant" + ], + [ + 155, + 159, + "DDNN", + "mutant" + ], + [ + 160, + 166, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 87, + "sent": "Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c).", + "section": "RESULTS", + "ner": [ + [ + 35, + 57, + "fluorescence intensity", + "evidence" + ], + [ + 65, + 74, + "uncleaved", + "protein_state" + ], + [ + 75, + 84, + "Regnase-1", + "protein" + ], + [ + 85, + 89, + "mRNA", + "chemical" + ], + [ + 105, + 112, + "mutants", + "protein_state" + ], + [ + 113, + 118, + "K184A", + "mutant" + ], + [ + 123, + 128, + "R214A", + "mutant" + ], + [ + 163, + 167, + "DDNN", + "mutant" + ], + [ + 168, + 174, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 88, + "sent": "Rescue of K184A and R214A by the DDNN mutant was also confirmed by a significant increase in the cleaved products.", + "section": "RESULTS", + "ner": [ + [ + 10, + 15, + "K184A", + "mutant" + ], + [ + 20, + 25, + "R214A", + "mutant" + ], + [ + 33, + 37, + "DDNN", + "mutant" + ], + [ + 38, + 44, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 89, + "sent": "This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN.", + "section": "RESULTS", + "ner": [ + [ + 60, + 64, + "K184", + "residue_name_number" + ], + [ + 69, + 73, + "R214", + "residue_name_number" + ], + [ + 81, + 88, + "primary", + "protein_state" + ], + [ + 89, + 92, + "PIN", + "structure_element" + ], + [ + 126, + 142, + "catalytic center", + "site" + ], + [ + 163, + 172, + "secondary", + "protein_state" + ], + [ + 173, + 176, + "PIN", + "structure_element" + ], + [ + 193, + 209, + "catalytic center", + "site" + ], + [ + 217, + 224, + "primary", + "protein_state" + ], + [ + 225, + 228, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 90, + "sent": "R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "R214", + "residue_name_number" + ], + [ + 33, + 38, + "dimer", + "oligomeric_state" + ], + [ + 80, + 85, + "R214A", + "mutant" + ], + [ + 93, + 102, + "secondary", + "protein_state" + ], + [ + 103, + 106, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 91, + "sent": "According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN.", + "section": "RESULTS", + "ner": [ + [ + 36, + 41, + "R214A", + "mutant" + ], + [ + 42, + 45, + "PIN", + "structure_element" + ], + [ + 69, + 74, + "dimer", + "oligomeric_state" + ], + [ + 84, + 88, + "DDNN", + "mutant" + ], + [ + 89, + 92, + "PIN", + "structure_element" + ], + [ + 105, + 114, + "secondary", + "protein_state" + ], + [ + 115, + 118, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 92, + "sent": "Taken together, the rescue experiments above support the proposed model in which the head-to-tail dimer is functional in vitro.", + "section": "RESULTS", + "ner": [ + [ + 85, + 97, + "head-to-tail", + "protein_state" + ], + [ + 98, + 103, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 93, + "sent": "We determined the individual domain structures of Regnase-1 by NMR and X-ray crystallography.", + "section": "DISCUSS", + "ner": [ + [ + 36, + 46, + "structures", + "evidence" + ], + [ + 50, + 59, + "Regnase-1", + "protein" + ], + [ + 63, + 66, + "NMR", + "experimental_method" + ], + [ + 71, + 92, + "X-ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 94, + "sent": "Although the function of the CTD remains elusive, we revealed the functions of the NTD, PIN, and ZF domains.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 32, + "CTD", + "structure_element" + ], + [ + 83, + 86, + "NTD", + "structure_element" + ], + [ + 88, + 91, + "PIN", + "structure_element" + ], + [ + 97, + 99, + "ZF", + "structure_element" + ] + ] + }, + { + "sid": 95, + "sent": "A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions.", + "section": "DISCUSS", + "ner": [ + [ + 2, + 11, + "Regnase-1", + "protein" + ], + [ + 36, + 39, + "PIN", + "structure_element" + ], + [ + 44, + 46, + "ZF", + "structure_element" + ], + [ + 68, + 80, + "Mus musculus", + "species" + ], + [ + 85, + 97, + "crystallized", + "experimental_method" + ], + [ + 112, + 128, + "electron density", + "evidence" + ], + [ + 136, + 138, + "ZF", + "structure_element" + ], + [ + 175, + 177, + "ZF", + "structure_element" + ], + [ + 188, + 201, + "highly mobile", + "protein_state" + ], + [ + 209, + 219, + "absence of", + "protein_state" + ], + [ + 227, + 231, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 96, + "sent": "Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10\u2009\u00b1\u20091.1\u2009\u03bcM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 7, + "NMR", + "experimental_method" + ], + [ + 52, + 54, + "ZF", + "structure_element" + ], + [ + 65, + 69, + "IL-6", + "protein_type" + ], + [ + 70, + 74, + "mRNA", + "chemical" + ], + [ + 82, + 84, + "Kd", + "evidence" + ], + [ + 117, + 141, + "in vitro gel shift assay", + "experimental_method" + ], + [ + 157, + 166, + "Regnase-1", + "protein" + ], + [ + 182, + 184, + "ZF", + "structure_element" + ], + [ + 208, + 212, + "mRNA", + "chemical" + ], + [ + 238, + 241, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 97, + "sent": "These results indicate that Regnase-1 directly binds to RNA and precipitates under such experimental conditions.", + "section": "DISCUSS", + "ner": [ + [ + 28, + 37, + "Regnase-1", + "protein" + ], + [ + 56, + 59, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 98, + "sent": "Due to this limitation, it is difficult to perform further structural analyses of mRNA-Regnase-1 complexes by X-ray crystallography or NMR.", + "section": "DISCUSS", + "ner": [ + [ + 59, + 78, + "structural analyses", + "experimental_method" + ], + [ + 82, + 96, + "mRNA-Regnase-1", + "complex_assembly" + ], + [ + 110, + 131, + "X-ray crystallography", + "experimental_method" + ], + [ + 135, + 138, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 99, + "sent": "The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2\u2009\u00c5. The amino acid sequences corresponding to PIN (residues 134\u2013295) are the two non-identical residues are substituted with similar amino acids.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 41, + "crystal structure", + "evidence" + ], + [ + 49, + 58, + "Regnase-1", + "protein" + ], + [ + 59, + 62, + "PIN", + "structure_element" + ], + [ + 83, + 95, + "Homo sapiens", + "species" + ], + [ + 140, + 152, + "Mus musculus", + "species" + ], + [ + 184, + 188, + "RMSD", + "evidence" + ], + [ + 241, + 244, + "PIN", + "structure_element" + ], + [ + 255, + 262, + "134\u2013295", + "residue_range" + ] + ] + }, + { + "sid": 100, + "sent": "Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 14, + "mouse", + "taxonomy_domain" + ], + [ + 19, + 24, + "human", + "species" + ], + [ + 25, + 28, + "PIN", + "structure_element" + ], + [ + 42, + 54, + "head-to-tail", + "protein_state" + ], + [ + 55, + 64, + "oligomers", + "oligomeric_state" + ], + [ + 83, + 96, + "crystal forms", + "evidence" + ] + ] + }, + { + "sid": 101, + "sent": "Rao and co-workers previously argued that PIN dimerization is likely to be a crystallographic artifact with no physiological significance, since monomers were dominant in their analytical ultra-centrifugation experiments.", + "section": "DISCUSS", + "ner": [ + [ + 42, + 45, + "PIN", + "structure_element" + ], + [ + 145, + 153, + "monomers", + "oligomeric_state" + ], + [ + 177, + 208, + "analytical ultra-centrifugation", + "experimental_method" + ] + ] + }, + { + "sid": 102, + "sent": "In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 31, + "gel filtration", + "experimental_method" + ], + [ + 38, + 57, + "mutational analyses", + "experimental_method" + ], + [ + 63, + 66, + "NMR", + "experimental_method" + ], + [ + 67, + 74, + "spectra", + "evidence" + ], + [ + 97, + 100, + "PIN", + "structure_element" + ], + [ + 116, + 128, + "head-to-tail", + "protein_state" + ], + [ + 129, + 134, + "dimer", + "oligomeric_state" + ], + [ + 174, + 191, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 103, + "sent": "This inconsistency might be due to difference in the analytical methods and/or protein concentrations used in each experiment, since the oligomer formation of PIN was dependent on the protein concentration in our study.", + "section": "DISCUSS", + "ner": [ + [ + 137, + 145, + "oligomer", + "oligomeric_state" + ], + [ + 159, + 162, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 104, + "sent": "Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 16, + "Single mutations", + "experimental_method" + ], + [ + 80, + 83, + "PIN", + "structure_element" + ], + [ + 84, + 95, + "monomerized", + "oligomeric_state" + ], + [ + 118, + 125, + "mutants", + "protein_state" + ], + [ + 137, + 142, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 105, + "sent": "Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c).", + "section": "DISCUSS", + "ner": [ + [ + 10, + 13, + "NMR", + "experimental_method" + ], + [ + 14, + 21, + "spectra", + "evidence" + ], + [ + 25, + 34, + "monomeric", + "oligomeric_state" + ], + [ + 35, + 42, + "mutants", + "protein_state" + ], + [ + 138, + 147, + "monomeric", + "oligomeric_state" + ], + [ + 148, + 155, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 106, + "sent": "Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro.", + "section": "DISCUSS", + "ner": [ + [ + 47, + 50, + "PIN", + "structure_element" + ], + [ + 51, + 54, + "PIN", + "structure_element" + ], + [ + 55, + 60, + "dimer", + "oligomeric_state" + ], + [ + 87, + 96, + "Regnase-1", + "protein" + ], + [ + 97, + 102, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 107, + "sent": "Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the \u201cprimary\u201d and \u201csecondary\u201d PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6).", + "section": "DISCUSS", + "ner": [ + [ + 11, + 28, + "crystal structure", + "evidence" + ], + [ + 36, + 39, + "PIN", + "structure_element" + ], + [ + 40, + 45, + "dimer", + "oligomeric_state" + ], + [ + 51, + 60, + "Regnase-1", + "protein" + ], + [ + 97, + 104, + "primary", + "protein_state" + ], + [ + 111, + 120, + "secondary", + "protein_state" + ], + [ + 122, + 126, + "PINs", + "structure_element" + ], + [ + 150, + 164, + "catalytic site", + "site" + ], + [ + 180, + 183, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 108, + "sent": "Moreover, our structure-based mutational analyses showed these two Regnase-1 specific basic regions were essential for target mRNA cleavage in vitro.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 49, + "structure-based mutational analyses", + "experimental_method" + ], + [ + 67, + 76, + "Regnase-1", + "protein" + ], + [ + 126, + 130, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 109, + "sent": "The cleavage assay also showed that the NTD is crucial for efficient mRNA cleavage.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 18, + "cleavage assay", + "experimental_method" + ], + [ + 40, + 43, + "NTD", + "structure_element" + ], + [ + 69, + 73, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 110, + "sent": "Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a).", + "section": "DISCUSS", + "ner": [ + [ + 28, + 31, + "NTD", + "structure_element" + ], + [ + 52, + 70, + "oligomeric surface", + "site" + ], + [ + 78, + 85, + "primary", + "protein_state" + ], + [ + 86, + 89, + "PIN", + "structure_element" + ], + [ + 104, + 109, + "helix", + "structure_element" + ], + [ + 127, + 145, + "catalytic residues", + "site" + ] + ] + }, + { + "sid": 111, + "sent": "Taken together, this suggests that the NTD and the PIN domain compete for a common binding site.", + "section": "DISCUSS", + "ner": [ + [ + 39, + 42, + "NTD", + "structure_element" + ], + [ + 51, + 54, + "PIN", + "structure_element" + ], + [ + 76, + 95, + "common binding site", + "site" + ] + ] + }, + { + "sid": 112, + "sent": "The affinity of the domain-domain interaction between two PIN domains (Kd\u2009=\u2009~10\u22124\u2009M) is similar to that of the NTD-PIN (Kd\u2009=\u2009110\u2009\u00b1\u20095.8\u2009\u03bcM) interactions; however, the covalent connection corresponding to residues 90\u2013133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 12, + "affinity", + "evidence" + ], + [ + 58, + 61, + "PIN", + "structure_element" + ], + [ + 71, + 73, + "Kd", + "evidence" + ], + [ + 111, + 114, + "NTD", + "structure_element" + ], + [ + 115, + 118, + "PIN", + "structure_element" + ], + [ + 120, + 122, + "Kd", + "evidence" + ], + [ + 212, + 218, + "90\u2013133", + "residue_range" + ], + [ + 231, + 234, + "NTD", + "structure_element" + ], + [ + 243, + 250, + "primary", + "protein_state" + ], + [ + 251, + 254, + "PIN", + "structure_element" + ], + [ + 329, + 340, + "full-length", + "protein_state" + ], + [ + 341, + 350, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 113, + "sent": "While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+.", + "section": "DISCUSS", + "ner": [ + [ + 74, + 116, + "docking and molecular dynamics simulations", + "experimental_method" + ], + [ + 131, + 134, + "NTD", + "structure_element" + ], + [ + 158, + 176, + "catalytic residues", + "site" + ], + [ + 184, + 187, + "PIN", + "structure_element" + ], + [ + 205, + 211, + "active", + "protein_state" + ], + [ + 246, + 250, + "Mg2+", + "chemical" + ] + ] + }, + { + "sid": 114, + "sent": "In this context, it is interesting that, in response to TCR stimulation, Malt1 cleaves Regnase-1 at R111 to control immune responses in vivo.", + "section": "DISCUSS", + "ner": [ + [ + 73, + 78, + "Malt1", + "protein" + ], + [ + 87, + 96, + "Regnase-1", + "protein" + ], + [ + 100, + 104, + "R111", + "residue_name_number" + ] + ] + }, + { + "sid": 115, + "sent": "This result is consistent with a model in which the NTD acts as an enhancer, and cleavage of the linker lowers enzymatic activity dramatically.", + "section": "DISCUSS", + "ner": [ + [ + 52, + 55, + "NTD", + "structure_element" + ], + [ + 97, + 103, + "linker", + "structure_element" + ] + ] + }, + { + "sid": 116, + "sent": "Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 49, + "structural and functional analyses", + "experimental_method" + ], + [ + 53, + 62, + "Regnase-1", + "protein" + ], + [ + 104, + 123, + "docking simulations", + "experimental_method" + ], + [ + 131, + 134, + "NTD", + "structure_element" + ], + [ + 136, + 139, + "PIN", + "structure_element" + ], + [ + 140, + 145, + "dimer", + "oligomeric_state" + ], + [ + 151, + 155, + "IL-6", + "protein_type" + ], + [ + 156, + 160, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 117, + "sent": "We incorporated information from the cleavage site of IL-6 mRNA in vitro is indicated by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 7a,b).", + "section": "DISCUSS", + "ner": [ + [ + 37, + 50, + "cleavage site", + "site" + ], + [ + 54, + 58, + "IL-6", + "protein_type" + ], + [ + 59, + 63, + "mRNA", + "chemical" + ], + [ + 100, + 134, + "polyacrylamide gel electrophoresis", + "experimental_method" + ] + ] + }, + { + "sid": 118, + "sent": "The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 11, + "docking", + "experimental_method" + ], + [ + 37, + 40, + "RNA", + "chemical" + ], + [ + 207, + 227, + "RNA-binding proteins", + "protein_type" + ], + [ + 247, + 251, + "loop", + "structure_element" + ], + [ + 277, + 286, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 119, + "sent": "The overall model of regulation of Regnase-1 RNase activity through domain-domain interactions in vitro is summarized in Fig. 6.", + "section": "DISCUSS", + "ner": [ + [ + 35, + 44, + "Regnase-1", + "protein" + ], + [ + 45, + 50, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 120, + "sent": "In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 17, + "absence of", + "protein_state" + ], + [ + 25, + 29, + "mRNA", + "chemical" + ], + [ + 35, + 38, + "PIN", + "structure_element" + ], + [ + 52, + 64, + "head-to-tail", + "protein_state" + ], + [ + 65, + 74, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 121, + "sent": "A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN).", + "section": "DISCUSS", + "ner": [ + [ + 2, + 14, + "fully active", + "protein_state" + ], + [ + 15, + 31, + "catalytic center", + "site" + ], + [ + 60, + 63, + "NTD", + "structure_element" + ], + [ + 84, + 92, + "oligomer", + "oligomeric_state" + ], + [ + 108, + 111, + "PIN", + "structure_element" + ], + [ + 141, + 153, + "head-to-tail", + "protein_state" + ], + [ + 154, + 162, + "oligomer", + "oligomeric_state" + ], + [ + 191, + 198, + "primary", + "protein_state" + ], + [ + 199, + 202, + "PIN", + "structure_element" + ], + [ + 217, + 227, + "functional", + "protein_state" + ], + [ + 228, + 233, + "dimer", + "oligomeric_state" + ], + [ + 264, + 267, + "PIN", + "structure_element" + ], + [ + 269, + 278, + "secondary", + "protein_state" + ], + [ + 279, + 282, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 122, + "sent": "While further investigations on the domain-domain interactions of Regnase-1 in vivo are necessary, these intramolecular and intermolecular domain interactions of Regnase-1 appear to structurally constrain Regnase-1activity, which, in turn, enables tight regulation of immune responses.", + "section": "DISCUSS", + "ner": [ + [ + 66, + 75, + "Regnase-1", + "protein" + ], + [ + 162, + 171, + "Regnase-1", + "protein" + ], + [ + 205, + 214, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 123, + "sent": "Structural and functional analyses of Regnase-1.", + "section": "FIG", + "ner": [ + [ + 0, + 34, + "Structural and functional analyses", + "experimental_method" + ], + [ + 38, + 47, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 124, + "sent": "(a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain.", + "section": "FIG", + "ner": [ + [ + 27, + 36, + "Regnase-1", + "protein" + ], + [ + 42, + 60, + "Solution structure", + "evidence" + ], + [ + 68, + 71, + "NTD", + "structure_element" + ], + [ + 77, + 94, + "Crystal structure", + "evidence" + ], + [ + 102, + 105, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 125, + "sent": "Catalytic Asp residues were shown in sticks.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Catalytic", + "protein_state" + ], + [ + 10, + 13, + "Asp", + "residue_name" + ] + ] + }, + { + "sid": 126, + "sent": "(d) Solution structure of the ZF domain.", + "section": "FIG", + "ner": [ + [ + 4, + 22, + "Solution structure", + "evidence" + ], + [ + 30, + 32, + "ZF", + "structure_element" + ] + ] + }, + { + "sid": 127, + "sent": "Three Cys residues and one His residue responsible for Zn2+-binding were shown in sticks.", + "section": "FIG", + "ner": [ + [ + 6, + 9, + "Cys", + "residue_name" + ], + [ + 27, + 30, + "His", + "residue_name" + ] + ] + }, + { + "sid": 128, + "sent": "(e) Solution structure of the CTD.", + "section": "FIG", + "ner": [ + [ + 4, + 22, + "Solution structure", + "evidence" + ], + [ + 30, + 33, + "CTD", + "structure_element" + ] + ] + }, + { + "sid": 129, + "sent": "All the structures were colored in rainbow from N-terminus (blue) to C-terminus (red).", + "section": "FIG", + "ner": [ + [ + 8, + 18, + "structures", + "evidence" + ] + ] + }, + { + "sid": 130, + "sent": "(f) In vitro gel shift binding assay between Regnase-1 and IL-6 mRNA.", + "section": "FIG", + "ner": [ + [ + 4, + 36, + "In vitro gel shift binding assay", + "experimental_method" + ], + [ + 45, + 54, + "Regnase-1", + "protein" + ], + [ + 59, + 63, + "IL-6", + "protein_type" + ], + [ + 64, + 68, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 131, + "sent": "Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1.", + "section": "FIG", + "ner": [ + [ + 0, + 22, + "Fluorescence intensity", + "evidence" + ], + [ + 30, + 34, + "free", + "protein_state" + ], + [ + 35, + 39, + "IL-6", + "protein_type" + ], + [ + 107, + 117, + "absence of", + "protein_state" + ], + [ + 118, + 127, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 132, + "sent": "(g) Binding of Regnase-1 and IL-6 mRNA was plotted.", + "section": "FIG", + "ner": [ + [ + 15, + 24, + "Regnase-1", + "protein" + ], + [ + 29, + 33, + "IL-6", + "protein_type" + ], + [ + 34, + 38, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 133, + "sent": "The percentage of the bound IL-6 was calculated based on the fluorescence intensities of the free IL-6 quantified in (f).", + "section": "FIG", + "ner": [ + [ + 28, + 32, + "IL-6", + "protein_type" + ], + [ + 61, + 85, + "fluorescence intensities", + "evidence" + ], + [ + 98, + 102, + "IL-6", + "protein_type" + ] + ] + }, + { + "sid": 134, + "sent": "(h) In vitro cleavage assay of Regnase-1 to IL-6 mRNA.", + "section": "FIG", + "ner": [ + [ + 4, + 27, + "In vitro cleavage assay", + "experimental_method" + ], + [ + 31, + 40, + "Regnase-1", + "protein" + ], + [ + 44, + 48, + "IL-6", + "protein_type" + ], + [ + 49, + 53, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 135, + "sent": "Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1.", + "section": "FIG", + "ner": [ + [ + 0, + 22, + "Fluorescence intensity", + "evidence" + ], + [ + 30, + 39, + "uncleaved", + "protein_state" + ], + [ + 40, + 44, + "IL-6", + "protein_type" + ], + [ + 45, + 49, + "mRNA", + "chemical" + ], + [ + 102, + 112, + "absence of", + "protein_state" + ], + [ + 113, + 122, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 136, + "sent": "Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1.", + "section": "FIG", + "ner": [ + [ + 0, + 12, + "Head-to-tail", + "protein_state" + ], + [ + 13, + 21, + "oligomer", + "oligomeric_state" + ], + [ + 39, + 42, + "PIN", + "structure_element" + ], + [ + 69, + 74, + "RNase", + "protein_type" + ], + [ + 87, + 96, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 137, + "sent": "(a) Gel filtration analyses of the PIN domain.", + "section": "FIG", + "ner": [ + [ + 4, + 27, + "Gel filtration analyses", + "experimental_method" + ], + [ + 35, + 38, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 138, + "sent": "(b) Dimer structure of the PIN domain.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "Dimer", + "oligomeric_state" + ], + [ + 10, + 19, + "structure", + "evidence" + ], + [ + 27, + 30, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 139, + "sent": "Two PIN molecules in the crystal were colored white and green, respectively.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "PIN", + "structure_element" + ], + [ + 25, + 32, + "crystal", + "evidence" + ] + ] + }, + { + "sid": 140, + "sent": "Catalytic residues and mutated residues were shown in sticks.", + "section": "FIG", + "ner": [ + [ + 0, + 18, + "Catalytic residues", + "site" + ] + ] + }, + { + "sid": 141, + "sent": "Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed.", + "section": "FIG", + "ner": [ + [ + 74, + 78, + "R215", + "residue_name_number" + ], + [ + 153, + 158, + "RNase", + "protein_type" + ], + [ + 171, + 180, + "monomeric", + "oligomeric_state" + ], + [ + 181, + 188, + "mutants", + "protein_state" + ], + [ + 193, + 197, + "IL-6", + "protein_type" + ], + [ + 198, + 202, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 142, + "sent": "Domain-domain interaction between the NTD and the PIN domain.", + "section": "FIG", + "ner": [ + [ + 38, + 41, + "NTD", + "structure_element" + ], + [ + 50, + 53, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 143, + "sent": "(a) NMR analyses of the NTD-binding to the PIN domain.", + "section": "FIG", + "ner": [ + [ + 4, + 16, + "NMR analyses", + "experimental_method" + ], + [ + 24, + 27, + "NTD", + "structure_element" + ], + [ + 43, + 46, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 144, + "sent": "The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red on the surface and ribbon structure of the PIN domain (right).", + "section": "FIG", + "ner": [ + [ + 73, + 81, + "overlaid", + "experimental_method" + ], + [ + 82, + 89, + "spectra", + "evidence" + ], + [ + 156, + 159, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 145, + "sent": "Pro and the residues without analysis were colored black and gray, respectively.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "Pro", + "residue_name" + ] + ] + }, + { + "sid": 146, + "sent": "(b) NMR analyses of the PIN-binding to the NTD.", + "section": "FIG", + "ner": [ + [ + 4, + 16, + "NMR analyses", + "experimental_method" + ], + [ + 24, + 27, + "PIN", + "structure_element" + ], + [ + 43, + 46, + "NTD", + "structure_element" + ] + ] + }, + { + "sid": 147, + "sent": "The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red, yellow, or green on the surface and ribbon structure of the NTD.", + "section": "FIG", + "ner": [ + [ + 18, + 52, + "significant chemical shift changes", + "evidence" + ], + [ + 73, + 81, + "overlaid", + "experimental_method" + ], + [ + 82, + 89, + "spectra", + "evidence" + ], + [ + 174, + 177, + "NTD", + "structure_element" + ] + ] + }, + { + "sid": 148, + "sent": "S62 was colored gray and excluded from the analysis, due to low signal intensity.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "S62", + "residue_name_number" + ] + ] + }, + { + "sid": 149, + "sent": "(c) Docking model of the NTD and the PIN domain.", + "section": "FIG", + "ner": [ + [ + 25, + 28, + "NTD", + "structure_element" + ], + [ + 37, + 40, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 150, + "sent": "The NTD and the PIN domain are shown in cyan and white, respectively.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "NTD", + "structure_element" + ], + [ + 16, + 19, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 151, + "sent": "Residues in close proximity (<5\u2009\u00c5) to each other in the docking structure were colored yellow.", + "section": "FIG", + "ner": [ + [ + 56, + 73, + "docking structure", + "evidence" + ] + ] + }, + { + "sid": 152, + "sent": "Catalytic residues of the PIN domain are shown in sticks, and the residues that exhibited significant chemical shift changes in (a,b) were labeled.", + "section": "FIG", + "ner": [ + [ + 0, + 18, + "Catalytic residues", + "site" + ], + [ + 26, + 29, + "PIN", + "structure_element" + ], + [ + 90, + 124, + "significant chemical shift changes", + "evidence" + ] + ] + }, + { + "sid": 153, + "sent": "Critical residues in the PIN domain for the RNase activity of Regnase-1.", + "section": "FIG", + "ner": [ + [ + 25, + 28, + "PIN", + "structure_element" + ], + [ + 44, + 49, + "RNase", + "protein_type" + ], + [ + 62, + 71, + "Regnase-1", + "protein" + ] + ] + }, + { + "sid": 154, + "sent": "(a) In vitro cleavage assay of basic residue mutants for IL-6 mRNA.", + "section": "FIG", + "ner": [ + [ + 4, + 27, + "In vitro cleavage assay", + "experimental_method" + ], + [ + 45, + 52, + "mutants", + "protein_state" + ], + [ + 57, + 61, + "IL-6", + "protein_type" + ], + [ + 62, + 66, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 155, + "sent": "(b) In vitro cleavage assay of basic residue mutants for Regnase-1 mRNA.", + "section": "FIG", + "ner": [ + [ + 4, + 27, + "In vitro cleavage assay", + "experimental_method" + ], + [ + 45, + 52, + "mutants", + "protein_state" + ], + [ + 57, + 66, + "Regnase-1", + "protein" + ], + [ + 67, + 71, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 156, + "sent": "The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure.", + "section": "FIG", + "ner": [ + [ + 4, + 26, + "fluorescence intensity", + "evidence" + ], + [ + 34, + 43, + "uncleaved", + "protein_state" + ], + [ + 44, + 48, + "mRNA", + "chemical" + ], + [ + 99, + 102, + "PIN", + "structure_element" + ], + [ + 103, + 108, + "dimer", + "oligomeric_state" + ], + [ + 109, + 118, + "structure", + "evidence" + ] + ] + }, + { + "sid": 157, + "sent": "Mutated basic residues were shown in sticks and those with significantly reduced RNase activities were colored red or yellow.", + "section": "FIG", + "ner": [ + [ + 81, + 86, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 158, + "sent": "Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity.", + "section": "FIG", + "ner": [ + [ + 44, + 53, + "Regnase-1", + "protein" + ], + [ + 68, + 75, + "mutants", + "protein_state" + ], + [ + 84, + 88, + "DDNN", + "mutant" + ], + [ + 89, + 95, + "mutant", + "protein_state" + ], + [ + 109, + 114, + "RNase", + "protein_type" + ] + ] + }, + { + "sid": 159, + "sent": "(a) Cartoon representation of the concept of the experiment. (b) In vitro cleavage assay of Regnase-1 for IL-6 mRNA.", + "section": "FIG", + "ner": [ + [ + 65, + 88, + "In vitro cleavage assay", + "experimental_method" + ], + [ + 92, + 101, + "Regnase-1", + "protein" + ], + [ + 106, + 110, + "IL-6", + "protein_type" + ], + [ + 111, + 115, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 160, + "sent": "(c) In vitro cleavage assay of Regnase-1 for Regnase-1 mRNA.", + "section": "FIG", + "ner": [ + [ + 4, + 27, + "In vitro cleavage assay", + "experimental_method" + ], + [ + 31, + 40, + "Regnase-1", + "protein" + ], + [ + 45, + 54, + "Regnase-1", + "protein" + ], + [ + 55, + 59, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 161, + "sent": "The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer.", + "section": "FIG", + "ner": [ + [ + 4, + 26, + "fluorescence intensity", + "evidence" + ], + [ + 34, + 43, + "uncleaved", + "protein_state" + ], + [ + 44, + 48, + "mRNA", + "chemical" + ], + [ + 99, + 102, + "PIN", + "structure_element" + ], + [ + 103, + 108, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 162, + "sent": "The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN.", + "section": "FIG", + "ner": [ + [ + 20, + 25, + "RNase", + "protein_type" + ], + [ + 63, + 74, + "presence of", + "protein_state" + ], + [ + 75, + 79, + "DDNN", + "mutant" + ], + [ + 80, + 86, + "mutant", + "protein_state" + ], + [ + 123, + 126, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 163, + "sent": "The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN.", + "section": "FIG", + "ner": [ + [ + 20, + 25, + "RNase", + "protein_type" + ], + [ + 58, + 69, + "presence of", + "protein_state" + ], + [ + 70, + 74, + "DDNN", + "mutant" + ], + [ + 75, + 81, + "mutant", + "protein_state" + ], + [ + 127, + 130, + "PIN", + "structure_element" + ] + ] + }, + { + "sid": 164, + "sent": "Schematic representation of regulation of the Regnase-1 catalytic activity through the domain-domain interactions.", + "section": "FIG", + "ner": [ + [ + 46, + 55, + "Regnase-1", + "protein" + ] + ] + } + ] + }, + "PMC4855620": { + "annotations": [ + { + "sid": 0, + "sent": "Structure\u2010activity relationship of the peptide binding\u2010motif mediating the BRCA2:RAD51 protein\u2013protein interaction", + "section": "TITLE", + "ner": [ + [ + 39, + 60, + "peptide binding\u2010motif", + "structure_element" + ], + [ + 75, + 86, + "BRCA2:RAD51", + "complex_assembly" + ] + ] + }, + { + "sid": 1, + "sent": " RAD51 is a recombinase involved in the homologous recombination of double\u2010strand breaks in DNA.", + "section": "ABSTRACT", + "ner": [ + [ + 1, + 6, + "RAD51", + "protein" + ], + [ + 12, + 23, + "recombinase", + "protein_type" + ] + ] + }, + { + "sid": 2, + "sent": "RAD51 forms oligomers by binding to another molecule of RAD51 via an \u2018FxxA\u2019 motif, and the same recognition sequence is similarly utilised to bind BRCA2.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 5, + "RAD51", + "protein" + ], + [ + 12, + 21, + "oligomers", + "oligomeric_state" + ], + [ + 56, + 61, + "RAD51", + "protein" + ], + [ + 70, + 74, + "FxxA", + "structure_element" + ], + [ + 147, + 152, + "BRCA2", + "protein" + ] + ] + }, + { + "sid": 3, + "sent": "We have tabulated the effects of mutation of this sequence, across a variety of experimental methods and from relevant mutations observed in the clinic.", + "section": "ABSTRACT", + "ner": [ + [ + 33, + 41, + "mutation", + "experimental_method" + ] + ] + }, + { + "sid": 4, + "sent": "We use mutants of a tetrapeptide sequence to probe the binding interaction, using both isothermal titration calorimetry and X\u2010ray crystallography.", + "section": "ABSTRACT", + "ner": [ + [ + 7, + 14, + "mutants", + "protein_state" + ], + [ + 20, + 32, + "tetrapeptide", + "chemical" + ], + [ + 87, + 119, + "isothermal titration calorimetry", + "experimental_method" + ], + [ + 124, + 145, + "X\u2010ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 5, + "sent": "Where possible, comparison between our tetrapeptide mutational study and the previously reported mutations is made, discrepancies are discussed and the importance of secondary structure in interpreting alanine scanning and mutational data of this nature is considered.", + "section": "ABSTRACT", + "ner": [ + [ + 39, + 68, + "tetrapeptide mutational study", + "experimental_method" + ], + [ + 202, + 218, + "alanine scanning", + "experimental_method" + ] + ] + }, + { + "sid": 6, + "sent": "Eukaryotic RAD51, archeal RadA and prokaryotic RecA are a family of ATP\u2010dependent recombinases involved in homologous recombination (HR) of double\u2010strand breaks in DNA 1.", + "section": "ABBR", + "ner": [ + [ + 0, + 10, + "Eukaryotic", + "taxonomy_domain" + ], + [ + 11, + 16, + "RAD51", + "protein" + ], + [ + 18, + 25, + "archeal", + "taxonomy_domain" + ], + [ + 26, + 30, + "RadA", + "protein" + ], + [ + 35, + 46, + "prokaryotic", + "taxonomy_domain" + ], + [ + 47, + 51, + "RecA", + "protein" + ], + [ + 68, + 94, + "ATP\u2010dependent recombinases", + "protein_type" + ] + ] + }, + { + "sid": 7, + "sent": "RAD51 interacts with BRCA2, and is thought to localise RAD51 to sites of DNA damage 2, 3.", + "section": "ABBR", + "ner": [ + [ + 0, + 5, + "RAD51", + "protein" + ], + [ + 21, + 26, + "BRCA2", + "protein" + ], + [ + 55, + 60, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 8, + "sent": "Both BRCA2 and RAD51 together are vital for helping to repair and maintain a high fidelity in DNA replication.", + "section": "ABBR", + "ner": [ + [ + 5, + 10, + "BRCA2", + "protein" + ], + [ + 15, + 20, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 9, + "sent": "BRCA2 especially has garnered much attention in a clinical context, as many mutations have been identified that drive an increased risk of cancer in individuals 4, 5.", + "section": "ABBR", + "ner": [ + [ + 0, + 5, + "BRCA2", + "protein" + ] + ] + }, + { + "sid": 10, + "sent": "Although the inactivation of the BRCA2:RAD51 DNA repair pathway can cause genomic instability and eventual tumour development, an inability to repair breaks in DNA may also engender a sensitivity to ionising radiation 6, 7.", + "section": "ABBR", + "ner": [ + [ + 33, + 44, + "BRCA2:RAD51", + "complex_assembly" + ] + ] + }, + { + "sid": 11, + "sent": "For this reason it is hypothesised that in tumour cells with an intact BRCA2:RAD51 repair pathway, small molecules which prevent the interaction between RAD51 and BRCA2 may confer radiosensitivity by disabling the HR pathway 8.", + "section": "ABBR", + "ner": [ + [ + 71, + 82, + "BRCA2:RAD51", + "complex_assembly" + ], + [ + 153, + 158, + "RAD51", + "protein" + ], + [ + 163, + 168, + "BRCA2", + "protein" + ] + ] + }, + { + "sid": 12, + "sent": "The interaction between the two proteins is mediated by eight BRC repeats, which are characterised by a conserved \u2018FxxA\u2019 motif 9.", + "section": "ABBR", + "ner": [ + [ + 62, + 73, + "BRC repeats", + "structure_element" + ], + [ + 104, + 113, + "conserved", + "protein_state" + ], + [ + 114, + 128, + "\u2018FxxA\u2019 motif 9", + "structure_element" + ] + ] + }, + { + "sid": 13, + "sent": "RAD51 and RadA proteins also contain an \u2018FxxA\u2019 sequence (FTTA for human RAD51) through which it can bind to other RAD51 and RadA molecules, and oligomerise to form higher order filament structures on DNA.", + "section": "ABBR", + "ner": [ + [ + 0, + 5, + "RAD51", + "protein" + ], + [ + 10, + 14, + "RadA", + "protein" + ], + [ + 41, + 45, + "FxxA", + "structure_element" + ], + [ + 57, + 61, + "FTTA", + "structure_element" + ], + [ + 66, + 71, + "human", + "species" + ], + [ + 72, + 77, + "RAD51", + "protein" + ], + [ + 114, + 119, + "RAD51", + "protein" + ], + [ + 124, + 128, + "RadA", + "protein" + ] + ] + }, + { + "sid": 14, + "sent": "The common FxxA motifs of both the BRC repeats and RAD51 oligomerisation sequence are recognised by the same FxxA\u2010binding site of RAD51.", + "section": "ABBR", + "ner": [ + [ + 11, + 15, + "FxxA", + "structure_element" + ], + [ + 35, + 46, + "BRC repeats", + "structure_element" + ], + [ + 51, + 56, + "RAD51", + "protein" + ], + [ + 57, + 81, + "oligomerisation sequence", + "structure_element" + ], + [ + 109, + 126, + "FxxA\u2010binding site", + "site" + ], + [ + 130, + 135, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 15, + "sent": "In general, the dominant contribution of certain residues to the overall binding energy of a protein\u2013protein interaction are known as \u2018hot\u2010spot\u2019 residues.", + "section": "ABBR", + "ner": [ + [ + 73, + 87, + "binding energy", + "evidence" + ], + [ + 135, + 143, + "hot\u2010spot", + "site" + ] + ] + }, + { + "sid": 16, + "sent": "Interestingly, small molecule inhibitors of PPIs are often found to occupy the same pockets which are otherwise occupied by hot\u2010spot residues in the native complex.", + "section": "ABBR", + "ner": [ + [ + 84, + 91, + "pockets", + "site" + ], + [ + 124, + 132, + "hot\u2010spot", + "site" + ], + [ + 149, + 155, + "native", + "protein_state" + ] + ] + }, + { + "sid": 17, + "sent": "It is therefore of great interest to identify hot\u2010spots in an effort to guide drug discovery efforts against a PPI.", + "section": "ABBR", + "ner": [ + [ + 46, + 55, + "hot\u2010spots", + "site" + ] + ] + }, + { + "sid": 18, + "sent": "Further, a correlation between residues that are strongly conserved and hot\u2010spot residues has been reported 10.", + "section": "ABBR", + "ner": [ + [ + 49, + 67, + "strongly conserved", + "protein_state" + ], + [ + 72, + 80, + "hot\u2010spot", + "site" + ] + ] + }, + { + "sid": 19, + "sent": "Purely based on the amino acid consensus sequence reported by Pellegrini et al., 11 phenylalanine and alanine would both be expected to be hot\u2010spots and to a lesser extent, threonine.", + "section": "ABBR", + "ner": [ + [ + 84, + 97, + "phenylalanine", + "residue_name" + ], + [ + 102, + 109, + "alanine", + "residue_name" + ], + [ + 139, + 148, + "hot\u2010spots", + "site" + ], + [ + 173, + 182, + "threonine", + "residue_name" + ] + ] + }, + { + "sid": 20, + "sent": "However, whilst the identification of highly conserved residues may be a good starting point for identifying hot\u2010spots, experimental validation by mutation of these sequences is vital.", + "section": "ABBR", + "ner": [ + [ + 38, + 54, + "highly conserved", + "protein_state" + ], + [ + 109, + 118, + "hot\u2010spots", + "site" + ], + [ + 147, + 155, + "mutation", + "experimental_method" + ] + ] + }, + { + "sid": 21, + "sent": "The importance of residues in the FxxA motif has been probed by a variety of techniques, collated in Table 1.", + "section": "ABBR", + "ner": [ + [ + 34, + 38, + "FxxA", + "structure_element" + ] + ] + }, + { + "sid": 22, + "sent": "Briefly, mutating phenylalanine to glutamic acid inactivated the BRC4 peptide and prevented RAD51 oligomerisation 11, 12.", + "section": "ABBR", + "ner": [ + [ + 9, + 17, + "mutating", + "experimental_method" + ], + [ + 18, + 31, + "phenylalanine", + "residue_name" + ], + [ + 35, + 48, + "glutamic acid", + "residue_name" + ], + [ + 49, + 60, + "inactivated", + "protein_state" + ], + [ + 65, + 69, + "BRC4", + "chemical" + ], + [ + 92, + 97, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 23, + "sent": "A phenylalanine\u2010truncated BRC4 is also found to be inactive 13, however, introducing a tryptophan for phenylalanine was found to have no significant effect on BRC4 affinity 12.", + "section": "ABBR", + "ner": [ + [ + 2, + 25, + "phenylalanine\u2010truncated", + "protein_state" + ], + [ + 26, + 30, + "BRC4", + "chemical" + ], + [ + 51, + 59, + "inactive", + "protein_state" + ], + [ + 73, + 84, + "introducing", + "experimental_method" + ], + [ + 87, + 97, + "tryptophan", + "residue_name" + ], + [ + 102, + 115, + "phenylalanine", + "residue_name" + ], + [ + 159, + 163, + "BRC4", + "chemical" + ], + [ + 164, + 172, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 24, + "sent": "A glutamine replacing the histidine in BRC4 maintains BRC4 activity 13.", + "section": "ABBR", + "ner": [ + [ + 2, + 11, + "glutamine", + "residue_name" + ], + [ + 12, + 21, + "replacing", + "experimental_method" + ], + [ + 26, + 35, + "histidine", + "residue_name" + ], + [ + 39, + 43, + "BRC4", + "chemical" + ], + [ + 54, + 58, + "BRC4", + "chemical" + ] + ] + }, + { + "sid": 25, + "sent": "The ability of BRC3 to interact with RAD51 nucleoprotein filaments is disrupted when threonine is mutated to an alanine 3.", + "section": "ABBR", + "ner": [ + [ + 15, + 19, + "BRC3", + "chemical" + ], + [ + 37, + 42, + "RAD51", + "protein" + ], + [ + 85, + 94, + "threonine", + "residue_name" + ], + [ + 98, + 105, + "mutated", + "experimental_method" + ], + [ + 112, + 119, + "alanine", + "residue_name" + ] + ] + }, + { + "sid": 26, + "sent": "Similarly, mutating alanine to glutamic acid in the RAD51 oligomerisation sequence 11 or to serine in BRC4 13 leads to loss of interaction in both cases.", + "section": "ABBR", + "ner": [ + [ + 11, + 19, + "mutating", + "experimental_method" + ], + [ + 20, + 27, + "alanine", + "residue_name" + ], + [ + 31, + 44, + "glutamic acid", + "residue_name" + ], + [ + 52, + 57, + "RAD51", + "protein" + ], + [ + 58, + 82, + "oligomerisation sequence", + "structure_element" + ], + [ + 92, + 98, + "serine", + "residue_name" + ], + [ + 102, + 106, + "BRC4", + "chemical" + ] + ] + }, + { + "sid": 27, + "sent": "The BRC5 repeat in humans has serine in the place of alanine, and is thought to be a nonbinding repeat 12.", + "section": "ABBR", + "ner": [ + [ + 4, + 8, + "BRC5", + "chemical" + ], + [ + 19, + 25, + "humans", + "species" + ], + [ + 30, + 36, + "serine", + "residue_name" + ], + [ + 53, + 60, + "alanine", + "residue_name" + ], + [ + 85, + 102, + "nonbinding repeat", + "structure_element" + ] + ] + }, + { + "sid": 28, + "sent": "Mutations identified in the clinic, in the FxxA region of the BRC repeats of BRCA2 are collated in Table 1 14.", + "section": "ABBR", + "ner": [ + [ + 43, + 47, + "FxxA", + "structure_element" + ], + [ + 62, + 73, + "BRC repeats", + "structure_element" + ], + [ + 77, + 82, + "BRCA2", + "protein" + ] + ] + }, + { + "sid": 29, + "sent": "For completeness, we present them here with this caveat, and to make the comment that these clinical mutations are consistent with abrogating the interaction with RAD51.", + "section": "ABBR", + "ner": [ + [ + 163, + 168, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 30, + "sent": "Summary of FxxA\u2010relevant mutations previously reported and degree of characterisation.", + "section": "TABLE", + "ner": [ + [ + 11, + 15, + "FxxA", + "structure_element" + ] + ] + }, + { + "sid": 31, + "sent": "The mutation, relevant peptide context, resulting FxxA motif sequence and experimental technique for each entry is given.", + "section": "TABLE", + "ner": [ + [ + 50, + 54, + "FxxA", + "structure_element" + ] + ] + }, + { + "sid": 32, + "sent": "Mutation contexta\tMutation\tFxxA motif\tTechnique used\tEffect\t \tRAD51 (FTTA)\tF86E\tETTA\tImmunoprecipitation 11\tNo binding\t \tBRC4 (FHTA)\tF1524E\tEHTA\tCompetitive ELISA 12\tPeptide inactive\t \tBRC4 (FHTA)\tF1524W\tWHTA\tCompetitive ELISA 12\tComparable activity to WT\t \tBRC4 (FHTA)\tF1524V\tVHTA\tBRCA2 mutations database 14\t\u2013\t \tBRC4 (FHTA)\t\u0394F1524\t\u2010HTA\tDissociation of RAD51\u2010DNA complex 13\tPeptide inactive\t \tBRC4 (FHTA)\tH1525Q\tFQTA\tDissociation of RAD51\u2010DNA complex 13\tComparable activity\t \tBRC7 (FSTA)\tS1979R\tFRTA\tBRCA2 mutations database 14\t\u2013\t \tBRC3 (FQTA)\tT1430A\tFQAA\tRAD51:DNA bandshift assay 3\tPeptide inactive\t \tBRC3 (FQTA)\tT1430A\tFQAA\tElectron microscopic visualisation of nucleoprotein filaments 3\tPeptide inactive\t \tBRC1 (FRTA)\tT1011R\tFRRA\tBRCA2 mutations database 14\t\u2013\t \tBRC2 (FYSA)\tS1221P\tFYPA\tBRCA2 mutations database 14\t\u2013\t \tBRC2 (FYSA)\tS1221Y\tFYYA\tBRCA2 mutations database 14\t\u2013\t \tRAD51 (FTTA)\tA89E\tFTTE\tImmunoprecipitation 11\tNo binding\t \tBRC4 (FHTA)\tA1527S\tFHTS\tDissociation of RAD51\u2010DNA complex 13\tPeptide inactive\t \t", + "section": "TABLE", + "ner": [ + [ + 27, + 31, + "FxxA", + "structure_element" + ], + [ + 62, + 67, + "RAD51", + "protein" + ], + [ + 69, + 73, + "FTTA", + "structure_element" + ], + [ + 75, + 79, + "F86E", + "mutant" + ], + [ + 80, + 84, + "ETTA", + "structure_element" + ], + [ + 85, + 104, + "Immunoprecipitation", + "experimental_method" + ], + [ + 121, + 125, + "BRC4", + "chemical" + ], + [ + 127, + 131, + "FHTA", + "structure_element" + ], + [ + 133, + 139, + "F1524E", + "mutant" + ], + [ + 140, + 144, + "EHTA", + "structure_element" + ], + [ + 145, + 162, + "Competitive ELISA", + "experimental_method" + ], + [ + 174, + 182, + "inactive", + "protein_state" + ], + [ + 185, + 189, + "BRC4", + "chemical" + ], + [ + 191, + 195, + "FHTA", + "structure_element" + ], + [ + 197, + 203, + "F1524W", + "mutant" + ], + [ + 204, + 208, + "WHTA", + "structure_element" + ], + [ + 209, + 226, + "Competitive ELISA", + "experimental_method" + ], + [ + 253, + 255, + "WT", + "protein_state" + ], + [ + 258, + 262, + "BRC4", + "chemical" + ], + [ + 264, + 268, + "FHTA", + "structure_element" + ], + [ + 270, + 276, + "F1524V", + "mutant" + ], + [ + 277, + 281, + "VHTA", + "structure_element" + ], + [ + 282, + 287, + "BRCA2", + "protein" + ], + [ + 314, + 318, + "BRC4", + "chemical" + ], + [ + 320, + 324, + "FHTA", + "structure_element" + ], + [ + 326, + 332, + "\u0394F1524", + "mutant" + ], + [ + 334, + 337, + "HTA", + "structure_element" + ], + [ + 354, + 363, + "RAD51\u2010DNA", + "complex_assembly" + ], + [ + 383, + 391, + "inactive", + "protein_state" + ], + [ + 394, + 398, + "BRC4", + "chemical" + ], + [ + 400, + 404, + "FHTA", + "structure_element" + ], + [ + 406, + 412, + "H1525Q", + "mutant" + ], + [ + 413, + 417, + "FQTA", + "structure_element" + ], + [ + 434, + 443, + "RAD51\u2010DNA", + "complex_assembly" + ], + [ + 477, + 481, + "BRC7", + "chemical" + ], + [ + 483, + 487, + "FSTA", + "structure_element" + ], + [ + 489, + 495, + "S1979R", + "mutant" + ], + [ + 496, + 500, + "FRTA", + "structure_element" + ], + [ + 501, + 506, + "BRCA2", + "protein" + ], + [ + 533, + 537, + "BRC3", + "chemical" + ], + [ + 539, + 543, + "FQTA", + "structure_element" + ], + [ + 545, + 551, + "T1430A", + "mutant" + ], + [ + 552, + 556, + "FQAA", + "structure_element" + ], + [ + 557, + 566, + "RAD51:DNA", + "complex_assembly" + ], + [ + 567, + 582, + "bandshift assay", + "experimental_method" + ], + [ + 593, + 601, + "inactive", + "protein_state" + ], + [ + 604, + 608, + "BRC3", + "chemical" + ], + [ + 610, + 614, + "FQTA", + "structure_element" + ], + [ + 616, + 622, + "T1430A", + "mutant" + ], + [ + 623, + 627, + "FQAA", + "structure_element" + ], + [ + 628, + 648, + "Electron microscopic", + "experimental_method" + ], + [ + 700, + 708, + "inactive", + "protein_state" + ], + [ + 711, + 715, + "BRC1", + "chemical" + ], + [ + 717, + 721, + "FRTA", + "structure_element" + ], + [ + 723, + 729, + "T1011R", + "mutant" + ], + [ + 730, + 734, + "FRRA", + "structure_element" + ], + [ + 735, + 740, + "BRCA2", + "protein" + ], + [ + 767, + 771, + "BRC2", + "chemical" + ], + [ + 773, + 777, + "FYSA", + "structure_element" + ], + [ + 779, + 785, + "S1221P", + "mutant" + ], + [ + 786, + 790, + "FYPA", + "structure_element" + ], + [ + 791, + 796, + "BRCA2", + "protein" + ], + [ + 823, + 827, + "BRC2", + "chemical" + ], + [ + 829, + 833, + "FYSA", + "structure_element" + ], + [ + 835, + 841, + "S1221Y", + "mutant" + ], + [ + 842, + 846, + "FYYA", + "structure_element" + ], + [ + 847, + 852, + "BRCA2", + "protein" + ], + [ + 879, + 884, + "RAD51", + "protein" + ], + [ + 886, + 890, + "FTTA", + "structure_element" + ], + [ + 892, + 896, + "A89E", + "mutant" + ], + [ + 897, + 901, + "FTTE", + "structure_element" + ], + [ + 902, + 921, + "Immunoprecipitation", + "experimental_method" + ], + [ + 938, + 942, + "BRC4", + "chemical" + ], + [ + 944, + 948, + "FHTA", + "structure_element" + ], + [ + 950, + 956, + "A1527S", + "mutant" + ], + [ + 957, + 961, + "FHTS", + "structure_element" + ], + [ + 978, + 987, + "RAD51\u2010DNA", + "complex_assembly" + ], + [ + 1007, + 1015, + "inactive", + "protein_state" + ] + ] + }, + { + "sid": 33, + "sent": "The wild\u2010type FxxA sequence is indicated in parenthesis.", + "section": "TABLE", + "ner": [ + [ + 4, + 13, + "wild\u2010type", + "protein_state" + ], + [ + 14, + 18, + "FxxA", + "structure_element" + ] + ] + }, + { + "sid": 34, + "sent": "In this work, we report the most detailed study of systematic mutations of peptides to probe the FxxA\u2010binding motif to date.", + "section": "ABBR", + "ner": [ + [ + 51, + 71, + "systematic mutations", + "experimental_method" + ], + [ + 97, + 115, + "FxxA\u2010binding motif", + "structure_element" + ] + ] + }, + { + "sid": 35, + "sent": "We have chosen to focus on tetrapeptides, which allows us to examine the effect of mutation on the fundamental unit of binding, FxxA, rather than in the context of either the BRC repeat or self\u2010oligomerisation sequence.", + "section": "ABBR", + "ner": [ + [ + 27, + 40, + "tetrapeptides", + "chemical" + ], + [ + 83, + 91, + "mutation", + "experimental_method" + ], + [ + 128, + 132, + "FxxA", + "structure_element" + ], + [ + 175, + 185, + "BRC repeat", + "structure_element" + ], + [ + 189, + 218, + "self\u2010oligomerisation sequence", + "structure_element" + ] + ] + }, + { + "sid": 36, + "sent": "Affinities of peptides were measured directly using Isothermal Titration Calorimetry (ITC) and the structures of many of the peptides bound to humanised RadA were determined by X\u2010ray crystallography.", + "section": "ABBR", + "ner": [ + [ + 0, + 10, + "Affinities", + "evidence" + ], + [ + 52, + 84, + "Isothermal Titration Calorimetry", + "experimental_method" + ], + [ + 86, + 89, + "ITC", + "experimental_method" + ], + [ + 99, + 109, + "structures", + "evidence" + ], + [ + 134, + 142, + "bound to", + "protein_state" + ], + [ + 143, + 152, + "humanised", + "protein_state" + ], + [ + 153, + 157, + "RadA", + "protein" + ], + [ + 177, + 198, + "X\u2010ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 37, + "sent": "The use of ITC is generally perceived as a gold\u2010standard in protein\u2013ligand characterisation, rather than a competitive assay which may be prone to aggregation artefacts.", + "section": "ABBR", + "ner": [ + [ + 11, + 14, + "ITC", + "experimental_method" + ], + [ + 107, + 124, + "competitive assay", + "experimental_method" + ] + ] + }, + { + "sid": 38, + "sent": "Wild\u2010type human RAD51, however, is a heterogeneous mixture of oligomers and when monomerised by mutation, is highly unstable.", + "section": "ABBR", + "ner": [ + [ + 0, + 9, + "Wild\u2010type", + "protein_state" + ], + [ + 10, + 15, + "human", + "species" + ], + [ + 16, + 21, + "RAD51", + "protein" + ], + [ + 62, + 71, + "oligomers", + "oligomeric_state" + ], + [ + 81, + 92, + "monomerised", + "oligomeric_state" + ], + [ + 96, + 104, + "mutation", + "experimental_method" + ], + [ + 109, + 124, + "highly unstable", + "protein_state" + ] + ] + }, + { + "sid": 39, + "sent": "In this context, we have previously reported the use of stable monomeric forms of RAD51, humanised from Pyrococcus furiosus homologue RadA, for ITC experiments and X\u2010ray crystallography 8, 15.", + "section": "ABBR", + "ner": [ + [ + 56, + 62, + "stable", + "protein_state" + ], + [ + 63, + 72, + "monomeric", + "oligomeric_state" + ], + [ + 82, + 87, + "RAD51", + "protein" + ], + [ + 89, + 98, + "humanised", + "protein_state" + ], + [ + 104, + 123, + "Pyrococcus furiosus", + "species" + ], + [ + 134, + 138, + "RadA", + "protein" + ], + [ + 144, + 147, + "ITC", + "experimental_method" + ], + [ + 164, + 185, + "X\u2010ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 40, + "sent": "Conservation of FxxA motif (A) BRC4 peptide (green cartoon) bound to truncated human RAD51 (grey surface) (PDB: 1n0w, 11).", + "section": "FIG", + "ner": [ + [ + 16, + 20, + "FxxA", + "structure_element" + ], + [ + 31, + 35, + "BRC4", + "chemical" + ], + [ + 60, + 68, + "bound to", + "protein_state" + ], + [ + 69, + 78, + "truncated", + "protein_state" + ], + [ + 79, + 84, + "human", + "species" + ], + [ + 85, + 90, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 41, + "sent": "The blue dashed box highlights the FxxA interaction pocket.", + "section": "FIG", + "ner": [ + [ + 35, + 58, + "FxxA interaction pocket", + "site" + ] + ] + }, + { + "sid": 42, + "sent": "(B) Two interacting protein molecules of RAD51 from Saccharomyces cerevisiae are shown.", + "section": "FIG", + "ner": [ + [ + 41, + 46, + "RAD51", + "protein" + ], + [ + 52, + 76, + "Saccharomyces cerevisiae", + "species" + ] + ] + }, + { + "sid": 43, + "sent": "One RAD51 (green cartoon) interacts with another molecule of RAD51 (grey and pink surface) via the FxxA pocket indicated by the dashed blue box.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "RAD51", + "protein" + ], + [ + 61, + 66, + "RAD51", + "protein" + ], + [ + 99, + 110, + "FxxA pocket", + "site" + ] + ] + }, + { + "sid": 44, + "sent": "The N\u2010terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation.", + "section": "FIG", + "ner": [ + [ + 29, + 34, + "RAD51", + "protein" + ], + [ + 35, + 43, + "protomer", + "oligomeric_state" + ], + [ + 130, + 138, + "protomer", + "oligomeric_state" + ], + [ + 141, + 170, + "FxxA oligomerisation sequence", + "structure_element" + ], + [ + 208, + 212, + "FxxA", + "structure_element" + ], + [ + 230, + 235, + "human", + "species" + ], + [ + 236, + 247, + "BRC repeats", + "structure_element" + ], + [ + 266, + 276, + "eukaryotic", + "taxonomy_domain" + ], + [ + 277, + 283, + "RAD51s", + "protein_type" + ], + [ + 291, + 296, + "RadAs", + "protein_type" + ] + ] + }, + { + "sid": 45, + "sent": "We have mutated and truncated the tetrapeptide epitope FHTA, and examined the effects both structurally and on the binding affinity with humanised RadA. As a comparative reference, we are using the FHTA sequence derived from the most tightly binding BRC repeat, BRC4 22.", + "section": "RESULTS", + "ner": [ + [ + 8, + 29, + "mutated and truncated", + "experimental_method" + ], + [ + 34, + 46, + "tetrapeptide", + "chemical" + ], + [ + 55, + 59, + "FHTA", + "structure_element" + ], + [ + 115, + 131, + "binding affinity", + "evidence" + ], + [ + 137, + 146, + "humanised", + "protein_state" + ], + [ + 147, + 151, + "RadA", + "protein" + ], + [ + 198, + 202, + "FHTA", + "structure_element" + ], + [ + 250, + 260, + "BRC repeat", + "structure_element" + ], + [ + 262, + 266, + "BRC4", + "chemical" + ] + ] + }, + { + "sid": 46, + "sent": "The peptides used are N\u2010acetylated and C\u2010amide terminated in order to provide the most relevant peptide in the context of a longer peptide chain.", + "section": "RESULTS", + "ner": [ + [ + 22, + 34, + "N\u2010acetylated", + "protein_state" + ] + ] + }, + { + "sid": 47, + "sent": "A summary of the peptide sequence, PDB codes and K D data measured by ITC with the corresponding \u0394H and T\u0394S values are collated in Table 2.", + "section": "RESULTS", + "ner": [ + [ + 49, + 52, + "K D", + "evidence" + ], + [ + 70, + 73, + "ITC", + "experimental_method" + ], + [ + 97, + 99, + "\u0394H", + "evidence" + ], + [ + 104, + 107, + "T\u0394S", + "evidence" + ] + ] + }, + { + "sid": 48, + "sent": "Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Phe1524", + "residue_name_number" + ], + [ + 11, + 15, + "BRC4", + "chemical" + ], + [ + 33, + 47, + "surface pocket", + "site" + ], + [ + 51, + 56, + "human", + "species" + ], + [ + 57, + 62, + "RAD51", + "protein" + ], + [ + 115, + 121, + "Met158", + "residue_name_number" + ], + [ + 123, + 129, + "Ile160", + "residue_name_number" + ], + [ + 131, + 137, + "Ala192", + "residue_name_number" + ], + [ + 139, + 145, + "Leu203", + "residue_name_number" + ], + [ + 150, + 156, + "Met210", + "residue_name_number" + ] + ] + }, + { + "sid": 49, + "sent": "The residue is highly conserved across BRC repeats and oligomerisation sequences.", + "section": "RESULTS", + "ner": [ + [ + 15, + 31, + "highly conserved", + "protein_state" + ], + [ + 39, + 50, + "BRC repeats", + "structure_element" + ], + [ + 55, + 80, + "oligomerisation sequences", + "structure_element" + ] + ] + }, + { + "sid": 50, + "sent": "Consistent with this, the truncated HTA tripeptide could not be detected to bind to humanised, monomeric RadA, HumRadA2 (Table 2, entry 13).", + "section": "RESULTS", + "ner": [ + [ + 26, + 35, + "truncated", + "protein_state" + ], + [ + 36, + 39, + "HTA", + "structure_element" + ], + [ + 40, + 50, + "tripeptide", + "chemical" + ], + [ + 84, + 93, + "humanised", + "protein_state" + ], + [ + 95, + 104, + "monomeric", + "oligomeric_state" + ], + [ + 105, + 109, + "RadA", + "protein" + ], + [ + 111, + 119, + "HumRadA2", + "mutant" + ] + ] + }, + { + "sid": 51, + "sent": "As previously discussed, there is some evidence that substituting a tryptophan for the phenylalanine at this position was tolerated in the context of BRC4 12.", + "section": "RESULTS", + "ner": [ + [ + 53, + 65, + "substituting", + "experimental_method" + ], + [ + 68, + 78, + "tryptophan", + "residue_name" + ], + [ + 87, + 100, + "phenylalanine", + "residue_name" + ], + [ + 150, + 154, + "BRC4", + "chemical" + ] + ] + }, + { + "sid": 52, + "sent": "Therefore, the WHTA peptide was tested and found to not only be tolerated, but to increase the binding affinity of the peptide approximately threefold.", + "section": "RESULTS", + "ner": [ + [ + 15, + 19, + "WHTA", + "structure_element" + ], + [ + 95, + 111, + "binding affinity", + "evidence" + ] + ] + }, + { + "sid": 53, + "sent": "The second position of the tetrapeptide was found to be largely invariant to changes in the side chains that were investigated.", + "section": "RESULTS", + "ner": [ + [ + 27, + 39, + "tetrapeptide", + "chemical" + ] + ] + }, + { + "sid": 54, + "sent": "The residue makes no interactions with the RAD51 protein, but may make an internal hydrogen bond with Thr1520 in the context of BRC4, Fig. 3A.", + "section": "RESULTS", + "ner": [ + [ + 43, + 48, + "RAD51", + "protein" + ], + [ + 83, + 96, + "hydrogen bond", + "bond_interaction" + ], + [ + 102, + 109, + "Thr1520", + "residue_name_number" + ], + [ + 128, + 132, + "BRC4", + "chemical" + ] + ] + }, + { + "sid": 55, + "sent": "Replacing the histidine with an asparagine, chosen to potentially mimic the hydrogen bond donor\u2013acceptor nature of histidine, resulted in a moderate, twofold decrease in potency (Table 2, entry 4).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Replacing", + "experimental_method" + ], + [ + 14, + 23, + "histidine", + "residue_name" + ], + [ + 32, + 42, + "asparagine", + "residue_name" + ], + [ + 76, + 89, + "hydrogen bond", + "bond_interaction" + ], + [ + 115, + 124, + "histidine", + "residue_name" + ] + ] + }, + { + "sid": 56, + "sent": "Mutating to an alanine, recapitulated the potency of FHTA, implying that the interactions made by histidine do not contribute overall to binding affinity (Table 2, entry 3).", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Mutating", + "experimental_method" + ], + [ + 15, + 22, + "alanine", + "residue_name" + ], + [ + 53, + 57, + "FHTA", + "structure_element" + ], + [ + 98, + 107, + "histidine", + "residue_name" + ], + [ + 137, + 153, + "binding affinity", + "evidence" + ] + ] + }, + { + "sid": 57, + "sent": "FPTA was also tested, but was found to have no affinity for the protein (Table 2, entry 5).", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "FPTA", + "structure_element" + ], + [ + 47, + 55, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 58, + "sent": "Modelling suggests that a proline in the second position would be expected to clash sterically with the surface of the protein, and provides a rationale for the lack of binding observed.", + "section": "RESULTS", + "ner": [ + [ + 26, + 33, + "proline", + "residue_name" + ] + ] + }, + { + "sid": 59, + "sent": "Threonine was mutated to an alanine, resulting in only a moderately weaker K D (twofold, Table 2, entry 7).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Threonine", + "residue_name" + ], + [ + 14, + 21, + "mutated", + "experimental_method" + ], + [ + 28, + 35, + "alanine", + "residue_name" + ], + [ + 75, + 78, + "K D", + "evidence" + ] + ] + }, + { + "sid": 60, + "sent": "In the context of a tetrapeptide at least, this result implies a lack of importance of a threonine at this position.", + "section": "RESULTS", + "ner": [ + [ + 20, + 32, + "tetrapeptide", + "chemical" + ], + [ + 89, + 98, + "threonine", + "residue_name" + ] + ] + }, + { + "sid": 61, + "sent": "Interestingly, it was found that a proline at this position improved the affinity almost threefold, to 113 \u03bcm (Table 2, entry 6).", + "section": "RESULTS", + "ner": [ + [ + 35, + 42, + "proline", + "residue_name" + ], + [ + 73, + 81, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 62, + "sent": "This beneficial mutation was incorporated with another previously identified variant to produce the peptide WHPA.", + "section": "RESULTS", + "ner": [ + [ + 16, + 24, + "mutation", + "experimental_method" + ], + [ + 108, + 112, + "WHPA", + "structure_element" + ] + ] + }, + { + "sid": 63, + "sent": "While the importance of the phenylalanine may be possible to predict from examination of the crystal structure, the alanine appears to be of much less importance in this regard.", + "section": "RESULTS", + "ner": [ + [ + 28, + 41, + "phenylalanine", + "residue_name" + ], + [ + 93, + 110, + "crystal structure", + "evidence" + ], + [ + 116, + 123, + "alanine", + "residue_name" + ] + ] + }, + { + "sid": 64, + "sent": "It is, however, a highly conserved residue and clearly of interest for systematic mutation.", + "section": "RESULTS", + "ner": [ + [ + 18, + 34, + "highly conserved", + "protein_state" + ] + ] + }, + { + "sid": 65, + "sent": "Removing the alanine residue entirely produced the truncated tripeptide FHT, which did not bind (Table 2, entry 12).", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Removing", + "experimental_method" + ], + [ + 13, + 20, + "alanine", + "residue_name" + ], + [ + 51, + 60, + "truncated", + "protein_state" + ], + [ + 61, + 71, + "tripeptide", + "chemical" + ], + [ + 72, + 75, + "FHT", + "structure_element" + ] + ] + }, + { + "sid": 66, + "sent": "The unnatural amino acid, \u03b1\u2010amino butyric acid (U), was introduced at the fourth position, positioning an ethyl group into the alanine pocket (Table 2, entry 9).", + "section": "RESULTS", + "ner": [ + [ + 26, + 46, + "\u03b1\u2010amino butyric acid", + "chemical" + ], + [ + 48, + 49, + "U", + "chemical" + ], + [ + 127, + 141, + "alanine pocket", + "site" + ] + ] + }, + { + "sid": 67, + "sent": "Perhaps surprisingly, it was accommodated and the affinity dropped only by twofold as compared to FHTA.", + "section": "RESULTS", + "ner": [ + [ + 50, + 58, + "affinity", + "evidence" + ], + [ + 98, + 102, + "FHTA", + "structure_element" + ] + ] + }, + { + "sid": 68, + "sent": "The effect of simply removing the \u03b2\u2010carbon of alanine, by mutation to glycine (FHTG), produced an approximately sixfold drop in binding affinity (Table 2, entry 8).", + "section": "RESULTS", + "ner": [ + [ + 21, + 29, + "removing", + "experimental_method" + ], + [ + 46, + 53, + "alanine", + "residue_name" + ], + [ + 58, + 69, + "mutation to", + "experimental_method" + ], + [ + 70, + 77, + "glycine", + "residue_name" + ], + [ + 79, + 83, + "FHTG", + "structure_element" + ], + [ + 128, + 144, + "binding affinity", + "evidence" + ] + ] + }, + { + "sid": 69, + "sent": "This is in line with the observation that alanine is not 100% conserved and some archeal RadA proteins contain a glycine in the place of alanine 23.", + "section": "RESULTS", + "ner": [ + [ + 42, + 49, + "alanine", + "residue_name" + ], + [ + 53, + 71, + "not 100% conserved", + "protein_state" + ], + [ + 81, + 88, + "archeal", + "taxonomy_domain" + ], + [ + 89, + 102, + "RadA proteins", + "protein_type" + ], + [ + 113, + 120, + "glycine", + "residue_name" + ], + [ + 137, + 144, + "alanine", + "residue_name" + ] + ] + }, + { + "sid": 70, + "sent": "Structural characterisation of peptide complexes", + "section": "RESULTS", + "ner": [ + [ + 0, + 27, + "Structural characterisation", + "experimental_method" + ] + ] + }, + { + "sid": 71, + "sent": "Structures of the key tetrapeptides were solved by soaking into crystals of a humanised form of RAD51, HumRadA1, which we have previously reported as a convenient surrogate system for RAD51 crystallography 15.", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 22, + 35, + "tetrapeptides", + "chemical" + ], + [ + 51, + 63, + "soaking into", + "experimental_method" + ], + [ + 64, + 72, + "crystals", + "evidence" + ], + [ + 78, + 87, + "humanised", + "protein_state" + ], + [ + 96, + 101, + "RAD51", + "protein" + ], + [ + 103, + 111, + "HumRadA1", + "mutant" + ], + [ + 184, + 189, + "RAD51", + "protein" + ], + [ + 190, + 205, + "crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 72, + "sent": "The corresponding PDB codes are indicated in Table 2 and crystallographic data are found in the Supporting Information.", + "section": "RESULTS", + "ner": [ + [ + 57, + 78, + "crystallographic data", + "evidence" + ] + ] + }, + { + "sid": 73, + "sent": "All structures are of high resolution (1.2\u20131.7 \u00c5) and the electron density for the peptide was clearly visible after the first refinement using unliganded RadA coordinates (Fig. S1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 58, + 74, + "electron density", + "evidence" + ], + [ + 144, + 154, + "unliganded", + "protein_state" + ], + [ + 155, + 159, + "RadA", + "protein" + ] + ] + }, + { + "sid": 74, + "sent": "Some of the SAR observed in the binding analysis can be interpreted in terms of these X\u2010ray crystal structures.", + "section": "RESULTS", + "ner": [ + [ + 32, + 48, + "binding analysis", + "experimental_method" + ], + [ + 86, + 91, + "X\u2010ray", + "experimental_method" + ], + [ + 92, + 110, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 75, + "sent": "For example, an overlay of the bound poses of the ligands FHTA and FHPA (Fig. 2B) reveals a high similarity in the binding modes, indicating that the conformational rigidity conferred by the proline is compatible with the FHTA\u2010binding mode, and a reduction in an entropic penalty of binding may be the source of the improvement in affinity.", + "section": "RESULTS", + "ner": [ + [ + 16, + 23, + "overlay", + "experimental_method" + ], + [ + 58, + 62, + "FHTA", + "structure_element" + ], + [ + 67, + 71, + "FHPA", + "structure_element" + ], + [ + 191, + 198, + "proline", + "residue_name" + ], + [ + 222, + 226, + "FHTA", + "structure_element" + ], + [ + 263, + 279, + "entropic penalty", + "evidence" + ], + [ + 331, + 339, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 76, + "sent": "WHTA peptide shows a relative dislocation when compared to FHTA (Fig 2A), with the entire ligand backbone of WHTA shifted to accommodate the change in the position of the main chain carbon of the first residue, as the larger indole side chain fills the Phe pocket.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "WHTA", + "structure_element" + ], + [ + 59, + 63, + "FHTA", + "structure_element" + ], + [ + 109, + 113, + "WHTA", + "structure_element" + ], + [ + 253, + 263, + "Phe pocket", + "site" + ] + ] + }, + { + "sid": 77, + "sent": "This shift is translated all the way to the alanine side chain.", + "section": "RESULTS", + "ner": [ + [ + 44, + 51, + "alanine", + "residue_name" + ] + ] + }, + { + "sid": 78, + "sent": "It is possible that this mutation is beneficial in the tetrapeptide context and neutral in the full\u2010length BRC4 context because the smaller peptide is less constrained and allowed to explore more conformations.", + "section": "RESULTS", + "ner": [ + [ + 25, + 33, + "mutation", + "experimental_method" + ], + [ + 55, + 67, + "tetrapeptide", + "chemical" + ], + [ + 95, + 106, + "full\u2010length", + "protein_state" + ], + [ + 107, + 111, + "BRC4", + "chemical" + ] + ] + }, + { + "sid": 79, + "sent": "An attempt to combine both the tryptophan and proline mutations, however, led to no improvement for WHPA peptide compared to FHTA.", + "section": "RESULTS", + "ner": [ + [ + 31, + 41, + "tryptophan", + "residue_name" + ], + [ + 46, + 53, + "proline", + "residue_name" + ], + [ + 54, + 63, + "mutations", + "experimental_method" + ], + [ + 100, + 104, + "WHPA", + "structure_element" + ], + [ + 125, + 129, + "FHTA", + "structure_element" + ] + ] + }, + { + "sid": 80, + "sent": "One possible explanation is that the \u2018shifted\u2019 binding mode observed in WHTA was not compatible with the conformational restriction that the proline of WHPA introduced.", + "section": "RESULTS", + "ner": [ + [ + 72, + 76, + "WHTA", + "structure_element" + ], + [ + 141, + 148, + "proline", + "residue_name" + ], + [ + 152, + 156, + "WHPA", + "structure_element" + ] + ] + }, + { + "sid": 81, + "sent": "Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan).", + "section": "FIG", + "ner": [ + [ + 46, + 53, + "Overlay", + "experimental_method" + ], + [ + 59, + 63, + "FHTA", + "structure_element" + ], + [ + 75, + 79, + "WHTA", + "structure_element" + ], + [ + 156, + 169, + "Superposition", + "experimental_method" + ], + [ + 173, + 177, + "FHTA", + "structure_element" + ], + [ + 189, + 193, + "FHPA", + "structure_element" + ], + [ + 253, + 260, + "Overlay", + "experimental_method" + ], + [ + 264, + 268, + "FHTU", + "structure_element" + ], + [ + 278, + 282, + "FHTA", + "structure_element" + ], + [ + 294, + 298, + "FHTG", + "structure_element" + ] + ] + }, + { + "sid": 82, + "sent": "The thermodynamic data of peptide binding are also shown in Table 2. Although we have both thermodynamic data and high\u2010quality X\u2010ray structural information for some of the mutant peptides, we do not attempt to interpret differences in thermodynamic profiles between ligands, that is, to analyse \u0394\u0394H and \u0394\u0394S.", + "section": "RESULTS", + "ner": [ + [ + 4, + 22, + "thermodynamic data", + "evidence" + ], + [ + 91, + 109, + "thermodynamic data", + "evidence" + ], + [ + 127, + 132, + "X\u2010ray", + "experimental_method" + ], + [ + 133, + 155, + "structural information", + "evidence" + ], + [ + 172, + 178, + "mutant", + "protein_state" + ], + [ + 179, + 187, + "peptides", + "chemical" + ], + [ + 235, + 257, + "thermodynamic profiles", + "evidence" + ], + [ + 295, + 298, + "\u0394\u0394H", + "evidence" + ], + [ + 303, + 306, + "\u0394\u0394S", + "evidence" + ] + ] + }, + { + "sid": 83, + "sent": "Although \u0394H and \u0394S are tabulated, the K Ds measured are relatively weak and necessarily performed under low c\u2010value conditions.", + "section": "RESULTS", + "ner": [ + [ + 9, + 11, + "\u0394H", + "evidence" + ], + [ + 16, + 18, + "\u0394S", + "evidence" + ], + [ + 38, + 42, + "K Ds", + "evidence" + ] + ] + }, + { + "sid": 84, + "sent": "In this experimental regime, nonsigmoidal curves are generated and therefore errors in \u0394H are expected to be much higher than the errors from model fitting given in Table 2 16.", + "section": "RESULTS", + "ner": [ + [ + 87, + 89, + "\u0394H", + "evidence" + ] + ] + }, + { + "sid": 85, + "sent": "As \u0394S is derived from \u0394G by subtracting \u0394H, errors in \u0394H will be correlated with errors in \u0394S, giving rise to a \u2018phantom\u2019 enthalpy\u2013entropy compensation.", + "section": "RESULTS", + "ner": [ + [ + 3, + 5, + "\u0394S", + "evidence" + ], + [ + 22, + 24, + "\u0394G", + "evidence" + ], + [ + 40, + 42, + "\u0394H", + "evidence" + ], + [ + 54, + 56, + "\u0394H", + "evidence" + ], + [ + 91, + 93, + "\u0394S", + "evidence" + ] + ] + }, + { + "sid": 86, + "sent": "Such effects have been discussed by Klebe 24 and Chodera and Mobley 25 and will frustrate attempts to interpret the measured \u0394\u0394H and \u0394\u0394S.", + "section": "RESULTS", + "ner": [ + [ + 125, + 128, + "\u0394\u0394H", + "evidence" + ], + [ + 133, + 136, + "\u0394\u0394S", + "evidence" + ] + ] + }, + { + "sid": 87, + "sent": "The conserved phenylalanine and alanine residues of the FHTA sequence were both found to be essential for binding by ITC.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "conserved", + "protein_state" + ], + [ + 14, + 27, + "phenylalanine", + "residue_name" + ], + [ + 32, + 39, + "alanine", + "residue_name" + ], + [ + 56, + 60, + "FHTA", + "structure_element" + ], + [ + 117, + 120, + "ITC", + "experimental_method" + ] + ] + }, + { + "sid": 88, + "sent": "Conversely the second position histidine residue, corresponding to the unconserved His1525 in the BRC4 sequence, could be mutated without significant effect on the peptide affinity.", + "section": "RESULTS", + "ner": [ + [ + 31, + 40, + "histidine", + "residue_name" + ], + [ + 71, + 82, + "unconserved", + "protein_state" + ], + [ + 83, + 90, + "His1525", + "residue_name_number" + ], + [ + 98, + 102, + "BRC4", + "chemical" + ], + [ + 122, + 129, + "mutated", + "experimental_method" + ], + [ + 164, + 180, + "peptide affinity", + "evidence" + ] + ] + }, + { + "sid": 89, + "sent": "The more general correlation between hot\u2010spot residues in protein\u2013protein interactions and the high conservation of such residues has been previously reported 10, 26.", + "section": "RESULTS", + "ner": [ + [ + 37, + 45, + "hot\u2010spot", + "site" + ], + [ + 95, + 112, + "high conservation", + "protein_state" + ] + ] + }, + { + "sid": 90, + "sent": "Interestingly, however, the highly conserved threonine residue could be mutated without affecting the peptide affinity.", + "section": "RESULTS", + "ner": [ + [ + 28, + 44, + "highly conserved", + "protein_state" + ], + [ + 45, + 54, + "threonine", + "residue_name" + ], + [ + 72, + 79, + "mutated", + "experimental_method" + ], + [ + 102, + 118, + "peptide affinity", + "evidence" + ] + ] + }, + { + "sid": 91, + "sent": "This unexpected result, in the light of its very high conservation in the BRC and oligomerisation sequences, begs the question of what the role of Thr1526 is and highlights a potential pitfall and need for caution in the experimental design of alanine mutation studies.", + "section": "RESULTS", + "ner": [ + [ + 49, + 66, + "high conservation", + "protein_state" + ], + [ + 74, + 77, + "BRC", + "structure_element" + ], + [ + 82, + 107, + "oligomerisation sequences", + "structure_element" + ], + [ + 147, + 154, + "Thr1526", + "residue_name_number" + ], + [ + 244, + 268, + "alanine mutation studies", + "experimental_method" + ] + ] + }, + { + "sid": 92, + "sent": "As the FHTA peptide is potentially a surrogate peptide for both the BRC repeat peptides and the RAD51 self\u2010oligomerisation peptide, it is useful to examine the role of Thr1526 (BRC4) and the analogous Thr87 (RAD51) in both binding contexts in more detail.", + "section": "RESULTS", + "ner": [ + [ + 7, + 11, + "FHTA", + "structure_element" + ], + [ + 12, + 19, + "peptide", + "chemical" + ], + [ + 68, + 78, + "BRC repeat", + "structure_element" + ], + [ + 96, + 101, + "RAD51", + "protein" + ], + [ + 102, + 130, + "self\u2010oligomerisation peptide", + "structure_element" + ], + [ + 168, + 175, + "Thr1526", + "residue_name_number" + ], + [ + 177, + 181, + "BRC4", + "chemical" + ], + [ + 201, + 206, + "Thr87", + "residue_name_number" + ], + [ + 208, + 213, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 93, + "sent": "Only one structure of BRC4 is published in complex with human RAD51 (PDB: 1n0w).", + "section": "RESULTS", + "ner": [ + [ + 9, + 18, + "structure", + "evidence" + ], + [ + 22, + 26, + "BRC4", + "chemical" + ], + [ + 40, + 55, + "in complex with", + "protein_state" + ], + [ + 56, + 61, + "human", + "species" + ], + [ + 62, + 67, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 94, + "sent": "Figure 3A shows the binding pose of BRC4 when bound to RAD51 and the intrapeptide hydrogen bonds that are made by BRC4.", + "section": "RESULTS", + "ner": [ + [ + 36, + 40, + "BRC4", + "chemical" + ], + [ + 46, + 54, + "bound to", + "protein_state" + ], + [ + 55, + 60, + "RAD51", + "protein" + ], + [ + 82, + 96, + "hydrogen bonds", + "bond_interaction" + ], + [ + 114, + 118, + "BRC4", + "chemical" + ] + ] + }, + { + "sid": 95, + "sent": "While Phe1524 and Ala1527 are buried in hydrophobic pockets on the surface, His1525 is close enough to form a hydrogen bond with the carbonyl of Thr1520, but the rotamer of His1525, supported by clearly positioned water molecules, is not compatible with hydrogen bonding.", + "section": "RESULTS", + "ner": [ + [ + 6, + 13, + "Phe1524", + "residue_name_number" + ], + [ + 18, + 25, + "Ala1527", + "residue_name_number" + ], + [ + 40, + 59, + "hydrophobic pockets", + "site" + ], + [ + 76, + 83, + "His1525", + "residue_name_number" + ], + [ + 110, + 123, + "hydrogen bond", + "bond_interaction" + ], + [ + 145, + 152, + "Thr1520", + "residue_name_number" + ], + [ + 173, + 180, + "His1525", + "residue_name_number" + ], + [ + 214, + 219, + "water", + "chemical" + ], + [ + 254, + 270, + "hydrogen bonding", + "bond_interaction" + ] + ] + }, + { + "sid": 96, + "sent": "Also, Thr1520 is constrained by crystal contacts in this structure.", + "section": "RESULTS", + "ner": [ + [ + 6, + 13, + "Thr1520", + "residue_name_number" + ], + [ + 57, + 66, + "structure", + "evidence" + ] + ] + }, + { + "sid": 97, + "sent": "Lack of conservation of this residue supports the idea that this interaction is not crucial for RAD51:BRC repeat binding.", + "section": "RESULTS", + "ner": [ + [ + 0, + 20, + "Lack of conservation", + "protein_state" + ], + [ + 96, + 112, + "RAD51:BRC repeat", + "complex_assembly" + ] + ] + }, + { + "sid": 98, + "sent": "(A) Highlight of intra\u2010BRC4 interactions when bound to RAD51 (omitted for clarity) (PDB: 1n0w), with key residues shown in colour. (B) Intrapeptide interactions from oligomerisation epitope of S. cerevisiae RAD51 when bound to next RAD51 in the filament (PDB: 1szp).", + "section": "FIG", + "ner": [ + [ + 23, + 27, + "BRC4", + "chemical" + ], + [ + 46, + 54, + "bound to", + "protein_state" + ], + [ + 55, + 60, + "RAD51", + "protein" + ], + [ + 166, + 189, + "oligomerisation epitope", + "structure_element" + ], + [ + 193, + 206, + "S. cerevisiae", + "species" + ], + [ + 208, + 213, + "RAD51", + "protein" + ], + [ + 219, + 227, + "bound to", + "protein_state" + ], + [ + 233, + 238, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 99, + "sent": "Residue numbering relates to the S. cerevisiae RAD51 protein, the corresponding human residues are in parentheses.", + "section": "FIG", + "ner": [ + [ + 33, + 46, + "S. cerevisiae", + "species" + ], + [ + 48, + 53, + "RAD51", + "protein" + ], + [ + 81, + 86, + "human", + "species" + ] + ] + }, + { + "sid": 100, + "sent": "Either a threonine or serine is most commonly found in the third position of the FxxA motif.", + "section": "RESULTS", + "ner": [ + [ + 9, + 18, + "threonine", + "residue_name" + ], + [ + 22, + 28, + "serine", + "residue_name" + ], + [ + 81, + 85, + "FxxA", + "structure_element" + ] + ] + }, + { + "sid": 101, + "sent": "Thr1526 makes no direct interactions with the RAD51 protein, but instead forms a hydrogen bond network with the highly conserved S1528 and K1530 (Fig. 1C).", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Thr1526", + "residue_name_number" + ], + [ + 46, + 51, + "RAD51", + "protein" + ], + [ + 81, + 102, + "hydrogen bond network", + "bond_interaction" + ], + [ + 112, + 128, + "highly conserved", + "protein_state" + ], + [ + 129, + 134, + "S1528", + "residue_name_number" + ], + [ + 139, + 144, + "K1530", + "residue_name_number" + ] + ] + }, + { + "sid": 102, + "sent": "The high degree of conservation of these three residues suggests an important possible role in facilitating a turn and stabilising the conformation of the peptide as it continues its way to a second interaction site on the side of RAD51.", + "section": "RESULTS", + "ner": [ + [ + 4, + 31, + "high degree of conservation", + "protein_state" + ], + [ + 199, + 215, + "interaction site", + "site" + ], + [ + 231, + 236, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 103, + "sent": "With respect to understanding the RAD51:RAD51 interaction, no human crystal structure has been published, however, several oligomeric structures of archaeal RadA as well that of Saccharomyces cerevisiae RAD51 have been reported 27, 28, 29.", + "section": "RESULTS", + "ner": [ + [ + 34, + 45, + "RAD51:RAD51", + "complex_assembly" + ], + [ + 62, + 67, + "human", + "species" + ], + [ + 68, + 85, + "crystal structure", + "evidence" + ], + [ + 134, + 144, + "structures", + "evidence" + ], + [ + 148, + 156, + "archaeal", + "taxonomy_domain" + ], + [ + 157, + 161, + "RadA", + "protein" + ], + [ + 178, + 202, + "Saccharomyces cerevisiae", + "species" + ], + [ + 203, + 208, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 104, + "sent": "Figure 3B shows a highlight of the FxxA portion of oligomerisation peptide from the S. cerevisiae RAD51 structure, with residues in parentheses corresponding to the human RAD51 protein.", + "section": "RESULTS", + "ner": [ + [ + 35, + 39, + "FxxA", + "structure_element" + ], + [ + 51, + 74, + "oligomerisation peptide", + "structure_element" + ], + [ + 84, + 97, + "S. cerevisiae", + "species" + ], + [ + 98, + 103, + "RAD51", + "protein" + ], + [ + 104, + 113, + "structure", + "evidence" + ], + [ + 165, + 170, + "human", + "species" + ], + [ + 171, + 176, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 105, + "sent": "The conserved threonine residue at the third position forms a hydrogen bond with the peptide backbone amide, which forms the base of an \u03b1\u2010helix.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "conserved", + "protein_state" + ], + [ + 14, + 23, + "threonine", + "residue_name" + ], + [ + 62, + 75, + "hydrogen bond", + "bond_interaction" + ], + [ + 136, + 143, + "\u03b1\u2010helix", + "structure_element" + ] + ] + }, + { + "sid": 106, + "sent": "In both structural contexts, the role of the third position threonine in FxxA seems to be in stabilising secondary structure; a \u03b2\u2010turn in the case of BRC binding and an \u03b1\u2010helix in the case of RAD51 oligomerisation.", + "section": "RESULTS", + "ner": [ + [ + 60, + 69, + "threonine", + "residue_name" + ], + [ + 73, + 77, + "FxxA", + "structure_element" + ], + [ + 128, + 134, + "\u03b2\u2010turn", + "structure_element" + ], + [ + 150, + 153, + "BRC", + "structure_element" + ], + [ + 169, + 176, + "\u03b1\u2010helix", + "structure_element" + ], + [ + 192, + 197, + "RAD51", + "protein" + ] + ] + }, + { + "sid": 107, + "sent": "In the tetrapeptide context these secondary interactions are not present and mutation of threonine to alanine would be expected to have little effect on affinity.", + "section": "RESULTS", + "ner": [ + [ + 7, + 19, + "tetrapeptide", + "chemical" + ], + [ + 77, + 85, + "mutation", + "experimental_method" + ], + [ + 89, + 98, + "threonine", + "residue_name" + ], + [ + 102, + 109, + "alanine", + "residue_name" + ], + [ + 153, + 161, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 108, + "sent": "In line with this, although we observe a slight twofold weakening of peptide affinity, the effect is far from being as drastic or inactivating as reported in longer peptide backgrounds 3. It would be interesting to investigate the importance of this residue in the context of the BRC4 peptide, and the oligomerisation peptide.", + "section": "RESULTS", + "ner": [ + [ + 69, + 85, + "peptide affinity", + "evidence" + ], + [ + 280, + 284, + "BRC4", + "chemical" + ], + [ + 302, + 325, + "oligomerisation peptide", + "structure_element" + ] + ] + }, + { + "sid": 109, + "sent": "Rather than indifference to alanine mutation, a significant effect, via lack of secondary structure stabilisation, would be predicted, as indeed has been reported for BRC3 3.", + "section": "RESULTS", + "ner": [ + [ + 28, + 35, + "alanine", + "residue_name" + ], + [ + 36, + 44, + "mutation", + "experimental_method" + ], + [ + 167, + 171, + "BRC3", + "chemical" + ] + ] + }, + { + "sid": 110, + "sent": "Two residues in the FxxA motif, phenylalanine and alanine, are highly conserved (Fig 4a).", + "section": "CONCL", + "ner": [ + [ + 20, + 24, + "FxxA", + "structure_element" + ], + [ + 32, + 45, + "phenylalanine", + "residue_name" + ], + [ + 50, + 57, + "alanine", + "residue_name" + ], + [ + 63, + 79, + "highly conserved", + "protein_state" + ] + ] + }, + { + "sid": 111, + "sent": "Phenylalanine mutated to tryptophan, in the context of the tetrapeptide improved potency, contrary to the reported result of comparable activity in the context of BRC4 12.", + "section": "CONCL", + "ner": [ + [ + 0, + 13, + "Phenylalanine", + "residue_name" + ], + [ + 14, + 24, + "mutated to", + "experimental_method" + ], + [ + 25, + 35, + "tryptophan", + "residue_name" + ], + [ + 59, + 71, + "tetrapeptide", + "chemical" + ], + [ + 163, + 167, + "BRC4", + "chemical" + ] + ] + }, + { + "sid": 112, + "sent": "Proline at the third position similarly improved potency.", + "section": "CONCL", + "ner": [ + [ + 0, + 7, + "Proline", + "residue_name" + ] + ] + }, + { + "sid": 113, + "sent": "Activity was lost by mutating the terminal alanine to glycine, but recovered somewhat with the novel \u03b1\u2010amino butyric acid (U).", + "section": "CONCL", + "ner": [ + [ + 21, + 29, + "mutating", + "experimental_method" + ], + [ + 43, + 50, + "alanine", + "residue_name" + ], + [ + 54, + 61, + "glycine", + "residue_name" + ], + [ + 101, + 121, + "\u03b1\u2010amino butyric acid", + "chemical" + ], + [ + 123, + 124, + "U", + "chemical" + ] + ] + }, + { + "sid": 114, + "sent": "Threonine was found to be relatively unimportant in the tetrapeptides but has been previously reported to be crucial in the context of BRC3.", + "section": "CONCL", + "ner": [ + [ + 0, + 9, + "Threonine", + "residue_name" + ], + [ + 56, + 69, + "tetrapeptides", + "chemical" + ], + [ + 135, + 139, + "BRC3", + "chemical" + ] + ] + }, + { + "sid": 115, + "sent": "The reason for this disconnection is suggested to be that threonine plays a role in stabilising the \u03b2\u2010turn in the BRC repeats, which is absent in the tetrapeptides studied.", + "section": "CONCL", + "ner": [ + [ + 58, + 67, + "threonine", + "residue_name" + ], + [ + 100, + 106, + "\u03b2\u2010turn", + "structure_element" + ], + [ + 114, + 125, + "BRC repeats", + "structure_element" + ], + [ + 150, + 163, + "tetrapeptides", + "chemical" + ] + ] + }, + { + "sid": 116, + "sent": "This may lead to a more general caution, that hot\u2010spot data should be interpreted by considering the bound interaction with the protein, as well as the potential role in stabilising the bound peptide secondary structure.", + "section": "CONCL", + "ner": [ + [ + 46, + 54, + "hot\u2010spot", + "site" + ] + ] + }, + { + "sid": 117, + "sent": "In either case, the requirement for structural data in correctly interpreting alanine\u2010scanning experiments is reinforced.", + "section": "CONCL", + "ner": [ + [ + 78, + 106, + "alanine\u2010scanning experiments", + "experimental_method" + ] + ] + }, + { + "sid": 118, + "sent": "Summary of key observations (A) FxxA motif sequence conservation of Rad51 oligomerisation sequences and BRC repeats. (B) Highlight of SAR identified for the tetrapeptide.", + "section": "FIG", + "ner": [ + [ + 32, + 36, + "FxxA", + "structure_element" + ], + [ + 68, + 73, + "Rad51", + "protein" + ], + [ + 104, + 115, + "BRC repeats", + "structure_element" + ], + [ + 157, + 169, + "tetrapeptide", + "chemical" + ] + ] + }, + { + "sid": 119, + "sent": "The differences in \u0394G for different peptide variants relative to FHTA are shown in the bar chart with colouring matching with the structural overlay below. (C) Overlay of tetrapeptide structures, with wild\u2010type FHTA peptide across the figure for reference and truncated segments of mutated residues shown in each panel.", + "section": "FIG", + "ner": [ + [ + 19, + 21, + "\u0394G", + "evidence" + ], + [ + 65, + 69, + "FHTA", + "structure_element" + ], + [ + 130, + 148, + "structural overlay", + "experimental_method" + ], + [ + 160, + 167, + "Overlay", + "experimental_method" + ], + [ + 171, + 183, + "tetrapeptide", + "chemical" + ], + [ + 184, + 194, + "structures", + "evidence" + ], + [ + 201, + 210, + "wild\u2010type", + "protein_state" + ], + [ + 211, + 215, + "FHTA", + "structure_element" + ] + ] + }, + { + "sid": 120, + "sent": "Purple carbon is WHTA, light blue is FATA, yellow is FHPA, cyan is FHTG and grey carbon is FHTA.", + "section": "FIG", + "ner": [ + [ + 17, + 21, + "WHTA", + "structure_element" + ], + [ + 37, + 41, + "FATA", + "structure_element" + ], + [ + 53, + 57, + "FHPA", + "structure_element" + ], + [ + 67, + 71, + "FHTG", + "structure_element" + ], + [ + 91, + 95, + "FHTA", + "structure_element" + ] + ] + }, + { + "sid": 121, + "sent": "Note the C\u2010terminal amide changes position in FHTG without the anchoring methyl group.", + "section": "FIG", + "ner": [ + [ + 46, + 50, + "FHTG", + "structure_element" + ] + ] + } + ] + }, + "PMC4850288": { + "annotations": [ + { + "sid": 0, + "sent": "Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome*", + "section": "TITLE", + "ner": [ + [ + 0, + 17, + "Crystal Structure", + "evidence" + ], + [ + 22, + 38, + "Activity Studies", + "experimental_method" + ], + [ + 46, + 49, + "C11", + "protein_type" + ], + [ + 50, + 68, + "Cysteine Peptidase", + "protein_type" + ], + [ + 74, + 96, + "Parabacteroides merdae", + "species" + ], + [ + 104, + 109, + "Human", + "species" + ] + ] + }, + { + "sid": 1, + "sent": "Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 27, + "Clan CD cysteine peptidases", + "protein_type" + ], + [ + 61, + 71, + "peptidases", + "protein_type" + ], + [ + 85, + 94, + "mammalian", + "taxonomy_domain" + ], + [ + 95, + 103, + "caspases", + "protein_type" + ] + ] + }, + { + "sid": 2, + "sent": "However, for the clostripain family (denoted C11), little is currently known.", + "section": "ABSTRACT", + "ner": [ + [ + 17, + 35, + "clostripain family", + "protein_type" + ], + [ + 45, + 48, + "C11", + "protein_type" + ] + ] + }, + { + "sid": 3, + "sent": "Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-\u212b resolution.", + "section": "ABSTRACT", + "ner": [ + [ + 28, + 45, + "crystal structure", + "evidence" + ], + [ + 51, + 54, + "C11", + "protein_type" + ], + [ + 72, + 77, + "human", + "species" + ], + [ + 82, + 91, + "bacterium", + "taxonomy_domain" + ], + [ + 93, + 115, + "Parabacteroides merdae", + "species" + ], + [ + 117, + 122, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 4, + "sent": "PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like \u03b1/\u03b2/\u03b1 sandwich and an unusual C-terminal domain.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 5, + "PmC11", + "protein" + ], + [ + 11, + 20, + "monomeric", + "oligomeric_state" + ], + [ + 21, + 39, + "cysteine peptidase", + "protein_type" + ], + [ + 58, + 94, + "extended caspase-like \u03b1/\u03b2/\u03b1 sandwich", + "structure_element" + ], + [ + 110, + 127, + "C-terminal domain", + "structure_element" + ] + ] + }, + { + "sid": 5, + "sent": "It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan.", + "section": "ABSTRACT", + "ner": [ + [ + 40, + 67, + "clan CD cysteine peptidases", + "protein_type" + ] + ] + }, + { + "sid": 6, + "sent": "These studies also revealed a well ordered break in the polypeptide chain at Lys147, resulting in a large conformational rearrangement close to the active site.", + "section": "ABSTRACT", + "ner": [ + [ + 77, + 83, + "Lys147", + "residue_name_number" + ], + [ + 148, + 159, + "active site", + "site" + ] + ] + }, + { + "sid": 7, + "sent": "Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 32, + "Biochemical and kinetic analysis", + "experimental_method" + ], + [ + 42, + 48, + "Lys147", + "residue_name_number" + ], + [ + 58, + 88, + "intramolecular processing site", + "site" + ], + [ + 98, + 106, + "cleavage", + "ptm" + ], + [ + 123, + 138, + "full activation", + "protein_state" + ], + [ + 146, + 152, + "enzyme", + "protein" + ] + ] + }, + { + "sid": 8, + "sent": "PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 5, + "PmC11", + "protein" + ], + [ + 13, + 34, + "acidic binding pocket", + "site" + ], + [ + 102, + 105, + "Arg", + "residue_name" + ], + [ + 110, + 113, + "Lys", + "residue_name" + ], + [ + 117, + 119, + "P1", + "residue_number" + ], + [ + 141, + 145, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 9, + "sent": "Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.", + "section": "ABSTRACT", + "ner": [ + [ + 77, + 82, + "PmC11", + "protein" + ], + [ + 123, + 137, + "C11 peptidases", + "protein_type" + ] + ] + }, + { + "sid": 10, + "sent": "Cysteine peptidases play crucial roles in the virulence of bacterial and other eukaryotic pathogens.", + "section": "INTRO", + "ner": [ + [ + 0, + 19, + "Cysteine peptidases", + "protein_type" + ], + [ + 59, + 68, + "bacterial", + "taxonomy_domain" + ], + [ + 79, + 89, + "eukaryotic", + "taxonomy_domain" + ] + ] + }, + { + "sid": 11, + "sent": "In the MEROPS peptidase database, clan CD contains groups (or families) of cysteine peptidases that share some highly conserved structural elements.", + "section": "INTRO", + "ner": [ + [ + 34, + 41, + "clan CD", + "protein_type" + ], + [ + 75, + 94, + "cysteine peptidases", + "protein_type" + ], + [ + 111, + 127, + "highly conserved", + "protein_state" + ] + ] + }, + { + "sid": 12, + "sent": "Clan CD families are typically described using the name of their archetypal, or founding, member and also given an identification number preceded by a \u201cC,\u201d to denote cysteine peptidase.", + "section": "INTRO", + "ner": [ + [ + 0, + 16, + "Clan CD families", + "protein_type" + ], + [ + 166, + 184, + "cysteine peptidase", + "protein_type" + ] + ] + }, + { + "sid": 13, + "sent": "Although seven families (C14 is additionally split into three subfamilies) have been described for this clan, crystal structures have only been determined from four: legumain (C13), caspase (C14a), paracaspase (C14b(P), metacaspase (C14b(M), gingipain (C25), and the cysteine peptidase domain (CPD) of various toxins (C80).", + "section": "INTRO", + "ner": [ + [ + 110, + 128, + "crystal structures", + "evidence" + ], + [ + 166, + 174, + "legumain", + "protein" + ], + [ + 176, + 179, + "C13", + "protein_type" + ], + [ + 182, + 189, + "caspase", + "protein" + ], + [ + 191, + 195, + "C14a", + "protein_type" + ], + [ + 198, + 209, + "paracaspase", + "protein" + ], + [ + 211, + 217, + "C14b(P", + "protein_type" + ], + [ + 220, + 231, + "metacaspase", + "protein" + ], + [ + 233, + 239, + "C14b(M", + "protein_type" + ], + [ + 242, + 251, + "gingipain", + "protein" + ], + [ + 253, + 256, + "C25", + "protein_type" + ], + [ + 267, + 292, + "cysteine peptidase domain", + "structure_element" + ], + [ + 294, + 297, + "CPD", + "structure_element" + ], + [ + 318, + 321, + "C80", + "protein_type" + ] + ] + }, + { + "sid": 14, + "sent": "No structural information is available for clostripain (C11), separase (C50), or PrtH-peptidase (C85).", + "section": "INTRO", + "ner": [ + [ + 43, + 54, + "clostripain", + "protein" + ], + [ + 56, + 59, + "C11", + "protein_type" + ], + [ + 62, + 70, + "separase", + "protein" + ], + [ + 72, + 75, + "C50", + "protein_type" + ], + [ + 81, + 95, + "PrtH-peptidase", + "protein" + ], + [ + 97, + 100, + "C85", + "protein_type" + ] + ] + }, + { + "sid": 15, + "sent": "Clan CD enzymes have a highly conserved His/Cys catalytic dyad and exhibit strict specificity for the P1 residue of their substrates.", + "section": "INTRO", + "ner": [ + [ + 0, + 15, + "Clan CD enzymes", + "protein_type" + ], + [ + 23, + 39, + "highly conserved", + "protein_state" + ], + [ + 40, + 62, + "His/Cys catalytic dyad", + "site" + ], + [ + 102, + 104, + "P1", + "residue_number" + ] + ] + }, + { + "sid": 16, + "sent": "However, despite these similarities, clan CD forms a functionally diverse group of enzymes: the overall structural diversity between (and at times within) the various families provides these peptidases with a wide variety of substrate specificities and activation mechanisms.", + "section": "INTRO", + "ner": [ + [ + 37, + 44, + "clan CD", + "protein_type" + ], + [ + 191, + 201, + "peptidases", + "protein_type" + ] + ] + }, + { + "sid": 17, + "sent": "The archetypal and arguably most notable family in the clan is that of the mammalian caspases (C14a), although clan CD members are distributed throughout the entire phylogenetic kingdom and are often required in fundamental biological processes.", + "section": "INTRO", + "ner": [ + [ + 75, + 84, + "mammalian", + "taxonomy_domain" + ], + [ + 85, + 93, + "caspases", + "protein_type" + ], + [ + 95, + 99, + "C14a", + "protein_type" + ], + [ + 111, + 118, + "clan CD", + "protein_type" + ] + ] + }, + { + "sid": 18, + "sent": "Interestingly, little is known about the structure or function of the C11 proteins, despite their widespread distribution and its archetypal member, clostripain from Clostridium histolyticum, first reported in the literature in 1938.", + "section": "INTRO", + "ner": [ + [ + 70, + 73, + "C11", + "protein_type" + ], + [ + 149, + 160, + "clostripain", + "protein" + ], + [ + 166, + 190, + "Clostridium histolyticum", + "species" + ] + ] + }, + { + "sid": 19, + "sent": "Clostripain has been described as an arginine-specific peptidase with a requirement for Ca2+ and loss of an internal nonapeptide for full activation; lack of structural information on the family appears to have prohibited further investigation.", + "section": "INTRO", + "ner": [ + [ + 0, + 11, + "Clostripain", + "protein" + ], + [ + 37, + 64, + "arginine-specific peptidase", + "protein_type" + ], + [ + 88, + 92, + "Ca2+", + "chemical" + ], + [ + 108, + 128, + "internal nonapeptide", + "structure_element" + ], + [ + 133, + 148, + "full activation", + "protein_state" + ] + ] + }, + { + "sid": 20, + "sent": "As part of an ongoing project to characterize commensal bacteria in the microbiome that inhabit the human gut, the structure of C11 peptidase, PmC11, from Parabacteroides merdae was determined using the Joint Center for Structural Genomics (JCSG)4 HTP structural biology pipeline.", + "section": "INTRO", + "ner": [ + [ + 56, + 64, + "bacteria", + "taxonomy_domain" + ], + [ + 100, + 105, + "human", + "species" + ], + [ + 115, + 124, + "structure", + "evidence" + ], + [ + 128, + 141, + "C11 peptidase", + "protein_type" + ], + [ + 143, + 148, + "PmC11", + "protein" + ], + [ + 155, + 177, + "Parabacteroides merdae", + "species" + ] + ] + }, + { + "sid": 21, + "sent": "The structure was analyzed, and the enzyme was biochemically characterized to provide the first structure/function correlation for a C11 peptidase.", + "section": "INTRO", + "ner": [ + [ + 4, + 26, + "structure was analyzed", + "experimental_method" + ], + [ + 47, + 74, + "biochemically characterized", + "experimental_method" + ], + [ + 133, + 146, + "C11 peptidase", + "protein_type" + ] + ] + }, + { + "sid": 22, + "sent": "Structure of PmC11", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 13, + 18, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 23, + "sent": "The crystal structure of the catalytically active form of PmC11 revealed an extended caspase-like \u03b1/\u03b2/\u03b1 sandwich architecture comprised of a central nine-stranded \u03b2-sheet, with an unusual C-terminal domain (CTD), starting at Lys250.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 29, + 49, + "catalytically active", + "protein_state" + ], + [ + 58, + 63, + "PmC11", + "protein" + ], + [ + 76, + 112, + "extended caspase-like \u03b1/\u03b2/\u03b1 sandwich", + "structure_element" + ], + [ + 149, + 170, + "nine-stranded \u03b2-sheet", + "structure_element" + ], + [ + 188, + 205, + "C-terminal domain", + "structure_element" + ], + [ + 207, + 210, + "CTD", + "structure_element" + ], + [ + 225, + 231, + "Lys250", + "residue_name_number" + ] + ] + }, + { + "sid": 24, + "sent": "A single cleavage was observed in the polypeptide chain at Lys147 (Fig. 1, A and B), where both ends of the cleavage site are fully visible and well ordered in the electron density.", + "section": "RESULTS", + "ner": [ + [ + 2, + 17, + "single cleavage", + "ptm" + ], + [ + 59, + 65, + "Lys147", + "residue_name_number" + ], + [ + 108, + 121, + "cleavage site", + "site" + ], + [ + 164, + 180, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 25, + "sent": "The central nine-stranded \u03b2-sheet (\u03b21\u2013\u03b29) of PmC11 consists of six parallel and three anti-parallel \u03b2-strands with 4\u21913\u21932\u21911\u21915\u21916\u21917\u21938\u21939\u2191 topology (Fig. 1A) and the overall structure includes 14 \u03b1-helices with six (\u03b11\u2013\u03b12 and \u03b14\u2013\u03b17) closely surrounding the \u03b2-sheet in an approximately parallel orientation.", + "section": "RESULTS", + "ner": [ + [ + 12, + 33, + "nine-stranded \u03b2-sheet", + "structure_element" + ], + [ + 35, + 40, + "\u03b21\u2013\u03b29", + "structure_element" + ], + [ + 45, + 50, + "PmC11", + "protein" + ], + [ + 67, + 75, + "parallel", + "structure_element" + ], + [ + 86, + 109, + "anti-parallel \u03b2-strands", + "structure_element" + ], + [ + 169, + 178, + "structure", + "evidence" + ], + [ + 191, + 200, + "\u03b1-helices", + "structure_element" + ], + [ + 211, + 216, + "\u03b11\u2013\u03b12", + "structure_element" + ], + [ + 221, + 226, + "\u03b14\u2013\u03b17", + "structure_element" + ], + [ + 252, + 259, + "\u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 26, + "sent": "Helices \u03b11, \u03b17, and \u03b16 are located on one side of the \u03b2-sheet with \u03b12, \u03b14, and \u03b15 on the opposite side (Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Helices", + "structure_element" + ], + [ + 8, + 10, + "\u03b11", + "structure_element" + ], + [ + 12, + 14, + "\u03b17", + "structure_element" + ], + [ + 20, + 22, + "\u03b16", + "structure_element" + ], + [ + 54, + 61, + "\u03b2-sheet", + "structure_element" + ], + [ + 67, + 69, + "\u03b12", + "structure_element" + ], + [ + 71, + 73, + "\u03b14", + "structure_element" + ], + [ + 79, + 81, + "\u03b15", + "structure_element" + ] + ] + }, + { + "sid": 27, + "sent": "Helix \u03b13 sits at the end of the loop following \u03b25 (L5), just preceding the Lys147 cleavage site, with both L5 and \u03b13 pointing away from the central \u03b2-sheet and toward the CTD, which starts with \u03b18.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "Helix", + "structure_element" + ], + [ + 6, + 8, + "\u03b13", + "structure_element" + ], + [ + 32, + 36, + "loop", + "structure_element" + ], + [ + 47, + 49, + "\u03b25", + "structure_element" + ], + [ + 51, + 53, + "L5", + "structure_element" + ], + [ + 75, + 81, + "Lys147", + "residue_name_number" + ], + [ + 82, + 95, + "cleavage site", + "site" + ], + [ + 107, + 109, + "L5", + "structure_element" + ], + [ + 114, + 116, + "\u03b13", + "structure_element" + ], + [ + 148, + 155, + "\u03b2-sheet", + "structure_element" + ], + [ + 171, + 174, + "CTD", + "structure_element" + ], + [ + 194, + 196, + "\u03b18", + "structure_element" + ] + ] + }, + { + "sid": 28, + "sent": "The structure also includes two short \u03b2-hairpins (\u03b2A\u2013\u03b2B and \u03b2D\u2013\u03b2E) and a small \u03b2-sheet (\u03b2C\u2013\u03b2F), which is formed from two distinct regions of the sequence (\u03b2C precedes \u03b111, \u03b112 and \u03b29, whereas \u03b2F follows the \u03b2D-\u03b2E hairpin) in the middle of the CTD (Fig. 1B).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 38, + 48, + "\u03b2-hairpins", + "structure_element" + ], + [ + 50, + 55, + "\u03b2A\u2013\u03b2B", + "structure_element" + ], + [ + 60, + 65, + "\u03b2D\u2013\u03b2E", + "structure_element" + ], + [ + 73, + 86, + "small \u03b2-sheet", + "structure_element" + ], + [ + 88, + 93, + "\u03b2C\u2013\u03b2F", + "structure_element" + ], + [ + 155, + 157, + "\u03b2C", + "structure_element" + ], + [ + 167, + 170, + "\u03b111", + "structure_element" + ], + [ + 172, + 175, + "\u03b112", + "structure_element" + ], + [ + 180, + 182, + "\u03b29", + "structure_element" + ], + [ + 192, + 194, + "\u03b2F", + "structure_element" + ], + [ + 207, + 212, + "\u03b2D-\u03b2E", + "structure_element" + ], + [ + 213, + 220, + "hairpin", + "structure_element" + ], + [ + 243, + 246, + "CTD", + "structure_element" + ] + ] + }, + { + "sid": 29, + "sent": "Crystal structure of a C11 peptidase from P. merdae.", + "section": "FIG", + "ner": [ + [ + 0, + 17, + "Crystal structure", + "evidence" + ], + [ + 23, + 36, + "C11 peptidase", + "protein_type" + ], + [ + 42, + 51, + "P. merdae", + "species" + ] + ] + }, + { + "sid": 30, + "sent": " A, primary sequence alignment of PmC11 (Uniprot ID A7A9N3) and clostripain (Uniprot ID P09870) from C. histolyticum with identical residues highlighted in gray shading.", + "section": "FIG", + "ner": [ + [ + 4, + 30, + "primary sequence alignment", + "experimental_method" + ], + [ + 34, + 39, + "PmC11", + "protein" + ], + [ + 64, + 75, + "clostripain", + "protein" + ], + [ + 101, + 116, + "C. histolyticum", + "species" + ] + ] + }, + { + "sid": 31, + "sent": "The secondary structure of PmC11 from the crystal structure is mapped onto its sequence with the position of the PmC11 catalytic dyad, autocatalytic cleavage site (Lys147), and S1 binding pocket Asp (Asp177) highlighted by a red star, a red downturned triangle, and a red upturned triangle, respectively.", + "section": "FIG", + "ner": [ + [ + 27, + 32, + "PmC11", + "protein" + ], + [ + 42, + 59, + "crystal structure", + "evidence" + ], + [ + 113, + 118, + "PmC11", + "protein" + ], + [ + 119, + 133, + "catalytic dyad", + "site" + ], + [ + 135, + 162, + "autocatalytic cleavage site", + "site" + ], + [ + 164, + 170, + "Lys147", + "residue_name_number" + ], + [ + 177, + 194, + "S1 binding pocket", + "site" + ], + [ + 195, + 198, + "Asp", + "residue_name" + ], + [ + 200, + 206, + "Asp177", + "residue_name_number" + ] + ] + }, + { + "sid": 32, + "sent": "Connecting loops are colored gray, the main \u03b2-sheet is in orange, with other strands in olive, \u03b1-helices are in blue, and the nonapeptide linker of clostripain that is excised upon autocleavage is underlined in red.", + "section": "FIG", + "ner": [ + [ + 11, + 16, + "loops", + "structure_element" + ], + [ + 44, + 51, + "\u03b2-sheet", + "structure_element" + ], + [ + 95, + 104, + "\u03b1-helices", + "structure_element" + ], + [ + 126, + 144, + "nonapeptide linker", + "structure_element" + ], + [ + 148, + 159, + "clostripain", + "protein" + ], + [ + 181, + 193, + "autocleavage", + "ptm" + ] + ] + }, + { + "sid": 33, + "sent": "Sequences around the catalytic site of clostripain and PmC11 align well.", + "section": "FIG", + "ner": [ + [ + 21, + 35, + "catalytic site", + "site" + ], + [ + 39, + 50, + "clostripain", + "protein" + ], + [ + 55, + 60, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 34, + "sent": "B, topology diagram of PmC11 colored as in A except that additional (non-core) \u03b2-strands are in yellow.", + "section": "FIG", + "ner": [ + [ + 23, + 28, + "PmC11", + "protein" + ], + [ + 79, + 88, + "\u03b2-strands", + "structure_element" + ] + ] + }, + { + "sid": 35, + "sent": "Helices found on either side of the central \u03b2-sheet are shown above and below the sheet, respectively.", + "section": "FIG", + "ner": [ + [ + 44, + 51, + "\u03b2-sheet", + "structure_element" + ], + [ + 82, + 87, + "sheet", + "structure_element" + ] + ] + }, + { + "sid": 36, + "sent": "The position of the catalytic dyad (H, C) and the processing site (Lys147) are highlighted.", + "section": "FIG", + "ner": [ + [ + 20, + 34, + "catalytic dyad", + "site" + ], + [ + 36, + 37, + "H", + "residue_name" + ], + [ + 39, + 40, + "C", + "residue_name" + ], + [ + 50, + 65, + "processing site", + "site" + ], + [ + 67, + 73, + "Lys147", + "residue_name_number" + ] + ] + }, + { + "sid": 37, + "sent": "Helices (1\u201314) and \u03b2-strands (1\u20139 and A-F) are numbered from the N terminus.", + "section": "FIG", + "ner": [ + [ + 19, + 28, + "\u03b2-strands", + "structure_element" + ] + ] + }, + { + "sid": 38, + "sent": "The core caspase-fold is highlighted in a box.", + "section": "FIG", + "ner": [ + [ + 4, + 21, + "core caspase-fold", + "structure_element" + ] + ] + }, + { + "sid": 39, + "sent": "C, tertiary structure of PmC11.", + "section": "FIG", + "ner": [ + [ + 25, + 30, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 40, + "sent": "The N and C termini (N and C) of PmC11 along with the central \u03b2-sheet (1\u20139), helix \u03b15, and helices \u03b18, \u03b111, and \u03b113 from the C-terminal domain, are all labeled.", + "section": "FIG", + "ner": [ + [ + 33, + 38, + "PmC11", + "protein" + ], + [ + 62, + 69, + "\u03b2-sheet", + "structure_element" + ], + [ + 77, + 82, + "helix", + "structure_element" + ], + [ + 83, + 85, + "\u03b15", + "structure_element" + ], + [ + 91, + 98, + "helices", + "structure_element" + ], + [ + 99, + 101, + "\u03b18", + "structure_element" + ], + [ + 103, + 106, + "\u03b111", + "structure_element" + ], + [ + 112, + 115, + "\u03b113", + "structure_element" + ], + [ + 125, + 142, + "C-terminal domain", + "structure_element" + ] + ] + }, + { + "sid": 41, + "sent": "Loops are colored gray, the main \u03b2-sheet is in orange, with other \u03b2-strands in yellow, and \u03b1-helices are in blue.", + "section": "FIG", + "ner": [ + [ + 33, + 40, + "\u03b2-sheet", + "structure_element" + ], + [ + 66, + 75, + "\u03b2-strands", + "structure_element" + ], + [ + 91, + 100, + "\u03b1-helices", + "structure_element" + ] + ] + }, + { + "sid": 42, + "sent": "The CTD of PmC11 is composed of a tight helical bundle formed from helices \u03b18\u2013\u03b114 and includes strands \u03b2C and \u03b2F, and \u03b2-hairpin \u03b2D\u2013\u03b2E. The CTD sits entirely on one side of the enzyme interacting only with \u03b13, \u03b15, \u03b29, and the loops surrounding \u03b28.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "CTD", + "structure_element" + ], + [ + 11, + 16, + "PmC11", + "protein" + ], + [ + 34, + 54, + "tight helical bundle", + "structure_element" + ], + [ + 67, + 74, + "helices", + "structure_element" + ], + [ + 75, + 81, + "\u03b18\u2013\u03b114", + "structure_element" + ], + [ + 95, + 102, + "strands", + "structure_element" + ], + [ + 103, + 105, + "\u03b2C", + "structure_element" + ], + [ + 110, + 112, + "\u03b2F", + "structure_element" + ], + [ + 118, + 127, + "\u03b2-hairpin", + "structure_element" + ], + [ + 128, + 133, + "\u03b2D\u2013\u03b2E", + "structure_element" + ], + [ + 139, + 142, + "CTD", + "structure_element" + ], + [ + 205, + 207, + "\u03b13", + "structure_element" + ], + [ + 209, + 211, + "\u03b15", + "structure_element" + ], + [ + 213, + 215, + "\u03b29", + "structure_element" + ], + [ + 225, + 230, + "loops", + "structure_element" + ], + [ + 243, + 245, + "\u03b28", + "structure_element" + ] + ] + }, + { + "sid": 43, + "sent": "Of the interacting secondary structure elements, \u03b15 is perhaps the most interesting.", + "section": "RESULTS", + "ner": [ + [ + 49, + 51, + "\u03b15", + "structure_element" + ] + ] + }, + { + "sid": 44, + "sent": "This helix makes a total of eight hydrogen bonds with the CTD, including one salt bridge (Arg191-Asp255) and is surrounded by the CTD on one side and the main core of the enzyme on the other, acting like a linchpin holding both components together (Fig. 1C).", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "This helix", + "structure_element" + ], + [ + 34, + 48, + "hydrogen bonds", + "bond_interaction" + ], + [ + 58, + 61, + "CTD", + "structure_element" + ], + [ + 77, + 88, + "salt bridge", + "bond_interaction" + ], + [ + 90, + 96, + "Arg191", + "residue_name_number" + ], + [ + 97, + 103, + "Asp255", + "residue_name_number" + ], + [ + 130, + 133, + "CTD", + "structure_element" + ], + [ + 154, + 163, + "main core", + "structure_element" + ] + ] + }, + { + "sid": 45, + "sent": "PmC11 is, as expected, most structurally similar to other members of clan CD with the top hits in a search of known structures being caspase-7, gingipain-K, and legumain (PBD codes 4hq0, 4tkx, and 4aw9, respectively) (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "PmC11", + "protein" + ], + [ + 69, + 76, + "clan CD", + "protein_type" + ], + [ + 116, + 126, + "structures", + "evidence" + ], + [ + 133, + 142, + "caspase-7", + "protein" + ], + [ + 144, + 155, + "gingipain-K", + "protein" + ], + [ + 161, + 169, + "legumain", + "protein" + ] + ] + }, + { + "sid": 46, + "sent": "The C-terminal domain is unique to PmC11 within clan CD and structure comparisons for this domain alone does not produce any hits in the PDB (DaliLite, PDBeFold), suggesting a completely novel fold.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "C-terminal domain", + "structure_element" + ], + [ + 35, + 40, + "PmC11", + "protein" + ], + [ + 48, + 55, + "clan CD", + "protein_type" + ], + [ + 60, + 81, + "structure comparisons", + "experimental_method" + ], + [ + 86, + 103, + "this domain alone", + "structure_element" + ], + [ + 142, + 150, + "DaliLite", + "experimental_method" + ], + [ + 152, + 160, + "PDBeFold", + "experimental_method" + ] + ] + }, + { + "sid": 47, + "sent": "As the archetypal and arguably most well studied member of clan CD, the caspases were used as the basis to investigate the structure/function relationships in PmC11, with caspase-7 as the representative member.", + "section": "RESULTS", + "ner": [ + [ + 59, + 66, + "clan CD", + "protein_type" + ], + [ + 72, + 80, + "caspases", + "protein_type" + ], + [ + 159, + 164, + "PmC11", + "protein" + ], + [ + 171, + 180, + "caspase-7", + "protein" + ] + ] + }, + { + "sid": 48, + "sent": "Six of the central \u03b2-strands in PmC11 (\u03b21\u2013\u03b22 and \u03b25\u2013\u03b28) share the same topology as the six-stranded \u03b2-sheet found in caspases, with strands \u03b23, \u03b24, and \u03b29 located on the outside of this core structure (Fig. 1B, box).", + "section": "RESULTS", + "ner": [ + [ + 19, + 28, + "\u03b2-strands", + "structure_element" + ], + [ + 32, + 37, + "PmC11", + "protein" + ], + [ + 39, + 44, + "\u03b21\u2013\u03b22", + "structure_element" + ], + [ + 49, + 54, + "\u03b25\u2013\u03b28", + "structure_element" + ], + [ + 87, + 107, + "six-stranded \u03b2-sheet", + "structure_element" + ], + [ + 117, + 125, + "caspases", + "protein_type" + ], + [ + 132, + 139, + "strands", + "structure_element" + ], + [ + 140, + 142, + "\u03b23", + "structure_element" + ], + [ + 144, + 146, + "\u03b24", + "structure_element" + ], + [ + 152, + 154, + "\u03b29", + "structure_element" + ], + [ + 186, + 200, + "core structure", + "structure_element" + ] + ] + }, + { + "sid": 49, + "sent": "His133 and Cys179 were found at locations structurally homologous to the caspase catalytic dyad, and other clan CD structures, at the C termini of strands \u03b25 and \u03b26, respectively (Figs. 1, A and B, and 2A).", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "His133", + "residue_name_number" + ], + [ + 11, + 17, + "Cys179", + "residue_name_number" + ], + [ + 73, + 80, + "caspase", + "protein_type" + ], + [ + 81, + 95, + "catalytic dyad", + "site" + ], + [ + 107, + 114, + "clan CD", + "protein_type" + ], + [ + 115, + 125, + "structures", + "evidence" + ], + [ + 147, + 154, + "strands", + "structure_element" + ], + [ + 155, + 157, + "\u03b25", + "structure_element" + ], + [ + 162, + 164, + "\u03b26", + "structure_element" + ] + ] + }, + { + "sid": 50, + "sent": "A multiple sequence alignment of C11 proteins revealed that these residues are highly conserved (data not shown).", + "section": "RESULTS", + "ner": [ + [ + 2, + 29, + "multiple sequence alignment", + "experimental_method" + ], + [ + 33, + 36, + "C11", + "protein_type" + ], + [ + 79, + 95, + "highly conserved", + "protein_state" + ] + ] + }, + { + "sid": 51, + "sent": "Summary of PDBeFOLD superposition of structures found to be most similar to PmC11 in the PBD based on DaliLite", + "section": "TABLE", + "ner": [ + [ + 11, + 33, + "PDBeFOLD superposition", + "experimental_method" + ], + [ + 76, + 81, + "PmC11", + "protein" + ], + [ + 102, + 110, + "DaliLite", + "experimental_method" + ] + ] + }, + { + "sid": 52, + "sent": "Biochemical and structural characterization of PmC11.", + "section": "FIG", + "ner": [ + [ + 0, + 43, + "Biochemical and structural characterization", + "experimental_method" + ], + [ + 47, + 52, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 53, + "sent": " A, ribbon representation of the overall structure of PmC11 illustrating the catalytic site, cleavage site displacement, and potential S1 binding site.", + "section": "FIG", + "ner": [ + [ + 54, + 59, + "PmC11", + "protein" + ], + [ + 77, + 91, + "catalytic site", + "site" + ], + [ + 135, + 150, + "S1 binding site", + "site" + ] + ] + }, + { + "sid": 54, + "sent": "The overall structure of PmC11 is shown in gray, looking down into the catalytic site with the catalytic dyad in red.", + "section": "FIG", + "ner": [ + [ + 12, + 21, + "structure", + "evidence" + ], + [ + 25, + 30, + "PmC11", + "protein" + ], + [ + 71, + 85, + "catalytic site", + "site" + ], + [ + 95, + 109, + "catalytic dyad", + "site" + ] + ] + }, + { + "sid": 55, + "sent": "The two ends of the autolytic cleavage site (Lys147 and Ala148, green) are displaced by 19.5 \u212b (thin black line) from one another and residues in the potential substrate binding pocket are highlighted in blue.", + "section": "FIG", + "ner": [ + [ + 20, + 43, + "autolytic cleavage site", + "site" + ], + [ + 45, + 51, + "Lys147", + "residue_name_number" + ], + [ + 56, + 62, + "Ala148", + "residue_name_number" + ], + [ + 160, + 184, + "substrate binding pocket", + "site" + ] + ] + }, + { + "sid": 56, + "sent": "B, size exclusion chromatography of PmC11.", + "section": "FIG", + "ner": [ + [ + 3, + 32, + "size exclusion chromatography", + "experimental_method" + ], + [ + 36, + 41, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 57, + "sent": "PmC11 migrates as a monomer with a molecular mass around 41 kDa calculated from protein standards of known molecular weights.", + "section": "FIG", + "ner": [ + [ + 20, + 27, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 58, + "sent": "Elution fractions across the major peak (1\u20136) were analyzed by SDS-PAGE on a 4\u201312% gel in MES buffer.", + "section": "FIG", + "ner": [ + [ + 63, + 71, + "SDS-PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 59, + "sent": "C, the active form of PmC11 and two mutants, PmC11C179A (C) and PmC11K147A (K), were examined by SDS-PAGE (lane 1) and Western blot analysis using an anti-His antibody (lane 2) show that PmC11 autoprocesses, whereas mutants, PmC11C179A and PmC11K147A, do not show autoprocessing in vitro.", + "section": "FIG", + "ner": [ + [ + 7, + 13, + "active", + "protein_state" + ], + [ + 22, + 27, + "PmC11", + "protein" + ], + [ + 45, + 55, + "PmC11C179A", + "mutant" + ], + [ + 64, + 74, + "PmC11K147A", + "mutant" + ], + [ + 97, + 105, + "SDS-PAGE", + "experimental_method" + ], + [ + 119, + 131, + "Western blot", + "experimental_method" + ], + [ + 187, + 192, + "PmC11", + "protein" + ], + [ + 193, + 206, + "autoprocesses", + "ptm" + ], + [ + 225, + 235, + "PmC11C179A", + "mutant" + ], + [ + 240, + 250, + "PmC11K147A", + "mutant" + ], + [ + 264, + 278, + "autoprocessing", + "ptm" + ] + ] + }, + { + "sid": 60, + "sent": "D, cysteine peptidase activity of PmC11.", + "section": "FIG", + "ner": [ + [ + 34, + 39, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 61, + "sent": "Km and Vmax of PmC11 and K147A mutant were determined by monitoring change in the fluorescence corresponding to AMC release from Bz-R-AMC.", + "section": "FIG", + "ner": [ + [ + 7, + 11, + "Vmax", + "evidence" + ], + [ + 15, + 20, + "PmC11", + "protein" + ], + [ + 25, + 30, + "K147A", + "mutant" + ], + [ + 129, + 137, + "Bz-R-AMC", + "chemical" + ] + ] + }, + { + "sid": 62, + "sent": "E, intermolecular processing of PmC11C179A by PmC11.", + "section": "FIG", + "ner": [ + [ + 3, + 28, + "intermolecular processing", + "ptm" + ], + [ + 32, + 42, + "PmC11C179A", + "mutant" + ], + [ + 46, + 51, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 63, + "sent": "PmC11C179A (20 \u03bcg) was incubated overnight at 37 \u00b0C with increasing amounts of processed PmC11 and analyzed on a 10% SDS-PAGE gel.", + "section": "FIG", + "ner": [ + [ + 89, + 94, + "PmC11", + "protein" + ], + [ + 117, + 125, + "SDS-PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 64, + "sent": "Inactive PmC11C179A was not processed to a major extent by active PmC11 until around a ratio of 1:4 (5 \u03bcg of active PmC11).", + "section": "FIG", + "ner": [ + [ + 9, + 19, + "PmC11C179A", + "mutant" + ], + [ + 59, + 65, + "active", + "protein_state" + ], + [ + 66, + 71, + "PmC11", + "protein" + ], + [ + 109, + 115, + "active", + "protein_state" + ], + [ + 116, + 121, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 65, + "sent": "A single lane of 20 \u03bcg of active PmC11 (labeled 20) is shown for comparison.", + "section": "FIG", + "ner": [ + [ + 26, + 32, + "active", + "protein_state" + ], + [ + 33, + 38, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 66, + "sent": "F, activity of PmC11 against basic substrates.", + "section": "FIG", + "ner": [ + [ + 3, + 11, + "activity", + "evidence" + ], + [ + 15, + 20, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 67, + "sent": "G, electrostatic surface potential of PmC11 shown in a similar orientation, where blue and red denote positively and negatively charged surface potential, respectively, contoured at \u00b15 kT/e.", + "section": "FIG", + "ner": [ + [ + 38, + 43, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 68, + "sent": "The position of the catalytic dyad, one potential key substrate binding residue Asp177, and the ends of the cleavage site Lys147 and Ala148 are indicated.", + "section": "FIG", + "ner": [ + [ + 20, + 34, + "catalytic dyad", + "site" + ], + [ + 50, + 79, + "key substrate binding residue", + "site" + ], + [ + 80, + 86, + "Asp177", + "residue_name_number" + ], + [ + 108, + 121, + "cleavage site", + "site" + ], + [ + 122, + 128, + "Lys147", + "residue_name_number" + ], + [ + 133, + 139, + "Ala148", + "residue_name_number" + ] + ] + }, + { + "sid": 69, + "sent": "Five of the \u03b1-helices surrounding the \u03b2-sheet of PmC11 (\u03b11, \u03b12, \u03b14, \u03b16, and \u03b17) are found in similar positions to the five structurally conserved helices in caspases and other members of clan CD, apart from family C80.", + "section": "RESULTS", + "ner": [ + [ + 12, + 21, + "\u03b1-helices", + "structure_element" + ], + [ + 38, + 45, + "\u03b2-sheet", + "structure_element" + ], + [ + 49, + 54, + "PmC11", + "protein" + ], + [ + 56, + 58, + "\u03b11", + "structure_element" + ], + [ + 60, + 62, + "\u03b12", + "structure_element" + ], + [ + 64, + 66, + "\u03b14", + "structure_element" + ], + [ + 68, + 70, + "\u03b16", + "structure_element" + ], + [ + 76, + 78, + "\u03b17", + "structure_element" + ], + [ + 123, + 145, + "structurally conserved", + "protein_state" + ], + [ + 146, + 153, + "helices", + "structure_element" + ], + [ + 157, + 165, + "caspases", + "protein_type" + ], + [ + 187, + 194, + "clan CD", + "protein_type" + ], + [ + 214, + 217, + "C80", + "protein_type" + ] + ] + }, + { + "sid": 70, + "sent": "Other than its more extended \u03b2-sheet, PmC11 differs most significantly from other clan CD members at its C terminus, where the CTD contains a further seven \u03b1-helices and four \u03b2-strands after \u03b28.", + "section": "RESULTS", + "ner": [ + [ + 20, + 36, + "extended \u03b2-sheet", + "structure_element" + ], + [ + 38, + 43, + "PmC11", + "protein" + ], + [ + 82, + 89, + "clan CD", + "protein_type" + ], + [ + 127, + 130, + "CTD", + "structure_element" + ], + [ + 156, + 165, + "\u03b1-helices", + "structure_element" + ], + [ + 175, + 184, + "\u03b2-strands", + "structure_element" + ], + [ + 191, + 193, + "\u03b28", + "structure_element" + ] + ] + }, + { + "sid": 71, + "sent": "Autoprocessing of PmC11", + "section": "RESULTS", + "ner": [ + [ + 0, + 14, + "Autoprocessing", + "ptm" + ], + [ + 18, + 23, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 72, + "sent": "Purification of recombinant PmC11 (molecular mass = 42.6 kDa) revealed partial processing into two cleavage products of 26.4 and 16.2 kDa, related to the observed cleavage at Lys147 in the crystal structure (Fig. 2A).", + "section": "RESULTS", + "ner": [ + [ + 0, + 12, + "Purification", + "experimental_method" + ], + [ + 28, + 33, + "PmC11", + "protein" + ], + [ + 163, + 171, + "cleavage", + "ptm" + ], + [ + 175, + 181, + "Lys147", + "residue_name_number" + ], + [ + 189, + 206, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 73, + "sent": "Incubation of PmC11 at 37 \u00b0C for 16 h, resulted in a fully processed enzyme that remained as an intact monomer when applied to a size-exclusion column (Fig. 2B).", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "Incubation", + "experimental_method" + ], + [ + 14, + 19, + "PmC11", + "protein" + ], + [ + 53, + 68, + "fully processed", + "protein_state" + ], + [ + 96, + 102, + "intact", + "protein_state" + ], + [ + 103, + 110, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 74, + "sent": "The single cleavage site of PmC11 at Lys147 is found immediately after \u03b13, in loop L5 within the central \u03b2-sheet (Figs. 1, A and B, and 2A).", + "section": "RESULTS", + "ner": [ + [ + 11, + 24, + "cleavage site", + "site" + ], + [ + 28, + 33, + "PmC11", + "protein" + ], + [ + 37, + 43, + "Lys147", + "residue_name_number" + ], + [ + 71, + 73, + "\u03b13", + "structure_element" + ], + [ + 78, + 82, + "loop", + "structure_element" + ], + [ + 83, + 85, + "L5", + "structure_element" + ], + [ + 105, + 112, + "\u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 75, + "sent": "The two ends of the cleavage site are remarkably well ordered in the crystal structure and displaced from one another by 19.5 \u212b (Fig. 2A).", + "section": "RESULTS", + "ner": [ + [ + 20, + 33, + "cleavage site", + "site" + ], + [ + 69, + 86, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 76, + "sent": "Moreover, the C-terminal side of the cleavage site resides near the catalytic dyad with Ala148 being 4.5 and 5.7 \u212b from His133 and Cys179, respectively.", + "section": "RESULTS", + "ner": [ + [ + 37, + 50, + "cleavage site", + "site" + ], + [ + 68, + 82, + "catalytic dyad", + "site" + ], + [ + 88, + 94, + "Ala148", + "residue_name_number" + ], + [ + 120, + 126, + "His133", + "residue_name_number" + ], + [ + 131, + 137, + "Cys179", + "residue_name_number" + ] + ] + }, + { + "sid": 77, + "sent": "Consequently, it appears feasible that the helix attached to Lys147 (\u03b13) could be responsible for steric autoinhibition of PmC11 when Lys147 is covalently bonded to Ala148.", + "section": "RESULTS", + "ner": [ + [ + 43, + 48, + "helix", + "structure_element" + ], + [ + 61, + 67, + "Lys147", + "residue_name_number" + ], + [ + 69, + 71, + "\u03b13", + "structure_element" + ], + [ + 123, + 128, + "PmC11", + "protein" + ], + [ + 134, + 140, + "Lys147", + "residue_name_number" + ], + [ + 165, + 171, + "Ala148", + "residue_name_number" + ] + ] + }, + { + "sid": 78, + "sent": "Thus, the cleavage would be required for full activation of PmC11.", + "section": "RESULTS", + "ner": [ + [ + 10, + 18, + "cleavage", + "ptm" + ], + [ + 41, + 56, + "full activation", + "protein_state" + ], + [ + 60, + 65, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 79, + "sent": "To investigate this possibility, two mutant forms of the enzyme were created: PmC11C179A (a catalytically inactive mutant) and PmC11K147A (a cleavage-site mutant).", + "section": "RESULTS", + "ner": [ + [ + 78, + 88, + "PmC11C179A", + "mutant" + ], + [ + 92, + 121, + "catalytically inactive mutant", + "protein_state" + ], + [ + 127, + 137, + "PmC11K147A", + "mutant" + ], + [ + 141, + 161, + "cleavage-site mutant", + "protein_state" + ] + ] + }, + { + "sid": 80, + "sent": "Initial SDS-PAGE and Western blot analysis of both mutants revealed no discernible processing occurred as compared with active PmC11 (Fig. 2C).", + "section": "RESULTS", + "ner": [ + [ + 8, + 16, + "SDS-PAGE", + "experimental_method" + ], + [ + 21, + 33, + "Western blot", + "experimental_method" + ], + [ + 120, + 126, + "active", + "protein_state" + ], + [ + 127, + 132, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 81, + "sent": "The PmC11K147A mutant enzyme had a markedly different reaction rate (Vmax) compared with WT, where the reaction velocity of PmC11 was 10 times greater than that of PmC11K147A (Fig. 2D).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "PmC11K147A", + "mutant" + ], + [ + 15, + 21, + "mutant", + "protein_state" + ], + [ + 54, + 67, + "reaction rate", + "evidence" + ], + [ + 69, + 73, + "Vmax", + "evidence" + ], + [ + 89, + 91, + "WT", + "protein_state" + ], + [ + 103, + 120, + "reaction velocity", + "evidence" + ], + [ + 124, + 129, + "PmC11", + "protein" + ], + [ + 164, + 174, + "PmC11K147A", + "mutant" + ] + ] + }, + { + "sid": 82, + "sent": "Taken together, these data reveal that PmC11 requires processing at Lys147 for optimum activity.", + "section": "RESULTS", + "ner": [ + [ + 39, + 44, + "PmC11", + "protein" + ], + [ + 68, + 74, + "Lys147", + "residue_name_number" + ] + ] + }, + { + "sid": 83, + "sent": "To investigate whether processing is a result of intra- or intermolecular cleavage, the PmC11C179A mutant was incubated with increasing concentrations of processed and activated PmC11.", + "section": "RESULTS", + "ner": [ + [ + 88, + 98, + "PmC11C179A", + "mutant" + ], + [ + 99, + 105, + "mutant", + "protein_state" + ], + [ + 110, + 150, + "incubated with increasing concentrations", + "experimental_method" + ], + [ + 154, + 163, + "processed", + "protein_state" + ], + [ + 168, + 177, + "activated", + "protein_state" + ], + [ + 178, + 183, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 84, + "sent": "These studies revealed that there was no apparent cleavage of PmC11C179A by the active enzyme at low concentrations of PmC11 and that only limited cleavage was observed when the ratio of active enzyme (PmC11:PmC11C179A) was increased to \u223c1:10 and 1:4, with complete cleavage observed at a ratio of 1:1 (Fig. 2E).", + "section": "RESULTS", + "ner": [ + [ + 62, + 72, + "PmC11C179A", + "mutant" + ], + [ + 80, + 86, + "active", + "protein_state" + ], + [ + 94, + 115, + "at low concentrations", + "experimental_method" + ], + [ + 119, + 124, + "PmC11", + "protein" + ], + [ + 187, + 193, + "active", + "protein_state" + ], + [ + 202, + 207, + "PmC11", + "protein" + ], + [ + 208, + 218, + "PmC11C179A", + "mutant" + ], + [ + 224, + 250, + "increased to \u223c1:10 and 1:4", + "experimental_method" + ], + [ + 289, + 301, + "ratio of 1:1", + "experimental_method" + ] + ] + }, + { + "sid": 85, + "sent": "This suggests that cleavage of PmC11C179A was most likely an effect of the increasing concentration of PmC11 and intermolecular cleavage.", + "section": "RESULTS", + "ner": [ + [ + 19, + 27, + "cleavage", + "ptm" + ], + [ + 31, + 41, + "PmC11C179A", + "mutant" + ], + [ + 103, + 108, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 86, + "sent": "Collectively, these data suggest that the pro-form of PmC11 is autoinhibited by a section of L5 blocking access to the active site, prior to intramolecular cleavage at Lys147.", + "section": "RESULTS", + "ner": [ + [ + 42, + 50, + "pro-form", + "protein_state" + ], + [ + 54, + 59, + "PmC11", + "protein" + ], + [ + 63, + 76, + "autoinhibited", + "protein_state" + ], + [ + 93, + 95, + "L5", + "structure_element" + ], + [ + 119, + 130, + "active site", + "site" + ], + [ + 141, + 164, + "intramolecular cleavage", + "ptm" + ], + [ + 168, + 174, + "Lys147", + "residue_name_number" + ] + ] + }, + { + "sid": 87, + "sent": "This cleavage subsequently allows movement of the region containing Lys147 and the active site to open up for substrate access.", + "section": "RESULTS", + "ner": [ + [ + 5, + 13, + "cleavage", + "ptm" + ], + [ + 68, + 74, + "Lys147", + "residue_name_number" + ], + [ + 83, + 94, + "active site", + "site" + ], + [ + 98, + 102, + "open", + "protein_state" + ] + ] + }, + { + "sid": 88, + "sent": "Substrate Specificity of PmC11", + "section": "RESULTS", + "ner": [ + [ + 25, + 30, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 89, + "sent": "The autocatalytic cleavage of PmC11 at Lys147 (sequence KLK\u2227A) demonstrates that the enzyme accepts substrates with Lys in the P1 position.", + "section": "RESULTS", + "ner": [ + [ + 4, + 26, + "autocatalytic cleavage", + "ptm" + ], + [ + 30, + 35, + "PmC11", + "protein" + ], + [ + 39, + 45, + "Lys147", + "residue_name_number" + ], + [ + 116, + 119, + "Lys", + "residue_name" + ], + [ + 127, + 129, + "P1", + "residue_number" + ] + ] + }, + { + "sid": 90, + "sent": "As expected, PmC11 showed no activity against substrates with Pro or Asp in P1 but was active toward substrates with a basic residue in P1 such as Bz-R-AMC, Z-GGR-AMC, and BOC-VLK-AMC.", + "section": "RESULTS", + "ner": [ + [ + 13, + 18, + "PmC11", + "protein" + ], + [ + 62, + 65, + "Pro", + "residue_name" + ], + [ + 69, + 72, + "Asp", + "residue_name" + ], + [ + 76, + 78, + "P1", + "residue_number" + ], + [ + 87, + 93, + "active", + "protein_state" + ], + [ + 136, + 138, + "P1", + "residue_number" + ], + [ + 147, + 155, + "Bz-R-AMC", + "chemical" + ], + [ + 157, + 166, + "Z-GGR-AMC", + "chemical" + ], + [ + 172, + 183, + "BOC-VLK-AMC", + "chemical" + ] + ] + }, + { + "sid": 91, + "sent": "The rate of cleavage was \u223c3-fold greater toward the single Arg substrate Bz-R-AMC than for the other two (Fig. 2F) and, unexpectedly, PmC11 showed no activity toward BOC-K-AMC.", + "section": "RESULTS", + "ner": [ + [ + 59, + 62, + "Arg", + "residue_name" + ], + [ + 73, + 81, + "Bz-R-AMC", + "chemical" + ], + [ + 134, + 139, + "PmC11", + "protein" + ], + [ + 166, + 175, + "BOC-K-AMC", + "chemical" + ] + ] + }, + { + "sid": 92, + "sent": "These results confirm that PmC11 accepts substrates containing Arg or Lys in P1 with a possible preference for Arg.", + "section": "RESULTS", + "ner": [ + [ + 27, + 32, + "PmC11", + "protein" + ], + [ + 63, + 66, + "Arg", + "residue_name" + ], + [ + 70, + 73, + "Lys", + "residue_name" + ], + [ + 77, + 79, + "P1", + "residue_number" + ], + [ + 111, + 114, + "Arg", + "residue_name" + ] + ] + }, + { + "sid": 93, + "sent": "The catalytic dyad of PmC11 sits near the bottom of an open pocket on the surface of the enzyme at a conserved location in the clan CD family.", + "section": "RESULTS", + "ner": [ + [ + 4, + 18, + "catalytic dyad", + "site" + ], + [ + 22, + 27, + "PmC11", + "protein" + ], + [ + 55, + 59, + "open", + "protein_state" + ], + [ + 60, + 66, + "pocket", + "site" + ], + [ + 101, + 119, + "conserved location", + "protein_state" + ], + [ + 132, + 141, + "CD family", + "protein_type" + ] + ] + }, + { + "sid": 94, + "sent": "The PmC11 structure reveals that the catalytic dyad forms part of a large acidic pocket (Fig. 2G), consistent with a binding site for a basic substrate.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "PmC11", + "protein" + ], + [ + 10, + 19, + "structure", + "evidence" + ], + [ + 37, + 51, + "catalytic dyad", + "site" + ], + [ + 74, + 87, + "acidic pocket", + "site" + ], + [ + 117, + 129, + "binding site", + "site" + ] + ] + }, + { + "sid": 95, + "sent": "This pocket is lined with the potential functional side chains of Asn50, Asp177, and Thr204 with Gly134, Asp207, and Met205 also contributing to the pocket (Fig. 2A).", + "section": "RESULTS", + "ner": [ + [ + 5, + 11, + "pocket", + "site" + ], + [ + 66, + 71, + "Asn50", + "residue_name_number" + ], + [ + 73, + 79, + "Asp177", + "residue_name_number" + ], + [ + 85, + 91, + "Thr204", + "residue_name_number" + ], + [ + 97, + 103, + "Gly134", + "residue_name_number" + ], + [ + 105, + 111, + "Asp207", + "residue_name_number" + ], + [ + 117, + 123, + "Met205", + "residue_name_number" + ], + [ + 149, + 155, + "pocket", + "site" + ] + ] + }, + { + "sid": 96, + "sent": "Interestingly, these residues are in regions that are structurally similar to those involved in the S1 binding pockets of other clan CD members (shown in Ref.).", + "section": "RESULTS", + "ner": [ + [ + 54, + 74, + "structurally similar", + "protein_state" + ], + [ + 100, + 118, + "S1 binding pockets", + "site" + ], + [ + 128, + 143, + "clan CD members", + "protein_type" + ] + ] + }, + { + "sid": 97, + "sent": "Because PmC11 recognizes basic substrates, the tetrapeptide inhibitor Z-VRPR-FMK was tested as an enzyme inhibitor and was found to inhibit both the autoprocessing and activity of PmC11 (Fig. 3A).", + "section": "RESULTS", + "ner": [ + [ + 8, + 13, + "PmC11", + "protein" + ], + [ + 70, + 80, + "Z-VRPR-FMK", + "chemical" + ], + [ + 132, + 139, + "inhibit", + "protein_state" + ], + [ + 149, + 163, + "autoprocessing", + "ptm" + ], + [ + 180, + 185, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 98, + "sent": "Z-VRPR-FMK was also shown to bind to the enzyme: a size-shift was observed, by SDS-PAGE analysis, in the larger processed product of PmC11 suggesting that the inhibitor bound to the active site (Fig. 3B).", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "Z-VRPR-FMK", + "chemical" + ], + [ + 51, + 61, + "size-shift", + "evidence" + ], + [ + 79, + 87, + "SDS-PAGE", + "experimental_method" + ], + [ + 133, + 138, + "PmC11", + "protein" + ], + [ + 159, + 174, + "inhibitor bound", + "protein_state" + ], + [ + 182, + 193, + "active site", + "site" + ] + ] + }, + { + "sid": 99, + "sent": "A structure overlay of PmC11 with the MALT1-paracacaspase (MALT1-P), in complex with Z-VRPR-FMK, revealed that the PmC11 dyad sits in a very similar position to that of active MALT1-P and that Asn50, Asp177, and Asp207 superimpose well with the principal MALT1-P inhibitor binding residues (Asp365, Asp462, and Glu500, respectively (VRPR-FMK from MALT1-P with the corresponding PmC11 residues from the structural overlay is shown in Fig. 1D), as described in Ref.).", + "section": "RESULTS", + "ner": [ + [ + 2, + 19, + "structure overlay", + "experimental_method" + ], + [ + 23, + 28, + "PmC11", + "protein" + ], + [ + 38, + 57, + "MALT1-paracacaspase", + "protein" + ], + [ + 59, + 66, + "MALT1-P", + "protein" + ], + [ + 72, + 79, + "complex", + "protein_state" + ], + [ + 85, + 95, + "Z-VRPR-FMK", + "chemical" + ], + [ + 115, + 120, + "PmC11", + "protein" + ], + [ + 121, + 125, + "dyad", + "site" + ], + [ + 169, + 175, + "active", + "protein_state" + ], + [ + 176, + 183, + "MALT1-P", + "protein" + ], + [ + 193, + 198, + "Asn50", + "residue_name_number" + ], + [ + 200, + 206, + "Asp177", + "residue_name_number" + ], + [ + 212, + 218, + "Asp207", + "residue_name_number" + ], + [ + 255, + 262, + "MALT1-P", + "protein" + ], + [ + 263, + 289, + "inhibitor binding residues", + "site" + ], + [ + 291, + 297, + "Asp365", + "residue_name_number" + ], + [ + 299, + 305, + "Asp462", + "residue_name_number" + ], + [ + 311, + 317, + "Glu500", + "residue_name_number" + ], + [ + 333, + 341, + "VRPR-FMK", + "chemical" + ], + [ + 347, + 354, + "MALT1-P", + "protein" + ], + [ + 378, + 383, + "PmC11", + "protein" + ], + [ + 402, + 420, + "structural overlay", + "experimental_method" + ] + ] + }, + { + "sid": 100, + "sent": "Asp177 is located near the catalytic cysteine and is conserved throughout the C11 family, suggesting it is the primary S1 binding site residue.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Asp177", + "residue_name_number" + ], + [ + 27, + 36, + "catalytic", + "protein_state" + ], + [ + 37, + 45, + "cysteine", + "residue_name" + ], + [ + 53, + 73, + "conserved throughout", + "protein_state" + ], + [ + 78, + 88, + "C11 family", + "protein_type" + ], + [ + 119, + 142, + "S1 binding site residue", + "site" + ] + ] + }, + { + "sid": 101, + "sent": "In the structure of PmC11, Asp207 resides on a flexible loop pointing away from the S1 binding pocket (Fig. 3C).", + "section": "RESULTS", + "ner": [ + [ + 7, + 16, + "structure", + "evidence" + ], + [ + 20, + 25, + "PmC11", + "protein" + ], + [ + 27, + 33, + "Asp207", + "residue_name_number" + ], + [ + 56, + 60, + "loop", + "structure_element" + ], + [ + 84, + 101, + "S1 binding pocket", + "site" + ] + ] + }, + { + "sid": 102, + "sent": "However, this loop has been shown to be important for substrate binding in clan CD and this residue could easily rotate and be involved in substrate binding in PmC11.", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "loop", + "structure_element" + ], + [ + 75, + 82, + "clan CD", + "protein_type" + ], + [ + 160, + 165, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 103, + "sent": "Thus, Asn50, Asp177, and Asp207 are most likely responsible for the substrate specificity of PmC11.", + "section": "RESULTS", + "ner": [ + [ + 6, + 11, + "Asn50", + "residue_name_number" + ], + [ + 13, + 19, + "Asp177", + "residue_name_number" + ], + [ + 25, + 31, + "Asp207", + "residue_name_number" + ], + [ + 93, + 98, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 104, + "sent": "Asp177 is highly conserved throughout the clan CD C11 peptidases and is thought to be primarily responsible for substrate specificity of the clan CD enzymes, as also illustrated from the proximity of these residues relative to the inhibitor Z-VRPR-FMK when PmC11 is overlaid on the MALT1-P structure (Fig. 3C).", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Asp177", + "residue_name_number" + ], + [ + 10, + 26, + "highly conserved", + "protein_state" + ], + [ + 42, + 64, + "clan CD C11 peptidases", + "protein_type" + ], + [ + 141, + 156, + "clan CD enzymes", + "protein_type" + ], + [ + 241, + 251, + "Z-VRPR-FMK", + "chemical" + ], + [ + 257, + 262, + "PmC11", + "protein" + ], + [ + 266, + 274, + "overlaid", + "experimental_method" + ], + [ + 282, + 289, + "MALT1-P", + "protein" + ], + [ + 290, + 299, + "structure", + "evidence" + ] + ] + }, + { + "sid": 105, + "sent": "PmC11 binds and is inhibited by Z-VRPR-FMK and does not require Ca2+ for activity.", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "PmC11", + "protein" + ], + [ + 32, + 42, + "Z-VRPR-FMK", + "chemical" + ], + [ + 64, + 68, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 106, + "sent": " A, PmC11 activity is inhibited by Z-VRPR-FMK.", + "section": "FIG", + "ner": [ + [ + 35, + 45, + "Z-VRPR-FMK", + "chemical" + ] + ] + }, + { + "sid": 107, + "sent": "Cleavage of Bz-R-AMC by PmC11 was measured in a fluorometric activity assay with (+, purple) and without (\u2212, red) Z-VRPR-FMK.", + "section": "FIG", + "ner": [ + [ + 12, + 20, + "Bz-R-AMC", + "chemical" + ], + [ + 24, + 29, + "PmC11", + "protein" + ], + [ + 48, + 75, + "fluorometric activity assay", + "experimental_method" + ], + [ + 114, + 124, + "Z-VRPR-FMK", + "chemical" + ] + ] + }, + { + "sid": 108, + "sent": "B, gel-shift assay reveals that Z-VRPR-FMK binds to PmC11.", + "section": "FIG", + "ner": [ + [ + 3, + 18, + "gel-shift assay", + "experimental_method" + ], + [ + 32, + 42, + "Z-VRPR-FMK", + "chemical" + ], + [ + 52, + 57, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 109, + "sent": "PmC11 was incubated with (+) or without (\u2212) Z-VRPR-FMK and the samples analyzed on a 10% SDS-PAGE gel.", + "section": "FIG", + "ner": [ + [ + 10, + 19, + "incubated", + "experimental_method" + ], + [ + 44, + 54, + "Z-VRPR-FMK", + "chemical" + ], + [ + 89, + 97, + "SDS-PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 110, + "sent": "A size shift can be observed in the larger processed product of PmC11 (26.1 kDa).", + "section": "FIG", + "ner": [ + [ + 2, + 12, + "size shift", + "evidence" + ], + [ + 64, + 69, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 111, + "sent": "C, PmC11 with the Z-VRPR-FMK from the MALT1-paracacaspase (MALT1-P) superimposed.", + "section": "FIG", + "ner": [ + [ + 3, + 8, + "PmC11", + "protein" + ], + [ + 18, + 28, + "Z-VRPR-FMK", + "chemical" + ], + [ + 38, + 57, + "MALT1-paracacaspase", + "protein" + ], + [ + 59, + 66, + "MALT1-P", + "protein" + ], + [ + 68, + 80, + "superimposed", + "experimental_method" + ] + ] + }, + { + "sid": 112, + "sent": "A three-dimensional structural overlay of Z-VRPR-FMK from the MALT1-P complex onto PmC11.", + "section": "FIG", + "ner": [ + [ + 2, + 38, + "three-dimensional structural overlay", + "experimental_method" + ], + [ + 42, + 52, + "Z-VRPR-FMK", + "chemical" + ], + [ + 62, + 69, + "MALT1-P", + "protein" + ], + [ + 83, + 88, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 113, + "sent": "The position and orientation of Z-VRPR-FMK was taken from superposition of the PmC11 and MALTI_P structures and indicates the presumed active site of PmC11.", + "section": "FIG", + "ner": [ + [ + 32, + 42, + "Z-VRPR-FMK", + "chemical" + ], + [ + 58, + 71, + "superposition", + "experimental_method" + ], + [ + 79, + 84, + "PmC11", + "protein" + ], + [ + 89, + 96, + "MALTI_P", + "protein" + ], + [ + 97, + 107, + "structures", + "evidence" + ], + [ + 135, + 146, + "active site", + "site" + ], + [ + 150, + 155, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 114, + "sent": "Residues surrounding the inhibitor are labeled and represent potentially important binding site residues, labeled in black and shown in an atomic representation.", + "section": "FIG", + "ner": [ + [ + 83, + 104, + "binding site residues", + "site" + ] + ] + }, + { + "sid": 115, + "sent": "C, divalent cations do not increase the activity of PmC11.", + "section": "FIG", + "ner": [ + [ + 52, + 57, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 116, + "sent": "The cleavage of Bz-R-AMC by PmC11 was measured in the presence of the cations Ca2+, Mn2+, Zn2+, Co2+, Cu2+, Mg2+, and Fe3+ with EGTA as a negative control, and relative fluorescence measured against time (min).", + "section": "FIG", + "ner": [ + [ + 16, + 24, + "Bz-R-AMC", + "chemical" + ], + [ + 28, + 33, + "PmC11", + "protein" + ], + [ + 78, + 82, + "Ca2+", + "chemical" + ], + [ + 84, + 88, + "Mn2+", + "chemical" + ], + [ + 90, + 94, + "Zn2+", + "chemical" + ], + [ + 96, + 100, + "Co2+", + "chemical" + ], + [ + 102, + 106, + "Cu2+", + "chemical" + ], + [ + 108, + 112, + "Mg2+", + "chemical" + ], + [ + 118, + 122, + "Fe3+", + "chemical" + ], + [ + 128, + 132, + "EGTA", + "chemical" + ], + [ + 160, + 203, + "relative fluorescence measured against time", + "experimental_method" + ] + ] + }, + { + "sid": 117, + "sent": "The addition of cations produced no improvement in activity of PmC11 when compared in the presence of EGTA, suggesting that PmC11 does not require metal ions for proteolytic activity.", + "section": "FIG", + "ner": [ + [ + 4, + 23, + "addition of cations", + "experimental_method" + ], + [ + 63, + 68, + "PmC11", + "protein" + ], + [ + 102, + 106, + "EGTA", + "chemical" + ], + [ + 124, + 129, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 118, + "sent": "Furthermore, Cu2+, Fe2+, and Zn2+ appear to inhibit PmC11.", + "section": "FIG", + "ner": [ + [ + 13, + 17, + "Cu2+", + "chemical" + ], + [ + 19, + 23, + "Fe2+", + "chemical" + ], + [ + 29, + 33, + "Zn2+", + "chemical" + ], + [ + 44, + 51, + "inhibit", + "protein_state" + ], + [ + 52, + 57, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 119, + "sent": "Comparison with Clostripain", + "section": "RESULTS", + "ner": [ + [ + 16, + 27, + "Clostripain", + "protein" + ] + ] + }, + { + "sid": 120, + "sent": "Clostripain from C. histolyticum is the founding member of the C11 family of peptidases and contains an additional 149 residues compared with PmC11.", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Clostripain", + "protein" + ], + [ + 17, + 32, + "C. histolyticum", + "species" + ], + [ + 63, + 73, + "C11 family", + "protein_type" + ], + [ + 77, + 87, + "peptidases", + "protein_type" + ], + [ + 115, + 127, + "149 residues", + "residue_range" + ], + [ + 142, + 147, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 121, + "sent": "A multiple sequence alignment revealed that most of the secondary structural elements are conserved between the two enzymes, although they are only \u223c23% identical (Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 2, + 29, + "multiple sequence alignment", + "experimental_method" + ], + [ + 56, + 85, + "secondary structural elements", + "structure_element" + ], + [ + 90, + 99, + "conserved", + "protein_state" + ] + ] + }, + { + "sid": 122, + "sent": "Nevertheless, PmC11 may be a good model for the core structure of clostripain.", + "section": "RESULTS", + "ner": [ + [ + 14, + 19, + "PmC11", + "protein" + ], + [ + 66, + 77, + "clostripain", + "protein" + ] + ] + }, + { + "sid": 123, + "sent": "The primary structural alignment also shows that the catalytic dyad in PmC11 is structurally conserved in clostripain (Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 4, + 32, + "primary structural alignment", + "experimental_method" + ], + [ + 53, + 67, + "catalytic dyad", + "site" + ], + [ + 71, + 76, + "PmC11", + "protein" + ], + [ + 80, + 102, + "structurally conserved", + "protein_state" + ], + [ + 106, + 117, + "clostripain", + "protein" + ] + ] + }, + { + "sid": 124, + "sent": "Unlike PmC11, clostripain has two cleavage sites (Arg181 and Arg190), which results in the removal of a nonapeptide, and is required for full activation of the enzyme (highlighted in Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 7, + 12, + "PmC11", + "protein" + ], + [ + 14, + 25, + "clostripain", + "protein" + ], + [ + 34, + 48, + "cleavage sites", + "site" + ], + [ + 50, + 56, + "Arg181", + "residue_name_number" + ], + [ + 61, + 67, + "Arg190", + "residue_name_number" + ], + [ + 104, + 115, + "nonapeptide", + "structure_element" + ], + [ + 137, + 152, + "full activation", + "protein_state" + ] + ] + }, + { + "sid": 125, + "sent": "Interestingly, Arg190 was found to align with Lys147 in PmC11.", + "section": "RESULTS", + "ner": [ + [ + 15, + 21, + "Arg190", + "residue_name_number" + ], + [ + 46, + 52, + "Lys147", + "residue_name_number" + ], + [ + 56, + 61, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 126, + "sent": "In addition, the predicted primary S1-binding residue in PmC11 Asp177 also overlays with the residue predicted to be the P1 specificity determining residue in clostripain (Asp229, Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 35, + 53, + "S1-binding residue", + "site" + ], + [ + 57, + 62, + "PmC11", + "protein" + ], + [ + 63, + 69, + "Asp177", + "residue_name_number" + ], + [ + 75, + 83, + "overlays", + "experimental_method" + ], + [ + 121, + 155, + "P1 specificity determining residue", + "site" + ], + [ + 159, + 170, + "clostripain", + "protein" + ], + [ + 172, + 178, + "Asp229", + "residue_name_number" + ] + ] + }, + { + "sid": 127, + "sent": "As studies on clostripain revealed addition of Ca2+ ions are required for full activation, the Ca2+ dependence of PmC11 was examined.", + "section": "RESULTS", + "ner": [ + [ + 14, + 25, + "clostripain", + "protein" + ], + [ + 47, + 51, + "Ca2+", + "chemical" + ], + [ + 74, + 89, + "full activation", + "protein_state" + ], + [ + 95, + 99, + "Ca2+", + "chemical" + ], + [ + 114, + 119, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 128, + "sent": "Surprisingly, Ca2+ did not enhance PmC11 activity and, furthermore, other divalent cations, Mg2+, Mn2+, Co2+, Fe2+, Zn2+, and Cu2+, were not necessary for PmC11 activity (Fig. 3D).", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "Ca2+", + "chemical" + ], + [ + 35, + 40, + "PmC11", + "protein" + ], + [ + 92, + 96, + "Mg2+", + "chemical" + ], + [ + 98, + 102, + "Mn2+", + "chemical" + ], + [ + 104, + 108, + "Co2+", + "chemical" + ], + [ + 110, + 114, + "Fe2+", + "chemical" + ], + [ + 116, + 120, + "Zn2+", + "chemical" + ], + [ + 126, + 130, + "Cu2+", + "chemical" + ], + [ + 155, + 160, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 129, + "sent": "In support of these findings, EGTA did not inhibit PmC11 suggesting that, unlike clostripain, PmC11 does not require Ca2+ or other divalent cations, for activity.", + "section": "RESULTS", + "ner": [ + [ + 30, + 34, + "EGTA", + "chemical" + ], + [ + 51, + 56, + "PmC11", + "protein" + ], + [ + 81, + 92, + "clostripain", + "protein" + ], + [ + 94, + 99, + "PmC11", + "protein" + ], + [ + 117, + 121, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 130, + "sent": "The crystal structure of PmC11 now provides three-dimensional information for a member of the clostripain C11 family of cysteine peptidases.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 25, + 30, + "PmC11", + "protein" + ], + [ + 94, + 105, + "clostripain", + "protein" + ], + [ + 106, + 116, + "C11 family", + "protein_type" + ], + [ + 120, + 139, + "cysteine peptidases", + "protein_type" + ] + ] + }, + { + "sid": 131, + "sent": "The enzyme exhibits all of the key structural elements of clan CD members, but is unusual in that it has a nine-stranded central \u03b2-sheet with a novel C-terminal domain.", + "section": "DISCUSS", + "ner": [ + [ + 58, + 73, + "clan CD members", + "protein_type" + ], + [ + 129, + 136, + "\u03b2-sheet", + "structure_element" + ], + [ + 150, + 167, + "C-terminal domain", + "structure_element" + ] + ] + }, + { + "sid": 132, + "sent": "The structural similarity of PmC11 with its nearest structural neighbors in the PDB is decidedly low, overlaying better with six-stranded caspase-7 than any of the other larger members of the clan (Table 2).", + "section": "DISCUSS", + "ner": [ + [ + 29, + 34, + "PmC11", + "protein" + ], + [ + 138, + 147, + "caspase-7", + "protein" + ] + ] + }, + { + "sid": 133, + "sent": "The substrate specificity of PmC11 is Arg/Lys and the crystal structure revealed an acidic pocket for specific binding of such basic substrates.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 34, + "PmC11", + "protein" + ], + [ + 38, + 41, + "Arg", + "residue_name" + ], + [ + 42, + 45, + "Lys", + "residue_name" + ], + [ + 54, + 71, + "crystal structure", + "evidence" + ], + [ + 84, + 97, + "acidic pocket", + "site" + ] + ] + }, + { + "sid": 134, + "sent": "In addition, the structure suggested a mechanism of self-inhibition in both PmC11 and clostripain and an activation mechanism that requires autoprocessing.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 26, + "structure", + "evidence" + ], + [ + 76, + 81, + "PmC11", + "protein" + ], + [ + 86, + 97, + "clostripain", + "protein" + ], + [ + 140, + 154, + "autoprocessing", + "ptm" + ] + ] + }, + { + "sid": 135, + "sent": "PmC11 differs from clostripain in that is does not appear to require divalent cations for activation.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "PmC11", + "protein" + ], + [ + 19, + 30, + "clostripain", + "protein" + ] + ] + }, + { + "sid": 136, + "sent": "Several other members of clan CD require processing for full activation including legumain, gingipain-R, MARTX-CPD, and the effector caspases, e.g. caspase-7.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 32, + "clan CD", + "protein_type" + ], + [ + 41, + 51, + "processing", + "ptm" + ], + [ + 56, + 71, + "full activation", + "protein_state" + ], + [ + 82, + 90, + "legumain", + "protein" + ], + [ + 92, + 103, + "gingipain-R", + "protein" + ], + [ + 105, + 114, + "MARTX-CPD", + "protein" + ], + [ + 124, + 141, + "effector caspases", + "protein_type" + ], + [ + 148, + 157, + "caspase-7", + "protein" + ] + ] + }, + { + "sid": 137, + "sent": "To date, the effector caspases are the only group of enzymes that require cleavage of a loop within the central \u03b2-sheet.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 30, + "effector caspases", + "protein_type" + ], + [ + 74, + 82, + "cleavage", + "ptm" + ], + [ + 88, + 92, + "loop", + "structure_element" + ], + [ + 112, + 119, + "\u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 138, + "sent": "This is also the case in PmC11, although the cleavage loop is structurally different to that found in the caspases and follows the catalytic His (Fig. 1A), as opposed to the Cys in the caspases.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 30, + "PmC11", + "protein" + ], + [ + 45, + 53, + "cleavage", + "ptm" + ], + [ + 54, + 58, + "loop", + "structure_element" + ], + [ + 106, + 114, + "caspases", + "protein_type" + ], + [ + 131, + 140, + "catalytic", + "protein_state" + ], + [ + 141, + 144, + "His", + "residue_name" + ], + [ + 174, + 177, + "Cys", + "residue_name" + ], + [ + 185, + 193, + "caspases", + "protein_type" + ] + ] + }, + { + "sid": 139, + "sent": "All other clan CD members requiring cleavage for full activation do so at sites external to their central sheets.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 25, + "clan CD members", + "protein_type" + ], + [ + 36, + 44, + "cleavage", + "ptm" + ], + [ + 49, + 64, + "full activation", + "protein_state" + ], + [ + 74, + 79, + "sites", + "site" + ], + [ + 106, + 112, + "sheets", + "structure_element" + ] + ] + }, + { + "sid": 140, + "sent": "The caspases and gingipain-R both undergo intermolecular (trans) cleavage and legumain and MARTX-CPD are reported to perform intramolecular (cis) cleavage.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 12, + "caspases", + "protein_type" + ], + [ + 17, + 28, + "gingipain-R", + "protein" + ], + [ + 42, + 73, + "intermolecular (trans) cleavage", + "ptm" + ], + [ + 78, + 86, + "legumain", + "protein" + ], + [ + 91, + 100, + "MARTX-CPD", + "protein" + ], + [ + 125, + 154, + "intramolecular (cis) cleavage", + "ptm" + ] + ] + }, + { + "sid": 141, + "sent": "In addition, several members of clan CD exhibit self-inhibition, whereby regions of the enzyme block access to the active site.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 39, + "clan CD", + "protein_type" + ], + [ + 73, + 80, + "regions", + "structure_element" + ], + [ + 115, + 126, + "active site", + "site" + ] + ] + }, + { + "sid": 142, + "sent": "Like PmC11, these structures show preformed catalytic machinery and, for a substrate to gain access, movement and/or cleavage of the blocking region is required.", + "section": "DISCUSS", + "ner": [ + [ + 5, + 10, + "PmC11", + "protein" + ], + [ + 117, + 125, + "cleavage", + "ptm" + ], + [ + 133, + 148, + "blocking region", + "structure_element" + ] + ] + }, + { + "sid": 143, + "sent": "The structure of PmC11 gives the first insight into this class of relatively unexplored family of proteins and should allow important catalytic and substrate binding residues to be identified in a variety of orthologues.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 17, + 22, + "PmC11", + "protein" + ] + ] + }, + { + "sid": 144, + "sent": "Indeed, insights gained from an analysis of the PmC11 structure revealed the identity of the Trypanosoma brucei PNT1 protein as a C11 cysteine peptidase with an essential role in organelle replication.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 53, + "PmC11", + "protein" + ], + [ + 54, + 63, + "structure", + "evidence" + ], + [ + 93, + 111, + "Trypanosoma brucei", + "species" + ], + [ + 112, + 116, + "PNT1", + "protein" + ], + [ + 130, + 152, + "C11 cysteine peptidase", + "protein_type" + ] + ] + }, + { + "sid": 145, + "sent": "The PmC11 structure should provide a good basis for structural modeling and, given the importance of other clan CD enzymes, this work should also advance the exploration of these peptidases and potentially identify new biologically important substrates.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 9, + "PmC11", + "protein" + ], + [ + 10, + 19, + "structure", + "evidence" + ], + [ + 52, + 71, + "structural modeling", + "experimental_method" + ], + [ + 107, + 122, + "clan CD enzymes", + "protein_type" + ], + [ + 179, + 189, + "peptidases", + "protein_type" + ] + ] + } + ] + }, + "PMC4872110": { + "annotations": [ + { + "sid": 0, + "sent": "Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans", + "section": "TITLE", + "ner": [ + [ + 0, + 26, + "Ribosome biogenesis factor", + "protein_type" + ], + [ + 27, + 31, + "Tsr3", + "protein" + ], + [ + 39, + 69, + "aminocarboxypropyl transferase", + "protein_type" + ], + [ + 86, + 94, + "18S rRNA", + "chemical" + ], + [ + 116, + 121, + "yeast", + "taxonomy_domain" + ], + [ + 126, + 132, + "humans", + "species" + ] + ] + }, + { + "sid": 1, + "sent": "The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3\u03a8) located next to the P-site tRNA on the small subunit 18S rRNA.", + "section": "ABSTRACT", + "ner": [ + [ + 44, + 54, + "eukaryotic", + "taxonomy_domain" + ], + [ + 55, + 59, + "rRNA", + "chemical" + ], + [ + 67, + 76, + "conserved", + "protein_state" + ], + [ + 77, + 90, + "hypermodified", + "protein_state" + ], + [ + 91, + 101, + "nucleotide", + "chemical" + ], + [ + 102, + 147, + "N1-methyl-N3-aminocarboxypropyl-pseudouridine", + "chemical" + ], + [ + 149, + 156, + "m1acp3\u03a8", + "chemical" + ], + [ + 178, + 184, + "P-site", + "site" + ], + [ + 185, + 189, + "tRNA", + "chemical" + ], + [ + 211, + 219, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 2, + "sent": "While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive.", + "section": "ABSTRACT", + "ner": [ + [ + 6, + 26, + "S-adenosylmethionine", + "chemical" + ], + [ + 63, + 81, + "aminocarboxypropyl", + "chemical" + ], + [ + 83, + 86, + "acp", + "chemical" + ], + [ + 143, + 146, + "acp", + "chemical" + ] + ] + }, + { + "sid": 3, + "sent": "Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells.", + "section": "ABSTRACT", + "ner": [ + [ + 61, + 65, + "Tsr3", + "protein" + ], + [ + 95, + 100, + "yeast", + "taxonomy_domain" + ], + [ + 105, + 110, + "human", + "species" + ] + ] + }, + { + "sid": 4, + "sent": "In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation.", + "section": "ABSTRACT", + "ner": [ + [ + 25, + 29, + "Tsr3", + "protein" + ], + [ + 30, + 37, + "mutants", + "protein_state" + ], + [ + 58, + 61, + "acp", + "chemical" + ], + [ + 110, + 122, + "20S pre-rRNA", + "chemical" + ] + ] + }, + { + "sid": 5, + "sent": "The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 25, + 33, + "archaeal", + "taxonomy_domain" + ], + [ + 34, + 38, + "Tsr3", + "protein" + ], + [ + 77, + 111, + "SPOUT-class RNA-methyltransferases", + "protein_type" + ], + [ + 127, + 143, + "SAM binding mode", + "site" + ] + ] + }, + { + "sid": 6, + "sent": "This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate.", + "section": "ABSTRACT", + "ner": [ + [ + 12, + 28, + "SAM binding mode", + "site" + ], + [ + 42, + 46, + "Tsr3", + "protein" + ], + [ + 61, + 64, + "acp", + "chemical" + ], + [ + 93, + 96, + "SAM", + "chemical" + ] + ] + }, + { + "sid": 7, + "sent": "Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes.", + "section": "ABSTRACT", + "ner": [ + [ + 14, + 18, + "Tsr3", + "protein" + ], + [ + 57, + 72, + "acp transferase", + "protein_type" + ] + ] + }, + { + "sid": 8, + "sent": "Eukaryotic ribosome biogenesis is highly complex and requires a large number of non-ribosomal proteins and small non-coding RNAs in addition to ribosomal RNAs (rRNAs) and proteins.", + "section": "INTRO", + "ner": [ + [ + 0, + 10, + "Eukaryotic", + "taxonomy_domain" + ], + [ + 107, + 128, + "small non-coding RNAs", + "chemical" + ], + [ + 144, + 158, + "ribosomal RNAs", + "chemical" + ], + [ + 160, + 165, + "rRNAs", + "chemical" + ] + ] + }, + { + "sid": 9, + "sent": "During eukaryotic ribosome biogenesis several dozens of rRNA nucleotides become chemically modified.", + "section": "INTRO", + "ner": [ + [ + 7, + 17, + "eukaryotic", + "taxonomy_domain" + ], + [ + 56, + 60, + "rRNA", + "chemical" + ], + [ + 61, + 72, + "nucleotides", + "chemical" + ] + ] + }, + { + "sid": 10, + "sent": "The most abundant rRNA modifications are methylations at the 2\u2032-OH ribose moieties and isomerizations of uridine residues to pseudouridine, catalyzed by small nucleolar ribonucleoprotein particles (snoRNPs).", + "section": "INTRO", + "ner": [ + [ + 18, + 22, + "rRNA", + "chemical" + ], + [ + 41, + 53, + "methylations", + "ptm" + ], + [ + 67, + 73, + "ribose", + "chemical" + ], + [ + 105, + 112, + "uridine", + "chemical" + ], + [ + 125, + 138, + "pseudouridine", + "chemical" + ], + [ + 153, + 196, + "small nucleolar ribonucleoprotein particles", + "complex_assembly" + ], + [ + 198, + 205, + "snoRNPs", + "complex_assembly" + ] + ] + }, + { + "sid": 11, + "sent": "In addition, 18S and 25S (yeast)/ 28S (humans) rRNAs contain several base modifications catalyzed by site-specific and snoRNA-independent enzymes.", + "section": "INTRO", + "ner": [ + [ + 13, + 16, + "18S", + "chemical" + ], + [ + 21, + 24, + "25S", + "chemical" + ], + [ + 26, + 31, + "yeast", + "taxonomy_domain" + ], + [ + 34, + 37, + "28S", + "chemical" + ], + [ + 39, + 45, + "humans", + "species" + ], + [ + 47, + 52, + "rRNAs", + "chemical" + ], + [ + 119, + 125, + "snoRNA", + "chemical" + ] + ] + }, + { + "sid": 12, + "sent": "In Saccharomyces cerevisiae 18S rRNA contains four base methylations, two acetylations and a single 3-amino-3-carboxypropyl (acp) modification, whereas six base methylations are present in the 25S rRNA.", + "section": "INTRO", + "ner": [ + [ + 3, + 27, + "Saccharomyces cerevisiae", + "species" + ], + [ + 28, + 36, + "18S rRNA", + "chemical" + ], + [ + 56, + 68, + "methylations", + "ptm" + ], + [ + 74, + 86, + "acetylations", + "ptm" + ], + [ + 100, + 123, + "3-amino-3-carboxypropyl", + "chemical" + ], + [ + 125, + 128, + "acp", + "chemical" + ], + [ + 161, + 173, + "methylations", + "ptm" + ], + [ + 193, + 201, + "25S rRNA", + "chemical" + ] + ] + }, + { + "sid": 13, + "sent": "While in humans the 18S rRNA base modifications are highly conserved, only three of the yeast base modifications catalyzed by ScRrp8/HsNML, ScRcm1/HsNSUN5 and ScNop2/HsNSUN1 are preserved in the corresponding human 28S rRNA.", + "section": "INTRO", + "ner": [ + [ + 9, + 15, + "humans", + "species" + ], + [ + 20, + 28, + "18S rRNA", + "chemical" + ], + [ + 52, + 68, + "highly conserved", + "protein_state" + ], + [ + 88, + 93, + "yeast", + "taxonomy_domain" + ], + [ + 126, + 132, + "ScRrp8", + "protein" + ], + [ + 133, + 138, + "HsNML", + "protein" + ], + [ + 140, + 146, + "ScRcm1", + "protein" + ], + [ + 147, + 154, + "HsNSUN5", + "protein" + ], + [ + 159, + 165, + "ScNop2", + "protein" + ], + [ + 166, + 173, + "HsNSUN1", + "protein" + ], + [ + 209, + 214, + "human", + "species" + ], + [ + 215, + 223, + "28S rRNA", + "chemical" + ] + ] + }, + { + "sid": 14, + "sent": "Ribosomal RNA modifications have been suggested to optimize ribosome function, although in most cases this remains to be clearly established.", + "section": "INTRO", + "ner": [ + [ + 0, + 13, + "Ribosomal RNA", + "chemical" + ] + ] + }, + { + "sid": 15, + "sent": "They might contribute to increased RNA stability by providing additional hydrogen bonds (pseudouridines), improved base stacking (pseudouridines and base methylations) or an increased resistance against hydrolysis (ribose methylations).", + "section": "INTRO", + "ner": [ + [ + 35, + 38, + "RNA", + "chemical" + ], + [ + 73, + 87, + "hydrogen bonds", + "bond_interaction" + ], + [ + 89, + 103, + "pseudouridines", + "chemical" + ], + [ + 115, + 128, + "base stacking", + "bond_interaction" + ], + [ + 130, + 144, + "pseudouridines", + "chemical" + ], + [ + 149, + 166, + "base methylations", + "ptm" + ], + [ + 215, + 234, + "ribose methylations", + "ptm" + ] + ] + }, + { + "sid": 16, + "sent": "Most modified rRNA nucleotides cluster in the vicinity of the decoding or the peptidyl transferase center, suggesting an influence on ribosome functionality and stability.", + "section": "INTRO", + "ner": [ + [ + 14, + 18, + "rRNA", + "chemical" + ], + [ + 19, + 30, + "nucleotides", + "chemical" + ], + [ + 62, + 70, + "decoding", + "site" + ], + [ + 78, + 105, + "peptidyl transferase center", + "site" + ] + ] + }, + { + "sid": 17, + "sent": "Defects of rRNA modification enzymes often lead to disturbed ribosome biogenesis or functionally impaired ribosomes, although the lack of individual rRNA modifications often has no or only a slight influence on the cell.", + "section": "INTRO", + "ner": [ + [ + 11, + 15, + "rRNA", + "chemical" + ], + [ + 149, + 153, + "rRNA", + "chemical" + ] + ] + }, + { + "sid": 18, + "sent": "The chemically most complex modification is located in the loop capping helix 31 of 18S rRNA (Supplementary Figure S1B).", + "section": "INTRO", + "ner": [ + [ + 59, + 80, + "loop capping helix 31", + "structure_element" + ], + [ + 84, + 92, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 19, + "sent": "There a uridine (U1191 in yeast) is modified to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3\u03a8, Figure 1A).", + "section": "INTRO", + "ner": [ + [ + 8, + 15, + "uridine", + "residue_name" + ], + [ + 17, + 22, + "U1191", + "residue_name_number" + ], + [ + 26, + 31, + "yeast", + "taxonomy_domain" + ], + [ + 48, + 98, + "1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine", + "chemical" + ], + [ + 100, + 107, + "m1acp3\u03a8", + "chemical" + ] + ] + }, + { + "sid": 20, + "sent": "This base modification was first described in 1968 for hamster cells and is conserved in eukaryotes.", + "section": "INTRO", + "ner": [ + [ + 55, + 62, + "hamster", + "taxonomy_domain" + ], + [ + 76, + 88, + "conserved in", + "protein_state" + ], + [ + 89, + 99, + "eukaryotes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 21, + "sent": "This hypermodified nucleotide, which is located at the P-site tRNA, is synthesized in three steps beginning with the snR35 H/ACA snoRNP guided conversion of uridine into pseudouridine.", + "section": "INTRO", + "ner": [ + [ + 5, + 18, + "hypermodified", + "protein_state" + ], + [ + 19, + 29, + "nucleotide", + "chemical" + ], + [ + 55, + 61, + "P-site", + "site" + ], + [ + 62, + 66, + "tRNA", + "chemical" + ], + [ + 117, + 122, + "snR35", + "chemical" + ], + [ + 123, + 128, + "H/ACA", + "structure_element" + ], + [ + 129, + 135, + "snoRNP", + "complex_assembly" + ], + [ + 157, + 164, + "uridine", + "chemical" + ], + [ + 170, + 183, + "pseudouridine", + "chemical" + ] + ] + }, + { + "sid": 22, + "sent": "In a second step, the essential SPOUT-class methyltransferase Nep1/Emg1 modifies the pseudouridine to N1-methylpseudouridine.", + "section": "INTRO", + "ner": [ + [ + 32, + 61, + "SPOUT-class methyltransferase", + "protein_type" + ], + [ + 62, + 66, + "Nep1", + "protein" + ], + [ + 67, + 71, + "Emg1", + "protein" + ], + [ + 85, + 98, + "pseudouridine", + "chemical" + ], + [ + 102, + 124, + "N1-methylpseudouridine", + "chemical" + ] + ] + }, + { + "sid": 23, + "sent": "Methylation can only occur once pseudouridylation has taken place, as the latter reaction generates the substrate for the former.", + "section": "INTRO", + "ner": [ + [ + 0, + 11, + "Methylation", + "ptm" + ], + [ + 32, + 49, + "pseudouridylation", + "ptm" + ] + ] + }, + { + "sid": 24, + "sent": "The final acp modification leading to N1-methyl-N3-aminocarboxypropyl-pseudouridine occurs late during 40S biogenesis in the cytoplasm, while the two former reactions are taking place in the nucleolus and nucleus, and is independent from pseudouridylation or methylation.", + "section": "INTRO", + "ner": [ + [ + 10, + 13, + "acp", + "chemical" + ], + [ + 38, + 83, + "N1-methyl-N3-aminocarboxypropyl-pseudouridine", + "chemical" + ], + [ + 103, + 106, + "40S", + "complex_assembly" + ], + [ + 238, + 255, + "pseudouridylation", + "ptm" + ] + ] + }, + { + "sid": 25, + "sent": "Both the methyl and the acp group are derived from S-adenosylmethionine (SAM), but the enzyme responsible for acp modification remained elusive for more than 40 years.", + "section": "INTRO", + "ner": [ + [ + 51, + 71, + "S-adenosylmethionine", + "chemical" + ], + [ + 73, + 76, + "SAM", + "chemical" + ], + [ + 110, + 113, + "acp", + "chemical" + ] + ] + }, + { + "sid": 26, + "sent": "Tsr3 is necessary for acp modification of 18S rRNA in yeast and human. (A) Hypermodified nucleotide m1acp3\u03a8 is synthesized in three steps: pseudouridylation catalyzed by snoRNP35, N1-methylation catalyzed by methyltransferase Nep1 and N3-acp modification catalyzed by Tsr3.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "Tsr3", + "protein" + ], + [ + 22, + 25, + "acp", + "chemical" + ], + [ + 42, + 50, + "18S rRNA", + "chemical" + ], + [ + 54, + 59, + "yeast", + "taxonomy_domain" + ], + [ + 64, + 69, + "human", + "species" + ], + [ + 75, + 88, + "Hypermodified", + "protein_state" + ], + [ + 89, + 99, + "nucleotide", + "chemical" + ], + [ + 100, + 107, + "m1acp3\u03a8", + "chemical" + ], + [ + 139, + 156, + "pseudouridylation", + "ptm" + ], + [ + 170, + 178, + "snoRNP35", + "complex_assembly" + ], + [ + 180, + 194, + "N1-methylation", + "ptm" + ], + [ + 208, + 225, + "methyltransferase", + "protein_type" + ], + [ + 226, + 230, + "Nep1", + "protein" + ], + [ + 238, + 241, + "acp", + "chemical" + ], + [ + 268, + 272, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 27, + "sent": "The asterisk indicates the C1-atom labeled in the 14C-incorporation assay.", + "section": "FIG", + "ner": [ + [ + 50, + 73, + "14C-incorporation assay", + "experimental_method" + ] + ] + }, + { + "sid": 28, + "sent": "(B) RP-HPLC elution profile of yeast 18S rRNA nucleosides.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "RP-HPLC", + "experimental_method" + ], + [ + 12, + 27, + "elution profile", + "evidence" + ], + [ + 31, + 36, + "yeast", + "taxonomy_domain" + ], + [ + 37, + 45, + "18S rRNA", + "chemical" + ], + [ + 46, + 57, + "nucleosides", + "chemical" + ] + ] + }, + { + "sid": 29, + "sent": "Hypermodified m1acp3\u03a8 elutes at 7.4 min (wild type, left profile) and is missing in \u0394tsr3 (middle profile) and \u0394nep1 \u0394nop6 mutants (right profile).", + "section": "FIG", + "ner": [ + [ + 0, + 13, + "Hypermodified", + "protein_state" + ], + [ + 14, + 21, + "m1acp3\u03a8", + "chemical" + ], + [ + 41, + 50, + "wild type", + "protein_state" + ], + [ + 84, + 89, + "\u0394tsr3", + "mutant" + ], + [ + 111, + 122, + "\u0394nep1 \u0394nop6", + "mutant" + ] + ] + }, + { + "sid": 30, + "sent": "(C) 14C-acp labeling of 18S rRNAs.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "14C-acp", + "chemical" + ], + [ + 24, + 33, + "18S rRNAs", + "chemical" + ] + ] + }, + { + "sid": 31, + "sent": "Wild type (WT) and plasmid encoded 18S rRNA (U1191U) show the 14C-acp signal, whereas the 14C-acp signal is missing in the\u00a0U1191A mutant plasmid encoded 18S rRNA (U1191A) and \u0394tsr3 mutants (\u0394tsr3).", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Wild type", + "protein_state" + ], + [ + 11, + 13, + "WT", + "protein_state" + ], + [ + 35, + 43, + "18S rRNA", + "chemical" + ], + [ + 45, + 51, + "U1191U", + "mutant" + ], + [ + 62, + 69, + "14C-acp", + "chemical" + ], + [ + 90, + 97, + "14C-acp", + "chemical" + ], + [ + 123, + 129, + "U1191A", + "mutant" + ], + [ + 130, + 136, + "mutant", + "protein_state" + ], + [ + 153, + 161, + "18S rRNA", + "chemical" + ], + [ + 163, + 169, + "U1191A", + "mutant" + ], + [ + 175, + 180, + "\u0394tsr3", + "mutant" + ], + [ + 190, + 195, + "\u0394tsr3", + "mutant" + ] + ] + }, + { + "sid": 32, + "sent": "Upper lanes show the ethidium bromide staining of the 18S rRNAs for quantification.", + "section": "FIG", + "ner": [ + [ + 21, + 37, + "ethidium bromide", + "chemical" + ], + [ + 54, + 63, + "18S rRNAs", + "chemical" + ] + ] + }, + { + "sid": 33, + "sent": "All samples were loaded on the gel with two different amounts of 5 and 10 \u03bcl. (D) Primer extension analysis of acp modification in yeast 18S rRNA (right gel) including a sequencing ladder (left gel).", + "section": "FIG", + "ner": [ + [ + 82, + 107, + "Primer extension analysis", + "experimental_method" + ], + [ + 111, + 114, + "acp", + "chemical" + ], + [ + 131, + 136, + "yeast", + "taxonomy_domain" + ], + [ + 137, + 145, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 34, + "sent": "The primer extension stop at nucleotide 1191 is missing exclusively in \u0394tsr3 mutants and \u0394tsr3 \u0394snr35 recombinants.", + "section": "FIG", + "ner": [ + [ + 40, + 44, + "1191", + "residue_number" + ], + [ + 71, + 76, + "\u0394tsr3", + "mutant" + ], + [ + 89, + 101, + "\u0394tsr3 \u0394snr35", + "mutant" + ] + ] + }, + { + "sid": 35, + "sent": "(E) Primer extension analysis of human 18S rRNA after siRNA knockdown of HsNEP1/EMG1 (541, 542 and 543) and HsTSR3 (544 and 545) (right gel), including a sequencing ladder (left gel).", + "section": "FIG", + "ner": [ + [ + 4, + 29, + "Primer extension analysis", + "experimental_method" + ], + [ + 33, + 38, + "human", + "species" + ], + [ + 39, + 47, + "18S rRNA", + "chemical" + ], + [ + 54, + 69, + "siRNA knockdown", + "experimental_method" + ], + [ + 73, + 79, + "HsNEP1", + "protein" + ], + [ + 80, + 84, + "EMG1", + "protein" + ], + [ + 108, + 114, + "HsTSR3", + "protein" + ] + ] + }, + { + "sid": 36, + "sent": "The primer extension arrest is reduced in HTC116 cells transfected with siRNAs 544 and 545.", + "section": "FIG", + "ner": [ + [ + 72, + 78, + "siRNAs", + "chemical" + ] + ] + }, + { + "sid": 37, + "sent": "The efficiency of siRNA mediated HsTSR3 repression correlates with the primer extension signals (see Supplementary Figure S2A).", + "section": "FIG", + "ner": [ + [ + 18, + 23, + "siRNA", + "chemical" + ], + [ + 33, + 39, + "HsTSR3", + "protein" + ], + [ + 71, + 95, + "primer extension signals", + "evidence" + ] + ] + }, + { + "sid": 38, + "sent": "Only a few acp transferring enzymes have been characterized until now.", + "section": "INTRO", + "ner": [ + [ + 11, + 14, + "acp", + "chemical" + ] + ] + }, + { + "sid": 39, + "sent": "During the biosynthesis of wybutosine, a tricyclic nucleoside present in eukaryotic and archaeal phenylalanine tRNA, Tyw2 (Trm12 in yeast) transfers an acp group from SAM to an acidic carbon atom.", + "section": "INTRO", + "ner": [ + [ + 27, + 37, + "wybutosine", + "chemical" + ], + [ + 51, + 61, + "nucleoside", + "chemical" + ], + [ + 73, + 83, + "eukaryotic", + "taxonomy_domain" + ], + [ + 88, + 96, + "archaeal", + "taxonomy_domain" + ], + [ + 97, + 110, + "phenylalanine", + "chemical" + ], + [ + 111, + 115, + "tRNA", + "chemical" + ], + [ + 117, + 121, + "Tyw2", + "protein" + ], + [ + 123, + 128, + "Trm12", + "protein" + ], + [ + 132, + 137, + "yeast", + "taxonomy_domain" + ], + [ + 152, + 155, + "acp", + "chemical" + ], + [ + 167, + 170, + "SAM", + "chemical" + ] + ] + }, + { + "sid": 40, + "sent": "Archaeal Tyw2 has a structure very similar to Rossmann-fold (class I) RNA-methyltransferases, but its distinctive SAM-binding mode enables the transfer of the acp group instead of the methyl group of the cofactor.", + "section": "INTRO", + "ner": [ + [ + 0, + 8, + "Archaeal", + "taxonomy_domain" + ], + [ + 9, + 13, + "Tyw2", + "protein" + ], + [ + 20, + 29, + "structure", + "evidence" + ], + [ + 46, + 92, + "Rossmann-fold (class I) RNA-methyltransferases", + "protein_type" + ], + [ + 114, + 130, + "SAM-binding mode", + "site" + ], + [ + 159, + 162, + "acp", + "chemical" + ] + ] + }, + { + "sid": 41, + "sent": "Another acp modification has been described in the diphtamide biosynthesis pathway, where an acp group is transferred from SAM to the carbon atom of a histidine residue of eukaryotic translation elongation factor 2 by use of a radical mechanism.", + "section": "INTRO", + "ner": [ + [ + 8, + 11, + "acp", + "chemical" + ], + [ + 51, + 61, + "diphtamide", + "chemical" + ], + [ + 93, + 96, + "acp", + "chemical" + ], + [ + 123, + 126, + "SAM", + "chemical" + ], + [ + 151, + 160, + "histidine", + "residue_name" + ], + [ + 172, + 182, + "eukaryotic", + "taxonomy_domain" + ], + [ + 183, + 214, + "translation elongation factor 2", + "protein_type" + ] + ] + }, + { + "sid": 42, + "sent": "In a recent bioinformatic study, the uncharacterized yeast gene YOR006c was predicted to be involved in ribosome biogenesis.", + "section": "INTRO", + "ner": [ + [ + 53, + 58, + "yeast", + "taxonomy_domain" + ], + [ + 64, + 71, + "YOR006c", + "gene" + ] + ] + }, + { + "sid": 43, + "sent": "It is highly conserved among eukaryotes and archaea (Supplementary Figure S1A) and its deletion leads to an accumulation of the 20S pre-rRNA precursor of 18S rRNA, suggesting an influence on D-site cleavage during the maturation of the small ribosomal subunit.", + "section": "INTRO", + "ner": [ + [ + 6, + 22, + "highly conserved", + "protein_state" + ], + [ + 29, + 39, + "eukaryotes", + "taxonomy_domain" + ], + [ + 44, + 51, + "archaea", + "taxonomy_domain" + ], + [ + 128, + 140, + "20S pre-rRNA", + "chemical" + ], + [ + 154, + 162, + "18S rRNA", + "chemical" + ], + [ + 191, + 197, + "D-site", + "site" + ] + ] + }, + { + "sid": 44, + "sent": "On this basis, YOR006C was renamed \u2018Twenty S rRNA accumulation 3\u2032 (TSR3).", + "section": "INTRO", + "ner": [ + [ + 15, + 22, + "YOR006C", + "gene" + ], + [ + 36, + 64, + "Twenty S rRNA accumulation 3", + "protein" + ], + [ + 67, + 71, + "TSR3", + "protein" + ] + ] + }, + { + "sid": 45, + "sent": "However, its function remained unclear although recently a putative nuclease function during 18S rRNA maturation was predicted.", + "section": "INTRO", + "ner": [ + [ + 93, + 101, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 46, + "sent": "Here, we identify Tsr3 as the long-sought acp transferase that catalyzes the last step in the biosynthesis of the hypermodified nucleotide m1acp3\u03a8 in yeast and human cells.", + "section": "INTRO", + "ner": [ + [ + 18, + 22, + "Tsr3", + "protein" + ], + [ + 42, + 57, + "acp transferase", + "protein_type" + ], + [ + 114, + 127, + "hypermodified", + "protein_state" + ], + [ + 128, + 138, + "nucleotide", + "chemical" + ], + [ + 139, + 146, + "m1acp3\u03a8", + "chemical" + ], + [ + 150, + 155, + "yeast", + "taxonomy_domain" + ], + [ + 160, + 165, + "human", + "species" + ] + ] + }, + { + "sid": 47, + "sent": "Furthermore using catalytically defective mutants of yeast Tsr3 we demonstrated that the acp modification is required for 18S rRNA maturation.", + "section": "INTRO", + "ner": [ + [ + 18, + 41, + "catalytically defective", + "protein_state" + ], + [ + 53, + 58, + "yeast", + "taxonomy_domain" + ], + [ + 59, + 63, + "Tsr3", + "protein" + ], + [ + 89, + 92, + "acp", + "chemical" + ], + [ + 122, + 130, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 48, + "sent": "Surprisingly, the crystal structures of archaeal homologs revealed that Tsr3 is structurally similar to the SPOUT-class RNA methyltransferases.", + "section": "INTRO", + "ner": [ + [ + 18, + 36, + "crystal structures", + "evidence" + ], + [ + 40, + 48, + "archaeal", + "taxonomy_domain" + ], + [ + 72, + 76, + "Tsr3", + "protein" + ], + [ + 108, + 142, + "SPOUT-class RNA methyltransferases", + "protein_type" + ] + ] + }, + { + "sid": 49, + "sent": "In contrast, the only other structurally characterized acp transferase enzyme Tyw2 belongs to the Rossmann-fold class of methyltransferase proteins.", + "section": "INTRO", + "ner": [ + [ + 55, + 70, + "acp transferase", + "protein_type" + ], + [ + 78, + 82, + "Tyw2", + "protein" + ], + [ + 98, + 147, + "Rossmann-fold class of methyltransferase proteins", + "protein_type" + ] + ] + }, + { + "sid": 50, + "sent": "Interestingly, the two structurally very different enzymes use similar strategies in binding the SAM-cofactor in order to ensure that in contrast to methyltransferases the acp and not the methyl group of SAM is transferred to the substrate.", + "section": "INTRO", + "ner": [ + [ + 97, + 100, + "SAM", + "chemical" + ], + [ + 149, + 167, + "methyltransferases", + "protein_type" + ], + [ + 172, + 175, + "acp", + "chemical" + ], + [ + 204, + 207, + "SAM", + "chemical" + ] + ] + }, + { + "sid": 51, + "sent": "Tsr3 is the enzyme responsible for 18S rRNA acp modification in yeast and humans", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "Tsr3", + "protein" + ], + [ + 35, + 43, + "18S rRNA", + "chemical" + ], + [ + 44, + 47, + "acp", + "chemical" + ], + [ + 64, + 69, + "yeast", + "taxonomy_domain" + ], + [ + 74, + 80, + "humans", + "species" + ] + ] + }, + { + "sid": 52, + "sent": "The S. cerevisiae 18S rRNA acp transferase was identified in a systematic genetic screen where numerous deletion mutants from the EUROSCARF strain collection (www.euroscarf.de) were analyzed by HPLC for alterations in 18S rRNA base modifications.", + "section": "RESULTS", + "ner": [ + [ + 4, + 17, + "S. cerevisiae", + "species" + ], + [ + 18, + 42, + "18S rRNA acp transferase", + "protein_type" + ], + [ + 194, + 198, + "HPLC", + "experimental_method" + ], + [ + 218, + 226, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 53, + "sent": "For the \u0394tsr3 deletion strain the HPLC elution profile of 18S rRNA nucleosides (Figure 1B) was very similar to that of the pseudouridine-N1 methyltransferase mutant \u0394nep1, where a shoulder at \u223c 7.4 min elution time was missing in the elution profile.", + "section": "RESULTS", + "ner": [ + [ + 8, + 13, + "\u0394tsr3", + "mutant" + ], + [ + 34, + 54, + "HPLC elution profile", + "evidence" + ], + [ + 58, + 66, + "18S rRNA", + "chemical" + ], + [ + 67, + 78, + "nucleosides", + "chemical" + ], + [ + 123, + 157, + "pseudouridine-N1 methyltransferase", + "protein_type" + ], + [ + 158, + 164, + "mutant", + "protein_state" + ], + [ + 165, + 170, + "\u0394nep1", + "mutant" + ] + ] + }, + { + "sid": 54, + "sent": "As previously reported this shoulder was identified by ESI-MS as corresponding to m1acp3\u03a8.", + "section": "RESULTS", + "ner": [ + [ + 55, + 61, + "ESI-MS", + "experimental_method" + ], + [ + 82, + 89, + "m1acp3\u03a8", + "chemical" + ] + ] + }, + { + "sid": 55, + "sent": "In order to directly analyze the presence of the acp modification of nucleotide 1191 we used an in vivo14C incorporation assay with 1-14C-methionine.", + "section": "RESULTS", + "ner": [ + [ + 49, + 52, + "acp", + "chemical" + ], + [ + 69, + 79, + "nucleotide", + "chemical" + ], + [ + 80, + 84, + "1191", + "residue_number" + ], + [ + 96, + 126, + "in vivo14C incorporation assay", + "experimental_method" + ], + [ + 132, + 148, + "1-14C-methionine", + "chemical" + ] + ] + }, + { + "sid": 56, + "sent": "Whereas the acp labeling of 18S rRNA was clearly present in the wild type strain no radioactive labeling could be observed in a \u0394tsr3 strain (Figure 1C).", + "section": "RESULTS", + "ner": [ + [ + 12, + 15, + "acp", + "chemical" + ], + [ + 28, + 36, + "18S rRNA", + "chemical" + ], + [ + 64, + 73, + "wild type", + "protein_state" + ], + [ + 128, + 133, + "\u0394tsr3", + "mutant" + ] + ] + }, + { + "sid": 57, + "sent": "No radioactive labeling was detected in the 18S U1191A mutant which served as a control for the specificity of the 14C-aminocarboxypropyl incorporation.", + "section": "RESULTS", + "ner": [ + [ + 44, + 54, + "18S U1191A", + "mutant" + ], + [ + 55, + 61, + "mutant", + "protein_state" + ], + [ + 115, + 137, + "14C-aminocarboxypropyl", + "chemical" + ] + ] + }, + { + "sid": 58, + "sent": "As previously shown, only the acp but none of the other modifications at U1191 of yeast 18S rRNA blocks reverse transcriptase activity.", + "section": "RESULTS", + "ner": [ + [ + 30, + 33, + "acp", + "chemical" + ], + [ + 73, + 78, + "U1191", + "residue_name_number" + ], + [ + 82, + 87, + "yeast", + "taxonomy_domain" + ], + [ + 88, + 96, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 59, + "sent": "Therefore the presence of the acp modification can be directly assessed by primer extension.", + "section": "RESULTS", + "ner": [ + [ + 30, + 33, + "acp", + "chemical" + ], + [ + 75, + 91, + "primer extension", + "experimental_method" + ] + ] + }, + { + "sid": 60, + "sent": "Indeed, in wild-type yeast a strong primer extension stop signal occurred at position 1192.", + "section": "RESULTS", + "ner": [ + [ + 11, + 20, + "wild-type", + "protein_state" + ], + [ + 21, + 26, + "yeast", + "taxonomy_domain" + ], + [ + 36, + 64, + "primer extension stop signal", + "evidence" + ], + [ + 86, + 90, + "1192", + "residue_number" + ] + ] + }, + { + "sid": 61, + "sent": "In contrast, in a \u0394tsr3 mutant no primer extension stop signal was present at this position.", + "section": "RESULTS", + "ner": [ + [ + 18, + 23, + "\u0394tsr3", + "mutant" + ], + [ + 24, + 30, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 62, + "sent": "As expected, in a \u0394snr35 deletion preventing pseudouridylation and N1-methylation (resulting in acp3U) as well as in a \u0394nep1 deletion strain where pseudouridine is not methylated (resulting in acp3\u03a8) a primer extension stop signal of similar intensity as in the wild type was observed.", + "section": "RESULTS", + "ner": [ + [ + 18, + 24, + "\u0394snr35", + "mutant" + ], + [ + 25, + 33, + "deletion", + "experimental_method" + ], + [ + 45, + 62, + "pseudouridylation", + "ptm" + ], + [ + 67, + 81, + "N1-methylation", + "ptm" + ], + [ + 96, + 101, + "acp3U", + "chemical" + ], + [ + 119, + 124, + "\u0394nep1", + "mutant" + ], + [ + 147, + 160, + "pseudouridine", + "chemical" + ], + [ + 164, + 178, + "not methylated", + "protein_state" + ], + [ + 193, + 198, + "acp3\u03a8", + "chemical" + ], + [ + 202, + 230, + "primer extension stop signal", + "evidence" + ], + [ + 262, + 271, + "wild type", + "protein_state" + ] + ] + }, + { + "sid": 63, + "sent": "In a \u0394tsr3 \u0394snr35 double deletion strain the 18S rRNA contains an unmodified U and the primer extension stop signal was missing (Figure 1D).", + "section": "RESULTS", + "ner": [ + [ + 5, + 17, + "\u0394tsr3 \u0394snr35", + "mutant" + ], + [ + 45, + 53, + "18S rRNA", + "chemical" + ], + [ + 66, + 76, + "unmodified", + "protein_state" + ], + [ + 77, + 78, + "U", + "chemical" + ] + ] + }, + { + "sid": 64, + "sent": "The Tsr3 protein is highly conserved in yeast and humans (50% identity).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "Tsr3", + "protein" + ], + [ + 20, + 36, + "highly conserved", + "protein_state" + ], + [ + 40, + 45, + "yeast", + "taxonomy_domain" + ], + [ + 50, + 56, + "humans", + "species" + ] + ] + }, + { + "sid": 65, + "sent": "Human 18S rRNA has also been shown to contain m1acp3\u03a8 in the 18S rRNA at position 1248.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "Human", + "species" + ], + [ + 6, + 14, + "18S rRNA", + "chemical" + ], + [ + 46, + 53, + "m1acp3\u03a8", + "ptm" + ], + [ + 61, + 69, + "18S rRNA", + "chemical" + ], + [ + 82, + 86, + "1248", + "residue_number" + ] + ] + }, + { + "sid": 66, + "sent": "After siRNA-mediated depletion of Tsr3 in human colon carcinoma HCT116(+/+) cells the acp primer extension arrest was reduced in comparison to cells transfected with a non-targeting scramble siRNA control (Figure 1E, compare lanes 544 and scramble).", + "section": "RESULTS", + "ner": [ + [ + 6, + 30, + "siRNA-mediated depletion", + "experimental_method" + ], + [ + 34, + 38, + "Tsr3", + "protein" + ], + [ + 42, + 47, + "human", + "species" + ], + [ + 86, + 113, + "acp primer extension arrest", + "evidence" + ], + [ + 191, + 196, + "siRNA", + "chemical" + ] + ] + }, + { + "sid": 67, + "sent": "The efficiency of siRNA-mediated depletion was established by RT-qPCR and found to be very high with siRNA 544 (Supplementary Figure S2A, remaining TSR3 mRNA level of 2%).", + "section": "RESULTS", + "ner": [ + [ + 18, + 23, + "siRNA", + "chemical" + ], + [ + 62, + 69, + "RT-qPCR", + "experimental_method" + ], + [ + 101, + 106, + "siRNA", + "chemical" + ], + [ + 148, + 152, + "TSR3", + "protein" + ] + ] + }, + { + "sid": 68, + "sent": "By comparison, treating cells with siRNA 545, which only reduced the TSR3 mRNA to 20%, did not markedly reduced the acp signal.", + "section": "RESULTS", + "ner": [ + [ + 35, + 40, + "siRNA", + "chemical" + ], + [ + 69, + 73, + "TSR3", + "protein" + ], + [ + 116, + 119, + "acp", + "chemical" + ] + ] + }, + { + "sid": 69, + "sent": "This suggests that low residual levels of HsTsr3 are sufficient to modify the RNA.", + "section": "RESULTS", + "ner": [ + [ + 42, + 48, + "HsTsr3", + "protein" + ], + [ + 78, + 81, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 70, + "sent": "Thus, HsTsr3 is also responsible for the acp modification of 18S rRNA nucleotide \u03a81248 in helix 31.", + "section": "RESULTS", + "ner": [ + [ + 6, + 12, + "HsTsr3", + "protein" + ], + [ + 41, + 44, + "acp", + "chemical" + ], + [ + 61, + 69, + "18S rRNA", + "chemical" + ], + [ + 70, + 80, + "nucleotide", + "chemical" + ], + [ + 81, + 86, + "\u03a81248", + "ptm" + ], + [ + 90, + 98, + "helix 31", + "structure_element" + ] + ] + }, + { + "sid": 71, + "sent": "Similar to yeast, siRNA-mediated depletion of the \u03a81248 N1-methyltransferase Nep1/Emg1 had no influence on the primer extension arrest (Figure 1E).", + "section": "RESULTS", + "ner": [ + [ + 11, + 16, + "yeast", + "taxonomy_domain" + ], + [ + 18, + 42, + "siRNA-mediated depletion", + "experimental_method" + ], + [ + 50, + 76, + "\u03a81248 N1-methyltransferase", + "protein_type" + ], + [ + 77, + 81, + "Nep1", + "protein" + ], + [ + 82, + 86, + "Emg1", + "protein" + ], + [ + 111, + 134, + "primer extension arrest", + "evidence" + ] + ] + }, + { + "sid": 72, + "sent": "Phenotypic characterization of \u0394tsr3 mutants", + "section": "RESULTS", + "ner": [ + [ + 31, + 36, + "\u0394tsr3", + "mutant" + ] + ] + }, + { + "sid": 73, + "sent": "Although the acp modification of 18S rRNA is highly conserved in eukaryotes, yeast \u0394tsr3 mutants showed only a minor growth defect.", + "section": "RESULTS", + "ner": [ + [ + 13, + 16, + "acp", + "chemical" + ], + [ + 33, + 41, + "18S rRNA", + "chemical" + ], + [ + 45, + 61, + "highly conserved", + "protein_state" + ], + [ + 65, + 75, + "eukaryotes", + "taxonomy_domain" + ], + [ + 77, + 82, + "yeast", + "taxonomy_domain" + ], + [ + 83, + 88, + "\u0394tsr3", + "mutant" + ] + ] + }, + { + "sid": 74, + "sent": "However, the \u0394tsr3 deletion was synthetic sick with a \u0394snr35 deletion preventing pseudouridylation and Nep1-catalyzed methylation of nucleotide 1191 (Figure 2A).", + "section": "RESULTS", + "ner": [ + [ + 13, + 18, + "\u0394tsr3", + "mutant" + ], + [ + 54, + 60, + "\u0394snr35", + "mutant" + ], + [ + 81, + 98, + "pseudouridylation", + "ptm" + ], + [ + 103, + 107, + "Nep1", + "protein" + ], + [ + 144, + 148, + "1191", + "residue_number" + ] + ] + }, + { + "sid": 75, + "sent": "Interestingly, no increased growth defect could be observed for \u0394tsr3 \u0394nep1 recombinants containing the nep1 suppressor mutation \u0394nop6 as well as for \u0394tsr3 \u0394snr35 \u0394nep1 recombinants with unmodified U1191 (Supplementary Figure S2D and E).", + "section": "RESULTS", + "ner": [ + [ + 64, + 75, + "\u0394tsr3 \u0394nep1", + "mutant" + ], + [ + 104, + 108, + "nep1", + "gene" + ], + [ + 129, + 134, + "\u0394nop6", + "mutant" + ], + [ + 150, + 168, + "\u0394tsr3 \u0394snr35 \u0394nep1", + "mutant" + ], + [ + 187, + 197, + "unmodified", + "protein_state" + ], + [ + 198, + 203, + "U1191", + "residue_name_number" + ] + ] + }, + { + "sid": 76, + "sent": "Phenotypic characterization of yeast TSR3 deletion (\u0394trs3) and human TSR3 depletion (siRNAs 544 and 545) and cellular localization of yeast Tsr3. (A) Growth of yeast wild type, \u0394tsr3, \u0394snr35 and \u0394tsr3 \u0394snr35 segregants after meiosis and tetrad dissection of \u0394tsr3/TSR3 \u0394snr35/SNR35 heterozygous diploids.", + "section": "FIG", + "ner": [ + [ + 31, + 36, + "yeast", + "taxonomy_domain" + ], + [ + 37, + 41, + "TSR3", + "protein" + ], + [ + 52, + 57, + "\u0394trs3", + "mutant" + ], + [ + 63, + 68, + "human", + "species" + ], + [ + 69, + 73, + "TSR3", + "protein" + ], + [ + 85, + 91, + "siRNAs", + "chemical" + ], + [ + 134, + 139, + "yeast", + "taxonomy_domain" + ], + [ + 140, + 144, + "Tsr3", + "protein" + ], + [ + 160, + 165, + "yeast", + "taxonomy_domain" + ], + [ + 166, + 175, + "wild type", + "protein_state" + ], + [ + 177, + 182, + "\u0394tsr3", + "mutant" + ], + [ + 184, + 190, + "\u0394snr35", + "mutant" + ], + [ + 195, + 207, + "\u0394tsr3 \u0394snr35", + "mutant" + ], + [ + 258, + 263, + "\u0394tsr3", + "mutant" + ], + [ + 264, + 268, + "TSR3", + "protein" + ], + [ + 269, + 275, + "\u0394snr35", + "mutant" + ], + [ + 276, + 281, + "SNR35", + "protein" + ] + ] + }, + { + "sid": 77, + "sent": "The \u0394tsr3 deletion is synthetic sick with a \u0394snr35 deletion preventing U1191 pseudouridylation.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "\u0394tsr3", + "mutant" + ], + [ + 44, + 50, + "\u0394snr35", + "mutant" + ], + [ + 71, + 76, + "U1191", + "residue_name_number" + ] + ] + }, + { + "sid": 78, + "sent": "(B) In agar diffusion assays the yeast \u0394tsr3 deletion mutant shows a hypersensitivity against paromomycin and hygromycin B which is further increased by recombination with \u0394snr35. (C) Northern blot analysis with\u00a0an ITS1 hybridization probe after siRNA depletion of HsTSR3 (siRNAs 544 and 545) and a scrambled siRNA as control.", + "section": "FIG", + "ner": [ + [ + 7, + 28, + "agar diffusion assays", + "experimental_method" + ], + [ + 33, + 38, + "yeast", + "taxonomy_domain" + ], + [ + 39, + 44, + "\u0394tsr3", + "mutant" + ], + [ + 45, + 60, + "deletion mutant", + "protein_state" + ], + [ + 94, + 105, + "paromomycin", + "chemical" + ], + [ + 110, + 122, + "hygromycin B", + "chemical" + ], + [ + 172, + 178, + "\u0394snr35", + "mutant" + ], + [ + 184, + 206, + "Northern blot analysis", + "experimental_method" + ], + [ + 246, + 261, + "siRNA depletion", + "experimental_method" + ], + [ + 265, + 271, + "HsTSR3", + "protein" + ], + [ + 273, + 279, + "siRNAs", + "chemical" + ], + [ + 309, + 314, + "siRNA", + "chemical" + ] + ] + }, + { + "sid": 79, + "sent": "The accumulation of 18SE and 47S and/or 45S pre-RNAs is enforced upon HsTSR3 depletion.", + "section": "FIG", + "ner": [ + [ + 20, + 24, + "18SE", + "chemical" + ], + [ + 29, + 32, + "47S", + "chemical" + ], + [ + 40, + 52, + "45S pre-RNAs", + "chemical" + ], + [ + 70, + 76, + "HsTSR3", + "protein" + ] + ] + }, + { + "sid": 80, + "sent": "Right gel: Ethidium bromide staining showing 18S and 28S rRNAs.", + "section": "FIG", + "ner": [ + [ + 45, + 48, + "18S", + "chemical" + ], + [ + 53, + 62, + "28S rRNAs", + "chemical" + ] + ] + }, + { + "sid": 81, + "sent": "(D) Cytoplasmic localization of yeast Tsr3 shown by fluorescence microscopy of GFP-fused Tsr3.", + "section": "FIG", + "ner": [ + [ + 32, + 37, + "yeast", + "taxonomy_domain" + ], + [ + 38, + 42, + "Tsr3", + "protein" + ], + [ + 52, + 75, + "fluorescence microscopy", + "experimental_method" + ], + [ + 79, + 93, + "GFP-fused Tsr3", + "mutant" + ] + ] + }, + { + "sid": 82, + "sent": "From left to right: differential interference contrast (DIC), green fluorescence of GFP-Tsr3, red fluorescence of Nop56-mRFP as nucleolar marker, and merge of GFP-Tsr3/Nop56-mRFP with DIC. (E) Elution profile (A254) after sucrose gradient separation of yeast ribosomal subunits and polysomes (upper part) and western blot analysis of 3xHA tagged Tsr3 (Tsr3-3xHA) after SDS-PAGE separation of polysome profile fractions taken every 20 s (lower part).", + "section": "FIG", + "ner": [ + [ + 20, + 54, + "differential interference contrast", + "experimental_method" + ], + [ + 56, + 59, + "DIC", + "experimental_method" + ], + [ + 84, + 92, + "GFP-Tsr3", + "mutant" + ], + [ + 114, + 124, + "Nop56-mRFP", + "mutant" + ], + [ + 159, + 167, + "GFP-Tsr3", + "mutant" + ], + [ + 168, + 178, + "Nop56-mRFP", + "mutant" + ], + [ + 184, + 187, + "DIC", + "experimental_method" + ], + [ + 193, + 208, + "Elution profile", + "evidence" + ], + [ + 222, + 249, + "sucrose gradient separation", + "experimental_method" + ], + [ + 253, + 258, + "yeast", + "taxonomy_domain" + ], + [ + 259, + 277, + "ribosomal subunits", + "complex_assembly" + ], + [ + 282, + 291, + "polysomes", + "complex_assembly" + ], + [ + 309, + 321, + "western blot", + "experimental_method" + ], + [ + 334, + 338, + "3xHA", + "chemical" + ], + [ + 346, + 350, + "Tsr3", + "protein" + ], + [ + 352, + 361, + "Tsr3-3xHA", + "mutant" + ], + [ + 369, + 377, + "SDS-PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 83, + "sent": "The TSR3 gene was genetically modified at its native locus, resulting in a C-terminal fusion of Tsr3 with a 3xHA epitope expressed by the native promotor in yeast strain CEN.BM258-5B.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "TSR3", + "protein" + ], + [ + 86, + 92, + "fusion", + "protein_state" + ], + [ + 96, + 100, + "Tsr3", + "protein" + ], + [ + 108, + 112, + "3xHA", + "chemical" + ], + [ + 157, + 162, + "yeast", + "taxonomy_domain" + ] + ] + }, + { + "sid": 84, + "sent": "The influence of the acp modification of nucleotide 1191 on ribosome function was analyzed by treating \u0394tsr3 mutants with protein synthesis inhibitors.", + "section": "RESULTS", + "ner": [ + [ + 21, + 24, + "acp", + "chemical" + ], + [ + 41, + 51, + "nucleotide", + "chemical" + ], + [ + 52, + 56, + "1191", + "residue_number" + ], + [ + 103, + 108, + "\u0394tsr3", + "mutant" + ] + ] + }, + { + "sid": 85, + "sent": "Similar to a temperature-sensitive nep1 mutant, the \u0394tsr3 deletion caused hypersensitivity to paromomycin and, to a lesser extent, to hygromycin B (Figure 2B), but not to G418 or cycloheximide (data not shown).", + "section": "RESULTS", + "ner": [ + [ + 35, + 39, + "nep1", + "gene" + ], + [ + 40, + 46, + "mutant", + "protein_state" + ], + [ + 52, + 57, + "\u0394tsr3", + "mutant" + ], + [ + 94, + 105, + "paromomycin", + "chemical" + ], + [ + 134, + 146, + "hygromycin B", + "chemical" + ], + [ + 171, + 175, + "G418", + "chemical" + ], + [ + 179, + 192, + "cycloheximide", + "chemical" + ] + ] + }, + { + "sid": 86, + "sent": "In accordance with the synthetic sick growth phenotype the paromomycin and hygromycin B hypersensitivity further increased in a \u0394tsr3 \u0394snr35 recombination strain (Figure 2B).", + "section": "RESULTS", + "ner": [ + [ + 59, + 70, + "paromomycin", + "chemical" + ], + [ + 75, + 87, + "hygromycin B", + "chemical" + ], + [ + 128, + 140, + "\u0394tsr3 \u0394snr35", + "mutant" + ] + ] + }, + { + "sid": 87, + "sent": "In a yeast \u0394tsr3 strain as well as in the \u0394tsr3 \u0394snr35 recombinant 20S pre-rRNA accumulated significantly and the level of mature 18S rRNA was reduced (Supplementary Figures S2C and S3D), as reported previously.", + "section": "RESULTS", + "ner": [ + [ + 5, + 10, + "yeast", + "taxonomy_domain" + ], + [ + 11, + 16, + "\u0394tsr3", + "mutant" + ], + [ + 42, + 54, + "\u0394tsr3 \u0394snr35", + "mutant" + ], + [ + 67, + 79, + "20S pre-rRNA", + "chemical" + ], + [ + 130, + 138, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 88, + "sent": "A minor effect on 20S rRNA accumulation was also observed for \u0394snr35, but - probably due to different strain backgrounds \u2013 to a weaker extent than described earlier.", + "section": "RESULTS", + "ner": [ + [ + 18, + 26, + "20S rRNA", + "chemical" + ], + [ + 62, + 68, + "\u0394snr35", + "mutant" + ] + ] + }, + { + "sid": 89, + "sent": "In human cells, the depletion of HsTsr3 in HCT116(+/+) cells caused an accumulation of the human 20S pre-rRNA equivalent 18S-E suggesting an evolutionary conserved role of Tsr3 in the late steps of 18S rRNA processing (Figure 2C and Supplementary Figure S2B).", + "section": "RESULTS", + "ner": [ + [ + 3, + 8, + "human", + "species" + ], + [ + 20, + 32, + "depletion of", + "experimental_method" + ], + [ + 33, + 39, + "HsTsr3", + "protein" + ], + [ + 91, + 96, + "human", + "species" + ], + [ + 97, + 109, + "20S pre-rRNA", + "chemical" + ], + [ + 121, + 126, + "18S-E", + "chemical" + ], + [ + 172, + 176, + "Tsr3", + "protein" + ], + [ + 198, + 206, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 90, + "sent": "Surprisingly, early nucleolar processing reactions were also inhibited, and this was observed in both yeast \u0394tsr3 cells (see accumulation of 35S in Supplementary Figure S2C) and Tsr3 depleted human cells (see 47S/45S accumulation in Figure 2C and Northern blot quantification in Supplementary Figure S2B).", + "section": "RESULTS", + "ner": [ + [ + 102, + 107, + "yeast", + "taxonomy_domain" + ], + [ + 108, + 113, + "\u0394tsr3", + "mutant" + ], + [ + 141, + 144, + "35S", + "complex_assembly" + ], + [ + 178, + 182, + "Tsr3", + "protein" + ], + [ + 192, + 197, + "human", + "species" + ], + [ + 209, + 212, + "47S", + "complex_assembly" + ], + [ + 213, + 216, + "45S", + "complex_assembly" + ], + [ + 247, + 260, + "Northern blot", + "experimental_method" + ] + ] + }, + { + "sid": 91, + "sent": "Consistent with its role in late 18S rRNA processing, TSR3 deletion leads to a ribosomal subunit imbalance with a reduced 40S to 60S ratio of 0.81 (\u03c3 = 0.024) which was further increased in a \u0394tsr3 \u0394snr35 recombinant to 0.73 (\u03c3 = 0.023) (Supplementary Figure S2F).", + "section": "RESULTS", + "ner": [ + [ + 33, + 41, + "18S rRNA", + "chemical" + ], + [ + 54, + 58, + "TSR3", + "protein" + ], + [ + 122, + 125, + "40S", + "complex_assembly" + ], + [ + 129, + 132, + "60S", + "complex_assembly" + ], + [ + 192, + 204, + "\u0394tsr3 \u0394snr35", + "mutant" + ] + ] + }, + { + "sid": 92, + "sent": "In polysome profiles, a reduced level of 80S ribosomes and a strong signal for free 60S subunits was observed in line with the 40S subunit deficiency (Supplementary Figure S2G).", + "section": "RESULTS", + "ner": [ + [ + 3, + 20, + "polysome profiles", + "evidence" + ], + [ + 41, + 54, + "80S ribosomes", + "complex_assembly" + ], + [ + 84, + 87, + "60S", + "complex_assembly" + ], + [ + 127, + 130, + "40S", + "complex_assembly" + ] + ] + }, + { + "sid": 93, + "sent": "Cellular localization of Tsr3 in S. cerevisiae", + "section": "RESULTS", + "ner": [ + [ + 25, + 29, + "Tsr3", + "protein" + ], + [ + 33, + 46, + "S. cerevisiae", + "species" + ] + ] + }, + { + "sid": 94, + "sent": "Fluorescence microscopy of GFP-tagged Tsr3 localized the fusion protein in the cytoplasm of yeast cells and no co-localization with the nucleolar marker protein Nop56 could be observed (Figure 2D).", + "section": "RESULTS", + "ner": [ + [ + 0, + 23, + "Fluorescence microscopy", + "experimental_method" + ], + [ + 27, + 37, + "GFP-tagged", + "protein_state" + ], + [ + 38, + 42, + "Tsr3", + "protein" + ], + [ + 92, + 97, + "yeast", + "taxonomy_domain" + ], + [ + 161, + 166, + "Nop56", + "protein" + ] + ] + }, + { + "sid": 95, + "sent": "This agrees with previous biochemical data suggesting that the acp modification of 18S rRNA occurs late during 40S subunit biogenesis in the cytoplasm, and makes an additional nuclear localization as reported in a previous large-scale analysis unlikely.", + "section": "RESULTS", + "ner": [ + [ + 63, + 66, + "acp", + "chemical" + ], + [ + 83, + 91, + "18S rRNA", + "chemical" + ], + [ + 111, + 114, + "40S", + "complex_assembly" + ] + ] + }, + { + "sid": 96, + "sent": "After polysome gradient separation C-terminally epitope-labeled Tsr3-3xHA was exclusively detectable in the low-density fraction (Figure 2E).", + "section": "RESULTS", + "ner": [ + [ + 6, + 34, + "polysome gradient separation", + "experimental_method" + ], + [ + 64, + 73, + "Tsr3-3xHA", + "mutant" + ] + ] + }, + { + "sid": 97, + "sent": "Such distribution on a density gradient suggests that Tsr3 only interacts transiently with pre-40S subunits, which presumably explains why it was not characterized in pre-ribosome affinity purifications.", + "section": "RESULTS", + "ner": [ + [ + 5, + 39, + "distribution on a density gradient", + "evidence" + ], + [ + 54, + 58, + "Tsr3", + "protein" + ], + [ + 91, + 107, + "pre-40S subunits", + "complex_assembly" + ], + [ + 167, + 202, + "pre-ribosome affinity purifications", + "experimental_method" + ] + ] + }, + { + "sid": 98, + "sent": "Structure of Tsr3", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 13, + 17, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 99, + "sent": "Searches for sequence homologs of S. cerevisiae Tsr3 (ScTsr3) by us and others revealed that the genomes of many archaea contain genes encoding Tsr3-like proteins.", + "section": "RESULTS", + "ner": [ + [ + 34, + 47, + "S. cerevisiae", + "species" + ], + [ + 48, + 52, + "Tsr3", + "protein" + ], + [ + 54, + 60, + "ScTsr3", + "protein" + ], + [ + 113, + 120, + "archaea", + "taxonomy_domain" + ], + [ + 144, + 162, + "Tsr3-like proteins", + "protein_type" + ] + ] + }, + { + "sid": 100, + "sent": "However, these archaeal homologs are significantly smaller than ScTsr3 (\u223c190 aa in archaea vs. 313 aa in yeast) due to shortened N- and C-termini (Supplementary Figure S1A).", + "section": "RESULTS", + "ner": [ + [ + 15, + 23, + "archaeal", + "taxonomy_domain" + ], + [ + 64, + 70, + "ScTsr3", + "protein" + ], + [ + 83, + 90, + "archaea", + "taxonomy_domain" + ], + [ + 105, + 110, + "yeast", + "taxonomy_domain" + ] + ] + }, + { + "sid": 101, + "sent": "To locate the domains most important for Tsr3 activity, ScTsr3 fragments of different lengths containing the highly conserved central part were expressed in a \u0394tsr3 mutant (Figure 3A) and analyzed by primer extension (Figure 3B) and Northern blotting (Figure 3C).", + "section": "RESULTS", + "ner": [ + [ + 41, + 45, + "Tsr3", + "protein" + ], + [ + 56, + 62, + "ScTsr3", + "protein" + ], + [ + 109, + 125, + "highly conserved", + "protein_state" + ], + [ + 144, + 153, + "expressed", + "experimental_method" + ], + [ + 159, + 164, + "\u0394tsr3", + "mutant" + ], + [ + 165, + 171, + "mutant", + "protein_state" + ], + [ + 200, + 216, + "primer extension", + "experimental_method" + ], + [ + 233, + 250, + "Northern blotting", + "experimental_method" + ] + ] + }, + { + "sid": 102, + "sent": "N-terminal truncations of up to 45 aa and C-terminal truncations of up to 76 aa mediated acp modification as efficiently as the full-length protein and no significant increased levels of 20S pre-RNA were detected.", + "section": "RESULTS", + "ner": [ + [ + 11, + 22, + "truncations", + "experimental_method" + ], + [ + 32, + 37, + "45 aa", + "residue_range" + ], + [ + 53, + 64, + "truncations", + "experimental_method" + ], + [ + 74, + 79, + "76 aa", + "residue_range" + ], + [ + 89, + 92, + "acp", + "chemical" + ], + [ + 128, + 139, + "full-length", + "protein_state" + ], + [ + 187, + 198, + "20S pre-RNA", + "chemical" + ] + ] + }, + { + "sid": 103, + "sent": "Even a Tsr3 fragment with a 90 aa C-terminal truncation showed a residual primer extension stop, whereas N-terminal truncations exceeding 46 aa almost completely abolished the primer extension arrest (Figure 3B).", + "section": "RESULTS", + "ner": [ + [ + 7, + 11, + "Tsr3", + "protein" + ], + [ + 28, + 33, + "90 aa", + "residue_range" + ], + [ + 138, + 143, + "46 aa", + "residue_range" + ] + ] + }, + { + "sid": 104, + "sent": "Domain characterization of yeast Tsr3 and correlation of acp modification with late 18S rRNA processing steps. (A) Scheme of the TSR3 gene with truncation positions in the open reading frame.", + "section": "FIG", + "ner": [ + [ + 27, + 32, + "yeast", + "taxonomy_domain" + ], + [ + 33, + 37, + "Tsr3", + "protein" + ], + [ + 57, + 60, + "acp", + "chemical" + ], + [ + 84, + 92, + "18S rRNA", + "chemical" + ], + [ + 129, + 133, + "TSR3", + "protein" + ] + ] + }, + { + "sid": 105, + "sent": "TSR3 fragments of different length were expressed under the native promotor from multicopy plasmids in a \u0394tsr3 deletion strain.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "TSR3", + "protein" + ], + [ + 105, + 110, + "\u0394tsr3", + "mutant" + ] + ] + }, + { + "sid": 106, + "sent": "(B) Primer extension analysis of 18S rRNA acp modification in yeast cells expressing the indicated TSR3 fragments.", + "section": "FIG", + "ner": [ + [ + 4, + 29, + "Primer extension analysis", + "experimental_method" + ], + [ + 33, + 41, + "18S rRNA", + "chemical" + ], + [ + 42, + 45, + "acp", + "chemical" + ], + [ + 62, + 67, + "yeast", + "taxonomy_domain" + ], + [ + 99, + 103, + "TSR3", + "protein" + ] + ] + }, + { + "sid": 107, + "sent": "N-terminal deletions of 36 or 45 amino acids and C-terminal deletions of 43 or 76 residues show a primer extension stop comparable to the wild type.", + "section": "FIG", + "ner": [ + [ + 11, + 20, + "deletions", + "experimental_method" + ], + [ + 24, + 26, + "36", + "residue_range" + ], + [ + 30, + 32, + "45", + "residue_range" + ], + [ + 60, + 69, + "deletions", + "experimental_method" + ], + [ + 73, + 75, + "43", + "residue_range" + ], + [ + 79, + 81, + "76", + "residue_range" + ], + [ + 98, + 119, + "primer extension stop", + "evidence" + ], + [ + 138, + 147, + "wild type", + "protein_state" + ] + ] + }, + { + "sid": 108, + "sent": "Tsr3 fragments 37\u2013223 or 46\u2013223 cause a nearly complete loss of the arrest signal.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "Tsr3", + "protein" + ], + [ + 15, + 21, + "37\u2013223", + "residue_range" + ], + [ + 25, + 31, + "46\u2013223", + "residue_range" + ] + ] + }, + { + "sid": 109, + "sent": "The box highlights the shortest Tsr3 fragment (aa 46\u2013270) with wild type activity (strong primer extension block). (C) Northern blot analysis of 20S pre-rRNA accumulation.", + "section": "FIG", + "ner": [ + [ + 32, + 36, + "Tsr3", + "protein" + ], + [ + 50, + 56, + "46\u2013270", + "residue_range" + ], + [ + 63, + 72, + "wild type", + "protein_state" + ], + [ + 90, + 112, + "primer extension block", + "evidence" + ], + [ + 119, + 132, + "Northern blot", + "experimental_method" + ], + [ + 145, + 157, + "20S pre-rRNA", + "chemical" + ] + ] + }, + { + "sid": 110, + "sent": "A weak 20S rRNA signal, indicating normal processing, is observed for Tsr3 fragment 46\u2013270 (highlighted in a box) showing its functionality.", + "section": "FIG", + "ner": [ + [ + 7, + 15, + "20S rRNA", + "chemical" + ], + [ + 70, + 74, + "Tsr3", + "protein" + ], + [ + 84, + 90, + "46\u2013270", + "residue_range" + ] + ] + }, + { + "sid": 111, + "sent": "Strong 20S rRNA accumulation similar to that of the \u0394tsr3 deletion is observed for Tsr3 fragments 37\u2013223 or 46\u2013223.", + "section": "FIG", + "ner": [ + [ + 52, + 57, + "\u0394tsr3", + "mutant" + ], + [ + 58, + 66, + "deletion", + "experimental_method" + ], + [ + 83, + 87, + "Tsr3", + "protein" + ], + [ + 98, + 104, + "37\u2013223", + "residue_range" + ], + [ + 108, + 114, + "46\u2013223", + "residue_range" + ] + ] + }, + { + "sid": 112, + "sent": "Thus, the archaeal homologs correspond to the functional core of Tsr3.", + "section": "RESULTS", + "ner": [ + [ + 10, + 18, + "archaeal", + "taxonomy_domain" + ], + [ + 65, + 69, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 113, + "sent": "In order to define the structural basis for Tsr3 function, homologs from thermophilic archaea were screened for crystallization.", + "section": "RESULTS", + "ner": [ + [ + 44, + 48, + "Tsr3", + "protein" + ], + [ + 73, + 93, + "thermophilic archaea", + "taxonomy_domain" + ], + [ + 112, + 127, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 114, + "sent": "We focused on archaeal species containing a putative Nep1 homolog suggesting that these species are in principle capable of synthesizing N1-methyl-N3-acp-pseudouridine.", + "section": "RESULTS", + "ner": [ + [ + 14, + 22, + "archaeal", + "taxonomy_domain" + ], + [ + 53, + 57, + "Nep1", + "protein" + ], + [ + 137, + 167, + "N1-methyl-N3-acp-pseudouridine", + "chemical" + ] + ] + }, + { + "sid": 115, + "sent": "Well diffracting crystals were obtained for Tsr3 homologs from the two crenarchaeal species Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) which share 36% (VdTsr3) and 38% (SsTsr3) identity with the ScTsr3 core region (ScTsr3 aa 46\u2013223).", + "section": "RESULTS", + "ner": [ + [ + 17, + 25, + "crystals", + "evidence" + ], + [ + 44, + 48, + "Tsr3", + "protein" + ], + [ + 71, + 83, + "crenarchaeal", + "taxonomy_domain" + ], + [ + 92, + 115, + "Vulcanisaeta distributa", + "species" + ], + [ + 117, + 123, + "VdTsr3", + "protein" + ], + [ + 129, + 152, + "Sulfolobus solfataricus", + "species" + ], + [ + 154, + 160, + "SsTsr3", + "protein" + ], + [ + 179, + 185, + "VdTsr3", + "protein" + ], + [ + 196, + 202, + "SsTsr3", + "protein" + ], + [ + 222, + 228, + "ScTsr3", + "protein" + ], + [ + 229, + 240, + "core region", + "structure_element" + ], + [ + 242, + 248, + "ScTsr3", + "protein" + ], + [ + 252, + 258, + "46\u2013223", + "residue_range" + ] + ] + }, + { + "sid": 116, + "sent": "While for S. solfataricus the existence of a modified nucleotide of unknown chemical composition in the loop capping helix 31 of its 16S rRNA has been demonstrated, no information regarding rRNA modifications is yet available for V. distributa.", + "section": "RESULTS", + "ner": [ + [ + 10, + 25, + "S. solfataricus", + "species" + ], + [ + 54, + 64, + "nucleotide", + "chemical" + ], + [ + 104, + 125, + "loop capping helix 31", + "structure_element" + ], + [ + 133, + 141, + "16S rRNA", + "chemical" + ], + [ + 230, + 243, + "V. distributa", + "species" + ] + ] + }, + { + "sid": 117, + "sent": "Crystals of VdTsr3 diffracted to a resolution of 1.6 \u00c5 whereas crystals of SsTsr3 diffracted to 2.25 \u00c5. Serendipitously, VdTsr3 was purified and crystallized in complex with endogenous (E. coli) SAM (Supplementary Figure S4) while SsTsr3 crystals contained the protein in the apo state.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Crystals", + "evidence" + ], + [ + 12, + 18, + "VdTsr3", + "protein" + ], + [ + 63, + 71, + "crystals", + "evidence" + ], + [ + 75, + 81, + "SsTsr3", + "protein" + ], + [ + 121, + 127, + "VdTsr3", + "protein" + ], + [ + 145, + 157, + "crystallized", + "experimental_method" + ], + [ + 158, + 173, + "in complex with", + "protein_state" + ], + [ + 174, + 184, + "endogenous", + "protein_state" + ], + [ + 186, + 193, + "E. coli", + "species" + ], + [ + 195, + 198, + "SAM", + "chemical" + ], + [ + 231, + 237, + "SsTsr3", + "protein" + ], + [ + 238, + 246, + "crystals", + "evidence" + ], + [ + 276, + 279, + "apo", + "protein_state" + ] + ] + }, + { + "sid": 118, + "sent": "The structure of VdTsr3 was solved ab initio, by single-wavelength anomalous diffraction phasing (Se-SAD) with Se containing derivatives (selenomethionine and seleno-substituted SAM).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 17, + 23, + "VdTsr3", + "protein" + ], + [ + 49, + 96, + "single-wavelength anomalous diffraction phasing", + "experimental_method" + ], + [ + 98, + 104, + "Se-SAD", + "experimental_method" + ], + [ + 111, + 113, + "Se", + "chemical" + ], + [ + 138, + 154, + "selenomethionine", + "chemical" + ], + [ + 159, + 181, + "seleno-substituted SAM", + "chemical" + ] + ] + }, + { + "sid": 119, + "sent": "The structure of SsTsr3 was solved by molecular replacement using VdTsr3 as a search model (see Supplementary Table S1 for data collection and refinement statistics).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 17, + 23, + "SsTsr3", + "protein" + ], + [ + 38, + 59, + "molecular replacement", + "experimental_method" + ], + [ + 66, + 72, + "VdTsr3", + "protein" + ] + ] + }, + { + "sid": 120, + "sent": "The structure of VdTsr3 can be divided into two domains (Figure 4A).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 17, + 23, + "VdTsr3", + "protein" + ] + ] + }, + { + "sid": 121, + "sent": "The N-terminal domain (aa 1\u201392) has a mixed \u03b1/\u03b2-structure centered around a five-stranded all-parallel \u03b2-sheet (Figure 4B) with the strand order \u03b25\u2191-\u03b23\u2191-\u03b24\u2191-\u03b21\u2191-\u03b22\u2191. The loops connecting \u03b21 and \u03b22, \u03b23 and \u03b24 and \u03b24 and \u03b25 include \u03b1-helices \u03b11, \u03b12 and \u03b13, respectively.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "N-terminal domain", + "structure_element" + ], + [ + 26, + 30, + "1\u201392", + "residue_range" + ], + [ + 44, + 57, + "\u03b1/\u03b2-structure", + "structure_element" + ], + [ + 76, + 110, + "five-stranded all-parallel \u03b2-sheet", + "structure_element" + ], + [ + 145, + 148, + "\u03b25\u2191", + "structure_element" + ], + [ + 149, + 152, + "\u03b23\u2191", + "structure_element" + ], + [ + 153, + 156, + "\u03b24\u2191", + "structure_element" + ], + [ + 157, + 160, + "\u03b21\u2191", + "structure_element" + ], + [ + 161, + 164, + "\u03b22\u2191", + "structure_element" + ], + [ + 170, + 175, + "loops", + "structure_element" + ], + [ + 187, + 189, + "\u03b21", + "structure_element" + ], + [ + 194, + 196, + "\u03b22", + "structure_element" + ], + [ + 198, + 200, + "\u03b23", + "structure_element" + ], + [ + 205, + 207, + "\u03b24", + "structure_element" + ], + [ + 212, + 214, + "\u03b24", + "structure_element" + ], + [ + 219, + 221, + "\u03b25", + "structure_element" + ], + [ + 230, + 239, + "\u03b1-helices", + "structure_element" + ], + [ + 240, + 242, + "\u03b11", + "structure_element" + ], + [ + 244, + 246, + "\u03b12", + "structure_element" + ], + [ + 251, + 253, + "\u03b13", + "structure_element" + ] + ] + }, + { + "sid": 122, + "sent": "The loop connecting \u03b22 and \u03b23 contains a single turn of a 310-helix. Helices \u03b11 and \u03b12 are located on one side of the five-stranded \u03b2-sheet while \u03b13 packs against the opposite \u03b2-sheet surface.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "loop", + "structure_element" + ], + [ + 20, + 22, + "\u03b22", + "structure_element" + ], + [ + 27, + 29, + "\u03b23", + "structure_element" + ], + [ + 58, + 67, + "310-helix", + "structure_element" + ], + [ + 69, + 76, + "Helices", + "structure_element" + ], + [ + 77, + 79, + "\u03b11", + "structure_element" + ], + [ + 84, + 86, + "\u03b12", + "structure_element" + ], + [ + 118, + 139, + "five-stranded \u03b2-sheet", + "structure_element" + ], + [ + 146, + 148, + "\u03b13", + "structure_element" + ], + [ + 176, + 183, + "\u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 123, + "sent": "The C-terminal domain (aa 93\u2013184) has a globular all \u03b1-helical structure comprising \u03b1-helices \u03b14 to \u03b19.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "C-terminal domain", + "structure_element" + ], + [ + 26, + 32, + "93\u2013184", + "residue_range" + ], + [ + 40, + 72, + "globular all \u03b1-helical structure", + "structure_element" + ], + [ + 84, + 93, + "\u03b1-helices", + "structure_element" + ], + [ + 94, + 102, + "\u03b14 to \u03b19", + "structure_element" + ] + ] + }, + { + "sid": 124, + "sent": "Remarkably, the entire C-terminal domain (92 aa) of the protein is threaded through the loop which connects \u03b2-strand \u03b23 and \u03b1-helix \u03b12 of the N-terminal domain.", + "section": "RESULTS", + "ner": [ + [ + 23, + 40, + "C-terminal domain", + "structure_element" + ], + [ + 42, + 47, + "92 aa", + "residue_range" + ], + [ + 88, + 92, + "loop", + "structure_element" + ], + [ + 108, + 116, + "\u03b2-strand", + "structure_element" + ], + [ + 117, + 119, + "\u03b23", + "structure_element" + ], + [ + 124, + 131, + "\u03b1-helix", + "structure_element" + ], + [ + 132, + 134, + "\u03b12", + "structure_element" + ], + [ + 142, + 159, + "N-terminal domain", + "structure_element" + ] + ] + }, + { + "sid": 125, + "sent": "Thus, the VdTsr3 structure contains a deep trefoil knot.", + "section": "RESULTS", + "ner": [ + [ + 10, + 16, + "VdTsr3", + "protein" + ], + [ + 17, + 26, + "structure", + "evidence" + ], + [ + 38, + 55, + "deep trefoil knot", + "structure_element" + ] + ] + }, + { + "sid": 126, + "sent": "The structure of SsTsr3 in the apo state is very similar to that of VdTsr3 (Figure 4C) with an RMSD for equivalent C\u03b1 atoms of 1.1 \u00c5. The only significant difference in the global structure of the two proteins is the presence of an extended \u03b1-helix \u03b18 and the absence of \u03b1-helix \u03b19 in SsTsr3.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 17, + 23, + "SsTsr3", + "protein" + ], + [ + 31, + 34, + "apo", + "protein_state" + ], + [ + 68, + 74, + "VdTsr3", + "protein" + ], + [ + 95, + 99, + "RMSD", + "evidence" + ], + [ + 180, + 189, + "structure", + "evidence" + ], + [ + 241, + 248, + "\u03b1-helix", + "structure_element" + ], + [ + 249, + 251, + "\u03b18", + "structure_element" + ], + [ + 260, + 270, + "absence of", + "protein_state" + ], + [ + 271, + 278, + "\u03b1-helix", + "structure_element" + ], + [ + 279, + 281, + "\u03b19", + "structure_element" + ], + [ + 285, + 291, + "SsTsr3", + "protein" + ] + ] + }, + { + "sid": 127, + "sent": "Tsr3 has a fold similar to SPOUT-class RNA methyltransferases. (A) Cartoon representation of the X-ray structure of VdTsr3 in two orientations.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "Tsr3", + "protein" + ], + [ + 27, + 61, + "SPOUT-class RNA methyltransferases", + "protein_type" + ], + [ + 97, + 112, + "X-ray structure", + "evidence" + ], + [ + 116, + 122, + "VdTsr3", + "protein" + ] + ] + }, + { + "sid": 128, + "sent": "\u03b2-strands are colored in crimson whereas \u03b1-helices in the N-terminal domain are colored light blue and \u03b1-helices in the C-terminal domain are colored dark blue.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "\u03b2-strands", + "structure_element" + ], + [ + 41, + 50, + "\u03b1-helices", + "structure_element" + ], + [ + 58, + 75, + "N-terminal domain", + "structure_element" + ], + [ + 103, + 112, + "\u03b1-helices", + "structure_element" + ], + [ + 120, + 137, + "C-terminal domain", + "structure_element" + ] + ] + }, + { + "sid": 129, + "sent": "The bound S-adenosylmethionine is shown in a stick representation and colored by atom type.", + "section": "FIG", + "ner": [ + [ + 10, + 30, + "S-adenosylmethionine", + "chemical" + ] + ] + }, + { + "sid": 130, + "sent": "A red arrow marks the location of the topological knot in the structure. (B) Secondary structure representation of the VdTsr3 structure.", + "section": "FIG", + "ner": [ + [ + 38, + 54, + "topological knot", + "structure_element" + ], + [ + 62, + 71, + "structure", + "evidence" + ], + [ + 119, + 125, + "VdTsr3", + "protein" + ], + [ + 126, + 135, + "structure", + "evidence" + ] + ] + }, + { + "sid": 131, + "sent": "The color coding is the same as in (A). (C) Structural superposition of the X-ray structures of VdTsr3 in the SAM-bound state (red) and SsTsr3 (blue) in the apo state.", + "section": "FIG", + "ner": [ + [ + 44, + 68, + "Structural superposition", + "experimental_method" + ], + [ + 76, + 92, + "X-ray structures", + "evidence" + ], + [ + 96, + 102, + "VdTsr3", + "protein" + ], + [ + 110, + 119, + "SAM-bound", + "protein_state" + ], + [ + 136, + 142, + "SsTsr3", + "protein" + ], + [ + 157, + 160, + "apo", + "protein_state" + ] + ] + }, + { + "sid": 132, + "sent": "The locations of the \u03b1-helix \u03b18 which is longer in SsTsr3 and of \u03b1-helix \u03b19 which is only present in VdTsr3 are indicated. (D) Secondary structure cartoon (left) of S. pombe Trm10 (pdb4jwf)\u2014the SPOUT-class RNA methyltransferase structurally most similar to Tsr3 and superposition of the VdTsr3 and Trm10 X-ray structures (right). (E) Analytical gel filtration profiles for VdTsr3 (red) and SsTsr3 (blue) show that both proteins are monomeric in solution.", + "section": "FIG", + "ner": [ + [ + 21, + 28, + "\u03b1-helix", + "structure_element" + ], + [ + 29, + 31, + "\u03b18", + "structure_element" + ], + [ + 51, + 57, + "SsTsr3", + "protein" + ], + [ + 65, + 72, + "\u03b1-helix", + "structure_element" + ], + [ + 73, + 75, + "\u03b19", + "structure_element" + ], + [ + 101, + 107, + "VdTsr3", + "protein" + ], + [ + 165, + 173, + "S. pombe", + "species" + ], + [ + 174, + 179, + "Trm10", + "protein" + ], + [ + 194, + 227, + "SPOUT-class RNA methyltransferase", + "protein_type" + ], + [ + 257, + 261, + "Tsr3", + "protein" + ], + [ + 266, + 279, + "superposition", + "experimental_method" + ], + [ + 287, + 293, + "VdTsr3", + "protein" + ], + [ + 298, + 303, + "Trm10", + "protein" + ], + [ + 304, + 320, + "X-ray structures", + "evidence" + ], + [ + 334, + 359, + "Analytical gel filtration", + "experimental_method" + ], + [ + 360, + 368, + "profiles", + "evidence" + ], + [ + 373, + 379, + "VdTsr3", + "protein" + ], + [ + 390, + 396, + "SsTsr3", + "protein" + ], + [ + 432, + 441, + "monomeric", + "oligomeric_state" + ] + ] + }, + { + "sid": 133, + "sent": "Vd, Vulcanisaeta distributa; Ss, Sulfolobus solfataricus.", + "section": "FIG", + "ner": [ + [ + 0, + 2, + "Vd", + "species" + ], + [ + 4, + 27, + "Vulcanisaeta distributa", + "species" + ], + [ + 29, + 31, + "Ss", + "species" + ], + [ + 33, + 56, + "Sulfolobus solfataricus", + "species" + ] + ] + }, + { + "sid": 134, + "sent": "Structure predictions suggested that Tsr3 might contain a so-called RLI domain which contains a \u2018bacterial like\u2019 ferredoxin fold and binds two iron-sulfur clusters through eight conserved cysteine residues.", + "section": "RESULTS", + "ner": [ + [ + 0, + 21, + "Structure predictions", + "experimental_method" + ], + [ + 37, + 41, + "Tsr3", + "protein" + ], + [ + 68, + 78, + "RLI domain", + "structure_element" + ], + [ + 97, + 128, + "bacterial like\u2019 ferredoxin fold", + "structure_element" + ], + [ + 178, + 187, + "conserved", + "protein_state" + ], + [ + 188, + 196, + "cysteine", + "residue_name" + ] + ] + }, + { + "sid": 135, + "sent": "However, no structural similarity to an RLI-domain was detectable.", + "section": "RESULTS", + "ner": [ + [ + 40, + 50, + "RLI-domain", + "structure_element" + ] + ] + }, + { + "sid": 136, + "sent": "This is in accordance with the functional analysis of alanine replacement mutations of cysteine residues in ScTsr3 (Supplementary Figure S3).", + "section": "RESULTS", + "ner": [ + [ + 54, + 83, + "alanine replacement mutations", + "experimental_method" + ], + [ + 87, + 95, + "cysteine", + "residue_name" + ], + [ + 108, + 114, + "ScTsr3", + "protein" + ] + ] + }, + { + "sid": 137, + "sent": "The \u03b2-strand topology and the deep C-terminal trefoil knot of archaeal Tsr3 are the structural hallmarks of the SPOUT-class RNA-methyltransferase fold.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "\u03b2-strand topology", + "structure_element" + ], + [ + 46, + 58, + "trefoil knot", + "structure_element" + ], + [ + 62, + 70, + "archaeal", + "taxonomy_domain" + ], + [ + 71, + 75, + "Tsr3", + "protein" + ], + [ + 112, + 145, + "SPOUT-class RNA-methyltransferase", + "protein_type" + ] + ] + }, + { + "sid": 138, + "sent": "The closest structural homolog identified in a DALI search is the tRNA methyltransferase Trm10 (DALI Z-score 6.8) which methylates the N1 nitrogen of G9/A9 in many archaeal and eukaryotic tRNAs by using SAM as the methyl group donor.", + "section": "RESULTS", + "ner": [ + [ + 47, + 58, + "DALI search", + "experimental_method" + ], + [ + 66, + 88, + "tRNA methyltransferase", + "protein_type" + ], + [ + 89, + 94, + "Trm10", + "protein" + ], + [ + 96, + 108, + "DALI Z-score", + "evidence" + ], + [ + 150, + 152, + "G9", + "residue_name_number" + ], + [ + 153, + 155, + "A9", + "residue_name_number" + ], + [ + 164, + 172, + "archaeal", + "taxonomy_domain" + ], + [ + 177, + 187, + "eukaryotic", + "taxonomy_domain" + ], + [ + 188, + 193, + "tRNAs", + "chemical" + ], + [ + 203, + 206, + "SAM", + "chemical" + ] + ] + }, + { + "sid": 139, + "sent": "In comparison to Tsr3 the central \u03b2-sheet element of Trm10 is extended by one additional \u03b2-strand pairing to \u03b22.", + "section": "RESULTS", + "ner": [ + [ + 17, + 21, + "Tsr3", + "protein" + ], + [ + 34, + 49, + "\u03b2-sheet element", + "structure_element" + ], + [ + 53, + 58, + "Trm10", + "protein" + ], + [ + 89, + 97, + "\u03b2-strand", + "structure_element" + ], + [ + 109, + 111, + "\u03b22", + "structure_element" + ] + ] + }, + { + "sid": 140, + "sent": "Furthermore, the trefoil knot of Trm10 is not as deep as that of Tsr3 (Figure 4D).", + "section": "RESULTS", + "ner": [ + [ + 17, + 29, + "trefoil knot", + "structure_element" + ], + [ + 33, + 38, + "Trm10", + "protein" + ], + [ + 65, + 69, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 141, + "sent": "Interestingly, Nep1\u2014the enzyme preceding Tsr3 in the biosynthetic pathway for the synthesis of m1acp3\u03a8\u2014also belongs to the SPOUT-class of RNA methyltransferases.", + "section": "RESULTS", + "ner": [ + [ + 15, + 19, + "Nep1", + "protein" + ], + [ + 41, + 45, + "Tsr3", + "protein" + ], + [ + 95, + 102, + "m1acp3\u03a8", + "chemical" + ], + [ + 123, + 160, + "SPOUT-class of RNA methyltransferases", + "protein_type" + ] + ] + }, + { + "sid": 142, + "sent": "However, the structural similarities between Nep1 and Tsr3 (DALI Z-score 4.4) are less pronounced than between Tsr3 and Trm10.", + "section": "RESULTS", + "ner": [ + [ + 45, + 49, + "Nep1", + "protein" + ], + [ + 54, + 58, + "Tsr3", + "protein" + ], + [ + 60, + 72, + "DALI Z-score", + "evidence" + ], + [ + 111, + 115, + "Tsr3", + "protein" + ], + [ + 120, + 125, + "Trm10", + "protein" + ] + ] + }, + { + "sid": 143, + "sent": "Most SPOUT-class RNA-methyltransferases are homodimers.", + "section": "RESULTS", + "ner": [ + [ + 5, + 39, + "SPOUT-class RNA-methyltransferases", + "protein_type" + ], + [ + 44, + 54, + "homodimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 144, + "sent": "A notable exception is Trm10.", + "section": "RESULTS", + "ner": [ + [ + 23, + 28, + "Trm10", + "protein" + ] + ] + }, + { + "sid": 145, + "sent": "Gel filtration experiments with both VdTsr3 and SsTsr3 (Figure 4E) showed that both proteins are monomeric in solution thereby extending the structural similarities to Trm10.", + "section": "RESULTS", + "ner": [ + [ + 0, + 14, + "Gel filtration", + "experimental_method" + ], + [ + 37, + 43, + "VdTsr3", + "protein" + ], + [ + 48, + 54, + "SsTsr3", + "protein" + ], + [ + 97, + 106, + "monomeric", + "oligomeric_state" + ], + [ + 168, + 173, + "Trm10", + "protein" + ] + ] + }, + { + "sid": 146, + "sent": "So far, structural information is only available for one other enzyme that transfers the acp group from SAM to an RNA nucleotide.", + "section": "RESULTS", + "ner": [ + [ + 89, + 92, + "acp", + "chemical" + ], + [ + 104, + 107, + "SAM", + "chemical" + ], + [ + 114, + 117, + "RNA", + "chemical" + ], + [ + 118, + 128, + "nucleotide", + "chemical" + ] + ] + }, + { + "sid": 147, + "sent": "This enzyme, Tyw2, is part of the biosynthesis pathway of wybutosine nucleotides in tRNAs.", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "Tyw2", + "protein" + ], + [ + 58, + 80, + "wybutosine nucleotides", + "chemical" + ], + [ + 84, + 89, + "tRNAs", + "chemical" + ] + ] + }, + { + "sid": 148, + "sent": "However, there are no structural similarities between Tsr3 and Tyw2, which contains an all-parallel \u03b2-sheet of a different topology and no knot structure.", + "section": "RESULTS", + "ner": [ + [ + 54, + 58, + "Tsr3", + "protein" + ], + [ + 63, + 67, + "Tyw2", + "protein" + ], + [ + 87, + 107, + "all-parallel \u03b2-sheet", + "structure_element" + ], + [ + 139, + 153, + "knot structure", + "structure_element" + ] + ] + }, + { + "sid": 149, + "sent": "Instead, Tyw2 has a fold typical for the class-I-or Rossmann-fold class of methyltransferases (Supplementary Figure S5B).", + "section": "RESULTS", + "ner": [ + [ + 9, + 13, + "Tyw2", + "protein" + ], + [ + 41, + 93, + "class-I-or Rossmann-fold class of methyltransferases", + "protein_type" + ] + ] + }, + { + "sid": 150, + "sent": "Cofactor binding of Tsr3", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 151, + "sent": "The SAM-binding site of Tsr3 is located in a deep crevice between the N- and C-terminal domains in the vicinity of the trefoil knot as typical for SPOUT-class RNA-methyltransferases (Figure 4A).", + "section": "RESULTS", + "ner": [ + [ + 4, + 20, + "SAM-binding site", + "site" + ], + [ + 24, + 28, + "Tsr3", + "protein" + ], + [ + 70, + 95, + "N- and C-terminal domains", + "structure_element" + ], + [ + 119, + 131, + "trefoil knot", + "structure_element" + ], + [ + 147, + 181, + "SPOUT-class RNA-methyltransferases", + "protein_type" + ] + ] + }, + { + "sid": 152, + "sent": "The adenine base of the cofactor is recognized by hydrogen bonds between its N1 nitrogen and the backbone amide of L93 directly preceding \u03b25 as well as between its N6-amino group and the backbone carbonyl group of Y108 located in the loop connecting \u03b25 in the N-terminal and \u03b14 in the C-terminal domain (Figure 5A).", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "adenine", + "chemical" + ], + [ + 50, + 64, + "hydrogen bonds", + "bond_interaction" + ], + [ + 115, + 118, + "L93", + "residue_name_number" + ], + [ + 138, + 140, + "\u03b25", + "structure_element" + ], + [ + 214, + 218, + "Y108", + "residue_name_number" + ], + [ + 234, + 238, + "loop", + "structure_element" + ], + [ + 250, + 252, + "\u03b25", + "structure_element" + ], + [ + 260, + 270, + "N-terminal", + "structure_element" + ], + [ + 275, + 277, + "\u03b14", + "structure_element" + ], + [ + 285, + 302, + "C-terminal domain", + "structure_element" + ] + ] + }, + { + "sid": 153, + "sent": "Furthermore, the adenine base of SAM is involved in hydrophobic packing interactions with the side chains of L45 (\u03b23), P47 and W73 (\u03b13) in the N-terminal domain as well as with L93, L110 (both in the loop connecting \u03b25 and \u03b14) and A115 (\u03b15) in the C-terminal domain.", + "section": "RESULTS", + "ner": [ + [ + 17, + 24, + "adenine", + "chemical" + ], + [ + 33, + 36, + "SAM", + "chemical" + ], + [ + 52, + 84, + "hydrophobic packing interactions", + "bond_interaction" + ], + [ + 109, + 112, + "L45", + "residue_name_number" + ], + [ + 114, + 116, + "\u03b23", + "structure_element" + ], + [ + 119, + 122, + "P47", + "residue_name_number" + ], + [ + 127, + 130, + "W73", + "residue_name_number" + ], + [ + 132, + 134, + "\u03b13", + "structure_element" + ], + [ + 143, + 160, + "N-terminal domain", + "structure_element" + ], + [ + 177, + 180, + "L93", + "residue_name_number" + ], + [ + 182, + 186, + "L110", + "residue_name_number" + ], + [ + 200, + 204, + "loop", + "structure_element" + ], + [ + 216, + 218, + "\u03b25", + "structure_element" + ], + [ + 223, + 225, + "\u03b14", + "structure_element" + ], + [ + 231, + 235, + "A115", + "residue_name_number" + ], + [ + 237, + 239, + "\u03b15", + "structure_element" + ], + [ + 248, + 265, + "C-terminal domain", + "structure_element" + ] + ] + }, + { + "sid": 154, + "sent": "The ribose 2\u2032 and 3\u2032 hydroxyl groups of SAM are hydrogen bonded to the backbone carbonyl group of I69.", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "ribose", + "chemical" + ], + [ + 40, + 43, + "SAM", + "chemical" + ], + [ + 48, + 63, + "hydrogen bonded", + "bond_interaction" + ], + [ + 98, + 101, + "I69", + "residue_name_number" + ] + ] + }, + { + "sid": 155, + "sent": "The acp side chain of SAM is fixed in position by hydrogen bonding of its carboxylate group to the backbone amide and the side chain hydroxyl group of T19 in \u03b11 as well as the backbone amide group of T112 in \u03b14 (C-terminal domain).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "acp", + "chemical" + ], + [ + 22, + 25, + "SAM", + "chemical" + ], + [ + 50, + 66, + "hydrogen bonding", + "bond_interaction" + ], + [ + 151, + 154, + "T19", + "residue_name_number" + ], + [ + 158, + 160, + "\u03b11", + "structure_element" + ], + [ + 200, + 204, + "T112", + "residue_name_number" + ], + [ + 208, + 210, + "\u03b14", + "structure_element" + ], + [ + 212, + 229, + "C-terminal domain", + "structure_element" + ] + ] + }, + { + "sid": 156, + "sent": "Most importantly, the methyl group of SAM is buried in a hydrophobic pocket formed by the sidechains of W73 and A76 both located in \u03b13 (Figure 5A and\u00a0B).", + "section": "RESULTS", + "ner": [ + [ + 38, + 41, + "SAM", + "chemical" + ], + [ + 57, + 75, + "hydrophobic pocket", + "site" + ], + [ + 104, + 107, + "W73", + "residue_name_number" + ], + [ + 112, + 115, + "A76", + "residue_name_number" + ], + [ + 132, + 134, + "\u03b13", + "structure_element" + ] + ] + }, + { + "sid": 157, + "sent": "W73 is highly conserved in all known Tsr3 proteins, whereas A76 can be replaced by other hydrophobic amino acids.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "W73", + "residue_name_number" + ], + [ + 7, + 23, + "highly conserved", + "protein_state" + ], + [ + 37, + 50, + "Tsr3 proteins", + "protein_type" + ], + [ + 60, + 63, + "A76", + "residue_name_number" + ], + [ + 101, + 112, + "amino acids", + "chemical" + ] + ] + }, + { + "sid": 158, + "sent": "Consequently, the accessibility of this methyl group for a nucleophilic attack is strongly reduced in comparison with RNA-methyltransferases such as Trm10 (Figure 5B, C).", + "section": "RESULTS", + "ner": [ + [ + 118, + 140, + "RNA-methyltransferases", + "protein_type" + ], + [ + 149, + 154, + "Trm10", + "protein" + ] + ] + }, + { + "sid": 159, + "sent": "In contrast, the acp side chain of SAM is accessible for reactions in the Tsr3-bound state (Figure 5B).", + "section": "RESULTS", + "ner": [ + [ + 17, + 20, + "acp", + "chemical" + ], + [ + 35, + 38, + "SAM", + "chemical" + ], + [ + 74, + 84, + "Tsr3-bound", + "protein_state" + ] + ] + }, + { + "sid": 160, + "sent": "SAM-binding by Tsr3.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "SAM", + "chemical" + ], + [ + 15, + 19, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 161, + "sent": "(A) Close-up view of the SAM-binding pocket of VdTsr3.", + "section": "FIG", + "ner": [ + [ + 25, + 43, + "SAM-binding pocket", + "site" + ], + [ + 47, + 53, + "VdTsr3", + "protein" + ] + ] + }, + { + "sid": 162, + "sent": "Nitrogen atoms are dark blue, oxygen atoms red, sulfur atoms orange, carbon atoms of the protein light blue and carbon atoms of SAM yellow.", + "section": "FIG", + "ner": [ + [ + 48, + 54, + "sulfur", + "chemical" + ], + [ + 128, + 131, + "SAM", + "chemical" + ] + ] + }, + { + "sid": 163, + "sent": "Hydrogen bonds are indicated by dashed lines.", + "section": "FIG", + "ner": [ + [ + 0, + 14, + "Hydrogen bonds", + "bond_interaction" + ] + ] + }, + { + "sid": 164, + "sent": "(B) Solvent accessibility of the acp group of SAM bound to VdTsr3.", + "section": "FIG", + "ner": [ + [ + 33, + 36, + "acp", + "chemical" + ], + [ + 46, + 49, + "SAM", + "chemical" + ], + [ + 50, + 58, + "bound to", + "protein_state" + ], + [ + 59, + 65, + "VdTsr3", + "protein" + ] + ] + }, + { + "sid": 165, + "sent": "The solvent accessible surface of the protein is shown in semitransparent gray whereas SAM is show in a stick representation.", + "section": "FIG", + "ner": [ + [ + 87, + 90, + "SAM", + "chemical" + ] + ] + }, + { + "sid": 166, + "sent": "A red arrow indicates the reactive CH2-moiety of the acp group. (C) Solvent accessibility of the SAM methyl group for SAM bound to the RNA methyltransferase Trm10.", + "section": "FIG", + "ner": [ + [ + 53, + 56, + "acp", + "chemical" + ], + [ + 97, + 100, + "SAM", + "chemical" + ], + [ + 118, + 121, + "SAM", + "chemical" + ], + [ + 122, + 130, + "bound to", + "protein_state" + ], + [ + 135, + 156, + "RNA methyltransferase", + "protein_type" + ], + [ + 157, + 162, + "Trm10", + "protein" + ] + ] + }, + { + "sid": 167, + "sent": "Bound SAM was modelled based on the X-ray structure of the Trm10/SAH-complex (pdb4jwf).", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "Bound", + "protein_state" + ], + [ + 6, + 9, + "SAM", + "chemical" + ], + [ + 36, + 51, + "X-ray structure", + "evidence" + ], + [ + 59, + 68, + "Trm10/SAH", + "complex_assembly" + ] + ] + }, + { + "sid": 168, + "sent": "A red arrow indicates the SAM methyl group. (D) Binding of SAM analogs to SsTsr3.", + "section": "FIG", + "ner": [ + [ + 26, + 29, + "SAM", + "chemical" + ], + [ + 59, + 62, + "SAM", + "chemical" + ], + [ + 74, + 80, + "SsTsr3", + "protein" + ] + ] + }, + { + "sid": 169, + "sent": "Tryptophan fluorescence quenching curves upon addition of SAM (blue), 5\u2032-methyl-thioadenosine (red) and SAH (black).", + "section": "FIG", + "ner": [ + [ + 0, + 40, + "Tryptophan fluorescence quenching curves", + "evidence" + ], + [ + 58, + 61, + "SAM", + "chemical" + ], + [ + 70, + 93, + "5\u2032-methyl-thioadenosine", + "chemical" + ], + [ + 104, + 107, + "SAH", + "chemical" + ] + ] + }, + { + "sid": 170, + "sent": "(E) Binding of 14C-labeled SAM to SsTsr3.", + "section": "FIG", + "ner": [ + [ + 15, + 30, + "14C-labeled SAM", + "chemical" + ], + [ + 34, + 40, + "SsTsr3", + "protein" + ] + ] + }, + { + "sid": 171, + "sent": "Radioactively labeled SAM is retained on a filter in the presence of SsTsr3.", + "section": "FIG", + "ner": [ + [ + 22, + 25, + "SAM", + "chemical" + ], + [ + 57, + 68, + "presence of", + "protein_state" + ], + [ + 69, + 75, + "SsTsr3", + "protein" + ] + ] + }, + { + "sid": 172, + "sent": "Addition of unlabeled SAM competes with the binding of labeled SAM.", + "section": "FIG", + "ner": [ + [ + 22, + 25, + "SAM", + "chemical" + ], + [ + 63, + 66, + "SAM", + "chemical" + ] + ] + }, + { + "sid": 173, + "sent": "A W66A-mutant of SsTsr3 (W73 in VdTsr3) does not bind SAM.", + "section": "FIG", + "ner": [ + [ + 2, + 6, + "W66A", + "mutant" + ], + [ + 7, + 13, + "mutant", + "protein_state" + ], + [ + 17, + 23, + "SsTsr3", + "protein" + ], + [ + 25, + 28, + "W73", + "residue_name_number" + ], + [ + 32, + 38, + "VdTsr3", + "protein" + ], + [ + 54, + 57, + "SAM", + "chemical" + ] + ] + }, + { + "sid": 174, + "sent": "(F) Primer extension (upper left) shows a strongly reduced acp modification of yeast 18S rRNA in \u0394tsr3 cells expressing Tsr3-S62D, -E111A or \u2013W114A.", + "section": "FIG", + "ner": [ + [ + 4, + 20, + "Primer extension", + "experimental_method" + ], + [ + 59, + 62, + "acp", + "chemical" + ], + [ + 79, + 84, + "yeast", + "taxonomy_domain" + ], + [ + 85, + 93, + "18S rRNA", + "chemical" + ], + [ + 97, + 102, + "\u0394tsr3", + "mutant" + ], + [ + 120, + 129, + "Tsr3-S62D", + "mutant" + ], + [ + 131, + 137, + "-E111A", + "mutant" + ], + [ + 141, + 147, + "\u2013W114A", + "mutant" + ] + ] + }, + { + "sid": 175, + "sent": "This correlates with a 20S pre-rRNA accumulation comparable to the \u0394tsr3 deletion (right: northern blot).", + "section": "FIG", + "ner": [ + [ + 23, + 35, + "20S pre-rRNA", + "chemical" + ], + [ + 67, + 72, + "\u0394tsr3", + "mutant" + ], + [ + 90, + 103, + "northern blot", + "experimental_method" + ] + ] + }, + { + "sid": 176, + "sent": "3xHA tagged Tsr3 mutants are expressed comparable to the wild type as shown by western blot (lower left).", + "section": "FIG", + "ner": [ + [ + 0, + 11, + "3xHA tagged", + "protein_state" + ], + [ + 12, + 16, + "Tsr3", + "protein" + ], + [ + 17, + 24, + "mutants", + "protein_state" + ], + [ + 57, + 66, + "wild type", + "protein_state" + ], + [ + 79, + 91, + "western blot", + "experimental_method" + ] + ] + }, + { + "sid": 177, + "sent": "Binding affinities for SAM and its analogs 5\u2032-methylthioadenosin and SAH to SsTsr3 were measured using tryptophan fluorescence quenching.", + "section": "RESULTS", + "ner": [ + [ + 0, + 18, + "Binding affinities", + "evidence" + ], + [ + 23, + 26, + "SAM", + "chemical" + ], + [ + 43, + 64, + "5\u2032-methylthioadenosin", + "chemical" + ], + [ + 69, + 72, + "SAH", + "chemical" + ], + [ + 76, + 82, + "SsTsr3", + "protein" + ], + [ + 103, + 136, + "tryptophan fluorescence quenching", + "experimental_method" + ] + ] + }, + { + "sid": 178, + "sent": "VdTsr3 could not be used in these experiments since we could not purify it in a stable SAM-free form.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "VdTsr3", + "protein" + ], + [ + 80, + 86, + "stable", + "protein_state" + ], + [ + 87, + 95, + "SAM-free", + "protein_state" + ] + ] + }, + { + "sid": 179, + "sent": "SsTsr3 bound SAM with a KD of 6.5 \u03bcM, which is similar to SAM-KD's reported for several SPOUT-class methyltransferases.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "SsTsr3", + "protein" + ], + [ + 7, + 12, + "bound", + "protein_state" + ], + [ + 13, + 16, + "SAM", + "chemical" + ], + [ + 24, + 26, + "KD", + "evidence" + ], + [ + 58, + 66, + "SAM-KD's", + "evidence" + ], + [ + 88, + 118, + "SPOUT-class methyltransferases", + "protein_type" + ] + ] + }, + { + "sid": 180, + "sent": "5\u2032-methylthioadenosin\u2014the reaction product after the acp-transfer\u2014binds only \u223c2.5-fold weaker (KD = 16.7 \u03bcM) compared to SAM.", + "section": "RESULTS", + "ner": [ + [ + 0, + 21, + "5\u2032-methylthioadenosin", + "chemical" + ], + [ + 53, + 56, + "acp", + "chemical" + ], + [ + 121, + 124, + "SAM", + "chemical" + ] + ] + }, + { + "sid": 181, + "sent": "S-adenosylhomocysteine which lacks the methyl group of SAM binds with significantly lower affinity (KD = 55.5 \u03bcM) (Figure 5D).", + "section": "RESULTS", + "ner": [ + [ + 0, + 22, + "S-adenosylhomocysteine", + "chemical" + ], + [ + 55, + 58, + "SAM", + "chemical" + ], + [ + 90, + 98, + "affinity", + "evidence" + ], + [ + 100, + 102, + "KD", + "evidence" + ] + ] + }, + { + "sid": 182, + "sent": "This suggests that the hydrophobic interaction between SAM's methyl group and the hydrophobic pocket of Tsr3 is thermodynamically important for the interaction.", + "section": "RESULTS", + "ner": [ + [ + 23, + 46, + "hydrophobic interaction", + "bond_interaction" + ], + [ + 55, + 58, + "SAM", + "chemical" + ], + [ + 82, + 100, + "hydrophobic pocket", + "site" + ], + [ + 104, + 108, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 183, + "sent": "On the other hand, the loss of hydrogen bonds between the acp sidechain carboxylate group and the protein appears to be thermodynamically less important but these hydrogen bonds might play a crucial role for the proper orientation of the cofactor side chain in the substrate binding pocket.", + "section": "RESULTS", + "ner": [ + [ + 31, + 45, + "hydrogen bonds", + "bond_interaction" + ], + [ + 58, + 61, + "acp", + "chemical" + ], + [ + 163, + 177, + "hydrogen bonds", + "bond_interaction" + ], + [ + 265, + 289, + "substrate binding pocket", + "site" + ] + ] + }, + { + "sid": 184, + "sent": "Accordingly, a W66A-mutation (W73 in VdTsr3) of SsTsr3 significantly diminished SAM-binding in a filter binding assay compared to the wild type (Figure 5E).", + "section": "RESULTS", + "ner": [ + [ + 15, + 19, + "W66A", + "mutant" + ], + [ + 20, + 28, + "mutation", + "experimental_method" + ], + [ + 30, + 33, + "W73", + "residue_name_number" + ], + [ + 37, + 43, + "VdTsr3", + "protein" + ], + [ + 48, + 54, + "SsTsr3", + "protein" + ], + [ + 80, + 91, + "SAM-binding", + "evidence" + ], + [ + 97, + 117, + "filter binding assay", + "experimental_method" + ], + [ + 134, + 143, + "wild type", + "protein_state" + ] + ] + }, + { + "sid": 185, + "sent": "Furthermore, a W to A mutation at the equivalent position W114 in ScTsr3 strongly reduced the in vivo acp transferase activity (Figure 5F).", + "section": "RESULTS", + "ner": [ + [ + 15, + 30, + "W to A mutation", + "experimental_method" + ], + [ + 58, + 62, + "W114", + "residue_name_number" + ], + [ + 66, + 72, + "ScTsr3", + "protein" + ], + [ + 102, + 117, + "acp transferase", + "protein_type" + ] + ] + }, + { + "sid": 186, + "sent": "The side chain hydroxyl group of T19 seems of minor importance for SAM binding since mutations of T17 (T19 in VdTsr3) to either A or D did not significantly influence the SAM-binding affinity of SsTsr3 (KD's = 3.9 or 11.2 mM, respectively).", + "section": "RESULTS", + "ner": [ + [ + 33, + 36, + "T19", + "residue_name_number" + ], + [ + 67, + 70, + "SAM", + "chemical" + ], + [ + 85, + 94, + "mutations", + "experimental_method" + ], + [ + 98, + 101, + "T17", + "residue_name_number" + ], + [ + 103, + 106, + "T19", + "residue_name_number" + ], + [ + 110, + 116, + "VdTsr3", + "protein" + ], + [ + 128, + 129, + "A", + "residue_name" + ], + [ + 133, + 134, + "D", + "residue_name" + ], + [ + 171, + 191, + "SAM-binding affinity", + "evidence" + ], + [ + 195, + 201, + "SsTsr3", + "protein" + ], + [ + 203, + 205, + "KD", + "evidence" + ] + ] + }, + { + "sid": 187, + "sent": "Nevertheless, a mutation of the equivalent position S62 of ScTsr3 to D, but not to A, resulted in reduced acp modification in vivo, as shown by primer extension analysis (Figure 5F).", + "section": "RESULTS", + "ner": [ + [ + 16, + 24, + "mutation", + "experimental_method" + ], + [ + 52, + 55, + "S62", + "residue_name_number" + ], + [ + 59, + 65, + "ScTsr3", + "protein" + ], + [ + 69, + 70, + "D", + "residue_name" + ], + [ + 83, + 84, + "A", + "residue_name" + ], + [ + 106, + 109, + "acp", + "chemical" + ], + [ + 144, + 169, + "primer extension analysis", + "experimental_method" + ] + ] + }, + { + "sid": 188, + "sent": "The acp-transfer reaction catalyzed by Tsr3 most likely requires the presence of a catalytic base in order to abstract a proton from the N3 imino group of the modified pseudouridine.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "acp", + "chemical" + ], + [ + 39, + 43, + "Tsr3", + "protein" + ], + [ + 168, + 181, + "pseudouridine", + "chemical" + ] + ] + }, + { + "sid": 189, + "sent": "The side chain of D70 (VdTsr3) located in \u03b24 is \u223c5 \u00c5 away from the SAM sulfur atom.", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "D70", + "residue_name_number" + ], + [ + 23, + 29, + "VdTsr3", + "protein" + ], + [ + 42, + 44, + "\u03b24", + "structure_element" + ], + [ + 67, + 70, + "SAM", + "chemical" + ] + ] + }, + { + "sid": 190, + "sent": "This residue is conserved as D or E both in archaeal and eukaryotic Tsr3 homologs.", + "section": "RESULTS", + "ner": [ + [ + 16, + 28, + "conserved as", + "protein_state" + ], + [ + 29, + 30, + "D", + "residue_name" + ], + [ + 34, + 35, + "E", + "residue_name" + ], + [ + 44, + 52, + "archaeal", + "taxonomy_domain" + ], + [ + 57, + 67, + "eukaryotic", + "taxonomy_domain" + ], + [ + 68, + 72, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 191, + "sent": "Mutations of the corresponding residue in SsTsr3 to A (D63) does not significantly alter the SAM-binding affinity of the protein (KD = 11.0 \u03bcM).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Mutations", + "experimental_method" + ], + [ + 42, + 48, + "SsTsr3", + "protein" + ], + [ + 52, + 53, + "A", + "residue_name" + ], + [ + 55, + 58, + "D63", + "residue_name_number" + ], + [ + 93, + 113, + "SAM-binding affinity", + "evidence" + ], + [ + 130, + 132, + "KD", + "evidence" + ] + ] + }, + { + "sid": 192, + "sent": "However, the mutation of the corresponding residue of ScTsr3 (E111A) leads to a significant decrease of the acp transferase activity in vivo (Figure 5F).", + "section": "RESULTS", + "ner": [ + [ + 13, + 21, + "mutation", + "experimental_method" + ], + [ + 54, + 60, + "ScTsr3", + "protein" + ], + [ + 62, + 67, + "E111A", + "mutant" + ], + [ + 108, + 123, + "acp transferase", + "protein_type" + ] + ] + }, + { + "sid": 193, + "sent": "RNA-binding of Tsr3", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "RNA", + "chemical" + ], + [ + 15, + 19, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 194, + "sent": "Analysis of the electrostatic surface properties of VdTsr3 clearly identified positively charged surface patches in the vicinity of the SAM-binding site suggesting a putative RNA-binding site (Figure 6A).", + "section": "RESULTS", + "ner": [ + [ + 0, + 48, + "Analysis of the electrostatic surface properties", + "experimental_method" + ], + [ + 52, + 58, + "VdTsr3", + "protein" + ], + [ + 78, + 112, + "positively charged surface patches", + "site" + ], + [ + 136, + 152, + "SAM-binding site", + "site" + ], + [ + 175, + 191, + "RNA-binding site", + "site" + ] + ] + }, + { + "sid": 195, + "sent": "Furthermore, a negatively charged MES-ion is found in the crystal structure of VdTsr3 complexed to the side chain of K22 in helix \u03b11.", + "section": "RESULTS", + "ner": [ + [ + 34, + 37, + "MES", + "chemical" + ], + [ + 58, + 75, + "crystal structure", + "evidence" + ], + [ + 79, + 85, + "VdTsr3", + "protein" + ], + [ + 86, + 98, + "complexed to", + "protein_state" + ], + [ + 117, + 120, + "K22", + "residue_name_number" + ], + [ + 124, + 129, + "helix", + "structure_element" + ], + [ + 130, + 132, + "\u03b11", + "structure_element" + ] + ] + }, + { + "sid": 196, + "sent": "Its negatively charged sulfate group might mimic an RNA backbone phosphate.", + "section": "RESULTS", + "ner": [ + [ + 23, + 30, + "sulfate", + "chemical" + ], + [ + 52, + 55, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 197, + "sent": "Helix \u03b11 contains two more positively charged amino acids K17 and R25 as does the loop preceding it (R9).", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "Helix", + "structure_element" + ], + [ + 6, + 8, + "\u03b11", + "structure_element" + ], + [ + 58, + 61, + "K17", + "residue_name_number" + ], + [ + 66, + 69, + "R25", + "residue_name_number" + ], + [ + 82, + 86, + "loop", + "structure_element" + ], + [ + 101, + 103, + "R9", + "residue_name_number" + ] + ] + }, + { + "sid": 198, + "sent": "A second cluster of positively charged residues is found in or near helix \u03b13 (K74, R75, K82, R85 and K87).", + "section": "RESULTS", + "ner": [ + [ + 68, + 73, + "helix", + "structure_element" + ], + [ + 74, + 76, + "\u03b13", + "structure_element" + ], + [ + 78, + 81, + "K74", + "residue_name_number" + ], + [ + 83, + 86, + "R75", + "residue_name_number" + ], + [ + 88, + 91, + "K82", + "residue_name_number" + ], + [ + 93, + 96, + "R85", + "residue_name_number" + ], + [ + 101, + 104, + "K87", + "residue_name_number" + ] + ] + }, + { + "sid": 199, + "sent": "Some of these amino acids are conserved between archaeal and eukaryotic Tsr3 (Supplementary Figure S1A).", + "section": "RESULTS", + "ner": [ + [ + 30, + 39, + "conserved", + "protein_state" + ], + [ + 48, + 56, + "archaeal", + "taxonomy_domain" + ], + [ + 61, + 71, + "eukaryotic", + "taxonomy_domain" + ], + [ + 72, + 76, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 200, + "sent": "In the C-terminal domain, the surface exposed \u03b1-helices \u03b15 and \u03b17 carry a significant amount of positively charged amino acids.", + "section": "RESULTS", + "ner": [ + [ + 7, + 24, + "C-terminal domain", + "structure_element" + ], + [ + 46, + 55, + "\u03b1-helices", + "structure_element" + ], + [ + 56, + 58, + "\u03b15", + "structure_element" + ], + [ + 63, + 65, + "\u03b17", + "structure_element" + ] + ] + }, + { + "sid": 201, + "sent": "A triple mutation of the conserved positively charged residues R60, K65 and R131 to A in ScTsr3 resulted in a protein with a significantly impaired acp transferase activity in vivo (Figure 6D) in line with an important functional role for these positively charged residues.", + "section": "RESULTS", + "ner": [ + [ + 2, + 17, + "triple mutation", + "experimental_method" + ], + [ + 25, + 34, + "conserved", + "protein_state" + ], + [ + 63, + 66, + "R60", + "residue_name_number" + ], + [ + 68, + 71, + "K65", + "residue_name_number" + ], + [ + 76, + 80, + "R131", + "residue_name_number" + ], + [ + 84, + 85, + "A", + "residue_name" + ], + [ + 89, + 95, + "ScTsr3", + "protein" + ], + [ + 148, + 163, + "acp transferase", + "protein_type" + ] + ] + }, + { + "sid": 202, + "sent": "RNA-binding of Tsr3.", + "section": "FIG", + "ner": [ + [ + 15, + 19, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 203, + "sent": "(A) Electrostatic charge distribution on the surface of VdTsr3.", + "section": "FIG", + "ner": [ + [ + 56, + 62, + "VdTsr3", + "protein" + ] + ] + }, + { + "sid": 204, + "sent": "SAM is shown in a stick representation.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "SAM", + "chemical" + ] + ] + }, + { + "sid": 205, + "sent": "Also shown in stick representation is a negatively charged MES ion.", + "section": "FIG", + "ner": [ + [ + 59, + 62, + "MES", + "chemical" + ] + ] + }, + { + "sid": 206, + "sent": "Conserved basic amino acids are labeled. (B) Comparison of the secondary structures of helix 31 from the small ribosomal subunit rRNAs in S. cerevisiae and S. solfataricus with the location of the hypermodified nucleotide indicated in red.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Conserved", + "protein_state" + ], + [ + 16, + 27, + "amino acids", + "chemical" + ], + [ + 87, + 95, + "helix 31", + "structure_element" + ], + [ + 129, + 134, + "rRNAs", + "chemical" + ], + [ + 138, + 151, + "S. cerevisiae", + "species" + ], + [ + 156, + 171, + "S. solfataricus", + "species" + ], + [ + 197, + 210, + "hypermodified", + "protein_state" + ], + [ + 211, + 221, + "nucleotide", + "chemical" + ] + ] + }, + { + "sid": 207, + "sent": "For S. solfataricus the chemical identity of the hypermodified nucleotide is not known but the existence of NEP1 and TSR3 homologs suggest that it is indeed N1-methyl-N3-acp-pseudouridine.", + "section": "FIG", + "ner": [ + [ + 4, + 19, + "S. solfataricus", + "species" + ], + [ + 49, + 62, + "hypermodified", + "protein_state" + ], + [ + 63, + 73, + "nucleotide", + "chemical" + ], + [ + 108, + 112, + "NEP1", + "protein" + ], + [ + 117, + 121, + "TSR3", + "protein" + ], + [ + 157, + 187, + "N1-methyl-N3-acp-pseudouridine", + "chemical" + ] + ] + }, + { + "sid": 208, + "sent": "(C) Binding of SsTsr3 to RNA.", + "section": "FIG", + "ner": [ + [ + 15, + 21, + "SsTsr3", + "protein" + ], + [ + 25, + 28, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 209, + "sent": "5\u2032-fluoresceine labeled RNA oligonucleotides corresponding either to the native (20mer \u2013 see inset) or a stabilized (20mer_GC - inset) helix 31 of the small ribosomal subunit rRNA from S. solfataricus were titrated with increasing amounts of SsTsr3 and the changes in the fluoresceine fluorescence anisotropy were measured and fitted to a binding curve (20mer \u2013 red, 20mer_GC \u2013 blue).", + "section": "FIG", + "ner": [ + [ + 3, + 15, + "fluoresceine", + "chemical" + ], + [ + 24, + 27, + "RNA", + "chemical" + ], + [ + 73, + 79, + "native", + "protein_state" + ], + [ + 81, + 86, + "20mer", + "oligomeric_state" + ], + [ + 105, + 115, + "stabilized", + "protein_state" + ], + [ + 117, + 125, + "20mer_GC", + "oligomeric_state" + ], + [ + 135, + 143, + "helix 31", + "structure_element" + ], + [ + 175, + 179, + "rRNA", + "chemical" + ], + [ + 185, + 200, + "S. solfataricus", + "species" + ], + [ + 206, + 238, + "titrated with increasing amounts", + "experimental_method" + ], + [ + 242, + 248, + "SsTsr3", + "protein" + ], + [ + 272, + 284, + "fluoresceine", + "chemical" + ], + [ + 285, + 308, + "fluorescence anisotropy", + "evidence" + ], + [ + 339, + 352, + "binding curve", + "evidence" + ], + [ + 354, + 359, + "20mer", + "oligomeric_state" + ], + [ + 367, + 375, + "20mer_GC", + "oligomeric_state" + ] + ] + }, + { + "sid": 210, + "sent": "Oligo-U9-RNA was used for comparison (black).", + "section": "FIG", + "ner": [ + [ + 0, + 12, + "Oligo-U9-RNA", + "chemical" + ] + ] + }, + { + "sid": 211, + "sent": "The 20mer_GC RNA was also titrated with SsTsr3 in the presence of 2 mM SAM (purple). (D) Mutants of ScTsr3 R60, K65 or R131 (equivalent to K17, K22 and R91 in VdTsr3) expressed in \u0394tsr3 yeast cells show a primer extension stop comparable to the wild type.", + "section": "FIG", + "ner": [ + [ + 4, + 12, + "20mer_GC", + "oligomeric_state" + ], + [ + 13, + 16, + "RNA", + "chemical" + ], + [ + 26, + 34, + "titrated", + "experimental_method" + ], + [ + 40, + 46, + "SsTsr3", + "protein" + ], + [ + 71, + 74, + "SAM", + "chemical" + ], + [ + 89, + 96, + "Mutants", + "protein_state" + ], + [ + 100, + 106, + "ScTsr3", + "protein" + ], + [ + 107, + 110, + "R60", + "residue_name_number" + ], + [ + 112, + 115, + "K65", + "residue_name_number" + ], + [ + 119, + 123, + "R131", + "residue_name_number" + ], + [ + 139, + 142, + "K17", + "residue_name_number" + ], + [ + 144, + 147, + "K22", + "residue_name_number" + ], + [ + 152, + 155, + "R91", + "residue_name_number" + ], + [ + 159, + 165, + "VdTsr3", + "protein" + ], + [ + 167, + 176, + "expressed", + "experimental_method" + ], + [ + 180, + 185, + "\u0394tsr3", + "mutant" + ], + [ + 186, + 191, + "yeast", + "taxonomy_domain" + ], + [ + 205, + 226, + "primer extension stop", + "evidence" + ], + [ + 245, + 254, + "wild type", + "protein_state" + ] + ] + }, + { + "sid": 212, + "sent": "Combination of the three point mutations (R60A/K65A/R131A) leads to a strongly reduced acp modification of 18S rRNA.", + "section": "FIG", + "ner": [ + [ + 0, + 40, + "Combination of the three point mutations", + "experimental_method" + ], + [ + 42, + 46, + "R60A", + "mutant" + ], + [ + 47, + 51, + "K65A", + "mutant" + ], + [ + 52, + 57, + "R131A", + "mutant" + ], + [ + 87, + 90, + "acp", + "chemical" + ], + [ + 107, + 115, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 213, + "sent": "In order to explore the RNA-ligand specificity of Tsr3 we titrated SsTsr3 prepared in RNase-free form with 5\u2032-fluoresceine-labeled RNA and determined the affinity by fluorescence anisotropy measurements.", + "section": "RESULTS", + "ner": [ + [ + 50, + 54, + "Tsr3", + "protein" + ], + [ + 58, + 66, + "titrated", + "experimental_method" + ], + [ + 67, + 73, + "SsTsr3", + "protein" + ], + [ + 86, + 96, + "RNase-free", + "protein_state" + ], + [ + 110, + 122, + "fluoresceine", + "chemical" + ], + [ + 131, + 134, + "RNA", + "chemical" + ], + [ + 154, + 162, + "affinity", + "evidence" + ], + [ + 166, + 202, + "fluorescence anisotropy measurements", + "experimental_method" + ] + ] + }, + { + "sid": 214, + "sent": "SsTsr3 in the apo state bound a 20mer RNA corresponding to helix 31 of S. solfataricus 16S rRNA (Figure 6B) with a KD of 1.9 \u03bcM and to a version of this hairpin stabilized by additional GC base pairs (20mer-GC) with a KD of 0.6 \u03bcM (Figure 6C).", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "SsTsr3", + "protein" + ], + [ + 14, + 17, + "apo", + "protein_state" + ], + [ + 24, + 29, + "bound", + "protein_state" + ], + [ + 32, + 37, + "20mer", + "oligomeric_state" + ], + [ + 38, + 41, + "RNA", + "chemical" + ], + [ + 59, + 67, + "helix 31", + "structure_element" + ], + [ + 71, + 86, + "S. solfataricus", + "species" + ], + [ + 87, + 95, + "16S rRNA", + "chemical" + ], + [ + 115, + 117, + "KD", + "evidence" + ], + [ + 153, + 160, + "hairpin", + "structure_element" + ], + [ + 201, + 209, + "20mer-GC", + "oligomeric_state" + ], + [ + 218, + 220, + "KD", + "evidence" + ] + ] + }, + { + "sid": 215, + "sent": "A single stranded oligoU-RNA bound with a 10-fold-reduced affinity (6.0 \u03bcM).", + "section": "RESULTS", + "ner": [ + [ + 18, + 28, + "oligoU-RNA", + "chemical" + ], + [ + 29, + 34, + "bound", + "protein_state" + ], + [ + 58, + 66, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 216, + "sent": "The presence of saturating amounts of SAM (2 mM) did not have a significant influence on the RNA-affinity of SsTsr3 (KD of 1.7 \u03bcM for the 20mer-GC-RNA) suggesting no cooperativity in substrate binding.", + "section": "RESULTS", + "ner": [ + [ + 38, + 41, + "SAM", + "chemical" + ], + [ + 93, + 105, + "RNA-affinity", + "evidence" + ], + [ + 109, + 115, + "SsTsr3", + "protein" + ], + [ + 117, + 119, + "KD", + "evidence" + ], + [ + 138, + 146, + "20mer-GC", + "oligomeric_state" + ], + [ + 147, + 150, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 217, + "sent": "U1191 is the only hypermodified base in the yeast 18S rRNA and is strongly conserved in eukaryotes.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "U1191", + "residue_name_number" + ], + [ + 18, + 31, + "hypermodified", + "protein_state" + ], + [ + 44, + 49, + "yeast", + "taxonomy_domain" + ], + [ + 50, + 58, + "18S rRNA", + "chemical" + ], + [ + 66, + 84, + "strongly conserved", + "protein_state" + ], + [ + 88, + 98, + "eukaryotes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 218, + "sent": "The formation of 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m1acp3\u03a8) is very complex requiring three successive modification reactions involving one H/ACA snoRNP (snR35) and two protein enzymes (Nep1/Emg1 and Tsr3).", + "section": "DISCUSS", + "ner": [ + [ + 17, + 67, + "1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine", + "chemical" + ], + [ + 69, + 76, + "m1acp3\u03a8", + "chemical" + ], + [ + 158, + 163, + "H/ACA", + "structure_element" + ], + [ + 164, + 170, + "snoRNP", + "complex_assembly" + ], + [ + 172, + 177, + "snR35", + "protein" + ], + [ + 204, + 208, + "Nep1", + "protein" + ], + [ + 209, + 213, + "Emg1", + "protein" + ], + [ + 218, + 222, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 219, + "sent": "This makes it unique in eukaryotic rRNA modification.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 34, + "eukaryotic", + "taxonomy_domain" + ], + [ + 35, + 39, + "rRNA", + "chemical" + ] + ] + }, + { + "sid": 220, + "sent": "The m1acp3\u03a8 base is located at the tip of helix 31 on the 18S rRNA (Supplementary Figure S1B) which, together with helices 18, 24, 34 and 44, contribute to building the decoding center of the small ribosomal subunit.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 11, + "m1acp3\u03a8", + "chemical" + ], + [ + 42, + 50, + "helix 31", + "structure_element" + ], + [ + 58, + 66, + "18S rRNA", + "chemical" + ], + [ + 115, + 140, + "helices 18, 24, 34 and 44", + "structure_element" + ] + ] + }, + { + "sid": 221, + "sent": "A similar modification (acp3U) was identified in Haloferax volcanii and corresponding modified nucleotides were also shown to occur in other archaea.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 29, + "acp3U", + "chemical" + ], + [ + 49, + 67, + "Haloferax volcanii", + "species" + ], + [ + 95, + 106, + "nucleotides", + "chemical" + ], + [ + 141, + 148, + "archaea", + "taxonomy_domain" + ] + ] + }, + { + "sid": 222, + "sent": "As shown here TSR3 encodes the transferase catalyzing the acp modification as the last step in the biosynthesis of m1acp3\u03a8 in yeast and human cells.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 18, + "TSR3", + "protein" + ], + [ + 58, + 61, + "acp", + "chemical" + ], + [ + 115, + 122, + "m1acp3\u03a8", + "chemical" + ], + [ + 126, + 131, + "yeast", + "taxonomy_domain" + ], + [ + 136, + 141, + "human", + "species" + ] + ] + }, + { + "sid": 223, + "sent": "Unexpectedly, archaeal Tsr3 has a structure similar to SPOUT-class RNA methyltransferases, and it is the first example for an enzyme of this class transferring an acp group, due to a modified SAM-binding pocket that exposes the acp instead of the methyl group of SAM to its RNA substrate.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 22, + "archaeal", + "taxonomy_domain" + ], + [ + 23, + 27, + "Tsr3", + "protein" + ], + [ + 34, + 43, + "structure", + "evidence" + ], + [ + 55, + 89, + "SPOUT-class RNA methyltransferases", + "protein_type" + ], + [ + 163, + 166, + "acp", + "chemical" + ], + [ + 192, + 210, + "SAM-binding pocket", + "site" + ], + [ + 228, + 231, + "acp", + "chemical" + ], + [ + 263, + 266, + "SAM", + "chemical" + ], + [ + 274, + 277, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 224, + "sent": "Similar to the structurally unrelated Rossmann-fold Tyw2 acp transferase, the SAM methyl group of Tsr3 is bound in an inaccessible hydrophobic pocket whereas the acp side chain becomes accessible for a nucleophilic attack by the N3 of pseudouridine.", + "section": "DISCUSS", + "ner": [ + [ + 38, + 72, + "Rossmann-fold Tyw2 acp transferase", + "protein_type" + ], + [ + 78, + 81, + "SAM", + "chemical" + ], + [ + 98, + 102, + "Tsr3", + "protein" + ], + [ + 131, + 149, + "hydrophobic pocket", + "site" + ], + [ + 162, + 165, + "acp", + "chemical" + ], + [ + 235, + 248, + "pseudouridine", + "chemical" + ] + ] + }, + { + "sid": 225, + "sent": "In contrast, in the structurally closely related RNA methyltransferase Trm10 the methyl group of the cofactor SAM is accessible whereas its acp side chain is buried inside the protein.", + "section": "DISCUSS", + "ner": [ + [ + 49, + 70, + "RNA methyltransferase", + "protein_type" + ], + [ + 71, + 76, + "Trm10", + "protein" + ], + [ + 110, + 113, + "SAM", + "chemical" + ], + [ + 140, + 143, + "acp", + "chemical" + ] + ] + }, + { + "sid": 226, + "sent": "This suggests that enzymes with a SAM-dependent acp transferase activity might have evolved from SAM-dependent methyltransferases by slight modifications of the SAM-binding pocket.", + "section": "DISCUSS", + "ner": [ + [ + 34, + 63, + "SAM-dependent acp transferase", + "protein_type" + ], + [ + 97, + 129, + "SAM-dependent methyltransferases", + "protein_type" + ], + [ + 161, + 179, + "SAM-binding pocket", + "site" + ] + ] + }, + { + "sid": 227, + "sent": "Thus, additional examples for acp transferase enzymes might be found with similarities to other structural classes of methyltransferases.", + "section": "DISCUSS", + "ner": [ + [ + 30, + 45, + "acp transferase", + "protein_type" + ], + [ + 118, + 136, + "methyltransferases", + "protein_type" + ] + ] + }, + { + "sid": 228, + "sent": "In contrast to Nep1, the enzyme preceding Tsr3 in the m1acp3\u03a8 biosynthesis pathway, Tsr3 binds rather weakly and with little specificity to its isolated substrate RNA.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 19, + "Nep1", + "protein" + ], + [ + 42, + 46, + "Tsr3", + "protein" + ], + [ + 54, + 61, + "m1acp3\u03a8", + "chemical" + ], + [ + 84, + 88, + "Tsr3", + "protein" + ], + [ + 163, + 166, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 229, + "sent": "This suggests that Tsr3 is not stably incorporated into pre-ribosomal particles and that its binding to the nascent ribosomal subunit possibly requires additional interactions with other pre-ribosomal components.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 23, + "Tsr3", + "protein" + ], + [ + 56, + 79, + "pre-ribosomal particles", + "complex_assembly" + ], + [ + 116, + 133, + "ribosomal subunit", + "complex_assembly" + ] + ] + }, + { + "sid": 230, + "sent": "Consistently, in sucrose gradient analysis, Tsr3 was found in low-molecular weight fractions rather than with pre-ribosome containing high-molecular weight fractions.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 42, + "sucrose gradient analysis", + "experimental_method" + ], + [ + 44, + 48, + "Tsr3", + "protein" + ], + [ + 110, + 122, + "pre-ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 231, + "sent": "In contrast to several enzymes that catalyze base specific modifications in rRNAs Tsr3 is not an essential protein.", + "section": "DISCUSS", + "ner": [ + [ + 76, + 81, + "rRNAs", + "chemical" + ], + [ + 82, + 86, + "Tsr3", + "protein" + ] + ] + }, + { + "sid": 232, + "sent": "Typically, other small subunit rRNA methyltransferases as Dim1, Bud23 and Nep1/Emg1 carry dual functions, in ribosome biogenesis and rRNA modification, and it is their involvement in pre-RNA processing that is essential rather than their RNA-methylating activity (, discussed in 7).", + "section": "DISCUSS", + "ner": [ + [ + 17, + 54, + "small subunit rRNA methyltransferases", + "protein_type" + ], + [ + 58, + 62, + "Dim1", + "protein" + ], + [ + 64, + 69, + "Bud23", + "protein" + ], + [ + 74, + 78, + "Nep1", + "protein" + ], + [ + 79, + 83, + "Emg1", + "protein" + ], + [ + 133, + 137, + "rRNA", + "chemical" + ], + [ + 183, + 190, + "pre-RNA", + "chemical" + ] + ] + }, + { + "sid": 233, + "sent": "In contrast, for several Tsr3 mutants (SAM-binding and cysteine mutations) we found a systematic correlation between the loss of acp modification and the efficiency of 18S rRNA maturation.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 29, + "Tsr3", + "protein" + ], + [ + 39, + 50, + "SAM-binding", + "protein_state" + ], + [ + 55, + 73, + "cysteine mutations", + "protein_state" + ], + [ + 129, + 132, + "acp", + "chemical" + ], + [ + 168, + 176, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 234, + "sent": "This demonstrates that, unlike the other small subunit rRNA base modifications, the acp modification is required for efficient pre-rRNA processing.", + "section": "DISCUSS", + "ner": [ + [ + 55, + 59, + "rRNA", + "chemical" + ], + [ + 84, + 87, + "acp", + "chemical" + ], + [ + 127, + 135, + "pre-rRNA", + "chemical" + ] + ] + }, + { + "sid": 235, + "sent": "Recently, structural, functional, and CRAC (cross-linking and cDNA analysis) experiments of late assembly factors involved in cytoplasmic processing of 40S subunits, along with cryo-EM studies of the late pre-40S subunits have provided important insights into late pre-40S processing.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 88, + "structural, functional, and CRAC (cross-linking and cDNA analysis) experiments", + "experimental_method" + ], + [ + 152, + 164, + "40S subunits", + "complex_assembly" + ], + [ + 177, + 184, + "cryo-EM", + "experimental_method" + ], + [ + 200, + 204, + "late", + "protein_state" + ], + [ + 205, + 221, + "pre-40S subunits", + "complex_assembly" + ], + [ + 265, + 272, + "pre-40S", + "complex_assembly" + ] + ] + }, + { + "sid": 236, + "sent": "Apart from most of the ribosomal proteins, cytoplasmic pre-40S particles contain 20S rRNA and at least seven non-ribosomal proteins including the D-site endonuclease Nob1 as well as Tsr1, a putative GTPase and Rio2 which block the mRNA channel and the initiator tRNA binding site, respectively, thus preventing translation initiation.", + "section": "DISCUSS", + "ner": [ + [ + 55, + 72, + "pre-40S particles", + "complex_assembly" + ], + [ + 81, + 89, + "20S rRNA", + "chemical" + ], + [ + 109, + 131, + "non-ribosomal proteins", + "protein_type" + ], + [ + 146, + 165, + "D-site endonuclease", + "protein_type" + ], + [ + 166, + 170, + "Nob1", + "protein" + ], + [ + 182, + 186, + "Tsr1", + "protein" + ], + [ + 199, + 205, + "GTPase", + "protein_type" + ], + [ + 210, + 214, + "Rio2", + "protein" + ], + [ + 231, + 243, + "mRNA channel", + "site" + ], + [ + 252, + 279, + "initiator tRNA binding site", + "site" + ] + ] + }, + { + "sid": 237, + "sent": "After structural changes, possibly driven by GTP hydrolysis, which go together with the formation of the decoding site, the 20S pre-rRNA becomes accessible for Nob1 cleavage at site D. This also involves joining of pre-40S and 60S subunits to 80S-like particles in a translation-like cycle promoted by eIF5B.", + "section": "DISCUSS", + "ner": [ + [ + 45, + 48, + "GTP", + "chemical" + ], + [ + 105, + 118, + "decoding site", + "site" + ], + [ + 124, + 136, + "20S pre-rRNA", + "chemical" + ], + [ + 160, + 164, + "Nob1", + "protein" + ], + [ + 177, + 183, + "site D", + "site" + ], + [ + 215, + 222, + "pre-40S", + "complex_assembly" + ], + [ + 227, + 239, + "60S subunits", + "complex_assembly" + ], + [ + 243, + 261, + "80S-like particles", + "complex_assembly" + ], + [ + 302, + 307, + "eIF5B", + "protein" + ] + ] + }, + { + "sid": 238, + "sent": "The cleavage step most likely acts as a quality control check that ensures the proper 40S subunit assembly with only completely processed precursors.", + "section": "DISCUSS", + "ner": [ + [ + 86, + 97, + "40S subunit", + "complex_assembly" + ] + ] + }, + { + "sid": 239, + "sent": "Finally, termination factor Rli1, an ATPase, promotes the dissociation of assembly factors and the 80S-like complex dissociates and releases the mature 40S subunit.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 27, + "termination factor", + "protein_type" + ], + [ + 28, + 32, + "Rli1", + "protein" + ], + [ + 37, + 43, + "ATPase", + "protein_type" + ], + [ + 99, + 115, + "80S-like complex", + "complex_assembly" + ], + [ + 145, + 151, + "mature", + "protein_state" + ], + [ + 152, + 163, + "40S subunit", + "complex_assembly" + ] + ] + }, + { + "sid": 240, + "sent": "Interestingly, differences in the level of acp modification were demonstrated for different steps of the cytoplasmic pre-40S subunit maturation after analyzing purified 20S pre-rRNAs using different purification bait proteins.", + "section": "DISCUSS", + "ner": [ + [ + 43, + 46, + "acp", + "chemical" + ], + [ + 117, + 132, + "pre-40S subunit", + "complex_assembly" + ], + [ + 169, + 182, + "20S pre-rRNAs", + "chemical" + ] + ] + }, + { + "sid": 241, + "sent": "Early cytoplasmic pre-40S subunits still containing the ribosome assembly factors Tsr1, Ltv1, Enp1 and Rio2 were not or only partially acp modified.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 34, + "pre-40S subunits", + "complex_assembly" + ], + [ + 56, + 81, + "ribosome assembly factors", + "protein_type" + ], + [ + 82, + 86, + "Tsr1", + "protein" + ], + [ + 88, + 92, + "Ltv1", + "protein" + ], + [ + 94, + 98, + "Enp1", + "protein" + ], + [ + 103, + 107, + "Rio2", + "protein" + ], + [ + 135, + 147, + "acp modified", + "protein_state" + ] + ] + }, + { + "sid": 242, + "sent": "In contrast, late pre-40S subunits containing Nob1 and Rio1 or already associated with 60S subunits in 80S-like particles showed acp modification levels comparable to mature 40S subunits.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 34, + "pre-40S subunits", + "complex_assembly" + ], + [ + 46, + 50, + "Nob1", + "protein" + ], + [ + 55, + 59, + "Rio1", + "protein" + ], + [ + 87, + 99, + "60S subunits", + "complex_assembly" + ], + [ + 103, + 121, + "80S-like particles", + "complex_assembly" + ], + [ + 129, + 132, + "acp", + "chemical" + ], + [ + 167, + 173, + "mature", + "protein_state" + ], + [ + 174, + 186, + "40S subunits", + "complex_assembly" + ] + ] + }, + { + "sid": 243, + "sent": "Thus, the acp transfer to m1\u03a81191 occurs during the step at which Rio2 leaves the pre-40S particle.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 13, + "acp", + "chemical" + ], + [ + 26, + 33, + "m1\u03a81191", + "residue_name_number" + ], + [ + 66, + 70, + "Rio2", + "protein" + ], + [ + 82, + 98, + "pre-40S particle", + "complex_assembly" + ] + ] + }, + { + "sid": 244, + "sent": "These data and the finding that a missing acp modification hinders pre-20S rRNA processing, suggest that the acp modification together with the release of Rio2 promotes the formation of the decoding site and thus D-site cleavage by Nob1.", + "section": "DISCUSS", + "ner": [ + [ + 42, + 45, + "acp", + "chemical" + ], + [ + 67, + 79, + "pre-20S rRNA", + "chemical" + ], + [ + 109, + 112, + "acp", + "chemical" + ], + [ + 155, + 159, + "Rio2", + "protein" + ], + [ + 190, + 203, + "decoding site", + "site" + ], + [ + 213, + 219, + "D-site", + "site" + ], + [ + 232, + 236, + "Nob1", + "protein" + ] + ] + }, + { + "sid": 245, + "sent": "The interrelation between acp modification and Rio2 release is also supported by CRAC analysis showing that Rio2 binds to helix 31 next to the \u03a81191 residue that receives the acp modification.", + "section": "DISCUSS", + "ner": [ + [ + 26, + 29, + "acp", + "chemical" + ], + [ + 47, + 51, + "Rio2", + "protein" + ], + [ + 81, + 94, + "CRAC analysis", + "experimental_method" + ], + [ + 108, + 112, + "Rio2", + "protein" + ], + [ + 122, + 130, + "helix 31", + "structure_element" + ], + [ + 143, + 148, + "\u03a81191", + "residue_name_number" + ], + [ + 175, + 178, + "acp", + "chemical" + ] + ] + }, + { + "sid": 246, + "sent": "Therefore, Rio2 either blocks the access of Tsr3 to helix 31, and acp modification can only occur after Rio2 is released, or the acp modification of m1\u03a81191 and putative subsequent conformational changes of 20S rRNA weaken the binding of Rio2 to helix 31 and support its release from the pre-rRNA.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 15, + "Rio2", + "protein" + ], + [ + 44, + 48, + "Tsr3", + "protein" + ], + [ + 52, + 60, + "helix 31", + "structure_element" + ], + [ + 66, + 69, + "acp", + "chemical" + ], + [ + 104, + 108, + "Rio2", + "protein" + ], + [ + 129, + 132, + "acp", + "chemical" + ], + [ + 149, + 156, + "m1\u03a81191", + "residue_name_number" + ], + [ + 207, + 215, + "20S rRNA", + "chemical" + ], + [ + 238, + 242, + "Rio2", + "protein" + ], + [ + 246, + 254, + "helix 31", + "structure_element" + ], + [ + 288, + 296, + "pre-rRNA", + "chemical" + ] + ] + }, + { + "sid": 247, + "sent": "In summary, by identifying Tsr3 as the enzyme responsible for introducing the acp group to the hypermodified m1acp3\u03a8 nucleotide at position 1191 (yeast)/ 1248 (humans) of 18S rRNA we added one of the last remaining pieces to the puzzle of eukaryotic small ribosomal subunit rRNA modifications.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 31, + "Tsr3", + "protein" + ], + [ + 78, + 81, + "acp", + "chemical" + ], + [ + 95, + 108, + "hypermodified", + "protein_state" + ], + [ + 109, + 116, + "m1acp3\u03a8", + "chemical" + ], + [ + 117, + 127, + "nucleotide", + "chemical" + ], + [ + 140, + 144, + "1191", + "residue_number" + ], + [ + 146, + 151, + "yeast", + "taxonomy_domain" + ], + [ + 154, + 158, + "1248", + "residue_number" + ], + [ + 160, + 166, + "humans", + "species" + ], + [ + 171, + 179, + "18S rRNA", + "chemical" + ], + [ + 239, + 249, + "eukaryotic", + "taxonomy_domain" + ], + [ + 250, + 278, + "small ribosomal subunit rRNA", + "chemical" + ] + ] + }, + { + "sid": 248, + "sent": "The current data together with the finding that acp modification takes place at the very last step in pre-40S subunit maturation indicate that the acp modification probably supports the formation of the decoding site and efficient 20S pre-rRNA D-site cleavage.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 51, + "acp", + "chemical" + ], + [ + 102, + 117, + "pre-40S subunit", + "complex_assembly" + ], + [ + 147, + 150, + "acp", + "chemical" + ], + [ + 203, + 216, + "decoding site", + "site" + ], + [ + 231, + 243, + "20S pre-rRNA", + "chemical" + ], + [ + 244, + 250, + "D-site", + "site" + ] + ] + }, + { + "sid": 249, + "sent": "Furthermore, our structural data unravelled how the regioselectivity of SAM-dependent group transfer reactions can be tuned by distinct small evolutionary adaptions of the ligand binding pocket of SAM-binding enzymes.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 32, + "structural data", + "evidence" + ], + [ + 72, + 75, + "SAM", + "chemical" + ], + [ + 172, + 193, + "ligand binding pocket", + "site" + ], + [ + 197, + 216, + "SAM-binding enzymes", + "protein_type" + ] + ] + } + ] + }, + "PMC4887326": { + "annotations": [ + { + "sid": 0, + "sent": "Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system", + "section": "TITLE", + "ner": [ + [ + 57, + 79, + "Pseudomonas aeruginosa", + "species" + ], + [ + 80, + 86, + "YfiBNR", + "complex_assembly" + ] + ] + }, + { + "sid": 1, + "sent": "YfiBNR is a recently identified bis-(3\u2019-5\u2019)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 6, + "YfiBNR", + "complex_assembly" + ], + [ + 32, + 62, + "bis-(3\u2019-5\u2019)-cyclic dimeric GMP", + "chemical" + ], + [ + 64, + 72, + "c-di-GMP", + "chemical" + ] + ] + }, + { + "sid": 2, + "sent": "In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production.", + "section": "ABSTRACT", + "ner": [ + [ + 28, + 32, + "YfiB", + "protein" + ], + [ + 93, + 97, + "YfiR", + "protein" + ], + [ + 127, + 131, + "YfiN", + "protein" + ], + [ + 209, + 213, + "YfiN", + "protein" + ], + [ + 224, + 232, + "c-di-GMP", + "chemical" + ] + ] + }, + { + "sid": 3, + "sent": "Here, we report the crystal\u00a0structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry.", + "section": "ABSTRACT", + "ner": [ + [ + 20, + 38, + "crystal\u00a0structures", + "evidence" + ], + [ + 42, + 46, + "YfiB", + "protein" + ], + [ + 47, + 52, + "alone", + "protein_state" + ], + [ + 63, + 69, + "active", + "protein_state" + ], + [ + 70, + 76, + "mutant", + "protein_state" + ], + [ + 77, + 85, + "YfiBL43P", + "mutant" + ], + [ + 86, + 100, + "complexed with", + "protein_state" + ], + [ + 101, + 105, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 4, + "sent": "Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the\u00a0peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 19, + "Structural analyses", + "experimental_method" + ], + [ + 53, + 73, + "compact conformation", + "protein_state" + ], + [ + 81, + 88, + "dimeric", + "oligomeric_state" + ], + [ + 89, + 93, + "YfiB", + "protein" + ], + [ + 94, + 99, + "alone", + "protein_state" + ], + [ + 101, + 109, + "YfiBL43P", + "mutant" + ], + [ + 119, + 141, + "stretched conformation", + "protein_state" + ], + [ + 151, + 160, + "activated", + "protein_state" + ], + [ + 161, + 165, + "YfiB", + "protein" + ], + [ + 183, + 196, + "peptidoglycan", + "chemical" + ], + [ + 198, + 200, + "PG", + "chemical" + ], + [ + 219, + 223, + "YfiR", + "protein" + ], + [ + 225, + 233, + "YfiBL43P", + "mutant" + ], + [ + 255, + 272, + "PG-binding pocket", + "site" + ], + [ + 289, + 308, + "PG binding affinity", + "evidence" + ], + [ + 314, + 323, + "wild-type", + "protein_state" + ], + [ + 324, + 328, + "YfiB", + "protein" + ], + [ + 384, + 388, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 5, + "sent": "In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation.", + "section": "ABSTRACT", + "ner": [ + [ + 17, + 42, + "crystallographic analyses", + "experimental_method" + ], + [ + 57, + 61, + "YfiR", + "protein" + ], + [ + 68, + 78, + "Vitamin B6", + "chemical" + ], + [ + 80, + 83, + "VB6", + "chemical" + ], + [ + 88, + 93, + "L-Trp", + "chemical" + ], + [ + 99, + 116, + "YfiB-binding site", + "site" + ], + [ + 131, + 134, + "VB6", + "chemical" + ], + [ + 139, + 144, + "L-Trp", + "chemical" + ], + [ + 164, + 172, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 6, + "sent": "Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.", + "section": "ABSTRACT", + "ner": [ + [ + 13, + 44, + "structural and biochemical data", + "evidence" + ], + [ + 92, + 98, + "YfiBNR", + "complex_assembly" + ] + ] + }, + { + "sid": 7, + "sent": "Bis-(3\u2019-5\u2019)-cyclic dimeric GMP (c-di-GMP) is a ubiquitous second messenger that bacteria use to facilitate behavioral adaptations to their ever-changing environment.", + "section": "INTRO", + "ner": [ + [ + 0, + 30, + "Bis-(3\u2019-5\u2019)-cyclic dimeric GMP", + "chemical" + ], + [ + 32, + 40, + "c-di-GMP", + "chemical" + ], + [ + 80, + 88, + "bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 8, + "sent": "An increase in c-di-GMP promotes biofilm formation, and a decrease results in biofilm degradation (Boehm et al.,; Duerig et al.,; Hickman et al.,; Jenal,; Romling et al.,).", + "section": "INTRO", + "ner": [ + [ + 15, + 23, + "c-di-GMP", + "chemical" + ] + ] + }, + { + "sid": 9, + "sent": "The c-di-GMP level is regulated by two reciprocal enzyme systems, namely, diguanylate cyclases (DGCs) that synthesize c-di-GMP and phosphodiesterases (PDEs) that hydrolyze c-di-GMP (Kulasakara et al.,; Ross et al.,; Ross et al.,). Many of these enzymes are multiple-domain proteins containing a variable N-terminal domain that commonly acts as a signal sensor or transduction module, followed by the relatively conserved GGDEF motif in DGCs or EAL/HD-GYP domains in PDEs (Hengge,; Navarro et al.,; Schirmer and Jenal,).", + "section": "INTRO", + "ner": [ + [ + 4, + 12, + "c-di-GMP", + "chemical" + ], + [ + 74, + 94, + "diguanylate cyclases", + "protein_type" + ], + [ + 96, + 100, + "DGCs", + "protein_type" + ], + [ + 118, + 126, + "c-di-GMP", + "chemical" + ], + [ + 131, + 149, + "phosphodiesterases", + "protein_type" + ], + [ + 151, + 155, + "PDEs", + "protein_type" + ], + [ + 172, + 180, + "c-di-GMP", + "chemical" + ], + [ + 304, + 321, + "N-terminal domain", + "structure_element" + ], + [ + 400, + 420, + "relatively conserved", + "protein_state" + ], + [ + 421, + 432, + "GGDEF motif", + "structure_element" + ], + [ + 436, + 440, + "DGCs", + "protein_type" + ], + [ + 444, + 462, + "EAL/HD-GYP domains", + "structure_element" + ], + [ + 466, + 470, + "PDEs", + "protein_type" + ] + ] + }, + { + "sid": 10, + "sent": "Intriguingly, studies in diverse species have revealed that a single bacterium can have dozens of DGCs and PDEs (Hickman et al.,; Kirillina et al.,; Kulasakara et al.,; Tamayo et al.,).", + "section": "INTRO", + "ner": [ + [ + 69, + 78, + "bacterium", + "taxonomy_domain" + ], + [ + 98, + 102, + "DGCs", + "protein_type" + ], + [ + 107, + 111, + "PDEs", + "protein_type" + ] + ] + }, + { + "sid": 11, + "sent": "In Pseudomonas aeruginosa in particular, 42 genes containing putative DGCs and/or PDEs were identified (Kulasakara et al.,).", + "section": "INTRO", + "ner": [ + [ + 3, + 25, + "Pseudomonas aeruginosa", + "species" + ], + [ + 70, + 74, + "DGCs", + "protein_type" + ], + [ + 82, + 86, + "PDEs", + "protein_type" + ] + ] + }, + { + "sid": 12, + "sent": "The functional role of a number of downstream effectors of c-di-GMP has been characterized as affecting exopolysaccharide (EPS) production, transcription, motility, and surface attachment (Caly et al.,; Camilli and Bassler,; Ha and O\u2019Toole,; Pesavento and Hengge,).", + "section": "INTRO", + "ner": [ + [ + 59, + 67, + "c-di-GMP", + "chemical" + ], + [ + 104, + 121, + "exopolysaccharide", + "chemical" + ], + [ + 123, + 126, + "EPS", + "chemical" + ] + ] + }, + { + "sid": 13, + "sent": "However, due to the intricacy of c-di-GMP signaling networks and the diversity of experimental cues, the detailed mechanisms by which these signaling pathways specifically sense and integrate different inputs remain largely elusive.", + "section": "INTRO", + "ner": [ + [ + 33, + 41, + "c-di-GMP", + "chemical" + ] + ] + }, + { + "sid": 14, + "sent": "Biofilm formation protects pathogenic bacteria from antibiotic treatment, and c-di-GMP-regulated biofilm formation has been extensively studied in P. aeruginosa (Evans,; Kirisits et al.,; Malone,; Reinhardt et al.,).", + "section": "INTRO", + "ner": [ + [ + 38, + 46, + "bacteria", + "taxonomy_domain" + ], + [ + 147, + 160, + "P. aeruginosa", + "species" + ] + ] + }, + { + "sid": 15, + "sent": "In the lungs of cystic fibrosis (CF) patients, adherent biofilm\u00a0formation and the appearance of small colony variant (SCV) morphologies of P. aeruginosa correlate with prolonged persistence of infection and poor lung function (Govan and Deretic,; Haussler et al.,; Haussler et al.,; Parsek and Singh,; Smith et al.,).", + "section": "INTRO", + "ner": [ + [ + 139, + 152, + "P. aeruginosa", + "species" + ] + ] + }, + { + "sid": 16, + "sent": "Recently, Malone and coworkers identified the tripartite c-di-GMP signaling module system YfiBNR (also known as AwsXRO (Beaumont et al.,; Giddens et al.,) or Tbp (Ueda and Wood,)) by genetic screening for mutants that displayed SCV phenotypes in P. aeruginosa PAO1 (Malone et al.,; Malone et al.,).", + "section": "INTRO", + "ner": [ + [ + 46, + 56, + "tripartite", + "protein_state" + ], + [ + 57, + 65, + "c-di-GMP", + "chemical" + ], + [ + 90, + 96, + "YfiBNR", + "complex_assembly" + ], + [ + 112, + 118, + "AwsXRO", + "complex_assembly" + ], + [ + 158, + 161, + "Tbp", + "complex_assembly" + ], + [ + 183, + 200, + "genetic screening", + "experimental_method" + ], + [ + 246, + 264, + "P. aeruginosa PAO1", + "species" + ] + ] + }, + { + "sid": 17, + "sent": "The YfiBNR system contains three protein members and modulates intracellular c-di-GMP levels in response to signals received in the periplasm (Malone et al.,).", + "section": "INTRO", + "ner": [ + [ + 4, + 10, + "YfiBNR", + "complex_assembly" + ], + [ + 77, + 85, + "c-di-GMP", + "chemical" + ] + ] + }, + { + "sid": 18, + "sent": "More recently, this system was also reported in other Gram-negative bacteria, such as Escherichia coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), Klebsiella pneumonia (Huertas et al.,) and Yersinia pestis (Ren et al.,).", + "section": "INTRO", + "ner": [ + [ + 54, + 76, + "Gram-negative bacteria", + "taxonomy_domain" + ], + [ + 86, + 102, + "Escherichia coli", + "species" + ], + [ + 165, + 185, + "Klebsiella pneumonia", + "species" + ], + [ + 208, + 223, + "Yersinia pestis", + "species" + ] + ] + }, + { + "sid": 19, + "sent": "YfiN is an integral inner-membrane protein with two potential transmembrane helices, a periplasmic Per-Arnt-Sim (PAS) domain, and cytosolic domains containing a HAMP domain (mediate input-output signaling in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and a C-terminal GGDEF domain indicating a DGC\u2019s function (Giardina et al.,; Malone et al.,).", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "YfiN", + "protein" + ], + [ + 62, + 83, + "transmembrane helices", + "structure_element" + ], + [ + 99, + 111, + "Per-Arnt-Sim", + "structure_element" + ], + [ + 113, + 116, + "PAS", + "structure_element" + ], + [ + 161, + 172, + "HAMP domain", + "structure_element" + ], + [ + 208, + 225, + "histidine kinases", + "protein_type" + ], + [ + 227, + 244, + "adenylyl cyclases", + "protein_type" + ], + [ + 246, + 282, + "methyl-accepting chemotaxis proteins", + "protein_type" + ], + [ + 288, + 300, + "phosphatases", + "protein_type" + ], + [ + 319, + 331, + "GGDEF domain", + "structure_element" + ], + [ + 345, + 348, + "DGC", + "protein_type" + ] + ] + }, + { + "sid": 20, + "sent": "YfiN is repressed by specific interaction between its periplasmic PAS domain and the periplasmic protein YfiR (Malone et al.,).", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "YfiN", + "protein" + ], + [ + 8, + 20, + "repressed by", + "protein_state" + ], + [ + 66, + 76, + "PAS domain", + "structure_element" + ], + [ + 105, + 109, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 21, + "sent": "YfiB is an OmpA/Pal-like outer-membrane lipoprotein (Parsons et al.,) that can activate YfiN by sequestering YfiR (Malone et al.,) in an unknown manner.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "YfiB", + "protein" + ], + [ + 11, + 24, + "OmpA/Pal-like", + "protein_type" + ], + [ + 40, + 51, + "lipoprotein", + "protein_type" + ], + [ + 88, + 92, + "YfiN", + "protein" + ], + [ + 109, + 113, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 22, + "sent": "Whether YfiB directly recruits YfiR or recruits YfiR via a third partner is an open question.", + "section": "INTRO", + "ner": [ + [ + 8, + 12, + "YfiB", + "protein" + ], + [ + 31, + 35, + "YfiR", + "protein" + ], + [ + 48, + 52, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 23, + "sent": "After the sequestration of YfiR by YfiB, the c-di-GMP produced by activated YfiN increases the biosynthesis of the Pel and Psl EPSs, resulting in the appearance of the SCV phenotype, which indicates enhanced biofilm formation (Malone et al.,).", + "section": "INTRO", + "ner": [ + [ + 27, + 31, + "YfiR", + "protein" + ], + [ + 35, + 39, + "YfiB", + "protein" + ], + [ + 45, + 53, + "c-di-GMP", + "chemical" + ], + [ + 66, + 75, + "activated", + "protein_state" + ], + [ + 76, + 80, + "YfiN", + "protein" + ], + [ + 115, + 118, + "Pel", + "chemical" + ], + [ + 123, + 126, + "Psl", + "chemical" + ], + [ + 127, + 131, + "EPSs", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "It has been reported that the activation of YfiN may be induced by redox-driven misfolding of YfiR (Giardina et al.,; Malone et al.,; Malone et al.,).", + "section": "INTRO", + "ner": [ + [ + 44, + 48, + "YfiN", + "protein" + ], + [ + 94, + 98, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 25, + "sent": "It is also proposed that the sequestration of YfiR by YfiB can be induced by certain YfiB-mediated cell wall stress, and mutagenesis studies revealed a number of activation residues of YfiB that were located in close proximity to the predicted first helix of the periplasmic domain (Malone et al.,).", + "section": "INTRO", + "ner": [ + [ + 46, + 50, + "YfiR", + "protein" + ], + [ + 54, + 58, + "YfiB", + "protein" + ], + [ + 85, + 89, + "YfiB", + "protein" + ], + [ + 121, + 140, + "mutagenesis studies", + "experimental_method" + ], + [ + 162, + 181, + "activation residues", + "structure_element" + ], + [ + 185, + 189, + "YfiB", + "protein" + ], + [ + 234, + 243, + "predicted", + "protein_state" + ], + [ + 244, + 255, + "first helix", + "structure_element" + ], + [ + 263, + 281, + "periplasmic domain", + "structure_element" + ] + ] + }, + { + "sid": 26, + "sent": "In addition, quorum sensing-related dephosphorylation of the PAS domain of YfiN may also be involved in the regulation (Ueda and Wood,; Xu et al.,).", + "section": "INTRO", + "ner": [ + [ + 61, + 71, + "PAS domain", + "structure_element" + ], + [ + 75, + 79, + "YfiN", + "protein" + ] + ] + }, + { + "sid": 27, + "sent": "Recently, we solved the crystal structure of YfiR in both the non-oxidized and the oxidized states, revealing breakage/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152), indicating that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR (Yang et al.,).", + "section": "INTRO", + "ner": [ + [ + 24, + 41, + "crystal structure", + "evidence" + ], + [ + 45, + 49, + "YfiR", + "protein" + ], + [ + 62, + 74, + "non-oxidized", + "protein_state" + ], + [ + 83, + 91, + "oxidized", + "protein_state" + ], + [ + 136, + 150, + "disulfide bond", + "ptm" + ], + [ + 152, + 157, + "Cys71", + "residue_name_number" + ], + [ + 158, + 164, + "Cys110", + "residue_name_number" + ], + [ + 220, + 226, + "Cys145", + "residue_name_number" + ], + [ + 227, + 233, + "Cys152", + "residue_name_number" + ], + [ + 252, + 258, + "Cys145", + "residue_name_number" + ], + [ + 259, + 265, + "Cys152", + "residue_name_number" + ], + [ + 328, + 332, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 28, + "sent": "In the present study, we solved the crystal structures of an N-terminal truncated form of YfiB (34\u2013168) and YfiR in complex with an active mutant YfiBL43P.", + "section": "INTRO", + "ner": [ + [ + 36, + 54, + "crystal structures", + "evidence" + ], + [ + 72, + 81, + "truncated", + "protein_state" + ], + [ + 90, + 94, + "YfiB", + "protein" + ], + [ + 96, + 102, + "34\u2013168", + "residue_range" + ], + [ + 108, + 112, + "YfiR", + "protein" + ], + [ + 113, + 128, + "in complex with", + "protein_state" + ], + [ + 132, + 138, + "active", + "protein_state" + ], + [ + 139, + 145, + "mutant", + "protein_state" + ], + [ + 146, + 154, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 29, + "sent": "Most recently, Li and coworkers reported the crystal structures of YfiB (27\u2013168) alone and YfiRC71S in complex with YfiB (59\u2013168) (Li et al.,).", + "section": "INTRO", + "ner": [ + [ + 45, + 63, + "crystal structures", + "evidence" + ], + [ + 67, + 71, + "YfiB", + "protein" + ], + [ + 73, + 79, + "27\u2013168", + "residue_range" + ], + [ + 81, + 86, + "alone", + "protein_state" + ], + [ + 91, + 99, + "YfiRC71S", + "mutant" + ], + [ + 100, + 115, + "in complex with", + "protein_state" + ], + [ + 116, + 120, + "YfiB", + "protein" + ], + [ + 122, + 128, + "59\u2013168", + "residue_range" + ] + ] + }, + { + "sid": 30, + "sent": "Compared with the reported complex structure, YfiBL43P in our YfiB-YfiR complex structure has additional visible N-terminal residues 44\u201358 that are shown to play essential roles in YfiB activation and biofilm formation.", + "section": "INTRO", + "ner": [ + [ + 46, + 54, + "YfiBL43P", + "mutant" + ], + [ + 62, + 71, + "YfiB-YfiR", + "complex_assembly" + ], + [ + 80, + 89, + "structure", + "evidence" + ], + [ + 133, + 138, + "44\u201358", + "residue_range" + ], + [ + 181, + 185, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 31, + "sent": "Therefore, we are able to visualize the detailed allosteric arrangement of the N-terminal structure of YfiB and its important role in YfiB-YfiR interaction.", + "section": "INTRO", + "ner": [ + [ + 103, + 107, + "YfiB", + "protein" + ], + [ + 134, + 143, + "YfiB-YfiR", + "complex_assembly" + ] + ] + }, + { + "sid": 32, + "sent": "In addition, we found that the YfiBL43P shows a much higher PG-binding affinity than wild-type YfiB, most likely due to its more compact PG-binding pocket.", + "section": "INTRO", + "ner": [ + [ + 31, + 39, + "YfiBL43P", + "mutant" + ], + [ + 60, + 79, + "PG-binding affinity", + "evidence" + ], + [ + 85, + 94, + "wild-type", + "protein_state" + ], + [ + 95, + 99, + "YfiB", + "protein" + ], + [ + 137, + 154, + "PG-binding pocket", + "site" + ] + ] + }, + { + "sid": 33, + "sent": "Moreover, we found that Vitamin B6 (VB6) or L-Trp can bind YfiR with an affinity in the ten millimolar range.", + "section": "INTRO", + "ner": [ + [ + 24, + 34, + "Vitamin B6", + "chemical" + ], + [ + 36, + 39, + "VB6", + "chemical" + ], + [ + 44, + 49, + "L-Trp", + "chemical" + ], + [ + 59, + 63, + "YfiR", + "protein" + ], + [ + 72, + 80, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 34, + "sent": "Together with functional data, these results provide new mechanistic insights into how activated YfiB sequesters YfiR and releases the suppression of YfiN. These findings may facilitate the development and optimization of anti-biofilm drugs for the treatment of chronic infections.", + "section": "INTRO", + "ner": [ + [ + 87, + 96, + "activated", + "protein_state" + ], + [ + 97, + 101, + "YfiB", + "protein" + ], + [ + 113, + 117, + "YfiR", + "protein" + ], + [ + 150, + 154, + "YfiN", + "protein" + ] + ] + }, + { + "sid": 35, + "sent": "Overall structure of YfiB", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 21, + 25, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 36, + "sent": "We obtained two crystal forms of YfiB (residues 34\u2013168, lacking the signal peptide from residues 1\u201326 and periplasmic residues 27\u201333), crystal forms I and II, belonging to space groups P21 and P41, respectively.", + "section": "RESULTS", + "ner": [ + [ + 16, + 29, + "crystal forms", + "evidence" + ], + [ + 33, + 37, + "YfiB", + "protein" + ], + [ + 48, + 54, + "34\u2013168", + "residue_range" + ], + [ + 56, + 63, + "lacking", + "protein_state" + ], + [ + 68, + 82, + "signal peptide", + "structure_element" + ], + [ + 97, + 101, + "1\u201326", + "residue_range" + ], + [ + 127, + 132, + "27\u201333", + "residue_range" + ] + ] + }, + { + "sid": 37, + "sent": "Overall structure of YfiB. (A) The overall structure of the YfiB monomer. (B) A topology diagram of the YfiB monomer. (C and D) The analytical ultracentrifugation experiment results for the wild-type YfiB and YfiBL43P ", + "section": "FIG", + "ner": [ + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 21, + 25, + "YfiB", + "protein" + ], + [ + 43, + 52, + "structure", + "evidence" + ], + [ + 60, + 64, + "YfiB", + "protein" + ], + [ + 65, + 72, + "monomer", + "oligomeric_state" + ], + [ + 104, + 108, + "YfiB", + "protein" + ], + [ + 109, + 116, + "monomer", + "oligomeric_state" + ], + [ + 132, + 162, + "analytical ultracentrifugation", + "experimental_method" + ], + [ + 190, + 199, + "wild-type", + "protein_state" + ], + [ + 200, + 204, + "YfiB", + "protein" + ], + [ + 209, + 217, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 38, + "sent": "Two dimeric types of YfiB dimer. (A\u2013C) The \u201chead to head\u201d dimer.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "dimeric", + "oligomeric_state" + ], + [ + 21, + 25, + "YfiB", + "protein" + ], + [ + 26, + 31, + "dimer", + "oligomeric_state" + ], + [ + 44, + 56, + "head to head", + "protein_state" + ], + [ + 58, + 63, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 39, + "sent": "The \u201cback to back\u201d dimer.", + "section": "FIG", + "ner": [ + [ + 5, + 17, + "back to back", + "protein_state" + ], + [ + 19, + 24, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 40, + "sent": "(A) and (E) indicate the front views of the two dimers, (B) and (F) indicate the top views of the two dimers, and (C) and (D) indicate the details of the two dimeric interfaces", + "section": "FIG", + "ner": [ + [ + 48, + 54, + "dimers", + "oligomeric_state" + ], + [ + 102, + 108, + "dimers", + "oligomeric_state" + ], + [ + 158, + 176, + "dimeric interfaces", + "site" + ] + ] + }, + { + "sid": 41, + "sent": "The crystal structure of YfiB monomer consists of a five-stranded \u03b2-sheet (\u03b21-2-5-3-4) flanked by five \u03b1-helices (\u03b11\u20135) on one side.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 25, + 29, + "YfiB", + "protein" + ], + [ + 30, + 37, + "monomer", + "oligomeric_state" + ], + [ + 52, + 73, + "five-stranded \u03b2-sheet", + "structure_element" + ], + [ + 75, + 85, + "\u03b21-2-5-3-4", + "structure_element" + ], + [ + 98, + 112, + "five \u03b1-helices", + "structure_element" + ], + [ + 114, + 118, + "\u03b11\u20135", + "structure_element" + ] + ] + }, + { + "sid": 42, + "sent": "In addition, there is a short helix turn connecting the \u03b24 strand and \u03b14 helix (Fig.\u00a01A and 1B).", + "section": "RESULTS", + "ner": [ + [ + 30, + 40, + "helix turn", + "structure_element" + ], + [ + 56, + 65, + "\u03b24 strand", + "structure_element" + ], + [ + 70, + 78, + "\u03b14 helix", + "structure_element" + ] + ] + }, + { + "sid": 43, + "sent": "Each crystal form contains three different dimeric types of YfiB, two of which are present in both, suggesting that the rest of the dimeric types may result from crystal packing.", + "section": "RESULTS", + "ner": [ + [ + 43, + 50, + "dimeric", + "oligomeric_state" + ], + [ + 60, + 64, + "YfiB", + "protein" + ], + [ + 132, + 139, + "dimeric", + "oligomeric_state" + ] + ] + }, + { + "sid": 44, + "sent": "Here, we refer to the two dimeric types as \u201chead to head\u201d and \u201cback to back\u201d according to the interacting mode (Fig.\u00a02A and 2E), with the total buried surface areas being 316.8 \u00c52 and 554.3 \u00c52, respectively.", + "section": "RESULTS", + "ner": [ + [ + 26, + 33, + "dimeric", + "oligomeric_state" + ], + [ + 44, + 56, + "head to head", + "protein_state" + ], + [ + 63, + 75, + "back to back", + "protein_state" + ] + ] + }, + { + "sid": 45, + "sent": "The \u201chead to head\u201d dimer exhibits a clamp shape.", + "section": "RESULTS", + "ner": [ + [ + 5, + 17, + "head to head", + "protein_state" + ], + [ + 19, + 24, + "dimer", + "oligomeric_state" + ], + [ + 36, + 47, + "clamp shape", + "protein_state" + ] + ] + }, + { + "sid": 46, + "sent": "The dimerization occurs mainly via hydrophobic interactions formed by A37 and I40 on the \u03b11 helices, L50 on the \u03b21 strands, and W55 on the \u03b22 strands of both molecules, making a hydrophobic interacting core (Fig.\u00a02A\u2013C).", + "section": "RESULTS", + "ner": [ + [ + 35, + 59, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 70, + 73, + "A37", + "residue_name_number" + ], + [ + 78, + 81, + "I40", + "residue_name_number" + ], + [ + 89, + 99, + "\u03b11 helices", + "structure_element" + ], + [ + 101, + 104, + "L50", + "residue_name_number" + ], + [ + 112, + 122, + "\u03b21 strands", + "structure_element" + ], + [ + 128, + 131, + "W55", + "residue_name_number" + ], + [ + 139, + 149, + "\u03b22 strands", + "structure_element" + ], + [ + 178, + 206, + "hydrophobic interacting core", + "site" + ] + ] + }, + { + "sid": 47, + "sent": "The \u201cback to back\u201d dimer presents a Y shape.", + "section": "RESULTS", + "ner": [ + [ + 5, + 17, + "back to back", + "protein_state" + ], + [ + 19, + 24, + "dimer", + "oligomeric_state" + ], + [ + 36, + 43, + "Y shape", + "protein_state" + ] + ] + }, + { + "sid": 48, + "sent": "The dimeric interaction is mainly hydrophilic, occurring among the main-chain and side-chain atoms of N68, L69, D70 and R71 on the \u03b12-\u03b13 loops and R116 and S120 on the \u03b14 helices of both molecules, resulting in a complex hydrogen bond network (Fig.\u00a02D\u2013F).", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "dimeric", + "oligomeric_state" + ], + [ + 12, + 45, + "interaction is mainly hydrophilic", + "bond_interaction" + ], + [ + 102, + 105, + "N68", + "residue_name_number" + ], + [ + 107, + 110, + "L69", + "residue_name_number" + ], + [ + 112, + 115, + "D70", + "residue_name_number" + ], + [ + 120, + 123, + "R71", + "residue_name_number" + ], + [ + 131, + 142, + "\u03b12-\u03b13 loops", + "structure_element" + ], + [ + 147, + 151, + "R116", + "residue_name_number" + ], + [ + 156, + 160, + "S120", + "residue_name_number" + ], + [ + 168, + 178, + "\u03b14 helices", + "structure_element" + ], + [ + 221, + 242, + "hydrogen bond network", + "site" + ] + ] + }, + { + "sid": 49, + "sent": "The YfiB-YfiR interaction", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "YfiB-YfiR", + "complex_assembly" + ] + ] + }, + { + "sid": 50, + "sent": "Overall structure of the YfiB-YfiR complex and the conserved surface in YfiR. (A) The overall structure of the YfiB-YfiR complex.", + "section": "FIG", + "ner": [ + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 25, + 34, + "YfiB-YfiR", + "complex_assembly" + ], + [ + 51, + 68, + "conserved surface", + "site" + ], + [ + 72, + 76, + "YfiR", + "protein" + ], + [ + 94, + 103, + "structure", + "evidence" + ], + [ + 111, + 120, + "YfiB-YfiR", + "complex_assembly" + ] + ] + }, + { + "sid": 51, + "sent": "The YfiBL43P molecules are shown in cyan and yellow.", + "section": "FIG", + "ner": [ + [ + 4, + 12, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 52, + "sent": "The YfiR molecules are shown in green and magenta.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 53, + "sent": "Two interacting regions are highlighted by red rectangles. (B) Structural superposition of apo YfiB and YfiR-bound YfiBL43P.", + "section": "FIG", + "ner": [ + [ + 63, + 87, + "Structural superposition", + "experimental_method" + ], + [ + 91, + 94, + "apo", + "protein_state" + ], + [ + 95, + 99, + "YfiB", + "protein" + ], + [ + 104, + 114, + "YfiR-bound", + "protein_state" + ], + [ + 115, + 123, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 54, + "sent": "To illustrate the differences between apo YfiB and YfiR-bound YfiBL43P, the apo YfiB is shown in pink, except residues 34\u201370 are shown in red, whereas the YfiR-bound YfiBL43P is shown in cyan, except residues 44\u201370 are shown in blue. (C) Close-up view of the differences between apo YfiB and YfiR-bound YfiBL43P.", + "section": "FIG", + "ner": [ + [ + 38, + 41, + "apo", + "protein_state" + ], + [ + 42, + 46, + "YfiB", + "protein" + ], + [ + 51, + 61, + "YfiR-bound", + "protein_state" + ], + [ + 62, + 70, + "YfiBL43P", + "mutant" + ], + [ + 76, + 79, + "apo", + "protein_state" + ], + [ + 80, + 84, + "YfiB", + "protein" + ], + [ + 119, + 124, + "34\u201370", + "residue_range" + ], + [ + 155, + 165, + "YfiR-bound", + "protein_state" + ], + [ + 166, + 174, + "YfiBL43P", + "mutant" + ], + [ + 209, + 214, + "44\u201370", + "residue_range" + ], + [ + 279, + 282, + "apo", + "protein_state" + ], + [ + 283, + 287, + "YfiB", + "protein" + ], + [ + 292, + 302, + "YfiR-bound", + "protein_state" + ], + [ + 303, + 311, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 55, + "sent": "The residues proposed to contribute to YfiB activation are illustrated in sticks.", + "section": "FIG", + "ner": [ + [ + 39, + 43, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 56, + "sent": "The key residues in apo YfiB are shown in red and those in YfiBL43P are shown in blue. (D) Close-up views showing interactions in regions I and II.", + "section": "FIG", + "ner": [ + [ + 20, + 23, + "apo", + "protein_state" + ], + [ + 24, + 28, + "YfiB", + "protein" + ], + [ + 59, + 67, + "YfiBL43P", + "mutant" + ], + [ + 130, + 146, + "regions I and II", + "structure_element" + ] + ] + }, + { + "sid": 57, + "sent": "YfiBL43P and YfiR are shown in cyan and green, respectively. (E and F) The conserved surface in YfiR contributes to the interaction with YfiB. (G) The residues of YfiR responsible for interacting with YfiB are shown in green sticks, and the proposed YfiN-interacting residues are shown in yellow sticks.", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "YfiBL43P", + "mutant" + ], + [ + 13, + 17, + "YfiR", + "protein" + ], + [ + 75, + 92, + "conserved surface", + "site" + ], + [ + 96, + 100, + "YfiR", + "protein" + ], + [ + 137, + 141, + "YfiB", + "protein" + ], + [ + 151, + 159, + "residues", + "structure_element" + ], + [ + 163, + 167, + "YfiR", + "protein" + ], + [ + 201, + 205, + "YfiB", + "protein" + ], + [ + 250, + 275, + "YfiN-interacting residues", + "site" + ] + ] + }, + { + "sid": 58, + "sent": "The red sticks, which represent the YfiB-interacting residues, are also responsible for the proposed interactions with YfiN", + "section": "FIG", + "ner": [ + [ + 36, + 61, + "YfiB-interacting residues", + "site" + ], + [ + 119, + 123, + "YfiN", + "protein" + ] + ] + }, + { + "sid": 59, + "sent": "To gain structural insights into the YfiB-YfiR interaction, we co-expressed YfiB (residues 34\u2013168) and YfiR (residues 35\u2013190, lacking the signal peptide), but failed to obtain the complex, in accordance with a previous report in which no stable complex of YfiB-YfiR was observed (Malone et al.,).", + "section": "RESULTS", + "ner": [ + [ + 37, + 46, + "YfiB-YfiR", + "complex_assembly" + ], + [ + 63, + 75, + "co-expressed", + "experimental_method" + ], + [ + 76, + 80, + "YfiB", + "protein" + ], + [ + 91, + 97, + "34\u2013168", + "residue_range" + ], + [ + 103, + 107, + "YfiR", + "protein" + ], + [ + 118, + 124, + "35\u2013190", + "residue_range" + ], + [ + 126, + 133, + "lacking", + "protein_state" + ], + [ + 138, + 152, + "signal peptide", + "structure_element" + ], + [ + 235, + 244, + "no stable", + "protein_state" + ], + [ + 256, + 265, + "YfiB-YfiR", + "complex_assembly" + ] + ] + }, + { + "sid": 60, + "sent": "It has been reported that single mutants of Q39, L43, F48 and W55 contribute to YfiB activation leading to the induction of the SCV phenotype in P. aeruginosa PAO1 (Malone et al.,).", + "section": "RESULTS", + "ner": [ + [ + 26, + 43, + "single mutants of", + "experimental_method" + ], + [ + 44, + 47, + "Q39", + "residue_name_number" + ], + [ + 49, + 52, + "L43", + "residue_name_number" + ], + [ + 54, + 57, + "F48", + "residue_name_number" + ], + [ + 62, + 65, + "W55", + "residue_name_number" + ], + [ + 80, + 84, + "YfiB", + "protein" + ], + [ + 145, + 163, + "P. aeruginosa PAO1", + "species" + ] + ] + }, + { + "sid": 61, + "sent": "It is likely that these residues may be involved in the conformational changes of YfiB that are related to YfiR sequestration (Fig.\u00a03C).", + "section": "RESULTS", + "ner": [ + [ + 82, + 86, + "YfiB", + "protein" + ], + [ + 107, + 111, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 62, + "sent": "Therefore, we constructed two such single mutants of YfiB (YfiBL43P and YfiBF48S).", + "section": "RESULTS", + "ner": [ + [ + 14, + 49, + "constructed two such single mutants", + "experimental_method" + ], + [ + 53, + 57, + "YfiB", + "protein" + ], + [ + 59, + 67, + "YfiBL43P", + "mutant" + ], + [ + 72, + 80, + "YfiBF48S", + "mutant" + ] + ] + }, + { + "sid": 63, + "sent": "As expected, both mutants form a stable complex with YfiR. Finally, we crystalized YfiR in complex with the YfiBL43P mutant and solved the structure at 1.78 \u00c5 resolution by molecular replacement using YfiR and YfiB as models.", + "section": "RESULTS", + "ner": [ + [ + 33, + 39, + "stable", + "protein_state" + ], + [ + 40, + 52, + "complex with", + "protein_state" + ], + [ + 53, + 57, + "YfiR", + "protein" + ], + [ + 71, + 82, + "crystalized", + "experimental_method" + ], + [ + 83, + 87, + "YfiR", + "protein" + ], + [ + 88, + 103, + "in complex with", + "protein_state" + ], + [ + 108, + 116, + "YfiBL43P", + "mutant" + ], + [ + 117, + 123, + "mutant", + "protein_state" + ], + [ + 139, + 148, + "structure", + "evidence" + ], + [ + 173, + 194, + "molecular replacement", + "experimental_method" + ], + [ + 201, + 205, + "YfiR", + "protein" + ], + [ + 210, + 214, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 64, + "sent": "The YfiB-YfiR complex is a 2:2 heterotetramer (Fig.\u00a03A) in which the YfiR dimer is clamped by two separated YfiBL43P molecules with a total buried surface area of 3161.2 \u00c52.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "YfiB-YfiR", + "complex_assembly" + ], + [ + 31, + 45, + "heterotetramer", + "oligomeric_state" + ], + [ + 69, + 73, + "YfiR", + "protein" + ], + [ + 74, + 79, + "dimer", + "oligomeric_state" + ], + [ + 108, + 116, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 65, + "sent": "The YfiR dimer in the complex is identical to the non-oxidized YfiR dimer alone (Yang et al.,), with only Cys145-Cys152 of the two disulfide bonds well formed, suggesting Cys71-Cys110 disulfide bond formation is not essential for forming YfiB-YfiR complex.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "YfiR", + "protein" + ], + [ + 9, + 14, + "dimer", + "oligomeric_state" + ], + [ + 50, + 62, + "non-oxidized", + "protein_state" + ], + [ + 63, + 67, + "YfiR", + "protein" + ], + [ + 68, + 73, + "dimer", + "oligomeric_state" + ], + [ + 74, + 79, + "alone", + "protein_state" + ], + [ + 106, + 112, + "Cys145", + "residue_name_number" + ], + [ + 113, + 119, + "Cys152", + "residue_name_number" + ], + [ + 131, + 146, + "disulfide bonds", + "ptm" + ], + [ + 171, + 176, + "Cys71", + "residue_name_number" + ], + [ + 177, + 183, + "Cys110", + "residue_name_number" + ], + [ + 184, + 198, + "disulfide bond", + "ptm" + ], + [ + 238, + 247, + "YfiB-YfiR", + "complex_assembly" + ] + ] + }, + { + "sid": 66, + "sent": "The N-terminal structural conformation of YfiBL43P, from the foremost N-terminus to residue D70, is significantly altered compared with that of the apo YfiB. The majority of the \u03b11 helix (residues 34\u201343) is invisible on the electron density map, and the \u03b12 helix and \u03b21 and \u03b22 strands are rearranged to form a long loop containing two short \u03b1-helix turns (Fig.\u00a03B and 3C), thus embracing the YfiR dimer.", + "section": "RESULTS", + "ner": [ + [ + 42, + 50, + "YfiBL43P", + "mutant" + ], + [ + 92, + 95, + "D70", + "residue_name_number" + ], + [ + 148, + 151, + "apo", + "protein_state" + ], + [ + 152, + 156, + "YfiB", + "protein" + ], + [ + 178, + 186, + "\u03b11 helix", + "structure_element" + ], + [ + 197, + 202, + "34\u201343", + "residue_range" + ], + [ + 224, + 244, + "electron density map", + "evidence" + ], + [ + 254, + 262, + "\u03b12 helix", + "structure_element" + ], + [ + 267, + 269, + "\u03b21", + "structure_element" + ], + [ + 274, + 284, + "\u03b22 strands", + "structure_element" + ], + [ + 315, + 319, + "loop", + "structure_element" + ], + [ + 341, + 354, + "\u03b1-helix turns", + "structure_element" + ], + [ + 392, + 396, + "YfiR", + "protein" + ], + [ + 397, + 402, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 67, + "sent": "The observed changes in conformation of YfiB and the results of mutagenesis suggest a mechanism by which YfiB sequesters YfiR.", + "section": "RESULTS", + "ner": [ + [ + 40, + 44, + "YfiB", + "protein" + ], + [ + 64, + 75, + "mutagenesis", + "experimental_method" + ], + [ + 105, + 109, + "YfiB", + "protein" + ], + [ + 121, + 125, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 68, + "sent": "The YfiB-YfiR interface can be divided into two regions (Fig.\u00a03A and 3D).", + "section": "RESULTS", + "ner": [ + [ + 4, + 23, + "YfiB-YfiR interface", + "site" + ] + ] + }, + { + "sid": 69, + "sent": "Region I is formed by numerous main-chain and side-chain hydrophilic interactions between residues E45, G47 and E53 from the N-terminal extended loop of YfiB and residues S57, R60, A89 and H177 from YfiR (Fig.\u00a03D-I(i)).", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Region I", + "structure_element" + ], + [ + 57, + 81, + "hydrophilic interactions", + "bond_interaction" + ], + [ + 99, + 102, + "E45", + "residue_name_number" + ], + [ + 104, + 107, + "G47", + "residue_name_number" + ], + [ + 112, + 115, + "E53", + "residue_name_number" + ], + [ + 145, + 149, + "loop", + "structure_element" + ], + [ + 153, + 157, + "YfiB", + "protein" + ], + [ + 171, + 174, + "S57", + "residue_name_number" + ], + [ + 176, + 179, + "R60", + "residue_name_number" + ], + [ + 181, + 184, + "A89", + "residue_name_number" + ], + [ + 189, + 193, + "H177", + "residue_name_number" + ], + [ + 199, + 203, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 70, + "sent": "Additionally, three hydrophobic anchoring sites exist in region I. The residues F48 and W55 of YfiB are inserted into the hydrophobic cores mainly formed by the main chain and side chain carbon atoms of residues S57/Q88/A89/N90 and R60/R175/H177 of YfiR, respectively; and F57 of YfiB is inserted into the hydrophobic pocket formed by L166/I169/V176/P178/L181 of YfiR (Fig.\u00a03D-I(ii)).", + "section": "RESULTS", + "ner": [ + [ + 20, + 47, + "hydrophobic anchoring sites", + "site" + ], + [ + 57, + 65, + "region I", + "structure_element" + ], + [ + 80, + 83, + "F48", + "residue_name_number" + ], + [ + 88, + 91, + "W55", + "residue_name_number" + ], + [ + 95, + 99, + "YfiB", + "protein" + ], + [ + 122, + 139, + "hydrophobic cores", + "site" + ], + [ + 212, + 215, + "S57", + "residue_name_number" + ], + [ + 216, + 219, + "Q88", + "residue_name_number" + ], + [ + 220, + 223, + "A89", + "residue_name_number" + ], + [ + 224, + 227, + "N90", + "residue_name_number" + ], + [ + 232, + 235, + "R60", + "residue_name_number" + ], + [ + 236, + 240, + "R175", + "residue_name_number" + ], + [ + 241, + 245, + "H177", + "residue_name_number" + ], + [ + 249, + 253, + "YfiR", + "protein" + ], + [ + 273, + 276, + "F57", + "residue_name_number" + ], + [ + 280, + 284, + "YfiB", + "protein" + ], + [ + 306, + 324, + "hydrophobic pocket", + "site" + ], + [ + 335, + 339, + "L166", + "residue_name_number" + ], + [ + 340, + 344, + "I169", + "residue_name_number" + ], + [ + 345, + 349, + "V176", + "residue_name_number" + ], + [ + 350, + 354, + "P178", + "residue_name_number" + ], + [ + 355, + 359, + "L181", + "residue_name_number" + ], + [ + 363, + 367, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 71, + "sent": "In region II, the side chains of R96, E98 and E157 from YfiB interact with the side chains of E163, S146 and R171 from YfiR, respectively.", + "section": "RESULTS", + "ner": [ + [ + 3, + 12, + "region II", + "structure_element" + ], + [ + 33, + 36, + "R96", + "residue_name_number" + ], + [ + 38, + 41, + "E98", + "residue_name_number" + ], + [ + 46, + 50, + "E157", + "residue_name_number" + ], + [ + 56, + 60, + "YfiB", + "protein" + ], + [ + 94, + 98, + "E163", + "residue_name_number" + ], + [ + 100, + 104, + "S146", + "residue_name_number" + ], + [ + 109, + 113, + "R171", + "residue_name_number" + ], + [ + 119, + 123, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 72, + "sent": "Additionally, the main chains of I163 and V165 from YfiB form hydrogen bonds with the main chains of L166 and A164 from YfiR, respectively, and the main chain of P166 from YfiB interacts with the side chain of R185 from YfiR (Fig.\u00a03D-II).", + "section": "RESULTS", + "ner": [ + [ + 33, + 37, + "I163", + "residue_name_number" + ], + [ + 42, + 46, + "V165", + "residue_name_number" + ], + [ + 52, + 56, + "YfiB", + "protein" + ], + [ + 62, + 76, + "hydrogen bonds", + "bond_interaction" + ], + [ + 101, + 105, + "L166", + "residue_name_number" + ], + [ + 110, + 114, + "A164", + "residue_name_number" + ], + [ + 120, + 124, + "YfiR", + "protein" + ], + [ + 162, + 166, + "P166", + "residue_name_number" + ], + [ + 172, + 176, + "YfiB", + "protein" + ], + [ + 210, + 214, + "R185", + "residue_name_number" + ], + [ + 220, + 224, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 73, + "sent": "These two regions contribute a robust hydrogen-bonding network to the YfiB-YfiR interface, resulting in a tightly bound complex.", + "section": "RESULTS", + "ner": [ + [ + 38, + 62, + "hydrogen-bonding network", + "site" + ], + [ + 70, + 89, + "YfiB-YfiR interface", + "site" + ] + ] + }, + { + "sid": 74, + "sent": "Based on the observations that two separated YfiBL43P molecules form a 2:2 complex structure with YfiR dimer, we performed an analytical ultracentrifugation experiment to check the oligomeric states of wild-type YfiB and YfiBL43P.", + "section": "RESULTS", + "ner": [ + [ + 45, + 53, + "YfiBL43P", + "mutant" + ], + [ + 83, + 92, + "structure", + "evidence" + ], + [ + 98, + 102, + "YfiR", + "protein" + ], + [ + 103, + 108, + "dimer", + "oligomeric_state" + ], + [ + 126, + 156, + "analytical ultracentrifugation", + "experimental_method" + ], + [ + 202, + 211, + "wild-type", + "protein_state" + ], + [ + 212, + 216, + "YfiB", + "protein" + ], + [ + 221, + 229, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 75, + "sent": "The results showed that wild-type YfiB exists in both monomeric and dimeric states in solution, while YfiBL43P primarily adopts the monomer state in solution (Fig.\u00a01C\u2013D).", + "section": "RESULTS", + "ner": [ + [ + 24, + 33, + "wild-type", + "protein_state" + ], + [ + 34, + 38, + "YfiB", + "protein" + ], + [ + 54, + 63, + "monomeric", + "oligomeric_state" + ], + [ + 68, + 75, + "dimeric", + "oligomeric_state" + ], + [ + 102, + 110, + "YfiBL43P", + "mutant" + ], + [ + 132, + 139, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 76, + "sent": "This suggests that the N-terminus of YfiB plays an important role in forming the dimeric YfiB in solution and that the conformational change of residue L43 is associated with the stretch of the N-terminus and opening of the dimer.", + "section": "RESULTS", + "ner": [ + [ + 37, + 41, + "YfiB", + "protein" + ], + [ + 81, + 88, + "dimeric", + "oligomeric_state" + ], + [ + 89, + 93, + "YfiB", + "protein" + ], + [ + 152, + 155, + "L43", + "residue_name_number" + ], + [ + 224, + 229, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 77, + "sent": "Therefore, it is possible that both dimeric types might exist in solution.", + "section": "RESULTS", + "ner": [ + [ + 36, + 43, + "dimeric", + "oligomeric_state" + ] + ] + }, + { + "sid": 78, + "sent": "For simplicity, we only discuss the \u201chead to head\u201d dimer in the following text.", + "section": "RESULTS", + "ner": [ + [ + 37, + 49, + "head to head", + "protein_state" + ], + [ + 51, + 56, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 79, + "sent": "The PG-binding site of YfiB", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "PG-binding site", + "site" + ], + [ + 23, + 27, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 80, + "sent": "The PG-binding site in YfiB. (A) Structural superposition of the PG-binding sites of the H. influenzae Pal/PG-P complex and YfiR-bound YfiBL43P complexed with sulfate ions.", + "section": "FIG", + "ner": [ + [ + 4, + 19, + "PG-binding site", + "site" + ], + [ + 23, + 27, + "YfiB", + "protein" + ], + [ + 33, + 57, + "Structural superposition", + "experimental_method" + ], + [ + 65, + 81, + "PG-binding sites", + "site" + ], + [ + 89, + 102, + "H. influenzae", + "species" + ], + [ + 103, + 111, + "Pal/PG-P", + "complex_assembly" + ], + [ + 124, + 134, + "YfiR-bound", + "protein_state" + ], + [ + 135, + 143, + "YfiBL43P", + "mutant" + ], + [ + 144, + 158, + "complexed with", + "protein_state" + ], + [ + 159, + 166, + "sulfate", + "chemical" + ] + ] + }, + { + "sid": 81, + "sent": "(B) Close-up view showing the key residues of Pal interacting with the m-Dap5 \u03b5-carboxylate group of PG-P. Pal is shown in wheat and PG-P is in magenta.", + "section": "FIG", + "ner": [ + [ + 46, + 49, + "Pal", + "protein_type" + ], + [ + 71, + 91, + "m-Dap5 \u03b5-carboxylate", + "chemical" + ], + [ + 101, + 105, + "PG-P", + "chemical" + ], + [ + 107, + 110, + "Pal", + "protein_type" + ], + [ + 133, + 137, + "PG-P", + "chemical" + ] + ] + }, + { + "sid": 82, + "sent": "(C) Close-up view showing the key residues of YfiR-bound YfiBL43P interacting with a sulfate ion.", + "section": "FIG", + "ner": [ + [ + 46, + 56, + "YfiR-bound", + "protein_state" + ], + [ + 57, + 65, + "YfiBL43P", + "mutant" + ], + [ + 85, + 92, + "sulfate", + "chemical" + ] + ] + }, + { + "sid": 83, + "sent": "YfiR-bound YfiBL43P is shown in cyan; the sulfate ion, in green; and the water molecule, in yellow. (D) Structural superposition of the PG-binding sites of apo YfiB and YfiR-bound YfiBL43P, the key residues are shown in stick.", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "YfiR-bound", + "protein_state" + ], + [ + 11, + 19, + "YfiBL43P", + "mutant" + ], + [ + 42, + 49, + "sulfate", + "chemical" + ], + [ + 73, + 78, + "water", + "chemical" + ], + [ + 104, + 128, + "Structural superposition", + "experimental_method" + ], + [ + 136, + 152, + "PG-binding sites", + "site" + ], + [ + 156, + 159, + "apo", + "protein_state" + ], + [ + 160, + 164, + "YfiB", + "protein" + ], + [ + 169, + 179, + "YfiR-bound", + "protein_state" + ], + [ + 180, + 188, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 84, + "sent": "Apo YfiB is shown in yellow and YfiR-bound YfiBL43P in cyan. (E and F) MST data and analysis for binding affinities of (E) YfiB wild-type and (F) YfiBL43P with PG. (G) The sequence alignment of P. aeruginosa and E. coli sources of YfiB, Pal and the periplasmic domain of OmpA", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "Apo", + "protein_state" + ], + [ + 4, + 8, + "YfiB", + "protein" + ], + [ + 32, + 42, + "YfiR-bound", + "protein_state" + ], + [ + 43, + 51, + "YfiBL43P", + "mutant" + ], + [ + 71, + 74, + "MST", + "experimental_method" + ], + [ + 97, + 115, + "binding affinities", + "evidence" + ], + [ + 123, + 127, + "YfiB", + "protein" + ], + [ + 128, + 137, + "wild-type", + "protein_state" + ], + [ + 146, + 154, + "YfiBL43P", + "mutant" + ], + [ + 160, + 162, + "PG", + "chemical" + ], + [ + 172, + 190, + "sequence alignment", + "experimental_method" + ], + [ + 194, + 207, + "P. aeruginosa", + "species" + ], + [ + 212, + 219, + "E. coli", + "species" + ], + [ + 231, + 235, + "YfiB", + "protein" + ], + [ + 237, + 240, + "Pal", + "protein_type" + ], + [ + 249, + 267, + "periplasmic domain", + "structure_element" + ], + [ + 271, + 275, + "OmpA", + "protein_type" + ] + ] + }, + { + "sid": 85, + "sent": "PG-associated lipoprotein (Pal) is highly conserved in Gram-negative bacteria and anchors to the outer membrane through an N-terminal lipid attachment and to PG layer through its periplasmic domain, which is implicated in maintaining outer membrane integrity.", + "section": "RESULTS", + "ner": [ + [ + 0, + 25, + "PG-associated lipoprotein", + "protein_type" + ], + [ + 27, + 30, + "Pal", + "protein_type" + ], + [ + 35, + 51, + "highly conserved", + "protein_state" + ], + [ + 55, + 77, + "Gram-negative bacteria", + "taxonomy_domain" + ], + [ + 158, + 160, + "PG", + "chemical" + ], + [ + 179, + 197, + "periplasmic domain", + "structure_element" + ] + ] + }, + { + "sid": 86, + "sent": "Previous homology modeling studies suggested that YfiB contains a Pal-like PG-binding site (Parsons et al.,), and the mutation of two residues at this site, D102 and G105, reduces the ability for biofilm formation and surface attachment (Malone et al.,).", + "section": "RESULTS", + "ner": [ + [ + 9, + 26, + "homology modeling", + "experimental_method" + ], + [ + 50, + 54, + "YfiB", + "protein" + ], + [ + 66, + 90, + "Pal-like PG-binding site", + "site" + ], + [ + 118, + 142, + "mutation of two residues", + "experimental_method" + ], + [ + 157, + 161, + "D102", + "residue_name_number" + ], + [ + 166, + 170, + "G105", + "residue_name_number" + ] + ] + }, + { + "sid": 87, + "sent": "In the YfiB-YfiR complex, one sulfate ion is found at the bottom of each YfiBL43P molecule (Fig.\u00a03A) and forms a strong hydrogen bond with D102 of YfiBL43P (Fig.\u00a04A and 4C).", + "section": "RESULTS", + "ner": [ + [ + 7, + 16, + "YfiB-YfiR", + "complex_assembly" + ], + [ + 30, + 37, + "sulfate", + "chemical" + ], + [ + 73, + 81, + "YfiBL43P", + "mutant" + ], + [ + 120, + 133, + "hydrogen bond", + "bond_interaction" + ], + [ + 139, + 143, + "D102", + "residue_name_number" + ], + [ + 147, + 155, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 88, + "sent": "Structural superposition between YfiBL43P and Haemophilus influenzae Pal complexed with biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-\u03b1-D-Glu-m-Dap-D-Ala-D-Ala (m-Dap is meso-diaminopimelate) (PDB code: 2aiz) (Parsons et al.,), revealed that the sulfate ion is located at the position of the m-Dap5 \u03f5-carboxylate group in the Pal/PG-P complex (Fig.\u00a04A).", + "section": "RESULTS", + "ner": [ + [ + 0, + 24, + "Structural superposition", + "experimental_method" + ], + [ + 33, + 41, + "YfiBL43P", + "mutant" + ], + [ + 46, + 68, + "Haemophilus influenzae", + "species" + ], + [ + 69, + 72, + "Pal", + "protein_type" + ], + [ + 73, + 87, + "complexed with", + "protein_state" + ], + [ + 101, + 124, + "peptidoglycan precursor", + "chemical" + ], + [ + 126, + 130, + "PG-P", + "chemical" + ], + [ + 133, + 184, + "UDP-N-acetylmuramyl-L-Ala-\u03b1-D-Glu-m-Dap-D-Ala-D-Ala", + "chemical" + ], + [ + 186, + 191, + "m-Dap", + "chemical" + ], + [ + 195, + 215, + "meso-diaminopimelate", + "chemical" + ], + [ + 271, + 278, + "sulfate", + "chemical" + ], + [ + 317, + 337, + "m-Dap5 \u03f5-carboxylate", + "chemical" + ], + [ + 351, + 359, + "Pal/PG-P", + "complex_assembly" + ] + ] + }, + { + "sid": 89, + "sent": "In the Pal/PG-P complex structure, the m-Dap5 \u03f5-carboxylate group interacts with the side-chain atoms of D71 and the main-chain amide of D37 (Fig.\u00a04B).", + "section": "RESULTS", + "ner": [ + [ + 7, + 15, + "Pal/PG-P", + "complex_assembly" + ], + [ + 24, + 33, + "structure", + "evidence" + ], + [ + 39, + 59, + "m-Dap5 \u03f5-carboxylate", + "chemical" + ], + [ + 105, + 108, + "D71", + "residue_name_number" + ], + [ + 137, + 140, + "D37", + "residue_name_number" + ] + ] + }, + { + "sid": 90, + "sent": "Similarly, in the YfiR-bound YfiBL43P structure, the sulfate ion interacts with the side-chain atoms of D102 (corresponding to D71 in Pal) and R117 (corresponding to R86 in Pal) and the main-chain amide of N68 (corresponding to D37 in Pal).", + "section": "RESULTS", + "ner": [ + [ + 18, + 28, + "YfiR-bound", + "protein_state" + ], + [ + 29, + 37, + "YfiBL43P", + "mutant" + ], + [ + 38, + 47, + "structure", + "evidence" + ], + [ + 53, + 60, + "sulfate", + "chemical" + ], + [ + 104, + 108, + "D102", + "residue_name_number" + ], + [ + 127, + 130, + "D71", + "residue_name_number" + ], + [ + 134, + 137, + "Pal", + "protein_type" + ], + [ + 143, + 147, + "R117", + "residue_name_number" + ], + [ + 166, + 169, + "R86", + "residue_name_number" + ], + [ + 173, + 176, + "Pal", + "protein_type" + ], + [ + 206, + 209, + "N68", + "residue_name_number" + ], + [ + 228, + 231, + "D37", + "residue_name_number" + ], + [ + 235, + 238, + "Pal", + "protein_type" + ] + ] + }, + { + "sid": 91, + "sent": "Moreover, a water molecule was found to bridge the sulfate ion and the side chains of N67 and D102, strengthening the hydrogen bond network (Fig.\u00a04C).", + "section": "RESULTS", + "ner": [ + [ + 12, + 17, + "water", + "chemical" + ], + [ + 51, + 58, + "sulfate", + "chemical" + ], + [ + 86, + 89, + "N67", + "residue_name_number" + ], + [ + 94, + 98, + "D102", + "residue_name_number" + ], + [ + 118, + 139, + "hydrogen bond network", + "site" + ] + ] + }, + { + "sid": 92, + "sent": "In addition, sequence alignment of YfiB with Pal and the periplasmic domain of OmpA (proteins containing PG-binding site) showed that N68 and D102 are highly conserved (Fig.\u00a04G, blue stars), suggesting that these residues contribute to the PG-binding ability of YfiB.", + "section": "RESULTS", + "ner": [ + [ + 13, + 31, + "sequence alignment", + "experimental_method" + ], + [ + 35, + 39, + "YfiB", + "protein" + ], + [ + 45, + 48, + "Pal", + "protein_type" + ], + [ + 57, + 75, + "periplasmic domain", + "structure_element" + ], + [ + 79, + 83, + "OmpA", + "protein_type" + ], + [ + 105, + 120, + "PG-binding site", + "site" + ], + [ + 134, + 137, + "N68", + "residue_name_number" + ], + [ + 142, + 146, + "D102", + "residue_name_number" + ], + [ + 151, + 167, + "highly conserved", + "protein_state" + ], + [ + 262, + 266, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 93, + "sent": "Interestingly, superposition of apo YfiB with YfiR-bound YfiBL43P revealed that the PG-binding region is largely altered mainly due to different conformation of the N68 containing loop.", + "section": "RESULTS", + "ner": [ + [ + 15, + 28, + "superposition", + "experimental_method" + ], + [ + 32, + 35, + "apo", + "protein_state" + ], + [ + 36, + 40, + "YfiB", + "protein" + ], + [ + 46, + 56, + "YfiR-bound", + "protein_state" + ], + [ + 57, + 65, + "YfiBL43P", + "mutant" + ], + [ + 84, + 101, + "PG-binding region", + "site" + ], + [ + 135, + 157, + "different conformation", + "protein_state" + ], + [ + 165, + 168, + "N68", + "residue_name_number" + ], + [ + 180, + 184, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 94, + "sent": "Compared to YfiBL43P, the N68-containing loop of the apo YfiB flips away about 7 \u00c5, and D102 and R117 swing slightly outward; thus, the PG-binding pocket is enlarged with no sulfate ion or water bound (Fig.\u00a04D).", + "section": "RESULTS", + "ner": [ + [ + 12, + 20, + "YfiBL43P", + "mutant" + ], + [ + 26, + 29, + "N68", + "residue_name_number" + ], + [ + 41, + 45, + "loop", + "structure_element" + ], + [ + 53, + 56, + "apo", + "protein_state" + ], + [ + 57, + 61, + "YfiB", + "protein" + ], + [ + 88, + 92, + "D102", + "residue_name_number" + ], + [ + 97, + 101, + "R117", + "residue_name_number" + ], + [ + 136, + 153, + "PG-binding pocket", + "site" + ], + [ + 174, + 181, + "sulfate", + "chemical" + ], + [ + 189, + 194, + "water", + "chemical" + ] + ] + }, + { + "sid": 95, + "sent": "Therefore, we proposed that the PG-binding ability of inactive YfiB might be weaker than that of active YfiB. To validate this, we performed a microscale thermophoresis (MST) assay to measure the binding affinities of PG to wild-type YfiB and YfiBL43P, respectively.", + "section": "RESULTS", + "ner": [ + [ + 32, + 34, + "PG", + "chemical" + ], + [ + 54, + 62, + "inactive", + "protein_state" + ], + [ + 63, + 67, + "YfiB", + "protein" + ], + [ + 97, + 103, + "active", + "protein_state" + ], + [ + 104, + 108, + "YfiB", + "protein" + ], + [ + 143, + 168, + "microscale thermophoresis", + "experimental_method" + ], + [ + 170, + 173, + "MST", + "experimental_method" + ], + [ + 196, + 214, + "binding affinities", + "evidence" + ], + [ + 218, + 220, + "PG", + "chemical" + ], + [ + 224, + 233, + "wild-type", + "protein_state" + ], + [ + 234, + 238, + "YfiB", + "protein" + ], + [ + 243, + 251, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 96, + "sent": "The results indicated that the PG-binding affinity of YfiBL43P is 65.5 \u03bcmol/L, which is about 16-fold stronger than that of wild-type YfiB (Kd = 1.1 mmol/L) (Fig.\u00a04E\u2013F).", + "section": "RESULTS", + "ner": [ + [ + 31, + 50, + "PG-binding affinity", + "evidence" + ], + [ + 54, + 62, + "YfiBL43P", + "mutant" + ], + [ + 124, + 133, + "wild-type", + "protein_state" + ], + [ + 134, + 138, + "YfiB", + "protein" + ], + [ + 140, + 142, + "Kd", + "evidence" + ] + ] + }, + { + "sid": 97, + "sent": "As the experiment is performed in the absence of YfiR, it suggests that an increase in the PG-binding affinity of YfiB is not a result of YfiB-YfiR interaction and is highly coupled to the activation of YfiB characterized by a stretched N-terminal conformation.", + "section": "RESULTS", + "ner": [ + [ + 31, + 48, + "in the absence of", + "protein_state" + ], + [ + 49, + 53, + "YfiR", + "protein" + ], + [ + 91, + 110, + "PG-binding affinity", + "evidence" + ], + [ + 114, + 118, + "YfiB", + "protein" + ], + [ + 138, + 147, + "YfiB-YfiR", + "complex_assembly" + ], + [ + 203, + 207, + "YfiB", + "protein" + ], + [ + 227, + 260, + "stretched N-terminal conformation", + "protein_state" + ] + ] + }, + { + "sid": 98, + "sent": "The conserved surface in YfiR is functional for binding YfiB and YfiN", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "conserved surface", + "site" + ], + [ + 25, + 29, + "YfiR", + "protein" + ], + [ + 56, + 60, + "YfiB", + "protein" + ], + [ + 65, + 69, + "YfiN", + "protein" + ] + ] + }, + { + "sid": 99, + "sent": "Calculation using the ConSurf Server (http://consurf.tau.ac.il/), which estimates the evolutionary conservation of amino acid positions and visualizes information on the structure surface, revealed a conserved surface on YfiR that contributes to the interaction with YfiB (Fig.\u00a03E and 3F).", + "section": "RESULTS", + "ner": [ + [ + 22, + 36, + "ConSurf Server", + "experimental_method" + ], + [ + 86, + 111, + "evolutionary conservation", + "evidence" + ], + [ + 170, + 187, + "structure surface", + "site" + ], + [ + 200, + 217, + "conserved surface", + "site" + ], + [ + 221, + 225, + "YfiR", + "protein" + ], + [ + 267, + 271, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 100, + "sent": "Interestingly, the majority of this conserved surface contributes to the interaction with YfiB (Fig.\u00a03E and 3F).", + "section": "RESULTS", + "ner": [ + [ + 36, + 53, + "conserved surface", + "site" + ], + [ + 90, + 94, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 101, + "sent": "Malone JG et al. have reported that F151, E163, I169 and Q187, located near the C-terminus of YfiR, comprise a putative YfiN binding site (Malone et al.,).", + "section": "RESULTS", + "ner": [ + [ + 36, + 40, + "F151", + "residue_name_number" + ], + [ + 42, + 46, + "E163", + "residue_name_number" + ], + [ + 48, + 52, + "I169", + "residue_name_number" + ], + [ + 57, + 61, + "Q187", + "residue_name_number" + ], + [ + 94, + 98, + "YfiR", + "protein" + ], + [ + 120, + 137, + "YfiN binding site", + "site" + ] + ] + }, + { + "sid": 102, + "sent": "Interestingly, these residues are part of the conserved surface of YfiR (Fig.\u00a03G).", + "section": "RESULTS", + "ner": [ + [ + 46, + 63, + "conserved surface", + "site" + ], + [ + 67, + 71, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 103, + "sent": "F151, E163 and I169 form a hydrophobic core while, Q187 is located at the end of the \u03b16 helix.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "F151", + "residue_name_number" + ], + [ + 6, + 10, + "E163", + "residue_name_number" + ], + [ + 15, + 19, + "I169", + "residue_name_number" + ], + [ + 27, + 43, + "hydrophobic core", + "site" + ], + [ + 51, + 55, + "Q187", + "residue_name_number" + ], + [ + 85, + 93, + "\u03b16 helix", + "structure_element" + ] + ] + }, + { + "sid": 104, + "sent": "E163 and I169 are YfiB-interacting residues of YfiR, in which E163 forms a hydrogen bond with R96 of YfiB (Fig.\u00a03D-II) and I169 is involved in forming the L166/I169/V176/P178/L181 hydrophobic core for anchoring F57 of YfiB (Fig.\u00a03D-I(ii)).", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "E163", + "residue_name_number" + ], + [ + 9, + 13, + "I169", + "residue_name_number" + ], + [ + 18, + 43, + "YfiB-interacting residues", + "site" + ], + [ + 47, + 51, + "YfiR", + "protein" + ], + [ + 62, + 66, + "E163", + "residue_name_number" + ], + [ + 75, + 88, + "hydrogen bond", + "bond_interaction" + ], + [ + 94, + 97, + "R96", + "residue_name_number" + ], + [ + 101, + 105, + "YfiB", + "protein" + ], + [ + 123, + 127, + "I169", + "residue_name_number" + ], + [ + 155, + 159, + "L166", + "residue_name_number" + ], + [ + 160, + 164, + "I169", + "residue_name_number" + ], + [ + 165, + 169, + "V176", + "residue_name_number" + ], + [ + 170, + 174, + "P178", + "residue_name_number" + ], + [ + 175, + 179, + "L181", + "residue_name_number" + ], + [ + 180, + 196, + "hydrophobic core", + "site" + ], + [ + 211, + 214, + "F57", + "residue_name_number" + ], + [ + 218, + 222, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 105, + "sent": "Collectively, a part of the YfiB-YfiR interface overlaps with the proposed YfiR-YfiN interface, suggesting alteration in the association-disassociation equilibrium of YfiR-YfiN and hence the ability of YfiB to sequester YfiR.", + "section": "RESULTS", + "ner": [ + [ + 28, + 47, + "YfiB-YfiR interface", + "site" + ], + [ + 75, + 94, + "YfiR-YfiN interface", + "site" + ], + [ + 167, + 171, + "YfiR", + "protein" + ], + [ + 172, + 176, + "YfiN", + "protein" + ], + [ + 202, + 206, + "YfiB", + "protein" + ], + [ + 220, + 224, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 106, + "sent": "YfiR binds small molecules", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 107, + "sent": "Previous studies indicated that YfiR constitutes a YfiB-independent sensing device that can activate YfiN in response to the redox status of the periplasm, and we have reported YfiR structures in both the non-oxidized and the oxidized states earlier, revealing that the Cys145-Cys152 disulfide bond plays an essential role in maintaining the correct folding of YfiR (Yang et al.,).", + "section": "RESULTS", + "ner": [ + [ + 32, + 36, + "YfiR", + "protein" + ], + [ + 51, + 55, + "YfiB", + "protein" + ], + [ + 101, + 105, + "YfiN", + "protein" + ], + [ + 177, + 181, + "YfiR", + "protein" + ], + [ + 182, + 192, + "structures", + "evidence" + ], + [ + 205, + 217, + "non-oxidized", + "protein_state" + ], + [ + 226, + 234, + "oxidized", + "protein_state" + ], + [ + 270, + 276, + "Cys145", + "residue_name_number" + ], + [ + 277, + 283, + "Cys152", + "residue_name_number" + ], + [ + 284, + 298, + "disulfide bond", + "ptm" + ], + [ + 361, + 365, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 108, + "sent": "However, whether YfiR is involved in other regulatory mechanisms is still an open question.", + "section": "RESULTS", + "ner": [ + [ + 17, + 21, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 109, + "sent": "Overall Structures of VB6-bound and Trp-bound YfiR. (A) Superposition of the overall structures of VB6-bound and Trp-bound YfiR. (B) Close-up views showing the key residues of YfiR that bind VB6 and L-Trp.", + "section": "FIG", + "ner": [ + [ + 8, + 18, + "Structures", + "evidence" + ], + [ + 22, + 31, + "VB6-bound", + "protein_state" + ], + [ + 36, + 45, + "Trp-bound", + "protein_state" + ], + [ + 46, + 50, + "YfiR", + "protein" + ], + [ + 56, + 69, + "Superposition", + "experimental_method" + ], + [ + 85, + 95, + "structures", + "evidence" + ], + [ + 99, + 108, + "VB6-bound", + "protein_state" + ], + [ + 113, + 122, + "Trp-bound", + "protein_state" + ], + [ + 123, + 127, + "YfiR", + "protein" + ], + [ + 176, + 180, + "YfiR", + "protein" + ], + [ + 191, + 194, + "VB6", + "chemical" + ], + [ + 199, + 204, + "L-Trp", + "chemical" + ] + ] + }, + { + "sid": 110, + "sent": "The electron densities of VB6 and Trp are countered at 3.0\u03c3 and 2.3\u03c3, respectively, in |Fo|-|Fc| maps. (C) Superposition of the hydrophobic pocket of YfiR with VB6, L-Trp and F57 of YfiB", + "section": "FIG", + "ner": [ + [ + 4, + 22, + "electron densities", + "evidence" + ], + [ + 26, + 29, + "VB6", + "chemical" + ], + [ + 34, + 37, + "Trp", + "chemical" + ], + [ + 87, + 101, + "|Fo|-|Fc| maps", + "evidence" + ], + [ + 107, + 120, + "Superposition", + "experimental_method" + ], + [ + 128, + 146, + "hydrophobic pocket", + "site" + ], + [ + 150, + 154, + "YfiR", + "protein" + ], + [ + 160, + 163, + "VB6", + "chemical" + ], + [ + 165, + 170, + "L-Trp", + "chemical" + ], + [ + 175, + 178, + "F57", + "residue_name_number" + ], + [ + 182, + 186, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 111, + "sent": "Intriguingly, a Dali search (Holm and Rosenstrom,) indicated that the closest homologs of YfiR shared the characteristic of being able to bind several structurally similar small molecules, such as L-Trp, L-Phe, B-group vitamins and their analogs, encouraging us to test whether YfiR can recognize these molecules.", + "section": "RESULTS", + "ner": [ + [ + 16, + 27, + "Dali search", + "experimental_method" + ], + [ + 90, + 94, + "YfiR", + "protein" + ], + [ + 197, + 202, + "L-Trp", + "chemical" + ], + [ + 204, + 209, + "L-Phe", + "chemical" + ], + [ + 278, + 282, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 112, + "sent": "For this purpose, we co-crystallized YfiR or soaked YfiR crystals with different small molecules, including L-Trp and B-group vitamins.", + "section": "RESULTS", + "ner": [ + [ + 21, + 36, + "co-crystallized", + "experimental_method" + ], + [ + 37, + 41, + "YfiR", + "protein" + ], + [ + 45, + 51, + "soaked", + "experimental_method" + ], + [ + 52, + 56, + "YfiR", + "protein" + ], + [ + 57, + 65, + "crystals", + "evidence" + ], + [ + 108, + 113, + "L-Trp", + "chemical" + ] + ] + }, + { + "sid": 113, + "sent": "Fortunately, we found obvious small-molecule density in the VB6-bound and Trp-bound YfiR crystal structures (Fig.\u00a05A and 5B), and in both structures, the YfiR dimers resemble the oxidized YfiR structure in which both two disulfide bonds are well formed (Yang et al.,).", + "section": "RESULTS", + "ner": [ + [ + 30, + 52, + "small-molecule density", + "evidence" + ], + [ + 60, + 69, + "VB6-bound", + "protein_state" + ], + [ + 74, + 83, + "Trp-bound", + "protein_state" + ], + [ + 84, + 88, + "YfiR", + "protein" + ], + [ + 89, + 107, + "crystal structures", + "evidence" + ], + [ + 138, + 148, + "structures", + "evidence" + ], + [ + 154, + 158, + "YfiR", + "protein" + ], + [ + 159, + 165, + "dimers", + "oligomeric_state" + ], + [ + 179, + 187, + "oxidized", + "protein_state" + ], + [ + 188, + 192, + "YfiR", + "protein" + ], + [ + 193, + 202, + "structure", + "evidence" + ], + [ + 221, + 236, + "disulfide bonds", + "ptm" + ] + ] + }, + { + "sid": 114, + "sent": "Functional analysis of VB6 and L-Trp. (A and B) The effect of increasing concentrations of VB6 or L-Trp on YfiBL43P-induced attachment (bars).", + "section": "FIG", + "ner": [ + [ + 23, + 26, + "VB6", + "chemical" + ], + [ + 31, + 36, + "L-Trp", + "chemical" + ], + [ + 52, + 87, + "effect of increasing concentrations", + "experimental_method" + ], + [ + 91, + 94, + "VB6", + "chemical" + ], + [ + 98, + 103, + "L-Trp", + "chemical" + ], + [ + 107, + 115, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 115, + "sent": "The relative optical density is represented as curves.", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "relative optical density", + "evidence" + ] + ] + }, + { + "sid": 116, + "sent": "Wild-type YfiB is used as negative control.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Wild-type", + "protein_state" + ], + [ + 10, + 14, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 117, + "sent": "(C and D) BIAcore data and analysis for binding affinities of (C) VB6 and (D) L-Trp with YfiR. (E\u2013G) ITC data and analysis for titration of (E) YfiB wild-type, (F) YfiBL43P, and (G) YfiBL43P/F57A into YfiR", + "section": "FIG", + "ner": [ + [ + 10, + 17, + "BIAcore", + "experimental_method" + ], + [ + 40, + 58, + "binding affinities", + "evidence" + ], + [ + 66, + 69, + "VB6", + "chemical" + ], + [ + 78, + 83, + "L-Trp", + "chemical" + ], + [ + 89, + 93, + "YfiR", + "protein" + ], + [ + 101, + 104, + "ITC", + "experimental_method" + ], + [ + 127, + 136, + "titration", + "experimental_method" + ], + [ + 144, + 148, + "YfiB", + "protein" + ], + [ + 149, + 158, + "wild-type", + "protein_state" + ], + [ + 182, + 190, + "YfiBL43P", + "mutant" + ], + [ + 191, + 195, + "F57A", + "mutant" + ], + [ + 201, + 205, + "YfiR", + "protein" + ] + ] + }, + { + "sid": 118, + "sent": "Structural analyses revealed that the VB6 and L-Trp molecules are bound at the periphery of the YfiR dimer, but not at the dimer interface.", + "section": "RESULTS", + "ner": [ + [ + 0, + 19, + "Structural analyses", + "experimental_method" + ], + [ + 38, + 41, + "VB6", + "chemical" + ], + [ + 46, + 51, + "L-Trp", + "chemical" + ], + [ + 66, + 74, + "bound at", + "protein_state" + ], + [ + 96, + 100, + "YfiR", + "protein" + ], + [ + 101, + 106, + "dimer", + "oligomeric_state" + ], + [ + 123, + 138, + "dimer interface", + "site" + ] + ] + }, + { + "sid": 119, + "sent": "Interestingly, VB6 and L-Trp were found to occupy the same hydrophobic pocket, formed by L166/I169/V176/P178/L181 of YfiR, which is also a binding pocket for F57 of YfiB, as observed in the YfiB-YfiR complex (Fig.\u00a05C).", + "section": "RESULTS", + "ner": [ + [ + 15, + 18, + "VB6", + "chemical" + ], + [ + 23, + 28, + "L-Trp", + "chemical" + ], + [ + 59, + 77, + "hydrophobic pocket", + "site" + ], + [ + 89, + 93, + "L166", + "residue_name_number" + ], + [ + 94, + 98, + "I169", + "residue_name_number" + ], + [ + 99, + 103, + "V176", + "residue_name_number" + ], + [ + 104, + 108, + "P178", + "residue_name_number" + ], + [ + 109, + 113, + "L181", + "residue_name_number" + ], + [ + 117, + 121, + "YfiR", + "protein" + ], + [ + 139, + 153, + "binding pocket", + "site" + ], + [ + 158, + 161, + "F57", + "residue_name_number" + ], + [ + 165, + 169, + "YfiB", + "protein" + ], + [ + 190, + 199, + "YfiB-YfiR", + "complex_assembly" + ] + ] + }, + { + "sid": 120, + "sent": "To evaluate the importance of F57 in YfiBL43P-YfiR interaction, the binding affinities of YfiBL43P and YfiBL43P/F57A for YfiR were measured by isothermal titration calorimetry (ITC).", + "section": "RESULTS", + "ner": [ + [ + 30, + 33, + "F57", + "residue_name_number" + ], + [ + 37, + 50, + "YfiBL43P-YfiR", + "complex_assembly" + ], + [ + 68, + 86, + "binding affinities", + "evidence" + ], + [ + 90, + 98, + "YfiBL43P", + "mutant" + ], + [ + 103, + 111, + "YfiBL43P", + "mutant" + ], + [ + 112, + 116, + "F57A", + "mutant" + ], + [ + 121, + 125, + "YfiR", + "protein" + ], + [ + 143, + 175, + "isothermal titration calorimetry", + "experimental_method" + ], + [ + 177, + 180, + "ITC", + "experimental_method" + ] + ] + }, + { + "sid": 121, + "sent": "The results showed Kd values of 1.4 \u00d7 10\u22127 mol/L and 5.3 \u00d7 10\u22127 mol/L for YfiBL43P and YfiBL43P/F57A, respectively, revealing that the YfiBL43P/F57A mutant caused a 3.8-fold reduction in the binding affinity compared with the YfiBL43P mutant (Fig.\u00a06F and 6G).", + "section": "RESULTS", + "ner": [ + [ + 19, + 21, + "Kd", + "evidence" + ], + [ + 74, + 82, + "YfiBL43P", + "mutant" + ], + [ + 87, + 95, + "YfiBL43P", + "mutant" + ], + [ + 96, + 100, + "F57A", + "mutant" + ], + [ + 135, + 143, + "YfiBL43P", + "mutant" + ], + [ + 144, + 148, + "F57A", + "mutant" + ], + [ + 149, + 155, + "mutant", + "protein_state" + ], + [ + 191, + 207, + "binding affinity", + "evidence" + ], + [ + 226, + 234, + "YfiBL43P", + "mutant" + ], + [ + 235, + 241, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 122, + "sent": "In parallel, to better understand the putative functional role of VB6 and L-Trp, yfiB was deleted in a PAO1 wild-type strain, and a construct expressing the YfiBL43P mutant was transformed into the PAO1 \u0394yfiB strain to trigger YfiBL43P-induced biofilm formation.", + "section": "RESULTS", + "ner": [ + [ + 66, + 69, + "VB6", + "chemical" + ], + [ + 74, + 79, + "L-Trp", + "chemical" + ], + [ + 81, + 85, + "yfiB", + "gene" + ], + [ + 90, + 97, + "deleted", + "experimental_method" + ], + [ + 103, + 107, + "PAO1", + "species" + ], + [ + 108, + 117, + "wild-type", + "protein_state" + ], + [ + 132, + 152, + "construct expressing", + "experimental_method" + ], + [ + 157, + 165, + "YfiBL43P", + "mutant" + ], + [ + 166, + 172, + "mutant", + "protein_state" + ], + [ + 177, + 193, + "transformed into", + "experimental_method" + ], + [ + 198, + 202, + "PAO1", + "species" + ], + [ + 203, + 208, + "\u0394yfiB", + "mutant" + ], + [ + 227, + 235, + "YfiBL43P", + "mutant" + ] + ] + }, + { + "sid": 123, + "sent": "Growth and surface attachment assays were carried out for the yfiB-L43P strain in the presence of increasing concentrations of VB6 or L-Trp.", + "section": "RESULTS", + "ner": [ + [ + 0, + 36, + "Growth and surface attachment assays", + "experimental_method" + ], + [ + 62, + 71, + "yfiB-L43P", + "mutant" + ], + [ + 98, + 123, + "increasing concentrations", + "experimental_method" + ], + [ + 127, + 130, + "VB6", + "chemical" + ], + [ + 134, + 139, + "L-Trp", + "chemical" + ] + ] + }, + { + "sid": 124, + "sent": "As shown in Fig.\u00a06A and 6B, the over-expression of YfiBL43P induced strong surface attachment and much slower growth of the yfiB-L43P strain, and as expected, a certain amount of VB6 or L-Trp (4\u20136 mmol/L for VB6 and 6\u201310 mmol/L for L-Trp) could reduce the surface attachment.", + "section": "RESULTS", + "ner": [ + [ + 32, + 47, + "over-expression", + "experimental_method" + ], + [ + 51, + 59, + "YfiBL43P", + "mutant" + ], + [ + 124, + 133, + "yfiB-L43P", + "mutant" + ], + [ + 179, + 182, + "VB6", + "chemical" + ], + [ + 186, + 191, + "L-Trp", + "chemical" + ], + [ + 208, + 211, + "VB6", + "chemical" + ], + [ + 232, + 237, + "L-Trp", + "chemical" + ] + ] + }, + { + "sid": 125, + "sent": "Interestingly, at a concentration higher than 8 mmol/L, VB6 lost its ability to inhibit biofilm formation, implying that the VB6-involving regulatory mechanism is highly complicated and remains to be further investigated.", + "section": "RESULTS", + "ner": [ + [ + 56, + 59, + "VB6", + "chemical" + ], + [ + 125, + 128, + "VB6", + "chemical" + ] + ] + }, + { + "sid": 126, + "sent": "Of note, both VB6 and L-Trp have been reported to correlate with biofilm formation in certain Gram-negative bacteria (Grubman et al.,; Shimazaki et al.,).", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "VB6", + "chemical" + ], + [ + 22, + 27, + "L-Trp", + "chemical" + ], + [ + 94, + 116, + "Gram-negative bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 127, + "sent": "In Helicobacter pylori in particular, VB6 biosynthetic enzymes act as novel virulence factors, and VB6 is required for full motility and virulence (Grubman et al.,).", + "section": "RESULTS", + "ner": [ + [ + 3, + 22, + "Helicobacter pylori", + "species" + ], + [ + 38, + 41, + "VB6", + "chemical" + ], + [ + 99, + 102, + "VB6", + "chemical" + ] + ] + }, + { + "sid": 128, + "sent": "\u00a0In E.\u00a0coli,\u00a0mutants with decreased tryptophan synthesis show greater biofilm formation, and matured biofilm is degraded by L-tryptophan addition (Shimazaki et al.,).", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "E.\u00a0coli", + "species" + ], + [ + 36, + 46, + "tryptophan", + "chemical" + ], + [ + 124, + 136, + "L-tryptophan", + "chemical" + ] + ] + }, + { + "sid": 129, + "sent": "To answer the question whether competition of VB6 or L-Trp for the YfiB F57-binding pocket of YfiR plays an essential role in inhibiting biofilm formation, we measured the binding affinities of VB6 and L-Trp for YfiR via BIAcore experiments.", + "section": "RESULTS", + "ner": [ + [ + 46, + 49, + "VB6", + "chemical" + ], + [ + 53, + 58, + "L-Trp", + "chemical" + ], + [ + 67, + 71, + "YfiB", + "protein" + ], + [ + 72, + 90, + "F57-binding pocket", + "site" + ], + [ + 94, + 98, + "YfiR", + "protein" + ], + [ + 172, + 190, + "binding affinities", + "evidence" + ], + [ + 194, + 197, + "VB6", + "chemical" + ], + [ + 202, + 207, + "L-Trp", + "chemical" + ], + [ + 212, + 216, + "YfiR", + "protein" + ], + [ + 221, + 228, + "BIAcore", + "experimental_method" + ] + ] + }, + { + "sid": 130, + "sent": "The results showed relatively weak Kd values of 35.2 mmol/L and 76.9 mmol/L for VB6 and L-Trp, respectively (Fig.\u00a06C and 6D).", + "section": "RESULTS", + "ner": [ + [ + 35, + 37, + "Kd", + "evidence" + ], + [ + 80, + 83, + "VB6", + "chemical" + ], + [ + 88, + 93, + "L-Trp", + "chemical" + ] + ] + }, + { + "sid": 131, + "sent": "Based on our results, we concluded that VB6 or L-Trp can bind to YfiR, however, VB6 or L-Trp alone may have little effects in interrupting the YfiB-YfiR interaction, the mechanism by which VB6 or L-Trp inhibits biofilm formation remains unclear and requires further investigation.", + "section": "RESULTS", + "ner": [ + [ + 40, + 43, + "VB6", + "chemical" + ], + [ + 47, + 52, + "L-Trp", + "chemical" + ], + [ + 65, + 69, + "YfiR", + "protein" + ], + [ + 80, + 83, + "VB6", + "chemical" + ], + [ + 87, + 92, + "L-Trp", + "chemical" + ], + [ + 93, + 98, + "alone", + "protein_state" + ], + [ + 143, + 152, + "YfiB-YfiR", + "complex_assembly" + ], + [ + 189, + 192, + "VB6", + "chemical" + ], + [ + 196, + 201, + "L-Trp", + "chemical" + ] + ] + }, + { + "sid": 132, + "sent": "Previous studies suggested that in response to cell stress, YfiB in the outer membrane sequesters the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus inducing the diguanylate cyclase activity of YfiN to allow c-di-GMP production (Giardina et al.,; Malone et al.,; Malone et al.,).", + "section": "DISCUSS", + "ner": [ + [ + 60, + 64, + "YfiB", + "protein" + ], + [ + 122, + 126, + "YfiR", + "protein" + ], + [ + 156, + 160, + "YfiN", + "protein" + ], + [ + 237, + 241, + "YfiN", + "protein" + ], + [ + 251, + 259, + "c-di-GMP", + "chemical" + ] + ] + }, + { + "sid": 133, + "sent": "Here, we report the crystal structures of YfiB alone and an active mutant YfiBL43P in complex with YfiR, indicating that YfiR forms a 2:2 complex with YfiB via a region composed of conserved residues.", + "section": "DISCUSS", + "ner": [ + [ + 20, + 38, + "crystal structures", + "evidence" + ], + [ + 42, + 46, + "YfiB", + "protein" + ], + [ + 47, + 52, + "alone", + "protein_state" + ], + [ + 60, + 66, + "active", + "protein_state" + ], + [ + 67, + 73, + "mutant", + "protein_state" + ], + [ + 74, + 82, + "YfiBL43P", + "mutant" + ], + [ + 83, + 98, + "in complex with", + "protein_state" + ], + [ + 99, + 103, + "YfiR", + "protein" + ], + [ + 121, + 125, + "YfiR", + "protein" + ], + [ + 138, + 150, + "complex with", + "protein_state" + ], + [ + 151, + 155, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 134, + "sent": "Our structural data analysis shows that the activated YfiB has an N-terminal portion that is largely altered, adopting a stretched conformation compared with the compact conformation of the apo YfiB. The apo YfiB structure constructed beginning at residue 34 has a compact conformation of approximately 45 \u00c5 in length.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 28, + "structural data analysis", + "experimental_method" + ], + [ + 44, + 53, + "activated", + "protein_state" + ], + [ + 54, + 58, + "YfiB", + "protein" + ], + [ + 66, + 84, + "N-terminal portion", + "structure_element" + ], + [ + 121, + 143, + "stretched conformation", + "protein_state" + ], + [ + 162, + 182, + "compact conformation", + "protein_state" + ], + [ + 190, + 193, + "apo", + "protein_state" + ], + [ + 194, + 198, + "YfiB", + "protein" + ], + [ + 204, + 207, + "apo", + "protein_state" + ], + [ + 208, + 212, + "YfiB", + "protein" + ], + [ + 213, + 222, + "structure", + "evidence" + ], + [ + 256, + 258, + "34", + "residue_number" + ], + [ + 265, + 285, + "compact conformation", + "protein_state" + ] + ] + }, + { + "sid": 135, + "sent": "In addition to the preceding 8 aa loop (from the lipid acceptor Cys26 to Gly34), the full length of the periplasmic portion of apo YfiB can reach approximately 60 \u00c5. It was reported that the distance between the outer membrane and the cell wall is approximately 50 \u00c5 and that the thickness of the PG layer is approximately 70 \u00c5 (Matias et al.,).", + "section": "DISCUSS", + "ner": [ + [ + 19, + 33, + "preceding 8 aa", + "residue_range" + ], + [ + 34, + 38, + "loop", + "structure_element" + ], + [ + 64, + 78, + "Cys26 to Gly34", + "residue_range" + ], + [ + 85, + 96, + "full length", + "protein_state" + ], + [ + 127, + 130, + "apo", + "protein_state" + ], + [ + 131, + 135, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 136, + "sent": "Thus, YfiB alone represents an inactive form that may only partially insert into the PG matrix.", + "section": "DISCUSS", + "ner": [ + [ + 6, + 10, + "YfiB", + "protein" + ], + [ + 11, + 16, + "alone", + "protein_state" + ], + [ + 31, + 39, + "inactive", + "protein_state" + ] + ] + }, + { + "sid": 137, + "sent": "By contrast, YfiR-bound YfiBL43P (residues 44\u2013168) has a stretched conformation of approximately 55 \u00c5 in length.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 23, + "YfiR-bound", + "protein_state" + ], + [ + 24, + 32, + "YfiBL43P", + "mutant" + ], + [ + 43, + 49, + "44\u2013168", + "residue_range" + ], + [ + 57, + 79, + "stretched conformation", + "protein_state" + ] + ] + }, + { + "sid": 138, + "sent": "In addition to the 17 preceding intracellular residues (from the lipid acceptor Cys26 to Leu43), the length of the intracellular portion of active YfiB may extend over 100 \u00c5, assuming a fully stretched conformation.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 54, + "17 preceding intracellular residues", + "residue_range" + ], + [ + 80, + 94, + "Cys26 to Leu43", + "residue_range" + ], + [ + 140, + 146, + "active", + "protein_state" + ], + [ + 147, + 151, + "YfiB", + "protein" + ], + [ + 186, + 214, + "fully stretched conformation", + "protein_state" + ] + ] + }, + { + "sid": 139, + "sent": "Provided that the diameter of the widest part of the YfiB dimer is approximately 64 \u00c5, which is slightly smaller than the smallest diameter of the PG pore (70 \u00c5) (Meroueh et al.,), the YfiB dimer should be able to penetrate the PG layer.", + "section": "DISCUSS", + "ner": [ + [ + 53, + 57, + "YfiB", + "protein" + ], + [ + 58, + 63, + "dimer", + "oligomeric_state" + ], + [ + 185, + 189, + "YfiB", + "protein" + ], + [ + 190, + 195, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 140, + "sent": "Regulatory model of the YfiBNR tripartite system.", + "section": "FIG", + "ner": [ + [ + 24, + 30, + "YfiBNR", + "complex_assembly" + ], + [ + 31, + 41, + "tripartite", + "protein_state" + ] + ] + }, + { + "sid": 141, + "sent": "The periplasmic domain of YfiB and the YfiB-YfiR complex are depicted according to the crystal structures.", + "section": "FIG", + "ner": [ + [ + 4, + 22, + "periplasmic domain", + "structure_element" + ], + [ + 26, + 30, + "YfiB", + "protein" + ], + [ + 39, + 48, + "YfiB-YfiR", + "complex_assembly" + ], + [ + 87, + 105, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 142, + "sent": "The lipid acceptor Cys26 is indicated as blue ball.", + "section": "FIG", + "ner": [ + [ + 19, + 24, + "Cys26", + "residue_name_number" + ] + ] + }, + { + "sid": 143, + "sent": "The loop connecting Cys26 and Gly34 of YfiB is modeled.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "loop", + "structure_element" + ], + [ + 20, + 25, + "Cys26", + "residue_name_number" + ], + [ + 30, + 35, + "Gly34", + "residue_name_number" + ], + [ + 39, + 43, + "YfiB", + "protein" + ] + ] + }, + { + "sid": 144, + "sent": "The PAS domain of YfiN is shown as pink oval.", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "PAS domain", + "structure_element" + ], + [ + 18, + 22, + "YfiN", + "protein" + ] + ] + }, + { + "sid": 145, + "sent": "Once activated by certain cell stress, the dimeric YfiB transforms from a compact conformation to a stretched conformation, allowing the periplasmic domain of the membrane-anchored YfiB to penetrate the cell wall and sequester the YfiR dimer, thus relieving the repression of YfiN", + "section": "FIG", + "ner": [ + [ + 5, + 14, + "activated", + "protein_state" + ], + [ + 43, + 50, + "dimeric", + "oligomeric_state" + ], + [ + 51, + 55, + "YfiB", + "protein" + ], + [ + 74, + 94, + "compact conformation", + "protein_state" + ], + [ + 100, + 122, + "stretched conformation", + "protein_state" + ], + [ + 137, + 155, + "periplasmic domain", + "structure_element" + ], + [ + 163, + 180, + "membrane-anchored", + "protein_state" + ], + [ + 181, + 185, + "YfiB", + "protein" + ], + [ + 231, + 235, + "YfiR", + "protein" + ], + [ + 236, + 241, + "dimer", + "oligomeric_state" + ], + [ + 276, + 280, + "YfiN", + "protein" + ] + ] + }, + { + "sid": 146, + "sent": "These results, together with our observation that activated YfiB has a much higher cell wall binding affinity, and previous mutagenesis data showing that (1) both PG binding and membrane anchoring are required for YfiB activity and (2) activating mutations possessing an altered N-terminal loop length are dominant over the loss of PG binding (Malone et al.,), suggest an updated regulatory model of the YfiBNR system (Fig.\u00a07).", + "section": "DISCUSS", + "ner": [ + [ + 50, + 59, + "activated", + "protein_state" + ], + [ + 60, + 64, + "YfiB", + "protein" + ], + [ + 83, + 109, + "cell wall binding affinity", + "evidence" + ], + [ + 163, + 165, + "PG", + "chemical" + ], + [ + 214, + 218, + "YfiB", + "protein" + ], + [ + 290, + 294, + "loop", + "structure_element" + ], + [ + 332, + 334, + "PG", + "chemical" + ], + [ + 404, + 410, + "YfiBNR", + "complex_assembly" + ] + ] + }, + { + "sid": 147, + "sent": "In this model, in response to a particular cell stress that is yet to be identified, the dimeric YfiB is activated from a compact, inactive conformation to a stretched conformation, which possesses increased PG binding affinity.", + "section": "DISCUSS", + "ner": [ + [ + 89, + 96, + "dimeric", + "oligomeric_state" + ], + [ + 97, + 101, + "YfiB", + "protein" + ], + [ + 105, + 114, + "activated", + "protein_state" + ], + [ + 122, + 129, + "compact", + "protein_state" + ], + [ + 131, + 139, + "inactive", + "protein_state" + ], + [ + 140, + 152, + "conformation", + "protein_state" + ], + [ + 158, + 180, + "stretched conformation", + "protein_state" + ], + [ + 208, + 210, + "PG", + "chemical" + ] + ] + }, + { + "sid": 148, + "sent": "This allows the C-terminal portion of the membrane-anchored YfiB to reach, bind and penetrate the cell wall and sequester the YfiR dimer.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 34, + "C-terminal portion", + "structure_element" + ], + [ + 42, + 59, + "membrane-anchored", + "protein_state" + ], + [ + 60, + 64, + "YfiB", + "protein" + ], + [ + 126, + 130, + "YfiR", + "protein" + ], + [ + 131, + 136, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 149, + "sent": "The YfiBNR system provides a good example of a delicate homeostatic system that integrates multiple signals to regulate the c-di-GMP level.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 10, + "YfiBNR", + "complex_assembly" + ], + [ + 124, + 132, + "c-di-GMP", + "chemical" + ] + ] + }, + { + "sid": 150, + "sent": "Homologs of the YfiBNR system are functionally conserved in P. aeruginosa (Malone et al.,; Malone et al.,), E. coli (Hufnagel et al.,; Raterman et al.,; Sanchez-Torres et al.,), K. pneumonia (Huertas et al.,) and Y. pestis (Ren et al.,), where they affect c-di-GMP production and biofilm formation.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 22, + "YfiBNR", + "complex_assembly" + ], + [ + 34, + 56, + "functionally conserved", + "protein_state" + ], + [ + 60, + 73, + "P. aeruginosa", + "species" + ], + [ + 108, + 115, + "E. coli", + "species" + ], + [ + 178, + 190, + "K. pneumonia", + "species" + ], + [ + 213, + 222, + "Y. pestis", + "species" + ], + [ + 256, + 264, + "c-di-GMP", + "chemical" + ] + ] + }, + { + "sid": 151, + "sent": "The mechanism by which activated YfiB relieves the repression of YfiN may be applicable to the YfiBNR system in other bacteria and to analogous outside-in signaling for c-di-GMP production, which in turn may be relevant to the development of drugs that can circumvent complicated antibiotic resistance.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 32, + "activated", + "protein_state" + ], + [ + 33, + 37, + "YfiB", + "protein" + ], + [ + 65, + 69, + "YfiN", + "protein" + ], + [ + 95, + 101, + "YfiBNR", + "complex_assembly" + ], + [ + 118, + 126, + "bacteria", + "taxonomy_domain" + ], + [ + 169, + 177, + "c-di-GMP", + "chemical" + ] + ] + } + ] + }, + "PMC4822050": { + "annotations": [ + { + "sid": 0, + "sent": "Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition", + "section": "TITLE", + "ner": [ + [ + 0, + 19, + "Hemi-methylated DNA", + "chemical" + ], + [ + 28, + 34, + "closed", + "protein_state" + ], + [ + 51, + 56, + "UHRF1", + "protein" + ], + [ + 75, + 82, + "histone", + "protein_type" + ] + ] + }, + { + "sid": 1, + "sent": "UHRF1 is an important epigenetic regulator for maintenance DNA methylation.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 5, + "UHRF1", + "protein" + ], + [ + 63, + 74, + "methylation", + "ptm" + ] + ] + }, + { + "sid": 2, + "sent": "UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 5, + "UHRF1", + "protein" + ], + [ + 17, + 36, + "hemi-methylated DNA", + "chemical" + ], + [ + 38, + 44, + "hm-DNA", + "chemical" + ], + [ + 50, + 64, + "trimethylation", + "ptm" + ], + [ + 68, + 75, + "histone", + "protein_type" + ], + [ + 76, + 78, + "H3", + "protein_type" + ], + [ + 78, + 80, + "K9", + "residue_name_number" + ], + [ + 82, + 84, + "H3", + "protein_type" + ], + [ + 84, + 89, + "K9me3", + "ptm" + ] + ] + }, + { + "sid": 3, + "sent": "Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition.", + "section": "ABSTRACT", + "ner": [ + [ + 18, + 23, + "UHRF1", + "protein" + ], + [ + 33, + 39, + "closed", + "protein_state" + ], + [ + 65, + 82, + "C-terminal region", + "structure_element" + ], + [ + 84, + 90, + "Spacer", + "structure_element" + ], + [ + 92, + 100, + "binds to", + "protein_state" + ], + [ + 105, + 124, + "tandem Tudor domain", + "structure_element" + ], + [ + 126, + 129, + "TTD", + "structure_element" + ], + [ + 144, + 146, + "H3", + "protein_type" + ], + [ + 146, + 151, + "K9me3", + "ptm" + ], + [ + 177, + 200, + "SET-and-RING-associated", + "structure_element" + ], + [ + 202, + 205, + "SRA", + "structure_element" + ], + [ + 214, + 222, + "binds to", + "protein_state" + ], + [ + 227, + 244, + "plant homeodomain", + "structure_element" + ], + [ + 246, + 249, + "PHD", + "structure_element" + ], + [ + 264, + 268, + "H3R2", + "site" + ] + ] + }, + { + "sid": 4, + "sent": "Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD\u2013PHD.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 6, + "Hm-DNA", + "chemical" + ], + [ + 60, + 62, + "H3", + "protein_type" + ], + [ + 62, + 67, + "K9me3", + "ptm" + ], + [ + 83, + 90, + "TTD\u2013PHD", + "structure_element" + ] + ] + }, + { + "sid": 5, + "sent": "The Spacer also facilitates UHRF1\u2013DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 10, + "Spacer", + "structure_element" + ], + [ + 28, + 39, + "UHRF1\u2013DNMT1", + "complex_assembly" + ], + [ + 65, + 88, + "hm-DNA-binding affinity", + "evidence" + ], + [ + 96, + 99, + "SRA", + "structure_element" + ] + ] + }, + { + "sid": 6, + "sent": "When TTD\u2013PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin.", + "section": "ABSTRACT", + "ner": [ + [ + 5, + 12, + "TTD\u2013PHD", + "structure_element" + ], + [ + 13, + 21, + "binds to", + "protein_state" + ], + [ + 22, + 24, + "H3", + "protein_type" + ], + [ + 24, + 29, + "K9me3", + "ptm" + ], + [ + 31, + 41, + "SRA-Spacer", + "structure_element" + ], + [ + 96, + 102, + "hm-DNA", + "chemical" + ], + [ + 115, + 120, + "DNMT1", + "protein" + ] + ] + }, + { + "sid": 7, + "sent": "Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1.", + "section": "ABSTRACT", + "ner": [ + [ + 50, + 52, + "H3", + "protein_type" + ], + [ + 52, + 57, + "K9me3", + "ptm" + ], + [ + 62, + 68, + "hm-DNA", + "chemical" + ], + [ + 84, + 89, + "URHF1", + "protein" + ] + ] + }, + { + "sid": 8, + "sent": " UHRF1 is involved in the maintenance of DNA methylation, but the regulatory mechanism of this epigenetic regulator is unclear.", + "section": "ABSTRACT", + "ner": [ 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+ ], + [ + 52, + 74, + "DNA methyltransferases", + "protein_type" + ], + [ + 75, + 84, + "DNMT3A/3B", + "protein" + ], + [ + 94, + 105, + "methylation", + "ptm" + ], + [ + 145, + 168, + "DNA methyltransferase 1", + "protein" + ], + [ + 170, + 175, + "DNMT1", + "protein" + ] + ] + }, + { + "sid": 12, + "sent": "Ubiquitin-like, containing PHD and RING fingers domains, 1 (UHRF1, also known as ICBP90 and NP95 in mouse) was shown to be essential for maintenance DNA methylation through recruiting DNMT1 to replication forks in S phase of the cell cycle.", + "section": "INTRO", + "ner": [ + [ + 0, + 58, + "Ubiquitin-like, containing PHD and RING fingers domains, 1", + "protein" + ], + [ + 60, + 65, + "UHRF1", + "protein" + ], + [ + 81, + 87, + "ICBP90", + "protein" + ], + [ + 92, + 96, + "NP95", + "protein" + ], + [ + 100, + 105, + "mouse", + "taxonomy_domain" + ], + [ + 153, + 164, + "methylation", + "ptm" + ], + [ + 184, + 189, + "DNMT1", + "protein" + ] + ] + }, + { + "sid": 13, + "sent": "UHRF1 is essential for S phase entry and is involved in heterochromatin formation.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 14, + "sent": "UHRF1 also plays an important role in promoting proliferation and is shown to be upregulated in a number of cancers, suggesting that UHRF1 may serve as a potential drug target for therapeutic applications.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "UHRF1", + "protein" + ], + [ + 133, + 138, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 15, + "sent": "UHRF1 is a multi-domain containing protein connecting histone modification and DNA methylation.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "UHRF1", + "protein" + ], + [ + 54, + 61, + "histone", + "protein_type" + ], + [ + 79, + 82, + "DNA", + "chemical" + ], + [ + 83, + 94, + "methylation", + "ptm" + ] + ] + }, + { + "sid": 16, + "sent": "As shown in Fig. 1a, UHRF1 is comprised of an N-terminal ubiquitin-like domain, followed by a tandem Tudor domain (TTD containing TTDN and TTDC sub-domains), a plant homeodomain (PHD), a SET-and-RING-associated (SRA) domain, and a C-terminal really interesting new gene (RING) domain.", + "section": "INTRO", + "ner": [ + [ + 21, + 26, + "UHRF1", + "protein" + ], + [ + 57, + 78, + "ubiquitin-like domain", + "structure_element" + ], + [ + 94, + 113, + "tandem Tudor domain", + "structure_element" + ], + [ + 115, + 118, + "TTD", + "structure_element" + ], + [ + 130, + 134, + "TTDN", + "structure_element" + ], + [ + 139, + 143, + "TTDC", + "structure_element" + ], + [ + 160, + 177, + "plant homeodomain", + "structure_element" + ], + [ + 179, + 182, + "PHD", + "structure_element" + ], + [ + 187, + 210, + "SET-and-RING-associated", + "structure_element" + ], + [ + 212, + 215, + "SRA", + "structure_element" + ], + [ + 242, + 269, + "really interesting new gene", + "structure_element" + ], + [ + 271, + 275, + "RING", + "structure_element" + ] + ] + }, + { + "sid": 17, + "sent": "We and other groups demonstrated that the TTD and the PHD coordinately recognize histone H3K9me3, in which residue R2 is recognized by the PHD and tri-methylation of residue K9 (K9me3) is recognized by the TTD.", + "section": "INTRO", + "ner": [ + [ + 42, + 45, + "TTD", + "structure_element" + ], + [ + 54, + 57, + "PHD", + "structure_element" + ], + [ + 81, + 88, + "histone", + "protein_type" + ], + [ + 89, + 91, + "H3", + "protein_type" + ], + [ + 91, + 96, + "K9me3", + "ptm" + ], + [ + 115, + 117, + "R2", + "residue_name_number" + ], + [ + 139, + 142, + "PHD", + "structure_element" + ], + [ + 147, + 162, + "tri-methylation", + "ptm" + ], + [ + 174, + 176, + "K9", + "residue_name_number" + ], + [ + 178, + 183, + "K9me3", + "ptm" + ], + [ + 206, + 209, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 18, + "sent": "The SRA preferentially binds to hemi-methylated DNA (hm-DNA).", + "section": "INTRO", + "ner": [ + [ + 4, + 7, + "SRA", + "structure_element" + ], + [ + 23, + 31, + "binds to", + "protein_state" + ], + [ + 32, + 51, + "hemi-methylated DNA", + "chemical" + ], + [ + 53, + 59, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 19, + "sent": "Recent studies show that the SRA directly binds to replication focus targeting sequence (RFTS) of DNMT1 (RFTSDNMT1).", + "section": "INTRO", + "ner": [ + [ + 29, + 32, + "SRA", + "structure_element" + ], + [ + 42, + 50, + "binds to", + "protein_state" + ], + [ + 51, + 87, + "replication focus targeting sequence", + "structure_element" + ], + [ + 89, + 93, + "RFTS", + "structure_element" + ], + [ + 98, + 103, + "DNMT1", + "protein" + ], + [ + 105, + 114, + "RFTSDNMT1", + "protein" + ] + ] + }, + { + "sid": 20, + "sent": "A spacer region (Fig. 1a, designated Spacer hereafter) connecting the SRA and the RING is rich in basic residues and predicted to be unstructured for unknown function.", + "section": "INTRO", + "ner": [ + [ + 2, + 15, + "spacer region", + "structure_element" + ], + [ + 37, + 43, + "Spacer", + "structure_element" + ], + [ + 70, + 73, + "SRA", + "structure_element" + ], + [ + 82, + 86, + "RING", + "structure_element" + ], + [ + 133, + 145, + "unstructured", + "protein_state" + ] + ] + }, + { + "sid": 21, + "sent": "Recent study shows that phosphatidylinostiol phosphate PI5P binds to the Spacer and induces a conformational change of UHRF1 to allow the TTD to recognize H3K9me3 (ref.).", + "section": "INTRO", + "ner": [ + [ + 24, + 54, + "phosphatidylinostiol phosphate", + "chemical" + ], + [ + 55, + 59, + "PI5P", + "chemical" + ], + [ + 60, + 68, + "binds to", + "protein_state" + ], + [ + 73, + 79, + "Spacer", + "structure_element" + ], + [ + 119, + 124, + "UHRF1", + "protein" + ], + [ + 138, + 141, + "TTD", + "structure_element" + ], + [ + 155, + 157, + "H3", + "protein_type" + ], + [ + 157, + 162, + "K9me3", + "ptm" + ] + ] + }, + { + "sid": 22, + "sent": "These studies indicate that UHRF1 connects dynamic regulation of DNA methylation and H3K9me3, which are positively correlated in human genome.", + "section": "INTRO", + "ner": [ + [ + 28, + 33, + "UHRF1", + "protein" + ], + [ + 65, + 68, + "DNA", + "chemical" + ], + [ + 69, + 80, + "methylation", + "ptm" + ], + [ + 85, + 87, + "H3", + "protein_type" + ], + [ + 87, + 92, + "K9me3", + "ptm" + ], + [ + 129, + 134, + "human", + "species" + ] + ] + }, + { + "sid": 23, + "sent": "However, how UHRF1 regulates the recognition of these two repressive epigenetic marks and recruits DNMT1 for chromatin localization remain largely unknown.", + "section": "INTRO", + "ner": [ + [ + 13, + 18, + "UHRF1", + "protein" + ], + [ + 99, + 104, + "DNMT1", + "protein" + ] + ] + }, + { + "sid": 24, + "sent": "Here we report that UHRF1 adopts a closed conformation, in which the C-terminal Spacer binds to the TTD and inhibits its recognition of H3K9me3, whereas the SRA binds to the PHD and inhibits its recognition of H3R2 (unmethylated histone H3 at residue R2).", + "section": "INTRO", + "ner": [ + [ + 20, + 25, + "UHRF1", + "protein" + ], + [ + 35, + 41, + "closed", + "protein_state" + ], + [ + 80, + 86, + "Spacer", + "structure_element" + ], + [ + 87, + 95, + "binds to", + "protein_state" + ], + [ + 100, + 103, + "TTD", + "structure_element" + ], + [ + 136, + 138, + "H3", + "protein_type" + ], + [ + 138, + 143, + "K9me3", + "ptm" + ], + [ + 157, + 160, + "SRA", + "structure_element" + ], + [ + 161, + 169, + "binds to", + "protein_state" + ], + [ + 174, + 177, + "PHD", + "structure_element" + ], + [ + 210, + 214, + "H3R2", + "site" + ], + [ + 216, + 228, + "unmethylated", + "protein_state" + ], + [ + 229, + 236, + "histone", + "protein_type" + ], + [ + 237, + 239, + "H3", + "protein_type" + ], + [ + 251, + 253, + "R2", + "residue_name_number" + ] + ] + }, + { + "sid": 25, + "sent": "Upon binding to hm-DNA, UHRF1 impairs the intramolecular interactions and promotes the H3K9me3 recognition by TTD\u2013PHD, which may further enhance its genomic localization.", + "section": "INTRO", + "ner": [ + [ + 5, + 15, + "binding to", + "protein_state" + ], + [ + 16, + 22, + "hm-DNA", + "chemical" + ], + [ + 24, + 29, + "UHRF1", + "protein" + ], + [ + 87, + 89, + "H3", + "protein_type" + ], + [ + 89, + 94, + "K9me3", + "ptm" + ], + [ + 110, + 117, + "TTD\u2013PHD", + "structure_element" + ] + ] + }, + { + "sid": 26, + "sent": "As a result, UHRF1 is locked in the open conformation by the association of H3K9me3 by TTD\u2013PHD, and thus SRA-Spacer either recognizes hm-DNA or recruits DNMT1 for DNA methylation.", + "section": "INTRO", + "ner": [ + [ + 13, + 18, + "UHRF1", + "protein" + ], + [ + 36, + 40, + "open", + "protein_state" + ], + [ + 76, + 78, + "H3", + "protein_type" + ], + [ + 78, + 83, + "K9me3", + "ptm" + ], + [ + 87, + 94, + "TTD\u2013PHD", + "structure_element" + ], + [ + 105, + 115, + "SRA-Spacer", + "structure_element" + ], + [ + 134, + 140, + "hm-DNA", + "chemical" + ], + [ + 153, + 158, + "DNMT1", + "protein" + ], + [ + 163, + 166, + "DNA", + "chemical" + ], + [ + 167, + 178, + "methylation", + "ptm" + ] + ] + }, + { + "sid": 27, + "sent": "Therefore, UHRF1 may engage in a sophisticated regulation for its chromatin localization and recruitment of DNMT1 through a mechanism yet to be fully elucidated.", + "section": "INTRO", + "ner": [ + [ + 11, + 16, + "UHRF1", + "protein" + ], + [ + 108, + 113, + "DNMT1", + "protein" + ] + ] + }, + { + "sid": 28, + "sent": "Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by UHRF1.", + "section": "INTRO", + "ner": [ + [ + 50, + 52, + "H3", + "protein_type" + ], + [ + 52, + 57, + "K9me3", + "ptm" + ], + [ + 62, + 68, + "hm-DNA", + "chemical" + ], + [ + 84, + 89, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 29, + "sent": "Hm-DNA facilitates histone H3K9me3 recognition by UHRF1", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Hm-DNA", + "chemical" + ], + [ + 19, + 26, + "histone", + "protein_type" + ], + [ + 27, + 29, + "H3", + "protein_type" + ], + [ + 29, + 34, + "K9me3", + "ptm" + ], + [ + 50, + 55, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 30, + "sent": "To investigate how UHRF1 coordinates the recognition of H3K9me3 and hm-DNA, we purified recombinant UHRF1 (truncations and mutations) proteins from bacteria.", + "section": "RESULTS", + "ner": [ + [ + 19, + 24, + "UHRF1", + "protein" + ], + [ + 56, + 58, + "H3", + "protein_type" + ], + [ + 58, + 63, + "K9me3", + "ptm" + ], + [ + 68, + 74, + "hm-DNA", + "chemical" + ], + [ + 100, + 105, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 31, + "sent": "We first performed an in vitro pull-down assay using biotinylated histone H3 peptides and hm-DNA (Supplementary Table 1).", + "section": "RESULTS", + "ner": [ + [ + 22, + 46, + "in vitro pull-down assay", + "experimental_method" + ], + [ + 53, + 65, + "biotinylated", + "protein_state" + ], + [ + 66, + 73, + "histone", + "protein_type" + ], + [ + 74, + 76, + "H3", + "protein_type" + ], + [ + 90, + 96, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 32, + "sent": "As shown in Fig. 1b, hm-DNA largely enhanced the interaction between full-length UHRF1 and unmethylated histone H3 (H3K9me0) or H3K9me3 peptide.", + "section": "RESULTS", + "ner": [ + [ + 21, + 27, + "hm-DNA", + "chemical" + ], + [ + 69, + 80, + "full-length", + "protein_state" + ], + [ + 81, + 86, + "UHRF1", + "protein" + ], + [ + 91, + 103, + "unmethylated", + "protein_state" + ], + [ + 104, + 111, + "histone", + "protein_type" + ], + [ + 112, + 114, + "H3", + "protein_type" + ], + [ + 116, + 118, + "H3", + "protein_type" + ], + [ + 118, + 123, + "K9me0", + "ptm" + ], + [ + 128, + 130, + "H3", + "protein_type" + ], + [ + 130, + 135, + "K9me3", + "ptm" + ] + ] + }, + { + "sid": 33, + "sent": "Compared with hm-DNA, um-DNA (unmethylated DNA) or fm-DNA (fully methylated DNA) showed marginal effect on facilitating the interaction between UHRF1 and histone peptides, which is consistent with previous studies that UHRF1 prefers hm-DNA for chromatin association (Supplementary Fig. 1a).", + "section": "RESULTS", + "ner": [ + [ + 14, + 20, + "hm-DNA", + "chemical" + ], + [ + 22, + 28, + "um-DNA", + "chemical" + ], + [ + 30, + 42, + "unmethylated", + "protein_state" + ], + [ + 43, + 46, + "DNA", + "chemical" + ], + [ + 51, + 57, + "fm-DNA", + "chemical" + ], + [ + 59, + 75, + "fully methylated", + "protein_state" + ], + [ + 76, + 79, + "DNA", + "chemical" + ], + [ + 144, + 149, + "UHRF1", + "protein" + ], + [ + 154, + 161, + "histone", + "protein_type" + ], + [ + 219, + 224, + "UHRF1", + "protein" + ], + [ + 233, + 239, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 34, + "sent": "In contrast, histone peptides showed no enhancement on the interaction between hm-DNA and UHRF1 (Fig. 1c).", + "section": "RESULTS", + "ner": [ + [ + 13, + 20, + "histone", + "protein_type" + ], + [ + 79, + 85, + "hm-DNA", + "chemical" + ], + [ + 90, + 95, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 35, + "sent": "These results suggest that hm-DNA facilitates histone recognition by UHRF1.", + "section": "RESULTS", + "ner": [ + [ + 27, + 33, + "hm-DNA", + "chemical" + ], + [ + 46, + 53, + "histone", + "protein_type" + ], + [ + 69, + 74, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 36, + "sent": "Our previous studies show that the PHD recognizes H3K9me0 and the TTD and the PHD together (TTD\u2013PHD) coordinately recognize H3K9me3 (refs.).", + "section": "RESULTS", + "ner": [ + [ + 35, + 38, + "PHD", + "structure_element" + ], + [ + 50, + 52, + "H3", + "protein_type" + ], + [ + 52, + 57, + "K9me0", + "ptm" + ], + [ + 66, + 69, + "TTD", + "structure_element" + ], + [ + 78, + 81, + "PHD", + "structure_element" + ], + [ + 92, + 99, + "TTD\u2013PHD", + "structure_element" + ], + [ + 124, + 126, + "H3", + "protein_type" + ], + [ + 126, + 131, + "K9me3", + "ptm" + ] + ] + }, + { + "sid": 37, + "sent": "We noticed that the isolated TTD\u2013PHD showed much higher (\u223c31-fold) binding affinity to H3K9me3 peptide than full-length UHRF1 (Fig. 1d and Supplementary Table 2), and the isolated PHD showed much higher (\u223c34-fold) binding affinity to H3K9me0 peptide than full-length UHRF1 (Fig. 1e).", + "section": "RESULTS", + "ner": [ + [ + 20, + 28, + "isolated", + "protein_state" + ], + [ + 29, + 36, + "TTD\u2013PHD", + "structure_element" + ], + [ + 67, + 83, + "binding affinity", + "evidence" + ], + [ + 87, + 89, + "H3", + "protein_type" + ], + [ + 89, + 94, + "K9me3", + "ptm" + ], + [ + 108, + 119, + "full-length", + "protein_state" + ], + [ + 120, + 125, + "UHRF1", + "protein" + ], + [ + 180, + 183, + "PHD", + "structure_element" + ], + [ + 214, + 230, + "binding affinity", + "evidence" + ], + [ + 234, + 236, + "H3", + "protein_type" + ], + [ + 236, + 241, + "K9me0", + "ptm" + ], + [ + 255, + 266, + "full-length", + "protein_state" + ], + [ + 267, + 272, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 38, + "sent": "The gel filtration analysis showed that UHRF1 is a monomer in solution (Supplementary Fig. 1b), indicating that the intramolecular (not intermolecular) interaction of UHRF1 regulates histone recognition.", + "section": "RESULTS", + "ner": [ + [ + 4, + 27, + "gel filtration analysis", + "experimental_method" + ], + [ + 40, + 45, + "UHRF1", + "protein" + ], + [ + 51, + 58, + "monomer", + "oligomeric_state" + ], + [ + 167, + 172, + "UHRF1", + "protein" + ], + [ + 183, + 190, + "histone", + "protein_type" + ] + ] + }, + { + "sid": 39, + "sent": "These results suggest that UHRF1 adopts an unfavourable conformation for histone H3 tails recognition, in which TTD\u2013PHD might be blocked by other regions of UHRF1, and hm-DNA impairs this intramolecular interaction to facilitate its recognition of histone H3 tails.", + "section": "RESULTS", + "ner": [ + [ + 27, + 32, + "UHRF1", + "protein" + ], + [ + 73, + 80, + "histone", + "protein_type" + ], + [ + 81, + 83, + "H3", + "protein_type" + ], + [ + 112, + 119, + "TTD\u2013PHD", + "structure_element" + ], + [ + 157, + 162, + "UHRF1", + "protein" + ], + [ + 168, + 174, + "hm-DNA", + "chemical" + ], + [ + 248, + 255, + "histone", + "protein_type" + ], + [ + 256, + 258, + "H3", + "protein_type" + ] + ] + }, + { + "sid": 40, + "sent": "Intramolecular interaction within UHRF1", + "section": "RESULTS", + "ner": [ + [ + 34, + 39, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 41, + "sent": "To test above hypothesis, we performed glutathione S-transferase (GST) pull-down assay using various truncations of UHRF1.", + "section": "RESULTS", + "ner": [ + [ + 39, + 86, + "glutathione S-transferase (GST) pull-down assay", + "experimental_method" + ], + [ + 101, + 112, + "truncations", + "experimental_method" + ], + [ + 116, + 121, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 42, + "sent": "Interestingly, the TTD directly bound to SRA-Spacer but not the SRA, suggesting that the Spacer (residues 587\u2013674) is important for the intramolecular interaction (Fig. 2a).", + "section": "RESULTS", + "ner": [ + [ + 19, + 22, + "TTD", + "structure_element" + ], + [ + 32, + 40, + "bound to", + "protein_state" + ], + [ + 41, + 51, + "SRA-Spacer", + "structure_element" + ], + [ + 64, + 67, + "SRA", + "structure_element" + ], + [ + 89, + 95, + "Spacer", + "structure_element" + ], + [ + 106, + 113, + "587\u2013674", + "residue_range" + ] + ] + }, + { + "sid": 43, + "sent": "The isothermal titration calorimetry (ITC) measurements show that the TTD bound to the Spacer (but not the SRA) in a 1:1 stoichiometry with a binding affinity (KD) of 1.59\u2009\u03bcM (Fig. 2b).", + "section": "RESULTS", + "ner": [ + [ + 4, + 36, + "isothermal titration calorimetry", + "experimental_method" + ], + [ + 38, + 41, + "ITC", + "experimental_method" + ], + [ + 70, + 73, + "TTD", + "structure_element" + ], + [ + 74, + 82, + "bound to", + "protein_state" + ], + [ + 87, + 93, + "Spacer", + "structure_element" + ], + [ + 107, + 110, + "SRA", + "structure_element" + ], + [ + 142, + 158, + "binding affinity", + "evidence" + ], + [ + 160, + 162, + "KD", + "evidence" + ] + ] + }, + { + "sid": 44, + "sent": "The presence of the Spacer markedly impaired the interaction between TTD\u2013PHD and H3K9me3 (Fig. 2c).", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "presence of", + "protein_state" + ], + [ + 20, + 26, + "Spacer", + "structure_element" + ], + [ + 69, + 76, + "TTD\u2013PHD", + "structure_element" + ], + [ + 81, + 83, + "H3", + "protein_type" + ], + [ + 83, + 88, + "K9me3", + "ptm" + ] + ] + }, + { + "sid": 45, + "sent": "The results indicate that the Spacer directly binds to the TTD and inhibits its interaction with H3K9me3.", + "section": "RESULTS", + "ner": [ + [ + 30, + 36, + "Spacer", + "structure_element" + ], + [ + 46, + 54, + "binds to", + "protein_state" + ], + [ + 59, + 62, + "TTD", + "structure_element" + ], + [ + 97, + 99, + "H3", + "protein_type" + ], + [ + 99, + 104, + "K9me3", + "ptm" + ] + ] + }, + { + "sid": 46, + "sent": "The GST pull-down assay also shows that the PHD bound to the SRA, which was further confirmed by the ITC measurements (KD=26.7\u2009\u03bcM; Fig. 2a,d).", + "section": "RESULTS", + "ner": [ + [ + 4, + 23, + "GST pull-down assay", + "experimental_method" + ], + [ + 44, + 47, + "PHD", + "structure_element" + ], + [ + 48, + 56, + "bound to", + "protein_state" + ], + [ + 61, + 64, + "SRA", + "structure_element" + ], + [ + 101, + 104, + "ITC", + "experimental_method" + ], + [ + 119, + 121, + "KD", + "evidence" + ] + ] + }, + { + "sid": 47, + "sent": "Compared with the PHD alone, PHD-SRA showed decreased binding affinity to H3K9me0 peptide by a factor of eight (Fig. 2e).", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "PHD", + "structure_element" + ], + [ + 22, + 27, + "alone", + "protein_state" + ], + [ + 29, + 36, + "PHD-SRA", + "structure_element" + ], + [ + 54, + 70, + "binding affinity", + "evidence" + ], + [ + 74, + 76, + "H3", + "protein_type" + ], + [ + 76, + 81, + "K9me0", + "ptm" + ] + ] + }, + { + "sid": 48, + "sent": "Pre-incubation of the SRA also modestly impaired PHD\u2013H3K9me0 interaction.", + "section": "RESULTS", + "ner": [ + [ + 0, + 14, + "Pre-incubation", + "experimental_method" + ], + [ + 22, + 25, + "SRA", + "structure_element" + ], + [ + 49, + 52, + "PHD", + "structure_element" + ], + [ + 53, + 55, + "H3", + "protein_type" + ], + [ + 55, + 60, + "K9me0", + "ptm" + ] + ] + }, + { + "sid": 49, + "sent": "These results indicate that the SRA directly binds to the PHD and inhibits its binding affinity to H3K9me0.", + "section": "RESULTS", + "ner": [ + [ + 32, + 35, + "SRA", + "structure_element" + ], + [ + 45, + 53, + "binds to", + "protein_state" + ], + [ + 58, + 61, + "PHD", + "structure_element" + ], + [ + 79, + 95, + "binding affinity", + "evidence" + ], + [ + 99, + 101, + "H3", + "protein_type" + ], + [ + 101, + 106, + "K9me0", + "ptm" + ] + ] + }, + { + "sid": 50, + "sent": "Taken together, UHRF1 seems to adopt a closed form through intramolecular interactions (TTD\u2013Spacer and PHD-SRA), which inhibit histone H3 tail recognition by UHRF1.", + "section": "RESULTS", + "ner": [ + [ + 16, + 21, + "UHRF1", + "protein" + ], + [ + 39, + 45, + "closed", + "protein_state" + ], + [ + 88, + 98, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 103, + 110, + "PHD-SRA", + "structure_element" + ], + [ + 127, + 134, + "histone", + "protein_type" + ], + [ + 135, + 137, + "H3", + "protein_type" + ], + [ + 158, + 163, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 51, + "sent": "Overall structure of TTD\u2013Spacer", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 21, + 31, + "TTD\u2013Spacer", + "structure_element" + ] + ] + }, + { + "sid": 52, + "sent": "To investigate the intramolecular interaction within UHRF1, we first mapped the minimal regions within the Spacer for the interaction with the TTD (Supplementary Fig. 2a).", + "section": "RESULTS", + "ner": [ + [ + 53, + 58, + "UHRF1", + "protein" + ], + [ + 107, + 113, + "Spacer", + "structure_element" + ], + [ + 143, + 146, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 53, + "sent": "Internal deletions of the Spacer, including Spacer\u0394660\u2013664, Spacer\u0394665\u2013669, Spacer\u0394670\u2013674 and Spacer642\u2013674, bound to the TTD with comparable binding affinities to that of the Spacer, whereas Spacer587\u2013641 showed no detectable interaction.", + "section": "RESULTS", + "ner": [ + [ + 9, + 18, + "deletions", + "experimental_method" + ], + [ + 26, + 32, + "Spacer", + "structure_element" + ], + [ + 44, + 58, + "Spacer\u0394660\u2013664", + "mutant" + ], + [ + 60, + 74, + "Spacer\u0394665\u2013669", + "mutant" + ], + [ + 76, + 90, + "Spacer\u0394670\u2013674", + "mutant" + ], + [ + 95, + 108, + "Spacer642\u2013674", + "mutant" + ], + [ + 110, + 118, + "bound to", + "protein_state" + ], + [ + 123, + 126, + "TTD", + "structure_element" + ], + [ + 143, + 161, + "binding affinities", + "evidence" + ], + [ + 177, + 183, + "Spacer", + "structure_element" + ], + [ + 193, + 206, + "Spacer587\u2013641", + "mutant" + ] + ] + }, + { + "sid": 54, + "sent": "Spacer\u0394642\u2013651, Spacer\u0394650\u2013654 and Spacer\u0394655\u2013659 also decreased binding affinities, indicating that residues 642\u2013674 are important for TTD\u2013Spacer interaction.", + "section": "RESULTS", + "ner": [ + [ + 0, + 14, + "Spacer\u0394642\u2013651", + "mutant" + ], + [ + 16, + 30, + "Spacer\u0394650\u2013654", + "mutant" + ], + [ + 35, + 49, + "Spacer\u0394655\u2013659", + "mutant" + ], + [ + 65, + 83, + "binding affinities", + "evidence" + ], + [ + 110, + 117, + "642\u2013674", + "residue_range" + ], + [ + 136, + 146, + "TTD\u2013Spacer", + "structure_element" + ] + ] + }, + { + "sid": 55, + "sent": "We next determined the solution structure of the TTD (residues 134\u2013285) bound to Spacer627\u2013674 by conventional NMR techniques (Supplementary Table 3 and Supplementary Fig. 3a,b).", + "section": "RESULTS", + "ner": [ + [ + 23, + 41, + "solution structure", + "evidence" + ], + [ + 49, + 52, + "TTD", + "structure_element" + ], + [ + 63, + 70, + "134\u2013285", + "residue_range" + ], + [ + 72, + 80, + "bound to", + "protein_state" + ], + [ + 81, + 94, + "Spacer627\u2013674", + "residue_range" + ], + [ + 111, + 114, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 56, + "sent": "In the complex structure, each Tudor domain adopts a \u2018Royal' fold containing a characteristic five-stranded \u03b2-sheet and the two Tudor domains tightly pack against each other with a buried area of 573\u2009\u00c52 (Fig. 3a).", + "section": "RESULTS", + "ner": [ + [ + 7, + 24, + "complex structure", + "evidence" + ], + [ + 31, + 43, + "Tudor domain", + "structure_element" + ], + [ + 53, + 65, + "\u2018Royal' fold", + "structure_element" + ], + [ + 94, + 115, + "five-stranded \u03b2-sheet", + "structure_element" + ], + [ + 128, + 141, + "Tudor domains", + "structure_element" + ] + ] + }, + { + "sid": 57, + "sent": "The TTD adopts similar fold to that in TTD\u2013PHD\u2013H3K9me3 complex structure (PDB: 4GY5) with a root-mean-square deviation of 1.09\u2009\u00c5 for 128 C\u03b1 atoms, indicating that the Spacer does not result in obvious conformational change of the TTD (Fig. 3b).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "TTD", + "structure_element" + ], + [ + 39, + 54, + "TTD\u2013PHD\u2013H3K9me3", + "complex_assembly" + ], + [ + 63, + 72, + "structure", + "evidence" + ], + [ + 92, + 118, + "root-mean-square deviation", + "evidence" + ], + [ + 167, + 173, + "Spacer", + "structure_element" + ], + [ + 230, + 233, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 58, + "sent": "The Spacer (residues 643\u2013655 were built in the model) adopts an extended conformation and binds to an acidic groove on the TTD (Fig. 3c).", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "Spacer", + "structure_element" + ], + [ + 21, + 28, + "643\u2013655", + "residue_range" + ], + [ + 64, + 85, + "extended conformation", + "protein_state" + ], + [ + 90, + 98, + "binds to", + "protein_state" + ], + [ + 102, + 115, + "acidic groove", + "site" + ], + [ + 123, + 126, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 59, + "sent": "The TTD\u2013Spacer interaction is mediated by a number of hydrogen bonds (Fig. 3d).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 54, + 68, + "hydrogen bonds", + "bond_interaction" + ] + ] + }, + { + "sid": 60, + "sent": "The side chain of residue K648 forms hydrogen bonds with the carbonyl oxygen atom of D189 and side chain of D190 of the TTD.", + "section": "RESULTS", + "ner": [ + [ + 26, + 30, + "K648", + "residue_name_number" + ], + [ + 37, + 51, + "hydrogen bonds", + "bond_interaction" + ], + [ + 85, + 89, + "D189", + "residue_name_number" + ], + [ + 108, + 112, + "D190", + "residue_name_number" + ], + [ + 120, + 123, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 61, + "sent": "The side chain of residue R649 packs against an acidic surface mainly formed by residues D142 and E153.", + "section": "RESULTS", + "ner": [ + [ + 26, + 30, + "R649", + "residue_name_number" + ], + [ + 31, + 44, + "packs against", + "bond_interaction" + ], + [ + 89, + 93, + "D142", + "residue_name_number" + ], + [ + 98, + 102, + "E153", + "residue_name_number" + ] + ] + }, + { + "sid": 62, + "sent": "Residue S651 forms hydrogen bonds with the main chain of residues G236 and W238.", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "S651", + "residue_name_number" + ], + [ + 19, + 33, + "hydrogen bonds", + "bond_interaction" + ], + [ + 66, + 70, + "G236", + "residue_name_number" + ], + [ + 75, + 79, + "W238", + "residue_name_number" + ] + ] + }, + { + "sid": 63, + "sent": "The interaction is further supported by hydrogen bonds formed between residues K650, A652, G653 and G654 of the Spacer and residues N228, G236 and W238 of the TTD, respectively.", + "section": "RESULTS", + "ner": [ + [ + 40, + 54, + "hydrogen bonds", + "bond_interaction" + ], + [ + 79, + 83, + "K650", + "residue_name_number" + ], + [ + 85, + 89, + "A652", + "residue_name_number" + ], + [ + 91, + 95, + "G653", + "residue_name_number" + ], + [ + 100, + 104, + "G654", + "residue_name_number" + ], + [ + 112, + 118, + "Spacer", + "structure_element" + ], + [ + 132, + 136, + "N228", + "residue_name_number" + ], + [ + 138, + 142, + "G236", + "residue_name_number" + ], + [ + 147, + 151, + "W238", + "residue_name_number" + ], + [ + 159, + 162, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 64, + "sent": "In support of above structural analyses, mutation D142A/E153A of the TTD abolished its interaction with the Spacer (Fig. 3e).", + "section": "RESULTS", + "ner": [ + [ + 20, + 39, + "structural analyses", + "experimental_method" + ], + [ + 41, + 49, + "mutation", + "experimental_method" + ], + [ + 50, + 55, + "D142A", + "mutant" + ], + [ + 56, + 61, + "E153A", + "mutant" + ], + [ + 69, + 72, + "TTD", + "structure_element" + ], + [ + 108, + 114, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 65, + "sent": "Mutations K648D and S651D of the Spacer decreased their binding affinities to the TTD, and mutation R649A of the Spacer showed more significant decrease (\u223c13-fold) in the binding affinity (Fig. 3f).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Mutations", + "experimental_method" + ], + [ + 10, + 15, + "K648D", + "mutant" + ], + [ + 20, + 25, + "S651D", + "mutant" + ], + [ + 33, + 39, + "Spacer", + "structure_element" + ], + [ + 56, + 74, + "binding affinities", + "evidence" + ], + [ + 82, + 85, + "TTD", + "structure_element" + ], + [ + 91, + 99, + "mutation", + "experimental_method" + ], + [ + 100, + 105, + "R649A", + "mutant" + ], + [ + 113, + 119, + "Spacer", + "structure_element" + ], + [ + 171, + 187, + "binding affinity", + "evidence" + ] + ] + }, + { + "sid": 66, + "sent": "As negative control, mutations S639D and S666D of the Spacer showed little effect on the interaction.", + "section": "RESULTS", + "ner": [ + [ + 21, + 30, + "mutations", + "experimental_method" + ], + [ + 31, + 36, + "S639D", + "mutant" + ], + [ + 41, + 46, + "S666D", + "mutant" + ], + [ + 54, + 60, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 67, + "sent": "Interestingly, phosphorylation at residue S651 of UHRF1 was observed in previous mass-spectrometry analyses.", + "section": "RESULTS", + "ner": [ + [ + 15, + 30, + "phosphorylation", + "ptm" + ], + [ + 42, + 46, + "S651", + "residue_name_number" + ], + [ + 50, + 55, + "UHRF1", + "protein" + ], + [ + 81, + 98, + "mass-spectrometry", + "experimental_method" + ] + ] + }, + { + "sid": 68, + "sent": "Compared with the unmodified peptide of Spacer642\u2013664, a phosphorylation at S651 markedly decreased the binding affinity to the TTD (Supplementary Fig. 2b), suggesting that the phosphorylation may regulate the intramolecular interaction within UHRF1.", + "section": "RESULTS", + "ner": [ + [ + 18, + 28, + "unmodified", + "protein_state" + ], + [ + 40, + 53, + "Spacer642\u2013664", + "mutant" + ], + [ + 57, + 72, + "phosphorylation", + "ptm" + ], + [ + 76, + 80, + "S651", + "residue_name_number" + ], + [ + 104, + 120, + "binding affinity", + "evidence" + ], + [ + 128, + 131, + "TTD", + "structure_element" + ], + [ + 177, + 192, + "phosphorylation", + "ptm" + ], + [ + 244, + 249, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 69, + "sent": "The spacer binds to the TTD by competing with the linker", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "spacer", + "structure_element" + ], + [ + 11, + 19, + "binds to", + "protein_state" + ], + [ + 24, + 27, + "TTD", + "structure_element" + ], + [ + 50, + 56, + "linker", + "structure_element" + ] + ] + }, + { + "sid": 70, + "sent": "Previous studies indicate that the TTD binds to a linker region connecting the TTD and PHD (residues 286\u2013306, designated Linker, Fig. 1a), and TTD\u2013Linker interaction is essential for H3K9me3 recognition by TTD\u2013PHD.", + "section": "RESULTS", + "ner": [ + [ + 35, + 38, + "TTD", + "structure_element" + ], + [ + 39, + 47, + "binds to", + "protein_state" + ], + [ + 50, + 63, + "linker region", + "structure_element" + ], + [ + 79, + 82, + "TTD", + "structure_element" + ], + [ + 87, + 90, + "PHD", + "structure_element" + ], + [ + 101, + 108, + "286\u2013306", + "residue_range" + ], + [ + 121, + 127, + "Linker", + "structure_element" + ], + [ + 143, + 153, + "TTD\u2013Linker", + "structure_element" + ], + [ + 183, + 185, + "H3", + "protein_type" + ], + [ + 185, + 190, + "K9me3", + "ptm" + ], + [ + 206, + 213, + "TTD\u2013PHD", + "structure_element" + ] + ] + }, + { + "sid": 71, + "sent": "Comparison of TTD\u2013Spacer and TTD\u2013PHD\u2013H3K9me3 (PDB: 4GY5) structures indicates that the Spacer and the Linker bind to the TTD in a similar manner in the two complexes (Fig. 3b).", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "Comparison", + "experimental_method" + ], + [ + 14, + 24, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 29, + 44, + "TTD\u2013PHD\u2013H3K9me3", + "complex_assembly" + ], + [ + 57, + 67, + "structures", + "evidence" + ], + [ + 87, + 93, + "Spacer", + "structure_element" + ], + [ + 102, + 108, + "Linker", + "structure_element" + ], + [ + 121, + 124, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 72, + "sent": "In TTD\u2013PHD\u2013H3K9me3 structure, residues R295, R296 and S298 of the Linker adopt almost identical conformation to residues K648, R649 and S651 of the Spacer in TTD\u2013Spacer structure, respectively.", + "section": "RESULTS", + "ner": [ + [ + 3, + 18, + "TTD\u2013PHD\u2013H3K9me3", + "complex_assembly" + ], + [ + 19, + 28, + "structure", + "evidence" + ], + [ + 39, + 43, + "R295", + "residue_name_number" + ], + [ + 45, + 49, + "R296", + "residue_name_number" + ], + [ + 54, + 58, + "S298", + "residue_name_number" + ], + [ + 66, + 72, + "Linker", + "structure_element" + ], + [ + 121, + 125, + "K648", + "residue_name_number" + ], + [ + 127, + 131, + "R649", + "residue_name_number" + ], + [ + 136, + 140, + "S651", + "residue_name_number" + ], + [ + 148, + 154, + "Spacer", + "structure_element" + ], + [ + 158, + 168, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 169, + 178, + "structure", + "evidence" + ] + ] + }, + { + "sid": 73, + "sent": "Similar intramolecular contacts (TTD\u2013Linker and TTD\u2013Spacer) were observed in the two structures (Fig. 3b,d and Supplementary Fig. 4a).", + "section": "RESULTS", + "ner": [ + [ + 33, + 43, + "TTD\u2013Linker", + "structure_element" + ], + [ + 48, + 58, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 85, + 95, + "structures", + "evidence" + ] + ] + }, + { + "sid": 74, + "sent": "Thus, the Spacer may disrupt the TTD\u2013Linker interaction and inhibits the recognition of H3K9me3 by TTD\u2013PHD.", + "section": "RESULTS", + "ner": [ + [ + 10, + 16, + "Spacer", + "structure_element" + ], + [ + 33, + 43, + "TTD\u2013Linker", + "structure_element" + ], + [ + 88, + 90, + "H3", + "protein_type" + ], + [ + 90, + 95, + "K9me3", + "ptm" + ], + [ + 99, + 106, + "TTD\u2013PHD", + "structure_element" + ] + ] + }, + { + "sid": 75, + "sent": "To test this hypothesis, we first investigated the potential competition between the Linker and the Spacer for their interaction with the TTD.", + "section": "RESULTS", + "ner": [ + [ + 85, + 91, + "Linker", + "structure_element" + ], + [ + 100, + 106, + "Spacer", + "structure_element" + ], + [ + 138, + 141, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 76, + "sent": "The ITC experiment shows that the Linker peptide (289\u2013306) bound to the TTD with a binding affinity of 24.04\u2009\u03bcM (Supplementary Fig. 4b), \u223c15-fold lower than that of the Spacer peptide (KD=1.59\u2009\u03bcM, Fig. 3e).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "ITC", + "experimental_method" + ], + [ + 34, + 40, + "Linker", + "structure_element" + ], + [ + 50, + 57, + "289\u2013306", + "residue_range" + ], + [ + 59, + 67, + "bound to", + "protein_state" + ], + [ + 72, + 75, + "TTD", + "structure_element" + ], + [ + 83, + 99, + "binding affinity", + "evidence" + ], + [ + 169, + 175, + "Spacer", + "structure_element" + ], + [ + 185, + 187, + "KD", + "evidence" + ] + ] + }, + { + "sid": 77, + "sent": "The competitive ITC experiments show that TTD\u2013Spacer binding affinity decreased by a factor of two in the presence of the Linker, whereas TTD\u2013Linker interaction was abolished in the presence of the Spacer (Supplementary Fig. 4c).", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "competitive ITC", + "experimental_method" + ], + [ + 42, + 69, + "TTD\u2013Spacer binding affinity", + "evidence" + ], + [ + 106, + 117, + "presence of", + "protein_state" + ], + [ + 122, + 128, + "Linker", + "structure_element" + ], + [ + 138, + 148, + "TTD\u2013Linker", + "structure_element" + ], + [ + 182, + 193, + "presence of", + "protein_state" + ], + [ + 198, + 204, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 78, + "sent": "Compared with TTD\u2013Spacer interaction (KD=1.48\u2009\u03bcM), TTD\u2013PHD decreased the binding affinity to the Spacer (KD=10.68\u2009\u03bcM), whereas mutation R295D/R296D (within the Linker and important for TTD\u2013Linker interaction) of TTD\u2013PHD showed minor decrease in the binding affinity (KD=2.69\u2009\u03bcM; Fig. 3g), indicating a competition between the Spacer and the Linker on the same binding site of the TTD.", + "section": "RESULTS", + "ner": [ + [ + 14, + 24, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 38, + 40, + "KD", + "evidence" + ], + [ + 51, + 58, + "TTD\u2013PHD", + "structure_element" + ], + [ + 73, + 89, + "binding affinity", + "evidence" + ], + [ + 97, + 103, + "Spacer", + "structure_element" + ], + [ + 105, + 107, + "KD", + "evidence" + ], + [ + 127, + 135, + "mutation", + "experimental_method" + ], + [ + 136, + 141, + "R295D", + "mutant" + ], + [ + 142, + 147, + "R296D", + "mutant" + ], + [ + 160, + 166, + "Linker", + "structure_element" + ], + [ + 185, + 195, + "TTD\u2013Linker", + "structure_element" + ], + [ + 212, + 219, + "TTD\u2013PHD", + "structure_element" + ], + [ + 249, + 265, + "binding affinity", + "evidence" + ], + [ + 267, + 269, + "KD", + "evidence" + ], + [ + 326, + 332, + "Spacer", + "structure_element" + ], + [ + 341, + 347, + "Linker", + "structure_element" + ], + [ + 360, + 372, + "binding site", + "site" + ], + [ + 380, + 383, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 79, + "sent": "Notably, although the Linker (in the context of TTD-PHD) impairs the TTD\u2013Spacer interaction to some extent, the isolated Spacer could still bind to TTD\u2013PHD with moderate binding affinity (KD=10.68\u2009\u03bcM), supporting the existence of the intramolecular interaction within UHRF1.", + "section": "RESULTS", + "ner": [ + [ + 22, + 28, + "Linker", + "structure_element" + ], + [ + 48, + 55, + "TTD-PHD", + "structure_element" + ], + [ + 69, + 79, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 121, + 127, + "Spacer", + "structure_element" + ], + [ + 148, + 155, + "TTD\u2013PHD", + "structure_element" + ], + [ + 170, + 186, + "binding affinity", + "evidence" + ], + [ + 188, + 190, + "KD", + "evidence" + ], + [ + 268, + 273, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 80, + "sent": "To test whether TTD\u2013Spacer association exists in the context of full-length UHRF1, we used various truncations of UHRF1 in the GST pull-down assay.", + "section": "RESULTS", + "ner": [ + [ + 16, + 26, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 64, + 75, + "full-length", + "protein_state" + ], + [ + 76, + 81, + "UHRF1", + "protein" + ], + [ + 99, + 110, + "truncations", + "experimental_method" + ], + [ + 114, + 119, + "UHRF1", + "protein" + ], + [ + 127, + 146, + "GST pull-down assay", + "experimental_method" + ] + ] + }, + { + "sid": 81, + "sent": "As indicated in Fig. 3h, full-length UHRF1 and UHRF1\u0394SRA showed no interaction with GST-tagged TTD, Linker or Spacer, suggesting that TTD\u2013Spacer interaction in-cis within full-length UHRF1 or UHRF1\u0394SRA prohibits TTD\u2013Spacer complex formation in-trans.", + "section": "RESULTS", + "ner": [ + [ + 25, + 36, + "full-length", + "protein_state" + ], + [ + 37, + 42, + "UHRF1", + "protein" + ], + [ + 47, + 56, + "UHRF1\u0394SRA", + "mutant" + ], + [ + 84, + 94, + "GST-tagged", + "protein_state" + ], + [ + 95, + 98, + "TTD", + "structure_element" + ], + [ + 100, + 106, + "Linker", + "structure_element" + ], + [ + 110, + 116, + "Spacer", + "structure_element" + ], + [ + 134, + 144, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 157, + 163, + "in-cis", + "protein_state" + ], + [ + 171, + 182, + "full-length", + "protein_state" + ], + [ + 183, + 188, + "UHRF1", + "protein" + ], + [ + 192, + 201, + "UHRF1\u0394SRA", + "mutant" + ], + [ + 212, + 222, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 241, + 249, + "in-trans", + "protein_state" + ] + ] + }, + { + "sid": 82, + "sent": "In contrast, UHRF1\u0394TTD bound to GST-TTD, and UHRF1\u0394627\u2013674 bound to GST-Spacer, indicating that lack of TTD\u2013Spacer interaction in-cis, TTD\u2013Spacer complex could form in-trans, supporting that the TTD binds to the Spacer in the context of full-length UHRF1.", + "section": "RESULTS", + "ner": [ + [ + 13, + 22, + "UHRF1\u0394TTD", + "mutant" + ], + [ + 23, + 31, + "bound to", + "protein_state" + ], + [ + 32, + 35, + "GST", + "experimental_method" + ], + [ + 36, + 39, + "TTD", + "structure_element" + ], + [ + 45, + 58, + "UHRF1\u0394627\u2013674", + "mutant" + ], + [ + 59, + 67, + "bound to", + "protein_state" + ], + [ + 68, + 71, + "GST", + "experimental_method" + ], + [ + 72, + 78, + "Spacer", + "structure_element" + ], + [ + 104, + 114, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 127, + 133, + "in-cis", + "protein_state" + ], + [ + 135, + 145, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 165, + 173, + "in-trans", + "protein_state" + ], + [ + 195, + 198, + "TTD", + "structure_element" + ], + [ + 199, + 207, + "binds to", + "protein_state" + ], + [ + 212, + 218, + "Spacer", + "structure_element" + ], + [ + 237, + 248, + "full-length", + "protein_state" + ], + [ + 249, + 254, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 83, + "sent": "Moreover, GST-Linker showed very weak if not undetectable interaction with wild-type or deletions of UHRF1, suggesting that TTD\u2013Linker interaction is much weaker than that of TTD\u2013Spacer.", + "section": "RESULTS", + "ner": [ + [ + 10, + 13, + "GST", + "experimental_method" + ], + [ + 14, + 20, + "Linker", + "structure_element" + ], + [ + 75, + 84, + "wild-type", + "protein_state" + ], + [ + 101, + 106, + "UHRF1", + "protein" + ], + [ + 124, + 134, + "TTD\u2013Linker", + "structure_element" + ], + [ + 175, + 185, + "TTD\u2013Spacer", + "structure_element" + ] + ] + }, + { + "sid": 84, + "sent": "Taken together, UHRF1 adopts a closed conformation, in which the Spacer binds to the TTD through competing with the Linker, and therefore inhibits H3K9me3 recognition by UHRF1.", + "section": "RESULTS", + "ner": [ + [ + 16, + 21, + "UHRF1", + "protein" + ], + [ + 31, + 37, + "closed", + "protein_state" + ], + [ + 65, + 71, + "Spacer", + "structure_element" + ], + [ + 72, + 80, + "binds to", + "protein_state" + ], + [ + 85, + 88, + "TTD", + "structure_element" + ], + [ + 116, + 122, + "Linker", + "structure_element" + ], + [ + 147, + 149, + "H3", + "protein_type" + ], + [ + 149, + 154, + "K9me3", + "ptm" + ], + [ + 170, + 175, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 85, + "sent": "The spacer inhibits H3K9me3 recognition by the isolated TTD", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "spacer", + "structure_element" + ], + [ + 20, + 22, + "H3", + "protein_type" + ], + [ + 22, + 27, + "K9me3", + "ptm" + ], + [ + 56, + 59, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 86, + "sent": "Our previous study indicates that H3K9me3 binds to the TTD in different manner in TTD\u2013PHD\u2013H3K9me3 (ref.) and TTD-H3K9me3 (PDB: 2L3R) structures.", + "section": "RESULTS", + "ner": [ + [ + 34, + 36, + "H3", + "protein_type" + ], + [ + 36, + 41, + "K9me3", + "ptm" + ], + [ + 42, + 50, + "binds to", + "protein_state" + ], + [ + 55, + 58, + "TTD", + "structure_element" + ], + [ + 82, + 97, + "TTD\u2013PHD\u2013H3K9me3", + "complex_assembly" + ], + [ + 109, + 120, + "TTD-H3K9me3", + "complex_assembly" + ], + [ + 133, + 143, + "structures", + "evidence" + ] + ] + }, + { + "sid": 87, + "sent": "Because the TTD is always associated with the PHD, whether the pattern of TTD\u2013H3K9me3 interaction exists in vivo remains unknown.", + "section": "RESULTS", + "ner": [ + [ + 12, + 15, + "TTD", + "structure_element" + ], + [ + 46, + 49, + "PHD", + "structure_element" + ], + [ + 74, + 85, + "TTD\u2013H3K9me3", + "complex_assembly" + ] + ] + }, + { + "sid": 88, + "sent": "Nevertheless, comparison of TTD\u2013H3K9me3 and TTD\u2013Spacer structures indicates that H3K9me3 and the Spacer overlap on the surface of the TTD (Supplementary Fig. 4d), suggesting that the Spacer might block the H3K9me3 recognition by the isolated TTD.", + "section": "RESULTS", + "ner": [ + [ + 14, + 24, + "comparison", + "experimental_method" + ], + [ + 28, + 39, + "TTD\u2013H3K9me3", + "complex_assembly" + ], + [ + 44, + 54, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 55, + 65, + "structures", + "evidence" + ], + [ + 81, + 83, + "H3", + "protein_type" + ], + [ + 83, + 88, + "K9me3", + "ptm" + ], + [ + 97, + 103, + "Spacer", + "structure_element" + ], + [ + 134, + 137, + "TTD", + "structure_element" + ], + [ + 183, + 189, + "Spacer", + "structure_element" + ], + [ + 206, + 208, + "H3", + "protein_type" + ], + [ + 208, + 213, + "K9me3", + "ptm" + ], + [ + 242, + 245, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 89, + "sent": "As shown in Supplementary Fig. 4e, the Spacer inhibited TTD\u2013H3K9me3 interaction, whereas its TTD-binding defective mutants of the Spacer or the SRA (a negative control) markedly decreased the inhibition.", + "section": "RESULTS", + "ner": [ + [ + 39, + 45, + "Spacer", + "structure_element" + ], + [ + 56, + 67, + "TTD\u2013H3K9me3", + "complex_assembly" + ], + [ + 93, + 114, + "TTD-binding defective", + "protein_state" + ], + [ + 115, + 122, + "mutants", + "protein_state" + ], + [ + 130, + 136, + "Spacer", + "structure_element" + ], + [ + 144, + 147, + "SRA", + "structure_element" + ] + ] + }, + { + "sid": 90, + "sent": "We next tested whether such inhibition also occurs in the context of full-length UHRF1.", + "section": "RESULTS", + "ner": [ + [ + 69, + 80, + "full-length", + "protein_state" + ], + [ + 81, + 86, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 91, + "sent": "Compared with full-length UHRF1, UHRF1\u0394627\u2013674 enhanced H3K9me3-binding affinity by a factor of four (Supplementary Fig. 4f).", + "section": "RESULTS", + "ner": [ + [ + 14, + 25, + "full-length", + "protein_state" + ], + [ + 26, + 31, + "UHRF1", + "protein" + ], + [ + 33, + 46, + "UHRF1\u0394627\u2013674", + "mutant" + ], + [ + 56, + 80, + "H3K9me3-binding affinity", + "evidence" + ] + ] + }, + { + "sid": 92, + "sent": "The restoration of H3K9me3-binding affinity is not dramatic because the PHD still binds to histone H3 in both proteins.", + "section": "RESULTS", + "ner": [ + [ + 19, + 43, + "H3K9me3-binding affinity", + "evidence" + ], + [ + 72, + 75, + "PHD", + "structure_element" + ], + [ + 82, + 90, + "binds to", + "protein_state" + ], + [ + 91, + 98, + "histone", + "protein_type" + ], + [ + 99, + 101, + "H3", + "protein_type" + ] + ] + }, + { + "sid": 93, + "sent": "To exclude this effect, we performed the assay using UHRF1D334A, which abolishes H3R2-binding affinity of the PHD.", + "section": "RESULTS", + "ner": [ + [ + 53, + 63, + "UHRF1D334A", + "mutant" + ], + [ + 71, + 80, + "abolishes", + "protein_state" + ], + [ + 81, + 102, + "H3R2-binding affinity", + "evidence" + ], + [ + 110, + 113, + "PHD", + "structure_element" + ] + ] + }, + { + "sid": 94, + "sent": "UHRF1D334A showed undetectable H3K9me3-binding affinity, whereas UHRF1D334A&\u0394627\u2013674 dramatically restored its H3K9me3-binding affinity (KD=8.69\u2009\u03bcM; Supplementary Fig. 4f), indicating that H3K9me3 recognition by the TTD is blocked by the Spacer through competitive interaction with the TTD.", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "UHRF1D334A", + "mutant" + ], + [ + 31, + 55, + "H3K9me3-binding affinity", + "evidence" + ], + [ + 65, + 75, + "UHRF1D334A", + "mutant" + ], + [ + 76, + 84, + "\u0394627\u2013674", + "mutant" + ], + [ + 111, + 135, + "H3K9me3-binding affinity", + "evidence" + ], + [ + 137, + 139, + "KD", + "evidence" + ], + [ + 189, + 191, + "H3", + "protein_type" + ], + [ + 191, + 196, + "K9me3", + "ptm" + ], + [ + 216, + 219, + "TTD", + "structure_element" + ], + [ + 238, + 244, + "Spacer", + "structure_element" + ], + [ + 286, + 289, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 95, + "sent": "Moreover, the R295D/R296D mutant of full-length UHRF1 showed decreased binding affinity to H3K9me3 (eightfold lower than wild type), suggesting that mutation of R295D/R296D favours TTD\u2013Spacer interaction and therefore promotes UHRF1 to exhibit a more stable closed conformation (Supplementary Fig. 4g).", + "section": "RESULTS", + "ner": [ + [ + 14, + 19, + "R295D", + "mutant" + ], + [ + 20, + 25, + "R296D", + "mutant" + ], + [ + 26, + 32, + "mutant", + "protein_state" + ], + [ + 36, + 47, + "full-length", + "protein_state" + ], + [ + 48, + 53, + "UHRF1", + "protein" + ], + [ + 71, + 87, + "binding affinity", + "evidence" + ], + [ + 91, + 93, + "H3", + "protein_type" + ], + [ + 93, + 98, + "K9me3", + "ptm" + ], + [ + 121, + 130, + "wild type", + "protein_state" + ], + [ + 149, + 157, + "mutation", + "experimental_method" + ], + [ + 161, + 166, + "R295D", + "mutant" + ], + [ + 167, + 172, + "R296D", + "mutant" + ], + [ + 181, + 191, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 227, + 232, + "UHRF1", + "protein" + ], + [ + 258, + 264, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 96, + "sent": "Taken together, the Spacer binds to the TTD and inhibits H3K9me3 recognition by UHRF1 through (i) disrupting TTD\u2013Linker interaction, which is essential for H3K9me3 recognition by TTD\u2013PHD, (ii) prohibiting H3K9me3 binding to the isolated TTD.", + "section": "RESULTS", + "ner": [ + [ + 20, + 26, + "Spacer", + "structure_element" + ], + [ + 27, + 35, + "binds to", + "protein_state" + ], + [ + 40, + 43, + "TTD", + "structure_element" + ], + [ + 57, + 59, + "H3", + "protein_type" + ], + [ + 59, + 64, + "K9me3", + "ptm" + ], + [ + 80, + 85, + "UHRF1", + "protein" + ], + [ + 109, + 119, + "TTD\u2013Linker", + "structure_element" + ], + [ + 156, + 158, + "H3", + "protein_type" + ], + [ + 158, + 163, + "K9me3", + "ptm" + ], + [ + 179, + 186, + "TTD\u2013PHD", + "structure_element" + ], + [ + 205, + 207, + "H3", + "protein_type" + ], + [ + 207, + 212, + "K9me3", + "ptm" + ], + [ + 237, + 240, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 97, + "sent": "TTD\u2013PHD\u2013H3K9me3 complex inhibits TTD\u2013spacer interaction", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "TTD\u2013PHD\u2013H3K9me3", + "complex_assembly" + ], + [ + 33, + 43, + "TTD\u2013spacer", + "structure_element" + ] + ] + }, + { + "sid": 98, + "sent": "Interestingly, pre-incubation of H3K9me3 peptide completely blocked the interaction between the Spacer and the TTD alone or TTD\u2013PHD (Supplementary Fig. 4h), whereas the presence of the Spacer partially impaired the interaction between TTD\u2013PHD and H3K9me3 (Fig. 2c).", + "section": "RESULTS", + "ner": [ + [ + 15, + 29, + "pre-incubation", + "experimental_method" + ], + [ + 33, + 35, + "H3", + "protein_type" + ], + [ + 35, + 40, + "K9me3", + "ptm" + ], + [ + 96, + 102, + "Spacer", + "structure_element" + ], + [ + 111, + 114, + "TTD", + "structure_element" + ], + [ + 115, + 120, + "alone", + "protein_state" + ], + [ + 124, + 131, + "TTD\u2013PHD", + "structure_element" + ], + [ + 169, + 180, + "presence of", + "protein_state" + ], + [ + 185, + 191, + "Spacer", + "structure_element" + ], + [ + 235, + 242, + "TTD\u2013PHD", + "structure_element" + ], + [ + 247, + 249, + "H3", + "protein_type" + ], + [ + 249, + 254, + "K9me3", + "ptm" + ] + ] + }, + { + "sid": 99, + "sent": "The results are also consistent with the previous observation that the interaction between TTD\u2013PHD and the Spacer is much weaker (KD=10.68\u2009\u03bcM, Fig. 3g) than that between TTD\u2013PHD and H3K9me3 (KD=0.15\u2009\u03bcM, Fig. 1d).", + "section": "RESULTS", + "ner": [ + [ + 91, + 98, + "TTD\u2013PHD", + "structure_element" + ], + [ + 107, + 113, + "Spacer", + "structure_element" + ], + [ + 130, + 132, + "KD", + "evidence" + ], + [ + 170, + 177, + "TTD\u2013PHD", + "structure_element" + ], + [ + 182, + 184, + "H3", + "protein_type" + ], + [ + 184, + 189, + "K9me3", + "ptm" + ], + [ + 191, + 193, + "KD", + "evidence" + ] + ] + }, + { + "sid": 100, + "sent": "These results suggest that once TTD\u2013PHD binds to H3K9me3, UHRF1 will be locked by H3K9me3 and the Spacer is unlikely to fold back for the intramolecular interaction.", + "section": "RESULTS", + "ner": [ + [ + 32, + 39, + "TTD\u2013PHD", + "structure_element" + ], + [ + 40, + 48, + "binds to", + "protein_state" + ], + [ + 49, + 51, + "H3", + "protein_type" + ], + [ + 51, + 56, + "K9me3", + "ptm" + ], + [ + 58, + 63, + "UHRF1", + "protein" + ], + [ + 82, + 84, + "H3", + "protein_type" + ], + [ + 84, + 89, + "K9me3", + "ptm" + ], + [ + 98, + 104, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 101, + "sent": "Hm-DNA disrupts intramolecular interaction within UHRF1", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Hm-DNA", + "chemical" + ], + [ + 50, + 55, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 102, + "sent": "To investigate whether hm-DNA could open the closed conformation of UHRF1, we first measured the intramolecular interaction using UHRF1 truncations in the presence or absence of hm-DNA.", + "section": "RESULTS", + "ner": [ + [ + 23, + 29, + "hm-DNA", + "chemical" + ], + [ + 36, + 40, + "open", + "protein_state" + ], + [ + 45, + 51, + "closed", + "protein_state" + ], + [ + 68, + 73, + "UHRF1", + "protein" + ], + [ + 130, + 135, + "UHRF1", + "protein" + ], + [ + 136, + 147, + "truncations", + "experimental_method" + ], + [ + 155, + 163, + "presence", + "protein_state" + ], + [ + 167, + 177, + "absence of", + "protein_state" + ], + [ + 178, + 184, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 103, + "sent": "The GST pull-down assays show that the PHD bound to the SRA and such interaction was impaired by the addition of hm-DNA (Fig. 4a).", + "section": "RESULTS", + "ner": [ + [ + 4, + 24, + "GST pull-down assays", + "experimental_method" + ], + [ + 39, + 42, + "PHD", + "structure_element" + ], + [ + 43, + 51, + "bound to", + "protein_state" + ], + [ + 56, + 59, + "SRA", + "structure_element" + ], + [ + 113, + 119, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 104, + "sent": "H3 peptide pull-down assays show that hm-DNA only enhanced the H3K9me0-binding affinities of UHRF1 truncations containing PHD-SRA, such as PHD-SRA, TTD-PHD-SRA, TTD-PHD-SRA-Spacer, UHRF1\u0394TTD and UHRF1\u0394Spacer (Fig. 4b).", + "section": "RESULTS", + "ner": [ + [ + 0, + 27, + "H3 peptide pull-down assays", + "experimental_method" + ], + [ + 38, + 44, + "hm-DNA", + "chemical" + ], + [ + 63, + 89, + "H3K9me0-binding affinities", + "evidence" + ], + [ + 93, + 98, + "UHRF1", + "protein" + ], + [ + 99, + 110, + "truncations", + "experimental_method" + ], + [ + 122, + 129, + "PHD-SRA", + "structure_element" + ], + [ + 139, + 146, + "PHD-SRA", + "structure_element" + ], + [ + 148, + 159, + "TTD-PHD-SRA", + "structure_element" + ], + [ + 161, + 179, + "TTD-PHD-SRA-Spacer", + "structure_element" + ], + [ + 181, + 190, + "UHRF1\u0394TTD", + "mutant" + ], + [ + 195, + 207, + "UHRF1\u0394Spacer", + "mutant" + ] + ] + }, + { + "sid": 105, + "sent": "The result indicates that hm-DNA disrupts PHD\u2013SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer.", + "section": "RESULTS", + "ner": [ + [ + 26, + 32, + "hm-DNA", + "chemical" + ], + [ + 42, + 49, + "PHD\u2013SRA", + "structure_element" + ], + [ + 78, + 102, + "H3K9me0-binding affinity", + "evidence" + ], + [ + 110, + 113, + "PHD", + "structure_element" + ], + [ + 145, + 148, + "TTD", + "structure_element" + ], + [ + 156, + 162, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 106, + "sent": "Moreover, the TTD or TTD\u2013PHD bound to SRA\u2013Spacer and the interaction was impaired by the addition of hm-DNA (Fig. 4c).", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "TTD", + "structure_element" + ], + [ + 21, + 28, + "TTD\u2013PHD", + "structure_element" + ], + [ + 29, + 37, + "bound to", + "protein_state" + ], + [ + 38, + 48, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 101, + 107, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 107, + "sent": "The ITC measurements show that the presence of hm-DNA markedly impaired the interaction between the TTD and SRA\u2013Spacer (Supplementary Fig. 5a).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "ITC", + "experimental_method" + ], + [ + 35, + 46, + "presence of", + "protein_state" + ], + [ + 47, + 53, + "hm-DNA", + "chemical" + ], + [ + 100, + 103, + "TTD", + "structure_element" + ], + [ + 108, + 118, + "SRA\u2013Spacer", + "structure_element" + ] + ] + }, + { + "sid": 108, + "sent": "However, the TTD\u2013Spacer interaction was not affected by the presence of the hm-DNA, indicating that hm-DNA displaces the Spacer from the TTD in a SRA-dependent manner (Supplementary Fig. 5b).", + "section": "RESULTS", + "ner": [ + [ + 13, + 23, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 60, + 71, + "presence of", + "protein_state" + ], + [ + 76, + 82, + "hm-DNA", + "chemical" + ], + [ + 100, + 106, + "hm-DNA", + "chemical" + ], + [ + 121, + 127, + "Spacer", + "structure_element" + ], + [ + 137, + 140, + "TTD", + "structure_element" + ], + [ + 146, + 149, + "SRA", + "structure_element" + ] + ] + }, + { + "sid": 109, + "sent": "To investigate whether hm-DNA disrupts TTD\u2013Spacer interaction in the context of full-length UHRF1, we monitored the conformational changes of UHRF1 using its histone-binding affinity as read-out.", + "section": "RESULTS", + "ner": [ + [ + 23, + 29, + "hm-DNA", + "chemical" + ], + [ + 39, + 49, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 80, + 91, + "full-length", + "protein_state" + ], + [ + 92, + 97, + "UHRF1", + "protein" + ], + [ + 142, + 147, + "UHRF1", + "protein" + ], + [ + 158, + 182, + "histone-binding affinity", + "evidence" + ] + ] + }, + { + "sid": 110, + "sent": "UHRF1D334A was used to exclude the effect of H3K9me0 recognition by the PHD.", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "UHRF1D334A", + "mutant" + ], + [ + 45, + 47, + "H3", + "protein_type" + ], + [ + 47, + 52, + "K9me0", + "ptm" + ], + [ + 72, + 75, + "PHD", + "structure_element" + ] + ] + }, + { + "sid": 111, + "sent": "As expected, all D334A-containing mutants showed undetectable interaction with H3K9me0 (Fig. 4d).", + "section": "RESULTS", + "ner": [ + [ + 17, + 22, + "D334A", + "mutant" + ], + [ + 34, + 41, + "mutants", + "protein_state" + ], + [ + 79, + 81, + "H3", + "protein_type" + ], + [ + 81, + 86, + "K9me0", + "ptm" + ] + ] + }, + { + "sid": 112, + "sent": "UHRF1D334A bound to H3K9me3 peptide in the presence of hm-DNA, but showed no interaction in the absence of hm-DNA, which is consistent with the ITC experiments (Supplementary Fig. 4f).", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "UHRF1D334A", + "mutant" + ], + [ + 11, + 19, + "bound to", + "protein_state" + ], + [ + 20, + 22, + "H3", + "protein_type" + ], + [ + 22, + 27, + "K9me3", + "ptm" + ], + [ + 43, + 54, + "presence of", + "protein_state" + ], + [ + 55, + 61, + "hm-DNA", + "chemical" + ], + [ + 96, + 106, + "absence of", + "protein_state" + ], + [ + 107, + 113, + "hm-DNA", + "chemical" + ], + [ + 144, + 147, + "ITC", + "experimental_method" + ] + ] + }, + { + "sid": 113, + "sent": "In contrast, UHRF1D334A&\u0394627\u2013674 strongly bound to H3K9me3 even in the absence of hm-DNA (Fig. 4d), indicating that the deletion of the Spacer releases otherwise blocked TTD\u2013PHD for H3K9me3 recognition.", + "section": "RESULTS", + "ner": [ + [ + 13, + 23, + "UHRF1D334A", + "mutant" + ], + [ + 24, + 32, + "\u0394627\u2013674", + "mutant" + ], + [ + 42, + 50, + "bound to", + "protein_state" + ], + [ + 51, + 53, + "H3", + "protein_type" + ], + [ + 53, + 58, + "K9me3", + "ptm" + ], + [ + 71, + 81, + "absence of", + "protein_state" + ], + [ + 82, + 88, + "hm-DNA", + "chemical" + ], + [ + 120, + 128, + "deletion", + "experimental_method" + ], + [ + 136, + 142, + "Spacer", + "structure_element" + ], + [ + 170, + 177, + "TTD\u2013PHD", + "structure_element" + ], + [ + 182, + 184, + "H3", + "protein_type" + ], + [ + 184, + 189, + "K9me3", + "ptm" + ] + ] + }, + { + "sid": 114, + "sent": "The results further support the conclusion that the Spacer binds to the TTD in the context of full-length UHRF1 and the intramolecular interactions are disrupted by hm-DNA.", + "section": "RESULTS", + "ner": [ + [ + 52, + 58, + "Spacer", + "structure_element" + ], + [ + 59, + 67, + "binds to", + "protein_state" + ], + [ + 72, + 75, + "TTD", + "structure_element" + ], + [ + 94, + 105, + "full-length", + "protein_state" + ], + [ + 106, + 111, + "UHRF1", + "protein" + ], + [ + 165, + 171, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 115, + "sent": "We next performed similar peptide pull-down assay using two mutants (N228C/G653C and R235C/G654C) generated on UHRF1D334A.", + "section": "RESULTS", + "ner": [ + [ + 26, + 49, + "peptide pull-down assay", + "experimental_method" + ], + [ + 60, + 67, + "mutants", + "protein_state" + ], + [ + 69, + 74, + "N228C", + "mutant" + ], + [ + 75, + 80, + "G653C", + "mutant" + ], + [ + 85, + 90, + "R235C", + "mutant" + ], + [ + 91, + 96, + "G654C", + "mutant" + ], + [ + 111, + 121, + "UHRF1D334A", + "mutant" + ] + ] + }, + { + "sid": 116, + "sent": "Residues N228/R235 from the TTD and G653/G654 from the Spacer were chosen according to the TTD\u2013Spacer complex structure (Supplementary Fig. 5c) so that the replaced Cysteine residues (one from the TTD and one from the Spacer) are physically close enough to each other to form a disulphide bond in the absence of reducing reagent (dithiothreitol, DTT).", + "section": "RESULTS", + "ner": [ + [ + 9, + 13, + "N228", + "residue_name_number" + ], + [ + 14, + 18, + "R235", + "residue_name_number" + ], + [ + 28, + 31, + "TTD", + "structure_element" + ], + [ + 36, + 40, + "G653", + "residue_name_number" + ], + [ + 41, + 45, + "G654", + "residue_name_number" + ], + [ + 55, + 61, + "Spacer", + "structure_element" + ], + [ + 91, + 101, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 110, + 119, + "structure", + "evidence" + ], + [ + 165, + 173, + "Cysteine", + "residue_name" + ], + [ + 197, + 200, + "TTD", + "structure_element" + ], + [ + 218, + 224, + "Spacer", + "structure_element" + ], + [ + 278, + 293, + "disulphide bond", + "ptm" + ], + [ + 301, + 311, + "absence of", + "protein_state" + ], + [ + 330, + 344, + "dithiothreitol", + "chemical" + ], + [ + 346, + 349, + "DTT", + "chemical" + ] + ] + }, + { + "sid": 117, + "sent": "As shown in Fig. 4d, hm-DNA largely enhanced the H3K9me3-binding affinities of both mutants in the presence of DTT, but not in the absence of DTT, indicating that the disulphide bond formation (in the absence of DTT) disallows hm-DNA to disrupt TTD\u2013Spacer interaction for H3K9me3 recognition.", + "section": "RESULTS", + "ner": [ + [ + 21, + 27, + "hm-DNA", + "chemical" + ], + [ + 49, + 75, + "H3K9me3-binding affinities", + "evidence" + ], + [ + 84, + 91, + "mutants", + "protein_state" + ], + [ + 99, + 110, + "presence of", + "protein_state" + ], + [ + 111, + 114, + "DTT", + "chemical" + ], + [ + 131, + 141, + "absence of", + "protein_state" + ], + [ + 142, + 145, + "DTT", + "chemical" + ], + [ + 167, + 182, + "disulphide bond", + "ptm" + ], + [ + 201, + 211, + "absence of", + "protein_state" + ], + [ + 212, + 215, + "DTT", + "chemical" + ], + [ + 227, + 233, + "hm-DNA", + "chemical" + ], + [ + 245, + 255, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 272, + 274, + "H3", + "protein_type" + ], + [ + 274, + 279, + "K9me3", + "ptm" + ] + ] + }, + { + "sid": 118, + "sent": "As negative controls, H3K9me3 recognition by UHRF1D334A or UHRF1D334A&\u0394627\u2013674 is not affected by DTT.", + "section": "RESULTS", + "ner": [ + [ + 22, + 24, + "H3", + "protein_type" + ], + [ + 24, + 29, + "K9me3", + "ptm" + ], + [ + 45, + 55, + "UHRF1D334A", + "mutant" + ], + [ + 59, + 69, + "UHRF1D334A", + "mutant" + ], + [ + 70, + 78, + "\u0394627\u2013674", + "mutant" + ], + [ + 98, + 101, + "DTT", + "chemical" + ] + ] + }, + { + "sid": 119, + "sent": "The above results collectively demonstrate that (i) full-length UHRF1 adopts a closed form, in which the Spacer binds to the TTD and H3K9me3 recognition is inhibited; (ii) hm-DNA displaces the Spacer from the TTD in the context of full-length UHRF1 and therefore largely enhances its histone H3K9me3-binding activity in a manner independent on the PHD (SRA is required).", + "section": "RESULTS", + "ner": [ + [ + 52, + 63, + "full-length", + "protein_state" + ], + [ + 64, + 69, + "UHRF1", + "protein" + ], + [ + 79, + 85, + "closed", + "protein_state" + ], + [ + 105, + 111, + "Spacer", + "structure_element" + ], + [ + 112, + 120, + "binds to", + "protein_state" + ], + [ + 125, + 128, + "TTD", + "structure_element" + ], + [ + 133, + 135, + "H3", + "protein_type" + ], + [ + 135, + 140, + "K9me3", + "ptm" + ], + [ + 172, + 178, + "hm-DNA", + "chemical" + ], + [ + 193, + 199, + "Spacer", + "structure_element" + ], + [ + 209, + 212, + "TTD", + "structure_element" + ], + [ + 231, + 242, + "full-length", + "protein_state" + ], + [ + 243, + 248, + "UHRF1", + "protein" + ], + [ + 284, + 291, + "histone", + "protein_type" + ], + [ + 292, + 294, + "H3", + "protein_type" + ], + [ + 294, + 299, + "K9me3", + "ptm" + ], + [ + 348, + 351, + "PHD", + "structure_element" + ], + [ + 353, + 356, + "SRA", + "structure_element" + ] + ] + }, + { + "sid": 120, + "sent": "We have previously demonstrated that hm-DNA also disrupts PHD\u2013SRA interaction and facilitates H3K9me0-binding affinity of the PHD in a manner independent on the TTD or the Spacer.", + "section": "RESULTS", + "ner": [ + [ + 37, + 43, + "hm-DNA", + "chemical" + ], + [ + 58, + 65, + "PHD\u2013SRA", + "structure_element" + ], + [ + 94, + 118, + "H3K9me0-binding affinity", + "evidence" + ], + [ + 126, + 129, + "PHD", + "structure_element" + ], + [ + 161, + 164, + "TTD", + "structure_element" + ], + [ + 172, + 178, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 121, + "sent": "Taken together, hm-DNA disrupts the intramolecular interactions within UHRF1, and therefore facilitates the coordinate recognition of H3K9me3 by TTD\u2013PHD.", + "section": "RESULTS", + "ner": [ + [ + 16, + 22, + "hm-DNA", + "chemical" + ], + [ + 71, + 76, + "UHRF1", + "protein" + ], + [ + 134, + 136, + "H3", + "protein_type" + ], + [ + 136, + 141, + "K9me3", + "ptm" + ], + [ + 145, + 152, + "TTD\u2013PHD", + "structure_element" + ] + ] + }, + { + "sid": 122, + "sent": "The spacer enhances hm-DNA-binding affinity of the SRA", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "spacer", + "structure_element" + ], + [ + 20, + 43, + "hm-DNA-binding affinity", + "evidence" + ], + [ + 51, + 54, + "SRA", + "structure_element" + ] + ] + }, + { + "sid": 123, + "sent": "To investigate how hm-DNA impairs TTD\u2013Spacer interaction, we tested whether the Spacer is involved in hm-DNA recognition by the SRA, which is the only known domain for hm-DNA recognition within UHRF1.", + "section": "RESULTS", + "ner": [ + [ + 19, + 25, + "hm-DNA", + "chemical" + ], + [ + 34, + 44, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 80, + 86, + "Spacer", + "structure_element" + ], + [ + 102, + 108, + "hm-DNA", + "chemical" + ], + [ + 128, + 131, + "SRA", + "structure_element" + ], + [ + 168, + 174, + "hm-DNA", + "chemical" + ], + [ + 194, + 199, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 124, + "sent": "In the electrophoretic mobility-shift assay, SRA\u2013Spacer showed higher hm-DNA-binding affinity than the SRA alone (Supplementary Fig. 6a).", + "section": "RESULTS", + "ner": [ + [ + 7, + 43, + "electrophoretic mobility-shift assay", + "experimental_method" + ], + [ + 45, + 55, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 70, + 93, + "hm-DNA-binding affinity", + "evidence" + ], + [ + 103, + 106, + "SRA", + "structure_element" + ], + [ + 107, + 112, + "alone", + "protein_state" + ] + ] + }, + { + "sid": 125, + "sent": "ITC measurements show that SRA\u2013Spacer bound to hm-DNA with a much higher binding affinity (KD=1.75\u2009\u03bcM) than the SRA (KD=25.12\u2009\u03bcM), whereas the Spacer alone showed no interaction with hm-DNA (Fig. 5a).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "ITC", + "experimental_method" + ], + [ + 27, + 37, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 38, + 46, + "bound to", + "protein_state" + ], + [ + 47, + 53, + "hm-DNA", + "chemical" + ], + [ + 73, + 89, + "binding affinity", + "evidence" + ], + [ + 91, + 93, + "KD", + "evidence" + ], + [ + 112, + 115, + "SRA", + "structure_element" + ], + [ + 117, + 119, + "KD", + "evidence" + ], + [ + 143, + 149, + "Spacer", + "structure_element" + ], + [ + 150, + 155, + "alone", + "protein_state" + ], + [ + 183, + 189, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 126, + "sent": "In the fluorescence polarization (FP) measurements, SRA\u2013Spacer, full-length UHRF1 and UHRF1\u0394TTD showed comparable hm-DNA-binding affinities (Fig. 5b and Supplementary Table 4), suggesting that UHRF1 binds to hm-DNA no matter UHRF1 adopts a closed form or not.", + "section": "RESULTS", + "ner": [ + [ + 7, + 36, + "fluorescence polarization (FP", + "experimental_method" + ], + [ + 52, + 62, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 64, + 75, + "full-length", + "protein_state" + ], + [ + 76, + 81, + "UHRF1", + "protein" + ], + [ + 86, + 95, + "UHRF1\u0394TTD", + "mutant" + ], + [ + 114, + 139, + "hm-DNA-binding affinities", + "evidence" + ], + [ + 193, + 198, + "UHRF1", + "protein" + ], + [ + 199, + 207, + "binds to", + "protein_state" + ], + [ + 208, + 214, + "hm-DNA", + "chemical" + ], + [ + 225, + 230, + "UHRF1", + "protein" + ], + [ + 240, + 246, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 127, + "sent": "In contrast, UHRF1\u0394SRA abolished hm-DNA-binding affinity, indicating that the SRA is essential for hm-DNA recognition.", + "section": "RESULTS", + "ner": [ + [ + 13, + 22, + "UHRF1\u0394SRA", + "mutant" + ], + [ + 33, + 56, + "hm-DNA-binding affinity", + "evidence" + ], + [ + 78, + 81, + "SRA", + "structure_element" + ], + [ + 99, + 105, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 128, + "sent": "Compared with full-length UHRF1, UHRF1\u0394627\u2013674 decreased the hm-DNA-binding affinity by a factor of 14 (Fig. 5b), further supporting that the Spacer plays an important role in hm-DNA recognition in the context of full-length UHRF1.", + "section": "RESULTS", + "ner": [ + [ + 14, + 25, + "full-length", + "protein_state" + ], + [ + 26, + 31, + "UHRF1", + "protein" + ], + [ + 33, + 46, + "UHRF1\u0394627\u2013674", + "mutant" + ], + [ + 61, + 84, + "hm-DNA-binding affinity", + "evidence" + ], + [ + 142, + 148, + "Spacer", + "structure_element" + ], + [ + 176, + 182, + "hm-DNA", + "chemical" + ], + [ + 213, + 224, + "full-length", + "protein_state" + ], + [ + 225, + 230, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 129, + "sent": "In addition, hm-DNA-binding affinities of SRA or SRA\u2013Spacer did not obviously vary upon the change of DNA lengths but did decrease with the increasing salt concentrations (Supplementary Fig. 6b,c and Supplementary Table 5).", + "section": "RESULTS", + "ner": [ + [ + 13, + 38, + "hm-DNA-binding affinities", + "evidence" + ], + [ + 42, + 45, + "SRA", + "structure_element" + ], + [ + 49, + 59, + "SRA\u2013Spacer", + "structure_element" + ] + ] + }, + { + "sid": 130, + "sent": "These results indicate that the Spacer not only binds to the TTD and inhibits H3K9me3 recognition when UHRF1 adopts closed conformation, but also facilitates hm-DNA recognition by the SRA when UHRF1 binds to hm-DNA.", + "section": "RESULTS", + "ner": [ + [ + 32, + 38, + "Spacer", + "structure_element" + ], + [ + 48, + 56, + "binds to", + "protein_state" + ], + [ + 61, + 64, + "TTD", + "structure_element" + ], + [ + 78, + 80, + "H3", + "protein_type" + ], + [ + 80, + 85, + "K9me3", + "ptm" + ], + [ + 103, + 108, + "UHRF1", + "protein" + ], + [ + 116, + 122, + "closed", + "protein_state" + ], + [ + 158, + 164, + "hm-DNA", + "chemical" + ], + [ + 184, + 187, + "SRA", + "structure_element" + ], + [ + 193, + 198, + "UHRF1", + "protein" + ], + [ + 199, + 207, + "binds to", + "protein_state" + ], + [ + 208, + 214, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 131, + "sent": "We next mapped the minimal region of the Spacer for the enhancement of hm-DNA-binding affinity.", + "section": "RESULTS", + "ner": [ + [ + 41, + 47, + "Spacer", + "structure_element" + ], + [ + 71, + 94, + "hm-DNA-binding affinity", + "evidence" + ] + ] + }, + { + "sid": 132, + "sent": "SRA\u2013Spacer-661 (residues 414\u2013661) still maintained strong hm-DNA-binding affinity comparable to that of SRA\u2013Spacer (residues 414\u2013674), whereas SRA\u2013Spacer-652 and SRA\u2013Spacer-642 markedly decreased their hm-DNA-binding affinities (Fig. 5c), indicating that residues 642\u2013661 are important for enhancing hm-DNA-binding affinity of the SRA.", + "section": "RESULTS", + "ner": [ + [ + 0, + 14, + "SRA\u2013Spacer-661", + "mutant" + ], + [ + 25, + 32, + "414\u2013661", + "residue_range" + ], + [ + 58, + 81, + "hm-DNA-binding affinity", + "evidence" + ], + [ + 104, + 114, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 125, + 132, + "414\u2013674", + "residue_range" + ], + [ + 143, + 157, + "SRA\u2013Spacer-652", + "mutant" + ], + [ + 162, + 176, + "SRA\u2013Spacer-642", + "mutant" + ], + [ + 202, + 227, + "hm-DNA-binding affinities", + "evidence" + ], + [ + 264, + 271, + "642\u2013661", + "residue_range" + ], + [ + 300, + 323, + "hm-DNA-binding affinity", + "evidence" + ], + [ + 331, + 334, + "SRA", + "structure_element" + ] + ] + }, + { + "sid": 133, + "sent": "This minimal region largely overlaps with the Spacer region (643\u2013655) essential for TTD interaction.", + "section": "RESULTS", + "ner": [ + [ + 46, + 52, + "Spacer", + "structure_element" + ], + [ + 61, + 68, + "643\u2013655", + "residue_range" + ], + [ + 84, + 87, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 134, + "sent": "We also determined the crystal structure of SRA\u2013Spacer bound to hm-DNA at 3.15\u2009\u00c5 resolution (Supplementary Table 6 and Supplementary Fig. 7a).", + "section": "RESULTS", + "ner": [ + [ + 23, + 40, + "crystal structure", + "evidence" + ], + [ + 44, + 54, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 55, + 63, + "bound to", + "protein_state" + ], + [ + 64, + 70, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 135, + "sent": "The structure shows that the SRA binds to hm-DNA in a manner similar to that observed in the previously reported SRA-hm-DNA structures.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 29, + 32, + "SRA", + "structure_element" + ], + [ + 33, + 41, + "binds to", + "protein_state" + ], + [ + 42, + 48, + "hm-DNA", + "chemical" + ], + [ + 113, + 123, + "SRA-hm-DNA", + "complex_assembly" + ], + [ + 124, + 134, + "structures", + "evidence" + ] + ] + }, + { + "sid": 136, + "sent": "Intriguingly, no electron density was observed for the Spacer.", + "section": "RESULTS", + "ner": [ + [ + 17, + 33, + "electron density", + "evidence" + ], + [ + 55, + 61, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 137, + "sent": "A possible explanation is that the Spacer facilitates SRA\u2013hm-DNA interaction through nonspecific salt bridge contacts because DNA is rich in acidic groups and the Spacer is rich in basic residues (Supplementary Fig. 7b).", + "section": "RESULTS", + "ner": [ + [ + 35, + 41, + "Spacer", + "structure_element" + ], + [ + 54, + 64, + "SRA\u2013hm-DNA", + "complex_assembly" + ], + [ + 97, + 108, + "salt bridge", + "bond_interaction" + ], + [ + 126, + 129, + "DNA", + "chemical" + ], + [ + 163, + 169, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 138, + "sent": "The nonspecific interaction is consistent with the previous observation that UHRF1 has no DNA sequence selectivity besides hm-CpG dinucleotide.", + "section": "RESULTS", + "ner": [ + [ + 77, + 82, + "UHRF1", + "protein" + ], + [ + 90, + 93, + "DNA", + "chemical" + ], + [ + 123, + 142, + "hm-CpG dinucleotide", + "chemical" + ] + ] + }, + { + "sid": 139, + "sent": "The spacer is important for PCH localization of UHRF1", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "spacer", + "structure_element" + ], + [ + 48, + 53, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 140, + "sent": "To investigate the role of the Spacer in the regulation of UHRF1 function, we transiently overexpressed GFP-tagged wild type or mutants of UHRF1 in NIH3T3 cells to determine their subcellular localization.", + "section": "RESULTS", + "ner": [ + [ + 31, + 37, + "Spacer", + "structure_element" + ], + [ + 59, + 64, + "UHRF1", + "protein" + ], + [ + 78, + 103, + "transiently overexpressed", + "experimental_method" + ], + [ + 104, + 114, + "GFP-tagged", + "protein_state" + ], + [ + 115, + 124, + "wild type", + "protein_state" + ], + [ + 128, + 135, + "mutants", + "protein_state" + ], + [ + 139, + 144, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 141, + "sent": "For the NIH3T3 cells expressing wild-type UHRF1, most cells (\u223c74.6%) showed a focal pattern of protein that is co-localized with 4,6-diamidino-2-phenylindole (DAPI) foci (Fig. 5d), whereas the rest cells showed a diffuse nuclear staining pattern.", + "section": "RESULTS", + "ner": [ + [ + 32, + 41, + "wild-type", + "protein_state" + ], + [ + 42, + 47, + "UHRF1", + "protein" + ], + [ + 129, + 157, + "4,6-diamidino-2-phenylindole", + "chemical" + ], + [ + 159, + 163, + "DAPI", + "chemical" + ] + ] + }, + { + "sid": 142, + "sent": "The result is consistent with the previous studies that UHRF1 is mainly localized to highly methylated pericentromeric heterochromatin (PCH).", + "section": "RESULTS", + "ner": [ + [ + 56, + 61, + "UHRF1", + "protein" + ], + [ + 85, + 102, + "highly methylated", + "protein_state" + ] + ] + }, + { + "sid": 143, + "sent": "In contrast, for the cells expressing UHRF1\u0394627\u2013674, a spacer deletion mutant with decreased hm-DNA-binding affinity (Fig. 5b), only \u223c22.1% cells showed co-localization with DAPI.", + "section": "RESULTS", + "ner": [ + [ + 38, + 51, + "UHRF1\u0394627\u2013674", + "mutant" + ], + [ + 55, + 77, + "spacer deletion mutant", + "protein_state" + ], + [ + 93, + 116, + "hm-DNA-binding affinity", + "evidence" + ], + [ + 174, + 178, + "DAPI", + "chemical" + ] + ] + }, + { + "sid": 144, + "sent": "Previous reports have shown that the H3K9me3 recognition of UHRF1 also plays an important role in its heterochromatin localization.", + "section": "RESULTS", + "ner": [ + [ + 37, + 39, + "H3", + "protein_type" + ], + [ + 39, + 44, + "K9me3", + "ptm" + ], + [ + 60, + 65, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 145, + "sent": "For example, UHRF1 mutant (within TTD domain) lacking H3K9me3-binding affinity largely reduces its co-localization with heterochromatin.", + "section": "RESULTS", + "ner": [ + [ + 13, + 18, + "UHRF1", + "protein" + ], + [ + 19, + 25, + "mutant", + "protein_state" + ], + [ + 34, + 37, + "TTD", + "structure_element" + ], + [ + 46, + 53, + "lacking", + "protein_state" + ], + [ + 54, + 78, + "H3K9me3-binding affinity", + "evidence" + ] + ] + }, + { + "sid": 146, + "sent": "Because manipulation of endogenous hm-DNA in cells is technically challenging, we used UHRF1\u0394SRA (lacks hm-DNA-binding affinity but maintains closed conformation, Figs 3h and 5b) to test whether closed conformation of UHRF1 exists in vivo.", + "section": "RESULTS", + "ner": [ + [ + 35, + 41, + "hm-DNA", + "chemical" + ], + [ + 87, + 96, + "UHRF1\u0394SRA", + "mutant" + ], + [ + 98, + 103, + "lacks", + "protein_state" + ], + [ + 104, + 127, + "hm-DNA-binding affinity", + "evidence" + ], + [ + 142, + 148, + "closed", + "protein_state" + ], + [ + 195, + 201, + "closed", + "protein_state" + ], + [ + 218, + 223, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 147, + "sent": "In NIH3T3 cells, UHRF1\u0394SRA largely decreased chromatin association (Fig. 5d).", + "section": "RESULTS", + "ner": [ + [ + 17, + 26, + "UHRF1\u0394SRA", + "mutant" + ] + ] + }, + { + "sid": 148, + "sent": "Only \u223c4.8% cells expressing UHRF1\u0394SRA showed an intermediate enrichment, but not characteristic focal pattern, at DAPI foci, whereas the majority of the cells showed a diffuse nuclear staining pattern.", + "section": "RESULTS", + "ner": [ + [ + 28, + 37, + "UHRF1\u0394SRA", + "mutant" + ], + [ + 114, + 118, + "DAPI", + "chemical" + ] + ] + }, + { + "sid": 149, + "sent": "The results suggest that UHRF1\u0394SRA adopts closed conformation so that H3K9me3 recognition by TTD\u2013PHD is blocked by the intramolecular interaction, and support the regulatory role of the Spacer in PCH localization of UHRF1 in vivo.", + "section": "RESULTS", + "ner": [ + [ + 25, + 34, + "UHRF1\u0394SRA", + "mutant" + ], + [ + 42, + 48, + "closed", + "protein_state" + ], + [ + 70, + 72, + "H3", + "protein_type" + ], + [ + 72, + 77, + "K9me3", + "ptm" + ], + [ + 93, + 100, + "TTD\u2013PHD", + "structure_element" + ], + [ + 186, + 192, + "Spacer", + "structure_element" + ], + [ + 216, + 221, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 150, + "sent": "The spacer facilitates UHRF1\u2013DNMT1 interaction", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "spacer", + "structure_element" + ], + [ + 23, + 34, + "UHRF1\u2013DNMT1", + "complex_assembly" + ] + ] + }, + { + "sid": 151, + "sent": "Previous studies show that UHRF1 recruits DNMT1 to hm-DNA for maintenance DNA methylation through the interaction between the SRA and RFTSDNMT1 (refs).", + "section": "RESULTS", + "ner": [ + [ + 27, + 32, + "UHRF1", + "protein" + ], + [ + 42, + 47, + "DNMT1", + "protein" + ], + [ + 51, + 57, + "hm-DNA", + "chemical" + ], + [ + 74, + 77, + "DNA", + "chemical" + ], + [ + 78, + 89, + "methylation", + "ptm" + ], + [ + 126, + 129, + "SRA", + "structure_element" + ], + [ + 134, + 143, + "RFTSDNMT1", + "protein" + ] + ] + }, + { + "sid": 152, + "sent": "We confirmed the direct interaction between RFTSDNMT1 and the SRA in a solution with low salt concentration (50\u2009mM NaCl), but observed weak or undetectable interaction in a solution with higher salt concentrations (100 or 150\u2009mM NaCl) (Supplementary Fig. 8a).", + "section": "RESULTS", + "ner": [ + [ + 44, + 53, + "RFTSDNMT1", + "protein" + ], + [ + 62, + 65, + "SRA", + "structure_element" + ], + [ + 115, + 119, + "NaCl", + "chemical" + ], + [ + 229, + 233, + "NaCl", + "chemical" + ] + ] + }, + { + "sid": 153, + "sent": "Compared with the SRA, SRA\u2013Spacer exhibited stronger interaction with RFTSDNMT1.", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "SRA", + "structure_element" + ], + [ + 23, + 33, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 70, + 79, + "RFTSDNMT1", + "protein" + ] + ] + }, + { + "sid": 154, + "sent": "In addition, RFTSDNMT1 bound to SRA\u2013Spacer with a binding affinity of 7.09\u2009\u03bcM, but showed no detectable interaction with the SRA (Supplementary Fig. 8b).", + "section": "RESULTS", + "ner": [ + [ + 13, + 22, + "RFTSDNMT1", + "protein" + ], + [ + 23, + 31, + "bound to", + "protein_state" + ], + [ + 32, + 42, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 50, + 66, + "binding affinity", + "evidence" + ], + [ + 125, + 128, + "SRA", + "structure_element" + ] + ] + }, + { + "sid": 155, + "sent": "Interestingly, the addition of hm-DNA abolished the interaction between RFTSDNMT1 and SRA\u2013Spacer, suggesting that hm-DNA also regulates UHRF1\u2013DNMT1 interaction (Supplementary Fig. 8c).", + "section": "RESULTS", + "ner": [ + [ + 31, + 37, + "hm-DNA", + "chemical" + ], + [ + 72, + 81, + "RFTSDNMT1", + "protein" + ], + [ + 86, + 96, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 114, + 120, + "hm-DNA", + "chemical" + ], + [ + 136, + 147, + "UHRF1\u2013DNMT1", + "complex_assembly" + ] + ] + }, + { + "sid": 156, + "sent": "These results indicate that the Spacer facilitates the interaction between RFTSDNMT1 and the SRA, and the interaction is impaired by the presence of hm-DNA.", + "section": "RESULTS", + "ner": [ + [ + 32, + 38, + "Spacer", + "structure_element" + ], + [ + 75, + 84, + "RFTSDNMT1", + "protein" + ], + [ + 93, + 96, + "SRA", + "structure_element" + ], + [ + 137, + 148, + "presence of", + "protein_state" + ], + [ + 149, + 155, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 157, + "sent": "We next tested whether the UHRF1\u2013DNMT1 interaction is regulated by the conformational change of UHRF1.", + "section": "RESULTS", + "ner": [ + [ + 27, + 38, + "UHRF1\u2013DNMT1", + "complex_assembly" + ], + [ + 96, + 101, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 158, + "sent": "Because the addition of hm-DNA disrupts the interaction between the SRA\u2013Spacer and RFTSDNMT1, we used various truncations to mimic open and closed forms of UHRF1.", + "section": "RESULTS", + "ner": [ + [ + 24, + 30, + "hm-DNA", + "chemical" + ], + [ + 68, + 78, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 83, + 92, + "RFTSDNMT1", + "protein" + ], + [ + 110, + 121, + "truncations", + "experimental_method" + ], + [ + 131, + 135, + "open", + "protein_state" + ], + [ + 140, + 146, + "closed", + "protein_state" + ], + [ + 156, + 161, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 159, + "sent": "In the absence of hm-DNA, only UHRF1\u0394TTD bound to RFTSDNMT1, whereas full-length UHRF1, UHRF1\u0394SRA and UHRF1\u0394627\u2013674 showed undetectable interaction (Fig. 5e).", + "section": "RESULTS", + "ner": [ + [ + 7, + 17, + "absence of", + "protein_state" + ], + [ + 18, + 24, + "hm-DNA", + "chemical" + ], + [ + 31, + 40, + "UHRF1\u0394TTD", + "mutant" + ], + [ + 41, + 49, + "bound to", + "protein_state" + ], + [ + 50, + 59, + "RFTSDNMT1", + "protein" + ], + [ + 69, + 80, + "full-length", + "protein_state" + ], + [ + 81, + 86, + "UHRF1", + "protein" + ], + [ + 88, + 97, + "UHRF1\u0394SRA", + "mutant" + ], + [ + 102, + 115, + "UHRF1\u0394627\u2013674", + "mutant" + ] + ] + }, + { + "sid": 160, + "sent": "As the deletion of the TTD allows UHRF1 to adopt an open conformation, the results suggest that RFTSDNMT1 binds to SRA\u2013Spacer when UHRF1 adopts an open conformation in the absence of hm-DNA.", + "section": "RESULTS", + "ner": [ + [ + 7, + 18, + "deletion of", + "experimental_method" + ], + [ + 23, + 26, + "TTD", + "structure_element" + ], + [ + 34, + 39, + "UHRF1", + "protein" + ], + [ + 52, + 56, + "open", + "protein_state" + ], + [ + 96, + 105, + "RFTSDNMT1", + "protein" + ], + [ + 106, + 114, + "binds to", + "protein_state" + ], + [ + 115, + 125, + "SRA\u2013Spacer", + "structure_element" + ], + [ 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UHRF1 conformation (Fig. 5f).", + "section": "DISCUSS", + "ner": [ + [ + 69, + 75, + "hm-DNA", + "chemical" + ], + [ + 99, + 104, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 163, + "sent": "In the absence of hm-DNA (A), UHRF1 prefers a closed conformation, in which the Spacer binds to the TTD by competing with the Linker and the SRA binds to the PHD.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 17, + "absence of", + "protein_state" + ], + [ + 18, + 24, + "hm-DNA", + "chemical" + ], + [ + 30, + 35, + "UHRF1", + "protein" + ], + [ + 46, + 52, + "closed", + "protein_state" + ], + [ + 80, + 86, + "Spacer", + "structure_element" + ], + [ + 87, + 95, + "binds to", + "protein_state" + ], + [ + 100, + 103, + "TTD", + "structure_element" + ], + [ + 126, + 132, + "Linker", + "structure_element" + ], + [ + 141, + 144, + "SRA", + "structure_element" + ], + [ + 145, + 153, + "binds to", + "protein_state" + ], + [ + 158, + 161, + "PHD", + "structure_element" + ] + ] + }, + { + "sid": 164, + "sent": "As a result, the recognition of histone H3K9me3 by the TTD is blocked by the Spacer, and recognition of unmodified histone H3 (H3R2) by the PHD is inhibited by the SRA.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 39, + "histone", + "protein_type" + ], + [ + 40, + 42, + "H3", + "protein_type" + ], + [ + 42, + 47, + "K9me3", + "ptm" + ], + [ + 55, + 58, + "TTD", + "structure_element" + ], + [ + 77, + 83, + "Spacer", + "structure_element" + ], + [ + 104, + 114, + "unmodified", + "protein_state" + ], + [ + 115, + 122, + "histone", + "protein_type" + ], + [ + 123, + 125, + "H3", + "protein_type" + ], + [ + 127, + 131, + "H3R2", + "site" + ], + [ + 140, + 143, + "PHD", + "structure_element" + ], + [ + 164, + 167, + "SRA", + "structure_element" + ] + ] + }, + { + "sid": 165, + "sent": "The interaction between UHRF1 and DNMT1 is also weak because the Spacer is unable to facilitate the intermolecular interaction.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 29, + "UHRF1", + "protein" + ], + [ + 34, + 39, + "DNMT1", + "protein" + ], + [ + 65, + 71, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 166, + "sent": "In the presence of hm-DNA (B), UHRF1 prefers an open conformation, in which the SRA binds to the hm-DNA; the Spacer dissociates from the TTD and facilitates the interaction between the SRA and hm-DNA; the Linker binds to the TTD and allows TTD\u2013PHD to recognize histone H3K9me3.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 18, + "presence of", + "protein_state" + ], + [ + 19, + 25, + "hm-DNA", + "chemical" + ], + [ + 31, + 36, + "UHRF1", + "protein" + ], + [ + 48, + 52, + "open", + "protein_state" + ], + [ + 80, + 83, + "SRA", + "structure_element" + ], + [ + 84, + 92, + "binds to", + "protein_state" + ], + [ + 97, + 103, + "hm-DNA", + "chemical" + ], + [ + 109, + 115, + "Spacer", + "structure_element" + ], + [ + 137, + 140, + "TTD", + "structure_element" + ], + [ + 185, + 188, + "SRA", + "structure_element" + ], + [ + 193, + 199, 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with the histone ubiquitylation and/or SRA-Spacer.", + "section": "DISCUSS", + "ner": [ + [ + 58, + 63, + "DNMT1", + "protein" + ], + [ + 72, + 78, + "hm-DNA", + "chemical" + ], + [ + 99, + 110, + "methylation", + "ptm" + ], + [ + 150, + 157, + "histone", + "protein_type" + ], + [ + 158, + 172, + "ubiquitylation", + "ptm" + ], + [ + 180, + 190, + "SRA-Spacer", + "structure_element" + ] + ] + }, + { + "sid": 170, + "sent": "The P(r) function obtained from small-angle X-ray scattering (SAXS) measurements of TTD\u2013PHD\u2013SRA\u2013Spacer\u2013hm-DNA complex showed a broader distribution than that of the TTD\u2013PHD\u2013SRA\u2013Spacer alone, supporting the proposed model that UHRF1 adopts an open conformation in the presence of hm-DNA (Supplementary Fig. 8e).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 17, + "P(r) function", + "evidence" + ], + [ + 32, + 60, + "small-angle X-ray scattering", + "experimental_method" + ], + [ + 62, + 66, + "SAXS", + "experimental_method" + ], + [ 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and decreases UHRF1 stability in the M phase of the cell cycle.", + "section": "DISCUSS", + "ner": [ + [ + 31, + 46, + "phosphorylation", + "ptm" + ], + [ + 50, + 54, + "S639", + "residue_name_number" + ], + [ + 66, + 72, + "Spacer", + "structure_element" + ], + [ + 102, + 107, + "UHRF1", + "protein" + ], + [ + 112, + 126, + "deubiquitylase", + "protein_type" + ], + [ + 127, + 131, + "USP7", + "protein" + ], + [ + 146, + 151, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 175, + "sent": "The Spacer was predicted to contain two nuclear localization signals, residues 581\u2013600 and 648-670 (ref.).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 10, + "Spacer", + "structure_element" + ], + [ + 40, + 68, + "nuclear localization signals", + "structure_element" + ], + [ + 79, + 86, + "581\u2013600", + "residue_range" + ], + [ + 91, + 98, + "648-670", + "residue_range" + ] + ] + }, + { + "sid": 176, + "sent": "In this report, we found that the Spacer (i) binds to the TTD in the closed form of UHRF1 and inhibits its interaction with H3K9me3; (ii) facilitates hm-DNA recognition by the SRA and (iii) facilitates the interaction between the SRA and RFTSDNMT1.", + "section": "DISCUSS", + "ner": [ + [ + 34, + 40, + "Spacer", + "structure_element" + ], + [ + 45, + 53, + "binds to", + "protein_state" + ], + [ + 58, + 61, + "TTD", + "structure_element" + ], + [ + 69, + 75, + "closed", + "protein_state" + ], + [ + 84, + 89, + "UHRF1", + "protein" + ], + [ + 124, + 126, + "H3", + "protein_type" + ], + [ + 126, + 131, + "K9me3", + "ptm" + ], + [ + 150, + 156, + "hm-DNA", + "chemical" + ], + [ + 176, + 179, + "SRA", + "structure_element" + ], + [ + 230, + 233, + "SRA", + "structure_element" + ], + [ + 238, + 247, + "RFTSDNMT1", + "protein" + ] + ] + }, + { + "sid": 177, + "sent": "These findings together indicate that the Spacer plays a very important role in the dynamic regulation of UHRF1.", + "section": "DISCUSS", + "ner": [ + [ + 42, + 48, + "Spacer", + "structure_element" + ], + [ + 106, + 111, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 178, + "sent": "When our manuscript was in preparation, Gelato et al. reported that binding of PI5P to the Spacer opens the closed conformation of UHRF1 and increases H3K9me3-binding affinity of the TTD.", + "section": "DISCUSS", + "ner": [ + [ + 79, + 83, + "PI5P", + "chemical" + ], + [ + 91, + 97, + "Spacer", + "structure_element" + ], + [ + 108, + 114, + "closed", + "protein_state" + ], + [ + 131, + 136, + "UHRF1", + "protein" + ], + [ + 151, + 175, + "H3K9me3-binding affinity", + "evidence" + ], + [ + 183, + 186, + "TTD", + "structure_element" + ] + ] + }, + { + "sid": 179, + "sent": "The result suggests that PI5P may facilitate the conformational change of UHRF1 induced by hm-DNA when UHRF1 is recruited to chromatin.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 29, + "PI5P", + "chemical" + ], + [ + 74, + 79, + "UHRF1", + "protein" + ], + [ + 91, + 97, + "hm-DNA", + "chemical" + ], + [ + 103, + 108, + 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"protein" + ], + [ + 79, + 85, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 182, + "sent": "Our study demonstrates that the Spacer markedly enhances the hm-DNA-binding affinity of the SRA and the deletion of the Spacer impairs heterochromatin localization of UHRF1, indicating that the Spacer is essential for recognition of hm-DNA in the context of full-length UHRF1.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 38, + "Spacer", + "structure_element" + ], + [ + 61, + 84, + "hm-DNA-binding affinity", + "evidence" + ], + [ + 92, + 95, + "SRA", + "structure_element" + ], + [ + 104, + 115, + "deletion of", + "experimental_method" + ], + [ + 120, + 126, + "Spacer", + "structure_element" + ], + [ + 167, + 172, + "UHRF1", + "protein" + ], + [ + 194, + 200, + "Spacer", + "structure_element" + ], + [ + 233, + 239, + "hm-DNA", + "chemical" + ], + [ + 258, + 269, + "full-length", + "protein_state" + ], + [ + 270, + 275, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 183, + "sent": "Interestingly, variant in methylation 1 (VIM1, a UHRF1 homologue in Arabidopsis) contains an equivalent spacer region, which was shown to be required for hm-DNA recognition by its SRA domain, suggesting a conserved regulatory mechanism in SRA domain-containing proteins.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 39, + "variant in methylation 1", + "protein" + ], + [ + 41, + 45, + "VIM1", + "protein" + ], + [ + 49, + 54, + "UHRF1", + "protein" + ], + [ + 68, + 79, + "Arabidopsis", + "taxonomy_domain" + ], + [ + 104, + 110, + "spacer", + "structure_element" + ], + [ + 154, + 160, + "hm-DNA", + "chemical" + ], + [ + 180, + 183, + "SRA", + "structure_element" + ], + [ + 239, + 242, + "SRA", + "structure_element" + ] + ] + }, + { + "sid": 184, + "sent": "Intriguingly, UHRF2 (the only mammalian homologue of UHRF1) and UHRF1 show very high sequence similarities for all the domains but very low similarity for the Spacer (Supplementary Fig. 7c).", + "section": "DISCUSS", + "ner": [ + [ + 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"DISCUSS", + "ner": [ + [ + 53, + 58, + "UHRF1", + "protein" + ], + [ + 75, + 81, + "hm-DNA", + "chemical" + ], + [ + 119, + 126, + "histone", + "protein_type" + ], + [ + 127, + 129, + "H3", + "protein_type" + ], + [ + 129, + 134, + "K9me3", + "ptm" + ], + [ + 151, + 157, + "hm-DNA", + "chemical" + ], + [ + 192, + 197, + "UHRF1", + "protein" + ], + [ + 219, + 222, + "DNA", + "chemical" + ], + [ + 223, + 234, + "methylation", + "ptm" + ] + ] + }, + { + "sid": 189, + "sent": "However, little is known about the crosstalk between these two epigenetic marks within UHRF1.", + "section": "DISCUSS", + "ner": [ + [ + 87, + 92, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 190, + "sent": "As shown in the proposed model, recognition of H3K9me3 by full-length UHRF1 is blocked to avoid its miss-localization to unmethylated genomic region, in which chromatin contains H3K9me3 (KD=4.61\u2009\u03bcM) or H3K9me0 (KD=25.99\u2009\u03bcM).", + "section": "DISCUSS", + "ner": [ + [ + 47, + 49, + "H3", + 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"structure_element" + ], + [ + 46, + 52, + "hm-DNA", + "chemical" + ], + [ + 57, + 65, + "binds to", + "protein_state" + ], + [ + 66, + 71, + "DNMT1", + "protein" + ], + [ + 108, + 113, + "UHRF1", + "protein" + ], + [ + 193, + 200, + "TTD\u2013PHD", + "structure_element" + ], + [ + 205, + 207, + "H3", + "protein_type" + ], + [ + 207, + 212, + "K9me3", + "ptm" + ], + [ + 217, + 220, + "PHD", + "structure_element" + ], + [ + 221, + 223, + "H3", + "protein_type" + ], + [ + 251, + 256, + "DNMT1", + "protein" + ], + [ + 274, + 277, + "DNA", + "chemical" + ], + [ + 292, + 303, + "methylation", + "ptm" + ] + ] + }, + { + "sid": 194, + "sent": "This explanation agrees nicely with previous observations and clarifies the importance of coordinate recognition of H3K9me3 and hm-DNA by UHRF1 for maintenance DNA methylation.", + "section": "DISCUSS", + "ner": [ + [ + 116, + 118, + "H3", + "protein_type" + ], + [ + 118, + 123, + "K9me3", + "ptm" + ], + [ + 128, + 134, + "hm-DNA", + "chemical" + ], + [ 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"sid": 197, + "sent": "Recent study indicates that histone tail association of UHRF1 (by the PHD domain) is required for histone H3 ubiquitylation, which is dependent on ubiquitin ligase activity of the RING domain of UHRF1 (ref.).", + "section": "DISCUSS", + "ner": [ + [ + 28, + 35, + "histone", + "protein_type" + ], + [ + 56, + 61, + "UHRF1", + "protein" + ], + [ + 70, + 73, + "PHD", + "structure_element" + ], + [ + 98, + 105, + "histone", + "protein_type" + ], + [ + 106, + 108, + "H3", + "protein_type" + ], + [ + 109, + 123, + "ubiquitylation", + "ptm" + ], + [ + 147, + 163, + "ubiquitin ligase", + "protein_type" + ], + [ + 180, + 184, + "RING", + "structure_element" + ], + [ + 195, + 200, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 198, + "sent": "DNMT1 binds to ubiquitylated histone H3 and ubiquitylation is required for maintenance of DNA methylation in vivo.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "DNMT1", + "protein" + ], + [ + 6, + 14, + "binds to", + "protein_state" + ], + [ + 15, + 28, + "ubiquitylated", + "protein_state" + ], + [ + 29, + 36, + "histone", + "protein_type" + ], + [ + 37, + 39, + "H3", + "protein_type" + ], + [ + 44, + 58, + "ubiquitylation", + "ptm" + ], + [ + 90, + 93, + "DNA", + "chemical" + ], + [ + 94, + 105, + "methylation", + "ptm" + ] + ] + }, + { + "sid": 199, + "sent": "In this study, we found that both TTD and PHD are regulated by hm-DNA to recognize histone tail.", + "section": "DISCUSS", + "ner": [ + [ + 34, + 37, + "TTD", + "structure_element" + ], + [ + 42, + 45, + "PHD", + "structure_element" + ], + [ + 63, + 69, + "hm-DNA", + "chemical" + ], + [ + 83, + 90, + "histone", + "protein_type" + ] + ] + }, + { + "sid": 200, + "sent": "Thus, the closed form UHRF1 may prevent miss localization of URHF1, whereas only the UHRF1 in open conformation (induced by hm-DNA) could properly binds to histone tail for ubiquitylation and subsequent DNA methylation.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 16, + "closed", + "protein_state" + ], + [ + 22, + 27, + "UHRF1", + "protein" + ], + [ + 61, + 66, + "URHF1", + "protein" + ], + [ + 85, + 90, + "UHRF1", + "protein" + ], + [ + 94, + 98, + "open", + "protein_state" + ], + [ + 124, + 130, + "hm-DNA", + "chemical" + ], + [ + 147, + 155, + "binds to", + "protein_state" + ], + [ + 156, + 163, + "histone", + "protein_type" + ], + [ + 173, + 187, + "ubiquitylation", + "ptm" + ], + [ + 203, + 206, + "DNA", + "chemical" + ], + [ + 207, + 218, + "methylation", + "ptm" + ] + ] + }, + { + "sid": 201, + "sent": "Moreover, structural analyses of DNMT1\u2013DNA and SRA\u2013DNA complexes also indicate that it is impossible for DNMT1 to methylate the hm-DNA that UHRF1 binds to because of steric hindrance.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 29, + "structural analyses", + "experimental_method" + ], + [ + 33, + 42, + "DNMT1\u2013DNA", + "complex_assembly" + ], + [ + 47, + 54, + "SRA\u2013DNA", + "complex_assembly" + ], + [ + 105, + 110, + "DNMT1", + "protein" + ], + [ + 128, + 134, + "hm-DNA", + "chemical" + ], + [ + 140, + 145, + "UHRF1", + "protein" + ], + [ + 146, + 154, + "binds to", + "protein_state" + ] + ] + }, + { + "sid": 202, + "sent": "In our in vitro assays, we could detect interaction between SRA\u2013Spacer and RFTSDNMT1, but not the interaction between full-length UHRF1 and RFTSDNMT1 (Supplementary Fig. 8a,b and Fig. 5e).", + "section": "DISCUSS", + "ner": [ + [ + 7, + 22, + "in vitro assays", + "experimental_method" + ], + [ + 60, + 70, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 75, + 84, + "RFTSDNMT1", + "protein" + ], + [ + 118, + 129, + "full-length", + "protein_state" + ], + [ + 130, + 135, + "UHRF1", + "protein" + ], + [ + 140, + 149, + "RFTSDNMT1", + "protein" + ] + ] + }, + { + "sid": 203, + "sent": "The results suggest that UHRF1 adopts multiple conformations.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 30, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 204, + "sent": "Binding of UHRF1 to hm-DNA may serve as a switch for its recruitment of DNMT1.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 16, + "UHRF1", + "protein" + ], + [ + 20, + 26, + "hm-DNA", + "chemical" + ], + [ + 72, + 77, + "DNMT1", + "protein" + ] + ] + }, + { + "sid": 205, + "sent": "The S phase-dependent interaction between UHRF1 and DNMT1 (refs) suggest that DNMT1 may also undergo conformation changes so that RFTSDNMT1 binds to UHRF1 and the catalytic domain of DNMT1 binds to hm-DNA for reaction.", + "section": "DISCUSS", + "ner": [ + [ + 42, + 47, + "UHRF1", + "protein" + ], + [ + 52, + 57, + "DNMT1", + "protein" + ], + [ + 78, + 83, + "DNMT1", + "protein" + ], + [ + 130, + 139, + "RFTSDNMT1", + "protein" + ], + [ + 140, + 148, + "binds to", + "protein_state" + ], + [ + 149, + 154, + "UHRF1", + "protein" + ], + [ + 163, + 179, + "catalytic domain", + "structure_element" + ], + [ + 183, + 188, + "DNMT1", + "protein" + ], + [ + 189, + 197, + "binds to", + "protein_state" + ], + [ + 198, + 204, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 206, + "sent": "Hm-DNA facilities histione tails recognition by full-length UHRF1.", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "Hm-DNA", + "chemical" + ], + [ + 18, + 26, + "histione", + "protein_type" + ], + [ + 48, + 59, + "full-length", + "protein_state" + ], + [ + 60, + 65, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 207, + "sent": "(a) Colour-coded domain structure of human UHRF1.", + "section": "FIG", + "ner": [ + [ + 37, + 42, + "human", + "species" + ], + [ + 43, + 48, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 208, + "sent": "Note that the conserved motif (green background) of the Linker (residues 286\u2013306) and the Spacer (residues 587\u2013674) bind to the TTD in a similar manner (Fig. 3b). (b) Hm-DNA facilities histone H3 and H3K9me3 recognition by UHRF1.", + "section": "FIG", + "ner": [ + [ + 14, + 23, + "conserved", + "protein_state" + ], + [ + 56, + 62, + "Linker", + "structure_element" + ], + [ + 73, + 80, + "286\u2013306", + "residue_range" + ], + [ + 90, + 96, + "Spacer", + "structure_element" + ], + [ + 107, + 114, + "587\u2013674", + "residue_range" + ], + [ + 128, + 131, + "TTD", + "structure_element" + ], + [ + 167, + 173, + "Hm-DNA", + "chemical" + ], + [ + 185, + 192, + "histone", + "protein_type" + ], + [ + 193, + 195, + "H3", + "protein_type" + ], + [ + 200, + 202, + "H3", + "protein_type" + ], + [ + 223, + 228, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 209, + "sent": "Purified full-length UHRF1 was incubated with biotinylated H3 (1\u201321) or H3K9me3 (1\u201321) peptides in the presence or absence of hm-DNA (molar ratio UHRF1/hm-DNA=1:2).", + "section": "FIG", + "ner": [ + [ + 9, + 20, + "full-length", + "protein_state" + ], + [ + 21, + 26, + "UHRF1", + "protein" + ], + [ + 46, + 58, + "biotinylated", + "protein_state" + ], + [ + 59, + 61, + "H3", + "protein_type" + ], + [ + 63, + 67, + "1\u201321", + "residue_range" + ], + [ + 72, + 74, + "H3", + "protein_type" + ], + [ + 74, + 79, + "K9me3", + "ptm" + ], + [ + 81, + 85, + "1\u201321", + "residue_range" + ], + [ + 115, + 125, + "absence of", + "protein_state" + ], + [ + 126, + 132, + "hm-DNA", + "chemical" + ], + [ + 146, + 151, + "UHRF1", + "protein" + ], + [ + 152, + 158, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 210, + "sent": "The bound proteins were analysed in SDS\u2013PAGE followed by Coomassie blue staining.", + "section": "FIG", + "ner": [ + [ + 36, + 44, + "SDS\u2013PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 211, + "sent": "Sequences of the peptides are indicated in Supplementary Table 1. (c) Histone peptides do not affect hm-DNA-binding affinity of UHRF1.", + "section": "FIG", + "ner": [ + [ + 70, + 77, + "Histone", + "protein_type" + ], + [ + 101, + 124, + "hm-DNA-binding affinity", + "evidence" + ], + [ + 128, + 133, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 212, + "sent": "Full-length UHRF1 was incubated with biotinylated hm-DNA in the presence or absence of H3 (1\u201317) or H3K9me3 (1\u201317) peptides and analysed as in b. (d,e) Superimposed ITC enthalpy plots for binding of H3K9me3 peptide (1\u201317) to TTD\u2013PHD and full-length UHRF1 (d), and H3 peptide (1\u201317) to the PHD and full-length UHRF1 (e).", + "section": "FIG", + "ner": [ + [ + 0, + 11, + "Full-length", + "protein_state" + ], + [ + 12, + 17, + "UHRF1", + "protein" + ], + [ + 22, + 36, + "incubated with", + "experimental_method" + ], + [ + 37, + 49, + "biotinylated", + "protein_state" + ], + [ + 50, + 56, + "hm-DNA", + "chemical" + ], + [ + 76, + 86, + "absence of", + "protein_state" + ], + [ + 87, + 89, + "H3", + "protein_type" + ], + [ + 91, + 95, + "1\u201317", + "residue_range" + ], + [ + 100, + 102, + "H3", + "protein_type" + ], + [ + 102, + 107, + "K9me3", + "ptm" + ], + [ + 109, + 113, + "1\u201317", + "residue_range" + ], + [ + 165, + 168, + "ITC", + "experimental_method" + ], + [ + 169, + 183, + "enthalpy plots", + "evidence" + ], + [ + 199, + 201, + "H3", + "protein_type" + ], + [ + 201, + 206, + "K9me3", + "ptm" + ], + [ + 216, + 220, + "1\u201317", + "residue_range" + ], + [ + 225, + 232, + "TTD\u2013PHD", + "structure_element" + ], + [ + 237, + 248, + "full-length", + "protein_state" + ], + [ + 249, + 254, + "UHRF1", + "protein" + ], + [ + 264, + 266, + "H3", + "protein_type" + ], + [ + 276, + 280, + "1\u201317", + "residue_range" + ], + [ + 289, + 292, + "PHD", + "structure_element" + ], + [ + 297, + 308, + "full-length", + "protein_state" + ], + [ + 309, + 314, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 213, + "sent": "The estimated binding affinities (KD) are listed.", + "section": "FIG", + "ner": [ + [ + 14, + 32, + "binding affinities", + "evidence" + ], + [ + 34, + 36, + "KD", + "evidence" + ] + ] + }, + { + "sid": 214, + "sent": "Intramolecular interactions inhibit histone recognition by UHRF1.", + "section": "FIG", + "ner": [ + [ + 36, + 43, + "histone", + "protein_type" + ], + [ + 59, + 64, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 215, + "sent": "(a) GST pull-down assays for the intramolecular interactions.", + "section": "FIG", + "ner": [ + [ + 4, + 24, + "GST pull-down assays", + "experimental_method" + ] + ] + }, + { + "sid": 216, + "sent": "The isolated domains of UHRF1 were incubated with GST-tagged TTD or PHD immobilized on glutathione resin.", + "section": "FIG", + "ner": [ + [ + 24, + 29, + "UHRF1", + "protein" + ], + [ + 35, + 44, + "incubated", + "experimental_method" + ], + [ + 50, + 60, + "GST-tagged", + "protein_state" + ], + [ + 61, + 64, + "TTD", + "structure_element" + ], + [ + 68, + 71, + "PHD", + "structure_element" + ] + ] + }, + { + "sid": 217, + "sent": "The bound proteins were analysed by SDS\u2013PAGE and Coomassie blue staining.", + "section": "FIG", + "ner": [ + [ + 36, + 44, + "SDS\u2013PAGE", + "experimental_method" + ], + [ + 36, + 44, + "SDS\u2013PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 218, + "sent": "(b,d) Superimposed ITC enthalpy plots for the intramolecular interactions of isolated UHRF1 domains.", + "section": "FIG", + "ner": [ + [ + 19, + 22, + "ITC", + "experimental_method" + ], + [ + 23, + 37, + "enthalpy plots", + "evidence" + ], + [ + 86, + 91, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 219, + "sent": "The estimated binding affinities (KD) were listed.", + "section": "FIG", + "ner": [ + [ + 14, + 32, + "binding affinities", + "evidence" + ], + [ + 34, + 36, + "KD", + "evidence" + ] + ] + }, + { + "sid": 220, + "sent": "ND, not detectable. (c) Superimposed ITC enthalpy plots for the binding of H3K9me3 to TTD\u2013PHD in the absence or presence of the Spacer (molar ratio TTD\u2013PHD/Spacer=1:2). (e) Superimposed ITC enthalpy plots for the binding of H3 to PHD\u2013SRA or PHD in the absence or presence of the SRA (molar ratio PHD/SRA=1:1 or 1:2).", + "section": "FIG", + "ner": [ + [ + 37, + 40, + "ITC", + "experimental_method" + ], + [ + 41, + 55, + "enthalpy plots", + "evidence" + ], + [ + 75, + 77, + "H3", + "protein_type" + ], + [ + 77, + 82, + "K9me3", + "ptm" + ], + [ + 86, + 93, + "TTD\u2013PHD", + "structure_element" + ], + [ + 101, + 108, + "absence", + "protein_state" + ], + [ + 112, + 123, + "presence of", + "protein_state" + ], + [ + 128, + 134, + "Spacer", + "structure_element" + ], + [ + 148, + 155, + "TTD\u2013PHD", + "structure_element" + ], + [ + 156, + 162, + "Spacer", + "structure_element" + ], + [ + 186, + 189, + "ITC", + "experimental_method" + ], + [ + 190, + 204, + "enthalpy plots", + "evidence" + ], + [ + 224, + 226, + "H3", + "protein_type" + ], + [ + 230, + 237, + "PHD\u2013SRA", + "structure_element" + ], + [ + 241, + 244, + "PHD", + "structure_element" + ], + [ + 252, + 259, + "absence", + "protein_state" + ], + [ + 263, + 274, + "presence of", + "protein_state" + ], + [ + 279, + 282, + "SRA", + "structure_element" + ], + [ + 296, + 299, + "PHD", + "structure_element" + ], + [ + 300, + 303, + "SRA", + "structure_element" + ] + ] + }, + { + "sid": 221, + "sent": "NMR structure of the TTD bound to the Spacer.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "NMR", + "experimental_method" + ], + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 21, + 24, + "TTD", + "structure_element" + ], + [ + 25, + 33, + "bound to", + "protein_state" + ], + [ + 38, + 44, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 222, + "sent": "(a) Ribbon representation of TTD\u2013Spacer structure.", + "section": "FIG", + "ner": [ + [ + 29, + 39, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 40, + 49, + "structure", + "evidence" + ] + ] + }, + { + "sid": 223, + "sent": "N- and C-termini of the Spacer are indicated.", + "section": "FIG", + "ner": [ + [ + 24, + 30, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 224, + "sent": "The TTD is coloured in green, and the Spacer is coloured in yellow.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "TTD", + "structure_element" + ], + [ + 38, + 44, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 225, + "sent": "(b) Superimposition of TTD\u2013Spacer and TTD\u2013PHD\u2013H3K9me3 (4GY5.PDB) structures shown in ribbon representations.", + "section": "FIG", + "ner": [ + [ + 4, + 19, + "Superimposition", + "experimental_method" + ], + [ + 23, + 33, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 38, + 53, + "TTD\u2013PHD\u2013H3K9me3", + "complex_assembly" + ], + [ + 65, + 75, + "structures", + "evidence" + ] + ] + }, + { + "sid": 226, + "sent": "The TTD is coloured in green and the Spacer in yellow in TTD\u2013Spacer structure.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "TTD", + "structure_element" + ], + [ + 37, + 43, + "Spacer", + "structure_element" + ], + [ + 57, + 67, + "TTD\u2013Spacer", + "structure_element" + ], + [ + 68, + 77, + "structure", + "evidence" + ] + ] + }, + { + "sid": 227, + "sent": "TTD\u2013PHD\u2013H3K9me3 complex is coloured in grey, and the PHD and H3K9me3 are omitted for simplicity.", + "section": "FIG", + "ner": [ + [ + 0, + 15, + "TTD\u2013PHD\u2013H3K9me3", + "complex_assembly" + ], + [ + 53, + 56, + "PHD", + "structure_element" + ], + [ + 61, + 63, + "H3", + "protein_type" + ], + [ + 63, + 68, + "K9me3", + "ptm" + ] + ] + }, + { + "sid": 228, + "sent": "(c) Electrostatic potential surface representation of the TTD with the Spacer shown in ribbon representation.", + "section": "FIG", + "ner": [ + [ + 58, + 61, + "TTD", + "structure_element" + ], + [ + 71, + 77, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 229, + "sent": "The critical residues on the Spacer for the interaction are shown in stick representation.", + "section": "FIG", + "ner": [ + [ + 29, + 35, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 230, + "sent": "(d) Close-up view of TTD\u2013Spacer interaction.", + "section": "FIG", + "ner": [ + [ + 21, + 31, + "TTD\u2013Spacer", + "structure_element" + ] + ] + }, + { + "sid": 231, + "sent": "Hydrogen bonds are indicated as dashed lines.", + "section": "FIG", + "ner": [ + [ + 0, + 14, + "Hydrogen bonds", + "bond_interaction" + ] + ] + }, + { + "sid": 232, + "sent": "(e\u2013g) Superimposed ITC enthalpy plots for the interaction between the Spacer and the TTD (or TTD\u2013PHD) with the estimated binding affinity (KD) indicated.", + "section": "FIG", + "ner": [ + [ + 19, + 22, + "ITC", + "experimental_method" + ], + [ + 23, + 37, + "enthalpy plots", + "evidence" + ], + [ + 70, + 76, + "Spacer", + "structure_element" + ], + [ + 85, + 88, + "TTD", + "structure_element" + ], + [ + 93, + 100, + "TTD\u2013PHD", + "structure_element" + ], + [ + 121, + 137, + "binding affinity", + "evidence" + ], + [ + 139, + 141, + "KD", + "evidence" + ] + ] + }, + { + "sid": 233, + "sent": "Wild-type and mutant proteins for the measurements are indicated.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Wild-type", + "protein_state" + ], + [ + 14, + 20, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 234, + "sent": "(h) GST pull-down assays for the intramolecular interactions.", + "section": "FIG", + "ner": [ + [ + 4, + 24, + "GST pull-down assays", + "experimental_method" + ] + ] + }, + { + "sid": 235, + "sent": "The wild-type or indicated truncations of UHRF1 were incubated with GST-tagged TTD, Linker or Spacer.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "wild-type", + "protein_state" + ], + [ + 42, + 47, + "UHRF1", + "protein" + ], + [ + 68, + 78, + "GST-tagged", + "protein_state" + ], + [ + 79, + 82, + "TTD", + "structure_element" + ], + [ + 84, + 90, + "Linker", + "structure_element" + ], + [ + 94, + 100, + "Spacer", + "structure_element" + ] + ] + }, + { + "sid": 236, + "sent": "Hm-DNA impairs the intramolecular interaction of UHRF1 and facilitates its histone recognition.", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "Hm-DNA", + "chemical" + ], + [ + 49, + 54, + "UHRF1", + "protein" + ], + [ + 75, + 82, + "histone", + "protein_type" + ] + ] + }, + { + "sid": 237, + "sent": "(a) Hm-DNA impairs the intramolecular interaction of PHD\u2013SRA.", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "Hm-DNA", + "chemical" + ], + [ + 53, + 60, + "PHD\u2013SRA", + "structure_element" + ] + ] + }, + { + "sid": 238, + "sent": "The SRA was incubated with GST-tagged PHD in the presence of increasing concentrations of hm-DNA and immobilized on glutathione resin.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "SRA", + "structure_element" + ], + [ + 12, + 21, + "incubated", + "experimental_method" + ], + [ + 27, + 37, + "GST-tagged", + "protein_state" + ], + [ + 38, + 41, + "PHD", + "structure_element" + ], + [ + 49, + 60, + "presence of", + "protein_state" + ], + [ + 90, + 96, + "hm-DNA", + "chemical" + ] + ] + }, + { + "sid": 239, + "sent": "The bound proteins were analysed in SDS\u2013PAGE and Coomassie blue staining (left) and quantified by band densitometry (right).", + "section": "FIG", + "ner": [ + [ + 36, + 44, + "SDS\u2013PAGE", + "experimental_method" + ], + [ + 49, + 72, + "Coomassie blue staining", + "experimental_method" + ], + [ + 98, + 115, + "band densitometry", + "experimental_method" + ] + ] + }, + { + "sid": 240, + "sent": "(b) Purified fragments of UHRF1 were analysed by histone peptide (H3K9me0) pull-down assay as described in Fig. 1b. (c) Hm-DNA impairs the intramolecular interaction of TTD\u2013Spacer.", + "section": "FIG", + "ner": [ + [ + 26, + 31, + "UHRF1", + "protein" + ], + [ + 49, + 64, + "histone peptide", + "experimental_method" + ], + [ + 66, + 68, + "H3", + "protein_type" + ], + [ + 68, + 73, + "K9me0", + "ptm" + ], + [ + 75, + 90, + "pull-down assay", + "experimental_method" + ], + [ + 120, + 126, + "Hm-DNA", + "chemical" + ], + [ + 169, + 179, + "TTD\u2013Spacer", + "structure_element" + ] + ] + }, + { + "sid": 241, + "sent": "SRA\u2013Spacer was incubated with GST-tagged TTD\u2013PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining.", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 15, + 24, + "incubated", + "experimental_method" + ], + [ + 30, + 40, + "GST-tagged", + "protein_state" + ], + [ + 41, + 48, + "TTD\u2013PHD", + "structure_element" + ], + [ + 52, + 55, + "TTD", + "structure_element" + ], + [ + 63, + 74, + "presence of", + "protein_state" + ], + [ + 104, + 110, + "hm-DNA", + "chemical" + ], + [ + 127, + 147, + "pull-down experiment", + "experimental_method" + ], + [ + 182, + 201, + "band densitometries", + "experimental_method" + ], + [ + 226, + 249, + "Coomassie blue staining", + "experimental_method" + ] + ] + }, + { + "sid": 242, + "sent": "(d) Histone peptide pull-down assay using UHRF1 mutants as indicated.", + "section": "FIG", + "ner": [ + [ + 4, + 35, + "Histone peptide pull-down assay", + "experimental_method" + ], + [ + 42, + 47, + "UHRF1", + "protein" + ], + [ + 48, + 55, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 243, + "sent": "The assays were performed in the presence (+DTT) or absence (\u2212DTT) of 15\u2009mM DTT.", + "section": "FIG", + "ner": [ + [ + 44, + 47, + "DTT", + "chemical" + ], + [ + 62, + 65, + "DTT", + "chemical" + ], + [ + 76, + 79, + "DTT", + "chemical" + ] + ] + }, + { + "sid": 244, + "sent": "The Spacer facilitates hm-DNA\u2013SRA interaction and DNMT1\u2013UHRF1 interaction.", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "Spacer", + "structure_element" + ], + [ + 23, + 29, + "hm-DNA", + "chemical" + ], + [ + 30, + 33, + "SRA", + "structure_element" + ], + [ + 50, + 61, + "DNMT1\u2013UHRF1", + "complex_assembly" + ] + ] + }, + { + "sid": 245, + "sent": "(a) Superimposed ITC enthalpy plots for hm-DNA-binding affinities of the SRA, the Spacer and SRA\u2013Spacer. (b,c) Superimposed fluorescence polarization (FP) plots for hm-DNA-binding affinities of truncations or full-length UHRF1.", + "section": "FIG", + "ner": [ + [ + 17, + 20, + "ITC", + "experimental_method" + ], + [ + 21, + 35, + "enthalpy plots", + "evidence" + ], + [ + 40, + 65, + "hm-DNA-binding affinities", + "evidence" + ], + [ + 73, + 76, + "SRA", + "structure_element" + ], + [ + 82, + 88, + "Spacer", + "structure_element" + ], + [ + 93, + 103, + "SRA\u2013Spacer", + "structure_element" + ], + [ + 124, + 160, + "fluorescence polarization (FP) plots", + "evidence" + ], + [ + 165, + 190, + "hm-DNA-binding affinities", + "evidence" + ], + [ + 209, + 220, + "full-length", + "protein_state" + ], + [ + 221, + 226, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 246, + "sent": "The estimated binding affinities (KD) are listed above. (d) Subcellular localization of GFP-tagged wild-type or indicated mutants of UHRF1 in NIH3T3 cells.", + "section": "FIG", + "ner": [ + [ + 14, + 32, + "binding affinities", + "evidence" + ], + [ + 34, + 36, + "KD", + "evidence" + ], + [ + 88, + 98, + "GFP-tagged", + "protein_state" + ], + [ + 99, + 108, + "wild-type", + "protein_state" + ], + [ + 122, + 129, + "mutants", + "protein_state" + ], + [ + 133, + 138, + "UHRF1", + "protein" + ] + ] + }, + { + "sid": 247, + "sent": "The percentages of cells showing co-localization with DAPI foci were counted from at least 100 cells and shown on the left of the corresponding representative confocal microscopy.", + "section": "FIG", + "ner": [ + [ + 54, + 58, + "DAPI", + "chemical" + ], + [ + 159, + 178, + "confocal microscopy", + "experimental_method" + ] + ] + }, + { + "sid": 248, + "sent": "Scale bar, 5\u2009\u03bcm. (e) GST pull-down experiment for the interactions between wild-type or truncations of UHRF1 and RFTSDNMT1 as described in Fig. 2a. (f) Working model for hm-DNA-mediated conformational changes of UHRF1, as described in the Discussion.", + "section": "FIG", + "ner": [ + [ + 21, + 45, + "GST pull-down experiment", + "experimental_method" + ], + [ + 75, + 84, + "wild-type", + "protein_state" + ], + [ + 88, + 99, + "truncations", + "experimental_method" + ], + [ + 103, + 108, + "UHRF1", + "protein" + ], + [ + 113, + 122, + "RFTSDNMT1", + "protein" + ], + [ + 170, + 176, + "hm-DNA", + "chemical" + ], + [ + 212, + 217, + "UHRF1", + "protein" + ] + ] + } + ] + }, + "PMC4831588": { + "annotations": [ + { + "sid": 0, + "sent": "X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an A\u03b217\u201336 \u03b2-Hairpin", + "section": "TITLE", + "ner": [ + [ + 0, + 33, + "X-ray Crystallographic Structures", + "evidence" + ], + [ + 39, + 45, + "Trimer", + "oligomeric_state" + ], + [ + 47, + 56, + "Dodecamer", + "oligomeric_state" + ], + [ + 62, + 74, + "Annular Pore", + "site" + ], + [ + 88, + 90, + "A\u03b2", + "protein" + ], + [ + 90, + 95, + "17\u201336", + "residue_range" + ], + [ + 96, + 105, + "\u03b2-Hairpin", + "structure_element" + ] + ] + }, + { + "sid": 1, + "sent": "High-resolution structures of oligomers formed by the \u03b2-amyloid peptide A\u03b2 are needed to understand the molecular basis of Alzheimer\u2019s disease and develop therapies.", + "section": "ABSTRACT", + "ner": [ + [ + 16, + 26, + "structures", + "evidence" + ], + [ + 30, + 39, + "oligomers", + "oligomeric_state" + ], + [ + 54, + 71, + "\u03b2-amyloid peptide", + "protein" + ], + [ + 72, + 74, + "A\u03b2", + "protein" + ] + ] + }, + { + "sid": 2, + "sent": "This paper presents the X-ray crystallographic structures of oligomers formed by a 20-residue peptide segment derived from A\u03b2.", + "section": "ABSTRACT", + "ner": [ + [ + 24, + 57, + "X-ray crystallographic structures", + "evidence" + ], + [ + 61, + 70, + "oligomers", + "oligomeric_state" + ], + [ + 83, + 109, + "20-residue peptide segment", + "residue_range" + ], + [ + 123, + 125, + "A\u03b2", + "protein" + ] + ] + }, + { + "sid": 3, + "sent": "The development of a peptide in which A\u03b217\u201336 is stabilized as a \u03b2-hairpin is described, and the X-ray crystallographic structures of oligomers it forms are reported.", + "section": "ABSTRACT", + "ner": [ + [ + 38, + 40, + "A\u03b2", + "protein" + ], + [ + 40, + 45, + "17\u201336", + "residue_range" + ], + [ + 65, + 74, + "\u03b2-hairpin", + "structure_element" + ], + [ + 97, + 130, + "X-ray crystallographic structures", + "evidence" + ], + [ + 134, + 143, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 4, + "sent": "Two covalent constraints act in tandem to stabilize the A\u03b217\u201336 peptide in a hairpin conformation: a \u03b4-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29.", + "section": "ABSTRACT", + "ner": [ + [ + 56, + 58, + "A\u03b2", + "protein" 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"folded", + "protein_state" + ], + [ + 45, + 54, + "\u03b2-hairpin", + "structure_element" + ], + [ + 94, + 109, + "crystal lattice", + "evidence" + ] + ] + }, + { + "sid": 7, + "sent": "Three \u03b2-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore.", + "section": "ABSTRACT", + "ner": [ + [ + 6, + 15, + "\u03b2-hairpin", + "structure_element" + ], + [ + 16, + 24, + "monomers", + "oligomeric_state" + ], + [ + 44, + 54, + "triangular", + "protein_state" + ], + [ + 55, + 61, + "trimer", + "oligomeric_state" + ], + [ + 68, + 75, + "trimers", + "oligomeric_state" + ], + [ + 124, + 133, + "dodecamer", + "oligomeric_state" + ], + [ + 144, + 154, + "dodecamers", + "oligomeric_state" + ], + [ + 180, + 192, + "annular pore", + "site" + ] + ] + }, + { + "sid": 8, + "sent": "This hierarchical assembly provides a model, in which full-length A\u03b2 transitions from an unfolded monomer to a folded \u03b2-hairpin, which assembles to form oligomers that further pack to form an annular pore.", + "section": "ABSTRACT", + "ner": [ + [ + 54, + 65, + "full-length", + "protein_state" + ], + [ + 66, + 68, + "A\u03b2", + "protein" + ], + [ + 89, + 97, + "unfolded", + "protein_state" + ], + [ + 98, + 105, + "monomer", + "oligomeric_state" + ], + [ + 111, + 117, + "folded", + "protein_state" + ], + [ + 118, + 127, + "\u03b2-hairpin", + "structure_element" + ], + [ + 153, + 162, + "oligomers", + "oligomeric_state" + ], + [ + 192, + 204, + "annular pore", + "site" + ] + ] + }, + { + "sid": 9, + "sent": "High-resolution structures of oligomers formed by the \u03b2-amyloid peptide A\u03b2 are desperately needed to understand the molecular basis of Alzheimer\u2019s disease and ultimately develop preventions or treatments.", + "section": "INTRO", + "ner": [ + [ + 16, + 26, + "structures", + "evidence" + ], + [ + 30, + 39, + "oligomers", + "oligomeric_state" + ], + [ + 54, + 71, + "\u03b2-amyloid peptide", + "protein" + ], + [ + 72, + 74, + "A\u03b2", + "protein" + ] + ] + }, + { + "sid": 10, + "sent": "In Alzheimer\u2019s disease, monomeric A\u03b2 aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils.", + "section": "INTRO", + "ner": [ + [ + 24, + 33, + "monomeric", + "oligomeric_state" + ], + [ + 34, + 36, + "A\u03b2", + "protein" + ], + [ + 85, + 94, + "oligomers", + "oligomeric_state" + ], + [ + 104, + 110, + "dimers", + "oligomeric_state" + ], + [ + 112, + 119, + "trimers", + "oligomeric_state" + ], + [ + 121, + 130, + "tetramers", + "oligomeric_state" + ], + [ + 132, + 140, + "hexamers", + "oligomeric_state" + ], + [ + 142, + 150, + "nonamers", + "oligomeric_state" + ], + [ + 156, + 166, + "dodecamers", + "oligomeric_state" + ], + [ + 221, + 241, + "annular protofibrils", + "complex_assembly" + ] + ] + }, + { + "sid": 11, + "sent": "Over the last two decades the role of A\u03b2 oligomers in the pathophysiology of Alzheimer\u2019s disease has begun to unfold.", + "section": "INTRO", + "ner": [ + [ + 38, + 40, + "A\u03b2", + "protein" + ], + [ + 41, + 50, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 12, + "sent": "Mouse models for Alzheimer\u2019s disease have helped shape our current understanding about the A\u03b2 oligomerization that precedes neurodegeneration.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "Mouse", + "taxonomy_domain" + ], + [ + 91, + 93, + "A\u03b2", + "protein" + ] + ] + }, + { + "sid": 13, + "sent": "A\u03b2 isolated from the brains of young plaque-free Tg2576 mice forms a mixture of low molecular weight oligomers.", + "section": "INTRO", + "ner": [ + [ + 0, + 2, + "A\u03b2", + "protein" + ], + [ + 56, + 60, + "mice", + "taxonomy_domain" + ], + [ + 101, + 110, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 14, + "sent": "A 56 kDa soluble oligomer identified by SDS-PAGE was found to be especially important within this mixture.", + "section": "INTRO", + "ner": [ + [ + 17, + 25, + "oligomer", + "oligomeric_state" + ], + [ + 40, + 48, + "SDS-PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 15, + "sent": "This oligomer was termed A\u03b2*56 and appears to be a dodecamer of A\u03b2.", + "section": "INTRO", + "ner": [ + [ + 5, + 13, + "oligomer", + "oligomeric_state" + ], + [ + 25, + 30, + "A\u03b2*56", + "complex_assembly" + ], + [ + 51, + 60, + "dodecamer", + "oligomeric_state" + ], + [ + 64, + 66, + "A\u03b2", + "protein" + ] + ] + }, + { + "sid": 16, + "sent": "Purified A\u03b2*56 injected intercranially into healthy rats was found to impair memory, providing evidence that this A\u03b2 oligomer may cause memory loss in Alzheimer\u2019s disease.", + "section": "INTRO", + "ner": [ + [ + 9, + 14, + "A\u03b2*56", + "complex_assembly" + ], + [ + 15, + 38, + "injected 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of the dodecamers, nonamers, and hexamers.", + "section": "INTRO", + "ner": [ + [ + 49, + 58, + "oligomers", + "oligomeric_state" + ], + [ + 64, + 85, + "hexafluoroisopropanol", + "chemical" + ], + [ + 131, + 141, + "dodecamers", + "oligomeric_state" + ], + [ + 143, + 151, + "nonamers", + "oligomeric_state" + ], + [ + 157, + 165, + "hexamers", + "oligomeric_state" + ], + [ + 171, + 178, + "trimers", + "oligomeric_state" + ], + [ + 183, + 191, + "monomers", + "oligomeric_state" + ], + [ + 209, + 216, + "trimers", + "oligomeric_state" + ], + [ + 250, + 260, + "dodecamers", + "oligomeric_state" + ], + [ + 262, + 270, + "nonamers", + "oligomeric_state" + ], + [ + 276, + 284, + "hexamers", + "oligomeric_state" + ] + ] + }, + { + "sid": 19, + "sent": "Recently, A\u03b2 trimers and A\u03b2*56 were identified in the brains of cognitively normal humans and were found to increase with age.", + "section": "INTRO", + "ner": [ + [ + 10, + 12, + "A\u03b2", + "protein" + ], + [ + 13, + 20, + "trimers", + "oligomeric_state" + ], + [ + 25, + 30, + "A\u03b2*56", + "complex_assembly" + ], + [ + 83, + 89, + "humans", + "species" + ] + ] + }, + { + "sid": 20, + "sent": "A type of large oligomers called annular protofibrils (APFs) have also been observed in the brains of transgenic mice and isolated from the brains of Alzheimer\u2019s patients.", + "section": "INTRO", + "ner": [ + [ + 16, + 25, + "oligomers", + "oligomeric_state" + ], + [ + 33, + 53, + "annular protofibrils", + "complex_assembly" + ], + [ + 55, + 59, + "APFs", + "complex_assembly" + ], + [ + 113, + 117, + "mice", + "taxonomy_domain" + ] + ] + }, + { + "sid": 21, + "sent": "APFs were first discovered in vitro using chemically synthesized A\u03b2 that aggregated into porelike structures that could be observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM).", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "APFs", + "complex_assembly" + ], + [ + 42, + 64, + "chemically synthesized", + "protein_state" + ], + [ + 65, + 67, + "A\u03b2", + "protein" + ], + [ + 89, + 108, + "porelike structures", + "structure_element" + ], + [ + 135, + 158, + "atomic force microscopy", + "experimental_method" + ], + [ + 160, + 163, + "AFM", + "experimental_method" + ], + [ + 169, + 201, + "transmission electron microscopy", + "experimental_method" + ], + [ + 203, + 206, + "TEM", + "experimental_method" + ] + ] + }, + { + "sid": 22, + "sent": "The sizes of APFs prepared in vitro vary among different studies.", + "section": "INTRO", + "ner": [ + [ + 13, + 17, + "APFs", + "complex_assembly" + ] + ] + }, + { + "sid": 23, + "sent": "Lashuel et al. observed APFs with an outer diameter that ranged from 7\u201310 nm and an inner diameter that ranged from 1.5\u20132 nm, consistent with molecular weights of 150\u2013250 kDa.", + "section": "INTRO", + "ner": [ + [ + 24, + 28, + "APFs", + "complex_assembly" + ] + ] + }, + { + "sid": 24, + "sent": "Quist et al. observed APFs with an outer diameter of 16 nm embedded in a lipid bilayer.", + "section": "INTRO", + "ner": [ + [ + 22, + 26, + "APFs", + "complex_assembly" + ] + ] + }, + { + "sid": 25, + "sent": "Kayed et al. observed APFs with an outer diameter that ranged from 8\u201325 nm, which were composed of small spherical A\u03b2 oligomers, 3\u20135 nm in diameter.", + "section": "INTRO", + "ner": [ + [ + 22, + 26, + "APFs", + "complex_assembly" + ], + [ + 99, + 114, + "small spherical", + "protein_state" + ], + [ + 115, + 117, + "A\u03b2", + "protein" + ], + [ + 118, + 127, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 26, + "sent": "Although the APFs in these studies differ in size, they share a similar annular morphology and appear to be composed of smaller oligomers.", + "section": "INTRO", + "ner": [ + [ + 13, + 17, + "APFs", + "complex_assembly" + ], + [ + 128, + 137, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 27, + "sent": "APFs have also been observed in the brains of APP23 transgenic mice by immunofluorescence with an anti-APF antibody and were found to accumulate in neuronal processes and synapses.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "APFs", + "complex_assembly" + ], + [ + 63, + 67, + "mice", + "taxonomy_domain" + ], + [ + 71, + 89, + "immunofluorescence", + "experimental_method" + ], + [ + 103, + 106, + "APF", + "complex_assembly" + ] + ] + }, + { + "sid": 28, + "sent": "In a subsequent study, APFs were isolated from the brains of Alzheimer\u2019s patients by immunoprecipitation with an anti-APF antibody.", + "section": "INTRO", + "ner": [ + [ + 23, + 27, + "APFs", + "complex_assembly" + ], + [ + 85, + 104, + "immunoprecipitation", + "experimental_method" + ], + [ + 118, + 121, + "APF", + "complex_assembly" + ] + ] + }, + { + "sid": 29, + "sent": "These APFs had an outer diameter that ranged from 11\u201314 nm and an inner diameter that ranged from 2.5\u20134 nm.", + "section": "INTRO", + "ner": [ + [ + 6, + 10, + "APFs", + "complex_assembly" + ] + ] + }, + { + "sid": 30, + "sent": "Dimers of A\u03b2 have also been isolated from the brains of Alzheimer\u2019s patients.\u2212 A\u03b2 dimers inhibit long-term potentiation in mice and promote hyperphosphorylation of the microtubule-associated protein tau, leading to neuritic damage.", + "section": "INTRO", + "ner": [ + [ + 0, + 6, + "Dimers", + "oligomeric_state" + ], + [ + 10, + 12, + "A\u03b2", + "protein" + ], + [ + 79, + 81, + "A\u03b2", + "protein" + ], + [ + 82, + 88, + "dimers", + "oligomeric_state" + ], + [ + 123, + 127, + "mice", + "taxonomy_domain" + ], + [ + 140, + 160, + "hyperphosphorylation", + "ptm" + ], + [ + 168, + 202, + "microtubule-associated protein tau", + "protein" + ] + ] + }, + { + "sid": 31, + "sent": "A\u03b2 dimers have only been isolated from human or transgenic mouse brains that contain the pathognomonic fibrillar A\u03b2 plaques associated with Alzheimer\u2019s disease.", + "section": "INTRO", + "ner": [ + [ + 0, + 2, + "A\u03b2", + "protein" + ], + [ + 3, + 9, + "dimers", + "oligomeric_state" + ], + [ + 39, + 44, + "human", + "species" + ], + [ + 59, + 64, + "mouse", + "taxonomy_domain" + ], + [ + 103, + 112, + "fibrillar", + "protein_state" + ], + [ + 113, + 115, + "A\u03b2", + "protein" + ] + ] + }, + { + "sid": 32, + "sent": "Furthermore, the endogenous rise of A\u03b2 dimers in the brains of Tg2576 and J20 transgenic mice coincides with the deposition of A\u03b2 plaques.", + "section": "INTRO", + "ner": [ + [ + 36, + 38, + "A\u03b2", + "protein" + ], + [ + 39, + 45, + "dimers", + "oligomeric_state" + ], + [ + 89, + 93, + "mice", + "taxonomy_domain" + ], + [ + 127, + 129, + "A\u03b2", + "protein" + ] + ] + }, + { + "sid": 33, + "sent": "These observations suggest that the A\u03b2 trimers, hexamers, dodecamers, and related assemblies may be associated with presymptomatic neurodegeneration, while A\u03b2 dimers are more closely associated with fibril formation and plaque deposition during symptomatic Alzheimer\u2019s disease.\u2212", + "section": "INTRO", + "ner": [ + [ + 36, + 38, + "A\u03b2", + "protein" + ], + [ + 39, + 46, + "trimers", + "oligomeric_state" + ], + [ + 48, + 56, + "hexamers", + "oligomeric_state" + ], + [ + 58, + 68, + "dodecamers", + "oligomeric_state" + ], + [ + 156, + 158, + "A\u03b2", + "protein" + ], + [ + 159, + 165, + "dimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 34, + "sent": "The approach of isolating and characterizing A\u03b2 oligomers has not provided any high-resolution structures of A\u03b2 oligomers.", + "section": "INTRO", + "ner": [ + [ + 45, + 47, + "A\u03b2", + "protein" + ], + [ + 48, + 57, + "oligomers", + "oligomeric_state" + ], + [ + 95, + 105, + "structures", + "evidence" + ], + [ + 109, + 111, + "A\u03b2", + "protein" + ], + [ + 112, + 121, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 35, + "sent": "Techniques such as SDS-PAGE, TEM, and AFM have only provided information about the molecular weights, sizes, morphologies, and stoichiometry of A\u03b2 oligomers.", + "section": "INTRO", + "ner": [ + [ + 19, + 27, + "SDS-PAGE", + "experimental_method" + ], + [ + 29, + 32, + "TEM", + "experimental_method" + ], + [ + 38, + 41, + "AFM", + "experimental_method" + ], + [ + 144, + 146, + "A\u03b2", + "protein" + ], + [ + 147, + 156, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 36, + "sent": "High-resolution structural studies of A\u03b2 have primarily focused on A\u03b2 fibrils and A\u03b2 monomers.", + "section": "INTRO", + "ner": [ + [ + 16, + 34, + "structural studies", + "experimental_method" + ], + [ + 38, + 40, + "A\u03b2", + "protein" + ], + [ + 67, + 69, + "A\u03b2", + "protein" + ], + [ + 70, + 77, + "fibrils", + "oligomeric_state" + ], + [ + 82, + 84, + "A\u03b2", + "protein" + ], + [ + 85, + 93, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 37, + "sent": "Solid-state NMR spectroscopy studies of A\u03b2 fibrils revealed that A\u03b2 fibrils are generally composed of extended networks of in-register parallel \u03b2-sheets.\u2212 X-ray crystallographic studies using fragments of A\u03b2 have provided additional information about how A\u03b2 fibrils pack.", + "section": "INTRO", + "ner": [ + [ + 0, + 28, + "Solid-state NMR spectroscopy", + "experimental_method" + ], + [ + 40, + 42, + "A\u03b2", + "protein" + ], + [ + 43, + 50, + "fibrils", + "oligomeric_state" + ], + [ + 65, + 67, + "A\u03b2", + "protein" + ], + [ + 68, + 75, + "fibrils", + "oligomeric_state" + ], + [ + 123, + 152, + "in-register parallel \u03b2-sheets", + "structure_element" + ], + [ + 155, + 185, + "X-ray crystallographic studies", + "experimental_method" + ], + [ + 205, + 207, + "A\u03b2", + "protein" + ], + [ + 255, + 257, + "A\u03b2", + "protein" + ], + [ + 258, + 265, + "fibrils", + "oligomeric_state" + ] + ] + }, + { + "sid": 38, + "sent": "Solution-phase NMR and solid-state NMR have been used to study the structures of the A\u03b2 monomers within oligomeric assemblies.\u2212 A major finding from these studies is that oligomeric assemblies of A\u03b2 are primarily composed of antiparallel \u03b2-sheets.", + "section": "INTRO", + "ner": [ + [ + 0, + 18, + "Solution-phase NMR", + "experimental_method" + ], + [ + 23, + 38, + "solid-state NMR", + "experimental_method" + ], + [ + 67, + 77, + "structures", + "evidence" + ], + [ + 85, + 87, + "A\u03b2", + "protein" + ], + [ + 88, + 96, + "monomers", + "oligomeric_state" + ], + [ + 196, + 198, + "A\u03b2", + "protein" + ], + [ + 225, + 246, + "antiparallel \u03b2-sheets", + "structure_element" + ] + ] + }, + { + "sid": 39, + "sent": "Many of these studies have reported the monomer subunit as adopting a \u03b2-hairpin conformation, in which the hydrophobic central and C-terminal regions form an antiparallel \u03b2-sheet.", + "section": "INTRO", + "ner": [ + [ + 40, + 47, + "monomer", + "oligomeric_state" + ], + [ + 48, + 55, + "subunit", + "structure_element" + ], + [ + 70, + 79, + "\u03b2-hairpin", + "structure_element" + ], + [ + 119, + 126, + "central", + "structure_element" + ], + [ + 131, + 149, + "C-terminal regions", + "structure_element" + ], + [ + 158, + 178, + "antiparallel \u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 40, + "sent": "In 2008, Hoyer et al. reported the NMR structure of an A\u03b2 monomer bound to an artificial binding protein called an affibody (PDB 2OTK).", + "section": "INTRO", + "ner": [ + [ + 35, + 38, + "NMR", + "experimental_method" + ], + [ + 39, + 48, + "structure", + "evidence" + ], + [ + 55, + 57, + "A\u03b2", + "protein" + ], + [ + 58, + 65, + "monomer", + "oligomeric_state" + ], + [ + 66, + 74, + "bound to", + "protein_state" + ], + [ + 78, + 104, + "artificial binding protein", + "chemical" + ], + [ + 115, + 123, + "affibody", + "chemical" + ] + ] + }, + { + "sid": 41, + "sent": "The structure revealed that monomeric A\u03b2 forms a \u03b2-hairpin when bound to the affibody.", + "section": "INTRO", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 28, + 37, + "monomeric", + "oligomeric_state" + ], + [ + 38, + 40, + "A\u03b2", + "protein" + ], + [ + 49, + 58, + "\u03b2-hairpin", + "structure_element" + ], + [ + 64, + 72, + "bound to", + "protein_state" + ], + [ + 77, + 85, + "affibody", + "chemical" + ] + ] + }, + { + "sid": 42, + "sent": "This A\u03b2 \u03b2-hairpin encompasses residues 17\u201337 and contains two \u03b2-strands comprising A\u03b217\u201324 and A\u03b230\u201337 connected by an A\u03b225\u201329 loop.", + "section": "INTRO", + "ner": [ + [ + 5, + 7, + "A\u03b2", + "protein" + ], + [ + 8, + 17, + "\u03b2-hairpin", + "structure_element" + ], + [ + 39, + 44, + "17\u201337", + "residue_range" + ], + [ + 62, + 71, + "\u03b2-strands", + "structure_element" + ], + [ + 83, + 85, + "A\u03b2", + "protein" + ], + [ + 85, + 90, + "17\u201324", + "residue_range" + ], + [ + 95, + 97, + "A\u03b2", + "protein" + ], + [ + 97, + 102, + "30\u201337", + "residue_range" + ], + [ + 119, + 121, + "A\u03b2", + "protein" + ], + [ + 121, + 126, + "25\u201329", + "residue_range" + ], + [ + 127, + 131, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 43, + "sent": "Sequestering A\u03b2 within the affibody prevents its fibrilization and reduces its neurotoxicity, providing evidence that the \u03b2-hairpin structure may contribute to the ability of A\u03b2 to form neurotoxic oligomers.", + "section": "INTRO", + "ner": [ + [ + 13, + 15, + "A\u03b2", + "protein" + ], + [ + 27, + 35, + "affibody", + "chemical" + ], + [ + 122, + 131, + "\u03b2-hairpin", + "structure_element" + ], + [ + 175, + 177, + "A\u03b2", + "protein" + ], + [ + 197, + 206, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 44, + "sent": "In a related study, Sandberg et al. constrained A\u03b2 in a \u03b2-hairpin conformation by mutating residues A21 and A30 to cysteine and forming an intramolecular disulfide bond.", + "section": "INTRO", + "ner": [ + [ + 48, + 50, + "A\u03b2", + "protein" + ], + [ + 56, + 65, + "\u03b2-hairpin", + "structure_element" + ], + [ + 82, + 90, + "mutating", + "experimental_method" + ], + [ + 100, + 103, + "A21", + "residue_name_number" + ], + [ + 108, + 111, + "A30", + "residue_name_number" + ], + [ + 115, + 123, + "cysteine", + "residue_name" + ], + [ + 154, + 168, + "disulfide bond", + "ptm" + ] + ] + }, + { + "sid": 45, + "sent": "Locking A\u03b2 into a \u03b2-hairpin structure resulted in the formation A\u03b2 oligomers, which were observed by size exclusion chromatography (SEC) and SDS-PAGE.", + "section": "INTRO", + "ner": [ + [ + 8, + 10, + "A\u03b2", + "protein" + ], + [ + 18, + 27, + "\u03b2-hairpin", + "structure_element" + ], + [ + 64, + 66, + "A\u03b2", + "protein" + ], + [ + 67, + 76, + "oligomers", + "oligomeric_state" + ], + [ + 101, + 130, + "size exclusion chromatography", + "experimental_method" + ], + [ + 132, + 135, + "SEC", + "experimental_method" + ], + [ + 141, + 149, + "SDS-PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 46, + "sent": "The oligomers with a molecular weight of \u223c100 kDa that were isolated by SEC were toxic toward neuronally derived SH-SY5Y cells.", + "section": "INTRO", + "ner": [ + [ + 4, + 13, + "oligomers", + "oligomeric_state" + ], + [ + 72, + 75, + "SEC", + "experimental_method" + ] + ] + }, + { + "sid": 47, + "sent": "This study provides evidence for the role of \u03b2-hairpin structure in A\u03b2 oligomerization and neurotoxicity.", + "section": "INTRO", + "ner": [ + [ + 45, + 54, + "\u03b2-hairpin", + "structure_element" + ], + [ + 68, + 70, + "A\u03b2", + "protein" + ] + ] + }, + { + "sid": 48, + "sent": "Inspired by these \u03b2-hairpin structures, our laboratory developed a macrocyclic \u03b2-sheet peptide derived from A\u03b217\u201336 designed to mimic an A\u03b2 \u03b2-hairpin and reported its X-ray crystallographic structure.", + "section": "INTRO", + "ner": [ + [ + 18, + 27, + "\u03b2-hairpin", + "structure_element" + ], + [ + 28, + 38, + "structures", + "evidence" + ], + [ + 79, + 86, + "\u03b2-sheet", + "structure_element" + ], + [ + 108, + 110, + "A\u03b2", + "protein" + ], + [ + 110, + 115, + "17\u201336", + "residue_range" + ], + [ + 137, + 139, + "A\u03b2", + "protein" + ], + [ + 140, + 149, + "\u03b2-hairpin", + "structure_element" + ], + [ + 167, + 199, + "X-ray crystallographic structure", + "evidence" + ] + ] + }, + { + "sid": 49, + "sent": "This peptide (peptide 1) consists of two \u03b2-strands comprising A\u03b217\u201323 and A\u03b230\u201336 covalently linked by two \u03b4-linked ornithine (\u03b4Orn) \u03b2-turn mimics.", + "section": "INTRO", + "ner": [ + [ + 14, + 23, + "peptide 1", + "mutant" + ], + [ + 41, + 50, + "\u03b2-strands", + "structure_element" + ], + [ + 62, + 64, + "A\u03b2", + "protein" + ], + [ + 64, + 69, + "17\u201323", + "residue_range" + ], + [ + 74, + 76, + "A\u03b2", + "protein" + ], + [ + 76, + 81, + "30\u201336", + "residue_range" + ], + [ + 107, + 115, + "\u03b4-linked", + "protein_state" + ], + [ + 116, + 125, + "ornithine", + "residue_name" + ], + [ + 127, + 131, + "\u03b4Orn", + "structure_element" + ], + [ + 133, + 139, + "\u03b2-turn", + "structure_element" + ] + ] + }, + { + "sid": 50, + "sent": "The \u03b4Orn that connects residues D23 and A30 replaces the A\u03b224\u201329 loop.", + "section": "INTRO", + "ner": [ + [ + 4, + 8, + "\u03b4Orn", + "structure_element" + ], + [ + 32, + 35, + "D23", + "residue_name_number" + ], + [ + 40, + 43, + "A30", + "residue_name_number" + ], + [ + 57, + 59, + "A\u03b2", + "protein" + ], + [ + 59, + 64, + "24\u201329", + "residue_range" + ], + [ + 65, + 69, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 51, + "sent": "The \u03b4Orn that connects residues L17 and V36 enforces \u03b2-hairpin structure.", + "section": "INTRO", + "ner": [ + [ + 4, + 8, + "\u03b4Orn", + "structure_element" + ], + [ + 32, + 35, + "L17", + "residue_name_number" + ], + [ + 40, + 43, + "V36", + "residue_name_number" + ], + [ + 53, + 62, + "\u03b2-hairpin", + "structure_element" + ] + ] + }, + { + "sid": 52, + "sent": "We incorporated an N-methyl group at position G33 to prevent uncontrolled aggregation and precipitation of the peptide.", + "section": "INTRO", + "ner": [ + [ + 46, + 49, + "G33", + "residue_name_number" + ] + ] + }, + { + "sid": 53, + "sent": "To improve the solubility of the peptide we replaced M35 with the hydrophilic isostere of methionine, ornithine (\u03b1-linked) (Figure 1B).", + "section": "INTRO", + "ner": [ + [ + 44, + 52, + "replaced", + "experimental_method" + ], + [ + 53, + 56, + "M35", + "residue_name_number" + ], + [ + 90, + 100, + "methionine", + "residue_name" + ], + [ + 102, + 111, + "ornithine", + "residue_name" + ], + [ + 113, + 121, + "\u03b1-linked", + "protein_state" + ] + ] + }, + { + "sid": 54, + "sent": "The X-ray crystallographic structure of peptide 1 reveals that it folds to form a \u03b2-hairpin that assembles to form trimers and that the trimers further assemble to form hexamers and dodecamers.", + "section": "INTRO", + "ner": [ + [ + 4, + 36, + "X-ray crystallographic structure", + "evidence" + ], + [ + 40, + 49, + "peptide 1", + "mutant" + ], + [ + 82, + 91, + "\u03b2-hairpin", + "structure_element" + ], + [ + 115, + 122, + "trimers", + "oligomeric_state" + ], + [ + 136, + 143, + "trimers", + "oligomeric_state" + ], + [ + 169, + 177, + "hexamers", + "oligomeric_state" + ], + [ + 182, + 192, + "dodecamers", + "oligomeric_state" + ] + ] + }, + { + "sid": 55, + "sent": "(A) Cartoon illustrating the design of peptides 1 and 2 and their relationship to an A\u03b217\u201336 \u03b2-hairpin.", + "section": "FIG", + "ner": [ + [ + 39, + 55, + "peptides 1 and 2", + "chemical" + ], + [ + 85, + 87, + "A\u03b2", + "protein" + ], + [ + 87, + 92, + "17\u201336", + "residue_range" + ], + [ + 93, + 102, + "\u03b2-hairpin", + "structure_element" + ] + ] + }, + { + "sid": 56, + "sent": " (B) Chemical structure of peptide 1 illustrating A\u03b217\u201323 and A\u03b230\u201336, M35Orn, the N-methyl group, and the \u03b4-linked ornithine turns. (C) Chemical structure of peptide 2 illustrating A\u03b217\u201336, the N-methyl group, the disulfide bond across positions 24 and 29, and the \u03b4-linked ornithine turn.", + "section": "FIG", + "ner": [ + [ + 27, + 36, + "peptide 1", + "mutant" + ], + [ + 50, + 52, + "A\u03b2", + "protein" + ], + [ + 62, + 64, + "A\u03b2", + "protein" + ], + [ + 107, + 115, + "\u03b4-linked", + "protein_state" + ], + [ + 116, + 125, + "ornithine", + "residue_name" + ], + [ + 126, + 131, + "turns", + "structure_element" + ], + [ + 159, + 168, + "peptide 2", + "mutant" + ], + [ + 182, + 184, + "A\u03b2", + "protein" + ], + [ + 215, + 229, + "disulfide bond", + "ptm" + ], + [ + 247, + 249, + "24", + "residue_number" + ], + [ + 254, + 256, + "29", + "residue_number" + ], + [ + 266, + 274, + "\u03b4-linked", + "protein_state" + ], + [ + 275, + 284, + "ornithine", + "residue_name" + ], + [ + 285, + 289, + "turn", + "structure_element" + ] + ] + }, + { + "sid": 57, + "sent": "Our design of peptide 1 omitted the A\u03b224\u201329 loop.", + "section": "INTRO", + "ner": [ + [ + 14, + 23, + "peptide 1", + "mutant" + ], + [ + 36, + 38, + "A\u03b2", + "protein" + ], + [ + 38, + 43, + "24\u201329", + "residue_range" + ], + [ + 44, + 48, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 58, + "sent": "To visualize the A\u03b224\u201329 loop, we performed replica-exchange molecular dynamics (REMD) simulations on A\u03b217\u201336 using the X-ray crystallographic coordinates of A\u03b217\u201323 and A\u03b230\u201336 from peptide 1.", + "section": "INTRO", + "ner": [ + [ + 17, + 19, + "A\u03b2", + "protein" + ], + [ + 19, + 24, + "24\u201329", + "residue_range" + ], + [ + 25, + 29, + "loop", + "structure_element" + ], + [ + 44, + 79, + "replica-exchange molecular dynamics", + "experimental_method" + ], + [ + 81, + 85, + "REMD", + "experimental_method" + ], + [ + 87, + 98, + "simulations", + "experimental_method" + ], + [ + 102, + 104, + "A\u03b2", + "protein" + ], + [ + 104, + 109, + "17\u201336", + "residue_range" + ], + [ + 120, + 154, + "X-ray crystallographic coordinates", + "evidence" + ], + [ + 158, + 160, + "A\u03b2", + "protein" + ], + [ + 160, + 165, + "17\u201323", + "residue_range" + ], + [ + 170, + 172, + "A\u03b2", + "protein" + ], + [ + 172, + 177, + "30\u201336", + "residue_range" + ], + [ + 183, + 192, + "peptide 1", + "mutant" + ] + ] + }, + { + "sid": 59, + "sent": "These studies provided a working model for a trimer of A\u03b217\u201336 \u03b2-hairpins and demonstrated that the trimer should be capable of accommodating the A\u03b224\u201329 loop.", + "section": "INTRO", + "ner": [ + [ + 45, + 51, + "trimer", + "oligomeric_state" + ], + [ + 55, + 57, + "A\u03b2", + "protein" + ], + [ + 57, + 62, + "17\u201336", + "residue_range" + ], + [ + 63, + 73, + "\u03b2-hairpins", + "structure_element" + ], + [ + 100, + 106, + "trimer", + "oligomeric_state" + ], + [ + 146, + 148, + "A\u03b2", + "protein" + ], + [ + 148, + 153, + "24\u201329", + "residue_range" + ], + [ + 154, + 158, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 60, + "sent": "In the current study we set out to restore the A\u03b224\u201329 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form.", + "section": "INTRO", + "ner": [ + [ + 35, + 42, + "restore", + "experimental_method" + ], + [ + 47, + 49, + "A\u03b2", + "protein" + ], + [ + 49, + 54, + "24\u201329", + "residue_range" + ], + [ + 55, + 59, + "loop", + "structure_element" + ], + [ + 61, + 72, + "reintroduce", + "experimental_method" + ], + [ + 77, + 87, + "methionine", + "residue_name" + ], + [ + 108, + 110, + "35", + "residue_number" + ], + [ + 130, + 163, + "X-ray crystallographic structures", + "evidence" + ], + [ + 167, + 176, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 61, + "sent": "We designed peptide 2 as a homologue of peptide 1 that embodies these ideas.", + "section": "INTRO", + "ner": [ + [ + 12, + 21, + "peptide 2", + "mutant" + ], + [ + 40, + 49, + "peptide 1", + "mutant" + ] + ] + }, + { + "sid": 62, + "sent": "Peptide 2 contains a methionine residue at position 35 and an A\u03b224\u201329 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C).", + "section": "INTRO", + "ner": [ + [ + 0, + 9, + "Peptide 2", + "mutant" + ], + [ + 21, + 31, + "methionine", + "residue_name" + ], + [ + 52, + 54, + "35", + "residue_number" + ], + [ + 62, + 64, + "A\u03b2", + "protein" + ], + [ + 64, + 69, + "24\u201329", + "residue_range" + ], + [ + 70, + 74, + "loop", + "structure_element" + ], + [ + 89, + 91, + "24", + "residue_number" + ], + [ + 96, + 98, + "29", + "residue_number" + ], + [ + 100, + 103, + "Val", + "residue_name" + ], + [ + 108, + 111, + "Gly", + "residue_name" + ], + [ + 113, + 120, + "mutated", + "experimental_method" + ], + [ + 124, + 132, + "cysteine", + "residue_name" + ], + [ + 149, + 163, + "disulfide bond", + "ptm" + ] + ] + }, + { + "sid": 63, + "sent": "Here, we describe the development of peptide 2 and report the X-ray crystallographic structures of the trimer, dodecamer, and annular pore observed within the crystal structure.", + "section": "INTRO", + "ner": [ + [ + 37, + 46, + "peptide 2", + "mutant" + ], + [ + 62, + 95, + "X-ray crystallographic structures", + "evidence" + ], + [ + 103, + 109, + "trimer", + "oligomeric_state" + ], + [ + 111, + 120, + "dodecamer", + "oligomeric_state" + ], + [ + 126, + 138, + "annular pore", + "site" + ], + [ + 159, + 176, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 64, + "sent": "Development of Peptide 2", + "section": "RESULTS", + "ner": [ + [ + 15, + 24, + "Peptide 2", + "mutant" + ] + ] + }, + { + "sid": 65, + "sent": "We developed peptide 2 from peptide 1 by an iterative process, in which we first attempted to restore the A\u03b224\u201329 loop without a disulfide linkage.", + "section": "RESULTS", + "ner": [ + [ + 13, + 22, + "peptide 2", + "mutant" + ], + [ + 28, + 37, + "peptide 1", + "mutant" + ], + [ + 106, + 108, + "A\u03b2", + "protein" + ], + [ + 108, + 113, + "24\u201329", + "residue_range" + ], + [ + 114, + 118, + "loop", + "structure_element" + ], + [ + 129, + 146, + "disulfide linkage", + "ptm" + ] + ] + }, + { + "sid": 66, + "sent": "We envisioned peptide 3 as a homologue of peptide 1 with the A\u03b224\u201329 loop in place of the \u03b4Orn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19.", + "section": "RESULTS", + "ner": [ + [ + 14, + 23, + "peptide 3", + "mutant" + ], + [ + 42, + 51, + "peptide 1", + "mutant" + ], + [ + 61, + 63, + "A\u03b2", + "protein" + ], + [ + 63, + 68, + "24\u201329", + "residue_range" + ], + [ + 69, + 73, + "loop", + "structure_element" + ], + [ + 90, + 94, + "\u03b4Orn", + "structure_element" + ], + [ + 109, + 112, + "D23", + "residue_name_number" + ], + [ + 117, + 120, + "A30", + "residue_name_number" + ], + [ + 125, + 144, + "p-iodophenylalanine", + "chemical" + ], + [ + 146, + 148, + "FI", + "chemical" + ], + [ + 162, + 165, + "F19", + "residue_name_number" + ] + ] + }, + { + "sid": 67, + "sent": "We routinely use p-iodophenylalanine to determine the X-ray crystallographic phases.", + "section": "RESULTS", + "ner": [ + [ + 17, + 36, + "p-iodophenylalanine", + "chemical" + ], + [ + 54, + 83, + "X-ray crystallographic phases", + "evidence" + ] + ] + }, + { + "sid": 68, + "sent": "After determining the X-ray crystallographic structure of the p-iodophenylalanine variant we attempt to determine the structure of the native phenylalanine compound by isomorphous replacement.", + "section": "RESULTS", + "ner": [ + [ + 22, + 54, + "X-ray crystallographic structure", + "evidence" + ], + [ + 62, + 81, + "p-iodophenylalanine", + "chemical" + ], + [ + 118, + 127, + "structure", + "evidence" + ], + [ + 142, + 155, + "phenylalanine", + "residue_name" + ], + [ + 168, + 191, + "isomorphous replacement", + "experimental_method" + ] + ] + }, + { + "sid": 69, + "sent": "Upon synthesizing peptide 3, we found that it formed an amorphous precipitate in most crystallization conditions screened and failed to afford crystals in any condition.", + "section": "RESULTS", + "ner": [ + [ + 18, + 27, + "peptide 3", + "mutant" + ], + [ + 143, + 151, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 70, + "sent": "We postulate that the loss of the \u03b4Orn constraint leads to conformational heterogeneity that prevents peptide 3 from crystallizing.", + "section": "RESULTS", + "ner": [ + [ + 34, + 38, + "\u03b4Orn", + "structure_element" + ], + [ + 102, + 111, + "peptide 3", + "mutant" + ] + ] + }, + { + "sid": 71, + "sent": "To address this issue, we next incorporated a disulfide bond between residues 24 and 29 as a conformational constraint that serves as a surrogate for \u03b4Orn.", + "section": "RESULTS", + "ner": [ + [ + 46, + 60, + "disulfide bond", + "ptm" + ], + [ + 78, + 80, + "24", + "residue_number" + ], + [ + 85, + 87, + "29", + "residue_number" + ], + [ + 150, + 154, + "\u03b4Orn", + "structure_element" + ] + ] + }, + { + "sid": 72, + "sent": "We designed peptide 4 to embody this idea, mutating Val24 and Gly29 to cysteine and forming an interstrand disulfide linkage.", + "section": "RESULTS", + "ner": [ + [ + 12, + 21, + "peptide 4", + "mutant" + ], + [ + 43, + 51, + "mutating", + "experimental_method" + ], + [ + 52, + 57, + "Val24", + "residue_name_number" + ], + [ + 62, + 67, + "Gly29", + "residue_name_number" + ], + [ + 71, + 79, + "cysteine", + "residue_name" + ], + [ + 107, + 124, + "disulfide linkage", + "ptm" + ] + ] + }, + { + "sid": 73, + "sent": "We mutated these residues because they occupy the same position as the \u03b4Orn that connects D23 and A30 in peptide 1.", + "section": "RESULTS", + "ner": [ + [ + 3, + 10, + "mutated", + "experimental_method" + ], + [ + 71, + 75, + "\u03b4Orn", + "structure_element" + ], + [ + 90, + 93, + "D23", + "residue_name_number" + ], + [ + 98, + 101, + "A30", + "residue_name_number" + ], + [ + 105, + 114, + "peptide 1", + "mutant" + ] + ] + }, + { + "sid": 74, + "sent": "Residues V24 and G29 form a non-hydrogen-bonded pair, which can readily accommodate disulfide linkages in antiparallel \u03b2-sheets.", + "section": "RESULTS", + "ner": [ + [ + 9, + 12, + "V24", + "residue_name_number" + ], + [ + 17, + 20, + "G29", + "residue_name_number" + ], + [ + 28, + 52, + "non-hydrogen-bonded pair", + "bond_interaction" + ], + [ + 84, + 102, + "disulfide linkages", + "ptm" + ], + [ + 106, + 127, + "antiparallel \u03b2-sheets", + "structure_element" + ] + ] + }, + { + "sid": 75, + "sent": "Disulfide bonds across non-hydrogen-bonded pairs stabilize \u03b2-hairpins, while disulfide bonds across hydrogen-bonded pairs do not.", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Disulfide bonds", + "ptm" + ], + [ + 23, + 48, + "non-hydrogen-bonded pairs", + "bond_interaction" + ], + [ + 59, + 69, + "\u03b2-hairpins", + "structure_element" + ], + [ + 77, + 92, + "disulfide bonds", + "ptm" + ], + [ + 100, + 121, + "hydrogen-bonded pairs", + "bond_interaction" + ] + ] + }, + { + "sid": 76, + "sent": "Although the disulfide bond between positions 24 and 29 helps stabilize the \u03b2-hairpin, it does not alter the charge or substantially change the hydrophobicity of the A\u03b217\u201336 \u03b2-hairpin.", + "section": "RESULTS", + "ner": [ + [ + 13, + 27, + "disulfide bond", + "ptm" + ], + [ + 46, + 48, + "24", + "residue_number" + ], + [ + 53, + 55, + "29", + "residue_number" + ], + [ + 76, + 85, + "\u03b2-hairpin", + "structure_element" + ], + [ + 166, + 168, + "A\u03b2", + "protein" + ], + [ + 168, + 173, + "17\u201336", + "residue_range" + ], + [ + 174, + 183, + "\u03b2-hairpin", + "structure_element" + ] + ] + }, + { + "sid": 77, + "sent": "We were gratified to find that peptide 4 afforded crystals suitable for X-ray crystallography.", + "section": "RESULTS", + "ner": [ + [ + 31, + 40, + "peptide 4", + "mutant" + ], + [ + 50, + 58, + "crystals", + "evidence" + ], + [ + 72, + 93, + "X-ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 78, + "sent": "As the next step in the iterative process, we determined the X-ray crystallographic structure of this peptide (PDB 5HOW).", + "section": "RESULTS", + "ner": [ + [ + 46, + 56, + "determined", + "experimental_method" + ], + [ + 61, + 93, + "X-ray crystallographic structure", + "evidence" + ] + ] + }, + { + "sid": 79, + "sent": "After determining the X-ray crystallographic structure of peptide 4 we reintroduced the native phenylalanine at position 19 and the methionine at position 35 to afford peptide 2.", + "section": "RESULTS", + "ner": [ + [ + 22, + 54, + "X-ray crystallographic structure", + "evidence" + ], + [ + 58, + 67, + "peptide 4", + "mutant" + ], + [ + 71, + 83, + "reintroduced", + "experimental_method" + ], + [ + 95, + 108, + "phenylalanine", + "residue_name" + ], + [ + 121, + 123, + "19", + "residue_number" + ], + [ + 132, + 142, + "methionine", + "residue_name" + ], + [ + 155, + 157, + "35", + "residue_number" + ], + [ + 168, + 177, + "peptide 2", + "mutant" + ] + ] + }, + { + "sid": 80, + "sent": "We completed the iterative process\u2014from 1 to 3 to 4 to 2\u2014by successfully determining the X-ray crystallographic structure of peptide 2 (PDB 5HOX and 5HOY).", + "section": "RESULTS", + "ner": [ + [ + 89, + 121, + "X-ray crystallographic structure", + "evidence" + ], + [ + 125, + 134, + "peptide 2", + "mutant" + ] + ] + }, + { + "sid": 81, + "sent": "The following sections describe the synthesis of peptides 2\u20134 and the X-ray crystallographic structure of peptide 2.", + "section": "RESULTS", + "ner": [ + [ + 49, + 61, + "peptides 2\u20134", + "mutant" + ], + [ + 70, + 102, + "X-ray crystallographic structure", + "evidence" + ], + [ + 106, + 115, + "peptide 2", + "mutant" + ] + ] + }, + { + "sid": 82, + "sent": "Synthesis of Peptides 2\u20134", + "section": "RESULTS", + "ner": [ + [ + 13, + 25, + "Peptides 2\u20134", + "mutant" + ] + ] + }, + { + "sid": 83, + "sent": "We synthesized peptides 2\u20134 by similar procedures to those we have developed for other macrocyclic peptides.", + "section": "RESULTS", + "ner": [ + [ + 15, + 27, + "peptides 2\u20134", + "mutant" + ] + ] + }, + { + "sid": 84, + "sent": "In synthesizing peptides 2 and 4 we formed the disulfide linkage after macrolactamization and deprotection of the acid-labile side chain protecting groups.", + "section": "RESULTS", + "ner": [ + [ + 16, + 32, + "peptides 2 and 4", + "mutant" + ], + [ + 47, + 64, + "disulfide linkage", + "ptm" + ] + ] + }, + { + "sid": 85, + "sent": "We used acid-stable Acm-protected cysteine residues at positions 24 and 29 and removed the Acm groups by oxidation with I2 in aqueous acetic acid to afford the disulfide linkage.", + "section": "RESULTS", + "ner": [ + [ + 8, + 19, + "acid-stable", + "protein_state" + ], + [ + 20, + 33, + "Acm-protected", + "protein_state" + ], + [ + 34, + 42, + "cysteine", + "residue_name" + ], + [ + 65, + 67, + "24", + "residue_number" + ], + [ + 72, + 74, + "29", + "residue_number" + ], + [ + 134, + 145, + "acetic acid", + "chemical" + ], + [ + 160, + 177, + "disulfide linkage", + "ptm" + ] + ] + }, + { + "sid": 86, + "sent": "Peptides 2\u20134 were purified by RP-HPLC.", + "section": "RESULTS", + "ner": [ + [ + 0, + 12, + "Peptides 2\u20134", + "mutant" + ], + [ + 30, + 37, + "RP-HPLC", + "experimental_method" + ] + ] + }, + { + "sid": 87, + "sent": "Crystallization, X-ray Crystallographic Data Collection, Data Processing, and Structure Determination of Peptides 2 and 4", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Crystallization", + "experimental_method" + ], + [ + 17, + 55, + "X-ray Crystallographic Data Collection", + "experimental_method" + ], + [ + 78, + 101, + "Structure Determination", + "experimental_method" + ], + [ + 105, + 121, + "Peptides 2 and 4", + "mutant" + ] + ] + }, + { + "sid": 88, + "sent": "We screened crystallization conditions for peptide 4 in a 96-well-plate format using three different Hampton Research crystallization kits (Crystal Screen, Index, and PEG/Ion) with three ratios of peptide and mother liquor per condition (864 experiments).", + "section": "RESULTS", + "ner": [ + [ + 3, + 38, + "screened crystallization conditions", + "experimental_method" + ], + [ + 43, + 52, + "peptide 4", + "mutant" + ] + ] + }, + { + "sid": 89, + "sent": "Peptide 4 afforded crystals in a single set of conditions containing HEPES buffer and Jeffamine M-600\u2014the same crystallization conditions that afforded crystals of peptide 1.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Peptide 4", + "mutant" + ], + [ + 19, + 27, + "crystals", + "evidence" + ], + [ + 86, + 101, + "Jeffamine M-600", + "chemical" + ], + [ + 152, + 160, + "crystals", + "evidence" + ], + [ + 164, + 173, + "peptide 1", + "mutant" + ] + ] + }, + { + "sid": 90, + "sent": "Peptide 2 also afforded crystals in these conditions.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Peptide 2", + "mutant" + ], + [ + 24, + 32, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 91, + "sent": "We further optimized these conditions to rapidly (\u223c72 h) yield crystals suitable for X-ray crystallography.", + "section": "RESULTS", + "ner": [ + [ + 63, + 71, + "crystals", + "evidence" + ], + [ + 85, + 106, + "X-ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 92, + "sent": "The optimized conditions consist of 0.1 M HEPES at pH 6.4 with 31% Jeffamine M-600 for peptide 4 and 0.1 M HEPES pH 7.1 with 29% Jeffamine M-600 for peptide 2.", + "section": "RESULTS", + "ner": [ + [ + 42, + 47, + "HEPES", + "chemical" + ], + [ + 67, + 82, + "Jeffamine M-600", + "chemical" + ], + [ + 87, + 96, + "peptide 4", + "mutant" + ], + [ + 107, + 112, + "HEPES", + "chemical" + ], + [ + 129, + 144, + "Jeffamine M-600", + "chemical" + ], + [ + 149, + 158, + "peptide 2", + "mutant" + ] + ] + }, + { + "sid": 93, + "sent": "Crystal diffraction data for peptides 4 and 2 were collected in-house with a Rigaku MicroMax 007HF X-ray diffractometer at 1.54 \u00c5 wavelength.", + "section": "RESULTS", + "ner": [ + [ + 0, + 24, + "Crystal diffraction data", + "evidence" + ], + [ + 29, + 45, + "peptides 4 and 2", + "mutant" + ] + ] + }, + { + "sid": 94, + "sent": "Crystal diffraction data for peptide 2 were also collected at the Advanced Light Source at Lawrence Berkeley National Laboratory with a synchrotron source at 1.00 \u00c5 wavelength to achieve higher resolution.", + "section": "RESULTS", + "ner": [ + [ + 0, + 24, + "Crystal diffraction data", + "evidence" + ], + [ + 29, + 38, + "peptide 2", + "mutant" + ] + ] + }, + { + "sid": 95, + "sent": "Data from peptides 4 and 2 suitable for refinement at 2.30 \u00c5 were obtained from the diffractometer; data from peptide 2 suitable for refinement at 1.90 \u00c5 were obtained from the synchrotron.", + "section": "RESULTS", + "ner": [ + [ + 10, + 26, + "peptides 4 and 2", + "mutant" + ], + [ + 110, + 119, + "peptide 2", + "mutant" + ] + ] + }, + { + "sid": 96, + "sent": "Data for peptides 4 and 2 were scaled and merged using XDS.", + "section": "RESULTS", + "ner": [ + [ + 9, + 25, + "peptides 4 and 2", + "mutant" + ] + ] + }, + { + "sid": 97, + "sent": "Phases for peptide 4 were determined by single-wavelength anomalous diffraction (SAD) phasing by using the coordinates of the iodine anomalous signal from p-iodophenylalanine.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Phases", + "evidence" + ], + [ + 11, + 20, + "peptide 4", + "mutant" + ], + [ + 40, + 79, + "single-wavelength anomalous diffraction", + "experimental_method" + ], + [ + 81, + 84, + "SAD", + "experimental_method" + ], + [ + 86, + 93, + "phasing", + "experimental_method" + ], + [ + 126, + 149, + "iodine anomalous signal", + "evidence" + ], + [ + 155, + 174, + "p-iodophenylalanine", + "chemical" + ] + ] + }, + { + "sid": 98, + "sent": "Phases for peptide 2 were determined by isomorphous replacement of peptide 4.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Phases", + "evidence" + ], + [ + 11, + 20, + "peptide 2", + "mutant" + ], + [ + 40, + 63, + "isomorphous replacement", + "experimental_method" + ], + [ + 67, + 76, + "peptide 4", + "mutant" + ] + ] + }, + { + "sid": 99, + "sent": "The structures of peptides 2 and 4 were solved and refined in the P6122 space group.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 18, + 34, + "peptides 2 and 4", + "mutant" + ], + [ + 40, + 46, + "solved", + "experimental_method" + ] + ] + }, + { + "sid": 100, + "sent": "The asymmetric unit of each peptide consists of six monomers, arranged as two trimers.", + "section": "RESULTS", + "ner": [ + [ + 28, + 35, + "peptide", + "chemical" + ], + [ + 52, + 60, + "monomers", + "oligomeric_state" + ], + [ + 78, + 85, + "trimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 101, + "sent": "Peptides 2 and 4 form morphologically identical structures and assemblies in the crystal lattice.", + "section": "RESULTS", + "ner": [ + [ + 0, + 16, + "Peptides 2 and 4", + "mutant" + ], + [ + 81, + 96, + "crystal lattice", + "evidence" + ] + ] + }, + { + "sid": 102, + "sent": "X-ray Crystallographic Structure of Peptide 2 and the Oligomers It Forms", + "section": "RESULTS", + "ner": [ + [ + 0, + 32, + "X-ray Crystallographic Structure", + "evidence" + ], + [ + 36, + 45, + "Peptide 2", + "mutant" + ], + [ + 54, + 63, + "Oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 103, + "sent": "The X-ray crystallographic structure of peptide 2 reveals that it folds to form a twisted \u03b2-hairpin comprising two \u03b2-strands connected by a loop (Figure 2A).", + "section": "RESULTS", + "ner": [ + [ + 4, + 36, + "X-ray crystallographic structure", + "evidence" + ], + [ + 40, + 49, + "peptide 2", + "mutant" + ], + [ + 82, + 99, + "twisted \u03b2-hairpin", + "structure_element" + ], + [ + 115, + 124, + "\u03b2-strands", + "structure_element" + ], + [ + 140, + 144, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 104, + "sent": "Eight residues make up each surface of the \u03b2-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface.", + "section": "RESULTS", + "ner": [ + [ + 43, + 52, + "\u03b2-hairpin", + "structure_element" + ], + [ + 54, + 57, + "L17", + "residue_name_number" + ], + [ + 59, + 62, + "F19", + "residue_name_number" + ], + [ + 64, + 67, + "A21", + "residue_name_number" + ], + [ + 69, + 72, + "D23", + "residue_name_number" + ], + [ + 74, + 77, + "A30", + "residue_name_number" + ], + [ + 79, + 82, + "I32", + "residue_name_number" + ], + [ + 84, + 87, + "L34", + "residue_name_number" + ], + [ + 93, + 96, + "V36", + "residue_name_number" + ], + [ + 118, + 121, + "V18", + "residue_name_number" + ], + [ + 123, + 126, + "F20", + "residue_name_number" + ], + [ + 128, + 131, + "E22", + "residue_name_number" + ], + [ + 133, + 136, + "C24", + "residue_name_number" + ], + [ + 138, + 141, + "C29", + "residue_name_number" + ], + [ + 143, + 146, + "I31", + "residue_name_number" + ], + [ + 148, + 151, + "G33", + "residue_name_number" + ], + [ + 157, + 160, + "M35", + "residue_name_number" + ] + ] + }, + { + "sid": 105, + "sent": "The \u03b2-strands of the monomers in the asymmetric unit are virtually identical, differing primarily in rotamers of F20, E22, C24, C29, I31, and M35 (Figure S1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "\u03b2-strands", + "structure_element" + ], + [ + 21, + 29, + "monomers", + "oligomeric_state" + ], + [ + 113, + 116, + "F20", + "residue_name_number" + ], + [ + 118, + 121, + "E22", + "residue_name_number" + ], + [ + 123, + 126, + "C24", + "residue_name_number" + ], + [ + 128, + 131, + "C29", + "residue_name_number" + ], + [ + 133, + 136, + "I31", + "residue_name_number" + ], + [ + 142, + 145, + "M35", + "residue_name_number" + ] + ] + }, + { + "sid": 106, + "sent": "The disulfide linkages suffered radiation damage under synchrotron radiation.", + "section": "RESULTS", + "ner": [ + [ + 4, + 22, + "disulfide linkages", + "ptm" + ] + ] + }, + { + "sid": 107, + "sent": "We refined three of the \u03b2-hairpins with intact disulfide linkages and three with thiols to represent cleaved disulfide linkages in the synchrotron data set (PDB 5HOX).", + "section": "RESULTS", + "ner": [ + [ + 3, + 10, + "refined", + "experimental_method" + ], + [ + 24, + 34, + "\u03b2-hairpins", + "structure_element" + ], + [ + 40, + 46, + "intact", + "protein_state" + ], + [ + 47, + 65, + "disulfide linkages", + "ptm" + ], + [ + 101, + 108, + "cleaved", + "protein_state" + ], + [ + 109, + 127, + "disulfide linkages", + "ptm" + ] + ] + }, + { + "sid": 108, + "sent": "No evidence for cleavage of the disulfides was observed in the refinement of the data set collected on the X-ray diffractometer, and we refined all disulfide linkages as intact (PDB 5HOY).", + "section": "RESULTS", + "ner": [ + [ + 32, + 42, + "disulfides", + "ptm" + ], + [ + 63, + 73, + "refinement", + "experimental_method" + ], + [ + 136, + 143, + "refined", + "experimental_method" + ], + [ + 148, + 166, + "disulfide linkages", + "ptm" + ], + [ + 170, + 176, + "intact", + "protein_state" + ] + ] + }, + { + "sid": 109, + "sent": "X-ray crystallographic structure of peptide 2 (PDB 5HOX, synchrotron data set). (A) X-ray crystallographic structure of a representative \u03b2-hairpin monomer formed by peptide 2. (B) Overlay of the six \u03b2-hairpin monomers in the asymmetric unit.", + "section": "FIG", + "ner": [ + [ + 36, + 45, + "peptide 2", + "mutant" + ], + [ + 84, + 116, + "X-ray crystallographic structure", + "evidence" + ], + [ + 137, + 146, + "\u03b2-hairpin", + "structure_element" + ], + [ + 147, + 154, + "monomer", + "oligomeric_state" + ], + [ + 165, + 174, + "peptide 2", + "mutant" + ], + [ + 180, + 187, + "Overlay", + "experimental_method" + ], + [ + 199, + 208, + "\u03b2-hairpin", + "structure_element" + ], + [ + 209, + 217, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 110, + "sent": "The \u03b2-hairpins are shown as cartoons to illustrate the differences in the A\u03b225\u201328 loops.", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "\u03b2-hairpins", + "structure_element" + ], + [ + 74, + 76, + "A\u03b2", + "protein" + ], + [ + 76, + 81, + "25\u201328", + "residue_range" + ], + [ + 82, + 87, + "loops", + "structure_element" + ] + ] + }, + { + "sid": 111, + "sent": "The A\u03b225\u201328 loops of the six monomers within the asymmetric unit vary substantially in backbone geometry and side chain rotamers (Figures 2B and S1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 6, + "A\u03b2", + "protein" + ], + [ + 6, + 11, + "25\u201328", + "residue_range" + ], + [ + 12, + 17, + "loops", + "structure_element" + ], + [ + 29, + 37, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 112, + "sent": "The electron density for the loops is weak and diffuse compared to the electron density for the \u03b2-strands.", + "section": "RESULTS", + "ner": [ + [ + 4, + 20, + "electron density", + "evidence" + ], + [ + 29, + 34, + "loops", + "structure_element" + ], + [ + 71, + 87, + "electron density", + "evidence" + ], + [ + 96, + 105, + "\u03b2-strands", + "structure_element" + ] + ] + }, + { + "sid": 113, + "sent": "The B values for the loops are large, indicating that the loops are dynamic and not well ordered.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "B values", + "evidence" + ], + [ + 21, + 26, + "loops", + "structure_element" + ], + [ + 58, + 63, + "loops", + "structure_element" + ] + ] + }, + { + "sid": 114, + "sent": "Thus, the differences in backbone geometry and side chain rotamers among the loops are likely of little significance and should be interpreted with caution.", + "section": "RESULTS", + "ner": [ + [ + 77, + 82, + "loops", + "structure_element" + ] + ] + }, + { + "sid": 115, + "sent": "Peptide 2 assembles into oligomers similar in morphology to those formed by peptide 1.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Peptide 2", + "mutant" + ], + [ + 25, + 34, + "oligomers", + "oligomeric_state" + ], + [ + 76, + 85, + "peptide 1", + "mutant" + ] + ] + }, + { + "sid": 116, + "sent": "Like peptide 1, peptide 2 forms a triangular trimer, and four trimers assemble to form a dodecamer.", + "section": "RESULTS", + "ner": [ + [ + 5, + 14, + "peptide 1", + "mutant" + ], + [ + 16, + 25, + "peptide 2", + "mutant" + ], + [ + 34, + 44, + "triangular", + "protein_state" + ], + [ + 45, + 51, + "trimer", + "oligomeric_state" + ], + [ + 62, + 69, + "trimers", + "oligomeric_state" + ], + [ + 89, + 98, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 117, + "sent": "In the higher-order assembly of the dodecamers formed by peptide 2 a new structure emerges, not seen in peptide 1, an annular pore consisting of five dodecamers.", + "section": "RESULTS", + "ner": [ + [ + 36, + 46, + "dodecamers", + "oligomeric_state" + ], + [ + 57, + 66, + "peptide 2", + "mutant" + ], + [ + 73, + 82, + "structure", + "evidence" + ], + [ + 104, + 113, + "peptide 1", + "mutant" + ], + [ + 118, + 130, + "annular pore", + "site" + ], + [ + 150, + 160, + "dodecamers", + "oligomeric_state" + ] + ] + }, + { + "sid": 118, + "sent": "Trimer", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 119, + "sent": "Peptide 2 forms a trimer, much like that which we observed previously for peptide 1, in which three \u03b2-hairpins assemble to form an equilateral triangle (Figure 3A).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Peptide 2", + "mutant" + ], + [ + 18, + 24, + "trimer", + "oligomeric_state" + ], + [ + 74, + 83, + "peptide 1", + "mutant" + ], + [ + 100, + 110, + "\u03b2-hairpins", + "structure_element" + ], + [ + 131, + 151, + "equilateral triangle", + "structure_element" + ] + ] + }, + { + "sid": 120, + "sent": "The trimer maintains all of the same stabilizing contacts as those of peptide 1.", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "trimer", + "oligomeric_state" + ], + [ + 70, + 79, + "peptide 1", + "mutant" + ] + ] + }, + { + "sid": 121, + "sent": "Hydrogen bonding and hydrophobic interactions between residues on the \u03b2-strands comprising A\u03b217\u201323 and A\u03b230\u201336 stabilize the core of the trimer.", + "section": "RESULTS", + "ner": [ + [ + 0, + 16, + "Hydrogen bonding", + "bond_interaction" + ], + [ + 21, + 45, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 70, + 79, + "\u03b2-strands", + "structure_element" + ], + [ + 91, + 93, + "A\u03b2", + "protein" + ], + [ + 93, + 98, + "17\u201323", + "residue_range" + ], + [ + 103, + 105, + "A\u03b2", + "protein" + ], + [ + 105, + 110, + "30\u201336", + "residue_range" + ], + [ + 125, + 129, + "core", + "structure_element" + ], + [ + 137, + 143, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 122, + "sent": "The disulfide bonds between residues 24 and 29 are adjacent to the structural core of the trimer and do not make any substantial intermolecular contacts.", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "disulfide bonds", + "ptm" + ], + [ + 37, + 39, + "24", + "residue_number" + ], + [ + 44, + 46, + "29", + "residue_number" + ], + [ + 67, + 82, + "structural core", + "structure_element" + ], + [ + 90, + 96, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 123, + "sent": "Two crystallographically distinct trimers comprise the peptide portion of the asymmetric unit.", + "section": "RESULTS", + "ner": [ + [ + 34, + 41, + "trimers", + "oligomeric_state" + ], + [ + 55, + 62, + "peptide", + "chemical" + ] + ] + }, + { + "sid": 124, + "sent": "The two trimers are almost identical in structure, differing slightly among side chain rotamers and loop conformations.", + "section": "RESULTS", + "ner": [ + [ + 8, + 15, + "trimers", + "oligomeric_state" + ], + [ + 100, + 104, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 125, + "sent": "X-ray crystallographic structure of the trimer formed by peptide 2. (A) Triangular trimer.", + "section": "FIG", + "ner": [ + [ + 0, + 32, + "X-ray crystallographic structure", + "evidence" + ], + [ + 40, + 46, + "trimer", + "oligomeric_state" + ], + [ + 57, + 66, + "peptide 2", + "mutant" + ], + [ + 72, + 82, + "Triangular", + "protein_state" + ], + [ + 83, + 89, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 126, + "sent": "The three water molecules in the center hole of the trimer are shown as spheres. (B) Detailed view of the intermolecular hydrogen bonds between the main chains of V18 and E22 and \u03b4Orn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The F20 face of the trimer, with key side chains as spheres.", + "section": "FIG", + "ner": [ + [ + 10, + 15, + "water", + "chemical" + ], + [ + 52, + 58, + "trimer", + "oligomeric_state" + ], + [ + 121, + 135, + "hydrogen bonds", + "bond_interaction" + ], + [ + 163, + 166, + "V18", + "residue_name_number" + ], + [ + 171, + 174, + "E22", + "residue_name_number" + ], + [ + 179, + 183, + "\u03b4Orn", + "structure_element" + ], + [ + 188, + 191, + "C24", + "residue_name_number" + ], + [ + 221, + 231, + "triangular", + "protein_state" + ], + [ + 232, + 238, + "trimer", + "oligomeric_state" + ], + [ + 248, + 251, + "F19", + "residue_name_number" + ], + [ + 264, + 270, + "trimer", + "oligomeric_state" + ], + [ + 319, + 322, + "F20", + "residue_name_number" + ], + [ + 335, + 341, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 127, + "sent": "A network of 18 intermolecular hydrogen bonds helps stabilize the trimer.", + "section": "RESULTS", + "ner": [ + [ + 31, + 45, + "hydrogen bonds", + "bond_interaction" + ], + [ + 66, + 72, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 128, + "sent": "At the corners of the trimer, the pairs of \u03b2-hairpin monomers form four hydrogen bonds: two between the main chains of V18 and E22 and two between \u03b4Orn and the main chain of C24 (Figure 3B).", + "section": "RESULTS", + "ner": [ + [ + 22, + 28, + "trimer", + "oligomeric_state" + ], + [ + 43, + 52, + "\u03b2-hairpin", + "structure_element" + ], + [ + 53, + 61, + "monomers", + "oligomeric_state" + ], + [ + 72, + 86, + "hydrogen bonds", + "bond_interaction" + ], + [ + 119, + 122, + "V18", + "residue_name_number" + ], + [ + 127, + 130, + "E22", + "residue_name_number" + ], + [ + 147, + 151, + "\u03b4Orn", + "structure_element" + ], + [ + 174, + 177, + "C24", + "residue_name_number" + ] + ] + }, + { + "sid": 129, + "sent": "Three ordered water molecules fill the hole in the center of the trimer, hydrogen bonding to each other and to the main chain of F20 (Figure 3A).", + "section": "RESULTS", + "ner": [ + [ + 14, + 19, + "water", + "chemical" + ], + [ + 65, + 71, + "trimer", + "oligomeric_state" + ], + [ + 73, + 89, + "hydrogen bonding", + "bond_interaction" + ], + [ + 129, + 132, + "F20", + "residue_name_number" + ] + ] + }, + { + "sid": 130, + "sent": "Hydrophobic contacts between residues at the three corners of the trimer, where the \u03b2-hairpins meet, further stabilize the trimer.", + "section": "RESULTS", + "ner": [ + [ + 0, + 20, + "Hydrophobic contacts", + "bond_interaction" + ], + [ + 66, + 72, + "trimer", + "oligomeric_state" + ], + [ + 84, + 94, + "\u03b2-hairpins", + "structure_element" + ], + [ + 123, + 129, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 131, + "sent": "At each corner, the side chains of residues L17, F19, and V36 of one \u03b2-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent \u03b2-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer.", + "section": "RESULTS", + "ner": [ + [ + 44, + 47, + "L17", + "residue_name_number" + ], + [ + 49, + 52, + "F19", + "residue_name_number" + ], + [ + 58, + 61, + "V36", + "residue_name_number" + ], + [ + 69, + 78, + "\u03b2-hairpin", + "structure_element" + ], + [ + 120, + 123, + "A21", + "residue_name_number" + ], + [ + 125, + 128, + "I32", + "residue_name_number" + ], + [ + 130, + 133, + "L34", + "residue_name_number" + ], + [ + 144, + 147, + "D23", + "residue_name_number" + ], + [ + 164, + 173, + "\u03b2-hairpin", + "structure_element" + ], + [ + 186, + 205, + "hydrophobic cluster", + "site" + ], + [ + 229, + 249, + "hydrophobic clusters", + "site" + ], + [ + 265, + 284, + "hydrophobic surface", + "site" + ], + [ + 304, + 310, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 132, + "sent": "The other face of the trimer displays a smaller hydrophobic surface, which includes the side chains of residues V18, F20, and I31 of the three \u03b2-hairpins (Figure 3D).", + "section": "RESULTS", + "ner": [ + [ + 22, + 28, + "trimer", + "oligomeric_state" + ], + [ + 48, + 67, + "hydrophobic surface", + "site" + ], + [ + 112, + 115, + "V18", + "residue_name_number" + ], + [ + 117, + 120, + "F20", + "residue_name_number" + ], + [ + 126, + 129, + "I31", + "residue_name_number" + ], + [ + 143, + 153, + "\u03b2-hairpins", + "structure_element" + ] + ] + }, + { + "sid": 133, + "sent": "In subsequent discussion, we designate the former surface the \u201cF19 face\u201d and the latter surface the \u201cF20 face\u201d.", + "section": "RESULTS", + "ner": [ + [ + 63, + 66, + "F19", + "residue_name_number" + ], + [ + 101, + 104, + "F20", + "residue_name_number" + ] + ] + }, + { + "sid": 134, + "sent": "Dodecamer", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 135, + "sent": "Four trimers assemble to form a dodecamer.", + "section": "RESULTS", + "ner": [ + [ + 5, + 12, + "trimers", + "oligomeric_state" + ], + [ + 32, + 41, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 136, + "sent": "The four trimers arrange in a tetrahedral fashion, creating a central cavity inside the dodecamer. Because each trimer is triangular, the resulting arrangement resembles an octahedron.", + "section": "RESULTS", + "ner": [ + [ + 9, + 16, + "trimers", + "oligomeric_state" + ], + [ + 30, + 41, + "tetrahedral", + "protein_state" + ], + [ + 62, + 76, + "central cavity", + "site" + ], + [ + 88, + 97, + "dodecamer", + "oligomeric_state" + ], + [ + 112, + 118, + "trimer", + "oligomeric_state" + ], + [ + 122, + 132, + "triangular", + "protein_state" + ], + [ + 173, + 183, + "octahedron", + "protein_state" + ] + ] + }, + { + "sid": 137, + "sent": "Each of the 12 \u03b2-hairpins constitutes an edge of the octahedron, and the triangular trimers occupy four of the eight faces of the octahedron.", + "section": "RESULTS", + "ner": [ + [ + 15, + 25, + "\u03b2-hairpins", + "structure_element" + ], + [ + 53, + 63, + "octahedron", + "protein_state" + ], + [ + 73, + 83, + "triangular", + "protein_state" + ], + [ + 84, + 91, + "trimers", + "oligomeric_state" + ], + [ + 130, + 140, + "octahedron", + "protein_state" + ] + ] + }, + { + "sid": 138, + "sent": "Figure 4A illustrates the octahedral shape of the dodecamer.", + "section": "RESULTS", + "ner": [ + [ + 26, + 36, + "octahedral", + "protein_state" + ], + [ + 50, + 59, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 139, + "sent": "Figure 4B illustrates the tetrahedral arrangement of the four trimers.", + "section": "RESULTS", + "ner": [ + [ + 26, + 37, + "tetrahedral", + "protein_state" + ], + [ + 62, + 69, + "trimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 140, + "sent": "X-ray crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral shape. (B) View of the dodecamer that illustrates the tetrahedral arrangement of the four trimers that comprise the dodecamer. (C) View of two trimer subunits from inside the cavity of the dodecamer.", + "section": "FIG", + "ner": [ + [ + 40, + 49, + "dodecamer", + "oligomeric_state" + ], + [ + 60, + 69, + "peptide 2", + "mutant" + ], + [ + 87, + 96, + "dodecamer", + "oligomeric_state" + ], + [ + 118, + 128, + "octahedral", + "protein_state" + ], + [ + 152, + 161, + "dodecamer", + "oligomeric_state" + ], + [ + 183, + 194, + "tetrahedral", + "protein_state" + ], + [ + 219, + 226, + "trimers", + "oligomeric_state" + ], + [ + 245, + 254, + "dodecamer", + "oligomeric_state" + ], + [ + 272, + 278, + "trimer", + "oligomeric_state" + ], + [ + 279, + 287, + "subunits", + "structure_element" + ], + [ + 304, + 310, + "cavity", + "site" + ], + [ + 318, + 327, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 141, + "sent": "Residues L17, L34, and V36 are shown as spheres, illustrating the hydrophobic packing that occurs at the six vertices of the dodecamer. (D) Detailed view of one of the six vertices of the dodecamer.", + "section": "FIG", + "ner": [ + [ + 9, + 12, + "L17", + "residue_name_number" + ], + [ + 14, + 17, + "L34", + "residue_name_number" + ], + [ + 23, + 26, + "V36", + "residue_name_number" + ], + [ + 66, + 85, + "hydrophobic packing", + "bond_interaction" + ], + [ + 125, + 134, + "dodecamer", + "oligomeric_state" + ], + [ + 188, + 197, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 142, + "sent": "The F19 faces of the trimers line the interior of the dodecamer.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "F19", + "residue_name_number" + ], + [ + 21, + 28, + "trimers", + "oligomeric_state" + ], + [ + 54, + 63, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 143, + "sent": "At the six vertices, hydrophobic packing between the side chains of L17, L34, and V36 helps stabilize the dodecamer (Figures 4C and D).", + "section": "RESULTS", + "ner": [ + [ + 21, + 40, + "hydrophobic packing", + "bond_interaction" + ], + [ + 68, + 71, + "L17", + "residue_name_number" + ], + [ + 73, + 76, + "L34", + "residue_name_number" + ], + [ + 82, + 85, + "V36", + "residue_name_number" + ], + [ + 106, + 115, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 144, + "sent": "Salt bridges between the side chains of D23 and \u03b4Orn at the vertices further stabilize the dodecamer.", + "section": "RESULTS", + "ner": [ + [ + 40, + 43, + "D23", + "residue_name_number" + ], + [ + 48, + 52, + "\u03b4Orn", + "structure_element" + ], + [ + 91, + 100, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 145, + "sent": "Each of the six vertices includes two A\u03b225\u201328 loops that extend past the core of the dodecamer without making any substantial intermolecular contacts.", + "section": "RESULTS", + "ner": [ + [ + 38, + 40, + "A\u03b2", + "protein" + ], + [ + 40, + 45, + "25\u201328", + "residue_range" + ], + [ + 46, + 51, + "loops", + "structure_element" + ], + [ + 73, + 77, + "core", + "structure_element" + ], + [ + 85, + 94, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 146, + "sent": "The exterior of the dodecamer displays four F20 faces (Figure S3).", + "section": "RESULTS", + "ner": [ + [ + 20, + 29, + "dodecamer", + "oligomeric_state" + ], + [ + 44, + 47, + "F20", + "residue_name_number" + ] + ] + }, + { + "sid": 147, + "sent": "In the crystal lattice, each F20 face of one dodecamer packs against an F20 face of another dodecamer.", + "section": "RESULTS", + "ner": [ + [ + 7, + 22, + "crystal lattice", + "evidence" + ], + [ + 29, + 32, + "F20", + "residue_name_number" + ], + [ + 45, + 54, + "dodecamer", + "oligomeric_state" + ], + [ + 72, + 75, + "F20", + "residue_name_number" + ], + [ + 92, + 101, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 148, + "sent": "Although the asymmetric unit comprises half a dodecamer, the crystal lattice may be thought of as being built of dodecamers.", + "section": "RESULTS", + "ner": [ + [ + 46, + 55, + "dodecamer", + "oligomeric_state" + ], + [ + 61, + 76, + "crystal lattice", + "evidence" + ], + [ + 113, + 123, + "dodecamers", + "oligomeric_state" + ] + ] + }, + { + "sid": 149, + "sent": "The electron density map for the X-ray crystallographic structure of peptide 2 has long tubes of electron density inside the central cavity of the dodecamer.", + "section": "RESULTS", + "ner": [ + [ + 4, + 24, + "electron density map", + "evidence" + ], + [ + 33, + 65, + "X-ray crystallographic structure", + "evidence" + ], + [ + 69, + 78, + "peptide 2", + "mutant" + ], + [ + 97, + 113, + "electron density", + "evidence" + ], + [ + 125, + 139, + "central cavity", + "site" + ], + [ + 147, + 156, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 150, + "sent": "The shape and length of the electron density is consistent with the structure of Jeffamine M-600, which is an essential component of the crystallization conditions.", + "section": "RESULTS", + "ner": [ + [ + 28, + 44, + "electron density", + "evidence" + ], + [ + 68, + 77, + "structure", + "evidence" + ], + [ + 81, + 96, + "Jeffamine M-600", + "chemical" + ] + ] + }, + { + "sid": 151, + "sent": "Jeffamine M-600 is a polypropylene glycol derivative with a 2-methoxyethoxy unit at one end and a 2-aminopropyl unit at the other end.", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Jeffamine M-600", + "chemical" + ] + ] + }, + { + "sid": 152, + "sent": "Although Jeffamine M-600 is a heterogeneous mixture with varying chain lengths and stereochemistry, we modeled a single stereoisomer with nine propylene glycol units (n = 9) to fit the electron density.", + "section": "RESULTS", + "ner": [ + [ + 9, + 24, + "Jeffamine M-600", + "chemical" + ], + [ + 185, + 201, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 153, + "sent": "The Jeffamine M-600 appears to stabilize the dodecamer by occupying the central cavity and making hydrophobic contacts with residues lining the cavity (Figure S3).", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "Jeffamine M-600", + "chemical" + ], + [ + 45, + 54, + "dodecamer", + "oligomeric_state" + ], + [ + 72, + 86, + "central cavity", + "site" + ], + [ + 98, + 118, + "hydrophobic contacts", + "bond_interaction" + ], + [ + 144, + 150, + "cavity", + "site" + ] + ] + }, + { + "sid": 154, + "sent": "In a dodecamer formed by full-length A\u03b2, the hydrophobic C-terminal residues (A\u03b237\u201340 or A\u03b237\u201342) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer.", + "section": "RESULTS", + "ner": [ + [ + 5, + 14, + "dodecamer", + "oligomeric_state" + ], + [ + 25, + 36, + "full-length", + "protein_state" + ], + [ + 37, + 39, + "A\u03b2", + "protein" + ], + [ + 78, + 80, + "A\u03b2", + "protein" + ], + [ + 80, + 85, + "37\u201340", + "residue_range" + ], + [ + 89, + 91, + "A\u03b2", + "protein" + ], + [ + 91, + 96, + "37\u201342", + "residue_range" + ], + [ + 139, + 148, + "dodecamer", + "oligomeric_state" + ], + [ + 174, + 190, + "hydrophobic core", + "site" + ], + [ + 202, + 216, + "central cavity", + "site" + ], + [ + 224, + 233, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 155, + "sent": "Annular Pore", + "section": "RESULTS", + "ner": [ + [ + 0, + 12, + "Annular Pore", + "site" + ] + ] + }, + { + "sid": 156, + "sent": "Five dodecamers assemble to form an annular porelike structure (Figure 5A).", + "section": "RESULTS", + "ner": [ + [ + 5, + 15, + "dodecamers", + "oligomeric_state" + ], + [ + 44, + 52, + "porelike", + "structure_element" + ] + ] + }, + { + "sid": 157, + "sent": "Hydrophobic packing between the F20 faces of trimers displayed on the outer surface of each dodecamer stabilizes the porelike assembly.", + "section": "RESULTS", + "ner": [ + [ + 0, + 19, + "Hydrophobic packing", + "bond_interaction" + ], + [ + 32, + 35, + "F20", + "residue_name_number" + ], + [ + 45, + 52, + "trimers", + "oligomeric_state" + ], + [ + 92, + 101, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 158, + "sent": "Two morphologically distinct interactions between trimers occur at the interfaces of the five dodecamers: one in which the trimers are eclipsed (Figure 5B), and one in which the trimers are staggered (Figure 5C).", + "section": "RESULTS", + "ner": [ + [ + 50, + 57, + "trimers", + "oligomeric_state" + ], + [ + 71, + 81, + "interfaces", + "site" + ], + [ + 94, + 104, + "dodecamers", + "oligomeric_state" + ], + [ + 123, + 130, + "trimers", + "oligomeric_state" + ], + [ + 135, + 143, + "eclipsed", + "protein_state" + ], + [ + 178, + 185, + "trimers", + "oligomeric_state" + ], + [ + 190, + 199, + "staggered", + "protein_state" + ] + ] + }, + { + "sid": 159, + "sent": "Hydrophobic packing between the side chains of F20, I31, and E22 stabilizes these interfaces (Figure 5D and E).", + "section": "RESULTS", + "ner": [ + [ + 0, + 19, + "Hydrophobic packing", + "bond_interaction" + ], + [ + 47, + 50, + "F20", + "residue_name_number" + ], + [ + 52, + 55, + "I31", + "residue_name_number" + ], + [ + 61, + 64, + "E22", + "residue_name_number" + ], + [ + 82, + 92, + "interfaces", + "site" + ] + ] + }, + { + "sid": 160, + "sent": "The annular pore contains three eclipsed interfaces and two staggered interfaces.", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "annular pore", + "site" + ], + [ + 32, + 40, + "eclipsed", + "protein_state" + ], + [ + 41, + 51, + "interfaces", + "site" + ], + [ + 60, + 69, + "staggered", + "protein_state" + ], + [ + 70, + 80, + "interfaces", + "site" + ] + ] + }, + { + "sid": 161, + "sent": "The eclipsed interfaces occur between dodecamers 1 and 2, 1 and 5, and 3 and 4, as shown in Figure 5A.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "eclipsed", + "protein_state" + ], + [ + 13, + 23, + "interfaces", + "site" + ], + [ + 38, + 56, + "dodecamers 1 and 2", + "structure_element" + ], + [ + 58, + 65, + "1 and 5", + "structure_element" + ], + [ + 71, + 78, + "3 and 4", + "structure_element" + ] + ] + }, + { + "sid": 162, + "sent": "The staggered interfaces occur between dodecamers 2 and 3 and 4 and 5.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "staggered", + "protein_state" + ], + [ + 14, + 24, + "interfaces", + "site" + ], + [ + 39, + 57, + "dodecamers 2 and 3", + "structure_element" + ], + [ + 62, + 69, + "4 and 5", + "structure_element" + ] + ] + }, + { + "sid": 163, + "sent": "The annular pore is not completely flat, instead, adopting a slightly puckered shape, which accommodates the eclipsed and staggered interfaces.", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "annular pore", + "site" + ], + [ + 109, + 117, + "eclipsed", + "protein_state" + ], + [ + 122, + 131, + "staggered", + "protein_state" + ], + [ + 132, + 142, + "interfaces", + "site" + ] + ] + }, + { + "sid": 164, + "sent": "Ten A\u03b225\u201328 loops from the vertices of the five dodecamers line the hole in the center of the pore.", + "section": "RESULTS", + "ner": [ + [ + 4, + 6, + "A\u03b2", + "protein" + ], + [ + 6, + 11, + "25\u201328", + "residue_range" + ], + [ + 12, + 17, + "loops", + "structure_element" + ], + [ + 48, + 58, + "dodecamers", + "oligomeric_state" + ], + [ + 94, + 98, + "pore", + "site" + ] + ] + }, + { + "sid": 165, + "sent": "The hydrophilic side chains of S26, N27, and K28 decorate the hole.", + "section": "RESULTS", + "ner": [ + [ + 31, + 34, + "S26", + "residue_name_number" + ], + [ + 36, + 39, + "N27", + "residue_name_number" + ], + [ + 45, + 48, + "K28", + "residue_name_number" + ] + ] + }, + { + "sid": 166, + "sent": "X-ray crystallographic structure of the annular pore formed by peptide 2. (A) Annular porelike structure illustrating the relationship of the five dodecamers that form the pore (top view).", + "section": "FIG", + "ner": [ + [ + 0, + 32, + "X-ray crystallographic structure", + "evidence" + ], + [ + 40, + 52, + "annular pore", + "site" + ], + [ + 63, + 72, + "peptide 2", + "mutant" + ], + [ + 78, + 94, + "Annular porelike", + "structure_element" + ], + [ + 95, + 104, + "structure", + "evidence" + ], + [ + 147, + 157, + "dodecamers", + "oligomeric_state" + ], + [ + 172, + 176, + "pore", + "site" + ] + ] + }, + { + "sid": 167, + "sent": " (B) Eclipsed interface between dodecamers 1 and 2 (side view).", + "section": "FIG", + "ner": [ + [ + 5, + 23, + "Eclipsed interface", + "site" + ], + [ + 32, + 50, + "dodecamers 1 and 2", + "structure_element" + ] + ] + }, + { + "sid": 168, + "sent": "The same eclipsed interface also occurs between dodecamers 1 and 5 and 3 and 4. (C) Staggered interface between dodecamers 2 and 3 (side view).", + "section": "FIG", + "ner": [ + [ + 9, + 27, + "eclipsed interface", + "site" + ], + [ + 48, + 66, + "dodecamers 1 and 5", + "structure_element" + ], + [ + 71, + 78, + "3 and 4", + "structure_element" + ], + [ + 84, + 103, + "Staggered interface", + "site" + ], + [ + 112, + 130, + "dodecamers 2 and 3", + "structure_element" + ] + ] + }, + { + "sid": 169, + "sent": "The same staggered interface also occurs between dodecamers 4 and 5. (D) Eclipsed interface between dodecamers 1 and 5 (top view).", + "section": "FIG", + "ner": [ + [ + 9, + 28, + "staggered interface", + "site" + ], + [ + 73, + 91, + "Eclipsed interface", + "site" + ], + [ + 100, + 118, + "dodecamers 1 and 5", + "structure_element" + ] + ] + }, + { + "sid": 170, + "sent": "The annular pore is comparable in size to other large protein assemblies.", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "annular pore", + "site" + ] + ] + }, + { + "sid": 171, + "sent": "The diameter of the hole in the center of the pore is \u223c2 nm.", + "section": "RESULTS", + "ner": [ + [ + 46, + 50, + "pore", + "site" + ] + ] + }, + { + "sid": 172, + "sent": "The thickness of the pore is \u223c5 nm, which is comparable to that of a lipid bilayer membrane.", + "section": "RESULTS", + "ner": [ + [ + 21, + 25, + "pore", + "site" + ] + ] + }, + { + "sid": 173, + "sent": "It is important to note that the annular pore formed by peptide 2 is not a discrete unit in the crystal lattice.", + "section": "RESULTS", + "ner": [ + [ + 33, + 45, + "annular pore", + "site" + ], + [ + 56, + 65, + "peptide 2", + "mutant" + ], + [ + 96, + 111, + "crystal lattice", + "evidence" + ] + ] + }, + { + "sid": 174, + "sent": "Rather, the crystal lattice is composed of conjoined annular pores in which all four F20 faces on the surface of each dodecamer contact F20 faces on other dodecamers (Figure S4).", + "section": "RESULTS", + "ner": [ + [ + 12, + 27, + "crystal lattice", + "evidence" + ], + [ + 53, + 66, + "annular pores", + "site" + ], + [ + 85, + 88, + "F20", + "residue_name_number" + ], + [ + 118, + 127, + "dodecamer", + "oligomeric_state" + ], + [ + 136, + 139, + "F20", + "residue_name_number" + ], + [ + 155, + 165, + "dodecamers", + "oligomeric_state" + ] + ] + }, + { + "sid": 175, + "sent": "The crystal lattice shows how the dodecamers can further assemble to form larger structures.", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "crystal lattice", + "evidence" + ], + [ + 34, + 44, + "dodecamers", + "oligomeric_state" + ] + ] + }, + { + "sid": 176, + "sent": "Each dodecamer may be thought of as a tetravalent building block with the potential to assemble on all four faces to form higher-order supramolecular assemblies.", + "section": "RESULTS", + "ner": [ + [ + 5, + 14, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 177, + "sent": "The X-ray crystallographic study of peptide 2 described here provides high-resolution structures of oligomers formed by an A\u03b217\u201336 \u03b2-hairpin.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 32, + "X-ray crystallographic study", + "experimental_method" + ], + [ + 36, + 45, + "peptide 2", + "mutant" + ], + [ + 86, + 96, + "structures", + "evidence" + ], + [ + 100, + 109, + "oligomers", + "oligomeric_state" + ], + [ + 123, + 125, + "A\u03b2", + "protein" + ], + [ + 125, + 130, + "17\u201336", + "residue_range" + ], + [ + 131, + 140, + "\u03b2-hairpin", + "structure_element" + ] + ] + }, + { + "sid": 178, + "sent": "The crystallographic assembly of peptide 2 into a trimer, dodecamer, and annular pore provides a model for the assembly of the full-length A\u03b2 peptide to form oligomers.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 29, + "crystallographic assembly", + "evidence" + ], + [ + 33, + 42, + "peptide 2", + "mutant" + ], + [ + 50, + 56, + "trimer", + "oligomeric_state" + ], + [ + 58, + 67, + "dodecamer", + "oligomeric_state" + ], + [ + 73, + 85, + "annular pore", + "site" + ], + [ + 127, + 138, + "full-length", + "protein_state" + ], + [ + 139, + 141, + "A\u03b2", + "protein" + ], + [ + 158, + 167, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 179, + "sent": "In this model A\u03b2 folds to form a \u03b2-hairpin comprising the hydrophobic central and C-terminal regions.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 16, + "A\u03b2", + "protein" + ], + [ + 33, + 42, + "\u03b2-hairpin", + "structure_element" + ], + [ + 70, + 100, + "central and 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"\u03b2-hairpin", + "structure_element" + ], + [ + 62, + 68, + "trimer", + "oligomeric_state" + ], + [ + 70, + 79, + "dodecamer", + "oligomeric_state" + ], + [ + 85, + 97, + "annular pore", + "site" + ], + [ + 140, + 149, + "peptide 2", + "mutant" + ] + ] + }, + { + "sid": 183, + "sent": "Monomeric A\u03b2 folds to form a \u03b2-hairpin in which the hydrophobic central and C-terminal regions form an antiparallel \u03b2-sheet.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Monomeric", + "oligomeric_state" + ], + [ + 10, + 12, + "A\u03b2", + "protein" + ], + [ + 29, + 38, + "\u03b2-hairpin", + "structure_element" + ], + [ + 64, + 71, + "central", + "structure_element" + ], + [ + 76, + 94, + "C-terminal regions", + "structure_element" + ], + [ + 103, + 123, + "antiparallel \u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 184, + "sent": "Three \u03b2-hairpin monomers assemble to form a triangular trimer.", + "section": "FIG", + "ner": [ + [ + 6, + 15, + "\u03b2-hairpin", + "structure_element" + ], + [ + 16, + 24, + "monomers", + "oligomeric_state" + ], + [ + 44, + 54, + "triangular", + "protein_state" + ], + [ + 55, + 61, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 185, + "sent": "Four triangular trimers assemble to form a dodecamer.", + "section": "FIG", + "ner": [ + [ + 5, + 15, + "triangular", + "protein_state" + ], + [ + 16, + 23, + "trimers", + "oligomeric_state" + ], + [ + 43, + 52, + "dodecamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 186, + "sent": "Five dodecamers assemble to form an annular pore.", + "section": "FIG", + "ner": [ + [ + 5, + 15, + "dodecamers", + "oligomeric_state" + ], + [ + 36, + 48, + "annular pore", + "site" + ] + ] + }, + { + "sid": 187, + "sent": "The molecular weights shown correspond to an A\u03b242 monomer (\u223c4.5 kDa), an A\u03b242 trimer (\u223c13.5 kDa), an A\u03b242 dodecamer (\u223c54 kDa), and an A\u03b242 annular pore composed of five dodecamers (\u223c270 kDa).", + "section": "FIG", + "ner": [ + [ + 45, + 49, + "A\u03b242", + "protein" + ], + [ + 50, + 57, + "monomer", + "oligomeric_state" + ], + [ + 73, + 77, + "A\u03b242", + "protein" + ], + [ + 78, + 84, + "trimer", + "oligomeric_state" + ], + [ + 101, + 105, + "A\u03b242", + "protein" + ], + [ + 106, + 115, + "dodecamer", + "oligomeric_state" + ], + [ + 134, + 138, + "A\u03b242", + "protein" + ], + [ + 139, + 151, + "annular pore", + "site" + ], + [ + 169, + 179, + "dodecamers", + "oligomeric_state" + ] + ] + }, + { + "sid": 188, + "sent": "The model put forth in Figure 6 is consistent with the current understanding of endogenous A\u03b2 oligomerization and explains at atomic resolution many key observations about A\u03b2 oligomers.", + "section": "DISCUSS", + "ner": [ + [ + 91, + 93, + "A\u03b2", + "protein" + ], + [ + 172, + 174, + "A\u03b2", + "protein" + ], + [ + 175, + 184, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 189, + "sent": "Two general types of endogenous A\u03b2 oligomers have been observed: A\u03b2 oligomers that occur on a pathway to fibrils, or \u201cfibrillar oligomers\u201d, and A\u03b2 oligomers that evade a fibrillar fate, or \u201cnonfibrillar oligomers\u201d.\u2212 Fibrillar oligomers accumulate in Alzheimer\u2019s disease later than nonfibrillar oligomers and coincide with the deposition of plaques.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 34, + "A\u03b2", + "protein" + ], + [ + 35, + 44, + "oligomers", + "oligomeric_state" + ], + [ + 65, + 67, + "A\u03b2", + "protein" + ], + [ + 68, + 77, + "oligomers", + "oligomeric_state" + ], + [ + 105, + 112, + "fibrils", + "oligomeric_state" + ], + [ + 118, + 127, + "fibrillar", + "protein_state" + ], + [ + 128, + 137, + "oligomers", + "oligomeric_state" + ], + [ + 144, + 146, + "A\u03b2", + "protein" + ], + [ + 147, + 156, + "oligomers", + "oligomeric_state" + ], + [ + 170, + 179, + "fibrillar", + "protein_state" + ], + [ + 190, + 202, + "nonfibrillar", + "protein_state" + ], + [ + 203, + 212, + 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166, + 174, + "oligomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 192, + "sent": "Fibrillar oligomers are recognized by the OC antibody but not the A11 antibody, whereas nonfibrillar oligomers are recognized by the A11 antibody but not the OC antibody.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 9, + "Fibrillar", + "protein_state" + ], + [ + 10, + 19, + "oligomers", + "oligomeric_state" + ], + [ + 88, + 100, + "nonfibrillar", + "protein_state" + ], + [ + 101, + 110, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 193, + "sent": "These criteria have been used to classify the A\u03b2 oligomers that accumulate in vivo.", + "section": "DISCUSS", + "ner": [ + [ + 46, + 48, + "A\u03b2", + "protein" + ], + [ + 49, + 58, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 194, + "sent": "A\u03b2 dimers have been classified as fibrillar oligomers, whereas A\u03b2 trimers, A\u03b2*56, and APFs have been classified as nonfibrillar oligomers.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 2, + "A\u03b2", + "protein" + ], + [ + 3, + 9, + "dimers", + "oligomeric_state" + ], + [ + 34, + 43, + "fibrillar", + "protein_state" + ], + [ + 44, + 53, + "oligomers", + "oligomeric_state" + ], + [ + 63, + 65, + "A\u03b2", + "protein" + ], + [ + 66, + 73, + "trimers", + "oligomeric_state" + ], + [ + 75, + 80, + "A\u03b2*56", + "complex_assembly" + ], + [ + 86, + 90, + "APFs", + "complex_assembly" + ], + [ + 115, + 127, + "nonfibrillar", + "protein_state" + ], + [ + 128, + 137, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 195, + "sent": "Larson and Lesn\u00e9 proposed a model for the endogenous production of nonfibrillar oligomers that explains these observations.", + "section": "DISCUSS", + "ner": [ + [ + 67, + 79, + "nonfibrillar", + "protein_state" + ], + [ + 80, + 89, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 196, + "sent": "In this model, folded A\u03b2 monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 21, + "folded", + "protein_state" + ], + [ + 22, + 24, + "A\u03b2", + "protein" + ], + [ + 25, + 32, + "monomer", + "oligomeric_state" + ], + [ + 50, + 56, + "trimer", + "oligomeric_state" + ], + [ + 62, + 68, + "trimer", + "oligomeric_state" + ], + [ + 92, + 100, + "hexamers", + "oligomeric_state" + ], + [ + 105, + 115, + "dodecamers", + "oligomeric_state" + ], + [ + 125, + 135, + "dodecamers", + "oligomeric_state" + ], + [ + 161, + 181, + "annular protofibrils", + "complex_assembly" + ] + ] + }, + { + "sid": 197, + "sent": "The hierarchical assembly of peptide 2 is consistent with this model; and the trimer, dodecamer, and annular pore formed by peptide 2 may share similarities to the trimers, A\u03b2*56, and APFs observed in vivo.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 38, + "peptide 2", + "mutant" + ], + [ + 78, + 84, + "trimer", + "oligomeric_state" + ], + [ + 86, + 95, + "dodecamer", + "oligomeric_state" + ], + [ + 101, + 113, + "annular pore", + "site" + ], + [ + 124, + 133, + "peptide 2", + "mutant" + ], + [ + 164, + 171, + "trimers", + "oligomeric_state" + ], + [ + 173, + 178, + "A\u03b2*56", + "complex_assembly" + ], + [ + 184, + 188, + "APFs", + "complex_assembly" + ] + ] + }, + { + "sid": 198, + "sent": "At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to A\u03b2 trimers and A\u03b2*56, as little is known about the structure of A\u03b2 trimers and A\u03b2*56.", + "section": "DISCUSS", + "ner": [ + [ + 49, + 55, + "trimer", + "oligomeric_state" + ], + [ + 60, + 69, + "dodecamer", + "oligomeric_state" + ], + [ + 80, + 89, + "peptide 2", + "mutant" + ], + [ + 123, + 125, + "A\u03b2", + "protein" + ], + [ + 126, + 133, + "trimers", + "oligomeric_state" + ], + [ + 138, + 143, + "A\u03b2*56", + "complex_assembly" + ], + [ + 174, 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+ "site" + ], + [ + 27, + 36, + "peptide 2", + "mutant" + ], + [ + 66, + 70, + "APFs", + "complex_assembly" + ] + ] + }, + { + "sid": 201, + "sent": "The varying sizes of APFs formed by full-length A\u03b2 might result from differences in the number of oligomer subunits comprising each APF.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 25, + "APFs", + "complex_assembly" + ], + [ + 36, + 47, + "full-length", + "protein_state" + ], + [ + 48, + 50, + "A\u03b2", + "protein" + ], + [ + 98, + 106, + "oligomer", + "oligomeric_state" + ], + [ + 107, + 115, + "subunits", + "structure_element" + ], + [ + 132, + 135, + "APF", + "complex_assembly" + ] + ] + }, + { + "sid": 202, + "sent": "Although the annular pore formed by peptide 2 contains five dodecamer subunits, pores containing fewer or more subunits can easily be envisioned.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 25, + "annular pore", + "site" + ], + [ + 36, + 45, + "peptide 2", + "mutant" + ], + [ + 60, + 69, + "dodecamer", + "oligomeric_state" + ], + [ + 70, + 78, + "subunits", + "structure_element" + ], + [ + 80, + 85, + "pores", + "site" + ], + [ + 111, + 119, + "subunits", + "structure_element" + ] + ] + }, + { + "sid": 203, + "sent": "The dodecamers that comprise the annular pore exhibit two modes of assembly\u2014eclipsed interactions and staggered interactions between the F20 faces of trimers within dodecamers.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 14, + "dodecamers", + "oligomeric_state" + ], + [ + 33, + 45, + "annular pore", + "site" + ], + [ + 76, + 84, + "eclipsed", + "protein_state" + ], + [ + 102, + 111, + "staggered", + "protein_state" + ], + [ + 137, + 140, + "F20", + "residue_name_number" + ], + [ + 150, + 157, + "trimers", + "oligomeric_state" + ], + [ + 165, + 175, + "dodecamers", + "oligomeric_state" + ] + ] + }, + { + "sid": 204, + "sent": "These two modes of assembly might reflect a dynamic interaction between dodecamers, which could permit assemblies of more dodecamers into larger annular pores.", + "section": "DISCUSS", + "ner": [ + [ + 72, + 82, + "dodecamers", + "oligomeric_state" + ], + [ + 122, + 132, + "dodecamers", + "oligomeric_state" + ], + [ + 145, + 158, + "annular pores", + "site" + ] + ] + }, + { + "sid": 205, + "sent": "Surface views of the annular pore formed by peptide 2. (A) Top view.", + "section": "FIG", + "ner": [ + [ + 21, + 33, + "annular pore", + "site" + ], + [ + 44, + 53, + "peptide 2", + "mutant" + ] + ] + }, + { + "sid": 206, + "sent": "Annular Pores Formed by A\u03b2 and Peptide 2", + "section": "TABLE", + "ner": [ + [ + 0, + 13, + "Annular Pores", + "site" + ], + [ + 24, + 26, + "A\u03b2", + "protein" + ], + [ + 31, + 40, + "Peptide 2", + "mutant" + ] + ] + }, + { + "sid": 207, + "sent": "annular pore\t \tsource\touter diameter\tinner diameter\tobservation\t \tmethod\t \tpeptide\u00a02\t \u223c11\u201312\u00a0nm\t\u223c2\u00a0nm\tX-ray\u00a0crystallography\t \tsynthetic A\u03b2\t7\u201310\u00a0nm\t1.5\u20132\u00a0nm\tTEM\t \tsynthetic A\u03b2\t16 nm\tnot reported\tAFM\t \tsynthetic A\u03b2\t8\u201325\u00a0nm\tnot reported\tTEM\t \tAlzheimer\u2019s brain\t11\u201314\u00a0nm\t2.5\u20134\u00a0nm\tTEM\t \t", + "section": "TABLE", + "ner": [ + [ + 0, + 12, + "annular pore", + "site" + ], + [ + 75, + 82, + "peptide", + "chemical" + ], + [ + 102, + 123, + "X-ray\u00a0crystallography", + "experimental_method" + ], + [ + 126, + 135, + "synthetic", + "protein_state" + ], + [ + 136, + 138, + "A\u03b2", + "protein" + ], + [ + 156, + 159, + "TEM", + "experimental_method" + ], + [ + 172, + 174, + "A\u03b2", + "protein" + ], + [ + 194, + 197, + "AFM", + "experimental_method" + ], + [ + 210, + 212, + "A\u03b2", + "protein" + ], + [ + 234, + 237, + "TEM", + "experimental_method" + ], + [ + 276, + 279, + "TEM", + "experimental_method" + ] + ] + }, + { + "sid": 208, + "sent": "Dot blot analysis shows that peptide 2 is reactive toward the A11 antibody (Figure S5).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 8, + "Dot blot", + "experimental_method" + ], + [ + 29, + 38, + "peptide 2", + "mutant" + ] + ] + }, + { + "sid": 209, + "sent": "This reactivity suggests that peptide 2 forms oligomers in solution that share structural similarities to the nonfibrillar oligomers formed by full-length A\u03b2.", + "section": "DISCUSS", + "ner": [ + [ + 30, + 39, + "peptide 2", + "mutant" + ], + [ + 46, + 55, + "oligomers", + "oligomeric_state" + ], + [ + 110, + 122, + "nonfibrillar", + "protein_state" + ], + [ + 123, + 132, + "oligomers", + "oligomeric_state" + ], + [ + 143, + 154, + "full-length", + "protein_state" + ], + [ + 155, + 157, + "A\u03b2", + "protein" + ] + ] + }, + { + "sid": 210, + "sent": "Further studies are needed to elucidate the species that peptide 2 forms in solution and to study their biological properties.", + "section": "DISCUSS", + "ner": [ + [ + 57, + 66, + "peptide 2", + "mutant" + ] + ] + }, + { + "sid": 211, + "sent": "Preliminary attempts to study these species by SEC and SDS-PAGE have not provided a clear measure of the structures formed in solution.", + "section": "DISCUSS", + "ner": [ + [ + 47, + 50, + "SEC", + "experimental_method" + ], + [ + 55, + 63, + "SDS-PAGE", + "experimental_method" + ], + [ + 105, + 115, + "structures", + "evidence" + ] + ] + }, + { + "sid": 212, + "sent": "The difficulty in studying the oligomers formed in solution may reflect the propensity of the dodecamer to assemble on all four F20 faces.", + "section": "DISCUSS", + "ner": [ + [ + 31, + 40, + "oligomers", + "oligomeric_state" + ], + [ + 94, + 103, + "dodecamer", + "oligomeric_state" + ], + [ + 128, + 131, + "F20", + "residue_name_number" + ] + ] + }, + { + "sid": 213, + "sent": "The X-ray crystallographic structure and A11 reactivity of peptide 2 support the model proposed by Larsen and Lesn\u00e9 and suggest that \u03b2-hairpins constitute a fundamental building block for nonfibrillar oligomers.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 36, + "X-ray crystallographic structure", + "evidence" + ], + [ + 59, + 68, + "peptide 2", + "mutant" + ], + [ + 133, + 143, + "\u03b2-hairpins", + "structure_element" + ], + [ + 188, + 200, + "nonfibrillar", + "protein_state" + ], + [ + 201, + 210, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 214, + "sent": "What makes \u03b2-hairpins special is that three \u03b2-hairpins can nestle together to form trimers, stabilized by a network of hydrogen bonds and hydrophobic interactions.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 21, + "\u03b2-hairpins", + "structure_element" + ], + [ + 44, + 54, + "\u03b2-hairpins", + "structure_element" + ], + [ + 83, + 90, + "trimers", + "oligomeric_state" + ], + [ + 119, + 133, + "hydrogen bonds", + "bond_interaction" + ], + [ + 138, + 162, + "hydrophobic interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 215, + "sent": "This mode of assembly is not unique to A\u03b2.", + "section": "DISCUSS", + "ner": [ + [ + 39, + 41, + "A\u03b2", + "protein" + ] + ] + }, + { + "sid": 216, + "sent": "The foldon domain of bacteriophage T4 fibritin is composed of three \u03b2-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 17, + "foldon domain", + "structure_element" + ], + [ + 21, + 37, + "bacteriophage T4", + "species" + ], + [ + 38, + 46, + "fibritin", + "protein" + ], + [ + 68, + 78, + "\u03b2-hairpins", + "structure_element" + ], + [ + 100, + 110, + "triangular", + "protein_state" + ], + [ + 111, + 117, + "trimer", + "oligomeric_state" + ], + [ + 133, + 143, + "triangular", + "protein_state" + ], + [ + 144, + 150, + "trimer", + "oligomeric_state" + ], + [ + 161, + 170, + "peptide 2", + "mutant" + ] + ] + }, + { + "sid": 217, + "sent": "Additionally, our research group has observed a similar assembly of a \u03b2-hairpin peptide derived from \u03b22-microglobulin.", + "section": "DISCUSS", + "ner": [ + [ + 70, + 79, + "\u03b2-hairpin", + "structure_element" + ], + [ + 101, + 117, + "\u03b22-microglobulin", + "protein" + ] + ] + }, + { + "sid": 218, + "sent": "Although we began these studies with a relatively simple hypothesis\u2014that the trimers and dodecamers formed by peptide 1 could accommodate the A\u03b224\u201329 loop\u2014an even more exciting finding has emerged\u2014that the dodecamers can assemble to form annular pores.", + "section": "CONCL", + "ner": [ + [ + 77, + 84, + "trimers", + "oligomeric_state" + ], + [ + 89, + 99, + "dodecamers", + "oligomeric_state" + ], + [ + 110, + 119, + "peptide 1", + "mutant" + ], + [ + 142, + 144, + "A\u03b2", + "protein" + ], + [ + 144, + 149, + "24\u201329", + "residue_range" + ], + [ + 150, + 154, + "loop", + "structure_element" + ], + [ + 206, + 216, + "dodecamers", + "oligomeric_state" + ], + [ + 238, + 251, + "annular pores", + "site" + ] + ] + }, + { + "sid": 219, + "sent": "This finding could not have been anticipated from the X-ray crystallographic structure of peptide 1 and reveals a new level of hierarchical assembly that recapitulates micrographic observations of annular protofibrils.", + "section": "CONCL", + "ner": [ + [ + 54, + 86, + "X-ray crystallographic structure", + "evidence" + ], + [ + 90, + 99, + "peptide 1", + "mutant" + ], + [ + 197, + 217, + "annular protofibrils", + "complex_assembly" + ] + ] + }, + { + "sid": 220, + "sent": "The crystallographically observed dodecamer, in turn, recapitulates the observation of A\u03b2*56, which appears to be a dodecamer of A\u03b2.", + "section": "CONCL", + "ner": [ + [ + 4, + 33, + "crystallographically observed", + "evidence" + ], + [ + 34, + 43, + "dodecamer", + "oligomeric_state" + ], + [ + 87, + 92, + "A\u03b2*56", + "complex_assembly" + ], + [ + 116, + 125, + "dodecamer", + "oligomeric_state" + ], + [ + 129, + 131, + "A\u03b2", + "protein" + ] + ] + }, + { + "sid": 221, + "sent": "The crystallographically observed trimer recapitulates the A\u03b2 trimers that are observed even before the onset of symptoms in Alzheimer\u2019s disease.", + "section": "CONCL", + "ner": [ + [ + 4, + 33, + "crystallographically observed", + "evidence" + ], + [ + 34, + 40, + "trimer", + "oligomeric_state" + ], + [ + 59, + 61, + "A\u03b2", + "protein" + ], + [ + 62, + 69, + "trimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 222, + "sent": "Our approach of constraining A\u03b217\u201336 into a \u03b2-hairpin conformation and blocking aggregation with an N-methyl group has allowed us to crystallize a large fragment of what is generally considered to be an uncrystallizable peptide.", + "section": "CONCL", + "ner": [ + [ + 29, + 31, + "A\u03b2", + "protein" + ], + [ + 31, + 36, + "17\u201336", + "residue_range" + ], + [ + 44, + 53, + "\u03b2-hairpin", + "structure_element" + ], + [ + 133, + 144, + "crystallize", + "experimental_method" + ] + ] + }, + { + "sid": 223, + "sent": "We believe this iterative, \u201cbottom up\u201d approach of identifying the minimal modification required to crystallize A\u03b2 peptides will ultimately allow larger fragments of A\u03b2 to be crystallized, thus providing greater insights into the structures of A\u03b2 oligomers.", + "section": "CONCL", + "ner": [ + [ + 100, + 111, + "crystallize", + "experimental_method" + ], + [ + 112, + 114, + "A\u03b2", + "protein" + ], + [ + 166, + 168, + "A\u03b2", + "protein" + ], + [ + 175, + 187, + "crystallized", + "experimental_method" + ], + [ + 230, + 240, + "structures", + "evidence" + ], + [ + 244, + 246, + "A\u03b2", + "protein" + ], + [ + 247, + 256, + "oligomers", + "oligomeric_state" + ] + ] + } + ] + }, + "PMC4848761": { + "annotations": [ + { + "sid": 0, + "sent": "Predictive features of ligand\u2010specific signaling through the estrogen receptor", + "section": "TITLE", + "ner": [ + [ + 61, + 78, + "estrogen receptor", + "protein_type" + ] + ] + }, + { + "sid": 1, + "sent": "Some estrogen receptor\u2010\u03b1 (ER\u03b1)\u2010targeted breast cancer therapies such as tamoxifen have tissue\u2010selective or cell\u2010specific activities, while others have similar activities in different cell types.", + "section": "ABSTRACT", + "ner": [ + [ + 5, + 24, + "estrogen receptor\u2010\u03b1", + "protein" + ], + [ + 26, + 29, + "ER\u03b1", + "protein" + ], + [ + 72, + 81, + "tamoxifen", + "chemical" + ] + ] + }, + { + "sid": 2, + "sent": "To identify biophysical determinants of cell\u2010specific signaling and breast cancer cell proliferation, we synthesized 241 ER\u03b1 ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ER\u03b1 activities and X\u2010ray crystallography.", + "section": "ABSTRACT", + "ner": [ + [ + 105, + 116, + "synthesized", + "experimental_method" + ], + [ + 121, + 124, + "ER\u03b1", + "protein" + ], + [ + 200, + 222, + "quantitative bioassays", + "experimental_method" + ], + [ + 237, + 240, + "ER\u03b1", + "protein" + ], + [ + 256, + 277, + "X\u2010ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 3, + "sent": "Ligands that regulate the dynamics and stability of the coactivator\u2010binding site in the C\u2010terminal ligand\u2010binding domain, called activation function\u20102 (AF\u20102), showed similar activity profiles in different cell types.", + "section": "ABSTRACT", + "ner": [ + [ + 56, + 80, + "coactivator\u2010binding site", + "site" + ], + [ + 99, + 120, + "ligand\u2010binding domain", + "structure_element" + ], + [ + 129, + 150, + "activation function\u20102", + "structure_element" + ], + [ + 152, + 156, + "AF\u20102", + "structure_element" + ] + ] + }, + { + "sid": 4, + "sent": "Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene.", + "section": "ABSTRACT", + "ner": [ + [ + 134, + 143, + "NCOA1/2/3", + "protein" + ], + [ + 165, + 170, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 5, + "sent": "For some ligand series, a single inter\u2010atomic distance in the ligand\u2010binding domain predicted their proliferative effects.", + "section": "ABSTRACT", + "ner": [ + [ + 33, + 54, + "inter\u2010atomic distance", + "evidence" + ], + [ + 62, + 83, + "ligand\u2010binding domain", + "structure_element" + ] + ] + }, + { + "sid": 6, + "sent": "In contrast, the N\u2010terminal coactivator\u2010binding site, activation function\u20101 (AF\u20101), determined cell\u2010specific signaling induced by ligands that used alternate mechanisms to control cell proliferation.", + "section": "ABSTRACT", + "ner": [ + [ + 28, + 52, + "coactivator\u2010binding site", + "site" + ], + [ + 54, + 75, + "activation function\u20101", + "structure_element" + ], + [ + 77, + 81, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 7, + "sent": "Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ER\u03b1.", + "section": "ABSTRACT", + "ner": [ + [ + 20, + 47, + "systems structural analyses", + "experimental_method" + ], + [ + 53, + 82, + "quantitative chemical biology", + "experimental_method" + ], + [ + 162, + 165, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 8, + "sent": "Many drugs are small\u2010molecule ligands of allosteric signaling proteins, including G protein\u2010coupled receptors (GPCRs) and nuclear receptors such as ER\u03b1.", + "section": "INTRO", + "ner": [ + [ + 82, + 109, + "G protein\u2010coupled receptors", + "protein_type" + ], + [ + 111, + 116, + "GPCRs", + "protein_type" + ], + [ + 122, + 139, + "nuclear receptors", + "protein_type" + ], + [ + 148, + 151, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 9, + "sent": "Small\u2010molecule ligands control receptor activity by modulating recruitment of effector enzymes to distal regions of the receptor, relative to the ligand\u2010binding site.", + "section": "INTRO", + "ner": [ + [ + 146, + 165, + "ligand\u2010binding site", + "site" + ] + ] + }, + { + "sid": 10, + "sent": "For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex\u00ae; AstraZeneca) or raloxifene (Evista\u00ae; Eli Lilly) (Fig\u00a01A) block the ER\u03b1\u2010mediated proliferative effects of the native estrogen, 17\u03b2\u2010estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et\u00a0al, 1996; Fisher et\u00a0al, 1998; McDonnell et\u00a0al, 2002; Jordan, 2003).", + "section": "INTRO", + "ner": [ + [ + 23, + 51, + "estrogen receptor modulators", + "protein_type" + ], + [ + 53, + 58, + "SERMs", + "protein_type" + ], + [ + 68, + 77, + "tamoxifen", + "chemical" + ], + [ + 79, + 88, + "Nolvadex\u00ae", + "chemical" + ], + [ + 106, + 116, + "raloxifene", + "chemical" + ], + [ + 118, + 125, + "Evista\u00ae", + "chemical" + ], + [ + 157, + 160, + "ER\u03b1", + "protein" + ], + [ + 206, + 214, + "estrogen", + "chemical" + ], + [ + 216, + 229, + "17\u03b2\u2010estradiol", + "chemical" + ], + [ + 231, + 233, + "E2", + "chemical" + ] + ] + }, + { + "sid": 11, + "sent": "Allosteric control of ER\u03b1 activity", + "section": "FIG", + "ner": [ + [ + 22, + 25, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 12, + "sent": "Chemical structures of some common ER\u03b1 ligands.", + "section": "FIG", + "ner": [ + [ + 35, + 38, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 13, + "sent": "E2\u2010rings are numbered A\u2010D. The E\u2010ring is the common site of attachment for BSC found in many SERMS.", + "section": "FIG", + "ner": [ + [ + 0, + 2, + "E2", + "chemical" + ], + [ + 93, + 98, + "SERMS", + "protein_type" + ] + ] + }, + { + "sid": 14, + "sent": "ER\u03b1 domain organization lettered, A\u2010F. DBD, DNA\u2010binding domain; LBD, ligand\u2010binding domain; AF, activation function", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "ER\u03b1", + "protein" + ], + [ + 39, + 42, + "DBD", + "structure_element" + ], + [ + 44, + 62, + "DNA\u2010binding domain", + "structure_element" + ], + [ + 64, + 67, + "LBD", + "structure_element" + ], + [ + 69, + 90, + "ligand\u2010binding domain", + "structure_element" + ], + [ + 92, + 94, + "AF", + "structure_element" + ], + [ + 96, + 115, + "activation function", + "structure_element" + ] + ] + }, + { + "sid": 15, + "sent": "Schematic illustration of the canonical ER\u03b1 signaling pathway.", + "section": "FIG", + "ner": [ + [ + 40, + 43, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 16, + "sent": "Linear causality model for ER\u03b1\u2010mediated cell proliferation.", + "section": "FIG", + "ner": [ + [ + 27, + 30, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 17, + "sent": "Branched causality model for ER\u03b1\u2010mediated cell proliferation.", + "section": "FIG", + "ner": [ + [ + 29, + 32, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 18, + "sent": "ER\u03b1 contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA\u2010binding domain (DBD) that is connected by a flexible hinge region to the ligand\u2010binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig\u00a01B).", + "section": "INTRO", + "ner": [ + [ + 0, + 3, + "ER\u03b1", + "protein" + ], + [ + 13, + 35, + "structurally conserved", + "protein_state" + ], + [ + 36, + 52, + "globular domains", + "structure_element" + ], + [ + 60, + 88, + "nuclear receptor superfamily", + "protein_type" + ], + [ + 102, + 120, + "DNA\u2010binding domain", + "structure_element" + ], + [ + 122, + 125, + "DBD", + "structure_element" + ], + [ + 150, + 158, + "flexible", + "protein_state" + ], + [ + 159, + 171, + "hinge region", + "structure_element" + ], + [ + 179, + 200, + "ligand\u2010binding domain", + "structure_element" + ], + [ + 202, + 205, + "LBD", + "structure_element" + ], + [ + 219, + 231, + "unstructured", + "protein_state" + ], + [ + 232, + 234, + "AB", + "structure_element" + ], + [ + 239, + 240, + "F", + "structure_element" + ] + ] + }, + { + "sid": 19, + "sent": "The LBD contains a ligand\u2010dependent coactivator\u2010binding site called activation function\u20102 (AF\u20102).", + "section": "INTRO", + "ner": [ + [ + 4, + 7, + "LBD", + "structure_element" + ], + [ + 36, + 60, + "coactivator\u2010binding site", + "site" + ], + [ + 68, + 89, + "activation function\u20102", + "structure_element" + ], + [ + 91, + 95, + "AF\u20102", + "structure_element" + ] + ] + }, + { + "sid": 20, + "sent": "However, the agonist activity of SERMs derives from activation function\u20101 (AF\u20101)\u2014a coactivator recruitment site located in the AB domain (Berry et\u00a0al, 1990; Shang & Brown, 2002; Abot et\u00a0al, 2013).", + "section": "INTRO", + "ner": [ + [ + 33, + 38, + "SERMs", + "protein_type" + ], + [ + 52, + 73, + "activation function\u20101", + "structure_element" + ], + [ + 75, + 79, + "AF\u20101", + "structure_element" + ], + [ + 83, + 111, + "coactivator recruitment site", + "site" + ], + [ + 127, + 129, + "AB", + "structure_element" + ] + ] + }, + { + "sid": 21, + "sent": "AF\u20101 and AF\u20102 bind distinct but overlapping sets of coregulators (Webb et\u00a0al, 1998; Endoh et\u00a0al, 1999; Delage\u2010Mourroux et\u00a0al, 2000; Yi et\u00a0al, 2015).", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "AF\u20101", + "structure_element" + ], + [ + 9, + 13, + "AF\u20102", + "structure_element" + ] + ] + }, + { + "sid": 22, + "sent": "AF\u20102 binds the signature LxxLL motif peptides of coactivators such as NCOA1/2/3 (also known as SRC\u20101/2/3).", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "AF\u20102", + "structure_element" + ], + [ + 25, + 36, + "LxxLL motif", + "structure_element" + ], + [ + 70, + 79, + "NCOA1/2/3", + "protein" + ], + [ + 95, + 104, + "SRC\u20101/2/3", + "protein" + ] + ] + }, + { + "sid": 23, + "sent": "AF\u20101 binds a separate surface on these coactivators (Webb et\u00a0al, 1998; Yi et\u00a0al, 2015).", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 24, + "sent": "Yet, it is unknown how different ER\u03b1 ligands control AF\u20101 through the LBD, and whether this inter\u2010domain communication is required for cell\u2010specific signaling or anti\u2010proliferative responses.", + "section": "INTRO", + "ner": [ + [ + 33, + 36, + "ER\u03b1", + "protein" + ], + [ + 53, + 57, + "AF\u20101", + "structure_element" + ], + [ + 70, + 73, + "LBD", + "structure_element" + ] + ] + }, + { + "sid": 25, + "sent": "In the canonical model of the ER\u03b1 signaling pathway (Fig\u00a01C), E2\u2010bound ER\u03b1 forms a homodimer that binds DNA at estrogen\u2010response elements (EREs), recruits NCOA1/2/3 (Metivier et\u00a0al, 2003; Johnson & O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ER\u03b1\u2010positive breast cancer cells (Ghosh et\u00a0al, 2000; Rae et\u00a0al, 2005; Deschenes et\u00a0al, 2007; Liu et\u00a0al, 2012; Srinivasan et\u00a0al, 2013).", + "section": "INTRO", + "ner": [ + [ + 30, + 33, + "ER\u03b1", + "protein" + ], + [ + 62, + 70, + "E2\u2010bound", + "protein_state" + ], + [ + 71, + 74, + "ER\u03b1", + "protein" + ], + [ + 83, + 92, + "homodimer", + "oligomeric_state" + ], + [ + 111, + 137, + "estrogen\u2010response elements", + "site" + ], + [ + 139, + 143, + "EREs", + "site" + ], + [ + 155, + 164, + "NCOA1/2/3", + "protein" + ], + [ + 233, + 238, + "GREB1", + "protein" + ], + [ + 284, + 287, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 26, + "sent": "However, ER\u03b1\u2010mediated proliferative responses vary in a ligand\u2010dependent manner (Srinivasan et\u00a0al, 2013); thus, it is not known whether this canonical model is widely applicable across diverse ER\u03b1 ligands.", + "section": "INTRO", + "ner": [ + [ + 9, + 12, + "ER\u03b1", + "protein" + ], + [ + 193, + 196, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 27, + "sent": "Our long\u2010term goal is to be able to predict proliferative or anti\u2010proliferative activity of a ligand in different tissues from its crystal structure by identifying different structural perturbations that lead to specific signaling outcomes.", + "section": "INTRO", + "ner": [ + [ + 131, + 148, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 28, + "sent": "The simplest response model for ligand\u2010specific proliferative effects is a linear causality model, where the degree of NCOA1/2/3 recruitment determines GREB1 expression, which in turn drives ligand\u2010specific cell proliferation (Fig\u00a01D).", + "section": "INTRO", + "ner": [ + [ + 119, + 128, + "NCOA1/2/3", + "protein" + ], + [ + 152, + 157, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 29, + "sent": "In this signaling model, multiple coregulator binding events and target genes (Won Jeong et\u00a0al, 2012; Nwachukwu et\u00a0al, 2014), LBD conformation, nucleocytoplasmic shuttling, the occupancy and dynamics of DNA binding, and other biophysical features could contribute independently to cell proliferation (Lickwar et\u00a0al, 2012).", + "section": "INTRO", + "ner": [ + [ + 126, + 129, + "LBD", + "structure_element" + ] + ] + }, + { + "sid": 30, + "sent": "To test these signaling models, we profiled a diverse library of ER\u03b1 ligands using systems biology approaches to X\u2010ray crystallography and chemical biology (Srinivasan et\u00a0al, 2013), including a series of quantitative bioassays for ER\u03b1 function that were statistically robust and reproducible, based on the Z\u2019\u2010statistic (Fig\u00a0EV1A and B; see Materials and Methods).", + "section": "INTRO", + "ner": [ + [ + 65, + 68, + "ER\u03b1", + "protein" + ], + [ + 113, + 134, + "X\u2010ray crystallography", + "experimental_method" + ], + [ + 139, + 155, + "chemical biology", + "experimental_method" + ], + [ + 231, + 234, + "ER\u03b1", + "protein" + ], + [ + 306, + 318, + "Z\u2019\u2010statistic", + "evidence" + ] + ] + }, + { + "sid": 31, + "sent": "We also determined the structures of 76 distinct ER\u03b1 LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ER\u03b1 LBD.", + "section": "INTRO", + "ner": [ + [ + 8, + 18, + "determined", + "experimental_method" + ], + [ + 23, + 33, + "structures", + "evidence" + ], + [ + 49, + 52, + "ER\u03b1", + "protein" + ], + [ + 53, + 56, + "LBD", + "structure_element" + ], + [ + 67, + 75, + "bound to", + "protein_state" + ], + [ + 172, + 178, + "active", + "protein_state" + ], + [ + 199, + 202, + "ER\u03b1", + "protein" + ], + [ + 203, + 206, + "LBD", + "structure_element" + ] + ] + }, + { + "sid": 32, + "sent": "Our findings here indicate that specific structural perturbations can be tied to ligand\u2010selective domain usage and signaling patterns, thus providing a framework for structure\u2010based design of improved breast cancer therapeutics, and understanding the different phenotypic effects of environmental estrogens.", + "section": "INTRO", + "ner": [ + [ + 297, + 306, + "estrogens", + "chemical" + ] + ] + }, + { + "sid": 33, + "sent": "High\u2010throughput screens for ER\u03b1 ligand profiling", + "section": "FIG", + "ner": [ + [ + 28, + 31, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 34, + "sent": "Summary of ligand screening assays used to measure ER\u2010mediated activities.", + "section": "FIG", + "ner": [ + [ + 11, + 34, + "ligand screening assays", + "experimental_method" + ] + ] + }, + { + "sid": 35, + "sent": "ERE, estrogen\u2010response element; Luc, luciferase reporter gene; M2H, mammalian 2\u2010hybrid; UAS, upstream\u2010activating sequence.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "ERE", + "structure_element" + ], + [ + 5, + 30, + "estrogen\u2010response element", + "structure_element" + ], + [ + 32, + 35, + "Luc", + "experimental_method" + ], + [ + 37, + 61, + "luciferase reporter gene", + "experimental_method" + ], + [ + 63, + 66, + "M2H", + "experimental_method" + ], + [ + 68, + 86, + "mammalian 2\u2010hybrid", + "experimental_method" + ], + [ + 88, + 91, + "UAS", + "structure_element" + ], + [ + 93, + 121, + "upstream\u2010activating sequence", + "structure_element" + ] + ] + }, + { + "sid": 36, + "sent": "Strength of AF\u20101 signaling does not determine cell\u2010specific signaling", + "section": "RESULTS", + "ner": [ + [ + 12, + 16, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 37, + "sent": "To compare ER\u03b1 signaling induced by diverse ligand types, we synthesized and assayed a library of 241 ER\u03b1 ligands containing 19 distinct molecular scaffolds.", + "section": "RESULTS", + "ner": [ + [ + 11, + 14, + "ER\u03b1", + "protein" + ], + [ + 61, + 84, + "synthesized and assayed", + "experimental_method" + ], + [ + 102, + 105, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 38, + "sent": "These include 15 indirect modulator series, which lack a SERM\u2010like side chain and modulate coactivator binding indirectly from the ligand\u2010binding pocket (Fig\u00a02A\u2013E; Dataset EV1) (Zheng et\u00a0al, 2012) (Zhu et\u00a0al, 2012) (Muthyala et\u00a0al, 2003; Seo et\u00a0al, 2006) (Srinivasan et\u00a0al, 2013) (Wang et\u00a0al, 2012) (Liao et\u00a0al, 2014) (Min et\u00a0al, 2013).", + "section": "RESULTS", + "ner": [ + [ + 50, + 54, + "lack", + "protein_state" + ], + [ + 57, + 66, + "SERM\u2010like", + "protein_type" + ], + [ + 131, + 152, + "ligand\u2010binding pocket", + "site" + ] + ] + }, + { + "sid": 39, + "sent": "We also generated four direct modulator series with side chains designed to directly dislocate h12 and thereby completely occlude the AF\u20102 surface (Fig\u00a02C and E; Dataset EV1) (Kieser et\u00a0al, 2010).", + "section": "RESULTS", + "ner": [ + [ + 95, + 98, + "h12", + "structure_element" + ], + [ + 134, + 146, + "AF\u20102 surface", + "site" + ] + ] + }, + { + "sid": 40, + "sent": "Ligand profiling using our quantitative bioassays revealed a wide range of ligand\u2010induced GREB1 expression, reporter gene activities, ER\u03b1\u2010coactivator interactions, and proliferative effects on MCF\u20107 breast cancer cells (Figs\u00a0EV1 and EV2A\u2013J).", + "section": "RESULTS", + "ner": [ + [ + 0, + 16, + "Ligand profiling", + "experimental_method" + ], + [ + 27, + 49, + "quantitative bioassays", + "experimental_method" + ], + [ + 90, + 95, + "GREB1", + "protein" + ], + [ + 134, + 137, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 41, + "sent": "This wide variance enabled us to probe specific features of ER\u03b1 signaling using ligand class analyses, and identify signaling patterns shared by specific ligand series or scaffolds.", + "section": "RESULTS", + "ner": [ + [ + 60, + 63, + "ER\u03b1", + "protein" + ], + [ + 80, + 101, + "ligand class analyses", + "experimental_method" + ] + ] + }, + { + "sid": 42, + "sent": "Classes of compounds in the ER\u03b1 ligand library", + "section": "FIG", + "ner": [ + [ + 28, + 31, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 43, + "sent": "Structure of the E2\u2010bound ER\u03b1 LBD in complex with an NCOA2 peptide of (PDB 1GWR).", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 17, + 25, + "E2\u2010bound", + "protein_state" + ], + [ + 26, + 29, + "ER\u03b1", + "protein" + ], + [ + 30, + 33, + "LBD", + "structure_element" + ], + [ + 34, + 49, + "in complex with", + "protein_state" + ], + [ + 53, + 58, + "NCOA2", + "protein" + ] + ] + }, + { + "sid": 44, + "sent": "Structural details of the ER\u03b1 LBD bound to the indicated ligands.", + "section": "FIG", + "ner": [ + [ + 26, + 29, + "ER\u03b1", + "protein" + ], + [ + 30, + 33, + "LBD", + "structure_element" + ], + [ + 34, + 42, + "bound to", + "protein_state" + ] + ] + }, + { + "sid": 45, + "sent": "Unlike E2 (PDB 1GWR), TAM is a direct modulator with a BSC that dislocates h12 to block the NCOA2\u2010binding site (PDB 3ERT).", + "section": "FIG", + "ner": [ + [ + 7, + 9, + "E2", + "chemical" + ], + [ + 22, + 25, + "TAM", + "chemical" + ], + [ + 75, + 78, + "h12", + "structure_element" + ], + [ + 92, + 110, + "NCOA2\u2010binding site", + "site" + ] + ] + }, + { + "sid": 46, + "sent": "OBHS is an indirect modulator that dislocates the h11 C\u2010terminus to destabilize the h11\u2013h12 interface (PDB 4ZN9).", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "OBHS", + "chemical" + ], + [ + 50, + 53, + "h11", + "structure_element" + ], + [ + 84, + 101, + "h11\u2013h12 interface", + "site" + ] + ] + }, + { + "sid": 47, + "sent": "The ER\u03b1 ligand library contains 241 ligands representing 15 indirect modulator scaffolds, plus 4 direct modulator scaffolds.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 48, + "sent": "ER\u03b1 ligands induced a range of agonist activity profiles", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 49, + "sent": "To this end, we compared the average ligand\u2010induced GREB1 mRNA levels in MCF\u20107 cells and 3\u00d7ERE\u2010Luc reporter gene activity in Ishikawa endometrial cancer cells (E\u2010Luc) or in HepG2 cells transfected with wild\u2010type ER\u03b1 (L\u2010Luc ER\u03b1\u2010WT) (Figs\u00a03A and EV2A\u2013C).", + "section": "RESULTS", + "ner": [ + [ + 52, + 57, + "GREB1", + "protein" + ], + [ + 89, + 98, + "3\u00d7ERE\u2010Luc", + "experimental_method" + ], + [ + 160, + 165, + "E\u2010Luc", + "experimental_method" + ], + [ + 202, + 211, + "wild\u2010type", + "protein_state" + ], + [ + 212, + 215, + "ER\u03b1", + "protein" + ], + [ + 217, + 222, + "L\u2010Luc", + "experimental_method" + ], + [ + 223, + 226, + "ER\u03b1", + "protein" + ], + [ + 227, + 229, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 50, + "sent": "Direct modulators showed significant differences in average activity between cell types except OBHS\u2010ASC analogs, which had similar low agonist activities in the three cell types.", + "section": "RESULTS", + "ner": [ + [ + 95, + 103, + "OBHS\u2010ASC", + "chemical" + ] + ] + }, + { + "sid": 51, + "sent": "While it was known that direct modulators such as tamoxifen drive cell\u2010specific signaling, these experiments reveal that indirect modulators also drive cell\u2010specific signaling, since eight of fourteen classes showed significant differences in average activity (Figs\u00a03A and EV2A\u2013C).", + "section": "RESULTS", + "ner": [ + [ + 50, + 59, + "tamoxifen", + "chemical" + ] + ] + }, + { + "sid": 52, + "sent": "Ligand\u2010specific signaling underlies ER\u03b1\u2010mediated cell proliferation", + "section": "FIG", + "ner": [ + [ + 36, + 39, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 53, + "sent": "(A) Ligand\u2010specific ER\u03b1 activities in HepG2, Ishikawa and MCF\u20107 cells.", + "section": "FIG", + "ner": [ + [ + 20, + 23, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 54, + "sent": "The ligand\u2010induced L\u2010Luc ER\u03b1\u2010WT and E\u2010Luc activities and GREB1 mRNA levels are shown by scaffold (mean\u00a0+\u00a0SD).", + "section": "FIG", + "ner": [ + [ + 19, + 24, + "L\u2010Luc", + "experimental_method" + ], + [ + 25, + 28, + "ER\u03b1", + "protein" + ], + [ + 29, + 31, + "WT", + "protein_state" + ], + [ + 36, + 41, + "E\u2010Luc", + "experimental_method" + ], + [ + 57, + 62, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 55, + "sent": "(B) Ligand class analysis of the L\u2010Luc ER\u03b1\u2010WT and ER\u03b1\u2010\u0394AB activities in HepG2 cells.", + "section": "FIG", + "ner": [ + [ + 11, + 25, + "class analysis", + "experimental_method" + ], + [ + 33, + 38, + "L\u2010Luc", + "experimental_method" + ], + [ + 39, + 42, + "ER\u03b1", + "protein" + ], + [ + 43, + 45, + "WT", + "protein_state" + ], + [ + 50, + 57, + "ER\u03b1\u2010\u0394AB", + "mutant" + ] + ] + }, + { + "sid": 56, + "sent": "Significant sensitivity to AB domain deletion was determined by Student's t\u2010test (n\u00a0=\u00a0number of ligands per scaffold in Fig\u00a02).", + "section": "FIG", + "ner": [ + [ + 27, + 29, + "AB", + "structure_element" + ], + [ + 64, + 80, + "Student's t\u2010test", + "experimental_method" + ] + ] + }, + { + "sid": 57, + "sent": "Correlation and regression analyses in a large test set.", + "section": "FIG", + "ner": [ + [ + 0, + 35, + "Correlation and regression analyses", + "experimental_method" + ] + ] + }, + { + "sid": 58, + "sent": "In cluster 1, the first three comparisons (rows) showed significant positive correlations (F\u2010test for nonzero slope, P\u00a0\u2264\u00a00.05).", + "section": "FIG", + "ner": [ + [ + 91, + 97, + "F\u2010test", + "experimental_method" + ], + [ + 117, + 118, + "P", + "evidence" + ] + ] + }, + { + "sid": 59, + "sent": "In cluster 2, only one of these comparisons revealed a significant positive correlation, while none was significant in cluster 3. +, statistically significant correlations gained by deletion of the AB or F domains.", + "section": "FIG", + "ner": [ + [ + 182, + 190, + "deletion", + "experimental_method" + ], + [ + 198, + 200, + "AB", + "structure_element" + ], + [ + 204, + 205, + "F", + "structure_element" + ] + ] + }, + { + "sid": 60, + "sent": "\u2212, significant correlations lost upon deletion of AB or F domains.", + "section": "FIG", + "ner": [ + [ + 50, + 52, + "AB", + "structure_element" + ], + [ + 56, + 57, + "F", + "structure_element" + ] + ] + }, + { + "sid": 61, + "sent": "Tamoxifen depends on AF\u20101 for its cell\u2010specific activity (Sakamoto et\u00a0al, 2002); therefore, we asked whether cell\u2010specific signaling observed here is due to a similar dependence on AF\u20101 for activity (Fig\u00a0EV1).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Tamoxifen", + "chemical" + ], + [ + 21, + 25, + "AF\u20101", + "structure_element" + ], + [ + 181, + 185, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 62, + "sent": "To test this idea, we compared the average L\u2010Luc activities of each scaffold in HepG2 cells co\u2010transfected with wild\u2010type ER\u03b1 or with ER\u03b1 lacking the AB domain (Figs\u00a01B and EV1).", + "section": "RESULTS", + "ner": [ + [ + 35, + 59, + "average L\u2010Luc activities", + "evidence" + ], + [ + 92, + 106, + "co\u2010transfected", + "experimental_method" + ], + [ + 112, + 121, + "wild\u2010type", + "protein_state" + ], + [ + 122, + 125, + "ER\u03b1", + "protein" + ], + [ + 134, + 137, + "ER\u03b1", + "protein" + ], + [ + 138, + 149, + "lacking the", + "protein_state" + ], + [ + 150, + 152, + "AB", + "structure_element" + ] + ] + }, + { + "sid": 63, + "sent": "While E2 showed similar L\u2010Luc ER\u03b1\u2010WT and ER\u03b1\u2010\u0394AB activities, tamoxifen showed complete loss of activity without the AB domain (Fig\u00a0EV1B).", + "section": "RESULTS", + "ner": [ + [ + 6, + 8, + "E2", + "chemical" + ], + [ + 24, + 29, + "L\u2010Luc", + "experimental_method" + ], + [ + 30, + 33, + "ER\u03b1", + "protein" + ], + [ + 34, + 36, + "WT", + "protein_state" + ], + [ + 41, + 48, + "ER\u03b1\u2010\u0394AB", + "mutant" + ], + [ + 61, + 70, + "tamoxifen", + "chemical" + ], + [ + 104, + 111, + "without", + "protein_state" + ], + [ + 116, + 118, + "AB", + "structure_element" + ] + ] + }, + { + "sid": 64, + "sent": "Deletion of the AB domain significantly reduced the average L\u2010Luc activities of 14 scaffolds (Student's t\u2010test, P\u00a0\u2264\u00a00.05) (Fig\u00a03B).", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Deletion of", + "experimental_method" + ], + [ + 16, + 18, + "AB", + "structure_element" + ], + [ + 52, + 76, + "average L\u2010Luc activities", + "evidence" + ], + [ + 94, + 110, + "Student's t\u2010test", + "experimental_method" + ], + [ + 112, + 113, + "P", + "evidence" + ] + ] + }, + { + "sid": 65, + "sent": "These \u201cAF\u20101\u2010sensitive\u201d activities were exhibited by both direct and indirect modulators, and were not limited to scaffolds that showed cell\u2010specific signaling (Fig\u00a03A and B).", + "section": "RESULTS", + "ner": [ + [ + 7, + 11, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 66, + "sent": "Thus, the strength of AF\u20101 signaling does not determine cell\u2010specific signaling.", + "section": "RESULTS", + "ner": [ + [ + 22, + 26, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 67, + "sent": "Identifying cell\u2010specific signaling clusters in ER\u03b1 ligand classes", + "section": "RESULTS", + "ner": [ + [ + 48, + 51, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 68, + "sent": "For each ligand class or scaffold, we calculated the Pearson's correlation coefficient, r, for pairwise comparison of activity profiles in breast (GREB1), liver (L\u2010Luc), and endometrial cells (E\u2010Luc).", + "section": "RESULTS", + "ner": [ + [ + 53, + 86, + "Pearson's correlation coefficient", + "evidence" + ], + [ + 88, + 89, + "r", + "evidence" + ], + [ + 147, + 152, + "GREB1", + "protein" + ], + [ + 162, + 167, + "L\u2010Luc", + "experimental_method" + ], + [ + 193, + 198, + "E\u2010Luc", + "experimental_method" + ] + ] + }, + { + "sid": 69, + "sent": "The value of r ranges from \u22121 to 1, and it defines the extent to which the data fit a straight line when compounds show similar agonist/antagonist activity profiles between cell types (Fig\u00a0EV3A).", + "section": "RESULTS", + "ner": [ + [ + 13, + 14, + "r", + "evidence" + ] + ] + }, + { + "sid": 70, + "sent": "We also calculated the coefficient of determination, r 2, which describes the percentage of variance in a dependent variable such as proliferation that can be predicted by an independent variable such as GREB1 expression.", + "section": "RESULTS", + "ner": [ + [ + 23, + 51, + "coefficient of determination", + "evidence" + ], + [ + 53, + 56, + "r 2", + "evidence" + ], + [ + 204, + 209, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 71, + "sent": "We present both calculations as r 2 to readily compare signaling specificities using a heat map on which the red\u2013yellow palette indicates significant positive correlations (P\u00a0\u2264\u00a00.05, F\u2010test for nonzero slope), while the blue palette denotes negative correlations (Fig\u00a03C\u2013F).", + "section": "RESULTS", + "ner": [ + [ + 32, + 35, + "r 2", + "evidence" + ], + [ + 173, + 174, + "P", + "evidence" + ], + [ + 183, + 189, + "F\u2010test", + "experimental_method" + ] + ] + }, + { + "sid": 72, + "sent": "The side chain of OBHS\u2010BSC analogs induces cell\u2010specific signaling", + "section": "FIG", + "ner": [ + [ + 18, + 26, + "OBHS\u2010BSC", + "chemical" + ] + ] + }, + { + "sid": 73, + "sent": "Correlation analysis of OBHS versus OBHS\u2010BSC activity across cell types.", + "section": "FIG", + "ner": [ + [ + 24, + 28, + "OBHS", + "chemical" + ], + [ + 36, + 44, + "OBHS\u2010BSC", + "chemical" + ] + ] + }, + { + "sid": 74, + "sent": "Correlation analysis of L\u2010Luc ER\u03b1\u2010\u0394AB activity versus endogenous ER\u03b1 activity of OBHS analogs.", + "section": "FIG", + "ner": [ + [ + 24, + 29, + "L\u2010Luc", + "experimental_method" + ], + [ + 30, + 37, + "ER\u03b1\u2010\u0394AB", + "mutant" + ], + [ + 65, + 68, + "ER\u03b1", + "protein" + ], + [ + 81, + 85, + "OBHS", + "chemical" + ] + ] + }, + { + "sid": 75, + "sent": "In panel (D), L\u2010Luc ER\u03b1\u2010WT activity from panel (B) is shown for comparison.", + "section": "FIG", + "ner": [ + [ + 14, + 19, + "L\u2010Luc", + "experimental_method" + ], + [ + 20, + 23, + "ER\u03b1", + "protein" + ], + [ + 24, + 26, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 76, + "sent": "Correlation analysis of L\u2010Luc ER\u03b1\u2010\u0394F activity versus endogenous ER\u03b1 activities of OBHS analogs.", + "section": "FIG", + "ner": [ + [ + 24, + 29, + "L\u2010Luc", + "experimental_method" + ], + [ + 30, + 36, + "ER\u03b1\u2010\u0394F", + "mutant" + ], + [ + 64, + 67, + "ER\u03b1", + "protein" + ], + [ + 82, + 86, + "OBHS", + "chemical" + ] + ] + }, + { + "sid": 77, + "sent": "Correlation analysis of MCF\u20107 cell proliferation versus NCOA2/3 recruitment or GREB1 levels observed in response to (G) OBHS\u2010N and (H) OBHS\u2010BSC analogs.", + "section": "FIG", + "ner": [ + [ + 56, + 63, + "NCOA2/3", + "protein" + ], + [ + 79, + 84, + "GREB1", + "protein" + ], + [ + 120, + 126, + "OBHS\u2010N", + "chemical" + ], + [ + 135, + 143, + "OBHS\u2010BSC", + "chemical" + ] + ] + }, + { + "sid": 78, + "sent": "Scaffolds in cluster 1 exhibited strongly correlated GREB1 levels, E\u2010Luc and L\u2010Luc activity profiles across the three cell types (Fig\u00a03C lanes 1\u20134), suggesting these ligands use similar ER\u03b1 signaling pathways in the breast, endometrial, and liver cell types.", + "section": "RESULTS", + "ner": [ + [ + 53, + 58, + "GREB1", + "protein" + ], + [ + 67, + 72, + "E\u2010Luc", + "experimental_method" + ], + [ + 77, + 82, + "L\u2010Luc", + "experimental_method" + ], + [ + 186, + 189, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 79, + "sent": "This cluster includes WAY\u2010C, OBHS, OBHS\u2010N, and triaryl\u2010ethylene analogs, all of which are indirect modulators.", + "section": "RESULTS", + "ner": [ + [ + 22, + 27, + "WAY\u2010C", + "chemical" + ], + [ + 29, + 33, + "OBHS", + "chemical" + ], + [ + 35, + 41, + "OBHS\u2010N", + "chemical" + ], + [ + 47, + 63, + "triaryl\u2010ethylene", + "chemical" + ] + ] + }, + { + "sid": 80, + "sent": "This cluster includes two classes of direct modulators (cyclofenil\u2010ASC and WAY dimer), and six classes of indirect modulators (2,5\u2010DTP, 3,4\u2010DTP, S\u2010OBHS\u20102 and S\u2010OBHS\u20103, furan, and WAY\u2010D).", + "section": "RESULTS", + "ner": [ + [ + 56, + 70, + "cyclofenil\u2010ASC", + "chemical" + ], + [ + 75, + 84, + "WAY dimer", + "chemical" + ], + [ + 127, + 134, + "2,5\u2010DTP", + "chemical" + ], + [ + 136, + 143, + "3,4\u2010DTP", + "chemical" + ], + [ + 145, + 153, + "S\u2010OBHS\u20102", + "chemical" + ], + [ + 158, + 166, + "S\u2010OBHS\u20103", + "chemical" + ], + [ + 168, + 173, + "furan", + "chemical" + ], + [ + 179, + 184, + "WAY\u2010D", + "chemical" + ] + ] + }, + { + "sid": 81, + "sent": "For example, 3,4\u2010DTP, furan, and S\u2010OBHS\u20102 drove positively correlated GREB1 levels and E\u2010Luc but not L\u2010Luc ER\u03b1\u2010WT activity (Fig\u00a03C lanes 5\u20137).", + "section": "RESULTS", + "ner": [ + [ + 13, + 20, + "3,4\u2010DTP", + "chemical" + ], + [ + 22, + 27, + "furan", + "chemical" + ], + [ + 33, + 41, + "S\u2010OBHS\u20102", + "chemical" + ], + [ + 70, + 75, + "GREB1", + "protein" + ], + [ + 87, + 92, + "E\u2010Luc", + "experimental_method" + ], + [ + 101, + 106, + "L\u2010Luc", + "experimental_method" + ], + [ + 107, + 110, + "ER\u03b1", + "protein" + ], + [ + 111, + 113, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 82, + "sent": "In\u00a0contrast, WAY dimer and WAY\u2010D analogs drove positively correlated GREB1 levels and L\u2010Luc ER\u03b1\u2010WT but not E\u2010Luc activity (Fig\u00a03C lanes 8 and 9).", + "section": "RESULTS", + "ner": [ + [ + 13, + 22, + "WAY dimer", + "chemical" + ], + [ + 27, + 32, + "WAY\u2010D", + "chemical" + ], + [ + 69, + 74, + "GREB1", + "protein" + ], + [ + 86, + 91, + "L\u2010Luc", + "experimental_method" + ], + [ + 92, + 95, + "ER\u03b1", + "protein" + ], + [ + 96, + 98, + "WT", + "protein_state" + ], + [ + 107, + 112, + "E\u2010Luc", + "experimental_method" + ] + ] + }, + { + "sid": 83, + "sent": "This cluster includes two direct modulator scaffolds (OBHS\u2010ASC and OBHS\u2010BSC), and five indirect modulator scaffolds (A\u2010CD, cyclofenil, 3,4\u2010DTPD, imine, and imidazopyridine).", + "section": "RESULTS", + "ner": [ + [ + 54, + 62, + "OBHS\u2010ASC", + "chemical" + ], + [ + 67, + 75, + "OBHS\u2010BSC", + "chemical" + ], + [ + 117, + 121, + "A\u2010CD", + "chemical" + ], + [ + 123, + 133, + "cyclofenil", + "chemical" + ], + [ + 135, + 143, + "3,4\u2010DTPD", + "chemical" + ], + [ + 145, + 150, + "imine", + "chemical" + ], + [ + 156, + 171, + "imidazopyridine", + "chemical" + ] + ] + }, + { + "sid": 84, + "sent": "These results suggest that addition of an extended side chain to an ER\u03b1 ligand scaffold is sufficient to induce cell\u2010specific signaling, where the relative activity profiles of the individual ligands change between cell types.", + "section": "RESULTS", + "ner": [ + [ + 68, + 71, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 85, + "sent": "This is demonstrated by directly comparing the signaling specificities of matched OBHS (indirect modulator, cluster 1) and OBHS\u2010BSC analogs (direct modulator, cluster 3), which differ only in the basic side chain (Fig\u00a02E).", + "section": "RESULTS", + "ner": [ + [ + 82, + 86, + "OBHS", + "chemical" + ], + [ + 123, + 131, + "OBHS\u2010BSC", + "chemical" + ] + ] + }, + { + "sid": 86, + "sent": "The activities of OBHS analogs were positively correlated across the three cell types, but the side chain of OBHS\u2010BSC analogs was sufficient to abolish these correlations (Figs\u00a03C lanes 1 and 19, and EV3A\u2013C).", + "section": "RESULTS", + "ner": [ + [ + 18, + 22, + "OBHS", + "chemical" + ], + [ + 109, + 117, + "OBHS\u2010BSC", + "chemical" + ] + ] + }, + { + "sid": 87, + "sent": "Thus, examining the correlated patterns of ER\u03b1 activity within each scaffold demonstrates that an extended side chain is not required for cell\u2010specific signaling.", + "section": "RESULTS", + "ner": [ + [ + 43, + 46, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 88, + "sent": "Modulation of signaling specificity by AF\u20101", + "section": "RESULTS", + "ner": [ + [ + 39, + 43, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 89, + "sent": "To evaluate the role of AF\u20101 and the F domain in ER\u03b1 signaling specificity, we compared activity of truncated ER\u03b1 constructs in HepG2 liver cells with endogenous ER\u03b1 activity in the other cell types.", + "section": "RESULTS", + "ner": [ + [ + 24, + 28, + "AF\u20101", + "structure_element" + ], + [ + 37, + 38, + "F", + "structure_element" + ], + [ + 49, + 52, + "ER\u03b1", + "protein" + ], + [ + 110, + 113, + "ER\u03b1", + "protein" + ], + [ + 162, + 165, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 90, + "sent": "The positive correlation between the L\u2010Luc and E\u2010Luc activities or GREB1 levels induced by scaffolds in cluster 1 was generally retained without the AB domain, or the F domain (Fig\u00a03D lanes 1\u20134).", + "section": "RESULTS", + "ner": [ + [ + 37, + 42, + "L\u2010Luc", + "experimental_method" + ], + [ + 47, + 52, + "E\u2010Luc", + "experimental_method" + ], + [ + 67, + 72, + "GREB1", + "protein" + ], + [ + 149, + 151, + "AB", + "structure_element" + ], + [ + 167, + 168, + "F", + "structure_element" + ] + ] + }, + { + "sid": 91, + "sent": "This demonstrates that the signaling specificities underlying these positive correlations are not modified by AF\u20101.", + "section": "RESULTS", + "ner": [ + [ + 110, + 114, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 92, + "sent": "OBHS analogs showed an average L\u2010Luc ER\u03b1\u2010\u0394AB activity of 3.2%\u00a0\u00b1\u00a03 (mean\u00a0+\u00a0SEM) relative to E2.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "OBHS", + "chemical" + ], + [ + 31, + 36, + "L\u2010Luc", + "experimental_method" + ], + [ + 37, + 44, + "ER\u03b1\u2010\u0394AB", + "mutant" + ], + [ + 91, + 93, + "E2", + "chemical" + ] + ] + }, + { + "sid": 93, + "sent": "Despite this nearly complete lack of activity, the pattern of L\u2010Luc ER\u03b1\u2010\u0394AB activity was still highly correlated with the E\u2010Luc activity and GREB1 expression (Fig\u00a0EV3D and E), demonstrating that very small AF\u20102 activities can be amplified by AF\u20101 to produce robust signals.", + "section": "RESULTS", + "ner": [ + [ + 62, + 67, + "L\u2010Luc", + "experimental_method" + ], + [ + 68, + 75, + "ER\u03b1\u2010\u0394AB", + "mutant" + ], + [ + 122, + 127, + "E\u2010Luc", + "experimental_method" + ], + [ + 141, + 146, + "GREB1", + "protein" + ], + [ + 206, + 210, + "AF\u20102", + "structure_element" + ], + [ + 242, + 246, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 94, + "sent": "Similarly, deletion of the F domain did not abolish correlations between the L\u2010Luc and E\u2010Luc or GREB1 levels induced by OBHS analogs (Fig\u00a0EV3F).", + "section": "RESULTS", + "ner": [ + [ + 11, + 22, + "deletion of", + "experimental_method" + ], + [ + 27, + 28, + "F", + "structure_element" + ], + [ + 77, + 82, + "L\u2010Luc", + "experimental_method" + ], + [ + 87, + 92, + "E\u2010Luc", + "experimental_method" + ], + [ + 96, + 101, + "GREB1", + "protein" + ], + [ + 120, + 124, + "OBHS", + "chemical" + ] + ] + }, + { + "sid": 95, + "sent": "These similar patterns of ligand activity in the wild\u2010type and deletion mutants suggest that AF\u20101 and the F domain purely amplify the AF\u20102 activities of ligands in cluster 1.", + "section": "RESULTS", + "ner": [ + [ + 49, + 58, + "wild\u2010type", + "protein_state" + ], + [ + 72, + 79, + "mutants", + "protein_state" + ], + [ + 93, + 97, + "AF\u20101", + "structure_element" + ], + [ + 106, + 107, + "F", + "structure_element" + ], + [ + 134, + 138, + "AF\u20102", + "structure_element" + ] + ] + }, + { + "sid": 96, + "sent": "In contrast, AF\u20101 was a determinant of signaling specificity for scaffolds in cluster 2.", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 97, + "sent": "Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5\u2010DTP, 3,4\u2010DTP, S\u2010OBHS\u20103, WAY\u2010D, WAY dimer, and cyclofenil\u2010ASC) (Fig\u00a03D lanes 5\u201312).", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Deletion of", + "experimental_method" + ], + [ + 16, + 18, + "AB", + "structure_element" + ], + [ + 22, + 23, + "F", + "structure_element" + ], + [ + 100, + 107, + "2,5\u2010DTP", + "chemical" + ], + [ + 109, + 116, + "3,4\u2010DTP", + "chemical" + ], + [ + 118, + 126, + "S\u2010OBHS\u20103", + "chemical" + ], + [ + 128, + 133, + "WAY\u2010D", + "chemical" + ], + [ + 135, + 144, + "WAY dimer", + "chemical" + ], + [ + 150, + 164, + "cyclofenil\u2010ASC", + "chemical" + ] + ] + }, + { + "sid": 98, + "sent": "Comparing Fig\u00a03C and D, the + and \u2212 signs indicate where the deletion mutant assays led to a gain or loss of statically significant correlation, respectively.", + "section": "RESULTS", + "ner": [ + [ + 61, + 83, + "deletion mutant assays", + "experimental_method" + ] + ] + }, + { + "sid": 99, + "sent": "Thus, in cluster 2, AF\u20101 substantially modulated the specificity of ligands with cell\u2010specific activity (Fig\u00a03D lanes 5\u201312).", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 100, + "sent": "For ligands in cluster 3, we could not eliminate a role for AF\u20101 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig\u00a03C), and deletion of the AB or F domain rarely induced such correlations (Fig\u00a03D), except for A\u2010CD and OBHS\u2010ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E\u2010Luc activity and/or GREB1 levels (Fig\u00a03D lanes 13 and 18).", + "section": "RESULTS", + "ner": [ + [ + 60, + 64, + "AF\u20101", + "structure_element" + ], + [ + 183, + 194, + "deletion of", + "experimental_method" + ], + [ + 199, + 201, + "AB", + "structure_element" + ], + [ + 205, + 206, + "F", + "structure_element" + ], + [ + 268, + 272, + "A\u2010CD", + "chemical" + ], + [ + 277, + 285, + "OBHS\u2010ASC", + "chemical" + ], + [ + 301, + 312, + "deletion of", + "experimental_method" + ], + [ + 317, + 319, + "AB", + "structure_element" + ], + [ + 330, + 331, + "F", + "structure_element" + ], + [ + 373, + 378, + "E\u2010Luc", + "experimental_method" + ], + [ + 395, + 400, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 101, + "sent": "Thus, ligands in cluster 2 rely on AF\u20101 for both activity (Fig\u00a03B) and signaling specificity (Fig\u00a03D).", + "section": "RESULTS", + "ner": [ + [ + 35, + 39, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 102, + "sent": "Ligand\u2010specific control of GREB1 expression", + "section": "RESULTS", + "ner": [ + [ + 27, + 32, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 103, + "sent": "To determine whether ligand classes control expression of native ER\u03b1 target genes through the canonical linear signaling pathway, we performed pairwise linear regression analyses using ER\u03b1\u2013NCOA1/2/3 interactions in M2H assay as independent predictors of GREB1 expression (the dependent variable) (Figs\u00a0EV1 and EV2A, F\u2013H).", + "section": "RESULTS", + "ner": [ + [ + 65, + 68, + "ER\u03b1", + "protein" + ], + [ + 143, + 178, + "pairwise linear regression analyses", + "experimental_method" + ], + [ + 185, + 198, + "ER\u03b1\u2013NCOA1/2/3", + "complex_assembly" + ], + [ + 215, + 224, + "M2H assay", + "experimental_method" + ], + [ + 254, + 259, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 104, + "sent": "In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY\u2010C, followed by triaryl\u2010ethylene, OBHS\u2010N, and OBHS series, while for NCOA3, OBHS\u2010N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig\u00a0EV2F\u2013H).", + "section": "RESULTS", + "ner": [ + [ + 33, + 38, + "NCOA1", + "protein" + ], + [ + 43, + 48, + "NCOA2", + "protein" + ], + [ + 65, + 70, + "WAY\u2010C", + "chemical" + ], + [ + 84, + 100, + "triaryl\u2010ethylene", + "chemical" + ], + [ + 102, + 108, + "OBHS\u2010N", + "chemical" + ], + [ + 114, + 118, + "OBHS", + "chemical" + ], + [ + 137, + 142, + "NCOA3", + "protein" + ], + [ + 144, + 150, + "OBHS\u2010N", + "chemical" + ], + [ + 194, + 198, + "OBHS", + "chemical" + ] + ] + }, + { + "sid": 105, + "sent": "The average induction of GREB1 by cluster 1 ligands showed greater variance, with a range between ~25 and ~75% for OBHS and a range from full agonist to inverse agonist for the others in cluster 1 (Fig\u00a0EV2A).", + "section": "RESULTS", + "ner": [ + [ + 25, + 30, + "GREB1", + "protein" + ], + [ + 115, + 119, + "OBHS", + "chemical" + ] + ] + }, + { + "sid": 106, + "sent": "GREB1 levels induced by OBHS analogs were determined by recruitment of NCOA1 but not NCOA2/3 (Fig\u00a03E lane 1), suggesting that there may be alternate or preferential use of these coactivators by different classes.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "GREB1", + "protein" + ], + [ + 24, + 28, + "OBHS", + "chemical" + ], + [ + 71, + 76, + "NCOA1", + "protein" + ], + [ + 85, + 92, + "NCOA2/3", + "protein" + ] + ] + }, + { + "sid": 107, + "sent": "However, in cluster 1, NCOA1/2/3 recruitment generally predicted GREB1 levels (Fig\u00a03E lanes 1\u20134), consistent with the canonical signaling model (Fig\u00a01D).", + "section": "RESULTS", + "ner": [ + [ + 23, + 32, + "NCOA1/2/3", + "protein" + ], + [ + 65, + 70, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 108, + "sent": "For clusters 2 and 3, GREB1 activity was generally not predicted by NCOA1/2/3 recruitment.", + "section": "RESULTS", + "ner": [ + [ + 22, + 27, + "GREB1", + "protein" + ], + [ + 68, + 77, + "NCOA1/2/3", + "protein" + ] + ] + }, + { + "sid": 109, + "sent": "Direct modulators showed low NCOA1/2/3 recruitment (Fig\u00a0EV2F\u2013H), but only OBHS\u2010ASC analogs had NCOA2 recruitment profiles that predicted a full range of effects on GREB1 levels (Figs\u00a03E lanes 9, 11, 18\u201319, and EV2A).", + "section": "RESULTS", + "ner": [ + [ + 29, + 38, + "NCOA1/2/3", + "protein" + ], + [ + 74, + 82, + "OBHS\u2010ASC", + "chemical" + ], + [ + 95, + 100, + "NCOA2", + "protein" + ], + [ + 164, + 169, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 110, + "sent": "The indirect modulators in clusters 2 and 3 stimulated NCOA1/2/3 recruitment and GREB1 expression with substantial variance (Figs\u00a03A and EV2F\u2013H).", + "section": "RESULTS", + "ner": [ + [ + 55, + 64, + "NCOA1/2/3", + "protein" + ], + [ + 81, + 86, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 111, + "sent": "However, ligand\u2010induced GREB1 levels were generally not determined by NCOA1/2/3 recruitment (Fig\u00a03E lanes 5\u201319), consistent with an alternate causality model (Fig\u00a01E).", + "section": "RESULTS", + "ner": [ + [ + 24, + 29, + "GREB1", + "protein" + ], + [ + 70, + 79, + "NCOA1/2/3", + "protein" + ] + ] + }, + { + "sid": 112, + "sent": "Out of 11 indirect modulator series in cluster 2 or 3, only the S\u2010OBHS\u20103 class had NCOA1/2/3 recruitment profiles that predicted GREB1 levels (Fig\u00a03E lane 12).", + "section": "RESULTS", + "ner": [ + [ + 64, + 72, + "S\u2010OBHS\u20103", + "chemical" + ], + [ + 83, + 92, + "NCOA1/2/3", + "protein" + ], + [ + 129, + 134, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 113, + "sent": "These results suggest that compounds that show cell\u2010specific signaling do not activate GREB1, or use coactivators other than NCOA1/2/3 to control GREB1 expression (Fig\u00a01E).", + "section": "RESULTS", + "ner": [ + [ + 87, + 92, + "GREB1", + "protein" + ], + [ + 125, + 134, + "NCOA1/2/3", + "protein" + ], + [ + 146, + 151, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 114, + "sent": "To determine mechanisms for ligand\u2010dependent control of breast cancer cell proliferation, we performed linear regression analyses across the 19 scaffolds using MCF\u20107 cell proliferation as the dependent variable, and the other activities as independent variables (Fig\u00a03F).", + "section": "RESULTS", + "ner": [ + [ + 103, + 129, + "linear regression analyses", + "experimental_method" + ] + ] + }, + { + "sid": 115, + "sent": "In cluster 1, E\u2010Luc and L\u2010Luc activities, NCOA1/2/3 recruitment, and GREB1 levels generally predicted the proliferative response (Fig\u00a03F lanes 2\u20134).", + "section": "RESULTS", + "ner": [ + [ + 14, + 19, + "E\u2010Luc", + "experimental_method" + ], + [ + 24, + 29, + "L\u2010Luc", + "experimental_method" + ], + [ + 42, + 51, + "NCOA1/2/3", + "protein" + ], + [ + 69, + 74, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 116, + "sent": "With the OBHS\u2010N compounds, NCOA3 and GREB1 showed near perfect prediction of proliferation (Fig\u00a0EV3G), with unexplained variance similar to the noise in the assays.", + "section": "RESULTS", + "ner": [ + [ + 9, + 15, + "OBHS\u2010N", + "chemical" + ], + [ + 27, + 32, + "NCOA3", + "protein" + ], + [ + 37, + 42, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 117, + "sent": "The lack of significant predictors for OBHS analogs (Fig\u00a03F lane 1) reflects their small range of proliferative effects on MCF\u20107 cells (Fig\u00a0EV2I).", + "section": "RESULTS", + "ner": [ + [ + 39, + 43, + "OBHS", + "chemical" + ] + ] + }, + { + "sid": 118, + "sent": "The significant correlations with GREB1 expression and NCOA1/2/3 recruitment observed in this cluster are consistent with the canonical signaling model (Fig\u00a01D), where NCOA1/2/3 recruitment determines GREB1 expression, which then drives proliferation.", + "section": "RESULTS", + "ner": [ + [ + 34, + 39, + "GREB1", + "protein" + ], + [ + 55, + 64, + "NCOA1/2/3", + "protein" + ], + [ + 168, + 177, + "NCOA1/2/3", + "protein" + ], + [ + 201, + 206, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 119, + "sent": "Despite this phenotypic variance, proliferation was not generally predicted by correlated NCOA1/2/3 recruitment and GREB1 induction (Figs\u00a03F lanes 5\u201319, and EV3H).", + "section": "RESULTS", + "ner": [ + [ + 90, + 99, + "NCOA1/2/3", + "protein" + ], + [ + 116, + 121, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 120, + "sent": "Out of 15 ligand series in these clusters, only 2,5\u2010DTP analogs induced a proliferative response that was predicted by GREB1 levels, which were not determined by NCOA1/2/3 recruitment (Fig\u00a03E and F lane 10).", + "section": "RESULTS", + "ner": [ + [ + 48, + 55, + "2,5\u2010DTP", + "chemical" + ], + [ + 119, + 124, + "GREB1", + "protein" + ], + [ + 162, + 171, + "NCOA1/2/3", + "protein" + ] + ] + }, + { + "sid": 121, + "sent": "3,4\u2010DTP, cyclofenil, 3,4\u2010DTPD, and imidazopyridine analogs had NCOA1/3 recruitment profiles that predicted their proliferative effects, without determining GREB1 levels (Fig\u00a03E and F, lanes 5 and 14\u201316).", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "3,4\u2010DTP", + "chemical" + ], + [ + 9, + 19, + "cyclofenil", + "chemical" + ], + [ + 21, + 29, + "3,4\u2010DTPD", + "chemical" + ], + [ + 35, + 50, + "imidazopyridine", + "chemical" + ], + [ + 63, + 70, + "NCOA1/3", + "protein" + ], + [ + 156, + 161, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 122, + "sent": "Similarly, S\u2010OBHS\u20103, cyclofenil\u2010ASC, and OBHS\u2010ASC had positively correlated NCOA1/2/3 recruitment and GREB1 levels, but none of these activities determined their proliferative effects (Fig\u00a03E and F lanes 11\u201312 and 18).", + "section": "RESULTS", + "ner": [ + [ + 11, + 19, + "S\u2010OBHS\u20103", + "chemical" + ], + [ + 21, + 35, + "cyclofenil\u2010ASC", + "chemical" + ], + [ + 41, + 49, + "OBHS\u2010ASC", + "chemical" + ], + [ + 76, + 85, + "NCOA1/2/3", + "protein" + ], + [ + 102, + 107, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 123, + "sent": "For ligands that show cell\u2010specific signaling, ER\u03b1\u2010mediated recruitment of other coregulators and activation of other target genes likely determine their proliferative effects on MCF\u20107 cells.", + "section": "RESULTS", + "ner": [ + [ + 47, + 50, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 124, + "sent": "NCOA3 occupancy at GREB1 did not predict the proliferative response", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "NCOA3", + "protein" + ], + [ + 19, + 24, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 125, + "sent": "We also questioned whether promoter occupancy by coactivators is statistically robust and reproducible for ligand class analysis using a chromatin immunoprecipitation (ChIP)\u2010based quantitative assay, and whether it has a better predictive power than the M2H assay.", + "section": "RESULTS", + "ner": [ + [ + 137, + 199, + "chromatin immunoprecipitation (ChIP)\u2010based quantitative assay,", + "experimental_method" + ], + [ + 254, + 263, + "M2H assay", + "experimental_method" + ] + ] + }, + { + "sid": 126, + "sent": "ER\u03b1 and NCOA3 cycle on and off the GREB1 promoter (Nwachukwu et\u00a0al, 2014).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "ER\u03b1", + "protein" + ], + [ + 8, + 13, + "NCOA3", + "protein" + ], + [ + 35, + 40, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 127, + "sent": "Therefore, we first performed a time\u2010course study, and found that E2 and the WAY\u2010C analog, AAPII\u2010151\u20104, induced recruitment of NCOA3 to the GREB1 promoter in a temporal cycle that peaked after 45\u00a0min in MCF\u20107 cells (Fig\u00a04A).", + "section": "RESULTS", + "ner": [ + [ + 32, + 49, + "time\u2010course study", + "experimental_method" + ], + [ + 66, + 68, + "E2", + "chemical" + ], + [ + 77, + 82, + "WAY\u2010C", + "chemical" + ], + [ + 91, + 102, + "AAPII\u2010151\u20104", + "chemical" + ], + [ + 127, + 132, + "NCOA3", + "protein" + ], + [ + 140, + 145, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 128, + "sent": "At this time point, other WAY\u2010C analogs also induced recruitment of NCOA3 at this site to varying degrees (Fig\u00a04B).", + "section": "RESULTS", + "ner": [ + [ + 26, + 31, + "WAY\u2010C", + "chemical" + ], + [ + 68, + 73, + "NCOA3", + "protein" + ] + ] + }, + { + "sid": 129, + "sent": "The Z\u2019 for this assay was 0.6, showing statistical robustness (see Materials and Methods).", + "section": "RESULTS", + "ner": [ + [ + 4, + 6, + "Z\u2019", + "evidence" + ] + ] + }, + { + "sid": 130, + "sent": "We prepared biological replicates with different cell passage numbers and separately prepared samples, which showed r 2 of 0.81, demonstrating high reproducibility (Fig\u00a04C).", + "section": "RESULTS", + "ner": [ + [ + 116, + 119, + "r 2", + "evidence" + ] + ] + }, + { + "sid": 131, + "sent": " NCOA3 occupancy at GREB1 is statistically robust but does not predict transcriptional activity", + "section": "FIG", + "ner": [ + [ + 1, + 6, + "NCOA3", + "protein" + ], + [ + 20, + 25, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 132, + "sent": "Kinetic ChIP assay examining recruitment of NCOA3 to the GREB1 gene in MCF\u20107 cells stimulated with E2 or the indicated WAY\u2010C analog.", + "section": "FIG", + "ner": [ + [ + 0, + 18, + "Kinetic ChIP assay", + "experimental_method" + ], + [ + 44, + 49, + "NCOA3", + "protein" + ], + [ + 57, + 62, + "GREB1", + "protein" + ], + [ + 99, + 101, + "E2", + "chemical" + ], + [ + 119, + 124, + "WAY\u2010C", + "chemical" + ] + ] + }, + { + "sid": 133, + "sent": "NCOA3 occupancy at GREB1 was compared by ChIP assay 45\u00a0min after stimulation with vehicle, E2, or the WAY\u2010C analogs.", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "NCOA3", + "protein" + ], + [ + 19, + 24, + "GREB1", + "protein" + ], + [ + 41, + 51, + "ChIP assay", + "experimental_method" + ], + [ + 91, + 93, + "E2", + "chemical" + ], + [ + 102, + 107, + "WAY\u2010C", + "chemical" + ] + ] + }, + { + "sid": 134, + "sent": "In panel (B), the average recruitment of two biological replicates are shown as mean\u00a0+\u00a0SEM, and the Z\u2010score is indicated.", + "section": "FIG", + "ner": [ + [ + 100, + 107, + "Z\u2010score", + "evidence" + ] + ] + }, + { + "sid": 135, + "sent": "In panel (C), correlation analysis was performed for two biological replicates.", + "section": "FIG", + "ner": [ + [ + 14, + 34, + "correlation analysis", + "experimental_method" + ] + ] + }, + { + "sid": 136, + "sent": "Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY\u2010C analogs. *Significant positive correlation (F\u2010test for nonzero slope, P\u2010value).", + "section": "FIG", + "ner": [ + [ + 0, + 26, + "Linear regression analyses", + "experimental_method" + ], + [ + 52, + 57, + "NCOA3", + "protein" + ], + [ + 83, + 87, + "ChIP", + "experimental_method" + ], + [ + 91, + 94, + "M2H", + "experimental_method" + ], + [ + 135, + 140, + "WAY\u2010C", + "chemical" + ], + [ + 185, + 191, + "F\u2010test", + "experimental_method" + ], + [ + 211, + 218, + "P\u2010value", + "evidence" + ] + ] + }, + { + "sid": 137, + "sent": "The M2H assay for NCOA3 recruitment broadly correlated with the other assays, and was predictive for GREB1 expression and cell proliferation (Fig\u00a03E).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "M2H assay", + "experimental_method" + ], + [ + 18, + 23, + "NCOA3", + "protein" + ], + [ + 101, + 106, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 138, + "sent": "However, the ChIP assays for WAY\u2010C\u2010induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY\u2010C activity profiles (Fig\u00a04D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2\u00a0=\u00a00.36 and P\u00a0=\u00a00.09 (F\u2010test for nonzero slope).", + "section": "RESULTS", + "ner": [ + [ + 13, + 24, + "ChIP assays", + "experimental_method" + ], + [ + 29, + 34, + "WAY\u2010C", + "chemical" + ], + [ + 58, + 63, + "NCOA3", + "protein" + ], + [ + 71, + 76, + "GREB1", + "protein" + ], + [ + 126, + 131, + "WAY\u2010C", + "chemical" + ], + [ + 202, + 213, + "ChIP assays", + "experimental_method" + ], + [ + 218, + 223, + "NCOA3", + "protein" + ], + [ + 240, + 249, + "M2H assay", + "experimental_method" + ], + [ + 290, + 293, + "r 2", + "evidence" + ], + [ + 305, + 306, + "P", + "evidence" + ], + [ + 315, + 321, + "F\u2010test", + "experimental_method" + ] + ] + }, + { + "sid": 139, + "sent": "Thus, the simplified coactivator\u2010binding assay showed much greater predictive power than the ChIP assay for ligand\u2010specific effects on GREB1 expression and cell proliferation.", + "section": "RESULTS", + "ner": [ + [ + 21, + 46, + "coactivator\u2010binding assay", + "experimental_method" + ], + [ + 93, + 103, + "ChIP assay", + "experimental_method" + ], + [ + 135, + 140, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 140, + "sent": "ER\u03b2 activity is not an independent predictor of cell\u2010specific activity", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "ER\u03b2", + "protein" + ] + ] + }, + { + "sid": 141, + "sent": "One difference between MCF\u20107 breast cancer cells and Ishikawa endometrial cancer cells is the contribution of ER\u03b2 to estrogenic response, as Ishikawa cells may express ER\u03b2 (Bhat & Pezzuto, 2001).", + "section": "RESULTS", + "ner": [ + [ + 110, + 113, + "ER\u03b2", + "protein" + ], + [ + 168, + 171, + "ER\u03b2", + "protein" + ] + ] + }, + { + "sid": 142, + "sent": "When overexpressed in MCF\u20107 cells, ER\u03b2 alters E2\u2010induced expression of only a subset of ER\u03b1\u2010target genes (Wu et\u00a0al, 2011), raising the possibility that ligand\u2010induced ER\u03b2 activity may contribute to E\u2010Luc activities, and thus underlie the lack of correlation between the E\u2010Luc and L\u2010Luc ER\u03b1\u2010WT activities or GREB1 levels induced by cell\u2010specific modulators in cluster 2 and cluster 3 (Fig\u00a03C).", + "section": "RESULTS", + "ner": [ + [ + 5, + 18, + "overexpressed", + "experimental_method" + ], + [ + 35, + 38, + "ER\u03b2", + "protein" + ], + [ + 46, + 48, + "E2", + "chemical" + ], + [ + 88, + 91, + "ER\u03b1", + "protein" + ], + [ + 167, + 170, + "ER\u03b2", + "protein" + ], + [ + 198, + 203, + "E\u2010Luc", + "experimental_method" + ], + [ + 270, + 275, + "E\u2010Luc", + "experimental_method" + ], + [ + 280, + 285, + "L\u2010Luc", + "experimental_method" + ], + [ + 286, + 289, + "ER\u03b1", + "protein" + ], + [ + 290, + 292, + "WT", + "protein_state" + ], + [ + 307, + 312, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 143, + "sent": "To test this idea, we determined the L\u2010Luc ER\u03b2 activity profiles of the ligands (Fig\u00a0EV1).", + "section": "RESULTS", + "ner": [ + [ + 37, + 42, + "L\u2010Luc", + "experimental_method" + ] + ] + }, + { + "sid": 144, + "sent": "All direct modulator and two indirect modulator scaffolds (OBHS and S\u2010OBHS\u20103) lacked ER\u03b2 agonist activity.", + "section": "RESULTS", + "ner": [ + [ + 59, + 63, + "OBHS", + "chemical" + ], + [ + 68, + 76, + "S\u2010OBHS\u20103", + "chemical" + ] + ] + }, + { + "sid": 145, + "sent": "For most scaffolds, L\u2010Luc ER\u03b2 and E\u2010Luc activities were not correlated, except for 2,5\u2010DTP and cyclofenil analogs, which showed moderate but significant correlations (Fig\u00a0EV4A).", + "section": "RESULTS", + "ner": [ + [ + 20, + 25, + "L\u2010Luc", + "experimental_method" + ], + [ + 34, + 39, + "E\u2010Luc", + "experimental_method" + ], + [ + 83, + 90, + "2,5\u2010DTP", + "chemical" + ], + [ + 95, + 105, + "cyclofenil", + "chemical" + ] + ] + }, + { + "sid": 146, + "sent": "Nevertheless, the E\u2010Luc activities of both 2,5\u2010DTP and cyclofenil analogs were better predicted by their L\u2010Luc ER\u03b1\u2010WT than L\u2010Luc ER\u03b2 activities (Fig\u00a0EV4A and B).", + "section": "RESULTS", + "ner": [ + [ + 18, + 23, + "E\u2010Luc", + "experimental_method" + ], + [ + 43, + 50, + "2,5\u2010DTP", + "chemical" + ], + [ + 55, + 65, + "cyclofenil", + "chemical" + ], + [ + 105, + 110, + "L\u2010Luc", + "experimental_method" + ], + [ + 111, + 114, + "ER\u03b1", + "protein" + ], + [ + 115, + 117, + "WT", + "protein_state" + ], + [ + 123, + 128, + "L\u2010Luc", + "experimental_method" + ], + [ + 129, + 132, + "ER\u03b2", + "protein" + ] + ] + }, + { + "sid": 147, + "sent": "ER\u03b2 activity is not an independent predictor of E\u2010Luc activity", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "ER\u03b2", + "protein" + ], + [ + 48, + 53, + "E\u2010Luc", + "experimental_method" + ] + ] + }, + { + "sid": 148, + "sent": "ER\u03b2 activity in HepG2 cells rarely correlates with E\u2010Luc activity.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "ER\u03b2", + "protein" + ], + [ + 51, + 56, + "E\u2010Luc", + "experimental_method" + ] + ] + }, + { + "sid": 149, + "sent": "ER\u03b1 activity of 2,5\u2010DTP and cyclofenil analogs correlates with E\u2010Luc activity.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "ER\u03b1", + "protein" + ], + [ + 16, + 23, + "2,5\u2010DTP", + "chemical" + ], + [ + 28, + 38, + "cyclofenil", + "chemical" + ], + [ + 63, + 68, + "E\u2010Luc", + "experimental_method" + ] + ] + }, + { + "sid": 150, + "sent": " Data information: The r 2 and P values for the indicated correlations are shown in both panels. *Significant positive correlation (F\u2010test for nonzero slope, P\u2010value)", + "section": "FIG", + "ner": [ + [ + 31, + 39, + "P values", + "evidence" + ], + [ + 132, + 138, + "F\u2010test", + "experimental_method" + ], + [ + 158, + 165, + "P\u2010value", + "evidence" + ] + ] + }, + { + "sid": 151, + "sent": "To overcome barriers to crystallization of ER\u03b1 LBD complexes, we developed a conformation\u2010trapping X\u2010ray crystallography approach using the ER\u03b1\u2010Y537S mutation (Nettles et\u00a0al, 2008; Bruning et\u00a0al, 2010; Srinivasan et\u00a0al, 2013).", + "section": "RESULTS", + "ner": [ + [ + 24, + 39, + "crystallization", + "experimental_method" + ], + [ + 43, + 46, + "ER\u03b1", + "protein" + ], + [ + 47, + 50, + "LBD", + "structure_element" + ], + [ + 77, + 120, + "conformation\u2010trapping X\u2010ray crystallography", + "experimental_method" + ], + [ + 140, + 149, + "ER\u03b1\u2010Y537S", + "mutant" + ] + ] + }, + { + "sid": 152, + "sent": "To further validate this approach, we solved the structure of the ER\u03b1\u2010Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild\u2010type and ER\u03b1\u2010Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix\u00a0Fig S1A and B) (Nettles et\u00a0al, 2008; Bruning et\u00a0al, 2010; Delfosse et\u00a0al, 2012).", + "section": "RESULTS", + "ner": [ + [ + 38, + 44, + "solved", + "experimental_method" + ], + [ + 49, + 58, + "structure", + "evidence" + ], + [ + 66, + 75, + "ER\u03b1\u2010Y537S", + "mutant" + ], + [ + 76, + 79, + "LBD", + "structure_element" + ], + [ + 80, + 95, + "in complex with", + "protein_state" + ], + [ + 96, + 114, + "diethylstilbestrol", + "chemical" + ], + [ + 116, + 119, + "DES", + "chemical" + ], + [ + 153, + 162, + "wild\u2010type", + "protein_state" + ], + [ + 167, + 176, + "ER\u03b1\u2010Y537S", + "mutant" + ], + [ + 177, + 181, + "LBDs", + "structure_element" + ], + [ + 241, + 244, + "h12", + "structure_element" + ] + ] + }, + { + "sid": 153, + "sent": "Using this approach, we solved 76 ER\u03b1 LBD structures in the active conformation and bound to ligands studied here (Appendix\u00a0Fig S1C).", + "section": "RESULTS", + "ner": [ + [ + 24, + 30, + "solved", + "experimental_method" + ], + [ + 34, + 37, + "ER\u03b1", + "protein" + ], + [ + 38, + 41, + "LBD", + "structure_element" + ], + [ + 42, + 52, + "structures", + "evidence" + ], + [ + 60, + 79, + "active conformation", + "protein_state" + ], + [ + 84, + 100, + "bound to ligands", + "protein_state" + ] + ] + }, + { + "sid": 154, + "sent": "Eleven of these structures have been published, while 65 are new, including the DES\u2010bound ER\u03b1\u2010Y537S LBD.", + "section": "RESULTS", + "ner": [ + [ + 16, + 26, + "structures", + "evidence" + ], + [ + 80, + 89, + "DES\u2010bound", + "protein_state" + ], + [ + 90, + 99, + "ER\u03b1\u2010Y537S", + "mutant" + ], + [ + 100, + 103, + "LBD", + "structure_element" + ] + ] + }, + { + "sid": 155, + "sent": "We present 57 of these new structures here (Dataset EV2), while the remaining eight new structures bound to OBHS\u2010N analogs will be published elsewhere (S. Srinivasan et al, in preparation).", + "section": "RESULTS", + "ner": [ + [ + 27, + 37, + "structures", + "evidence" + ], + [ + 88, + 98, + "structures", + "evidence" + ], + [ + 99, + 107, + "bound to", + "protein_state" + ], + [ + 108, + 114, + "OBHS\u2010N", + "chemical" + ] + ] + }, + { + "sid": 156, + "sent": "Examining many closely related structures allows us to visualize subtle structural differences, in effect using X\u2010ray crystallography as a systems biology tool.", + "section": "RESULTS", + "ner": [ + [ + 31, + 41, + "structures", + "evidence" + ], + [ + 112, + 133, + "X\u2010ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 157, + "sent": "The indirect modulator scaffolds in cluster 1 did not show cell\u2010specific signaling (Fig\u00a03C), but shared common structural perturbations that we designed to modulate h12 dynamics.", + "section": "RESULTS", + "ner": [ + [ + 165, + 168, + "h12", + "structure_element" + ] + ] + }, + { + "sid": 158, + "sent": "Based on our original OBHS structure, the OBHS, OBHS\u2010N, and triaryl\u2010ethylene compounds were modified with h11\u2010directed pendant groups (Zheng et\u00a0al, 2012; Zhu et\u00a0al, 2012; Liao et\u00a0al, 2014).", + "section": "RESULTS", + "ner": [ + [ + 22, + 26, + "OBHS", + "chemical" + ], + [ + 27, + 36, + "structure", + "evidence" + ], + [ + 42, + 46, + "OBHS", + "chemical" + ], + [ + 48, + 54, + "OBHS\u2010N", + "chemical" + ], + [ + 60, + 76, + "triaryl\u2010ethylene", + "chemical" + ], + [ + 106, + 109, + "h11", + "structure_element" + ] + ] + }, + { + "sid": 159, + "sent": "Superposing the LBDs based on the class of bound ligands provides an ensemble view of the structural variance and clarifies what part of the ligand\u2010binding pocket is differentially perturbed or targeted.", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Superposing", + "experimental_method" + ], + [ + 16, + 20, + "LBDs", + "structure_element" + ], + [ + 141, + 162, + "ligand\u2010binding pocket", + "site" + ] + ] + }, + { + "sid": 160, + "sent": "The 24 structures containing OBHS, OBHS\u2010N, or triaryl\u2010ethylene analogs showed structural diversity in the same part of the scaffolds (Figs\u00a05A and EV5A), and the same region of the LBD\u2014the C\u2010terminal end of h11 (Figs\u00a05B and C, and EV5B), which in turn nudges h12 (Fig\u00a05C and D).", + "section": "RESULTS", + "ner": [ + [ + 7, + 17, + "structures", + "evidence" + ], + [ + 29, + 33, + "OBHS", + "chemical" + ], + [ + 35, + 41, + "OBHS\u2010N", + "chemical" + ], + [ + 46, + 62, + "triaryl\u2010ethylene", + "chemical" + ], + [ + 180, + 183, + "LBD", + "structure_element" + ], + [ + 206, + 209, + "h11", + "structure_element" + ], + [ + 258, + 261, + "h12", + "structure_element" + ] + ] + }, + { + "sid": 161, + "sent": "We observed that the OBHS\u2010N analogs displaced h11 along a vector away from Leu354 in a region of h3 that is unaffected by the ligands, and toward the dimer interface.", + "section": "RESULTS", + "ner": [ + [ + 21, + 27, + "OBHS\u2010N", + "chemical" + ], + [ + 46, + 49, + "h11", + "structure_element" + ], + [ + 75, + 81, + "Leu354", + "residue_name_number" + ], + [ + 97, + 99, + "h3", + "structure_element" + ], + [ + 150, + 165, + "dimer interface", + "site" + ] + ] + }, + { + "sid": 162, + "sent": "For the triaryl\u2010ethylene analogs, the displacement of h11 was in a perpendicular direction, away from Ile424 in h8 and toward h12.", + "section": "RESULTS", + "ner": [ + [ + 8, + 24, + "triaryl\u2010ethylene", + "chemical" + ], + [ + 54, + 57, + "h11", + "structure_element" + ], + [ + 102, + 108, + "Ile424", + "residue_name_number" + ], + [ + 112, + 114, + "h8", + "structure_element" + ], + [ + 126, + 129, + "h12", + "structure_element" + ] + ] + }, + { + "sid": 163, + "sent": "Remarkably, these individual inter\u2010atomic distances showed a ligand class\u2010specific ability to significantly predict proliferative effects (Fig\u00a05E and F), demonstrating the feasibility of developing a minimal set of activity predictors from crystal structures.", + "section": "RESULTS", + "ner": [ + [ + 29, + 51, + "inter\u2010atomic distances", + "evidence" + ], + [ + 240, + 258, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 164, + "sent": "Structure\u2010class analysis of triaryl\u2010ethylene analogs.", + "section": "FIG", + "ner": [ + [ + 0, + 24, + "Structure\u2010class analysis", + "experimental_method" + ], + [ + 28, + 44, + "triaryl\u2010ethylene", + "chemical" + ] + ] + }, + { + "sid": 165, + "sent": "Triaryl\u2010ethylene analogs bound to the superposed crystal structures of the ER\u03b1 LBD are shown.", + "section": "FIG", + "ner": [ + [ + 0, + 16, + "Triaryl\u2010ethylene", + "chemical" + ], + [ + 25, + 33, + "bound to", + "protein_state" + ], + [ + 38, + 48, + "superposed", + "experimental_method" + ], + [ + 49, + 67, + "crystal structures", + "evidence" + ], + [ + 75, + 78, + "ER\u03b1", + "protein" + ], + [ + 79, + 82, + "LBD", + "structure_element" + ] + ] + }, + { + "sid": 166, + "sent": "Arrows indicate chemical variance in the orientation of the different h11\u2010directed ligand side groups (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF and 5DP0).", + "section": "FIG", + "ner": [ + [ + 70, + 73, + "h11", + "structure_element" + ] + ] + }, + { + "sid": 167, + "sent": "Triaryl\u2010ethylene analogs induce variance of ER\u03b1 conformations at the C\u2010terminal region of h11.", + "section": "FIG", + "ner": [ + [ + 0, + 16, + "Triaryl\u2010ethylene", + "chemical" + ], + [ + 44, + 47, + "ER\u03b1", + "protein" + ], + [ + 90, + 93, + "h11", + "structure_element" + ] + ] + }, + { + "sid": 168, + "sent": "Panel\u00a0(B) shows the crystal structure of a triaryl\u2010ethylene analog\u2010bound ER\u03b1 LBD (PDB 5DLR).", + "section": "FIG", + "ner": [ + [ + 20, + 37, + "crystal structure", + "evidence" + ], + [ + 43, + 59, + "triaryl\u2010ethylene", + "chemical" + ], + [ + 73, + 76, + "ER\u03b1", + "protein" + ], + [ + 77, + 80, + "LBD", + "structure_element" + ] + ] + }, + { + "sid": 169, + "sent": "The h11\u2013h12 interface (circled) includes the C\u2010terminal part of h11.", + "section": "FIG", + "ner": [ + [ + 4, + 21, + "h11\u2013h12 interface", + "site" + ], + [ + 64, + 67, + "h11", + "structure_element" + ] + ] + }, + { + "sid": 170, + "sent": "This region was expanded in panel (C), where the 10 triaryl\u2010ethylene analog\u2010bound ER\u03b1 LBD structures (see Datasets EV1 and EV2) were superposed to show variations in the h11 C\u2010terminus (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, and 5DP0).", + "section": "FIG", + "ner": [ + [ + 52, + 68, + "triaryl\u2010ethylene", + "chemical" + ], + [ + 82, + 85, + "ER\u03b1", + "protein" + ], + [ + 86, + 89, + "LBD", + "structure_element" + ], + [ + 90, + 100, + "structures", + "evidence" + ], + [ + 133, + 143, + "superposed", + "experimental_method" + ], + [ + 170, + 173, + "h11", + "structure_element" + ] + ] + }, + { + "sid": 171, + "sent": "ER\u03b1 LBDs in complex with diethylstilbestrol (DES) or a triaryl\u2010ethylene analog were superposed to show that the ligand\u2010induced difference in h11 conformation is transmitted to the C\u2010terminus of h12 (PDB 4ZN7, 5DMC).", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "ER\u03b1", + "protein" + ], + [ + 4, + 8, + "LBDs", + "structure_element" + ], + [ + 9, + 24, + "in complex with", + "protein_state" + ], + [ + 25, + 43, + "diethylstilbestrol", + "chemical" + ], + [ + 45, + 48, + "DES", + "chemical" + ], + [ + 55, + 71, + "triaryl\u2010ethylene", + "chemical" + ], + [ + 84, + 94, + "superposed", + "experimental_method" + ], + [ + 141, + 144, + "h11", + "structure_element" + ], + [ + 194, + 197, + "h12", + "structure_element" + ] + ] + }, + { + "sid": 172, + "sent": "Inter\u2010atomic distances predict the proliferative effects of specific ligand series.", + "section": "FIG", + "ner": [ + [ + 0, + 22, + "Inter\u2010atomic distances", + "evidence" + ] + ] + }, + { + "sid": 173, + "sent": "Ile424\u2013His524 distance measured in the crystal structures correlates with the proliferative effect of triaryl\u2010ethylene analogs in MCF\u20107 cells.", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "Ile424", + "residue_name_number" + ], + [ + 7, + 13, + "His524", + "residue_name_number" + ], + [ + 14, + 22, + "distance", + "evidence" + ], + [ + 39, + 57, + "crystal structures", + "evidence" + ], + [ + 102, + 118, + "triaryl\u2010ethylene", + "chemical" + ] + ] + }, + { + "sid": 174, + "sent": "In contrast, the Leu354\u2013Leu525 distance correlates with the proliferative effects of OBHS\u2010N analogs in MCF\u20107 cells.", + "section": "FIG", + "ner": [ + [ + 17, + 23, + "Leu354", + "residue_name_number" + ], + [ + 24, + 30, + "Leu525", + "residue_name_number" + ], + [ + 31, + 39, + "distance", + "evidence" + ], + [ + 85, + 91, + "OBHS\u2010N", + "chemical" + ] + ] + }, + { + "sid": 175, + "sent": "Structure\u2010class analysis of WAY\u2010C analogs.", + "section": "FIG", + "ner": [ + [ + 0, + 24, + "Structure\u2010class analysis", + "experimental_method" + ], + [ + 28, + 33, + "WAY\u2010C", + "chemical" + ] + ] + }, + { + "sid": 176, + "sent": "WAY\u2010C side groups subtly nudge h12 Leu540.", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "WAY\u2010C", + "chemical" + ], + [ + 31, + 34, + "h12", + "structure_element" + ], + [ + 35, + 41, + "Leu540", + "residue_name_number" + ] + ] + }, + { + "sid": 177, + "sent": "ER\u03b1 LBD structures bound to 4 distinct WAY\u2010C analogs were superposed (PDB 4\u00a0IU7, 4IV4, 4IVW, 4IW6) (see Datasets EV1 and EV2).", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "ER\u03b1", + "protein" + ], + [ + 4, + 7, + "LBD", + "structure_element" + ], + [ + 8, + 18, + "structures", + "evidence" + ], + [ + 19, + 27, + "bound to", + "protein_state" + ], + [ + 39, + 44, + "WAY\u2010C", + "chemical" + ], + [ + 58, + 68, + "superposed", + "experimental_method" + ] + ] + }, + { + "sid": 178, + "sent": "Structure\u2010class analysis of indirect modulators", + "section": "FIG", + "ner": [ + [ + 0, + 24, + "Structure\u2010class analysis", + "experimental_method" + ] + ] + }, + { + "sid": 179, + "sent": "Structure\u2010class analysis of indirect modulators in cluster 1.", + "section": "FIG", + "ner": [ + [ + 0, + 24, + "Structure\u2010class analysis", + "experimental_method" + ] + ] + }, + { + "sid": 180, + "sent": "Crystal structures of the ER\u03b1 LBD bound to OBHS and OBHS\u2010N analogs were superposed.", + "section": "FIG", + "ner": [ + [ + 0, + 18, + "Crystal structures", + "evidence" + ], + [ + 26, + 29, + "ER\u03b1", + "protein" + ], + [ + 30, + 33, + "LBD", + "structure_element" + ], + [ + 34, + 42, + "bound to", + "protein_state" + ], + [ + 43, + 47, + "OBHS", + "chemical" + ], + [ + 52, + 58, + "OBHS\u2010N", + "chemical" + ], + [ + 72, + 82, + "superposed", + "experimental_method" + ] + ] + }, + { + "sid": 181, + "sent": "Arrows indicate chemical variance in the orientation of the different h11\u2010directed ligand side groups.", + "section": "FIG", + "ner": [ + [ + 70, + 73, + "h11", + "structure_element" + ] + ] + }, + { + "sid": 182, + "sent": "Panel\u00a0(B) shows the ligand\u2010induced conformational variation at the C\u2010terminal region of h11 (OBHS: PDB 4ZN9, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, and 4ZNW; OBHS\u2010N: PDB 4ZUB, 4ZUC, 4ZWH, 4ZWK, 5BNU, 5BP6, 5BPR, and 5BQ4).", + "section": "FIG", + "ner": [ + [ + 88, + 91, + "h11", + "structure_element" + ], + [ + 93, + 97, + "OBHS", + "chemical" + ], + [ + 149, + 155, + "OBHS\u2010N", + "chemical" + ] + ] + }, + { + "sid": 183, + "sent": "Structure\u2010class analysis of indirect modulators in clusters 2 and 3.", + "section": "FIG", + "ner": [ + [ + 0, + 24, + "Structure\u2010class analysis", + "experimental_method" + ] + ] + }, + { + "sid": 184, + "sent": "Crystal structures of the ER\u03b1 LBD bound to ligands with cell\u2010specific activities were superposed.", + "section": "FIG", + "ner": [ + [ + 0, + 18, + "Crystal structures", + "evidence" + ], + [ + 26, + 29, + "ER\u03b1", + "protein" + ], + [ + 30, + 33, + "LBD", + "structure_element" + ], + [ + 34, + 42, + "bound to", + "protein_state" + ], + [ + 86, + 96, + "superposed", + "experimental_method" + ] + ] + }, + { + "sid": 185, + "sent": "The bound ligands are shown, and arrows indicate considerable variation in the orientation of the different h3\u2010, h8\u2010, h11\u2010, or h12\u2010directed ligand side groups.", + "section": "FIG", + "ner": [ + [ + 108, + 110, + "h3", + "structure_element" + ], + [ + 113, + 115, + "h8", + "structure_element" + ], + [ + 118, + 121, + "h11", + "structure_element" + ], + [ + 127, + 130, + "h12", + "structure_element" + ] + ] + }, + { + "sid": 186, + "sent": "As visualized in four LBD structures (Srinivasan et\u00a0al, 2013), WAY\u2010C analogs were designed with small substitutions that slightly nudge h12 Leu540, without exiting the ligand\u2010binding pocket (Fig\u00a05G and H).", + "section": "RESULTS", + "ner": [ + [ + 22, + 25, + "LBD", + "structure_element" + ], + [ + 26, + 36, + "structures", + "evidence" + ], + [ + 63, + 68, + "WAY\u2010C", + "chemical" + ], + [ + 136, + 139, + "h12", + "structure_element" + ], + [ + 140, + 146, + "Leu540", + "residue_name_number" + ], + [ + 168, + 189, + "ligand\u2010binding pocket", + "site" + ] + ] + }, + { + "sid": 187, + "sent": "Therefore, changing h12 dynamics maintains the canonical signaling pathway defined by E2 (Fig\u00a01D) to support AF\u20102\u2010driven signaling and recruit NCOA1/2/3 for GREB1\u2010stimulated proliferation.", + "section": "RESULTS", + "ner": [ + [ + 20, + 23, + "h12", + "structure_element" + ], + [ + 86, + 88, + "E2", + "chemical" + ], + [ + 109, + 113, + "AF\u20102", + "structure_element" + ], + [ + 143, + 152, + "NCOA1/2/3", + "protein" + ], + [ + 157, + 162, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 188, + "sent": "Ligands with cell\u2010specific activity alter the shape of the AF\u20102 surface", + "section": "RESULTS", + "ner": [ + [ + 59, + 71, + "AF\u20102 surface", + "site" + ] + ] + }, + { + "sid": 189, + "sent": "Direct modulators like tamoxifen drive AF\u20101\u2010dependent cell\u2010specific activity by completely occluding AF\u20102, but it is not known how indirect modulators produce cell\u2010specific ER\u03b1 activity.", + "section": "RESULTS", + "ner": [ + [ + 23, + 32, + "tamoxifen", + "chemical" + ], + [ + 39, + 43, + "AF\u20101", + "structure_element" + ], + [ + 101, + 105, + "AF\u20102", + "structure_element" + ], + [ + 173, + 176, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 190, + "sent": "Therefore, we examined another 50 LBD structures containing ligands in clusters 2 and 3.", + "section": "RESULTS", + "ner": [ + [ + 34, + 37, + "LBD", + "structure_element" + ], + [ + 38, + 48, + "structures", + "evidence" + ] + ] + }, + { + "sid": 191, + "sent": "These structures demonstrated that cell\u2010specific activity derived from altering the shape of the AF\u20102 surface without an extended side chain.", + "section": "RESULTS", + "ner": [ + [ + 6, + 16, + "structures", + "evidence" + ], + [ + 97, + 109, + "AF\u20102 surface", + "site" + ] + ] + }, + { + "sid": 192, + "sent": "Ligands in cluster 2 and cluster 3 showed conformational heterogeneity in parts of the scaffold that were directed toward multiple regions of the receptor including h3, h8, h11, h12, and/or the \u03b2\u2010sheets (Fig\u00a0EV5C\u2013G).", + "section": "RESULTS", + "ner": [ + [ + 165, + 167, + "h3", + "structure_element" + ], + [ + 169, + 171, + "h8", + "structure_element" + ], + [ + 173, + 176, + "h11", + "structure_element" + ], + [ + 178, + 181, + "h12", + "structure_element" + ], + [ + 194, + 202, + "\u03b2\u2010sheets", + "structure_element" + ] + ] + }, + { + "sid": 193, + "sent": "For instance, S\u2010OBHS\u20102 and S\u2010OBHS\u20103 analogs (Fig\u00a02) had similar ER\u03b1 activity profiles in the different cell types (Fig\u00a0EV2A\u2013C), but the 2\u2010 versus 3\u2010methyl substituted phenol rings altered the correlated signaling patterns in different cell types (Fig\u00a03B lanes 7 and 12).", + "section": "RESULTS", + "ner": [ + [ + 14, + 22, + "S\u2010OBHS\u20102", + "chemical" + ], + [ + 27, + 35, + "S\u2010OBHS\u20103", + "chemical" + ], + [ + 64, + 67, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 194, + "sent": "This difference in ligand positioning altered the AF\u20102 surface via a shift in the N\u2010terminus of h12, which directly contacts the coactivator.", + "section": "RESULTS", + "ner": [ + [ + 50, + 62, + "AF\u20102 surface", + "site" + ], + [ + 96, + 99, + "h12", + "structure_element" + ] + ] + }, + { + "sid": 195, + "sent": "This effect is evident in a single structure due to its 1 \u00c5 magnitude (Fig\u00a06A and B).", + "section": "RESULTS", + "ner": [ + [ + 35, + 44, + "structure", + "evidence" + ] + ] + }, + { + "sid": 196, + "sent": "The shifts in h12 residues Asp538 and Leu539 led to rotation of the coactivator peptide (Fig\u00a06C).", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "h12", + "structure_element" + ], + [ + 27, + 33, + "Asp538", + "residue_name_number" + ], + [ + 38, + 44, + "Leu539", + "residue_name_number" + ] + ] + }, + { + "sid": 197, + "sent": "Thus, cell\u2010specific activity can stem from perturbation of the AF\u20102 surface without an\u00a0extended side chain, which presumably alters the receptor\u2013coregulator interaction profile.", + "section": "RESULTS", + "ner": [ + [ + 63, + 75, + "AF\u20102 surface", + "site" + ] + ] + }, + { + "sid": 198, + "sent": "S\u2010OBHS\u20102/3 analogs subtly distort the AF\u20102 surface.", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "S\u2010OBHS\u20102/3", + "chemical" + ], + [ + 38, + 50, + "AF\u20102 surface", + "site" + ] + ] + }, + { + "sid": 199, + "sent": "Panel\u00a0(A) shows the crystal structure of an S\u2010OBHS\u20103\u2010bound ER\u03b1 LBD (PDB 5DUH).", + "section": "FIG", + "ner": [ + [ + 20, + 37, + "crystal structure", + "evidence" + ], + [ + 44, + 58, + "S\u2010OBHS\u20103\u2010bound", + "protein_state" + ], + [ + 59, + 62, + "ER\u03b1", + "protein" + ], + [ + 63, + 66, + "LBD", + "structure_element" + ] + ] + }, + { + "sid": 200, + "sent": "The h3\u2013h12 interface (circled) at AF\u20102 (pink) was expanded in panels (B, C).", + "section": "FIG", + "ner": [ + [ + 4, + 20, + "h3\u2013h12 interface", + "site" + ], + [ + 34, + 38, + "AF\u20102", + "structure_element" + ] + ] + }, + { + "sid": 201, + "sent": "The S\u2010OBHS\u20102/3\u2010bound ER\u03b1 LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF\u20102 surface (panel C).", + "section": "FIG", + "ner": [ + [ + 4, + 20, + "S\u2010OBHS\u20102/3\u2010bound", + "protein_state" + ], + [ + 21, + 24, + "ER\u03b1", + "protein" + ], + [ + 25, + 29, + "LBDs", + "structure_element" + ], + [ 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"protein_state" + ], + [ + 80, + 89, + "structure", + "evidence" + ] + ] + }, + { + "sid": 204, + "sent": "Average (mean\u00a0+\u00a0SEM) \u03b1\u2010carbon distance measured from h3 Thr347 to h11 Leu525 of A\u2010CD\u2010, 2,5\u2010DTP\u2010, and 3,4\u2010DTPD\u2010bound ER\u03b1 LBDs.", + "section": "FIG", + "ner": [ + [ + 21, + 38, + "\u03b1\u2010carbon distance", + "evidence" + ], + [ + 53, + 55, + "h3", + "structure_element" + ], + [ + 56, + 62, + "Thr347", + "residue_name_number" + ], + [ + 66, + 69, + "h11", + "structure_element" + ], + [ + 70, + 76, + "Leu525", + "residue_name_number" + ], + [ + 80, + 115, + "A\u2010CD\u2010, 2,5\u2010DTP\u2010, and 3,4\u2010DTPD\u2010bound", + "protein_state" + ], + [ + 116, + 119, + "ER\u03b1", + "protein" + ], + [ + 120, + 124, + "LBDs", + "structure_element" + ] + ] + }, + { + "sid": 205, + "sent": "*Two\u2010tailed Student's t\u2010test, P\u00a0=\u00a00.002 (PDB A\u2010CD: 5DI7, 5DID, 5DIE, 5DIG, and 4PPS; 2,5\u2010DTP: 4IWC, 5DRM, and 5DRJ; 3,4\u2010DTPD: 5DTV and 5DU5).", + "section": "FIG", + "ner": [ + [ + 12, + 28, + "Student's t\u2010test", + "experimental_method" + ], + [ + 30, + 31, + "P", + "evidence" + ], + [ + 45, + 49, + "A\u2010CD", + "chemical" + ], + [ + 85, + 92, + "2,5\u2010DTP", + "chemical" + ], + [ + 116, + 124, + "3,4\u2010DTPD", + "chemical" + ] + ] + }, + { + "sid": 206, + "sent": "Crystal structures show that a 3,4\u2010DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A\u2010CD\u2010ring estrogen (PDB 4PPS, 5DTV).", + "section": "FIG", + "ner": [ + [ + 0, + 18, + "Crystal structures", + "evidence" + ], + [ + 31, + 39, + "3,4\u2010DTPD", + "chemical" + ], + [ + 54, + 56, + "h3", + "structure_element" + ], + [ + 58, + 59, + "F", + "structure_element" + ], + [ + 69, + 74, + "NCOA2", + "protein" + ], + [ + 102, + 106, + "A\u2010CD", + "chemical" + ], + [ + 112, + 120, + "estrogen", + "chemical" + ] + ] + }, + { + "sid": 207, + "sent": "Hierarchical clustering of ligand\u2010specific binding of 154 interacting peptides to the ER\u03b1 LBD was performed in triplicate by MARCoNI analysis.", + "section": "FIG", + "ner": [ + [ + 0, + 23, + "Hierarchical clustering", + "experimental_method" + ], + [ + 86, + 89, + "ER\u03b1", + "protein" + ], + [ + 90, + 93, + "LBD", + "structure_element" + ], + [ + 125, + 141, + "MARCoNI analysis", + "experimental_method" + ] + ] + }, + { + "sid": 208, + "sent": "The 2,5\u2010DTP analogs showed perturbation of h11, as well as h3, which forms part of the AF\u20102 surface.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "2,5\u2010DTP", + "chemical" + ], + [ + 43, + 46, + "h11", + "structure_element" + ], + [ + 59, + 61, + "h3", + "structure_element" + ], + [ + 87, + 99, + "AF\u20102 surface", + "site" + ] + ] + }, + { + "sid": 209, + "sent": "These compounds bind the LBD in an unusual fashion because they have a phenol\u2010to\u2010phenol length of ~12 \u00c5, which is longer than steroids and other prototypical ER\u03b1 agonists that are ~10 \u00c5 in length.", + "section": "RESULTS", + "ner": [ + [ + 25, + 28, + "LBD", + "structure_element" + ], + [ + 158, + 161, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 210, + "sent": "One phenol pushed further toward h3 (Fig\u00a06D), while the other phenol pushed toward the C\u2010terminus of h11 to a greater extent than A\u2010CD\u2010ring estrogens (Nwachukwu et\u00a0al, 2014), which are close structural analogs of E2 that lack a B\u2010ring (Fig\u00a02).", + "section": "RESULTS", + "ner": [ + [ + 33, + 35, + "h3", + "structure_element" + ], + [ + 101, + 104, + "h11", + "structure_element" + ], + [ + 130, + 134, + "A\u2010CD", + "chemical" + ], + [ + 140, + 149, + "estrogens", + "chemical" + ], + [ + 213, + 215, + "E2", + "chemical" + ] + ] + }, + { + "sid": 211, + "sent": "To quantify this difference, we compared the distance between \u03b1\u2010carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5\u2010DTP analogs (n\u00a0=\u00a03) or A\u2010CD\u2010ring analogs (n\u00a0=\u00a05) (Fig\u00a06E).", + "section": "RESULTS", + "ner": [ + [ + 45, + 53, + "distance", + "evidence" + ], + [ + 75, + 77, + "h3", + "structure_element" + ], + [ + 78, + 84, + "Thr347", + "residue_name_number" + ], + [ + 89, + 92, + "h11", + "structure_element" + ], + [ + 93, + 99, + "Leu525", + "residue_name_number" + ], + [ + 114, + 124, + "structures", + "evidence" + ], + [ + 136, + 143, + "2,5\u2010DTP", + "chemical" + ], + [ + 163, + 167, + "A\u2010CD", + "chemical" + ] + ] + }, + { + "sid": 212, + "sent": "We observed a difference of 0.4 \u00c5 that was significant (two\u2010tailed Student's t\u2010test, P\u00a0=\u00a00.002) due to the very tight clustering of the 2,5\u2010DTP\u2010induced LBD conformation.", + "section": "RESULTS", + "ner": [ + [ + 67, + 83, + "Student's t\u2010test", + "experimental_method" + ], + [ + 85, + 86, + "P", + "evidence" + ], + [ + 136, + 143, + "2,5\u2010DTP", + "chemical" + ], + [ + 152, + 155, + "LBD", + "structure_element" + ] + ] + }, + { + "sid": 213, + "sent": "The shifts in h3 suggest these compounds are positioned to alter coregulator preferences.", + "section": "RESULTS", + "ner": [ + [ + 14, + 16, + "h3", + "structure_element" + ] + ] + }, + { + "sid": 214, + "sent": "The 2,5\u2010DTP and 3,4\u2010DTP scaffolds are isomeric, but with aryl groups at obtuse and acute angles, respectively (Fig\u00a02).", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "2,5\u2010DTP", + "chemical" + ], + [ + 16, + 23, + "3,4\u2010DTP", + "chemical" + ] + ] + }, + { + "sid": 215, + "sent": "The crystal structure of ER\u03b1 in complex with a 3,4\u2010DTP is unknown; however, we solved two crystal structures of ER\u03b1 bound to 3,4\u2010DTPD analogs and one structure containing a furan ligand\u2014all of which have a 3,4\u2010diaryl configuration (Fig\u00a02; Datasets EV1 and EV2).", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 25, + 28, + "ER\u03b1", + "protein" + ], + [ + 29, + 44, + "in complex with", + "protein_state" + ], + [ + 47, + 54, + "3,4\u2010DTP", + "chemical" + ], + [ + 79, + 85, + "solved", + "experimental_method" + ], + [ + 90, + 108, + "crystal structures", + "evidence" + ], + [ + 112, + 115, + "ER\u03b1", + "protein" + ], + [ + 116, + 124, + "bound to", + "protein_state" + ], + [ + 125, + 133, + "3,4\u2010DTPD", + "chemical" + ], + [ + 150, + 159, + "structure", + "evidence" + ], + [ + 173, + 178, + "furan", + "chemical" + ] + ] + }, + { + "sid": 216, + "sent": "In these structures, the A\u2010ring mimetic of the 3,4\u2010DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ER\u03b1 ligand\u2010binding pocket (Fig\u00a06F).", + "section": "RESULTS", + "ner": [ + [ + 9, + 19, + "structures", + "evidence" + ], + [ + 47, + 55, + "3,4\u2010DTPD", + "chemical" + ], + [ + 71, + 73, + "h3", + "structure_element" + ], + [ + 74, + 80, + "Glu353", + "residue_name_number" + ], + [ + 130, + 132, + "h3", + "structure_element" + ], + [ + 143, + 156, + "hydrogen bond", + "bond_interaction" + ], + [ + 162, + 168, + "Thr347", + "residue_name_number" + ], + [ + 217, + 220, + "ER\u03b1", + "protein" + ], + [ + 221, + 242, + "ligand\u2010binding pocket", + "site" + ] + ] + }, + { + "sid": 217, + "sent": "The 3,4\u2010DTPD analogs also induced a shift in h3 positioning, which translated again into a shift in the bound coactivator peptide (Fig\u00a06F).", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "3,4\u2010DTPD", + "chemical" + ], + [ + 45, + 47, + "h3", + "structure_element" + ] + ] + }, + { + "sid": 218, + "sent": "Therefore, these indirect modulators, including S\u2010OBHS\u20102, S\u2010OBHS\u20103, 2,5\u2010DTP, and 3,4\u2010DTPD analogs\u2014all of which show cell\u2010specific activity profiles\u2014induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF\u20102 surface.", + "section": "RESULTS", + "ner": [ + [ + 48, + 56, + "S\u2010OBHS\u20102", + "chemical" + ], + [ + 58, + 66, + "S\u2010OBHS\u20103", + "chemical" + ], + [ + 68, + 75, + "2,5\u2010DTP", + "chemical" + ], + [ + 81, + 89, + "3,4\u2010DTPD", + "chemical" + ], + [ + 166, + 168, + "h3", + "structure_element" + ], + [ + 173, + 176, + "h12", + "structure_element" + ], + [ + 241, + 253, + "AF\u20102 surface", + "site" + ] + ] + }, + { + "sid": 219, + "sent": "To test whether the AF\u20102 surface shows changes in shape in solution, we used the microarray assay for real\u2010time coregulator\u2013nuclear receptor interaction (MARCoNI) analysis (Aarts et\u00a0al, 2013).", + "section": "RESULTS", + "ner": [ + [ + 20, + 32, + "AF\u20102 surface", + "site" + ], + [ + 81, + 152, + "microarray assay for real\u2010time coregulator\u2013nuclear receptor interaction", + "experimental_method" + ], + [ + 154, + 161, + "MARCoNI", + "experimental_method" + ] + ] + }, + { + "sid": 220, + "sent": "Here, the ligand\u2010dependent interactions of the ER\u03b1 LBD with over 150 distinct LxxLL motif peptides were assayed to define structural fingerprints for the AF\u20102 surface, in a manner similar to the use of phage display peptides as structural probes (Connor et\u00a0al, 2001).", + "section": "RESULTS", + "ner": [ + [ + 47, + 50, + "ER\u03b1", + "protein" + ], + [ + 51, + 54, + "LBD", + "structure_element" + ], + [ + 78, + 89, + "LxxLL motif", + "structure_element" + ], + [ + 154, + 166, + "AF\u20102 surface", + "site" + ], + [ + 202, + 224, + "phage display peptides", + "experimental_method" + ] + ] + }, + { + "sid": 221, + "sent": "Despite the similar average activities of these ligand classes (Fig\u00a03A and B), 2,5\u2010DTP and 3,4\u2010DTP analogs displayed remarkably different peptide recruitment patterns (Fig\u00a06H), consistent with the structural analyses.", + "section": "RESULTS", + "ner": [ + [ + 79, + 86, + "2,5\u2010DTP", + "chemical" + ], + [ + 91, + 98, + "3,4\u2010DTP", + "chemical" + ], + [ + 197, + 216, + "structural analyses", + "experimental_method" + ] + ] + }, + { + "sid": 222, + "sent": "Hierarchical clustering revealed that many of the 2,5\u2010DTP analogs recapitulated most of the peptide recruitment and dismissal patterns observed with E2 (Fig\u00a06H).", + "section": "RESULTS", + "ner": [ + [ + 0, + 23, + "Hierarchical clustering", + "experimental_method" + ], + [ + 50, + 57, + "2,5\u2010DTP", + "chemical" + ], + [ + 149, + 151, + "E2", + "chemical" + ] + ] + }, + { + "sid": 223, + "sent": "However, there was a unique cluster of peptides that were recruited by E2 but not the 2,5\u2010DTP analogs.", + "section": "RESULTS", + "ner": [ + [ + 71, + 73, + "E2", + "chemical" + ], + [ + 86, + 93, + "2,5\u2010DTP", + "chemical" + ] + ] + }, + { + "sid": 224, + "sent": "In contrast, 3,4\u2010DTP analogs dismissed most of the peptides from the AF\u20102 surface (Fig\u00a06H).", + "section": "RESULTS", + "ner": [ + [ + 13, + 20, + "3,4\u2010DTP", + "chemical" + ], + [ + 69, + 81, + "AF\u20102 surface", + "site" + ] + ] + }, + { + "sid": 225, + "sent": "Thus, the isomeric attachment of diaryl groups to the thiophene core changed the AF\u20102 surface from inside the ligand\u2010binding pocket, as predicted by the crystal structures.", + "section": "RESULTS", + "ner": [ + [ + 54, + 63, + "thiophene", + "chemical" + ], + [ + 81, + 93, + "AF\u20102 surface", + "site" + ], + [ + 110, + 131, + "ligand\u2010binding pocket", + "site" + ], + [ + 153, + 171, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 226, + "sent": "Together, these findings suggest that without an extended side chain, cell\u2010specific activity stems from different coregulator recruitment profiles, due to unique ligand\u2010induced conformations of the AF\u20102 surface, in addition to differential usage of AF\u20101.", + "section": "RESULTS", + "ner": [ + [ + 198, + 210, + "AF\u20102 surface", + "site" + ], + [ + 249, + 253, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 227, + "sent": "Indirect modulators in cluster 1 avoid this by perturbing the h11\u2013h12 interface, and modulating the dynamics of h12 without changing the shape of AF\u20102 when stabilized.", + "section": "RESULTS", + "ner": [ + [ + 62, + 79, + "h11\u2013h12 interface", + "site" + ], + [ + 112, + 115, + "h12", + "structure_element" + ], + [ + 146, + 150, + "AF\u20102", + "structure_element" + ] + ] + }, + { + "sid": 228, + "sent": "Our goal was to identify a minimal set of predictors that would link specific structural perturbations to ER\u03b1 signaling pathways that control cell\u2010specific signaling and proliferation.", + "section": "DISCUSS", + "ner": [ + [ + 106, + 109, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 229, + "sent": "We found a very strong set of predictors, where ligands in cluster 1, defined by similar signaling across cell types, showed indirect modulation of h12 dynamics via the h11\u201312 interface or slight contact with h12.", + "section": "DISCUSS", + "ner": [ + [ + 148, + 151, + "h12", + "structure_element" + ], + [ + 169, + 185, + "h11\u201312 interface", + "site" + ], + [ + 209, + 212, + "h12", + "structure_element" + ] + ] + }, + { + "sid": 230, + "sent": "This perturbation determined proliferation that correlated strongly with AF\u20102 activity, recruitment of NCOA1/2/3 family members, and induction of the GREB1 gene, consistent with the canonical ER\u03b1 signaling pathway (Fig\u00a01D).", + "section": "DISCUSS", + "ner": [ + [ + 73, + 77, + "AF\u20102", + "structure_element" + ], + [ + 103, + 112, + "NCOA1/2/3", + "protein" + ], + [ + 150, + 155, + "GREB1", + "protein" + ], + [ + 192, + 195, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 231, + "sent": "For ligands in cluster 1, deletion of AF\u20101 reduced activity to varying degrees, but did not change the underlying signaling patterns established through AF\u20102.", + "section": "DISCUSS", + "ner": [ + [ + 26, + 34, + "deletion", + "experimental_method" + ], + [ + 38, + 42, + "AF\u20101", + "structure_element" + ], + [ + 153, + 157, + "AF\u20102", + "structure_element" + ] + ] + }, + { + "sid": 232, + "sent": "In contrast, an extended side chain designed to directly reposition h12 and completely disrupt the AF\u20102 surface results in cell\u2010specific signaling.", + "section": "DISCUSS", + "ner": [ + [ + 68, + 71, + "h12", + "structure_element" + ], + [ + 99, + 111, + "AF\u20102 surface", + "site" + ] + ] + }, + { + "sid": 233, + "sent": "Compared to cluster 1, the structural rules are less clear in clusters 2 and 3, but a number of indirect modulator classes perturbed the LBD conformation at the intersection of h3, the h12\u00a0N\u2010terminus, and the AF\u20102 surface.", + "section": "DISCUSS", + "ner": [ + [ + 137, + 140, + "LBD", + "structure_element" + ], + [ + 177, + 179, + "h3", + "structure_element" + ], + [ + 185, + 188, + "h12", + "structure_element" + ], + [ + 209, + 221, + "AF\u20102 surface", + "site" + ] + ] + }, + { + "sid": 234, + "sent": "Ligands in these classes altered the shape of AF\u20102 to affect coregulator preferences.", + "section": "DISCUSS", + "ner": [ + [ + 46, + 50, + "AF\u20102", + "structure_element" + ] + ] + }, + { + "sid": 235, + "sent": "For direct and indirect modulators in cluster 2 or 3, the canonical ER\u03b1 signaling pathway involving recruitment of NCOA1/2/3 and induction of GREB1 did not generally predict their proliferative effects, indicating an alternate causal model (Fig\u00a01E).", + "section": "DISCUSS", + "ner": [ + [ + 68, + 71, + "ER\u03b1", + "protein" + ], + [ + 115, + 124, + "NCOA1/2/3", + "protein" + ], + [ + 142, + 147, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 236, + "sent": "These principles outlined above provide a structural basis for how the ligand\u2013receptor interface leads to different signaling specificities through AF\u20101 and AF\u20102.", + "section": "DISCUSS", + "ner": [ + [ + 71, + 96, + "ligand\u2013receptor interface", + "site" + ], + [ + 148, + 152, + "AF\u20101", + "structure_element" + ], + [ + 157, + 161, + "AF\u20102", + "structure_element" + ] + ] + }, + { + "sid": 237, + "sent": "It is noteworthy that regulation of h12 dynamics indirectly through h11 can virtually abolish AF\u20102 activity, and yet still drive robust transcriptional activity through AF\u20101, as demonstrated with the OBHS series.", + "section": "DISCUSS", + "ner": [ + [ + 36, + 39, + "h12", + "structure_element" + ], + [ + 68, + 71, + "h11", + "structure_element" + ], + [ + 94, + 98, + "AF\u20102", + "structure_element" + ], + [ + 169, + 173, + "AF\u20101", + "structure_element" + ], + [ + 200, + 204, + "OBHS", + "chemical" + ] + ] + }, + { + "sid": 238, + "sent": "This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF\u20101 and AF\u20102 (McInerney et\u00a0al, 1996; Webb et\u00a0al, 1998), which allows ligands to nucleate ER\u03b1\u2013NCOA1/2/3 interaction through AF\u20102, and reinforce this interaction with additional binding to AF\u20101.", + "section": "DISCUSS", + "ner": [ + [ + 47, + 56, + "NCOA1/2/3", + "protein" + ], + [ + 74, + 87, + "binding sites", + "site" + ], + [ + 109, + 113, + "AF\u20101", + "structure_element" + ], + [ + 118, + 122, + "AF\u20102", + "structure_element" + ], + [ + 199, + 212, + "ER\u03b1\u2013NCOA1/2/3", + "complex_assembly" + ], + [ + 233, + 237, + "AF\u20102", + "structure_element" + ], + [ + 297, + 301, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 239, + "sent": "Completely blocking AF\u20102 with an extended side chain or altering the shape of AF\u20102 changes the preference away from NCOA1/2/3 for determining GREB1 levels and proliferation of breast cancer cells.", + "section": "DISCUSS", + "ner": [ + [ + 20, + 24, + "AF\u20102", + "structure_element" + ], + [ + 78, + 82, + "AF\u20102", + "structure_element" + ], + [ + 116, + 125, + "NCOA1/2/3", + "protein" + ], + [ + 142, + 147, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 240, + "sent": "AF\u20102 blockade also allows AF\u20101 to function independently, which is important since AF\u20101 drives tissue\u2010selective effects in\u00a0vivo.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "AF\u20102", + "structure_element" + ], + [ + 26, + 30, + "AF\u20101", + "structure_element" + ], + [ + 83, + 87, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 241, + "sent": "This was demonstrated with AF\u20101 knockout mice that show E2\u2010dependent vascular protection, but not uterine proliferation, thus highlighting the role of AF\u20101 in tissue\u2010selective or cell\u2010specific signaling (Billon\u2010Gales et\u00a0al, 2009; Abot et\u00a0al, 2013).", + "section": "DISCUSS", + "ner": [ + [ + 27, + 31, + "AF\u20101", + "structure_element" + ], + [ + 56, + 58, + "E2", + "chemical" + ], + [ + 151, + 155, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 242, + "sent": "Here, we examined many LBD structures and tested several variables that were not predictive, including ER\u03b2 activity, the strength of AF\u20101 signaling, and NCOA3 occupancy at the GREB1 gene.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 26, + "LBD", + "structure_element" + ], + [ + 27, + 37, + "structures", + "evidence" + ], + [ + 103, + 106, + "ER\u03b2", + "protein" + ], + [ + 133, + 137, + "AF\u20101", + "structure_element" + ], + [ + 153, + 158, + "NCOA3", + "protein" + ], + [ + 176, + 181, + "GREB1", + "protein" + ] + ] + }, + { + "sid": 243, + "sent": "Similarly, we visualized structures to identify patterns.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 35, + "structures", + "evidence" + ] + ] + }, + { + "sid": 244, + "sent": "For example, phage display was used to identify the androgen receptor interactome, which was cloned into an M2H library and used to identify clusters of ligand\u2010selective interactions (Norris et\u00a0al, 2009).", + "section": "DISCUSS", + "ner": [ + [ + 13, + 26, + "phage display", + "experimental_method" + ], + [ + 108, + 111, + "M2H", + "experimental_method" + ] + ] + }, + { + "sid": 245, + "sent": "Also, we have used siRNA screening to identify a number of coregulators required for ER\u03b1\u2010mediated repression of the IL\u20106 gene (Nwachukwu et\u00a0al, 2014).", + "section": "DISCUSS", + "ner": [ + [ + 19, + 34, + "siRNA screening", + "experimental_method" + ], + [ + 85, + 88, + "ER\u03b1", + "protein" + ] + ] + }, + { + "sid": 246, + "sent": "If we calculated inter\u2010atomic distance matrices containing 4,000 atoms per structure \u00d7\u00a076 ligand\u2013receptor complexes, we would have 3\u00a0\u00d7\u00a0105 predictions.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 47, + "inter\u2010atomic distance matrices", + "evidence" + ] + ] + }, + { + "sid": 247, + "sent": "We have identified atomic vectors for the OBHS\u2010N and triaryl\u2010ethylene classes that predict ligand response (Fig\u00a05E and\u00a0F).", + "section": "DISCUSS", + "ner": [ + [ + 19, + 33, + "atomic vectors", + "evidence" + ], + [ + 42, + 48, + "OBHS\u2010N", + "chemical" + ], + [ + 53, + 69, + "triaryl\u2010ethylene", + "chemical" + ] + ] + }, + { + "sid": 248, + "sent": "Indeed, the most anti\u2010proliferative compound in the OBHS\u2010N series had a fulvestrant\u2010like profile across a battery of assays (S. Srinivasan et al, in preparation).", + "section": "DISCUSS", + "ner": [ + [ + 52, + 58, + "OBHS\u2010N", + "chemical" + ] + ] + }, + { + "sid": 249, + "sent": "Secondly, our finding that WAY\u2010C compounds do not rely of AF\u20101 for signaling efficacy may derive from the slight contacts with h12 observed in crystal structures (Figs\u00a03B and 5H), unlike other compounds in cluster 1 that dislocate h11 and rely on AF\u20101 for signaling efficacy (Figs\u00a03B and 5C, and EV5B).", + "section": "DISCUSS", + "ner": [ + [ + 27, + 32, + "WAY\u2010C", + "chemical" + ], + [ + 58, + 62, + "AF\u20101", + "structure_element" + ], + [ + 127, + 130, + "h12", + "structure_element" + ], + [ + 143, + 161, + "crystal structures", + "evidence" + ], + [ + 231, + 234, + "h11", + "structure_element" + ], + [ + 247, + 251, + "AF\u20101", + "structure_element" + ] + ] + }, + { + "sid": 250, + "sent": "Some of these ligands altered the shape of the AF\u20102 surface by perturbing the h3\u2013h12 interface, thus providing a route to new SERM\u2010like activity profiles by combining indirect and direct modulation of receptor structure.", + "section": "DISCUSS", + "ner": [ + [ + 47, + 59, + "AF\u20102 surface", + "site" + ], + [ + 78, + 94, + "h3\u2013h12 interface", + "site" + ] + ] + }, + { + "sid": 251, + "sent": "Incorporation of statistical approaches to understand relationships between structure and signaling variables moves us toward predictive models for complex ER\u03b1\u2010mediated responses such as in\u00a0vivo uterine proliferation or tumor growth, and more generally toward structure\u2010based design for other allosteric drug targets including GPCRs and other nuclear receptors.", + "section": "DISCUSS", + "ner": [ + [ + 156, + 159, + "ER\u03b1", + "protein" + ], + [ + 327, + 332, + "GPCRs", + "protein_type" + ], + [ + 343, + 360, + "nuclear receptors", + "protein_type" + ] + ] + } + ] + }, + "PMC4822561": { + "annotations": [ + { + "sid": 0, + "sent": "Structure of a quinolone-stabilized cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex", + "section": "TITLE", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 56, + 72, + "topoisomerase IV", + "complex_assembly" + ], + [ + 78, + 99, + "Klebsiella pneumoniae", + "species" + ], + [ + 130, + 154, + "Streptococcus pneumoniae", + "species" + ] + ] + }, + { + "sid": 1, + "sent": "Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 18, + "Crystal structures", + "evidence" + ], + [ + 48, + 64, + "topoisomerase IV", + "complex_assembly" + ], + [ + 70, + 83, + "Gram-negative", + "taxonomy_domain" + ], + [ + 85, + 98, + "K. pneumoniae", + "species" + ], + [ + 104, + 117, + "Gram-positive", + "taxonomy_domain" + ], + [ + 119, + 132, + "S. pneumoniae", + "species" + ], + [ + 134, + 143, + "bacterial", + "taxonomy_domain" + ], + [ + 212, + 224, + "levofloxacin", + "chemical" + ] + ] + }, + { + "sid": 2, + "sent": "For K. pneumoniae, this is the first high-resolution cleavage complex structure to be reported.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 17, + "K. pneumoniae", + "species" + ], + [ + 70, + 79, + "structure", + "evidence" + ] + ] + }, + { + "sid": 3, + "sent": " Klebsiella pneumoniae is a Gram-negative bacterium that is responsible for a range of common infections, including pulmonary pneumonia, bloodstream infections and meningitis.", + "section": "ABSTRACT", + "ner": [ + [ + 1, + 22, + "Klebsiella pneumoniae", + "species" + ], + [ + 28, + 51, + "Gram-negative bacterium", + "taxonomy_domain" + ] + ] + }, + { + "sid": 4, + "sent": "Certain strains of Klebsiella have become highly resistant to antibiotics.", + "section": "ABSTRACT", + "ner": [ + [ + 19, + 29, + "Klebsiella", + "taxonomy_domain" + ] + ] + }, + { + "sid": 5, + "sent": "Despite the vast amount of research carried out on this class of bacteria, the molecular structure of its topoisomerase IV, a type II topoisomerase essential for catalysing chromosomal segregation, had remained unknown.", + "section": "ABSTRACT", + "ner": [ + [ + 65, + 73, + "bacteria", + "taxonomy_domain" + ], + [ + 89, + 98, + "structure", + "evidence" + ], + [ + 106, + 122, + "topoisomerase IV", + "complex_assembly" + ], + [ + 126, + 147, + "type II topoisomerase", + "protein_type" + ] + ] + }, + { + "sid": 6, + "sent": "In this paper, the structure of its DNA-cleavage complex is reported at 3.35\u2005\u00c5 resolution.", + "section": "ABSTRACT", + "ner": [ + [ + 19, + 28, + "structure", + "evidence" + ], + [ + 36, + 39, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 7, + "sent": "The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent.", + "section": "ABSTRACT", + "ner": [ + [ + 28, + 32, + "ParC", + "protein" + ], + [ + 33, + 49, + "breakage-reunion", + "structure_element" + ], + [ + 54, + 58, + "ParE", + "protein" + ], + [ + 59, + 65, + "TOPRIM", + "structure_element" + ], + [ + 77, + 90, + "K. pneumoniae", + "species" + ], + [ + 91, + 107, + "topoisomerase IV", + "complex_assembly" + ], + [ + 113, + 116, + "DNA", + "chemical" + ], + [ + 131, + 143, + "levofloxacin", + "chemical" + ], + [ + 162, + 177, + "fluoroquinolone", + "chemical" + ] + ] + }, + { + "sid": 8, + "sent": "This complex is compared with a similar complex from Streptococcus pneumoniae, which has recently been solved.", + "section": "ABSTRACT", + "ner": [ + [ + 53, + 77, + "Streptococcus pneumoniae", + "species" + ] + ] + }, + { + "sid": 9, + "sent": " Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica.", + "section": "INTRO", + "ner": [ + [ + 1, + 11, + "Klebsiella", + "taxonomy_domain" + ], + [ + 40, + 58, + "Enterobacteriaceae", + "taxonomy_domain" + ], + [ + 69, + 90, + "Gram-negative bacilli", + "taxonomy_domain" + ], + [ + 162, + 165, + "DNA", + "chemical" + ], + [ + 176, + 189, + "K. pneumoniae", + "species" + ], + [ + 191, + 201, + "K. ozaenae", + "species" + ], + [ + 203, + 222, + "K. rhinoscleromatis", + "species" + ], + [ + 224, + 234, + "K. oxytoca", + "species" + ], + [ + 236, + 249, + "K. planticola", + "species" + ], + [ + 251, + 263, + "K. terrigena", + "species" + ], + [ + 268, + 286, + "K. ornithinolytica", + "species" + ] + ] + }, + { + "sid": 10, + "sent": "K. pneumoniae is the most medically important species of the genus owing to its high resistance to antibiotics.", + "section": "INTRO", + "ner": [ + [ + 0, + 13, + "K. pneumoniae", + "species" + ] + ] + }, + { + "sid": 11, + "sent": "Significant morbidity and mortality has been associated with an emerging, highly drug-resistant strain of K. pneumoniae characterized as producing the carbapenemase enzyme (KPC-producing bacteria; Nordmann et al., 2009).", + "section": "INTRO", + "ner": [ + [ + 106, + 119, + "K. pneumoniae", + "species" + ], + [ + 151, + 164, + "carbapenemase", + "protein_type" + ], + [ + 187, + 195, + "bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 12, + "sent": "However, common treatments (based on in vitro susceptibility testing) are the polymyxins, tigecycline and, less frequently, aminoglycoside antibiotics (Arnold et al., 2011).", + "section": "INTRO", + "ner": [ + [ + 37, + 68, + "in vitro susceptibility testing", + "experimental_method" + ], + [ + 78, + 88, + "polymyxins", + "chemical" + ], + [ + 90, + 101, + "tigecycline", + "chemical" + ], + [ + 124, + 138, + "aminoglycoside", + "chemical" + ] + ] + }, + { + "sid": 13, + "sent": "Another effective strategy involves the limited use of certain antimicrobials, specifically fluoroquinolones and cephalo\u00adsporins (Gasink et al., 2009).", + "section": "INTRO", + "ner": [ + [ + 92, + 108, + "fluoroquinolones", + "chemical" + ], + [ + 113, + 128, + "cephalo\u00adsporins", + "chemical" + ] + ] + }, + { + "sid": 14, + "sent": "These include combinations of existing \u03b2-lactam antibiotics with new \u03b2-lactamase inhibitors able to circumvent KPC resistance.", + "section": "INTRO", + "ner": [ + [ + 69, + 80, + "\u03b2-lactamase", + "protein_type" + ] + ] + }, + { + "sid": 15, + "sent": "Neoglycosides are novel aminoglycosides that have activity against KPC-producing bacteria that are also being developed (Chen et al., 2012).", + "section": "INTRO", + "ner": [ + [ + 0, + 13, + "Neoglycosides", + "chemical" + ], + [ + 24, + 39, + "aminoglycosides", + "chemical" + ], + [ + 81, + 89, + "bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 16, + "sent": "Type II topoisomerase enzymes play important roles in prokaryotic and eukaryotic DNA replication, recombination and transcription (Drlica et al., 2008; Laponogov et al., 2013; Lee et al., 2013; Nitiss, 2009a ,b ; Schoeffler & Berger, 2008; Sissi & Palumbo, 2009; Vos et al., 2011; Wendorff et al., 2012; Wu et al., 2011, 2013).", + "section": "INTRO", + "ner": [ + [ + 0, + 29, + "Type II topoisomerase enzymes", + "protein_type" + ], + [ + 54, + 65, + "prokaryotic", + "taxonomy_domain" + ], + [ + 70, + 80, + "eukaryotic", + "taxonomy_domain" + ], + [ + 81, + 84, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 17, + "sent": "In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks.", + "section": "INTRO", + "ner": [ + [ + 3, + 11, + "bacteria", + "taxonomy_domain" + ], + [ + 13, + 29, + "topoisomerase IV", + "complex_assembly" + ], + [ + 33, + 41, + "tetramer", + "oligomeric_state" + ], + [ + 49, + 53, + "ParC", + "protein" + ], + [ + 62, + 66, + "ParE", + "protein" + ], + [ + 157, + 163, + "gyrase", + "protein_type" + ], + [ + 167, + 177, + "GyrA2GyrB2", + "complex_assembly" + ], + [ + 178, + 186, + "tetramer", + "oligomeric_state" + ], + [ + 199, + 202, + "DNA", + "chemical" + ], + [ + 220, + 223, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 18, + "sent": "Both enzymes act via a double-strand DNA break involving a cleavage complex and are targets for quinolone antimicrobials that act by trapping these enzymes at the DNA-cleavage stage and preventing strand re-joining (Drlica et al., 2008).", + "section": "INTRO", + "ner": [ + [ + 37, + 40, + "DNA", + "chemical" + ], + [ + 163, + 166, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 19, + "sent": "Levofloxacin is a broad-spectrum third-generation fluoro\u00adquinolone antibiotic.", + "section": "INTRO", + "ner": [ + [ + 0, + 12, + "Levofloxacin", + "chemical" + ] + ] + }, + { + "sid": 20, + "sent": "It is active against Gram-positive and Gram-negative bacteria and functions by inhibiting gyrase and topoisomerase IV (Drlica & Zhao, 1997; Laponogov et al., 2010).", + "section": "INTRO", + "ner": [ + [ + 21, + 34, + "Gram-positive", + "taxonomy_domain" + ], + [ + 39, + 61, + "Gram-negative bacteria", + "taxonomy_domain" + ], + [ + 90, + 96, + "gyrase", + "protein_type" + ], + [ + 101, + 117, + "topoisomerase IV", + "complex_assembly" + ] + ] + }, + { + "sid": 21, + "sent": "Acquiring a deep structural and functional understanding of the mode of action of fluoroquinolones (Toma\u0161i\u0107 & Ma\u0161i\u010d, 2014) and the development of new drugs targeted against topoisomerase IV and gyrase from a wide range of Gram-positive and Gram-negative pathogenic bacteria are highly active areas of current research directed at overcoming the vexed problem of drug resistance (Bax et al., 2010; Chan et al., 2015; Drlica et al., 2014; Mutsaev et al., 2014; Pommier, 2013; Srikannathasan et al., 2015).", + "section": "INTRO", + "ner": [ + [ + 82, + 98, + "fluoroquinolones", + "chemical" + ], + [ + 173, + 189, + "topoisomerase IV", + "complex_assembly" + ], + [ + 194, + 200, + "gyrase", + "protein_type" + ], + [ + 222, + 235, + "Gram-positive", + "taxonomy_domain" + ], + [ + 240, + 253, + "Gram-negative", + "taxonomy_domain" + ], + [ + 265, + 273, + "bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 22, + "sent": "Here, we report the first three-dimensional X-ray structure of a K. pneumoniae topoisomerase IV ParC/ParE cleavage complex with DNA stabilized by levofloxacin.", + "section": "INTRO", + "ner": [ + [ + 44, + 59, + "X-ray structure", + "evidence" + ], + [ + 65, + 78, + "K. pneumoniae", + "species" + ], + [ + 79, + 95, + "topoisomerase IV", + "complex_assembly" + ], + [ + 96, + 105, + "ParC/ParE", + "complex_assembly" + ], + [ + 128, + 131, + "DNA", + "chemical" + ], + [ + 146, + 158, + "levofloxacin", + "chemical" + ] + ] + }, + { + "sid": 23, + "sent": "The crystal structure provides structural information on topoisomerase IV from K. pneumoniae, a pathogen for which drug resistance is a serious concern.", + "section": "INTRO", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 57, + 73, + "topoisomerase IV", + "complex_assembly" + ], + [ + 79, + 92, + "K. pneumoniae", + "species" + ] + ] + }, + { + "sid": 24, + "sent": "The structure of the ParC/ParE\u2013DNA\u2013levofloxacin binding site highlights the details of the cleavage-complex assembly that are essential for the rational design of Klebsiella topoisomerase inhibitors.", + "section": "INTRO", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 21, + 30, + "ParC/ParE", + "complex_assembly" + ], + [ + 31, + 60, + "DNA\u2013levofloxacin binding site", + "site" + ], + [ + 163, + 173, + "Klebsiella", + "taxonomy_domain" + ], + [ + 174, + 187, + "topoisomerase", + "protein_type" + ] + ] + }, + { + "sid": 25, + "sent": "We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1\u2013490) and ParE TOPRIM domain (ParE30; residues 390\u2013631) with a precut 34\u2005bp DNA duplex (the E-site), stabilized by levofloxacin.", + "section": "RESULTS", + "ner": [ + [ + 8, + 23, + "co-crystallized", + "experimental_method" + ], + [ + 28, + 41, + "K. pneumoniae", + "species" + ], + [ + 42, + 58, + "topoisomerase IV", + "complex_assembly" + ], + [ + 59, + 68, + "ParC/ParE", + "complex_assembly" + ], + [ + 69, + 85, + "breakage-reunion", + "structure_element" + ], + [ + 94, + 100, + "ParC55", + "protein" + ], + [ + 111, + 116, + "1\u2013490", + "residue_range" + ], + [ + 122, + 126, + "ParE", + "protein" + ], + [ + 127, + 133, + "TOPRIM", + "structure_element" + ], + [ + 142, + 148, + "ParE30", + "protein" + ], + [ + 159, + 166, + "390\u2013631", + "residue_range" + ], + [ + 188, + 191, + "DNA", + "chemical" + ], + [ + 204, + 210, + "E-site", + "site" + ], + [ + 227, + 239, + "levofloxacin", + "chemical" + ] + ] + }, + { + "sid": 26, + "sent": "The X-ray crystal structure of the complex was determined to 3.35\u2005\u00c5 resolution, revealing a closed ParC55 dimer flanked by two ParE30 monomers (Figs. 1 \u25b8, 2 \u25b8 and 3 \u25b8).", + "section": "RESULTS", + "ner": [ + [ + 4, + 27, + "X-ray crystal structure", + "evidence" + ], + [ + 92, + 98, + "closed", + "protein_state" + ], + [ + 99, + 105, + "ParC55", + "protein" + ], + [ + 106, + 111, + "dimer", + "oligomeric_state" + ], + [ + 127, + 133, + "ParE30", + "protein" + ], + [ + 134, + 142, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 27, + "sent": "The overall architecture of this complex is similar to that found for S. pneumoniae topoisomerase\u2013DNA\u2013drug complexes (Laponogov et al., 2009, 2010).", + "section": "RESULTS", + "ner": [ + [ + 70, + 83, + "S. pneumoniae", + "species" + ] + ] + }, + { + "sid": 28, + "sent": "Residues 6\u201330 of the N-terminal \u03b1-helix \u03b11 of the ParC subunit again embrace the ParE subunit, \u2018hugging\u2019 the ParE subunits close to either side of the ParC dimer (Laponogov et al., 2010).", + "section": "RESULTS", + "ner": [ + [ + 9, + 13, + "6\u201330", + "residue_range" + ], + [ + 32, + 39, + "\u03b1-helix", + "structure_element" + ], + [ + 40, + 42, + "\u03b11", + "structure_element" + ], + [ + 50, + 54, + "ParC", + "protein" + ], + [ + 81, + 85, + "ParE", + "protein" + ], + [ + 109, + 113, + "ParE", + "protein" + ], + [ + 151, + 155, + "ParC", + "protein" + ], + [ + 156, + 161, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 29, + "sent": "Deletion of this \u2018arm\u2019 \u03b11 results in loss of DNA-cleavage activity (Laponogov et al., 2007) and is clearly very important in complex stability (Fig. 3 \u25b8).", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Deletion of", + "experimental_method" + ], + [ + 18, + 21, + "arm", + "structure_element" + ], + [ + 23, + 25, + "\u03b11", + "structure_element" + ], + [ + 37, + 66, + "loss of DNA-cleavage activity", + "protein_state" + ] + ] + }, + { + "sid": 30, + "sent": "This structural feature was absent in our original ParC55 structure (Laponogov et al., 2007; Sohi et al., 2008).", + "section": "RESULTS", + "ner": [ + [ + 51, + 57, + "ParC55", + "protein" + ], + [ + 58, + 67, + "structure", + "evidence" + ] + ] + }, + { + "sid": 31, + "sent": "The upper region of the topoisomerase complex consists of the E-subunit TOPRIM metal-binding domain formed of four parallel \u03b2-sheets and the surrounding \u03b1-helices.", + "section": "RESULTS", + "ner": [ + [ + 24, + 37, + "topoisomerase", + "protein_type" + ], + [ + 62, + 71, + "E-subunit", + "protein" + ], + [ + 72, + 99, + "TOPRIM metal-binding domain", + "structure_element" + ], + [ + 115, + 132, + "parallel \u03b2-sheets", + "structure_element" + ], + [ + 153, + 162, + "\u03b1-helices", + "structure_element" + ] + ] + }, + { + "sid": 32, + "sent": "The C-subunit provides the WHD (winged-helix domain; a CAP-like structure; McKay & Steitz, 1981) and the \u2018tower\u2019 which form the U groove-shaped protein region into which the G-gate DNA binds with an induced U-shaped bend.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "C-subunit", + "protein" + ], + [ + 27, + 30, + "WHD", + "structure_element" + ], + [ + 32, + 51, + "winged-helix domain", + "structure_element" + ], + [ + 55, + 73, + "CAP-like structure", + "structure_element" + ], + [ + 106, + 111, + "tower", + "structure_element" + ], + [ + 128, + 136, + "U groove", + "structure_element" + ], + [ + 174, + 180, + "G-gate", + "structure_element" + ], + [ + 181, + 184, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 33, + "sent": "The lower C-gate region (Fig. 3 \u25b8) consists of the same disposition of pairs of two long \u03b1-helices terminated by a spanning short \u03b1-helix forming a 30\u2005\u00c5 wide DNA-accommodating cavity through which the T-gate DNA passes as found in the S. pneumoniae complex.", + "section": "RESULTS", + "ner": [ + [ + 10, + 16, + "C-gate", + "structure_element" + ], + [ + 84, + 98, + "long \u03b1-helices", + "structure_element" + ], + [ + 124, + 137, + "short \u03b1-helix", + "structure_element" + ], + [ + 158, + 182, + "DNA-accommodating cavity", + "site" + ], + [ + 201, + 207, + "T-gate", + "structure_element" + ], + [ + 208, + 211, + "DNA", + "chemical" + ], + [ + 235, + 248, + "S. pneumoniae", + "species" + ] + ] + }, + { + "sid": 34, + "sent": "Owing to the structural similarity, it appears that the topo\u00adisomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 \u25b8; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD \u03b2-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013).", + "section": "RESULTS", + "ner": [ + [ + 56, + 74, + "topo\u00adisomerases IV", + "complex_assembly" + ], + [ + 80, + 93, + "K. pneumoniae", + "species" + ], + [ + 98, + 111, + "S. pneumoniae", + "species" + ], + [ + 151, + 164, + "topoisomerase", + "protein_type" + ], + [ + 260, + 269, + "structure", + "evidence" + ], + [ + 277, + 289, + "full complex", + "protein_state" + ], + [ + 295, + 305, + "holoenzyme", + "protein_state" + ], + [ + 315, + 329, + "CTD \u03b2-pinwheel", + "structure_element" + ], + [ + 347, + 360, + "ATPase domain", + "structure_element" + ], + [ + 368, + 372, + "open", + "protein_state" + ] + ] + }, + { + "sid": 35, + "sent": "The G-gate DNA for the S. pneumoniae complex consists of an 18-base-pair E-site sequence (our designation for a DNA site which we first found from DNA-mapping studies; Leo et al., 2005; Arnoldi et al., 2013; Fig. 1 \u25b8).", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "G-gate", + "structure_element" + ], + [ + 11, + 14, + "DNA", + "chemical" + ], + [ + 23, + 36, + "S. pneumoniae", + "species" + ], + [ + 73, + 79, + "E-site", + "site" + ], + [ + 112, + 120, + "DNA site", + "site" + ], + [ + 147, + 166, + "DNA-mapping studies", + "experimental_method" + ] + ] + }, + { + "sid": 36, + "sent": "The crystallized complex was formed by turning over the topoisomerase tetramer in the presence of DNA and levofloxacin and crystallizing the product.", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "crystallized", + "experimental_method" + ], + [ + 56, + 69, + "topoisomerase", + "protein_type" + ], + [ + 70, + 78, + "tetramer", + "oligomeric_state" + ], + [ + 86, + 97, + "presence of", + "protein_state" + ], + [ + 98, + 101, + "DNA", + "chemical" + ], + [ + 106, + 118, + "levofloxacin", + "chemical" + ], + [ + 123, + 136, + "crystallizing", + "experimental_method" + ] + ] + }, + { + "sid": 37, + "sent": "In contrast, the K. pneumoniae complex was formed by co-crystallizing the topoisomerase tetramer complex in the presence of a 34-base-pair pre-cleaved DNA in the presence of levofloxacin.", + "section": "RESULTS", + "ner": [ + [ + 17, + 30, + "K. pneumoniae", + "species" + ], + [ + 53, + 69, + "co-crystallizing", + "experimental_method" + ], + [ + 74, + 87, + "topoisomerase", + "protein_type" + ], + [ + 88, + 96, + "tetramer", + "oligomeric_state" + ], + [ + 112, + 123, + "presence of", + "protein_state" + ], + [ + 139, + 150, + "pre-cleaved", + "protein_state" + ], + [ + 151, + 154, + "DNA", + "chemical" + ], + [ + 162, + 173, + "presence of", + "protein_state" + ], + [ + 174, + 186, + "levofloxacin", + "chemical" + ] + ] + }, + { + "sid": 38, + "sent": "In both cases the DNA is bent into a U-form and bound snugly against the protein of the G-gate.", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "DNA", + "chemical" + ], + [ + 37, + 43, + "U-form", + "protein_state" + ], + [ + 48, + 53, + "bound", + "protein_state" + ], + [ + 88, + 94, + "G-gate", + "structure_element" + ] + ] + }, + { + "sid": 39, + "sent": "We have been able to unambiguously read off the DNA sequences in the electron-density maps.", + "section": "RESULTS", + "ner": [ + [ + 48, + 51, + "DNA", + "chemical" + ], + [ + 69, + 90, + "electron-density maps", + "evidence" + ] + ] + }, + { + "sid": 40, + "sent": "There is 41.6% sequence identity and 54.4% sequence homology between the ParE subunit of K. pneumoniae and that of S. pneumoniae.", + "section": "RESULTS", + "ner": [ + [ + 73, + 77, + "ParE", + "protein" + ], + [ + 89, + 102, + "K. pneumoniae", + "species" + ], + [ + 115, + 128, + "S. pneumoniae", + "species" + ] + ] + }, + { + "sid": 41, + "sent": "For the ParC subunits, the figures are 40.8 identity and 55.6% homology between the two organisms.", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "ParC", + "protein" + ] + ] + }, + { + "sid": 42, + "sent": "The sequence alignment is given in Supplementary Fig. S1, with the key metal-binding residues and those which give rise to quinolone resistance highlighted.", + "section": "RESULTS", + "ner": [ + [ + 4, + 22, + "sequence alignment", + "experimental_method" + ], + [ + 71, + 93, + "metal-binding residues", + "site" + ] + ] + }, + { + "sid": 43, + "sent": "The binding of levofloxacin in the K. pneumoniae complex is shown in Figs. 2 \u25b8, 3 \u25b8 and 5 \u25b8 and is hemi-intercalated into the DNA and stacked against the DNA bases at the cleavage site (positions \u22121 and +1 of the four-base-pair staggered cut in the 34-mer DNA) which is similar to that found for the S. pneumoniae complex.", + "section": "RESULTS", + "ner": [ + [ + 15, + 27, + "levofloxacin", + "chemical" + ], + [ + 35, + 48, + "K. pneumoniae", + "species" + ], + [ + 126, + 129, + "DNA", + "chemical" + ], + [ + 154, + 157, + "DNA", + "chemical" + ], + [ + 171, + 184, + "cleavage site", + "site" + ], + [ + 196, + 198, + "\u22121", + "residue_number" + ], + [ + 203, + 205, + "+1", + "residue_number" + ], + [ + 256, + 259, + "DNA", + "chemical" + ], + [ + 300, + 313, + "S. pneumoniae", + "species" + ] + ] + }, + { + "sid": 44, + "sent": "Fig. 5 \u25b8 presents side-by-side views of the K. pneumoniae and S. pneumoniae active sites which shows that levofloxacin binds in a very similar manner in these two complexes with extensive \u03c0\u2013\u03c0 stacking interaction between the bases and the drug.", + "section": "RESULTS", + "ner": [ + [ + 44, + 57, + "K. pneumoniae", + "species" + ], + [ + 62, + 75, + "S. pneumoniae", + "species" + ], + [ + 76, + 88, + "active sites", + "site" + ], + [ + 106, + 118, + "levofloxacin", + "chemical" + ], + [ + 188, + 212, + "\u03c0\u2013\u03c0 stacking interaction", + "bond_interaction" + ] + ] + }, + { + "sid": 45, + "sent": "The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain.", + "section": "RESULTS", + "ner": [ + [ + 4, + 20, + "methylpiperazine", + "chemical" + ], + [ + 51, + 60, + "quinolone", + "chemical" + ], + [ + 136, + 142, + "Glu474", + "residue_name_number" + ], + [ + 147, + 153, + "Glu475", + "residue_name_number" + ], + [ + 158, + 171, + "S. pneumoniae", + "species" + ], + [ + 184, + 190, + "Gln460", + "residue_name_number" + ], + [ + 195, + 201, + "Glu461", + "residue_name_number" + ], + [ + 206, + 219, + "K. pneumoniae", + "species" + ], + [ + 231, + 247, + "glutamate at 474", + "residue_name_number" + ], + [ + 268, + 284, + "glutamine at 460", + "residue_name_number" + ], + [ + 292, + 302, + "Klebsiella", + "taxonomy_domain" + ] + ] + }, + { + "sid": 46, + "sent": "Interestingly, for S. pneumoniae we observe only one possible orientation of the C7 groups in both sub\u00adunits, while for K. pneumoniae we can see two: one with the same orientation as in S. pneumoniae and other rotated 180\u00b0 away.", + "section": "RESULTS", + "ner": [ + [ + 19, + 32, + "S. pneumoniae", + "species" + ], + [ + 120, + 133, + "K. pneumoniae", + "species" + ], + [ + 186, + 199, + "S. pneumoniae", + "species" + ] + ] + }, + { + "sid": 47, + "sent": "They both exist within the same crystal in the two different dimers in the asymmetric unit.", + "section": "RESULTS", + "ner": [ + [ + 32, + 39, + "crystal", + "evidence" + ], + [ + 61, + 67, + "dimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 48, + "sent": "The side chains surrounding them in ParE are quite disordered and are more defined in K. pneumoniae (even though this complex is at lower resolution) than in S. pneumoniae.", + "section": "RESULTS", + "ner": [ + [ + 36, + 40, + "ParE", + "protein" + ], + [ + 86, + 99, + "K. pneumoniae", + "species" + ], + [ + 158, + 171, + "S. pneumoniae", + "species" + ] + ] + }, + { + "sid": 49, + "sent": "There are no direct hydrogen bonds from the drug to these residues (although it is possible that some are formed through water, which cannot be observed at this resolution).", + "section": "RESULTS", + "ner": [ + [ + 20, + 34, + "hydrogen bonds", + "bond_interaction" + ], + [ + 121, + 126, + "water", + "chemical" + ] + ] + }, + { + "sid": 50, + "sent": "Obviously, the drug\u2013ParE interaction in this region is less strong compared with PD 0305970 binding to the S. pneumoniae DNA complex, where PD 0305970 forms a hydrogen bond to ParE residue Asp475 and can form one to Asp474 if the bond rotates (Laponogov et al., 2010).", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "ParE", + "protein" + ], + [ + 81, + 91, + "PD 0305970", + "chemical" + ], + [ + 107, + 120, + "S. pneumoniae", + "species" + ], + [ + 121, + 124, + "DNA", + "chemical" + ], + [ + 140, + 150, + "PD 0305970", + "chemical" + ], + [ + 159, + 172, + "hydrogen bond", + "bond_interaction" + ], + [ + 176, + 180, + "ParE", + "protein" + ], + [ + 189, + 195, + "Asp475", + "residue_name_number" + ], + [ + 216, + 222, + "Asp474", + "residue_name_number" + ] + ] + }, + { + "sid": 51, + "sent": "This may explain why drug-resistance mutations for levofloxacin are more likely to form in the ParC subunits rather than in the ParE subunits.", + "section": "RESULTS", + "ner": [ + [ + 51, + 63, + "levofloxacin", + "chemical" + ], + [ + 95, + 99, + "ParC", + "protein" + ], + [ + 128, + 132, + "ParE", + "protein" + ] + ] + }, + { + "sid": 52, + "sent": "For both complexes there is a Mg2+ ion bound to levofloxacin between the carbonyl group at position 4 of the quinolone and the carboxyl at position 6 (Figs. 2 \u25b8 and 5 \u25b8 and Supplementary Fig. 2 \u25b8).", + "section": "RESULTS", + "ner": [ + [ + 30, + 34, + "Mg2+", + "chemical" + ], + [ + 39, + 47, + "bound to", + "protein_state" + ], + [ + 48, + 60, + "levofloxacin", + "chemical" + ], + [ + 109, + 118, + "quinolone", + "chemical" + ] + ] + }, + { + "sid": 53, + "sent": "For S. pneumoniae topoisomerase IV, one of the O atoms of the carboxyl of Asp83 points towards the Mg2+ ion and is within hydrogen-bonding distance (5.04\u2005\u00c5) through an Mg2+-coordinated water.", + "section": "RESULTS", + "ner": [ + [ + 4, + 17, + "S. pneumoniae", + "species" + ], + [ + 18, + 34, + "topoisomerase IV", + "complex_assembly" + ], + [ + 74, + 79, + "Asp83", + "residue_name_number" + ], + [ + 99, + 103, + "Mg2+", + "chemical" + ], + [ + 122, + 138, + "hydrogen-bonding", + "bond_interaction" + ], + [ + 168, + 172, + "Mg2+", + "chemical" + ], + [ + 185, + 190, + "water", + "chemical" + ] + ] + }, + { + "sid": 54, + "sent": "For K. pneumoniae both of the carboxyl O atoms are pointing towards the Mg2+ ion at distances of 4.86 and 4.23\u2005\u00c5. These residues are ordered in only one of the two dimers in the K. pneumoniae crystal (the one in which the C7 group is pointing towards the DNA away from ParE, although the conformations of these two groups on the drug are probably not correlated).", + "section": "RESULTS", + "ner": [ + [ + 4, + 17, + "K. pneumoniae", + "species" + ], + [ + 72, + 76, + "Mg2+", + "chemical" + ], + [ + 164, + 170, + "dimers", + "oligomeric_state" + ], + [ + 178, + 191, + "K. pneumoniae", + "species" + ], + [ + 192, + 199, + "crystal", + "evidence" + ], + [ + 255, + 258, + "DNA", + "chemical" + ], + [ + 269, + 273, + "ParE", + "protein" + ] + ] + }, + { + "sid": 55, + "sent": "The topoisomerase IV ParE27-ParC55 fusion protein from K. pneumoniae was fully active in promoting levofloxacin-mediated cleavage of DNA (Fig. 6 \u25b8).", + "section": "RESULTS", + "ner": [ + [ + 4, + 20, + "topoisomerase IV", + "complex_assembly" + ], + [ + 21, + 34, + "ParE27-ParC55", + "complex_assembly" + ], + [ + 55, + 68, + "K. pneumoniae", + "species" + ], + [ + 99, + 111, + "levofloxacin", + "chemical" + ], + [ + 133, + 136, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 56, + "sent": "In the absence of the drug and ATP, the protein converted supercoiled pBR322 into a ladder of bands corresponding to relaxed DNA.", + "section": "RESULTS", + "ner": [ + [ + 7, + 17, + "absence of", + "protein_state" + ], + [ + 22, + 26, + "drug", + "chemical" + ], + [ + 31, + 34, + "ATP", + "chemical" + ], + [ + 125, + 128, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 57, + "sent": "The inclusion of levofloxacin produced linear DNA in a dose-dependent and ATP-independent fashion.", + "section": "RESULTS", + "ner": [ + [ + 17, + 29, + "levofloxacin", + "chemical" + ], + [ + 46, + 49, + "DNA", + "chemical" + ], + [ + 74, + 77, + "ATP", + "chemical" + ] + ] + }, + { + "sid": 58, + "sent": "Similar behaviour was observed for the S. pneumoniae topo\u00adisomerase IV ParE30-ParC55 fusion protein.", + "section": "RESULTS", + "ner": [ + [ + 39, + 52, + "S. pneumoniae", + "species" + ], + [ + 53, + 70, + "topo\u00adisomerase IV", + "complex_assembly" + ], + [ + 71, + 84, + "ParE30-ParC55", + "complex_assembly" + ] + ] + }, + { + "sid": 59, + "sent": "The CC25 (the drug concentration that converted 25% of the supercoiled DNA substrate to a linear form) was 0.5\u2005\u00b5M for the Klebsiella enzyme and 1\u2005\u00b5M for the pneumococcal enzyme.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "CC25", + "evidence" + ], + [ + 71, + 74, + "DNA", + "chemical" + ], + [ + 122, + 132, + "Klebsiella", + "taxonomy_domain" + ], + [ + 157, + 169, + "pneumococcal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 60, + "sent": "Interestingly, K. pneumoniae strains are much more susceptible to levofloxacin than S. pneumoniae, with typical MIC values of 0.016 and 1\u2005mg\u2005l\u22121, respectively (Odenholt & Cars, 2006), reflecting differences in multiple factors (in addition to binding affinity) that influence drug responses, including membrane, peptidoglycan structure, drug-uptake and efflux mechanisms.", + "section": "RESULTS", + "ner": [ + [ + 15, + 28, + "K. pneumoniae", + "species" + ], + [ + 66, + 78, + "levofloxacin", + "chemical" + ], + [ + 84, + 97, + "S. pneumoniae", + "species" + ], + [ + 243, + 259, + "binding affinity", + "evidence" + ] + ] + }, + { + "sid": 61, + "sent": "Moreover, although topoisomerase IV is primarily the target of levofloxacin in S. pneumoniae, it is likely to be gyrase in the Gram-negative K. pneumoniae.", + "section": "RESULTS", + "ner": [ + [ + 19, + 35, + "topoisomerase IV", + "complex_assembly" + ], + [ + 63, + 75, + "levofloxacin", + "chemical" + ], + [ + 79, + 92, + "S. pneumoniae", + "species" + ], + [ + 113, + 119, + "gyrase", + "protein_type" + ], + [ + 127, + 140, + "Gram-negative", + "taxonomy_domain" + ], + [ + 141, + 154, + "K. pneumoniae", + "species" + ] + ] + }, + { + "sid": 62, + "sent": "In summary, we have determined the first structure of a quinolone\u2013DNA cleavage complex involving a type II topo\u00adisomerase from K. pneumoniae.", + "section": "RESULTS", + "ner": [ + [ + 41, + 50, + "structure", + "evidence" + ], + [ + 56, + 65, + "quinolone", + "chemical" + ], + [ + 66, + 69, + "DNA", + "chemical" + ], + [ + 99, + 121, + "type II topo\u00adisomerase", + "protein_type" + ], + [ + 127, + 140, + "K. pneumoniae", + "species" + ] + ] + }, + { + "sid": 63, + "sent": "Given the current concerns about drug-resistant strains of Klebsiella, the structure reported here provides key information in understanding the action of currently used quinolones and should aid in the development of other topoisomerase-targeting therapeutics active against this major human pathogen.", + "section": "RESULTS", + "ner": [ + [ + 59, + 69, + "Klebsiella", + "taxonomy_domain" + ], + [ + 75, + 84, + "structure", + "evidence" + ], + [ + 170, + 180, + "quinolones", + "chemical" + ], + [ + 224, + 237, + "topoisomerase", + "protein_type" + ], + [ + 287, + 292, + "human", + "species" + ] + ] + }, + { + "sid": 64, + "sent": "Protein and DNA used in the co-crystallization experiment.", + "section": "FIG", + "ner": [ + [ + 12, + 15, + "DNA", + "chemical" + ], + [ + 28, + 46, + "co-crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 65, + "sent": "(a) Coloured diagram of the protein constructs used in crystallization.", + "section": "FIG", + "ner": [ + [ + 55, + 70, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 66, + "sent": "(b) DNA sequences used in crystallization.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "DNA", + "chemical" + ], + [ + 26, + 41, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 67, + "sent": "Chemical structure of levofloxacin (a) and its conformations observed within the active sites of S. pneumoniae topoisomerase IV (b) and K.\u00a0pneumoniae topoisomerase IV (c, d).", + "section": "FIG", + "ner": [ + [ + 22, + 34, + "levofloxacin", + "chemical" + ], + [ + 81, + 93, + "active sites", + "site" + ], + [ + 97, + 110, + "S. pneumoniae", + "species" + ], + [ + 111, + 127, + "topoisomerase IV", + "complex_assembly" + ], + [ + 136, + 149, + "K.\u00a0pneumoniae", + "species" + ], + [ + 150, + 166, + "topoisomerase IV", + "complex_assembly" + ] + ] + }, + { + "sid": 68, + "sent": "Electron-density maps (2F obs \u2212 F calc) are shown as meshes for the drug molecules contoured at 1.5\u03c3 and are limited to a distance of 2.3\u2005\u00c5 from the drug atoms.", + "section": "FIG", + "ner": [ + [ + 0, + 21, + "Electron-density maps", + "evidence" + ] + ] + }, + { + "sid": 69, + "sent": "Overall orthogonal views of the cleavage complex of topoisomerase IV from K. pneumoniae in surface (left) and cartoon (right) representations.", + "section": "FIG", + "ner": [ + [ + 52, + 68, + "topoisomerase IV", + "complex_assembly" + ], + [ + 74, + 87, + "K. pneumoniae", + "species" + ] + ] + }, + { + "sid": 70, + "sent": "The ParC subunit is in blue, ParE is in yellow and DNA is in cyan.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "ParC", + "protein" + ], + [ + 29, + 33, + "ParE", + "protein" + ], + [ + 51, + 54, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 71, + "sent": "The bound quinolone molecules (levofloxacin) are in red and are shown using van der Waals representation.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "bound", + "protein_state" + ], + [ + 10, + 19, + "quinolone", + "chemical" + ], + [ + 31, + 43, + "levofloxacin", + "chemical" + ] + ] + }, + { + "sid": 72, + "sent": "Schematic representation of the catalytic cycle of type II topoisomerases.", + "section": "FIG", + "ner": [ + [ + 51, + 73, + "type II topoisomerases", + "protein_type" + ] + ] + }, + { + "sid": 73, + "sent": "The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal \u03b2-\u00adpinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "ParC", + "protein" + ], + [ + 28, + 34, + "ParC55", + "protein" + ], + [ + 52, + 56, + "ParC", + "protein" + ], + [ + 68, + 86, + "\u03b2-\u00adpinwheel domain", + "structure_element" + ], + [ + 105, + 109, + "ParE", + "protein" + ], + [ + 121, + 134, + "ATPase domain", + "structure_element" + ], + [ + 150, + 154, + "ParE", + "protein" + ], + [ + 155, + 172, + "C-terminal domain", + "structure_element" + ], + [ + 174, + 180, + "ParE30", + "protein" + ], + [ + 200, + 206, + "G-gate", + "structure_element" + ], + [ + 207, + 210, + "DNA", + "chemical" + ], + [ + 231, + 240, + "T-segment", + "structure_element" + ], + [ + 241, + 244, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 74, + "sent": "Bound ATP is indicated by pink circles in the ATPase domains (reproduced with permission from Fig. 1 of Lapanogov et al., 2013).", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "Bound", + "protein_state" + ], + [ + 6, + 9, + "ATP", + "chemical" + ], + [ + 46, + 60, + "ATPase domains", + "structure_element" + ] + ] + }, + { + "sid": 75, + "sent": "Detailed views of the active sites of topoisomerase IV from S. pneumoniae and K. pneumoniae with quinolone molecules bound.", + "section": "FIG", + "ner": [ + [ + 22, + 34, + "active sites", + "site" + ], + [ + 38, + 54, + "topoisomerase IV", + "complex_assembly" + ], + [ + 60, + 73, + "S. pneumoniae", + "species" + ], + [ + 78, + 91, + "K. pneumoniae", + "species" + ], + [ + 97, + 106, + "quinolone", + "chemical" + ], + [ + 117, + 122, + "bound", + "protein_state" + ] + ] + }, + { + "sid": 76, + "sent": "The magnesium ions and their coordinating amino acids are shown in purple.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "magnesium", + "chemical" + ] + ] + }, + { + "sid": 77, + "sent": "The active-site tyrosine and arginine are in orange.", + "section": "FIG", + "ner": [ + [ + 4, + 15, + "active-site", + "site" + ], + [ + 16, + 24, + "tyrosine", + "residue_name" + ], + [ + 29, + 37, + "arginine", + "residue_name" + ] + ] + }, + { + "sid": 78, + "sent": "The DNA is shown in silver/cyan.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 79, + "sent": "The ParC and ParE backbones are shown in blue and yellow, respectively.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "ParC", + "protein" + ], + [ + 13, + 17, + "ParE", + "protein" + ] + ] + }, + { + "sid": 80, + "sent": "Comparison of DNA cleavage by topoisomerase IV core ParE-ParC fusion proteins from K. pneumoniae (KP) and S.\u00a0pneumoniae (SP) promoted by levofloxacin.", + "section": "FIG", + "ner": [ + [ + 14, + 17, + "DNA", + "chemical" + ], + [ + 30, + 46, + "topoisomerase IV", + "complex_assembly" + ], + [ + 52, + 61, + "ParE-ParC", + "complex_assembly" + ], + [ + 83, + 96, + "K. pneumoniae", + "species" + ], + [ + 98, + 100, + "KP", + "species" + ], + [ + 106, + 119, + "S.\u00a0pneumoniae", + "species" + ], + [ + 121, + 123, + "SP", + "species" + ], + [ + 137, + 149, + "levofloxacin", + "chemical" + ] + ] + }, + { + "sid": 81, + "sent": "Supercoiled plasmid pBR322 (400\u2005ng) was incubated with topoisomerase IV proteins (400\u2005ng) in the absence or presence of levofloxacin at the indicated concentrations.", + "section": "FIG", + "ner": [ + [ + 55, + 71, + "topoisomerase IV", + "complex_assembly" + ], + [ + 108, + 119, + "presence of", + "protein_state" + ], + [ + 120, + 132, + "levofloxacin", + "chemical" + ] + ] + }, + { + "sid": 82, + "sent": "After 60\u2005min incubation, samples were treated with SDS and proteinase K to remove proteins covalent bound to DNA, and the DNA products were examined by gel electrophoresis in 1% agarose.", + "section": "FIG", + "ner": [ + [ + 109, + 112, + "DNA", + "chemical" + ], + [ + 122, + 125, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 83, + "sent": "Lane A, supercoiled pBR322 DNA; N, L and S, nicked, linear and supercoiled pBR322, respectively.", + "section": "FIG", + "ner": [ + [ + 27, + 30, + "DNA", + "chemical" + ] + ] + } + ] + }, + "PMC4885502": { + "annotations": [ + { + "sid": 0, + "sent": "Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase", + "section": "TITLE", + "ner": [ + [ + 6, + 13, + "Cryo-EM", + "experimental_method" + ], + [ + 78, + 101, + "Glutamate Dehydrogenase", + "protein_type" + ] + ] + }, + { + "sid": 1, + "sent": "Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 24, + "Cryo-electron microscopy", + "experimental_method" + ], + [ + 26, + 33, + "cryo-EM", + "experimental_method" + ], + [ + 75, + 85, + "structures", + "evidence" + ] + ] + }, + { + "sid": 2, + "sent": "We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate.", + "section": "ABSTRACT", + "ner": [ + [ + 30, + 53, + "glutamate dehydrogenase", + "protein_type" + ], + [ + 55, + 58, + "GDH", + "protein_type" + ], + [ + 132, + 141, + "glutamate", + "chemical" + ] + ] + }, + { + "sid": 3, + "sent": "Dysregulation of GDH leads to a variety of metabolic and neurologic disorders.", + "section": "ABSTRACT", + "ner": [ + [ + 17, + 20, + "GDH", + "protein_type" + ] + ] + }, + { + "sid": 4, + "sent": "Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 \u00c5 to 3.6 \u00c5 for GDH complexes, including complexes for which crystal structures are not available.", + "section": "ABSTRACT", + "ner": [ + [ + 39, + 46, + "cryo-EM", + "experimental_method" + ], + [ + 47, + 57, + "structures", + "evidence" + ], + [ + 106, + 109, + "GDH", + "protein_type" + ], + [ + 151, + 169, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 5, + "sent": "We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an \u201copen\u201d or \u201cclosed\u201d state.", + "section": "ABSTRACT", + "ner": [ + [ + 41, + 45, + "NADH", + "chemical" + ], + [ + 71, + 74, + "GTP", + "chemical" + ], + [ + 140, + 144, + "open", + "protein_state" + ], + [ + 150, + 156, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 6, + "sent": "Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site.", + "section": "ABSTRACT", + "ner": [ + [ + 12, + 21, + "structure", + "evidence" + ], + [ + 25, + 29, + "NADH", + "chemical" + ], + [ + 37, + 48, + "active site", + "site" + ], + [ + 72, + 76, + "open", + "protein_state" + ], + [ + 81, + 87, + "closed", + "protein_state" + ], + [ + 132, + 147, + "regulatory site", + "site" + ] + ] + }, + { + "sid": 7, + "sent": "Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.", + "section": "ABSTRACT", + "ner": [ + [ + 117, + 138, + "X-ray crystallography", + "experimental_method" + ], + [ + 140, + 147, + "cryo-EM", + "experimental_method" + ] + ] + }, + { + "sid": 8, + "sent": "Recent advances in cryo-electron microscopy (cryo-EM) allow determination of structures of small protein complexes and membrane proteins at near-atomic resolution, marking a critical shift in the structural biology field.", + "section": "INTRO", + "ner": [ + [ + 19, + 43, + "cryo-electron microscopy", + "experimental_method" + ], + [ + 45, + 52, + "cryo-EM", + "experimental_method" + ], + [ + 77, + 87, + "structures", + "evidence" + ] + ] + }, + { + "sid": 9, + "sent": "One specific area of broad general interest in drug discovery is the localization of bound ligands and cofactors under conditions in which efforts at crystallization have not been successful because of structural heterogeneity.", + "section": "INTRO", + "ner": [ + [ + 150, + 165, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 10, + "sent": "Recent cryo-EM analyses have already demonstrated that it is now possible to use single-particle cryo-EM methods to localize small bound ligands or inhibitors on target proteins.", + "section": "INTRO", + "ner": [ + [ + 7, + 14, + "cryo-EM", + "experimental_method" + ], + [ + 81, + 104, + "single-particle cryo-EM", + "experimental_method" + ] + ] + }, + { + "sid": 11, + "sent": "Here, we address this question using mammalian glutamate dehydrogenase as an example.", + "section": "INTRO", + "ner": [ + [ + 37, + 46, + "mammalian", + "taxonomy_domain" + ], + [ + 47, + 70, + "glutamate dehydrogenase", + "protein_type" + ] + ] + }, + { + "sid": 12, + "sent": "Glutamate dehydrogenase (GDH) is a highly conserved enzyme expressed in most organisms.", + "section": "INTRO", + "ner": [ + [ + 0, + 23, + "Glutamate dehydrogenase", + "protein_type" + ], + [ + 25, + 28, + "GDH", + "protein_type" + ], + [ + 35, + 51, + "highly conserved", + "protein_state" + ] + ] + }, + { + "sid": 13, + "sent": "GDH plays a central role in glutamate metabolism by catalyzing the reversible oxidative deamination of glutamate to generate \u03b1-ketoglutarate and ammonia, with the concomitant transfer of a pair of electrons to either NAD+ or NADP+.", + "section": "INTRO", + "ner": [ + [ + 0, + 3, + "GDH", + "protein_type" + ], + [ + 28, + 37, + "glutamate", + "chemical" + ], + [ + 103, + 112, + "glutamate", + "chemical" + ], + [ + 125, + 140, + "\u03b1-ketoglutarate", + "chemical" + ], + [ + 145, + 152, + "ammonia", + "chemical" + ], + [ + 217, + 221, + "NAD+", + "chemical" + ], + [ + 225, + 230, + "NADP+", + "chemical" + ] + ] + }, + { + "sid": 14, + "sent": "Regulation of GDH is tightly controlled through multiple allosteric mechanisms.", + "section": "INTRO", + "ner": [ + [ + 14, + 17, + "GDH", + "protein_type" + ] + ] + }, + { + "sid": 15, + "sent": "Extensive biochemical and crystallographic studies have characterized the enzymatic activity of GDH and its modulation by a chemically diverse group of compounds such as nucleotides, amino acids, steroid hormones, antipsychotic drugs, and natural products.", + "section": "INTRO", + "ner": [ + [ + 10, + 50, + "biochemical and crystallographic studies", + "experimental_method" + ], + [ + 96, + 99, + "GDH", + "protein_type" + ] + ] + }, + { + "sid": 16, + "sent": "X-ray crystallographic studies have shown that the functional unit of GDH is a homohexamer composed of a trimer of dimers, with a 3-fold axis and an equatorial plane that define its D3 symmetry (Fig. 1A).", + "section": "INTRO", + "ner": [ + [ + 0, + 30, + "X-ray crystallographic studies", + "experimental_method" + ], + [ + 70, + 73, + "GDH", + "protein_type" + ], + [ + 79, + 90, + "homohexamer", + "oligomeric_state" + ], + [ + 105, + 111, + "trimer", + "oligomeric_state" + ], + [ + 115, + 121, + "dimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 17, + "sent": "Each 56-kDa protomer consists of three domains.", + "section": "INTRO", + "ner": [ + [ + 12, + 20, + "protomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 18, + "sent": "The first is located near the dimer interface and forms the core of the hexamer.", + "section": "INTRO", + "ner": [ + [ + 30, + 45, + "dimer interface", + "site" + ], + [ + 72, + 79, + "hexamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 19, + "sent": "The second, a nucleotide-binding domain (NBD) with a Rossmann fold, defines one face of the catalytic cleft bounded by the core domain.", + "section": "INTRO", + "ner": [ + [ + 14, + 39, + "nucleotide-binding domain", + "structure_element" + ], + [ + 41, + 44, + "NBD", + "structure_element" + ], + [ + 53, + 66, + "Rossmann fold", + "structure_element" + ], + [ + 92, + 107, + "catalytic cleft", + "site" + ] + ] + }, + { + "sid": 20, + "sent": "During the catalytic cycle, the NBD executes a large movement, hinged around a \u201cpivot\u201d helix, that closes the catalytic cleft, and drives a large conformational change in the hexamer from open to closed states (Fig. 1B).", + "section": "INTRO", + "ner": [ + [ + 32, + 35, + "NBD", + "structure_element" + ], + [ + 79, + 92, + "\u201cpivot\u201d helix", + "structure_element" + ], + [ + 110, + 125, + "catalytic cleft", + "site" + ], + [ + 175, + 182, + "hexamer", + "oligomeric_state" + ], + [ + 188, + 192, + "open", + "protein_state" + ], + [ + 196, + 202, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 21, + "sent": "The third domain, dubbed the \u201cantenna,\u201d is an evolutionary acquisition in protista and animals.", + "section": "INTRO", + "ner": [ + [ + 30, + 37, + "antenna", + "structure_element" + ], + [ + 74, + 82, + "protista", + "taxonomy_domain" + ], + [ + 87, + 94, + "animals", + "taxonomy_domain" + ] + ] + }, + { + "sid": 22, + "sent": "Antennae of adjacent protomers in each trimer intercalate to form a bundle, perpendicular to the pivot helices, that protrudes along the distal extremes of the 3-fold axis.", + "section": "INTRO", + "ner": [ + [ + 0, + 8, + "Antennae", + "structure_element" + ], + [ + 21, + 30, + "protomers", + "oligomeric_state" + ], + [ + 39, + 45, + "trimer", + "oligomeric_state" + ], + [ + 97, + 110, + "pivot helices", + "structure_element" + ] + ] + }, + { + "sid": 23, + "sent": "When a protomer undergoes a conformational change, the rotation of its pivot helix is transferred through the antenna to the adjacent subunit.", + "section": "INTRO", + "ner": [ + [ + 7, + 15, + "protomer", + "oligomeric_state" + ], + [ + 71, + 82, + "pivot helix", + "structure_element" + ], + [ + 110, + 117, + "antenna", + "structure_element" + ], + [ + 134, + 141, + "subunit", + "structure_element" + ] + ] + }, + { + "sid": 24, + "sent": "The influence of the antenna, present only in protozoan and metazoan enzymes, has been proposed to explain its cooperative behavior, which is absent in bacterial homologs.", + "section": "INTRO", + "ner": [ + [ + 21, + 28, + "antenna", + "structure_element" + ], + [ + 46, + 55, + "protozoan", + "taxonomy_domain" + ], + [ + 60, + 68, + "metazoan", + "taxonomy_domain" + ], + [ + 152, + 161, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 25, + "sent": "Structure and quaternary conformational changes in GDH. (A) Views of open (PDB ID 1NR7) and closed (PDB 3MW9) states of the GDH hexamer, shown in ribbon representation perpendicular to the 2-fold symmetry axis (side view, top) and 3-fold symmetry axis (top view, bottom).", + "section": "FIG", + "ner": [ + [ + 51, + 54, + "GDH", + "protein_type" + ], + [ + 69, + 73, + "open", + "protein_state" + ], + [ + 92, + 98, + "closed", + "protein_state" + ], + [ + 124, + 127, + "GDH", + "protein_type" + ], + [ + 128, + 135, + "hexamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 26, + "sent": "Only three protomers are shown in the top view for purposes of visual clarity.", + "section": "FIG", + "ner": [ + [ + 11, + 20, + "protomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 27, + "sent": "The dashed lines and arrows, respectively, highlight the slight extension in length, and twist in shape that occurs with transition from open to the closed state.", + "section": "FIG", + "ner": [ + [ + 137, + 141, + "open", + "protein_state" + ], + [ + 149, + 155, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 28, + "sent": "The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "open", + "protein_state" + ], + [ + 28, + 38, + "unliganded", + "protein_state" + ], + [ + 39, + 42, + "GDH", + "protein_type" + ], + [ + 56, + 62, + "closed", + "protein_state" + ], + [ + 73, + 77, + "NADH", + "chemical" + ], + [ + 79, + 82, + "GTP", + "chemical" + ], + [ + 88, + 97, + "glutamate", + "chemical" + ], + [ + 98, + 103, + "bound", + "protein_state" + ], + [ + 109, + 122, + "Superposition", + "experimental_method" + ], + [ + 126, + 136, + "structures", + "evidence" + ], + [ + 141, + 147, + "closed", + "protein_state" + ], + [ + 152, + 156, + "open", + "protein_state" + ] + ] + }, + { + "sid": 29, + "sent": "The transition between \u201cclosed\u201d and \u201copen\u201d states of GDH is modulated by two allosteric sites in each protomer (Fig. 1A), which are differentially bound by GTP (an inhibitor) and ADP (an activator).", + "section": "INTRO", + "ner": [ + [ + 24, + 30, + "closed", + "protein_state" + ], + [ + 37, + 41, + "open", + "protein_state" + ], + [ + 53, + 56, + "GDH", + "protein_type" + ], + [ + 77, + 93, + "allosteric sites", + "site" + ], + [ + 102, + 110, + "protomer", + "oligomeric_state" + ], + [ + 147, + 155, + "bound by", + "protein_state" + ], + [ + 156, + 159, + "GTP", + "chemical" + ], + [ + 179, + 182, + "ADP", + "chemical" + ] + ] + }, + { + "sid": 30, + "sent": "These allosteric modulators tightly control GDH function in vivo.", + "section": "INTRO", + "ner": [ + [ + 44, + 47, + "GDH", + "protein_type" + ] + ] + }, + { + "sid": 31, + "sent": "In the first site, which sits next to the pivot helix at the base of the antenna (the \u201cGTP binding site\u201d), GTP binding is known to act as an inhibitor, preventing release of the reaction product from the catalytic site by stabilizing the closed conformation of the catalytic cleft.", + "section": "INTRO", + "ner": [ + [ + 42, + 53, + "pivot helix", + "structure_element" + ], + [ + 73, + 80, + "antenna", + "structure_element" + ], + [ + 87, + 103, + "GTP binding site", + "site" + ], + [ + 107, + 110, + "GTP", + "chemical" + ], + [ + 204, + 218, + "catalytic site", + "site" + ], + [ + 238, + 244, + "closed", + "protein_state" + ], + [ + 265, + 280, + "catalytic cleft", + "site" + ] + ] + }, + { + "sid": 32, + "sent": "In the second \u201cregulatory site\u201d, which is situated near the pivot helix between adjacent protomers, ADP acts as an activator of enzymatic activity, presumably by hastening the opening of the catalytic cleft that leads to the release of the reaction product.", + "section": "INTRO", + "ner": [ + [ + 15, + 30, + "regulatory site", + "site" + ], + [ + 60, + 71, + "pivot helix", + "structure_element" + ], + [ + 89, + 98, + "protomers", + "oligomeric_state" + ], + [ + 100, + 103, + "ADP", + "chemical" + ], + [ + 191, + 206, + "catalytic cleft", + "site" + ] + ] + }, + { + "sid": 33, + "sent": "Interestingly, it has also been shown that the coenzyme NADH can bind to the regulatory site (also bound by the activator ADP), exerting a converse, inhibitory effect on GDH product release, although the role this may play in vivo is not entirely clear.", + "section": "INTRO", + "ner": [ + [ + 56, + 60, + "NADH", + "chemical" + ], + [ + 77, + 92, + "regulatory site", + "site" + ], + [ + 99, + 107, + "bound by", + "protein_state" + ], + [ + 122, + 125, + "ADP", + "chemical" + ], + [ + 170, + 173, + "GDH", + "protein_type" + ] + ] + }, + { + "sid": 34, + "sent": "Although there are numerous crystal structures available for GDH in complex with cofactors and nucleotides, they are limited to the combinations that have been amenable to crystallization.", + "section": "INTRO", + "ner": [ + [ + 28, + 46, + "crystal structures", + "evidence" + ], + [ + 61, + 64, + "GDH", + "protein_type" + ], + [ + 65, + 80, + "in complex with", + "protein_state" + ], + [ + 172, + 187, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 35, + "sent": "Nearly all X-ray structures of mammalian GDH are in the closed conformation, and the few structures that are in the open conformation are at lower resolution (Table 1).", + "section": "INTRO", + "ner": [ + [ + 11, + 16, + "X-ray", + "experimental_method" + ], + [ + 17, + 27, + "structures", + "evidence" + ], + [ + 31, + 40, + "mammalian", + "taxonomy_domain" + ], + [ + 41, + 44, + "GDH", + "protein_type" + ], + [ + 56, + 62, + "closed", + "protein_state" + ], + [ + 89, + 99, + "structures", + "evidence" + ], + [ + 116, + 120, + "open", + "protein_state" + ] + ] + }, + { + "sid": 36, + "sent": "Of those structures in the closed conformation, most include NAD[P]H, GTP, and glutamate (or, alternately, NAD+, GTP, and \u03b1-ketoglutarate).", + "section": "INTRO", + "ner": [ + [ + 9, + 19, + "structures", + "evidence" + ], + [ + 27, + 33, + "closed", + "protein_state" + ], + [ + 61, + 68, + "NAD[P]H", + "chemical" + ], + [ + 70, + 73, + "GTP", + "chemical" + ], + [ + 79, + 88, + "glutamate", + "chemical" + ], + [ + 107, + 111, + "NAD+", + "chemical" + ], + [ + 113, + 116, + "GTP", + "chemical" + ], + [ + 122, + 137, + "\u03b1-ketoglutarate", + "chemical" + ] + ] + }, + { + "sid": 37, + "sent": "However, the effects of coenzyme and GTP, bound alone or in concert in the absence of glutamate, have not been analyzed by crystallographic methods.", + "section": "INTRO", + "ner": [ + [ + 37, + 40, + "GTP", + "chemical" + ], + [ + 42, + 53, + "bound alone", + "protein_state" + ], + [ + 75, + 85, + "absence of", + "protein_state" + ], + [ + 86, + 95, + "glutamate", + "chemical" + ] + ] + }, + { + "sid": 38, + "sent": "Here, we report single-particle cryo-electron microscopy (cryo-EM) studies that show that under these conditions enzyme complexes coexist in both closed and open conformations.", + "section": "INTRO", + "ner": [ + [ + 16, + 56, + "single-particle cryo-electron microscopy", + "experimental_method" + ], + [ + 58, + 65, + "cryo-EM", + "experimental_method" + ], + [ + 146, + 152, + "closed", + "protein_state" + ], + [ + 157, + 161, + "open", + "protein_state" + ] + ] + }, + { + "sid": 39, + "sent": "We show that the structures in both states can be resolved at near-atomic resolution, suggesting a molecular mechanism for synergistic inhibition of GDH by NADH and GTP (see Table 2 for detailed information on all cryo-EM-derived structures that we report in this work).", + "section": "INTRO", + "ner": [ + [ + 17, + 27, + "structures", + "evidence" + ], + [ + 149, + 152, + "GDH", + "protein_type" + ], + [ + 156, + 160, + "NADH", + "chemical" + ], + [ + 165, + 168, + "GTP", + "chemical" + ], + [ + 214, + 221, + "cryo-EM", + "experimental_method" + ], + [ + 230, + 240, + "structures", + "evidence" + ] + ] + }, + { + "sid": 40, + "sent": "X-ray structures of mammalian GDH reported in both the open and closed conformations", + "section": "TABLE", + "ner": [ + [ + 0, + 5, + "X-ray", + "experimental_method" + ], + [ + 6, + 16, + "structures", + "evidence" + ], + [ + 20, + 29, + "mammalian", + "taxonomy_domain" + ], + [ + 30, + 33, + "GDH", + "protein_type" + ], + [ + 55, + 59, + "open", + "protein_state" + ], + [ + 64, + 70, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 41, + "sent": "GDH\tLigands\tPDB ID\tConformation\tResolution\t \tWT\tNADH + GLU + GTP\t3MW9\tClosed\t2.4\t \tWT\tGlu, GTP, NADPH, and Bithionol\t3ETD\tClosed\t2.5\t \tWT\tGlu, NADPH, GTP + GW5074\t3ETG\tClosed\t2.5\t \tWT\tapo\t1L1F\tOpen\t2.7\t \tWT\tNADPH, glutamate, and GTP\t1HWZ\tClosed\t2.8\t \tWT\tNADPH + GLU + GTP + Zinc\t3MVQ\tClosed\t2.94\t \tWT\tNADPH, Glu, GTP, Hexachlorophene\t3ETE\tClosed\t3\t \tWT\tNAD, PO4, and 2-oxoglutarate\t1HWY\tClosed\t3.2\t \tWT\tNADPH + GLU + Eu\t3MVO\tClosed\t3.23\t \tR463A mutant\tapo\t1NR1\tOpen\t3.3\t \tWT\tapo\t1NR7\tOpen\t3.3\t \tWT\tADP\t1NQT\tOpen\t3.5\t \tWT\tNADPH and Epicatechin-3-gallate (Ecg)\t3QMU\tOpen\t3.62\t \t", + "section": "TABLE", + "ner": [ + [ + 0, + 3, + "GDH", + "protein" + ], + [ + 45, + 47, + "WT", + "protein_state" + ], + [ + 48, + 52, + "NADH", + "chemical" + ], + [ + 55, + 58, + "GLU", + "chemical" + ], + [ + 61, + 64, + "GTP", + "chemical" + ], + [ + 70, + 76, + "Closed", + "protein_state" + ], + [ + 83, + 85, + "WT", + "protein_state" + ], + [ + 86, + 89, + "Glu", + "chemical" + ], + [ + 91, + 94, + "GTP", + "chemical" + ], + [ + 96, + 101, + "NADPH", + "chemical" + ], + [ + 122, + 128, + "Closed", + "protein_state" + ], + [ + 135, + 137, + "WT", + "protein_state" + ], + [ + 138, + 141, + "Glu", + "chemical" + ], + [ + 143, + 148, + "NADPH", + "chemical" + ], + [ + 150, + 153, + "GTP", + "chemical" + ], + [ + 168, + 174, + "Closed", + "protein_state" + ], + [ + 181, + 183, + "WT", + "protein_state" + ], + [ + 184, + 187, + "apo", + "protein_state" + ], + [ + 193, + 197, + "Open", + "protein_state" + ], + [ + 204, + 206, + "WT", + "protein_state" + ], + [ + 207, + 212, + "NADPH", + "chemical" + ], + [ + 214, + 223, + "glutamate", + "chemical" + ], + [ + 229, + 232, + "GTP", + "chemical" + ], + [ + 238, + 244, + "Closed", + "protein_state" + ], + [ + 251, + 253, + "WT", + "protein_state" + ], + [ + 254, + 259, + "NADPH", + "chemical" + ], + [ + 262, + 265, + "GLU", + "chemical" + ], + [ + 268, + 271, + "GTP", + "chemical" + ], + [ + 274, + 278, + "Zinc", + "chemical" + ], + [ + 284, + 290, + "Closed", + "protein_state" + ], + [ + 298, + 300, + "WT", + "protein_state" + ], + [ + 301, + 306, + "NADPH", + "chemical" + ], + [ + 308, + 311, + "Glu", + "chemical" + ], + [ + 313, + 316, + "GTP", + "chemical" + ], + [ + 339, + 345, + "Closed", + "protein_state" + ], + [ + 350, + 352, + "WT", + "protein_state" + ], + [ + 353, + 356, + "NAD", + "chemical" + ], + [ + 358, + 361, + "PO4", + "chemical" + ], + [ + 367, + 381, + "2-oxoglutarate", + "chemical" + ], + [ + 387, + 393, + "Closed", + "protein_state" + ], + [ + 400, + 402, + "WT", + "protein_state" + ], + [ + 403, + 408, + "NADPH", + "chemical" + ], + [ + 411, + 414, + "GLU", + "chemical" + ], + [ + 425, + 431, + "Closed", + "protein_state" + ], + [ + 445, + 451, + "mutant", + "protein_state" + ], + [ + 452, + 455, + "apo", + "protein_state" + ], + [ + 461, + 465, + "Open", + "protein_state" + ], + [ + 472, + 474, + "WT", + "protein_state" + ], + [ + 475, + 478, + "apo", + "protein_state" + ], + [ + 484, + 488, + "Open", + "protein_state" + ], + [ + 495, + 497, + "WT", + "protein_state" + ], + [ + 498, + 501, + "ADP", + "chemical" + ], + [ + 507, + 511, + "Open", + "protein_state" + ], + [ + 518, + 520, + "WT", + "protein_state" + ], + [ + 521, + 526, + "NADPH", + "chemical" + ], + [ + 564, + 568, + "Open", + "protein_state" + ] + ] + }, + { + "sid": 42, + "sent": "Cryo-EM structures of mammalian GDH determined for this study", + "section": "TABLE", + "ner": [ + [ + 0, + 7, + "Cryo-EM", + "experimental_method" + ], + [ + 8, + 18, + "structures", + "evidence" + ], + [ + 22, + 31, + "mammalian", + "taxonomy_domain" + ], + [ + 32, + 35, + "GDH", + "protein_type" + ] + ] + }, + { + "sid": 43, + "sent": "GDH\tLigands\tEMDB ID\tPDB ID\tConformation\tResolution\tParticles\t \tWT\tapo\tEMD-6630\t3JCZ\tOpen\t3.26\t22462\t \tWT\tGTP\tEMD-6631\t3JD0\tOpen\t3.47\t39439\t \tWT\tNADH\tEMD-6635\t3JD2\tOpen\t3.27\t34716\t \tWT\tNADH\tEMD-6634\t3JD1\tClosed\t3.27\t34926\t \tWT\tNADH + GTP\tEMD-6632\t3JD3\tOpen\t3.55\t14793\t \tWT\tNADH + GTP\tEMD-6633\t3JD4\tClosed\t3.40\t20429\t \t", + "section": "TABLE", + "ner": [ + [ + 0, + 3, + "GDH", + "protein_type" + ], + [ + 63, + 65, + "WT", + "protein_state" + ], + [ + 66, + 69, + "apo", + "protein_state" + ], + [ + 84, + 88, + "Open", + "protein_state" + ], + [ + 102, + 104, + "WT", + "protein_state" + ], + [ + 105, + 108, + "GTP", + "chemical" + ], + [ + 123, + 127, + "Open", + "protein_state" + ], + [ + 141, + 143, + "WT", + "protein_state" + ], + [ + 144, + 148, + "NADH", + "chemical" + ], + [ + 163, + 167, + "Open", + "protein_state" + ], + [ + 181, + 183, + "WT", + "protein_state" + ], + [ + 184, + 188, + "NADH", + "chemical" + ], + [ + 203, + 209, + "Closed", + "protein_state" + ], + [ + 223, + 225, + "WT", + "protein_state" + ], + [ + 226, + 230, + "NADH", + "chemical" + ], + [ + 233, + 236, + "GTP", + "chemical" + ], + [ + 251, + 255, + "Open", + "protein_state" + ], + [ + 269, + 271, + "WT", + "protein_state" + ], + [ + 272, + 276, + "NADH", + "chemical" + ], + [ + 279, + 282, + "GTP", + "chemical" + ], + [ + 297, + 303, + "Closed", + "protein_state" + ] + ] + }, + { + "sid": 44, + "sent": "To explore the conformational landscape of apo-GDH, we first determined its structure in the absence of any added ligands (Supplemental Fig. 1, Fig. 2, A\u2013C).", + "section": "RESULTS", + "ner": [ + [ + 43, + 46, + "apo", + "protein_state" + ], + [ + 47, + 50, + "GDH", + "protein" + ], + [ + 76, + 85, + "structure", + "evidence" + ], + [ + 93, + 103, + "absence of", + "protein_state" + ] + ] + }, + { + "sid": 45, + "sent": "The density map, refined to an average resolution of \u223c3.0 \u00c5 (Supplemental Fig. 2), is in the open conformation and closely matches the model of unliganded GDH derived by X-ray crystallography at 3.3 \u00c5 resolution (PDB ID 1NR7).", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "density map", + "evidence" + ], + [ + 93, + 97, + "open", + "protein_state" + ], + [ + 144, + 154, + "unliganded", + "protein_state" + ], + [ + 155, + 158, + "GDH", + "protein" + ], + [ + 170, + 191, + "X-ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 46, + "sent": "The variation in local resolution from the core to the periphery, as reported by ResMap (Supplemental Fig. 3D), is consistent with the B-factor gradient observed in the crystal structure (Supplemental Fig. 3A).", + "section": "RESULTS", + "ner": [ + [ + 81, + 87, + "ResMap", + "experimental_method" + ], + [ + 135, + 152, + "B-factor gradient", + "evidence" + ], + [ + 169, + 186, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 47, + "sent": "Extensive classification without imposing symmetry yielded only open structures and failed to detect any closed catalytic cleft in the unliganded enzyme, suggesting that all six protomers are in the open conformation.", + "section": "RESULTS", + "ner": [ + [ + 64, + 68, + "open", + "protein_state" + ], + [ + 69, + 79, + "structures", + "evidence" + ], + [ + 105, + 111, + "closed", + "protein_state" + ], + [ + 112, + 127, + "catalytic cleft", + "site" + ], + [ + 135, + 145, + "unliganded", + "protein_state" + ], + [ + 178, + 187, + "protomers", + "oligomeric_state" + ], + [ + 199, + 203, + "open", + "protein_state" + ] + ] + }, + { + "sid": 48, + "sent": "Consistent with this conclusion, the loops connecting the \u03b2-strands of the Rossmann fold are well-defined (Fig. 2B), implying that there is little movement at the NBD, as the transition between closed and open states is associated with NBD movement (Fig. 1B).", + "section": "RESULTS", + "ner": [ + [ + 37, + 42, + "loops", + "structure_element" + ], + [ + 58, + 67, + "\u03b2-strands", + "structure_element" + ], + [ + 75, + 88, + "Rossmann fold", + "structure_element" + ], + [ + 163, + 166, + "NBD", + "structure_element" + ], + [ + 194, + 200, + "closed", + "protein_state" + ], + [ + 205, + 209, + "open", + "protein_state" + ], + [ + 236, + 239, + "NBD", + "structure_element" + ] + ] + }, + { + "sid": 49, + "sent": "Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at \u223c3 \u00c5 resolution.", + "section": "FIG", + "ner": [ + [ + 0, + 7, + "Cryo-EM", + "experimental_method" + ], + [ + 8, + 18, + "structures", + "evidence" + ], + [ + 22, + 25, + "GDH", + "protein" + ], + [ + 29, + 39, + "unliganded", + "protein_state" + ], + [ + 44, + 54, + "NADH-bound", + "protein_state" + ], + [ + 75, + 82, + "cryo-EM", + "experimental_method" + ], + [ + 83, + 86, + "map", + "evidence" + ], + [ + 90, + 100, + "unliganded", + "protein_state" + ], + [ + 101, + 104, + "GDH", + "protein" + ] + ] + }, + { + "sid": 50, + "sent": "(B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH.", + "section": "FIG", + "ner": [ + [ + 23, + 34, + "density map", + "evidence" + ], + [ + 67, + 99, + "Rossmann nucleotide binding fold", + "structure_element" + ], + [ + 105, + 130, + "pivot and antenna helices", + "structure_element" + ], + [ + 142, + 152, + "unliganded", + "protein_state" + ], + [ + 153, + 156, + "GDH", + "protein" + ], + [ + 157, + 160, + "map", + "evidence" + ], + [ + 166, + 173, + "Cryo-EM", + "experimental_method" + ], + [ + 182, + 194, + "density maps", + "evidence" + ], + [ + 250, + 253, + "GDH", + "protein" + ], + [ + 257, + 265, + "bound to", + "protein_state" + ], + [ + 279, + 283, + "NADH", + "chemical" + ] + ] + }, + { + "sid": 51, + "sent": "Each protomer is shown in a different color and densities for NADH bound in both regulatory (red) and catalytic (purple) sites on one protomer are indicated.", + "section": "FIG", + "ner": [ + [ + 5, + 13, + "protomer", + "oligomeric_state" + ], + [ + 48, + 57, + "densities", + "evidence" + ], + [ + 62, + 66, + "NADH", + "chemical" + ], + [ + 67, + 75, + "bound in", + "protein_state" + ], + [ + 81, + 91, + "regulatory", + "site" + ], + [ + 102, + 111, + "catalytic", + "site" + ], + [ + 121, + 126, + "sites", + "site" + ], + [ + 134, + 142, + "protomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 52, + "sent": "The overall quaternary structures of the two conformations are essentially the same as that of the open and closed states observed by X-ray crystallography.", + "section": "FIG", + "ner": [ + [ + 99, + 103, + "open", + "protein_state" + ], + [ + 108, + 114, + "closed", + "protein_state" + ], + [ + 134, + 155, + "X-ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 53, + "sent": "When GDH is bound to NADH, GTP, and glutamate, the enzyme adopts a closed conformation; this \u201cabortive complex\u201d has been determined to 2.4-\u00c5 resolution by X-ray crystallography (PDB 3MW9).", + "section": "RESULTS", + "ner": [ + [ + 5, + 8, + "GDH", + "protein" + ], + [ + 12, + 20, + "bound to", + "protein_state" + ], + [ + 21, + 25, + "NADH", + "chemical" + ], + [ + 27, + 30, + "GTP", + "chemical" + ], + [ + 36, + 45, + "glutamate", + "chemical" + ], + [ + 67, + 73, + "closed", + "protein_state" + ], + [ + 155, + 176, + "X-ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 54, + "sent": "However, crystal structures of GDH bound only to NADH or to GTP have not yet been reported.", + "section": "RESULTS", + "ner": [ + [ + 9, + 27, + "crystal structures", + "evidence" + ], + [ + 31, + 34, + "GDH", + "protein" + ], + [ + 35, + 48, + "bound only to", + "protein_state" + ], + [ + 49, + 53, + "NADH", + "chemical" + ], + [ + 57, + 59, + "to", + "protein_state" + ], + [ + 60, + 63, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 55, + "sent": "To test the effect of NADH binding on GDH conformation in solution, we determined the structure of this binary complex using cryo-EM methods combined with three-dimensional classification.", + "section": "RESULTS", + "ner": [ + [ + 22, + 26, + "NADH", + "chemical" + ], + [ + 38, + 41, + "GDH", + "protein" + ], + [ + 86, + 95, + "structure", + "evidence" + ], + [ + 125, + 132, + "cryo-EM", + "experimental_method" + ], + [ + 155, + 187, + "three-dimensional classification", + "experimental_method" + ] + ] + }, + { + "sid": 56, + "sent": "Two dominant conformational states, in an all open or all closed conformation were detected, segregated (Fig. 2D), and further refined to near-atomic resolution (\u223c3.3 \u00c5; Supplemental Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 46, + 50, + "open", + "protein_state" + ], + [ + 58, + 64, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 57, + "sent": "Densities for 12 molecules of bound NADH were identified in maps of both open and closed states (Supplemental Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Densities", + "evidence" + ], + [ + 30, + 35, + "bound", + "protein_state" + ], + [ + 36, + 40, + "NADH", + "chemical" + ], + [ + 60, + 64, + "maps", + "evidence" + ], + [ + 73, + 77, + "open", + "protein_state" + ], + [ + 82, + 88, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 58, + "sent": "The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "NADH-bound", + "protein_state" + ], + [ + 15, + 21, + "closed", + "protein_state" + ], + [ + 47, + 56, + "structure", + "evidence" + ], + [ + 95, + 116, + "X-ray crystallography", + "experimental_method" + ], + [ + 142, + 149, + "density", + "evidence" + ], + [ + 167, + 170, + "GTP", + "chemical" + ], + [ + 175, + 184, + "glutamate", + "chemical" + ], + [ + 203, + 210, + "cryo-EM", + "experimental_method" + ], + [ + 219, + 222, + "map", + "evidence" + ] + ] + }, + { + "sid": 59, + "sent": "Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by \u223c7\u00b0, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D).", + "section": "RESULTS", + "ner": [ + [ + 18, + 28, + "NADH-bound", + "protein_state" + ], + [ + 29, + 35, + "closed", + "protein_state" + ], + [ + 56, + 66, + "NADH-bound", + "protein_state" + ], + [ + 67, + 71, + "open", + "protein_state" + ], + [ + 114, + 129, + "catalytic cleft", + "site" + ], + [ + 133, + 139, + "closed", + "protein_state" + ], + [ + 148, + 152, + "NBDs", + "structure_element" + ], + [ + 229, + 240, + "pivot helix", + "structure_element" + ], + [ + 303, + 311, + "antennae", + "structure_element" + ] + ] + }, + { + "sid": 60, + "sent": "A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171\u2013186), as well as a tilt of the core \u03b2-sheets relative to the equatorial plane of the enzyme (residues 57\u201397, 122\u2013130) and \u03b1-helix 2 (residues 36\u201354), and a bending of the N-terminal helix.", + "section": "RESULTS", + "ner": [ + [ + 21, + 31, + "NADH-bound", + "protein_state" + ], + [ + 32, + 36, + "open", + "protein_state" + ], + [ + 41, + 47, + "closed", + "protein_state" + ], + [ + 94, + 101, + "helix 5", + "structure_element" + ], + [ + 112, + 119, + "171\u2013186", + "residue_range" + ], + [ + 152, + 160, + "\u03b2-sheets", + "structure_element" + ], + [ + 218, + 223, + "57\u201397", + "residue_range" + ], + [ + 225, + 232, + "122\u2013130", + "residue_range" + ], + [ + 238, + 247, + "\u03b1-helix 2", + "structure_element" + ], + [ + 258, + 263, + "36\u201354", + "residue_range" + ], + [ + 298, + 303, + "helix", + "structure_element" + ] + ] + }, + { + "sid": 61, + "sent": "Thus, closure of the catalytic cleft is accompanied by a quaternary structural change that can be described as a global bending of the structure about an axis that runs parallel to the pivot helix, accompanied by an expansion of the core (Figs. 1A and 2D).", + "section": "RESULTS", + "ner": [ + [ + 21, + 36, + "catalytic cleft", + "site" + ], + [ + 135, + 144, + "structure", + "evidence" + ], + [ + 185, + 196, + "pivot helix", + "structure_element" + ] + ] + }, + { + "sid": 62, + "sent": "Detailed analysis of the GDH/NADH structures shows that both the adenosine and nicotinamide moieties of NADH bind to the catalytic site within the NBD in nearly the same orientation in both the open and the closed states, and display closely comparable interactions with the Rossmann fold (Fig. 3, A and B).", + "section": "RESULTS", + "ner": [ + [ + 25, + 33, + "GDH/NADH", + "complex_assembly" + ], + [ + 34, + 44, + "structures", + "evidence" + ], + [ + 104, + 108, + "NADH", + "chemical" + ], + [ + 121, + 135, + "catalytic site", + "site" + ], + [ + 147, + 150, + "NBD", + "structure_element" + ], + [ + 194, + 198, + "open", + "protein_state" + ], + [ + 207, + 213, + "closed", + "protein_state" + ], + [ + 275, + 288, + "Rossmann fold", + "structure_element" + ] + ] + }, + { + "sid": 63, + "sent": "At the regulatory site, where either ADP can bind as an activator or NADH can bind as an inhibitor, the binding of the adenine moiety of NADH is nearly identical between the two conformers.", + "section": "RESULTS", + "ner": [ + [ + 7, + 22, + "regulatory site", + "site" + ], + [ + 37, + 40, + "ADP", + "chemical" + ], + [ + 69, + 73, + "NADH", + "chemical" + ], + [ + 137, + 141, + "NADH", + "chemical" + ] + ] + }, + { + "sid": 64, + "sent": "In the closed state, the nicotinamide group is oriented toward the center of the hexamer, inserted into a narrow cavity between two adjacent subunits in the trimer.", + "section": "RESULTS", + "ner": [ + [ + 7, + 13, + "closed", + "protein_state" + ], + [ + 81, + 88, + "hexamer", + "oligomeric_state" + ], + [ + 113, + 119, + "cavity", + "site" + ], + [ + 141, + 149, + "subunits", + "structure_element" + ], + [ + 157, + 163, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 65, + "sent": "There are extensive interactions between NADH and the residues lining this cavity, which may explain the well-defined density of this portion of NADH in the closed state.", + "section": "RESULTS", + "ner": [ + [ + 41, + 45, + "NADH", + "chemical" + ], + [ + 75, + 81, + "cavity", + "site" + ], + [ + 118, + 125, + "density", + "evidence" + ], + [ + 145, + 149, + "NADH", + "chemical" + ], + [ + 157, + 163, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 66, + "sent": "In contrast, in the open conformation, the cavity present in the closed state becomes too narrow for the nicotinamide group; instead, the group is oriented in the opposite direction, parallel to the pivot helix with the amido group extending toward the C-terminal end of the helix.", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "open", + "protein_state" + ], + [ + 43, + 49, + "cavity", + "site" + ], + [ + 65, + 71, + "closed", + "protein_state" + ], + [ + 199, + 210, + "pivot helix", + "structure_element" + ], + [ + 275, + 280, + "helix", + "structure_element" + ] + ] + }, + { + "sid": 67, + "sent": "Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states.", + "section": "FIG", + "ner": [ + [ + 17, + 21, + "NADH", + "chemical" + ], + [ + 38, + 68, + "catalytic and regulatory sites", + "site" + ], + [ + 77, + 81, + "NADH", + "chemical" + ], + [ + 82, + 89, + "density", + "evidence" + ], + [ + 123, + 138, + "catalytic sites", + "site" + ], + [ + 142, + 148, + "closed", + "protein_state" + ], + [ + 157, + 161, + "open", + "protein_state" + ], + [ + 181, + 185, + "NADH", + "chemical" + ], + [ + 186, + 193, + "density", + "evidence" + ], + [ + 224, + 240, + "regulatory sites", + "site" + ], + [ + 244, + 250, + "closed", + "protein_state" + ], + [ + 259, + 263, + "open", + "protein_state" + ] + ] + }, + { + "sid": 68, + "sent": "Although there is a difference in orientation of the nicotinamide moiety between the closed and open states in the regulatory site, in both structures the adenine portion of NADH has a similar binding pocket and is located in almost exactly the same position as ADP, a potent activator of GDH function (Supplemental Fig. 5).", + "section": "RESULTS", + "ner": [ + [ + 85, + 91, + "closed", + "protein_state" + ], + [ + 96, + 100, + "open", + "protein_state" + ], + [ + 115, + 130, + "regulatory site", + "site" + ], + [ + 140, + 150, + "structures", + "evidence" + ], + [ + 174, + 178, + "NADH", + "chemical" + ], + [ + 193, + 207, + "binding pocket", + "site" + ], + [ + 262, + 265, + "ADP", + "chemical" + ], + [ + 289, + 292, + "GDH", + "protein" + ] + ] + }, + { + "sid": 69, + "sent": "In the open state, the binding of ADP or NADH is further stabilized by His209, a residue that undergoes a large movement during the transition from open to closed conformation (Fig. 3, C and D).", + "section": "RESULTS", + "ner": [ + [ + 7, + 11, + "open", + "protein_state" + ], + [ + 34, + 37, + "ADP", + "chemical" + ], + [ + 41, + 45, + "NADH", + "chemical" + ], + [ + 71, + 77, + "His209", + "residue_name_number" + ], + [ + 148, + 152, + "open", + "protein_state" + ], + [ + 156, + 162, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 70, + "sent": "In the open conformation, the distance between His209 and the \u03b1-phosphate of NADH is \u223c4.4 \u00c5, which is comparable with the corresponding distance in the ADP-bound conformation.", + "section": "RESULTS", + "ner": [ + [ + 7, + 11, + "open", + "protein_state" + ], + [ + 47, + 53, + "His209", + "residue_name_number" + ], + [ + 77, + 81, + "NADH", + "chemical" + ], + [ + 152, + 161, + "ADP-bound", + "protein_state" + ] + ] + }, + { + "sid": 71, + "sent": "In the closed conformation, however, this key histidine residue is >10.5 \u00c5 away from the nearest phosphate group on NADH, altering a critical stabilization point within the regulatory site.", + "section": "RESULTS", + "ner": [ + [ + 7, + 13, + "closed", + "protein_state" + ], + [ + 46, + 55, + "histidine", + "residue_name" + ], + [ + 116, + 120, + "NADH", + "chemical" + ], + [ + 173, + 188, + "regulatory site", + "site" + ] + ] + }, + { + "sid": 72, + "sent": "This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity.", + "section": "RESULTS", + "ner": [ + [ + 48, + 52, + "NADH", + "chemical" + ], + [ + 60, + 64, + "open", + "protein_state" + ], + [ + 71, + 86, + "regulatory site", + "site" + ], + [ + 122, + 125, + "ADP", + "chemical" + ], + [ + 147, + 151, + "NADH", + "chemical" + ], + [ + 159, + 165, + "closed", + "protein_state" + ], + [ + 172, + 187, + "regulatory site", + "site" + ], + [ + 276, + 280, + "NADH", + "chemical" + ], + [ + 285, + 288, + "ADP", + "chemical" + ], + [ + 292, + 295, + "GDH", + "protein" + ] + ] + }, + { + "sid": 73, + "sent": "In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of \u223c20 \u03bcM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 \u00c5, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state.", + "section": "RESULTS", + "ner": [ + [ + 7, + 17, + "absence of", + "protein_state" + ], + [ + 18, + 22, + "NADH", + "chemical" + ], + [ + 24, + 27, + "GTP", + "chemical" + ], + [ + 44, + 47, + "GDH", + "protein" + ], + [ + 55, + 76, + "dissociation constant", + "evidence" + ], + [ + 88, + 95, + "Cryo-EM", + "experimental_method" + ], + [ + 108, + 111, + "GDH", + "protein" + ], + [ + 112, + 126, + "incubated with", + "protein_state" + ], + [ + 127, + 130, + "GTP", + "chemical" + ], + [ + 145, + 154, + "structure", + "evidence" + ], + [ + 215, + 219, + "open", + "protein_state" + ], + [ + 265, + 269, + "NBDs", + "structure_element" + ], + [ + 277, + 281, + "open", + "protein_state" + ] + ] + }, + { + "sid": 74, + "sent": "The density for GTP is not very well defined, suggesting considerable wobble in the binding site.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "density", + "evidence" + ], + [ + 16, + 19, + "GTP", + "chemical" + ], + [ + 84, + 96, + "binding site", + "site" + ] + ] + }, + { + "sid": 75, + "sent": "Subtraction of the GTP-bound map with that of the apo state shows that GTP binding can nevertheless be visualized specifically in the GTP binding site (Supplemental Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Subtraction", + "experimental_method" + ], + [ + 19, + 28, + "GTP-bound", + "protein_state" + ], + [ + 29, + 32, + "map", + "evidence" + ], + [ + 50, + 53, + "apo", + "protein_state" + ], + [ + 71, + 74, + "GTP", + "chemical" + ], + [ + 134, + 150, + "GTP binding site", + "site" + ] + ] + }, + { + "sid": 76, + "sent": "Importantly, the binding of GTP alone does not appear to drive the transition from the open to the closed state of GDH.", + "section": "RESULTS", + "ner": [ + [ + 28, + 31, + "GTP", + "chemical" + ], 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+ "chemical" + ] + ] + }, + { + "sid": 78, + "sent": "When NADH and GTP are both present, classification reveals the presence of both closed and open GDH conformations, similar to the condition when only NADH is present (Fig. 4, A and B).", + "section": "RESULTS", + "ner": [ + [ + 5, + 9, + "NADH", + "chemical" + ], + [ + 14, + 17, + "GTP", + "chemical" + ], + [ + 36, + 50, + "classification", + "experimental_method" + ], + [ + 63, + 74, + "presence of", + "protein_state" + ], + [ + 80, + 86, + "closed", + "protein_state" + ], + [ + 91, + 95, + "open", + "protein_state" + ], + [ + 96, + 99, + "GDH", + "protein" + ], + [ + 150, + 154, + "NADH", + "chemical" + ] + ] + }, + { + "sid": 79, + "sent": "Reconstruction without classification, however, yields a structure clearly in the closed conformation, suggesting that, in coordination with NADH, GTP may further stabilize the closed conformation.", + "section": "RESULTS", + "ner": [ + [ + 0, + 37, + "Reconstruction without classification", + "experimental_method" + ], + [ + 57, + 66, + "structure", + "evidence" + ], + [ + 82, + 88, + "closed", + "protein_state" + ], + [ + 141, + 145, + "NADH", + "chemical" + ], + [ + 147, + 150, + "GTP", + "chemical" + ], + [ + 177, + 183, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 80, + "sent": "The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate.", + "section": "RESULTS", + "ner": [ + [ + 16, + 19, + "GTP", + "chemical" + ], + [ + 27, + 31, + "open", + "protein_state" + ], + [ + 36, + 42, + "closed", + "protein_state" + ], + [ + 57, + 69, + "GDH/NADH/GTP", + "complex_assembly" + ], + [ + 104, + 121, + "crystal structure", + "evidence" + ], + [ + 138, + 149, + "presence of", + "protein_state" + ], + [ + 150, + 154, + "NADH", + "chemical" + ], + [ + 156, + 159, + "GTP", + "chemical" + ], + [ + 165, + 174, + "glutamate", + "chemical" + ] + ] + }, + { + "sid": 81, + "sent": "Likewise, the position of NADH in the open and closed states closely resembles the position of NADH in the GDH/NADH open and closed structures.", + "section": "RESULTS", + "ner": [ + [ + 26, + 30, + "NADH", + "chemical" + ], + [ + 38, + 42, + "open", + "protein_state" + ], + [ + 47, + 53, + "closed", + "protein_state" + ], + [ + 95, + 99, + "NADH", + "chemical" + ], + [ + 107, + 115, + "GDH/NADH", + "complex_assembly" + ], + [ + 116, + 120, + "open", + "protein_state" + ], + [ + 125, + 131, + "closed", + "protein_state" + ], + [ + 132, + 142, + "structures", + "evidence" + ] + ] + }, + { + "sid": 82, + "sent": "One key difference between the open and closed states of these structures is the position of the His209 residue: As mentioned above, His209 swings away from the adenine moiety of NADH in the closed state.", + "section": "RESULTS", + "ner": [ + [ + 31, + 35, + "open", + "protein_state" + ], + [ + 40, + 46, + "closed", + "protein_state" + ], + [ + 63, + 73, + "structures", + "evidence" + ], + [ + 97, + 103, + "His209", + "residue_name_number" + ], + [ + 133, + 139, + "His209", + "residue_name_number" + ], + [ + 179, + 183, + "NADH", + "chemical" + ], + [ + 191, + 197, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 83, + "sent": "When GTP is present in the GTP binding site, His209 instead interacts with GTP, probably stabilizing the closed conformation (Fig. 4, C and D).", + "section": "RESULTS", + "ner": [ + [ + 5, + 8, + "GTP", + "chemical" + ], + [ + 27, + 43, + "GTP binding site", + "site" + ], + [ + 45, + 51, + "His209", + "residue_name_number" + ], + [ + 75, + 78, + "GTP", + "chemical" + ], + [ + 105, + 111, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 84, + "sent": "Thus, GTP binding to GDH appears synergistic with NADH and displaces the conformational landscape toward the closed state.", + "section": "RESULTS", + "ner": [ + [ + 6, + 9, + "GTP", + "chemical" + ], + [ + 21, + 24, + "GDH", + "protein" + ], + [ + 50, + 54, + "NADH", + "chemical" + ], + [ + 109, + 115, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 85, + "sent": "Cryo-EM structure of GDH bound to both NADH and GTP.", + "section": "FIG", + "ner": [ + [ + 0, + 7, + "Cryo-EM", + "experimental_method" + ], + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 21, + 24, + "GDH", + "protein" + ], + [ + 25, + 33, + "bound to", + "protein_state" + ], + [ + 39, + 43, + "NADH", + "chemical" + ], + [ + 48, + 51, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 86, + "sent": "(A, B) Observation of co-existing open (A) and closed (B) conformations in the GDH-NADH-GTP ternary complex.", + "section": "FIG", + "ner": [ + [ + 34, + 38, + "open", + "protein_state" + ], + [ + 47, + 53, + "closed", + "protein_state" + ], + [ + 79, + 91, + "GDH-NADH-GTP", + "complex_assembly" + ] + ] + }, + { + "sid": 87, + "sent": "Densities for GTP (yellow) as well as NADH bound to both catalytic (purple) and regulatory (red) sites in each protomer are shown.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Densities", + "evidence" + ], + [ + 14, + 17, + "GTP", + "chemical" + ], + [ + 38, + 42, + "NADH", + "chemical" + ], + [ + 43, + 51, + "bound to", + "protein_state" + ], + [ + 57, + 66, + "catalytic", + "site" + ], + [ + 80, + 90, + "regulatory", + "site" + ], + [ + 97, + 102, + "sites", + "site" + ], + [ + 111, + 119, + "protomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 88, + "sent": "(C, D) Detailed inspection of the interactions near the regulatory site show that the orientation of His209 switches between the two states, which may allow interactions with bound GTP in the closed (D), but not open (C) conformation.", + "section": "FIG", + "ner": [ + [ + 56, + 71, + "regulatory site", + "site" + ], + [ + 101, + 107, + "His209", + "residue_name_number" + ], + [ + 175, + 180, + "bound", + "protein_state" + ], + [ + 181, + 184, + "GTP", + "chemical" + ], + [ + 192, + 198, + "closed", + "protein_state" + ], + [ + 212, + 216, + "open", + "protein_state" + ] + ] + }, + { + "sid": 89, + "sent": "Our structural studies thus establish that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states.", + "section": "RESULTS", + "ner": [ + [ + 4, + 22, + "structural studies", + "experimental_method" + ], + [ + 58, + 61, + "GTP", + "chemical" + ], + [ + 65, + 70, + "bound", + "protein_state" + ], + [ + 72, + 76, + "NADH", + "chemical" + ], + [ + 102, + 132, + "catalytic and regulatory sites", + "site" + ], + [ + 146, + 150, + "open", + "protein_state" + ], + [ + 155, + 161, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 90, + "sent": "Whereas the orientation in which NADH binds at the catalytic site is similar for both conformations, the orientation of the nicotinamide portion of NADH in the regulatory site is different between the open and closed conformations (Figs. 3 and 4).", + "section": "RESULTS", + "ner": [ + [ + 33, + 37, + "NADH", + "chemical" + ], + [ + 51, + 65, + "catalytic site", + "site" + ], + [ + 148, + 152, + "NADH", + "chemical" + ], + [ + 160, + 175, + "regulatory site", + "site" + ], + [ + 201, + 205, + "open", + "protein_state" + ], + [ + 210, + 216, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 91, + "sent": "In the closed state, the nicotinamide moiety is inserted into a well-defined cavity at the interface between two adjacent protomers in the trimer.", + "section": "RESULTS", + "ner": [ + [ + 7, + 13, + "closed", + "protein_state" + ], + [ + 77, + 83, + "cavity", + "site" + ], + [ + 91, + 100, + "interface", + "site" + ], + [ + 122, + 131, + "protomers", + "oligomeric_state" + ], + [ + 139, + 145, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 92, + "sent": "As mentioned above, this cavity is much narrower in the open state, suggesting that this cavity may be unavailable to the NADH nicotinamide moiety when the enzyme is in the open conformation.", + "section": "RESULTS", + "ner": [ + [ + 25, + 31, + "cavity", + "site" + ], + [ + 56, + 60, + "open", + "protein_state" + ], + [ + 89, + 95, + "cavity", + "site" + ], + [ + 122, + 126, + "NADH", + "chemical" + ], + [ + 173, + 177, + "open", + "protein_state" + ] + ] + }, + { + "sid": 93, + "sent": "These structural features provide a potential explanation of the weaker density for the nicotinamide moiety of NADH in the open state, and may account for the higher reported affinity of NADH for the closed state.", + "section": "RESULTS", + "ner": [ + [ + 72, + 79, + "density", + "evidence" + ], + [ + 111, + 115, + "NADH", + "chemical" + ], + [ + 123, + 127, + "open", + "protein_state" + ], + [ + 187, + 191, + "NADH", + "chemical" + ], + [ + 200, + 206, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 94, + "sent": "The role of the nicotinamide moiety in acting as a wedge that prevents the transition to the open conformation also suggests a structural explanation of the mechanism by which NADH binding inhibits the activity of the enzyme by stabilizing the closed conformation state.", + "section": "RESULTS", + "ner": [ + [ + 93, + 97, + "open", + "protein_state" + ], + [ + 176, + 180, + "NADH", + "chemical" + ], + [ + 244, + 250, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 95, + "sent": "The rapid emergence of cryo-EM as a tool for near-atomic resolution structure determination provides new opportunities for complementing atomic resolution information from X-ray crystallography, as illustrated here with GDH.", + "section": "RESULTS", + "ner": [ + [ + 23, + 30, + "cryo-EM", + "experimental_method" + ], + [ + 68, + 91, + "structure determination", + "experimental_method" + ], + [ + 172, + 193, + "X-ray crystallography", + "experimental_method" + ], + [ + 220, + 223, + "GDH", + "protein" + ] + ] + }, + { + "sid": 96, + "sent": "Perhaps the most important contribution of these methods is the prospect that when there are discrete subpopulations present, the structure of each state can be determined at near-atomic resolution. What we demonstrate here with GDH is that by employing three-dimensional image classification approaches, we not only can isolate distinct, coexisting conformations, but we can also localize small molecule ligands in each of these conformations.", + "section": "RESULTS", + "ner": [ + [ + 130, + 139, + "structure", + "evidence" + ], + [ + 229, + 232, + "GDH", + "protein" + ], + [ + 254, + 303, + "three-dimensional image classification approaches", + "experimental_method" + ] + ] + } + ] + }, + "PMC4919469": { + "annotations": [ + { + "sid": 0, + "sent": "Investigation of the Interaction between Cdc42 and Its Effector TOCA1", + "section": "TITLE", + "ner": [ + [ + 41, + 46, + "Cdc42", + "protein" + ], + [ + 64, + 69, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 1, + "sent": "Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 54, + "Transducer of Cdc42-dependent actin assembly protein 1", + "protein" + ], + [ + 56, + 61, + "TOCA1", + "protein" + ], + [ + 85, + 111, + "Rho family small G protein", + "protein_type" + ], + [ + 112, + 117, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 2, + "sent": "It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain.", + "section": "ABSTRACT", + "ner": [ + [ + 33, + 38, + "F-BAR", + "structure_element" + ], + [ + 59, + 73, + "Src homology 3", + "structure_element" + ], + [ + 75, + 78, + "SH3", + "structure_element" + ], + [ + 93, + 128, + "G protein-binding homology region 1", + "structure_element" + ], + [ + 130, + 133, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 3, + "sent": "TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 5, + "TOCA1", + "protein" + ], + [ + 17, + 22, + "Cdc42", + "protein" + ], + [ + 32, + 37, + "actin", + "protein_type" + ] + ] + }, + { + "sid": 4, + "sent": "We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein.", + "section": "ABSTRACT", + "ner": [ + [ + 8, + 14, + "solved", + "experimental_method" + ], + [ + 19, + 28, + "structure", + "evidence" + ], + [ + 36, + 39, + "HR1", + "structure_element" + ], + [ + 50, + 55, + "TOCA1", + "protein" + ], + [ + 77, + 92, + "structural data", + "evidence" + ] + ] + }, + { + "sid": 5, + "sent": "We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains.", + "section": "ABSTRACT", + "ner": [ + [ + 23, + 28, + "TOCA1", + "protein" + ], + [ + 29, + 32, + "HR1", + "structure_element" + ], + [ + 59, + 63, + "CIP4", + "protein" + ], + [ + 64, + 67, + "HR1", + "structure_element" + ], + [ + 136, + 139, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 6, + "sent": "We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP.", + "section": "ABSTRACT", + "ner": [ + [ + 45, + 49, + "TOCA", + "protein" + ], + [ + 50, + 53, + "HR1", + "structure_element" + ], + [ + 64, + 69, + "Cdc42", + "protein" + ], + [ + 112, + 117, + "Cdc42", + "protein" + ], + [ + 126, + 151, + "G protein-binding regions", + "site" + ], + [ + 155, + 160, + "TOCA1", + "protein" + ], + [ + 181, + 220, + "Wiskott-Aldrich syndrome protein family", + "protein_type" + ], + [ + 222, + 228, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 7, + "sent": "TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 5, + "TOCA1", + "protein" + ], + [ + 12, + 17, + "Cdc42", + "protein" + ], + [ + 89, + 95, + "N-WASP", + "protein" + ], + [ + 96, + 120, + "G protein-binding region", + "site" + ], + [ + 125, + 130, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 8, + "sent": "NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 3, + "NMR", + "experimental_method" + ], + [ + 30, + 50, + "Cdc42-binding domain", + "site" + ], + [ + 56, + 62, + "N-WASP", + "protein" + ], + [ + 83, + 88, + "TOCA1", + "protein" + ], + [ + 89, + 92, + "HR1", + "structure_element" + ], + [ + 98, + 103, + "Cdc42", + "protein" + ], + [ + 117, + 123, + "N-WASP", + "protein" + ], + [ + 143, + 148, + "TOCA1", + "protein" + ], + [ + 149, + 152, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 9, + "sent": "This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery.", + "section": "ABSTRACT", + "ner": [ + [ + 19, + 24, + "TOCA1", + "protein" + ], + [ + 36, + 41, + "Cdc42", + "protein" + ], + [ + 66, + 71, + "Cdc42", + "protein" + ], + [ + 140, + 158, + "binding affinities", + "evidence" + ], + [ + 203, + 208, + "TOCA1", + "protein" + ], + [ + 212, + 218, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 10, + "sent": "The Ras superfamily of small GTPases comprises over 150 members that regulate a multitude of cellular processes in eukaryotes.", + "section": "INTRO", + "ner": [ + [ + 4, + 19, + "Ras superfamily", + "protein_type" + ], + [ + 23, + 36, + "small GTPases", + "protein_type" + ], + [ + 115, + 125, + "eukaryotes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 11, + "sent": "The superfamily can be divided into five families based on structural and functional similarities: Ras, Rho, Rab, Arf, and Ran.", + "section": "INTRO", + "ner": [ + [ + 99, + 102, + "Ras", + "protein_type" + ], + [ + 104, + 107, + "Rho", + "protein_type" + ], + [ + 109, + 112, + "Rab", + "protein_type" + ], + [ + 114, + 117, + "Arf", + "protein_type" + ], + [ + 123, + 126, + "Ran", + "protein_type" + ] + ] + }, + { + "sid": 12, + "sent": "All members share a well defined core structure of \u223c20 kDa known as the G domain, which is responsible for guanine nucleotide binding.", + "section": "INTRO", + "ner": [ + [ + 72, + 80, + "G domain", + "structure_element" + ], + [ + 107, + 125, + "guanine nucleotide", + "chemical" + ] + ] + }, + { + "sid": 13, + "sent": "These molecular switches cycle between active, GTP-bound, and inactive, GDP-bound, states with the help of auxiliary proteins.", + "section": "INTRO", + "ner": [ + [ + 39, + 45, + "active", + "protein_state" + ], + [ + 47, + 56, + "GTP-bound", + "protein_state" + ], + [ + 62, + 70, + "inactive", + "protein_state" + ], + [ + 72, + 81, + "GDP-bound", + "protein_state" + ] + ] + }, + { + "sid": 14, + "sent": "The guanine nucleotide exchange factors mediate formation of the active state by promoting the dissociation of GDP, allowing GTP to bind.", + "section": "INTRO", + "ner": [ + [ + 4, + 39, + "guanine nucleotide exchange factors", + "protein_type" + ], + [ + 65, + 71, + "active", + "protein_state" + ], + [ + 111, + 114, + "GDP", + "chemical" + ], + [ + 125, + 128, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 15, + "sent": "The GTPase-activating proteins stimulate the rate of intrinsic GTP hydrolysis, mediating the return to the inactive state (reviewed in Ref.).", + "section": "INTRO", + "ner": [ + [ + 4, + 30, + "GTPase-activating proteins", + "protein_type" + ], + [ + 63, + 66, + "GTP", + "chemical" + ], + [ + 107, + 115, + "inactive", + "protein_state" + ] + ] + }, + { + "sid": 16, + "sent": "The overall conformation of small G proteins in the active and inactive states is similar, but they differ significantly in two main regions known as switch I and switch II.", + "section": "INTRO", + "ner": [ + [ + 28, + 44, + "small G proteins", + "protein_type" + ], + [ + 52, + 58, + "active", + "protein_state" + ], + [ + 63, + 71, + "inactive", + "protein_state" + ], + [ + 150, + 158, + "switch I", + "site" + ], + [ + 163, + 172, + "switch II", + "site" + ] + ] + }, + { + "sid": 17, + "sent": "These regions are responsible for \u201csensing\u201d the nucleotide state, with the GTP-bound state showing greater rigidity and the GDP-bound state adopting a more relaxed conformation (reviewed in Ref.).", + "section": "INTRO", + "ner": [ + [ + 75, + 84, + "GTP-bound", + "protein_state" + ], + [ + 124, + 133, + "GDP-bound", + "protein_state" + ] + ] + }, + { + "sid": 18, + "sent": "In the active state, G proteins bind to an array of downstream effectors, through which they exert their extensive roles within the cell.", + "section": "INTRO", + "ner": [ + [ + 7, + 13, + "active", + "protein_state" + ], + [ + 21, + 31, + "G proteins", + "protein_type" + ] + ] + }, + { + "sid": 19, + "sent": "The structures of more than 60 small G protein\u00b7effector complexes have been solved, and, not surprisingly, the switch regions have been implicated in a large proportion of the G protein-effector interactions (reviewed in Ref.).", + "section": "INTRO", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 37, + 46, + "G protein", + "protein_type" + ], + [ + 76, + 82, + "solved", + "experimental_method" + ], + [ + 111, + 125, + "switch regions", + "site" + ], + [ + 176, + 185, + "G protein", + "protein_type" + ] + ] + }, + { + "sid": 20, + "sent": "However, because each of the 150 members of the superfamily interacts with multiple effectors, there are still a huge number of known G protein-effector interactions that have not yet been studied structurally.", + "section": "INTRO", + "ner": [ + [ + 134, + 143, + "G protein", + "protein_type" + ] + ] + }, + { + "sid": 21, + "sent": "The Rho family comprises 20 members, of which three, RhoA, Rac1, and Cdc42, have been relatively well studied.", + "section": "INTRO", + "ner": [ + [ + 4, + 14, + "Rho family", + "protein_type" + ], + [ + 53, + 57, + "RhoA", + "protein" + ], + [ + 59, + 63, + "Rac1", + "protein" + ], + [ + 69, + 74, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 22, + "sent": "RhoA acts to rearrange existing actin structures to form stress fibers, whereas Rac1 and Cdc42 promote de novo actin polymerization to form lamellipodia and filopodia, respectively.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "RhoA", + "protein" + ], + [ + 80, + 84, + "Rac1", + "protein" + ], + [ + 89, + 94, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 23, + "sent": "A number of RhoA and Rac1 effector proteins, including the formins and members of the protein kinase C-related kinase (PRK)6 family, along with Cdc42 effectors, including the Wiskott-Aldrich syndrome (WASP) family and the transducer of Cdc42-dependent actin assembly (TOCA) family, have also been linked to the pathways that govern cytoskeletal dynamics.", + "section": "INTRO", + "ner": [ + [ + 12, + 16, + "RhoA", + "protein" + ], + [ + 21, + 25, + "Rac1", + "protein" + ], + [ + 86, + 117, + "protein kinase C-related kinase", + "protein_type" + ], + [ + 119, + 122, + "PRK", + "protein_type" + ], + [ + 123, + 124, + "6", + "protein_type" + ], + [ + 144, + 149, + "Cdc42", + "protein" + ], + [ + 175, + 199, + "Wiskott-Aldrich syndrome", + "protein_type" + ], + [ + 201, + 205, + "WASP", + "protein_type" + ], + [ + 236, + 266, + "Cdc42-dependent actin assembly", + "protein_type" + ], + [ + 268, + 272, + "TOCA", + "protein_type" + ] + ] + }, + { + "sid": 24, + "sent": "Cdc42 effectors, TOCA1 and the ubiquitously expressed member of the WASP family, N-WASP, have been implicated in the regulation of actin polymerization downstream of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2).", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "Cdc42", + "protein" + ], + [ + 17, + 22, + "TOCA1", + "protein" + ], + [ + 68, + 79, + "WASP family", + "protein_type" + ], + [ + 81, + 87, + "N-WASP", + "protein" + ], + [ + 166, + 171, + "Cdc42", + "protein" + ], + [ + 176, + 213, + "phosphatidylinositol 4,5-bisphosphate", + "chemical" + ], + [ + 215, + 224, + "PI(4,5)P2", + "chemical" + ] + ] + }, + { + "sid": 25, + "sent": "N-WASP exists in an autoinhibited conformation, which is released upon PI(4,5)P2 and Cdc42 binding or by other factors, such as phosphorylation.", + "section": "INTRO", + "ner": [ + [ + 0, + 6, + "N-WASP", + "protein" + ], + [ + 20, + 46, + "autoinhibited conformation", + "protein_state" + ], + [ + 71, + 80, + "PI(4,5)P2", + "chemical" + ], + [ + 85, + 90, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 26, + "sent": "Following their release, the C-terminal regions of N-WASP are free to interact with G-actin and a known nucleator of actin assembly, the Arp2/3 complex.", + "section": "INTRO", + "ner": [ + [ + 29, + 47, + "C-terminal regions", + "structure_element" + ], + [ + 51, + 57, + "N-WASP", + "protein" + ], + [ + 84, + 91, + "G-actin", + "protein_type" + ], + [ + 137, + 143, + "Arp2/3", + "complex_assembly" + ] + ] + }, + { + "sid": 27, + "sent": "The importance of TOCA1 in actin polymerization has been demonstrated in a range of in vitro and in vivo studies, but the exact role of TOCA1 in the many pathways involving actin assembly remains unclear.", + "section": "INTRO", + "ner": [ + [ + 18, + 23, + "TOCA1", + "protein" + ], + [ + 136, + 141, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 28, + "sent": "The most widely studied role of TOCA1 is in membrane invagination and endocytosis, although it has also been implicated in filopodia formation, neurite elongation, transcriptional reprogramming via nuclear actin, and interaction with ZO-1 at tight junctions.", + "section": "INTRO", + "ner": [ + [ + 32, + 37, + "TOCA1", + "protein" + ], + [ + 206, + 211, + "actin", + "protein_type" + ], + [ + 234, + 238, + "ZO-1", + "protein" + ] + ] + }, + { + "sid": 29, + "sent": "TOCA1 comprises an N-terminal F-BAR domain, a central homology region 1 (HR1) domain, and a C-terminal SH3 domain.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "TOCA1", + "protein" + ], + [ + 30, + 35, + "F-BAR", + "structure_element" + ], + [ + 46, + 71, + "central homology region 1", + "structure_element" + ], + [ + 73, + 76, + "HR1", + "structure_element" + ], + [ + 103, + 106, + "SH3", + "structure_element" + ] + ] + }, + { + "sid": 30, + "sent": "The F-BAR domain is a known dimerization, membrane-binding, and membrane-deforming module found in a number of cell signaling proteins.", + "section": "INTRO", + "ner": [ + [ + 4, + 9, + "F-BAR", + "structure_element" + ] + ] + }, + { + "sid": 31, + "sent": "The TOCA1 SH3 domain has many known binding partners, including N-WASP and dynamin.", + "section": "INTRO", + "ner": [ + [ + 4, + 9, + "TOCA1", + "protein" + ], + [ + 10, + 13, + "SH3", + "structure_element" + ], + [ + 64, + 70, + "N-WASP", + "protein" + ], + [ + 75, + 82, + "dynamin", + "protein" + ] + ] + }, + { + "sid": 32, + "sent": "The HR1 domain has been directly implicated in the interaction between TOCA1 and Cdc42, representing the first Cdc42-HR1 domain interaction to be identified.", + "section": "INTRO", + "ner": [ + [ + 4, + 7, + "HR1", + "structure_element" + ], + [ + 71, + 76, + "TOCA1", + "protein" + ], + [ + 81, + 86, + "Cdc42", + "protein" + ], + [ + 111, + 116, + "Cdc42", + "protein" + ], + [ + 117, + 120, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 33, + "sent": "Other HR1 domains studied so far, including those from the PRK family, have been found to bind their cognate Rho family G protein-binding partner with high specificity and affinities in the nanomolar range.", + "section": "INTRO", + "ner": [ + [ + 6, + 9, + "HR1", + "structure_element" + ], + [ + 59, + 69, + "PRK family", + "protein_type" + ], + [ + 120, + 129, + "G protein", + "protein_type" + ], + [ + 172, + 182, + "affinities", + "evidence" + ] + ] + }, + { + "sid": 34, + "sent": "The structures of the PRK1 HR1a domain in complex with RhoA and the HR1b domain in complex with Rac1 show that the HR1 domain comprises an anti-parallel coiled-coil that interacts with its G protein binding partner via both helices.", + "section": "INTRO", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 22, + 26, + "PRK1", + "protein" + ], + [ + 27, + 31, + "HR1a", + "structure_element" + ], + [ + 42, + 54, + "complex with", + "protein_state" + ], + [ + 55, + 59, + "RhoA", + "protein" + ], + [ + 68, + 72, + "HR1b", + "structure_element" + ], + [ + 83, + 95, + "complex with", + "protein_state" + ], + [ + 96, + 100, + "Rac1", + "protein" + ], + [ + 115, + 118, + "HR1", + "structure_element" + ], + [ + 139, + 164, + "anti-parallel coiled-coil", + "structure_element" + ], + [ + 189, + 198, + "G protein", + "protein_type" + ], + [ + 224, + 231, + "helices", + "structure_element" + ] + ] + }, + { + "sid": 35, + "sent": "Both of the G protein switch regions are involved in the interaction.", + "section": "INTRO", + "ner": [ + [ + 12, + 36, + "G protein switch regions", + "site" + ] + ] + }, + { + "sid": 36, + "sent": "The coiled-coil fold is shared by the HR1 domain of the TOCA family protein, CIP4, and, based on sequence homology, by TOCA1 itself.", + "section": "INTRO", + "ner": [ + [ + 4, + 20, + "coiled-coil fold", + "structure_element" + ], + [ + 38, + 41, + "HR1", + "structure_element" + ], + [ + 56, + 75, + "TOCA family protein", + "protein_type" + ], + [ + 77, + 81, + "CIP4", + "protein" + ], + [ + 119, + 124, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 37, + "sent": "These HR1 domains, however, show specificity for Cdc42, rather than RhoA or Rac1.", + "section": "INTRO", + "ner": [ + [ + 6, + 9, + "HR1", + "structure_element" + ], + [ + 49, + 54, + "Cdc42", + "protein" + ], + [ + 68, + 72, + "RhoA", + "protein" + ], + [ + 76, + 80, + "Rac1", + "protein" + ] + ] + }, + { + "sid": 38, + "sent": "How different HR1 domain proteins distinguish their specific G protein partners remains only partially understood, and structural characterization of a novel G protein-HR1 domain interaction would add to the growing body of information pertaining to these protein complexes.", + "section": "INTRO", + "ner": [ + [ + 14, + 17, + "HR1", + "structure_element" + ], + [ + 61, + 70, + "G protein", + "protein_type" + ], + [ + 158, + 167, + "G protein", + "protein_type" + ], + [ + 168, + 171, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 39, + "sent": "Furthermore, the biological function of the interaction between TOCA1 and Cdc42 remains poorly understood, and so far there has been no biophysical or structural insight.", + "section": "INTRO", + "ner": [ + [ + 64, + 69, + "TOCA1", + "protein" + ], + [ + 74, + 79, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 40, + "sent": "The interactions of TOCA1 and N-WASP with Cdc42 as well as with each other have raised questions as to whether the two Cdc42 effectors can interact with a single molecule of Cdc42 simultaneously.", + "section": "INTRO", + "ner": [ + [ + 20, + 25, + "TOCA1", + "protein" + ], + [ + 30, + 36, + "N-WASP", + "protein" + ], + [ + 42, + 47, + "Cdc42", + "protein" + ], + [ + 119, + 124, + "Cdc42", + "protein" + ], + [ + 174, + 179, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 41, + "sent": "There is some evidence for a ternary complex between Cdc42, N-WASP, and TOCA1, but there was no direct demonstration of simultaneous contacts between the two effectors and a single molecule of Cdc42.", + "section": "INTRO", + "ner": [ + [ + 53, + 58, + "Cdc42", + "protein" + ], + [ + 60, + 66, + "N-WASP", + "protein" + ], + [ + 72, + 77, + "TOCA1", + "protein" + ], + [ + 193, + 198, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 42, + "sent": "Nonetheless, the substantial difference between the structures of the G protein-binding regions of the two effectors is intriguing and implies that they bind to Cdc42 quite differently, providing motivation for investigating the possibility that Cdc42 can bind both effectors concurrently.", + "section": "INTRO", + "ner": [ + [ + 52, + 62, + "structures", + "evidence" + ], + [ + 70, + 95, + "G protein-binding regions", + "site" + ], + [ + 161, + 166, + "Cdc42", + "protein" + ], + [ + 246, + 251, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 43, + "sent": "WASP interacts with Cdc42 via a conserved, unstructured binding motif known as the Cdc42- and Rac-interactive binding region (CRIB), which forms an intermolecular \u03b2-sheet, expanding the anti-parallel \u03b22 and \u03b23 strands of Cdc42.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "WASP", + "protein_type" + ], + [ + 20, + 25, + "Cdc42", + "protein" + ], + [ + 32, + 41, + "conserved", + "protein_state" + ], + [ + 43, + 69, + "unstructured binding motif", + "structure_element" + ], + [ + 83, + 124, + "Cdc42- and Rac-interactive binding region", + "structure_element" + ], + [ + 126, + 130, + "CRIB", + "structure_element" + ], + [ + 148, + 170, + "intermolecular \u03b2-sheet", + "structure_element" + ], + [ + 200, + 217, + "\u03b22 and \u03b23 strands", + "structure_element" + ], + [ + 221, + 226, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 44, + "sent": "In contrast, the TOCA family proteins are thought to interact via the HR1 domain, which may form a triple coiled-coil with switch II of Rac1, like the HR1b domain of PRK1.", + "section": "INTRO", + "ner": [ + [ + 17, + 37, + "TOCA family proteins", + "protein_type" + ], + [ + 70, + 73, + "HR1", + "structure_element" + ], + [ + 99, + 117, + "triple coiled-coil", + "structure_element" + ], + [ + 123, + 132, + "switch II", + "site" + ], + [ + 136, + 140, + "Rac1", + "protein" + ], + [ + 151, + 155, + "HR1b", + "structure_element" + ], + [ + 166, + 170, + "PRK1", + "protein" + ] + ] + }, + { + "sid": 45, + "sent": "Here, we present the solution NMR structure of the HR1 domain of TOCA1, providing the first structural data for this protein.", + "section": "INTRO", + "ner": [ + [ + 21, + 33, + "solution NMR", + "experimental_method" + ], + [ + 34, + 43, + "structure", + "evidence" + ], + [ + 51, + 54, + "HR1", + "structure_element" + ], + [ + 65, + 70, + "TOCA1", + "protein" + ], + [ + 92, + 107, + "structural data", + "evidence" + ] + ] + }, + { + "sid": 46, + "sent": "We also present data pertaining to binding of the TOCA HR1 domain to Cdc42, which is the first biophysical description of an HR1 domain binding this particular Rho family small G protein.", + "section": "INTRO", + "ner": [ + [ + 50, + 54, + "TOCA", + "protein_type" + ], + [ + 55, + 58, + "HR1", + "structure_element" + ], + [ + 69, + 74, + "Cdc42", + "protein" + ], + [ + 125, + 128, + "HR1", + "structure_element" + ], + [ + 160, + 186, + "Rho family small G protein", + "protein_type" + ] + ] + }, + { + "sid": 47, + "sent": "Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics.", + "section": "INTRO", + "ner": [ + [ + 62, + 67, + "Cdc42", + "protein" + ], + [ + 76, + 101, + "G protein-binding regions", + "site" + ], + [ + 105, + 110, + "TOCA1", + "protein" + ], + [ + 115, + 121, + "N-WASP", + "protein" + ], + [ + 160, + 169, + "G protein", + "protein_type" + ], + [ + 216, + 221, + "Cdc42", + "protein" + ], + [ + 223, + 229, + "N-WASP", + "protein" + ], + [ + 235, + 240, + "TOCA1", + "protein" + ], + [ + 269, + 274, + "actin", + "protein_type" + ] + ] + }, + { + "sid": 48, + "sent": "Cdc42-TOCA1 Binding", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "Cdc42", + "protein" + ], + [ + 6, + 11, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 49, + "sent": "TOCA1 was identified in Xenopus extracts as a protein necessary for Cdc42-dependent actin assembly and was shown to bind to Cdc42\u00b7GTP\u03b3S but not to Cdc42\u00b7GDP or to Rac1 and RhoA. Given its homology to other Rho family binding modules, it is likely that the HR1 domain of TOCA1 is sufficient to bind Cdc42.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "TOCA1", + "protein" + ], + [ + 24, + 31, + "Xenopus", + "taxonomy_domain" + ], + [ + 68, + 73, + "Cdc42", + "protein" + ], + [ + 124, + 135, + "Cdc42\u00b7GTP\u03b3S", + "complex_assembly" + ], + [ + 147, + 156, + "Cdc42\u00b7GDP", + "complex_assembly" + ], + [ + 163, + 167, + "Rac1", + "protein" + ], + [ + 172, + 176, + "RhoA", + "protein" + ], + [ + 206, + 232, + "Rho family binding modules", + "site" + ], + [ + 256, + 259, + "HR1", + "structure_element" + ], + [ + 270, + 275, + "TOCA1", + "protein" + ], + [ + 298, + 303, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 50, + "sent": "The C. elegans TOCA1 orthologues also bind to Cdc42 via their consensus HR1 domain.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "C. elegans", + "species" + ], + [ + 15, + 20, + "TOCA1", + "protein" + ], + [ + 46, + 51, + "Cdc42", + "protein" + ], + [ + 72, + 75, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 51, + "sent": "The HR1 domains from the PRK family bind their G protein partners with a high affinity, exhibiting a range of submicromolar dissociation constants (Kd) as low as 26 nm.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "HR1", + "structure_element" + ], + [ + 25, + 35, + "PRK family", + "protein_type" + ], + [ + 47, + 56, + "G protein", + "protein_type" + ], + [ + 124, + 146, + "dissociation constants", + "evidence" + ], + [ + 148, + 150, + "Kd", + "evidence" + ] + ] + }, + { + "sid": 52, + "sent": "A Kd in the nanomolar range was therefore expected for the interaction of the TOCA1 HR1 domain with Cdc42.", + "section": "RESULTS", + "ner": [ + [ + 2, + 4, + "Kd", + "evidence" + ], + [ + 78, + 83, + "TOCA1", + "protein" + ], + [ + 84, + 87, + "HR1", + "structure_element" + ], + [ + 100, + 105, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 53, + "sent": "We generated an X. tropicalis TOCA1 HR1 domain construct encompassing residues 330\u2013426.", + "section": "RESULTS", + "ner": [ + [ + 16, + 29, + "X. tropicalis", + "species" + ], + [ + 30, + 35, + "TOCA1", + "protein" + ], + [ + 36, + 39, + "HR1", + "structure_element" + ], + [ + 79, + 86, + "330\u2013426", + "residue_range" + ] + ] + }, + { + "sid": 54, + "sent": "This region comprises the complete HR1 domain based on secondary structure predictions and sequence alignments with another TOCA family member, CIP4, whose structure has been determined.", + "section": "RESULTS", + "ner": [ + [ + 35, + 38, + "HR1", + "structure_element" + ], + [ + 91, + 110, + "sequence alignments", + "experimental_method" + ], + [ + 124, + 135, + "TOCA family", + "protein_type" + ], + [ + 144, + 148, + "CIP4", + "protein" + ], + [ + 156, + 165, + "structure", + "evidence" + ] + ] + }, + { + "sid": 55, + "sent": "The interaction between [3H]GTP\u00b7Cdc42 and a C-terminally His-tagged TOCA1 HR1 domain construct was investigated using SPA.", + "section": "RESULTS", + "ner": [ + [ + 24, + 37, + "[3H]GTP\u00b7Cdc42", + "complex_assembly" + ], + [ + 57, + 67, + "His-tagged", + "protein_state" + ], + [ + 68, + 73, + "TOCA1", + "protein" + ], + [ + 74, + 77, + "HR1", + "structure_element" + ], + [ + 118, + 121, + "SPA", + "experimental_method" + ] + ] + }, + { + "sid": 56, + "sent": "The binding isotherm for the interaction is shown in Fig. 1A, together with the Cdc42-PAK interaction as a positive control.", + "section": "RESULTS", + "ner": [ + [ + 4, + 20, + "binding isotherm", + "evidence" + ], + [ + 80, + 85, + "Cdc42", + "protein" + ], + [ + 86, + 89, + "PAK", + "protein" + ] + ] + }, + { + "sid": 57, + "sent": "The binding of TOCA1 HR1 to Cdc42 was unexpectedly weak, with a Kd of >1 \u03bcm.", + "section": "RESULTS", + "ner": [ + [ + 15, + 20, + "TOCA1", + "protein" + ], + [ + 21, + 24, + "HR1", + "structure_element" + ], + [ + 28, + 33, + "Cdc42", + "protein" + ], + [ + 64, + 66, + "Kd", + "evidence" + ] + ] + }, + { + "sid": 58, + "sent": "It was not possible to estimate the Kd more accurately using direct SPA experiments, because saturation could not be reached due to nonspecific signal at higher protein concentrations.", + "section": "RESULTS", + "ner": [ + [ + 36, + 38, + "Kd", + "evidence" + ], + [ + 68, + 71, + "SPA", + "experimental_method" + ] + ] + }, + { + "sid": 59, + "sent": "The TOCA1 HR1-Cdc42 interaction is low affinity.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "TOCA1", + "protein" + ], + [ + 10, + 13, + "HR1", + "structure_element" + ], + [ + 14, + 19, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 60, + "sent": " A, curves derived from direct binding assays in which the indicated concentrations of Cdc42\u03947Q61L\u00b7[3H]GTP were incubated with 30 nm GST-PAK or HR1-His6 in SPAs.", + "section": "FIG", + "ner": [ + [ + 24, + 45, + "direct binding assays", + "experimental_method" + ], + [ + 87, + 106, + "Cdc42\u03947Q61L\u00b7[3H]GTP", + "complex_assembly" + ], + [ + 112, + 121, + "incubated", + "experimental_method" + ], + [ + 133, + 140, + "GST-PAK", + "mutant" + ], + [ + 144, + 152, + "HR1-His6", + "mutant" + ], + [ + 156, + 160, + "SPAs", + "experimental_method" + ] + ] + }, + { + "sid": 61, + "sent": "The SPA signal was corrected by subtraction of control data with no GST-PAK or HR1-His6.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "SPA", + "experimental_method" + ], + [ + 68, + 75, + "GST-PAK", + "mutant" + ], + [ + 79, + 87, + "HR1-His6", + "mutant" + ] + ] + }, + { + "sid": 62, + "sent": "The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal; B and C, competition SPA experiments were carried out with the indicated concentrations of ACK GBD (B) or HR1 domain (C) titrated into 30 nm GST-ACK and either 30 nm Cdc42\u03947Q61L\u00b7[3H]GTP or full-length Cdc42Q61L\u00b7[3H]GTP.", + "section": "FIG", + "ner": [ + [ + 26, + 42, + "binding isotherm", + "evidence" + ], + [ + 63, + 65, + "Kd", + "evidence" + ], + [ + 132, + 147, + "competition SPA", + "experimental_method" + ], + [ + 214, + 217, + "ACK", + "protein" + ], + [ + 218, + 221, + "GBD", + "structure_element" + ], + [ + 229, + 232, + "HR1", + "structure_element" + ], + [ + 244, + 252, + "titrated", + "experimental_method" + ], + [ + 264, + 271, + "GST-ACK", + "mutant" + ], + [ + 289, + 308, + "Cdc42\u03947Q61L\u00b7[3H]GTP", + "complex_assembly" + ], + [ + 312, + 323, + "full-length", + "protein_state" + ], + [ + 324, + 341, + "Cdc42Q61L\u00b7[3H]GTP", + "complex_assembly" + ] + ] + }, + { + "sid": 63, + "sent": "The Kd values derived for the ACK GBD with Cdc42\u03947 and full-length Cdc42 were 0.032 \u00b1 0.01 and 0.011 \u00b1 0.01 \u03bcm, respectively.", + "section": "FIG", + "ner": [ + [ + 4, + 6, + "Kd", + "evidence" + ], + [ + 30, + 33, + "ACK", + "protein" + ], + [ + 34, + 37, + "GBD", + "structure_element" + ], + [ + 43, + 50, + "Cdc42\u03947", + "mutant" + ], + [ + 55, + 66, + "full-length", + "protein_state" + ], + [ + 67, + 72, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 64, + "sent": "The Kd values derived for the TOCA1 HR1 with Cdc42\u03947 and full-length Cdc42 were 6.05 \u00b1 1.96 and 5.39 \u00b1 1.69 \u03bcm, respectively.", + "section": "FIG", + "ner": [ + [ + 4, + 6, + "Kd", + "evidence" + ], + [ + 30, + 35, + "TOCA1", + "protein" + ], + [ + 36, + 39, + "HR1", + "structure_element" + ], + [ + 45, + 52, + "Cdc42\u03947", + "mutant" + ], + [ + 57, + 68, + "full-length", + "protein_state" + ], + [ + 69, + 74, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 65, + "sent": "It was possible that the low affinity observed was due to negative effects of immobilization of the HR1 domain, so other methods were employed, which utilized untagged proteins.", + "section": "RESULTS", + "ner": [ + [ + 100, + 103, + "HR1", + "structure_element" + ], + [ + 159, + 167, + "untagged", + "protein_state" + ] + ] + }, + { + "sid": 66, + "sent": "Isothermal titration calorimetry was carried out, but no heat changes were observed at a range of concentrations and temperatures (data not shown), suggesting that the interaction is predominantly entropically driven.", + "section": "RESULTS", + "ner": [ + [ + 0, + 32, + "Isothermal titration calorimetry", + "experimental_method" + ] + ] + }, + { + "sid": 67, + "sent": "Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.7 Infrared interferometry with immobilized Cdc42 was also attempted but was unsuccessful for both TOCA1 HR1 and for the positive control, ACK.", + "section": "RESULTS", + "ner": [ + [ + 6, + 15, + "G protein", + "protein_type" + ], + [ + 16, + 19, + "HR1", + "structure_element" + ], + [ + 93, + 116, + "Infrared interferometry", + "experimental_method" + ], + [ + 122, + 133, + "immobilized", + "protein_state" + ], + [ + 134, + 139, + "Cdc42", + "protein" + ], + [ + 189, + 194, + "TOCA1", + "protein" + ], + [ + 195, + 198, + "HR1", + "structure_element" + ], + [ + 229, + 232, + "ACK", + "protein" + ] + ] + }, + { + "sid": 68, + "sent": "The affinity was therefore determined using competition SPAs.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "affinity", + "evidence" + ], + [ + 44, + 60, + "competition SPAs", + "experimental_method" + ] + ] + }, + { + "sid": 69, + "sent": "A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42, with radiolabeled [3H]GTP\u00b7Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined.", + "section": "RESULTS", + "ner": [ + [ + 15, + 25, + "GST fusion", + "experimental_method" + ], + [ + 33, + 36, + "GBD", + "structure_element" + ], + [ + 40, + 43, + "ACK", + "protein" + ], + [ + 81, + 86, + "Cdc42", + "protein" + ], + [ + 106, + 119, + "[3H]GTP\u00b7Cdc42", + "complex_assembly" + ], + [ + 153, + 178, + "increasing concentrations", + "experimental_method" + ], + [ + 182, + 190, + "untagged", + "protein_state" + ], + [ + 191, + 196, + "TOCA1", + "protein" + ], + [ + 197, + 200, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 70, + "sent": "Competition of GST-ACK GBD bound to [3H]GTP\u00b7Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced.", + "section": "RESULTS", + "ner": [ + [ + 15, + 22, + "GST-ACK", + "mutant" + ], + [ + 23, + 26, + "GBD", + "structure_element" + ], + [ + 27, + 35, + "bound to", + "protein_state" + ], + [ + 36, + 49, + "[3H]GTP\u00b7Cdc42", + "complex_assembly" + ], + [ + 53, + 57, + "free", + "protein_state" + ], + [ + 58, + 61, + "ACK", + "protein" + ], + [ + 62, + 65, + "GBD", + "structure_element" + ], + [ + 141, + 146, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 71, + "sent": "The data were fitted to a binding isotherm describing competition.", + "section": "RESULTS", + "ner": [ + [ + 26, + 42, + "binding isotherm", + "evidence" + ] + ] + }, + { + "sid": 72, + "sent": "Free ACK competed with itself with an affinity of 32 nm, similar to the value obtained by direct binding of 23 nm.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "Free", + "protein_state" + ], + [ + 5, + 8, + "ACK", + "protein" + ], + [ + 38, + 46, + "affinity", + "evidence" + ], + [ + 90, + 104, + "direct binding", + "experimental_method" + ] + ] + }, + { + "sid": 73, + "sent": "The TOCA1 HR1 domain also fully competed with the GST-ACK but bound with an affinity of 6 \u03bcm (Fig. 1, B and C), in agreement with the low affinity observed in the direct binding experiments.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "TOCA1", + "protein" + ], + [ + 10, + 13, + "HR1", + "structure_element" + ], + [ + 50, + 57, + "GST-ACK", + "mutant" + ], + [ + 62, + 67, + "bound", + "protein_state" + ], + [ + 76, + 84, + "affinity", + "evidence" + ], + [ + 138, + 146, + "affinity", + "evidence" + ], + [ + 163, + 189, + "direct binding experiments", + "experimental_method" + ] + ] + }, + { + "sid": 74, + "sent": "The Cdc42 construct used in the binding assays has seven residues deleted from the C terminus to facilitate purification.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "Cdc42", + "protein" + ], + [ + 32, + 46, + "binding assays", + "experimental_method" + ], + [ + 51, + 65, + "seven residues", + "residue_range" + ], + [ + 66, + 73, + "deleted", + "experimental_method" + ] + ] + }, + { + "sid": 75, + "sent": "These residues are not generally required for G protein-effector interactions, including the interaction between RhoA and the PRK1 HR1a domain.", + "section": "RESULTS", + "ner": [ + [ + 46, + 55, + "G protein", + "protein_type" + ], + [ + 113, + 117, + "RhoA", + "protein" + ], + [ + 126, + 130, + "PRK1", + "protein" + ], + [ + 131, + 135, + "HR1a", + "structure_element" + ] + ] + }, + { + "sid": 76, + "sent": "In contrast, the C terminus of Rac1 contains a polybasic sequence, which is crucial for Rac1 binding to the HR1b domain from PRK1.", + "section": "RESULTS", + "ner": [ + [ + 31, + 35, + "Rac1", + "protein" + ], + [ + 88, + 92, + "Rac1", + "protein" + ], + [ + 108, + 112, + "HR1b", + "structure_element" + ], + [ + 125, + 129, + "PRK1", + "protein" + ] + ] + }, + { + "sid": 77, + "sent": "As the observed affinity between TOCA1 HR1 and Cdc42 was much lower than expected, we reasoned that the C terminus of Cdc42 might be necessary for a high affinity interaction.", + "section": "RESULTS", + "ner": [ + [ + 16, + 24, + "affinity", + "evidence" + ], + [ + 33, + 38, + "TOCA1", + "protein" + ], + [ + 39, + 42, + "HR1", + "structure_element" + ], + [ + 47, + 52, + "Cdc42", + "protein" + ], + [ + 118, + 123, + "Cdc42", + "protein" + ], + [ + 154, + 162, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 78, + "sent": "The binding experiments were repeated with full-length [3H]GTP\u00b7Cdc42, but the affinity of the HR1 domain for full-length Cdc42 was similar to its affinity for truncated Cdc42 (Kd \u2248 5 \u03bcm; Fig. 1C).", + "section": "RESULTS", + "ner": [ + [ + 4, + 23, + "binding experiments", + "experimental_method" + ], + [ + 43, + 54, + "full-length", + "protein_state" + ], + [ + 55, + 68, + "[3H]GTP\u00b7Cdc42", + "complex_assembly" + ], + [ + 78, + 86, + "affinity", + "evidence" + ], + [ + 94, + 97, + "HR1", + "structure_element" + ], + [ + 109, + 120, + "full-length", + "protein_state" + ], + [ + 121, + 126, + "Cdc42", + "protein" + ], + [ + 146, + 154, + "affinity", + "evidence" + ], + [ + 159, + 168, + "truncated", + "protein_state" + ], + [ + 169, + 174, + "Cdc42", + "protein" + ], + [ + 176, + 178, + "Kd", + "evidence" + ] + ] + }, + { + "sid": 79, + "sent": "Thus, the C-terminal region of Cdc42 is not required for maximal binding of TOCA1 HR1.", + "section": "RESULTS", + "ner": [ + [ + 10, + 27, + "C-terminal region", + "structure_element" + ], + [ + 31, + 36, + "Cdc42", + "protein" + ], + [ + 76, + 81, + "TOCA1", + "protein" + ], + [ + 82, + 85, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 80, + "sent": "Another possible explanation for the low affinities observed was that the HR1 domain alone is not sufficient for maximal binding of the TOCA proteins to Cdc42 and that the other domains are required.", + "section": "RESULTS", + "ner": [ + [ + 41, + 51, + "affinities", + "evidence" + ], + [ + 74, + 77, + "HR1", + "structure_element" + ], + [ + 85, + 90, + "alone", + "protein_state" + ], + [ + 136, + 149, + "TOCA proteins", + "protein_type" + ], + [ + 153, + 158, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 81, + "sent": "Indeed, GST pull-downs performed with in vitro translated human TOCA1 fragments had suggested that residues N-terminal to the HR1 domain may be required to stabilize the HR1 domain structure.", + "section": "RESULTS", + "ner": [ + [ + 8, + 22, + "GST pull-downs", + "experimental_method" + ], + [ + 58, + 63, + "human", + "species" + ], + [ + 64, + 69, + "TOCA1", + "protein" + ], + [ + 126, + 129, + "HR1", + "structure_element" + ], + [ + 170, + 173, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 82, + "sent": "Furthermore, both BAR and SH3 domains have been implicated in interactions with small G proteins (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1), while an SH3 domain mediates the interaction between Rac1 and the guanine nucleotide exchange factor, \u03b2-PIX.", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "BAR", + "structure_element" + ], + [ + 26, + 29, + "SH3", + "structure_element" + ], + [ + 86, + 96, + "G proteins", + "protein_type" + ], + [ + 107, + 110, + "BAR", + "structure_element" + ], + [ + 121, + 130, + "Arfaptin2", + "protein" + ], + [ + 140, + 144, + "Rac1", + "protein" + ], + [ + 149, + 153, + "Arl1", + "protein" + ], + [ + 165, + 168, + "SH3", + "structure_element" + ], + [ + 209, + 213, + "Rac1", + "protein" + ], + [ + 222, + 256, + "guanine nucleotide exchange factor", + "protein" + ], + [ + 258, + 263, + "\u03b2-PIX", + "protein" + ] + ] + }, + { + "sid": 83, + "sent": "TOCA1 dimerizes via its F-BAR domain, which could also affect Cdc42 binding, for example by presenting two HR1 domains for Cdc42 interactions.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "TOCA1", + "protein" + ], + [ + 6, + 11, + "dimer", + "oligomeric_state" + ], + [ + 24, + 29, + "F-BAR", + "structure_element" + ], + [ + 62, + 67, + "Cdc42", + "protein" + ], + [ + 107, + 110, + "HR1", + "structure_element" + ], + [ + 123, + 128, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 84, + "sent": "Various TOCA1 fragments (Fig. 2A) were therefore assessed for binding to full-length Cdc42 by direct SPA.", + "section": "RESULTS", + "ner": [ + [ + 8, + 13, + "TOCA1", + "protein" + ], + [ + 73, + 84, + "full-length", + "protein_state" + ], + [ + 85, + 90, + "Cdc42", + "protein" + ], + [ + 101, + 104, + "SPA", + "experimental_method" + ] + ] + }, + { + "sid": 85, + "sent": "The isolated F-BAR domain showed no binding to full-length Cdc42 (Fig. 2B).", + "section": "RESULTS", + "ner": [ + [ + 13, + 18, + "F-BAR", + "structure_element" + ], + [ + 47, + 58, + "full-length", + "protein_state" + ], + [ + 59, + 64, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 86, + "sent": "Full-length TOCA1 and \u0394SH3 TOCA1 bound with micromolar affinity (Fig. 2B), in a similar manner to the isolated HR1 domain (Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Full-length", + "protein_state" + ], + [ + 12, + 17, + "TOCA1", + "protein" + ], + [ + 22, + 26, + "\u0394SH3", + "mutant" + ], + [ + 27, + 32, + "TOCA1", + "protein" + ], + [ + 33, + 38, + "bound", + "protein_state" + ], + [ + 111, + 114, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 87, + "sent": "The HR1-SH3 protein could not be purified to homogeneity as a fusion protein, so it was assayed in competition assays after cleavage of the His tag.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "HR1-SH3", + "mutant" + ], + [ + 99, + 117, + "competition assays", + "experimental_method" + ] + ] + }, + { + "sid": 88, + "sent": "This construct competed with GST-ACK GBD to give a similar affinity to the HR1 domain alone (Kd = 4.6 \u00b1 4 \u03bcm; Fig. 2C).", + "section": "RESULTS", + "ner": [ + [ + 29, + 36, + "GST-ACK", + "mutant" + ], + [ + 37, + 40, + "GBD", + "structure_element" + ], + [ + 75, + 78, + "HR1", + "structure_element" + ], + [ + 86, + 91, + "alone", + "protein_state" + ], + [ + 93, + 95, + "Kd", + "evidence" + ] + ] + }, + { + "sid": 89, + "sent": "Taken together, these data suggest that the TOCA1 HR1 domain is sufficient for maximal binding and that this binding is of a relatively low affinity compared with many other Cdc42\u00b7effector complexes.", + "section": "RESULTS", + "ner": [ + [ + 44, + 49, + "TOCA1", + "protein" + ], + [ + 50, + 53, + "HR1", + "structure_element" + ], + [ + 174, + 179, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 90, + "sent": "The Cdc42-HR1 interaction is of low affinity in the context of full-length protein and in TOCA1 paralogues.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "Cdc42-HR1", + "complex_assembly" + ], + [ + 63, + 74, + "full-length", + "protein_state" + ], + [ + 90, + 95, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 91, + "sent": " A, diagram illustrating the TOCA1 constructs assayed for Cdc42 binding.", + "section": "FIG", + "ner": [ + [ + 29, + 34, + "TOCA1", + "protein" + ], + [ + 58, + 63, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 92, + "sent": "Domain boundaries are derived from secondary structure predictions; B, binding curves derived from direct binding assays, in which the indicated concentrations of Cdc42\u03947Q61L\u00b7[3H]GTP were incubated with 30 nm GST-ACK or His-tagged TOCA1 constructs, as indicated, in SPAs.", + "section": "FIG", + "ner": [ + [ + 71, + 85, + "binding curves", + "evidence" + ], + [ + 99, + 120, + "direct binding assays", + "experimental_method" + ], + [ + 163, + 182, + "Cdc42\u03947Q61L\u00b7[3H]GTP", + "complex_assembly" + ], + [ + 188, + 197, + "incubated", + "experimental_method" + ], + [ + 209, + 216, + "GST-ACK", + "mutant" + ], + [ + 220, + 230, + "His-tagged", + "protein_state" + ], + [ + 231, + 236, + "TOCA1", + "protein" + ], + [ + 266, + 270, + "SPAs", + "experimental_method" + ] + ] + }, + { + "sid": 93, + "sent": "The SPA signal was corrected by subtraction of control data with no fusion protein.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "SPA", + "experimental_method" + ] + ] + }, + { + "sid": 94, + "sent": "The data were fitted to a binding isotherm to give an apparent Kd and are expressed as a percentage of the maximum signal.", + "section": "FIG", + "ner": [ + [ + 26, + 42, + "binding isotherm", + "evidence" + ], + [ + 63, + 65, + "Kd", + "evidence" + ] + ] + }, + { + "sid": 95, + "sent": "C\u2013E, representative examples of competition SPA experiments carried out with the indicated concentrations of the TOCA1 HR1-SH3 construct titrated into 30 nm GST-ACK and 30 nm Cdc42\u03947Q61L\u00b7[3H]GTP (C) or HR1CIP4 (D) or HR1FBP17 (E) titrated into 30 nm GST-ACK and 30 nm Cdc42FLQ61L\u00b7[3H]GTP.", + "section": "FIG", + "ner": [ + [ + 32, + 47, + "competition SPA", + "experimental_method" + ], + [ + 113, + 118, + "TOCA1", + "protein" + ], + [ + 119, + 126, + "HR1-SH3", + "mutant" + ], + [ + 137, + 145, + "titrated", + "experimental_method" + ], + [ + 157, + 164, + "GST-ACK", + "mutant" + ], + [ + 175, + 194, + "Cdc42\u03947Q61L\u00b7[3H]GTP", + "complex_assembly" + ], + [ + 202, + 205, + "HR1", + "structure_element" + ], + [ + 217, + 220, + "HR1", + "structure_element" + ], + [ + 230, + 238, + "titrated", + "experimental_method" + ], + [ + 250, + 257, + "GST-ACK", + "mutant" + ], + [ + 268, + 287, + "Cdc42FLQ61L\u00b7[3H]GTP", + "complex_assembly" + ] + ] + }, + { + "sid": 96, + "sent": "The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17 and CIP4, also bind weakly.", + "section": "RESULTS", + "ner": [ + [ + 24, + 29, + "TOCA1", + "protein" + ], + [ + 30, + 33, + "HR1", + "structure_element" + ], + [ + 34, + 39, + "Cdc42", + "protein" + ], + [ + 99, + 104, + "Cdc42", + "protein" + ], + [ + 113, + 133, + "TOCA family proteins", + "protein_type" + ], + [ + 135, + 140, + "FBP17", + "protein" + ], + [ + 145, + 149, + "CIP4", + "protein" + ] + ] + }, + { + "sid": 97, + "sent": "The HR1 domains from FBP17 and CIP4 were purified and assayed for Cdc42 binding in competition SPAs, analogous to those carried out with the TOCA1 HR1 domain.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "HR1", + "structure_element" + ], + [ + 21, + 26, + "FBP17", + "protein" + ], + [ + 31, + 35, + "CIP4", + "protein" + ], + [ + 41, + 49, + "purified", + "experimental_method" + ], + [ + 66, + 71, + "Cdc42", + "protein" + ], + [ + 83, + 99, + "competition SPAs", + "experimental_method" + ], + [ + 141, + 146, + "TOCA1", + "protein" + ], + [ + 147, + 150, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 98, + "sent": "The affinities of both the FBP17 and CIP4 HR1 domains were also in the low micromolar range (10 and 5 \u03bcm, respectively) (Fig. 2, D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "affinities", + "evidence" + ], + [ + 27, + 32, + "FBP17", + "protein" + ], + [ + 37, + 41, + "CIP4", + "protein" + ], + [ + 42, + 45, + "HR1", + "structure_element" + ], + [ + 186, + 191, + "Cdc42", + "protein" + ], + [ + 224, + 235, + "TOCA family", + "protein_type" + ] + ] + }, + { + "sid": 99, + "sent": "Structure of the TOCA1 HR1 Domain", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 17, + 22, + "TOCA1", + "protein" + ], + [ + 23, + 26, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 100, + "sent": "Because the TOCA1 HR1 domain was sufficient for maximal Cdc42-binding, we used this construct for structural studies.", + "section": "RESULTS", + "ner": [ + [ + 12, + 17, + "TOCA1", + "protein" + ], + [ + 18, + 21, + "HR1", + "structure_element" + ], + [ + 56, + 61, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 101, + "sent": "Initial experiments were performed with TOCA1 residues 324\u2013426, but we observed that the N terminus was cleaved during purification to yield a new N terminus at residue 330 (data not shown).", + "section": "RESULTS", + "ner": [ + [ + 40, + 45, + "TOCA1", + "protein" + ], + [ + 55, + 62, + "324\u2013426", + "residue_range" + ], + [ + 169, + 172, + "330", + "residue_number" + ] + ] + }, + { + "sid": 102, + "sent": "We therefore engineered a construct comprising residues 330\u2013426 to produce the minimal, stable HR1 domain.", + "section": "RESULTS", + "ner": [ + [ + 56, + 63, + "330\u2013426", + "residue_range" + ], + [ + 79, + 86, + "minimal", + "protein_state" + ], + [ + 88, + 94, + "stable", + "protein_state" + ], + [ + 95, + 98, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 103, + "sent": "2,778 non-degenerate NOE restraints were used in initial structure calculations (1,791 unambiguous and 987 ambiguous), derived from three-dimensional 15N-separated NOESY and 13C-separated NOESY experiments.", + "section": "RESULTS", + "ner": [ + [ + 21, + 35, + "NOE restraints", + "evidence" + ], + [ + 57, + 79, + "structure calculations", + "experimental_method" + ], + [ + 150, + 169, + "15N-separated NOESY", + "experimental_method" + ], + [ + 174, + 193, + "13C-separated NOESY", + "experimental_method" + ] + ] + }, + { + "sid": 104, + "sent": "There were 1,845 unambiguous NOEs and 757 ambiguous NOEs after eight iterations.", + "section": "RESULTS", + "ner": [ + [ + 29, + 33, + "NOEs", + "evidence" + ], + [ + 52, + 56, + "NOEs", + "evidence" + ] + ] + }, + { + "sid": 105, + "sent": "100 structures were calculated in the final iteration; the 50 lowest energy structures were water-refined; and of these, the 35 lowest energy structures were analyzed.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 20, + 30, + "calculated", + "experimental_method" + ], + [ + 76, + 86, + "structures", + "evidence" + ], + [ + 142, + 152, + "structures", + "evidence" + ] + ] + }, + { + "sid": 106, + "sent": "Table 1 indicates that the HR1 domain structure is well defined by the NMR data.", + "section": "RESULTS", + "ner": [ + [ + 27, + 30, + "HR1", + "structure_element" + ], + [ + 38, + 47, + "structure", + "evidence" + ], + [ + 71, + 74, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 107, + "sent": "a , the average root mean square deviations for the ensemble \u00b1 S.D.", + "section": "TABLE", + "ner": [ + [ + 12, + 47, + "average root mean square deviations", + "evidence" + ] + ] + }, + { + "sid": 108, + "sent": "b c, values for the structure that is closest to the mean.", + "section": "TABLE", + "ner": [ + [ + 24, + 33, + "structure", + "evidence" + ] + ] + }, + { + "sid": 109, + "sent": "The structure closest to the mean is shown in Fig. 3A.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ] + ] + }, + { + "sid": 110, + "sent": "The two \u03b1-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (PDB code 2KE4) and PRK1 (PDB codes 1CXZ and 1URF).", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "\u03b1-helices", + "structure_element" + ], + [ + 25, + 28, + "HR1", + "structure_element" + ], + [ + 56, + 81, + "anti-parallel coiled-coil", + "structure_element" + ], + [ + 134, + 137, + "HR1", + "structure_element" + ], + [ + 149, + 153, + "CIP4", + "protein" + ], + [ + 174, + 178, + "PRK1", + "protein" + ] + ] + }, + { + "sid": 111, + "sent": "A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B.", + "section": "RESULTS", + "ner": [ + [ + 2, + 20, + "sequence alignment", + "experimental_method" + ], + [ + 74, + 79, + "TOCA1", + "protein" + ], + [ + 84, + 88, + "CIP4", + "protein" + ], + [ + 89, + 92, + "HR1", + "structure_element" + ], + [ + 109, + 113, + "HR1a", + "structure_element" + ], + [ + 118, + 122, + "HR1b", + "structure_element" + ], + [ + 136, + 140, + "PRK1", + "protein" + ] + ] + }, + { + "sid": 112, + "sent": "The structure of the TOCA1 HR1 domain.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 21, + 26, + "TOCA1", + "protein" + ], + [ + 27, + 30, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 113, + "sent": " A, the backbone trace of the 35 lowest energy structures of the HR1 domain overlaid with the structure closest to the mean is shown alongside a schematic representation of the structure closest to the mean.", + "section": "FIG", + "ner": [ + [ + 17, + 22, + "trace", + "evidence" + ], + [ + 47, + 57, + "structures", + "evidence" + ], + [ + 65, + 68, + "HR1", + "structure_element" + ], + [ + 94, + 103, + "structure", + "evidence" + ], + [ + 177, + 186, + "structure", + "evidence" + ] + ] + }, + { + "sid": 114, + "sent": "Flexible regions at the N and C termini (residues 330\u2013333 and 421\u2013426) are omitted for clarity.", + "section": "FIG", + "ner": [ + [ + 50, + 57, + "330\u2013333", + "residue_range" + ], + [ + 62, + 69, + "421\u2013426", + "residue_range" + ] + ] + }, + { + "sid": 115, + "sent": "B, a sequence alignment of the HR1 domains from TOCA1, CIP4, and PRK1.", + "section": "FIG", + "ner": [ + [ + 5, + 23, + "sequence alignment", + "experimental_method" + ], + [ + 31, + 34, + "HR1", + "structure_element" + ], + [ + 48, + 53, + "TOCA1", + "protein" + ], + [ + 55, + 59, + "CIP4", + "protein" + ], + [ + 65, + 69, + "PRK1", + "protein" + ] + ] + }, + { + "sid": 116, + "sent": "The secondary structure was deduced using Stride, based on the Ramachandran angles, and is indicated as follows: gray, turn; yellow, \u03b1-helix; blue, 310 helix; white, coil.", + "section": "FIG", + "ner": [ + [ + 42, + 48, + "Stride", + "experimental_method" + ], + [ + 63, + 82, + "Ramachandran angles", + "evidence" + ], + [ + 133, + 140, + "\u03b1-helix", + "structure_element" + ], + [ + 148, + 157, + "310 helix", + "structure_element" + ] + ] + }, + { + "sid": 117, + "sent": "C, a close-up of the N-terminal region of TOCA1 HR1, indicating some of the NOEs defining its position with respect to the two \u03b1-helices.", + "section": "FIG", + "ner": [ + [ + 42, + 47, + "TOCA1", + "protein" + ], + [ + 48, + 51, + "HR1", + "structure_element" + ], + [ + 76, + 80, + "NOEs", + "evidence" + ], + [ + 127, + 136, + "\u03b1-helices", + "structure_element" + ] + ] + }, + { + "sid": 118, + "sent": "Dotted lines, NOE restraints.", + "section": "FIG", + "ner": [ + [ + 14, + 28, + "NOE restraints", + "evidence" + ] + ] + }, + { + "sid": 119, + "sent": "D, a close-up of the interhelix loop region showing some of the contacts between the loop and helix 1.", + "section": "FIG", + "ner": [ + [ + 21, + 36, + "interhelix loop", + "structure_element" + ], + [ + 85, + 89, + "loop", + "structure_element" + ], + [ + 94, + 101, + "helix 1", + "structure_element" + ] + ] + }, + { + "sid": 120, + "sent": "In the HR1a domain of PRK1, a region N-terminal to helix 1 forms a short \u03b1-helix, which packs against both helices of the HR1 domain.", + "section": "RESULTS", + "ner": [ + [ + 7, + 11, + "HR1a", + "structure_element" + ], + [ + 22, + 26, + "PRK1", + "protein" + ], + [ + 51, + 58, + "helix 1", + "structure_element" + ], + [ + 67, + 80, + "short \u03b1-helix", + "structure_element" + ], + [ + 122, + 125, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 121, + "sent": "This region of TOCA1 HR1 (residues 334\u2013340) is well defined in the family of structures (Fig. 3A) but does not form an \u03b1-helix.", + "section": "RESULTS", + "ner": [ + [ + 15, + 20, + "TOCA1", + "protein" + ], + [ + 21, + 24, + "HR1", + "structure_element" + ], + [ + 35, + 42, + "334\u2013340", + "residue_range" + ], + [ + 77, + 87, + "structures", + "evidence" + ], + [ + 119, + 126, + "\u03b1-helix", + "structure_element" + ] + ] + }, + { + "sid": 122, + "sent": "It instead forms a series of turns, defined by NOE restraints observed between residues separated by one (residues 332\u2013334, 333\u2013335, etc.) or two (residues 337\u2013340) residues in the sequence and the \u03c6 and \u03c8 angles, assessed using Stride.", + "section": "RESULTS", + "ner": [ + [ + 47, + 61, + "NOE restraints", + "evidence" + ], + [ + 115, + 122, + "332\u2013334", + "residue_range" + ], + [ + 124, + 131, + "333\u2013335", + "residue_range" + ], + [ + 156, + 163, + "337\u2013340", + "residue_range" + ], + [ + 198, + 212, + "\u03c6 and \u03c8 angles", + "evidence" + ], + [ + 229, + 235, + "Stride", + "experimental_method" + ] + ] + }, + { + "sid": 123, + "sent": "These turns cause the chain to reverse direction, allowing the N-terminal segment (residues 334\u2013340) to contact both helices of the HR1 domain.", + "section": "RESULTS", + "ner": [ + [ + 92, + 99, + "334\u2013340", + "residue_range" + ], + [ + 132, + 135, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 124, + "sent": "Long range NOEs were observed linking Leu-334, Glu-335, and Asp-336 with Trp-413 of helix 2, Leu-334 with Lys-409 of helix 2, and Phe-337 and Ser-338 with Arg-345, Arg-348, and Leu-349 of helix 1.", + "section": "RESULTS", + "ner": [ + [ + 11, + 15, + "NOEs", + "evidence" + ], + [ + 38, + 45, + "Leu-334", + "residue_name_number" + ], + [ + 47, + 54, + "Glu-335", + "residue_name_number" + ], + [ + 60, + 67, + "Asp-336", + "residue_name_number" + ], + [ + 73, + 80, + "Trp-413", + "residue_name_number" + ], + [ + 84, + 91, + "helix 2", + "structure_element" + ], + [ + 93, + 100, + "Leu-334", + "residue_name_number" + ], + [ + 106, + 113, + "Lys-409", + "residue_name_number" + ], + [ + 117, + 124, + "helix 2", + "structure_element" + ], + [ + 130, + 137, + "Phe-337", + "residue_name_number" + ], + [ + 142, + 149, + "Ser-338", + "residue_name_number" + ], + [ + 155, + 162, + "Arg-345", + "residue_name_number" + ], + [ + 164, + 171, + "Arg-348", + "residue_name_number" + ], + [ + 177, + 184, + "Leu-349", + "residue_name_number" + ], + [ + 188, + 195, + "helix 1", + "structure_element" + ] + ] + }, + { + "sid": 125, + "sent": "The two \u03b1-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380\u2013389) that contains two short 310 helices (residues 381\u2013383 and 386\u2013389).", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "\u03b1-helices", + "structure_element" + ], + [ + 21, + 26, + "TOCA1", + "protein" + ], + [ + 27, + 30, + "HR1", + "structure_element" + ], + [ + 55, + 59, + "loop", + "structure_element" + ], + [ + 85, + 92, + "380\u2013389", + "residue_range" + ], + [ + 112, + 129, + "short 310 helices", + "structure_element" + ], + [ + 140, + 147, + "381\u2013383", + "residue_range" + ], + [ + 152, + 159, + "386\u2013389", + "residue_range" + ] + ] + }, + { + "sid": 126, + "sent": "Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (Fig. 3D).", + "section": "RESULTS", + "ner": [ + [ + 50, + 61, + "loop region", + "structure_element" + ], + [ + 80, + 87, + "helix 1", + "structure_element" + ], + [ + 162, + 169, + "Asn-380", + "residue_name_number" + ], + [ + 174, + 181, + "Met-383", + "residue_name_number" + ], + [ + 189, + 200, + "loop region", + "structure_element" + ], + [ + 205, + 212, + "Tyr-377", + "residue_name_number" + ], + [ + 217, + 224, + "Val-376", + "residue_name_number" + ], + [ + 228, + 235, + "helix 1", + "structure_element" + ] + ] + }, + { + "sid": 127, + "sent": "The backbone NH and CH\u03b1 groups of Gly-384 and Asp-385 also show NOEs with the side chain of Tyr-377.", + "section": "RESULTS", + "ner": [ + [ + 34, + 41, + "Gly-384", + "residue_name_number" + ], + [ + 46, + 53, + "Asp-385", + "residue_name_number" + ], + [ + 92, + 99, + "Tyr-377", + "residue_name_number" + ] + ] + }, + { + "sid": 128, + "sent": "Mapping the TOCA1 and Cdc42 Binding Interfaces", + "section": "RESULTS", + "ner": [ + [ + 12, + 17, + "TOCA1", + "protein" + ], + [ + 22, + 46, + "Cdc42 Binding Interfaces", + "site" + ] + ] + }, + { + "sid": 129, + "sent": "The HR1TOCA1-Cdc42 interface was investigated using NMR spectroscopy.", + "section": "RESULTS", + "ner": [ + [ + 4, + 28, + "HR1TOCA1-Cdc42 interface", + "site" + ], + [ + 52, + 68, + "NMR spectroscopy", + "experimental_method" + ] + ] + }, + { + "sid": 130, + "sent": "A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42\u03947Q61L\u00b7GMPPNP to map the Cdc42-binding surface.", + "section": "RESULTS", + "ner": [ + [ + 12, + 20, + "15N HSQC", + "experimental_method" + ], + [ + 49, + 52, + "15N", + "chemical" + ], + [ + 53, + 60, + "labeled", + "protein_state" + ], + [ + 61, + 66, + "TOCA1", + "protein" + ], + [ + 67, + 70, + "HR1", + "structure_element" + ], + [ + 85, + 96, + "presence of", + "protein_state" + ], + [ + 97, + 122, + "increasing concentrations", + "experimental_method" + ], + [ + 126, + 135, + "unlabeled", + "protein_state" + ], + [ + 136, + 154, + "Cdc42\u03947Q61L\u00b7GMPPNP", + "complex_assembly" + ], + [ + 166, + 187, + "Cdc42-binding surface", + "site" + ] + ] + }, + { + "sid": 131, + "sent": "A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (Fig. 4A).", + "section": "RESULTS", + "ner": [ + [ + 20, + 28, + "15N HSQC", + "experimental_method" + ], + [ + 29, + 36, + "spectra", + "evidence" + ], + [ + 40, + 44, + "free", + "protein_state" + ], + [ + 45, + 48, + "HR1", + "structure_element" + ], + [ + 53, + 56, + "HR1", + "structure_element" + ], + [ + 64, + 75, + "presence of", + "protein_state" + ], + [ + 83, + 88, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 132, + "sent": "This behavior cannot be explained by the increase in molecular mass (from 12 to 33 kDa) when Cdc42 binds and is more likely to be due to conformational exchange.", + "section": "RESULTS", + "ner": [ + [ + 93, + 98, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 133, + "sent": "Overall chemical shift perturbations (CSPs) were calculated for each residue, whereas those that had disappeared were assigned a shift change of 0.2 (Fig. 4B).", + "section": "RESULTS", + "ner": [ + [ + 8, + 36, + "chemical shift perturbations", + "experimental_method" + ], + [ + 38, + 42, + "CSPs", + "experimental_method" + ] + ] + }, + { + "sid": 134, + "sent": "A peak that disappeared or had a CSP above the mean CSP for the spectrum was considered to be significantly affected.", + "section": "RESULTS", + "ner": [ + [ + 33, + 36, + "CSP", + "experimental_method" + ], + [ + 52, + 55, + "CSP", + "experimental_method" + ] + ] + }, + { + "sid": 135, + "sent": "Mapping the binding surface of Cdc42 onto the TOCA1 HR1 domain.", + "section": "FIG", + "ner": [ + [ + 12, + 27, + "binding surface", + "site" + ], + [ + 31, + 36, + "Cdc42", + "protein" + ], + [ + 46, + 51, + "TOCA1", + "protein" + ], + [ + 52, + 55, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 136, + "sent": " A, the 15N HSQC of 200 \u03bcm TOCA1 HR1 domain is shown in the free form (black) and in the presence of a 4-fold molar excess of Cdc42\u03947Q61L\u00b7GMPPNP (red).", + "section": "FIG", + "ner": [ + [ + 8, + 16, + "15N HSQC", + "experimental_method" + ], + [ + 27, + 32, + "TOCA1", + "protein" + ], + [ + 33, + 36, + "HR1", + "structure_element" + ], + [ + 60, + 69, + "free form", + "protein_state" + ], + [ + 89, + 100, + "presence of", + "protein_state" + ], + [ + 126, + 144, + "Cdc42\u03947Q61L\u00b7GMPPNP", + "complex_assembly" + ] + ] + }, + { + "sid": 137, + "sent": "B, CSPs were calculated as described under \u201cExperimental Procedures\u201d and are shown for backbone and side chain NH groups.", + "section": "FIG", + "ner": [ + [ + 3, + 7, + "CSPs", + "experimental_method" + ] + ] + }, + { + "sid": 138, + "sent": "The mean CSP is marked with a red line.", + "section": "FIG", + "ner": [ + [ + 9, + 12, + "CSP", + "experimental_method" + ] + ] + }, + { + "sid": 139, + "sent": "Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.2 but were excluded when calculating the mean CSP and are indicated with open bars.", + "section": "FIG", + "ner": [ + [ + 33, + 44, + "presence of", + "protein_state" + ], + [ + 45, + 50, + "Cdc42", + "protein" + ], + [ + 67, + 70, + "CSP", + "experimental_method" + ], + [ + 122, + 125, + "CSP", + "experimental_method" + ] + ] + }, + { + "sid": 140, + "sent": "Those that were not traceable due to spectral overlap were assigned a CSP of zero and are marked with an asterisk below the bar.", + "section": "FIG", + "ner": [ + [ + 70, + 73, + "CSP", + "experimental_method" + ] + ] + }, + { + "sid": 141, + "sent": "Residues with affected side chain CSPs derived from 13C HSQCs are marked with green asterisks above the bars.", + "section": "FIG", + "ner": [ + [ + 34, + 38, + "CSPs", + "experimental_method" + ], + [ + 52, + 61, + "13C HSQCs", + "experimental_method" + ] + ] + }, + { + "sid": 142, + "sent": "C, a schematic representation of the HR1 domain.", + "section": "FIG", + "ner": [ + [ + 37, + 40, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 143, + "sent": "Residues with significantly affected backbone or side chain chemical shifts when Cdc42 bound and that are buried are colored dark blue, whereas those that are solvent-accessible are colored yellow.", + "section": "FIG", + "ner": [ + [ + 81, + 92, + "Cdc42 bound", + "protein_state" + ], + [ + 159, + 177, + "solvent-accessible", + "protein_state" + ] + ] + }, + { + "sid": 144, + "sent": "Residues with significantly affected backbone and side chain groups that are solvent-accessible are colored red.", + "section": "FIG", + "ner": [ + [ + 77, + 95, + "solvent-accessible", + "protein_state" + ] + ] + }, + { + "sid": 145, + "sent": "A close-up of the binding region is shown, with affected side chain heavy atoms shown as sticks.", + "section": "FIG", + "ner": [ + [ + 18, + 32, + "binding region", + "site" + ] + ] + }, + { + "sid": 146, + "sent": "D, the G protein-binding region is marked in red onto structures of the HR1 domains as indicated.", + "section": "FIG", + "ner": [ + [ + 7, + 31, + "G protein-binding region", + "site" + ], + [ + 54, + 64, + "structures", + "evidence" + ], + [ + 72, + 75, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 147, + "sent": "15N HSQC shift mapping experiments report on changes to amide groups, which are mainly inaccessible because they are buried inside the helices and are involved in hydrogen bonds.", + "section": "RESULTS", + "ner": [ + [ + 0, + 22, + "15N HSQC shift mapping", + "experimental_method" + ], + [ + 135, + 142, + "helices", + "structure_element" + ], + [ + 163, + 177, + "hydrogen bonds", + "bond_interaction" + ] + ] + }, + { + "sid": 148, + "sent": "Therefore, 13C HSQC and methyl-selective SOFAST-HMQC experiments were also recorded on 15N,13C-labeled TOCA1 HR1 to yield more information on side chain involvement.", + "section": "RESULTS", + "ner": [ + [ + 11, + 19, + "13C HSQC", + "experimental_method" + ], + [ + 24, + 52, + "methyl-selective SOFAST-HMQC", + "experimental_method" + ], + [ + 87, + 90, + "15N", + "chemical" + ], + [ + 91, + 94, + "13C", + "chemical" + ], + [ + 95, + 102, + "labeled", + "protein_state" + ], + [ + 103, + 108, + "TOCA1", + "protein" + ], + [ + 109, + 112, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 149, + "sent": "Side chains whose CH groups disappeared in the presence of Cdc42 are marked on the graph in Fig. 4B with green asterisks.", + "section": "RESULTS", + "ner": [ + [ + 47, + 58, + "presence of", + "protein_state" + ], + [ + 59, + 64, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 150, + "sent": "TOCA1 residues whose signals were affected by Cdc42 binding were mapped onto the structure of TOCA1 HR1 (Fig. 4C).", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "TOCA1", + "protein" + ], + [ + 46, + 51, + "Cdc42", + "protein" + ], + [ + 81, + 90, + "structure", + "evidence" + ], + [ + 94, + 99, + "TOCA1", + "protein" + ], + [ + 100, + 103, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 151, + "sent": "The changes were localized to one end of the coiled-coil, and the binding site appeared to include residues from both \u03b1-helices and the loop region that joins them.", + "section": "RESULTS", + "ner": [ + [ + 45, + 56, + "coiled-coil", + "structure_element" + ], + [ + 66, + 78, + "binding site", + "site" + ], + [ + 118, + 127, + "\u03b1-helices", + "structure_element" + ], + [ + 136, + 147, + "loop region", + "structure_element" + ] + ] + }, + { + "sid": 152, + "sent": "The residues in the interhelical loop and helix 1 that contact each other (Fig. 3D) show shift changes in their backbone NH and side chains in the presence of Cdc42.", + "section": "RESULTS", + "ner": [ + [ + 20, + 37, + "interhelical loop", + "structure_element" + ], + [ + 42, + 49, + "helix 1", + "structure_element" + ], + [ + 147, + 158, + "presence of", + "protein_state" + ], + [ + 159, + 164, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 153, + "sent": "For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation.", + "section": "RESULTS", + "ner": [ + [ + 31, + 38, + "Asn-380", + "residue_name_number" + ], + [ + 60, + 67, + "Val-376", + "residue_name_number" + ], + [ + 72, + 79, + "Tyr-377", + "residue_name_number" + ], + [ + 134, + 138, + "free", + "protein_state" + ], + [ + 139, + 144, + "TOCA1", + "protein" + ], + [ + 145, + 148, + "HR1", + "structure_element" + ], + [ + 149, + 158, + "structure", + "evidence" + ], + [ + 212, + 216, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 154, + "sent": "The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (Figs. 3B and 4D).", + "section": "RESULTS", + "ner": [ + [ + 4, + 26, + "chemical shift mapping", + "experimental_method" + ], + [ + 50, + 74, + "G protein-binding region", + "site" + ], + [ + 82, + 87, + "TOCA1", + "protein" + ], + [ + 88, + 91, + "HR1", + "structure_element" + ], + [ + 133, + 137, + "CIP4", + "protein" + ], + [ + 142, + 146, + "PRK1", + "protein" + ], + [ + 147, + 150, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 155, + "sent": "The corresponding 15N and 13C NMR experiments were also recorded on 15N-Cdc42\u03947Q61L\u00b7GMPPNP or 15N/13C -Cdc42\u03947Q61L\u00b7GMPPNP in the presence of unlabeled HR1 domain.", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "15N", + "experimental_method" + ], + [ + 26, + 33, + "13C NMR", + "experimental_method" + ], + [ + 68, + 71, + "15N", + "chemical" + ], + [ + 72, + 90, + "Cdc42\u03947Q61L\u00b7GMPPNP", + "complex_assembly" + ], + [ + 94, + 97, + "15N", + "chemical" + ], + [ + 98, + 101, + "13C", + "chemical" + ], + [ + 103, + 121, + "Cdc42\u03947Q61L\u00b7GMPPNP", + "complex_assembly" + ], + [ + 129, + 140, + "presence of", + "protein_state" + ], + [ + 141, + 150, + "unlabeled", + "protein_state" + ], + [ + 151, + 154, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 156, + "sent": "The overall CSP was calculated for each residue.", + "section": "RESULTS", + "ner": [ + [ + 12, + 15, + "CSP", + "experimental_method" + ] + ] + }, + { + "sid": 157, + "sent": "As was the case when labeled HR1 was observed, several peaks were shifted in the complex, but many disappeared, indicating exchange on an unfavorable, millisecond time scale (Fig. 5A).", + "section": "RESULTS", + "ner": [ + [ + 21, + 28, + "labeled", + "protein_state" + ], + [ + 29, + 32, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 158, + "sent": "Detailed side chain data could not be obtained for all residues due to spectral overlap, but constant time 13C HSQC and methyl-selective SOFAST-HMQC experiments provided further information on certain well resolved side chains (marked with green asterisks in Fig. 5B).", + "section": "RESULTS", + "ner": [ + [ + 93, + 115, + "constant time 13C HSQC", + "experimental_method" + ], + [ + 120, + 148, + "methyl-selective SOFAST-HMQC", + "experimental_method" + ] + ] + }, + { + "sid": 159, + "sent": "Mapping the binding surface of the HR1 domain onto Cdc42.", + "section": "FIG", + "ner": [ + [ + 12, + 27, + "binding surface", + "site" + ], + [ + 35, + 38, + "HR1", + "structure_element" + ], + [ + 51, + 56, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 160, + "sent": " A, the 15N HSQC of Cdc42\u03947Q61L\u00b7GMPPNP is shown in its free form (black) and in the presence of excess TOCA1 HR1 domain (1:2.2, red).", + "section": "FIG", + "ner": [ + [ + 8, + 16, + "15N HSQC", + "experimental_method" + ], + [ + 20, + 38, + "Cdc42\u03947Q61L\u00b7GMPPNP", + "complex_assembly" + ], + [ + 55, + 64, + "free form", + "protein_state" + ], + [ + 84, + 95, + "presence of", + "protein_state" + ], + [ + 103, + 108, + "TOCA1", + "protein" + ], + [ + 109, + 112, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 161, + "sent": "B, CSPs are shown for backbone NH groups.", + "section": "FIG", + "ner": [ + [ + 3, + 7, + "CSPs", + "experimental_method" + ] + ] + }, + { + "sid": 162, + "sent": "The red line indicates the mean CSP, plus one S.D. Residues that disappeared in the presence of Cdc42 were assigned a CSP of 0.1 and are indicated with open bars.", + "section": "FIG", + "ner": [ + [ + 32, + 35, + "CSP", + "experimental_method" + ], + [ + 84, + 95, + "presence of", + "protein_state" + ], + [ + 96, + 101, + "Cdc42", + "protein" + ], + [ + 118, + 121, + "CSP", + "experimental_method" + ] + ] + }, + { + "sid": 163, + "sent": "Residues with disappeared peaks in 13C HSQC experiments are marked on the chart with green asterisks.", + "section": "FIG", + "ner": [ + [ + 35, + 43, + "13C HSQC", + "experimental_method" + ] + ] + }, + { + "sid": 164, + "sent": "C, the residues with significantly affected backbone and side chain groups are highlighted on an NMR structure of free Cdc42\u03947Q61L\u00b7GMPPNP; those that are buried are colored dark blue, whereas those that are solvent-accessible are colored red.", + "section": "FIG", + "ner": [ + [ + 97, + 100, + "NMR", + "experimental_method" + ], + [ + 101, + 110, + "structure", + "evidence" + ], + [ + 114, + 118, + "free", + "protein_state" + ], + [ + 119, + 137, + "Cdc42\u03947Q61L\u00b7GMPPNP", + "complex_assembly" + ], + [ + 207, + 225, + "solvent-accessible", + "protein_state" + ] + ] + }, + { + "sid": 165, + "sent": "Residues with either side chain or backbone groups affected are colored blue if buried and yellow if solvent-accessible.", + "section": "FIG", + "ner": [ + [ + 101, + 119, + "solvent-accessible", + "protein_state" + ] + ] + }, + { + "sid": 166, + "sent": "Residues without information from shift mapping are colored gray.", + "section": "FIG", + "ner": [ + [ + 34, + 47, + "shift mapping", + "experimental_method" + ] + ] + }, + { + "sid": 167, + "sent": "The flexible switch regions are circled.", + "section": "FIG", + "ner": [ + [ + 4, + 12, + "flexible", + "protein_state" + ], + [ + 13, + 27, + "switch regions", + "site" + ] + ] + }, + { + "sid": 168, + "sent": "As many of the peaks disappeared, the mean chemical shift change was relatively low, so a threshold of the mean plus one S.D. value was used to define a significant CSP.", + "section": "RESULTS", + "ner": [ + [ + 38, + 64, + "mean chemical shift change", + "evidence" + ], + [ + 165, + 168, + "CSP", + "experimental_method" + ] + ] + }, + { + "sid": 169, + "sent": "Parts of the switch regions (Fig. 5, B and C) are invisible in NMR spectra recorded on free Cdc42 due to conformational exchange.", + "section": "RESULTS", + "ner": [ + [ + 13, + 27, + "switch regions", + "site" + ], + [ + 63, + 66, + "NMR", + "experimental_method" + ], + [ + 67, + 74, + "spectra", + "evidence" + ], + [ + 87, + 91, + "free", + "protein_state" + ], + [ + 92, + 97, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 170, + "sent": "These switch regions become visible in Cdc42 and other small G protein\u00b7effector complexes due to a decrease in conformational freedom upon complex formation.", + "section": "RESULTS", + "ner": [ + [ + 6, + 20, + "switch regions", + "site" + ], + [ + 39, + 44, + "Cdc42", + "protein" + ], + [ + 61, + 70, + "G protein", + "protein_type" + ] + ] + }, + { + "sid": 171, + "sent": "The switch regions of Cdc42 did not, however, become visible in the presence of the TOCA1 HR1 domain.", + "section": "RESULTS", + "ner": [ + [ + 4, + 18, + "switch regions", + "site" + ], + [ + 22, + 27, + "Cdc42", + "protein" + ], + [ + 68, + 79, + "presence of", + "protein_state" + ], + [ + 84, + 89, + "TOCA1", + "protein" + ], + [ + 90, + 93, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 172, + "sent": "Indeed, Ser-30 of switch I and Arg-66, Arg-68, Leu-70, and Ser-71 of switch II are visible in free Cdc42 but disappear in the presence of the HR1 domain.", + "section": "RESULTS", + "ner": [ + [ + 8, + 14, + "Ser-30", + "residue_name_number" + ], + [ + 18, + 26, + "switch I", + "site" + ], + [ + 31, + 37, + "Arg-66", + "residue_name_number" + ], + [ + 39, + 45, + "Arg-68", + "residue_name_number" + ], + [ + 47, + 53, + "Leu-70", + "residue_name_number" + ], + [ + 59, + 65, + "Ser-71", + "residue_name_number" + ], + [ + 69, + 78, + "switch II", + "site" + ], + [ + 94, + 98, + "free", + "protein_state" + ], + [ + 99, + 104, + "Cdc42", + "protein" + ], + [ + 126, + 137, + "presence of", + "protein_state" + ], + [ + 142, + 145, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 173, + "sent": "This suggests that the switch regions are not rigidified in the HR1 complex and are still in conformational exchange.", + "section": "RESULTS", + "ner": [ + [ + 23, + 37, + "switch regions", + "site" + ], + [ + 64, + 67, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 174, + "sent": "Nevertheless, mapping of the affected residues onto the NMR structure of free Cdc42\u03947Q61L\u00b7GMPPNP (Fig. 5C)8 shows that, although they are relatively widespread compared with changes in the HR1 domain, in general, they are on the face of the protein that includes the switches.", + "section": "RESULTS", + "ner": [ + [ + 56, + 59, + "NMR", + "experimental_method" + ], + [ + 60, + 69, + "structure", + "evidence" + ], + [ + 73, + 77, + "free", + "protein_state" + ], + [ + 78, + 96, + "Cdc42\u03947Q61L\u00b7GMPPNP", + "complex_assembly" + ], + [ + 189, + 192, + "HR1", + "structure_element" + ], + [ + 267, + 275, + "switches", + "site" + ] + ] + }, + { + "sid": 175, + "sent": "Although the binding interface may be overestimated, this suggests that the switch regions are involved in binding to TOCA1.", + "section": "RESULTS", + "ner": [ + [ + 13, + 30, + "binding interface", + "site" + ], + [ + 76, + 90, + "switch regions", + "site" + ], + [ + 118, + 123, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 176, + "sent": "Modeling the Cdc42\u00b7TOCA1 HR1 Complex", + "section": "RESULTS", + "ner": [ + [ + 13, + 28, + "Cdc42\u00b7TOCA1 HR1", + "complex_assembly" + ] + ] + }, + { + "sid": 177, + "sent": "The Cdc42\u00b7HR1TOCA1 complex was not amenable to full structural analysis due to the weak interaction and the extensive exchange broadening seen in the NMR experiments.", + "section": "RESULTS", + "ner": [ + [ + 4, + 18, + "Cdc42\u00b7HR1TOCA1", + "complex_assembly" + ], + [ + 150, + 153, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 178, + "sent": "HADDOCK was therefore used to perform rigid body docking based on the structures of free HR1 domain and Cdc42 and ambiguous interaction restraints derived from the titration experiments described above.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "HADDOCK", + "experimental_method" + ], + [ + 38, + 56, + "rigid body docking", + "experimental_method" + ], + [ + 70, + 80, + "structures", + "evidence" + ], + [ + 84, + 88, + "free", + "protein_state" + ], + [ + 89, + 92, + "HR1", + "structure_element" + ], + [ + 104, + 109, + "Cdc42", + "protein" + ], + [ + 164, + 185, + "titration experiments", + "experimental_method" + ] + ] + }, + { + "sid": 179, + "sent": "The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42.", + "section": "RESULTS", + "ner": [ + [ + 23, + 26, + "HR1", + "structure_element" + ], + [ + 50, + 55, + "Cdc42", + "protein" + ], + [ + 147, + 154, + "HADDOCK", + "experimental_method" + ], + [ + 193, + 196, + "HR1", + "structure_element" + ], + [ + 233, + 238, + "Cdc42", + "protein" + ], + [ + 286, + 291, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 180, + "sent": "The cluster with the lowest root mean square deviation from the lowest energy structure is assumed to be the best model.", + "section": "RESULTS", + "ner": [ + [ + 28, + 54, + "root mean square deviation", + "evidence" + ], + [ + 78, + 87, + "structure", + "evidence" + ] + ] + }, + { + "sid": 181, + "sent": "By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1.", + "section": "RESULTS", + "ner": [ + [ + 42, + 45, + "HR1", + "structure_element" + ], + [ + 88, + 92, + "HR1a", + "structure_element" + ], + [ + 103, + 107, + "PRK1", + "protein" + ], + [ + 108, + 116, + "bound to", + "protein_state" + ], + [ + 117, + 121, + "RhoA", + "protein" + ], + [ + 130, + 134, + "HR1b", + "structure_element" + ], + [ + 142, + 150, + "bound to", + "protein_state" + ], + [ + 151, + 155, + "Rac1", + "protein" + ] + ] + }, + { + "sid": 182, + "sent": "A representative model from this cluster is shown in Fig. 6A alongside the Rac1-HR1b structure (PDB code 2RMK) in Fig. 6B.", + "section": "RESULTS", + "ner": [ + [ + 75, + 84, + "Rac1-HR1b", + "complex_assembly" + ], + [ + 85, + 94, + "structure", + "evidence" + ] + ] + }, + { + "sid": 183, + "sent": "Model of Cdc42\u00b7HR1 complex.", + "section": "FIG", + "ner": [ + [ + 9, + 18, + "Cdc42\u00b7HR1", + "complex_assembly" + ] + ] + }, + { + "sid": 184, + "sent": " A, a representative model of the Cdc42\u00b7HR1 complex from the cluster closest to the lowest energy model produced using HADDOCK.", + "section": "FIG", + "ner": [ + [ + 34, + 43, + "Cdc42\u00b7HR1", + "complex_assembly" + ], + [ + 119, + 126, + "HADDOCK", + "experimental_method" + ] + ] + }, + { + "sid": 185, + "sent": "Residues of Cdc42 that are affected in the presence of the HR1 domain but are not in close proximity to it are colored in red and labeled.", + "section": "FIG", + "ner": [ + [ + 12, + 17, + "Cdc42", + "protein" + ], + [ + 43, + 54, + "presence of", + "protein_state" + ], + [ + 59, + 62, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 186, + "sent": "B, structure of Rac1 in complex with the HR1b domain of PRK1 (PDB code 2RMK).", + "section": "FIG", + "ner": [ + [ + 3, + 12, + "structure", + "evidence" + ], + [ + 16, + 20, + "Rac1", + "protein" + ], + [ + 21, + 36, + "in complex with", + "protein_state" + ], + [ + 41, + 45, + "HR1b", + "structure_element" + ], + [ + 56, + 60, + "PRK1", + "protein" + ] + ] + }, + { + "sid": 187, + "sent": "C, sequence alignment of RhoA, Cdc42 and Rac1.", + "section": "FIG", + "ner": [ + [ + 3, + 21, + "sequence alignment", + "experimental_method" + ], + [ + 25, + 29, + "RhoA", + "protein" + ], + [ + 31, + 36, + "Cdc42", + "protein" + ], + [ + 41, + 45, + "Rac1", + "protein" + ] + ] + }, + { + "sid": 188, + "sent": "Contact residues of RhoA and Rac1 to PRK1 HR1a and HR1b, respectively, are colored cyan.", + "section": "FIG", + "ner": [ + [ + 20, + 24, + "RhoA", + "protein" + ], + [ + 29, + 33, + "Rac1", + "protein" + ], + [ + 37, + 41, + "PRK1", + "protein" + ], + [ + 42, + 46, + "HR1a", + "structure_element" + ], + [ + 51, + 55, + "HR1b", + "structure_element" + ] + ] + }, + { + "sid": 189, + "sent": "Residues of Cdc42 that disappear or show chemical shift changes in the presence of TOCA1 are colored cyan if also identified as contacts in RhoA and Rac1 and yellow if they are not.", + "section": "FIG", + "ner": [ + [ + 12, + 17, + "Cdc42", + "protein" + ], + [ + 71, + 82, + "presence of", + "protein_state" + ], + [ + 83, + 88, + "TOCA1", + "protein" + ], + [ + 140, + 144, + "RhoA", + "protein" + ], + [ + 149, + 153, + "Rac1", + "protein" + ] + ] + }, + { + "sid": 190, + "sent": "Residues equivalent to Rac1 and RhoA contact sites but that are invisible in free Cdc42 are gray.", + "section": "FIG", + "ner": [ + [ + 23, + 27, + "Rac1", + "protein" + ], + [ + 32, + 36, + "RhoA", + "protein" + ], + [ + 37, + 50, + "contact sites", + "site" + ], + [ + 77, + 81, + "free", + "protein_state" + ], + [ + 82, + 87, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 191, + "sent": "D, regions of interest of the Cdc42\u00b7HR1 domain model.", + "section": "FIG", + "ner": [ + [ + 30, + 39, + "Cdc42\u00b7HR1", + "complex_assembly" + ] + ] + }, + { + "sid": 192, + "sent": "The four lowest energy structures in the chosen HADDOCK cluster are shown overlaid, with the residues of interest shown as sticks and labeled.", + "section": "FIG", + "ner": [ + [ + 23, + 33, + "structures", + "evidence" + ], + [ + 48, + 55, + "HADDOCK", + "experimental_method" + ] + ] + }, + { + "sid": 193, + "sent": "Cdc42 is shown in cyan, and TOCA1 is shown in purple.", + "section": "FIG", + "ner": [ + [ + 28, + 33, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 194, + "sent": "A sequence alignment of RhoA, Cdc42, and Rac1 is shown in Fig. 6C.", + "section": "RESULTS", + "ner": [ + [ + 2, + 20, + "sequence alignment", + "experimental_method" + ], + [ + 24, + 28, + "RhoA", + "protein" + ], + [ + 30, + 35, + "Cdc42", + "protein" + ], + [ + 41, + 45, + "Rac1", + "protein" + ] + ] + }, + { + "sid": 195, + "sent": "The RhoA and Rac1 contact residues in the switch regions are invisible in the spectra of Cdc42, but they are generally conserved between all three G proteins.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "RhoA", + "protein" + ], + [ + 13, + 17, + "Rac1", + "protein" + ], + [ + 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"residue_name_number" + ], + [ + 54, + 59, + "Cdc42", + "protein" + ], + [ + 61, + 68, + "Leu-160", + "residue_name_number" + ], + [ + 68, + 73, + "Cdc42", + "protein" + ], + [ + 79, + 86, + "Lys-163", + "residue_name_number" + ], + [ + 86, + 91, + "Cdc42", + "protein" + ], + [ + 108, + 116, + "switch I", + "site" + ], + [ + 184, + 190, + "switch", + "site" + ], + [ + 198, + 204, + "Glu-95", + "residue_name_number" + ], + [ + 204, + 209, + "Cdc42", + "protein" + ], + [ + 214, + 220, + "Lys-96", + "residue_name_number" + ], + [ + 220, + 225, + "Cdc42", + "protein" + ], + [ + 237, + 242, + "helix", + "structure_element" + ], + [ + 250, + 259, + "switch II", + "site" + ] + ] + }, + { + "sid": 198, + "sent": "Other residues that are affected in the Cdc42\u00b7TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42.", + "section": "RESULTS", + "ner": [ + [ + 40, + 51, + "Cdc42\u00b7TOCA1", + "complex_assembly" + ], + [ + 110, + 114, + "RhoA", + "protein" + ], + [ + 118, + 122, + "Rac1", + "protein" + ], + [ + 141, + 146, + "Gln-2", + "residue_name_number" + ], + [ + 146, + 151, + "Cdc42", + "protein" + ], + [ + 153, + 159, + "Lys-16", + "residue_name_number" + ], + [ + 159, + 164, + "Cdc42", + "protein" + ], + [ + 166, + 172, + "Thr-52", + "residue_name_number" + ], + [ + 172, + 177, + "Cdc42", + "protein" + ], + [ + 183, + 189, + "Arg-68", + "residue_name_number" + ], + [ + 189, + 194, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 199, + "sent": "Lys-16Cdc42 is unlikely to be a contact residue because it is involved in nucleotide binding, but the others may represent specific Cdc42-TOCA1 contacts.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Lys-16", + "residue_name_number" + ], + [ + 6, + 11, + "Cdc42", + "protein" + ], + [ + 132, + 143, + "Cdc42-TOCA1", + "complex_assembly" + ] + ] + }, + { + "sid": 200, + "sent": "Competition between N-WASP and TOCA1", + "section": "RESULTS", + "ner": [ + [ + 20, + 26, + "N-WASP", + "protein" + ], + [ + 31, + 36, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 201, + "sent": "From the known interactions and effects of the proteins in biological systems, it has been suggested that TOCA1 and N-WASP could bind Cdc42 simultaneously.", + "section": "RESULTS", + "ner": [ + [ + 106, + 111, + "TOCA1", + "protein" + ], + [ + 116, + 122, + "N-WASP", + "protein" + ], + [ + 134, + 139, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 202, + "sent": "Studies in CHO cells indicated that a Cdc42\u00b7N-WASP\u00b7TOCA1 complex existed because FRET was observed between RFP-TOCA1 and GFP-N-WASP, and the efficiency was decreased when an N-WASP mutant was used that no longer binds Cdc42.", + "section": "RESULTS", + "ner": [ + [ + 38, + 56, + "Cdc42\u00b7N-WASP\u00b7TOCA1", + "complex_assembly" + ], + [ + 81, + 85, + "FRET", + "evidence" + ], + [ + 107, + 110, + "RFP", + "chemical" + ], + [ + 111, + 116, + "TOCA1", + "protein" + ], + [ + 121, + 124, + "GFP", + "chemical" + ], + [ + 125, + 131, + "N-WASP", + "protein" + ], + [ + 174, + 180, + "N-WASP", + "protein" + ], + [ + 181, + 187, + "mutant", + "protein_state" + ], + [ + 218, + 223, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 203, + "sent": "An overlay of the HADDOCK model of the Cdc42\u00b7HR1TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (PDB code 1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (Fig. 7A).", + "section": "RESULTS", + "ner": [ + [ + 3, + 10, + "overlay", + "experimental_method" + ], + [ + 18, + 25, + "HADDOCK", + "experimental_method" + ], + [ + 26, + 31, + "model", + "evidence" + ], + [ + 39, + 53, + "Cdc42\u00b7HR1TOCA1", + "complex_assembly" + ], + [ + 70, + 79, + "structure", + "evidence" + ], + [ + 83, + 88, + "Cdc42", + "protein" + ], + [ + 89, + 104, + "in complex with", + "protein_state" + ], + 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+ "protein" + ], + [ + 246, + 249, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 205, + "sent": "A basic region in WASP including three lysines (residues 230\u2013232), N-terminal to the core CRIB, has been implicated in an electrostatic steering mechanism, and these residues would be free to bind in the presence of TOCA1 HR1 (Fig. 7A).", + "section": "RESULTS", + "ner": [ + [ + 18, + 22, + "WASP", + "protein" + ], + [ + 39, + 46, + "lysines", + "residue_name" + ], + [ + 57, + 64, + "230\u2013232", + "residue_range" + ], + [ + 90, + 94, + "CRIB", + "structure_element" + ], + [ + 204, + 215, + "presence of", + "protein_state" + ], + [ + 216, + 221, + "TOCA1", + "protein" + ], + [ + 222, + 225, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 206, + "sent": "The N-WASP GBD displaces the TOCA1 HR1 domain.", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "N-WASP", + "protein" + ], + [ + 11, + 14, + "GBD", + "structure_element" + ], + [ + 29, + 34, + "TOCA1", + "protein" + ], + [ + 35, + 38, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 207, + "sent": " A, the model of the Cdc42\u00b7TOCA1 HR1 domain complex overlaid with the Cdc42-WASP structure.", + "section": "FIG", + "ner": [ + [ + 21, + 32, + "Cdc42\u00b7TOCA1", + "complex_assembly" + ], + [ + 33, + 36, + "HR1", + "structure_element" + ], + [ + 70, + 80, + "Cdc42-WASP", + "complex_assembly" + ], + [ + 81, + 90, + "structure", + "evidence" + ] + ] + }, + { + "sid": 208, + "sent": "Cdc42 is shown in green, and TOCA1 is shown in purple.", + "section": "FIG", + "ner": [ + [ + 29, + 34, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 209, + "sent": "The core CRIB region of WASP is shown in red, whereas its basic region is shown in orange and the C-terminal region required for maximal affinity is shown in cyan.", + "section": "FIG", + "ner": [ + [ + 9, + 13, + "CRIB", + "structure_element" + ], + [ + 24, + 28, + "WASP", + "protein" + ] + ] + }, + { + "sid": 210, + "sent": "A semitransparent surface representation of Cdc42 and WASP is shown overlaid with the schematic.", + "section": "FIG", + "ner": [ + [ + 44, + 49, + "Cdc42", + "protein" + ], + [ + 54, + 58, + "WASP", + "protein" + ] + ] + }, + { + "sid": 211, + "sent": "B, competition SPA experiments carried out with indicated concentrations of the N-WASP GBD construct titrated into 30 nm GST-ACK or GST-WASP GBD and 30 nm Cdc42\u03947Q61L\u00b7[3H]GTP.", + "section": "FIG", + "ner": [ + [ + 3, + 18, + "competition SPA", + "experimental_method" + ], + [ + 80, + 86, + "N-WASP", + "protein" + ], + [ + 87, + 90, + "GBD", + "structure_element" + ], + [ + 101, + 109, + "titrated", + "experimental_method" + ], + [ + 121, + 128, + "GST-ACK", + "mutant" + ], + [ + 132, + 140, + "GST-WASP", + "mutant" + ], + [ + 141, + 144, + "GBD", + "structure_element" + ], + [ + 155, + 174, + "Cdc42\u03947Q61L\u00b7[3H]GTP", + "complex_assembly" + ] + ] + }, + { + "sid": 212, + "sent": "C, Selected regions of the 15N HSQC of 145 \u03bcm Cdc42\u03947Q61L\u00b7GMPPNP with the indicated ratios of the TOCA1 HR1 domain, the N-WASP GBD, or both, showing that the TOCA HR1 domain does not displace the N-WASP GBD.", + "section": "FIG", + "ner": [ + [ + 27, + 35, + "15N HSQC", + "experimental_method" + ], + [ + 46, + 64, + "Cdc42\u03947Q61L\u00b7GMPPNP", + "complex_assembly" + ], + [ + 98, + 103, + "TOCA1", + "protein" + ], + [ + 104, + 107, + "HR1", + "structure_element" + ], + [ + 120, + 126, + "N-WASP", + "protein" + ], + [ + 127, + 130, + "GBD", + "structure_element" + ], + [ + 158, + 162, + "TOCA", + "protein" + ], + [ + 163, + 166, + "HR1", + "structure_element" + ], + [ + 196, + 202, + "N-WASP", + "protein" + ], + [ + 203, + 206, + "GBD", + "structure_element" + ] + ] + }, + { + "sid": 213, + "sent": "D, selected regions of the 15N HSQC of 600 \u03bcm TOCA1 HR1 domain in complex with Cdc42 in the absence and presence of the N-WASP GBD, showing displacement of Cdc42 from the HR1 domain by N-WASP.", + "section": "FIG", + "ner": [ + [ + 27, + 35, + "15N HSQC", + "experimental_method" + ], + [ + 46, + 51, + "TOCA1", + "protein" + ], + [ + 52, + 55, + "HR1", + "structure_element" + ], + [ + 63, + 78, + "in complex with", + "protein_state" + ], + [ + 79, + 84, + "Cdc42", + "protein" + ], + [ + 92, + 99, + "absence", + "protein_state" + ], + [ + 104, + 115, + "presence of", + "protein_state" + ], + [ + 120, + 126, + "N-WASP", + "protein" + ], + [ + 127, + 130, + "GBD", + "structure_element" + ], + [ + 156, + 161, + "Cdc42", + "protein" + ], + [ + 171, + 174, + "HR1", + "structure_element" + ], + [ + 185, + 191, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 214, + "sent": "An N-WASP GBD construct was produced, and its affinity for Cdc42 was measured by competition SPA (Fig. 7B).", + "section": "RESULTS", + "ner": [ + [ + 3, + 9, + "N-WASP", + "protein" + ], + [ + 10, + 13, + "GBD", + "structure_element" + ], + [ + 46, + 54, + "affinity", + "evidence" + ], + [ + 59, + 64, + "Cdc42", + "protein" + ], + [ + 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Cdc42\u00b7N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Unlabeled", + "protein_state" + ], + [ + 10, + 13, + "HR1", + "structure_element" + ], + [ + 13, + 18, + "TOCA1", + "protein" + ], + [ + 41, + 53, + "Cdc42\u00b7N-WASP", + "complex_assembly" + ], + [ + 109, + 115, + "N-WASP", + "protein" + ], + [ + 116, + 119, + "GBD", + "structure_element" + ], + [ + 150, + 161, + "presence of", + "protein_state" + ], + [ + 181, + 184, + "HR1", + "structure_element" + ], + [ + 184, + 189, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 218, + "sent": "These experiments were recorded at sufficiently high protein concentrations (145 \u03bcm Cdc42, 145 \u03bcm N-WASP GBD, 725 \u03bcm TOCA1 HR1 domain) to be far in excess of the Kd values of the individual interactions (TOCA1 Kd \u2248 5 \u03bcm, N-WASP Kd = 37 nm).", + "section": "RESULTS", + "ner": [ + [ + 84, + 89, + "Cdc42", + "protein" + ], + [ + 98, + 104, + "N-WASP", + "protein" + ], + [ + 105, + 108, + "GBD", + "structure_element" + ], + [ + 117, + 122, + "TOCA1", + "protein" + ], + [ + 123, + 126, + "HR1", + "structure_element" + ], + [ + 162, + 164, + "Kd", + "evidence" + ], + [ + 204, + 209, + "TOCA1", + "protein" + ], + [ + 210, + 212, + "Kd", + "evidence" + ], + [ + 221, + 227, + "N-WASP", + "protein" + ], + [ + 228, + 230, + "Kd", + "evidence" + ] + ] + }, + { + "sid": 219, + "sent": "A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (Fig. 7C).", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "HSQC", + "experimental_method" + ], + [ + 49, + 52, + "15N", + "chemical" + ], + [ + 53, + 58, + "Cdc42", + "protein" + ], + [ + 59, + 64, + "alone", + "protein_state" + ], + [ + 73, + 84, + "presence of", + "protein_state" + ], + [ + 85, + 90, + "TOCA1", + "protein" + ], + [ + 91, + 94, + "HR1", + "structure_element" + ], + [ + 96, + 102, + "N-WASP", + "protein" + ], + [ + 103, + 106, + "GBD", + "structure_element" + ], + [ + 132, + 139, + "spectra", + "evidence" + ], + [ + 147, + 158, + "presence of", + "protein_state" + ], + [ + 159, + 165, + "N-WASP", + "protein" + ], + [ + 177, + 188, + "presence of", + "protein_state" + ], + [ + 194, + 200, + "N-WASP", + "protein" + ], + [ + 205, + 210, + "TOCA1", + "protein" + ], + [ + 211, + 214, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 220, + "sent": "Furthermore, 15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42\u03947Q61L\u00b7GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD.", + "section": "RESULTS", + "ner": [ + [ + 13, + 16, + "15N", + "chemical" + ], + [ + 17, + 22, + "TOCA1", + "protein" + ], + [ + 23, + 26, + "HR1", + "structure_element" + ], + [ + 48, + 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"N-WASP", + "protein" + ], + [ + 183, + 188, + "TOCA1", + "protein" + ], + [ + 189, + 192, + "HR1", + "structure_element" + ], + [ + 193, + 197, + "free", + "protein_state" + ], + [ + 202, + 209, + "complex", + "protein_state" + ], + [ + 210, + 217, + "spectra", + "evidence" + ] + ] + }, + { + "sid": 222, + "sent": "When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain.", + "section": "RESULTS", + "ner": [ + [ + 27, + 30, + "NMR", + "experimental_method" + ], + [ + 87, + 91, + "free", + "protein_state" + ], + [ + 96, + 101, + "bound", + "protein_state" + ], + [ + 130, + 138, + "spectrum", + "evidence" + ], + [ + 192, + 196, + "free", + "protein_state" + ], + [ + 201, + 206, + "bound", + "protein_state" + ], + [ + 207, + 210, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 223, + "sent": "Again, the experiments were recorded on protein samples far in excess of the individual Kd values (600 \u03bcm each protein).", + "section": "RESULTS", + "ner": [ + [ + 88, + 90, + "Kd", + "evidence" + ] + ] + }, + { + "sid": 224, + "sent": "These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed.", + "section": "RESULTS", + "ner": [ + [ + 29, + 32, + "HR1", + "structure_element" + ], + [ + 58, + 63, + "Cdc42", + "protein" + ], + [ + 67, + 73, + "N-WASP", + "protein" + ], + [ + 112, + 117, + "TOCA1", + "protein" + ], + [ + 118, + 121, + "HR1", + "structure_element" + ], + [ + 123, + 129, + "N-WASP", + "protein" + ], + [ + 130, + 133, + "GBD", + "structure_element" + ], + [ + 139, + 144, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 225, + "sent": "Taken together, the data in Fig. 7, C and D, indicate unidirectional competition for Cdc42 binding in which the N-WASP GBD displaces TOCA1 HR1 but not vice versa.", + "section": "RESULTS", + "ner": [ + [ + 85, + 90, + "Cdc42", + "protein" + ], + [ + 112, + 118, + "N-WASP", + "protein" + ], + [ + 119, + 122, + "GBD", + "structure_element" + ], + [ + 133, + 138, + "TOCA1", + "protein" + ], + [ + 139, + 142, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 226, + "sent": "To extend these studies to a more complex system and to assess the ability of TOCA1 HR1 to compete with full-length N-WASP, pyrene actin assays were employed.", + "section": "RESULTS", + "ner": [ + [ + 78, + 83, + "TOCA1", + "protein" + ], + [ + 84, + 87, + "HR1", + "structure_element" + ], + [ + 104, + 115, + "full-length", + "protein_state" + ], + [ + 116, + 122, + "N-WASP", + "protein" + ], + [ + 124, + 143, + "pyrene actin assays", + "experimental_method" + ] + ] + }, + { + "sid": 227, + "sent": "These assays, described in detail elsewhere, were carried out using pyrene actin-supplemented Xenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization.", + "section": "RESULTS", + "ner": [ + [ + 68, + 80, + "pyrene actin", + "chemical" + ], + [ + 94, + 101, + "Xenopus", + "taxonomy_domain" + ], + [ + 132, + 137, + "TOCA1", + "protein" + ], + [ + 138, + 141, + "HR1", + "structure_element" + ], + [ + 152, + 158, + "N-WASP", + "protein" + ], + [ + 159, + 162, + "GBD", + "structure_element" + ], + [ + 201, + 206, + "actin", + "protein_type" + ] + ] + }, + { + "sid": 228, + "sent": "Actin polymerization in all cases was initiated by the addition of PI(4,5)P2-containing liposomes.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "Actin", + "protein_type" + ], + [ + 67, + 76, + "PI(4,5)P2", + "chemical" + ] + ] + }, + { + "sid": 229, + "sent": "Actin polymerization triggered by the addition of PI(4,5)P2-containing liposomes has previously been shown to depend on TOCA1 and N-WASP.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "Actin", + "protein_type" + ], + [ + 50, + 59, + "PI(4,5)P2", + "chemical" + ], + [ + 120, + 125, + "TOCA1", + "protein" + ], + [ + 130, + 136, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 230, + "sent": "Endogenous N-WASP is present at \u223c100 nm in Xenopus extracts, whereas TOCA1 is present at a 10-fold lower concentration than N-WASP.", + "section": "RESULTS", + "ner": [ + [ + 11, + 17, + "N-WASP", + "protein" + ], + [ + 43, + 50, + "Xenopus", + "taxonomy_domain" + ], + [ + 69, + 74, + "TOCA1", + "protein" + ], + [ + 124, + 130, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 231, + "sent": "The addition of the isolated N-WASP GBD significantly inhibited the polymerization of actin at concentrations as low as 100 nm and completely abolished polymerization at higher concentrations (Fig. 8).", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "addition", + "experimental_method" + ], + [ + 29, + 35, + "N-WASP", + "protein" + ], + [ + 36, + 39, + "GBD", + "structure_element" + ], + [ + 86, + 91, + "actin", + "protein_type" + ] + ] + }, + { + "sid": 232, + "sent": "The GBD presumably acts as a dominant negative, sequestering endogenous Cdc42 and preventing endogenous full-length N-WASP from binding and becoming activated.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "GBD", + "structure_element" + ], + [ + 72, + 77, + "Cdc42", + "protein" + ], + [ + 93, + 103, + "endogenous", + "protein_state" + ], + [ + 104, + 115, + "full-length", + "protein_state" + ], + [ + 116, + 122, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 233, + "sent": "The addition of the TOCA1 HR1 domain to 100 \u03bcm had no significant effect on the rate of actin polymerization or maximum fluorescence.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "addition", + "experimental_method" + ], + [ + 20, + 25, + "TOCA1", + "protein" + ], + [ + 26, + 29, + "HR1", + "structure_element" + ], + [ + 88, + 93, + "actin", + "protein_type" + ], + [ + 112, + 132, + "maximum fluorescence", + "evidence" + ] + ] + }, + { + "sid": 234, + "sent": "This is consistent with endogenous N-WASP, activated by other components of the assay, outcompeting the TOCA1 HR1 domain for Cdc42 binding.", + "section": "RESULTS", + "ner": [ + [ + 24, + 34, + "endogenous", + "protein_state" + ], + [ + 35, + 41, + "N-WASP", + "protein" + ], + [ + 104, + 109, + "TOCA1", + "protein" + ], + [ + 110, + 113, + "HR1", + "structure_element" + ], + [ + 125, + 130, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 235, + "sent": "Actin polymerization downstream of Cdc42\u00b7N-WASP\u00b7TOCA1 is inhibited by excess N-WASP GBD but not by the TOCA1 HR1 domain.", + "section": "FIG", + "ner": [ + [ + 35, + 53, + "Cdc42\u00b7N-WASP\u00b7TOCA1", + "complex_assembly" + ], + [ + 57, + 66, + "inhibited", + "protein_state" + ], + [ + 77, + 83, + "N-WASP", + "protein" + ], + [ + 84, + 87, + "GBD", + "structure_element" + ], + [ + 103, + 108, + "TOCA1", + "protein" + ], + [ + 109, + 112, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 236, + "sent": "Fluorescence curves show actin polymerization in the presence of increasing concentrations of N-WASP GBD or TOCA1 HR1 domain as indicated.", + "section": "FIG", + "ner": [ + [ + 0, + 19, + "Fluorescence curves", + "evidence" + ], + [ + 53, + 64, + "presence of", + "protein_state" + ], + [ + 65, + 90, + "increasing concentrations", + "experimental_method" + ], + [ + 94, + 100, + "N-WASP", + "protein" + ], + [ + 101, + 104, + "GBD", + "structure_element" + ], + [ + 108, + 113, + "TOCA1", + "protein" + ], + [ + 114, + 117, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 237, + "sent": "The Cdc42-TOCA1 Interaction", + "section": "DISCUSS", + "ner": [ + [ + 4, + 9, + "Cdc42", + "protein" + ], + [ + 10, + 15, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 238, + "sent": "The TOCA1 HR1 domain alone is sufficient for Cdc42 binding in vitro, yet the affinity of the TOCA1 HR1 domain for Cdc42 is remarkably low (Kd \u2248 5 \u03bcm).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 9, + "TOCA1", + "protein" + ], + [ + 10, + 13, + "HR1", + "structure_element" + ], + [ + 21, + 26, + "alone", + "protein_state" + ], + [ + 45, + 50, + "Cdc42", + "protein" + ], + [ + 77, + 85, + "affinity", + "evidence" + ], + [ + 93, + 98, + "TOCA1", + "protein" + ], + [ + 99, + 102, + "HR1", + "structure_element" + ], + [ + 114, + 119, + "Cdc42", + "protein" + ], + [ + 139, + 141, + "Kd", + "evidence" + ] + ] + }, + { + "sid": 239, + "sent": "This is over 100 times lower than that of the N-WASP GBD (Kd = 37 nm) and considerably lower than other known G protein-HR1 domain interactions.", + "section": "DISCUSS", + "ner": [ + [ + 46, + 52, + "N-WASP", + "protein" + ], + [ + 53, + 56, + "GBD", + "structure_element" + ], + [ + 58, + 60, + "Kd", + "evidence" + ], + [ + 110, + 119, + "G protein", + "protein_type" + ], + [ + 120, + 123, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 240, + "sent": "The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction.", + "section": "DISCUSS", + "ner": [ + [ + 31, + 48, + "C-terminal region", + "structure_element" + ], + [ + 52, + 57, + "Cdc42", + "protein" + ], + [ + 104, + 109, + "TOCA1", + "protein" + ], + [ + 159, + 163, + "Rac1", + "protein" + ], + [ + 172, + 176, + "HR1b", + "structure_element" + ], + [ + 187, + 191, + "PRK1", + "protein" + ], + [ + 216, + 220, + "PRK1", + "protein" + ], + [ + 221, + 225, + "HR1a", + "structure_element" + ], + [ + 226, + 230, + "RhoA", + "protein" + ] + ] + }, + { + "sid": 241, + "sent": "A single binding interface on both the HR1 domain and Cdc42 can be concluded from the data presented here.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 26, + "binding interface", + "site" + ], + [ + 39, + 42, + "HR1", + "structure_element" + ], + [ + 54, + 59, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 242, + "sent": "Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions (Fig. 4), and the lowest energy model produced in rigid body docking resembles previously studied G protein\u00b7HR1 complexes (Fig. 6).", + "section": "DISCUSS", + "ner": [ + [ + 17, + 27, + "interfaces", + "site" + ], + [ + 63, + 72, + "G protein", + "protein_type" + ], + [ + 73, + 76, + "HR1", + "structure_element" + ], + [ + 122, + 127, + "model", + "evidence" + ], + [ + 140, + 158, + "rigid body docking", + "experimental_method" + ], + [ + 188, + 201, + "G protein\u00b7HR1", + "complex_assembly" + ] + ] + }, + { + "sid": 243, + "sent": "It seems, therefore, that the interaction, despite its relatively low affinity, is specific and sterically similar to other HR1 domain-G protein interactions.", + "section": "DISCUSS", + "ner": [ + [ + 124, + 127, + "HR1", + "structure_element" + ], + [ + 135, + 144, + "G protein", + "protein_type" + ] + ] + }, + { + "sid": 244, + "sent": "The TOCA1 HR1 domain is a left-handed coiled-coil comparable with other known HR1 domains.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 9, + "TOCA1", + "protein" + ], + [ + 10, + 13, + "HR1", + "structure_element" + ], + [ + 38, + 49, + "coiled-coil", + "structure_element" + ], + [ + 78, + 81, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 245, + "sent": "A short region N-terminal to the coiled-coil exhibits a series of turns and contacts residues of both helices of the coiled-coil (Fig. 3).", + "section": "DISCUSS", + "ner": [ + [ + 33, + 44, + "coiled-coil", + "structure_element" + ], + [ + 117, + 128, + "coiled-coil", + "structure_element" + ] + ] + }, + { + "sid": 246, + "sent": "The corresponding sequence in CIP4 also includes a series of turns but is flexible, whereas in the HR1a domain of PRK1, the equivalent region adopts an \u03b1-helical structure that packs against the coiled-coil.", + "section": "DISCUSS", + "ner": [ + [ + 30, + 34, + "CIP4", + "protein" + ], + [ + 99, + 103, + "HR1a", + "structure_element" + ], + [ + 114, + 118, + "PRK1", + "protein" + ], + [ + 152, + 171, + "\u03b1-helical structure", + "structure_element" + ], + [ + 195, + 206, + "coiled-coil", + "structure_element" + ] + ] + }, + { + "sid": 247, + "sent": "The contacts between the N-terminal region and the coiled-coil are predominantly hydrophobic in both cases, but sequence-specific contacts do not appear to be conserved.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 12, + "contacts", + "bond_interaction" + ], + [ + 51, + 62, + "coiled-coil", + "structure_element" + ], + [ + 81, + 92, + "hydrophobic", + "bond_interaction" + ] + ] + }, + { + "sid": 248, + "sent": "This region is distant from the G protein-binding interface of the HR1 domains, so the structural differences may relate to the structure and regulation of these domains rather than their G protein interactions.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 59, + "G protein-binding interface", + "site" + ], + [ + 67, + 70, + "HR1", + "structure_element" + ], + [ + 188, + 197, + "G protein", + "protein_type" + ] + ] + }, + { + "sid": 249, + "sent": "The interhelical loops of TOCA1 and CIP4 differ from the same region in the HR1 domains of PRK1 in that they are longer and contain two short stretches of 310-helix.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 22, + "interhelical loops", + "structure_element" + ], + [ + 26, + 31, + "TOCA1", + "protein" + ], + [ + 36, + 40, + "CIP4", + "protein" + ], + [ + 76, + 79, + "HR1", + "structure_element" + ], + [ + 91, + 95, + "PRK1", + "protein" + ], + [ + 155, + 164, + "310-helix", + "structure_element" + ] + ] + }, + { + "sid": 250, + "sent": "This region lies within the G protein-binding surface of all of the HR1 domains (Fig. 4D).", + "section": "DISCUSS", + "ner": [ + [ + 28, + 53, + "G protein-binding surface", + "site" + ], + [ + 68, + 71, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 251, + "sent": "TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "TOCA1", + "protein" + ], + [ + 10, + 14, + "CIP4", + "protein" + ], + [ + 35, + 40, + "Cdc42", + "protein" + ], + [ + 54, + 58, + "HR1a", + "structure_element" + ], + [ + 69, + 73, + "PRK1", + "protein" + ], + [ + 91, + 95, + "RhoA", + "protein" + ], + [ + 100, + 104, + "Rac1", + "protein" + ], + [ + 114, + 118, + "HR1b", + "structure_element" + ], + [ + 135, + 139, + "Rac1", + "protein" + ] + ] + }, + { + "sid": 252, + "sent": "The structural features shared by TOCA1 and CIP4 may therefore be related to Cdc42 binding specificity and the low affinities.", + "section": "DISCUSS", + "ner": [ + [ + 34, + 39, + "TOCA1", + "protein" + ], + [ + 44, + 48, + "CIP4", + "protein" + ], + [ + 77, + 82, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 253, + "sent": "In free TOCA1, the side chains of the interhelical region make extensive contacts with residues in helix 1.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 7, + "free", + "protein_state" + ], + [ + 8, + 13, + "TOCA1", + "protein" + ], + [ + 38, + 57, + "interhelical region", + "structure_element" + ], + [ + 99, + 106, + "helix 1", + "structure_element" + ] + ] + }, + { + "sid": 254, + "sent": "Many of these residues are significantly affected in the presence of Cdc42, so it is likely that the conformation of this loop is altered in the Cdc42 complex.", + "section": "DISCUSS", + "ner": [ + [ + 57, + 68, + "presence of", + "protein_state" + ], + [ + 69, + 74, + "Cdc42", + "protein" + ], + [ + 122, + 126, + "loop", + "structure_element" + ], + [ + 145, + 150, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 255, + "sent": "These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42.", + "section": "DISCUSS", + "ner": [ + [ + 67, + 75, + "mutation", + "experimental_method" + ], + [ + 79, + 85, + "Met383", + "residue_name_number" + ], + [ + 86, + 92, + "Gly384", + "residue_name_number" + ], + [ + 93, + 99, + "Asp385", + "residue_name_number" + ], + [ + 103, + 109, + "Ile383", + "residue_name_number" + ], + [ + 110, + 116, + "Ser384", + "residue_name_number" + ], + [ + 117, + 123, + "Thr385", + "residue_name_number" + ], + [ + 134, + 139, + "TOCA1", + "protein" + ], + [ + 151, + 156, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 256, + "sent": "The lowest energy model produced by HADDOCK using ambiguous interaction restraints from the titration data resembled the NMR structures of RhoA and Rac1 in complex with their HR1 domain partners.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 23, + "model", + "evidence" + ], + [ + 36, + 43, + "HADDOCK", + "experimental_method" + ], + [ + 92, + 101, + "titration", + "evidence" + ], + [ + 121, + 124, + "NMR", + "experimental_method" + ], + [ + 125, + 135, + "structures", + "evidence" + ], + [ + 139, + 143, + "RhoA", + "protein" + ], + [ + 148, + 152, + "Rac1", + "protein" + ], + [ + 153, + 168, + "in complex with", + "protein_state" + ], + [ + 175, + 178, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 257, + "sent": "For example, Phe-56Cdc42, which is not visible in free Cdc42 or Cdc42\u00b7HR1TOCA1, is close to the TOCA1 HR1 (Fig. 6A).", + "section": "DISCUSS", + "ner": [ + [ + 13, + 19, + "Phe-56", + "residue_name_number" + ], + [ + 19, + 24, + "Cdc42", + "protein" + ], + [ + 50, + 54, + "free", + "protein_state" + ], + [ + 55, + 60, + "Cdc42", + "protein" + ], + [ + 64, + 78, + "Cdc42\u00b7HR1TOCA1", + "complex_assembly" + ], + [ + 96, + 101, + "TOCA1", + "protein" + ], + [ + 102, + 105, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 258, + "sent": "Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (Fig. 6C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 6, + "Phe-56", + "residue_name_number" + ], + [ + 6, + 11, + "Cdc42", + "protein" + ], + [ + 24, + 27, + "Trp", + "residue_name" + ], + [ + 36, + 40, + "Rac1", + "protein" + ], + [ + 45, + 49, + "RhoA", + "protein" + ], + [ + 87, + 95, + "switch I", + "site" + ], + [ + 101, + 106, + "Cdc42", + "protein" + ], + [ + 122, + 125, + "ACK", + "protein" + ] + ] + }, + { + "sid": 259, + "sent": "This residue has also been identified as important for Cdc42-WASP binding.", + "section": "DISCUSS", + "ner": [ + [ + 55, + 60, + "Cdc42", + "protein" + ], + [ + 61, + 65, + "WASP", + "protein" + ] + ] + }, + { + "sid": 260, + "sent": "Phe-56Cdc42 is therefore likely to be involved in the Cdc42-TOCA1 interaction, probably by stabilizing the position of switch I.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 6, + "Phe-56", + "residue_name_number" + ], + [ + 6, + 11, + "Cdc42", + "protein" + ], + [ + 54, + 59, + "Cdc42", + "protein" + ], + [ + 60, + 65, + "TOCA1", + "protein" + ], + [ + 119, + 127, + "switch I", + "site" + ] + ] + }, + { + "sid": 261, + "sent": "Some residues that are affected in the Cdc42\u00b7HR1TOCA1 complex but do not correspond to contact residues of RhoA or Rac1 (Fig. 6C) may contact HR1TOCA1 directly (Fig. 6D).", + "section": "DISCUSS", + "ner": [ + [ + 39, + 53, + "Cdc42\u00b7HR1TOCA1", + "complex_assembly" + ], + [ + 107, + 111, + "RhoA", + "protein" + ], + [ + 115, + 119, + "Rac1", + "protein" + ], + [ + 142, + 145, + "HR1", + "structure_element" + ], + [ + 145, + 150, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 262, + "sent": "Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42\u00b7ACK complex, contacts Val-376TOCA1 and Asn-380TOCA1 in the model and disrupts the contacts between the interhelical loop and the first helix of the TOCA1 coiled-coil.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "Gln-2", + "residue_name_number" + ], + [ + 5, + 10, + "Cdc42", + "protein" + ], + [ + 71, + 80, + "Cdc42\u00b7ACK", + "complex_assembly" + ], + [ + 99, + 106, + "Val-376", + "residue_name_number" + ], + [ + 106, + 111, + "TOCA1", + "protein" + ], + [ + 116, + 123, + "Asn-380", + "residue_name_number" + ], + [ + 123, + 128, + "TOCA1", + "protein" + ], + [ + 180, + 197, + "interhelical loop", + "structure_element" + ], + [ + 206, + 217, + "first helix", + "structure_element" + ], + [ + 225, + 230, + "TOCA1", + "protein" + ], + [ + 231, + 242, + "coiled-coil", + "structure_element" + ] + ] + }, + { + "sid": 263, + "sent": "Thr-52Cdc42, which has also been identified as making minor contacts with ACK, falls near the side chains of HR1TOCA1 helix 1, particularly Lys-372TOCA1, whereas the equivalent position in Rac1 is Asn-52Rac1.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 6, + "Thr-52", + "residue_name_number" + ], + [ + 6, + 11, + "Cdc42", + "protein" + ], + [ + 74, + 77, + "ACK", + "protein" + ], + [ + 109, + 112, + "HR1", + "structure_element" + ], + [ + 112, + 117, + "TOCA1", + "protein" + ], + [ + 118, + 125, + "helix 1", + "structure_element" + ], + [ + 140, + 147, + "Lys-372", + "residue_name_number" + ], + [ + 147, + 152, + "TOCA1", + "protein" + ], + [ + 189, + 193, + "Rac1", + "protein" + ], + [ + 197, + 203, + "Asn-52", + "residue_name_number" + ], + [ + 203, + 207, + "Rac1", + "protein" + ] + ] + }, + { + "sid": 264, + "sent": "N52T is one of a combination of seven residues found to confer ACK binding on Rac1 and so may represent a specific Cdc42-effector contact residue.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "N52T", + "mutant" + ], + [ + 63, + 66, + "ACK", + "protein" + ], + [ + 78, + 82, + "Rac1", + "protein" + ], + [ + 115, + 120, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 265, + "sent": "The position equivalent to Lys-372TOCA1 in PRK1 is Glu-58HR1a or Gln-151HR1b.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 34, + "Lys-372", + "residue_name_number" + ], + [ + 34, + 39, + "TOCA1", + "protein" + ], + [ + 43, + 47, + "PRK1", + "protein" + ], + [ + 51, + 57, + "Glu-58", + "residue_name_number" + ], + [ + 57, + 61, + "HR1a", + "structure_element" + ], + [ + 65, + 72, + "Gln-151", + "residue_name_number" + ], + [ + 72, + 76, + "HR1b", + "structure_element" + ] + ] + }, + { + "sid": 266, + "sent": "Thr-52Cdc42-Lys-372TOCA1 may therefore represent a specific Cdc42-HR1TOCA1 contact.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 6, + "Thr-52", + "residue_name_number" + ], + [ + 6, + 11, + "Cdc42", + "protein" + ], + [ + 12, + 19, + "Lys-372", + "residue_name_number" + ], + [ + 19, + 24, + "TOCA1", + "protein" + ], + [ + 60, + 65, + "Cdc42", + "protein" + ], + [ + 66, + 69, + "HR1", + "structure_element" + ], + [ + 69, + 74, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 267, + "sent": "Arg-68Cdc42 of switch II is positioned close to Glu-395TOCA1 (Fig. 6D), suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 6, + "Arg-68", + "residue_name_number" + ], + [ + 6, + 11, + "Cdc42", + "protein" + ], + [ + 15, + 24, + "switch II", + "site" + ], + [ + 48, + 55, + "Glu-395", + "residue_name_number" + ], + [ + 55, + 60, + "TOCA1", + "protein" + ], + [ + 122, + 131, + "switch II", + "site" + ], + [ + 135, + 140, + "Cdc42", + "protein" + ], + [ + 145, + 152, + "helix 2", + "structure_element" + ], + [ + 160, + 163, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 268, + "sent": "The equivalent Arg in Rac1 and RhoA is pointing away from the HR1 domains of PRK1.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 18, + "Arg", + "residue_name" + ], + [ + 22, + 26, + "Rac1", + "protein" + ], + [ + 31, + 35, + "RhoA", + "protein" + ], + [ + 62, + 65, + "HR1", + "structure_element" + ], + [ + 77, + 81, + "PRK1", + "protein" + ] + ] + }, + { + "sid": 269, + "sent": "The importance of this residue in the Cdc42-TOCA1 interaction remains unclear, although its mutation reduces binding to RhoGAP, suggesting that it can be involved in Cdc42 interactions.", + "section": "DISCUSS", + "ner": [ + [ + 38, + 43, + "Cdc42", + "protein" + ], + [ + 44, + 49, + "TOCA1", + "protein" + ], + [ + 92, + 100, + "mutation", + "experimental_method" + ], + [ + 120, + 126, + "RhoGAP", + "protein" + ], + [ + 166, + 171, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 270, + "sent": "The solution structure of the TOCA1 HR1 domain presented here, along with the model of the HR1TOCA1\u00b7Cdc42 complex is consistent with a conserved mode of binding across the known HR1 domain-Rho family interactions, despite their differing affinities.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 22, + "solution structure", + "evidence" + ], + [ + 30, + 35, + "TOCA1", + "protein" + ], + [ + 36, + 39, + "HR1", + "structure_element" + ], + [ + 91, + 105, + "HR1TOCA1\u00b7Cdc42", + "complex_assembly" + ], + [ + 178, + 181, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 271, + "sent": "The weak binding prevented detailed structural and thermodynamic studies of the complex.", + "section": "DISCUSS", + "ner": [ + [ + 36, + 72, + "structural and thermodynamic studies", + "experimental_method" + ] + ] + }, + { + "sid": 272, + "sent": "Nonetheless, structural studies of the TOCA1 HR1 domain, combined with chemical shift mapping, have highlighted some potentially interesting differences between Cdc42-HR1TOCA1 and RhoA/Rac1-HR1PRK1 binding.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 31, + "structural studies", + "experimental_method" + ], + [ + 39, + 44, + "TOCA1", + "protein" + ], + [ + 45, + 48, + "HR1", + "structure_element" + ], + [ + 71, + 93, + "chemical shift mapping", + "experimental_method" + ], + [ + 161, + 166, + "Cdc42", + "protein" + ], + [ + 167, + 170, + "HR1", + "structure_element" + ], + [ + 170, + 175, + "TOCA1", + "protein" + ], + [ + 180, + 184, + "RhoA", + "protein" + ], + [ + 185, + 189, + "Rac1", + "protein" + ], + [ + 190, + 193, + "HR1", + "structure_element" + ], + [ + 193, + 197, + "PRK1", + "protein" + ] + ] + }, + { + "sid": 273, + "sent": "We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures (Tm) and Rac binders being more rigid.", + "section": "DISCUSS", + "ner": [ + [ + 63, + 66, + "HR1", + "structure_element" + ], + [ + 125, + 146, + "Rho family G proteins", + "protein_type" + ], + [ + 165, + 168, + "HR1", + "structure_element" + ], + [ + 236, + 256, + "melting temperatures", + "evidence" + ], + [ + 258, + 260, + "Tm", + "evidence" + ], + [ + 266, + 269, + "Rac", + "protein_type" + ] + ] + }, + { + "sid": 274, + "sent": "The Tm of the TOCA1 HR1 domain is 61.9 \u00b0C (data not shown), which is the highest Tm that we have measured for an HR1 domain thus far.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 6, + "Tm", + "evidence" + ], + [ + 14, + 19, + "TOCA1", + "protein" + ], + [ + 20, + 23, + "HR1", + "structure_element" + ], + [ + 81, + 83, + "Tm", + "evidence" + ], + [ + 113, + 116, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 275, + "sent": "As such, the ability of the TOCA1 HR1 domain to bind to Cdc42 (a close relative of Rac1 rather than RhoA) fits this trend.", + "section": "DISCUSS", + "ner": [ + [ + 28, + 33, + "TOCA1", + "protein" + ], + [ + 34, + 37, + "HR1", + "structure_element" + ], + [ + 56, + 61, + "Cdc42", + "protein" + ], + [ + 83, + 87, + "Rac1", + "protein" + ], + [ + 100, + 104, + "RhoA", + "protein" + ] + ] + }, + { + "sid": 276, + "sent": "An investigation into the local motions, particularly in the G protein-binding regions, may offer further insight into the differential specificities and affinities of G protein-HR1 domain interactions.", + "section": "DISCUSS", + "ner": [ + [ + 61, + 86, + "G protein-binding regions", + "site" + ], + [ + 168, + 177, + "G protein", + "protein_type" + ], + [ + 178, + 181, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 277, + "sent": "The low affinity of the Cdc42-HR1TOCA1 interaction is consistent with a tightly spatially and temporally regulated pathway, requiring combinatorial signals leading to a series of coincident weak interactions that elicit full activation.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 29, + "Cdc42", + "protein" + ], + [ + 30, + 33, + "HR1", + "structure_element" + ], + [ + 33, + 38, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 278, + "sent": "The HR1 domains from other TOCA family members, CIP4 and FBP17, also bind at low micromolar affinities to Cdc42, so the low affinity interaction appears to be commonplace among this family of HR1 domain proteins, in contrast to the PRK family.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 7, + "HR1", + "structure_element" + ], + [ + 27, + 46, + "TOCA family members", + "protein_type" + ], + [ + 48, + 52, + "CIP4", + "protein" + ], + [ + 57, + 62, + "FBP17", + "protein" + ], + [ + 106, + 111, + "Cdc42", + "protein" + ], + [ + 192, + 211, + "HR1 domain proteins", + "protein_type" + ], + [ + 232, + 242, + "PRK family", + "protein_type" + ] + ] + }, + { + "sid": 279, + "sent": "The low affinity of the HR1TOCA1-Cdc42 interaction in the context of the physiological concentration of TOCA1 in Xenopus extracts (\u223c10 nm) suggests that binding between TOCA1 and Cdc42 is likely to occur in vivo only when TOCA1 is at high local concentrations and membrane-localized and therefore in close proximity to activated Cdc42.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 32, + "HR1TOCA1", + "structure_element" + ], + [ + 33, + 38, + "Cdc42", + "protein" + ], + [ + 104, + 109, + "TOCA1", + "protein" + ], + [ + 113, + 120, + "Xenopus", + "taxonomy_domain" + ], + [ + 169, + 174, + "TOCA1", + "protein" + ], + [ + 179, + 184, + "Cdc42", + "protein" + ], + [ + 222, + 227, + "TOCA1", + "protein" + ], + [ + 319, + 328, + "activated", + "protein_state" + ], + [ + 329, + 334, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 280, + "sent": "Evidence suggests that the TOCA family of proteins are recruited to the membrane via an interaction between their F-BAR domain and specific signaling lipids.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 38, + "TOCA family", + "protein_type" + ], + [ + 114, + 119, + "F-BAR", + "structure_element" + ] + ] + }, + { + "sid": 281, + "sent": "For example, electrostatic interactions between the F-BAR domain and the membrane are required for TOCA1 recruitment to membrane vesicles and tubules, and TOCA1-dependent actin polymerization is known to depend specifically on PI(4,5)P2.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 39, + "electrostatic interactions", + "bond_interaction" + ], + [ + 52, + 57, + "F-BAR", + "structure_element" + ], + [ + 99, + 104, + "TOCA1", + "protein" + ], + [ + 155, + 160, + "TOCA1", + "protein" + ], + [ + 227, + 236, + "PI(4,5)P2", + "chemical" + ] + ] + }, + { + "sid": 282, + "sent": "Furthermore, the isolated F-BAR domain of FBP17 has been shown to induce membrane tubulation of brain liposomes and BAR domain proteins that promote tubulation cluster on membranes at high densities.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 25, + "isolated", + "experimental_method" + ], + [ + 26, + 31, + "F-BAR", + "structure_element" + ], + [ + 42, + 47, + "FBP17", + "protein" + ], + [ + 116, + 119, + "BAR", + "structure_element" + ] + ] + }, + { + "sid": 283, + "sent": "Once at the membrane, high local concentrations of TOCA1 could exceed the Kd of F-BAR dimerization (likely to be comparable with that of the FCHo2 F-BAR domain (2.5 \u03bcm)) and that of the Cdc42-HR1TOCA1 interaction.", + "section": "DISCUSS", + "ner": [ + [ + 51, + 56, + "TOCA1", + "protein" + ], + [ + 74, + 76, + "Kd", + "evidence" + ], + [ + 80, + 85, + "F-BAR", + "structure_element" + ], + [ + 86, + 91, + "dimer", + "oligomeric_state" + ], + [ + 141, + 146, + "FCHo2", + "protein" + ], + [ + 147, + 152, + "F-BAR", + "structure_element" + ], + [ + 186, + 191, + "Cdc42", + "protein" + ], + [ + 192, + 195, + "HR1", + "structure_element" + ], + [ + 195, + 200, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 284, + "sent": "Cdc42-HR1TOCA1 binding would then be favorable, as long as coincident activation of Cdc42 had occurred, leading to stabilization of TOCA1 at the membrane and downstream activation of N-WASP.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "Cdc42", + "protein" + ], + [ + 6, + 9, + "HR1", + "structure_element" + ], + [ + 9, + 14, + "TOCA1", + "protein" + ], + [ + 84, + 89, + "Cdc42", + "protein" + ], + [ + 132, + 137, + "TOCA1", + "protein" + ], + [ + 183, + 189, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 285, + "sent": "It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced.", + "section": "DISCUSS", + "ner": [ + [ + 28, + 32, + "WASP", + "protein_type" + ], + [ + 37, + 43, + "N-WASP", + "protein" + ], + [ + 73, + 79, + "folded", + "protein_state" + ], + [ + 81, + 89, + "inactive", + "protein_state" + ], + [ + 95, + 103, + "unfolded", + "protein_state" + ], + [ + 105, + 111, + "active", + "protein_state" + ], + [ + 128, + 136, + "affinity", + "evidence" + ], + [ + 140, + 145, + "Cdc42", + "protein" + ], + [ + 154, + 162, + "unfolded", + "protein_state" + ], + [ + 163, + 167, + "WASP", + "protein_type" + ] + ] + }, + { + "sid": 286, + "sent": "The unfolded, high affinity state of WASP is represented by a short peptide, the GBD, which binds with a low nanomolar affinity to Cdc42.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 12, + "unfolded", + "protein_state" + ], + [ + 37, + 41, + "WASP", + "protein_type" + ], + [ + 68, + 75, + "peptide", + "chemical" + ], + [ + 81, + 84, + "GBD", + "structure_element" + ], + [ + 131, + 136, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 287, + "sent": "In contrast, the best estimate of the affinity of full-length WASP for Cdc42 is low micromolar.", + "section": "DISCUSS", + "ner": [ + [ + 38, + 46, + "affinity", + "evidence" + ], + [ + 50, + 61, + "full-length", + "protein_state" + ], + [ + 62, + 66, + "WASP", + "protein_type" + ], + [ + 71, + 76, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 288, + "sent": "In the inactive state of WASP, the actin- and Arp2/3-binding VCA domain contacts the GBD, competing for Cdc42 binding.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 15, + "inactive", + "protein_state" + ], + [ + 25, + 29, + "WASP", + "protein_type" + ], + [ + 46, + 52, + "Arp2/3", + "complex_assembly" + ], + [ + 61, + 64, + "VCA", + "structure_element" + ], + [ + 85, + 88, + "GBD", + "structure_element" + ], + [ + 104, + 109, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 289, + "sent": "The high affinity of Cdc42 for the unfolded, active form pushes the equilibrium in favor of (N-)WASP activation.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 26, + "Cdc42", + "protein" + ], + [ + 35, + 43, + "unfolded", + "protein_state" + ], + [ + 45, + 51, + "active", + "protein_state" + ], + [ + 92, + 100, + "(N-)WASP", + "protein" + ] + ] + }, + { + "sid": 290, + "sent": "Binding of PI(4,5)P2 to the basic region just N-terminal to the GBD further favors the active conformation.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 20, + "PI(4,5)P2", + "chemical" + ], + [ + 64, + 67, + "GBD", + "structure_element" + ], + [ + 87, + 93, + "active", + "protein_state" + ] + ] + }, + { + "sid": 291, + "sent": "A substantial body of data has illuminated the complex regulation of WASP/N-WASP proteins, and current evidence suggests that these allosteric activation mechanisms and oligomerization combine to regulate WASP activity, allowing the synchronization and integration of multiple potential activation signals (reviewed in Ref.).", + "section": "DISCUSS", + "ner": [ + [ + 69, + 89, + "WASP/N-WASP proteins", + "protein_type" + ], + [ + 205, + 209, + "WASP", + "protein_type" + ] + ] + }, + { + "sid": 292, + "sent": "We envisage that TOCA1 is first recruited to the appropriate membrane in response to PI(4,5)P2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 22, + "TOCA1", + "protein" + ], + [ + 85, + 94, + "PI(4,5)P2", + "chemical" + ], + [ + 103, + 108, + "F-BAR", + "structure_element" + ], + [ + 166, + 171, + "F-BAR", + "structure_element" + ], + [ + 181, + 186, + "dimer", + "oligomeric_state" + ], + [ + 197, + 202, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 293, + "sent": "Cdc42 is activated in response to co-incident signals and can then bind to TOCA1, further stabilizing TOCA1 at the membrane.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "Cdc42", + "protein" + ], + [ + 75, + 80, + "TOCA1", + "protein" + ], + [ + 102, + 107, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 294, + "sent": "TOCA1 can then recruit N-WASP via an interaction between its SH3 domain and the N-WASP proline-rich region.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "TOCA1", + "protein" + ], + [ + 23, + 29, + "N-WASP", + "protein" + ], + [ + 61, + 64, + "SH3", + "structure_element" + ], + [ + 80, + 86, + "N-WASP", + "protein" + ], + [ + 87, + 106, + "proline-rich region", + "structure_element" + ] + ] + }, + { + "sid": 295, + "sent": "The recruitment of N-WASP alone and of the N-WASP\u00b7WIP complex by TOCA1 and FBP17 has been demonstrated.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 25, + "N-WASP", + "protein" + ], + [ + 26, + 31, + "alone", + "protein_state" + ], + [ + 43, + 53, + "N-WASP\u00b7WIP", + "complex_assembly" + ], + [ + 65, + 70, + "TOCA1", + "protein" + ], + [ + 75, + 80, + "FBP17", + "protein" + ] + ] + }, + { + "sid": 296, + "sent": "WIP inhibits the activation of N-WASP by Cdc42, an effect that is reversed by TOCA1.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 3, + "WIP", + "protein" + ], + [ + 31, + 37, + "N-WASP", + "protein" + ], + [ + 41, + 46, + "Cdc42", + "protein" + ], + [ + 78, + 83, + "TOCA1", + "protein" + ] + ] + }, + { + "sid": 297, + "sent": "It may therefore be envisaged that WIP and TOCA1 exert opposing allosteric effects on N-WASP, with TOCA1 favoring the unfolded, active conformation of N-WASP and increasing its affinity for Cdc42.", + "section": "DISCUSS", + "ner": [ + [ + 35, + 38, + "WIP", + "protein" + ], + [ + 43, + 48, + "TOCA1", + "protein" + ], + [ + 86, + 92, + "N-WASP", + "protein" + ], + [ + 99, + 104, + "TOCA1", + "protein" + ], + [ + 118, + 126, + "unfolded", + "protein_state" + ], + [ + 128, + 134, + "active", + "protein_state" + ], + [ + 151, + 157, + "N-WASP", + "protein" + ], + [ + 190, + 195, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 298, + "sent": "TOCA1 may also activate N-WASP by effective oligomerization because clustering of TOCA1 at the membrane following coincident interactions with PI(4,5)P2 and Cdc42 would in turn lead to clustering of N-WASP, in addition to pushing the equilibrium toward the unfolded, active state.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "TOCA1", + "protein" + ], + [ + 24, + 30, + "N-WASP", + "protein" + ], + [ + 82, + 87, + "TOCA1", + "protein" + ], + [ + 143, + 152, + "PI(4,5)P2", + "chemical" + ], + [ + 157, + 162, + "Cdc42", + "protein" + ], + [ + 199, + 205, + "N-WASP", + "protein" + ], + [ + 257, + 265, + "unfolded", + "protein_state" + ], + [ + 267, + 273, + "active", + "protein_state" + ] + ] + }, + { + "sid": 299, + "sent": "In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 34, + "full-length", + "protein_state" + ], + [ + 35, + 40, + "TOCA1", + "protein" + ], + [ + 45, + 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+ ], + [ + 193, + 198, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 302, + "sent": "A combination of allosteric activation by PI(4,5)P2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization.", + "section": "DISCUSS", + "ner": [ + [ + 42, + 51, + "PI(4,5)P2", + "chemical" + ], + [ + 53, + 62, + "activated", + "protein_state" + ], + [ + 63, + 68, + "Cdc42", + "protein" + ], + [ + 73, + 78, + "TOCA1", + "protein" + ], + [ + 121, + 126, + "TOCA1", + "protein" + ], + [ + 141, + 156, + "full activation", + "protein_state" + ], + [ + 160, + 166, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 303, + "sent": "In such an array of molecules localized to a discrete region of the membrane, it is plausible that WASP could bind to a second Cdc42 molecule rather than displacing TOCA1 from its cognate Cdc42.", + "section": "DISCUSS", + "ner": [ + [ + 99, + 103, + "WASP", + "protein" + ], + [ + 127, + 132, + "Cdc42", + "protein" + ], + [ + 165, + 170, + "TOCA1", + "protein" + ], + [ + 188, + 193, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 304, + "sent": "Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 7, + "NMR", + "experimental_method" + ], + [ + 12, + 25, + "affinity data", + "evidence" + ], + [ + 76, + 81, + "TOCA1", + "protein" + ], + [ + 82, + 85, + "HR1", + "structure_element" + ], + [ + 93, + 99, + "N-WASP", + "protein" + ], + [ + 100, + 103, + "GBD", + "structure_element" + ] + ] + }, + { + "sid": 305, + "sent": "Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP\u00b7WIP, implying that it may not be possible for Cdc42 to bind and activate N-WASP prior to TOCA1-Cdc42 binding.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 18, + "TOCA1", + "protein" + ], + [ + 35, + 40, + "Cdc42", + "protein" + ], + [ + 64, + 74, + "N-WASP\u00b7WIP", + "complex_assembly" + ], + [ + 117, + 122, + "Cdc42", + "protein" + ], + [ + 144, + 150, + "N-WASP", + "protein" + ], + [ + 160, + 165, + "TOCA1", + "protein" + ], + [ + 166, + 171, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 306, + "sent": "The commonly used MGD \u2192 IST (Cdc42-binding deficient) mutant of TOCA1 has a reduced ability to activate the N-WASP\u00b7WIP complex, further indicating the importance of the Cdc42-HR1TOCA1 interaction prior to downstream activation of N-WASP.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 27, + "MGD \u2192 IST", + "mutant" + ], + [ + 29, + 52, + "Cdc42-binding deficient", + "protein_state" + ], + [ + 64, + 69, + "TOCA1", + "protein" + ], + [ + 108, + 118, + "N-WASP\u00b7WIP", + "complex_assembly" + ], + [ + 169, + 174, + "Cdc42", + "protein" + ], + [ + 175, + 178, + "HR1", + "structure_element" + ], + [ + 178, + 183, + "TOCA1", + "protein" + ], + [ + 230, + 236, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 307, + "sent": "In light of this, we favor an \u201ceffector handover\u201d scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule.", + "section": "DISCUSS", + "ner": [ + [ + 65, + 70, + "TOCA1", + "protein" + ], + [ + 86, + 91, + "Cdc42", + "protein" + ], + [ + 101, + 107, + "N-WASP", + "protein" + ], + [ + 132, + 138, + "N-WASP", + "protein" + ], + [ + 149, + 154, + "TOCA1", + "protein" + ], + [ + 164, + 169, + "bound", + "protein_state" + ], + [ + 170, + 175, + "Cdc42", + "protein" + ], + [ + 191, + 206, + "fully activated", + "protein_state" + ], + [ + 236, + 241, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 308, + "sent": "Potentially, the TOCA1-Cdc42 interaction functions to position N-WASP and Cdc42 such that they are poised to interact with high affinity.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 22, + "TOCA1", + "protein" + ], + [ + 23, + 28, + "Cdc42", + "protein" + ], + [ + 63, + 69, + "N-WASP", + "protein" + ], + [ + 74, + 79, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 309, + "sent": "The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP\u00b7WIP-induced actin polymerization.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 32, + "TOCA1", + "protein" + ], + [ + 38, + 43, + "Cdc42", + "protein" + ], + [ + 56, + 64, + "bound to", + "protein_state" + ], + [ + 65, + 71, + "N-WASP", + "protein" + ], + [ + 107, + 112, + "TOCA1", + "protein" + ], + [ + 133, + 143, + "N-WASP\u00b7WIP", + "complex_assembly" + ] + ] + }, + { + "sid": 310, + "sent": "There is an advantage to such an effector handover, in that N-WASP would only be robustly recruited when F-BAR domains are already present.", + "section": "DISCUSS", + "ner": [ + [ + 60, + 66, + "N-WASP", + "protein" + ], + [ + 105, + 110, + "F-BAR", + "structure_element" + ] + ] + }, + { + "sid": 311, + "sent": "Hence, actin polymerization cannot occur until F-BAR domains are poised for membrane distortion.", + "section": "DISCUSS", + "ner": [ + [ + 47, + 52, + "F-BAR", + "structure_element" + ] + ] + }, + { + "sid": 312, + "sent": "Our model of the Cdc42\u00b7HR1TOCA1 complex indicates a mechanism by which such a handover could take place (Fig. 9) because it shows that the effector binding sites only partially overlap on Cdc42.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 31, + "Cdc42\u00b7HR1TOCA1", + "complex_assembly" + ], + [ + 139, + 161, + "effector binding sites", + "site" + ], + [ + 188, + 193, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 313, + "sent": "The lysine residues thought to be involved in an electrostatic steering mechanism in WASP-Cdc42 binding are conserved in N-WASP and would be able to interact with Cdc42 even when the TOCA1 HR1 domain is already bound.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 10, + "lysine", + "residue_name" + ], + [ + 85, + 89, + "WASP", + "protein" + ], + [ + 90, + 95, + "Cdc42", + "protein" + ], + [ + 121, + 127, + "N-WASP", + "protein" + ], + [ + 163, + 168, + "Cdc42", + "protein" + ], + [ + 183, + 188, + "TOCA1", + "protein" + ], + [ + 189, + 192, + "HR1", + "structure_element" + ], + [ + 211, + 216, + "bound", + "protein_state" + ] + ] + }, + { + "sid": 314, + "sent": "It has been postulated that the initial interactions between this basic region and Cdc42 could stabilize the active conformation of WASP, leading to high affinity binding between the core CRIB and Cdc42.", + "section": "DISCUSS", + "ner": [ + [ + 83, + 88, + "Cdc42", + "protein" + ], + [ + 109, + 115, + "active", + "protein_state" + ], + [ + 132, + 136, + "WASP", + "protein" + ], + [ + 188, + 192, + "CRIB", + "structure_element" + ], + [ + 197, + 202, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 315, + "sent": "The region C-terminal to the core CRIB, required for maximal affinity binding, would then fully displace the TOCA1 HR1.", + "section": "DISCUSS", + "ner": [ + [ + 34, + 38, + "CRIB", + "structure_element" + ], + [ + 109, + 114, + "TOCA1", + "protein" + ], + [ + 115, + 118, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 316, + "sent": "A simplified model of the early stages of Cdc42\u00b7N-WASP\u00b7TOCA1-dependent actin polymerization.", + "section": "FIG", + "ner": [ + [ + 42, + 60, + "Cdc42\u00b7N-WASP\u00b7TOCA1", + "complex_assembly" + ] + ] + }, + { + "sid": 317, + "sent": " Step 1, TOCA1 is recruited to the membrane via its F-BAR domain and/or Cdc42 interactions.", + "section": "FIG", + "ner": [ + [ + 9, + 14, + "TOCA1", + "protein" + ], + [ + 52, + 57, + "F-BAR", + "structure_element" + ], + [ + 72, + 77, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 318, + "sent": "F-BAR oligomerization is expected to occur following membrane binding, but a single monomer is shown for clarity.", + "section": "FIG", + "ner": [ + [ + 84, + 91, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 319, + "sent": "Step 2, N-WASP exists in an inactive, folded conformation.", + "section": "FIG", + "ner": [ + [ + 8, + 14, + "N-WASP", + "protein" + ], + [ + 28, + 36, + "inactive", + "protein_state" + ], + [ + 38, + 44, + "folded", + "protein_state" + ] + ] + }, + { + "sid": 320, + "sent": "The TOCA1 SH3 domain interacts with N-WASP, causing an activatory allosteric effect.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "TOCA1", + "protein" + ], + [ + 10, + 13, + "SH3", + "structure_element" + ], + [ + 36, + 42, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 321, + "sent": "The HR1TOCA1-Cdc42 and SH3TOCA1-N-WASP interactions position Cdc42 and N-WASP for binding.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "HR1", + "structure_element" + ], + [ + 23, + 26, + "SH3", + "structure_element" + ], + [ + 61, + 66, + "Cdc42", + "protein" + ], + [ + 71, + 77, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 322, + "sent": "Step 3, electrostatic interactions between Cdc42 and the basic region upstream of the CRIB initiate Cdc42\u00b7N-WASP binding.", + "section": "FIG", + "ner": [ + [ + 8, + 34, + "electrostatic interactions", + "bond_interaction" + ], + [ + 43, + 48, + "Cdc42", + "protein" + ], + [ + 86, + 90, + "CRIB", + "structure_element" + ], + [ + 100, + 112, + "Cdc42\u00b7N-WASP", + "complex_assembly" + ] + ] + }, + { + "sid": 323, + "sent": "Step 4, the core CRIB binds with high affinity while the region C-terminal to the CRIB displaces the TOCA1 HR1 domain and increases the affinity of the N-WASP-Cdc42 interaction further.", + "section": "FIG", + "ner": [ + [ + 17, + 21, + "CRIB", + "structure_element" + ], + [ + 82, + 86, + "CRIB", + "structure_element" + ], + [ + 101, + 106, + "TOCA1", + "protein" + ], + [ + 107, + 110, + "HR1", + "structure_element" + ], + [ + 152, + 158, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 324, + "sent": "The VCA domain is released for downstream interactions, and actin polymerization proceeds.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "VCA", + "structure_element" + ] + ] + }, + { + "sid": 325, + "sent": "WH1, WASP homology 1 domain; PP, proline-rich region; VCA, verprolin homology, cofilin homology, acidic region.", + "section": "FIG", + "ner": [ + [ + 5, + 27, + "WASP homology 1 domain", + "structure_element" + ], + [ + 29, + 31, + "PP", + "structure_element" + ], + [ + 33, + 52, + "proline-rich region", + "structure_element" + ], + [ + 54, + 57, + "VCA", + "structure_element" + ], + [ + 59, + 110, + "verprolin homology, cofilin homology, acidic region", + "structure_element" + ] + ] + }, + { + "sid": 326, + "sent": "In conclusion, the data presented here show that the TOCA1 HR1 domain is sufficient for Cdc42 binding in vitro and that the interaction is of micromolar affinity, lower than that of other G protein-HR1 domain interactions.", + "section": "DISCUSS", + "ner": [ + [ + 53, + 58, + "TOCA1", + "protein" + ], + [ + 59, + 62, + "HR1", + "structure_element" + ], + [ + 88, + 93, + "Cdc42", + "protein" + ], + [ + 188, + 197, + "G protein", + "protein_type" + ], + [ + 198, + 201, + "HR1", + "structure_element" + ] + ] + }, + { + "sid": 327, + "sent": "The analogous HR1 domains from other TOCA1 family members, FBP17 and CIP4, also exhibit micromolar affinity for Cdc42.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 17, + "HR1", + "structure_element" + ], + [ + 37, + 49, + "TOCA1 family", + "protein_type" + ], + [ + 59, + 64, + "FBP17", + "protein" + ], + [ + 69, + 73, + "CIP4", + "protein" + ], + [ + 112, + 117, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 328, + "sent": "A role for the TOCA1-, FBP17-, and CIP4-Cdc42 interactions in the recruitment of these proteins to the membrane therefore appears unlikely.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 20, + "TOCA1", + "protein" + ], + [ + 23, + 28, + "FBP17", + "protein" + ], + [ + 35, + 39, + "CIP4", + "protein" + ], + [ + 40, + 45, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 329, + "sent": "Instead, our findings agree with earlier suggestions that the F-BAR domain is responsible for membrane recruitment.", + "section": "DISCUSS", + "ner": [ + [ + 62, + 67, + "F-BAR", + "structure_element" + ] + ] + }, + { + "sid": 330, + "sent": "The role of the Cdc42-TOCA1 interaction remains somewhat elusive, but it may serve to position activated Cdc42 and N-WASP to allow full activation of N-WASP and as such serve to couple F-BAR-mediated membrane deformation with N-WASP activation.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 21, + "Cdc42", + "protein" + ], + [ + 22, + 27, + "TOCA1", + "protein" + ], + [ + 95, + 104, + "activated", + "protein_state" + ], + [ + 105, + 110, + "Cdc42", + "protein" + ], + [ + 115, + 121, + "N-WASP", + "protein" + ], + [ + 131, + 146, + "full activation", + "protein_state" + ], + [ + 150, + 156, + "N-WASP", + "protein" + ], + [ + 185, + 190, + "F-BAR", + "structure_element" + ], + [ + 226, + 232, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 331, + "sent": "We envisage a complex interplay of equilibria between free and bound, active and inactive Cdc42, TOCA family, and WASP family proteins, facilitating a tightly spatially and temporally regulated pathway requiring numerous simultaneous events in order to achieve appropriate and robust activation of the downstream pathway.", + "section": "DISCUSS", + "ner": [ + [ + 54, + 58, + "free", + "protein_state" + ], + [ + 63, + 68, + "bound", + "protein_state" + ], + [ + 70, + 76, + "active", + "protein_state" + ], + [ + 81, + 89, + "inactive", + "protein_state" + ], + [ + 90, + 95, + "Cdc42", + "protein" + ], + [ + 97, + 108, + "TOCA family", + "protein_type" + ], + [ + 114, + 118, + "WASP", + "protein_type" + ] + ] + }, + { + "sid": 332, + "sent": "Our data are therefore easily reconciled with the dynamic instability models described in relation to the formation of endocytic vesicles and with the current data pertaining to the complex activation of WASP/N-WASP pathways by allosteric and oligomeric effects.", + "section": "DISCUSS", + "ner": [ + [ + 204, + 208, + "WASP", + "protein_type" + ], + [ + 209, + 215, + "N-WASP", + "protein" + ] + ] + }, + { + "sid": 333, + "sent": "It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding.", + "section": "DISCUSS", + "ner": [ + [ + 46, + 51, + "TOCA1", + "protein" + ], + [ + 56, + 62, + "N-WASP", + "protein" + ], + [ + 75, + 80, + "Cdc42", + "protein" + ], + [ + 105, + 111, + "N-WASP", + "protein" + ], + [ + 136, + 141, + "TOCA1", + "protein" + ], + [ + 146, + 151, + "Cdc42", + "protein" + ] + ] + }, + { + "sid": 334, + "sent": "We therefore postulate an effector handover mechanism based on current evidence surrounding WASP/N-WASP activation and our model of the Cdc42\u00b7HR1TOCA1 complex.", + "section": "DISCUSS", + "ner": [ + [ + 92, + 96, + "WASP", + "protein" + ], + [ + 97, + 103, + "N-WASP", + "protein" + ], + [ + 136, + 150, + "Cdc42\u00b7HR1TOCA1", + "complex_assembly" + ] + ] + }, + { + "sid": 335, + "sent": "The displacement of the TOCA1 HR1 domain from Cdc42 by N-WASP may represent a unidirectional step in the pathway of Cdc42\u00b7N-WASP\u00b7TOCA1-dependent actin assembly.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 29, + "TOCA1", + "protein" + ], + [ + 30, + 33, + "HR1", + "structure_element" + ], + [ + 46, + 51, + "Cdc42", + "protein" + ], + [ + 55, + 61, + "N-WASP", + "protein" + ], + [ + 116, + 134, + "Cdc42\u00b7N-WASP\u00b7TOCA1", + "complex_assembly" + ] + ] + } + ] + }, + "PMC4833862": { + "annotations": [ + { + "sid": 0, + "sent": "The dynamic organization of fungal acetyl-CoA carboxylase", + "section": "TITLE", + "ner": [ + [ + 4, + 11, + "dynamic", + "protein_state" + ], + [ + 28, + 34, + "fungal", + "taxonomy_domain" + ], + [ + 35, + 57, + "acetyl-CoA carboxylase", + "protein_type" + ] + ] + }, + { + "sid": 1, + "sent": "Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 23, + "Acetyl-CoA carboxylases", + "protein_type" + ], + [ + 25, + 29, + "ACCs", + "protein_type" + ], + [ + 91, + 94, + "ATP", + "chemical" + ], + [ + 122, + 132, + "acetyl-CoA", + "chemical" + ], + [ + 136, + 147, + "malonyl-CoA", + "chemical" + ] + ] + }, + { + "sid": 2, + "sent": "Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 10, + "Eukaryotic", + "taxonomy_domain" + ], + [ + 11, + 15, + "ACCs", + "protein_type" + ], + [ + 20, + 45, + "single-chain multienzymes", + "protein_type" + ], + [ + 72, + 85, + "non-catalytic", + "protein_state" + ], + [ + 86, + 100, + "central domain", + "structure_element" + ], + [ + 102, + 104, + "CD", + "structure_element" + ], + [ + 121, + 124, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 3, + "sent": "Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization.", + "section": "ABSTRACT", + "ner": [ + [ + 19, + 36, + "crystal structure", + "evidence" + ], + [ + 44, + 49, + "yeast", + "taxonomy_domain" + ], + [ + 50, + 53, + "ACC", + "protein_type" + ], + [ + 54, + 56, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 4, + "sent": "A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD.", + "section": "ABSTRACT", + "ner": [ + [ + 2, + 17, + "regulatory loop", + "structure_element" + ], + [ + 28, + 42, + "phosphorylated", + "protein_state" + ], + [ + 65, + 85, + "phosphorylation site", + "site" + ], + [ + 89, + 95, + "fungal", + "taxonomy_domain" + ], + [ + 96, + 99, + "ACC", + "protein_type" + ], + [ + 146, + 148, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 5, + "sent": "Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture.", + "section": "ABSTRACT", + "ner": [ + [ + 14, + 19, + "yeast", + "taxonomy_domain" + ], + [ + 20, + 22, + "CD", + "structure_element" + ], + [ + 23, + 32, + "structure", + "evidence" + ], + [ + 78, + 94, + "larger fragments", + "mutant" + ], + [ + 101, + 107, + "intact", + "protein_state" + ], + [ + 108, + 112, + "ACCs", + "protein_type" + ], + [ + 162, + 169, + "dynamic", + "protein_state" + ], + [ + 170, + 176, + "fungal", + "taxonomy_domain" + ], + [ + 177, + 180, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 6, + "sent": "In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.", + "section": "ABSTRACT", + "ner": [ + [ + 23, + 35, + "carboxylases", + "protein_type" + ], + [ + 133, + 135, + "CD", + "structure_element" + ], + [ + 142, + 157, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 7, + "sent": " Acetyl-CoA carboxylases are central regulatory hubs of fatty acid metabolism and are important targets for drug development in obesity and cancer.", + "section": "ABSTRACT", + "ner": [ + [ + 1, + 24, + "Acetyl-CoA carboxylases", + "protein_type" + ] + ] + }, + { + "sid": 8, + "sent": "Here, the authors demonstrate that the regulation of these highly dynamic enzymes in fungi is governed by a mechanism based on phosphorylation-dependent conformational variability.", + "section": "ABSTRACT", + "ner": [ + [ + 59, + 73, + "highly dynamic", + "protein_state" + ], + [ + 74, + 81, + "enzymes", + "protein_type" + ], + [ + 85, + 90, + "fungi", + "taxonomy_domain" + ], + [ + 127, + 142, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 9, + "sent": "Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase.", + "section": "INTRO", + "ner": [ + [ + 0, + 40, + "Biotin-dependent acetyl-CoA carboxylases", + "protein_type" + ], + [ + 42, + 46, + "ACCs", + "protein_type" + ], + [ + 88, + 91, + "ATP", + "chemical" + ], + [ + 119, + 129, + "acetyl-CoA", + "chemical" + ], + [ + 133, + 144, + "malonyl-CoA", + "chemical" + ], + [ + 227, + 238, + "fatty acids", + "chemical" + ], + [ + 243, + 262, + "fatty-acid synthase", + "protein_type" + ] + ] + }, + { + "sid": 10, + "sent": "By catalysing this rate-limiting step in fatty-acid biosynthesis, ACC plays a key role in anabolic metabolism.", + "section": "INTRO", + "ner": [ + [ + 66, + 69, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 11, + "sent": "ACC inhibition and knock-out studies show the potential of targeting ACC for treatment of the metabolic syndrome.", + "section": "INTRO", + "ner": [ + [ + 0, + 36, + "ACC inhibition and knock-out studies", + "experimental_method" + ], + [ + 69, + 72, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 12, + "sent": "Furthermore, elevated ACC activity is observed in malignant tumours.", + "section": "INTRO", + "ner": [ + [ + 22, + 25, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 13, + "sent": "A direct link between ACC and cancer is provided by cancer-associated mutations in the breast cancer susceptibility gene 1 (BRCA1), which relieve inhibitory interactions of BRCA1 with ACC.", + "section": "INTRO", + "ner": [ + [ + 22, + 25, + "ACC", + "protein_type" + ], + [ + 70, + 79, + "mutations", + "mutant" + ], + [ + 87, + 122, + "breast cancer susceptibility gene 1", + "protein" + ], + [ + 124, + 129, + "BRCA1", + "protein" + ], + [ + 173, + 178, + "BRCA1", + "protein" + ], + [ + 184, + 187, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 14, + "sent": "Thus, ACC is a relevant drug target for type 2 diabetes and cancer.", + "section": "INTRO", + "ner": [ + [ + 6, + 9, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 15, + "sent": "Microbial ACCs are also the principal target of antifungal and antibiotic compounds, such as Soraphen A.", + "section": "INTRO", + "ner": [ + [ + 0, + 9, + "Microbial", + "taxonomy_domain" + ], + [ + 10, + 14, + "ACCs", + "protein_type" + ], + [ + 93, + 103, + "Soraphen A", + "chemical" + ] + ] + }, + { + "sid": 16, + "sent": "The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP).", + "section": "INTRO", + "ner": [ + [ + 47, + 51, + "ACCs", + "protein_type" + ], + [ + 102, + 118, + "Escherichia coli", + "species" + ], + [ + 120, + 127, + "E. coli", + "species" + ], + [ + 129, + 132, + "ACC", + "protein_type" + ], + [ + 134, + 152, + "Biotin carboxylase", + "protein_type" + ], + [ + 154, + 156, + "BC", + "protein_type" + ], + [ + 172, + 175, + "ATP", + "chemical" + ], + [ + 205, + 211, + "biotin", + "chemical" + ], + [ + 254, + 285, + "biotin carboxyl carrier protein", + "protein_type" + ], + [ + 287, + 291, + "BCCP", + "protein_type" + ] + ] + }, + { + "sid": 17, + "sent": "Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits.", + "section": "INTRO", + "ner": [ + [ + 0, + 19, + "Carboxyltransferase", + "protein_type" + ], + [ + 21, + 23, + "CT", + "protein_type" + ], + [ + 49, + 57, + "carboxyl", + "chemical" + ], + [ + 69, + 82, + "carboxybiotin", + "chemical" + ], + [ + 86, + 96, + "acetyl-CoA", + "chemical" + ], + [ + 106, + 117, + "malonyl-CoA", + "chemical" + ], + [ + 119, + 130, + "Prokaryotic", + "taxonomy_domain" + ], + [ + 131, + 135, + "ACCs", + "protein_type" + ], + [ + 140, + 149, + "transient", + "protein_state" + ], + [ + 175, + 177, + "BC", + "protein_type" + ], + [ + 179, + 181, + "CT", + "protein_type" + ], + [ + 186, + 190, + "BCCP", + "protein_type" + ] + ] + }, + { + "sid": 18, + "sent": "Eukaryotic ACCs, instead, are multienzymes, which integrate all functional components into a single polypeptide chain of \u223c2,300 amino acids.", + "section": "INTRO", + "ner": [ + [ + 0, + 10, + "Eukaryotic", + "taxonomy_domain" + ], + [ + 11, + 15, + "ACCs", + "protein_type" + ], + [ + 30, + 42, + "multienzymes", + "protein_type" + ] + ] + }, + { + "sid": 19, + "sent": "Human ACC occurs in two closely related isoforms, ACC1 and 2, located in the cytosol and at the outer mitochondrial membrane, respectively.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "Human", + "species" + ], + [ + 6, + 9, + "ACC", + "protein_type" + ], + [ + 40, + 48, + "isoforms", + "protein_state" + ], + [ + 50, + 54, + "ACC1", + "protein" + ], + [ + 59, + 60, + "2", + "protein" + ] + ] + }, + { + "sid": 20, + "sent": "In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC\u2013CT interaction domain (BT).", + "section": "INTRO", + "ner": [ + [ + 29, + 43, + "ACC components", + "structure_element" + ], + [ + 45, + 55, + "eukaryotic", + "taxonomy_domain" + ], + [ + 56, + 60, + "ACCs", + "protein_type" + ], + [ + 73, + 86, + "non-catalytic", + "protein_state" + ], + [ + 87, + 94, + "regions", + "structure_element" + ], + [ + 106, + 120, + "central domain", + "structure_element" + ], + [ + 122, + 124, + "CD", + "structure_element" + ], + [ + 134, + 158, + "BC\u2013CT interaction domain", + "structure_element" + ], + [ + 160, + 162, + "BT", + "structure_element" + ] + ] + }, + { + "sid": 21, + "sent": "The CD comprises one-third of the protein and is a unique feature of eukaryotic ACCs without homologues in other proteins.", + "section": "INTRO", + "ner": [ + [ + 4, + 6, + "CD", + "structure_element" + ], + [ + 51, + 68, + "unique feature of", + "protein_state" + ], + [ + 69, + 79, + "eukaryotic", + "taxonomy_domain" + ], + [ + 80, + 84, + "ACCs", + "protein_type" + ] + ] + }, + { + "sid": 22, + "sent": "The function of this domain remains poorly characterized, although phosphorylation of several serine residues in the CD regulates ACC activity.", + "section": "INTRO", + "ner": [ + [ + 67, + 82, + "phosphorylation", + "ptm" + ], + [ + 94, + 100, + "serine", + "residue_name" + ], + [ + 117, + 119, + "CD", + "structure_element" + ], + [ + 130, + 133, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 23, + "sent": "The BT domain has been visualized in bacterial carboxylases, where it mediates contacts between \u03b1- and \u03b2-subunits.", + "section": "INTRO", + "ner": [ + [ + 4, + 6, + "BT", + "structure_element" + ], + [ + 37, + 46, + "bacterial", + "taxonomy_domain" + ], + [ + 47, + 59, + "carboxylases", + "protein_type" + ], + [ + 96, + 98, + "\u03b1-", + "structure_element" + ], + [ + 103, + 113, + "\u03b2-subunits", + "structure_element" + ] + ] + }, + { + "sid": 24, + "sent": "Structural studies on the functional architecture of intact ACCs have been hindered by their huge size and pronounced dynamics, as well as the transient assembly mode of bacterial ACCs.", + "section": "INTRO", + "ner": [ + [ + 0, + 18, + "Structural studies", + "experimental_method" + ], + [ + 53, + 59, + "intact", + "protein_state" + ], + [ + 60, + 64, + "ACCs", + "protein_type" + ], + [ + 143, + 152, + "transient", + "protein_state" + ], + [ + 170, + 179, + "bacterial", + "taxonomy_domain" + ], + [ + 180, + 184, + "ACCs", + "protein_type" + ] + ] + }, + { + "sid": 25, + "sent": "However, crystal structures of individual components or domains from prokaryotic and eukaryotic ACCs, respectively, have been solved.", + "section": "INTRO", + "ner": [ + [ + 9, + 27, + "crystal structures", + "evidence" + ], + [ + 69, + 80, + "prokaryotic", + "taxonomy_domain" + ], + [ + 85, + 95, + "eukaryotic", + "taxonomy_domain" + ], + [ + 96, + 100, + "ACCs", + "protein_type" + ] + ] + }, + { + "sid": 26, + "sent": "The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components.", + "section": "INTRO", + "ner": [ + [ + 4, + 27, + "structure determination", + "experimental_method" + ], + [ + 35, + 46, + "holoenzymes", + "protein_state" + ], + [ + 50, + 59, + "bacterial", + "taxonomy_domain" + ], + [ + 60, + 89, + "biotin-dependent carboxylases", + "protein_type" + ], + [ + 97, + 101, + "lack", + "protein_state" + ], + [ + 121, + 123, + "CD", + "structure_element" + ], + [ + 137, + 157, + "pyruvate carboxylase", + "protein_type" + ], + [ + 159, + 161, + "PC", + "protein_type" + ], + [ + 164, + 189, + "propionyl-CoA carboxylase", + "protein_type" + ], + [ + 191, + 224, + "3-methyl-crotonyl-CoA carboxylase", + "protein_type" + ], + [ + 231, + 262, + "long-chain acyl-CoA carboxylase", + "protein_type" + ] + ] + }, + { + "sid": 27, + "sent": "In these structures, the BC and CT active sites are at distances between 40 and 80\u2009\u00c5, such that substrate transfer could be mediated solely by the mobility of the flexibly tethered BCCP.", + "section": "INTRO", + "ner": [ + [ + 9, + 19, + "structures", + "evidence" + ], + [ + 25, + 27, + "BC", + "protein_type" + ], + [ + 32, + 34, + "CT", + "protein_type" + ], + [ + 35, + 47, + "active sites", + "site" + ], + [ + 163, + 180, + "flexibly tethered", + "protein_state" + ], + [ + 181, + 185, + "BCCP", + "protein_type" + ] + ] + }, + { + "sid": 28, + "sent": "Human ACC1 is regulated allosterically, via specific protein\u2013protein interactions, and by reversible phosphorylation.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "Human", + "species" + ], + [ + 6, + 10, + "ACC1", + "protein" + ], + [ + 14, + 38, + "regulated allosterically", + "protein_state" + ], + [ + 101, + 116, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 29, + "sent": "Dynamic polymerization of human ACC1 is linked to increased activity and is regulated allosterically by the activator citrate and the inhibitor palmitate, or by binding of the small protein MIG-12 (ref.).", + "section": "INTRO", + "ner": [ + [ + 26, + 31, + "human", + "species" + ], + [ + 32, + 36, + "ACC1", + "protein" + ], + [ + 76, + 100, + "regulated allosterically", + "protein_state" + ], + [ + 118, + 125, + "citrate", + "chemical" + ], + [ + 144, + 153, + "palmitate", + "chemical" + ], + [ + 190, + 196, + "MIG-12", + "protein" + ] + ] + }, + { + "sid": 30, + "sent": "Human ACC1 is further regulated by specific phosphorylation-dependent binding of BRCA1 to Ser1263 in the CD.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "Human", + "species" + ], + [ + 6, + 10, + "ACC1", + "protein" + ], + [ + 44, + 59, + "phosphorylation", + "ptm" + ], + [ + 81, + 86, + "BRCA1", + "protein" + ], + [ + 90, + 97, + "Ser1263", + "residue_name_number" + ], + [ + 105, + 107, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 31, + "sent": "BRCA1 binds only to the phosphorylated form of ACC1 and prevents ACC activation by phosphatase-mediated dephosphorylation.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "BRCA1", + "protein" + ], + [ + 24, + 38, + "phosphorylated", + "protein_state" + ], + [ + 47, + 51, + "ACC1", + "protein" + ], + [ + 65, + 68, + "ACC", + "protein_type" + ], + [ + 83, + 94, + "phosphatase", + "protein_type" + ] + ] + }, + { + "sid": 32, + "sent": "Furthermore, phosphorylation by AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA) leads to a decrease in ACC1 activity.", + "section": "INTRO", + "ner": [ + [ + 13, + 28, + "phosphorylation", + "ptm" + ], + [ + 32, + 60, + "AMP-activated protein kinase", + "protein" + ], + [ + 62, + 66, + "AMPK", + "protein" + ], + [ + 72, + 101, + "cAMP-dependent protein kinase", + "protein" + ], + [ + 103, + 106, + "PKA", + "protein" + ], + [ + 131, + 135, + "ACC1", + "protein" + ] + ] + }, + { + "sid": 33, + "sent": "AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "AMPK", + "protein" + ], + [ + 20, + 24, + "ACC1", + "protein" + ], + [ + 37, + 42, + "Ser80", + "residue_name_number" + ], + [ + 44, + 51, + "Ser1201", + "residue_name_number" + ], + [ + 56, + 63, + "Ser1216", + "residue_name_number" + ], + [ + 68, + 71, + "PKA", + "protein" + ], + [ + 75, + 80, + "Ser78", + "residue_name_number" + ], + [ + 85, + 92, + "Ser1201", + "residue_name_number" + ] + ] + }, + { + "sid": 34, + "sent": "However, regulatory effects on ACC1 activity are mainly mediated by phosphorylation of Ser80 and Ser1201 (refs).", + "section": "INTRO", + "ner": [ + [ + 31, + 35, + "ACC1", + "protein" + ], + [ + 68, + 83, + "phosphorylation", + "ptm" + ], + [ + 87, + 92, + "Ser80", + "residue_name_number" + ], + [ + 97, + 104, + "Ser1201", + "residue_name_number" + ] + ] + }, + { + "sid": 35, + "sent": "Phosphorylated Ser80, which is highly conserved only in higher eukaryotes, presumably binds into the Soraphen A-binding pocket.", + "section": "INTRO", + "ner": [ + [ + 0, + 14, + "Phosphorylated", + "protein_state" + ], + [ + 15, + 20, + "Ser80", + "residue_name_number" + ], + [ + 31, + 47, + "highly conserved", + "protein_state" + ], + [ + 56, + 73, + "higher eukaryotes", + "taxonomy_domain" + ], + [ + 101, + 126, + "Soraphen A-binding pocket", + "site" + ] + ] + }, + { + "sid": 36, + "sent": "The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes.", + "section": "INTRO", + "ner": [ + [ + 15, + 22, + "Ser1201", + "residue_name_number" + ], + [ + 34, + 55, + "moderate conservation", + "protein_state" + ], + [ + 63, + 80, + "higher eukaryotes", + "taxonomy_domain" + ], + [ + 92, + 106, + "phosphorylated", + "protein_state" + ], + [ + 107, + 114, + "Ser1216", + "residue_name_number" + ], + [ + 118, + 134, + "highly conserved", + "protein_state" + ], + [ + 146, + 156, + "eukaryotes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 37, + "sent": "However, no effect of Ser1216 phosphorylation on ACC activity has been reported in higher eukaryotes.", + "section": "INTRO", + "ner": [ + [ + 22, + 29, + "Ser1216", + "residue_name_number" + ], + [ + 30, + 45, + "phosphorylation", + "ptm" + ], + [ + 49, + 52, + "ACC", + "protein_type" + ], + [ + 83, + 100, + "higher eukaryotes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 38, + "sent": "For fungal ACC, neither spontaneous nor inducible polymerization has been detected despite considerable sequence conservation to human ACC1.", + "section": "INTRO", + "ner": [ + [ + 4, + 10, + "fungal", + "taxonomy_domain" + ], + [ + 11, + 14, + "ACC", + "protein_type" + ], + [ + 129, + 134, + "human", + "species" + ], + [ + 135, + 139, + "ACC1", + "protein" + ] + ] + }, + { + "sid": 39, + "sent": "The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein\u2013protein interactions of fungal ACC have been described.", + "section": "INTRO", + "ner": [ + [ + 4, + 9, + "BRCA1", + "protein" + ], + [ + 22, + 35, + "phosphoserine", + "residue_name" + ], + [ + 48, + 61, + "not conserved", + "protein_state" + ], + [ + 65, + 71, + "fungal", + "taxonomy_domain" + ], + [ + 72, + 75, + "ACC", + "protein_type" + ], + [ + 140, + 146, + "fungal", + "taxonomy_domain" + ], + [ + 147, + 150, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 40, + "sent": "In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.).", + "section": "INTRO", + "ner": [ + [ + 3, + 8, + "yeast", + "taxonomy_domain" + ], + [ + 9, + 12, + "ACC", + "protein_type" + ], + [ + 14, + 35, + "phosphorylation sites", + "site" + ], + [ + 60, + 64, + "Ser2", + "residue_name_number" + ], + [ + 66, + 72, + "Ser735", + "residue_name_number" + ], + [ + 74, + 81, + "Ser1148", + "residue_name_number" + ], + [ + 83, + 90, + "Ser1157", + "residue_name_number" + ], + [ + 95, + 102, + "Ser1162", + "residue_name_number" + ] + ] + }, + { + "sid": 41, + "sent": "Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1.", + "section": "INTRO", + "ner": [ + [ + 15, + 22, + "Ser1157", + "residue_name_number" + ], + [ + 26, + 42, + "highly conserved", + "protein_state" + ], + [ + 46, + 52, + "fungal", + "taxonomy_domain" + ], + [ + 53, + 56, + "ACC", + "protein_type" + ], + [ + 61, + 70, + "aligns to", + "experimental_method" + ], + [ + 71, + 78, + "Ser1216", + "residue_name_number" + ], + [ + 82, + 87, + "human", + "species" + ], + [ + 88, + 92, + "ACC1", + "protein" + ] + ] + }, + { + "sid": 42, + "sent": "Its phosphorylation by the AMPK homologue SNF1 results in strongly reduced ACC activity.", + "section": "INTRO", + "ner": [ + [ + 4, + 19, + "phosphorylation", + "ptm" + ], + [ + 27, + 31, + "AMPK", + "protein" + ], + [ + 42, + 46, + "SNF1", + "protein" + ], + [ + 75, + 78, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 43, + "sent": "Despite the outstanding relevance of ACC in primary metabolism and disease, the dynamic organization and regulation of the giant eukaryotic, and in particular fungal ACC, remain poorly characterized.", + "section": "INTRO", + "ner": [ + [ + 37, + 40, + "ACC", + "protein_type" + ], + [ + 129, + 139, + "eukaryotic", + "taxonomy_domain" + ], + [ + 159, + 165, + "fungal", + "taxonomy_domain" + ], + [ + 166, + 169, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 44, + "sent": "Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a).", + "section": "INTRO", + "ner": [ + [ + 20, + 29, + "structure", + "evidence" + ], + [ + 33, + 57, + "Saccharomyces cerevisiae", + "species" + ], + [ + 59, + 62, + "Sce", + "species" + ], + [ + 64, + 67, + "ACC", + "protein_type" + ], + [ + 68, + 70, + "CD", + "structure_element" + ], + [ + 105, + 115, + "structures", + "evidence" + ], + [ + 119, + 124, + "human", + "species" + ], + [ + 126, + 129, + "Hsa", + "species" + ], + [ + 131, + 134, + "ACC", + "protein_type" + ], + [ + 135, + 137, + "CD", + "structure_element" + ], + [ + 142, + 158, + "larger fragments", + "mutant" + ], + [ + 162, + 168, + "fungal", + "taxonomy_domain" + ], + [ + 169, + 172, + "ACC", + "protein_type" + ], + [ + 178, + 201, + "Chaetomium thermophilum", + "species" + ], + [ + 203, + 206, + "Cth", + "species" + ] + ] + }, + { + "sid": 45, + "sent": "Integrating these data with small-angle X-ray scattering (SAXS) and electron microscopy (EM) observations yield a comprehensive representation of the dynamic structure and regulation of fungal ACC.", + "section": "INTRO", + "ner": [ + [ + 28, + 56, + "small-angle X-ray scattering", + "experimental_method" + ], + [ + 58, + 62, + "SAXS", + "experimental_method" + ], + [ + 68, + 87, + "electron microscopy", + "experimental_method" + ], + [ + 89, + 91, + "EM", + "experimental_method" + ], + [ + 186, + 192, + "fungal", + "taxonomy_domain" + ], + [ + 193, + 196, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 46, + "sent": "The organization of the yeast ACC CD", + "section": "RESULTS", + "ner": [ + [ + 24, + 29, + "yeast", + "taxonomy_domain" + ], + [ + 30, + 33, + "ACC", + "protein_type" + ], + [ + 34, + 36, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 47, + "sent": "First, we focused on structure determination of the 82-kDa CD.", + "section": "RESULTS", + "ner": [ + [ + 21, + 44, + "structure determination", + "experimental_method" + ], + [ + 59, + 61, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 48, + "sent": "The crystal structure of the CD of SceACC (SceCD) was determined at 3.0\u2009\u00c5 resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 29, + 31, + "CD", + "structure_element" + ], + [ + 35, + 41, + "SceACC", + "protein" + ], + [ + 43, + 46, + "Sce", + "species" + ], + [ + 46, + 48, + "CD", + "structure_element" + ], + [ + 88, + 108, + "experimental phasing", + "experimental_method" + ], + [ + 113, + 120, + "refined", + "experimental_method" + ], + [ + 124, + 129, + "Rwork", + "evidence" + ], + [ + 130, + 135, + "Rfree", + "evidence" + ] + ] + }, + { + "sid": 49, + "sent": "The overall extent of the SceCD is 70 by 75\u2009\u00c5 (Fig. 1b and Supplementary Fig. 1a,b), and the attachment points of the N-terminal 26-residue linker to the BCCP domain and the C-terminal CT domain are separated by 46\u2009\u00c5 (the N- and C termini are indicated with spheres in Fig. 1b).", + "section": "RESULTS", + "ner": [ + [ + 26, + 29, + "Sce", + "species" + ], + [ + 29, + 31, + "CD", + "structure_element" + ], + [ + 129, + 146, + "26-residue linker", + "structure_element" + ], + [ + 154, + 158, + "BCCP", + "structure_element" + ], + [ + 185, + 187, + "CT", + "structure_element" + ] + ] + }, + { + "sid": 50, + "sent": "SceCD comprises four distinct domains, an N-terminal \u03b1-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two \u03b1\u2013\u03b2-fold C-terminal domains (CDC1/CDC2).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "Sce", + "species" + ], + [ + 3, + 5, + "CD", + "structure_element" + ], + [ + 53, + 69, + "\u03b1-helical domain", + "structure_element" + ], + [ + 71, + 74, + "CDN", + "structure_element" + ], + [ + 91, + 122, + "four-helix bundle linker domain", + "structure_element" + ], + [ + 124, + 127, + "CDL", + "structure_element" + ], + [ + 146, + 173, + "\u03b1\u2013\u03b2-fold C-terminal domains", + "structure_element" + ], + [ + 175, + 179, + "CDC1", + "structure_element" + ], + [ + 180, + 184, + "CDC2", + "structure_element" + ] + ] + }, + { + "sid": 51, + "sent": "CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (N\u03b13-6), the other end is a helical hairpin (N\u03b18,9) and the bridging region comprises six helices (N\u03b11,2,7,10\u201312).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "CDN", + "structure_element" + ], + [ + 20, + 27, + "C shape", + "protein_state" + ], + [ + 56, + 81, + "regular four-helix bundle", + "structure_element" + ], + [ + 83, + 88, + "N\u03b13-6", + "structure_element" + ], + [ + 110, + 125, + "helical hairpin", + "structure_element" + ], + [ + 127, + 132, + "N\u03b18,9", + "structure_element" + ], + [ + 142, + 157, + "bridging region", + "structure_element" + ], + [ + 172, + 179, + "helices", + "structure_element" + ], + [ + 181, + 194, + "N\u03b11,2,7,10\u201312", + "structure_element" + ] + ] + }, + { + "sid": 52, + "sent": "CDL is composed of a small, irregular four-helix bundle (L\u03b11\u20134) and tightly interacts with the open face of CDC1 via an interface of 1,300\u2009\u00c52 involving helices L\u03b13 and L\u03b14.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "CDL", + "structure_element" + ], + [ + 21, + 55, + "small, irregular four-helix bundle", + "structure_element" + ], + [ + 57, + 62, + "L\u03b11\u20134", + "structure_element" + ], + [ + 108, + 112, + "CDC1", + "structure_element" + ], + [ + 120, + 129, + "interface", + "site" + ], + [ + 152, + 159, + "helices", + "structure_element" + ], + [ + 160, + 163, + "L\u03b13", + "structure_element" + ], + [ + 168, + 171, + "L\u03b14", + "structure_element" + ] + ] + }, + { + "sid": 53, + "sent": "CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between L\u03b12/\u03b13 and the N-terminal end of L\u03b11, with an interface area of 400\u2009\u00c52.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "CDL", + "structure_element" + ], + [ + 27, + 30, + "CDN", + "structure_element" + ], + [ + 97, + 101, + "CDC2", + "structure_element" + ], + [ + 108, + 112, + "loop", + "structure_element" + ], + [ + 121, + 127, + "L\u03b12/\u03b13", + "structure_element" + ], + [ + 154, + 157, + "L\u03b11", + "structure_element" + ], + [ + 167, + 176, + "interface", + "site" + ] + ] + }, + { + "sid": 54, + "sent": "CDC1/CDC2 share a common fold; they are composed of six-stranded \u03b2-sheets flanked on one side by two long, bent helices inserted between strands \u03b23/\u03b24 and \u03b24/\u03b25.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "CDC1", + "structure_element" + ], + [ + 5, + 9, + "CDC2", + "structure_element" + ], + [ + 52, + 73, + "six-stranded \u03b2-sheets", + "structure_element" + ], + [ + 101, + 119, + "long, bent helices", + "structure_element" + ], + [ + 137, + 144, + "strands", + "structure_element" + ], + [ + 145, + 150, + "\u03b23/\u03b24", + "structure_element" + ], + [ + 155, + 160, + "\u03b24/\u03b25", + "structure_element" + ] + ] + }, + { + "sid": 55, + "sent": "CDC2 is extended at its C terminus by an additional \u03b2-strand and an irregular \u03b2-hairpin.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "CDC2", + "structure_element" + ], + [ + 8, + 16, + "extended", + "protein_state" + ], + [ + 52, + 60, + "\u03b2-strand", + "structure_element" + ], + [ + 68, + 87, + "irregular \u03b2-hairpin", + "structure_element" + ] + ] + }, + { + "sid": 56, + "sent": "On the basis of a root mean square deviation of main chain atom positions of 2.2\u2009\u00c5, CDC1/CDC2 are structurally more closely related to each other than to any other protein (Fig. 1c); they may thus have evolved by duplication.", + "section": "RESULTS", + "ner": [ + [ + 18, + 44, + "root mean square deviation", + "evidence" + ], + [ + 84, + 88, + "CDC1", + "structure_element" + ], + [ + 89, + 93, + "CDC2", + "structure_element" + ] + ] + }, + { + "sid": 57, + "sent": "Close structural homologues could not be found for the CDN or the CDC domains.", + "section": "RESULTS", + "ner": [ + [ + 55, + 58, + "CDN", + "structure_element" + ], + [ + 66, + 69, + "CDC", + "structure_element" + ] + ] + }, + { + "sid": 58, + "sent": "A regulatory loop mediates interdomain interactions", + "section": "RESULTS", + "ner": [ + [ + 2, + 17, + "regulatory loop", + "structure_element" + ] + ] + }, + { + "sid": 59, + "sent": "To define the functional state of insect-cell-expressed ACC variants, we employed mass spectrometry (MS) for phosphorylation site detection.", + "section": "RESULTS", + "ner": [ + [ + 34, + 55, + "insect-cell-expressed", + "experimental_method" + ], + [ + 56, + 59, + "ACC", + "protein_type" + ], + [ + 82, + 99, + "mass spectrometry", + "experimental_method" + ], + [ + 101, + 103, + "MS", + "experimental_method" + ], + [ + 109, + 139, + "phosphorylation site detection", + "experimental_method" + ] + ] + }, + { + "sid": 60, + "sent": "In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae.", + "section": "RESULTS", + "ner": [ + [ + 3, + 24, + "insect-cell-expressed", + "experimental_method" + ], + [ + 25, + 36, + "full-length", + "protein_state" + ], + [ + 37, + 43, + "SceACC", + "protein" + ], + [ + 49, + 65, + "highly conserved", + "protein_state" + ], + [ + 66, + 73, + "Ser1157", + "residue_name_number" + ], + [ + 86, + 100, + "fully occupied", + "protein_state" + ], + [ + 101, + 121, + "phosphorylation site", + "site" + ], + [ + 151, + 164, + "S. cerevisiae", + "species" + ] + ] + }, + { + "sid": 61, + "sent": "Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast.", + "section": "RESULTS", + "ner": [ + [ + 11, + 26, + "phosphorylation", + "ptm" + ], + [ + 44, + 51, + "Ser2101", + "residue_name_number" + ], + [ + 56, + 63, + "Tyr2179", + "residue_name_number" + ], + [ + 90, + 107, + "neither conserved", + "protein_state" + ], + [ + 115, + 121, + "fungal", + "taxonomy_domain" + ], + [ + 122, + 125, + "ACC", + "protein_type" + ], + [ + 126, + 153, + "nor natively phosphorylated", + "protein_state" + ], + [ + 157, + 162, + "yeast", + "taxonomy_domain" + ] + ] + }, + { + "sid": 62, + "sent": "MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals.", + "section": "RESULTS", + "ner": [ + [ + 0, + 2, + "MS", + "experimental_method" + ], + [ + 15, + 33, + "dissolved crystals", + "experimental_method" + ], + [ + 48, + 62, + "phosphorylated", + "protein_state" + ], + [ + 72, + 79, + "Ser1157", + "residue_name_number" + ], + [ + 88, + 91, + "Sce", + "species" + ], + [ + 91, + 93, + "CD", + "structure_element" + ], + [ + 94, + 102, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 63, + "sent": "The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "Sce", + "species" + ], + [ + 7, + 9, + "CD", + "structure_element" + ], + [ + 10, + 19, + "structure", + "evidence" + ], + [ + 63, + 69, + "SceACC", + "protein" + ], + [ + 81, + 87, + "enzyme", + "protein" + ], + [ + 91, + 100, + "inhibited", + "protein_state" + ], + [ + 104, + 134, + "SNF1-dependent phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 64, + "sent": "In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands \u03b22 and \u03b23 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here.", + "section": "RESULTS", + "ner": [ + [ + 7, + 10, + "Sce", + "species" + ], + [ + 10, + 12, + "CD", + "structure_element" + ], + [ + 13, + 30, + "crystal structure", + "evidence" + ], + [ + 36, + 50, + "phosphorylated", + "protein_state" + ], + [ + 51, + 58, + "Ser1157", + "residue_name_number" + ], + [ + 72, + 101, + "regulatory 36-amino-acid loop", + "structure_element" + ], + [ + 110, + 117, + "strands", + "structure_element" + ], + [ + 118, + 120, + "\u03b22", + "structure_element" + ], + [ + 125, + 127, + "\u03b23", + "structure_element" + ], + [ + 131, + 135, + "CDC1", + "structure_element" + ], + [ + 179, + 193, + "less-conserved", + "protein_state" + ], + [ + 194, + 215, + "phosphorylation sites", + "site" + ], + [ + 217, + 224, + "Ser1148", + "residue_name_number" + ], + [ + 229, + 236, + "Ser1162", + "residue_name_number" + ], + [ + 251, + 256, + "yeast", + "taxonomy_domain" + ] + ] + }, + { + "sid": 65, + "sent": "This regulatory loop wedges between the CDC1 and CDC2 domains and provides the largest contribution to the interdomain interface.", + "section": "RESULTS", + "ner": [ + [ + 5, + 20, + "regulatory loop", + "structure_element" + ], + [ + 40, + 44, + "CDC1", + "structure_element" + ], + [ + 49, + 53, + "CDC2", + "structure_element" + ], + [ + 107, + 128, + "interdomain interface", + "site" + ] + ] + }, + { + "sid": 66, + "sent": "The N-terminal region of the regulatory loop also directly contacts the C-terminal region of CDC2 leading into CT.", + "section": "RESULTS", + "ner": [ + [ + 29, + 44, + "regulatory loop", + "structure_element" + ], + [ + 93, + 97, + "CDC2", + "structure_element" + ], + [ + 111, + 113, + "CT", + "structure_element" + ] + ] + }, + { + "sid": 67, + "sent": "Phosphoserine 1157 is tightly bound by two highly conserved arginines (Arg1173 and Arg1260) of CDC1 (Fig. 1d).", + "section": "RESULTS", + "ner": [ + [ + 0, + 18, + "Phosphoserine 1157", + "residue_name_number" + ], + [ + 43, + 59, + "highly conserved", + "protein_state" + ], + [ + 60, + 69, + "arginines", + "residue_name" + ], + [ + 71, + 78, + "Arg1173", + "residue_name_number" + ], + [ + 83, + 90, + "Arg1260", + "residue_name_number" + ], + [ + 95, + 99, + "CDC1", + "structure_element" + ] + ] + }, + { + "sid": 68, + "sent": "Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains.", + "section": "RESULTS", + "ner": [ + [ + 23, + 37, + "phosphorylated", + "protein_state" + ], + [ + 38, + 45, + "Ser1157", + "residue_name_number" + ], + [ + 72, + 87, + "regulatory loop", + "structure_element" + ], + [ + 116, + 137, + "phosphorylation sites", + "site" + ], + [ + 138, + 145, + "Ser1148", + "residue_name_number" + ], + [ + 150, + 157, + "Ser1162", + "residue_name_number" + ], + [ + 165, + 174, + "same loop", + "structure_element" + ], + [ + 236, + 251, + "regulatory loop", + "structure_element" + ], + [ + 260, + 264, + "CDC1", + "structure_element" + ], + [ + 269, + 273, + "CDC2", + "structure_element" + ] + ] + }, + { + "sid": 69, + "sent": "Phosphorylation of the regulatory loop thus determines interdomain interactions of CDC1 and CDC2, suggesting that it may exert its regulatory function by modifying the overall structure and dynamics of the CD.", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Phosphorylation", + "ptm" + ], + [ + 23, + 38, + "regulatory loop", + "structure_element" + ], + [ + 83, + 87, + "CDC1", + "structure_element" + ], + [ + 92, + 96, + "CDC2", + "structure_element" + ], + [ + 206, + 208, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 70, + "sent": "The functional role of Ser1157 was confirmed by an activity assay based on the incorporation of radioactive carbonate into acid non-volatile material.", + "section": "RESULTS", + "ner": [ + [ + 23, + 30, + "Ser1157", + "residue_name_number" + ], + [ + 51, + 65, + "activity assay", + "experimental_method" + ] + ] + }, + { + "sid": 71, + "sent": "Phosphorylated SceACC shows only residual activity (kcat=0.4\u00b10.2\u2009s\u22121, s.d. based on five replicate measurements), which increases 16-fold (kcat=6.5\u00b10.3\u2009s\u22121) after dephosphorylation with \u03bb protein phosphatase.", + "section": "RESULTS", + "ner": [ + [ + 0, + 14, + "Phosphorylated", + "protein_state" + ], + [ + 15, + 21, + "SceACC", + "protein" + ], + [ + 52, + 56, + "kcat", + "evidence" + ], + [ + 139, + 143, + "kcat", + "evidence" + ], + [ + 186, + 207, + "\u03bb protein phosphatase", + "protein_type" + ] + ] + }, + { + "sid": 72, + "sent": "The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli.", + "section": "RESULTS", + "ner": [ + [ + 24, + 40, + "dephosphorylated", + "protein_state" + ], + [ + 41, + 47, + "SceACC", + "protein" + ], + [ + 90, + 108, + "non-phosphorylated", + "protein_state" + ], + [ + 109, + 114, + "yeast", + "taxonomy_domain" + ], + [ + 115, + 118, + "ACC", + "protein_type" + ], + [ + 119, + 131, + "expressed in", + "experimental_method" + ], + [ + 132, + 139, + "E. coli", + "species" + ] + ] + }, + { + "sid": 73, + "sent": "The variable CD is conserved between yeast and human", + "section": "RESULTS", + "ner": [ + [ + 13, + 15, + "CD", + "structure_element" + ], + [ + 19, + 28, + "conserved", + "protein_state" + ], + [ + 37, + 42, + "yeast", + "taxonomy_domain" + ], + [ + 47, + 52, + "human", + "species" + ] + ] + }, + { + "sid": 74, + "sent": "To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a).", + "section": "RESULTS", + "ner": [ + [ + 31, + 37, + "fungal", + "taxonomy_domain" + ], + [ + 42, + 47, + "human", + "species" + ], + [ + 48, + 51, + "ACC", + "protein_type" + ], + [ + 52, + 54, + "CD", + "structure_element" + ], + [ + 59, + 83, + "determined the structure", + "experimental_method" + ], + [ + 89, + 94, + "human", + "species" + ], + [ + 95, + 108, + "ACC1 fragment", + "mutant" + ], + [ + 128, + 130, + "BT", + "structure_element" + ], + [ + 135, + 137, + "CD", + "structure_element" + ], + [ + 147, + 155, + "HsaBT-CD", + "mutant" + ], + [ + 162, + 167, + "lacks", + "protein_state" + ], + [ + 179, + 183, + "BCCP", + "structure_element" + ] + ] + }, + { + "sid": 75, + "sent": "An experimentally phased map was obtained at 3.7\u2009\u00c5 resolution for a cadmium-derivatized crystal and was interpreted by a poly-alanine model (Fig. 1e and Table 1).", + "section": "RESULTS", + "ner": [ + [ + 3, + 28, + "experimentally phased map", + "evidence" + ], + [ + 68, + 75, + "cadmium", + "chemical" + ] + ] + }, + { + "sid": 76, + "sent": "Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures.", + "section": "RESULTS", + "ner": [ + [ + 17, + 19, + "CD", + "structure_element" + ], + [ + 31, + 39, + "HsaBT-CD", + "mutant" + ], + [ + 81, + 84, + "Sce", + "species" + ], + [ + 84, + 86, + "CD", + "structure_element" + ], + [ + 104, + 109, + "human", + "species" + ], + [ + 114, + 119, + "yeast", + "taxonomy_domain" + ], + [ + 120, + 123, + "CDs", + "structure_element" + ], + [ + 149, + 159, + "structures", + "evidence" + ] + ] + }, + { + "sid": 77, + "sent": "In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30\u00b0 based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c).", + "section": "RESULTS", + "ner": [ + [ + 45, + 48, + "Sce", + "species" + ], + [ + 48, + 50, + "CD", + "structure_element" + ], + [ + 88, + 91, + "CDL", + "structure_element" + ], + [ + 96, + 100, + "CDC1", + "structure_element" + ], + [ + 117, + 125, + "HsaBT-CD", + "mutant" + ], + [ + 135, + 140, + "human", + "species" + ], + [ + 141, + 144, + "CDL", + "structure_element" + ], + [ + 145, + 149, + "CDC1", + "structure_element" + ], + [ + 187, + 200, + "superposition", + "experimental_method" + ], + [ + 204, + 209, + "human", + "species" + ], + [ + 214, + 219, + "yeast", + "taxonomy_domain" + ], + [ + 220, + 224, + "CDC2", + "structure_element" + ] + ] + }, + { + "sid": 78, + "sent": "As a result, the N terminus of CDL at helix L\u03b11, which connects to CDN, is shifted by 12\u2009\u00c5. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD.", + "section": "RESULTS", + "ner": [ + [ + 31, + 34, + "CDL", + "structure_element" + ], + [ + 38, + 43, + "helix", + "structure_element" + ], + [ + 44, + 47, + "L\u03b11", + "structure_element" + ], + [ + 67, + 70, + "CDN", + "structure_element" + ], + [ + 104, + 107, + "CDN", + "structure_element" + ], + [ + 111, + 119, + "HsaBT-CD", + "mutant" + ], + [ + 176, + 179, + "Sce", + "species" + ], + [ + 179, + 181, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 79, + "sent": "With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160\u00b0 around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d).", + "section": "RESULTS", + "ner": [ + [ + 5, + 8, + "CDL", + "structure_element" + ], + [ + 9, + 13, + "CDC1", + "structure_element" + ], + [ + 14, + 24, + "superposed", + "experimental_method" + ], + [ + 26, + 29, + "CDN", + "structure_element" + ], + [ + 33, + 41, + "HsaBT-CD", + "mutant" + ], + [ + 70, + 75, + "hinge", + "structure_element" + ], + [ + 97, + 100, + "CDN", + "structure_element" + ], + [ + 101, + 104, + "CDL", + "structure_element" + ] + ] + }, + { + "sid": 80, + "sent": "This rotation displaces the N terminus of CDN in HsaBT-CD by 51\u2009\u00c5 compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67\u2009\u00c5 (the attachment points are indicated with spheres in Fig. 1e).", + "section": "RESULTS", + "ner": [ + [ + 42, + 45, + "CDN", + "structure_element" + ], + [ + 49, + 57, + "HsaBT-CD", + "mutant" + ], + [ + 80, + 83, + "Sce", + "species" + ], + [ + 83, + 85, + "CD", + "structure_element" + ], + [ + 156, + 162, + "linker", + "structure_element" + ], + [ + 170, + 181, + "BCCP domain", + "structure_element" + ], + [ + 201, + 203, + "CT", + "structure_element" + ] + ] + }, + { + "sid": 81, + "sent": "The BT domain of HsaBT-CD consists of a helix that is surrounded at its N terminus by an antiparallel eight-stranded \u03b2-barrel.", + "section": "RESULTS", + "ner": [ + [ + 4, + 6, + "BT", + "structure_element" + ], + [ + 17, + 25, + "HsaBT-CD", + "mutant" + ], + [ + 40, + 45, + "helix", + "structure_element" + ], + [ + 89, + 125, + "antiparallel eight-stranded \u03b2-barrel", + "structure_element" + ] + ] + }, + { + "sid": 82, + "sent": "It resembles the BT of propionyl-CoA carboxylase; only the four C-terminal strands of the \u03b2-barrel are slightly tilted.", + "section": "RESULTS", + "ner": [ + [ + 17, + 19, + "BT", + "structure_element" + ], + [ + 23, + 48, + "propionyl-CoA carboxylase", + "protein_type" + ], + [ + 75, + 98, + "strands of the \u03b2-barrel", + "structure_element" + ] + ] + }, + { + "sid": 83, + "sent": "On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%).", + "section": "RESULTS", + "ner": [ + [ + 16, + 18, + "MS", + "experimental_method" + ], + [ + 31, + 52, + "insect-cell-expressed", + "experimental_method" + ], + [ + 53, + 58, + "human", + "species" + ], + [ + 59, + 70, + "full-length", + "protein_state" + ], + [ + 71, + 74, + "ACC", + "protein_type" + ], + [ + 76, + 81, + "Ser80", + "residue_name_number" + ], + [ + 110, + 125, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 84, + "sent": "Ser29 and Ser1263, implicated in insulin-dependent phosphorylation and BRCA1 binding, respectively, are phosphorylated at intermediate levels (40%).", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "Ser29", + "residue_name_number" + ], + [ + 10, + 17, + "Ser1263", + "residue_name_number" + ], + [ + 33, + 66, + "insulin-dependent phosphorylation", + "ptm" + ], + [ + 71, + 76, + "BRCA1", + "protein" + ], + [ + 104, + 118, + "phosphorylated", + "protein_state" + ] + ] + }, + { + "sid": 85, + "sent": "The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated.", + "section": "RESULTS", + "ner": [ + [ + 4, + 20, + "highly conserved", + "protein_state" + ], + [ + 21, + 28, + "Ser1216", + "residue_name_number" + ], + [ + 47, + 60, + "S. cerevisiae", + "species" + ], + [ + 61, + 68, + "Ser1157", + "residue_name_number" + ], + [ + 82, + 89, + "Ser1201", + "residue_name_number" + ], + [ + 103, + 118, + "regulatory loop", + "structure_element" + ], + [ + 140, + 158, + "not phosphorylated", + "protein_state" + ] + ] + }, + { + "sid": 86, + "sent": "However, residual phosphorylation levels were detected for Ser1204 (7%) and Ser1218 (7%) in the same loop.", + "section": "RESULTS", + "ner": [ + [ + 18, + 33, + "phosphorylation", + "ptm" + ], + [ + 59, + 66, + "Ser1204", + "residue_name_number" + ], + [ + 76, + 83, + "Ser1218", + "residue_name_number" + ], + [ + 96, + 105, + "same loop", + "structure_element" + ] + ] + }, + { + "sid": 87, + "sent": "MS analysis of the HsaBT-CD crystallization sample reveals partial proteolytic digestion of the regulatory loop.", + "section": "RESULTS", + "ner": [ + [ + 0, + 2, + "MS", + "experimental_method" + ], + [ + 19, + 27, + "HsaBT-CD", + "mutant" + ], + [ + 28, + 50, + "crystallization sample", + "evidence" + ], + [ + 96, + 111, + "regulatory loop", + "structure_element" + ] + ] + }, + { + "sid": 88, + "sent": "Accordingly, most of this loop is not represented in the HsaBT-CD crystal structure.", + "section": "RESULTS", + "ner": [ + [ + 21, + 30, + "this loop", + "structure_element" + ], + [ + 57, + 65, + "HsaBT-CD", + "mutant" + ], + [ + 66, + 83, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 89, + "sent": "The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "absence of", + "protein_state" + ], + [ + 19, + 34, + "regulatory loop", + "structure_element" + ], + [ + 58, + 73, + "less-restrained", + "protein_state" + ], + [ + 74, + 83, + "interface", + "site" + ], + [ + 87, + 90, + "CDL", + "structure_element" + ], + [ + 91, + 95, + "CDC1", + "structure_element" + ], + [ + 100, + 104, + "CDC2", + "structure_element" + ], + [ + 148, + 155, + "domains", + "structure_element" + ] + ] + }, + { + "sid": 90, + "sent": "Besides the regulatory loop, also the phosphopeptide target region for BRCA1 interaction is not resolved presumably because of pronounced flexibility.", + "section": "RESULTS", + "ner": [ + [ + 12, + 27, + "regulatory loop", + "structure_element" + ], + [ + 38, + 66, + "phosphopeptide target region", + "site" + ], + [ + 71, + 76, + "BRCA1", + "protein" + ] + ] + }, + { + "sid": 91, + "sent": "At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region.", + "section": "RESULTS", + "ner": [ + [ + 16, + 24, + "isolated", + "experimental_method" + ], + [ + 25, + 30, + "yeast", + "taxonomy_domain" + ], + [ + 35, + 40, + "human", + "species" + ], + [ + 41, + 43, + "CD", + "structure_element" + ], + [ + 49, + 68, + "structural analysis", + "experimental_method" + ], + [ + 108, + 114, + "hinges", + "structure_element" + ], + [ + 156, + 174, + "CDN/CDL connection", + "structure_element" + ], + [ + 216, + 219, + "CDL", + "structure_element" + ], + [ + 220, + 224, + "CDC1", + "structure_element" + ], + [ + 229, + 233, + "CDC2", + "structure_element" + ], + [ + 257, + 272, + "phosphorylation", + "ptm" + ], + [ + 280, + 295, + "regulatory loop", + "structure_element" + ] + ] + }, + { + "sid": 92, + "sent": "The integration of CD into the fungal ACC multienzyme", + "section": "RESULTS", + "ner": [ + [ + 19, + 21, + "CD", + "structure_element" + ], + [ + 31, + 37, + "fungal", + "taxonomy_domain" + ], + [ + 38, + 53, + "ACC multienzyme", + "protein_type" + ] + ] + }, + { + "sid": 93, + "sent": "To further obtain insights into the functional architecture of fungal ACC, we characterized larger multidomain fragments up to the intact enzymes.", + "section": "RESULTS", + "ner": [ + [ + 63, + 69, + "fungal", + "taxonomy_domain" + ], + [ + 70, + 73, + "ACC", + "protein_type" + ], + [ + 92, + 120, + "larger multidomain fragments", + "mutant" + ], + [ + 131, + 137, + "intact", + "protein_state" + ], + [ + 138, + 145, + "enzymes", + "protein" + ] + ] + }, + { + "sid": 94, + "sent": "Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5\u2009\u00c5 (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2\u2009\u00c5 resolution (CthCD-CT; Figs 1a and 2, Table 1).", + "section": "RESULTS", + "ner": [ + [ + 6, + 27, + "molecular replacement", + "experimental_method" + ], + [ + 37, + 43, + "fungal", + "taxonomy_domain" + ], + [ + 44, + 47, + "ACC", + "protein_type" + ], + [ + 48, + 50, + "CD", + "structure_element" + ], + [ + 55, + 57, + "CT", + "structure_element" + ], + [ + 78, + 88, + "structures", + "evidence" + ], + [ + 94, + 101, + "variant", + "mutant" + ], + [ + 113, + 116, + "Cth", + "species" + ], + [ + 116, + 118, + "CT", + "structure_element" + ], + [ + 123, + 127, + "CDC1", + "structure_element" + ], + [ + 128, + 132, + "CDC2", + "structure_element" + ], + [ + 136, + 153, + "two crystal forms", + "evidence" + ], + [ + 187, + 202, + "CthCD-CTCter1/2", + "mutant" + ], + [ + 235, + 238, + "Cth", + "species" + ], + [ + 238, + 240, + "CT", + "structure_element" + ], + [ + 262, + 264, + "CD", + "structure_element" + ], + [ + 286, + 294, + "CthCD-CT", + "mutant" + ] + ] + }, + { + "sid": 95, + "sent": "No crystals diffracting to sufficient resolution were obtained for larger BC-containing fragments, or for full-length Cth or SceACC.", + "section": "RESULTS", + "ner": [ + [ + 67, + 97, + "larger BC-containing fragments", + "mutant" + ], + [ + 106, + 117, + "full-length", + "protein_state" + ], + [ + 118, + 121, + "Cth", + "species" + ], + [ + 125, + 131, + "SceACC", + "protein" + ] + ] + }, + { + "sid": 96, + "sent": "To improve crystallizability, we generated \u0394BCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a\u2013c).", + "section": "RESULTS", + "ner": [ + [ + 3, + 28, + "improve crystallizability", + "experimental_method" + ], + [ + 33, + 42, + "generated", + "experimental_method" + ], + [ + 43, + 57, + "\u0394BCCP variants", + "mutant" + ], + [ + 61, + 72, + "full-length", + "protein_state" + ], + [ + 73, + 76, + "ACC", + "protein_type" + ], + [ + 94, + 107, + "SAXS analysis", + "experimental_method" + ], + [ + 132, + 138, + "intact", + "protein_state" + ], + [ + 139, + 142, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 97, + "sent": "For Cth\u0394BCCP, crystals diffracting to 8.4\u2009\u00c5 resolution were obtained.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "Cth\u0394BCCP", + "mutant" + ], + [ + 14, + 22, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 98, + "sent": "However, molecular replacement did not reveal a unique positioning of the BC domain.", + "section": "RESULTS", + "ner": [ + [ + 9, + 30, + "molecular replacement", + "experimental_method" + ], + [ + 74, + 76, + "BC", + "structure_element" + ] + ] + }, + { + "sid": 99, + "sent": "Owing to the limited resolution the discussion of structures of CthCD-CT and Cth\u0394BCCP is restricted to the analysis of domain localization.", + "section": "RESULTS", + "ner": [ + [ + 50, + 60, + "structures", + "evidence" + ], + [ + 64, + 72, + "CthCD-CT", + "mutant" + ], + [ + 77, + 85, + "Cth\u0394BCCP", + "mutant" + ] + ] + }, + { + "sid": 100, + "sent": "Still, these structures contribute considerably to the visualization of an intrinsically dynamic fungal ACC.", + "section": "RESULTS", + "ner": [ + [ + 7, + 23, + "these structures", + "evidence" + ], + [ + 89, + 96, + "dynamic", + "protein_state" + ], + [ + 97, + 103, + "fungal", + "taxonomy_domain" + ], + [ + 104, + 107, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 101, + "sent": "In all these crystal structures, the CT domains build a canonical head-to-tail dimer, with active sites formed by contributions from both protomers (Fig. 2 and Supplementary Fig. 3a).", + "section": "RESULTS", + "ner": [ + [ + 13, + 31, + "crystal structures", + "evidence" + ], + [ + 37, + 39, + "CT", + "structure_element" + ], + [ + 66, + 78, + "head-to-tail", + "protein_state" + ], + [ + 79, + 84, + "dimer", + "oligomeric_state" + ], + [ + 91, + 103, + "active sites", + "site" + ], + [ + 138, + 147, + "protomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 102, + "sent": "The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular \u03b2-hairpin/\u03b2-strand extension of CDC2 (Supplementary Fig. 3b).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "connection", + "structure_element" + ], + [ + 18, + 20, + "CD", + "structure_element" + ], + [ + 25, + 27, + "CT", + "structure_element" + ], + [ + 45, + 71, + "10-residue peptide stretch", + "residue_range" + ], + [ + 103, + 105, + "CT", + "structure_element" + ], + [ + 113, + 151, + "irregular \u03b2-hairpin/\u03b2-strand extension", + "structure_element" + ], + [ + 155, + 159, + "CDC2", + "structure_element" + ] + ] + }, + { + "sid": 103, + "sent": "The connecting region is remarkably similar in isolated CD and CthCD-CTCter structures, indicating inherent conformational stability.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "connecting region", + "structure_element" + ], + [ + 47, + 55, + "isolated", + "protein_state" + ], + [ + 56, + 58, + "CD", + "structure_element" + ], + [ + 63, + 75, + "CthCD-CTCter", + "mutant" + ], + [ + 76, + 86, + "structures", + "evidence" + ] + ] + }, + { + "sid": 104, + "sent": "CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the \u03b2-hairpin extension of CDC2 as well as the loop between strands \u03b22/\u03b23 of the CT N-lobe, which contains a conserved RxxGxN motif.", + "section": "RESULTS", + "ner": [ + [ + 0, + 2, + "CD", + "structure_element" + ], + [ + 3, + 5, + "CT", + "structure_element" + ], + [ + 90, + 109, + "\u03b2-hairpin extension", + "structure_element" + ], + [ + 113, + 117, + "CDC2", + "structure_element" + ], + [ + 133, + 137, + "loop", + "structure_element" + ], + [ + 146, + 159, + "strands \u03b22/\u03b23", + "structure_element" + ], + [ + 167, + 176, + "CT N-lobe", + "structure_element" + ], + [ + 195, + 204, + "conserved", + "protein_state" + ], + [ + 205, + 217, + "RxxGxN motif", + "structure_element" + ] + ] + }, + { + "sid": 105, + "sent": "The neighbouring loop on the CT side (between CT \u03b21/\u03b22) is displaced by 2.5\u2009\u00c5 compared to isolated CT structures (Supplementary Fig. 3c).", + "section": "RESULTS", + "ner": [ + [ + 17, + 21, + "loop", + "structure_element" + ], + [ + 29, + 31, + "CT", + "structure_element" + ], + [ + 46, + 48, + "CT", + "structure_element" + ], + [ + 49, + 51, + "\u03b21", + "structure_element" + ], + [ + 52, + 54, + "\u03b22", + "structure_element" + ], + [ + 90, + 98, + "isolated", + "protein_state" + ], + [ + 99, + 101, + "CT", + "structure_element" + ], + [ + 102, + 112, + "structures", + "evidence" + ] + ] + }, + { + "sid": 106, + "sent": "On the basis of an interface area of \u223c600\u2009\u00c52 and its edge-to-edge connection characteristics, the interface between CT and CD might be classified as conformationally variable.", + "section": "RESULTS", + "ner": [ + [ + 98, + 107, + "interface", + "site" + ], + [ + 116, + 118, + "CT", + "structure_element" + ], + [ + 123, + 125, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 107, + "sent": "Indeed, the comparison of the positioning of eight instances of the C-terminal part of CD relative to CT in crystal structures determined here, reveals flexible interdomain linking (Fig. 3a).", + "section": "RESULTS", + "ner": [ + [ + 87, + 89, + "CD", + "structure_element" + ], + [ + 102, + 104, + "CT", + "structure_element" + ], + [ + 108, + 126, + "crystal structures", + "evidence" + ], + [ + 127, + 137, + "determined", + "experimental_method" + ] + ] + }, + { + "sid": 108, + "sent": "The CDC2/CT interface acts as a true hinge with observed rotation up to 16\u00b0, which results in a translocation of the distal end of CDC2 by 8\u2009\u00c5.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "CDC2/CT interface", + "site" + ], + [ + 32, + 42, + "true hinge", + "structure_element" + ], + [ + 131, + 135, + "CDC2", + "structure_element" + ] + ] + }, + { + "sid": 109, + "sent": "The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD\u2013CT junction, and permits only limited rotation and tilting (Fig. 3b).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "interface", + "site" + ], + [ + 22, + 26, + "CDC2", + "structure_element" + ], + [ + 31, + 34, + "CDL", + "structure_element" + ], + [ + 35, + 39, + "CDC1", + "structure_element" + ], + [ + 66, + 80, + "phosphorylated", + "protein_state" + ], + [ + 81, + 96, + "regulatory loop", + "structure_element" + ], + [ + 104, + 107, + "Sce", + "species" + ], + [ + 107, + 109, + "CD", + "structure_element" + ], + [ + 110, + 119, + "structure", + "evidence" + ], + [ + 147, + 161, + "CD\u2013CT junction", + "structure_element" + ] + ] + }, + { + "sid": 110, + "sent": "Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution.", + "section": "RESULTS", + "ner": [ + [ + 26, + 41, + "phosphorylation", + "ptm" + ], + [ + 49, + 58, + "interface", + "site" + ], + [ + 67, + 71, + "CDC2", + "structure_element" + ], + [ + 76, + 79, + "CDL", + "structure_element" + ], + [ + 80, + 84, + "CDC1", + "structure_element" + ], + [ + 88, + 102, + "CthACC variant", + "mutant" + ], + [ + 103, + 113, + "structures", + "evidence" + ] + ] + }, + { + "sid": 111, + "sent": "However, MS analysis of CthCD-CT and Cth\u0394BCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157).", + "section": "RESULTS", + "ner": [ + [ + 9, + 11, + "MS", + "experimental_method" + ], + [ + 24, + 32, + "CthCD-CT", + "mutant" + ], + [ + 37, + 45, + "Cth\u0394BCCP", + "mutant" + ], + [ + 85, + 100, + "phosphorylation", + "ptm" + ], + [ + 104, + 111, + "Ser1170", + "residue_name_number" + ], + [ + 130, + 136, + "SceACC", + "protein" + ], + [ + 137, + 144, + "Ser1157", + "residue_name_number" + ] + ] + }, + { + "sid": 112, + "sent": "The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23\u2009\u00c5 (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of Cth\u0394BCCP, denoted as CthCD-CT1/2 and Cth\u0394BCCP1, respectively).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "CDN", + "structure_element" + ], + [ + 39, + 42, + "CDL", + "structure_element" + ], + [ + 43, + 47, + "CDC1", + "structure_element" + ], + [ + 112, + 122, + "structures", + "evidence" + ], + [ + 126, + 129, + "Sce", + "species" + ], + [ + 129, + 131, + "CD", + "structure_element" + ], + [ + 140, + 163, + "larger CthACC fragments", + "mutant" + ], + [ + 165, + 168, + "CDN", + "structure_element" + ], + [ + 257, + 266, + "protomers", + "oligomeric_state" + ], + [ + 270, + 278, + "CthCD-CT", + "mutant" + ], + [ + 287, + 295, + "protomer", + "oligomeric_state" + ], + [ + 299, + 307, + "Cth\u0394BCCP", + "mutant" + ], + [ + 320, + 331, + "CthCD-CT1/2", + "mutant" + ], + [ + 336, + 345, + "Cth\u0394BCCP1", + "mutant" + ] + ] + }, + { + "sid": 113, + "sent": "In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70\u00b0 (Fig. 4b, observed in the second protomer of Cth\u0394BCCP, denoted as Cth\u0394BCCP2) and 160\u00b0 (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40\u2009\u00c5, respectively.", + "section": "RESULTS", + "ner": [ + [ + 13, + 16, + "CDN", + "structure_element" + ], + [ + 35, + 41, + "hinges", + "structure_element" + ], + [ + 68, + 71, + "CDN", + "structure_element" + ], + [ + 72, + 75, + "CDL", + "structure_element" + ], + [ + 116, + 124, + "protomer", + "oligomeric_state" + ], + [ + 128, + 136, + "Cth\u0394BCCP", + "mutant" + ], + [ + 149, + 158, + "Cth\u0394BCCP2", + "mutant" + ], + [ + 191, + 194, + "Sce", + "species" + ], + [ + 194, + 196, + "CD", + "structure_element" + ], + [ + 229, + 240, + "anchor site", + "site" + ], + [ + 249, + 260, + "BCCP linker", + "structure_element" + ] + ] + }, + { + "sid": 114, + "sent": "Conformational variability in the CD thus contributes considerably to variations in the spacing between the BC and CT domains, and may extend to distance variations beyond the mobility range of the flexibly tethered BCCP.", + "section": "RESULTS", + "ner": [ + [ + 34, + 36, + "CD", + "structure_element" + ], + [ + 108, + 110, + "BC", + "structure_element" + ], + [ + 115, + 117, + "CT", + "structure_element" + ], + [ + 198, + 215, + "flexibly tethered", + "protein_state" + ], + [ + 216, + 220, + "BCCP", + "structure_element" + ] + ] + }, + { + "sid": 115, + "sent": "On the basis of the occurrence of related conformational changes between fungal and human ACC fragments, the observed set of conformations may well represent general states present in all eukaryotic ACCs.", + "section": "RESULTS", + "ner": [ + [ + 73, + 79, + "fungal", + "taxonomy_domain" + ], + [ + 84, + 89, + "human", + "species" + ], + [ + 90, + 103, + "ACC fragments", + "mutant" + ], + [ + 188, + 198, + "eukaryotic", + "taxonomy_domain" + ], + [ + 199, + 203, + "ACCs", + "protein_type" + ] + ] + }, + { + "sid": 116, + "sent": "Large-scale conformational variability of fungal ACC", + "section": "RESULTS", + "ner": [ + [ + 42, + 48, + "fungal", + "taxonomy_domain" + ], + [ + 49, + 52, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 117, + "sent": "To obtain a comprehensive view of fungal ACC dynamics in solution, we employed SAXS and EM.", + "section": "RESULTS", + "ner": [ + [ + 34, + 40, + "fungal", + "taxonomy_domain" + ], + [ + 41, + 44, + "ACC", + "protein_type" + ], + [ + 54, + 65, + "in solution", + "protein_state" + ], + [ + 79, + 83, + "SAXS", + "experimental_method" + ], + [ + 88, + 90, + "EM", + "experimental_method" + ] + ] + }, + { + "sid": 118, + "sent": "SAXS analysis of CthACC agrees with a dimeric state and an elongated shape with a maximum extent of 350\u2009\u00c5 (Supplementary Table 1).", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "SAXS", + "experimental_method" + ], + [ + 17, + 23, + "CthACC", + "protein" + ], + [ + 38, + 45, + "dimeric", + "oligomeric_state" + ], + [ + 59, + 74, + "elongated shape", + "protein_state" + ] + ] + }, + { + "sid": 119, + "sent": "The smooth appearance of scattering curves and derived distance distributions might indicate substantial interdomain flexibility (Supplementary Fig. 2a\u2013c).", + "section": "RESULTS", + "ner": [ + [ + 25, + 42, + "scattering curves", + "evidence" + ], + [ + 47, + 77, + "derived distance distributions", + "evidence" + ] + ] + }, + { + "sid": 120, + "sent": "Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles.", + "section": "RESULTS", + "ner": [ + [ + 33, + 44, + "full-length", + "protein_state" + ], + [ + 45, + 51, + "CthACC", + "protein" + ], + [ + 52, + 61, + "particles", + "evidence" + ], + [ + 76, + 78, + "MS", + "experimental_method" + ], + [ + 106, + 120, + "phosphorylated", + "protein_state" + ], + [ + 121, + 139, + "low-activity state", + "protein_state" + ], + [ + 144, + 161, + "negative stain EM", + "experimental_method" + ], + [ + 204, + 221, + "rod-like extended", + "protein_state" + ], + [ + 225, + 233, + "U-shaped", + "protein_state" + ], + [ + 234, + 243, + "particles", + "evidence" + ] + ] + }, + { + "sid": 121, + "sent": "Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC\u2013BCCP\u2013CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer.", + "section": "RESULTS", + "ner": [ + [ + 0, + 14, + "Class averages", + "evidence" + ], + [ + 28, + 88, + "maximum-likelihood-based two-dimensional (2D) classification", + "experimental_method" + ], + [ + 109, + 116, + "dimeric", + "oligomeric_state" + ], + [ + 117, + 119, + "CT", + "structure_element" + ], + [ + 135, + 139, + "full", + "protein_state" + ], + [ + 140, + 150, + "BC\u2013BCCP\u2013CD", + "mutant" + ], + [ + 170, + 178, + "protomer", + "oligomeric_state" + ], + [ + 230, + 232, + "BC", + "structure_element" + ], + [ + 233, + 235, + "CD", + "structure_element" + ], + [ + 260, + 262, + "CT", + "structure_element" + ], + [ + 263, + 268, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 122, + "sent": "They identify the connections between CDN/CDL and between CDC2/CT as major contributors to conformational heterogeneity (Supplementary Fig. 4a,b).", + "section": "RESULTS", + "ner": [ + [ + 38, + 41, + "CDN", + "structure_element" + ], + [ + 42, + 45, + "CDL", + "structure_element" + ], + [ + 58, + 62, + "CDC2", + "structure_element" + ], + [ + 63, + 65, + "CT", + "structure_element" + ] + ] + }, + { + "sid": 123, + "sent": "The flexibility in the CDC2/CT hinge appears substantially larger than the variations observed in the set of crystal structures.", + "section": "RESULTS", + "ner": [ + [ + 23, + 36, + "CDC2/CT hinge", + "structure_element" + ], + [ + 109, + 127, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 124, + "sent": "The BC domain is not completely disordered, but laterally attached to BT/CDN in a generally conserved position, albeit with increased flexibility.", + "section": "RESULTS", + "ner": [ + [ + 4, + 6, + "BC", + "structure_element" + ], + [ + 70, + 72, + "BT", + "structure_element" + ], + [ + 73, + 76, + "CDN", + "structure_element" + ], + [ + 82, + 110, + "generally conserved position", + "protein_state" + ] + ] + }, + { + "sid": 125, + "sent": "Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110\u2009\u00c5. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase.", + "section": "RESULTS", + "ner": [ + [ + 26, + 59, + "linear and U-shaped conformations", + "protein_state" + ], + [ + 99, + 101, + "BC", + "structure_element" + ], + [ + 106, + 108, + "CT", + "structure_element" + ], + [ + 109, + 121, + "active sites", + "site" + ], + [ + 211, + 217, + "static", + "protein_state" + ], + [ + 218, + 228, + "structures", + "evidence" + ], + [ + 250, + 278, + "biotin-dependent carboxylase", + "protein_type" + ] + ] + }, + { + "sid": 126, + "sent": "Furthermore, based on an average length of the BCCP\u2013CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT.", + "section": "RESULTS", + "ner": [ + [ + 47, + 61, + "BCCP\u2013CD linker", + "structure_element" + ], + [ + 65, + 71, + "fungal", + "taxonomy_domain" + ], + [ + 72, + 75, + "ACC", + "protein_type" + ], + [ + 79, + 93, + "26 amino acids", + "residue_range" + ], + [ + 111, + 115, + "BCCP", + "structure_element" + ], + [ + 160, + 172, + "active sites", + "site" + ], + [ + 176, + 178, + "BC", + "structure_element" + ], + [ + 183, + 185, + "CT", + "structure_element" + ] + ] + }, + { + "sid": 127, + "sent": "The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure.", + "section": "RESULTS", + "ner": [ + [ + 130, + 149, + "CDC1/CDC2 interface", + "site" + ], + [ + 178, + 185, + "Ser1157", + "residue_name_number" + ], + [ + 186, + 200, + "phosphorylated", + "protein_state" + ], + [ + 201, + 216, + "regulatory loop", + "structure_element" + ], + [ + 237, + 240, + "Sce", + "species" + ], + [ + 240, + 242, + "CD", + "structure_element" + ], + [ + 243, + 260, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 128, + "sent": "Altogether, the architecture of fungal ACC is based on the central dimeric CT domain (Fig. 4d).", + "section": "DISCUSS", + "ner": [ + [ + 32, + 38, + "fungal", + "taxonomy_domain" + ], + [ + 39, + 42, + "ACC", + "protein_type" + ], + [ + 67, + 74, + "dimeric", + "oligomeric_state" + ], + [ + 75, + 77, + "CT", + "structure_element" + ] + ] + }, + { + "sid": 129, + "sent": "The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 6, + "CD", + "structure_element" + ], + [ + 33, + 43, + "subdomains", + "structure_element" + ], + [ + 74, + 76, + "CT", + "structure_element" + ], + [ + 84, + 90, + "mobile", + "protein_state" + ], + [ + 91, + 95, + "BCCP", + "structure_element" + ], + [ + 103, + 111, + "oriented", + "protein_state" + ], + [ + 112, + 114, + "BC", + "structure_element" + ] + ] + }, + { + "sid": 130, + "sent": "The CD has no direct role in substrate recognition or catalysis but contributes to the regulation of all eukaryotic ACCs.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 6, + "CD", + "structure_element" + ], + [ + 105, + 115, + "eukaryotic", + "taxonomy_domain" + ], + [ + 116, + 120, + "ACCs", + "protein_type" + ] + ] + }, + { + "sid": 131, + "sent": "In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 20, + "higher eukaryotic", + "taxonomy_domain" + ], + [ + 21, + 25, + "ACCs", + "protein_type" + ], + [ + 42, + 57, + "phosphorylation", + "ptm" + ], + [ + 98, + 113, + "phosphorylation", + "ptm" + ], + [ + 117, + 122, + "Ser80", + "residue_name_number" + ], + [ + 124, + 131, + "Ser1201", + "residue_name_number" + ], + [ + 136, + 143, + "Ser1263", + "residue_name_number" + ] + ] + }, + { + "sid": 132, + "sent": "In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 9, + "fungal", + "taxonomy_domain" + ], + [ + 10, + 13, + "ACC", + "protein_type" + ], + [ + 24, + 31, + "Ser1157", + "residue_name_number" + ], + [ + 39, + 54, + "regulatory loop", + "structure_element" + ], + [ + 62, + 64, + "CD", + "structure_element" + ], + [ + 77, + 97, + "phosphorylation site", + "site" + ], + [ + 136, + 150, + "phosphorylated", + "protein_state" + ], + [ + 193, + 196, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 133, + "sent": "In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 21, + "phosphorylated", + "protein_state" + ], + [ + 33, + 48, + "regulatory loop", + "structure_element" + ], + [ + 60, + 67, + "Ser1157", + "residue_name_number" + ], + [ + 83, + 87, + "CDC1", + "structure_element" + ], + [ + 88, + 92, + "CDC2", + "structure_element" + ], + [ + 119, + 141, + "conformational freedom", + "protein_state" + ], + [ + 150, + 171, + "interdomain interface", + "site" + ] + ] + }, + { + "sid": 134, + "sent": "However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 34, + "hinge", + "structure_element" + ], + [ + 55, + 72, + "full ACC activity", + "protein_state" + ], + [ + 103, + 121, + "BCCP anchor points", + "structure_element" + ], + [ + 130, + 142, + "active sites", + "site" + ], + [ + 146, + 148, + "BC", + "structure_element" + ], + [ + 153, + 155, + "CT", + "structure_element" + ], + [ + 206, + 210, + "BCCP", + "structure_element" + ] + ] + }, + { + "sid": 135, + "sent": "The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT.", + "section": "DISCUSS", + "ner": [ + [ + 49, + 55, + "fungal", + "taxonomy_domain" + ], + [ + 56, + 59, + "ACC", + "protein_type" + ], + [ + 107, + 113, + "unique", + "protein_state" + ], + [ + 114, + 116, + "CD", + "structure_element" + ], + [ + 175, + 187, + "active sites", + "site" + ], + [ + 191, + 193, + "BC", + "structure_element" + ], + [ + 198, + 200, + "CT", + "structure_element" + ] + ] + }, + { + "sid": 136, + "sent": "A comparison between fungal and human ACC will help to further discriminate mechanistic differences that contribute to the extended control and polymerization of human ACC.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 27, + "fungal", + "taxonomy_domain" + ], + [ + 32, + 37, + "human", + "species" + ], + [ + 38, + 41, + "ACC", + "protein_type" + ], + [ + 162, + 167, + "human", + "species" + ], + [ + 168, + 171, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 137, + "sent": "Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 34, + "crystal structure", + "evidence" + ], + [ + 38, + 54, + "near full-length", + "protein_state" + ], + [ + 55, + 73, + "non-phosphorylated", + "protein_state" + ], + [ + 74, + 77, + "ACC", + "protein_type" + ], + [ + 83, + 95, + "S. cerevisae", + "species" + ], + [ + 97, + 109, + "lacking only", + "protein_state" + ], + [ + 110, + 112, + "21", + "residue_range" + ], + [ + 153, + 158, + "flACC", + "protein" + ] + ] + }, + { + "sid": 138, + "sent": "In flACC, the ACC dimer obeys twofold symmetry and assembles in a triangular architecture with dimeric BC domains (Supplementary Fig. 5a).", + "section": "DISCUSS", + "ner": [ + [ + 3, + 8, + "flACC", + "protein" + ], + [ + 14, + 17, + "ACC", + "protein_type" + ], + [ + 18, + 23, + "dimer", + "oligomeric_state" + ], + [ + 66, + 89, + "triangular architecture", + "protein_state" + ], + [ + 95, + 102, + "dimeric", + "oligomeric_state" + ], + [ + 103, + 105, + "BC", + "structure_element" + ] + ] + }, + { + "sid": 139, + "sent": "In their study, mutational data indicate a requirement for BC dimerization for catalytic activity.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 31, + "mutational data", + "experimental_method" + ] + ] + }, + { + "sid": 140, + "sent": "The transition from the elongated open shape, observed in our experiments, towards a compact triangular shape is based on an intricate interplay of several hinge-bending motions in the CD (Fig. 4d).", + "section": "DISCUSS", + "ner": [ + [ + 24, + 44, + "elongated open shape", + "protein_state" + ], + [ + 85, + 109, + "compact triangular shape", + "protein_state" + ], + [ + 185, + 187, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 141, + "sent": "Comparison of flACC with our Cth\u0394BCCP structure reveals the CDC2/CT hinge as a major contributor to conformational flexibility (Supplementary Fig. 5b,c).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 10, + "Comparison", + "experimental_method" + ], + [ + 14, + 19, + "flACC", + "protein" + ], + [ + 29, + 37, + "Cth\u0394BCCP", + "mutant" + ], + [ + 38, + 47, + "structure", + "evidence" + ], + [ + 60, + 73, + "CDC2/CT hinge", + "structure_element" + ] + ] + }, + { + "sid": 142, + "sent": "In flACC, CDC2 rotates \u223c120\u00b0 with respect to the CT domain.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 8, + "flACC", + "protein" + ], + [ + 10, + 14, + "CDC2", + "structure_element" + ], + [ + 49, + 51, + "CT", + "structure_element" + ] + ] + }, + { + "sid": 143, + "sent": "A second hinge can be identified between CDC1/CDC2.", + "section": "DISCUSS", + "ner": [ + [ + 2, + 14, + "second hinge", + "structure_element" + ], + [ + 41, + 45, + "CDC1", + "structure_element" + ], + [ + 46, + 50, + "CDC2", + "structure_element" + ] + ] + }, + { + "sid": 144, + "sent": "On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30\u00b0 relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d).", + "section": "DISCUSS", + "ner": [ + [ + 18, + 31, + "superposition", + "experimental_method" + ], + [ + 35, + 39, + "CDC2", + "structure_element" + ], + [ + 41, + 45, + "CDC1", + "structure_element" + ], + [ + 53, + 67, + "phosphorylated", + "protein_state" + ], + [ + 68, + 71, + "Sce", + "species" + ], + [ + 71, + 73, + "CD", + "structure_element" + ], + [ + 104, + 108, + "CDC1", + "structure_element" + ], + [ + 116, + 134, + "non-phosphorylated", + "protein_state" + ], + [ + 135, + 140, + "flACC", + "protein" + ], + [ + 207, + 225, + "non-phosphorylated", + "protein_state" + ], + [ + 226, + 234, + "HsaBT-CD", + "mutant" + ] + ] + }, + { + "sid": 145, + "sent": "When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations.", + "section": "DISCUSS", + "ner": [ + [ + 5, + 15, + "inspecting", + "experimental_method" + ], + [ + 31, + 39, + "protomer", + "oligomeric_state" + ], + [ + 44, + 52, + "fragment", + "mutant" + ], + [ + 53, + 63, + "structures", + "evidence" + ], + [ + 111, + 130, + "CDN/CDC1 connection", + "structure_element" + ], + [ + 136, + 151, + "highly flexible", + "protein_state" + ], + [ + 152, + 157, + "hinge", + "structure_element" + ] + ] + }, + { + "sid": 146, + "sent": "The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 29, + "regulatory", + "protein_state" + ], + [ + 30, + 49, + "phophorylation site", + "site" + ], + [ + 53, + 59, + "fungal", + "taxonomy_domain" + ], + [ + 60, + 63, + "ACC", + "protein_type" + ], + [ + 71, + 86, + "regulatory loop", + "structure_element" + ], + [ + 116, + 120, + "CDC1", + "structure_element" + ], + [ + 121, + 125, + "CDC2", + "structure_element" + ], + [ + 170, + 188, + "hinge conformation", + "structure_element" + ] + ] + }, + { + "sid": 147, + "sent": "In flACC, the regulatory loop is mostly disordered, illustrating the increased flexibility due to the absence of the phosphoryl group.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 8, + "flACC", + "protein" + ], + [ + 14, + 29, + "regulatory loop", + "structure_element" + ], + [ + 33, + 50, + "mostly disordered", + "protein_state" + ], + [ + 117, + 127, + "phosphoryl", + "chemical" + ] + ] + }, + { + "sid": 148, + "sent": "Only in three out of eight observed protomers a short peptide stretch (including Ser1157) was modelled.", + "section": "DISCUSS", + "ner": [ + [ + 36, + 45, + "protomers", + "oligomeric_state" + ], + [ + 48, + 61, + "short peptide", + "structure_element" + ], + [ + 81, + 88, + "Ser1157", + "residue_name_number" + ], + [ + 94, + 102, + "modelled", + "evidence" + ] + ] + }, + { + "sid": 149, + "sent": "In those instances the Ser1157 residue is located at a distance of 14\u201320\u2009\u00c5 away from the location of the phosphorylated serine observed here, based on superposition of either CDC1 or CDC2.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 30, + "Ser1157", + "residue_name_number" + ], + [ + 105, + 119, + "phosphorylated", + "protein_state" + ], + [ + 120, + 126, + "serine", + "residue_name" + ], + [ + 151, + 164, + "superposition", + "experimental_method" + ], + [ + 175, + 179, + "CDC1", + "structure_element" + ], + [ + 183, + 187, + "CDC2", + "structure_element" + ] + ] + }, + { + "sid": 150, + "sent": "Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 8, + "Applying", + "experimental_method" + ], + [ + 33, + 48, + "CDC1/CDC2 hinge", + "structure_element" + ], + [ + 61, + 64, + "Sce", + "species" + ], + [ + 64, + 66, + "CD", + "structure_element" + ], + [ + 70, + 75, + "flACC", + "protein" + ], + [ + 85, + 88, + "CDN", + "structure_element" + ], + [ + 114, + 118, + "CDC2", + "structure_element" + ], + [ + 123, + 125, + "BT", + "structure_element" + ], + [ + 126, + 129, + "CDN", + "structure_element" + ], + [ + 144, + 146, + "CT", + "structure_element" + ] + ] + }, + { + "sid": 151, + "sent": "Thus, in accordance with the results presented here, phosphorylation of Ser1157 in SceACC most likely limits flexibility in the CDC1/CDC2 hinge such that activation through BC dimerization is not possible (Fig. 4d), which however does not exclude intermolecular dimerization.", + "section": "DISCUSS", + "ner": [ + [ + 53, + 68, + "phosphorylation", + "ptm" + ], + [ + 72, + 79, + "Ser1157", + "residue_name_number" + ], + [ + 83, + 89, + "SceACC", + "protein" + ], + [ + 128, + 143, + "CDC1/CDC2 hinge", + "structure_element" + ], + [ + 173, + 175, + "BC", + "structure_element" + ] + ] + }, + { + "sid": 152, + "sent": "In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7).", + "section": "DISCUSS", + "ner": [ + [ + 13, + 15, + "EM", + "experimental_method" + ], + [ + 16, + 27, + "micrographs", + "evidence" + ], + [ + 31, + 45, + "phosphorylated", + "protein_state" + ], + [ + 50, + 66, + "dephosphorylated", + "protein_state" + ], + [ + 67, + 73, + "SceACC", + "protein" + ], + [ + 106, + 142, + "elongated and U-shaped conformations", + "protein_state" + ], + [ + 181, + 209, + "particle shape distributions", + "evidence" + ] + ] + }, + { + "sid": 153, + "sent": "This implicates that the triangular shape with dimeric BC domains has a low population also in the active form, even though a biasing influence of grid preparation cannot be excluded completely.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 41, + "triangular shape", + "protein_state" + ], + [ + 47, + 54, + "dimeric", + "oligomeric_state" + ], + [ + 55, + 57, + "BC", + "structure_element" + ], + [ + 99, + 110, + "active form", + "protein_state" + ] + ] + }, + { + "sid": 154, + "sent": "Large-scale conformational variability has also been observed in most other carrier protein-based multienzymes, including polyketide and fatty-acid synthases (with the exception of fungal-type fatty-acid synthases), non-ribosomal peptide synthetases and the pyruvate dehydrogenase complexes, although based on completely different architectures.", + "section": "DISCUSS", + "ner": [ + [ + 76, + 110, + "carrier protein-based multienzymes", + "protein_type" + ], + [ + 122, + 157, + "polyketide and fatty-acid synthases", + "protein_type" + ], + [ + 181, + 213, + "fungal-type fatty-acid synthases", + "protein_type" + ], + [ + 216, + 249, + "non-ribosomal peptide synthetases", + "protein_type" + ], + [ + 258, + 290, + "pyruvate dehydrogenase complexes", + "protein_type" + ] + ] + }, + { + "sid": 155, + "sent": "Together, this structural information suggests that variable carrier protein tethering is not sufficient for efficient substrate transfer and catalysis in any of these systems.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 37, + "structural information", + "evidence" + ] + ] + }, + { + "sid": 156, + "sent": "The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 29, + "determination of a set of", + "experimental_method" + ], + [ + 30, + 48, + "crystal structures", + "evidence" + ], + [ + 52, + 58, + "SceACC", + "protein" + ], + [ + 74, + 90, + "unphosphorylated", + "protein_state" + ], + [ + 95, + 109, + "phosphorylated", + "protein_state" + ], + [ + 117, + 138, + "major regulatory site", + "site" + ], + [ + 139, + 146, + "Ser1157", + "residue_name_number" + ] + ] + }, + { + "sid": 157, + "sent": "The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 18, + "phosphorylated", + "protein_state" + ], + [ + 19, + 34, + "regulatory loop", + "structure_element" + ], + [ + 47, + 62, + "allosteric site", + "site" + ], + [ + 70, + 79, + "interface", + "site" + ], + [ + 87, + 100, + "non-catalytic", + "protein_state" + ], + [ + 157, + 163, + "hinges", + "structure_element" + ], + [ + 171, + 178, + "dynamic", + "protein_state" + ], + [ + 179, + 182, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 158, + "sent": "It disfavours the adoption of a rare, compact conformation, in which intramolecular dimerization of the BC domains results in catalytic turnover.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 58, + "rare, compact conformation", + "protein_state" + ], + [ + 104, + 106, + "BC", + "structure_element" + ] + ] + }, + { + "sid": 159, + "sent": "The regulation of activity thus results from restrained large-scale conformational dynamics rather than a direct or indirect influence on active site structure.", + "section": "DISCUSS", + "ner": [ + [ + 138, + 159, + "active site structure", + "site" + ] + ] + }, + { + "sid": 160, + "sent": "To our best knowledge, ACC is the first multienzyme for which such a phosphorylation-dependent mechanical control mechanism has been visualized.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 26, + "ACC", + "protein_type" + ], + [ + 40, + 51, + "multienzyme", + "protein_type" + ], + [ + 69, + 84, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 161, + "sent": "However, the example of ACC now demonstrates the possibility of regulating activity by controlled dynamics of non-enzymatic linker regions also in other families of carrier-dependent multienzymes.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 27, + "ACC", + "protein_type" + ], + [ + 110, + 138, + "non-enzymatic linker regions", + "structure_element" + ], + [ + 165, + 195, + "carrier-dependent multienzymes", + "protein_type" + ] + ] + }, + { + "sid": 162, + "sent": "The phosphorylated central domain of yeast ACC.", + "section": "FIG", + "ner": [ + [ + 4, + 18, + "phosphorylated", + "protein_state" + ], + [ + 19, + 33, + "central domain", + "structure_element" + ], + [ + 37, + 42, + "yeast", + "taxonomy_domain" + ], + [ + 43, + 46, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 163, + "sent": "(a) Schematic overview of the domain organization of eukaryotic ACCs.", + "section": "FIG", + "ner": [ + [ + 53, + 63, + "eukaryotic", + "taxonomy_domain" + ], + [ + 64, + 68, + "ACCs", + "protein_type" + ] + ] + }, + { + "sid": 164, + "sent": "Crystallized constructs are indicated.", + "section": "FIG", + "ner": [ + [ + 0, + 23, + "Crystallized constructs", + "evidence" + ] + ] + }, + { + "sid": 165, + "sent": "(b) Cartoon representation of the SceCD crystal structure.", + "section": "FIG", + "ner": [ + [ + 34, + 37, + "Sce", + "species" + ], + [ + 37, + 39, + "CD", + "structure_element" + ], + [ + 40, + 57, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 166, + "sent": "CDN is linked by a four-helix bundle (CDL) to two \u03b1\u2013\u03b2-fold domains (CDC1 and CDC2).", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "CDN", + "structure_element" + ], + [ + 19, + 36, + "four-helix bundle", + "structure_element" + ], 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(d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group.", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "Superposition", + "experimental_method" + ], + [ + 21, + 25, + "CDC1", + "structure_element" + ], + [ + 30, + 34, + "CDC2", + "structure_element" + ], + [ + 43, + 59, + "highly conserved", + "protein_state" + ], + [ + 60, + 65, + "folds", + "structure_element" + ], + [ + 75, + 90, + "regulatory loop", + "structure_element" + ], + [ + 100, + 114, + "phosphorylated", + "protein_state" + ], + [ + 115, + 122, + "Ser1157", + "residue_name_number" + ], + [ + 155, + 159, + "CDC1", + "structure_element" + ], + [ + 164, + 168, + "CDC2", + "structure_element" + ], + [ + 174, + 183, + "conserved", + "protein_state" + ], + [ + 193, + 200, + "Arg1173", + "residue_name_number" + ], + [ + 205, + 212, + "Arg1260", + "residue_name_number" + ], + [ + 228, + 238, + "phosphoryl", + "chemical" + ] + ] + }, + { + "sid": 169, + "sent": "(e) Structural overview of HsaBT-CD.", + "section": "FIG", + "ner": [ + [ + 27, + 35, + "HsaBT-CD", + "mutant" + ] + ] + }, + { + "sid": 170, + "sent": "The attachment points to the N-terminal BCCP domain and the C-terminal CT domain are indicated with spheres.", + "section": "FIG", + "ner": [ + [ + 40, + 44, + "BCCP", + "structure_element" + ], + [ + 71, + 73, + "CT", + "structure_element" + ] + ] + }, + { + "sid": 171, + "sent": "Architecture of the CD\u2013CT core of fungal ACC.", + "section": "FIG", + "ner": [ + [ + 20, + 22, + "CD", + "structure_element" + ], + [ + 23, + 25, + "CT", + "structure_element" + ], + [ + 34, + 40, + "fungal", + "taxonomy_domain" + ], + [ + 41, + 44, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 172, + "sent": "Cartoon representation of crystal structures of multidomain constructs of CthACC.", + "section": "FIG", + "ner": [ + [ + 26, + 44, + "crystal structures", + "evidence" + ], + [ + 48, + 70, + "multidomain constructs", + "mutant" + ], + [ + 74, + 80, + "CthACC", + "protein" + ] + ] + }, + { + "sid": 173, + "sent": "One protomer is shown in colour and one in grey.", + "section": "FIG", + "ner": [ + [ + 4, + 12, + "protomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 174, + "sent": "Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively.", + "section": "FIG", + "ner": [ + [ + 37, + 48, + "active site", + "site" + ], + [ + 52, + 54, + "CT", + "structure_element" + ], + [ + 79, + 88, + "conserved", + "protein_state" + ], + [ + 89, + 99, + "regulatory", + "protein_state" + ], + [ + 100, + 118, + "phosphoserine site", + "site" + ], + [ + 128, + 131, + "Sce", + "species" + ], + [ + 131, + 133, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 175, + "sent": "Variability of the connections of CDC2 to CT and CDC1 in fungal ACC.", + "section": "FIG", + "ner": [ + [ + 34, + 38, + "CDC2", + "structure_element" + ], + [ + 42, + 44, + "CT", + "structure_element" + ], + [ + 49, + 53, + "CDC1", + "structure_element" + ], + [ + 57, + 63, + "fungal", + "taxonomy_domain" + ], + [ + 64, + 67, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 176, + "sent": "(a) Hinge properties of the CDC2\u2013CT connection analysed by a CT-based superposition of eight instances of the CDC2-CT segment.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "Hinge", + "structure_element" + ], + [ + 28, + 46, + "CDC2\u2013CT connection", + "structure_element" + ], + [ + 61, + 83, + "CT-based superposition", + "experimental_method" + ], + [ + 110, + 125, + "CDC2-CT segment", + "mutant" + ] + ] + }, + { + "sid": 177, + "sent": "For clarity, only one protomer of CthCD-CTCter1 is shown in full colour as reference.", + "section": "FIG", + "ner": [ + [ + 22, + 30, + "protomer", + "oligomeric_state" + ], + [ + 34, + 47, + "CthCD-CTCter1", + "mutant" + ] + ] + }, + { + "sid": 178, + "sent": "For other instances, CDC2 domains are shown in transparent tube representation with only one helix each highlighted.", + "section": "FIG", + "ner": [ + [ + 21, + 25, + "CDC2", + "structure_element" + ] + ] + }, + { + "sid": 179, + "sent": "The range of hinge bending is indicated and the connection points between CDC2 and CT (blue) as well as between CDC1 and CDC2 (green and grey) are marked as spheres.", + "section": "FIG", + "ner": [ + [ + 74, + 78, + "CDC2", + "structure_element" + ], + [ + 83, + 85, + "CT", + "structure_element" + ], + [ + 112, + 116, + "CDC1", + "structure_element" + ], + [ + 121, + 125, + "CDC2", + "structure_element" + ] + ] + }, + { + "sid": 180, + "sent": "(b) The interdomain interface of CDC1 and CDC2 exhibits only limited plasticity.", + "section": "FIG", + "ner": [ + [ + 8, + 29, + "interdomain interface", + "site" + ], + [ + 33, + 37, + "CDC1", + "structure_element" + ], + [ + 42, + 46, + "CDC2", + "structure_element" + ] + ] + }, + { + "sid": 181, + "sent": "Representation as in a, but the CDC1 and CDC2 are superposed based on CDC2.", + "section": "FIG", + "ner": [ + [ + 32, + 36, + "CDC1", + "structure_element" + ], + [ + 41, + 45, + "CDC2", + "structure_element" + ], + [ + 50, + 60, + "superposed", + "experimental_method" + ], + [ + 70, + 74, + "CDC2", + "structure_element" + ] + ] + }, + { + "sid": 182, + "sent": "One protomer of Cth\u0394BCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle.", + "section": "FIG", + "ner": [ + [ + 4, + 12, + "protomer", + "oligomeric_state" + ], + [ + 16, + 24, + "Cth\u0394BCCP", + "mutant" + ], + [ + 49, + 52, + "CDL", + "structure_element" + ], + [ + 109, + 123, + "phosphorylated", + "protein_state" + ], + [ + 124, + 130, + "serine", + "residue_name" + ], + [ + 140, + 143, + "Sce", + "species" + ], + [ + 143, + 145, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 183, + "sent": "The connection points from CDC1 to CDC2 and to CDL are represented by green spheres.", + "section": "FIG", + "ner": [ + [ + 27, + 31, + "CDC1", + "structure_element" + ], + [ + 35, + 39, + "CDC2", + "structure_element" + ], + [ + 47, + 50, + "CDL", + "structure_element" + ] + ] + }, + { + "sid": 184, + "sent": "The conformational dynamics of fungal ACC.", + "section": "FIG", + "ner": [ + [ + 31, + 37, + "fungal", + "taxonomy_domain" + ], + [ + 38, + 41, + "ACC", + "protein_type" + ] + ] + }, + { + "sid": 185, + "sent": "(a\u2013c) Large-scale conformational variability of the CDN domain relative to the CDL/CDC1 domain.", + "section": "FIG", + "ner": [ + [ + 52, + 55, + "CDN", + "structure_element" + ], + [ + 79, + 82, + "CDL", + "structure_element" + ], + [ + 83, + 87, + "CDC1", + "structure_element" + ] + ] + }, + { + "sid": 186, + "sent": "CthCD-CT1 (in colour) serves as reference, the compared structures (as indicated, numbers after construct name differentiate between individual protomers) are shown in grey.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "CthCD-CT1", + "mutant" + ], + [ + 47, + 66, + "compared structures", + "experimental_method" + ], + [ + 144, + 153, + "protomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 187, + "sent": "Domains other than CDN and CDL/CDC1 are omitted for clarity.", + "section": "FIG", + "ner": [ + [ + 19, + 22, + "CDN", + "structure_element" + ], + [ + 27, + 30, + "CDL", + "structure_element" + ], + [ + 31, + 35, + "CDC1", + "structure_element" + ] + ] + }, + { + "sid": 188, + "sent": "The domains are labelled and the distances between the N termini of CDN (spheres) in the compared structures are indicated.", + "section": "FIG", + "ner": [ + [ + 68, + 71, + "CDN", + "structure_element" + ] + ] + }, + { + "sid": 189, + "sent": "(d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state.", + "section": "FIG", + "ner": [ + [ + 23, + 29, + "fungal", + "taxonomy_domain" + ], + [ + 30, + 33, + "ACC", + "protein_type" + ], + [ + 82, + 84, + "CD", + "structure_element" + ], + [ + 92, + 106, + "phosphorylated", + "protein_state" + ], + [ + 107, + 116, + "inhibited", + "protein_state" + ], + [ + 124, + 142, + "non-phosphorylated", + "protein_state" + ], + [ + 143, + 152, + "activated", + "protein_state" + ] + ] + }, + { + "sid": 190, + "sent": "Flexibility of the CDC2/CT and CDN/CDL hinges is illustrated by arrows.", + "section": "FIG", + "ner": [ + [ + 19, + 23, + "CDC2", + "structure_element" + ], + [ + 24, + 26, + "CT", + "structure_element" + ], + [ + 31, + 34, + "CDN", + "structure_element" + ], + [ + 35, + 38, + "CDL", + "structure_element" + ], + [ + 39, + 45, + "hinges", + "structure_element" + ] + ] + }, + { + "sid": 191, + "sent": "The Ser1157 phosphorylation site and the regulatory loop are schematically indicated in magenta.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "Ser1157", + "residue_name_number" + ], + [ + 12, + 27, + "phosphorylation", + "ptm" + ], + [ + 41, + 56, + "regulatory loop", + "structure_element" + ] + ] + } + ] + }, + "PMC4746701": { + "annotations": [ + { + "sid": 0, + "sent": "Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway", + "section": "TITLE", + "ner": [ + [ + 0, + 17, + "Crystal structure", + "evidence" + ], + [ + 21, + 26, + "SEL1L", + "protein" + ], + [ + 54, + 57, + "SLR", + "structure_element" + ] + ] + }, + { + "sid": 1, + "sent": "SEL1L, a component of the ERAD machinery, plays an important role in selecting and transporting ERAD substrates for degradation.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 5, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 2, + "sent": "We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent).", + "section": "ABSTRACT", + "ner": [ + [ + 23, + 40, + "crystal structure", + "evidence" + ], + [ + 48, + 53, + "mouse", + "taxonomy_domain" + ], + [ + 54, + 59, + "SEL1L", + "protein" + ], + [ + 60, + 74, + "central domain", + "structure_element" + ], + [ + 91, + 108, + "Sel1-Like Repeats", + "structure_element" + ], + [ + 110, + 127, + "SLR motifs 5 to 9", + "structure_element" + ], + [ + 146, + 155, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 3, + "sent": "Strikingly, SEL1Lcent forms a homodimer with two-fold symmetry in a head-to-tail manner.", + "section": "ABSTRACT", + "ner": [ + [ + 12, + 21, + "SEL1Lcent", + "structure_element" + ], + [ + 30, + 39, + "homodimer", + "oligomeric_state" + ], + [ + 68, + 80, + "head-to-tail", + "protein_state" + ] + ] + }, + { + "sid": 4, + "sent": "Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface.", + "section": "ABSTRACT", + "ner": [ + [ + 18, + 29, + "SLR motif 9", + "structure_element" + ], + [ + 57, + 62, + "dimer", + "oligomeric_state" + ], + [ + 87, + 101, + "domain-swapped", + "protein_state" + ], + [ + 139, + 156, + "dimeric interface", + "site" + ] + ] + }, + { + "sid": 5, + "sent": "We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells.", + "section": "ABSTRACT", + "ner": [ + [ + 23, + 34, + "full-length", + "protein_state" + ], + [ + 35, + 40, + "SEL1L", + "protein" + ], + [ + 49, + 62, + "self-oligomer", + "oligomeric_state" + ], + [ + 75, + 84, + "SEL1Lcent", + "structure_element" + ], + [ + 95, + 104, + "mammalian", + "taxonomy_domain" + ] + ] + }, + { + "sid": 6, + "sent": "Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1.", + "section": "ABSTRACT", + "ner": [ + [ + 36, + 41, + "SLR-C", + "structure_element" + ], + [ + 54, + 74, + "SLR motifs 10 and 11", + "structure_element" + ], + [ + 79, + 84, + "SEL1L", + "protein" + ], + [ + 124, + 137, + "luminal loops", + "structure_element" + ], + [ + 141, + 145, + "HRD1", + "protein" + ] + ] + }, + { + "sid": 7, + "sent": "Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery.", + "section": "ABSTRACT", + "ner": [ + [ + 35, + 38, + "SLR", + "structure_element" + ], + [ + 49, + 54, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 8, + "sent": "Protein quality control in the endoplasmic reticulum (ER) is essential for maintenance of cellular homeostasis in eukaryotes and is implicated in many severe diseases.", + "section": "INTRO", + "ner": [ + [ + 114, + 124, + "eukaryotes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 9, + "sent": "Terminally misfolded proteins in the lumen or membrane of the ER are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome.", + "section": "INTRO", + "ner": [ + [ + 105, + 122, + "polyubiquitinated", + "protein_state" + ], + [ + 144, + 154, + "proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 10, + "sent": "The process is called ER-associated protein degradation (ERAD) and is conserved in all eukaryotes.", + "section": "INTRO", + "ner": [ + [ + 70, + 79, + "conserved", + "protein_state" + ], + [ + 87, + 97, + "eukaryotes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 11, + "sent": "Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans.", + "section": "INTRO", + "ner": [ + [ + 72, + 76, + "HRD1", + "protein" + ], + [ + 78, + 83, + "SEL1L", + "protein" + ], + [ + 85, + 90, + "Hrd3p", + "protein" + ], + [ + 93, + 109, + "Derlin-1, -2, -3", + "protein" + ], + [ + 111, + 116, + "Der1p", + "protein" + ], + [ + 119, + 129, + "HERP-1, -2", + "protein" + ], + [ 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substrate ubiquitination via its cytosolic RING finger domain.", + "section": "INTRO", + "ner": [ + [ + 4, + 8, + "HRD1", + "protein" + ], + [ + 9, + 28, + "E3 ubiquitin ligase", + "protein_type" + ], + [ + 194, + 212, + "RING finger domain", + "structure_element" + ] + ] + }, + { + "sid": 14, + "sent": "SEL1L, the mammalian homolog of Hrd3p, associates with HRD1, mediates HRD1 interactions with the ER luminal lectin OS9, and recognizes substrates to be degraded.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "SEL1L", + "protein" + ], + [ + 11, + 20, + "mammalian", + "taxonomy_domain" + ], + [ + 32, + 37, + "Hrd3p", + "protein" + ], + [ + 55, + 59, + "HRD1", + "protein" + ], + [ + 70, + 74, + "HRD1", + "protein" + ], + [ + 108, + 114, + "lectin", + "protein_type" + ], + [ + 115, + 118, + "OS9", + "protein" + ] + ] + }, + { + "sid": 15, + "sent": "In particular, SEL1L is crucial for translocation of Class I major histocompatibility complex (MHC) heavy chains (HCs).", + "section": "INTRO", + "ner": [ + [ + 15, + 20, + "SEL1L", + "protein" + ], + [ + 53, + 93, + "Class I major histocompatibility complex", + "complex_assembly" + ], + [ + 95, + 98, + "MHC", + "complex_assembly" + ], + [ + 100, + 112, + "heavy chains", + "protein_type" + ], + [ + 114, + 117, + "HCs", + "protein_type" + ] + ] + }, + { + "sid": 16, + "sent": "Recent research based on the inducible Sel1l knockout mouse model highlights the physiological functions of SEL1L.", + "section": "INTRO", + "ner": [ + [ + 39, + 44, + "Sel1l", + "gene" + ], + [ + 45, + 59, + "knockout mouse", + "experimental_method" + ], + [ + 108, + 113, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 17, + "sent": "SEL1L is required for ER homeostasis, which is essential for protein translation, pancreatic function, and cellular and organismal survival.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 18, + "sent": "However, despite the functional importance of SEL1L, the molecular structure of SEL1L has not been solved.", + "section": "INTRO", + "ner": [ + [ + 46, + 51, + "SEL1L", + "protein" + ], + [ + 67, + 76, + "structure", + "evidence" + ], + [ + 80, + 85, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 19, + "sent": "Previous biochemical studies reveal that SEL1L is a type I transmembrane protein and has a large luminal domain comprising sets of repeated Sel1-like (SLR) motifs.", + "section": "INTRO", + "ner": [ + [ + 9, + 28, + "biochemical studies", + "experimental_method" + ], + [ + 41, + 46, + "SEL1L", + "protein" + ], + [ + 52, + 80, + "type I transmembrane protein", + "protein_type" + ], + [ + 97, + 111, + "luminal domain", + "structure_element" + ], + [ + 131, + 149, + "repeated Sel1-like", + "structure_element" + ], + [ + 151, + 154, + "SLR", + "structure_element" + ] + ] + }, + { + "sid": 20, + "sent": "The SLR motif is a structural motif that closely resembles the tetratricopeptide-repeat (TPR) motif, which is a protein-protein interaction module.", + "section": "INTRO", + "ner": [ + [ + 4, + 7, + "SLR", + "structure_element" + ], + [ + 63, + 87, + "tetratricopeptide-repeat", + "structure_element" + ], + [ + 89, + 92, + "TPR", + "structure_element" + ] + ] + }, + { + "sid": 21, + "sent": "Although there is evidence that the luminal domain of SEL1L is involved in substrate recognition or in forming complexes with chaperones, it is not known how the unique structure of the repeated SLR motifs contributes to the molecular function of the HRD1-SEL1L E3 ligase complex and affects ERAD at the molecular level.", + "section": "INTRO", + "ner": [ + [ + 36, + 50, + "luminal domain", + "structure_element" + ], + [ + 54, + 59, + "SEL1L", + "protein" + ], + [ + 126, + 136, + "chaperones", + "protein_type" + ], + [ + 195, + 198, + "SLR", + "structure_element" + ], + [ + 251, + 261, + "HRD1-SEL1L", + "complex_assembly" + ], + [ + 262, + 271, + "E3 ligase", + "protein_type" + ] + ] + }, + { + "sid": 22, + "sent": "Furthermore, while repeated SLR motifs are commonly found in tandem arrays, the SLR motifs in SEL1L are, according to the primary structure prediction of SEL1L, interspersed among other sequences in the luminal domain and form three SLR domain clusters.", + "section": "INTRO", + "ner": [ + [ + 28, + 31, + "SLR", + "structure_element" + ], + [ + 80, + 83, + "SLR", + "structure_element" + ], + [ + 94, + 99, + "SEL1L", + "protein" + ], + [ + 154, + 159, + "SEL1L", + "protein" + ], + [ + 203, + 217, + "luminal domain", + "structure_element" + ], + [ + 233, + 236, + "SLR", + "structure_element" + ] + ] + }, + { + "sid": 23, + "sent": "Therefore, the way in which these unique structural features of SEL1L are related to its critical function in ERAD remains to be elucidated.", + "section": "INTRO", + "ner": [ + [ + 64, + 69, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 24, + "sent": "To clearly understand the biochemical role of the SLR domains of SEL1L in ERAD, we determined the crystal structure of the central SLR domain of SEL1L.", + "section": "INTRO", + "ner": [ + [ + 50, + 53, + "SLR", + "structure_element" + ], + [ + 65, + 70, + "SEL1L", + "protein" + ], + [ + 98, + 115, + "crystal structure", + "evidence" + ], + [ + 131, + 134, + "SLR", + "structure_element" + ], + [ + 145, + 150, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 25, + "sent": "We found that the central domain of SEL1L, comprising SLR motifs 5 through 9 (SEL1Lcent), forms a tight dimer with two-fold symmetry due to domain swapping of the SLR motif 9.", + "section": "INTRO", + "ner": [ + [ + 18, + 32, + "central domain", + "structure_element" + ], + [ + 36, + 41, + "SEL1L", + "protein" + ], + [ + 54, + 76, + "SLR motifs 5 through 9", + "structure_element" + ], + [ + 78, + 87, + "SEL1Lcent", + "structure_element" + ], + [ + 104, + 109, + "dimer", + "oligomeric_state" + ], + [ + 163, + 174, + "SLR motif 9", + "structure_element" + ] + ] + }, + { + "sid": 26, + "sent": "We also found that SLR-C, consisting of SLR motifs 10 and 11, directly interacts with the N-terminus luminal loop of HRD1.", + "section": "INTRO", + "ner": [ + [ + 19, + 24, + "SLR-C", + "structure_element" + ], + [ + 40, + 60, + "SLR motifs 10 and 11", + "structure_element" + ], + [ + 101, + 113, + "luminal loop", + "structure_element" + ], + [ + 117, + 121, + "HRD1", + "protein" + ] + ] + }, + { + "sid": 27, + "sent": "Based on these observations, we propose a model wherein the SLR domains of SEL1L contribute to the formation of stable oligomers of the ERAD translocation machinery, which is indispensable for ERAD.", + "section": "INTRO", + "ner": [ + [ + 60, + 63, + "SLR", + "structure_element" + ], + [ + 75, + 80, + "SEL1L", + "protein" + ], + [ + 112, + 118, + "stable", + "protein_state" + ], + [ + 119, + 128, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 28, + "sent": "Structure Determination of SEL1Lcent", + "section": "RESULTS", + "ner": [ + [ + 0, + 23, + "Structure Determination", + "experimental_method" + ], + [ + 27, + 36, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 29, + "sent": "The Mus musculus SEL1L protein contains 790 amino acids and has 17% sequence identity to its yeast homolog, Hrd3p.", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "Mus musculus", + "species" + ], + [ + 17, + 22, + "SEL1L", + "protein" + ], + [ + 93, + 98, + "yeast", + "taxonomy_domain" + ], + [ + 108, + 113, + "Hrd3p", + "protein" + ] + ] + }, + { + "sid": 30, + "sent": "Mouse SEL1L contains a fibronectin type II domain at the N-terminus, followed by 11 SLR motifs and a single transmembrane domain at the C-terminus (Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "Mouse", + "taxonomy_domain" + ], + [ + 6, + 11, + "SEL1L", + "protein" + ], + [ + 23, + 49, + "fibronectin type II domain", + "structure_element" + ], + [ + 84, + 87, + "SLR", + "structure_element" + ], + [ + 108, + 128, + "transmembrane domain", + "structure_element" + ] + ] + }, + { + "sid": 31, + "sent": "The 11 SLR motifs are located in the ER lumen and account for more than two thirds of the mass of full-length SEL1L.", + "section": "RESULTS", + "ner": [ + [ + 7, + 10, + "SLR", + "structure_element" + ], + [ + 98, + 109, + "full-length", + "protein_state" + ], + [ + 110, + 115, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 32, + "sent": "The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "SLR", + "structure_element" + ], + [ + 72, + 88, + "linker sequences", + "structure_element" + ], + [ + 109, + 112, + "SLR", + "structure_element" + ], + [ + 121, + 126, + "SLR-N", + "structure_element" + ], + [ + 128, + 145, + "SLR motifs 1 to 4", + "structure_element" + ], + [ + 148, + 153, + "SLR-M", + "structure_element" + ], + [ + 155, + 172, + "SLR motifs 5 to 9", + "structure_element" + ], + [ + 179, + 184, + "SLR-C", + "structure_element" + ], + [ + 186, + 205, + "SLR motifs 10 to 11", + "structure_element" + ] + ] + }, + { + "sid": 33, + "sent": "Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336\u2013345) between SLR-N and SLR-M and a long linker sequence (residues 528\u2013635) between SLR-M and SLR-C (Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 0, + 18, + "Sequence alignment", + "experimental_method" + ], + [ + 26, + 29, + "SLR", + "structure_element" + ], + [ + 68, + 83, + "linker sequence", + "structure_element" + ], + [ + 94, + 101, + "336\u2013345", + "residue_range" + ], + [ + 111, + 116, + "SLR-N", + "structure_element" + ], + [ + 121, + 126, + "SLR-M", + "structure_element" + ], + [ + 138, + 153, + "linker sequence", + "structure_element" + ], + [ + 164, + 171, + "528\u2013635", + "residue_range" + ], + [ + 181, + 186, + "SLR-M", + "structure_element" + ], + [ + 191, + 196, + "SLR-C", + "structure_element" + ] + ] + }, + { + "sid": 34, + "sent": "We first tried to prepare the full-length mouse SEL1L protein, excluding the transmembrane domain at the C-terminus (residues 735\u2013755), by expression in bacteria.", + "section": "RESULTS", + "ner": [ + [ + 30, + 41, + "full-length", + "protein_state" + ], + [ + 42, + 47, + "mouse", + "taxonomy_domain" + ], + [ + 48, + 53, + "SEL1L", + "protein" + ], + [ + 77, + 97, + "transmembrane domain", + "structure_element" + ], + [ + 126, + 133, + "735\u2013755", + "residue_range" + ], + [ + 139, + 161, + "expression in bacteria", + "experimental_method" + ] + ] + }, + { + "sid": 35, + "sent": "However, the full-length SEL1L protein aggregated in solution and produced no soluble protein.", + "section": "RESULTS", + "ner": [ + [ + 13, + 24, + "full-length", + "protein_state" + ], + [ + 25, + 30, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 36, + "sent": "To identify a soluble form of SEL1L, we generated serial truncation constructs of SEL1L based on the predicted SLR motifs and highly conserved regions across several different species.", + "section": "RESULTS", + "ner": [ + [ + 30, + 35, + "SEL1L", + "protein" + ], + [ + 50, + 78, + "serial truncation constructs", + "experimental_method" + ], + [ + 82, + 87, + "SEL1L", + "protein" + ], + [ + 111, + 114, + "SLR", + "structure_element" + ], + [ + 126, + 142, + "highly conserved", + "protein_state" + ] + ] + }, + { + "sid": 37, + "sent": "Both SLR-N (residues 194\u2013343) and SLR-C (residues 639\u2013719) alone could be solubilized with an MBP tag at the N-terminus, but appeared to be polydisperse when analyzed by size-exclusion chromatography.", + "section": "RESULTS", + "ner": [ + [ + 5, + 10, + "SLR-N", + "structure_element" + ], + [ + 21, + 28, + "194\u2013343", + "residue_range" + ], + [ + 34, + 39, + "SLR-C", + "structure_element" + ], + [ + 50, + 57, + "639\u2013719", + "residue_range" + ], + [ + 94, + 119, + "MBP tag at the N-terminus", + "experimental_method" + ], + [ + 170, + 199, + "size-exclusion chromatography", + "experimental_method" + ] + ] + }, + { + "sid": 38, + "sent": "However, the central region of SEL1L, comprising residues 337\u2013554, was soluble and homogenous in size, as determined by size-exclusion chromatography.", + "section": "RESULTS", + "ner": [ + [ + 13, + 27, + "central region", + "structure_element" + ], + [ + 31, + 36, + "SEL1L", + "protein" + ], + [ + 58, + 65, + "337\u2013554", + "residue_range" + ], + [ + 120, + 149, + "size-exclusion chromatography", + "experimental_method" + ] + ] + }, + { + "sid": 39, + "sent": "To define compact domain boundaries for the central region of SEL1L, we digested the protein with trypsin and analyzed the proteolysis products by SDS-PAGE and N-terminal sequencing.", + "section": "RESULTS", + "ner": [ + [ + 44, + 58, + "central region", + "structure_element" + ], + [ + 62, + 67, + "SEL1L", + "protein" + ], + [ + 72, + 105, + "digested the protein with trypsin", + "experimental_method" + ], + [ + 147, + 155, + "SDS-PAGE", + "experimental_method" + ], + [ + 160, + 181, + "N-terminal sequencing", + "experimental_method" + ] + ] + }, + { + "sid": 40, + "sent": "The results of this preliminary biochemical analysis suggested that SEL1L residues 348\u2013533 (SEL1Lcent) would be amenable to structural analysis (Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 68, + 73, + "SEL1L", + "protein" + ], + [ + 83, + 90, + "348\u2013533", + "residue_range" + ], + [ + 92, + 101, + "SEL1Lcent", + "structure_element" + ], + [ + 124, + 143, + "structural analysis", + "experimental_method" + ] + ] + }, + { + "sid": 41, + "sent": "Crystals of SEL1Lcent grew in space group P21 with four copies of SEL1Lcent (a total of 82\u2009kDa) in the asymmetric unit.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Crystals", + "evidence" + ], + [ + 12, + 21, + "SEL1Lcent", + "structure_element" + ], + [ + 66, + 75, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 42, + "sent": "The structure was determined by the single-wavelength anomalous diffraction (SAD) method using selenium as the anomalous scatterer (Table 1 and Methods).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 36, + 75, + "single-wavelength anomalous diffraction", + "experimental_method" + ], + [ + 77, + 80, + "SAD", + "experimental_method" + ], + [ + 95, + 103, + "selenium", + "chemical" + ] + ] + }, + { + "sid": 43, + "sent": "The assignment of residues during model building was aided by the selenium atom positions, and the structure was refined with native data to 2.6\u2009\u00c5 resolution with Rwork/Rfree values of 20.7/27.7%.", + "section": "RESULTS", + "ner": [ + [ + 66, + 74, + "selenium", + "chemical" + ], + [ + 99, + 108, + "structure", + "evidence" + ], + [ + 163, + 174, + "Rwork/Rfree", + "evidence" + ] + ] + }, + { + "sid": 44, + "sent": "Overall Structure of SEL1Lcent", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "Structure", + "evidence" + ], + [ + 21, + 30, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 45, + "sent": "The mouse SEL1Lcent crystallized as a homodimer, and there were two homodimers in the crystal asymmetric unit (Fig. 1B,C, Supplementary Fig. 1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "mouse", + "taxonomy_domain" + ], + [ + 10, + 19, + "SEL1Lcent", + "structure_element" + ], + [ + 20, + 32, + "crystallized", + "experimental_method" + ], + [ + 38, + 47, + "homodimer", + "oligomeric_state" + ], + [ + 68, + 78, + "homodimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 46, + "sent": "The two SEL1Lcent molecules dimerize in a head-to-tail manner through a two-fold symmetry interface resulting in a cosmos-like shaped structure (Fig. 1B).", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "SEL1Lcent", + "structure_element" + ], + [ + 28, + 36, + "dimerize", + "oligomeric_state" + ], + [ + 42, + 54, + "head-to-tail", + "protein_state" + ], + [ + 72, + 99, + "two-fold symmetry interface", + "site" + ], + [ + 134, + 143, + "structure", + "evidence" + ] + ] + }, + { + "sid": 47, + "sent": "The resulting structure resembles the yin-yang symbol with overall dimensions of 60\u2009\u00d7\u200960\u2009\u00d7\u200925\u2009\u00c5, where a SEL1Lcent monomer corresponds to half the symbol.", + "section": "RESULTS", + "ner": [ + [ + 14, + 23, + "structure", + "evidence" + ], + [ + 105, + 114, + "SEL1Lcent", + "structure_element" + ], + [ + 115, + 122, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 48, + "sent": "The dimer formation buries a surface area of 1670\u2009\u00c52 for each monomer, and no significant differences between the protomers were displayed (final root mean square deviation (RMSD) of 0.6\u2009\u00c5 for all C\u03b1 atoms).", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "dimer", + "oligomeric_state" + ], + [ + 62, + 69, + "monomer", + "oligomeric_state" + ], + [ + 114, + 123, + "protomers", + "oligomeric_state" + ], + [ + 146, + 172, + "root mean square deviation", + "evidence" + ], + [ + 174, + 178, + "RMSD", + "evidence" + ] + ] + }, + { + "sid": 49, + "sent": "Each protomer is composed of ten \u03b1-helices, which form the five SLRs, resulting in an elongated curved structure, confirming the primary structure prediction (Fig. 1D).", + "section": "RESULTS", + "ner": [ + [ + 5, + 13, + "protomer", + "oligomeric_state" + ], + [ + 33, + 42, + "\u03b1-helices", + "structure_element" + ], + [ + 64, + 68, + "SLRs", + "structure_element" + ] + ] + }, + { + "sid": 50, + "sent": "The \u03b1-helices subdivide the structure into five pairs (A and B) as shown in a number of TPRs and SLRs.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "\u03b1-helices", + "structure_element" + ], + [ + 28, + 37, + "structure", + "evidence" + ], + [ + 55, + 56, + "A", + "structure_element" + ], + [ + 61, + 62, + "B", + "structure_element" + ], + [ + 88, + 92, + "TPRs", + "structure_element" + ], + [ + 97, + 101, + "SLRs", + "structure_element" + ] + ] + }, + { + "sid": 51, + "sent": "Helices A and B are 14 and 13 residues long, respectively, and the two helices are connected by a short turn and loop (Fig. 1D).", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Helices A and B", + "structure_element" + ], + [ + 71, + 78, + "helices", + "structure_element" + ], + [ + 104, + 108, + "turn", + "structure_element" + ], + [ + 113, + 117, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 52, + "sent": "In addition, a longer loop, consisting of approximately eight amino acids, is inserted between helix B of one SLR and helix A of the next SLR.", + "section": "RESULTS", + "ner": [ + [ + 22, + 26, + "loop", + "structure_element" + ], + [ + 95, + 102, + "helix B", + "structure_element" + ], + [ + 110, + 113, + "SLR", + "structure_element" + ], + [ + 118, + 125, + "helix A", + "structure_element" + ], + [ + 138, + 141, + "SLR", + "structure_element" + ] + ] + }, + { + "sid": 53, + "sent": "This arrangement is a unique feature for SLRs among the major classes of repeats containing an \u03b1-solenoid.", + "section": "RESULTS", + "ner": [ + [ + 41, + 45, + "SLRs", + "structure_element" + ], + [ + 95, + 105, + "\u03b1-solenoid", + "structure_element" + ] + ] + }, + { + "sid": 54, + "sent": "Starting from its N-terminus, the \u03b1-solenoid of SEL1L extends across a semi-circle in a right-handed superhelix fashion along the rotation axis of the yin-yang circle.", + "section": "RESULTS", + "ner": [ + [ + 34, + 44, + "\u03b1-solenoid", + "structure_element" + ], + [ + 48, + 53, + "SEL1L", + "protein" + ], + [ + 151, + 166, + "yin-yang circle", + "structure_element" + ] + ] + }, + { + "sid": 55, + "sent": "However, the last helix, 9B, at the C-terminus adopts a different conformation, lying parallel to the long axis of helix 9A instead of forming an antiparallel SLR.", + "section": "RESULTS", + "ner": [ + [ + 25, + 27, + "9B", + "structure_element" + ], + [ + 115, + 123, + "helix 9A", + "structure_element" + ], + [ + 159, + 162, + "SLR", + "structure_element" + ] + ] + }, + { + "sid": 56, + "sent": "This unique conformation of helix 9B most likely contributes to formation of the dimer structure of SEL1Lcent, as detailed below.", + "section": "RESULTS", + "ner": [ + [ + 28, + 36, + "helix 9B", + "structure_element" + ], + [ + 81, + 86, + "dimer", + "oligomeric_state" + ], + [ + 100, + 109, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 57, + "sent": "With the exception of the last SLR, the four \u03b1-helix pairs possess similar conformations, with RMSD values of 0.7\u2009\u00c5 for all C\u03b1 atoms.", + "section": "RESULTS", + "ner": [ + [ + 31, + 34, + "SLR", + "structure_element" + ], + [ + 45, + 52, + "\u03b1-helix", + "structure_element" + ], + [ + 95, + 99, + "RMSD", + "evidence" + ] + ] + }, + { + "sid": 58, + "sent": "Although the sequence similarity for the pairwise alignments varies between 25% and 35%, all the residues present in the SLR motifs are conserved among the five pairs.", + "section": "RESULTS", + "ner": [ + [ + 41, + 60, + "pairwise alignments", + "experimental_method" + ], + [ + 121, + 124, + "SLR", + "structure_element" + ], + [ + 136, + 145, + "conserved", + "protein_state" + ] + ] + }, + { + "sid": 59, + "sent": "The SLR domain of SLR-M ends at residue 524, and C-terminal amino acids 525\u2013533 of the protein are not visible in the electron density map, suggesting that this region is highly flexible.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "SLR", + "structure_element" + ], + [ + 18, + 23, + "SLR-M", + "structure_element" + ], + [ + 40, + 43, + "524", + "residue_number" + ], + [ + 72, + 79, + "525\u2013533", + "residue_range" + ], + [ + 118, + 138, + "electron density map", + "evidence" + ], + [ + 171, + 186, + "highly flexible", + "protein_state" + ] + ] + }, + { + "sid": 60, + "sent": "Since no information regarding dimer formation by SEL1L through its SLR motifs is available, we tested whether the SEL1Lcent dimer shown in our crystal structure is a biological unit.", + "section": "RESULTS", + "ner": [ + [ + 31, + 36, + "dimer", + "oligomeric_state" + ], + [ + 50, + 55, + "SEL1L", + "protein" + ], + [ + 68, + 71, + "SLR", + "structure_element" + ], + [ + 115, + 124, + "SEL1Lcent", + "structure_element" + ], + [ + 125, + 130, + "dimer", + "oligomeric_state" + ], + [ + 144, + 161, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 61, + "sent": "First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337\u2013554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE.", + "section": "RESULTS", + "ner": [ + [ + 10, + 22, + "cross-linked", + "experimental_method" + ], + [ + 23, + 32, + "SEL1Lcent", + "structure_element" + ], + [ + 58, + 63, + "SEL1L", + "protein" + ], + [ + 65, + 74, + "SEL1Llong", + "mutant" + ], + [ + 85, + 92, + "337\u2013554", + "residue_range" + ], + [ + 126, + 140, + "glutaraldehyde", + "chemical" + ], + [ + 142, + 144, + "GA", + "chemical" + ], + [ + 149, + 170, + "dimethyl suberimidate", + "chemical" + ], + [ + 172, + 175, + "DMS", + "chemical" + ], + [ + 206, + 214, + "SDS-PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 62, + "sent": "We detected bands at the mass of a dimer for both SEL1Lcent and SEL1Llong when cross-linked with low concentrations of GA (0.005%) or DMS (0.3\u2009mM) (Supplementary Fig. 2A,B).", + "section": "RESULTS", + "ner": [ + [ + 35, + 40, + "dimer", + "oligomeric_state" + ], + [ + 50, + 59, + "SEL1Lcent", + "structure_element" + ], + [ + 64, + 73, + "SEL1Llong", + "mutant" + ], + [ + 79, + 91, + "cross-linked", + "experimental_method" + ], + [ + 119, + 121, + "GA", + "chemical" + ], + [ + 134, + 137, + "DMS", + "chemical" + ] + ] + }, + { + "sid": 63, + "sent": "Next, we conducted analytical ultracentrifugation of SEL1Lcent.", + "section": "RESULTS", + "ner": [ + [ + 19, + 49, + "analytical ultracentrifugation", + "experimental_method" + ], + [ + 53, + 62, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 64, + "sent": "Consistent with the cross-linking data, analytical ultracentrifugation revealed that the molecular weight of SEL1Lcent corresponds to a dimer (Supplementary Fig. 2C).", + "section": "RESULTS", + "ner": [ + [ + 20, + 33, + "cross-linking", + "experimental_method" + ], + [ + 40, + 70, + "analytical ultracentrifugation", + "experimental_method" + ], + [ + 89, + 105, + "molecular weight", + "evidence" + ], + [ + 109, + 118, + "SEL1Lcent", + "structure_element" + ], + [ + 136, + 141, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 65, + "sent": "Taken together, these data indicate that some kind of dimer is formed in solution.", + "section": "RESULTS", + "ner": [ + [ + 54, + 59, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 66, + "sent": "Dimer Interface of SEL1Lcent", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Dimer Interface", + "site" + ], + [ + 19, + 28, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 67, + "sent": "In contrast to a previously described SLR motif containing proteins that exist as monomers in solution, SEL1Lcent forms an intimate two-fold homotypic dimer interface (Figs 1B and 2A).", + "section": "RESULTS", + "ner": [ + [ + 38, + 67, + "SLR motif containing proteins", + "protein_type" + ], + [ + 82, + 90, + "monomers", + "oligomeric_state" + ], + [ + 104, + 113, + "SEL1Lcent", + "structure_element" + ], + [ + 132, + 166, + "two-fold homotypic dimer interface", + "site" + ] + ] + }, + { + "sid": 68, + "sent": "The concave surface of each SEL1L domain comprising helix 5A to 9A encircles its dimer counterpart in an interlocking clasp-like arrangement.", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "concave surface", + "site" + ], + [ + 28, + 33, + "SEL1L", + "protein" + ], + [ + 52, + 66, + "helix 5A to 9A", + "structure_element" + ], + [ + 81, + 86, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 69, + "sent": "However, no interactions were seen between the two-fold-related protomers through the concave inner surfaces themselves.", + "section": "RESULTS", + "ner": [ + [ + 64, + 73, + "protomers", + "oligomeric_state" + ], + [ + 86, + 108, + "concave inner surfaces", + "site" + ] + ] + }, + { + "sid": 70, + "sent": "Rather, the unique structure of SLR motif 9, consisting of two parallel helices (9A and 9B), is located in the space generated by the concave surface and provides an extensive dimerization interface between the two-fold-related molecules (Fig. 2A).", + "section": "RESULTS", + "ner": [ + [ + 32, + 43, + "SLR motif 9", + "structure_element" + ], + [ + 81, + 83, + "9A", + "structure_element" + ], + [ + 88, + 90, + "9B", + "structure_element" + ], + [ + 134, + 149, + "concave surface", + "site" + ], + [ + 176, + 198, + "dimerization interface", + "site" + ] + ] + }, + { + "sid": 71, + "sent": "Helix 9B from one protomer inserts into the empty space surrounded by the concave region in the other monomer, forming a domain-swapped conformation.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Helix 9B", + "structure_element" + ], + [ + 18, + 26, + "protomer", + "oligomeric_state" + ], + [ + 74, + 88, + "concave region", + "site" + ], + [ + 102, + 109, + "monomer", + "oligomeric_state" + ], + [ + 121, + 135, + "domain-swapped", + "protein_state" + ] + ] + }, + { + "sid": 72, + "sent": "Three major contact interfaces are involved in the interactions, and all interfaces are symmetrically related between the dimer subunits (Fig. 2A).", + "section": "RESULTS", + "ner": [ + [ + 12, + 30, + "contact interfaces", + "site" + ], + [ + 73, + 83, + "interfaces", + "site" + ], + [ + 122, + 127, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 73, + "sent": "Structure-based sequence alignment of 135 SEL1L phylogenetic sequences using a ConSurf server revealed that the surface residues in the dimer interfaces were highly conserved among the SEL1L orthologs (Fig. 1E).", + "section": "RESULTS", + "ner": [ + [ + 0, + 34, + "Structure-based sequence alignment", + "experimental_method" + ], + [ + 42, + 47, + "SEL1L", + "protein" + ], + [ + 79, + 93, + "ConSurf server", + "experimental_method" + ], + [ + 136, + 152, + "dimer interfaces", + "site" + ], + [ + 158, + 174, + "highly conserved", + "protein_state" + ], + [ + 185, + 190, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 74, + "sent": "First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR \u03b1-helices (5B, 6B, 7B, and 8B) from its counterpart.", + "section": "RESULTS", + "ner": [ + [ + 7, + 15, + "helix 9B", + "structure_element" + ], + [ + 24, + 33, + "SEL1Lcent", + "structure_element" + ], + [ + 77, + 89, + "inner groove", + "site" + ], + [ + 99, + 102, + "SLR", + "structure_element" + ], + [ + 103, + 112, + "\u03b1-helices", + "structure_element" + ], + [ + 114, + 116, + "5B", + "structure_element" + ], + [ + 118, + 120, + "6B", + "structure_element" + ], + [ + 122, + 124, + "7B", + "structure_element" + ], + [ + 130, + 132, + "8B", + "structure_element" + ] + ] + }, + { + "sid": 75, + "sent": "In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail).", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "interface", + "site" + ], + [ + 19, + 26, + "Leu 516", + "residue_name_number" + ], + [ + 31, + 38, + "Tyr 519", + "residue_name_number" + ], + [ + 42, + 50, + "helix 9B", + "structure_element" + ], + [ + 85, + 109, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 115, + 122, + "Trp 478", + "residue_name_number" + ], + [ + 126, + 134, + "helix 8B", + "structure_element" + ], + [ + 136, + 143, + "Val 444", + "residue_name_number" + ], + [ + 147, + 155, + "helix 7B", + "structure_element" + ], + [ + 157, + 164, + "Phe 411", + "residue_name_number" + ], + [ + 168, + 176, + "helix 6B", + "structure_element" + ], + [ + 182, + 189, + "Leu 380", + "residue_name_number" + ], + [ + 193, + 201, + "helix 5B", + "structure_element" + ], + [ + 211, + 220, + "SEL1Lcent", + "structure_element" + ], + [ + 243, + 254, + "Interface 1", + "site" + ] + ] + }, + { + "sid": 76, + "sent": "In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer.", + "section": "RESULTS", + "ner": [ + [ + 15, + 39, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 74, + 81, + "Tyr 519", + "residue_name_number" + ], + [ + 111, + 118, + "Ile 515", + "residue_name_number" + ], + [ + 124, + 131, + "H-bonds", + "bond_interaction" + ], + [ + 157, + 166, + "conserved", + "protein_state" + ], + [ + 167, + 174, + "Gln 377", + "residue_name_number" + ], + [ + 179, + 186, + "His 381", + "residue_name_number" + ], + [ + 190, + 198, + "helix 5B", + "structure_element" + ], + [ + 223, + 231, + "protomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 77, + "sent": "The side chain of Gln 523 forms an H-bond to the side chain of Asp 480 on the two-fold-related protomer (Fig. 2A, Interface 1 detail).", + "section": "RESULTS", + "ner": [ + [ + 18, + 25, + "Gln 523", + "residue_name_number" + ], + [ + 35, + 41, + "H-bond", + "bond_interaction" + ], + [ + 63, + 70, + "Asp 480", + "residue_name_number" + ], + [ + 95, + 103, + "protomer", + "oligomeric_state" + ], + [ + 114, + 125, + "Interface 1", + "site" + ] + ] + }, + { + "sid": 78, + "sent": "Second, the residues from helix 9A interact with the residues from helix 5A of its counterpart in a head-to-tail orientation.", + "section": "RESULTS", + "ner": [ + [ + 26, + 34, + "helix 9A", + "structure_element" + ], + [ + 67, + 75, + "helix 5A", + "structure_element" + ], + [ + 100, + 112, + "head-to-tail", + "protein_state" + ] + ] + }, + { + "sid": 79, + "sent": "In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363.", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "interface", + "site" + ], + [ + 47, + 55, + "helix 9A", + "structure_element" + ], + [ + 67, + 74, + "Leu 503", + "residue_name_number" + ], + [ + 76, + 83, + "Tyr 499", + "residue_name_number" + ], + [ + 117, + 124, + "Lys 500", + "residue_name_number" + ], + [ + 155, + 177, + "van der Waals contacts", + "bond_interaction" + ], + [ + 227, + 235, + "helix 5A", + "structure_element" + ], + [ + 247, + 254, + "Tyr 360", + "residue_name_number" + ], + [ + 256, + 263, + "Leu 356", + "residue_name_number" + ], + [ + 265, + 272, + "Tyr 359", + "residue_name_number" + ], + [ + 278, + 285, + "Leu 363", + "residue_name_number" + ] + ] + }, + { + "sid": 80, + "sent": "In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail).", + "section": "RESULTS", + "ner": [ + [ + 15, + 39, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 60, + 67, + "Asn 507", + "residue_name_number" + ], + [ + 72, + 79, + "Ser 510", + "residue_name_number" + ], + [ + 83, + 91, + "helix 9A", + "structure_element" + ], + [ + 97, + 104, + "H-bonds", + "bond_interaction" + ], + [ + 110, + 126, + "highly conserved", + "protein_state" + ], + [ + 127, + 134, + "Arg 384", + "residue_name_number" + ], + [ + 142, + 146, + "loop", + "structure_element" + ], + [ + 155, + 163, + "helix 5B", + "structure_element" + ], + [ + 168, + 170, + "6A", + "structure_element" + ], + [ + 197, + 205, + "protomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 81, + "sent": "Third, the helix 9B from each protomer is involved in the dimer interaction by forming a two-fold antiparallel symmetry.", + "section": "RESULTS", + "ner": [ + [ + 11, + 19, + "helix 9B", + "structure_element" + ], + [ + 30, + 38, + "protomer", + "oligomeric_state" + ], + [ + 58, + 63, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 82, + "sent": "In particular, the side chains of hydrophobic residues, including Phe 518, Leu 521, and Met 524, are directed toward each other, where they make both inter- and intramolecular contacts (Fig. 2A, Interface 3 detail).", + "section": "RESULTS", + "ner": [ + [ + 66, + 73, + "Phe 518", + "residue_name_number" + ], + [ + 75, + 82, + "Leu 521", + "residue_name_number" + ], + [ + 88, + 95, + "Met 524", + "residue_name_number" + ], + [ + 195, + 206, + "Interface 3", + "site" + ] + ] + }, + { + "sid": 83, + "sent": "To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L348\u2013497) lacking SLR motif 9 (helix 9A and 9B) from SEL1Lcent for comparative analysis.", + "section": "RESULTS", + "ner": [ + [ + 56, + 73, + "crystal structure", + "evidence" + ], + [ + 101, + 116, + "deletion mutant", + "protein_state" + ], + [ + 118, + 130, + "SEL1L348\u2013497", + "mutant" + ], + [ + 132, + 139, + "lacking", + "protein_state" + ], + [ + 140, + 151, + "SLR motif 9", + "structure_element" + ], + [ + 153, + 161, + "helix 9A", + "structure_element" + ], + [ + 166, + 168, + "9B", + "structure_element" + ], + [ + 175, + 184, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 84, + "sent": "The deletion mutant and the wild-type SEL1Lcent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1Lcent (Supplementary Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "deletion mutant", + "protein_state" + ], + [ + 28, + 37, + "wild-type", + "protein_state" + ], + [ + 38, + 47, + "SEL1Lcent", + "structure_element" + ], + [ + 72, + 79, + "spectra", + "evidence" + ], + [ + 83, + 98, + "CD spectroscopy", + "experimental_method" + ], + [ + 120, + 128, + "deletion", + "experimental_method" + ], + [ + 136, + 147, + "SLR motif 9", + "structure_element" + ], + [ + 190, + 199, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 85, + "sent": "However, the mutant behaved as a monomer in size-exclusion chromatography and analytical ultracentrifugation experiments (Fig. 2B, Supplementary Fig. 2C).", + "section": "RESULTS", + "ner": [ + [ + 13, + 19, + "mutant", + "protein_state" + ], + [ + 33, + 40, + "monomer", + "oligomeric_state" + ], + [ + 44, + 73, + "size-exclusion chromatography", + "experimental_method" + ], + [ + 78, + 108, + "analytical ultracentrifugation", + "experimental_method" + ] + ] + }, + { + "sid": 86, + "sent": "Additionally, to further validate the key residues involved in dimer formation, we generated a triple point mutant (Interface 1, I515A, L516A, and Y519A) of the hydrophobic residues that are involved in dimerization.", + "section": "RESULTS", + "ner": [ + [ + 63, + 68, + "dimer", + "oligomeric_state" + ], + [ + 95, + 114, + "triple point mutant", + "protein_state" + ], + [ + 116, + 127, + "Interface 1", + "site" + ], + [ + 129, + 134, + "I515A", + "mutant" + ], + [ + 136, + 141, + "L516A", + "mutant" + ], + [ + 147, + 152, + "Y519A", + "mutant" + ], + [ + 203, + 215, + "dimerization", + "oligomeric_state" + ] + ] + }, + { + "sid": 87, + "sent": "The triple point mutant eluted at the monomer position upon size-exclusion chromatography, while the negative control point mutant (Q460A) eluted at the same position as the wild-type.", + "section": "RESULTS", + "ner": [ + [ + 4, + 23, + "triple point mutant", + "protein_state" + ], + [ + 38, + 45, + "monomer", + "oligomeric_state" + ], + [ + 60, + 89, + "size-exclusion chromatography", + "experimental_method" + ], + [ + 118, + 130, + "point mutant", + "protein_state" + ], + [ + 132, + 137, + "Q460A", + "mutant" + ], + [ + 174, + 183, + "wild-type", + "protein_state" + ] + ] + }, + { + "sid": 88, + "sent": "Notably, a single-residue mutation (L521A in interface 3) abolished the dimerization of SEL1Lcent (Fig. 2B).", + "section": "RESULTS", + "ner": [ + [ + 11, + 34, + "single-residue mutation", + "experimental_method" + ], + [ + 36, + 41, + "L521A", + "mutant" + ], + [ + 45, + 56, + "interface 3", + "site" + ], + [ + 58, + 84, + "abolished the dimerization", + "protein_state" + ], + [ + 88, + 97, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 89, + "sent": "Leu 521 is located in the dimerization center of the antiparallel 9B helices in the SEL1Lcent dimer.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Leu 521", + "residue_name_number" + ], + [ + 26, + 45, + "dimerization center", + "site" + ], + [ + 66, + 76, + "9B helices", + "structure_element" + ], + [ + 84, + 93, + "SEL1Lcent", + "structure_element" + ], + [ + 94, + 99, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 90, + "sent": "Taken together, these structural and biochemical data demonstrate that SEL1Lcent exists as a dimer in solution and that SLR motif 9 in SEL1Lcent plays an important role in generating a two-fold dimerization interface.", + "section": "RESULTS", + "ner": [ + [ + 22, + 53, + "structural and biochemical data", + "evidence" + ], + [ + 71, + 80, + "SEL1Lcent", + "structure_element" + ], + [ + 93, + 98, + "dimer", + "oligomeric_state" + ], + [ + 120, + 131, + "SLR motif 9", + "structure_element" + ], + [ + 135, + 144, + "SEL1Lcent", + "structure_element" + ], + [ + 194, + 216, + "dimerization interface", + "site" + ] + ] + }, + { + "sid": 91, + "sent": "The Two Glycine Residues (G512 and G513) Create a Hinge for Domain Swapping of SLR Motif 9", + "section": "RESULTS", + "ner": [ + [ + 8, + 15, + "Glycine", + "residue_name" + ], + [ + 26, + 30, + "G512", + "residue_name_number" + ], + [ + 35, + 39, + "G513", + "residue_name_number" + ], + [ + 50, + 55, + "Hinge", + "structure_element" + ], + [ + 79, + 90, + "SLR Motif 9", + "structure_element" + ] + ] + }, + { + "sid": 92, + "sent": "SLRs of mouse SEL1L were predicted using the TPRpred server.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "SLRs", + "structure_element" + ], + [ + 8, + 13, + "mouse", + "taxonomy_domain" + ], + [ + 14, + 19, + "SEL1L", + "protein" + ], + [ + 45, + 59, + "TPRpred server", + "experimental_method" + ] + ] + }, + { + "sid": 93, + "sent": "Based on the prediction, full-length SEL1L contains a total of 11 SLR motifs, and our construct corresponds to SLR motifs 5 through 9.", + "section": "RESULTS", + "ner": [ + [ + 25, + 36, + "full-length", + "protein_state" + ], + [ + 37, + 42, + "SEL1L", + "protein" + ], + [ + 66, + 69, + "SLR", + "structure_element" + ], + [ + 111, + 133, + "SLR motifs 5 through 9", + "structure_element" + ] + ] + }, + { + "sid": 94, + "sent": "Although amino acid sequences from helix 9A and 9B correctly aligned with the regular SLR repeats and corresponded to SLR motif 9 (Fig. 3A), the structural arrangement of the two helices deviated from the common structure for the SLR motif.", + "section": "RESULTS", + "ner": [ + [ + 35, + 43, + "helix 9A", + "structure_element" + ], + [ + 48, + 50, + "9B", + "structure_element" + ], + [ + 86, + 97, + "SLR repeats", + "structure_element" + ], + [ + 118, + 129, + "SLR motif 9", + "structure_element" + ], + [ + 179, + 186, + "helices", + "structure_element" + ], + [ + 230, + 233, + "SLR", + "structure_element" + ] + ] + }, + { + "sid": 95, + "sent": "According to our crystal structure, the central axis of helix 9B is almost parallel to that of helix 9A (Fig. 3B).", + "section": "RESULTS", + "ner": [ + [ + 17, + 34, + "crystal structure", + "evidence" + ], + [ + 56, + 64, + "helix 9B", + "structure_element" + ], + [ + 95, + 103, + "helix 9A", + "structure_element" + ] + ] + }, + { + "sid": 96, + "sent": "However, this unusual conformation of SLR motif 9 seems to be essential for dimer formation, as described earlier.", + "section": "RESULTS", + "ner": [ + [ + 38, + 49, + "SLR motif 9", + "structure_element" + ], + [ + 76, + 81, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 97, + "sent": "For this structural geometry, two adjacent residues, Gly 512 and Gly 513, in SEL1L confer flexibility at this position by adopting main-chain dihedral angles that are disallowed for non-glycine residues.", + "section": "RESULTS", + "ner": [ + [ + 53, + 60, + "Gly 512", + "residue_name_number" + ], + [ + 65, + 72, + "Gly 513", + "residue_name_number" + ], + [ + 77, + 82, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 98, + "sent": "The phi and psi dihedrals are 100\u00b0 and 20\u00b0 for Gly 512, and 110\u00b0 and \u221220\u00b0 for Gly 513, respectively (Fig. 3C).", + "section": "RESULTS", + "ner": [ + [ + 47, + 54, + "Gly 512", + "residue_name_number" + ], + [ + 78, + 85, + "Gly 513", + "residue_name_number" + ] + ] + }, + { + "sid": 99, + "sent": "Gly 513 is conserved among other SLR motifs in the SEL1Lcent, but Gly 512 is present only in the SLR motif 9 of SEL1Lcent (Fig. 3A).", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Gly 513", + "residue_name_number" + ], + [ + 11, + 20, + "conserved", + "protein_state" + ], + [ + 33, + 36, + "SLR", + "structure_element" + ], + [ + 51, + 60, + "SEL1Lcent", + "structure_element" + ], + [ + 66, + 73, + "Gly 512", + "residue_name_number" + ], + [ + 97, + 108, + "SLR motif 9", + "structure_element" + ], + [ + 112, + 121, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 100, + "sent": "Thus, the Gly-Gly residues generate an unusual sharp bend at the C-terminal SLR motif 9.", + "section": "RESULTS", + "ner": [ + [ + 10, + 17, + "Gly-Gly", + "structure_element" + ], + [ + 76, + 87, + "SLR motif 9", + "structure_element" + ] + ] + }, + { + "sid": 101, + "sent": "The involvement of a glycine residue in forming a hinge for domain swapping has been reported previously.", + "section": "RESULTS", + "ner": [ + [ + 21, + 28, + "glycine", + "residue_name" + ], + [ + 50, + 55, + "hinge", + "structure_element" + ] + ] + }, + { + "sid": 102, + "sent": "The significance of Gly 513 is further highlighted by its absolute conservation among different species, including the budding yeast homolog Hrd3p.", + "section": "RESULTS", + "ner": [ + [ + 20, + 27, + "Gly 513", + "residue_name_number" + ], + [ + 58, + 79, + "absolute conservation", + "protein_state" + ], + [ + 119, + 132, + "budding yeast", + "taxonomy_domain" + ], + [ + 141, + 146, + "Hrd3p", + "protein" + ] + ] + }, + { + "sid": 103, + "sent": "To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C).", + "section": "RESULTS", + "ner": [ + [ + 41, + 48, + "Gly 512", + "residue_name_number" + ], + [ + 53, + 60, + "Gly 513", + "residue_name_number" + ], + [ + 76, + 79, + "SLR", + "structure_element" + ], + [ + 111, + 125, + "point mutation", + "experimental_method" + ], + [ + 127, + 137, + "Gly to Ala", + "mutant" + ], + [ + 186, + 193, + "Gly 512", + "residue_name_number" + ], + [ + 198, + 205, + "Gly 513", + "residue_name_number" + ], + [ + 241, + 249, + "helix 9B", + "structure_element" + ], + [ + 267, + 275, + "protomer", + "oligomeric_state" + ], + [ + 321, + 328, + "alanine", + "residue_name" + ], + [ + 389, + 397, + "mutation", + "experimental_method" + ] + ] + }, + { + "sid": 104, + "sent": "This means that the effect of the mutation is mainly to generate a more restricted geometry at the hinge region.", + "section": "RESULTS", + "ner": [ + [ + 34, + 42, + "mutation", + "experimental_method" + ], + [ + 99, + 104, + "hinge", + "structure_element" + ] + ] + }, + { + "sid": 105, + "sent": "G512A or G513A alone showed no differences from wild-type in terms of the size-exclusion chromatography elution profile (Fig. 3D), suggesting that the restriction for single glycine flexibility would not be enough to break the swapped structure of helix 9B.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "G512A", + "mutant" + ], + [ + 9, + 14, + "G513A", + "mutant" + ], + [ + 48, + 57, + "wild-type", + "protein_state" + ], + [ + 74, + 103, + "size-exclusion chromatography", + "experimental_method" + ], + [ + 174, + 181, + "glycine", + "residue_name" + ], + [ + 248, + 256, + "helix 9B", + "structure_element" + ] + ] + }, + { + "sid": 106, + "sent": "However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 13, + 26, + "double mutant", + "protein_state" + ], + [ + 28, + 33, + "G512A", + "mutant" + ], + [ + 34, + 39, + "G513A", + "mutant" + ], + [ + 93, + 102, + "wild-type", + "protein_state" + ], + [ + 120, + 128, + "mutation", + "experimental_method" + ], + [ + 161, + 166, + "hinge", + "structure_element" + ], + [ + 175, + 183, + "helix 9A", + "structure_element" + ], + [ + 188, + 190, + "9B", + "structure_element" + ], + [ + 230, + 238, + "helix 9B", + "structure_element" + ], + [ + 325, + 334, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 107, + "sent": "When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely.", + "section": "RESULTS", + "ner": [ + [ + 23, + 33, + "mutated to", + "experimental_method" + ], + [ + 34, + 40, + "lysine", + "residue_name" + ], + [ + 42, + 47, + "G512K", + "mutant" + ], + [ + 48, + 53, + "G513K", + "mutant" + ], + [ + 60, + 66, + "mutant", + "protein_state" + ], + [ + 119, + 124, + "hinge", + "structure_element" + ], + [ + 197, + 205, + "protomer", + "oligomeric_state" + ], + [ + 209, + 218, + "SEL1Lcent", + "structure_element" + ], + [ + 259, + 268, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 108, + "sent": "The G512K/G513K double mutant eluted at the monomer position in size-exclusion chromatography (Fig. 3D).", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "G512K", + "mutant" + ], + [ + 10, + 15, + "G513K", + "mutant" + ], + [ + 16, + 29, + "double mutant", + "protein_state" + ], + [ + 44, + 51, + "monomer", + "oligomeric_state" + ], + [ + 64, + 93, + "size-exclusion chromatography", + "experimental_method" + ] + ] + }, + { + "sid": 109, + "sent": "A previous study shows that induction of steric hindrance by mutation destabilizes the dimerization interface of a different protein, ClC transporter.", + "section": "RESULTS", + "ner": [ + [ + 61, + 69, + "mutation", + "experimental_method" + ], + [ + 87, + 109, + "dimerization interface", + "site" + ], + [ + 134, + 149, + "ClC transporter", + "protein_type" + ] + ] + }, + { + "sid": 110, + "sent": "Collectively, these data suggest that the Gly 512 and Gly 513 at the connection between helix 9A and 9B play a crucial role in forming the domain-swapped conformation that enables dimer formation.", + "section": "RESULTS", + "ner": [ + [ + 42, + 49, + "Gly 512", + "residue_name_number" + ], + [ + 54, + 61, + "Gly 513", + "residue_name_number" + ], + [ + 88, + 96, + "helix 9A", + "structure_element" + ], + [ + 101, + 103, + "9B", + "structure_element" + ], + [ + 139, + 153, + "domain-swapped", + "protein_state" + ], + [ + 180, + 185, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 111, + "sent": "SEL1L Forms Self-oligomers through SEL1Lcent domain in vivo", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "SEL1L", + "protein" + ], + [ + 12, + 26, + "Self-oligomers", + "oligomeric_state" + ], + [ + 35, + 44, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 112, + "sent": "Next, we examined if SEL1L also forms self-oligomers in vivo using HEK293T cells.", + "section": "RESULTS", + "ner": [ + [ + 21, + 26, + "SEL1L", + "protein" + ], + [ + 38, + 52, + "self-oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 113, + "sent": "We generated full-length SEL1L-HA and SEL1L-FLAG fusion constructs and co-transfected the constructs into HEK293T cells.", + "section": "RESULTS", + "ner": [ + [ + 13, + 24, + "full-length", + "protein_state" + ], + [ + 25, + 30, + "SEL1L", + "protein" + ], + [ + 31, + 33, + "HA", + "experimental_method" + ], + [ + 38, + 43, + "SEL1L", + "protein" + ], + [ + 44, + 48, + "FLAG", + "experimental_method" + ], + [ + 49, + 66, + "fusion constructs", + "experimental_method" + ], + [ + 71, + 85, + "co-transfected", + "experimental_method" + ] + ] + }, + { + "sid": 114, + "sent": "A co-immunoprecipitation assay using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody showed that full-length SEL1L forms self-oligomers in vivo (Fig. 4A).", + "section": "RESULTS", + "ner": [ + [ + 2, + 30, + "co-immunoprecipitation assay", + "experimental_method" + ], + [ + 45, + 49, + "FLAG", + "experimental_method" + ], + [ + 71, + 83, + "Western blot", + "experimental_method" + ], + [ + 107, + 109, + "HA", + "experimental_method" + ], + [ + 131, + 142, + "full-length", + "protein_state" + ], + [ + 143, + 148, + "SEL1L", + "protein" + ], + [ + 155, + 169, + "self-oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 115, + "sent": "To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348\u2013497) construct, which were fused to the C-terminus of SEL1L signal peptides.", + "section": "RESULTS", + "ner": [ + [ + 31, + 40, + "SEL1Lcent", + "structure_element" + ], + [ + 90, + 101, + "full-length", + "protein_state" + ], + [ + 102, + 107, + "SEL1L", + "protein" + ], + [ + 122, + 131, + "SEL1Lcent", + "structure_element" + ], + [ + 136, + 147, + "SLR motif 9", + "structure_element" + ], + [ + 148, + 156, + "deletion", + "experimental_method" + ], + [ + 158, + 170, + "SEL1L348\u2013497", + "mutant" + ], + [ + 194, + 202, + "fused to", + "experimental_method" + ], + [ + 221, + 226, + "SEL1L", + "protein" + ], + [ + 227, + 242, + "signal peptides", + "structure_element" + ] + ] + }, + { + "sid": 116, + "sent": "Co-immunoprecipitation analysis showed that the SEL1Lcent was sufficient to physically interact with the full-length SEL1L, while SEL1L348\u2013497 failed to do so (Fig. 4A).", + "section": "RESULTS", + "ner": [ + [ + 0, + 31, + "Co-immunoprecipitation analysis", + "experimental_method" + ], + [ + 48, + 57, + "SEL1Lcent", + "structure_element" + ], + [ + 105, + 116, + "full-length", + "protein_state" + ], + [ + 117, + 122, + "SEL1L", + "protein" + ], + [ + 130, + 142, + "SEL1L348\u2013497", + "mutant" + ] + ] + }, + { + "sid": 117, + "sent": "Interestingly, however, the expression level of SEL1L348\u2013497 was consistently lower than that of SEL1Lcent (Fig. 4A,B).", + "section": "RESULTS", + "ner": [ + [ + 48, + 60, + "SEL1L348\u2013497", + "mutant" + ], + [ + 97, + 106, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 118, + "sent": "Semi-quantitative RT-PCR revealed no significant difference in transcriptional levels of the two constructs (data not shown).", + "section": "RESULTS", + "ner": [ + [ + 0, + 24, + "Semi-quantitative RT-PCR", + "experimental_method" + ] + ] + }, + { + "sid": 119, + "sent": "We speculated that SEL1L348\u2013497 could be secreted while the SEL1Lcent is retained in the ER by association with the endogenous ERAD complex.", + "section": "RESULTS", + "ner": [ + [ + 19, + 31, + "SEL1L348\u2013497", + "mutant" + ], + [ + 60, + 69, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 120, + "sent": "Indeed, immunoprecipitation followed by western blot analysis using the culture medium detected secreted SEL1L348\u2013497 fragment, but not SEL1Lcent (Fig. 4B).", + "section": "RESULTS", + "ner": [ + [ + 8, + 27, + "immunoprecipitation", + "experimental_method" + ], + [ + 40, + 52, + "western blot", + "experimental_method" + ], + [ + 105, + 117, + "SEL1L348\u2013497", + "mutant" + ], + [ + 136, + 145, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 121, + "sent": "We next examined if the reason why SEL1L348\u2013497 failed to bind to the full-length SEL1L may be because of the lower level of SEL1L348\u2013497 in the ER lumen compared to SEL1Lcent fragment.", + "section": "RESULTS", + "ner": [ + [ + 35, + 47, + "SEL1L348\u2013497", + "mutant" + ], + [ + 70, + 81, + "full-length", + "protein_state" + ], + [ + 82, + 87, + "SEL1L", + "protein" + ], + [ + 125, + 137, + "SEL1L348\u2013497", + "mutant" + ], + [ + 166, + 175, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 122, + "sent": "In order to retain two SEL1L fragments in the ER lumen, we added KDEL ER retention sequence to the C-terminus of both fragments.", + "section": "RESULTS", + "ner": [ + [ + 23, + 28, + "SEL1L", + "protein" + ], + [ + 65, + 69, + "KDEL", + "structure_element" + ], + [ + 70, + 91, + "ER retention sequence", + "structure_element" + ] + ] + }, + { + "sid": 123, + "sent": "Indeed, the addition of KDEL peptide increased the level of SEL1L348\u2013497 in the ER lumen (Fig. 4D,E) and the immunostaining analysis showed both constructs were well localized to the ER (Fig. 4C).", + "section": "RESULTS", + "ner": [ + [ + 24, + 28, + "KDEL", + "structure_element" + ], + [ + 60, + 72, + "SEL1L348\u2013497", + "mutant" + ], + [ + 109, + 123, + "immunostaining", + "experimental_method" + ] + ] + }, + { + "sid": 124, + "sent": "We further analyzed whether SEL1Lcent may competitively inhibit the self-oligomerization of SEL1L in vivo.", + "section": "RESULTS", + "ner": [ + [ + 28, + 37, + "SEL1Lcent", + "structure_element" + ], + [ + 92, + 97, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 125, + "sent": "To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348\u2013497-KDEL or SEL1Lcent (L521A)-KDEL, respectively.", + "section": "RESULTS", + "ner": [ + [ + 16, + 30, + "co-transfected", + "experimental_method" + ], + [ + 50, + 56, + "tagged", + "protein_state" + ], + [ + 57, + 68, + "full-length", + "protein_state" + ], + [ + 69, + 74, + "SEL1L", + "protein" + ], + [ + 76, + 81, + "SEL1L", + "protein" + ], + [ + 82, + 84, + "HA", + "experimental_method" + ], + [ + 89, + 94, + "SEL1L", + "protein" + ], + [ + 95, + 99, + "FLAG", + "experimental_method" + ], + [ + 105, + 121, + "increasing doses", + "experimental_method" + ], + [ + 125, + 139, + "SEL1Lcent-KDEL", + "mutant" + ], + [ + 141, + 158, + "SEL1L348\u2013497-KDEL", + "mutant" + ], + [ + 162, + 184, + "SEL1Lcent (L521A)-KDEL", + "mutant" + ] + ] + }, + { + "sid": 126, + "sent": "Co-immunoprecipitation assay revealed that wild-type SEL1Lcent-KDEL, indeed, competitively disrupted the self-association of the full-length SEL1L (Fig. 4E).", + "section": "RESULTS", + "ner": [ + [ + 0, + 28, + "Co-immunoprecipitation assay", + "experimental_method" + ], + [ + 43, + 52, + "wild-type", + "protein_state" + ], + [ + 53, + 67, + "SEL1Lcent-KDEL", + "mutant" + ], + [ + 129, + 140, + "full-length", + "protein_state" + ] + ] + }, + { + "sid": 127, + "sent": "In contrast, SEL1L348\u2013497-KDEL and the single-residue mutation L521A in SEL1Lcent did not competitively inhibit the self-association of full-length SEL1L (Fig. 4E,F).", + "section": "RESULTS", + "ner": [ + [ + 13, + 30, + "SEL1L348\u2013497-KDEL", + "mutant" + ], + [ + 63, + 68, + "L521A", + "mutant" + ], + [ + 72, + 81, + "SEL1Lcent", + "structure_element" + ], + [ + 136, + 147, + "full-length", + "protein_state" + ], + [ + 148, + 153, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 128, + "sent": "These data suggest that the SEL1L forms self-oligomers and the oligomerization is mediated by the SEL1Lcent domain in vivo.", + "section": "RESULTS", + "ner": [ + [ + 28, + 33, + "SEL1L", + "protein" + ], + [ + 40, + 54, + "self-oligomers", + "oligomeric_state" + ], + [ + 98, + 107, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 129, + "sent": "Structural Comparison of SEL1L SLRs with TPRs or SLRs of Other Proteins", + "section": "RESULTS", + "ner": [ + [ + 0, + 21, + "Structural Comparison", + "experimental_method" + ], + [ + 25, + 30, + "SEL1L", + "protein" + ], + [ + 31, + 35, + "SLRs", + "structure_element" + ], + [ + 41, + 45, + "TPRs", + "structure_element" + ], + [ + 49, + 53, + "SLRs", + "structure_element" + ] + ] + }, + { + "sid": 130, + "sent": "Previous studies reveal that TPRs and SLRs have similar consensus sequences, suggesting that their three-dimensional structures are also similar.", + "section": "RESULTS", + "ner": [ + [ + 29, + 33, + "TPRs", + "structure_element" + ], + [ + 38, + 42, + "SLRs", + "structure_element" + ] + ] + }, + { + "sid": 131, + "sent": "The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1\u2009\u00c5, confirming that the isolated repeats are indeed similar.", + "section": "RESULTS", + "ner": [ + [ + 4, + 17, + "superposition", + "experimental_method" + ], + [ + 30, + 34, + "TPRs", + "structure_element" + ], + [ + 40, + 45, + "Cdc23", + "protein" + ], + [ + 47, + 55, + "S. pombe", + "species" + ], + [ + 57, + 79, + "cell division cycle 23", + "protein" + ], + [ + 108, + 112, + "SLRs", + "structure_element" + ], + [ + 118, + 122, + "HcpC", + "protein" + ], + [ + 124, + 160, + "Helicobacter Cysteine-rich Protein C", + "protein" + ], + [ + 184, + 189, + "RMSDs", + "evidence" + ] + ] + }, + { + "sid": 132, + "sent": "This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6\u2009\u00c5 for all C\u03b1 chains) from Cdc23N-term and SLRs (RMSD 0.6\u2009\u00c5 for all C\u03b1 chains) from HcpC (Fig. 5A).", + "section": "RESULTS", + "ner": [ + [ + 20, + 23, + "SLR", + "structure_element" + ], + [ + 34, + 39, + "SEL1L", + "protein" + ], + [ + 53, + 56, + "SLR", + "structure_element" + ], + [ + 69, + 78, + "SEL1Lcent", + "structure_element" + ], + [ + 91, + 111, + "structural alignment", + "experimental_method" + ], + [ + 126, + 130, + "TPRs", + "structure_element" + ], + [ + 132, + 136, + "RMSD", + "evidence" + ], + [ + 167, + 172, + "Cdc23", + "protein" + ], + [ + 183, + 187, + "SLRs", + "structure_element" + ], + [ + 189, + 193, + "RMSD", + "evidence" + ], + [ + 224, + 228, + "HcpC", + "protein" + ] + ] + }, + { + "sid": 133, + "sent": "However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of >2.5\u2009\u00c5 for C\u03b1 atoms) (Fig. 5B).", + "section": "RESULTS", + "ner": [ + [ + 9, + 22, + "superimposing", + "experimental_method" + ], + [ + 27, + 36, + "structure", + "evidence" + ], + [ + 40, + 57, + "SLR motifs 5 to 9", + "structure_element" + ], + [ + 63, + 72, + "SEL1Lcent", + "structure_element" + ], + [ + 90, + 95, + "Cdc23", + "protein" + ], + [ + 105, + 116, + "full-length", + "protein_state" + ], + [ + 117, + 121, + "HcpC", + "protein" + ], + [ + 122, + 132, + "structures", + "evidence" + ], + [ + 147, + 164, + "SLR motifs 5 to 9", + "structure_element" + ], + [ + 168, + 177, + "SEL1Lcent", + "structure_element" + ], + [ + 230, + 235, + "Cdc23", + "protein" + ], + [ + 239, + 243, + "HcpC", + "protein" + ], + [ + 245, + 249, + "RMSD", + "evidence" + ] + ] + }, + { + "sid": 134, + "sent": "The differences may result from the differing numbers of residues in the loops and differences in antiparallel helix packing.", + "section": "RESULTS", + "ner": [ + [ + 73, + 78, + "loops", + "structure_element" + ], + [ + 98, + 116, + "antiparallel helix", + "structure_element" + ] + ] + }, + { + "sid": 135, + "sent": "Moreover, there are conserved disulfide bonds in the SLR motifs of HcpC and HcpB, but no such bonds are observed in SEL1Lcent.", + "section": "RESULTS", + "ner": [ + [ + 20, + 29, + "conserved", + "protein_state" + ], + [ + 30, + 45, + "disulfide bonds", + "ptm" + ], + [ + 53, + 56, + "SLR", + "structure_element" + ], + [ + 67, + 71, + "HcpC", + "protein" + ], + [ + 76, + 80, + "HcpB", + "protein" + ], + [ + 116, + 125, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 136, + "sent": "These factors contribute to the differences in the overall conformation of the SLR motifs in SEL1L and other SLR or TPR motif-containing proteins.", + "section": "RESULTS", + "ner": [ + [ + 79, + 82, + "SLR", + "structure_element" + ], + [ + 93, + 98, + "SEL1L", + "protein" + ], + [ + 109, + 145, + "SLR or TPR motif-containing proteins", + "protein_type" + ] + ] + }, + { + "sid": 137, + "sent": "Another major difference in the structure of SLR motifs between SEL1L and HcpC is the oligomeric state of proteins.", + "section": "RESULTS", + "ner": [ + [ + 32, + 41, + "structure", + "evidence" + ], + [ + 45, + 48, + "SLR", + "structure_element" + ], + [ + 64, + 69, + "SEL1L", + "protein" + ], + [ + 74, + 78, + "HcpC", + "protein" + ] + ] + }, + { + "sid": 138, + "sent": "The TPR motif is involved in the dimerization of proteins such as Cdc23, Cdc16, and Cdc27.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "TPR", + "structure_element" + ], + [ + 33, + 45, + "dimerization", + "oligomeric_state" + ], + [ + 66, + 71, + "Cdc23", + "protein" + ], + [ + 73, + 78, + "Cdc16", + "protein" + ], + [ + 84, + 89, + "Cdc27", + "protein" + ] + ] + }, + { + "sid": 139, + "sent": "In particular, the N-terminal domain of Cdc23 (Cdc23N-term) has a TPR-motif organization similar to that of the SLR motif in SEL1Lcent.", + "section": "RESULTS", + "ner": [ + [ + 40, + 45, + "Cdc23", + "protein" + ], + [ + 47, + 52, + "Cdc23", + "protein" + ], + [ + 66, + 69, + "TPR", + "structure_element" + ], + [ + 112, + 115, + "SLR", + "structure_element" + ], + [ + 125, + 134, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 140, + "sent": "The seven TPR motifs of Cdc23N-term are assembled into a superhelical structure, generating a hollow surface and encircling its dimer counterpart in an interlocking clasp-like arrangement (Fig. 5C).", + "section": "RESULTS", + "ner": [ + [ + 10, + 13, + "TPR", + "structure_element" + ], + [ + 24, + 29, + "Cdc23", + "protein" + ], + [ + 57, + 79, + "superhelical structure", + "structure_element" + ], + [ + 128, + 133, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 141, + "sent": "The TPR motif 1 (TPR1) of each Cdc23N-term subunit is located in the hollow surface of the counter subunit and interacts with residues lining the inner groove TPR \u03b1-helices, generating two-fold symmetry homotype interactions.", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "TPR motif 1", + "structure_element" + ], + [ + 17, + 21, + "TPR1", + "structure_element" + ], + [ + 31, + 36, + "Cdc23", + "protein" + ], + [ + 146, + 158, + "inner groove", + "site" + ], + [ + 159, + 162, + "TPR", + "structure_element" + ], + [ + 163, + 172, + "\u03b1-helices", + "structure_element" + ] + ] + }, + { + "sid": 142, + "sent": "However, in this structure, a conformational change in the TPR motif itself is not observed.", + "section": "RESULTS", + "ner": [ + [ + 17, + 26, + "structure", + "evidence" + ], + [ + 59, + 62, + "TPR", + "structure_element" + ] + ] + }, + { + "sid": 143, + "sent": "Self-association of HcpC has not been reported, and there is no domain-swapped structure in the SLR motifs of HcpC, in contrast to that observed in SEL1Lcent.", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "HcpC", + "protein" + ], + [ + 64, + 78, + "domain-swapped", + "protein_state" + ], + [ + 96, + 99, + "SLR", + "structure_element" + ], + [ + 110, + 114, + "HcpC", + "protein" + ], + [ + 148, + 157, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 144, + "sent": "Although SEL1L contains a number of SLR motifs comparable to HcpC, the SLR motifs in SEL1L are interrupted by other sequences, making three SLR motif clusters (Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 9, + 14, + "SEL1L", + "protein" + ], + [ + 36, + 39, + "SLR", + "structure_element" + ], + [ + 61, + 65, + "HcpC", + "protein" + ], + [ + 71, + 74, + "SLR", + "structure_element" + ], + [ + 85, + 90, + "SEL1L", + "protein" + ], + [ + 140, + 143, + "SLR", + "structure_element" + ] + ] + }, + { + "sid": 145, + "sent": "The interrupted SLR motifs may be required for dimerization of SEL1Lcent, as five SLR motifs are more than enough to form the semicircle of the yin-yang symbol (Fig. 1B).", + "section": "RESULTS", + "ner": [ + [ + 16, + 19, + "SLR", + "structure_element" + ], + [ + 47, + 59, + "dimerization", + "oligomeric_state" + ], + [ + 63, + 72, + "SEL1Lcent", + "structure_element" + ], + [ + 82, + 85, + "SLR", + "structure_element" + ], + [ + 126, + 152, + "semicircle of the yin-yang", + "structure_element" + ] + ] + }, + { + "sid": 146, + "sent": "Helix 5A from SLR motif 5 meets helix 9A from SLR motif 9 of the counterpart SEL1L.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Helix 5A", + "structure_element" + ], + [ + 14, + 25, + "SLR motif 5", + "structure_element" + ], + [ + 32, + 40, + "helix 9A", + "structure_element" + ], + [ + 46, + 57, + "SLR motif 9", + "structure_element" + ], + [ + 77, + 82, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 147, + "sent": "If the SLR motifs 5 to 9 were not isolated from other SLR motifs, steric hindrance could interfere with dimerization of SEL1L.", + "section": "RESULTS", + "ner": [ + [ + 7, + 24, + "SLR motifs 5 to 9", + "structure_element" + ], + [ + 54, + 57, + "SLR", + "structure_element" + ], + [ + 104, + 116, + "dimerization", + "oligomeric_state" + ], + [ + 120, + 125, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 148, + "sent": "This is one of the biggest differences from TPRs in Cdc23 and from the SLRs in HcpC, where the motifs exist in tandem.", + "section": "RESULTS", + "ner": [ + [ + 44, + 48, + "TPRs", + "structure_element" + ], + [ + 52, + 57, + "Cdc23", + "protein" + ], + [ + 71, + 75, + "SLRs", + "structure_element" + ], + [ + 79, + 83, + "HcpC", + "protein" + ] + ] + }, + { + "sid": 149, + "sent": "TPR and SLR motifs are generally involved in protein-protein interaction modules, and the sequences between the SLR motifs of SEL1L might actually facilitate the self-association of this protein.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "TPR", + "structure_element" + ], + [ + 8, + 11, + "SLR", + "structure_element" + ], + [ + 112, + 115, + "SLR", + "structure_element" + ], + [ + 126, + 131, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 150, + "sent": "SLR-C of SEL1L Binds HRD1 N-terminus Luminal Loop", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "SLR-C", + "structure_element" + ], + [ + 9, + 14, + "SEL1L", + "protein" + ], + [ + 21, + 25, + "HRD1", + "protein" + ], + [ + 37, + 49, + "Luminal Loop", + "structure_element" + ] + ] + }, + { + "sid": 151, + "sent": "Based on the structural data presented herein, a possible arrangement of membrane-associated ERAD components in mammals, highlighting the molecular functions of SLR domains in SEL1L, is shown in Fig. 6C.", + "section": "RESULTS", + "ner": [ + [ + 13, + 28, + "structural data", + "evidence" + ], + [ + 112, + 119, + "mammals", + "taxonomy_domain" + ], + [ + 161, + 164, + "SLR", + "structure_element" + ], + [ + 176, + 181, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 152, + "sent": "We suggest that the middle SLR domains are involved in the dimerization of SEL1L based on the crystal structure and biochemical data.", + "section": "RESULTS", + "ner": [ + [ + 27, + 30, + "SLR", + "structure_element" + ], + [ + 59, + 71, + "dimerization", + "oligomeric_state" + ], + [ + 75, + 80, + "SEL1L", + "protein" + ], + [ + 94, + 111, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 153, + "sent": "SLR-C, which contains SLR motifs 10 to 11, might be involved in the interaction with HRD1.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "SLR-C", + "structure_element" + ], + [ + 22, + 41, + "SLR motifs 10 to 11", + "structure_element" + ], + [ + 85, + 89, + "HRD1", + "protein" + ] + ] + }, + { + "sid": 154, + "sent": "Indirect evidence from a previous yeast study shows that the circumscribed region of C-terminal Hrd3p, specifically residues 664\u2013695, forms contacts with the Hrd1 luminal loops.", + "section": "RESULTS", + "ner": [ + [ + 34, + 39, + "yeast", + "taxonomy_domain" + ], + [ + 96, + 101, + "Hrd3p", + "protein" + ], + [ + 125, + 132, + "664\u2013695", + "residue_range" + ], + [ + 158, + 162, + "Hrd1", + "protein" + ], + [ + 163, + 176, + "luminal loops", + "structure_element" + ] + ] + }, + { + "sid": 155, + "sent": "The Hrd3p residues 664\u2013695 correspond to mouse SEL1L residues 696\u2013727, which include the entire helix 11B (residue 697\u2013709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "Hrd3p", + "protein" + ], + [ + 19, + 26, + "664\u2013695", + "residue_range" + ], + [ + 41, + 46, + "mouse", + "taxonomy_domain" + ], + [ + 47, + 52, + "SEL1L", + "protein" + ], + [ + 62, + 69, + "696\u2013727", + "residue_range" + ], + [ + 96, + 105, + "helix 11B", + "structure_element" + ], + [ + 115, + 122, + "697\u2013709", + "residue_range" + ], + [ + 127, + 139, + "SLR motif 11", + "structure_element" + ], + [ + 146, + 160, + "well-conserved", + "protein_state" + ] + ] + }, + { + "sid": 156, + "sent": "This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5).", + "section": "RESULTS", + "ner": [ + [ + 76, + 94, + "SLR motif 10 to 11", + "structure_element" + ], + [ + 123, + 159, + "structure-guided SLR motif alignment", + "experimental_method" + ], + [ + 182, + 197, + "structure study", + "experimental_method" + ], + [ + 227, + 248, + "sequence conservation", + "protein_state" + ], + [ + 257, + 266, + "mammalian", + "taxonomy_domain" + ], + [ + 267, + 272, + "SEL1L", + "protein" + ], + [ + 277, + 282, + "yeast", + "taxonomy_domain" + ], + [ + 283, + 288, + "Hrd3p", + "protein" + ], + [ + 296, + 315, + "SLR motifs 10 to 11", + "structure_element" + ], + [ + 352, + 356, + "HRD1", + "protein" + ], + [ + 358, + 363, + "Hrd1p", + "protein" + ] + ] + }, + { + "sid": 157, + "sent": "To address this hypothesis, we prepared constructs encoding mouse HRD1 luminal fragments fused to GST as shown in Fig. 6A, and tested their ability to bind certain SLR motifs in SEL1L.", + "section": "RESULTS", + "ner": [ + [ + 60, + 65, + "mouse", + "taxonomy_domain" + ], + [ + 66, + 70, + "HRD1", + "protein" + ], + [ + 89, + 101, + "fused to GST", + "experimental_method" + ], + [ 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+ [ + 106, + 118, + "luminal loop", + "structure_element" + ], + [ + 129, + 134, + "21\u201342", + "residue_range" + ], + [ + 139, + 143, + "HRD1", + "protein" + ] + ] + }, + { + "sid": 160, + "sent": "The molecular functions of SLR-N are unclear.", + "section": "RESULTS", + "ner": [ + [ + 27, + 32, + "SLR-N", + "structure_element" + ] + ] + }, + { + "sid": 161, + "sent": "One possibility is that SLR-N contributes to substrate recognition of proteins to be degraded because there are a couple of putative glycosylation sites within the SLR-N domain (Fig. 1A).", + "section": "RESULTS", + "ner": [ + [ + 24, + 29, + "SLR-N", + "structure_element" + ], + [ + 133, + 152, + "glycosylation sites", + "site" + ], + [ + 164, + 169, + "SLR-N", + "structure_element" + ] + ] + }, + { + "sid": 162, + "sent": "SEL1Lcent contains a putative N-glycosylation site, Asn 427, which is highly conserved among different species and structurally exposed to the surface of the SEL1L dimer according to the crystal structure (Fig. 6C).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "SEL1Lcent", + "structure_element" + ], + [ + 30, + 50, + "N-glycosylation site", + "site" + ], + [ + 52, + 59, + "Asn 427", + "residue_name_number" + ], + [ + 70, + 86, + "highly conserved", + "protein_state" + ], + [ + 158, + 163, + "SEL1L", + "protein" + ], + [ + 164, + 169, + "dimer", + "oligomeric_state" + ], + [ + 187, + 204, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 163, + "sent": "Many reports demonstrate that membrane-bound ERAD machinery proteins in yeast, such as Hrd1p, Der1p, and Usa1p, are involved in oligomerization of ERAD components.", + "section": "DISCUSS", + "ner": [ + [ + 72, + 77, + "yeast", + "taxonomy_domain" + ], + [ + 87, + 92, + "Hrd1p", + "protein" + ], + [ + 94, + 99, + "Der1p", + "protein" + ], + [ + 105, + 110, + "Usa1p", + "protein" + ] + ] + }, + { + "sid": 164, + "sent": "The Hrd1p complex forms dimers upon sucrose gradient sedimentation and size-exclusion chromatography.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 9, + "Hrd1p", + "protein" + ], + [ + 24, + 30, + "dimers", + "oligomeric_state" + ], + [ + 36, + 66, + "sucrose gradient sedimentation", + "experimental_method" + ], + [ + 71, + 100, + "size-exclusion chromatography", + "experimental_method" + ] + ] + }, + { + "sid": 165, + "sent": "Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 41, + "HA-epitope-tagged", + "protein_state" + ], + [ + 42, + 47, + "Hrd3p", + "protein" + ], + [ + 51, + 56, + "Hrd1p", + "protein" + ], + [ + 89, + 99, + "unmodified", + "protein_state" + ], + [ + 100, + 105, + "Hrd3p", + "protein" + ], + [ + 110, + 115, + "Hrd1p", + "protein" + ], + [ + 152, + 157, + "Hrd1p", + "protein" + ], + [ + 162, + 167, + "Hrd3p", + "protein" + ], + [ + 168, + 178, + "homodimers", + "oligomeric_state" + ], + [ + 219, + 222, + "Hrd", + "complex_assembly" + ] + ] + }, + { + "sid": 166, + "sent": "Considering that the functional and structural composition of ERAD components are conserved in both yeast and mammals, we propose that the mammalian ERAD components also form self-associating oligomers.", + "section": "DISCUSS", + "ner": [ + [ + 100, + 105, + "yeast", + "taxonomy_domain" + ], + [ + 110, + 117, + "mammals", + "taxonomy_domain" + ], + [ + 139, + 148, + "mammalian", + "taxonomy_domain" + ], + [ + 192, + 201, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 167, + "sent": "This hypothesis is supported by cross-linking data suggesting that human HRD1 forms a homodimer.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 50, + "cross-linking data", + "experimental_method" + ], + [ + 67, + 72, + "human", + "species" + ], + [ + 73, + 77, + "HRD1", + "protein" + ], + [ + 86, + 95, + "homodimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 168, + "sent": "Consistent with the previous data, our crystal structure and biochemical data demonstrate that mouse SEL1Lcent exists as a homodimer in the ER lumen via domain swapping of SLR motif 9.", + "section": "DISCUSS", + "ner": [ + [ + 39, + 56, + "crystal structure", + "evidence" + ], + [ + 61, + 77, + "biochemical data", + "evidence" + ], + [ + 95, + 100, + "mouse", + "taxonomy_domain" + ], + [ + 101, + 110, + "SEL1Lcent", + "structure_element" + ], + [ + 123, + 132, + "homodimer", + "oligomeric_state" + ], + [ + 172, + 183, + "SLR motif 9", + "structure_element" + ] + ] + }, + { + "sid": 169, + "sent": "We need to further test whether there are contacts involved in dimer formation in SEL1L in addition to those in the SLR-M region.", + "section": "DISCUSS", + "ner": [ + [ + 63, + 68, + "dimer", + "oligomeric_state" + ], + [ + 82, + 87, + "SEL1L", + "protein" + ], + [ + 116, + 121, + "SLR-M", + "structure_element" + ] + ] + }, + { + "sid": 170, + "sent": "In yeast, Usa1p acts as a scaffold for Hrd1p and Der1p, in which the N-terminus of Usa1p interacts with the C-terminal 34 amino acids of Hrd1p in the cytosol to induce oligomerization of Hrd1p, which is essential for its activity.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 8, + "yeast", + "taxonomy_domain" + ], + [ + 10, + 15, + "Usa1p", + "protein" + ], + [ + 39, + 44, + "Hrd1p", + "protein" + ], + [ + 49, + 54, + "Der1p", + "protein" + ], + [ + 83, + 88, + "Usa1p", + "protein" + ], + [ + 137, + 142, + "Hrd1p", + "protein" + ], + [ + 187, + 192, + "Hrd1p", + "protein" + ] + ] + }, + { + "sid": 171, + "sent": "However, metazoans lack a clear Usa1p homolog.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 18, + "metazoans", + "taxonomy_domain" + ], + [ + 32, + 37, + "Usa1p", + "protein" + ] + ] + }, + { + "sid": 172, + "sent": "Although mammalian HERP has sequences and domains that are conserved in Usa1p, the molecular function of HERP is not clearly related to that of Usa1p.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 18, + "mammalian", + "taxonomy_domain" + ], + [ + 19, + 23, + "HERP", + "protein_type" + ], + [ + 59, + 71, + "conserved in", + "protein_state" + ], + [ + 72, + 77, + "Usa1p", + "protein" + ], + [ + 105, + 109, + "HERP", + "protein_type" + ], + [ + 144, + 149, + "Usa1p", + "protein" + ] + ] + }, + { + "sid": 173, + "sent": "Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate \u03b1-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites.", + "section": "DISCUSS", + "ner": [ + [ + 37, + 58, + "transiently expressed", + "protein_state" + ], + [ + 59, + 69, + "HRD1-SEL1L", + "complex_assembly" + ], + [ + 109, + 116, + "lectins", + "protein_type" + ], + [ + 117, + 120, + "OS9", + "protein" + ], + [ + 124, + 129, + "XTP-B", + "protein" + ], + [ + 229, + 257, + "\u03b1-antitrypsin null Hong-Kong", + "protein" + ], + [ + 259, + 262, + "NHK", + "protein" + ], + [ + 281, + 288, + "NHK-QQQ", + "mutant" + ], + [ + 296, + 301, + "lacks", + "protein_state" + ], + [ + 306, + 327, + "N-glycosylation sites", + "site" + ] + ] + }, + { + "sid": 174, + "sent": "Assuming that the correct oligomerization of ERAD components may be critical for their function, we hypothesize that homodimer formation of SEL1L in the ER lumen may stabilize oligomerization of the HRD complex, given that SEL1L forms a stoichiometric complex with HRD1.", + "section": "DISCUSS", + "ner": [ + [ + 117, + 126, + "homodimer", + "oligomeric_state" + ], + [ + 140, + 145, + "SEL1L", + "protein" + ], + [ + 199, + 202, + "HRD", + "complex_assembly" + ], + [ + 223, + 228, + "SEL1L", + "protein" + ], + [ + 252, + 264, + "complex with", + "protein_state" + ], + [ + 265, + 269, + "HRD1", + "protein" + ] + ] + }, + { + "sid": 175, + "sent": "This is further supported by our data showing that the SLR-C of SEL1L directly interacts with the luminal fragment of HRD1 in the ER lumen.", + "section": "DISCUSS", + "ner": [ + [ + 55, + 60, + "SLR-C", + "structure_element" + ], + [ + 64, + 69, + "SEL1L", + "protein" + ], + [ + 118, + 122, + "HRD1", + "protein" + ] + ] + }, + { + "sid": 176, + "sent": "Although the organization of membrane-bound HRD complex components may be very similar between metazoans and yeast, the molecular details of interactions between the components may not necessarily be conserved.", + "section": "DISCUSS", + "ner": [ + [ + 44, + 47, + "HRD", + "complex_assembly" + ], + [ + 95, + 104, + "metazoans", + "taxonomy_domain" + ], + [ + 109, + 114, + "yeast", + "taxonomy_domain" + ] + ] + }, + { + "sid": 177, + "sent": "In yeast, it is unclear whether self-association of Hrd3p is due to SLR motifs because the sequence of Hrd3p does not align precisely with the SLR motifs in SEL1L.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 8, + "yeast", + "taxonomy_domain" + ], + [ + 52, + 57, + "Hrd3p", + "protein" + ], + [ + 68, + 71, + "SLR", + "structure_element" + ], + [ + 103, + 108, + "Hrd3p", + "protein" + ], + [ + 143, + 146, + "SLR", + "structure_element" + ], + [ + 157, + 162, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 178, + "sent": "Furthermore, we are uncertain whether self-association of Hrd3p contributes to formation of the active form of the Hrd1p complex.", + "section": "DISCUSS", + "ner": [ + [ + 58, + 63, + "Hrd3p", + "protein" + ], + [ + 96, + 102, + "active", + "protein_state" + ], + [ + 115, + 120, + "Hrd1p", + "protein" + ] + ] + }, + { + "sid": 179, + "sent": "Recently, a truncated version of Yos9 was shown to form a dimer in the ER lumen and to contribute to the dimeric state of the Hrd1p complex.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 21, + "truncated", + "protein_state" + ], + [ + 33, + 37, + "Yos9", + "protein" + ], + [ + 58, + 63, + "dimer", + "oligomeric_state" + ], + [ + 105, + 112, + "dimeric", + "oligomeric_state" + ], + [ + 126, + 131, + "Hrd1p", + "protein" + ] + ] + }, + { + "sid": 180, + "sent": "This interaction seems to be weak because direct Yos9-Yos9 interactions were not detected in immunoprecipitation experiments from yeast cell extracts containing different epitope-tagged variants of Yos9.", + "section": "DISCUSS", + "ner": [ + [ + 49, + 53, + "Yos9", + "protein" + ], + [ + 54, + 58, + "Yos9", + "protein" + ], + [ + 93, + 124, + "immunoprecipitation experiments", + "experimental_method" + ], + [ + 130, + 135, + "yeast", + "taxonomy_domain" + ], + [ + 171, + 185, + "epitope-tagged", + "protein_state" + ], + [ + 198, + 202, + "Yos9", + "protein" + ] + ] + }, + { + "sid": 181, + "sent": "However, the dimerization of Yos9 could provide a higher stability for the Hrd1p complex oligomer.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 25, + "dimerization", + "oligomeric_state" + ], + [ + 29, + 33, + "Yos9", + "protein" + ], + [ + 75, + 80, + "Hrd1p", + "protein" + ], + [ + 89, + 97, + "oligomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 182, + "sent": "Likewise, the dimerization of SEL1L might provide stability for the mammalian HRD oligomer complex.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 26, + "dimerization", + "oligomeric_state" + ], + [ + 30, + 35, + "SEL1L", + "protein" + ], + [ + 68, + 77, + "mammalian", + "taxonomy_domain" + ], + [ + 78, + 81, + "HRD", + "complex_assembly" + ], + [ + 82, + 90, + "oligomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 183, + "sent": "Further cell biological studies are required to clarify whether SEL1L (Hrd3p) dimerization could be cooperative with the oligomerization of the HRD complex.", + "section": "DISCUSS", + "ner": [ + [ + 64, + 69, + "SEL1L", + "protein" + ], + [ + 71, + 76, + "Hrd3p", + "protein" + ], + [ + 78, + 90, + "dimerization", + "oligomeric_state" + ], + [ + 144, + 147, + "HRD", + "complex_assembly" + ] + ] + }, + { + "sid": 184, + "sent": "Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans.", + "section": "DISCUSS", + "ner": [ + [ + 62, + 65, + "HRD", + "complex_assembly" + ], + [ + 106, + 115, + "oligomers", + "oligomeric_state" + ], + [ + 157, + 162, + "SEL1L", + "protein" + ], + [ + 198, + 204, + "active", + "protein_state" + ], + [ + 218, + 221, + "HRD", + "complex_assembly" + ], + [ + 243, + 253, + "absence of", + "protein_state" + ], + [ + 254, + 259, + "Usa1p", + "protein" + ], + [ + 264, + 273, + "metazoans", + "taxonomy_domain" + ] + ] + }, + { + "sid": 185, + "sent": "These findings should provide a foundation for molecular-level studies to understand the membrane-associated HRD complex assembly in ERAD.", + "section": "DISCUSS", + "ner": [ + [ + 109, + 112, + "HRD", + "complex_assembly" + ] + ] + }, + { + "sid": 186, + "sent": "Crystal Structure of SEL1Lcent.", + "section": "FIG", + "ner": [ + [ + 0, + 17, + "Crystal Structure", + "evidence" + ], + [ + 21, + 30, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 187, + "sent": "(A) The diagram shows the domain structure of Mus musculus SEL1L, as defined by proteolytic mapping and sequence/structure analysis.", + "section": "FIG", + "ner": [ + [ + 46, + 58, + "Mus musculus", + "species" + ], + [ + 59, + 64, + "SEL1L", + "protein" + ], + [ + 80, + 99, + "proteolytic mapping", + "experimental_method" + ], + [ + 104, + 131, + "sequence/structure analysis", + "experimental_method" + ] + ] + }, + { + "sid": 188, + "sent": "The 11 SLR motifs were divided into three groups (SLR-N, SLR-M, and SLR-C) due to the presence of linker sequences that are not predicted SLR motifs.", + "section": "FIG", + "ner": [ + [ + 7, + 10, + "SLR", + "structure_element" + ], + [ + 50, + 55, + "SLR-N", + "structure_element" + ], + [ + 57, + 62, + "SLR-M", + "structure_element" + ], + [ + 68, + 73, + "SLR-C", + "structure_element" + ], + [ + 98, + 114, + "linker sequences", + "structure_element" + ], + [ + 138, + 141, + "SLR", + "structure_element" + ] + ] + }, + { + "sid": 189, + "sent": "Putative N-glycosylation sites are indicated by black triangles.", + "section": "FIG", + "ner": [ + [ + 9, + 30, + "N-glycosylation sites", + "site" + ] + ] + }, + { + "sid": 190, + "sent": "We determined the crystal structure of the SLR-M, residues 348\u2013533. (B) Ribbon diagram of the biological unit of the SEL1Lcent, viewed along the two-fold NCS axis.", + "section": "FIG", + "ner": [ + [ + 18, + 35, + "crystal structure", + "evidence" + ], + [ + 43, + 48, + "SLR-M", + "structure_element" + ], + [ + 59, + 66, + "348\u2013533", + "residue_range" + ], + [ + 117, + 126, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 191, + "sent": "The crystal structure was determined by SAD phasing using selenium as the anomalous scatterer and refined to 2.6 \u00c5 resolution (Table 1).", + "section": "FIG", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 40, + 51, + "SAD phasing", + "experimental_method" + ], + [ + 58, + 66, + "selenium", + "chemical" + ] + ] + }, + { + "sid": 192, + "sent": "(C) SEL1Lcent ribbon diagram rotated 90\u00b0 around a horizontal axis relative to (B).", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 193, + "sent": "(D) One protomer of the SEL1Lcent dimer.", + "section": "FIG", + "ner": [ + [ + 8, + 16, + "protomer", + "oligomeric_state" + ], + [ + 24, + 33, + "SEL1Lcent", + "structure_element" + ], + [ + 34, + 39, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 194, + "sent": "Starting from the N-terminus, SEL1Lcent has five SLR motifs comprising ten \u03b1 helices.", + "section": "FIG", + "ner": [ + [ + 30, + 39, + "SEL1Lcent", + "structure_element" + ], + [ + 49, + 52, + "SLR", + "structure_element" + ], + [ + 75, + 84, + "\u03b1 helices", + "structure_element" + ] + ] + }, + { + "sid": 195, + "sent": "Each SLR motif (from 5 to 9) is indicated in a different color. (E) Evolutionary conservation of surface residues in SEL1Lcent, calculated using ConSurf, from a structure-based alignment of 135 SEL1L sequences.", + "section": "FIG", + "ner": [ + [ + 5, + 8, + "SLR", + "structure_element" + ], + [ + 117, + 126, + "SEL1Lcent", + "structure_element" + ], + [ + 145, + 152, + "ConSurf", + "experimental_method" + ], + [ + 161, + 186, + "structure-based alignment", + "experimental_method" + ], + [ + 194, + 199, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 196, + "sent": "The surface is colored from red (high) to white (poor) according to the degree of conservation in the SEL1L phylogenetic orthologs.", + "section": "FIG", + "ner": [ + [ + 102, + 107, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 197, + "sent": "The ribbon diagram of the counterpart protomer is drawn to show the orientation of the SEL1Lcent dimer.", + "section": "FIG", + "ner": [ + [ + 38, + 46, + "protomer", + "oligomeric_state" + ], + [ + 87, + 96, + "SEL1Lcent", + "structure_element" + ], + [ + 97, + 102, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 198, + "sent": "Dimer Interface of SEL1Lcent.", + "section": "FIG", + "ner": [ + [ + 0, + 15, + "Dimer Interface", + "site" + ], + [ + 19, + 28, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 199, + "sent": "(A) The diagram on the left shows the SEL1Lcent dimer viewed along the two-fold symmetry axis.", + "section": "FIG", + "ner": [ + [ + 38, + 47, + "SEL1Lcent", + "structure_element" + ], + [ + 48, + 53, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 200, + "sent": "Three distinct contact regions are indicated with labeled boxes.", + "section": "FIG", + "ner": [ + [ + 15, + 30, + "contact regions", + "site" + ] + ] + }, + { + "sid": 201, + "sent": "The close-up view on the right shows the residues of SEL1Lcent that contribute to dimer formation via the three contact interfaces.", + "section": "FIG", + "ner": [ + [ + 53, + 62, + "SEL1Lcent", + "structure_element" + ], + [ + 82, + 87, + "dimer", + "oligomeric_state" + ], + [ + 112, + 130, + "contact interfaces", + "site" + ] + ] + }, + { + "sid": 202, + "sent": "The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins.", + "section": "FIG", + "ner": [ + [ + 48, + 62, + "hydrogen bonds", + "bond_interaction" + ], + [ + 75, + 84, + "protomers", + "oligomeric_state" + ], + [ + 88, + 97, + "SEL1Lcent", + "structure_element" + ], + [ + 103, + 132, + "Size-exclusion chromatography", + "experimental_method" + ], + [ + 134, + 137, + "SEC", + "experimental_method" + ], + [ + 155, + 164, + "wild-type", + "protein_state" + ], + [ + 169, + 186, + "dimeric interface", + "site" + ], + [ + 187, + 196, + "SEL1Lcent", + "structure_element" + ], + [ + 197, + 204, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 203, + "sent": "The standard molecular masses for the SEC experiments (top) were obtained from the following proteins: aldolase, 158\u2009kDa; cobalbumin, 75\u2009kDa; ovalbumin, 44\u2009kDa; and carbonic anhydrase, 29\u2009kDa.", + "section": "FIG", + "ner": [ + [ + 38, + 41, + "SEC", + "experimental_method" + ] + ] + }, + { + "sid": 204, + "sent": "The elution fractions, indicated by the gray shading, were run on SDS-PAGE and are shown below the gel-filtration elution profile.", + "section": "FIG", + "ner": [ + [ + 66, + 74, + "SDS-PAGE", + "experimental_method" + ], + [ + 99, + 129, + "gel-filtration elution profile", + "evidence" + ] + ] + }, + { + "sid": 205, + "sent": "The schematic diagrams representing the protein constructs used in the SEC are shown on the left of each SDS-PAGE profile.", + "section": "FIG", + "ner": [ + [ + 71, + 74, + "SEC", + "experimental_method" + ], + [ + 105, + 113, + "SDS-PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 206, + "sent": "Domain Swapping for Dimerization of SEL1Lcent.", + "section": "FIG", + "ner": [ + [ + 20, + 32, + "Dimerization", + "oligomeric_state" + ], + [ + 36, + 45, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 207, + "sent": "(A) Sequence alignment of the SLR motifs in SEL1L.", + "section": "FIG", + "ner": [ + [ + 4, + 22, + "Sequence alignment", + "experimental_method" + ], + [ + 30, + 33, + "SLR", + "structure_element" + ], + [ + 44, + 49, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 208, + "sent": "The 11 SLR motifs were aligned based on the present crystal structure of SEL1Lcent.", + "section": "FIG", + "ner": [ + [ + 7, + 10, + "SLR", + "structure_element" + ], + [ + 23, + 30, + "aligned", + "experimental_method" + ], + [ + 52, + 69, + "crystal structure", + "evidence" + ], + [ + 73, + 82, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 209, + "sent": "The sequences of SEL1Lcent included in the crystal structure are highlighted by the blue box.", + "section": "FIG", + "ner": [ + [ + 17, + 26, + "SEL1Lcent", + "structure_element" + ], + [ + 43, + 60, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 210, + "sent": "The secondary structure elements are indicated above the sequences, with helices depicted as cylinders.", + "section": "FIG", + "ner": [ + [ + 73, + 80, + "helices", + "structure_element" + ] + ] + }, + { + "sid": 211, + "sent": "The GG sequence in SLR motif 9, which creates the hinge for domain swapping (see text), is shaded yellow.", + "section": "FIG", + "ner": [ + [ + 4, + 6, + "GG", + "structure_element" + ], + [ + 19, + 30, + "SLR motif 9", + "structure_element" + ], + [ + 50, + 55, + "hinge", + "structure_element" + ] + ] + }, + { + "sid": 212, + "sent": "Stars below the sequences indicate the specific residues that commonly appear in SLRs.", + "section": "FIG", + "ner": [ + [ + 81, + 85, + "SLRs", + "structure_element" + ] + ] + }, + { + "sid": 213, + "sent": "(B) Structure alignment of five SLR motifs in SEL1Lcent is shown to highlight the unusual geometry of SLR motif 9.", + "section": "FIG", + "ner": [ + [ + 4, + 23, + "Structure alignment", + "experimental_method" + ], + [ + 32, + 35, + "SLR", + "structure_element" + ], + [ + 46, + 55, + "SEL1Lcent", + "structure_element" + ], + [ + 102, + 113, + "SLR motif 9", + "structure_element" + ] + ] + }, + { + "sid": 214, + "sent": "Each SLR motif is shown in a different color.", + "section": "FIG", + "ner": [ + [ + 5, + 8, + "SLR", + "structure_element" + ] + ] + }, + { + "sid": 215, + "sent": "In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an \u03b1-hairpin structure. (C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer.", + "section": "FIG", + "ner": [ + [ + 3, + 14, + "SLR motif 9", + "structure_element" + ], + [ + 37, + 44, + "helices", + "structure_element" + ], + [ + 82, + 85, + "SLR", + "structure_element" + ], + [ + 102, + 111, + "\u03b1-hairpin", + "structure_element" + ], + [ + 154, + 161, + "Gly 512", + "residue_name_number" + ], + [ + 166, + 173, + "Gly 513", + "residue_name_number" + ], + [ + 227, + 235, + "helix 9B", + "structure_element" + ], + [ + 257, + 262, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 216, + "sent": "The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "Gly 512", + "residue_name_number" + ], + [ + 16, + 23, + "Gly 513", + "residue_name_number" + ], + [ + 92, + 107, + "point mutations", + "experimental_method" + ], + [ + 150, + 157, + "Gly 512", + "residue_name_number" + ], + [ + 162, + 169, + "Gly 513", + "residue_name_number" + ], + [ + 206, + 211, + "hinge", + "structure_element" + ], + [ + 215, + 226, + "SLR motif 9", + "structure_element" + ], + [ + 228, + 233, + "G512A", + "mutant" + ], + [ + 235, + 240, + "G513A", + "mutant" + ], + [ + 242, + 247, + "G512A", + "mutant" + ], + [ + 248, + 253, + "G513A", + "mutant" + ], + [ + 259, + 264, + "G512K", + "mutant" + ], + [ + 265, + 270, + "G513K", + "mutant" + ] + ] + }, + { + "sid": 217, + "sent": "Size-exclusion chromatography was conducted as described in Fig. 2B.", + "section": "FIG", + "ner": [ + [ + 0, + 29, + "Size-exclusion chromatography", + "experimental_method" + ] + ] + }, + { + "sid": 218, + "sent": "SEL1L forms self-oligomer mediated by the SEL1Lcent domain in vivo.", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "SEL1L", + "protein" + ], + [ + 12, + 25, + "self-oligomer", + "oligomeric_state" + ], + [ + 42, + 51, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 219, + "sent": "(A) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody.", + "section": "FIG", + "ner": [ + [ + 94, + 112, + "immunoprecipitated", + "experimental_method" + ], + [ + 126, + 130, + "FLAG", + "experimental_method" + ], + [ + 152, + 164, + "western blot", + "experimental_method" + ], + [ + 188, + 190, + "HA", + "experimental_method" + ] + ] + }, + { + "sid": 220, + "sent": "The full-length SEL1L-FLAG was co-immunoprecipitated with the full-length SEL1L-HA.", + "section": "FIG", + "ner": [ + [ + 4, + 15, + "full-length", + "protein_state" + ], + [ + 16, + 21, + "SEL1L", + "protein" + ], + [ + 22, + 26, + "FLAG", + "experimental_method" + ], + [ + 31, + 52, + "co-immunoprecipitated", + "experimental_method" + ], + [ + 62, + 73, + "full-length", + "protein_state" + ], + [ + 74, + 79, + "SEL1L", + "protein" + ], + [ + 80, + 82, + "HA", + "experimental_method" + ] + ] + }, + { + "sid": 221, + "sent": "Also, SEL1Lcent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively.", + "section": "FIG", + "ner": [ + [ + 6, + 15, + "SEL1Lcent", + "structure_element" + ], + [ + 20, + 41, + "co-immunoprecipitated", + "experimental_method" + ], + [ + 51, + 62, + "full-length", + "protein_state" + ], + [ + 63, + 68, + "SEL1L", + "protein" + ], + [ + 79, + 90, + "SLR motif 9", + "structure_element" + ], + [ + 91, + 99, + "deletion", + "experimental_method" + ], + [ + 249, + 261, + "western blot", + "experimental_method" + ], + [ + 275, + 294, + "immunoprecipitation", + "experimental_method" + ] + ] + }, + { + "sid": 222, + "sent": "The SEL1L348\u2013497 fragment was secreted to the culture media but the SEL1Lcent was retained in the ER. (C) SEL1Lcent-FLAG-KDEL and SEL1L348\u2013497-FLAG-KDEL localized to the ER.", + "section": "FIG", + "ner": [ + [ + 4, + 16, + "SEL1L348\u2013497", + "mutant" + ], + [ + 68, + 77, + "SEL1Lcent", + "structure_element" + ], + [ + 106, + 125, + "SEL1Lcent-FLAG-KDEL", + "mutant" + ], + [ + 130, + 152, + "SEL1L348\u2013497-FLAG-KDEL", + "mutant" + ] + ] + }, + { + "sid": 223, + "sent": "The SEL1L fragments were stained in red. (D) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-HA antibody followed by Western blot analysis using an anti-FLAG antibody.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "SEL1L", + "protein" + ], + [ + 135, + 153, + "immunoprecipitated", + "experimental_method" + ], + [ + 167, + 169, + "HA", + "experimental_method" + ], + [ + 191, + 203, + "Western blot", + "experimental_method" + ], + [ + 227, + 231, + "FLAG", + "experimental_method" + ] + ] + }, + { + "sid": 224, + "sent": "The full-length SEL1L forms self-oligomers and the SEL1Lcent-FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA.", + "section": "FIG", + "ner": [ + [ + 4, + 15, + "full-length", + "protein_state" + ], + [ + 16, + 21, + "SEL1L", + "protein" + ], + [ + 28, + 42, + "self-oligomers", + "oligomeric_state" + ], + [ + 51, + 70, + "SEL1Lcent-FLAG-KDEL", + "mutant" + ], + [ + 75, + 96, + "co-immunoprecipitated", + "experimental_method" + ], + [ + 102, + 113, + "full-length", + "protein_state" + ], + [ + 114, + 119, + "SEL1L", + "protein" + ], + [ + 120, + 122, + "HA", + "experimental_method" + ] + ] + }, + { + "sid": 225, + "sent": "The red asterisk indicates the expected signal for SEL1L348\u2013497-FLAG-KDEL.", + "section": "FIG", + "ner": [ + [ + 51, + 73, + "SEL1L348\u2013497-FLAG-KDEL", + "mutant" + ] + ] + }, + { + "sid": 226, + "sent": "SEL1L348\u2013497-FLAG-KDEL did not co-immunoprecipitate with full-length SEL1L-HA.", + "section": "FIG", + "ner": [ + [ + 0, + 22, + "SEL1L348\u2013497-FLAG-KDEL", + "mutant" + ], + [ + 57, + 68, + "full-length", + "protein_state" + ], + [ + 69, + 74, + "SEL1L", + "protein" + ], + [ + 75, + 77, + "HA", + "experimental_method" + ] + ] + }, + { + "sid": 227, + "sent": "The white asterisks indicate non-specific bands. (E) SEL1Lcent-HA-KDEL competitively inhibited self-oligomerization of full-length SEL1L.", + "section": "FIG", + "ner": [ + [ + 53, + 70, + "SEL1Lcent-HA-KDEL", + "mutant" + ], + [ + 119, + 130, + "full-length", + "protein_state" + ], + [ + 131, + 136, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 228, + "sent": "The indicated plasmid constructs were transfected and immunoprecipitation assay was performed using an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody.", + "section": "FIG", + "ner": [ + [ + 54, + 79, + "immunoprecipitation assay", + "experimental_method" + ], + [ + 108, + 112, + "FLAG", + "experimental_method" + ], + [ + 134, + 146, + "western blot", + "experimental_method" + ], + [ + 170, + 172, + "HA", + "experimental_method" + ] + ] + }, + { + "sid": 229, + "sent": "The red rectangle indicates competitively inhibited SEL1L self-oligomer formation by the increasing doses of SEL1Lcent-HA-KDEL. (F) L521A point mutant in SEL1Lcent did not inhibit the self-association of SEL1L.", + "section": "FIG", + "ner": [ + [ + 52, + 57, + "SEL1L", + "protein" + ], + [ + 63, + 71, + "oligomer", + "oligomeric_state" + ], + [ + 109, + 126, + "SEL1Lcent-HA-KDEL", + "mutant" + ], + [ + 132, + 137, + "L521A", + "mutant" + ], + [ + 138, + 150, + "point mutant", + "protein_state" + ], + [ + 154, + 163, + "SEL1Lcent", + "structure_element" + ], + [ + 204, + 209, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 230, + "sent": "Comparison of SLR in SEL1L with TPR or Other SLR-Containing Proteins.", + "section": "FIG", + "ner": [ + [ + 14, + 17, + "SLR", + "structure_element" + ], + [ + 21, + 26, + "SEL1L", + "protein" + ], + [ + 32, + 35, + "TPR", + "structure_element" + ], + [ + 45, + 68, + "SLR-Containing Proteins", + "protein_type" + ] + ] + }, + { + "sid": 231, + "sent": "(A) Ribbon diagram showing superimposition of an isolated TPR motif from Cdc23 and an SLR motif from SEL1Lcent (left), and SLR motifs in HcpC and SEL1Lcent (right).", + "section": "FIG", + "ner": [ + [ + 27, + 42, + "superimposition", + "experimental_method" + ], + [ + 58, + 61, + "TPR", + "structure_element" + ], + [ + 73, + 78, + "Cdc23", + "protein" + ], + [ + 86, + 89, + "SLR", + "structure_element" + ], + [ + 101, + 110, + "SEL1Lcent", + "structure_element" + ], + [ + 123, + 126, + "SLR", + "structure_element" + ], + [ + 137, + 141, + "HcpC", + "protein" + ], + [ + 146, + 155, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 232, + "sent": "The SEL1L, Cdc23, and HcpC are colored magenta, green and cyan, respectively.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "SEL1L", + "protein" + ], + [ + 11, + 16, + "Cdc23", + "protein" + ], + [ + 22, + 26, + "HcpC", + "protein" + ] + ] + }, + { + "sid": 233, + "sent": "The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the \u03b1-solenoid domain.", + "section": "FIG", + "ner": [ + [ + 24, + 39, + "disulfide bonds", + "ptm" + ], + [ + 47, + 51, + "HcpC", + "protein" + ], + [ + 57, + 60, + "Cys", + "residue_name" + ], + [ + 82, + 99, + "disulfide bonding", + "ptm" + ], + [ + 162, + 177, + "superimposition", + "experimental_method" + ], + [ + 181, + 186, + "Cdc23", + "protein" + ], + [ + 191, + 200, + "SEL1Lcent", + "structure_element" + ], + [ + 211, + 215, + "HcpC", + "protein" + ], + [ + 220, + 229, + "SEL1Lcent", + "structure_element" + ], + [ + 281, + 298, + "\u03b1-solenoid domain", + "structure_element" + ] + ] + }, + { + "sid": 234, + "sent": "Both SEL1Lcent schematics are identically oriented for comparison.", + "section": "FIG", + "ner": [ + [ + 5, + 14, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 235, + "sent": "The C\u03b1 atoms of the residues in each \u03b1-solenoid domain are superimposed with a root-mean-squared deviation of 3.3\u2009\u00c5 for Cdc23 and SEL1Lcent (left), and 2.5\u2009\u00c5 for HcpC and SEL1Lcent (right).", + "section": "FIG", + "ner": [ + [ + 37, + 54, + "\u03b1-solenoid domain", + "structure_element" + ], + [ + 59, + 71, + "superimposed", + "experimental_method" + ], + [ + 79, + 106, + "root-mean-squared deviation", + "evidence" + ], + [ + 120, + 125, + "Cdc23", + "protein" + ], + [ + 130, + 139, + "SEL1Lcent", + "structure_element" + ], + [ + 162, + 166, + "HcpC", + "protein" + ], + [ + 171, + 180, + "SEL1Lcent", + "structure_element" + ] + ] + }, + { + "sid": 236, + "sent": "SEL1Lcent, Cdc23, and HcpC are colored as in (A). (C) Ribbon diagram showing the overall structure of Cdc23N-term (left) and SEL1Lcent (right) to compare their similarities regarding dimer formation through domain swapping.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "SEL1Lcent", + "structure_element" + ], + [ + 11, + 16, + "Cdc23", + "protein" + ], + [ + 22, + 26, + "HcpC", + "protein" + ], + [ + 89, + 98, + "structure", + "evidence" + ], + [ + 102, + 107, + "Cdc23", + "protein" + ], + [ + 125, + 134, + "SEL1Lcent", + "structure_element" + ], + [ + 183, + 188, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 237, + "sent": "The Role of SLR-C in ERAD machinery and Model for the Organization of Proteins in Membrane-Associated ERAD Components.", + "section": "FIG", + "ner": [ + [ + 12, + 17, + "SLR-C", + "structure_element" + ] + ] + }, + { + "sid": 238, + "sent": "(A) Schematic diagram shows three HRD1 fragment constructs used in the GST pull-down experiment. (B) Pull-down experiments to examine the interactions between HRD luminal loops and certain SLR motifs of SEL1L.", + "section": "FIG", + "ner": [ + [ + 34, + 38, + "HRD1", + "protein" + ], + [ + 71, + 84, + "GST pull-down", + "experimental_method" + ], + [ + 101, + 122, + "Pull-down experiments", + "experimental_method" + ], + [ + 159, + 162, + "HRD", + "complex_assembly" + ], + [ + 163, + 176, + "luminal loops", + "structure_element" + ], + [ + 189, + 192, + "SLR", + "structure_element" + ], + [ + 203, + 208, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 239, + "sent": "Fragments of the luminal loop of HRD1 fused to GST were immobilized on glutathione sepharose beads and incubated with purified three clusters of SLR motifs and monomer form of SLR-M (SLR-ML521A, right panel) in SEL1L.", + "section": "FIG", + "ner": [ + [ + 17, + 29, + "luminal loop", + "structure_element" + ], + [ + 33, + 37, + "HRD1", + "protein" + ], + [ + 47, + 50, + "GST", + "chemical" + ], + [ + 145, + 148, + "SLR", + "structure_element" + ], + [ + 160, + 167, + "monomer", + "oligomeric_state" + ], + [ + 176, + 181, + "SLR-M", + "structure_element" + ], + [ + 183, + 193, + "SLR-ML521A", + "mutant" + ], + [ + 211, + 216, + "SEL1L", + "protein" + ] + ] + }, + { + "sid": 240, + "sent": "Proteins were analyzed by 12% SDS-PAGE and Coomassie blue staining.", + "section": "FIG", + "ner": [ + [ + 30, + 38, + "SDS-PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 241, + "sent": "(C) Schematic representation of the organization of metazoan ERAD components in the ER membrane.", + "section": "FIG", + "ner": [ + [ + 52, + 60, + "metazoan", + "taxonomy_domain" + ] + ] + }, + { + "sid": 242, + "sent": "The 11 SLR motifs of SEL1L were expressed with red cylinders and grouped into three parts (SLR-N, SLR-M, and SLR-C) based on the sequence alignment across the motifs and the crystal structure presented herein.", + "section": "FIG", + "ner": [ + [ + 7, + 10, + "SLR", + "structure_element" + ], + [ + 21, + 26, + "SEL1L", + "protein" + ], + [ + 91, + 96, + "SLR-N", + "structure_element" + ], + [ + 98, + 103, + "SLR-M", + "structure_element" + ], + [ + 109, + 114, + "SLR-C", + "structure_element" + ], + [ + 129, + 147, + "sequence alignment", + "experimental_method" + ], + [ + 174, + 191, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 243, + "sent": "We hypothesized that the interrupted SLR motifs of SEL1L have distinct functions such that the SLR-M is important for dimer formation of the protein, and SLR-C is involved in the interaction with HRD1 in the ER lumen.", + "section": "FIG", + "ner": [ + [ + 37, + 40, + "SLR", + "structure_element" + ], + [ + 51, + 56, + "SEL1L", + "protein" + ], + [ + 95, + 100, + "SLR-M", + "structure_element" + ], + [ + 118, + 123, + "dimer", + "oligomeric_state" + ], + [ + 154, + 159, + "SLR-C", + "structure_element" + ], + [ + 196, + 200, + "HRD1", + "protein" + ] + ] + }, + { + "sid": 244, + "sent": "The surface representation of SEL1Lcent is placed in the same orientation as that shown in the schematic model to show that the putative N-glycosylation site, residue N427 (indicated in yellow), is exposed on the surface of the protein.", + "section": "FIG", + "ner": [ + [ + 30, + 39, + "SEL1Lcent", + "structure_element" + ], + [ + 137, + 157, + "N-glycosylation site", + "site" + ], + [ + 167, + 171, + "N427", + "residue_name_number" + ] + ] + } + ] + }, + "PMC4880283": { + "annotations": [ + { + "sid": 0, + "sent": "Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana", + "section": "TITLE", + "ner": [ + [ + 0, + 18, + "Crystal Structures", + "evidence" + ], + [ + 31, + 44, + "Sugar Kinases", + "protein_type" + ], + [ + 50, + 82, + "Synechococcus Elongatus PCC 7942", + "species" + ], + [ + 87, + 107, + "Arabidopsis Thaliana", + "species" + ] + ] + }, + { + "sid": 1, + "sent": "The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana.", + "section": "ABSTRACT", + "ner": [ + [ + 18, + 57, + "Synechococcus elongatus strain PCC 7942", + "species" + ], + [ + 77, + 89, + "sugar kinase", + "protein_type" + ], + [ + 91, + 96, + "SePSK", + "protein" + ], + [ + 145, + 162, + "xylulose kinase-1", + "protein" + ], + [ + 164, + 170, + "AtXK-1", + "protein" + ], + [ + 177, + 197, + "Arabidopsis thaliana", + "species" + ] + ] + }, + { + "sid": 2, + "sent": "Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 18, + "Sequence alignment", + "experimental_method" + ], + [ + 38, + 45, + "kinases", + "protein_type" + ], + [ + 60, + 98, + "ribulokinase-like carbohydrate kinases", + "protein_type" + ], + [ + 116, + 148, + "FGGY family carbohydrate kinases", + "protein_type" + ] + ] + }, + { + "sid": 3, + "sent": "Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates.", + "section": "ABSTRACT", + "ner": [ + [ + 8, + 14, + "solved", + "experimental_method" + ], + [ + 19, + 29, + "structures", + "evidence" + ], + [ + 33, + 38, + "SePSK", + "protein" + ], + [ + 43, + 49, + "AtXK-1", + "protein" + ], + [ + 64, + 67, + "apo", + "protein_state" + ], + [ + 78, + 93, + "in complex with", + "protein_state" + ], + [ + 94, + 104, + "nucleotide", + "chemical" + ] + ] + }, + { + "sid": 4, + "sent": "The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates.", + "section": "ABSTRACT", + "ner": [ + [ + 73, + 80, + "kinases", + "protein_type" + ], + [ + 92, + 95, + "ATP", + "chemical" + ], + [ + 123, + 144, + "absence of substrates", + "protein_state" + ] + ] + }, + { + "sid": 5, + "sent": "In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose.", + "section": "ABSTRACT", + "ner": [ + [ + 17, + 33, + "enzymatic assays", + "experimental_method" + ], + [ + 49, + 54, + "SePSK", + "protein" + ], + [ + 91, + 101, + "D-ribulose", + "chemical" + ] + ] + }, + { + "sid": 6, + "sent": "In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK.", + "section": "ABSTRACT", + "ner": [ + [ + 50, + 55, + "SePSK", + "protein" + ], + [ + 60, + 66, + "solved", + "experimental_method" + ], + [ + 71, + 80, + "structure", + "evidence" + ], + [ + 84, + 89, + "SePSK", + "protein" + ], + [ + 90, + 105, + "in complex with", + "protein_state" + ], + [ + 106, + 116, + "D-ribulose", + "chemical" + ], + [ + 141, + 166, + "substrate binding pockets", + "site" + ], + [ + 170, + 175, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 7, + "sent": "Using mutation and activity analysis, we further verified the key residues important for its catalytic activity.", + "section": "ABSTRACT", + "ner": [ + [ + 6, + 36, + "mutation and activity analysis", + "experimental_method" + ] + ] + }, + { + "sid": 8, + "sent": "Moreover, our structural comparison with other family members suggests that there are major conformational changes in SePSK upon substrate binding, facilitating the catalytic process.", + "section": "ABSTRACT", + "ner": [ + [ + 14, + 35, + "structural comparison", + "experimental_method" + ], + [ + 118, + 123, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 9, + "sent": "Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1.", + "section": "ABSTRACT", + "ner": [ + [ + 169, + 174, + "SePSK", + "protein" + ], + [ + 211, + 216, + "plant", + "taxonomy_domain" + ], + [ + 227, + 233, + "AtXK-1", + "protein" + ] + ] + }, + { + "sid": 10, + "sent": "Carbohydrates are essential cellular compounds involved in the metabolic processes present in all organisms.", + "section": "INTRO", + "ner": [ + [ + 0, + 13, + "Carbohydrates", + "chemical" + ] + ] + }, + { + "sid": 11, + "sent": "Phosphorylation is one of the various pivotal modifications of carbohydrates, and is catalyzed by specific sugar kinases.", + "section": "INTRO", + "ner": [ + [ + 0, + 15, + "Phosphorylation", + "ptm" + ], + [ + 63, + 76, + "carbohydrates", + "chemical" + ], + [ + 107, + 120, + "sugar kinases", + "protein_type" + ] + ] + }, + { + "sid": 12, + "sent": "These kinases exhibit considerable differences in their folding pattern and substrate specificity.", + "section": "INTRO", + "ner": [ + [ + 6, + 13, + "kinases", + "protein_type" + ] + ] + }, + { + "sid": 13, + "sent": "Based on sequence analysis, they can be divided into four families, namely HSP 70_NBD family, FGGY family, Mer_B like family and Parm_like family.", + "section": "INTRO", + "ner": [ + [ + 9, + 26, + "sequence analysis", + "experimental_method" + ], + [ + 75, + 92, + "HSP 70_NBD family", + "protein_type" + ], + [ + 94, + 105, + "FGGY family", + "protein_type" + ], + [ + 107, + 124, + "Mer_B like family", + "protein_type" + ], + [ + 129, + 145, + "Parm_like family", + "protein_type" + ] + ] + }, + { + "sid": 14, + "sent": "The FGGY family carbohydrate kinases contain different types of sugar kinases, all of which possess different catalytic substrates with preferences for short-chained sugar substrates, ranging from triose to heptose.", + "section": "INTRO", + "ner": [ + [ + 4, + 36, + "FGGY family carbohydrate kinases", + "protein_type" + ], + [ + 64, + 77, + "sugar kinases", + "protein_type" + ], + [ + 166, + 171, + "sugar", + "chemical" + ], + [ + 197, + 203, + "triose", + "chemical" + ], + [ + 207, + 214, + "heptose", + "chemical" + ] + ] + }, + { + "sid": 15, + "sent": "These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose.", + "section": "INTRO", + "ner": [ + [ + 6, + 11, + "sugar", + "chemical" + ], + [ + 31, + 41, + "L-ribulose", + "chemical" + ], + [ + 43, + 53, + "erythritol", + "chemical" + ], + [ + 55, + 65, + "L-fuculose", + "chemical" + ], + [ + 67, + 77, + "D-glycerol", + "chemical" + ], + [ + 79, + 90, + "D-gluconate", + "chemical" + ], + [ + 92, + 102, + "L-xylulose", + "chemical" + ], + [ + 104, + 114, + "D-ribulose", + "chemical" + ], + [ + 116, + 128, + "L-rhamnulose", + "chemical" + ], + [ + 133, + 143, + "D-xylulose", + "chemical" + ] + ] + }, + { + "sid": 16, + "sent": "Structures reported in the Protein Data Bank of the FGGY family carbohydrate kinases exhibit a similar overall architecture containing two protein domains, one of which is responsible for the binding of substrate, while the second is used for binding cofactor ATP.", + "section": "INTRO", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 52, + 84, + "FGGY family carbohydrate kinases", + "protein_type" + ], + [ + 260, + 263, + "ATP", + "chemical" + ] + ] + }, + { + "sid": 17, + "sent": "While the binding pockets for substrates are at the same position, each FGGY family carbohydrate kinases uses different substrate-binding residues, resulting in high substrate specificity.", + "section": "INTRO", + "ner": [ + [ + 10, + 25, + "binding pockets", + "site" + ], + [ + 72, + 104, + "FGGY family carbohydrate kinases", + "protein_type" + ], + [ + 120, + 146, + "substrate-binding residues", + "site" + ] + ] + }, + { + "sid": 18, + "sent": "Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids.", + "section": "INTRO", + "ner": [ + [ + 0, + 15, + "Synpcc7942_2462", + "gene" + ], + [ + 25, + 38, + "cyanobacteria", + "taxonomy_domain" + ], + [ + 39, + 71, + "Synechococcus elongatus PCC 7942", + "species" + ], + [ + 91, + 103, + "sugar kinase", + "protein_type" + ], + [ + 105, + 110, + "SePSK", + "protein" + ], + [ + 122, + 128, + "kinase", + "protein_type" + ], + [ + 138, + 141, + "426", + "residue_range" + ] + ] + }, + { + "sid": 19, + "sent": "The At2g21370 gene product from Arabidopsis thaliana, xylulose kinase-1 (AtXK-1), whose mature form contains 436 amino acids, is located in the chloroplast (ChloroP 1.1 Server).", + "section": "INTRO", + "ner": [ + [ + 4, + 13, + "At2g21370", + "gene" + ], + [ + 32, + 52, + "Arabidopsis thaliana", + "species" + ], + [ + 54, + 71, + "xylulose kinase-1", + "protein" + ], + [ + 73, + 79, + "AtXK-1", + "protein" + ], + [ + 88, + 99, + "mature form", + "protein_state" + ], + [ + 109, + 112, + "436", + "residue_range" + ] + ] + }, + { + "sid": 20, + "sent": "SePSK and AtXK-1 display a sequence identity of 44.9%, and belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "SePSK", + "protein" + ], + [ + 10, + 16, + "AtXK-1", + "protein" + ], + [ + 73, + 111, + "ribulokinase-like carbohydrate kinases", + "protein_type" + ], + [ + 129, + 161, + "FGGY family carbohydrate kinases", + "protein_type" + ] + ] + }, + { + "sid": 21, + "sent": "Members of this sub-family are responsible for the phosphorylation of sugars similar to L-ribulose and D-ribulose.", + "section": "INTRO", + "ner": [ + [ + 51, + 66, + "phosphorylation", + "ptm" + ], + [ + 70, + 76, + "sugars", + "chemical" + ], + [ + 88, + 98, + "L-ribulose", + "chemical" + ], + [ + 103, + 113, + "D-ribulose", + "chemical" + ] + ] + }, + { + "sid": 22, + "sent": "The sequence and the substrate specificity of ribulokinase-like carbohydrate kinases are different, but they share the common folding feature with two domains.", + "section": "INTRO", + "ner": [ + [ + 46, + 84, + "ribulokinase-like carbohydrate kinases", + "protein_type" + ] + ] + }, + { + "sid": 23, + "sent": "Domain I exhibits a ribonuclease H-like folding pattern, and is responsible for the substrate binding, while domain II possesses an actin-like ATPase domain that binds cofactor ATP.", + "section": "INTRO", + "ner": [ + [ + 0, + 8, + "Domain I", + "structure_element" + ], + [ + 20, + 55, + "ribonuclease H-like folding pattern", + "structure_element" + ], + [ + 109, + 118, + "domain II", + "structure_element" + ], + [ + 132, + 156, + "actin-like ATPase domain", + "structure_element" + ], + [ + 177, + 180, + "ATP", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "Two possible xylulose kinases (xylulose kinase-1: XK-1 and xylulose kinase-2: XK-2) from Arabidopsis thaliana were previously proposed.", + "section": "INTRO", + "ner": [ + [ + 13, + 29, + "xylulose kinases", + "protein_type" + ], + [ + 31, + 48, + "xylulose kinase-1", + "protein" + ], + [ + 50, + 54, + "XK-1", + "protein" + ], + [ + 59, + 76, + "xylulose kinase-2", + "protein" + ], + [ + 78, + 82, + "XK-2", + "protein" + ], + [ + 89, + 109, + "Arabidopsis thaliana", + "species" + ] + ] + }, + { + "sid": 25, + "sent": "It was shown that XK-2 (At5g49650) located in the cytosol is indeed xylulose kinase.", + "section": "INTRO", + "ner": [ + [ + 18, + 22, + "XK-2", + "protein" + ], + [ + 24, + 33, + "At5g49650", + "gene" + ], + [ + 68, + 83, + "xylulose kinase", + "protein_type" + ] + ] + }, + { + "sid": 26, + "sent": "However, the function of XK-1 (At2g21370) inside the chloroplast stroma has remained unknown.", + "section": "INTRO", + "ner": [ + [ + 25, + 29, + "XK-1", + "protein" + ], + [ + 31, + 40, + "At2g21370", + "gene" + ] + ] + }, + { + "sid": 27, + "sent": "SePSK from Synechococcus elongatus strain PCC 7942 is the homolog of AtXK-1, though its physiological function and substrates remain unclear.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "SePSK", + "protein" + ], + [ + 11, + 50, + "Synechococcus elongatus strain PCC 7942", + "species" + ], + [ + 69, + 75, + "AtXK-1", + "protein" + ] + ] + }, + { + "sid": 28, + "sent": "In order to obtain functional and structural information about these two proteins, here we reported the crystal structures of SePSK and AtXK-1.", + "section": "INTRO", + "ner": [ + [ + 104, + 122, + "crystal structures", + "evidence" + ], + [ + 126, + 131, + "SePSK", + "protein" + ], + [ + 136, + 142, + "AtXK-1", + "protein" + ] + ] + }, + { + "sid": 29, + "sent": "Our findings provide new details of the catalytic mechanism of SePSK and lay the foundation for future studies into its homologs in eukaryotes.", + "section": "INTRO", + "ner": [ + [ + 63, + 68, + "SePSK", + "protein" + ], + [ + 132, + 142, + "eukaryotes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 30, + "sent": "Overall structures of apo-SePSK and apo-AtXK-1", + "section": "RESULTS", + "ner": [ + [ + 8, + 18, + "structures", + "evidence" + ], + [ + 22, + 25, + "apo", + "protein_state" + ], + [ + 26, + 31, + "SePSK", + "protein" + ], + [ + 36, + 39, + "apo", + "protein_state" + ], + [ + 40, + 46, + "AtXK-1", + "protein" + ] + ] + }, + { + "sid": 31, + "sent": "The attempt to solve the SePSK structure by molecular replacement method failed with ribulokinase from Bacillus halodurans (PDB code: 3QDK, 15.7% sequence identity) as an initial model.", + "section": "RESULTS", + "ner": [ + [ + 25, + 30, + "SePSK", + "protein" + ], + [ + 31, + 40, + "structure", + "evidence" + ], + [ + 44, + 72, + "molecular replacement method", + "experimental_method" + ], + [ + 85, + 97, + "ribulokinase", + "protein" + ], + [ + 103, + 122, + "Bacillus halodurans", + "species" + ] + ] + }, + { + "sid": 32, + "sent": "We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 \u00c5. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study.", + "section": "RESULTS", + "ner": [ + [ + 18, + 76, + "single isomorphous replacement anomalous scattering method", + "experimental_method" + ], + [ + 78, + 83, + "SIRAS", + "experimental_method" + ], + [ + 116, + 119, + "apo", + "protein_state" + ], + [ + 120, + 125, + "SePSK", + "protein" + ], + [ + 126, + 135, + "structure", + "evidence" + ], + [ + 180, + 183, + "apo", + "protein_state" + ], + [ + 184, + 189, + "SePSK", + "protein" + ], + [ + 190, + 199, + "structure", + "evidence" + ], + [ + 212, + 239, + "molecular replacement model", + "experimental_method" + ], + [ + 259, + 269, + "structures", + "evidence" + ] + ] + }, + { + "sid": 33, + "sent": "Our structural analysis showed that apo-SePSK consists of one SePSK protein molecule in an asymmetric unit.", + "section": "RESULTS", + "ner": [ + [ + 4, + 23, + "structural analysis", + "experimental_method" + ], + [ + 36, + 39, + "apo", + "protein_state" + ], + [ + 40, + 45, + "SePSK", + "protein" + ], + [ + 62, + 67, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 34, + "sent": "The amino-acid residues were traced from Val2 to His419, except for the Met1 residue and the seven residues at the C-termini.", + "section": "RESULTS", + "ner": [ + [ + 41, + 45, + "Val2", + "residue_name_number" + ], + [ + 49, + 55, + "His419", + "residue_name_number" + ], + [ + 72, + 76, + "Met1", + "residue_name_number" + ] + ] + }, + { + "sid": 35, + "sent": "Apo-SePSK contains two domains referred to further on as domain I and domain II (Fig 1A).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "Apo", + "protein_state" + ], + [ + 4, + 9, + "SePSK", + "protein" + ], + [ + 57, + 65, + "domain I", + "structure_element" + ], + [ + 70, + 79, + "domain II", + "structure_element" + ] + ] + }, + { + "sid": 36, + "sent": "Domain I consists of non-contiguous portions of the polypeptide chains (aa.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Domain I", + "structure_element" + ] + ] + }, + { + "sid": 37, + "sent": "2\u2013228 and aa.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "2\u2013228", + "residue_range" + ] + ] + }, + { + "sid": 38, + "sent": "402\u2013419), exhibiting 11 \u03b1-helices and 11 \u03b2-sheets.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "402\u2013419", + "residue_range" + ], + [ + 24, + 33, + "\u03b1-helices", + "structure_element" + ], + [ + 41, + 49, + "\u03b2-sheets", + "structure_element" + ] + ] + }, + { + "sid": 39, + "sent": "Among all these structural elements, \u03b14/\u03b15/\u03b111/\u03b118, \u03b23/\u03b22/\u03b21/\u03b26/\u03b219/\u03b220/\u03b217 and \u03b121/\u03b132 form three patches, referred to as A1, B1 and A2, exhibiting the core region.", + "section": "RESULTS", + "ner": [ + [ + 37, + 39, + "\u03b14", + "structure_element" + ], + [ + 40, + 42, + "\u03b15", + "structure_element" + ], + [ + 43, + 46, + "\u03b111", + "structure_element" + ], + [ + 47, + 50, + "\u03b118", + "structure_element" + ], + [ + 52, + 54, + "\u03b23", + "structure_element" + ], + [ + 55, + 57, + "\u03b22", + "structure_element" + ], + [ + 58, + 60, + "\u03b21", + "structure_element" + ], + [ + 61, + 63, + "\u03b26", + "structure_element" + ], + [ + 64, + 67, + "\u03b219", + "structure_element" + ], + [ + 68, + 71, + "\u03b220", + "structure_element" + ], + [ + 72, + 75, + "\u03b217", + "structure_element" + ], + [ + 80, + 83, + "\u03b121", + "structure_element" + ], + [ + 84, + 87, + "\u03b132", + "structure_element" + ], + [ + 123, + 125, + "A1", + "structure_element" + ], + [ + 127, + 129, + "B1", + "structure_element" + ], + [ + 134, + 136, + "A2", + "structure_element" + ], + [ + 153, + 164, + "core region", + "structure_element" + ] + ] + }, + { + "sid": 40, + "sent": "In addition, four \u03b2-sheets (\u03b27, \u03b210, \u03b212 and \u03b216) and five \u03b1-helices (\u03b18, \u03b19, \u03b113, \u03b114 and \u03b115) flank the left side of the core region.", + "section": "RESULTS", + "ner": [ + [ + 18, + 26, + "\u03b2-sheets", + "structure_element" + ], + [ + 28, + 30, + "\u03b27", + "structure_element" + ], + [ + 32, + 35, + "\u03b210", + "structure_element" + ], + [ + 37, + 40, + "\u03b212", + "structure_element" + ], + [ + 45, + 48, + "\u03b216", + "structure_element" + ], + [ + 59, + 68, + "\u03b1-helices", + "structure_element" + ], + [ + 70, + 72, + "\u03b18", + "structure_element" + ], + [ + 74, + 76, + "\u03b19", + "structure_element" + ], + [ + 78, + 81, + "\u03b113", + "structure_element" + ], + [ + 83, + 86, + "\u03b114", + "structure_element" + ], + [ + 91, + 94, + "\u03b115", + "structure_element" + ], + [ + 123, + 134, + "core region", + "structure_element" + ] + ] + }, + { + "sid": 41, + "sent": "Domain II is comprised of aa.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Domain II", + "structure_element" + ] + ] + }, + { + "sid": 42, + "sent": "229\u2013401 and classified into B2 (\u03b231/\u03b229/\u03b222/\u03b223/\u03b225/\u03b224) and A3 (\u03b126/\u03b127/\u03b128/\u03b130) (Fig 1A and S1 Fig).", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "229\u2013401", + "residue_range" + ], + [ + 28, + 30, + "B2", + "structure_element" + ], + [ + 32, + 35, + "\u03b231", + "structure_element" + ], + [ + 36, + 39, + "\u03b229", + "structure_element" + ], + [ + 40, + 43, + "\u03b222", + "structure_element" + ], + [ + 44, + 47, + "\u03b223", + "structure_element" + ], + [ + 48, + 51, + "\u03b225", + "structure_element" + ], + [ + 52, + 55, + "\u03b224", + "structure_element" + ], + [ + 61, + 63, + "A3", + "structure_element" + ], + [ + 65, + 68, + "\u03b126", + "structure_element" + ], + [ + 69, + 72, + "\u03b127", + "structure_element" + ], + [ + 73, + 76, + "\u03b128", + "structure_element" + ], + [ + 77, + 80, + "\u03b130", + "structure_element" + ] + ] + }, + { + "sid": 43, + "sent": "In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (\u03b1/\u03b2/\u03b1/\u03b2/\u03b1) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig).", + "section": "RESULTS", + "ner": [ + [ + 7, + 12, + "SePSK", + "protein" + ], + [ + 13, + 22, + "structure", + "evidence" + ], + [ + 24, + 26, + "B1", + "structure_element" + ], + [ + 31, + 33, + "B2", + "structure_element" + ], + [ + 52, + 54, + "A1", + "structure_element" + ], + [ + 56, + 58, + "A2", + "structure_element" + ], + [ + 63, + 65, + "A3", + "structure_element" + ], + [ + 81, + 90, + "structure", + "evidence" + ], + [ + 101, + 103, + "A1", + "structure_element" + ], + [ + 104, + 106, + "B1", + "structure_element" + ], + [ + 107, + 109, + "A2", + "structure_element" + ], + [ + 110, + 112, + "B2", + "structure_element" + ], + [ + 113, + 115, + "A3", + "structure_element" + ], + [ + 117, + 118, + "\u03b1", + "structure_element" + ], + [ + 119, + 120, + "\u03b2", + "structure_element" + ], + [ + 121, + 122, + "\u03b1", + "structure_element" + ], + [ + 123, + 124, + "\u03b2", + "structure_element" + ], + [ + 125, + 126, + "\u03b1", + "structure_element" + ], + [ + 186, + 218, + "FGGY family carbohydrate kinases", + "protein_type" + ] + ] + }, + { + "sid": 44, + "sent": "The overall folding of SePSK resembles a clip, with A2 of domain I acting as a hinge region.", + "section": "RESULTS", + "ner": [ + [ + 23, + 28, + "SePSK", + "protein" + ], + [ + 52, + 54, + "A2", + "structure_element" + ], + [ + 58, + 66, + "domain I", + "structure_element" + ], + [ + 79, + 91, + "hinge region", + "structure_element" + ] + ] + }, + { + "sid": 45, + "sent": "Overall structures of SePSK and AtXK-1.", + "section": "FIG", + "ner": [ + [ + 8, + 18, + "structures", + "evidence" + ], + [ + 22, + 27, + "SePSK", + "protein" + ], + [ + 32, + 38, + "AtXK-1", + "protein" + ] + ] + }, + { + "sid": 46, + "sent": "(A) Three-dimensional structure of apo-SePSK.", + "section": "FIG", + "ner": [ + [ + 22, + 31, + "structure", + "evidence" + ], + [ + 35, + 38, + "apo", + "protein_state" + ], + [ + 39, + 44, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 47, + "sent": "The secondary structural elements are indicated (\u03b1-helix: cyan, \u03b2-sheet: yellow).", + "section": "FIG", + "ner": [ + [ + 49, + 56, + "\u03b1-helix", + "structure_element" + ], + [ + 64, + 71, + "\u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 48, + "sent": "(B) Three-dimensional structure of apo-AtXK-1.", + "section": "FIG", + "ner": [ + [ + 22, + 31, + "structure", + "evidence" + ], + [ + 35, + 38, + "apo", + "protein_state" + ], + [ + 39, + 45, + "AtXK-1", + "protein" + ] + ] + }, + { + "sid": 49, + "sent": "The secondary structural elements are indicated (\u03b1-helix: green, \u03b2-sheet: wheat).", + "section": "FIG", + "ner": [ + [ + 49, + 56, + "\u03b1-helix", + "structure_element" + ], + [ + 65, + 72, + "\u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 50, + "sent": "Apo-AtXK-1 exhibits a folding pattern similar to that of SePSK in line with their high sequence identity (Fig 1B and S1 Fig).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "Apo", + "protein_state" + ], + [ + 4, + 10, + "AtXK-1", + "protein" + ], + [ + 57, + 62, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 51, + "sent": "However, superposition of structures of AtXK-1 and SePSK shows some differences, especially at the loop regions.", + "section": "RESULTS", + "ner": [ + [ + 9, + 22, + "superposition", + "experimental_method" + ], + [ + 26, + 36, + "structures", + "evidence" + ], + [ + 40, + 46, + "AtXK-1", + "protein" + ], + [ + 51, + 56, + "SePSK", + "protein" + ], + [ + 99, + 111, + "loop regions", + "structure_element" + ] + ] + }, + { + "sid": 52, + "sent": "A considerable difference is found in the loop3 linking \u03b23 and \u03b14, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part.", + "section": "RESULTS", + "ner": [ + [ + 42, + 47, + "loop3", + "structure_element" + ], + [ + 56, + 58, + "\u03b23", + "structure_element" + ], + [ + 63, + 65, + "\u03b14", + "structure_element" + ], + [ + 97, + 103, + "AtXK-1", + "protein" + ], + [ + 104, + 113, + "structure", + "evidence" + ], + [ + 128, + 133, + "SePSK", + "protein" + ], + [ + 134, + 143, + "structure", + "evidence" + ] + ] + }, + { + "sid": 53, + "sent": "The corresponding residues between these two structures (SePSK-Lys35 and AtXK-1-Lys48) have a distance of 15.4 \u00c5 (S3 Fig).", + "section": "RESULTS", + "ner": [ + [ + 45, + 55, + "structures", + "evidence" + ], + [ + 57, + 62, + "SePSK", + "protein" + ], + [ + 63, + 68, + "Lys35", + "residue_name_number" + ], + [ + 73, + 79, + "AtXK-1", + "protein" + ], + [ + 80, + 85, + "Lys48", + "residue_name_number" + ] + ] + }, + { + "sid": 54, + "sent": "Activity assays of SePSK and AtXK-1", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Activity assays", + "experimental_method" + ], + [ + 19, + 24, + "SePSK", + "protein" + ], + [ + 29, + 35, + "AtXK-1", + "protein" + ] + ] + }, + { + "sid": 55, + "sent": "In order to understand the function of these two kinases, we performed structural comparison using Dali server.", + "section": "RESULTS", + "ner": [ + [ + 71, + 92, + "structural comparison", + "experimental_method" + ], + [ + 99, + 110, + "Dali server", + "experimental_method" + ] + ] + }, + { + "sid": 56, + "sent": "The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 39, + 44, + "SePSK", + "protein" + ], + [ + 49, + 64, + "xylulose kinase", + "protein_type" + ], + [ + 66, + 81, + "glycerol kinase", + "protein_type" + ], + [ + 86, + 101, + "ribulose kinase", + "protein_type" + ], + [ + 117, + 122, + "SePSK", + "protein" + ], + [ + 127, + 133, + "AtXK-1", + "protein" + ], + [ + 168, + 175, + "kinases", + "protein_type" + ] + ] + }, + { + "sid": 57, + "sent": "We first tested whether both enzymes possessed ATP hydrolysis activity in the absence of substrates.", + "section": "RESULTS", + "ner": [ + [ + 47, + 50, + "ATP", + "chemical" + ], + [ + 78, + 88, + "absence of", + "protein_state" + ] + ] + }, + { + "sid": 58, + "sent": "As shown in Fig 2A, both SePSK and AtXK-1 exhibited ATP hydrolysis activity.", + "section": "RESULTS", + "ner": [ + [ + 25, + 30, + "SePSK", + "protein" + ], + [ + 35, + 41, + "AtXK-1", + "protein" + ], + [ + 52, + 55, + "ATP", + "chemical" + ] + ] + }, + { + "sid": 59, + "sent": "This finding is in agreement with a previous result showing that xylulose kinase (PDB code: 2ITM) possessed ATP hydrolysis activity without adding substrate.", + "section": "RESULTS", + "ner": [ + [ + 65, + 80, + "xylulose kinase", + "protein_type" + ], + [ + 108, + 111, + "ATP", + "chemical" + ] + ] + }, + { + "sid": 60, + "sent": "To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays.", + "section": "RESULTS", + "ner": [ + [ + 44, + 49, + "SePSK", + "protein" + ], + [ + 54, + 60, + "AtXK-1", + "protein" + ], + [ + 104, + 114, + "D-ribulose", + "chemical" + ], + [ + 116, + 126, + "L-ribulose", + "chemical" + ], + [ + 128, + 138, + "D-xylulose", + "chemical" + ], + [ + 140, + 150, + "L-xylulose", + "chemical" + ], + [ + 155, + 163, + "Glycerol", + "chemical" + ], + [ + 178, + 203, + "enzymatic activity assays", + "experimental_method" + ] + ] + }, + { + "sid": 61, + "sent": "As shown in Fig 2B, the ATP hydrolysis activity of SePSK greatly increased upon adding D-ribulose than adding other potential substrates, suggesting that it has D-ribulose kinase activity.", + "section": "RESULTS", + "ner": [ + [ + 24, + 27, + "ATP", + "chemical" + ], + [ + 51, + 56, + "SePSK", + "protein" + ], + [ + 87, + 97, + "D-ribulose", + "chemical" + ], + [ + 161, + 178, + "D-ribulose kinase", + "protein_type" + ] + ] + }, + { + "sid": 62, + "sent": "In contrary, limited increasing of ATP hydrolysis activity was detected for AtXK-1 upon addition of D-ribulose (Fig 2C), despite its structural similarity with SePSK.", + "section": "RESULTS", + "ner": [ + [ + 35, + 38, + "ATP", + "chemical" + ], + [ + 76, + 82, + "AtXK-1", + "protein" + ], + [ + 100, + 110, + "D-ribulose", + "chemical" + ], + [ + 160, + 165, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 63, + "sent": "The enzymatic activity assays of SePSK and AtXK-1.", + "section": "FIG", + "ner": [ + [ + 4, + 29, + "enzymatic activity assays", + "experimental_method" + ], + [ + 33, + 38, + "SePSK", + "protein" + ], + [ + 43, + 49, + "AtXK-1", + "protein" + ] + ] + }, + { + "sid": 64, + "sent": "(A) The ATP hydrolysis activity of SePSK and AtXK-1.", + "section": "FIG", + "ner": [ + [ + 8, + 11, + "ATP", + "chemical" + ], + [ + 35, + 40, + "SePSK", + "protein" + ], + [ + 45, + 51, + "AtXK-1", + "protein" + ] + ] + }, + { + "sid": 65, + "sent": "Both SePSK and AtXK-1 showed ATP hydrolysis activity in the absence of substrate.", + "section": "FIG", + "ner": [ + [ + 5, + 10, + "SePSK", + "protein" + ], + [ + 15, + 21, + "AtXK-1", + "protein" + ], + [ + 29, + 32, + "ATP", + "chemical" + ], + [ + 60, + 70, + "absence of", + "protein_state" + ] + ] + }, + { + "sid": 66, + "sent": "While the ATP hydrolysis activity of SePSK greatly increases upon addition of D-ribulose (DR).", + "section": "FIG", + "ner": [ + [ + 10, + 13, + "ATP", + "chemical" + ], + [ + 37, + 42, + "SePSK", + "protein" + ], + [ + 78, + 88, + "D-ribulose", + "chemical" + ], + [ + 90, + 92, + "DR", + "chemical" + ] + ] + }, + { + "sid": 67, + "sent": "(B) The ATP hydrolysis activity of SePSK with addition of five different substrates.", + "section": "FIG", + "ner": [ + [ + 8, + 11, + "ATP", + "chemical" + ], + [ + 35, + 40, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 68, + "sent": "The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK.", + "section": "FIG", + "ner": [ + [ + 19, + 21, + "DR", + "chemical" + ], + [ + 23, + 33, + "D-ribulose", + "chemical" + ], + [ + 36, + 38, + "LR", + "chemical" + ], + [ + 40, + 50, + "L-ribulose", + "chemical" + ], + [ + 53, + 55, + "DX", + "chemical" + ], + [ + 57, + 67, + "D-xylulose", + "chemical" + ], + [ + 70, + 72, + "LX", + "chemical" + ], + [ + 74, + 84, + "L-xylulose", + "chemical" + ], + [ + 90, + 93, + "GLY", + "chemical" + ], + [ + 95, + 103, + "Glycerol", + "chemical" + ], + [ + 114, + 117, + "ATP", + "chemical" + ], + [ + 141, + 146, + "SePSK", + "protein" + ], + [ + 151, + 157, + "AtXK-1", + "protein" + ], + [ + 174, + 184, + "D-ribulose", + "chemical" + ], + [ + 194, + 197, + "ATP", + "chemical" + ], + [ + 221, + 230, + "wild-type", + "protein_state" + ], + [ + 232, + 234, + "WT", + "protein_state" + ], + [ + 263, + 268, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 69, + "sent": "Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK.", + "section": "FIG", + "ner": [ + [ + 29, + 34, + "SePSK", + "protein" + ], + [ + 39, + 42, + "D8A", + "mutant" + ], + [ + 43, + 48, + "SePSK", + "protein" + ], + [ + 50, + 54, + "T11A", + "mutant" + ], + [ + 55, + 60, + "SePSK", + "protein" + ], + [ + 65, + 70, + "D221A", + "mutant" + ], + [ + 71, + 76, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 70, + "sent": "The ATP hydrolysis activity measured via luminescent ADP-Glo assay (Promega).", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "ATP", + "chemical" + ], + [ + 41, + 66, + "luminescent ADP-Glo assay", + "experimental_method" + ] + ] + }, + { + "sid": 71, + "sent": "To understand the catalytic mechanism of SePSK, we performed structural comparisons among xylulose kinase, glycerol kinase, ribulose kinase and SePSK.", + "section": "RESULTS", + "ner": [ + [ + 41, + 46, + "SePSK", + "protein" + ], + [ + 61, + 83, + "structural comparisons", + "experimental_method" + ], + [ + 90, + 105, + "xylulose kinase", + "protein_type" + ], + [ + 107, + 122, + "glycerol kinase", + "protein_type" + ], + [ + 124, + 139, + "ribulose kinase", + "protein_type" + ], + [ + 144, + 149, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 72, + "sent": "Our results suggested that three conserved residues (D8, T11 and D221 of SePSK) play an important role in SePSK function.", + "section": "RESULTS", + "ner": [ + [ + 53, + 55, + "D8", + "residue_name_number" + ], + [ + 57, + 60, + "T11", + "residue_name_number" + ], + [ + 65, + 69, + "D221", + "residue_name_number" + ], + [ + 73, + 78, + "SePSK", + "protein" + ], + [ + 106, + 111, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 73, + "sent": "Mutations of the corresponding residue in xylulose kinase and glycerol kinase from Escherichia coli greatly reduced their activity.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Mutations", + "experimental_method" + ], + [ + 42, + 57, + "xylulose kinase", + "protein_type" + ], + [ + 62, + 77, + "glycerol kinase", + "protein_type" + ], + [ + 83, + 99, + "Escherichia coli", + "species" + ] + ] + }, + { + "sid": 74, + "sent": "To identify the function of these three residues of SePSK, we constructed D8A, T11A and D221A mutants.", + "section": "RESULTS", + "ner": [ + [ + 52, + 57, + "SePSK", + "protein" + ], + [ + 74, + 77, + "D8A", + "mutant" + ], + [ + 79, + 83, + "T11A", + "mutant" + ], + [ + 88, + 93, + "D221A", + "mutant" + ], + [ + 94, + 101, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 75, + "sent": "Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D).", + "section": "RESULTS", + "ner": [ + [ + 6, + 31, + "enzymatic activity assays", + "experimental_method" + ], + [ + 99, + 102, + "ATP", + "chemical" + ], + [ + 127, + 137, + "D-ribulose", + "chemical" + ], + [ + 151, + 160, + "wild type", + "protein_state" + ], + [ + 256, + 271, + "phosphorylation", + "ptm" + ], + [ + 272, + 282, + "D-ribulose", + "chemical" + ], + [ + 317, + 322, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 76, + "sent": "SePSK and AtXK-1 possess a similar ATP binding site", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "SePSK", + "protein" + ], + [ + 10, + 16, + "AtXK-1", + "protein" + ], + [ + 35, + 51, + "ATP binding site", + "site" + ] + ] + }, + { + "sid": 77, + "sent": "To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 \u00c5 and 1.8 \u00c5, respectively.", + "section": "RESULTS", + "ner": [ + [ + 39, + 44, + "SePSK", + "protein" + ], + [ + 49, + 55, + "AtXK-1", + "protein" + ], + [ + 56, + 71, + "in complex with", + "protein_state" + ], + [ + 72, + 75, + "ATP", + "chemical" + ], + [ + 80, + 86, + "soaked", + "experimental_method" + ], + [ + 91, + 94, + "apo", + "protein_state" + ], + [ + 95, + 103, + "crystals", + "evidence" + ], + [ + 137, + 140, + "ATP", + "chemical" + ], + [ + 159, + 169, + "structures", + "evidence" + ], + [ + 173, + 178, + "SePSK", + "protein" + ], + [ + 183, + 189, + "AtXK-1", + "protein" + ], + [ + 190, + 200, + "bound with", + "protein_state" + ], + [ + 201, + 204, + "ATP", + "chemical" + ] + ] + }, + { + "sid": 78, + "sent": "In both structures, a strong electron density was found in the conserved ATP binding pocket, but can only be fitted with an ADP molecule (S4 Fig).", + "section": "RESULTS", + "ner": [ + [ + 8, + 18, + "structures", + "evidence" + ], + [ + 29, + 45, + "electron density", + "evidence" + ], + [ + 63, + 72, + "conserved", + "protein_state" + ], + [ + 73, + 91, + "ATP binding pocket", + "site" + ], + [ + 124, + 127, + "ADP", + "chemical" + ] + ] + }, + { + "sid": 79, + "sent": "Thus the two structures were named ADP-SePSK and ADP-AtXK-1, respectively.", + "section": "RESULTS", + "ner": [ + [ + 13, + 23, + "structures", + "evidence" + ], + [ + 35, + 44, + "ADP-SePSK", + "complex_assembly" + ], + [ + 49, + 59, + "ADP-AtXK-1", + "complex_assembly" + ] + ] + }, + { + "sid": 80, + "sent": "The extremely weak electron densities of ATP \u03b3-phosphate in both structures suggest that the \u03b3-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1.", + "section": "RESULTS", + "ner": [ + [ + 19, + 37, + "electron densities", + "evidence" + ], + [ + 47, + 56, + "phosphate", + "chemical" + ], + [ + 65, + 75, + "structures", + "evidence" + ], + [ + 95, + 104, + "phosphate", + "chemical" + ], + [ + 114, + 117, + "ATP", + "chemical" + ], + [ + 154, + 159, + "SePSK", + "protein" + ], + [ + 164, + 170, + "AtXK-1", + "protein" + ] + ] + }, + { + "sid": 81, + "sent": "This result was consistent with our enzymatic activity assays where SePSK and AtXK-1 showed ATP hydrolysis activity without adding any substrates (Fig 2A and 2C).", + "section": "RESULTS", + "ner": [ + [ + 36, + 61, + "enzymatic activity assays", + "experimental_method" + ], + [ + 68, + 73, + "SePSK", + "protein" + ], + [ + 78, + 84, + "AtXK-1", + "protein" + ], + [ + 92, + 95, + "ATP", + "chemical" + ] + ] + }, + { + "sid": 82, + "sent": "To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP.", + "section": "RESULTS", + "ner": [ + [ + 23, + 26, + "ATP", + "chemical" + ], + [ + 31, + 37, + "soaked", + "experimental_method" + ], + [ + 42, + 50, + "crystals", + "evidence" + ], + [ + 54, + 57, + "apo", + "protein_state" + ], + [ + 58, + 63, + "SePSK", + "protein" + ], + [ + 68, + 71, + "apo", + "protein_state" + ], + [ + 72, + 78, + "AtXK-1", + "protein" + ], + [ + 105, + 112, + "AMP-PNP", + "chemical" + ] + ] + }, + { + "sid": 83, + "sent": "However, we found that the electron densities of \u03b3-phosphate group of AMP-PNP (AMP-PNP \u03b3-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-\u03b3-phosphate.", + "section": "RESULTS", + "ner": [ + [ + 27, + 45, + "electron densities", + "evidence" + ], + [ + 51, + 60, + "phosphate", + "chemical" + ], + [ + 70, + 77, + "AMP-PNP", + "chemical" + ], + [ + 79, + 86, + "AMP-PNP", + "chemical" + ], + [ + 89, + 98, + "phosphate", + "chemical" + ], + [ + 122, + 135, + "AMP-PNP-SePSK", + "complex_assembly" + ], + [ + 140, + 154, + "AMP-PNP-AtXK-1", + "complex_assembly" + ], + [ + 155, + 165, + "structures", + "evidence" + ], + [ + 198, + 201, + "ATP", + "chemical" + ], + [ + 204, + 213, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 84, + "sent": "The \u03b3-phosphate group of ATP is transferred to the sugar substrate during the reaction process, so this flexibility might be important for the ability of these kinases.", + "section": "RESULTS", + "ner": [ + [ + 6, + 15, + "phosphate", + "chemical" + ], + [ + 25, + 28, + "ATP", + "chemical" + ], + [ + 51, + 56, + "sugar", + "chemical" + ], + [ + 160, + 167, + "kinases", + "protein_type" + ] + ] + }, + { + "sid": 85, + "sent": "The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members.", + "section": "RESULTS", + "ner": [ + [ + 12, + 22, + "structures", + "evidence" + ], + [ + 60, + 63, + "ADP", + "chemical" + ], + [ + 68, + 75, + "AMP-PNP", + "chemical" + ], + [ + 83, + 97, + "AMP-PNP-AtXK-1", + "complex_assembly" + ], + [ + 99, + 109, + "ADP-AtXK-1", + "complex_assembly" + ], + [ + 111, + 120, + "ADP-SePSK", + "complex_assembly" + ], + [ + 125, + 138, + "AMP-PNP-SePSK", + "complex_assembly" + ], + [ + 139, + 149, + "structures", + "evidence" + ], + [ + 195, + 204, + "structure", + "evidence" + ], + [ + 208, + 221, + "AMP-PNP-SePSK", + "complex_assembly" + ] + ] + }, + { + "sid": 86, + "sent": "As shown in Fig 3A, one SePSK protein molecule is in an asymmetric unit with one AMP-PNP molecule.", + "section": "RESULTS", + "ner": [ + [ + 24, + 29, + "SePSK", + "protein" + ], + [ + 81, + 88, + "AMP-PNP", + "chemical" + ] + ] + }, + { + "sid": 87, + "sent": "The AMP-PNP is bound at the domain II, where it fits well inside a positively charged groove.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "AMP-PNP", + "chemical" + ], + [ + 28, + 37, + "domain II", + "structure_element" + ], + [ + 67, + 92, + "positively charged groove", + "site" + ] + ] + }, + { + "sid": 88, + "sent": "The AMP-PNP binding pocket consists of four \u03b1-helices (\u03b126, \u03b128, \u03b127 and \u03b130) and forms a shape resembling a half-fist (Fig 3A and 3B).", + "section": "RESULTS", + "ner": [ + [ + 4, + 26, + "AMP-PNP binding pocket", + "site" + ], + [ + 39, + 53, + "four \u03b1-helices", + "structure_element" + ], + [ + 55, + 58, + "\u03b126", + "structure_element" + ], + [ + 60, + 63, + "\u03b128", + "structure_element" + ], + [ + 65, + 68, + "\u03b127", + "structure_element" + ], + [ + 73, + 76, + "\u03b130", + "structure_element" + ], + [ + 90, + 118, + "shape resembling a half-fist", + "protein_state" + ] + ] + }, + { + "sid": 89, + "sent": "The head group of the AMP-PNP is embedded in a pocket surrounded by Trp383, Asn380, Gly376 and Gly377.", + "section": "RESULTS", + "ner": [ + [ + 22, + 29, + "AMP-PNP", + "chemical" + ], + [ + 47, + 53, + "pocket", + "site" + ], + [ + 68, + 74, + "Trp383", + "residue_name_number" + ], + [ + 76, + 82, + "Asn380", + "residue_name_number" + ], + [ + 84, + 90, + "Gly376", + "residue_name_number" + ], + [ + 95, + 101, + "Gly377", + "residue_name_number" + ] + ] + }, + { + "sid": 90, + "sent": "The purine ring of AMP-PNP is positioned in parallel to the indole ring of Trp383.", + "section": "RESULTS", + "ner": [ + [ + 19, + 26, + "AMP-PNP", + "chemical" + ], + [ + 75, + 81, + "Trp383", + "residue_name_number" + ] + ] + }, + { + "sid": 91, + "sent": "In addition, it is hydrogen-bonded with the side chain amide of Asn380 (Fig 3B).", + "section": "RESULTS", + "ner": [ + [ + 19, + 34, + "hydrogen-bonded", + "bond_interaction" + ], + [ + 64, + 70, + "Asn380", + "residue_name_number" + ] + ] + }, + { + "sid": 92, + "sent": "The tail of AMP-PNP points to the hinge region of SePSK, and its \u03b1-phosphate and \u03b2-phosphate groups are stabilized by Gly376 and Ser243, respectively.", + "section": "RESULTS", + "ner": [ + [ + 12, + 19, + "AMP-PNP", + "chemical" + ], + [ + 34, + 46, + "hinge region", + "structure_element" + ], + [ + 50, + 55, + "SePSK", + "protein" + ], + [ + 67, + 76, + "phosphate", + "chemical" + ], + [ + 83, + 92, + "phosphate", + "chemical" + ], + [ + 118, + 124, + "Gly376", + "residue_name_number" + ], + [ + 129, + 135, + "Ser243", + "residue_name_number" + ] + ] + }, + { + "sid": 93, + "sent": "Together, this structure clearly shows that the AMP-PNP-\u03b2-phosphate is sticking out of the ATP binding pocket, thus the \u03b3-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein.", + "section": "RESULTS", + "ner": [ + [ + 15, + 24, + "structure", + "evidence" + ], + [ + 48, + 55, + "AMP-PNP", + "chemical" + ], + [ + 58, + 67, + "phosphate", + "chemical" + ], + [ + 91, + 109, + "ATP binding pocket", + "site" + ], + [ + 122, + 131, + "phosphate", + "chemical" + ], + [ + 168, + 176, + "domain I", + "structure_element" + ], + [ + 181, + 190, + "domain II", + "structure_element" + ] + ] + }, + { + "sid": 94, + "sent": "Structure of SePSK in complex with AMP-PNP.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 13, + 18, + "SePSK", + "protein" + ], + [ + 19, + 34, + "in complex with", + "protein_state" + ], + [ + 35, + 42, + "AMP-PNP", + "chemical" + ] + ] + }, + { + "sid": 95, + "sent": "(A) The electron density of AMP-PNP.", + "section": "FIG", + "ner": [ + [ + 8, + 24, + "electron density", + "evidence" + ], + [ + 28, + 35, + "AMP-PNP", + "chemical" + ] + ] + }, + { + "sid": 96, + "sent": "The SePSK structure is shown in the electrostatic potential surface mode.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "SePSK", + "protein" + ], + [ + 10, + 19, + "structure", + "evidence" + ] + ] + }, + { + "sid": 97, + "sent": "The AMP-PNP is depicted as sticks with its \u01c0Fo\u01c0-\u01c0Fc\u01c0 map contoured at 3 \u03c3 shown as cyan mesh.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "AMP-PNP", + "chemical" + ], + [ + 43, + 56, + "\u01c0Fo\u01c0-\u01c0Fc\u01c0 map", + "evidence" + ] + ] + }, + { + "sid": 98, + "sent": "(B) The AMP-PNP binding pocket.", + "section": "FIG", + "ner": [ + [ + 8, + 30, + "AMP-PNP binding pocket", + "site" + ] + ] + }, + { + "sid": 99, + "sent": "The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383).", + "section": "FIG", + "ner": [ + [ + 12, + 19, + "AMP-PNP", + "chemical" + ], + [ + 23, + 36, + "sandwiched by", + "bond_interaction" + ], + [ + 52, + 58, + "Leu293", + "residue_name_number" + ], + [ + 60, + 66, + "Gly376", + "residue_name_number" + ], + [ + 68, + 74, + "Gly377", + "residue_name_number" + ], + [ + 79, + 85, + "Trp383", + "residue_name_number" + ] + ] + }, + { + "sid": 100, + "sent": "The four \u03b1-helices (\u03b126, \u03b128, \u03b127 and \u03b130) are labeled in red.", + "section": "FIG", + "ner": [ + [ + 9, + 18, + "\u03b1-helices", + "structure_element" + ], + [ + 20, + 23, + "\u03b126", + "structure_element" + ], + [ + 25, + 28, + "\u03b128", + "structure_element" + ], + [ + 30, + 33, + "\u03b127", + "structure_element" + ], + [ + 38, + 41, + "\u03b130", + "structure_element" + ] + ] + }, + { + "sid": 101, + "sent": "The AMP-PNP and coordinated residues are shown as sticks.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "AMP-PNP", + "chemical" + ] + ] + }, + { + "sid": 102, + "sent": "The potential substrate binding site in SePSK", + "section": "RESULTS", + "ner": [ + [ + 14, + 36, + "substrate binding site", + "site" + ], + [ + 40, + 45, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 103, + "sent": "The results from our activity assays suggested that SePSK has D-ribulose kinase activity.", + "section": "RESULTS", + "ner": [ + [ + 21, + 36, + "activity assays", + "experimental_method" + ], + [ + 52, + 57, + "SePSK", + "protein" + ], + [ + 62, + 79, + "D-ribulose kinase", + "protein_type" + ] + ] + }, + { + "sid": 104, + "sent": "To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved.", + "section": "RESULTS", + "ner": [ + [ + 53, + 58, + "SePSK", + "protein" + ], + [ + 63, + 73, + "D-ribulose", + "chemical" + ], + [ + 79, + 82, + "apo", + "protein_state" + ], + [ + 83, + 88, + "SePSK", + "protein" + ], + [ + 89, + 114, + "crystals were soaked into", + "experimental_method" + ], + [ + 119, + 128, + "reservoir", + "experimental_method" + ], + [ + 140, + 150, + "D-ribulose", + "chemical" + ], + [ + 152, + 155, + "RBL", + "chemical" + ], + [ + 165, + 174, + "RBL-SePSK", + "complex_assembly" + ], + [ + 175, + 184, + "structure", + "evidence" + ], + [ + 189, + 195, + "solved", + "experimental_method" + ] + ] + }, + { + "sid": 105, + "sent": "As shown in S6 Fig, two residual electron densities are visible in domain I, which can be interpreted as two D-ribulose molecules with reasonable fit.", + "section": "RESULTS", + "ner": [ + [ + 33, + 51, + "electron densities", + "evidence" + ], + [ + 67, + 75, + "domain I", + "structure_element" + ], + [ + 109, + 119, + "D-ribulose", + "chemical" + ] + ] + }, + { + "sid": 106, + "sent": "As shown in Fig 4A, the nearest distance between the carbon skeleton of two D-ribulose molecules are approx.", + "section": "RESULTS", + "ner": [ + [ + 76, + 86, + "D-ribulose", + "chemical" + ] + ] + }, + { + "sid": 107, + "sent": "7.1 \u00c5 (RBL1-C4 and RBL2-C1).", + "section": "RESULTS", + "ner": [ + [ + 7, + 11, + "RBL1", + "residue_name_number" + ], + [ + 19, + 23, + "RBL2", + "residue_name_number" + ] + ] + }, + { + "sid": 108, + "sent": "RBL1 is located in the pocket consisting of \u03b121 and the loop between \u03b26 and \u03b27.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "RBL1", + "residue_name_number" + ], + [ + 23, + 29, + "pocket", + "site" + ], + [ + 44, + 47, + "\u03b121", + "structure_element" + ], + [ + 56, + 60, + "loop", + "structure_element" + ], + [ + 69, + 78, + "\u03b26 and \u03b27", + "structure_element" + ] + ] + }, + { + "sid": 109, + "sent": "The O4 and O5 of RBL1 are coordinated with the side chain carboxyl group of Asp221.", + "section": "RESULTS", + "ner": [ + [ + 17, + 21, + "RBL1", + "residue_name_number" + ], + [ + 26, + 42, + "coordinated with", + "bond_interaction" + ], + [ + 76, + 82, + "Asp221", + "residue_name_number" + ] + ] + }, + { + "sid": 110, + "sent": "Furthermore, the O2 of RBL1 interacts with the main chain amide nitrogen of Ser72 (Fig 4B).", + "section": "RESULTS", + "ner": [ + [ + 23, + 27, + "RBL1", + "residue_name_number" + ], + [ + 28, + 42, + "interacts with", + "bond_interaction" + ], + [ + 76, + 81, + "Ser72", + "residue_name_number" + ] + ] + }, + { + "sid": 111, + "sent": "This pocket is at a similar position of substrate binding site of other sugar kinase, such as L-ribulokinase (PDB code: 3QDK) (S7 Fig).", + "section": "RESULTS", + "ner": [ + [ + 5, + 11, + "pocket", + "site" + ], + [ + 40, + 62, + "substrate binding site", + "site" + ], + [ + 72, + 84, + "sugar kinase", + "protein_type" + ], + [ + 94, + 108, + "L-ribulokinase", + "protein" + ] + ] + }, + { + "sid": 112, + "sent": "However, structural comparison shows that the substrate ligating residues between the two structures are not strictly conserved.", + "section": "RESULTS", + "ner": [ + [ + 9, + 30, + "structural comparison", + "experimental_method" + ], + [ + 90, + 100, + "structures", + "evidence" + ], + [ + 105, + 127, + "not strictly conserved", + "protein_state" + ] + ] + }, + { + "sid": 113, + "sent": "Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig).", + "section": "RESULTS", + "ner": [ + [ + 13, + 23, + "structures", + "evidence" + ], + [ + 50, + 54, + "RBL1", + "residue_name_number" + ], + [ + 58, + 67, + "RBL-SePSK", + "complex_assembly" + ], + [ + 68, + 77, + "structure", + "evidence" + ], + [ + 82, + 87, + "Ser72", + "residue_name_number" + ], + [ + 89, + 95, + "Asp221", + "residue_name_number" + ], + [ + 100, + 106, + "Ser222", + "residue_name_number" + ], + [ + 140, + 150, + "L-ribulose", + "chemical" + ], + [ + 156, + 170, + "L-ribulokinase", + "protein" + ], + [ + 175, + 180, + "Ala96", + "residue_name_number" + ], + [ + 182, + 188, + "Lys208", + "residue_name_number" + ], + [ + 190, + 196, + "Asp274", + "residue_name_number" + ], + [ + 201, + 207, + "Glu329", + "residue_name_number" + ] + ] + }, + { + "sid": 114, + "sent": "Glu329 in 3QDK has no counterpart in RBL-SePSK structure.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Glu329", + "residue_name_number" + ], + [ + 37, + 46, + "RBL-SePSK", + "complex_assembly" + ], + [ + 47, + 56, + "structure", + "evidence" + ] + ] + }, + { + "sid": 115, + "sent": "In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two \u03b1-helices (\u03b19 and \u03b113) of SePSK.", + "section": "RESULTS", + "ner": [ + [ + 22, + 28, + "Lys208", + "residue_name_number" + ], + [ + 32, + 46, + "L-ribulokinase", + "protein" + ], + [ + 78, + 84, + "Lys163", + "residue_name_number" + ], + [ + 89, + 98, + "RBL-SePSK", + "complex_assembly" + ], + [ + 99, + 108, + "structure", + "evidence" + ], + [ + 114, + 127, + "hydrogen bond", + "bond_interaction" + ], + [ + 131, + 137, + "Lys163", + "residue_name_number" + ], + [ + 192, + 201, + "\u03b1-helices", + "structure_element" + ], + [ + 203, + 205, + "\u03b19", + "structure_element" + ], + [ + 210, + 213, + "\u03b113", + "structure_element" + ], + [ + 218, + 223, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 116, + "sent": "The binding of D-ribulose (RBL) with SePSK.", + "section": "FIG", + "ner": [ + [ + 15, + 25, + "D-ribulose", + "chemical" + ], + [ + 27, + 30, + "RBL", + "chemical" + ], + [ + 37, + 42, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 117, + "sent": "(A) The electrostatic potential surface map of RBL-SePSK and a zoom-in view of RBL binding site.", + "section": "FIG", + "ner": [ + [ + 8, + 43, + "electrostatic potential surface map", + "evidence" + ], + [ + 47, + 56, + "RBL-SePSK", + "complex_assembly" + ], + [ + 79, + 95, + "RBL binding site", + "site" + ] + ] + }, + { + "sid": 118, + "sent": "The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "RBL1", + "residue_name_number" + ], + [ + 13, + 17, + "RBL2", + "residue_name_number" + ], + [ + 65, + 75, + "D-ribulose", + "chemical" + ], + [ + 87, + 91, + "RBL1", + "residue_name_number" + ], + [ + 96, + 100, + "RBL2", + "residue_name_number" + ], + [ + 107, + 112, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 119, + "sent": "The RBL molecules (carbon atoms colored yellow) and amino acid residues of SePSK (carbon atoms colored green) involved in RBL interaction are shown as sticks.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "RBL", + "chemical" + ], + [ + 75, + 80, + "SePSK", + "protein" + ], + [ + 122, + 125, + "RBL", + "chemical" + ] + ] + }, + { + "sid": 120, + "sent": "The hydrogen bonds are indicated by the black dashed lines and the numbers near the dashed lines are the distances (\u00c5). (C) The binding affinity assays of SePSK with D-ribulose.", + "section": "FIG", + "ner": [ + [ + 4, + 18, + "hydrogen bonds", + "bond_interaction" + ], + [ + 128, + 151, + "binding affinity assays", + "experimental_method" + ], + [ + 155, + 160, + "SePSK", + "protein" + ], + [ + 166, + 176, + "D-ribulose", + "chemical" + ] + ] + }, + { + "sid": 121, + "sent": "Single-cycle kinetic data are reflecting the interaction of SePSK and D8A-SePSK with D-ribulose.", + "section": "FIG", + "ner": [ + [ + 0, + 25, + "Single-cycle kinetic data", + "experimental_method" + ], + [ + 60, + 65, + "SePSK", + "protein" + ], + [ + 70, + 73, + "D8A", + "mutant" + ], + [ + 74, + 79, + "SePSK", + "protein" + ], + [ + 85, + 95, + "D-ribulose", + "chemical" + ] + ] + }, + { + "sid": 122, + "sent": "It shows two experimental sensorgrams after minus the empty sensorgrams.", + "section": "FIG", + "ner": [ + [ + 26, + 37, + "sensorgrams", + "evidence" + ], + [ + 60, + 71, + "sensorgrams", + "evidence" + ] + ] + }, + { + "sid": 123, + "sent": "The original data is shown as black curve, and the fitted data is shown as different color (wild type SePSK: red curve, D8A-SePSK: green curve).", + "section": "FIG", + "ner": [ + [ + 92, + 101, + "wild type", + "protein_state" + ], + [ + 102, + 107, + "SePSK", + "protein" + ], + [ + 120, + 123, + "D8A", + "mutant" + ], + [ + 124, + 129, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 124, + "sent": "Dissociation rate constant of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively.", + "section": "FIG", + "ner": [ + [ + 0, + 26, + "Dissociation rate constant", + "evidence" + ], + [ + 30, + 39, + "wild type", + "protein_state" + ], + [ + 44, + 47, + "D8A", + "mutant" + ], + [ + 48, + 53, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 125, + "sent": "The binding pocket of RBL2 with relatively weak electron density is near the N-terminal region of SePSK and is negatively charged.", + "section": "RESULTS", + "ner": [ + [ + 4, + 18, + "binding pocket", + "site" + ], + [ + 22, + 26, + "RBL2", + "residue_name_number" + ], + [ + 48, + 64, + "electron density", + "evidence" + ], + [ + 98, + 103, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 126, + "sent": "The side chain of Asp8 interacts strongly with O3 and O4 of RBL2.", + "section": "RESULTS", + "ner": [ + [ + 18, + 22, + "Asp8", + "residue_name_number" + ], + [ + 23, + 46, + "interacts strongly with", + "bond_interaction" + ], + [ + 60, + 64, + "RBL2", + "residue_name_number" + ] + ] + }, + { + "sid": 127, + "sent": "The hydroxyl group of Ser12 coordinates with O2 of RBL2.", + "section": "RESULTS", + "ner": [ + [ + 22, + 27, + "Ser12", + "residue_name_number" + ], + [ + 28, + 44, + "coordinates with", + "bond_interaction" + ], + [ + 51, + 55, + "RBL2", + "residue_name_number" + ] + ] + }, + { + "sid": 128, + "sent": "The backbone amide nitrogens of Gly13 and Arg15 also keep hydrogen bonds with RBL2 (Fig 4B).", + "section": "RESULTS", + "ner": [ + [ + 32, + 37, + "Gly13", + "residue_name_number" + ], + [ + 42, + 47, + "Arg15", + "residue_name_number" + ], + [ + 58, + 72, + "hydrogen bonds", + "bond_interaction" + ], + [ + 78, + 82, + "RBL2", + "residue_name_number" + ] + ] + }, + { + "sid": 129, + "sent": "Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins.", + "section": "RESULTS", + "ner": [ + [ + 0, + 21, + "Structural comparison", + "experimental_method" + ], + [ + 25, + 30, + "SePSK", + "protein" + ], + [ + 35, + 41, + "AtXK-1", + "protein" + ], + [ + 64, + 83, + "RBL1 binding pocket", + "site" + ], + [ + 87, + 96, + "conserved", + "protein_state" + ], + [ + 102, + 113, + "RBL2 pocket", + "site" + ], + [ + 130, + 136, + "AtXK-1", + "protein" + ], + [ + 137, + 146, + "structure", + "evidence" + ], + [ + 200, + 204, + "RBL2", + "residue_name_number" + ], + [ + 209, + 225, + "highly conserved", + "protein_state" + ] + ] + }, + { + "sid": 130, + "sent": "In the RBL-SePSK structure, a 2.6 \u00c5 hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken.", + "section": "RESULTS", + "ner": [ + [ + 7, + 16, + "RBL-SePSK", + "complex_assembly" + ], + [ + 17, + 26, + "structure", + "evidence" + ], + [ + 36, + 49, + "hydrogen bond", + "bond_interaction" + ], + [ + 69, + 73, + "RBL2", + "residue_name_number" + ], + [ + 78, + 83, + "Ser12", + "residue_name_number" + ], + [ + 107, + 113, + "AtXK-1", + "protein" + ], + [ + 114, + 123, + "structure", + "evidence" + ], + [ + 129, + 142, + "hydrogen bond", + "bond_interaction" + ], + [ + 175, + 180, + "Ser22", + "residue_name_number" + ] + ] + }, + { + "sid": 131, + "sent": "This break is probably induced by the conformational change of the two \u03b2-sheets (\u03b21 and \u03b22), with the result that the linking loop (loop 1) is located further away from the RBL2 binding site.", + "section": "RESULTS", + "ner": [ + [ + 71, + 79, + "\u03b2-sheets", + "structure_element" + ], + [ + 81, + 83, + "\u03b21", + "structure_element" + ], + [ + 88, + 90, + "\u03b22", + "structure_element" + ], + [ + 118, + 130, + "linking loop", + "structure_element" + ], + [ + 132, + 138, + "loop 1", + "structure_element" + ], + [ + 173, + 190, + "RBL2 binding site", + "site" + ] + ] + }, + { + "sid": 132, + "sent": "This change might be the reason that AtXK-1 only shows limited increasing in its ATP hydrolysis ability upon adding D-ribulose as a substrate after comparing with SePSK (Fig 2C).", + "section": "RESULTS", + "ner": [ + [ + 37, + 43, + "AtXK-1", + "protein" + ], + [ + 81, + 84, + "ATP", + "chemical" + ], + [ + 116, + 126, + "D-ribulose", + "chemical" + ], + [ + 163, + 168, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 133, + "sent": "Our SePSK structure shows that the Asp8 residue forms strong hydrogen bond with RBL2 (Fig 4B).", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "SePSK", + "protein" + ], + [ + 10, + 19, + "structure", + "evidence" + ], + [ + 35, + 39, + "Asp8", + "residue_name_number" + ], + [ + 61, + 74, + "hydrogen bond", + "bond_interaction" + ], + [ + 80, + 84, + "RBL2", + "residue_name_number" + ] + ] + }, + { + "sid": 134, + "sent": "In addition, our enzymatic assays indicated that Asp8 is important for the activity of SePSK (Fig 2D).", + "section": "RESULTS", + "ner": [ + [ + 17, + 33, + "enzymatic assays", + "experimental_method" + ], + [ + 49, + 53, + "Asp8", + "residue_name_number" + ], + [ + 87, + 92, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 135, + "sent": "To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method.", + "section": "RESULTS", + "ner": [ + [ + 49, + 65, + "binding affinity", + "evidence" + ], + [ + 70, + 80, + "D-ribulose", + "chemical" + ], + [ + 89, + 98, + "wild type", + "protein_state" + ], + [ + 100, + 102, + "WT", + "protein_state" + ], + [ + 108, + 111, + "D8A", + "mutant" + ], + [ + 112, + 118, + "mutant", + "protein_state" + ], + [ + 122, + 127, + "SePSK", + "protein" + ], + [ + 136, + 168, + "surface plasmon resonance method", + "experimental_method" + ] + ] + }, + { + "sid": 136, + "sent": "The results showed that the affinity of D8A-SePSK with D-ribulose is weaker than that of WT with a reduction of approx.", + "section": "RESULTS", + "ner": [ + [ + 28, + 36, + "affinity", + "evidence" + ], + [ + 40, + 43, + "D8A", + "mutant" + ], + [ + 44, + 49, + "SePSK", + "protein" + ], + [ + 55, + 65, + "D-ribulose", + "chemical" + ], + [ + 89, + 91, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 137, + "sent": "Dissociation rate constant (Kd) of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively.", + "section": "RESULTS", + "ner": [ + [ + 0, + 26, + "Dissociation rate constant", + "evidence" + ], + [ + 28, + 30, + "Kd", + "evidence" + ], + [ + 35, + 44, + "wild type", + "protein_state" + ], + [ + 49, + 52, + "D8A", + "mutant" + ], + [ + 53, + 58, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 138, + "sent": "The results implied that the second RBL binding site plays a role in the D-ribulose kinase function of SePSK.", + "section": "RESULTS", + "ner": [ + [ + 29, + 52, + "second RBL binding site", + "site" + ], + [ + 73, + 90, + "D-ribulose kinase", + "protein_type" + ], + [ + 103, + 108, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 139, + "sent": "However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact.", + "section": "RESULTS", + "ner": [ + [ + 47, + 57, + "D-ribulose", + "chemical" + ], + [ + 67, + 82, + "crystal soaking", + "experimental_method" + ], + [ + 115, + 131, + "electron density", + "evidence" + ], + [ + 135, + 139, + "RBL2", + "residue_name_number" + ], + [ + 170, + 189, + "second binding site", + "site" + ], + [ + 193, + 203, + "D-ribulose", + "chemical" + ], + [ + 207, + 212, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 140, + "sent": "Simulated conformational change of SePSK during the catalytic process", + "section": "RESULTS", + "ner": [ + [ + 35, + 40, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 141, + "sent": "It was reported earlier that the crossing angle between the domain I and domain II in FGGY family carbohydrate kinases is different.", + "section": "RESULTS", + "ner": [ + [ + 60, + 68, + "domain I", + "structure_element" + ], + [ + 73, + 82, + "domain II", + "structure_element" + ], + [ + 86, + 118, + "FGGY family carbohydrate kinases", + "protein_type" + ] + ] + }, + { + "sid": 142, + "sent": "In addition, this difference may be caused by the binding of substrates and/or ATP.", + "section": "RESULTS", + "ner": [ + [ + 79, + 82, + "ATP", + "chemical" + ] + ] + }, + { + "sid": 143, + "sent": "As reported previously, members of the sugar kinase family undergo a conformational change to narrow the crossing angle between two domains and reduce the distance between substrate and ATP in order to facilitate the catalytic reaction of phosphorylation of sugar substrates.", + "section": "RESULTS", + "ner": [ + [ + 39, + 51, + "sugar kinase", + "protein_type" + ], + [ + 186, + 189, + "ATP", + "chemical" + ], + [ + 239, + 254, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 144, + "sent": "After comparing structures of apo-SePSK, RBL-SePSK and AMP-PNP-SePSK, we noticed that these structures presented here are similar.", + "section": "RESULTS", + "ner": [ + [ + 16, + 26, + "structures", + "evidence" + ], + [ + 30, + 33, + "apo", + "protein_state" + ], + [ + 34, + 39, + "SePSK", + "protein" + ], + [ + 41, + 50, + "RBL-SePSK", + "complex_assembly" + ], + [ + 55, + 68, + "AMP-PNP-SePSK", + "complex_assembly" + ], + [ + 92, + 102, + "structures", + "evidence" + ] + ] + }, + { + "sid": 145, + "sent": "Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP \u03b3-phosphate and RBL1/RBL2 is 7.5 \u00c5 (RBL1-O5)/6.7 \u00c5 (RBL2-O1) (S8 Fig).", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Superposing", + "experimental_method" + ], + [ + 16, + 26, + "structures", + "evidence" + ], + [ + 30, + 39, + "RBL-SePSK", + "complex_assembly" + ], + [ + 44, + 57, + "AMP-PNP-SePSK", + "complex_assembly" + ], + [ + 110, + 117, + "AMP-PNP", + "chemical" + ], + [ + 120, + 129, + "phosphate", + "chemical" + ], + [ + 134, + 138, + "RBL1", + "residue_name_number" + ], + [ + 139, + 143, + "RBL2", + "residue_name_number" + ], + [ + 154, + 158, + "RBL1", + "residue_name_number" + ], + [ + 170, + 174, + "RBL2", + "residue_name_number" + ] + ] + }, + { + "sid": 146, + "sent": "This distance is too long to transfer the \u03b3-phosphate group from ATP to the substrate.", + "section": "RESULTS", + "ner": [ + [ + 44, + 53, + "phosphate", + "chemical" + ], + [ + 65, + 68, + "ATP", + "chemical" + ] + ] + }, + { + "sid": 147, + "sent": "Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates.", + "section": "RESULTS", + "ner": [ + [ + 25, + 30, + "SePSK", + "protein" + ], + [ + 60, + 69, + "structure", + "evidence" + ], + [ + 95, + 105, + "structures", + "evidence" + ], + [ + 109, + 114, + "SePSK", + "protein" + ], + [ + 129, + 133, + "open", + "protein_state" + ], + [ + 208, + 214, + "closed", + "protein_state" + ], + [ + 269, + 284, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 148, + "sent": "For studying such potential conformational change, a simulation on the Hingeprot Server was performed to predict the movement of different SePSK domains.", + "section": "RESULTS", + "ner": [ + [ + 53, + 63, + "simulation", + "experimental_method" + ], + [ + 71, + 87, + "Hingeprot Server", + "experimental_method" + ], + [ + 139, + 144, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 149, + "sent": "The results showed that domain I and domain II are closer to each other with Ala228 and Thr401 in A2 as Hinge-residues.", + "section": "RESULTS", + "ner": [ + [ + 24, + 32, + "domain I", + "structure_element" + ], + [ + 37, + 46, + "domain II", + "structure_element" + ], + [ + 77, + 83, + "Ala228", + "residue_name_number" + ], + [ + 88, + 94, + "Thr401", + "residue_name_number" + ], + [ + 98, + 100, + "A2", + "structure_element" + ], + [ + 104, + 118, + "Hinge-residues", + "structure_element" + ] + ] + }, + { + "sid": 150, + "sent": "Based on the above results, SePSK is divided into two rigid parts.", + "section": "RESULTS", + "ner": [ + [ + 28, + 33, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 151, + "sent": "The domain I of RBL-SePSK (aa. 1\u2013228, aa. 402\u2013421) and the domain II of AMP-PNP-SePSK (aa. 229\u2013401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ).", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "domain I", + "structure_element" + ], + [ + 16, + 25, + "RBL-SePSK", + "complex_assembly" + ], + [ + 31, + 36, + "1\u2013228", + "residue_range" + ], + [ + 42, + 49, + "402\u2013421", + "residue_range" + ], + [ + 59, + 68, + "domain II", + "structure_element" + ], + [ + 72, + 85, + "AMP-PNP-SePSK", + "complex_assembly" + ], + [ + 91, + 98, + "229\u2013401", + "residue_range" + ], + [ + 105, + 115, + "superposed", + "experimental_method" + ], + [ + 121, + 131, + "structures", + "evidence" + ], + [ + 143, + 146, + "apo", + "protein_state" + ], + [ + 147, + 153, + "AtXK-1", + "protein" + ], + [ + 155, + 158, + "apo", + "protein_state" + ], + [ + 159, + 164, + "SePSK", + "protein" + ], + [ + 166, + 181, + "xylulose kinase", + "protein_type" + ], + [ + 187, + 212, + "Lactobacillus acidophilus", + "species" + ], + [ + 238, + 242, + "S58W", + "mutant" + ], + [ + 243, + 249, + "mutant", + "protein_state" + ], + [ + 258, + 273, + "glycerol kinase", + "protein_type" + ], + [ + 279, + 295, + "Escherichia coli", + "species" + ] + ] + }, + { + "sid": 152, + "sent": "The results of superposition displayed different crossing angle between these two domains.", + "section": "RESULTS", + "ner": [ + [ + 15, + 28, + "superposition", + "experimental_method" + ] + ] + }, + { + "sid": 153, + "sent": "After superposition, the distances of AMP-PNP \u03b3-phosphate and the fifth hydroxyl group of RBL1 are 7.9 \u00c5 (superposed with AtXK-1), 7.4 \u00c5 (superposed with SePSK), 6.6 \u00c5 (superposed with 3LL3) and 6.1 \u00c5 (superposed with 1GLJ).", + "section": "RESULTS", + "ner": [ + [ + 6, + 19, + "superposition", + "experimental_method" + ], + [ + 38, + 45, + "AMP-PNP", + "chemical" + ], + [ + 48, + 57, + "phosphate", + "chemical" + ], + [ + 90, + 94, + "RBL1", + "residue_name_number" + ], + [ + 106, + 116, + "superposed", + "experimental_method" + ], + [ + 122, + 128, + "AtXK-1", + "protein" + ], + [ + 138, + 148, + "superposed", + "experimental_method" + ], + [ + 154, + 159, + "SePSK", + "protein" + ], + [ + 169, + 179, + "superposed", + "experimental_method" + ], + [ + 202, + 212, + "superposed", + "experimental_method" + ] + ] + }, + { + "sid": 154, + "sent": "Meanwhile, the distances of AMP-PNP \u03b3-phosphate and the first hydroxyl group of RBL2 are 7.2 \u00c5 (superposed with AtXK-1), 6.7 \u00c5 (superposed with SePSK), 3.7 \u00c5 (superposed with 3LL3), until AMP-PNP \u03b3-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5).", + "section": "RESULTS", + "ner": [ + [ + 28, + 35, + "AMP-PNP", + "chemical" + ], + [ + 38, + 47, + "phosphate", + "chemical" + ], + [ + 80, + 84, + "RBL2", + "residue_name_number" + ], + [ + 96, + 106, + "superposed", + "experimental_method" + ], + [ + 112, + 118, + "AtXK-1", + "protein" + ], + [ + 128, + 138, + "superposed", + "experimental_method" + ], + [ + 144, + 149, + "SePSK", + "protein" + ], + [ + 159, + 169, + "superposed", + "experimental_method" + ], + [ + 188, + 195, + "AMP-PNP", + "chemical" + ], + [ + 198, + 207, + "phosphate", + "chemical" + ], + [ + 223, + 227, + "RBL2", + "residue_name_number" + ], + [ + 234, + 247, + "superposition", + "experimental_method" + ] + ] + }, + { + "sid": 155, + "sent": "This distance between RBL2 and AMP-PNP-\u03b3-phosphate is close enough to facilitate phosphate transferring.", + "section": "RESULTS", + "ner": [ + [ + 22, + 26, + "RBL2", + "residue_name_number" + ], + [ + 31, + 38, + "AMP-PNP", + "chemical" + ], + [ + 41, + 50, + "phosphate", + "chemical" + ], + [ + 81, + 90, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 156, + "sent": "Together, our superposition results provided snapshots of the conformational changes at different catalytic stages of SePSK and potentially revealed the closed form of SePSK.", + "section": "RESULTS", + "ner": [ + [ + 14, + 27, + "superposition", + "experimental_method" + ], + [ + 118, + 123, + "SePSK", + "protein" + ], + [ + 153, + 159, + "closed", + "protein_state" + ], + [ + 168, + 173, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 157, + "sent": "Simulated conformational change of SePSK during the catalytic process.", + "section": "FIG", + "ner": [ + [ + 35, + 40, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 158, + "sent": "The structures are shown as cartoon and the ligands are shown as sticks.", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ] + ] + }, + { + "sid": 159, + "sent": "Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively.", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "Domain I", + "structure_element" + ], + [ + 14, + 30, + "D-ribulose-SePSK", + "complex_assembly" + ], + [ + 43, + 52, + "Domain II", + "structure_element" + ], + [ + 58, + 71, + "AMP-PNP-SePSK", + "complex_assembly" + ], + [ + 83, + 93, + "superposed", + "experimental_method" + ], + [ + 99, + 102, + "apo", + "protein_state" + ], + [ + 103, + 109, + "AtXK-1", + "protein" + ], + [ + 117, + 120, + "apo", + "protein_state" + ], + [ + 121, + 126, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 160, + "sent": "The numbers near the black dashed lines show the distances (\u00c5) between two nearest atoms of RBL and AMP-PNP.", + "section": "FIG", + "ner": [ + [ + 92, + 95, + "RBL", + "chemical" + ], + [ + 100, + 107, + "AMP-PNP", + "chemical" + ] + ] + }, + { + "sid": 161, + "sent": "In summary, our structural and enzymatic analyses provide evidence that SePSK shows D-ribulose kinase activity, and exhibits the conserved features of FGGY family carbohydrate kinases.", + "section": "RESULTS", + "ner": [ + [ + 16, + 49, + "structural and enzymatic analyses", + "experimental_method" + ], + [ + 72, + 77, + "SePSK", + "protein" + ], + [ + 84, + 101, + "D-ribulose kinase", + "protein_type" + ], + [ + 151, + 183, + "FGGY family carbohydrate kinases", + "protein_type" + ] + ] + }, + { + "sid": 162, + "sent": "Three conserved residues in SePSK were identified to be essential for this function.", + "section": "RESULTS", + "ner": [ + [ + 6, + 15, + "conserved", + "site" + ], + [ + 28, + 33, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 163, + "sent": "Our results provide the detailed information about the interaction of SePSK with ATP and substrates.", + "section": "RESULTS", + "ner": [ + [ + 70, + 75, + "SePSK", + "protein" + ], + [ + 81, + 84, + "ATP", + "chemical" + ] + ] + }, + { + "sid": 164, + "sent": "Moreover, structural superposition results enable us to visualize the conformational change of SePSK during the catalytic process.", + "section": "RESULTS", + "ner": [ + [ + 10, + 34, + "structural superposition", + "experimental_method" + ], + [ + 95, + 100, + "SePSK", + "protein" + ] + ] + }, + { + "sid": 165, + "sent": "In conclusion, our results provide important information for a more detailed understanding of the mechanisms of SePSK and other members of FGGY family carbohydrate kinases.", + "section": "RESULTS", + "ner": [ + [ + 112, + 117, + "SePSK", + "protein" + ], + [ + 139, + 171, + "FGGY family carbohydrate kinases", + "protein_type" + ] + ] + } + ] + }, + "PMC4832331": { + "annotations": [ + { + "sid": 0, + "sent": "Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA", + "section": "TITLE", + "ner": [ + [ + 29, + 45, + "Escherichia coli", + "species" + ], + [ + 46, + 67, + "lysine decarboxylases", + "protein_type" + ], + [ + 119, + 130, + "AAA+ ATPase", + "protein_type" + ], + [ + 131, + 135, + "RavA", + "protein" + ] + ] + }, + { + "sid": 1, + "sent": "The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 13, + "inducible", + "protein_state" + ], + [ + 14, + 34, + "lysine decarboxylase", + "protein_type" + ], + [ + 35, + 39, + "LdcI", + "protein" + ], + [ + 56, + 71, + "enterobacterial", + "taxonomy_domain" + ], + [ + 72, + 99, + "acid stress response enzyme", + "protein_type" + ], + [ + 108, + 112, + "LdcC", + "protein" + ] + ] + }, + { + "sid": 2, + "sent": "A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation.", + "section": "ABSTRACT", + "ner": [ + [ + 43, + 51, + "decamers", + "oligomeric_state" + ], + [ + 59, + 75, + "Escherichia coli", + "species" + ], + [ + 76, + 80, + "LdcI", + "protein" + ], + [ + 90, + 98, + "hexamers", + "oligomeric_state" + ], + [ + 106, + 117, + "AAA+ ATPase", + "protein_type" + ], + [ + 118, + 122, + "RavA", + "protein" + ] + ] + }, + { + "sid": 3, + "sent": "Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer.", + "section": "ABSTRACT", + "ner": [ + [ + 26, + 44, + "pseudoatomic model", + "evidence" + ], + [ + 52, + 61, + "LdcI-RavA", + "complex_assembly" + ], + [ + 80, + 104, + "cryo-electron microscopy", + "experimental_method" + ], + [ + 105, + 108, + "map", + "evidence" + ], + [ + 113, + 131, + "crystal structures", + "evidence" + ], + [ + 138, + 146, + "inactive", + "protein_state" + ], + [ + 147, + 151, + "LdcI", + "protein" + ], + [ + 152, + 159, + "decamer", + "oligomeric_state" + ], + [ + 166, + 170, + "RavA", + "protein" + ], + [ + 171, + 178, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 4, + "sent": "We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity.", + "section": "ABSTRACT", + "ner": [ + [ + 15, + 39, + "cryo-electron microscopy", + "experimental_method" + ], + [ + 40, + 58, + "3D reconstructions", + "evidence" + ], + [ + 66, + 73, + "E. coli", + "species" + ], + [ + 74, + 78, + "LdcI", + "protein" + ], + [ + 83, + 87, + "LdcC", + "protein" + ], + [ + 96, + 108, + "improved map", + "evidence" + ], + [ + 116, + 120, + "LdcI", + "protein" + ], + [ + 121, + 129, + "bound to", + "protein_state" + ], + [ + 134, + 145, + "LARA domain", + "structure_element" + ], + [ + 149, + 153, + "RavA", + "protein" + ], + [ + 158, + 168, + "pH optimal", + "protein_state" + ] + ] + }, + { + "sid": 5, + "sent": "Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 10, + "Comparison", + "experimental_method" + ], + [ + 46, + 56, + "structures", + "evidence" + ], + [ + 86, + 90, + "LdcI", + "protein" + ], + [ + 95, + 99, + "LdcC", + "protein" + ], + [ + 124, + 151, + "acid stress response enzyme", + "protein_type" + ], + [ + 174, + 178, + "RavA", + "protein" + ], + [ + 234, + 246, + "pH-dependent", + "protein_state" + ], + [ + 278, + 282, + "RavA", + "protein" + ] + ] + }, + { + "sid": 6, + "sent": "Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 27, + "Multiple sequence alignment", + "experimental_method" + ], + [ + 41, + 62, + "phylogenetic analysis", + "experimental_method" + ], + [ + 84, + 98, + "enterobacteria", + "taxonomy_domain" + ], + [ + 134, + 154, + "lysine decarboxylase", + "protein_type" + ], + [ + 191, + 195, + "RavA", + "protein" + ] + ] + }, + { + "sid": 7, + "sent": "Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the \u03b1-family of pyridoxal-5\u2032-phosphate (PLP)-dependent enzymes.", + "section": "INTRO", + "ner": [ + [ + 0, + 15, + "Enterobacterial", + "taxonomy_domain" + ], + [ + 16, + 25, + "inducible", + "protein_state" + ], + [ + 26, + 40, + "decarboxylases", + "protein_type" + ], + [ + 44, + 49, + "basic", + "protein_state" + ], + [ + 50, + 61, + "amino acids", + "chemical" + ], + [ + 62, + 68, + "lysine", + "residue_name" + ], + [ + 70, + 78, + "arginine", + "residue_name" + ], + [ + 83, + 92, + "ornithine", + "residue_name" + ], + [ + 145, + 153, + "\u03b1-family", + "protein_type" + ], + [ + 157, + 179, + "pyridoxal-5\u2032-phosphate", + "chemical" + ], + [ + 181, + 184, + "PLP", + "chemical" + ] + ] + }, + { + "sid": 8, + "sent": "They counteract acid stress experienced by the bacterium in the host digestive and urinary tract, and in particular in the extremely acidic stomach.", + "section": "INTRO", + "ner": [ + [ + 47, + 56, + "bacterium", + "taxonomy_domain" + ] + ] + }, + { + "sid": 9, + "sent": "Each decarboxylase is induced by an excess of the target amino acid and a specific range of extracellular pH, and works in conjunction with a cognate inner membrane antiporter.", + "section": "INTRO", + "ner": [ + [ + 5, + 18, + "decarboxylase", + "protein_type" + ], + [ + 57, + 67, + "amino acid", + "chemical" + ], + [ + 150, + 175, + "inner membrane antiporter", + "protein_type" + ] + ] + }, + { + "sid": 10, + "sent": "Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate.", + "section": "INTRO", + "ner": [ + [ + 23, + 33, + "amino acid", + "chemical" + ], + [ + 41, + 50, + "polyamine", + "chemical" + ], + [ + 69, + 72, + "PLP", + "chemical" + ], + [ + 134, + 140, + "proton", + "chemical" + ], + [ + 156, + 159, + "CO2", + "chemical" + ], + [ + 216, + 225, + "polyamine", + "chemical" + ], + [ + 245, + 255, + "antiporter", + "protein_type" + ], + [ + 278, + 288, + "amino acid", + "chemical" + ] + ] + }, + { + "sid": 11, + "sent": "Consequently, these enzymes buffer both the bacterial cytoplasm and the local extracellular environment.", + "section": "INTRO", + "ner": [ + [ + 44, + 53, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 12, + "sent": "These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH.", + "section": "INTRO", + "ner": [ + [ + 6, + 31, + "amino acid decarboxylases", + "protein_type" + ], + [ + 65, + 74, + "inducible", + "protein_state" + ], + [ + 78, + 92, + "biodegradative", + "protein_state" + ], + [ + 124, + 136, + "biosynthetic", + "protein_state" + ], + [ + 137, + 171, + "lysine and ornithine decarboxylase", + "protein_type" + ], + [ + 234, + 243, + "polyamine", + "chemical" + ], + [ + 258, + 268, + "neutral pH", + "protein_state" + ] + ] + }, + { + "sid": 13, + "sent": "Inducible enterobacterial amino acid decarboxylases have been intensively studied since the early 1940 because the ability of bacteria to withstand acid stress can be linked to their pathogenicity in humans.", + "section": "INTRO", + "ner": [ + [ + 0, + 9, + "Inducible", + "protein_state" + ], + [ + 10, + 25, + "enterobacterial", + "taxonomy_domain" + ], + [ + 26, + 51, + "amino acid decarboxylases", + "protein_type" + ], + [ + 126, + 134, + "bacteria", + "taxonomy_domain" + ], + [ + 200, + 206, + "humans", + "species" + ] + ] + }, + { + "sid": 14, + "sent": "In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions.", + "section": "INTRO", + "ner": [ + [ + 19, + 28, + "inducible", + "protein_state" + ], + [ + 29, + 49, + "lysine decarboxylase", + "protein_type" + ], + [ + 50, + 54, + "LdcI", + "protein" + ], + [ + 59, + 63, + "CadA", + "protein" + ], + [ + 95, + 109, + "broad pH range", + "protein_state" + ], + [ + 184, + 198, + "enterobacteria", + "taxonomy_domain" + ], + [ + 207, + 246, + "Salmonella enterica serovar Typhimurium", + "species" + ], + [ + 248, + 263, + "Vibrio cholerae", + "species" + ], + [ + 268, + 285, + "Vibrio vulnificus", + "species" + ] + ] + }, + { + "sid": 15, + "sent": "Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI).", + "section": "INTRO", + "ner": [ + [ + 18, + 22, + "LdcI", + "protein" + ], + [ + 31, + 43, + "biosynthetic", + "protein_state" + ], + [ + 44, + 64, + "lysine decarboxylase", + "protein_type" + ], + [ + 65, + 69, + "LdcC", + "protein" + ], + [ + 73, + 103, + "uropathogenic Escherichia coli", + "species" + ], + [ + 105, + 109, + "UPEC", + "species" + ], + [ + 219, + 231, + "nitric oxide", + "chemical" + ] + ] + }, + { + "sid": 16, + "sent": "This effect is attributed to cadaverine, the diamine produced by decarboxylation of lysine by LdcI and LdcC, that was shown to enhance UPEC colonisation of the bladder.", + "section": "INTRO", + "ner": [ + [ + 29, + 39, + "cadaverine", + "chemical" + ], + [ + 84, + 90, + "lysine", + "residue_name" + ], + [ + 94, + 98, + "LdcI", + "protein" + ], + [ + 103, + 107, + "LdcC", + "protein" + ], + [ + 135, + 139, + "UPEC", + "species" + ] + ] + }, + { + "sid": 17, + "sent": "In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established.", + "section": "INTRO", + "ner": [ + [ + 17, + 29, + "biosynthetic", + "protein_state" + ], + [ + 30, + 37, + "E. coli", + "species" + ], + [ + 38, + 58, + "lysine decarboxylase", + "protein_type" + ], + [ + 59, + 63, + "LdcC", + "protein" + ], + [ + 172, + 188, + "fluoroquinolones", + "chemical" + ], + [ + 212, + 216, + "RpoS", + "protein" + ], + [ + 260, + 270, + "cadaverine", + "chemical" + ], + [ + 293, + 300, + "E. coli", + "species" + ] + ] + }, + { + "sid": 18, + "sent": "Both acid pH and cadaverine induce closure of outer membrane porins thereby contributing to bacterial protection from acid stress, but also from certain antibiotics, by reduction in membrane permeability.", + "section": "INTRO", + "ner": [ + [ + 5, + 12, + "acid pH", + "protein_state" + ], + [ + 17, + 27, + "cadaverine", + "chemical" + ], + [ + 61, + 67, + "porins", + "protein_type" + ], + [ + 92, + 101, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 19, + "sent": "The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings.", + "section": "INTRO", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 29, + 36, + "E. coli", + "species" + ], + [ + 37, + 41, + "LdcI", + "protein" + ], + [ + 92, + 111, + "electron microscopy", + "experimental_method" + ], + [ + 113, + 115, + "EM", + "experimental_method" + ], + [ + 137, + 144, + "decamer", + "oligomeric_state" + ], + [ + 157, + 167, + "pentameric", + "oligomeric_state" + ], + [ + 168, + 173, + "rings", + "structure_element" + ] + ] + }, + { + "sid": 20, + "sent": "Each monomer is composed of three domains \u2013 an N-terminal wing domain (residues 1\u2013129), a PLP-binding core domain (residues 130\u2013563), and a C-terminal domain (CTD, residues 564\u2013715).", + "section": "INTRO", + "ner": [ + [ + 5, + 12, + "monomer", + "oligomeric_state" + ], + [ + 58, + 69, + "wing domain", + "structure_element" + ], + [ + 80, + 85, + "1\u2013129", + "residue_range" + ], + [ + 90, + 113, + "PLP-binding core domain", + "structure_element" + ], + [ + 124, + 131, + "130\u2013563", + "residue_range" + ], + [ + 140, + 157, + "C-terminal domain", + "structure_element" + ], + [ + 159, + 162, + "CTD", + "structure_element" + ], + [ + 173, + 180, + "564\u2013715", + "residue_range" + ] + ] + }, + { + "sid": 21, + "sent": "Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery.", + "section": "INTRO", + "ner": [ + [ + 0, + 8, + "Monomers", + "oligomeric_state" + ], + [ + 37, + 49, + "core domains", + "structure_element" + ], + [ + 55, + 73, + "2-fold symmetrical", + "protein_state" + ], + [ + 74, + 80, + "dimers", + "oligomeric_state" + ], + [ + 99, + 111, + "active sites", + "site" + ], + [ + 133, + 166, + "toroidal D5-symmetrical structure", + "structure_element" + ], + [ + 179, + 183, + "wing", + "structure_element" + ], + [ + 188, + 199, + "core domain", + "structure_element" + ], + [ + 224, + 236, + "central pore", + "structure_element" + ], + [ + 247, + 251, + "CTDs", + "structure_element" + ] + ] + }, + { + "sid": 22, + "sent": "Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response.", + "section": "INTRO", + "ner": [ + [ + 33, + 40, + "E. coli", + "species" + ], + [ + 41, + 52, + "AAA+ ATPase", + "protein_type" + ], + [ + 53, + 57, + "RavA", + "protein" + ], + [ + 130, + 134, + "LdcI", + "protein" + ], + [ + 169, + 173, + "LdcC", + "protein" + ], + [ + 203, + 213, + "pentameric", + "oligomeric_state" + ], + [ + 214, + 219, + "rings", + "structure_element" + ], + [ + 227, + 231, + "LdcI", + "protein" + ], + [ + 260, + 269, + "hexameric", + "oligomeric_state" + ], + [ + 270, + 275, + "rings", + "structure_element" + ], + [ + 279, + 283, + "RavA", + "protein" + ], + [ + 341, + 350, + "bacterium", + "taxonomy_domain" + ] + ] + }, + { + "sid": 23, + "sent": "Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins.", + "section": "INTRO", + "ner": [ + [ + 25, + 45, + "solved the structure", + "experimental_method" + ], + [ + 53, + 60, + "E. coli", + "species" + ], + [ + 61, + 70, + "LdcI-RavA", + "complex_assembly" + ], + [ + 82, + 106, + "cryo-electron microscopy", + "experimental_method" + ], + [ + 108, + 114, + "cryoEM", + "experimental_method" + ], + [ + 141, + 159, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 24, + "sent": "This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation.", + "section": "INTRO", + "ner": [ + [ + 26, + 44, + "pseudoatomic model", + "evidence" + ], + [ + 85, + 91, + "cryoEM", + "experimental_method" + ], + [ + 92, + 95, + "map", + "evidence" + ], + [ + 103, + 112, + "LdcI-LARA", + "complex_assembly" + ], + [ + 127, + 131, + "LARA", + "structure_element" + ], + [ + 145, + 176, + "LdcI associating domain of RavA", + "structure_element" + ] + ] + }, + { + "sid": 25, + "sent": "The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal \u03b2-sheet of LdcI.", + "section": "INTRO", + "ner": [ + [ + 29, + 38, + "LdcI-RavA", + "complex_assembly" + ], + [ + 83, + 87, + "loop", + "structure_element" + ], + [ + 95, + 106, + "LARA domain", + "structure_element" + ], + [ + 110, + 114, + "RavA", + "protein" + ], + [ + 134, + 141, + "\u03b2-sheet", + "structure_element" + ], + [ + 145, + 149, + "LdcI", + "protein" + ] + ] + }, + { + "sid": 26, + "sent": "In spite of this wealth of structural information, the fact that LdcC does not interact with RavA, although the two lysine decarboxylases are 69% identical and 84% similar, and the physiological significance of the absence of this interaction remained unexplored.", + "section": "INTRO", + "ner": [ + [ + 27, + 49, + "structural information", + "evidence" + ], + [ + 65, + 69, + "LdcC", + "protein" + ], + [ + 93, + 97, + "RavA", + "protein" + ], + [ + 116, + 137, + "lysine decarboxylases", + "protein_type" + ] + ] + }, + { + "sid": 27, + "sent": "To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures.", + "section": "INTRO", + "ner": [ + [ + 84, + 90, + "cryoEM", + "experimental_method" + ], + [ + 91, + 105, + "reconstruction", + "evidence" + ], + [ + 109, + 113, + "LdcC", + "protein" + ], + [ + 149, + 153, + "LdcI", + "protein" + ], + [ + 158, + 167, + "LdcI-RavA", + "complex_assembly" + ], + [ + 168, + 178, + "structures", + "evidence" + ] + ] + }, + { + "sid": 28, + "sent": "Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2).", + "section": "INTRO", + "ner": [ + [ + 15, + 19, + "LdcI", + "protein" + ], + [ + 20, + 38, + "crystal structures", + "evidence" + ], + [ + 56, + 63, + "high pH", + "protein_state" + ], + [ + 84, + 92, + "inactive", + "protein_state" + ], + [ + 94, + 99, + "LdcIi", + "protein" + ], + [ + 101, + 107, + "pH 8.5", + "protein_state" + ], + [ + 122, + 128, + "cryoEM", + "experimental_method" + ], + [ + 129, + 144, + "reconstructions", + "evidence" + ], + [ + 148, + 157, + "LdcI-RavA", + "complex_assembly" + ], + [ + 162, + 171, + "LdcI-LARA", + "complex_assembly" + ], + [ + 185, + 202, + "acidic pH optimal", + "protein_state" + ], + [ + 279, + 296, + "3D reconstruction", + "evidence" + ], + [ + 304, + 308, + "LdcI", + "protein" + ], + [ + 312, + 321, + "active pH", + "protein_state" + ], + [ + 323, + 328, + "LdcIa", + "protein" + ], + [ + 330, + 336, + "pH 6.2", + "protein_state" + ] + ] + }, + { + "sid": 29, + "sent": "This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal \u03b2-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal \u03b2-sheets in question.", + "section": "INTRO", + "ner": [ + [ + 51, + 65, + "biodegradative", + "protein_state" + ], + [ + 74, + 86, + "biosynthetic", + "protein_state" + ], + [ + 87, + 108, + "lysine decarboxylases", + "protein_type" + ], + [ + 166, + 178, + "pH-dependent", + "protein_state" + ], + [ + 201, + 205, + "RavA", + "protein" + ], + [ + 240, + 257, + "RavA binding site", + "site" + ], + [ + 289, + 296, + "\u03b2-sheet", + "structure_element" + ], + [ + 300, + 304, + "LdcI", + "protein" + ], + [ + 364, + 382, + "LdcI-LdcC chimeras", + "mutant" + ], + [ + 392, + 404, + "interchanged", + "experimental_method" + ], + [ + 420, + 428, + "\u03b2-sheets", + "structure_element" + ] + ] + }, + { + "sid": 30, + "sent": "Finally, we performed multiple sequence alignment of 22 lysine decarboxylases from Enterobacteriaceae containing the ravA-viaA operon in their genome.", + "section": "INTRO", + "ner": [ + [ + 22, + 49, + "multiple sequence alignment", + "experimental_method" + ], + [ + 56, + 77, + "lysine decarboxylases", + "protein_type" + ], + [ + 83, + 101, + "Enterobacteriaceae", + "taxonomy_domain" + ], + [ + 117, + 133, + "ravA-viaA operon", + "gene" + ] + ] + }, + { + "sid": 31, + "sent": "Remarkably, this analysis revealed that several specific residues in the above-mentioned \u03b2-sheet, independently of the rest of the protein sequence, are sufficient to define if a particular lysine decarboxylase should be classified as an \u201cLdcC-like\u201d or an \u201cLdcI-like\u201d.", + "section": "INTRO", + "ner": [ + [ + 48, + 65, + "specific residues", + "structure_element" + ], + [ + 89, + 96, + "\u03b2-sheet", + "structure_element" + ], + [ + 190, + 210, + "lysine decarboxylase", + "protein_type" + ], + [ + 239, + 248, + "LdcC-like", + "protein_type" + ], + [ + 257, + 266, + "LdcI-like", + "protein_type" + ] + ] + }, + { + "sid": 32, + "sent": "This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA.", + "section": "INTRO", + "ner": [ + [ + 56, + 60, + "RavA", + "protein" + ], + [ + 103, + 118, + "enterobacterial", + "taxonomy_domain" + ], + [ + 119, + 139, + "lysine decarboxylase", + "protein_type" + ], + [ + 156, + 183, + "high degree of conservation", + "protein_state" + ], + [ + 192, + 214, + "small structural motif", + "structure_element" + ], + [ + 279, + 293, + "biodegradative", + "protein_state" + ], + [ + 294, + 309, + "enterobacterial", + "taxonomy_domain" + ], + [ + 310, + 331, + "lysine decarboxylases", + "protein_type" + ], + [ + 340, + 351, + "AAA+ ATPase", + "protein_type" + ], + [ + 352, + 356, + "RavA", + "protein" + ] + ] + }, + { + "sid": 33, + "sent": "CryoEM 3D reconstructions of LdcC, LdcIa and LdcI-LARA", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "CryoEM", + "experimental_method" + ], + [ + 7, + 25, + "3D reconstructions", + "evidence" + ], + [ + 29, + 33, + "LdcC", + "protein" + ], + [ + 35, + 40, + "LdcIa", + "protein" + ], + [ + 45, + 54, + "LdcI-LARA", + "complex_assembly" + ] + ] + }, + { + "sid": 34, + "sent": "In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity \u2013 a 5.5\u2009\u00c5 resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1\u2009\u00c5 resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2).", + "section": "RESULTS", + "ner": [ + [ + 73, + 79, + "cryoEM", + "experimental_method" + ], + [ + 80, + 95, + "reconstructions", + "evidence" + ], + [ + 103, + 110, + "E. coli", + "species" + ], + [ + 111, + 132, + "lysine decarboxylases", + "protein_type" + ], + [ + 136, + 146, + "pH optimal", + "protein_state" + ], + [ + 197, + 203, + "cryoEM", + "experimental_method" + ], + [ + 204, + 207, + "map", + "evidence" + ], + [ + 215, + 219, + "LdcC", + "protein" + ], + [ + 221, + 227, + "pH 7.5", + "protein_state" + ], + [ + 341, + 347, + "cryoEM", + "experimental_method" + ], + [ + 348, + 351, + "map", + "evidence" + ], + [ + 359, + 364, + "LdcIa", + "protein" + ], + [ + 367, + 373, + "pH 6.2", + "protein_state" + ] + ] + }, + { + "sid": 35, + "sent": "In addition, we improved our earlier cryoEM map of the LdcI-LARA complex from 7.5\u2009\u00c5 to 6.2\u2009\u00c5 resolution (Figs 1E,F and S3).", + "section": "RESULTS", + "ner": [ + [ + 37, + 43, + "cryoEM", + "experimental_method" + ], + [ + 44, + 47, + "map", + "evidence" + ], + [ + 55, + 64, + "LdcI-LARA", + "complex_assembly" + ] + ] + }, + { + "sid": 36, + "sent": "Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1).", + "section": "RESULTS", + "ner": [ + [ + 15, + 30, + "reconstructions", + "evidence" + ], + [ + 41, + 60, + "pseudoatomic models", + "evidence" + ], + [ + 102, + 121, + "flexible fitting of", + "experimental_method" + ], + [ + 133, + 150, + "crystal structure", + "evidence" + ], + [ + 154, + 159, + "LdcIi", + "protein" + ], + [ + 173, + 198, + "structural homology model", + "experimental_method" + ], + [ + 202, + 206, + "LdcC", + "protein" + ] + ] + }, + { + "sid": 37, + "sent": "Significant differences between these pseudoatomic models can be interpreted as movements between specific biological states of the proteins as described below.", + "section": "RESULTS", + "ner": [ + [ + 38, + 57, + "pseudoatomic models", + "evidence" + ] + ] + }, + { + "sid": 38, + "sent": "The wing domains as a stable anchor at the center of the double-ring", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "wing domains", + "structure_element" + ], + [ + 57, + 68, + "double-ring", + "structure_element" + ] + ] + }, + { + "sid": 39, + "sent": "As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1).", + "section": "RESULTS", + "ner": [ + [ + 46, + 58, + "superimposed", + "experimental_method" + ], + [ + 69, + 75, + "cryoEM", + "experimental_method" + ], + [ + 76, + 91, + "reconstructions", + "evidence" + ], + [ + 93, + 98, + "LdcIa", + "protein" + ], + [ + 100, + 109, + "LdcI-LARA", + "complex_assembly" + ], + [ + 114, + 118, + "LdcC", + "protein" + ], + [ + 128, + 145, + "crystal structure", + "evidence" + ], + [ + 153, + 158, + "LdcIi", + "protein" + ], + [ + 159, + 166, + "decamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 40, + "sent": "This superposition reveals that the densities lining the central hole of the toroid are roughly at the same location, while the rest of the structure exhibits noticeable changes.", + "section": "RESULTS", + "ner": [ + [ + 5, + 18, + "superposition", + "experimental_method" + ], + [ + 36, + 45, + "densities", + "evidence" + ], + [ + 57, + 69, + "central hole", + "structure_element" + ], + [ + 77, + 83, + "toroid", + "structure_element" + ], + [ + 140, + 149, + "structure", + "evidence" + ] + ] + }, + { + "sid": 41, + "sent": "Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2\u2009\u00c5 for the C\u03b1 atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2\u2009\u00c5) (Table S2).", + "section": "RESULTS", + "ner": [ + [ + 35, + 46, + "double-ring", + "structure_element" + ], + [ + 51, + 63, + "wing domains", + "structure_element" + ], + [ + 92, + 101, + "conserved", + "protein_state" + ], + [ + 134, + 167, + "lowest root mean square deviation", + "evidence" + ], + [ + 169, + 173, + "RMSD", + "evidence" + ], + [ + 243, + 247, + "CTDs", + "structure_element" + ], + [ + 263, + 285, + "RavA binding interface", + "site" + ], + [ + 307, + 311, + "RMSD", + "evidence" + ] + ] + }, + { + "sid": 42, + "sent": "In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1\u2009\u00c5. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2\u2009\u00c5 can serve as a baseline below which differences may be assumed as insignificant.", + "section": "RESULTS", + "ner": [ + [ + 17, + 29, + "wing domains", + "structure_element" + ], + [ + 37, + 47, + "structures", + "evidence" + ], + [ + 75, + 79, + "RMSD", + "evidence" + ], + [ + 116, + 123, + "RMSDmin", + "evidence" + ], + [ + 185, + 191, + "cryoEM", + "experimental_method" + ], + [ + 192, + 196, + "maps", + "evidence" + ], + [ + 232, + 244, + "wing domains", + "structure_element" + ], + [ + 261, + 271, + "structures", + "evidence" + ], + [ + 332, + 336, + "RMSD", + "evidence" + ] + ] + }, + { + "sid": 43, + "sent": "This preservation of the central part of the double-ring assembly may help the enzymes to maintain their decameric state upon activation and incorporation into the LdcI-RavA cage.", + "section": "RESULTS", + "ner": [ + [ + 25, + 37, + "central part", + "structure_element" + ], + [ + 105, + 114, + "decameric", + "oligomeric_state" + ], + [ + 164, + 173, + "LdcI-RavA", + "complex_assembly" + ] + ] + }, + { + "sid": 44, + "sent": "The core domain and the active site rearrangements upon pH-dependent enzyme activation and LARA binding", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "core domain", + "structure_element" + ], + [ + 24, + 35, + "active site", + "site" + ], + [ + 56, + 68, + "pH-dependent", + "protein_state" + ] + ] + }, + { + "sid": 45, + "sent": "Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi).", + "section": "RESULTS", + "ner": [ + [ + 5, + 22, + "visual inspection", + "experimental_method" + ], + [ + 36, + 53, + "RMSD calculations", + "experimental_method" + ], + [ + 94, + 104, + "structures", + "evidence" + ], + [ + 108, + 117, + "active pH", + "protein_state" + ], + [ + 119, + 124, + "LdcIa", + "protein" + ], + [ + 126, + 135, + "LdcI-LARA", + "complex_assembly" + ], + [ + 140, + 144, + "LdcC", + "protein" + ], + [ + 213, + 220, + "high pH", + "protein_state" + ], + [ + 233, + 238, + "LdcIi", + "protein" + ] + ] + }, + { + "sid": 46, + "sent": "The decameric enzyme is built of five dimers associating into a 5-fold symmetrical double-ring (two monomers making a dimer are delineated in Fig. 1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "decameric", + "oligomeric_state" + ], + [ + 38, + 44, + "dimers", + "oligomeric_state" + ], + [ + 64, + 94, + "5-fold symmetrical double-ring", + "structure_element" + ], + [ + 100, + 108, + "monomers", + "oligomeric_state" + ], + [ + 118, + 123, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 47, + "sent": "As common for the \u03b1 family of the PLP-dependent decarboxylases, dimerization is required for the enzymatic activity because the active site is buried in the dimer interface (Fig. 3A,B).", + "section": "RESULTS", + "ner": [ + [ + 18, + 26, + "\u03b1 family", + "protein_type" + ], + [ + 34, + 62, + "PLP-dependent decarboxylases", + "protein_type" + ], + [ + 128, + 139, + "active site", + "site" + ], + [ + 157, + 172, + "dimer interface", + "site" + ] + ] + }, + { + "sid": 48, + "sent": "This interface is formed essentially by the core domains with some contribution of the CTDs.", + "section": "RESULTS", + "ner": [ + [ + 5, + 14, + "interface", + "site" + ], + [ + 44, + 56, + "core domains", + "structure_element" + ], + [ + 87, + 91, + "CTDs", + "structure_element" + ] + ] + }, + { + "sid": 49, + "sent": "The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184\u2013417) flanked by two smaller subdomains rich in partly disordered loops \u2013 the linker region (residues 130\u2013183) and the subdomain 4 (residues 418\u2013563).", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "core domain", + "structure_element" + ], + [ + 32, + 53, + "PLP-binding subdomain", + "structure_element" + ], + [ + 55, + 61, + "PLP-SD", + "structure_element" + ], + [ + 72, + 79, + "184\u2013417", + "residue_range" + ], + [ + 104, + 114, + "subdomains", + "structure_element" + ], + [ + 123, + 140, + "partly disordered", + "protein_state" + ], + [ + 141, + 146, + "loops", + "structure_element" + ], + [ + 153, + 166, + "linker region", + "structure_element" + ], + [ + 177, + 184, + "130\u2013183", + "residue_range" + ], + [ + 194, + 205, + "subdomain 4", + "structure_element" + ], + [ + 216, + 223, + "418\u2013563", + "residue_range" + ] + ] + }, + { + "sid": 50, + "sent": "Zooming in the variations in the PLP-SD shows that most of the structural changes concern displacements in the active site (Fig. 3C\u2013F).", + "section": "RESULTS", + "ner": [ + [ + 33, + 39, + "PLP-SD", + "structure_element" + ], + [ + 111, + 122, + "active site", + "site" + ] + ] + }, + { + "sid": 51, + "sent": "The most conspicuous differences between the PLP-SDs can be linked to the pH-dependent activation of the enzymes.", + "section": "RESULTS", + "ner": [ + [ + 45, + 52, + "PLP-SDs", + "structure_element" + ], + [ + 74, + 86, + "pH-dependent", + "protein_state" + ] + ] + }, + { + "sid": 52, + "sent": "The resolution of the cryoEM maps does not allow modeling the position of the PLP moiety and calls for caution in detailed mechanistic interpretations in terms of individual amino acids.", + "section": "RESULTS", + "ner": [ + [ + 22, + 28, + "cryoEM", + "experimental_method" + ], + [ + 29, + 33, + "maps", + "evidence" + ], + [ + 78, + 81, + "PLP", + "chemical" + ], + [ + 174, + 185, + "amino acids", + "chemical" + ] + ] + }, + { + "sid": 53, + "sent": "In particular, transition from LdcIi to LdcI-LARA involves ~3.5\u2009\u00c5 and ~4.5\u2009\u00c5 shifts away from the 5-fold axis in the active site \u03b1-helices spanning residues 218\u2013232 and 246\u2013254 respectively (Fig. 3C\u2013E).", + "section": "RESULTS", + "ner": [ + [ + 31, + 36, + "LdcIi", + "protein" + ], + [ + 40, + 49, + "LdcI-LARA", + "complex_assembly" + ], + [ + 117, + 128, + "active site", + "site" + ], + [ + 129, + 138, + "\u03b1-helices", + "structure_element" + ], + [ + 157, + 164, + "218\u2013232", + "residue_range" + ], + [ + 169, + 176, + "246\u2013254", + "residue_range" + ] + ] + }, + { + "sid": 54, + "sent": "Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin\u2009=\u20090.9\u2009\u00c5, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity.", + "section": "RESULTS", + "ner": [ + [ + 32, + 39, + "PLP-SDs", + "structure_element" + ], + [ + 43, + 48, + "LdcIa", + "protein" + ], + [ + 53, + 57, + "LdcC", + "protein" + ], + [ + 97, + 104, + "decamer", + "oligomeric_state" + ], + [ + 131, + 138, + "RMSDmin", + "evidence" + ], + [ + 194, + 204, + "optimal pH", + "protein_state" + ], + [ + 212, + 233, + "lysine decarboxylases", + "protein_type" + ] + ] + }, + { + "sid": 55, + "sent": "In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA.", + "section": "RESULTS", + "ner": [ + [ + 25, + 48, + "biochemical observation", + "experimental_method" + ], + [ + 80, + 85, + "LdcIa", + "protein" + ], + [ + 103, + 107, + "RavA", + "protein" + ], + [ + 181, + 192, + "active site", + "site" + ], + [ + 214, + 219, + "LdcIa", + "protein" + ], + [ + 223, + 232, + "LdcI-LARA", + "complex_assembly" + ] + ] + }, + { + "sid": 56, + "sent": "Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.).", + "section": "RESULTS", + "ner": [ + [ + 47, + 64, + "crystal structure", + "evidence" + ], + [ + 68, + 73, + "LdcIi", + "protein" + ], + [ + 91, + 100, + "inducible", + "protein_state" + ], + [ + 101, + 123, + "arginine decarboxylase", + "protein_type" + ], + [ + 124, + 128, + "AdiA", + "protein" + ], + [ + 138, + 155, + "high conservation", + "protein_state" + ], + [ + 163, + 188, + "PLP-coordinating residues", + "site" + ], + [ + 206, + 242, + "patch of negatively charged residues", + "site" + ], + [ + 254, + 273, + "active site channel", + "site" + ], + [ + 289, + 301, + "binding site", + "site" + ], + [ + 317, + 327, + "amino acid", + "chemical" + ] + ] + }, + { + "sid": 57, + "sent": "Rearrangements of the ppGpp binding pocket upon pH-dependent enzyme activation and LARA binding", + "section": "RESULTS", + "ner": [ + [ + 22, + 42, + "ppGpp binding pocket", + "site" + ], + [ + 48, + 60, + "pH-dependent", + "protein_state" + ], + [ + 83, + 87, + "LARA", + "structure_element" + ] + ] + }, + { + "sid": 58, + "sent": "An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4).", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "LdcI", + "protein" + ], + [ + 29, + 33, + "LdcC", + "protein" + ], + [ + 48, + 75, + "stringent response alarmone", + "chemical" + ], + [ + 76, + 81, + "ppGpp", + "chemical" + ], + [ + 107, + 116, + "interface", + "site" + ], + [ + 137, + 145, + "monomers", + "oligomeric_state" + ], + [ + 158, + 162, + "ring", + "structure_element" + ] + ] + }, + { + "sid": 59, + "sent": "The ppGpp binding pocket is made up by residues from all domains and is located approximately 30\u2009\u00c5 away from the PLP moiety.", + "section": "RESULTS", + "ner": [ + [ + 4, + 24, + "ppGpp binding pocket", + "site" + ], + [ + 113, + 116, + "PLP", + "chemical" + ] + ] + }, + { + "sid": 60, + "sent": "Whereas the crystal structure of the ppGpp-LdcIi was solved to 2\u2009\u00c5 resolution, only a 4.1\u2009\u00c5 resolution structure of the ppGpp-free LdcIi could be obtained.", + "section": "RESULTS", + "ner": [ + [ + 12, + 29, + "crystal structure", + "evidence" + ], + [ + 37, + 48, + "ppGpp-LdcIi", + "complex_assembly" + ], + [ + 53, + 59, + "solved", + "experimental_method" + ], + [ + 103, + 112, + "structure", + "evidence" + ], + [ + 120, + 130, + "ppGpp-free", + "protein_state" + ], + [ + 131, + 136, + "LdcIi", + "protein" + ] + ] + }, + { + "sid": 61, + "sent": "At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ).", + "section": "RESULTS", + "ner": [ + [ + 24, + 27, + "apo", + "protein_state" + ], + [ + 28, + 33, + "LdcIi", + "protein" + ], + [ + 38, + 49, + "ppGpp-LdcIi", + "complex_assembly" + ], + [ + 50, + 60, + "structures", + "evidence" + ], + [ + 77, + 83, + "pH 8.5", + "protein_state" + ], + [ + 139, + 144, + "ppGpp", + "chemical" + ] + ] + }, + { + "sid": 62, + "sent": "Thus, we speculated that inhibition of LdcI by ppGpp would be accompanied by a transduction of subtle structural changes at the level of individual amino acid side chains between the ppGpp binding pocket and the active site of the enzyme.", + "section": "RESULTS", + "ner": [ + [ + 39, + 43, + "LdcI", + "protein" + ], + [ + 47, + 52, + "ppGpp", + "chemical" + ], + [ + 148, + 158, + "amino acid", + "chemical" + ], + [ + 183, + 203, + "ppGpp binding pocket", + "site" + ], + [ + 212, + 223, + "active site", + "site" + ] + ] + }, + { + "sid": 63, + "sent": "All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study.", + "section": "RESULTS", + "ner": [ + [ + 16, + 22, + "cryoEM", + "experimental_method" + ], + [ + 23, + 38, + "reconstructions", + "evidence" + ], + [ + 46, + 67, + "lysine decarboxylases", + "protein_type" + ], + [ + 89, + 99, + "absence of", + "protein_state" + ], + [ + 100, + 105, + "ppGpp", + "chemical" + ], + [ + 135, + 141, + "active", + "protein_state" + ] + ] + }, + { + "sid": 64, + "sent": "While differences in the ppGpp binding site could indeed be visualized (Fig. S4), the level of resolution warns against speculations about their significance.", + "section": "RESULTS", + "ner": [ + [ + 25, + 43, + "ppGpp binding site", + "site" + ] + ] + }, + { + "sid": 65, + "sent": "The fact that interaction with RavA reduces the ppGpp affinity for LdcI despite the long distance of ~30\u2009\u00c5 between the LARA domain binding site and the closest ppGpp binding pocket (Fig. S5) seems to favor an allosteric regulation mechanism.", + "section": "RESULTS", + "ner": [ + [ + 31, + 35, + "RavA", + "protein" + ], + [ + 48, + 53, + "ppGpp", + "chemical" + ], + [ + 67, + 71, + "LdcI", + "protein" + ], + [ + 119, + 143, + "LARA domain binding site", + "site" + ], + [ + 160, + 180, + "ppGpp binding pocket", + "site" + ] + ] + }, + { + "sid": 66, + "sent": "Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated.", + "section": "RESULTS", + "ner": [ + [ + 36, + 58, + "ppGpp binding residues", + "site" + ], + [ + 63, + 81, + "strictly conserved", + "protein_state" + ], + [ + 90, + 94, + "LdcI", + "protein" + ], + [ + 99, + 103, + "AdiA", + "protein" + ], + [ + 120, + 128, + "decamers", + "oligomeric_state" + ], + [ + 132, + 146, + "low pH optimal", + "protein_state" + ], + [ + 155, + 177, + "arginine decarboxylase", + "protein_type" + ], + [ + 191, + 196, + "ppGpp", + "chemical" + ], + [ + 211, + 215, + "AdiA", + "protein" + ] + ] + }, + { + "sid": 67, + "sent": "Swinging and stretching of the CTDs upon pH-dependent LdcI activation and LARA binding", + "section": "RESULTS", + "ner": [ + [ + 31, + 35, + "CTDs", + "structure_element" + ], + [ + 41, + 53, + "pH-dependent", + "protein_state" + ], + [ + 54, + 58, + "LdcI", + "protein" + ], + [ + 74, + 78, + "LARA", + "structure_element" + ] + ] + }, + { + "sid": 68, + "sent": "Inspection of the superimposed decameric structures (Figs 2 and S6) suggests a depiction of the wing domains as an anchor around which the peripheral CTDs swing.", + "section": "RESULTS", + "ner": [ + [ + 18, + 30, + "superimposed", + "experimental_method" + ], + [ + 31, + 40, + "decameric", + "oligomeric_state" + ], + [ + 41, + 51, + "structures", + "evidence" + ], + [ + 96, + 108, + "wing domains", + "structure_element" + ], + [ + 150, + 154, + "CTDs", + "structure_element" + ] + ] + }, + { + "sid": 69, + "sent": "This swinging movement seems to be mediated by the core domains and is accompanied by a stretching of the whole LdcI subunits attracted by the RavA magnets.", + "section": "RESULTS", + "ner": [ + [ + 51, + 63, + "core domains", + "structure_element" + ], + [ + 112, + 116, + "LdcI", + "protein" + ], + [ + 117, + 125, + "subunits", + "structure_element" + ], + [ + 143, + 147, + "RavA", + "protein" + ] + ] + }, + { + "sid": 70, + "sent": "Indeed, all CTDs have very similar structures (RMSDmin <1\u2009\u00c5).", + "section": "RESULTS", + "ner": [ + [ + 12, + 16, + "CTDs", + "structure_element" + ], + [ + 47, + 54, + "RMSDmin", + "evidence" + ] + ] + }, + { + "sid": 71, + "sent": "Yet the superposition of the decamers lays bare a progressive movement of the CTD as a whole upon enzyme activation by pH and the binding of LARA.", + "section": "RESULTS", + "ner": [ + [ + 8, + 21, + "superposition", + "experimental_method" + ], + [ + 29, + 37, + "decamers", + "oligomeric_state" + ], + [ + 78, + 81, + "CTD", + "structure_element" + ], + [ + 141, + 145, + "LARA", + "structure_element" + ] + ] + }, + { + "sid": 72, + "sent": "The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4).", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "LdcIi", + "protein" + ], + [ + 10, + 17, + "monomer", + "oligomeric_state" + ], + [ + 25, + 37, + "most compact", + "protein_state" + ], + [ + 47, + 52, + "LdcIa", + "protein" + ], + [ + 68, + 77, + "LdcI-LARA", + "complex_assembly" + ], + [ + 78, + 94, + "gradually extend", + "protein_state" + ], + [ + 101, + 105, + "CTDs", + "structure_element" + ], + [ + 118, + 129, + "LARA domain", + "structure_element" + ], + [ + 133, + 137, + "RavA", + "protein" + ] + ] + }, + { + "sid": 73, + "sent": "These small but noticeable swinging and stretching (up to ~4\u2009\u00c5) may be related to the incorporation of the LdcI decamer into the LdcI-RavA cage.", + "section": "RESULTS", + "ner": [ + [ + 107, + 111, + "LdcI", + "protein" + ], + [ + 112, + 119, + "decamer", + "oligomeric_state" + ], + [ + 129, + 138, + "LdcI-RavA", + "complex_assembly" + ] + ] + }, + { + "sid": 74, + "sent": "The C-terminal \u03b2-sheet of a lysine decarboxylase as a major determinant of the interaction with RavA", + "section": "RESULTS", + "ner": [ + [ + 15, + 22, + "\u03b2-sheet", + "structure_element" + ], + [ + 28, + 48, + "lysine decarboxylase", + "protein_type" + ], + [ + 96, + 100, + "RavA", + "protein" + ] + ] + }, + { + "sid": 75, + "sent": "In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded \u03b2-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases.", + "section": "RESULTS", + "ner": [ + [ + 54, + 59, + "LdcIi", + "protein" + ], + [ + 68, + 72, + "LARA", + "structure_element" + ], + [ + 73, + 91, + "crystal structures", + "evidence" + ], + [ + 101, + 110, + "LdcI-LARA", + "complex_assembly" + ], + [ + 111, + 117, + "cryoEM", + "experimental_method" + ], + [ + 118, + 125, + "density", + "evidence" + ], + [ + 149, + 158, + "LdcI-RavA", + "complex_assembly" + ], + [ + 201, + 221, + "two-stranded \u03b2-sheet", + "structure_element" + ], + [ + 229, + 233, + "LdcI", + "protein" + ], + [ + 247, + 253, + "cryoEM", + "experimental_method" + ], + [ + 254, + 258, + "maps", + "evidence" + ], + [ + 263, + 282, + "pseudoatomic models", + "evidence" + ], + [ + 355, + 364, + "inducible", + "protein_state" + ], + [ + 373, + 385, + "constitutive", + "protein_state" + ], + [ + 386, + 407, + "lysine decarboxylases", + "protein_type" + ] + ] + }, + { + "sid": 76, + "sent": "Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant \u03b2-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal \u03b2-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal \u03b2-sheet of LdcI) (Fig. 5A\u2013C).", + "section": "RESULTS", + "ner": [ + [ + 137, + 141, + "RavA", + "protein" + ], + [ + 159, + 166, + "swapped", + "experimental_method" + ], + [ + 180, + 188, + "\u03b2-sheets", + "structure_element" + ], + [ + 228, + 236, + "chimeras", + "mutant" + ], + [ + 245, + 250, + "LdcIC", + "mutant" + ], + [ + 257, + 261, + "LdcI", + "protein" + ], + [ + 282, + 289, + "\u03b2-sheet", + "structure_element" + ], + [ + 293, + 297, + "LdcC", + "protein" + ], + [ + 303, + 308, + "LdcCI", + "mutant" + ], + [ + 315, + 319, + "LdcC", + "protein" + ], + [ + 340, + 347, + "\u03b2-sheet", + "structure_element" + ], + [ + 351, + 355, + "LdcI", + "protein" + ] + ] + }, + { + "sid": 77, + "sent": "Both constructs could be purified and could form decamers visually indistinguishable from the wild-type proteins.", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Both constructs", + "mutant" + ], + [ + 49, + 57, + "decamers", + "oligomeric_state" + ], + [ + 94, + 103, + "wild-type", + "protein_state" + ] + ] + }, + { + "sid": 78, + "sent": "As expected, binding of LdcI to RavA was completely abolished by this procedure and no LdcIC-RavA complex could be detected.", + "section": "RESULTS", + "ner": [ + [ + 24, + 28, + "LdcI", + "protein" + ], + [ + 32, + 36, + "RavA", + "protein" + ], + [ + 87, + 97, + "LdcIC-RavA", + "complex_assembly" + ] + ] + }, + { + "sid": 79, + "sent": "On the contrary, introduction of the C-terminal \u03b2-sheet of LdcI into LdcC led to an assembly of the LdcCI-RavA complex.", + "section": "RESULTS", + "ner": [ + [ + 17, + 29, + "introduction", + "experimental_method" + ], + [ + 48, + 55, + "\u03b2-sheet", + "structure_element" + ], + [ + 59, + 63, + "LdcI", + "protein" + ], + [ + 69, + 73, + "LdcC", + "protein" + ], + [ + 100, + 110, + "LdcCI-RavA", + "complex_assembly" + ] + ] + }, + { + "sid": 80, + "sent": "On the negative stain EM grid, the chimeric cages appeared less rigid than the native LdcI-RavA, which probably means that the environment of the \u03b2-sheet contributes to the efficiency of the interaction and the stability of the entire architecture (Fig. 5D\u2013F).", + "section": "RESULTS", + "ner": [ + [ + 7, + 29, + "negative stain EM grid", + "experimental_method" + ], + [ + 35, + 43, + "chimeric", + "protein_state" + ], + [ + 79, + 85, + "native", + "protein_state" + ], + [ + 86, + 95, + "LdcI-RavA", + "complex_assembly" + ], + [ + 146, + 153, + "\u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 81, + "sent": "The C-terminal \u03b2-sheet of a lysine decarboxylase is a highly conserved signature allowing to distinguish between LdcI and LdcC", + "section": "RESULTS", + "ner": [ + [ + 15, + 22, + "\u03b2-sheet", + "structure_element" + ], + [ + 28, + 48, + "lysine decarboxylase", + "protein_type" + ], + [ + 54, + 70, + "highly conserved", + "protein_state" + ], + [ + 113, + 117, + "LdcI", + "protein" + ], + [ + 122, + 126, + "LdcC", + "protein" + ] + ] + }, + { + "sid": 82, + "sent": "Alignment of the primary sequences of the E. coli LdcI and LdcC shows that some amino acid residues of the C-terminal \u03b2-sheet are the same in the two proteins, whereas others are notably different in chemical nature.", + "section": "RESULTS", + "ner": [ + [ + 0, + 34, + "Alignment of the primary sequences", + "experimental_method" + ], + [ + 42, + 49, + "E. coli", + "species" + ], + [ + 50, + 54, + "LdcI", + "protein" + ], + [ + 59, + 63, + "LdcC", + "protein" + ], + [ + 118, + 125, + "\u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 83, + "sent": "Importantly, most of the amino acid differences between the two enzymes are located in this very region.", + "section": "RESULTS", + "ner": [ + [ + 92, + 103, + "very region", + "structure_element" + ] + ] + }, + { + "sid": 84, + "sent": "Thus, to advance beyond our experimental confirmation of the C-terminal \u03b2-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this \u03b2-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome.", + "section": "RESULTS", + "ner": [ + [ + 72, + 79, + "\u03b2-sheet", + "structure_element" + ], + [ + 135, + 155, + "lysine decarboxylase", + "protein_type" + ], + [ + 176, + 180, + "RavA", + "protein" + ], + [ + 216, + 232, + "certain residues", + "structure_element" + ], + [ + 241, + 248, + "\u03b2-sheet", + "structure_element" + ], + [ + 253, + 262, + "conserved", + "protein_state" + ], + [ + 266, + 287, + "lysine decarboxylases", + "protein_type" + ], + [ + 301, + 315, + "enterobacteria", + "taxonomy_domain" + ], + [ + 330, + 346, + "ravA-viaA operon", + "gene" + ] + ] + }, + { + "sid": 85, + "sent": "We inspected the genetic environment of lysine decarboxylases from 22 enterobacterial species referenced in the NCBI database, corrected the gene annotation where necessary (Tables S3 and S4), and performed multiple sequence alignment coupled to a phylogenetic analysis (see Methods).", + "section": "RESULTS", + "ner": [ + [ + 3, + 36, + "inspected the genetic environment", + "experimental_method" + ], + [ + 40, + 61, + "lysine decarboxylases", + "protein_type" + ], + [ + 70, + 85, + "enterobacterial", + "taxonomy_domain" + ], + [ + 207, + 234, + "multiple sequence alignment", + "experimental_method" + ], + [ + 248, + 269, + "phylogenetic analysis", + "experimental_method" + ] + ] + }, + { + "sid": 86, + "sent": "First of all, consensus sequence for the entire lysine decarboxylase family was derived.", + "section": "RESULTS", + "ner": [ + [ + 14, + 32, + "consensus sequence", + "evidence" + ], + [ + 48, + 68, + "lysine decarboxylase", + "protein_type" + ] + ] + }, + { + "sid": 87, + "sent": "Second, the phylogenetic analysis clearly split the lysine decarboxylases into two groups (Fig. 6A).", + "section": "RESULTS", + "ner": [ + [ + 12, + 33, + "phylogenetic analysis", + "experimental_method" + ], + [ + 52, + 73, + "lysine decarboxylases", + "protein_type" + ] + ] + }, + { + "sid": 88, + "sent": "All lysine decarboxylases predicted to be \u201cLdcI-like\u201d or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as \u201cLdcC-like\u201d or biosynthetic partitioned into another group.", + "section": "RESULTS", + "ner": [ + [ + 4, + 25, + "lysine decarboxylases", + "protein_type" + ], + [ + 43, + 52, + "LdcI-like", + "protein_type" + ], + [ + 57, + 70, + "biodegradable", + "protein_state" + ], + [ + 179, + 183, + "CadB", + "protein" + ], + [ + 184, + 194, + "antiporter", + "protein_type" + ], + [ + 247, + 254, + "enzymes", + "protein_type" + ], + [ + 269, + 278, + "LdcC-like", + "protein_type" + ], + [ + 283, + 295, + "biosynthetic", + "protein_state" + ] + ] + }, + { + "sid": 89, + "sent": "Thus, consensus sequences could also be determined for each of the two groups (Figs 6B,C and S7).", + "section": "RESULTS", + "ner": [ + [ + 6, + 25, + "consensus sequences", + "evidence" + ] + ] + }, + { + "sid": 90, + "sent": "Inspection of these consensus sequences revealed important differences between the groups regarding charge, size and hydrophobicity of several residues precisely at the level of the C-terminal \u03b2-sheet that is responsible for the interaction with RavA (Fig. 6B\u2013D).", + "section": "RESULTS", + "ner": [ + [ + 20, + 39, + "consensus sequences", + "evidence" + ], + [ + 193, + 200, + "\u03b2-sheet", + "structure_element" + ], + [ + 246, + 250, + "RavA", + "protein" + ] + ] + }, + { + "sid": 91, + "sent": "For example, in our previous study, site-directed mutations identified Y697 as critically required for the RavA binding.", + "section": "RESULTS", + "ner": [ + [ + 36, + 59, + "site-directed mutations", + "experimental_method" + ], + [ + 71, + 75, + "Y697", + "residue_name_number" + ], + [ + 107, + 111, + "RavA", + "protein" + ] + ] + }, + { + "sid": 92, + "sent": "Our current analysis shows that Y697 is strictly conserved in the \u201cLdcI-like\u201d group whereas the \u201cLdcC-like\u201d enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis.", + "section": "RESULTS", + "ner": [ + [ + 32, + 36, + "Y697", + "residue_name_number" + ], + [ + 40, + 58, + "strictly conserved", + "protein_state" + ], + [ + 67, + 76, + "LdcI-like", + "protein_type" + ], + [ + 97, + 106, + "LdcC-like", + "protein_type" + ], + [ + 116, + 127, + "always have", + "protein_state" + ], + [ + 130, + 136, + "lysine", + "residue_name" + ], + [ + 242, + 246, + "RavA", + "protein" + ], + [ + 277, + 302, + "site-directed mutagenesis", + "experimental_method" + ] + ] + }, + { + "sid": 93, + "sent": "The third and most remarkable finding was that exactly the same separation into \u201cLdcI-like\u201d and \u201cLdcC\u201d-like groups can be obtained based on a comparison of the C-terminal \u03b2-sheets only, without taking the rest of the primary sequence into account.", + "section": "RESULTS", + "ner": [ + [ + 81, + 90, + "LdcI-like", + "protein_type" + ], + [ + 97, + 107, + "LdcC\u201d-like", + "protein_type" + ], + [ + 171, + 179, + "\u03b2-sheets", + "structure_element" + ] + ] + }, + { + "sid": 94, + "sent": "Therefore the C-terminal \u03b2-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the \u201cLdcI-like\u201d and \u201cLdcC-like\u201d enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7).", + "section": "RESULTS", + "ner": [ + [ + 25, + 32, + "\u03b2-sheet", + "structure_element" + ], + [ + 52, + 68, + "highly conserved", + "protein_state" + ], + [ + 69, + 87, + "signature sequence", + "structure_element" + ], + [ + 143, + 152, + "LdcI-like", + "protein_type" + ], + [ + 159, + 168, + "LdcC-like", + "protein_type" + ], + [ + 170, + 185, + "enterobacterial", + "taxonomy_domain" + ], + [ + 186, + 207, + "lysine decarboxylases", + "protein_type" + ] + ] + }, + { + "sid": 95, + "sent": "Our structures show that this motif is not involved in the enzymatic activity or the oligomeric state of the proteins.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 25, + 35, + "this motif", + "structure_element" + ] + ] + }, + { + "sid": 96, + "sent": "Thus, enterobacteria identified here (Fig. 6, Table S4) appear to exert evolutionary pressure on the biodegradative lysine decarboxylase towards the RavA binding.", + "section": "RESULTS", + "ner": [ + [ + 6, + 20, + "enterobacteria", + "taxonomy_domain" + ], + [ + 101, + 115, + "biodegradative", + "protein_state" + ], + [ + 116, + 136, + "lysine decarboxylase", + "protein_type" + ], + [ + 149, + 153, + "RavA", + "protein" + ] + ] + }, + { + "sid": 97, + "sent": "One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp.", + "section": "RESULTS", + "ner": [ + [ + 35, + 44, + "LdcI-RavA", + "complex_assembly" + ], + [ + 65, + 69, + "LdcI", + "protein" + ], + [ + 99, + 114, + "enterobacterial", + "taxonomy_domain" + ], + [ + 140, + 144, + "LdcI", + "protein" + ], + [ + 163, + 190, + "stringent response alarmone", + "chemical" + ], + [ + 191, + 196, + "ppGpp", + "chemical" + ] + ] + }, + { + "sid": 98, + "sent": "Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5\u2009MDa assembly.", + "section": "RESULTS", + "ner": [ + [ + 57, + 61, + "LdcI", + "protein" + ], + [ + 66, + 70, + "RavA", + "protein" + ], + [ + 85, + 93, + "subunits", + "structure_element" + ], + [ + 101, + 122, + "respiratory complex I", + "protein_type" + ], + [ + 169, + 173, + "RavA", + "protein" + ], + [ + 201, + 221, + "iron-sulfur proteins", + "protein_type" + ] + ] + }, + { + "sid": 99, + "sent": "The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to \u201cLdcI-like\u201d or \u201cLdcC-like\u201d proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage.", + "section": "RESULTS", + "ner": [ + [ + 37, + 41, + "LdcI", + "protein" + ], + [ + 69, + 73, + "RavA", + "protein" + ], + [ + 167, + 176, + "LdcI-RavA", + "complex_assembly" + ], + [ + 250, + 265, + "enterobacterial", + "taxonomy_domain" + ], + [ + 266, + 286, + "lysine decarboxylase", + "protein_type" + ], + [ + 299, + 308, + "LdcI-like", + "protein_type" + ], + [ + 314, + 323, + "LdcC-like", + "protein_type" + ], + [ + 397, + 406, + "LdcI-RavA", + "complex_assembly" + ] + ] + }, + { + "sid": 100, + "sent": "Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity.", + "section": "RESULTS", + "ner": [ + [ + 13, + 23, + "structures", + "evidence" + ], + [ + 32, + 51, + "pseudoatomic models", + "evidence" + ], + [ + 59, + 65, + "active", + "protein_state" + ], + [ + 66, + 76, + "ppGpp-free", + "protein_state" + ], + [ + 96, + 110, + "biodegradative", + "protein_state" + ], + [ + 119, + 131, + "biosynthetic", + "protein_state" + ], + [ + 132, + 139, + "E. coli", + "species" + ], + [ + 140, + 161, + "lysine decarboxylases", + "protein_type" + ], + [ + 217, + 221, + "UPEC", + "species" + ] + ] + }, + { + "sid": 101, + "sent": "Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development.", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "apo", + "protein_state" + ], + [ + 22, + 26, + "LdcI", + "protein" + ], + [ + 31, + 42, + "ppGpp-LdcIi", + "complex_assembly" + ], + [ + 43, + 61, + "crystal structures", + "evidence" + ], + [ + 67, + 73, + "cryoEM", + "experimental_method" + ], + [ + 74, + 89, + "reconstructions", + "evidence" + ], + [ + 179, + 200, + "lysine decarboxylases", + "protein_type" + ], + [ + 212, + 226, + "enterobacteria", + "taxonomy_domain" + ], + [ + 264, + 282, + "Enterobacteriaceae", + "taxonomy_domain" + ], + [ + 301, + 321, + "lysine decarboxylase", + "protein_type" + ], + [ + 325, + 344, + "Eikenella corrodens", + "species" + ] + ] + }, + { + "sid": 102, + "sent": "Finally, cadaverine being an important platform chemical for the production of industrial polymers such as nylon, structural information is valuable for optimisation of bacterial lysine decarboxylases used for its production in biotechnology.", + "section": "RESULTS", + "ner": [ + [ + 9, + 19, + "cadaverine", + "chemical" + ], + [ + 169, + 178, + "bacterial", + "taxonomy_domain" + ], + [ + 179, + 200, + "lysine decarboxylases", + "protein_type" + ] + ] + }, + { + "sid": 103, + "sent": "3D cryoEM reconstructions of LdcC, LdcI-LARA and LdcIa.", + "section": "FIG", + "ner": [ + [ + 3, + 9, + "cryoEM", + "experimental_method" + ], + [ + 10, + 25, + "reconstructions", + "evidence" + ], + [ + 29, + 33, + "LdcC", + "protein" + ], + [ + 35, + 44, + "LdcI-LARA", + "complex_assembly" + ], + [ + 49, + 54, + "LdcIa", + "protein" + ] + ] + }, + { + "sid": 104, + "sent": "(A,C,E) cryoEM map of the LdcC (A), LdcIa (C) and LdcI-LARA (E) decamers with one protomer in light grey.", + "section": "FIG", + "ner": [ + [ + 8, + 14, + "cryoEM", + "experimental_method" + ], + [ + 15, + 18, + "map", + "evidence" + ], + [ + 26, + 30, + "LdcC", + "protein" + ], + [ + 36, + 41, + "LdcIa", + "protein" + ], + [ + 50, + 59, + "LdcI-LARA", + "complex_assembly" + ], + [ + 64, + 72, + "decamers", + "oligomeric_state" + ], + [ + 82, + 90, + "protomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 105, + "sent": "In the rest of the protomers, the wing, core and C-terminal domains are colored from light to dark in shades of green for LdcC (A), pink for LdcIa (C) and blue for LdcI in LdcI-LARA (E).", + "section": "FIG", + "ner": [ + [ + 19, + 28, + "protomers", + "oligomeric_state" + ], + [ + 34, + 38, + "wing", + "structure_element" + ], + [ + 40, + 44, + "core", + "structure_element" + ], + [ + 49, + 67, + "C-terminal domains", + "structure_element" + ], + [ + 122, + 126, + "LdcC", + "protein" + ], + [ + 141, + 146, + "LdcIa", + "protein" + ], + [ + 164, + 168, + "LdcI", + "protein" + ], + [ + 172, + 181, + "LdcI-LARA", + "complex_assembly" + ] + ] + }, + { + "sid": 106, + "sent": " In (E), the LARA domain density is shown in dark grey.", + "section": "FIG", + "ner": [ + [ + 13, + 24, + "LARA domain", + "structure_element" + ] + ] + }, + { + "sid": 107, + "sent": "Two monomers making a dimer are delineated.", + "section": "FIG", + "ner": [ + [ + 4, + 12, + "monomers", + "oligomeric_state" + ], + [ + 22, + 27, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 108, + "sent": "Scale bar 50\u2009\u00c5. (B,D,F) One protomer from the cryoEM map of the LdcC (B), LdcIa (D) and LdcI-LARA (F) in light grey with the pseudoatomic model represented as cartoons and colored as the densities in (A,C,E).", + "section": "FIG", + "ner": [ + [ + 28, + 36, + "protomer", + "oligomeric_state" + ], + [ + 46, + 52, + "cryoEM", + "experimental_method" + ], + [ + 53, + 56, + "map", + "evidence" + ], + [ + 64, + 68, + "LdcC", + "protein" + ], + [ + 74, + 79, + "LdcIa", + "protein" + ], + [ + 88, + 97, + "LdcI-LARA", + "complex_assembly" + ], + [ + 125, + 143, + "pseudoatomic model", + "evidence" + ] + ] + }, + { + "sid": 109, + "sent": "Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and LdcIa colored as in Fig. 1, and the crystal structure of LdcIi in shades of yellow.", + "section": "FIG", + "ner": [ + [ + 0, + 13, + "Superposition", + "experimental_method" + ], + [ + 21, + 40, + "pseudoatomic models", + "evidence" + ], + [ + 44, + 48, + "LdcC", + "protein" + ], + [ + 50, + 54, + "LdcI", + "protein" + ], + [ + 60, + 69, + "LdcI-LARA", + "complex_assembly" + ], + [ + 74, + 79, + "LdcIa", + "protein" + ], + [ + 110, + 127, + "crystal structure", + "evidence" + ], + [ + 131, + 136, + "LdcIi", + "protein" + ] + ] + }, + { + "sid": 110, + "sent": "Only one of the two rings of the double toroid is shown for clarity.", + "section": "FIG", + "ner": [ + [ + 20, + 25, + "rings", + "structure_element" + ], + [ + 33, + 46, + "double toroid", + "structure_element" + ] + ] + }, + { + "sid": 111, + "sent": "The dashed circle indicates the central region that remains virtually unchanged between all the structures, while the periphery undergoes visible movements.", + "section": "FIG", + "ner": [ + [ + 40, + 46, + "region", + "structure_element" + ], + [ + 96, + 106, + "structures", + "evidence" + ] + ] + }, + { + "sid": 112, + "sent": "Conformational rearrangements in the enzyme active site.", + "section": "FIG", + "ner": [ + [ + 44, + 55, + "active site", + "site" + ] + ] + }, + { + "sid": 113, + "sent": "(A) LdcIi crystal structure, with one ring represented as a grey surface and the second as a cartoon.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "LdcIi", + "protein" + ], + [ + 10, + 27, + "crystal structure", + "evidence" + ], + [ + 38, + 42, + "ring", + "structure_element" + ] + ] + }, + { + "sid": 114, + "sent": "A monomer with its PLP cofactor is delineated.", + "section": "FIG", + "ner": [ + [ + 2, + 9, + "monomer", + "oligomeric_state" + ], + [ + 19, + 22, + "PLP", + "chemical" + ] + ] + }, + { + "sid": 115, + "sent": "The PLP moieties of the cartoon ring are shown in red.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "PLP", + "chemical" + ], + [ + 32, + 36, + "ring", + "structure_element" + ] + ] + }, + { + "sid": 116, + "sent": " (B) The LdcIi dimer extracted from the crystal structure of the decamer.", + "section": "FIG", + "ner": [ + [ + 9, + 14, + "LdcIi", + "protein" + ], + [ + 15, + 20, + "dimer", + "oligomeric_state" + ], + [ + 40, + 57, + "crystal structure", + "evidence" + ], + [ + 65, + 72, + "decamer", + "oligomeric_state" + ] + ] + }, + { + "sid": 117, + "sent": "One monomer is colored in shades of yellow as in Figs 1 and 2, while the monomer related by C2 symmetry is grey.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "monomer", + "oligomeric_state" + ], + [ + 73, + 80, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 118, + "sent": "The PLP is red.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "PLP", + "chemical" + ] + ] + }, + { + "sid": 119, + "sent": "The active site is boxed.", + "section": "FIG", + "ner": [ + [ + 4, + 15, + "active site", + "site" + ] + ] + }, + { + "sid": 120, + "sent": "Stretching of the LdcI monomer upon pH-dependent enzyme activation and LARA binding.", + "section": "FIG", + "ner": [ + [ + 18, + 22, + "LdcI", + "protein" + ], + [ + 23, + 30, + "monomer", + "oligomeric_state" + ], + [ + 36, + 48, + "pH-dependent", + "protein_state" + ], + [ + 71, + 75, + "LARA", + "structure_element" + ] + ] + }, + { + "sid": 121, + "sent": "(A\u2013C) A slice through the pseudoatomic models of the LdcI monomers extracted from the superimposed decamers (Fig. 2) The rectangle indicates the regions enlarged in (D\u2013F).", + "section": "FIG", + "ner": [ + [ + 26, + 45, + "pseudoatomic models", + "evidence" + ], + [ + 53, + 57, + "LdcI", + "protein" + ], + [ + 58, + 66, + "monomers", + "oligomeric_state" + ], + [ + 86, + 98, + "superimposed", + "experimental_method" + ], + [ + 99, + 107, + "decamers", + "oligomeric_state" + ] + ] + }, + { + "sid": 122, + "sent": "(A) compares LdcIi (yellow) and LdcIa (pink), (B) compares LdcIa (pink) and LdcI-LARA (blue), and (C) compares LdcIi (yellow), LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to show the progressive stretching described in the text.", + "section": "FIG", + "ner": [ + [ + 13, + 18, + "LdcIi", + "protein" + ], + [ + 32, + 37, + "LdcIa", + "protein" + ], + [ + 59, + 64, + "LdcIa", + "protein" + ], + [ + 76, + 85, + "LdcI-LARA", + "complex_assembly" + ], + [ + 111, + 116, + "LdcIi", + "protein" + ], + [ + 127, + 132, + "LdcIa", + "protein" + ], + [ + 144, + 153, + "LdcI-LARA", + "complex_assembly" + ] + ] + }, + { + "sid": 123, + "sent": "The cryoEM density of the LARA domain is represented as a grey surface to show the position of the binding site and the direction of the movement.", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "cryoEM", + "experimental_method" + ], + [ + 11, + 18, + "density", + "evidence" + ], + [ + 26, + 37, + "LARA domain", + "structure_element" + ], + [ + 99, + 111, + "binding site", + "site" + ] + ] + }, + { + "sid": 124, + "sent": "(D\u2013F) Inserts zooming at the CTD part in proximity of the LARA binding site.", + "section": "FIG", + "ner": [ + [ + 29, + 32, + "CTD", + "structure_element" + ], + [ + 58, + 75, + "LARA binding site", + "site" + ] + ] + }, + { + "sid": 125, + "sent": "Analysis of the LdcIC and LdcCI chimeras.", + "section": "FIG", + "ner": [ + [ + 16, + 21, + "LdcIC", + "mutant" + ], + [ + 26, + 31, + "LdcCI", + "mutant" + ], + [ + 32, + 40, + "chimeras", + "mutant" + ] + ] + }, + { + "sid": 126, + "sent": "(A) A slice through the pseudoatomic models of the LdcIa (purple) and LdcC (green) monomers extracted from the superimposed decamers (Fig. 2). (B) The C-terminal \u03b2-sheet in LdcIa and LdcC enlarged from (A,C) Exchanged primary sequences (capital letters) and their immediate vicinity (lower case letters) colored as in (A,B), with the corresponding secondary structure elements and the amino acid numbering shown.", + "section": "FIG", + "ner": [ + [ + 24, + 43, + "pseudoatomic models", + "evidence" + ], + [ + 51, + 56, + "LdcIa", + "protein" + ], + [ + 70, + 74, + "LdcC", + "protein" + ], + [ + 83, + 91, + "monomers", + "oligomeric_state" + ], + [ + 111, + 123, + "superimposed", + "experimental_method" + ], + [ + 124, + 132, + "decamers", + "oligomeric_state" + ], + [ + 162, + 169, + "\u03b2-sheet", + "structure_element" + ], + [ + 173, + 178, + "LdcIa", + "protein" + ], + [ + 183, + 187, + "LdcC", + "protein" + ] + ] + }, + { + "sid": 127, + "sent": "(D,E) A gallery of negative stain EM images of (D) the wild type LdcI-RavA cage and (E) the LdcCI-RavA cage-like particles. (F) Some representative class averages of the LdcCI-RavA cage-like particles.", + "section": "FIG", + "ner": [ + [ + 55, + 64, + "wild type", + "protein_state" + ], + [ + 65, + 74, + "LdcI-RavA", + "complex_assembly" + ], + [ + 92, + 122, + "LdcCI-RavA cage-like particles", + "mutant" + ], + [ + 170, + 200, + "LdcCI-RavA cage-like particles", + "mutant" + ] + ] + }, + { + "sid": 128, + "sent": "Sequence analysis of enterobacterial lysine decarboxylases.", + "section": "FIG", + "ner": [ + [ + 0, + 17, + "Sequence analysis", + "experimental_method" + ], + [ + 21, + 36, + "enterobacterial", + "taxonomy_domain" + ], + [ + 37, + 58, + "lysine decarboxylases", + "protein_type" + ] + ] + }, + { + "sid": 129, + "sent": "(A) Maximum likelihood tree with the \u201cLdcC-like\u201d and the \u201cLdcI-like\u201d groups highlighted in green and pink, respectively.", + "section": "FIG", + "ner": [ + [ + 4, + 27, + "Maximum likelihood tree", + "evidence" + ], + [ + 38, + 47, + "LdcC-like", + "protein_type" + ], + [ + 58, + 67, + "LdcI-like", + "protein_type" + ] + ] + }, + { + "sid": 130, + "sent": "(B) Analysis of consensus \u201cLdcI-like\u201d and \u201cLdcC-like\u201d sequences around the first and second C-terminal \u03b2-strands.", + "section": "FIG", + "ner": [ + [ + 27, + 36, + "LdcI-like", + "protein_type" + ], + [ + 43, + 52, + "LdcC-like", + "protein_type" + ], + [ + 103, + 112, + "\u03b2-strands", + "structure_element" + ] + ] + }, + { + "sid": 131, + "sent": "Numbering as in E. coli.", + "section": "FIG", + "ner": [ + [ + 16, + 23, + "E. coli", + "species" + ] + ] + }, + { + "sid": 132, + "sent": " (C) Signature sequences of LdcI and LdcC in the C-terminal \u03b2-sheet.", + "section": "FIG", + "ner": [ + [ + 28, + 32, + "LdcI", + "protein" + ], + [ + 37, + 41, + "LdcC", + "protein" + ], + [ + 60, + 67, + "\u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 133, + "sent": "Polarity differences are highlighted. (D) Position and nature of these differences at the surface of the respective cryoEM maps with the color code as in B. See also Fig. S7 and Tables S3 and S4.", + "section": "FIG", + "ner": [ + [ + 116, + 122, + "cryoEM", + "experimental_method" + ], + [ + 123, + 127, + "maps", + "evidence" + ] + ] + } + ] + }, + "PMC4852598": { + "annotations": [ + { + "sid": 0, + "sent": "Structural basis for Mep2 ammonium transceptor activation by phosphorylation", + "section": "TITLE", + "ner": [ + [ + 21, + 25, + "Mep2", + "protein_type" + ], + [ + 26, + 46, + "ammonium transceptor", + "protein_type" + ], + [ + 61, + 76, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 1, + "sent": "Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 13, + "Mep2 proteins", + "protein_type" + ], + [ + 18, + 24, + "fungal", + "taxonomy_domain" + ], + [ + 25, + 37, + "transceptors", + "protein_type" + ], + [ + 69, + 77, + "ammonium", + "chemical" + ], + [ + 89, + 95, + "fungal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 2, + "sent": "Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 4, + "Mep2", + "protein_type" + ], + [ + 38, + 53, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 3, + "sent": "Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations.", + "section": "ABSTRACT", + "ner": [ + [ + 15, + 39, + "X-ray crystal structures", + "evidence" + ], + [ + 47, + 51, + "Mep2", + "protein_type" + ], + [ + 69, + 93, + "Saccharomyces cerevisiae", + "species" + ], + [ + 98, + 114, + "Candida albicans", + "species" + ], + [ + 170, + 182, + "transporters", + "protein_type" + ], + [ + 187, + 205, + "not phosphorylated", + "protein_state" + ], + [ + 221, + 227, + "closed", + "protein_state" + ], + [ + 229, + 237, + "inactive", + "protein_state" + ] + ] + }, + { + "sid": 4, + "sent": "Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif.", + "section": "ABSTRACT", + "ner": [ + [ + 16, + 20, + "open", + "protein_state" + ], + [ + 21, + 30, + "bacterial", + "taxonomy_domain" + ], + [ + 31, + 52, + "ammonium transporters", + "protein_type" + ], + [ + 54, + 72, + "non-phosphorylated", + "protein_state" + ], + [ + 73, + 77, + "Mep2", + "protein_type" + ], + [ + 97, + 114, + "cytoplasmic loops", + "structure_element" + ], + [ + 123, + 140, + "C-terminal region", + "structure_element" + ], + [ + 142, + 145, + "CTR", + "structure_element" + ], + [ + 174, + 178, + "exit", + "site" + ], + [ + 186, + 193, + "channel", + "site" + ], + [ + 215, + 219, + "His2", + "residue_name_number" + ], + [ + 227, + 241, + "twin-His motif", + "structure_element" + ] + ] + }, + { + "sid": 5, + "sent": "The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket \u223c30\u2009\u00c5 away from the channel exit.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 24, + "phosphorylation site", + "site" + ], + [ + 32, + 35, + "CTR", + "structure_element" + ], + [ + 39, + 57, + "solvent accessible", + "protein_state" + ], + [ + 75, + 100, + "negatively charged pocket", + "site" + ], + [ + 121, + 133, + "channel exit", + "site" + ] + ] + }, + { + "sid": 6, + "sent": "The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 25, + 50, + "phosphorylation-mimicking", + "protein_state" + ], + [ + 51, + 64, + "Mep2 variants", + "mutant" + ], + [ + 70, + 81, + "C. albicans", + "species" + ], + [ + 121, + 130, + "conserved", + "protein_state" + ], + [ + 172, + 175, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 7, + "sent": "The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.", + "section": "ABSTRACT", + "ner": [ + [ + 58, + 68, + "eukaryotic", + "taxonomy_domain" + ], + [ + 69, + 77, + "ammonium", + "chemical" + ], + [ + 91, + 106, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 8, + "sent": " Mep2 proteins are tightly regulated fungal ammonium transporters.", + "section": "ABSTRACT", + "ner": [ + [ + 1, + 14, + "Mep2 proteins", + "protein_type" + ], + [ + 37, + 43, + "fungal", + "taxonomy_domain" + ], + [ + 44, + 65, + "ammonium transporters", + "protein_type" + ] + ] + }, + { + "sid": 9, + "sent": "Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria.", + "section": "ABSTRACT", + "ner": [ + [ + 29, + 47, + "crystal structures", + "evidence" + ], + [ + 51, + 57, + "closed", + "protein_state" + ], + [ + 68, + 81, + "Mep2 proteins", + "protein_type" + ], + [ + 123, + 145, + "by comparing them with", + "experimental_method" + ], + [ + 150, + 154, + "open", + "protein_state" + ], + [ + 155, + 176, + "ammonium transporters", + "protein_type" + ], + [ + 180, + 188, + "bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 10, + "sent": "Transceptors are membrane proteins that function not only as transporters but also as receptors/sensors during nutrient sensing to activate downstream signalling pathways.", + "section": "INTRO", + "ner": [ + [ + 0, + 12, + "Transceptors", + "protein_type" + ], + [ + 17, + 34, + "membrane proteins", + "protein_type" + ] + ] + }, + { + "sid": 11, + "sent": "A common feature of transceptors is that they are induced when cells are starved for their substrate.", + "section": "INTRO", + "ner": [ + [ + 20, + 32, + "transceptors", + "protein_type" + ] + ] + }, + { + "sid": 12, + "sent": "While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter).", + "section": "INTRO", + "ner": [ + [ + 39, + 63, + "Saccharomyces cerevisiae", + "species" + ], + [ + 64, + 76, + "transceptors", + "protein_type" + ], + [ + 81, + 90, + "phosphate", + "chemical" + ], + [ + 92, + 97, + "Pho84", + "protein" + ], + [ + 100, + 111, + "amino acids", + "chemical" + ], + [ + 113, + 117, + "Gap1", + "protein" + ], + [ + 123, + 131, + "ammonium", + "chemical" + ], + [ + 133, + 137, + "Mep2", + "protein" + ], + [ + 140, + 152, + "transceptors", + "protein_type" + ], + [ + 166, + 183, + "higher eukaryotes", + "taxonomy_domain" + ], + [ + 210, + 219, + "mammalian", + "taxonomy_domain" + ], + [ + 220, + 225, + "SNAT2", + "protein" + ], + [ + 226, + 248, + "amino-acid transporter", + "protein_type" + ], + [ + 257, + 262, + "GLUT2", + "protein" + ], + [ + 263, + 282, + "glucose transporter", + "protein_type" + ] + ] + }, + { + "sid": 13, + "sent": "One of the most important unresolved questions in the field is how the transceptors couple to downstream signalling pathways.", + "section": "INTRO", + "ner": [ + [ + 71, + 83, + "transceptors", + "protein_type" + ] + ] + }, + { + "sid": 14, + "sent": "One hypothesis is that downstream signalling is dependent on a specific conformation of the transporter.", + "section": "INTRO", + "ner": [ + [ + 92, + 103, + "transporter", + "protein_type" + ] + ] + }, + { + "sid": 15, + "sent": "Mep2 (methylammonium (MA) permease) proteins are ammonium transceptors that are ubiquitous in fungi.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "Mep2", + "protein_type" + ], + [ + 5, + 44, + "(methylammonium (MA) permease) proteins", + "protein_type" + ], + [ + 49, + 70, + "ammonium transceptors", + "protein_type" + ], + [ + 94, + 99, + "fungi", + "taxonomy_domain" + ] + ] + }, + { + "sid": 16, + "sent": "They belong to the Amt/Mep/Rh family of transporters that are present in all kingdoms of life and they take up ammonium from the extracellular environment.", + "section": "INTRO", + "ner": [ + [ + 19, + 52, + "Amt/Mep/Rh family of transporters", + "protein_type" + ], + [ + 73, + 93, + "all kingdoms of life", + "taxonomy_domain" + ], + [ + 111, + 119, + "ammonium", + "chemical" + ] + ] + }, + { + "sid": 17, + "sent": "Fungi typically have more than one Mep paralogue, for example, Mep1-3 in S. cerevisiae.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "Fungi", + "taxonomy_domain" + ], + [ + 35, + 38, + "Mep", + "protein_type" + ], + [ + 63, + 69, + "Mep1-3", + "protein" + ], + [ + 73, + 86, + "S. cerevisiae", + "species" + ] + ] + }, + { + "sid": 18, + "sent": "Of these, only Mep2 proteins function as ammonium receptors/sensors in fungal development.", + "section": "INTRO", + "ner": [ + [ + 15, + 28, + "Mep2 proteins", + "protein_type" + ], + [ + 41, + 49, + "ammonium", + "chemical" + ], + [ + 71, + 77, + "fungal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 19, + "sent": "Under conditions of nitrogen limitation, Mep2 initiates a signalling cascade that results in a switch from the yeast form to filamentous (pseudohyphal) growth that may be required for fungal pathogenicity.", + "section": "INTRO", + "ner": [ + [ + 41, + 45, + "Mep2", + "protein" + ], + [ + 184, + 190, + "fungal", + "taxonomy_domain" + ] + ] + }, + { + "sid": 20, + "sent": "As is the case for other transceptors, it is not clear how Mep2 interacts with downstream signalling partners, but the protein kinase A and mitogen-activated protein kinase pathways have been proposed as downstream effectors of Mep2 (refs).", + "section": "INTRO", + "ner": [ + [ + 25, + 37, + "transceptors", + "protein_type" + ], + [ + 59, + 63, + "Mep2", + "protein" + ], + [ + 228, + 232, + "Mep2", + "protein" + ] + ] + }, + { + "sid": 21, + "sent": "Compared with Mep1 and Mep3, Mep2 is highly expressed and functions as a low-capacity, high-affinity transporter in the uptake of MA.", + "section": "INTRO", + "ner": [ + [ + 14, + 18, + "Mep1", + "protein" + ], + [ + 23, + 27, + "Mep3", + "protein" + ], + [ + 29, + 33, + "Mep2", + "protein" + ], + [ + 37, + 53, + "highly expressed", + "protein_state" + ], + [ + 130, + 132, + "MA", + "chemical" + ] + ] + }, + { + "sid": 22, + "sent": "In addition, Mep2 is also important for uptake of ammonium produced by growth on other nitrogen sources.", + "section": "INTRO", + "ner": [ + [ + 13, + 17, + "Mep2", + "protein" + ], + [ + 50, + 58, + "ammonium", + "chemical" + ], + [ + 87, + 95, + "nitrogen", + "chemical" + ] + ] + }, + { + "sid": 23, + "sent": "With the exception of the human RhCG structure, no structural information is available for eukaryotic ammonium transporters.", + "section": "INTRO", + "ner": [ + [ + 26, + 31, + "human", + "species" + ], + [ + 32, + 36, + "RhCG", + "protein" + ], + [ + 37, + 46, + "structure", + "evidence" + ], + [ + 91, + 101, + "eukaryotic", + "taxonomy_domain" + ], + [ + 102, + 123, + "ammonium transporters", + "protein_type" + ] + ] + }, + { + "sid": 24, + "sent": "By contrast, several bacterial Amt orthologues have been characterized in detail via high-resolution crystal structures and a number of molecular dynamics (MD) studies.", + "section": "INTRO", + "ner": [ + [ + 21, + 30, + "bacterial", + "taxonomy_domain" + ], + [ + 31, + 34, + "Amt", + "protein_type" + ], + [ + 101, + 119, + "crystal structures", + "evidence" + ], + [ + 136, + 154, + "molecular dynamics", + "experimental_method" + ], + [ + 156, + 158, + "MD", + "experimental_method" + ] + ] + }, + { + "sid": 25, + "sent": "All the solved structures including that of RhCG are very similar, establishing the basic architecture of ammonium transporters.", + "section": "INTRO", + "ner": [ + [ + 15, + 25, + "structures", + "evidence" + ], + [ + 44, + 48, + "RhCG", + "protein" + ], + [ + 106, + 127, + "ammonium transporters", + "protein_type" + ] + ] + }, + { + "sid": 26, + "sent": "The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium.", + "section": "INTRO", + "ner": [ + [ + 18, + 24, + "stable", + "protein_state" + ], + [ + 25, + 32, + "trimers", + "oligomeric_state" + ], + [ + 44, + 51, + "monomer", + "oligomeric_state" + ], + [ + 62, + 75, + "transmembrane", + "structure_element" + ], + [ + 77, + 79, + "TM", + "structure_element" + ], + [ + 81, + 88, + "helices", + "structure_element" + ], + [ + 95, + 110, + "central channel", + "site" + ], + [ + 132, + 140, + "ammonium", + "chemical" + ] + ] + }, + { + "sid": 27, + "sent": "All structures show the transporters in open conformations.", + "section": "INTRO", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 24, + 36, + "transporters", + "protein_type" + ], + [ + 40, + 44, + "open", + "protein_state" + ] + ] + }, + { + "sid": 28, + "sent": "Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport).", + "section": "INTRO", + "ner": [ + [ + 48, + 55, + "ammonia", + "chemical" + ], + [ + 117, + 133, + "Amt/Mep proteins", + "protein_type" + ], + [ + 146, + 152, + "active", + "protein_state" + ], + [ + 154, + 179, + "electrogenic transporters", + "protein_type" + ], + [ + 190, + 194, + "NH4+", + "chemical" + ], + [ + 208, + 211, + "NH3", + "chemical" + ], + [ + 212, + 214, + "H+", + "chemical" + ] + ] + }, + { + "sid": 29, + "sent": "A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport.", + "section": "INTRO", + "ner": [ + [ + 2, + 18, + "highly conserved", + "protein_state" + ], + [ + 27, + 34, + "channel", + "site" + ], + [ + 42, + 51, + "histidine", + "residue_name" + ], + [ + 72, + 86, + "twin-His motif", + "structure_element" + ], + [ + 128, + 131, + "NH3", + "chemical" + ], + [ + 150, + 157, + "channel", + "site" + ], + [ + 165, + 168, + "NH3", + "chemical" + ], + [ + 169, + 171, + "H+", + "chemical" + ] + ] + }, + { + "sid": 30, + "sent": "Ammonium transport is tightly regulated.", + "section": "INTRO", + "ner": [ + [ + 0, + 8, + "Ammonium", + "chemical" + ] + ] + }, + { + "sid": 31, + "sent": "In animals, this is due to toxicity of elevated intracellular ammonium levels, whereas for microorganisms ammonium is a preferred nitrogen source.", + "section": "INTRO", + "ner": [ + [ + 3, + 10, + "animals", + "taxonomy_domain" + ], + [ + 62, + 70, + "ammonium", + "chemical" + ], + [ + 91, + 105, + "microorganisms", + "taxonomy_domain" + ], + [ + 106, + 114, + "ammonium", + "chemical" + ] + ] + }, + { + "sid": 32, + "sent": "In bacteria, amt genes are present in an operon with glnK, encoding a PII-like signal transduction class protein.", + "section": "INTRO", + "ner": [ + [ + 3, + 11, + "bacteria", + "taxonomy_domain" + ], + [ + 13, + 16, + "amt", + "gene" + ], + [ + 53, + 57, + "glnK", + "gene" + ], + [ + 70, + 112, + "PII-like signal transduction class protein", + "protein_type" + ] + ] + }, + { + "sid": 33, + "sent": "By binding tightly to Amt proteins without inducing a conformational change in the transporter, GlnK sterically blocks ammonium conductance when nitrogen levels are sufficient.", + "section": "INTRO", + "ner": [ + [ + 22, + 34, + "Amt proteins", + "protein_type" + ], + [ + 83, + 94, + "transporter", + "protein_type" + ], + [ + 96, + 100, + "GlnK", + "protein_type" + ], + [ + 119, + 127, + "ammonium", + "chemical" + ] + ] + }, + { + "sid": 34, + "sent": "Under conditions of nitrogen limitation, GlnK becomes uridylated, blocking its ability to bind and inhibit Amt proteins.", + "section": "INTRO", + "ner": [ + [ + 20, + 28, + "nitrogen", + "chemical" + ], + [ + 41, + 45, + "GlnK", + "protein_type" + ], + [ + 54, + 64, + "uridylated", + "protein_state" + ], + [ + 107, + 119, + "Amt proteins", + "protein_type" + ] + ] + }, + { + "sid": 35, + "sent": "Importantly, eukaryotes do not have GlnK orthologues and have a different mechanism for regulation of ammonium transport activity.", + "section": "INTRO", + "ner": [ + [ + 13, + 23, + "eukaryotes", + "taxonomy_domain" + ], + [ + 36, + 40, + "GlnK", + "protein_type" + ], + [ + 102, + 110, + "ammonium", + "chemical" + ] + ] + }, + { + "sid": 36, + "sent": "In plants, transporter phosphorylation and dephosphorylation are known to regulate activity.", + "section": "INTRO", + "ner": [ + [ + 3, + 9, + "plants", + "taxonomy_domain" + ], + [ + 11, + 22, + "transporter", + "protein_type" + ], + [ + 23, + 38, + "phosphorylation", + "ptm" + ], + [ + 43, + 60, + "dephosphorylation", + "ptm" + ] + ] + }, + { + "sid": 37, + "sent": "In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change.", + "section": "INTRO", + "ner": [ + [ + 3, + 16, + "S. cerevisiae", + "species" + ], + [ + 18, + 33, + "phosphorylation", + "ptm" + ], + [ + 37, + 43, + "Ser457", + "residue_name_number" + ], + [ + 55, + 72, + "C-terminal region", + "structure_element" + ], + [ + 74, + 77, + "CTR", + "structure_element" + ], + [ + 127, + 131, + "Mep2", + "protein_type" + ] + ] + }, + { + "sid": 38, + "sent": "To elucidate the mechanism of Mep2 transport regulation, we present here X-ray crystal structures of the Mep2 transceptors from S. cerevisiae and C. albicans.", + "section": "INTRO", + "ner": [ + [ + 30, + 34, + "Mep2", + "protein_type" + ], + [ + 73, + 97, + "X-ray crystal structures", + "evidence" + ], + [ + 105, + 122, + "Mep2 transceptors", + "protein_type" + ], + [ + 128, + 141, + "S. cerevisiae", + "species" + ], + [ + 146, + 157, + "C. albicans", + "species" + ] + ] + }, + { + "sid": 39, + "sent": "The structures are similar to each other but show considerable differences to all other ammonium transporter structures.", + "section": "INTRO", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 88, + 108, + "ammonium transporter", + "protein_type" + ], + [ + 109, + 119, + "structures", + "evidence" + ] + ] + }, + { + "sid": 40, + "sent": "The most striking difference is the fact that the Mep2 proteins have closed conformations.", + "section": "INTRO", + "ner": [ + [ + 50, + 63, + "Mep2 proteins", + "protein_type" + ], + [ + 69, + 75, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 41, + "sent": "The putative phosphorylation site is solvent accessible and located in a negatively charged pocket \u223c30\u2009\u00c5 away from the channel exit.", + "section": "INTRO", + "ner": [ + [ + 13, + 33, + "phosphorylation site", + "site" + ], + [ + 37, + 55, + "solvent accessible", + "protein_state" + ], + [ + 73, + 98, + "negatively charged pocket", + "site" + ], + [ + 119, + 131, + "channel exit", + "site" + ] + ] + }, + { + "sid": 42, + "sent": "The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR.", + "section": "INTRO", + "ner": [ + [ + 4, + 12, + "channels", + "site" + ], + [ + 16, + 49, + "phosphorylation-mimicking mutants", + "protein_state" + ], + [ + 53, + 64, + "C. albicans", + "species" + ], + [ + 65, + 69, + "Mep2", + "protein" + ], + [ + 80, + 86, + "closed", + "protein_state" + ], + [ + 134, + 143, + "conserved", + "protein_state" + ], + [ + 156, + 159, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 43, + "sent": "Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport.", + "section": "INTRO", + "ner": [ + [ + 16, + 25, + "structure", + "evidence" + ], + [ + 42, + 54, + "Mep2 variant", + "mutant" + ], + [ + 55, + 62, + "lacking", + "protein_state" + ], + [ + 67, + 74, + "segment", + "structure_element" + ], + [ + 90, + 110, + "phosphorylation site", + "site" + ], + [ + 203, + 213, + "eukaryotic", + "taxonomy_domain" + ], + [ + 214, + 222, + "ammonium", + "chemical" + ] + ] + }, + { + "sid": 44, + "sent": "General architecture of Mep2 ammonium transceptors", + "section": "RESULTS", + "ner": [ + [ + 24, + 28, + "Mep2", + "protein_type" + ], + [ + 29, + 50, + "ammonium transceptors", + "protein_type" + ] + ] + }, + { + "sid": 45, + "sent": "The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2\u2009\u00c5 resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "Mep2", + "protein" + ], + [ + 20, + 33, + "S. cerevisiae", + "species" + ], + [ + 35, + 41, + "ScMep2", + "protein" + ], + [ + 47, + 60, + "overexpressed", + "experimental_method" + ], + [ + 64, + 77, + "S. cerevisiae", + "species" + ], + [ + 103, + 126, + "structure determination", + "experimental_method" + ], + [ + 130, + 151, + "X-ray crystallography", + "experimental_method" + ], + [ + 186, + 207, + "molecular replacement", + "experimental_method" + ], + [ + 209, + 211, + "MR", + "experimental_method" + ], + [ + 222, + 237, + "archaebacterial", + "taxonomy_domain" + ], + [ + 238, + 243, + "Amt-1", + "protein" + ], + [ + 244, + 253, + "structure", + "evidence" + ] + ] + }, + { + "sid": 46, + "sent": "Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure\u2013function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals.", + "section": "RESULTS", + "ner": [ + [ + 40, + 49, + "structure", + "evidence" + ], + [ + 89, + 95, + "ScMep2", + "protein" + ], + [ + 120, + 146, + "structure\u2013function studies", + "experimental_method" + ], + [ + 162, + 168, + "fungal", + "taxonomy_domain" + ], + [ + 169, + 173, + "Mep2", + "protein_type" + ], + [ + 204, + 234, + "overexpressed and screened for", + "experimental_method" + ], + [ + 255, + 263, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 47, + "sent": "Of these, Mep2 from C. albicans (CaMep2) showed superior stability in relatively harsh detergents such as nonyl-glucoside, allowing structure determination in two different crystal forms to high resolution (up to 1.5\u2009\u00c5).", + "section": "RESULTS", + "ner": [ + [ + 10, + 14, + "Mep2", + "protein" + ], + [ + 20, + 31, + "C. albicans", + "species" + ], + [ + 33, + 39, + "CaMep2", + "protein" + ], + [ + 132, + 155, + "structure determination", + "experimental_method" + ], + [ + 173, + 186, + "crystal forms", + "evidence" + ] + ] + }, + { + "sid": 48, + "sent": "Despite different crystal packing (Supplementary Table 1), the two CaMep2 structures are identical to each other and very similar to ScMep2 (C\u03b1 r.m.s.d.", + "section": "RESULTS", + "ner": [ + [ + 67, + 73, + "CaMep2", + "protein" + ], + [ + 74, + 84, + "structures", + "evidence" + ], + [ + 133, + 139, + "ScMep2", + "protein" + ], + [ + 144, + 152, + "r.m.s.d.", + "evidence" + ] + ] + }, + { + "sid": 49, + "sent": "(root mean square deviation)=0.7\u2009\u00c5 for 434 residues), with the main differences confined to the N terminus and the CTR (Fig. 1).", + "section": "RESULTS", + "ner": [ + [ + 1, + 27, + "root mean square deviation", + "evidence" + ], + [ + 115, + 118, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 50, + "sent": "Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered.", + "section": "RESULTS", + "ner": [ + [ + 0, + 16, + "Electron density", + "evidence" + ], + [ + 100, + 102, + "43", + "residue_range" + ], + [ + 104, + 110, + "ScMep2", + "protein" + ], + [ + 116, + 118, + "25", + "residue_range" + ], + [ + 129, + 135, + "CaMep2", + "protein" + ], + [ + 148, + 164, + "poorly conserved", + "protein_state" + ], + [ + 180, + 190, + "disordered", + "protein_state" + ] + ] + }, + { + "sid": 51, + "sent": "Both Mep2 proteins show the archetypal trimeric assemblies in which each monomer consists of 11 TM helices surrounding a central pore.", + "section": "RESULTS", + "ner": [ + [ + 5, + 18, + "Mep2 proteins", + "protein_type" + ], + [ + 39, + 47, + "trimeric", + "oligomeric_state" + ], + [ + 73, + 80, + "monomer", + "oligomeric_state" + ], + [ + 96, + 106, + "TM helices", + "structure_element" + ], + [ + 121, + 133, + "central pore", + "structure_element" + ] + ] + }, + { + "sid": 52, + "sent": "Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG.", + "section": "RESULTS", + "ner": [ + [ + 56, + 77, + "ammonium binding site", + "site" + ], + [ + 83, + 91, + "Phe gate", + "site" + ], + [ + 100, + 114, + "twin-His motif", + "structure_element" + ], + [ + 126, + 145, + "hydrophobic channel", + "site" + ], + [ + 191, + 200, + "bacterial", + "taxonomy_domain" + ], + [ + 201, + 213, + "transporters", + "protein_type" + ], + [ + 218, + 222, + "RhCG", + "protein" + ] + ] + }, + { + "sid": 53, + "sent": "In the remainder of the manuscript, we will specifically discuss CaMep2 due to the superior resolution of the structure.", + "section": "RESULTS", + "ner": [ + [ + 65, + 71, + "CaMep2", + "protein" + ], + [ + 110, + 119, + "structure", + "evidence" + ] + ] + }, + { + "sid": 54, + "sent": "Unless specifically stated, the drawn conclusions also apply to ScMep2.", + "section": "RESULTS", + "ner": [ + [ + 64, + 70, + "ScMep2", + "protein" + ] + ] + }, + { + "sid": 55, + "sent": "While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (C\u03b1 r.m.s.d. with Amt-1=1.4\u2009\u00c5 for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR.", + "section": "RESULTS", + "ner": [ + [ + 34, + 38, + "Mep2", + "protein" + ], + [ + 65, + 76, + "prokaryotic", + "taxonomy_domain" + ], + [ + 77, + 89, + "transporters", + "protein_type" + ], + [ + 94, + 102, + "r.m.s.d.", + "evidence" + ], + [ + 108, + 113, + "Amt-1", + "protein" + ], + [ + 190, + 209, + "intracellular loops", + "structure_element" + ], + [ + 211, + 215, + "ICLs", + "structure_element" + ], + [ + 217, + 221, + "ICL1", + "structure_element" + ], + [ + 226, + 230, + "ICL3", + "structure_element" + ], + [ + 240, + 243, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 56, + "sent": "The N termini of the Mep2 proteins are \u223c20\u201325 residues longer compared with their bacterial counterparts (Figs 1 and 2), substantially increasing the size of the extracellular domain.", + "section": "RESULTS", + "ner": [ + [ + 21, + 34, + "Mep2 proteins", + "protein_type" + ], + [ + 40, + 45, + "20\u201325", + "residue_range" + ], + [ + 82, + 91, + "bacterial", + "taxonomy_domain" + ], + [ + 162, + 182, + "extracellular domain", + "structure_element" + ] + ] + }, + { + "sid": 57, + "sent": "Moreover, the N terminus of one monomer interacts with the extended extracellular loop ECL5 of a neighbouring monomer.", + "section": "RESULTS", + "ner": [ + [ + 32, + 39, + "monomer", + "oligomeric_state" + ], + [ + 68, + 86, + "extracellular loop", + "structure_element" + ], + [ + 87, + 91, + "ECL5", + "structure_element" + ], + [ + 110, + 117, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 58, + "sent": "Together with additional, smaller differences in other extracellular loops, these changes generate a distinct vestibule leading to the ammonium binding site that is much more pronounced than in the bacterial proteins.", + "section": "RESULTS", + "ner": [ + [ + 55, + 74, + "extracellular loops", + "structure_element" + ], + [ + 110, + 119, + "vestibule", + "structure_element" + ], + [ + 135, + 156, + "ammonium binding site", + "site" + ], + [ + 198, + 207, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 59, + "sent": "The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins.", + "section": "RESULTS", + "ner": [ + [ + 15, + 24, + "vestibule", + "structure_element" + ], + [ + 49, + 56, + "monomer", + "oligomeric_state" + ], + [ + 107, + 111, + "Mep2", + "protein" + ], + [ + 112, + 118, + "trimer", + "oligomeric_state" + ], + [ + 143, + 148, + "plant", + "taxonomy_domain" + ], + [ + 149, + 161, + "AMT proteins", + "protein_type" + ] + ] + }, + { + "sid": 60, + "sent": "However, given that an N-terminal deletion mutant (2-27\u0394) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear.", + "section": "RESULTS", + "ner": [ + [ + 34, + 49, + "deletion mutant", + "protein_state" + ], + [ + 51, + 56, + "2-27\u0394", + "mutant" + ], + [ + 75, + 84, + "wild-type", + "protein_state" + ], + [ + 86, + 88, + "WT", + "protein_state" + ], + [ + 90, + 94, + "Mep2", + "protein" + ], + [ + 106, + 114, + "ammonium", + "chemical" + ], + [ + 194, + 198, + "Mep2", + "protein" + ] + ] + }, + { + "sid": 61, + "sent": "Mep2 channels are closed by a two-tier channel block", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "Mep2", + "protein" + ], + [ + 5, + 13, + "channels", + "site" + ], + [ + 18, + 24, + "closed", + "protein_state" + ], + [ + 39, + 52, + "channel block", + "structure_element" + ] + ] + }, + { + "sid": 62, + "sent": "The largest differences between the Mep2 structures and the other known ammonium transporter structures are located on the intracellular side of the membrane.", + "section": "RESULTS", + "ner": [ + [ + 36, + 40, + "Mep2", + "protein" + ], + [ + 41, + 51, + "structures", + "evidence" + ], + [ + 72, + 92, + "ammonium transporter", + "protein_type" + ], + [ + 93, + 103, + "structures", + "evidence" + ] + ] + }, + { + "sid": 63, + "sent": "In the vicinity of the Mep2 channel exit, the cytoplasmic end of TM2 has unwound, generating a longer ICL1 even though there are no insertions in this region compared to the bacterial proteins (Figs 2 and 4).", + "section": "RESULTS", + "ner": [ + [ + 23, + 27, + "Mep2", + "protein" + ], + [ + 28, + 40, + "channel exit", + "site" + ], + [ + 65, + 68, + "TM2", + "structure_element" + ], + [ + 102, + 106, + "ICL1", + "structure_element" + ], + [ + 174, + 183, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 64, + "sent": "ICL1 has also moved inwards relative to its position in the bacterial Amts.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "ICL1", + "structure_element" + ], + [ + 60, + 69, + "bacterial", + "taxonomy_domain" + ], + [ + 70, + 74, + "Amts", + "protein_type" + ] + ] + }, + { + "sid": 65, + "sent": "The largest backbone movements of equivalent residues within ICL1 are \u223c10\u2009\u00c5, markedly affecting the conserved basic RxK motif (Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 61, + 65, + "ICL1", + "structure_element" + ], + [ + 100, + 109, + "conserved", + "protein_state" + ], + [ + 110, + 115, + "basic", + "protein_state" + ], + [ + 116, + 125, + "RxK motif", + "structure_element" + ] + ] + }, + { + "sid": 66, + "sent": "The head group of Arg54 has moved \u223c11\u2009\u00c5 relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20\u2009\u00c5. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 18, + 23, + "Arg54", + "residue_name_number" + ], + [ + 60, + 65, + "Amt-1", + "protein" + ], + [ + 119, + 124, + "Lys55", + "residue_name_number" + ], + [ + 167, + 172, + "Lys56", + "residue_name_number" + ], + [ + 180, + 185, + "basic", + "protein_state" + ], + [ + 186, + 191, + "motif", + "structure_element" + ], + [ + 231, + 235, + "Mep2", + "protein" + ], + [ + 236, + 246, + "structures", + "evidence" + ], + [ + 283, + 288, + "Amt-1", + "protein" + ] + ] + }, + { + "sid": 67, + "sent": "In addition to changing the RxK motif, the movement of ICL1 has another, crucial functional consequence.", + "section": "RESULTS", + "ner": [ + [ + 28, + 37, + "RxK motif", + "structure_element" + ], + [ + 55, + 59, + "ICL1", + "structure_element" + ] + ] + }, + { + "sid": 68, + "sent": "At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the \u025b2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5).", + "section": "RESULTS", + "ner": [ + [ + 25, + 28, + "TM1", + "structure_element" + ], + [ + 67, + 87, + "relatively conserved", + "protein_state" + ], + [ + 88, + 93, + "Tyr49", + "residue_name_number" + ], + [ + 95, + 100, + "Tyr53", + "residue_name_number" + ], + [ + 104, + 110, + "ScMep2", + "protein" + ], + [ + 127, + 140, + "hydrogen bond", + "bond_interaction" + ], + [ + 174, + 194, + "absolutely conserved", + "protein_state" + ], + [ + 195, + 201, + "His342", + "residue_name_number" + ], + [ + 209, + 223, + "twin-His motif", + "structure_element" + ], + [ + 225, + 231, + "His348", + "residue_name_number" + ], + [ + 235, + 241, + "ScMep2", + "protein" + ], + [ + 256, + 263, + "channel", + "site" + ] + ] + }, + { + "sid": 69, + "sent": "In bacterial Amt proteins, this Tyr side chain is rotated \u223c4\u2009\u00c5 away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 3, + 12, + "bacterial", + "taxonomy_domain" + ], + [ + 13, + 25, + "Amt proteins", + "protein_type" + ], + [ + 32, + 35, + "Tyr", + "residue_name" + ], + [ + 113, + 116, + "TM1", + "structure_element" + ], + [ + 130, + 137, + "channel", + "site" + ], + [ + 138, + 142, + "open", + "protein_state" + ], + [ + 151, + 160, + "histidine", + "residue_name" + ] + ] + }, + { + "sid": 70, + "sent": "Compared with ICL1, the backbone conformational changes observed for the neighbouring ICL2 are smaller, but large shifts are nevertheless observed for the conserved residues Glu140 and Arg141 (Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "ICL1", + "structure_element" + ], + [ + 86, + 90, + "ICL2", + "structure_element" + ], + [ + 155, + 164, + "conserved", + "protein_state" + ], + [ + 174, + 180, + "Glu140", + "residue_name_number" + ], + [ + 185, + 191, + "Arg141", + "residue_name_number" + ] + ] + }, + { + "sid": 71, + "sent": "Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to \u223c10\u2009\u00c5 and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5).", + "section": "RESULTS", + "ner": [ + [ + 23, + 27, + "ICL3", + "structure_element" + ], + [ + 40, + 65, + "pseudo-symmetrical halves", + "structure_element" + ], + [ + 67, + 72, + "TM1-5", + "structure_element" + ], + [ + 77, + 83, + "TM6-10", + "structure_element" + ], + [ + 92, + 103, + "transporter", + "protein_type" + ], + [ + 180, + 187, + "channel", + "site" + ] + ] + }, + { + "sid": 72, + "sent": "This two-tier channel block likely ensures that very little ammonium transport will take place under nitrogen-sufficient conditions.", + "section": "RESULTS", + "ner": [ + [ + 14, + 27, + "channel block", + "structure_element" + ], + [ + 60, + 68, + "ammonium", + "chemical" + ], + [ + 101, + 109, + "nitrogen", + "chemical" + ] + ] + }, + { + "sid": 73, + "sent": "The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif.", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "closed", + "protein_state" + ], + [ + 24, + 31, + "channel", + "site" + ], + [ + 55, + 65, + "no density", + "evidence" + ], + [ + 93, + 101, + "ammonium", + "chemical" + ], + [ + 106, + 111, + "water", + "chemical" + ], + [ + 157, + 161, + "Mep2", + "protein" + ], + [ + 162, + 169, + "channel", + "site" + ], + [ + 183, + 197, + "twin-His motif", + "structure_element" + ] + ] + }, + { + "sid": 74, + "sent": "Significantly, this is also true for ScMep2, which was crystallized in the presence of 0.2\u2009M ammonium ions (see Methods section).", + "section": "RESULTS", + "ner": [ + [ + 37, + 43, + "ScMep2", + "protein" + ], + [ + 55, + 67, + "crystallized", + "experimental_method" + ], + [ + 93, + 101, + "ammonium", + "chemical" + ] + ] + }, + { + "sid": 75, + "sent": "The final region in Mep2 that shows large differences compared with the bacterial transporters is the CTR.", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "Mep2", + "protein" + ], + [ + 72, + 81, + "bacterial", + "taxonomy_domain" + ], + [ + 82, + 94, + "transporters", + "protein_type" + ], + [ + 102, + 105, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 76, + "sent": "In Mep2, the CTR has moved away and makes relatively few contacts with the main body of the transporter, generating a more elongated protein (Figs 1 and 4).", + "section": "RESULTS", + "ner": [ + [ + 3, + 7, + "Mep2", + "protein" + ], + [ + 13, + 16, + "CTR", + "structure_element" + ], + [ + 75, + 84, + "main body", + "structure_element" + ], + [ + 92, + 103, + "transporter", + "protein_type" + ], + [ + 123, + 132, + "elongated", + "protein_state" + ] + ] + }, + { + "sid": 77, + "sent": "By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure.", + "section": "RESULTS", + "ner": [ + [ + 20, + 30, + "structures", + "evidence" + ], + [ + 34, + 43, + "bacterial", + "taxonomy_domain" + ], + [ + 58, + 61, + "CTR", + "structure_element" + ], + [ + 89, + 104, + "N-terminal half", + "structure_element" + ], + [ + 112, + 124, + "transporters", + "protein_type" + ], + [ + 143, + 148, + "TM1-5", + "structure_element" + ], + [ + 171, + 178, + "compact", + "protein_state" + ], + [ + 179, + 188, + "structure", + "evidence" + ] + ] + }, + { + "sid": 78, + "sent": "This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 49, + 70, + "universally conserved", + "protein_state" + ], + [ + 91, + 94, + "CTR", + "structure_element" + ], + [ + 105, + 111, + "Arg415", + "residue_name_number" + ], + [ + 112, + 115, + "370", + "residue_number" + ], + [ + 118, + 124, + "Glu421", + "residue_name_number" + ], + [ + 125, + 128, + "376", + "residue_number" + ], + [ + 131, + 137, + "Gly424", + "residue_name_number" + ], + [ + 138, + 141, + "379", + "residue_number" + ], + [ + 144, + 150, + "Asp426", + "residue_name_number" + ], + [ + 151, + 154, + "381", + "residue_number" + ], + [ + 160, + 167, + "Tyr 435", + "residue_name_number" + ], + [ + 168, + 171, + "390", + "residue_number" + ], + [ + 176, + 182, + "CaMep2", + "protein" + ], + [ + 183, + 188, + "Amt-1", + "protein" + ] + ] + }, + { + "sid": 79, + "sent": "These residues include those of the \u2018ExxGxD' motif, which when mutated generate inactive transporters.", + "section": "RESULTS", + "ner": [ + [ + 36, + 50, + "\u2018ExxGxD' motif", + "structure_element" + ], + [ + 63, + 70, + "mutated", + "experimental_method" + ], + [ + 80, + 88, + "inactive", + "protein_state" + ], + [ + 89, + 101, + "transporters", + "protein_type" + ] + ] + }, + { + "sid": 80, + "sent": "In Amt-1 and other bacterial ammonium transporters, these CTR residues interact with residues within the N-terminal half of the protein.", + "section": "RESULTS", + "ner": [ + [ + 3, + 8, + "Amt-1", + "protein" + ], + [ + 19, + 28, + "bacterial", + "taxonomy_domain" + ], + [ + 29, + 50, + "ammonium transporters", + "protein_type" + ], + [ + 58, + 61, + "CTR", + "structure_element" + ], + [ + 105, + 120, + "N-terminal half", + "structure_element" + ] + ] + }, + { + "sid": 81, + "sent": "On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3.", + "section": "RESULTS", + "ner": [ + [ + 17, + 23, + "Tyr390", + "residue_name_number" + ], + [ + 36, + 41, + "Amt-1", + "protein" + ], + [ + 45, + 60, + "hydrogen bonded", + "bond_interaction" + ], + [ + 88, + 97, + "conserved", + "protein_state" + ], + [ + 98, + 104, + "His185", + "residue_name_number" + ], + [ + 130, + 134, + "loop", + "structure_element" + ], + [ + 135, + 139, + "ICL3", + "structure_element" + ] + ] + }, + { + "sid": 82, + "sent": "At the other end of ICL3, the backbone carbonyl groups of Gly172 and Lys173 are hydrogen bonded to the side chain of Arg370.", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "ICL3", + "structure_element" + ], + [ + 58, + 64, + "Gly172", + "residue_name_number" + ], + [ + 69, + 75, + "Lys173", + "residue_name_number" + ], + [ + 80, + 95, + "hydrogen bonded", + "bond_interaction" + ], + [ + 117, + 123, + "Arg370", + "residue_name_number" + ] + ] + }, + { + "sid": 83, + "sent": "Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71).", + "section": "RESULTS", + "ner": [ + [ + 31, + 39, + "modelled", + "experimental_method" + ], + [ + 47, + 53, + "active", + "protein_state" + ], + [ + 55, + 73, + "non-phosphorylated", + "protein_state" + ], + [ + 74, + 79, + "plant", + "taxonomy_domain" + ], + [ + 80, + 89, + "AtAmt-1;1", + "protein" + ], + [ + 90, + 99, + "structure", + "evidence" + ], + [ + 114, + 118, + "Y467", + "residue_name_number" + ], + [ + 119, + 123, + "H239", + "residue_name_number" + ], + [ + 128, + 132, + "D458", + "residue_name_number" + ], + [ + 133, + 136, + "K71", + "residue_name_number" + ] + ] + }, + { + "sid": 84, + "sent": "The result of these interactions is that the CTR \u2018hugs' the N-terminal half of the transporters (Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 45, + 48, + "CTR", + "structure_element" + ], + [ + 60, + 75, + "N-terminal half", + "structure_element" + ], + [ + 83, + 95, + "transporters", + "protein_type" + ] + ] + }, + { + "sid": 85, + "sent": "Also noteworthy is Asp381, the side chain of which interacts strongly with the positive dipole on the N-terminal end of TM2.", + "section": "RESULTS", + "ner": [ + [ + 19, + 25, + "Asp381", + "residue_name_number" + ], + [ + 120, + 123, + "TM2", + "structure_element" + ] + ] + }, + { + "sid": 86, + "sent": "This interaction in the centre of the protein may be particularly important to stabilize the open conformations of ammonium transporters.", + "section": "RESULTS", + "ner": [ + [ + 93, + 97, + "open", + "protein_state" + ], + [ + 115, + 136, + "ammonium transporters", + "protein_type" + ] + ] + }, + { + "sid": 87, + "sent": "In the Mep2 structures, none of the interactions mentioned above are present.", + "section": "RESULTS", + "ner": [ + [ + 7, + 11, + "Mep2", + "protein" + ], + [ + 12, + 22, + "structures", + "evidence" + ] + ] + }, + { + "sid": 88, + "sent": "Phosphorylation target site is at the periphery of Mep2", + "section": "RESULTS", + "ner": [ + [ + 0, + 27, + "Phosphorylation target site", + "site" + ], + [ + 51, + 55, + "Mep2", + "protein" + ] + ] + }, + { + "sid": 89, + "sent": "Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions.", + "section": "RESULTS", + "ner": [ + [ + 51, + 57, + "Ser457", + "residue_name_number" + ], + [ + 61, + 67, + "ScMep2", + "protein" + ], + [ + 86, + 92, + "Ser453", + "residue_name_number" + ], + [ + 96, + 102, + "CaMep2", + "protein" + ], + [ + 107, + 121, + "phosphorylated", + "protein_state" + ], + [ + 129, + 150, + "TORC1 effector kinase", + "protein_type" + ], + [ + 151, + 155, + "Npr1", + "protein" + ], + [ + 162, + 170, + "nitrogen", + "chemical" + ] + ] + }, + { + "sid": 90, + "sent": "In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3\u0394 strain (triple mep\u0394) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1).", + "section": "RESULTS", + "ner": [ + [ + 7, + 17, + "absence of", + "protein_state" + ], + [ + 18, + 22, + "Npr1", + "protein" + ], + [ + 24, + 39, + "plasmid-encoded", + "experimental_method" + ], + [ + 40, + 42, + "WT", + "protein_state" + ], + [ + 43, + 47, + "Mep2", + "protein" + ], + [ + 53, + 66, + "S. cerevisiae", + "species" + ], + [ + 67, + 74, + "mep1-3\u0394", + "mutant" + ], + [ + 83, + 94, + "triple mep\u0394", + "mutant" + ], + [ + 143, + 151, + "ammonium", + "chemical" + ], + [ + 173, + 184, + "transporter", + "protein_type" + ], + [ + 188, + 196, + "inactive", + "protein_state" + ] + ] + }, + { + "sid": 91, + "sent": "Conversely, the phosphorylation-mimicking S457D variant is active both in the triple mep\u0394 background and in a triple mep\u0394 npr1\u0394 strain (Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 16, + 41, + "phosphorylation-mimicking", + "protein_state" + ], + [ + 42, + 47, + "S457D", + "mutant" + ], + [ + 59, + 65, + "active", + "protein_state" + ], + [ + 78, + 89, + "triple mep\u0394", + "mutant" + ], + [ + 110, + 127, + "triple mep\u0394 npr1\u0394", + "mutant" + ] + ] + }, + { + "sid": 92, + "sent": "Mutation of other potential phosphorylation sites in the CTR did not support growth in the npr1\u0394 background.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Mutation", + "experimental_method" + ], + [ + 28, + 49, + "phosphorylation sites", + "site" + ], + [ + 57, + 60, + "CTR", + "structure_element" + ], + [ + 91, + 96, + "npr1\u0394", + "mutant" + ] + ] + }, + { + "sid": 93, + "sent": "Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake.", + "section": "RESULTS", + "ner": [ + [ + 38, + 53, + "phosphorylation", + "ptm" + ], + [ + 57, + 63, + "Ser457", + "residue_name_number" + ], + [ + 57, + 63, + "Ser457", + "residue_name_number" + ], + [ + 74, + 78, + "Mep2", + "protein" + ], + [ + 79, + 86, + "channel", + "site" + ], + [ + 96, + 104, + "ammonium", + "chemical" + ] + ] + }, + { + "sid": 94, + "sent": "Ser457 is located in a part of the CTR that is conserved in a subgroup of Mep2 proteins, but which is not present in bacterial proteins (Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Ser457", + "residue_name_number" + ], + [ + 35, + 38, + "CTR", + "structure_element" + ], + [ + 47, + 56, + "conserved", + "protein_state" + ], + [ + 74, + 87, + "Mep2 proteins", + "protein_type" + ], + [ + 117, + 126, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 95, + "sent": "This segment (residues 450\u2013457 in ScMep2 and 446\u2013453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 5, + 12, + "segment", + "structure_element" + ], + [ + 23, + 30, + "450\u2013457", + "residue_range" + ], + [ + 34, + 40, + "ScMep2", + "protein" + ], + [ + 45, + 52, + "446\u2013453", + "residue_range" + ], + [ + 56, + 62, + "CaMep2", + "protein" + ], + [ + 78, + 104, + "autoinhibitory (AI) region", + "structure_element" + ], + [ + 132, + 139, + "removal", + "experimental_method" + ], + [ + 153, + 159, + "active", + "protein_state" + ], + [ + 160, + 171, + "transporter", + "protein_type" + ], + [ + 179, + 189, + "absence of", + "protein_state" + ], + [ + 190, + 194, + "Npr1", + "protein" + ] + ] + }, + { + "sid": 96, + "sent": "Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 13, + 22, + "AI region", + "structure_element" + ], + [ + 31, + 35, + "Npr1", + "protein" + ], + [ + 36, + 56, + "phosphorylation site", + "site" + ], + [ + 70, + 80, + "structures", + "evidence" + ], + [ + 111, + 120, + "AI region", + "structure_element" + ], + [ + 145, + 148, + "CTR", + "structure_element" + ], + [ + 191, + 197, + "trimer", + "oligomeric_state" + ], + [ + 219, + 228, + "bacterial", + "taxonomy_domain" + ], + [ + 229, + 239, + "structures", + "evidence" + ] + ] + }, + { + "sid": 97, + "sent": "The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "AI region", + "structure_element" + ], + [ + 52, + 55, + "TM2", + "structure_element" + ], + [ + 60, + 63, + "TM4", + "structure_element" + ], + [ + 88, + 97, + "main body", + "structure_element" + ], + [ + 105, + 116, + "transporter", + "protein_type" + ], + [ + 126, + 129, + "CTR", + "structure_element" + ], + [ + 189, + 195, + "Val447", + "residue_name_number" + ], + [ + 197, + 203, + "Asp449", + "residue_name_number" + ], + [ + 205, + 211, + "Pro450", + "residue_name_number" + ], + [ + 216, + 222, + "Arg452", + "residue_name_number" + ] + ] + }, + { + "sid": 98, + "sent": "The AI regions have very similar conformations in CaMep2 and ScMep2, despite considerable differences in the rest of the CTR (Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "AI regions", + "structure_element" + ], + [ + 50, + 56, + "CaMep2", + "protein" + ], + [ + 61, + 67, + "ScMep2", + "protein" + ], + [ + 121, + 124, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 99, + "sent": "Strikingly, the Npr1 target serine residue is located at the periphery of the trimer, far away (\u223c30\u2009\u00c5) from any channel exit (Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 16, + 34, + "Npr1 target serine", + "site" + ], + [ + 78, + 84, + "trimer", + "oligomeric_state" + ], + [ + 112, + 124, + "channel exit", + "site" + ] + ] + }, + { + "sid": 100, + "sent": "Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 45, + 51, + "trimer", + "oligomeric_state" + ], + [ + 57, + 73, + "electron density", + "evidence" + ], + [ + 82, + 88, + "serine", + "residue_name" + ], + [ + 113, + 117, + "Mep2", + "protein" + ], + [ + 118, + 128, + "structures", + "evidence" + ], + [ + 152, + 170, + "non-phosphorylated", + "protein_state" + ] + ] + }, + { + "sid": 101, + "sent": "This makes sense since the proteins were expressed in rich medium and confirms the recent suggestion by Boeckstaens et al. that the non-phosphorylated form of Mep2 corresponds to the inactive state.", + "section": "RESULTS", + "ner": [ + [ + 132, + 150, + "non-phosphorylated", + "protein_state" + ], + [ + 159, + 163, + "Mep2", + "protein" + ], + [ + 183, + 191, + "inactive", + "protein_state" + ] + ] + }, + { + "sid": 102, + "sent": "For ScMep2, Ser457 is the most C-terminal residue for which electron density is visible, indicating that the region beyond Ser457 is disordered.", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "ScMep2", + "protein" + ], + [ + 12, + 18, + "Ser457", + "residue_name_number" + ], + [ + 60, + 76, + "electron density", + "evidence" + ], + [ + 123, + 129, + "Ser457", + "residue_name_number" + ], + [ + 133, + 143, + "disordered", + "protein_state" + ] + ] + }, + { + "sid": 103, + "sent": "In CaMep2, the visible part of the sequence extends for two residues beyond Ser453 (Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 3, + 9, + "CaMep2", + "protein" + ], + [ + 76, + 82, + "Ser453", + "residue_name_number" + ] + ] + }, + { + "sid": 104, + "sent": "The peripheral location and disorder of the CTR beyond the kinase target site should facilitate the phosphorylation by Npr1.", + "section": "RESULTS", + "ner": [ + [ + 28, + 36, + "disorder", + "protein_state" + ], + [ + 44, + 47, + "CTR", + "structure_element" + ], + [ + 59, + 77, + "kinase target site", + "site" + ], + [ + 100, + 115, + "phosphorylation", + "ptm" + ], + [ + 119, + 123, + "Npr1", + "protein" + ] + ] + }, + { + "sid": 105, + "sent": "The disordered part of the CTR is not conserved in ammonium transporters (Fig. 2), suggesting that it is not important for transport.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "disordered", + "protein_state" + ], + [ + 27, + 30, + "CTR", + "structure_element" + ], + [ + 34, + 47, + "not conserved", + "protein_state" + ], + [ + 51, + 72, + "ammonium transporters", + "protein_type" + ] + ] + }, + { + "sid": 106, + "sent": "Interestingly, a ScMep2 457\u0394 truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1.", + "section": "RESULTS", + "ner": [ + [ + 17, + 23, + "ScMep2", + "protein" + ], + [ + 24, + 28, + "457\u0394", + "mutant" + ], + [ + 29, + 46, + "truncation mutant", + "protein_state" + ], + [ + 83, + 89, + "Ser457", + "residue_name_number" + ], + [ + 118, + 130, + "low activity", + "protein_state" + ], + [ + 211, + 226, + "phosphorylation", + "ptm" + ], + [ + 230, + 234, + "Npr1", + "protein" + ] + ] + }, + { + "sid": 107, + "sent": "The same mutant lacking the His-tag has WT properties (Supplementary Fig. 1b), confirming that the region following the phosphorylation site is dispensable for function.", + "section": "RESULTS", + "ner": [ + [ + 9, + 15, + "mutant", + "mutant" + ], + [ + 16, + 35, + "lacking the His-tag", + "protein_state" + ], + [ + 40, + 42, + "WT", + "protein_state" + ], + [ + 120, + 140, + "phosphorylation site", + "site" + ] + ] + }, + { + "sid": 108, + "sent": "Mep2 lacking the AI region is conformationally heterogeneous", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "Mep2", + "protein" + ], + [ + 5, + 12, + "lacking", + "protein_state" + ], + [ + 17, + 26, + "AI region", + "structure_element" + ], + [ + 30, + 60, + "conformationally heterogeneous", + "protein_state" + ] + ] + }, + { + "sid": 109, + "sent": "Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter.", + "section": "RESULTS", + "ner": [ + [ + 11, + 17, + "Ser457", + "residue_name_number" + ], + [ + 18, + 21, + "453", + "residue_number" + ], + [ + 38, + 50, + "channel exit", + "site" + ], + [ + 89, + 104, + "phosphorylation", + "ptm" + ], + [ + 115, + 119, + "Mep2", + "protein" + ], + [ + 120, + 127, + "channel", + "site" + ], + [ + 143, + 149, + "active", + "protein_state" + ], + [ + 150, + 161, + "transporter", + "protein_type" + ] + ] + }, + { + "sid": 110, + "sent": "Boeckstaens et al. proposed that phosphorylation does not affect channel activity directly, but instead relieves inhibition by the AI region.", + "section": "RESULTS", + "ner": [ + [ + 33, + 48, + "phosphorylation", + "ptm" + ], + [ + 131, + 140, + "AI region", + "structure_element" + ] + ] + }, + { + "sid": 111, + "sent": "The data behind this hypothesis is the observation that a ScMep2 449-485\u0394 deletion mutant lacking the AI region is highly active in MA uptake both in the triple mep\u0394 and triple mep\u0394 npr1\u0394 backgrounds, implying that this Mep2 variant has a constitutively open channel.", + "section": "RESULTS", + "ner": [ + [ + 58, + 64, + "ScMep2", + "protein" + ], + [ + 65, + 73, + "449-485\u0394", + "mutant" + ], + [ + 74, + 89, + "deletion mutant", + "protein_state" + ], + [ + 90, + 97, + "lacking", + "protein_state" + ], + [ + 102, + 111, + "AI region", + "structure_element" + ], + [ + 115, + 128, + "highly active", + "protein_state" + ], + [ + 132, + 134, + "MA", + "chemical" + ], + [ + 154, + 165, + "triple mep\u0394", + "mutant" + ], + [ + 170, + 187, + "triple mep\u0394 npr1\u0394", + "mutant" + ], + [ + 220, + 232, + "Mep2 variant", + "mutant" + ], + [ + 239, + 258, + "constitutively open", + "protein_state" + ], + [ + 259, + 266, + "channel", + "site" + ] + ] + }, + { + "sid": 112, + "sent": "We obtained a similar result for ammonium uptake by the 446\u0394 mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442\u0394 truncation mutant and determined the crystal structure using data to 3.4\u2009\u00c5 resolution.", + "section": "RESULTS", + "ner": [ + [ + 56, + 60, + "446\u0394", + "mutant" + ], + [ + 61, + 67, + "mutant", + "protein_state" + ], + [ + 125, + 149, + "constructed and purified", + "experimental_method" + ], + [ + 164, + 170, + "CaMep2", + "protein" + ], + [ + 171, + 175, + "442\u0394", + "mutant" + ], + [ + 176, + 193, + "truncation mutant", + "protein_state" + ], + [ + 198, + 208, + "determined", + "experimental_method" + ], + [ + 213, + 230, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 113, + "sent": "The structure shows that removal of the AI region markedly increases the dynamics of the cytoplasmic parts of the transporter.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 25, + 35, + "removal of", + "experimental_method" + ], + [ + 40, + 49, + "AI region", + "structure_element" + ], + [ + 89, + 106, + "cytoplasmic parts", + "structure_element" + ], + [ + 114, + 125, + "transporter", + "protein_type" + ] + ] + }, + { + "sid": 114, + "sent": "This is not unexpected given the fact that the AI region bridges the CTR and the main body of Mep2 (Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 47, + 56, + "AI region", + "structure_element" + ], + [ + 69, + 72, + "CTR", + "structure_element" + ], + [ + 81, + 90, + "main body", + "structure_element" + ], + [ + 94, + 98, + "Mep2", + "protein" + ] + ] + }, + { + "sid": 115, + "sent": "Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442\u0394 mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7).", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Density", + "evidence" + ], + [ + 12, + 16, + "ICL3", + "structure_element" + ], + [ + 25, + 28, + "CTR", + "structure_element" + ], + [ + 44, + 50, + "Arg415", + "residue_name_number" + ], + [ + 69, + 73, + "442\u0394", + "mutant" + ], + [ + 74, + 80, + "mutant", + "protein_state" + ], + [ + 90, + 97, + "density", + "evidence" + ], + [ + 112, + 116, + "ICLs", + "structure_element" + ], + [ + 127, + 131, + "ICL1", + "structure_element" + ], + [ + 176, + 185, + "structure", + "evidence" + ] + ] + }, + { + "sid": 116, + "sent": "Interestingly, however, the Tyr49-His342 hydrogen bond that closes the channel in the WT protein is still present (Fig. 7 and Supplementary Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 28, + 33, + "Tyr49", + "residue_name_number" + ], + [ + 34, + 40, + "His342", + "residue_name_number" + ], + [ + 41, + 54, + "hydrogen bond", + "bond_interaction" + ], + [ + 86, + 88, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 117, + "sent": "Why then does this mutant appear to be constitutively active? We propose two possibilities.", + "section": "RESULTS", + "ner": [ + [ + 54, + 60, + "active", + "protein_state" + ] + ] + }, + { + "sid": 118, + "sent": "The first one is that the open state is disfavoured by crystallization because of lower stability or due to crystal packing constraints.", + "section": "RESULTS", + "ner": [ + [ + 26, + 30, + "open", + "protein_state" + ], + [ + 55, + 70, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 119, + "sent": "The second possibility is that the Tyr\u2013His hydrogen bond has to be disrupted by the incoming substrate to open the channel.", + "section": "RESULTS", + "ner": [ + [ + 35, + 56, + "Tyr\u2013His hydrogen bond", + "site" + ], + [ + 106, + 110, + "open", + "protein_state" + ] + ] + }, + { + "sid": 120, + "sent": "The latter model would fit well with the NH3/H+ symport model in which the proton is relayed by the twin-His motif.", + "section": "RESULTS", + "ner": [ + [ + 41, + 44, + "NH3", + "chemical" + ], + [ + 45, + 47, + "H+", + "chemical" + ], + [ + 100, + 114, + "twin-His motif", + "structure_element" + ] + ] + }, + { + "sid": 121, + "sent": "The importance of the Tyr\u2013His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 22, + 43, + "Tyr\u2013His hydrogen bond", + "site" + ], + [ + 80, + 87, + "removal", + "experimental_method" + ], + [ + 95, + 101, + "ScMep2", + "protein" + ], + [ + 102, + 106, + "Y53A", + "mutant" + ], + [ + 107, + 113, + "mutant", + "protein_state" + ], + [ + 127, + 148, + "constitutively active", + "protein_state" + ], + [ + 149, + 160, + "transporter", + "protein_type" + ] + ] + }, + { + "sid": 122, + "sent": "Phosphorylation causes a conformational change in the CTR", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Phosphorylation", + "ptm" + ], + [ + 54, + 57, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 123, + "sent": "Do the Mep2 structures provide any clues regarding the potential effect of phosphorylation?", + "section": "RESULTS", + "ner": [ + [ + 7, + 11, + "Mep2", + "protein" + ], + [ + 12, + 22, + "structures", + "evidence" + ], + [ + 75, + 90, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 124, + "sent": "The side-chain hydroxyl of Ser457/453 is located in a well-defined electronegative pocket that is solvent accessible (Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 27, + 33, + "Ser457", + "residue_name_number" + ], + [ + 34, + 37, + "453", + "residue_number" + ], + [ + 67, + 89, + "electronegative pocket", + "site" + ], + [ + 98, + 116, + "solvent accessible", + "protein_state" + ] + ] + }, + { + "sid": 125, + "sent": "The closest atoms to the serine hydroxyl group are the backbone carbonyl atoms of Asp419, Glu420 and Glu421, which are 3\u20134\u2009\u00c5 away.", + "section": "RESULTS", + "ner": [ + [ + 25, + 31, + "serine", + "residue_name" + ], + [ + 82, + 88, + "Asp419", + "residue_name_number" + ], + [ + 90, + 96, + "Glu420", + "residue_name_number" + ], + [ + 101, + 107, + "Glu421", + "residue_name_number" + ] + ] + }, + { + "sid": 126, + "sent": "We therefore predict that phosphorylation of Ser453 will result in steric clashes as well as electrostatic repulsion, which in turn might cause substantial conformational changes within the CTR.", + "section": "RESULTS", + "ner": [ + [ + 26, + 41, + "phosphorylation", + "ptm" + ], + [ + 45, + 51, + "Ser453", + "residue_name_number" + ], + [ + 190, + 193, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 127, + "sent": "To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed \u2018DD mutant'), using data to a resolution of 2.4\u2009\u00c5. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6).", + "section": "RESULTS", + "ner": [ + [ + 28, + 38, + "determined", + "experimental_method" + ], + [ + 43, + 52, + "structure", + "evidence" + ], + [ + 60, + 85, + "phosphorylation-mimicking", + "protein_state" + ], + [ + 86, + 97, + "R452D/S453D", + "mutant" + ], + [ + 125, + 134, + "DD mutant", + "mutant" + ], + [ + 179, + 201, + "additional mutation of", + "experimental_method" + ], + [ + 206, + 214, + "arginine", + "residue_name" + ], + [ + 229, + 249, + "phosphorylation site", + "site" + ], + [ + 342, + 351, + "phosphate", + "chemical" + ], + [ + 423, + 432, + "AI region", + "structure_element" + ], + [ + 442, + 451, + "main body", + "structure_element" + ], + [ + 459, + 470, + "transporter", + "protein_type" + ] + ] + }, + { + "sid": 128, + "sent": "The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mep\u0394 background (Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "ammonium", + "chemical" + ], + [ + 36, + 49, + "S. cerevisiae", + "species" + ], + [ + 65, + 74, + "DD mutant", + "mutant" + ], + [ + 98, + 100, + "WT", + "protein_state" + ], + [ + 101, + 105, + "Mep2", + "protein" + ], + [ + 114, + 119, + "S453D", + "mutant" + ], + [ + 120, + 126, + "mutant", + "protein_state" + ], + [ + 205, + 216, + "triple mep\u0394", + "mutant" + ] + ] + }, + { + "sid": 129, + "sent": "Unexpectedly, the AI segment containing the mutated residues has only undergone a slight shift compared with the WT protein (Fig. 8 and Supplementary Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 18, + 28, + "AI segment", + "structure_element" + ], + [ + 113, + 115, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 130, + "sent": "By contrast, the conserved part of the CTR has undergone a large conformational change involving formation of a 12-residue-long \u03b1-helix from Leu427 to Asp438.", + "section": "RESULTS", + "ner": [ + [ + 17, + 26, + "conserved", + "protein_state" + ], + [ + 39, + 42, + "CTR", + "structure_element" + ], + [ + 112, + 135, + "12-residue-long \u03b1-helix", + "structure_element" + ], + [ + 141, + 157, + "Leu427 to Asp438", + "residue_range" + ] + ] + }, + { + "sid": 131, + "sent": "In addition, residues Glu420-Leu423 including Glu421 of the ExxGxD motif are now disordered (Fig. 8 and Supplementary Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 22, + 35, + "Glu420-Leu423", + "residue_range" + ], + [ + 46, + 52, + "Glu421", + "residue_name_number" + ], + [ + 60, + 72, + "ExxGxD motif", + "structure_element" + ], + [ + 81, + 91, + "disordered", + "protein_state" + ] + ] + }, + { + "sid": 132, + "sent": "This is the first time a large conformational change has been observed in an ammonium transporter as a result of a mutation, and confirms previous hypotheses that phosphorylation causes structural changes in the CTR.", + "section": "RESULTS", + "ner": [ + [ + 77, + 97, + "ammonium transporter", + "protein_type" + ], + [ + 115, + 123, + "mutation", + "experimental_method" + ], + [ + 163, + 178, + "phosphorylation", + "ptm" + ], + [ + 212, + 215, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 133, + "sent": "To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the \u2018single D' S453D mutant.", + "section": "RESULTS", + "ner": [ + [ + 47, + 52, + "R452D", + "mutant" + ], + [ + 111, + 121, + "determined", + "experimental_method" + ], + [ + 126, + 135, + "structure", + "evidence" + ], + [ + 144, + 152, + "single D", + "mutant" + ], + [ + 154, + 159, + "S453D", + "mutant" + ], + [ + 160, + 166, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 134, + "sent": "As shown in Supplementary Fig. 4, the consequence of the single D mutation is very similar to that of the DD substitution, with conformational changes and increased dynamics confined to the conserved part of the CTR (Supplementary Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 57, + 65, + "single D", + "mutant" + ], + [ + 66, + 74, + "mutation", + "experimental_method" + ], + [ + 106, + 121, + "DD substitution", + "mutant" + ], + [ + 190, + 199, + "conserved", + "protein_state" + ], + [ + 212, + 215, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 135, + "sent": "To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J).", + "section": "RESULTS", + "ner": [ + [ + 18, + 36, + "crystal structures", + "evidence" + ], + [ + 56, + 65, + "modelling", + "experimental_method" + ], + [ + 70, + 72, + "MD", + "experimental_method" + ], + [ + 84, + 86, + "WT", + "protein_state" + ], + [ + 87, + 93, + "CaMep2", + "protein" + ], + [ + 99, + 108, + "DD mutant", + "mutant" + ], + [ + 113, + 127, + "phosphorylated", + "protein_state" + ], + [ + 137, + 142, + "S453J", + "mutant" + ] + ] + }, + { + "sid": 136, + "sent": "In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453.", + "section": "RESULTS", + "ner": [ + [ + 7, + 9, + "WT", + "protein_state" + ], + [ + 10, + 19, + "structure", + "evidence" + ], + [ + 41, + 47, + "Asp419", + "residue_name_number" + ], + [ + 49, + 55, + "Glu420", + "residue_name_number" + ], + [ + 60, + 66, + "Glu421", + "residue_name_number" + ], + [ + 78, + 94, + "hydrogen bonding", + "bond_interaction" + ], + [ + 107, + 113, + "Ser453", + "residue_name_number" + ] + ] + }, + { + "sid": 137, + "sent": "After 200\u2009ns of MD simulation, the interactions between these residues and Ser453 remain intact.", + "section": "RESULTS", + "ner": [ + [ + 16, + 18, + "MD", + "experimental_method" + ], + [ + 19, + 29, + "simulation", + "experimental_method" + ], + [ + 75, + 81, + "Ser453", + "residue_name_number" + ] + ] + }, + { + "sid": 138, + "sent": "The protein backbone has an average r.m.s.d. of only \u223c3\u2009\u00c5 during the 200-ns simulation, indicating that the protein is stable.", + "section": "RESULTS", + "ner": [ + [ + 36, + 44, + "r.m.s.d.", + "evidence" + ], + [ + 76, + 86, + "simulation", + "experimental_method" + ], + [ + 119, + 125, + "stable", + "protein_state" + ] + ] + }, + { + "sid": 139, + "sent": "There is flexibility in the side chains of the acidic residues so that they are able to form stable hydrogen bonds with Ser453.", + "section": "RESULTS", + "ner": [ + [ + 93, + 99, + "stable", + "protein_state" + ], + [ + 100, + 114, + "hydrogen bonds", + "bond_interaction" + ], + [ + 120, + 126, + "Ser453", + "residue_name_number" + ] + ] + }, + { + "sid": 140, + "sent": "In particular, persistent hydrogen bonds are observed between the Ser453 hydroxyl group and the acidic group of Glu420, and also between the amine group of Ser453 and the backbone carbonyl of Glu420 (Supplementary Fig. 5).", + "section": "RESULTS", + "ner": [ + [ + 26, + 40, + "hydrogen bonds", + "bond_interaction" + ], + [ + 66, + 72, + "Ser453", + "residue_name_number" + ], + [ + 112, + 118, + "Glu420", + "residue_name_number" + ], + [ + 156, + 162, + "Ser453", + "residue_name_number" + ], + [ + 192, + 198, + "Glu420", + "residue_name_number" + ] + ] + }, + { + "sid": 141, + "sent": "The DD mutant is also stable during the simulations, but the average backbone r.m.s.d of \u223c3.6\u2009\u00c5 suggests slightly more conformational flexibility than WT.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "DD mutant", + "mutant" + ], + [ + 22, + 28, + "stable", + "protein_state" + ], + [ + 40, + 51, + "simulations", + "experimental_method" + ], + [ + 78, + 85, + "r.m.s.d", + "evidence" + ], + [ + 151, + 153, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 142, + "sent": "As the simulation proceeds, the side chains of the acidic residues move away from Asp452 and Asp453, presumably to avoid electrostatic repulsion.", + "section": "RESULTS", + "ner": [ + [ + 7, + 17, + "simulation", + "experimental_method" + ], + [ + 82, + 88, + "Asp452", + "residue_name_number" + ], + [ + 93, + 99, + "Asp453", + "residue_name_number" + ] + ] + }, + { + "sid": 143, + "sent": "For example, the distance between the Asp453 acidic oxygens and the Glu420 acidic oxygens increases from \u223c7 to >22\u2009\u00c5 after 200\u2009ns simulations, and thus these residues are not interacting.", + "section": "RESULTS", + "ner": [ + [ + 17, + 25, + "distance", + "evidence" + ], + [ + 38, + 44, + "Asp453", + "residue_name_number" + ], + [ + 68, + 74, + "Glu420", + "residue_name_number" + ], + [ + 130, + 141, + "simulations", + "experimental_method" + ] + ] + }, + { + "sid": 144, + "sent": "The protein is structurally stable throughout the simulation with little deviation in the other parts of the protein.", + "section": "RESULTS", + "ner": [ + [ + 15, + 34, + "structurally stable", + "protein_state" + ], + [ + 50, + 60, + "simulation", + "experimental_method" + ] + ] + }, + { + "sid": 145, + "sent": "Finally, the S453J mutant is also stable throughout the 200-ns simulation and has an average backbone deviation of \u223c3.8\u2009\u00c5, which is similar to the DD mutant.", + "section": "RESULTS", + "ner": [ + [ + 13, + 18, + "S453J", + "mutant" + ], + [ + 19, + 25, + "mutant", + "protein_state" + ], + [ + 34, + 40, + "stable", + "protein_state" + ], + [ + 63, + 73, + "simulation", + "experimental_method" + ], + [ + 147, + 156, + "DD mutant", + "mutant" + ] + ] + }, + { + "sid": 146, + "sent": "The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant.", + "section": "RESULTS", + "ner": [ + [ + 46, + 52, + "Arg452", + "residue_name_number" + ], + [ + 57, + 63, + "Sep453", + "residue_name_number" + ], + [ + 91, + 101, + "simulation", + "experimental_method" + ], + [ + 144, + 150, + "Asp452", + "residue_name_number" + ], + [ + 155, + 161, + "Asp453", + "residue_name_number" + ], + [ + 169, + 178, + "DD mutant", + "mutant" + ] + ] + }, + { + "sid": 147, + "sent": "The distance between the phosphate of Sep453 and the acidic oxygen atoms of Glu420 is initially \u223c11\u2009\u00c5, but increases to >30\u2009\u00c5 after 200\u2009ns.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "distance", + "evidence" + ], + [ + 25, + 34, + "phosphate", + "chemical" + ], + [ + 38, + 44, + "Sep453", + "residue_name_number" + ], + [ + 76, + 82, + "Glu420", + "residue_name_number" + ] + ] + }, + { + "sid": 148, + "sent": "The short helix formed by residues Leu427 to Asp438 unravels during the simulations to a disordered state.", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "short helix", + "structure_element" + ], + [ + 35, + 51, + "Leu427 to Asp438", + "residue_range" + ], + [ + 72, + 83, + "simulations", + "experimental_method" + ], + [ + 89, + 99, + "disordered", + "protein_state" + ] + ] + }, + { + "sid": 149, + "sent": "Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR.", + "section": "RESULTS", + "ner": [ + [ + 10, + 12, + "MD", + "experimental_method" + ], + [ + 13, + 24, + "simulations", + "experimental_method" + ], + [ + 53, + 71, + "crystal structures", + "evidence" + ], + [ + 77, + 92, + "phosphorylation", + "ptm" + ], + [ + 133, + 142, + "conserved", + "protein_state" + ], + [ + 155, + 158, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 150, + "sent": "However, the conformational changes for the phosphomimetic mutants in the crystals are confined to the CTR (Fig. 8), and the channels are still closed (Supplementary Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 44, + 66, + "phosphomimetic mutants", + "mutant" + ], + [ + 74, + 82, + "crystals", + "evidence" + ], + [ + 103, + 106, + "CTR", + "structure_element" + ], + [ + 125, + 133, + "channels", + "site" + ], + [ + 144, + 150, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 151, + "sent": "One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 37, + 44, + "mutants", + "mutant" + ], + [ + 71, + 84, + "phosphoserine", + "residue_name" + ], + [ + 115, + 120, + "S453D", + "mutant" + ], + [ + 125, + 135, + "DD mutants", + "mutant" + ], + [ + 140, + 152, + "fully active", + "protein_state" + ], + [ + 160, + 170, + "absence of", + "protein_state" + ], + [ + 171, + 175, + "Npr1", + "protein" + ], + [ + 194, + 203, + "mutations", + "experimental_method" + ], + [ + 227, + 242, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 152, + "sent": "The fact that the S453D structure was obtained in the presence of 10\u2009mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels.", + "section": "RESULTS", + "ner": [ + [ + 18, + 23, + "S453D", + "mutant" + ], + [ + 24, + 33, + "structure", + "evidence" + ], + [ + 72, + 80, + "ammonium", + "chemical" + ], + [ + 104, + 119, + "crystallization", + "experimental_method" + ], + [ + 136, + 142, + "closed", + "protein_state" + ], + [ + 157, + 161, + "Mep2", + "protein" + ], + [ + 162, + 170, + "channels", + "site" + ] + ] + }, + { + "sid": 153, + "sent": "Knowledge about ammonium transporter structure has been obtained from experimental and theoretical studies on bacterial family members.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 36, + "ammonium transporter", + "protein_type" + ], + [ + 37, + 46, + "structure", + "evidence" + ], + [ + 110, + 119, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 154, + "sent": "In addition, a number of biochemical and genetic studies are available for bacterial, fungal and plant proteins.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 56, + "biochemical and genetic studies", + "experimental_method" + ], + [ + 75, + 84, + "bacterial", + "taxonomy_domain" + ], + [ + 86, + 92, + "fungal", + "taxonomy_domain" + ], + [ + 97, + 102, + "plant", + "taxonomy_domain" + ] + ] + }, + { + "sid": 155, + "sent": "These efforts have advanced our knowledge considerably but have not yet yielded atomic-level answers to several important mechanistic questions, including how ammonium transport is regulated in eukaryotes and the mechanism of ammonium signalling.", + "section": "DISCUSS", + "ner": [ + [ + 159, + 167, + "ammonium", + "chemical" + ], + [ + 194, + 204, + "eukaryotes", + "taxonomy_domain" + ], + [ + 226, + 234, + "ammonium", + "chemical" + ] + ] + }, + { + "sid": 156, + "sent": "In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 23, + "Arabidopsis thaliana", + "species" + ], + [ + 24, + 31, + "Amt-1;1", + "protein" + ], + [ + 33, + 48, + "phosphorylation", + "ptm" + ], + [ + 56, + 59, + "CTR", + "structure_element" + ], + [ + 68, + 72, + "T460", + "residue_name_number" + ], + [ + 98, + 106, + "ammonium", + "chemical" + ], + [ + 158, + 176, + "non-phosphorylated", + "protein_state" + ], + [ + 191, + 196, + "plant", + "taxonomy_domain" + ], + [ + 197, + 208, + "transporter", + "protein_type" + ], + [ + 212, + 216, + "open", + "protein_state" + ] + ] + }, + { + "sid": 157, + "sent": "Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 39, + "phosphomimetic mutations", + "mutant" + ], + [ + 60, + 67, + "monomer", + "oligomeric_state" + ], + [ + 90, + 96, + "trimer", + "oligomeric_state" + ], + [ + 158, + 161, + "CTR", + "structure_element" + ], + [ + 196, + 204, + "ammonium", + "chemical" + ], + [ + 228, + 243, + "phosphorylation", + "ptm" + ] + ] + }, + { + "sid": 158, + "sent": "Owing to the lack of structural information for plant AMTs, the details of channel closure and inter-monomer crosstalk are not yet clear.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 53, + "plant", + "taxonomy_domain" + ], + [ + 54, + 58, + "AMTs", + "protein_type" + ], + [ + 75, + 82, + "channel", + "site" + ] + ] + }, + { + "sid": 159, + "sent": "Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 26, + "plant", + "taxonomy_domain" + ], + [ + 27, + 39, + "transporters", + "protein_type" + ], + [ + 45, + 53, + "inactive", + "protein_state" + ], + [ + 64, + 77, + "Mep2 proteins", + "protein_type" + ], + [ + 103, + 111, + "ammonium", + "chemical" + ], + [ + 116, + 134, + "non-phosphorylated", + "protein_state" + ], + [ + 141, + 149, + "channels", + "site" + ], + [ + 159, + 165, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 160, + "sent": "The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 35, + "transporters", + "protein_type" + ], + [ + 44, + 55, + "A. thaliana", + "species" + ], + [ + 56, + 63, + "Amt-1;1", + "protein" + ], + [ + 68, + 72, + "Mep2", + "protein" + ], + [ + 107, + 122, + "phosphorylation", + "ptm" + ], + [ + 124, + 136, + "inactivation", + "protein_state" + ], + [ + 140, + 146, + "plants", + "taxonomy_domain" + ], + [ + 151, + 161, + "activation", + "protein_state" + ], + [ + 165, + 170, + "fungi", + "taxonomy_domain" + ] + ] + }, + { + "sid": 161, + "sent": "In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 8, + "fungi", + "taxonomy_domain" + ], + [ + 21, + 29, + "ammonium", + "chemical" + ], + [ + 59, + 80, + "ammonium transporters", + "protein_type" + ], + [ + 111, + 119, + "ammonium", + "chemical" + ], + [ + 145, + 153, + "ammonium", + "chemical" + ], + [ + 168, + 171, + "Ato", + "protein_type" + ], + [ + 172, + 184, + "transporters", + "protein_type" + ] + ] + }, + { + "sid": 162, + "sent": "By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 35, + "structures", + "evidence" + ], + [ + 39, + 45, + "closed", + "protein_state" + ], + [ + 46, + 67, + "ammonium transporters", + "protein_type" + ], + [ + 72, + 81, + "comparing", + "experimental_method" + ], + [ + 88, + 98, + "structures", + "evidence" + ], + [ + 108, + 124, + "permanently open", + "protein_state" + ], + [ + 125, + 134, + "bacterial", + "taxonomy_domain" + ], + [ + 165, + 169, + "Mep2", + "protein_type" + ], + [ + 170, + 177, + "channel", + "site" + ], + [ + 220, + 223, + "CTR", + "structure_element" + ], + [ + 228, + 232, + "ICL1", + "structure_element" + ], + [ + 237, + 241, + "ICL3", + "structure_element" + ] + ] + }, + { + "sid": 163, + "sent": "More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a).", + "section": "DISCUSS", + "ner": [ + [ + 54, + 57, + "CTR", + "structure_element" + ], + [ + 62, + 66, + "ICL1", + "structure_element" + ], + [ + 67, + 71, + "ICL3", + "structure_element" + ], + [ + 83, + 87, + "open", + "protein_state" + ], + [ + 88, + 100, + "transporters", + "protein_type" + ], + [ + 124, + 128, + "ICL3", + "structure_element" + ], + [ + 160, + 167, + "channel", + "site" + ] + ] + }, + { + "sid": 164, + "sent": "In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 17, + "ICL1", + "structure_element" + ], + [ + 59, + 66, + "channel", + "site" + ], + [ + 87, + 91, + "His2", + "residue_name_number" + ], + [ + 101, + 115, + "twin-His motif", + "structure_element" + ], + [ + 120, + 136, + "hydrogen bonding", + "bond_interaction" + ], + [ + 144, + 160, + "highly conserved", + "protein_state" + ], + [ + 161, + 169, + "tyrosine", + "residue_name" + ] + ] + }, + { + "sid": 165, + "sent": "Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9).", + "section": "DISCUSS", + "ner": [ + [ + 5, + 20, + "phosphorylation", + "ptm" + ], + [ + 28, + 32, + "Npr1", + "protein" + ], + [ + 33, + 39, + "kinase", + "protein_type" + ], + [ + 55, + 63, + "nitrogen", + "chemical" + ], + [ + 98, + 107, + "conserved", + "protein_state" + ], + [ + 108, + 120, + "ExxGxD motif", + "structure_element" + ], + [ + 170, + 177, + "channel", + "site" + ] + ] + }, + { + "sid": 166, + "sent": "Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 40, + "structural similarities", + "evidence" + ], + [ + 48, + 56, + "TM parts", + "structure_element" + ], + [ + 60, + 64, + "Mep2", + "protein" + ], + [ + 69, + 76, + "AfAmt-1", + "protein" + ], + [ + 100, + 107, + "channel", + "site" + ], + [ + 188, + 195, + "channel", + "site" + ] + ] + }, + { + "sid": 167, + "sent": "How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 23, + "channel", + "site" + ], + [ + 79, + 83, + "open", + "protein_state" + ], + [ + 133, + 136, + "CTR", + "structure_element" + ], + [ + 171, + 175, + "ICL3", + "structure_element" + ], + [ + 196, + 203, + "monomer", + "oligomeric_state" + ], + [ + 265, + 272, + "channel", + "site" + ], + [ + 300, + 304, + "ICL3", + "structure_element" + ] + ] + }, + { + "sid": 168, + "sent": "Owing to the crosstalk between monomers, a single phosphorylation event might lead to opening of the entire trimer, although this has not yet been tested (Fig. 9b).", + "section": "DISCUSS", + "ner": [ + [ + 31, + 39, + "monomers", + "oligomeric_state" + ], + [ + 50, + 65, + "phosphorylation", + "ptm" + ], + [ + 108, + 114, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 169, + "sent": "Whether or not Mep2 channel opening requires, in addition to phosphorylation, disruption of the Tyr\u2013His2 interaction by the ammonium substrate is not yet clear.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 19, + "Mep2", + "protein_type" + ], + [ + 20, + 27, + "channel", + "site" + ], + [ + 61, + 76, + "phosphorylation", + "ptm" + ], + [ + 96, + 116, + "Tyr\u2013His2 interaction", + "site" + ], + [ + 124, + 132, + "ammonium", + "chemical" + ] + ] + }, + { + "sid": 170, + "sent": "Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states.", + "section": "DISCUSS", + "ner": [ + [ + 40, + 44, + "Mep2", + "protein" + ], + [ + 45, + 53, + "channels", + "site" + ], + [ + 70, + 80, + "eukaryotic", + "taxonomy_domain" + ], + [ + 81, + 102, + "ammonium transporters", + "protein_type" + ], + [ + 108, + 123, + "structural data", + "evidence" + ], + [ + 185, + 188, + "CTR", + "structure_element" + ], + [ + 193, + 210, + "cytoplasmic loops", + "structure_element" + ], + [ + 237, + 243, + "closed", + "protein_state" + ], + [ + 248, + 252, + "open", + "protein_state" + ] + ] + }, + { + "sid": 171, + "sent": "However, even the otherwise highly similar Mep2 proteins of S. cerevisiae and C. albicans have different structures for their CTRs (Fig. 1 and Supplementary Fig. 6).", + "section": "DISCUSS", + "ner": [ + [ + 43, + 56, + "Mep2 proteins", + "protein_type" + ], + [ + 60, + 73, + "S. cerevisiae", + "species" + ], + [ + 78, + 89, + "C. albicans", + "species" + ], + [ + 105, + 115, + "structures", + "evidence" + ], + [ + 126, + 130, + "CTRs", + "structure_element" + ] + ] + }, + { + "sid": 172, + "sent": "In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 26, + "AI region", + "structure_element" + ], + [ + 34, + 37, + "CTR", + "structure_element" + ], + [ + 53, + 69, + "Npr1 kinase site", + "site" + ], + [ + 73, + 82, + "conserved", + "protein_state" + ], + [ + 103, + 109, + "fungal", + "taxonomy_domain" + ], + [ + 110, + 122, + "transporters", + "protein_type" + ] + ] + }, + { + "sid": 173, + "sent": "Nevertheless, given the central role of absolutely conserved residues within the ICL1-ICL3-CTR interaction network (Fig. 4), we propose that the structural basics of fungal ammonium transporter activation are conserved.", + "section": "DISCUSS", + "ner": [ + [ + 40, + 60, + "absolutely conserved", + "protein_state" + ], + [ + 81, + 114, + "ICL1-ICL3-CTR interaction network", + "site" + ], + [ + 166, + 172, + "fungal", + "taxonomy_domain" + ], + [ + 173, + 181, + "ammonium", + "chemical" + ], + [ + 209, + 218, + "conserved", + "protein_state" + ] + ] + }, + { + "sid": 174, + "sent": "The fact that Mep2 orthologues of distantly related fungi are fully functional in ammonium transport and signalling in S. cerevisiae supports this notion.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 18, + "Mep2", + "protein_type" + ], + [ + 52, + 57, + "fungi", + "taxonomy_domain" + ], + [ + 82, + 90, + "ammonium", + "chemical" + ], + [ + 119, + 132, + "S. cerevisiae", + "species" + ] + ] + }, + { + "sid": 175, + "sent": "It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr\u2013His2 hydrogen bond might be a general way to close Mep2 proteins.", + "section": "DISCUSS", + "ner": [ + [ + 33, + 41, + "tyrosine", + "residue_name" + ], + [ + 67, + 71, + "His2", + "residue_name_number" + ], + [ + 75, + 91, + "highly conserved", + "protein_state" + ], + [ + 95, + 101, + "fungal", + "taxonomy_domain" + ], + [ + 102, + 106, + "Mep2", + "protein_type" + ], + [ + 140, + 162, + "Tyr\u2013His2 hydrogen bond", + "site" + ], + [ + 189, + 194, + "close", + "protein_state" + ], + [ + 195, + 208, + "Mep2 proteins", + "protein_type" + ] + ] + }, + { + "sid": 176, + "sent": "With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 21, + "plant", + "taxonomy_domain" + ], + [ + 22, + 26, + "AMTs", + "protein_type" + ], + [ + 54, + 69, + "phosphorylation", + "ptm" + ], + [ + 73, + 77, + "T460", + "residue_name_number" + ], + [ + 145, + 149, + "pore", + "site" + ], + [ + 158, + 168, + "C terminus", + "structure_element" + ] + ] + }, + { + "sid": 177, + "sent": "This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer.", + "section": "DISCUSS", + "ner": [ + [ + 38, + 52, + "homology model", + "experimental_method" + ], + [ + 57, + 64, + "Amt-1;1", + "protein" + ], + [ + 79, + 83, + "open", + "protein_state" + ], + [ + 85, + 100, + "archaebacterial", + "taxonomy_domain" + ], + [ + 101, + 108, + "AfAmt-1", + "protein" + ], + [ + 109, + 118, + "structure", + "evidence" + ], + [ + 145, + 155, + "C terminus", + "structure_element" + ], + [ + 159, + 166, + "Amt-1;1", + "protein" + ], + [ + 208, + 215, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 178, + "sent": "Our Mep2 structures show that this assumption may not be correct (Fig. 4 and Supplementary Fig. 6).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 8, + "Mep2", + "protein" + ], + [ + 9, + 19, + "structures", + "evidence" + ] + ] + }, + { + "sid": 179, + "sent": "In addition, the considerable differences between structurally resolved CTR domains means that the exact environment of T460 in Amt-1;1 is also not known (Supplementary Fig. 6).", + "section": "DISCUSS", + "ner": [ + [ + 72, + 75, + "CTR", + "structure_element" + ], + [ + 120, + 124, + "T460", + "residue_name_number" + ], + [ + 128, + 135, + "Amt-1;1", + "protein" + ] + ] + }, + { + "sid": 180, + "sent": "Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71).", + "section": "DISCUSS", + "ner": [ + [ + 23, + 45, + "structural information", + "evidence" + ], + [ + 120, + 127, + "Amt-1;1", + "protein" + ], + [ + 195, + 198, + "CTR", + "structure_element" + ], + [ + 203, + 207, + "ICL1", + "structure_element" + ], + [ + 208, + 212, + "ICL3", + "structure_element" + ], + [ + 227, + 231, + "Y467", + "residue_name_number" + ], + [ + 232, + 236, + "H239", + "residue_name_number" + ], + [ + 241, + 245, + "D458", + "residue_name_number" + ], + [ + 246, + 249, + "K71", + "residue_name_number" + ] + ] + }, + { + "sid": 181, + "sent": "There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr\u2013His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters.", + "section": "DISCUSS", + "ner": [ + [ + 37, + 43, + "CaMep2", + "protein" + ], + [ + 44, + 49, + "Tyr49", + "residue_name_number" + ], + [ + 53, + 58, + "plant", + "taxonomy_domain" + ], + [ + 59, + 63, + "AMTs", + "protein_type" + ], + [ + 83, + 105, + "Tyr\u2013His2 hydrogen bond", + "site" + ], + [ + 121, + 125, + "Mep2", + "protein" + ], + [ + 152, + 158, + "closed", + "protein_state" + ], + [ + 168, + 173, + "plant", + "taxonomy_domain" + ], + [ + 174, + 186, + "transporters", + "protein_type" + ] + ] + }, + { + "sid": 182, + "sent": "We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of \u2018intra-' interactions leads to inactive states.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 58, + "intra-monomeric CTR-ICL1/ICL3 interactions", + "site" + ], + [ + 98, + 104, + "fungal", + "taxonomy_domain" + ], + [ + 109, + 114, + "plant", + "taxonomy_domain" + ], + [ + 115, + 136, + "ammonium transporters", + "protein_type" + ], + [ + 166, + 170, + "open", + "protein_state" + ], + [ + 171, + 179, + "channels", + "site" + ], + [ + 193, + 200, + "lack of", + "protein_state" + ], + [ + 232, + 240, + "inactive", + "protein_state" + ] + ] + }, + { + "sid": 183, + "sent": "The need to regulate in opposite ways may be the reason why the phosphorylation sites are in different parts of the CTR, that is, centrally located close to the ExxGxD motif in AMTs and peripherally in Mep2.", + "section": "DISCUSS", + "ner": [ + [ + 64, + 85, + "phosphorylation sites", + "site" + ], + [ + 116, + 119, + "CTR", + "structure_element" + ], + [ + 161, + 173, + "ExxGxD motif", + "structure_element" + ], + [ + 177, + 181, + "AMTs", + "protein_type" + ], + [ + 202, + 206, + "Mep2", + "protein" + ] + ] + }, + { + "sid": 184, + "sent": "In this way, phosphorylation can either lead to channel closing (in the case of AMTs) or channel opening in the case of Mep2.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 28, + "phosphorylation", + "ptm" + ], + [ + 48, + 55, + "channel", + "site" + ], + [ + 80, + 84, + "AMTs", + "protein_type" + ], + [ + 89, + 96, + "channel", + "site" + ], + [ + 120, + 124, + "Mep2", + "protein" + ] + ] + }, + { + "sid": 185, + "sent": "Our model also provides an explanation for the observation that certain mutations within the CTR completely abolish transport activity.", + "section": "DISCUSS", + "ner": [ + [ + 64, + 81, + "certain mutations", + "mutant" + ], + [ + 93, + 96, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 186, + "sent": "An example of an inactivating residue is the glycine of the ExxGxD motif of the CTR.", + "section": "DISCUSS", + "ner": [ + [ + 45, + 52, + "glycine", + "residue_name" + ], + [ + 60, + 72, + "ExxGxD motif", + "structure_element" + ], + [ + 80, + 83, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 187, + "sent": "Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 8, + "Mutation", + "experimental_method" + ], + [ + 26, + 30, + "G393", + "residue_name_number" + ], + [ + 34, + 40, + "EcAmtB", + "protein" + ], + [ + 42, + 46, + "G456", + "residue_name_number" + ], + [ + 50, + 59, + "AtAmt-1;1", + "protein" + ], + [ + 73, + 85, + "transporters", + "protein_type" + ], + [ + 100, + 116, + "Escherichia coli", + "species" + ], + [ + 117, + 121, + "AmtB", + "protein" + ], + [ + 126, + 137, + "A. thaliana", + "species" + ], + [ + 138, + 145, + "Amt-1;1", + "protein" + ] + ] + }, + { + "sid": 188, + "sent": "Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2.", + "section": "DISCUSS", + "ner": [ + [ + 54, + 57, + "CTR", + "structure_element" + ], + [ + 98, + 101, + "CTR", + "structure_element" + ], + [ + 106, + 110, + "ICL1", + "structure_element" + ], + [ + 111, + 115, + "ICL3", + "structure_element" + ], + [ + 139, + 145, + "closed", + "protein_state" + ], + [ + 192, + 196, + "Mep2", + "protein" + ] + ] + }, + { + "sid": 189, + "sent": "Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters.", + "section": "DISCUSS", + "ner": [ + [ + 51, + 66, + "phosphorylation", + "ptm" + ], + [ + 102, + 112, + "aquaporins", + "protein_type" + ], + [ + 117, + 134, + "urea transporters", + "protein_type" + ], + [ + 175, + 185, + "eukaryotic", + "taxonomy_domain" + ], + [ + 186, + 194, + "channels", + "protein_type" + ], + [ + 199, + 211, + "transporters", + "protein_type" + ] + ] + }, + { + "sid": 190, + "sent": "Recently, phosphorylation was also shown to modulate substrate affinity in nitrate transporters.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 25, + "phosphorylation", + "ptm" + ], + [ + 75, + 95, + "nitrate transporters", + "protein_type" + ] + ] + }, + { + "sid": 191, + "sent": "With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs).", + "section": "DISCUSS", + "ner": [ + [ + 16, + 24, + "ammonium", + "chemical" + ], + [ + 36, + 51, + "phosphorylation", + "ptm" + ], + [ + 85, + 96, + "A. thaliana", + "species" + ], + [ + 97, + 101, + "AMTs", + "protein_type" + ], + [ + 110, + 123, + "S. cerevisiae", + "species" + ], + [ + 124, + 128, + "Mep2", + "protein" + ] + ] + }, + { + "sid": 192, + "sent": "However, the absence of GlnK proteins in eukaryotes suggests that phosphorylation-based regulation of ammonium transport may be widespread.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 23, + "absence of", + "protein_state" + ], + [ + 24, + 37, + "GlnK proteins", + "protein_type" + ], + [ + 41, + 51, + "eukaryotes", + "taxonomy_domain" + ], + [ + 66, + 81, + "phosphorylation", + "ptm" + ], + [ + 102, + 110, + "ammonium", + "chemical" + ] + ] + }, + { + "sid": 193, + "sent": "With respect to Mep2-mediated signalling to induce pseudohyphal growth, two models have been put forward as to how this occurs and why it is specific to Mep2 proteins.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 20, + "Mep2", + "protein_type" + ], + [ + 153, + 166, + "Mep2 proteins", + "protein_type" + ] + ] + }, + { + "sid": 194, + "sent": "In one model, signalling is proposed to depend on the nature of the transported substrate, which might be different in certain subfamilies of ammonium transporters (for example, Mep1/Mep3 versus Mep2).", + "section": "DISCUSS", + "ner": [ + [ + 142, + 163, + "ammonium transporters", + "protein_type" + ], + [ + 178, + 182, + "Mep1", + "protein" + ], + [ + 183, + 187, + "Mep3", + "protein" + ], + [ + 195, + 199, + "Mep2", + "protein" + ] + ] + }, + { + "sid": 195, + "sent": "For example, NH3 uniport or symport of NH3/H+ might result in changes in local pH, but NH4+ uniport might not, and this difference might determine signalling.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 16, + "NH3", + "chemical" + ], + [ + 39, + 42, + "NH3", + "chemical" + ], + [ + 43, + 45, + "H+", + "chemical" + ], + [ + 87, + 91, + "NH4+", + "chemical" + ] + ] + }, + { + "sid": 196, + "sent": "In the other model, signalling is thought to require a distinct conformation of the Mep2 transporter occurring during the transport cycle.", + "section": "DISCUSS", + "ner": [ + [ + 84, + 88, + "Mep2", + "protein" + ], + [ + 89, + 100, + "transporter", + "protein_type" + ] + ] + }, + { + "sid": 197, + "sent": "While the current study does not specifically address the mechanism of signalling underlying pseudohyphal growth, our structures do show that Mep2 proteins can assume different conformations.", + "section": "DISCUSS", + "ner": [ + [ + 118, + 128, + "structures", + "evidence" + ], + [ + 142, + 155, + "Mep2 proteins", + "protein_type" + ] + ] + }, + { + "sid": 198, + "sent": "It is clear that ammonium transport across biomembranes remains a fascinating and challenging field in large part due to the unique properties of the substrate.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 25, + "ammonium", + "chemical" + ] + ] + }, + { + "sid": 199, + "sent": "Our Mep2 structural work now provides a foundation for future studies to uncover the details of the structural changes that occur during eukaryotic ammonium transport and signaling, and to assess the possibility to utilize small molecules to shut down ammonium sensing and downstream signalling pathways in pathogenic fungi.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 8, + "Mep2", + "protein" + ], + [ + 137, + 147, + "eukaryotic", + "taxonomy_domain" + ], + [ + 148, + 156, + "ammonium", + "chemical" + ], + [ + 252, + 260, + "ammonium", + "chemical" + ], + [ + 318, + 323, + "fungi", + "taxonomy_domain" + ] + ] + }, + { + "sid": 200, + "sent": "X-ray crystal structures of Mep2 transceptors.", + "section": "FIG", + "ner": [ + [ + 0, + 24, + "X-ray crystal structures", + "evidence" + ], + [ + 28, + 32, + "Mep2", + "protein" + ], + [ + 33, + 45, + "transceptors", + "protein_type" + ] + ] + }, + { + "sid": 201, + "sent": "(a) Monomer cartoon models viewed from the side for (left) A. fulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and C. albicans Mep2 (right).", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "Monomer", + "oligomeric_state" + ], + [ + 71, + 76, + "Amt-1", + "protein" + ], + [ + 92, + 105, + "S. cerevisiae", + "species" + ], + [ + 106, + 110, + "Mep2", + "protein" + ], + [ + 124, + 135, + "C. albicans", + "species" + ], + [ + 136, + 140, + "Mep2", + "protein" + ] + ] + }, + { + "sid": 202, + "sent": "The region showing ICL1 (blue), ICL3 (green) and the CTR (red) is boxed for comparison.", + "section": "FIG", + "ner": [ + [ + 19, + 23, + "ICL1", + "structure_element" + ], + [ + 32, + 36, + "ICL3", + "structure_element" + ], + [ + 53, + 56, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 203, + "sent": "(b) CaMep2 trimer viewed from the intracellular side (right).", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "CaMep2", + "protein" + ], + [ + 11, + 17, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 204, + "sent": "One monomer is coloured as in a and one monomer is coloured by B-factor (blue, low; red; high).", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "monomer", + "oligomeric_state" + ], + [ + 40, + 47, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 205, + "sent": "The CTR is boxed.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 206, + "sent": " (c) Overlay of ScMep2 (grey) and CaMep2 (rainbow), illustrating the differences in the CTRs.", + "section": "FIG", + "ner": [ + [ + 5, + 12, + "Overlay", + "experimental_method" + ], + [ + 16, + 22, + "ScMep2", + "protein" + ], + [ + 34, + 40, + "CaMep2", + "protein" + ], + [ + 88, + 92, + "CTRs", + "structure_element" + ] + ] + }, + { + "sid": 207, + "sent": "Sequence conservation in ammonium transporters.", + "section": "FIG", + "ner": [ + [ + 0, + 21, + "Sequence conservation", + "evidence" + ], + [ + 25, + 46, + "ammonium transporters", + "protein_type" + ] + ] + }, + { + "sid": 208, + "sent": "ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E. coli AmtB and A. thaliana Amt-1;1.", + "section": "FIG", + "ner": [ + [ + 0, + 18, + "ClustalW alignment", + "experimental_method" + ], + [ + 22, + 28, + "CaMep2", + "protein" + ], + [ + 30, + 36, + "ScMep2", + "protein" + ], + [ + 38, + 49, + "A. fulgidus", + "species" + ], + [ + 50, + 55, + "Amt-1", + "protein" + ], + [ + 65, + 69, + "AmtB", + "protein" + ], + [ + 74, + 85, + "A. thaliana", + "species" + ], + [ + 86, + 93, + "Amt-1;1", + "protein" + ] + ] + }, + { + "sid": 209, + "sent": "The secondary structure elements observed for CaMep2 are indicated, with the numbers corresponding to the centre of the TM segment.", + "section": "FIG", + "ner": [ + [ + 46, + 52, + "CaMep2", + "protein" + ], + [ + 120, + 130, + "TM segment", + "structure_element" + ] + ] + }, + { + "sid": 210, + "sent": "The conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, the conserved ExxGxD motif of the CTR in red and the AI region in yellow.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "conserved", + "protein_state" + ], + [ + 14, + 23, + "RxK motif", + "structure_element" + ], + [ + 27, + 31, + "ICL1", + "structure_element" + ], + [ + 54, + 62, + "ER motif", + "structure_element" + ], + [ + 66, + 70, + "ICL2", + "structure_element" + ], + [ + 84, + 93, + "conserved", + "protein_state" + ], + [ + 94, + 106, + "ExxGxD motif", + "structure_element" + ], + [ + 114, + 117, + "CTR", + "structure_element" + ], + [ + 133, + 142, + "AI region", + "structure_element" + ] + ] + }, + { + "sid": 211, + "sent": " Coloured residues are functionally important and correspond to those of the Phe gate (blue), the binding site Trp residue (magenta) and the twin-His motif (red).", + "section": "FIG", + "ner": [ + [ + 77, + 85, + "Phe gate", + "site" + ], + [ + 98, + 110, + "binding site", + "site" + ], + [ + 111, + 114, + "Trp", + "residue_name" + ] + ] + }, + { + "sid": 212, + "sent": "The Npr1 kinase site in the AI region is highlighted pink.", + "section": "FIG", + "ner": [ + [ + 4, + 20, + "Npr1 kinase site", + "site" + ], + [ + 28, + 37, + "AI region", + "structure_element" + ] + ] + }, + { + "sid": 213, + "sent": "The grey sequences at the C termini of CaMep2 and ScMep2 are not visible in the structures and are likely disordered.", + "section": "FIG", + "ner": [ + [ + 39, + 45, + "CaMep2", + "protein" + ], + [ + 50, + 56, + "ScMep2", + "protein" + ], + [ + 80, + 90, + "structures", + "evidence" + ], + [ + 99, + 116, + "likely disordered", + "protein_state" + ] + ] + }, + { + "sid": 214, + "sent": "Growth of ScMep2 variants on low ammonium medium.", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "Growth", + "experimental_method" + ], + [ + 10, + 25, + "ScMep2 variants", + "mutant" + ] + ] + }, + { + "sid": 215, + "sent": "(a) The triple mep\u0394 strain (black) and triple mep\u0394 npr1\u0394 strain (grey) containing plasmids expressing WT and variant ScMep2 were grown on minimal medium containing 1\u2009mM ammonium sulphate.", + "section": "FIG", + "ner": [ + [ + 8, + 19, + "triple mep\u0394", + "mutant" + ], + [ + 102, + 104, + "WT", + "protein_state" + ], + [ + 109, + 123, + "variant ScMep2", + "mutant" + ], + [ + 129, + 152, + "grown on minimal medium", + "experimental_method" + ], + [ + 169, + 186, + "ammonium sulphate", + "chemical" + ] + ] + }, + { + "sid": 216, + "sent": "The quantified cell density reflects logarithmic growth after 24\u2009h. Error bars are the s.d. for three replicates of each strain (b) The strains used in a were also serially diluted and spotted onto minimal agar plates containing glutamate (0.1%) or ammonium sulphate (1\u2009mM), and grown for 3 days at 30\u2009\u00b0C.", + "section": "FIG", + "ner": [ + [ + 15, + 27, + "cell density", + "evidence" + ], + [ + 229, + 238, + "glutamate", + "chemical" + ], + [ + 249, + 266, + "ammonium sulphate", + "chemical" + ] + ] + }, + { + "sid": 217, + "sent": "Structural differences between Mep2 and bacterial ammonium transporters.", + "section": "FIG", + "ner": [ + [ + 31, + 35, + "Mep2", + "protein" + ], + [ + 40, + 49, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 218, + "sent": "(a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing unwinding and inward movement in the fungal protein. (b) Stereo diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in AfAmt-1 (light colours) and CaMep2 (dark colours).", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "ICL1", + "structure_element" + ], + [ + 12, + 19, + "AfAmt-1", + "protein" + ], + [ + 37, + 43, + "CaMep2", + "protein" + ], + [ + 102, + 108, + "fungal", + "taxonomy_domain" + ], + [ + 164, + 168, + "ICL1", + "structure_element" + ], + [ + 170, + 174, + "ICL3", + "structure_element" + ], + [ + 191, + 194, + "CTR", + "structure_element" + ], + [ + 204, + 211, + "AfAmt-1", + "protein" + ], + [ + 232, + 238, + "CaMep2", + "protein" + ] + ] + }, + { + "sid": 219, + "sent": "The side chains of residues in the RxK motif as well as those of Tyr49 and His342 are labelled.", + "section": "FIG", + "ner": [ + [ + 35, + 44, + "RxK motif", + "structure_element" + ], + [ + 65, + 70, + "Tyr49", + "residue_name_number" + ], + [ + 75, + 81, + "His342", + "residue_name_number" + ] + ] + }, + { + "sid": 220, + "sent": " The numbering is for CaMep2.", + "section": "FIG", + "ner": [ + [ + 22, + 28, + "CaMep2", + "protein" + ] + ] + }, + { + "sid": 221, + "sent": "(c) Conserved residues in ICL1-3 and the CTR.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "Conserved", + "protein_state" + ], + [ + 26, + 32, + "ICL1-3", + "structure_element" + ], + [ + 41, + 44, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 222, + "sent": "Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the large differences in conformation of the conserved residues in ICL1 (RxK motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red).", + "section": "FIG", + "ner": [ + [ + 27, + 33, + "CaMep2", + "protein" + ], + [ + 45, + 52, + "AfAmt-1", + "protein" + ], + [ + 112, + 121, + "conserved", + "protein_state" + ], + [ + 134, + 138, + "ICL1", + "structure_element" + ], + [ + 158, + 162, + "ICL2", + "structure_element" + ], + [ + 164, + 172, + "ER motif", + "structure_element" + ], + [ + 181, + 185, + "ICL3", + "structure_element" + ], + [ + 202, + 205, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 223, + "sent": "The labelled residues are analogous within both structures.", + "section": "FIG", + "ner": [ + [ + 48, + 58, + "structures", + "evidence" + ] + ] + }, + { + "sid": 224, + "sent": "In b and c, the centre of the trimer is at top.", + "section": "FIG", + "ner": [ + [ + 30, + 36, + "trimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 225, + "sent": "Channel closures in Mep2.", + "section": "FIG", + "ner": [ + [ + 20, + 24, + "Mep2", + "protein" + ] + ] + }, + { + "sid": 226, + "sent": "(a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2.", + "section": "FIG", + "ner": [ + [ + 11, + 24, + "superposition", + "experimental_method" + ], + [ + 28, + 35, + "AfAmt-1", + "protein" + ], + [ + 40, + 46, + "CaMep2", + "protein" + ], + [ + 75, + 83, + "Phe gate", + "site" + ], + [ + 85, + 89, + "His2", + "residue_name_number" + ], + [ + 97, + 111, + "twin-His motif", + "structure_element" + ], + [ + 120, + 128, + "tyrosine", + "residue_name" + ], + [ + 137, + 140, + "Y49", + "residue_name_number" + ], + [ + 144, + 147, + "TM1", + "structure_element" + ], + [ + 161, + 174, + "hydrogen bond", + "bond_interaction" + ], + [ + 180, + 184, + "His2", + "residue_name_number" + ], + [ + 188, + 194, + "CaMep2", + "protein" + ], + [ + 271, + 284, + "channel block", + "structure_element" + ], + [ + 314, + 320, + "CaMep2", + "protein" + ] + ] + }, + { + "sid": 227, + "sent": "The Npr1 kinase target Ser453 is dephosphorylated and located in an electronegative pocket.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "Npr1", + "protein" + ], + [ + 9, + 15, + "kinase", + "protein_type" + ], + [ + 23, + 29, + "Ser453", + "residue_name_number" + ], + [ + 33, + 49, + "dephosphorylated", + "protein_state" + ], + [ + 68, + 90, + "electronegative pocket", + "site" + ] + ] + }, + { + "sid": 228, + "sent": "(a) Stereoviews of CaMep2 showing 2Fo\u2013Fc electron density (contoured at 1.0\u2009\u03c3) for CTR residues Asp419-Met422 and for Tyr446-Thr455 of the AI region.", + "section": "FIG", + "ner": [ + [ + 19, + 25, + "CaMep2", + "protein" + ], + [ + 83, + 86, + "CTR", + "structure_element" + ], + [ + 96, + 109, + "Asp419-Met422", + "residue_range" + ], + [ + 118, + 131, + "Tyr446-Thr455", + "residue_range" + ], + [ + 139, + 148, + "AI region", + "structure_element" + ] + ] + }, + { + "sid": 229, + "sent": "The phosphorylation target residue Ser453 is labelled in bold.", + "section": "FIG", + "ner": [ + [ + 4, + 19, + "phosphorylation", + "ptm" + ], + [ + 35, + 41, + "Ser453", + "residue_name_number" + ] + ] + }, + { + "sid": 230, + "sent": "(b) Overlay of the CTRs of ScMep2 (grey) and CaMep2 (green), showing the similar electronegative environment surrounding the phosphorylation site (P).", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "Overlay", + "experimental_method" + ], + [ + 19, + 23, + "CTRs", + "structure_element" + ], + [ + 27, + 33, + "ScMep2", + "protein" + ], + [ + 45, + 51, + "CaMep2", + "protein" + ], + [ + 125, + 145, + "phosphorylation site", + "site" + ] + ] + }, + { + "sid": 231, + "sent": "The AI regions are coloured magenta.", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "AI regions", + "structure_element" + ] + ] + }, + { + "sid": 232, + "sent": "(c) Cytoplasmic view of the Mep2 trimer indicating the large distance between Ser453 and the channel exits (circles; Ile52 lining the channel exit is shown).", + "section": "FIG", + "ner": [ + [ + 28, + 32, + "Mep2", + "protein" + ], + [ + 33, + 39, + "trimer", + "oligomeric_state" + ], + [ + 78, + 84, + "Ser453", + "residue_name_number" + ], + [ + 93, + 106, + "channel exits", + "site" + ], + [ + 117, + 122, + "Ile52", + "residue_name_number" + ], + [ + 134, + 146, + "channel exit", + "site" + ] + ] + }, + { + "sid": 233, + "sent": "Effect of removal of the AI region on Mep2 structure.", + "section": "FIG", + "ner": [ + [ + 10, + 17, + "removal", + "experimental_method" + ], + [ + 25, + 34, + "AI region", + "structure_element" + ], + [ + 38, + 42, + "Mep2", + "protein" + ], + [ + 43, + 52, + "structure", + "evidence" + ] + ] + }, + { + "sid": 234, + "sent": "(a) Side views for WT CaMep2 (left) and the truncation mutant 442\u0394 (right).", + "section": "FIG", + "ner": [ + [ + 19, + 21, + "WT", + "protein_state" + ], + [ + 22, + 28, + "CaMep2", + "protein" + ], + [ + 44, + 61, + "truncation mutant", + "protein_state" + ], + [ + 62, + 66, + "442\u0394", + "mutant" + ] + ] + }, + { + "sid": 235, + "sent": "The latter is shown as a putty model according to B-factors to illustrate the disorder in the protein on the cytoplasmic side.", + "section": "FIG", + "ner": [ + [ + 78, + 86, + "disorder", + "protein_state" + ] + ] + }, + { + "sid": 236, + "sent": " Missing regions are labelled. (b) Stereo superpositions of WT CaMep2 and the truncation mutant.", + "section": "FIG", + "ner": [ + [ + 42, + 56, + "superpositions", + "experimental_method" + ], + [ + 60, + 62, + "WT", + "protein_state" + ], + [ + 63, + 69, + "CaMep2", + "protein" + ], + [ + 78, + 95, + "truncation mutant", + "protein_state" + ] + ] + }, + { + "sid": 237, + "sent": "2Fo\u2013Fc electron density (contoured at 1.0\u2009\u03c3) for residues Tyr49 and His342 is shown for the truncation mutant.", + "section": "FIG", + "ner": [ + [ + 58, + 63, + "Tyr49", + "residue_name_number" + ], + [ + 68, + 74, + "His342", + "residue_name_number" + ], + [ + 92, + 109, + "truncation mutant", + "protein_state" + ] + ] + }, + { + "sid": 238, + "sent": "Phosphorylation causes conformational changes in the CTR.", + "section": "FIG", + "ner": [ + [ + 0, + 15, + "Phosphorylation", + "ptm" + ], + [ + 53, + 56, + "CTR", + "structure_element" + ] + ] + }, + { + "sid": 239, + "sent": "(a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2 superposed in grey for one of the monomers.", + "section": "FIG", + "ner": [ + [ + 28, + 37, + "DD mutant", + "mutant" + ], + [ + 38, + 44, + "trimer", + "oligomeric_state" + ], + [ + 51, + 53, + "WT", + "protein_state" + ], + [ + 54, + 60, + "CaMep2", + "protein" + ], + [ + 61, + 71, + "superposed", + "experimental_method" + ], + [ + 95, + 103, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 240, + "sent": "The arrow indicates the phosphorylation site.", + "section": "FIG", + "ner": [ + [ + 24, + 44, + "phosphorylation site", + "site" + ] + ] + }, + { + "sid": 241, + "sent": "The AI region is coloured magenta.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "AI region", + "structure_element" + ] + ] + }, + { + "sid": 242, + "sent": "(b) Monomer side-view superposition of WT CaMep2 and the DD mutant, showing the conformational change and disorder around the ExxGxD motif.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "Monomer", + "oligomeric_state" + ], + [ + 22, + 35, + "superposition", + "experimental_method" + ], + [ + 39, + 41, + "WT", + "protein_state" + ], + [ + 42, + 48, + "CaMep2", + "protein" + ], + [ + 57, + 66, + "DD mutant", + "mutant" + ], + [ + 126, + 138, + "ExxGxD motif", + "structure_element" + ] + ] + }, + { + "sid": 243, + "sent": "Side chains for residues 452 and 453 are shown as stick models.", + "section": "FIG", + "ner": [ + [ + 25, + 28, + "452", + "residue_number" + ], + [ + 33, + 36, + "453", + "residue_number" + ] + ] + }, + { + "sid": 244, + "sent": "Schematic model for phosphorylation-based regulation of Mep2 ammonium transporters.", + "section": "FIG", + "ner": [ + [ + 56, + 60, + "Mep2", + "protein" + ] + ] + }, + { + "sid": 245, + "sent": "(a) In the closed, non-phosphorylated state (i), the CTR (magenta) and ICL3 (green) are far apart with the latter blocking the intracellular channel exit (indicated with a hatched circle).", + "section": "FIG", + "ner": [ + [ + 11, + 17, + "closed", + "protein_state" + ], + [ + 19, + 37, + "non-phosphorylated", + "protein_state" + ], + [ + 53, + 56, + "CTR", + "structure_element" + ], + [ + 71, + 75, + "ICL3", + "structure_element" + ], + [ + 141, + 153, + "channel exit", + "site" + ] + ] + }, + { + "sid": 246, + "sent": "Upon phosphorylation and mimicked by the CaMep2 S453D and DD mutants (ii), the region around the ExxGxD motif undergoes a conformational change that results in the CTR interacting with the inward-moving ICL3, opening the channel (full circle) (iii).", + "section": "FIG", + "ner": [ + [ + 5, + 20, + "phosphorylation", + "ptm" + ], + [ + 25, + 33, + "mimicked", + "protein_state" + ], + [ + 41, + 47, + "CaMep2", + "protein" + ], + [ + 48, + 53, + "S453D", + "mutant" + ], + [ + 58, + 68, + "DD mutants", + "mutant" + ], + [ + 97, + 109, + "ExxGxD motif", + "structure_element" + ], + [ + 164, + 167, + "CTR", + "structure_element" + ], + [ + 203, + 207, + "ICL3", + "structure_element" + ], + [ + 221, + 228, + "channel", + "site" + ] + ] + }, + { + "sid": 247, + "sent": "The open-channel Mep2 structure is represented by archaebacterial Amt-1 and shown in lighter colours consistent with Fig. 4.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "open", + "protein_state" + ], + [ + 9, + 16, + "channel", + "site" + ], + [ + 17, + 21, + "Mep2", + "protein" + ], + [ + 22, + 31, + "structure", + "evidence" + ], + [ + 50, + 65, + "archaebacterial", + "taxonomy_domain" + ], + [ + 66, + 71, + "Amt-1", + "protein" + ] + ] + }, + { + "sid": 248, + "sent": "As discussed in the text, similar structural arrangements may occur in plant AMTs.", + "section": "FIG", + "ner": [ + [ + 71, + 76, + "plant", + "taxonomy_domain" + ], + [ + 77, + 81, + "AMTs", + "protein_type" + ] + ] + }, + { + "sid": 249, + "sent": "In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR\u2013ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow).", + "section": "FIG", + "ner": [ + [ + 26, + 30, + "open", + "protein_state" + ], + [ + 31, + 38, + "channel", + "site" + ], + [ + 58, + 76, + "non-phosphorylated", + "protein_state" + ], + [ + 84, + 99, + "phosphorylation", + "ptm" + ], + [ + 144, + 151, + "channel", + "site" + ], + [ + 216, + 231, + "phosphorylation", + "ptm" + ], + [ + 237, + 241, + "Mep2", + "protein" + ], + [ + 242, + 249, + "monomer", + "oligomeric_state" + ], + [ + 271, + 275, + "open", + "protein_state" + ], + [ + 276, + 284, + "channels", + "site" + ], + [ + 299, + 305, + "trimer", + "oligomeric_state" + ], + [ + 353, + 356, + "CTR", + "structure_element" + ], + [ + 361, + 365, + "ICL3", + "structure_element" + ], + [ + 384, + 391, + "monomer", + "oligomeric_state" + ] + ] + } + ] + }, + "PMC4820378": { + "annotations": [ + { + "sid": 0, + "sent": "Template-dependent nucleotide addition in the reverse (3\u2032-5\u2032) direction by Thg1-like protein", + "section": "TITLE", + "ner": [ + [ + 75, + 92, + "Thg1-like protein", + "protein_type" + ] + ] + }, + { + "sid": 1, + "sent": "Structures of Thg1-like proteins provide insight into the template-dependent nucleotide addition in the reverse (3\u2032-5\u2032) direction.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 14, + 32, + "Thg1-like proteins", + "protein_type" + ] + ] + }, + { + "sid": 2, + "sent": "Thg1-like protein (TLP) catalyzes the addition of a nucleotide to the 5\u2032-end of truncated transfer RNA (tRNA) species in a Watson-Crick template\u2013dependent manner.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 17, + "Thg1-like protein", + "protein_type" + ], + [ + 19, + 22, + "TLP", + "protein_type" + ], + [ + 90, + 102, + "transfer RNA", + "chemical" 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second-step reactions in the vicinity of one reaction center consisting of two Mg2+ ions, and the two reactions are executed at the same reaction center in a stepwise fashion.", + "section": "ABSTRACT", + "ner": [ + [ + 23, + 46, + "substrate binding sites", + "site" + ], + [ + 111, + 126, + "reaction center", + "site" + ], + [ + 145, + 149, + "Mg2+", + "chemical" + ], + [ + 203, + 218, + "reaction center", + "site" + ] + ] + }, + { + "sid": 6, + "sent": "When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3\u2032-OH of the incoming nucleotide and the 5\u2032-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred.", + "section": "ABSTRACT", + "ner": [ + [ + 18, + 28, + "nucleotide", + "chemical" + ], + [ + 32, + 40, + "bound to", + "protein_state" + ], + [ + 45, + 64, + "second binding site", + "site" + ], + [ + 70, + 97, + "Watson-Crick hydrogen bonds", + "bond_interaction" + ], + [ + 144, + 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] + ] + }, + { + "sid": 10, + "sent": "Furthermore, mutational analyses show that tRNAHis is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode.", + "section": "ABSTRACT", + "ner": [ + [ + 13, + 32, + "mutational analyses", + "experimental_method" + ], + [ + 43, + 50, + "tRNAHis", + "chemical" + ], + [ + 54, + 62, + "bound to", + "protein_state" + ], + [ + 63, + 66, + "TLP", + "protein_type" + ], + [ + 90, + 94, + "Thg1", + "protein" + ], + [ + 117, + 120, + "TLP", + "protein_type" + ] + ] + }, + { + "sid": 11, + "sent": "All polynucleotide chain elongation reactions, whether with DNA or RNA, proceed in the 5\u2032-3\u2032 direction.", + "section": "INTRO", + "ner": [ + [ + 60, + 63, + "DNA", + "chemical" + ], + [ + 67, + 70, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 12, + "sent": "This reaction involves the nucleophilic attack of a 3\u2032-OH of the terminal nucleotide in the elongating chain on the \u03b1-phosphate of an incoming nucleotide.", + "section": "INTRO", + "ner": [ + [ + 118, + 127, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 13, + "sent": "This elongation reaction of DNA/RNA chains is in clear contrast to the elongation of protein chains in which the high energy of the incoming aminoacyl-tRNA is not used for its own addition but for the addition of the next monomer (termed head polymerization).", + "section": "INTRO", + "ner": [ + [ + 28, + 31, + "DNA", + "chemical" + ], + [ + 32, + 35, + "RNA", + "chemical" + ], + [ + 141, + 155, + "aminoacyl-tRNA", + "chemical" + ], + [ + 222, + 229, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 14, + "sent": "However, recent studies have shown that the Thg1/Thg1-like protein (TLP) family of proteins extends tRNA nucleotide chains in the reverse (3\u2032-5\u2032) direction.", + "section": "INTRO", + "ner": [ + [ + 44, + 48, + "Thg1", + "protein" + ], + [ + 49, + 66, + "Thg1-like protein", + "protein_type" + ], + [ + 68, + 71, + "TLP", + "protein_type" + ], + [ + 100, + 104, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 15, + "sent": "In this case, the 5\u2032-end of tRNA is first activated using adenosine 5\u2032-triphosphate (ATP)/guanosine 5\u2032-triphosphate (GTP), followed by nucleophilic attack of a 3\u2032-OH on the incoming nucleotide [nucleoside 5\u2032-triphosphate (NTP)] to yield pppN-tRNA.", + "section": "INTRO", + "ner": [ + [ + 28, + 32, + "tRNA", + "chemical" + ], + [ + 58, + 83, + "adenosine 5\u2032-triphosphate", + "chemical" + ], + [ + 85, + 88, + "ATP", + "chemical" + ], + [ + 90, + 115, + "guanosine 5\u2032-triphosphate", + "chemical" + ], + [ + 117, + 120, + "GTP", + "chemical" + ], + [ + 194, + 220, + "nucleoside 5\u2032-triphosphate", + "chemical" + ], + [ + 222, + 225, + "NTP", + "chemical" + ], + [ + 237, + 246, + "pppN-tRNA", + "chemical" + ] + ] + }, + { + "sid": 16, + "sent": "Thus, the energy in the triphosphate bond of the incoming nucleotide is not used for its own addition but is reserved for subsequent polymerization (that is, head polymerization) (Fig. 1).", + "section": "INTRO", + "ner": [ + [ + 24, + 36, + "triphosphate", + "chemical" + ] + ] + }, + { + "sid": 17, + "sent": "Top: Reaction scheme of 3\u2032-5\u2032 elongation by Thg1/TLP family proteins.", + "section": "FIG", + "ner": [ + [ + 44, + 48, + "Thg1", + "protein" + ], + [ + 49, + 52, + "TLP", + "protein_type" + ] + ] + }, + { + "sid": 18, + "sent": "Bottom: Reaction scheme of 5\u2032-3\u2032 elongation by DNA/RNA polymerases.", + "section": "FIG", + "ner": [ + [ + 47, + 66, + "DNA/RNA polymerases", + "protein_type" + ] + ] + }, + { + "sid": 19, + "sent": "In 3\u2032-5\u2032 elongation by Thg1/TLP family proteins, the 5\u2032-monophosphate of the tRNA is first activated by ATP/GTP, followed by the actual elongation reaction.", + "section": "FIG", + "ner": [ + [ + 23, + 27, + "Thg1", + "protein" + ], + [ + 28, + 31, + "TLP", + "protein_type" + ], + [ + 53, + 69, + "5\u2032-monophosphate", + "chemical" + ], + [ + 77, + 81, + "tRNA", + "chemical" + ], + [ + 104, + 107, + "ATP", + "chemical" + ], + [ + 108, + 111, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 20, + "sent": "In 5\u2032-3\u2032 elongation by DNA/RNA polymerases, the energy of the incoming nucleotide is used for its own addition (tail polymerization).", + "section": "FIG", + "ner": [ + [ + 23, + 42, + "DNA/RNA polymerases", + "protein_type" + ] + ] + }, + { + "sid": 21, + "sent": "The best-characterized member of this family of proteins is eukaryotic Thg1 (tRNAHis guanylyltransferase), which catalyzes the nontemplated addition of a guanylate to the 5\u2032-end of immature tRNAHis.", + "section": "INTRO", + "ner": [ + [ + 60, + 70, + "eukaryotic", + "taxonomy_domain" + ], + [ + 71, + 75, + "Thg1", + "protein" + ], + [ + 77, + 104, + "tRNAHis guanylyltransferase", + "protein_type" + ], + [ + 190, + 197, + "tRNAHis", + "chemical" + ] + ] + }, + { + "sid": 22, + "sent": "This guanosine at position \u22121 (G\u22121) of tRNAHis is a critical identity element for recognition by the histidyl-tRNA synthase.", + "section": "INTRO", + "ner": [ + [ + 5, + 14, + "guanosine", + "chemical" + ], + [ + 27, + 29, + "\u22121", + "residue_number" + ], + [ + 31, + 34, + "G\u22121", + "residue_name_number" + ], + [ + 39, + 46, + "tRNAHis", + "chemical" + ], + [ + 101, + 123, + "histidyl-tRNA synthase", + "protein_type" + ] + ] + }, + { + "sid": 23, + "sent": "Therefore, Thg1 is essential to the fidelity of protein synthesis in eukaryotes.", + "section": "INTRO", + "ner": [ + [ + 11, + 15, + "Thg1", + "protein" + ], + [ + 69, + 79, + "eukaryotes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 24, + "sent": "However, Thg1 homologs or TLPs are found in organisms in which G\u22121 is genetically encoded, and thus, posttranscriptional modification is not required.", + "section": "INTRO", + "ner": [ + [ + 9, + 13, + "Thg1", + "protein" + ], + [ + 26, + 30, + "TLPs", + "protein_type" + ], + [ + 63, + 66, + "G\u22121", + "residue_name_number" + ] + ] + }, + { + "sid": 25, + "sent": "This finding suggests that TLPs may have potential functions other than tRNAHis maturation.", + "section": "INTRO", + "ner": [ + [ + 27, + 31, + "TLPs", + "protein_type" + ], + [ + 72, + 79, + "tRNAHis", + "chemical" + ] + ] + }, + { + "sid": 26, + "sent": "TLPs have been shown to catalyze 5\u2032-end nucleotide addition to truncated tRNA species in vitro in a Watson-Crick template\u2013dependent manner.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "TLPs", + "protein_type" + ], + [ + 73, + 77, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 27, + "sent": "This function of TLPs is not limited to tRNAHis but occurs efficiently with other tRNAs.", + "section": "INTRO", + "ner": [ + [ + 17, + 21, + "TLPs", + "protein_type" + ], + [ + 40, + 47, + "tRNAHis", + "chemical" + ], + [ + 82, + 87, + "tRNAs", + "chemical" + ] + ] + }, + { + "sid": 28, + "sent": "Furthermore, the yeast homolog (Thg1p) has been shown to interact with the replication origin recognition complex for DNA replication, and the plant homolog (ICA1) was identified as a protein affecting the capacity to repair DNA damage.", + "section": "INTRO", + "ner": [ + [ + 17, + 22, + "yeast", + "taxonomy_domain" + ], + [ + 32, + 37, + "Thg1p", + "protein" + ], + [ + 118, + 121, + "DNA", + "chemical" + ], + [ + 143, + 148, + "plant", + "taxonomy_domain" + ], + [ + 158, + 162, + "ICA1", + "protein" + ], + [ + 225, + 228, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 29, + "sent": "These observations suggest that TLPs may have more general functions in DNA/RNA repair.", + "section": "INTRO", + "ner": [ + [ + 32, + 36, + "TLPs", + "protein_type" + ], + [ + 72, + 75, + "DNA", + "chemical" + ], + [ + 76, + 79, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 30, + "sent": "The 3\u2032-5\u2032 addition reaction catalyzed by Thg1 occurs through three reaction steps.", + "section": "INTRO", + "ner": [ + [ + 41, + 45, + "Thg1", + "protein" + ] + ] + }, + { + "sid": 31, + "sent": "In the first step, the 5\u2032-monophosphorylated tRNAHis, which is cleaved by ribonuclease P from pre-tRNAHis, is activated by ATP, creating a 5\u2032-adenylylated tRNAHis intermediate.", + "section": "INTRO", + "ner": [ + [ + 45, + 52, + "tRNAHis", + "chemical" + ], + [ + 74, + 88, + "ribonuclease P", + "protein_type" + ], + [ + 94, + 105, + "pre-tRNAHis", + "chemical" + ], + [ + 123, + 126, + "ATP", + "chemical" + ], + [ + 155, + 162, + "tRNAHis", + "chemical" + ] + ] + }, + { + "sid": 32, + "sent": "In the second step, the 3\u2032-OH of the incoming GTP attacks the activated intermediate, yielding pppG\u22121-tRNAHis.", + "section": "INTRO", + "ner": [ + [ + 46, + 49, + "GTP", + "chemical" + ], + [ + 95, + 109, + "pppG\u22121-tRNAHis", + "chemical" + ] + ] + }, + { + "sid": 33, + "sent": "Finally, the pyrophosphate is removed, and mature pG\u22121-tRNAHis is created.", + "section": "INTRO", + "ner": [ + [ + 13, + 26, + "pyrophosphate", + "chemical" + ], + [ + 50, + 62, + "pG\u22121-tRNAHis", + "chemical" + ] + ] + }, + { + "sid": 34, + "sent": "The crystal structure of human Thg1 (HsThg1) shows that its catalytic core shares structural homology with canonical 5\u2032-3\u2032 nucleotide polymerases, such as T7 DNA/RNA polymerases.", + "section": "INTRO", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 25, + 30, + "human", + "species" + ], + [ + 31, + 35, + "Thg1", + "protein" + ], + [ + 37, + 43, + "HsThg1", + "protein" + ], + [ + 60, + 74, + "catalytic core", + "site" + ], + [ + 117, + 145, + "5\u2032-3\u2032 nucleotide polymerases", + "protein_type" + ], + [ + 155, + 177, + "T7 DNA/RNA polymerases", + "protein_type" + ] + ] + }, + { + "sid": 35, + "sent": "This finding suggests that 3\u2032-5\u2032 elongation enzymes are related to 5\u2032-3\u2032 polymerases and raises important questions on why 5\u2032-3\u2032 polymerases predominate in nature.", + "section": "INTRO", + "ner": [ + [ + 27, + 51, + "3\u2032-5\u2032 elongation enzymes", + "protein_type" + ], + [ + 67, + 84, + "5\u2032-3\u2032 polymerases", + "protein_type" + ], + [ + 123, + 140, + "5\u2032-3\u2032 polymerases", + "protein_type" + ] + ] + }, + { + "sid": 36, + "sent": "The crystal structure of TLP from Bacillus thuringiensis shows that it shares a similar tetrameric assembly and active-site architecture with HsThg1.", + "section": "INTRO", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 25, + 28, + "TLP", + "protein_type" + ], + [ + 34, + 56, + "Bacillus thuringiensis", + "species" + ], + [ + 88, + 98, + "tetrameric", + "oligomeric_state" + ], + [ + 112, + 123, + "active-site", + "site" + ], + [ + 142, + 148, + "HsThg1", + "protein" + ] + ] + }, + { + "sid": 37, + "sent": "Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase.", + "section": "INTRO", + "ner": [ + [ + 17, + 26, + "structure", + "evidence" + ], + [ + 30, + 46, + "Candida albicans", + "species" + ], + [ + 47, + 51, + "Thg1", + "protein" + ], + [ + 53, + 59, + "CaThg1", + "protein" + ], + [ + 61, + 75, + "complexed with", + "protein_state" + ], + [ + 76, + 83, + "tRNAHis", + "chemical" + ], + [ + 101, + 105, + "tRNA", + "chemical" + ], + [ + 129, + 144, + "reaction center", + "site" + ], + [ + 192, + 210, + "DNA/RNA polymerase", + "protein_type" + ] + ] + }, + { + "sid": 38, + "sent": "However, in this structural analysis, the 5\u2032-end of tRNAHis was not activated and the second substrate (GTP) was not bound.", + "section": "INTRO", + "ner": [ + [ + 17, + 36, + "structural analysis", + "experimental_method" + ], + [ + 52, + 59, + "tRNAHis", + "chemical" + ], + [ + 104, + 107, + "GTP", + "chemical" + ], + [ + 113, + 122, + "not bound", + "protein_state" + ] + ] + }, + { + "sid": 39, + "sent": "Here, we successfully solved the structure of TLP from the methanogenic archaeon Methanosarcina acetivorans (MaTLP) in complex with ppptRNAPhe\u03941, which mimics the activated intermediate of the repair substrate.", + "section": "INTRO", + "ner": [ + [ + 22, + 28, + "solved", + "experimental_method" + ], + [ + 33, + 42, + "structure", + "evidence" + ], + [ + 46, + 49, + "TLP", + "protein_type" + ], + [ + 59, + 80, + "methanogenic archaeon", + "taxonomy_domain" + ], + [ + 81, + 107, + "Methanosarcina acetivorans", + "species" + ], + [ + 109, + 114, + "MaTLP", + "protein" + ], + [ + 116, + 131, + "in complex with", + "protein_state" + ], + [ + 132, + 144, + "ppptRNAPhe\u03941", + "chemical" + ] + ] + }, + { + "sid": 40, + "sent": "Although TLP and Thg1 have similar tetrameric organization, the mode of tRNA binding is different in TLP.", + "section": "INTRO", + "ner": [ + [ + 9, + 12, + "TLP", + "protein_type" + ], + [ + 17, + 21, + "Thg1", + "protein" + ], + [ + 35, + 45, + "tetrameric", + "oligomeric_state" + ], + [ + 72, + 76, + "tRNA", + "chemical" + ], + [ + 101, + 104, + "TLP", + "protein_type" + ] + ] + }, + { + "sid": 41, + "sent": "Furthermore, we obtained the structure in which the GTP analog (GDPNP) was inserted into this complex to form a Watson-Crick base pair with C72 at the 3\u2032-end region of the tRNA.", + "section": "INTRO", + "ner": [ + [ + 29, + 38, + "structure", + "evidence" + ], + [ + 52, + 55, + "GTP", + "chemical" + ], + [ + 64, + 69, + "GDPNP", + "chemical" + ], + [ + 112, + 134, + "Watson-Crick base pair", + "bond_interaction" + ], + [ + 140, + 143, + "C72", + "residue_name_number" + ], + [ + 172, + 176, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 42, + "sent": "On the basis of these structures, we discuss the reaction mechanism of template-dependent reverse (3\u2032-5\u2032) polymerization in comparison with canonical 5\u2032-3\u2032 polymerization.", + "section": "INTRO", + "ner": [ + [ + 22, + 32, + "structures", + "evidence" + ] + ] + }, + { + "sid": 43, + "sent": "Anticodon-independent binding of ppptRNAPhe\u03941 to MaTLP", + "section": "RESULTS", + "ner": [ + [ + 33, + 45, + "ppptRNAPhe\u03941", + "chemical" + ], + [ + 49, + 54, + "MaTLP", + "protein" + ] + ] + }, + { + "sid": 44, + "sent": "Previous biochemical experiments have suggested that ppptRNAPhe\u03941, in which the 5\u2032-end of tRNAPhe was triphosphorylated and G1 was deleted, can be an efficient substrate for the repair reaction (guanylyl transfer) of Thg1/TLP.", + "section": "RESULTS", + "ner": [ + [ + 9, + 32, + "biochemical experiments", + "experimental_method" + ], + [ + 53, + 65, + "ppptRNAPhe\u03941", + "chemical" + ], + [ + 90, + 97, + "tRNAPhe", + "chemical" + ], + [ + 124, + 126, + "G1", + "residue_name_number" + ], + [ + 131, + 138, + "deleted", + "experimental_method" + ], + [ + 217, + 221, + "Thg1", + "protein" + ], + [ + 222, + 225, + "TLP", + "protein_type" + ] + ] + }, + { + "sid": 45, + "sent": "Therefore, we prepared a crystal of MaTLP complexed with ppptRNAPhe\u03941 and solved its structure to study the template-directed 3\u2032-5\u2032 elongation reaction by TLP (fig. S1).", + "section": "RESULTS", + "ner": [ + [ + 25, + 32, + "crystal", + "evidence" + ], + [ + 36, + 41, + "MaTLP", + "protein" + ], + [ + 42, + 56, + "complexed with", + "protein_state" + ], + [ + 57, + 69, + "ppptRNAPhe\u03941", + "chemical" + ], + [ + 74, + 80, + "solved", + "experimental_method" + ], + [ + 85, + 94, + "structure", + "evidence" + ], + [ + 155, + 158, + "TLP", + "protein_type" + ] + ] + }, + { + "sid": 46, + "sent": "The crystal contained a dimer of TLP (A and B molecules) and one tRNA in an asymmetric unit.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "crystal", + "evidence" + ], + [ + 24, + 29, + "dimer", + "oligomeric_state" + ], + [ + 33, + 36, + "TLP", + "protein_type" + ], + [ + 38, + 39, + "A", + "structure_element" + ], + [ + 44, + 45, + "B", + "structure_element" + ], + [ + 65, + 69, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 47, + "sent": "Two dimers in the crystal further assembled as a dimer of dimers by the crystallographic twofold axis (Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "dimers", + "oligomeric_state" + ], + [ + 18, + 25, + "crystal", + "evidence" + ], + [ + 49, + 54, + "dimer", + "oligomeric_state" + ], + [ + 58, + 64, + "dimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 48, + "sent": "This tetrameric structure and 4:2 stoichiometry of the TLP-tRNA complex are the same as those of the CaThg1-tRNA complex.", + "section": "RESULTS", + "ner": [ + [ + 5, + 15, + "tetrameric", + "oligomeric_state" + ], + [ + 16, + 25, + "structure", + "evidence" + ], + [ + 55, + 63, + "TLP-tRNA", + "complex_assembly" + ], + [ + 101, + 112, + "CaThg1-tRNA", + "complex_assembly" + ] + ] + }, + { + "sid": 49, + "sent": "However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region.", + "section": "RESULTS", + "ner": [ + [ + 21, + 23, + "AB", + "structure_element" + ], + [ + 28, + 30, + "CD", + "structure_element" + ], + [ + 31, + 37, + "dimers", + "oligomeric_state" + ], + [ + 41, + 51, + "tetrameric", + "oligomeric_state" + ], + [ + 52, + 58, + "CaThg1", + "protein" + ], + [ + 110, + 123, + "accepter stem", + "structure_element" + ], + [ + 141, + 148, + "tRNAHis", + "chemical" + ], + [ + 154, + 156, + "AB", + "structure_element" + ], + [ + 157, + 162, + "dimer", + "oligomeric_state" + ], + [ + 186, + 188, + "CD", + "structure_element" + ], + [ + 189, + 194, + "dimer", + "oligomeric_state" + ], + [ + 199, + 209, + "tetrameric", + "oligomeric_state" + ], + [ + 210, + 215, + "MaTLP", + "protein" + ], + [ + 251, + 255, + "tRNA", + "chemical" + ], + [ + 283, + 287, + "tRNA", + "chemical" + ], + [ + 288, + 301, + "accepter stem", + "structure_element" + ], + [ + 306, + 318, + "elbow region", + "structure_element" + ] + ] + }, + { + "sid": 50, + "sent": "Thus, consistent with the notion that MaTLP is an anticodon-independent repair enzyme, the anticodon was not recognized in the MaTLP-tRNA complex, whereas the binding mode of CaThg1 is for the G\u22121 addition reaction, therefore the His anticodon has to be recognized (see \u201cDual binding mode for tRNA repair\u201d).", + "section": "RESULTS", + "ner": [ + [ + 38, + 43, + "MaTLP", + "protein" + ], + [ + 50, + 85, + "anticodon-independent repair enzyme", + "protein_type" + ], + [ + 127, + 137, + "MaTLP-tRNA", + "complex_assembly" + ], + [ + 175, + 181, + "CaThg1", + "protein" + ], + [ + 193, + 196, + "G\u22121", + "residue_name_number" + ], + [ + 230, + 233, + "His", + "residue_name" + ], + [ + 293, + 297, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 51, + "sent": "Structure of the MaTLP complex with ppptRNAPhe\u03941.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 17, + 22, + "MaTLP", + "protein" + ], + [ + 23, + 35, + "complex with", + "protein_state" + ], + [ + 36, + 48, + "ppptRNAPhe\u03941", + "chemical" + ] + ] + }, + { + "sid": 52, + "sent": "Left: One molecule of the tRNA substrate (ppptRNAPhe\u03941) is bound to the MaTLP dimer.", + "section": "FIG", + "ner": [ + [ + 26, + 30, + "tRNA", + "chemical" + ], + [ + 42, + 54, + "ppptRNAPhe\u03941", + "chemical" + ], + [ + 59, + 67, + "bound to", + "protein_state" + ], + [ + 72, + 77, + "MaTLP", + "protein" + ], + [ + 78, + 83, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 53, + "sent": "The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers).", + "section": "FIG", + "ner": [ + [ + 4, + 6, + "AB", + "structure_element" + ], + [ + 11, + 13, + "CD", + "structure_element" + ], + [ + 14, + 20, + "dimers", + "oligomeric_state" + ], + [ + 33, + 42, + "dimerized", + "oligomeric_state" + ], + [ + 90, + 100, + "tetrameric", + "oligomeric_state" + ], + [ + 101, + 110, + "structure", + "evidence" + ], + [ + 112, + 117, + "dimer", + "oligomeric_state" + ], + [ + 121, + 127, + "dimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 54, + "sent": "The CD dimer is omitted for clarity.", + "section": "FIG", + "ner": [ + [ + 4, + 6, + "CD", + "structure_element" + ], + [ + 7, + 12, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 55, + "sent": "The accepter stem of the tRNA is recognized by molecule A (yellow), and the elbow region by molecule B (blue).", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "accepter stem", + "structure_element" + ], + [ + 25, + 29, + "tRNA", + "chemical" + ], + [ + 76, + 88, + "elbow region", + "structure_element" + ] + ] + }, + { + "sid": 56, + "sent": "The \u03b2-hairpin region of molecule B is shown in red.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "\u03b2-hairpin", + "structure_element" + ] + ] + }, + { + "sid": 57, + "sent": "The elbow region of the tRNA substrate was recognized by the \u03b2-hairpin of molecule B of MaTLP.", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "elbow region", + "structure_element" + ], + [ + 24, + 28, + "tRNA", + "chemical" + ], + [ + 61, + 70, + "\u03b2-hairpin", + "structure_element" + ], + [ + 88, + 93, + "MaTLP", + "protein" + ] + ] + }, + { + "sid": 58, + "sent": "The N atoms in the side chain of R215 in the \u03b2-hairpin region of MaTLP were hydrogen-bonded to the phosphate groups of U55 and G57.", + "section": "RESULTS", + "ner": [ + [ + 33, + 37, + "R215", + "residue_name_number" + ], + [ + 45, + 54, + "\u03b2-hairpin", + "structure_element" + ], + [ + 65, + 70, + "MaTLP", + "protein" + ], + [ + 76, + 91, + "hydrogen-bonded", + "bond_interaction" + ], + [ + 99, + 108, + "phosphate", + "chemical" + ], + [ + 119, + 122, + "U55", + "residue_name_number" + ], + [ + 127, + 130, + "G57", + "residue_name_number" + ] + ] + }, + { + "sid": 59, + "sent": "The O atom on the S213 side chain was also hydrogen-bonded to the phosphate moiety of G57 of the tRNA (Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 18, + 22, + "S213", + "residue_name_number" + ], + [ + 43, + 58, + "hydrogen-bonded", + "bond_interaction" + ], + [ + 66, + 75, + "phosphate", + "chemical" + ], + [ + 86, + 89, + "G57", + "residue_name_number" + ], + [ + 97, + 101, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 60, + "sent": "This \u03b2-hairpin region was disordered in the crystal structure of the CaThg1-tRNA complex.", + "section": "RESULTS", + "ner": [ + [ + 5, + 14, + "\u03b2-hairpin", + "structure_element" + ], + [ + 26, + 36, + "disordered", + "protein_state" + ], + [ + 44, + 61, + "crystal structure", + "evidence" + ], + [ + 69, + 80, + "CaThg1-tRNA", + "complex_assembly" + ] + ] + }, + { + "sid": 61, + "sent": "The accepter stem of the tRNA substrate was recognized by molecule A of MaTLP.", + "section": "RESULTS", + "ner": [ + [ + 4, + 17, + "accepter stem", + "structure_element" + ], + [ + 25, + 29, + "tRNA", + "chemical" + ], + [ + 72, + 77, + "MaTLP", + "protein" + ] + ] + }, + { + "sid": 62, + "sent": "The N7 atom of G2 at the 5\u2032-end was hydrogen-bonded to the N atom of the R136 side chain, whereas the \u03b1-phosphate was bonded to the N137 side chain (Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 15, + 17, + "G2", + "residue_name_number" + ], + [ + 36, + 51, + "hydrogen-bonded", + "bond_interaction" + ], + [ + 73, + 77, + "R136", + "residue_name_number" + ], + [ + 104, + 113, + "phosphate", + "chemical" + ], + [ + 132, + 136, + "N137", + "residue_name_number" + ] + ] + }, + { + "sid": 63, + "sent": "R136 was also hydrogen-bonded to the base of C72 (the Watson-Crick bond partner of \u0394G1).", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "R136", + "residue_name_number" + ], + [ + 14, + 29, + "hydrogen-bonded", + "bond_interaction" + ], + [ + 45, + 48, + "C72", + "residue_name_number" + ] + ] + }, + { + "sid": 64, + "sent": "The triphosphate moiety at the 5\u2032-end of the tRNA was bonded to the D21-K26 region.", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "triphosphate", + "chemical" + ], + [ + 45, + 49, + "tRNA", + "chemical" + ], + [ + 68, + 75, + "D21-K26", + "residue_range" + ] + ] + }, + { + "sid": 65, + "sent": "These phosphates were also coordinated to two metal ions, presumably Mg2+ (Mg2+A and Mg2+B) because they were observed at the same positions (figs. S3 and S4) previously identified by CaThg1 and HsThg1 structures.", + "section": "RESULTS", + "ner": [ + [ + 6, + 16, + "phosphates", + "chemical" + ], + [ + 27, + 41, + "coordinated to", + "bond_interaction" + ], + [ + 69, + 73, + "Mg2+", + "chemical" + ], + [ + 75, + 79, + "Mg2+", + "chemical" + ], + [ + 85, + 89, + "Mg2+", + "chemical" + ], + [ + 184, + 190, + "CaThg1", + "protein" + ], + [ + 195, + 201, + "HsThg1", + "protein" + ], + [ + 202, + 212, + "structures", + "evidence" + ] + ] + }, + { + "sid": 66, + "sent": "These ions were in turn coordinated by the O atoms of the side chains of D21 and D69 and the main-chain O of G22 (fig. S3A).", + "section": "RESULTS", + "ner": [ + [ + 24, + 38, + "coordinated by", + "bond_interaction" + ], + [ + 73, + 76, + "D21", + "residue_name_number" + ], + [ + 81, + 84, + "D69", + "residue_name_number" + ], + [ + 109, + 112, + "G22", + "residue_name_number" + ] + ] + }, + { + "sid": 67, + "sent": "Mutation of D29 and D76 in HsThg1 (corresponding to D21 and D69 of MaTLP) has been shown to markedly decrease G\u22121 addition activity.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Mutation", + "experimental_method" + ], + [ + 12, + 15, + "D29", + "residue_name_number" + ], + [ + 20, + 23, + "D76", + "residue_name_number" + ], + [ + 27, + 33, + "HsThg1", + "protein" + ], + [ + 52, + 55, + "D21", + "residue_name_number" + ], + [ + 60, + 63, + "D69", + "residue_name_number" + ], + [ + 67, + 72, + "MaTLP", + "protein" + ], + [ + 110, + 113, + "G\u22121", + "residue_name_number" + ] + ] + }, + { + "sid": 68, + "sent": "Template-dependent binding of the GTP analog to the MaTLP-ppptRNAPhe\u03941 complex", + "section": "RESULTS", + "ner": [ + [ + 34, + 37, + "GTP", + "chemical" + ], + [ + 52, + 70, + "MaTLP-ppptRNAPhe\u03941", + "complex_assembly" + ] + ] + }, + { + "sid": 69, + "sent": "Here, we successfully obtained the structure of the ternary complex of MaTLP, 5\u2032-activated tRNA (ppptRNAPhe\u03941), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPhe\u03941 complex crystal in a solution containing GDPNP.", + "section": "RESULTS", + "ner": [ + [ + 35, + 44, + "structure", + "evidence" + ], + [ + 71, + 76, + "MaTLP", + "protein" + ], + [ + 91, + 95, + "tRNA", + "chemical" + ], + [ + 97, + 109, + "ppptRNAPhe\u03941", + "chemical" + ], + [ + 120, + 123, + "GTP", + "chemical" + ], + [ + 132, + 137, + "GDPNP", + "chemical" + ], + [ + 163, + 170, + "soaking", + "experimental_method" + ], + [ + 175, + 193, + "MaTLP-ppptRNAPhe\u03941", + "complex_assembly" + ], + [ + 202, + 209, + "crystal", + "evidence" + ], + [ + 235, + 240, + "GDPNP", + "chemical" + ] + ] + }, + { + "sid": 70, + "sent": "The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme.", + "section": "RESULTS", + "ner": [ + [ + 13, + 22, + "structure", + "evidence" + ], + [ + 39, + 46, + "guanine", + "chemical" + ], + [ + 68, + 73, + "GDPNP", + "chemical" + ], + [ + 81, + 108, + "Watson-Crick hydrogen bonds", + "bond_interaction" + ], + [ + 114, + 117, + "C72", + "residue_name_number" + ], + [ + 134, + 160, + "base-stacking interactions", + "bond_interaction" + ], + [ + 166, + 168, + "G2", + "residue_name_number" + ], + [ + 176, + 180, + "tRNA", + "chemical" + ], + [ + 240, + 247, + "guanine", + "chemical" + ] + ] + }, + { + "sid": 71, + "sent": "The 5\u2032-end (position 2) of the tRNA moved significantly (Fig. 3C) due to the insertion of GDPNP.", + "section": "RESULTS", + "ner": [ + [ + 31, + 35, + "tRNA", + "chemical" + ], + [ + 90, + 95, + "GDPNP", + "chemical" + ] + ] + }, + { + "sid": 72, + "sent": "Surprisingly, the 5\u2032-triphosphate moiety after movement occupied the GTP/ATP triphosphate position during the activation step (Fig. 3D).", + "section": "RESULTS", + "ner": [ + [ + 18, + 33, + "5\u2032-triphosphate", + "chemical" + ], + [ + 69, + 72, + "GTP", + "chemical" + ], + [ + 73, + 76, + "ATP", + "chemical" + ], + [ + 77, + 89, + "triphosphate", + "chemical" + ] + ] + }, + { + "sid": 73, + "sent": "Together with the observation that the 3\u2032-OH of the incoming GTP analog was within coordination distance (2.8 \u00c5) to Mg2+A (fig. S3B) and was able to execute a nucleophilic attack on the \u03b1-phosphate of the 5\u2032-end, this structure indicates that the elongation reaction (second reaction) takes place at the same reaction center where the activation reaction (first reaction) occurs.", + "section": "RESULTS", + "ner": [ + [ + 61, + 64, + "GTP", + "chemical" + ], + [ + 116, + 120, + "Mg2+", + "chemical" + ], + [ + 188, + 197, + "phosphate", + "chemical" + ], + [ + 218, + 227, + "structure", + "evidence" + ], + [ + 309, + 324, + "reaction center", + "site" + ] + ] + }, + { + "sid": 74, + "sent": "Structural change of the tRNA (ppptRNAPhe\u03941).", + "section": "FIG", + "ner": [ + [ + 25, + 29, + "tRNA", + "chemical" + ], + [ + 31, + 43, + "ppptRNAPhe\u03941", + "chemical" + ] + ] + }, + { + "sid": 75, + "sent": "Structural change of the tRNA (ppptRNAPhe\u03941) accepter stem in MaTLP caused by insertion of GDPNP. (A) Structure before GDPNP binding.", + "section": "FIG", + "ner": [ + [ + 25, + 29, + "tRNA", + "chemical" + ], + [ + 31, + 43, + "ppptRNAPhe\u03941", + "chemical" + ], + [ + 45, + 58, + "accepter stem", + "structure_element" + ], + [ + 62, + 67, + "MaTLP", + "protein" + ], + [ + 91, + 96, + "GDPNP", + "chemical" + ], + [ + 102, + 111, + "Structure", + "evidence" + ], + [ + 119, + 124, + "GDPNP", + "chemical" + ] + ] + }, + { + "sid": 76, + "sent": "(B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5\u2032-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5\u2032-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "Structure", + "evidence" + ], + [ + 20, + 25, + "GDPNP", + "chemical" + ], + [ + 39, + 52, + "Superposition", + "experimental_method" + ], + [ + 64, + 74, + "structures", + "evidence" + ], + [ + 113, + 117, + "tRNA", + "chemical" + ], + [ + 161, + 166, + "GDPNP", + "chemical" + ], + [ + 172, + 185, + "Superposition", + "experimental_method" + ], + [ + 207, + 211, + "tRNA", + "chemical" + ], + [ + 218, + 223, + "GDPNP", + "chemical" + ], + [ + 245, + 248, + "GTP", + "chemical" + ], + [ + 299, + 311, + "triphosphate", + "chemical" + ] + ] + }, + { + "sid": 77, + "sent": "The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B).", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "triphosphate", + "chemical" + ], + [ + 27, + 32, + "GDPNP", + "chemical" + ], + [ + 44, + 53, + "interface", + "site" + ], + [ + 72, + 73, + "A", + "structure_element" + ], + [ + 78, + 79, + "B", + "structure_element" + ], + [ + 147, + 150, + "R19", + "residue_name_number" + ], + [ + 161, + 162, + "A", + "structure_element" + ], + [ + 165, + 168, + "R83", + "residue_name_number" + ], + [ + 179, + 180, + "B", + "structure_element" + ], + [ + 183, + 186, + "K86", + "residue_name_number" + ], + [ + 197, + 198, + "B", + "structure_element" + ], + [ + 205, + 209, + "R114", + "residue_name_number" + ], + [ + 220, + 221, + "A", + "structure_element" + ] + ] + }, + { + "sid": 78, + "sent": "All of these residues are well conserved (fig. S5), and mutation of corresponding residues in ScThg1 (R27, R93, K96, and R133) decreased the catalytic efficiency of G\u22121 addition.", + "section": "RESULTS", + "ner": [ + [ + 26, + 40, + "well conserved", + "protein_state" + ], + [ + 56, + 64, + "mutation", + "experimental_method" + ], + [ + 94, + 100, + "ScThg1", + "protein" + ], + [ + 102, + 105, + "R27", + "residue_name_number" + ], + [ + 107, + 110, + "R93", + "residue_name_number" + ], + [ + 112, + 115, + "K96", + "residue_name_number" + ], + [ + 121, + 125, + "R133", + "residue_name_number" + ], + [ + 165, + 168, + "G\u22121", + "residue_name_number" + ] + ] + }, + { + "sid": 79, + "sent": "The triphosphate of the GDPNP was also bonded to the third Mg2+ (Mg2+C), which, unlike Mg2+A and Mg2+B, is not coordinated by the TLP molecule (fig. S3B).", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "triphosphate", + "chemical" + ], + [ + 24, + 29, + "GDPNP", + "chemical" + ], + [ + 59, + 63, + "Mg2+", + "chemical" + ], + [ + 65, + 69, + "Mg2+", + "chemical" + ], + [ + 87, + 91, + "Mg2+", + "chemical" + ], + [ + 97, + 101, + "Mg2+", + "chemical" + ], + [ + 111, + 125, + "coordinated by", + "bond_interaction" + ], + [ + 130, + 133, + "TLP", + "protein_type" + ] + ] + }, + { + "sid": 80, + "sent": "This triphosphate binding mode is the same as that for the second nucleotide binding site in Thg1.", + "section": "RESULTS", + "ner": [ + [ + 5, + 17, + "triphosphate", + "chemical" + ], + [ + 59, + 89, + "second nucleotide binding site", + "site" + ], + [ + 93, + 97, + "Thg1", + "protein" + ] + ] + }, + { + "sid": 81, + "sent": "However, in previous analyses, the base moiety at the second site was either invisible or far beyond the reaction distance of the phosphate, and therefore, flipping of the base was expected to occur.", + "section": "RESULTS", + "ner": [ + [ + 54, + 65, + "second site", + "site" + ], + [ + 130, + 139, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 82, + "sent": "tRNA binding and repair experiments of the \u03b2-hairpin mutants", + "section": "RESULTS", + "ner": [ + [ + 0, + 35, + "tRNA binding and repair experiments", + "experimental_method" + ], + [ + 43, + 52, + "\u03b2-hairpin", + "structure_element" + ], + [ + 53, + 60, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 83, + "sent": "To confirm tRNA recognition by the \u03b2-hairpin, we created mutation variants with altered residues in the \u03b2-hairpin region.", + "section": "RESULTS", + "ner": [ + [ + 11, + 15, + "tRNA", + "chemical" + ], + [ + 35, + 44, + "\u03b2-hairpin", + "structure_element" + ], + [ + 49, + 74, + "created mutation variants", + "experimental_method" + ], + [ + 104, + 113, + "\u03b2-hairpin", + "structure_element" + ] + ] + }, + { + "sid": 84, + "sent": "Then, tRNA binding and enzymatic activities were measured.", + "section": "RESULTS", + "ner": [ + [ + 6, + 57, + "tRNA binding and enzymatic activities were measured", + "experimental_method" + ] + ] + }, + { + "sid": 85, + "sent": "\u03b2-Hairpin deletion variant delR198-R215 almost completely abolished the binding of tRNAPhe\u03941 (fig. S6).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "\u03b2-Hairpin", + "structure_element" + ], + [ + 10, + 26, + "deletion variant", + "protein_state" + ], + [ + 27, + 39, + "delR198-R215", + "mutant" + ], + [ + 83, + 92, + "tRNAPhe\u03941", + "chemical" + ] + ] + }, + { + "sid": 86, + "sent": "Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A).", + "section": "RESULTS", + "ner": [ + [ + 41, + 53, + "delR198-R215", + "mutant" + ], + [ + 58, + 70, + "delG202-E210", + "mutant" + ], + [ + 130, + 139, + "wild type", + "protein_state" + ], + [ + 149, + 158, + "mutations", + "experimental_method" + ], + [ + 160, + 165, + "N179A", + "mutant" + ], + [ + 170, + 175, + "F174A", + "mutant" + ], + [ + 176, + 181, + "N179A", + "mutant" + ], + [ + 182, + 187, + "R188A", + "mutant" + ], + [ + 196, + 222, + "anticodon recognition site", + "site" + ], + [ + 241, + 253, + "Thg1-tRNAHis", + "complex_assembly" + ], + [ + 262, + 271, + "structure", + "evidence" + ] + ] + }, + { + "sid": 87, + "sent": "Experiments using the tRNAHis\u03941 substrate gave similar results (Fig. 4A).", + "section": "RESULTS", + "ner": [ + [ + 22, + 31, + "tRNAHis\u03941", + "chemical" + ] + ] + }, + { + "sid": 88, + "sent": "All these results are consistent with the crystal structure and suggest that the \u03b2-hairpin plays an important role in anticodon-independent binding of the tRNA substrate.", + "section": "RESULTS", + "ner": [ + [ + 42, + 59, + "crystal structure", + "evidence" + ], + [ + 81, + 90, + "\u03b2-hairpin", + "structure_element" + ], + [ + 155, + 159, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 89, + "sent": "Residues in the \u03b2-hairpin are not well conserved, except for R215 (fig. S5).", + "section": "RESULTS", + "ner": [ + [ + 16, + 25, + "\u03b2-hairpin", + "structure_element" + ], + [ + 30, + 48, + "not well conserved", + "protein_state" + ], + [ + 61, + 65, + "R215", + "residue_name_number" + ] + ] + }, + { + "sid": 90, + "sent": "Mutants R215A and R215A/S213A, in which the completely conserved R215 was changed to alanine, showed a moderate effect on the activity (27.3 and 16.3%, respectively).", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Mutants", + "protein_state" + ], + [ + 8, + 13, + "R215A", + "mutant" + ], + [ + 18, + 23, + "R215A", + "mutant" + ], + [ + 24, + 29, + "S213A", + "mutant" + ], + [ + 44, + 64, + "completely conserved", + "protein_state" + ], + [ + 65, + 69, + "R215", + "residue_name_number" + ], + [ + 74, + 81, + "changed", + "experimental_method" + ], + [ + 85, + 92, + "alanine", + "residue_name" + ] + ] + }, + { + "sid": 91, + "sent": "Thus, specific interactions with the conserved R215 and van der Waals contacts to residues in the \u03b2-hairpin would be important for tRNA recognition.", + "section": "RESULTS", + "ner": [ + [ + 37, + 46, + "conserved", + "protein_state" + ], + [ + 47, + 51, + "R215", + "residue_name_number" + ], + [ + 56, + 78, + "van der Waals contacts", + "bond_interaction" + ], + [ + 98, + 107, + "\u03b2-hairpin", + "structure_element" + ], + [ + 131, + 135, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 92, + "sent": "Mutational analysis of the \u03b2-hairpin and anticodon binding region.", + "section": "FIG", + "ner": [ + [ + 0, + 19, + "Mutational analysis", + "experimental_method" + ], + [ + 27, + 36, + "\u03b2-hairpin", + "structure_element" + ], + [ + 41, + 65, + "anticodon binding region", + "site" + ] + ] + }, + { + "sid": 93, + "sent": "(A) Guanylylation of ppptRNAPhe\u03941 and ppptRNAHis\u03941 by various TLP mutants.", + "section": "FIG", + "ner": [ + [ + 21, + 33, + "ppptRNAPhe\u03941", + "chemical" + ], + [ + 38, + 50, + "ppptRNAHis\u03941", + "chemical" + ], + [ + 62, + 65, + "TLP", + "protein_type" + ], + [ + 66, + 73, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 94, + "sent": "The activity using [\u03b1-32P]GTP, wild-type MaTLP, and ppptRNAPhe\u03941 is denoted as 100. (B) Guanylylation of tRNAPhe\u03941, tRNAPhe, and tRNAHis\u0394\u22121 by various TLP mutants.", + "section": "FIG", + "ner": [ + [ + 19, + 29, + "[\u03b1-32P]GTP", + "chemical" + ], + [ + 31, + 40, + "wild-type", + "protein_state" + ], + [ + 41, + 46, + "MaTLP", + "protein" + ], + [ + 52, + 64, + "ppptRNAPhe\u03941", + "chemical" + ], + [ + 105, + 114, + "tRNAPhe\u03941", + "chemical" + ], + [ + 116, + 123, + "tRNAPhe", + "chemical" + ], + [ + 129, + 139, + "tRNAHis\u0394\u22121", + "chemical" + ], + [ + 151, + 154, + "TLP", + "protein_type" + ], + [ + 155, + 162, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 95, + "sent": "The activity to tRNAPhe\u03941 is about 10% of ppptRNAPhe\u03941.", + "section": "FIG", + "ner": [ + [ + 16, + 25, + "tRNAPhe\u03941", + "chemical" + ], + [ + 42, + 54, + "ppptRNAPhe\u03941", + "chemical" + ] + ] + }, + { + "sid": 96, + "sent": "Termination of the elongation reaction by measuring the accepter stem", + "section": "RESULTS", + "ner": [ + [ + 56, + 69, + "accepter stem", + "structure_element" + ] + ] + }, + { + "sid": 97, + "sent": "TLPs catalyze the Watson-Crick template\u2013dependent elongation or repair reaction for 5\u2032-end truncated tRNAPhe substrates lacking G1 only (tRNAPhe\u03941), or lacking both G1 and G2 (tRNAPhe\u03941,2), whereas they do not show any activity with intact tRNAPhe (thus, repair is unnecessary).", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "TLPs", + "protein_type" + ], + [ + 101, + 108, + "tRNAPhe", + "chemical" + ], + [ + 128, + 130, + "G1", + "residue_name_number" + ], + [ + 137, + 146, + "tRNAPhe\u03941", + "chemical" + ], + [ + 165, + 167, + "G1", + "residue_name_number" + ], + [ + 172, + 174, + "G2", + "residue_name_number" + ], + [ + 176, + 187, + "tRNAPhe\u03941,2", + "chemical" + ], + [ + 240, + 247, + "tRNAPhe", + "chemical" + ] + ] + }, + { + "sid": 98, + "sent": "How TLP distinguishes between tRNAs that need 5\u2032-end repair from ones that do not, or in other words, how the elongation reaction is properly terminated, remains unknown.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "TLP", + "protein_type" + ], + [ + 30, + 35, + "tRNAs", + "chemical" + ] + ] + }, + { + "sid": 99, + "sent": "The present structure of the MaTLP-ppptRNAPhe\u03941 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the \u03b2-hairpin of molecule B and the 5\u2032-end by molecule A. Therefore, we speculated that the flexible nature of the \u03b2-hairpin enables the recognition of tRNA substrates with different accepter stem lengths.", + "section": "RESULTS", + "ner": [ + [ + 12, + 21, + "structure", + "evidence" + ], + [ + 29, + 47, + "MaTLP-ppptRNAPhe\u03941", + "complex_assembly" + ], + [ + 75, + 79, + "Thg1", + "protein" + ], + [ + 85, + 88, + "TLP", + "protein_type" + ], + [ + 89, + 94, + "dimer", + "oligomeric_state" + ], + [ + 117, + 121, + "tRNA", + "chemical" + ], + [ + 141, + 153, + "elbow region", + "structure_element" + ], + [ + 161, + 170, + "\u03b2-hairpin", + "structure_element" + ], + [ + 183, + 184, + "B", + "structure_element" + ], + [ + 212, + 213, + "A", + "structure_element" + ], + [ + 249, + 257, + "flexible", + "protein_state" + ], + [ + 272, + 281, + "\u03b2-hairpin", + "structure_element" + ], + [ + 309, + 313, + "tRNA", + "chemical" + ], + [ + 340, + 353, + "accepter stem", + "structure_element" + ] + ] + }, + { + "sid": 100, + "sent": "To confirm this speculation, we used computer graphics to examine whether the \u03b2-hairpin region was able to bind tRNA substrates with different accepter stem lengths when the 5\u2032-end was properly placed in the reaction site.", + "section": "RESULTS", + "ner": [ + [ + 32, + 65, + "used computer graphics to examine", + "experimental_method" + ], + [ + 78, + 87, + "\u03b2-hairpin", + "structure_element" + ], + [ + 112, + 116, + "tRNA", + "chemical" + ], + [ + 143, + 156, + "accepter stem", + "structure_element" + ], + [ + 208, + 221, + "reaction site", + "site" + ] + ] + }, + { + "sid": 101, + "sent": "When the 5\u2032-end was placed in the reaction site, the body of the tRNA molecule shifted in a manner dependent on the accepter stem length.", + "section": "RESULTS", + "ner": [ + [ + 65, + 69, + "tRNA", + "chemical" + ], + [ + 116, + 129, + "accepter stem", + "structure_element" + ] + ] + }, + { + "sid": 102, + "sent": "The tRNA body also rotated because of the helical nature of the accepter stem (fig. S7).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "tRNA", + "chemical" + ], + [ + 64, + 77, + "accepter stem", + "structure_element" + ] + ] + }, + { + "sid": 103, + "sent": "This model structure showed that the accepter stem of intact tRNAPhe was too long for the \u03b2-hairpin to recognize its elbow region, whereas tRNAPhe\u03941 and tRNAPhe\u03941,2 were recognized by the \u03b2-hairpin region (fig. S7), which is consistent with previous experiments.", + "section": "RESULTS", + "ner": [ + [ + 11, + 20, + "structure", + "evidence" + ], + [ + 37, + 50, + "accepter stem", + "structure_element" + ], + [ + 61, + 68, + "tRNAPhe", + "chemical" + ], + [ + 90, + 99, + "\u03b2-hairpin", + "structure_element" + ], + [ + 117, + 129, + "elbow region", + "structure_element" + ], + [ + 139, + 148, + "tRNAPhe\u03941", + "chemical" + ], + [ + 153, + 164, + "tRNAPhe\u03941,2", + "chemical" + ], + [ + 188, + 197, + "\u03b2-hairpin", + "structure_element" + ] + ] + }, + { + "sid": 104, + "sent": "On the basis of these model structures, we concluded that the TLP molecule can properly terminate elongation by measuring the accepter stem length of tRNA substrates.", + "section": "RESULTS", + "ner": [ + [ + 28, + 38, + "structures", + "evidence" + ], + [ + 62, + 65, + "TLP", + "protein_type" + ], + [ + 126, + 139, + "accepter stem", + "structure_element" + ], + [ + 150, + 154, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 105, + "sent": "Dual binding mode for tRNA repair", + "section": "RESULTS", + "ner": [ + [ + 22, + 26, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 106, + "sent": "The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer.", + "section": "RESULTS", + "ner": [ + [ + 12, + 31, + "structural analysis", + "experimental_method" + ], + [ + 55, + 58, + "TLP", + "protein_type" + ], + [ + 63, + 67, + "Thg1", + "protein" + ], + [ + 83, + 93, + "tetrameric", + "oligomeric_state" + ], + [ + 146, + 151, + "tRNAs", + "chemical" + ], + [ + 153, + 157, + "Thg1", + "protein" + ], + [ + 161, + 169, + "bound to", + "protein_state" + ], + [ + 170, + 177, + "tRNAHis", + "chemical" + ], + [ + 183, + 191, + "tetramer", + "oligomeric_state" + ], + [ + 201, + 204, + "TLP", + "protein_type" + ], + [ + 208, + 216, + "bound to", + "protein_state" + ], + [ + 217, + 224, + "tRNAPhe", + "chemical" + ], + [ + 230, + 235, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 107, + "sent": "This difference in the tRNA binding modes is closely related to their enzymatic functions.", + "section": "RESULTS", + "ner": [ + [ + 23, + 27, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 108, + "sent": "The tRNAHis-specific G\u22121 addition enzyme Thg1 needs to recognize both the accepter stem and anticodon of tRNAHis.", + "section": "RESULTS", + "ner": [ + [ + 4, + 40, + "tRNAHis-specific G\u22121 addition enzyme", + "protein_type" + ], + [ + 41, + 45, + "Thg1", + "protein" + ], + [ + 74, + 87, + "accepter stem", + "structure_element" + ], + [ + 92, + 101, + "anticodon", + "structure_element" + ], + [ + 105, + 112, + "tRNAHis", + "chemical" + ] + ] + }, + { + "sid": 109, + "sent": "The tetrameric architecture of the Thg1 molecule allows it to access both regions located at the opposite side of the tRNA molecule [the AB dimer recognizes the accepter stem and CD dimer anticodon ].", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "tetrameric", + "oligomeric_state" + ], + [ + 35, + 39, + "Thg1", + "protein" + ], + [ + 118, + 122, + "tRNA", + "chemical" + ], + [ + 137, + 139, + "AB", + "structure_element" + ], + [ + 140, + 145, + "dimer", + "oligomeric_state" + ], + [ + 161, + 174, + "accepter stem", + "structure_element" + ], + [ + 179, + 181, + "CD", + "structure_element" + ], + [ + 182, + 187, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 110, + "sent": "In contrast, the binding mode of TLP corresponds to the anticodon-independent repair reactions of 5\u2032-truncated general tRNAs.", + "section": "RESULTS", + "ner": [ + [ + 33, + 36, + "TLP", + "protein_type" + ], + [ + 119, + 124, + "tRNAs", + "chemical" + ] + ] + }, + { + "sid": 111, + "sent": "This binding mode is also suitable for the correct termination of the elongation or repair reaction by measuring the length of the accepter stem by the flexible \u03b2-hairpin.", + "section": "RESULTS", + "ner": [ + [ + 131, + 144, + "accepter stem", + "structure_element" + ], + [ + 152, + 160, + "flexible", + "protein_state" + ], + [ + 161, + 170, + "\u03b2-hairpin", + "structure_element" + ] + ] + }, + { + "sid": 112, + "sent": "Because tRNAHis requires an extra guanosine (G\u22121) at the 5\u2032-end, the repair enzyme has to extend the 5\u2032-end by one more nucleotide than other tRNAs.", + "section": "RESULTS", + "ner": [ + [ + 8, + 15, + "tRNAHis", + "chemical" + ], + [ + 34, + 43, + "guanosine", + "chemical" + ], + [ + 45, + 48, + "G\u22121", + "residue_name_number" + ], + [ + 142, + 147, + "tRNAs", + "chemical" + ] + ] + }, + { + "sid": 113, + "sent": "TLP has been shown to confer such catalytic activity on tRNAHis\u0394\u22121 (Fig. 4B).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "TLP", + "protein_type" + ], + [ + 56, + 66, + "tRNAHis\u0394\u22121", + "chemical" + ] + ] + }, + { + "sid": 114, + "sent": "Here, we showed that the TLP mutants, wherein the \u03b2-hairpin is truncated and tRNAPhe\u03941 binding ability is lost, can still bind to tRNAPhe (GUG) whose anticodon is changed to that for His (fig. S6, C, H, and I).", + "section": "RESULTS", + "ner": [ + [ + 25, + 28, + "TLP", + "protein_type" + ], + [ + 29, + 36, + "mutants", + "protein_state" + ], + [ + 50, + 59, + "\u03b2-hairpin", + "structure_element" + ], + [ + 63, + 72, + "truncated", + "protein_state" + ], + [ + 77, + 86, + "tRNAPhe\u03941", + "chemical" + ], + [ + 130, + 137, + "tRNAPhe", + "chemical" + ], + [ + 139, + 142, + "GUG", + "chemical" + ], + [ + 183, + 186, + "His", + "residue_name" + ] + ] + }, + { + "sid": 115, + "sent": "Also, the intact tRNAPhe, which is not recognized by TLP (Fig. 4B and fig. S6E), can be recognized when its anticodon is changed to that for His (fig. S6D).", + "section": "RESULTS", + "ner": [ + [ + 17, + 24, + "tRNAPhe", + "chemical" + ], + [ + 53, + 56, + "TLP", + "protein_type" + ], + [ + 141, + 144, + "His", + "residue_name" + ] + ] + }, + { + "sid": 116, + "sent": "Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHis\u0394\u22121 (Fig. 4B).", + "section": "RESULTS", + "ner": [ + [ + 17, + 20, + "TLP", + "protein_type" + ], + [ + 21, + 28, + "variant", + "protein_state" + ], + [ + 30, + 35, + "F174A", + "mutant" + ], + [ + 36, + 41, + "N179A", + "mutant" + ], + [ + 42, + 47, + "R188A", + "mutant" + ], + [ + 55, + 81, + "anticodon recognition site", + "site" + ], + [ + 100, + 112, + "Thg1-tRNAHis", + "complex_assembly" + ], + [ + 121, + 130, + "structure", + "evidence" + ], + [ + 201, + 211, + "tRNAHis\u0394\u22121", + "chemical" + ] + ] + }, + { + "sid": 117, + "sent": "All these experimental results indicate that TLP recognizes and binds tRNAs carrying the His anticodon in the same way that Thg1 recognizes tRNAHis.", + "section": "RESULTS", + "ner": [ + [ + 45, + 48, + "TLP", + "protein_type" + ], + [ + 70, + 75, + "tRNAs", + "chemical" + ], + [ + 89, + 92, + "His", + "residue_name" + ], + [ + 124, + 128, + "Thg1", + "protein" + ], + [ + 140, + 147, + "tRNAHis", + "chemical" + ] + ] + }, + { + "sid": 118, + "sent": "Thus, we concluded that TLP has two tRNA binding modes that are selectively used, depending on both the length of the accepter stem and the anticodon.", + "section": "RESULTS", + "ner": [ + [ + 24, + 27, + "TLP", + "protein_type" + ], + [ + 36, + 40, + "tRNA", + "chemical" + ], + [ + 118, + 131, + "accepter stem", + "structure_element" + ], + [ + 140, + 149, + "anticodon", + "structure_element" + ] + ] + }, + { + "sid": 119, + "sent": "The elongation or repair reaction normally terminates when the 5\u2032-end reaches position 1, but when the His anticodon is present, TLP binds the tRNA in the second mode by recognizing the anticodon to execute the G\u22121 addition reaction.", + "section": "RESULTS", + "ner": [ + [ + 103, + 106, + "His", + "residue_name" + ], + [ + 129, + 132, + "TLP", + "protein_type" + ], + [ + 143, + 147, + "tRNA", + "chemical" + ], + [ + 211, + 214, + "G\u22121", + "residue_name_number" + ] + ] + }, + { + "sid": 120, + "sent": "By having two different binding modes, TLP can manage this special feature of tRNAHis.", + "section": "RESULTS", + "ner": [ + [ + 39, + 42, + "TLP", + "protein_type" + ], + [ + 78, + 85, + "tRNAHis", + "chemical" + ] + ] + }, + { + "sid": 121, + "sent": "The Thg1/TLP family of proteins extends tRNA chains in the 3\u2032-5\u2032 direction.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 8, + "Thg1", + "protein" + ], + [ + 9, + 12, + "TLP", + "protein_type" + ], + [ + 40, + 44, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 122, + "sent": "First, the 5\u2032-phosphate is activated by GTP/ATP.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 23, + "5\u2032-phosphate", + "chemical" + ], + [ + 40, + 43, + "GTP", + "chemical" + ], + [ + 44, + 47, + "ATP", + "chemical" + ] + ] + }, + { + "sid": 123, + "sent": "Then, the activated phosphate is attacked by the incoming nucleotide, resulting in an extension by one nucleotide at the 5\u2032-end.", + "section": "DISCUSS", + "ner": [ + [ + 20, + 29, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 124, + "sent": "Here, we successfully solved for the first time the intermediate structures of the template-dependent 3\u2032-5\u2032 elongation complex of MaTLP.", + "section": "DISCUSS", + "ner": [ + [ + 22, + 28, + "solved", + "experimental_method" + ], + [ + 65, + 75, + "structures", + "evidence" + ], + [ + 130, + 135, + "MaTLP", + "protein" + ] + ] + }, + { + "sid": 125, + "sent": "On the basis of these structures, we will discuss the 3\u2032-5\u2032 addition reaction compared with canonical 5\u2032-3\u2032 elongation by DNA/RNA polymerases.", + "section": "DISCUSS", + "ner": [ + [ + 22, + 32, + "structures", + "evidence" + ], + [ + 122, + 141, + "DNA/RNA polymerases", + "protein_type" + ] + ] + }, + { + "sid": 126, + "sent": "Figure 5 is a schematic diagram of the 3\u2032-5\u2032 addition reaction of TLP.", + "section": "DISCUSS", + "ner": [ + [ + 66, + 69, + "TLP", + "protein_type" + ] + ] + }, + { + "sid": 127, + "sent": "This enzyme has two triphosphate binding sites and one reaction center at the position overlapping these two binding sites (Fig. 5A).", + "section": "DISCUSS", + "ner": [ + [ + 20, + 46, + "triphosphate binding sites", + "site" + ], + [ + 55, + 70, + "reaction center", + "site" + ], + [ + 109, + 122, + "binding sites", + "site" + ] + ] + }, + { + "sid": 128, + "sent": "In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5\u2032-phosphate of the tRNA is deprotonated by Mg2+A and attacks the \u03b1-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C).", + "section": "DISCUSS", + "ner": [ + [ + 35, + 38, + "GTP", + "chemical" + ], + [ + 39, + 42, + "ATP", + "chemical" + ], + [ + 46, + 54, + "bound to", + "protein_state" + ], + [ + 55, + 61, + "site 1", + "site" + ], + [ + 77, + 89, + "5\u2032-phosphate", + "chemical" + ], + [ + 97, + 101, + "tRNA", + "chemical" + ], + [ + 121, + 125, + "Mg2+", + "chemical" + ], + [ + 145, + 154, + "phosphate", + "chemical" + ], + [ + 162, + 165, + "GTP", + "chemical" + ], + [ + 166, + 169, + "ATP", + "chemical" + ] + ] + }, + { + "sid": 129, + "sent": "The structure of the MaTLP-ppptRNAPhe\u03941 complex, wherein \u03b2- and \u03b3-phosphates coordinate with Mg2+A and Mg2+B, respectively (Figs. 3A and 5C\u2032), may represent this activated intermediate.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 21, + 39, + "MaTLP-ppptRNAPhe\u03941", + "complex_assembly" + ], + [ + 66, + 76, + "phosphates", + "chemical" + ], + [ + 77, + 92, + "coordinate with", + "bond_interaction" + ], + [ + 93, + 97, + "Mg2+", + "chemical" + ], + [ + 103, + 107, + "Mg2+", + "chemical" + ] + ] + }, + { + "sid": 130, + "sent": "Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3\u2032-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5\u2032-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D).", + "section": "DISCUSS", + "ner": [ + [ + 34, + 44, + "nucleotide", + "chemical" + ], + [ + 48, + 54, + "site 2", + "site" + ], + [ + 84, + 106, + "Watson-Crick base pair", + "bond_interaction" + ], + [ + 114, + 124, + "nucleotide", + "chemical" + ], + [ + 182, + 192, + "nucleotide", + "chemical" + ], + [ + 229, + 233, + "Mg2+", + "chemical" + ], + [ + 243, + 258, + "5\u2032-triphosphate", + "chemical" + ], + [ + 266, + 270, + "tRNA", + "chemical" + ], + [ + 278, + 293, + "reaction center", + "site" + ] + ] + }, + { + "sid": 131, + "sent": "Thus, the present structure shows that this 3\u2032-5\u2032 elongation enzyme utilizes a reaction center homologous to that of 5\u2032-3\u2032 elongation enzymes for both activation and elongation in a stepwise fashion.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 27, + "structure", + "evidence" + ], + [ + 44, + 67, + "3\u2032-5\u2032 elongation enzyme", + "protein_type" + ], + [ + 79, + 94, + "reaction center", + "site" + ], + [ + 117, + 141, + "5\u2032-3\u2032 elongation enzymes", + "protein_type" + ] + ] + }, + { + "sid": 132, + "sent": "It should be noted that TLP has evolved to allow the occurrence of these two elaborate reaction steps within one reaction center.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 27, + "TLP", + "protein_type" + ], + [ + 113, + 128, + "reaction center", + "site" + ] + ] + }, + { + "sid": 133, + "sent": "(A) The reaction center overlapped with two triphosphate binding sites.", + "section": "FIG", + "ner": [ + [ + 8, + 23, + "reaction center", + "site" + ], + [ + 44, + 70, + "triphosphate binding sites", + "site" + ] + ] + }, + { + "sid": 134, + "sent": "A, B, and C (in green) represent binding sites for Mg2+A, Mg2+B, and Mg2+C. P (in blue) represents the phosphate binding sites; O\u2212 (in red) is the binding site for the deprotonated OH group.", + "section": "FIG", + "ner": [ + [ + 33, + 46, + "binding sites", + "site" + ], + [ + 51, + 55, + "Mg2+", + "chemical" + ], + [ + 58, + 62, + "Mg2+", + "chemical" + ], + [ + 69, + 73, + "Mg2+", + "chemical" + ], + [ + 76, + 77, + "P", + "site" + ], + [ + 103, + 126, + "phosphate binding sites", + "site" + ], + [ + 147, + 159, + "binding site", + "site" + ] + ] + }, + { + "sid": 135, + "sent": "Important TLP residues for tRNA and Mg2+ binding are also shown. (B) Structure of the activation complex (corresponding to fig. S8).", + "section": "FIG", + "ner": [ + [ + 10, + 13, + "TLP", + "protein_type" + ], + [ + 27, + 31, + "tRNA", + "chemical" + ], + [ + 36, + 40, + "Mg2+", + "chemical" + ], + [ + 69, + 78, + "Structure", + "evidence" + ] + ] + }, + { + "sid": 136, + "sent": "GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5\u2032-phosphate attacks the \u03b1-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C\u2032).", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "GTP", + "chemical" + ], + [ + 4, + 7, + "ATP", + "chemical" + ], + [ + 17, + 44, + "triphosphate binding site 1", + "site" + ], + [ + 79, + 91, + "5\u2032-phosphate", + "chemical" + ], + [ + 106, + 115, + "phosphate", + "chemical" + ], + [ + 119, + 122, + "GTP", + "chemical" + ], + [ + 123, + 126, + "ATP", + "chemical" + ], + [ + 132, + 135, + "PPi", + "chemical" + ], + [ + 137, + 160, + "inorganic pyrophosphate", + "chemical" + ], + [ + 188, + 197, + "structure", + "evidence" + ], + [ + 246, + 255, + "structure", + "evidence" + ] + ] + }, + { + "sid": 137, + "sent": "(C\u2032) Structure before the elongation reaction (corresponding to Fig. 3A).", + "section": "FIG", + "ner": [ + [ + 5, + 14, + "Structure", + "evidence" + ] + ] + }, + { + "sid": 138, + "sent": "The 5\u2032-triphosphate of the tRNA binds to the same site as for activation of the 5\u2032-terminus of the tRNA in (B).", + "section": "FIG", + "ner": [ + [ + 4, + 19, + "5\u2032-triphosphate", + "chemical" + ], + [ + 27, + 31, + "tRNA", + "chemical" + ], + [ + 99, + 103, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 139, + "sent": "(D) Structure of initiation of the elongation reaction (corresponding to Fig. 3B).", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "Structure", + "evidence" + ] + ] + }, + { + "sid": 140, + "sent": "The base of the incoming GTP forms a Watson-Crick hydrogen bond with the nucleotide at position 72 in the template chain and a base-stacking interaction with a neighboring base (G2).", + "section": "FIG", + "ner": [ + [ + 25, + 28, + "GTP", + "chemical" + ], + [ + 37, + 63, + "Watson-Crick hydrogen bond", + "bond_interaction" + ], + [ + 73, + 83, + "nucleotide", + "chemical" + ], + [ + 96, + 98, + "72", + "residue_number" + ], + [ + 127, + 152, + "base-stacking interaction", + "bond_interaction" + ], + [ + 178, + 180, + "G2", + "residue_name_number" + ] + ] + }, + { + "sid": 141, + "sent": "Movement of the 5\u2032-terminal chain leaves the 5\u2032-triphosphate of the tRNA in the same site as the activation step in (B).", + "section": "FIG", + "ner": [ + [ + 45, + 60, + "5\u2032-triphosphate", + "chemical" + ], + [ + 68, + 72, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 142, + "sent": "The 3\u2032-OH of the incoming GTP is deprotonated by Mg2+A and attacks the \u03b1-phosphate to form a covalent bond. (E) After the elongation reaction, the triphosphate of the new nucleotide is placed on site 1, as in (C\u2032), and is ready for the next reaction.", + "section": "FIG", + "ner": [ + [ + 26, + 29, + "GTP", + "chemical" + ], + [ + 49, + 53, + "Mg2+", + "chemical" + ], + [ + 73, + 82, + "phosphate", + "chemical" + ], + [ + 147, + 159, + "triphosphate", + "chemical" + ], + [ + 171, + 181, + "nucleotide", + "chemical" + ], + [ + 195, + 201, + "site 1", + "site" + ] + ] + }, + { + "sid": 143, + "sent": "Figure 6 compares the 3\u2032-5\u2032 and 5\u2032-3\u2032 elongation mechanisms, showing the symmetrical nature of both elongation reactions using a similar reaction center composed of Mg2+A and Mg2+B in the conserved catalytic core.", + "section": "DISCUSS", + "ner": [ + [ + 137, + 152, + "reaction center", + "site" + ], + [ + 165, + 169, + "Mg2+", + "chemical" + ], + [ + 175, + 179, + "Mg2+", + "chemical" + ], + [ + 188, + 197, + "conserved", + "protein_state" + ], + [ + 198, + 212, + "catalytic core", + "site" + ] + ] + }, + { + "sid": 144, + "sent": "In TLP, which carries out 3\u2032-5\u2032 elongation, the 3\u2032-OH of the incoming nucleotide attacks the 5\u2032-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the T7 RNA polymerase, a representative 5\u2032-3\u2032 DNA/RNA polymerase, the 3\u2032-OH of the 3\u2032-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 6, + "TLP", + "protein_type" + ], + [ + 106, + 115, + "phosphate", + "chemical" + ], + [ + 123, + 127, + "tRNA", + "chemical" + ], + [ + 174, + 191, + "T7 RNA polymerase", + "protein" + ], + [ + 210, + 234, + "5\u2032-3\u2032 DNA/RNA polymerase", + "protein_type" + ], + [ + 283, + 286, + "RNA", + "chemical" + ], + [ + 309, + 318, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 145, + "sent": "In these reactions, the roles of the two Mg ions are identical.", + "section": "DISCUSS", + "ner": [ + [ + 41, + 43, + "Mg", + "chemical" + ] + ] + }, + { + "sid": 146, + "sent": "Mg2+A activates the 3\u2032-OH of the incoming nucleotide in TLP and the 3\u2032-OH of the 3\u2032-end of the RNA chain in T7 RNA polymerase.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "Mg2+", + "chemical" + ], + [ + 56, + 59, + "TLP", + "protein_type" + ], + [ + 95, + 98, + "RNA", + "chemical" + ], + [ + 108, + 125, + "T7 RNA polymerase", + "protein" + ] + ] + }, + { + "sid": 147, + "sent": "The role of Mg2+B is to position the 5\u2032-triphosphate of the tRNA in TLP and the incoming nucleotide in T7 RNA polymerase.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 16, + "Mg2+", + "chemical" + ], + [ + 37, + 52, + "5\u2032-triphosphate", + "chemical" + ], + [ + 60, + 64, + "tRNA", + "chemical" + ], + [ + 68, + 71, + "TLP", + "protein_type" + ], + [ + 103, + 120, + "T7 RNA polymerase", + "protein" + ] + ] + }, + { + "sid": 148, + "sent": "These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 14, + "Mg2+", + "chemical" + ], + [ + 24, + 38, + "coordinated by", + "bond_interaction" + ], + [ + 41, + 50, + "conserved", + "protein_state" + ], + [ + 51, + 54, + "Asp", + "residue_name" + ], + [ + 56, + 59, + "D21", + "residue_name_number" + ], + [ + 64, + 67, + "D69", + "residue_name_number" + ], + [ + 71, + 74, + "TLP", + "protein_type" + ], + [ + 83, + 92, + "conserved", + "protein_state" + ], + [ + 93, + 107, + "catalytic core", + "site" + ] + ] + }, + { + "sid": 149, + "sent": "Structures of template-dependent nucleotide elongation in the 3\u2032-5\u2032 and 5\u2032-3\u2032 directions.", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ] + ] + }, + { + "sid": 150, + "sent": "Symmetrical relationship between 3\u2032-5\u2032 elongation by TLP (this study) (left) and 5\u2032-3\u2032 elongation by T7 RNA polymerase [Protein Data Bank (PDB) ID: 1S76] (right).", + "section": "FIG", + "ner": [ + [ + 53, + 56, + "TLP", + "protein_type" + ], + [ + 101, + 118, + "T7 RNA polymerase", + "protein" + ] + ] + }, + { + "sid": 151, + "sent": "In the 3\u2032-5\u2032 elongation reaction, the 3\u2032-OH of the incoming nucleotide attacks the 5\u2032-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the 5\u2032-3\u2032 elongation reaction, the 3\u2032-OH of the 3\u2032-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond.", + "section": "FIG", + "ner": [ + [ + 96, + 105, + "phosphate", + "chemical" + ], + [ + 113, + 117, + "tRNA", + "chemical" + ], + [ + 238, + 241, + "RNA", + "chemical" + ], + [ + 264, + 273, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 152, + "sent": "Green spheres represent Mg2+ ions.", + "section": "FIG", + "ner": [ + [ + 24, + 28, + "Mg2+", + "chemical" + ] + ] + }, + { + "sid": 153, + "sent": "Because the chemical roles of tRNA and the incoming nucleotide are reversed in these two reactions, these two substrates are inserted into a similar reaction center from opposite directions (Fig. 6).", + "section": "DISCUSS", + "ner": [ + [ + 30, + 34, + "tRNA", + "chemical" + ], + [ + 149, + 164, + "reaction center", + "site" + ] + ] + }, + { + "sid": 154, + "sent": "However, from an energetic viewpoint, these two reactions are clearly different: Whereas the high energy of the incoming nucleotide is used for its own addition in DNA/RNA polymerases, the high energy of the incoming nucleotide is used for subsequent addition in TLP.", + "section": "DISCUSS", + "ner": [ + [ + 164, + 183, + "DNA/RNA polymerases", + "protein_type" + ], + [ + 263, + 266, + "TLP", + "protein_type" + ] + ] + }, + { + "sid": 155, + "sent": "For this reason, TLP requires a mechanism that activates the 5\u2032-terminus of the tRNA during the initial step of the reaction.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 20, + "TLP", + "protein_type" + ], + [ + 80, + 84, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 156, + "sent": "Our analysis showed that the initial activation and subsequent elongation reactions occur sequentially at one reaction center.", + "section": "DISCUSS", + "ner": [ + [ + 110, + 125, + "reaction center", + "site" + ] + ] + }, + { + "sid": 157, + "sent": "In this case, the enzyme needs to create two substrate binding sites for two different reactions in the vicinities of one reaction center.", + "section": "DISCUSS", + "ner": [ + [ + 45, + 68, + "substrate binding sites", + "site" + ], + [ + 122, + 137, + "reaction center", + "site" + ] + ] + }, + { + "sid": 158, + "sent": "TLP has successfully created such sites by utilizing a conformational change in the tRNA through Watson-Crick base pairing (Fig. 3).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 3, + "TLP", + "protein_type" + ], + [ + 84, + 88, + "tRNA", + "chemical" + ], + [ + 97, + 122, + "Watson-Crick base pairing", + "bond_interaction" + ] + ] + }, + { + "sid": 159, + "sent": "These structural features of the TLP molecule suggest that development of an activation reaction site is a prerequisite for developing the 3\u2032-5\u2032 elongation enzyme.", + "section": "DISCUSS", + "ner": [ + [ + 33, + 36, + "TLP", + "protein_type" + ], + [ + 77, + 101, + "activation reaction site", + "site" + ], + [ + 139, + 162, + "3\u2032-5\u2032 elongation enzyme", + "protein_type" + ] + ] + }, + { + "sid": 160, + "sent": "This is clearly more difficult than developing the 5\u2032-3\u2032 elongation enzyme, wherein the activation reaction site is not necessary, and which may be the primary reason why the 5\u2032-3\u2032 elongation enzyme has been exclusively developed.", + "section": "DISCUSS", + "ner": [ + [ + 51, + 74, + "5\u2032-3\u2032 elongation enzyme", + "protein_type" + ], + [ + 88, + 112, + "activation reaction site", + "site" + ], + [ + 175, + 198, + "5\u2032-3\u2032 elongation enzyme", + "protein_type" + ] + ] + }, + { + "sid": 161, + "sent": "Here, we established a structural basis for 3\u2032-5\u2032 nucleotide elongation and showed that TLP has evolved to acquire a two-step Watson-Crick template\u2013dependent 3\u2032-5\u2032 elongation reaction using the catalytic center homologous to 5\u2032-3\u2032 elongation enzymes.", + "section": "DISCUSS", + "ner": [ + [ + 88, + 91, + "TLP", + "protein_type" + ], + [ + 194, + 210, + "catalytic center", + "site" + ], + [ + 225, + 249, + "5\u2032-3\u2032 elongation enzymes", + "protein_type" + ] + ] + }, + { + "sid": 162, + "sent": "The active site of this enzyme is created at the dimerization interface.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 15, + "active site", + "site" + ], + [ + 49, + 71, + "dimerization interface", + "site" + ] + ] + }, + { + "sid": 163, + "sent": "The dimerization also endows this protein with the ability to measure the length of the accepter stem of the tRNA substrate, so that the enzyme can properly terminate the elongation reaction.", + "section": "DISCUSS", + "ner": [ + [ + 88, + 101, + "accepter stem", + "structure_element" + ], + [ + 109, + 113, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 164, + "sent": "Furthermore, the dual binding mode of this protein suggests that it has further evolved to cover G\u22121 addition of tRNAHis by additional dimerization (dimer of dimers).", + "section": "DISCUSS", + "ner": [ + [ + 97, + 100, + "G\u22121", + "residue_name_number" + ], + [ + 113, + 120, + "tRNAHis", + "chemical" + ], + [ + 149, + 154, + "dimer", + "oligomeric_state" + ], + [ + 158, + 164, + "dimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 165, + "sent": "Thus, the present structural analysis is consistent with the scenario in which TLP began as a 5\u2032-end repair enzyme and evolved into a tRNAHis-specific G\u22121 addition enzyme.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 37, + "structural analysis", + "experimental_method" + ], + [ + 79, + 82, + "TLP", + "protein_type" + ], + [ + 134, + 170, + "tRNAHis-specific G\u22121 addition enzyme", + "protein_type" + ] + ] + }, + { + "sid": 166, + "sent": "The detailed molecular mechanism of the Thg1/TLP family established by our analysis will open up new perspectives in our understanding of 3\u2032-5\u2032 versus 5\u2032-3\u2032 polymerization and the molecular evolution of template-dependent polymerases.", + "section": "DISCUSS", + "ner": [ + [ + 40, + 44, + "Thg1", + "protein" + ], + [ + 45, + 48, + "TLP", + "protein_type" + ], + [ + 203, + 233, + "template-dependent polymerases", + "protein_type" + ] + ] + }, + { + "sid": 167, + "sent": "Transcribed tRNAs were purified by a HiTrap DEAE FF column (GE Healthcare) as previously described.", + "section": "METHODS", + "ner": [ + [ + 12, + 17, + "tRNAs", + "chemical" + ] + ] + }, + { + "sid": 168, + "sent": "Pooled tRNAs were precipitated with isopropanol and dissolved in buffer E [20 mM Hepes-NaOH (pH 7.5), 100 mM NaCl, and 10 mM MgCl2].", + "section": "METHODS", + "ner": [ + [ + 7, + 12, + "tRNAs", + "chemical" + ] + ] + }, + { + "sid": 169, + "sent": "The highly conserved tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex and is required for the G2/M phase transition in the yeast Saccharomyces cerevisiae", + "section": "REF", + "ner": [ + [ + 49, + 54, + "Thg1p", + "protein" + ] + ] + } + ] + }, + "PMC4968113": { + "annotations": [ + { + "sid": 0, + "sent": "Structural diversity in a human antibody germline library", + "section": "TITLE", + "ner": [ + [ + 26, + 31, + "human", + "species" + ], + [ + 32, + 40, + "antibody", + "protein_type" + ] + ] + }, + { + "sid": 1, + "sent": "To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined.", + "section": "ABSTRACT", + "ner": [ + [ + 11, + 19, + "antibody", + "protein_type" + ], + [ + 49, + 67, + "crystal structures", + "evidence" + ], + [ + 125, + 143, + "kappa light chains", + "structure_element" + ], + [ + 168, + 180, + "heavy chains", + "structure_element" + ] + ] + }, + { + "sid": 2, + "sent": "All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure.", + "section": "ABSTRACT", + "ner": [ + [ + 9, + 21, + "heavy chains", + "structure_element" + ], + [ + 29, + 54, + "antigen-binding fragments", + "structure_element" + ], + [ + 56, + 60, + "Fabs", + "structure_element" + ], + [ + 76, + 110, + "complementarity-determining region", + "structure_element" + ], + [ + 112, + 115, + "CDR", + "structure_element" + ], + [ + 117, + 119, + "H3", + "structure_element" + ], + [ + 152, + 155, + "Fab", + "structure_element" + ], + [ + 156, + 165, + "structure", + "evidence" + ] + ] + }, + { + "sid": 3, + "sent": "The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 22, + "structure analyses", + "experimental_method" + ], + [ + 58, + 68, + "structures", + "evidence" + ], + [ + 80, + 90, + "structures", + "evidence" + ], + [ + 98, + 102, + "CDRs", + "structure_element" + ], + [ + 111, + 116, + "VH:VL", + "complex_assembly" + ], + [ + 117, + 137, + "packing interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 4, + "sent": "The CDR conformations for the most part are tightly clustered, especially for the ones with shorter lengths.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 7, + "CDR", + "structure_element" + ] + ] + }, + { + "sid": 5, + "sent": "The longer CDRs with tandem glycines or serines have more conformational diversity than the others.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 10, + "longer", + "protein_state" + ], + [ + 11, + 15, + "CDRs", + "structure_element" + ], + [ + 28, + 36, + "glycines", + "residue_name" + ], + [ + 40, + 47, + "serines", + "residue_name" + ] + ] + }, + { + "sid": 6, + "sent": "CDR H3, despite having the same amino acid sequence, exhibits the largest conformational diversity.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 3, + "CDR", + "structure_element" + ], + [ + 4, + 6, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 7, + "sent": "About half of the structures have CDR H3 conformations similar to that of the parent; the others diverge significantly.", + "section": "ABSTRACT", + "ner": [ + [ + 18, + 28, + "structures", + "evidence" + ], + [ + 34, + 37, + "CDR", + "structure_element" + ], + [ + 38, + 40, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 8, + "sent": "One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing.", + "section": "ABSTRACT", + "ner": [ + [ + 27, + 30, + "CDR", + "structure_element" + ], + [ + 31, + 33, + "H3", + "structure_element" + ], + [ + 148, + 153, + "heavy", + "structure_element" + ], + [ + 158, + 169, + "light chain", + "structure_element" + ] + ] + }, + { + "sid": 9, + "sent": "The stem regions of 14 of the variant pairs are in the \u2018kinked\u2019 conformation, and only 2 are in the extended conformation.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 16, + "stem regions", + "structure_element" + ], + [ + 56, + 62, + "kinked", + "protein_state" + ], + [ + 100, + 108, + "extended", + "protein_state" + ] + ] + }, + { + "sid": 10, + "sent": "The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees.", + "section": "ABSTRACT", + "ner": [ + [ + 19, + 21, + "VH", + "structure_element" + ], + [ + 26, + 28, + "VL", + "structure_element" + ], + [ + 73, + 81, + "antibody", + "protein_type" + ], + [ + 82, + 91, + "structure", + "evidence" + ], + [ + 101, + 112, + "tilt angles", + "evidence" + ] + ] + }, + { + "sid": 11, + "sent": "Two of 16 structures showed particularly large variations in the tilt angles when compared with the other pairings.", + "section": "ABSTRACT", + "ner": [ + [ + 10, + 20, + "structures", + "evidence" + ], + [ + 65, + 76, + "tilt angles", + "evidence" + ] + ] + }, + { + "sid": 12, + "sent": "The structures and their analyses provide a rich foundation for future antibody modeling and engineering efforts.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 71, + 79, + "antibody", + "protein_type" + ] + ] + }, + { + "sid": 13, + "sent": "At present, therapeutic antibodies are the largest class of biotherapeutic proteins that are in clinical trials.", + "section": "INTRO", + "ner": [ + [ + 24, + 34, + "antibodies", + "protein_type" + ] + ] + }, + { + "sid": 14, + "sent": "The use of monoclonal antibodies as therapeutics began in the early 1980s, and their composition has transitioned from murine antibodies to generally less immunogenic humanized and human antibodies.", + "section": "INTRO", + "ner": [ + [ + 22, + 32, + "antibodies", + "protein_type" + ], + [ + 119, + 125, + "murine", + "taxonomy_domain" + ], + [ + 126, + 136, + "antibodies", + "protein_type" + ], + [ + 181, + 186, + "human", + "species" + ], + [ + 187, + 197, + "antibodies", + "protein_type" + ] + ] + }, + { + "sid": 15, + "sent": "The technologies currently used to obtain human antibodies include transgenic mice containing human antibody repertoires, cloning directly from human B cells, and in vitro selection from antibody libraries using various display technologies.", + "section": "INTRO", + "ner": [ + [ + 42, + 47, + "human", + "species" + ], + [ + 48, + 58, + "antibodies", + "protein_type" + ], + [ + 78, + 82, + "mice", + "taxonomy_domain" + ], + [ + 94, + 99, + "human", + "species" + ], + [ + 100, + 108, + "antibody", + "protein_type" + ], + [ + 144, + 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"INTRO", + "ner": [ + [ + 21, + 38, + "crystal structure", + "evidence" + ], + [ + 55, + 63, + "antibody", + "protein_type" + ] + ] + }, + { + "sid": 19, + "sent": "Today's antibody modeling approaches, which normally focus on the variable region, are being developed by the application of structural principles and insights that are evolving as our knowledge of antibody structures continues to expand.", + "section": "INTRO", + "ner": [ + [ + 8, + 16, + "antibody", + "protein_type" + ], + [ + 66, + 81, + "variable region", + "structure_element" + ], + [ + 198, + 206, + "antibody", + "protein_type" + ], + [ + 207, + 217, + "structures", + "evidence" + ] + ] + }, + { + "sid": 20, + "sent": "Our current structural knowledge of antibodies is based on a multitude of studies that used many techniques to gain insight into the functional and structural properties of this class of macromolecule.", + "section": "INTRO", + "ner": [ + [ + 36, + 46, + "antibodies", + "protein_type" + ] + ] + }, + { + "sid": 21, + "sent": "Five different antibody isotypes occur, IgG, IgD, IgE, IgA and IgM, and each isotype has a unique role in the adaptive immune system.", + "section": "INTRO", + "ner": [ + [ + 15, + 23, + "antibody", + "protein_type" + ], + [ + 40, + 43, + "IgG", + "protein" + ], + [ + 45, + 48, + "IgD", + "protein" + ], + [ + 50, + 53, + "IgE", + "protein" + ], + [ + 55, + 58, + "IgA", + "protein" + ], + [ + 63, + 66, + "IgM", + "protein" + ] + ] + }, + { + "sid": 22, + "sent": "IgG, IgD and IgE isotypes are composed of 2 heavy chains (HCs) and 2 light chains (LCs) linked through disulfide bonds, while IgA and IgM are double and quintuple versions of antibodies, respectively.", + "section": "INTRO", + "ner": [ + [ + 0, + 3, + "IgG", + "protein" + ], + [ + 5, + 8, + "IgD", + "protein" + ], + [ + 13, + 16, + "IgE", + "protein" + ], + [ + 44, + 56, + "heavy chains", + "structure_element" + ], + [ + 58, + 61, + "HCs", + "structure_element" + ], + [ + 69, + 81, + "light chains", + 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] + ] + }, + { + "sid": 24, + "sent": "These multimeric forms are linked with an additional J chain.", + "section": "INTRO", + "ner": [ + [ + 53, + 54, + "J", + "structure_element" + ] + ] + }, + { + "sid": 25, + "sent": "The LCs that associate with the HCs are divided into 2 functionally indistinguishable classes, \u03ba and \u03bb.", + "section": "INTRO", + "ner": [ + [ + 4, + 7, + "LCs", + "structure_element" + ], + [ + 32, + 35, + "HCs", + "structure_element" + ], + [ + 95, + 96, + "\u03ba", + "structure_element" + ], + [ + 101, + 102, + "\u03bb", + "structure_element" + ] + ] + }, + { + "sid": 26, + "sent": "Both \u03ba and \u03bb polypeptide chains are composed of a single V domain and a single C domain.", + "section": "INTRO", + "ner": [ + [ + 5, + 6, + "\u03ba", + "structure_element" + ], + [ + 11, + 12, + "\u03bb", + "structure_element" + ], + [ + 57, + 65, + "V domain", + "structure_element" + ], + [ + 79, + 87, + "C domain", + "structure_element" + ] + ] + }, + { + "sid": 27, + "sent": "The heavy and light chains are composed of structural domains that have \u223c110 amino acid residues.", + "section": "INTRO", + "ner": [ + [ + 4, + 9, + "heavy", + "structure_element" + ], + [ + 14, + 26, + "light chains", + "structure_element" + ], + [ + 43, + 61, + "structural domains", + "structure_element" + ], + [ + 72, + 96, + "\u223c110 amino acid residues", + "residue_range" + ] + ] + }, + { + "sid": 28, + "sent": "These domains have a common folding pattern often referred to as the \u201cimmunoglobulin fold,\u201d formed by the packing together of 2 anti-parallel \u03b2-sheets.", + "section": "INTRO", + "ner": [ + [ + 70, + 89, + "immunoglobulin fold", + "structure_element" + ], + [ + 128, + 150, + "anti-parallel \u03b2-sheets", + "structure_element" + ] + ] + }, + { + "sid": 29, + "sent": "All immunoglobulin chains have an N-terminal V domain followed by 1 to 4 C domains, depending upon the chain type.", + "section": "INTRO", + "ner": [ + [ + 4, + 25, + "immunoglobulin chains", + "protein_type" + ], + [ + 45, + 53, + "V domain", + "structure_element" + ], + [ + 73, + 82, + "C domains", + "structure_element" + ] + ] + }, + { + "sid": 30, + "sent": "In antibodies, the heavy and light chain V domains pack together forming the antigen combining site.", + "section": "INTRO", + "ner": [ + [ + 3, + 13, + "antibodies", + "protein_type" + ], + [ + 19, + 40, + "heavy and light chain", + "structure_element" + ], + [ + 41, + 50, + "V domains", + "structure_element" + ], + [ + 77, + 99, + "antigen combining site", + "site" + ] + ] + }, + { + "sid": 31, + "sent": "This site, which interacts with the antigen (or target), is the focus of current antibody modeling efforts.", + "section": "INTRO", + "ner": [ + [ + 81, + 89, + "antibody", + "protein_type" + ] + ] + }, + { + "sid": 32, + "sent": "This interaction site is composed of 6 complementarity-determining regions (CDRs) that were identified in early antibody amino acid sequence analyses to be hypervariable in nature, and thus are responsible for the sequence and structural diversity of our antibody repertoire.", + "section": "INTRO", + "ner": [ + [ + 5, + 21, + "interaction site", + "site" + ], + [ + 39, + 74, + "complementarity-determining regions", + "structure_element" + ], + [ + 76, + 80, + "CDRs", + "structure_element" + ], + [ + 112, + 149, + "antibody amino acid sequence analyses", + "experimental_method" + ], + [ + 156, + 169, + "hypervariable", + "protein_state" + ], + [ + 255, + 263, + "antibody", + "protein_type" + ] + ] + }, + { + "sid": 33, + "sent": "The sequence diversity of the CDR regions presents a substantial challenge to antibody modeling.", + "section": "INTRO", + "ner": [ + [ + 30, + 41, + "CDR regions", + "structure_element" + ], + [ + 78, + 86, + "antibody", + "protein_type" + ] + ] + }, + { + "sid": 34, + "sent": "However, an initial structural analysis of the combining sites of the small set of structures of immunoglobulin fragments available in the 1980s found that 5 of the 6 hypervariable loops or CDRs had canonical structures (a limited set of main-chain conformations).", + "section": "INTRO", + "ner": [ + [ + 20, + 39, + "structural analysis", + "experimental_method" + ], + [ + 47, + 62, + "combining sites", + "site" + ], + [ + 83, + 93, + "structures", + "evidence" + ], + [ + 167, + 186, + "hypervariable loops", + "structure_element" + ], + [ + 190, + 194, + "CDRs", + "structure_element" + ] + ] + }, + { + "sid": 35, + "sent": "A CDR canonical structure is defined by its length and conserved residues located in the hypervariable loop and framework residues (V-region residues that are not part of the CDRs).", + "section": "INTRO", + "ner": [ + [ + 2, + 5, + "CDR", + "structure_element" + ], + [ + 89, + 107, + "hypervariable loop", + "structure_element" + ], + [ + 112, + 130, + "framework residues", + "structure_element" + ], + [ + 132, + 140, + "V-region", + "structure_element" + ], + [ + 175, + 179, + "CDRs", + "structure_element" + ] + ] + }, + { + "sid": 36, + "sent": "Furthermore, studies of antibody sequences revealed that the total number of canonical structures are limited for each CDR, indicating possibly that antigen recognition may be affected by structural restrictions at the antigen-binding site.", + "section": "INTRO", + "ner": [ + [ + 24, + 32, + "antibody", + "protein_type" + ], + [ + 119, + 122, + "CDR", + "structure_element" + ], + [ + 219, + 239, + "antigen-binding site", + "site" + ] + ] + }, + { + "sid": 37, + "sent": "Later studies found that the CDR loop length is the primary determining factor of antigen-binding site topography because it is the primary factor for determining a canonical structure.", + "section": "INTRO", + "ner": [ + [ + 29, + 37, + "CDR loop", + "structure_element" + ], + [ + 82, + 102, + "antigen-binding site", + "site" + ] + ] + }, + { + "sid": 38, + "sent": "Additional efforts have led to our current understanding that the LC CDRs L1, L2, and L3 have preferred sets of canonical structures based on length and amino acid sequence composition.", + "section": "INTRO", + "ner": [ + [ + 66, + 68, + "LC", + "structure_element" + ], + [ + 69, + 73, + "CDRs", + "structure_element" + ], + [ + 74, + 76, + "L1", + "structure_element" + ], + [ + 78, + 80, + "L2", + "structure_element" + ], + [ + 86, + 88, + "L3", + "structure_element" + ] + ] + }, + { + "sid": 39, + "sent": "This was also found to be the case for the H1 and H2 CDRs.", + "section": "INTRO", + "ner": [ + [ + 43, + 45, + "H1", + "structure_element" + ], + [ + 50, + 52, + "H2", + "structure_element" + ], + [ + 53, + 57, + "CDRs", + "structure_element" + ] + ] + }, + { + "sid": 40, + "sent": "Classification schemes for the canonical structures of these 5 CDRs have emerged and evolved as the number of depositions in the Protein Data Bank of Fab fragments of antibodies grow.", + "section": "INTRO", + "ner": [ + [ + 63, + 67, + "CDRs", + "structure_element" + ], + [ + 150, + 153, + "Fab", + "structure_element" + ], + [ + 167, + 177, + "antibodies", + "protein_type" + ] + ] + }, + { + "sid": 41, + "sent": "Recently, a comprehensive CDR classification scheme was reported identifying 72 clusters of conformations observed in antibody structures.", + "section": "INTRO", + "ner": [ + [ + 26, + 29, + "CDR", + "structure_element" + ], + [ + 118, + 126, + "antibody", + "protein_type" + ], + [ + 127, + 137, + "structures", + "evidence" + ] + ] + }, + { + "sid": 42, + "sent": "The knowledge and predictability of these CDR canonical structures have greatly advanced antibody modeling efforts.", + "section": "INTRO", + "ner": [ + [ + 42, + 45, + "CDR", + "structure_element" + ], + [ + 56, + 66, + "structures", + "evidence" + ], + [ + 89, + 97, + "antibody", + "protein_type" + ] + ] + }, + { + "sid": 43, + "sent": "In contrast to CDRs L1, L2, L3, H1 and H2, no canonical structures have been observed for CDR H3, which is the most variable in length and amino acid sequence.", + 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predicting the conformation of the anchor region of CDR H3 continue to be refined, producing new insight into the CDR H3 conformations and new tools for antibody engineering.", + "section": "INTRO", + "ner": [ + [ + 83, + 96, + "anchor region", + "structure_element" + ], + [ + 100, + 103, + "CDR", + "structure_element" + ], + [ + 104, + 106, + "H3", + "structure_element" + ], + [ + 162, + 165, + "CDR", + "structure_element" + ], + [ + 166, + 168, + "H3", + "structure_element" + ], + [ + 201, + 209, + "antibody", + "protein_type" + ] + ] + }, + { + "sid": 48, + "sent": "Current antibody modeling approaches take advantage of the most recent advances in homology modeling, the evolving understanding of the CDR canonical structures, the emerging rules for CDR H3 modeling and the growing body of antibody structural data available from the PDB.", + "section": "INTRO", + "ner": [ + [ + 8, + 16, + "antibody", + "protein_type" + ], + [ + 83, + 100, + "homology modeling", + "experimental_method" 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}, + { + "sid": 53, + "sent": "To support antibody engineering and therapeutic development efforts, a phage library was designed and constructed based on a limited number of scaffolds built with frequently used human germ-line IGV and IGJ gene segments that encode antigen combining sites suitable for recognition of peptides and proteins.", + "section": "INTRO", + "ner": [ + [ + 11, + 19, + "antibody", + "protein_type" + ], + [ + 71, + 84, + "phage library", + "experimental_method" + ], + [ + 180, + 185, + "human", + "species" + ], + [ + 196, + 199, + "IGV", + "structure_element" + ], + [ + 204, + 207, + "IGJ", + "structure_element" + ], + [ + 234, + 257, + "antigen combining sites", + "site" + ] + ] + }, + { + "sid": 54, + "sent": "This Fab library is composed of 3 HC germlines, IGHV1-69 (H1-69), IGHV3-23 (H3-23) and IGHV5-51(H5-51), and 4 LC germlines (all \u03ba), IGKV1-39 (L1-39), IGKV3-11 (L3-11), IGKV3-20 (L3-20) and IGKV4-1 (L4-1).", + "section": "INTRO", + "ner": [ + [ + 5, + 8, 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"refinement statistics", + "evidence" + ] + ] + }, + { + "sid": 64, + "sent": "(Continued) Crystal data, X-ray data, and refinement statistics.", + "section": "TABLE", + "ner": [ + [ + 12, + 24, + "Crystal data", + "evidence" + ], + [ + 26, + 36, + "X-ray data", + "evidence" + ], + [ + 42, + 63, + "refinement statistics", + "evidence" + ], + [ + 12, + 24, + "Crystal data", + "evidence" + ], + [ + 26, + 36, + "X-ray data", + "evidence" + ], + [ + 42, + 63, + "refinement statistics", + "evidence" + ], + [ + 12, + 24, + "Crystal data", + "evidence" + ], + [ + 26, + 36, + "X-ray data", + "evidence" + ], + [ + 42, + 63, + "refinement statistics", + "evidence" + ] + ] + }, + { + "sid": 65, + "sent": "The crystal structures of a germline library composed of 16 Fabs generated by combining 4 HCs (H1-69, H3-23, H3-53 and H5-51) and 4 LCs (L1-39, L3-11, L3-20 and L4-1) have been determined.", + "section": "RESULTS", + "ner": [ + [ + 4, + 22, + "crystal structures", + "evidence" + ], + [ + 28, + 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"RESULTS", + "ner": [ + [ + 19, + 22, + "HCs", + "structure_element" + ], + [ + 41, + 44, + "CDR", + "structure_element" + ], + [ + 45, + 47, + "H3", + "structure_element" + ], + [ + 58, + 70, + "ARYDGIYGELDF", + "structure_element" + ] + ] + }, + { + "sid": 68, + "sent": "Crystallization of the 16 Fabs was previously reported.", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Crystallization", + "experimental_method" + ], + [ + 26, + 30, + "Fabs", + "structure_element" + ] + ] + }, + { + "sid": 69, + "sent": "Three sets of the crystals were isomorphous with nearly identical unit cells (Table\u00a01).", + "section": "RESULTS", + "ner": [ + [ + 18, + 26, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 70, + "sent": "These include (1) H3-23:L3-11 and H3-23:L4-1 in P212121, (2) H3-53:L1-39, H3-53:L3-11 and H3-53:L3-20 in P6522, and (3) H5-51:L1-39, H5-51:L3-11 and H5-51:L3-20 in P212121.", + "section": "RESULTS", + "ner": [ + [ + 18, + 29, + "H3-23:L3-11", + "complex_assembly" + ], + [ + 34, + 44, + "H3-23:L4-1", + "complex_assembly" + ], + [ + 61, + 72, + "H3-53:L1-39", + "complex_assembly" + ], + [ + 74, + 85, + "H3-53:L3-11", + "complex_assembly" + ], + [ + 90, + 101, + "H3-53:L3-20", + "complex_assembly" + ], + [ + 120, + 131, + "H5-51:L1-39", + "complex_assembly" + ], + [ + 133, + 144, + "H5-51:L3-11", + "complex_assembly" + ], + [ + 149, + 160, + "H5-51:L3-20", + "complex_assembly" + ] + ] + }, + { + "sid": 71, + "sent": "Variations occur in the pH (buffer) and the additives, and, in group 3, PEG 3350 is the precipitant for one variants while ammonium sulfate is the precipitant for the other two.", + "section": "RESULTS", + "ner": [ + [ + 72, + 80, + "PEG 3350", + "chemical" + ], + [ + 123, + 139, + "ammonium sulfate", + "chemical" + ] + ] + }, + { + "sid": 72, + "sent": "The similarity in the crystal forms is attributed in part to cross-seeding using the microseed matrix screening for groups 2 and 3.", + "section": "RESULTS", + "ner": [ + [ + 22, + 35, + "crystal forms", + "evidence" + ], + [ + 85, + 111, + "microseed matrix screening", + "experimental_method" + ] + ] + }, + { + "sid": 73, + "sent": "The crystal structures of the 16 Fabs have been determined at resolutions ranging from 3.3 \u212b to 1.65 \u212b (Table\u00a01).", + "section": "RESULTS", + "ner": [ + [ + 4, + 22, + "crystal structures", + "evidence" + ], + [ + 33, + 37, + "Fabs", + "structure_element" + ] + ] + }, + { + "sid": 74, + "sent": "The number of Fab molecules in the crystallographic asymmetric unit varies from 1 (for 12 Fabs) to 2 (for 4 Fabs).", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "Fab", + "structure_element" + ], + [ + 90, + 94, + "Fabs", + "structure_element" + ], + [ + 108, + 112, + "Fabs", + "structure_element" + ] + ] + }, + { + "sid": 75, + "sent": "Overall the structures are fairly complete, and, as can be expected, the models for the higher resolution structures are more complete than those for the lower resolution structures (Table\u00a0S1).", + "section": "RESULTS", + "ner": [ + [ + 12, + 22, + "structures", + "evidence" + ], + [ + 106, + 116, + "structures", + "evidence" + ], + [ + 171, + 181, + "structures", + "evidence" + ] + ] + }, + { + "sid": 76, + "sent": "Invariably, the HCs have more disorder than the LCs.", + "section": "RESULTS", + "ner": [ + [ + 16, + 19, + "HCs", + "structure_element" + ], + [ + 30, + 38, + "disorder", + "protein_state" + ], + [ + 48, + 51, + "LCs", + "structure_element" + ] + ] + }, + { + "sid": 77, + "sent": "For the LC, the disorder is observed at 2 of the C-terminal residues with few exceptions.", + "section": "RESULTS", + "ner": [ + [ + 8, + 10, + "LC", + "structure_element" + ], + [ + 16, + 24, + "disorder", + "protein_state" + ] + ] + }, + { + "sid": 78, + "sent": "Apart from the C-terminus, only a few surface residues in LC are disordered.", + "section": "RESULTS", + "ner": [ + [ + 58, + 60, + "LC", + "structure_element" + ], + [ + 65, + 75, + "disordered", + "protein_state" + ] + ] + }, + { + "sid": 79, + "sent": "The HCs feature the largest number of disordered residues, with the lower resolution structures having the most.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "HCs", + "structure_element" + ], + [ + 38, + 48, + "disordered", + "protein_state" + ], + [ + 85, + 95, + "structures", + "evidence" + ] + ] + }, + { + "sid": 80, + "sent": "The C-terminal residues including the 6xHis tags are disordered in all 16 structures.", + "section": "RESULTS", + "ner": [ + [ + 53, + 63, + "disordered", + "protein_state" + ], + [ + 74, + 84, + "structures", + "evidence" + ] + ] + }, + { + "sid": 81, + "sent": "In addition to these, 2 primary disordered stretches of residues are observed in a number of structures (Table\u00a0S1).", + "section": "RESULTS", + "ner": [ + [ + 93, + 103, + "structures", + "evidence" + ] + ] + }, + { + "sid": 82, + "sent": "One involves the loop connecting the first 2 \u03b2-strands of the constant domain (in all Fabs except H3-23:L1-39, H3-23:L3-11 and H3-53:L1-39).", + "section": "RESULTS", + "ner": [ + [ + 17, + 21, + "loop", + "structure_element" + ], + [ + 45, + 54, + "\u03b2-strands", + "structure_element" + ], + [ + 62, + 77, + "constant domain", + "structure_element" + ], + [ + 86, + 90, + "Fabs", + "structure_element" + ], + [ + 98, + 109, + "H3-23:L1-39", + "complex_assembly" + ], + [ + 111, + 122, + "H3-23:L3-11", + "complex_assembly" + ], + [ + 127, + 138, + "H3-53:L1-39", + "complex_assembly" + ] + ] + }, + { + "sid": 83, + "sent": "The other is located in CDR H3 (in H5-51:L3-11, H5-51:L3-20 and in one of 2 copies of H3-23:L4-1).", + "section": "RESULTS", + "ner": [ + [ + 24, + 27, + "CDR", + "structure_element" + ], + [ + 28, + 30, + "H3", + "structure_element" + ], + [ + 35, + 46, + "H5-51:L3-11", + "complex_assembly" + ], + [ + 48, + 59, + "H5-51:L3-20", + "complex_assembly" + ], + [ + 86, + 96, + "H3-23:L4-1", + "complex_assembly" + ] + ] + }, + { + "sid": 84, + "sent": "CDR H1 and CDR H2 also show some degree of disorder, but to a lesser extent.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "CDR", + "structure_element" + ], + [ + 4, + 6, + "H1", + "structure_element" + ], + [ + 11, + 14, + "CDR", + "structure_element" + ], + [ + 15, + 17, + "H2", + "structure_element" + ], + [ + 43, + 51, + "disorder", + "protein_state" + ] + ] + }, + { + "sid": 85, + "sent": "CDR canonical structures", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "CDR", + "structure_element" + ], + [ + 14, + 24, + "structures", + "evidence" + ] + ] + }, + { + "sid": 86, + "sent": "Several CDR definitions have evolved over decades of antibody research.", + "section": "RESULTS", + "ner": [ + [ + 8, + 11, + "CDR", + "structure_element" + ], + [ + 53, + 61, + "antibody", + "protein_type" + ] + ] + }, + { + "sid": 87, + "sent": "Depending on the focus of the study, the CDR boundaries differ slightly between various definitions.", + "section": "RESULTS", + "ner": [ + [ + 41, + 44, + "CDR", + "structure_element" + ] + ] + }, + { + "sid": 88, + "sent": "In this work, we use the CDR definition of North et\u00a0al., which is similar to that of Martin with the following exceptions: 1) CDRs H1 and H3 begin immediately after the Cys; and 2) CDR L2 includes an additional residue at the N-terminal side, typically Tyr.", + "section": "RESULTS", + "ner": [ + [ + 25, + 28, + "CDR", + "structure_element" + ], + [ + 126, + 130, + "CDRs", + "structure_element" + ], + [ + 131, + 133, + "H1", + "structure_element" + ], + [ + 138, + 140, + "H3", + "structure_element" + ], + [ + 169, + 172, + "Cys", + "residue_name" + ], + [ + 181, + 184, + "CDR", + "structure_element" + ], + [ + 185, + 187, + "L2", + "structure_element" + ], + [ + 253, + 256, + "Tyr", + "residue_name" + ] + ] + }, + { + "sid": 89, + "sent": "CDR H1", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "CDR", + "structure_element" + ], + [ + 4, + 6, + "H1", + "structure_element" + ] + ] + }, + { + "sid": 90, + "sent": "The superposition of CDR H1 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51.", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "superposition", + "experimental_method" + ], + [ + 21, + 24, + "CDR", + "structure_element" + ], + [ + 25, + 27, + "H1", + "structure_element" + ], + [ + 46, + 51, + "HC:LC", + "complex_assembly" + ], + [ + 63, + 75, + "heavy chains", + "structure_element" + ], + [ + 81, + 86, + "H1-69", + "mutant" + ], + [ + 92, + 97, + "H3-23", + "mutant" + ], + [ + 103, + 108, + "H3-53", + "mutant" + ], + [ + 117, + 122, + "H5-51", + "mutant" + ] + ] + }, + { + "sid": 91, + "sent": "CDRs are defined using the Dunbrack convention [12].", + "section": "TABLE", + "ner": [ + [ + 0, + 4, + "CDRs", + "structure_element" + ] + ] + }, + { + "sid": 92, + "sent": "Assignments for 2 copies of the Fab in the asymmetric unit are given for 5 structures.", + "section": "TABLE", + "ner": [ + [ + 32, + 35, + "Fab", + "structure_element" + ], + [ + 75, + 85, + "structures", + "evidence" + ] + ] + }, + { + "sid": 93, + "sent": "No assignment (NA) for CDRs with missing residues.", + "section": "TABLE", + "ner": [ + [ + 23, + 27, + "CDRs", + "structure_element" + ] + ] + }, + { + "sid": 94, + "sent": "The four HCs feature CDR H1 of the same length, and their sequences are highly similar (Table\u00a02).", + "section": "RESULTS", + "ner": [ + [ + 9, + 12, + "HCs", + "structure_element" + ], + [ + 21, + 24, + "CDR", + "structure_element" + ], + [ + 25, + 27, + "H1", + "structure_element" + ] + ] + }, + { + "sid": 95, + "sent": "The CDR H1 backbone conformations for all variants for each of the HCs are shown in Fig.\u00a01.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "CDR", + "structure_element" + ], + [ + 8, + 10, + "H1", + "structure_element" + ], + [ + 67, + 70, + "HCs", + "structure_element" + ] + ] + }, + { + "sid": 96, + "sent": "Three of the HCs, H3-23, H3-53 and H5-51, have the same canonical structure, H1-13-1, and the backbone conformations are tightly clustered for each set of Fab structures as reflected in the rmsd values (Fig.\u00a01B-D).", + "section": "RESULTS", + "ner": [ + [ + 13, + 16, + "HCs", + "structure_element" + ], + [ + 18, + 23, + "H3-23", + "mutant" + ], + [ + 25, + 30, + "H3-53", + "mutant" + ], + [ + 35, + 40, + "H5-51", + "mutant" + ], + [ + 77, + 84, + "H1-13-1", + "mutant" + ], + [ + 155, + 158, + "Fab", + "structure_element" + ], + [ + 159, + 169, + "structures", + "evidence" + ], + [ + 190, + 201, + "rmsd values", + "evidence" + ] + ] + }, + { + "sid": 97, + "sent": "Some deviation is observed for H3-53, mostly due to H3-53:L4-1, which exhibits a significant degree of disorder in CDR H1.", + "section": "RESULTS", + "ner": [ + [ + 31, + 36, + "H3-53", + "mutant" + ], + [ + 52, + 62, + "H3-53:L4-1", + "complex_assembly" + ], + [ + 115, + 118, + "CDR", + "structure_element" + ], + [ + 119, + 121, + "H1", + "structure_element" + ] + ] + }, + { + "sid": 98, + "sent": "The electron density for the backbone is weak and discontinuous, and completely missing for several side chains.", + "section": "RESULTS", + "ner": [ + [ + 4, + 20, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 99, + "sent": "The CDR H1 structures with H1-69 shown in Fig.\u00a01A are quite variable, both for the structures with different LCs and for the copies of the same Fab in the asymmetric unit, H1-69:L3-11 and H1-69:L3-20.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "CDR", + "structure_element" + ], + [ + 8, + 10, + "H1", + "structure_element" + ], + [ + 11, + 21, + "structures", + "evidence" + ], + [ + 27, + 32, + "H1-69", + "mutant" + ], + [ + 83, + 93, + "structures", + "evidence" + ], + [ + 109, + 112, + "LCs", + "structure_element" + ], + [ + 144, + 147, + "Fab", + "structure_element" + ], + [ + 172, + 183, + "H1-69:L3-11", + "complex_assembly" + ], + [ + 188, + 199, + "H1-69:L3-20", + "complex_assembly" + ] + ] + }, + { + "sid": 100, + "sent": "In total, 6 independent Fab structures produce 5 different canonical structures, namely H1-13-1, H1-13-3, H1-13-4, H1-13-6 and H1-13-10.", + "section": "RESULTS", + "ner": [ + [ + 24, + 27, + "Fab", + "structure_element" + ], + [ + 28, + 38, + "structures", + "evidence" + ], + [ + 69, + 79, + "structures", + "evidence" + ], + [ + 88, + 95, + "H1-13-1", + "mutant" + ], + [ + 97, + 104, + "H1-13-3", + "mutant" + ], + [ + 106, + 113, + "H1-13-4", + "mutant" + ], + [ + 115, + 122, + "H1-13-6", + "mutant" + ], + [ + 127, + 135, + "H1-13-10", + "mutant" + ] + ] + }, + { + "sid": 101, + "sent": "A major difference of H1-69 from the other germlines in the experimental data set is the presence of Gly instead of Phe or Tyr at position 27 (residue 5 of 13 in CDR H1).", + "section": "RESULTS", + "ner": [ + [ + 22, + 27, + "H1-69", + "mutant" + ], + [ + 101, + 104, + "Gly", + "residue_name" + ], + [ + 116, + 119, + "Phe", + "residue_name" + ], + [ + 123, + 126, + "Tyr", + "residue_name" + ], + [ + 139, + 141, + "27", + "residue_number" + ], + [ + 162, + 165, + "CDR", + "structure_element" + ], + [ + 166, + 168, + "H1", + "structure_element" + ] + ] + }, + { + "sid": 102, + "sent": "Glycine introduces the possibility of a higher degree of conformational flexibility that undoubtedly translates to the differences observed, and contributes to the elevated thermal parameters for the atoms in the amino acid residues in this region.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Glycine", + "residue_name" + ] + ] + }, + { + "sid": 103, + "sent": "CDR H2", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "CDR", + "structure_element" + ], + [ + 4, + 6, + "H2", + "structure_element" + ] + ] + }, + { + "sid": 104, + "sent": "The superposition of CDR H2 backbones for all HC:LC pairs with heavy chains: (A) H1-69, (B) H3-23, (C) H3-53 and (D) H5-51.", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "superposition", + "experimental_method" + ], + [ + 21, + 24, + "CDR", + "structure_element" + ], + [ + 25, + 27, + "H2", + "structure_element" + ], + [ + 46, + 51, + "HC:LC", + "complex_assembly" + ], + [ + 63, + 75, + "heavy chains", + "structure_element" + ], + [ + 81, + 86, + "H1-69", + "mutant" + ], + [ + 92, + 97, + "H3-23", + "mutant" + ], + [ + 103, + 108, + "H3-53", + "mutant" + ], + [ + 117, + 122, + "H5-51", + "mutant" + ] + ] + }, + { + "sid": 105, + "sent": "The canonical structures of CDR H2 have fairly consistent conformations (Table\u00a02, Fig.\u00a02).", + "section": "RESULTS", + "ner": [ + [ + 28, + 31, + "CDR", + "structure_element" + ], + [ + 32, + 34, + "H2", + "structure_element" + ] + ] + }, + { + "sid": 106, + "sent": "Each of the 4 HCs adopts only one canonical structure regardless of the pairing LC.", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "HCs", + "structure_element" + ], + [ + 80, + 82, + "LC", + "structure_element" + ] + ] + }, + { + "sid": 107, + "sent": "Germlines H1-69 and H5-51 have the same canonical structure assignment H2-10-1, H3-23 has H2-10-2, and H3-53 has H2-9-3.", + "section": "RESULTS", + "ner": [ + [ + 10, + 15, + "H1-69", + "mutant" + ], + [ + 20, + 25, + "H5-51", + "mutant" + ], + [ + 71, + 78, + "H2-10-1", + "mutant" + ], + [ + 80, + 85, + "H3-23", + "mutant" + ], + [ + 90, + 97, + "H2-10-2", + "mutant" + ], + [ + 103, + 108, + "H3-53", + "mutant" + ], + [ + 113, + 119, + "H2-9-3", + "mutant" + ] + ] + }, + { + "sid": 108, + "sent": "The conformations for all of these CDR H2s are tightly clustered (Fig.\u00a02).", + "section": "RESULTS", + "ner": [ + [ + 35, + 38, + "CDR", + "structure_element" + ], + [ + 39, + 42, + "H2s", + "structure_element" + ] + ] + }, + { + "sid": 109, + "sent": "In one case, in the second Fab of H1-69:L3-20, CDR H2 is partially disordered (\u039455-60).", + "section": "RESULTS", + "ner": [ + [ + 27, + 30, + "Fab", + "structure_element" + ], + [ + 34, + 45, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 47, + 50, + "CDR", + "structure_element" + ], + [ + 51, + 53, + "H2", + "structure_element" + ], + [ + 57, + 77, + "partially disordered", + "protein_state" + ], + [ + 79, + 85, + "\u039455-60", + "mutant" + ] + ] + }, + { + "sid": 110, + "sent": "Although three of the germlines have CDR H2 of the same length, 10 residues, they adopt 2 distinctively different conformations depending mostly on the residue at position 71 from the so-called CDR H4.", + "section": "RESULTS", + "ner": [ + [ + 37, + 40, + "CDR", + "structure_element" + ], + [ + 41, + 43, + "H2", + "structure_element" + ], + [ + 64, + 75, + "10 residues", + "residue_range" + ], + [ + 172, + 174, + "71", + "residue_number" + ], + [ + 194, + 197, + "CDR", + "structure_element" + ], + [ + 198, + 200, + "H4", + "structure_element" + ] + ] + }, + { + "sid": 111, + "sent": "Arg71 in H3-23 fills the space between CDRs H2 and H4, and defines the conformation of the tip of CDR H2 so that residue 54 points away from the antigen binding site.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "Arg71", + "residue_name_number" + ], + [ + 9, + 14, + "H3-23", + "mutant" + ], + [ + 39, + 43, + "CDRs", + "structure_element" + ], + [ + 44, + 46, + "H2", + "structure_element" + ], + [ + 51, + 53, + "H4", + "structure_element" + ], + [ + 98, + 101, + "CDR", + "structure_element" + ], + [ + 102, + 104, + "H2", + "structure_element" + ], + [ + 121, + 123, + "54", + "residue_number" + ], + [ + 145, + 165, + "antigen binding site", + "site" + ] + ] + }, + { + "sid": 112, + "sent": "Germlines H1-69 and H5-51 are unique in the human repertoire in having an Ala at position 71 that leaves enough space for H-Pro52a to pack deeper against CDR H4 so that the following residues 53 and 54 point toward the putative antigen.", + "section": "RESULTS", + "ner": [ + [ + 10, + 15, + "H1-69", + "mutant" + ], + [ + 20, + 25, + "H5-51", + "mutant" + ], + [ + 44, + 49, + "human", + "species" + ], + [ + 74, + 77, + "Ala", + "residue_name" + ], + [ + 90, + 92, + "71", + 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"structure_element" + ], + [ + 65, + 67, + "H3", + "structure_element" + ], + [ + 142, + 146, + "CDRs", + "structure_element" + ] + ] + }, + { + "sid": 152, + "sent": "Three of the 21 Fab structures (including multiple copies in the asymmetric unit), H5-51:L3-11, H551:L3-20 and H3-23:L4-1 (one of the 2 Fabs), have missing (disordered) residues at the apex of the CDR loop.", + "section": "RESULTS", + "ner": [ + [ + 16, + 19, + "Fab", + "structure_element" + ], + [ + 20, + 30, + "structures", + "evidence" + ], + [ + 83, + 94, + "H5-51:L3-11", + "complex_assembly" + ], + [ + 96, + 106, + "H551:L3-20", + "complex_assembly" + ], + [ + 111, + 121, + "H3-23:L4-1", + "complex_assembly" + ], + [ + 136, + 140, + "Fabs", + "structure_element" + ], + [ + 148, + 155, + "missing", + "protein_state" + ], + [ + 157, + 167, + "disordered", + "protein_state" + ], + [ + 197, + 205, + "CDR loop", + "structure_element" + ] + ] + }, + { + "sid": 153, + "sent": "Another four of the Fabs, H3-23:L1-39, H3-53:L1-39, H3-53:L3-11 and H3-53:L4-1 have missing side-chain atoms.", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "Fabs", + "structure_element" + ], + [ + 26, + 37, + "H3-23:L1-39", + "complex_assembly" + ], + [ + 39, + 50, + "H3-53:L1-39", + "complex_assembly" + ], + [ + 52, + 63, + "H3-53:L3-11", + "complex_assembly" + ], + [ + 68, + 78, + "H3-53:L4-1", + "complex_assembly" + ] + ] + }, + { + "sid": 154, + "sent": "The variations in CDR H3 conformation are illustrated in Fig.\u00a06 for the 18 Fab structures that have ordered backbone atoms.", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "CDR", + "structure_element" + ], + [ + 22, + 24, + "H3", + "structure_element" + ], + [ + 75, + 78, + "Fab", + "structure_element" + ], + [ + 79, + 89, + "structures", + "evidence" + ] + ] + }, + { + "sid": 155, + "sent": "A comparison of representatives of the \u201ckinked\u201d and \u201cextended\u201d structures.", + "section": "FIG", + "ner": [ + [ + 40, + 46, + "kinked", + "protein_state" + ], + [ + 53, + 61, + "extended", + "protein_state" + ], + [ + 63, + 73, + "structures", + "evidence" + ] + ] + }, + { + "sid": 156, + "sent": "(A) The \u201ckinked\u201d CDR H3 of H1-69:L3-11 with purple carbon atoms and yellow dashed lines connecting the H-bond pairs for Leu100b O and Trp103 NE1, Arg94 NE and Asp101 OD1, and Arg94 NH2 and Asp101 OD2.", + "section": "FIG", + "ner": [ + [ + 9, + 15, + "kinked", + "protein_state" + ], + [ + 17, + 20, + "CDR", + "structure_element" + ], + [ + 21, + 23, + "H3", + "structure_element" + ], + [ + 27, + 38, + "H1-69:L3-11", + "complex_assembly" + ], + [ + 120, + 127, + "Leu100b", + "residue_name_number" + ], + [ + 134, + 140, + "Trp103", + "residue_name_number" + ], + [ + 146, + 151, + "Arg94", + "residue_name_number" + ], + [ + 159, + 165, + "Asp101", + "residue_name_number" + ], + [ + 175, + 180, + "Arg94", + "residue_name_number" + ], + [ + 189, + 195, + "Asp101", + "residue_name_number" + ] + ] + }, + { + "sid": 157, + "sent": "(B) The \u201cextended\u201d CDR H3 of H1-69:L3-20 with green carbon atoms and yellow dashed lines connecting the H-bond pairs for Asp101 OD1 and OD2 and Trp103 NE1.", + "section": "FIG", + "ner": [ + [ + 9, + 17, + "extended", + "protein_state" + ], + [ + 19, + 22, + "CDR", + "structure_element" + ], + [ + 23, + 25, + "H3", + "structure_element" + ], + [ + 29, + 40, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 121, + 127, + "Asp101", + "residue_name_number" + ], + [ + 144, + 150, + "Trp103", + "residue_name_number" + ] + ] + }, + { + "sid": 158, + "sent": "In 10 of the 18 Fab structures, H1-69:L1-39, H1-69:L3-11 (2 Fabs), H1-69:L4-1, H3-23:L3-11 (2 Fabs), H3-23:L3-20, H3-53:L3-11, H3-53:L3-20 and H5-51:L1-39, the CDRs have similar conformations to that found in 4DN3.", + "section": "RESULTS", + "ner": [ + [ + 16, + 19, + "Fab", + "structure_element" + ], + [ + 20, + 30, + "structures", + "evidence" + ], + [ + 32, + 43, + "H1-69:L1-39", + "complex_assembly" + ], + [ + 45, + 56, + "H1-69:L3-11", + "complex_assembly" + ], + [ + 60, + 64, + "Fabs", + "structure_element" + ], + [ + 67, + 77, + "H1-69:L4-1", + "complex_assembly" + ], + [ + 79, + 90, + "H3-23:L3-11", + "complex_assembly" + ], + [ + 94, + 98, + "Fabs", + "structure_element" + ], + [ + 101, + 112, + "H3-23:L3-20", + "complex_assembly" + ], + [ + 114, + 125, + "H3-53:L3-11", + "complex_assembly" + ], + [ + 127, + 138, + "H3-53:L3-20", + "complex_assembly" + ], + [ + 143, + 154, + "H5-51:L1-39", + "complex_assembly" + ], + [ + 160, + 164, + "CDRs", + "structure_element" + ] + ] + }, + { + "sid": 159, + "sent": "The bases of these structures have the \u2018kinked\u2019 conformation with the H-bond between Trp103 and Leu100b.", + "section": "RESULTS", + "ner": [ + [ + 19, + 29, + "structures", + "evidence" + ], + [ + 40, + 46, + "kinked", + "protein_state" + ], + [ + 85, + 91, + "Trp103", + "residue_name_number" + ], + [ + 96, + 103, + "Leu100b", + "residue_name_number" + ] + ] + }, + { + "sid": 160, + "sent": "A representative CDR H3 structure for H1-69:L1-39 illustrating this is shown in Fig.\u00a07A.", + "section": "RESULTS", + "ner": [ + [ + 17, + 20, + "CDR", + "structure_element" + ], + [ + 21, + 23, + "H3", + "structure_element" + ], + [ + 24, + 33, + "structure", + "evidence" + ], + [ + 38, + 49, + "H1-69:L1-39", + "complex_assembly" + ] + ] + }, + { + "sid": 161, + "sent": "The largest backbone conformational deviation for the set is at Tyr99, where the C=O is rotated by 90\u00b0 relative to that observed in 4DN3.", + "section": "RESULTS", + "ner": [ + [ + 64, + 69, + "Tyr99", + "residue_name_number" + ] + ] + }, + { + "sid": 162, + "sent": "Also, it is worth noting that only one of these structures, H1-69:L4-1, has the conserved water molecule in CDR H3 observed in the 4DN3 and 4DN4 structures.", + "section": "RESULTS", + "ner": [ + [ + 48, + 58, + "structures", + "evidence" + ], + [ + 60, + 70, + "H1-69:L4-1", + "complex_assembly" + ], + [ + 80, + 89, + "conserved", + "protein_state" + ], + [ + 90, + 95, + "water", + "chemical" + ], + [ + 108, + 111, + "CDR", + "structure_element" + ], + [ + 112, + 114, + "H3", + "structure_element" + ], + [ + 145, + 155, + "structures", + "evidence" + ] + ] + }, + { + "sid": 163, + "sent": "In fact, it is the only Fab in the set that has a water molecule present at this site.", + "section": "RESULTS", + "ner": [ + [ + 24, + 27, + "Fab", + "structure_element" + ], + [ + 50, + 55, + "water", + "chemical" + ] + ] + }, + { + "sid": 164, + "sent": "The CDR H3 for this structure is shown in Fig.\u00a0S3.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "CDR", + "structure_element" + ], + [ + 8, + 10, + "H3", + "structure_element" + ], + [ + 20, + 29, + "structure", + "evidence" + ] + ] + }, + { + "sid": 165, + "sent": "The remaining 8 Fabs can be grouped into 5 different conformational classes.", + "section": "RESULTS", + "ner": [ + [ + 16, + 20, + "Fabs", + "structure_element" + ] + ] + }, + { + "sid": 166, + "sent": "Three of the Fabs, H3-23:L1-39, H3-23:L4-1 and H3-53:L1-39, have distinctive conformations.", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "Fabs", + "structure_element" + ], + [ + 19, + 30, + "H3-23:L1-39", + "complex_assembly" + ], + [ + 32, + 42, + "H3-23:L4-1", + "complex_assembly" + ], + [ + 47, + 58, + "H3-53:L1-39", + "complex_assembly" + ] + ] + }, + { + "sid": 167, + "sent": "The stem regions in these 3 cases are in the \u2018kinked\u2019 conformation consistent with that observed for 4DN3.", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "stem regions", + "structure_element" + ], + [ + 46, + 52, + "kinked", + "protein_state" + ] + ] + }, + { + "sid": 168, + "sent": "The five remaining Fabs, H5-51:L4-1 (2 copies), H1-69:L3-20 (2 copies) and H3-53:L4-1, have 3 different CDR H3 conformations (Fig.\u00a0S4).", + "section": "RESULTS", + "ner": [ + [ + 19, + 23, + "Fabs", + "structure_element" + ], + [ + 25, + 35, + "H5-51:L4-1", + "complex_assembly" + ], + [ + 48, + 59, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 75, + 85, + "H3-53:L4-1", + "complex_assembly" + ], + [ + 104, + 107, + "CDR", + "structure_element" + ], + [ + 108, + 110, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 169, + "sent": "The stem regions of CDR H3 for the H5-51:L4-1 Fabs are in the \u2018kinked\u2019 conformation while, surprisingly, those of the H1-69:L3-20 pair and H3-53:L4-1 are in the \u2018extended\u2019 conformation (Fig.\u00a07B).", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "stem regions", + "structure_element" + ], + [ + 20, + 23, + "CDR", + "structure_element" + ], + [ + 24, + 26, + "H3", + "structure_element" + ], + [ + 35, + 45, + "H5-51:L4-1", + "complex_assembly" + ], + [ + 46, + 50, + "Fabs", + "structure_element" + ], + [ + 63, + 69, + "kinked", + "protein_state" + ], + [ + 118, + 129, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 139, + 149, + "H3-53:L4-1", + "complex_assembly" + ], + [ + 162, + 170, + "extended", + "protein_state" + ] + ] + }, + { + "sid": 170, + "sent": "VH:VL domain packing", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "VH:VL", + "complex_assembly" + ] + ] + }, + { + "sid": 171, + "sent": "The VH and VL domains have a \u03b2-sandwich structure (also often referred as a Greek key motif) and each is composed of a 4-stranded and a 5-stranded antiparallel \u03b2-sheets.", + "section": "RESULTS", + "ner": [ + [ + 4, + 6, + "VH", + "structure_element" + ], + [ + 11, + 13, + "VL", + "structure_element" + ], + [ + 29, + 49, + "\u03b2-sandwich structure", + "structure_element" + ], + [ + 76, + 91, + "Greek key motif", + "structure_element" + ], + [ + 119, + 168, + "4-stranded and a 5-stranded antiparallel \u03b2-sheets", + "structure_element" + ] + ] + }, + { + "sid": 172, + "sent": "The two domains pack together such that the 5-stranded \u03b2-sheets, which have hydrophobic surfaces, interact with each other bringing the CDRs from both the VH and VL domains into close proximity.", + "section": "RESULTS", + "ner": [ + [ + 44, + 63, + "5-stranded \u03b2-sheets", + "structure_element" + ], + [ + 136, + 140, + "CDRs", + "structure_element" + ], + [ + 155, + 157, + "VH", + "structure_element" + ], + [ + 162, + 164, + "VL", + "structure_element" + ] + ] + }, + { + "sid": 173, + "sent": "The domain packing of the variants was assessed by computing the domain interface interactions, the VH:VL tilt angles, the buried surface area and surface complementarity.", + "section": "RESULTS", + "ner": [ + [ + 65, + 81, + "domain interface", + "site" + ], + [ + 100, + 105, + "VH:VL", + "complex_assembly" + ], + [ + 106, + 117, + "tilt angles", + "evidence" + ] + ] + }, + { + "sid": 174, + "sent": "VH:VL interface amino acid residue interactions", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "VH:VL interface", + "site" + ] + ] + }, + { + "sid": 175, + "sent": "The conserved VH:VL interactions as viewed along the VH/VL axis.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "conserved", + "protein_state" + ], + [ + 14, + 19, + "VH:VL", + "complex_assembly" + ], + [ + 53, + 55, + "VH", + "structure_element" + ], + [ + 56, + 58, + "VL", + "structure_element" + ] + ] + }, + { + "sid": 176, + "sent": "The VH residues are in blue, the VL residues are in orange.", + "section": "FIG", + "ner": [ + [ + 4, + 6, + "VH", + "structure_element" + ], + [ + 33, + 35, + "VL", + "structure_element" + ] + ] + }, + { + "sid": 177, + "sent": "The VH:VL interface is pseudosymmetric, and involves 2 stretches of the polypeptide chain from each domain, namely CDR3 and the framework region between CDRs 1 and 2.", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "VH:VL interface", + "site" + ], + [ + 23, + 38, + "pseudosymmetric", + "protein_state" + ], + [ + 115, + 119, + "CDR3", + "structure_element" + ], + [ + 128, + 144, + "framework region", + "structure_element" + ], + [ + 153, + 165, + "CDRs 1 and 2", + "structure_element" + ] + ] + }, + { + "sid": 178, + "sent": "These stretches form antiparallel \u03b2-hairpins within the internal 5-stranded \u03b2-sheet.", + "section": "RESULTS", + "ner": [ + [ + 21, + 44, + "antiparallel \u03b2-hairpins", + "structure_element" + ], + [ + 65, + 83, + "5-stranded \u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 179, + "sent": "There are a few principal inter-domain interactions that are conserved not only in the experimental set of 16 Fabs, but in all human antibodies.", + "section": "RESULTS", + "ner": [ + [ + 110, + 114, + "Fabs", + "structure_element" + ], + [ + 127, + 132, + "human", + "species" + ], + [ + 133, + 143, + "antibodies", + "protein_type" + ] + ] + }, + { + "sid": 180, + "sent": "They include: 1) a bidentate hydrogen bond between L-Gln38 and H-Gln39; 2) H-Leu45 in a hydrophobic pocket between L-Phe98, L-Tyr87 and L-Pro44; 3) L-Pro44 stacked against H-Trp103; and 4) L-Ala43 opposite the face of H-Tyr91 (Fig.\u00a08).", + "section": "RESULTS", + "ner": [ + [ + 29, + 42, + "hydrogen bond", + "bond_interaction" + ], + [ + 51, + 52, + "L", + "structure_element" + ], + [ + 53, + 58, + "Gln38", + "residue_name_number" + ], + [ + 63, + 64, + "H", + "structure_element" + ], + [ + 65, + 70, + "Gln39", + "residue_name_number" + ], + [ + 75, + 76, + "H", + "structure_element" + ], + [ + 77, + 82, + "Leu45", + "residue_name_number" + ], + [ + 88, + 106, + "hydrophobic pocket", + "site" + ], + [ + 115, + 116, + "L", + "structure_element" + ], + [ + 117, + 122, + "Phe98", + "residue_name_number" + ], + [ + 124, + 125, + "L", + "structure_element" + ], + [ + 126, + 131, + "Tyr87", + "residue_name_number" + ], + [ + 136, + 137, + "L", + "structure_element" + ], + [ + 138, + 143, + "Pro44", + "residue_name_number" + ], + [ + 148, + 149, + "L", + "structure_element" + ], + [ + 150, + 155, + "Pro44", + "residue_name_number" + ], + [ + 172, + 173, + "H", + "structure_element" + ], + [ + 174, + 180, + "Trp103", + "residue_name_number" + ], + [ + 189, + 190, + "L", + "structure_element" + ], + [ + 191, + 196, + "Ala43", + "residue_name_number" + ], + [ + 218, + 219, + "H", + "structure_element" + ], + [ + 220, + 225, + "Tyr91", + "residue_name_number" + ] + ] + }, + { + "sid": 181, + "sent": "With the exception of L-Ala43, all other residues are conserved in human germlines.", + "section": "RESULTS", + "ner": [ + [ + 22, + 23, + "L", + "structure_element" + ], + [ + 24, + 29, + "Ala43", + "residue_name_number" + ], + [ + 67, + 72, + "human", + "species" + ] + ] + }, + { + "sid": 182, + "sent": "Position 43 may be alternatively occupied by Ser, Val or Pro (as in L4-1), but the hydrophobic interaction with H-Tyr91 is preserved.", + "section": "RESULTS", + "ner": [ + [ + 9, + 11, + "43", + "residue_number" + ], + [ + 45, + 48, + "Ser", + "residue_name" + ], + [ + 50, + 53, + "Val", + "residue_name" + ], + [ + 57, + 60, + "Pro", + "residue_name" + ], + [ + 68, + 72, + "L4-1", + "mutant" + ], + [ + 83, + 106, + "hydrophobic interaction", + "bond_interaction" + ], + [ + 112, + 113, + "H", + "structure_element" + ], + [ + 114, + 119, + "Tyr91", + "residue_name_number" + ] + ] + }, + { + "sid": 183, + "sent": "These core interactions provide enough stability to the VH:VL dimer so that additional VH-VL contacts can tolerate amino acid sequence variations in CDRs H3 and L3 that form part of the VH:VL interface.", + "section": "RESULTS", + "ner": [ + [ + 56, + 61, + "VH:VL", + "complex_assembly" + ], + [ + 62, + 67, + "dimer", + "oligomeric_state" + ], + [ + 87, + 101, + "VH-VL contacts", + "site" + ], + [ + 149, + 153, + "CDRs", + "structure_element" + ], + [ + 154, + 156, + "H3", + "structure_element" + ], + [ + 161, + 163, + "L3", + "structure_element" + ], + [ + 186, + 201, + "VH:VL interface", + "site" + ] + ] + }, + { + "sid": 184, + "sent": "In total, about 20 residues are involved in the VH:VL interactions on each side (Fig.\u00a0S5).", + "section": "RESULTS", + "ner": [ + [ + 16, + 27, + "20 residues", + "residue_range" + ], + [ + 48, + 53, + "VH:VL", + "complex_assembly" + ] + ] + }, + { + "sid": 185, + "sent": "Half of them are in the framework regions and those residues (except residue 61 in HC, which is actually in CDR2 in Kabat's definition) are conserved in the set of 16 Fabs.", + "section": "RESULTS", + "ner": [ + [ + 24, + 41, + "framework regions", + "structure_element" + ], + [ + 77, + 79, + "61", + "residue_number" + ], + [ + 83, + 85, + "HC", + "structure_element" + ], + [ + 108, + 112, + "CDR2", + "structure_element" + ], + [ + 167, + 171, + "Fabs", + "structure_element" + ] + ] + }, + { + "sid": 186, + "sent": "One notable exception is H-Trp47, which exhibits 2 conformations of the indole ring.", + "section": "RESULTS", + "ner": [ + [ + 25, + 26, + "H", + "structure_element" + ], + [ + 27, + 32, + "Trp47", + "residue_name_number" + ] + ] + }, + { + "sid": 187, + "sent": "In most of the structures, it has the \u03c72 angle of \u223c80\u00b0, while the ring is flipped over (\u03c72 = \u2212100\u00b0) in H5-51:L3:11 and H5-51:L3-20.", + "section": "RESULTS", + "ner": [ + [ + 15, + 25, + "structures", + "evidence" + ], + [ + 38, + 40, + "\u03c72", + "evidence" + ], + [ + 88, + 90, + "\u03c72", + "evidence" + ], + [ + 103, + 114, + "H5-51:L3:11", + "complex_assembly" + ], + [ + 119, + 130, + "H5-51:L3-20", + "complex_assembly" + ] + ] + }, + { + "sid": 188, + "sent": "Interestingly, these are the only 2 structures with residues missing in CDR H3 because of disorder, although both structures are determined at high resolution and the rest of the structure is well defined.", + "section": "RESULTS", + "ner": [ + [ + 36, + 46, + "structures", + "evidence" + ], + [ + 61, + 68, + "missing", + "protein_state" + ], + [ + 72, + 75, + "CDR", + "structure_element" + ], + [ + 76, + 78, + "H3", + "structure_element" + ], + [ + 114, + 124, + "structures", + "evidence" + ], + [ + 179, + 188, + "structure", + "evidence" + ] + ] + }, + { + "sid": 189, + "sent": "Apparently, residues flanking CDR H3 in the 2 VH:VL pairings are inconsistent with any stable conformation of CDR H3, which translates into a less restricted conformational space for some of them, including H-Trp47.", + "section": "RESULTS", + "ner": [ + [ + 30, + 33, + "CDR", + "structure_element" + ], + [ + 34, + 36, + "H3", + "structure_element" + ], + [ + 46, + 51, + "VH:VL", + "complex_assembly" + ], + [ + 87, + 93, + "stable", + "protein_state" + ], + [ + 110, + 113, + "CDR", + "structure_element" + ], + [ + 114, + 116, + "H3", + "structure_element" + ], + [ + 207, + 208, + "H", + "structure_element" + ], + [ + 209, + 214, + "Trp47", + "residue_name_number" + ] + ] + }, + { + "sid": 190, + "sent": "VH:VL tilt angles", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "VH:VL", + "complex_assembly" + ], + [ + 6, + 17, + "tilt angles", + "evidence" + ] + ] + }, + { + "sid": 191, + "sent": "The relative orientation of VH and VL has been measured in a number of different ways.", + "section": "RESULTS", + "ner": [ + [ + 28, + 30, + "VH", + "structure_element" + ], + [ + 35, + 37, + "VL", + "structure_element" + ] + ] + }, + { + "sid": 192, + "sent": "The first approach uses ABangles, the results of which are shown in Table\u00a0S2.", + "section": "RESULTS", + "ner": [ + [ + 24, + 32, + "ABangles", + "experimental_method" + ] + ] + }, + { + "sid": 193, + "sent": "The four LCs all are classified as Type A because they have a proline at position 44, and the results for each orientation parameter are within the range of values of this type reported by Dunbar and co-workers.", + "section": "RESULTS", + "ner": [ + [ + 9, + 12, + "LCs", + "structure_element" + ], + [ + 62, + 69, + "proline", + "residue_name" + ], + [ + 82, + 84, + "44", + "residue_number" + ], + [ + 111, + 132, + "orientation parameter", + "evidence" + ] + ] + }, + { + "sid": 194, + "sent": "In fact, the parameter values for the set of 16 Fabs are in the middle of the distribution observed for 351 non-redundant antibody structures determined at 3.0 \u212b resolution or better.", + "section": "RESULTS", + "ner": [ + [ + 48, + 52, + "Fabs", + "structure_element" + ], + [ + 122, + 130, + "antibody", + "protein_type" + ], + [ + 131, + 141, + "structures", + "evidence" + ] + ] + }, + { + "sid": 195, + "sent": "The only exception is HC1, which is shifted toward smaller angles with the mean value of 70.8\u00b0 as compared to the distribution centered at 72\u00b0 for the entire PDB.", + "section": "RESULTS", + "ner": [ + [ + 22, + 25, + "HC1", + "structure_element" + ] + ] + }, + { + "sid": 196, + "sent": "This probably reflects the invariance of CDR H3 in the current set as opposed to the CDR H3 diversity in the PDB.", + "section": "RESULTS", + "ner": [ + [ + 41, + 44, + "CDR", + "structure_element" + ], + [ + 45, + 47, + "H3", + "structure_element" + ], + [ + 85, + 88, + "CDR", + "structure_element" + ], + [ + 89, + 91, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 197, + "sent": "The second approach used for comparing tilt angles involved computing the difference in the tilt angles between all pairs of structures.", + "section": "RESULTS", + "ner": [ + [ + 39, + 50, + "tilt angles", + "evidence" + ], + [ + 74, + 84, + "difference", + "evidence" + ], + [ + 92, + 103, + "tilt angles", + "evidence" + ], + [ + 125, + 135, + "structures", + "evidence" + ] + ] + }, + { + "sid": 198, + "sent": "For structures with 2 copies of the Fab in the asymmetric unit, only one structure was used.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 36, + 39, + "Fab", + "structure_element" + ], + [ + 73, + 82, + "structure", + "evidence" + ] + ] + }, + { + "sid": 199, + "sent": "The differences between independent Fabs in the same structure are 4.9\u00b0 for H1-69:L3-20, 1.6\u00b0 for H1-69:L3-11, 1.4\u00b0 for H3-23:L4-1, 3.3\u00b0 for H3-23:L3-11, and 2.5\u00b0 for H5-51:L4-1.", + "section": "RESULTS", + "ner": [ + [ + 36, + 40, + "Fabs", + "structure_element" + ], + [ + 53, + 62, + "structure", + "evidence" + ], + [ + 76, + 87, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 98, + 109, + "H1-69:L3-11", + "complex_assembly" + ], + [ + 120, + 130, + "H3-23:L4-1", + "complex_assembly" + ], + [ + 141, + 152, + "H3-23:L3-11", + "complex_assembly" + ], + [ + 167, + 177, + "H5-51:L4-1", + "complex_assembly" + ] + ] + }, + { + "sid": 200, + "sent": "With the exception of H1-69:L3-20, the angles are within the range of 2-3\u00b0 as are observed in the identical structures in the PDB.", + "section": "RESULTS", + "ner": [ + [ + 22, + 33, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 108, + 118, + "structures", + "evidence" + ] + ] + }, + { + "sid": 201, + "sent": "In H1-69:L3-20, one of the Fabs is substantially disordered so that part of CDR H2 (the outer \u03b2-strand, residues 55-60) is completely missing.", + "section": "RESULTS", + "ner": [ + [ + 3, + 14, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 27, + 31, + "Fabs", + "structure_element" + ], + [ + 49, + 59, + "disordered", + "protein_state" + ], + [ + 76, + 79, + "CDR", + "structure_element" + ], + [ + 80, + 82, + "H2", + "structure_element" + ], + [ + 94, + 102, + "\u03b2-strand", + "structure_element" + ], + [ + 113, + 118, + "55-60", + "residue_range" + ] + ] + }, + { + "sid": 202, + "sent": "This kind of disorder may compromise the integrity of the VH domain and its interaction with the VL.", + "section": "RESULTS", + "ner": [ + [ + 58, + 60, + "VH", + "structure_element" + ], + [ + 97, + 99, + "VL", + "structure_element" + ] + ] + }, + { + "sid": 203, + "sent": "Indeed, this Fab has the largest twist angle HC2 within the experimental set that exceeds the mean value by 2.5 standard deviations (Table\u00a0S2).", + "section": "RESULTS", + "ner": [ + [ + 13, + 16, + "Fab", + "structure_element" + ], + [ + 33, + 44, + "twist angle", + "evidence" + ], + [ + 45, + 48, + "HC2", + "structure_element" + ] + ] + }, + { + "sid": 204, + "sent": "An illustration of the difference in tilt angle for 2 pairs of variants by the superposition of the VH domains of (A) H1-69:L3-20 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 10.5\u00b0) and (B) H1-69:L4-1 on that of H5-51:L1-39 (the VL domain is off by a rigid-body roatation of 1.6\u00b0).", + "section": "FIG", + "ner": [ + [ + 79, + 92, + "superposition", + "experimental_method" + ], + [ + 100, + 102, + "VH", + "structure_element" + ], + [ + 118, + 129, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 141, + 152, + "H5-51:L1-39", + "complex_assembly" + ], + [ + 158, + 160, + "VL", + "structure_element" + ], + [ + 219, + 229, + "H1-69:L4-1", + "complex_assembly" + ], + [ + 241, + 252, + "H5-51:L1-39", + "complex_assembly" + ], + [ + 258, + 260, + "VL", + "structure_element" + ] + ] + }, + { + "sid": 205, + "sent": "Differences in VH:VL tilt angles.", + "section": "TABLE", + "ner": [ + [ + 15, + 20, + "VH:VL", + "complex_assembly" + ], + [ + 21, + 32, + "tilt angles", + "evidence" + ] + ] + }, + { + "sid": 206, + "sent": "The differences in the tilt angle are shown for all pairs of V regions in Table\u00a03.", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "differences", + "evidence" + ], + [ + 23, + 33, + "tilt angle", + "evidence" + ], + [ + 61, + 70, + "V regions", + "structure_element" + ] + ] + }, + { + "sid": 207, + "sent": "The smallest differences in the tilt angle are between the Fabs in isomorphous crystal forms.", + "section": "RESULTS", + "ner": [ + [ + 32, + 42, + "tilt angle", + "evidence" + ], + [ + 59, + 63, + "Fabs", + "structure_element" + ], + [ + 79, + 92, + "crystal forms", + "evidence" + ] + ] + }, + { + "sid": 208, + "sent": "The largest deviations in the tilt angle, up to 11.0\u00b0, are found for 2 structures, H1-69:L3-20 and H3-23:L3-20, that stand out from the other Fabs.", + "section": "RESULTS", + "ner": [ + [ + 30, + 40, + "tilt angle", + "evidence" + ], + [ + 71, + 81, + "structures", + "evidence" + ], + [ + 83, + 94, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 99, + 110, + "H3-23:L3-20", + "complex_assembly" + ], + [ + 142, + 146, + "Fabs", + "structure_element" + ] + ] + }, + { + "sid": 209, + "sent": "One of the 2 structures, H1-69:L3-20, has its CDR H3 in the \u2018extended\u2019 conformation; the other structure has it in the \u2018kinked\u2019 conformation.", + "section": "RESULTS", + "ner": [ + [ + 13, + 23, + "structures", + "evidence" + ], + [ + 25, + 36, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 46, + 49, + "CDR", + "structure_element" + ], + [ + 50, + 52, + "H3", + "structure_element" + ], + [ + 61, + 69, + "extended", + "protein_state" + ], + [ + 120, + 126, + "kinked", + "protein_state" + ] + ] + }, + { + "sid": 210, + "sent": "Two examples illustrating large (10.5\u00b0) and small (1.6\u00b0) differences in the tilt angles are shown in Fig.\u00a09.", + "section": "RESULTS", + "ner": [ + [ + 76, + 87, + "tilt angles", + "evidence" + ] + ] + }, + { + "sid": 211, + "sent": "VH:VL buried surface area and complementarity", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "VH:VL", + "complex_assembly" + ] + ] + }, + { + "sid": 212, + "sent": "VH:VL surface areas and surface complementarity.", + "section": "TABLE", + "ner": [ + [ + 0, + 5, + "VH:VL", + "complex_assembly" + ] + ] + }, + { + "sid": 213, + "sent": "Some side chain atoms in CDR H3 are missing.", + "section": "TABLE", + "ner": [ + [ + 25, + 28, + "CDR", + "structure_element" + ], + [ + 29, + 31, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 214, + "sent": "Residues in CDR H3 are missing: YGE in H5-51:L3-11, GIY in H5-51:L3-20.", + "section": "TABLE", + "ner": [ + [ + 12, + 15, + "CDR", + "structure_element" + ], + [ + 16, + 18, + "H3", + "structure_element" + ], + [ + 32, + 35, + "YGE", + "structure_element" + ], + [ + 39, + 50, + "H5-51:L3-11", + "complex_assembly" + ], + [ + 52, + 55, + "GIY", + "structure_element" + ], + [ + 59, + 70, + "H5-51:L3-20", + "complex_assembly" + ] + ] + }, + { + "sid": 215, + "sent": "The results of the PISA contact surface calculation and surface complementarity calculation are shown in Table\u00a04.", + "section": "RESULTS", + "ner": [ + [ + 19, + 23, + "PISA", + "experimental_method" + ], + [ + 24, + 51, + "contact surface calculation", + "experimental_method" + ], + [ + 56, + 91, + "surface complementarity calculation", + "experimental_method" + ] + ] + }, + { + "sid": 216, + "sent": "The interface areas are calculated as the average of the VH and VL contact surfaces.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "interface", + "site" + ], + [ + 57, + 83, + "VH and VL contact surfaces", + "site" + ] + ] + }, + { + "sid": 217, + "sent": "Six of the 16 structures have CDR H3 side chains or complete residues missing, and therefore their interfaces are much smaller than in the other 10 structures with complete CDRs (the results are provided for all Fabs for completeness).", + "section": "RESULTS", + "ner": [ + [ + 14, + 24, + "structures", + "evidence" + ], + [ + 30, + 33, + "CDR", + "structure_element" + ], + [ + 34, + 36, + "H3", + "structure_element" + ], + [ + 70, + 77, + "missing", + "protein_state" + ], + [ + 99, + 109, + "interfaces", + "site" + ], + [ + 148, + 158, + "structures", + "evidence" + ], + [ + 164, + 172, + "complete", + "protein_state" + ], + [ + 173, + 177, + "CDRs", + "structure_element" + ], + [ + 212, + 216, + "Fabs", + "structure_element" + ] + ] + }, + { + "sid": 218, + "sent": "Among the complete structures, the interface areas range from 684 to 836 \u212b2.", + "section": "RESULTS", + "ner": [ + [ + 10, + 18, + "complete", + "protein_state" + ], + [ + 19, + 29, + "structures", + "evidence" + ], + [ + 35, + 44, + "interface", + "site" + ] + ] + }, + { + "sid": 219, + "sent": "Interestingly, the 2 structures that have the largest tilt angle differences with the other variants, H3-23:L3-20 and H1-69:L3-20, have the smallest VH:VL interfaces, 684 and 725 \u212b2, respectively.", + "section": "RESULTS", + "ner": [ + [ + 21, + 31, + "structures", + "evidence" + ], + [ + 54, + 76, + "tilt angle differences", + "evidence" + ], + [ + 102, + 113, + "H3-23:L3-20", + "complex_assembly" + ], + [ + 118, + 129, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 149, + 165, + "VH:VL interfaces", + "site" + ] + ] + }, + { + "sid": 220, + "sent": "H3-23:L3-20 is also unique in that it has the lowest value (0.676) of surface complementarity.", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "H3-23:L3-20", + "complex_assembly" + ], + [ + 70, + 93, + "surface complementarity", + "evidence" + ] + ] + }, + { + "sid": 221, + "sent": "Melting temperatures for the 16 Fabs.", + "section": "TABLE", + "ner": [ + [ + 0, + 20, + "Melting temperatures", + "evidence" + ], + [ + 32, + 36, + "Fabs", + "structure_element" + ] + ] + }, + { + "sid": 222, + "sent": "Colors: blue (Tm < 70\u00b0C), green (70\u00b0C < Tm < 73\u00b0C), yellow (73\u00b0C < Tm < 78\u00b0C), orange (Tm > 78\u00b0C).", + "section": "TABLE", + "ner": [ + [ + 14, + 16, + "Tm", + "evidence" + ], + [ + 40, + 42, + "Tm", + "evidence" + ], + [ + 67, + 69, + "Tm", + "evidence" + ], + [ + 87, + 89, + "Tm", + "evidence" + ] + ] + }, + { + "sid": 223, + "sent": "Melting temperatures (Tm) were measured for all Fabs using differential scanning calorimetry (Table\u00a05).", + "section": "RESULTS", + "ner": [ + [ + 0, + 20, + "Melting temperatures", + "evidence" + ], + [ + 22, + 24, + "Tm", + "evidence" + ], + [ + 48, + 52, + "Fabs", + "structure_element" + ], + [ + 59, + 92, + "differential scanning calorimetry", + "experimental_method" + ] + ] + }, + { + "sid": 224, + "sent": "It appears that for each given LC, the Fabs with germlines H1-69 and H3-23 are substantially more stable than those with germlines H3-53 and H5-51.", + "section": "RESULTS", + "ner": [ + [ + 31, + 33, + "LC", + "structure_element" + ], + [ + 39, + 43, + "Fabs", + "structure_element" + ], + [ + 59, + 64, + "H1-69", + "mutant" + ], + [ + 69, + 74, + "H3-23", + "mutant" + ], + [ + 98, + 104, + "stable", + "protein_state" + ], + [ + 131, + 136, + "H3-53", + "mutant" + ], + [ + 141, + 146, + "H5-51", + "mutant" + ] + ] + }, + { + "sid": 225, + "sent": "In addition, L1-39 provides a much higher degree of stabilization than the other 3 LC germlines when combined with any of the HCs.", + "section": "RESULTS", + "ner": [ + [ + 13, + 18, + "L1-39", + "mutant" + ], + [ + 83, + 85, + "LC", + "structure_element" + ], + [ + 126, + 129, + "HCs", + "structure_element" + ] + ] + }, + { + "sid": 226, + "sent": "As a result, the Tm for pairs H1-69:L1-39 and H3-23:L1-39 is 12-13\u00b0 higher than for pairs H3-53:L3-20, H3-53:L4-1, H5-51:L3-20 and H5-51:L4-1.", + "section": "RESULTS", + "ner": [ + [ + 17, + 19, + "Tm", + "evidence" + ], + [ + 30, + 41, + "H1-69:L1-39", + "complex_assembly" + ], + [ + 46, + 57, + "H3-23:L1-39", + "complex_assembly" + ], + [ + 90, + 101, + "H3-53:L3-20", + "complex_assembly" + ], + [ + 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"evidence" + ] + ] + }, + { + "sid": 229, + "sent": "No electron density is observed for a number of side chains in CDRs H3 and L3 in all Fabs with germline H3-53, which indicates loose packing of the variable domains.", + "section": "RESULTS", + "ner": [ + [ + 3, + 19, + "electron density", + "evidence" + ], + [ + 63, + 67, + "CDRs", + "structure_element" + ], + [ + 68, + 70, + "H3", + "structure_element" + ], + [ + 75, + 77, + "L3", + "structure_element" + ], + [ + 85, + 89, + "Fabs", + "structure_element" + ], + [ + 104, + 109, + "H3-53", + "mutant" + ], + [ + 148, + 164, + "variable domains", + "structure_element" + ] + ] + }, + { + "sid": 230, + "sent": "All those molecules are relatively unstable, as is reflected in their low Tms.", + "section": "RESULTS", + "ner": [ + [ + 74, + 77, + "Tms", + "evidence" + ] + ] + }, + { + "sid": 231, + "sent": "This is the first report of a systematic structural investigation of a phage germline library.", + "section": "DISCUSS", + "ner": [ + [ 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conformations for all members of a set are virtually identical, for others subtle changes occur in a few members of a set, and in some cases larger deviations are observed within a set.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 21, + "CDR", + "structure_element" + ] + ] + }, + { + "sid": 236, + "sent": "The five variants that crystallized with 2 copies of the Fab in the asymmetric unit serve somewhat as controls for the influence of crystal packing on the conformations of the CDRs.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 35, + "crystallized", + "experimental_method" + ], + [ + 57, + 60, + "Fab", + "structure_element" + ], + [ + 176, + 180, + "CDRs", + "structure_element" + ] + ] + }, + { + "sid": 237, + "sent": "In four of the 5 structures the CDR conformations are consistent.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 27, + "structures", + "evidence" + ], + [ + 32, + 35, + "CDR", + "structure_element" + ] + ] + }, + { + "sid": 238, + "sent": "In only one case, that of H1-69:L3-20 (the lowest resolution structure), do we see differences in the conformations of the 2 copies of CDRs H1 and L1.", + "section": "DISCUSS", + "ner": [ + [ + 26, + 37, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 61, + 70, + "structure", + "evidence" + ], + [ + 135, + 139, + "CDRs", + "structure_element" + ], + [ + 140, + 142, + "H1", + "structure_element" + ], + [ + 147, + 149, + "L1", + "structure_element" + ] + ] + }, + { + "sid": 239, + "sent": "This variability is likely a result of 2 factors, crystal packing interactions and internal instability of the variable domain.", + "section": "DISCUSS", + "ner": [ + [ + 111, + 126, + "variable domain", + "structure_element" + ] + ] + }, + { + "sid": 240, + "sent": "For the CDRs with canonical structures, the largest changes in conformation occur for CDR H1 of H1-69 and H3-53.", + "section": "DISCUSS", + "ner": [ + [ + 8, + 12, + "CDRs", + "structure_element" + ], + [ + 86, + 89, + "CDR", + "structure_element" + ], + [ + 90, + 92, + "H1", + "structure_element" + ], + [ + 96, + 101, + "H1-69", + "mutant" + ], + [ + 106, + 111, + "H3-53", + "mutant" + ] + ] + }, + { + "sid": 241, + "sent": "The other 2 HCs, H3-23 and H5-51, have canonical structures that are remarkably well conserved (Fig.\u00a01).", + "section": "DISCUSS", + "ner": [ + [ + 12, + 15, + "HCs", + "structure_element" + ], + [ + 17, + 22, + "H3-23", + "mutant" + ], + [ + 27, + 32, + "H5-51", + "mutant" + ], + [ + 69, + 94, + "remarkably well conserved", + "protein_state" + ] + ] + }, + { + "sid": 242, + "sent": "Of the 4 HCs, H1-69 has the greatest number of canonical structure assignments (Table\u00a02).", + "section": "DISCUSS", + "ner": [ + [ + 9, + 12, + "HCs", + "structure_element" + ], + [ + 14, + 19, + "H1-69", + "mutant" + ] + ] + }, + { + "sid": 243, + "sent": "H1-69 is unique in having a pair of glycine residues at positions 26 and 27, which provide more conformational freedom in CDR H1.", + "section": "DISCUSS", + "ner": [ + [ 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same CDR H3 provides some insights into why molecular modeling efforts of CDR H3 have proven so difficult.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 19, + "VH:VL", + "complex_assembly" + ], + [ + 40, + 43, + "CDR", + "structure_element" + ], + [ + 44, + 46, + "H3", + "structure_element" + ], + [ + 109, + 112, + "CDR", + "structure_element" + ], + [ + 113, + 115, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 246, + "sent": "As mentioned in the Results section, this data set is composed of 21 Fabs, since 5 of the 16 variants have 2 Fab copies in the asymmetric unit.", + "section": "DISCUSS", + "ner": [ + [ + 69, + 73, + "Fabs", + "structure_element" + ], + [ + 109, + 112, + "Fab", + "structure_element" + ] + ] + }, + { + "sid": 247, + "sent": "For the 18 Fabs with complete backbone atoms for CDR H3, 10 have conformations similar to that of the parent, while the others have significantly different conformations (Fig.\u00a06).", + "section": "DISCUSS", + "ner": [ + [ + 11, + 15, + "Fabs", + "structure_element" + ], + [ + 49, + 52, + "CDR", + "structure_element" + ], + [ + 53, + 55, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 248, + "sent": "Thus, it is likely that the CDR H3 conformation is dependent upon 2 dominating factors: 1) amino acid sequence; and 2) VH and VL context.", + "section": "DISCUSS", + "ner": [ + [ + 28, + 31, + "CDR", + "structure_element" + ], + [ + 32, + 34, + "H3", + "structure_element" + ], + [ + 119, + 121, + "VH", + "structure_element" + ], + [ + 126, + 128, + "VL", + "structure_element" + ] + ] + }, + { + "sid": 249, + "sent": "More than half of the variants retain the conformation of the parent despite having differences in the VH:VL pairing.", + "section": "DISCUSS", + "ner": [ + [ + 103, + 108, + "VH:VL", + "complex_assembly" + ] + ] + }, + { + "sid": 250, + "sent": "This subset includes 2 structures with 2 copies of the Fab in the asymmetric unit, all of which are nearly identical in conformation.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 33, + "structures", + "evidence" + ], + [ + 55, + 58, + "Fab", + "structure_element" + ] + ] + }, + { + "sid": 251, + "sent": "The remaining 8 structures exhibit \u201cnon-parental\u201d conformations, indicating that the VH and VL context can also be a dominating factor influencing CDR H3.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 26, + "structures", + "evidence" + ], + [ + 85, + 87, + "VH", + "structure_element" + ], + [ + 92, + 94, + "VL", + "structure_element" + ], + [ + 147, + 150, + "CDR", + "structure_element" + ], + [ + 151, + 153, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 252, + "sent": "This subset also has 2 structures with 2 Fab copies in the asymmetric unit.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 33, + "structures", + "evidence" + ], + [ + 41, + 44, + "Fab", + "structure_element" + ] + ] + }, + { + "sid": 253, + "sent": "Interestingly, as described earlier, these 2 pairs differ in the stem regions with the H1-69:L3-20 pair in the \u2018extended\u2019 conformation and H5-51:L4-1 pair in the \u2018kinked\u2019 conformation.", + "section": "DISCUSS", + "ner": [ + [ + 65, + 77, + "stem regions", + "structure_element" + ], + [ + 87, + 98, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 112, + 120, + "extended", + "protein_state" + ], + [ + 139, + 149, + "H5-51:L4-1", + "complex_assembly" + ], + [ + 163, + 169, + "kinked", + "protein_state" + ] + ] + }, + { + "sid": 254, + "sent": "The CDR H3 conformational analysis shows that, for each set of variants of one HC paired with the 4 different LCs, both \u201cparental\u201d and \u201cnon-parental\u201d conformations are observed.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 7, + "CDR", + "structure_element" + ], + [ + 8, + 10, + "H3", + "structure_element" + ], + [ + 11, + 34, + "conformational analysis", + "experimental_method" + ], + [ + 79, + 81, + "HC", + "structure_element" + ], + [ + 110, + 113, + "LCs", + "structure_element" + ] + ] + }, + { + "sid": 255, + "sent": "The same variability is observed for the sets of variants composed of one LC paired with each of the 4 HCs.", + "section": "DISCUSS", + "ner": [ + [ + 74, + 76, + "LC", + "structure_element" + ], + [ + 103, + 106, + "HCs", + "structure_element" + ] + ] + }, + { + "sid": 256, + "sent": "Thus, no patterns of conformational preference for a particular HC or LC emerge to shed any direct light on what drives the conformational differences.", + "section": "DISCUSS", + "ner": [ + [ + 64, + 66, + "HC", + "structure_element" + ], + [ + 70, + 72, + "LC", + "structure_element" + ] + ] + }, + { + "sid": 257, + "sent": "This finding supports the hypothesis of Weitzner et\u00a0al. that the H3 conformation is controlled both by its sequence and its environment.", + "section": "DISCUSS", + "ner": [ + [ + 65, + 67, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 258, + "sent": "In looking at a possible correlation between the tilt angle and the conformation of CDR H3, no clear trends are observed.", + "section": "DISCUSS", + "ner": [ + [ + 49, + 59, + "tilt angle", + "evidence" + ], + [ + 84, + 87, + "CDR", + "structure_element" + ], + [ + 88, + 90, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 259, + "sent": "Two variants, H1-69:L3-20 and H3-23:L3-20, have the largest differences in the tilt angles compared to other variants as seen in Table\u00a03.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 25, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 30, + 41, + "H3-23:L3-20", + "complex_assembly" + ] + ] + }, + { + "sid": 260, + "sent": "The absolute VH:VL orientation parameters for the 2 Fabs (Table\u00a0S2) show significant deviation in HL, LC1 and HC2 values (2-3 standard deviations from the mean).", + "section": "DISCUSS", + "ner": [ + [ + 13, + 18, + "VH:VL", + "complex_assembly" + ], + [ + 19, + 41, + "orientation parameters", + "evidence" + ], + [ + 52, + 56, + "Fabs", + "structure_element" + ], + [ + 85, + 94, + "deviation", + "evidence" + ], + [ + 98, + 100, + "HL", + "structure_element" + ], + [ + 102, + 105, + "LC1", + "structure_element" + ], + [ + 110, + 113, + "HC2", + "structure_element" + ] + ] + }, + { + "sid": 261, + "sent": "One of the variants, H3-23:L3-20, has the CDR H3 conformation similar to the parent, but the other, H1-69:L3-20, is different.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 32, + "H3-23:L3-20", + "complex_assembly" + ], + [ + 42, + 45, + "CDR", + "structure_element" + ], + [ + 46, + 48, + "H3", + "structure_element" + ], + [ + 100, + 111, + "H1-69:L3-20", + "complex_assembly" + ] + ] + }, + { + "sid": 262, + "sent": "As noted in the Results section, the 2 variants, H1-69:L3-20 and H3-23:L3-20, are outliers in terms of the tilt angle; at the same time, both have the smallest VH:VL interface.", + "section": "DISCUSS", + "ner": [ + [ + 49, + 60, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 65, + 76, + "H3-23:L3-20", + "complex_assembly" + ], + [ + 107, + 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(Fig.\u00a01A) and CDR L1 (Fig.\u00a03C), especially with the tandem glycines and multiple serines present, respectively.", + "section": "DISCUSS", + "ner": [ + [ + 64, + 68, + "CDRs", + "structure_element" + ], + [ + 84, + 87, + "CDR", + "structure_element" + ], + [ + 88, + 90, + "H1", + "structure_element" + ], + [ + 105, + 108, + "CDR", + "structure_element" + ], + [ + 109, + 111, + "L1", + "structure_element" + ], + [ + 150, + 158, + "glycines", + "residue_name" + ], + [ + 172, + 179, + "serines", + "residue_name" + ] + ] + }, + { + "sid": 266, + "sent": "Pairing of different germlines yields antibodies with various degrees of stability.", + "section": "DISCUSS", + "ner": [ + [ + 38, + 48, + "antibodies", + "protein_type" + ] + ] + }, + { + "sid": 267, + "sent": "As indicated by the melting temperatures, germlines H1-69 and H3-23 for HC and germline L1-39 for LC produce more stable Fabs compared to the other germlines in the experimental set.", + "section": "DISCUSS", + "ner": [ + [ + 20, + 40, + "melting temperatures", + "evidence" + ], + [ + 52, + 57, + "H1-69", + "mutant" + ], + [ + 62, + 67, + "H3-23", + "mutant" + ], + [ + 72, + 74, + "HC", + "structure_element" + ], + [ + 88, + 93, + "L1-39", + "mutant" + ], + [ + 98, + 100, + "LC", + "structure_element" + ], + [ + 114, + 120, + "stable", + "protein_state" + ], + [ + 121, + 125, + "Fabs", + "structure_element" + ] + ] + }, + { + "sid": 268, + "sent": "One possible explanation of the clear preference of LC germline L1-39 is that CDR L3 has smaller residues at positions 91 and 94, allowing for more room to accommodate CDR H3.", + "section": "DISCUSS", + "ner": [ + [ + 52, + 54, + "LC", + "structure_element" + ], + [ + 64, + 69, + "L1-39", + "mutant" + ], + [ + 78, + 81, + "CDR", + "structure_element" + ], + [ + 82, + 84, + "L3", + "structure_element" + ], + [ + 119, + 121, + "91", + "residue_number" + ], + [ + 126, + 128, + "94", + "residue_number" + ], + [ + 168, + 171, + "CDR", + "structure_element" + ], + [ + 172, + 174, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 269, + "sent": "Other germlines have bulky residues, Tyr, Arg and Trp, at these positions, whereas L1-39 has Ser and Thr.", + "section": "DISCUSS", + "ner": [ + [ + 37, + 40, + "Tyr", + "residue_name" + ], + [ + 42, + 45, + "Arg", + "residue_name" + ], + [ + 50, + 53, + "Trp", + "residue_name" + ], + [ + 83, + 88, + "L1-39", + "mutant" + ], + [ + 93, + 96, + "Ser", + "residue_name" + ], + [ + 101, + 104, + "Thr", + "residue_name" + ] + ] + }, + { + "sid": 270, + "sent": "Various combinations of germline sequences for VL and VH impose certain constraints on CDR H3, which has to adapt to the environment.", + "section": "DISCUSS", + "ner": [ + [ + 47, + 49, + "VL", + "structure_element" + ], + [ + 54, + 56, + "VH", + "structure_element" + ], + [ + 87, + 90, + "CDR", + "structure_element" + ], + [ + 91, + 93, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 271, + "sent": "A more compact CDR L3 may be beneficial in this situation.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 14, + "compact", + "protein_state" + ], + [ + 15, + 18, + "CDR", + "structure_element" + ], + [ + 19, + 21, + "L3", + "structure_element" + ] + ] + }, + { + "sid": 272, + "sent": "At the other end of the stability range is LC germline L3-20, which yields antibodies with the lowest Tms.", + "section": "DISCUSS", + "ner": [ + [ + 43, + 45, + "LC", + "structure_element" + ], + [ + 55, + 60, + "L3-20", + "mutant" + ], + [ + 75, + 85, + "antibodies", + "protein_type" + ], + [ + 102, + 105, + "Tms", + "evidence" + ] + ] + }, + { + "sid": 273, + "sent": "While pairings with H3-53 and H5-51 may be safely called a mismatch, those with H1-69 and H3-23 have Tms about 5-6\u00b0 higher.", + "section": "DISCUSS", + "ner": [ + [ + 20, + 25, + "H3-53", + "mutant" + ], + [ + 30, + 35, + "H5-51", + "mutant" + ], + [ + 80, + 85, + "H1-69", + "mutant" + ], + [ + 90, + 95, + "H3-23", + "mutant" + ], + [ + 101, + 104, + "Tms", + "evidence" + ] + ] + }, + { + "sid": 274, + "sent": "Curiously, the 2 Fabs, H1-69:L3-20 and H3-23:L3-20, deviate markedly in their tilt angles from the rest of the panel.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 21, + "Fabs", + "structure_element" + ], + [ + 23, + 34, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 39, + 50, + "H3-23:L3-20", + "complex_assembly" + ], + [ + 78, + 89, + "tilt angles", + "evidence" + ] + ] + }, + { + "sid": 275, + "sent": "It is possible that by adopting extreme tilt angles the structure modulates CDR H3 and its environment, which apparently cannot be achieved solely by conformational rearrangement of the CDR.", + "section": "DISCUSS", + "ner": [ + [ + 40, + 51, + "tilt angles", + "evidence" + ], + [ + 56, + 65, + "structure", + "evidence" + ], + [ + 76, + 79, + "CDR", + "structure_element" + ], + [ + 80, + 82, + "H3", + "structure_element" + ], + [ + 186, + 189, + "CDR", + "structure_element" + ] + ] + }, + { + "sid": 276, + "sent": "Note that most of the VH:VL interface residues are invariant; therefore, significant change of the tilt angle must come with a penalty in free energy.", + "section": "DISCUSS", + "ner": [ + [ + 22, + 37, + "VH:VL interface", + "site" + ] + ] + }, + { + "sid": 277, + "sent": "Yet, for the 2 antibodies, the total gain in stability merits the domain repacking.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 25, + "antibodies", + "protein_type" + ] + ] + }, + { + "sid": 278, + "sent": "Overall, the stability of the Fab, as measured by Tm, is a result of the mutual adjustment of the HC and LC variable domains and adjustment of CDR H3 to the VH:VL cleft.", + "section": "DISCUSS", + "ner": [ + [ + 30, + 33, + "Fab", + "structure_element" + ], + [ + 50, + 52, + "Tm", + "evidence" + ], + [ + 98, + 100, + "HC", + "structure_element" + ], + [ + 105, + 107, + "LC", + "structure_element" + ], + [ + 108, + 124, + "variable domains", + "structure_element" + ], + [ + 143, + 146, + "CDR", + "structure_element" + ], + [ + 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"structure_element" + ], + [ + 136, + 138, + "H3", + "structure_element" + ], + [ + 173, + 181, + "antibody", + "protein_type" + ], + [ + 182, + 191, + "structure", + "evidence" + ], + [ + 276, + 278, + "L1", + "structure_element" + ], + [ + 280, + 282, + "L2", + "structure_element" + ], + [ + 284, + 286, + "L3", + "structure_element" + ], + [ + 288, + 290, + "H1", + "structure_element" + ], + [ + 295, + 297, + "H2", + "structure_element" + ], + [ + 298, + 302, + "CDRs", + "structure_element" + ] + ] + }, + { + "sid": 281, + "sent": "Comparison of the CDR H3s reveals a large set of variants with conformations similar to the parent, while a second set has significant conformational variability, indicating that both the sequence and the structural context define the CDR H3 conformation.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 21, + "CDR", + "structure_element" + ], + [ + 22, + 25, + "H3s", + "structure_element" + ], + [ + 235, + 238, + "CDR", + "structure_element" + ], + [ + 239, + 241, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 282, + "sent": "Quite unexpectedly, 2 of the variants, H1-69:L3-20 and H3-53:L4-1, have the \u2018extended\u2019 stem region differing from the other 14 that have a \u2018kinked\u2019 stem region.", + "section": "DISCUSS", + "ner": [ + [ + 39, + 50, + "H1-69:L3-20", + "complex_assembly" + ], + [ + 55, + 65, + "H3-53:L4-1", + "complex_assembly" + ], + [ + 77, + 85, + "extended", + "protein_state" + ], + [ + 87, + 98, + "stem region", + "structure_element" + ], + [ + 140, + 146, + "kinked", + "protein_state" + ], + [ + 148, + 159, + "stem region", + "structure_element" + ] + ] + }, + { + "sid": 283, + "sent": "These data reveal the difficulty of modeling CDR H3 accurately, as shown again in Antibody Modeling Assessment II.", + "section": "DISCUSS", + "ner": [ + [ + 45, + 48, + "CDR", + "structure_element" + ], + [ + 49, + 51, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 284, + "sent": "Furthermore, antibody CDRs, H3 in particular, may go through conformational changes upon binding their targets, making structural prediction for docking purposes an even more difficult task.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 21, + "antibody", + "protein_type" + ], + [ + 22, + 26, + "CDRs", + "structure_element" + ], + [ + 28, + 30, + "H3", + "structure_element" + ] + ] + }, + { + "sid": 285, + "sent": "Fortunately, for most applications of antibody modeling, such as engineering affinity and biophysical properties, an accurate CDR H3 structure is not always necessary.", + "section": "DISCUSS", + "ner": [ + [ + 38, + 46, + "antibody", + "protein_type" + ], + [ + 126, + 129, + "CDR", + "structure_element" + ], + [ + 130, + 132, + "H3", + "structure_element" + ], + [ + 133, + 142, + "structure", + "evidence" + ] + ] + }, + { + "sid": 286, + "sent": "For those applications where accurate CDR structures are essential, such as docking, the results in this work demonstrate the importance of experimental structures.", + "section": "DISCUSS", + "ner": [ + [ + 38, + 41, + "CDR", + "structure_element" + ], + [ + 42, + 52, + "structures", + "evidence" + ], + [ + 153, + 163, + "structures", + "evidence" + ] + ] + }, + { + "sid": 287, + "sent": "With the recent advances in expression and crystallization methods, Fab structures can be obtained rapidly.", + "section": "DISCUSS", + "ner": [ + [ + 28, + 66, + "expression and crystallization methods", + "experimental_method" + ], + [ + 68, + 71, + "Fab", + "structure_element" + ], + [ + 72, + 82, + "structures", + "evidence" + ] + ] + }, + { + "sid": 288, + "sent": "The set of 16 germline Fab structures offers a unique dataset to facilitate software development for antibody modeling.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 26, + "Fab", + "structure_element" + ], + [ + 27, + 37, + "structures", + "evidence" + ], + [ + 101, + 109, + "antibody", + "protein_type" + ] + ] + }, + { + "sid": 289, + "sent": "The results essentially support the underlying idea of canonical structures, indicating that most CDRs with germline sequences tend to adopt predefined conformations.", + "section": "DISCUSS", + "ner": [ + [ + 65, + 75, + "structures", + "evidence" + ], + [ + 98, + 102, + "CDRs", + "structure_element" + ] + ] + }, + { + "sid": 290, + "sent": "From this point of view, a novel approach to design combinatorial antibody libraries would be to cover the range of CDR conformations that may not necessarily coincide with the germline usage in the human repertoire.", + "section": "DISCUSS", + "ner": [ + [ + 66, + 74, + "antibody", + "protein_type" + ], + [ + 116, + 119, + "CDR", + "structure_element" + ], + [ + 199, + 204, + "human", + "species" + ] + ] + }, + { + "sid": 291, + "sent": "This would insure more structural diversity, leading to a more diverse panel of antibodies that would bind to a broad spectrum of targets.", + "section": "DISCUSS", + "ner": [ + [ + 80, + 90, + "antibodies", + "protein_type" + ] + ] + } + ] + }, + "PMC4773095": { + "annotations": [ + { + "sid": 0, + "sent": "Structure of the Response Regulator NsrR from Streptococcus agalactiae, Which Is Involved in Lantibiotic Resistance", + "section": "TITLE", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 17, + 35, + "Response Regulator", + "protein_type" + ], + [ + 36, + 40, + "NsrR", + "protein" + ], + [ + 46, + 70, + "Streptococcus agalactiae", + "species" + ], + [ + 93, + 104, + "Lantibiotic", + "chemical" + ] + ] + }, + { + "sid": 1, + "sent": "Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 12, + "Lantibiotics", + "chemical" + ], + [ + 17, + 39, + "antimicrobial peptides", + "chemical" + ], + [ + 52, + 74, + "Gram-positive bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 2, + "sent": "Interestingly, several clinically relevant and human pathogenic strains are inherently resistant towards lantibiotics.", + "section": "ABSTRACT", + "ner": [ + [ + 47, + 52, + "human", + "species" + ], + [ + 105, + 117, + "lantibiotics", + "chemical" + ] + ] + }, + { + "sid": 3, + "sent": "The expression of the genes responsible for lantibiotic resistance is regulated by a specific two-component system consisting of a histidine kinase and a response regulator.", + "section": "ABSTRACT", + "ner": [ + [ + 44, + 55, + "lantibiotic", + "chemical" + ], + [ + 94, + 114, + "two-component system", + "complex_assembly" + ], + [ + 131, + 147, + "histidine kinase", + "protein_type" + ], + [ + 154, + 172, + "response regulator", + "protein_type" + ] + ] + }, + { + "sid": 4, + "sent": "Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain.", + "section": "ABSTRACT", + "ner": [ + [ + 22, + 40, + "response regulator", + "protein_type" + ], + [ + 53, + 64, + "lantibiotic", + "chemical" + ], + [ + 77, + 81, + "NsrR", + "protein" + ], + [ + 87, + 111, + "Streptococcus agalactiae", + "species" + ], + [ + 132, + 150, + "crystal structures", + "evidence" + ], + [ + 169, + 184, + "receiver domain", + "structure_element" + ], + [ + 200, + 227, + "DNA-binding effector domain", + "structure_element" + ] + ] + }, + { + "sid": 5, + "sent": "The C-terminal domain exhibits a fold that classifies NsrR as a member of the OmpR/PhoB subfamily of regulators.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 21, + "C-terminal domain", + "structure_element" + ], + [ + 54, + 58, + "NsrR", + "protein" + ], + [ + 78, + 97, + "OmpR/PhoB subfamily", + "protein_type" + ] + ] + }, + { + "sid": 6, + "sent": "Amino acids involved in phosphorylation, dimerization, and DNA-binding were identified and demonstrated to be conserved in lantibiotic resistance regulators.", + "section": "ABSTRACT", + "ner": [ + [ + 24, + 39, + "phosphorylation", + "ptm" + ], + [ + 59, + 62, + "DNA", + "chemical" + ], + [ + 110, + 119, + "conserved", + "protein_state" + ], + [ + 123, + 156, + "lantibiotic resistance regulators", + "protein_type" + ] + ] + }, + { + "sid": 7, + "sent": "Finally, a model of the full-length NsrR in the active and inactive state provides insights into protein dimerization and DNA-binding.", + "section": "ABSTRACT", + "ner": [ + [ + 24, + 35, + "full-length", + "protein_state" + ], + [ + 36, + 40, + "NsrR", + "protein" + ], + [ + 48, + 54, + "active", + "protein_state" + ], + [ + 59, + 67, + "inactive", + "protein_state" + ], + [ + 122, + 125, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 8, + "sent": "This has led to the search for novel antibiotics that can be used as pharmaceuticals against human pathogenic bacteria.", + "section": "INTRO", + "ner": [ + [ + 93, + 98, + "human", + "species" + ], + [ + 110, + 118, + "bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 9, + "sent": "One of the potential antibiotic alternatives are lantibiotics.", + "section": "INTRO", + "ner": [ + [ + 49, + 61, + "lantibiotics", + "chemical" + ] + ] + }, + { + "sid": 10, + "sent": "Lantibiotics are small antimicrobial peptides (30\u201350 amino acids in length), which are produced by several Gram-positive bacterial strains.", + "section": "INTRO", + "ner": [ + [ + 0, + 12, + "Lantibiotics", + "chemical" + ], + [ + 23, + 45, + "antimicrobial peptides", + "chemical" + ], + [ + 107, + 130, + "Gram-positive bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 11, + "sent": "They are post-translationally modified and contain specific lanthionine/methyl-lanthionine rings, which are crucial for their high antimicrobial activity.", + "section": "INTRO", + "ner": [ + [ + 60, + 71, + "lanthionine", + "chemical" + ], + [ + 72, + 90, + "methyl-lanthionine", + "chemical" + ] + ] + }, + { + "sid": 12, + "sent": "Lantibiotics are for example highly effective against various Gram-positive, human pathogenic bacteria including Streptococcus pneumoniae and several methicillin-resistant Staphylococcus aureus (MRSA) strains.", + "section": "INTRO", + "ner": [ + [ + 0, + 12, + "Lantibiotics", + "chemical" + ], + [ + 62, + 75, + "Gram-positive", + "taxonomy_domain" + ], + [ + 77, + 82, + "human", + "species" + ], + [ + 94, + 102, + "bacteria", + "taxonomy_domain" + ], + [ + 113, + 137, + "Streptococcus pneumoniae", + "species" + ], + [ + 150, + 193, + "methicillin-resistant Staphylococcus aureus", + "species" + ], + [ + 195, + 199, + "MRSA", + "species" + ] + ] + }, + { + "sid": 13, + "sent": "The high potency of lantibiotics for medical usage has already been noticed, and several lantibiotics are already included in clinical trials.", + "section": "INTRO", + "ner": [ + [ + 20, + 32, + "lantibiotics", + "chemical" + ], + [ + 89, + 101, + "lantibiotics", + "chemical" + ] + ] + }, + { + "sid": 14, + "sent": "Nisin is the most prominent member of the lantibiotic family and is able to inhibit cell growth, penetrates the membranes of various Gram-positive bacteria, and is characterized by five specific (methyl-)lanthionine rings, which are crucial for stability and activity in the nanomolar range.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "Nisin", + "chemical" + ], + [ + 42, + 53, + "lantibiotic", + "chemical" + ], + [ + 133, + 155, + "Gram-positive bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 15, + "sent": "Thus, the lantibiotic producer strains have an inbuilt self-protection mechanism (immunity) to prevent cell death caused due to the action of its cognate lantibiotic.", + "section": "INTRO", + "ner": [ + [ + 10, + 21, + "lantibiotic", + "chemical" + ], + [ + 154, + 165, + "lantibiotic", + "chemical" + ] + ] + }, + { + "sid": 16, + "sent": "This immunity system consists of a membrane\u2013associated lipoprotein (usually referred to as LanI) and/or an ABC transporter (termed as LanFEG and comprising three subunits).", + "section": "INTRO", + "ner": [ + [ + 35, + 66, + "membrane\u2013associated lipoprotein", + "protein_type" + ], + [ + 91, + 95, + "LanI", + "protein_type" + ], + [ + 107, + 122, + "ABC transporter", + "protein_type" + ], + [ + 134, + 140, + "LanFEG", + "protein_type" + ] + ] + }, + { + "sid": 17, + "sent": "Although some lantibiotics such as Pep5, epicidin, epilancin, and lactocin S only require LanI for immunity, other lantibiotics with a dual mode of action involving pore formation and lipid II binding such as nisin, subtilin, epidermin, gallidermin, and lacticin 3147 require additionally the presence of LanFEG.", + "section": "INTRO", + "ner": [ + [ + 14, + 26, + "lantibiotics", + "chemical" + ], + [ + 35, + 39, + "Pep5", + "chemical" + ], + [ + 41, + 49, + "epicidin", + "chemical" + ], + [ + 51, + 60, + "epilancin", + "chemical" + ], + [ + 66, + 76, + "lactocin S", + "chemical" + ], + [ + 90, + 94, + "LanI", + "protein_type" + ], + [ + 115, + 127, + "lantibiotics", + "chemical" + ], + [ + 209, + 214, + "nisin", + "chemical" + ], + [ + 216, + 224, + "subtilin", + "chemical" + ], + [ + 226, + 235, + "epidermin", + "chemical" + ], + [ + 237, + 248, + "gallidermin", + "chemical" + ], + [ + 254, + 267, + "lacticin 3147", + "chemical" + ], + [ + 305, + 311, + "LanFEG", + "protein_type" + ] + ] + }, + { + "sid": 18, + "sent": "Examples for LanFEG are NisI and NisFEG of the nisin system, SpaI and SpaFEG conferring immunity towards subtilin, and PepI constituting the immunity system of Pep5 producing strains.", + "section": "INTRO", + "ner": [ + [ + 13, + 19, + "LanFEG", + "protein_type" + ], + [ + 24, + 28, + "NisI", + "protein" + ], + [ + 33, + 39, + "NisFEG", + "protein" + ], + [ + 47, + 52, + "nisin", + "chemical" + ], + [ + 61, + 65, + "SpaI", + "protein" + ], + [ + 70, + 76, + "SpaFEG", + "protein" + ], + [ + 105, + 113, + "subtilin", + "chemical" + ], + [ + 119, + 123, + "PepI", + "protein" + ], + [ + 160, + 164, + "Pep5", + "chemical" + ] + ] + }, + { + "sid": 19, + "sent": "Structural data are reported for the immunity proteins NisI from Lactococcus lactis, SpaI from Bacillus subtilis and MlbQ from the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024.", + "section": "INTRO", + "ner": [ + [ + 0, + 15, + "Structural data", + "evidence" + ], + [ + 37, + 54, + "immunity proteins", + "protein_type" + ], + [ + 55, + 59, + "NisI", + "protein" + ], + [ + 65, + 83, + "Lactococcus lactis", + "species" + ], + [ + 85, + 89, + "SpaI", + "protein" + ], + [ + 95, + 112, + "Bacillus subtilis", + "species" + ], + [ + 117, + 121, + "MlbQ", + "protein" + ], + [ + 131, + 142, + "lantibiotic", + "chemical" + ], + [ + 143, + 150, + "NAI-107", + "chemical" + ], + [ + 167, + 193, + "Microbispora ATCC PTA-5024", + "species" + ] + ] + }, + { + "sid": 20, + "sent": "Recently, gene clusters were identified in certain clinically relevant human pathogenic strains such as Streptococcus agalactiae, S. aureus, and others that confer inherent resistance against specific lantibiotics such as nisin and resemble the genetic architecture of the lantibiotic immunity genes found in the producing strains.", + "section": "INTRO", + "ner": [ + [ + 71, + 76, + "human", + "species" + ], + [ + 104, + 128, + "Streptococcus agalactiae", + "species" + ], + [ + 130, + 139, + "S. aureus", + "species" + ], + [ + 201, + 213, + "lantibiotics", + "chemical" + ], + [ + 222, + 227, + "nisin", + "chemical" + ] + ] + }, + { + "sid": 21, + "sent": "Within these resistance operons, genes encoding for a membrane-associated protease and an ABC transporter were identified.", + "section": "INTRO", + "ner": [ + [ + 54, + 82, + "membrane-associated protease", + "protein_type" + ], + [ + 90, + 105, + "ABC transporter", + "protein_type" + ] + ] + }, + { + "sid": 22, + "sent": "Expression of these proteins provides resistance against lantibiotics.", + "section": "INTRO", + "ner": [ + [ + 57, + 69, + "lantibiotics", + "chemical" + ] + ] + }, + { + "sid": 23, + "sent": "Recently, the structure of SaNSR from S. agalactiae was solved which provides resistance against nisin by a protease activity.", + "section": "INTRO", + "ner": [ + [ + 14, + 23, + "structure", + "evidence" + ], + [ + 27, + 32, + "SaNSR", + "protein" + ], + [ + 38, + 51, + "S. agalactiae", + "species" + ], + [ + 97, + 102, + "nisin", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "Furthermore, the upregulation of these genes is mediated by a specific two-component system (TCS) similar to the one found in lantibiotic producing strains, consisting of a sensor histidine kinase (HK) and a response regulator (RR), apparently mediate the expression of the resistance proteins: HK senses the external lantibiotic and, upon receiving the stimuli, auto-phosphorylates at a conserved histidine residue within the cytosol; this high-energetic phosphoryl group is then transferred to the associated RR inducing a conformational change there, which activates the RR to evoke the cellular response.", + "section": "INTRO", + "ner": [ + [ + 71, + 91, + "two-component system", + "complex_assembly" + ], + [ + 93, + 96, + "TCS", + "complex_assembly" + ], + [ + 126, + 137, + "lantibiotic", + "chemical" + ], + [ + 180, + 196, + "histidine kinase", + "protein_type" + ], + [ + 198, + 200, + "HK", + "protein_type" + ], + [ + 208, + 226, + "response regulator", + "protein_type" + ], + [ + 228, + 230, + "RR", + "protein_type" + ], + [ + 295, + 297, + "HK", + "protein_type" + ], + [ + 318, + 329, + "lantibiotic", + "chemical" + ], + [ + 363, + 382, + "auto-phosphorylates", + "ptm" + ], + [ + 388, + 397, + "conserved", + "protein_state" + ], + [ + 398, + 407, + "histidine", + "residue_name" + ], + [ + 511, + 513, + "RR", + "protein_type" + ], + [ + 574, + 576, + "RR", + "protein_type" + ] + ] + }, + { + "sid": 25, + "sent": "Bacteria have the ability to sense and survive various environmental stimuli through adaptive responses, which are regulated by TCSs.", + "section": "INTRO", + "ner": [ + [ + 0, + 8, + "Bacteria", + "taxonomy_domain" + ], + [ + 128, + 132, + "TCSs", + "complex_assembly" + ] + ] + }, + { + "sid": 26, + "sent": "The absence of TCSs within mammals makes them unique targets for novel antimicrobial drugs.", + "section": "INTRO", + "ner": [ + [ + 4, + 14, + "absence of", + "protein_state" + ], + [ + 15, + 19, + "TCSs", + "complex_assembly" + ], + [ + 27, + 34, + "mammals", + "taxonomy_domain" + ] + ] + }, + { + "sid": 27, + "sent": "The expression of the lantibiotic-resistance genes via TCS is generally regulated by microorganism-specific lantibiotics, which act via external stimuli.", + "section": "INTRO", + "ner": [ + [ + 22, + 33, + "lantibiotic", + "chemical" + ], + [ + 55, + 58, + "TCS", + "complex_assembly" + ], + [ + 108, + 120, + "lantibiotics", + "chemical" + ] + ] + }, + { + "sid": 28, + "sent": "Some examples of TCS are: BraRS in S. aureus which is induced by bacitracin, nisin and nukacin-ISK-1 resistance, BceRS in Bacillus spp. that is induced by actagardine and mersacidin resistance, LcrRS in Streptococcus mutans induced by nukacin-ISK-1 and lacticin 481 and LisRK of Listeria monocytogenes induced by nisin resistance.", + "section": "INTRO", + "ner": [ + [ + 17, + 20, + "TCS", + "complex_assembly" + ], + [ + 26, + 31, + "BraRS", + "protein" + ], + [ + 35, + 44, + "S. aureus", + "species" + ], + [ + 65, + 75, + "bacitracin", + "chemical" + ], + [ + 77, + 82, + "nisin", + "chemical" + ], + [ + 87, + 100, + "nukacin-ISK-1", + "chemical" + ], + [ + 113, + 118, + "BceRS", + "protein" + ], + [ + 122, + 134, + "Bacillus spp", + "taxonomy_domain" + ], + [ + 155, + 166, + "actagardine", + "chemical" + ], + [ + 171, + 181, + "mersacidin", + "chemical" + ], + [ + 194, + 199, + "LcrRS", + "protein" + ], + [ + 203, + 223, + "Streptococcus mutans", + "species" + ], + [ + 235, + 248, + "nukacin-ISK-1", + "chemical" + ], + [ + 253, + 265, + "lacticin 481", + "chemical" + ], + [ + 270, + 275, + "LisRK", + "protein" + ], + [ + 279, + 301, + "Listeria monocytogenes", + "species" + ], + [ + 313, + 318, + "nisin", + "chemical" + ] + ] + }, + { + "sid": 29, + "sent": "Furthermore, multiple lantibiotics can induce the TCS CprRK from Clostridium difficile, leading to the expression of the genes localized on the cpr operon, resulting in resistance against several lantibiotics of which nisin, gallidermin, subtilin, and mutacin 1140 are some examples.", + "section": "INTRO", + "ner": [ + [ + 22, + 34, + "lantibiotics", + "chemical" + ], + [ + 50, + 53, + "TCS", + "complex_assembly" + ], + [ + 54, + 59, + "CprRK", + "protein" + ], + [ + 65, + 86, + "Clostridium difficile", + "species" + ], + [ + 144, + 147, + "cpr", + "gene" + ], + [ + 196, + 208, + "lantibiotics", + "chemical" + ], + [ + 218, + 223, + "nisin", + "chemical" + ], + [ + 225, + 236, + "gallidermin", + "chemical" + ], + [ + 238, + 246, + "subtilin", + "chemical" + ], + [ + 252, + 264, + "mutacin 1140", + "chemical" + ] + ] + }, + { + "sid": 30, + "sent": "Interestingly, the histidine kinase contains two-transmembrane helices but lacks an extracellular sensory domain, and are therefore known as \u2018intramembrane-sensing\u2019 histidine kinases.", + "section": "INTRO", + "ner": [ + [ + 19, + 35, + "histidine kinase", + "protein_type" + ], + [ + 45, + 70, + "two-transmembrane helices", + "structure_element" + ], + [ + 84, + 112, + "extracellular sensory domain", + "structure_element" + ], + [ + 141, + 182, + "\u2018intramembrane-sensing\u2019 histidine kinases", + "protein_type" + ] + ] + }, + { + "sid": 31, + "sent": "It has been suggested that in addition to conferring general resistance against lantibiotics, the BceAB-type transporters assist in signalling as via the presence of a large extracellular domain within the transmembrane segment indicated by experimental evidence from various systems.", + "section": "INTRO", + "ner": [ + [ + 80, + 92, + "lantibiotics", + "chemical" + ], + [ + 98, + 121, + "BceAB-type transporters", + "protein_type" + ], + [ + 174, + 194, + "extracellular domain", + "structure_element" + ], + [ + 206, + 227, + "transmembrane segment", + "structure_element" + ] + ] + }, + { + "sid": 32, + "sent": "The recently discovered nsr gene cluster of the human pathogen S. agalactiae encodes for the resistance protein NSR and the ABC transporter NsrFP, both conferring resistance against nisin.", + "section": "INTRO", + "ner": [ + [ + 24, + 27, + "nsr", + "gene" + ], + [ + 48, + 53, + "human", + "species" + ], + [ + 63, + 76, + "S. agalactiae", + "species" + ], + [ + 93, + 111, + "resistance protein", + "protein_type" + ], + [ + 112, + 115, + "NSR", + "protein" + ], + [ + 124, + 139, + "ABC transporter", + "protein_type" + ], + [ + 140, + 145, + "NsrFP", + "protein" + ], + [ + 182, + 187, + "nisin", + "chemical" + ] + ] + }, + { + "sid": 33, + "sent": "Homologous operons have been identified in various human pathogenic strains such as Staphylococcus epidermis and Streptococcus ictaluri based on the high sequence identity of NSR and NsrFP.", + "section": "INTRO", + "ner": [ + [ + 51, + 56, + "human", + "species" + ], + [ + 84, + 108, + "Staphylococcus epidermis", + "species" + ], + [ + 113, + 135, + "Streptococcus ictaluri", + "species" + ], + [ + 175, + 178, + "NSR", + "protein" + ], + [ + 183, + 188, + "NsrFP", + "protein" + ] + ] + }, + { + "sid": 34, + "sent": "In this gene cluster, the TCS NsrRK is responsible for the expression of the nsr and nsrFP genes.", + "section": "INTRO", + "ner": [ + [ + 26, + 29, + "TCS", + "complex_assembly" + ], + [ + 30, + 35, + "NsrRK", + "protein" + ], + [ + 77, + 80, + "nsr", + "gene" + ], + [ + 85, + 90, + "nsrFP", + "gene" + ] + ] + }, + { + "sid": 35, + "sent": "The similarity of the TCS within all the described nisin resistance operons suggests an expression specifically induced by nisin.", + "section": "INTRO", + "ner": [ + [ + 22, + 25, + "TCS", + "complex_assembly" + ], + [ + 51, + 56, + "nisin", + "chemical" + ], + [ + 123, + 128, + "nisin", + "chemical" + ] + ] + }, + { + "sid": 36, + "sent": "Thus, NsrRK might be a useful target to combat inherently pathogenic lantibiotic-resistant strains.", + "section": "INTRO", + "ner": [ + [ + 6, + 11, + "NsrRK", + "protein" + ], + [ + 69, + 80, + "lantibiotic", + "chemical" + ] + ] + }, + { + "sid": 37, + "sent": "Generally, RRs consist of two distinct structural domains, a receiver domain (RD) and an effector domain (ED), that are separated from each other by a flexible linker.", + "section": "INTRO", + "ner": [ + [ + 11, + 14, + "RRs", + "protein_type" + ], + [ + 61, + 76, + "receiver domain", + "structure_element" + ], + [ + 78, + 80, + "RD", + "structure_element" + ], + [ + 89, + 104, + "effector domain", + "structure_element" + ], + [ + 106, + 108, + "ED", + "structure_element" + ], + [ + 151, + 159, + "flexible", + "protein_state" + ], + [ + 160, + 166, + "linker", + "structure_element" + ] + ] + }, + { + "sid": 38, + "sent": "RDs contain a highly conserved aspartate residue, which acts as a phosphoryl acceptor that becomes phosphorylated by the kinase domain of the histidine kinase upon reception of an external signal.", + "section": "INTRO", + "ner": [ + [ + 0, + 3, + "RDs", + "structure_element" + ], + [ + 14, + 30, + "highly conserved", + "protein_state" + ], + [ + 31, + 40, + "aspartate", + "residue_name" + ], + [ + 99, + 113, + "phosphorylated", + "protein_state" + ], + [ + 121, + 134, + "kinase domain", + "structure_element" + ], + [ + 142, + 158, + "histidine kinase", + "protein_type" + ] + ] + }, + { + "sid": 39, + "sent": "The ED is thereby activated and binds to the designated promoters, thus initiating transcription of the target genes.", + "section": "INTRO", + "ner": [ + [ + 4, + 6, + "ED", + "structure_element" + ] + ] + }, + { + "sid": 40, + "sent": "The RRs are classified into different subfamilies depending on the three-dimensional structure of their EDs.", + "section": "INTRO", + "ner": [ + [ + 4, + 7, + "RRs", + "protein_type" + ], + [ + 104, + 107, + "EDs", + "structure_element" + ] + ] + }, + { + "sid": 41, + "sent": "The OmpR/PhoB subfamily is the largest subgroup of RRs and comprises approximately 40% of all response regulators in bacteria.", + "section": "INTRO", + "ner": [ + [ + 4, + 23, + "OmpR/PhoB subfamily", + "protein_type" + ], + [ + 51, + 54, + "RRs", + "protein_type" + ], + [ + 94, + 113, + "response regulators", + "protein_type" + ], + [ + 117, + 125, + "bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 42, + "sent": "All their members are characterized by a winged helix-turn-helix (wHTH) motif.", + "section": "INTRO", + "ner": [ + [ + 41, + 64, + "winged helix-turn-helix", + "structure_element" + ], + [ + 66, + 70, + "wHTH", + "structure_element" + ] + ] + }, + { + "sid": 43, + "sent": "Although numerous structures of the single domains are known, only a few structures of full-length OmpR/PhoB-type RRs have been determined: RegX3 (PDB code: 2OQR), MtrA (PDB code: 2GWR), PrrA (PDB code: 1YS6) and PhoP (PDB code: 3R0J) from Mycobacterium tuberculosis; DrrB (PDB code: 1P2F) and DrrD (PDB code: 1KGS) from Thermotoga maritima; and KdpE from Escherichia coli (PDB code: 4KNY).", + "section": "INTRO", + "ner": [ + [ + 18, + 28, + "structures", + "evidence" + ], + [ + 73, + 83, + "structures", + "evidence" + ], + [ + 87, + 98, + "full-length", + "protein_state" + ], + [ + 99, + 117, + "OmpR/PhoB-type RRs", + "protein_type" + ], + [ + 140, + 145, + "RegX3", + "protein" + ], + [ + 164, + 168, + "MtrA", + "protein" + ], + [ + 187, + 191, + "PrrA", + "protein" + ], + [ + 213, + 217, + "PhoP", + "protein" + ], + [ + 240, + 266, + "Mycobacterium tuberculosis", + "species" + ], + [ + 268, + 272, + "DrrB", + "protein" + ], + [ + 294, + 298, + "DrrD", + "protein" + ], + [ + 321, + 340, + "Thermotoga maritima", + "species" + ], + [ + 346, + 350, + "KdpE", + "protein" + ], + [ + 356, + 372, + "Escherichia coli", + "species" + ] + ] + }, + { + "sid": 44, + "sent": "The various structures of RRs reveal that in addition to being in either \u201cinactive\u201d or \u201cactive\u201d state, the RRs can also exist in two distinct conformations: \u201copen\u201d and \u201cclosed\u201d.", + "section": "INTRO", + "ner": [ + [ + 12, + 22, + "structures", + "evidence" + ], + [ + 26, + 29, + "RRs", + "protein_type" + ], + [ + 74, + 82, + "inactive", + "protein_state" + ], + [ + 88, + 94, + "active", + "protein_state" + ], + [ + 107, + 110, + "RRs", + "protein_type" + ], + [ + 158, + 162, + "open", + "protein_state" + ], + [ + 169, + 175, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 45, + "sent": "MtrA and PrrA exhibit a very compact, closed structure with the DNA-binding sequence, called recognition helix, of the ED being inaccessible to DNA.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "MtrA", + "protein" + ], + [ + 9, + 13, + "PrrA", + "protein" + ], + [ + 24, + 36, + "very compact", + "protein_state" + ], + [ + 38, + 44, + "closed", + "protein_state" + ], + [ + 45, + 54, + "structure", + "evidence" + ], + [ + 64, + 84, + "DNA-binding sequence", + "structure_element" + ], + [ + 93, + 110, + "recognition helix", + "structure_element" + ], + [ + 119, + 121, + "ED", + "structure_element" + ], + [ + 144, + 147, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 46, + "sent": "The structures of DrrD and DrrB exist in an open conformation, here the recognition helix is fully exposed, suggesting that RRs are flexible in solution and can adopt multiple conformations.", + "section": "INTRO", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 18, + 22, + "DrrD", + "protein" + ], + [ + 27, + 31, + "DrrB", + "protein" + ], + [ + 44, + 48, + "open", + "protein_state" + ], + [ + 72, + 89, + "recognition helix", + "structure_element" + ], + [ + 93, + 106, + "fully exposed", + "protein_state" + ], + [ + 124, + 127, + "RRs", + "protein_type" + ], + [ + 132, + 140, + "flexible", + "protein_state" + ] + ] + }, + { + "sid": 47, + "sent": "Here, we describe the crystal structures of the N-terminal RD and the C-terminal ED of the lantibiotic resistance-associated RR NsrR from S. agalactiae.", + "section": "INTRO", + "ner": [ + [ + 22, + 40, + "crystal structures", + "evidence" + ], + [ + 59, + 61, + "RD", + "structure_element" + ], + [ + 81, + 83, + "ED", + "structure_element" + ], + [ + 91, + 127, + "lantibiotic resistance-associated RR", + "protein_type" + ], + [ + 128, + 132, + "NsrR", + "protein" + ], + [ + 138, + 151, + "S. agalactiae", + "species" + ] + ] + }, + { + "sid": 48, + "sent": "NsrR is part of the nisin resistance operon.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "NsrR", + "protein" + ], + [ + 20, + 25, + "nisin", + "chemical" + ] + ] + }, + { + "sid": 49, + "sent": "The expression of the genes of this operon is induced by a TCS consisting of the HK NsrK and the RR NsrR. Based on the crystal structures of both the domains, modeling was employed to shed light on the putative DNA-bound state of full-length NsrR.", + "section": "INTRO", + "ner": [ + [ + 59, + 62, + "TCS", + "complex_assembly" + ], + [ + 81, + 83, + "HK", + "protein_type" + ], + [ + 84, + 88, + "NsrK", + "protein" + ], + [ + 97, + 99, + "RR", + "protein_type" + ], + [ + 100, + 104, + "NsrR", + "protein" + ], + [ + 119, + 137, + "crystal structures", + "evidence" + ], + [ + 211, + 220, + "DNA-bound", + "protein_state" + ], + [ + 230, + 241, + "full-length", + "protein_state" + ], + [ + 242, + 246, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 50, + "sent": "NsrR was expressed and purified as described, resulting in a homogenous protein as observed by size exclusion chromatography (Fig 1A), with a yield of 2 mg per liter of cell culture.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "NsrR", + "protein" + ], + [ + 9, + 31, + "expressed and purified", + "experimental_method" + ], + [ + 95, + 124, + "size exclusion chromatography", + "experimental_method" + ] + ] + }, + { + "sid": 51, + "sent": "By calibrating the column with proteins of known molecular weight the NsrR full length protein elutes as a dimer.", + "section": "RESULTS", + "ner": [ + [ + 70, + 74, + "NsrR", + "protein" + ], + [ + 75, + 86, + "full length", + "protein_state" + ], + [ + 107, + 112, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 52, + "sent": "The purified NsrR protein has a theoretical molecular mass of 27.7 kDa and was >98% pure as assessed by SDS-PAGE (Fig 1B, indicated by *).", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "NsrR", + "protein" + ], + [ + 44, + 58, + "molecular mass", + "evidence" + ], + [ + 104, + 112, + "SDS-PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 53, + "sent": "Surprisingly, over time NsrR degraded into two distinct fragments as visible on SDS-PAGE analysis using the same purified protein sample after one week (Fig 1C, indicated by ** and ***, respectively).", + "section": "RESULTS", + "ner": [ + [ + 24, + 28, + "NsrR", + "protein" + ], + [ + 80, + 88, + "SDS-PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 54, + "sent": "This was also observed by size exclusion chromatography where a peak at an elution time of 18 min appeared (Fig 1A).", + "section": "RESULTS", + "ner": [ + [ + 26, + 55, + "size exclusion chromatography", + "experimental_method" + ] + ] + }, + { + "sid": 55, + "sent": "Both bands were subjected to mass spectrometry analysis.", + "section": "RESULTS", + "ner": [ + [ + 29, + 55, + "mass spectrometry analysis", + "experimental_method" + ] + ] + }, + { + "sid": 56, + "sent": "The analysis revealed that the larger fragment (**) represents the N-terminal receiver domain (residues 1\u2013119; referred to as NsrR-RD) whereas the smaller fragment (***) contained the C-terminal DNA-binding effector domain of NsrR (residues 129\u2013243 including 21 amino acids derived from the expression tag; referred to as NsrR-ED) (Fig 1C).", + "section": "RESULTS", + "ner": [ + [ + 78, + 93, + "receiver domain", + "structure_element" + ], + [ + 104, + 109, + "1\u2013119", + "residue_range" + ], + [ + 126, + 130, + "NsrR", + "protein" + ], + [ + 131, + 133, + "RD", + "structure_element" + ], + [ + 195, + 222, + "DNA-binding effector domain", + "structure_element" + ], + [ + 226, + 230, + "NsrR", + "protein" + ], + [ + 241, + 248, + "129\u2013243", + "residue_range" + ], + [ + 322, + 326, + "NsrR", + "protein" + ], + [ + 327, + 329, + "ED", + "structure_element" + ] + ] + }, + { + "sid": 57, + "sent": "Residues 120\u2013128 form the linker connecting the RD and ED.", + "section": "RESULTS", + "ner": [ + [ + 9, + 16, + "120\u2013128", + "residue_range" + ], + [ + 26, + 32, + "linker", + "structure_element" + ], + [ + 48, + 50, + "RD", + "structure_element" + ], + [ + 55, + 57, + "ED", + "structure_element" + ] + ] + }, + { + "sid": 58, + "sent": "Such a cleavage of the full-length RR into two specific domains is not unusual and has been previously reported for other RRs as well.", + "section": "RESULTS", + "ner": [ + [ + 23, + 34, + "full-length", + "protein_state" + ], + [ + 35, + 37, + "RR", + "protein_type" + ], + [ + 122, + 125, + "RRs", + "protein_type" + ] + ] + }, + { + "sid": 59, + "sent": "Mass spectrometry analysis did not reveal the presence of any specific protease in the purified NsrR sample.", + "section": "RESULTS", + "ner": [ + [ + 0, + 26, + "Mass spectrometry analysis", + "experimental_method" + ], + [ + 96, + 100, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 60, + "sent": "Furthermore, addition of a protease inhibitor, such as PMSF (Phenylmethylsulfonyl fluoride) and AEBSF {4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride}, even at high concentrations, did not inhibit proteolysis (data not shown).", + "section": "RESULTS", + "ner": [ + [ + 55, + 59, + "PMSF", + "chemical" + ], + [ + 61, + 90, + "Phenylmethylsulfonyl fluoride", + "chemical" + ], + [ + 96, + 101, + "AEBSF", + "chemical" + ], + [ + 103, + 158, + "4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride", + "chemical" + ] + ] + }, + { + "sid": 61, + "sent": "Purification of NsrR and SDS PAGE analysis of purified NsrR directly and one week after purification.", + "section": "FIG", + "ner": [ + [ + 0, + 12, + "Purification", + "experimental_method" + ], + [ + 16, + 20, + "NsrR", + "protein" + ], + [ + 25, + 33, + "SDS PAGE", + "experimental_method" + ], + [ + 55, + 59, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 62, + "sent": "(a) Elution profile of size-exclusion chromatography step of NsrR. The y-axis represents the UV absorption of the protein at 280 nm, while the x-axis represents the elution volume.", + "section": "FIG", + "ner": [ + [ + 4, + 19, + "Elution profile", + "evidence" + ], + [ + 23, + 52, + "size-exclusion chromatography", + "experimental_method" + ], + [ + 61, + 65, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 63, + "sent": "The bold line represents the chromatogram of freshly purified NsrR while the dashed line shows the chromatogram of the same NsrR protein after one week.", + "section": "FIG", + "ner": [ + [ + 29, + 41, + "chromatogram", + "evidence" + ], + [ + 62, + 66, + "NsrR", + "protein" + ], + [ + 99, + 111, + "chromatogram", + "evidence" + ], + [ + 124, + 128, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 64, + "sent": "(b) Freshly purified NsrR protein, and (c) NsrR protein after one week.", + "section": "FIG", + "ner": [ + [ + 21, + 25, + "NsrR", + "protein" + ], + [ + 43, + 47, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 65, + "sent": "Lanes: M represents the PAGE Ruler Unstained Ladder; 1: NsrR after a two-step purification; 2: NsrR one week after purification.", + "section": "FIG", + "ner": [ + [ + 56, + 60, + "NsrR", + "protein" + ], + [ + 95, + 99, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 66, + "sent": "* corresponds to full-length NsrR protein at 27 kDa, while ** and *** correspond to the NsrR-RD and NsrR-ED domain at around 13 kDa, respectively.", + "section": "FIG", + "ner": [ + [ + 17, + 28, + "full-length", + "protein_state" + ], + [ + 29, + 33, + "NsrR", + "protein" + ], + [ + 88, + 92, + "NsrR", + "protein" + ], + [ + 93, + 95, + "RD", + "structure_element" + ], + [ + 100, + 104, + "NsrR", + "protein" + ], + [ + 105, + 107, + "ED", + "structure_element" + ] + ] + }, + { + "sid": 67, + "sent": "Since formation of the crystals took around one month, it is not surprising that this cleavage also occurred in the crystallization drop.", + "section": "RESULTS", + "ner": [ + [ + 23, + 31, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 68, + "sent": "NsrR was crystallized yielding two crystal forms, which were distinguishable by visual inspection.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "NsrR", + "protein" + ], + [ + 9, + 21, + "crystallized", + "experimental_method" + ] + ] + }, + { + "sid": 69, + "sent": "Initially, we tried to solve the structure of NsrR by molecular replacement, which was not successful.", + "section": "RESULTS", + "ner": [ + [ + 33, + 42, + "structure", + "evidence" + ], + [ + 46, + 50, + "NsrR", + "protein" + ], + [ + 54, + 75, + "molecular replacement", + "experimental_method" + ] + ] + }, + { + "sid": 70, + "sent": "Therefore, we tried heavy atom phasing using a platinum compound.", + "section": "RESULTS", + "ner": [ + [ + 20, + 38, + "heavy atom phasing", + "experimental_method" + ], + [ + 47, + 55, + "platinum", + "chemical" + ] + ] + }, + { + "sid": 71, + "sent": "This succeeded for the rectangular plate-shaped crystals.", + "section": "RESULTS", + "ner": [ + [ + 48, + 56, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 72, + "sent": "After the structure was solved, it became evident that these crystals contained two monomers of the ED of NsrR in the asymmetric unit.", + "section": "RESULTS", + "ner": [ + [ + 10, + 19, + "structure", + "evidence" + ], + [ + 61, + 69, + "crystals", + "evidence" + ], + [ + 84, + 92, + "monomers", + "oligomeric_state" + ], + [ + 100, + 102, + "ED", + "structure_element" + ], + [ + 106, + 110, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 73, + "sent": "We also tried to solve the structure of the thin plate-shaped crystals with this template, but the resulting model generated was not sufficient.", + "section": "RESULTS", + "ner": [ + [ + 27, + 36, + "structure", + "evidence" + ], + [ + 62, + 70, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 74, + "sent": "Therefore, we thought that these crystals contained the N-terminal domain of NsrR and successfully phased this dataset using molecular replacement with the N-terminal domain of PhoB (PDB code: 1B00; as a template.", + "section": "RESULTS", + "ner": [ + [ + 33, + 41, + "crystals", + "evidence" + ], + [ + 56, + 73, + "N-terminal domain", + "structure_element" + ], + [ + 77, + 81, + "NsrR", + "protein" + ], + [ + 125, + 146, + "molecular replacement", + "experimental_method" + ], + [ + 156, + 173, + "N-terminal domain", + "structure_element" + ], + [ + 177, + 181, + "PhoB", + "protein" + ] + ] + }, + { + "sid": 75, + "sent": "This approach revealed that this crystal form indeed contained two monomers of the RD of NsrR in the asymmetric unit.", + "section": "RESULTS", + "ner": [ + [ + 67, + 75, + "monomers", + "oligomeric_state" + ], + [ + 83, + 85, + "RD", + "structure_element" + ], + [ + 89, + 93, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 76, + "sent": "Since both crystals forms were obtained in the same drop it is not surprising that, when dissolving several crystals and performing subsequent mass-spectrometry to identify the protein in the crystals, it yielded peptide fragments throughout the NsrR sequence.", + "section": "RESULTS", + "ner": [ + [ + 108, + 116, + "crystals", + "evidence" + ], + [ + 143, + 160, + "mass-spectrometry", + "experimental_method" + ], + [ + 192, + 200, + "crystals", + "evidence" + ], + [ + 246, + 250, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 77, + "sent": "In summary, the two crystal forms contained one of the two domains, respectively, such that both domains were successfully crystallized.", + "section": "RESULTS", + "ner": [ + [ + 20, + 33, + "crystal forms", + "evidence" + ], + [ + 123, + 135, + "crystallized", + "experimental_method" + ] + ] + }, + { + "sid": 78, + "sent": "We determined the crystal structures of NsrR-RD and NsrR-ED separately.", + "section": "RESULTS", + "ner": [ + [ + 18, + 36, + "crystal structures", + "evidence" + ], + [ + 40, + 44, + "NsrR", + "protein" + ], + [ + 45, + 47, + "RD", + "structure_element" + ], + [ + 52, + 56, + "NsrR", + "protein" + ], + [ + 57, + 59, + "ED", + "structure_element" + ] + ] + }, + { + "sid": 79, + "sent": "However, a part of the linker region (residues 120\u2013128; 120RRSQQFIQQ128; underlined are the amino acid residues not visible in either domain) could not be traced in the electron density.", + "section": "RESULTS", + "ner": [ + [ + 23, + 36, + "linker region", + "structure_element" + ], + [ + 47, + 54, + "120\u2013128", + "residue_range" + ], + [ + 56, + 71, + "120RRSQQFIQQ128", + "structure_element" + ], + [ + 169, + 185, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 80, + "sent": "Overall structure of the N-terminal NsrR receiver domain (NsrR-RD)", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 36, + 40, + "NsrR", + "protein" + ], + [ + 41, + 56, + "receiver domain", + "structure_element" + ], + [ + 58, + 62, + "NsrR", + "protein" + ], + [ + 63, + 65, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 81, + "sent": "The structure of the NsrR-RD was determined at a resolution of 1.8 \u00c5 (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 21, + 25, + "NsrR", + "protein" + ], + [ + 26, + 28, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 82, + "sent": "The Rwork and Rfree values after refinement were 0.17 and 0.22, respectively.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "Rwork", + "evidence" + ], + [ + 14, + 19, + "Rfree", + "evidence" + ] + ] + }, + { + "sid": 83, + "sent": "Ramachandran validation revealed that all residues (100%, 236 amino acids) were in the preferred or allowed regions.", + "section": "RESULTS", + "ner": [ + [ + 0, + 23, + "Ramachandran validation", + "evidence" + ] + ] + }, + { + "sid": 84, + "sent": "The structure contained many ethylene glycol molecules arising from the cryo-protecting procedure.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 29, + 44, + "ethylene glycol", + "chemical" + ] + ] + }, + { + "sid": 85, + "sent": "The asymmetric unit contains two copies of NsrR-RD.", + "section": "RESULTS", + "ner": [ + [ + 43, + 47, + "NsrR", + "protein" + ], + [ + 48, + 50, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 86, + "sent": "Although the entire N-terminal receiver domain is composed of residues Met1-Leu119, only residues Asn4 to Arg121 of chain A (including residues Arg120 and Arg121 of the linker) and Gln5 to Ser122 of chain B (including residues Arg120 until Ser122 of the linker) could be traced in the electron density of NsrR-RD.", + "section": "RESULTS", + "ner": [ + [ + 31, + 46, + "receiver domain", + "structure_element" + ], + [ + 71, + 82, + "Met1-Leu119", + "residue_range" + ], + [ + 98, + 112, + "Asn4 to Arg121", + "residue_range" + ], + [ + 116, + 123, + "chain A", + "structure_element" + ], + [ + 144, + 150, + "Arg120", + "residue_name_number" + ], + [ + 155, + 161, + "Arg121", + "residue_name_number" + ], + [ + 169, + 175, + "linker", + "structure_element" + ], + [ + 181, + 195, + "Gln5 to Ser122", + "residue_range" + ], + [ + 199, + 206, + "chain B", + "structure_element" + ], + [ + 227, + 246, + "Arg120 until Ser122", + "residue_range" + ], + [ + 254, + 260, + "linker", + "structure_element" + ], + [ + 285, + 301, + "electron density", + "evidence" + ], + [ + 305, + 309, + "NsrR", + "protein" + ], + [ + 310, + 312, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 87, + "sent": "For Asn85, Asp86, and Glu87 of chain A, poor electron density was observed for the side chains and, thus, these side chains were deleted during refinement and are not present in the final structure.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "Asn85", + "residue_name_number" + ], + [ + 11, + 16, + "Asp86", + "residue_name_number" + ], + [ + 22, + 27, + "Glu87", + "residue_name_number" + ], + [ + 31, + 38, + "chain A", + "structure_element" + ], + [ + 45, + 61, + "electron density", + "evidence" + ], + [ + 188, + 197, + "structure", + "evidence" + ] + ] + }, + { + "sid": 88, + "sent": "Since the two monomers of NsrR-RD were virtually identical (rmsd of 0.6 \u00c5 over 116 C\u03b1 atoms for the two monomers).", + "section": "RESULTS", + "ner": [ + [ + 14, + 22, + "monomers", + "oligomeric_state" + ], + [ + 26, + 30, + "NsrR", + "protein" + ], + [ + 31, + 33, + "RD", + "structure_element" + ], + [ + 60, + 64, + "rmsd", + "evidence" + ], + [ + 104, + 112, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 89, + "sent": "Therefore, the overall structure is described for monomer A only.", + "section": "RESULTS", + "ner": [ + [ + 23, + 32, + "structure", + "evidence" + ], + [ + 50, + 57, + "monomer", + "oligomeric_state" + ], + [ + 58, + 59, + "A", + "structure_element" + ] + ] + }, + { + "sid": 90, + "sent": "NsrR-RD structurally adopts a \u03b1\u03b2 doubly-wound fold previously observed in OmpR/PhoB type regulators.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "NsrR", + "protein" + ], + [ + 5, + 7, + "RD", + "structure_element" + ], + [ + 30, + 50, + "\u03b1\u03b2 doubly-wound fold", + "structure_element" + ], + [ + 74, + 99, + "OmpR/PhoB type regulators", + "protein_type" + ] + ] + }, + { + "sid": 91, + "sent": "Five \u03b2-strands (\u03b21-\u03b25) are arranged in a parallel fashion constituting the central core of the structure, which is surrounded by two \u03b1-helices (\u03b11 and \u03b15) on one and three helices (\u03b12, \u03b13, \u03b14) on the other side (Fig 2).", + "section": "RESULTS", + "ner": [ + [ + 5, + 14, + "\u03b2-strands", + "structure_element" + ], + [ + 16, + 21, + "\u03b21-\u03b25", + "structure_element" + ], + [ + 95, + 104, + "structure", + "evidence" + ], + [ + 133, + 142, + "\u03b1-helices", + "structure_element" + ], + [ + 144, + 146, + "\u03b11", + "structure_element" + ], + [ + 151, + 153, + "\u03b15", + "structure_element" + ], + [ + 172, + 179, + "helices", + "structure_element" + ], + [ + 181, + 183, + "\u03b12", + "structure_element" + ], + [ + 185, + 187, + "\u03b13", + "structure_element" + ], + [ + 189, + 191, + "\u03b14", + "structure_element" + ] + ] + }, + { + "sid": 92, + "sent": "The NsrR-RD structure shows a \u03b21-\u03b11-\u03b22-\u03b12-\u03b23-\u03b13-\u03b24-\u03b14-\u03b25-\u03b15 topology as also observed for other RRs.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "NsrR", + "protein" + ], + [ + 9, + 11, + "RD", + "structure_element" + ], + [ + 12, + 21, + "structure", + "evidence" + ], + [ + 30, + 59, + "\u03b21-\u03b11-\u03b22-\u03b12-\u03b23-\u03b13-\u03b24-\u03b14-\u03b25-\u03b15", + "structure_element" + ], + [ + 96, + 99, + "RRs", + "protein_type" + ] + ] + }, + { + "sid": 93, + "sent": "Structure of NsrR-RD.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 13, + 17, + "NsrR", + "protein" + ], + [ + 18, + 20, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 94, + "sent": "Cartoon representation of the helices (\u03b11 \u2013 \u03b15) and \u03b2-sheets (\u03b21 - \u03b25).", + "section": "FIG", + "ner": [ + [ + 30, + 37, + "helices", + "structure_element" + ], + [ + 39, + 46, + "\u03b11 \u2013 \u03b15", + "structure_element" + ], + [ + 52, + 60, + "\u03b2-sheets", + "structure_element" + ], + [ + 62, + 69, + "\u03b21 - \u03b25", + "structure_element" + ] + ] + }, + { + "sid": 95, + "sent": "Structural areas with the highest variations to the receiver domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1B00) are marked in separate boxes.", + "section": "FIG", + "ner": [ + [ + 52, + 68, + "receiver domains", + "structure_element" + ], + [ + 72, + 76, + "DrrB", + "protein" + ], + [ + 91, + 95, + "MtrA", + "protein" + ], + [ + 114, + 118, + "PhoB", + "protein" + ] + ] + }, + { + "sid": 96, + "sent": "Comparison with structures of other receiver domains", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "Comparison", + "experimental_method" + ], + [ + 16, + 26, + "structures", + "evidence" + ], + [ + 36, + 52, + "receiver domains", + "structure_element" + ] + ] + }, + { + "sid": 97, + "sent": "NsrR belongs to the OmpR/PhoB family of RRs.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "NsrR", + "protein" + ], + [ + 20, + 36, + "OmpR/PhoB family", + "protein_type" + ], + [ + 40, + 43, + "RRs", + "protein_type" + ] + ] + }, + { + "sid": 98, + "sent": "The receiver domain of NsrR was superimposed with other structurally characterized receiver domains from the OmpR/PhoB family, such as DrrB, KdpE, MtrA, and the crystal structure of only the receiver domain of PhoB. The rmsd of the overlays and the corresponding PDB codes used are highlighted in Table 2.", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "receiver domain", + "structure_element" + ], + [ + 23, + 27, + "NsrR", + "protein" + ], + [ + 32, + 44, + "superimposed", + "experimental_method" + ], + [ + 83, + 99, + "receiver domains", + "structure_element" + ], + [ + 109, + 125, + "OmpR/PhoB family", + "protein_type" + ], + [ + 135, + 139, + "DrrB", + "protein" + ], + [ + 141, + 145, + "KdpE", + "protein" + ], + [ + 147, + 151, + "MtrA", + "protein" + ], + [ + 161, + 178, + "crystal structure", + "evidence" + ], + [ + 191, + 206, + "receiver domain", + "structure_element" + ], + [ + 210, + 214, + "PhoB", + "protein" + ], + [ + 220, + 224, + "rmsd", + "evidence" + ], + [ + 232, + 240, + "overlays", + "experimental_method" + ] + ] + }, + { + "sid": 99, + "sent": "Superimposition of the structures revealed that helix \u03b14 is slightly rotated outward in NsrR-RD (Fig 2).", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Superimposition", + "experimental_method" + ], + [ + 23, + 33, + "structures", + "evidence" + ], + [ + 48, + 53, + "helix", + "structure_element" + ], + [ + 54, + 56, + "\u03b14", + "structure_element" + ], + [ + 88, + 92, + "NsrR", + "protein" + ], + [ + 93, + 95, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 100, + "sent": "In receiver domains of response regulators, helix \u03b14 has been shown to be a crucial part of the dimerization interface.", + "section": "RESULTS", + "ner": [ + [ + 3, + 19, + "receiver domains", + "structure_element" + ], + [ + 23, + 42, + "response regulators", + "protein_type" + ], + [ + 44, + 49, + "helix", + "structure_element" + ], + [ + 50, + 52, + "\u03b14", + "structure_element" + ], + [ + 96, + 118, + "dimerization interface", + "site" + ] + ] + }, + { + "sid": 101, + "sent": "Furthermore, helix \u03b14 in NsrR is shorter than in other RRs.", + "section": "RESULTS", + "ner": [ + [ + 13, + 18, + "helix", + "structure_element" + ], + [ + 19, + 21, + "\u03b14", + "structure_element" + ], + [ + 25, + 29, + "NsrR", + "protein" + ], + [ + 55, + 58, + "RRs", + "protein_type" + ] + ] + }, + { + "sid": 102, + "sent": "The first helical turn is unwound and adopts an unstructured region (see Fig 2).", + "section": "RESULTS", + "ner": [ + [ + 4, + 22, + "first helical turn", + "structure_element" + ], + [ + 26, + 33, + "unwound", + "protein_state" + ], + [ + 48, + 60, + "unstructured", + "protein_state" + ] + ] + }, + { + "sid": 103, + "sent": "A slightly outward rotation or unwinding of helix \u03b14 has been observed in the structures of other RD of regulators.", + "section": "RESULTS", + "ner": [ + [ + 44, + 49, + "helix", + "structure_element" + ], + [ + 50, + 52, + "\u03b14", + "structure_element" + ], + [ + 78, + 88, + "structures", + "evidence" + ], + [ + 98, + 100, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 104, + "sent": "For example, the structure of BaeR and RegX3 displayed a completely unwound helix \u03b14.", + "section": "RESULTS", + "ner": [ + [ + 17, + 26, + "structure", + "evidence" + ], + [ + 30, + 34, + "BaeR", + "protein" + ], + [ + 39, + 44, + "RegX3", + "protein" + ], + [ + 68, + 75, + "unwound", + "protein_state" + ], + [ + 76, + 81, + "helix", + "structure_element" + ], + [ + 82, + 84, + "\u03b14", + "structure_element" + ] + ] + }, + { + "sid": 105, + "sent": "In the structure of DrrD, helix \u03b14 is only partially displaced.", + "section": "RESULTS", + "ner": [ + [ + 7, + 16, + "structure", + "evidence" + ], + [ + 20, + 24, + "DrrD", + "protein" + ], + [ + 26, + 31, + "helix", + "structure_element" + ], + [ + 32, + 34, + "\u03b14", + "structure_element" + ] + ] + }, + { + "sid": 106, + "sent": "In the receiver domain of NsrR, helix \u03b14 is also partially displaced but in a different direction (S1 Fig).", + "section": "RESULTS", + "ner": [ + [ + 7, + 22, + "receiver domain", + "structure_element" + ], + [ + 26, + 30, + "NsrR", + "protein" + ], + [ + 32, + 37, + "helix", + "structure_element" + ], + [ + 38, + 40, + "\u03b14", + "structure_element" + ] + ] + }, + { + "sid": 107, + "sent": "Inspection of the crystal contacts revealed no major interactions in this region that could have influenced the orientation of helix \u03b14.", + "section": "RESULTS", + "ner": [ + [ + 127, + 132, + "helix", + "structure_element" + ], + [ + 133, + 135, + "\u03b14", + "structure_element" + ] + ] + }, + { + "sid": 108, + "sent": "Furthermore, NsrR is crystallized as a monomer, and investigation of the symmetry-related molecules did not reveal a functional dimer within the crystal.", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "NsrR", + "protein" + ], + [ + 21, + 33, + "crystallized", + "experimental_method" + ], + [ + 39, + 46, + "monomer", + "oligomeric_state" + ], + [ + 128, + 133, + "dimer", + "oligomeric_state" + ], + [ + 145, + 152, + "crystal", + "evidence" + ] + ] + }, + { + "sid": 109, + "sent": "This could explain the flexibility and thereby the different orientation of helix \u03b14 in NsrR.", + "section": "RESULTS", + "ner": [ + [ + 76, + 81, + "helix", + "structure_element" + ], + [ + 82, + 84, + "\u03b14", + "structure_element" + ], + [ + 88, + 92, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 110, + "sent": "The structures of the RD and ED domains of NsrR aligned to other response regulators.", + "section": "TABLE", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 22, + 24, + "RD", + "structure_element" + ], + [ + 29, + 31, + "ED", + "structure_element" + ], + [ + 43, + 47, + "NsrR", + "protein" + ], + [ + 48, + 55, + "aligned", + "experimental_method" + ], + [ + 65, + 84, + "response regulators", + "protein_type" + ] + ] + }, + { + "sid": 111, + "sent": "The rmsd values of the superimpositions of the structures of NsrR-RD and NsrR-ED with the available structures of members of the OmpR/PhoB subfamily are highlighted. *Seq ID (%) corresponds to the full-length protein sequence.", + "section": "TABLE", + "ner": [ + [ + 4, + 8, + "rmsd", + "evidence" + ], + [ + 23, + 39, + "superimpositions", + "experimental_method" + ], + [ + 47, + 57, + "structures", + "evidence" + ], + [ + 61, + 65, + "NsrR", + "protein" + ], + [ + 66, + 68, + "RD", + "structure_element" + ], + [ + 73, + 77, + "NsrR", + "protein" + ], + [ + 78, + 80, + "ED", + "structure_element" + ], + [ + 100, + 110, + "structures", + "evidence" + ], + [ + 129, + 148, + "OmpR/PhoB subfamily", + "protein_type" + ], + [ + 197, + 208, + "full-length", + "protein_state" + ] + ] + }, + { + "sid": 112, + "sent": "Based on the Dali server, the NsrR-RD domain is structurally closely related to KdpE (PDB code: 4KNY) from E. coli, displaying a sequence identity of 28%.", + "section": "RESULTS", + "ner": [ + [ + 13, + 24, + "Dali server", + "experimental_method" + ], + [ + 30, + 34, + "NsrR", + "protein" + ], + [ + 35, + 37, + "RD", + "structure_element" + ], + [ + 80, + 84, + "KdpE", + "protein" + ], + [ + 107, + 114, + "E. coli", + "species" + ] + ] + }, + { + "sid": 113, + "sent": "This structural homology is also reflected by the low rmsd of 1.9 \u00c5 over 117 C\u03b1 atoms after superimposition of the receiver domains of NsrR and KdpE (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 54, + 58, + "rmsd", + "evidence" + ], + [ + 92, + 107, + "superimposition", + "experimental_method" + ], + [ + 115, + 131, + "receiver domains", + "structure_element" + ], + [ + 135, + 139, + "NsrR", + "protein" + ], + [ + 144, + 148, + "KdpE", + "protein" + ] + ] + }, + { + "sid": 114, + "sent": "Furthermore, the orientation of the helix \u03b14 in NsrR is close to that present in KdpE (S1 Fig).", + "section": "RESULTS", + "ner": [ + [ + 36, + 41, + "helix", + "structure_element" + ], + [ + 42, + 44, + "\u03b14", + "structure_element" + ], + [ + 48, + 52, + "NsrR", + "protein" + ], + [ + 81, + 85, + "KdpE", + "protein" + ] + ] + }, + { + "sid": 115, + "sent": "Active site residues and dimerization", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Active site", + "site" + ] + ] + }, + { + "sid": 116, + "sent": "All RRs contain a highly conserved aspartate residue in the active site (Fig 3; shown in red).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "RRs", + "protein_type" + ], + [ + 18, + 34, + "highly conserved", + "protein_state" + ], + [ + 35, + 44, + "aspartate", + "residue_name" + ], + [ + 60, + 71, + "active site", + "site" + ] + ] + }, + { + "sid": 117, + "sent": "Phosphorylation of this aspartate residue induces a conformational change leading to the activation of the effector domain that binds DNA and regulates the transcription of target genes.", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Phosphorylation", + "ptm" + ], + [ + 24, + 33, + "aspartate", + "residue_name" + ], + [ + 107, + 122, + "effector domain", + "structure_element" + ], + [ + 134, + 137, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 118, + "sent": "This site of phosphorylation is conserved throughout the family of response regulators, including the lantibiotic resistance-associated RRs such as BraR from L. monocytogenes, BceR from Bacillus subtilis, CprR from C. difficile, GraR from S. aureus, LcrR from S. mutans, LisR, and VirR from L. monocytogenes (Fig 3).", + "section": "RESULTS", + "ner": [ + [ + 13, + 28, + "phosphorylation", + "ptm" + ], + [ + 32, + 41, + "conserved", + "protein_state" + ], + [ + 67, + 86, + "response regulators", + "protein_type" + ], + [ + 102, + 139, + "lantibiotic resistance-associated RRs", + "protein_type" + ], + [ + 148, + 152, + "BraR", + "protein" + ], + [ + 158, + 174, + "L. monocytogenes", + "species" + ], + [ + 176, + 180, + "BceR", + "protein" + ], + [ + 186, + 203, + "Bacillus subtilis", + "species" + ], + [ + 205, + 209, + "CprR", + "protein" + ], + [ + 215, + 227, + "C. difficile", + "species" + ], + [ + 229, + 233, + "GraR", + "protein" + ], + [ + 239, + 248, + "S. aureus", + "species" + ], + [ + 250, + 254, + "LcrR", + "protein" + ], + [ + 260, + 269, + "S. mutans", + "species" + ], + [ + 271, + 275, + "LisR", + "protein" + ], + [ + 281, + 285, + "VirR", + "protein" + ], + [ + 291, + 307, + "L. monocytogenes", + "species" + ] + ] + }, + { + "sid": 119, + "sent": "Sequence alignment of NsrR protein with other response regulators.", + "section": "FIG", + "ner": [ + [ + 0, + 18, + "Sequence alignment", + "experimental_method" + ], + [ + 22, + 26, + "NsrR", + "protein" + ], + [ + 46, + 65, + "response regulators", + "protein_type" + ] + ] + }, + { + "sid": 120, + "sent": "A sequence alignment of NsrR with RRs belonging to the OmpR/PhoB subfamily (marked in grey) and RRs involved in lantibiotic resistance (black) is shown.", + "section": "FIG", + "ner": [ + [ + 2, + 20, + "sequence alignment", + "experimental_method" + ], + [ + 24, + 28, + "NsrR", + "protein" + ], + [ + 34, + 37, + "RRs", + "protein_type" + ], + [ + 55, + 74, + "OmpR/PhoB subfamily", + "protein_type" + ], + [ + 96, + 99, + "RRs", + "protein_type" + ], + [ + 112, + 123, + "lantibiotic", + "chemical" + ] + ] + }, + { + "sid": 121, + "sent": "The active site aspartate residue (highlighted in red), the residues forming the acidic pocket surrounding it (highlighted in pink), the switch residues (highlighted in blue), the conserved lysine residue (highlighted in green), the highly conserved residues of the linker region (colored in purple), the residues involved in dimer interface of receiver domain (highlighted in yellow), residues involved in interdomain interactions (shown in orange boxes and in cyan) and the residues involved in interaction with DNA (colored in blue) are shown.", + "section": "FIG", + "ner": [ + [ + 4, + 15, + "active site", + "site" + ], + [ + 16, + 25, + "aspartate", + "residue_name" + ], + [ + 81, + 94, + "acidic pocket", + "site" + ], + [ + 137, + 152, + "switch residues", + "site" + ], + [ + 180, + 189, + "conserved", + "protein_state" + ], + [ + 190, + 196, + "lysine", + "residue_name" + ], + [ + 233, + 249, + "highly conserved", + "protein_state" + ], + [ + 266, + 279, + "linker region", + "structure_element" + ], + [ + 326, + 341, + "dimer interface", + "site" + ], + [ + 345, + 360, + "receiver domain", + "structure_element" + ], + [ + 514, + 517, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 122, + "sent": "The linker region of the known structures is underlined within the sequence.", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "linker region", + "structure_element" + ], + [ + 31, + 41, + "structures", + "evidence" + ] + ] + }, + { + "sid": 123, + "sent": "The putative phosphorylation site of NsrR is Asp55, which is localized at the end of strand \u03b23 (Fig 3, shown in red; Fig 4) and lies within an acidic environment composed of the side chains of Glu12 and Asp13 (Fig 3, highlighted in pink).", + "section": "RESULTS", + "ner": [ + [ + 13, + 33, + "phosphorylation site", + "site" + ], + [ + 37, + 41, + "NsrR", + "protein" + ], + [ + 45, + 50, + "Asp55", + "residue_name_number" + ], + [ + 85, + 91, + "strand", + "structure_element" + ], + [ + 92, + 94, + "\u03b23", + "structure_element" + ], + [ + 193, + 198, + "Glu12", + "residue_name_number" + ], + [ + 203, + 208, + "Asp13", + "residue_name_number" + ] + ] + }, + { + "sid": 124, + "sent": "This pocket is similar to the acidic active site observed within most structures of RRs such as PhoB from E. coli, PhoP from M. tuberculosis, and DivK from Caulobacter crescentus.", + "section": "RESULTS", + "ner": [ + [ + 5, + 11, + "pocket", + "site" + ], + [ + 30, + 36, + "acidic", + "protein_state" + ], + [ + 37, + 48, + "active site", + "site" + ], + [ + 70, + 80, + "structures", + "evidence" + ], + [ + 84, + 87, + "RRs", + "protein_type" + ], + [ + 96, + 100, + "PhoB", + "protein" + ], + [ + 106, + 113, + "E. coli", + "species" + ], + [ + 115, + 119, + "PhoP", + "protein" + ], + [ + 125, + 140, + "M. tuberculosis", + "species" + ], + [ + 146, + 150, + "DivK", + "protein" + ], + [ + 156, + 178, + "Caulobacter crescentus", + "species" + ] + ] + }, + { + "sid": 125, + "sent": "In NsrR, Glu12, Asp13, and Asp55 are in close proximity of a highly conserved Lys104 residue (highlighted in green in Fig 3).", + "section": "RESULTS", + "ner": [ + [ + 3, + 7, + "NsrR", + "protein" + ], + [ + 9, + 14, + "Glu12", + "residue_name_number" + ], + [ + 16, + 21, + "Asp13", + "residue_name_number" + ], + [ + 27, + 32, + "Asp55", + "residue_name_number" + ], + [ + 61, + 77, + "highly conserved", + "protein_state" + ], + [ + 78, + 84, + "Lys104", + "residue_name_number" + ] + ] + }, + { + "sid": 126, + "sent": "Location of the highly conserved Asp55 and inactive state conformation of the key switch residues, Ser82 and Phe101 in NsrR-RD.", + "section": "FIG", + "ner": [ + [ + 16, + 32, + "highly conserved", + "protein_state" + ], + [ + 33, + 38, + "Asp55", + "residue_name_number" + ], + [ + 43, + 51, + "inactive", + "protein_state" + ], + [ + 82, + 97, + "switch residues", + "site" + ], + [ + 99, + 104, + "Ser82", + "residue_name_number" + ], + [ + 109, + 115, + "Phe101", + "residue_name_number" + ], + [ + 119, + 123, + "NsrR", + "protein" + ], + [ + 124, + 126, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 127, + "sent": "NsrR (represented in yellow) displays a geometry representing the inactive state as deduced from the inactive state structure of PhoB (shown in brown, PDB code 1B00) (a).", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "NsrR", + "protein" + ], + [ + 66, + 74, + "inactive", + "protein_state" + ], + [ + 101, + 109, + "inactive", + "protein_state" + ], + [ + 116, + 125, + "structure", + "evidence" + ], + [ + 129, + 133, + "PhoB", + "protein" + ] + ] + }, + { + "sid": 128, + "sent": "The inactive conformation of NsrR differs from the active state structure of PhoB (light blue, PDB code 1ZES) (b) in the orientation of the corresponding switch residues, Ser82 and Phe101, which adopt a conformation pointing away from the active site (Asp55 in NsrR).", + "section": "FIG", + "ner": [ + [ + 4, + 12, + "inactive", + "protein_state" + ], + [ + 29, + 33, + "NsrR", + "protein" + ], + [ + 51, + 57, + "active", + "protein_state" + ], + [ + 64, + 73, + "structure", + "evidence" + ], + [ + 77, + 81, + "PhoB", + "protein" + ], + [ + 154, + 169, + "switch residues", + "site" + ], + [ + 171, + 176, + "Ser82", + "residue_name_number" + ], + [ + 181, + 187, + "Phe101", + "residue_name_number" + ], + [ + 239, + 250, + "active site", + "site" + ], + [ + 252, + 257, + "Asp55", + "residue_name_number" + ], + [ + 261, + 265, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 129, + "sent": "A divalent metal ion is usually bound in this acidic environment and is essential for phosphorylation and de-phosphorylation of RRs.", + "section": "RESULTS", + "ner": [ + [ + 86, + 101, + "phosphorylation", + "ptm" + ], + [ + 106, + 124, + "de-phosphorylation", + "ptm" + ], + [ + 128, + 131, + "RRs", + "protein_type" + ] + ] + }, + { + "sid": 130, + "sent": "In some RRs like CheY, Mg2+ is observed in the structure, bound near the phosphorylation site.", + "section": "RESULTS", + "ner": [ + [ + 8, + 11, + "RRs", + "protein_type" + ], + [ + 17, + 21, + "CheY", + "protein" + ], + [ + 23, + 27, + "Mg2+", + "chemical" + ], + [ + 47, + 56, + "structure", + "evidence" + ], + [ + 58, + 63, + "bound", + "protein_state" + ], + [ + 73, + 93, + "phosphorylation site", + "site" + ] + ] + }, + { + "sid": 131, + "sent": "In the KdpE regulator from E. coli that is involved in osmoregulation, a divalent calcium ion is present.", + "section": "RESULTS", + "ner": [ + [ + 7, + 11, + "KdpE", + "protein" + ], + [ + 12, + 21, + "regulator", + "protein_type" + ], + [ + 27, + 34, + "E. coli", + "species" + ], + [ + 82, + 89, + "calcium", + "chemical" + ] + ] + }, + { + "sid": 132, + "sent": "However, the structure of NsrR-RD did not contain any divalent ion.", + "section": "RESULTS", + "ner": [ + [ + 13, + 22, + "structure", + "evidence" + ], + [ + 26, + 30, + "NsrR", + "protein" + ], + [ + 31, + 33, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 133, + "sent": "Instead, a water molecule is present, which interacts with Glu12 of the acidic pocket, Lys104, and another water molecule in the vicinity.", + "section": "RESULTS", + "ner": [ + [ + 11, + 16, + "water", + "chemical" + ], + [ + 59, + 64, + "Glu12", + "residue_name_number" + ], + [ + 72, + 85, + "acidic pocket", + "site" + ], + [ + 87, + 93, + "Lys104", + "residue_name_number" + ], + [ + 107, + 112, + "water", + "chemical" + ] + ] + }, + { + "sid": 134, + "sent": "Within the \u03b24-\u03b14 loop and in \u03b25 of the RD of RRs, specific amino acids are crucial for signal transduction from the RD to the ED via conformational changes that are a consequence of phosphorylation of the RD.", + "section": "RESULTS", + "ner": [ + [ + 11, + 21, + "\u03b24-\u03b14 loop", + "structure_element" + ], + [ + 29, + 31, + "\u03b25", + "structure_element" + ], + [ + 39, + 41, + "RD", + "structure_element" + ], + [ + 45, + 48, + "RRs", + "protein_type" + ], + [ + 116, + 118, + "RD", + "structure_element" + ], + [ + 126, + 128, + "ED", + "structure_element" + ], + [ + 182, + 197, + "phosphorylation", + "ptm" + ], + [ + 205, + 207, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 135, + "sent": "These amino acids are Ser/Thr and Phe/Tyr located at the end of \u03b24 and before \u03b25, respectively, and designated as \u201csignature switch residues\u201d.", + "section": "RESULTS", + "ner": [ + [ + 22, + 25, + "Ser", + "residue_name" + ], + [ + 26, + 29, + "Thr", + "residue_name" + ], + [ + 34, + 37, + "Phe", + "residue_name" + ], + [ + 38, + 41, + "Tyr", + "residue_name" + ], + [ + 64, + 66, + "\u03b24", + "structure_element" + ], + [ + 78, + 80, + "\u03b25", + "structure_element" + ], + [ + 115, + 140, + "signature switch residues", + "site" + ] + ] + }, + { + "sid": 136, + "sent": "As seen in the alignment (Fig 3, highlighted in blue), these signature residues (Ser/Thr and Phe/Tyr) are highly conserved in the lantibiotic resistance-associated RRs.", + "section": "RESULTS", + "ner": [ + [ + 15, + 24, + "alignment", + "experimental_method" + ], + [ + 81, + 84, + "Ser", + "residue_name" + ], + [ + 85, + 88, + "Thr", + "residue_name" + ], + [ + 93, + 96, + "Phe", + "residue_name" + ], + [ + 97, + 100, + "Tyr", + "residue_name" + ], + [ + 106, + 122, + "highly conserved", + "protein_state" + ], + [ + 130, + 167, + "lantibiotic resistance-associated RRs", + "protein_type" + ] + ] + }, + { + "sid": 137, + "sent": "The orientation of the side chains of these residues determines whether the RD is in an active or inactive state.", + "section": "RESULTS", + "ner": [ + [ + 76, + 78, + "RD", + "structure_element" + ], + [ + 88, + 94, + "active", + "protein_state" + ], + [ + 98, + 106, + "inactive", + "protein_state" + ] + ] + }, + { + "sid": 138, + "sent": "In the inactive state, the phenylalanine or tyrosine residue faces away from the active site, and the corresponding serine or threonine residue adopts an outward-facing conformation as well (Fig 4A).", + "section": "RESULTS", + "ner": [ + [ + 7, + 15, + "inactive", + "protein_state" + ], + [ + 27, + 40, + "phenylalanine", + "residue_name" + ], + [ + 44, + 52, + "tyrosine", + "residue_name" + ], + [ + 81, + 92, + "active site", + "site" + ], + [ + 116, + 122, + "serine", + "residue_name" + ], + [ + 126, + 135, + "threonine", + "residue_name" + ], + [ + 154, + 168, + "outward-facing", + "protein_state" + ] + ] + }, + { + "sid": 139, + "sent": "In contrast, the switch residues face towards the active site in the active state conformation (Fig 4B).", + "section": "RESULTS", + "ner": [ + [ + 17, + 32, + "switch residues", + "site" + ], + [ + 50, + 61, + "active site", + "site" + ], + [ + 69, + 75, + "active", + "protein_state" + ] + ] + }, + { + "sid": 140, + "sent": "By sequence alignment with other lantibiotic resistance-associated RRs, these \u201csignature switch residues\u201d are identified as Ser82 and Phe101 in NsrR (see above).", + "section": "RESULTS", + "ner": [ + [ + 3, + 21, + "sequence alignment", + "experimental_method" + ], + [ + 33, + 70, + "lantibiotic resistance-associated RRs", + "protein_type" + ], + [ + 79, + 104, + "signature switch residues", + "site" + ], + [ + 124, + 129, + "Ser82", + "residue_name_number" + ], + [ + 134, + 140, + "Phe101", + "residue_name_number" + ], + [ + 144, + 148, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 141, + "sent": "Although some RRs such as KdpE, BraR, BceR, GraR, and VirR contain a serine residue as the first switch residue, the others possess a threonine instead.", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "RRs", + "protein_type" + ], + [ + 26, + 30, + "KdpE", + "protein" + ], + [ + 32, + 36, + "BraR", + "protein" + ], + [ + 38, + 42, + "BceR", + "protein" + ], + [ + 44, + 48, + "GraR", + "protein" + ], + [ + 54, + 58, + "VirR", + "protein" + ], + [ + 69, + 75, + "serine", + "residue_name" + ], + [ + 91, + 111, + "first switch residue", + "site" + ], + [ + 134, + 143, + "threonine", + "residue_name" + ] + ] + }, + { + "sid": 142, + "sent": "Furthermore, the second switch residue is mostly a tyrosine, with NsrR, BraR, and BceR being the only exceptions containing a phenylalanine at that position.", + "section": "RESULTS", + "ner": [ + [ + 17, + 38, + "second switch residue", + "site" + ], + [ + 51, + 59, + "tyrosine", + "residue_name" + ], + [ + 66, + 70, + "NsrR", + "protein" + ], + [ + 72, + 76, + "BraR", + "protein" + ], + [ + 82, + 86, + "BceR", + "protein" + ], + [ + 126, + 139, + "phenylalanine", + "residue_name" + ] + ] + }, + { + "sid": 143, + "sent": "A comparison of the NsrR-RD structure with the available structures of PhoB (Fig 4) in the active (PDB code: 1ZES) and inactive (PDB code: 1B00) states demonstrates that Ser82 (NsrR-RD) is oriented away from the active site Asp55, and that Phe101 is also in an outward conformation suggesting an inactive state of the NsrR-RD (Fig 4A).", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "NsrR", + "protein" + ], + [ + 25, + 27, + "RD", + "structure_element" + ], + [ + 28, + 37, + "structure", + "evidence" + ], + [ + 57, + 67, + "structures", + "evidence" + ], + [ + 71, + 75, + "PhoB", + "protein" + ], + [ + 91, + 97, + "active", + "protein_state" + ], + [ + 119, + 127, + "inactive", + "protein_state" + ], + [ + 170, + 175, + "Ser82", + "residue_name_number" + ], + [ + 177, + 181, + "NsrR", + "protein" + ], + [ + 182, + 184, + "RD", + "structure_element" + ], + [ + 212, + 223, + "active site", + "site" + ], + [ + 224, + 229, + "Asp55", + "residue_name_number" + ], + [ + 240, + 246, + "Phe101", + "residue_name_number" + ], + [ + 261, + 268, + "outward", + "protein_state" + ], + [ + 296, + 304, + "inactive", + "protein_state" + ], + [ + 318, + 322, + "NsrR", + "protein" + ], + [ + 323, + 325, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 144, + "sent": "As mentioned above, RRs contain a phosphorylation-activated switch and normally exist in equilibrium between the active and inactive conformations.", + "section": "RESULTS", + "ner": [ + [ + 20, + 23, + "RRs", + "protein_type" + ], + [ + 34, + 59, + "phosphorylation-activated", + "protein_state" + ], + [ + 60, + 66, + "switch", + "site" + ], + [ + 113, + 119, + "active", + "protein_state" + ], + [ + 124, + 132, + "inactive", + "protein_state" + ] + ] + }, + { + "sid": 145, + "sent": "Phosphorylation shifts the equilibrium towards the active conformation and induces the formation of rotationally symmetric dimers on the \u03b14-\u03b25-\u03b15 interface of RDs.", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Phosphorylation", + "ptm" + ], + [ + 51, + 57, + "active", + "protein_state" + ], + [ + 123, + 129, + "dimers", + "oligomeric_state" + ], + [ + 137, + 155, + "\u03b14-\u03b25-\u03b15 interface", + "site" + ], + [ + 159, + 162, + "RDs", + "structure_element" + ] + ] + }, + { + "sid": 146, + "sent": "It has been suggested that dimerization is crucial for DNA-binding of RRs of the OmpR/PhoB subfamily.", + "section": "RESULTS", + "ner": [ + [ + 55, + 58, + "DNA", + "chemical" + ], + [ + 70, + 73, + "RRs", + "protein_type" + ], + [ + 81, + 100, + "OmpR/PhoB subfamily", + "protein_type" + ] + ] + }, + { + "sid": 147, + "sent": "The RD domain of NsrR was crystallized with two separate monomers in the asymmetric unit.", + "section": "RESULTS", + "ner": [ + [ + 4, + 6, + "RD", + "structure_element" + ], + [ + 17, + 21, + "NsrR", + "protein" + ], + [ + 26, + 38, + "crystallized", + "experimental_method" + ], + [ + 57, + 65, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 148, + "sent": "Therefore, we performed a DALI search and focused on RD domains that were structurally determined as functional dimers.", + "section": "RESULTS", + "ner": [ + [ + 26, + 37, + "DALI search", + "experimental_method" + ], + [ + 53, + 55, + "RD", + "structure_element" + ], + [ + 101, + 111, + "functional", + "protein_state" + ], + [ + 112, + 118, + "dimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 149, + "sent": "In this context, the dimer of full-length KdpE from E. coli (Z-score 18.8, rmsd 1.9 \u00c5 over 117 C\u03b1 atoms) (PDB code: 4KNY) and the structure of the functional dimer of the RD of KdpE from E. coli (PDB code: 1ZH2) represent the most structurally related structures.", + "section": "RESULTS", + "ner": [ + [ + 21, + 26, + "dimer", + "oligomeric_state" + ], + [ + 30, + 41, + "full-length", + "protein_state" + ], + [ + 42, + 46, + "KdpE", + "protein" + ], + [ + 52, + 59, + "E. coli", + "species" + ], + [ + 61, + 68, + "Z-score", + "evidence" + ], + [ + 75, + 79, + "rmsd", + "evidence" + ], + [ + 130, + 139, + "structure", + "evidence" + ], + [ + 147, + 157, + "functional", + "protein_state" + ], + [ + 158, + 163, + "dimer", + "oligomeric_state" + ], + [ + 171, + 173, + "RD", + "structure_element" + ], + [ + 177, + 181, + "KdpE", + "protein" + ], + [ + 187, + 194, + "E. coli", + "species" + ], + [ + 252, + 262, + "structures", + "evidence" + ] + ] + }, + { + "sid": 150, + "sent": "We aligned NsrR-RD on both monomers of the RD of KdpE. Since helix \u03b14 of NsrR-RD is orientated slightly different when compared with other structures of RDs (Fig 2), helix \u03b14 and the N-terminal loop of one monomer were clashing with the second monomer (S2A Fig).", + "section": "RESULTS", + "ner": [ + [ + 3, + 10, + "aligned", + "experimental_method" + ], + [ + 11, + 15, + "NsrR", + "protein" + ], + [ + 16, + 18, + "RD", + "structure_element" + ], + [ + 27, + 35, + "monomers", + "oligomeric_state" + ], + [ + 43, + 45, + "RD", + "structure_element" + ], + [ + 49, + 53, + "KdpE", + "protein" + ], + [ + 61, + 66, + "helix", + "structure_element" + ], + [ + 67, + 69, + "\u03b14", + "structure_element" + ], + [ + 73, + 77, + "NsrR", + "protein" + ], + [ + 78, + 80, + "RD", + "structure_element" + ], + [ + 139, + 149, + "structures", + "evidence" + ], + [ + 153, + 156, + "RDs", + "structure_element" + ], + [ + 166, + 171, + "helix", + "structure_element" + ], + [ + 172, + 174, + "\u03b14", + "structure_element" + ], + [ + 194, + 198, + "loop", + "structure_element" + ], + [ + 206, + 213, + "monomer", + "oligomeric_state" + ], + [ + 244, + 251, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 151, + "sent": "Therefore, helix \u03b14 and the N-terminal loop were shifted to the position of KdpE by primarily modifying backbone torsion angles in the region immediately C-terminal to helix \u03b14.", + "section": "RESULTS", + "ner": [ + [ + 11, + 16, + "helix", + "structure_element" + ], + [ + 17, + 19, + "\u03b14", + "structure_element" + ], + [ + 39, + 43, + "loop", + "structure_element" + ], + [ + 76, + 80, + "KdpE", + "protein" + ], + [ + 168, + 173, + "helix", + "structure_element" + ], + [ + 174, + 176, + "\u03b14", + "structure_element" + ] + ] + }, + { + "sid": 152, + "sent": "Afterwards, helix \u03b14 and the adjacent loops were energy minimized with the MAB force field as implemented in the program Moloc; all other atoms of NsrR-RD were kept fixed.", + "section": "RESULTS", + "ner": [ + [ + 12, + 17, + "helix", + "structure_element" + ], + [ + 18, + 20, + "\u03b14", + "structure_element" + ], + [ + 38, + 43, + "loops", + "structure_element" + ], + [ + 49, + 90, + "energy minimized with the MAB force field", + "experimental_method" + ], + [ + 147, + 151, + "NsrR", + "protein" + ], + [ + 152, + 154, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 153, + "sent": "The result is highlighted in S2B Fig. The energy minimized structure of NsrR-RD was then superimposed on the dimeric structure of KdpE.", + "section": "RESULTS", + "ner": [ + [ + 42, + 58, + "energy minimized", + "protein_state" + ], + [ + 59, + 68, + "structure", + "evidence" + ], + [ + 72, + 76, + "NsrR", + "protein" + ], + [ + 77, + 79, + "RD", + "structure_element" + ], + [ + 89, + 101, + "superimposed", + "experimental_method" + ], + [ + 109, + 116, + "dimeric", + "oligomeric_state" + ], + [ + 117, + 126, + "structure", + "evidence" + ], + [ + 130, + 134, + "KdpE", + "protein" + ] + ] + }, + { + "sid": 154, + "sent": "The putative functional dimer of NsrR-RD is depicted in Fig 5.", + "section": "RESULTS", + "ner": [ + [ + 24, + 29, + "dimer", + "oligomeric_state" + ], + [ + 33, + 37, + "NsrR", + "protein" + ], + [ + 38, + 40, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 155, + "sent": "The dimeric interface is formed by \u03b14-\u03b25-\u03b15 of RD (Fig 5A), as previously observed in other RRs.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "dimeric interface", + "site" + ], + [ + 35, + 43, + "\u03b14-\u03b25-\u03b15", + "structure_element" + ], + [ + 47, + 49, + "RD", + "structure_element" + ], + [ + 92, + 95, + "RRs", + "protein_type" + ] + ] + }, + { + "sid": 156, + "sent": "In KdpE, a network of salt bridges and other electrostatic interactions stabilize the interface within a single monomer as well as between the monomers.", + "section": "RESULTS", + "ner": [ + [ + 3, + 7, + "KdpE", + "protein" + ], + [ + 22, + 34, + "salt bridges", + "bond_interaction" + ], + [ + 45, + 71, + "electrostatic interactions", + "bond_interaction" + ], + [ + 86, + 95, + "interface", + "site" + ], + [ + 112, + 119, + "monomer", + "oligomeric_state" + ], + [ + 143, + 151, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 157, + "sent": "Majority of these interactions involve residues that are highly conserved within the OmpR/PhoB subfamily of RRs.", + "section": "RESULTS", + "ner": [ + [ + 57, + 73, + "highly conserved", + "protein_state" + ], + [ + 85, + 104, + "OmpR/PhoB subfamily", + "protein_type" + ], + [ + 108, + 111, + "RRs", + "protein_type" + ] + ] + }, + { + "sid": 158, + "sent": "In addition, the dimeric interface of KdpE is characterized by hydrophobic patch formed by residues Ile88 (\u03b14), Leu91 (\u03b14), Ala110 (\u03b15), and Val114 (\u03b15).", + "section": "RESULTS", + "ner": [ + [ + 17, + 34, + "dimeric interface", + "site" + ], + [ + 38, + 42, + "KdpE", + "protein" + ], + [ + 63, + 80, + "hydrophobic patch", + "site" + ], + [ + 100, + 105, + "Ile88", + "residue_name_number" + ], + [ + 107, + 109, + "\u03b14", + "structure_element" + ], + [ + 112, + 117, + "Leu91", + "residue_name_number" + ], + [ + 119, + 121, + "\u03b14", + "structure_element" + ], + [ + 124, + 130, + "Ala110", + "residue_name_number" + ], + [ + 132, + 134, + "\u03b15", + "structure_element" + ], + [ + 141, + 147, + "Val114", + "residue_name_number" + ], + [ + 149, + 151, + "\u03b15", + "structure_element" + ] + ] + }, + { + "sid": 159, + "sent": "Structurally, a similar set of residues is also found in NsrR: Leu94 (\u03b14), Val110 (\u03b15) and Ala113 (\u03b15), respectively (depicted as spheres in Fig 5B), which are conserved to some extent on sequence level (highlighted in yellow; Fig 3).", + "section": "RESULTS", + "ner": [ + [ + 57, + 61, + "NsrR", + "protein" + ], + [ + 63, + 68, + "Leu94", + "residue_name_number" + ], + [ + 70, + 72, + "\u03b14", + "structure_element" + ], + [ + 75, + 81, + "Val110", + "residue_name_number" + ], + [ + 83, + 85, + "\u03b15", + "structure_element" + ], + [ + 91, + 97, + "Ala113", + "residue_name_number" + ], + [ + 99, + 101, + "\u03b15", + "structure_element" + ], + [ + 160, + 169, + "conserved", + "protein_state" + ] + ] + }, + { + "sid": 160, + "sent": "Functional dimer orientation of the RDs of NsrR.", + "section": "FIG", + "ner": [ + [ + 11, + 16, + "dimer", + "oligomeric_state" + ], + [ + 36, + 39, + "RDs", + "structure_element" + ], + [ + 43, + 47, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 161, + "sent": "Dimeric structure of the RD of NsrR aligned to the structure of KdpE (PDB code 1ZH2, not shown).", + "section": "FIG", + "ner": [ + [ + 0, + 7, + "Dimeric", + "oligomeric_state" + ], + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 25, + 27, + "RD", + "structure_element" + ], + [ + 31, + 35, + "NsrR", + "protein" + ], + [ + 51, + 60, + "structure", + "evidence" + ], + [ + 64, + 68, + "KdpE", + "protein" + ] + ] + }, + { + "sid": 162, + "sent": "(a) The two monomers of NsrR as functional dimers are represented in a cartoon representation displayed in cyan and yellow colors.", + "section": "FIG", + "ner": [ + [ + 12, + 20, + "monomers", + "oligomeric_state" + ], + [ + 24, + 28, + "NsrR", + "protein" + ], + [ + 43, + 49, + "dimers", + "oligomeric_state" + ] + ] + }, + { + "sid": 163, + "sent": "(b) Zoom-in of the dimeric interface mediated by \u03b14-\u03b25-\u03b15.", + "section": "FIG", + "ner": [ + [ + 19, + 36, + "dimeric interface", + "site" + ], + [ + 49, + 57, + "\u03b14-\u03b25-\u03b15", + "structure_element" + ] + ] + }, + { + "sid": 164, + "sent": "The monomer-monomer interactions are facilitated by hydrophobic residues (displayed as spheres), inter- and intra-domain interactions (displayed as sticks).", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "monomer", + "oligomeric_state" + ], + [ + 12, + 19, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 165, + "sent": "Conserved intermolecular electrostatic interactions further stabilize the monomer-monomer interaction of KdpE and are formed between Asp97 (\u03b25) and Arg111 (\u03b15), Asp96 (\u03b14\u2013\u03b25 loop) and Arg118 (\u03b15), and Asp92 (\u03b14) and Arg113 (\u03b15).", + "section": "RESULTS", + "ner": [ + [ + 25, + 51, + "electrostatic interactions", + "bond_interaction" + ], + [ + 74, + 81, + "monomer", + "oligomeric_state" + ], + [ + 82, + 89, + "monomer", + "oligomeric_state" + ], + [ + 105, + 109, + "KdpE", + "protein" + ], + [ + 133, + 138, + "Asp97", + "residue_name_number" + ], + [ + 140, + 142, + "\u03b25", + "structure_element" + ], + [ + 148, + 154, + "Arg111", + "residue_name_number" + ], + [ + 156, + 158, + "\u03b15", + "structure_element" + ], + [ + 161, + 166, + "Asp96", + "residue_name_number" + ], + [ + 168, + 178, + "\u03b14\u2013\u03b25 loop", + "structure_element" + ], + [ + 184, + 190, + "Arg118", + "residue_name_number" + ], + [ + 192, + 194, + "\u03b15", + "structure_element" + ], + [ + 201, + 206, + "Asp92", + "residue_name_number" + ], + [ + 208, + 210, + "\u03b14", + "structure_element" + ], + [ + 216, + 222, + "Arg113", + "residue_name_number" + ], + [ + 224, + 226, + "\u03b15", + "structure_element" + ] + ] + }, + { + "sid": 166, + "sent": "Some of these interactions can also be identified in the dimeric model of NsrR-RD.", + "section": "RESULTS", + "ner": [ + [ + 57, + 64, + "dimeric", + "oligomeric_state" + ], + [ + 74, + 78, + "NsrR", + "protein" + ], + [ + 79, + 81, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 167, + "sent": "Here, Asp100 (\u03b25) and Lys114 (\u03b15) form an interaction within one monomer, and an intermolecular interaction can be observed between Asn95 (\u03b14) of one monomer with Thr116 (\u03b15) of the other monomer (Fig 3, shown in cyan).", + "section": "RESULTS", + "ner": [ + [ + 6, + 12, + "Asp100", + "residue_name_number" + ], + [ + 14, + 16, + "\u03b25", + "structure_element" + ], + [ + 22, + 28, + "Lys114", + "residue_name_number" + ], + [ + 30, + 32, + "\u03b15", + "structure_element" + ], + [ + 65, + 72, + "monomer", + "oligomeric_state" + ], + [ + 132, + 137, + "Asn95", + "residue_name_number" + ], + [ + 139, + 141, + "\u03b14", + "structure_element" + ], + [ + 150, + 157, + "monomer", + "oligomeric_state" + ], + [ + 163, + 169, + "Thr116", + "residue_name_number" + ], + [ + 171, + 173, + "\u03b15", + "structure_element" + ], + [ + 188, + 195, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 168, + "sent": "Asp99 (\u03b14\u2013\u03b25 loop; Fig 3, shown in cyan) points toward the side chain of Arg121.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "Asp99", + "residue_name_number" + ], + [ + 7, + 17, + "\u03b14\u2013\u03b25 loop", + "structure_element" + ], + [ + 73, + 79, + "Arg121", + "residue_name_number" + ] + ] + }, + { + "sid": 169, + "sent": "This interaction is also observed in KdpE (Asp96 (\u03b14\u2013\u03b25 loop) and Arg118 (\u03b15)).", + "section": "RESULTS", + "ner": [ + [ + 37, + 41, + "KdpE", + "protein" + ], + [ + 43, + 48, + "Asp96", + "residue_name_number" + ], + [ + 50, + 60, + "\u03b14\u2013\u03b25 loop", + "structure_element" + ], + [ + 66, + 72, + "Arg118", + "residue_name_number" + ], + [ + 74, + 76, + "\u03b15", + "structure_element" + ] + ] + }, + { + "sid": 170, + "sent": "In KdpE, Arg111 is additionally stabilized by another intra-molecular salt bridge with Glu107 (\u03b15).", + "section": "RESULTS", + "ner": [ + [ + 3, + 7, + "KdpE", + "protein" + ], + [ + 9, + 15, + "Arg111", + "residue_name_number" + ], + [ + 70, + 81, + "salt bridge", + "bond_interaction" + ], + [ + 87, + 93, + "Glu107", + "residue_name_number" + ], + [ + 95, + 97, + "\u03b15", + "structure_element" + ] + ] + }, + { + "sid": 171, + "sent": "Interestingly, in NsrR-RD this amino acid corresponds to Val110 (highlighted in yellow in Fig 3).", + "section": "RESULTS", + "ner": [ + [ + 18, + 22, + "NsrR", + "protein" + ], + [ + 23, + 25, + "RD", + "structure_element" + ], + [ + 57, + 63, + "Val110", + "residue_name_number" + ] + ] + }, + { + "sid": 172, + "sent": "As observed in this alignment, the above-mentioned arginine residue (Arg111 in KdpE) is either an arginine or a lysine residue (Lys114 in NsrR) in all RRs used in the alignment (Fig 3, shown in cyan).", + "section": "RESULTS", + "ner": [ + [ + 20, + 29, + "alignment", + "experimental_method" + ], + [ + 51, + 59, + "arginine", + "residue_name" + ], + [ + 69, + 75, + "Arg111", + "residue_name_number" + ], + [ + 79, + 83, + "KdpE", + "protein" + ], + [ + 98, + 106, + "arginine", + "residue_name" + ], + [ + 112, + 118, + "lysine", + "residue_name" + ], + [ + 128, + 134, + "Lys114", + "residue_name_number" + ], + [ + 138, + 142, + "NsrR", + "protein" + ], + [ + 151, + 154, + "RRs", + "protein_type" + ], + [ + 167, + 176, + "alignment", + "experimental_method" + ] + ] + }, + { + "sid": 173, + "sent": "Interestingly, whenever an arginine is present at this position (Arg111 in KdpE), a glutamate (Glu107 in KdpE) is present as well, presumably stabilizing the arginine side chain.", + "section": "RESULTS", + "ner": [ + [ + 27, + 35, + "arginine", + "residue_name" + ], + [ + 65, + 71, + "Arg111", + "residue_name_number" + ], + [ + 75, + 79, + "KdpE", + "protein" + ], + [ + 84, + 93, + "glutamate", + "residue_name" + ], + [ + 95, + 101, + "Glu107", + "residue_name_number" + ], + [ + 105, + 109, + "KdpE", + "protein" + ], + [ + 158, + 166, + "arginine", + "residue_name" + ] + ] + }, + { + "sid": 174, + "sent": "However, when a lysine is present at this position, the glutamate is exchanged to a hydrophobic residue contributing to the hydrophobic patch described above.", + "section": "RESULTS", + "ner": [ + [ + 16, + 22, + "lysine", + "residue_name" + ], + [ + 56, + 65, + "glutamate", + "residue_name" + ], + [ + 124, + 141, + "hydrophobic patch", + "site" + ] + ] + }, + { + "sid": 175, + "sent": "Additionally, it has been shown for PhoB from E. coli and PhoP from B. subtilis that mutating the corresponding residues involved in dimerisation (residues Asp100, Val110 and Lys114 in NsrR) results in monomeric form of response regulator which has lost the ability to dimerize as well as display reduced DNA binding capabilities.", + "section": "RESULTS", + "ner": [ + [ + 36, + 40, + "PhoB", + "protein" + ], + [ + 46, + 53, + "E. coli", + "species" + ], + [ + 58, + 62, + "PhoP", + "protein" + ], + [ + 68, + 79, + "B. subtilis", + "species" + ], + [ + 85, + 93, + "mutating", + "experimental_method" + ], + [ + 156, + 162, + "Asp100", + "residue_name_number" + ], + [ + 164, + 170, + "Val110", + "residue_name_number" + ], + [ + 175, + 181, + "Lys114", + "residue_name_number" + ], + [ + 185, + 189, + "NsrR", + "protein" + ], + [ + 202, + 211, + "monomeric", + "oligomeric_state" + ], + [ + 220, + 238, + "response regulator", + "protein_type" + ], + [ + 249, + 277, + "lost the ability to dimerize", + "protein_state" + ], + [ + 305, + 308, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 176, + "sent": "Overall Structure of C-terminal DNA-binding effector domain of NsrR", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "Structure", + "evidence" + ], + [ + 32, + 59, + "DNA-binding effector domain", + "structure_element" + ], + [ + 63, + 67, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 177, + "sent": "The structure of NsrR-ED from S. agalactiae was determined using experimental phases from a single-wavelength anomalous dispersion dataset from the rectangular plate-shaped crystal derivatized with platinum at a resolution of 1.6 \u00c5 in space group P21212.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 17, + 21, + "NsrR", + "protein" + ], + [ + 22, + 24, + "ED", + "structure_element" + ], + [ + 30, + 43, + "S. agalactiae", + "species" + ], + [ + 92, + 138, + "single-wavelength anomalous dispersion dataset", + "experimental_method" + ], + [ + 198, + 206, + "platinum", + "chemical" + ] + ] + }, + { + "sid": 178, + "sent": "The Rwork and Rfree values after refinement were 0.18 and 0.22, respectively.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "Rwork", + "evidence" + ], + [ + 14, + 19, + "Rfree", + "evidence" + ] + ] + }, + { + "sid": 179, + "sent": "Ramachandran validation was done using MolProbity.", + "section": "RESULTS", + "ner": [ + [ + 0, + 23, + "Ramachandran validation", + "evidence" + ] + ] + }, + { + "sid": 180, + "sent": "The latter is Glu128 (last residue of the linker region) of chain B that is involved in crystal contacts and, therefore, likely adopts an unfavorable conformation.", + "section": "RESULTS", + "ner": [ + [ + 14, + 20, + "Glu128", + "residue_name_number" + ], + [ + 42, + 55, + "linker region", + "structure_element" + ], + [ + 60, + 67, + "chain B", + "structure_element" + ] + ] + }, + { + "sid": 181, + "sent": "The structure contained a few ethylene glycol molecules introduced by the cryo-protecting procedure.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 30, + 45, + "ethylene glycol", + "chemical" + ] + ] + }, + { + "sid": 182, + "sent": "The C-terminal effector DNA-binding domain of NsrR is about 13 kDa in size and consists of residues 129\u2013243 (including 21 amino acid residues of the expression tag).", + "section": "RESULTS", + "ner": [ + [ + 15, + 42, + "effector DNA-binding domain", + "structure_element" + ], + [ + 46, + 50, + "NsrR", + "protein" + ], + [ + 100, + 107, + "129\u2013243", + "residue_range" + ] + ] + }, + { + "sid": 183, + "sent": "Monomer A contains residue 129\u2013224 and monomer B contain residues 128\u2013225.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Monomer", + "oligomeric_state" + ], + [ + 8, + 9, + "A", + "structure_element" + ], + [ + 27, + 34, + "129\u2013224", + "residue_range" + ], + [ + 39, + 46, + "monomer", + "oligomeric_state" + ], + [ + 47, + 48, + "B", + "structure_element" + ], + [ + 66, + 73, + "128\u2013225", + "residue_range" + ] + ] + }, + { + "sid": 184, + "sent": "For Asp147 of chain A and Glu174 of chain B, poor electron density was observed for the side chains and, thus, these side chains were removed during refinement.", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "Asp147", + "residue_name_number" + ], + [ + 14, + 21, + "chain A", + "structure_element" + ], + [ + 26, + 32, + "Glu174", + "residue_name_number" + ], + [ + 36, + 43, + "chain B", + "structure_element" + ], + [ + 50, + 66, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 185, + "sent": "The asymmetric unit contains two copies of NsrR-ED related by two-fold rotational symmetry.", + "section": "RESULTS", + "ner": [ + [ + 43, + 47, + "NsrR", + "protein" + ], + [ + 48, + 50, + "ED", + "structure_element" + ] + ] + }, + { + "sid": 186, + "sent": "An overlay revealed that both monomers display high similarity in their overall structure with an rmsd of 0.5 \u00c5 over 95 C\u03b1 atoms.", + "section": "RESULTS", + "ner": [ + [ + 3, + 10, + "overlay", + "experimental_method" + ], + [ + 30, + 38, + "monomers", + "oligomeric_state" + ], + [ + 80, + 89, + "structure", + "evidence" + ], + [ + 98, + 102, + "rmsd", + "evidence" + ] + ] + }, + { + "sid": 187, + "sent": "We therefore describe for the overall structure only monomer A.", + "section": "RESULTS", + "ner": [ + [ + 38, + 47, + "structure", + "evidence" + ], + [ + 53, + 60, + "monomer", + "oligomeric_state" + ], + [ + 61, + 62, + "A", + "structure_element" + ] + ] + }, + { + "sid": 188, + "sent": "The ED domain of NsrR consists of six \u03b2-strands and three \u03b1-helices in a \u03b26-\u03b27-\u03b28-\u03b29-\u03b16-\u03b17-\u03b18-\u03b210-\u03b211 topology (the secondary structure elements are counted in continuation of those of the RD).", + "section": "RESULTS", + "ner": [ + [ + 4, + 6, + "ED", + "structure_element" + ], + [ + 17, + 21, + "NsrR", + "protein" + ], + [ + 38, + 47, + "\u03b2-strands", + "structure_element" + ], + [ + 58, + 67, + "\u03b1-helices", + "structure_element" + ], + [ + 73, + 101, + "\u03b26-\u03b27-\u03b28-\u03b29-\u03b16-\u03b17-\u03b18-\u03b210-\u03b211", + "structure_element" + ], + [ + 189, + 191, + "RD", + "structure_element" + ] + ] + }, + { + "sid": 189, + "sent": "The effector domain starts with a 4-stranded antiparallel \u03b2-sheet, followed by three \u03b1-helices and eventually ends in a C-terminal \u03b2-hairpin (Fig 6).", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "effector domain", + "structure_element" + ], + [ + 34, + 65, + "4-stranded antiparallel \u03b2-sheet", + "structure_element" + ], + [ + 85, + 94, + "\u03b1-helices", + "structure_element" + ], + [ + 131, + 140, + "\u03b2-hairpin", + "structure_element" + ] + ] + }, + { + "sid": 190, + "sent": "The two \u03b2-sheets sandwich the three \u03b1-helices.", + "section": "RESULTS", + "ner": [ + [ + 8, + 16, + "\u03b2-sheets", + "structure_element" + ], + [ + 36, + 45, + "\u03b1-helices", + "structure_element" + ] + ] + }, + { + "sid": 191, + "sent": "Structure of the C-terminal effector domain of NsrR.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 28, + 43, + "effector domain", + "structure_element" + ], + [ + 47, + 51, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 192, + "sent": "Cartoon representation of the C-terminal effector domain of NsrR (green; recognition helix in cyan).", + "section": "FIG", + "ner": [ + [ + 41, + 56, + "effector domain", + "structure_element" + ], + [ + 60, + 64, + "NsrR", + "protein" + ], + [ + 73, + 90, + "recognition helix", + "structure_element" + ] + ] + }, + { + "sid": 193, + "sent": "The structural areas with the highest variations compared to the effector domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1GXQ) are marked.", + "section": "FIG", + "ner": [ + [ + 65, + 81, + "effector domains", + "structure_element" + ], + [ + 85, + 89, + "DrrB", + "protein" + ], + [ + 104, + 108, + "MtrA", + "protein" + ], + [ + 127, + 131, + "PhoB", + "protein" + ] + ] + }, + { + "sid": 194, + "sent": "The transactivation loop of MtrA is missing in the structure, therefore, the two termini are connected by a dashed line.", + "section": "FIG", + "ner": [ + [ + 4, + 24, + "transactivation loop", + "structure_element" + ], + [ + 28, + 32, + "MtrA", + "protein" + ], + [ + 36, + 43, + "missing", + "protein_state" + ], + [ + 51, + 60, + "structure", + "evidence" + ] + ] + }, + { + "sid": 195, + "sent": "The characteristic feature of the OmpR/PhoB subfamily of RRs is a winged helix-turn-helix (wHTH) fold that is adopted by the \u03b17-loop-\u03b18 segment in full-length and single effector domain structures of RRs of this subfamily.", + "section": "RESULTS", + "ner": [ + [ + 34, + 53, + "OmpR/PhoB subfamily", + "protein_type" + ], + [ + 57, + 60, + "RRs", + "protein_type" + ], + [ + 66, + 89, + "winged helix-turn-helix", + "structure_element" + ], + [ + 91, + 95, + "wHTH", + "structure_element" + ], + [ + 125, + 143, + "\u03b17-loop-\u03b18 segment", + "structure_element" + ], + [ + 147, + 158, + "full-length", + "protein_state" + ], + [ + 170, + 185, + "effector domain", + "structure_element" + ], + [ + 186, + 196, + "structures", + "evidence" + ], + [ + 200, + 203, + "RRs", + "protein_type" + ] + ] + }, + { + "sid": 196, + "sent": "The structure of NsrR-ED also contains such a wHTH motif built up by helices \u03b17 and \u03b18 (Fig 6).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 17, + 21, + "NsrR", + "protein" + ], + [ + 22, + 24, + "ED", + "structure_element" + ], + [ + 46, + 50, + "wHTH", + "structure_element" + ], + [ + 69, + 76, + "helices", + "structure_element" + ], + [ + 77, + 79, + "\u03b17", + "structure_element" + ], + [ + 84, + 86, + "\u03b18", + "structure_element" + ] + ] + }, + { + "sid": 197, + "sent": "The second helix of the wHTH motif is important for DNA-binding and, therefore, is termed \u201crecognition helix\u201d (shown in cyan in Fig 6).", + "section": "RESULTS", + "ner": [ + [ + 11, + 16, + "helix", + "structure_element" + ], + [ + 24, + 28, + "wHTH", + "structure_element" + ], + [ + 52, + 55, + "DNA", + "chemical" + ], + [ + 91, + 108, + "recognition helix", + "structure_element" + ] + ] + }, + { + "sid": 198, + "sent": "Furthermore, a helix within the HTH motif, named \u201cpositioning helix\u201d, is important for proper orientation and positioning of the loop between these two helices and is referred to as \u201ctransactivation loop\u201d (also called \u03b1-loop; Fig 6).", + "section": "RESULTS", + "ner": [ + [ + 15, + 20, + "helix", + "structure_element" + ], + [ + 32, + 35, + "HTH", + "structure_element" + ], + [ + 50, + 67, + "positioning helix", + "structure_element" + ], + [ + 129, + 133, + "loop", + "structure_element" + ], + [ + 183, + 203, + "transactivation loop", + "structure_element" + ], + [ + 218, + 224, + "\u03b1-loop", + "structure_element" + ] + ] + }, + { + "sid": 199, + "sent": "In the structure of NsrR-ED, helix \u03b18 is identified as the recognition helix, \u03b17 as the positioning helix, and the loop region between helices \u03b17-\u03b18 as transactivation loop as observed in other RRs (Fig 6).", + "section": "RESULTS", + "ner": [ + [ + 7, + 16, + "structure", + "evidence" + ], + [ + 20, + 24, + "NsrR", + "protein" + ], + [ + 25, + 27, + "ED", + "structure_element" + ], + [ + 29, + 34, + "helix", + "structure_element" + ], + [ + 35, + 37, + "\u03b18", + "structure_element" + ], + [ + 59, + 76, + "recognition helix", + "structure_element" + ], + [ + 78, + 80, + "\u03b17", + "structure_element" + ], + [ + 88, + 105, + "positioning helix", + "structure_element" + ], + [ + 115, + 126, + "loop region", + "structure_element" + ], + [ + 143, + 148, + "\u03b17-\u03b18", + "structure_element" + ], + [ + 152, + 172, + "transactivation loop", + "structure_element" + ], + [ + 194, + 197, + "RRs", + "protein_type" + ] + ] + }, + { + "sid": 200, + "sent": "The 16-residue long, solvent-exposed recognition helix \u03b18 of NsrR-ED contains four positively charged residues that can potentially interact with DNA.", + "section": "RESULTS", + "ner": [ + [ + 21, + 36, + "solvent-exposed", + "protein_state" + ], + [ + 37, + 54, + "recognition helix", + "structure_element" + ], + [ + 55, + 57, + "\u03b18", + "structure_element" + ], + [ + 61, + 65, + "NsrR", + "protein" + ], + [ + 66, + 68, + "ED", + "structure_element" + ], + [ + 146, + 149, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 201, + "sent": "These are Arg198, Arg200, Lys201, and Lys202.", + "section": "RESULTS", + "ner": [ + [ + 10, + 16, + "Arg198", + "residue_name_number" + ], + [ + 18, + 24, + "Arg200", + "residue_name_number" + ], + [ + 26, + 32, + "Lys201", + "residue_name_number" + ], + [ + 38, + 44, + "Lys202", + "residue_name_number" + ] + ] + }, + { + "sid": 202, + "sent": "When comparing the sequence of NsrR with PhoB, KdpE, and MtrA, the alignment (Fig 3, colored in blue) emphasizes the variations at these positions, except for Arg200, which is conserved throughout the lantibiotic resistance RRs.", + "section": "RESULTS", + "ner": [ + [ + 31, + 35, + "NsrR", + "protein" + ], + [ + 41, + 45, + "PhoB", + "protein" + ], + [ + 47, + 51, + "KdpE", + "protein" + ], + [ + 57, + 61, + "MtrA", + "protein" + ], + [ + 67, + 76, + "alignment", + "experimental_method" + ], + [ + 159, + 165, + "Arg200", + "residue_name_number" + ], + [ + 176, + 185, + "conserved", + "protein_state" + ], + [ + 201, + 227, + "lantibiotic resistance RRs", + "protein_type" + ] + ] + }, + { + "sid": 203, + "sent": "Additionally, Lys202 is also highly conserved throughout the family of RRs except PhoB, clearly reflecting differences in the sequences of DNA to be bound.", + "section": "RESULTS", + "ner": [ + [ + 14, + 20, + "Lys202", + "residue_name_number" + ], + [ + 29, + 45, + "highly conserved", + "protein_state" + ], + [ + 71, + 74, + "RRs", + "protein_type" + ], + [ + 82, + 86, + "PhoB", + "protein" + ], + [ + 139, + 142, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 204, + "sent": "Comparison with structures of other effector domains", + "section": "RESULTS", + "ner": [ + [ + 16, + 26, + "structures", + "evidence" + ], + [ + 36, + 52, + "effector domains", + "structure_element" + ] + ] + }, + { + "sid": 205, + "sent": "We performed a DALI search to identify structurally related proteins to NsrR-ED.", + "section": "RESULTS", + "ner": [ + [ + 15, + 26, + "DALI search", + "experimental_method" + ], + [ + 72, + 76, + "NsrR", + "protein" + ], + [ + 77, + 79, + "ED", + "structure_element" + ] + ] + }, + { + "sid": 206, + "sent": "Here the structure of the effector domain of PhoB from E. coli (PDB code: 1GXQ) (Z-score of 13.7) is structurally the most closely related.", + "section": "RESULTS", + "ner": [ + [ + 9, + 18, + "structure", + "evidence" + ], + [ + 26, + 41, + "effector domain", + "structure_element" + ], + [ + 45, + 49, + "PhoB", + "protein" + ], + [ + 55, + 62, + "E. coli", + "species" + ], + [ + 81, + 88, + "Z-score", + "evidence" + ] + ] + }, + { + "sid": 207, + "sent": "Similar to the PhoB effector domain, a 9-residues long loop (amino acid 182\u2013189) is also present in the structure of NsrR-ED that connects helices \u03b17 and \u03b18.", + "section": "RESULTS", + "ner": [ + [ + 15, + 19, + "PhoB", + "protein" + ], + [ + 20, + 35, + "effector domain", + "structure_element" + ], + [ + 55, + 59, + "loop", + "structure_element" + ], + [ + 72, + 79, + "182\u2013189", + "residue_range" + ], + [ + 104, + 113, + "structure", + "evidence" + ], + [ + 117, + 121, + "NsrR", + "protein" + ], + [ + 122, + 124, + "ED", + "structure_element" + ], + [ + 139, + 146, + "helices", + "structure_element" + ], + [ + 147, + 149, + "\u03b17", + "structure_element" + ], + [ + 154, + 156, + "\u03b18", + "structure_element" + ] + ] + }, + { + "sid": 208, + "sent": "The rmsd between the three helices of the effector domain (including the two helices forming the wHTH motif) of PhoB and NsrR-ED is 1.6 \u00c5 over 47 C\u03b1 atoms, clearly indicating that NsrR belongs to the OmpR/PhoB family of RRs.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "rmsd", + "evidence" + ], + [ + 42, + 57, + "effector domain", + "structure_element" + ], + [ + 77, + 84, + "helices", + "structure_element" + ], + [ + 97, + 101, + "wHTH", + "structure_element" + ], + [ + 112, + 116, + "PhoB", + "protein" + ], + [ + 121, + 125, + "NsrR", + "protein" + ], + [ + 126, + 128, + "ED", + "structure_element" + ], + [ + 180, + 184, + "NsrR", + "protein" + ], + [ + 200, + 223, + "OmpR/PhoB family of RRs", + "protein_type" + ] + ] + }, + { + "sid": 209, + "sent": "Therefore, we superimposed the C\u03b1 traces of the effector domain of NsrR (NsrR-ED) with other previously determined effector domains from the OmpR/PhoB family such as DrrB, MtrA and of only the effector domain structure of PhoB from E. coli.", + "section": "RESULTS", + "ner": [ + [ + 14, + 26, + "superimposed", + "experimental_method" + ], + [ + 48, + 63, + "effector domain", + "structure_element" + ], + [ + 67, + 71, + "NsrR", + "protein" + ], + [ + 73, + 77, + "NsrR", + "protein" + ], + [ + 78, + 80, + "ED", + "structure_element" + ], + [ + 115, + 131, + "effector domains", + "structure_element" + ], + [ + 141, + 157, + "OmpR/PhoB family", + "protein_type" + ], + [ + 166, + 170, + "DrrB", + "protein" + ], + [ + 172, + 176, + "MtrA", + "protein" + ], + [ + 193, + 208, + "effector domain", + "structure_element" + ], + [ + 209, + 218, + "structure", + "evidence" + ], + [ + 222, + 226, + "PhoB", + "protein" + ], + [ + 232, + 239, + "E. coli", + "species" + ] + ] + }, + { + "sid": 210, + "sent": "Overall, the structures are very similar with rmsd\u2019s ranging from 1.7 to 2.6 \u00c5 (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 13, + 23, + "structures", + "evidence" + ], + [ + 46, + 50, + "rmsd", + "evidence" + ] + ] + }, + { + "sid": 211, + "sent": "The highest variations (Fig 6) are visible in in the loop regions \u03b17-\u03b18, which corresponds to the transactivation loop.", + "section": "RESULTS", + "ner": [ + [ + 53, + 57, + "loop", + "structure_element" + ], + [ + 66, + 71, + "\u03b17-\u03b18", + "structure_element" + ], + [ + 98, + 118, + "transactivation loop", + "structure_element" + ] + ] + }, + { + "sid": 212, + "sent": "In many RRs this transactivation loop along with the recognition helix \u03b18, form inter-domain contacts in the inactive state and are only exposed upon activation of the RRs via a conformational change where the N- and C-terminal domains move away from each other.", + "section": "RESULTS", + "ner": [ + [ + 8, + 11, + "RRs", + "protein_type" + ], + [ + 17, + 37, + "transactivation loop", + "structure_element" + ], + [ + 53, + 70, + "recognition helix", + "structure_element" + ], + [ + 71, + 73, + "\u03b18", + "structure_element" + ], + [ + 109, + 117, + "inactive", + "protein_state" + ], + [ + 168, + 171, + "RRs", + "protein_type" + ] + ] + }, + { + "sid": 213, + "sent": "Linker region", + "section": "RESULTS", + "ner": [ + [ + 0, + 13, + "Linker region", + "structure_element" + ] + ] + }, + { + "sid": 214, + "sent": "The linkers that connect the RDs and EDs in response regulators are highly variable with respect to both length and sequence.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "linkers", + "structure_element" + ], + [ + 29, + 32, + "RDs", + "structure_element" + ], + [ + 37, + 40, + "EDs", + "structure_element" + ], + [ + 44, + 63, + "response regulators", + "protein_type" + ], + [ + 68, + 83, + "highly variable", + "protein_state" + ] + ] + }, + { + "sid": 215, + "sent": "The exact boundaries of these linkers are difficult to predict from sequence alignments in the absence of structural information of the distinct RR.", + "section": "RESULTS", + "ner": [ + [ + 30, + 37, + "linkers", + "structure_element" + ], + [ + 68, + 87, + "sequence alignments", + "experimental_method" + ], + [ + 95, + 105, + "absence of", + "protein_state" + ], + [ + 145, + 147, + "RR", + "protein_type" + ] + ] + }, + { + "sid": 216, + "sent": "Linker lengths in OmpR/PhoB proteins of unknown structure have been estimated by comparing the number of residues between conserved landmark residues in the regulatory and effector domains to those from structurally characterized family members.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Linker", + "structure_element" + ], + [ + 18, + 36, + "OmpR/PhoB proteins", + "protein_type" + ], + [ + 157, + 188, + "regulatory and effector domains", + "structure_element" + ] + ] + }, + { + "sid": 217, + "sent": "Similar to the OmpR/PhoB family, the lantibiotic resistance-associated family of response regulators also displays diverse linker regions, which are recognized in sequence alignments by the introduction of gaps (Fig 3).", + "section": "RESULTS", + "ner": [ + [ + 15, + 31, + "OmpR/PhoB family", + "protein_type" + ], + [ + 37, + 100, + "lantibiotic resistance-associated family of response regulators", + "protein_type" + ], + [ + 123, + 137, + "linker regions", + "structure_element" + ], + [ + 163, + 182, + "sequence alignments", + "experimental_method" + ] + ] + }, + { + "sid": 218, + "sent": "Interestingly, two arginine residues (Arg120 and Arg121 in NsrR; Fig 3, shown in purple) at the end of the RDs seem to be strictly conserved throughout the family of response regulators in both the OmpR/PhoB and lantibiotic resistance-associated RRs, indicating a conserved similarity.", + "section": "RESULTS", + "ner": [ + [ + 19, + 27, + "arginine", + "residue_name" + ], + [ + 38, + 44, + "Arg120", + "residue_name_number" + ], + [ + 49, + 55, + "Arg121", + "residue_name_number" + ], + [ + 59, + 63, + "NsrR", + "protein" + ], + [ + 107, + 110, + "RDs", + "structure_element" + ], + [ + 122, + 140, + "strictly conserved", + "protein_state" + ], + [ + 166, + 185, + "response regulators", + "protein_type" + ], + [ + 198, + 249, + "OmpR/PhoB and lantibiotic resistance-associated RRs", + "protein_type" + ], + [ + 264, + 273, + "conserved", + "protein_state" + ] + ] + }, + { + "sid": 219, + "sent": "As seen in the structures of MtrA and KdpE, this arginine residue residing at the end of \u03b15 participates in the active state dimer interface of the RD through a salt bridge interaction with an aspartate residue.", + "section": "RESULTS", + "ner": [ + [ + 15, + 25, + "structures", + "evidence" + ], + [ + 29, + 33, + "MtrA", + "protein" + ], + [ + 38, + 42, + "KdpE", + "protein" + ], + [ + 49, + 57, + "arginine", + "residue_name" + ], + [ + 89, + 91, + "\u03b15", + "structure_element" + ], + [ + 112, + 118, + "active", + "protein_state" + ], + [ + 125, + 140, + "dimer interface", + "site" + ], + [ + 148, + 150, + "RD", + "structure_element" + ], + [ + 161, + 172, + "salt bridge", + "bond_interaction" + ], + [ + 193, + 202, + "aspartate", + "residue_name" + ] + ] + }, + { + "sid": 220, + "sent": "This aspartate residue is identified in NsrR as Asp99 (see above).", + "section": "RESULTS", + "ner": [ + [ + 5, + 14, + "aspartate", + "residue_name" + ], + [ + 40, + 44, + "NsrR", + "protein" + ], + [ + 48, + 53, + "Asp99", + "residue_name_number" + ] + ] + }, + { + "sid": 221, + "sent": "Arginine 121 of NsrR points towards this Asp99 residue however, the distance for a salt bridge interaction is too large.", + "section": "RESULTS", + "ner": [ + [ + 0, + 12, + "Arginine 121", + "residue_name_number" + ], + [ + 16, + 20, + "NsrR", + "protein" + ], + [ + 41, + 46, + "Asp99", + "residue_name_number" + ], + [ + 83, + 94, + "salt bridge", + "bond_interaction" + ] + ] + }, + { + "sid": 222, + "sent": "Although we aimed at crystallizing full-length NsrR, this endeavor failed due to proteolytic cleavage within the linker region during the time period of crystallization.", + "section": "RESULTS", + "ner": [ + [ + 21, + 34, + "crystallizing", + "experimental_method" + ], + [ + 35, + 46, + "full-length", + "protein_state" + ], + [ + 47, + 51, + "NsrR", + "protein" + ], + [ + 113, + 126, + "linker region", + "structure_element" + ], + [ + 153, + 168, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 223, + "sent": "Nonetheless, the structures of NsrR-RD and NsrR-ED together provide the required structural knowledge to predict the linker region that joins the receiver and effector domains.", + "section": "RESULTS", + "ner": [ + [ + 17, + 27, + "structures", + "evidence" + ], + [ + 31, + 35, + "NsrR", + "protein" + ], + [ + 36, + 38, + "RD", + "structure_element" + ], + [ + 43, + 47, + "NsrR", + "protein" + ], + [ + 48, + 50, + "ED", + "structure_element" + ], + [ + 117, + 130, + "linker region", + "structure_element" + ], + [ + 146, + 175, + "receiver and effector domains", + "structure_element" + ] + ] + }, + { + "sid": 224, + "sent": "The linker region of NsrR consists of approximately nine residues (Fig 3), comprising 120RRSQQFIQQ128 (underlined residues are neither present in the structure of RD nor in ED of NsrR) and contains two positively charged amino acids.", + "section": "RESULTS", + "ner": [ + [ + 4, + 17, + "linker region", + "structure_element" + ], + [ + 21, + 25, + "NsrR", + "protein" + ], + [ + 86, + 101, + "120RRSQQFIQQ128", + "structure_element" + ], + [ + 150, + 159, + "structure", + "evidence" + ], + [ + 163, + 165, + "RD", + "structure_element" + ], + [ + 173, + 175, + "ED", + "structure_element" + ], + [ + 179, + 183, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 225, + "sent": "DNA-binding mode of NsrR using a full-length model", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "DNA", + "chemical" + ], + [ + 20, + 24, + "NsrR", + "protein" + ], + [ + 33, + 44, + "full-length", + "protein_state" + ] + ] + }, + { + "sid": 226, + "sent": "Since the structures of both domains of NsrR were determined, we used this structural information together with the available crystal structures of related proteins to create a model of the full-length NsrR in its active and inactive state.", + "section": "RESULTS", + "ner": [ + [ + 10, + 20, + "structures", + "evidence" + ], + [ + 40, + 44, + "NsrR", + "protein" + ], + [ + 75, + 97, + "structural information", + "evidence" + ], + [ + 126, + 144, + "crystal structures", + "evidence" + ], + [ + 190, + 201, + "full-length", + "protein_state" + ], + [ + 202, + 206, + "NsrR", + "protein" + ], + [ + 214, + 220, + "active", + "protein_state" + ], + [ + 225, + 233, + "inactive", + "protein_state" + ] + ] + }, + { + "sid": 227, + "sent": "To achieve this, we first carefully analyzed the outcome of the Dali search for each domain and identified structurally highly similar proteins (based on Z-scores and rmsd values) and choose the full-length structures previously reported.", + "section": "RESULTS", + "ner": [ + [ + 64, + 75, + "Dali search", + "experimental_method" + ], + [ + 154, + 162, + "Z-scores", + "evidence" + ], + [ + 167, + 171, + "rmsd", + "evidence" + ], + [ + 195, + 206, + "full-length", + "protein_state" + ], + [ + 207, + 217, + "structures", + "evidence" + ] + ] + }, + { + "sid": 228, + "sent": "This resulted in a list of possible templates for modeling the full-length structure of NsrR (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 63, + 74, + "full-length", + "protein_state" + ], + [ + 75, + 84, + "structure", + "evidence" + ], + [ + 88, + 92, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 229, + "sent": "In solution, RRs exist in equilibrium between the active and inactive state, which is shifted towards the active state upon phosphorylation of the ED.", + "section": "RESULTS", + "ner": [ + [ + 13, + 16, + "RRs", + "protein_type" + ], + [ + 50, + 56, + "active", + "protein_state" + ], + [ + 61, + 69, + "inactive", + "protein_state" + ], + [ + 106, + 112, + "active", + "protein_state" + ], + [ + 124, + 139, + "phosphorylation", + "ptm" + ], + [ + 147, + 149, + "ED", + "structure_element" + ] + ] + }, + { + "sid": 230, + "sent": "This results in oligomerization of the RR and a higher affinity towards DNA.", + "section": "RESULTS", + "ner": [ + [ + 39, + 41, + "RR", + "protein_type" + ], + [ + 72, + 75, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 231, + "sent": "Based on the above-mentioned criteria, the structure of MtrA from M. tuberculosis, crystallized in an inactive and non-phosphorylated state, seemed best suited for modeling purposes.", + "section": "RESULTS", + "ner": [ + [ + 43, + 52, + "structure", + "evidence" + ], + [ + 56, + 60, + "MtrA", + "protein" + ], + [ + 66, + 81, + "M. tuberculosis", + "species" + ], + [ + 83, + 95, + "crystallized", + "experimental_method" + ], + [ + 102, + 110, + "inactive", + "protein_state" + ], + [ + 115, + 133, + "non-phosphorylated", + "protein_state" + ] + ] + }, + { + "sid": 232, + "sent": "Furthermore, the linker between the two domains of MtrA contains nine amino acids and is of similar length as the linker of NsrR. We aligned the NsrR-RD and -ED to the corresponding MtrA domains and evaluated the structure.", + "section": "RESULTS", + "ner": [ + [ + 17, + 23, + "linker", + "structure_element" + ], + [ + 51, + 55, + "MtrA", + "protein" + ], + [ + 114, + 120, + "linker", + "structure_element" + ], + [ + 124, + 128, + "NsrR", + "protein" + ], + [ + 133, + 140, + "aligned", + "experimental_method" + ], + [ + 145, + 149, + "NsrR", + "protein" + ], + [ + 150, + 152, + "RD", + "structure_element" + ], + [ + 158, + 160, + "ED", + "structure_element" + ], + [ + 182, + 186, + "MtrA", + "protein" + ], + [ + 213, + 222, + "structure", + "evidence" + ] + ] + }, + { + "sid": 233, + "sent": "This mimics the closed inactive conformation of NsrR (Fig 7A; the missing linker is represented as dotted line).", + "section": "RESULTS", + "ner": [ + [ + 16, + 22, + "closed", + "protein_state" + ], + [ + 23, + 31, + "inactive", + "protein_state" + ], + [ + 48, + 52, + "NsrR", + "protein" + ], + [ + 66, + 73, + "missing", + "protein_state" + ], + [ + 74, + 80, + "linker", + "structure_element" + ] + ] + }, + { + "sid": 234, + "sent": "Model of full-length NsrR in its inactive state and active state.", + "section": "FIG", + "ner": [ + [ + 9, + 20, + "full-length", + "protein_state" + ], + [ + 21, + 25, + "NsrR", + "protein" + ], + [ + 33, + 41, + "inactive", + "protein_state" + ], + [ + 52, + 58, + "active", + "protein_state" + ] + ] + }, + { + "sid": 235, + "sent": "The RD domain of NsrR is highlighted in yellow and the ED domain in green with the \u201crecognition helix\u201d colored in cyan. (a) Inactive state conformation: Both domains of NsrR were aligned to the structure of MtrA (not shown), which adopts a closed inactive conformation, to obtain a model of full-length NsrR. Phe101 and Asp187 stabilize this closed conformation.", + "section": "FIG", + "ner": [ + [ + 4, + 6, + "RD", + "structure_element" + ], + [ + 17, + 21, + "NsrR", + "protein" + ], + [ + 55, + 57, + "ED", + "structure_element" + ], + [ + 84, + 101, + "recognition helix", + "structure_element" + ], + [ + 124, + 132, + "Inactive", + "protein_state" + ], + [ + 169, + 173, + "NsrR", + "protein" + ], + [ + 179, + 186, + "aligned", + "experimental_method" + ], + [ + 194, + 203, + "structure", + "evidence" + ], + [ + 207, + 211, + "MtrA", + "protein" + ], + [ + 240, + 246, + "closed", + "protein_state" + ], + [ + 247, + 255, + "inactive", + "protein_state" + ], + [ + 291, + 302, + "full-length", + "protein_state" + ], + [ + 303, + 307, + "NsrR", + "protein" + ], + [ + 309, + 315, + "Phe101", + "residue_name_number" + ], + [ + 320, + 326, + "Asp187", + "residue_name_number" + ], + [ + 342, + 348, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 236, + "sent": "The missing linker is represented by a dotted line.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "missing", + "protein_state" + ], + [ + 12, + 18, + "linker", + "structure_element" + ] + ] + }, + { + "sid": 237, + "sent": "(b) Active state conformation: A model of full-length NsrR in active conformation based on the alignment of both the domains of NsrR to the structure of DNA bound structure of KdpE (PDB code: 4KNY), adopting an active open conformation, where the other molecule of NsrR is shown in shades of blue with the recognition helix colored in green.", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "Active", + "protein_state" + ], + [ + 42, + 53, + "full-length", + "protein_state" + ], + [ + 54, + 58, + "NsrR", + "protein" + ], + [ + 62, + 68, + "active", + "protein_state" + ], + [ + 95, + 104, + "alignment", + "experimental_method" + ], + [ + 128, + 132, + "NsrR", + "protein" + ], + [ + 140, + 149, + "structure", + "evidence" + ], + [ + 153, + 162, + "DNA bound", + "protein_state" + ], + [ + 163, + 172, + "structure", + "evidence" + ], + [ + 176, + 180, + "KdpE", + "protein" + ], + [ + 211, + 217, + "active", + "protein_state" + ], + [ + 218, + 222, + "open", + "protein_state" + ], + [ + 265, + 269, + "NsrR", + "protein" + ], + [ + 306, + 323, + "recognition helix", + "structure_element" + ] + ] + }, + { + "sid": 238, + "sent": "In MtrA, the two domains interact via the \u03b14-\u03b25-\u03b15 interface of the receiver domain and the end of \u03b17, \u03b17-\u03b18 loop and \u03b18 of the effector domain.", + "section": "RESULTS", + "ner": [ + [ + 3, + 7, + "MtrA", + "protein" + ], + [ + 42, + 60, + "\u03b14-\u03b25-\u03b15 interface", + "site" + ], + [ + 68, + 83, + "receiver domain", + "structure_element" + ], + [ + 99, + 101, + "\u03b17", + "structure_element" + ], + [ + 103, + 113, + "\u03b17-\u03b18 loop", + "structure_element" + ], + [ + 118, + 120, + "\u03b18", + "structure_element" + ], + [ + 128, + 143, + "effector domain", + "structure_element" + ] + ] + }, + { + "sid": 239, + "sent": "Both interfaces have been shown to form functionally important contact areas in the active state within members of the OmpR/PhoB subfamily.", + "section": "RESULTS", + "ner": [ + [ + 5, + 15, + "interfaces", + "site" + ], + [ + 84, + 90, + "active", + "protein_state" + ], + [ + 119, + 138, + "OmpR/PhoB subfamily", + "protein_type" + ] + ] + }, + { + "sid": 240, + "sent": "In our model of full-length NsrR, a similar orientation between the domains is observed, contributing to the inter-domain interactions.", + "section": "RESULTS", + "ner": [ + [ + 16, + 27, + "full-length", + "protein_state" + ], + [ + 28, + 32, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 241, + "sent": "The inactive conformation of MtrA is supported by the orientation of the side chain of Tyr102, which points away from the active Asp56 residue, while the side chain of Tyr102 interacts with Asp190 of the RD of MtrA, thereby stabilizing its closed conformation.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "inactive", + "protein_state" + ], + [ + 29, + 33, + "MtrA", + "protein" + ], + [ + 87, + 93, + "Tyr102", + "residue_name_number" + ], + [ + 122, + 128, + "active", + "protein_state" + ], + [ + 129, + 134, + "Asp56", + "residue_name_number" + ], + [ + 168, + 174, + "Tyr102", + "residue_name_number" + ], + [ + 190, + 196, + "Asp190", + "residue_name_number" + ], + [ + 204, + 206, + "RD", + "structure_element" + ], + [ + 210, + 214, + "MtrA", + "protein" + ], + [ + 240, + 246, + "closed", + "protein_state" + ] + ] + }, + { + "sid": 242, + "sent": "In the model of NsrR, similar amino acids are present, Phe101 (switch residue) and Asp188 (Fig 3, represented by orange boxes) forming a likewise similar network of interaction.", + "section": "RESULTS", + "ner": [ + [ + 16, + 20, + "NsrR", + "protein" + ], + [ + 55, + 61, + "Phe101", + "residue_name_number" + ], + [ + 63, + 77, + "switch residue", + "site" + ], + [ + 83, + 89, + "Asp188", + "residue_name_number" + ] + ] + }, + { + "sid": 243, + "sent": "Next, we were interested in the active conformation of the NsrR protein adopting an active \u201copen\u201d conformation in the dimeric state.", + "section": "RESULTS", + "ner": [ + [ + 32, + 38, + "active", + "protein_state" + ], + [ + 59, + 63, + "NsrR", + "protein" + ], + [ + 84, + 90, + "active", + "protein_state" + ], + [ + 92, + 96, + "open", + "protein_state" + ], + [ + 118, + 125, + "dimeric", + "oligomeric_state" + ] + ] + }, + { + "sid": 244, + "sent": "We compared and aligned the NsrR-RD and ED on the dimeric structure of KdpE that was solved in the DNA-bound state (Fig 7B).", + "section": "RESULTS", + "ner": [ + [ + 3, + 23, + "compared and aligned", + "experimental_method" + ], + [ + 28, + 32, + "NsrR", + "protein" + ], + [ + 33, + 35, + "RD", + "structure_element" + ], + [ + 40, + 42, + "ED", + "structure_element" + ], + [ + 50, + 57, + "dimeric", + "oligomeric_state" + ], + [ + 58, + 67, + "structure", + "evidence" + ], + [ + 71, + 75, + "KdpE", + "protein" + ], + [ + 85, + 91, + "solved", + "experimental_method" + ], + [ + 99, + 108, + "DNA-bound", + "protein_state" + ] + ] + }, + { + "sid": 245, + "sent": "Also the linker region of KdpE is of similar length as of NsrR, which suggests that the distance in the DNA-bound state between the RD and ED of NsrR will be similar to that in the KdpE active dimer.", + "section": "RESULTS", + "ner": [ + [ + 9, + 22, + "linker region", + "structure_element" + ], + [ + 26, + 30, + "KdpE", + "protein" + ], + [ + 58, + 62, + "NsrR", + "protein" + ], + [ + 104, + 113, + "DNA-bound", + "protein_state" + ], + [ + 132, + 134, + "RD", + "structure_element" + ], + [ + 139, + 141, + "ED", + "structure_element" + ], + [ + 145, + 149, + "NsrR", + "protein" + ], + [ + 181, + 185, + "KdpE", + "protein" + ], + [ + 186, + 192, + "active", + "protein_state" + ], + [ + 193, + 198, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 246, + "sent": "We superimposed the ED of NsrR with the DNA-binding domain of KdpE resulting in a reasonably well-aligned structure (rmsd of 2.6\u00c5 over 86 C\u03b1 atoms; Table 2).", + "section": "RESULTS", + "ner": [ + [ + 3, + 15, + "superimposed", + "experimental_method" + ], + [ + 20, + 22, + "ED", + "structure_element" + ], + [ + 26, + 30, + "NsrR", + "protein" + ], + [ + 40, + 58, + "DNA-binding domain", + "structure_element" + ], + [ + 62, + 66, + "KdpE", + "protein" + ], + [ + 106, + 115, + "structure", + "evidence" + ], + [ + 117, + 121, + "rmsd", + "evidence" + ] + ] + }, + { + "sid": 247, + "sent": "As a result, a highly positive groove is created by the two ED domains of NsrR which likely represents the DNA binding site as observed in KdpE. A prediction of the putative promoter sequence that NsrR binds via the BPROM online server was performed (S3 Fig).", + "section": "RESULTS", + "ner": [ + [ + 15, + 37, + "highly positive groove", + "site" + ], + [ + 60, + 62, + "ED", + "structure_element" + ], + [ + 74, + 78, + "NsrR", + "protein" + ], + [ + 107, + 123, + "DNA binding site", + "site" + ], + [ + 139, + 143, + "KdpE", + "protein" + ], + [ + 197, + 201, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 248, + "sent": "A promoter region was identified upstream of the nsr operon.", + "section": "RESULTS", + "ner": [ + [ + 49, + 52, + "nsr", + "gene" + ] + ] + }, + { + "sid": 249, + "sent": "However, the regulation of the predicted promoter and the DNA binding by NsrR has to be confirmed.", + "section": "RESULTS", + "ner": [ + [ + 58, + 61, + "DNA", + "chemical" + ], + [ + 73, + 77, + "NsrR", + "protein" + ] + ] + }, + { + "sid": 250, + "sent": "In numerous pathogenic bacteria such as S. agalactiae, S. aureus, and C. difficile that apparently do not produce a lantibiotic, a gene cluster is present to provide resistance against lantibiotics such as nisin, nukacin ISK-1, lacticin 481 gallidermin, actagardine, or mersacidin.", + "section": "RESULTS", + "ner": [ + [ + 23, + 31, + "bacteria", + "taxonomy_domain" + ], + [ + 40, + 53, + "S. agalactiae", + "species" + ], + [ + 55, + 64, + "S. aureus", + "species" + ], + [ + 70, + 82, + "C. difficile", + "species" + ], + [ + 116, + 127, + "lantibiotic", + "chemical" + ], + [ + 185, + 197, + "lantibiotics", + "chemical" + ], + [ + 206, + 211, + "nisin", + "chemical" + ], + [ + 213, + 226, + "nukacin ISK-1", + "chemical" + ], + [ + 228, + 240, + "lacticin 481", + "chemical" + ], + [ + 241, + 252, + "gallidermin", + "chemical" + ], + [ + 254, + 265, + "actagardine", + "chemical" + ], + [ + 270, + 280, + "mersacidin", + "chemical" + ] + ] + }, + { + "sid": 251, + "sent": "The structure of the response regulator NsrR from S. agalactiae presented in this study is the first structural information available for the subgroup of lantibiotic resistance-associated RRs.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 21, + 39, + "response regulator", + "protein_type" + ], + [ + 40, + 44, + "NsrR", + "protein" + ], + [ + 50, + 63, + "S. agalactiae", + "species" + ], + [ + 154, + 191, + "lantibiotic resistance-associated RRs", + "protein_type" + ] + ] + } + ] + }, + "PMC4937829": { + "annotations": [ + { + "sid": 0, + "sent": "Visualizing chaperone-assisted protein folding", + "section": "TITLE", + "ner": [ + [ + 12, + 21, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 1, + "sent": "Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes.", + "section": "ABSTRACT", + "ner": [ + [ + 30, + 40, + "structures", + "evidence" + ] + ] + }, + { + "sid": 2, + "sent": "One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state.", + "section": "ABSTRACT", + "ner": [ + [ + 20, + 29, + "chaperone", + "protein_type" + ], + [ + 96, + 105, + "chaperone", + "protein_type" + ], + [ + 154, + 164, + "chaperones", + "protein_type" + ] + ] + }, + { + "sid": 3, + "sent": "To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ).", + "section": "ABSTRACT", + "ner": [ + [ + 81, + 102, + "X-ray crystallography", + "experimental_method" + ], + [ + 111, + 150, + "Residual Electron and Anomalous Density", + "experimental_method" + ], + [ + 152, + 156, + "READ", + "experimental_method" + ] + ] + }, + { + "sid": 4, + "sent": "READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 4, + "READ", + "experimental_method" + ], + [ + 92, + 110, + "immunity protein 7", + "protein" + ], + [ + 112, + 115, + "Im7", + "protein" + ], + [ + 117, + 132, + "in complex with", + "protein_state" + ], + [ + 137, + 144, + "E. coli", + "species" + ], + [ + 145, + 154, + "chaperone", + "protein_type" + ], + [ + 155, + 158, + "Spy", + "protein" + ] + ] + }, + { + "sid": 5, + "sent": "This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy.", + "section": "ABSTRACT", + "ner": [ + [ + 85, + 88, + "Im7", + "protein" + ], + [ + 95, + 103, + "bound to", + "protein_state" + ], + [ + 104, + 107, + "Spy", + "protein" + ] + ] + }, + { + "sid": 6, + "sent": "The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone.", + "section": "ABSTRACT", + "ner": [ + [ + 24, + 38, + "Spy-associated", + "protein_state" + ], + [ + 39, + 42, + "Im7", + "protein" + ], + [ + 78, + 86, + "unfolded", + "protein_state" + ], + [ + 100, + 106, + "folded", + "protein_state" + ], + [ + 111, + 117, + "native", + "protein_state" + ], + [ + 199, + 207, + "bound to", + "protein_state" + ], + [ + 210, + 219, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 7, + "sent": "High-resolution structural models of protein-protein interactions are critical for obtaining mechanistic insights into biological processes.", + "section": "INTRO", + "ner": [ + [ + 16, + 33, + "structural models", + "evidence" + ] + ] + }, + { + "sid": 8, + "sent": "However, many protein-protein interactions are highly dynamic, making it difficult to obtain high-resolution data.", + "section": "INTRO", + "ner": [ + [ + 47, + 61, + "highly dynamic", + "protein_state" + ] + ] + }, + { + "sid": 9, + "sent": "Particularly challenging are interactions of intrinsically or conditionally disordered sections of proteins with their partner proteins.", + "section": "INTRO", + "ner": [ + [ + 45, + 86, + "intrinsically or conditionally disordered", + "protein_state" + ] + ] + }, + { + "sid": 10, + "sent": "Recent advances in X-ray crystallography and NMR spectroscopy continue to improve our ability to analyze biomolecules that exist in multiple conformations.", + "section": "INTRO", + "ner": [ + [ + 19, + 40, + "X-ray crystallography", + "experimental_method" + ], + [ + 45, + 61, + "NMR spectroscopy", + "experimental_method" + ] + ] + }, + { + "sid": 11, + "sent": "X-ray crystallography has historically provided valuable information on small-scale conformational changes, but observing large-amplitude heterogeneous conformational changes often falls beyond the reach of current crystallographic techniques.", + "section": "INTRO", + "ner": [ + [ + 0, + 21, + "X-ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 12, + "sent": "NMR can theoretically be used to determine heterogeneous ensembles, but in practice, this proves to be very challenging.", + "section": "INTRO", + "ner": [ + [ + 0, + 3, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 13, + "sent": "It is clear that molecular chaperones aid in protein folding.", + "section": "INTRO", + "ner": [ + [ + 27, + 37, + "chaperones", + "protein_type" + ] + ] + }, + { + "sid": 14, + "sent": "Structural characterization of chaperone-assisted protein folding likely would help bring clarity to this question.", + "section": "INTRO", + "ner": [ + [ + 31, + 40, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 15, + "sent": "Structural models of chaperone-substrate complexes have recently begun to provide information as to how a chaperone can recognize its substrate.", + "section": "INTRO", + "ner": [ + [ + 0, + 17, + "Structural models", + "evidence" + ], + [ + 21, + 30, + "chaperone", + "protein_type" + ], + [ + 106, + 115, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 16, + "sent": "However, the impact that chaperones have on their substrates, and how these interactions affect the folding process remain largely unknown.", + "section": "INTRO", + "ner": [ + [ + 25, + 35, + "chaperones", + "protein_type" + ] + ] + }, + { + "sid": 17, + "sent": "For most chaperones, it is still unclear whether the chaperone actively participates in and affects the folding of the substrate proteins, or merely provides a suitable microenvironment enabling the substrate to fold on its own.", + "section": "INTRO", + "ner": [ + [ + 9, + 19, + "chaperones", + "protein_type" + ], + [ + 53, + 62, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 18, + "sent": "This is a truly fundamental question in the chaperone field, and one that has eluded the community largely because of the highly dynamic nature of the chaperone-substrate complexes.", + "section": "INTRO", + "ner": [ + [ + 44, + 53, + "chaperone", + "protein_type" + ], + [ + 122, + 136, + "highly dynamic", + "protein_state" + ], + [ + 151, + 160, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 19, + "sent": "To address this question, we investigated the ATP-independent Escherichia coli periplasmic chaperone Spy.", + "section": "INTRO", + "ner": [ + [ + 46, + 61, + "ATP-independent", + "protein_state" + ], + [ + 62, + 78, + "Escherichia coli", + "species" + ], + [ + 91, + 100, + "chaperone", + "protein_type" + ], + [ + 101, + 104, + "Spy", + "protein" + ] + ] + }, + { + "sid": 20, + "sent": "Spy prevents protein aggregation and aids in protein folding under various stress conditions, including treatment with tannin and butanol.", + "section": "INTRO", + "ner": [ + [ + 0, + 3, + "Spy", + "protein" + ], + [ + 119, + 125, + "tannin", + "chemical" + ], + [ + 130, + 137, + "butanol", + "chemical" + ] + ] + }, + { + "sid": 21, + "sent": "We originally discovered Spy by its ability to stabilize the protein-folding model Im7 in vivo and recently demonstrated that Im7 folds while associated with Spy.", + "section": "INTRO", + "ner": [ + [ + 25, + 28, + "Spy", + "protein" + ], + [ + 83, + 86, + "Im7", + "protein" + ], + [ + 126, + 129, + "Im7", + "protein" + ], + [ + 158, + 161, + "Spy", + "protein" + ] + ] + }, + { + "sid": 22, + "sent": "The crystal structure of Spy revealed that it forms a thin \u03b1-helical homodimeric cradle.", + "section": "INTRO", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 25, + 28, + "Spy", + "protein" + ], + [ + 69, + 80, + "homodimeric", + "oligomeric_state" + ], + [ + 81, + 87, + "cradle", + "site" + ] + ] + }, + { + "sid": 23, + "sent": "Crosslinking and genetic experiments suggested that Spy interacts with substrates somewhere on its concave side.", + "section": "INTRO", + "ner": [ + [ + 0, + 36, + "Crosslinking and genetic experiments", + "experimental_method" + ], + [ + 52, + 55, + "Spy", + "protein" + ] + ] + }, + { + "sid": 24, + "sent": "By using a novel X-ray crystallography-based approach to model disorder in crystal structures, we have now determined the high-resolution ensemble of the dynamic Spy:Im7 complex.", + "section": "INTRO", + "ner": [ + [ + 17, + 38, + "X-ray crystallography", + "experimental_method" + ], + [ + 75, + 93, + "crystal structures", + "evidence" + ], + [ + 138, + 146, + "ensemble", + "evidence" + ], + [ + 154, + 161, + "dynamic", + "protein_state" + ], + [ + 162, + 169, + "Spy:Im7", + "complex_assembly" + ] + ] + }, + { + "sid": 25, + "sent": "This work provides a detailed view of chaperone-mediated protein folding and shows how substrates like Im7 find their native fold while bound to their chaperones.", + "section": "INTRO", + "ner": [ + [ + 38, + 47, + "chaperone", + "protein_type" + ], + [ + 103, + 106, + "Im7", + "protein" + ], + [ + 136, + 144, + "bound to", + "protein_state" + ], + [ + 151, + 161, + "chaperones", + "protein_type" + ] + ] + }, + { + "sid": 26, + "sent": "Crystallizing the Spy:Im7 complex", + "section": "RESULTS", + "ner": [ + [ + 0, + 13, + "Crystallizing", + "experimental_method" + ], + [ + 18, + 25, + "Spy:Im7", + "complex_assembly" + ] + ] + }, + { + "sid": 27, + "sent": "We reasoned that to obtain crystals of complexes between Spy (domain boundaries in Supplementary Fig. 1) and its substrate proteins, our best approach was to identify crystallization conditions that yielded Spy crystals in the presence of protein substrates but not in their absence.", + "section": "RESULTS", + "ner": [ + [ + 27, + 35, + "crystals", + "evidence" + ], + [ + 57, + 60, + "Spy", + "protein" + ], + [ + 167, + 193, + "crystallization conditions", + "experimental_method" + ], + [ + 207, + 210, + "Spy", + "protein" + ], + [ + 211, + 219, + "crystals", + "evidence" + ], + [ + 227, + 238, + "presence of", + "protein_state" + ], + [ + 275, + 282, + "absence", + "protein_state" + ] + ] + }, + { + "sid": 28, + "sent": "We therefore screened crystallization conditions for Spy with four different substrate proteins: a fragment of the largely unfolded bovine \u03b1-casein protein, wild-type (WT) E. coli Im7, an unfolded variant of Im7 (L18A L19A L37A), and the N-terminal half of Im7 (Im76-45), which encompasses the entire Spy-binding portion of Im7.", + "section": "RESULTS", + "ner": [ + [ + 13, + 21, + "screened", + "experimental_method" + ], + [ + 22, + 48, + "crystallization conditions", + "experimental_method" + ], + [ + 53, + 56, + "Spy", + "protein" + ], + [ + 123, + 131, + "unfolded", + "protein_state" + ], + [ + 132, + 138, + "bovine", + "taxonomy_domain" + ], + [ + 139, + 147, + "\u03b1-casein", + "chemical" + ], + [ + 157, + 166, + "wild-type", + "protein_state" + ], + [ + 168, + 170, + "WT", + "protein_state" + ], + [ + 172, + 179, + "E. coli", + "species" + ], + [ + 180, + 183, + "Im7", + "protein" + ], + [ + 188, + 196, + "unfolded", + "protein_state" + ], + [ + 208, + 211, + "Im7", + "protein" + ], + [ + 213, + 217, + "L18A", + "mutant" + ], + [ + 218, + 222, + "L19A", + "mutant" + ], + [ + 223, + 227, + "L37A", + "mutant" + ], + [ + 238, + 253, + "N-terminal half", + "structure_element" + ], + [ + 257, + 260, + "Im7", + "protein" + ], + [ + 262, + 269, + "Im76-45", + "mutant" + ], + [ + 301, + 320, + "Spy-binding portion", + "structure_element" + ], + [ + 324, + 327, + "Im7", + "protein" + ] + ] + }, + { + "sid": 29, + "sent": "We found conditions in which all four substrates co-crystallized with Spy, but in which Spy alone did not yield crystals.", + "section": "RESULTS", + "ner": [ + [ + 49, + 64, + "co-crystallized", + "experimental_method" + ], + [ + 65, + 69, + "with", + "protein_state" + ], + [ + 70, + 73, + "Spy", + "protein" + ], + [ + 88, + 91, + "Spy", + "protein" + ], + [ + 92, + 97, + "alone", + "protein_state" + ], + [ + 112, + 120, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 30, + "sent": "Subsequent crystal washing and dissolution experiments confirmed the presence of the substrates in the co-crystals (Supplementary Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 11, + 42, + "crystal washing and dissolution", + "experimental_method" + ], + [ + 103, + 114, + "co-crystals", + "experimental_method" + ] + ] + }, + { + "sid": 31, + "sent": "The crystals diffracted to ~1.8 \u00c5 resolution.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 32, + "sent": "We used Spy:Im76-45 selenomethionine crystals for phasing with single-wavelength anomalous diffraction (SAD) experiments, and used this solution to build the well-ordered Spy portions of all four complexes.", + "section": "RESULTS", + "ner": [ + [ + 8, + 19, + "Spy:Im76-45", + "complex_assembly" + ], + [ + 20, + 36, + "selenomethionine", + "chemical" + ], + [ + 37, + 45, + "crystals", + "evidence" + ], + [ + 63, + 102, + "single-wavelength anomalous diffraction", + "experimental_method" + ], + [ + 104, + 107, + "SAD", + "experimental_method" + ], + [ + 171, + 174, + "Spy", + "protein" + ] + ] + }, + { + "sid": 33, + "sent": "However, modeling of the substrate in the complex proved to be a substantial challenge, as the electron density of the substrate was discontinuous and fragmented.", + "section": "RESULTS", + "ner": [ + [ + 95, + 111, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 34, + "sent": "Even the minimal binding portion of Im7 (Im76-45) showed highly dispersed electron density (Fig. 1a).", + "section": "RESULTS", + "ner": [ + [ + 9, + 32, + "minimal binding portion", + "structure_element" + ], + [ + 36, + 39, + "Im7", + "protein" + ], + [ + 41, + 48, + "Im76-45", + "mutant" + ], + [ + 74, + 90, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 35, + "sent": "We hypothesized that the fragmented density was due to multiple, partially occupied conformations of the substrate bound within the crystal.", + "section": "RESULTS", + "ner": [ + [ + 36, + 43, + "density", + "evidence" + ], + [ + 132, + 139, + "crystal", + "evidence" + ] + ] + }, + { + "sid": 36, + "sent": "Such residual density is typically not considered usable by traditional X-ray crystallography methods.", + "section": "RESULTS", + "ner": [ + [ + 72, + 93, + "X-ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 37, + "sent": "Thus, we developed a new approach to interpret the chaperone-bound substrate in multiple conformations.", + "section": "RESULTS", + "ner": [ + [ + 51, + 66, + "chaperone-bound", + "protein_state" + ] + ] + }, + { + "sid": 38, + "sent": "READ: a strategy to visualize heterogeneous and dynamic biomolecules", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "READ", + "experimental_method" + ] + ] + }, + { + "sid": 39, + "sent": "To determine the structure of the substrate portion of these Spy:substrate complexes, we conceived of an approach that we term READ, for Residual Electron and Anomalous Density.", + "section": "RESULTS", + "ner": [ + [ + 17, + 26, + "structure", + "evidence" + ], + [ + 61, + 64, + "Spy", + "protein" + ], + [ + 127, + 131, + "READ", + "experimental_method" + ], + [ + 137, + 176, + "Residual Electron and Anomalous Density", + "experimental_method" + ] + ] + }, + { + "sid": 40, + "sent": "We split this approach into five steps: (1) By using a well-diffracting Spy:substrate co-crystal, we first determined the structure of the folded domain of Spy and obtained high quality residual electron density within the dynamic regions of the substrate.", + "section": "RESULTS", + "ner": [ + [ + 72, + 75, + "Spy", + "protein" + ], + [ + 86, + 96, + "co-crystal", + "evidence" + ], + [ + 122, + 131, + "structure", + "evidence" + ], + [ + 139, + 145, + "folded", + "protein_state" + ], + [ + 146, + 152, + "domain", + "structure_element" + ], + [ + 156, + 159, + "Spy", + "protein" + ], + [ + 186, + 211, + "residual electron density", + "evidence" + ], + [ + 223, + 230, + "dynamic", + "protein_state" + ] + ] + }, + { + "sid": 41, + "sent": "(2) We then labeled individual residues in the flexible regions of the substrate with the strong anomalous scatterer iodine, which serves to locate these residues in three-dimensional space using their anomalous density.", + "section": "RESULTS", + "ner": [ + [ + 47, + 55, + "flexible", + "protein_state" + ], + [ + 117, + 123, + "iodine", + "chemical" + ], + [ + 202, + 219, + "anomalous density", + "evidence" + ] + ] + }, + { + "sid": 42, + "sent": "(3) We performed molecular dynamics (MD) simulations to generate a pool of energetically reasonable conformations of the dynamic complex and (4) applied a sample-and-select algorithm to determine the minimal set of substrate conformations that fit both the residual and anomalous density.", + "section": "RESULTS", + "ner": [ + [ + 17, + 35, + "molecular dynamics", + "experimental_method" + ], + [ + 37, + 39, + "MD", + "experimental_method" + ], + [ + 41, + 52, + "simulations", + "experimental_method" + ], + [ + 121, + 128, + "dynamic", + "protein_state" + ], + [ + 155, + 182, + "sample-and-select algorithm", + "experimental_method" + ], + [ + 257, + 287, + "residual and anomalous density", + "evidence" + ] + ] + }, + { + "sid": 43, + "sent": "Importantly, even though we only labeled a subset of the residues in the flexible regions of the substrate with iodine, the residual electron density can provide spatial information on many of the other flexible residues.", + "section": "RESULTS", + "ner": [ + [ + 73, + 81, + "flexible", + "protein_state" + ], + [ + 112, + 118, + "iodine", + "chemical" + ], + [ + 124, + 149, + "residual electron density", + "evidence" + ], + [ + 203, + 211, + "flexible", + "protein_state" + ] + ] + }, + { + "sid": 44, + "sent": "The electron density then allowed us to connect the labeled residues of the substrate by confining the protein chain within regions of detectable density.", + "section": "RESULTS", + "ner": [ + [ + 4, + 20, + "electron density", + "evidence" + ], + [ + 146, + 153, + "density", + "evidence" + ] + ] + }, + { + "sid": 45, + "sent": "In this way, the two forms of data together were able to describe multiple conformations of the substrate within the crystal.", + "section": "RESULTS", + "ner": [ + [ + 117, + 124, + "crystal", + "evidence" + ] + ] + }, + { + "sid": 46, + "sent": "As described in detail below, we developed the READ method to uncover the ensemble of conformations that the Spy-binding domain of Im7 (i.e., Im76-45) adopts while bound to Spy.", + "section": "RESULTS", + "ner": [ + [ + 47, + 51, + "READ", + "experimental_method" + ], + [ + 109, + 127, + "Spy-binding domain", + "structure_element" + ], + [ + 131, + 134, + "Im7", + "protein" + ], + [ + 142, + 149, + "Im76-45", + "mutant" + ], + [ + 164, + 172, + "bound to", + "protein_state" + ], + [ + 173, + 176, + "Spy", + "protein" + ] + ] + }, + { + "sid": 47, + "sent": "However, we believe that READ will prove generally applicable to visualizing heterogeneous and dynamic complexes that have previously escaped detailed structural analysis.", + "section": "RESULTS", + "ner": [ + [ + 25, + 29, + "READ", + "experimental_method" + ] + ] + }, + { + "sid": 48, + "sent": "Collecting READ data for the Spy:Im76-45 complex", + "section": "RESULTS", + "ner": [ + [ + 11, + 15, + "READ", + "experimental_method" + ], + [ + 29, + 40, + "Spy:Im76-45", + "complex_assembly" + ] + ] + }, + { + "sid": 49, + "sent": "To apply the READ technique to the folding mechanism employed by the chaperone Spy, we selected Im76-45 for further investigation because NMR data suggested that Im76-45 could recapitulate unfolded, partially folded, and native-like states of Im7 (Supplementary Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 13, + 27, + "READ technique", + "experimental_method" + ], + [ + 69, + 78, + "chaperone", + "protein_type" + ], + [ + 79, + 82, + "Spy", + "protein" + ], + [ + 96, + 103, + "Im76-45", + "mutant" + ], + [ + 138, + 141, + "NMR", + "experimental_method" + ], + [ + 162, + 169, + "Im76-45", + "mutant" + ], + [ + 189, + 197, + "unfolded", + "protein_state" + ], + [ + 209, + 215, + "folded", + "protein_state" + ], + [ + 243, + 246, + "Im7", + "protein" + ] + ] + }, + { + "sid": 50, + "sent": "Moreover, binding experiments indicated that Im76-45 comprises the entire Spy-binding region.", + "section": "RESULTS", + "ner": [ + [ + 10, + 29, + "binding experiments", + "experimental_method" + ], + [ + 45, + 52, + "Im76-45", + "mutant" + ], + [ + 74, + 92, + "Spy-binding region", + "site" + ] + ] + }, + { + "sid": 51, + "sent": "To introduce the anomalous scatterer iodine, we replaced eight Im76-45 residues with the non-canonical amino acid 4-iodophenylalanine (pI-Phe).", + "section": "RESULTS", + "ner": [ + [ + 37, + 43, + "iodine", + "chemical" + ], + [ + 48, + 56, + "replaced", + "experimental_method" + ], + [ + 63, + 70, + "Im76-45", + "mutant" + ], + [ + 114, + 133, + "4-iodophenylalanine", + "chemical" + ], + [ + 135, + 141, + "pI-Phe", + "chemical" + ] + ] + }, + { + "sid": 52, + "sent": "Its strong anomalous scattering allowed us to track the positions of these individual Im76-45 residues one at a time, potentially even if the residue was found in several locations in the same crystal.", + "section": "RESULTS", + "ner": [ + [ + 11, + 31, + "anomalous scattering", + "evidence" + ], + [ + 86, + 93, + "Im76-45", + "mutant" + ], + [ + 193, + 200, + "crystal", + "evidence" + ] + ] + }, + { + "sid": 53, + "sent": "We then co-crystallized Spy and the eight Im76-45 peptides, each of which harbored an individual pI-Phe substitution at one distinct position, and collected anomalous data for all eight Spy:Im76-45 complexes (Fig. 1B, Supplementary Table 1 Supplementary Dataset 1, and Supplementary Table 2).", + "section": "RESULTS", + "ner": [ + [ + 8, + 23, + "co-crystallized", + "experimental_method" + ], + [ + 24, + 27, + "Spy", + "protein" + ], + [ + 42, + 49, + "Im76-45", + "mutant" + ], + [ + 97, + 103, + "pI-Phe", + "chemical" + ], + [ + 104, + 116, + "substitution", + "experimental_method" + ], + [ + 147, + 156, + "collected", + "experimental_method" + ], + [ + 157, + 171, + "anomalous data", + "evidence" + ], + [ + 186, + 197, + "Spy:Im76-45", + "complex_assembly" + ] + ] + }, + { + "sid": 54, + "sent": "Consistent with our electron density map, we found that the majority of anomalous signals emerged in the cradle of Spy, implying that this is the likely Im7 substrate binding site.", + "section": "RESULTS", + "ner": [ + [ + 20, + 40, + "electron density map", + "evidence" + ], + [ + 72, + 89, + "anomalous signals", + "evidence" + ], + [ + 105, + 111, + "cradle", + "site" + ], + [ + 115, + 118, + "Spy", + "protein" + ], + [ + 153, + 156, + "Im7", + "protein" + ], + [ + 157, + 179, + "substrate binding site", + "site" + ] + ] + }, + { + "sid": 55, + "sent": "Consistent with the fragmented density, however, we observed multiple iodine positions for seven of the eight substituted residues.", + "section": "RESULTS", + "ner": [ + [ + 31, + 38, + "density", + "evidence" + ], + [ + 70, + 76, + "iodine", + "chemical" + ] + ] + }, + { + "sid": 56, + "sent": "Together, these results indicated that the Im7 substrate binds Spy in multiple conformations.", + "section": "RESULTS", + "ner": [ + [ + 43, + 46, + "Im7", + "protein" + ], + [ + 63, + 66, + "Spy", + "protein" + ] + ] + }, + { + "sid": 57, + "sent": "READ sample-and-select procedure", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "READ", + "experimental_method" + ], + [ + 5, + 22, + "sample-and-select", + "experimental_method" + ] + ] + }, + { + "sid": 58, + "sent": "To determine the structural ensemble that Im76-45 adopts while bound to Spy, we combined the residual electron density and the anomalous signals from our pI-Phe substituted Spy:Im76-45 complexes.", + "section": "RESULTS", + "ner": [ + [ + 42, + 49, + "Im76-45", + "mutant" + ], + [ + 63, + 71, + "bound to", + "protein_state" + ], + [ + 72, + 75, + "Spy", + "protein" + ], + [ + 93, + 118, + "residual electron density", + "evidence" + ], + [ + 127, + 144, + "anomalous signals", + "evidence" + ], + [ + 154, + 160, + "pI-Phe", + "chemical" + ], + [ + 173, + 184, + "Spy:Im76-45", + "complex_assembly" + ] + ] + }, + { + "sid": 59, + "sent": "To generate an accurate depiction of the chaperone-substrate interactions, we devised a selection protocol based on a sample-and-select procedure employed in NMR spectroscopy.", + "section": "RESULTS", + "ner": [ + [ + 41, + 50, + "chaperone", + "protein_type" + ], + [ + 118, + 135, + "sample-and-select", + "experimental_method" + ], + [ + 158, + 174, + "NMR spectroscopy", + "experimental_method" + ] + ] + }, + { + "sid": 60, + "sent": "During each round of the selection, a genetic algorithm alters the ensemble and its agreement to the experimental data is re-evaluated.", + "section": "RESULTS", + "ner": [ + [ + 38, + 55, + "genetic algorithm", + "experimental_method" + ] + ] + }, + { + "sid": 61, + "sent": "If successful, the selection identifies the smallest group of specific conformations that best fits the residual electron density and anomalous signals.", + "section": "RESULTS", + "ner": [ + [ + 104, + 129, + "residual electron density", + "evidence" + ], + [ + 134, + 151, + "anomalous signals", + "evidence" + ] + ] + }, + { + "sid": 62, + "sent": "The READ sample-and-select algorithm is diagrammed in Fig. 2.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "READ", + "experimental_method" + ], + [ + 9, + 36, + "sample-and-select algorithm", + "experimental_method" + ] + ] + }, + { + "sid": 63, + "sent": "Prior to performing the selection, we generated a large and diverse pool of chaperone-substrate complexes using coarse-grained MD simulations in a pseudo-crystal environment (Fig. 2 and Supplementary Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 76, + 85, + "chaperone", + "protein_type" + ], + [ + 112, + 141, + "coarse-grained MD simulations", + "experimental_method" + ], + [ + 147, + 173, + "pseudo-crystal environment", + "experimental_method" + ] + ] + }, + { + "sid": 64, + "sent": "The coarse-grained simulations are based on a single-residue resolution model for protein folding and were extended here to describe Spy-Im76-45 binding events (Online Methods).", + "section": "RESULTS", + "ner": [ + [ + 4, + 30, + "coarse-grained simulations", + "experimental_method" + ], + [ + 133, + 144, + "Spy-Im76-45", + "complex_assembly" + ] + ] + }, + { + "sid": 65, + "sent": "The initial conditions of the binding simulations are not biased toward a particular conformation of the substrate or any specific chaperone-substrate interaction (Online Methods).", + "section": "RESULTS", + "ner": [ + [ + 30, + 49, + "binding simulations", + "experimental_method" + ], + [ + 131, + 140, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 66, + "sent": "Im76-45 binds and unbinds to Spy throughout the simulations.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Im76-45", + "mutant" + ], + [ + 29, + 32, + "Spy", + "protein" + ], + [ + 48, + 59, + "simulations", + "experimental_method" + ] + ] + }, + { + "sid": 67, + "sent": "This strategy allows a wide range of substrate conformations to interact with the chaperone.", + "section": "RESULTS", + "ner": [ + [ + 82, + 91, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 68, + "sent": "From the MD simulations, we extracted ~10,000 diverse Spy:Im76-45 complexes to be used by the ensuing selection.", + "section": "RESULTS", + "ner": [ + [ + 9, + 11, + "MD", + "experimental_method" + ], + [ + 12, + 23, + "simulations", + "experimental_method" + ], + [ + 54, + 65, + "Spy:Im76-45", + "complex_assembly" + ] + ] + }, + { + "sid": 69, + "sent": "Each complex within this pool comprises one Spy dimer bound to a single Im76-45 substrate.", + "section": "RESULTS", + "ner": [ + [ + 44, + 47, + "Spy", + "protein" + ], + [ + 48, + 53, + "dimer", + "oligomeric_state" + ], + [ + 54, + 62, + "bound to", + "protein_state" + ], + [ + 72, + 79, + "Im76-45", + "mutant" + ] + ] + }, + { + "sid": 70, + "sent": "This pool was then used by the selection algorithm to identify the minimal ensemble that best satisfies both the residual electron and anomalous crystallographic data.", + "section": "RESULTS", + "ner": [ + [ + 113, + 166, + "residual electron and anomalous crystallographic data", + "evidence" + ] + ] + }, + { + "sid": 71, + "sent": "The anomalous scattering portion of the selection uses our basic knowledge of pI-Phe geometry: the iodine is separated from its respective C\u03b1 atom in each coarse-grained conformer by 6.5 \u00c5. The selection then picks ensembles that best reproduce the collection of iodine anomalous signals.", + "section": "RESULTS", + "ner": [ + [ + 4, + 24, + "anomalous scattering", + "evidence" + ], + [ + 78, + 84, + "pI-Phe", + "chemical" + ], + [ + 99, + 105, + "iodine", + "chemical" + ], + [ + 263, + 269, + "iodine", + "chemical" + ], + [ + 270, + 287, + "anomalous signals", + "evidence" + ] + ] + }, + { + "sid": 72, + "sent": "Simultaneously, it uses the residual electron density to help choose ensembles.", + "section": "RESULTS", + "ner": [ + [ + 28, + 53, + "residual electron density", + "evidence" + ] + ] + }, + { + "sid": 73, + "sent": "To make the electron density selection practical, we needed to develop a method to rapidly evaluate the agreement between the selected sub-ensembles and the experimental electron density on-the-fly during the selection procedure.", + "section": "RESULTS", + "ner": [ + [ + 12, + 38, + "electron density selection", + "experimental_method" + ], + [ + 170, + 186, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 74, + "sent": "To accomplish this task, we generated a compressed version of the experimental 2mFo\u2212DFc electron density map for use in the selection.", + "section": "RESULTS", + "ner": [ + [ + 79, + 108, + "2mFo\u2212DFc electron density map", + "evidence" + ] + ] + }, + { + "sid": 75, + "sent": "This process provided us with a target map that the ensuing selection tried to recapitulate.", + "section": "RESULTS", + "ner": [ + [ + 39, + 42, + "map", + "evidence" + ] + ] + }, + { + "sid": 76, + "sent": "To reduce the extent of 3D space to be explored, this compressed map was created by only using density from regions of space significantly sampled by Im76-45 in the Spy:Im76-45 MD simulations.", + "section": "RESULTS", + "ner": [ + [ + 65, + 68, + "map", + "evidence" + ], + [ + 95, + 102, + "density", + "evidence" + ], + [ + 150, + 157, + "Im76-45", + "mutant" + ], + [ + 165, + 176, + "Spy:Im76-45", + "complex_assembly" + ], + [ + 177, + 179, + "MD", + "experimental_method" + ], + [ + 180, + 191, + "simulations", + "experimental_method" + ] + ] + }, + { + "sid": 77, + "sent": "For each of the ~10,000 complexes in the coarse-grained MD pool, the electron density at the C\u03b1 positions of Im76-45 was extracted and used to construct an electron density map (Online Methods).", + "section": "RESULTS", + "ner": [ + [ + 41, + 55, + "coarse-grained", + "experimental_method" + ], + [ + 56, + 58, + "MD", + "experimental_method" + ], + [ + 69, + 85, + "electron density", + "evidence" + ], + [ + 109, + 116, + "Im76-45", + "mutant" + ], + [ + 156, + 176, + "electron density map", + "evidence" + ] + ] + }, + { + "sid": 78, + "sent": "These individual electron density maps from the separate conformers could then be combined (Fig. 2) and compared to the averaged experimental electron density map as part of the selection algorithm.", + "section": "RESULTS", + "ner": [ + [ + 17, + 38, + "electron density maps", + "evidence" + ], + [ + 142, + 162, + "electron density map", + "evidence" + ] + ] + }, + { + "sid": 79, + "sent": "This approach allowed us to simultaneously use both the iodine anomalous signals and the residual electron density in the selection procedure.", + "section": "RESULTS", + "ner": [ + [ + 56, + 62, + "iodine", + "chemical" + ], + [ + 63, + 80, + "anomalous signals", + "evidence" + ], + [ + 89, + 114, + "residual electron density", + "evidence" + ] + ] + }, + { + "sid": 80, + "sent": "The selection resulted in small ensembles from the MD pool that best fit the READ data (Fig. 1c,d).", + "section": "RESULTS", + "ner": [ + [ + 51, + 53, + "MD", + "experimental_method" + ], + [ + 77, + 81, + "READ", + "experimental_method" + ] + ] + }, + { + "sid": 81, + "sent": "Before analyzing the details of the Spy:Im76-45 complex, we first engaged in a series of validation tests to verify the ensemble and selection procedure (Supplementary Note 1, Figures 1c,d, Supplemental Figures 5-7).", + "section": "RESULTS", + "ner": [ + [ + 36, + 47, + "Spy:Im76-45", + "complex_assembly" + ] + ] + }, + { + "sid": 82, + "sent": "Of note, the final six-membered ensemble was the largest ensemble that could simultaneously decrease the RFree and pass the 10-fold cross-validation test.", + "section": "RESULTS", + "ner": [ + [ + 105, + 110, + "RFree", + "evidence" + ], + [ + 124, + 153, + "10-fold cross-validation test", + "experimental_method" + ] + ] + }, + { + "sid": 83, + "sent": "This ensemble depicts the conformations that the substrate Im76-45 adopts while bound to the chaperone Spy (Fig. 3 Supplementary Movie 1, and Table 1).", + "section": "RESULTS", + "ner": [ + [ + 59, + 66, + "Im76-45", + "mutant" + ], + [ + 80, + 88, + "bound to", + "protein_state" + ], + [ + 93, + 102, + "chaperone", + "protein_type" + ], + [ + 103, + 106, + "Spy", + "protein" + ] + ] + }, + { + "sid": 84, + "sent": "Folding and interactions of Im7 while bound to Spy", + "section": "RESULTS", + "ner": [ + [ + 28, + 31, + "Im7", + "protein" + ], + [ + 38, + 46, + "bound to", + "protein_state" + ], + [ + 47, + 50, + "Spy", + "protein" + ] + ] + }, + { + "sid": 85, + "sent": "Our results showed that by using this novel READ approach, we were able to obtain structural information about the dynamic interaction of a chaperone with its substrate protein.", + "section": "RESULTS", + "ner": [ + [ + 44, + 48, + "READ", + "experimental_method" + ], + [ + 140, + 149, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 86, + "sent": "We were particularly interested in finding answers to one of the most fundamental questions in chaperone biology\u2014how does chaperone binding affect substrate structure and vice versa.", + "section": "RESULTS", + "ner": [ + [ + 95, + 104, + "chaperone", + "protein_type" + ], + [ + 122, + 131, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 87, + "sent": "By analyzing the individual structures of the six-member ensemble of Im76-45 bound to Spy, we observed that Im76-45 takes on several different conformations while bound.", + "section": "RESULTS", + "ner": [ + [ + 28, + 38, + "structures", + "evidence" + ], + [ + 69, + 76, + "Im76-45", + "mutant" + ], + [ + 77, + 85, + "bound to", + "protein_state" + ], + [ + 86, + 89, + "Spy", + "protein" + ], + [ + 108, + 115, + "Im76-45", + "mutant" + ], + [ + 163, + 168, + "bound", + "protein_state" + ] + ] + }, + { + "sid": 88, + "sent": "We found these conformations to be highly heterogeneous and to include unfolded, partially folded, and native-like states (Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 71, + 79, + "unfolded", + "protein_state" + ], + [ + 81, + 97, + "partially folded", + "protein_state" + ], + [ + 103, + 114, + "native-like", + "protein_state" + ] + ] + }, + { + "sid": 89, + "sent": "The ensemble primarily encompasses Im76-45 laying diagonally within the Spy cradle in several different orientations, but some conformations traverse as far as the tips or even extend over the side of the cradle (Figs. 3,4a).", + "section": "RESULTS", + "ner": [ + [ + 35, + 42, + "Im76-45", + "mutant" + ], + [ + 72, + 75, + "Spy", + "protein" + ], + [ + 76, + 82, + "cradle", + "site" + ], + [ + 205, + 211, + "cradle", + "site" + ] + ] + }, + { + "sid": 90, + "sent": "We constructed a contact map of the complex, which shows the frequency of interactions for chaperone-substrate residue pairs (Fig. 4).", + "section": "RESULTS", + "ner": [ + [ + 17, + 28, + "contact map", + "evidence" + ], + [ + 91, + 100, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 91, + "sent": "We found that the primary interaction sites on Spy reside at the N and C termini (Arg122, Thr124, and Phe29) as well as on the concave face of the chaperone (Arg61, Arg43, Lys47, His96, and Met46).", + "section": "RESULTS", + "ner": [ + [ + 26, + 43, + "interaction sites", + "site" + ], + [ + 47, + 50, + "Spy", + "protein" + ], + [ + 82, + 88, + "Arg122", + "residue_name_number" + ], + [ + 90, + 96, + "Thr124", + "residue_name_number" + ], + [ + 102, + 107, + "Phe29", + "residue_name_number" + ], + [ + 147, + 156, + "chaperone", + "protein_type" + ], + [ + 158, + 163, + "Arg61", + "residue_name_number" + ], + [ + 165, + 170, + "Arg43", + "residue_name_number" + ], + [ + 172, + 177, + "Lys47", + "residue_name_number" + ], + [ + 179, + 184, + "His96", + "residue_name_number" + ], + [ + 190, + 195, + "Met46", + "residue_name_number" + ] + ] + }, + { + "sid": 92, + "sent": "The Spy-contacting residues comprise a mixture of charged, polar, and hydrophobic residues.", + "section": "RESULTS", + "ner": [ + [ + 4, + 27, + "Spy-contacting residues", + "site" + ] + ] + }, + { + "sid": 93, + "sent": "Surprisingly, we noted that in the ensemble, Im76-45 interacts with only 38% of the hydrophobic residues in the Spy cradle, but interacts with 61% of the hydrophilic residues in the cradle.", + "section": "RESULTS", + "ner": [ + [ + 45, + 52, + "Im76-45", + "mutant" + ], + [ + 112, + 115, + "Spy", + "protein" + ], + [ + 116, + 122, + "cradle", + "site" + ], + [ + 182, + 188, + "cradle", + "site" + ] + ] + }, + { + "sid": 94, + "sent": "This mixture suggests the importance of both electrostatic and hydrophobic components in binding the Im76-45 ensemble.", + "section": "RESULTS", + "ner": [ + [ + 101, + 108, + "Im76-45", + "mutant" + ] + ] + }, + { + "sid": 95, + "sent": "With respect to the substrate, we observed that nearly every residue in Im76-45 is in contact with Spy (Fig. 4a).", + "section": "RESULTS", + "ner": [ + [ + 72, + 79, + "Im76-45", + "mutant" + ], + [ + 99, + 102, + "Spy", + "protein" + ] + ] + }, + { + "sid": 96, + "sent": "However, we did notice that despite this uniformity, regions of Im76-45 preferentially interact with different regions in Spy (Fig. 4b).", + "section": "RESULTS", + "ner": [ + [ + 64, + 71, + "Im76-45", + "mutant" + ], + [ + 122, + 125, + "Spy", + "protein" + ] + ] + }, + { + "sid": 97, + "sent": "For example, the N-terminal half of Im76-45 binds more consistently in the Spy cradle, whereas the C-terminal half predominantly binds to the outer edges of Spy\u2019s concave surface.", + "section": "RESULTS", + "ner": [ + [ + 17, + 32, + "N-terminal half", + "structure_element" + ], + [ + 36, + 43, + "Im76-45", + "mutant" + ], + [ + 75, + 78, + "Spy", + "protein" + ], + [ + 79, + 85, + "cradle", + "site" + ], + [ + 99, + 114, + "C-terminal half", + "structure_element" + ], + [ + 157, + 160, + "Spy", + "protein" + ], + [ + 163, + 178, + "concave surface", + "site" + ] + ] + }, + { + "sid": 98, + "sent": "Not unexpectedly, we found that as Im76-45 progresses from the unfolded to the native state, its interactions with Spy shift accordingly.", + "section": "RESULTS", + "ner": [ + [ + 35, + 42, + "Im76-45", + "mutant" + ], + [ + 63, + 71, + "unfolded", + "protein_state" + ], + [ + 79, + 85, + "native", + "protein_state" + ], + [ + 115, + 118, + "Spy", + "protein" + ] + ] + }, + { + "sid": 99, + "sent": "Whereas the least-folded Im76-45 pose in the ensemble forms the most hydrophobic contacts with Spy (Fig. 3), the two most-folded conformations form the fewest hydrophobic contacts (Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 12, + 24, + "least-folded", + "protein_state" + ], + [ + 25, + 32, + "Im76-45", + "mutant" + ], + [ + 95, + 98, + "Spy", + "protein" + ], + [ + 117, + 128, + "most-folded", + "protein_state" + ] + ] + }, + { + "sid": 100, + "sent": "This shift in contacts is likely due to hydrophobic residues of Im76-45 preferentially forming intra-molecular contacts upon folding (i.e., hydrophobic collapse), effectively removing themselves from the interaction sites.", + "section": "RESULTS", + "ner": [ + [ + 64, + 71, + "Im76-45", + "mutant" + ], + [ + 204, + 221, + "interaction sites", + "site" + ] + ] + }, + { + "sid": 101, + "sent": "The diversity of conformations and binding sites observed here emphasizes the dynamic and heterogeneous nature of the chaperone-substrate ensemble.", + "section": "RESULTS", + "ner": [ + [ + 35, + 48, + "binding sites", + "site" + ], + [ + 118, + 127, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 102, + "sent": "Although we do not yet have time resolution data of these various snapshots of Im76-45, this ensemble illustrates how a substrate samples its folding landscape while bound to a chaperone.", + "section": "RESULTS", + "ner": [ + [ + 79, + 86, + "Im76-45", + "mutant" + ], + [ + 166, + 174, + "bound to", + "protein_state" + ], + [ + 177, + 186, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 103, + "sent": "Spy changes conformation upon substrate binding", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "Spy", + "protein" + ] + ] + }, + { + "sid": 104, + "sent": "Comparing the structure of Spy in its substrate-bound and apo states revealed that the Spy dimer also undergoes significant conformational changes upon substrate binding (Fig. 5a and Supplementary Movie 2).", + "section": "RESULTS", + "ner": [ + [ + 14, + 23, + "structure", + "evidence" + ], + [ + 27, + 30, + "Spy", + "protein" + ], + [ + 38, + 53, + "substrate-bound", + "protein_state" + ], + [ + 58, + 61, + "apo", + "protein_state" + ], + [ + 87, + 90, + "Spy", + "protein" + ], + [ + 91, + 96, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 105, + "sent": "Upon substrate binding, the Spy dimer twists 9\u00b0 about its center relative to its apo form.", + "section": "RESULTS", + "ner": [ + [ + 28, + 31, + "Spy", + "protein" + ], + [ + 32, + 37, + "dimer", + "oligomeric_state" + ], + [ + 81, + 84, + "apo", + "protein_state" + ] + ] + }, + { + "sid": 106, + "sent": "This twist yields asymmetry and results in substantially different interaction patterns in the two Spy monomers (Fig. 4b).", + "section": "RESULTS", + "ner": [ + [ + 99, + 102, + "Spy", + "protein" + ], + [ + 103, + 111, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 107, + "sent": "It is possible that this twist serves to increase heterogeneity in Spy by providing more binding poses.", + "section": "RESULTS", + "ner": [ + [ + 67, + 70, + "Spy", + "protein" + ] + ] + }, + { + "sid": 108, + "sent": "Additionally, we observed that the linker region (residues 47\u201357) of Spy, which participates in substrate interaction, becomes mostly disordered upon binding the substrate.", + "section": "RESULTS", + "ner": [ + [ + 35, + 48, + "linker region", + "structure_element" + ], + [ + 59, + 64, + "47\u201357", + "residue_range" + ], + [ + 69, + 72, + "Spy", + "protein" + ], + [ + 134, + 144, + "disordered", + "protein_state" + ] + ] + }, + { + "sid": 109, + "sent": "This increased disorder might explain how Spy is able to recognize and bind different substrates and/or differing conformations of the same substrate.", + "section": "RESULTS", + "ner": [ + [ + 42, + 45, + "Spy", + "protein" + ] + ] + }, + { + "sid": 110, + "sent": "Importantly, we observed the same structural changes in Spy regardless of which of the four substrates was bound (Fig. 5b, Table 1).", + "section": "RESULTS", + "ner": [ + [ + 56, + 59, + "Spy", + "protein" + ] + ] + }, + { + "sid": 111, + "sent": "The RMSD between the well-folded sections of Spy in the four chaperone-substrate complexes was very small, less than 0.3 \u00c5. Combined with competition experiments showing that the substrates compete in solution for Spy binding (Fig. 5c and Supplementary Fig. 8), we conclude that all the tested substrates share the same overall Spy binding site.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "RMSD", + "evidence" + ], + [ + 21, + 32, + "well-folded", + "protein_state" + ], + [ + 45, + 48, + "Spy", + "protein" + ], + [ + 61, + 70, + "chaperone", + "protein_type" + ], + [ + 138, + 161, + "competition experiments", + "experimental_method" + ], + [ + 214, + 217, + "Spy", + "protein" + ], + [ + 328, + 344, + "Spy binding site", + "site" + ] + ] + }, + { + "sid": 112, + "sent": "To shed light on how chaperones interact with their substrates, we developed a novel structural biology method (READ) and applied it to determine a conformational ensemble of the chaperone Spy bound to substrate.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 31, + "chaperones", + "protein_type" + ], + [ + 112, + 116, + "READ", + "experimental_method" + ], + [ + 148, + 171, + "conformational ensemble", + "evidence" + ], + [ + 179, + 188, + "chaperone", + "protein_type" + ], + [ + 189, + 192, + "Spy", + "protein" + ], + [ + 193, + 211, + "bound to substrate", + "protein_state" + ] + ] + }, + { + "sid": 113, + "sent": "As a substrate, we used Im76-45, the chaperone-interacting portion of the protein-folding model protein Im7.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 31, + "Im76-45", + "mutant" + ], + [ + 37, + 66, + "chaperone-interacting portion", + "structure_element" + ], + [ + 104, + 107, + "Im7", + "protein" + ] + ] + }, + { + "sid": 114, + "sent": "In the chaperone-bound ensemble, Im76-45 samples unfolded, partially folded, and native-like states.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 22, + "chaperone-bound", + "protein_state" + ], + [ + 33, + 40, + "Im76-45", + "mutant" + ], + [ + 49, + 57, + "unfolded", + "protein_state" + ], + [ + 69, + 75, + "folded", + "protein_state" + ], + [ + 81, + 87, + "native", + "protein_state" + ] + ] + }, + { + "sid": 115, + "sent": "The ensemble provides an unprecedented description of the conformations that a substrate assumes while exploring its chaperone-associated folding landscape.", + "section": "DISCUSS", + "ner": [ + [ + 117, + 126, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 116, + "sent": "This substrate-chaperone ensemble helps accomplish the longstanding goal of obtaining a detailed view of how a chaperone aids protein folding.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 24, + "chaperone", + "protein_type" + ], + [ + 111, + 120, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 117, + "sent": "We recently showed that Im7 can fold while remaining continuously bound to Spy.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 27, + "Im7", + "protein" + ], + [ + 53, + 74, + "continuously bound to", + "protein_state" + ], + [ + 75, + 78, + "Spy", + "protein" + ] + ] + }, + { + "sid": 118, + "sent": "The high-resolution ensemble obtained here now provides insight into exactly how this occurs.", + "section": "DISCUSS", + "ner": [ + [ + 20, + 28, + "ensemble", + "evidence" + ] + ] + }, + { + "sid": 119, + "sent": "The structures of our ensemble agree well with lower-resolution crosslinking data, which indicate that chaperone-substrate interactions primarily occur on the concave surface of Spy.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 22, + 30, + "ensemble", + "evidence" + ], + [ + 103, + 112, + "chaperone", + "protein_type" + ], + [ + 159, + 174, + "concave surface", + "site" + ], + [ + 178, + 181, + "Spy", + "protein" + ] + ] + }, + { + "sid": 120, + "sent": "The ensemble suggests a model in which Spy provides an amphipathic surface that allows substrate proteins to assume different conformations while bound to the chaperone.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 12, + "ensemble", + "evidence" + ], + [ + 39, + 42, + "Spy", + "protein" + ], + [ + 55, + 74, + "amphipathic surface", + "site" + ], + [ + 146, + 154, + "bound to", + "protein_state" + ], + [ + 159, + 168, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 121, + "sent": "This model is consistent with previous studies postulating that the flexible binding of chaperones allows for substrate protein folding.", + "section": "DISCUSS", + "ner": [ + [ + 88, + 98, + "chaperones", + "protein_type" + ] + ] + }, + { + "sid": 122, + "sent": "The amphipathic concave surface of Spy likely facilitates this flexible binding and may be a crucial feature for Spy and potentially other chaperones, allowing them to bind multiple conformations of many different substrates.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 31, + "concave surface", + "site" + ], + [ + 35, + 38, + "Spy", + "protein" + ], + [ + 113, + 116, + "Spy", + "protein" + ], + [ + 139, + 149, + "chaperones", + "protein_type" + ] + ] + }, + { + "sid": 123, + "sent": "In contrast to Spy\u2019s binding hotspots, Im76-45 displays substantially less specificity in its binding sites.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 18, + "Spy", + "protein" + ], + [ + 21, + 37, + "binding hotspots", + "site" + ], + [ + 39, + 46, + "Im76-45", + "mutant" + ], + [ + 94, + 107, + "binding sites", + "site" + ] + ] + }, + { + "sid": 124, + "sent": "Nearly all Im76-45 residues come in contact with Spy.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 18, + "Im76-45", + "mutant" + ], + [ + 49, + 52, + "Spy", + "protein" + ] + ] + }, + { + "sid": 125, + "sent": "Unfolded substrate conformers interact with Spy through both hydrophobic and hydrophilic interactions, whereas the binding of native-like states is mainly hydrophilic.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 8, + "Unfolded", + "protein_state" + ], + [ + 44, + 47, + "Spy", + "protein" + ], + [ + 61, + 101, + "hydrophobic and hydrophilic interactions", + "bond_interaction" + ], + [ + 126, + 137, + "native-like", + "protein_state" + ] + ] + }, + { + "sid": 126, + "sent": "This trend suggests that complex formation between an ATP-independent chaperone and its unfolded substrate may initially involve hydrophobic interactions, effectively shielding the exposed aggregation-sensitive hydrophobic regions in the substrate.", + "section": "DISCUSS", + "ner": [ + [ + 54, + 69, + "ATP-independent", + "protein_state" + ], + [ + 70, + 79, + "chaperone", + "protein_type" + ], + [ + 88, + 96, + "unfolded", + "protein_state" + ], + [ + 129, + 153, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 211, + 230, + "hydrophobic regions", + "site" + ] + ] + }, + { + "sid": 127, + "sent": "Once the substrate begins to fold within this protected environment, it progressively buries its own hydrophobic residues, and its interactions with the chaperone shift towards becoming more electrostatic.", + "section": "DISCUSS", + "ner": [ + [ + 153, + 162, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 128, + "sent": "Notably, the most frequent contacts between Spy and Im76-45 are charge-charge interactions.", + "section": "DISCUSS", + "ner": [ + [ + 44, + 47, + "Spy", + "protein" + ], + [ + 52, + 59, + "Im76-45", + "mutant" + ], + [ + 64, + 90, + "charge-charge interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 129, + "sent": "The negatively charged Im7 residues Glu21, Asp32, and Asp35 reside on the surface of Im7 and form interactions with Spy\u2019s positively charged cradle in both the unfolded and native-like states.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 26, + "Im7", + "protein" + ], + [ + 36, + 41, + "Glu21", + "residue_name_number" + ], + [ + 43, + 48, + "Asp32", + "residue_name_number" + ], + [ + 54, + 59, + "Asp35", + "residue_name_number" + ], + [ + 85, + 88, + "Im7", + "protein" + ], + [ + 116, + 119, + "Spy", + "protein" + ], + [ + 141, + 147, + "cradle", + "site" + ], + [ + 160, + 168, + "unfolded", + "protein_state" + ], + [ + 173, + 184, + "native-like", + "protein_state" + ] + ] + }, + { + "sid": 130, + "sent": "Residues Asp32 and Asp35 are close to each other in the folded state of Im7.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 14, + "Asp32", + "residue_name_number" + ], + [ + 19, + 24, + "Asp35", + "residue_name_number" + ], + [ + 56, + 62, + "folded", + "protein_state" + ], + [ + 72, + 75, + "Im7", + "protein" + ] + ] + }, + { + "sid": 131, + "sent": "This proximity likely causes electrostatic repulsion that destabilizes Im7\u2019s native state.", + "section": "DISCUSS", + "ner": [ + [ + 71, + 74, + "Im7", + "protein" + ], + [ + 77, + 83, + "native", + "protein_state" + ] + ] + }, + { + "sid": 132, + "sent": "Interaction with Spy\u2019s positively-charged residues likely relieves the charge repulsion between Asp32 and Asp35, promoting their compaction into a helical conformation.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 20, + "Spy", + "protein" + ], + [ + 96, + 101, + "Asp32", + "residue_name_number" + ], + [ + 106, + 111, + "Asp35", + "residue_name_number" + ], + [ + 147, + 167, + "helical conformation", + "protein_state" + ] + ] + }, + { + "sid": 133, + "sent": "As inter-molecular hydrophobic interactions between Spy and the substrate become progressively replaced by intra-molecular interactions within the substrate, the affinity between chaperone and substrates could decrease, eventually leading to release of the folded client protein.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 43, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 52, + 55, + "Spy", + "protein" + ], + [ + 179, + 188, + "chaperone", + "protein_type" + ], + [ + 257, + 263, + "folded", + "protein_state" + ] + ] + }, + { + "sid": 134, + "sent": "Recently, we employed a genetic selection system to improve the chaperone activity of Spy.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 48, + "genetic selection system", + "experimental_method" + ], + [ + 64, + 73, + "chaperone", + "protein_type" + ], + [ + 86, + 89, + "Spy", + "protein" + ] + ] + }, + { + "sid": 135, + "sent": "This selection resulted in \u201cSuper Spy\u201d variants that were more effective at both preventing aggregation and promoting protein folding.", + "section": "DISCUSS", + "ner": [ + [ + 34, + 37, + "Spy", + "protein" + ], + [ + 39, + 47, + "variants", + "protein_state" + ] + ] + }, + { + "sid": 136, + "sent": "In conjunction with our bound Im76-45 ensemble, these mutants now allowed us to investigate structural features important to chaperone function.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 29, + "bound", + "protein_state" + ], + [ + 30, + 37, + "Im76-45", + "mutant" + ], + [ + 38, + 46, + "ensemble", + "evidence" + ], + [ + 125, + 134, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 137, + "sent": "Previous analysis revealed that the Super Spy variants either bound Im7 tighter than WT Spy, increased chaperone flexibility as measured via H/D exchange, or both.", + "section": "DISCUSS", + "ner": [ + [ + 42, + 45, + "Spy", + "protein" + ], + [ + 46, + 54, + "variants", + "protein_state" + ], + [ + 62, + 67, + "bound", + "protein_state" + ], + [ + 68, + 71, + "Im7", + "protein" + ], + [ + 85, + 87, + "WT", + "protein_state" + ], + [ + 88, + 91, + "Spy", + "protein" + ], + [ + 103, + 112, + "chaperone", + "protein_type" + ], + [ + 141, + 153, + "H/D exchange", + "experimental_method" + ] + ] + }, + { + "sid": 138, + "sent": "Our ensemble revealed that two of the Super Spy mutations (H96L and Q100L) form part of the chaperone contact surface that binds to Im76-45 (Fig. 4a).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 12, + "ensemble", + "evidence" + ], + [ + 44, + 47, + "Spy", + "protein" + ], + [ + 48, + 57, + "mutations", + "protein_state" + ], + [ + 59, + 63, + "H96L", + "mutant" + ], + [ + 68, + 73, + "Q100L", + "mutant" + ], + [ + 92, + 117, + "chaperone contact surface", + "site" + ], + [ + 132, + 139, + "Im76-45", + "mutant" + ] + ] + }, + { + "sid": 139, + "sent": "Moreover, our co-structure suggests that the L32P substitution, which increases Spy\u2019s flexibility, could operate by unhinging the N-terminal helix and effectively expanding the size of the disordered linker.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 26, + "co-structure", + "evidence" + ], + [ + 45, + 49, + "L32P", + "mutant" + ], + [ + 80, + 83, + "Spy", + "protein" + ], + [ + 130, + 146, + "N-terminal helix", + "structure_element" + ], + [ + 189, + 199, + "disordered", + "protein_state" + ], + [ + 200, + 206, + "linker", + "structure_element" + ] + ] + }, + { + "sid": 140, + "sent": "This possibility is supported by the Spy:substrate structures, in which the linker region becomes more flexible compared to the apo state (Fig. 6a).", + "section": "DISCUSS", + "ner": [ + [ + 37, + 40, + "Spy", + "protein" + ], + [ + 51, + 61, + "structures", + "evidence" + ], + [ + 76, + 89, + "linker region", + "structure_element" + ], + [ + 128, + 131, + "apo", + "protein_state" + ] + ] + }, + { + "sid": 141, + "sent": "By sampling multiple conformations, this linker region may allow diverse substrate conformations to be accommodated.", + "section": "DISCUSS", + "ner": [ + [ + 41, + 54, + "linker region", + "structure_element" + ] + ] + }, + { + "sid": 142, + "sent": "Other Super Spy mutations (F115I and F115L) caused increased flexibility but not tighter substrate binding.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 15, + "Spy", + "protein" + ], + [ + 16, + 25, + "mutations", + "protein_state" + ], + [ + 27, + 32, + "F115I", + "mutant" + ], + [ + 37, + 42, + "F115L", + "mutant" + ] + ] + }, + { + "sid": 143, + "sent": "This residue does not directly contact Im76-45 in our READ-derived ensemble.", + "section": "DISCUSS", + "ner": [ + [ + 39, + 46, + "Im76-45", + "mutant" + ], + [ + 54, + 58, + "READ", + "experimental_method" + ], + [ + 67, + 75, + "ensemble", + "evidence" + ] + ] + }, + { + "sid": 144, + "sent": "Instead, when Spy is bound to substrate, F115 engages in close CH\u22ef\u03c0 hydrogen bonds with Tyr104 (Fig. 6b).", + "section": "DISCUSS", + "ner": [ + [ + 14, + 17, + "Spy", + "protein" + ], + [ + 21, + 29, + "bound to", + "protein_state" + ], + [ + 41, + 45, + "F115", + "residue_name_number" + ], + [ + 68, + 82, + "hydrogen bonds", + "bond_interaction" + ], + [ + 88, + 94, + "Tyr104", + "residue_name_number" + ] + ] + }, + { + "sid": 145, + "sent": "This interaction presumably reduces the mobility of the C-terminal helix.", + "section": "DISCUSS", + "ner": [ + [ + 56, + 72, + "C-terminal helix", + "structure_element" + ] + ] + }, + { + "sid": 146, + "sent": "The F115I/L substitutions would replace these hydrogen bonds with hydrophobic interactions that have little angular dependence.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 9, + "F115I", + "mutant" + ], + [ + 10, + 11, + "L", + "mutant" + ], + [ + 46, + 60, + "hydrogen bonds", + "bond_interaction" + ], + [ + 66, + 90, + "hydrophobic interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 147, + "sent": "As a result, the C-terminus, and possibly also the flexible linker, is likely to become more flexible and thus more accommodating of different conformations of substrates.", + "section": "DISCUSS", + "ner": [ + [ + 51, + 59, + "flexible", + "protein_state" + ], + [ + 60, + 66, + "linker", + "structure_element" + ] + ] + }, + { + "sid": 148, + "sent": "Overall, comparison of our ensemble to the Super Spy variants provides specific examples to corroborate the importance of conformational flexibility in chaperone-substrate interactions.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 35, + "ensemble", + "evidence" + ], + [ + 49, + 52, + "Spy", + "protein" + ], + [ + 53, + 61, + "variants", + "protein_state" + ], + [ + 152, + 161, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 149, + "sent": "Despite extensive studies, exactly how complex chaperone machines help proteins fold remains controversial.", + "section": "DISCUSS", + "ner": [ + [ + 47, + 56, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 150, + "sent": "Our study indicates that the chaperone Spy employs a simple surface binding approach that allows the substrate to explore various conformations and form transiently favorable interactions while being protected from aggregation.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 38, + "chaperone", + "protein_type" + ], + [ + 39, + 42, + "Spy", + "protein" + ] + ] + }, + { + "sid": 151, + "sent": "We speculate that many other chaperones could utilize a similar strategy.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 39, + "chaperones", + "protein_type" + ] + ] + }, + { + "sid": 152, + "sent": "ATP and co-chaperone dependencies may have emerged later through evolution to better modulate and control chaperone action.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 3, + "ATP", + "chemical" + ], + [ + 11, + 20, + "chaperone", + "protein_type" + ], + [ + 106, + 115, + "chaperone", + "protein_type" + ] + ] + }, + { + "sid": 153, + "sent": "In addition to insights into chaperone function, this work presents a new method for determining heterogeneous structural ensembles via a hybrid methodology of X-ray crystallography and computational modeling.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 38, + "chaperone", + "protein_type" + ], + [ + 160, + 181, + "X-ray crystallography", + "experimental_method" + ], + [ + 186, + 208, + "computational modeling", + "experimental_method" + ] + ] + }, + { + "sid": 154, + "sent": "Heterogeneous dynamic complexes or disordered regions of single proteins, once considered solely approachable by NMR spectroscopy, can now be visualized through X-ray crystallography.", + "section": "DISCUSS", + "ner": [ + [ + 35, + 45, + "disordered", + "protein_state" + ], + [ + 113, + 129, + "NMR spectroscopy", + "experimental_method" + ], + [ + 161, + 182, + "X-ray crystallography", + "experimental_method" + ] + ] + }, + { + "sid": 155, + "sent": "Crystallographic data and ensemble selection. (a) 2mFo\u2212DFc omit map of residual Im76-45 and flexible linker electron density contoured at 0.5 \u03c3.", + "section": "FIG", + "ner": [ + [ + 50, + 67, + "2mFo\u2212DFc omit map", + "evidence" + ], + [ + 80, + 87, + "Im76-45", + "mutant" + ], + [ + 92, + 107, + "flexible linker", + "structure_element" + ], + [ + 108, + 124, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 156, + "sent": "This is the residual density that is used in the READ selection.", + "section": "FIG", + "ner": [ + [ + 21, + 28, + "density", + "evidence" + ], + [ + 49, + 53, + "READ", + "experimental_method" + ] + ] + }, + { + "sid": 157, + "sent": "(b) Composites of iodine positions detected from anomalous signals using pI-Phe substitutions, colored and numbered by sequence.", + "section": "FIG", + "ner": [ + [ + 18, + 24, + "iodine", + "chemical" + ], + [ + 49, + 66, + "anomalous signals", + "evidence" + ], + [ + 73, + 79, + "pI-Phe", + "chemical" + ], + [ + 80, + 93, + "substitutions", + "experimental_method" + ] + ] + }, + { + "sid": 158, + "sent": "Multiple iodine positions were detected for most residues.", + "section": "FIG", + "ner": [ + [ + 9, + 15, + "iodine", + "chemical" + ] + ] + }, + { + "sid": 159, + "sent": "Agreement to the residual Im76-45 electron density (c) and anomalous iodine signals (d) for ensembles of varying size generated by randomly choosing from the MD pool (blue) and from the selection procedure (black).", + "section": "FIG", + "ner": [ + [ + 26, + 33, + "Im76-45", + "mutant" + ], + [ + 34, + 50, + "electron density", + "evidence" + ], + [ + 59, + 83, + "anomalous iodine signals", + "evidence" + ], + [ + 158, + 160, + "MD", + "experimental_method" + ] + ] + }, + { + "sid": 160, + "sent": "The cost function, \u03c72, decreases as the agreement to the experimental data increases and is defined in the Online Methods.", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "cost function", + "evidence" + ], + [ + 19, + 21, + "\u03c72", + "evidence" + ] + ] + }, + { + "sid": 161, + "sent": "Flowchart of the READ sample-and-select process.", + "section": "FIG", + "ner": [ + [ + 17, + 21, + "READ", + "experimental_method" + ], + [ + 22, + 39, + "sample-and-select", + "experimental_method" + ] + ] + }, + { + "sid": 162, + "sent": "Spy:Im76-45 ensemble, arranged by RMSD to native state of Im76-45. Although the six-membered ensemble from the READ selection should be considered only as an ensemble, for clarity, the individual conformers are shown separately here.", + "section": "FIG", + "ner": [ + [ + 0, + 11, + "Spy:Im76-45", + "complex_assembly" + ], + [ + 34, + 38, + "RMSD", + "evidence" + ], + [ + 42, + 48, + "native", + "protein_state" + ], + [ + 58, + 65, + "Im76-45", + "mutant" + ], + [ + 111, + 115, + "READ", + "experimental_method" + ] + ] + }, + { + "sid": 163, + "sent": "Spy is depicted as a gray surface and the Im76-45 conformer is shown as orange balls.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "Spy", + "protein" + ], + [ + 42, + 49, + "Im76-45", + "mutant" + ] + ] + }, + { + "sid": 164, + "sent": "Atoms that were either not directly selected in the READ procedure, or whose position could not be justified based on agreement with the residual electron density were removed, leading to non-contiguous sections.", + "section": "FIG", + "ner": [ + [ + 52, + 56, + "READ", + "experimental_method" + ], + [ + 137, + 162, + "residual electron density", + "evidence" + ] + ] + }, + { + "sid": 165, + "sent": "Dashed lines connect non-contiguous segments of the Im76-45 substrate.", + "section": "FIG", + "ner": [ + [ + 52, + 59, + "Im76-45", + "mutant" + ] + ] + }, + { + "sid": 166, + "sent": "Residues of the Spy flexible linker region that fit the residual electron density are shown as larger gray spheres.", + "section": "FIG", + "ner": [ + [ + 16, + 19, + "Spy", + "protein" + ], + [ + 29, + 42, + "linker region", + "structure_element" + ], + [ + 56, + 81, + "residual electron density", + "evidence" + ] + ] + }, + { + "sid": 167, + "sent": "Shown below each ensemble member is the RMSD of each conformer to the native state of Im76-45, as well as the percentage of contacts between Im76-45 and Spy that are hydrophobic.", + "section": "FIG", + "ner": [ + [ + 40, + 44, + "RMSD", + "evidence" + ], + [ + 70, + 76, + "native", + "protein_state" + ], + [ + 86, + 93, + "Im76-45", + "mutant" + ], + [ + 141, + 148, + "Im76-45", + "mutant" + ], + [ + 153, + 156, + "Spy", + "protein" + ] + ] + }, + { + "sid": 168, + "sent": "Contact maps of Spy:Im76-45 complex.", + "section": "FIG", + "ner": [ + [ + 0, + 12, + "Contact maps", + "evidence" + ], + [ + 16, + 27, + "Spy:Im76-45", + "complex_assembly" + ] + ] + }, + { + "sid": 169, + "sent": "(a) Spy:Im76-45 contact map projected onto the bound Spy dimer (above) and Im76-45 (below) structures.", + "section": "FIG", + "ner": [ + [ + 4, + 15, + "Spy:Im76-45", + "complex_assembly" + ], + [ + 16, + 27, + "contact map", + "evidence" + ], + [ + 47, + 52, + "bound", + "protein_state" + ], + [ + 53, + 56, + "Spy", + "protein" + ], + [ + 57, + 62, + "dimer", + "oligomeric_state" + ], + [ + 75, + 82, + "Im76-45", + "mutant" + ], + [ + 91, + 101, + "structures", + "evidence" + ] + ] + }, + { + "sid": 170, + "sent": "For clarity, Im76-45 is represented with a single conformation.", + "section": "FIG", + "ner": [ + [ + 13, + 20, + "Im76-45", + "mutant" + ] + ] + }, + { + "sid": 171, + "sent": "The frequency plotted is calculated as the average contact frequency from Spy to every residue of Im76-45 and vice-versa.", + "section": "FIG", + "ner": [ + [ + 51, + 68, + "contact frequency", + "evidence" + ], + [ + 74, + 77, + "Spy", + "protein" + ], + [ + 98, + 105, + "Im76-45", + "mutant" + ] + ] + }, + { + "sid": 172, + "sent": "As the residues involved in contacts are more evenly distributed in Im76-45 compared to Spy, its contact map was amplified. (b) Detailed contact maps of Spy:Im76-45.", + "section": "FIG", + "ner": [ + [ + 68, + 75, + "Im76-45", + "mutant" + ], + [ + 88, + 91, + "Spy", + "protein" + ], + [ + 97, + 108, + "contact map", + "evidence" + ], + [ + 137, + 149, + "contact maps", + "evidence" + ], + [ + 153, + 164, + "Spy:Im76-45", + "complex_assembly" + ] + ] + }, + { + "sid": 173, + "sent": "Contacts to the two Spy monomers are depicted separately.", + "section": "FIG", + "ner": [ + [ + 20, + 23, + "Spy", + "protein" + ], + [ + 24, + 32, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 174, + "sent": "Note that the flexible linker region of Spy (residues 47\u201357) is not represented in the 2D contact maps.", + "section": "FIG", + "ner": [ + [ + 14, + 22, + "flexible", + "protein_state" + ], + [ + 23, + 36, + "linker region", + "structure_element" + ], + [ + 40, + 43, + "Spy", + "protein" + ], + [ + 54, + 59, + "47\u201357", + "residue_range" + ], + [ + 90, + 102, + "contact maps", + "evidence" + ] + ] + }, + { + "sid": 175, + "sent": "Spy conformation changes upon substrate binding.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "Spy", + "protein" + ] + ] + }, + { + "sid": 176, + "sent": "(a) Overlay of apo Spy (PDB ID: 3O39, gray) and bound Spy (green). (b) Overlay of WT Spy bound to Im76-45 (green), H96L Spy bound to Im7 L18A L19 AL13A (blue), H96L Spy bound to WT Im7 (yellow), and WT Spy bound to casein (salmon). (c) Competition assay showing Im76-45 competes with Im7 L18A L19A L37A H40W for the same binding site on Spy (further substrate competition assays are shown in Supplementary Fig. 8).", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "Overlay", + "experimental_method" + ], + [ + 15, + 18, + "apo", + "protein_state" + ], + [ + 19, + 22, + "Spy", + "protein" + ], + [ + 48, + 53, + "bound", + "protein_state" + ], + [ + 54, + 57, + "Spy", + "protein" + ], + [ + 71, + 78, + "Overlay", + "experimental_method" + ], + [ + 82, + 84, + "WT", + "protein_state" + ], + [ + 85, + 88, + "Spy", + "protein" + ], + [ + 89, + 97, + "bound to", + "protein_state" + ], + [ + 98, + 105, + "Im76-45", + "mutant" + ], + [ + 115, + 119, + "H96L", + "mutant" + ], + [ + 120, + 123, + "Spy", + "protein" + ], + [ + 124, + 132, + "bound to", + "protein_state" + ], + [ + 133, + 136, + "Im7", + "protein" + ], + [ + 137, + 141, + "L18A", + "mutant" + ], + [ + 142, + 147, + "L19 A", + "mutant" + ], + [ + 147, + 151, + "L13A", + "mutant" + ], + [ + 160, + 164, + "H96L", + "mutant" + ], + [ + 165, + 168, + "Spy", + "protein" + ], + [ + 169, + 177, + "bound to", + "protein_state" + ], + [ + 178, + 180, + "WT", + "protein_state" + ], + [ + 181, + 184, + "Im7", + "protein" + ], + [ + 199, + 201, + "WT", + "protein_state" + ], + [ + 202, + 205, + "Spy", + "protein" + ], + [ + 206, + 214, + "bound to", + "protein_state" + ], + [ + 215, + 221, + "casein", + "chemical" + ], + [ + 236, + 253, + "Competition assay", + "experimental_method" + ], + [ + 262, + 269, + "Im76-45", + "mutant" + ], + [ + 284, + 287, + "Im7", + "protein" + ], + [ + 288, + 292, + "L18A", + "mutant" + ], + [ + 293, + 297, + "L19A", + "mutant" + ], + [ + 298, + 302, + "L37A", + "mutant" + ], + [ + 303, + 307, + "H40W", + "mutant" + ], + [ + 321, + 333, + "binding site", + "site" + ], + [ + 337, + 340, + "Spy", + "protein" + ], + [ + 350, + 378, + "substrate competition assays", + "experimental_method" + ] + ] + }, + { + "sid": 177, + "sent": "Flexibility of Spy linker region and effect of Super Spy mutants. (a) The Spy linker region adopts one dominant conformation in its apo state (PDB ID 3039, gray), but expands and adopts multiple conformations in bound states (green).", + "section": "FIG", + "ner": [ + [ + 15, + 18, + "Spy", + "protein" + ], + [ + 19, + 32, + "linker region", + "structure_element" + ], + [ + 53, + 56, + "Spy", + "protein" + ], + [ + 74, + 77, + "Spy", + "protein" + ], + [ + 78, + 91, + "linker region", + "structure_element" + ], + [ + 132, + 135, + "apo", + "protein_state" + ], + [ + 212, + 217, + "bound", + "protein_state" + ] + ] + }, + { + "sid": 178, + "sent": "(b) F115 and L32 tether Spy\u2019s linker region to its cradle, decreasing Spy activity by limiting linker region flexibility.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "F115", + "residue_name_number" + ], + [ + 13, + 16, + "L32", + "residue_name_number" + ], + [ + 24, + 27, + "Spy", + "protein" + ], + [ + 30, + 43, + "linker region", + "structure_element" + ], + [ + 51, + 57, + "cradle", + "site" + ], + [ + 70, + 73, + "Spy", + "protein" + ], + [ + 95, + 108, + "linker region", + "structure_element" + ] + ] + }, + { + "sid": 179, + "sent": "The Super Spy mutants F115L, F115I, and L32P are proposed to gain activity by increasing the flexibility or size of this linker region.", + "section": "FIG", + "ner": [ + [ + 10, + 13, + "Spy", + "protein" + ], + [ + 22, + 27, + "F115L", + "mutant" + ], + [ + 29, + 34, + "F115I", + "mutant" + ], + [ + 40, + 44, + "L32P", + "mutant" + ], + [ + 121, + 134, + "linker region", + "structure_element" + ] + ] + }, + { + "sid": 180, + "sent": "L32, F115, and Y104 are rendered in purple to illustrate residues that are most affected by Super Spy mutations; CH\u22ef\u03c0 hydrogen bonds are depicted by orange dashes.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "L32", + "residue_name_number" + ], + [ + 5, + 9, + "F115", + "residue_name_number" + ], + [ + 15, + 19, + "Y104", + "residue_name_number" + ], + [ + 98, + 101, + "Spy", + "protein" + ], + [ + 102, + 111, + "mutations", + "protein_state" + ], + [ + 118, + 132, + "hydrogen bonds", + "bond_interaction" + ] + ] + } + ] + }, + "PMC4857006": { + "annotations": [ + { + "sid": 0, + "sent": "Reversal of DNA damage induced Topoisomerase 2 DNA\u2013protein crosslinks by Tdp2", + "section": "TITLE", + "ner": [ + [ + 12, + 15, + "DNA", + "chemical" + ], + [ + 31, + 46, + "Topoisomerase 2", + "protein_type" + ], + [ + 47, + 50, + "DNA", + "chemical" + ], + [ + 73, + 77, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 1, + "sent": "Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA\u2013protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 9, + "Mammalian", + "taxonomy_domain" + ], + [ + 10, + 41, + "Tyrosyl-DNA phosphodiesterase 2", + "protein" + ], + [ + 43, + 47, + "Tdp2", + "protein" + ], + [ + 58, + 73, + "Topoisomerase 2", + "protein_type" + ], + [ + 75, + 79, + "Top2", + "protein_type" + ], + [ + 81, + 84, + "DNA", + "chemical" + ], + [ + 117, + 121, + "Top2", + "protein_type" + ], + [ + 136, + 139, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 2, + "sent": "Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 4, + "Tdp2", + "protein" + ], + [ + 81, + 85, + "Top2", + "protein_type" + ] + ] + }, + { + "sid": 3, + "sent": "Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions.", + "section": "ABSTRACT", + "ner": [ + [ + 18, + 42, + "X-ray crystal structures", + "evidence" + ], + [ + 46, + 57, + "ligand-free", + "protein_state" + ], + [ + 58, + 62, + "Tdp2", + "protein" + ], + [ + 67, + 75, + "Tdp2-DNA", + "complex_assembly" + ], + [ + 112, + 115, + "DNA", + "chemical" + ], + [ + 130, + 137, + "dynamic", + "protein_state" + ], + [ + 138, + 142, + "Tdp2", + "protein" + ], + [ + 143, + 158, + "active site lid", + "structure_element" + ], + [ + 168, + 192, + "substrate binding trench", + "site" + ], + [ + 230, + 233, + "DNA", + "chemical" + ], + [ + 262, + 266, + "Top2", + "protein_type" + ] + ] + }, + { + "sid": 4, + "sent": "Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-\u03c0 interface.", + "section": "ABSTRACT", + "ner": [ + [ + 23, + 27, + "Tdp2", + "protein" + ], + [ + 63, + 74, + "mutagenesis", + "experimental_method" + ], + [ + 79, + 98, + "biochemical studies", + "experimental_method" + ], + [ + 116, + 120, + "Mg2+", + "chemical" + ], + [ + 149, + 191, + "phosphotyrosyl-arginine cation-\u03c0 interface", + "site" + ] + ] + }, + { + "sid": 5, + "sent": "We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide.", + "section": "ABSTRACT", + "ner": [ + [ + 22, + 26, + "Tdp2", + "protein" + ], + [ + 27, + 38, + "active site", + "site" + ], + [ + 48, + 55, + "ablates", + "protein_state" + ], + [ + 56, + 60, + "Tdp2", + "protein" + ], + [ + 61, + 65, + "Mg2+", + "chemical" + ], + [ + 106, + 110, + "Tdp2", + "protein" + ], + [ + 128, + 136, + "tyrosine", + "residue_name" + ], + [ + 206, + 215, + "etoposide", + "chemical" + ] + ] + }, + { + "sid": 6, + "sent": "Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.", + "section": "ABSTRACT", + "ner": [ + [ + 61, + 65, + "Tdp2", + "protein" + ], + [ + 94, + 97, + "DNA", + "chemical" + ], + [ + 117, + 121, + "Top2", + "protein_type" + ], + [ + 165, + 169, + "Tdp2", + "protein" + ], + [ + 287, + 291, + "Top2", + "protein_type" + ] + ] + }, + { + "sid": 7, + "sent": "Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling\u2014over- and under-winding of DNA strands, respectively\u2014and the linking together (catenation) of DNA strands.", + "section": "INTRO", + "ner": [ + [ + 8, + 11, + "DNA", + "chemical" + ], + [ + 41, + 44, + "DNA", + "chemical" + ], + [ + 49, + 64, + "RNA polymerases", + "protein_type" + ], + [ + 94, + 97, + "DNA", + "chemical" + ], + [ + 138, + 141, + "DNA", + "chemical" + ], + [ + 205, + 208, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 8, + "sent": "Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division.", + "section": "INTRO", + "ner": [ + [ + 0, + 14, + "Topoisomerases", + "protein_type" + ], + [ + 35, + 38, + "DNA", + "chemical" + ], + [ + 94, + 97, + "DNA", + "chemical" + ], + [ + 121, + 124, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 9, + "sent": "The mammalian type II topoisomerases Top2\u03b1 and Top2\u03b2 enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions.", + "section": "INTRO", + "ner": [ + [ + 4, + 13, + "mammalian", + "taxonomy_domain" + ], + [ + 14, + 36, + "type II topoisomerases", + "protein_type" + ], + [ + 37, + 42, + "Top2\u03b1", + "protein" + ], + [ + 47, + 52, + "Top2\u03b2", + "protein" + ], + [ + 92, + 95, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 10, + "sent": "Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5\u2032-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc).", + "section": "INTRO", + "ner": [ + [ + 17, + 21, + "Top2", + "protein_type" + ], + [ + 22, + 25, + "DNA", + "chemical" + ], + [ + 92, + 115, + "phosphotyrosyl linkages", + "ptm" + ], + [ + 128, + 140, + "5\u2032-phosphate", + "chemical" + ], + [ + 175, + 186, + "active site", + "site" + ], + [ + 187, + 191, + "Top2", + "protein_type" + ], + [ + 192, + 200, + "tyrosine", + "residue_name" + ], + [ + 215, + 219, + "Top2", + "protein_type" + ], + [ + 240, + 246, + "Top2cc", + "complex_assembly" + ] + ] + }, + { + "sid": 11, + "sent": "The Top2cc protein\u2013DNA adduct is a unique threat to genomic integrity which must be resolved to prevent catastrophic Top2cc collisions with the cellular replication and transcription machineries.", + "section": "INTRO", + "ner": [ + [ + 4, + 10, + "Top2cc", + "complex_assembly" + ], + [ + 19, + 22, + "DNA", + "chemical" + ], + [ + 117, + 123, + "Top2cc", + "complex_assembly" + ] + ] + }, + { + "sid": 12, + "sent": "To promote cancer cell death, Top2 reactions are \u2018poisoned\u2019 by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin.", + "section": "INTRO", + "ner": [ + [ + 30, + 34, + "Top2", + "protein_type" + ], + [ + 111, + 120, + "etoposide", + "chemical" + ], + [ + 122, + 132, + "teniposide", + "chemical" + ], + [ + 137, + 148, + "doxorubicin", + "chemical" + ] + ] + }, + { + "sid": 13, + "sent": "Importantly, Top2 is also poisoned when it engages abundant endogenous DNA damage not limited to but including ribonucleotides, abasic sites and alkylation damage such as exocyclic DNA adducts arising from bioactivation of the vinyl chloride carcinogen (Figure 1A).", + "section": "INTRO", + "ner": [ + [ + 13, + 17, + "Top2", + "protein_type" + ], + [ + 71, + 74, + "DNA", + "chemical" + ], + [ + 181, + 184, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 14, + "sent": "In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5\u2032-linked Top2 DNA\u2013protein crosslink.", + "section": "INTRO", + "ner": [ + [ + 15, + 18, + "DNA", + "chemical" + ], + [ + 36, + 42, + "Top2cc", + "complex_assembly" + ], + [ + 53, + 56, + "DNA", + "chemical" + ], + [ + 117, + 120, + "DNA", + "chemical" + ], + [ + 160, + 164, + "Top2", + "protein_type" + ], + [ + 165, + 168, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 15, + "sent": "The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc.", + "section": "INTRO", + "ner": [ + [ + 27, + 30, + "DNA", + "chemical" + ], + [ + 46, + 52, + "Top2cc", + "complex_assembly" + ], + [ + 71, + 74, + "DNA", + "chemical" + ], + [ + 145, + 148, + "DNA", + "chemical" + ], + [ + 221, + 227, + "Top2cc", + "complex_assembly" + ] + ] + }, + { + "sid": 16, + "sent": "Precisely how the cellular DNA repair machinery navigates these complex lesions is an important aspect of Top2cc repair that has not yet been explored.", + "section": "INTRO", + "ner": [ + [ + 27, + 30, + "DNA", + "chemical" + ], + [ + 106, + 112, + "Top2cc", + "complex_assembly" + ] + ] + }, + { + "sid": 17, + "sent": "Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "Tdp2", + "protein" + ], + [ + 15, + 38, + "phosphotyrosyl linkages", + "ptm" + ], + [ + 50, + 53, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 18, + "sent": "(A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5\u2032\u2013Top2 protein adduct linked by a phosphotyrosine bond.", + "section": "FIG", + "ner": [ + [ + 15, + 18, + "DNA", + "chemical" + ], + [ + 65, + 68, + "DNA", + "chemical" + ], + [ + 121, + 125, + "Top2", + "protein_type" + ], + [ + 135, + 139, + "Top2", + "protein_type" + ], + [ + 158, + 164, + "Top2cc", + "complex_assembly" + ], + [ + 196, + 200, + "Top2", + "protein_type" + ], + [ + 228, + 243, + "phosphotyrosine", + "residue_name" + ] + ] + }, + { + "sid": 19, + "sent": "Tdp2 hydrolyzes the 5\u2032\u2013phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5\u2032-phosphate, which facilitates DNA end joining through the NHEJ pathway.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "Tdp2", + "protein" + ], + [ + 23, + 38, + "phosphotyrosine", + "residue_name" + ], + [ + 68, + 72, + "Top2", + "protein_type" + ], + [ + 81, + 84, + "DNA", + "chemical" + ], + [ + 97, + 109, + "5\u2032-phosphate", + "chemical" + ], + [ + 129, + 132, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 20, + "sent": "(B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (\u03f5A) or an abasic site (THF) and a 5\u2032\u2013nitrophenol moiety.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "DNA", + "chemical" + ], + [ + 85, + 103, + "Tdp2 enzyme assays", + "experimental_method" + ], + [ + 112, + 124, + "deoxyadenine", + "chemical" + ], + [ + 126, + 128, + "dA", + "chemical" + ], + [ + 131, + 144, + "Ethenoadenine", + "chemical" + ], + [ + 146, + 148, + "\u03f5A", + "chemical" + ], + [ + 156, + 167, + "abasic site", + "site" + ], + [ + 169, + 172, + "THF", + "chemical" + ] + ] + }, + { + "sid": 21, + "sent": "Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p\u2013nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s\u22121 produced by mTdp2cat.", + "section": "FIG", + "ner": [ + [ + 0, + 14, + "Phosphotyrosyl", + "ptm" + ], + [ + 44, + 52, + "mTdp2cat", + "structure_element" + ], + [ + 62, + 75, + "p-nitrophenol", + "chemical" + ], + [ + 134, + 142, + "mTdp2cat", + "structure_element" + ], + [ + 143, + 157, + "reaction rates", + "evidence" + ], + [ + 161, + 174, + "p\u2013nitrophenol", + "chemical" + ], + [ + 184, + 187, + "DNA", + "chemical" + ], + [ + 252, + 255, + "PNP", + "chemical" + ], + [ + 272, + 280, + "mTdp2cat", + "structure_element" + ] + ] + }, + { + "sid": 22, + "sent": "P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5\u2032-phosphate DNA (product complex) containing \u03f5A (yellow).", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "P-values", + "evidence" + ], + [ + 37, + 43, + "t-test", + "experimental_method" + ], + [ + 111, + 120, + "Structure", + "evidence" + ], + [ + 124, + 132, + "mTdp2cat", + "structure_element" + ], + [ + 133, + 141, + "bound to", + "protein_state" + ], + [ + 142, + 158, + "5\u2032-phosphate DNA", + "chemical" + ], + [ + 188, + 190, + "\u03f5A", + "chemical" + ] + ] + }, + { + "sid": 23, + "sent": "DNA binding \u03b22H\u03b2\u2013grasp (tan) and cap elements engage the 5\u2032-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA.", + "section": "FIG", + "ner": [ + [ + 0, + 22, + "DNA binding \u03b22H\u03b2\u2013grasp", + "site" + ], + [ + 128, + 131, + "DNA", + "chemical" + ], + [ + 0, + 22, + "DNA binding \u03b22H\u03b2\u2013grasp", + "site" + ], + [ + 128, + 131, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5\u2032-phosphate DNA (product complex) containing THF (yellow).", + "section": "FIG", + "ner": [ + [ + 51, + 60, + "Structure", + "evidence" + ], + [ + 64, + 72, + "mTdp2cat", + "structure_element" + ], + [ + 73, + 81, + "bound to", + "protein_state" + ], + [ + 82, + 98, + "5\u2032-phosphate DNA", + "chemical" + ], + [ + 128, + 131, + "THF", + "chemical" + ] + ] + }, + { + "sid": 25, + "sent": "PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.", + "section": "FIG", + "ner": [ + [ + 51, + 60, + "Structure", + "evidence" + ], + [ + 64, + 72, + "mTdp2cat", + "structure_element" + ], + [ + 80, + 90, + "absence of", + "protein_state" + ], + [ + 91, + 94, + "DNA", + "chemical" + ], + [ + 107, + 115, + "extended", + "protein_state" + ], + [ + 116, + 128, + "3-helix loop", + "structure_element" + ], + [ + 135, + 139, + "open", + "protein_state" + ], + [ + 160, + 177, + "DNA-binding grasp", + "site" + ], + [ + 189, + 196, + "monomer", + "oligomeric_state" + ], + [ + 197, + 198, + "E", + "structure_element" + ], + [ + 206, + 209, + "apo", + "protein_state" + ], + [ + 210, + 219, + "structure", + "evidence" + ] + ] + }, + { + "sid": 26, + "sent": "Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5\u2032-phosphotyrosyl (5\u2032-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons.", + "section": "INTRO", + "ner": [ + [ + 0, + 31, + "Tyrosyl DNA phosphodiesterase 2", + "protein" + ], + [ + 33, + 37, + "Tdp2", + "protein" + ], + [ + 59, + 76, + "5\u2032-phosphotyrosyl", + "ptm" + ], + [ + 78, + 82, + "5\u2032-Y", + "ptm" + ], + [ + 84, + 92, + "linkages", + "ptm" + ], + [ + 160, + 164, + "Top2", + "protein_type" + ] + ] + }, + { + "sid": 27, + "sent": "Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear \u03b3H2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "Tdp2", + "protein" + ], + [ + 5, + 14, + "knockdown", + "experimental_method" + ], + [ + 52, + 61, + "etoposide", + "chemical" + ], + [ + 158, + 162, + "Tdp2", + "protein" + ], + [ + 175, + 181, + "Top2cc", + "complex_assembly" + ] + ] + }, + { + "sid": 28, + "sent": "Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "Tdp2", + "protein" + ], + [ + 76, + 82, + "mutant", + "protein_state" + ], + [ + 83, + 86, + "p53", + "protein" + ], + [ + 106, + 112, + "mutant", + "protein_state" + ], + [ + 113, + 116, + "p53", + "protein" + ] + ] + }, + { + "sid": 29, + "sent": "The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2\u03b2-dependent transcription.", + "section": "INTRO", + "ner": [ + [ + 18, + 22, + "Tdp2", + "protein" + ], + [ + 36, + 49, + "topoisomerase", + "protein_type" + ], + [ + 98, + 103, + "human", + "species" + ], + [ + 104, + 108, + "TDP2", + "protein" + ], + [ + 237, + 244, + "loss of", + "protein_state" + ], + [ + 245, + 249, + "Tdp2", + "protein" + ], + [ + 259, + 264, + "Top2\u03b2", + "protein" + ] + ] + }, + { + "sid": 30, + "sent": "It is possible that TDP2 single nucleotide polymorphisms (SNPs) encode mutations that impact Tdp2 function, but the molecular underpinnings for such Tdp2 deficiencies are not understood.", + "section": "INTRO", + "ner": [ + [ + 20, + 24, + "TDP2", + "protein" + ], + [ + 93, + 97, + "Tdp2", + "protein" + ], + [ + 149, + 153, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 31, + "sent": "Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5\u2032-phosphorylated reaction product.", + "section": "INTRO", + "ner": [ + [ + 39, + 44, + "X-ray", + "experimental_method" + ], + [ + 45, + 63, + "crystal structures", + "evidence" + ], + [ + 71, + 99, + "minimal catalytically active", + "protein_state" + ], + [ + 100, + 136, + "endonuclease/exonuclease/phosphatase", + "structure_element" + ], + [ + 138, + 141, + "EEP", + "structure_element" + ], + [ + 153, + 158, + "mouse", + "taxonomy_domain" + ], + [ + 159, + 163, + "Tdp2", + "protein" + ], + [ + 165, + 173, + "mTdp2cat", + "structure_element" + ], + [ + 175, + 183, + "bound to", + "protein_state" + ], + [ + 186, + 189, + "DNA", + "chemical" + ], + [ + 213, + 230, + "5\u2032-phosphorylated", + "protein_state" + ] + ] + }, + { + "sid": 32, + "sent": "However, important questions regarding the mechanism of Tdp2 engagement and processing of DNA damage remain.", + "section": "INTRO", + "ner": [ + [ + 56, + 60, + "Tdp2", + "protein" + ], + [ + 90, + 93, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 33, + "sent": "First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site.", + "section": "INTRO", + "ner": [ + [ + 24, + 28, + "Tdp2", + "protein" + ], + [ + 39, + 62, + "phosphotyrosyl linkages", + "ptm" + ], + [ + 81, + 84, + "DNA", + "chemical" + ], + [ + 106, + 112, + "Top2cc", + "complex_assembly" + ], + [ + 169, + 172, + "DNA", + "chemical" + ], + [ + 191, + 202, + "active site", + "site" + ] + ] + }, + { + "sid": 34, + "sent": "Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization.", + "section": "INTRO", + "ner": [ + [ + 9, + 20, + "metal-bound", + "protein_state" + ], + [ + 21, + 25, + "Tdp2", + "protein" + ], + [ + 26, + 36, + "structures", + "evidence" + ], + [ + 64, + 68, + "Mg2+", + "chemical" + ] + ] + }, + { + "sid": 35, + "sent": "Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage.", + "section": "INTRO", + "ner": [ + [ + 32, + 56, + "structure-function study", + "experimental_method" + ], + [ + 64, + 68, + "Tdp2", + "protein" + ], + [ + 120, + 125, + "X-ray", + "experimental_method" + ], + [ + 126, + 136, + "structures", + "evidence" + ], + [ + 140, + 151, + "ligand-free", + "protein_state" + ], + [ + 152, + 156, + "Tdp2", + "protein" + ], + [ + 162, + 166, + "Tdp2", + "protein" + ], + [ + 167, + 175, + "bound to", + "protein_state" + ], + [ + 198, + 217, + "1-N6-etheno-adenine", + "chemical" + ], + [ + 219, + 222, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 36, + "sent": "Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage.", + "section": "INTRO", + "ner": [ + [ + 28, + 47, + "structural analysis", + "experimental_method" + ], + [ + 49, + 60, + "mutagenesis", + "experimental_method" + ], + [ + 62, + 79, + "functional assays", + "experimental_method" + ], + [ + 84, + 121, + "quanyum mechanics/molecular mechanics", + "experimental_method" + ], + [ + 123, + 128, + "QM/MM", + "experimental_method" + ], + [ + 130, + 138, + "modeling", + "experimental_method" + ], + [ + 146, + 150, + "Tdp2", + "protein" + ], + [ + 194, + 198, + "Tdp2", + "protein" + ], + [ + 227, + 256, + "tyrosyl DNA phosphodiesterase", + "protein_type" + ], + [ + 295, + 298, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 37, + "sent": "We further establish that DNA damage binding in the Tdp2 active site is linked to conformational change and binding of metal cofactor.", + "section": "INTRO", + "ner": [ + [ + 26, + 29, + "DNA", + "chemical" + ], + [ + 52, + 56, + "Tdp2", + "protein" + ], + [ + 57, + 68, + "active site", + "site" + ] + ] + }, + { + "sid": 38, + "sent": "Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells.", + "section": "INTRO", + "ner": [ + [ + 27, + 31, + "Tdp2", + "protein" + ], + [ + 41, + 48, + "ablates", + "protein_state" + ], + [ + 53, + 57, + "Tdp2", + "protein" + ], + [ + 58, + 83, + "single metal binding site", + "site" + ], + [ + 88, + 92, + "Tdp2", + "protein" + ], + [ + 147, + 151, + "Top2", + "protein_type" + ], + [ + 172, + 181, + "mammalian", + "taxonomy_domain" + ] + ] + }, + { + "sid": 39, + "sent": "Tdp2 processing of compound DNA damage", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "Tdp2", + "protein" + ], + [ + 28, + 31, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 40, + "sent": "Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, \u03f5A) and abundant abasic sites (Figure 1A).", + "section": "RESULTS", + "ner": [ + [ + 11, + 15, + "Top2", + "protein_type" + ], + [ + 48, + 51, + "DNA", + "chemical" + ], + [ + 69, + 72, + "DNA", + "chemical" + ], + [ + 92, + 110, + "1-N6-ethenoadenine", + "chemical" + ], + [ + 112, + 114, + "\u03f5A", + "chemical" + ] + ] + }, + { + "sid": 41, + "sent": "Whether Tdp2 processes phosphotyrosyl linkages within these diverse structural contexts is not known.", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "Tdp2", + "protein" + ], + [ + 23, + 46, + "phosphotyrosyl linkages", + "ptm" + ] + ] + }, + { + "sid": 42, + "sent": "To test this, we adapted an EDC coupling method to generate 5\u2032-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5\u2032-nucleotide position (see Materials and Methods).", + "section": "RESULTS", + "ner": [ + [ + 28, + 47, + "EDC coupling method", + "experimental_method" + ], + [ + 72, + 85, + "p-nitrophenol", + "chemical" + ], + [ + 87, + 90, + "PNP", + "chemical" + ], + [ + 137, + 140, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 43, + "sent": "We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5\u2032-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B).", + "section": "RESULTS", + "ner": [ + [ + 56, + 61, + "mouse", + "taxonomy_domain" + ], + [ + 62, + 66, + "Tdp2", + "protein" + ], + [ + 67, + 83, + "catalytic domain", + "structure_element" + ], + [ + 85, + 93, + "mTdp2cat", + "structure_element" + ], + [ + 106, + 109, + "PNP", + "chemical" + ], + [ + 135, + 148, + "topoisomerase", + "protein_type" + ], + [ + 149, + 157, + "tyrosine", + "residue_name" + ], + [ + 200, + 203, + "DNA", + "chemical" + ], + [ + 223, + 241, + "colorimetric assay", + "experimental_method" + ] + ] + }, + { + "sid": 44, + "sent": "We observe robust Tdp2-dependent release of PNP from 5\u2032-modified oligonucleotides in the context of dA-PNP, \u03f5A-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C).", + "section": "RESULTS", + "ner": [ + [ + 18, + 22, + "Tdp2", + "protein" + ], + [ + 44, + 47, + "PNP", + "chemical" + ], + [ + 100, + 106, + "dA-PNP", + "chemical" + ], + [ + 108, + 114, + "\u03f5A-PNP", + "chemical" + ], + [ + 141, + 163, + "tetrahydrofuran spacer", + "chemical" + ], + [ + 165, + 168, + "THF", + "chemical" + ] + ] + }, + { + "sid": 45, + "sent": "Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5\u2032 lesions composed of abasic or bulky DNA base adduct DNA damage.", + "section": "RESULTS", + "ner": [ + [ + 6, + 10, + "Tdp2", + "protein" + ], + [ + 31, + 54, + "phosphotyrosyl linkages", + "ptm" + ], + [ + 123, + 126, + "DNA", + "chemical" + ], + [ + 139, + 142, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 46, + "sent": "To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5\u2032-phosphate DNA (product complex) with a 5\u2032-\u03f5A at 1.43 \u00c5 resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5\u2032-THF at 2.15 \u00c5 resolution (PDB entry 5INK; Figure 1D and\u00a0E, Table 1).", + "section": "RESULTS", + "ner": [ + [ + 38, + 42, + "Tdp2", + "protein" + ], + [ + 57, + 63, + "Top2cc", + "complex_assembly" + ], + [ + 82, + 85, + "DNA", + "chemical" + ], + [ + 97, + 124, + "crystallized and determined", + "experimental_method" + ], + [ + 125, + 130, + "X-ray", + "experimental_method" + ], + [ + 131, + 149, + "crystal structures", + "evidence" + ], + [ + 153, + 161, + "mTdp2cat", + "structure_element" + ], + [ + 162, + 170, + "bound to", + "protein_state" + ], + [ + 171, + 187, + "5\u2032-phosphate DNA", + "chemical" + ], + [ + 213, + 218, + "5\u2032-\u03f5A", + "chemical" + ], + [ + 272, + 275, + "DNA", + "chemical" + ], + [ + 289, + 295, + "5\u2032-THF", + "chemical" + ] + ] + }, + { + "sid": 47, + "sent": "In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed \u03b1-\u03b2 fold typified by a central 12-stranded anti-parallel \u03b2-sandwich enveloped by several helical elements that mold the Tdp2 active site.", + "section": "RESULTS", + "ner": [ + [ + 9, + 17, + "Tdp2-DNA", + "complex_assembly" + ], + [ + 26, + 36, + "structures", + "evidence" + ], + [ + 38, + 46, + "mTdp2cat", + "structure_element" + ], + [ + 56, + 70, + "mixed \u03b1-\u03b2 fold", + "structure_element" + ], + [ + 93, + 129, + "12-stranded anti-parallel \u03b2-sandwich", + "structure_element" + ], + [ + 182, + 186, + "Tdp2", + "protein" + ], + [ + 187, + 198, + "active site", + "site" + ] + ] + }, + { + "sid": 48, + "sent": "One half of the molecule contributes to formation of the walls of the DNA-binding cleft that embraces the terminal position of the damaged DNA substrate.", + "section": "RESULTS", + "ner": [ + [ + 70, + 87, + "DNA-binding cleft", + "site" + ], + [ + 139, + 142, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 49, + "sent": "In the DNA lesion-bound state, two key DNA binding elements, the \u03b2-2-helix-\u03b2 (\u03b22H\u03b2) \u2018grasp\u2019, and \u2018helical cap\u2019 mold the substrate binding trench and direct the ssDNA of a 5\u2032-overhang substrate into the active site.", + "section": "RESULTS", + "ner": [ + [ + 7, + 23, + "DNA lesion-bound", + "protein_state" + ], + [ + 39, + 42, + "DNA", + "chemical" + ], + [ + 65, + 76, + "\u03b2-2-helix-\u03b2", + "structure_element" + ], + [ + 78, + 82, + "\u03b22H\u03b2", + "structure_element" + ], + [ + 85, + 90, + "grasp", + "structure_element" + ], + [ + 98, + 109, + "helical cap", + "structure_element" + ], + [ + 120, + 144, + "substrate binding trench", + "site" + ], + [ + 160, + 165, + "ssDNA", + "chemical" + ], + [ + 202, + 213, + "active site", + "site" + ] + ] + }, + { + "sid": 50, + "sent": "A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates.", + "section": "RESULTS", + "ner": [ + [ + 34, + 43, + "structure", + "evidence" + ], + [ + 47, + 55, + "DNA-free", + "protein_state" + ], + [ + 56, + 60, + "Tdp2", + "protein" + ], + [ + 62, + 65, + "apo", + "protein_state" + ], + [ + 100, + 104, + "loop", + "structure_element" + ], + [ + 108, + 131, + "conformationally mobile", + "protein_state" + ], + [ + 159, + 162, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 51, + "sent": "The mode of engagement of the 5\u2032-nucleobase of the bulky \u03f5A adduct describes a mechanism for Tdp2 to bind 5\u2032-tyrosylated substrates that contain diverse forms of DNA damage.", + "section": "RESULTS", + "ner": [ + [ + 57, + 59, + "\u03f5A", + "chemical" + ], + [ + 93, + 97, + "Tdp2", + "protein" + ], + [ + 106, + 120, + "5\u2032-tyrosylated", + "protein_state" + ], + [ + 162, + 165, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 52, + "sent": "The 5\u2032-\u03f5A nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the \u2018hydrophobic wall\u2019 that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and\u00a0B).", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "5\u2032-\u03f5A", + "chemical" + ], + [ + 50, + 54, + "Tdp2", + "protein" + ], + [ + 55, + 88, + "van Der Waals interaction surface", + "site" + ], + [ + 115, + 131, + "hydrophobic wall", + "site" + ], + [ + 183, + 189, + "Leu315", + "residue_name_number" + ], + [ + 194, + 200, + "Ile317", + "residue_name_number" + ] + ] + }, + { + "sid": 53, + "sent": "Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning.", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 14, + 22, + "mTdp2cat", + "structure_element" + ], + [ + 23, + 31, + "bound to", + "protein_state" + ], + [ + 32, + 35, + "DNA", + "chemical" + ], + [ + 57, + 61, + "Top2", + "protein_type" + ] + ] + }, + { + "sid": 54, + "sent": "(A) Structure of mTdp2cat bound to 5\u2032-phosphate DNA (product complex) containing \u03f5A (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "Structure", + "evidence" + ], + [ + 17, + 25, + "mTdp2cat", + "structure_element" + ], + [ + 26, + 34, + "bound to", + "protein_state" + ], + [ + 35, + 51, + "5\u2032-phosphate DNA", + "chemical" + ], + [ + 81, + 83, + "\u03f5A", + "chemical" + ], + [ + 94, + 98, + "Mg2+", + "chemical" + ], + [ + 130, + 136, + "waters", + "chemical" + ] + ] + }, + { + "sid": 55, + "sent": "mTdp2cat is colored by electrostatic surface potential (red = negative, blue = positive, gray = neutral/hydrophobic).", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "mTdp2cat", + "structure_element" + ] + ] + }, + { + "sid": 56, + "sent": "(B) \u03c3-A weighted 2Fo-Fc electron density map (at 1.43 \u00c5 resolution, contoured at 2.0 \u03c3) for the \u03f5A DNA complex.", + "section": "FIG", + "ner": [ + [ + 4, + 44, + "\u03c3-A weighted 2Fo-Fc electron density map", + "evidence" + ], + [ + 96, + 102, + "\u03f5A DNA", + "chemical" + ] + ] + }, + { + "sid": 57, + "sent": "The \u03f5A nucleotide is shown in yellow and a hydrogen bond from the \u03f5A O4\u2032 to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5\u2032-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).", + "section": "FIG", + "ner": [ + [ + 4, + 6, + "\u03f5A", + "chemical" + ], + [ + 43, + 56, + "hydrogen bond", + "bond_interaction" + ], + [ + 66, + 68, + "\u03f5A", + "chemical" + ], + [ + 89, + 94, + "water", + "chemical" + ], + [ + 124, + 133, + "Structure", + "evidence" + ], + [ + 137, + 145, + "mTdp2cat", + "structure_element" + ], + [ + 146, + 154, + "bound to", + "protein_state" + ], + [ + 155, + 171, + "5\u2032-phosphate DNA", + "chemical" + ], + [ + 201, + 204, + "THF", + "chemical" + ], + [ + 215, + 219, + "Mg2+", + "chemical" + ], + [ + 251, + 257, + "waters", + "chemical" + ] + ] + }, + { + "sid": 58, + "sent": "mTdp2cat is colored with red (electronegative), blue (electropositive) and gray (hydrophobic) electrostatic surface potential displayed.", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "mTdp2cat", + "structure_element" + ] + ] + }, + { + "sid": 59, + "sent": "PDB entry 5INK is displayed. (D) \u03c3-A weighted 2Fo-Fc electron density map (at 2.15 \u00c5 resolution, contoured at 2.0 \u03c3) for THF-DNA complex.", + "section": "FIG", + "ner": [ + [ + 33, + 73, + "\u03c3-A weighted 2Fo-Fc electron density map", + "evidence" + ], + [ + 121, + 128, + "THF-DNA", + "complex_assembly" + ] + ] + }, + { + "sid": 60, + "sent": "The THF is shown in yellow and a hydrogen bond from the THF O4\u2032 to inner-sphere water is shown as gray dashes.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "THF", + "chemical" + ], + [ + 33, + 46, + "hydrogen bond", + "bond_interaction" + ], + [ + 56, + 59, + "THF", + "chemical" + ], + [ + 80, + 85, + "water", + "chemical" + ] + ] + }, + { + "sid": 61, + "sent": "For comparison, we also determined a structure of an undamaged 5\u2032-adenine (5\u2032-dA) bound to Tdp2 at 1.55 \u00c5 (PDB entry 5INL).", + "section": "RESULTS", + "ner": [ + [ + 24, + 34, + "determined", + "experimental_method" + ], + [ + 37, + 46, + "structure", + "evidence" + ], + [ + 63, + 73, + "5\u2032-adenine", + "chemical" + ], + [ + 75, + 80, + "5\u2032-dA", + "chemical" + ], + [ + 82, + 90, + "bound to", + "protein_state" + ], + [ + 91, + 95, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 62, + "sent": "A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare \u03f5A, dA and dC, Supplementary Figure S1A\u2013D).", + "section": "RESULTS", + "ner": [ + [ + 2, + 20, + "structural overlay", + "experimental_method" + ], + [ + 127, + 132, + "bound", + "protein_state" + ], + [ + 147, + 150, + "DNA", + "chemical" + ], + [ + 167, + 169, + "\u03f5A", + "chemical" + ], + [ + 171, + 173, + "dA", + "chemical" + ], + [ + 178, + 180, + "dC", + "chemical" + ] + ] + }, + { + "sid": 63, + "sent": "Therefore, structurally diverse undamaged or alkylated bases (e.g. \u03f5G, \u03f5T) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the \u03b22H\u03b2 motif.", + "section": "RESULTS", + "ner": [ + [ + 67, + 69, + "\u03f5G", + "chemical" + ], + [ + 71, + 73, + "\u03f5T", + "chemical" + ], + [ + 111, + 115, + "Tdp2", + "protein" + ], + [ + 116, + 127, + "active site", + "site" + ], + [ + 132, + 152, + "planar base stacking", + "bond_interaction" + ], + [ + 162, + 173, + "active site", + "site" + ], + [ + 181, + 197, + "hydrophobic wall", + "site" + ], + [ + 205, + 209, + "\u03b22H\u03b2", + "structure_element" + ] + ] + }, + { + "sid": 64, + "sent": "Likewise, the abasic deoxyribose analog THF substrate binds similar to the alkylated and non-alkylated substrates, but with a slight alteration in the approach of the 5\u2032-terminus (Figure 2C).", + "section": "RESULTS", + "ner": [ + [ + 14, + 32, + "abasic deoxyribose", + "chemical" + ], + [ + 40, + 43, + "THF", + "chemical" + ] + ] + }, + { + "sid": 65, + "sent": "Interestingly, in the absence of a nucleobase, O4\u2032 of the THF ring adopts a close approach (2.8 \u00c5) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D).", + "section": "RESULTS", + "ner": [ + [ + 22, + 32, + "absence of", + "protein_state" + ], + [ + 58, + 61, + "THF", + "chemical" + ], + [ + 104, + 109, + "water", + "chemical" + ], + [ + 173, + 177, + "Mg2+", + "chemical" + ], + [ + 178, + 200, + "ion coordination shell", + "bond_interaction" + ] + ] + }, + { + "sid": 66, + "sent": "These collective differences may explain the slight, but statistically significant elevated activity on the THF substrate (Figure 1C).", + "section": "RESULTS", + "ner": [ + [ + 108, + 111, + "THF", + "chemical" + ] + ] + }, + { + "sid": 67, + "sent": "Structural plasticity in the Tdp2 DNA binding trench", + "section": "RESULTS", + "ner": [ + [ + 29, + 33, + "Tdp2", + "protein" + ], + [ + 34, + 52, + "DNA binding trench", + "site" + ] + ] + }, + { + "sid": 68, + "sent": "An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein\u2013water\u2013protein contacts that span a gap between the catalytic core and the DNA binding \u03b22H\u03b2-grasp (Supplementary Figure S2).", + "section": "RESULTS", + "ner": [ + [ + 29, + 45, + "DNA-damage bound", + "protein_state" + ], + [ + 66, + 70, + "Tdp2", + "protein" + ], + [ + 71, + 82, + "active site", + "site" + ], + [ + 119, + 124, + "water", + "chemical" + ], + [ + 170, + 184, + "catalytic core", + "site" + ], + [ + 193, + 215, + "DNA binding \u03b22H\u03b2-grasp", + "site" + ] + ] + }, + { + "sid": 69, + "sent": "In this arrangement, six solvent molecules form a channel under the \u03b22H\u03b2-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358.", + "section": "RESULTS", + "ner": [ + [ + 68, + 78, + "\u03b22H\u03b2-grasp", + "site" + ], + [ + 92, + 106, + "hydrogen bonds", + "bond_interaction" + ], + [ + 138, + 142, + "Mg2+", + "chemical" + ], + [ + 150, + 156, + "Asp358", + "residue_name_number" + ] + ] + }, + { + "sid": 70, + "sent": "The paucity of hydrophobic interactions stabilizing the \u03b22H\u03b2 DNA-bound conformation suggests that conformational plasticity in the \u03b22H\u03b2 might be a feature of DNA damage and metal cofactor engagement.", + "section": "RESULTS", + "ner": [ + [ + 15, + 39, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 56, + 60, + "\u03b22H\u03b2", + "structure_element" + ], + [ + 61, + 70, + "DNA-bound", + "protein_state" + ], + [ + 131, + 135, + "\u03b22H\u03b2", + "structure_element" + ], + [ + 158, + 161, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 71, + "sent": "To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 \u00c5 resolution (PDB entry 5INM; Figures 1F and\u00a03A).", + "section": "RESULTS", + "ner": [ + [ + 28, + 40, + "crystallized", + "experimental_method" + ], + [ + 41, + 45, + "Tdp2", + "protein" + ], + [ + 53, + 63, + "absence of", + "protein_state" + ], + [ + 64, + 67, + "DNA", + "chemical" + ], + [ + 85, + 93, + "DNA free", + "protein_state" + ], + [ + 94, + 98, + "Tdp2", + "protein" + ], + [ + 99, + 108, + "structure", + "evidence" + ] + ] + }, + { + "sid": 72, + "sent": "Conformational plasticity in the Tdp2 active site.", + "section": "FIG", + "ner": [ + [ + 33, + 37, + "Tdp2", + "protein" + ], + [ + 38, + 49, + "active site", + "site" + ] + ] + }, + { + "sid": 73, + "sent": "(A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core.", + "section": "FIG", + "ner": [ + [ + 8, + 12, + "open", + "protein_state" + ], + [ + 14, + 21, + "3-helix", + "structure_element" + ], + [ + 44, + 52, + "flexible", + "protein_state" + ], + [ + 53, + 69, + "active-site loop", + "structure_element" + ], + [ + 82, + 89, + "monomer", + "oligomeric_state" + ], + [ + 90, + 91, + "E", + "structure_element" + ], + [ + 99, + 107, + "DNA-free", + "protein_state" + ], + [ + 108, + 116, + "mTdp2cat", + "structure_element" + ], + [ + 117, + 126, + "structure", + "evidence" + ], + [ + 160, + 164, + "T309", + "residue_name_number" + ], + [ + 198, + 201, + "EEP", + "structure_element" + ] + ] + }, + { + "sid": 74, + "sent": "The \u03b22H\u03b2 docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed.", + "section": "FIG", + "ner": [ + [ + 4, + 23, + "\u03b22H\u03b2 docking pocket", + "site" + ], + [ + 61, + 65, + "N312", + "residue_name_number" + ], + [ + 67, + 71, + "N314", + "residue_name_number" + ], + [ + 76, + 80, + "L315", + "residue_name_number" + ], + [ + 94, + 109, + "solvent-exposed", + "protein_state" + ] + ] + }, + { + "sid": 75, + "sent": "Wall-eyed stereo view is displayed. (B) The closed \u03b22H\u03b2 conformation in the mTdp2cat\u2013DNA product structure containing 5\u2032-\u03f5A (yellow, PDB entry 5HT2). T309 (green) is an integral part of the \u03b22H\u03b2 DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the \u03b22H\u03b2 docking pocket.", + "section": "FIG", + "ner": [ + [ + 44, + 50, + "closed", + "protein_state" + ], + [ + 51, + 55, + "\u03b22H\u03b2", + "structure_element" + ], + [ + 76, + 88, + "mTdp2cat\u2013DNA", + "complex_assembly" + ], + [ + 97, + 106, + "structure", + "evidence" + ], + [ + 118, + 123, + "5\u2032-\u03f5A", + "chemical" + ], + [ + 150, + 154, + "T309", + "residue_name_number" + ], + [ + 190, + 212, + "\u03b22H\u03b2 DNA-binding grasp", + "site" + ], + [ + 223, + 237, + "hydrogen bonds", + "bond_interaction" + ], + [ + 257, + 261, + "Y321", + "residue_name_number" + ], + [ + 269, + 273, + "N314", + "residue_name_number" + ], + [ + 296, + 315, + "\u03b22H\u03b2 docking pocket", + "site" + ] + ] + }, + { + "sid": 76, + "sent": "Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as \u2018\u223c\u2019 (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop.", + "section": "FIG", + "ner": [ + [ + 53, + 69, + "active site loop", + "structure_element" + ], + [ + 99, + 108, + "promoters", + "oligomeric_state" + ], + [ + 116, + 124, + "DNA-free", + "protein_state" + ], + [ + 125, + 133, + "mTdp2cat", + "structure_element" + ], + [ + 208, + 226, + "sequence alignment", + "experimental_method" + ], + [ + 264, + 280, + "electron density", + "evidence" + ], + [ + 301, + 328, + "Limited trypsin proteolysis", + "experimental_method" + ], + [ + 369, + 377, + "flexible", + "protein_state" + ], + [ + 378, + 394, + "active-site loop", + "structure_element" + ] + ] + }, + { + "sid": 77, + "sent": "mTdp2cat WT (lanes 1\u201313) or mTdp2cat D358N (lanes 14\u201326) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5\u2032-phosphorylated DNA (substrate \u201812 nt\u2019 in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng \u03bcl\u22121 of trypsin.", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "mTdp2cat", + "structure_element" + ], + [ + 9, + 11, + "WT", + "protein_state" + ], + [ + 28, + 36, + "mTdp2cat", + "structure_element" + ], + [ + 37, + 42, + "D358N", + "mutant" + ], + [ + 79, + 87, + "presence", + "protein_state" + ], + [ + 91, + 101, + "absence of", + "protein_state" + ], + [ + 102, + 106, + "Mg2+", + "chemical" + ], + [ + 156, + 159, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 78, + "sent": "Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop.", + "section": "FIG", + "ner": [ + [ + 28, + 36, + "SDS-PAGE", + "experimental_method" + ], + [ + 98, + 130, + "Limited chymotrypsin proteolysis", + "experimental_method" + ], + [ + 171, + 179, + "flexible", + "protein_state" + ], + [ + 180, + 196, + "active-site loop", + "structure_element" + ] + ] + }, + { + "sid": 79, + "sent": "Experiments performed as in panel D for mTdp2cat WT (lanes 27\u201339) or mTdp2cat D358N (lanes 40\u201352), but with chymotrypsin instead of trypsin.", + "section": "FIG", + "ner": [ + [ + 40, + 48, + "mTdp2cat", + "structure_element" + ], + [ + 49, + 51, + "WT", + "protein_state" + ], + [ + 69, + 77, + "mTdp2cat", + "structure_element" + ], + [ + 78, + 83, + "D358N", + "mutant" + ], + [ + 108, + 120, + "chymotrypsin", + "experimental_method" + ], + [ + 132, + 139, + "trypsin", + "experimental_method" + ] + ] + }, + { + "sid": 80, + "sent": "This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers.", + "section": "RESULTS", + "ner": [ + [ + 29, + 33, + "Tdp2", + "protein" + ], + [ + 95, + 106, + "active site", + "site" + ], + [ + 107, + 111, + "Mg2+", + "chemical" + ], + [ + 126, + 149, + "substrate binding loops", + "structure_element" + ], + [ + 178, + 187, + "protomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 81, + "sent": "The most striking feature of the DNA ligand-free state is that the active site \u03b22H\u03b2-grasp can adopt alternative structures that are distinct from the DNA-bound, closed \u03b22H\u03b2 DNA binding grasp (Figure 3A and\u00a0B).", + "section": "RESULTS", + "ner": [ + [ + 33, + 48, + "DNA ligand-free", + "protein_state" + ], + [ + 67, + 89, + "active site \u03b22H\u03b2-grasp", + "site" + ], + [ + 150, + 159, + "DNA-bound", + "protein_state" + ], + [ + 161, + 167, + "closed", + "protein_state" + ], + [ + 168, + 190, + "\u03b22H\u03b2 DNA binding grasp", + "site" + ] + ] + }, + { + "sid": 82, + "sent": "In one monomer (chain \u2018E\u2019), the grasp adopts an \u2018open\u2019 3-helix loop conformation that projects away from the EEP catalytic core.", + "section": "RESULTS", + "ner": [ + [ + 7, + 14, + "monomer", + "oligomeric_state" + ], + [ + 16, + 25, + "chain \u2018E\u2019", + "structure_element" + ], + [ + 32, + 37, + "grasp", + "structure_element" + ], + [ + 49, + 53, + "open", + "protein_state" + ], + [ + 55, + 67, + "3-helix loop", + "structure_element" + ], + [ + 109, + 112, + "EEP", + "structure_element" + ], + [ + 113, + 127, + "catalytic core", + "site" + ] + ] + }, + { + "sid": 83, + "sent": "Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "monomers", + "oligomeric_state" + ], + [ + 27, + 37, + "disordered", + "protein_state" + ], + [ + 67, + 83, + "DNA binding loop", + "structure_element" + ], + [ + 106, + 122, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 84, + "sent": "The remaining two molecules in the DNA-free crystal form are closed \u03b22H\u03b2 conformers similar to the DNA bound structures (Figure 3C).", + "section": "RESULTS", + "ner": [ + [ + 35, + 43, + "DNA-free", + "protein_state" + ], + [ + 44, + 56, + "crystal form", + "evidence" + ], + [ + 61, + 67, + "closed", + "protein_state" + ], + [ + 68, + 72, + "\u03b22H\u03b2", + "structure_element" + ], + [ + 99, + 108, + "DNA bound", + "protein_state" + ], + [ + 109, + 119, + "structures", + "evidence" + ] + ] + }, + { + "sid": 85, + "sent": "Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the \u03b22H\u03b2 grasp, rather than inducing closure upon binding.", + "section": "RESULTS", + "ner": [ + [ + 20, + 24, + "Tdp2", + "protein" + ], + [ + 25, + 28, + "DNA", + "chemical" + ], + [ + 66, + 72, + "closed", + "protein_state" + ], + [ + 85, + 95, + "\u03b22H\u03b2 grasp", + "site" + ] + ] + }, + { + "sid": 86, + "sent": "A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes.", + "section": "RESULTS", + "ner": [ + [ + 27, + 35, + "extended", + "protein_state" + ], + [ + 36, + 43, + "3-helix", + "structure_element" + ], + [ + 72, + 94, + "substrate-binding loop", + "structure_element" + ] + ] + }, + { + "sid": 87, + "sent": "In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop.", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "open", + "protein_state" + ], + [ + 28, + 41, + "Asn312-Leu315", + "residue_range" + ], + [ + 62, + 73, + "active site", + "site" + ], + [ + 78, + 93, + "solvent-exposed", + "protein_state" + ], + [ + 128, + 134, + "Thr309", + "residue_name_number" + ], + [ + 183, + 189, + "pocket", + "site" + ], + [ + 197, + 200, + "EEP", + "structure_element" + ], + [ + 220, + 224, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 88, + "sent": "Burial of Thr309 is enabled by an unusual main chain cis\u2013peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the \u03b22H\u03b2 fold.", + "section": "RESULTS", + "ner": [ + [ + 10, + 16, + "Thr309", + "residue_name_number" + ], + [ + 53, + 69, + "cis\u2013peptide bond", + "bond_interaction" + ], + [ + 78, + 84, + "Asp308", + "residue_name_number" + ], + [ + 85, + 91, + "Thr309", + "residue_name_number" + ], + [ + 115, + 145, + "short antiparallel beta-strand", + "structure_element" + ], + [ + 153, + 157, + "\u03b22H\u03b2", + "structure_element" + ] + ] + }, + { + "sid": 89, + "sent": "By comparison, the closed \u03b22H\u03b2 grasp conformer is stabilized by Asn312 and Asn314 binding into two \u03b22H\u03b2 docking pockets, and Leu315 engagement of the 5\u2032-terminal nucleobase (Figure 3B).", + "section": "RESULTS", + "ner": [ + [ + 19, + 25, + "closed", + "protein_state" + ], + [ + 26, + 36, + "\u03b22H\u03b2 grasp", + "site" + ], + [ + 64, + 70, + "Asn312", + "residue_name_number" + ], + [ + 75, + 81, + "Asn314", + "residue_name_number" + ], + [ + 99, + 119, + "\u03b22H\u03b2 docking pockets", + "site" + ], + [ + 125, + 131, + "Leu315", + "residue_name_number" + ] + ] + }, + { + "sid": 90, + "sent": "To transition into the closed \u03b22H\u03b2 conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the \u03b22H\u03b2 antiparallel \u03b2-sheet.", + "section": "RESULTS", + "ner": [ + [ + 23, + 29, + "closed", + "protein_state" + ], + [ + 30, + 34, + "\u03b22H\u03b2", + "structure_element" + ], + [ + 49, + 55, + "Thr309", + "residue_name_number" + ], + [ + 76, + 79, + "EEP", + "structure_element" + ], + [ + 87, + 93, + "pocket", + "site" + ], + [ + 168, + 172, + "\u03b22H\u03b2", + "structure_element" + ], + [ + 173, + 193, + "antiparallel \u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 91, + "sent": "Stabilization of the closed \u03b22H\u03b2-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358.", + "section": "RESULTS", + "ner": [ + [ + 21, + 27, + "closed", + "protein_state" + ], + [ + 28, + 38, + "\u03b22H\u03b2-grasp", + "site" + ], + [ + 69, + 80, + "active site", + "site" + ], + [ + 91, + 104, + "hydrogen bond", + "bond_interaction" + ], + [ + 113, + 119, + "Trp307", + "residue_name_number" + ], + [ + 128, + 153, + "Mg2+ coordinating residue", + "site" + ], + [ + 154, + 160, + "Asp358", + "residue_name_number" + ] + ] + }, + { + "sid": 92, + "sent": "Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site.", + "section": "RESULTS", + "ner": [ + [ + 20, + 28, + "DNA free", + "protein_state" + ], + [ + 29, + 38, + "structure", + "evidence" + ], + [ + 71, + 77, + "closed", + "protein_state" + ], + [ + 78, + 86, + "monomers", + "oligomeric_state" + ], + [ + 103, + 107, + "Mg2+", + "chemical" + ], + [ + 121, + 133, + "active sites", + "site" + ], + [ + 145, + 153, + "monomers", + "oligomeric_state" + ], + [ + 159, + 163, + "open", + "protein_state" + ], + [ + 210, + 228, + "metal binding site", + "site" + ] + ] + }, + { + "sid": 93, + "sent": "Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the \u03b22H\u03b2 DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site.", + "section": "RESULTS", + "ner": [ + [ + 71, + 74, + "DNA", + "chemical" + ], + [ + 147, + 169, + "\u03b22H\u03b2 DNA binding grasp", + "site" + ], + [ + 217, + 221, + "Mg2+", + "chemical" + ], + [ + 237, + 241, + "Tdp2", + "protein" + ], + [ + 242, + 253, + "active site", + "site" + ] + ] + }, + { + "sid": 94, + "sent": "To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C\u2013E).", + "section": "RESULTS", + "ner": [ + [ + 12, + 16, + "Mg2+", + "chemical" + ], + [ + 21, + 24, + "DNA", + "chemical" + ], + [ + 35, + 39, + "Tdp2", + "protein" + ], + [ + 81, + 89, + "mTdp2cat", + "structure_element" + ], + [ + 110, + 154, + "limited trypsin and chymotrypsin proteolysis", + "experimental_method" + ] + ] + }, + { + "sid": 95, + "sent": "In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315).", + "section": "RESULTS", + "ner": [ + [ + 0, + 17, + "In the absence of", + "protein_state" + ], + [ + 18, + 21, + "DNA", + "chemical" + ], + [ + 25, + 30, + "Mg2+,", + "chemical" + ], + [ + 31, + 39, + "mTdp2cat", + "structure_element" + ], + [ + 82, + 99, + "DNA binding grasp", + "site" + ], + [ + 115, + 122, + "trypsin", + "experimental_method" + ], + [ + 124, + 130, + "Arg316", + "residue_name_number" + ], + [ + 156, + 168, + "chymotrypsin", + "experimental_method" + ], + [ + 170, + 176, + "Trp307", + "residue_name_number" + ], + [ + 181, + 187, + "Leu315", + "residue_name_number" + ] + ] + }, + { + "sid": 96, + "sent": "By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage.", + "section": "RESULTS", + "ner": [ + [ + 15, + 20, + "Mg2+,", + "chemical" + ], + [ + 45, + 49, + "Mg2+", + "chemical" + ], + [ + 50, + 53, + "DNA", + "chemical" + ], + [ + 109, + 117, + "mTdp2cat", + "structure_element" + ] + ] + }, + { + "sid": 97, + "sent": "Interestingly, addition of Mg2+ alone protects against proteolysis as well.", + "section": "RESULTS", + "ner": [ + [ + 27, + 31, + "Mg2+", + "chemical" + ] + ] + }, + { + "sid": 98, + "sent": "This is consistent with Mg2+ stabilizing the closed conformation of the \u03b22H\u03b2-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the \u03b22H\u03b2-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs).", + "section": "RESULTS", + "ner": [ + [ + 24, + 28, + "Mg2+", + "chemical" + ], + [ + 45, + 51, + "closed", + "protein_state" + ], + [ + 72, + 82, + "\u03b22H\u03b2-grasp", + "site" + ], + [ + 103, + 127, + "hydrogen-bonding network", + "bond_interaction" + ], + [ + 133, + 139, + "Asp358", + "residue_name_number" + ], + [ + 167, + 177, + "\u03b22H\u03b2-grasp", + "site" + ], + [ + 186, + 192, + "Trp307", + "residue_name_number" + ], + [ + 219, + 223, + "Tdp2", + "protein" + ], + [ + 224, + 235, + "active site", + "site" + ] + ] + }, + { + "sid": 99, + "sent": "To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 \u00c5 resolution structure of the human Tdp2cat domain bound to a DNA 5\u2032-PO4 terminus product complex (PDB entry 5INO).", + "section": "RESULTS", + "ner": [ + [ + 37, + 41, + "Tdp2", + "protein" + ], + [ + 73, + 78, + "human", + "species" + ], + [ + 83, + 88, + "mouse", + "taxonomy_domain" + ], + [ + 89, + 93, + "Tdp2", + "protein" + ], + [ + 103, + 113, + "determined", + "experimental_method" + ], + [ + 133, + 142, + "structure", + "evidence" + ], + [ + 150, + 155, + "human", + "species" + ], + [ + 156, + 163, + "Tdp2cat", + "structure_element" + ], + [ + 171, + 179, + "bound to", + "protein_state" + ], + [ + 182, + 192, + "DNA 5\u2032-PO4", + "chemical" + ] + ] + }, + { + "sid": 100, + "sent": "Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A).", + "section": "RESULTS", + "ner": [ + [ + 19, + 24, + "human", + "species" + ], + [ + 25, + 37, + "hTdp2cat-DNA", + "complex_assembly" + ], + [ + 46, + 55, + "structure", + "evidence" + ], + [ + 63, + 71, + "mTdp2cat", + "structure_element" + ], + [ + 72, + 81, + "DNA bound", + "protein_state" + ], + [ + 129, + 138, + "DNA-bound", + "protein_state" + ] + ] + }, + { + "sid": 101, + "sent": "Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B).", + "section": "RESULTS", + "ner": [ + [ + 21, + 29, + "mTdp2cat", + "structure_element" + ], + [ + 61, + 69, + "hTdp2cat", + "structure_element" + ], + [ + 70, + 92, + "substrate binding loop", + "structure_element" + ], + [ + 117, + 121, + "Mg2+", + "chemical" + ], + [ + 126, + 129, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 102, + "sent": "Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction.", + "section": "RESULTS", + "ner": [ + [ + 6, + 11, + "X-ray", + "experimental_method" + ], + [ + 12, + 22, + "structures", + "evidence" + ], + [ + 27, + 55, + "limited proteolysis analysis", + "experimental_method" + ], + [ + 70, + 73, + "DNA", + "chemical" + ], + [ + 122, + 131, + "conserved", + "protein_state" + ], + [ + 147, + 157, + "vertebrate", + "taxonomy_domain" + ], + [ + 158, + 162, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 103, + "sent": "Tdp2 metal ion dependence", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 104, + "sent": "Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site.", + "section": "RESULTS", + "ner": [ + [ + 32, + 57, + "X-ray structural analyses", + "experimental_method" + ], + [ + 90, + 94, + "Mg2+", + "chemical" + ], + [ + 101, + 109, + "bound in", + "protein_state" + ], + [ + 114, + 118, + "Tdp2", + "protein" + ], + [ + 119, + 130, + "active site", + "site" + ] + ] + }, + { + "sid": 105, + "sent": "This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA).", + "section": "RESULTS", + "ner": [ + [ + 18, + 26, + "DNA-free", + "protein_state" + ], + [ + 40, + 56, + "DNA damage bound", + "protein_state" + ], + [ + 73, + 95, + "reaction product-bound", + "protein_state" + ], + [ + 96, + 109, + "crystal forms", + "evidence" + ], + [ + 113, + 118, + "mouse", + "taxonomy_domain" + ], + [ + 138, + 146, + "D. rerio", + "species" + ], + [ + 168, + 178, + "C. elegans", + "species" + ], + [ + 179, + 183, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 106, + "sent": "However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction.", + "section": "RESULTS", + "ner": [ + [ + 18, + 38, + "biochemical analysis", + "experimental_method" + ], + [ + 100, + 104, + "Tdp2", + "protein" + ], + [ + 105, + 137, + "phosphotyrosyl phosphodiesterase", + "protein_type" + ] + ] + }, + { + "sid": 107, + "sent": "In these experiments, at limiting Mg2+ concentrations, Ca2+ addition to Tdp2 reactions stimulated activity.", + "section": "RESULTS", + "ner": [ + [ + 34, + 38, + "Mg2+", + "chemical" + ], + [ + 55, + 59, + "Ca2+", + "chemical" + ], + [ + 72, + 76, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 108, + "sent": "While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site.", + "section": "RESULTS", + "ner": [ + [ + 64, + 78, + "phosphotyrosyl", + "ptm" + ], + [ + 96, + 100, + "Tdp2", + "protein" + ], + [ + 164, + 187, + "metal ion binding sites", + "site" + ], + [ + 203, + 214, + "active site", + "site" + ] + ] + }, + { + "sid": 109, + "sent": "In fact, divalent metals have been observed in the Tdp2 protein\u2013DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies.", + "section": "RESULTS", + "ner": [ + [ + 9, + 24, + "divalent metals", + "chemical" + ], + [ + 51, + 55, + "Tdp2", + "protein" + ], + [ + 64, + 67, + "DNA", + "chemical" + ], + [ + 109, + 122, + "active center", + "site" + ] + ] + }, + { + "sid": 110, + "sent": "To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).", + "section": "RESULTS", + "ner": [ + [ + 49, + 53, + "Tdp2", + "protein" + ], + [ + 54, + 71, + "phosphodiesterase", + "protein_type" + ], + [ + 95, + 119, + "metal ion binding assays", + "experimental_method" + ], + [ + 132, + 150, + "crystal structures", + "evidence" + ], + [ + 158, + 169, + "presence of", + "protein_state" + ], + [ + 177, + 192, + "divalent metals", + "chemical" + ], + [ + 194, + 198, + "Mn2+", + "chemical" + ], + [ + 203, + 208, + "Ca2+)", + "chemical" + ], + [ + 251, + 255, + "Tdp2", + "protein" + ], + [ + 256, + 288, + "phosphotyrosyl phosphodiesterase", + "protein_type" + ] + ] + }, + { + "sid": 111, + "sent": "Metal cofactor interactions with Tdp2. (A) Intrinsic tryptophan fluorescence of mTdp2cat was used to monitor a conformational response to divalent metal ion binding.", + "section": "FIG", + "ner": [ + [ + 33, + 37, + "Tdp2", + "protein" + ], + [ + 43, + 76, + "Intrinsic tryptophan fluorescence", + "evidence" + ], + [ + 80, + 88, + "mTdp2cat", + "structure_element" + ] + ] + }, + { + "sid": 112, + "sent": "Either Mg2+ or Ca2+ were titrated in the presence or absence of 5\u2032-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters.", + "section": "FIG", + "ner": [ + [ + 7, + 11, + "Mg2+", + "chemical" + ], + [ + 15, + 19, + "Ca2+", + "chemical" + ], + [ + 25, + 33, + "titrated", + "experimental_method" + ], + [ + 41, + 49, + "presence", + "protein_state" + ], + [ + 53, + 63, + "absence of", + "protein_state" + ], + [ + 64, + 72, + "5\u2032-P DNA", + "chemical" + ], + [ + 82, + 105, + "tryptophan fluorescence", + "evidence" + ] + ] + }, + { + "sid": 113, + "sent": "Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.", + "section": "FIG", + "ner": [ + [ + 5, + 9, + "Mg2+", + "chemical" + ], + [ + 14, + 18, + "Ca2+", + "chemical" + ], + [ + 79, + 102, + "tryptophan fluorescence", + "evidence" + ], + [ + 106, + 114, + "mTdp2cat", + "structure_element" + ], + [ + 122, + 130, + "presence", + "protein_state" + ], + [ + 135, + 145, + "absence of", + "protein_state" + ], + [ + 146, + 149, + "DNA", + "chemical" + ], + [ + 157, + 162, + "D358N", + "mutant" + ], + [ + 163, + 174, + "active site", + "site" + ], + [ + 175, + 181, + "mutant", + "protein_state" + ], + [ + 185, + 193, + "mTdp2cat", + "structure_element" + ], + [ + 197, + 209, + "unresponsive", + "protein_state" + ], + [ + 213, + 217, + "Mg2+", + "chemical" + ], + [ + 223, + 231, + "mTdp2cat", + "structure_element" + ], + [ + 254, + 259, + "T5PNP", + "chemical" + ], + [ + 287, + 291, + "Mg2+", + "chemical" + ], + [ + 296, + 300, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 114, + "sent": "PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) \u03c3-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat\u2013DNA\u2013Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "PNP", + "chemical" + ], + [ + 65, + 69, + "Mg2+", + "chemical" + ], + [ + 95, + 102, + "absence", + "protein_state" + ], + [ + 106, + 117, + "presence of", + "protein_state" + ], + [ + 129, + 133, + "Ca2+", + "chemical" + ], + [ + 172, + 212, + "\u03c3-A weighted 2Fo-Fc electron density map", + "evidence" + ], + [ + 224, + 265, + "model-phased anomalous difference Fourier", + "evidence" + ], + [ + 276, + 280, + "maps", + "evidence" + ], + [ + 289, + 306, + "mTdp2cat\u2013DNA\u2013Mn2+", + "complex_assembly" + ], + [ + 346, + 350, + "Mn2+", + "chemical" + ] + ] + }, + { + "sid": 115, + "sent": "A 53\u03c3 peak in the anomalous difference Fourier map (data collected at \u03bb = 1.5418 \u00c5) supports Mn2+ as the identity of this atom.", + "section": "FIG", + "ner": [ + [ + 18, + 50, + "anomalous difference Fourier map", + "evidence" + ], + [ + 93, + 97, + "Mn2+", + "chemical" + ] + ] + }, + { + "sid": 116, + "sent": "(D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat\u2013DNA structures shows that Ca2+ distorts the 5\u2032-phosphate binding mode.", + "section": "FIG", + "ner": [ + [ + 18, + 22, + "Ca2+", + "chemical" + ], + [ + 30, + 34, + "Ca2+", + "chemical" + ], + [ + 47, + 50, + "DNA", + "chemical" + ], + [ + 74, + 78, + "Mg2+", + "chemical" + ], + [ + 88, + 92, + "Mg2+", + "chemical" + ], + [ + 105, + 108, + "DNA", + "chemical" + ], + [ + 127, + 139, + "mTdp2cat\u2013DNA", + "complex_assembly" + ], + [ + 140, + 150, + "structures", + "evidence" + ], + [ + 162, + 166, + "Ca2+", + "chemical" + ], + [ + 180, + 205, + "5\u2032-phosphate binding mode", + "site" + ] + ] + }, + { + "sid": 117, + "sent": "Our proteolysis results indicate a Mg2+-dependent Tdp2 conformational response to metal binding.", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "proteolysis", + "experimental_method" + ], + [ + 35, + 39, + "Mg2+", + "chemical" + ], + [ + 50, + 54, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 118, + "sent": "The Tdp2 active site has three tryptophan residues within 10 \u00c5 of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "Tdp2", + "protein" + ], + [ + 9, + 20, + "active site", + "site" + ], + [ + 31, + 41, + "tryptophan", + "residue_name" + ], + [ + 70, + 90, + "metal binding center", + "site" + ], + [ + 106, + 139, + "intrinsic tryptophan fluorescence", + "evidence" + ], + [ + 190, + 198, + "mTdp2cat", + "structure_element" + ] + ] + }, + { + "sid": 119, + "sent": "These data were an excellent fit to a single-site binding model both in the presence and absence of DNA (Figure 4A).", + "section": "RESULTS", + "ner": [ + [ + 76, + 84, + "presence", + "protein_state" + ], + [ + 89, + 99, + "absence of", + "protein_state" + ], + [ + 100, + 103, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 120, + "sent": "This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2).", + "section": "RESULTS", + "ner": [ + [ + 23, + 27, + "Mg2+", + "chemical" + ], + [ + 28, + 30, + "Kd", + "evidence" + ], + [ + 70, + 87, + "Hill coefficients", + "evidence" + ], + [ + 124, + 142, + "metal binding site", + "site" + ], + [ + 155, + 163, + "presence", + "protein_state" + ], + [ + 168, + 178, + "absence of", + "protein_state" + ], + [ + 179, + 182, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 121, + "sent": "We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5\u2032-phosphate by mTdp2cat.", + "section": "RESULTS", + "ner": [ + [ + 56, + 60, + "Tdp2", + "protein" + ], + [ + 73, + 109, + "p-nitrophenyl-thymidine-5\u2032-phosphate", + "chemical" + ], + [ + 113, + 121, + "mTdp2cat", + "structure_element" + ] + ] + }, + { + "sid": 122, + "sent": "This small molecule substrate is not expected to be influenced by metal\u2013DNA coordination outside of the active site.", + "section": "RESULTS", + "ner": [ + [ + 72, + 75, + "DNA", + "chemical" + ], + [ + 104, + 115, + "active site", + "site" + ] + ] + }, + { + "sid": 123, + "sent": "Inclusion of ultrapure Ca2+ (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting Mg2+ (Figure 4B).", + "section": "RESULTS", + "ner": [ + [ + 23, + 27, + "Ca2+", + "chemical" + ], + [ + 103, + 107, + "Tdp2", + "protein" + ], + [ + 149, + 153, + "Mg2+", + "chemical" + ] + ] + }, + { + "sid": 124, + "sent": "We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+.", + "section": "RESULTS", + "ner": [ + [ + 22, + 32, + "titrations", + "experimental_method" + ], + [ + 38, + 43, + "human", + "species" + ], + [ + 44, + 51, + "hTdp2FL", + "protein" + ], + [ + 56, + 64, + "hTdp2cat", + "structure_element" + ], + [ + 136, + 140, + "Mg2+", + "chemical" + ], + [ + 159, + 163, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 125, + "sent": "Overall, these metal binding analyses are consistent with a single metal ion mediated reaction.", + "section": "RESULTS", + "ner": [ + [ + 15, + 37, + "metal binding analyses", + "experimental_method" + ] + ] + }, + { + "sid": 126, + "sent": "To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ).", + "section": "RESULTS", + "ner": [ + [ + 72, + 76, + "Tdp2", + "protein" + ], + [ + 77, + 88, + "active site", + "site" + ], + [ + 104, + 122, + "crystal structures", + "evidence" + ], + [ + 126, + 142, + "soaking crystals", + "experimental_method" + ], + [ + 176, + 183, + "support", + "protein_state" + ], + [ + 185, + 189, + "Mn2+", + "chemical" + ], + [ + 194, + 201, + "inhibit", + "protein_state" + ], + [ + 203, + 207, + "Ca2+", + "chemical" + ], + [ + 224, + 228, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 127, + "sent": "Anomalous difference Fourier maps of the Tdp2\u2013DNA\u2013Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3).", + "section": "RESULTS", + "ner": [ + [ + 0, + 33, + "Anomalous difference Fourier maps", + "evidence" + ], + [ + 41, + 54, + "Tdp2\u2013DNA\u2013Mn2+", + "complex_assembly" + ], + [ + 77, + 89, + "binding site", + "site" + ], + [ + 94, + 98, + "Mn2+", + "chemical" + ], + [ + 107, + 111, + "Tdp2", + "protein" + ], + [ + 112, + 123, + "active site", + "site" + ], + [ + 142, + 165, + "octahedral coordination", + "bond_interaction" + ], + [ + 195, + 199, + "Mn2+", + "chemical" + ] + ] + }, + { + "sid": 128, + "sent": "The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "Mn2+", + "chemical" + ], + [ + 34, + 38, + "Tdp2", + "protein" + ], + [ + 39, + 50, + "active site", + "site" + ], + [ + 66, + 76, + "Mg2+-bound", + "protein_state" + ], + [ + 138, + 142, + "Mn2+", + "chemical" + ], + [ + 161, + 165, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 129, + "sent": "In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted \u223c1 \u00c5 from the Mg2+ site.", + "section": "RESULTS", + "ner": [ + [ + 19, + 40, + "co-complex structures", + "evidence" + ], + [ + 46, + 50, + "Ca2+", + "chemical" + ], + [ + 81, + 85, + "Ca2+", + "chemical" + ], + [ + 148, + 157, + "Mg2+ site", + "site" + ] + ] + }, + { + "sid": 130, + "sent": "Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site.", + "section": "RESULTS", + "ner": [ + [ + 9, + 13, + "Ca2+", + "chemical" + ], + [ + 22, + 46, + "octahedrally coordinated", + "bond_interaction" + ], + [ + 76, + 80, + "Ca2+", + "chemical" + ], + [ + 124, + 128, + "Ca2+", + "chemical" + ], + [ + 149, + 162, + "Mg2+ ion site", + "site" + ] + ] + }, + { + "sid": 131, + "sent": "Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5\u2032-PO4 out of the Tdp2 active site (Figure 4D).", + "section": "RESULTS", + "ner": [ + [ + 15, + 53, + "bi-dentate inner sphere metal contacts", + "bond_interaction" + ], + [ + 63, + 67, + "Ca2+", + "chemical" + ], + [ + 75, + 81, + "Glu162", + "residue_name_number" + ], + [ + 94, + 128, + "active site phosphate-binding mode", + "site" + ], + [ + 147, + 153, + "5\u2032-PO4", + "chemical" + ], + [ + 165, + 169, + "Tdp2", + "protein" + ], + [ + 170, + 181, + "active site", + "site" + ] + ] + }, + { + "sid": 132, + "sent": "Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction.", + "section": "RESULTS", + "ner": [ + [ + 71, + 75, + "Ca2+", + "chemical" + ], + [ + 112, + 116, + "Tdp2", + "protein" + ], + [ + 131, + 151, + "divalent metal bound", + "protein_state" + ], + [ + 152, + 156, + "Tdp2", + "protein" + ], + [ + 157, + 167, + "structures", + "evidence" + ], + [ + 192, + 196, + "Ca2+", + "chemical" + ], + [ + 224, + 228, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 133, + "sent": "Modeling the Tdp2 reaction coordinate", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 134, + "sent": "Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides.", + "section": "RESULTS", + "ner": [ + [ + 56, + 60, + "Mg2+", + "chemical" + ], + [ + 75, + 84, + "simulated", + "experimental_method" + ], + [ + 89, + 93, + "Tdp2", + "protein" + ], + [ + 119, + 140, + "hybrid QM/MM modeling", + "experimental_method" + ], + [ + 147, + 151, + "Tdp2", + "protein" + ], + [ + 152, + 169, + "substrate analog-", + "protein_state" + ], + [ + 174, + 187, + "product-bound", + "protein_state" + ], + [ + 188, + 198, + "structures", + "evidence" + ] + ] + }, + { + "sid": 135, + "sent": "Previous structural analyses showed that the superposition of a DNA substrate mimic (5\u2032-aminohexanol) and product (5\u2032-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5\u2032-phosphorus.", + "section": "RESULTS", + "ner": [ + [ + 9, + 28, + "structural analyses", + "experimental_method" + ], + [ + 45, + 58, + "superposition", + "experimental_method" + ], + [ + 64, + 67, + "DNA", + "chemical" + ], + [ + 85, + 100, + "5\u2032-aminohexanol", + "chemical" + ], + [ + 115, + 121, + "5\u2032-PO4", + "chemical" + ], + [ + 155, + 159, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 136, + "sent": "In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack \u223c180\u00b0 opposite of the P\u2013O bond of the 5\u2032-Tyr adduct.", + "section": "RESULTS", + "ner": [ + [ + 53, + 58, + "water", + "chemical" + ], + [ + 76, + 91, + "hydrogen bonded", + "bond_interaction" + ], + [ + 95, + 101, + "Asp272", + "residue_name_number" + ], + [ + 106, + 112, + "Asn274", + "residue_name_number" + ] + ] + }, + { + "sid": 137, + "sent": "Structure-function analysis of the Tdp2 reaction mechanism.", + "section": "FIG", + "ner": [ + [ + 35, + 39, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 138, + "sent": "(A) Proposed mechanism for hydrolysis of phosphotyrosine bond by Tdp2.", + "section": "FIG", + "ner": [ + [ + 41, + 56, + "phosphotyrosine", + "residue_name" + ], + [ + 65, + 69, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 139, + "sent": "Residues in green form the binding-site for the 5\u2032-tyrosine (red) and phosphate, yellow bind the 5\u2032 nucleotide and blue bind nucleotides 2\u20133.", + "section": "FIG", + "ner": [ + [ + 27, + 39, + "binding-site", + "site" + ], + [ + 48, + 59, + "5\u2032-tyrosine", + "residue_name" + ], + [ + 70, + 79, + "phosphate", + "chemical" + ] + ] + }, + { + "sid": 140, + "sent": "Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5\u2032-phosphorus atom.", + "section": "FIG", + "ner": [ + [ + 34, + 39, + "mTdp2", + "protein" + ], + [ + 53, + 64, + "Free energy", + "evidence" + ], + [ + 76, + 92, + "QM/MM simulation", + "experimental_method" + ], + [ + 144, + 149, + "water", + "chemical" + ] + ] + }, + { + "sid": 141, + "sent": "Reaction proceeds from right to left. (C) Models for the mTdp2cat-DNA complex during the QM/MM reaction path simulation showing the substrate (left, tan), transition state intermediate (center, cyan) and product (right, pink) states.", + "section": "FIG", + "ner": [ + [ + 57, + 69, + "mTdp2cat-DNA", + "complex_assembly" + ], + [ + 89, + 119, + "QM/MM reaction path simulation", + "experimental_method" + ] + ] + }, + { + "sid": 142, + "sent": "Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5\u2032-phosphotyrosine in isolation (upper panel) and in the presence of a cation\u2013\u03c0 interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction.", + "section": "FIG", + "ner": [ + [ + 34, + 39, + "mTdp2", + "protein" + ], + [ + 53, + 84, + "Electrostatic surface potential", + "evidence" + ], + [ + 100, + 118, + "5\u2032-phosphotyrosine", + "residue_name" + ], + [ + 157, + 168, + "presence of", + "protein_state" + ], + [ + 171, + 191, + "cation\u2013\u03c0 interaction", + "bond_interaction" + ], + [ + 222, + 228, + "Arg216", + "residue_name_number" + ] + ] + }, + { + "sid": 143, + "sent": "Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates.", + "section": "FIG", + "ner": [ + [ + 0, + 23, + "Electrostatic potential", + "evidence" + ], + [ + 161, + 170, + "wild-type", + "protein_state" + ], + [ + 175, + 181, + "mutant", + "protein_state" + ], + [ + 182, + 187, + "human", + "species" + ], + [ + 188, + 191, + "MBP", + "experimental_method" + ], + [ + 192, + 200, + "hTdp2cat", + "structure_element" + ], + [ + 201, + 216, + "fusion proteins", + "experimental_method" + ] + ] + }, + { + "sid": 144, + "sent": "Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm.", + "section": "FIG", + "ner": [ + [ + 11, + 14, + "PNP", + "chemical" + ], + [ + 20, + 23, + "PNP", + "chemical" + ], + [ + 24, + 33, + "phosphate", + "chemical" + ], + [ + 38, + 43, + "T5PNP", + "chemical" + ] + ] + }, + { + "sid": 145, + "sent": "Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d.", + "section": "FIG", + "ner": [ + [ + 0, + 14, + "Reaction rates", + "evidence" + ], + [ + 68, + 76, + "wildtype", + "protein_state" + ], + [ + 77, + 80, + "MBP", + "experimental_method" + ], + [ + 81, + 89, + "hTdp2cat", + "structure_element" + ] + ] + }, + { + "sid": 146, + "sent": "Mutants of hTdp2 (black) and the equivalent residue in mTdp2 (tan) are indicated.", + "section": "FIG", + "ner": [ + [ + 11, + 16, + "hTdp2", + "protein" + ], + [ + 55, + 60, + "mTdp2", + "protein" + ] + ] + }, + { + "sid": 147, + "sent": "We examined the energy profile of the nucleophilic attack of the water molecule by using the distance between the water oxygen and the P atom on the phosphate moiety as the sole reaction coordinate in the present calculation (Figure 5B and\u00a0C).", + "section": "RESULTS", + "ner": [ + [ + 65, + 70, + "water", + "chemical" + ], + [ + 114, + 119, + "water", + "chemical" + ] + ] + }, + { + "sid": 148, + "sent": "A starting model was generated from atomic coordinates of the mTdp2cat 5\u2032\u2013aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5\u2032-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium.", + "section": "RESULTS", + "ner": [ + [ + 62, + 70, + "mTdp2cat", + "structure_element" + ], + [ + 71, + 86, + "5\u2032\u2013aminohexanol", + "chemical" + ], + [ + 104, + 113, + "structure", + "evidence" + ], + [ + 132, + 140, + "tyrosine", + "residue_name" + ], + [ + 155, + 170, + "5\u2032-aminohexanol", + "chemical" + ], + [ + 187, + 191, + "Mg2+", + "chemical" + ], + [ + 209, + 215, + "waters", + "chemical" + ], + [ + 225, + 234, + "mTdp2-DNA", + "complex_assembly" + ], + [ + 243, + 252, + "structure", + "evidence" + ], + [ + 298, + 327, + "molecular dynamics simulation", + "experimental_method" + ] + ] + }, + { + "sid": 149, + "sent": "After QM/MM optimization of this model (Figure 5C, \u2018i-substrate\u2019), the O\u2013P distance is 3.4 \u00c5, which is in agreement with the range of distances observed in the mTdp2cat 5\u2032-aminohexanol substrate analog structure (3.2\u20133.4 \u00c5).", + "section": "RESULTS", + "ner": [ + [ + 6, + 24, + "QM/MM optimization", + "experimental_method" + ], + [ + 160, + 168, + "mTdp2cat", + "structure_element" + ], + [ + 169, + 184, + "5\u2032-aminohexanol", + "chemical" + ], + [ + 202, + 211, + "structure", + "evidence" + ] + ] + }, + { + "sid": 150, + "sent": "Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 \u00c5) and the phosphotyrosyl O\u2013P distance is stretched to 1.77 \u00c5, which is 0.1 \u00c5 beyond an equilibrium bond length.", + "section": "RESULTS", + "ner": [ + [ + 10, + 15, + "water", + "chemical" + ], + [ + 48, + 54, + "Asp272", + "residue_name_number" + ], + [ + 80, + 93, + "hydrogen bond", + "bond_interaction" + ], + [ + 123, + 137, + "phosphotyrosyl", + "ptm" + ] + ] + }, + { + "sid": 151, + "sent": "In the subsequent two steps of the simulation, as the water-phosphate O\u2013P distance reduces to 1.98 \u00c5, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 \u00c5 as the water H\u2013O bond approaches the point of dissociation.", + "section": "RESULTS", + "ner": [ + [ + 35, + 45, + "simulation", + "experimental_method" + ], + [ + 54, + 59, + "water", + "chemical" + ], + [ + 108, + 121, + "hydrogen bond", + "bond_interaction" + ], + [ + 147, + 152, + "water", + "chemical" + ], + [ + 157, + 163, + "Asp272", + "residue_name_number" + ], + [ + 190, + 195, + "water", + "chemical" + ] + ] + }, + { + "sid": 152, + "sent": "The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction.", + "section": "RESULTS", + "ner": [ + [ + 25, + 30, + "water", + "chemical" + ], + [ + 62, + 75, + "hydrogen bond", + "bond_interaction" + ], + [ + 81, + 87, + "Asn274", + "residue_name_number" + ], + [ + 155, + 160, + "water", + "chemical" + ] + ] + }, + { + "sid": 153, + "sent": "Concomitant with this, the phosphotyrosyl O\u2013P bond weakens (d = 1.89 \u00c5), and the formation of the penta-covalent transition state (Figure 5C \u2018ii-transition state\u2019) is observed.", + "section": "RESULTS", + "ner": [ + [ + 27, + 41, + "phosphotyrosyl", + "ptm" + ] + ] + }, + { + "sid": 154, + "sent": "The final steps show inversion of stereochemistry at the phosphate, along with lengthening and breaking of the phosphotyrosyl O\u2013P bond.", + "section": "RESULTS", + "ner": [ + [ + 57, + 66, + "phosphate", + "chemical" + ], + [ + 111, + 125, + "phosphotyrosyl", + "ptm" + ] + ] + }, + { + "sid": 155, + "sent": "Product formation is coupled to a transfer of a proton from the nucleophillic water to Asp272, consistent with the proposed function for this residue as the catalytic base.", + "section": "RESULTS", + "ner": [ + [ + 78, + 83, + "water", + "chemical" + ], + [ + 87, + 93, + "Asp272", + "residue_name_number" + ] + ] + }, + { + "sid": 156, + "sent": "Of note, both nitrogens of the imidazole side chain of His 359 require protonation for stability of the simulation.", + "section": "RESULTS", + "ner": [ + [ + 55, + 62, + "His 359", + "residue_name_number" + ], + [ + 104, + 114, + "simulation", + "experimental_method" + ] + ] + }, + { + "sid": 157, + "sent": "Asp 326 makes a hydrogen bond to N\u22021 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Asp 326", + "residue_name_number" + ], + [ + 16, + 29, + "hydrogen bond", + "bond_interaction" + ], + [ + 40, + 46, + "His359", + "residue_name_number" + ], + [ + 69, + 80, + "salt bridge", + "bond_interaction" + ], + [ + 101, + 111, + "protonated", + "protein_state" + ], + [ + 120, + 126, + "His359", + "residue_name_number" + ], + [ + 170, + 173, + "Asp", + "residue_name" + ], + [ + 174, + 177, + "His", + "residue_name" + ], + [ + 190, + 193, + "EEP", + "structure_element" + ], + [ + 204, + 208, + "APE1", + "protein" + ], + [ + 229, + 232, + "pKa", + "evidence" + ], + [ + 241, + 244, + "His", + "residue_name" + ], + [ + 302, + 315, + "hydrogen bond", + "bond_interaction" + ], + [ + 328, + 345, + "doubly protonated", + "protein_state" + ], + [ + 346, + 352, + "His359", + "residue_name_number" + ], + [ + 361, + 370, + "phosphate", + "chemical" + ], + [ + 426, + 430, + "Mg2+", + "chemical" + ], + [ + 449, + 455, + "His359", + "residue_name_number" + ], + [ + 485, + 491, + "H-bond", + "bond_interaction" + ], + [ + 501, + 507, + "Asp326", + "residue_name_number" + ] + ] + }, + { + "sid": 158, + "sent": "In the final optimized structure, the observed product state (Figure 5C, \u2018iii-product\u2019) is found in a conformation that is 7.4 kcal mol\u22121 more stable than the initial reactive state (Figure 5B).", + "section": "RESULTS", + "ner": [ + [ + 23, + 32, + "structure", + "evidence" + ] + ] + }, + { + "sid": 159, + "sent": "The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 \u00c5 distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion.", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "tyrosine", + "residue_name" + ], + [ + 34, + 48, + "coordinated to", + "bond_interaction" + ], + [ + 53, + 57, + "Mg2+", + "chemical" + ], + [ + 118, + 122, + "Mg2+", + "chemical" + ], + [ + 148, + 153, + "water", + "chemical" + ], + [ + 196, + 205, + "phosphate", + "chemical" + ], + [ + 220, + 226, + "Glu162", + "residue_name_number" + ], + [ + 259, + 263, + "Mg2+", + "chemical" + ] + ] + }, + { + "sid": 160, + "sent": "An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group.", + "section": "RESULTS", + "ner": [ + [ + 48, + 62, + "QM/MM modeling", + "experimental_method" + ], + [ + 99, + 103, + "Top2", + "protein_type" + ], + [ + 104, + 112, + "tyrosine", + "residue_name" + ] + ] + }, + { + "sid": 161, + "sent": "A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket.", + "section": "RESULTS", + "ner": [ + [ + 10, + 19, + "conserved", + "protein_state" + ], + [ + 30, + 37, + "Tyr 188", + "residue_name_number" + ], + [ + 39, + 46, + "Arg 216", + "residue_name_number" + ], + [ + 51, + 58, + "Ser 239", + "residue_name_number" + ], + [ + 81, + 90, + "conserved", + "protein_state" + ], + [ + 91, + 95, + "Top2", + "protein_type" + ], + [ + 96, + 119, + "tyrosine binding pocket", + "site" + ] + ] + }, + { + "sid": 162, + "sent": "We propose this cation\u2013\u03c0 interaction further contributes to tuned stabilization of the negatively charged phenolate reaction product.", + "section": "RESULTS", + "ner": [ + [ + 16, + 36, + "cation\u2013\u03c0 interaction", + "bond_interaction" + ] + ] + }, + { + "sid": 163, + "sent": "Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D).", + "section": "RESULTS", + "ner": [ + [ + 34, + 57, + "electrostatic potential", + "evidence" + ], + [ + 65, + 79, + "phosphotyrosyl", + "ptm" + ], + [ + 116, + 124, + "presence", + "protein_state" + ], + [ + 129, + 139, + "absence of", + "protein_state" + ], + [ + 144, + 150, + "Arg216", + "residue_name_number" + ], + [ + 171, + 177, + "Arg216", + "residue_name_number" + ] + ] + }, + { + "sid": 164, + "sent": "We further examined the contribution of this cation\u2013\u03c0 interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or \u223c0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A).", + "section": "RESULTS", + "ner": [ + [ + 45, + 65, + "cation\u2013\u03c0 interaction", + "bond_interaction" + ], + [ + 127, + 133, + "Arg216", + "residue_name_number" + ], + [ + 143, + 145, + "QM", + "experimental_method" + ], + [ + 160, + 162, + "MM", + "experimental_method" + ], + [ + 223, + 239, + "energy penalties", + "evidence" + ] + ] + }, + { + "sid": 165, + "sent": "Removing Arg216 from the quantum subsystem incurs an \u223c2 kcal mol\u22121 penalty in the transition state and product complex.", + "section": "RESULTS", + "ner": [ + [ + 9, + 15, + "Arg216", + "residue_name_number" + ] + ] + }, + { + "sid": 166, + "sent": "Removing the +1 charge on the Arg216 has a minimal impact on the transition state, but incurs an additional \u223c2 kcal mol\u22121 penalty in the product complex.", + "section": "RESULTS", + "ner": [ + [ + 30, + 36, + "Arg216", + "residue_name_number" + ] + ] + }, + { + "sid": 167, + "sent": "Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5\u2032-phosphotyrosine bond hydrolysis.", + "section": "RESULTS", + "ner": [ + [ + 12, + 26, + "QM/MM modeling", + "experimental_method" + ], + [ + 62, + 66, + "Tdp2", + "protein" + ], + [ + 114, + 118, + "Mg2+", + "chemical" + ], + [ + 170, + 174, + "Tdp2", + "protein" + ], + [ + 185, + 203, + "5\u2032-phosphotyrosine", + "residue_name" + ] + ] + }, + { + "sid": 168, + "sent": "Tdp2 mutational analysis", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "Tdp2", + "protein" + ], + [ + 5, + 24, + "mutational analysis", + "experimental_method" + ] + ] + }, + { + "sid": 169, + "sent": "To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering \u2018m\u2019 for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and \u2018h\u2019 for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5\u2032-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5\u2032-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).", + "section": "RESULTS", + "ner": [ + [ + 27, + 31, + "Tdp2", + "protein" + ], + [ + 95, + 100, + "mouse", + "taxonomy_domain" + ], + [ + 101, + 105, + "Tdp2", + "protein" + ], + [ + 106, + 124, + "crystal structures", + "evidence" + ], + [ + 186, + 191, + "mouse", + "taxonomy_domain" + ], + [ + 205, + 228, + "engineered and purified", + "experimental_method" + ], + [ + 238, + 243, + "human", + "species" + ], + [ + 244, + 247, + "MBP", + "experimental_method" + ], + [ + 248, + 256, + "hTdp2cat", + "structure_element" + ], + [ + 257, + 263, + "mutant", + "protein_state" + ], + [ + 325, + 330, + "human", + "species" + ], + [ + 367, + 376, + "mutations", + "experimental_method" + ], + [ + 380, + 384, + "Tdp2", + "protein" + ], + [ + 457, + 468, + "tyrosylated", + "protein_state" + ], + [ + 469, + 472, + "DNA", + "chemical" + ], + [ + 484, + 488, + "5\u2032-Y", + "ptm" + ], + [ + 491, + 514, + "p-nitrophenyl phosphate", + "chemical" + ], + [ + 516, + 520, + "PNPP", + "chemical" + ], + [ + 526, + 572, + "thymidine 5\u2032-monophosphate p-nitrophenyl ester", + "chemical" + ], + [ + 574, + 579, + "T5PNP", + "chemical" + ] + ] + }, + { + "sid": 170, + "sent": "By analyzing activities on this nested set of chemically related substrates we aimed to dissect structure-activity relationships of Tdp2 catalysis.", + "section": "RESULTS", + "ner": [ + [ + 132, + 136, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 171, + "sent": "For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5\u2032-Y and T5PNP but not PNPP that lacks a nucleobase.", + "section": "RESULTS", + "ner": [ + [ + 13, + 22, + "mutations", + "experimental_method" + ], + [ + 33, + 37, + "Tdp2", + "protein" + ], + [ + 38, + 49, + "active site", + "site" + ], + [ + 64, + 78, + "phosphotyrosyl", + "ptm" + ], + [ + 174, + 177, + "DNA", + "chemical" + ], + [ + 224, + 228, + "5\u2032-Y", + "ptm" + ], + [ + 233, + 238, + "T5PNP", + "chemical" + ], + [ + 247, + 251, + "PNPP", + "chemical" + ] + ] + }, + { + "sid": 172, + "sent": "Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage.", + "section": "RESULTS", + "ner": [ + [ + 0, + 18, + "Structural results", + "evidence" + ], + [ + 23, + 37, + "QM/MM modeling", + "experimental_method" + ], + [ + 47, + 54, + "mAsp272", + "residue_name_number" + ], + [ + 67, + 72, + "water", + "chemical" + ], + [ + 130, + 152, + "phosphotyrosyl linkage", + "ptm" + ] + ] + }, + { + "sid": 173, + "sent": "To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site.", + "section": "RESULTS", + "ner": [ + [ + 74, + 81, + "mutated", + "experimental_method" + ], + [ + 85, + 87, + "to", + "experimental_method" + ], + [ + 90, + 93, + "His", + "residue_name" + ], + [ + 183, + 186, + "Leu", + "residue_name" + ], + [ + 191, + 194, + "Met", + "residue_name" + ], + [ + 228, + 246, + "water-binding site", + "site" + ] + ] + }, + { + "sid": 174, + "sent": "Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis.", + "section": "RESULTS", + "ner": [ + [ + 38, + 44, + "hD262N", + "mutant" + ], + [ + 65, + 78, + "substitutions", + "experimental_method" + ], + [ + 127, + 134, + "hAsp262", + "residue_name_number" + ], + [ + 136, + 143, + "mAsp272", + "residue_name_number" + ] + ] + }, + { + "sid": 175, + "sent": "Next, we mutated key elements of the mobile loop (\u03b22H\u03b2 hydrophobic wall, Figure 2A and\u00a0C).", + "section": "RESULTS", + "ner": [ + [ + 9, + 16, + "mutated", + "experimental_method" + ], + [ + 50, + 71, + "\u03b22H\u03b2 hydrophobic wall", + "site" + ] + ] + }, + { + "sid": 176, + "sent": "Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Mutations", + "experimental_method" + ], + [ + 10, + 16, + "hI307A", + "mutant" + ], + [ + 18, + 24, + "hL305A", + "mutant" + ], + [ + 26, + 32, + "hL305F", + "mutant" + ], + [ + 37, + 43, + "hL305W", + "mutant" + ] + ] + }, + { + "sid": 177, + "sent": "The hL305W substitution that we expect to have the most distorting impact on conformation of the \u03b22H\u03b2 hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5\u2032-Y. By comparison, as predicted by our model where \u03b22H\u03b2 dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity).", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "hL305W", + "mutant" + ], + [ + 97, + 118, + "\u03b22H\u03b2 hydrophobic wall", + "site" + ], + [ + 167, + 170, + "DNA", + "chemical" + ], + [ + 181, + 185, + "5\u2032-Y", + "ptm" + ], + [ + 234, + 238, + "\u03b22H\u03b2", + "structure_element" + ], + [ + 318, + 331, + "substitutions", + "experimental_method" + ], + [ + 354, + 358, + "PNPP", + "chemical" + ] + ] + }, + { + "sid": 178, + "sent": "Third, we altered properties of the proposed enzyme substrate cation\u2013\u03c0 interface.", + "section": "RESULTS", + "ner": [ + [ + 45, + 80, + "enzyme substrate cation\u2013\u03c0 interface", + "site" + ] + ] + }, + { + "sid": 179, + "sent": "No activity was detected for a mutant that removes the positive charge at this position (hR206A).", + "section": "RESULTS", + "ner": [ + [ + 31, + 37, + "mutant", + "protein_state" + ], + [ + 89, + 95, + "hR206A", + "mutant" + ] + ] + }, + { + "sid": 180, + "sent": "The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5\u2032-Y, no detectable activity on T5PNP or PNPP).", + "section": "RESULTS", + "ner": [ + [ + 29, + 35, + "pocket", + "site" + ], + [ + 70, + 81, + "replacement", + "experimental_method" + ], + [ + 85, + 92, + "hArg206", + "residue_name_number" + ], + [ + 94, + 101, + "mArg216", + "residue_name_number" + ], + [ + 110, + 116, + "lysine", + "residue_name" + ], + [ + 183, + 187, + "5\u2032-Y", + "ptm" + ], + [ + 215, + 220, + "T5PNP", + "chemical" + ], + [ + 224, + 228, + "PNPP", + "chemical" + ] + ] + }, + { + "sid": 181, + "sent": "Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates.", + "section": "RESULTS", + "ner": [ + [ + 11, + 19, + "mutation", + "experimental_method" + ], + [ + 23, + 30, + "hTyr178", + "residue_name_number" + ], + [ + 63, + 70, + "hArg206", + "residue_name_number" + ], + [ + 72, + 79, + "mArg216", + "residue_name_number" + ], + [ + 135, + 140, + "Y178F", + "mutant" + ], + [ + 145, + 150, + "Y178W", + "mutant" + ] + ] + }, + { + "sid": 182, + "sent": "Fourth, we evaluated roles for the hHis351\u2013hAsp316 (mAsp326\u2013mHis359) transition state stabilization charge pair.", + "section": "RESULTS", + "ner": [ + [ + 35, + 42, + "hHis351", + "residue_name_number" + ], + [ + 43, + 50, + "hAsp316", + "residue_name_number" + ], + [ + 52, + 59, + "mAsp326", + "residue_name_number" + ], + [ + 60, + 67, + "mHis359", + "residue_name_number" + ] + ] + }, + { + "sid": 183, + "sent": "We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis.", + "section": "RESULTS", + "ner": [ + [ + 14, + 23, + "mutations", + "experimental_method" + ], + [ + 29, + 36, + "removed", + "experimental_method" + ], + [ + 76, + 89, + "hydrogen bond", + "bond_interaction" + ], + [ + 91, + 97, + "hH351Q", + "mutant" + ], + [ + 131, + 134, + "pKa", + "evidence" + ], + [ + 142, + 151, + "Histidine", + "residue_name" + ], + [ + 153, + 159, + "hD316N", + "mutant" + ] + ] + }, + { + "sid": 184, + "sent": "Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme\u2013substrate cation\u2013\u03c0 interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis.", + "section": "RESULTS", + "ner": [ + [ + 63, + 74, + "active site", + "site" + ], + [ + 86, + 95, + "aspartate", + "residue_name" + ], + [ + 97, + 103, + "mobile", + "protein_state" + ], + [ + 104, + 130, + "substrate engagement loops", + "structure_element" + ], + [ + 149, + 170, + "cation\u2013\u03c0 interactions", + "bond_interaction" + ], + [ + 176, + 187, + "active site", + "site" + ], + [ + 217, + 235, + "charge interaction", + "bond_interaction" + ], + [ + 250, + 254, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 185, + "sent": "A Tdp2 active site single nucleotide polymorphism impairs Tdp2 function", + "section": "RESULTS", + "ner": [ + [ + 2, + 6, + "Tdp2", + "protein" + ], + [ + 7, + 18, + "active site", + "site" + ], + [ + 58, + 62, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 186, + "sent": "Recently, it was found that inactivation of TDP2 by a splice-site mutation is associated with neurological disease and confers hypersensitivity to Top2 poisons.", + "section": "RESULTS", + "ner": [ + [ + 44, + 48, + "TDP2", + "protein" + ], + [ + 147, + 151, + "Top2", + "protein_type" + ] + ] + }, + { + "sid": 187, + "sent": "We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA\u2013protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini.", + "section": "RESULTS", + "ner": [ + [ + 22, + 27, + "human", + "species" + ], + [ + 78, + 82, + "Tdp2", + "protein" + ], + [ + 83, + 86, + "DNA", + "chemical" + ], + [ + 150, + 154, + "Tdp2", + "protein" + ], + [ + 180, + 183, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 188, + "sent": "We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A).", + "section": "RESULTS", + "ner": [ + [ + 26, + 31, + "human", + "species" + ], + [ + 32, + 36, + "TDP2", + "protein" + ], + [ + 115, + 141, + "DNA processing active site", + "site" + ], + [ + 143, + 154, + "rs199602263", + "gene" + ], + [ + 206, + 213, + "hAsp350", + "residue_name_number" + ], + [ + 218, + 221, + "Asn", + "residue_name" + ], + [ + 227, + 237, + "rs77273535", + "gene" + ], + [ + 287, + 294, + "hIle307", + "residue_name_number" + ], + [ + 299, + 302, + "Val", + "residue_name" + ] + ] + }, + { + "sid": 189, + "sent": "We show the hD350N substitution severely impairs activity on all substrates tested in vitro, whereas hI307V only has a mild impact on catalysis (Figure 6B\u2013D).", + "section": "RESULTS", + "ner": [ + [ + 12, + 18, + "hD350N", + "mutant" + ], + [ + 19, + 31, + "substitution", + "experimental_method" + ], + [ + 101, + 107, + "hI307V", + "mutant" + ] + ] + }, + { + "sid": 190, + "sent": "To better understand the basis for the D350N catalytic defect, we analyzed the structural environment of this substitution based on the high-resolution structures of mTdp2cat (Figure 6A).", + "section": "RESULTS", + "ner": [ + [ + 39, + 44, + "D350N", + "mutant" + ], + [ + 152, + 162, + "structures", + "evidence" + ], + [ + 166, + 174, + "mTdp2cat", + "structure_element" + ] + ] + }, + { + "sid": 191, + "sent": "Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the \u03b22H\u03b2 substrate-binding loop through hydrogen bonding to mTrp307.", + "section": "RESULTS", + "ner": [ + [ + 19, + 23, + "Tdp2", + "protein" + ], + [ + 31, + 35, + "Mg2+", + "chemical" + ], + [ + 40, + 69, + "octahedral coordination shell", + "bond_interaction" + ], + [ + 96, + 120, + "hydrogen-bonding network", + "bond_interaction" + ], + [ + 133, + 140, + "hAsp350", + "residue_name_number" + ], + [ + 142, + 149, + "mAsp358", + "residue_name_number" + ], + [ + 171, + 180, + "DNA-bound", + "protein_state" + ], + [ + 201, + 228, + "\u03b22H\u03b2 substrate-binding loop", + "structure_element" + ], + [ + 237, + 253, + "hydrogen bonding", + "bond_interaction" + ], + [ + 257, + 264, + "mTrp307", + "residue_name_number" + ] + ] + }, + { + "sid": 192, + "sent": "Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations.", + "section": "RESULTS", + "ner": [ + [ + 6, + 13, + "hAsp350", + "residue_name_number" + ], + [ + 15, + 22, + "mAsp358", + "residue_name_number" + ], + [ + 61, + 72, + "active site", + "site" + ], + [ + 90, + 112, + "substrate binding loop", + "structure_element" + ] + ] + }, + { + "sid": 193, + "sent": "Tdp2 SNPs impair function. (A) Active site residues mutated by TDP2 SNPs.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "Tdp2", + "protein" + ], + [ + 31, + 42, + "Active site", + "site" + ], + [ + 63, + 67, + "TDP2", + "protein" + ] + ] + }, + { + "sid": 194, + "sent": "D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1).", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "D350N", + "mutant" + ], + [ + 7, + 12, + "mTdp2", + "protein" + ], + [ + 13, + 18, + "D358N", + "mutant" + ], + [ + 24, + 29, + "I307V", + "mutant" + ], + [ + 31, + 36, + "mTdp2", + "protein" + ], + [ + 37, + 42, + "I317V", + "mutant" + ], + [ + 44, + 57, + "substitutions", + "experimental_method" + ], + [ + 78, + 82, + "Tdp2", + "protein" + ], + [ + 83, + 94, + "active site", + "site" + ], + [ + 118, + 126, + "mTdp2cat", + "structure_element" + ], + [ + 127, + 136, + "structure", + "evidence" + ] + ] + }, + { + "sid": 195, + "sent": "(B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5\u2032\u2013phosphotyrosyl\u2013DNA oligonucleotides with 3\u2032-fluorescein label.", + "section": "FIG", + "ner": [ + [ + 27, + 35, + "SDS-PAGE", + "experimental_method" + ], + [ + 52, + 54, + "WT", + "protein_state" + ], + [ + 59, + 65, + "mutant", + "protein_state" + ], + [ + 66, + 69, + "MBP", + "experimental_method" + ], + [ + 70, + 78, + "hTdp2cat", + "structure_element" + ], + [ + 139, + 141, + "WT", + "protein_state" + ], + [ + 146, + 152, + "mutant", + "protein_state" + ], + [ + 153, + 156, + "MBP", + "experimental_method" + ], + [ + 157, + 165, + "hTdp2cat", + "structure_element" + ], + [ + 180, + 218, + "5\u2032\u2013phosphotyrosyl\u2013DNA oligonucleotides", + "chemical" + ], + [ + 227, + 238, + "fluorescein", + "chemical" + ] + ] + }, + { + "sid": 196, + "sent": "Samples were withdrawn from reactions, neutralized with TBE-urea loading dye at the indicated timepoints, and electrophoresed on a 20% TBE-urea PAGE.", + "section": "FIG", + "ner": [ + [ + 135, + 148, + "TBE-urea PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 197, + "sent": "(D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates.", + "section": "FIG", + "ner": [ + [ + 25, + 27, + "WT", + "protein_state" + ], + [ + 42, + 48, + "mutant", + "protein_state" + ], + [ + 49, + 54, + "human", + "species" + ], + [ + 55, + 58, + "MBP", + "experimental_method" + ], + [ + 59, + 67, + "hTdp2cat", + "structure_element" + ], + [ + 99, + 103, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 198, + "sent": "Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5\u2032-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed.", + "section": "FIG", + "ner": [ + [ + 26, + 29, + "MBP", + "experimental_method" + ], + [ + 30, + 38, + "hTdp2cat", + "structure_element" + ], + [ + 60, + 62, + "WT", + "protein_state" + ], + [ + 79, + 103, + "5\u2032-Y DNA oligonucleotide", + "chemical" + ], + [ + 127, + 132, + "T5PNP", + "chemical" + ], + [ + 148, + 152, + "PNPP", + "chemical" + ] + ] + }, + { + "sid": 199, + "sent": "Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5\u2032-Y substrate is quantification of activity in a gel based assay shown in Figure 6C.", + "section": "FIG", + "ner": [ + [ + 11, + 14, + "PNP", + "chemical" + ], + [ + 20, + 33, + "PNP phosphate", + "chemical" + ], + [ + 35, + 39, + "PNPP", + "chemical" + ], + [ + 110, + 114, + "5\u2032-Y", + "ptm" + ], + [ + 160, + 175, + "gel based assay", + "experimental_method" + ] + ] + }, + { + "sid": 200, + "sent": "To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8\u00c5 resolution (PDB entry 5INN).", + "section": "RESULTS", + "ner": [ + [ + 38, + 44, + "hD350N", + "mutant" + ], + [ + 46, + 52, + "mD358N", + "mutant" + ], + [ + 65, + 92, + "crystallized and determined", + "experimental_method" + ], + [ + 97, + 106, + "structure", + "evidence" + ], + [ + 114, + 122, + "DNA-free", + "protein_state" + ], + [ + 135, + 141, + "mD358N", + "mutant" + ] + ] + }, + { + "sid": 201, + "sent": "This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307.", + "section": "RESULTS", + "ner": [ + [ + 5, + 14, + "structure", + "evidence" + ], + [ + 25, + 30, + "D358N", + "mutant" + ], + [ + 31, + 39, + "mutation", + "experimental_method" + ], + [ + 53, + 66, + "hydrogen bond", + "bond_interaction" + ], + [ + 75, + 81, + "Asp358", + "residue_name_number" + ], + [ + 86, + 92, + "Trp307", + "residue_name_number" + ], + [ + 117, + 123, + "Asn358", + "residue_name_number" + ], + [ + 141, + 147, + "Trp307", + "residue_name_number" + ] + ] + }, + { + "sid": 202, + "sent": "Consequently, poor electron density is visible for the \u03b22H\u03b2 loop which is mostly disordered (Supplementary Figure S6).", + "section": "RESULTS", + "ner": [ + [ + 19, + 35, + "electron density", + "evidence" + ], + [ + 55, + 64, + "\u03b22H\u03b2 loop", + "structure_element" + ], + [ + 81, + 91, + "disordered", + "protein_state" + ] + ] + }, + { + "sid": 203, + "sent": "Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals.", + "section": "RESULTS", + "ner": [ + [ + 9, + 13, + "Mg2+", + "chemical" + ], + [ + 58, + 60, + "WT", + "protein_state" + ], + [ + 61, + 68, + "mTdpcat", + "protein" + ], + [ + 69, + 77, + "crystals", + "evidence" + ], + [ + 99, + 109, + "metal site", + "site" + ], + [ + 113, + 123, + "unoccupied", + "protein_state" + ], + [ + 131, + 137, + "mD358N", + "mutant" + ], + [ + 138, + 146, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 204, + "sent": "Therefore, metal-regulated opening/closure of the active site may modulate Tdp2 activity, and D350N is sufficient to block both metal binding and conformational change.", + "section": "RESULTS", + "ner": [ + [ + 50, + 61, + "active site", + "site" + ], + [ + 75, + 79, + "Tdp2", + "protein" + ], + [ + 94, + 99, + "D350N", + "mutant" + ] + ] + }, + { + "sid": 205, + "sent": "In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D).", + "section": "RESULTS", + "ner": [ + [ + 38, + 44, + "hD350N", + "mutant" + ], + [ + 46, + 52, + "mD358N", + "mutant" + ], + [ + 62, + 66, + "Mg2+", + "chemical" + ], + [ + 90, + 123, + "intrinsic tryptophan fluorescence", + "evidence" + ], + [ + 151, + 155, + "Mg2+", + "chemical" + ], + [ + 167, + 178, + "active site", + "site" + ], + [ + 258, + 262, + "Tdp2", + "protein" + ], + [ + 275, + 279, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 206, + "sent": "Tdp2 facilitates NHEJ repair of 5\u2032-phosphotyrosine adducted DSBs", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "Tdp2", + "protein" + ], + [ + 32, + 50, + "5\u2032-phosphotyrosine", + "residue_name" + ] + ] + }, + { + "sid": 207, + "sent": "Overall, our Tdp2 structure/activity studies reveal a tuned, 5\u2032-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway.", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "Tdp2", + "protein" + ], + [ + 18, + 44, + "structure/activity studies", + "experimental_method" + ], + [ + 61, + 78, + "5\u2032-detyrosylation", + "ptm" + ], + [ + 79, + 82, + "DNA", + "chemical" + ], + [ + 141, + 145, + "Tdp2", + "protein" + ], + [ + 169, + 173, + "Top2", + "protein_type" + ] + ] + }, + { + "sid": 208, + "sent": "Accordingly, we demonstrate here that 5\u2032-tyrosylated ends are sufficient to severely impair an in vitro reconstituted mammalian NHEJ reaction (Figure 7A, lanes 3 and 6), unless supplemented with catalytic quantities of hTdp2FL (Figure 7A, lane 8).", + "section": "RESULTS", + "ner": [ + [ + 38, + 52, + "5\u2032-tyrosylated", + "protein_state" + ], + [ + 118, + 127, + "mammalian", + "taxonomy_domain" + ], + [ + 219, + 226, + "hTdp2FL", + "protein" + ] + ] + }, + { + "sid": 209, + "sent": "Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A).", + "section": "RESULTS", + "ner": [ + [ + 15, + 23, + "hTdp2cat", + "structure_element" + ], + [ + 56, + 63, + "hTdp2FL", + "protein" + ], + [ + 108, + 131, + "catalytically deficient", + "protein_state" + ], + [ + 132, + 137, + "E152Q", + "mutant" + ], + [ + 138, + 144, + "mutant", + "protein_state" + ], + [ + 149, + 157, + "inactive", + "protein_state" + ], + [ + 200, + 204, + "Tdp2", + "protein" + ], + [ + 255, + 269, + "phosphotyrosyl", + "ptm" + ] + ] + }, + { + "sid": 210, + "sent": "We confirmed that efficient joining of the same tyrosine-adducted substrate in cells (Figure 7B) was dependent on both NHEJ (reduced over 10-fold in ligase IV deficient HCT 116 cells; Supplementary Figure S7B), and Tdp2 (reduced 5-fold in Tdp2 deficient MEFs; Figure 7C).", + "section": "RESULTS", + "ner": [ + [ + 48, + 56, + "tyrosine", + "residue_name" + ], + [ + 215, + 219, + "Tdp2", + "protein" + ], + [ + 239, + 243, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 211, + "sent": "Moreover, products with error (i.e. junctions have missing sequence flanking the adducted terminus) are twice as frequent in cells deficient in Tdp2 (Figure 7D).", + "section": "RESULTS", + "ner": [ + [ + 144, + 148, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 212, + "sent": "Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex).", + "section": "RESULTS", + "ner": [ + [ + 52, + 60, + "tyrosine", + "residue_name" + ], + [ + 81, + 85, + "Tdp2", + "protein" + ], + [ + 95, + 109, + "detyrosylation", + "ptm" + ], + [ + 213, + 220, + "Artemis", + "protein" + ], + [ + 228, + 244, + "Mre11/Rad50/Nbs1", + "complex_assembly" + ] + ] + }, + { + "sid": 213, + "sent": "Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5\u2032-phosphate termini (Lanes 1\u20134) or 5\u2032-tyrosylated termini (Lanes 5\u20139) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37\u00b0C.", + "section": "FIG", + "ner": [ + [ + 11, + 15, + "Tdp2", + "protein" + ], + [ + 16, + 27, + "active site", + "site" + ], + [ + 60, + 64, + "Tdp2", + "protein" + ], + [ + 80, + 83, + "Cy5", + "chemical" + ], + [ + 108, + 120, + "5\u2032-phosphate", + "chemical" + ], + [ + 144, + 158, + "5\u2032-tyrosylated", + "protein_state" + ], + [ + 199, + 201, + "Ku", + "protein" + ], + [ + 207, + 218, + "NHEJ ligase", + "protein_type" + ], + [ + 220, + 225, + "XRCC4", + "protein" + ], + [ + 227, + 236, + "ligase IV", + "protein" + ], + [ + 241, + 244, + "XLF", + "protein" + ], + [ + 262, + 269, + "hTdp2FL", + "protein" + ] + ] + }, + { + "sid": 214, + "sent": "Concatemer ligation products were detected by 5% native PAGE.", + "section": "FIG", + "ner": [ + [ + 49, + 60, + "native PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 215, + "sent": "(B) Workflow diagram of cellular end joining assays.", + "section": "FIG", + "ner": [ + [ + 24, + 51, + "cellular end joining assays", + "experimental_method" + ] + ] + }, + { + "sid": 216, + "sent": "DNA substrates with 5\u2032-phosphotyrosine adducts and 4 nucleotide 5\u2032 overhangs were electroporated into cultured mammalian cells.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "DNA", + "chemical" + ], + [ + 20, + 38, + "5\u2032-phosphotyrosine", + "residue_name" + ], + [ + 111, + 120, + "mammalian", + "taxonomy_domain" + ] + ] + }, + { + "sid": 217, + "sent": "After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2\u2212/\u2212 cells and Tdp2\u2212/\u2212 cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.", + "section": "FIG", + "ner": [ + [ + 11, + 14, + "DNA", + "chemical" + ], + [ + 65, + 69, + "qPCR", + "experimental_method" + ], + [ + 73, + 83, + "sequencing", + "experimental_method" + ], + [ + 102, + 106, + "qPCR", + "experimental_method" + ], + [ + 160, + 171, + "tyrosylated", + "protein_state" + ], + [ + 205, + 213, + "wildtype", + "protein_state" + ], + [ + 227, + 231, + "Tdp2", + "protein" + ], + [ + 245, + 249, + "Tdp2", + "protein" + ], + [ + 277, + 285, + "wildtype", + "protein_state" + ], + [ + 299, + 306, + "hTDP2FL", + "protein" + ], + [ + 390, + 392, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 218, + "sent": "Error bars, s.d, n = 3. (D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate.", + "section": "FIG", + "ner": [ + [ + 53, + 80, + "cellular end-joining assays", + "experimental_method" + ], + [ + 127, + 137, + "sequencing", + "experimental_method" + ], + [ + 152, + 174, + "end-joining error rate", + "evidence" + ] + ] + }, + { + "sid": 219, + "sent": "Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3.", + "section": "FIG", + "ner": [ + [ + 28, + 53, + "Clonogenic survival assay", + "experimental_method" + ], + [ + 57, + 59, + "WT", + "protein_state" + ], + [ + 61, + 65, + "Tdp2", + "protein" + ], + [ + 151, + 160, + "etoposide", + "chemical" + ] + ] + }, + { + "sid": 220, + "sent": "We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C).", + "section": "RESULTS", + "ner": [ + [ + 32, + 41, + "wild-type", + "protein_state" + ], + [ + 46, + 52, + "mutant", + "protein_state" + ], + [ + 53, + 60, + "hTdp2FL", + "protein" + ], + [ + 84, + 88, + "Tdp2", + "protein" + ], + [ + 99, + 104, + "mouse", + "taxonomy_domain" + ] + ] + }, + { + "sid": 221, + "sent": "Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.", + "section": "RESULTS", + "ner": [ + [ + 28, + 31, + "DNA", + "chemical" + ], + [ + 37, + 52, + "phosphotyrosine", + "residue_name" + ], + [ + 177, + 186, + "wild-type", + "protein_state" + ], + [ + 187, + 192, + "mouse", + "taxonomy_domain" + ], + [ + 206, + 210, + "Tdp2", + "protein" + ], + [ + 214, + 219, + "mouse", + "taxonomy_domain" + ], + [ + 235, + 244, + "wild-type", + "protein_state" + ], + [ + 245, + 250, + "human", + "species" + ], + [ + 251, + 255, + "Tdp2", + "protein" + ], + [ + 261, + 265, + "Tdp2", + "protein" + ], + [ + 294, + 299, + "I307V", + "mutant" + ], + [ + 300, + 307, + "variant", + "protein_state" + ], + [ + 308, + 313, + "human", + "species" + ], + [ + 314, + 318, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 222, + "sent": "In contrast, joining of 5\u2032 phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N.", + "section": "RESULTS", + "ner": [ + [ + 78, + 82, + "Tdp2", + "protein" + ], + [ + 133, + 137, + "Tdp2", + "protein" + ], + [ + 161, + 165, + "Tdp2", + "protein" + ], + [ + 166, + 171, + "D350N", + "mutant" + ] + ] + }, + { + "sid": 223, + "sent": "Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D).", + "section": "RESULTS", + "ner": [ + [ + 71, + 75, + "Tdp2", + "protein" + ], + [ + 96, + 100, + "Tdp2", + "protein" + ], + [ + 132, + 137, + "D350N", + "mutant" + ], + [ + 168, + 177, + "wild type", + "protein_state" + ], + [ + 178, + 182, + "Tdp2", + "protein" + ], + [ + 186, + 191, + "hTdp2", + "protein" + ], + [ + 192, + 197, + "I307V", + "mutant" + ] + ] + }, + { + "sid": 224, + "sent": "Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E).", + "section": "RESULTS", + "ner": [ + [ + 14, + 23, + "wild type", + "protein_state" + ], + [ + 27, + 32, + "I307V", + "mutant" + ], + [ + 33, + 38, + "human", + "species" + ], + [ + 39, + 43, + "Tdp2", + "protein" + ], + [ + 47, + 51, + "Tdp2", + "protein" + ], + [ + 114, + 123, + "etoposide", + "chemical" + ], + [ + 150, + 159, + "wild-type", + "protein_state" + ], + [ + 176, + 190, + "overexpression", + "experimental_method" + ], + [ + 194, + 199, + "human", + "species" + ], + [ + 200, + 205, + "D350N", + "mutant" + ], + [ + 206, + 210, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 225, + "sent": "The rare D350N variant is thus inactive by all metrics analyzed.", + "section": "RESULTS", + "ner": [ + [ + 9, + 14, + "D350N", + "mutant" + ], + [ + 15, + 22, + "variant", + "protein_state" + ], + [ + 31, + 39, + "inactive", + "protein_state" + ] + ] + }, + { + "sid": 226, + "sent": "By comparison the more frequent I307V has only mild effects on in vitro activity, and no detectable impact on cellular assays.", + "section": "RESULTS", + "ner": [ + [ + 32, + 37, + "I307V", + "mutant" + ] + ] + }, + { + "sid": 227, + "sent": "Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "Top2", + "protein_type" + ], + [ + 99, + 102, + "DNA", + "chemical" + ], + [ + 115, + 119, + "Top2", + "protein_type" + ], + [ + 120, + 123, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 228, + "sent": "Understanding how cells cope with complex DNA breaks bearing topoisomerase\u2013DNA protein crosslinks is key to deciphering individual responses to chemotherapeutic outcomes and genotoxic agents that poison Top2.", + "section": "DISCUSS", + "ner": [ + [ + 42, + 45, + "DNA", + "chemical" + ], + [ + 75, + 78, + "DNA", + "chemical" + ], + [ + 203, + 207, + "Top2", + "protein_type" + ] + ] + }, + { + "sid": 229, + "sent": "Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 25, + "mutagenesis", + "experimental_method" + ], + [ + 30, + 47, + "functional assays", + "experimental_method" + ], + [ + 57, + 61, + "Tdp2", + "protein" + ], + [ + 62, + 72, + "structures", + "evidence" + ], + [ + 73, + 90, + "in the absence of", + "protein_state" + ], + [ + 91, + 98, + "ligands", + "chemical" + ], + [ + 103, + 118, + "in complex with", + "protein_state" + ], + [ + 119, + 122, + "DNA", + "chemical" + ], + [ + 158, + 162, + "Tdp2", + "protein" + ], + [ + 163, + 166, + "DNA", + "chemical" + ], + [ + 205, + 209, + "Tdp2", + "protein" + ], + [ + 210, + 221, + "active site", + "site" + ], + [ + 268, + 271, + "DNA", + "chemical" + ], + [ + 320, + 323, + "DNA", + "chemical" + ], + [ + 345, + 349, + "Top2", + "protein_type" + ], + [ + 382, + 401, + "structural analysis", + "experimental_method" + ], + [ + 415, + 433, + "mutational studies", + "experimental_method" + ], + [ + 438, + 462, + "QM/MM molecular modeling", + "experimental_method" + ], + [ + 470, + 474, + "Tdp2", + "protein" + ], + [ + 557, + 560, + "EEP", + "structure_element" + ], + [ + 578, + 598, + "phosphoryl hydrolase", + "protein_type" + ], + [ + 620, + 624, + "Tdp2", + "protein" + ], + [ + 625, + 636, + "active site", + "site" + ], + [ + 640, + 664, + "conformationally plastic", + "protein_state" + ], + [ + 710, + 713, + "DNA", + "chemical" + ], + [ + 718, + 722, + "Mg2+", + "chemical" + ], + [ + 769, + 773, + "Tdp2", + "protein" + ], + [ + 793, + 797, + "Tdp2", + "protein" + ], + [ + 798, + 809, + "active site", + "site" + ] + ] + }, + { + "sid": 230, + "sent": "This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing.", + "section": "DISCUSS", + "ner": [ + [ + 31, + 35, + "Tdp2", + "protein" + ], + [ + 62, + 65, + "DNA", + "chemical" + ], + [ + 149, + 153, + "Tdp2", + "protein" + ], + [ + 154, + 157, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 231, + "sent": "Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "Tdp2", + "protein" + ], + [ + 74, + 78, + "Top1", + "protein_type" + ], + [ + 83, + 87, + "Top2", + "protein_type" + ], + [ + 188, + 192, + "Tdp2", + "protein" + ], + [ + 215, + 218, + "DNA", + "chemical" + ], + [ + 238, + 242, + "Top2", + "protein_type" + ], + [ + 257, + 261, + "Top2", + "protein_type" + ], + [ + 262, + 265, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 232, + "sent": "The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis.", + "section": "DISCUSS", + "ner": [ + [ + 26, + 29, + "DNA", + "chemical" + ], + [ + 52, + 56, + "Top2", + "protein_type" + ], + [ + 57, + 60, + "DNA", + "chemical" + ], + [ + 97, + 101, + "Tdp2", + "protein" + ], + [ + 156, + 169, + "topoisomerase", + "protein_type" + ], + [ + 185, + 196, + "active site", + "site" + ] + ] + }, + { + "sid": 233, + "sent": "The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 8, + "Tdp2", + "protein" + ], + [ + 9, + 37, + "substrate interaction groove", + "site" + ], + [ + 50, + 53, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 234, + "sent": "First, the nucleic acid binding trench is assembled by a dynamic \u03b22H\u03b2 DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as \u03f5A. This is achieved by binding of nucleic acid \u2018bases out\u2019 by an extended base-stacking hydrophobic wall of the \u03b22H\u03b2-loop.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 38, + "nucleic acid binding trench", + "site" + ], + [ + 57, + 64, + "dynamic", + "protein_state" + ], + [ + 65, + 93, + "\u03b22H\u03b2 DNA damage-binding loop", + "structure_element" + ], + [ + 148, + 171, + "phosphotyrosyl linkages", + "ptm" + ], + [ + 217, + 219, + "\u03f5A", + "chemical" + ], + [ + 292, + 305, + "base-stacking", + "bond_interaction" + ], + [ + 306, + 322, + "hydrophobic wall", + "site" + ], + [ + 330, + 339, + "\u03b22H\u03b2-loop", + "structure_element" + ] + ] + }, + { + "sid": 235, + "sent": "Secondly, our QM/MM analysis further highlights an enzyme\u2013substrate cation\u2013\u03c0 interaction as an additional key feature of the Tdp2 protein\u2013DNA crosslink binding and reversal.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 19, + "QM/MM", + "experimental_method" + ], + [ + 68, + 88, + "cation\u2013\u03c0 interaction", + "bond_interaction" + ], + [ + 125, + 129, + "Tdp2", + "protein" + ], + [ + 138, + 141, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 236, + "sent": "The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 22, + "strictly conserved", + "protein_state" + ], + [ + 23, + 34, + "active site", + "site" + ], + [ + 35, + 41, + "Arg216", + "residue_name_number" + ], + [ + 137, + 151, + "phosphotyrosyl", + "ptm" + ] + ] + }, + { + "sid": 237, + "sent": "To our knowledge, this is the first proposed example of a substrate cation\u2013\u03c0 interface exploited to promote a phosphoryl-transfer reaction.", + "section": "DISCUSS", + "ner": [ + [ + 58, + 86, + "substrate cation\u2013\u03c0 interface", + "site" + ] + ] + }, + { + "sid": 238, + "sent": "This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein\u2013RNA and Hepatitis B Virus (HBV) protein\u2013DNA processing intermediates.", + "section": "DISCUSS", + "ner": [ + [ + 85, + 89, + "Tdp2", + "protein" + ], + [ + 166, + 172, + "Top2cc", + "complex_assembly" + ], + [ + 174, + 186, + "picornaviral", + "taxonomy_domain" + ], + [ + 195, + 198, + "RNA", + "chemical" + ], + [ + 203, + 220, + "Hepatitis B Virus", + "taxonomy_domain" + ], + [ + 222, + 225, + "HBV", + "taxonomy_domain" + ], + [ + 235, + 238, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 239, + "sent": "By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 24, + "EEP", + "structure_element" + ], + [ + 25, + 34, + "nucleases", + "protein_type" + ], + [ + 43, + 47, + "Ape1", + "protein" + ], + [ + 52, + 56, + "Ape2", + "protein" + ], + [ + 77, + 80, + "DNA", + "chemical" + ], + [ + 159, + 163, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 240, + "sent": "The dynamic nature of the Tdp2 active site presents opportunities for enzyme regulation.", + "section": "DISCUSS", + "ner": [ + [ + 26, + 30, + "Tdp2", + "protein" + ], + [ + 31, + 42, + "active site", + "site" + ] + ] + }, + { + "sid": 241, + "sent": "However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 \u03b22H\u03b2-loop is unknown.", + "section": "DISCUSS", + "ner": [ + [ + 56, + 60, + "Tdp2", + "protein" + ], + [ + 102, + 106, + "Tdp2", + "protein" + ], + [ + 107, + 116, + "\u03b22H\u03b2-loop", + "structure_element" + ] + ] + }, + { + "sid": 242, + "sent": "We hypothesize that binding of the Top2 protein component of a DNA\u2013protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed.", + "section": "DISCUSS", + "ner": [ + [ + 35, + 39, + "Top2", + "protein_type" + ], + [ + 63, + 66, + "DNA", + "chemical" + ], + [ + 132, + 136, + "Tdp2", + "protein" + ], + [ + 137, + 148, + "active site", + "site" + ], + [ + 178, + 182, + "Tdp2", + "protein" + ], + [ + 222, + 226, + "Top2", + "protein_type" + ], + [ + 306, + 310, + "Top2", + "protein_type" + ] + ] + }, + { + "sid": 243, + "sent": "Furthermore, high-resolution structures of mouse (Figures 3 and\u00a04) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 39, + "structures", + "evidence" + ], + [ + 43, + 48, + "mouse", + "taxonomy_domain" + ], + [ + 71, + 81, + "C. elegans", + "species" + ], + [ + 82, + 86, + "Tdp2", + "protein" + ], + [ + 129, + 133, + "Tdp2", + "protein" + ], + [ + 134, + 145, + "active site", + "site" + ], + [ + 151, + 156, + "worms", + "taxonomy_domain" + ], + [ + 160, + 163, + "man", + "taxonomy_domain" + ] + ] + }, + { + "sid": 244, + "sent": "Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism.", + "section": "DISCUSS", + "ner": [ + [ + 83, + 116, + "intrinsic tryptophan fluorescence", + "experimental_method" + ], + [ + 118, + 143, + "crystallographic analysis", + "experimental_method" + ], + [ + 180, + 191, + "mutagenesis", + "experimental_method" + ], + [ + 193, + 216, + "Ca2+ inhibition studies", + "experimental_method" + ], + [ + 221, + 235, + "QM/MM analysis", + "experimental_method" + ], + [ + 271, + 275, + "Mg2+", + "chemical" + ], + [ + 285, + 289, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 245, + "sent": "Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 9, + "Etoposide", + "chemical" + ], + [ + 20, + 24, + "Top2", + "protein_type" + ], + [ + 74, + 78, + "Tdp2", + "protein" + ], + [ + 107, + 112, + "human", + "species" + ], + [ + 151, + 155, + "Top2", + "protein_type" + ], + [ + 174, + 183, + "etoposide", + "chemical" + ], + [ + 188, + 199, + "doxyrubicin", + "chemical" + ] + ] + }, + { + "sid": 246, + "sent": "Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection.", + "section": "DISCUSS", + "ner": [ + [ + 6, + 10, + "Tdp2", + "protein" + ], + [ + 28, + 33, + "human", + "species" + ], + [ + 117, + 121, + "TDP2", + "protein" + ], + [ + 259, + 263, + "Top2", + "protein_type" + ], + [ + 277, + 282, + "viral", + "taxonomy_domain" + ] + ] + }, + { + "sid": 247, + "sent": "Lastly, Tdp2 inhibitors may synergize or potentiate cytotoxic effects of current anticancer treatments that target Tdp2.", + "section": "DISCUSS", + "ner": [ + [ + 8, + 12, + "Tdp2", + "protein" + ], + [ + 115, + 119, + "Tdp2", + "protein" + ] + ] + }, + { + "sid": 248, + "sent": "Thus, we anticipate this atomic-level and mechanistic definition of the molecular determinants of Tdp2 catalysis and conformational changes driven by DNA\u2013protein and protein\u2013protein interactions will foster unique strategies for the development of Tdp2 targeted small molecule interventions.", + "section": "DISCUSS", + "ner": [ + [ + 98, + 102, + "Tdp2", + "protein" + ], + [ + 150, + 153, + "DNA", + "chemical" + ], + [ + 248, + 252, + "Tdp2", + "protein" + ] + ] + } + ] + }, + "PMC4918766": { + "annotations": [ + { + "sid": 0, + "sent": "Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger", + "section": "TITLE", + "ner": [ + [ + 44, + 56, + "binding-site", + "site" + ], + [ + 74, + 98, + "sodium-calcium exchanger", + "protein_type" + ] + ] + }, + { + "sid": 1, + "sent": "Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 19, + "Na+/Ca2+ exchangers", + "protein_type" + ], + [ + 32, + 35, + "Na+", + "chemical" + ], + [ + 113, + 117, + "Ca2+", + "chemical" + ], + [ + 146, + 150, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 2, + "sent": "Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state.", + "section": "ABSTRACT", + "ner": [ + [ + 92, + 111, + "structural analysis", + "experimental_method" + ], + [ + 119, + 128, + "exchanger", + "protein_type" + ], + [ + 134, + 158, + "Methanococcus jannaschii", + "species" + ], + [ + 160, + 166, + "NCX_Mj", + "protein" + ], + [ + 168, + 176, + "bound to", + "protein_state" + ], + [ + 177, + 180, + "Na+", + "chemical" + ], + [ + 182, + 186, + "Ca2+", + "chemical" + ], + [ + 190, + 194, + "Sr2+", + "chemical" + ], + [ + 228, + 231, + "apo", + "protein_state" + ] + ] + }, + { + "sid": 3, + "sent": "This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism.", + "section": "ABSTRACT", + "ner": [ + [ + 130, + 133, + "Na+", + "chemical" + ], + [ + 135, + 139, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 4, + "sent": "An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations.", + "section": "ABSTRACT", + "ner": [ + [ + 44, + 80, + "conformational free-energy landscape", + "evidence" + ], + [ + 84, + 90, + "NCX_Mj", + "protein" + ], + [ + 104, + 117, + "ion-occupancy", + "protein_state" + ], + [ + 135, + 183, + "enhanced-sampling molecular-dynamics simulations", + "experimental_method" + ], + [ + 207, + 225, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 5, + "sent": "These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport.", + "section": "ABSTRACT", + "ner": [ + [ + 6, + 18, + "calculations", + "experimental_method" + ], + [ + 58, + 65, + "outward", + "protein_state" + ], + [ + 69, + 75, + "inward", + "protein_state" + ], + [ + 182, + 185, + "Na+", + "chemical" + ], + [ + 186, + 190, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 6, + "sent": "Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis.", + "section": "INTRO", + "ner": [ + [ + 0, + 19, + "Na+/Ca2+ exchangers", + "protein_type" + ], + [ + 21, + 24, + "NCX", + "protein_type" + ], + [ + 66, + 70, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 7, + "sent": "NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry.", + "section": "INTRO", + "ner": [ + [ + 0, + 3, + "NCX", + "protein_type" + ], + [ + 52, + 56, + "Ca2+", + "chemical" + ], + [ + 138, + 141, + "Na+", + "chemical" + ], + [ + 166, + 169, + "Na+", + "chemical" + ], + [ + 175, + 179, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 8, + "sent": "The mechanism of NCX proteins is therefore highly likely to be consistent with the alternating-access model of secondary-active transport.", + "section": "INTRO", + "ner": [ + [ + 17, + 20, + "NCX", + "protein_type" + ] + ] + }, + { + "sid": 9, + "sent": "The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves.", + "section": "INTRO", + "ner": [ + [ + 47, + 50, + "NCX", + "protein_type" + ], + [ + 67, + 93, + "membrane-spanning segments", + "structure_element" + ], + [ + 121, + 127, + "halves", + "structure_element" + ] + ] + }, + { + "sid": 10, + "sent": "Each of these halves contains a highly conserved region, referred to as \u03b1-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1).", + "section": "INTRO", + "ner": [ + [ + 14, + 20, + "halves", + "structure_element" + ], + [ + 32, + 48, + "highly conserved", + "protein_state" + ], + [ + 72, + 80, + "\u03b1-repeat", + "structure_element" + ], + [ + 142, + 152, + "eukaryotic", + "taxonomy_domain" + ], + [ + 153, + 156, + "NCX", + "protein_type" + ], + [ + 166, + 172, + "halves", + "structure_element" + ], + [ + 198, + 229, + "intracellular regulatory domain", + "structure_element" + ], + [ + 240, + 246, + "absent", + "protein_state" + ], + [ + 250, + 259, + "microbial", + "taxonomy_domain" + ], + [ + 260, + 263, + "NCX", + "protein_type" + ] + ] + }, + { + "sid": 11, + "sent": "Despite a long history of physiological and functional studies, the molecular mechanism of NCX has been elusive, owing to the lack of structural information.", + "section": "INTRO", + "ner": [ + [ + 91, + 94, + "NCX", + "protein_type" + ] + ] + }, + { + "sid": 12, + "sent": "Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein.", + "section": "INTRO", + "ner": [ + [ + 29, + 38, + "structure", + "evidence" + ], + [ + 42, + 48, + "NCX_Mj", + "protein" + ], + [ + 54, + 78, + "Methanococcus jannaschii", + "species" + ], + [ + 138, + 141, + "NCX", + "protein_type" + ] + ] + }, + { + "sid": 13, + "sent": "This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).", + "section": "INTRO", + "ner": [ + [ + 5, + 14, + "structure", + "evidence" + ], + [ + 25, + 34, + "exchanger", + "protein_type" + ], + [ + 41, + 55, + "outward-facing", + "protein_state" + ], + [ + 95, + 112, + "ion-binding sites", + "site" + ], + [ + 126, + 134, + "internal", + "site" + ], + [ + 136, + 140, + "Sint", + "site" + ], + [ + 143, + 151, + "external", + "site" + ], + [ + 153, + 157, + "Sext", + "site" + ], + [ + 160, + 172, + "Ca2+-binding", + "site" + ], + [ + 174, + 177, + "SCa", + "site" + ], + [ + 183, + 189, + "middle", + "site" + ], + [ + 191, + 195, + "Smid", + "site" + ], + [ + 241, + 254, + "occluded from", + "protein_state" + ] + ] + }, + { + "sid": 14, + "sent": "With similar ion exchange properties to those of its eukaryotic counterparts, NCX_Mj provides a compelling model system to investigate the structural basis for the specificity, stoichiometry and mechanism of the ion-exchange reaction catalyzed by NCX.", + "section": "INTRO", + "ner": [ + [ + 53, + 63, + "eukaryotic", + "taxonomy_domain" + ], + [ + 78, + 84, + "NCX_Mj", + "protein" + ], + [ + 247, + 250, + "NCX", + "protein_type" + ] + ] + }, + { + "sid": 15, + "sent": "In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations.", + "section": "INTRO", + "ner": [ + [ + 43, + 53, + "structures", + "evidence" + ], + [ + 57, + 71, + "outward-facing", + "protein_state" + ], + [ + 72, + 81, + "wild-type", + "protein_state" + ], + [ + 82, + 88, + "NCX_Mj", + "protein" + ], + [ + 89, + 104, + "in complex with", + "protein_state" + ], + [ + 105, + 108, + "Na+", + "chemical" + ], + [ + 110, + 114, + "Ca2+", + "chemical" + ], + [ + 119, + 123, + "Sr2+", + "chemical" + ] + ] + }, + { + "sid": 16, + "sent": "These structures reveal the mode of recognition of these ions, their relative affinities, and the mechanism of extracellular ion exchange, for a well-defined, functional conformation in a membrane-like environment.", + "section": "INTRO", + "ner": [ + [ + 6, + 16, + "structures", + "evidence" + ] + ] + }, + { + "sid": 17, + "sent": "An independent analysis based on molecular-dynamics simulations demonstrates that the structures capture mechanistically relevant states.", + "section": "INTRO", + "ner": [ + [ + 33, + 63, + "molecular-dynamics simulations", + "experimental_method" + ], + [ + 86, + 96, + "structures", + "evidence" + ] + ] + }, + { + "sid": 18, + "sent": "These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism.", + "section": "INTRO", + "ner": [ + [ + 6, + 18, + "calculations", + "experimental_method" + ], + [ + 66, + 80, + "outward-facing", + "protein_state" + ], + [ + 81, + 90, + "exchanger", + "protein_type" + ], + [ + 143, + 156, + "inward-facing", + "protein_state" + ] + ] + }, + { + "sid": 19, + "sent": "Extracellular Na+ binding", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 20, + "sent": "The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals.", + "section": "RESULTS", + "ner": [ + [ + 27, + 48, + "central binding sites", + "site" + ], + [ + 87, + 93, + "NCX_Mj", + "protein" + ], + [ + 94, + 103, + "structure", + "evidence" + ], + [ + 141, + 144, + "Na+", + "chemical" + ], + [ + 149, + 153, + "Ca2+", + "chemical" + ], + [ + 169, + 177, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 21, + "sent": "To conclusively clarify this assignment, we first set out to examine the Na+ occupancy of these sites without Ca2+.", + "section": "RESULTS", + "ner": [ + [ + 73, + 76, + "Na+", + "chemical" + ], + [ + 110, + 114, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 22, + "sent": "Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Crystals", + "evidence" + ], + [ + 30, + 34, + "NaCl", + "chemical" + ], + [ + 45, + 64, + "lipidic cubic phase", + "experimental_method" + ], + [ + 66, + 69, + "LCP", + "experimental_method" + ] + ] + }, + { + "sid": 23, + "sent": "The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods).", + "section": "RESULTS", + "ner": [ + [ + 41, + 44, + "LCP", + "experimental_method" + ], + [ + 132, + 136, + "NaCl", + "chemical" + ], + [ + 141, + 145, + "EGTA", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c).", + "section": "RESULTS", + "ner": [ + [ + 0, + 17, + "X-ray diffraction", + "experimental_method" + ], + [ + 34, + 42, + "crystals", + "evidence" + ], + [ + 54, + 57, + "Na+", + "chemical" + ], + [ + 85, + 114, + "electron-density distribution", + "evidence" + ], + [ + 124, + 128, + "Sext", + "site" + ], + [ + 130, + 133, + "SCa", + "site" + ], + [ + 138, + 142, + "Sint", + "site" + ], + [ + 157, + 160, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 25, + "sent": "Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 0, + 20, + "Occupancy refinement", + "experimental_method" + ], + [ + 35, + 38, + "Na+", + "chemical" + ], + [ + 52, + 56, + "Sint", + "site" + ], + [ + 61, + 64, + "SCa", + "site" + ], + [ + 72, + 75, + "Na+", + "chemical" + ], + [ + 131, + 135, + "Sint", + "site" + ] + ] + }, + { + "sid": 26, + "sent": "Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c).", + "section": "RESULTS", + "ner": [ + [ + 19, + 22, + "Na+", + "chemical" + ], + [ + 26, + 30, + "Sext", + "site" + ], + [ + 70, + 77, + "density", + "evidence" + ], + [ + 106, + 109, + "Na+", + "chemical" + ], + [ + 130, + 134, + "Sext", + "site" + ], + [ + 174, + 177, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 27, + "sent": "The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "Na+", + "chemical" + ], + [ + 22, + 25, + "SCa", + "site" + ], + [ + 57, + 60, + "Na+", + "chemical" + ], + [ + 62, + 66, + "Ca2+", + "chemical" + ], + [ + 121, + 125, + "Smid", + "site" + ] + ] + }, + { + "sid": 28, + "sent": "Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study.", + "section": "RESULTS", + "ner": [ + [ + 41, + 46, + "water", + "chemical" + ], + [ + 70, + 73, + "Na+", + "chemical" + ], + [ + 87, + 91, + "Smid", + "site" + ], + [ + 122, + 132, + "simulation", + "experimental_method" + ] + ] + }, + { + "sid": 29, + "sent": "First, the electron density at Smid does not depend significantly on the Na+ concentration.", + "section": "RESULTS", + "ner": [ + [ + 11, + 27, + "electron density", + "evidence" + ], + [ + 31, + 35, + "Smid", + "site" + ], + [ + 73, + 76, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 30, + "sent": "Second, the protein coordination geometry at Smid is clearly suboptimal for Na+ (Supplementary Fig. 1d).", + "section": "RESULTS", + "ner": [ + [ + 45, + 49, + "Smid", + "site" + ], + [ + 76, + 79, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 31, + "sent": "The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "water", + "chemical" + ], + [ + 22, + 26, + "Smid", + "site" + ], + [ + 33, + 47, + "hydrogen-bonds", + "bond_interaction" + ], + [ + 57, + 73, + "highly conserved", + "protein_state" + ], + [ + 74, + 79, + "Glu54", + "residue_name_number" + ], + [ + 84, + 90, + "Glu213", + "residue_name_number" + ], + [ + 158, + 168, + "coordinate", + "bond_interaction" + ], + [ + 178, + 181, + "Na+", + "chemical" + ], + [ + 190, + 194, + "Sext", + "site" + ], + [ + 196, + 199, + "SCa", + "site" + ], + [ + 204, + 208, + "Sint", + "site" + ] + ] + }, + { + "sid": 32, + "sent": "It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study.", + "section": "RESULTS", + "ner": [ + [ + 45, + 50, + "Glu54", + "residue_name_number" + ], + [ + 55, + 61, + "Glu213", + "residue_name_number" + ], + [ + 81, + 87, + "Asp240", + "residue_name_number" + ], + [ + 102, + 106, + "Smid", + "site" + ], + [ + 127, + 130, + "Asn", + "residue_name" + ], + [ + 134, + 144, + "eukaryotic", + "taxonomy_domain" + ], + [ + 145, + 148, + "NCX", + "protein_type" + ], + [ + 159, + 169, + "protonated", + "protein_state" + ], + [ + 206, + 216, + "simulation", + "experimental_method" + ] + ] + }, + { + "sid": 33, + "sent": "Na+-dependent conformational change", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 34, + "sent": "The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "NCX_Mj", + "protein" + ], + [ + 11, + 21, + "structures", + "evidence" + ], + [ + 33, + 36, + "Na+", + "chemical" + ], + [ + 69, + 72, + "Na+", + "chemical" + ], + [ + 84, + 88, + "Sext", + "site" + ] + ] + }, + { + "sid": 35, + "sent": "When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a).", + "section": "RESULTS", + "ner": [ + [ + 5, + 8, + "Na+", + "chemical" + ], + [ + 18, + 22, + "Sext", + "site" + ], + [ + 26, + 30, + "high", + "protein_state" + ], + [ + 51, + 66, + "N-terminal half", + "structure_element" + ], + [ + 70, + 73, + "TM7", + "structure_element" + ], + [ + 91, + 104, + "short helices", + "structure_element" + ], + [ + 106, + 110, + "TM7a", + "structure_element" + ], + [ + 115, + 119, + "TM7b", + "structure_element" + ] + ] + }, + { + "sid": 36, + "sent": "TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d).", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "TM7b", + "structure_element" + ], + [ + 23, + 44, + "central binding sites", + "site" + ], + [ + 103, + 109, + "Ala206", + "residue_name_number" + ], + [ + 110, + 122, + "coordinating", + "bond_interaction" + ], + [ + 127, + 130, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 37, + "sent": "However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d).", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "Sext", + "site" + ], + [ + 27, + 32, + "empty", + "protein_state" + ], + [ + 36, + 39, + "low", + "protein_state" + ], + [ + 40, + 43, + "Na+", + "chemical" + ], + [ + 60, + 64, + "TM7a", + "structure_element" + ], + [ + 69, + 73, + "TM7b", + "structure_element" + ], + [ + 103, + 108, + "helix", + "structure_element" + ], + [ + 146, + 152, + "Ala206", + "residue_name_number" + ] + ] + }, + { + "sid": 38, + "sent": "TM7a also forms hydrophobic contacts with the C-terminal half of TM6.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "TM7a", + "structure_element" + ], + [ + 16, + 36, + "hydrophobic contacts", + "bond_interaction" + ], + [ + 46, + 61, + "C-terminal half", + "structure_element" + ], + [ + 65, + 68, + "TM6", + "structure_element" + ] + ] + }, + { + "sid": 39, + "sent": "These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b).", + "section": "RESULTS", + "ner": [ + [ + 33, + 42, + "structure", + "evidence" + ], + [ + 48, + 51, + "Na+", + "chemical" + ], + [ + 55, + 59, + "Sext", + "site" + ], + [ + 107, + 114, + "helices", + "structure_element" + ] + ] + }, + { + "sid": 40, + "sent": "This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation.", + "section": "RESULTS", + "ner": [ + [ + 38, + 41, + "TM6", + "structure_element" + ], + [ + 46, + 49, + "TM1", + "structure_element" + ], + [ + 138, + 149, + "transporter", + "protein_type" + ], + [ + 166, + 179, + "inward-facing", + "protein_state" + ] + ] + }, + { + "sid": 41, + "sent": "The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d).", + "section": "RESULTS", + "ner": [ + [ + 21, + 26, + "TM7ab", + "structure_element" + ], + [ + 84, + 88, + "Sext", + "site" + ], + [ + 93, + 97, + "Smid", + "site" + ], + [ + 105, + 108, + "SCa", + "site" + ], + [ + 113, + 117, + "Sint", + "site" + ], + [ + 125, + 133, + "occluded", + "protein_state" + ] + ] + }, + { + "sid": 42, + "sent": "Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively.", + "section": "RESULTS", + "ner": [ + [ + 10, + 20, + "structures", + "evidence" + ], + [ + 24, + 28, + "high", + "protein_state" + ], + [ + 33, + 36, + "low", + "protein_state" + ], + [ + 37, + 40, + "Na+", + "chemical" + ], + [ + 70, + 84, + "outward-facing", + "protein_state" + ], + [ + 85, + 93, + "occluded", + "protein_state" + ], + [ + 98, + 112, + "partially open", + "protein_state" + ] + ] + }, + { + "sid": 43, + "sent": "This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa.", + "section": "RESULTS", + "ner": [ + [ + 47, + 50, + "Na+", + "chemical" + ], + [ + 64, + 68, + "Sext", + "site" + ], + [ + 85, + 88, + "Na+", + "chemical" + ], + [ + 106, + 110, + "Sint", + "site" + ], + [ + 115, + 118, + "SCa", + "site" + ] + ] + }, + { + "sid": 44, + "sent": "Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations.", + "section": "RESULTS", + "ner": [ + [ + 4, + 41, + "crystallographic titration experiment", + "experimental_method" + ], + [ + 61, + 65, + "K1/2", + "evidence" + ], + [ + 74, + 77, + "Na+", + "chemical" + ], + [ + 145, + 149, + "Sext", + "site" + ], + [ + 153, + 171, + "partially occupied", + "protein_state" + ], + [ + 180, + 186, + "NCX_Mj", + "protein" + ], + [ + 187, + 194, + "crystal", + "evidence" + ], + [ + 220, + 228, + "occluded", + "protein_state" + ], + [ + 233, + 247, + "partially open", + "protein_state" + ] + ] + }, + { + "sid": 45, + "sent": "This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes.", + "section": "RESULTS", + "ner": [ + [ + 26, + 38, + "Na+ affinity", + "evidence" + ], + [ + 69, + 72, + "Na+", + "chemical" + ], + [ + 100, + 103, + "NCX", + "protein_type" + ], + [ + 118, + 128, + "eukaryotes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 46, + "sent": "The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX.", + "section": "RESULTS", + "ner": [ + [ + 21, + 24, + "Na+", + "chemical" + ], + [ + 105, + 116, + "transporter", + "protein_type" + ], + [ + 150, + 166, + "Hill coefficient", + "evidence" + ], + [ + 174, + 177, + "Na+", + "chemical" + ], + [ + 210, + 220, + "eukaryotic", + "taxonomy_domain" + ], + [ + 221, + 224, + "NCX", + "protein_type" + ] + ] + }, + { + "sid": 47, + "sent": "Extracellular Ca2+ and Sr2+ binding and their competition with Na+", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "Ca2+", + "chemical" + ], + [ + 23, + 27, + "Sr2+", + "chemical" + ], + [ + 63, + 66, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 48, + "sent": "To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods).", + "section": "RESULTS", + "ner": [ + [ + 17, + 21, + "Ca2+", + "chemical" + ], + [ + 31, + 37, + "NCX_Mj", + "protein" + ], + [ + 56, + 59, + "Na+", + "chemical" + ], + [ + 70, + 91, + "titrated the crystals", + "experimental_method" + ], + [ + 97, + 101, + "Sr2+", + "chemical" + ] + ] + }, + { + "sid": 49, + "sent": "Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "Sr2+", + "chemical" + ], + [ + 23, + 26, + "NCX", + "protein_type" + ], + [ + 40, + 44, + "Ca2+", + "chemical" + ], + [ + 75, + 78, + "Na+", + "chemical" + ], + [ + 94, + 120, + "electron-density intensity", + "evidence" + ] + ] + }, + { + "sid": 50, + "sent": "Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a).", + "section": "RESULTS", + "ner": [ + [ + 0, + 23, + "Protein crystals soaked", + "experimental_method" + ], + [ + 35, + 39, + "Sr2+", + "chemical" + ], + [ + 51, + 54, + "Na+", + "chemical" + ], + [ + 73, + 94, + "electron-density peak", + "evidence" + ], + [ + 103, + 106, + "SCa", + "site" + ], + [ + 139, + 143, + "Sr2+", + "chemical" + ] + ] + }, + { + "sid": 51, + "sent": "The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations.", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "Sr2+-loaded", + "protein_state" + ], + [ + 16, + 22, + "NCX_Mj", + "protein" + ], + [ + 23, + 32, + "structure", + "evidence" + ], + [ + 44, + 58, + "partially open", + "protein_state" + ], + [ + 88, + 91, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 52, + "sent": "Binding of Sr2+, however, excludes Na+ entirely.", + "section": "RESULTS", + "ner": [ + [ + 11, + 15, + "Sr2+", + "chemical" + ], + [ + 35, + 38, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 53, + "sent": "Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b).", + "section": "RESULTS", + "ner": [ + [ + 0, + 18, + "Crystal titrations", + "experimental_method" + ], + [ + 24, + 34, + "decreasing", + "experimental_method" + ], + [ + 35, + 39, + "Sr2+", + "chemical" + ], + [ + 43, + 53, + "increasing", + "experimental_method" + ], + [ + 54, + 57, + "Na+", + "chemical" + ], + [ + 76, + 80, + "Sr2+", + "chemical" + ], + [ + 94, + 108, + "outward-facing", + "protein_state" + ], + [ + 109, + 115, + "NCX_Mj", + "protein" + ], + [ + 170, + 173, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 54, + "sent": "Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state.", + "section": "RESULTS", + "ner": [ + [ + 16, + 19, + "Na+", + "chemical" + ], + [ + 30, + 34, + "Sr2+", + "chemical" + ], + [ + 36, + 39, + "Na+", + "chemical" + ], + [ + 60, + 64, + "Sr2+", + "chemical" + ], + [ + 88, + 94, + "NCX_Mj", + "protein" + ], + [ + 102, + 112, + "Na+-loaded", + "protein_state" + ], + [ + 114, + 128, + "fully occluded", + "protein_state" + ] + ] + }, + { + "sid": 55, + "sent": "Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).", + "section": "RESULTS", + "ner": [ + [ + 8, + 29, + "titration experiments", + "experimental_method" + ], + [ + 42, + 46, + "Ca2+", + "chemical" + ], + [ + 51, + 55, + "Sr2+", + "chemical" + ], + [ + 67, + 73, + "NCX_Mj", + "protein" + ], + [ + 100, + 129, + "electron density distribution", + "evidence" + ], + [ + 135, + 153, + "crystals soaked in", + "experimental_method" + ], + [ + 154, + 158, + "high", + "protein_state" + ], + [ + 159, + 163, + "Ca2+", + "chemical" + ], + [ + 168, + 171, + "low", + "protein_state" + ], + [ + 172, + 175, + "Na+", + "chemical" + ], + [ + 192, + 196, + "Ca2+", + "chemical" + ], + [ + 209, + 213, + "Smid", + "site" + ], + [ + 225, + 228, + "SCa", + "site" + ], + [ + 252, + 255, + "SCa", + "site" + ] + ] + }, + { + "sid": 56, + "sent": "Binding of Ca2+ to both sites simultaneously is highly improbable due to their close proximity, and at least one water molecule can be discerned coordinating the ion (Fig. 3b).", + "section": "RESULTS", + "ner": [ + [ + 11, + 15, + "Ca2+", + "chemical" + ], + [ + 113, + 118, + "water", + "chemical" + ], + [ + 145, + 157, + "coordinating", + "bond_interaction" + ] + ] + }, + { + "sid": 57, + "sent": "The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "partial", + "protein_state" + ], + [ + 12, + 16, + "Ca2+", + "chemical" + ], + [ + 17, + 26, + "occupancy", + "protein_state" + ], + [ + 30, + 34, + "Smid", + "site" + ], + [ + 55, + 61, + "Asp240", + "residue_name_number" + ], + [ + 107, + 117, + "coordinate", + "bond_interaction" + ], + [ + 118, + 122, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 58, + "sent": "Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport.", + "section": "RESULTS", + "ner": [ + [ + 9, + 45, + "functional and computational studies", + "experimental_method" + ], + [ + 65, + 71, + "Asp240", + "residue_name_number" + ], + [ + 80, + 90, + "protonated", + "protein_state" + ] + ] + }, + { + "sid": 59, + "sent": "Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid.", + "section": "RESULTS", + "ner": [ + [ + 16, + 19, + "NCX", + "protein_type" + ], + [ + 29, + 35, + "Asp240", + "residue_name_number" + ], + [ + 39, + 50, + "substituted", + "experimental_method" + ], + [ + 54, + 57, + "Asn", + "residue_name" + ], + [ + 97, + 101, + "Ca2+", + "chemical" + ], + [ + 113, + 117, + "Smid", + "site" + ] + ] + }, + { + "sid": 60, + "sent": "SCa is therefore the functional Ca2+ site.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "SCa", + "site" + ], + [ + 32, + 41, + "Ca2+ site", + "site" + ] + ] + }, + { + "sid": 61, + "sent": "Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c).", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "Sr2+", + "chemical" + ], + [ + 19, + 23, + "Ca2+", + "chemical" + ], + [ + 39, + 47, + "affinity", + "evidence" + ], + [ + 51, + 65, + "outward-facing", + "protein_state" + ], + [ + 66, + 72, + "NCX_Mj", + "protein" + ], + [ + 105, + 108, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 62, + "sent": "This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side.", + "section": "RESULTS", + "ner": [ + [ + 32, + 66, + "physiological and biochemical data", + "evidence" + ], + [ + 76, + 86, + "eukaryotic", + "taxonomy_domain" + ], + [ + 87, + 90, + "NCX", + "protein_type" + ], + [ + 95, + 101, + "NCX_Mj", + "protein" + ], + [ + 131, + 144, + "Ca2+ affinity", + "evidence" + ] + ] + }, + { + "sid": 63, + "sent": "Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM).", + "section": "RESULTS", + "ner": [ + [ + 18, + 50, + "crystallographic titration assay", + "experimental_method" + ], + [ + 61, + 65, + "Ca2+", + "chemical" + ], + [ + 92, + 100, + "affinity", + "evidence" + ], + [ + 147, + 162, + "Ca2+ affinities", + "evidence" + ], + [ + 196, + 199, + "NCX", + "protein_type" + ], + [ + 201, + 203, + "Km", + "evidence" + ], + [ + 219, + 225, + "NCX_Mj", + "protein" + ], + [ + 227, + 229, + "Km", + "evidence" + ] + ] + }, + { + "sid": 64, + "sent": "Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.", + "section": "RESULTS", + "ner": [ + [ + 22, + 51, + "crystal titration experiments", + "experimental_method" + ], + [ + 78, + 91, + "binding sites", + "site" + ], + [ + 95, + 109, + "outward-facing", + "protein_state" + ], + [ + 110, + 116, + "NCX_Mj", + "protein" + ], + [ + 148, + 152, + "Sint", + "site" + ], + [ + 157, + 161, + "Sext", + "site" + ], + [ + 166, + 169, + "Na+", + "chemical" + ], + [ + 187, + 190, + "SCa", + "site" + ], + [ + 222, + 226, + "Ca2+", + "chemical" + ], + [ + 251, + 254, + "Na+", + "chemical" + ], + [ + 279, + 289, + "simulation", + "experimental_method" + ], + [ + 308, + 312, + "Sr2+", + "chemical" + ], + [ + 314, + 318, + "Smid", + "site" + ], + [ + 352, + 356, + "Ca2+", + "chemical" + ], + [ + 378, + 382, + "Smid", + "site" + ], + [ + 410, + 415, + "water", + "chemical" + ] + ] + }, + { + "sid": 65, + "sent": "The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "ion-binding sites", + "site" + ], + [ + 25, + 31, + "NCX_Mj", + "protein" + ], + [ + 70, + 73, + "Na+", + "chemical" + ], + [ + 122, + 125, + "Na+", + "chemical" + ], + [ + 130, + 134, + "Ca2+", + "chemical" + ], + [ + 139, + 143, + "Sr2+", + "chemical" + ], + [ + 188, + 198, + "eukaryotic", + "taxonomy_domain" + ], + [ + 199, + 209, + "exchangers", + "protein_type" + ] + ] + }, + { + "sid": 66, + "sent": "A structure of NCX_Mj without Na+ or Ca2+ bound", + "section": "RESULTS", + "ner": [ + [ + 2, + 11, + "structure", + "evidence" + ], + [ + 15, + 21, + "NCX_Mj", + "protein" + ], + [ + 22, + 29, + "without", + "protein_state" + ], + [ + 30, + 33, + "Na+", + "chemical" + ], + [ + 37, + 41, + "Ca2+", + "chemical" + ], + [ + 42, + 47, + "bound", + "protein_state" + ] + ] + }, + { + "sid": 67, + "sent": "An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM).", + "section": "RESULTS", + "ner": [ + [ + 3, + 6, + "apo", + "protein_state" + ], + [ + 16, + 30, + "outward-facing", + "protein_state" + ], + [ + 31, + 37, + "NCX_Mj", + "protein" + ], + [ + 140, + 143, + "Na+", + "chemical" + ], + [ + 158, + 162, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 68, + "sent": "We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods).", + "section": "RESULTS", + "ner": [ + [ + 29, + 32, + "apo", + "protein_state" + ], + [ + 39, + 48, + "structure", + "evidence" + ], + [ + 52, + 58, + "NCX_Mj", + "protein" + ], + [ + 63, + 76, + "crystallizing", + "experimental_method" + ], + [ + 92, + 100, + "lower pH", + "protein_state" + ], + [ + 112, + 122, + "absence of", + "protein_state" + ], + [ + 123, + 126, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 69, + "sent": "This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences.", + "section": "RESULTS", + "ner": [ + [ + 5, + 14, + "structure", + "evidence" + ], + [ + 33, + 47, + "partially open", + "protein_state" + ], + [ + 48, + 57, + "structure", + "evidence" + ], + [ + 67, + 70, + "Na+", + "chemical" + ], + [ + 85, + 89, + "Ca2+", + "chemical" + ], + [ + 97, + 101, + "Sr2+", + "chemical" + ] + ] + }, + { + "sid": 70, + "sent": "First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites.", + "section": "RESULTS", + "ner": [ + [ + 7, + 12, + "TM7ab", + "structure_element" + ], + [ + 28, + 46, + "extracellular half", + "structure_element" + ], + [ + 54, + 57, + "TM6", + "structure_element" + ], + [ + 62, + 65, + "TM1", + "structure_element" + ], + [ + 168, + 181, + "binding sites", + "site" + ] + ] + }, + { + "sid": 71, + "sent": "Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d).", + "section": "RESULTS", + "ner": [ + [ + 8, + 13, + "Glu54", + "residue_name_number" + ], + [ + 18, + 24, + "Glu213", + "residue_name_number" + ], + [ + 58, + 71, + "binding sites", + "site" + ], + [ + 91, + 105, + "hydrogen-bonds", + "bond_interaction" + ], + [ + 132, + 148, + "ion coordination", + "bond_interaction" + ], + [ + 156, + 172, + "fully Na+-loaded", + "protein_state" + ], + [ + 173, + 182, + "structure", + "evidence" + ] + ] + }, + { + "sid": 72, + "sent": "Although the binding sites are thus fully accessible to the external solution (Fig. 3e), the lack of electron density therein indicates no ions or ordered solvent molecules.", + "section": "RESULTS", + "ner": [ + [ + 13, + 26, + "binding sites", + "site" + ], + [ + 36, + 52, + "fully accessible", + "protein_state" + ], + [ + 101, + 117, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 73, + "sent": "This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj.", + "section": "RESULTS", + "ner": [ + [ + 5, + 8, + "apo", + "protein_state" + ], + [ + 9, + 18, + "structure", + "evidence" + ], + [ + 49, + 57, + "unloaded", + "protein_state" + ], + [ + 59, + 63, + "open", + "protein_state" + ], + [ + 73, + 87, + "outward-facing", + "protein_state" + ], + [ + 88, + 94, + "NCX_Mj", + "protein" + ] + ] + }, + { + "sid": 74, + "sent": "Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind.", + "section": "RESULTS", + "ner": [ + [ + 20, + 29, + "structure", + "evidence" + ], + [ + 46, + 62, + "fully protonated", + "protein_state" + ], + [ + 76, + 87, + "transporter", + "protein_type" + ], + [ + 98, + 101, + "Na+", + "chemical" + ], + [ + 106, + 110, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 75, + "sent": "Such interpretation would be consistent with the computer simulations reported below.", + "section": "RESULTS", + "ner": [ + [ + 49, + 69, + "computer simulations", + "experimental_method" + ] + ] + }, + { + "sid": 76, + "sent": "Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle.", + "section": "RESULTS", + "ner": [ + [ + 8, + 24, + "transport assays", + "experimental_method" + ], + [ + 28, + 34, + "NCX_Mj", + "protein" + ], + [ + 63, + 74, + "presence of", + "protein_state" + ], + [ + 75, + 78, + "Na+", + "chemical" + ], + [ + 82, + 86, + "Ca2+", + "chemical" + ], + [ + 88, + 94, + "low pH", + "protein_state" + ], + [ + 95, + 106, + "inactivates", + "protein_state" + ] + ] + }, + { + "sid": 77, + "sent": "Ion occupancy determines the free-energy landscape of NCX_Mj", + "section": "RESULTS", + "ner": [ + [ + 54, + 60, + "NCX_Mj", + "protein" + ] + ] + }, + { + "sid": 78, + "sent": "That secondary-active transporters are able to harness an electrochemical gradient of one substrate to power the uphill transport of another relies on a seemingly simple principle: they must not transition between outward- and inward-open conformations unless in two precise substrate occupancy states.", + "section": "RESULTS", + "ner": [ + [ + 5, + 34, + "secondary-active transporters", + "protein_type" + ], + [ + 214, + 222, + "outward-", + "protein_state" + ], + [ + 227, + 238, + "inward-open", + "protein_state" + ] + ] + }, + { + "sid": 79, + "sent": "NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "NCX", + "protein_type" + ], + [ + 33, + 36, + "Na+", + "chemical" + ], + [ + 42, + 46, + "Ca2+", + "chemical" + ], + [ + 78, + 88, + "antiporter", + "protein_type" + ], + [ + 90, + 100, + "symporters", + "protein_type" + ], + [ + 217, + 222, + "bound", + "protein_state" + ], + [ + 234, + 237, + "apo", + "protein_state" + ] + ] + }, + { + "sid": 80, + "sent": "To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations.", + "section": "RESULTS", + "ner": [ + [ + 64, + 100, + "conformational free-energy landscape", + "evidence" + ], + [ + 104, + 110, + "NCX_Mj", + "protein" + ], + [ + 175, + 193, + "molecular dynamics", + "experimental_method" + ], + [ + 195, + 197, + "MD", + "experimental_method" + ], + [ + 199, + 210, + "simulations", + "experimental_method" + ] + ] + }, + { + "sid": 81, + "sent": "This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above.", + "section": "RESULTS", + "ner": [ + [ + 62, + 71, + "structure", + "evidence" + ], + [ + 75, + 81, + "NCX_Mj", + "protein" + ], + [ + 104, + 128, + "crystallographic studies", + "experimental_method" + ] + ] + }, + { + "sid": 82, + "sent": "As it happens, the results confirm that the structures now available are representing interconverting states of the functional cycle of NCX_Mj, while revealing how the alternating-access mechanism is controlled by the ion-occupancy state.", + "section": "RESULTS", + "ner": [ + [ + 44, + 54, + "structures", + "evidence" + ], + [ + 136, + 142, + "NCX_Mj", + "protein" + ] + ] + }, + { + "sid": 83, + "sent": "A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy.", + "section": "RESULTS", + "ner": [ + [ + 24, + 38, + "MD simulations", + "experimental_method" + ], + [ + 97, + 103, + "NCX_Mj", + "protein" + ], + [ + 104, + 113, + "structure", + "evidence" + ], + [ + 134, + 151, + "ion-binding sites", + "site" + ] + ] + }, + { + "sid": 84, + "sent": "Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b).", + "section": "RESULTS", + "ner": [ + [ + 23, + 32, + "simulated", + "experimental_method" + ], + [ + 37, + 53, + "outward-occluded", + "protein_state" + ], + [ + 131, + 139, + "high-Na+", + "protein_state" + ], + [ + 140, + 157, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 85, + "sent": "That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid.", + "section": "RESULTS", + "ner": [ + [ + 9, + 12, + "Na+", + "chemical" + ], + [ + 25, + 29, + "Sext", + "site" + ], + [ + 31, + 34, + "SCa", + "site" + ], + [ + 40, + 44, + "Sint", + "site" + ], + [ + 52, + 56, + "D240", + "residue_name_number" + ], + [ + 60, + 70, + "protonated", + "protein_state" + ], + [ + 77, + 82, + "water", + "chemical" + ], + [ + 101, + 105, + "Smid", + "site" + ] + ] + }, + { + "sid": 86, + "sent": "The Na+ ion at Sext was then relocated from the site to the bulk solution (Methods), and this system was then allowed to evolve freely in time.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "Na+", + "chemical" + ], + [ + 15, + 19, + "Sext", + "site" + ] + ] + }, + { + "sid": 87, + "sent": "The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "Na+", + "chemical" + ], + [ + 16, + 19, + "SCa", + "site" + ], + [ + 24, + 28, + "Sint", + "site" + ], + [ + 75, + 85, + "simulation", + "experimental_method" + ] + ] + }, + { + "sid": 88, + "sent": "These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures.", + "section": "RESULTS", + "ner": [ + [ + 14, + 25, + "simulations", + "experimental_method" + ], + [ + 61, + 72, + "transporter", + "protein_type" + ], + [ + 116, + 134, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 89, + "sent": "The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a).", + "section": "RESULTS", + "ner": [ + [ + 45, + 48, + "Na+", + "chemical" + ], + [ + 54, + 58, + "Sext", + "site" + ], + [ + 84, + 89, + "TM7ab", + "structure_element" + ] + ] + }, + { + "sid": 90, + "sent": "When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear \u03b1-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b).", + "section": "RESULTS", + "ner": [ + [ + 7, + 10, + "Na+", + "chemical" + ], + [ + 20, + 25, + "bound", + "protein_state" + ], + [ + 27, + 32, + "TM7ab", + "structure_element" + ], + [ + 80, + 99, + "\u03b1-helical fragments", + "structure_element" + ], + [ + 150, + 164, + "hydrogen-bonds", + "bond_interaction" + ], + [ + 173, + 177, + "F202", + "residue_name_number" + ], + [ + 182, + 186, + "A206", + "residue_name_number" + ], + [ + 192, + 196, + "T203", + "residue_name_number" + ], + [ + 201, + 205, + "F207", + "residue_name_number" + ] + ] + }, + { + "sid": 91, + "sent": "This distortion occludes Sext from the exterior (Fig. 4d, 4h-i) and appears to be induced by the Na+ ion itself, which pulls the carbonyl group of A206 into its coordination sphere (Fig. 4g).", + "section": "RESULTS", + "ner": [ + [ + 25, + 29, + "Sext", + "site" + ], + [ + 97, + 100, + "Na+", + "chemical" + ], + [ + 147, + 151, + "A206", + "residue_name_number" + ] + ] + }, + { + "sid": 92, + "sent": "With Sext empty, however, TM7ab forms a canonical \u03b1-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded.", + "section": "RESULTS", + "ner": [ + [ + 5, + 9, + "Sext", + "site" + ], + [ + 10, + 15, + "empty", + "protein_state" + ], + [ + 26, + 31, + "TM7ab", + "structure_element" + ], + [ + 50, + 57, + "\u03b1-helix", + "structure_element" + ], + [ + 111, + 114, + "TM3", + "structure_element" + ], + [ + 119, + 122, + "TM7", + "structure_element" + ], + [ + 145, + 150, + "water", + "chemical" + ], + [ + 202, + 206, + "Sext", + "site" + ], + [ + 233, + 244, + "transporter", + "protein_type" + ], + [ + 248, + 266, + "no longer occluded", + "protein_state" + ] + ] + }, + { + "sid": 93, + "sent": "Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c).", + "section": "RESULTS", + "ner": [ + [ + 16, + 19, + "Na+", + "chemical" + ], + [ + 25, + 28, + "SCa", + "site" + ], + [ + 33, + 37, + "Sint", + "site" + ] + ] + }, + { + "sid": 94, + "sent": "The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e).", + "section": "RESULTS", + "ner": [ + [ + 55, + 58, + "TM7", + "structure_element" + ], + [ + 63, + 66, + "TM2", + "structure_element" + ], + [ + 151, + 154, + "Na+", + "chemical" + ], + [ + 162, + 165, + "SCa", + "site" + ] + ] + }, + { + "sid": 95, + "sent": "TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "TM1", + "structure_element" + ], + [ + 8, + 11, + "TM6", + "structure_element" + ], + [ + 76, + 92, + "outward-occluded", + "protein_state" + ] + ] + }, + { + "sid": 96, + "sent": "Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i).", + "section": "RESULTS", + "ner": [ + [ + 38, + 53, + "aqueous channel", + "site" + ], + [ + 76, + 79, + "SCa", + "site" + ], + [ + 84, + 88, + "Sint", + "site" + ] + ] + }, + { + "sid": 97, + "sent": "The transporter thus becomes fully outward-open.", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "transporter", + "protein_type" + ], + [ + 29, + 47, + "fully outward-open", + "protein_state" + ] + ] + }, + { + "sid": 98, + "sent": "To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods).", + "section": "RESULTS", + "ner": [ + [ + 111, + 120, + "exchanger", + "protein_type" + ], + [ + 167, + 182, + "MD calculations", + "experimental_method" + ], + [ + 242, + 258, + "outward-occluded", + "protein_state" + ], + [ + 263, + 281, + "fully outward-open", + "protein_state" + ], + [ + 312, + 333, + "free-energy landscape", + "evidence" + ] + ] + }, + { + "sid": 99, + "sent": "As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+.", + "section": "RESULTS", + "ner": [ + [ + 68, + 71, + "Na+", + "chemical" + ], + [ + 75, + 79, + "Sext", + "site" + ], + [ + 81, + 84, + "SCa", + "site" + ], + [ + 89, + 93, + "Sint", + "site" + ], + [ + 100, + 103, + "Na+", + "chemical" + ], + [ + 112, + 115, + "SCa", + "site" + ], + [ + 120, + 124, + "Sint", + "site" + ], + [ + 130, + 137, + "without", + "protein_state" + ], + [ + 138, + 141, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 100, + "sent": "These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape.", + "section": "RESULTS", + "ner": [ + [ + 6, + 18, + "calculations", + "experimental_method" + ], + [ + 40, + 43, + "Na+", + "chemical" + ], + [ + 67, + 78, + "transporter", + "protein_type" + ], + [ + 108, + 144, + "conformational free-energy landscape", + "evidence" + ] + ] + }, + { + "sid": 101, + "sent": "When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b).", + "section": "RESULTS", + "ner": [ + [ + 9, + 18, + "Na+ sites", + "site" + ], + [ + 44, + 63, + "free-energy minimum", + "evidence" + ], + [ + 168, + 173, + "TM7ab", + "structure_element" + ], + [ + 205, + 208, + "TM2", + "structure_element" + ], + [ + 213, + 216, + "TM3", + "structure_element" + ], + [ + 229, + 242, + "binding sites", + "site" + ] + ] + }, + { + "sid": 102, + "sent": "At a small energetic cost, however, the transporter can adopt a metastable \u2018half-open\u2019 conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b).", + "section": "RESULTS", + "ner": [ + [ + 40, + 51, + "transporter", + "protein_type" + ], + [ + 64, + 74, + "metastable", + "protein_state" + ], + [ + 76, + 85, + "half-open", + "protein_state" + ], + [ + 109, + 114, + "TM7ab", + "structure_element" + ], + [ + 142, + 146, + "Sext", + "site" + ], + [ + 150, + 154, + "open", + "protein_state" + ] + ] + }, + { + "sid": 103, + "sent": "The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O\u2013A206:N hydrogen-bond (Fig. 5c).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "Na+", + "chemical" + ], + [ + 15, + 19, + "Sext", + "site" + ], + [ + 28, + 45, + "fully coordinated", + "protein_state" + ], + [ + 62, + 67, + "water", + "chemical" + ], + [ + 111, + 115, + "A206", + "residue_name_number" + ], + [ + 147, + 151, + "F202", + "residue_name_number" + ], + [ + 154, + 158, + "A206", + "residue_name_number" + ], + [ + 161, + 174, + "hydrogen-bond", + "bond_interaction" + ] + ] + }, + { + "sid": 104, + "sent": "This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 \u00d7 Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation.", + "section": "RESULTS", + "ner": [ + [ + 5, + 14, + "semi-open", + "protein_state" + ], + [ + 91, + 94, + "Na+", + "chemical" + ], + [ + 109, + 112, + "SCa", + "site" + ], + [ + 117, + 121, + "Sint", + "site" + ], + [ + 127, + 130, + "Na+", + "chemical" + ], + [ + 185, + 188, + "Na+", + "chemical" + ], + [ + 192, + 196, + "Sext", + "site" + ], + [ + 212, + 222, + "metastable", + "protein_state" + ] + ] + }, + { + "sid": 105, + "sent": "Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c).", + "section": "RESULTS", + "ner": [ + [ + 92, + 107, + "aqueous channel", + "site" + ], + [ + 141, + 144, + "SCa", + "site" + ], + [ + 153, + 156, + "TM7", + "structure_element" + ], + [ + 161, + 164, + "TM2", + "structure_element" + ], + [ + 178, + 192, + "gating helices", + "structure_element" + ], + [ + 193, + 196, + "TM1", + "structure_element" + ], + [ + 201, + 204, + "TM6", + "structure_element" + ], + [ + 219, + 223, + "open", + "protein_state" + ] + ] + }, + { + "sid": 106, + "sent": "Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a).", + "section": "RESULTS", + "ner": [ + [ + 23, + 44, + "free-energy landscape", + "evidence" + ], + [ + 54, + 72, + "partially occupied", + "protein_state" + ], + [ + 101, + 109, + "occluded", + "protein_state" + ] + ] + }, + { + "sid": 107, + "sent": "Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c).", + "section": "RESULTS", + "ner": [ + [ + 34, + 37, + "Na+", + "chemical" + ], + [ + 48, + 51, + "SCa", + "site" + ], + [ + 56, + 60, + "Sint", + "site" + ], + [ + 82, + 103, + "free-energy landscape", + "evidence" + ], + [ + 111, + 122, + "transporter", + "protein_type" + ], + [ + 170, + 180, + "fully open", + "protein_state" + ], + [ + 205, + 221, + "aqueous channels", + "site" + ] + ] + }, + { + "sid": 108, + "sent": "The transition to the occluded state in this apo state is again energetically unfeasible.", + "section": "RESULTS", + "ner": [ + [ + 22, + 30, + "occluded", + "protein_state" + ], + [ + 45, + 48, + "apo", + "protein_state" + ] + ] + }, + { + "sid": 109, + "sent": "From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete.", + "section": "RESULTS", + "ner": [ + [ + 67, + 71, + "open", + "protein_state" + ], + [ + 76, + 85, + "semi-open", + "protein_state" + ], + [ + 132, + 135, + "Na+", + "chemical" + ], + [ + 175, + 178, + "Na+", + "chemical" + ], + [ + 287, + 308, + "free-energy landscape", + "evidence" + ], + [ + 316, + 327, + "transporter", + "protein_type" + ], + [ + 346, + 354, + "occluded", + "protein_state" + ], + [ + 392, + 417, + "Na+ occupancy is complete", + "protein_state" + ] + ] + }, + { + "sid": 110, + "sent": "This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state.", + "section": "RESULTS", + "ner": [ + [ + 41, + 44, + "Na+", + "chemical" + ], + [ + 93, + 103, + "Ca2+-bound", + "protein_state" + ], + [ + 144, + 155, + "transporter", + "protein_type" + ], + [ + 223, + 235, + "outward-open", + "protein_state" + ], + [ + 276, + 280, + "Ca2+", + "chemical" + ], + [ + 295, + 303, + "occluded", + "protein_state" + ], + [ + 331, + 340, + "semi-open", + "protein_state" + ], + [ + 385, + 396, + "inward-open", + "protein_state" + ] + ] + }, + { + "sid": 111, + "sent": "By contrast, occupancy by H+, which as mentioned are not transported, might be compatible with a semi-open state as well as with the fully open conformation, but should not be conducive to occlusion.", + "section": "RESULTS", + "ner": [ + [ + 26, + 28, + "H+", + "chemical" + ], + [ + 97, + 106, + "semi-open", + "protein_state" + ], + [ + 133, + 143, + "fully open", + "protein_state" + ] + ] + }, + { + "sid": 112, + "sent": "To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted).", + "section": "RESULTS", + "ner": [ + [ + 42, + 71, + "enhanced-sampling simulations", + "experimental_method" + ], + [ + 80, + 84, + "Ca2+", + "protein_state" + ], + [ + 89, + 97, + "H+-bound", + "protein_state" + ], + [ + 108, + 122, + "outward-facing", + "protein_state" + ], + [ + 123, + 129, + "NCX_Mj", + "protein" + ], + [ + 169, + 172, + "Na+", + "chemical" + ], + [ + 249, + 259, + "structures", + "evidence" + ], + [ + 267, + 277, + "Ca2+-bound", + "protein_state" + ] + ] + }, + { + "sid": 113, + "sent": "The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 \u00d7 Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "calculated", + "experimental_method" + ], + [ + 15, + 36, + "free-energy landscape", + "evidence" + ], + [ + 41, + 51, + "Ca2+-bound", + "protein_state" + ], + [ + 52, + 58, + "NCX_Mj", + "protein" + ], + [ + 103, + 107, + "Ca2+", + "chemical" + ], + [ + 149, + 155, + "NCX_Mj", + "protein" + ], + [ + 176, + 180, + "Ca2+", + "chemical" + ], + [ + 186, + 194, + "occluded", + "protein_state" + ], + [ + 196, + 206, + "dehydrated", + "protein_state" + ], + [ + 274, + 283, + "exchanger", + "protein_type" + ], + [ + 303, + 312, + "semi-open", + "protein_state" + ], + [ + 317, + 321, + "open", + "protein_state" + ], + [ + 356, + 360, + "Ca2+", + "chemical" + ], + [ + 373, + 376, + "Na+", + "chemical" + ], + [ + 402, + 425, + "aqueous access channels", + "site" + ], + [ + 439, + 443, + "Sext", + "site" + ], + [ + 448, + 451, + "SCa", + "site" + ] + ] + }, + { + "sid": 114, + "sent": "By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 \u00d7 H+, Fig. 6a-c).", + "section": "RESULTS", + "ner": [ + [ + 13, + 24, + "protonation", + "protein_state" + ], + [ + 28, + 33, + "Glu54", + "residue_name_number" + ], + [ + 38, + 44, + "Glu213", + "residue_name_number" + ], + [ + 55, + 63, + "occluded", + "protein_state" + ], + [ + 133, + 139, + "NCX_Mj", + "protein" + ], + [ + 159, + 166, + "protons", + "chemical" + ], + [ + 176, + 184, + "H+-bound", + "protein_state" + ], + [ + 204, + 213, + "exchanger", + "protein_type" + ], + [ + 228, + 237, + "semi-open", + "protein_state" + ], + [ + 267, + 273, + "low pH", + "protein_state" + ], + [ + 275, + 278, + "apo", + "protein_state" + ], + [ + 279, + 296, + "crystal structure", + "evidence" + ], + [ + 302, + 304, + "H+", + "chemical" + ] + ] + }, + { + "sid": 115, + "sent": "Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible.", + "section": "RESULTS", + "ner": [ + [ + 21, + 54, + "systematic computational analysis", + "experimental_method" + ], + [ + 58, + 72, + "outward-facing", + "protein_state" + ], + [ + 73, + 79, + "NCX_Mj", + "protein" + ], + [ + 168, + 186, + "Na+/Ca2+ exchanger", + "protein_type" + ], + [ + 253, + 274, + "free-energy landscape", + "evidence" + ], + [ + 335, + 343, + "occluded", + "protein_state" + ] + ] + }, + { + "sid": 116, + "sent": "This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites.", + "section": "RESULTS", + "ner": [ + [ + 5, + 13, + "occluded", + "protein_state" + ], + [ + 74, + 81, + "outward", + "protein_state" + ], + [ + 86, + 97, + "inward-open", + "protein_state" + ], + [ + 137, + 148, + "dehydration", + "protein_state" + ], + [ + 189, + 207, + "complete occupancy", + "protein_state" + ], + [ + 215, + 228, + "binding sites", + "site" + ] + ] + }, + { + "sid": 117, + "sent": "The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized.", + "section": "DISCUSS", + "ner": [ + [ + 78, + 85, + "outward", + "protein_state" + ], + [ + 91, + 102, + "inward-open", + "protein_state" + ], + [ + 128, + 144, + "secondary-active", + "protein_state" + ], + [ + 145, + 156, + "transporter", + "protein_type" + ] + ] + }, + { + "sid": 118, + "sent": "It is however also non-trivial: antiporters, for example, do not undergo the alternating-access transition without a cargo, but this is precisely how membrane symporters reset their transport cycles.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 43, + "antiporters", + "protein_type" + ], + [ + 150, + 169, + "membrane symporters", + "protein_type" + ] + ] + }, + { + "sid": 119, + "sent": "Similarly puzzling is that a given antiporter will undergo this transition upon recognition of substrates of different charge, size and number.", + "section": "DISCUSS", + "ner": [ + [ + 35, + 45, + "antiporter", + "protein_type" + ] + ] + }, + { + "sid": 120, + "sent": "Yet, when multiple species are to be co-translocated, by either an antiporter or a symporter, partial occupancies must not be conducive to the alternating-access switch.", + "section": "DISCUSS", + "ner": [ + [ + 67, + 77, + "antiporter", + "protein_type" + ], + [ + 83, + 92, + "symporter", + "protein_type" + ], + [ + 143, + 168, + "alternating-access switch", + "site" + ] + ] + }, + { + "sid": 121, + "sent": "Here, we have provided novel insights into this intriguing mechanism of conformational control through structural studies and quantitative molecular simulations of a Na+/Ca2+ exchanger.", + "section": "DISCUSS", + "ner": [ + [ + 103, + 121, + "structural studies", + "experimental_method" + ], + [ + 126, + 160, + "quantitative molecular simulations", + "experimental_method" + ], + [ + 166, + 184, + "Na+/Ca2+ exchanger", + "protein_type" + ] + ] + }, + { + "sid": 122, + "sent": "Specifically, our studies of NCX_Mj reveal the mechanism of forward ion exchange (Fig. 7).", + "section": "DISCUSS", + "ner": [ + [ + 29, + 35, + "NCX_Mj", + "protein" + ] + ] + }, + { + "sid": 123, + "sent": "The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 39, + "outward-facing", + "protein_state" + ], + [ + 40, + 46, + "NCX_Mj", + "protein" + ], + [ + 55, + 68, + "inward-facing", + "protein_state" + ], + [ + 69, + 87, + "crystal structures", + "evidence" + ], + [ + 99, + 117, + "Ca2+/H+ exchangers", + "protein_type" + ], + [ + 168, + 171, + "NCX", + "protein_type" + ], + [ + 209, + 212, + "TM1", + "structure_element" + ], + [ + 217, + 220, + "TM6", + "structure_element" + ], + [ + 249, + 260, + "transporter", + "protein_type" + ] + ] + }, + { + "sid": 124, + "sent": "Here, we demonstrate that conformational changes in the extracellular region of the TM2-TM3 and TM7-TM8 bundle precede and are necessary for the transition, and are associated with ion recognition and/or release.", + "section": "DISCUSS", + "ner": [ + [ + 56, + 76, + "extracellular region", + "structure_element" + ], + [ + 84, + 91, + "TM2-TM3", + "structure_element" + ], + [ + 96, + 110, + "TM7-TM8 bundle", + "structure_element" + ] + ] + }, + { + "sid": 125, + "sent": "The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 63, + "N-terminal half", + "structure_element" + ], + [ + 67, + 70, + "TM7", + "structure_element" + ], + [ + 72, + 77, + "TM7ab", + "structure_element" + ], + [ + 123, + 126, + "TM2", + "structure_element" + ], + [ + 131, + 134, + "TM3", + "structure_element" + ], + [ + 151, + 156, + "TM7ab", + "structure_element" + ], + [ + 213, + 229, + "aqueous channels", + "site" + ], + [ + 247, + 264, + "ion-binding sites", + "site" + ] + ] + }, + { + "sid": 126, + "sent": "Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation.", + "section": "DISCUSS", + "ner": [ + [ + 30, + 33, + "TM7", + "structure_element" + ], + [ + 71, + 88, + "ion-binding sites", + "site" + ], + [ + 123, + 126, + "TM6", + "structure_element" + ], + [ + 145, + 148, + "TM6", + "structure_element" + ], + [ + 153, + 156, + "TM1", + "structure_element" + ], + [ + 180, + 193, + "inward-facing", + "protein_state" + ] + ] + }, + { + "sid": 127, + "sent": "The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 22, + "crystal structures", + "evidence" + ], + [ + 26, + 32, + "NCX_Mj", + "protein" + ], + [ + 60, + 64, + "Na+,", + "chemical" + ], + [ + 65, + 70, + "Ca2+,", + "chemical" + ], + [ + 71, + 75, + "Sr2+", + "chemical" + ], + [ + 79, + 81, + "H+", + "chemical" + ], + [ + 82, + 87, + "bound", + "protein_state" + ], + [ + 101, + 110, + "exchanger", + "protein_type" + ] + ] + }, + { + "sid": 128, + "sent": "These states can only represent a subset among all possible, but they ought to reflect inherent preferences of the transporter, modulated by the experimental conditions.", + "section": "DISCUSS", + "ner": [ + [ + 115, + 126, + "transporter", + "protein_type" + ] + ] + }, + { + "sid": 129, + "sent": "For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).", + "section": "DISCUSS", + "ner": [ + [ + 20, + 27, + "crystal", + "evidence" + ], + [ + 31, + 37, + "NCX_Mj", + "protein" + ], + [ + 41, + 44, + "LCP", + "experimental_method" + ], + [ + 50, + 68, + "extracellular half", + "structure_element" + ], + [ + 76, + 90, + "gating helices", + "structure_element" + ], + [ + 92, + 95, + "TM6", + "structure_element" + ], + [ + 100, + 103, + "TM1", + "structure_element" + ], + [ + 190, + 207, + "ion-binding sites", + "site" + ], + [ + 235, + 238, + "apo", + "protein_state" + ], + [ + 240, + 246, + "low pH", + "protein_state" + ], + [ + 247, + 256, + "structure", + "evidence" + ] + ] + }, + { + "sid": 130, + "sent": "Nonetheless, the calculated free-energy landscapes, derived without knowledge of the experimental data, reassuringly confirm that the crystallized structures correspond to mechanistically relevant, interconverting states.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 50, + "calculated free-energy landscapes", + "evidence" + ], + [ + 134, + 157, + "crystallized structures", + "evidence" + ] + ] + }, + { + "sid": 131, + "sent": "The simulations also demonstrate how this landscape is drastically re-shaped upon each ion-binding event.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 15, + "simulations", + "experimental_method" + ] + ] + }, + { + "sid": 132, + "sent": "Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry.", + "section": "DISCUSS", + "ner": [ + [ + 58, + 66, + "occluded", + "protein_state" + ], + [ + 130, + 136, + "NCX_Mj", + "protein" + ], + [ + 146, + 149, + "Na+", + "chemical" + ], + [ + 151, + 155, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 133, + "sent": "We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms.", + "section": "DISCUSS", + "ner": [ + [ + 89, + 101, + "transporters", + "protein_type" + ], + [ + 140, + 150, + "symporters", + "protein_type" + ], + [ + 155, + 166, + "antiporters", + "protein_type" + ], + [ + 197, + 205, + "occluded", + "protein_state" + ], + [ + 228, + 272, + "protein conformational free-energy landscape", + "evidence" + ] + ] + }, + { + "sid": 134, + "sent": "In multiple ways, our findings provide an explanation for, existing functional, biochemical and biophysical data for both NCX_Mj and its eukaryotic homologues.", + "section": "DISCUSS", + "ner": [ + [ + 122, + 128, + "NCX_Mj", + "protein" + ], + [ + 137, + 147, + "eukaryotic", + "taxonomy_domain" + ] + ] + }, + { + "sid": 135, + "sent": "The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 70, + "ion-binding affinities", + "evidence" + ], + [ + 89, + 116, + "crystallographic titrations", + "experimental_method" + ], + [ + 125, + 127, + "Km", + "evidence" + ], + [ + 132, + 143, + "K1/2 values", + "evidence" + ], + [ + 168, + 185, + "functional assays", + "experimental_method" + ] + ] + }, + { + "sid": 136, + "sent": "Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d).", + "section": "DISCUSS", + "ner": [ + [ + 113, + 119, + "NCX_Mj", + "protein" + ], + [ + 132, + 135, + "NCX", + "protein_type" + ], + [ + 193, + 199, + "NCX_Mj", + "protein" + ], + [ + 200, + 210, + "structures", + "evidence" + ] + ] + }, + { + "sid": 137, + "sent": "The crystallographic data also provides the long-sought structural basis for the \u2018two-site\u2019 model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 25, + "crystallographic data", + "evidence" + ], + [ + 149, + 159, + "eukaryotic", + "taxonomy_domain" + ], + [ + 160, + 163, + "NCX", + "protein_type" + ], + [ + 212, + 218, + "NCX_Mj", + "protein" + ], + [ + 237, + 255, + "Na+/Ca2+ exchanger", + "protein_type" + ] + ] + }, + { + "sid": 138, + "sent": "Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 36, + "crystal titrations", + "experimental_method" + ], + [ + 66, + 69, + "Na+", + "chemical" + ], + [ + 70, + 74, + "Ca2+", + "chemical" + ], + [ + 91, + 95, + "Sint", + "site" + ], + [ + 100, + 103, + "SCa", + "site" + ], + [ + 111, + 115, + "Ca2+", + "chemical" + ], + [ + 120, + 123, + "Na+", + "chemical" + ], + [ + 162, + 165, + "Na+", + "chemical" + ], + [ + 211, + 214, + "Na+", + "chemical" + ], + [ + 239, + 242, + "Na+", + "chemical" + ], + [ + 261, + 266, + "water", + "chemical" + ], + [ + 280, + 284, + "Smid", + "site" + ], + [ + 313, + 317, + "Ca2+", + "chemical" + ], + [ + 334, + 350, + "Hill coefficient", + "evidence" + ], + [ + 361, + 364, + "Na+", + "chemical" + ], + [ + 389, + 393, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 139, + "sent": "The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 8, + "Sext", + "site" + ], + [ + 51, + 66, + "activation site", + "site" + ], + [ + 78, + 81, + "Na+", + "chemical" + ], + [ + 127, + 130, + "Na+", + "chemical" + ], + [ + 149, + 152, + "Na+", + "chemical" + ], + [ + 199, + 207, + "occluded", + "protein_state" + ] + ] + }, + { + "sid": 140, + "sent": "Interestingly, binding of Ca2+ to Smid appears to be also possible, but available evidence indicates that this event transiently blocks the exchange cycle.", + "section": "DISCUSS", + "ner": [ + [ + 26, + 30, + "Ca2+", + "chemical" + ], + [ + 34, + 38, + "Smid", + "site" + ] + ] + }, + { + "sid": 141, + "sent": "Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium.", + "section": "DISCUSS", + "ner": [ + [ + 8, + 18, + "structures", + "evidence" + ], + [ + 22, + 28, + "NCX_Mj", + "protein" + ], + [ + 29, + 37, + "bound to", + "protein_state" + ], + [ + 38, + 42, + "Cd2+", + "chemical" + ], + [ + 46, + 50, + "Mn2+", + "chemical" + ], + [ + 104, + 108, + "Smid", + "site" + ], + [ + 123, + 127, + "Sr2+", + "chemical" + ], + [ + 142, + 145, + "SCa", + "site" + ], + [ + 182, + 185, + "NCX", + "protein_type" + ], + [ + 199, + 206, + "calcium", + "chemical" + ] + ] + }, + { + "sid": 142, + "sent": "Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations.", + "section": "DISCUSS", + "ner": [ + [ + 37, + 43, + "NCX_Mj", + "protein" + ], + [ + 72, + 76, + "Ca2+", + "chemical" + ], + [ + 80, + 83, + "Na+", + "chemical" + ], + [ + 147, + 174, + "hydrogen-deuterium exchange", + "experimental_method" + ], + [ + 176, + 179, + "HDX", + "experimental_method" + ], + [ + 184, + 190, + "NCX_Mj", + "protein" + ], + [ + 199, + 207, + "presence", + "protein_state" + ], + [ + 211, + 221, + "absence of", + "protein_state" + ], + [ + 259, + 273, + "outward-facing", + "protein_state" + ] + ] + }, + { + "sid": 143, + "sent": "Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the \u03b1-repeat helices.", + "section": "DISCUSS", + "ner": [ + [ + 36, + 40, + "Ca2+", + "chemical" + ], + [ + 44, + 47, + "Na+", + "chemical" + ], + [ + 89, + 97, + "HDX rate", + "evidence" + ], + [ + 132, + 148, + "\u03b1-repeat helices", + "structure_element" + ] + ] + }, + { + "sid": 144, + "sent": "We interpret these observations as reflecting that the solvent accessibility of the protein interior is diminished upon ion recognition, consistent with our finding that opening and closing of extracellular aqueous pathways to the ion-binding sites depend on ion occupancy state.", + "section": "DISCUSS", + "ner": [ + [ + 231, + 248, + "ion-binding sites", + "site" + ] + ] + }, + { + "sid": 145, + "sent": "In addition, the increased compactness of the protein tertiary structure in the occluded state would also slow down the dynamics of the secondary-structure elements, and thus further reduce the HDX rate.", + "section": "DISCUSS", + "ner": [ + [ + 80, + 88, + "occluded", + "protein_state" + ], + [ + 194, + 202, + "HDX rate", + "evidence" + ] + ] + }, + { + "sid": 146, + "sent": "Our data would also explain the observation that the reduction in the HDX rate is comparable for Na+ and Ca2+, as well as the finding that the degree of deuterium incorporation remains non-negligible even under saturating ion concentrations.", + "section": "DISCUSS", + "ner": [ + [ + 70, + 78, + "HDX rate", + "evidence" + ], + [ + 97, + 100, + "Na+", + "chemical" + ], + [ + 105, + 109, + "Ca2+", + "chemical" + ] + ] + }, + { + "sid": 147, + "sent": "As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 40, + "calculated free-energy landscapes", + "evidence" + ], + [ + 47, + 50, + "Na+", + "chemical" + ], + [ + 55, + 59, + "Ca2+", + "chemical" + ], + [ + 88, + 99, + "transporter", + "protein_type" + ], + [ + 136, + 145, + "ion-bound", + "protein_state" + ], + [ + 219, + 229, + "fully open", + "protein_state" + ] + ] + }, + { + "sid": 148, + "sent": "Na+ binding to outward-facing NCX_Mj.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "Na+", + "chemical" + ], + [ + 15, + 29, + "outward-facing", + "protein_state" + ], + [ + 30, + 36, + "NCX_Mj", + "protein" + ] + ] + }, + { + "sid": 149, + "sent": "(a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+.", + "section": "FIG", + "ner": [ + [ + 12, + 21, + "structure", + "evidence" + ], + [ + 25, + 31, + "native", + "protein_state" + ], + [ + 32, + 46, + "outward-facing", + "protein_state" + ], + [ + 47, + 53, + "NCX_Mj", + "protein" + ], + [ + 59, + 73, + "crystals grown", + "experimental_method" + ], + [ + 84, + 87, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 150, + "sent": "Colored spheres represent the bound Na+ (green) and water (red).", + "section": "FIG", + "ner": [ + [ + 36, + 39, + "Na+", + "chemical" + ], + [ + 52, + 57, + "water", + "chemical" + ] + ] + }, + { + "sid": 151, + "sent": "(b) Structural details and definition of the four central binding sites.", + "section": "FIG", + "ner": [ + [ + 50, + 71, + "central binding sites", + "site" + ] + ] + }, + { + "sid": 152, + "sent": "The electron density (grey mesh, 1.9 \u00c5 Fo-Fc ion omit map contoured at 4\u03c3) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres).", + "section": "FIG", + "ner": [ + [ + 4, + 20, + "electron density", + "evidence" + ], + [ + 39, + 57, + "Fo-Fc ion omit map", + "evidence" + ], + [ + 78, + 82, + "Smid", + "site" + ], + [ + 98, + 103, + "water", + "chemical" + ], + [ + 130, + 134, + "Sext", + "site" + ], + [ + 136, + 139, + "SCa", + "site" + ], + [ + 144, + 148, + "Sint", + "site" + ], + [ + 152, + 155, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 153, + "sent": "Further details are shown in Supplementary Fig. 1. (c) Concentration-dependent change in Na+ occupancy (see also Table 1).", + "section": "FIG", + "ner": [ + [ + 89, + 92, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 154, + "sent": "All Fo \u2013 Fc ion-omit maps are calculated to 2.4 \u00c5 and contoured at 3\u03c3 for comparison.", + "section": "FIG", + "ner": [ + [ + 4, + 25, + "Fo \u2013 Fc ion-omit maps", + "evidence" + ] + ] + }, + { + "sid": 155, + "sent": "The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively).", + "section": "FIG", + "ner": [ + [ + 20, + 24, + "A206", + "residue_name_number" + ], + [ + 39, + 42, + "Na+", + "chemical" + ], + [ + 85, + 99, + "partially open", + "protein_state" + ], + [ + 107, + 115, + "occluded", + "protein_state" + ], + [ + 148, + 151, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 156, + "sent": "At 20 mM Na+, both conformations co-exist.", + "section": "FIG", + "ner": [ + [ + 9, + 12, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 157, + "sent": "No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites.", + "section": "FIG", + "ner": [ + [ + 75, + 80, + "water", + "chemical" + ], + [ + 101, + 104, + "SCa", + "site" + ], + [ + 106, + 110, + "Sint", + "site" + ], + [ + 115, + 119, + "Smid", + "site" + ] + ] + }, + { + "sid": 158, + "sent": "Na+-occupancy dependent conformational change in NCX_Mj.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "Na+", + "chemical" + ], + [ + 49, + 55, + "NCX_Mj", + "protein" + ] + ] + }, + { + "sid": 159, + "sent": "(a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations.", + "section": "FIG", + "ner": [ + [ + 4, + 19, + "Superimposition", + "experimental_method" + ], + [ + 27, + 33, + "NCX_Mj", + "protein" + ], + [ + 34, + 52, + "crystal structures", + "evidence" + ], + [ + 128, + 131, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 160, + "sent": "(b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively).", + "section": "FIG", + "ner": [ + [ + 25, + 34, + "interface", + "site" + ], + [ + 43, + 46, + "TM6", + "structure_element" + ], + [ + 51, + 56, + "TM7ab", + "structure_element" + ], + [ + 64, + 70, + "NCX_Mj", + "protein" + ], + [ + 71, + 81, + "structures", + "evidence" + ], + [ + 107, + 110, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 161, + "sent": "Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail.", + "section": "FIG", + "ner": [ + [ + 47, + 56, + "structure", + "evidence" + ], + [ + 60, + 63, + "low", + "protein_state" + ], + [ + 64, + 67, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 162, + "sent": "(c) Close-up view of the Na+-binding sites.", + "section": "FIG", + "ner": [ + [ + 25, + 42, + "Na+-binding sites", + "site" + ] + ] + }, + { + "sid": 163, + "sent": "The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere.", + "section": "FIG", + "ner": [ + [ + 11, + 15, + "Sext", + "site" + ], + [ + 28, + 37, + "structure", + "evidence" + ], + [ + 41, + 44, + "low", + "protein_state" + ], + [ + 45, + 48, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 164, + "sent": "Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen.", + "section": "FIG", + "ner": [ + [ + 56, + 60, + "A206", + "residue_name_number" + ], + [ + 78, + 89, + "coordinates", + "bond_interaction" + ], + [ + 90, + 93, + "Na+", + "chemical" + ], + [ + 97, + 101, + "Sext", + "site" + ] + ] + }, + { + "sid": 165, + "sent": "(d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+].", + "section": "FIG", + "ner": [ + [ + 47, + 64, + "ion binding sites", + "site" + ], + [ + 72, + 82, + "structures", + "evidence" + ], + [ + 86, + 90, + "high", + "protein_state" + ], + [ + 95, + 98, + "low", + "protein_state" + ], + [ + 100, + 103, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 166, + "sent": "Putative solvent channels are represented as light-purple surfaces.", + "section": "FIG", + "ner": [ + [ + 9, + 25, + "solvent channels", + "site" + ] + ] + }, + { + "sid": 167, + "sent": "Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).", + "section": "FIG", + "ner": [ + [ + 28, + 31, + "apo", + "protein_state" + ], + [ + 32, + 41, + "structure", + "evidence" + ], + [ + 45, + 51, + "NCX_Mj", + "protein" + ], + [ + 66, + 70, + "Sr2+", + "chemical" + ], + [ + 99, + 102, + "SCa", + "site" + ], + [ + 106, + 123, + "crystals titrated", + "experimental_method" + ], + [ + 135, + 139, + "Sr2+", + "chemical" + ], + [ + 151, + 154, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 168, + "sent": "Residues involved in Sr2+ coordination are labeled.", + "section": "FIG", + "ner": [ + [ + 21, + 25, + "Sr2+", + "chemical" + ] + ] + }, + { + "sid": 169, + "sent": "There are no significant changes in the side-chains involved in ion coordination, relative to the Na+-bound state.", + "section": "FIG", + "ner": [ + [ + 98, + 107, + "Na+-bound", + "protein_state" + ] + ] + }, + { + "sid": 170, + "sent": "T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "T50", + "residue_name_number" + ], + [ + 8, + 12, + "T209", + "residue_name_number" + ], + [ + 30, + 40, + "coordinate", + "bond_interaction" + ], + [ + 41, + 45, + "Sr2+", + "chemical" + ] + ] + }, + { + "sid": 171, + "sent": "High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel).", + "section": "FIG", + "ner": [ + [ + 5, + 8, + "Na+", + "chemical" + ], + [ + 53, + 57, + "Sr2+", + "chemical" + ], + [ + 70, + 76, + "NCX_Mj", + "protein" + ], + [ + 84, + 92, + "occluded", + "protein_state" + ] + ] + }, + { + "sid": 172, + "sent": "The contour level of the Fo \u2013 Fc omit map of the structure at high Na+ concentration was lowered (to 4\u03c3) so as to visualize the density from Na+ ions and H2O.", + "section": "FIG", + "ner": [ + [ + 25, + 41, + "Fo \u2013 Fc omit map", + "evidence" + ], + [ + 49, + 58, + "structure", + "evidence" + ], + [ + 67, + 70, + "Na+", + "chemical" + ], + [ + 128, + 135, + "density", + "evidence" + ], + [ + 141, + 144, + "Na+", + "chemical" + ], + [ + 154, + 157, + "H2O", + "chemical" + ] + ] + }, + { + "sid": 173, + "sent": "(b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "Ca2+", + "chemical" + ], + [ + 42, + 45, + "SCa", + "site" + ], + [ + 49, + 53, + "Smid", + "site" + ], + [ + 57, + 74, + "crystals titrated", + "experimental_method" + ], + [ + 86, + 90, + "Ca2+", + "chemical" + ], + [ + 102, + 105, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 174, + "sent": "The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.", + "section": "FIG", + "ner": [ + [ + 60, + 75, + "Superimposition", + "experimental_method" + ], + [ + 79, + 85, + "NCX_Mj", + "protein" + ], + [ + 86, + 96, + "structures", + "evidence" + ], + [ + 113, + 116, + "Na+", + "chemical" + ], + [ + 169, + 179, + "absence of", + "protein_state" + ], + [ + 180, + 183, + "Na+", + "chemical" + ], + [ + 188, + 192, + "pH 4", + "protein_state" + ], + [ + 223, + 226, + "apo", + "protein_state" + ], + [ + 259, + 276, + "ion-binding sites", + "site" + ], + [ + 284, + 287, + "apo", + "protein_state" + ], + [ + 292, + 299, + "high H+", + "protein_state" + ] + ] + }, + { + "sid": 175, + "sent": "The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison.", + "section": "FIG", + "ner": [ + [ + 19, + 22, + "E54", + "residue_name_number" + ], + [ + 27, + 31, + "E213", + "residue_name_number" + ], + [ + 41, + 48, + "low Na+", + "protein_state" + ], + [ + 49, + 58, + "structure", + "evidence" + ] + ] + }, + { + "sid": 176, + "sent": "White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj.", + "section": "FIG", + "ner": [ + [ + 36, + 40, + "Sint", + "site" + ], + [ + 42, + 46, + "Smid", + "site" + ], + [ + 47, + 50, + "SCa", + "site" + ], + [ + 99, + 116, + "ion-binding sites", + "site" + ], + [ + 120, + 123, + "apo", + "protein_state" + ], + [ + 124, + 130, + "NCX_Mj", + "protein" + ] + ] + }, + { + "sid": 177, + "sent": "Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+.", + "section": "FIG", + "ner": [ + [ + 27, + 36, + "structure", + "evidence" + ], + [ + 40, + 56, + "outward-occluded", + "protein_state" + ], + [ + 58, + 76, + "fully Na+-occupied", + "protein_state" + ], + [ + 77, + 83, + "NCX_Mj", + "protein" + ], + [ + 132, + 135, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 178, + "sent": "(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).", + "section": "FIG", + "ner": [ + [ + 19, + 29, + "simulation", + "experimental_method" + ], + [ + 43, + 49, + "NCX_Mj", + "protein" + ], + [ + 65, + 68, + "Na+", + "chemical" + ], + [ + 69, + 77, + "bound at", + "protein_state" + ], + [ + 78, + 82, + "Sext", + "site" + ], + [ + 84, + 87, + "SCa", + "site" + ], + [ + 92, + 96, + "Sint", + "site" + ], + [ + 139, + 142, + "Na+", + "chemical" + ], + [ + 143, + 156, + "bound only at", + "protein_state" + ], + [ + 157, + 160, + "SCa", + "site" + ], + [ + 165, + 169, + "Sint", + "site" + ], + [ + 240, + 255, + "N-terminal half", + "structure_element" + ], + [ + 259, + 262, + "TM7", + "structure_element" + ], + [ + 264, + 269, + "TM7ab", + "structure_element" + ], + [ + 284, + 287, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 179, + "sent": "(c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons).", + "section": "FIG", + "ner": [ + [ + 19, + 39, + "simulation snapshots", + "evidence" + ], + [ + 61, + 64, + "Na+", + "chemical" + ], + [ + 65, + 73, + "bound at", + "protein_state" + ], + [ + 74, + 77, + "SCa", + "site" + ], + [ + 82, + 86, + "Sint", + "site" + ], + [ + 125, + 132, + "without", + "protein_state" + ], + [ + 137, + 140, + "Na+", + "chemical" + ], + [ + 141, + 146, + "bound", + "protein_state" + ] + ] + }, + { + "sid": 180, + "sent": "(d) Close-up of the ion-binding region in the fully Na+-occupied state.", + "section": "FIG", + "ner": [ + [ + 20, + 38, + "ion-binding region", + "site" + ], + [ + 46, + 64, + "fully Na+-occupied", + "protein_state" + ] + ] + }, + { + "sid": 181, + "sent": "Approximate distances between TM2, TM3 and TM7 are indicated in \u00c5. (e) Close-up of the ion-binding region in the partially Na+-occupied state.", + "section": "FIG", + "ner": [ + [ + 30, + 33, + "TM2", + "structure_element" + ], + [ + 35, + 38, + "TM3", + "structure_element" + ], + [ + 43, + 46, + "TM7", + "structure_element" + ], + [ + 87, + 105, + "ion-binding region", + "site" + ], + [ + 113, + 135, + "partially Na+-occupied", + "protein_state" + ] + ] + }, + { + "sid": 182, + "sent": "(f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f).", + "section": "FIG", + "ner": [ + [ + 20, + 38, + "ion-binding region", + "site" + ], + [ + 46, + 54, + "Na+-free", + "protein_state" + ], + [ + 142, + 153, + "simulations", + "experimental_method" + ], + [ + 162, + 175, + "Na+-occupancy", + "protein_state" + ] + ] + }, + { + "sid": 183, + "sent": "These descriptors were employed as collective variables in the Bias-Exchange Metadynamics simulations (Methods).", + "section": "FIG", + "ner": [ + [ + 63, + 101, + "Bias-Exchange Metadynamics simulations", + "experimental_method" + ] + ] + }, + { + "sid": 184, + "sent": "(g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1).", + "section": "FIG", + "ner": [ + [ + 4, + 29, + "Probability distributions", + "evidence" + ], + [ + 74, + 90, + "hydrogen-bonding", + "bond_interaction" + ], + [ + 102, + 107, + "TM7ab", + "structure_element" + ], + [ + 223, + 227, + "Sext", + "site" + ], + [ + 348, + 351, + "SCa", + "site" + ] + ] + }, + { + "sid": 185, + "sent": "Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound.", + "section": "FIG", + "ner": [ + [ + 108, + 111, + "NCX", + "protein_type" + ], + [ + 117, + 165, + "Calculated conformational free-energy landscapes", + "evidence" + ], + [ + 170, + 184, + "outward-facing", + "protein_state" + ], + [ + 185, + 191, + "NCX_Mj", + "protein" + ], + [ + 211, + 214, + "Na+", + "chemical" + ], + [ + 254, + 267, + "no ions bound", + "protein_state" + ] + ] + }, + { + "sid": 186, + "sent": "The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods).", + "section": "FIG", + "ner": [ + [ + 4, + 15, + "free energy", + "evidence" + ], + [ + 106, + 122, + "aqueous channels", + "site" + ], + [ + 138, + 142, + "Sext", + "site" + ], + [ + 147, + 150, + "SCa", + "site" + ] + ] + }, + { + "sid": 187, + "sent": "Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study.", + "section": "FIG", + "ner": [ + [ + 22, + 38, + "X-ray structures", + "evidence" + ], + [ + 42, + 48, + "NCX_Mj", + "protein" + ], + [ + 61, + 65, + "high", + "protein_state" + ], + [ + 70, + 73, + "low", + "protein_state" + ], + [ + 74, + 77, + "Na+", + "chemical" + ], + [ + 112, + 118, + "low pH", + "protein_state" + ] + ] + }, + { + "sid": 188, + "sent": "(b) Density isosurfaces for water molecules within 12 \u00c5 of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state.", + "section": "FIG", + "ner": [ + [ + 4, + 23, + "Density isosurfaces", + "evidence" + ], + [ + 28, + 33, + "water", + "chemical" + ], + [ + 63, + 81, + "ion-binding region", + "site" + ], + [ + 120, + 153, + "conformational free-energy minima", + "evidence" + ] + ] + }, + { + "sid": 189, + "sent": "Na+ ions are shown as green spheres.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 190, + "sent": "The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine).", + "section": "FIG", + "ner": [ + [ + 8, + 33, + "inverted-topology repeats", + "structure_element" + ], + [ + 41, + 52, + "transporter", + "protein_type" + ], + [ + 53, + 62, + "structure", + "evidence" + ], + [ + 111, + 116, + "TM1-5", + "structure_element" + ], + [ + 126, + 132, + "TM6-10", + "structure_element" + ] + ] + }, + { + "sid": 191, + "sent": "(c) Close-up views of the ion-binding region in the same conformational free-energy minima.", + "section": "FIG", + "ner": [ + [ + 26, + 44, + "ion-binding region", + "site" + ], + [ + 57, + 90, + "conformational free-energy minima", + "evidence" + ] + ] + }, + { + "sid": 192, + "sent": "Key residues involved in Na+ and water coordination (W) are highlighted (sticks, black lines).", + "section": "FIG", + "ner": [ + [ + 25, + 28, + "Na+", + "chemical" + ], + [ + 33, + 38, + "water", + "chemical" + ] + ] + }, + { + "sid": 193, + "sent": "The water-density maps in (b) is shown here as a grey mesh.", + "section": "FIG", + "ner": [ + [ + 4, + 22, + "water-density maps", + "evidence" + ] + ] + }, + { + "sid": 194, + "sent": "Note D240 is protonated, while E54 and E213 are ionized.", + "section": "FIG", + "ner": [ + [ + 5, + 9, + "D240", + "residue_name_number" + ], + [ + 31, + 34, + "E54", + "residue_name_number" + ], + [ + 39, + 43, + "E213", + "residue_name_number" + ] + ] + }, + { + "sid": 195, + "sent": "Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state.", + "section": "FIG", + "ner": [ + [ + 108, + 111, + "NCX", + "protein_type" + ], + [ + 117, + 150, + "Calculated free-energy landscapes", + "evidence" + ], + [ + 155, + 169, + "outward-facing", + "protein_state" + ], + [ + 170, + 176, + "NCX_Mj", + "protein" + ], + [ + 186, + 190, + "Ca2+", + "chemical" + ], + [ + 199, + 215, + "fully protonated", + "protein_state" + ] + ] + }, + { + "sid": 196, + "sent": "The free energy is plotted as in Fig. 5.", + "section": "FIG", + "ner": [ + [ + 4, + 15, + "free energy", + "evidence" + ] + ] + }, + { + "sid": 197, + "sent": "For Ca2+, a map is shown in which a correction for the charge-transfer between the ion and the protein is introduced, alongside the uncorrected map (see Supplementary Notes 3-4 and Supplementary Fig. 5-6).", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "Ca2+", + "chemical" + ], + [ + 12, + 15, + "map", + "evidence" + ], + [ + 144, + 147, + "map", + "evidence" + ] + ] + }, + { + "sid": 198, + "sent": "The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+).", + "section": "FIG", + "ner": [ + [ + 16, + 19, + "map", + "evidence" + ], + [ + 39, + 43, + "open", + "protein_state" + ], + [ + 66, + 75, + "semi-open", + "protein_state" + ], + [ + 80, + 88, + "occluded", + "protein_state" + ], + [ + 166, + 174, + "bound to", + "protein_state" + ], + [ + 218, + 221, + "Na+", + "chemical" + ] + ] + }, + { + "sid": 199, + "sent": "Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study.", + "section": "FIG", + "ner": [ + [ + 22, + 40, + "crystal structures", + "evidence" + ], + [ + 58, + 62, + "Ca2+", + "chemical" + ], + [ + 84, + 90, + "low pH", + "protein_state" + ], + [ + 95, + 102, + "high H+", + "protein_state" + ] + ] + }, + { + "sid": 200, + "sent": "(b) Water-density isosurfaces analogous to those in Fig. 5 are shown for each of the major conformational free-energy minima in the free-energy maps.", + "section": "FIG", + "ner": [ + [ + 4, + 29, + "Water-density isosurfaces", + "evidence" + ], + [ + 106, + 124, + "free-energy minima", + "evidence" + ], + [ + 132, + 148, + "free-energy maps", + "evidence" + ] + ] + }, + { + "sid": 201, + "sent": "The Ca2+ ion is shown as a red sphere; the protein is shown as in Fig. 5. (c) Close-up views of the ion-binding region in the same conformational free-energy minima.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "Ca2+", + "chemical" + ], + [ + 100, + 118, + "ion-binding region", + "site" + ], + [ + 131, + 164, + "conformational free-energy minima", + "evidence" + ] + ] + }, + { + "sid": 202, + "sent": "Key residues involved in Ca2+ and water coordination (W) are highlighted (sticks, black lines).", + "section": "FIG", + "ner": [ + [ + 25, + 29, + "Ca2+", + "chemical" + ], + [ + 34, + 39, + "water", + "chemical" + ] + ] + }, + { + "sid": 203, + "sent": "The water-density maps in (b) are shown here as a grey mesh.", + "section": "FIG", + "ner": [ + [ + 4, + 22, + "water-density maps", + "evidence" + ] + ] + }, + { + "sid": 204, + "sent": "In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210.", + "section": "FIG", + "ner": [ + [ + 7, + 15, + "occluded", + "protein_state" + ], + [ + 27, + 31, + "Ca2+", + "chemical" + ], + [ + 32, + 37, + "bound", + "protein_state" + ], + [ + 39, + 44, + "helix", + "structure_element" + ], + [ + 45, + 50, + "TM7ab", + "structure_element" + ], + [ + 83, + 97, + "fully occupied", + "protein_state" + ], + [ + 98, + 101, + "Na+", + "chemical" + ], + [ + 128, + 134, + "Ala206", + "residue_name_number" + ], + [ + 143, + 171, + "hydrogen-bonding interaction", + "bond_interaction" + ], + [ + 177, + 183, + "Ser210", + "residue_name_number" + ] + ] + }, + { + "sid": 205, + "sent": "Structural mechanism of extracellular forward ion exchange in NCX.", + "section": "FIG", + "ner": [ + [ + 62, + 65, + "NCX", + "protein_type" + ] + ] + }, + { + "sid": 206, + "sent": "The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes.", + "section": "FIG", + "ner": [ + [ + 23, + 28, + "Ala47", + "residue_name_number" + ], + [ + 33, + 37, + "TM2b", + "structure_element" + ], + [ + 43, + 49, + "Ala206", + "residue_name_number" + ], + [ + 54, + 58, + "TM7b", + "structure_element" + ], + [ + 84, + 89, + "Glu54", + "residue_name_number" + ], + [ + 94, + 98, + "TM2c", + "structure_element" + ], + [ + 104, + 110, + "Glu213", + "residue_name_number" + ], + [ + 115, + 119, + "TM7c", + "structure_element" + ] + ] + }, + { + "sid": 207, + "sent": "The green open cylinders represent the gating helices TM1 and TM6.", + "section": "FIG", + "ner": [ + [ + 39, + 53, + "gating helices", + "structure_element" + ], + [ + 54, + 57, + "TM1", + "structure_element" + ], + [ + 62, + 65, + "TM6", + "structure_element" + ] + ] + }, + { + "sid": 208, + "sent": "Asterisks mark the states whose crystal structures have been determined in this study.", + "section": "FIG", + "ner": [ + [ + 32, + 50, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 209, + "sent": "These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state.", + "section": "FIG", + "ner": [ + [ + 65, + 98, + "calculated free-energy landscapes", + "evidence" + ], + [ + 120, + 131, + "Ca2+-loaded", + "protein_state" + ], + [ + 132, + 146, + "outward-facing", + "protein_state" + ], + [ + 147, + 155, + "occluded", + "protein_state" + ], + [ + 170, + 178, + "unloaded", + "protein_state" + ], + [ + 180, + 190, + "fully open", + "protein_state" + ] + ] + } + ] + }, + "PMC4786784": { + "annotations": [ + { + "sid": 0, + "sent": "An extended U2AF65\u2013RNA-binding domain recognizes the 3\u2032 splice site signal", + "section": "TITLE", + "ner": [ + [ + 3, + 11, + "extended", + "protein_state" + ], + [ + 12, + 37, + "U2AF65\u2013RNA-binding domain", + "structure_element" + ], + [ + 53, + 67, + "3\u2032 splice site", + "site" + ] + ] + }, + { + "sid": 1, + "sent": "How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3\u2032 splice sites in human gene transcripts remains incompletely understood.", + "section": "ABSTRACT", + "ner": [ + [ + 18, + 42, + "pre-mRNA splicing factor", + "protein_type" + ], + [ + 43, + 49, + "U2AF65", + "protein" + ], + [ + 65, + 79, + "polypyrimidine", + "chemical" + ], + [ + 81, + 83, + "Py", + "chemical" + ], + [ + 115, + 130, + "3\u2032 splice sites", + "site" + ], + [ + 134, + 139, + "human", + "species" + ] + ] + }, + { + "sid": 2, + "sent": "We determined four structures of an extended U2AF65\u2013RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5\u2009\u00c5. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract.", + "section": "ABSTRACT", + "ner": [ + [ + 3, + 29, + "determined four structures", + "experimental_method" + ], + [ + 36, + 44, + "extended", + "protein_state" + ], + [ + 45, + 70, + "U2AF65\u2013RNA-binding domain", + "structure_element" + ], + [ + 71, + 79, + "bound to", + "protein_state" + ], + [ + 80, + 105, + "Py-tract oligonucleotides", + "chemical" + ], + [ + 150, + 160, + "structures", + "evidence" + ], + [ + 175, + 206, + "RNA binding and splicing assays", + "experimental_method" + ], + [ + 235, + 241, + "U2AF65", + "protein" + ], + [ + 242, + 263, + "inter-domain residues", + "site" + ], + [ + 281, + 291, + "contiguous", + "structure_element" + ], + [ + 298, + 308, + "nucleotide", + "chemical" + ], + [ + 309, + 317, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 3, + "sent": "The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3\u2032 terminus and third nucleotide.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 10, + "U2AF65", + "protein" + ], + [ + 11, + 17, + "linker", + "structure_element" + ], + [ + 44, + 66, + "RNA recognition motifs", + "structure_element" + ], + [ + 68, + 72, + "RRMs", + "structure_element" + ], + [ + 96, + 106, + "nucleotide", + "chemical" + ], + [ + 138, + 152, + "RRM extensions", + "structure_element" + ], + [ + 167, + 178, + "3\u2032 terminus", + "site" + ], + [ + 183, + 188, + "third", + "residue_number" + ], + [ + 189, + 199, + "nucleotide", + "chemical" + ] + ] + }, + { + "sid": 4, + "sent": "Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 20, + "Single-molecule FRET", + "experimental_method" + ], + [ + 94, + 100, + "U2AF65", + "protein" + ], + [ + 101, + 105, + "RRMs", + "structure_element" + ], + [ + 139, + 147, + "Py-tract", + "chemical" + ] + ] + }, + { + "sid": 5, + "sent": "Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.", + "section": "ABSTRACT", + "ner": [ + [ + 110, + 121, + "splice site", + "site" + ] + ] + }, + { + "sid": 6, + "sent": " The pre-mRNA splicing factor U2AF65 recognizes 3\u2032 splice sites in human gene transcripts, but the details are not fully understood.", + "section": "ABSTRACT", + "ner": [ + [ + 5, + 29, + "pre-mRNA splicing factor", + "protein_type" + ], + [ + 30, + 36, + "U2AF65", + "protein" + ], + [ + 48, + 63, + "3\u2032 splice sites", + "site" + ], + [ + 67, + 72, + "human", + "species" + ] + ] + }, + { + "sid": 7, + "sent": "Here, the authors report U2AF65 structures and single molecule FRET that reveal mechanistic insights into splice site recognition.", + "section": "ABSTRACT", + "ner": [ + [ + 25, + 31, + "U2AF65", + "protein" + ], + [ + 32, + 42, + "structures", + "evidence" + ], + [ + 47, + 67, + "single molecule FRET", + "experimental_method" + ], + [ + 106, + 117, + "splice site", + "site" + ] + ] + }, + { + "sid": 8, + "sent": "The differential skipping or inclusion of alternatively spliced pre-mRNA regions is a major source of diversity for nearly all human gene transcripts.", + "section": "INTRO", + "ner": [ + [ + 64, + 80, + "pre-mRNA regions", + "structure_element" + ], + [ + 127, + 132, + "human", + "species" + ] + ] + }, + { + "sid": 9, + "sent": "The splice sites are marked by relatively short consensus sequences and are regulated by additional pre-mRNA motifs (reviewed in ref.).", + "section": "INTRO", + "ner": [ + [ + 4, + 16, + "splice sites", + "site" + ], + [ + 42, + 67, + "short consensus sequences", + "structure_element" + ], + [ + 100, + 115, + "pre-mRNA motifs", + "structure_element" + ] + ] + }, + { + "sid": 10, + "sent": "At the 3\u2032 splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3\u2032 splice site junction.", + "section": "INTRO", + "ner": [ + [ + 7, + 21, + "3\u2032 splice site", + "site" + ], + [ + 65, + 90, + "polypyrimidine (Py) tract", + "chemical" + ], + [ + 112, + 113, + "U", + "residue_name" + ], + [ + 118, + 119, + "C", + "residue_name" + ], + [ + 145, + 166, + "branch point sequence", + "site" + ], + [ + 168, + 171, + "BPS", + "site" + ], + [ + 247, + 262, + "AG-dinucleotide", + "chemical" + ], + [ + 270, + 284, + "3\u2032 splice site", + "site" + ] + ] + }, + { + "sid": 11, + "sent": "Disease-causing mutations often compromise pre-mRNA splicing (reviewed in refs), yet a priori predictions of splice sites and the consequences of their mutations are challenged by the brevity and degeneracy of known splice site sequences.", + "section": "INTRO", + "ner": [ + [ + 43, + 51, + "pre-mRNA", + "chemical" + ], + [ + 109, + 121, + "splice sites", + "site" + ], + [ + 216, + 227, + "splice site", + "site" + ] + ] + }, + { + "sid": 12, + "sent": "High-resolution structures of intact splicing factor\u2013RNA complexes would offer key insights regarding the juxtaposition of the distinct splice site consensus sequences and their relationship to disease-causing point mutations.", + "section": "INTRO", + "ner": [ + [ + 16, + 26, + "structures", + "evidence" + ], + [ + 30, + 36, + "intact", + "protein_state" + ], + [ + 37, + 56, + "splicing factor\u2013RNA", + "complex_assembly" + ], + [ + 136, + 147, + "splice site", + "site" + ] + ] + }, + { + "sid": 13, + "sent": "The early-stage pre-mRNA splicing factor U2AF65 is essential for viability in vertebrates and other model organisms (for example, ref.).", + "section": "INTRO", + "ner": [ + [ + 16, + 40, + "pre-mRNA splicing factor", + "protein_type" + ], + [ + 41, + 47, + "U2AF65", + "protein" + ], + [ + 78, + 89, + "vertebrates", + "taxonomy_domain" + ] + ] + }, + { + "sid": 14, + "sent": "A tightly controlled assembly among U2AF65, the pre-mRNA, and partner proteins sequentially identifies the 3\u2032 splice site and promotes association of the spliceosome, which ultimately accomplishes the task of splicing.", + "section": "INTRO", + "ner": [ + [ + 21, + 29, + "assembly", + "complex_assembly" + ], + [ + 36, + 42, + "U2AF65", + "protein" + ], + [ + 48, + 56, + "pre-mRNA", + "chemical" + ], + [ + 107, + 121, + "3\u2032 splice site", + "site" + ], + [ + 154, + 165, + "spliceosome", + "complex_assembly" + ] + ] + }, + { + "sid": 15, + "sent": "Initially U2AF65 recognizes the Py-tract splice site signal.", + "section": "INTRO", + "ner": [ + [ + 10, + 16, + "U2AF65", + "protein" + ], + [ + 32, + 40, + "Py-tract", + "chemical" + ], + [ + 41, + 52, + "splice site", + "site" + ] + ] + }, + { + "sid": 16, + "sent": "In turn, the ternary complex of U2AF65 with SF1 and U2AF35 identifies the surrounding BPS and 3\u2032 splice site junctions.", + "section": "INTRO", + "ner": [ + [ + 13, + 28, + "ternary complex", + "complex_assembly" + ], + [ + 32, + 38, + "U2AF65", + "protein" + ], + [ + 44, + 47, + "SF1", + "protein" + ], + [ + 52, + 58, + "U2AF35", + "protein" + ], + [ + 86, + 89, + "BPS", + "site" + ], + [ + 94, + 108, + "3\u2032 splice site", + "site" + ] + ] + }, + { + "sid": 17, + "sent": "Subsequently U2AF65 recruits the U2 small nuclear ribonucleoprotein particle (snRNP) and ultimately dissociates from the active spliceosome.", + "section": "INTRO", + "ner": [ + [ + 13, + 19, + "U2AF65", + "protein" + ], + [ + 33, + 76, + "U2 small nuclear ribonucleoprotein particle", + "complex_assembly" + ], + [ + 78, + 83, + "snRNP", + "complex_assembly" + ], + [ + 121, + 127, + "active", + "protein_state" + ], + [ + 128, + 139, + "spliceosome", + "complex_assembly" + ] + ] + }, + { + "sid": 18, + "sent": "Biochemical characterizations of U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract (Fig. 1a).", + "section": "INTRO", + "ner": [ + [ + 0, + 29, + "Biochemical characterizations", + "experimental_method" + ], + [ + 33, + 39, + "U2AF65", + "protein" + ], + [ + 65, + 87, + "RNA recognition motifs", + "structure_element" + ], + [ + 89, + 93, + "RRM1", + "structure_element" + ], + [ + 98, + 102, + "RRM2", + "structure_element" + ], + [ + 118, + 126, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 19, + "sent": "Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs.", + "section": "INTRO", + "ner": [ + [ + 10, + 28, + "crystal structures", + "evidence" + ], + [ + 36, + 40, + "core", + "protein_state" + ], + [ + 41, + 47, + "U2AF65", + "protein" + ], + [ + 48, + 52, + "RRM1", + "structure_element" + ], + [ + 57, + 61, + "RRM2", + "structure_element" + ], + [ + 77, + 86, + "shortened", + "protein_state" + ], + [ + 87, + 103, + "inter-RRM linker", + "structure_element" + ], + [ + 105, + 115, + "dU2AF651,2", + "mutant" + ], + [ + 182, + 188, + "U2AF65", + "protein" + ], + [ + 189, + 193, + "RRMs", + "structure_element" + ] + ] + }, + { + "sid": 20, + "sent": "A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation.", + "section": "INTRO", + "ner": [ + [ + 13, + 16, + "NMR", + "experimental_method" + ], + [ + 17, + 26, + "structure", + "evidence" + ], + [ + 45, + 57, + "side-by-side", + "protein_state" + ], + [ + 77, + 84, + "minimal", + "protein_state" + ], + [ + 85, + 91, + "U2AF65", + "protein" + ], + [ + 92, + 96, + "RRM1", + "structure_element" + ], + [ + 101, + 105, + "RRM2", + "structure_element" + ], + [ + 121, + 127, + "linker", + "structure_element" + ], + [ + 131, + 145, + "natural length", + "protein_state" + ], + [ + 147, + 156, + "U2AF651,2", + "mutant" + ], + [ + 179, + 189, + "dU2AF651,2", + "mutant" + ], + [ + 190, + 208, + "crystal structures", + "evidence" + ], + [ + 213, + 216, + "RNA", + "chemical" + ], + [ + 261, + 277, + "inter-RRM linker", + "structure_element" + ] + ] + }, + { + "sid": 21, + "sent": "As such, the molecular mechanisms for Py-tract recognition by the intact U2AF65\u2013RNA-binding domain remained unknown.", + "section": "INTRO", + "ner": [ + [ + 38, + 46, + "Py-tract", + "chemical" + ], + [ + 66, + 72, + "intact", + "protein_state" + ], + [ + 73, + 98, + "U2AF65\u2013RNA-binding domain", + "structure_element" + ] + ] + }, + { + "sid": 22, + "sent": "Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs.", + "section": "INTRO", + "ner": [ + [ + 13, + 34, + "X-ray crystallography", + "experimental_method" + ], + [ + 39, + 58, + "biochemical studies", + "experimental_method" + ], + [ + 82, + 90, + "Py-tract", + "chemical" + ], + [ + 111, + 127, + "inter-RRM linker", + "structure_element" + ], + [ + 161, + 165, + "core", + "protein_state" + ], + [ + 166, + 172, + "U2AF65", + "protein" + ], + [ + 173, + 177, + "RRMs", + "structure_element" + ] + ] + }, + { + "sid": 23, + "sent": "We use single-molecule F\u00f6rster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65\u2013RNA-binding domain during Py-tract recognition.", + "section": "INTRO", + "ner": [ + [ + 7, + 56, + "single-molecule F\u00f6rster resonance energy transfer", + "experimental_method" + ], + [ + 58, + 64, + "smFRET", + "experimental_method" + ], + [ + 86, + 109, + "conformational dynamics", + "evidence" + ], + [ + 118, + 126, + "extended", + "protein_state" + ], + [ + 127, + 152, + "U2AF65\u2013RNA-binding domain", + "structure_element" + ], + [ + 160, + 168, + "Py-tract", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "Cognate U2AF65\u2013Py-tract recognition requires RRM extensions", + "section": "RESULTS", + "ner": [ + [ + 8, + 14, + "U2AF65", + "protein" + ], + [ + 15, + 23, + "Py-tract", + "chemical" + ], + [ + 45, + 59, + "RRM extensions", + "structure_element" + ] + ] + }, + { + "sid": 25, + "sent": "The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1\u2013RRM2 folds (U2AF651,2, residues 148\u2013336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "RNA affinity", + "evidence" + ], + [ + 24, + 31, + "minimal", + "protein_state" + ], + [ + 32, + 41, + "U2AF651,2", + "mutant" + ], + [ + 64, + 68, + "core", + "protein_state" + ], + [ + 69, + 73, + "RRM1", + "structure_element" + ], + [ + 74, + 78, + "RRM2", + "structure_element" + ], + [ + 79, + 84, + "folds", + "structure_element" + ], + [ + 86, + 95, + "U2AF651,2", + "mutant" + ], + [ + 106, + 113, + "148\u2013336", + "residue_range" + ], + [ + 148, + 159, + "full-length", + "protein_state" + ], + [ + 160, + 166, + "U2AF65", + "protein" + ] + ] + }, + { + "sid": 26, + "sent": "Historically, this difference was attributed to the U2AF65 arginine\u2013serine rich domain, which contacts pre-mRNA\u2013U2 snRNA duplexes outside of the Py tract.", + "section": "RESULTS", + "ner": [ + [ + 52, + 58, + "U2AF65", + "protein" + ], + [ + 59, + 86, + "arginine\u2013serine rich domain", + "structure_element" + ], + [ + 103, + 129, + "pre-mRNA\u2013U2 snRNA duplexes", + "complex_assembly" + ], + [ + 145, + 153, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 27, + "sent": "We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141\u2013342; Fig. 1a).", + "section": "RESULTS", + "ner": [ + [ + 20, + 40, + "RNA-binding affinity", + "evidence" + ], + [ + 48, + 57, + "U2AF651,2", + "mutant" + ], + [ + 93, + 127, + "addition of seven and six residues", + "experimental_method" + ], + [ + 169, + 176, + "minimal", + "protein_state" + ], + [ + 177, + 181, + "RRM1", + "structure_element" + ], + [ + 186, + 190, + "RRM2", + "structure_element" + ], + [ + 192, + 202, + "U2AF651,2L", + "mutant" + ], + [ + 213, + 220, + "141\u2013342", + "residue_range" + ] + ] + }, + { + "sid": 28, + "sent": "In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a\u2013d).", + "section": "RESULTS", + "ner": [ + [ + 5, + 34, + "fluorescence anisotropy assay", + "experimental_method" + ], + [ + 64, + 72, + "Py tract", + "chemical" + ], + [ + 109, + 120, + "splice site", + "site" + ], + [ + 128, + 158, + "adenovirus major late promoter", + "gene" + ], + [ + 160, + 164, + "AdML", + "gene" + ], + [ + 171, + 183, + "RNA affinity", + "evidence" + ], + [ + 187, + 197, + "U2AF651,2L", + "mutant" + ], + [ + 232, + 241, + "U2AF651,2", + "mutant" + ], + [ + 266, + 277, + "full-length", + "protein_state" + ], + [ + 278, + 284, + "U2AF65", + "protein" + ] + ] + }, + { + "sid": 29, + "sent": "Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5\u2032-region of the Py tract and promiscuity for C-rich regions in the 3\u2032-region (Fig. 1c, Supplementary Fig. 1e\u2013h).", + "section": "RESULTS", + "ner": [ + [ + 15, + 25, + "U2AF651,2L", + "mutant" + ], + [ + 30, + 41, + "full-length", + "protein_state" + ], + [ + 42, + 48, + "U2AF65", + "protein" + ], + [ + 64, + 84, + "sequence specificity", + "evidence" + ], + [ + 89, + 105, + "U-rich stretches", + "structure_element" + ], + [ + 113, + 122, + "5\u2032-region", + "site" + ], + [ + 130, + 138, + "Py tract", + "chemical" + ], + [ + 159, + 173, + "C-rich regions", + "structure_element" + ], + [ + 181, + 190, + "3\u2032-region", + "site" + ] + ] + }, + { + "sid": 30, + "sent": "U2AF65-bound Py tract comprises nine contiguous nucleotides", + "section": "RESULTS", + "ner": [ + [ + 0, + 12, + "U2AF65-bound", + "protein_state" + ], + [ + 13, + 21, + "Py tract", + "chemical" + ], + [ + 37, + 47, + "contiguous", + "structure_element" + ], + [ + 48, + 59, + "nucleotides", + "chemical" + ] + ] + }, + { + "sid": 31, + "sent": "To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1).", + "section": "RESULTS", + "ner": [ + [ + 48, + 54, + "U2AF65", + "protein" + ], + [ + 72, + 82, + "contiguous", + "structure_element" + ], + [ + 83, + 91, + "Py tract", + "chemical" + ], + [ + 96, + 106, + "determined", + "experimental_method" + ], + [ + 112, + 130, + "crystal structures", + "evidence" + ], + [ + 134, + 144, + "U2AF651,2L", + "mutant" + ], + [ + 145, + 153, + "bound to", + "protein_state" + ], + [ + 154, + 179, + "Py-tract oligonucleotides", + "chemical" + ] + ] + }, + { + "sid": 32, + "sent": "By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5\u2009\u00c5 resolution.", + "section": "RESULTS", + "ner": [ + [ + 3, + 28, + "sequential boot strapping", + "experimental_method" + ], + [ + 57, + 72, + "oligonucleotide", + "chemical" + ], + [ + 99, + 104, + "Br-dU", + "chemical" + ], + [ + 139, + 149, + "nucleotide", + "chemical" + ], + [ + 151, + 153, + "rU", + "residue_name" + ], + [ + 155, + 157, + "dU", + "residue_name" + ], + [ + 162, + 164, + "rC", + "residue_name" + ], + [ + 191, + 201, + "U2AF651,2L", + "mutant" + ], + [ + 202, + 210, + "bound to", + "protein_state" + ], + [ + 211, + 221, + "contiguous", + "structure_element" + ], + [ + 222, + 231, + "Py tracts", + "chemical" + ] + ] + }, + { + "sid": 33, + "sent": "The protein and oligonucleotide conformations are nearly identical among the four new U2AF651,2L structures (Supplementary Fig. 2a).", + "section": "RESULTS", + "ner": [ + [ + 16, + 31, + "oligonucleotide", + "chemical" + ], + [ + 86, + 96, + "U2AF651,2L", + "mutant" + ], + [ + 97, + 107, + "structures", + "evidence" + ] + ] + }, + { + "sid": 34, + "sent": "The U2AF651,2L RRM1 and RRM2 associate with the Py tract in a parallel, side-by-side arrangement (shown for representative structure iv in Fig. 2b,c; Supplementary Movie 1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "U2AF651,2L", + "mutant" + ], + [ + 15, + 19, + "RRM1", + "structure_element" + ], + [ + 24, + 28, + "RRM2", + "structure_element" + ], + [ + 48, + 56, + "Py tract", + "chemical" + ], + [ + 62, + 70, + "parallel", + "protein_state" + ], + [ + 72, + 84, + "side-by-side", + "protein_state" + ] + ] + }, + { + "sid": 35, + "sent": "An extended conformation of the U2AF65 inter-RRM linker traverses across the \u03b1-helical surface of RRM1 and the central \u03b2-strands of RRM2 and is well defined in the electron density (Fig. 2b).", + "section": "RESULTS", + "ner": [ + [ + 3, + 24, + "extended conformation", + "protein_state" + ], + [ + 32, + 38, + "U2AF65", + "protein" + ], + [ + 39, + 55, + "inter-RRM linker", + "structure_element" + ], + [ + 77, + 94, + "\u03b1-helical surface", + "structure_element" + ], + [ + 98, + 102, + "RRM1", + "structure_element" + ], + [ + 119, + 128, + "\u03b2-strands", + "structure_element" + ], + [ + 132, + 136, + "RRM2", + "structure_element" + ], + [ + 164, + 180, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 36, + "sent": "The extensions at the N terminus of RRM1 and C terminus of RRM2 adopt well-ordered \u03b1-helices.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "extensions", + "structure_element" + ], + [ + 36, + 40, + "RRM1", + "structure_element" + ], + [ + 59, + 63, + "RRM2", + "structure_element" + ], + [ + 83, + 92, + "\u03b1-helices", + "structure_element" + ] + ] + }, + { + "sid": 37, + "sent": "Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide.", + "section": "RESULTS", + "ner": [ + [ + 5, + 9, + "RRM1", + "structure_element" + ], + [ + 10, + 14, + "RRM2", + "structure_element" + ], + [ + 15, + 25, + "extensions", + "structure_element" + ], + [ + 34, + 50, + "inter-RRM linker", + "structure_element" + ], + [ + 54, + 64, + "U2AF651,2L", + "mutant" + ], + [ + 88, + 93, + "bound", + "protein_state" + ], + [ + 94, + 109, + "oligonucleotide", + "chemical" + ] + ] + }, + { + "sid": 38, + "sent": "We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2.", + "section": "RESULTS", + "ner": [ + [ + 42, + 52, + "U2AF651,2L", + "mutant" + ], + [ + 53, + 63, + "structures", + "evidence" + ], + [ + 79, + 89, + "dU2AF651,2", + "mutant" + ], + [ + 90, + 107, + "crystal structure", + "evidence" + ], + [ + 112, + 121, + "U2AF651,2", + "mutant" + ], + [ + 122, + 125, + "NMR", + "experimental_method" + ], + [ + 126, + 135, + "structure", + "evidence" + ] + ] + }, + { + "sid": 39, + "sent": "The discovery of nine U2AF65-binding sites for contiguous Py-tract nucleotides was unexpected.", + "section": "RESULTS", + "ner": [ + [ + 22, + 42, + "U2AF65-binding sites", + "site" + ], + [ + 47, + 57, + "contiguous", + "structure_element" + ], + [ + 58, + 78, + "Py-tract nucleotides", + "chemical" + ] + ] + }, + { + "sid": 40, + "sent": "Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures.", + "section": "RESULTS", + "ner": [ + [ + 9, + 19, + "dU2AF651,2", + "mutant" + ], + [ + 20, + 30, + "structures", + "evidence" + ], + [ + 68, + 74, + "U2AF65", + "protein" + ], + [ + 75, + 79, + "RRMs", + "structure_element" + ], + [ + 95, + 102, + "minimal", + "protein_state" + ], + [ + 109, + 120, + "nucleotides", + "chemical" + ], + [ + 139, + 149, + "structures", + "evidence" + ] + ] + }, + { + "sid": 41, + "sent": "Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3\u2032-terminal nucleotide (Fig. 2c; Supplementary Movie 1).", + "section": "RESULTS", + "ner": [ + [ + 18, + 32, + "RRM2 extension", + "structure_element" + ], + [ + 33, + 49, + "inter-RRM linker", + "structure_element" + ], + [ + 73, + 97, + "nucleotide-binding sites", + "site" + ], + [ + 107, + 125, + "RRM1/RRM2 junction", + "site" + ], + [ + 134, + 148, + "RRM1 extension", + "structure_element" + ], + [ + 176, + 186, + "nucleotide", + "chemical" + ] + ] + }, + { + "sid": 42, + "sent": "The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2\u2032-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "U2AF651,2L", + "mutant" + ], + [ + 15, + 25, + "structures", + "evidence" + ], + [ + 39, + 45, + "ribose", + "chemical" + ], + [ + 47, + 48, + "r", + "chemical" + ], + [ + 50, + 61, + "nucleotides", + "chemical" + ], + [ + 76, + 89, + "binding sites", + "site" + ], + [ + 101, + 108, + "seventh", + "residue_number" + ], + [ + 113, + 119, + "eighth", + "residue_number" + ], + [ + 120, + 130, + "deoxy-(d)U", + "chemical" + ], + [ + 191, + 200, + "RNA-bound", + "protein_state" + ], + [ + 201, + 211, + "dU2AF651,2", + "mutant" + ], + [ + 212, + 221, + "structure", + "evidence" + ] + ] + }, + { + "sid": 43, + "sent": "Qualitatively, a subset of the U2AF651,2L-nucleotide-binding sites (sites 1\u20133 and 7\u20139) share similar locations to those of the dU2AF651,2 structures (Supplementary Figs 2c,d and 3).", + "section": "RESULTS", + "ner": [ + [ + 31, + 66, + "U2AF651,2L-nucleotide-binding sites", + "site" + ], + [ + 68, + 77, + "sites 1\u20133", + "site" + ], + [ + 82, + 85, + "7\u20139", + "site" + ], + [ + 127, + 137, + "dU2AF651,2", + "mutant" + ], + [ + 138, + 148, + "structures", + "evidence" + ] + ] + }, + { + "sid": 44, + "sent": "Yet, only the U2AF651,2L interactions at sites 1 and 7 are nearly identical to those of the dU2AF651,2 structures (Supplementary Fig. 3a,f).", + "section": "RESULTS", + "ner": [ + [ + 14, + 24, + "U2AF651,2L", + "mutant" + ], + [ + 41, + 54, + "sites 1 and 7", + "site" + ], + [ + 92, + 102, + "dU2AF651,2", + "mutant" + ], + [ + 103, + 113, + "structures", + "evidence" + ] + ] + }, + { + "sid": 45, + "sent": "In striking departures from prior partial views, the U2AF651,2L structures reveal three unanticipated nucleotide-binding sites at the centre of the Py tract, as well as numerous new interactions that underlie cognate recognition of the Py tract (Fig. 3a\u2013h).", + "section": "RESULTS", + "ner": [ + [ + 53, + 63, + "U2AF651,2L", + "mutant" + ], + [ + 64, + 74, + "structures", + "evidence" + ], + [ + 102, + 126, + "nucleotide-binding sites", + "site" + ], + [ + 148, + 156, + "Py tract", + "chemical" + ], + [ + 236, + 244, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 46, + "sent": "U2AF65 inter-RRM linker interacts with the Py tract", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "U2AF65", + "protein" + ], + [ + 7, + 23, + "inter-RRM linker", + "structure_element" + ], + [ + 43, + 51, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 47, + "sent": "The U2AF651,2L RRM2, the inter-RRM linker and RRM1 concomitantly recognize the three central nucleotides of the Py tract, which are likely to coordinate the conformational arrangement of these disparate portions of the protein.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "U2AF651,2L", + "mutant" + ], + [ + 15, + 19, + "RRM2", + "structure_element" + ], + [ + 25, + 41, + "inter-RRM linker", + "structure_element" + ], + [ + 46, + 50, + "RRM1", + "structure_element" + ], + [ + 93, + 104, + "nucleotides", + "chemical" + ], + [ + 112, + 120, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 48, + "sent": "Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d).", + "section": "RESULTS", + "ner": [ + [ + 16, + 33, + "C-terminal region", + "structure_element" + ], + [ + 41, + 47, + "U2AF65", + "protein" + ], + [ + 48, + 64, + "inter-RRM linker", + "structure_element" + ], + [ + 94, + 106, + "binding site", + "site" + ], + [ + 115, + 120, + "fifth", + "residue_number" + ], + [ + 121, + 131, + "nucleotide", + "chemical" + ], + [ + 139, + 151, + "RRM2 surface", + "site" + ], + [ + 169, + 188, + "RRM1/RRM2 interface", + "site" + ] + ] + }, + { + "sid": 49, + "sent": "The backbone amide of the linker V254 and the carbonyl of T252 engage in hydrogen bonds with the rU5-O4 and -N3H atoms.", + "section": "RESULTS", + "ner": [ + [ + 26, + 32, + "linker", + "structure_element" + ], + [ + 33, + 37, + "V254", + "residue_name_number" + ], + [ + 58, + 62, + "T252", + "residue_name_number" + ], + [ + 73, + 87, + "hydrogen bonds", + "bond_interaction" + ], + [ + 97, + 100, + "rU5", + "residue_name_number" + ] + ] + }, + { + "sid": 50, + "sent": "In the C-terminal \u03b2-strand of RRM1, the side chains of K225 and R227 donate additional hydrogen bonds to the rU5-O2 lone pair electrons.", + "section": "RESULTS", + "ner": [ + [ + 18, + 26, + "\u03b2-strand", + "structure_element" + ], + [ + 30, + 34, + "RRM1", + "structure_element" + ], + [ + 55, + 59, + "K225", + "residue_name_number" + ], + [ + 64, + 68, + "R227", + "residue_name_number" + ], + [ + 87, + 101, + "hydrogen bonds", + "bond_interaction" + ], + [ + 109, + 112, + "rU5", + "residue_name_number" + ] + ] + }, + { + "sid": 51, + "sent": "The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c).", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "C-terminal region", + "structure_element" + ], + [ + 29, + 45, + "inter-RRM linker", + "structure_element" + ], + [ + 81, + 97, + "rU4-binding site", + "site" + ], + [ + 109, + 113, + "V254", + "residue_name_number" + ], + [ + 136, + 140, + "D256", + "residue_name_number" + ], + [ + 166, + 170, + "K260", + "residue_name_number" + ], + [ + 185, + 198, + "hydrogen bond", + "bond_interaction" + ], + [ + 208, + 211, + "rU4", + "residue_name_number" + ] + ] + }, + { + "sid": 52, + "sent": "Otherwise, the rU4 nucleotide packs against F304 in the signature ribonucleoprotein consensus motif (RNP)-2 of RRM2.", + "section": "RESULTS", + "ner": [ + [ + 15, + 18, + "rU4", + "residue_name_number" + ], + [ + 19, + 29, + "nucleotide", + "chemical" + ], + [ + 44, + 48, + "F304", + "residue_name_number" + ], + [ + 66, + 107, + "ribonucleoprotein consensus motif (RNP)-2", + "structure_element" + ], + [ + 111, + 115, + "RRM2", + "structure_element" + ] + ] + }, + { + "sid": 53, + "sent": "At the opposite side of the central fifth nucleotide, the sixth rU6 nucleotide is located at the inter-RRM1/RRM2 interface (Fig. 3e; Supplementary Movie 1).", + "section": "RESULTS", + "ner": [ + [ + 36, + 41, + "fifth", + "residue_number" + ], + [ + 42, + 52, + "nucleotide", + "chemical" + ], + [ + 58, + 63, + "sixth", + "residue_number" + ], + [ + 64, + 67, + "rU6", + "residue_name_number" + ], + [ + 68, + 78, + "nucleotide", + "chemical" + ], + [ + 97, + 122, + "inter-RRM1/RRM2 interface", + "site" + ] + ] + }, + { + "sid": 54, + "sent": "This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the \u03b22/\u03b23 loops of RRM1 and RRM2.", + "section": "RESULTS", + "ner": [ + [ + 5, + 15, + "nucleotide", + "chemical" + ], + [ + 45, + 51, + "U2AF65", + "protein" + ], + [ + 52, + 58, + "linker", + "structure_element" + ], + [ + 83, + 86, + "rU6", + "residue_name_number" + ], + [ + 87, + 93, + "uracil", + "residue_name" + ], + [ + 122, + 133, + "\u03b22/\u03b23 loops", + "structure_element" + ], + [ + 137, + 141, + "RRM1", + "structure_element" + ], + [ + 146, + 150, + "RRM2", + "structure_element" + ] + ] + }, + { + "sid": 55, + "sent": "The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "rU6", + "residue_name_number" + ], + [ + 32, + 47, + "solvent exposed", + "protein_state" + ], + [ + 66, + 69, + "rU6", + "residue_name_number" + ], + [ + 70, + 84, + "hydrogen bonds", + "bond_interaction" + ], + [ + 90, + 96, + "U2AF65", + "protein" + ], + [ + 101, + 106, + "water", + "chemical" + ], + [ + 162, + 166, + "RRM1", + "structure_element" + ], + [ + 167, + 171, + "N196", + "residue_name_number" + ] + ] + }, + { + "sid": 56, + "sent": "We tested the contribution of the U2AF651,2L interactions with the new central nucleotide to Py-tract affinity (Fig. 3i; Supplementary Fig. 4a,b).", + "section": "RESULTS", + "ner": [ + [ + 3, + 26, + "tested the contribution", + "experimental_method" + ], + [ + 34, + 44, + "U2AF651,2L", + "mutant" + ], + [ + 79, + 89, + "nucleotide", + "chemical" + ], + [ + 93, + 110, + "Py-tract affinity", + "evidence" + ] + ] + }, + { + "sid": 57, + "sent": "Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1\u2013R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively.", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Mutagenesis", + "experimental_method" + ], + [ + 22, + 26, + "V254", + "residue_name_number" + ], + [ + 34, + 40, + "U2AF65", + "protein" + ], + [ + 41, + 57, + "inter-RRM linker", + "structure_element" + ], + [ + 61, + 68, + "proline", + "residue_name" + ], + [ + 72, + 76, + "RRM1", + "structure_element" + ], + [ + 77, + 81, + "R227", + "residue_name_number" + ], + [ + 85, + 92, + "alanine", + "residue_name" + ], + [ + 111, + 124, + "hydrogen bond", + "bond_interaction" + ], + [ + 134, + 139, + "fifth", + "residue_number" + ], + [ + 140, + 146, + "uracil", + "residue_name" + ], + [ + 170, + 180, + "affinities", + "evidence" + ], + [ + 184, + 194, + "U2AF651,2L", + "mutant" + ], + [ + 218, + 222, + "AdML", + "gene" + ], + [ + 223, + 231, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 58, + "sent": "The energetic penalties due to these mutations (\u0394\u0394G 0.8\u20130.9\u2009kcal\u2009mol\u22121) are consistent with the loss of each hydrogen bond with the rU5 base and support the relevance of the central nucleotide interactions observed in the U2AF651,2L structures.", + "section": "RESULTS", + "ner": [ + [ + 48, + 51, + "\u0394\u0394G", + "evidence" + ], + [ + 109, + 122, + "hydrogen bond", + "bond_interaction" + ], + [ + 132, + 135, + "rU5", + "residue_name_number" + ], + [ + 222, + 232, + "U2AF651,2L", + "mutant" + ], + [ + 233, + 243, + "structures", + "evidence" + ] + ] + }, + { + "sid": 59, + "sent": "U2AF65 RRM extensions interact with the Py tract", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "U2AF65", + "protein" + ], + [ + 7, + 21, + "RRM extensions", + "structure_element" + ], + [ + 40, + 48, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 60, + "sent": "The N- and C-terminal extensions of the U2AF65 RRM1 and RRM2 directly contact the bound Py tract.", + "section": "RESULTS", + "ner": [ + [ + 4, + 32, + "N- and C-terminal extensions", + "structure_element" + ], + [ + 40, + 46, + "U2AF65", + "protein" + ], + [ + 47, + 51, + "RRM1", + "structure_element" + ], + [ + 56, + 60, + "RRM2", + "structure_element" + ], + [ + 82, + 87, + "bound", + "protein_state" + ], + [ + 88, + 96, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 61, + "sent": "Rather than interacting with a new 5\u2032-terminal nucleotide as we had hypothesized, the C-terminal \u03b1-helix of RRM2 instead folds across one surface of rU3 in the third binding site (Fig. 3b).", + "section": "RESULTS", + "ner": [ + [ + 47, + 57, + "nucleotide", + "chemical" + ], + [ + 97, + 104, + "\u03b1-helix", + "structure_element" + ], + [ + 108, + 112, + "RRM2", + "structure_element" + ], + [ + 149, + 152, + "rU3", + "residue_name_number" + ], + [ + 160, + 178, + "third binding site", + "site" + ] + ] + }, + { + "sid": 62, + "sent": "There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension.", + "section": "RESULTS", + "ner": [ + [ + 9, + 20, + "salt bridge", + "bond_interaction" + ], + [ + 33, + 37, + "K340", + "residue_name_number" + ], + [ + 53, + 63, + "nucleotide", + "chemical" + ], + [ + 86, + 90, + "G338", + "residue_name_number" + ], + [ + 96, + 104, + "stacking", + "bond_interaction" + ], + [ + 111, + 124, + "hydrogen bond", + "bond_interaction" + ], + [ + 155, + 159, + "G338", + "residue_name_number" + ], + [ + 168, + 171, + "rU3", + "residue_name_number" + ], + [ + 187, + 201, + "RRM2 extension", + "structure_element" + ] + ] + }, + { + "sid": 63, + "sent": "Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal \u03b2-strand of RRM2.", + "section": "RESULTS", + "ner": [ + [ + 45, + 50, + "third", + "residue_number" + ], + [ + 51, + 61, + "nucleotide", + "chemical" + ], + [ + 72, + 75, + "rU2", + "residue_name_number" + ], + [ + 76, + 86, + "nucleotide", + "chemical" + ], + [ + 94, + 113, + "second binding site", + "site" + ], + [ + 139, + 147, + "\u03b2-strand", + "structure_element" + ], + [ + 151, + 155, + "RRM2", + "structure_element" + ] + ] + }, + { + "sid": 64, + "sent": "Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b).", + "section": "RESULTS", + "ner": [ + [ + 18, + 34, + "U2AF651,2L-bound", + "protein_state" + ], + [ + 35, + 38, + "rU2", + "residue_name_number" + ], + [ + 61, + 75, + "hydrogen bonds", + "bond_interaction" + ], + [ + 85, + 89, + "K329", + "residue_name_number" + ], + [ + 137, + 150, + "hydrogen bond", + "bond_interaction" + ], + [ + 160, + 164, + "K329", + "residue_name_number" + ], + [ + 192, + 202, + "dU2AF651,2", + "mutant" + ], + [ + 203, + 212, + "structure", + "evidence" + ] + ] + }, + { + "sid": 65, + "sent": "At the N terminus, the \u03b1-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3\u2032 terminus of the Py tract (Fig. 3f\u2013h).", + "section": "RESULTS", + "ner": [ + [ + 23, + 42, + "\u03b1-helical extension", + "structure_element" + ], + [ + 46, + 52, + "U2AF65", + "protein" + ], + [ + 53, + 57, + "RRM1", + "structure_element" + ], + [ + 72, + 76, + "Q147", + "residue_name_number" + ], + [ + 102, + 108, + "eighth", + "residue_number" + ], + [ + 113, + 118, + "ninth", + "residue_number" + ], + [ + 119, + 130, + "nucleotides", + "chemical" + ], + [ + 138, + 149, + "3\u2032 terminus", + "site" + ], + [ + 157, + 165, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 66, + "sent": "The Q147 residue participates in hydrogen bonds with the -N3H of the eighth uracil and -O2 of the ninth pyrimidine.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "Q147", + "residue_name_number" + ], + [ + 33, + 47, + "hydrogen bonds", + "bond_interaction" + ], + [ + 69, + 75, + "eighth", + "residue_number" + ], + [ + 76, + 82, + "uracil", + "residue_name" + ], + [ + 98, + 103, + "ninth", + "residue_number" + ], + [ + 104, + 114, + "pyrimidine", + "chemical" + ] + ] + }, + { + "sid": 67, + "sent": "The adjacent R146 guanidinium group donates hydrogen bonds to the 3\u2032-terminal ribose-O2\u2032 and O3\u2032 atoms, where it could form a salt bridge with a phospho-diester group in the context of a longer pre-mRNA.", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "R146", + "residue_name_number" + ], + [ + 44, + 58, + "hydrogen bonds", + "bond_interaction" + ], + [ + 78, + 84, + "ribose", + "chemical" + ], + [ + 126, + 137, + "salt bridge", + "bond_interaction" + ], + [ + 194, + 202, + "pre-mRNA", + "chemical" + ] + ] + }, + { + "sid": 68, + "sent": "Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (\u0394\u0394G 1.0\u2009kcal\u2009mol\u22121), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c).", + "section": "RESULTS", + "ner": [ + [ + 26, + 39, + "hydrogen bond", + "bond_interaction" + ], + [ + 49, + 54, + "ninth", + "residue_number" + ], + [ + 55, + 65, + "pyrimidine", + "chemical" + ], + [ + 70, + 73, + "\u0394\u0394G", + "evidence" + ], + [ + 91, + 99, + "mutation", + "experimental_method" + ], + [ + 107, + 111, + "Q147", + "residue_name_number" + ], + [ + 118, + 125, + "alanine", + "residue_name" + ], + [ + 134, + 153, + "U2AF651,2L affinity", + "evidence" + ], + [ + 162, + 166, + "AdML", + "gene" + ], + [ + 167, + 175, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 69, + "sent": "We compare U2AF65 interactions with uracil relative to cytosine pyrimidines at the ninth binding site in Fig. 3g,h and the Supplementary Discussion.", + "section": "RESULTS", + "ner": [ + [ + 3, + 10, + "compare", + "experimental_method" + ], + [ + 11, + 17, + "U2AF65", + "protein" + ], + [ + 36, + 42, + "uracil", + "residue_name" + ], + [ + 55, + 63, + "cytosine", + "residue_name" + ], + [ + 64, + 75, + "pyrimidines", + "chemical" + ], + [ + 83, + 101, + "ninth binding site", + "site" + ] + ] + }, + { + "sid": 70, + "sent": "Versatile primary sequence of the U2AF65 inter-RRM linker", + "section": "RESULTS", + "ner": [ + [ + 34, + 40, + "U2AF65", + "protein" + ], + [ + 41, + 57, + "inter-RRM linker", + "structure_element" + ] + ] + }, + { + "sid": 71, + "sent": "The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second \u03b1-helix of RRM1, the \u03b22/\u03b23 strands of RRM2 and the N-terminal \u03b1-helical extension of RRM1.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "U2AF651,2L", + "mutant" + ], + [ + 15, + 25, + "structures", + "evidence" + ], + [ + 42, + 58, + "inter-RRM linker", + "structure_element" + ], + [ + 71, + 90, + "extensive interface", + "site" + ], + [ + 107, + 114, + "\u03b1-helix", + "structure_element" + ], + [ + 118, + 122, + "RRM1", + "structure_element" + ], + [ + 128, + 141, + "\u03b22/\u03b23 strands", + "structure_element" + ], + [ + 145, + 149, + "RRM2", + "structure_element" + ], + [ + 169, + 188, + "\u03b1-helical extension", + "structure_element" + ], + [ + 192, + 196, + "RRM1", + "structure_element" + ] + ] + }, + { + "sid": 72, + "sent": "Altogether, the U2AF65 inter-RRM linker residues (R228\u2013K260) bury 2,800\u2009\u00c52 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900\u2009\u00c52 for a typical protein\u2013protein complex.", + "section": "RESULTS", + "ner": [ + [ + 16, + 22, + "U2AF65", + "protein" + ], + [ + 23, + 39, + "inter-RRM linker", + "structure_element" + ], + [ + 50, + 59, + "R228\u2013K260", + "residue_range" + ], + [ + 98, + 108, + "U2AF651,2L", + "mutant" + ], + [ + 109, + 121, + "holo-protein", + "protein_state" + ], + [ + 139, + 156, + "cognate interface", + "site" + ] + ] + }, + { + "sid": 73, + "sent": "The path of the linker initiates at P229 following the core RRM1 \u03b2-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right).", + "section": "RESULTS", + "ner": [ + [ + 16, + 22, + "linker", + "structure_element" + ], + [ + 36, + 40, + "P229", + "residue_name_number" + ], + [ + 55, + 59, + "core", + "protein_state" + ], + [ + 60, + 64, + "RRM1", + "structure_element" + ], + [ + 65, + 73, + "\u03b2-strand", + "structure_element" + ], + [ + 80, + 84, + "kink", + "structure_element" + ], + [ + 107, + 131, + "intra-molecular stacking", + "bond_interaction" + ], + [ + 154, + 158, + "R228", + "residue_name_number" + ], + [ + 160, + 164, + "Y232", + "residue_name_number" + ], + [ + 169, + 173, + "P234", + "residue_name_number" + ] + ] + }, + { + "sid": 74, + "sent": "A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal \u03b1-helical RRM1 extension and the core RRM1 \u03b12-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site.", + "section": "RESULTS", + "ner": [ + [ + 2, + 13, + "second kink", + "structure_element" + ], + [ + 17, + 21, + "P236", + "residue_name_number" + ], + [ + 62, + 66, + "L235", + "residue_name_number" + ], + [ + 71, + 75, + "M238", + "residue_name_number" + ], + [ + 106, + 130, + "\u03b1-helical RRM1 extension", + "structure_element" + ], + [ + 139, + 143, + "core", + "protein_state" + ], + [ + 144, + 148, + "RRM1", + "structure_element" + ], + [ + 149, + 157, + "\u03b12-helix", + "structure_element" + ], + [ + 189, + 205, + "inter-RRM linker", + "structure_element" + ], + [ + 218, + 237, + "RRM1/RRM2 interface", + "site" + ], + [ + 256, + 272, + "RNA-binding site", + "site" + ] + ] + }, + { + "sid": 75, + "sent": "In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two \u03b1-helices of the core RRM1.", + "section": "RESULTS", + "ner": [ + [ + 41, + 47, + "linker", + "structure_element" + ], + [ + 53, + 57, + "V244", + "residue_name_number" + ], + [ + 62, + 66, + "V246", + "residue_name_number" + ], + [ + 89, + 107, + "hydrophobic pocket", + "site" + ], + [ + 120, + 129, + "\u03b1-helices", + "structure_element" + ], + [ + 137, + 141, + "core", + "protein_state" + ], + [ + 142, + 146, + "RRM1", + "structure_element" + ] + ] + }, + { + "sid": 76, + "sent": "The adjacent V249 and V250 are notable for their respective interactions that connect RRM1 and RRM2 at this distal interface from the RNA-binding site (Fig. 4a, top).", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "V249", + "residue_name_number" + ], + [ + 22, + 26, + "V250", + "residue_name_number" + ], + [ + 86, + 90, + "RRM1", + "structure_element" + ], + [ + 95, + 99, + "RRM2", + "structure_element" + ], + [ + 115, + 124, + "interface", + "site" + ], + [ + 134, + 150, + "RNA-binding site", + "site" + ] + ] + }, + { + "sid": 77, + "sent": "A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA.", + "section": "RESULTS", + "ner": [ + [ + 2, + 12, + "third kink", + "structure_element" + ], + [ + 13, + 19, + "stacks", + "bond_interaction" + ], + [ + 20, + 24, + "P247", + "residue_name_number" + ], + [ + 29, + 33, + "G248", + "residue_name_number" + ], + [ + 39, + 43, + "Y245", + "residue_name_number" + ], + [ + 63, + 80, + "C-terminal region", + "structure_element" + ], + [ + 88, + 94, + "linker", + "structure_element" + ], + [ + 107, + 111, + "RRM2", + "structure_element" + ], + [ + 116, + 121, + "bound", + "protein_state" + ], + [ + 122, + 125, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 78, + "sent": "At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the \u03b2-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left).", + "section": "RESULTS", + "ner": [ + [ + 7, + 10, + "RNA", + "chemical" + ], + [ + 28, + 32, + "V254", + "residue_name_number" + ], + [ + 53, + 58, + "fifth", + "residue_number" + ], + [ + 59, + 65, + "uracil", + "residue_name" + ], + [ + 81, + 101, + "hydrophobic contacts", + "bond_interaction" + ], + [ + 133, + 148, + "\u03b2-sheet surface", + "structure_element" + ], + [ + 152, + 156, + "RRM2", + "structure_element" + ], + [ + 180, + 184, + "RNP1", + "structure_element" + ], + [ + 185, + 189, + "F304", + "residue_name_number" + ], + [ + 203, + 209, + "stacks", + "bond_interaction" + ], + [ + 219, + 225, + "fourth", + "residue_number" + ], + [ + 226, + 232, + "uracil", + "residue_name" + ] + ] + }, + { + "sid": 79, + "sent": "Few direct contacts are made between the remaining residues of the linker and the U2AF65 RRM2; instead, the C-terminal conformation of the linker appears primarily RNA mediated (Fig. 3c,d).", + "section": "RESULTS", + "ner": [ + [ + 67, + 73, + "linker", + "structure_element" + ], + [ + 82, + 88, + "U2AF65", + "protein" + ], + [ + 89, + 93, + "RRM2", + "structure_element" + ], + [ + 139, + 145, + "linker", + "structure_element" + ], + [ + 164, + 167, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 80, + "sent": "We investigated whether the observed contacts between the RRMs and linker were critical for RNA binding by structure-guided mutagenesis (Fig. 4b).", + "section": "RESULTS", + "ner": [ + [ + 58, + 62, + "RRMs", + "structure_element" + ], + [ + 67, + 73, + "linker", + "structure_element" + ], + [ + 107, + 135, + "structure-guided mutagenesis", + "experimental_method" + ] + ] + }, + { + "sid": 81, + "sent": "We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d\u2013h).", + "section": "RESULTS", + "ner": [ + [ + 3, + 11, + "titrated", + "experimental_method" + ], + [ + 18, + 24, + "mutant", + "protein_state" + ], + [ + 25, + 35, + "U2AF651,2L", + "mutant" + ], + [ + 50, + 61, + "fluorescein", + "chemical" + ], + [ + 71, + 75, + "AdML", + "gene" + ], + [ + 76, + 88, + "Py-tract RNA", + "chemical" + ], + [ + 101, + 132, + "fluorescence anisotropy changes", + "evidence" + ], + [ + 156, + 178, + "equilibrium affinities", + "evidence" + ] + ] + }, + { + "sid": 82, + "sent": "We introduced glycine substitutions to maximally reduce the buried surface area without directly interfering with its hydrogen bonds between backbone atoms and the base.", + "section": "RESULTS", + "ner": [ + [ + 14, + 21, + "glycine", + "residue_name" + ], + [ + 22, + 35, + "substitutions", + "experimental_method" + ], + [ + 118, + 132, + "hydrogen bonds", + "bond_interaction" + ] + ] + }, + { + "sid": 83, + "sent": "First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly).", + "section": "RESULTS", + "ner": [ + [ + 10, + 18, + "replaced", + "experimental_method" + ], + [ + 19, + 23, + "V249", + "residue_name_number" + ], + [ + 28, + 32, + "V250", + "residue_name_number" + ], + [ + 40, + 59, + "RRM1/RRM2 interface", + "site" + ], + [ + 64, + 68, + "V254", + "residue_name_number" + ], + [ + 76, + 81, + "bound", + "protein_state" + ], + [ + 82, + 85, + "RNA", + "chemical" + ], + [ + 96, + 103, + "glycine", + "residue_name" + ], + [ + 105, + 109, + "3Gly", + "mutant" + ] + ] + }, + { + "sid": 84, + "sent": "However, the resulting decrease in the AdML RNA affinity of the U2AF651,2L-3Gly mutant relative to wild-type protein was not significant (Fig. 4b).", + "section": "RESULTS", + "ner": [ + [ + 39, + 43, + "AdML", + "gene" + ], + [ + 44, + 56, + "RNA affinity", + "evidence" + ], + [ + 64, + 79, + "U2AF651,2L-3Gly", + "mutant" + ], + [ + 80, + 86, + "mutant", + "protein_state" + ], + [ + 99, + 108, + "wild-type", + "protein_state" + ], + [ + 109, + 116, + "protein", + "protein" + ] + ] + }, + { + "sid": 85, + "sent": "In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein.", + "section": "RESULTS", + "ner": [ + [ + 16, + 24, + "replaced", + "experimental_method" + ], + [ + 30, + 45, + "linker residues", + "structure_element" + ], + [ + 47, + 51, + "S251", + "residue_name_number" + ], + [ + 53, + 57, + "T252", + "residue_name_number" + ], + [ + 59, + 63, + "V253", + "residue_name_number" + ], + [ + 65, + 69, + "V254", + "residue_name_number" + ], + [ + 74, + 78, + "P255", + "residue_name_number" + ], + [ + 87, + 116, + "fifth nucleotide-binding site", + "site" + ], + [ + 122, + 130, + "glycines", + "residue_name" + ], + [ + 132, + 136, + "5Gly", + "mutant" + ], + [ + 162, + 174, + "RNA affinity", + "evidence" + ], + [ + 182, + 197, + "U2AF651,2L-5Gly", + "mutant" + ], + [ + 198, + 204, + "mutant", + "protein_state" + ], + [ + 250, + 259, + "wild-type", + "protein_state" + ], + [ + 260, + 267, + "protein", + "protein" + ] + ] + }, + { + "sid": 86, + "sent": "A more conservative substitution of these five residues (251\u2013255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract.", + "section": "RESULTS", + "ner": [ + [ + 7, + 32, + "conservative substitution", + "experimental_method" + ], + [ + 57, + 64, + "251\u2013255", + "residue_range" + ], + [ + 122, + 136, + "hydrogen bonds", + "bond_interaction" + ], + [ + 138, + 149, + "STVVP>NLALA", + "mutant" + ], + [ + 197, + 215, + "inter-RRM sequence", + "structure_element" + ], + [ + 219, + 227, + "affinity", + "evidence" + ], + [ + 236, + 240, + "AdML", + "gene" + ], + [ + 241, + 249, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 87, + "sent": "Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly).", + "section": "RESULTS", + "ner": [ + [ + 81, + 87, + "linker", + "structure_element" + ], + [ + 88, + 91, + "RRM", + "structure_element" + ], + [ + 105, + 116, + "substituted", + "experimental_method" + ], + [ + 117, + 124, + "glycine", + "residue_name" + ], + [ + 180, + 196, + "inter-RRM linker", + "structure_element" + ], + [ + 208, + 212, + "M144", + "residue_name_number" + ], + [ + 214, + 218, + "L235", + "residue_name_number" + ], + [ + 220, + 224, + "M238", + "residue_name_number" + ], + [ + 226, + 230, + "V244", + "residue_name_number" + ], + [ + 232, + 236, + "V246", + "residue_name_number" + ], + [ + 238, + 242, + "V249", + "residue_name_number" + ], + [ + 244, + 248, + "V250", + "residue_name_number" + ], + [ + 250, + 254, + "S251", + "residue_name_number" + ], + [ + 256, + 260, + "T252", + "residue_name_number" + ], + [ + 262, + 266, + "V253", + "residue_name_number" + ], + [ + 268, + 272, + "V254", + "residue_name_number" + ], + [ + 274, + 278, + "P255", + "residue_name_number" + ], + [ + 287, + 292, + "12Gly", + "mutant" + ] + ] + }, + { + "sid": 88, + "sent": "Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i).", + "section": "RESULTS", + "ner": [ + [ + 8, + 31, + "12 concurrent mutations", + "experimental_method" + ], + [ + 37, + 41, + "AdML", + "gene" + ], + [ + 42, + 54, + "RNA affinity", + "evidence" + ], + [ + 62, + 78, + "U2AF651,2L-12Gly", + "mutant" + ], + [ + 79, + 86, + "variant", + "protein_state" + ], + [ + 134, + 144, + "unmodified", + "protein_state" + ], + [ + 145, + 152, + "protein", + "protein" + ], + [ + 202, + 207, + "V254P", + "mutant" + ], + [ + 235, + 238, + "rU5", + "residue_name_number" + ], + [ + 239, + 252, + "hydrogen bond", + "bond_interaction" + ] + ] + }, + { + "sid": 89, + "sent": "To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L).", + "section": "RESULTS", + "ner": [ + [ + 29, + 35, + "U2AF65", + "protein" + ], + [ + 36, + 52, + "inter-RRM linker", + "structure_element" + ], + [ + 80, + 94, + "RRM extensions", + "structure_element" + ], + [ + 99, + 110, + "constructed", + "experimental_method" + ], + [ + 123, + 138, + "linker deletion", + "experimental_method" + ], + [ + 142, + 153, + "20-residues", + "residue_range" + ], + [ + 165, + 173, + "extended", + "protein_state" + ], + [ + 174, + 192, + "RNA-binding domain", + "structure_element" + ], + [ + 194, + 205, + "dU2AF651,2L", + "mutant" + ] + ] + }, + { + "sid": 90, + "sent": "We found that the affinity of dU2AF651,2L for the AdML RNA was significantly reduced relative to U2AF651,2L (four-fold, Figs 1b and 4b; Supplementary Fig. 4i).", + "section": "RESULTS", + "ner": [ + [ + 18, + 26, + "affinity", + "evidence" + ], + [ + 30, + 41, + "dU2AF651,2L", + "mutant" + ], + [ + 50, + 54, + "AdML", + "gene" + ], + [ + 55, + 58, + "RNA", + "chemical" + ], + [ + 97, + 107, + "U2AF651,2L", + "mutant" + ] + ] + }, + { + "sid": 91, + "sent": "Yet, it is well known that the linker deletion in the context of the minimal RRM1\u2013RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j).", + "section": "RESULTS", + "ner": [ + [ + 31, + 46, + "linker deletion", + "experimental_method" + ], + [ + 69, + 76, + "minimal", + "protein_state" + ], + [ + 77, + 81, + "RRM1", + "structure_element" + ], + [ + 82, + 86, + "RRM2", + "structure_element" + ], + [ + 130, + 144, + "RNA affinities", + "evidence" + ], + [ + 148, + 158, + "dU2AF651,2", + "mutant" + ], + [ + 173, + 182, + "U2AF651,2", + "mutant" + ] + ] + }, + { + "sid": 92, + "sent": "The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24\u2009\u00c5 distance between U2AF651,2L R227-C\u03b1\u2013H259-C\u03b1 atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "U2AF651,2L", + "mutant" + ], + [ + 15, + 25, + "structures", + "evidence" + ], + [ + 42, + 63, + "extended conformation", + "protein_state" + ], + [ + 71, + 80, + "truncated", + "protein_state" + ], + [ + 81, + 91, + "dU2AF651,2", + "mutant" + ], + [ + 92, + 108, + "inter-RRM linker", + "structure_element" + ], + [ + 138, + 148, + "U2AF651,2L", + "mutant" + ], + [ + 149, + 153, + "RRM1", + "structure_element" + ], + [ + 186, + 190, + "RRM2", + "structure_element" + ], + [ + 214, + 224, + "U2AF651,2L", + "mutant" + ], + [ + 225, + 229, + "R227", + "residue_name_number" + ], + [ + 233, + 237, + "H259", + "residue_name_number" + ], + [ + 279, + 293, + "RNA affinities", + "evidence" + ], + [ + 297, + 307, + "dU2AF651,2", + "mutant" + ], + [ + 312, + 321, + "U2AF651,2", + "mutant" + ], + [ + 322, + 326, + "dual", + "protein_state" + ], + [ + 327, + 331, + "RRMs", + "structure_element" + ], + [ + 350, + 360, + "individual", + "protein_state" + ], + [ + 361, + 367, + "U2AF65", + "protein" + ], + [ + 368, + 372, + "RRMs", + "structure_element" + ] + ] + }, + { + "sid": 93, + "sent": "However, stretching of the truncated dU2AF651,2L linker to connect the RRM termini is expected to disrupt its nucleotide interactions.", + "section": "RESULTS", + "ner": [ + [ + 27, + 36, + "truncated", + "protein_state" + ], + [ + 37, + 48, + "dU2AF651,2L", + "mutant" + ], + [ + 49, + 55, + "linker", + "structure_element" + ], + [ + 71, + 82, + "RRM termini", + "structure_element" + ] + ] + }, + { + "sid": 94, + "sent": "Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L.", + "section": "RESULTS", + "ner": [ + [ + 10, + 18, + "deletion", + "experimental_method" + ], + [ + 37, + 51, + "RRM1 extension", + "structure_element" + ], + [ + 59, + 68, + "shortened", + "protein_state" + ], + [ + 132, + 138, + "linker", + "structure_element" + ], + [ + 144, + 155, + "kinked turn", + "structure_element" + ], + [ + 166, + 170, + "P229", + "residue_name_number" + ], + [ + 208, + 222, + "RNA affinities", + "evidence" + ], + [ + 226, + 237, + "dU2AF651,2L", + "mutant" + ], + [ + 239, + 249, + "dU2AF651,2", + "mutant" + ], + [ + 254, + 263, + "U2AF651,2", + "mutant" + ], + [ + 278, + 288, + "U2AF651,2L", + "mutant" + ] + ] + }, + { + "sid": 95, + "sent": "To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d).", + "section": "RESULTS", + "ner": [ + [ + 38, + 44, + "U2AF65", + "protein" + ], + [ + 45, + 59, + "RRM extensions", + "structure_element" + ], + [ + 64, + 80, + "inter-RRM linker", + "structure_element" + ], + [ + 135, + 140, + "Q147A", + "mutant" + ], + [ + 141, + 146, + "V254P", + "mutant" + ], + [ + 147, + 152, + "R227A", + "mutant" + ], + [ + 153, + 161, + "mutation", + "experimental_method" + ], + [ + 163, + 178, + "U2AF651,2L-3Mut", + "mutant" + ] + ] + }, + { + "sid": 96, + "sent": "Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the \u0394\u0394G's for the single mutations.", + "section": "RESULTS", + "ner": [ + [ + 13, + 18, + "Q147A", + "mutant" + ], + [ + 19, + 24, + "V254P", + "mutant" + ], + [ + 25, + 30, + "R227A", + "mutant" + ], + [ + 31, + 39, + "mutation", + "experimental_method" + ], + [ + 52, + 64, + "RNA affinity", + "evidence" + ], + [ + 72, + 87, + "U2AF651,2L-3Mut", + "mutant" + ], + [ + 167, + 170, + "\u0394\u0394G", + "evidence" + ] + ] + }, + { + "sid": 97, + "sent": "This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract.", + "section": "RESULTS", + "ner": [ + [ + 35, + 51, + "linearly distant", + "protein_state" + ], + [ + 52, + 59, + "regions", + "structure_element" + ], + [ + 67, + 73, + "U2AF65", + "protein" + ], + [ + 102, + 106, + "Q147", + "residue_name_number" + ], + [ + 125, + 139, + "RRM1 extension", + "structure_element" + ], + [ + 144, + 148, + "R227", + "residue_name_number" + ], + [ + 149, + 153, + "V254", + "residue_name_number" + ], + [ + 175, + 189, + "linker regions", + "structure_element" + ], + [ + 197, + 218, + "fifth nucleotide site", + "site" + ], + [ + 248, + 256, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 98, + "sent": "Altogether, we conclude that the conformation of the U2AF65 inter-RRM linker is key for recognizing RNA and is positioned by the RRM extension but otherwise relatively independent of the side chain composition.", + "section": "RESULTS", + "ner": [ + [ + 53, + 59, + "U2AF65", + "protein" + ], + [ + 60, + 76, + "inter-RRM linker", + "structure_element" + ], + [ + 100, + 103, + "RNA", + "chemical" + ], + [ + 129, + 142, + "RRM extension", + "structure_element" + ] + ] + }, + { + "sid": 99, + "sent": "The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs.", + "section": "RESULTS", + "ner": [ + [ + 32, + 37, + "Q147A", + "mutant" + ], + [ + 38, + 43, + "V254P", + "mutant" + ], + [ + 44, + 49, + "R227A", + "mutant" + ], + [ + 50, + 65, + "triple mutation", + "experimental_method" + ], + [ + 127, + 133, + "U2AF65", + "protein" + ], + [ + 134, + 149, + "linker deletion", + "experimental_method" + ], + [ + 215, + 221, + "U2AF65", + "protein" + ], + [ + 222, + 228, + "linker", + "structure_element" + ], + [ + 237, + 265, + "N- and C-terminal extensions", + "structure_element" + ], + [ + 279, + 283, + "core", + "protein_state" + ], + [ + 284, + 288, + "RRMs", + "structure_element" + ] + ] + }, + { + "sid": 100, + "sent": "Importance of U2AF65\u2013RNA contacts for pre-mRNA splicing", + "section": "RESULTS", + "ner": [ + [ + 14, + 24, + "U2AF65\u2013RNA", + "complex_assembly" + ], + [ + 38, + 46, + "pre-mRNA", + "chemical" + ] + ] + }, + { + "sid": 101, + "sent": "We proceeded to test the importance of new U2AF65\u2013Py-tract interactions for splicing of a model pre-mRNA substrate in a human cell line (Fig. 5; Supplementary Fig. 5).", + "section": "RESULTS", + "ner": [ + [ + 43, + 58, + "U2AF65\u2013Py-tract", + "complex_assembly" + ], + [ + 96, + 104, + "pre-mRNA", + "chemical" + ], + [ + 120, + 125, + "human", + "species" + ] + ] + }, + { + "sid": 102, + "sent": "As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a).", + "section": "RESULTS", + "ner": [ + [ + 73, + 99, + "minigene splicing reporter", + "chemical" + ], + [ + 108, + 112, + "pyPY", + "chemical" + ], + [ + 154, + 156, + "py", + "chemical" + ], + [ + 179, + 185, + "U-rich", + "structure_element" + ], + [ + 187, + 189, + "PY", + "chemical" + ], + [ + 191, + 212, + "polypyrimidine tracts", + "chemical" + ], + [ + 239, + 251, + "splice sites", + "site" + ] + ] + }, + { + "sid": 103, + "sent": "When transfected into HEK293T cells containing only endogenous U2AF65, the PY splice site is used and the remaining transcript remains unspliced.", + "section": "RESULTS", + "ner": [ + [ + 5, + 16, + "transfected", + "experimental_method" + ], + [ + 52, + 62, + "endogenous", + "protein_state" + ], + [ + 63, + 69, + "U2AF65", + "protein" + ], + [ + 75, + 89, + "PY splice site", + "site" + ] + ] + }, + { + "sid": 104, + "sent": "When co-transfected with an expression plasmid for wild-type U2AF65, use of the py splice site significantly increases (by more than five-fold) and as documented converts a fraction of the unspliced to spliced transcript.", + "section": "RESULTS", + "ner": [ + [ + 5, + 19, + "co-transfected", + "experimental_method" + ], + [ + 28, + 46, + "expression plasmid", + "experimental_method" + ], + [ + 51, + 60, + "wild-type", + "protein_state" + ], + [ + 61, + 67, + "U2AF65", + "protein" + ], + [ + 80, + 94, + "py splice site", + "site" + ] + ] + }, + { + "sid": 105, + "sent": "The strong PY splice site is insensitive to added U2AF65, suggesting that endogenous U2AF65 levels are sufficient to saturate this site (Supplementary Fig. 5b).", + "section": "RESULTS", + "ner": [ + [ + 11, + 25, + "PY splice site", + "site" + ], + [ + 50, + 56, + "U2AF65", + "protein" + ], + [ + 74, + 84, + "endogenous", + "protein_state" + ], + [ + 85, + 91, + "U2AF65", + "protein" + ] + ] + }, + { + "sid": 106, + "sent": "We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut).", + "section": "RESULTS", + "ner": [ + [ + 18, + 33, + "triple mutation", + "experimental_method" + ], + [ + 35, + 40, + "V254P", + "mutant" + ], + [ + 41, + 46, + "R227A", + "mutant" + ], + [ + 47, + 52, + "Q147A", + "mutant" + ], + [ + 81, + 91, + "U2AF651,2L", + "mutant" + ], + [ + 113, + 121, + "Py tract", + "chemical" + ], + [ + 150, + 161, + "full-length", + "protein_state" + ], + [ + 162, + 168, + "U2AF65", + "protein" + ], + [ + 170, + 181, + "U2AF65-3Mut", + "mutant" + ] + ] + }, + { + "sid": 107, + "sent": "Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak \u2018py' splice site relative to wild-type U2AF65 (Fig. 5b,c).", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Co-transfection", + "experimental_method" + ], + [ + 23, + 34, + "U2AF65-3Mut", + "mutant" + ], + [ + 44, + 48, + "pyPY", + "chemical" + ], + [ + 111, + 127, + "\u2018py' splice site", + "site" + ], + [ + 140, + 149, + "wild-type", + "protein_state" + ], + [ + 150, + 156, + "U2AF65", + "protein" + ] + ] + }, + { + "sid": 108, + "sent": "We conclude that the Py-tract interactions with these residues of the U2AF65 inter-RRM linker and RRM extensions are important for splicing as well as for binding a representative of the major U2-class of splice sites.", + "section": "RESULTS", + "ner": [ + [ + 21, + 29, + "Py-tract", + "chemical" + ], + [ + 70, + 76, + "U2AF65", + "protein" + ], + [ + 77, + 93, + "inter-RRM linker", + "structure_element" + ], + [ + 98, + 112, + "RRM extensions", + "structure_element" + ], + [ + 187, + 217, + "major U2-class of splice sites", + "structure_element" + ] + ] + }, + { + "sid": 109, + "sent": "Sparse inter-RRM contacts underlie apo-U2AF65 dynamics", + "section": "RESULTS", + "ner": [ + [ + 7, + 16, + "inter-RRM", + "structure_element" + ], + [ + 35, + 38, + "apo", + "protein_state" + ], + [ + 39, + 45, + "U2AF65", + "protein" + ] + ] + }, + { + "sid": 110, + "sent": "The direct interface between U2AF651,2L RRM1 and RRM2 is minor, burying 265\u2009\u00c52 of solvent accessible surface area compared with 570\u2009\u00c52 on average for a crystal packing interface.", + "section": "RESULTS", + "ner": [ + [ + 11, + 20, + "interface", + "site" + ], + [ + 29, + 39, + "U2AF651,2L", + "mutant" + ], + [ + 40, + 44, + "RRM1", + "structure_element" + ], + [ + 49, + 53, + "RRM2", + "structure_element" + ] + ] + }, + { + "sid": 111, + "sent": "A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c).", + "section": "RESULTS", + "ner": [ + [ + 13, + 22, + "inter-RRM", + "structure_element" + ], + [ + 23, + 37, + "hydrogen bonds", + "bond_interaction" + ], + [ + 78, + 82, + "RRM1", + "structure_element" + ], + [ + 83, + 87, + "N155", + "residue_name_number" + ], + [ + 92, + 96, + "RRM2", + "structure_element" + ], + [ + 97, + 101, + "K292", + "residue_name_number" + ], + [ + 103, + 107, + "RRM1", + "structure_element" + ], + [ + 108, + 112, + "N155", + "residue_name_number" + ], + [ + 117, + 121, + "RRM2", + "structure_element" + ], + [ + 122, + 126, + "D272", + "residue_name_number" + ], + [ + 160, + 164, + "RRM1", + "structure_element" + ], + [ + 165, + 169, + "G221", + "residue_name_number" + ], + [ + 174, + 178, + "RRM2", + "structure_element" + ], + [ + 179, + 183, + "D273", + "residue_name_number" + ] + ] + }, + { + "sid": 112, + "sent": "This minor U2AF65 RRM1/RRM2 interface, coupled with the versatile sequence of the inter-RRM linker, highlighted the potential role for inter-RRM conformational dynamics in U2AF65-splice site recognition.", + "section": "RESULTS", + "ner": [ + [ + 11, + 17, + "U2AF65", + "protein" + ], + [ + 18, + 37, + "RRM1/RRM2 interface", + "site" + ], + [ + 82, + 98, + "inter-RRM linker", + "structure_element" + ], + [ + 135, + 144, + "inter-RRM", + "structure_element" + ], + [ + 172, + 178, + "U2AF65", + "protein" + ] + ] + }, + { + "sid": 113, + "sent": "Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or \u2018closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor \u2018open' conformation resembling the RNA-bound inter-RRM arrangement.", + "section": "RESULTS", + "ner": [ + [ + 0, + 34, + "Paramagnetic resonance enhancement", + "experimental_method" + ], + [ + 36, + 39, + "PRE", + "experimental_method" + ], + [ + 93, + 105, + "back-to-back", + "protein_state" + ], + [ + 111, + 117, + "closed", + "protein_state" + ], + [ + 139, + 142, + "apo", + "protein_state" + ], + [ + 143, + 152, + "U2AF651,2", + "mutant" + ], + [ + 153, + 157, + "RRM1", + "structure_element" + ], + [ + 162, + 166, + "RRM2", + "structure_element" + ], + [ + 196, + 200, + "open", + "protein_state" + ], + [ + 230, + 239, + "RNA-bound", + "protein_state" + ], + [ + 240, + 249, + "inter-RRM", + "structure_element" + ] + ] + }, + { + "sid": 114, + "sent": "Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the \u2018closed' and \u2018open' conformations.", + "section": "RESULTS", + "ner": [ + [ + 5, + 33, + "small-angle X-ray scattering", + "experimental_method" + ], + [ + 35, + 39, + "SAXS", + "experimental_method" + ], + [ + 70, + 77, + "minimal", + "protein_state" + ], + [ + 78, + 87, + "U2AF651,2", + "mutant" + ], + [ + 121, + 162, + "highly diverse continuum of conformations", + "protein_state" + ], + [ + 170, + 180, + "absence of", + "protein_state" + ], + [ + 181, + 184, + "RNA", + "chemical" + ], + [ + 204, + 210, + "closed", + "protein_state" + ], + [ + 217, + 221, + "open", + "protein_state" + ] + ] + }, + { + "sid": 115, + "sent": "To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution.", + "section": "RESULTS", + "ner": [ + [ + 38, + 48, + "U2AF651,2L", + "mutant" + ], + [ + 49, + 58, + "structure", + "evidence" + ], + [ + 85, + 106, + "X-ray crystallography", + "experimental_method" + ], + [ + 116, + 122, + "smFRET", + "experimental_method" + ], + [ + 143, + 177, + "probability distribution functions", + "evidence" + ], + [ + 201, + 207, + "U2AF65", + "protein" + ], + [ + 208, + 217, + "inter-RRM", + "structure_element" + ] + ] + }, + { + "sid": 116, + "sent": "The inter-RRM dynamics of U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2 (Fig. 6a,b, Methods).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "inter-RRM", + "structure_element" + ], + [ + 26, + 32, + "U2AF65", + "protein" + ], + [ + 53, + 57, + "FRET", + "experimental_method" + ], + [ + 66, + 78, + "fluorophores", + "chemical" + ], + [ + 91, + 95, + "RRM1", + "structure_element" + ], + [ + 100, + 104, + "RRM2", + "structure_element" + ] + ] + }, + { + "sid": 117, + "sent": "The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the \u2018closed' model of apo-U2AF651,2.", + "section": "RESULTS", + "ner": [ + [ + 24, + 32, + "cysteine", + "residue_name" + ], + [ + 33, + 42, + "mutations", + "experimental_method" + ], + [ + 47, + 58, + "fluorophore", + "chemical" + ], + [ + 71, + 76, + "A181C", + "mutant" + ], + [ + 80, + 84, + "RRM1", + "structure_element" + ], + [ + 89, + 94, + "Q324C", + "mutant" + ], + [ + 98, + 102, + "RRM2", + "structure_element" + ], + [ + 143, + 153, + "U2AF651,2L", + "mutant" + ], + [ + 154, + 164, + "structures", + "evidence" + ], + [ + 174, + 180, + "closed", + "protein_state" + ], + [ + 191, + 194, + "apo", + "protein_state" + ], + [ + 195, + 204, + "U2AF651,2", + "mutant" + ] + ] + }, + { + "sid": 118, + "sent": "Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50\u2009\u00c5 for U2AF651,2L\u2013Py tract and 30\u2009\u00c5 for the closed apo-model) that are expected to be near the F\u00f6rster radius (Ro) for the Cy3/Cy5 pair (56\u2009\u00c5), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the \u2018closed' apo-model as opposed to the \u2018open' RNA-bound structures (by \u223c30%).", + "section": "RESULTS", + "ner": [ + [ + 107, + 114, + "RRM/RNA", + "complex_assembly" + ], + [ + 118, + 138, + "inter-RRM interfaces", + "site" + ], + [ + 175, + 194, + "U2AF651,2L\u2013Py tract", + "complex_assembly" + ], + [ + 212, + 218, + "closed", + "protein_state" + ], + [ + 219, + 222, + "apo", + "protein_state" + ], + [ + 263, + 282, + "F\u00f6rster radius (Ro)", + "experimental_method" + ], + [ + 291, + 294, + "Cy3", + "chemical" + ], + [ + 295, + 298, + "Cy5", + "chemical" + ], + [ + 405, + 422, + "FRET efficiencies", + "evidence" + ], + [ + 480, + 486, + "closed", + "protein_state" + ], + [ + 488, + 491, + "apo", + "protein_state" + ], + [ + 517, + 521, + "open", + "protein_state" + ], + [ + 523, + 532, + "RNA-bound", + "protein_state" + ], + [ + 533, + 543, + "structures", + "evidence" + ] + ] + }, + { + "sid": 119, + "sent": "The FRET efficiencies of either of these structurally characterized conformations also are expected to be significantly greater than elongated U2AF65 conformations that lack inter-RRM contacts.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "FRET efficiencies", + "evidence" + ], + [ + 133, + 142, + "elongated", + "protein_state" + ], + [ + 143, + 149, + "U2AF65", + "protein" + ], + [ + 169, + 173, + "lack", + "protein_state" + ], + [ + 180, + 183, + "RRM", + "structure_element" + ] + ] + }, + { + "sid": 120, + "sent": "Double-cysteine variant of U2AF651,2 was modified with equimolar amount of Cy3 and Cy5.", + "section": "RESULTS", + "ner": [ + [ + 7, + 15, + "cysteine", + "residue_name" + ], + [ + 16, + 23, + "variant", + "protein_state" + ], + [ + 27, + 36, + "U2AF651,2", + "mutant" + ], + [ + 41, + 49, + "modified", + "experimental_method" + ], + [ + 75, + 78, + "Cy3", + "chemical" + ], + [ + 83, + 86, + "Cy5", + "chemical" + ] + ] + }, + { + "sid": 121, + "sent": "Only traces that showed single photobleaching events for both donor and acceptor dyes and anti-correlated changes in acceptor and donor fluorescence were included in smFRET data analysis.", + "section": "RESULTS", + "ner": [ + [ + 5, + 11, + "traces", + "evidence" + ], + [ + 166, + 172, + "smFRET", + "experimental_method" + ] + ] + }, + { + "sid": 122, + "sent": "We first characterized the conformational dynamics spectrum of U2AF65 in the absence of RNA (Fig. 6c,d; Supplementary Fig. 7a,b).", + "section": "RESULTS", + "ner": [ + [ + 63, + 69, + "U2AF65", + "protein" + ], + [ + 77, + 87, + "absence of", + "protein_state" + ], + [ + 88, + 91, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 123, + "sent": "The double-labelled U2AF651,2LFRET(Cy3/Cy5) protein was tethered to a slide via biotin-NTA/Ni+2 resin.", + "section": "RESULTS", + "ner": [ + [ + 20, + 34, + "U2AF651,2LFRET", + "mutant" + ], + [ + 35, + 38, + "Cy3", + "chemical" + ], + [ + 39, + 42, + "Cy5", + "chemical" + ], + [ + 56, + 64, + "tethered", + "protein_state" + ], + [ + 80, + 101, + "biotin-NTA/Ni+2 resin", + "chemical" + ] + ] + }, + { + "sid": 124, + "sent": "Virtually no fluorescent molecules were detected in the absence of biotin-NTA/Ni+2, which demonstrates the absence of detectable non-specific binding of U2AF651,2LFRET to the slide.", + "section": "RESULTS", + "ner": [ + [ + 56, + 66, + "absence of", + "protein_state" + ], + [ + 67, + 82, + "biotin-NTA/Ni+2", + "chemical" + ], + [ + 107, + 117, + "absence of", + "protein_state" + ], + [ + 153, + 167, + "U2AF651,2LFRET", + "mutant" + ] + ] + }, + { + "sid": 125, + "sent": "The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of \u223c0.4 (Fig. 6d).", + "section": "RESULTS", + "ner": [ + [ + 4, + 31, + "FRET distribution histogram", + "evidence" + ], + [ + 64, + 70, + "traces", + "evidence" + ], + [ + 74, + 88, + "U2AF651,2LFRET", + "mutant" + ], + [ + 89, + 92, + "Cy3", + "chemical" + ], + [ + 93, + 96, + "Cy5", + "chemical" + ], + [ + 105, + 115, + "absence of", + "protein_state" + ], + [ + 116, + 122, + "ligand", + "chemical" + ], + [ + 175, + 190, + "FRET efficiency", + "evidence" + ] + ] + }, + { + "sid": 126, + "sent": "Approximately 40% of the smFRET traces showed apparent transitions between multiple FRET values (for example, Fig. 6c).", + "section": "RESULTS", + "ner": [ + [ + 25, + 31, + "smFRET", + "experimental_method" + ], + [ + 32, + 38, + "traces", + "evidence" + ], + [ + 84, + 95, + "FRET values", + "evidence" + ] + ] + }, + { + "sid": 127, + "sent": "Despite the large width of the FRET-distribution histogram, the majority (80%) of traces that showed fluctuations sampled only two distinct FRET states (for example, Supplementary Fig. 7a).", + "section": "RESULTS", + "ner": [ + [ + 31, + 58, + "FRET-distribution histogram", + "evidence" + ], + [ + 82, + 88, + "traces", + "evidence" + ], + [ + 140, + 151, + "FRET states", + "evidence" + ] + ] + }, + { + "sid": 128, + "sent": "Approximately 70% of observed fluctuations were interchanges between the \u223c0.65 and \u223c0.45 FRET values (Supplementary Fig. 7b).", + "section": "RESULTS", + "ner": [ + [ + 89, + 100, + "FRET values", + "evidence" + ] + ] + }, + { + "sid": 129, + "sent": "We cannot exclude a possibility that tethering of U2AF651,2LFRET(Cy3/Cy5) to the microscope slide introduces structural heterogeneity into the protein and, thus, contributes to the breadth of the FRET distribution histogram.", + "section": "RESULTS", + "ner": [ + [ + 50, + 64, + "U2AF651,2LFRET", + "mutant" + ], + [ + 65, + 68, + "Cy3", + "chemical" + ], + [ + 69, + 72, + "Cy5", + "chemical" + ], + [ + 196, + 223, + "FRET distribution histogram", + "evidence" + ] + ] + }, + { + "sid": 130, + "sent": "However, the presence of repetitive fluctuations between particular FRET values supports the hypothesis that RNA-free U2AF65 samples several distinct conformations.", + "section": "RESULTS", + "ner": [ + [ + 68, + 79, + "FRET values", + "evidence" + ], + [ + 109, + 117, + "RNA-free", + "protein_state" + ], + [ + 118, + 124, + "U2AF65", + "protein" + ] + ] + }, + { + "sid": 131, + "sent": "This result is consistent with the broad ensembles of extended solution conformations that best fit the SAXS data collected for U2AF651,2 as well as for a longer construct (residues 136\u2013347).", + "section": "RESULTS", + "ner": [ + [ + 54, + 62, + "extended", + "protein_state" + ], + [ + 104, + 108, + "SAXS", + "experimental_method" + ], + [ + 128, + 137, + "U2AF651,2", + "mutant" + ], + [ + 182, + 189, + "136\u2013347", + "residue_range" + ] + ] + }, + { + "sid": 132, + "sent": "We conclude that weak contacts between the U2AF65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA.", + "section": "RESULTS", + "ner": [ + [ + 43, + 49, + "U2AF65", + "protein" + ], + [ + 50, + 54, + "RRM1", + "structure_element" + ], + [ + 59, + 63, + "RRM2", + "structure_element" + ], + [ + 93, + 97, + "RRMs", + "structure_element" + ], + [ + 105, + 115, + "absence of", + "protein_state" + ], + [ + 116, + 119, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 133, + "sent": "U2AF65 conformational selection and induced fit by bound RNA", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "U2AF65", + "protein" + ], + [ + 51, + 56, + "bound", + "protein_state" + ], + [ + 57, + 60, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 134, + "sent": "We next used smFRET to probe the conformational selection of distinct inter-RRM arrangements following association of U2AF65 with the AdML Py-tract prototype.", + "section": "RESULTS", + "ner": [ + [ + 13, + 19, + "smFRET", + "experimental_method" + ], + [ + 70, + 79, + "inter-RRM", + "structure_element" + ], + [ + 118, + 124, + "U2AF65", + "protein" + ], + [ + 134, + 138, + "AdML", + "gene" + ], + [ + 139, + 147, + "Py-tract", + "chemical" + ] + ] + }, + { + "sid": 135, + "sent": "Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an \u223c0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f).", + "section": "RESULTS", + "ner": [ + [ + 16, + 20, + "AdML", + "gene" + ], + [ + 21, + 24, + "RNA", + "chemical" + ], + [ + 28, + 36, + "tethered", + "protein_state" + ], + [ + 37, + 51, + "U2AF651,2LFRET", + "mutant" + ], + [ + 52, + 55, + "Cy3", + "chemical" + ], + [ + 56, + 59, + "Cy5", + "chemical" + ], + [ + 133, + 148, + "FRET efficiency", + "evidence" + ], + [ + 242, + 252, + "FRET state", + "evidence" + ] + ] + }, + { + "sid": 136, + "sent": "To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme.", + "section": "RESULTS", + "ner": [ + [ + 40, + 48, + "RNA-free", + "protein_state" + ], + [ + 66, + 72, + "U2AF65", + "protein" + ], + [ + 119, + 128, + "tethering", + "experimental_method" + ], + [ + 132, + 146, + "U2AF651,2LFRET", + "mutant" + ], + [ + 147, + 150, + "Cy3", + "chemical" + ], + [ + 151, + 154, + "Cy5", + "chemical" + ], + [ + 185, + 212, + "distribution of FRET values", + "evidence" + ], + [ + 217, + 251, + "reversed the immobilization scheme", + "experimental_method" + ] + ] + }, + { + "sid": 137, + "sent": "We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c\u2013g).", + "section": "RESULTS", + "ner": [ + [ + 3, + 11, + "tethered", + "protein_state" + ], + [ + 16, + 20, + "AdML", + "gene" + ], + [ + 21, + 24, + "RNA", + "chemical" + ], + [ + 44, + 76, + "biotinylated oligonucleotide DNA", + "chemical" + ], + [ + 88, + 93, + "added", + "experimental_method" + ], + [ + 94, + 108, + "U2AF651,2LFRET", + "mutant" + ], + [ + 109, + 112, + "Cy3", + "chemical" + ], + [ + 113, + 116, + "Cy5", + "chemical" + ], + [ + 125, + 135, + "absence of", + "protein_state" + ], + [ + 136, + 152, + "biotin-NTA resin", + "chemical" + ] + ] + }, + { + "sid": 138, + "sent": "A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5).", + "section": "RESULTS", + "ner": [ + [ + 7, + 17, + "FRET value", + "evidence" + ], + [ + 62, + 71, + "RNA-bound", + "protein_state" + ], + [ + 117, + 127, + "untethered", + "protein_state" + ], + [ + 132, + 140, + "tethered", + "protein_state" + ], + [ + 141, + 155, + "U2AF651,2LFRET", + "mutant" + ], + [ + 156, + 159, + "Cy3", + "chemical" + ], + [ + 160, + 163, + "Cy5", + "chemical" + ] + ] + }, + { + "sid": 139, + "sent": "We examined the effect on U2AF651,2L conformations of purine interruptions that often occur in relatively degenerate human Py tracts.", + "section": "RESULTS", + "ner": [ + [ + 26, + 36, + "U2AF651,2L", + "mutant" + ], + [ + 54, + 74, + "purine interruptions", + "experimental_method" + ], + [ + 117, + 122, + "human", + "species" + ], + [ + 123, + 132, + "Py tracts", + "chemical" + ] + ] + }, + { + "sid": 140, + "sent": "We introduced an rArA purine dinucleotide within a variant of the AdML Py tract (detailed in Methods).", + "section": "RESULTS", + "ner": [ + [ + 3, + 13, + "introduced", + "experimental_method" + ], + [ + 17, + 21, + "rArA", + "chemical" + ], + [ + 22, + 41, + "purine dinucleotide", + "chemical" + ], + [ + 66, + 70, + "AdML", + "gene" + ], + [ + 71, + 79, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 141, + "sent": "Insertion of adenine nucleotides decreased binding affinity of U2AF65 to RNA by approximately five-fold.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Insertion", + "experimental_method" + ], + [ + 13, + 32, + "adenine nucleotides", + "chemical" + ], + [ + 43, + 59, + "binding affinity", + "evidence" + ], + [ + 63, + 69, + "U2AF65", + "protein" + ], + [ + 73, + 76, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 142, + "sent": "Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent \u223c0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract.", + "section": "RESULTS", + "ner": [ + [ + 62, + 66, + "rArA", + "chemical" + ], + [ + 79, + 82, + "RNA", + "chemical" + ], + [ + 83, + 97, + "slide-tethered", + "protein_state" + ], + [ + 98, + 112, + "U2AF651,2LFRET", + "mutant" + ], + [ + 113, + 116, + "Cy3", + "chemical" + ], + [ + 117, + 120, + "Cy5", + "chemical" + ], + [ + 156, + 166, + "FRET value", + "evidence" + ], + [ + 237, + 245, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 143, + "sent": "Therefore, RRM1-to-RRM2 distance remains similar regardless of whether U2AF65 is bound to interrupted or continuous Py tract.", + "section": "RESULTS", + "ner": [ + [ + 11, + 15, + "RRM1", + "structure_element" + ], + [ + 19, + 23, + "RRM2", + "structure_element" + ], + [ + 71, + 77, + "U2AF65", + "protein" + ], + [ + 81, + 89, + "bound to", + "protein_state" + ], + [ + 116, + 124, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 144, + "sent": "The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the \u03b1-carbon atoms of the respective residues in the crystal structures of U2AF651,2L bound to Py-tract oligonucleotides.", + "section": "RESULTS", + "ner": [ + [ + 4, + 31, + "inter-fluorophore distances", + "evidence" + ], + [ + 63, + 73, + "FRET state", + "evidence" + ], + [ + 160, + 178, + "crystal structures", + "evidence" + ], + [ + 182, + 192, + "U2AF651,2L", + "mutant" + ], + [ + 193, + 201, + "bound to", + "protein_state" + ], + [ + 202, + 227, + "Py-tract oligonucleotides", + "chemical" + ] + ] + }, + { + "sid": 145, + "sent": "It should be noted that inferring distances from FRET values is prone to significant error because of uncertainties in the determination of fluorophore orientation factor \u03ba2 and F\u00f6rster radius R0, the parameters used in distance calculations.", + "section": "RESULTS", + "ner": [ + [ + 49, + 60, + "FRET values", + "evidence" + ] + ] + }, + { + "sid": 146, + "sent": "Nevertheless, the predominant 0.45 FRET state in the presence of RNA agrees with the Py-tract-bound crystal structure of U2AF651,2L.", + "section": "RESULTS", + "ner": [ + [ + 35, + 45, + "FRET state", + "evidence" + ], + [ + 65, + 68, + "RNA", + "chemical" + ], + [ + 85, + 99, + "Py-tract-bound", + "protein_state" + ], + [ + 100, + 117, + "crystal structure", + "evidence" + ], + [ + 121, + 131, + "U2AF651,2L", + "mutant" + ] + ] + }, + { + "sid": 147, + "sent": "Importantly, the majority of traces (\u223c70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a \u223c0.45 FRET value (for example, Fig. 6g).", + "section": "RESULTS", + "ner": [ + [ + 29, + 35, + "traces", + "evidence" + ], + [ + 46, + 60, + "U2AF651,2LFRET", + "mutant" + ], + [ + 61, + 64, + "Cy3", + "chemical" + ], + [ + 65, + 68, + "Cy5", + "chemical" + ], + [ + 70, + 78, + "bound to", + "protein_state" + ], + [ + 98, + 101, + "RNA", + "chemical" + ], + [ + 163, + 173, + "FRET value", + "evidence" + ] + ] + }, + { + "sid": 148, + "sent": "The remaining \u223c30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values.", + "section": "RESULTS", + "ner": [ + [ + 22, + 28, + "traces", + "evidence" + ], + [ + 33, + 47, + "U2AF651,2LFRET", + "mutant" + ], + [ + 48, + 51, + "Cy3", + "chemical" + ], + [ + 52, + 55, + "Cy5", + "chemical" + ], + [ + 57, + 65, + "bound to", + "protein_state" + ], + [ + 85, + 88, + "RNA", + "chemical" + ], + [ + 126, + 137, + "FRET values", + "evidence" + ] + ] + }, + { + "sid": 149, + "sent": "The majority of traces that show fluctuations began at high (0.65\u20130.8) FRET value and transitioned to a \u223c0.45 FRET value (Supplementary Fig. 7c\u2013g).", + "section": "RESULTS", + "ner": [ + [ + 16, + 22, + "traces", + "evidence" + ], + [ + 71, + 81, + "FRET value", + "evidence" + ], + [ + 110, + 120, + "FRET value", + "evidence" + ] + ] + }, + { + "sid": 150, + "sent": "Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to \u223c0.7\u20130.8 and \u223c0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state.", + "section": "RESULTS", + "ner": [ + [ + 0, + 32, + "Hidden Markov modelling analysis", + "experimental_method" + ], + [ + 36, + 42, + "smFRET", + "experimental_method" + ], + [ + 43, + 49, + "traces", + "evidence" + ], + [ + 64, + 73, + "RNA-bound", + "protein_state" + ], + [ + 74, + 84, + "U2AF651,2L", + "mutant" + ], + [ + 164, + 175, + "FRET values", + "evidence" + ], + [ + 246, + 256, + "FRET state", + "evidence" + ] + ] + }, + { + "sid": 151, + "sent": "Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to \u223c0.7\u20130.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to \u223c0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures.", + "section": "RESULTS", + "ner": [ + [ + 63, + 73, + "U2AF651,2L", + "mutant" + ], + [ + 100, + 111, + "FRET values", + "evidence" + ], + [ + 121, + 124, + "RNA", + "chemical" + ], + [ + 129, + 132, + "RNA", + "chemical" + ], + [ + 148, + 155, + "compact", + "protein_state" + ], + [ + 173, + 183, + "U2AF651,2L", + "mutant" + ], + [ + 257, + 267, + "FRET value", + "evidence" + ], + [ + 297, + 309, + "side-by-side", + "protein_state" + ], + [ + 310, + 319, + "inter-RRM", + "structure_element" + ], + [ + 339, + 349, + "U2AF651,2L", + "mutant" + ], + [ + 350, + 368, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 152, + "sent": "Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c\u2013g) suggests that a \u2018conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by \u2018induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final \u2018side-by-side' conformation), as discussed below.", + "section": "RESULTS", + "ner": [ + [ + 51, + 57, + "U2AF65", + "protein" + ], + [ + 70, + 76, + "smFRET", + "experimental_method" + ], + [ + 77, + 83, + "traces", + "evidence" + ], + [ + 166, + 174, + "Py-tract", + "chemical" + ], + [ + 227, + 241, + "pre-configured", + "protein_state" + ], + [ + 242, + 248, + "U2AF65", + "protein" + ], + [ + 339, + 345, + "U2AF65", + "protein" + ], + [ + 346, + 350, + "RRMs", + "structure_element" + ], + [ + 373, + 385, + "side-by-side", + "protein_state" + ] + ] + }, + { + "sid": 153, + "sent": "The U2AF65 structures and analyses presented here represent a successful step towards defining a molecular map of the 3\u2032 splice site.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 10, + "U2AF65", + "protein" + ], + [ + 11, + 21, + "structures", + "evidence" + ], + [ + 26, + 34, + "analyses", + "evidence" + ], + [ + 118, + 132, + "3\u2032 splice site", + "site" + ] + ] + }, + { + "sid": 154, + "sent": "Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65\u2013Py-tract recognition.", + "section": "DISCUSS", + "ner": [ + [ + 97, + 113, + "inter-RRM linker", + "structure_element" + ], + [ + 118, + 122, + "RRM1", + "structure_element" + ], + [ + 124, + 128, + "RRM2", + "structure_element" + ], + [ + 149, + 163, + "RRM1 extension", + "structure_element" + ], + [ + 192, + 198, + "U2AF65", + "protein" + ], + [ + 199, + 207, + "Py-tract", + "chemical" + ] + ] + }, + { + "sid": 155, + "sent": "Truncation of U2AF65 to the core RRM1\u2013RRM2 region reduces its RNA affinity by 100-fold.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 10, + "Truncation", + "experimental_method" + ], + [ + 14, + 20, + "U2AF65", + "protein" + ], + [ + 28, + 32, + "core", + "protein_state" + ], + [ + 33, + 49, + "RRM1\u2013RRM2 region", + "structure_element" + ], + [ + 62, + 74, + "RNA affinity", + "evidence" + ] + ] + }, + { + "sid": 156, + "sent": "Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65\u2013RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 18, + "deletion", + "experimental_method" + ], + [ + 22, + 24, + "20", + "residue_range" + ], + [ + 25, + 50, + "inter-RRM linker residues", + "structure_element" + ], + [ + 73, + 79, + "U2AF65", + "protein" + ], + [ + 80, + 83, + "RNA", + "chemical" + ], + [ + 135, + 141, + "longer", + "protein_state" + ], + [ + 142, + 152, + "U2AF651,2L", + "mutant" + ], + [ + 178, + 192, + "RRM extensions", + "structure_element" + ], + [ + 221, + 227, + "linker", + "structure_element" + ], + [ + 232, + 235, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 157, + "sent": "Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 26, + "triple mutation", + "protein_state" + ], + [ + 46, + 51, + "V254P", + "mutant" + ], + [ + 53, + 58, + "Q147A", + "mutant" + ], + [ + 63, + 68, + "R227A", + "mutant" + ], + [ + 88, + 104, + "inter-RRM linker", + "structure_element" + ], + [ + 106, + 134, + "N- and C-terminal extensions", + "structure_element" + ], + [ + 157, + 168, + "RNA binding", + "evidence" + ] + ] + }, + { + "sid": 158, + "sent": "Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition.", + "section": "DISCUSS", + "ner": [ + [ + 60, + 66, + "U2AF65", + "protein" + ], + [ + 67, + 71, + "RRM1", + "structure_element" + ], + [ + 72, + 76, + "RRM2", + "structure_element" + ], + [ + 78, + 94, + "inter-RRM linker", + "structure_element" + ], + [ + 96, + 123, + "N-and C-terminal extensions", + "structure_element" + ], + [ + 165, + 173, + "Py-tract", + "chemical" + ] + ] + }, + { + "sid": 159, + "sent": "The implications of this finding for U2AF65 conservation and Py-tract recognition are detailed in the Supplementary Discussion.", + "section": "DISCUSS", + "ner": [ + [ + 37, + 43, + "U2AF65", + "protein" + ], + [ + 61, + 69, + "Py-tract", + "chemical" + ] + ] + }, + { + "sid": 160, + "sent": "Recently, high-throughput sequencing studies have shown that somatic mutations in pre-mRNA splicing factors occur in the majority of patients with myelodysplastic syndrome (MDS).", + "section": "DISCUSS", + "ner": [ + [ + 10, + 44, + "high-throughput sequencing studies", + "experimental_method" + ], + [ + 82, + 107, + "pre-mRNA splicing factors", + "protein_type" + ] + ] + }, + { + "sid": 161, + "sent": "MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)\u2014possibly due to the selective versus nearly universal requirements of these factors for splicing.", + "section": "DISCUSS", + "ner": [ + [ + 41, + 46, + "small", + "protein_state" + ], + [ + 47, + 59, + "U2AF subunit", + "protein_type" + ], + [ + 61, + 67, + "U2AF35", + "protein" + ], + [ + 72, + 77, + "U2AF1", + "protein" + ], + [ + 115, + 120, + "large", + "protein_state" + ], + [ + 121, + 127, + "U2AF65", + "protein" + ], + [ + 149, + 154, + "U2AF2", + "protein" + ] + ] + }, + { + "sid": 162, + "sent": "A confirmed somatic mutation of U2AF65 in patients with MDS, L187V, is located on a solvent-exposed surface of RRM1 that is distinct from the RNA interface (Fig. 7a).", + "section": "DISCUSS", + "ner": [ + [ + 32, + 38, + "U2AF65", + "protein" + ], + [ + 61, + 66, + "L187V", + "mutant" + ], + [ + 84, + 107, + "solvent-exposed surface", + "site" + ], + [ + 111, + 115, + "RRM1", + "structure_element" + ], + [ + 142, + 155, + "RNA interface", + "site" + ] + ] + }, + { + "sid": 163, + "sent": "This L187 surface is oriented towards the N terminus of the U2AF651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer.", + "section": "DISCUSS", + "ner": [ + [ + 5, + 9, + "L187", + "residue_name_number" + ], + [ + 60, + 70, + "U2AF651,2L", + "mutant" + ], + [ + 115, + 134, + "U2AF35-binding site", + "site" + ], + [ + 157, + 168, + "full-length", + "protein_state" + ], + [ + 169, + 173, + "U2AF", + "protein" + ], + [ + 174, + 185, + "heterodimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 164, + "sent": "Likewise, an unconfirmed M144I mutation reported by the same group corresponds to the N-terminal residue of U2AF651,2L, which is separated by only \u223c20 residues from the U2AF35-binding site.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 30, + "M144I", + "mutant" + ], + [ + 108, + 118, + "U2AF651,2L", + "mutant" + ], + [ + 169, + 188, + "U2AF35-binding site", + "site" + ] + ] + }, + { + "sid": 165, + "sent": "As such, we suggest that the MDS-relevant U2AF65 mutations contribute to MDS progression indirectly, by destabilizing a relevant conformation of the conjoined U2AF35 subunit rather than affecting U2AF65 functions in RNA binding or spliceosome recruitment per se.", + "section": "DISCUSS", + "ner": [ + [ + 42, + 48, + "U2AF65", + "protein" + ], + [ + 159, + 165, + "U2AF35", + "protein" + ], + [ + 196, + 202, + "U2AF65", + "protein" + ], + [ + 216, + 219, + "RNA", + "chemical" + ], + [ + 231, + 242, + "spliceosome", + "complex_assembly" + ] + ] + }, + { + "sid": 166, + "sent": "Our smFRET results agree with prior NMR/PRE evidence for multi-domain conformational selection as one mechanistic basis for U2AF65\u2013RNA association (Fig. 7b).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 10, + "smFRET", + "experimental_method" + ], + [ + 36, + 39, + "NMR", + "experimental_method" + ], + [ + 40, + 43, + "PRE", + "experimental_method" + ], + [ + 124, + 130, + "U2AF65", + "protein" + ], + [ + 131, + 134, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 167, + "sent": "An \u223c0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 19, + "FRET value", + "evidence" + ], + [ + 51, + 57, + "U2AF65", + "protein" + ], + [ + 89, + 99, + "U2AF651,2L", + "mutant" + ], + [ + 100, + 118, + "crystal structures", + "evidence" + ], + [ + 133, + 137, + "RRM1", + "structure_element" + ], + [ + 142, + 146, + "RRM2", + "structure_element" + ], + [ + 152, + 164, + "side-by-side", + "protein_state" + ], + [ + 172, + 196, + "Py-tract oligonucleotide", + "chemical" + ] + ] + }, + { + "sid": 168, + "sent": "The lesser 0.65\u20130.8 and 0.2\u20130.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the \u2018closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 43, + "FRET values", + "evidence" + ], + [ + 51, + 61, + "untethered", + "protein_state" + ], + [ + 62, + 76, + "U2AF651,2LFRET", + "mutant" + ], + [ + 77, + 80, + "Cy3", + "chemical" + ], + [ + 81, + 84, + "Cy5", + "chemical" + ], + [ + 145, + 151, + "closed", + "protein_state" + ], + [ + 154, + 166, + "back-to-back", + "protein_state" + ], + [ + 167, + 173, + "U2AF65", + "protein" + ], + [ + 205, + 208, + "NMR", + "experimental_method" + ], + [ + 209, + 212, + "PRE", + "experimental_method" + ], + [ + 225, + 233, + "extended", + "protein_state" + ], + [ + 234, + 240, + "U2AF65", + "protein" + ], + [ + 284, + 288, + "RRM1", + "structure_element" + ], + [ + 289, + 293, + "RRM2", + "structure_element" + ], + [ + 328, + 335, + "protein", + "protein" + ], + [ + 339, + 347, + "bound to", + "protein_state" + ], + [ + 348, + 351, + "RNA", + "chemical" + ], + [ + 356, + 362, + "single", + "protein_state" + ], + [ + 363, + 367, + "RRMs", + "structure_element" + ] + ] + }, + { + "sid": 169, + "sent": "An increased prevalence of the \u223c0.45 FRET value following U2AF65\u2013RNA binding, coupled with the apparent absence of transitions in many \u223c0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the \u223c0.45 FRET value.", + "section": "DISCUSS", + "ner": [ + [ + 37, + 47, + "FRET value", + "evidence" + ], + [ + 58, + 64, + "U2AF65", + "protein" + ], + [ + 65, + 68, + "RNA", + "chemical" + ], + [ + 104, + 114, + "absence of", + "protein_state" + ], + [ + 163, + 169, + "traces", + "evidence" + ], + [ + 231, + 234, + "RNA", + "chemical" + ], + [ + 282, + 296, + "pre-configured", + "protein_state" + ], + [ + 297, + 306, + "inter-RRM", + "structure_element" + ], + [ + 358, + 368, + "FRET value", + "evidence" + ] + ] + }, + { + "sid": 170, + "sent": "Notably, our smFRET results reveal that U2AF65\u2013Py-tract recognition can be characterized by an \u2018extended conformational selection' model (Fig. 7b).", + "section": "DISCUSS", + "ner": [ + [ + 13, + 19, + "smFRET", + "experimental_method" + ], + [ + 40, + 46, + "U2AF65", + "protein" + ], + [ + 47, + 55, + "Py-tract", + "chemical" + ] + ] + }, + { + "sid": 171, + "sent": "Examples of \u2018extended conformational selection' during ligand binding have been characterized for a growing number of macromolecules (for example, adenylate kinase, LAO-binding protein, poly-ubiquitin, maltose-binding protein and the preQ1 riboswitch, among others).", + "section": "DISCUSS", + "ner": [ + [ + 13, + 21, + "extended", + "protein_state" + ], + [ + 147, + 163, + "adenylate kinase", + "protein_type" + ], + [ + 165, + 184, + "LAO-binding protein", + "protein_type" + ], + [ + 186, + 200, + "poly-ubiquitin", + "protein_type" + ], + [ + 202, + 225, + "maltose-binding protein", + "protein_type" + ], + [ + 234, + 250, + "preQ1 riboswitch", + "protein_type" + ] + ] + }, + { + "sid": 172, + "sent": "Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65\u20130.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c\u2013g).", + "section": "DISCUSS", + "ner": [ + [ + 33, + 39, + "smFRET", + "experimental_method" + ], + [ + 40, + 46, + "traces", + "evidence" + ], + [ + 51, + 65, + "U2AF651,2LFRET", + "mutant" + ], + [ + 66, + 69, + "Cy3", + "chemical" + ], + [ + 70, + 73, + "Cy5", + "chemical" + ], + [ + 75, + 83, + "bound to", + "protein_state" + ], + [ + 99, + 102, + "RNA", + "chemical" + ], + [ + 128, + 138, + "FRET value", + "evidence" + ], + [ + 178, + 188, + "FRET value", + "evidence" + ] + ] + }, + { + "sid": 173, + "sent": "These transitions could correspond to rearrangement from the \u2018closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure.", + "section": "DISCUSS", + "ner": [ + [ + 62, + 68, + "closed", + "protein_state" + ], + [ + 70, + 73, + "NMR", + "experimental_method" + ], + [ + 74, + 77, + "PRE", + "experimental_method" + ], + [ + 84, + 90, + "U2AF65", + "protein" + ], + [ + 117, + 136, + "RNA-binding surface", + "site" + ], + [ + 147, + 153, + "single", + "protein_state" + ], + [ + 154, + 157, + "RRM", + "structure_element" + ], + [ + 236, + 248, + "side-by-side", + "protein_state" + ], + [ + 250, + 259, + "RNA-bound", + "protein_state" + ], + [ + 260, + 277, + "crystal structure", + "evidence" + ] + ] + }, + { + "sid": 174, + "sent": "As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 19, + "smFRET", + "experimental_method" + ], + [ + 116, + 119, + "NMR", + "experimental_method" + ], + [ + 120, + 123, + "PRE", + "experimental_method" + ], + [ + 168, + 177, + "inter-RRM", + "structure_element" + ], + [ + 204, + 208, + "SAXS", + "experimental_method" + ], + [ + 222, + 225, + "apo", + "protein_state" + ], + [ + 226, + 233, + "protein", + "protein" + ] + ] + }, + { + "sid": 175, + "sent": "Similar interdisciplinary structural approaches are likely to illuminate whether similar mechanistic bases for RNA binding are widespread among other members of the vast multi-RRM family.", + "section": "DISCUSS", + "ner": [ + [ + 176, + 179, + "RRM", + "structure_element" + ] + ] + }, + { + "sid": 176, + "sent": "The finding that U2AF65 recognizes a nine base pair Py tract contributes to an elusive \u2018code' for predicting splicing patterns from primary sequences in the post-genomic era (reviewed in ref.).", + "section": "DISCUSS", + "ner": [ + [ + 17, + 23, + "U2AF65", + "protein" + ], + [ + 52, + 60, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 177, + "sent": "Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 35, + "RNA affinities", + "evidence" + ], + [ + 39, + 45, + "U2AF65", + "protein" + ], + [ + 50, + 60, + "U2AF651,2L", + "mutant" + ], + [ + 110, + 120, + "U2AF651,2L", + "mutant" + ], + [ + 121, + 131, + "structures", + "evidence" + ], + [ + 188, + 197, + "penalties", + "evidence" + ], + [ + 201, + 227, + "structure-guided mutations", + "experimental_method" + ], + [ + 231, + 262, + "RNA binding and splicing assays", + "experimental_method" + ], + [ + 284, + 292, + "extended", + "protein_state" + ], + [ + 293, + 310, + "inter-RRM regions", + "structure_element" + ], + [ + 318, + 328, + "U2AF651,2L", + "mutant" + ], + [ + 329, + 339, + "structures", + "evidence" + ], + [ + 357, + 365, + "Py-tract", + "chemical" + ], + [ + 385, + 396, + "full-length", + "protein_state" + ], + [ + 397, + 403, + "U2AF65", + "protein" + ] + ] + }, + { + "sid": 178, + "sent": "Further research will be needed to understand the roles of SF1 and U2AF35 subunits in the conformational equilibria underlying U2AF65 association with Py tracts.", + "section": "DISCUSS", + "ner": [ + [ + 59, + 62, + "SF1", + "protein" + ], + [ + 67, + 73, + "U2AF35", + "protein" + ], + [ + 127, + 133, + "U2AF65", + "protein" + ], + [ + 151, + 160, + "Py tracts", + "chemical" + ] + ] + }, + { + "sid": 179, + "sent": "Moreover, structural differences among U2AF65 homologues and paralogues may regulate splice site selection.", + "section": "DISCUSS", + "ner": [ + [ + 39, + 45, + "U2AF65", + "protein" + ], + [ + 85, + 96, + "splice site", + "site" + ] + ] + }, + { + "sid": 180, + "sent": "Ultimately, these guidelines will assist the identification of 3\u2032 splice sites and the relationship of disease-causing mutations to penalties for U2AF65 association.", + "section": "DISCUSS", + "ner": [ + [ + 63, + 78, + "3\u2032 splice sites", + "site" + ], + [ + 146, + 152, + "U2AF65", + "protein" + ] + ] + }, + { + "sid": 181, + "sent": "The intact U2AF65 RRM1/RRM2-containing domain and flanking residues are required for binding contiguous Py tracts.", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "intact", + "protein_state" + ], + [ + 11, + 17, + "U2AF65", + "protein" + ], + [ + 18, + 22, + "RRM1", + "structure_element" + ], + [ + 23, + 27, + "RRM2", + "structure_element" + ], + [ + 93, + 103, + "contiguous", + "structure_element" + ], + [ + 104, + 113, + "Py tracts", + "chemical" + ] + ] + }, + { + "sid": 182, + "sent": "(a) Domain organization of full-length (fl) U2AF65 and constructs used for RNA binding and structural experiments.", + "section": "FIG", + "ner": [ + [ + 27, + 38, + "full-length", + "protein_state" + ], + [ + 40, + 42, + "fl", + "protein_state" + ], + [ + 44, + 50, + "U2AF65", + "protein" + ], + [ + 75, + 78, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 183, + "sent": "An internal deletion (d, \u0394) of residues 238\u2013257 removes a portion of the inter-RRM linker from the dU2AF651,2 and dU2AF651,2L constructs.", + "section": "FIG", + "ner": [ + [ + 22, + 23, + "d", + "mutant" + ], + [ + 25, + 26, + "\u0394", + "mutant" + ], + [ + 40, + 47, + "238\u2013257", + "residue_range" + ], + [ + 73, + 89, + "inter-RRM linker", + "structure_element" + ], + [ + 99, + 109, + "dU2AF651,2", + "mutant" + ], + [ + 114, + 125, + "dU2AF651,2L", + "mutant" + ] + ] + }, + { + "sid": 184, + "sent": "(b) Comparison of the apparent equilibrium affinities of various U2AF65 constructs for binding the prototypical AdML Py tract (5\u2032-CCCUUUUUUUUCC-3\u2032).", + "section": "FIG", + "ner": [ + [ + 31, + 53, + "equilibrium affinities", + "evidence" + ], + [ + 65, + 71, + "U2AF65", + "protein" + ], + [ + 112, + 116, + "AdML", + "gene" + ], + [ + 117, + 125, + "Py tract", + "chemical" + ], + [ + 127, + 146, + "5\u2032-CCCUUUUUUUUCC-3\u2032", + "chemical" + ] + ] + }, + { + "sid": 185, + "sent": "The flU2AF65 protein includes a heterodimerization domain of the U2AF35 subunit to promote solubility and folding.", + "section": "FIG", + "ner": [ + [ + 4, + 12, + "flU2AF65", + "protein" + ], + [ + 32, + 57, + "heterodimerization domain", + "structure_element" + ], + [ + 65, + 71, + "U2AF35", + "protein" + ] + ] + }, + { + "sid": 186, + "sent": "The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30\u00b13\u2009nM; U2AF651,2L, 35\u00b16\u2009nM; U2AF651,2, 3,600\u00b1300\u2009nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5\u2032- (light grey fill) or 3\u2032- (dark grey fill) regions.", + "section": "FIG", + "ner": [ + [ + 13, + 47, + "equilibrium dissociation constants", + "evidence" + ], + [ + 49, + 51, + "KD", + "evidence" + ], + [ + 69, + 73, + "AdML", + "gene" + ], + [ + 96, + 104, + "flU2AF65", + "protein" + ], + [ + 115, + 125, + "U2AF651,2L", + "mutant" + ], + [ + 136, + 145, + "U2AF651,2", + "mutant" + ], + [ + 183, + 209, + "RNA sequence specificities", + "evidence" + ], + [ + 213, + 221, + "flU2AF65", + "protein" + ], + [ + 226, + 236, + "U2AF651,2L", + "mutant" + ], + [ + 256, + 262, + "C-rich", + "structure_element" + ], + [ + 263, + 272, + "Py tracts", + "chemical" + ] + ] + }, + { + "sid": 187, + "sent": "The KD's for binding 5\u2032-CCUUUUCCCCCCC-3\u2032 are: flU2AF65, 41\u00b12\u2009nM; U2AF651,2L, 31\u00b13\u2009nM. The KD's for binding 5\u2032-CCCCCCCUUUUCC-3\u2032 are: flU2AF65, 414\u00b112\u2009nM; U2AF651,2L, 417\u00b110\u2009nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted.", + "section": "FIG", + "ner": [ + [ + 4, + 6, + "KD", + "evidence" + ], + [ + 21, + 40, + "5\u2032-CCUUUUCCCCCCC-3\u2032", + "chemical" + ], + [ + 46, + 54, + "flU2AF65", + "protein" + ], + [ + 65, + 75, + "U2AF651,2L", + "mutant" + ], + [ + 90, + 92, + "KD", + "evidence" + ], + [ + 107, + 126, + "5\u2032-CCCCCCCUUUUCC-3\u2032", + "chemical" + ], + [ + 132, + 140, + "flU2AF65", + "protein" + ], + [ + 153, + 163, + "U2AF651,2L", + "mutant" + ], + [ + 239, + 276, + "average apparent equilibrium affinity", + "evidence" + ], + [ + 278, + 280, + "KA", + "evidence" + ] + ] + }, + { + "sid": 188, + "sent": "The purified protein and average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 1.", + "section": "FIG", + "ner": [ + [ + 25, + 82, + "average fitted fluorescence anisotropy RNA-binding curves", + "evidence" + ] + ] + }, + { + "sid": 189, + "sent": "RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "RRM", + "structure_element" + ], + [ + 5, + 26, + "RNA recognition motif", + "structure_element" + ], + [ + 28, + 30, + "RS", + "structure_element" + ], + [ + 32, + 52, + "arginine-serine rich", + "structure_element" + ], + [ + 54, + 57, + "UHM", + "structure_element" + ], + [ + 59, + 78, + "U2AF homology motif", + "structure_element" + ], + [ + 80, + 83, + "ULM", + "structure_element" + ], + [ + 85, + 102, + "U2AF ligand motif", + "structure_element" + ] + ] + }, + { + "sid": 190, + "sent": "Structures of U2AF651,2L recognizing a contiguous Py tract.", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 14, + 24, + "U2AF651,2L", + "mutant" + ], + [ + 39, + 49, + "contiguous", + "structure_element" + ], + [ + 50, + 58, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 191, + "sent": "(a) Alignment of oligonucleotide sequences that were co-crystallized in the indicated U2AF651,2L structures.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "Alignment", + "experimental_method" + ], + [ + 17, + 32, + "oligonucleotide", + "chemical" + ], + [ + 53, + 68, + "co-crystallized", + "experimental_method" + ], + [ + 86, + 96, + "U2AF651,2L", + "mutant" + ], + [ + 97, + 107, + "structures", + "evidence" + ] + ] + }, + { + "sid": 192, + "sent": "The regions of RRM1, RRM2 and linker contacts are indicated above by respective black and blue arrows from N- to C-terminus.", + "section": "FIG", + "ner": [ + [ + 15, + 19, + "RRM1", + "structure_element" + ], + [ + 21, + 25, + "RRM2", + "structure_element" + ], + [ + 30, + 36, + "linker", + "structure_element" + ] + ] + }, + { + "sid": 193, + "sent": "For clarity, we consistently number the U2AF651,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty.", + "section": "FIG", + "ner": [ + [ + 40, + 50, + "U2AF651,2L", + "mutant" + ], + [ + 51, + 75, + "nucleotide-binding sites", + "site" + ], + [ + 121, + 136, + "co-crystallized", + "experimental_method" + ], + [ + 137, + 152, + "oligonucleotide", + "chemical" + ], + [ + 169, + 180, + "nucleotides", + "chemical" + ], + [ + 204, + 222, + "first binding site", + "site" + ] + ] + }, + { + "sid": 194, + "sent": "The prior dU2AF651,2 nucleotide-binding sites are given in parentheses (site 4' interacts with dU2AF65 RRM1 and RRM2 by crystallographic symmetry).", + "section": "FIG", + "ner": [ + [ + 10, + 20, + "dU2AF651,2", + "mutant" + ], + [ + 21, + 45, + "nucleotide-binding sites", + "site" + ], + [ + 95, + 102, + "dU2AF65", + "mutant" + ], + [ + 103, + 107, + "RRM1", + "structure_element" + ], + [ + 112, + 116, + "RRM2", + "structure_element" + ] + ] + }, + { + "sid": 195, + "sent": "(b) Stereo views of a \u2018kicked' 2|Fo|\u2212|Fc| electron density map contoured at 1\u03c3 for the inter-RRM linker, N- and C-terminal residues (blue) or bound oligonucleotide of a representative U2AF651,2L structure (structure iv, bound to 5\u2032-(P)rUrUrUdUrUrU(BrdU)dUrC) (magenta).", + "section": "FIG", + "ner": [ + [ + 31, + 62, + "2|Fo|\u2212|Fc| electron density map", + "evidence" + ], + [ + 87, + 103, + "inter-RRM linker", + "structure_element" + ], + [ + 148, + 163, + "oligonucleotide", + "chemical" + ], + [ + 184, + 194, + "U2AF651,2L", + "mutant" + ], + [ + 220, + 228, + "bound to", + "protein_state" + ] + ] + }, + { + "sid": 196, + "sent": "Crystallographic statistics are given in Table 1 and the overall conformations of U2AF651,2L and prior dU2AF651,2/U2AF651,2 structures are compared in Supplementary Fig. 2.", + "section": "FIG", + "ner": [ + [ + 82, + 92, + "U2AF651,2L", + "mutant" + ], + [ + 103, + 113, + "dU2AF651,2", + "mutant" + ], + [ + 114, + 123, + "U2AF651,2", + "mutant" + ], + [ + 124, + 134, + "structures", + "evidence" + ] + ] + }, + { + "sid": 197, + "sent": "BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5\u2032-phosphorylation; r, ribose.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "BrdU", + "chemical" + ], + [ + 6, + 27, + "5-bromo-deoxy-uridine", + "chemical" + ], + [ + 29, + 30, + "d", + "chemical" + ], + [ + 32, + 44, + "deoxy-ribose", + "chemical" + ], + [ + 46, + 48, + "P-", + "chemical" + ], + [ + 50, + 68, + "5\u2032-phosphorylation", + "chemical" + ], + [ + 70, + 71, + "r", + "chemical" + ], + [ + 73, + 79, + "ribose", + "chemical" + ] + ] + }, + { + "sid": 198, + "sent": "Representative views of the U2AF651,2L interactions with each new nucleotide of the bound Py tract.", + "section": "FIG", + "ner": [ + [ + 28, + 38, + "U2AF651,2L", + "mutant" + ], + [ + 66, + 76, + "nucleotide", + "chemical" + ], + [ + 84, + 89, + "bound", + "protein_state" + ], + [ + 90, + 98, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 199, + "sent": "New residues of the U2AF651,2L structures are coloured a darker shade of blue, apart from residues that were tested by site-directed mutagenesis, which are coloured yellow.", + "section": "FIG", + "ner": [ + [ + 20, + 30, + "U2AF651,2L", + "mutant" + ], + [ + 31, + 41, + "structures", + "evidence" + ], + [ + 119, + 144, + "site-directed mutagenesis", + "experimental_method" + ] + ] + }, + { + "sid": 200, + "sent": "The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a\u2013h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2\u2013RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv.", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "nucleotide-binding sites", + "site" + ], + [ + 36, + 46, + "U2AF651,2L", + "mutant" + ], + [ + 57, + 67, + "dU2AF651,2", + "mutant" + ], + [ + 68, + 77, + "structure", + "evidence" + ], + [ + 123, + 165, + "first and seventh U2AF651,2L-binding sites", + "site" + ], + [ + 195, + 209, + "dU2AF651,2\u2013RNA", + "complex_assembly" + ], + [ + 210, + 219, + "structure", + "evidence" + ], + [ + 275, + 285, + "U2AF651,2L", + "mutant" + ], + [ + 286, + 296, + "structures", + "evidence" + ], + [ + 362, + 372, + "ninth site", + "site" + ], + [ + 438, + 448, + "U2AF651,2L", + "mutant" + ], + [ + 449, + 458, + "structure", + "evidence" + ], + [ + 499, + 516, + "ribose nucleotide", + "chemical" + ], + [ + 540, + 543, + "rU2", + "residue_name_number" + ], + [ + 565, + 568, + "rU3", + "residue_name_number" + ], + [ + 591, + 594, + "rU4", + "residue_name_number" + ], + [ + 615, + 618, + "rU5", + "residue_name_number" + ], + [ + 641, + 644, + "rU6", + "residue_name_number" + ], + [ + 666, + 669, + "dU8", + "residue_name_number" + ], + [ + 692, + 695, + "dU9", + "residue_name_number" + ], + [ + 718, + 721, + "rC9", + "residue_name_number" + ] + ] + }, + { + "sid": 201, + "sent": "(i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5\u2032-CCCUUUUUUUUCC-3\u2032).", + "section": "FIG", + "ner": [ + [ + 26, + 48, + "equilibrium affinities", + "evidence" + ], + [ + 50, + 52, + "KA", + "evidence" + ], + [ + 61, + 70, + "wild type", + "protein_state" + ], + [ + 96, + 102, + "mutant", + "protein_state" + ], + [ + 112, + 122, + "U2AF651,2L", + "mutant" + ], + [ + 144, + 148, + "AdML", + "gene" + ], + [ + 149, + 157, + "Py tract", + "chemical" + ], + [ + 159, + 178, + "5\u2032-CCCUUUUUUUUCC-3\u2032", + "chemical" + ] + ] + }, + { + "sid": 202, + "sent": "The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35\u00b16\u2009nM; R227A, 166\u00b12\u2009nM; V254P, 137\u00b110\u2009nM; Q147A, 171\u00b121\u2009nM. The average KA and s.e.m. for three independent titrations are plotted.", + "section": "FIG", + "ner": [ + [ + 13, + 47, + "equilibrium dissociation constants", + "evidence" + ], + [ + 49, + 51, + "KD", + "evidence" + ], + [ + 60, + 70, + "U2AF651,2L", + "mutant" + ], + [ + 71, + 77, + "mutant", + "protein_state" + ], + [ + 92, + 101, + "wild type", + "protein_state" + ], + [ + 103, + 105, + "WT", + "protein_state" + ], + [ + 117, + 122, + "R227A", + "mutant" + ], + [ + 134, + 139, + "V254P", + "mutant" + ], + [ + 152, + 157, + "Q147A", + "mutant" + ], + [ + 182, + 184, + "KA", + "evidence" + ] + ] + }, + { + "sid": 203, + "sent": "The average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4a\u2013c.", + "section": "FIG", + "ner": [ + [ + 43, + 61, + "RNA-binding curves", + "evidence" + ] + ] + }, + { + "sid": 204, + "sent": "The U2AF65 linker/RRM and inter-RRM interactions.", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "U2AF65", + "protein" + ], + [ + 11, + 17, + "linker", + "structure_element" + ], + [ + 18, + 21, + "RRM", + "structure_element" + ], + [ + 32, + 35, + "RRM", + "structure_element" + ] + ] + }, + { + "sid": 205, + "sent": "(a) Contacts of the U2AF65 inter-RRM linker with the RRMs.", + "section": "FIG", + "ner": [ + [ + 20, + 26, + "U2AF65", + "protein" + ], + [ + 27, + 43, + "inter-RRM linker", + "structure_element" + ], + [ + 53, + 57, + "RRMs", + "structure_element" + ] + ] + }, + { + "sid": 206, + "sent": "A semi-transparent space-filling surface is shown for the RRM1 (green) and RRM2 (light blue).", + "section": "FIG", + "ner": [ + [ + 58, + 62, + "RRM1", + "structure_element" + ], + [ + 75, + 79, + "RRM2", + "structure_element" + ] + ] + }, + { + "sid": 207, + "sent": "Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant.", + "section": "FIG", + "ner": [ + [ + 9, + 13, + "V249", + "residue_name_number" + ], + [ + 15, + 19, + "V250", + "residue_name_number" + ], + [ + 21, + 25, + "V254", + "residue_name_number" + ], + [ + 39, + 46, + "mutated", + "experimental_method" + ], + [ + 50, + 55, + "V249G", + "mutant" + ], + [ + 56, + 61, + "V250G", + "mutant" + ], + [ + 62, + 67, + "V254G", + "mutant" + ], + [ + 75, + 86, + "3Gly mutant", + "mutant" + ], + [ + 97, + 101, + "S251", + "residue_name_number" + ], + [ + 103, + 107, + "T252", + "residue_name_number" + ], + [ + 109, + 113, + "V253", + "residue_name_number" + ], + [ + 115, + 119, + "P255", + "residue_name_number" + ], + [ + 137, + 141, + "V254", + "residue_name_number" + ], + [ + 146, + 153, + "mutated", + "experimental_method" + ], + [ + 157, + 162, + "S251G", + "mutant" + ], + [ + 163, + 168, + "T252G", + "mutant" + ], + [ + 169, + 174, + "V253G", + "mutant" + ], + [ + 175, + 180, + "V254G", + "mutant" + ], + [ + 181, + 186, + "P255G", + "mutant" + ], + [ + 194, + 205, + "5Gly mutant", + "mutant" + ], + [ + 212, + 217, + "S251N", + "mutant" + ], + [ + 218, + 223, + "T252L", + "mutant" + ], + [ + 224, + 229, + "V253A", + "mutant" + ], + [ + 230, + 235, + "V254L", + "mutant" + ], + [ + 236, + 241, + "P255A", + "mutant" + ], + [ + 249, + 261, + "NLALA mutant", + "mutant" + ], + [ + 272, + 276, + "M144", + "residue_name_number" + ], + [ + 278, + 282, + "L235", + "residue_name_number" + ], + [ + 284, + 288, + "M238", + "residue_name_number" + ], + [ + 290, + 294, + "V244", + "residue_name_number" + ], + [ + 296, + 300, + "V246", + "residue_name_number" + ], + [ + 321, + 325, + "V249", + "residue_name_number" + ], + [ + 327, + 331, + "V250", + "residue_name_number" + ], + [ + 333, + 337, + "S251", + "residue_name_number" + ], + [ + 339, + 343, + "T252", + "residue_name_number" + ], + [ + 345, + 349, + "V253", + "residue_name_number" + ], + [ + 351, + 355, + "V254", + "residue_name_number" + ], + [ + 357, + 361, + "P255", + "residue_name_number" + ], + [ + 366, + 373, + "mutated", + "experimental_method" + ], + [ + 377, + 382, + "M144G", + "mutant" + ], + [ + 383, + 388, + "L235G", + "mutant" + ], + [ + 389, + 394, + "M238G", + "mutant" + ], + [ + 395, + 400, + "V244G", + "mutant" + ], + [ + 401, + 406, + "V246G", + "mutant" + ], + [ + 407, + 412, + "V249G", + "mutant" + ], + [ + 414, + 419, + "V250G", + "mutant" + ], + [ + 420, + 425, + "S251G", + "mutant" + ], + [ + 426, + 431, + "T252G", + "mutant" + ], + [ + 432, + 437, + "V253G", + "mutant" + ], + [ + 438, + 443, + "V254G", + "mutant" + ], + [ + 444, + 449, + "P255G", + "mutant" + ], + [ + 457, + 469, + "12Gly mutant", + "mutant" + ] + ] + }, + { + "sid": 208, + "sent": "Other linker residues are coloured either dark blue for new residues in the U2AF651,2L structure or light blue for the remaining inter-RRM residues.", + "section": "FIG", + "ner": [ + [ + 6, + 12, + "linker", + "structure_element" + ], + [ + 76, + 86, + "U2AF651,2L", + "mutant" + ], + [ + 129, + 138, + "inter-RRM", + "structure_element" + ] + ] + }, + { + "sid": 209, + "sent": "The central panel shows an overall view with stick diagrams for mutated residues; boxed regions are expanded to show the C-terminal (bottom left) and central linker regions (top) at the inter-RRM interfaces, and N-terminal linker region contacts with RRM1 (bottom right).", + "section": "FIG", + "ner": [ + [ + 150, + 172, + "central linker regions", + "structure_element" + ], + [ + 186, + 206, + "inter-RRM interfaces", + "structure_element" + ], + [ + 251, + 255, + "RRM1", + "structure_element" + ] + ] + }, + { + "sid": 210, + "sent": "(b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5\u2032-CCCUUUUUUUUCC-3\u2032) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1\u2013RRM2 region (residues 148\u2013237, 258\u2013336) or dU2AF651,2L (residues 141\u2013237, 258\u2013342).", + "section": "FIG", + "ner": [ + [ + 26, + 48, + "equilibrium affinities", + "evidence" + ], + [ + 50, + 52, + "KA", + "evidence" + ], + [ + 62, + 66, + "AdML", + "gene" + ], + [ + 67, + 75, + "Py tract", + "chemical" + ], + [ + 77, + 96, + "5\u2032-CCCUUUUUUUUCC-3\u2032", + "chemical" + ], + [ + 105, + 114, + "wild-type", + "protein_state" + ], + [ + 122, + 132, + "U2AF651,2L", + "mutant" + ], + [ + 193, + 197, + "3Gly", + "mutant" + ], + [ + 208, + 212, + "5Gly", + "mutant" + ], + [ + 220, + 225, + "NLALA", + "mutant" + ], + [ + 241, + 246, + "12Gly", + "mutant" + ], + [ + 264, + 280, + "linker deletions", + "experimental_method" + ], + [ + 281, + 291, + "dU2AF651,2", + "mutant" + ], + [ + 299, + 306, + "minimal", + "protein_state" + ], + [ + 307, + 323, + "RRM1\u2013RRM2 region", + "structure_element" + ], + [ + 334, + 341, + "148\u2013237", + "residue_range" + ], + [ + 343, + 350, + "258\u2013336", + "residue_range" + ], + [ + 355, + 366, + "dU2AF651,2L", + "mutant" + ], + [ + 377, + 384, + "141\u2013237", + "residue_range" + ], + [ + 386, + 393, + "258\u2013342", + "residue_range" + ] + ] + }, + { + "sid": 211, + "sent": "The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35\u00b16\u2009nM; 3Gly, 47\u00b14\u2009nM; 5Gly, 61\u00b13\u2009nM; 12Gly, 88\u00b121\u2009nM; NLALA, 45\u00b13\u2009nM; dU2AF651,2L, 123\u00b15\u2009nM; dU2AF651,2, 5000\u00b1100\u2009nM; 3Mut, 5630\u00b170\u2009nM. The average KA and s.e.m. for three independent titrations are plotted.", + "section": "FIG", + "ner": [ + [ + 13, + 47, + "equilibrium dissociation constants", + "evidence" + ], + [ + 49, + 51, + "KD", + "evidence" + ], + [ + 60, + 70, + "U2AF651,2L", + "mutant" + ], + [ + 71, + 77, + "mutant", + "protein_state" + ], + [ + 92, + 101, + "wild type", + "protein_state" + ], + [ + 103, + 105, + "WT", + "protein_state" + ], + [ + 117, + 121, + "3Gly", + "mutant" + ], + [ + 132, + 136, + "5Gly", + "mutant" + ], + [ + 147, + 152, + "12Gly", + "mutant" + ], + [ + 164, + 169, + "NLALA", + "mutant" + ], + [ + 180, + 191, + "dU2AF651,2L", + "mutant" + ], + [ + 203, + 213, + "dU2AF651,2", + "mutant" + ], + [ + 228, + 232, + "3Mut", + "mutant" + ], + [ + 258, + 260, + "KA", + "evidence" + ] + ] + }, + { + "sid": 212, + "sent": "The fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4d\u2013j. (c) Close view of the U2AF65 RRM1/RRM2 interface following a two-fold rotation about the x-axis relative to a.", + "section": "FIG", + "ner": [ + [ + 35, + 53, + "RNA-binding curves", + "evidence" + ], + [ + 114, + 120, + "U2AF65", + "protein" + ], + [ + 121, + 140, + "RRM1/RRM2 interface", + "site" + ] + ] + }, + { + "sid": 213, + "sent": "U2AF65 inter-domain residues are important for splicing a representative pre-mRNA substrate in human cells.", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "U2AF65", + "protein" + ], + [ + 73, + 81, + "pre-mRNA", + "chemical" + ], + [ + 95, + 100, + "human", + "species" + ] + ] + }, + { + "sid": 214, + "sent": "(a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset).", + "section": "FIG", + "ner": [ + [ + 29, + 33, + "pyPY", + "chemical" + ], + [ + 89, + 101, + "splice sites", + "site" + ], + [ + 134, + 142, + "Py tract", + "chemical" + ], + [ + 144, + 146, + "py", + "chemical" + ], + [ + 162, + 166, + "AdML", + "gene" + ], + [ + 167, + 175, + "Py tract", + "chemical" + ], + [ + 177, + 179, + "PY", + "chemical" + ] + ] + }, + { + "sid": 215, + "sent": "(b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65).", + "section": "FIG", + "ner": [ + [ + 19, + 25, + "RT-PCR", + "experimental_method" + ], + [ + 29, + 33, + "pyPY", + "chemical" + ], + [ + 65, + 79, + "co-transfected", + "experimental_method" + ], + [ + 109, + 113, + "pyPY", + "chemical" + ], + [ + 134, + 143, + "wild-type", + "protein_state" + ], + [ + 145, + 147, + "WT", + "protein_state" + ], + [ + 149, + 155, + "U2AF65", + "protein" + ], + [ + 168, + 174, + "U2AF65", + "protein" + ], + [ + 175, + 181, + "mutant", + "protein_state" + ], + [ + 183, + 187, + "3Mut", + "mutant" + ], + [ + 192, + 197, + "Q147A", + "mutant" + ], + [ + 199, + 204, + "R227A", + "mutant" + ], + [ + 209, + 214, + "V254P", + "mutant" + ], + [ + 274, + 276, + "py", + "chemical" + ], + [ + 285, + 289, + "mRNA", + "chemical" + ], + [ + 317, + 321, + "pyPY", + "chemical" + ], + [ + 401, + 407, + "U2AF65", + "protein" + ], + [ + 422, + 424, + "WT", + "protein_state" + ], + [ + 425, + 431, + "U2AF65", + "protein" + ], + [ + 439, + 443, + "3Mut", + "mutant" + ], + [ + 444, + 450, + "U2AF65", + "protein" + ] + ] + }, + { + "sid": 216, + "sent": "Protein overexpression and qRT-PCR results are shown in Supplementary Fig. 5.", + "section": "FIG", + "ner": [ + [ + 0, + 22, + "Protein overexpression", + "experimental_method" + ], + [ + 27, + 34, + "qRT-PCR", + "experimental_method" + ] + ] + }, + { + "sid": 217, + "sent": "RNA binding stabilizes the side-by-side conformation of U2AF65 RRMs.", + "section": "FIG", + "ner": [ + [ + 27, + 39, + "side-by-side", + "protein_state" + ], + [ + 56, + 62, + "U2AF65", + "protein" + ], + [ + 63, + 67, + "RRMs", + "structure_element" + ] + ] + }, + { + "sid": 218, + "sent": "(a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of \u2018side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or \u2018closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2.", + "section": "FIG", + "ner": [ + [ + 15, + 19, + "FRET", + "experimental_method" + ], + [ + 68, + 72, + "RRM1", + "structure_element" + ], + [ + 77, + 81, + "RRM2", + "structure_element" + ], + [ + 89, + 106, + "crystal structure", + "evidence" + ], + [ + 111, + 123, + "side-by-side", + "protein_state" + ], + [ + 125, + 135, + "U2AF651,2L", + "mutant" + ], + [ + 136, + 140, + "RRMs", + "structure_element" + ], + [ + 141, + 149, + "bound to", + "protein_state" + ], + [ + 152, + 176, + "Py-tract oligonucleotide", + "chemical" + ], + [ + 214, + 220, + "closed", + "protein_state" + ], + [ + 222, + 225, + "NMR", + "experimental_method" + ], + [ + 226, + 229, + "PRE", + "experimental_method" + ], + [ + 245, + 254, + "U2AF651,2", + "mutant" + ], + [ + 301, + 305, + "RRM2", + "structure_element" + ] + ] + }, + { + "sid": 219, + "sent": "The U2AF651,2LFRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site.", + "section": "FIG", + "ner": [ + [ + 4, + 18, + "U2AF651,2LFRET", + "mutant" + ], + [ + 52, + 57, + "A181C", + "mutant" + ], + [ + 58, + 63, + "Q324C", + "mutant" + ], + [ + 87, + 90, + "Cy3", + "chemical" + ], + [ + 91, + 94, + "Cy5", + "chemical" + ], + [ + 95, + 107, + "fluorophores", + "chemical" + ] + ] + }, + { + "sid": 220, + "sent": "(c\u2013f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5\u2009\u03bcM AdML Py-tract RNA (5\u2032-CCUUUUUUUUCC-3\u2032) (e,f), or in the presence of 10\u2009\u03bcM adenosine-interrupted variant RNA (5\u2032-CUUUUUAAUUUCCA-3\u2032) (i,j).", + "section": "FIG", + "ner": [ + [ + 14, + 28, + "U2AF651,2LFRET", + "mutant" + ], + [ + 29, + 32, + "Cy3", + "chemical" + ], + [ + 33, + 36, + "Cy5", + "chemical" + ], + [ + 90, + 105, + "biotin-NTA/Ni+2", + "chemical" + ], + [ + 217, + 227, + "absence of", + "protein_state" + ], + [ + 228, + 235, + "ligands", + "chemical" + ], + [ + 267, + 271, + "AdML", + "gene" + ], + [ + 272, + 284, + "Py-tract RNA", + "chemical" + ], + [ + 286, + 304, + "5\u2032-CCUUUUUUUUCC-3\u2032", + "chemical" + ], + [ + 341, + 350, + "adenosine", + "residue_name" + ], + [ + 371, + 374, + "RNA", + "chemical" + ], + [ + 376, + 396, + "5\u2032-CUUUUUAAUUUCCA-3\u2032", + "chemical" + ] + ] + }, + { + "sid": 221, + "sent": "The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1\u2009nM) was added to AdML RNA\u2013polyethylene-glycol-linker\u2013DNA oligonucleotide (10\u2009nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line).", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "untethered", + "protein_state" + ], + [ + 15, + 29, + "U2AF651,2LFRET", + "mutant" + ], + [ + 30, + 33, + "Cy3", + "chemical" + ], + [ + 34, + 37, + "Cy5", + "chemical" + ], + [ + 67, + 71, + "AdML", + "gene" + ], + [ + 72, + 122, + "RNA\u2013polyethylene-glycol-linker\u2013DNA oligonucleotide", + "chemical" + ], + [ + 212, + 240, + "biotinyl-DNA oligonucleotide", + "chemical" + ] + ] + }, + { + "sid": 222, + "sent": "Typical single-molecule FRET traces (c,e,g,i) show fluorescence intensities from Cy3 (green) and Cy5 (red) and the calculated apparent FRET efficiency (blue).", + "section": "FIG", + "ner": [ + [ + 8, + 28, + "single-molecule FRET", + "experimental_method" + ], + [ + 29, + 35, + "traces", + "evidence" + ], + [ + 81, + 84, + "Cy3", + "chemical" + ], + [ + 97, + 100, + "Cy5", + "chemical" + ], + [ + 115, + 150, + "calculated apparent FRET efficiency", + "evidence" + ] + ] + }, + { + "sid": 223, + "sent": "Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j).", + "section": "FIG", + "ner": [ + [ + 11, + 17, + "traces", + "evidence" + ], + [ + 22, + 32, + "untethered", + "protein_state" + ], + [ + 34, + 43, + "RNA-bound", + "protein_state" + ], + [ + 44, + 58, + "U2AF651,2LFRET", + "mutant" + ], + [ + 59, + 62, + "Cy3", + "chemical" + ], + [ + 63, + 66, + "Cy5", + "chemical" + ], + [ + 106, + 116, + "Histograms", + "evidence" + ], + [ + 136, + 163, + "distribution of FRET values", + "evidence" + ], + [ + 167, + 175, + "RNA-free", + "protein_state" + ], + [ + 177, + 191, + "slide-tethered", + "protein_state" + ], + [ + 192, + 206, + "U2AF651,2LFRET", + "mutant" + ], + [ + 207, + 210, + "Cy3", + "chemical" + ], + [ + 211, + 214, + "Cy5", 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"section": "FIG", + "ner": [ + [ + 35, + 41, + "traces", + "evidence" + ], + [ + 60, + 69, + "histogram", + "evidence" + ] + ] + }, + { + "sid": 225, + "sent": "Schematic models of U2AF65 recognizing the Py tract.", + "section": "FIG", + "ner": [ + [ + 20, + 26, + "U2AF65", + "protein" + ], + [ + 43, + 51, + "Py tract", + "chemical" + ] + ] + }, + { + "sid": 226, + "sent": "(a) Diagram of the U2AF65, SF1 and U2AF35 splicing factors bound to the consensus elements of the 3\u2032 splice site.", + "section": "FIG", + "ner": [ + [ + 19, + 25, + "U2AF65", + "protein" + ], + [ + 27, + 30, + "SF1", + "protein" + ], + [ + 35, + 41, + "U2AF35", + "protein" + ], + [ + 59, + 67, + "bound to", + "protein_state" + ], + [ + 98, + 112, + "3\u2032 splice site", + "site" + ] + ] + }, + { + "sid": 227, + "sent": "A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3\u2032 splice site are unknown.", + "section": "FIG", + "ner": [ + [ + 28, + 38, + "U2AF651,2L", + "mutant" + ], + [ + 48, + 56, + "bound to", + "protein_state" + ], + [ + 62, + 73, + "nucleotides", + "chemical" + ], + [ + 128, + 149, + "branch point sequence", + "site" + ], + [ + 151, + 154, + "BPS", + "site" + ], + [ + 170, + 185, + "AG dinucleotide", + "chemical" + ], + [ + 193, + 207, + "3\u2032 splice site", + "site" + ] + ] + }, + { + "sid": 228, + "sent": "MDS-relevant mutated residues of U2AF65 are shown as yellow spheres (L187 and M144).", + "section": "FIG", + "ner": [ + [ + 33, + 39, + "U2AF65", + "protein" + ], + [ + 69, + 73, + "L187", + "residue_name_number" + ], + [ + 78, + 82, + "M144", + "residue_name_number" + ] + ] + }, + { + "sid": 229, + "sent": "(b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the \u2018closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to \u223c0.45 FRET value, which is consistent with \u2018open', side-by-side RRMs such as the U2AF651,2L crystal structures.", + "section": "FIG", + "ner": [ + [ + 29, + 41, + "Py-tract RNA", + "chemical" + ], + [ + 75, + 84, + "high FRET", + "evidence" + ], + [ + 110, + 116, + "closed", + "protein_state" + ], + [ + 119, + 131, + "back-to-back", + "protein_state" + ], + [ + 132, + 135, + "apo", + "protein_state" + ], + [ + 136, + 142, + "U2AF65", + "protein" + ], + [ + 164, + 167, + "PRE", + "experimental_method" + ], + [ + 168, + 171, + "NMR", + "experimental_method" + ], + [ + 262, + 272, + "FRET value", + "evidence" + ], + [ + 300, + 304, + "open", + "protein_state" + ], + [ + 307, + 319, + "side-by-side", + "protein_state" + ], + [ + 320, + 324, + "RRMs", + "structure_element" + ], + [ + 337, + 347, + "U2AF651,2L", + "mutant" + ], + [ + 348, + 366, + "crystal structures", + "evidence" + ] + ] + }, + { + "sid": 230, + "sent": "Alternatively, a conformation of U2AF65 corresponding to \u223c0.45 FRET value can directly bind to RNA; RNA binding stabilizes the \u2018open', side-by-side conformation and thus shifts the U2AF65 population towards the \u223c0.45 FRET value.", + "section": "FIG", + "ner": [ + [ + 33, + 39, + "U2AF65", + "protein" + ], + [ + 63, + 73, + "FRET value", + "evidence" + ], + [ + 95, + 98, + "RNA", + "chemical" + ], + [ + 100, + 103, + "RNA", + "chemical" + ], + [ + 128, + 132, + "open", + "protein_state" + ], + [ + 135, + 147, + "side-by-side", + "protein_state" + ], + [ + 181, + 187, + "U2AF65", + "protein" + ], + [ + 217, + 227, + "FRET value", + "evidence" + ] + ] + }, + { + "sid": 231, + "sent": "RRM1, green; RRM2, pale blue; RRM extensions/linker, blue.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "RRM1", + "structure_element" + ], + [ + 13, + 17, + "RRM2", + "structure_element" + ], + [ + 30, + 44, + "RRM extensions", + "structure_element" + ], + [ + 45, + 51, + "linker", + "structure_element" + ] + ] + } + ] + }, + "PMC4854314": { + "annotations": [ + { + "sid": 0, + "sent": "RNA protects a nucleoprotein complex against radiation damage", + "section": "TITLE", + "ner": [ + [ + 0, + 3, + "RNA", + "chemical" + ], + [ + 15, + 28, + "nucleoprotein", + "complex_assembly" + ] + ] + }, + { + "sid": 1, + "sent": "Systematic analysis of radiation damage within a protein\u2013RNA complex over a large dose range (1.3\u201325\u2005MGy) reveals significant differential susceptibility of RNA and protein.", + "section": "ABSTRACT", + "ner": [ + [ + 49, + 60, + "protein\u2013RNA", + "complex_assembly" + ], + [ + 157, + 160, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 2, + "sent": "A new method of difference electron-density quantification is presented.", + "section": "ABSTRACT", + "ner": [ + [ + 16, + 58, + "difference electron-density quantification", + "experimental_method" + ] + ] + }, + { + "sid": 3, + "sent": "Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations.", + "section": "ABSTRACT", + "ner": [ + [ + 24, + 77, + "macromolecular X-ray crystallographic data collection", + "experimental_method" + ], + [ + 116, + 155, + "macromolecular structure determinations", + "experimental_method" + ] + ] + }, + { + "sid": 4, + "sent": "Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes.", + "section": "ABSTRACT", + "ner": [ + [ + 57, + 65, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 5, + "sent": "Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3\u201325.0\u2005MGy) range and at higher resolution (1.98\u2005\u00c5) than the previous systematic specific damage study on a protein\u2013DNA complex.", + "section": "ABSTRACT", + "ner": [ + [ + 47, + 71, + "per-atom density changes", + "evidence" + ], + [ + 244, + 247, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 6, + "sent": "Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein.", + "section": "ABSTRACT", + "ner": [ + [ + 64, + 99, + "trp RNA-binding attenuation protein", + "protein_type" + ], + [ + 101, + 105, + "TRAP", + "complex_assembly" + ], + [ + 107, + 115, + "bound to", + "protein_state" + ], + [ + 134, + 137, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 7, + "sent": "Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein.", + "section": "ABSTRACT", + "ner": [ + [ + 29, + 32, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 8, + "sent": "The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility.", + "section": "ABSTRACT", + "ner": [ + [ + 24, + 28, + "TRAP", + "complex_assembly" + ], + [ + 91, + 96, + "bound", + "protein_state" + ], + [ + 97, + 100, + "RNA", + "chemical" + ], + [ + 160, + 163, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 9, + "sent": "The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule.", + "section": "ABSTRACT", + "ner": [ + [ + 33, + 37, + "TRAP", + "complex_assembly" + ], + [ + 38, + 42, + "ring", + "structure_element" + ], + [ + 95, + 98, + "Glu", + "residue_name" + ], + [ + 103, + 106, + "Asp", + "residue_name" + ], + [ + 129, + 132, + "RNA", + "chemical" + ], + [ + 236, + 255, + "RNA-binding pockets", + "site" + ] + ] + }, + { + "sid": 10, + "sent": "Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density.", + "section": "ABSTRACT", + "ner": [ + [ + 88, + 91, + "Lys", + "residue_name" + ], + [ + 96, + 99, + "Phe", + "residue_name" + ], + [ + 117, + 120, + "RNA", + "chemical" + ], + [ + 147, + 163, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 11, + "sent": "With the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100\u2005K).", + "section": "INTRO", + "ner": [ + [ + 150, + 173, + "structure determination", + "experimental_method" + ], + [ + 180, + 210, + "macromolecular crystallography", + "experimental_method" + ], + [ + 212, + 214, + "MX", + "experimental_method" + ] + ] + }, + { + "sid": 12, + "sent": "Significant progress has been made in recent years in understanding the inevitable manifestations of X-ray-induced RD within protein crystals, and there is now a body of literature on possible strategies to mitigate the effects of RD (e.g. Zeldin, Brockhauser et al., 2013; Bourenkov & Popov, 2010).", + "section": "INTRO", + "ner": [ + [ + 133, + 141, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 13, + "sent": "However, there is still no general consensus within the field on how to minimize RD during MX data collection, and debates on the dependence of RD progression on incident X-ray energy (Shimizu et al., 2007; Liebschner et al., 2015) and the efficacy of radical scavengers (Allan et al., 2013) have yet to be resolved.", + "section": "INTRO", + "ner": [ + [ + 91, + 93, + "MX", + "experimental_method" + ] + ] + }, + { + "sid": 14, + "sent": "Global radiation damage is observed within reciprocal space as the overall decay of the summed intensity of reflections detected within the diffraction pattern as dose increases (Garman, 2010; Murray & Garman, 2002).", + "section": "INTRO", + "ner": [ + [ + 140, + 159, + "diffraction pattern", + "evidence" + ] + ] + }, + { + "sid": 15, + "sent": "Dose is defined as the absorbed energy per unit mass of crystal in grays (Gy; 1\u2005Gy = 1\u2005J\u2005kg\u22121), and is the metric against which damage progression should be monitored during MX data collection, as opposed to time.", + "section": "INTRO", + "ner": [ + [ + 174, + 176, + "MX", + "experimental_method" + ] + ] + }, + { + "sid": 16, + "sent": "At 100\u2005K, an experimental dose limit of 30\u2005MGy has been recommended as an upper limit beyond which the biological information derived from any macromolecular crystal may be compromised (Owen et al., 2006).", + "section": "INTRO", + "ner": [ + [ + 158, + 165, + "crystal", + "evidence" + ] + ] + }, + { + "sid": 17, + "sent": " Specific radiation damage (SRD) is observed in the real-space electron density, and has been detected at much lower doses than any observable decay in the intensity of reflections.", + "section": "INTRO", + "ner": [ + [ + 1, + 26, + "Specific radiation damage", + "experimental_method" + ], + [ + 28, + 31, + "SRD", + "experimental_method" + ], + [ + 52, + 79, + "real-space electron density", + "evidence" + ] + ] + }, + { + "sid": 18, + "sent": "Indeed, the C\u2014Se bond in selenomethionine, the stability of which is key for the success of experimental phasing methods, can be cleaved at a dose as low as 2\u2005MGy for a crystal maintained at 100\u2005K (Holton, 2007).", + "section": "INTRO", + "ner": [ + [ + 14, + 16, + "Se", + "chemical" + ], + [ + 25, + 41, + "selenomethionine", + "chemical" + ], + [ + 169, + 176, + "crystal", + "evidence" + ] + ] + }, + { + "sid": 19, + "sent": "SRD has been well characterized in a large range of proteins, and is seen to follow a reproducible order: metallo-centre reduction, disulfide-bond cleavage, acidic residue decarboxylation and methionine methylthio cleavage (Ravelli & McSweeney, 2000; Burmeister, 2000; Weik et al., 2000; Yano et al., 2005).", + "section": "INTRO", + "ner": [ + [ + 132, + 146, + "disulfide-bond", + "ptm" + ] + ] + }, + { + "sid": 20, + "sent": "There are a number of cases where SRD manifestations have compromised the biological information extracted from MX-determined structures at much lower doses than the recommended 30\u2005MGy limit, leading to false structural interpretations of protein mechanisms.", + "section": "INTRO", + "ner": [ + [ + 112, + 125, + "MX-determined", + "experimental_method" + ], + [ + 126, + 136, + "structures", + "evidence" + ] + ] + }, + { + "sid": 21, + "sent": "Active-site residues appear to be particularly susceptible, particularly for photosensitive proteins and in instances where chemical strain is an intrinsic feature of the reaction mechanism.", + "section": "INTRO", + "ner": [ + [ + 0, + 20, + "Active-site residues", + "site" + ] + ] + }, + { + "sid": 22, + "sent": "For instance, structure determination of the purple membrane protein bacterio\u00adrhodopsin required careful corrections for radiation-induced structural changes before the correct photosensitive intermediate states could be isolated (Matsui et al., 2002).", + "section": "INTRO", + "ner": [ + [ + 14, + 37, + "structure determination", + "experimental_method" + ], + [ + 69, + 87, + "bacterio\u00adrhodopsin", + "protein_type" + ] + ] + }, + { + "sid": 23, + "sent": "The significant chemical strain required for catalysis within the active site of phosphoserine aminotransferase has been observed to diminish during X-ray exposure (Dubnovitsky et al., 2005).", + "section": "INTRO", + "ner": [ + [ + 66, + 77, + "active site", + "site" + ], + [ + 81, + 111, + "phosphoserine aminotransferase", + "protein_type" + ] + ] + }, + { + "sid": 24, + "sent": "Since the majority of SRD studies to date have focused on proteins, much less is known about the effects of X-ray irradiation on the wider class of crystalline nucleoprotein complexes or how to correct for such radiation-induced structural changes.", + "section": "INTRO", + "ner": [ + [ + 22, + 33, + "SRD studies", + "experimental_method" + ], + [ + 160, + 173, + "nucleoprotein", + "complex_assembly" + ] + ] + }, + { + "sid": 25, + "sent": "Understanding RD to such complexes is crucial, since DNA is rarely naked within a cell, instead dynamically interacting with proteins, facilitating replication, transcription, modification and DNA repair.", + "section": "INTRO", + "ner": [ + [ + 53, + 56, + "DNA", + "chemical" + ], + [ + 193, + 196, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 26, + "sent": "As of early 2016, >5400 nucleoprotein complex structures have been deposited within the PDB, with 91% solved by MX.", + "section": "INTRO", + "ner": [ + [ + 24, + 37, + "nucleoprotein", + "complex_assembly" + ], + [ + 46, + 56, + "structures", + "evidence" + ], + [ + 112, + 114, + "MX", + "experimental_method" + ] + ] + }, + { + "sid": 27, + "sent": "It is essential to understand how these increasingly complex macromolecular structures are affected by the radiation used to solve them.", + "section": "INTRO", + "ner": [ + [ + 76, + 86, + "structures", + "evidence" + ] + ] + }, + { + "sid": 28, + "sent": "Nucleoproteins also represent one of the main targets of radiotherapy, and an insight into the damage mechanisms induced by X-ray irradiation could inform innovative treatments.", + "section": "INTRO", + "ner": [ + [ + 0, + 14, + "Nucleoproteins", + "complex_assembly" + ] + ] + }, + { + "sid": 29, + "sent": "Investigations on sub-ionization-level LEEs (0\u201315\u2005eV) interacting with both dried and aqueous oligonucleotides (Alizadeh & Sanche, 2014; Simons, 2006) concluded that resonant electron attachment to DNA bases and the sugar-phosphate backbone could lead to the preferential cleavage of strong (\u223c4\u2005eV, 385\u2005kJ\u2005mol\u22121) sugar-phosphate C\u2014O covalent bonds within the DNA backbone and then base-sugar N1\u2014C bonds, eventually leading to single-strand breakages (SSBs; Ptasi\u0144ska & Sanche, 2007).", + "section": "INTRO", + "ner": [ + [ + 198, + 201, + "DNA", + "chemical" + ], + [ + 359, + 362, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 30, + "sent": "Electrons have been shown to be mobile at 77\u2005K by electron spin resonance spectroscopy studies (Symons, 1997; Jones et al., 1987), with rapid electron quantum tunnelling and positive hole migration along the protein backbone and through stacked DNA bases indicated as a dominant mechanism by which oxidative and reductive damage localizes at distances from initial ionization sites at 100\u2005K (O\u2019Neill et al., 2002).", + "section": "INTRO", + "ner": [ + [ + 50, + 86, + "electron spin resonance spectroscopy", + "experimental_method" + ], + [ + 245, + 248, + "DNA", + "chemical" + ], + [ + 365, + 381, + "ionization sites", + "site" + ] + ] + }, + { + "sid": 31, + "sent": "The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein\u2013DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8\u2005\u00c5; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1\u201344.6\u2005MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015).", + "section": "INTRO", + "ner": [ + [ + 187, + 199, + "crystallized", + "experimental_method" + ], + [ + 247, + 256, + "bacterial", + "taxonomy_domain" + ], + [ + 265, + 268, + "DNA", + "chemical" + ], + [ + 278, + 288, + "C.Esp1396I", + "complex_assembly" + ], + [ + 460, + 463, + "DNA", + "chemical" + ], + [ + 475, + 482, + "crystal", + "evidence" + ] + ] + }, + { + "sid": 32, + "sent": "Only at doses above 20\u2005MGy were precursors of phosphodiester-bond cleavage observed within AT-rich regions of the 35-mer DNA.", + "section": "INTRO", + "ner": [ + [ + 91, + 106, + "AT-rich regions", + "structure_element" + ], + [ + 121, + 124, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 33, + "sent": "For crystalline complexes such as C.Esp1396I, whether the protein is intrinsically more susceptible to X-ray-induced damage or whether the protein scavenges electrons to protect the DNA remains unclear in the absence of a non-nucleic acid-bound protein control obtained under exactly the same crystallization and data-collection conditions.", + "section": "INTRO", + "ner": [ + [ + 34, + 44, + "C.Esp1396I", + "complex_assembly" + ], + [ + 182, + 185, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 34, + "sent": "To monitor the effects of nucleic acid binding on protein damage susceptibility, a crystal containing two protein molecules per asymmetric unit, only one of which was bound to RNA, is reported here (Fig. 1 \u25b8).", + "section": "INTRO", + "ner": [ + [ + 83, + 90, + "crystal", + "evidence" + ], + [ + 167, + 175, + "bound to", + "protein_state" + ], + [ + 176, + 179, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 35, + "sent": "Using newly developed methodology, we present a controlled SRD investigation at 1.98\u2005\u00c5 resolution using a large (\u223c91\u2005kDa) crystalline protein\u2013RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53\u2005bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002).", + "section": "INTRO", + "ner": [ + [ + 48, + 62, + "controlled SRD", + "experimental_method" + ], + [ + 134, + 145, + "protein\u2013RNA", + "complex_assembly" + ], + [ + 155, + 190, + "trp RNA-binding attenuation protein", + "protein_type" + ], + [ + 192, + 196, + "TRAP", + "complex_assembly" + ], + [ + 198, + 206, + "bound to", + "protein_state" + ], + [ + 215, + 218, + "RNA", + "chemical" + ], + [ + 228, + 240, + "(GAGUU)10GAG", + "chemical" + ] + ] + }, + { + "sid": 36, + "sent": "TRAP consists of 11 identical subunits assembled into a ring with 11-fold rotational symmetry.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "TRAP", + "complex_assembly" + ], + [ + 30, + 38, + "subunits", + "structure_element" + ], + [ + 56, + 60, + "ring", + "structure_element" + ] + ] + }, + { + "sid": 37, + "sent": "It binds with high affinity (K d \u2243 1.0\u2005nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999).", + "section": "INTRO", + "ner": [ + [ + 29, + 32, + "K d", + "evidence" + ], + [ + 46, + 49, + "RNA", + "chemical" + ], + [ + 73, + 89, + "GAG/UAG triplets", + "structure_element" + ], + [ + 116, + 134, + "spacer nucleotides", + "structure_element" + ], + [ + 191, + 201, + "tryptophan", + "chemical" + ], + [ + 224, + 241, + "Bacillus subtilis", + "species" + ] + ] + }, + { + "sid": 38, + "sent": "In this structure, the bases of the G1-A2-G3 nucleotides form direct hydrogen bonds to the protein, unlike the U4-U5 nucleotides, which appear to be more flexible.", + "section": "INTRO", + "ner": [ + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 36, + 44, + "G1-A2-G3", + "chemical" + ], + [ + 69, + 83, + "hydrogen bonds", + "bond_interaction" + ], + [ + 111, + 116, + "U4-U5", + "chemical" + ] + ] + }, + { + "sid": 39, + "sent": "Ten successive 1.98\u2005\u00c5 resolution MX data sets were collected from the same TRAP\u2013RNA crystal to analyse X-ray-induced structural changes over a large dose range (d 1 = 1.3\u2005MGy to d 10 = 25.0\u2005MGy).", + "section": "INTRO", + "ner": [ + [ + 33, + 35, + "MX", + "experimental_method" + ], + [ + 75, + 83, + "TRAP\u2013RNA", + "complex_assembly" + ], + [ + 84, + 91, + "crystal", + "evidence" + ] + ] + }, + { + "sid": 40, + "sent": "To avoid the previous necessity for visual inspection of electron-density maps to detect SRD sites, a computational approach was designed to quantify the electron-density change for each refined atom with increasing dose, thus providing a rapid systematic method for SRD study on such large multimeric complexes.", + "section": "INTRO", + "ner": [ + [ + 57, + 78, + "electron-density maps", + "evidence" + ], + [ + 89, + 98, + "SRD sites", + "site" + ], + [ + 154, + 177, + "electron-density change", + "evidence" + ] + ] + }, + { + "sid": 41, + "sent": "By employing the high 11-fold structural symmetry within each TRAP macromolecule, this approach permitted a thorough statistical quantification of the RD effects of RNA binding to TRAP.", + "section": "INTRO", + "ner": [ + [ + 62, + 66, + "TRAP", + "complex_assembly" + ], + [ + 165, + 168, + "RNA", + "chemical" + ], + [ + 180, + 184, + "TRAP", + "complex_assembly" + ] + ] + }, + { + "sid": 42, + "sent": "Per-atom quantification of electron density \u00a0 ", + "section": "RESULTS", + "ner": [ + [ + 0, + 43, + "Per-atom quantification of electron density", + "experimental_method" + ] + ] + }, + { + "sid": 43, + "sent": "To quantify the exact effects of nucleic acid binding to a protein on SRD susceptibility, a high-throughput and automated pipeline was created to systematically calculate the electron-density change for every refined atom within the TRAP\u2013RNA structure as a function of dose.", + "section": "RESULTS", + "ner": [ + [ + 175, + 198, + "electron-density change", + "evidence" + ], + [ + 233, + 241, + "TRAP\u2013RNA", + "complex_assembly" + ], + [ + 242, + 251, + "structure", + "evidence" + ] + ] + }, + { + "sid": 44, + "sent": "This provides an atom-specific quantification of density\u2013dose dynamics, which was previously lacking within the field.", + "section": "RESULTS", + "ner": [ + [ + 49, + 70, + "density\u2013dose dynamics", + "evidence" + ] + ] + }, + { + "sid": 45, + "sent": "Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) \u2212 F obs(d 1) Fourier difference map peaks in terms of the sigma (\u03c3) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible.", + "section": "RESULTS", + "ner": [ + [ + 36, + 45, + "SRD sites", + "site" + ], + [ + 73, + 125, + "F obs(d n) \u2212 F obs(d 1) Fourier difference map peaks", + "evidence" + ], + [ + 142, + 147, + "sigma", + "evidence" + ], + [ + 149, + 150, + "\u03c3", + "evidence" + ], + [ + 181, + 200, + "standard deviations", + "evidence" + ], + [ + 210, + 241, + "mean map electron-density value", + "evidence" + ] + ] + }, + { + "sid": 46, + "sent": "However, these \u03c3 levels depend on the standard deviation values of the map, which can deviate between data sets, and are thus unsuitable for quantitative comparison of density between different dose data sets.", + "section": "RESULTS", + "ner": [ + [ + 15, + 16, + "\u03c3", + "evidence" + ], + [ + 38, + 56, + "standard deviation", + "evidence" + ], + [ + 71, + 74, + "map", + "evidence" + ], + [ + 168, + 175, + "density", + "evidence" + ] + ] + }, + { + "sid": 47, + "sent": "Instead, we use here a maximum density-loss metric (D loss), which is the per-atom equivalent of the magnitude of these negative Fourier difference map peaks in units of e\u2005\u00c5\u22123.", + "section": "RESULTS", + "ner": [ + [ + 23, + 50, + "maximum density-loss metric", + "evidence" + ], + [ + 52, + 58, + "D loss", + "evidence" + ], + [ + 120, + 157, + "negative Fourier difference map peaks", + "evidence" + ] + ] + }, + { + "sid": 48, + "sent": "Large positive D loss values indicate radiation-induced atomic disordering reproducibly throughout the unit cells with respect to the initial low-dose data set.", + "section": "RESULTS", + "ner": [ + [ + 15, + 21, + "D loss", + "evidence" + ] + ] + }, + { + "sid": 49, + "sent": "For each TRAP\u2013RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 \u25b8 a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9\u2005MGy; Fig. 3 \u25b8 a).", + "section": "RESULTS", + "ner": [ + [ + 9, + 17, + "TRAP\u2013RNA", + "complex_assembly" + ], + [ + 32, + 45, + "D loss metric", + "evidence" + ], + [ + 102, + 105, + "SRD", + "experimental_method" + ], + [ + 131, + 134, + "Glu", + "residue_name" + ], + [ + 139, + 142, + "Asp", + "residue_name" + ], + [ + 188, + 202, + "difference map", + "evidence" + ] + ] + }, + { + "sid": 50, + "sent": "The main sequence of TRAP does not contain any Trp and Cys residues (and thus contains no disulfide bonds).", + "section": "RESULTS", + "ner": [ + [ + 21, + 25, + "TRAP", + "complex_assembly" + ], + [ + 47, + 50, + "Trp", + "residue_name" + ], + [ + 55, + 58, + "Cys", + "residue_name" + ] + ] + }, + { + "sid": 51, + "sent": "The substrate Trp amino-acid ligands also exhibited disordering of the free terminal carboxyl groups at higher doses (Fig. 2 \u25b8 a); however, no clear Fourier difference peaks could be observed visually.", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "Trp", + "chemical" + ], + [ + 149, + 173, + "Fourier difference peaks", + "evidence" + ] + ] + }, + { + "sid": 52, + "sent": "Even for radiation-insensitive residues (e.g. Gly) the average D loss increases with dose: this is the effect of global radiation damage, since as dose increases the electron density associated with each refined atom becomes weaker as the atomic occupancy decreases (Fig. 2 \u25b8 b).", + "section": "RESULTS", + "ner": [ + [ + 46, + 49, + "Gly", + "residue_name" + ], + [ + 63, + 69, + "D loss", + "evidence" + ], + [ + 166, + 182, + "electron density", + "evidence" + ] + ] + }, + { + "sid": 53, + "sent": "Only Glu and Asp residues exhibit a rate of D loss increase that consistently exceeds the average decay (Fig. 2 \u25b8 b, dashed line) at each dose.", + "section": "RESULTS", + "ner": [ + [ + 5, + 8, + "Glu", + "residue_name" + ], + [ + 13, + 16, + "Asp", + "residue_name" + ], + [ + 44, + 50, + "D loss", + "evidence" + ] + ] + }, + { + "sid": 54, + "sent": "The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 \u25b8 b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8\u2005kJ\u2005mol\u22121 from oxidized Glu residues than from their Asp counterparts.", + "section": "RESULTS", + "ner": [ + [ + 12, + 18, + "D loss", + "evidence" + ], + [ + 90, + 93, + "Glu", + "residue_name" + ], + [ + 108, + 111, + "Asp", + "residue_name" + ], + [ + 321, + 337, + "hydrogen-bonding", + "bond_interaction" + ], + [ + 352, + 355, + "CO2", + "chemical" + ], + [ + 406, + 414, + "oxidized", + "protein_state" + ], + [ + 415, + 418, + "Glu", + "residue_name" + ], + [ + 444, + 447, + "Asp", + "residue_name" + ] + ] + }, + { + "sid": 55, + "sent": "RNA is less susceptible to electron-density loss than protein within the TRAP\u2013RNA complex \u00a0 ", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "RNA", + "chemical" + ], + [ + 27, + 43, + "electron-density", + "evidence" + ], + [ + 73, + 81, + "TRAP\u2013RNA", + "complex_assembly" + ] + ] + }, + { + "sid": 56, + "sent": "Visual inspection of Fourier difference maps illustrated the clear lack of RNA electron-density degradation with increasing dose compared with the obvious protein damage manifestations (Figs. 3 \u25b8 b and 3 \u25b8 c).", + "section": "RESULTS", + "ner": [ + [ + 0, + 20, + "Visual inspection of", + "experimental_method" + ], + [ + 21, + 44, + "Fourier difference maps", + "evidence" + ], + [ + 75, + 78, + "RNA", + "chemical" + ], + [ + 79, + 107, + "electron-density degradation", + "evidence" + ] + ] + }, + { + "sid": 57, + "sent": "Only at the highest doses investigated (>20\u2005MGy) was density loss observed at the RNA phosphate and C\u2014O bonds of the phosphodiester backbone.", + "section": "RESULTS", + "ner": [ + [ + 82, + 85, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 58, + "sent": "However, the median D loss was lower by a factor of >2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0\u2005MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly C\u03b1 atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3).", + "section": "RESULTS", + "ner": [ + [ + 20, + 26, + "D loss", + "evidence" + ], + [ + 59, + 62, + "RNA", + "chemical" + ], + [ + 80, + 83, + "Glu", + "residue_name" + ], + [ + 88, + 91, + "Asp", + "residue_name" + ], + [ + 207, + 210, + "Gly", + "residue_name" + ], + [ + 227, + 231, + "TRAP", + "complex_assembly" + ] + ] + }, + { + "sid": 59, + "sent": "RNA binding protects radiation-sensitive residues \u00a0 ", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 60, + "sent": "For the large number of acidic residues per TRAP ring (four Asp and six Glu residues per protein monomer), a strong dependence of decarboxylation susceptibility on local environment was observed (Fig. 4 \u25b8).", + "section": "RESULTS", + "ner": [ + [ + 44, + 48, + "TRAP", + "complex_assembly" + ], + [ + 49, + 53, + "ring", + "structure_element" + ], + [ + 60, + 63, + "Asp", + "residue_name" + ], + [ + 72, + 75, + "Glu", + "residue_name" + ], + [ + 97, + 104, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 61, + "sent": "For each Glu\u2005C\u03b4 or Asp\u2005C\u03b3 atom, D loss provided a direct measure of the rate of side-chain carboxyl-group disordering and subsequent decarboxylation.", + "section": "RESULTS", + "ner": [ + [ + 9, + 12, + "Glu", + "residue_name" + ], + [ + 19, + 22, + "Asp", + "residue_name" + ], + [ + 32, + 38, + "D loss", + "evidence" + ] + ] + }, + { + "sid": 62, + "sent": "For acidic residues with no differing interactions between nonbound and bound TRAP (Fig. 4 \u25b8 a), similar damage was apparent between the two rings within the asymmetric unit, as expected.", + "section": "RESULTS", + "ner": [ + [ + 59, + 67, + "nonbound", + "protein_state" + ], + [ + 72, + 77, + "bound", + "protein_state" + ], + [ + 78, + 82, + "TRAP", + "complex_assembly" + ] + ] + }, + { + "sid": 63, + "sent": "However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 \u25b8 b), and for residues at the ring\u2013ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 \u25b8 c).", + "section": "RESULTS", + "ner": [ + [ + 9, + 13, + "TRAP", + "complex_assembly" + ], + [ + 39, + 61, + "RNA-binding interfaces", + "site" + ], + [ + 103, + 111, + "nonbound", + "protein_state" + ], + [ + 112, + 116, + "TRAP", + "complex_assembly" + ], + [ + 155, + 175, + "ring\u2013ring interfaces", + "site" + ], + [ + 215, + 220, + "bound", + "protein_state" + ], + [ + 221, + 225, + "TRAP", + "complex_assembly" + ] + ] + }, + { + "sid": 64, + "sent": "Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 \u25b8 shows their density changes with increasing dose.", + "section": "RESULTS", + "ner": [ + [ + 23, + 28, + "Glu36", + "residue_name_number" + ], + [ + 30, + 35, + "Asp39", + "residue_name_number" + ], + [ + 40, + 45, + "Glu42", + "residue_name_number" + ], + [ + 63, + 66, + "RNA", + "chemical" + ], + [ + 102, + 106, + "TRAP", + "complex_assembly" + ], + [ + 107, + 111, + "ring", + "structure_element" + ], + [ + 112, + 120, + "subunits", + "structure_element" + ], + [ + 147, + 162, + "density changes", + "evidence" + ] + ] + }, + { + "sid": 65, + "sent": "Hotelling\u2019s T-squared test (the multivariate counterpart of Student\u2019s t-test) was used to reject the null hypothesis that the means of the D loss metric were equal for the bound and nonbound groups in Fig. 5 \u25b8.", + "section": "RESULTS", + "ner": [ + [ + 0, + 26, + "Hotelling\u2019s T-squared test", + "experimental_method" + ], + [ + 60, + 76, + "Student\u2019s t-test", + "experimental_method" + ], + [ + 139, + 152, + "D loss metric", + "evidence" + ], + [ + 172, + 177, + "bound", + "protein_state" + ], + [ + 182, + 190, + "nonbound", + "protein_state" + ] + ] + }, + { + "sid": 66, + "sent": "A significant reduction in D loss is seen for Glu36 in RNA-bound compared with nonbound TRAP, indicative of a lower rate of side-chain decarboxylation (Fig. 5 \u25b8 a; p = 6.06 \u00d7 10\u22125).", + "section": "RESULTS", + "ner": [ + [ + 27, + 33, + "D loss", + "evidence" + ], + [ + 46, + 51, + "Glu36", + "residue_name_number" + ], + [ + 55, + 64, + "RNA-bound", + "protein_state" + ], + [ + 79, + 87, + "nonbound", + "protein_state" + ], + [ + 88, + 92, + "TRAP", + "complex_assembly" + ] + ] + }, + { + "sid": 67, + "sent": "For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base.", + "section": "RESULTS", + "ner": [ + [ + 9, + 13, + "TRAP", + "complex_assembly" + ], + [ + 14, + 18, + "ring", + "structure_element" + ], + [ + 19, + 26, + "subunit", + "structure_element" + ], + [ + 32, + 37, + "Glu36", + "residue_name_number" + ], + [ + 82, + 96, + "hydrogen bonds", + "bond_interaction" + ], + [ + 125, + 127, + "G3", + "residue_name_number" + ], + [ + 128, + 131, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 68, + "sent": "In our analysis, Asp39 in the TRAP\u2013(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP\u2013RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 \u25b8 b, p = 0.0925).", + "section": "RESULTS", + "ner": [ + [ + 17, + 22, + "Asp39", + "residue_name_number" + ], + [ + 30, + 47, + "TRAP\u2013(GAGUU)10GAG", + "complex_assembly" + ], + [ + 48, + 57, + "structure", + "evidence" + ], + [ + 90, + 104, + "hydrogen bonds", + "bond_interaction" + ], + [ + 112, + 114, + "G1", + "residue_name_number" + ], + [ + 142, + 161, + "TRAP\u2013RNA interfaces", + "site" + ], + [ + 171, + 176, + "Glu36", + "residue_name_number" + ], + [ + 180, + 182, + "G3", + "residue_name_number" + ], + [ + 210, + 217, + "density", + "evidence" + ], + [ + 235, + 238, + "RNA", + "chemical" + ], + [ + 275, + 280, + "Asp39", + "residue_name_number" + ], + [ + 290, + 295, + "Glu36", + "residue_name_number" + ] + ] + }, + { + "sid": 69, + "sent": "RNA binding reduces radiation-induced disorder on the atomic scale \u00a0 ", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 70, + "sent": "One oxygen (O\u220a1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O\u220a2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999).", + "section": "RESULTS", + "ner": [ + [ + 20, + 25, + "Glu42", + "residue_name_number" + ], + [ + 44, + 57, + "hydrogen bond", + "bond_interaction" + ], + [ + 70, + 75, + "water", + "chemical" + ], + [ + 88, + 111, + "TRAP RNA-binding pocket", + "site" + ], + [ + 154, + 165, + "salt-bridge", + "bond_interaction" + ], + [ + 183, + 188, + "Arg58", + "residue_name_number" + ] + ] + }, + { + "sid": 71, + "sent": "Salt-bridge interactions have previously been suggested to reduce the glutamate decarboxylation rate within the large (\u223c62.4\u2005kDa) myrosinase protein structure (Burmeister, 2000).", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Salt-bridge", + "bond_interaction" + ], + [ + 70, + 79, + "glutamate", + "residue_name" + ], + [ + 130, + 140, + "myrosinase", + "protein_type" + ], + [ + 149, + 158, + "structure", + "evidence" + ] + ] + }, + { + "sid": 72, + "sent": "A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42\u2005O\u220a1 atoms (Fig. 5 \u25b8 c; p\u00a0=\u00a00.007) but not for the Glu42\u2005O\u220a2 atoms (Fig. 5 \u25b8 d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA.", + "section": "RESULTS", + "ner": [ + [ + 50, + 65, + "D loss dynamics", + "evidence" + ], + [ + 74, + 82, + "nonbound", + "protein_state" + ], + [ + 83, + 88, + "bound", + "protein_state" + ], + [ + 89, + 94, + "Glu42", + "residue_name_number" + ], + [ + 145, + 150, + "Glu42", + "residue_name_number" + ], + [ + 235, + 246, + "salt-bridge", + "bond_interaction" + ], + [ + 278, + 281, + "RNA", + "chemical" + ], + [ + 303, + 308, + "water", + "chemical" + ], + [ + 318, + 331, + "hydrogen bond", + "bond_interaction" + ], + [ + 399, + 409, + "absence of", + "protein_state" + ], + [ + 410, + 413, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 73, + "sent": "The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group C\u03b4 atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate.", + "section": "RESULTS", + "ner": [ + [ + 4, + 27, + "density-change dynamics", + "evidence" + ], + [ + 73, + 78, + "bound", + "protein_state" + ], + [ + 83, + 91, + "nonbound", + "protein_state" + ], + [ + 92, + 96, + "TRAP", + "complex_assembly" + ], + [ + 106, + 111, + "Glu42", + "residue_name_number" + ], + [ + 169, + 172, + "RNA", + "chemical" + ], + [ + 195, + 206, + "salt-bridge", + "bond_interaction" + ], + [ + 251, + 256, + "Glu42", + "residue_name_number" + ] + ] + }, + { + "sid": 74, + "sent": "The RNA-stabilizing effect was not restricted to radiation-sensitive acidic residues.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 75, + "sent": "The side chain of Phe32 stacks against the G3 base within the 11 TRAP RNA-binding interfaces (Antson et al., 1999).", + "section": "RESULTS", + "ner": [ + [ + 18, + 23, + "Phe32", + "residue_name_number" + ], + [ + 43, + 45, + "G3", + "residue_name_number" + ], + [ + 65, + 92, + "TRAP RNA-binding interfaces", + "site" + ] + ] + }, + { + "sid": 76, + "sent": "With increasing dose, the D loss associated with the Phe32 side chain was significantly reduced upon RNA binding (Fig. 5 \u25b8 e; Phe32\u2005C\u03b6; p = 0.0014), an indication that radiation-induced conformation disordering of Phe32 had been reduced.", + "section": "RESULTS", + "ner": [ + [ + 26, + 32, + "D loss", + "evidence" + ], + [ + 53, + 58, + "Phe32", + "residue_name_number" + ], + [ + 101, + 104, + "RNA", + "chemical" + ], + [ + 126, + 131, + "Phe32", + "residue_name_number" + ], + [ + 214, + 219, + "Phe32", + "residue_name_number" + ] + ] + }, + { + "sid": 77, + "sent": "The extended aliphatic Lys37 side chain stacks against the nearby G1 base, making a series of nonpolar contacts within each RNA-binding interface.", + "section": "RESULTS", + "ner": [ + [ + 23, + 28, + "Lys37", + "residue_name_number" + ], + [ + 66, + 68, + "G1", + "residue_name_number" + ], + [ + 94, + 111, + "nonpolar contacts", + "bond_interaction" + ], + [ + 124, + 145, + "RNA-binding interface", + "site" + ] + ] + }, + { + "sid": 78, + "sent": "The D loss for Lys37 side-chain atoms was also reduced when stacked against the G1 base (Fig. 5 \u25b8 f; p = 0.0243 for Lys37\u2005C\u220a atoms).", + "section": "RESULTS", + "ner": [ + [ + 4, + 10, + "D loss", + "evidence" + ], + [ + 15, + 20, + "Lys37", + "residue_name_number" + ], + [ + 60, + 67, + "stacked", + "bond_interaction" + ], + [ + 80, + 82, + "G1", + "residue_name_number" + ], + [ + 116, + 121, + "Lys37", + "residue_name_number" + ] + ] + }, + { + "sid": 79, + "sent": "Representative Phe32 and Lys37 atoms were selected to illustrate these trends.", + "section": "RESULTS", + "ner": [ + [ + 15, + 20, + "Phe32", + "residue_name_number" + ], + [ + 25, + 30, + "Lys37", + "residue_name_number" + ] + ] + }, + { + "sid": 80, + "sent": "Here, MX radiation-induced specific structural changes within the large TRAP\u2013RNA assembly over a large dose range (1.3\u201325.0\u2005MGy) have been analysed using a high-throughput quantitative approach, providing a measure of the electron-density distribution for each refined atom with increasing dose, D loss.", + "section": "DISCUSS", + "ner": [ + [ + 6, + 8, + "MX", + "experimental_method" + ], + [ + 72, + 80, + "TRAP\u2013RNA", + "complex_assembly" + ], + [ + 222, + 251, + "electron-density distribution", + "evidence" + ], + [ + 296, + 302, + "D loss", + "evidence" + ] + ] + }, + { + "sid": 81, + "sent": "Compared with previous studies, the results provide a further step in the detailed characterization of SRD effects in MX.", + "section": "DISCUSS", + "ner": [ + [ + 118, + 120, + "MX", + "experimental_method" + ] + ] + }, + { + "sid": 82, + "sent": "Our method\u00adology, which eliminated tedious and error-prone visual inspection, permitted the determination on a per-atom basis of the most damaged sites, as characterized by F obs(d n) \u2212 F obs(d 1) Fourier difference map peaks between successive data sets collected from the same crystal.", + "section": "DISCUSS", + "ner": [ + [ + 173, + 225, + "F obs(d n) \u2212 F obs(d 1) Fourier difference map peaks", + "evidence" + ], + [ + 279, + 286, + "crystal", + "evidence" + ] + ] + }, + { + "sid": 83, + "sent": "Here, it provided the precision required to quantify the role of RNA in the damage susceptibilities of equivalent atoms between RNA-bound and nonbound TRAP, but it is applicable to any MX SRD study.", + "section": "DISCUSS", + "ner": [ + [ + 65, + 68, + "RNA", + "chemical" + ], + [ + 128, + 137, + "RNA-bound", + "protein_state" + ], + [ + 142, + 150, + "nonbound", + "protein_state" + ], + [ + 151, + 155, + "TRAP", + "complex_assembly" + ], + [ + 185, + 187, + "MX", + "experimental_method" + ] + ] + }, + { + "sid": 84, + "sent": "The RNA was found to be substantially more radiation-resistant than the protein, even at the highest doses investigated (\u223c25.0\u2005MGy), which is in strong concurrence with our previous SRD investigation of the C.Esp1396I protein\u2013DNA complex (Bury et al., 2015).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 7, + "RNA", + "chemical" + ], + [ + 43, + 62, + "radiation-resistant", + "protein_state" + ], + [ + 182, + 199, + "SRD investigation", + "experimental_method" + ], + [ + 207, + 217, + "C.Esp1396I", + "complex_assembly" + ], + [ + 226, + 229, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 85, + "sent": "Consistent with that study, at high doses of above \u223c20\u2005MGy, F obs(d n) \u2212 F obs(d 1) map density was detected around P, O3\u2032 and O5\u2032 atoms of the RNA backbone, with no significant difference density localized to RNA ribose and basic subunits.", + "section": "DISCUSS", + "ner": [ + [ + 60, + 95, + "F obs(d n) \u2212 F obs(d 1) map density", + "evidence" + ], + [ + 144, + 147, + "RNA", + "chemical" + ], + [ + 178, + 196, + "difference density", + "evidence" + ], + [ + 210, + 213, + "RNA", + "chemical" + ], + [ + 231, + 239, + "subunits", + "structure_element" + ] + ] + }, + { + "sid": 86, + "sent": "RNA backbone disordering thus appears to be the main radiation-induced effect in RNA, with the protein\u2013base interactions maintained even at high doses (>20\u2005MGy).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 3, + "RNA", + "chemical" + ], + [ + 81, + 84, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 87, + "sent": "The U4 phosphate exhibited marginally larger D loss values above 20\u2005MGy than G1, A2 and G3 (Supplementary Fig. S4).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 6, + "U4", + "residue_name_number" + ], + [ + 7, + 16, + "phosphate", + "chemical" + ], + [ + 45, + 51, + "D loss", + "evidence" + ], + [ + 77, + 79, + "G1", + "residue_name_number" + ], + [ + 81, + 83, + "A2", + "residue_name_number" + ], + [ + 88, + 90, + "G3", + "residue_name_number" + ] + ] + }, + { + "sid": 88, + "sent": "Since U4 is the only refined nucleotide not to exhibit significant base\u2013protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility.", + "section": "DISCUSS", + "ner": [ + [ + 6, + 8, + "U4", + "residue_name_number" + ], + [ + 100, + 104, + "TRAP", + "complex_assembly" + ], + [ + 113, + 118, + "water", + "chemical" + ], + [ + 128, + 141, + "hydrogen bond", + "bond_interaction" + ], + [ + 175, + 183, + "subunits", + "structure_element" + ], + [ + 197, + 202, + "Arg58", + "residue_name_number" + ], + [ + 203, + 216, + "hydrogen bond", + "bond_interaction" + ], + [ + 245, + 253, + "subunits", + "structure_element" + ], + [ + 271, + 273, + "U4", + "residue_name_number" + ], + [ + 274, + 280, + "D loss", + "evidence" + ] + ] + }, + { + "sid": 89, + "sent": "At 25.0\u2005MGy, the magnitude of the RNA backbone D loss was of the same order as for the radiation-insensitive Gly\u2005C\u03b1 atoms and on average less than half that of the acidic residues of the protein (Supplementary Fig. S3).", + "section": "DISCUSS", + "ner": [ + [ + 34, + 37, + "RNA", + "chemical" + ], + [ + 47, + 53, + "D loss", + "evidence" + ], + [ + 109, + 112, + "Gly", + "residue_name" + ] + ] + }, + { + "sid": 90, + "sent": "Consequently, no clear single-strand breaks could be located, and since RNA-binding within the current TRAP\u2013(GAGUU)10GAG complex is mediated predominantly through base\u2013protein interactions, the biological integrity of the RNA complex was dictated by the rate at which protein decarboxylation occurred.", + "section": "DISCUSS", + "ner": [ + [ + 72, + 75, + "RNA", + "chemical" + ], + [ + 103, + 120, + "TRAP\u2013(GAGUU)10GAG", + "complex_assembly" + ], + [ + 222, + 225, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 91, + "sent": "RNA interacting with TRAP was shown to offer significant protection against radiation-induced structural changes.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 3, + "RNA", + "chemical" + ], + [ + 21, + 25, + "TRAP", + "complex_assembly" + ] + ] + }, + { + "sid": 92, + "sent": "Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively).", + "section": "DISCUSS", + "ner": [ + [ + 5, + 10, + "Glu36", + "residue_name_number" + ], + [ + 15, + 20, + "Asp39", + "residue_name_number" + ], + [ + 38, + 41, + "RNA", + "chemical" + ], + [ + 60, + 74, + "hydrogen bonds", + "bond_interaction" + ], + [ + 78, + 85, + "guanine", + "chemical" + ], + [ + 93, + 95, + "G3", + "residue_name_number" + ], + [ + 100, + 102, + "G1", + "residue_name_number" + ] + ] + }, + { + "sid": 93, + "sent": "However, compared with Asp39, Glu36 is strikingly less decarboxylated when bound to RNA (Fig. 4 \u25b8).", + "section": "DISCUSS", + "ner": [ + [ + 23, + 28, + "Asp39", + "residue_name_number" + ], + [ + 30, + 35, + "Glu36", + "residue_name_number" + ], + [ + 75, + 83, + "bound to", + "protein_state" + ], + [ + 84, + 87, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 94, + "sent": "This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d \u2243 1.1 \u00b1 0.4\u2005nM).", + "section": "DISCUSS", + "ner": [ + [ + 40, + 83, + "mutagenesis and nucleoside analogue studies", + "experimental_method" + ], + [ + 133, + 135, + "G1", + "residue_name_number" + ], + [ + 164, + 168, + "TRAP", + "complex_assembly" + ], + [ + 187, + 189, + "A2", + "residue_name_number" + ], + [ + 194, + 196, + "G3", + "residue_name_number" + ], + [ + 232, + 252, + "RNA-binding affinity", + "evidence" + ], + [ + 256, + 260, + "TRAP", + "complex_assembly" + ], + [ + 262, + 265, + "K d", + "evidence" + ] + ] + }, + { + "sid": 95, + "sent": "For Glu36 and Asp39, no direct quantitative correlation could be established between hydrogen-bond length and D loss (linear R 2 of <0.23 for all doses; Supplementary Fig. S5).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 9, + "Glu36", + "residue_name_number" + ], + [ + 14, + 19, + "Asp39", + "residue_name_number" + ], + [ + 85, + 98, + "hydrogen-bond", + "bond_interaction" + ], + [ + 110, + 116, + "D loss", + "evidence" + ], + [ + 118, + 128, + "linear R 2", + "evidence" + ] + ] + }, + { + "sid": 96, + "sent": "Thus, another factor must be responsible for this clear reduction in Glu36 CO2 decarboxyl\u00adation in RNA-bound TRAP.", + "section": "DISCUSS", + "ner": [ + [ + 69, + 74, + "Glu36", + "residue_name_number" + ], + [ + 99, + 108, + "RNA-bound", + "protein_state" + ], + [ + 109, + 113, + "TRAP", + "complex_assembly" + ] + ] + }, + { + "sid": 97, + "sent": "The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 9, + "Glu36", + "residue_name_number" + ], + [ + 53, + 67, + "hydrogen bonds", + "bond_interaction" + ], + [ + 71, + 76, + "His34", + "residue_name_number" + ], + [ + 81, + 86, + "Lys56", + "residue_name_number" + ], + [ + 121, + 130, + "conserved", + "protein_state" + ], + [ + 147, + 149, + "G3", + "residue_name_number" + ], + [ + 231, + 236, + "Glu36", + "residue_name_number" + ], + [ + 240, + 249, + "RNA-bound", + "protein_state" + ], + [ + 250, + 254, + "TRAP", + "complex_assembly" + ] + ] + }, + { + "sid": 98, + "sent": "When bound to RNA, the average solvent-accessible area of the Glu36 side-chain O atoms is reduced from \u223c15 to 0\u2005\u00c52.", + "section": "DISCUSS", + "ner": [ + [ + 5, + 13, + "bound to", + "protein_state" + ], + [ + 14, + 17, + "RNA", + "chemical" + ], + [ + 62, + 67, + "Glu36", + "residue_name_number" + ] + ] + }, + { + "sid": 99, + "sent": "We propose that with no solvent accessibility Glu36 decarboxylation is inhibited, since the CO2-formation rate K 2 is greatly reduced, and suggest that steric hindrance prevents each radicalized Glu36 CO2 group from achieving the planar conformation required for complete dissociation from TRAP.", + "section": "DISCUSS", + "ner": [ + [ + 46, + 51, + "Glu36", + "residue_name_number" + ], + [ + 92, + 114, + "CO2-formation rate K 2", + "evidence" + ], + [ + 195, + 200, + "Glu36", + "residue_name_number" + ], + [ + 290, + 294, + "TRAP", + "complex_assembly" + ] + ] + }, + { + "sid": 100, + "sent": "The electron-recombination rate K \u22121 remains high, however, owing to rapid electron migration through the protein\u2013RNA complex to refill the Glu36 positive hole (the precursor for Glu decarboxylation).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 36, + "electron-recombination rate K \u22121", + "evidence" + ], + [ + 106, + 117, + "protein\u2013RNA", + "complex_assembly" + ], + [ + 140, + 145, + "Glu36", + "residue_name_number" + ], + [ + 146, + 159, + "positive hole", + "site" + ], + [ + 179, + 182, + "Glu", + "residue_name" + ] + ] + }, + { + "sid": 101, + "sent": "Upon RNA binding, the Asp39 side-chain carboxyl group solvent-accessible area changes from \u223c75 to 35\u2005\u00c52, still allowing a high CO2-formation rate K 2.", + "section": "DISCUSS", + "ner": [ + [ + 5, + 8, + "RNA", + "chemical" + ], + [ + 22, + 27, + "Asp39", + "residue_name_number" + ], + [ + 127, + 130, + "CO2", + "chemical" + ], + [ + 141, + 149, + "rate K 2", + "evidence" + ] + ] + }, + { + "sid": 102, + "sent": "The prevalence of radical attack from solvent channels surrounding the protein in the crystal is a questionable cause, considering previous observations indicating that the strongly oxidizing hydroxyl radical is immobile at 100\u2005K (Allan et al., 2013; Owen et al., 2012).", + "section": "DISCUSS", + "ner": [ + [ + 86, + 93, + "crystal", + "evidence" + ] + ] + }, + { + "sid": 103, + "sent": "By comparing equivalent acidic residues with and without RNA, we have now deconvoluted the role of solvent accessibility from other local protein environment factors, and thus propose a suitable mechanism by which exceptionally low solvent accessibility can reduce the rate of decarboxylation.", + "section": "DISCUSS", + "ner": [ + [ + 40, + 44, + "with", + "protein_state" + ], + [ + 49, + 56, + "without", + "protein_state" + ], + [ + 57, + 60, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 104, + "sent": "Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 \u25b8 c).", + "section": "DISCUSS", + "ner": [ + [ + 17, + 39, + "RNA-binding interfaces", + "site" + ], + [ + 41, + 44, + "RNA", + "chemical" + ], + [ + 102, + 107, + "Glu50", + "residue_name_number" + ], + [ + 109, + 114, + "Glu71", + "residue_name_number" + ], + [ + 119, + 124, + "Glu73", + "residue_name_number" + ], + [ + 180, + 184, + "TRAP", + "complex_assembly" + ], + [ + 185, + 190, + "rings", + "structure_element" + ] + ] + }, + { + "sid": 105, + "sent": "However, for each of these residues the exact crystal contacts are not preserved between bound and nonbound TRAP or even between monomers within one TRAP ring.", + "section": "DISCUSS", + "ner": [ + [ + 89, + 94, + "bound", + "protein_state" + ], + [ + 99, + 107, + "nonbound", + "protein_state" + ], + [ + 108, + 112, + "TRAP", + "complex_assembly" + ], + [ + 149, + 153, + "TRAP", + "complex_assembly" + ], + [ + 154, + 158, + "ring", + "structure_element" + ] + ] + }, + { + "sid": 106, + "sent": "For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 21, + "bound", + "protein_state" + ], + [ + 22, + 26, + "TRAP", + "complex_assembly" + ], + [ + 28, + 33, + "Glu73", + "residue_name_number" + ], + [ + 61, + 67, + "lysine", + "residue_name" + ], + [ + 86, + 94, + "subunits", + "structure_element" + ], + [ + 107, + 115, + "nonbound", + "protein_state" + ], + [ + 116, + 120, + "TRAP", + "complex_assembly" + ], + [ + 152, + 157, + "Glu73", + "residue_name_number" + ], + [ + 202, + 208, + "waters", + "chemical" + ], + [ + 217, + 224, + "subunit", + "structure_element" + ] + ] + }, + { + "sid": 107, + "sent": "Radiation-induced side-chain conformational changes have been poorly characterized in previous SRD investigations owing to their strong dependence on packing density and geometric strain.", + "section": "DISCUSS", + "ner": [ + [ + 95, + 113, + "SRD investigations", + "experimental_method" + ] + ] + }, + { + "sid": 108, + "sent": "Such structural changes are known to have significant roles within enzymatic pathways, and experimenters must be aware of these possible confounding factors when assigning true functional mechanisms using MX.", + "section": "DISCUSS", + "ner": [ + [ + 205, + 207, + "MX", + "experimental_method" + ] + ] + }, + { + "sid": 109, + "sent": "Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively.", + "section": "DISCUSS", + "ner": [ + [ + 22, + 25, + "RNA", + "chemical" + ], + [ + 37, + 41, + "TRAP", + "complex_assembly" + ], + [ + 95, + 99, + "TRAP", + "complex_assembly" + ], + [ + 128, + 133, + "Lys37", + "residue_name_number" + ], + [ + 138, + 143, + "Phe32", + "residue_name_number" + ], + [ + 169, + 171, + "G1", + "residue_name_number" + ], + [ + 176, + 178, + "G3", + "residue_name_number" + ] + ] + }, + { + "sid": 110, + "sent": "It has been suggested (Burmeister, 2000) that Tyr residues can lose their aromatic \u2013OH group owing to radiation-induced effects; however, no energetically favourable pathway for \u2013OH cleavage exists and this has not been detected in aqueous radiation-chemistry studies.", + "section": "DISCUSS", + "ner": [ + [ + 46, + 49, + "Tyr", + "residue_name" + ] + ] + }, + { + "sid": 111, + "sent": "In TRAP, D loss increased at a similar rate for both the Tyr O atoms and aromatic ring atoms, suggesting that full ring conformational disordering is more likely.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 7, + "TRAP", + "complex_assembly" + ], + [ + 9, + 15, + "D loss", + "evidence" + ], + [ + 57, + 60, + "Tyr", + "residue_name" + ], + [ + 82, + 86, + "ring", + "structure_element" + ], + [ + 115, + 119, + "ring", + "structure_element" + ] + ] + }, + { + "sid": 112, + "sent": "Indeed, no convincing reproducible Fourier difference peaks above the background map noise were observed around any Tyr terminal \u2013OH groups.", + "section": "DISCUSS", + "ner": [ + [ + 35, + 59, + "Fourier difference peaks", + "evidence" + ], + [ + 81, + 84, + "map", + "evidence" + ], + [ + 116, + 119, + "Tyr", + "residue_name" + ] + ] + }, + { + "sid": 113, + "sent": "The RNA-stabilization effects on protein are observed at short ranges and are restricted to within the RNA-binding interfaces around the TRAP ring.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 7, + "RNA", + "chemical" + ], + [ + 103, + 125, + "RNA-binding interfaces", + "site" + ], + [ + 137, + 141, + "TRAP", + "complex_assembly" + ], + [ + 142, + 146, + "ring", + "structure_element" + ] + ] + }, + { + "sid": 114, + "sent": "For example, Asp17 is located \u223c6.8\u2005\u00c5 from the G1 base, outside the RNA-binding interfaces, and has indistinguishable C\u03b3 atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p > 0.9).", + "section": "DISCUSS", + "ner": [ + [ + 13, + 18, + "Asp17", + "residue_name_number" + ], + [ + 46, + 48, + "G1", + "residue_name_number" + ], + [ + 67, + 89, + "RNA-binding interfaces", + "site" + ], + [ + 127, + 145, + "loss dose-dynamics", + "evidence" + ], + [ + 154, + 163, + "RNA-bound", + "protein_state" + ], + [ + 168, + 176, + "nonbound", + "protein_state" + ], + [ + 177, + 181, + "TRAP", + "complex_assembly" + ] + ] + }, + { + "sid": 115, + "sent": "An increase in the dose at which functionally important residues remain intact has biological ramifications for understanding the mechanisms at which ionizing radiation damage is mitigated within naturally forming DNA\u2013protein and RNA\u2013protein complexes.", + "section": "DISCUSS", + "ner": [ + [ + 214, + 225, + "DNA\u2013protein", + "complex_assembly" + ], + [ + 230, + 241, + "RNA\u2013protein", + "complex_assembly" + ] + ] + }, + { + "sid": 116, + "sent": "Observations of lower protein radiation-sensitivity in DNA-bound forms have been recorded in solution at RT at much lower doses (\u223c1\u2005kGy) than those used for typical MX experiments [e.g. an oestrogen response element\u2013receptor complex (St\u00edsov\u00e1 et al., 2006) and a DNA glycosylase and its abasic DNA target site (Gillard et al., 2004)].", + "section": "DISCUSS", + "ner": [ + [ + 55, + 64, + "DNA-bound", + "protein_state" + ], + [ + 165, + 167, + "MX", + "experimental_method" + ], + [ + 262, + 277, + "DNA glycosylase", + "protein_type" + ], + [ + 286, + 308, + "abasic DNA target site", + "site" + ] + ] + }, + { + "sid": 117, + "sent": "In these studies, the main damaging species is predicted to be the oxidizing hydroxyl radical produced through solvent irradiation, which is known to add to double covalent bonds within both DNA and RNA bases to induce strand breaks and base modification (Spotheim-Maurizot & Dav\u00eddkov\u00e1, 2011; Chance et al., 1997).", + "section": "DISCUSS", + "ner": [ + [ + 191, + 194, + "DNA", + "chemical" + ], + [ + 199, + 202, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 118, + "sent": "It was suggested that physical screening of DNA by protein shielded the DNA\u2013protein interaction sites from radical damage, yielding an extended life-dose for the nucleoprotein complex compared with separate protein and DNA constituents at RT.", + "section": "DISCUSS", + "ner": [ + [ + 44, + 47, + "DNA", + "chemical" + ], + [ + 72, + 101, + "DNA\u2013protein interaction sites", + "site" + ], + [ + 219, + 222, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 119, + "sent": "However, in the current MX study at 100\u2005K, the main damaging species are believed to be migrating LEEs and holes produced directly within the protein\u2013RNA components or in closely associated solvent.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 26, + "MX", + "experimental_method" + ], + [ + 142, + 153, + "protein\u2013RNA", + "complex_assembly" + ] + ] + }, + { + "sid": 120, + "sent": "The results presented here suggest that biologically relevant nucleoprotein complexes also exhibit prolonged life-doses under the effect of LEE-induced structural changes, involving direct physical protection of key RNA-binding residues.", + "section": "DISCUSS", + "ner": [ + [ + 62, + 75, + "nucleoprotein", + "complex_assembly" + ], + [ + 216, + 236, + "RNA-binding residues", + "site" + ] + ] + }, + { + "sid": 121, + "sent": "Such reduced radiation-sensitivity in this case ensures that the interacting protein remains bound long enough to the RNA to complete its function, even whilst exposed to ionizing radiation.", + "section": "DISCUSS", + "ner": [ + [ + 93, + 98, + "bound", + "protein_state" + ], + [ + 118, + 121, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 122, + "sent": "Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 \u25b8).", + "section": "DISCUSS", + "ner": [ + [ + 11, + 19, + "nonbound", + "protein_state" + ], + [ + 20, + 24, + "TRAP", + "complex_assembly" + ], + [ + 82, + 104, + "RNA-binding interfaces", + "site" + ], + [ + 106, + 111, + "Asp39", + "residue_name_number" + ], + [ + 113, + 118, + "Glu36", + "residue_name_number" + ], + [ + 120, + 125, + "Glu42", + "residue_name_number" + ] + ] + }, + { + "sid": 123, + "sent": "When exposed to X-rays, these residues will be preferentially damaged by X-rays and subsequently reduce the affinity with which TRAP binds to RNA.", + "section": "DISCUSS", + "ner": [ + [ + 128, + 132, + "TRAP", + "complex_assembly" + ], + [ + 142, + 145, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 124, + "sent": "Within the cellular environment, this mechanism could reduce the risk that radiation-damaged proteins might bind to RNA, thus avoiding the detrimental introduction of incorrect DNA-repair, transcriptional and base-modification pathways.", + "section": "DISCUSS", + "ner": [ + [ + 116, + 119, + "RNA", + "chemical" + ], + [ + 177, + 180, + "DNA", + "chemical" + ] + ] + }, + { + "sid": 125, + "sent": "The TRAP\u2013(GAGUU)10GAG complex asymmetric unit (PDB entry 1gtf; Hopcroft et al., 2002).", + "section": "FIG", + "ner": [ + [ + 4, + 21, + "TRAP\u2013(GAGUU)10GAG", + "complex_assembly" + ] + ] + }, + { + "sid": 126, + "sent": "Bound tryptophan ligands are represented as coloured spheres.", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "Bound", + "protein_state" + ], + [ + 6, + 16, + "tryptophan", + "chemical" + ] + ] + }, + { + "sid": 127, + "sent": "RNA is shown is yellow.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "RNA", + "chemical" + ] + ] + }, + { + "sid": 128, + "sent": "(a) Electron-density loss sites as indicated by D loss in the TRAP\u2013RNA complex crystal by residue/nucleotide type for five doses [sites determined above the 4\u00d7 average D loss threshold, calculated over the TRAP\u2013RNA structure for the first difference map: F obs(d 2) \u2212 F obs(d 1)].", + "section": "FIG", + "ner": [ + [ + 62, + 70, + "TRAP\u2013RNA", + "complex_assembly" + ], + [ + 79, + 86, + "crystal", + "evidence" + ], + [ + 206, + 214, + "TRAP\u2013RNA", + "complex_assembly" + ], + [ + 215, + 224, + "structure", + "evidence" + ], + [ + 239, + 253, + "difference map", + "evidence" + ] + ] + }, + { + "sid": 129, + "sent": "(b) Average D loss for each residue/nucleotide type with respect to the DWD (diffraction-weighted dose; Zeldin, Brock\u00adhauser et al., 2013).", + "section": "FIG", + "ner": [ + [ + 72, + 75, + "DWD", + "evidence" + ], + [ + 77, + 102, + "diffraction-weighted dose", + "evidence" + ] + ] + }, + { + "sid": 130, + "sent": "Only a subset of key TRAP residue types are included.", + "section": "FIG", + "ner": [ + [ + 21, + 25, + "TRAP", + "complex_assembly" + ] + ] + }, + { + "sid": 131, + "sent": "The average D loss (calculated over the whole TRAP asymmetric unit) is shown at each dose (dashed line).", + "section": "FIG", + "ner": [ + [ + 46, + 50, + "TRAP", + "complex_assembly" + ] + ] + }, + { + "sid": 132, + "sent": "In (a) clear difference density is observed around the Glu42 carboxyl side chain in chain H, within the lowest dose difference map at d 2 = 3.9\u2005MGy.", + "section": "FIG", + "ner": [ + [ + 13, + 31, + "difference density", + "evidence" + ], + [ + 55, + 60, + "Glu42", + "residue_name_number" + ], + [ + 104, + 130, + "lowest dose difference map", + "evidence" + ] + ] + }, + { + "sid": 133, + "sent": "Radiation-induced protein disordering is evident across the large dose range (b, c); in comparison, no clear deterioration of the RNA density was observed.", + "section": "FIG", + "ner": [ + [ + 130, + 133, + "RNA", + "chemical" + ], + [ + 134, + 141, + "density", + "evidence" + ] + ] + }, + { + "sid": 134, + "sent": " D loss calculated for all side-chain carboxyl group Glu\u2005C\u03b4 and Asp\u2005C\u03b3 atoms within the TRAP\u2013RNA complex for a dose of 19.3\u2005MGy (d 8).", + "section": "FIG", + "ner": [ + [ + 53, + 56, + "Glu", + "residue_name" + ], + [ + 64, + 67, + "Asp", + "residue_name" + ], + [ + 88, + 96, + "TRAP\u2013RNA", + "complex_assembly" + ] + ] + }, + { + "sid": 135, + "sent": "Residues have been grouped by amino-acid number, and split into bound and nonbound groupings, with each bar representing the mean calculated over 11 equivalent atoms around a TRAP ring.", + "section": "FIG", + "ner": [ + [ + 64, + 69, + "bound", + "protein_state" + ], + [ + 74, + 82, + "nonbound", + "protein_state" + ], + [ + 175, + 179, + "TRAP", + "complex_assembly" + ], + [ + 180, + 184, + "ring", + "structure_element" + ] + ] + }, + { + "sid": 136, + "sent": " D loss against dose for (a) Glu36\u2005C\u03b4, (b) Asp39\u2005C\u03b3, (c) Glu42\u2005O\u220a1, (d) Glu42\u2005O\u220a2, (e) Phe32\u2005C\u03b6 and (f) Lys37\u2005C\u220a atoms.", + "section": "FIG", + "ner": [ + [ + 29, + 34, + "Glu36", + "residue_name_number" + ], + [ + 43, + 48, + "Asp39", + "residue_name_number" + ], + [ + 57, + 62, + "Glu42", + "residue_name_number" + ], + [ + 72, + 77, + "Glu42", + "residue_name_number" + ], + [ + 87, + 92, + "Phe32", + "residue_name_number" + ], + [ + 104, + 109, + "Lys37", + "residue_name_number" + ] + ] + }, + { + "sid": 137, + "sent": "95% CI are included for each set of 11 equivalent atoms grouped as bound/nonbound.", + "section": "FIG", + "ner": [ + [ + 67, + 72, + "bound", + "protein_state" + ], + [ + 73, + 81, + "nonbound", + "protein_state" + ] + ] + }, + { + "sid": 138, + "sent": "RNA-binding interface interactions are shown for TRAP chain N, with the F obs(d 7) \u2212 F obs(d 1) Fourier difference map (dose 16.7\u2005MGy) overlaid and contoured at a \u00b14\u03c3 level.", + "section": "FIG", + "ner": [ + [ + 0, + 21, + "RNA-binding interface", + "site" + ], + [ + 49, + 53, + "TRAP", + "complex_assembly" + ] + ] + } + ] + }, + "PMC4802042": { + "annotations": [ + { + "sid": 0, + "sent": "A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation", + "section": "TITLE", + "ner": [ + [ + 21, + 55, + "JNK/p38-specific MAPK phosphatases", + "protein_type" + ], + [ + 77, + 81, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 1, + "sent": "Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs).", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 33, + "Mitogen-activated protein kinases", + "protein_type" + ], + [ + 35, + 40, + "MAPKs", + "protein_type" + ], + [ + 133, + 148, + "protein kinases", + "protein_type" + ], + [ + 156, + 173, + "MAPK phosphatases", + "protein_type" + ], + [ + 175, + 179, + "MKPs", + "protein_type" + ] + ] + }, + { + "sid": 2, + "sent": "MAPK signalling efficiency and specificity is modulated by protein\u2013protein interactions between individual MAPKs and the docking motifs in cognate binding partners.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 4, + "MAPK", + "protein_type" + ], + [ + 107, + 112, + "MAPKs", + "protein_type" + ], + [ + 121, + 135, + "docking motifs", + "structure_element" + ] + ] + }, + { + "sid": 3, + "sent": "Two types of docking interactions have been identified: D-motif-mediated interaction and FXF-docking interaction.", + "section": "ABSTRACT", + "ner": [ + [ + 56, + 63, + "D-motif", + "structure_element" + ], + [ + 89, + 112, + "FXF-docking interaction", + "site" + ] + ] + }, + { + "sid": 4, + "sent": "Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-\u00c5 resolution, providing high-resolution structural insight into the FXF-docking interaction.", + "section": "ABSTRACT", + "ner": [ + [ + 19, + 36, + "crystal structure", + "evidence" + ], + [ + 40, + 44, + "JNK1", + "protein" + ], + [ + 45, + 53, + "bound to", + "protein_state" + ], + [ + 58, + 74, + "catalytic domain", + "structure_element" + ], + [ + 78, + 82, + "MKP7", + "protein" + ], + [ + 158, + 181, + "FXF-docking interaction", + "site" + ] + ] + }, + { + "sid": 5, + "sent": "The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix \u03b1G. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 22, + "285FNFL288 segment", + "structure_element" + ], + [ + 26, + 30, + "MKP7", + "protein" + ], + [ + 51, + 67, + "hydrophobic site", + "site" + ], + [ + 71, + 75, + "JNK1", + "protein" + ], + [ + 93, + 97, + "MAPK", + "protein_type" + ], + [ + 112, + 117, + "helix", + "structure_element" + ], + [ + 118, + 120, + "\u03b1G", + "structure_element" + ], + [ + 122, + 141, + "Biochemical studies", + "experimental_method" + ], + [ + 167, + 183, + "highly conserved", + "protein_state" + ], + [ + 184, + 200, + "structural motif", + "structure_element" + ], + [ + 234, + 244, + "MKP family", + "protein_type" + ], + [ + 305, + 308, + "MKP", + "protein_type" + ], + [ + 309, + 313, + "MAPK", + "protein_type" + ] + ] + }, + { + "sid": 6, + "sent": " The important MAPK family of signalling proteins is controlled by MAPK phosphatases (MKPs).", + "section": "ABSTRACT", + "ner": [ + [ + 15, + 26, + "MAPK family", + "protein_type" + ], + [ + 67, + 84, + "MAPK phosphatases", + "protein_type" + ], + [ + 86, + 90, + "MKPs", + "protein_type" + ] + ] + }, + { + "sid": 7, + "sent": "Here, the authors report the structure of MKP7 bound to JNK1 and characterise the conserved MKP-MAPK interaction.", + "section": "ABSTRACT", + "ner": [ + [ + 29, + 38, + "structure", + "evidence" + ], + [ + 42, + 46, + "MKP7", + "protein" + ], + [ + 47, + 55, + "bound to", + "protein_state" + ], + [ + 56, + 60, + "JNK1", + "protein" + ], + [ + 82, + 91, + "conserved", + "protein_state" + ], + [ + 92, + 95, + "MKP", + "protein_type" + ], + [ + 96, + 100, + "MAPK", + "protein_type" + ] + ] + }, + { + "sid": 8, + "sent": "The mitogen-activated protein kinases (MAPKs) are central components of the signal-transduction pathways, which mediate the cellular response to a variety of extracellular stimuli, ranging from growth factors to environmental stresses.", + "section": "INTRO", + "ner": [ + [ + 4, + 37, + "mitogen-activated protein kinases", + "protein_type" + ], + [ + 39, + 44, + "MAPKs", + "protein_type" + ] + ] + }, + { + "sid": 9, + "sent": "The MAPK signalling pathways are evolutionally highly conserved.", + "section": "INTRO", + "ner": [ + [ + 4, + 8, + "MAPK", + "protein_type" + ] + ] + }, + { + "sid": 10, + "sent": "The basic assembly of MAPK pathways is a three-tier kinase module that establishes a sequential activation cascade: a MAPK kinase kinase activates a MAPK kinase, which in turn activates a MAPK.", + "section": "INTRO", + "ner": [ + [ + 22, + 26, + "MAPK", + "protein_type" + ], + [ + 52, + 58, + "kinase", + "protein_type" + ], + [ + 118, + 136, + "MAPK kinase kinase", + "protein_type" + ], + [ + 149, + 160, + "MAPK kinase", + "protein_type" + ], + [ + 188, + 192, + "MAPK", + "protein_type" + ] + ] + }, + { + "sid": 11, + "sent": "The three best-characterized MAPK signalling pathways are mediated by the kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38.", + "section": "INTRO", + "ner": [ + [ + 29, + 33, + "MAPK", + "protein_type" + ], + [ + 74, + 81, + "kinases", + "protein_type" + ], + [ + 82, + 119, + "extracellular signal-regulated kinase", + "protein_type" + ], + [ + 121, + 124, + "ERK", + "protein_type" + ], + [ + 127, + 150, + "c-Jun N-terminal kinase", + "protein_type" + ], + [ + 152, + 155, + "JNK", + "protein_type" + ], + [ + 161, + 164, + "p38", + "protein_type" + ] + ] + }, + { + "sid": 12, + "sent": "The ERK pathway is activated by various mitogens and phorbol esters, whereas the JNK and p38 pathways are stimulated mainly by environmental stress and inflammatory cytokines.", + "section": "INTRO", + "ner": [ + [ + 4, + 7, + "ERK", + "protein_type" + ], + [ + 81, + 84, + "JNK", + "protein_type" + ], + [ + 89, + 92, + "p38", + "protein_type" + ], + [ + 165, + 174, + "cytokines", + "protein_type" + ] + ] + }, + { + "sid": 13, + "sent": "The MAPKs are activated by MAPK kinases that phosphorylate the MAPKs at conserved threonine and tyrosine residues within their activation loop.", + "section": "INTRO", + "ner": [ + [ + 4, + 9, + "MAPKs", + "protein_type" + ], + [ + 27, + 39, + "MAPK kinases", + "protein_type" + ], + [ + 63, + 68, + "MAPKs", + "protein_type" + ], + [ + 72, + 81, + "conserved", + "protein_state" + ], + [ + 82, + 91, + "threonine", + "residue_name" + ], + [ + 96, + 104, + "tyrosine", + "residue_name" + ], + [ + 127, + 142, + "activation loop", + "structure_element" + ] + ] + }, + { + "sid": 14, + "sent": "After activation, each MAPK phosphorylates a distinct set of protein substrates, which act as the critical effectors that enable cells to mount the appropriate responses to varied stimuli.", + "section": "INTRO", + "ner": [ + [ + 23, + 27, + "MAPK", + "protein_type" + ] + ] + }, + { + "sid": 15, + "sent": "MAPKs lie at the bottom of conserved three-component phosphorylation cascades and utilize docking interactions to link module components and bind substrates.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "MAPKs", + "protein_type" + ] + ] + }, + { + "sid": 16, + "sent": "Two types of docking motifs have been identified in MAPK substrates and cognate proteins: kinase-interacting motif (D-motif) and FXF-motif (also called DEF motif, docking site for ERK FXF).", + "section": "INTRO", + "ner": [ + [ + 13, + 27, + "docking motifs", + "structure_element" + ], + [ + 52, + 56, + "MAPK", + "protein_type" + ], + [ + 90, + 114, + "kinase-interacting motif", + "structure_element" + ], + [ + 116, + 123, + "D-motif", + "structure_element" + ], + [ + 129, + 138, + "FXF-motif", + "structure_element" + ], + [ + 152, + 161, + "DEF motif", + "structure_element" + ], + [ + 163, + 175, + "docking site", + "site" + ], + [ + 180, + 183, + "ERK", + "protein_type" + ], + [ + 184, + 187, + "FXF", + "structure_element" + ] + ] + }, + { + "sid": 17, + "sent": "The best-studied docking interactions are those between MAP kinases and \u2018D-motifs', which consists of two or more basic residues followed by a short linker and a cluster of hydrophobic residues.", + "section": "INTRO", + "ner": [ + [ + 56, + 67, + "MAP kinases", + "protein_type" + ], + [ + 73, + 81, + "D-motifs", + "structure_element" + ], + [ + 143, + 155, + "short linker", + "structure_element" + ] + ] + }, + { + "sid": 18, + "sent": "The D-motif-docking site (D-site) in MAPKs is situated in a noncatalytic region opposite of the kinase catalytic pocket and is comprised of a highly acidic patch and a hydrophobic groove.", + "section": "INTRO", + "ner": [ + [ + 4, + 24, + "D-motif-docking site", + "site" + ], + [ + 26, + 32, + "D-site", + "site" + ], + [ + 37, + 42, + "MAPKs", + "protein_type" + ], + [ + 60, + 79, + "noncatalytic region", + "site" + ], + [ + 96, + 102, + "kinase", + "protein_type" + ], + [ + 103, + 119, + "catalytic pocket", + "site" + ], + [ + 142, + 161, + "highly acidic patch", + "site" + ], + [ + 168, + 186, + "hydrophobic groove", + "site" + ] + ] + }, + { + "sid": 19, + "sent": "D-motifs are found in many MAPK-interacting proteins, including substrates, activating kinases and inactivating phosphatases, as well as scaffolding proteins.", + "section": "INTRO", + "ner": [ + [ + 0, + 8, + "D-motifs", + "structure_element" + ], + [ + 27, + 52, + "MAPK-interacting proteins", + "protein_type" + ], + [ + 87, + 94, + "kinases", + "protein_type" + ], + [ + 112, + 124, + "phosphatases", + "protein_type" + ] + ] + }, + { + "sid": 20, + "sent": "A second docking motif for MAPKs consists of two Phe residues separated by one residue (FXF-motif).", + "section": "INTRO", + "ner": [ + [ + 2, + 22, + "second docking motif", + "structure_element" + ], + [ + 27, + 32, + "MAPKs", + "protein_type" + ], + [ + 49, + 52, + "Phe", + "residue_name" + ], + [ + 88, + 97, + "FXF-motif", + "structure_element" + ] + ] + }, + { + "sid": 21, + "sent": "This motif has been observed in several MAPK substrates.", + "section": "INTRO", + "ner": [ + [ + 40, + 44, + "MAPK", + "protein_type" + ] + ] + }, + { + "sid": 22, + "sent": "The FXF-motif-binding site of ERK2 has been mapped to a hydrophobic pocket formed between the P+1 site, \u03b1G helix and the MAPK insert.", + "section": "INTRO", + "ner": [ + [ + 4, + 26, + "FXF-motif-binding site", + "site" + ], + [ + 30, + 34, + "ERK2", + "protein" + ], + [ + 56, + 74, + "hydrophobic pocket", + "site" + ], + [ + 94, + 102, + "P+1 site", + "site" + ], + [ + 104, + 112, + "\u03b1G helix", + "structure_element" + ], + [ + 121, + 132, + "MAPK insert", + "structure_element" + ] + ] + }, + { + "sid": 23, + "sent": "However, the generality and mechanism of the FXF-mediated interaction is unclear.", + "section": "INTRO", + "ner": [ + [ + 45, + 48, + "FXF", + "structure_element" + ] + ] + }, + { + "sid": 24, + "sent": "The physiological outcome of MAPK signalling depends on both the magnitude and the duration of kinase activation.", + "section": "INTRO", + "ner": [ + [ + 29, + 33, + "MAPK", + "protein_type" + ] + ] + }, + { + "sid": 25, + "sent": "Downregulation of MAPK activity can be achieved through direct dephosphorylation of the phospho-threonine and/or tyrosine residues by various serine/threonine phosphatases, tyrosine phosphatases and dual-specificity phosphatases (DUSPs) termed MKPs.", + "section": "INTRO", + "ner": [ + [ + 18, + 22, + "MAPK", + "protein_type" + ], + [ + 88, + 121, + "phospho-threonine and/or tyrosine", + "residue_name" + ], + [ + 142, + 171, + "serine/threonine phosphatases", + "protein_type" + ], + [ + 173, + 194, + "tyrosine phosphatases", + "protein_type" + ], + [ + 199, + 228, + "dual-specificity phosphatases", + "protein_type" + ], + [ + 230, + 235, + "DUSPs", + "protein_type" + ], + [ + 244, + 248, + "MKPs", + "protein_type" + ] + ] + }, + { + "sid": 26, + "sent": "MKPs constitute a group of DUSPs that are characterized by their ability to dephosphorylate both phosphotyrosine and phosphoserine/phospho-threonine residues within a substrate.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "MKPs", + "protein_type" + ], + [ + 27, + 32, + "DUSPs", + "protein_type" + ], + [ + 97, + 112, + "phosphotyrosine", + "residue_name" + ], + [ + 117, + 130, + "phosphoserine", + "residue_name" + ], + [ + 131, + 148, + "phospho-threonine", + "residue_name" + ] + ] + }, + { + "sid": 27, + "sent": "Dysregulated expression of MKPs has been associated with pathogenesis of various diseases, and understanding their precise recognition mechanism presents an important challenge and opportunity for drug development.", + "section": "INTRO", + "ner": [ + [ + 27, + 31, + "MKPs", + "protein_type" + ] + ] + }, + { + "sid": 28, + "sent": "Here, we present the crystal structure of JNK1 in complex with the catalytic domain of MKP7.", + "section": "INTRO", + "ner": [ + [ + 21, + 38, + "crystal structure", + "evidence" + ], + [ + 42, + 46, + "JNK1", + "protein" + ], + [ + 47, + 62, + "in complex with", + "protein_state" + ], + [ + 67, + 83, + "catalytic domain", + "structure_element" + ], + [ + 87, + 91, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 29, + "sent": "This structure reveals the molecular mechanism underlying the docking interaction between MKP7 and JNK1.", + "section": "INTRO", + "ner": [ + [ + 5, + 14, + "structure", + "evidence" + ], + [ + 90, + 94, + "MKP7", + "protein" + ], + [ + 99, + 103, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 30, + "sent": "In the JNK1\u2013MKP7 complex, a hydrophobic motif (285FNFL288) that initiates the helix \u03b15 in the MKP7 catalytic domain directly binds to the FXF-motif-binding site on JNK1, providing the structural insight into the classic FXF-type docking interaction.", + "section": "INTRO", + "ner": [ + [ + 7, + 16, + "JNK1\u2013MKP7", + "complex_assembly" + ], + [ + 28, + 45, + "hydrophobic motif", + "structure_element" + ], + [ + 47, + 57, + "285FNFL288", + "structure_element" + ], + [ + 78, + 83, + "helix", + "structure_element" + ], + [ + 84, + 86, + "\u03b15", + "structure_element" + ], + [ + 94, + 98, + "MKP7", + "protein" + ], + [ + 99, + 115, + "catalytic domain", + "structure_element" + ], + [ + 138, + 160, + "FXF-motif-binding site", + "site" + ], + [ + 164, + 168, + "JNK1", + "protein" + ], + [ + 220, + 248, + "FXF-type docking interaction", + "site" + ] + ] + }, + { + "sid": 31, + "sent": "Biochemical and modelling studies further demonstrate that the molecular interactions mediate this key element for substrate recognition are highly conserved among all MKP-family members.", + "section": "INTRO", + "ner": [ + [ + 0, + 33, + "Biochemical and modelling studies", + "experimental_method" + ], + [ + 168, + 186, + "MKP-family members", + "protein_type" + ] + ] + }, + { + "sid": 32, + "sent": "Thus, our study reveals a hitherto unrecognized interaction mode for encoding complex target specificity among MAPK isoforms.", + "section": "INTRO", + "ner": [ + [ + 111, + 124, + "MAPK isoforms", + "protein_type" + ] + ] + }, + { + "sid": 33, + "sent": "Interaction of JNK1 with the MKP7 catalytic domain", + "section": "RESULTS", + "ner": [ + [ + 15, + 19, + "JNK1", + "protein" + ], + [ + 29, + 33, + "MKP7", + "protein" + ], + [ + 34, + 50, + "catalytic domain", + "structure_element" + ] + ] + }, + { + "sid": 34, + "sent": "DUSPs belong to the protein-tyrosine phosphatases (PTPase) superfamily, which is defined by the PTPase-signature motif CXXGXXR.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "DUSPs", + "protein_type" + ], + [ + 20, + 49, + "protein-tyrosine phosphatases", + "protein_type" + ], + [ + 51, + 57, + "PTPase", + "protein_type" + ], + [ + 96, + 102, + "PTPase", + "protein_type" + ], + [ + 119, + 126, + "CXXGXXR", + "structure_element" + ] + ] + }, + { + "sid": 35, + "sent": "MKPs represent a distinct subfamily within a larger group of DUSPs.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "MKPs", + "protein_type" + ], + [ + 61, + 66, + "DUSPs", + "protein_type" + ] + ] + }, + { + "sid": 36, + "sent": "In mammalian cells, the MKP subfamily includes 10 distinct catalytically active MKPs.", + "section": "RESULTS", + "ner": [ + [ + 3, + 12, + "mammalian", + "taxonomy_domain" + ], + [ + 24, + 37, + "MKP subfamily", + "protein_type" + ], + [ + 59, + 79, + "catalytically active", + "protein_state" + ], + [ + 80, + 84, + "MKPs", + "protein_type" + ] + ] + }, + { + "sid": 37, + "sent": "All MKPs contain a highly conserved C-terminal catalytic domain (CD) and an N-terminal kinase-binding domain (KBD).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "MKPs", + "protein_type" + ], + [ + 19, + 35, + "highly conserved", + "protein_state" + ], + [ + 47, + 63, + "catalytic domain", + "structure_element" + ], + [ + 65, + 67, + "CD", + "structure_element" + ], + [ + 87, + 108, + "kinase-binding domain", + "structure_element" + ], + [ + 110, + 113, + "KBD", + "structure_element" + ] + ] + }, + { + "sid": 38, + "sent": "The KBD is homologous to the rhodanese family and contains an intervening cluster of basic amino acids, which has been suggested to be important for interacting with the target MAPKs.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "KBD", + "structure_element" + ], + [ + 29, + 45, + "rhodanese family", + "protein_type" + ], + [ + 177, + 182, + "MAPKs", + "protein_type" + ] + ] + }, + { + "sid": 39, + "sent": "On the basis of sequence similarity, substrate specificity and predominant subcellular localization, the MKP family can be further divided into three groups (Fig. 1).", + "section": "RESULTS", + "ner": [ + [ + 105, + 115, + "MKP family", + "protein_type" + ] + ] + }, + { + "sid": 40, + "sent": "Biochemical and structural studies have revealed that the KBD of MKPs is critical for MKP3 docking to ERK2, and MKP5 binding to p38\u03b1, although their binding mechanisms are completely different.", + "section": "RESULTS", + "ner": [ + [ + 0, + 34, + "Biochemical and structural studies", + "experimental_method" + ], + [ + 58, + 61, + "KBD", + "structure_element" + ], + [ + 65, + 69, + "MKPs", + "protein_type" + ], + [ + 86, + 90, + "MKP3", + "protein" + ], + [ + 102, + 106, + "ERK2", + "protein" + ], + [ + 112, + 116, + "MKP5", + "protein" + ], + [ + 128, + 132, + "p38\u03b1", + "protein" + ] + ] + }, + { + "sid": 41, + "sent": "However, it is unknown if other MAPKs can interact with the KBD of their cognate phosphatases in the same manner as observed for recognition of ERK2 and p38\u03b1 by their MKPs, or whether they recognize distinct docking motifs of MKPs.", + "section": "RESULTS", + "ner": [ + [ + 32, + 37, + "MAPKs", + "protein_type" + ], + [ + 60, + 63, + "KBD", + "structure_element" + ], + [ + 81, + 93, + "phosphatases", + "protein_type" + ], + [ + 144, + 148, + "ERK2", + "protein" + ], + [ + 153, + 157, + "p38\u03b1", + "protein" + ], + [ + 167, + 171, + "MKPs", + "protein_type" + ], + [ + 208, + 222, + "docking motifs", + "structure_element" + ], + [ + 226, + 230, + "MKPs", + "protein_type" + ] + ] + }, + { + "sid": 42, + "sent": "MKP7, the biggest molecule in the MKP family, selectively inactivates JNK and p38 following stress activation.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "MKP7", + "protein" + ], + [ + 34, + 44, + "MKP family", + "protein_type" + ], + [ + 70, + 73, + "JNK", + "protein_type" + ], + [ + 78, + 81, + "p38", + "protein_type" + ] + ] + }, + { + "sid": 43, + "sent": "In addition to the CD and KBD, MKP7 has a long C-terminal region that contains both nuclear localization and export sequences by which MKP7 shuttles between the nucleus and the cytoplasm (Fig. 2a).", + "section": "RESULTS", + "ner": [ + [ + 19, + 21, + "CD", + "structure_element" + ], + [ + 26, + 29, + "KBD", + "structure_element" + ], + [ + 31, + 35, + "MKP7", + "protein" + ], + [ + 47, + 64, + "C-terminal region", + "structure_element" + ], + [ + 135, + 139, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 44, + "sent": "To quantitatively assess the contribution of the N-terminal domain to the MKP7-catalysed JNK1 dephosphorylation, we first measured the kinetic parameters of the C-terminal truncation of MKP7 (MKP7\u0394C304, residues 5\u2013303) and MKP7-CD (residues 156\u2013301) towards phosphorylated JNK1 (pJNK1).", + "section": "RESULTS", + "ner": [ + [ + 49, + 66, + "N-terminal domain", + "structure_element" + ], + [ + 74, + 78, + "MKP7", + "protein" + ], + [ + 89, + 93, + "JNK1", + "protein" + ], + [ + 94, + 111, + "dephosphorylation", + "ptm" + ], + [ + 135, + 142, + "kinetic", + "evidence" + ], + [ + 172, + 182, + "truncation", + "experimental_method" + ], + [ + 186, + 190, + "MKP7", + "protein" + ], + [ + 192, + 201, + "MKP7\u0394C304", + "mutant" + ], + [ + 212, + 217, + "5\u2013303", + "residue_range" + ], + [ + 223, + 227, + "MKP7", + "protein" + ], + [ + 228, + 230, + "CD", + "structure_element" + ], + [ + 241, + 248, + "156\u2013301", + "residue_range" + ], + [ + 258, + 272, + "phosphorylated", + "protein_state" + ], + [ + 273, + 277, + "JNK1", + "protein" + ], + [ + 279, + 280, + "p", + "protein_state" + ], + [ + 280, + 284, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 45, + "sent": "Figure 2b shows the variation of initial rates of the MKP7\u0394C304 and MKP7-CD-catalysed reaction with the concentration of phospho-JNK1. Because the concentrations of MKP7 and pJNK1 were comparable in the reaction, the assumption that the free-substrate concentration is equal to the total substrate concentration is not valid.", + "section": "RESULTS", + "ner": [ + [ + 20, + 46, + "variation of initial rates", + "evidence" + ], + [ + 54, + 63, + "MKP7\u0394C304", + "mutant" + ], + [ + 68, + 72, + "MKP7", + "protein" + ], + [ + 73, + 75, + "CD", + "structure_element" + ], + [ + 121, + 128, + "phospho", + "protein_state" + ], + [ + 129, + 133, + "JNK1", + "protein" + ], + [ + 165, + 169, + "MKP7", + "protein" + ], + [ + 174, + 175, + "p", + "protein_state" + ], + [ + 175, + 179, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 46, + "sent": "Thus, the kinetic data were analysed using the general initial velocity equation, taking substrate depletion into account:", + "section": "RESULTS", + "ner": [ + [ + 10, + 22, + "kinetic data", + "evidence" + ], + [ + 55, + 80, + "initial velocity equation", + "evidence" + ] + ] + }, + { + "sid": 47, + "sent": "The kcat and Km of the MKP7-CD (0.028\u2009s\u22121 and 0.26\u2009\u03bcM) so determined were nearly identical to those of MKP7\u0394C304 (0.029\u2009s\u22121 and 0.27\u2009\u03bcM), indicating that the MKP7-KBD has no effect on enzyme catalysis.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "kcat", + "evidence" + ], + [ + 13, + 15, + "Km", + "evidence" + ], + [ + 23, + 27, + "MKP7", + "protein" + ], + [ + 28, + 30, + "CD", + "structure_element" + ], + [ + 103, + 112, + "MKP7\u0394C304", + "mutant" + ], + [ + 158, + 162, + "MKP7", + "protein" + ], + [ + 163, + 166, + "KBD", + "structure_element" + ] + ] + }, + { + "sid": 48, + "sent": "We next examined the interaction of JNK1 with the CD and KBD of MKP7 by gel filtration analysis.", + "section": "RESULTS", + "ner": [ + [ + 36, + 40, + "JNK1", + "protein" + ], + [ + 50, + 52, + "CD", + "structure_element" + ], + [ + 57, + 60, + "KBD", + "structure_element" + ], + [ + 64, + 68, + "MKP7", + "protein" + ], + [ + 72, + 95, + "gel filtration analysis", + "experimental_method" + ] + ] + }, + { + "sid": 49, + "sent": "When 3\u2009molar equivalents of CD were mixed with 1\u2009molar equivalent of JNK1, a significant amount of CD co-migrated with JNK1 to earlier fractions, and the excess amount of CD was eluted from the size exclusion column as a monomer, indicating stable complex formation (Fig. 2c).", + "section": "RESULTS", + "ner": [ + [ + 28, + 30, + "CD", + "structure_element" + ], + [ + 69, + 73, + "JNK1", + "protein" + ], + [ + 99, + 101, + "CD", + "structure_element" + ], + [ + 119, + 123, + "JNK1", + "protein" + ], + [ + 171, + 173, + "CD", + "structure_element" + ], + [ + 221, + 228, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 50, + "sent": "In contrast, no KBD\u2013JNK1 complex was detected when 3\u2009molar equivalents of KBD were mixed with 1\u2009molar equivalent of JNK1.", + "section": "RESULTS", + "ner": [ + [ + 16, + 24, + "KBD\u2013JNK1", + "complex_assembly" + ], + [ + 74, + 77, + "KBD", + "structure_element" + ], + [ + 116, + 120, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 51, + "sent": "To further confirm the JNK1\u2013MKP7-CD interaction, we performed a pull-down assay using the purified proteins.", + "section": "RESULTS", + "ner": [ + [ + 23, + 35, + "JNK1\u2013MKP7-CD", + "complex_assembly" + ], + [ + 64, + 79, + "pull-down assay", + "experimental_method" + ] + ] + }, + { + "sid": 52, + "sent": "As shown in Fig. 2d, the CD of MKP7 can be pulled down by JNK1, while the KBD failed to bind to the counterpart protein.", + "section": "RESULTS", + "ner": [ + [ + 25, + 27, + "CD", + "structure_element" + ], + [ + 31, + 35, + "MKP7", + "protein" + ], + [ + 58, + 62, + "JNK1", + "protein" + ], + [ + 74, + 77, + "KBD", + "structure_element" + ] + ] + }, + { + "sid": 53, + "sent": "Taken together, our data indicate that the CD of MKP7, but not the KBD domain, is responsible for JNK substrate-binding and enzymatic specificity.", + "section": "RESULTS", + "ner": [ + [ + 43, + 45, + "CD", + "structure_element" + ], + [ + 49, + 53, + "MKP7", + "protein" + ], + [ + 67, + 70, + "KBD", + "structure_element" + ], + [ + 98, + 101, + "JNK", + "protein_type" + ] + ] + }, + { + "sid": 54, + "sent": "Crystal structure of JNK1 in complex with the MKP7-CD", + "section": "RESULTS", + "ner": [ + [ + 0, + 17, + "Crystal structure", + "evidence" + ], + [ + 21, + 25, + "JNK1", + "protein" + ], + [ + 26, + 41, + "in complex with", + "protein_state" + ], + [ + 46, + 50, + "MKP7", + "protein" + ], + [ + 51, + 53, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 55, + "sent": "To understand the molecular basis of JNK1 recognition by MKP7, we determined the crystal structure of unphosphorylated JNK1 in complex with the MKP7-CD (Fig. 3a, Supplementary Fig. 1a and Table 1).", + "section": "RESULTS", + "ner": [ + [ + 37, + 41, + "JNK1", + "protein" + ], + [ + 57, + 61, + "MKP7", + "protein" + ], + [ + 81, + 98, + "crystal structure", + "evidence" + ], + [ + 102, + 118, + "unphosphorylated", + "protein_state" + ], + [ + 119, + 123, + "JNK1", + "protein" + ], + [ + 124, + 139, + "in complex with", + "protein_state" + ], + [ + 144, + 148, + "MKP7", + "protein" + ], + [ + 149, + 151, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 56, + "sent": "In the complex, JNK1 has its characteristic bilobal structure comprising an N-terminal lobe rich in \u03b2-sheet and a C-terminal lobe that is mostly \u03b1-helical.", + "section": "RESULTS", + "ner": [ + [ + 16, + 20, + "JNK1", + "protein" + ], + [ + 76, + 91, + "N-terminal lobe", + "structure_element" + ], + [ + 100, + 107, + "\u03b2-sheet", + "structure_element" + ], + [ + 114, + 129, + "C-terminal lobe", + "structure_element" + ], + [ + 145, + 154, + "\u03b1-helical", + "structure_element" + ] + ] + }, + { + "sid": 57, + "sent": "The overall folding of MKP7-CD is typical of DUSPs, with a central twisted five-stranded \u03b2-sheet surrounded by six \u03b1-helices.", + "section": "RESULTS", + "ner": [ + [ + 23, + 27, + "MKP7", + "protein" + ], + [ + 28, + 30, + "CD", + "structure_element" + ], + [ + 45, + 50, + "DUSPs", + "protein_type" + ], + [ + 67, + 96, + "twisted five-stranded \u03b2-sheet", + "structure_element" + ], + [ + 115, + 124, + "\u03b1-helices", + "structure_element" + ] + ] + }, + { + "sid": 58, + "sent": "One side of the \u03b2-sheet is covered with two \u03b1-helices and the other is covered with four \u03b1-helices (Fig. 3b).", + "section": "RESULTS", + "ner": [ + [ + 16, + 23, + "\u03b2-sheet", + "structure_element" + ], + [ + 44, + 53, + "\u03b1-helices", + "structure_element" + ], + [ + 89, + 98, + "\u03b1-helices", + "structure_element" + ] + ] + }, + { + "sid": 59, + "sent": "The catalytic domain of MKP7 interacts with JNK1 through a contiguous surface area that is remote from the active site.", + "section": "RESULTS", + "ner": [ + [ + 4, + 20, + "catalytic domain", + "structure_element" + ], + [ + 24, + 28, + "MKP7", + "protein" + ], + [ + 44, + 48, + "JNK1", + "protein" + ], + [ + 107, + 118, + "active site", + "site" + ] + ] + }, + { + "sid": 60, + "sent": "MKP7-CD is positioned onto the JNK1 molecule so that the active site of the phosphatase faces towards the activation segment.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "MKP7", + "protein" + ], + [ + 5, + 7, + "CD", + "structure_element" + ], + [ + 31, + 35, + "JNK1", + "protein" + ], + [ + 57, + 68, + "active site", + "site" + ], + [ + 76, + 87, + "phosphatase", + "protein_type" + ], + [ + 106, + 124, + "activation segment", + "structure_element" + ] + ] + }, + { + "sid": 61, + "sent": "In an alignment of the structure of MKP7-CD with that of VHR, an atypical \u2018MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (root mean squared deviation) of 1.05\u2009\u00c5 (Fig. 3c).", + "section": "RESULTS", + "ner": [ + [ + 6, + 15, + "alignment", + "experimental_method" + ], + [ + 23, + 32, + "structure", + "evidence" + ], + [ + 36, + 40, + "MKP7", + "protein" + ], + [ + 41, + 43, + "CD", + "structure_element" + ], + [ + 57, + 60, + "VHR", + "protein" + ], + [ + 75, + 78, + "MKP", + "protein_type" + ], + [ + 101, + 117, + "catalytic domain", + "structure_element" + ], + [ + 130, + 134, + "MKP7", + "protein" + ], + [ + 135, + 137, + "CD", + "structure_element" + ], + [ + 156, + 168, + "superimposed", + "experimental_method" + ], + [ + 176, + 184, + "r.m.s.d.", + "evidence" + ], + [ + 186, + 213, + "root mean squared deviation", + "evidence" + ] + ] + }, + { + "sid": 62, + "sent": "The most striking difference is that helix \u03b10 and loop \u03b10\u2013\u03b21 of VHR are absent in MKP7-CD.", + "section": "RESULTS", + "ner": [ + [ + 37, + 42, + "helix", + "structure_element" + ], + [ + 43, + 45, + "\u03b10", + "structure_element" + ], + [ + 50, + 54, + "loop", + "structure_element" + ], + [ + 55, + 60, + "\u03b10\u2013\u03b21", + "structure_element" + ], + [ + 64, + 67, + "VHR", + "protein" + ], + [ + 82, + 86, + "MKP7", + "protein" + ], + [ + 87, + 89, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 63, + "sent": "Another region that cannot be aligned with VHR is found in loop \u03b23\u2013\u03b24.", + "section": "RESULTS", + "ner": [ + [ + 43, + 46, + "VHR", + "protein" + ], + [ + 59, + 63, + "loop", + "structure_element" + ], + [ + 64, + 69, + "\u03b23\u2013\u03b24", + "structure_element" + ] + ] + }, + { + "sid": 64, + "sent": "This loop is shortened by nine residues in MKP7-CD compared with that in VHR.", + "section": "RESULTS", + "ner": [ + [ + 5, + 9, + "loop", + "structure_element" + ], + [ + 43, + 47, + "MKP7", + "protein" + ], + [ + 48, + 50, + "CD", + "structure_element" + ], + [ + 73, + 76, + "VHR", + "protein" + ] + ] + }, + { + "sid": 65, + "sent": "Since helix \u03b10 and the following loop \u03b10\u2013\u03b21 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7.", + "section": "RESULTS", + "ner": [ + [ + 6, + 11, + "helix", + "structure_element" + ], + [ + 12, + 14, + "\u03b10", + "structure_element" + ], + [ + 33, + 37, + "loop", + "structure_element" + ], + [ + 38, + 43, + "\u03b10\u2013\u03b21", + "structure_element" + ], + [ + 60, + 87, + "substrate-recognition motif", + "site" + ], + [ + 91, + 94, + "VHR", + "protein" + ], + [ + 105, + 117, + "phosphatases", + "protein_type" + ], + [ + 198, + 202, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 66, + "sent": "The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b).", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "active site", + "site" + ], + [ + 19, + 23, + "MKP7", + "protein" + ], + [ + 40, + 62, + "phosphate-binding loop", + "structure_element" + ], + [ + 64, + 70, + "P-loop", + "structure_element" + ], + [ + 72, + 78, + "Cys244", + "residue_name_number" + ], + [ + 79, + 85, + "Leu245", + "residue_name_number" + ], + [ + 86, + 92, + "Ala246", + "residue_name_number" + ], + [ + 93, + 99, + "Gly247", + "residue_name_number" + ], + [ + 100, + 106, + "Ile248", + "residue_name_number" + ], + [ + 107, + 113, + "Ser249", + "residue_name_number" + ], + [ + 114, + 120, + "Arg250", + "residue_name_number" + ], + [ + 127, + 133, + "Asp213", + "residue_name_number" + ], + [ + 141, + 158, + "general acid loop", + "structure_element" + ] + ] + }, + { + "sid": 67, + "sent": "The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs.", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "MKP7", + "protein" + ], + [ + 9, + 11, + "CD", + "structure_element" + ], + [ + 12, + 21, + "structure", + "evidence" + ], + [ + 31, + 42, + "active site", + "site" + ], + [ + 62, + 81, + "active conformation", + "protein_state" + ], + [ + 94, + 97, + "VHR", + "protein" + ], + [ + 108, + 112, + "PTPs", + "protein_type" + ] + ] + }, + { + "sid": 68, + "sent": "The catalytic residue, Cys244, is located just after strand \u03b25 and optimally positioned for nucleophilic attack.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "catalytic residue", + "site" + ], + [ + 23, + 29, + "Cys244", + "residue_name_number" + ], + [ + 53, + 59, + "strand", + "structure_element" + ], + [ + 60, + 62, + "\u03b25", + "structure_element" + ] + ] + }, + { + "sid": 69, + "sent": "Asp213 in MKP7 also adopts a position similar to that of Asp92 in VHR (Supplementary Fig. 1c), indicating that Asp213 is likely to function as the general acid in MKP7.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Asp213", + "residue_name_number" + ], + [ + 10, + 14, + "MKP7", + "protein" + ], + [ + 57, + 62, + "Asp92", + "residue_name_number" + ], + [ + 66, + 69, + "VHR", + "protein" + ], + [ + 111, + 117, + "Asp213", + "residue_name_number" + ], + [ + 163, + 167, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 70, + "sent": "We also observed the binding of a chloride ion in the active site of MKP7-CD.", + "section": "RESULTS", + "ner": [ + [ + 34, + 42, + "chloride", + "chemical" + ], + [ + 54, + 65, + "active site", + "site" + ], + [ + 69, + 73, + "MKP7", + "protein" + ], + [ + 74, + 76, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 71, + "sent": "It is located 3.36\u2009\u00c5 from the Cys244 side chain and makes electrostatic interactions with the dipole moment of helix \u03b13 and with several main-chain amide groups.", + "section": "RESULTS", + "ner": [ + [ + 30, + 36, + "Cys244", + "residue_name_number" + ], + [ + 58, + 84, + "electrostatic interactions", + "bond_interaction" + ], + [ + 111, + 116, + "helix", + "structure_element" + ], + [ + 117, + 119, + "\u03b13", + "structure_element" + ] + ] + }, + { + "sid": 72, + "sent": "The side chain of strictly conserved Arg250 is oriented towards the negatively charged chloride, similar to the canonical phosphate-coordinating conformation.", + "section": "RESULTS", + "ner": [ + [ + 18, + 36, + "strictly conserved", + "protein_state" + ], + [ + 37, + 43, + "Arg250", + "residue_name_number" + ], + [ + 87, + 95, + "chloride", + "chemical" + ], + [ + 122, + 157, + "phosphate-coordinating conformation", + "structure_element" + ] + ] + }, + { + "sid": 73, + "sent": "Thus this chloride ion is a mimic for the phosphate group of the substrate, as revealed by a comparison with the structure of PTP1B in complex with phosphotyrosine (Supplementary Fig. 1d).", + "section": "RESULTS", + "ner": [ + [ + 10, + 18, + "chloride", + "chemical" + ], + [ + 42, + 51, + "phosphate", + "chemical" + ], + [ + 113, + 122, + "structure", + "evidence" + ], + [ + 126, + 131, + "PTP1B", + "protein" + ], + [ + 132, + 147, + "in complex with", + "protein_state" + ], + [ + 148, + 163, + "phosphotyrosine", + "residue_name" + ] + ] + }, + { + "sid": 74, + "sent": "Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c).", + "section": "RESULTS", + "ner": [ + [ + 49, + 53, + "MKP7", + "protein" + ], + [ + 54, + 56, + "CD", + "structure_element" + ], + [ + 88, + 91, + "VHR", + "protein" + ], + [ + 113, + 119, + "P-loop", + "structure_element" + ], + [ + 123, + 127, + "MKP7", + "protein" + ], + [ + 192, + 195, + "VHR", + "protein" + ], + [ + 197, + 203, + "Cys124", + "residue_name_number" + ], + [ + 204, + 210, + "Arg125", + "residue_name_number" + ], + [ + 211, + 217, + "Glu126", + "residue_name_number" + ], + [ + 218, + 224, + "Gly127", + "residue_name_number" + ], + [ + 225, + 231, + "Tyr128", + "residue_name_number" + ], + [ + 232, + 238, + "Gly129", + "residue_name_number" + ], + [ + 239, + 245, + "Arg130", + "residue_name_number" + ] + ] + }, + { + "sid": 75, + "sent": "The difference in the polarity/hydrophobicity of the surface may also point to the origin of the differences in the substrate-recognition mechanism for these two phosphatases (Supplementary Fig. 1e,f).", + "section": "RESULTS", + "ner": [ + [ + 162, + 174, + "phosphatases", + "protein_type" + ] + ] + }, + { + "sid": 76, + "sent": "In the complex, MKP7-CD and JNK1 form extensive protein\u2013protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e).", + "section": "RESULTS", + "ner": [ + [ + 16, + 20, + "MKP7", + "protein" + ], + [ + 21, + 23, + "CD", + "structure_element" + ], + [ + 28, + 32, + "JNK1", + "protein" + ], + [ + 91, + 109, + "C-terminal helices", + "structure_element" + ], + [ + 113, + 117, + "MKP7", + "protein" + ], + [ + 118, + 120, + "CD", + "structure_element" + ], + [ + 125, + 131, + "C-lobe", + "structure_element" + ], + [ + 135, + 139, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 77, + "sent": "As a result, the buried solvent-accessible surface area is \u223c1,315\u2009\u00c5. In the C-terminal domain, JNK1 has an insertion after the helix \u03b1G. This insertion consists of two helices (\u03b11L14 and \u03b12L14) that are common to all members of the MAPK family.", + "section": "RESULTS", + "ner": [ + [ + 76, + 93, + "C-terminal domain", + "structure_element" + ], + [ + 95, + 99, + "JNK1", + "protein" + ], + [ + 127, + 132, + "helix", + "structure_element" + ], + [ + 133, + 135, + "\u03b1G", + "structure_element" + ], + [ + 168, + 175, + "helices", + "structure_element" + ], + [ + 177, + 182, + "\u03b11L14", + "structure_element" + ], + [ + 187, + 192, + "\u03b12L14", + "structure_element" + ], + [ + 232, + 243, + "MAPK family", + "protein_type" + ] + ] + }, + { + "sid": 78, + "sent": "The interactive surface in JNK1, formed by the helices \u03b1G and \u03b12L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d).", + "section": "RESULTS", + "ner": [ + [ + 4, + 23, + "interactive surface", + "site" + ], + [ + 27, + 31, + "JNK1", + "protein" + ], + [ + 47, + 54, + "helices", + "structure_element" + ], + [ + 55, + 57, + "\u03b1G", + "structure_element" + ], + [ + 62, + 67, + "\u03b12L14", + "structure_element" + ], + [ + 80, + 98, + "hydrophobic region", + "site" + ], + [ + 111, + 117, + "Trp234", + "residue_name_number" + ] + ] + }, + { + "sid": 79, + "sent": "The MKP7-docking region includes two helices, \u03b14 and \u03b15, and the general acid loop.", + "section": "RESULTS", + "ner": [ + [ + 4, + 23, + "MKP7-docking region", + "site" + ], + [ + 37, + 44, + "helices", + "structure_element" + ], + [ + 46, + 48, + "\u03b14", + "structure_element" + ], + [ + 53, + 55, + "\u03b15", + "structure_element" + ], + [ + 65, + 82, + "general acid loop", + "structure_element" + ] + ] + }, + { + "sid": 80, + "sent": "The aromatic ring of Phe285 on MKP7 \u03b15-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g).", + "section": "RESULTS", + "ner": [ + [ + 21, + 27, + "Phe285", + "residue_name_number" + ], + [ + 31, + 35, + "MKP7", + "protein" + ], + [ + 36, + 44, + "\u03b15-helix", + "structure_element" + ], + [ + 61, + 79, + "hydrophobic pocket", + "site" + ], + [ + 83, + 87, + "JNK1", + "protein" + ], + [ + 114, + 120, + "Ile197", + "residue_name_number" + ], + [ + 122, + 128, + "Leu198", + "residue_name_number" + ], + [ + 130, + 136, + "Ile231", + "residue_name_number" + ], + [ + 138, + 144, + "Trp234", + "residue_name_number" + ], + [ + 146, + 152, + "Val256", + "residue_name_number" + ], + [ + 154, + 160, + "Tyr259", + "residue_name_number" + ], + [ + 162, + 168, + "Val260", + "residue_name_number" + ], + [ + 198, + 204, + "His230", + "residue_name_number" + ] + ] + }, + { + "sid": 81, + "sent": "In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1.", + "section": "RESULTS", + "ner": [ + [ + 23, + 37, + "hydrogen bonds", + "bond_interaction" + ], + [ + 46, + 52, + "Ser282", + "residue_name_number" + ], + [ + 57, + 63, + "Asn286", + "residue_name_number" + ], + [ + 67, + 71, + "MKP7", + "protein" + ], + [ + 76, + 82, + "His230", + "residue_name_number" + ], + [ + 87, + 93, + "Thr255", + "residue_name_number" + ], + [ + 97, + 101, + "JNK1", + "protein" + ], + [ + 125, + 131, + "Phe215", + "residue_name_number" + ], + [ + 139, + 156, + "general acid loop", + "structure_element" + ], + [ + 160, + 164, + "MKP7", + "protein" + ], + [ + 168, + 183, + "hydrogen-bonded", + "bond_interaction" + ], + [ + 205, + 211, + "Gln253", + "residue_name_number" + ], + [ + 215, + 219, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 82, + "sent": "The second interactive area involves the \u03b14 helix of MKP7 and charged/polar residues of JNK1 (Fig. 3e).", + "section": "RESULTS", + "ner": [ + [ + 4, + 27, + "second interactive area", + "site" + ], + [ + 41, + 49, + "\u03b14 helix", + "structure_element" + ], + [ + 53, + 57, + "MKP7", + "protein" + ], + [ + 88, + 92, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 83, + "sent": "The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively.", + "section": "RESULTS", + "ner": [ + [ + 19, + 25, + "Asp268", + "residue_name_number" + ], + [ + 29, + 33, + "MKP7", + "protein" + ], + [ + 42, + 53, + "salt bridge", + "bond_interaction" + ], + [ + 73, + 79, + "Arg263", + "residue_name_number" + ], + [ + 83, + 87, + "JNK1", + "protein" + ], + [ + 93, + 99, + "Lys275", + "residue_name_number" + ], + [ + 103, + 107, + "MKP7", + "protein" + ], + [ + 116, + 129, + "hydrogen bond", + "bond_interaction" + ], + [ + 136, + 147, + "salt bridge", + "bond_interaction" + ], + [ + 153, + 159, + "Thr228", + "residue_name_number" + ], + [ + 164, + 170, + "Asp229", + "residue_name_number" + ], + [ + 174, + 178, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 84, + "sent": "Mutational analysis of the JNK1\u2013MKP7 docking interface", + "section": "RESULTS", + "ner": [ + [ + 0, + 19, + "Mutational analysis", + "experimental_method" + ], + [ + 27, + 54, + "JNK1\u2013MKP7 docking interface", + "site" + ] + ] + }, + { + "sid": 85, + "sent": "To assess the importance of the aforementioned interactions, we generated a series of point mutations on the MKP7-CD and examined their effect on the MKP7-catalysed JNK1 dephosphorylation (Fig. 4a).", + "section": "RESULTS", + "ner": [ + [ + 86, + 101, + "point mutations", + "experimental_method" + ], + [ + 109, + 113, + "MKP7", + "protein" + ], + [ + 114, + 116, + "CD", + "structure_element" + ], + [ + 150, + 154, + "MKP7", + "protein" + ], + [ + 165, + 169, + "JNK1", + "protein" + ], + [ + 170, + 187, + "dephosphorylation", + "ptm" + ] + ] + }, + { + "sid": 86, + "sent": "When the hydrophobic residues Phe285 and Phe287 on the \u03b15 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased \u223c10-fold.", + "section": "RESULTS", + "ner": [ + [ + 30, + 36, + "Phe285", + "residue_name_number" + ], + [ + 41, + 47, + "Phe287", + "residue_name_number" + ], + [ + 55, + 57, + "\u03b15", + "structure_element" + ], + [ + 61, + 65, + "MKP7", + "protein" + ], + [ + 66, + 68, + "CD", + "structure_element" + ], + [ + 74, + 82, + "replaced", + "experimental_method" + ], + [ + 86, + 89, + "Asp", + "residue_name" + ], + [ + 93, + 96, + "Ala", + "residue_name" + ], + [ + 131, + 135, + "JNK1", + "protein" + ], + [ + 136, + 153, + "dephosphorylation", + "ptm" + ] + ] + }, + { + "sid": 87, + "sent": "In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP.", + "section": "RESULTS", + "ner": [ + [ + 15, + 26, + "replacement", + "experimental_method" + ], + [ + 50, + 56, + "Phe215", + "residue_name_number" + ], + [ + 58, + 64, + "Asp268", + "residue_name_number" + ], + [ + 66, + 72, + "Lys275", + "residue_name_number" + ], + [ + 74, + 80, + "Ser282", + "residue_name_number" + ], + [ + 82, + 88, + "Asn286", + "residue_name_number" + ], + [ + 93, + 99, + "Leu292", + "residue_name_number" + ], + [ + 109, + 112, + "Ala", + "residue_name" + ], + [ + 116, + 119, + "Asp", + "residue_name" + ], + [ + 251, + 254, + "MKP", + "protein_type" + ] + ] + }, + { + "sid": 88, + "sent": "Mutation of Leu288 markedly reduced its solubility when expressed in Escherichia coli, resulting in the insoluble aggregation of the mutant protein.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Mutation", + "experimental_method" + ], + [ + 12, + 18, + "Leu288", + "residue_name_number" + ], + [ + 69, + 85, + "Escherichia coli", + "species" + ], + [ + 133, + 139, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 89, + "sent": "Gel filtration analysis further confirmed the key role of Phe285 in the MKP7\u2013JNK1 interaction: no F285D\u2013JNK1 complex was detected when 3\u2009molar equivalents of MKP7-CD (F285D) were mixed with 1\u2009molar equivalent of JNK1 (Fig. 4b).", + "section": "RESULTS", + "ner": [ + [ + 0, + 23, + "Gel filtration analysis", + "experimental_method" + ], + [ + 58, + 64, + "Phe285", + "residue_name_number" + ], + [ + 72, + 76, + "MKP7", + "protein" + ], + [ + 77, + 81, + "JNK1", + "protein" + ], + [ + 98, + 108, + "F285D\u2013JNK1", + "complex_assembly" + ], + [ + 158, + 162, + "MKP7", + "protein" + ], + [ + 163, + 165, + "CD", + "structure_element" + ], + [ + 167, + 172, + "F285D", + "mutant" + ], + [ + 212, + 216, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 90, + "sent": "Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a).", + "section": "RESULTS", + "ner": [ + [ + 15, + 23, + "mutation", + "experimental_method" + ], + [ + 27, + 33, + "Phe287", + "residue_name_number" + ], + [ + 85, + 86, + "p", + "protein_state" + ], + [ + 86, + 90, + "JNK1", + "protein" + ], + [ + 112, + 120, + "affinity", + "evidence" + ], + [ + 124, + 128, + "MKP7", + "protein" + ], + [ + 129, + 131, + "CD", + "structure_element" + ], + [ + 136, + 140, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 91, + "sent": "We also generated a series of point mutations in the JNK1 and assessed the effect on JNK1 binding using the GST pull-down assay (Fig. 4c).", + "section": "RESULTS", + "ner": [ + [ + 30, + 45, + "point mutations", + "experimental_method" + ], + [ + 53, + 57, + "JNK1", + "protein" + ], + [ + 85, + 89, + "JNK1", + "protein" + ], + [ + 108, + 127, + "GST pull-down assay", + "experimental_method" + ] + ] + }, + { + "sid": 92, + "sent": "Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK.", + "section": "RESULTS", + "ner": [ + [ + 0, + 12, + "Substitution", + "experimental_method" + ], + [ + 16, + 22, + "Asp229", + "residue_name_number" + ], + [ + 24, + 30, + "Trp234", + "residue_name_number" + ], + [ + 32, + 38, + "Thr255", + "residue_name_number" + ], + [ + 40, + 46, + "Val256", + "residue_name_number" + ], + [ + 48, + 54, + "Tyr259", + "residue_name_number" + ], + [ + 59, + 65, + "Val260", + "residue_name_number" + ], + [ + 92, + 108, + "binding affinity", + "evidence" + ], + [ + 112, + 116, + "MKP7", + "protein" + ], + [ + 117, + 119, + "CD", + "structure_element" + ], + [ + 124, + 127, + "JNK", + "protein_type" + ] + ] + }, + { + "sid": 93, + "sent": "To determine whether the deficiencies in their abilities to bind partner proteins or carry out catalytic function are owing to misfolding of the purified mutant proteins, we also examined the folding properties of the JNK1 and MKP7 mutants with circular dichroism.", + "section": "RESULTS", + "ner": [ + [ + 154, + 160, + "mutant", + "protein_state" + ], + [ + 218, + 222, + "JNK1", + "protein" + ], + [ + 227, + 231, + "MKP7", + "protein" + ], + [ + 232, + 239, + "mutants", + "protein_state" + ], + [ + 245, + 263, + "circular dichroism", + "experimental_method" + ] + ] + }, + { + "sid": 94, + "sent": "The spectra of these mutants are similar to the wild-type proteins, indicating that these mutants fold as well as the wild-type proteins (Fig. 4d,e).", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "spectra", + "evidence" + ], + [ + 21, + 28, + "mutants", + "protein_state" + ], + [ + 48, + 57, + "wild-type", + "protein_state" + ], + [ + 90, + 97, + "mutants", + "protein_state" + ], + [ + 118, + 127, + "wild-type", + "protein_state" + ] + ] + }, + { + "sid": 95, + "sent": "Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme\u2013substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1.", + "section": "RESULTS", + "ner": [ + [ + 62, + 84, + "crystallographic model", + "evidence" + ], + [ + 103, + 123, + "hydrophobic contacts", + "bond_interaction" + ], + [ + 136, + 140, + "MKP7", + "protein" + ], + [ + 141, + 157, + "catalytic domain", + "structure_element" + ], + [ + 162, + 166, + "JNK1", + "protein" + ], + [ + 252, + 258, + "Phe285", + "residue_name_number" + ], + [ + 266, + 270, + "MKP7", + "protein" + ], + [ + 271, + 273, + "CD", + "structure_element" + ], + [ + 324, + 328, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 96, + "sent": "It has previously been reported that several cytosolic and inducible nuclear MKPs undergo catalytic activation upon interaction with the MAPK substrates.", + "section": "RESULTS", + "ner": [ + [ + 77, + 81, + "MKPs", + "protein_type" + ], + [ + 137, + 141, + "MAPK", + "protein_type" + ] + ] + }, + { + "sid": 97, + "sent": "This allosteric activation of MKP3 has been well-documented in vitro using pNPP, a small-molecule phosphotyrosine analogue of its normal substrate.", + "section": "RESULTS", + "ner": [ + [ + 30, + 34, + "MKP3", + "protein" + ], + [ + 75, + 79, + "pNPP", + "chemical" + ], + [ + 98, + 113, + "phosphotyrosine", + "residue_name" + ] + ] + }, + { + "sid": 98, + "sent": "We then assayed pNPPase activities of MKP7\u0394C304 and MKP7-CD in the presence of JNK1.", + "section": "RESULTS", + "ner": [ + [ + 16, + 23, + "pNPPase", + "protein_type" + ], + [ + 38, + 47, + "MKP7\u0394C304", + "mutant" + ], + [ + 52, + 56, + "MKP7", + "protein" + ], + [ + 57, + 59, + "CD", + "structure_element" + ], + [ + 67, + 78, + "presence of", + "protein_state" + ], + [ + 79, + 83, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 99, + "sent": "Incubation of MKP7 with JNK1 did not markedly stimulate the phosphatase activity, which is consistent with previous results that MKP7 solely possesses the intrinsic activity (Supplementary Fig. 2b).", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "Incubation", + "experimental_method" + ], + [ + 14, + 18, + "MKP7", + "protein" + ], + [ + 24, + 28, + "JNK1", + "protein" + ], + [ + 60, + 71, + "phosphatase", + "protein_type" + ], + [ + 129, + 133, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 100, + "sent": "The small pNPP molecule binds directly at the enzyme active site and can be used to probe the reaction mechanism of protein phosphatases.", + "section": "RESULTS", + "ner": [ + [ + 10, + 14, + "pNPP", + "chemical" + ], + [ + 53, + 64, + "active site", + "site" + ], + [ + 116, + 136, + "protein phosphatases", + "protein_type" + ] + ] + }, + { + "sid": 101, + "sent": "We therefore examined the effects of the MKP7-CD mutants on their pNPPase activities.", + "section": "RESULTS", + "ner": [ + [ + 41, + 45, + "MKP7", + "protein" + ], + [ + 46, + 48, + "CD", + "structure_element" + ], + [ + 49, + 56, + "mutants", + "protein_state" + ], + [ + 66, + 73, + "pNPPase", + "protein_type" + ] + ] + }, + { + "sid": 102, + "sent": "As shown in Fig. 4f, all the mutants, except F287D/A, showed little or no activity change compared with the wild-type MKP7-CD.", + "section": "RESULTS", + "ner": [ + [ + 29, + 36, + "mutants", + "protein_state" + ], + [ + 45, + 52, + "F287D/A", + "mutant" + ], + [ + 108, + 117, + "wild-type", + "protein_state" + ], + [ + 118, + 122, + "MKP7", + "protein" + ], + [ + 123, + 125, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 103, + "sent": "In the JNK1/MKP7-CD complex structure, Phe287 of MKP7 does not make contacts with JNK1 substrate.", + "section": "RESULTS", + "ner": [ + [ + 7, + 19, + "JNK1/MKP7-CD", + "complex_assembly" + ], + [ + 28, + 37, + "structure", + "evidence" + ], + [ + 39, + 45, + "Phe287", + "residue_name_number" + ], + [ + 49, + 53, + "MKP7", + "protein" + ], + [ + 82, + 86, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 104, + "sent": "It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis (Supplementary Fig. 2c).", + "section": "RESULTS", + "ner": [ + [ + 21, + 27, + "pocket", + "site" + ], + [ + 56, + 62, + "P-loop", + "structure_element" + ], + [ + 67, + 84, + "general acid loop", + "structure_element" + ], + [ + 95, + 115, + "hydrophobic contacts", + "bond_interaction" + ], + [ + 162, + 168, + "Arg250", + "residue_name_number" + ], + [ + 170, + 176, + "Glu217", + "residue_name_number" + ], + [ + 181, + 187, + "Ile219", + "residue_name_number" + ], + [ + 205, + 211, + "Phe287", + "residue_name_number" + ], + [ + 215, + 219, + "MKP7", + "protein" + ], + [ + 291, + 295, + "PTPs", + "protein_type" + ], + [ + 297, + 303, + "Gln266", + "residue_name_number" + ], + [ + 307, + 312, + "PTP1B", + "protein" + ], + [ + 318, + 321, + "VHR", + "protein" + ], + [ + 323, + 329, + "Phe166", + "residue_name_number" + ], + [ + 333, + 336, + "VHR", + "protein" + ], + [ + 366, + 386, + "active-site residues", + "site" + ], + [ + 390, + 394, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 105, + "sent": "Kinase-associated phosphatase (KAP), a member of the DUSP family, plays a crucial role in cell cycle regulation by dephosphorylating the pThr160 residue of CDK2 (cyclin-dependent kinase 2).", + "section": "RESULTS", + "ner": [ + [ + 0, + 29, + "Kinase-associated phosphatase", + "protein" + ], + [ + 31, + 34, + "KAP", + "protein" + ], + [ + 53, + 64, + "DUSP family", + "protein_type" + ], + [ + 137, + 144, + "pThr160", + "ptm" + ], + [ + 156, + 160, + "CDK2", + "protein" + ], + [ + 162, + 187, + "cyclin-dependent kinase 2", + "protein" + ] + ] + }, + { + "sid": 106, + "sent": "The crystal structure of the CDK2/KAP complex has been determined at 3.0\u2009\u00c5 (Fig. 5a).", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 29, + 37, + "CDK2/KAP", + "complex_assembly" + ] + ] + }, + { + "sid": 107, + "sent": "The interface between these two proteins consists of three discontinuous contact regions.", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "interface", + "site" + ] + ] + }, + { + "sid": 108, + "sent": "Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the \u03b1G helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b).", + "section": "RESULTS", + "ner": [ + [ + 72, + 75, + "KAP", + "protein" + ], + [ + 80, + 84, + "CDK2", + "protein" + ], + [ + 102, + 118, + "recognition site", + "site" + ], + [ + 130, + 134, + "CDK2", + "protein" + ], + [ + 153, + 161, + "\u03b1G helix", + "structure_element" + ], + [ + 166, + 174, + "L14 loop", + "structure_element" + ], + [ + 183, + 208, + "N-terminal helical region", + "structure_element" + ], + [ + 212, + 215, + "KAP", + "protein" + ] + ] + }, + { + "sid": 109, + "sent": "There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2.", + "section": "RESULTS", + "ner": [ + [ + 11, + 24, + "hydrogen bond", + "bond_interaction" + ], + [ + 60, + 66, + "Ile183", + "residue_name_number" + ], + [ + 68, + 71, + "KAP", + "protein" + ], + [ + 98, + 104, + "Glu208", + "residue_name_number" + ], + [ + 106, + 110, + "CDK2", + "protein" + ], + [ + 138, + 144, + "Lys184", + "residue_name_number" + ], + [ + 148, + 151, + "KAP", + "protein" + ], + [ + 156, + 162, + "Asp235", + "residue_name_number" + ], + [ + 166, + 170, + "CDK2", + "protein" + ] + ] + }, + { + "sid": 110, + "sent": "Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c).", + "section": "RESULTS", + "ner": [ + [ + 0, + 19, + "Structural analysis", + "experimental_method" + ], + [ + 24, + 42, + "sequence alignment", + "experimental_method" + ], + [ + 90, + 94, + "MKP7", + "protein" + ], + [ + 95, + 97, + "CD", + "structure_element" + ], + [ + 102, + 105, + "KAP", + "protein" + ], + [ + 113, + 137, + "substrate-binding region", + "site" + ], + [ + 167, + 171, + "FNFL", + "structure_element" + ], + [ + 175, + 179, + "MKP7", + "protein" + ], + [ + 180, + 182, + "CD", + "structure_element" + ], + [ + 205, + 209, + "IKQY", + "structure_element" + ], + [ + 213, + 216, + "KAP", + "protein" + ] + ] + }, + { + "sid": 111, + "sent": "The substitution of the two hydrophobic residues with charged/polar residues (F285I/N286K) seriously disrupts the hydrophobic interaction required for MKP7 binding on JNK1 (Fig. 4a).", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "substitution", + "experimental_method" + ], + [ + 78, + 83, + "F285I", + "mutant" + ], + [ + 84, + 89, + "N286K", + "mutant" + ], + [ + 114, + 137, + "hydrophobic interaction", + "bond_interaction" + ], + [ + 151, + 155, + "MKP7", + "protein" + ], + [ + 167, + 171, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 112, + "sent": "In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK.", + "section": "RESULTS", + "ner": [ + [ + 13, + 19, + "His230", + "residue_name_number" + ], + [ + 24, + 30, + "Val256", + "residue_name_number" + ], + [ + 34, + 38, + "JNK1", + "protein" + ], + [ + 87, + 93, + "Glu208", + "residue_name_number" + ], + [ + 98, + 104, + "Asp235", + "residue_name_number" + ], + [ + 108, + 112, + "CDK2", + "protein" + ], + [ + 159, + 163, + "CDK2", + "protein" + ], + [ + 164, + 183, + "interactive surface", + "site" + ], + [ + 216, + 219, + "JNK", + "protein_type" + ] + ] + }, + { + "sid": 113, + "sent": "These data indicated that a unique hydrophobic pocket formed between the MAPK insert and \u03b1G helix plays a major role in the substrate recognition by MKPs.", + "section": "RESULTS", + "ner": [ + [ + 35, + 53, + "hydrophobic pocket", + "site" + ], + [ + 73, + 84, + "MAPK insert", + "structure_element" + ], + [ + 89, + 97, + "\u03b1G helix", + "structure_element" + ], + [ + 149, + 153, + "MKPs", + "protein_type" + ] + ] + }, + { + "sid": 114, + "sent": "F-site interaction is crucial for JNK1 inactivation in vivo", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "F-site", + "site" + ], + [ + 34, + 38, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 115, + "sent": "JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "JNK", + "protein_type" + ], + [ + 82, + 92, + "anisomycin", + "chemical" + ], + [ + 94, + 98, + "H2O2", + "chemical" + ], + [ + 119, + 127, + "sorbitol", + "chemical" + ], + [ + 188, + 197, + "etoposide", + "chemical" + ], + [ + 199, + 208, + "cisplatin", + "chemical" + ], + [ + 213, + 218, + "taxol", + "chemical" + ] + ] + }, + { + "sid": 116, + "sent": "To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7\u0394C304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment.", + "section": "RESULTS", + "ner": [ + [ + 25, + 29, + "MKP7", + "protein" + ], + [ + 38, + 45, + "mutants", + "protein_state" + ], + [ + 78, + 81, + "JNK", + "protein_type" + ], + [ + 148, + 157, + "HA-tagged", + "protein_state" + ], + [ + 173, + 184, + "full-length", + "protein_state" + ], + [ + 185, + 189, + "MKP7", + "protein" + ], + [ + 191, + 200, + "MKP7\u0394C304", + "mutant" + ], + [ + 205, + 209, + "MKP7", + "protein" + ], + [ + 210, + 212, + "CD", + "structure_element" + ], + [ + 216, + 220, + "MKP7", + "protein" + ], + [ + 221, + 228, + "mutants", + "protein_state" + ], + [ + 265, + 274, + "etoposide", + "chemical" + ] + ] + }, + { + "sid": 117, + "sent": "As shown in Fig. 6a\u2013c, immunobloting showed similar expression levels for the different MKP7 constructs in all the cells.", + "section": "RESULTS", + "ner": [ + [ + 23, + 36, + "immunobloting", + "experimental_method" + ], + [ + 88, + 92, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 118, + "sent": "Overexpressed full-length MKP7, MKP7\u0394C304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells.", + "section": "RESULTS", + "ner": [ + [ + 0, + 13, + "Overexpressed", + "experimental_method" + ], + [ + 14, + 25, + "full-length", + "protein_state" + ], + [ + 26, + 30, + "MKP7", + "protein" + ], + [ + 32, + 41, + "MKP7\u0394C304", + "mutant" + ], + [ + 46, + 50, + "MKP7", + "protein" + ], + [ + 51, + 53, + "CD", + "structure_element" + ], + [ + 100, + 114, + "phosphorylated", + "protein_state" + ], + [ + 115, + 118, + "JNK", + "protein_type" + ] + ] + }, + { + "sid": 119, + "sent": "Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo.", + "section": "RESULTS", + "ner": [ + [ + 45, + 52, + "D-motif", + "structure_element" + ], + [ + 53, + 60, + "mutants", + "protein_state" + ], + [ + 62, + 66, + "R56A", + "mutant" + ], + [ + 67, + 71, + "R57A", + "mutant" + ], + [ + 76, + 80, + "V63A", + "mutant" + ], + [ + 81, + 85, + "I65A", + "mutant" + ], + [ + 87, + 103, + "dephosphorylated", + "protein_state" + ], + [ + 104, + 107, + "JNK", + "protein_type" + ], + [ + 119, + 128, + "wild type", + "protein_state" + ], + [ + 184, + 188, + "MKP7", + "protein" + ], + [ + 189, + 192, + "KBD", + "structure_element" + ], + [ + 217, + 220, + "JNK", + "protein_type" + ] + ] + }, + { + "sid": 120, + "sent": "Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK.", + "section": "RESULTS", + "ner": [ + [ + 48, + 62, + "phosphorylated", + "protein_state" + ], + [ + 63, + 66, + "JNK", + "protein_type" + ], + [ + 96, + 100, + "MKP7", + "protein" + ], + [ + 101, + 110, + "FXF-motif", + "structure_element" + ], + [ + 111, + 118, + "mutants", + "protein_state" + ], + [ + 120, + 125, + "F285D", + "mutant" + ], + [ + 127, + 132, + "F287D", + "mutant" + ], + [ + 137, + 142, + "L288D", + "mutant" + ], + [ + 171, + 175, + "MKP7", + "protein" + ], + [ + 176, + 181, + "D268A", + "mutant" + ], + [ + 186, + 191, + "N286A", + "mutant" + ], + [ + 192, + 199, + "mutants", + "protein_state" + ], + [ + 261, + 264, + "JNK", + "protein_type" + ] + ] + }, + { + "sid": 121, + "sent": "We next tested in vivo interactions between JNK1 mutants and full-length MKP7 by coimmunoprecipitation experiments under unstimulated conditions.", + "section": "RESULTS", + "ner": [ + [ + 44, + 48, + "JNK1", + "protein" + ], + [ + 49, + 56, + "mutants", + "protein_state" + ], + [ + 61, + 72, + "full-length", + "protein_state" + ], + [ + 73, + 77, + "MKP7", + "protein" + ], + [ + 81, + 114, + "coimmunoprecipitation experiments", + "experimental_method" + ] + ] + }, + { + "sid": 122, + "sent": "When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo.", + "section": "RESULTS", + "ner": [ + [ + 5, + 17, + "co-expressed", + "experimental_method" + ], + [ + 36, + 45, + "wild-type", + "protein_state" + ], + [ + 51, + 55, + "JNK1", + "protein" + ], + [ + 92, + 96, + "MKP7", + "protein" + ], + [ + 124, + 128, + "MKP7", + "protein" + ], + [ + 135, + 151, + "dephosphorylated", + "protein_state" + ], + [ + 152, + 156, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 123, + "sent": "In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1\u2013MKP7 interaction (Fig. 6d and Supplementary Fig. 3a).", + "section": "RESULTS", + "ner": [ + [ + 22, + 40, + "in vitro pull-down", + "experimental_method" + ], + [ + 54, + 61, + "mutants", + "protein_state" + ], + [ + 62, + 67, + "D229A", + "mutant" + ], + [ + 69, + 74, + "W234D", + "mutant" + ], + [ + 79, + 84, + "Y259D", + "mutant" + ], + [ + 115, + 119, + "MKP7", + "protein" + ], + [ + 129, + 134, + "I231D", + "mutant" + ], + [ + 135, + 141, + "mutant", + "protein_state" + ], + [ + 172, + 181, + "JNK1\u2013MKP7", + "complex_assembly" + ] + ] + }, + { + "sid": 124, + "sent": "Activation of the JNK signalling pathway is frequently associated with apoptotic cell death, and inhibition of JNK can prevent apoptotic death of multiple cells.", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "JNK", + "protein_type" + ], + [ + 111, + 114, + "JNK", + "protein_type" + ] + ] + }, + { + "sid": 125, + "sent": "To examine whether the inhibition of JNK activity by MKP7 would provide protections against the apoptosis, we analysed the rate of apoptosis in ultraviolet-irradiated cells transfected with MKP7 (wild type or mutants) by flow cytometry.", + "section": "RESULTS", + "ner": [ + [ + 37, + 40, + "JNK", + "protein_type" + ], + [ + 53, + 57, + "MKP7", + "protein" + ], + [ + 190, + 194, + "MKP7", + "protein" + ], + [ + 196, + 205, + "wild type", + "protein_state" + ], + [ + 209, + 216, + "mutants", + "protein_state" + ], + [ + 221, + 235, + "flow cytometry", + "experimental_method" + ] + ] + }, + { + "sid": 126, + "sent": "The results showed similar apoptotic rates between cells transfected with blank vector or with MKP7 (wild type or mutants) under unstimulated conditions (Supplementary Fig. 3b), while ultraviolet-irradiation significantly increased apoptotic rate in cells transfected with blank vector (Fig. 6e).", + "section": "RESULTS", + "ner": [ + [ + 95, + 99, + "MKP7", + "protein" + ], + [ + 101, + 110, + "wild type", + "protein_state" + ], + [ + 114, + 121, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 127, + "sent": "Expressions of wild-type MKP7, MKP7\u0394C304 and MKP7-CD significantly decreased the proportion of apoptotic cells after ultraviolet treatment.", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Expressions", + "experimental_method" + ], + [ + 15, + 24, + "wild-type", + "protein_state" + ], + [ + 25, + 29, + "MKP7", + "protein" + ], + [ + 31, + 40, + "MKP7\u0394C304", + "mutant" + ], + [ + 45, + 49, + "MKP7", + "protein" + ], + [ + 50, + 52, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 128, + "sent": "Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did.", + "section": "RESULTS", + "ner": [ + [ + 40, + 44, + "MKP7", + "protein" + ], + [ + 45, + 48, + "KBD", + "structure_element" + ], + [ + 49, + 56, + "mutants", + "protein_state" + ], + [ + 58, + 62, + "R56A", + "mutant" + ], + [ + 63, + 67, + "R57A", + "mutant" + ], + [ + 72, + 76, + "V63A", + "mutant" + ], + [ + 77, + 81, + "I65A", + "mutant" + ], + [ + 136, + 140, + "MKP7", + "protein" + ], + [ + 141, + 150, + "wild type", + "protein_state" + ] + ] + }, + { + "sid": 129, + "sent": "In contrast, cells transfected with the MKP7 FXF-motif mutants (F285D, F287D and L288D) showed little protective effect after ultraviolet treatment and similar levels of apoptosis rates were detected to cells transfected with control vectors (Fig. 6e,f).", + "section": "RESULTS", + "ner": [ + [ + 40, + 44, + "MKP7", + "protein" + ], + [ + 45, + 54, + "FXF-motif", + "structure_element" + ], + [ + 55, + 62, + "mutants", + "protein_state" + ], + [ + 64, + 69, + "F285D", + "mutant" + ], + [ + 71, + 76, + "F287D", + "mutant" + ], + [ + 81, + 86, + "L288D", + "mutant" + ] + ] + }, + { + "sid": 130, + "sent": "Taken together, our results suggested that FXF-motif-mediated, rather than KBD-mediated, interaction is essential for MKP7 to block ultraviolet-induced apoptosis.", + "section": "RESULTS", + "ner": [ + [ + 43, + 52, + "FXF-motif", + "structure_element" + ], + [ + 75, + 78, + "KBD", + "structure_element" + ], + [ + 118, + 122, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 131, + "sent": "A similar docking mechanism for JNK1 recognition by MKP5", + "section": "RESULTS", + "ner": [ + [ + 32, + 36, + "JNK1", + "protein" + ], + [ + 52, + 56, + "MKP5", + "protein" + ] + ] + }, + { + "sid": 132, + "sent": "MKP5 belongs to the same subfamily as MKP7.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "MKP5", + "protein" + ], + [ + 38, + 42, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 133, + "sent": "MKP5 is unique among the MKPs in possessing an additional domain of unknown function at the N-terminus (Fig. 7a).", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "MKP5", + "protein" + ], + [ + 25, + 29, + "MKPs", + "protein_type" + ] + ] + }, + { + "sid": 134, + "sent": "The KBD of MKP5 interacts with the D-site of p38\u03b1 to mediate the enzyme\u2013substrate interaction.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "KBD", + "structure_element" + ], + [ + 11, + 15, + "MKP5", + "protein" + ], + [ + 35, + 41, + "D-site", + "site" + ], + [ + 45, + 49, + "p38\u03b1", + "protein" + ] + ] + }, + { + "sid": 135, + "sent": "Deletion of the KBD in MKP5 leads to a 280-fold increase in Km for p38\u03b1 substrate.", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Deletion of", + "experimental_method" + ], + [ + 16, + 19, + "KBD", + "structure_element" + ], + [ + 23, + 27, + "MKP5", + "protein" + ], + [ + 60, + 62, + "Km", + "evidence" + ], + [ + 67, + 71, + "p38\u03b1", + "protein" + ] + ] + }, + { + "sid": 136, + "sent": "In contrast to p38\u03b1 substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b).", + "section": "RESULTS", + "ner": [ + [ + 15, + 19, + "p38\u03b1", + "protein" + ], + [ + 31, + 42, + "deletion of", + "experimental_method" + ], + [ + 47, + 51, + "MKP5", + "protein" + ], + [ + 52, + 55, + "KBD", + "structure_element" + ], + [ + 109, + 113, + "JNK1", + "protein" + ], + [ + 153, + 156, + "KBD", + "structure_element" + ], + [ + 160, + 164, + "MKP5", + "protein" + ], + [ + 189, + 193, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 137, + "sent": "The substrate specificity constant kcat /Km value for MKP5-CD was calculated as 1.0 \u00d7 105\u2009M\u22121\u2009s\u22121, which is very close to that of MKP7-CD (1.07 \u00d7 105\u2009M\u22121\u2009s\u22121).", + "section": "RESULTS", + "ner": [ + [ + 4, + 34, + "substrate specificity constant", + "evidence" + ], + [ + 35, + 43, + "kcat /Km", + "evidence" + ], + [ + 54, + 58, + "MKP5", + "protein" + ], + [ + 59, + 61, + "CD", + "structure_element" + ], + [ + 130, + 134, + "MKP7", + "protein" + ], + [ + 135, + 137, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 138, + "sent": "The crystal structure of human MKP5-CD has been determined.", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "crystal structure", + "evidence" + ], + [ + 25, + 30, + "human", + "species" + ], + [ + 31, + 35, + "MKP5", + "protein" + ], + [ + 36, + 38, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 139, + "sent": "Comparisons between catalytic domains structures of MKP5 and MKP7 reveal that the overall folds of the two proteins are highly similar, with only a few regions exhibiting small deviations (r.m.s.d. of 0.79\u2009\u00c5; Fig. 7c).", + "section": "RESULTS", + "ner": [ + [ + 20, + 37, + "catalytic domains", + "structure_element" + ], + [ + 38, + 48, + "structures", + "evidence" + ], + [ + 52, + 56, + "MKP5", + "protein" + ], + [ + 61, + 65, + "MKP7", + "protein" + ], + [ + 189, + 197, + "r.m.s.d.", + "evidence" + ] + ] + }, + { + "sid": 140, + "sent": "Given the distinct interaction mode revealed by the crystal structure of JNK1\u2013MKP7-CD, one obvious question is whether this is a general mechanism used by all members of the JNK-specific MKPs.", + "section": "RESULTS", + "ner": [ + [ + 52, + 69, + "crystal structure", + "evidence" + ], + [ + 73, + 85, + "JNK1\u2013MKP7-CD", + "complex_assembly" + ], + [ + 174, + 191, + "JNK-specific MKPs", + "protein_type" + ] + ] + }, + { + "sid": 141, + "sent": "To address this issue, we first examined the docking ability of JNK1 to the KBD and CD of MKP5 using gel filtration analysis and pull-down assays.", + "section": "RESULTS", + "ner": [ + [ + 64, + 68, + "JNK1", + "protein" + ], + [ + 76, + 79, + "KBD", + "structure_element" + ], + [ + 84, + 86, + "CD", + "structure_element" + ], + [ + 90, + 94, + "MKP5", + "protein" + ], + [ + 101, + 124, + "gel filtration analysis", + "experimental_method" + ], + [ + 129, + 145, + "pull-down assays", + "experimental_method" + ] + ] + }, + { + "sid": 142, + "sent": "It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d).", + "section": "RESULTS", + "ner": [ + [ + 20, + 46, + "gel filtration experiments", + "experimental_method" + ], + [ + 52, + 56, + "JNK1", + "protein" + ], + [ + 69, + 75, + "stable", + "protein_state" + ], + [ + 76, + 87, + "heterodimer", + "oligomeric_state" + ], + [ + 93, + 97, + "MKP5", + "protein" + ], + [ + 98, + 100, + "CD", + "structure_element" + ], + [ + 163, + 166, + "KBD", + "structure_element" + ] + ] + }, + { + "sid": 143, + "sent": "Pull-down assays also confirmed the protein\u2013protein interactions observed above.", + "section": "RESULTS", + "ner": [ + [ + 0, + 16, + "Pull-down assays", + "experimental_method" + ] + ] + }, + { + "sid": 144, + "sent": "The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs.", + "section": "RESULTS", + "ner": [ + [ + 4, + 20, + "catalytic domain", + "structure_element" + ], + [ + 24, + 28, + "MKP5", + "protein" + ], + [ + 42, + 45, + "KBD", + "structure_element" + ], + [ + 92, + 96, + "JNK1", + "protein" + ], + [ + 169, + 172, + "p38", + "protein_type" + ], + [ + 177, + 180, + "JNK", + "protein_type" + ], + [ + 181, + 186, + "MAPKs", + "protein_type" + ] + ] + }, + { + "sid": 145, + "sent": "To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity.", + "section": "RESULTS", + "ner": [ + [ + 54, + 58, + "MKP5", + "protein" + ], + [ + 59, + 61, + "CD", + "structure_element" + ], + [ + 70, + 79, + "mutations", + "experimental_method" + ], + [ + 120, + 124, + "MKP7", + "protein" + ], + [ + 125, + 127, + "CD", + "structure_element" + ], + [ + 144, + 177, + "sequence and structural alignment", + "experimental_method" + ], + [ + 212, + 223, + "phosphatase", + "protein_type" + ] + ] + }, + { + "sid": 146, + "sent": "As shown in Fig. 7f, the T432A and L449F MKP5 mutant showed little or no difference in phosphatase activity, whereas the other mutants showed reduced specific activities of MKP5.", + "section": "RESULTS", + "ner": [ + [ + 25, + 30, + "T432A", + "mutant" + ], + [ + 35, + 40, + "L449F", + "mutant" + ], + [ + 41, + 45, + "MKP5", + "protein" + ], + [ + 46, + 52, + "mutant", + "protein_state" + ], + [ + 127, + 134, + "mutants", + "protein_state" + ], + [ + 173, + 177, + "MKP5", + "protein" + ] + ] + }, + { + "sid": 147, + "sent": "As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h).", + "section": "RESULTS", + "ner": [ + [ + 18, + 22, + "MKP7", + "protein" + ], + [ + 32, + 39, + "mutants", + "protein_state" + ], + [ + 48, + 55, + "F451D/A", + "mutant" + ], + [ + 67, + 74, + "pNPPase", + "protein_type" + ], + [ + 110, + 119, + "wild-type", + "protein_state" + ], + [ + 120, + 124, + "MKP5", + "protein" + ], + [ + 125, + 127, + "CD", + "structure_element" + ], + [ + 147, + 162, + "point mutations", + "experimental_method" + ], + [ + 166, + 170, + "JNK1", + "protein" + ], + [ + 188, + 204, + "binding affinity", + "evidence" + ], + [ + 208, + 212, + "MKP5", + "protein" + ], + [ + 213, + 215, + "CD", + "structure_element" + ], + [ + 220, + 224, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 148, + "sent": "In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a).", + "section": "RESULTS", + "ner": [ + [ + 58, + 68, + "CD spectra", + "evidence" + ], + [ + 77, + 86, + "wild-type", + "protein_state" + ], + [ + 91, + 97, + "mutant", + "protein_state" + ], + [ + 136, + 146, + "structures", + "evidence" + ], + [ + 156, + 163, + "mutants", + "protein_state" + ], + [ + 206, + 215, + "wild-type", + "protein_state" + ], + [ + 216, + 220, + "MKP5", + "protein" + ] + ] + }, + { + "sid": 149, + "sent": "Taken together, our results suggest that MKP5 binds JNK1 in a docking mode similar to that in the JNK1\u2013MKP7 complex, and the detailed interaction model can be generated using molecular dynamics simulation based on the structure of JNK1\u2013MKP7-CD complex (Supplementary Fig. 4b,c).", + "section": "RESULTS", + "ner": [ + [ + 41, + 45, + "MKP5", + "protein" + ], + [ + 52, + 56, + "JNK1", + "protein" + ], + [ + 98, + 107, + "JNK1\u2013MKP7", + "complex_assembly" + ], + [ + 175, + 204, + "molecular dynamics simulation", + "experimental_method" + ], + [ + 218, + 227, + "structure", + "evidence" + ], + [ + 231, + 243, + "JNK1\u2013MKP7-CD", + "complex_assembly" + ] + ] + }, + { + "sid": 150, + "sent": "In this model, the MKP5-CD adopts a conformation nearly identical to that in its unbound form, suggesting that the conformation of the catalytic domain undergoes little change, if any at all, upon JNK1 binding.", + "section": "RESULTS", + "ner": [ + [ + 19, + 23, + "MKP5", + "protein" + ], + [ + 24, + 26, + "CD", + "structure_element" + ], + [ + 81, + 88, + "unbound", + "protein_state" + ], + [ + 135, + 151, + "catalytic domain", + "structure_element" + ], + [ + 197, + 201, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 151, + "sent": "In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1\u2013MKP7-CD complex (Supplementary Fig. 4d).", + "section": "RESULTS", + "ner": [ + [ + 15, + 21, + "Leu449", + "residue_name_number" + ], + [ + 25, + 29, + "MKP5", + "protein" + ], + [ + 70, + 76, + "Phe285", + "residue_name_number" + ], + [ + 80, + 84, + "MKP7", + "protein" + ], + [ + 111, + 129, + "hydrophobic pocket", + "site" + ], + [ + 133, + 137, + "JNK1", + "protein" + ], + [ + 157, + 163, + "Phe285", + "residue_name_number" + ], + [ + 171, + 183, + "JNK1\u2013MKP7-CD", + "complex_assembly" + ] + ] + }, + { + "sid": 152, + "sent": "Despite the strong similarities between JNK1\u2013MKP5-CD and JNK1\u2013MKP7-CD, however, there are differences.", + "section": "RESULTS", + "ner": [ + [ + 40, + 44, + "JNK1", + "protein" + ], + [ + 45, + 49, + "MKP5", + "protein" + ], + [ + 50, + 52, + "CD", + "structure_element" + ], + [ + 57, + 69, + "JNK1\u2013MKP7-CD", + "complex_assembly" + ] + ] + }, + { + "sid": 153, + "sent": "The JNK1\u2013MKP7-CD interaction is better and more extensive.", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "JNK1\u2013MKP7-CD", + "complex_assembly" + ] + ] + }, + { + "sid": 154, + "sent": "Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Asp268", + "residue_name_number" + ], + [ + 10, + 14, + "MKP7", + "protein" + ], + [ + 15, + 17, + "CD", + "structure_element" + ], + [ + 24, + 35, + "salt bridge", + "bond_interaction" + ], + [ + 41, + 45, + "JNK1", + "protein" + ], + [ + 46, + 52, + "Arg263", + "residue_name_number" + ], + [ + 88, + 94, + "Thr432", + "residue_name_number" + ], + [ + 98, + 102, + "MKP5", + "protein" + ], + [ + 103, + 105, + "CD", + "structure_element" + ], + [ + 128, + 132, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 155, + "sent": "In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1.", + "section": "RESULTS", + "ner": [ + [ + 45, + 49, + "MKP7", + "protein" + ], + [ + 50, + 52, + "CD", + "structure_element" + ], + [ + 54, + 60, + "Phe215", + "residue_name_number" + ], + [ + 62, + 68, + "Leu267", + "residue_name_number" + ], + [ + 73, + 79, + "Leu288", + "residue_name_number" + ], + [ + 124, + 130, + "Asn379", + "residue_name_number" + ], + [ + 132, + 138, + "Met431", + "residue_name_number" + ], + [ + 143, + 149, + "Met452", + "residue_name_number" + ], + [ + 153, + 157, + "MKP5", + "protein" + ], + [ + 158, + 160, + "CD", + "structure_element" + ], + [ + 213, + 237, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 246, + 250, + "MKP5", + "protein" + ], + [ + 251, + 253, + "CD", + "structure_element" + ], + [ + 258, + 262, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 156, + "sent": "This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is \u223c20-fold higher than that of MKP7-CD).", + "section": "RESULTS", + "ner": [ + [ + 66, + 70, + "JNK1", + "protein" + ], + [ + 80, + 84, + "MKP7", + "protein" + ], + [ + 85, + 87, + "CD", + "structure_element" + ], + [ + 111, + 115, + "MKP5", + "protein" + ], + [ + 116, + 118, + "CD", + "structure_element" + ], + [ + 132, + 136, + "MKP5", + "protein" + ], + [ + 137, + 139, + "CD", + "structure_element" + ], + [ + 144, + 145, + "p", + "protein_state" + ], + [ + 145, + 149, + "JNK1", + "protein" + ], + [ + 192, + 196, + "MKP7", + "protein" + ], + [ + 197, + 199, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 157, + "sent": "The MAPKs p38, ERK and JNK, are central to evolutionarily conserved signalling pathways that are present in all eukaryotic cells.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 9, + "MAPKs", + "protein_type" + ], + [ + 10, + 13, + "p38", + "protein_type" + ], + [ + 15, + 18, + "ERK", + "protein_type" + ], + [ + 23, + 26, + "JNK", + "protein_type" + ], + [ + 112, + 122, + "eukaryotic", + "taxonomy_domain" + ] + ] + }, + { + "sid": 158, + "sent": "Each MAPK cascade is activated in response to a diverse array of extracellular signals and culminates in the dual-phosphorylation of a threonine and a tyrosine residue in the MAPK-activation loop.", + "section": "DISCUSS", + "ner": [ + [ + 5, + 9, + "MAPK", + "protein_type" + ], + [ + 109, + 129, + "dual-phosphorylation", + "ptm" + ], + [ + 135, + 144, + "threonine", + "residue_name" + ], + [ + 151, + 159, + "tyrosine", + "residue_name" + ], + [ + 175, + 195, + "MAPK-activation loop", + "structure_element" + ] + ] + }, + { + "sid": 159, + "sent": "The propagation of MAPK signals is attenuated through the actions of the MKPs.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 23, + "MAPK", + "protein_type" + ], + [ + 73, + 77, + "MKPs", + "protein_type" + ] + ] + }, + { + "sid": 160, + "sent": "Most studies have focused on the dephosphorylation of MAPKs by phosphatases containing the \u2018kinase-interaction motif ' (D-motif), including a group of DUSPs (MKPs) and a distinct subfamily of tyrosine phosphatases (HePTP, STEP and PTP-SL).", + "section": "DISCUSS", + "ner": [ + [ + 54, + 59, + "MAPKs", + "protein_type" + ], + [ + 63, + 75, + "phosphatases", + "protein_type" + ], + [ + 92, + 116, + "kinase-interaction motif", + "structure_element" + ], + [ + 120, + 127, + "D-motif", + "structure_element" + ], + [ + 151, + 156, + "DUSPs", + "protein_type" + ], + [ + 158, + 162, + "MKPs", + "protein_type" + ], + [ + 192, + 213, + "tyrosine phosphatases", + "protein_type" + ], + [ + 215, + 220, + "HePTP", + "protein" + ], + [ + 222, + 226, + "STEP", + "protein" + ], + [ + 231, + 237, + "PTP-SL", + "protein" + ] + ] + }, + { + "sid": 161, + "sent": "Crystal structures of ERK2 bound with the D-motif sequences derived from MKP3 and HePTP have been reported.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 18, + "Crystal structures", + "evidence" + ], + [ + 22, + 26, + "ERK2", + "protein" + ], + [ + 27, + 37, + "bound with", + "protein_state" + ], + [ + 42, + 49, + "D-motif", + "structure_element" + ], + [ + 73, + 77, + "MKP3", + "protein" + ], + [ + 82, + 87, + "HePTP", + "protein" + ] + ] + }, + { + "sid": 162, + "sent": "These structures revealed that linear docking motifs in interacting proteins bind to a common docking site on MAPKs outside the kinase active site.", + "section": "DISCUSS", + "ner": [ + [ + 6, + 16, + "structures", + "evidence" + ], + [ + 31, + 52, + "linear docking motifs", + "structure_element" + ], + [ + 94, + 106, + "docking site", + "site" + ], + [ + 110, + 115, + "MAPKs", + "protein_type" + ], + [ + 128, + 134, + "kinase", + "protein_type" + ], + [ + 135, + 146, + "active site", + "site" + ] + ] + }, + { + "sid": 163, + "sent": "The particular amino acids and their spacing within D-motif sequences and amino acid composition of the docking sites on MAPKs appear to determine the specificity of D-motifs for individual MAPKs.", + "section": "DISCUSS", + "ner": [ + [ + 52, + 59, + "D-motif", + "structure_element" + ], + [ + 104, + 117, + "docking sites", + "site" + ], + [ + 121, + 126, + "MAPKs", + "protein_type" + ], + [ + 166, + 174, + "D-motifs", + "structure_element" + ], + [ + 190, + 195, + "MAPKs", + "protein_type" + ] + ] + }, + { + "sid": 164, + "sent": "Recently, the crystal structure of a complex between the KBD of MKP5 and p38\u03b1 has been obtained.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 31, + "crystal structure", + "evidence" + ], + [ + 57, + 60, + "KBD", + "structure_element" + ], + [ + 64, + 68, + "MKP5", + "protein" + ], + [ + 73, + 77, + "p38\u03b1", + "protein" + ] + ] + }, + { + "sid": 165, + "sent": "This complex has revealed a distinct interaction mode for MKP5.", + "section": "DISCUSS", + "ner": [ + [ + 58, + 62, + "MKP5", + "protein" + ] + ] + }, + { + "sid": 166, + "sent": "The KBD of MKP5 binds to p38\u03b1 in the opposite polypeptide direction compared with how the D-motif of MKP3 binds to ERK2.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 7, + "KBD", + "structure_element" + ], + [ + 11, + 15, + "MKP5", + "protein" + ], + [ + 25, + 29, + "p38\u03b1", + "protein" + ], + [ + 90, + 97, + "D-motif", + "structure_element" + ], + [ + 101, + 105, + "MKP3", + "protein" + ], + [ + 115, + 119, + "ERK2", + "protein" + ] + ] + }, + { + "sid": 167, + "sent": "In contrast to the canonical D-motif-binding mode, separate helices, \u03b12 and \u03b13\u2032, in the KBD of MKP5 engage the p38\u03b1-docking site.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 49, + "D-motif-binding mode", + "site" + ], + [ + 60, + 67, + "helices", + "structure_element" + ], + [ + 69, + 71, + "\u03b12", + "structure_element" + ], + [ + 76, + 79, + "\u03b13\u2032", + "structure_element" + ], + [ + 88, + 91, + "KBD", + "structure_element" + ], + [ + 95, + 99, + "MKP5", + "protein" + ], + [ + 111, + 128, + "p38\u03b1-docking site", + "site" + ] + ] + }, + { + "sid": 168, + "sent": "Further structural and biochemical studies indicate that KBD of MKP7 may interact with p38\u03b1 in a similar manner to that of MKP5.", + "section": "DISCUSS", + "ner": [ + [ + 8, + 42, + "structural and biochemical studies", + "experimental_method" + ], + [ + 57, + 60, + "KBD", + "structure_element" + ], + [ + 64, + 68, + "MKP7", + "protein" + ], + [ + 87, + 91, + "p38\u03b1", + "protein" + ], + [ + 123, + 127, + "MKP5", + "protein" + ] + ] + }, + { + "sid": 169, + "sent": "In contrast to MKP5, removal of the KBD domain from MKP7 does not drastically affect enzyme catalysis, and the kinetic parameters of MKP7-CD for p38\u03b1 substrate are very similar to those for JNK1 substrate.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 19, + "MKP5", + "protein" + ], + [ + 21, + 31, + "removal of", + "experimental_method" + ], + [ + 36, + 39, + "KBD", + "structure_element" + ], + [ + 52, + 56, + "MKP7", + "protein" + ], + [ + 133, + 137, + "MKP7", + "protein" + ], + [ + 138, + 140, + "CD", + "structure_element" + ], + [ + 145, + 149, + "p38\u03b1", + "protein" + ], + [ + 190, + 194, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 170, + "sent": "Taken together, these results suggest that MKP7 utilizes a bipartite recognition mechanism to achieve the efficiency and fidelity of p38\u03b1 signalling.", + "section": "DISCUSS", + "ner": [ + [ + 43, + 47, + "MKP7", + "protein" + ], + [ + 133, + 137, + "p38\u03b1", + "protein" + ] + ] + }, + { + "sid": 171, + "sent": "The MKP7-KBD docks to the D-site located on the back side of the p38\u03b1 catalytic pocket for high-affinity association, whereas the interaction of the MKP7-CD with another p38\u03b1 structural region, which is close to the activation loop, may not only stabilize binding but also provide contacts crucial for organizing the MKP7 active site with respect to the phosphoreceptor in the p38\u03b1 substrate for efficient dephosphorylation.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 8, + "MKP7", + "protein" + ], + [ + 9, + 12, + "KBD", + "structure_element" + ], + [ + 26, + 32, + "D-site", + "site" + ], + [ + 65, + 69, + "p38\u03b1", + "protein" + ], + [ + 70, + 86, + "catalytic pocket", + "site" + ], + [ + 149, + 153, + "MKP7", + "protein" + ], + [ + 154, + 156, + "CD", + "structure_element" + ], + [ + 170, + 174, + "p38\u03b1", + "protein" + ], + [ + 216, + 231, + "activation loop", + "structure_element" + ], + [ + 317, + 321, + "MKP7", + "protein" + ], + [ + 322, + 333, + "active site", + "site" + ], + [ + 377, + 381, + "p38\u03b1", + "protein" + ] + ] + }, + { + "sid": 172, + "sent": "In addition to the canonical D-site, the MAPK ERK2 contains a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif-binding site (also called F-site), that is exposed in active ERK2 and the D-motif peptide-induced conformation of MAPKs.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 35, + "D-site", + "site" + ], + [ + 41, + 45, + "MAPK", + "protein_type" + ], + [ + 46, + 50, + "ERK2", + "protein" + ], + [ + 62, + 81, + "second binding site", + "site" + ], + [ + 130, + 142, + "phosphatases", + "protein_type" + ], + [ + 148, + 170, + "FXF-motif-binding site", + "site" + ], + [ + 184, + 190, + "F-site", + "site" + ], + [ + 212, + 218, + "active", + "protein_state" + ], + [ + 219, + 223, + "ERK2", + "protein" + ], + [ + 232, + 239, + "D-motif", + "structure_element" + ], + [ + 272, + 277, + "MAPKs", + "protein_type" + ] + ] + }, + { + "sid": 173, + "sent": "This hydrophobic site was first identified by changes in deuterium exchange profiles, and is near the MAPK insertion and helix \u03b1G. Interestingly, many of the equivalent residues in JNK1, important for MKP7-CD recognition, are also used for substrate binding by ERK2 (ref.), indicating that this site is overlapped with the DEF-site previously identified in ERK2 (Fig. 5d).", + "section": "DISCUSS", + "ner": [ + [ + 5, + 21, + "hydrophobic site", + "site" + ], + [ + 46, + 84, + "changes in deuterium exchange profiles", + "evidence" + ], + [ + 102, + 116, + "MAPK insertion", + "structure_element" + ], + [ + 121, + 126, + "helix", + "structure_element" + ], + [ + 127, + 129, + "\u03b1G", + "structure_element" + ], + [ + 181, + 185, + "JNK1", + "protein" + ], + [ + 201, + 205, + "MKP7", + "protein" + ], + [ + 206, + 208, + "CD", + "structure_element" + ], + [ + 261, + 265, + "ERK2", + "protein" + ], + [ + 323, + 331, + "DEF-site", + "site" + ], + [ + 357, + 361, + "ERK2", + "protein" + ] + ] + }, + { + "sid": 174, + "sent": "MKP3 is highly specific in dephosphorylating and inactivating ERK2, and the phosphatase activity of the MKP3-catalysed pNPP reaction can be markedly increased in the presence of ERK2 (refs).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "MKP3", + "protein" + ], + [ + 62, + 66, + "ERK2", + "protein" + ], + [ + 104, + 108, + "MKP3", + "protein" + ], + [ + 119, + 123, + "pNPP", + "chemical" + ], + [ + 166, + 177, + "presence of", + "protein_state" + ], + [ + 178, + 182, + "ERK2", + "protein" + ] + ] + }, + { + "sid": 175, + "sent": "Sequence alignment of all MKPs reveals a high degree of conservation of residues surrounding the interacting region observed in JNK1\u2013MKP7-CD complex (Supplementary Fig. 5).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 18, + "Sequence alignment", + "experimental_method" + ], + [ + 26, + 30, + "MKPs", + "protein_type" + ], + [ + 97, + 115, + "interacting region", + "site" + ], + [ + 128, + 140, + "JNK1\u2013MKP7-CD", + "complex_assembly" + ] + ] + }, + { + "sid": 176, + "sent": "Therefore, it is tempting to speculate that the catalytic domain of MKP3 may bind to ERK2 in a manner analogous to the way by which MKP7-CD binds to JNK1.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 64, + "catalytic domain", + "structure_element" + ], + [ + 68, + 72, + "MKP3", + "protein" + ], + [ + 85, + 89, + "ERK2", + "protein" + ], + [ + 132, + 136, + "MKP7", + "protein" + ], + [ + 137, + 139, + "CD", + "structure_element" + ], + [ + 149, + 153, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 177, + "sent": "A comprehensive examination of the molecular basis of the specific ERK2 recognition by MKP3 is underway.", + "section": "DISCUSS", + "ner": [ + [ + 67, + 71, + "ERK2", + "protein" + ], + [ + 87, + 91, + "MKP3", + "protein" + ] + ] + }, + { + "sid": 178, + "sent": "The ongoing work demonstrates that although the overall interaction modes are similar between the JNK1\u2013MKP7-CD and ERK2\u2013MKP3-CD complexes, the ERK2\u2013MKP3-CD interaction is less extensive and helix \u03b14 from MKP3-CD does not interact directly with ERK2.", + "section": "DISCUSS", + "ner": [ + [ + 98, + 110, + "JNK1\u2013MKP7-CD", + "complex_assembly" + ], + [ + 115, + 127, + "ERK2\u2013MKP3-CD", + "complex_assembly" + ], + [ + 143, + 155, + "ERK2\u2013MKP3-CD", + "complex_assembly" + ], + [ + 190, + 195, + "helix", + "structure_element" + ], + [ + 196, + 198, + "\u03b14", + "structure_element" + ], + [ + 204, + 208, + "MKP3", + "protein" + ], + [ + 209, + 211, + "CD", + "structure_element" + ], + [ + 244, + 248, + "ERK2", + "protein" + ] + ] + }, + { + "sid": 179, + "sent": "The FXF-motif-mediated interaction is critical for both pERK2 inactivation and ERK2-induced MKP3 activation (manuscript in preparation).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 13, + "FXF-motif", + "structure_element" + ], + [ + 56, + 57, + "p", + "protein_state" + ], + [ + 57, + 61, + "ERK2", + "protein" + ], + [ + 79, + 83, + "ERK2", + "protein" + ], + [ + 92, + 96, + "MKP3", + "protein" + ] + ] + }, + { + "sid": 180, + "sent": "In summary, we have resolved the structure of JNK1 in complex with the catalytic domain of MKP7.", + "section": "DISCUSS", + "ner": [ + [ + 33, + 42, + "structure", + "evidence" + ], + [ + 46, + 50, + "JNK1", + "protein" + ], + [ + 51, + 66, + "in complex with", + "protein_state" + ], + [ + 71, + 87, + "catalytic domain", + "structure_element" + ], + [ + 91, + 95, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 181, + "sent": "This structure reveals an FXF-docking interaction mode between MAPK and MKP.", + "section": "DISCUSS", + "ner": [ + [ + 5, + 14, + "structure", + "evidence" + ], + [ + 26, + 54, + "FXF-docking interaction mode", + "site" + ], + [ + 63, + 67, + "MAPK", + "protein_type" + ], + [ + 72, + 75, + "MKP", + "protein_type" + ] + ] + }, + { + "sid": 182, + "sent": "Results from biochemical characterization of the Phe285 and Phe287 MKP7 mutants combined with structural information support the conclusion that the two Phe residues serve different roles in the catalytic reaction.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 41, + "biochemical characterization", + "experimental_method" + ], + [ + 49, + 55, + "Phe285", + "residue_name_number" + ], + [ + 60, + 66, + "Phe287", + "residue_name_number" + ], + [ + 67, + 71, + "MKP7", + "protein" + ], + [ + 72, + 79, + "mutants", + "protein_state" + ], + [ + 94, + 116, + "structural information", + "evidence" + ], + [ + 153, + 156, + "Phe", + "residue_name" + ] + ] + }, + { + "sid": 183, + "sent": "Phe285 is essential for JNK1 substrate binding, whereas Phe287 plays a role for the precise alignment of active-site residues, which are important for transition-state stabilization.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 6, + "Phe285", + "residue_name_number" + ], + [ + 24, + 28, + "JNK1", + "protein" + ], + [ + 56, + 62, + "Phe287", + "residue_name_number" + ], + [ + 105, + 125, + "active-site residues", + "site" + ] + ] + }, + { + "sid": 184, + "sent": "This newly identified FXF-type motif is present in all MKPs, except that the residue at the first position in MKP5 is an equivalent hydrophobic leucine residue (see also Fig. 7f,g), suggesting that these two Phe residues would play a similar role in facilitating substrate recognition and catalysis, respectively.", + "section": "DISCUSS", + "ner": [ + [ + 22, + 36, + "FXF-type motif", + "structure_element" + ], + [ + 55, + 59, + "MKPs", + "protein_type" + ], + [ + 110, + 114, + "MKP5", + "protein" + ], + [ + 144, + 151, + "leucine", + "residue_name" + ], + [ + 208, + 211, + "Phe", + "residue_name" + ] + ] + }, + { + "sid": 185, + "sent": "An important feature of MKP\u2013JNK1 interactions is that MKP7 or MKP5 only interact with the F-site of JNK1.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 27, + "MKP", + "protein_type" + ], + [ + 28, + 32, + "JNK1", + "protein" + ], + [ + 54, + 58, + "MKP7", + "protein" + ], + [ + 62, + 66, + "MKP5", + "protein" + ], + [ + 90, + 96, + "F-site", + "site" + ], + [ + 100, + 104, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 186, + "sent": "One possible explanation is that JNK1 needs to use the D-site to interact with JIP-1, a scaffold protein for JNK signalling.", + "section": "DISCUSS", + "ner": [ + [ + 33, + 37, + "JNK1", + "protein" + ], + [ + 55, + 61, + "D-site", + "site" + ], + [ + 79, + 84, + "JIP-1", + "protein" + ], + [ + 109, + 112, + "JNK", + "protein_type" + ] + ] + }, + { + "sid": 187, + "sent": "The N-terminal JNK-binding domain of JIP-1 interacts with the D-site on JNK and this interaction is required for JIP-1-mediated enhancement of JNK activation.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 33, + "JNK-binding domain", + "structure_element" + ], + [ + 37, + 42, + "JIP-1", + "protein" + ], + [ + 62, + 68, + "D-site", + "site" + ], + [ + 72, + 75, + "JNK", + "protein_type" + ], + [ + 113, + 118, + "JIP-1", + "protein" + ], + [ + 143, + 146, + "JNK", + "protein_type" + ] + ] + }, + { + "sid": 188, + "sent": "In addition, JIP-1 can also associate with MKP7 via the C-terminal region of MKP7 (ref.).", + "section": "DISCUSS", + "ner": [ + [ + 13, + 18, + "JIP-1", + "protein" + ], + [ + 43, + 47, + "MKP7", + "protein" + ], + [ + 56, + 73, + "C-terminal region", + "structure_element" + ], + [ + 77, + 81, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 189, + "sent": "When MKP7 is bound to JIP-1, it reduces JNK activation, leading to reduced phosphorylation of the JNK target c-Jun.", + "section": "DISCUSS", + "ner": [ + [ + 5, + 9, + "MKP7", + "protein" + ], + [ + 13, + 21, + "bound to", + "protein_state" + ], + [ + 22, + 27, + "JIP-1", + "protein" + ], + [ + 40, + 43, + "JNK", + "protein_type" + ], + [ + 98, + 101, + "JNK", + "protein_type" + ], + [ + 109, + 114, + "c-Jun", + "protein_type" + ] + ] + }, + { + "sid": 190, + "sent": "Thus, our biochemical and structural data allow us to present a model for the JNK1\u2013JIP-1\u2013MKP7 ternary complex and provide an important insight into the assembly and function of JNK signalling modules (Supplementary Fig. 6).", + "section": "DISCUSS", + "ner": [ + [ + 10, + 41, + "biochemical and structural data", + "evidence" + ], + [ + 78, + 93, + "JNK1\u2013JIP-1\u2013MKP7", + "complex_assembly" + ], + [ + 177, + 180, + "JNK", + "protein_type" + ] + ] + }, + { + "sid": 191, + "sent": "Domain structures of ten human MKPs and the atypical VHR.", + "section": "FIG", + "ner": [ + [ + 7, + 17, + "structures", + "evidence" + ], + [ + 25, + 30, + "human", + "species" + ], + [ + 31, + 35, + "MKPs", + "protein_type" + ], + [ + 53, + 56, + "VHR", + "protein" + ] + ] + }, + { + "sid": 192, + "sent": "On the basis of sequence similarity, protein structure, substrate specificity and subcellular localization, the ten members of MKP family can be divided into three groups.", + "section": "FIG", + "ner": [ + [ + 45, + 54, + "structure", + "evidence" + ], + [ + 127, + 137, + "MKP family", + "protein_type" + ] + ] + }, + { + "sid": 193, + "sent": "The first subfamily comprises MKP1, MKP2, PAC1 and hVH3, which are inducible nuclear phosphatases and can dephosphorylate ERK (and JNK, p38) MAPKs.", + "section": "FIG", + "ner": [ + [ + 30, + 34, + "MKP1", + "protein" + ], + [ + 36, + 40, + "MKP2", + "protein" + ], + [ + 42, + 46, + "PAC1", + "protein" + ], + [ + 51, + 55, + "hVH3", + "protein" + ], + [ + 67, + 76, + "inducible", + "protein_state" + ], + [ + 77, + 97, + "nuclear phosphatases", + "protein_type" + ], + [ + 122, + 125, + "ERK", + "protein_type" + ], + [ + 131, + 134, + "JNK", + "protein_type" + ], + [ + 136, + 139, + "p38", + "protein_type" + ], + [ + 141, + 146, + "MAPKs", + "protein_type" + ] + ] + }, + { + "sid": 194, + "sent": "The second subfamily contains MKP3, MKP4 and MKPX, which are cytoplasmic ERK-specific MKPs.", + "section": "FIG", + "ner": [ + [ + 30, + 34, + "MKP3", + "protein" + ], + [ + 36, + 40, + "MKP4", + "protein" + ], + [ + 45, + 49, + "MKPX", + "protein" + ], + [ + 73, + 90, + "ERK-specific MKPs", + "protein_type" + ] + ] + }, + { + "sid": 195, + "sent": "The third subfamily comprises MKP5, MKP7 and hVH5, which were located in both nucleus and cytoplasm, and selectively inactivate JNK and p38.", + "section": "FIG", + "ner": [ + [ + 30, + 34, + "MKP5", + "protein" + ], + [ + 36, + 40, + "MKP7", + "protein" + ], + [ + 45, + 49, + "hVH5", + "protein" + ], + [ + 128, + 131, + "JNK", + "protein_type" + ], + [ + 136, + 139, + "p38", + "protein_type" + ] + ] + }, + { + "sid": 196, + "sent": "All MKPs contain both the CD and KBD domains, whereas VHR, an atypical MKP, only contains a highly conserved catalytic domain.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "MKPs", + "protein_type" + ], + [ + 26, + 28, + "CD", + "structure_element" + ], + [ + 33, + 36, + "KBD", + "structure_element" + ], + [ + 54, + 57, + "VHR", + "protein" + ], + [ + 71, + 74, + "MKP", + "protein_type" + ], + [ + 92, + 108, + "highly conserved", + "protein_state" + ], + [ + 109, + 125, + "catalytic domain", + "structure_element" + ] + ] + }, + { + "sid": 197, + "sent": "In addition to the CD and KBD, MKP7 contains a unique long C-terminal region that contains NES, NLS and PEST motifs, which has no effect on the binding ability and phosphatase activity of MKP7 toward MAPKs.", + "section": "FIG", + "ner": [ + [ + 19, + 21, + "CD", + "structure_element" + ], + [ + 26, + 29, + "KBD", + "structure_element" + ], + [ + 31, + 35, + "MKP7", + "protein" + ], + [ + 59, + 76, + "C-terminal region", + "structure_element" + ], + [ + 91, + 94, + "NES", + "structure_element" + ], + [ + 96, + 99, + "NLS", + "structure_element" + ], + [ + 104, + 115, + "PEST motifs", + "structure_element" + ], + [ + 188, + 192, + "MKP7", + "protein" + ], + [ + 200, + 205, + "MAPKs", + "protein_type" + ] + ] + }, + { + "sid": 198, + "sent": "NES, nuclear export signal; NLS, nuclear localization signal; PEST, C-terminal sequence rich in prolines, glutamates, serines and threonines.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "NES", + "structure_element" + ], + [ + 5, + 26, + "nuclear export signal", + "structure_element" + ], + [ + 28, + 31, + "NLS", + "structure_element" + ], + [ + 33, + 60, + "nuclear localization signal", + "structure_element" + ], + [ + 62, + 66, + "PEST", + "structure_element" + ], + [ + 68, + 92, + "C-terminal sequence rich", + "structure_element" + ], + [ + 96, + 104, + "prolines", + "residue_name" + ], + [ + 106, + 116, + "glutamates", + "residue_name" + ], + [ + 118, + 125, + "serines", + "residue_name" + ], + [ + 130, + 140, + "threonines", + "residue_name" + ] + ] + }, + { + "sid": 199, + "sent": "MKP7-CD is crucial for JNK1 binding and enzyme catalysis.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "MKP7", + "protein" + ], + [ + 5, + 7, + "CD", + "structure_element" + ], + [ + 23, + 27, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 200, + "sent": "(a) Domain organization of human MKP7 and JNK1.", + "section": "FIG", + "ner": [ + [ + 27, + 32, + "human", + "species" + ], + [ + 33, + 37, + "MKP7", + "protein" + ], + [ + 42, + 46, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 201, + "sent": "The KBD and CD of MKP7 are shown in green and blue, and the N-lobe and C-lobe of JNK1 are coloured in lemon and yellow, respectively.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "KBD", + "structure_element" + ], + [ + 12, + 14, + "CD", + "structure_element" + ], + [ + 18, + 22, + "MKP7", + "protein" + ], + [ + 60, + 66, + "N-lobe", + "structure_element" + ], + [ + 71, + 77, + "C-lobe", + "structure_element" + ], + [ + 81, + 85, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 202, + "sent": "The colour scheme is the same in the following figures unless indicated otherwise. (b) Plots of initial velocity of the MKP7-catalysed reaction versus phospho-JNK1 concentration.", + "section": "FIG", + "ner": [ + [ + 87, + 112, + "Plots of initial velocity", + "evidence" + ], + [ + 120, + 124, + "MKP7", + "protein" + ], + [ + 151, + 158, + "phospho", + "ptm" + ], + [ + 159, + 163, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 203, + "sent": "The error bars represent s.e.m. (c) Gel filtration analysis for interaction of JNK1 with MKP7-CD and MKP7-KBD.", + "section": "FIG", + "ner": [ + [ + 36, + 59, + "Gel filtration analysis", + "experimental_method" + ], + [ + 79, + 83, + "JNK1", + "protein" + ], + [ + 89, + 93, + "MKP7", + "protein" + ], + [ + 94, + 96, + "CD", + "structure_element" + ], + [ + 101, + 105, + "MKP7", + "protein" + ], + [ + 106, + 109, + "KBD", + "structure_element" + ] + ] + }, + { + "sid": 204, + "sent": "(d) GST-mediated pull-down assay for interaction of JNK1 with MKP7-CD and MKP7-KBD.", + "section": "FIG", + "ner": [ + [ + 4, + 32, + "GST-mediated pull-down assay", + "experimental_method" + ], + [ + 52, + 56, + "JNK1", + "protein" + ], + [ + 62, + 66, + "MKP7", + "protein" + ], + [ + 67, + 69, + "CD", + "structure_element" + ], + [ + 74, + 78, + "MKP7", + "protein" + ], + [ + 79, + 82, + "KBD", + "structure_element" + ] + ] + }, + { + "sid": 205, + "sent": "The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays.", + "section": "FIG", + "ner": [ + [ + 33, + 43, + "affinities", + "evidence" + ], + [ + 47, + 51, + "MKP7", + "protein" + ], + [ + 52, + 54, + "CD", + "structure_element" + ], + [ + 59, + 63, + "MKP7", + "protein" + ], + [ + 64, + 67, + "KBD", + "structure_element" + ], + [ + 71, + 75, + "JNK1", + "protein" + ], + [ + 86, + 94, + "affinity", + "evidence" + ], + [ + 98, + 102, + "MKP7", + "protein" + ], + [ + 103, + 105, + "CD", + "structure_element" + ], + [ + 174, + 178, + "MKP7", + "protein" + ], + [ + 183, + 187, + "JNK1", + "protein" + ], + [ + 194, + 214, + "GST pull-down assays", + "experimental_method" + ] + ] + }, + { + "sid": 206, + "sent": "The protein amounts of MKP7 used are shown at the bottom.", + "section": "FIG", + "ner": [ + [ + 23, + 27, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 207, + "sent": "Structure of JNK1 in complex with MKP7-CD.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 13, + 17, + "JNK1", + "protein" + ], + [ + 18, + 33, + "in complex with", + "protein_state" + ], + [ + 34, + 38, + "MKP7", + "protein" + ], + [ + 39, + 41, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 208, + "sent": "(a) Ribbon diagram of JNK1\u2013MKP7-CD complex in two views related by a 45\u00b0 rotation around a vertical axis. (b) Structure of MKP7-CD with its active site highlight in cyan.", + "section": "FIG", + "ner": [ + [ + 22, + 34, + "JNK1\u2013MKP7-CD", + "complex_assembly" + ], + [ + 110, + 119, + "Structure", + "evidence" + ], + [ + 123, + 127, + "MKP7", + "protein" + ], + [ + 128, + 130, + "CD", + "structure_element" + ], + [ + 140, + 151, + "active site", + "site" + ] + ] + }, + { + "sid": 209, + "sent": "The 2Fo\u2212Fc omit map (contoured at 1.5\u03c3) for the P-loop of MKP7-CD is shown at inset of b. (c) Structure of VHR with its active site highlighted in marine blue. (d) Close-up view of the JNK1\u2013MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan).", + "section": "FIG", + "ner": [ + [ + 4, + 19, + "2Fo\u2212Fc omit map", + "evidence" + ], + [ + 48, + 54, + "P-loop", + "structure_element" + ], + [ + 58, + 62, + "MKP7", + "protein" + ], + [ + 63, + 65, + "CD", + "structure_element" + ], + [ + 94, + 103, + "Structure", + "evidence" + ], + [ + 107, + 110, + "VHR", + "protein" + ], + [ + 120, + 131, + "active site", + "site" + ], + [ + 185, + 204, + "JNK1\u2013MKP7 interface", + "site" + ], + [ + 240, + 244, + "JNK1", + "protein" + ], + [ + 258, + 262, + "MKP7", + "protein" + ], + [ + 263, + 265, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 210, + "sent": "The JNK1 is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red).", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 211, + "sent": "(e) Interaction networks mainly involving helices \u03b14 and \u03b15 from MKP7-CD, and \u03b1G and \u03b12L14 of JNK1.", + "section": "FIG", + "ner": [ + [ + 4, + 24, + "Interaction networks", + "site" + ], + [ + 42, + 49, + "helices", + "structure_element" + ], + [ + 50, + 52, + "\u03b14", + "structure_element" + ], + [ + 57, + 59, + "\u03b15", + "structure_element" + ], + [ + 65, + 69, + "MKP7", + "protein" + ], + [ + 70, + 72, + "CD", + "structure_element" + ], + [ + 78, + 80, + "\u03b1G", + "structure_element" + ], + [ + 85, + 90, + "\u03b12L14", + "structure_element" + ], + [ + 94, + 98, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 212, + "sent": "MKP7-CD is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red).", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "MKP7", + "protein" + ], + [ + 5, + 7, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 213, + "sent": "Blue dashed lines represent polar interactions.", + "section": "FIG", + "ner": [ + [ + 28, + 46, + "polar interactions", + "bond_interaction" + ], + [ + 28, + 46, + "polar interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 214, + "sent": "(f) The 2Fo\u2212Fc omit map (contoured at 1.5\u03c3) clearly shows electron density for the 285FNFL288 segment of MKP7-CD.", + "section": "FIG", + "ner": [ + [ + 8, + 23, + "2Fo\u2212Fc omit map", + "evidence" + ], + [ + 58, + 74, + "electron density", + "evidence" + ], + [ + 83, + 101, + "285FNFL288 segment", + "structure_element" + ], + [ + 105, + 109, + "MKP7", + "protein" + ], + [ + 110, + 112, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 215, + "sent": "Mutational analysis on interactions between MKP7-CD and JNK1.", + "section": "FIG", + "ner": [ + [ + 0, + 19, + "Mutational analysis", + "experimental_method" + ], + [ + 44, + 48, + "MKP7", + "protein" + ], + [ + 49, + 51, + "CD", + "structure_element" + ], + [ + 56, + 60, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 216, + "sent": "(a) Effects of mutations in MKP7-CD on the JNK1 dephosphorylation (mean\u00b1s.e.m., n=3).", + "section": "FIG", + "ner": [ + [ + 28, + 32, + "MKP7", + "protein" + ], + [ + 33, + 35, + "CD", + "structure_element" + ], + [ + 43, + 47, + "JNK1", + "protein" + ], + [ + 48, + 65, + "dephosphorylation", + "ptm" + ] + ] + }, + { + "sid": 217, + "sent": "Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. (b) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D.", + "section": "FIG", + "ner": [ + [ + 21, + 57, + "hydrophobic and hydrophilic contacts", + "bond_interaction" + ], + [ + 106, + 129, + "Gel filtration analysis", + "experimental_method" + ], + [ + 149, + 153, + "JNK1", + "protein" + ], + [ + 159, + 163, + "MKP7", + "protein" + ], + [ + 164, + 166, + "CD", + "structure_element" + ], + [ + 167, + 173, + "mutant", + "protein_state" + ], + [ + 174, + 179, + "F285D", + "mutant" + ] + ] + }, + { + "sid": 218, + "sent": "Mutant F285D and JNK1 were eluted as monomers, with the molecular masses of \u223c17 and 44\u2009kDa, respectively.", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "Mutant", + "protein_state" + ], + [ + 7, + 12, + "F285D", + "mutant" + ], + [ + 17, + 21, + "JNK1", + "protein" + ], + [ + 37, + 45, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 219, + "sent": "However, in contrast to the wild-type MKP7-CD, mutant F285D did not co-migrate with JNK1.", + "section": "FIG", + "ner": [ + [ + 28, + 37, + "wild-type", + "protein_state" + ], + [ + 38, + 42, + "MKP7", + "protein" + ], + [ + 43, + 45, + "CD", + "structure_element" + ], + [ + 47, + 53, + "mutant", + "protein_state" + ], + [ + 54, + 59, + "F285D", + "mutant" + ], + [ + 84, + 88, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 220, + "sent": "(c) Pull-down assays of MKP7-CD by GST-tagged JNK1 mutants.", + "section": "FIG", + "ner": [ + [ + 4, + 20, + "Pull-down assays", + "experimental_method" + ], + [ + 24, + 28, + "MKP7", + "protein" + ], + [ + 29, + 31, + "CD", + "structure_element" + ], + [ + 35, + 45, + "GST-tagged", + "protein_state" + ], + [ + 46, + 50, + "JNK1", + "protein" + ], + [ + 51, + 58, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 221, + "sent": "The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays.", + "section": "FIG", + "ner": [ + [ + 33, + 43, + "affinities", + "evidence" + ], + [ + 47, + 51, + "MKP7", + "protein" + ], + [ + 52, + 54, + "CD", + "structure_element" + ], + [ + 58, + 62, + "JNK1", + "protein" + ], + [ + 63, + 70, + "mutants", + "protein_state" + ], + [ + 81, + 89, + "affinity", + "evidence" + ], + [ + 93, + 102, + "wild-type", + "protein_state" + ], + [ + 103, + 107, + "JNK1", + "protein" + ], + [ + 176, + 180, + "MKP7", + "protein" + ], + [ + 181, + 183, + "CD", + "structure_element" + ], + [ + 188, + 192, + "JNK1", + "protein" + ], + [ + 193, + 200, + "mutants", + "protein_state" + ], + [ + 207, + 227, + "GST pull-down assays", + "experimental_method" + ] + ] + }, + { + "sid": 222, + "sent": "The protein amounts of MKP7-CD used are shown at the bottom. (d) Circular dichroism spectra for MKP7-CD wild type and mutants.", + "section": "FIG", + "ner": [ + [ + 23, + 27, + "MKP7", + "protein" + ], + [ + 28, + 30, + "CD", + "structure_element" + ], + [ + 65, + 83, + "Circular dichroism", + "experimental_method" + ], + [ + 84, + 91, + "spectra", + "evidence" + ], + [ + 96, + 100, + "MKP7", + "protein" + ], + [ + 101, + 103, + "CD", + "structure_element" + ], + [ + 104, + 113, + "wild type", + "protein_state" + ], + [ + 118, + 125, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 223, + "sent": "Measurements were averaged for three scans. (e) Circular dichroism spectra for JNK1 wild type and mutants.", + "section": "FIG", + "ner": [ + [ + 48, + 66, + "Circular dichroism", + "experimental_method" + ], + [ + 67, + 74, + "spectra", + "evidence" + ], + [ + 79, + 83, + "JNK1", + "protein" + ], + [ + 84, + 93, + "wild type", + "protein_state" + ], + [ + 98, + 105, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 224, + "sent": "(f) Effects of mutations in MKP7-CD on the pNPP hydrolysis reaction (mean\u00b1s.e.m., n=3).", + "section": "FIG", + "ner": [ + [ + 15, + 24, + "mutations", + "experimental_method" + ], + [ + 28, + 32, + "MKP7", + "protein" + ], + [ + 33, + 35, + "CD", + "structure_element" + ], + [ + 43, + 47, + "pNPP", + "chemical" + ] + ] + }, + { + "sid": 225, + "sent": "Comparison of CDK2-KAP and JNK1\u2013MKP7-CD.", + "section": "FIG", + "ner": [ + [ + 14, + 22, + "CDK2-KAP", + "complex_assembly" + ], + [ + 27, + 39, + "JNK1\u2013MKP7-CD", + "complex_assembly" + ] + ] + }, + { + "sid": 226, + "sent": "(a) Superposition of the complex structures of CDK2-KAP (PDB 1FQ1) and JNK1\u2013MKP7-CD.", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "Superposition", + "experimental_method" + ], + [ + 33, + 43, + "structures", + "evidence" + ], + [ + 47, + 55, + "CDK2-KAP", + "complex_assembly" + ], + [ + 71, + 83, + "JNK1\u2013MKP7-CD", + "complex_assembly" + ] + ] + }, + { + "sid": 227, + "sent": "The N-lobe and C-lobe of CDK2 are coloured in grey and pink, respectively, and KAP is coloured in green.", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "N-lobe", + "structure_element" + ], + [ + 15, + 21, + "C-lobe", + "structure_element" + ], + [ + 25, + 29, + "CDK2", + "protein" + ], + [ + 79, + 82, + "KAP", + "protein" + ] + ] + }, + { + "sid": 228, + "sent": "The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I).", + "section": "FIG", + "ner": [ + [ + 75, + 90, + "contact regions", + "site" + ], + [ + 116, + 130, + "hydrogen bonds", + "bond_interaction" + ], + [ + 143, + 150, + "pThr160", + "ptm" + ], + [ + 154, + 158, + "CDK2", + "protein" + ], + [ + 167, + 178, + "active site", + "site" + ], + [ + 182, + 185, + "KAP", + "protein" + ], + [ + 187, + 195, + "region I", + "structure_element" + ] + ] + }, + { + "sid": 229, + "sent": "Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II).", + "section": "FIG", + "ner": [ + [ + 34, + 38, + "CDK2", + "protein" + ], + [ + 42, + 45, + "KAP", + "protein" + ], + [ + 72, + 81, + "interface", + "site" + ], + [ + 117, + 121, + "JNK1", + "protein" + ], + [ + 126, + 130, + "MKP7", + "protein" + ], + [ + 131, + 133, + "CD", + "structure_element" + ], + [ + 135, + 144, + "region II", + "structure_element" + ] + ] + }, + { + "sid": 230, + "sent": "(b) Interactions networks at the auxiliary region II mainly involving helix \u03b17 from KAP and the \u03b1G helix and following L14 loop of CDK2.", + "section": "FIG", + "ner": [ + [ + 33, + 52, + "auxiliary region II", + "structure_element" + ], + [ + 70, + 75, + "helix", + "structure_element" + ], + [ + 76, + 78, + "\u03b17", + "structure_element" + ], + [ + 84, + 87, + "KAP", + "protein" + ], + [ + 96, + 104, + "\u03b1G helix", + "structure_element" + ], + [ + 119, + 127, + "L14 loop", + "structure_element" + ], + [ + 131, + 135, + "CDK2", + "protein" + ] + ] + }, + { + "sid": 231, + "sent": "The CDK2 is shown in surface representation coloured according to the electrostatic potential (positive, blue; negative, red).", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "CDK2", + "protein" + ] + ] + }, + { + "sid": 232, + "sent": "Residues of KAP and CDK2 are highlighted as green and red sticks, respectively.", + "section": "FIG", + "ner": [ + [ + 12, + 15, + "KAP", + "protein" + ], + [ + 20, + 24, + "CDK2", + "protein" + ] + ] + }, + { + "sid": 233, + "sent": "One remarkable difference between these two kinase-phosphatase complexes is that helix \u03b16 of KAP (corresponding to helix \u03b14 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. (c) Sequence alignment of the JNK-interacting regions on MKPs.", + "section": "FIG", + "ner": [ + [ + 81, + 86, + "helix", + "structure_element" + ], + [ + 87, + 89, + "\u03b16", + "structure_element" + ], + [ + 93, + 96, + "KAP", + "protein" + ], + [ + 115, + 120, + "helix", + "structure_element" + ], + [ + 121, + 123, + "\u03b14", + "structure_element" + ], + [ + 127, + 131, + "MKP7", + "protein" + ], + [ + 132, + 134, + "CD", + "structure_element" + ], + [ + 185, + 191, + "stable", + "protein_state" + ], + [ + 192, + 203, + "heterodimer", + "oligomeric_state" + ], + [ + 207, + 211, + "CDK2", + "protein" + ], + [ + 216, + 219, + "KAP", + "protein" + ], + [ + 225, + 243, + "Sequence alignment", + "experimental_method" + ], + [ + 251, + 274, + "JNK-interacting regions", + "site" + ], + [ + 278, + 282, + "MKPs", + "protein_type" + ] + ] + }, + { + "sid": 234, + "sent": "Residues of MKP7-CD involved in JNK1 recognition are indicated by cyan asterisks, and the conserved FXF-motif is highlighted in cyan.", + "section": "FIG", + "ner": [ + [ + 12, + 16, + "MKP7", + "protein" + ], + [ + 17, + 19, + "CD", + "structure_element" + ], + [ + 32, + 36, + "JNK1", + "protein" + ], + [ + 90, + 99, + "conserved", + "protein_state" + ], + [ + 100, + 109, + "FXF-motif", + "structure_element" + ] + ] + }, + { + "sid": 235, + "sent": "The secondary structure assignments of MKP7-CD and KAP are shown above and below each sequence.", + "section": "FIG", + "ner": [ + [ + 39, + 43, + "MKP7", + "protein" + ], + [ + 44, + 46, + "CD", + "structure_element" + ], + [ + 51, + 54, + "KAP", + "protein" + ] + ] + }, + { + "sid": 236, + "sent": "(d) Sequence alignment of the F-site regions on MAPKs.", + "section": "FIG", + "ner": [ + [ + 4, + 22, + "Sequence alignment", + "experimental_method" + ], + [ + 30, + 44, + "F-site regions", + "structure_element" + ], + [ + 48, + 53, + "MAPKs", + "protein_type" + ] + ] + }, + { + "sid": 237, + "sent": "Residues of JNK1 involved in recognition of MKP7 are indicated by orange asterisks, and those forming the F-site are highlighted in yellow.", + "section": "FIG", + "ner": [ + [ + 12, + 16, + "JNK1", + "protein" + ], + [ + 44, + 48, + "MKP7", + "protein" + ], + [ + 106, + 112, + "F-site", + "site" + ] + ] + }, + { + "sid": 238, + "sent": "FXF-motif is critical for controlling the phosphorylation of JNK and ultraviolet-induced apoptosis.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "FXF-motif", + "structure_element" + ], + [ + 42, + 57, + "phosphorylation", + "ptm" + ], + [ + 61, + 64, + "JNK", + "protein_type" + ] + ] + }, + { + "sid": 239, + "sent": "(a\u2013c) FXF-motif is essential for the dephosphorylation of JNK by MKP7.", + "section": "FIG", + "ner": [ + [ + 6, + 15, + "FXF-motif", + "structure_element" + ], + [ + 58, + 61, + "JNK", + "protein_type" + ], + [ + 65, + 69, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 240, + "sent": "HEK293T cells were infected with lentiviruses expressing MKP7 and its mutants (1.0\u2009\u03bcg).", + "section": "FIG", + "ner": [ + [ + 33, + 45, + "lentiviruses", + "taxonomy_domain" + ], + [ + 57, + 61, + "MKP7", + "protein" + ], + [ + 70, + 77, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 241, + "sent": "After 36\u2009h infection, cells were untreated in a, stimulated with 30\u2009\u03bcM etoposide for 3\u2009h in b or irradiated with 25\u2009J\u2009m\u22122 ultraviolet light at 30\u2009min before lysis in c. Whole-cell extracts were then immunoblotted with antibody indicated.", + "section": "FIG", + "ner": [ + [ + 71, + 80, + "etoposide", + "chemical" + ] + ] + }, + { + "sid": 242, + "sent": "Shown is a typical immunoblot for phosphorylated JNK from three independent experiments.", + "section": "FIG", + "ner": [ + [ + 34, + 48, + "phosphorylated", + "protein_state" + ], + [ + 49, + 52, + "JNK", + "protein_type" + ] + ] + }, + { + "sid": 243, + "sent": "(d) F-site is required for JNK1 to interact with MKP7.", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "F-site", + "site" + ], + [ + 27, + 31, + "JNK1", + "protein" + ], + [ + 49, + 53, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 244, + "sent": "HEK293T cells were co-transfected with MKP7 full-length (1.0\u2009\u03bcg) and JNK1 (wild type or mutants as indicated, 1.0\u2009\u03bcg).", + "section": "FIG", + "ner": [ + [ + 19, + 33, + "co-transfected", + "experimental_method" + ], + [ + 39, + 43, + "MKP7", + "protein" + ], + [ + 44, + 55, + "full-length", + "protein_state" + ], + [ + 69, + 73, + "JNK1", + "protein" + ], + [ + 75, + 84, + "wild type", + "protein_state" + ], + [ + 88, + 95, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 245, + "sent": "Whole-cell extracts were then immunoprecipitated with antibody against Myc for MKP7; immunobloting was carried out with antibodies indicated.", + "section": "FIG", + "ner": [ + [ + 30, + 48, + "immunoprecipitated", + "experimental_method" + ], + [ + 79, + 83, + "MKP7", + "protein" + ] + ] + }, + { + "sid": 246, + "sent": "IP, immunoprecipitation; TCL, total cell lysate.", + "section": "FIG", + "ner": [ + [ + 0, + 2, + "IP", + "experimental_method" + ], + [ + 4, + 23, + "immunoprecipitation", + "experimental_method" + ] + ] + }, + { + "sid": 247, + "sent": "(e) Effect of MKP7 (wild type or mutants) expression on ultraviolet-induced apoptosis.", + "section": "FIG", + "ner": [ + [ + 14, + 18, + "MKP7", + "protein" + ], + [ + 20, + 29, + "wild type", + "protein_state" + ], + [ + 33, + 40, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 248, + "sent": "HeLa cells were infected with lentiviruses expressing MKP7 full-length and its mutants.", + "section": "FIG", + "ner": [ + [ + 30, + 42, + "lentiviruses", + "taxonomy_domain" + ], + [ + 54, + 58, + "MKP7", + "protein" + ], + [ + 59, + 70, + "full-length", + "protein_state" + ], + [ + 79, + 86, + "mutants", + "protein_state" + ] + ] + }, + { + "sid": 249, + "sent": "Cells were then subjected to flow cytometry analysis.", + "section": "FIG", + "ner": [ + [ + 29, + 43, + "flow cytometry", + "experimental_method" + ] + ] + }, + { + "sid": 250, + "sent": "Apoptotic cells were determined by Annexin-V-APC/PI staining.", + "section": "FIG", + "ner": [ + [ + 35, + 48, + "Annexin-V-APC", + "chemical" + ], + [ + 49, + 51, + "PI", + "chemical" + ] + ] + }, + { + "sid": 251, + "sent": "The results using Annexin-V stain for membrane phosphatidylserine eversion, combined with propidium iodide (PI) uptake to evaluate cells whose membranes had been compromised.", + "section": "FIG", + "ner": [ + [ + 18, + 27, + "Annexin-V", + "chemical" + ], + [ + 90, + 106, + "propidium iodide", + "chemical" + ], + [ + 108, + 110, + "PI", + "chemical" + ] + ] + }, + { + "sid": 252, + "sent": "Staining with both Annexin-V and PI indicate apoptosis (upper right quadrant).", + "section": "FIG", + "ner": [ + [ + 19, + 28, + "Annexin-V", + "chemical" + ], + [ + 33, + 35, + "PI", + "chemical" + ] + ] + }, + { + "sid": 253, + "sent": "(f) Statistical analysis of apoptotic cells (mean\u00b1s.e.m., n=3), *P<0.05, ***P<0.001 (ANOVA followed by Tukey's test).", + "section": "FIG", + "ner": [ + [ + 64, + 66, + "*P", + "evidence" + ], + [ + 73, + 77, + "***P", + "evidence" + ], + [ + 85, + 90, + "ANOVA", + "experimental_method" + ], + [ + 103, + 115, + "Tukey's test", + "experimental_method" + ] + ] + }, + { + "sid": 254, + "sent": "MKP5-CD is crucial for JNK1 binding and enzyme catalysis.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "MKP5", + "protein" + ], + [ + 5, + 7, + "CD", + "structure_element" + ], + [ + 23, + 27, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 255, + "sent": "(a) Domain organization of human MKP5.", + "section": "FIG", + "ner": [ + [ + 27, + 32, + "human", + "species" + ], + [ + 33, + 37, + "MKP5", + "protein" + ] + ] + }, + { + "sid": 256, + "sent": "The KBD and CD of MKP5 are shown in brown and grey, respectively. (b) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "KBD", + "structure_element" + ], + [ + 12, + 14, + "CD", + "structure_element" + ], + [ + 18, + 22, + "MKP5", + "protein" + ], + [ + 70, + 95, + "Plots of initial velocity", + "evidence" + ], + [ + 103, + 107, + "MKP5", + "protein" + ], + [ + 134, + 141, + "phospho", + "protein_state" + ], + [ + 142, + 146, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 257, + "sent": "The solid lines are best-fitting results according to the Michaelis\u2013Menten equation with Km and kcat values indicated.", + "section": "FIG", + "ner": [ + [ + 89, + 91, + "Km", + "evidence" + ], + [ + 96, + 100, + "kcat", + "evidence" + ] + ] + }, + { + "sid": 258, + "sent": "The error bars represent s.e.m. (c) Structural comparison of the JNK-interacting residues on MKP5-CD (PDB 1ZZW) and MKP7-CD.", + "section": "FIG", + "ner": [ + [ + 36, + 57, + "Structural comparison", + "experimental_method" + ], + [ + 65, + 89, + "JNK-interacting residues", + "site" + ], + [ + 93, + 97, + "MKP5", + "protein" + ], + [ + 98, + 100, + "CD", + "structure_element" + ], + [ + 116, + 120, + "MKP7", + "protein" + ], + [ + 121, + 123, + "CD", + "structure_element" + ] + ] + }, + { + "sid": 259, + "sent": "The corresponding residues on MKP5 are depicted as orange sticks, and MKP5 residues numbers are in parentheses.", + "section": "FIG", + "ner": [ + [ + 30, + 34, + "MKP5", + "protein" + ], + [ + 70, + 74, + "MKP5", + "protein" + ] + ] + }, + { + "sid": 260, + "sent": "(d) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. (e) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD.", + "section": "FIG", + "ner": [ + [ + 4, + 27, + "Gel filtration analysis", + "experimental_method" + ], + [ + 47, + 51, + "JNK1", + "protein" + ], + [ + 57, + 61, + "MKP5", + "protein" + ], + [ + 62, + 64, + "CD", + "structure_element" + ], + [ + 69, + 73, + "MKP5", + "protein" + ], + [ + 74, + 77, + "KBD", + "structure_element" + ], + [ + 83, + 112, + "GST-mediated pull-down assays", + "experimental_method" + ], + [ + 132, + 136, + "JNK1", + "protein" + ], + [ + 142, + 146, + "MKP5", + "protein" + ], + [ + 147, + 149, + "CD", + "structure_element" + ], + [ + 154, + 158, + "MKP5", + "protein" + ], + [ + 159, + 162, + "KBD", + "structure_element" + ] + ] + }, + { + "sid": 261, + "sent": "The panels are arranged the same as in Fig. 2d. (f) Effects of mutations in MKP5-CD on the JNK1 dephosphorylation (mean\u00b1s.e.m., n=3).", + "section": "FIG", + "ner": [ + [ + 63, + 72, + "mutations", + "experimental_method" + ], + [ + 76, + 80, + "MKP5", + "protein" + ], + [ + 81, + 83, + "CD", + "structure_element" + ], + [ + 91, + 95, + "JNK1", + "protein" + ] + ] + }, + { + "sid": 262, + "sent": "(g) Effects of mutations in MKP5-CD on the pNPP hydrolysis reaction (mean\u00b1s.e.m., n=3).", + "section": "FIG", + "ner": [ + [ + 15, + 24, + "mutations", + "experimental_method" + ], + [ + 28, + 32, + "MKP5", + "protein" + ], + [ + 33, + 35, + "CD", + "structure_element" + ], + [ + 43, + 47, + "pNPP", + "chemical" + ] + ] + }, + { + "sid": 263, + "sent": "(h) Pull-down assays of MKP5-CD by GST-tagged JNK1 mutants.", + "section": "FIG", + "ner": [ + [ + 4, + 20, + "Pull-down assays", + "experimental_method" + ], + [ + 24, + 28, + "MKP5", + "protein" + ], + [ + 29, + 31, + "CD", + "structure_element" + ], + [ + 35, + 45, + "GST-tagged", + "protein_state" + ], + [ + 46, + 50, + "JNK1", + "protein" + ], + [ + 51, + 58, + "mutants", + "protein_state" + ] + ] + } + ] + }, + "PMC4848090": { + "annotations": [ + { + "sid": 0, + "sent": "Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission", + "section": "TITLE", + "ner": [ + [ + 27, + 42, + "peptide hormone", + "protein_type" + ] + ] + }, + { + "sid": 1, + "sent": "Plants constantly renew during their life cycle and thus require to shed senescent and damaged organs.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 6, + "Plants", + "taxonomy_domain" + ] + ] + }, + { + "sid": 2, + "sent": "Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA.", + "section": "ABSTRACT", + "ner": [ + [ + 39, + 74, + "leucine-rich repeat receptor kinase", + "protein_type" + ], + [ + 76, + 82, + "LRR-RK", + "protein_type" + ], + [ + 84, + 89, + "HAESA", + "protein" + ], + [ + 98, + 113, + "peptide hormone", + "protein_type" + ], + [ + 114, + 117, + "IDA", + "protein" + ] + ] + }, + { + "sid": 3, + "sent": "It is unknown how expression of IDA in the abscission zone leads to HAESA activation.", + "section": "ABSTRACT", + "ner": [ + [ + 32, + 35, + "IDA", + "protein" + ], + [ + 68, + 73, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 4, + "sent": "Here we show that IDA is sensed directly by the HAESA ectodomain.", + "section": "ABSTRACT", + "ner": [ + [ + 18, + 21, + "IDA", + "protein" + ], + [ + 48, + 53, + "HAESA", + "protein" + ], + [ + 54, + 64, + "ectodomain", + "structure_element" + ] + ] + }, + { + "sid": 5, + "sent": "Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 18, + "Crystal structures", + "evidence" + ], + [ + 22, + 27, + "HAESA", + "protein" + ], + [ + 28, + 43, + "in complex with", + "protein_state" + ], + [ + 44, + 47, + "IDA", + "protein" + ], + [ + 57, + 79, + "hormone binding pocket", + "site" + ], + [ + 101, + 107, + "active", + "protein_state" + ], + [ + 108, + 117, + "dodecamer", + "structure_element" + ], + [ + 118, + 125, + "peptide", + "chemical" + ] + ] + }, + { + "sid": 6, + "sent": "A central hydroxyproline residue anchors IDA to the receptor.", + "section": "ABSTRACT", + "ner": [ + [ + 10, + 24, + "hydroxyproline", + "residue_name" + ], + [ + 41, + 44, + "IDA", + "protein" + ] + ] + }, + { + "sid": 7, + "sent": "The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 9, + "HAESA", + "protein" + ], + [ + 10, + 21, + "co-receptor", + "protein_type" + ], + [ + 22, + 27, + "SERK1", + "protein" + ], + [ + 124, + 139, + "peptide hormone", + "protein_type" + ], + [ + 157, + 174, + "Arg-His-Asn motif", + "structure_element" + ], + [ + 178, + 181, + "IDA", + "protein" + ] + ] + }, + { + "sid": 8, + "sent": "This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism.", + "section": "ABSTRACT", + "ner": [ + [ + 25, + 34, + "conserved", + "protein_state" + ], + [ + 49, + 54, + "plant", + "taxonomy_domain" + ], + [ + 55, + 63, + "peptides", + "chemical" + ], + [ + 81, + 86, + "plant", + "taxonomy_domain" + ], + [ + 87, + 112, + "peptide hormone receptors", + "protein_type" + ] + ] + }, + { + "sid": 9, + "sent": "Plants can shed their leaves, flowers or other organs when they no longer need them. But how does a leaf or a flower know when to let go? A receptor protein called HAESA is found on the surface of the cells that surround a future break point on the plant. When its time to shed an organ, a hormone called IDA instructs HAESA to trigger the shedding process.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 6, + "Plants", + "taxonomy_domain" + ], + [ + 140, + 156, + "receptor protein", + "protein_type" + ], + [ + 164, + 169, + "HAESA", + "protein" + ], + [ + 290, + 297, + "hormone", + "chemical" + ], + [ + 305, + 308, + "IDA", + "protein" + ], + [ + 319, + 324, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 10, + "sent": "However, the molecular details of how IDA triggers organ shedding are not clear.", + "section": "ABSTRACT", + "ner": [ + [ + 38, + 41, + "IDA", + "protein" + ] + ] + }, + { + "sid": 11, + "sent": "The shedding of floral organs (or leaves) can be easily studied in a model plant called Arabidopsis.", + "section": "ABSTRACT", + "ner": [ + [ + 75, + 80, + "plant", + "taxonomy_domain" + ], + [ + 88, + 99, + "Arabidopsis", + "taxonomy_domain" + ] + ] + }, + { + "sid": 12, + "sent": "Santiago et al. used protein biochemistry, structural biology and genetics to uncover how the IDA hormone activates HAESA.", + "section": "ABSTRACT", + "ner": [ + [ + 21, + 41, + "protein biochemistry", + "experimental_method" + ], + [ + 43, + 61, + "structural biology", + "experimental_method" + ], + [ + 66, + 74, + "genetics", + "experimental_method" + ], + [ + 94, + 97, + "IDA", + "protein" + ], + [ + 98, + 105, + "hormone", + "chemical" + ], + [ + 116, + 121, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 13, + "sent": "The experiments show that IDA binds directly to a canyon shaped pocket in HAESA that extends out from the surface of the cell.", + "section": "ABSTRACT", + "ner": [ + [ + 26, + 29, + "IDA", + "protein" + ], + [ + 30, + 47, + "binds directly to", + "protein_state" + ], + [ + 50, + 63, + "canyon shaped", + "protein_state" + ], + [ + 64, + 70, + "pocket", + "site" + ], + [ + 74, + 79, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 14, + "sent": "IDA binding to HAESA allows another receptor protein called SERK1 to bind to HAESA, which results in the release of signals inside the cell that trigger the shedding of organs.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 3, + "IDA", + "protein" + ], + [ + 15, + 20, + "HAESA", + "protein" + ], + [ + 36, + 52, + "receptor protein", + "protein_type" + ], + [ + 60, + 65, + "SERK1", + "protein" + ], + [ + 66, + 76, + "to bind to", + "protein_state" + ], + [ + 77, + 82, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 15, + "sent": "The next step following on from this work is to understand what signals are produced when IDA activates HAESA.", + "section": "ABSTRACT", + "ner": [ + [ + 90, + 93, + "IDA", + "protein" + ], + [ + 104, + 109, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 16, + "sent": "Another challenge will be to find out where IDA is produced in the plant and what causes it to accumulate in specific places in preparation for organ shedding.", + "section": "ABSTRACT", + "ner": [ + [ + 44, + 47, + "IDA", + "protein" + ], + [ + 67, + 72, + "plant", + "taxonomy_domain" + ] + ] + }, + { + "sid": 17, + "sent": "The HAESA ectodomain folds into a superhelical assembly of 21 leucine-rich repeats.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "HAESA", + "protein" + ], + [ + 10, + 20, + "ectodomain", + "structure_element" + ], + [ + 34, + 55, + "superhelical assembly", + "structure_element" + ], + [ + 62, + 82, + "leucine-rich repeats", + "structure_element" + ] + ] + }, + { + "sid": 18, + "sent": "(A) SDS PAGE analysis of the purified Arabidopsis thaliana HAESA ectodomain (residues 20\u2013620) obtained by secreted expression in insect cells.", + "section": "FIG", + "ner": [ + [ + 4, + 12, + "SDS PAGE", + "experimental_method" + ], + [ + 38, + 58, + "Arabidopsis thaliana", + "species" + ], + [ + 59, + 64, + "HAESA", + "protein" + ], + [ + 65, + 75, + "ectodomain", + "structure_element" + ], + [ + 86, + 92, + "20\u2013620", + "residue_range" + ], + [ + 106, + 141, + "secreted expression in insect cells", + "experimental_method" + ] + ] + }, + { + "sid": 19, + "sent": "The calculated molecular mass is 65.7 kDa, the actual molecular mass obtained by mass spectrometry is 74,896 Da, accounting for the N-glycans. (B) Ribbon diagrams showing front (left panel) and side views (right panel) of the isolated HAESA LRR domain.", + "section": "FIG", + "ner": [ + [ + 81, + 98, + "mass spectrometry", + "experimental_method" + ], + [ + 132, + 141, + "N-glycans", + "chemical" + ], + [ + 235, + 240, + "HAESA", + "protein" + ], + [ + 241, + 251, + "LRR domain", + "structure_element" + ] + ] + }, + { + "sid": 20, + "sent": "The N- (residues 20\u201388) and C-terminal (residues 593\u2013615) capping domains are shown in yellow, the central 21 LRR motifs are in blue and disulphide bonds are highlighted in green (in bonds representation). (C) Structure based sequence alignment of the 21 leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison.", + "section": "FIG", + "ner": [ + [ + 17, + 22, + "20\u201388", + "residue_range" + ], + [ + 49, + 56, + "593\u2013615", + "residue_range" + ], + [ + 58, + 73, + "capping domains", + "structure_element" + ], + [ + 110, + 120, + "LRR motifs", + "structure_element" + ], + [ + 137, + 153, + "disulphide bonds", + "ptm" + ], + [ + 210, + 244, + "Structure based sequence alignment", + "experimental_method" + ], + [ + 255, + 275, + "leucine-rich repeats", + "structure_element" + ], + [ + 279, + 284, + "HAESA", + "protein" + ], + [ + 294, + 299, + "plant", + "taxonomy_domain" + ], + [ + 300, + 303, + "LRR", + "structure_element" + ] + ] + }, + { + "sid": 21, + "sent": "Conserved hydrophobic residues are shaded in gray, N-glycosylation sites visible in our structures are highlighted in blue, cysteine residues involved in disulphide bridge formation in green. (D) Asn-linked glycans mask the N-terminal portion of the HAESA ectodomain.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Conserved", + "protein_state" + ], + [ + 10, + 21, + "hydrophobic", + "protein_state" + ], + [ + 22, + 30, + "residues", + "structure_element" + ], + [ + 51, + 72, + "N-glycosylation sites", + "site" + ], + [ + 88, + 98, + "structures", + "evidence" + ], + [ + 124, + 132, + "cysteine", + "residue_name" + ], + [ + 154, + 171, + "disulphide bridge", + "ptm" + ], + [ + 196, + 214, + "Asn-linked glycans", + "ptm" + ], + [ + 250, + 255, + "HAESA", + "protein" + ], + [ + 256, + 266, + "ectodomain", + "structure_element" + ] + ] + }, + { + "sid": 22, + "sent": "Oligomannose core structures (containing two N-actylglucosamines and three terminal mannose units) as found in Trichoplusia ni cells and in plants were modeled onto the seven glycosylation sites observed in our HAESA structures, to visualize the surface areas potentially not masked by carbohydrate.", + "section": "FIG", + "ner": [ + [ + 0, + 12, + "Oligomannose", + "chemical" + ], + [ + 45, + 64, + "N-actylglucosamines", + "chemical" + ], + [ + 84, + 91, + "mannose", + "chemical" + ], + [ + 111, + 126, + "Trichoplusia ni", + "species" + ], + [ + 140, + 146, + "plants", + "taxonomy_domain" + ], + [ + 175, + 194, + "glycosylation sites", + "site" + ], + [ + 211, + 216, + "HAESA", + "protein" + ], + [ + 217, + 227, + "structures", + "evidence" + ], + [ + 286, + 298, + "carbohydrate", + "chemical" + ] + ] + }, + { + "sid": 23, + "sent": "The HAESA ectodomain is shown in blue (in surface representation), the glycan structures are shown in yellow.", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "HAESA", + "protein" + ], + [ + 10, + 20, + "ectodomain", + "structure_element" + ], + [ + 71, + 77, + "glycan", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "Hydrophobic contacts and a hydrogen-bond network mediate the interaction between HAESA and the peptide hormone IDA.", + "section": "FIG", + "ner": [ + [ + 0, + 20, + "Hydrophobic contacts", + "bond_interaction" + ], + [ + 27, + 48, + "hydrogen-bond network", + "site" + ], + [ + 81, + 86, + "HAESA", + "protein" + ], + [ + 95, + 110, + "peptide hormone", + "protein_type" + ], + [ + 111, + 114, + "IDA", + "protein" + ] + ] + }, + { + "sid": 25, + "sent": "(A) Details of the IDA binding pocket.", + "section": "FIG", + "ner": [ + [ + 19, + 37, + "IDA binding pocket", + "site" + ] + ] + }, + { + "sid": 26, + "sent": "HAESA is shown in blue (ribbon diagram), the C-terminal Arg-His-Asn motif (left panel), the central Hyp anchor (center) and the N-terminal Pro-rich motif in IDA (right panel) are shown in yellow (in bonds representation).", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "HAESA", + "protein" + ], + [ + 56, + 73, + "Arg-His-Asn motif", + "structure_element" + ], + [ + 100, + 110, + "Hyp anchor", + "structure_element" + ], + [ + 139, + 153, + "Pro-rich motif", + "structure_element" + ], + [ + 157, + 160, + "IDA", + "protein" + ] + ] + }, + { + "sid": 27, + "sent": "HAESA interface residues are shown as sticks, selected hydrogen bond interactions are denoted as dotted lines (in magenta). (B) View of the complete IDA (in bonds representation, in yellow) binding pocket in HAESA (surface view, in blue).", + "section": "FIG", + "ner": [ + [ + 0, + 24, + "HAESA interface residues", + "site" + ], + [ + 55, + 81, + "hydrogen bond interactions", + "bond_interaction" + ], + [ + 149, + 152, + "IDA", + "protein" + ], + [ + 190, + 204, + "binding pocket", + "site" + ], + [ + 208, + 213, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 28, + "sent": "Orientation as in (A). (C) Structure based sequence alignment of leucine-rich repeats in HAESA with the plant LRR consensus sequence shown for comparison.", + "section": "FIG", + "ner": [ + [ + 27, + 61, + "Structure based sequence alignment", + "experimental_method" + ], + [ + 65, + 85, + "leucine-rich repeats", + "structure_element" + ], + [ + 89, + 94, + "HAESA", + "protein" + ], + [ + 104, + 109, + "plant", + "taxonomy_domain" + ], + [ + 110, + 113, + "LRR", + "structure_element" + ], + [ + 114, + 132, + "consensus sequence", + "evidence" + ] + ] + }, + { + "sid": 29, + "sent": "Residues mediating hydrophobic interactions with the IDA peptide are highlighted in blue, residues contributing to hydrogen bond interactions and/or salt bridges are shown in red.", + "section": "FIG", + "ner": [ + [ + 19, + 43, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 53, + 64, + "IDA peptide", + "chemical" + ], + [ + 115, + 141, + "hydrogen bond interactions", + "bond_interaction" + ], + [ + 149, + 161, + "salt bridges", + "bond_interaction" + ] + ] + }, + { + "sid": 30, + "sent": "The IDA binding pocket covers LRRs 2\u201314 and all residues originate from the inner surface of the HAESA superhelix.", + "section": "FIG", + "ner": [ + [ + 4, + 22, + "IDA binding pocket", + "site" + ], + [ + 30, + 39, + "LRRs 2\u201314", + "structure_element" + ], + [ + 97, + 102, + "HAESA", + "protein" + ], + [ + 103, + 113, + "superhelix", + "structure_element" + ] + ] + }, + { + "sid": 31, + "sent": "The IDA-HAESA and SERK1-HAESA complex interfaces are conserved among HAESA and HAESA-like\u00a0proteins from different plant species.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "IDA-HAESA", + "complex_assembly" + ], + [ + 18, + 29, + "SERK1-HAESA", + "complex_assembly" + ], + [ + 38, + 48, + "interfaces", + "site" + ], + [ + 53, + 62, + "conserved", + "protein_state" + ], + [ + 69, + 74, + "HAESA", + "protein" + ], + [ + 79, + 98, + "HAESA-like\u00a0proteins", + "protein_type" + ], + [ + 114, + 119, + "plant", + "taxonomy_domain" + ] + ] + }, + { + "sid": 32, + "sent": "Structure-based sequence alignment of the HAESA family members: Arabidopsis thaliana HAESA (Uniprot (http://www.uniprot.org) ID P47735), Arabidopsis thaliana HSL2 (Uniprot ID C0LGX3), Capsella rubella HAESA (Uniprot ID R0F2U6), Citrus clementina HSL2 (Uniprot ID V4U227), Vitis vinifera HAESA (Uniprot ID F6HM39).", + "section": "FIG", + "ner": [ + [ + 0, + 34, + "Structure-based sequence alignment", + "experimental_method" + ], + [ + 42, + 62, + "HAESA family members", + "protein_type" + ], + [ + 64, + 84, + "Arabidopsis thaliana", + "species" + ], + [ + 85, + 90, + "HAESA", + "protein" + ], + [ + 137, + 157, + "Arabidopsis thaliana", + "species" + ], + [ + 158, + 162, + "HSL2", + "protein" + ], + [ + 184, + 200, + "Capsella rubella", + "species" + ], + [ + 201, + 206, + "HAESA", + "protein" + ], + [ + 228, + 245, + "Citrus clementina", + "species" + ], + [ + 246, + 250, + "HSL2", + "protein" + ], + [ + 272, + 286, + "Vitis vinifera", + "species" + ], + [ + 287, + 292, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 33, + "sent": "The alignment includes a secondary structure assignment calculated with the program DSSP and colored according to Figure 1, with the N- and C-terminal caps and the 21 LRR motifs indicated in orange and blue, respectively.", + "section": "FIG", + "ner": [ + [ + 151, + 155, + "caps", + "structure_element" + ], + [ + 167, + 177, + "LRR motifs", + "structure_element" + ] + ] + }, + { + "sid": 34, + "sent": "Cysteine residues engaged in disulphide bonds are depicted in green.", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "Cysteine", + "residue_name" + ], + [ + 29, + 45, + "disulphide bonds", + "ptm" + ] + ] + }, + { + "sid": 35, + "sent": "HAESA residues interacting with the IDA peptide and/or the SERK1 co-receptor kinase ectodomain are highlighted in blue and orange, respectively.", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "HAESA", + "protein" + ], + [ + 36, + 47, + "IDA peptide", + "chemical" + ], + [ + 59, + 64, + "SERK1", + "protein" + ], + [ + 65, + 83, + "co-receptor kinase", + "protein_type" + ], + [ + 84, + 94, + "ectodomain", + "structure_element" + ] + ] + }, + { + "sid": 36, + "sent": "The peptide hormone IDA binds to the HAESA LRR ectodomain.", + "section": "FIG", + "ner": [ + [ + 4, + 19, + "peptide hormone", + "protein_type" + ], + [ + 20, + 23, + "IDA", + "protein" + ], + [ + 37, + 42, + "HAESA", + "protein" + ], + [ + 43, + 57, + "LRR ectodomain", + "structure_element" + ] + ] + }, + { + "sid": 37, + "sent": "(A) Multiple sequence alignment of selected IDA family members.", + "section": "FIG", + "ner": [ + [ + 4, + 31, + "Multiple sequence alignment", + "experimental_method" + ], + [ + 44, + 62, + "IDA family members", + "protein_type" + ] + ] + }, + { + "sid": 38, + "sent": "The conserved PIP motif is highlighted in yellow, the central Hyp in blue.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "conserved", + "protein_state" + ], + [ + 14, + 23, + "PIP motif", + "structure_element" + ], + [ + 62, + 65, + "Hyp", + "residue_name" + ] + ] + }, + { + "sid": 39, + "sent": "The PKGV motif present in our N-terminally extended IDA peptide is highlighted in red. (B) Isothermal titration calorimetry of the HAESA ectodomain vs. IDA and including the synthetic peptide sequence.", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "PKGV motif", + "structure_element" + ], + [ + 30, + 51, + "N-terminally extended", + "protein_state" + ], + [ + 52, + 63, + "IDA peptide", + "chemical" + ], + [ + 91, + 123, + "Isothermal titration calorimetry", + "experimental_method" + ], + [ + 131, + 136, + "HAESA", + "protein" + ], + [ + 137, + 147, + "ectodomain", + "structure_element" + ], + [ + 152, + 155, + "IDA", + "protein" + ], + [ + 174, + 183, + "synthetic", + "protein_state" + ], + [ + 184, + 191, + "peptide", + "chemical" + ] + ] + }, + { + "sid": 40, + "sent": "(C) Structure of the HAESA \u2013 IDA complex with HAESA shown in blue (ribbon diagram).", + "section": "FIG", + "ner": [ + [ + 21, + 32, + "HAESA \u2013 IDA", + "complex_assembly" + ], + [ + 46, + 51, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 41, + "sent": "IDA (in bonds representation, surface view included) is depicted in yellow.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "IDA", + "protein" + ] + ] + }, + { + "sid": 42, + "sent": "The peptide binding pocket covers HAESA LRRs 2\u201314. (D) Close-up view of the entire IDA (in yellow) peptide binding site in HAESA (in blue).", + "section": "FIG", + "ner": [ + [ + 4, + 26, + "peptide binding pocket", + "site" + ], + [ + 34, + 39, + "HAESA", + "protein" + ], + [ + 40, + 49, + "LRRs 2\u201314", + "structure_element" + ], + [ + 83, + 86, + "IDA", + "protein" + ], + [ + 99, + 119, + "peptide binding site", + "site" + ], + [ + 123, + 128, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 43, + "sent": "Details of the interactions between the central Hyp anchor in IDA and the C-terminal Arg-His-Asn motif with HAESA\u00a0are highlighted in (E) and (F), respectively.", + "section": "FIG", + "ner": [ + [ + 48, + 58, + "Hyp anchor", + "structure_element" + ], + [ + 62, + 65, + "IDA", + "protein" + ], + [ + 85, + 102, + "Arg-His-Asn motif", + "structure_element" + ], + [ + 108, + 113, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 44, + "sent": "Hydrogren bonds are depicted as dotted lines (in magenta), a water molecule is shown as a red sphere.", + "section": "FIG", + "ner": [ + [ + 61, + 66, + "water", + "chemical" + ] + ] + }, + { + "sid": 45, + "sent": "During their growth, development and reproduction plants use cell separation processes to detach no-longer required, damaged or senescent organs.", + "section": "INTRO", + "ner": [ + [ + 50, + 56, + "plants", + "taxonomy_domain" + ] + ] + }, + { + "sid": 46, + "sent": "Abscission of floral organs in Arabidopsis is a model system to study these cell separation processes in molecular detail.", + "section": "INTRO", + "ner": [ + [ + 31, + 42, + "Arabidopsis", + "taxonomy_domain" + ] + ] + }, + { + "sid": 47, + "sent": "The LRR-RKs HAESA (greek: to adhere to) and HAESA-LIKE 2 (HSL2) redundantly control floral abscission.", + "section": "INTRO", + "ner": [ + [ + 4, + 11, + "LRR-RKs", + "structure_element" + ], + [ + 12, + 17, + "HAESA", + "protein" + ], + [ + 44, + 56, + "HAESA-LIKE 2", + "protein" + ], + [ + 58, + 62, + "HSL2", + "protein" + ] + ] + }, + { + "sid": 48, + "sent": "Loss-of-function of the secreted small protein INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) causes floral organs to remain attached while its over-expression leads to premature shedding.", + "section": "INTRO", + "ner": [ + [ + 47, + 84, + "INFLORESCENCE DEFICIENT IN ABSCISSION", + "protein" + ], + [ + 86, + 89, + "IDA", + "protein" + ] + ] + }, + { + "sid": 49, + "sent": "Full-length IDA is proteolytically processed and a conserved stretch of 20 amino-acids (termed EPIP) can rescue the IDA loss-of-function phenotype (Figure 1A).", + "section": "INTRO", + "ner": [ + [ + 0, + 11, + "Full-length", + "protein_state" + ], + [ + 12, + 15, + "IDA", + "protein" + ], + [ + 19, + 44, + "proteolytically processed", + "ptm" + ], + [ + 51, + 60, + "conserved", + "protein_state" + ], + [ + 61, + 86, + "stretch of 20 amino-acids", + "residue_range" + ], + [ + 95, + 99, + "EPIP", + "structure_element" + ], + [ + 116, + 119, + "IDA", + "protein" + ] + ] + }, + { + "sid": 50, + "sent": "It has been demonstrated that a dodecamer peptide within EPIP is able to activate HAESA and\u00a0HSL2 in transient assays in tobacco cells.", + "section": "INTRO", + "ner": [ + [ + 32, + 41, + "dodecamer", + "structure_element" + ], + [ + 42, + 49, + "peptide", + "chemical" + ], + [ + 57, + 61, + "EPIP", + "structure_element" + ], + [ + 82, + 87, + "HAESA", + "protein" + ], + [ + 92, + 96, + "HSL2", + "protein" + ], + [ + 100, + 116, + "transient assays", + "experimental_method" + ], + [ + 120, + 127, + "tobacco", + "taxonomy_domain" + ] + ] + }, + { + "sid": 51, + "sent": "This sequence motif is highly conserved among IDA family members (IDA-LIKE PROTEINS, IDLs) and contains a central Pro residue, presumed to be post-translationally modified to hydroxyproline (Hyp; Figure 1A).", + "section": "INTRO", + "ner": [ + [ + 0, + 19, + "This sequence motif", + "structure_element" + ], + [ + 23, + 39, + "highly conserved", + "protein_state" + ], + [ + 46, + 64, + "IDA family members", + "protein_type" + ], + [ + 66, + 83, + "IDA-LIKE PROTEINS", + "protein_type" + ], + [ + 85, + 89, + "IDLs", + "protein_type" + ], + [ + 114, + 117, + "Pro", + "residue_name" + ], + [ + 142, + 171, + "post-translationally modified", + "protein_state" + ], + [ + 175, + 189, + "hydroxyproline", + "residue_name" + ], + [ + 191, + 194, + "Hyp", + "residue_name" + ] + ] + }, + { + "sid": 52, + "sent": "The available genetic and biochemical evidence suggests that IDA and HAESA together control floral abscission, but it is poorly understood if IDA is directly sensed by the receptor kinase HAESA and how IDA binding at the cell surface would activate the receptor.", + "section": "INTRO", + "ner": [ + [ + 61, + 64, + "IDA", + "protein" + ], + [ + 69, + 74, + "HAESA", + "protein" + ], + [ + 142, + 145, + "IDA", + "protein" + ], + [ + 172, + 187, + "receptor kinase", + "protein_type" + ], + [ + 188, + 193, + "HAESA", + "protein" + ], + [ + 202, + 205, + "IDA", + "protein" + ] + ] + }, + { + "sid": 53, + "sent": "IDA directly binds to the LRR domain of HAESA", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "IDA", + "protein" + ], + [ + 26, + 36, + "LRR domain", + "structure_element" + ], + [ + 40, + 45, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 54, + "sent": "Active IDA-family peptide hormones are hydroxyprolinated dodecamers.", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "Active", + "protein_state" + ], + [ + 7, + 34, + "IDA-family peptide hormones", + "protein_type" + ], + [ + 39, + 56, + "hydroxyprolinated", + "protein_state" + ], + [ + 57, + 67, + "dodecamers", + "structure_element" + ] + ] + }, + { + "sid": 55, + "sent": "Close-up views of (A) IDA, (B) the N-terminally extended PKGV-IDA and (C) IDL1 bound to the HAESA hormone binding pocket (in bonds representation, in yellow) and including simulated annealing 2Fo\u2013Fc omit electron density maps contoured at 1.0 \u03c3.", + "section": "FIG", + "ner": [ + [ + 22, + 25, + "IDA", + "protein" + ], + [ + 35, + 56, + "N-terminally extended", + "protein_state" + ], + [ + 57, + 65, + "PKGV-IDA", + "mutant" + ], + [ + 74, + 78, + "IDL1", + "protein" + ], + [ + 79, + 87, + "bound to", + "protein_state" + ], + [ + 92, + 97, + "HAESA", + "protein" + ], + [ + 98, + 120, + "hormone binding pocket", + "site" + ], + [ + 172, + 191, + "simulated annealing", + "experimental_method" + ], + [ + 192, + 225, + "2Fo\u2013Fc omit electron density maps", + "evidence" + ] + ] + }, + { + "sid": 56, + "sent": "Note that Pro58IDA and Leu67IDA are the first residues defined by electron density when bound to the HAESA ectodomain. (D) Table summaries for equilibrium dissociation constants (Kd), binding enthalpies (\u0394H), binding entropies (\u0394S) and stoichoimetries (N) for different IDA peptides binding to the HAESA ectodomain ( \u00b1 fitting errors; n.d.", + "section": "FIG", + "ner": [ + [ + 10, + 15, + "Pro58", + "residue_name_number" + ], + [ + 15, + 18, + "IDA", + "protein" + ], + [ + 23, + 28, + "Leu67", + "residue_name_number" + ], + [ + 28, + 31, + "IDA", + "protein" + ], + [ + 66, + 82, + "electron density", + "evidence" + ], + [ + 88, + 96, + "bound to", + "protein_state" + ], + [ + 101, + 106, + "HAESA", + "protein" + ], + [ + 107, + 117, + "ectodomain", + "structure_element" + ], + [ + 143, + 177, + "equilibrium dissociation constants", + "evidence" + ], + [ + 179, + 181, + "Kd", + "evidence" + ], + [ + 184, + 202, + "binding enthalpies", + "evidence" + ], + [ + 204, + 206, + "\u0394H", + "evidence" + ], + [ + 209, + 226, + "binding entropies", + "evidence" + ], + [ + 228, + 230, + "\u0394S", + "evidence" + ], + [ + 270, + 282, + "IDA peptides", + "chemical" + ], + [ + 298, + 303, + "HAESA", + "protein" + ], + [ + 304, + 314, + "ectodomain", + "structure_element" + ] + ] + }, + { + "sid": 57, + "sent": "no detectable binding). (E) Structural superposition of the active IDA (in bonds representation, in gray) and IDL1 peptide (in yellow) hormones bound to the HAESA ectodomain.", + "section": "FIG", + "ner": [ + [ + 28, + 52, + "Structural superposition", + "experimental_method" + ], + [ + 60, + 66, + "active", + "protein_state" + ], + [ + 67, + 70, + "IDA", + "protein" + ], + [ + 110, + 122, + "IDL1 peptide", + "chemical" + ], + [ + 144, + 152, + "bound to", + "protein_state" + ], + [ + 157, + 162, + "HAESA", + "protein" + ], + [ + 163, + 173, + "ectodomain", + "structure_element" + ] + ] + }, + { + "sid": 58, + "sent": "Root mean square deviation (r.m.s.d.) is 1.0 \u00c5 comparing 100 corresponding atoms.", + "section": "FIG", + "ner": [ + [ + 0, + 26, + "Root mean square deviation", + "evidence" + ], + [ + 28, + 36, + "r.m.s.d.", + "evidence" + ] + ] + }, + { + "sid": 59, + "sent": "The receptor kinase SERK1 acts as a HAESA co-receptor and promotes high-affinity IDA sensing.", + "section": "FIG", + "ner": [ + [ + 4, + 19, + "receptor kinase", + "protein_type" + ], + [ + 20, + 25, + "SERK1", + "protein" + ], + [ + 36, + 53, + "HAESA co-receptor", + "protein_type" + ], + [ + 81, + 84, + "IDA", + "protein" + ] + ] + }, + { + "sid": 60, + "sent": "(A) Petal break-strength assays measure the force (expressed in gram equivalents) required to remove the petals from the flower of serk mutant plants compared to haesa/hsl2 mutant and Col-0 wild-type flowers.", + "section": "FIG", + "ner": [ + [ + 4, + 31, + "Petal break-strength assays", + "experimental_method" + ], + [ + 131, + 135, + "serk", + "gene" + ], + [ + 136, + 142, + "mutant", + "protein_state" + ], + [ + 143, + 149, + "plants", + "taxonomy_domain" + ], + [ + 162, + 167, + "haesa", + "gene" + ], + [ + 168, + 172, + "hsl2", + "gene" + ], + [ + 173, + 179, + "mutant", + "protein_state" + ], + [ + 190, + 199, + "wild-type", + "protein_state" + ] + ] + }, + { + "sid": 61, + "sent": "Petal break-strength was found significantly increased in almost all positions (indicated with a *) for haesa/hsl2 and serk1-1 mutant plants with respect to the Col-0 control.", + "section": "FIG", + "ner": [ + [ + 104, + 109, + "haesa", + "gene" + ], + [ + 110, + 114, + "hsl2", + "gene" + ], + [ + 119, + 126, + "serk1-1", + "gene" + ], + [ + 127, + 133, + "mutant", + "protein_state" + ], + [ + 134, + 140, + "plants", + "taxonomy_domain" + ] + ] + }, + { + "sid": 62, + "sent": "(B) Analytical size-exclusion chromatography.", + "section": "FIG", + "ner": [ + [ + 4, + 44, + "Analytical size-exclusion chromatography", + "experimental_method" + ] + ] + }, + { + "sid": 63, + "sent": "The HAESA LRR domain elutes as a monomer (black dotted line), as does the isolated SERK1 ectodomain (blue dotted line).", + "section": "FIG", + "ner": [ + [ + 4, + 9, + "HAESA", + "protein" + ], + [ + 10, + 20, + "LRR domain", + "structure_element" + ], + [ + 33, + 40, + "monomer", + "oligomeric_state" + ], + [ + 83, + 88, + "SERK1", + "protein" + ], + [ + 89, + 99, + "ectodomain", + "structure_element" + ] + ] + }, + { + "sid": 64, + "sent": "A HAESA \u2013 IDA \u2013 SERK1 complex elutes as an apparent heterodimer (red line), while a mixture of HAESA and SERK1 yields two isolated peaks that correspond to monomeric HAESA and SERK1, respectively (black line).", + "section": "FIG", + "ner": [ + [ + 2, + 21, + "HAESA \u2013 IDA \u2013 SERK1", + "complex_assembly" + ], + [ + 52, + 63, + "heterodimer", + "oligomeric_state" + ], + [ + 95, + 100, + "HAESA", + "protein" + ], + [ + 105, + 110, + "SERK1", + "protein" + ], + [ + 156, + 165, + "monomeric", + "oligomeric_state" + ], + [ + 166, + 171, + "HAESA", + "protein" + ], + [ + 176, + 181, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 65, + "sent": "Void (V0) volume and total volume (Vt) are shown, together with elution volumes for molecular mass standards (A, Thyroglobulin, 669,000 Da; B, Ferritin, 440,00 Da, C, Aldolase, 158,000 Da; D, Conalbumin, 75,000 Da; E, Ovalbumin, 44,000 Da; F, Carbonic anhydrase, 29,000 Da).", + "section": "FIG", + "ner": [ + [ + 113, + 126, + "Thyroglobulin", + "protein" + ], + [ + 143, + 151, + "Ferritin", + "protein" + ], + [ + 167, + 175, + "Aldolase", + "protein" + ], + [ + 192, + 202, + "Conalbumin", + "protein" + ], + [ + 218, + 227, + "Ovalbumin", + "protein" + ], + [ + 243, + 261, + "Carbonic anhydrase", + "protein" + ], + [ + 113, + 126, + "Thyroglobulin", + "protein" + ], + [ + 143, + 151, + "Ferritin", + "protein" + ], + [ + 167, + 175, + "Aldolase", + "protein" + ], + [ + 192, + 202, + "Conalbumin", + "protein" + ], + [ + 218, + 227, + "Ovalbumin", + "protein" + ], + [ + 243, + 261, + "Carbonic anhydrase", + "protein" + ] + ] + }, + { + "sid": 66, + "sent": "A SDS PAGE of the peak fractions is shown alongside.", + "section": "FIG", + "ner": [ + [ + 2, + 10, + "SDS PAGE", + "experimental_method" + ], + [ + 2, + 10, + "SDS PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 67, + "sent": "Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively. (C) Isothermal titration calorimetry of wild-type and Hyp64\u2192Pro IDA versus the HAESA and SERK1 ectodomains.", + "section": "FIG", + "ner": [ + [ + 9, + 14, + "HAESA", + "protein" + ], + [ + 19, + 24, + "SERK1", + "protein" + ], + [ + 64, + 96, + "Isothermal titration calorimetry", + "experimental_method" + ], + [ + 100, + 109, + "wild-type", + "protein_state" + ], + [ + 114, + 123, + "Hyp64\u2192Pro", + "ptm" + ], + [ + 124, + 127, + "IDA", + "protein" + ], + [ + 139, + 144, + "HAESA", + "protein" + ], + [ + 149, + 154, + "SERK1", + "protein" + ], + [ + 155, + 166, + "ectodomains", + "structure_element" + ] + ] + }, + { + "sid": 68, + "sent": "The titration of IDA wild-type versus the isolated HAESA ectodomain from Figure 1B is shown for comparison (red line; n.d.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "titration", + "experimental_method" + ], + [ + 17, + 20, + "IDA", + "protein" + ], + [ + 21, + 30, + "wild-type", + "protein_state" + ], + [ + 51, + 56, + "HAESA", + "protein" + ], + [ + 57, + 67, + "ectodomain", + "structure_element" + ] + ] + }, + { + "sid": 69, + "sent": "no detectable binding) (D) Analytical size-exclusion chromatography in the presence of the IDA Hyp64\u2192Pro mutant peptide reveals no complex formation between HAESA and SERK1 ectodomains.", + "section": "FIG", + "ner": [ + [ + 27, + 67, + "Analytical size-exclusion chromatography", + "experimental_method" + ], + [ + 75, + 86, + "presence of", + "protein_state" + ], + [ + 91, + 94, + "IDA", + "protein" + ], + [ + 95, + 104, + "Hyp64\u2192Pro", + "ptm" + ], + [ + 105, + 111, + "mutant", + "protein_state" + ], + [ + 112, + 119, + "peptide", + "chemical" + ], + [ + 157, + 162, + "HAESA", + "protein" + ], + [ + 167, + 172, + "SERK1", + "protein" + ], + [ + 173, + 184, + "ectodomains", + "structure_element" + ] + ] + }, + { + "sid": 70, + "sent": "(E) In vitro kinase assays of the HAESA and SERK1 kinase domains.", + "section": "FIG", + "ner": [ + [ + 4, + 26, + "In vitro kinase assays", + "experimental_method" + ], + [ + 34, + 39, + "HAESA", + "protein" + ], + [ + 44, + 49, + "SERK1", + "protein" + ], + [ + 50, + 64, + "kinase domains", + "structure_element" + ] + ] + }, + { + "sid": 71, + "sent": "Wild-type HAESA and SERK1 kinase domains (KDs) exhibit auto-phosphorylation activities (lanes 1 + 3).", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Wild-type", + "protein_state" + ], + [ + 10, + 15, + "HAESA", + "protein" + ], + [ + 20, + 25, + "SERK1", + "protein" + ], + [ + 26, + 40, + "kinase domains", + "structure_element" + ], + [ + 42, + 45, + "KDs", + "structure_element" + ] + ] + }, + { + "sid": 72, + "sent": "Mutant (m)\u00a0versions, which carry point mutations in their active sites (Asp837HAESA\u2192Asn, Asp447SERK1\u2192Asn) possess no autophosphorylation activity (lanes 2+4).", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "Mutant", + "protein_state" + ], + [ + 33, + 48, + "point mutations", + "experimental_method" + ], + [ + 58, + 70, + "active sites", + "site" + ], + [ + 72, + 87, + "Asp837HAESA\u2192Asn", + "mutant" + ], + [ + 89, + 104, + "Asp447SERK1\u2192Asn", + "mutant" + ] + ] + }, + { + "sid": 73, + "sent": "Transphosphorylation activity from the active kinase to the mutated form can be observed in both directions (lanes 5+6).", + "section": "FIG", + "ner": [ + [ + 39, + 45, + "active", + "protein_state" + ], + [ + 60, + 67, + "mutated", + "protein_state" + ] + ] + }, + { + "sid": 74, + "sent": "We purified the HAESA ectodomain (residues 20\u2013620) from baculovirus-infected insect cells (Figure 1\u2014figure supplement 1A, see Materials\u00a0and\u00a0methods) and quantified the interaction of the ~75\u2009kDa glycoprotein with synthetic IDA peptides using isothermal titration calorimetry (ITC).", + "section": "RESULTS", + "ner": [ + [ + 3, + 11, + "purified", + "experimental_method" + ], + [ + 16, + 21, + "HAESA", + "protein" + ], + [ + 22, + 32, + "ectodomain", + "structure_element" + ], + [ + 43, + 49, + "20\u2013620", + "residue_range" + ], + [ + 56, + 89, + "baculovirus-infected insect cells", + "experimental_method" + ], + [ + 195, + 207, + "glycoprotein", + "protein_type" + ], + [ + 213, + 222, + "synthetic", + "protein_state" + ], + [ + 223, + 235, + "IDA peptides", + "chemical" + ], + [ + 242, + 274, + "isothermal titration calorimetry", + "experimental_method" + ], + [ + 276, + 279, + "ITC", + "experimental_method" + ] + ] + }, + { + "sid": 75, + "sent": "A Hyp-modified dodecamer comprising the highly conserved PIP motif in IDA (Figure 1A) interacts with HAESA with 1:1 stoichiometry (N) and with a dissociation constant (Kd) of ~20\u2009\u03bcM (Figure 1B).", + "section": "RESULTS", + "ner": [ + [ + 2, + 14, + "Hyp-modified", + "protein_state" + ], + [ + 15, + 24, + "dodecamer", + "structure_element" + ], + [ + 40, + 56, + "highly conserved", + "protein_state" + ], + [ + 57, + 66, + "PIP motif", + "structure_element" + ], + [ + 70, + 73, + "IDA", + "protein" + ], + [ + 101, + 106, + "HAESA", + "protein" + ], + [ + 145, + 166, + "dissociation constant", + "evidence" + ], + [ + 168, + 170, + "Kd", + "evidence" + ] + ] + }, + { + "sid": 76, + "sent": "We next determined crystal structures of the apo HAESA ectodomain and of a HAESA-IDA complex, at 1.74 and 1.86\u2009\u00c5 resolution, respectively (Figure 1C; Figure 1\u2014figure supplement 1B\u2013D; Tables 1,2).", + "section": "RESULTS", + "ner": [ + [ + 19, + 37, + "crystal structures", + "evidence" + ], + [ + 45, + 48, + "apo", + "protein_state" + ], + [ + 49, + 54, + "HAESA", + "protein" + ], + [ + 55, + 65, + "ectodomain", + "structure_element" + ], + [ + 75, + 84, + "HAESA-IDA", + "complex_assembly" + ] + ] + }, + { + "sid": 77, + "sent": "IDA binds in a completely extended conformation along the inner surface of the HAESA ectodomain, covering LRRs 2\u201314 (Figure 1C,D, Figure 1\u2014figure supplement 2).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "IDA", + "protein" + ], + [ + 15, + 47, + "completely extended conformation", + "protein_state" + ], + [ + 79, + 84, + "HAESA", + "protein" + ], + [ + 85, + 95, + "ectodomain", + "structure_element" + ], + [ + 106, + 115, + "LRRs 2\u201314", + "structure_element" + ] + ] + }, + { + "sid": 78, + "sent": "The central Hyp64IDA is buried in a specific pocket formed by HAESA LRRs 8\u201310, with its hydroxyl group establishing hydrogen bonds with the strictly conserved Glu266HAESA and with a water molecule, which in turn is coordinated by the main chain oxygens of Phe289HAESA and Ser311HAESA (Figure 1E; Figure 1\u2014figure supplement 3).", + "section": "RESULTS", + "ner": [ + [ + 12, + 17, + "Hyp64", + "ptm" + ], + [ + 17, + 20, + "IDA", + "protein" + ], + [ + 45, + 51, + "pocket", + "site" + ], + [ + 62, + 67, + "HAESA", + "protein" + ], + [ + 68, + 77, + "LRRs 8\u201310", + "structure_element" + ], + [ + 116, + 130, + "hydrogen bonds", + "bond_interaction" + ], + [ + 140, + 158, + "strictly conserved", + "protein_state" + ], + [ + 159, + 165, + "Glu266", + "residue_name_number" + ], + [ + 165, + 170, + "HAESA", + "protein" + ], + [ + 182, + 187, + "water", + "chemical" + ], + [ + 256, + 262, + "Phe289", + "residue_name_number" + ], + [ + 262, + 267, + "HAESA", + "protein" + ], + [ + 272, + 278, + "Ser311", + "residue_name_number" + ], + [ + 278, + 283, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 79, + "sent": "The restricted size of the Hyp pocket suggests that IDA does not require arabinosylation of Hyp64IDA for activity in vivo, a modification that has been reported for Hyp residues in plant CLE peptide hormones.", + "section": "RESULTS", + "ner": [ + [ + 27, + 37, + "Hyp pocket", + "site" + ], + [ + 52, + 55, + "IDA", + "protein" + ], + [ + 73, + 88, + "arabinosylation", + "ptm" + ], + [ + 92, + 97, + "Hyp64", + "ptm" + ], + [ + 97, + 100, + "IDA", + "protein" + ], + [ + 165, + 168, + "Hyp", + "residue_name" + ], + [ + 181, + 186, + "plant", + "taxonomy_domain" + ], + [ + 187, + 207, + "CLE peptide hormones", + "protein_type" + ] + ] + }, + { + "sid": 80, + "sent": "The C-terminal Arg-His-Asn motif in IDA maps to a cavity formed by HAESA LRRs 11\u201314 (Figure 1D,F).", + "section": "RESULTS", + "ner": [ + [ + 15, + 32, + "Arg-His-Asn motif", + "structure_element" + ], + [ + 36, + 39, + "IDA", + "protein" + ], + [ + 50, + 56, + "cavity", + "site" + ], + [ + 67, + 72, + "HAESA", + "protein" + ], + [ + 73, + 83, + "LRRs 11\u201314", + "structure_element" + ] + ] + }, + { + "sid": 81, + "sent": "The COO- group of Asn69IDA is in direct contact with Arg407HAESA and Arg409HAESA and HAESA cannot bind a C-terminally extended IDA-SFVN peptide (Figures 1D,F, 2D).", + "section": "RESULTS", + "ner": [ + [ + 18, + 23, + "Asn69", + "residue_name_number" + ], + [ + 23, + 26, + "IDA", + "protein" + ], + [ + 53, + 59, + "Arg407", + "residue_name_number" + ], + [ + 59, + 64, + "HAESA", + "protein" + ], + [ + 69, + 75, + "Arg409", + "residue_name_number" + ], + [ + 75, + 80, + "HAESA", + "protein" + ], + [ + 85, + 90, + "HAESA", + "protein" + ], + [ + 105, + 126, + "C-terminally extended", + "protein_state" + ], + [ + 127, + 135, + "IDA-SFVN", + "mutant" + ] + ] + }, + { + "sid": 82, + "sent": "This suggests that the conserved Asn69IDA may constitute the very C-terminus of the mature IDA peptide in planta and that active IDA is generated by proteolytic processing from a longer pre-protein.", + "section": "RESULTS", + "ner": [ + [ + 23, + 32, + "conserved", + "protein_state" + ], + [ + 33, + 38, + "Asn69", + "residue_name_number" + ], + [ + 38, + 41, + "IDA", + "protein" + ], + [ + 84, + 90, + "mature", + "protein_state" + ], + [ + 91, + 102, + "IDA peptide", + "chemical" + ], + [ + 106, + 112, + "planta", + "taxonomy_domain" + ], + [ + 122, + 128, + "active", + "protein_state" + ], + [ + 129, + 132, + "IDA", + "protein" + ] + ] + }, + { + "sid": 83, + "sent": "Mutation of Arg417HSL2 (which corresponds to Arg409HAESA) causes a loss-of-function phenotype in HSL2, which indicates that the peptide binding pockets in different HAESA receptors have common structural and sequence features.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Mutation", + "experimental_method" + ], + [ + 12, + 18, + "Arg417", + "residue_name_number" + ], + [ + 18, + 22, + "HSL2", + "protein" + ], + [ + 45, + 51, + "Arg409", + "residue_name_number" + ], + [ + 51, + 56, + "HAESA", + "protein" + ], + [ + 97, + 101, + "HSL2", + "protein" + ], + [ + 128, + 151, + "peptide binding pockets", + "site" + ], + [ + 165, + 180, + "HAESA receptors", + "protein_type" + ] + ] + }, + { + "sid": 84, + "sent": "Indeed, we find many of the residues contributing to the formation of the IDA binding surface in HAESA to be conserved in HSL2 and in\u00a0other HAESA-type receptors in different plant species (Figure 1\u2014figure supplement 3).", + "section": "RESULTS", + "ner": [ + [ + 74, + 93, + "IDA binding surface", + "site" + ], + [ + 97, + 102, + "HAESA", + "protein" + ], + [ + 109, + 118, + "conserved", + "protein_state" + ], + [ + 122, + 126, + "HSL2", + "protein" + ], + [ + 140, + 160, + "HAESA-type receptors", + "protein_type" + ], + [ + 174, + 179, + "plant", + "taxonomy_domain" + ] + ] + }, + { + "sid": 85, + "sent": "A N-terminal Pro-rich motif in IDA makes contacts with\u00a0LRRs 2\u20136 of the receptor (Figure 1D, Figure 1\u2014figure supplement 2A\u2013C).", + "section": "RESULTS", + "ner": [ + [ + 13, + 27, + "Pro-rich motif", + "structure_element" + ], + [ + 31, + 34, + "IDA", + "protein" + ], + [ + 55, + 63, + "LRRs 2\u20136", + "structure_element" + ] + ] + }, + { + "sid": 86, + "sent": "Other hydrophobic and polar interactions are mediated by Ser62IDA, Ser65IDA and by backbone atoms along the IDA peptide (Figure 1D, Figure 1\u2014figure supplement 2A\u2013C).", + "section": "RESULTS", + "ner": [ + [ + 6, + 40, + "hydrophobic and polar interactions", + "bond_interaction" + ], + [ + 57, + 62, + "Ser62", + "residue_name_number" + ], + [ + 62, + 65, + "IDA", + "protein" + ], + [ + 67, + 72, + "Ser65", + "residue_name_number" + ], + [ + 72, + 75, + "IDA", + "protein" + ], + [ + 108, + 119, + "IDA peptide", + "chemical" + ] + ] + }, + { + "sid": 87, + "sent": "HAESA specifically senses IDA-family dodecamer peptides", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "HAESA", + "protein" + ], + [ + 26, + 36, + "IDA-family", + "protein_type" + ], + [ + 37, + 46, + "dodecamer", + "structure_element" + ], + [ + 47, + 55, + "peptides", + "chemical" + ] + ] + }, + { + "sid": 88, + "sent": "We next investigated whether HAESA binds N-terminally extended versions of IDA.", + "section": "RESULTS", + "ner": [ + [ + 29, + 34, + "HAESA", + "protein" + ], + [ + 41, + 62, + "N-terminally extended", + "protein_state" + ], + [ + 75, + 78, + "IDA", + "protein" + ] + ] + }, + { + "sid": 89, + "sent": "We obtained a structure of HAESA in complex with a PKGV-IDA peptide at 1.94\u2009\u00c5 resolution (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 14, + 23, + "structure", + "evidence" + ], + [ + 27, + 32, + "HAESA", + "protein" + ], + [ + 33, + 48, + "in complex with", + "protein_state" + ], + [ + 51, + 59, + "PKGV-IDA", + "mutant" + ], + [ + 60, + 67, + "peptide", + "chemical" + ] + ] + }, + { + "sid": 90, + "sent": "In this structure, no additional electron density accounts for the PKGV motif at the IDA N-terminus (Figure 2A,B).", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 33, + 49, + "electron density", + "evidence" + ], + [ + 67, + 77, + "PKGV motif", + "structure_element" + ], + [ + 85, + 88, + "IDA", + "protein" + ] + ] + }, + { + "sid": 91, + "sent": "Consistently, PKGV-IDA and IDA have similar binding affinities in our ITC assays, further indicating that HAESA senses a dodecamer peptide comprising residues 58-69IDA (Figure 2D).", + "section": "RESULTS", + "ner": [ + [ + 14, + 22, + "PKGV-IDA", + "mutant" + ], + [ + 27, + 30, + "IDA", + "protein" + ], + [ + 44, + 62, + "binding affinities", + "evidence" + ], + [ + 70, + 80, + "ITC assays", + "experimental_method" + ], + [ + 106, + 111, + "HAESA", + "protein" + ], + [ + 121, + 130, + "dodecamer", + "structure_element" + ], + [ + 131, + 138, + "peptide", + "chemical" + ], + [ + 159, + 164, + "58-69", + "residue_range" + ], + [ + 164, + 167, + "IDA", + "protein" + ] + ] + }, + { + "sid": 92, + "sent": "We next tested if HAESA binds other IDA peptide family members.", + "section": "RESULTS", + "ner": [ + [ + 18, + 23, + "HAESA", + "protein" + ], + [ + 36, + 62, + "IDA peptide family members", + "chemical" + ] + ] + }, + { + "sid": 93, + "sent": "IDL1, which can rescue IDA loss-of-function mutants when introduced in abscission zone cells, can also be sensed by HAESA, albeit with lower affinity (Figure 2D).", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "IDL1", + "protein" + ], + [ + 23, + 26, + "IDA", + "protein" + ], + [ + 116, + 121, + "HAESA", + "protein" + ], + [ + 141, + 149, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 94, + "sent": "A 2.56\u2009\u00c5 co-crystal structure with IDL1 reveals that different IDA family members use a common binding mode to interact with HAESA-type receptors (Figure 2A\u2013C,E, Table 2).", + "section": "RESULTS", + "ner": [ + [ + 9, + 29, + "co-crystal structure", + "evidence" + ], + [ + 35, + 39, + "IDL1", + "protein" + ], + [ + 63, + 81, + "IDA family members", + "protein_type" + ], + [ + 125, + 145, + "HAESA-type receptors", + "protein_type" + ] + ] + }, + { + "sid": 95, + "sent": "We do not detect interaction between HAESA and a synthetic peptide missing the C-terminal Asn69IDA (\u0394N69), highlighting the importance of the polar interactions between the IDA carboxy-terminus and Arg407HAESA/Arg409HAESA (Figures 1F, 2D).", + "section": "RESULTS", + "ner": [ + [ + 37, + 42, + "HAESA", + "protein" + ], + [ + 49, + 58, + "synthetic", + "protein_state" + ], + [ + 59, + 66, + "peptide", + "chemical" + ], + [ + 67, + 89, + "missing the C-terminal", + "protein_state" + ], + [ + 90, + 95, + "Asn69", + "residue_name_number" + ], + [ + 95, + 98, + "IDA", + "protein" + ], + [ + 100, + 104, + "\u0394N69", + "mutant" + ], + [ + 142, + 160, + "polar interactions", + "bond_interaction" + ], + [ + 173, + 176, + "IDA", + "protein" + ], + [ + 198, + 204, + "Arg407", + "residue_name_number" + ], + [ + 204, + 209, + "HAESA", + "protein" + ], + [ + 210, + 216, + "Arg409", + "residue_name_number" + ], + [ + 216, + 221, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 96, + "sent": "Replacing Hyp64IDA, which is common to all IDLs, with proline impairs the interaction with the receptor, as does the Lys66IDA/Arg67IDA \u2192 Ala double-mutant discussed below (Figure 1A, 2D).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Replacing", + "experimental_method" + ], + [ + 10, + 15, + "Hyp64", + "ptm" + ], + [ + 15, + 18, + "IDA", + "protein" + ], + [ + 43, + 47, + "IDLs", + "protein_type" + ], + [ + 54, + 61, + "proline", + "residue_name" + ], + [ + 117, + 140, + "Lys66IDA/Arg67IDA \u2192 Ala", + "mutant" + ], + [ + 141, + 154, + "double-mutant", + "protein_state" + ] + ] + }, + { + "sid": 97, + "sent": "Notably, HAESA can discriminate between IDLs and functionally unrelated dodecamer peptides with Hyp modifications, such as CLV3 (Figures 2D, 7).", + "section": "RESULTS", + "ner": [ + [ + 9, + 14, + "HAESA", + "protein" + ], + [ + 40, + 44, + "IDLs", + "protein_type" + ], + [ + 49, + 71, + "functionally unrelated", + "protein_state" + ], + [ + 72, + 81, + "dodecamer", + "structure_element" + ], + [ + 82, + 90, + "peptides", + "chemical" + ], + [ + 96, + 113, + "Hyp modifications", + "ptm" + ], + [ + 123, + 127, + "CLV3", + "protein" + ] + ] + }, + { + "sid": 98, + "sent": "The co-receptor kinase SERK1 allows for high-affinity IDA sensing", + "section": "RESULTS", + "ner": [ + [ + 4, + 22, + "co-receptor kinase", + "protein_type" + ], + [ + 23, + 28, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 99, + "sent": "Our binding assays reveal that IDA family\u00a0peptides are sensed by the isolated HAESA ectodomain with relatively weak binding affinities (Figures 1B, 2A\u2013D).", + "section": "RESULTS", + "ner": [ + [ + 4, + 18, + "binding assays", + "experimental_method" + ], + [ + 31, + 50, + "IDA family\u00a0peptides", + "chemical" + ], + [ + 69, + 77, + "isolated", + "protein_state" + ], + [ + 78, + 83, + "HAESA", + "protein" + ], + [ + 84, + 94, + "ectodomain", + "structure_element" + ], + [ + 116, + 134, + "binding affinities", + "evidence" + ] + ] + }, + { + "sid": 100, + "sent": "It has been recently reported that SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) are positive regulators of floral abscission and can interact with HAESA and HSL2 in an IDA-dependent manner.", + "section": "RESULTS", + "ner": [ + [ + 35, + 73, + "SOMATIC EMBRYOGENESIS RECEPTOR KINASES", + "protein_type" + ], + [ + 75, + 80, + "SERKs", + "protein_type" + ], + [ + 149, + 154, + "HAESA", + "protein" + ], + [ + 159, + 163, + "HSL2", + "protein" + ] + ] + }, + { + "sid": 101, + "sent": "As all five SERK family members appear to be expressed in the Arabidopsis abscission zone, we quantified their relative contribution to floral abscission in Arabidopsis using a petal break-strength assay.", + "section": "RESULTS", + "ner": [ + [ + 12, + 31, + "SERK family members", + "protein_type" + ], + [ + 62, + 73, + "Arabidopsis", + "taxonomy_domain" + ], + [ + 157, + 168, + "Arabidopsis", + "taxonomy_domain" + ], + [ + 177, + 203, + "petal break-strength assay", + "experimental_method" + ] + ] + }, + { + "sid": 102, + "sent": "Our experiments suggest that among the SERK family members, SERK1 is a positive regulator of floral abscission.", + "section": "RESULTS", + "ner": [ + [ + 39, + 58, + "SERK family members", + "protein_type" + ], + [ + 60, + 65, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 103, + "sent": "We found that the force required to remove the petals of serk1-1 mutants is significantly higher than that needed for wild-type plants, as previously observed for haesa/hsl2 mutants, and that floral abscission is delayed in serk1-1 (Figure 3A).", + "section": "RESULTS", + "ner": [ + [ + 57, + 64, + "serk1-1", + "gene" + ], + [ + 65, + 72, + "mutants", + "protein_state" + ], + [ + 118, + 127, + "wild-type", + "protein_state" + ], + [ + 128, + 134, + "plants", + "taxonomy_domain" + ], + [ + 163, + 168, + "haesa", + "gene" + ], + [ + 169, + 173, + "hsl2", + "gene" + ], + [ + 174, + 181, + "mutants", + "protein_state" + ], + [ + 224, + 231, + "serk1-1", + "gene" + ] + ] + }, + { + "sid": 104, + "sent": "The serk2-2, serk3-1, serk4-1 and serk5-1 mutant lines showed a petal break-strength profile not significantly different from wild-type plants.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "serk2-2", + "gene" + ], + [ + 13, + 20, + "serk3-1", + "gene" + ], + [ + 22, + 29, + "serk4-1", + "gene" + ], + [ + 34, + 41, + "serk5-1", + "gene" + ], + [ + 42, + 48, + "mutant", + "protein_state" + ], + [ + 126, + 135, + "wild-type", + "protein_state" + ], + [ + 136, + 142, + "plants", + "taxonomy_domain" + ] + ] + }, + { + "sid": 105, + "sent": "Possibly because SERKs have additional roles in plant development such as in pollen formation and brassinosteroid signaling, we found that higher-order SERK mutants exhibit pleiotropic phenotypes in the flower, rendering their analysis and comparison by quantitative petal break-strength assays difficult.", + "section": "RESULTS", + "ner": [ + [ + 17, + 22, + "SERKs", + "protein_type" + ], + [ + 254, + 294, + "quantitative petal break-strength assays", + "experimental_method" + ] + ] + }, + { + "sid": 106, + "sent": "We thus focused on analyzing the contribution of SERK1 to HAESA ligand sensing and receptor activation.", + "section": "RESULTS", + "ner": [ + [ + 49, + 54, + "SERK1", + "protein" + ], + [ + 58, + 63, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 107, + "sent": "In vitro, the LRR ectodomain of SERK1 (residues 24\u2013213) forms stable, IDA-dependent heterodimeric complexes with HAESA in size exclusion chromatography experiments (Figure 3B).", + "section": "RESULTS", + "ner": [ + [ + 14, + 28, + "LRR ectodomain", + "structure_element" + ], + [ + 32, + 37, + "SERK1", + "protein" + ], + [ + 48, + 54, + "24\u2013213", + "residue_range" + ], + [ + 62, + 68, + "stable", + "protein_state" + ], + [ + 70, + 83, + "IDA-dependent", + "protein_state" + ], + [ + 84, + 97, + "heterodimeric", + "oligomeric_state" + ], + [ + 98, + 112, + "complexes with", + "protein_state" + ], + [ + 113, + 118, + "HAESA", + "protein" + ], + [ + 122, + 151, + "size exclusion chromatography", + "experimental_method" + ] + ] + }, + { + "sid": 108, + "sent": "We next quantified the contribution of SERK1 to IDA recognition by HAESA.", + "section": "RESULTS", + "ner": [ + [ + 39, + 44, + "SERK1", + "protein" + ], + [ + 48, + 51, + "IDA", + "protein" + ], + [ + 67, + 72, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 109, + "sent": "We found that HAESA senses IDA with a ~60\u00a0fold higher binding affinity in the presence of SERK1, suggesting that SERK1 is involved in the specific recognition of the peptide hormone (Figure 3C).", + "section": "RESULTS", + "ner": [ + [ + 14, + 19, + "HAESA", + "protein" + ], + [ + 27, + 30, + "IDA", + "protein" + ], + [ + 54, + 70, + "binding affinity", + "evidence" + ], + [ + 78, + 89, + "presence of", + "protein_state" + ], + [ + 90, + 95, + "SERK1", + "protein" + ], + [ + 113, + 118, + "SERK1", + "protein" + ], + [ + 166, + 181, + "peptide hormone", + "protein_type" + ] + ] + }, + { + "sid": 110, + "sent": "We next titrated SERK1 into a solution containing only the HAESA ectodomain.", + "section": "RESULTS", + "ner": [ + [ + 8, + 16, + "titrated", + "experimental_method" + ], + [ + 17, + 22, + "SERK1", + "protein" + ], + [ + 59, + 64, + "HAESA", + "protein" + ], + [ + 65, + 75, + "ectodomain", + "structure_element" + ] + ] + }, + { + "sid": 111, + "sent": "In this case, there was no detectable interaction between receptor and co-receptor, while in the presence of IDA, SERK1 strongly binds HAESA with a dissociation constant in the mid-nanomolar range (Figure 3C).", + "section": "RESULTS", + "ner": [ + [ + 97, + 108, + "presence of", + "protein_state" + ], + [ + 109, + 112, + "IDA", + "protein" + ], + [ + 114, + 119, + "SERK1", + "protein" + ], + [ + 135, + 140, + "HAESA", + "protein" + ], + [ + 148, + 169, + "dissociation constant", + "evidence" + ] + ] + }, + { + "sid": 112, + "sent": "This suggests that IDA itself promotes receptor \u2013 co-receptor association, as previously described for the steroid hormone brassinolide and for other LRR-RK complexes.", + "section": "RESULTS", + "ner": [ + [ + 19, + 22, + "IDA", + "protein" + ], + [ + 107, + 122, + "steroid hormone", + "chemical" + ], + [ + 123, + 135, + "brassinolide", + "chemical" + ], + [ + 150, + 156, + "LRR-RK", + "complex_assembly" + ] + ] + }, + { + "sid": 113, + "sent": "Importantly, hydroxyprolination of IDA is critical for HAESA-IDA-SERK1 complex formation (Figure 3C,D).", + "section": "RESULTS", + "ner": [ + [ + 13, + 31, + "hydroxyprolination", + "ptm" + ], + [ + 35, + 38, + "IDA", + "protein" + ], + [ + 55, + 70, + "HAESA-IDA-SERK1", + "complex_assembly" + ] + ] + }, + { + "sid": 114, + "sent": "Our calorimetry experiments now reveal that SERKs may render HAESA, and potentially other receptor kinases, competent for high-affinity sensing of their cognate ligands.", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "calorimetry", + "experimental_method" + ], + [ + 44, + 49, + "SERKs", + "protein_type" + ], + [ + 61, + 66, + "HAESA", + "protein" + ], + [ + 90, + 106, + "receptor kinases", + "protein_type" + ] + ] + }, + { + "sid": 115, + "sent": "Upon IDA binding at the cell surface, the kinase domains of HAESA and SERK1, which have been shown to be active protein kinases, may interact in the cytoplasm to activate each other.", + "section": "RESULTS", + "ner": [ + [ + 5, + 8, + "IDA", + "protein" + ], + [ + 42, + 56, + "kinase domains", + "structure_element" + ], + [ + 60, + 65, + "HAESA", + "protein" + ], + [ + 70, + 75, + "SERK1", + "protein" + ], + [ + 105, + 111, + "active", + "protein_state" + ], + [ + 112, + 127, + "protein kinases", + "protein_type" + ] + ] + }, + { + "sid": 116, + "sent": "Consistently, the HAESA kinase domain can transphosphorylate SERK1 and vice versa in in vitro transphosphorylation assays (Figure 3E).", + "section": "RESULTS", + "ner": [ + [ + 18, + 23, + "HAESA", + "protein" + ], + [ + 24, + 37, + "kinase domain", + "structure_element" + ], + [ + 61, + 66, + "SERK1", + "protein" + ], + [ + 94, + 121, + "transphosphorylation assays", + "experimental_method" + ] + ] + }, + { + "sid": 117, + "sent": "Together, our genetic and biochemical experiments implicate SERK1 as a HAESA co-receptor in the Arabidopsis abscission zone.", + "section": "RESULTS", + "ner": [ + [ + 14, + 49, + "genetic and biochemical experiments", + "experimental_method" + ], + [ + 60, + 65, + "SERK1", + "protein" + ], + [ + 71, + 88, + "HAESA co-receptor", + "protein_type" + ], + [ + 96, + 107, + "Arabidopsis", + "taxonomy_domain" + ] + ] + }, + { + "sid": 118, + "sent": "SERK1 senses a conserved motif in IDA family peptides", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "SERK1", + "protein" + ], + [ + 15, + 24, + "conserved", + "protein_state" + ], + [ + 25, + 30, + "motif", + "structure_element" + ], + [ + 34, + 53, + "IDA family peptides", + "chemical" + ] + ] + }, + { + "sid": 119, + "sent": "Crystal structure of a HAESA \u2013 IDA \u2013 SERK1 signaling complex.", + "section": "FIG", + "ner": [ + [ + 0, + 17, + "Crystal structure", + "evidence" + ], + [ + 23, + 42, + "HAESA \u2013 IDA \u2013 SERK1", + "complex_assembly" + ] + ] + }, + { + "sid": 120, + "sent": "(A) Overview of the ternary complex with HAESA in blue (surface representation), IDA in yellow (bonds representation) and SERK1 in orange (surface view). (B) The HAESA ectodomain undergoes a conformational change upon SERK1 co-receptor binding.", + "section": "FIG", + "ner": [ + [ + 41, + 46, + "HAESA", + "protein" + ], + [ + 81, + 84, + "IDA", + "protein" + ], + [ + 122, + 127, + "SERK1", + "protein" + ], + [ + 162, + 167, + "HAESA", + "protein" + ], + [ + 168, + 178, + "ectodomain", + "structure_element" + ], + [ + 218, + 223, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 121, + "sent": "Shown are C\u03b1 traces of a structural superposition of the unbound (yellow) and SERK1-bound (blue) HAESA ectodomains (r.m.s.d. is 1.5 \u00c5 between 572 corresponding C\u03b1 atoms).", + "section": "FIG", + "ner": [ + [ + 25, + 49, + "structural superposition", + "experimental_method" + ], + [ + 57, + 64, + "unbound", + "protein_state" + ], + [ + 78, + 89, + "SERK1-bound", + "protein_state" + ], + [ + 97, + 102, + "HAESA", + "protein" + ], + [ + 103, + 114, + "ectodomains", + "structure_element" + ], + [ + 116, + 124, + "r.m.s.d.", + "evidence" + ] + ] + }, + { + "sid": 122, + "sent": "SERK1 (in orange) and IDA (in red) are shown alongside.", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "SERK1", + "protein" + ], + [ + 22, + 25, + "IDA", + "protein" + ] + ] + }, + { + "sid": 123, + "sent": "The conformational change in the C-terminal LRRs and capping domain is indicated by an arrow. (C) SERK1 forms an integral part of the receptor's peptide binding pocket.", + "section": "FIG", + "ner": [ + [ + 44, + 48, + "LRRs", + "structure_element" + ], + [ + 53, + 67, + "capping domain", + "structure_element" + ], + [ + 98, + 103, + "SERK1", + "protein" + ], + [ + 145, + 167, + "peptide binding pocket", + "site" + ] + ] + }, + { + "sid": 124, + "sent": "The N-terminal capping domain of SERK1 (in orange) directly contacts the C-terminal part of IDA (in yellow, in bonds representation) and the receptor HAESA (in blue).", + "section": "FIG", + "ner": [ + [ + 15, + 29, + "capping domain", + "structure_element" + ], + [ + 33, + 38, + "SERK1", + "protein" + ], + [ + 92, + 95, + "IDA", + "protein" + ], + [ + 141, + 149, + "receptor", + "protein_type" + ], + [ + 150, + 155, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 125, + "sent": "Polar contacts of SERK1 with IDA are shown in magenta, with the HAESA LRR domain in gray. (D) Details of the zipper-like SERK1-HAESA interface.", + "section": "FIG", + "ner": [ + [ + 0, + 14, + "Polar contacts", + "bond_interaction" + ], + [ + 18, + 23, + "SERK1", + "protein" + ], + [ + 29, + 32, + "IDA", + "protein" + ], + [ + 64, + 69, + "HAESA", + "protein" + ], + [ + 70, + 80, + "LRR domain", + "structure_element" + ], + [ + 109, + 120, + "zipper-like", + "structure_element" + ], + [ + 121, + 142, + "SERK1-HAESA interface", + "site" + ] + ] + }, + { + "sid": 126, + "sent": "Ribbon diagrams of HAESA (in blue) and SERK1 (in orange) are shown with selected interface residues (in bonds representation).", + "section": "FIG", + "ner": [ + [ + 19, + 24, + "HAESA", + "protein" + ], + [ + 39, + 44, + "SERK1", + "protein" + ], + [ + 81, + 99, + "interface residues", + "site" + ] + ] + }, + { + "sid": 127, + "sent": "Polar interactions are highlighted as dotted lines (in magenta).", + "section": "FIG", + "ner": [ + [ + 0, + 18, + "Polar interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 128, + "sent": "To understand in molecular terms how SERK1 contributes to high-affinity IDA recognition, we solved a 2.43\u2009\u00c5 crystal structure of the ternary HAESA \u2013 IDA \u2013 SERK1 complex (Figure 4A, Table 2).", + "section": "RESULTS", + "ner": [ + [ + 37, + 42, + "SERK1", + "protein" + ], + [ + 72, + 75, + "IDA", + "protein" + ], + [ + 108, + 125, + "crystal structure", + "evidence" + ], + [ + 141, + 160, + "HAESA \u2013 IDA \u2013 SERK1", + "complex_assembly" + ] + ] + }, + { + "sid": 129, + "sent": "HAESA LRRs 16\u201321 and its C-terminal capping domain undergo a conformational change upon SERK1 binding (Figure 4B).", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "HAESA", + "protein" + ], + [ + 6, + 16, + "LRRs 16\u201321", + "structure_element" + ], + [ + 36, + 50, + "capping domain", + "structure_element" + ], + [ + 88, + 93, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 130, + "sent": "The SERK1 ectodomain interacts with the IDA peptide binding site using a loop region (residues 51-59SERK1) from its N-terminal cap (Figure 4A,C).", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "SERK1", + "protein" + ], + [ + 10, + 20, + "ectodomain", + "structure_element" + ], + [ + 40, + 64, + "IDA peptide binding site", + "site" + ], + [ + 73, + 84, + "loop region", + "structure_element" + ], + [ + 95, + 100, + "51-59", + "residue_range" + ], + [ + 100, + 105, + "SERK1", + "protein" + ], + [ + 127, + 130, + "cap", + "structure_element" + ] + ] + }, + { + "sid": 131, + "sent": "SERK1 loop residues establish multiple hydrophobic and polar contacts with Lys66IDA and the C-terminal Arg-His-Asn motif in IDA (Figure 4C).", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "SERK1", + "protein" + ], + [ + 6, + 10, + "loop", + "structure_element" + ], + [ + 39, + 69, + "hydrophobic and polar contacts", + "bond_interaction" + ], + [ + 75, + 80, + "Lys66", + "residue_name_number" + ], + [ + 80, + 83, + "IDA", + "protein" + ], + [ + 103, + 120, + "Arg-His-Asn motif", + "structure_element" + ], + [ + 124, + 127, + "IDA", + "protein" + ] + ] + }, + { + "sid": 132, + "sent": "SERK1 LRRs 1\u20135 and its C-terminal capping domain form an additional zipper-like interface with residues originating from HAESA LRRs 15\u201321 and from the HAESA C-terminal cap (Figure 4D).", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "SERK1", + "protein" + ], + [ + 6, + 14, + "LRRs 1\u20135", + "structure_element" + ], + [ + 34, + 48, + "capping domain", + "structure_element" + ], + [ + 68, + 79, + "zipper-like", + "structure_element" + ], + [ + 80, + 89, + "interface", + "site" + ], + [ + 121, + 126, + "HAESA", + "protein" + ], + [ + 127, + 137, + "LRRs 15\u201321", + "structure_element" + ], + [ + 151, + 156, + "HAESA", + "protein" + ], + [ + 168, + 171, + "cap", + "structure_element" + ] + ] + }, + { + "sid": 133, + "sent": "SERK1 binds HAESA using these two distinct interaction surfaces (Figure 1\u2014figure supplement 3), with the N-cap of the SERK1 LRR domain partially covering the IDA peptide binding cleft.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "SERK1", + "protein" + ], + [ + 12, + 17, + "HAESA", + "protein" + ], + [ + 43, + 63, + "interaction surfaces", + "site" + ], + [ + 105, + 110, + "N-cap", + "structure_element" + ], + [ + 118, + 123, + "SERK1", + "protein" + ], + [ + 124, + 134, + "LRR domain", + "structure_element" + ], + [ + 158, + 183, + "IDA peptide binding cleft", + "site" + ] + ] + }, + { + "sid": 134, + "sent": "The IDA C-terminal motif is required for HAESA-SERK1 complex formation and for IDA bioactivity.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "IDA", + "protein" + ], + [ + 8, + 24, + "C-terminal motif", + "structure_element" + ], + [ + 41, + 52, + "HAESA-SERK1", + "complex_assembly" + ] + ] + }, + { + "sid": 135, + "sent": "(A) Size exclusion chromatography experiments similar to Figure 3B,D reveal that IDA mutant peptides targeting the C-terminal motif do not form biochemically stable HAESA-IDA-SERK1 complexes.", + "section": "FIG", + "ner": [ + [ + 4, + 33, + "Size exclusion chromatography", + "experimental_method" + ], + [ + 81, + 84, + "IDA", + "protein" + ], + [ + 85, + 91, + "mutant", + "protein_state" + ], + [ + 92, + 100, + "peptides", + "chemical" + ], + [ + 115, + 131, + "C-terminal motif", + "structure_element" + ], + [ + 144, + 164, + "biochemically stable", + "protein_state" + ], + [ + 165, + 180, + "HAESA-IDA-SERK1", + "complex_assembly" + ] + ] + }, + { + "sid": 136, + "sent": "Deletion of the C-terminal Asn69IDA completely inhibits complex formation.", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "Deletion", + "experimental_method" + ], + [ + 27, + 32, + "Asn69", + "residue_name_number" + ], + [ + 32, + 35, + "IDA", + "protein" + ], + [ + 47, + 55, + "inhibits", + "protein_state" + ] + ] + }, + { + "sid": 137, + "sent": "Purified HAESA and SERK1 are ~75 and ~28 kDa, respectively.", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "Purified", + "experimental_method" + ], + [ + 9, + 14, + "HAESA", + "protein" + ], + [ + 19, + 24, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 138, + "sent": "Left panel: IDA K66A/R67A; center: IDA\u00a0\u0394N69, right panel: SDS-PAGE of peak fractions.", + "section": "FIG", + "ner": [ + [ + 12, + 25, + "IDA K66A/R67A", + "mutant" + ], + [ + 35, + 43, + "IDA\u00a0\u0394N69", + "mutant" + ], + [ + 58, + 66, + "SDS-PAGE", + "experimental_method" + ] + ] + }, + { + "sid": 139, + "sent": "Note that the HAESA and SERK1 input lanes have already been shown in Figure 3D. (B) Isothermal titration thermographs of wild-type and mutant IDA peptides titrated into a HAESA - SERK1 mixture in the cell.", + "section": "FIG", + "ner": [ + [ + 14, + 19, + "HAESA", + "protein" + ], + [ + 24, + 29, + "SERK1", + "protein" + ], + [ + 84, + 117, + "Isothermal titration thermographs", + "evidence" + ], + [ + 121, + 130, + "wild-type", + "protein_state" + ], + [ + 135, + 141, + "mutant", + "protein_state" + ], + [ + 142, + 154, + "IDA peptides", + "chemical" + ], + [ + 155, + 163, + "titrated", + "experimental_method" + ], + [ + 171, + 176, + "HAESA", + "protein" + ], + [ + 179, + 184, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 140, + "sent": "Table summaries for calorimetric binding constants and stoichoimetries for different IDA peptides binding to the HAESA \u2013 SERK1 ectodomain mixture ( \u00b1 fitting errors; n.d.", + "section": "FIG", + "ner": [ + [ + 20, + 50, + "calorimetric binding constants", + "evidence" + ], + [ + 85, + 97, + "IDA peptides", + "chemical" + ], + [ + 113, + 118, + "HAESA", + "protein" + ], + [ + 121, + 126, + "SERK1", + "protein" + ], + [ + 127, + 137, + "ectodomain", + "structure_element" + ] + ] + }, + { + "sid": 141, + "sent": "(C) Quantitative petal break-strength assay for Col-0 wild-type flowers and 35S::IDA wild-type and 35S::IDA K66A/R67A mutant flowers.", + "section": "FIG", + "ner": [ + [ + 17, + 43, + "petal break-strength assay", + "experimental_method" + ], + [ + 54, + 63, + "wild-type", + "protein_state" + ], + [ + 76, + 79, + "35S", + "gene" + ], + [ + 81, + 84, + "IDA", + "protein" + ], + [ + 85, + 94, + "wild-type", + "protein_state" + ], + [ + 99, + 102, + "35S", + "gene" + ], + [ + 104, + 117, + "IDA K66A/R67A", + "mutant" + ], + [ + 118, + 124, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 142, + "sent": "35S::IDA plants showed significantly increased abscission compared to Col-0 controls in inflorescence positions 2 and 3 (a).", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "35S", + "gene" + ], + [ + 5, + 8, + "IDA", + "protein" + ], + [ + 9, + 15, + "plants", + "taxonomy_domain" + ] + ] + }, + { + "sid": 143, + "sent": "Up to inflorescence position 4, petal break in 35S::IDA K66A/R67A mutant plants was significantly increased compared to both Col-0 control plants (b) and 35S::IDA plants (c) (D) Normalized expression levels (relative expression \u00b1 standard error; ida: -0.02 \u00b1 0.001; Col-0: 1 \u00b1 0.11; 35S::IDA 124 \u00b1 0.75; 35S::IDA K66A/R67A: 159 \u00b1 0.58) of IDA wild-type and mutant transcripts in the 35S promoter over-expression lines analyzed in (C). (E) Magnified view of representative abscission zones from 35S::IDA, Col-0 wild-type and 35S::IDA K66A/R67A double-mutant T3 transgenic lines.", + "section": "FIG", + "ner": [ + [ + 47, + 50, + "35S", + "gene" + ], + [ + 52, + 65, + "IDA K66A/R67A", + "mutant" + ], + [ + 66, + 72, + "mutant", + "protein_state" + ], + [ + 73, + 79, + "plants", + "taxonomy_domain" + ], + [ + 139, + 145, + "plants", + "taxonomy_domain" + ], + [ + 154, + 157, + "35S", + "gene" + ], + [ + 159, + 162, + "IDA", + "protein" + ], + [ + 163, + 169, + "plants", + "taxonomy_domain" + ], + [ + 283, + 286, + "35S", + "gene" + ], + [ + 288, + 291, + "IDA", + "protein" + ], + [ + 304, + 307, + "35S", + "gene" + ], + [ + 309, + 322, + "IDA K66A/R67A", + "mutant" + ], + [ + 339, + 342, + "IDA", + "protein" + ], + [ + 343, + 352, + "wild-type", + "protein_state" + ], + [ + 357, + 363, + "mutant", + "protein_state" + ], + [ + 383, + 417, + "35S promoter over-expression lines", + "experimental_method" + ], + [ + 494, + 497, + "35S", + "gene" + ], + [ + 499, + 502, + "IDA", + "protein" + ], + [ + 510, + 519, + "wild-type", + "protein_state" + ], + [ + 524, + 527, + "35S", + "gene" + ], + [ + 529, + 542, + "IDA K66A/R67A", + "mutant" + ], + [ + 543, + 556, + "double-mutant", + "protein_state" + ], + [ + 557, + 576, + "T3 transgenic lines", + "experimental_method" + ] + ] + }, + { + "sid": 144, + "sent": "15 out of 15 35S::IDA plants, 0 out of 15 Col-0 plants and 0 out of 15 35S::IDA K66A/R67A double-mutant plants, showed an enlarged abscission zone, respectively (3 independent lines were analyzed).", + "section": "FIG", + "ner": [ + [ + 13, + 16, + "35S", + "gene" + ], + [ + 18, + 21, + "IDA", + "protein" + ], + [ + 22, + 28, + "plants", + "taxonomy_domain" + ], + [ + 48, + 54, + "plants", + "taxonomy_domain" + ], + [ + 71, + 74, + "35S", + "gene" + ], + [ + 76, + 89, + "IDA K66A/R67A", + "mutant" + ], + [ + 90, + 103, + "double-mutant", + "protein_state" + ], + [ + 104, + 110, + "plants", + "taxonomy_domain" + ] + ] + }, + { + "sid": 145, + "sent": "The four C-terminal residues in IDA (Lys66IDA-Asn69IDA) are conserved among IDA family members and are in direct contact with SERK1 (Figures 1A, 4C).", + "section": "RESULTS", + "ner": [ + [ + 32, + 35, + "IDA", + "protein" + ], + [ + 37, + 54, + "Lys66IDA-Asn69IDA", + "residue_range" + ], + [ + 60, + 69, + "conserved", + "protein_state" + ], + [ + 76, + 94, + "IDA family members", + "protein_type" + ], + [ + 126, + 131, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 146, + "sent": "We thus assessed their contribution to HAESA \u2013 SERK1 complex formation.", + "section": "RESULTS", + "ner": [ + [ + 39, + 52, + "HAESA \u2013 SERK1", + "complex_assembly" + ] + ] + }, + { + "sid": 147, + "sent": "Deletion of the buried Asn69IDA completely inhibits receptor \u2013 co-receptor complex formation and HSL2 activation (Figure 5A,B).", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Deletion", + "experimental_method" + ], + [ + 23, + 28, + "Asn69", + "residue_name_number" + ], + [ + 28, + 31, + "IDA", + "protein" + ], + [ + 32, + 51, + "completely inhibits", + "protein_state" + ] + ] + }, + { + "sid": 148, + "sent": "A synthetic Lys66IDA/Arg67IDA \u2192 Ala mutant peptide (IDA K66A/R66A) showed a 10 fold reduced binding affinity when titrated in a HAESA/SERK1 protein solution (Figures 5A,B, 2D).", + "section": "RESULTS", + "ner": [ + [ + 2, + 11, + "synthetic", + "protein_state" + ], + [ + 12, + 35, + "Lys66IDA/Arg67IDA \u2192 Ala", + "mutant" + ], + [ + 36, + 42, + "mutant", + "protein_state" + ], + [ + 43, + 50, + "peptide", + "chemical" + ], + [ + 52, + 65, + "IDA K66A/R66A", + "mutant" + ], + [ + 92, + 108, + "binding affinity", + "evidence" + ], + [ + 114, + 122, + "titrated", + "experimental_method" + ], + [ + 128, + 133, + "HAESA", + "protein" + ], + [ + 134, + 139, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 149, + "sent": "We over-expressed full-length wild-type IDA or this Lys66IDA/Arg67IDA \u2192 Ala double-mutant to similar levels in Col-0 Arabidopsis plants (Figure 5D).", + "section": "RESULTS", + "ner": [ + [ + 3, + 17, + "over-expressed", + "experimental_method" + ], + [ + 18, + 29, + "full-length", + "protein_state" + ], + [ + 30, + 39, + "wild-type", + "protein_state" + ], + [ + 40, + 43, + "IDA", + "protein" + ], + [ + 52, + 75, + "Lys66IDA/Arg67IDA \u2192 Ala", + "mutant" + ], + [ + 76, + 89, + "double-mutant", + "protein_state" + ], + [ + 117, + 128, + "Arabidopsis", + "taxonomy_domain" + ], + [ + 129, + 135, + "plants", + "taxonomy_domain" + ] + ] + }, + { + "sid": 150, + "sent": "We found that over-expression of wild-type IDA leads to early floral abscission and an enlargement of the abscission zone (Figure 5C\u2013E).", + "section": "RESULTS", + "ner": [ + [ + 14, + 29, + "over-expression", + "experimental_method" + ], + [ + 33, + 42, + "wild-type", + "protein_state" + ], + [ + 43, + 46, + "IDA", + "protein" + ] + ] + }, + { + "sid": 151, + "sent": "In contrast, over-expression of the IDA Lys66IDA/Arg67IDA \u2192 Ala double mutant significantly delays floral abscission when compared to wild-type control plants, suggesting that the mutant IDA peptide has reduced activity in planta (Figure 5C\u2013E).", + "section": "RESULTS", + "ner": [ + [ + 13, + 28, + "over-expression", + "experimental_method" + ], + [ + 36, + 63, + "IDA Lys66IDA/Arg67IDA \u2192 Ala", + "mutant" + ], + [ + 64, + 77, + "double mutant", + "protein_state" + ], + [ + 134, + 143, + "wild-type", + "protein_state" + ], + [ + 152, + 158, + "plants", + "taxonomy_domain" + ], + [ + 180, + 186, + "mutant", + "protein_state" + ], + [ + 187, + 198, + "IDA peptide", + "chemical" + ], + [ + 223, + 229, + "planta", + "taxonomy_domain" + ] + ] + }, + { + "sid": 152, + "sent": "Comparison of 35S::IDA wild-type and mutant plants further indicates that mutation of Lys66IDA/Arg67IDA \u2192 Ala may cause a weak dominant negative effect (Figure 5C\u2013E).", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "35S", + "gene" + ], + [ + 19, + 22, + "IDA", + "protein" + ], + [ + 23, + 32, + "wild-type", + "protein_state" + ], + [ + 37, + 43, + "mutant", + "protein_state" + ], + [ + 44, + 50, + "plants", + "taxonomy_domain" + ], + [ + 74, + 82, + "mutation", + "experimental_method" + ], + [ + 86, + 109, + "Lys66IDA/Arg67IDA \u2192 Ala", + "mutant" + ] + ] + }, + { + "sid": 153, + "sent": "In agreement with our structures and biochemical assays, this experiment suggests a role of the conserved IDA C-terminus in the control of floral abscission.", + "section": "RESULTS", + "ner": [ + [ + 22, + 32, + "structures", + "evidence" + ], + [ + 37, + 55, + "biochemical assays", + "experimental_method" + ], + [ + 96, + 105, + "conserved", + "protein_state" + ], + [ + 106, + 109, + "IDA", + "protein" + ] + ] + }, + { + "sid": 154, + "sent": "In contrast to animal LRR receptors, plant LRR-RKs harbor spiral-shaped ectodomains and thus they require shape-complementary co-receptor proteins for receptor activation.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 21, + "animal", + "taxonomy_domain" + ], + [ + 22, + 35, + "LRR receptors", + "protein_type" + ], + [ + 37, + 42, + "plant", + "taxonomy_domain" + ], + [ + 43, + 50, + "LRR-RKs", + "structure_element" + ], + [ + 58, + 71, + "spiral-shaped", + "protein_state" + ], + [ + 72, + 83, + "ectodomains", + "structure_element" + ], + [ + 106, + 125, + "shape-complementary", + "protein_state" + ], + [ + 126, + 146, + "co-receptor proteins", + "protein_type" + ] + ] + }, + { + "sid": 155, + "sent": "For a rapidly growing number of plant signaling pathways, SERK proteins act as these essential co-receptors (;\u00a0).", + "section": "DISCUSS", + "ner": [ + [ + 32, + 37, + "plant", + "taxonomy_domain" + ], + [ + 58, + 71, + "SERK proteins", + "protein_type" + ], + [ + 95, + 107, + "co-receptors", + "protein_type" + ] + ] + }, + { + "sid": 156, + "sent": "\u00a0SERK1 has been previously reported as a positive regulator in plant embryogenesis, male sporogenesis, brassinosteroid signaling and in phytosulfokine perception.", + "section": "DISCUSS", + "ner": [ + [ + 63, + 68, + "plant", + "taxonomy_domain" + ] + ] + }, + { + "sid": 157, + "sent": "Recent findings by and our mechanistic studies now also support a positive role for SERK1 in floral abscission.", + "section": "DISCUSS", + "ner": [ + [ + 84, + 89, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 158, + "sent": "As serk1-1 mutant plants show intermediate abscission phenotypes when compared to haesa/hsl2 mutants, SERK1 likely acts redundantly with other SERKs in the abscission zone (Figure 3A).", + "section": "DISCUSS", + "ner": [ + [ + 3, + 10, + "serk1-1", + "gene" + ], + [ + 11, + 17, + "mutant", + "protein_state" + ], + [ + 18, + 24, + "plants", + "taxonomy_domain" + ], + [ + 82, + 87, + "haesa", + "gene" + ], + [ + 93, + 100, + "mutants", + "protein_state" + ], + [ + 102, + 107, + "SERK1", + "protein" + ], + [ + 143, + 148, + "SERKs", + "protein_type" + ] + ] + }, + { + "sid": 159, + "sent": "It has been previously suggested that SERK1 can inhibit cell separation.", + "section": "DISCUSS", + "ner": [ + [ + 38, + 43, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 160, + "sent": "However our results show that SERK1 also can activate this process upon IDA sensing, indicating that SERKs may fulfill several different functions in the course of the abscission process.", + "section": "DISCUSS", + "ner": [ + [ + 30, + 35, + "SERK1", + "protein" + ], + [ + 72, + 75, + "IDA", + "protein" + ], + [ + 101, + 106, + "SERKs", + "protein_type" + ] + ] + }, + { + "sid": 161, + "sent": "While the sequence of the mature IDA peptide has not been experimentally determined in planta, our HAESA-IDA complex structures and calorimetry assays suggest that active IDLs are hydroxyprolinated dodecamers.", + "section": "DISCUSS", + "ner": [ + [ + 26, + 32, + "mature", + "protein_state" + ], + [ + 33, + 44, + "IDA peptide", + "chemical" + ], + [ + 87, + 93, + "planta", + "taxonomy_domain" + ], + [ + 99, + 108, + "HAESA-IDA", + "complex_assembly" + ], + [ + 117, + 127, + "structures", + "evidence" + ], + [ + 132, + 150, + "calorimetry assays", + "evidence" + ], + [ + 164, + 170, + "active", + "protein_state" + ], + [ + 171, + 175, + "IDLs", + "protein_type" + ], + [ + 180, + 197, + "hydroxyprolinated", + "protein_state" + ], + [ + 198, + 208, + "dodecamers", + "structure_element" + ] + ] + }, + { + "sid": 162, + "sent": "It will be thus interesting to see if proteolytic processing of full-length IDA in vivo is regulated in a cell-type or tissue-specific manner.", + "section": "DISCUSS", + "ner": [ + [ + 64, + 75, + "full-length", + "protein_state" + ], + [ + 76, + 79, + "IDA", + "protein" + ] + ] + }, + { + "sid": 163, + "sent": "The central Hyp residue in IDA is found buried in the HAESA peptide binding surface and thus this post-translational modification may regulate IDA bioactivity.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 15, + "Hyp", + "residue_name" + ], + [ + 27, + 30, + "IDA", + "protein" + ], + [ + 54, + 59, + "HAESA", + "protein" + ], + [ + 60, + 83, + "peptide binding surface", + "site" + ], + [ + 143, + 146, + "IDA", + "protein" + ] + ] + }, + { + "sid": 164, + "sent": "Our comparative structural and biochemical analysis further suggests that IDLs share a common receptor binding mode, but may preferably bind to HAESA, HSL1 or HSL2 in different plant tissues and organs.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 51, + "comparative structural and biochemical analysis", + "experimental_method" + ], + [ + 74, + 78, + "IDLs", + "protein_type" + ], + [ + 144, + 149, + "HAESA", + "protein" + ], + [ + 151, + 155, + "HSL1", + "protein" + ], + [ + 159, + 163, + "HSL2", + "protein" + ], + [ + 177, + 182, + "plant", + "taxonomy_domain" + ] + ] + }, + { + "sid": 165, + "sent": "In our quantitative biochemical assays, the presence of SERK1 dramatically increases the HAESA binding specificity and affinity for IDA.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 38, + "quantitative biochemical assays", + "experimental_method" + ], + [ + 44, + 55, + "presence of", + "protein_state" + ], + [ + 56, + 61, + "SERK1", + "protein" + ], + [ + 89, + 94, + "HAESA", + "protein" + ], + [ + 132, + 135, + "IDA", + "protein" + ] + ] + }, + { + "sid": 166, + "sent": "This observation is consistent with our complex structure in which receptor and co-receptor together form the IDA binding pocket.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 57, + "structure", + "evidence" + ], + [ + 110, + 128, + "IDA binding pocket", + "site" + ] + ] + }, + { + "sid": 167, + "sent": "The fact that SERK1 specifically interacts with the very C-terminus of IDLs may allow for the rational design of peptide hormone antagonists, as previously demonstrated for the brassinosteroid pathway.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 19, + "SERK1", + "protein" + ], + [ + 71, + 75, + "IDLs", + "protein_type" + ], + [ + 113, + 140, + "peptide hormone antagonists", + "chemical" + ] + ] + }, + { + "sid": 168, + "sent": "Importantly, our calorimetry assays reveal that the SERK1 ectodomain binds HAESA with nanomolar affinity, but only in the presence of IDA (Figure 3C).", + "section": "DISCUSS", + "ner": [ + [ + 17, + 35, + "calorimetry assays", + "experimental_method" + ], + [ + 52, + 57, + "SERK1", + "protein" + ], + [ + 58, + 68, + "ectodomain", + "structure_element" + ], + [ + 69, + 74, + "binds", + "protein_state" + ], + [ + 75, + 80, + "HAESA", + "protein" + ], + [ + 122, + 133, + "presence of", + "protein_state" + ], + [ + 134, + 137, + "IDA", + "protein" + ] + ] + }, + { + "sid": 169, + "sent": "This ligand-induced formation of a receptor \u2013 co-receptor complex may allow the HAESA and SERK1 kinase domains to efficiently trans-phosphorylate and activate each other in the cytoplasm.", + "section": "DISCUSS", + "ner": [ + [ + 80, + 85, + "HAESA", + "protein" + ], + [ + 90, + 95, + "SERK1", + "protein" + ], + [ + 96, + 110, + "kinase domains", + "structure_element" + ] + ] + }, + { + "sid": 170, + "sent": "It is of note that our reported binding affinities for IDA and SERK1 have been measured using synthetic peptides and the isolated HAESA and SERK1 ectodomains, and thus\u00a0might differ in the context of the full-length, membrane-embedded signaling complex.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 50, + "binding affinities", + "evidence" + ], + [ + 55, + 58, + "IDA", + "protein" + ], + [ + 63, + 68, + "SERK1", + "protein" + ], + [ + 94, + 103, + "synthetic", + "protein_state" + ], + [ + 104, + 112, + "peptides", + "chemical" + ], + [ + 121, + 129, + "isolated", + "experimental_method" + ], + [ + 130, + 135, + "HAESA", + "protein" + ], + [ + 140, + 145, + "SERK1", + "protein" + ], + [ + 146, + 157, + "ectodomains", + "structure_element" + ], + [ + 203, + 214, + "full-length", + "protein_state" + ], + [ + 216, + 233, + "membrane-embedded", + "protein_state" + ] + ] + }, + { + "sid": 171, + "sent": "SERK1 uses partially overlapping surface areas to activate different plant signaling receptors.", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "SERK1", + "protein" + ], + [ + 69, + 74, + "plant", + "taxonomy_domain" + ], + [ + 75, + 94, + "signaling receptors", + "protein_type" + ] + ] + }, + { + "sid": 172, + "sent": "(A) Structural comparison of plant steroid and peptide hormone membrane signaling complexes.", + "section": "FIG", + "ner": [ + [ + 4, + 25, + "Structural comparison", + "experimental_method" + ], + [ + 29, + 34, + "plant", + "taxonomy_domain" + ], + [ + 35, + 42, + "steroid", + "chemical" + ], + [ + 47, + 62, + "peptide hormone", + "protein_type" + ], + [ + 63, + 91, + "membrane signaling complexes", + "protein_type" + ] + ] + }, + { + "sid": 173, + "sent": "Left panel: Ribbon diagram of HAESA (in blue), SERK1 (in orange) and IDA (in bonds and surface represention).", + "section": "FIG", + "ner": [ + [ + 30, + 35, + "HAESA", + "protein" + ], + [ + 47, + 52, + "SERK1", + "protein" + ], + [ + 69, + 72, + "IDA", + "protein" + ] + ] + }, + { + "sid": 174, + "sent": "Right panel: Ribbon diagram of the plant steroid receptor BRI1 (in blue) bound to brassinolide (in gray, in bonds representation) and to SERK1, shown in the same orientation (PDB-ID. 4lsx).", + "section": "FIG", + "ner": [ + [ + 35, + 40, + "plant", + "taxonomy_domain" + ], + [ + 41, + 57, + "steroid receptor", + "protein_type" + ], + [ + 58, + 62, + "BRI1", + "protein" + ], + [ + 73, + 81, + "bound to", + "protein_state" + ], + [ + 82, + 94, + "brassinolide", + "chemical" + ], + [ + 137, + 142, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 175, + "sent": "(B) View of the inner surface of the SERK1 LRR domain (PDB-ID 4lsc, surface representation, in gray).", + "section": "FIG", + "ner": [ + [ + 37, + 42, + "SERK1", + "protein" + ], + [ + 43, + 53, + "LRR domain", + "structure_element" + ] + ] + }, + { + "sid": 176, + "sent": "A ribbon diagram of SERK1 in the same orientation is shown alongside.", + "section": "FIG", + "ner": [ + [ + 20, + 25, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 177, + "sent": "Residues interacting with the HAESA or BRI1 LRR domains are shown in orange or magenta, respectively.", + "section": "FIG", + "ner": [ + [ + 30, + 35, + "HAESA", + "protein" + ], + [ + 39, + 43, + "BRI1", + "protein" + ], + [ + 44, + 55, + "LRR domains", + "structure_element" + ] + ] + }, + { + "sid": 178, + "sent": "Comparison of our HAESA \u2013 IDA \u2013 SERK1 structure with the brassinosteroid receptor signaling complex, where SERK1 also acts as co-receptor, reveals an overall conserved mode of SERK1 binding, while the ligand binding pockets map to very different areas in the corresponding receptors (LRRs 2 \u2013 14; HAESA; LRRs 21 \u2013 25, BRI1) and may involve an island domain (BRI1) or not (HAESA) (Figure 6A).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 10, + "Comparison", + "experimental_method" + ], + [ + 18, + 37, + "HAESA \u2013 IDA \u2013 SERK1", + "complex_assembly" + ], + [ + 38, + 47, + "structure", + "evidence" + ], + [ + 107, + 112, + "SERK1", + "protein" + ], + [ + 126, + 137, + "co-receptor", + "protein_type" + ], + [ + 158, + 167, + "conserved", + "protein_state" + ], + [ + 176, + 181, + "SERK1", + "protein" + ], + [ + 201, + 223, + "ligand binding pockets", + "site" + ], + [ + 284, + 295, + "LRRs 2 \u2013 14", + "structure_element" + ], + [ + 297, + 302, + "HAESA", + "protein" + ], + [ + 304, + 316, + "LRRs 21 \u2013 25", + "structure_element" + ], + [ + 318, + 322, + "BRI1", + "protein" + ], + [ + 358, + 362, + "BRI1", + "protein" + ], + [ + 372, + 377, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 179, + "sent": "Several residues in the SERK1\u2009N-terminal capping domain (Thr59SERK1, Phe61SERK1) and the\u00a0LRR inner surface (Asp75SERK1, Tyr101SERK1, SER121SERK1, Phe145SERK1) contribute to the formation of both complexes (Figures 4C,D, 6B).", + "section": "DISCUSS", + "ner": [ + [ + 24, + 29, + "SERK1", + "protein" + ], + [ + 41, + 55, + "capping domain", + "structure_element" + ], + [ + 57, + 62, + "Thr59", + "residue_name_number" + ], + [ + 62, + 67, + "SERK1", + "protein" + ], + [ + 69, + 74, + "Phe61", + "residue_name_number" + ], + [ + 74, + 79, + "SERK1", + "protein" + ], + [ + 89, + 106, + "LRR inner surface", + "site" + ], + [ + 108, + 113, + "Asp75", + "residue_name_number" + ], + [ + 113, + 118, + "SERK1", + "protein" + ], + [ + 120, + 126, + "Tyr101", + "residue_name_number" + ], + [ + 126, + 131, + "SERK1", + "protein" + ], + [ + 133, + 139, + "SER121", + "residue_name_number" + ], + [ + 139, + 144, + "SERK1", + "protein" + ], + [ + 146, + 152, + "Phe145", + "residue_name_number" + ], + [ + 152, + 157, + "SERK1", + "protein" + ] + ] + }, + { + "sid": 180, + "sent": "In addition, residues 53-55SERK1 from the SERK1\u2009N-terminal cap mediate specific interactions with the IDA peptide (Figures 4C, 6B).", + "section": "DISCUSS", + "ner": [ + [ + 22, + 27, + "53-55", + "residue_range" + ], + [ + 27, + 32, + "SERK1", + "protein" + ], + [ + 42, + 47, + "SERK1", + "protein" + ], + [ + 59, + 62, + "cap", + "structure_element" + ], + [ + 102, + 113, + "IDA peptide", + "chemical" + ] + ] + }, + { + "sid": 181, + "sent": "These residues are not involved in the sensing of the steroid hormone brassinolide.", + "section": "DISCUSS", + "ner": [ + [ + 54, + 69, + "steroid hormone", + "chemical" + ], + [ + 70, + 82, + "brassinolide", + "chemical" + ] + ] + }, + { + "sid": 182, + "sent": "In both cases however, the co-receptor completes the hormone binding pocket.", + "section": "DISCUSS", + "ner": [ + [ + 53, + 75, + "hormone binding pocket", + "site" + ] + ] + }, + { + "sid": 183, + "sent": "This fact together with the largely overlapping SERK1 binding surfaces in HAESA and BRI1 allows us to speculate that SERK1 may promote high-affinity peptide hormone and brassinosteroid sensing by simply slowing down dissociation of the ligand from its cognate receptor.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 70, + "SERK1 binding surfaces", + "site" + ], + [ + 74, + 79, + "HAESA", + "protein" + ], + [ + 84, + 88, + "BRI1", + "protein" + ], + [ + 117, + 122, + "SERK1", + "protein" + ], + [ + 149, + 164, + "peptide hormone", + "protein_type" + ] + ] + }, + { + "sid": 184, + "sent": "Different plant peptide hormone families contain a C-terminal (Arg)-His-Asn motif, which in IDA represents the co-receptor recognition site.", + "section": "FIG", + "ner": [ + [ + 10, + 15, + "plant", + "taxonomy_domain" + ], + [ + 16, + 40, + "peptide hormone families", + "protein_type" + ], + [ + 62, + 81, + "(Arg)-His-Asn motif", + "structure_element" + ], + [ + 92, + 95, + "IDA", + "protein" + ], + [ + 111, + 139, + "co-receptor recognition site", + "site" + ] + ] + }, + { + "sid": 185, + "sent": "Structure-guided multiple sequence alignment of IDA and IDA-like peptides with other plant peptide hormone families, including CLAVATA3 \u2013 EMBRYO SURROUNDING REGION-RELATED (CLV3/CLE), ROOT GROWTH FACTOR \u2013 GOLVEN (RGF/GLV), PRECURSOR GENE PROPEP1 (PEP1) from Arabidopsis thaliana.", + "section": "FIG", + "ner": [ + [ + 0, + 44, + "Structure-guided multiple sequence alignment", + "experimental_method" + ], + [ + 48, + 51, + "IDA", + "protein" + ], + [ + 56, + 73, + "IDA-like peptides", + "chemical" + ], + [ + 85, + 90, + "plant", + "taxonomy_domain" + ], + [ + 91, + 115, + "peptide hormone families", + "protein_type" + ], + [ + 127, + 171, + "CLAVATA3 \u2013 EMBRYO SURROUNDING REGION-RELATED", + "protein_type" + ], + [ + 173, + 181, + "CLV3/CLE", + "protein_type" + ], + [ + 184, + 211, + "ROOT GROWTH FACTOR \u2013 GOLVEN", + "protein_type" + ], + [ + 213, + 220, + "RGF/GLV", + "protein_type" + ], + [ + 223, + 245, + "PRECURSOR GENE PROPEP1", + "protein_type" + ], + [ + 247, + 251, + "PEP1", + "protein_type" + ], + [ + 258, + 278, + "Arabidopsis thaliana", + "species" + ] + ] + }, + { + "sid": 186, + "sent": "The conserved (Arg)-His-Asn motif is highlighted in red, the central Hyp residue in IDLs and CLEs is marked in blue.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "conserved", + "protein_state" + ], + [ + 14, + 33, + "(Arg)-His-Asn motif", + "structure_element" + ], + [ + 69, + 72, + "Hyp", + "residue_name" + ], + [ + 84, + 88, + "IDLs", + "protein_type" + ], + [ + 93, + 97, + "CLEs", + "protein_type" + ] + ] + }, + { + "sid": 187, + "sent": "Our experiments reveal that SERK1 recognizes a C-terminal Arg-His-Asn motif in IDA.", + "section": "DISCUSS", + "ner": [ + [ + 28, + 33, + "SERK1", + "protein" + ], + [ + 58, + 75, + "Arg-His-Asn motif", + "structure_element" + ], + [ + 79, + 82, + "IDA", + "protein" + ] + ] + }, + { + "sid": 188, + "sent": "Importantly, this motif can also be found in other peptide hormone families (Figure 7).", + "section": "DISCUSS", + "ner": [ + [ + 13, + 23, + "this motif", + "structure_element" + ], + [ + 51, + 75, + "peptide hormone families", + "protein_type" + ] + ] + }, + { + "sid": 189, + "sent": "Among these are the CLE peptides regulating stem cell maintenance in the shoot and the root.", + "section": "DISCUSS", + "ner": [ + [ + 20, + 32, + "CLE peptides", + "chemical" + ] + ] + }, + { + "sid": 190, + "sent": "It is interesting to note, that CLEs in their mature form are also hydroxyprolinated dodecamers, which bind to a surface area in the BARELY ANY MERISTEM 1 receptor that would correspond to part of the IDA binding cleft in HAESA.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 36, + "CLEs", + "protein_type" + ], + [ + 46, + 57, + "mature form", + "protein_state" + ], + [ + 67, + 84, + "hydroxyprolinated", + "protein_state" + ], + [ + 85, + 95, + "dodecamers", + "structure_element" + ], + [ + 113, + 125, + "surface area", + "site" + ], + [ + 133, + 163, + "BARELY ANY MERISTEM 1 receptor", + "protein_type" + ], + [ + 201, + 218, + "IDA binding cleft", + "site" + ], + [ + 222, + 227, + "HAESA", + "protein" + ] + ] + }, + { + "sid": 191, + "sent": "Diverse plant peptide hormones may thus also bind their LRR-RK receptors in an extended conformation along the inner surface of the LRR domain and may also use small, shape-complementary co-receptors for high-affinity ligand binding and receptor activation.", + "section": "DISCUSS", + "ner": [ + [ + 8, + 13, + "plant", + "taxonomy_domain" + ], + [ + 14, + 30, + "peptide hormones", + "protein_type" + ], + [ + 56, + 72, + "LRR-RK receptors", + "protein_type" + ], + [ + 79, + 100, + "extended conformation", + "protein_state" + ], + [ + 132, + 142, + "LRR domain", + "structure_element" + ], + [ + 160, + 165, + "small", + "protein_state" + ], + [ + 167, + 186, + "shape-complementary", + "protein_state" + ], + [ + 187, + 199, + "co-receptors", + "protein_type" + ] + ] + } + ] + }, + "PMC4896748": { + "annotations": [ + { + "sid": 0, + "sent": "Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome", + "section": "TITLE", + "ner": [ + [ + 9, + 16, + "cryo-EM", + "experimental_method" + ], + [ + 26, + 34, + "inchworm", + "protein_state" + ], + [ + 59, + 64, + "viral", + "taxonomy_domain" + ], + [ + 65, + 69, + "IRES", + "site" + ], + [ + 82, + 90, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 1, + "sent": "Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 29, + "Internal ribosome entry sites", + "site" + ], + [ + 31, + 36, + "IRESs", + "site" + ], + [ + 77, + 82, + "viral", + "taxonomy_domain" + ], + [ + 83, + 88, + "mRNAs", + "chemical" + ] + ] + }, + { + "sid": 2, + "sent": "Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2\u2022GTP bound with sordarin.", + "section": "ABSTRACT", + "ner": [ + [ + 6, + 30, + "electron cryo-microscopy", + "experimental_method" + ], + [ + 69, + 77, + "ribosome", + "complex_assembly" + ], + [ + 78, + 88, + "structures", + "evidence" + ], + [ + 105, + 125, + "Taura syndrome virus", + "species" + ], + [ + 126, + 130, + "IRES", + "site" + ], + [ + 135, + 146, + "translocase", + "protein_type" + ], + [ + 147, + 155, + "eEF2\u2022GTP", + "complex_assembly" + ], + [ + 156, + 166, + "bound with", + "protein_state" + ], + [ + 167, + 175, + "sordarin", + "chemical" + ] + ] + }, + { + "sid": 3, + "sent": "The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 39, + 43, + "IRES", + "site" + ], + [ + 84, + 94, + "initiation", + "protein_state" + ], + [ + 133, + 137, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 4, + "sent": "The IRES rearranges from extended to bent to extended conformations.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 8, + "IRES", + "site" + ], + [ + 25, + 33, + "extended", + "protein_state" + ], + [ + 37, + 41, + "bent", + "protein_state" + ], + [ + 45, + 53, + "extended", + "protein_state" + ] + ] + }, + { + "sid": 5, + "sent": "This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel.", + "section": "ABSTRACT", + "ner": [ + [ + 5, + 13, + "inchworm", + "protein_state" + ], + [ + 81, + 84, + "40S", + "complex_assembly" + ], + [ + 85, + 89, + "head", + "structure_element" + ] + ] + }, + { + "sid": 6, + "sent": "eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 4, + "eEF2", + "protein" + ], + [ + 22, + 25, + "60S", + "complex_assembly" + ], + [ + 26, + 33, + "subunit", + "structure_element" + ], + [ + 61, + 64, + "40S", + "complex_assembly" + ], + [ + 65, + 72, + "subunit", + "structure_element" + ], + [ + 65, + 72, + "subunit", + "structure_element" + ], + [ + 86, + 92, + "A site", + "site" + ] + ] + }, + { + "sid": 7, + "sent": "Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 15, + "diphthamide", + "ptm" + ], + [ + 38, + 40, + "IV", + "structure_element" + ], + [ + 55, + 82, + "tRNA-mRNA-like pseudoknot I", + "structure_element" + ], + [ + 84, + 87, + "PKI", + "structure_element" + ], + [ + 96, + 100, + "IRES", + "site" + ], + [ + 110, + 125, + "decoding center", + "site" + ] + ] + }, + { + "sid": 8, + "sent": "This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites.", + "section": "ABSTRACT", + "ner": [ + [ + 13, + 16, + "40S", + "complex_assembly" + ], + [ + 39, + 43, + "head", + "structure_element" + ], + [ + 63, + 67, + "IRES", + "site" + ], + [ + 122, + 125, + "PKI", + "structure_element" + ], + [ + 147, + 160, + "A and P sites", + "site" + ] + ] 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"sid": 46, + "sent": "The second large-scale rearrangement involves rotation, or swiveling, of the head of the small subunit relative to the body.", + "section": "INTRO", + "ner": [ + [ + 77, + 81, + "head", + "structure_element" + ], + [ + 89, + 102, + "small subunit", + "structure_element" + ], + [ + 119, + 123, + "body", + "structure_element" + ] + ] + }, + { + "sid": 47, + "sent": "The head can rotate by up to ~20\u00b0 around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA or in the presence of a single P/E tRNA and eEF2 or EF-G. F\u00f6rster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA\u2022mRNA.", + "section": "INTRO", + "ner": [ + [ + 4, + 8, + "head", + "structure_element" + ], + [ + 109, + 119, + "absence of", + "protein_state" + ], + [ + 120, + 124, + "tRNA", + "chemical" + ], + [ + 135, + 146, + "presence of", + "protein_state" + ], + [ + 156, + 157, + "P", + "site" + ], + [ + 158, + 159, + "E", + "site" + ], + [ + 160, + 164, + "tRNA", + "chemical" + ], + [ + 169, + 173, + "eEF2", + "protein" + ], + [ + 177, + 181, + "EF-G", + "protein" + ], + [ + 183, + 216, + "F\u00f6rster resonance energy transfer", + "experimental_method" + ], + [ + 218, + 222, + "FRET", + "experimental_method" + ], + [ + 242, + 246, + "head", + "structure_element" + ], + [ + 261, + 268, + "rotated", + "protein_state" + ], + [ + 269, + 282, + "small subunit", + "structure_element" + ], + [ + 295, + 299, + "EF-G", + "protein" + ], + [ + 321, + 331, + "2tRNA\u2022mRNA", + "complex_assembly" + ] + ] + }, + { + "sid": 48, + "sent": "Structures of the 70S\u2022EF-G complex bound with two nearly translocated tRNAs, exhibit a large 18\u00b0 to 21\u00b0 head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S\u20222tRNA\u2022EF-G structures.", + "section": "INTRO", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 18, + 26, + "70S\u2022EF-G", + "complex_assembly" + ], + [ + 35, + 45, + "bound with", + "protein_state" + ], + [ + 50, + 69, + "nearly translocated", + "protein_state" + ], + [ + 70, + 75, + "tRNAs", + "chemical" + ], + [ + 104, + 108, + "head", + "structure_element" + ], + [ + 121, + 132, + "mid-rotated", + "protein_state" + ], + [ + 133, + 140, + "subunit", + "structure_element" + ], + [ + 153, + 157, + "head", + "structure_element" + ], + [ + 184, + 197, + "fully rotated", + "protein_state" + ], + [ + 198, + 215, + "pre-translocation", + "protein_state" + ], + [ + 226, + 237, + "non-rotated", + "protein_state" + ], + [ + 238, + 256, + "post-translocation", + "protein_state" + ], + [ + 257, + 271, + "70S\u20222tRNA\u2022EF-G", + "complex_assembly" + ], + [ + 272, + 282, + "structures", + "evidence" + ] + ] + }, + { + "sid": 49, + "sent": "The structural role of head swivel is not fully understood.", + "section": "INTRO", + "ner": [ + [ + 23, + 27, + "head", + "structure_element" + ] + ] + }, + { + "sid": 50, + "sent": "The head swivel was proposed to facilitate transition of the tRNA from the P to E site by widening a constriction between these sites on the 30S subunit.", + "section": "INTRO", + "ner": [ + [ + 4, + 8, + "head", + "structure_element" + ], + [ + 61, + 65, + "tRNA", + "chemical" + ], + [ + 75, + 86, + "P to E site", + "site" + ], + [ + 101, + 113, + "constriction", + "site" + ], + [ + 141, + 144, + "30S", + "complex_assembly" + ], + [ + 145, + 152, + "subunit", + "structure_element" + ], + [ + 145, + 152, + "subunit", + "structure_element" + ] + ] + }, + { + "sid": 51, + "sent": "This widening\u00a0allows the ASL to sample positions between the P and E sites.", + "section": "INTRO", + "ner": [ + [ + 25, + 28, + "ASL", + "structure_element" + ], + [ + 61, + 74, + "P and E sites", + "site" + ] + ] + }, + { + "sid": 52, + "sent": "Whether and how the head swivel mediates tRNA transition from the A to P site remains unknown.", + "section": "INTRO", + "ner": [ + [ + 20, + 24, + "head", + "structure_element" + ], + [ + 41, + 45, + "tRNA", + "chemical" + ], + [ + 66, + 77, + "A to P site", + "site" + ] + ] + }, + { + "sid": 53, + "sent": "Comparison of 70S\u20222tRNA\u2022mRNA and 80S\u2022IRES translocation complexes.", + "section": "FIG", + "ner": [ + [ + 14, + 28, + "70S\u20222tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 33, + 41, + "80S\u2022IRES", + "complex_assembly" + ] + ] + }, + { + "sid": 54, + "sent": "(a) Structures of bacterial 70S\u20222tRNA\u2022mRNA translocation complexes, ordered according to the position of the translocating A->P tRNA (orange).", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "Structures", + "evidence" + ], + [ + 18, + 27, + "bacterial", + "taxonomy_domain" + ], + [ + 28, + 42, + "70S\u20222tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 123, + 127, + "A->P", + "site" + ], + [ + 128, + 132, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 55, + "sent": "The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body), elongation factor G (EF-G) is shown in green.", + "section": "FIG", + "ner": [ + [ + 20, + 27, + "subunit", + "structure_element" + ], + [ + 50, + 63, + "small subunit", + "structure_element" + ], + [ + 81, + 85, + "head", + "structure_element" + ], + [ + 105, + 109, + "body", + "structure_element" + ], + [ + 112, + 131, + "elongation factor G", + "protein" + ], + [ + 133, + 137, + "EF-G", + "protein" + ] + ] + }, + { + "sid": 56, + "sent": "Nucleotides C1054, G966 and G693 of 16S rRNA are shown in black to denote the A, P and E sites, respectively.", + "section": "FIG", + "ner": [ + [ + 12, + 17, + "C1054", + "residue_name_number" + ], + [ + 19, + 23, + "G966", + "residue_name_number" + ], + [ + 28, + 32, + "G693", + "residue_name_number" + ], + [ + 36, + 44, + "16S rRNA", + "chemical" + ], + [ + 78, + 94, + "A, P and E sites", + "site" + ] + ] + }, + { + "sid": 57, + "sent": "The extents of the 30S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows.", + "section": "FIG", + "ner": [ + [ + 19, + 22, + "30S", + "complex_assembly" + ], + [ + 23, + 30, + "subunit", + "structure_element" + ], + [ + 44, + 48, + "head", + "structure_element" + ], + [ + 91, + 109, + "post-translocation", + "protein_state" + ], + [ + 110, + 119, + "structure", + "evidence" + ] + ] + }, + { + "sid": 58, + "sent": "References and PDB codes of the structures are shown.", + "section": "FIG", + "ner": [ + [ + 32, + 42, + "structures", + "evidence" + ] + ] + }, + { + "sid": 59, + "sent": "(b) Structures of the 80S\u2022IRES complexes in the absence and presence of eEF2 (this work).", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "Structures", + "evidence" + ], + [ + 22, + 30, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 48, + 55, + "absence", + "protein_state" + ], + [ + 60, + 71, + "presence of", + "protein_state" + ], + [ + 72, + 76, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 60, + "sent": "The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green.", + "section": "FIG", + "ner": [ + [ + 20, + 27, + "subunit", + "structure_element" + ], + [ + 50, + 63, + "small subunit", + "structure_element" + ], + [ + 81, + 85, + "head", + "structure_element" + ], + [ + 105, + 109, + "body", + "structure_element" + ], + [ + 116, + 119, + "TSV", + "species" + ], + [ + 120, + 124, + "IRES", + "site" + ], + [ + 133, + 137, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 61, + "sent": "Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively.", + "section": "FIG", + "ner": [ + [ + 12, + 17, + "C1274", + "residue_name_number" + ], + [ + 19, + 24, + "U1191", + "residue_name_number" + ], + [ + 32, + 35, + "40S", + "complex_assembly" + ], + [ + 36, + 40, + "head", + "structure_element" + ], + [ + 45, + 49, + "G904", + "residue_name_number" + ], + [ + 57, + 65, + "platform", + "structure_element" + ], + [ + 84, + 89, + "C1054", + "residue_name_number" + ], + [ + 91, + 95, + "G966", + "residue_name_number" + ], + [ + 100, + 104, + "G693", + "residue_name_number" + ], + [ + 108, + 115, + "E. coli", + "species" + ], + [ + 116, + 124, + "16S rRNA", + "chemical" + ], + [ + 159, + 175, + "A, P and E sites", + "site" + ] + ] + }, + { + "sid": 62, + "sent": "Unresolved regions of the IRES in densities for Structures III and V are shown in gray.", + "section": "FIG", + "ner": [ + [ + 26, + 30, + "IRES", + "site" + ], + [ + 34, + 43, + "densities", + "evidence" + ], + [ + 48, + 68, + "Structures III and V", + "evidence" + ], + [ + 26, + 30, + "IRES", + "site" + ], + [ + 34, + 43, + "densities", + "evidence" + ], + [ + 48, + 68, + "Structures III and V", + "evidence" + ] + ] + }, + { + "sid": 63, + "sent": "The extents of the 40S subunit rotation and head swivel relative to their positions in the post-translocation structure are shown with arrows.", + "section": "FIG", + "ner": [ + [ + 19, + 22, + "40S", + "complex_assembly" + ], + [ + 23, + 30, + "subunit", + "structure_element" + ], + [ + 44, + 48, + "head", + "structure_element" + ], + [ + 91, + 109, + "post-translocation", + "protein_state" + ], + [ + 110, + 119, + "structure", + "evidence" + ] + ] + }, + { + "sid": 64, + "sent": "Schematic of cryo-EM refinement and classification procedures.", + "section": "FIG", + "ner": [ + [ + 13, + 20, + "cryo-EM", + "experimental_method" + ] + ] + }, + { + "sid": 65, + "sent": "All particles were initially aligned to a single model.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "particles", + "experimental_method" + ] + ] + }, + { + "sid": 66, + "sent": "3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2.", + "section": "FIG", + "ner": [ + [ + 0, + 17, + "3D classification", + "experimental_method" + ], + [ + 26, + 33, + "3D mask", + "evidence" + ], + [ + 45, + 48, + "40S", + "complex_assembly" + ], + [ + 49, + 53, + "head", + "structure_element" + ], + [ + 55, + 58, + "TSV", + "species" + ], + [ + 59, + 63, + "IRES", + "site" + ], + [ + 68, + 72, + "eEF2", + "protein" + ], + [ + 91, + 96, + "stack", + "bond_interaction" + ], + [ + 118, + 127, + "particles", + "experimental_method" + ], + [ + 148, + 152, + "IRES", + "site" + ], + [ + 157, + 161, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 67, + "sent": "Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses).", + "section": "FIG", + "ner": [ + [ + 11, + 28, + "3D classification", + "experimental_method" + ], + [ + 37, + 44, + "2D mask", + "evidence" + ], + [ + 56, + 59, + "PKI", + "structure_element" + ], + [ + 71, + 73, + "IV", + "structure_element" + ], + [ + 77, + 81, + "eEF2", + "protein" + ], + [ + 124, + 146, + "Structures I through V", + "evidence" + ], + [ + 148, + 166, + "Sub-classification", + "experimental_method" + ], + [ + 234, + 241, + "density", + "evidence" + ], + [ + 249, + 252, + "PKI", + "structure_element" + ], + [ + 313, + 322, + "particles", + "experimental_method" + ], + [ + 330, + 344, + "sub-classified", + "experimental_method" + ], + [ + 345, + 359, + "reconstruction", + "evidence" + ] + ] + }, + { + "sid": 68, + "sent": "Cryo-EM density of Structures I-V.", + "section": "FIG", + "ner": [ + [ + 0, + 7, + "Cryo-EM", + "experimental_method" + ], + [ + 8, + 15, + "density", + "evidence" + ], + [ + 19, + 33, + "Structures I-V", + "evidence" + ] + ] + }, + { + "sid": 69, + "sent": "In panels (a-e), the maps are segmented and colored as in Figure 1.", + "section": "FIG", + "ner": [ + [ + 21, + 25, + "maps", + "evidence" + ] + ] + }, + { + "sid": 70, + "sent": "The maps in all panels were B-softened by applying a B-factor of 30 \u00c52.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "maps", + "evidence" + ] + ] + }, + { + "sid": 71, + "sent": "(a-e) Cryo-EM map of Structures I, II, III, IV and V. (f-j) Local resolution of unfiltered and unmasked cryo-EM reconstructions, assessed using Blocres from the BSoft package, for Structures I, II, III, IV and V. (k-o) Cryo-EM density for the TSV IRES (red model) and eEF2 (green model) in Structures I, II, III, IV and V. (p) Fourier shell correlation (FSC) curves for Structures I-V. The horizontal axis is labeled with spatial frequency \u00c5-1 and with \u00c5. The resolutions stated in the text correspond to an FSC threshold value of 0.143, shown as a dotted line, for the FREALIGN-derived FSC ('Part_FSC').", + "section": "FIG", + "ner": [ + [ + 6, + 13, + "Cryo-EM", + "experimental_method" + ], + [ + 14, + 17, + "map", + "evidence" + ], + [ + 21, + 52, + "Structures I, II, III, IV and V", + "evidence" + ], + [ + 104, + 111, + "cryo-EM", + "experimental_method" + ], + [ + 112, + 127, + "reconstructions", + "evidence" + ], + [ + 144, + 151, + "Blocres", + "experimental_method" + ], + [ + 180, + 211, + "Structures I, II, III, IV and V", + "evidence" + ], + [ + 219, + 226, + "Cryo-EM", + "experimental_method" + ], + [ + 227, + 234, + "density", + "evidence" + ], + [ + 243, + 246, + "TSV", + "species" + ], + [ + 247, + 251, + "IRES", + "site" + ], + [ + 268, + 272, + "eEF2", + "protein" + ], + [ + 290, + 321, + "Structures I, II, III, IV and V", + "evidence" + ], + [ + 327, + 352, + "Fourier shell correlation", + "evidence" + ], + [ + 354, + 357, + "FSC", + "evidence" + ], + [ + 359, + 365, + "curves", + "evidence" + ], + [ + 370, + 384, + "Structures I-V", + "evidence" + ], + [ + 508, + 511, + "FSC", + "evidence" + ], + [ + 570, + 578, + "FREALIGN", + "experimental_method" + ], + [ + 587, + 590, + "FSC", + "evidence" + ] + ] + }, + { + "sid": 72, + "sent": "Cryo-EM structures of the 80S\u2022TSV IRES bound with eEF2\u2022GDP\u2022sordarin.", + "section": "FIG", + "ner": [ + [ + 0, + 7, + "Cryo-EM", + "experimental_method" + ], + [ + 8, + 18, + "structures", + "evidence" + ], + [ + 26, + 38, + "80S\u2022TSV IRES", + "complex_assembly" + ], + [ + 39, + 49, + "bound with", + "protein_state" + ], + [ + 50, + 67, + "eEF2\u2022GDP\u2022sordarin", + "complex_assembly" + ] + ] + }, + { + "sid": 73, + "sent": "(a)\u00a0Structures I through V. In all panels, the large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green.", + "section": "FIG", + "ner": [ + [ + 4, + 26, + "Structures I through V", + "evidence" + ], + [ + 47, + 70, + "large ribosomal subunit", + "structure_element" + ], + [ + 93, + 106, + "small subunit", + "structure_element" + ], + [ + 124, + 128, + "head", + "structure_element" + ], + [ + 148, + 152, + "body", + "structure_element" + ], + [ + 159, + 162, + "TSV", + "species" + ], + [ + 163, + 167, + "IRES", + "site" + ], + [ + 176, + 180, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 74, + "sent": "Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively.", + "section": "FIG", + "ner": [ + [ + 12, + 17, + "C1274", + "residue_name_number" + ], + [ + 19, + 24, + "U1191", + "residue_name_number" + ], + [ + 32, + 35, + "40S", + "complex_assembly" + ], + [ + 36, + 40, + "head", + "structure_element" + ], + [ + 45, + 49, + "G904", + "residue_name_number" + ], + [ + 57, + 65, + "platform", + "site" + ], + [ + 67, + 72, + "C1054", + "residue_name_number" + ], + [ + 74, + 78, + "G966", + "residue_name_number" + ], + [ + 83, + 87, + "G693", + "residue_name_number" + ], + [ + 91, + 98, + "E. coli", + "species" + ], + [ + 99, + 107, + "16S rRNA", + "chemical" + ], + [ + 142, + 158, + "A, P and E sites", + "site" + ] + ] + }, + { + "sid": 75, + "sent": "(b) Schematic representation of the structures shown in panel a, denoting the conformations of the small subunit relative to the large subunit.", + "section": "FIG", + "ner": [ + [ + 36, + 46, + "structures", + "evidence" + ], + [ + 99, + 112, + "small subunit", + "structure_element" + ], + [ + 129, + 142, + "large subunit", + "structure_element" + ] + ] + }, + { + "sid": 76, + "sent": "A, P and E sites are shown as rectangles.", + "section": "FIG", + "ner": [ + [ + 0, + 16, + "A, P and E sites", + "site" + ] + ] + }, + { + "sid": 77, + "sent": "All measurements are relative to the non-rotated 80S\u20222tRNA\u2022mRNA structure.", + "section": "FIG", + "ner": [ + [ + 37, + 48, + "non-rotated", + "protein_state" + ], + [ + 49, + 63, + "80S\u20222tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 64, + 73, + "structure", + "evidence" + ], + [ + 37, + 48, + "non-rotated", + "protein_state" + ], + [ + 49, + 63, + "80S\u20222tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 64, + 73, + "structure", + "evidence" + ] + ] + }, + { + "sid": 78, + "sent": "We sought to address the following questions by structural visualization of 80S\u2022IRES\u2022eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation?", + "section": "INTRO", + "ner": [ + [ + 48, + 72, + "structural visualization", + "experimental_method" + ], + [ + 76, + 89, + "80S\u2022IRES\u2022eEF2", + "complex_assembly" + ], + [ + 136, + 140, + "IRES", + "site" + ], + [ + 141, + 144, + "RNA", + "chemical" + ], + [ + 202, + 205, + "PKI", + "structure_element" + ], + [ + 215, + 226, + "A to P site", + "site" + ], + [ + 234, + 247, + "small subunit", + "structure_element" + ], + [ + 262, + 266, + "eEF2", + "protein" + ], + [ + 275, + 279, + "IRES", + "site" + ], + [ + 304, + 308, + "IRES", + "site" + ], + [ + 359, + 367, + "ribosome", + "complex_assembly" + ], + [ + 380, + 384, + "tRNA", + "chemical" + ], + [ + 445, + 448, + "40S", + "complex_assembly" + ], + [ + 449, + 453, + "head", + "structure_element" + ], + [ + 466, + 470, + "IRES", + "site" + ] + ] + }, + { + "sid": 79, + "sent": "We used cryo-EM to visualize 80S\u2022TSV IRES complexes formed in the presence of eEF2\u2022GTP and the translation inhibitor sordarin, which stabilizes eEF2 on the ribosome.", + "section": "INTRO", + "ner": [ + [ + 8, + 15, + "cryo-EM", + "experimental_method" + ], + [ + 29, + 41, + "80S\u2022TSV IRES", + "complex_assembly" + ], + [ + 66, + 77, + "presence of", + "protein_state" + ], + [ + 78, + 86, + "eEF2\u2022GTP", + "complex_assembly" + ], + [ + 117, + 125, + "sordarin", + "chemical" + ], + [ + 144, + 148, + "eEF2", + "protein" + ], + [ + 156, + 164, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 80, + "sent": "Although the mechanism of sordarin action is not fully understood, the inhibitor does not affect the conformation of eEF2\u2022GDPNP on the ribosome, rendering it an excellent tool in translocation studies.", + "section": "INTRO", + "ner": [ + [ + 26, + 34, + "sordarin", + "chemical" + ], + [ + 117, + 127, + "eEF2\u2022GDPNP", + "complex_assembly" + ], + [ + 135, + 143, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 81, + "sent": "Maximum-likelihood classification using FREALIGN identified five IRES-eEF2-bound ribosome structures within a single sample (Figures 1 and 2).", + "section": "INTRO", + "ner": [ + [ + 0, + 33, + "Maximum-likelihood classification", + "experimental_method" + ], + [ + 40, + 48, + "FREALIGN", + "experimental_method" + ], + [ + 65, + 80, + "IRES-eEF2-bound", + "protein_state" + ], + [ + 81, + 89, + "ribosome", + "complex_assembly" + ], + [ + 90, + 100, + "structures", + "evidence" + ] + ] + }, + { + "sid": 82, + "sent": "The structures differ in the positions and conformations of ribosomal subunits (Figures 1b and 2), IRES RNA (Figures 3 and 4) and eEF2 (Figures 5 and 6).", + "section": "INTRO", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 99, + 103, + "IRES", + "site" + ], + [ + 104, + 107, + "RNA", + "chemical" + ], + [ + 130, + 134, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 83, + "sent": "This ensemble of structures allowed us to reconstruct a sequence of steps in IRES translocation induced by eEF2.", + "section": "INTRO", + "ner": [ + [ + 17, + 27, + "structures", + "evidence" + ], + [ + 77, + 81, + "IRES", + "site" + ], + [ + 107, + 111, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 84, + "sent": "We used single-particle cryo-EM and maximum-likelihood image classification in FREALIGN to obtain three-dimensional density maps from a single specimen.", + "section": "RESULTS", + "ner": [ + [ + 8, + 31, + "single-particle cryo-EM", + "experimental_method" + ], + [ + 36, + 75, + "maximum-likelihood image classification", + "experimental_method" + ], + [ + 79, + 87, + "FREALIGN", + "experimental_method" + ], + [ + 116, + 128, + "density maps", + "evidence" + ] + ] + }, + { + "sid": 85, + "sent": "The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin.", + "section": "RESULTS", + "ner": [ + [ + 43, + 56, + "S. cerevisiae", + "species" + ], + [ + 57, + 70, + "80S ribosomes", + "complex_assembly" + ], + [ + 72, + 92, + "Taura syndrome virus", + "species" + ], + [ + 93, + 97, + "IRES", + "site" + ], + [ + 103, + 116, + "S. cerevisiae", + "species" + ], + [ + 117, + 121, + "eEF2", + "protein" + ], + [ + 129, + 140, + "presence of", + "protein_state" + ], + [ + 141, + 144, + "GTP", + "chemical" + ], + [ + 153, + 157, + "eEF2", + "protein" + ], + [ + 188, + 196, + "sordarin", + "chemical" + ] + ] + }, + { + "sid": 86, + "sent": "Unsupervised cryo-EM data classification was combined with the use of three-dimensional and two-dimensional masking around the ribosomal A site (Figure 1\u2014figure supplement 2).", + "section": "RESULTS", + "ner": [ + [ + 0, + 40, + "Unsupervised cryo-EM data classification", + "experimental_method" + ], + [ + 70, + 115, + "three-dimensional and two-dimensional masking", + "experimental_method" + ], + [ + 137, + 143, + "A site", + "site" + ] + ] + }, + { + "sid": 87, + "sent": "This approach revealed five 80S\u2022IRES\u2022eEF2\u2022GDP structures at average resolutions of 3.5 to 4.2\u2009\u00c5, sufficient to locate IRES domains and to resolve individual residues in the core regions of the ribosome and eEF2 (Figures 3c,d, and\u00a05f,h; see also Figure 1\u2014figure supplement 2 and Figure 5\u2014figure supplement 2), including the post-translational modification diphthamide 699 (Figure 3c).", + "section": "RESULTS", + "ner": [ + [ + 28, + 45, + "80S\u2022IRES\u2022eEF2\u2022GDP", + "complex_assembly" + ], + [ + 46, + 56, + "structures", + "evidence" + ], + [ + 118, + 122, + "IRES", + "site" + ], + [ + 193, + 201, + "ribosome", + "complex_assembly" + ], + [ + 206, + 210, + "eEF2", + "protein" + ], + [ + 355, + 370, + "diphthamide 699", + "ptm" + ] + ] + }, + { + "sid": 88, + "sent": "Large-scale rearrangements in Structures I through V, coupled with the movement of PKI from the A to P site and eEF2 entry into the A site.", + "section": "FIG", + "ner": [ + [ + 30, + 52, + "Structures I through V", + "evidence" + ], + [ + 83, + 86, + "PKI", + "structure_element" + ], + [ + 96, + 107, + "A to P site", + "site" + ], + [ + 112, + 116, + "eEF2", + "protein" + ], + [ + 132, + 138, + "A site", + "site" + ], + [ + 30, + 52, + "Structures I through V", + "evidence" + ], + [ + 83, + 86, + "PKI", + "structure_element" + ], + [ + 96, + 107, + "A to P site", + "site" + ], + [ + 112, + 116, + "eEF2", + "protein" + ], + [ + 132, + 138, + "A site", + "site" + ] + ] + }, + { + "sid": 89, + "sent": "(a) Rotational states of the 40S subunit in the 80S\u2022IRES structure (INIT; PDB 3J6Y) and in 80S\u2022IRES\u2022eEF2 Structures I, II, III, IV and V (this work).", + "section": "FIG", + "ner": [ + [ + 29, + 32, + "40S", + "complex_assembly" + ], + [ + 33, + 40, + "subunit", + "structure_element" + ], + [ + 48, + 56, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 57, + 66, + "structure", + "evidence" + ], + [ + 68, + 72, + "INIT", + "complex_assembly" + ], + [ + 91, + 104, + "80S\u2022IRES\u2022eEF2", + "complex_assembly" + ], + [ + 105, + 136, + "Structures I, II, III, IV and V", + "evidence" + ] + ] + }, + { + "sid": 90, + "sent": "For each structure, the triangle outlines the contours of the 40S body; the lower angle illustrates the extent of intersubunit (body) rotation.", + "section": "FIG", + "ner": [ + [ + 9, + 18, + "structure", + "evidence" + ], + [ + 62, + 65, + "40S", + "complex_assembly" + ], + [ + 66, + 70, + "body", + "structure_element" + ], + [ + 128, + 132, + "body", + "structure_element" + ] + ] + }, + { + "sid": 91, + "sent": "The sizes of the arrows correspond to the extent of the head swivel (yellow) and subunit rotation (black).", + "section": "FIG", + "ner": [ + [ + 56, + 60, + "head", + "structure_element" + ], + [ + 81, + 88, + "subunit", + "structure_element" + ] + ] + }, + { + "sid": 92, + "sent": "The views were obtained by structural alignment of the 25S rRNAs; the sarcin-ricin loop (SRL) of 25S rRNA is shown in gray for reference.", + "section": "FIG", + "ner": [ + [ + 27, + 47, + "structural alignment", + "experimental_method" + ], + [ + 55, + 64, + "25S rRNAs", + "chemical" + ], + [ + 70, + 87, + "sarcin-ricin loop", + "structure_element" + ], + [ + 89, + 92, + "SRL", + "structure_element" + ], + [ + 97, + 105, + "25S rRNA", + "chemical" + ] + ] + }, + { + "sid": 93, + "sent": "(b) Solvent view (opposite from that shown in (a)) of the 40S subunit in the 80S\u2022IRES structure (INIT; PDB 3J6Y) and in 80S\u2022IRES\u2022eEF2 Structures I, II, III, IV and V (this work).", + "section": "FIG", + "ner": [ + [ + 58, + 61, + "40S", + "complex_assembly" + ], + [ + 62, + 69, + "subunit", + "structure_element" + ], + [ + 77, + 85, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 86, + 95, + "structure", + "evidence" + ], + [ + 97, + 101, + "INIT", + "complex_assembly" + ], + [ + 120, + 133, + "80S\u2022IRES\u2022eEF2", + "complex_assembly" + ], + [ + 134, + 165, + "Structures I, II, III, IV and V", + "evidence" + ] + ] + }, + { + "sid": 94, + "sent": "The structures are colored as in Figure 1.", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ] + ] + }, + { + "sid": 95, + "sent": "(a) Comparison of the 40S-subunit rotational states in Structures I through V, sampling a ~10\u00b0 range between Structure I (fully rotated) and Structure V (non-rotated).", + "section": "FIG", + "ner": [ + [ + 22, + 25, + "40S", + "complex_assembly" + ], + [ + 26, + 33, + "subunit", + "structure_element" + ], + [ + 55, + 77, + "Structures I through V", + "evidence" + ], + [ + 109, + 120, + "Structure I", + "evidence" + ], + [ + 122, + 135, + "fully rotated", + "protein_state" + ], + [ + 141, + 152, + "Structure V", + "evidence" + ], + [ + 154, + 165, + "non-rotated", + "protein_state" + ] + ] + }, + { + "sid": 96, + "sent": "18S ribosomal RNA is shown and ribosomal proteins are omitted for clarity.", + "section": "FIG", + "ner": [ + [ + 0, + 17, + "18S ribosomal RNA", + "chemical" + ] + ] + }, + { + "sid": 97, + "sent": "The superpositions of Structures I-V were performed by structural alignments of the 25S ribosomal RNAs.", + "section": "FIG", + "ner": [ + [ + 4, + 18, + "superpositions", + "experimental_method" + ], + [ + 22, + 36, + "Structures I-V", + "evidence" + ], + [ + 55, + 76, + "structural alignments", + "experimental_method" + ], + [ + 84, + 102, + "25S ribosomal RNAs", + "chemical" + ] + ] + }, + { + "sid": 98, + "sent": "(b)\u00a0Bar graph of the angles characterizing the 40S rotational and 40S head swiveling states in Structures I through V. Measurements for the two 80S\u2022IRES (INIT) structures are included for comparison.", + "section": "FIG", + "ner": [ + [ + 47, + 50, + "40S", + "complex_assembly" + ], + [ + 66, + 69, + "40S", + "complex_assembly" + ], + [ + 70, + 74, + "head", + "structure_element" + ], + [ + 95, + 117, + "Structures I through V", + "evidence" + ], + [ + 144, + 152, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 154, + 158, + "INIT", + "complex_assembly" + ], + [ + 160, + 170, + "structures", + "evidence" + ] + ] + }, + { + "sid": 99, + "sent": "(c) Comparison of the 40S conformations in Structures I through V shows distinct positions of the head relative to the body of the 40S subunit (head swivel).", + "section": "FIG", + "ner": [ + [ + 22, + 25, + "40S", + "complex_assembly" + ], + [ + 43, + 65, + "Structures I through V", + "evidence" + ], + [ + 98, + 102, + "head", + "structure_element" + ], + [ + 119, + 123, + "body", + "structure_element" + ], + [ + 131, + 134, + "40S", + "complex_assembly" + ], + [ + 135, + 142, + "subunit", + "structure_element" + ], + [ + 144, + 148, + "head", + "structure_element" + ] + ] + }, + { + "sid": 100, + "sent": "Conformation of the non-swiveled 40S subunit in the S. cerevisiae 80S ribosome bound with two tRNAs is shown for reference (blue).", + "section": "FIG", + "ner": [ + [ + 20, + 32, + "non-swiveled", + "protein_state" + ], + [ + 33, + 36, + "40S", + "complex_assembly" + ], + [ + 37, + 44, + "subunit", + "structure_element" + ], + [ + 52, + 65, + "S. cerevisiae", + "species" + ], + [ + 66, + 78, + "80S ribosome", + "complex_assembly" + ], + [ + 79, + 89, + "bound with", + "protein_state" + ], + [ + 94, + 99, + "tRNAs", + "chemical" + ] + ] + }, + { + "sid": 101, + "sent": "(d) Comparison of conformations of the L1 and P stalks of the large subunit in Structures I through V with those in the 80S\u2022IRES and tRNA-bound 80S structures.", + "section": "FIG", + "ner": [ + [ + 39, + 41, + "L1", + "structure_element" + ], + [ + 46, + 54, + "P stalks", + "structure_element" + ], + [ + 62, + 75, + "large subunit", + "structure_element" + ], + [ + 79, + 101, + "Structures I through V", + "evidence" + ], + [ + 120, + 128, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 133, + 143, + "tRNA-bound", + "protein_state" + ], + [ + 144, + 147, + "80S", + "complex_assembly" + ], + [ + 148, + 158, + "structures", + "evidence" + ] + ] + }, + { + "sid": 102, + "sent": "Superpositions were performed by structural alignments of 25S ribosomal RNAs.", + "section": "FIG", + "ner": [ + [ + 0, + 14, + "Superpositions", + "experimental_method" + ], + [ + 33, + 54, + "structural alignments", + "experimental_method" + ], + [ + 58, + 76, + "25S ribosomal RNAs", + "chemical" + ] + ] + }, + { + "sid": 103, + "sent": "The central protuberance (CP) is labeled.", + "section": "FIG", + "ner": [ + [ + 4, + 24, + "central protuberance", + "structure_element" + ], + [ + 26, + 28, + "CP", + "structure_element" + ] + ] + }, + { + "sid": 104, + "sent": "\u00a0(e) Bar graph of the positions of PKI and domain IV of eEF2 relative to the P site residues of the head (U1191) and body (C1637) in Structures I through V. (f and g)\u00a0Close-up view of rearrangements in the A and P sites from the initiation state (INIT: PDB ID 3J6Y) to the post-translocation Structure V. The fragment shown within a rectangle in panel f is magnified in panel g. Nucleotides of the 40S body are shown in orange, 40S head in yellow.", + "section": "FIG", + "ner": [ + [ + 35, + 38, + "PKI", + "structure_element" + ], + [ + 50, + 52, + "IV", + "structure_element" + ], + [ + 56, + 60, + "eEF2", + "protein" + ], + [ + 77, + 83, + "P site", + "site" + ], + [ + 100, + 104, + "head", + "structure_element" + ], + [ + 106, + 111, + "U1191", + "residue_name_number" + ], + [ + 117, + 121, + "body", + "structure_element" + ], + [ + 123, + 128, + "C1637", + "residue_name_number" + ], + [ + 133, + 155, + "Structures I through V", + "evidence" + ], + [ + 206, + 219, + "A and P sites", + "site" + ], + [ + 229, + 239, + "initiation", + "protein_state" + ], + [ + 247, + 251, + "INIT", + "complex_assembly" + ], + [ + 273, + 291, + "post-translocation", + "protein_state" + ], + [ + 292, + 303, + "Structure V", + "evidence" + ], + [ + 398, + 401, + "40S", + "complex_assembly" + ], + [ + 402, + 406, + "body", + "structure_element" + ], + [ + 428, + 431, + "40S", + "complex_assembly" + ], + [ + 432, + 436, + "head", + "structure_element" + ] + ] + }, + { + "sid": 105, + "sent": "The superpositions of structures were performed by structural alignments of the 18S ribosomal RNAs excluding the head region (nt 1150\u20131620).", + "section": "FIG", + "ner": [ + [ + 4, + 18, + "superpositions", + "experimental_method" + ], + [ + 22, + 32, + "structures", + "evidence" + ], + [ + 51, + 72, + "structural alignments", + "experimental_method" + ], + [ + 80, + 98, + "18S ribosomal RNAs", + "chemical" + ], + [ + 113, + 117, + "head", + "structure_element" + ], + [ + 129, + 138, + "1150\u20131620", + "residue_range" + ] + ] + }, + { + "sid": 106, + "sent": "Our structures represent hitherto uncharacterized translocation complexes of the TSV IRES captured within globally distinct 80S conformations (Figures 1b and 2).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 81, + 84, + "TSV", + "species" + ], + [ + 85, + 89, + "IRES", + "site" + ], + [ + 124, + 127, + "80S", + "complex_assembly" + ] + ] + }, + { + "sid": 107, + "sent": "We numbered the structures from I to V, according to the position of the tRNA-mRNA-like PKI on the 40S subunit (Figure 2\u2014source data 1).", + "section": "RESULTS", + "ner": [ + [ + 16, + 38, + "structures from I to V", + "evidence" + ], + [ + 73, + 82, + "tRNA-mRNA", + "complex_assembly" + ], + [ + 88, + 91, + "PKI", + "structure_element" + ], + [ + 99, + 102, + "40S", + "complex_assembly" + ], + [ + 103, + 110, + "subunit", + "structure_element" + ] + ] + }, + { + "sid": 108, + "sent": "Specifically, PKI is partially withdrawn from the A site in Structure I, and fully translocated to the P site in Structure V (Figure 4; see also Figure 3\u2014figure supplement 1).", + "section": "RESULTS", + "ner": [ + [ + 14, + 17, + "PKI", + "structure_element" + ], + [ + 50, + 56, + "A site", + "site" + ], + [ + 60, + 71, + "Structure I", + "evidence" + ], + [ + 77, + 95, + "fully translocated", + "protein_state" + ], + [ + 103, + 109, + "P site", + "site" + ], + [ + 113, + 124, + "Structure V", + "evidence" + ] + ] + }, + { + "sid": 109, + "sent": "Thus Structures I to IV represent different positions of PKI between the A and P sites (Figure 2\u2014source data 1), suggesting that these structures describe intermediate states of translocation.", + "section": "RESULTS", + "ner": [ + [ + 5, + 23, + "Structures I to IV", + "evidence" + ], + [ + 57, + 60, + "PKI", + "structure_element" + ], + [ + 73, + 86, + "A and P sites", + "site" + ], + [ + 135, + 145, + "structures", + "evidence" + ] + ] + }, + { + "sid": 110, + "sent": "Structure V corresponds to the post-translocation state.", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Structure V", + "evidence" + ], + [ + 31, + 49, + "post-translocation", + "protein_state" + ] + ] + }, + { + "sid": 111, + "sent": "Changes in ribosome conformation and eEF2 positions are coupled with IRES movement through the ribosome", + "section": "RESULTS", + "ner": [ + [ + 11, + 19, + "ribosome", + "complex_assembly" + ], + [ + 37, + 41, + "eEF2", + "protein" + ], + [ + 69, + 73, + "IRES", + "site" + ], + [ + 95, + 103, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 112, + "sent": "Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S\u20222tRNA\u2022mRNA), in which both the subunit rotation and the head-body swivel are 0\u00b0, we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1\u2014figure supplement 1 and Figure 2\u2014source data 1).", + "section": "RESULTS", + "ner": [ + [ + 10, + 28, + "post-translocation", + "protein_state" + ], + [ + 29, + 42, + "S. cerevisiae", + "species" + ], + [ + 43, + 55, + "80S ribosome", + "complex_assembly" + ], + [ + 56, + 66, + "bound with", + "protein_state" + ], + [ + 71, + 83, + "P and E site", + "site" + ], + [ + 84, + 89, + "tRNAs", + "chemical" + ], + [ + 106, + 120, + "80S\u20222tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 141, + 148, + "subunit", + "structure_element" + ], + [ + 166, + 170, + "head", + "structure_element" + ], + [ + 171, + 175, + "body", + "structure_element" + ], + [ + 209, + 217, + "ribosome", + "complex_assembly" + ], + [ + 265, + 287, + "Structures I through V", + "evidence" + ] + ] + }, + { + "sid": 113, + "sent": "Structure I comprises the most rotated ribosome conformation (~10\u00b0), characteristic of pre-translocation hybrid-tRNA states.", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Structure I", + "evidence" + ], + [ + 26, + 38, + "most rotated", + "protein_state" + ], + [ + 39, + 47, + "ribosome", + "complex_assembly" + ], + [ + 87, + 104, + "pre-translocation", + "protein_state" + ], + [ + 105, + 116, + "hybrid-tRNA", + "protein_state" + ] + ] + }, + { + "sid": 114, + "sent": "From Structure I to V, the body of the small subunit undergoes backward (reverse) rotation (Figure 2b; see also Figure 1\u2014figure supplement 2 and Figure 2\u2014figure supplement 1).", + "section": "RESULTS", + "ner": [ + [ + 5, + 21, + "Structure I to V", + "evidence" + ], + [ + 27, + 31, + "body", + "structure_element" + ], + [ + 39, + 52, + "small subunit", + "structure_element" + ] + ] + }, + { + "sid": 115, + "sent": "Structures II and III are in mid-rotation conformations (~5\u00b0).", + "section": "RESULTS", + "ner": [ + [ + 0, + 21, + "Structures II and III", + "evidence" + ], + [ + 29, + 41, + "mid-rotation", + "protein_state" + ] + ] + }, + { + "sid": 116, + "sent": "Structure IV adopts a slightly rotated conformation (~1\u00b0).", + "section": "RESULTS", + "ner": [ + [ + 0, + 12, + "Structure IV", + "evidence" + ], + [ + 22, + 38, + "slightly rotated", + "protein_state" + ] + ] + }, + { + "sid": 117, + "sent": "Structure V is in a nearly non-rotated conformation (0.5\u00b0), very similar to that of post-translocation ribosome-tRNA complexes.", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Structure V", + "evidence" + ], + [ + 27, + 38, + "non-rotated", + "protein_state" + ], + [ + 84, + 102, + "post-translocation", + "protein_state" + ], + [ + 103, + 116, + "ribosome-tRNA", + "complex_assembly" + ] + ] + }, + { + "sid": 118, + "sent": "Thus, intersubunit rotation of ~9\u00b0 from Structure I to V covers a nearly complete range of relative subunit positions, similar to what was reported for tRNA-bound yeast, bacterial and mammalian ribosomes.", + "section": "RESULTS", + "ner": [ + [ + 40, + 56, + "Structure I to V", + "evidence" + ], + [ + 100, + 107, + "subunit", + "structure_element" + ], + [ + 152, + 162, + "tRNA-bound", + "protein_state" + ], + [ + 163, + 168, + "yeast", + "taxonomy_domain" + ], + [ + 170, + 179, + "bacterial", + "taxonomy_domain" + ], + [ + 184, + 193, + "mammalian", + "taxonomy_domain" + ], + [ + 194, + 203, + "ribosomes", + "complex_assembly" + ] + ] + }, + { + "sid": 119, + "sent": "40S head swivel", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "40S", + "complex_assembly" + ], + [ + 4, + 8, + "head", + "structure_element" + ] + ] + }, + { + "sid": 120, + "sent": "The pattern of 40S head swivel between the structures is different from that of intersubunit rotation (Figures 2c and d; see also Figure 2\u2014source data 1).", + "section": "RESULTS", + "ner": [ + [ + 15, + 18, + "40S", + "complex_assembly" + ], + [ + 19, + 23, + "head", + "structure_element" + ], + [ + 43, + 53, + "structures", + "evidence" + ] + ] + }, + { + "sid": 121, + "sent": "As with the intersubunit rotation, the small head swivel (~1\u00b0) in the non-rotated Structure V is closest to that in the 80S\u20222tRNA\u2022mRNA post-translocation ribosome.", + "section": "RESULTS", + "ner": [ + [ + 45, + 49, + "head", + "structure_element" + ], + [ + 70, + 81, + "non-rotated", + "protein_state" + ], + [ + 82, + 93, + "Structure V", + "evidence" + ], + [ + 120, + 134, + "80S\u20222tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 135, + 153, + "post-translocation", + "protein_state" + ], + [ + 154, + 162, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 122, + "sent": "However in the pre-translocation intermediates (from Structure I to IV), the beak of the head domain first turns toward the large subunit and then backs off (Figure 2\u2014figure supplement 1).", + "section": "RESULTS", + "ner": [ + [ + 15, + 32, + "pre-translocation", + "protein_state" + ], + [ + 53, + 70, + "Structure I to IV", + "evidence" + ], + [ + 89, + 93, + "head", + "structure_element" + ], + [ + 124, + 137, + "large subunit", + "structure_element" + ] + ] + }, + { + "sid": 123, + "sent": "The head samples a mid-swiveled position in Structure I (12\u00b0), then a highly-swiveled position in Structures II and III (17\u00b0) and a less swiveled position in Structure IV (14\u00b0).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "head", + "structure_element" + ], + [ + 19, + 31, + "mid-swiveled", + "protein_state" + ], + [ + 44, + 55, + "Structure I", + "evidence" + ], + [ + 70, + 85, + "highly-swiveled", + "protein_state" + ], + [ + 98, + 119, + "Structures II and III", + "evidence" + ], + [ + 132, + 145, + "less swiveled", + "protein_state" + ], + [ + 158, + 170, + "Structure IV", + "evidence" + ] + ] + }, + { + "sid": 124, + "sent": "The maximum head swivel is observed in the mid-rotated complexes II and III, in which PKI transitions from the A to P site, while eEF2 occupies the A site partially.", + "section": "RESULTS", + "ner": [ + [ + 12, + 16, + "head", + "structure_element" + ], + [ + 43, + 54, + "mid-rotated", + "protein_state" + ], + [ + 65, + 75, + "II and III", + "evidence" + ], + [ + 86, + 89, + "PKI", + "structure_element" + ], + [ + 111, + 122, + "A to P site", + "site" + ], + [ + 130, + 134, + "eEF2", + "protein" + ], + [ + 148, + 154, + "A site", + "site" + ] + ] + }, + { + "sid": 125, + "sent": "By comparison, the similarly mid-rotated (4\u00b0) 80S\u2022TSV IRES initiation complex, in the absence of eEF2, adopts a mid-swiveled position (~10\u00b0) (Figure 2c).", + "section": "RESULTS", + "ner": [ + [ + 29, + 40, + "mid-rotated", + "protein_state" + ], + [ + 46, + 58, + "80S\u2022TSV IRES", + "complex_assembly" + ], + [ + 59, + 69, + "initiation", + "protein_state" + ], + [ + 86, + 96, + "absence of", + "protein_state" + ], + [ + 97, + 101, + "eEF2", + "protein" + ], + [ + 112, + 124, + "mid-swiveled", + "protein_state" + ] + ] + }, + { + "sid": 126, + "sent": "These observations suggest that eEF2 is necessary for inducing or stabilizing the large head swivel of the 40S subunit characteristic for IRES translocation intermediates.", + "section": "RESULTS", + "ner": [ + [ + 32, + 36, + "eEF2", + "protein" + ], + [ + 88, + 92, + "head", + "structure_element" + ], + [ + 107, + 110, + "40S", + "complex_assembly" + ], + [ + 111, + 118, + "subunit", + "structure_element" + ], + [ + 138, + 142, + "IRES", + "site" + ] + ] + }, + { + "sid": 127, + "sent": "IRES rearrangements", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "IRES", + "site" + ] + ] + }, + { + "sid": 128, + "sent": "Comparison of the TSV IRES and eEF2 positions in Structures I through V.", + "section": "FIG", + "ner": [ + [ + 18, + 21, + "TSV", + "species" + ], + [ + 22, + 26, + "IRES", + "site" + ], + [ + 31, + 35, + "eEF2", + "protein" + ], + [ + 49, + 71, + "Structures I through V", + "evidence" + ] + ] + }, + { + "sid": 129, + "sent": "(a) Positions of the IRES and eEF2 in the initiation, pre-translocation (I) and post-translocation (V) states, relative to the body of the 40S subunit (not shown) (b) Positions of the IRES and eEF2 in the initiation state (INIT) and intermediate steps of translocation (II, III and IV), relative to the body of the 40S subunit (not shown).", + "section": "FIG", + "ner": [ + [ + 21, + 25, + "IRES", + "site" + ], + [ + 30, + 34, + "eEF2", + "protein" + ], + [ + 42, + 52, + "initiation", + "protein_state" + ], + [ + 54, + 71, + "pre-translocation", + "protein_state" + ], + [ + 73, + 74, + "I", + "evidence" + ], + [ + 80, + 98, + "post-translocation", + "protein_state" + ], + [ + 100, + 101, + "V", + "evidence" + ], + [ + 127, + 131, + "body", + "structure_element" + ], + [ + 139, + 142, + "40S", + "complex_assembly" + ], + [ + 143, + 150, + "subunit", + "structure_element" + ], + [ + 143, + 150, + "subunit", + "structure_element" + ], + [ + 184, + 188, + "IRES", + "site" + ], + [ + 193, + 197, + "eEF2", + "protein" + ], + [ + 205, + 215, + "initiation", + "protein_state" + ], + [ + 223, + 227, + "INIT", + "complex_assembly" + ], + [ + 270, + 284, + "II, III and IV", + "evidence" + ], + [ + 303, + 307, + "body", + "structure_element" + ], + [ + 315, + 318, + "40S", + "complex_assembly" + ], + [ + 319, + 326, + "subunit", + "structure_element" + ], + [ + 319, + 326, + "subunit", + "structure_element" + ] + ] + }, + { + "sid": 130, + "sent": "\u00a0Superpositions were obtained by structural alignments of the 18S rRNAs excluding the head domains (nt 1150\u20131620).", + "section": "FIG", + "ner": [ + [ + 33, + 54, + "structural alignments", + "experimental_method" + ], + [ + 62, + 71, + "18S rRNAs", + "chemical" + ], + [ + 86, + 90, + "head", + "structure_element" + ], + [ + 103, + 112, + "1150\u20131620", + "residue_range" + ] + ] + }, + { + "sid": 131, + "sent": "Positions of the IRES relative to proteins uS7, uS11 and eS25.", + "section": "FIG", + "ner": [ + [ + 17, + 21, + "IRES", + "site" + ], + [ + 43, + 46, + "uS7", + "protein" + ], + [ + 48, + 52, + "uS11", + "protein" + ], + [ + 57, + 61, + "eS25", + "protein" + ] + ] + }, + { + "sid": 132, + "sent": "(a) Intra-IRES rearrangements from the 80S*IRES initiation structure (INIT; PDB 3J6Y,) to Structures I through V. For each structure (shown in red), the conformation from a preceding structure is shown in light red for comparison.", + "section": "FIG", + "ner": [ + [ + 10, + 14, + "IRES", + "site" + ], + [ + 39, + 47, + "80S*IRES", + "complex_assembly" + ], + [ + 48, + 58, + "initiation", + "protein_state" + ], + [ + 59, + 68, + "structure", + "evidence" + ], + [ + 70, + 74, + "INIT", + "complex_assembly" + ], + [ + 90, + 112, + "Structures I through V", + "evidence" + ], + [ + 123, + 132, + "structure", + "evidence" + ], + [ + 183, + 192, + "structure", + "evidence" + ] + ] + }, + { + "sid": 133, + "sent": "Superpositions were obtained by structural alignments of 18S rRNA.", + "section": "FIG", + "ner": [ + [ + 0, + 14, + "Superpositions", + "experimental_method" + ], + [ + 32, + 53, + "structural alignments", + "experimental_method" + ], + [ + 57, + 65, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 134, + "sent": "(b) Positions of the IRES and eEF2 relative to those of classical P- and E-site tRNAs in the 80S\u2022tRNA complex. (c) Positions of the IRES relative to proteins uS11 (40S platform) and uS7 and eS25 (40S head), which interact with the 5\u2032 domain of the IRES in the initiation state (left panel).", + "section": "FIG", + "ner": [ + [ + 21, + 25, + "IRES", + "site" + ], + [ + 30, + 34, + "eEF2", + "protein" + ], + [ + 66, + 79, + "P- and E-site", + "site" + ], + [ + 80, + 85, + "tRNAs", + "chemical" + ], + [ + 93, + 101, + "80S\u2022tRNA", + "complex_assembly" + ], + [ + 132, + 136, + "IRES", + "site" + ], + [ + 158, + 162, + "uS11", + "protein" + ], + [ + 164, + 176, + "40S platform", + "site" + ], + [ + 182, + 185, + "uS7", + "protein" + ], + [ + 190, + 194, + "eS25", + "protein" + ], + [ + 196, + 199, + "40S", + "complex_assembly" + ], + [ + 200, + 204, + "head", + "structure_element" + ], + [ + 231, + 240, + "5\u2032 domain", + "structure_element" + ], + [ + 248, + 252, + "IRES", + "site" + ], + [ + 260, + 270, + "initiation", + "protein_state" + ] + ] + }, + { + "sid": 135, + "sent": "In all panels, superpositions were obtained by structural alignments of the 18S rRNAs.", + "section": "FIG", + "ner": [ + [ + 15, + 29, + "superpositions", + "experimental_method" + ], + [ + 47, + 68, + "structural alignments", + "experimental_method" + ], + [ + 76, + 85, + "18S rRNAs", + "chemical" + ] + ] + }, + { + "sid": 136, + "sent": "Ribosomal proteins of the initiation state are shown in gray for comparison.", + "section": "FIG", + "ner": [ + [ + 26, + 36, + "initiation", + "protein_state" + ] + ] + }, + { + "sid": 137, + "sent": "Positions of the L1stalk, tRNA and TSV IRES relative to proteins uS7 and eS25, in 80S\u2022tRNA structures and 80S\u2022IRES structures I and V (this work).", + "section": "FIG", + "ner": [ + [ + 17, + 24, + "L1stalk", + "structure_element" + ], + [ + 26, + 30, + "tRNA", + "chemical" + ], + [ + 35, + 38, + "TSV", + "species" + ], + [ + 39, + 43, + "IRES", + "site" + ], + [ + 65, + 68, + "uS7", + "protein" + ], + [ + 73, + 77, + "eS25", + "protein" + ], + [ + 82, + 90, + "80S\u2022tRNA", + "complex_assembly" + ], + [ + 91, + 101, + "structures", + "evidence" + ], + [ + 106, + 114, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 115, + 133, + "structures I and V", + "evidence" + ] + ] + }, + { + "sid": 138, + "sent": "The view shows the vicinity of the ribosomal E site.", + "section": "FIG", + "ner": [ + [ + 45, + 51, + "E site", + "site" + ] + ] + }, + { + "sid": 139, + "sent": "Loop 1.1 and stem loops 4 and 5 of the IRES are labeled.", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "Loop 1.1", + "structure_element" + ], + [ + 13, + 31, + "stem loops 4 and 5", + "structure_element" + ], + [ + 39, + 43, + "IRES", + "site" + ] + ] + }, + { + "sid": 140, + "sent": "Interactions of the stem loops 4 and 5 of the TSV with proteins uS7 and eS25.", + "section": "FIG", + "ner": [ + [ + 20, + 38, + "stem loops 4 and 5", + "structure_element" + ], + [ + 46, + 49, + "TSV", + "species" + ], + [ + 64, + 67, + "uS7", + "protein" + ], + [ + 72, + 76, + "eS25", + "protein" + ] + ] + }, + { + "sid": 141, + "sent": "Position and interactions of loop 3 (variable loop region) of the PKI domain in Structure V (this work) resembles those of the anticodon stem loop of the E-site tRNA (blue) in the 80S\u20222tRNA\u2022mRNA complex.", + "section": "FIG", + "ner": [ + [ + 29, + 35, + "loop 3", + "structure_element" + ], + [ + 37, + 57, + "variable loop region", + "structure_element" + ], + [ + 66, + 69, + "PKI", + "structure_element" + ], + [ + 80, + 91, + "Structure V", + "evidence" + ], + [ + 127, + 146, + "anticodon stem loop", + "structure_element" + ], + [ + 154, + 160, + "E-site", + "site" + ], + [ + 161, + 165, + "tRNA", + "chemical" + ], + [ + 180, + 194, + "80S\u20222tRNA\u2022mRNA", + "complex_assembly" + ] + ] + }, + { + "sid": 142, + "sent": "Positions of tRNAs and the TSV IRES relative to the A-site finger (blue, nt 1008\u20131043 of 25S rRNA) and the P site of the large subunit, comprising helix 84 of 25S rRNA (nt.", + "section": "FIG", + "ner": [ + [ + 13, + 18, + "tRNAs", + "chemical" + ], + [ + 27, + 30, + "TSV", + "species" + ], + [ + 31, + 35, + "IRES", + "site" + ], + [ + 52, + 65, + "A-site finger", + "structure_element" + ], + [ + 76, + 85, + "1008\u20131043", + "residue_range" + ], + [ + 89, + 97, + "25S rRNA", + "chemical" + ], + [ + 107, + 113, + "P site", + "site" + ], + [ + 121, + 134, + "large subunit", + "structure_element" + ], + [ + 147, + 155, + "helix 84", + "structure_element" + ], + [ + 159, + 167, + "25S rRNA", + "chemical" + ] + ] + }, + { + "sid": 143, + "sent": "2668\u20132687) and protein uL5 (collectively labeled as central protuberance, CP, in the upper-row first figure, and individually labeled in the lower-row first figure).", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "2668\u20132687", + "residue_range" + ], + [ + 23, + 26, + "uL5", + "protein" + ], + [ + 52, + 72, + "central protuberance", + "structure_element" + ], + [ + 74, + 76, + "CP", + "structure_element" + ] + ] + }, + { + "sid": 144, + "sent": "Structures of translocation complexes of the bacterial 70S ribosome bound with two tRNAs and yeast 80S complexes with tRNAs are shown in the upper row and labeled.", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 45, + 54, + "bacterial", + "taxonomy_domain" + ], + [ + 55, + 67, + "70S ribosome", + "complex_assembly" + ], + [ + 68, + 78, + "bound with", + "protein_state" + ], + [ + 83, + 88, + "tRNAs", + "chemical" + ], + [ + 93, + 98, + "yeast", + "taxonomy_domain" + ], + [ + 99, + 102, + "80S", + "complex_assembly" + ], + [ + 103, + 117, + "complexes with", + "protein_state" + ], + [ + 118, + 123, + "tRNAs", + "chemical" + ] + ] + }, + { + "sid": 145, + "sent": "Structures of 80S\u2022IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the lower row and labeled.", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 14, + 22, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 40, + 50, + "absence of", + "protein_state" + ], + [ + 51, + 55, + "eEF2", + "protein" + ], + [ + 57, + 61, + "INIT", + "complex_assembly" + ], + [ + 85, + 96, + "presence of", + "protein_state" + ], + [ + 97, + 101, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 146, + "sent": "Interactions of the TSV IRES with uL5 and eL42.", + "section": "FIG", + "ner": [ + [ + 20, + 23, + "TSV", + "species" + ], + [ + 24, + 28, + "IRES", + "site" + ], + [ + 34, + 37, + "uL5", + "protein" + ], + [ + 42, + 46, + "eL42", + "protein" + ] + ] + }, + { + "sid": 147, + "sent": "Structures of 80S\u2022IRES complexes in the absence of eEF2 (INIT; PDB 3J6Y,) and in the presence of eEF2 (this work) are shown in the upper row and labeled.", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 14, + 22, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 40, + 50, + "absence of", + "protein_state" + ], + [ + 51, + 55, + "eEF2", + "protein" + ], + [ + 57, + 61, + "INIT", + "complex_assembly" + ], + [ + 85, + 96, + "presence of", + "protein_state" + ], + [ + 97, + 101, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 148, + "sent": "Structures of the 80S complexes with tRNAs are shown in the lower row in a view similar to that for the 80S\u2022IRES complex.", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 18, + 21, + "80S", + "complex_assembly" + ], + [ + 22, + 36, + "complexes with", + "protein_state" + ], + [ + 37, + 42, + "tRNAs", + "chemical" + ], + [ + 104, + 112, + "80S\u2022IRES", + "complex_assembly" + ] + ] + }, + { + "sid": 149, + "sent": "Positions of the IRES relative to eEF2 and elements of the ribosome in Structures I through V.", + "section": "FIG", + "ner": [ + [ + 17, + 21, + "IRES", + "site" + ], + [ + 34, + 38, + "eEF2", + "protein" + ], + [ + 59, + 67, + "ribosome", + "complex_assembly" + ], + [ + 71, + 93, + "Structures I through V", + "evidence" + ] + ] + }, + { + "sid": 150, + "sent": "(a) Secondary structure of the TSV IRES.", + "section": "FIG", + "ner": [ + [ + 14, + 23, + "structure", + "evidence" + ], + [ + 31, + 34, + "TSV", + "species" + ], + [ + 35, + 39, + "IRES", + "site" + ] + ] + }, + { + "sid": 151, + "sent": "\u00a0The TSV IRES comprises two domains: the 5' domain (blue) and the PKI domain (red).", + "section": "FIG", + "ner": [ + [ + 5, + 8, + "TSV", + "species" + ], + [ + 9, + 13, + "IRES", + "site" + ], + [ + 41, + 50, + "5' domain", + "structure_element" + ], + [ + 66, + 69, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 152, + "sent": "The open reading frame (gray) is immediately following pseudoknot I (PKI).", + "section": "FIG", + "ner": [ + [ + 4, + 22, + "open reading frame", + "structure_element" + ], + [ + 55, + 67, + "pseudoknot I", + "structure_element" + ], + [ + 69, + 72, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 153, + "sent": "(b) Three-dimensional structure of the TSV IRES (Structure II).", + "section": "FIG", + "ner": [ + [ + 22, + 31, + "structure", + "evidence" + ], + [ + 39, + 42, + "TSV", + "species" + ], + [ + 43, + 47, + "IRES", + "site" + ], + [ + 49, + 61, + "Structure II", + "evidence" + ] + ] + }, + { + "sid": 154, + "sent": "Pseudoknots and stem loops are labeled and colored as in (a).", + "section": "FIG", + "ner": [ + [ + 16, + 26, + "stem loops", + "structure_element" + ] + ] + }, + { + "sid": 155, + "sent": "(c) Positions of the IRES and eEF2 on the small subunit in Structures I to V. The initiation-state IRES is shown in gray.", + "section": "FIG", + "ner": [ + [ + 21, + 25, + "IRES", + "site" + ], + [ + 30, + 34, + "eEF2", + "protein" + ], + [ + 42, + 55, + "small subunit", + "structure_element" + ], + [ + 59, + 76, + "Structures I to V", + "evidence" + ], + [ + 82, + 92, + "initiation", + "protein_state" + ], + [ + 99, + 103, + "IRES", + "site" + ] + ] + }, + { + "sid": 156, + "sent": "\u00a0The insert shows density for interaction of diphthamide 699 (eEF2; green) with the codon-anticodon-like helix (PKI; red) in Structure V. (d and e) Density of the P site in Structure V shows that interactions of PKI with the 18S rRNA nucleotides (c) are nearly identical to those in the P site of the 2tRNA\u2022mRNA-bound 70S ribosome (d).", + "section": "FIG", + "ner": [ + [ + 61, + 65, + "eEF2", + "protein" + ], + [ + 111, + 114, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 157, + "sent": "In each structure, the TSV IRES adopts a distinct conformation in the intersubunit space of the ribosome (Figures 3 and 4).", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "structure", + "evidence" + ], + [ + 23, + 26, + "TSV", + "species" + ], + [ + 27, + 31, + "IRES", + "site" + ], + [ + 96, + 104, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 158, + "sent": "The IRES (nt 6758\u20136952) consists of two globular parts (Figure 3a): the 5\u2019-region (domains I and II, nt 6758\u20136888) and the PKI domain (domain III, nt 6889\u20136952).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "IRES", + "site" + ], + [ + 13, + 22, + "6758\u20136952", + "residue_range" + ], + [ + 72, + 81, + "5\u2019-region", + "structure_element" + ], + [ + 91, + 92, + "I", + "structure_element" + ], + [ + 97, + 99, + "II", + "structure_element" + ], + [ + 104, + 113, + "6758\u20136888", + "residue_range" + ], + [ + 123, + 126, + "PKI", + "structure_element" + ], + [ + 142, + 145, + "III", + "structure_element" + ], + [ + 150, + 159, + "6889\u20136952", + "residue_range" + ] + ] + }, + { + "sid": 159, + "sent": "We collectively term domains I and II the 5\u2019 domain.", + "section": "RESULTS", + "ner": [ + [ + 29, + 30, + "I", + "structure_element" + ], + [ + 35, + 37, + "II", + "structure_element" + ], + [ + 42, + 51, + "5\u2019 domain", + "structure_element" + ] + ] + }, + { + "sid": 160, + "sent": "The PKI domain comprises PKI and stem loop 3 (SL3), which stacks on top of the stem of PKI.", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "PKI", + "structure_element" + ], + [ + 25, + 28, + "PKI", + "structure_element" + ], + [ + 33, + 44, + "stem loop 3", + "structure_element" + ], + [ + 46, + 49, + "SL3", + "structure_element" + ], + [ + 87, + 90, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 161, + "sent": "The 6953GCU triplet immediately following the PKI domain is the first codon of the open reading frame.", + "section": "RESULTS", + "ner": [ + [ + 46, + 49, + "PKI", + "structure_element" + ], + [ + 83, + 101, + "open reading frame", + "structure_element" + ] + ] + }, + { + "sid": 162, + "sent": "In the eEF2-free 80S\u2022IRES initiation complex (INIT), the bulk of the 5\u2019-domain (nt.", + "section": "RESULTS", + "ner": [ + [ + 7, + 16, + "eEF2-free", + "protein_state" + ], + [ + 17, + 25, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 26, + 36, + "initiation", + "protein_state" + ], + [ + 46, + 50, + "INIT", + "complex_assembly" + ], + [ + 69, + 78, + "5\u2019-domain", + "structure_element" + ] + ] + }, + { + "sid": 163, + "sent": "6758\u20136888) binds near the E site, contacting the ribosome mostly by means of three protruding structural elements: the L1.1 region and stem loops 4 and 5 (SL4 and SL5).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "6758\u20136888", + "residue_range" + ], + [ + 26, + 32, + "E site", + "site" + ], + [ + 49, + 57, + "ribosome", + "complex_assembly" + ], + [ + 119, + 130, + "L1.1 region", + "structure_element" + ], + [ + 135, + 153, + "stem loops 4 and 5", + "structure_element" + ], + [ + 155, + 158, + "SL4", + "structure_element" + ], + [ + 163, + 166, + "SL5", + "structure_element" + ] + ] + }, + { + "sid": 164, + "sent": "In Structures I to IV, these contacts remain as in the initiation complex (Figure 1a).", + "section": "RESULTS", + "ner": [ + [ + 3, + 21, + "Structures I to IV", + "evidence" + ], + [ + 55, + 73, + "initiation complex", + "complex_assembly" + ] + ] + }, + { + "sid": 165, + "sent": "Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3\u2014figure supplement 2 and Figure 3\u2014figure supplement 3; ribosomal proteins are termed according to).", + "section": "RESULTS", + "ner": [ + [ + 18, + 29, + "L1.1 region", + "structure_element" + ], + [ + 49, + 57, + "L1 stalk", + "structure_element" + ], + [ + 65, + 78, + "large subunit", + "structure_element" + ], + [ + 86, + 89, + "SL4", + "structure_element" + ], + [ + 94, + 97, + "SL5", + "structure_element" + ], + [ + 122, + 125, + "40S", + "complex_assembly" + ], + [ + 126, + 130, + "head", + "structure_element" + ], + [ + 158, + 161, + "uS7", + "protein" + ], + [ + 163, + 167, + "uS11", + "protein" + ], + [ + 172, + 176, + "eS25", + "protein" + ] + ] + }, + { + "sid": 166, + "sent": "In Structures I-IV, the minor groove of SL4 (at nt 6840\u20136846) binds next to an \u03b1-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222).", + "section": "RESULTS", + "ner": [ + [ + 3, + 18, + "Structures I-IV", + "evidence" + ], + [ + 24, + 36, + "minor groove", + "site" + ], + [ + 40, + 43, + "SL4", + "structure_element" + ], + [ + 51, + 60, + "6840\u20136846", + "residue_range" + ], + [ + 79, + 86, + "\u03b1-helix", + "structure_element" + ], + [ + 90, + 93, + "uS7", + "protein" + ], + [ + 141, + 145, + "K212", + "residue_name_number" + ], + [ + 147, + 151, + "K213", + "residue_name_number" + ], + [ + 153, + 157, + "R219", + "residue_name_number" + ], + [ + 162, + 166, + "K222", + "residue_name_number" + ] + ] + }, + { + "sid": 167, + "sent": "The tip of SL4 binds in the vicinity of R157 in the \u03b2-hairpin of uS7 and of Y58 in uS11.", + "section": "RESULTS", + "ner": [ + [ + 11, + 14, + "SL4", + "structure_element" + ], + [ + 40, + 44, + "R157", + "residue_name_number" + ], + [ + 52, + 61, + "\u03b2-hairpin", + "structure_element" + ], + [ + 65, + 68, + "uS7", + "protein" + ], + [ + 76, + 79, + "Y58", + "residue_name_number" + ], + [ + 83, + 87, + "uS11", + "protein" + ] + ] + }, + { + "sid": 168, + "sent": "The minor groove of SL5 (at nt 6862\u20136868) contacts the positively charged region of eS25 (R49, R58 and R68) (Figure 3\u2014figure supplement 4).", + "section": "RESULTS", + "ner": [ + [ + 4, + 16, + "minor groove", + "site" + ], + [ + 20, + 23, + "SL5", + "structure_element" + ], + [ + 31, + 40, + "6862\u20136868", + "residue_range" + ], + [ + 84, + 88, + "eS25", + "protein" + ], + [ + 90, + 93, + "R49", + "residue_name_number" + ], + [ + 95, + 98, + "R58", + "residue_name_number" + ], + [ + 103, + 106, + "R68", + "residue_name_number" + ] + ] + }, + { + "sid": 169, + "sent": "In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20\u2009\u00c5 away from its initial position (Figure 3\u2014figure supplement 2c).", + "section": "RESULTS", + "ner": [ + [ + 3, + 14, + "Structure V", + "evidence" + ], + [ + 29, + 36, + "density", + "evidence" + ], + [ + 41, + 44, + "SL5", + "structure_element" + ], + [ + 72, + 75, + "SL5", + "structure_element" + ], + [ + 79, + 85, + "mobile", + "protein_state" + ], + [ + 98, + 101, + "SL4", + "structure_element" + ], + [ + 102, + 109, + "density", + "evidence" + ], + [ + 124, + 127, + "SL4", + "structure_element" + ], + [ + 160, + 163, + "uS7", + "protein" + ] + ] + }, + { + "sid": 170, + "sent": "The L1.1 region remains in contact with the L1 stalk (Figure 3\u2014figure supplement 3).", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "L1.1 region", + "structure_element" + ], + [ + 44, + 52, + "L1 stalk", + "structure_element" + ] + ] + }, + { + "sid": 171, + "sent": "Inchworm-like translocation of the TSV IRES.", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "Inchworm", + "protein_state" + ], + [ + 35, + 38, + "TSV", + "species" + ], + [ + 39, + 43, + "IRES", + "site" + ] + ] + }, + { + "sid": 172, + "sent": "Conformations and positions of the IRES in the initiation state and in Structures I-V are shown relative to those of the A-, P- and E-site tRNAs.", + "section": "FIG", + "ner": [ + [ + 35, + 39, + "IRES", + "site" + ], + [ + 47, + 57, + "initiation", + "protein_state" + ], + [ + 71, + 85, + "Structures I-V", + "evidence" + ], + [ + 121, + 138, + "A-, P- and E-site", + "site" + ], + [ + 139, + 144, + "tRNAs", + "chemical" + ] + ] + }, + { + "sid": 173, + "sent": "The view was obtained by structural alignment of the body domains of 18S rRNAs of the corresponding 80S structures.", + "section": "FIG", + "ner": [ + [ + 25, + 45, + "structural alignment", + "experimental_method" + ], + [ + 53, + 57, + "body", + "structure_element" + ], + [ + 69, + 78, + "18S rRNAs", + "chemical" + ], + [ + 100, + 103, + "80S", + "complex_assembly" + ], + [ + 104, + 114, + "structures", + "evidence" + ] + ] + }, + { + "sid": 174, + "sent": "Distances between nucleotides 6848 and 6913 in SL4 and PKI, respectively, are shown (see also Figure 2\u2014source data 1).", + "section": "FIG", + "ner": [ + [ + 30, + 34, + "6848", + "residue_number" + ], + [ + 39, + 43, + "6913", + "residue_number" + ], + [ + 47, + 50, + "SL4", + "structure_element" + ], + [ + 55, + 58, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 175, + "sent": "The shape of the IRES changes considerably from the initiation state to Structures I through V, from an extended to compact to extended conformation (Figure 4; see also Figure 3\u2014figure supplement 2a).", + "section": "RESULTS", + "ner": [ + [ + 17, + 21, + "IRES", + "site" + ], + [ + 52, + 62, + "initiation", + "protein_state" + ], + [ + 72, + 94, + "Structures I through V", + "evidence" + ], + [ + 104, + 112, + "extended", + "protein_state" + ], + [ + 116, + 123, + "compact", + "protein_state" + ], + [ + 127, + 135, + "extended", + "protein_state" + ] + ] + }, + { + "sid": 176, + "sent": "Because in Structures I to IV the PKI domain shifts toward the P site, while the 5\u2019 remains unchanged near the E site, the distance between the domains shortens (Figure 4).", + "section": "RESULTS", + "ner": [ + [ + 11, + 29, + "Structures I to IV", + "evidence" + ], + [ + 34, + 37, + "PKI", + "structure_element" + ], + [ + 63, + 69, + "P site", + "site" + ], + [ + 111, + 117, + "E site", + "site" + ] + ] + }, + { + "sid": 177, + "sent": "In the 80S\u2022IRES initiation state, the A-site-bound PKI is separated from SL4 by almost 50\u2009\u00c5 (Figure 4).", + "section": "RESULTS", + "ner": [ + [ + 7, + 15, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 16, + 26, + "initiation", + "protein_state" + ], + [ + 38, + 50, + "A-site-bound", + "protein_state" + ], + [ + 51, + 54, + "PKI", + "structure_element" + ], + [ + 73, + 76, + "SL4", + "structure_element" + ] + ] + }, + { + "sid": 178, + "sent": "In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35\u2009\u00c5. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25\u2009\u00c5 of SL4.", + "section": "RESULTS", + "ner": [ + [ + 3, + 22, + "Structures I and II", + "evidence" + ], + [ + 28, + 31, + "PKI", + "structure_element" + ], + [ + 64, + 70, + "A site", + "site" + ], + [ + 93, + 96, + "SL4", + "structure_element" + ], + [ + 119, + 122, + "PKI", + "structure_element" + ], + [ + 140, + 146, + "P site", + "site" + ], + [ + 150, + 171, + "Structures III and IV", + "evidence" + ], + [ + 177, + 180, + "PKI", + "structure_element" + ], + [ + 218, + 221, + "SL4", + "structure_element" + ] + ] + }, + { + "sid": 179, + "sent": "Because the 5\u2019-domain in the following structure (V) moves by ~20\u2009\u00c5 along the 40S head, the IRES returns to an extended conformation (~45\u2009\u00c5) that is similar to that in the 80S\u2022IRES initiation complex.", + "section": "RESULTS", + "ner": [ + [ + 12, + 21, + "5\u2019-domain", + "structure_element" + ], + [ + 39, + 52, + "structure (V)", + "evidence" + ], + [ + 78, + 81, + "40S", + "complex_assembly" + ], + [ + 82, + 86, + "head", + "structure_element" + ], + [ + 92, + 96, + "IRES", + "site" + ], + [ + 111, + 119, + "extended", + "protein_state" + ], + [ + 172, + 180, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 181, + 191, + "initiation", + "protein_state" + ] + ] + }, + { + "sid": 180, + "sent": "Rearrangements of the IRES involve restructuring of several interactions with the ribosome.", + "section": "RESULTS", + "ner": [ + [ + 22, + 26, + "IRES", + "site" + ], + [ + 82, + 90, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 181, + "sent": "In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008\u20131043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt.", + "section": "RESULTS", + "ner": [ + [ + 3, + 14, + "Structure I", + "evidence" + ], + [ + 16, + 19, + "SL3", + "structure_element" + ], + [ + 27, + 30, + "PKI", + "structure_element" + ], + [ + 64, + 77, + "A-site finger", + "structure_element" + ], + [ + 82, + 91, + "1008\u20131043", + "residue_range" + ], + [ + 95, + 103, + "25S rRNA", + "chemical" + ], + [ + 113, + 119, + "P site", + "site" + ], + [ + 127, + 130, + "60S", + "complex_assembly" + ], + [ + 131, + 138, + "subunit", + "structure_element" + ], + [ + 151, + 159, + "helix 84", + "structure_element" + ], + [ + 163, + 171, + "25S rRNA", + "chemical" + ] + ] + }, + { + "sid": 182, + "sent": "2668\u20132687) and protein uL5 (Figure 3\u2014figure supplement 6).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "2668\u20132687", + "residue_range" + ], + [ + 23, + 26, + "uL5", + "protein" + ] + ] + }, + { + "sid": 183, + "sent": "This position of SL3 is ~25\u2009\u00c5 away from that in the 80S\u2022IRES initiation state, in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively.", + "section": "RESULTS", + "ner": [ + [ + 17, + 20, + "SL3", + "structure_element" + ], + [ + 52, + 60, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 61, + 71, + "initiation", + "protein_state" + ], + [ + 88, + 91, + "PKI", + "structure_element" + ], + [ + 96, + 99, + "SL3", + "structure_element" + ], + [ + 118, + 121, + "ASL", + "structure_element" + ], + [ + 126, + 131, + "elbow", + "structure_element" + ], + [ + 139, + 145, + "A-site", + "site" + ], + [ + 146, + 150, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 184, + "sent": "As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA.", + "section": "RESULTS", + "ner": [ + [ + 33, + 43, + "initiation", + "protein_state" + ], + [ + 53, + 64, + "Structure I", + "evidence" + ], + [ + 91, + 94, + "SL3", + "structure_element" + ], + [ + 106, + 119, + "A-site finger", + "structure_element" + ], + [ + 159, + 176, + "pre-translocation", + "protein_state" + ], 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3\u2014figure supplement 7).", + "section": "RESULTS", + "ner": [ + [ + 7, + 18, + "highly bent", + "protein_state" + ], + [ + 19, + 40, + "Structures III and IV", + "evidence" + ], + [ + 46, + 58, + "hinge region", + "structure_element" + ], + [ + 78, + 99, + "universally conserved", + "protein_state" + ], + [ + 100, + 103, + "uL5", + "protein" + ], + [ + 112, + 127, + "C-terminal tail", + "structure_element" + ], + [ + 131, + 135, + "eL42", + "protein" + ] + ] + }, + { + "sid": 187, + "sent": "However, in the extended conformations, these parts of the IRES and the 60S subunit are separated by more than 10\u2009\u00c5, suggesting that an interaction between them stabilizes the bent conformations but not the extended ones.", + "section": "RESULTS", + "ner": [ + [ + 16, + 24, + "extended", + "protein_state" + ], + [ + 59, + 63, + "IRES", + "site" + ], + [ + 72, + 75, + "60S", + "complex_assembly" + ], + [ + 76, + 83, + "subunit", + "structure_element" + ], + [ + 176, + 180, + "bent", + "protein_state" + ], + [ + 207, + 215, + "extended", + "protein_state" + ] + ] + }, + { + "sid": 188, + "sent": "Another local rearrangement concerns loop 3, also known as the variable loop region\u00a0, which connects the ASL- and mRNA-like parts of PKI.", + "section": "RESULTS", + "ner": [ + [ + 37, + 43, + "loop 3", + "structure_element" + ], + [ + 63, + 83, + "variable loop region", + "structure_element" + ], + [ + 105, + 129, + "ASL- and mRNA-like parts", + "structure_element" + ], + [ + 133, + 136, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 189, + "sent": "This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI and biochemical studies of unbound IRESs.", + "section": "RESULTS", + "ner": [ + [ + 5, + 9, + "loop", + "structure_element" + ], + [ + 32, + 55, + "Structures I through IV", + "evidence" + ], + [ + 113, + 131, + "structural studies", + "experimental_method" + ], + 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"Elements of the 80S ribosome that contact eEF2 in Structures I through V.", + "section": "FIG", + "ner": [ + [ + 16, + 28, + "80S ribosome", + "complex_assembly" + ], + [ + 42, + 46, + "eEF2", + "protein" + ], + [ + 50, + 72, + "Structures I through V", + "evidence" + ] + ] + }, + { + "sid": 197, + "sent": "The view and colors are as in Figure 5b: eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal.", + "section": "FIG", + "ner": [ + [ + 41, + 45, + "eEF2", + "protein" + ], + [ + 65, + 69, + "IRES", + "site" + ], + [ + 70, + 73, + "RNA", + "chemical" + ], + [ + 82, + 85, + "40S", + "complex_assembly" + ], + [ + 86, + 93, + "subunit", + "structure_element" + ], + [ + 86, + 93, + "subunit", + "structure_element" + ], + [ + 114, + 117, + "60S", + "complex_assembly" + ] + ] + }, + { + "sid": 198, + "sent": "Cryo-EM density of the GTPase region in Structures I and II.", + "section": "FIG", + "ner": [ + [ + 0, + 7, + "Cryo-EM", + "experimental_method" + ], + [ + 8, + 15, + "density", + "evidence" + ], + [ + 23, + 36, + "GTPase region", + "structure_element" + ], + [ + 40, + 59, + "Structures I and II", + "evidence" + ] + ] + }, + { + "sid": 199, + "sent": "The switch loop I in Structure I is shown in blue.", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "switch loop I", + "structure_element" + ], + [ + 21, + 32, + "Structure I", + "evidence" + ] + ] + }, + { + "sid": 200, + "sent": "The putative position of the switch loop I, unresolved in the density of Structure II, is shown with a dashed line.", + "section": "FIG", + "ner": [ + [ + 29, + 42, + "switch loop I", + "structure_element" + ], + [ + 62, + 69, + "density", + "evidence" + ], + [ + 73, + 85, + "Structure II", + "evidence" + ] + ] + }, + { + "sid": 201, + "sent": "Colors for the ribosome and eEF2 are as in Figure 1.", + "section": "FIG", + "ner": [ + [ + 15, + 23, + "ribosome", + "complex_assembly" + ], + [ + 28, + 32, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 202, + "sent": "Conformations and interactions of eEF2.", + "section": "FIG", + "ner": [ + [ + 34, + 38, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 203, + "sent": "(a) Conformations of eEF2 in Structures I-V and domain organization of eEF2 are shown.", + "section": "FIG", + "ner": [ + [ + 21, + 25, + "eEF2", + "protein" + ], + [ + 29, + 43, + "Structures I-V", + "evidence" + ], + [ + 71, + 75, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 204, + "sent": "Roman numerals denote eEF2 domains.", + "section": "FIG", + "ner": [ + [ + 22, + 26, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 205, + "sent": "Superposition was obtained by structural alignment of domains I and II.", + "section": "FIG", + "ner": [ + [ + 0, + 13, + "Superposition", + "experimental_method" + ], + [ + 30, + 50, + "structural alignment", + "experimental_method" + ], + [ + 62, + 63, + "I", + "structure_element" + ], + [ + 68, + 70, + "II", + "structure_element" + ] + ] + }, + { + "sid": 206, + "sent": "(b) Elements of the 80S ribosome in Structures I and V that contact eEF2.", + "section": "FIG", + "ner": [ + [ + 20, + 32, + "80S ribosome", + "complex_assembly" + ], + [ + 36, + 54, + "Structures I and V", + "evidence" + ], + [ + 68, + 72, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 207, + "sent": "eEF2 is shown in green, IRES RNA in red, 40S subunit elements in orange, 60S in cyan/teal.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "eEF2", + "protein" + ], + [ + 24, + 28, + "IRES", + "site" + ], + [ + 29, + 32, + "RNA", + "chemical" + ], + [ + 41, + 44, + "40S", + "complex_assembly" + ], + [ + 45, + 52, + "subunit", + "structure_element" + ], + [ + 73, + 76, + "60S", + "complex_assembly" + ] + ] + }, + { + "sid": 208, + "sent": "(c) Comparison of conformations of eEF2\u2022sordarin in Structure I (light green) with those of free apo-eEF2 (magenta) and eEF2\u2022sordarin (teal).", + "section": "FIG", + "ner": [ + [ + 35, + 48, + "eEF2\u2022sordarin", + "complex_assembly" + ], + [ + 52, + 63, + 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"Switch loop I (SWI) in Structure I is in blue; dashed line shows the putative location of unresolved switch loop I in Structure II.", + "section": "FIG", + "ner": [ + [ + 0, + 13, + "Switch loop I", + "structure_element" + ], + [ + 15, + 18, + "SWI", + "structure_element" + ], + [ + 23, + 34, + "Structure I", + "evidence" + ], + [ + 101, + 114, + "switch loop I", + "structure_element" + ], + [ + 118, + 130, + "Structure II", + "evidence" + ] + ] + }, + { + "sid": 211, + "sent": "Superposition was obtained by structural alignment of the 25S rRNAs.", + "section": "FIG", + "ner": [ + [ + 0, + 13, + "Superposition", + "experimental_method" + ], + [ + 30, + 50, + "structural alignment", + "experimental_method" + ], + [ + 58, + 67, + "25S rRNAs", + "chemical" + ] + ] + }, + { + "sid": 212, + "sent": "(e) Comparison of the GTP-like conformation of eEF2\u2022GDP in Structure I (light green) with those of 70S-bound elongation factors EF-Tu\u2022GDPCP (teal) and EF-G\u2022GDP\u2022fusidic acid (magenta; fusidic acid not shown). (f) Cryo-EM density showing guanosine diphosphate bound in the GTPase center (green) next to the sarcin-ricin loop of 25S rRNA (cyan) of Structure II. (g) Comparison of the sordarin-binding sites in the ribosome-bound (light green; Structure II) and isolated eEF2 (teal).", + "section": "FIG", + "ner": [ + [ + 22, + 30, + "GTP-like", + "protein_state" + ], + [ + 47, + 55, + "eEF2\u2022GDP", + "complex_assembly" + ], + [ + 59, + 70, + "Structure I", + "evidence" + ], + [ + 99, + 108, + "70S-bound", + "protein_state" + ], + [ + 109, + 127, + "elongation factors", + "protein_type" + ], + [ + 128, + 139, + "EF-Tu\u2022GDPCP", + "complex_assembly" + ], + [ + 151, + 172, + "EF-G\u2022GDP\u2022fusidic acid", + "complex_assembly" + ], + [ + 212, + 219, + "Cryo-EM", + "experimental_method" + ], + [ + 220, + 227, + "density", + "evidence" + ], + [ + 236, + 257, + "guanosine diphosphate", + "chemical" + ], + [ + 258, + 266, + "bound in", + "protein_state" + ], + [ + 271, + 284, + "GTPase center", + "site" + ], + [ + 305, + 322, + "sarcin-ricin loop", + "structure_element" + ], + [ + 326, + 334, + "25S rRNA", + "chemical" + ], + [ + 345, + 357, + "Structure II", + "evidence" + ], + [ + 381, + 403, + "sordarin-binding sites", + "site" + ], + [ + 411, + 425, + "ribosome-bound", + "protein_state" + ], + [ + 440, + 452, + "Structure II", + "evidence" + ], + [ + 467, + 471, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 213, + "sent": "(h) Cryo-EM density showing the sordarin-binding pocket of eEF2 (Structure II).", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "Cryo-EM", + "experimental_method" + ], + [ + 12, + 19, + "density", + "evidence" + ], + [ + 32, + 55, + "sordarin-binding pocket", + "site" + ], + [ + 59, + 63, + "eEF2", + "protein" + ], + [ + 65, + 77, + "Structure II", + "evidence" + ] + ] + }, + { + "sid": 214, + "sent": "Sordarin is shown in pink with oxygen atoms in red.", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "Sordarin", + "chemical" + ] + ] + }, + { + "sid": 215, + "sent": "Elongation factor eEF2 in all five structures is bound with GDP and sordarin (Figure 5).", + "section": "RESULTS", + "ner": [ + [ + 0, + 17, + "Elongation factor", + "protein_type" + ], + [ + 18, + 22, + "eEF2", + "protein" + ], + [ + 35, + 45, + "structures", + "evidence" + ], + [ + 49, + 59, + "bound with", + "protein_state" + ], + [ + 60, + 63, + "GDP", + "chemical" + ], + [ + 68, + 76, + "sordarin", + "chemical" + ] + ] + }, + { + "sid": 216, + "sent": "The elongation factor consists of three dynamic superdomains: an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a).", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "elongation factor", + "protein_type" + ], + [ + 48, + 60, + "superdomains", + "structure_element" + ], + [ + 85, + 96, + "superdomain", + "structure_element" + ], + [ + 111, + 128, + "G (GTPase) domain", + "structure_element" + ], + [ + 137, + 138, + "I", + "structure_element" + ], + [ + 151, + 153, + "II", + "structure_element" + ], + [ + 157, + 174, + "linker domain III", + "structure_element" + ], + [ + 193, + 204, + "superdomain", + "structure_element" + ], + [ + 224, + 226, + "IV", + "structure_element" + ], + [ + 231, + 232, + "V", + "structure_element" + ] + ] + }, + { + "sid": 217, + "sent": "Domain IV extends from the main body and is critical for translocation catalyzed by eEF2\u00a0or\u00a0EF-G. ADP-ribosylation of eEF2 at the tip of domain IV or deletion of domain IV from EF-G abrogate translocation.", + "section": "RESULTS", + "ner": [ + [ + 7, + 9, + "IV", + "structure_element" + ], + [ + 32, + 36, + "body", + "structure_element" + ], + [ + 84, + 88, + "eEF2", + "protein" + ], + [ + 92, + 96, + "EF-G", + "protein" + ], + [ + 98, + 114, + "ADP-ribosylation", + "ptm" + ], + [ + 118, + 122, + "eEF2", + "protein" + ], + [ + 144, + 146, + "IV", + "structure_element" + ], + [ + 150, + 158, + "deletion", + "experimental_method" + ], + [ + 169, + 171, + "IV", + "structure_element" + ], + [ + 177, + 181, + "EF-G", + "protein" + ] + ] + }, + { + "sid": 218, + "sent": "In post-translocation-like 80S\u2022tRNA\u2022eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site.", + "section": "RESULTS", + "ner": [ + [ + 3, + 21, + "post-translocation", + "protein_state" + ], + [ + 27, + 40, + "80S\u2022tRNA\u2022eEF2", + "complex_assembly" + ], + [ + 59, + 61, + "IV", + "structure_element" + ], + [ + 75, + 78, + "40S", + "complex_assembly" + ], + [ + 79, + 85, + "A site", + "site" + ], + [ + 127, + 129, + "IV", + "structure_element" + ], + [ + 150, + 154, + "tRNA", + "chemical" + ], + [ + 164, + 175, + "A to P site", + "site" + ] + ] + }, + { + "sid": 219, + "sent": "GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the \u03b2-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2\u2022sordarin complex.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "GDP", + "chemical" + ], + [ + 11, + 21, + "structures", + "evidence" + ], + [ + 25, + 33, + "bound in", + "protein_state" + ], + [ + 38, + 51, + "GTPase center", + "site" + ], + [ + 78, + 86, + "sordarin", + "chemical" + ], + [ + 113, + 124, + "\u03b2-platforms", + "structure_element" + ], + [ + 136, + 139, + "III", + "structure_element" + ], + [ + 144, + 145, + "V", + "structure_element" + ], + [ + 176, + 185, + "structure", + "evidence" + ], + [ + 189, + 193, + "free", + "protein_state" + ], + [ + 194, + 207, + "eEF2\u2022sordarin", + "complex_assembly" + ] + ] + }, + { + "sid": 220, + "sent": "The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD \u2264\u00a02\u00a0\u00c5), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2\u2014source data 1).", + "section": "RESULTS", + "ner": [ + [ + 28, + 32, + "eEF2", + "protein" + ], + [ + 66, + 76, + "structures", + "evidence" + ], + [ + 87, + 91, + "RMSD", + "evidence" + ], + [ + 121, + 125, + "eEF2", + "protein" + ], + [ + 142, + 145, + "40S", + "complex_assembly" + ], + [ + 146, + 153, + "subunit", + "structure_element" + ], + [ + 190, + 193, + "40S", + "complex_assembly" + ], + [ + 194, + 201, + "subunit", + "structure_element" + ] + ] + }, + { + "sid": 221, + "sent": "From Structure I to V, eEF2 is rigidly attached to the GTPase-associated center of the 60S subunit.", + "section": "RESULTS", + "ner": [ + [ + 5, + 21, + "Structure I to V", + "evidence" + ], + [ + 23, + 27, + "eEF2", + "protein" + ], + [ + 55, + 79, + "GTPase-associated center", + "site" + ], + [ + 87, + 90, + "60S", + "complex_assembly" + ], + [ + 91, + 98, + "subunit", + "structure_element" + ] + ] + }, + { + "sid": 222, + "sent": "The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012\u20133042).", + "section": "RESULTS", + "ner": [ + [ + 4, + 28, + "GTPase-associated center", + "site" + ], + [ + 43, + 50, + "P stalk", + "structure_element" + ], + [ + 52, + 55, + "L11", + "structure_element" + ], + [ + 60, + 62, + "L7", + "structure_element" + ], + [ + 63, + 66, + "L12", + "structure_element" + ], + [ + 67, + 72, + "stalk", + "structure_element" + ], + [ + 76, + 84, + "bacteria", + "taxonomy_domain" + ], + [ + 94, + 111, + "sarcin-ricin loop", + "structure_element" + ], + [ + 113, + 116, + "SRL", + "structure_element" + ], + [ + 121, + 130, + "3012\u20133042", + "residue_range" + ] + ] + }, + { + "sid": 223, + "sent": "The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An \u03b1\u03b2\u03b2 motif of the eukaryote-specific protein P0 (aa 126\u2013154) packs in the crevice between the long \u03b1-helix D (aa 172\u2013188) of the GTPase domain and the \u03b2-sheet region (aa 246\u2013263) of the GTPase domain insert (or G\u2019 insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G are the same).", + "section": "RESULTS", + "ner": [ + [ + 12, + 20, + "25S rRNA", + "chemical" + ], + [ + 21, + 38, + "helices 43 and 44", + "structure_element" + ], + [ + 46, + 53, + "P stalk", + "structure_element" + ], + [ + 67, + 72, + "G1242", + "residue_name_number" + ], + [ + 77, + 82, + "A1270", + "residue_name_number" + ], + [ + 98, + 103, + "stack", + "bond_interaction" + ], + [ + 107, + 111, + "V754", + "residue_name_number" + ], + [ + 116, + 120, + "Y744", + "residue_name_number" + ], + [ + 131, + 132, + "V", + "structure_element" + ], + [ + 137, + 146, + "\u03b1\u03b2\u03b2 motif", + "structure_element" + ], + [ + 154, + 163, + "eukaryote", + "taxonomy_domain" + ], + [ + 181, + 183, + "P0", + "protein" + ], + [ + 188, + 195, + "126\u2013154", + "residue_range" + ], + [ + 230, + 244, + "long \u03b1-helix D", + "structure_element" + ], + [ + 249, + 256, + "172\u2013188", + "residue_range" + ], + [ + 265, + 278, + "GTPase domain", + "structure_element" + ], + [ + 287, + 301, + "\u03b2-sheet region", + "structure_element" + ], + [ + 306, + 313, + "246\u2013263", + "residue_range" + ], + [ + 322, + 342, + "GTPase domain insert", + "structure_element" + ], + [ + 347, + 356, + "G\u2019 insert", + "structure_element" + ], + [ + 361, + 365, + "eEF2", + "protein" + ], + [ + 405, + 409, + "eEF2", + "protein" + ], + [ + 414, + 418, + "EF-G", + "protein" + ] + ] + }, + { + "sid": 224, + "sent": "Although the P/L11 stalk is known to be dynamic, its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223\u20131286) are within 2.5\u2009\u00c5. However, with respect to its position in the 80S\u2022IRES complex in the absence of eEF2 and in the 80S\u20222tRNA\u2022mRNA complex, the P stalk is shifted by ~13\u2009\u00c5 toward the A site (Figure 2d).", + "section": "RESULTS", + "ner": [ + [ + 13, + 24, + "P/L11 stalk", + "structure_element" + ], + [ + 85, + 101, + "Structure I to V", + "evidence" + ], + [ + 112, + 140, + "root-mean-square differences", + "evidence" + ], + [ + 149, + 157, + "25S rRNA", + "chemical" + ], + [ + 165, + 172, + "P stalk", + "structure_element" + ], + [ + 177, + 186, + "1223\u20131286", + "residue_range" + ], + [ + 251, + 259, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 275, + 285, + "absence of", + "protein_state" + ], + [ + 286, + 290, + "eEF2", + "protein" + ], + [ + 302, + 316, + "80S\u20222tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 330, + 337, + "P stalk", + "structure_element" + ], + [ + 369, + 375, + "A site", + "site" + ] + ] + }, + { + "sid": 225, + "sent": "The sarcin-ricin loop interacts with the GTP-binding site of eEF2 (Figures 5d and f).", + "section": "RESULTS", + "ner": [ + [ + 4, + 21, + "sarcin-ricin loop", + "structure_element" + ], + [ + 41, + 57, + "GTP-binding site", + "site" + ], + [ + 61, + 65, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 226, + "sent": "While the overall mode of this interaction is similar to that seen in 70S\u2022EF-G crystal structures, there is an important local difference between Structure I and Structures II-V in switch loop I, as discussed below.", + "section": "RESULTS", + "ner": [ + [ + 70, + 78, + "70S\u2022EF-G", + "complex_assembly" + ], + [ + 79, + 97, + "crystal structures", + "evidence" + ], + [ + 146, + 157, + "Structure I", + "evidence" + ], + [ + 162, + 177, + "Structures II-V", + "evidence" + ], + [ + 181, + 194, + "switch loop I", + "structure_element" + ] + ] + }, + { + "sid": 227, + "sent": "Repositioning (sliding) of the positively-charged cluster of domain IV of eEF2 over the phosphate backbone (red) of the 18S helices 33 and 34.", + "section": "FIG", + "ner": [ + [ + 31, + 57, + "positively-charged cluster", + "site" + ], + [ + 68, + 70, + "IV", + "structure_element" + ], + [ + 74, + 78, + "eEF2", + "protein" + ], + [ + 120, + 141, + "18S helices 33 and 34", + "structure_element" + ] + ] + }, + { + "sid": 228, + "sent": "Structures I through V are shown.", + "section": "FIG", + "ner": [ + [ + 0, + 22, + "Structures I through V", + "evidence" + ] + ] + }, + { + "sid": 229, + "sent": "Electrostatic surface of eEF2 is shown; negatively and positively charged regions are shown in red and blue, respectively.", + "section": "FIG", + "ner": [ + [ + 25, + 29, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 230, + "sent": "The view was obtained by structural alignment of the 18S rRNAs.", + "section": "FIG", + "ner": [ + [ + 25, + 45, + "structural alignment", + "experimental_method" + ], + [ + 53, + 62, + "18S rRNAs", + "chemical" + ] + ] + }, + { + "sid": 231, + "sent": "Interactions of eEF2 with the 40S subunit.", + "section": "FIG", + "ner": [ + [ + 16, + 20, + "eEF2", + "protein" + ], + [ + 30, + 33, + "40S", + "complex_assembly" + ], + [ + 34, + 41, + "subunit", + "structure_element" + ] + ] + }, + { + "sid": 232, + "sent": "(a)\u00a0eEF2 (green) interacts only with the body in Structure I (eEF2 domains are labeled with roman numerals in white), and with both the head and body in Structures II through V. Colors are as in Figure 1, except for the 40S structural elements that contact eEF2, which are labeled and shown in purple. (b)\u00a0Entry of eEF2 into the 40S A site, from Structure I through V. Distances to the A-site accommodated eEF2 (Structure V) are shown.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "eEF2", + "protein" + ], + [ + 41, + 45, + "body", + "structure_element" + ], + [ + 49, + 60, + "Structure I", + "evidence" + ], + [ + 62, + 66, + "eEF2", + "protein" + ], + [ + 136, + 140, + "head", + "structure_element" + ], + [ + 145, + 149, + "body", + "structure_element" + ], + [ + 153, + 176, + "Structures II through V", + "evidence" + ], + [ + 220, + 223, + "40S", + "complex_assembly" + ], + [ + 257, + 261, + "eEF2", + "protein" + ], + [ + 315, + 319, + "eEF2", + "protein" + ], + [ + 329, + 332, + "40S", + "complex_assembly" + ], + [ + 333, + 339, + "A site", + "site" + ], + [ + 346, + 367, + "Structure I through V", + "evidence" + ], + [ + 386, + 392, + "A-site", + "site" + ], + [ + 406, + 410, + "eEF2", + "protein" + ], + [ + 412, + 423, + "Structure V", + "evidence" + ] + ] + }, + { + "sid": 233, + "sent": "The view was obtained by superpositions of the body domains of 18S rRNAs.", + "section": "FIG", + "ner": [ + [ + 25, + 39, + "superpositions", + "experimental_method" + ], + [ + 47, + 51, + "body", + "structure_element" + ], + [ + 63, + 72, + "18S rRNAs", + "chemical" + ] + ] + }, + { + "sid": 234, + "sent": "(c)\u00a0Rearrangements, from Structure I through V, of a positively charged cluster of eEF2 (K613, R617 and R631) positioned over the phosphate backbone of 18S helices 33 and 34, suggesting a role of electrostatic interactions in eEF2 diffusion over the 40S surface.", + "section": "FIG", + "ner": [ + [ + 25, + 46, + "Structure I through V", + "evidence" + ], + [ + 83, + 87, + "eEF2", + "protein" + ], + [ + 89, + 93, + "K613", + "residue_name_number" + ], + [ + 95, + 99, + "R617", + "residue_name_number" + ], + [ + 104, + 108, + "R631", + "residue_name_number" + ], + [ + 152, + 173, + "18S helices 33 and 34", + "structure_element" + ], + [ + 196, + 222, + "electrostatic interactions", + "bond_interaction" + ], + [ + 226, + 230, + "eEF2", + "protein" + ], + [ + 250, + 253, + "40S", + "complex_assembly" + ] + ] + }, + { + "sid": 235, + "sent": "(d) Shift of the tip of domain III of eEF2, interacting with uS12 upon reverse subunit rotation from Structure I to Structure V. Structure I colored as in Figure 1, except uS12, which is in purple; Structure V is in gray.", + "section": "FIG", + "ner": [ + [ + 31, + 34, + "III", + "structure_element" + ], + [ + 38, + 42, + "eEF2", + "protein" + ], + [ + 61, + 65, + "uS12", + "protein" + ], + [ + 79, + 86, + "subunit", + "structure_element" + ], + [ + 101, + 127, + "Structure I to Structure V", + "evidence" + ], + [ + 129, + 140, + "Structure I", + "evidence" + ], + [ + 172, + 176, + "uS12", + "protein" + ], + [ + 198, + 209, + "Structure V", + "evidence" + ] + ] + }, + { + "sid": 236, + "sent": "There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50\u2009\u00c5 of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c), eEF2 undergoes moderate repositioning of domain IV (~3\u2009\u00c5; Figure 5a) and domain III (~5\u2009\u00c5; Figure 6d).", + "section": "RESULTS", + "ner": [ + [ + 66, + 84, + "Structures I and V", + "evidence" + ], + [ + 96, + 100, + "free", + "protein_state" + ], + [ + 101, + 105, + "eEF2", + "protein" + ], + [ + 175, + 186, + "superdomain", + "structure_element" + ], + [ + 214, + 225, + "superdomain", + "structure_element" + ], + [ + 239, + 243, + "eEF2", + "protein" + ], + [ + 287, + 289, + "IV", + "structure_element" + ], + [ + 319, + 322, + "III", + "structure_element" + ] + ] + }, + { + "sid": 237, + "sent": "This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit.", + "section": "RESULTS", + "ner": [ + [ + 32, + 46, + "ribosome-bound", + "protein_state" + ], + [ + 47, + 51, + "eEF2", + "protein" + ], + [ + 101, + 105, + "eEF2", + "protein" + ], + [ + 106, + 118, + "superdomains", + "structure_element" + ], + [ + 132, + 133, + "I", + "structure_element" + ], + [ + 138, + 139, + "V", + "structure_element" + ], + [ + 148, + 172, + "GTPase-associated center", + "site" + ], + [ + 180, + 193, + "large subunit", + "structure_element" + ] + ] + }, + { + "sid": 238, + "sent": "Domain IV of eEF2 binds at the 40S A site in Structures I to V but the mode of interaction differs in each complex (Figure 6).", + "section": "RESULTS", + "ner": [ + [ + 7, + 9, + "IV", + "structure_element" + ], + [ + 13, + 17, + "eEF2", + "protein" + ], + [ + 31, + 34, + "40S", + "complex_assembly" + ], + [ + 35, + 41, + "A site", + "site" + ], + [ + 45, + 62, + "Structures I to V", + "evidence" + ] + ] + }, + { + "sid": 239, + "sent": "Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel.", + "section": "RESULTS", + "ner": [ + [ + 8, + 12, + "eEF2", + "protein" + ], + [ + 40, + 43, + "60S", + "complex_assembly" + ], + [ + 44, + 51, + "subunit", + "structure_element" + ], + [ + 85, + 92, + "subunit", + "structure_element" + ], + [ + 133, + 135, + "IV", + "structure_element" + ], + [ + 145, + 151, + "A site", + "site" + ], + [ + 160, + 178, + "Structures I and V", + "evidence" + ], + [ + 189, + 192, + "40S", + "complex_assembly" + ], + [ + 193, + 200, + "subunit", + "structure_element" + ], + [ + 214, + 218, + "head", + "structure_element" + ] + ] + }, + { + "sid": 240, + "sent": "eEF2 settles into the A site from Structure I to V, as the tip of domain IV shifts by ~10\u2009\u00c5 relative to the body and by ~20\u2009\u00c5 relative to the swiveling head.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "eEF2", + "protein" + ], + [ + 22, + 28, + "A site", + "site" + ], + [ + 34, + 50, + "Structure I to V", + "evidence" + ], + [ + 73, + 75, + "IV", + "structure_element" + ], + [ + 108, + 112, + "body", + "structure_element" + ], + [ + 152, + 156, + "head", + "structure_element" + ] + ] + }, + { + "sid": 241, + "sent": "Modest intra-eEF2 shifts of domain IV between Structures I to V outline a stochastic trajectory (Figure 5a), consistent with local adjustments of the domain in the A site.", + "section": "RESULTS", + "ner": [ + [ + 13, + 17, + "eEF2", + "protein" + ], + [ + 35, + 37, + "IV", + "structure_element" + ], + [ + 46, + 63, + "Structures I to V", + "evidence" + ], + [ + 164, + 170, + "A site", + "site" + ] + ] + }, + { + "sid": 242, + "sent": "At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48\u201352 and 429\u2013432 of 18S rRNA helix 5 and uS12).", + "section": "RESULTS", + "ner": [ + [ + 25, + 29, + "eEF2", + "protein" + ], + [ + 39, + 41, + "II", + "structure_element" + ], + [ + 46, + 49, + "III", + "structure_element" + ], + [ + 62, + 65, + "40S", + "complex_assembly" + ], + [ + 66, + 70, + "body", + "structure_element" + ], + [ + 94, + 99, + "48\u201352", + "residue_range" + ], + [ + 104, + 111, + "429\u2013432", + "residue_range" + ], + [ + 115, + 123, + "18S rRNA", + "chemical" + ], + [ + 124, + 131, + "helix 5", + "structure_element" + ], + [ + 136, + 140, + "uS12", + "protein" + ] + ] + }, + { + "sid": 243, + "sent": "From Structure I to V, these central domains migrate by ~10\u2009\u00c5 along the 40S surface (Figure 6c).", + "section": "RESULTS", + "ner": [ + [ + 5, + 21, + "Structure I to V", + "evidence" + ], + [ + 72, + 75, + "40S", + "complex_assembly" + ] + ] + }, + { + "sid": 244, + "sent": "Comparison of eEF2 conformations reveals that in Structure V, domain III is displaced as a result of interaction with uS12, as discussed below.", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "eEF2", + "protein" + ], + [ + 49, + 60, + "Structure V", + "evidence" + ], + [ + 69, + 72, + "III", + "structure_element" + ], + [ + 118, + 122, + "uS12", + "protein" + ] + ] + }, + { + "sid": 245, + "sent": "In summary, between Structures I and V, a step-wise translocation of PKI by ~15\u2009\u00c5 from the A to P site - within the 40S subunit \u2013 occurs simultaneously with the ~11\u2009\u00c5 side-way entry of domain IV into the A site coupled with ~3 to 5\u2009\u00c5 inter-domain rearrangements in eEF2.", + "section": "RESULTS", + "ner": [ + [ + 20, + 38, + "Structures I and V", + "evidence" + ], + [ + 69, + 72, + "PKI", + "structure_element" + ], + [ + 91, + 102, + "A to P site", + "site" + ], + [ + 116, + 119, + "40S", + "complex_assembly" + ], + [ + 120, + 127, + "subunit", + "structure_element" + ], + [ + 192, + 194, + "IV", + "structure_element" + ], + [ + 204, + 210, + "A site", + "site" + ], + [ + 265, + 269, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 246, + "sent": "These shifts occur during the reverse rotation of the 40S body coupled with the forward-then-reverse head swivel.", + "section": "RESULTS", + "ner": [ + [ + 54, + 57, + "40S", + "complex_assembly" + ], + [ + 58, + 62, + "body", + "structure_element" + ], + [ + 101, + 105, + "head", + "structure_element" + ] + ] + }, + { + "sid": 247, + "sent": "To elucidate the detailed structural mechanism of IRES translocation and the roles of eEF2 and ribosome rearrangements, we describe in the following sections the interactions of PKI and eEF2 with the ribosomal A and P sites in Structures I through V (Figure 2g; see also Figure 1\u2014figure supplement 1).", + "section": "RESULTS", + "ner": [ + [ + 50, + 54, + "IRES", + "site" + ], + [ + 86, + 90, + "eEF2", + "protein" + ], + [ + 95, + 103, + "ribosome", + "complex_assembly" + ], + [ + 178, + 181, + "PKI", + "structure_element" + ], + [ + 186, + 190, + "eEF2", + "protein" + ], + [ + 210, + 223, + "A and P sites", + "site" + ], + [ + 227, + 249, + "Structures I through V", + "evidence" + ] + ] + }, + { + "sid": 248, + "sent": "Structure I represents a pre-translocation IRES and initial entry of eEF2 in a GTP-like state", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Structure I", + "evidence" + ], + [ + 25, + 42, + "pre-translocation", + "protein_state" + ], + [ + 43, + 47, + "IRES", + "site" + ], + [ + 69, + 73, + "eEF2", + "protein" + ], + [ + 79, + 82, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 249, + "sent": "In the fully rotated Structure I, PKI is shifted toward the P site by ~3\u2009\u00c5 relative to its position in the initiation complex but maintains interactions with the partially swiveled head.", + "section": "RESULTS", + "ner": [ + [ + 7, + 20, + "fully rotated", + "protein_state" + ], + [ + 21, + 32, + "Structure I", + "evidence" + ], + [ + 34, + 37, + "PKI", + "structure_element" + ], + [ + 60, + 66, + "P site", + "site" + ], + [ + 107, + 125, + "initiation complex", + "complex_assembly" + ], + [ + 162, + 180, + "partially swiveled", + "protein_state" + ], + [ + 181, + 185, + "head", + "structure_element" + ] + ] + }, + { + "sid": 250, + "sent": "At the head, C1274 of the 18S rRNA (C1054 in E. coli) base pairs with the first nucleotide of the ORF immediately downstream of PKI.", + "section": "RESULTS", + "ner": [ + [ + 7, + 11, + "head", + "structure_element" + ], + [ + 13, + 18, + "C1274", + "residue_name_number" + ], + [ + 26, + 34, + "18S rRNA", + "chemical" + ], + [ + 36, + 41, + "C1054", + "residue_name_number" + ], + [ + 45, + 52, + "E. coli", + "species" + ], + [ + 98, + 101, + "ORF", + "structure_element" + ], + [ + 128, + 131, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 251, + "sent": "The C1274:G6953 base pair provides a stacking platform for the codon-anticodon\u2013like helix of PKI.", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "C1274", + "residue_name_number" + ], + [ + 10, + 15, + "G6953", + "residue_name_number" + ], + [ + 37, + 54, + "stacking platform", + "site" + ], + [ + 63, + 89, + "codon-anticodon\u2013like helix", + "structure_element" + ], + [ + 93, + 96, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 252, + "sent": "We therefore define C1274 as the foundation of the 'head A site'.", + "section": "RESULTS", + "ner": [ + [ + 20, + 25, + "C1274", + "residue_name_number" + ], + [ + 52, + 56, + "head", + "structure_element" + ], + [ + 57, + 63, + "A site", + "site" + ] + ] + }, + { + "sid": 253, + "sent": "Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures and in our post-translocation Structure V discussed below.", + "section": "RESULTS", + "ner": [ + [ + 20, + 25, + "U1191", + "residue_name_number" + ], + [ + 27, + 31, + "G966", + "residue_name_number" + ], + [ + 35, + 42, + "E. coli", + "species" + ], + [ + 48, + 53, + "C1637", + "residue_name_number" + ], + [ + 55, + 60, + "C1400", + "residue_name_number" + ], + [ + 64, + 71, + "E. coli", + "species" + ], + [ + 105, + 109, + "head", + "structure_element" + ], + [ + 110, + 116, + "P site", + "site" + ], + [ + 123, + 127, + "body", + "structure_element" + ], + [ + 128, + 134, + "P site", + "site" + ], + [ + 224, + 242, + "fully translocated", + "protein_state" + ], + [ + 243, + 258, + "mRNA-tRNA helix", + "structure_element" + ], + [ + 262, + 272, + "tRNA-bound", + "protein_state" + ], + [ + 273, + 283, + "structures", + "evidence" + ], + [ + 295, + 313, + "post-translocation", + "protein_state" + ], + [ + 314, + 325, + "Structure V", + "evidence" + ] + ] + }, + { + "sid": 254, + "sent": "Interactions of the residues at the eEF2 tip with the decoding center of the IRES-bound ribosome.", + "section": "FIG", + "ner": [ + [ + 36, + 40, + "eEF2", + "protein" + ], + [ + 54, + 69, + "decoding center", + "site" + ], + [ + 77, + 87, + "IRES-bound", + "protein_state" + ], + [ + 88, + 96, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 255, + "sent": "Key elements of the decoding center of the 'locked' initiation structure, 'unlocked' Structure I, and post-translocation Structure V (this work) are shown.", + "section": "FIG", + "ner": [ + [ + 20, + 35, + "decoding center", + "site" + ], + [ + 44, + 50, + "locked", + "protein_state" + ], + [ + 52, + 62, + "initiation", + "protein_state" + ], + [ + 63, + 72, + "structure", + "evidence" + ], + [ + 75, + 83, + "unlocked", + "protein_state" + ], + [ + 85, + 96, + "Structure I", + "evidence" + ], + [ + 102, + 120, + "post-translocation", + "protein_state" + ], + [ + 121, + 132, + "Structure V", + "evidence" + ] + ] + }, + { + "sid": 256, + "sent": "The histidine-diphthamide tip of eEF2 is shown in green.", + "section": "FIG", + "ner": [ + [ + 4, + 29, + "histidine-diphthamide tip", + "site" + ], + [ + 33, + 37, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 257, + "sent": "The codon-anticodon-like helix of PKI is shown in red, the downstream first codon of the ORF in magenta.", + "section": "FIG", + "ner": [ + [ + 4, + 30, + "codon-anticodon-like helix", + "structure_element" + ], + [ + 34, + 37, + "PKI", + "structure_element" + ], + [ + 89, + 92, + "ORF", + "structure_element" + ] + ] + }, + { + "sid": 258, + "sent": "Nucleotides of the 18S rRNA body are in orange and head in yellow; 25S rRNA nucleotide A2256 is blue.", + "section": "FIG", + "ner": [ + [ + 19, + 27, + "18S rRNA", + "chemical" + ], + [ + 28, + 32, + "body", + "structure_element" + ], + [ + 51, + 55, + "head", + "structure_element" + ], + [ + 67, + 75, + "25S rRNA", + "chemical" + ], + [ + 87, + 92, + "A2256", + "residue_name_number" + ] + ] + }, + { + "sid": 259, + "sent": "A and P sites are schematically demarcated by dotted lines.", + "section": "FIG", + "ner": [ + [ + 0, + 13, + "A and P sites", + "site" + ] + ] + }, + { + "sid": 260, + "sent": "The interaction of PKI with the 40S body is substantially rearranged relative to that in the initiation state.", + "section": "RESULTS", + "ner": [ + [ + 19, + 22, + "PKI", + "structure_element" + ], + [ + 32, + 35, + "40S", + "complex_assembly" + ], + [ + 36, + 40, + "body", + "structure_element" + ], + [ + 93, + 103, + "initiation", + "protein_state" + ] + ] + }, + { + "sid": 261, + "sent": "In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes.", + "section": "RESULTS", + "ner": [ + [ + 15, + 18, + "PKI", + "structure_element" + ], + [ + 58, + 79, + "universally conserved", + "protein_state" + ], + [ + 80, + 95, + "decoding-center", + "site" + ], + [ + 108, + 112, + "G577", + "residue_name_number" + ], + [ + 114, + 119, + "A1755", + "residue_name_number" + ], + [ + 124, + 129, + "A1756", + "residue_name_number" + ], + [ + 132, + 136, + "body", + "structure_element" + ], + [ + 137, + 143, + "A site", + "site" + ], + [ + 157, + 163, + "A-site", + "site" + ], + [ + 164, + 174, + "tRNA bound", + "protein_state" + ] + ] + }, + { + "sid": 262, + "sent": "In Structure I, PKI does not contact these nucleotides (Figures 2g and 7).", + "section": "RESULTS", + "ner": [ + [ + 3, + 14, + "Structure I", + "evidence" + ], + [ + 16, + 19, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 263, + "sent": "The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5\u2014figure supplement 1).", + "section": "RESULTS", + "ner": [ + [ + 16, + 20, + "eEF2", + "protein" + ], + [ + 28, + 31, + "40S", + "complex_assembly" + ], + [ + 32, + 39, + "subunit", + "structure_element" + ], + [ + 32, + 39, + "subunit", + "structure_element" + ], + [ + 43, + 54, + "Structure I", + "evidence" + ], + [ + 90, + 108, + "Structures II to V", + "evidence" + ], + [ + 114, + 125, + "translocase", + "protein_type" + ], + [ + 145, + 148, + "40S", + "complex_assembly" + ], + [ + 149, + 153, + "body", + "structure_element" + ], + [ + 179, + 183, + "head", + "structure_element" + ] + ] + }, + { + "sid": 264, + "sent": "Domain IV is partially engaged with the body A site.", + "section": "RESULTS", + "ner": [ + [ + 7, + 9, + "IV", + "structure_element" + ], + [ + 40, + 44, + "body", + "structure_element" + ], + [ + 45, + 51, + "A site", + "site" + ] + ] + }, + { + "sid": 265, + "sent": "The tip of domain IV is wedged between PKI and decoding-center nucleotides A1755 and A1756, which are bulged out of h44.", + "section": "RESULTS", + "ner": [ + [ + 18, + 20, + "IV", + "structure_element" + ], + [ + 39, + 42, + "PKI", + "structure_element" + ], + [ + 47, + 62, + "decoding-center", + "site" + ], + [ + 75, + 80, + "A1755", + "residue_name_number" + ], + [ + 85, + 90, + "A1756", + "residue_name_number" + ] + ] + }, + { + "sid": 266, + "sent": "This tip contains the histidine-diphthamide triad (H583, H694 and Diph699), which interacts with the codon-anticodon-like helix of PKI and A1756 (Figure 7).", + "section": "RESULTS", + "ner": [ + [ + 22, + 49, + "histidine-diphthamide triad", + "site" + ], + [ + 51, + 55, + "H583", + "residue_name_number" + ], + [ + 57, + 61, + "H694", + "residue_name_number" + ], + [ + 66, + 73, + "Diph699", + "ptm" + ], + [ + 101, + 127, + "codon-anticodon-like helix", + "structure_element" + ], + [ + 131, + 134, + "PKI", + "structure_element" + ], + [ + 139, + 144, + "A1756", + "residue_name_number" + ] + ] + }, + { + "sid": 267, + "sent": "Histidines 583 and 694 interact with the phosphate backbone of the anticodon-like strand (at G6907 and C6908).", + "section": "RESULTS", + "ner": [ + [ + 0, + 22, + "Histidines 583 and 694", + "residue_name_number" + ], + [ + 67, + 88, + "anticodon-like strand", + "structure_element" + ], + [ + 93, + 98, + "G6907", + "residue_name_number" + ], + [ + 103, + 108, + "C6908", + "residue_name_number" + ] + ] + }, + { + "sid": 268, + "sent": "Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-\u00c5 long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1.", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Diphthamide", + "ptm" + ], + [ + 55, + 64, + "conserved", + "protein_state" + ], + [ + 68, + 76, + "archaeal", + "taxonomy_domain" + ], + [ + 81, + 91, + "eukaryotic", + "taxonomy_domain" + ], + [ + 92, + 95, + "EF2", + "protein" + ], + [ + 108, + 111, + "699", + "residue_number" + ], + [ + 115, + 128, + "S. cerevisiae", + "species" + ], + [ + 222, + 231, + "histidine", + "residue_name" + ] + ] + }, + { + "sid": 269, + "sent": "The trimethylamino end of Diph699 packs over A1756 (Figure 7).", + "section": "RESULTS", + "ner": [ + [ + 26, + 33, + "Diph699", + "ptm" + ], + [ + 45, + 50, + "A1756", + "residue_name_number" + ] + ] + }, + { + "sid": 270, + "sent": "The opposite surface of the tail is oriented toward the minor-groove side of the second base pair of the codon-anticodon helix (G6906:C6951).", + "section": "RESULTS", + "ner": [ + [ + 56, + 68, + "minor-groove", + "site" + ], + [ + 105, + 126, + "codon-anticodon helix", + "structure_element" + ], + [ + 128, + 133, + "G6906", + "residue_name_number" + ], + [ + 134, + 139, + "C6951", + "residue_name_number" + ] + ] + }, + { + "sid": 271, + "sent": "Thus, in comparison with the initiation state, the histidine-diphthamide tip of eEF2 replaces the codon-anticodon\u2013like helix of PKI.", + "section": "RESULTS", + "ner": [ + [ + 29, + 39, + "initiation", + "protein_state" + ], + [ + 51, + 76, + "histidine-diphthamide tip", + "site" + ], + [ + 80, + 84, + "eEF2", + "protein" + ], + [ + 98, + 124, + "codon-anticodon\u2013like helix", + "structure_element" + ], + [ + 128, + 131, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 272, + "sent": "The splitting of the interaction of A1755-A1756 and PKI is achieved by providing the histidine-diphthamine tip as a binding partner for both A1756 and the minor groove of the codon-anticodon helix (Figure 7).", + "section": "RESULTS", + "ner": [ + [ + 36, + 41, + "A1755", + "residue_name_number" + ], + [ + 42, + 47, + "A1756", + "residue_name_number" + ], + [ + 52, + 55, + "PKI", + "structure_element" + ], + [ + 85, + 110, + "histidine-diphthamine tip", + "site" + ], + [ + 141, + 146, + "A1756", + "residue_name_number" + ], + [ + 155, + 167, + "minor groove", + "site" + ], + [ + 175, + 196, + "codon-anticodon helix", + "structure_element" + ] + ] + }, + { + "sid": 273, + "sent": "Unlike in Structures II to V, the conformation of the eEF2 GTPase center in Structure I resembles that of a GTP-bound translocase (Figure 5e).", + "section": "RESULTS", + "ner": [ + [ + 10, + 28, + "Structures II to V", + "evidence" + ], + [ + 54, + 58, + "eEF2", + "protein" + ], + [ + 59, + 72, + "GTPase center", + "site" + ], + [ + 76, + 87, + "Structure I", + "evidence" + ], + [ + 108, + 117, + "GTP-bound", + "protein_state" + ], + [ + 118, + 129, + "translocase", + "protein_type" + ] + ] + }, + { + "sid": 274, + "sent": "In translational GTPases, switch loops I and II are involved in the GTPase activity (reviewed in).", + "section": "RESULTS", + "ner": [ + [ + 3, + 24, + "translational GTPases", + "protein_type" + ], + [ + 26, + 47, + "switch loops I and II", + "structure_element" + ], + [ + 68, + 74, + "GTPase", + "protein_type" + ] + ] + }, + { + "sid": 275, + "sent": "Switch loop II (aa 105\u2013110), which carries the catalytic H108 (H92 in E. coli EF-G; is well resolved in all five structures.", + "section": "RESULTS", + "ner": [ + [ + 0, + 14, + "Switch loop II", + "structure_element" + ], + [ + 19, + 26, + "105\u2013110", + "residue_range" + ], + [ + 47, + 56, + "catalytic", + "protein_state" + ], + [ + 57, + 61, + "H108", + "residue_name_number" + ], + [ + 63, + 66, + "H92", + "residue_name_number" + ], + [ + 70, + 77, + "E. coli", + "species" + ], + [ + 78, + 82, + "EF-G", + "protein" + ], + [ + 113, + 123, + "structures", + "evidence" + ] + ] + }, + { + "sid": 276, + "sent": "The histidine resides next to the backbone of G3028 of the sarcin-ricin loop and near the diphosphate of GDP (Figure 5e).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "histidine", + "residue_name" + ], + [ + 46, + 51, + "G3028", + "residue_name_number" + ], + [ + 59, + 76, + "sarcin-ricin loop", + "structure_element" + ], + [ + 105, + 108, + "GDP", + "chemical" + ] + ] + }, + { + "sid": 277, + "sent": "By contrast, switch loop I (aa 50\u201370 in S. cerevisiae eEF2) is resolved only in Structure I (Figure 5\u2014figure supplement 2).", + "section": "RESULTS", + "ner": [ + [ + 13, + 26, + "switch loop I", + "structure_element" + ], + [ + 31, + 36, + "50\u201370", + "residue_range" + ], + [ + 40, + 53, + "S. cerevisiae", + "species" + ], + [ + 54, + 58, + "eEF2", + "protein" + ], + [ + 80, + 91, + "Structure I", + "evidence" + ] + ] + }, + { + "sid": 278, + "sent": "The N-terminal part of the loop (aa 50\u201360) is sandwiched between the tip of helix 14 (415CAAA418) of the 18S rRNA of the 40S subunit and helix A (aa 32\u201342) of eEF2 (Figure 5d).", + "section": "RESULTS", + "ner": [ + [ + 27, + 31, + "loop", + "structure_element" + ], + [ + 36, + 41, + "50\u201360", + "residue_range" + ], + [ + 76, + 84, + "helix 14", + "structure_element" + ], + [ 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[ + 40, + 51, + "switch loop", + "structure_element" + ], + [ + 56, + 61, + "61\u201367", + "residue_range" + ], + [ + 72, + 84, + "helical fold", + "protein_state" + ] + ] + }, + { + "sid": 281, + "sent": "As such, the conformations of SWI and the GTPase center in general are similar to those observed in ribosome-bound EF-Tu and EF-G in the presence of GTP analogs.", + "section": "RESULTS", + "ner": [ + [ + 30, + 33, + "SWI", + "structure_element" + ], + [ + 42, + 55, + "GTPase center", + "site" + ], + [ + 100, + 114, + "ribosome-bound", + "protein_state" + ], + [ + 115, + 120, + "EF-Tu", + "protein" + ], + [ + 125, + 129, + "EF-G", + "protein" + ], + [ + 137, + 148, + "presence of", + "protein_state" + ], + [ + 149, + 152, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 282, + "sent": "Structure II reveals PKI between the body A and P sites and eEF2 partially advanced into the A site", + "section": "RESULTS", + "ner": [ + [ + 0, + 12, + "Structure II", + "evidence" + ], + [ + 21, + 24, + "PKI", + "structure_element" + ], + [ + 37, + 41, + "body", + "structure_element" + ], + [ + 42, + 55, + "A and P sites", + "site" + ], + [ + 60, + 64, + "eEF2", + "protein" + ], + [ + 93, + 99, + "A site", + "site" + ] + ] + }, + { + "sid": 283, + "sent": "In Structure II, relative to Structure I, PKI is further shifted along the 40S body, traversing ~4\u2009\u00c5 toward the P site (Figures 2e, f, and g), while stacking on C1274 at the head A site.", + "section": "RESULTS", + "ner": [ + [ + 3, + 15, + "Structure II", + "evidence" + ], + [ + 29, + 40, + "Structure I", + "evidence" + ], + [ + 42, + 45, + "PKI", + "structure_element" + ], + [ + 75, + 78, + "40S", + "complex_assembly" + ], + [ + 79, + 83, + "body", + "structure_element" + ], + [ + 112, + 118, + "P site", + "site" + ], + [ + 149, + 157, + "stacking", + "bond_interaction" + ], + [ + 161, + 166, + "C1274", + "residue_name_number" + ], + [ + 174, + 178, + "head", + "structure_element" + ], + [ + 179, + 185, + "A site", + "site" + ] + ] + }, + { + "sid": 284, + "sent": "Thus, the intermediate position of PKI is possible due to a large swivel of the head relative to the body, which brings the head A site close to the body P site.", + "section": "RESULTS", + "ner": [ + [ + 35, + 38, + "PKI", + "structure_element" + ], + [ + 80, + 84, + "head", + "structure_element" + ], + [ + 101, + 105, + "body", + "structure_element" + ], + [ + 124, + 128, + "head", + "structure_element" + ], + [ + 129, + 135, + "A site", + "site" + ], + [ + 149, + 153, + "body", + "structure_element" + ], + [ + 154, + 160, + "P site", + "site" + ] + ] + }, + { + "sid": 285, + "sent": "Domain IV of eEF2 is further entrenched in the A site by ~3\u2009\u00c5 relative to the body and ~8\u2009\u00c5 relative to the head, preserving its interactions with PKI.", + "section": "RESULTS", + "ner": [ + [ + 7, + 9, + "IV", + "structure_element" + ], + [ + 13, + 17, + "eEF2", + "protein" + ], + [ + 47, + 53, + "A site", + "site" + ], + [ + 78, + 82, + "body", + "structure_element" + ], + [ + 108, + 112, + "head", + "structure_element" + ], + [ + 147, + 150, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 286, + "sent": "The decoding center residues A1755 and A1756 are rearranged to pack inside helix 44, making room for eEF2.", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "decoding center", + "site" + ], + [ + 29, + 34, + "A1755", + "residue_name_number" + ], + [ + 39, + 44, + "A1756", + "residue_name_number" + ], + [ + 75, + 83, + "helix 44", + "structure_element" + ], + [ + 101, + 105, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 287, + "sent": "This conformation of decoding center residues is also observed in the absence of A-site ligands.", + "section": "RESULTS", + "ner": [ + [ + 21, + 36, + "decoding center", + "site" + ], + [ + 70, + 80, + "absence of", + "protein_state" + ], + [ + 81, + 87, + "A-site", + "site" + ] + ] + }, + { + "sid": 288, + "sent": "The head interface of domain IV interacts with the 40S head (Figure 6a).", + "section": "RESULTS", + "ner": [ + [ + 4, + 18, + "head interface", + "site" + ], + [ + 29, + 31, + "IV", + "structure_element" + ], + [ + 51, + 54, + "40S", + "complex_assembly" + ], + [ + 55, + 59, + "head", + "structure_element" + ] + ] + }, + { + "sid": 289, + "sent": "Here, a positively charged surface of\u00a0eEF2, formed by K613, R617 and R631 contacts the phosphate backbone of helix 33 (Figures 6c; see also Figure 6\u2014figure supplement 1).", + "section": "RESULTS", + "ner": [ + [ + 8, + 34, + "positively charged surface", + "site" + ], + [ + 38, + 42, + "eEF2", + "protein" + ], + [ + 54, + 58, + "K613", + "residue_name_number" + ], + [ + 60, + 64, + "R617", + "residue_name_number" + ], + [ + 69, + 73, + "R631", + "residue_name_number" + ], + [ + 109, + 117, + "helix 33", + "structure_element" + ] + ] + }, + { + "sid": 290, + "sent": "Structure III represents a highly bent IRES with PKI captured between the head A and P sites", + "section": "RESULTS", + "ner": [ + [ + 0, + 13, + "Structure III", + "evidence" + ], + [ + 27, + 38, + "highly bent", + "protein_state" + ], + [ + 39, + 43, + "IRES", + "site" + ], + [ + 49, + 52, + "PKI", + "structure_element" + ], + [ + 74, + 78, + "head", + "structure_element" + ], + [ + 79, + 92, + "A and P sites", + "site" + ] + ] + }, + { + "sid": 291, + "sent": "Consistent with the similar head swivels in Structure III and Structure II, relative positions of the 40S\u00a0head A site and body P site remain as in Structure II.", + "section": "RESULTS", + "ner": [ + [ + 28, + 32, + "head", + "structure_element" + ], + [ + 44, + 57, + "Structure III", + "evidence" + ], + [ + 62, + 74, + "Structure II", + "evidence" + ], + [ + 102, + 105, + "40S", + "complex_assembly" + ], + [ + 106, + 110, + "head", + "structure_element" + ], + [ + 111, + 117, + "A site", + "site" + ], + [ + 122, + 126, + "body", + "structure_element" + ], + [ + 127, + 133, + "P site", + "site" + ], + [ + 147, + 159, + "Structure II", + "evidence" + ] + ] + }, + { + "sid": 292, + "sent": "Among the five structures, the PKI domain is least ordered in Structure III and lacks density for SL3.", + "section": "RESULTS", + "ner": [ + [ + 15, + 25, + "structures", + "evidence" + ], + [ + 31, + 34, + "PKI", + "structure_element" + ], + [ + 62, + 75, + "Structure III", + "evidence" + ], + [ + 86, + 93, + "density", + "evidence" + ], + [ + 98, + 101, + "SL3", + "structure_element" + ] + ] + }, + { + "sid": 293, + "sent": "The map allows placement of PKI at the body P site (Figure 1\u2014figure supplement 3).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "map", + "evidence" + ], + [ + 28, + 31, + "PKI", + "structure_element" + ], + [ + 39, + 43, + "body", + "structure_element" + ], + [ + 44, + 50, + "P site", + "site" + ] + ] + }, + { + "sid": 294, + "sent": "Thus, in Structure III, PKI has translocated along the 40S body, but the head remains fully swiveled so that PKI is between the head A and P sites.", + "section": "RESULTS", + "ner": [ + [ + 9, + 22, + "Structure III", + "evidence" + ], + [ + 24, + 27, + "PKI", + "structure_element" + ], + [ + 55, + 58, + "40S", + "complex_assembly" + ], + [ + 59, + 63, + "body", + "structure_element" + ], + [ + 73, + 77, + "head", + "structure_element" + ], + [ + 86, + 100, + "fully swiveled", + "protein_state" + ], + [ + 109, + 112, + "PKI", + "structure_element" + ], + [ + 128, + 132, + "head", + "structure_element" + ], + [ + 133, + 146, + "A and P sites", + "site" + ] + ] + }, + { + "sid": 295, + "sent": "Lower resolution of the map in this region suggests that PKI is somewhat destabilized in the vicinity of the body P site in the absence of stacking with the foundations of the head A site (C1274) or P site (U1191).", + "section": "RESULTS", + "ner": [ + [ + 24, + 27, + "map", + "evidence" + ], + [ + 57, + 60, + "PKI", + "structure_element" + ], + [ + 109, + 113, + "body", + "structure_element" + ], + [ + 114, + 120, + "P site", + 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"head", + "structure_element" + ], + [ + 32, + 36, + "head", + "structure_element" + ], + [ + 37, + 43, + "P-site", + "site" + ], + [ + 52, + 57, + "U1191", + "residue_name_number" + ], + [ + 62, + 66, + "body", + "structure_element" + ], + [ + 67, + 73, + "P-site", + "site" + ], + [ + 82, + 87, + "C1637", + "residue_name_number" + ], + [ + 139, + 142, + "40S", + "complex_assembly" + ], + [ + 143, + 149, + "P site", + "site" + ] + ] + }, + { + "sid": 300, + "sent": "Whereas C1637 forms a stacking platform for the last base pair of PKI, U1191 does not yet stack on PKI because the head remains partially swiveled.", + "section": "RESULTS", + "ner": [ + [ + 8, + 13, + "C1637", + "residue_name_number" + ], + [ + 22, + 39, + "stacking platform", + "site" + ], + [ + 66, + 69, + "PKI", + "structure_element" + ], + [ + 71, + 76, + "U1191", + "residue_name_number" + ], + [ + 90, + 95, + "stack", + "bond_interaction" + ], + [ + 99, + 102, + "PKI", + "structure_element" + ], + [ + 115, + 119, + "head", + "structure_element" + ] + ] + }, + { + "sid": 301, + "sent": "This renders PKI partially accommodated in the P site (Figure 2g).", + "section": "RESULTS", + "ner": [ + [ + 13, + 16, + "PKI", + "structure_element" + ], + [ + 47, + 53, + "P site", + "site" + ] + ] + }, + { + "sid": 302, + "sent": "Unwinding of the 40S head also positions the head A site closer to the body A site.", + "section": "RESULTS", + "ner": [ + [ + 17, + 20, + "40S", + "complex_assembly" + ], + [ + 21, + 25, + "head", + "structure_element" + ], + [ + 45, + 49, + "head", + "structure_element" + ], + [ + 50, + 56, + "A site", + "site" + ], + [ + 71, + 75, + "body", + "structure_element" + ], + [ + 76, + 82, + "A site", + "site" + ] + ] + }, + { + "sid": 303, + "sent": "This results in rearrangements of eEF2 interactions with the head, allowing eEF2 to advance further into the A site.", + "section": "RESULTS", + "ner": [ + [ + 34, + 38, + "eEF2", + "protein" + ], + [ + 61, + 65, + "head", + "structure_element" + ], + [ + 76, + 80, + "eEF2", + "protein" + ], + [ + 109, + 115, + "A site", + "site" + ] + ] + }, + { + "sid": 304, + "sent": "To this end, the head-interacting interface of domain IV slides along the surface of the head by 5\u2009\u00c5. Helix A of domain IV is positioned next to the backbone of h34, with positively charged residues K613, R617 and R631 rearranged from the backbone of h33 (Figure 6c; see also Figure 6\u2014figure supplement 1).", + "section": "RESULTS", + "ner": [ + [ + 17, + 43, + "head-interacting interface", + "site" + ], + [ + 54, + 56, + "IV", + "structure_element" + ], + [ + 89, + 93, + "head", + "structure_element" + ], + [ + 102, + 109, + "Helix A", + "structure_element" + ], + [ + 120, + 122, + "IV", + "structure_element" + ], + [ + 161, + 164, + "h34", + "structure_element" + ], + [ + 199, + 203, + "K613", + "residue_name_number" + ], + [ + 205, + 209, + "R617", + "residue_name_number" + ], + [ + 214, + 218, + "R631", + "residue_name_number" + ], + [ + 251, + 254, + "h33", + "structure_element" + ] + ] + }, + { + "sid": 305, + "sent": "Structure V represents an extended IRES with PKI fully accommodated in the P site and domain IV of eEF2 in the A site", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Structure V", + "evidence" + ], + [ + 26, + 34, + "extended", + "protein_state" + ], + [ + 35, + 39, + "IRES", + "site" + ], + [ + 45, + 48, + "PKI", + "structure_element" + ], + [ + 75, + 81, + "P site", + "site" + ], + [ + 93, + 95, + "IV", + "structure_element" + ], + [ + 99, + 103, + "eEF2", + "protein" + ], + [ + 111, + 117, + "A site", + "site" + ] + ] + }, + { + "sid": 306, + "sent": "In the nearly non-rotated and non-swiveled ribosome conformation in Structure V closely resembling that of the post-translocation 80S\u20222tRNA\u2022mRNA complex, PKI is fully accommodated in the P site.", + "section": "RESULTS", + "ner": [ + [ + 7, + 25, + "nearly non-rotated", + "protein_state" + ], + [ + 30, + 42, + "non-swiveled", + "protein_state" + ], + [ + 43, + 51, + "ribosome", + "complex_assembly" + ], + [ + 68, + 79, + "Structure V", + "evidence" + ], + [ + 111, + 129, + "post-translocation", + "protein_state" + ], + [ + 130, + 144, + "80S\u20222tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 154, + 157, + "PKI", + "structure_element" + ], + [ + 187, + 193, + "P site", + "site" + ] + ] + }, + { + "sid": 307, + "sent": "The codon-anticodon\u2013like helix is stacked on P-site residues U1191 and C1637 (Figure 3d), analogous to stacking of the tRNA-mRNA helix (Figure 3e).", + "section": "RESULTS", + "ner": [ + [ + 4, + 30, + "codon-anticodon\u2013like helix", + "structure_element" + ], + [ + 45, + 51, + "P-site", + "site" + ], + [ + 61, + 66, + "U1191", + "residue_name_number" + ], + [ + 71, + 76, + "C1637", + "residue_name_number" + ], + [ + 103, + 111, + "stacking", + "bond_interaction" + ], + [ + 119, + 128, + "tRNA-mRNA", + "complex_assembly" + ], + [ + 129, + 134, + "helix", + "structure_element" + ] + ] + }, + { + "sid": 308, + "sent": "A notable conformational change in eEF2 from that in the preceding Structures is visible in the position of domain III, which contacts uS12 (Figure 6d).", + "section": "RESULTS", + "ner": [ + [ + 35, + 39, + "eEF2", + "protein" + ], + [ + 67, + 77, + "Structures", + "evidence" + ], + [ + 115, + 118, + "III", + "structure_element" + ], + [ + 135, + 139, + "uS12", + "protein" + ] + ] + }, + { + "sid": 309, + "sent": "In Structure V, protein uS12 is shifted along with the 40S body as a result of intersubunit rotation.", + "section": "RESULTS", + "ner": [ + [ + 3, + 14, + "Structure V", + "evidence" + ], + [ + 24, + 28, + "uS12", + "protein" + ], + [ + 55, + 58, + "40S", + "complex_assembly" + ], + [ + 59, + 63, + "body", + "structure_element" + ] + ] + }, + { + "sid": 310, + "sent": "In this position, uS12 forms extensive interactions with eEF2 domains II and III.", + "section": "RESULTS", + "ner": [ + [ + 18, + 22, + "uS12", + "protein" + ], + [ + 57, + 61, + "eEF2", + "protein" + ], + [ + 70, + 72, + "II", + "structure_element" + ], + [ + 77, + 80, + "III", + "structure_element" + ] + ] + }, + { + "sid": 311, + "sent": "Specifically, the C-terminal tail of uS12 packs against the \u03b2-barrel of domain II, while the \u03b2-barrel of uS12 packs against helix A of domain III.", + "section": "RESULTS", + "ner": [ + [ + 18, + 33, + "C-terminal tail", + "structure_element" + ], + [ + 37, + 41, + "uS12", + "protein" + ], + [ + 60, + 68, + "\u03b2-barrel", + "structure_element" + ], + [ + 79, + 81, + "II", + "structure_element" + ], + [ + 93, + 101, + "\u03b2-barrel", + "structure_element" + ], + [ + 105, + 109, + "uS12", + "protein" + ], + [ + 124, + 131, + "helix A", + "structure_element" + ], + [ + 142, + 145, + "III", + "structure_element" + ] + ] + }, + { + "sid": 312, + "sent": "This shifts the tip of helix A of domain III (at aa 500) by ~5\u2009\u00c5 (relative to that in Structure I) toward domain I. Although domain III remains in contact with domain V, the shift occurs in the direction that could eventually disconnect the \u03b2-platforms of these domains.", + "section": "RESULTS", + "ner": [ + [ + 23, + 30, + "helix A", + "structure_element" + ], + [ + 41, + 44, + "III", + "structure_element" + ], + [ + 52, + 55, + "500", + "residue_number" + ], + [ + 86, + 97, + "Structure I", + "evidence" + ], + [ + 113, + 114, + "I", + "structure_element" + ], + [ + 132, + 135, + "III", + "structure_element" + ], + [ + 167, + 168, + "V", + "structure_element" + ], + [ + 241, + 252, + "\u03b2-platforms", + "structure_element" + ] + ] + }, + { + "sid": 313, + "sent": "Domain IV of eEF2 is fully accommodated in the A site.", + "section": "RESULTS", + "ner": [ + [ + 7, + 9, + "IV", + "structure_element" + ], + [ + 13, + 17, + "eEF2", + "protein" + ], + [ + 47, + 53, + "A site", + "site" + ] + ] + }, + { + "sid": 314, + "sent": "The first codon of the open reading frame is also positioned in the A site, with bases exposed toward eEF2 (Figure 7), resembling the conformations of the A-site codons in EF-G-bound 70S complexes.", + "section": "RESULTS", + "ner": [ + [ + 23, + 41, + "open reading frame", + "structure_element" + ], + [ + 68, + 74, + "A site", + "site" + ], + [ + 102, + 106, + "eEF2", + "protein" + ], + [ + 155, + 161, + "A-site", + "site" + ], + [ + 172, + 182, + "EF-G-bound", + "protein_state" + ], + [ + 183, + 186, + "70S", + "complex_assembly" + ] + ] + }, + { + "sid": 315, + "sent": "As in the preceding Structures, the histidine-diphthamide tip is bound in the minor groove of the P-site codon-anticodon helix.", + "section": "RESULTS", + "ner": [ + [ + 20, + 30, + "Structures", + "evidence" + ], + [ + 36, + 61, + "histidine-diphthamide tip", + "site" + ], + [ + 65, + 73, + "bound in", + "protein_state" + ], + [ + 78, + 90, + "minor groove", + "site" + ], + [ + 98, + 104, + "P-site", + "site" + ], + [ + 105, + 126, + "codon-anticodon helix", + "structure_element" + ] + ] + }, + { + "sid": 316, + "sent": "Diph699 slightly rearranges, relative to that in Structure I (Figure 7), and interacts with four out of six codon-anticodon nucleotides.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Diph699", + "ptm" + ], + [ + 49, + 60, + "Structure I", + "evidence" + ] + ] + }, + { + "sid": 317, + "sent": "The imidazole moiety stacks on G6907 (corresponding to nt 36 in the tRNA anticodon) and hydrogen bonds with O2\u2019 of G6906 (nt 35 of tRNA).", + "section": "RESULTS", + "ner": [ + [ + 31, + 36, + "G6907", + "residue_name_number" + ], + [ + 68, + 72, + "tRNA", + "chemical" + ], + [ + 88, + 102, + "hydrogen bonds", + "bond_interaction" + ], + [ + 115, + 120, + "G6906", + "residue_name_number" + ], + [ + 131, + 135, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 318, + "sent": "The amide at the diphthamide end interacts with N2 of G6906 and O2 and O2\u2019 of C6951 (corresponding to nt 2 of the codon).", + "section": "RESULTS", + "ner": [ + [ + 17, + 28, + "diphthamide", + "ptm" + ], + [ + 54, + 59, + "G6906", + "residue_name_number" + ], + [ + 78, + 83, + "C6951", + "residue_name_number" + ] + ] + }, + { + "sid": 319, + "sent": "The trimethylamino-group is positioned over the ribose of C6952 (codon nt 3).", + "section": "RESULTS", + "ner": [ + [ + 58, + 63, + "C6952", + "residue_name_number" + ] + ] + }, + { + "sid": 320, + "sent": "IRES translocation mechanism", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "IRES", + "site" + ] + ] + }, + { + "sid": 321, + "sent": "Animation showing the transition from the initiation 80S\u2022TSV IRES structures (Koh et al., 2014) to eEF2-bound Structures I through V (this work).", + "section": "FIG", + "ner": [ + [ + 42, + 52, + "initiation", + "protein_state" + ], + [ + 53, + 65, + "80S\u2022TSV IRES", + "complex_assembly" + ], + [ + 66, + 76, + "structures", + "evidence" + ], + [ + 99, + 109, + "eEF2-bound", + "protein_state" + ], + [ + 110, + 132, + "Structures I through V", + "evidence" + ] + ] + }, + { + "sid": 322, + "sent": "Four views (scenes) are shown: (1) A view down the intersubunit space, with the head of the 40S subunit oriented toward a viewer, as in Figure 1a; (2) A view at the solvent side of the 40S subunit, with the 40S head shown at the top, as in Figure 2\u2014figure supplement 1; (3) A view down at the subunit interface of the 40S subunit; (4) A close-up view of the decoding center (A site) and the P site, as in Figure 2g. Each scene is shown twice.", + "section": "FIG", + "ner": [ + [ + 80, + 84, + "head", + "structure_element" + ], + [ + 92, + 95, + "40S", + "complex_assembly" + ], + [ + 96, + 103, + "subunit", + "structure_element" + ], + [ + 185, + 188, + "40S", + "complex_assembly" + ], + [ + 189, + 196, + "subunit", + "structure_element" + ], + [ + 207, + 210, + "40S", + "complex_assembly" + ], + [ + 211, + 215, + "head", + "structure_element" + ], + [ + 318, + 321, + "40S", + "complex_assembly" + ], + [ + 322, + 329, + "subunit", + "structure_element" + ], + [ + 358, + 373, + "decoding center", + "site" + ], + [ + 375, + 381, + "A site", + "site" + ], + [ + 391, + 397, + "P site", + "site" + ] + ] + }, + { + "sid": 323, + "sent": "In scenes 1, 2 and 3, nucleotides C1274, U1191 of the 40S head and G904 of the 40S platform are shown in black to denote the A, P and E sites, respectively.", + "section": "FIG", + "ner": [ + [ + 34, + 39, + "C1274", + "residue_name_number" + ], + [ + 41, + 46, + "U1191", + "residue_name_number" + ], + [ + 54, + 57, + "40S", + "complex_assembly" + ], + [ + 58, + 62, + "head", + "structure_element" + ], + [ + 67, + 71, + "G904", + "residue_name_number" + ], + [ + 79, + 91, + "40S platform", + "site" + ], + [ + 125, + 141, + "A, P and E sites", + "site" + ] + ] + }, + { + "sid": 324, + "sent": "In scene 4, C1274 and U1191 are labeled and shown in yellow; G577, A1755 and A1756 of the 40S body A site and C1637 of the body P site are labeled and shown in orange.", + "section": "FIG", + "ner": [ + [ + 12, + 17, + "C1274", + "residue_name_number" + ], + [ + 22, + 27, + "U1191", + "residue_name_number" + ], + [ + 61, + 65, + "G577", + "residue_name_number" + ], + [ + 67, + 72, + "A1755", + "residue_name_number" + ], + [ + 77, + 82, + "A1756", + "residue_name_number" + ], + [ + 90, + 93, + "40S", + "complex_assembly" + ], + [ + 94, + 98, + "body", + "structure_element" + ], + [ + 99, + 105, + "A site", + "site" + ], + [ + 110, + 115, + "C1637", + "residue_name_number" + ], + [ + 123, + 127, + "body", + "structure_element" + ], + [ + 128, + 134, + "P site", + "site" + ] + ] + }, + { + "sid": 325, + "sent": "In this work we have captured the structures of the TSV IRES, whose PKI samples positions between the A and P sites (Structures I\u2013IV), as well as in the P site (Structure V).", + "section": "DISCUSS", + "ner": [ + [ + 34, + 44, + "structures", + "evidence" + ], + [ + 52, + 55, + "TSV", + "species" + ], + [ + 56, + 60, + "IRES", + "site" + ], + [ + 68, + 71, + "PKI", + "structure_element" + ], + [ + 102, + 115, + "A and P sites", + "site" + ], + [ + 117, + 132, + "Structures I\u2013IV", + "evidence" + ], + [ + 153, + 159, + "P site", + "site" + ], + [ + 161, + 172, + "Structure V", + "evidence" + ] + ] + }, + { + "sid": 326, + "sent": "We propose that together with the previously reported initiation state, these structures represent the trajectory of eEF2-induced IRES translocation (shown as an animation in http://labs.umassmed.edu/korostelevlab/msc/iresmovie.gif and Video 1).", + "section": "DISCUSS", + "ner": [ + [ + 54, + 64, + "initiation", + "protein_state" + ], + [ + 78, + 88, + "structures", + "evidence" + ], + [ + 117, + 121, + "eEF2", + "protein" + ], + [ + 130, + 134, + "IRES", + "site" + ] + ] + }, + { + "sid": 327, + "sent": "Our structures reveal previously unseen intermediate states of eEF2 or EF-G engagement with the A site, providing the structural basis for the mechanism of translocase action.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 63, + 67, + "eEF2", + "protein" + ], + [ + 71, + 75, + "EF-G", + "protein" + ], + [ + 96, + 102, + "A site", + "site" + ], + [ + 156, + 167, + "translocase", + "protein_type" + ] + ] + }, + { + "sid": 328, + "sent": "Furthermore, they provide insight into the mechanism of eEF2\u2022GTP association with the pre-translocation ribosome and eEF2\u2022GDP dissociation from the post-translocation ribosome, also delineating the mechanism of translation inhibition by the antifungal drug sordarin.", + "section": "DISCUSS", + "ner": [ + [ + 56, + 64, + "eEF2\u2022GTP", + "complex_assembly" + ], + [ + 86, + 103, + "pre-translocation", + "protein_state" + ], + [ + 104, + 112, + "ribosome", + "complex_assembly" + ], + [ + 117, + 125, + "eEF2\u2022GDP", + "complex_assembly" + ], + [ + 148, + 166, + "post-translocation", + "protein_state" + ], + [ + 167, + 175, + "ribosome", + "complex_assembly" + ], + [ + 257, + 265, + "sordarin", + "chemical" + ] + ] + }, + { + "sid": 329, + "sent": "In summary, the reported ensemble of structures substantially enhances our understanding of the translocation mechanism, including that of tRNAs as discussed below.", + "section": "DISCUSS", + "ner": [ + [ + 37, + 47, + "structures", + "evidence" + ], + [ + 139, + 144, + "tRNAs", + "chemical" + ] + ] + }, + { + "sid": 330, + "sent": "Translocation of the TSV IRES on the 40S subunit globally resembles a step of an inchworm (Figure 4; see also Figure 3\u2014figure supplement 2).", + "section": "DISCUSS", + "ner": [ + [ + 21, + 24, + "TSV", + "species" + ], + [ + 25, + 29, + "IRES", + "site" + ], + [ + 37, + 40, + "40S", + "complex_assembly" + ], + [ + 41, + 48, + "subunit", + "structure_element" + ], + [ + 81, + 89, + "inchworm", + "protein_state" + ] + ] + }, + { + "sid": 331, + "sent": "At the start (initiation state), the IRES adopts an extended conformation (extended inchworm).", + "section": "DISCUSS", + "ner": [ + [ + 14, + 24, + "initiation", + "protein_state" + ], + [ + 37, + 41, + "IRES", + "site" + ], + [ + 52, + 60, + "extended", + "protein_state" + ], + [ + 75, + 92, + "extended inchworm", + "protein_state" + ] + ] + }, + { + "sid": 332, + "sent": "The front 'legs' (SL4 and SL5) of the 5\u2019-domain (front end) are attached to the 40S head proteins uS7, uS11 and eS25 (Figure 3\u2014figure supplement 2).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 15, + "front 'legs", + "structure_element" + ], + [ + 18, + 21, + "SL4", + "structure_element" + ], + [ + 26, + 29, + "SL5", + "structure_element" + ], + [ + 38, + 47, + "5\u2019-domain", + "structure_element" + ], + [ + 49, + 58, + "front end", + "structure_element" + ], + [ + 80, + 83, + "40S", + "complex_assembly" + ], + [ + 84, + 88, + "head", + "structure_element" + ], + [ + 98, + 101, + "uS7", + "protein" + ], + [ + 103, + 107, + "uS11", + "protein" + ], + [ + 112, + 116, + "eS25", + "protein" + ] + ] + }, + { + "sid": 333, + "sent": "PKI, representing the hind end, is bound in the A site.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 3, + "PKI", + "structure_element" + ], + [ + 22, + 30, + "hind end", + "structure_element" + ], + [ + 35, + 43, + "bound in", + "protein_state" + ], + [ + 48, + 54, + "A site", + "site" + ] + ] + }, + { + "sid": 334, + "sent": "In the first sub-step (Structures I to IV), the hind end advances from the A to the P site and approaches the front end, which remains attached to the 40S surface.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 41, + "Structures I to IV", + "evidence" + ], + [ + 48, + 56, + "hind end", + "structure_element" + ], + [ + 75, + 90, + "A to the P site", + "site" + ], + [ + 110, + 119, + "front end", + "structure_element" + ], + [ + 151, + 154, + "40S", + "complex_assembly" + ] + ] + }, + { + "sid": 335, + "sent": "This shortens the distance between PKI and SL4 by up to 20\u2009\u00c5 relative to the initiating IRES structure, resulting in a bent IRES conformation (bent inchworm).", + "section": "DISCUSS", + "ner": [ + [ + 35, + 38, + "PKI", + "structure_element" + ], + [ + 43, + 46, + "SL4", + "structure_element" + ], + [ + 88, + 92, + "IRES", + "site" + ], + [ + 93, + 102, + "structure", + "evidence" + ], + [ + 119, + 123, + "bent", + "protein_state" + ], + [ + 124, + 128, + "IRES", + "site" + ], + [ + 143, + 156, + "bent inchworm", + "protein_state" + ] + ] + }, + { + "sid": 336, + "sent": "Finally (Structures IV to V), as the hind end is accommodated in the P site, the front 'legs' advance by departing from their initial binding sites.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 27, + "Structures IV to V", + "evidence" + ], + [ + 37, + 45, + "hind end", + "structure_element" + ], + [ + 69, + 75, + "P site", + "site" + ], + [ + 81, + 93, + "front 'legs'", + "structure_element" + ], + [ + 126, + 147, + "initial binding sites", + "site" + ] + ] + }, + { + "sid": 337, + "sent": "This converts the IRES into an extended conformation, rendering the inchworm prepared for the next translocation step.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 22, + "IRES", + "site" + ], + [ + 31, + 39, + "extended", + "protein_state" + ], + [ + 68, + 76, + "inchworm", + "protein_state" + ] + ] + }, + { + "sid": 338, + "sent": "Notably, at all steps, the head of the IRES inchworm (L1.1 region) is supported by the mobile L1 stalk.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 31, + "head", + "structure_element" + ], + [ + 39, + 43, + "IRES", + "site" + ], + [ + 44, + 52, + "inchworm", + "protein_state" + ], + [ + 54, + 65, + "L1.1 region", + "structure_element" + ], + [ + 87, + 93, + "mobile", + "protein_state" + ], + [ + 94, + 102, + "L1 stalk", + "structure_element" + ] + ] + }, + { + "sid": 339, + "sent": "In the post-translocation CrPV IRES structure, the 5\u2019-domain similarly protrudes between the subunits and interacts with the L1 stalk, as in the initiation state for this IRES.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 25, + "post-translocation", + "protein_state" + ], + [ + 26, + 30, + "CrPV", + "species" + ], + [ + 31, + 35, + "IRES", + "site" + ], + [ + 36, + 45, + "structure", + "evidence" + ], + [ + 51, + 60, + "5\u2019-domain", + "structure_element" + ], + [ + 125, + 133, + "L1 stalk", + "structure_element" + ], + [ + 145, + 155, + "initiation", + "protein_state" + ], + [ + 171, + 175, + "IRES", + "site" + ] + ] + }, + { + "sid": 340, + "sent": "This underlines structural similarity for the TSV and CrPV IRES translocation mechanisms.", + "section": "DISCUSS", + "ner": [ + [ + 46, + 49, + "TSV", + "species" + ], + [ + 54, + 58, + "CrPV", + "species" + ], + [ + 59, + 63, + "IRES", + "site" + ] + ] + }, + { + "sid": 341, + "sent": "Upon translocation, the GCU start codon is positioned in the A site (Structure V), ready for interaction with Ala-tRNAAla upon eEF2 departure.", + "section": "DISCUSS", + "ner": [ + [ + 61, + 67, + "A site", + "site" + ], + [ + 69, + 80, + "Structure V", + "evidence" + ], + [ + 110, + 121, + "Ala-tRNAAla", + "chemical" + ], + [ + 127, + 131, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 342, + "sent": "Recent studies have shown that in some cases a fraction of IGR IRES-driven translation results from an alternative reading frame, which is shifted by one nucleotide relative to the normal ORF.", + "section": "DISCUSS", + "ner": [ + [ + 59, + 62, + "IGR", + "structure_element" + ], + [ + 63, + 67, + "IRES", + "site" + ], + [ + 188, + 191, + "ORF", + "structure_element" + ] + ] + }, + { + "sid": 343, + "sent": "One of the mechanistic scenarios (discussed in) involves binding of the first aminoacyl-tRNA to the post-translocated IRES mRNA frame shifted by one nucleotide (predominantly a +1 frame shift).", + "section": "DISCUSS", + "ner": [ + [ + 78, + 92, + "aminoacyl-tRNA", + "chemical" + ], + [ + 100, + 117, + "post-translocated", + "protein_state" + ], + [ + 118, + 122, + "IRES", + "site" + ], + [ + 123, + 127, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 344, + "sent": "In our structures, the IRES presents to the decoding center a pre-translocated or fully translocated ORF, rather than a +1 (more translocated) ORF, suggesting that eEF2 does not induce a highly populated fraction of +1 shifted IRES mRNAs.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 17, + "structures", + "evidence" + ], + [ + 23, + 27, + "IRES", + "site" + ], + [ + 44, + 59, + "decoding center", + "site" + ], + [ + 62, + 78, + "pre-translocated", + "protein_state" + ], + [ + 82, + 100, + "fully translocated", + "protein_state" + ], + [ + 101, + 104, + "ORF", + "structure_element" + ], + [ + 143, + 146, + "ORF", + "structure_element" + ], + [ + 164, + 168, + "eEF2", + "protein" + ], + [ + 227, + 231, + "IRES", + "site" + ], + [ + 232, + 237, + "mRNAs", + "chemical" + ] + ] + }, + { + "sid": 345, + "sent": "It is likely that alternative frame setting occurs following eEF2 release and that this depends on transient displacement of the start codon in the decoding center, allowing binding of the corresponding amino acyl-tRNA to an off-frame codon.", + "section": "DISCUSS", + "ner": [ + [ + 61, + 65, + "eEF2", + "protein" + ], + [ + 148, + 163, + "decoding center", + "site" + ], + [ + 203, + 218, + "amino acyl-tRNA", + "chemical" + ] + ] + }, + { + "sid": 346, + "sent": "Further structural studies involving 80S\u2022IRES\u2022tRNA complexes are necessary to understand the mechanisms underlying alternative reading frame selection.", + "section": "DISCUSS", + "ner": [ + [ + 8, + 26, + "structural studies", + "experimental_method" + ], + [ + 37, + 50, + "80S\u2022IRES\u2022tRNA", + "complex_assembly" + ] + ] + }, + { + "sid": 347, + "sent": "The presence of several translocation complexes in a single sample suggests that the structures represent equilibrium states of forward and reverse translocation of the IRES, which interconvert among each other.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 15, + "presence of", + "protein_state" + ], + [ + 85, + 95, + "structures", + "evidence" + ], + [ + 169, + 173, + "IRES", + "site" + ] + ] + }, + { + "sid": 348, + "sent": "This is consistent with the observations that the intergenic IRESs are prone to reverse translocation.", + "section": "DISCUSS", + "ner": [ + [ + 61, + 66, + "IRESs", + "site" + ] + ] + }, + { + "sid": 349, + "sent": "Specifically, biochemical toe-printing studies in the presence of eEF2\u2022GTP identified IRES in a non-translocated position unless eEF1a\u2022aa-tRNA is also present.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 46, + "biochemical toe-printing studies", + "experimental_method" + ], + [ + 54, + 65, + "presence of", + "protein_state" + ], + [ + 66, + 74, + "eEF2\u2022GTP", + "complex_assembly" + ], + [ + 86, + 90, + "IRES", + "site" + ], + [ + 96, + 112, + "non-translocated", + "protein_state" + ], + [ + 129, + 142, + "eEF1a\u2022aa-tRNA", + "complex_assembly" + ] + ] + }, + { + "sid": 350, + "sent": "These findings indicate that IRES translocation by eEF2 is futile: the IRES returns to the A site upon releasing eEF2\u2022GDP unless an amino-acyl tRNA enters the A site and blocks IRES back-translocation.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 33, + "IRES", + "site" + ], + [ + 51, + 55, + "eEF2", + "protein" + ], + [ + 71, + 75, + "IRES", + "site" + ], + [ + 91, + 97, + "A site", + "site" + ], + [ + 113, + 121, + "eEF2\u2022GDP", + "complex_assembly" + ], + [ + 132, + 147, + "amino-acyl tRNA", + "chemical" + ], + [ + 159, + 165, + "A site", + "site" + ], + [ + 177, + 181, + "IRES", + "site" + ] + ] + }, + { + "sid": 351, + "sent": "This contrasts with the post-translocated 2tRNA\u2022mRNA complex, in which the classical P and E-site tRNAs are stabilized in the non-rotated ribosome after translocase release.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 41, + "post-translocated", + "protein_state" + ], + [ + 42, + 52, + "2tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 85, + 97, + "P and E-site", + "site" + ], + [ + 98, + 103, + "tRNAs", + "chemical" + ], + [ + 126, + 137, + "non-rotated", + "protein_state" + ], + [ + 138, + 146, + "ribosome", + "complex_assembly" + ], + [ + 153, + 164, + "translocase", + "protein_type" + ] + ] + }, + { + "sid": 352, + "sent": "Thus, the meta-stability of the post-translocation IRES is likely due to the absence of stabilizing structural features present in the 2tRNA\u2022mRNA complex.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 50, + "post-translocation", + "protein_state" + ], + [ + 51, + 55, + "IRES", + "site" + ], + [ + 77, + 87, + "absence of", + "protein_state" + ], + [ + 135, + 145, + "2tRNA\u2022mRNA", + "complex_assembly" + ] + ] + }, + { + "sid": 353, + "sent": "In the initiation state, the IRES resembles a pre-translocation 2tRNA\u2022mRNA complex reduced to the A/P-tRNA anticodon-stem loop and elbow in the A site and the P/E-tRNA elbow contacting the L1 stalk.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 17, + "initiation", + "protein_state" + ], + [ + 29, + 33, + "IRES", + "site" + ], + [ + 46, + 63, + "pre-translocation", + "protein_state" + ], + [ + 64, + 74, + "2tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 98, + 101, + "A/P", + "site" + ], + [ + 102, + 106, + "tRNA", + "chemical" + ], + [ + 107, + 126, + "anticodon-stem loop", + "structure_element" + ], + [ + 131, + 136, + "elbow", + "structure_element" + ], + [ + 144, + 150, + "A site", + "site" + ], + [ + 159, + 162, + "P/E", + "site" + ], + [ + 163, + 167, + "tRNA", + "chemical" + ], + [ + 168, + 173, + "elbow", + "structure_element" + ], + [ + 189, + 197, + "L1 stalk", + "structure_element" + ] + ] + }, + { + "sid": 354, + "sent": "Because the anticodon-stem loop of the A-tRNA is sufficient for translocation completion, we ascribe the meta-stability of the post-translocation IRES to the absence of the P/E-tRNA elements, either the ASL or the acceptor arm, or both.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 31, + "anticodon-stem loop", + "structure_element" + ], + [ + 39, + 40, + "A", + "site" + ], + [ + 41, + 45, + "tRNA", + "chemical" + ], + [ + 127, + 145, + "post-translocation", + "protein_state" + ], + [ + 146, + 150, + "IRES", + "site" + ], + [ + 158, + 168, + "absence of", + "protein_state" + ], + [ + 173, + 176, + "P/E", + "site" + ], + [ + 177, + 181, + "tRNA", + "chemical" + ], + [ + 203, + 206, + "ASL", + "structure_element" + ] + ] + }, + { + "sid": 355, + "sent": "Furthermore, interactions of SL4 and SL5 with the 40S subunit likely contribute to stabilization of pre-translocation structures.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 32, + "SL4", + "structure_element" + ], + [ + 37, + 40, + "SL5", + "structure_element" + ], + [ + 50, + 53, + "40S", + "complex_assembly" + ], + [ + 54, + 61, + "subunit", + "structure_element" + ], + [ + 100, + 117, + "pre-translocation", + "protein_state" + ], + [ + 118, + 128, + "structures", + "evidence" + ] + ] + }, + { + "sid": 356, + "sent": "Partitioned roles of 40S subunit rearrangements", + "section": "DISCUSS", + "ner": [ + [ + 21, + 24, + "40S", + "complex_assembly" + ], + [ + 25, + 32, + "subunit", + "structure_element" + ] + ] + }, + { + "sid": 357, + "sent": "Our structures delineate the mechanistic functions for intersubunit rotation and head swivel in translocation.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 81, + 85, + "head", + "structure_element" + ] + ] + }, + { + "sid": 358, + "sent": "Specifically, intersubunit rotation allows eEF2 entry into the A site, while the head swivel mediates PKI translocation.", + "section": "DISCUSS", + "ner": [ + [ + 43, + 47, + "eEF2", + "protein" + ], + [ + 63, + 69, + "A site", + "site" + ], + [ + 81, + 85, + "head", + "structure_element" + ], + [ + 102, + 105, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 359, + "sent": "Various degrees of intersubunit rotation have been observed in cryo-EM studies of the 80S\u2022IRES initiation complexes.", + "section": "DISCUSS", + "ner": [ + [ + 63, + 78, + "cryo-EM studies", + "experimental_method" + ], + [ + 86, + 94, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 95, + 105, + "initiation", + "protein_state" + ] + ] + }, + { + "sid": 360, + "sent": "This suggests that the subunits are capable of spontaneous rotation, as is the case for tRNA-bound pre-translocation complexes.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 31, + "subunits", + "structure_element" + ], + [ + 88, + 98, + "tRNA-bound", + "protein_state" + ], + [ + 99, + 116, + "pre-translocation", + "protein_state" + ] + ] + }, + { + "sid": 361, + "sent": "The pre-translocation Structure I with eEF2 least advanced into the A site adopts a fully rotated conformation.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 21, + "pre-translocation", + "protein_state" + ], + [ + 22, + 33, + "Structure I", + "evidence" + ], + [ + 39, + 43, + "eEF2", + "protein" + ], + [ + 68, + 74, + "A site", + "site" + ], + [ + 84, + 110, + "fully rotated conformation", + "protein_state" + ] + ] + }, + { + "sid": 362, + "sent": "Reverse intersubunit rotation from Structure I to V shifts the translocation tunnel (the tunnel between the A, P and E sites) toward eEF2, which is rigidly attached to the 60S subunit.", + "section": "DISCUSS", + "ner": [ + [ + 35, + 51, + "Structure I to V", + "evidence" + ], + [ + 63, + 83, + "translocation tunnel", + "site" + ], + [ + 89, + 95, + "tunnel", + "site" + ], + [ + 108, + 124, + "A, P and E sites", + "site" + ], + [ + 133, + 137, + "eEF2", + "protein" + ], + [ + 172, + 175, + "60S", + "complex_assembly" + ], + [ + 176, + 183, + "subunit", + "structure_element" + ] + ] + }, + { + "sid": 363, + "sent": "This allows eEF2 to move into the A site.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 16, + "eEF2", + "protein" + ], + [ + 34, + 40, + "A site", + "site" + ] + ] + }, + { + "sid": 364, + "sent": "As such, reverse intersubunit rotation facilitates full docking of eEF2 in the A site.", + "section": "DISCUSS", + "ner": [ + [ + 67, + 71, + "eEF2", + "protein" + ], + [ + 79, + 85, + "A site", + "site" + ] + ] + }, + { + "sid": 365, + "sent": "Because the histidine-diphthamide tip of eEF2 (H583, H694 and Diph699) attaches to the codon-anticodon-like helix of PKI, eEF2 appears to directly force PKI out of the A site.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 37, + "histidine-diphthamide tip", + "site" + ], + [ + 41, + 45, + "eEF2", + "protein" + ], + [ + 47, + 51, + "H583", + "residue_name_number" + ], + [ + 53, + 57, + "H694", + "residue_name_number" + ], + [ + 62, + 69, + "Diph699", + "ptm" + ], + [ + 87, + 113, + "codon-anticodon-like helix", + "structure_element" + ], + [ + 117, + 120, + "PKI", + "structure_element" + ], + [ + 122, + 126, + "eEF2", + "protein" + ], + [ + 153, + 156, + "PKI", + "structure_element" + ], + [ + 168, + 174, + "A site", + "site" + ] + ] + }, + { + "sid": 366, + "sent": "The head swivel allows gradual translocation of PKI to the P site, first with respect to the body and then to the head.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 8, + "head", + "structure_element" + ], + [ + 48, + 51, + "PKI", + "structure_element" + ], + [ + 59, + 65, + "P site", + "site" + ], + [ + 93, + 97, + "body", + "structure_element" + ], + [ + 114, + 118, + "head", + "structure_element" + ] + ] + }, + { + "sid": 367, + "sent": "The fully swiveled conformations of Structures II and III represent the mid-point of translocation, in which PKI relocates between the head A site and body P site.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 18, + "fully swiveled", + "protein_state" + ], + [ + 36, + 57, + "Structures II and III", + "evidence" + ], + [ + 109, + 112, + "PKI", + "structure_element" + ], + [ + 135, + 139, + "head", + "structure_element" + ], + [ + 140, + 146, + "A site", + "site" + ], + [ + 151, + 155, + "body", + "structure_element" + ], + [ + 156, + 162, + "P site", + "site" + ] + ] + }, + { + "sid": 368, + "sent": "We note that such mid-states have not been observed for 2tRNA\u2022mRNA, but their formation can explain the formation of subsequent pe/E hybrid and ap/P chimeric structures (Figure 1\u2014figure supplement 1).", + "section": "DISCUSS", + "ner": [ + [ + 56, + 66, + "2tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 128, + 139, + "pe/E hybrid", + "protein_state" + ], + [ + 144, + 157, + "ap/P chimeric", + "protein_state" + ], + [ + 158, + 168, + "structures", + "evidence" + ] + ] + }, + { + "sid": 369, + "sent": "Reverse swivel from Structure III to V brings the head to the non-swiveled position, restoring the A and P sites on the small subunit.", + "section": "DISCUSS", + "ner": [ + [ + 20, + 38, + "Structure III to V", + "evidence" + ], + [ + 50, + 54, + "head", + "structure_element" + ], + [ + 62, + 74, + "non-swiveled", + "protein_state" + ], + [ + 99, + 112, + "A and P sites", + "site" + ], + [ + 120, + 133, + "small subunit", + "structure_element" + ] + ] + }, + { + "sid": 370, + "sent": "The functions of eEF2 in translocation", + "section": "DISCUSS", + "ner": [ + [ + 17, + 21, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 371, + "sent": "To our knowledge, our work provides the first high-resolution view of the dynamics of a ribosomal translocase that is inferred from an ensemble of structures sampled under uniform conditions.", + "section": "DISCUSS", + "ner": [ + [ + 88, + 109, + "ribosomal translocase", + "protein_type" + ], + [ + 147, + 157, + "structures", + "evidence" + ] + ] + }, + { + "sid": 372, + "sent": "The structures, therefore, offer a unique opportunity to address the role of the elongation factors during translocation.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 81, + 99, + "elongation factors", + "protein_type" + ] + ] + }, + { + "sid": 373, + "sent": "Translocases are efficient enzymes.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 12, + "Translocases", + "protein_type" + ] + ] + }, + { + "sid": 374, + "sent": "While the ribosome itself has the capacity to translocate in the absence of the translocase, spontaneous translocation is slow.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 18, + "ribosome", + "complex_assembly" + ], + [ + 65, + 75, + "absence of", + "protein_state" + ], + [ + 80, + 91, + "translocase", + "protein_type" + ] + ] + }, + { + "sid": 375, + "sent": "EF-G enhances the translocation rate by several orders of magnitude, aided by an additional 2- to 50-fold boost from GTP hydrolysis.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "EF-G", + "protein" + ], + [ + 117, + 120, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 376, + "sent": "Due to the lack of structures of translocation intermediates, the mechanistic role of eEF2/EF-G is not fully understood.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 29, + "structures", + "evidence" + ], + [ + 86, + 90, + "eEF2", + "protein" + ], + [ + 91, + 95, + "EF-G", + "protein" + ] + ] + }, + { + "sid": 377, + "sent": "The 80S\u2022IRES\u2022eEF2 structures reported here suggest two main roles for eEF2 in translocation.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 17, + "80S\u2022IRES\u2022eEF2", + "complex_assembly" + ], + [ + 18, + 28, + "structures", + "evidence" + ], + [ + 70, + 74, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 378, + "sent": "As discussed above, the first role is to directly shift PKI out of the A site upon spontaneous reverse intersubunit rotation.", + "section": "DISCUSS", + "ner": [ + [ + 56, + 59, + "PKI", + "structure_element" + ], + [ + 71, + 77, + "A site", + "site" + ] + ] + }, + { + "sid": 379, + "sent": "In our structures, the tip of domain IV docks next to PKI, with diphthamide 699 fit into the minor groove of the codon-anticodon-like helix of PKI (Figure 7).", + "section": "DISCUSS", + "ner": [ + [ + 7, + 17, + "structures", + "evidence" + ], + [ + 37, + 39, + "IV", + "structure_element" + ], + [ + 54, + 57, + "PKI", + "structure_element" + ], + [ + 64, + 79, + "diphthamide 699", + "ptm" + ], + [ + 93, + 105, + "minor groove", + "site" + ], + [ + 113, + 139, + "codon-anticodon-like helix", + "structure_element" + ], + [ + 143, + 146, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 380, + "sent": "This arrangement rationalizes inactivation of eEF2 by diphtheria toxin, which catalyzes ADP-ribosylation of the diphthamide (reviewed in).", + "section": "DISCUSS", + "ner": [ + [ + 46, + 50, + "eEF2", + "protein" + ], + [ + 54, + 70, + "diphtheria toxin", + "protein_type" + ], + [ + 88, + 104, + "ADP-ribosylation", + "ptm" + ], + [ + 112, + 123, + "diphthamide", + "ptm" + ] + ] + }, + { + "sid": 381, + "sent": "The enzyme ADP-ribosylates the NE2 atom of the imidazole ring, which in our structures interacts with the first two residues of the anticodon-like strand of PKI.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 26, + "ADP-ribosylates", + "ptm" + ], + [ + 76, + 86, + "structures", + "evidence" + ], + [ + 132, + 153, + "anticodon-like strand", + "structure_element" + ], + [ + 157, + 160, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 382, + "sent": "The bulky ADP-ribosyl moiety at this position would disrupt the interaction, rendering eEF2 unable to bind to the A site and/or stalled on ribosomes in a non-productive conformation.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 13, + "ADP", + "chemical" + ], + [ + 87, + 91, + "eEF2", + "protein" + ], + [ + 114, + 120, + "A site", + "site" + ], + [ + 139, + 148, + "ribosomes", + "complex_assembly" + ] + ] + }, + { + "sid": 383, + "sent": "As eEF2 shifts PKI toward the P site in the course of reverse intersubunit rotation, the 60S-attached translocase migrates along the surface of the 40S subunit, guided by electrostatic interactions.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 7, + "eEF2", + "protein" + ], + [ + 15, + 18, + "PKI", + "structure_element" + ], + [ + 30, + 36, + "P site", + "site" + ], + [ + 89, + 101, + "60S-attached", + "protein_state" + ], + [ + 102, + 113, + "translocase", + "protein_type" + ], + [ + 148, + 151, + "40S", + "complex_assembly" + ], + [ + 152, + 159, + "subunit", + "structure_element" + ], + [ + 171, + 197, + "electrostatic interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 384, + "sent": "Positively-charged patches of domains II and III (R391, K394, R433, R510) and IV (K613, R617, R609, R631, K651) slide over rRNA of the 40S body (h5) and head (h18 and h33/h34), respectively.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 26, + "Positively-charged patches", + "site" + ], + [ + 38, + 40, + "II", + "structure_element" + ], + [ + 45, + 48, + "III", + "structure_element" + ], + [ + 50, + 54, + "R391", + "residue_name_number" + ], + [ + 56, + 60, + "K394", + "residue_name_number" + ], + [ + 62, + 66, + "R433", + "residue_name_number" + ], + [ + 68, + 72, + "R510", + "residue_name_number" + ], + [ + 78, + 80, + "IV", + "structure_element" + ], + [ + 82, + 86, + "K613", + "residue_name_number" + ], + [ + 88, + 92, + "R617", + "residue_name_number" + ], + [ + 94, + 98, + "R609", + "residue_name_number" + ], + [ + 100, + 104, + "R631", + "residue_name_number" + ], + [ + 106, + 110, + "K651", + "residue_name_number" + ], + [ + 123, + 127, + "rRNA", + "chemical" + ], + [ + 135, + 138, + "40S", + "complex_assembly" + ], + [ + 139, + 143, + "body", + "structure_element" + ], + [ + 145, + 147, + "h5", + "structure_element" + ], + [ + 153, + 157, + "head", + "structure_element" + ], + [ + 159, + 162, + "h18", + "structure_element" + ], + [ + 167, + 170, + "h33", + "structure_element" + ], + [ + 171, + 174, + "h34", + "structure_element" + ] + ] + }, + { + "sid": 385, + "sent": "The Structures reveal hopping of the positive clusters over rRNA helices.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 14, + "Structures", + "evidence" + ], + [ + 60, + 64, + "rRNA", + "chemical" + ], + [ + 65, + 72, + "helices", + "structure_element" + ] + ] + }, + { + "sid": 386, + "sent": "For example, between Structures II and V, the K613/R617/R631 cluster of domain IV hops by ~19\u2009\u00c5 (for C\u03b1 of R617) from the phosphate backbone of h33 (at nt 1261\u20131264) to that of the neighboring h34 (at nt 1442\u20131445).", + "section": "DISCUSS", + "ner": [ + [ + 21, + 40, + "Structures II and V", + "evidence" + ], + [ + 46, + 50, + "K613", + "residue_name_number" + ], + [ + 51, + 55, + "R617", + "residue_name_number" + ], + [ + 56, + 60, + "R631", + "residue_name_number" + ], + [ + 79, + 81, + "IV", + "structure_element" + ], + [ + 107, + 111, + "R617", + "residue_name_number" + ], + [ + 144, + 147, + "h33", + "structure_element" + ], + [ + 155, + 164, + "1261\u20131264", + "residue_range" + ], + [ + 193, + 196, + "h34", + "structure_element" + ], + [ + 204, + 213, + "1442\u20131445", + "residue_range" + ] + ] + }, + { + "sid": 387, + "sent": "Thus, sliding of eEF2 involves reorganization of electrostatic, perhaps isoenergetic interactions, echoing those implied in extraordinarily fast ribosome inactivation rates by the small-protein ribotoxins and in fast protein association and diffusion along DNA.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 21, + "eEF2", + "protein" + ], + [ + 49, + 97, + "electrostatic, perhaps isoenergetic interactions", + "bond_interaction" + ], + [ + 145, + 153, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 388, + "sent": "Comparison of our structures with the 80S\u2022IRES initiation structure reveals the structural basis for the second key function of the translocase: 'unlocking' of intrasubunit rearrangements that are required for step-wise translocation of PKI on the small subunit.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 10, + "Comparison", + "experimental_method" + ], + [ + 18, + 28, + "structures", + "evidence" + ], + [ + 38, + 46, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 47, + 57, + "initiation", + "protein_state" + ], + [ + 58, + 67, + "structure", + "evidence" + ], + [ + 132, + 143, + "translocase", + "protein_type" + ], + [ + 237, + 240, + "PKI", + "structure_element" + ], + [ + 248, + 261, + "small subunit", + "structure_element" + ] + ] + }, + { + "sid": 389, + "sent": "The unlocking model of the ribosome\u20222tRNA\u2022mRNA pre-translocation complex has been proposed decades ago and functional requirement of the translocase in this process has been implicated.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 46, + "ribosome\u20222tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 47, + 64, + "pre-translocation", + "protein_state" + ], + [ + 137, + 148, + "translocase", + "protein_type" + ] + ] + }, + { + "sid": 390, + "sent": "However, the structural and mechanistic definitions of the locked and unlocked states have remained unclear, ranging from the globally distinct ribosome conformations to unknown local rearrangements, e.g. those in the decoding center.", + "section": "DISCUSS", + "ner": [ + [ + 59, + 65, + "locked", + "protein_state" + ], + [ + 70, + 78, + "unlocked", + "protein_state" + ], + [ + 144, + 152, + "ribosome", + "complex_assembly" + ], + [ + 218, + 233, + "decoding center", + "site" + ] + ] + }, + { + "sid": 391, + "sent": "FRET data indicate that translocation of 2tRNA\u2022mRNA on the 70S ribosome requires a forward-and-reverse head swivel, which may be related to the unlocking phenomenon.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 9, + "FRET data", + "evidence" + ], + [ + 41, + 51, + "2tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 59, + 71, + "70S ribosome", + "complex_assembly" + ], + [ + 103, + 107, + "head", + "structure_element" + ] + ] + }, + { + "sid": 392, + "sent": "Whereas intersubunit rotation of the pre-translocation complex occurs spontaneously, the head swivel is induced by the eEF2/EF-G translocase, consistent with requirement of eEF2 for unlocking.", + "section": "DISCUSS", + "ner": [ + [ + 37, + 54, + "pre-translocation", + "protein_state" + ], + [ + 89, + 93, + "head", + "structure_element" + ], + [ + 119, + 123, + "eEF2", + "protein" + ], + [ + 124, + 128, + "EF-G", + "protein" + ], + [ + 129, + 140, + "translocase", + "protein_type" + ], + [ + 173, + 177, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 393, + "sent": "Structural studies revealed large head swivels in various 70S\u2022tRNA\u2022EF-G and 80S\u2022tRNA\u2022eEF2 complexes, but not in 'locked' complexes with the A site occupied by the tRNA in the absence of the translocase.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 18, + "Structural studies", + "experimental_method" + ], + [ + 34, + 38, + "head", + "structure_element" + ], + [ + 58, + 71, + "70S\u2022tRNA\u2022EF-G", + "complex_assembly" + ], + [ + 76, + 89, + "80S\u2022tRNA\u2022eEF2", + "complex_assembly" + ], + [ + 113, + 119, + "locked", + "protein_state" + ], + [ + 121, + 135, + "complexes with", + "protein_state" + ], + [ + 140, + 146, + "A site", + "site" + ], + [ + 163, + 167, + "tRNA", + "chemical" + ], + [ + 175, + 185, + "absence of", + "protein_state" + ], + [ + 190, + 201, + "translocase", + "protein_type" + ] + ] + }, + { + "sid": 394, + "sent": "Our structures suggest that eEF2 induces head swivel by 'unlocking' the head-body interactions (Figure 7).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 28, + 32, + "eEF2", + "protein" + ], + [ + 41, + 45, + "head", + "structure_element" + ], + [ + 72, + 76, + "head", + "structure_element" + ], + [ + 77, + 81, + "body", + "structure_element" + ] + ] + }, + { + "sid": 395, + "sent": "Binding of the ASL to the A site is known from structural studies of bacterial ribosomes to result in 'domain closure' of the small subunit, i.e. closer association of the head, shoulder and body domains.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 18, + "ASL", + "structure_element" + ], + [ + 26, + 32, + "A site", + "site" + ], + [ + 47, + 65, + "structural studies", + "experimental_method" + ], + [ + 69, + 78, + "bacterial", + "taxonomy_domain" + ], + [ + 79, + 88, + "ribosomes", + "complex_assembly" + ], + [ + 103, + 117, + "domain closure", + "protein_state" + ], + [ + 126, + 139, + "small subunit", + "structure_element" + ], + [ + 172, + 176, + "head", + "structure_element" + ], + [ + 178, + 186, + "shoulder", + "structure_element" + ], + [ + 191, + 195, + "body", + "structure_element" + ] + ] + }, + { + "sid": 396, + "sent": "The domain closure 'locks' cognate tRNA in the A site via stacking on the head A site (C1274 in S. cerevisiae or C1054 in E. coli) and interactions with the body A-site nucleotides A1755 and A1756 (A1492 and A1493 in E. coli).", + "section": "DISCUSS", + "ner": [ + [ + 35, + 39, + "tRNA", + "chemical" + ], + [ + 47, + 53, + "A site", + "site" + ], + [ + 58, + 66, + "stacking", + "bond_interaction" + ], + [ + 74, + 78, + "head", + "structure_element" + ], + [ + 79, + 85, + "A site", + "site" + ], + [ + 87, + 92, + "C1274", + "residue_name_number" + ], + [ + 96, + 109, + "S. cerevisiae", + "species" + ], + [ + 113, + 118, + "C1054", + "residue_name_number" + ], + [ + 122, + 129, + "E. coli", + "species" + ], + [ + 157, + 161, + "body", + "structure_element" + ], + [ + 162, + 168, + "A-site", + "site" + ], + [ + 181, + 186, + "A1755", + "residue_name_number" + ], + [ + 191, + 196, + "A1756", + "residue_name_number" + ], + [ + 198, + 203, + "A1492", + "residue_name_number" + ], + [ + 208, + 213, + "A1493", + "residue_name_number" + ], + [ + 217, + 224, + "E. coli", + "species" + ] + ] + }, + { + "sid": 397, + "sent": "This 'locked' state is identical to that observed for PKI in the 80S\u2022IRES initiation structures in the absence of eEF2.", + "section": "DISCUSS", + "ner": [ + [ + 6, + 12, + "locked", + "protein_state" + ], + [ + 54, + 57, + "PKI", + "structure_element" + ], + [ + 65, + 73, + "80S\u2022IRES", + "complex_assembly" + ], + [ + 74, + 84, + "initiation", + "protein_state" + ], + [ + 85, + 95, + "structures", + "evidence" + ], + [ + 103, + 113, + "absence of", + "protein_state" + ], + [ + 114, + 118, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 398, + "sent": "Structure I demonstrates that at an early pre-translocation step, the histidine-diphthamide tip of eEF2 is wedged between A1755 and A1756 and PKI.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 11, + "Structure I", + "evidence" + ], + [ + 42, + 59, + "pre-translocation", + "protein_state" + ], + [ + 70, + 95, + "histidine-diphthamide tip", + "site" + ], + [ + 99, + 103, + "eEF2", + "protein" + ], + [ + 122, + 127, + "A1755", + "residue_name_number" + ], + [ + 132, + 137, + "A1756", + "residue_name_number" + ], + [ + 142, + 145, + "PKI", + "structure_element" + ] + ] + }, + { + "sid": 399, + "sent": "This destabilization allows PKI to detach from the body A site upon spontaneous reverse 40S body rotation, while maintaining interactions with the head A site.", + "section": "DISCUSS", + "ner": [ + [ + 28, + 31, + "PKI", + "structure_element" + ], + [ + 51, + 55, + "body", + "structure_element" + ], + [ + 56, + 62, + "A site", + "site" + ], + [ + 88, + 91, + "40S", + "complex_assembly" + ], + [ + 92, + 96, + "body", + "structure_element" + ], + [ + 147, + 151, + "head", + "structure_element" + ], + [ + 152, + 158, + "A site", + "site" + ] + ] + }, + { + "sid": 400, + "sent": "Destabilization of the head-bound PKI at the body A site thus allows mobility of the head relative to the body.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 33, + "head-bound", + "protein_state" + ], + [ + 34, + 37, + "PKI", + "structure_element" + ], + [ + 45, + 49, + "body", + "structure_element" + ], + [ + 50, + 56, + "A site", + "site" + ], + [ + 85, + 89, + "head", + "structure_element" + ], + [ + 106, + 110, + "body", + "structure_element" + ] + ] + }, + { + "sid": 401, + "sent": "The histidine-diphthamide-induced disengagement of PKI from A1755 and A1756 therefore provides the structural definition for the 'unlocking' mode of eEF2 action.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 25, + "histidine-diphthamide", + "ptm" + ], + [ + 51, + 54, + "PKI", + "structure_element" + ], + [ + 60, + 65, + "A1755", + "residue_name_number" + ], + [ + 70, + 75, + "A1756", + "residue_name_number" + ], + [ + 149, + 153, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 402, + "sent": "In summary, our structures are consistent with a model of eEF2-induced translocation in which both PKI and eEF2 passively migrate into the P and A site, respectively, during spontaneous 40S body rotation and head swivel, the latter being allowed by 'unlocking' of the A site by eEF2.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 26, + "structures", + "evidence" + ], + [ + 58, + 62, + "eEF2", + "protein" + ], + [ + 99, + 102, + "PKI", + "structure_element" + ], + [ + 107, + 111, + "eEF2", + "protein" + ], + [ + 139, + 151, + "P and A site", + "site" + ], + [ + 186, + 189, + "40S", + "complex_assembly" + ], + [ + 190, + 194, + "body", + "structure_element" + ], + [ + 208, + 212, + "head", + "structure_element" + ], + [ + 268, + 274, + "A site", + "site" + ], + [ + 278, + 282, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 403, + "sent": "Observation of different PKI conformations sampling a range of positions between the A and P sites in the presence of eEF2\u2022GDP implies that thermal fluctuations of the 40S head domain are sufficient for translocation along the energetically flat trajectory.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 28, + "PKI", + "structure_element" + ], + [ + 85, + 98, + "A and P sites", + "site" + ], + [ + 106, + 117, + "presence of", + "protein_state" + ], + [ + 118, + 126, + "eEF2\u2022GDP", + "complex_assembly" + ], + [ + 168, + 171, + "40S", + "complex_assembly" + ], + [ + 172, + 176, + "head", + "structure_element" + ] + ] + }, + { + "sid": 404, + "sent": "Insights into eEF2 association with and dissociation from the ribosome", + "section": "DISCUSS", + "ner": [ + [ + 14, + 18, + "eEF2", + "protein" + ], + [ + 62, + 70, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 405, + "sent": "The conformational rearrangements in eEF2 from Structure I through Structure V provide insights into the mechanisms of eEF2 association with the pre-translocation ribosome and dissociation from the post-translocation ribosome.", + "section": "DISCUSS", + "ner": [ + [ + 37, + 41, + "eEF2", + "protein" + ], + [ + 47, + 58, + "Structure I", + "evidence" + ], + [ + 67, + 78, + "Structure V", + "evidence" + ], + [ + 119, + 123, + "eEF2", + "protein" + ], + [ + 145, + 162, + "pre-translocation", + "protein_state" + ], + [ + 163, + 171, + "ribosome", + "complex_assembly" + ], + [ + 198, + 216, + "post-translocation", + "protein_state" + ], + [ + 217, + 225, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 406, + "sent": "In all five structures, the GTPase domain is attached to the P stalk and the sarcin-ricin loop.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 22, + "structures", + "evidence" + ], + [ + 28, + 41, + "GTPase domain", + "structure_element" + ], + [ + 61, + 68, + "P stalk", + "structure_element" + ], + [ + 77, + 94, + "sarcin-ricin loop", + "structure_element" + ] + ] + }, + { + "sid": 407, + "sent": "In the fully-rotated pre-translocation-like Structure I, an additional interaction exists.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 20, + "fully-rotated", + "protein_state" + ], + [ + 21, + 38, + "pre-translocation", + "protein_state" + ], + [ + 44, + 55, + "Structure I", + "evidence" + ] + ] + }, + { + "sid": 408, + "sent": "Here, switch loop I interacts with helix 14 (415CAAA418) of the 18S rRNA.", + "section": "DISCUSS", + "ner": [ + [ + 6, + 19, + "switch loop I", + "structure_element" + ], + [ + 35, + 43, + "helix 14", + "structure_element" + ], + [ + 45, + 55, + "415CAAA418", + "structure_element" + ], + [ + 64, + 72, + "18S rRNA", + "chemical" + ] + ] + }, + { + "sid": 409, + "sent": "This stabilization renders the GTPase center to adopt a GTP-bound conformation, similar to those observed in other translational GTPases in the presence of GTP analogs and in the 80S\u2022eEF2 complex bound with a transition-state mimic GDP\u2022AlF4\u2013. The switch loop contacts the base of A416 (invariable A344 in E. coli and A463 in H. sapiens).", + "section": "DISCUSS", + "ner": [ + [ + 31, + 44, + "GTPase center", + "site" + ], + [ + 56, + 65, + "GTP-bound", + "protein_state" + ], + [ + 115, + 136, + "translational GTPases", + "protein_type" + ], + [ + 144, + 155, + "presence of", + "protein_state" + ], + [ + 156, + 159, + "GTP", + "chemical" + ], + [ + 179, + 187, + "80S\u2022eEF2", + "complex_assembly" + ], + [ + 196, + 206, + "bound with", + "protein_state" + ], + [ + 232, + 241, + "GDP\u2022AlF4\u2013", + "complex_assembly" + ], + [ + 247, + 258, + "switch loop", + "structure_element" + ], + [ + 280, + 284, + "A416", + "residue_name_number" + ], + [ + 286, + 296, + "invariable", + "protein_state" + ], + [ + 297, + 301, + "A344", + "residue_name_number" + ], + [ + 305, + 312, + "E. coli", + "species" + ], + [ + 317, + 321, + "A463", + "residue_name_number" + ], + [ + 325, + 335, + "H. sapiens", + "species" + ] + ] + }, + { + "sid": 410, + "sent": "Mutations of residues flanking A344 in E. coli 16S rRNA modestly inhibit translation but do not specifically affect EF-G-mediated translocation.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 9, + "Mutations", + "experimental_method" + ], + [ + 31, + 35, + "A344", + "residue_name_number" + ], + [ + 39, + 46, + "E. coli", + "species" + ], + [ + 47, + 55, + "16S rRNA", + "chemical" + ], + [ + 116, + 120, + "EF-G", + "protein" + ] + ] + }, + { + "sid": 411, + "sent": "However, the effect of A344 mutation on translation was not addressed in that study, leaving the question open whether this residue is critical for eEF2/EF-G function.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 27, + "A344", + "residue_name_number" + ], + [ + 28, + 36, + "mutation", + "experimental_method" + ], + [ + 148, + 152, + "eEF2", + "protein" + ], + [ + 153, + 157, + "EF-G", + "protein" + ] + ] + }, + { + "sid": 412, + "sent": "The interaction between h14 and switch loop I is not resolved in Structures II to V, in all of which the small subunit is partially rotated or non-rotated, so that helix 14 is placed at least 6\u2009\u00c5 farther from eEF2 (Figure 5d).", + "section": "DISCUSS", + "ner": [ + [ + 24, + 27, + "h14", + "structure_element" + ], + [ + 32, + 45, + "switch loop I", + "structure_element" + ], + [ + 65, + 83, + "Structures II to V", + "evidence" + ], + [ + 105, + 118, + "small subunit", + "structure_element" + ], + [ + 122, + 139, + "partially rotated", + "protein_state" + ], + [ + 143, + 154, + "non-rotated", + "protein_state" + ], + [ + 164, + 172, + "helix 14", + "structure_element" + ], + [ + 209, + 213, + "eEF2", + "protein" + ] + ] + }, + { + "sid": 413, + "sent": "We conclude that unlike other conformations of the ribosome, the fully rotated 40S subunit of the pre-translocation ribosome provides an interaction surface, complementing the P stalk and SRL, for binding of the GTP-bound translocase.", + "section": "DISCUSS", + "ner": [ + [ + 51, + 59, + "ribosome", + "complex_assembly" + ], + [ + 65, + 78, + "fully rotated", + "protein_state" + ], + [ + 79, + 82, + "40S", + "complex_assembly" + ], + [ + 83, + 90, + "subunit", + "structure_element" + ], + [ + 98, + 115, + "pre-translocation", + "protein_state" + ], + [ + 116, + 124, + "ribosome", + "complex_assembly" + ], + [ + 137, + 156, + "interaction surface", + "site" + ], + [ + 176, + 183, + "P stalk", + "structure_element" + ], + [ + 188, + 191, + "SRL", + "structure_element" + ], + [ + 212, + 221, + "GTP-bound", + "protein_state" + ], + [ + 222, + 233, + "translocase", + "protein_type" + ] + ] + }, + { + "sid": 414, + "sent": "This structural basis rationalizes the observation of transient stabilization of the rotated 70S ribosome upon EF-G\u2022GTP binding and prior to translocation.", + "section": "DISCUSS", + "ner": [ + [ + 85, + 92, + "rotated", + "protein_state" + ], + [ + 93, + 105, + "70S ribosome", + "complex_assembly" + ], + [ + 111, + 119, + "EF-G\u2022GTP", + "complex_assembly" + ] + ] + }, + { + "sid": 415, + "sent": "The least rotated conformation of the post-translocation Structure V suggests conformational changes that may trigger eEF2 release from the ribosome at the end of translocation.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 17, + "least rotated", + "protein_state" + ], + [ + 38, + 56, + "post-translocation", + "protein_state" + ], + [ + 57, + 68, + "Structure V", + "evidence" + ], + [ + 118, + 122, + "eEF2", + "protein" + ], + [ + 140, + 148, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 416, + "sent": "The most pronounced inter-domain rearrangement in eEF2 involves movement of domain III.", + "section": "DISCUSS", + "ner": [ + [ + 50, + 54, + "eEF2", + "protein" + ], + [ + 83, + 86, + "III", + "structure_element" + ] + ] + }, + { + "sid": 417, + "sent": "In the rotated or mid-rotated Structures I through III, this domain remains rigidly associated with domain V and the N-terminal superdomain and does not undergo noticeable rearrangements.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 14, + "rotated", + "protein_state" + ], + [ + 18, + 29, + "mid-rotated", + "protein_state" + ], + [ + 30, + 54, + "Structures I through III", + "evidence" + ], + [ + 107, + 108, + "V", + "structure_element" + ], + [ + 128, + 139, + "superdomain", + "structure_element" + ] + ] + }, + { + "sid": 418, + "sent": "In Structure V, however, the tip of helix A of domain III is displaced toward domain I by ~5\u2009\u00c5 relative to that in mid-rotated or fully rotated structures.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 14, + "Structure V", + "evidence" + ], + [ + 36, + 43, + "helix A", + "structure_element" + ], + [ + 54, + 57, + "III", + "structure_element" + ], + [ + 85, + 86, + "I", + "structure_element" + ], + [ + 115, + 126, + "mid-rotated", + "protein_state" + ], + [ + 130, + 143, + "fully rotated", + "protein_state" + ], + [ + 144, + 154, + "structures", + "evidence" + ] + ] + }, + { + "sid": 419, + "sent": "This displacement is caused by the 8\u2009\u00c5 movement of the 40S body protein uS12 upon reverse intersubunit rotation from Structure I to V (Figure 6d).", + "section": "DISCUSS", + "ner": [ + [ + 55, + 58, + "40S", + "complex_assembly" + ], + [ + 59, + 63, + "body", + "structure_element" + ], + [ + 72, + 76, + "uS12", + "protein" + ], + [ + 117, + 133, + "Structure I to V", + "evidence" + ] + ] + }, + { + "sid": 420, + "sent": "We propose that the shift of domain III by uS12 initiates intra-domain rearrangements in eEF2, which unstack the \u03b2-platform of domain III from that of domain V. This would result in a conformation characteristic of free eEF2 and EF-G in which the \u03b2-platforms are nearly perpendicular.", + "section": "DISCUSS", + "ner": [ + [ + 36, + 39, + "III", + "structure_element" + ], + [ + 43, + 47, + "uS12", + "protein" + ], + [ + 89, + 93, + "eEF2", + "protein" + ], + [ + 113, + 123, + "\u03b2-platform", + "structure_element" + ], + [ + 134, + 137, + "III", + "structure_element" + ], + [ + 158, + 159, + "V", + "structure_element" + ], + [ + 215, + 219, + "free", + "protein_state" + ], + [ + 220, + 224, + "eEF2", + "protein" + ], + [ + 229, + 233, + "EF-G", + "protein" + ], + [ + 247, + 258, + "\u03b2-platforms", + "structure_element" + ] + ] + }, + { + "sid": 421, + "sent": "As we discuss below, Structure V captures a 'pre-unstacking' state due to stabilization of the interface between domains III and V by sordarin.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 32, + "Structure V", + "evidence" + ], + [ + 45, + 59, + "pre-unstacking", + "protein_state" + ], + [ + 95, + 104, + "interface", + "site" + ], + [ + 121, + 124, + "III", + "structure_element" + ], + [ + 129, + 130, + "V", + "structure_element" + ], + [ + 134, + 142, + "sordarin", + "chemical" + ] + ] + }, + { + "sid": 422, + "sent": "Sordarin stabilizes GDP-bound eEF2 on the ribosome", + "section": "DISCUSS", + "ner": [ + [ + 0, + 8, + "Sordarin", + "chemical" + ], + [ + 20, + 29, + "GDP-bound", + "protein_state" + ], + [ + 30, + 34, + "eEF2", + "protein" + ], + [ + 42, + 50, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 423, + "sent": "Sordarin is a potent antifungal antibiotic that inhibits translation.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 8, + "Sordarin", + "chemical" + ] + ] + }, + { + "sid": 424, + "sent": "Based on biochemical experiments, two alternative mechanisms of action were proposed: sordarin either prevents eEF2 departure by inhibiting GTP hydrolysis or acts after GTP hydrolysis.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 32, + "biochemical experiments", + "experimental_method" + ], + [ + 86, + 94, + "sordarin", + "chemical" + ], + [ + 111, + 115, + "eEF2", + "protein" + ], + [ + 140, + 143, + "GTP", + "chemical" + ], + [ + 169, + 172, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 425, + "sent": "Although our complex was assembled using eEF2\u2022GTP, density maps clearly show GDP and Mg2+ in each structure (Figure 5g).", + "section": "DISCUSS", + "ner": [ + [ + 41, + 49, + "eEF2\u2022GTP", + "complex_assembly" + ], + [ + 51, + 63, + "density maps", + "evidence" + ], + [ + 77, + 80, + "GDP", + "chemical" + ], + [ + 85, + 89, + "Mg2+", + "chemical" + ], + [ + 98, + 107, + "structure", + "evidence" + ] + ] + }, + { + "sid": 426, + "sent": "Our structures therefore indicate that sordarin stalls eEF2 on the ribosome in the GDP-bound form, i.e. following GTP hydrolysis and phosphate release.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 39, + 47, + "sordarin", + "chemical" + ], + [ + 55, + 59, + "eEF2", + "protein" + ], + [ + 67, + 75, + "ribosome", + "complex_assembly" + ], + [ + 83, + 92, + "GDP-bound", + "protein_state" + ], + [ + 114, + 117, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 427, + "sent": "The mechanism of stalling is suggested by comparison of pre-translocation and post-translocation structures in our ensemble.", + "section": "DISCUSS", + "ner": [ + [ + 56, + 73, + "pre-translocation", + "protein_state" + ], + [ + 78, + 96, + "post-translocation", + "protein_state" + ], + [ + 97, + 107, + "structures", + "evidence" + ] + ] + }, + { + "sid": 428, + "sent": "In all five structures, sordarin is bound between domains III and V of eEF2, stabilized by hydrophobic interactions identical to those in the isolated eEF2\u2022sordarin complex (Figures 5g and h).", + "section": "DISCUSS", + "ner": [ + [ + 12, + 22, + "structures", + "evidence" + ], + [ + 24, + 32, + "sordarin", + "chemical" + ], + [ + 36, + 41, + "bound", + "protein_state" + ], + [ + 58, + 61, + "III", + "structure_element" + ], + [ + 66, + 67, + "V", + "structure_element" + ], + [ + 71, + 75, + "eEF2", + "protein" + ], + [ + 91, + 115, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 142, + 150, + "isolated", + "protein_state" + ], + [ + 151, + 164, + "eEF2\u2022sordarin", + "complex_assembly" + ] + ] + }, + { + "sid": 429, + "sent": "In the nearly non-rotated post-translocation Structure V, the tip of domain III is shifted, however the interface between domains III and V remains unchanged, suggesting strong stabilization of this interface by sordarin.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 25, + "nearly non-rotated", + "protein_state" + ], + [ + 26, + 44, + "post-translocation", + "protein_state" + ], + [ + 45, + 56, + "Structure V", + "evidence" + ], + [ + 76, + 79, + "III", + "structure_element" + ], + [ + 104, + 113, + "interface", + "site" + ], + [ + 130, + 133, + "III", + "structure_element" + ], + [ + 138, + 139, + "V", + "structure_element" + ], + [ + 199, + 208, + "interface", + "site" + ], + [ + 212, + 220, + "sordarin", + "chemical" + ] + ] + }, + { + "sid": 430, + "sent": "We note that Structure V is slightly more rotated than the 80S\u20222tRNA\u2022mRNA complex in the absence of eEF2\u2022sordarin, implying that sordarin interferes with the final stages of reverse rotation of the post-translocation ribosome.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 24, + "Structure V", + "evidence" + ], + [ + 59, + 73, + "80S\u20222tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 89, + 99, + "absence of", + "protein_state" + ], + [ + 100, + 113, + "eEF2\u2022sordarin", + "complex_assembly" + ], + [ + 129, + 137, + "sordarin", + "chemical" + ], + [ + 198, + 216, + "post-translocation", + "protein_state" + ], + [ + 217, + 225, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 431, + "sent": "We propose that sordarin acts to prevent full reverse rotation and release of eEF2\u2022GDP by stabilizing the interdomain interface and thus blocking uS12-induced disengagement of domain III from domain V.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 24, + "sordarin", + "chemical" + ], + [ + 78, + 86, + "eEF2\u2022GDP", + "complex_assembly" + ], + [ + 106, + 127, + "interdomain interface", + "site" + ], + [ + 146, + 150, + "uS12", + "protein" + ], + [ + 183, + 186, + "III", + "structure_element" + ], + [ + 199, + 200, + "V", + "structure_element" + ] + ] + }, + { + "sid": 432, + "sent": "Implications for tRNA and mRNA translocation during translation", + "section": "DISCUSS", + "ner": [ + [ + 17, + 21, + "tRNA", + "chemical" + ], + [ + 26, + 30, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 433, + "sent": "Because translocation of tRNA must involve large-scale dynamics, this step has long been regarded as the most puzzling step of translation.", + "section": "DISCUSS", + "ner": [ + [ + 25, + 29, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 434, + "sent": "Intersubunit rearrangements and tRNA hybrid states have been proposed to play key roles half a century ago.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 36, + "tRNA", + "chemical" + ], + [ + 37, + 43, + "hybrid", + "protein_state" + ] + ] + }, + { + "sid": 435, + "sent": "Despite an impressive body of biochemical, fluorescence and structural data accumulated since then, translocation remains the least understood step of elongation.", + "section": "DISCUSS", + "ner": [ + [ + 22, + 26, + "body", + "structure_element" + ], + [ + 30, + 75, + "biochemical, fluorescence and structural data", + "evidence" + ] + ] + }, + { + "sid": 436, + "sent": "The structural understanding of ribosome and tRNA dynamics has been greatly aided by a wealth of X-ray and cryo-EM structures (reviewed in).", + "section": "DISCUSS", + "ner": [ + [ + 32, + 40, + "ribosome", + "complex_assembly" + ], + [ + 45, + 49, + "tRNA", + "chemical" + ], + [ + 97, + 102, + "X-ray", + "experimental_method" + ], + [ + 107, + 114, + "cryo-EM", + "experimental_method" + ], + [ + 115, + 125, + "structures", + "evidence" + ] + ] + }, + { + "sid": 437, + "sent": "However, visualization of the eEF2/EF-G-induced translocation is confined to very early pre-EF-G-entry states and late (almost translocated or fully translocated) states, leaving most of the path from the A to the P site uncharacterized (Figure 1\u2014figure supplement 1).", + "section": "DISCUSS", + "ner": [ + [ + 30, + 34, + "eEF2", + "protein" + ], + [ + 35, + 39, + "EF-G", + "protein" + ], + [ + 88, + 102, + "pre-EF-G-entry", + "protein_state" + ], + [ + 120, + 139, + "almost translocated", + "protein_state" + ], + [ + 143, + 161, + "fully translocated", + "protein_state" + ], + [ + 205, + 220, + "A to the P site", + "site" + ] + ] + }, + { + "sid": 438, + "sent": "Our study provides new insights into the structural understanding of tRNA translocation.", + "section": "DISCUSS", + "ner": [ + [ + 69, + 73, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 439, + "sent": "First, we propose that tRNA and IRES translocations occur via the same general trajectory.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 27, + "tRNA", + "chemical" + ], + [ + 32, + 36, + "IRES", + "site" + ] + ] + }, + { + "sid": 440, + "sent": "This is evident from the fact that ribosome rearrangements in translocation are inherent to the ribosome and likely occur in similar ways in both cases.", + "section": "DISCUSS", + "ner": [ + [ + 35, + 43, + "ribosome", + "complex_assembly" + ], + [ + 96, + 104, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 441, + "sent": "Furthermore, the step-wise coupling of ribosome dynamics with IRES translocation is overall consistent with that observed for 2tRNA\u2022mRNA translocation in solution.", + "section": "DISCUSS", + "ner": [ + [ + 39, + 47, + "ribosome", + "complex_assembly" + ], + [ + 62, + 66, + "IRES", + "site" + ], + [ + 126, + 136, + "2tRNA\u2022mRNA", + "complex_assembly" + ] + ] + }, + { + "sid": 442, + "sent": "For example, fluorescence and biochemical studies revealed that the early pre-translocation EF-G-bound ribosomes are fully rotated and translocation of the tRNA-mRNA complex occurs during reverse rotation of the small subunit, coupled with head swivel.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 49, + "fluorescence and biochemical studies", + "experimental_method" + ], + [ + 74, + 91, + "pre-translocation", + "protein_state" + ], + [ + 92, + 102, + "EF-G-bound", + "protein_state" + ], + [ + 103, + 112, + "ribosomes", + "complex_assembly" + ], + [ + 117, + 130, + "fully rotated", + "protein_state" + ], + [ + 156, + 165, + "tRNA-mRNA", + "complex_assembly" + ], + [ + 212, + 225, + "small subunit", + "structure_element" + ], + [ + 240, + 244, + "head", + "structure_element" + ] + ] + }, + { + "sid": 443, + "sent": "The sequence of ribosome rearrangements during IRES translocation also agrees with that inferred from 70S\u2022EF-G structures, including those in which the A-to-P-site translocating tRNA was not present.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 24, + "ribosome", + "complex_assembly" + ], + [ + 47, + 51, + "IRES", + "site" + ], + [ + 102, + 110, + "70S\u2022EF-G", + "complex_assembly" + ], + [ + 111, + 121, + "structures", + "evidence" + ], + [ + 152, + 163, + "A-to-P-site", + "site" + ], + [ + 178, + 182, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 444, + "sent": "Specifically, an earlier translocation intermediate ribosome (TIpre) was proposed to adopt a rotated (7\u20139\u00b0) body and a partly rotated head (5\u20137.5\u00b0), in agreement with the conformation of our Structure I. The most swiveled head (18\u201321\u00b0) was observed in a mid-rotated ribosome (3\u20135\u00b0) of a later translocation intermediate TIpost, similar to the conformation of our Structure III.", + "section": "DISCUSS", + "ner": [ + [ + 52, + 60, + "ribosome", + "complex_assembly" + ], + [ + 93, + 100, + "rotated", + "protein_state" + ], + [ + 108, + 112, + "body", + "structure_element" + ], + [ + 119, + 133, + "partly rotated", + "protein_state" + ], + [ + 134, + 138, + "head", + "structure_element" + ], + [ + 191, + 202, + "Structure I", + "evidence" + ], + [ + 208, + 221, + "most swiveled", + "protein_state" + ], + [ + 222, + 226, + "head", + "structure_element" + ], + [ + 254, + 265, + "mid-rotated", + "protein_state" + ], + [ + 266, + 274, + "ribosome", + "complex_assembly" + ], + [ + 363, + 376, + "Structure III", + "evidence" + ] + ] + }, + { + "sid": 445, + "sent": "Overall, these correlations suggest that the intermediate locations of the elusive A-to-P-site translocating tRNA are similar to those of PKI in our structures.", + "section": "DISCUSS", + "ner": [ + [ + 83, + 94, + "A-to-P-site", + "site" + ], + [ + 109, + 113, + "tRNA", + "chemical" + ], + [ + 138, + 141, + "PKI", + "structure_element" + ], + [ + 149, + 159, + "structures", + "evidence" + ] + ] + }, + { + "sid": 446, + "sent": "Second, the structures clarify the structural basis of the often-used but structurally undefined terms 'locking' and 'unlocking' with respect to the pre-translocation complex (Figure 6f).", + "section": "DISCUSS", + "ner": [ + [ + 12, + 22, + "structures", + "evidence" + ], + [ + 149, + 166, + "pre-translocation", + "protein_state" + ] + ] + }, + { + "sid": 447, + "sent": "We deem the pre-translocation complex locked, because the A-site bound ASL-mRNA is stabilized by interactions with the decoding center.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 29, + "pre-translocation", + "protein_state" + ], + [ + 38, + 44, + "locked", + "protein_state" + ], + [ + 58, + 70, + "A-site bound", + "protein_state" + ], + [ + 75, + 79, + "mRNA", + "chemical" + ], + [ + 119, + 134, + "decoding center", + "site" + ] + ] + }, + { + "sid": 448, + "sent": "These interactions are maintained for the classical- and hybrid-state tRNAs in the spontaneously sampled non-rotated and rotated ribosomes, respectively.", + "section": "DISCUSS", + "ner": [ + [ + 42, + 51, + "classical", + "protein_state" + ], + [ + 57, + 63, + "hybrid", + "protein_state" + ], + [ + 70, + 75, + "tRNAs", + "chemical" + ], + [ + 105, + 116, + "non-rotated", + "protein_state" + ], + [ + 121, + 128, + "rotated", + "protein_state" + ], + [ + 129, + 138, + "ribosomes", + "complex_assembly" + ] + ] + }, + { + "sid": 449, + "sent": "Unlocking involves separation of the codon-anticodon helix from the decoding center residues by the protruding tip of eEF2/EF-G (Figure 7), occurring in the fully rotated ribosome at an early pre-translocation step.", + "section": "DISCUSS", + "ner": [ + [ + 37, + 58, + "codon-anticodon helix", + "structure_element" + ], + [ + 68, + 83, + "decoding center", + "site" + ], + [ + 118, + 122, + "eEF2", + "protein" + ], + [ + 123, + 127, + "EF-G", + "protein" + ], + [ + 157, + 170, + "fully rotated", + "protein_state" + ], + [ + 171, + 179, + "ribosome", + "complex_assembly" + ], + [ + 192, + 209, + "pre-translocation", + "protein_state" + ] + ] + }, + { + "sid": 450, + "sent": "This unlatches the head, allowing creation of hitherto elusive intermediate tRNA positions during spontaneous reverse body rotation.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 23, + "head", + "structure_element" + ], + [ + 76, + 80, + "tRNA", + "chemical" + ], + [ + 118, + 122, + "body", + "structure_element" + ] + ] + }, + { + "sid": 451, + "sent": "Third, our findings uncover a new role of the head swivel.", + "section": "DISCUSS", + "ner": [ + [ + 46, + 50, + "head", + "structure_element" + ] + ] + }, + { + "sid": 452, + "sent": "Previous studies showed that this movement widens the constriction ('gate') between the P and E sites, thus allowing the P-tRNA passage to the E site.", + "section": "DISCUSS", + "ner": [ + [ + 54, + 66, + "constriction", + "site" + ], + [ + 69, + 73, + "gate", + "site" + ], + [ + 88, + 101, + "P and E sites", + "site" + ], + [ + 121, + 122, + "P", + "site" + ], + [ + 123, + 127, + "tRNA", + "chemical" + ], + [ + 143, + 149, + "E site", + "site" + ] + ] + }, + { + "sid": 453, + "sent": "In addition to the 'gate-opening' role, we now show that the head swivel brings the head A site to the body P site, allowing a step-wise conveying of the codon-anticodon helix between the A and P sites.", + "section": "DISCUSS", + "ner": [ + [ + 20, + 24, + "gate", + "site" + ], + [ + 61, + 65, + "head", + "structure_element" + ], + [ + 84, + 88, + "head", + "structure_element" + ], + [ + 89, + 95, + "A site", + "site" + ], + [ + 103, + 107, + "body", + "structure_element" + ], + [ + 108, + 114, + "P site", + "site" + ], + [ + 154, + 175, + "codon-anticodon helix", + "structure_element" + ], + [ + 188, + 201, + "A and P sites", + "site" + ] + ] + }, + { + "sid": 454, + "sent": "Finally, the similar populations of particles (within a 2X range) in our 80S\u2022IRES\u2022eEF2 reconstructions (Figure 1\u2014figure supplement 2) suggest that the intermediate translocation states sample several energetically similar and interconverting conformations.", + "section": "DISCUSS", + "ner": [ + [ + 36, + 45, + "particles", + "experimental_method" + ], + [ + 73, + 86, + "80S\u2022IRES\u2022eEF2", + "complex_assembly" + ], + [ + 87, + 102, + "reconstructions", + "evidence" + ] + ] + }, + { + "sid": 455, + "sent": "This is consistent with the idea of a rather flat energy landscape of translocation, suggested by recent work that measured mechanical work produced by the ribosome during translocation.", + "section": "DISCUSS", + "ner": [ + [ + 156, + 164, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 456, + "sent": "Our findings implicate, however, that the energy landscape is not completely flat and contains local minima for transient positions of the codon-anticodon helix between the A and P sites.", + "section": "DISCUSS", + "ner": [ + [ + 139, + 160, + "codon-anticodon helix", + "structure_element" + ], + [ + 173, + 186, + "A and P sites", + "site" + ] + ] + }, + { + "sid": 457, + "sent": "The shift of the PKI with respect to the body occurs during forward head swivel in two major sub-steps of ~4\u2009\u00c5 each (initiation complex to I, and I to II), after which PKI undergoes small shifts to settle in the body P site in Structures III, IV and V (Figure 2\u2014source data 1).", + "section": "DISCUSS", + "ner": [ + [ + 17, + 20, + "PKI", + "structure_element" + ], + [ + 41, + 45, + "body", + "structure_element" + ], + [ + 68, + 72, + "head", + "structure_element" + ], + [ + 117, + 135, + "initiation complex", + "complex_assembly" + ], + [ + 139, + 140, + "I", + "evidence" + ], + [ + 146, + 147, + "I", + "evidence" + ], + [ + 151, + 153, + "II", + "evidence" + ], + [ + 168, + 171, + "PKI", + "structure_element" + ], + [ + 212, + 216, + "body", + "structure_element" + ], + [ + 217, + 223, + "P site", + "site" + ], + [ + 227, + 251, + "Structures III, IV and V", + "evidence" + ] + ] + }, + { + "sid": 458, + "sent": "Movement of PKI relative to the head occurs during the subsequent reverse swivel in three 3\u20137\u2009\u00c5 sub-steps (II to III to IV to V).", + "section": "DISCUSS", + "ner": [ + [ + 12, + 15, + "PKI", + "structure_element" + ], + [ + 32, + 36, + "head", + "structure_element" + ], + [ + 107, + 127, + "II to III to IV to V", + "evidence" + ] + ] + }, + { + "sid": 459, + "sent": "We note that four of our near-atomic resolution maps comprised ~30,000 particles each, the minimum number required for a near-atomic-resolution reconstruction of the ribosome.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 52, + "maps", + "evidence" + ], + [ + 71, + 80, + "particles", + "experimental_method" + ], + [ + 121, + 158, + "near-atomic-resolution reconstruction", + "evidence" + ], + [ + 166, + 174, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 460, + "sent": "Translation of viral mRNA", + "section": "DISCUSS", + "ner": [ + [ + 15, + 20, + "viral", + "taxonomy_domain" + ], + [ + 21, + 25, + "mRNA", + "chemical" + ] + ] + }, + { + "sid": 461, + "sent": "Our work sheds light on the dynamic mechanism of cap-independent translation by IGR IRESs, tightly coupled with the universally conserved dynamic properties of the ribosome.", + "section": "DISCUSS", + "ner": [ + [ + 80, + 83, + "IGR", + "structure_element" + ], + [ + 84, + 89, + "IRESs", + "site" + ], + [ + 116, + 137, + "universally conserved", + "protein_state" + ], + [ + 164, + 172, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 462, + "sent": "The cryo-EM structures demonstrate that the TSV IRES structurally and dynamically represents a chimera of the 2tRNA\u2022mRNA translocating complex (A/P-tRNA \u2022 P/E-tRNA \u2022 mRNA).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 11, + "cryo-EM", + "experimental_method" + ], + [ + 12, + 22, + "structures", + "evidence" + ], + [ + 44, + 47, + "TSV", + "species" + ], + [ + 48, + 52, + "IRES", + "site" + ], + [ + 110, + 120, + "2tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 144, + 170, + "A/P-tRNA \u2022 P/E-tRNA \u2022 mRNA", + "complex_assembly" + ] + ] + }, + { + "sid": 463, + "sent": "Like in the 2tRNA\u2022mRNA translocating complex in which the two tRNAs move independently of each other, the PKI domain moves relative to the 5\u00b4-domain, causing the IRES to undergo an inchworm-walk translocation.", + "section": "DISCUSS", + "ner": [ + [ + 12, + 22, + "2tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 62, + 67, + "tRNAs", + "chemical" + ], + [ + 106, + 109, + "PKI", + "structure_element" + ], + [ + 139, + 148, + "5\u00b4-domain", + "structure_element" + ], + [ + 162, + 166, + "IRES", + "site" + ], + [ + 181, + 189, + "inchworm", + "protein_state" + ] + ] + }, + { + "sid": 464, + "sent": "A large structural difference between the IRES and the 2tRNA\u2022mRNA complex exists, however, in that the IRES lacks three out of six tRNA-like domains involved in tRNA translocation.", + "section": "DISCUSS", + "ner": [ + [ + 42, + 46, + "IRES", + "site" + ], + [ + 55, + 65, + "2tRNA\u2022mRNA", + "complex_assembly" + ], + [ + 103, + 107, + "IRES", + "site" + ], + [ + 108, + 113, + "lacks", + "protein_state" + ], + [ + 131, + 148, + "tRNA-like domains", + "structure_element" + ], + [ + 161, + 165, + "tRNA", + "chemical" + ] + ] + }, + { + "sid": 465, + "sent": "This difference likely accounts for the inefficient translocation of the IRES, which is difficult to stabilize in the post-translocation state and therefore is prone to reverse translocation.", + "section": "DISCUSS", + "ner": [ + [ + 73, + 77, + "IRES", + "site" + ], + [ + 118, + 136, + "post-translocation", + "protein_state" + ] + ] + }, + { + "sid": 466, + "sent": "Although structurally handicapped, the TSV IRES manages to translocate by employing ribosome dynamics that are remarkably similar to that in 2tRNA\u2022mRNA translocation.", + "section": "DISCUSS", + "ner": [ + [ + 39, + 42, + "TSV", + "species" + ], + [ + 43, + 47, + "IRES", + "site" + ], + [ + 84, + 92, + "ribosome", + "complex_assembly" + ], + [ + 141, + 151, + "2tRNA\u2022mRNA", + "complex_assembly" + ] + ] + }, + { + "sid": 467, + "sent": "The uniformity of ribosome dynamics underscores the idea that translocation is an inherent and structurally-optimized property of the ribosome, supported also by translocation activity in the absence of the elongation factor.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 26, + "ribosome", + "complex_assembly" + ], + [ + 134, + 142, + "ribosome", + "complex_assembly" + ], + [ + 192, + 202, + "absence of", + "protein_state" + ], + [ + 207, + 224, + "elongation factor", + "protein_type" + ] + ] + }, + { + "sid": 468, + "sent": "This property is rendered by the relative mobility of the three major building blocks, the 60S subunit and the 40S head and body, assisted by ligand-interacting extensions including the L1 stalk and the P stalk.", + "section": "DISCUSS", + "ner": [ + [ + 91, + 94, + "60S", + "complex_assembly" + ], + [ + 95, + 102, + "subunit", + "structure_element" + ], + [ + 111, + 114, + "40S", + "complex_assembly" + ], + [ + 115, + 119, + "head", + "structure_element" + ], + [ + 124, + 128, + "body", + "structure_element" + ], + [ + 142, + 171, + "ligand-interacting extensions", + "structure_element" + ], + [ + 186, + 194, + "L1 stalk", + "structure_element" + ], + [ + 203, + 210, + "P stalk", + "structure_element" + ] + ] + }, + { + "sid": 469, + "sent": "Intergenic IRESs, in turn, represent a striking example of convergent molecular evolution.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 16, + "IRESs", + "site" + ] + ] + }, + { + "sid": 470, + "sent": "Viral mRNAs have evolved to adopt an atypical structure to employ the inherent ribosome dynamics, to be able to hijack the host translational machinery in a simple fashion.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "Viral", + "taxonomy_domain" + ], + [ + 6, + 11, + "mRNAs", + "chemical" + ], + [ + 46, + 55, + "structure", + "evidence" + ], + [ + 79, + 87, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 471, + "sent": "Ensemble cryo-EM", + "section": "DISCUSS", + "ner": [ + [ + 9, + 16, + "cryo-EM", + "experimental_method" + ] + ] + }, + { + "sid": 472, + "sent": "Our current understanding of macromolecular machines, such as the ribosome, is often limited by a gap between biophysical/biochemical studies and structural studies.", + "section": "DISCUSS", + "ner": [ + [ + 66, + 74, + "ribosome", + "complex_assembly" + ], + [ + 110, + 141, + "biophysical/biochemical studies", + "experimental_method" + ], + [ + 146, + 164, + "structural studies", + "experimental_method" + ] + ] + }, + { + "sid": 473, + "sent": "For example, F\u00f6rster resonance energy transfer can provide insight into the macromolecular dynamics of an assembly at the single-molecule level but is limited to specifically labeled locations within the assembly.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 46, + "F\u00f6rster resonance energy transfer", + "experimental_method" + ] + ] + }, + { + "sid": 474, + "sent": "High-resolution crystal structures, on the other hand, can provide static images of an assembly, and the structural dynamics can only be inferred by comparing structures that are usually obtained in different experiments and under different, often non-native, conditions.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 34, + "crystal structures", + "evidence" + ], + [ + 159, + 169, + "structures", + "evidence" + ] + ] + }, + { + "sid": 475, + "sent": "Cryo-EM offers the possibility of obtaining integrated information of both structure and dynamics as demonstrated in lower-resolution studies of bacterial ribosome complexes.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 7, + "Cryo-EM", + "experimental_method" + ], + [ + 75, + 84, + "structure", + "evidence" + ], + [ + 145, + 154, + "bacterial", + "taxonomy_domain" + ], + [ + 155, + 163, + "ribosome", + "complex_assembly" + ] + ] + }, + { + "sid": 476, + "sent": "This is presumably one of the reasons why most recent studies of ribosome complexes have focused on a single high-resolution structure despite the non-uniform local resolution of the maps that likely reflects structural heterogeneity.", + "section": "DISCUSS", + "ner": [ + [ + 65, + 73, + "ribosome", + "complex_assembly" + ], + [ + 125, + 134, + "structure", + "evidence" + ], + [ + 183, + 187, + "maps", + "evidence" + ] + ] + }, + { + "sid": 477, + "sent": "The computational efficiency of FREALIGN has allowed us to classify a relatively large dataset (1.1 million particles) into 15 classes (Figure 1\u2014figure supplement 2) and obtain eight near-atomic-resolution structures from it.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 40, + "FREALIGN", + "experimental_method" + ], + [ + 108, + 117, + "particles", + "experimental_method" + ], + [ + 206, + 216, + "structures", + "evidence" + ] + ] + }, + { + "sid": 478, + "sent": "The classification, which followed an initial alignment of all particles to a single reference, required about 130,000 CPU hours or about five to six full days on a 1000-CPU cluster.", + "section": "DISCUSS", + "ner": [ + [ + 63, + 72, + "particles", + "experimental_method" + ] + ] + }, + { + "sid": 479, + "sent": "Therefore, cryo-EM has the potential to become a standard tool for uncovering detailed dynamic pathways of complex macromolecular machines.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 18, + "cryo-EM", + "experimental_method" + ] + ] + } + ] + }, + "PMC4792962": { + "annotations": [ + { + "sid": 0, + "sent": "A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome", + "section": "TITLE", + "ner": [ + [ + 40, + 64, + "autocatalytic activation", + "ptm" + ], + [ + 72, + 86, + "20S proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 1, + "sent": "Biogenesis of the 20S proteasome is tightly regulated.", + "section": "ABSTRACT", + "ner": [ + [ + 18, + 32, + "20S proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 2, + "sent": "The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly.", + "section": "ABSTRACT", + "ner": [ + [ + 15, + 26, + "propeptides", + "structure_element" + ], + [ + 42, + 53, + "active-site", + "site" + ], + [ + 54, + 64, + "threonines", + "residue_name" + ], + [ + 69, + 86, + "autocatalytically", + "ptm" + ] + ] + }, + { + "sid": 3, + "sent": "However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic.", + "section": "ABSTRACT", + "ner": [ + [ + 68, + 87, + "strict conservation", + "protein_state" + ], + [ + 91, + 100, + "threonine", + "residue_name" + ] + ] + }, + { + "sid": 4, + "sent": "Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad.", + "section": "ABSTRACT", + "ner": [ + [ + 12, + 23, + "mutagenesis", + "experimental_method" + ], + [ + 25, + 46, + "X-ray crystallography", + "experimental_method" + ], + [ + 51, + 69, + "biochemical assays", + "experimental_method" + ], + [ + 86, + 91, + "Lys33", + "residue_name_number" + ], + [ + 129, + 139, + "propeptide", + "structure_element" + ], + [ + 161, + 165, + "Thr1", + "residue_name_number" + ], + [ + 218, + 223, + "Asp17", + "residue_name_number" + ], + [ + 238, + 253, + "catalytic triad", + "site" + ] + ] + }, + { + "sid": 5, + "sent": "Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 12, + "Substitution", + "experimental_method" + ], + [ + 16, + 20, + "Thr1", + "residue_name_number" + ], + [ + 24, + 27, + "Cys", + "residue_name" + ], + [ + 58, + 63, + "Lys33", + "residue_name_number" + ], + [ + 68, + 79, + "inactivates", + "protein_state" + ], + [ + 84, + 94, + "proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 6, + "sent": "Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates.", + "section": "ABSTRACT", + "ner": [ + [ + 11, + 18, + "Thr1Ser", + "mutant" + ], + [ + 19, + 25, + "mutant", + "protein_state" + ], + [ + 29, + 35, + "active", + "protein_state" + ], + [ + 72, + 81, + "wild type", + "protein_state" + ], + [ + 125, + 129, + "Ser1", + "residue_name_number" + ] + ] + }, + { + "sid": 7, + "sent": "This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.", + "section": "ABSTRACT", + "ner": [ + [ + 72, + 92, + "propeptide autolysis", + "ptm" + ], + [ + 147, + 157, + "proteasome", + "complex_assembly" + ], + [ + 170, + 188, + "threonine protease", + "protein_type" + ] + ] + }, + { + "sid": 8, + "sent": " The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear.", + "section": "ABSTRACT", + "ner": [ + [ + 5, + 15, + "proteasome", + "complex_assembly" + ], + [ + 54, + 72, + "threonine protease", + "protein_type" + ] + ] + }, + { + "sid": 9, + "sent": "Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity.", + "section": "ABSTRACT", + "ner": [ + [ + 85, + 95, + "proteasome", + "complex_assembly" + ], + [ + 96, + 107, + "active site", + "site" + ] + ] + }, + { + "sid": 10, + "sent": "The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells.", + "section": "INTRO", + "ner": [ + [ + 4, + 32, + "20S proteasome core particle", + "complex_assembly" + ], + [ + 34, + 36, + "CP", + "complex_assembly" + ], + [ + 49, + 71, + "non-lysosomal protease", + "protein_type" + ], + [ + 75, + 85, + "eukaryotic", + "taxonomy_domain" + ] + ] + }, + { + "sid": 11, + "sent": "Its seven different \u03b1 and seven different \u03b2 subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder.", + "section": "INTRO", + "ner": [ + [ + 20, + 21, + "\u03b1", + "protein" + ], + [ + 42, + 52, + "\u03b2 subunits", + "protein" + ], + [ + 72, + 82, + "heptameric", + "oligomeric_state" + ], + [ + 83, + 88, + "rings", + "structure_element" + ], + [ + 130, + 145, + "hollow cylinder", + "structure_element" + ] + ] + }, + { + "sid": 12, + "sent": "While the inactive \u03b1 subunits build the two outer rings, the \u03b2 subunits form the inner rings.", + "section": "INTRO", + "ner": [ + [ + 10, + 18, + "inactive", + "protein_state" + ], + [ + 19, + 29, + "\u03b1 subunits", + "protein" + ], + [ + 50, + 55, + "rings", + "structure_element" + ], + [ + 61, + 71, + "\u03b2 subunits", + "protein" + ], + [ + 87, + 92, + "rings", + "structure_element" + ] + ] + }, + { + "sid": 13, + "sent": "Only three out of the seven different \u03b2 subunits, namely \u03b21, \u03b22 and \u03b25, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides.", + "section": "INTRO", + "ner": [ + [ + 38, + 48, + "\u03b2 subunits", + "protein" + ], + [ + 57, + 59, + "\u03b21", + "protein" + ], + [ + 61, + 63, + "\u03b22", + "protein" + ], + [ + 68, + 70, + "\u03b25", + "protein" + ], + [ + 88, + 114, + "proteolytic active centres", + "site" + ], + [ + 127, + 129, + "CP", + "complex_assembly" + ], + [ + 164, + 175, + "propeptides", + "structure_element" + ] + ] + }, + { + "sid": 14, + "sent": "In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1).", + "section": "INTRO", + "ner": [ + [ + 21, + 23, + "CP", + "complex_assembly" + ], + [ + 40, + 51, + "prosegments", + "structure_element" + ], + [ + 56, + 81, + "autocatalytically removed", + "ptm" + ], + [ + 117, + 136, + "active site residue", + "site" + ], + [ + 137, + 141, + "Thr1", + "residue_name_number" + ], + [ + 182, + 189, + "Gly(-1)", + "residue_name_number" + ] + ] + }, + { + "sid": 15, + "sent": "Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides.", + "section": "INTRO", + "ner": [ + [ + 15, + 26, + "propeptides", + "structure_element" + ], + [ + 50, + 56, + "active", + "protein_state" + ], + [ + 57, + 59, + "CP", + "complex_assembly" + ] + ] + }, + { + "sid": 16, + "sent": "Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active \u03b2 subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds.", + "section": "INTRO", + "ner": [ + [ + 36, + 61, + "substrate-binding channel", + "site" + ], + [ + 129, + 135, + "active", + "protein_state" + ], + [ + 136, + 146, + "\u03b2 subunits", + "protein" + ], + [ + 152, + 164, + "active sites", + "site" + ] + ] + }, + { + "sid": 17, + "sent": "Nucleophilic attack of Thr1O\u03b3 on the carbonyl carbon atom of the scissile peptide bond creates a first cleavage product and a covalent acyl-enzyme intermediate.", + "section": "INTRO", + "ner": [ + [ + 23, + 27, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 18, + "sent": "Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment.", + "section": "INTRO", + "ner": [ + [ + 19, + 26, + "complex", + "complex_assembly" + ], + [ + 61, + 66, + "water", + "chemical" + ], + [ + 92, + 98, + "enzyme", + "complex_assembly" + ], + [ + 123, + 130, + "peptide", + "chemical" + ] + ] + }, + { + "sid": 19, + "sent": "The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1O\u03b3 for peptide-bond cleavage.", + "section": "INTRO", + "ner": [ + [ + 4, + 14, + "proteasome", + "complex_assembly" + ], + [ + 40, + 80, + "N-terminal nucleophilic (Ntn) hydrolases", + "protein_type" + ], + [ + 90, + 94, + "free", + "protein_state" + ], + [ + 121, + 125, + "Thr1", + "residue_name_number" + ], + [ + 158, + 162, + "Thr1", + "residue_name_number" + ], + [ + 205, + 209, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 20, + "sent": "This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed.", + "section": "INTRO", + "ner": [ + [ + 40, + 74, + "autocatalytic precursor processing", + "ptm" + ], + [ + 90, + 98, + "immature", + "protein_state" + ], + [ + 99, + 111, + "active sites", + "site" + ], + [ + 113, + 117, + "Thr1", + "residue_name_number" + ], + [ + 152, + 159, + "Gly(-1)", + "residue_name_number" + ] + ] + }, + { + "sid": 21, + "sent": "An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7\u2009\u00c5).", + "section": "INTRO", + "ner": [ + [ + 47, + 51, + "Thr1", + "residue_name_number" + ], + [ + 88, + 93, + "Lys33", + "residue_name_number" + ], + [ + 110, + 126, + "hydrogen-bonding", + "bond_interaction" + ], + [ + 139, + 143, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 22, + "sent": "In principle it could function as the general base during both autocatalytic removal of the propeptide and protein substrate cleavage.", + "section": "INTRO", + "ner": [ + [ + 63, + 84, + "autocatalytic removal", + "ptm" + ], + [ + 92, + 102, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 23, + "sent": "Here we provide experimental evidences for this distinct view of the proteasome active-site mechanism.", + "section": "INTRO", + "ner": [ + [ + 69, + 79, + "proteasome", + "complex_assembly" + ], + [ + 80, + 91, + "active-site", + "site" + ] + ] + }, + { + "sid": 24, + "sent": "Data from biochemical and structural analyses of proteasome variants with mutations in the \u03b25 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome.", + "section": "INTRO", + "ner": [ + [ + 10, + 45, + "biochemical and structural analyses", + "experimental_method" + ], + [ + 91, + 93, + "\u03b25", + "protein" + ], + [ + 94, + 104, + "propeptide", + "structure_element" + ], + [ + 113, + 124, + "active site", + "site" + ], + [ + 228, + 252, + "autocatalytic activation", + "ptm" + ], + [ + 260, + 270, + "proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 25, + "sent": "Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays.", + "section": "INTRO", + "ner": [ + [ + 44, + 47, + "Thr", + "residue_name" + ], + [ + 53, + 56, + "Cys", + "residue_name" + ], + [ + 60, + 63, + "Ser", + "residue_name" + ], + [ + 101, + 122, + "X-ray crystallography", + "experimental_method" + ], + [ + 137, + 167, + "activity and inhibition assays", + "experimental_method" + ] + ] + }, + { + "sid": 26, + "sent": "Inactivation of proteasome subunits by T1A mutations", + "section": "RESULTS", + "ner": [ + [ + 16, + 26, + "proteasome", + "complex_assembly" + ], + [ + 27, + 35, + "subunits", + "protein" + ], + [ + 39, + 42, + "T1A", + "mutant" + ], + [ + 43, + 52, + "mutations", + "experimental_method" + ] + ] + }, + { + "sid": 27, + "sent": "Proteasome-mediated degradation of cell-cycle regulators and potentially toxic misfolded proteins is required for the viability of eukaryotic cells.", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "Proteasome", + "complex_assembly" + ], + [ + 131, + 141, + "eukaryotic", + "taxonomy_domain" + ] + ] + }, + { + "sid": 28, + "sent": "Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites.", + "section": "RESULTS", + "ner": [ + [ + 20, + 31, + "active site", + "site" + ], + [ + 32, + 36, + "Thr1", + "residue_name_number" + ], + [ + 40, + 51, + "mutation to", + "experimental_method" + ], + [ + 52, + 55, + "Ala", + "residue_name" + ], + [ + 126, + 136, + "proteasome", + "complex_assembly" + ], + [ + 137, + 149, + "active sites", + "site" + ] + ] + }, + { + "sid": 29, + "sent": "Yeast strains carrying the single mutations \u03b21-T1A or \u03b22-T1A, or both, are viable, even though one or two of the three distinct catalytic \u03b2 subunits are disabled and carry remnants of their N-terminal propeptides (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "Yeast", + "taxonomy_domain" + ], + [ + 44, + 50, + "\u03b21-T1A", + "mutant" + ], + [ + 54, + 60, + "\u03b22-T1A", + "mutant" + ], + [ + 128, + 137, + "catalytic", + "protein_state" + ], + [ + 138, + 148, + "\u03b2 subunits", + "protein" + ], + [ + 153, + 161, + "disabled", + "protein_state" + ], + [ + 166, + 183, + "carry remnants of", + "protein_state" + ], + [ + 201, + 212, + "propeptides", + "structure_element" + ] + ] + }, + { + "sid": 30, + "sent": "These results indicate that the \u03b21 and \u03b22 proteolytic activities are not essential for cell survival.", + "section": "RESULTS", + "ner": [ + [ + 32, + 34, + "\u03b21", + "protein" + ], + [ + 39, + 41, + "\u03b22", + "protein" + ] + ] + }, + { + "sid": 31, + "sent": "By contrast, the T1A mutation in subunit \u03b25 has been reported to be lethal or nearly so.", + "section": "RESULTS", + "ner": [ + [ + 17, + 20, + "T1A", + "mutant" + ], + [ + 41, + 43, + "\u03b25", + "protein" + ] + ] + }, + { + "sid": 32, + "sent": "Viability is restored if the \u03b25-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (\u03b25-T1A pp trans), although substantial phenotypic impairment remains (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 29, + 35, + "\u03b25-T1A", + "mutant" + ], + [ + 52, + 62, + "propeptide", + "structure_element" + ], + [ + 64, + 66, + "pp", + "chemical" + ], + [ + 68, + 100, + "deleted but expressed separately", + "experimental_method" + ], + [ + 104, + 109, + "trans", + "protein_state" + ], + [ + 111, + 117, + "\u03b25-T1A", + "mutant" + ], + [ + 118, + 120, + "pp", + "chemical" + ], + [ + 121, + 126, + "trans", + "protein_state" + ] + ] + }, + { + "sid": 33, + "sent": "Our present crystallographic analysis of the \u03b25-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed \u03b25 propeptide is not bound in the \u03b25 substrate-binding channel (Supplementary Fig. 1a).", + "section": "RESULTS", + "ner": [ + [ + 12, + 37, + "crystallographic analysis", + "experimental_method" + ], + [ + 45, + 51, + "\u03b25-T1A", + "mutant" + ], + [ + 52, + 54, + "pp", + "chemical" + ], + [ + 55, + 60, + "trans", + "protein_state" + ], + [ + 61, + 67, + "mutant", + "protein_state" + ], + [ + 90, + 98, + "mutation", + "experimental_method" + ], + [ + 138, + 159, + "catalytic active site", + "site" + ], + [ + 173, + 188, + "trans-expressed", + "experimental_method" + ], + [ + 189, + 191, + "\u03b25", + "protein" + ], + [ + 192, + 202, + "propeptide", + "structure_element" + ], + [ + 206, + 215, + "not bound", + "protein_state" + ], + [ + 223, + 225, + "\u03b25", + "protein" + ], + [ + 226, + 251, + "substrate-binding channel", + "site" + ] + ] + }, + { + "sid": 34, + "sent": "The extremely weak growth of the \u03b25-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy.", + "section": "RESULTS", + "ner": [ + [ + 33, + 39, + "\u03b25-T1A", + "mutant" + ], + [ + 40, + 46, + "mutant", + "protein_state" + ], + [ + 47, + 49, + "pp", + "chemical" + ], + [ + 50, + 53, + "cis", + "protein_state" + ] + ] + }, + { + "sid": 35, + "sent": "Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the \u03b25-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4\u20135 days for initial colony formation (Table 1 and Supplementary Methods).", + "section": "RESULTS", + "ner": [ + [ + 0, + 26, + "Sequencing of the plasmids", + "experimental_method" + ], + [ + 59, + 64, + "yeast", + "taxonomy_domain" + ], + [ + 88, + 113, + "site-directed mutagenesis", + "experimental_method" + ], + [ + 132, + 138, + "\u03b25-T1A", + "mutant" + ], + [ + 139, + 145, + "mutant", + "protein_state" + ], + [ + 146, + 148, + "pp", + "chemical" + ], + [ + 149, + 152, + "cis", + "protein_state" + ] + ] + }, + { + "sid": 36, + "sent": "We also identified an additional point mutation K81R in subunit \u03b25 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits \u03b14, \u03b24 and \u03b25 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts.", + "section": "RESULTS", + "ner": [ + [ + 48, + 52, + "K81R", + "mutant" + ], + [ + 64, + 66, + "\u03b25", + "protein" + ], + [ + 112, + 143, + "This single amino-acid exchange", + "experimental_method" + ], + [ + 162, + 171, + "interface", + "site" + ], + [ + 188, + 190, + "\u03b14", + "protein" + ], + [ + 192, + 194, + "\u03b24", + "protein" + ], + [ + 199, + 201, + "\u03b25", + "protein" + ], + [ + 251, + 253, + "CP", + "complex_assembly" + ] + ] + }, + { + "sid": 37, + "sent": "The slightly better growth of the \u03b25-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the \u03b25-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1).", + "section": "RESULTS", + "ner": [ + [ + 34, + 45, + "\u03b25-T1A-K81R", + "mutant" + ], + [ + 46, + 52, + "mutant", + "protein_state" + ], + [ + 77, + 94, + "crystal structure", + "evidence" + ], + [ + 100, + 105, + "yeast", + "taxonomy_domain" + ], + [ + 106, + 116, + "proteasome", + "complex_assembly" + ], + [ + 118, + 121, + "yCP", + "complex_assembly" + ], + [ + 132, + 138, + "\u03b25-T1A", + "mutant" + ] + ] + }, + { + "sid": 38, + "sent": "Propeptide conformation and triggering of autolysis", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "Propeptide", + "structure_element" + ], + [ + 42, + 51, + "autolysis", + "ptm" + ] + ] + }, + { + "sid": 39, + "sent": "In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature \u03b2-subunit domains.", + "section": "RESULTS", + "ner": [ + [ + 22, + 32, + "proteasome", + "complex_assembly" + ], + [ + 49, + 60, + "propeptides", + "structure_element" + ], + [ + 65, + 90, + "autocatalytically cleaved", + "ptm" + ], + [ + 100, + 106, + "mature", + "protein_state" + ], + [ + 107, + 124, + "\u03b2-subunit domains", + "protein" + ] + ] + }, + { + "sid": 40, + "sent": "For subunit \u03b21, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2).", + "section": "RESULTS", + "ner": [ + [ + 12, + 14, + "\u03b21", + "protein" + ], + [ + 73, + 83, + "propeptide", + "structure_element" + ], + [ + 104, + 108, + "(-2)", + "residue_number" + ], + [ + 147, + 168, + "S1 specificity pocket", + "site" + ], + [ + 176, + 201, + "substrate-binding channel", + "site" + ], + [ + 223, + 225, + "45", + "residue_number" + ] + ] + }, + { + "sid": 41, + "sent": "Furthermore, it was observed that the prosegment forms an antiparallel \u03b2-sheet in the active site, and that Gly(-1) adopts a \u03b3-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.).", + "section": "RESULTS", + "ner": [ + [ + 38, + 48, + "prosegment", + "structure_element" + ], + [ + 58, + 78, + "antiparallel \u03b2-sheet", + "structure_element" + ], + [ + 86, + 97, + "active site", + "site" + ], + [ + 108, + 115, + "Gly(-1)", + "residue_name_number" + ], + [ + 125, + 144, + "\u03b3-turn conformation", + "structure_element" + ], + [ + 188, + 201, + "hydrogen bond", + "bond_interaction" + ], + [ + 210, + 217, + "Leu(-2)", + "residue_name_number" + ], + [ + 223, + 227, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 42, + "sent": "Here we again analysed the \u03b21-T1A mutant crystallographically but in addition determined the structures of the \u03b22-T1A single and \u03b21-T1A-\u03b22-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1).", + "section": "RESULTS", + "ner": [ + [ + 27, + 33, + "\u03b21-T1A", + "mutant" + ], + [ + 34, + 40, + "mutant", + "protein_state" + ], + [ + 41, + 61, + "crystallographically", + "experimental_method" + ], + [ + 93, + 103, + "structures", + "evidence" + ], + [ + 111, + 117, + "\u03b22-T1A", + "mutant" + ], + [ + 129, + 142, + "\u03b21-T1A-\u03b22-T1A", + "mutant" + ] + ] + }, + { + "sid": 43, + "sent": "In subunit \u03b21, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a).", + "section": "RESULTS", + "ner": [ + [ + 11, + 13, + "\u03b21", + "protein" + ], + [ + 29, + 36, + "Gly(-1)", + "residue_name_number" + ], + [ + 52, + 62, + "sharp turn", + "structure_element" + ], + [ + 81, + 100, + "prosegment cleavage", + "ptm" + ] + ] + }, + { + "sid": 44, + "sent": "However, the \u03b3-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1.", + "section": "RESULTS", + "ner": [ + [ + 13, + 32, + "\u03b3-turn conformation", + "structure_element" + ], + [ + 52, + 65, + "hydrogen bond", + "bond_interaction" + ], + [ + 198, + 205, + "Gly(-1)", + "residue_name_number" + ], + [ + 233, + 237, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 45, + "sent": "Regarding the \u03b22 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in \u03b21, which might be due to the rather large \u03b22-S1 pocket created by Gly45.", + "section": "RESULTS", + "ner": [ + [ + 14, + 16, + "\u03b22", + "protein" + ], + [ + 17, + 27, + "propeptide", + "structure_element" + ], + [ + 29, + 36, + "Thr(-2)", + "residue_name_number" + ], + [ + 50, + 59, + "S1 pocket", + "site" + ], + [ + 102, + 109, + "Leu(-2)", + "residue_name_number" + ], + [ + 113, + 115, + "\u03b21", + "protein" + ], + [ + 156, + 158, + "\u03b22", + "protein" + ], + [ + 159, + 168, + "S1 pocket", + "site" + ], + [ + 180, + 185, + "Gly45", + "residue_name_number" + ] + ] + }, + { + "sid": 46, + "sent": "Thr(-2) positions Gly(-1)O via hydrogen bonding (\u223c2.8\u2009\u00c5) in a perfect trajectory for the nucleophilic attack by Thr1O\u03b3 (Fig. 1b and Supplementary Fig. 2b).", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Thr(-2)", + "residue_name_number" + ], + [ + 18, + 25, + "Gly(-1)", + "residue_name_number" + ], + [ + 31, + 47, + "hydrogen bonding", + "bond_interaction" + ], + [ + 112, + 116, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 47, + "sent": "Next, we examined the position of the \u03b25 propeptide in the \u03b25-T1A-K81R mutant.", + "section": "RESULTS", + "ner": [ + [ + 38, + 40, + "\u03b25", + "protein" + ], + [ + 41, + 51, + "propeptide", + "structure_element" + ], + [ + 59, + 70, + "\u03b25-T1A-K81R", + "mutant" + ], + [ + 71, + 77, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 48, + "sent": "Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel \u03b2-sheet.", + "section": "RESULTS", + "ner": [ + [ + 14, + 21, + "Gly(-1)", + "residue_name_number" + ], + [ + 60, + 69, + "histidine", + "residue_name" + ], + [ + 93, + 97, + "(-2)", + "residue_number" + ], + [ + 112, + 114, + "S2", + "site" + ], + [ + 130, + 139, + "S1 pocket", + "site" + ], + [ + 164, + 184, + "antiparallel \u03b2-sheet", + "structure_element" + ] + ] + }, + { + "sid": 49, + "sent": "Nonetheless, the carbonyl carbon of Gly(-1) would be ideally placed for nucleophilic attack by Thr1O\u03b3 (Fig. 1c and Supplementary Fig. 2c,d).", + "section": "RESULTS", + "ner": [ + [ + 36, + 43, + "Gly(-1)", + "residue_name_number" + ], + [ + 95, + 99, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 50, + "sent": "As the K81R mutation is located far from the active site (Thr1C\u03b1\u2013Arg81C\u03b1: 24\u2009\u00c5), any influence on propeptide conformation can be excluded.", + "section": "RESULTS", + "ner": [ + [ + 7, + 11, + "K81R", + "mutant" + ], + [ + 45, + 56, + "active site", + "site" + ], + [ + 58, + 62, + "Thr1", + "residue_name_number" + ], + [ + 65, + 70, + "Arg81", + "residue_name_number" + ], + [ + 98, + 108, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 51, + "sent": "Instead, the plasticity of the \u03b25 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis.", + "section": "RESULTS", + "ner": [ + [ + 31, + 33, + "\u03b25", + "protein" + ], + [ + 34, + 43, + "S1 pocket", + "site" + ], + [ + 84, + 89, + "Met45", + "residue_name_number" + ], + [ + 128, + 135, + "His(-2)", + "residue_name_number" + ], + [ + 143, + 150, + "S1 site", + "site" + ], + [ + 201, + 210, + "autolysis", + "ptm" + ] + ] + }, + { + "sid": 52, + "sent": "Processing of \u03b2-subunit precursors requires deprotonation of Thr1OH; however, the general base initiating autolysis is unknown.", + "section": "RESULTS", + "ner": [ + [ + 61, + 65, + "Thr1", + "residue_name_number" + ], + [ + 106, + 115, + "autolysis", + "ptm" + ] + ] + }, + { + "sid": 53, + "sent": "Remarkably, eukaryotic proteasomal \u03b25 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a).", + "section": "RESULTS", + "ner": [ + [ + 12, + 22, + "eukaryotic", + "taxonomy_domain" + ], + [ + 35, + 37, + "\u03b25", + "protein" + ], + [ + 54, + 57, + "His", + "residue_name" + ], + [ + 78, + 82, + "(-2)", + "residue_number" + ], + [ + 90, + 100, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 54, + "sent": "As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the \u03b25 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30\u2009\u00b0C, but suffered from growth defects at 37\u2009\u00b0C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).", + "section": "RESULTS", + "ner": [ + [ + 3, + 12, + "histidine", + "residue_name" + ], + [ + 59, + 75, + "catalytic triads", + "site" + ], + [ + 79, + 95, + "serine proteases", + "protein_type" + ], + [ + 125, + 132, + "His(-2)", + "residue_name_number" + ], + [ + 154, + 156, + "\u03b25", + "protein" + ], + [ + 157, + 167, + "propeptide", + "structure_element" + ], + [ + 171, + 188, + "exchanging it for", + "experimental_method" + ], + [ + 189, + 192, + "Asn", + "residue_name" + ], + [ + 194, + 197, + "Lys", + "residue_name" + ], + [ + 199, + 202, + "Phe", + "residue_name" + ], + [ + 207, + 210, + "Ala", + "residue_name" + ], + [ + 216, + 221, + "yeast", + "taxonomy_domain" + ], + [ + 303, + 309, + "H(-2)N", + "mutant" + ], + [ + 314, + 320, + "H(-2)F", + "mutant" + ] + ] + }, + { + "sid": 55, + "sent": "In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c).", + "section": "RESULTS", + "ner": [ + [ + 56, + 62, + "H(-2)N", + "mutant" + ], + [ + 67, + 73, + "H(-2)F", + "mutant" + ], + [ + 74, + 80, + "mutant", + "protein_state" + ], + [ + 81, + 85, + "yCPs", + "complex_assembly" + ] + ] + }, + { + "sid": 56, + "sent": "Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO\u2013FC electron densities.", + "section": "RESULTS", + "ner": [ + [ + 0, + 19, + "Structural analyses", + "experimental_method" + ], + [ + 38, + 49, + "propeptides", + "structure_element" + ], + [ + 57, + 63, + "mutant", + "protein_state" + ], + [ + 64, + 68, + "yCPs", + "complex_assembly" + ], + [ + 85, + 110, + "2FO\u2013FC electron densities", + "evidence" + ] + ] + }, + { + "sid": 57, + "sent": "Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Gly(-1)", + "residue_name_number" + ], + [ + 12, + 15, + "Phe", + "residue_name" + ], + [ + 16, + 23, + "Lys(-2)", + "residue_name_number" + ], + [ + 64, + 67, + "Ala", + "residue_name" + ], + [ + 68, + 75, + "Asn(-2)", + "residue_name_number" + ] + ] + }, + { + "sid": 58, + "sent": "This observation indicates a mixture of processed and unprocessed \u03b25 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base.", + "section": "RESULTS", + "ner": [ + [ + 40, + 49, + "processed", + "protein_state" + ], + [ + 54, + 65, + "unprocessed", + "protein_state" + ], + [ + 66, + 68, + "\u03b25", + "protein" + ], + [ + 101, + 110, + "autolysis", + "ptm" + ], + [ + 160, + 164, + "(-2)", + "residue_number" + ] + ] + }, + { + "sid": 59, + "sent": "Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions.", + "section": "RESULTS", + "ner": [ + [ + 40, + 44, + "(-2)", + "residue_number" + ], + [ + 71, + 81, + "propeptide", + "structure_element" + ], + [ + 85, + 114, + "creating mutants that combine", + "experimental_method" + ], + [ + 119, + 122, + "T1A", + "mutant" + ], + [ + 124, + 128, + "K81R", + "mutant" + ], + [ + 130, + 141, + "mutation(s)", + "experimental_method" + ], + [ + 147, + 153, + "H(-2)L", + "mutant" + ], + [ + 155, + 161, + "H(-2)T", + "mutant" + ], + [ + 165, + 171, + "H(-2)A", + "mutant" + ], + [ + 172, + 185, + "substitutions", + "experimental_method" + ] + ] + }, + { + "sid": 60, + "sent": "Leu(-2) is encoded in the yeast \u03b21 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of \u03b22-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the \u03b25-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate \u2018\u03b21-like' propeptide positioning.", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Leu(-2)", + "residue_name_number" + ], + [ + 26, + 31, + "yeast", + "taxonomy_domain" + ], + [ + 32, + 34, + "\u03b21", + "protein" + ], + [ + 78, + 85, + "Thr(-2)", + "residue_name_number" + ], + [ + 107, + 109, + "\u03b22", + "protein" + ], + [ + 110, + 121, + "propeptides", + "structure_element" + ], + [ + 151, + 158, + "Ala(-2)", + "residue_name_number" + ], + [ + 183, + 185, + "\u03b25", + "protein" + ], + [ + 186, + 195, + "S1 pocket", + "site" + ], + [ + 239, + 244, + "Met45", + "residue_name_number" + ] + ] + }, + { + "sid": 61, + "sent": "As expected from \u03b25-T1A mutants, the yeasts show severe growth phenotypes, with minor variations (Supplementary Fig. 4a and Table 1).", + "section": "RESULTS", + "ner": [ + [ + 17, + 23, + "\u03b25-T1A", + "mutant" + ], + [ + 37, + 43, + "yeasts", + "taxonomy_domain" + ] + ] + }, + { + "sid": 62, + "sent": "We determined crystal structures of the \u03b25-H(-2)L-T1A, \u03b25-H(-2)T-T1A and the \u03b25-H(-2)A-T1A-K81R mutants (Supplementary Table 1).", + "section": "RESULTS", + "ner": [ + [ + 14, + 32, + "crystal structures", + "evidence" + ], + [ + 40, + 53, + "\u03b25-H(-2)L-T1A", + "mutant" + ], + [ + 55, + 68, + "\u03b25-H(-2)T-T1A", + "mutant" + ], + [ + 77, + 95, + "\u03b25-H(-2)A-T1A-K81R", + "mutant" + ] + ] + }, + { + "sid": 63, + "sent": "For the \u03b25-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d).", + "section": "RESULTS", + "ner": [ + [ + 8, + 26, + "\u03b25-H(-2)A-T1A-K81R", + "mutant" + ], + [ + 54, + 61, + "Gly(-1)", + "residue_name_number" + ], + [ + 66, + 73, + "Ala(-2)", + "residue_name_number" + ], + [ + 111, + 118, + "Ala(-2)", + "residue_name_number" + ], + [ + 162, + 172, + "propeptide", + "structure_element" + ], + [ + 180, + 205, + "substrate-binding channel", + "site" + ] + ] + }, + { + "sid": 64, + "sent": "By contrast, the prosegments of the \u03b25-H(-2)L-T1A and the \u03b25-H(-2)T-T1A mutants were significantly better resolved in the 2FO\u2013FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable.", + "section": "RESULTS", + "ner": [ + [ + 17, + 28, + "prosegments", + "structure_element" + ], + [ + 36, + 49, + "\u03b25-H(-2)L-T1A", + "mutant" + ], + [ + 58, + 71, + "\u03b25-H(-2)T-T1A", + "mutant" + ], + [ + 122, + 150, + "2FO\u2013FC electron-density maps", + "evidence" + ], + [ + 258, + 268, + "propeptide", + "structure_element" + ], + [ + 277, + 284, + "His(-2)", + "residue_name_number" + ] + ] + }, + { + "sid": 65, + "sent": "Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f\u2013h).", + "section": "RESULTS", + "ner": [ + [ + 19, + 26, + "Leu(-2)", + "residue_name_number" + ], + [ + 31, + 38, + "Thr(-2)", + "residue_name_number" + ], + [ + 64, + 85, + "S1 specificity pocket", + "site" + ], + [ + 96, + 101, + "Met45", + "residue_name_number" + ] + ] + }, + { + "sid": 66, + "sent": "This result proves that the naturally occurring His(-2) of the \u03b25 propeptide does not stably fit into the S1 site.", + "section": "RESULTS", + "ner": [ + [ + 48, + 55, + "His(-2)", + "residue_name_number" + ], + [ + 63, + 65, + "\u03b25", + "protein" + ], + [ + 66, + 76, + "propeptide", + "structure_element" + ], + [ + 106, + 113, + "S1 site", + "site" + ] + ] + }, + { + "sid": 67, + "sent": "Since Gly(-1) adopts the same position in both wild-type (WT) and mutant \u03b25 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1O\u03b3 (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel \u03b2-sheet is essential for autolysis of the propeptide.", + "section": "RESULTS", + "ner": [ + [ + 6, + 13, + "Gly(-1)", + "residue_name_number" + ], + [ + 47, + 56, + "wild-type", + "protein_state" + ], + [ + 58, + 60, + "WT", + "protein_state" + ], + [ + 66, + 72, + "mutant", + "protein_state" + ], + [ + 73, + 75, + "\u03b25", + "protein" + ], + [ + 76, + 87, + "propeptides", + "structure_element" + ], + [ + 179, + 183, + "Thr1", + "residue_name_number" + ], + [ + 240, + 244, + "(-2)", + "residue_number" + ], + [ + 252, + 261, + "S1 pocket", + "site" + ], + [ + 283, + 303, + "antiparallel \u03b2-sheet", + "structure_element" + ], + [ + 321, + 330, + "autolysis", + "ptm" + ], + [ + 338, + 348, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 68, + "sent": "Next, we determined the crystal structure of a chimeric yCP having the yeast \u03b21-propeptide replaced by its \u03b25 counterpart.", + "section": "RESULTS", + "ner": [ + [ + 24, + 41, + "crystal structure", + "evidence" + ], + [ + 47, + 55, + "chimeric", + "protein_state" + ], + [ + 56, + 59, + "yCP", + "complex_assembly" + ], + [ + 71, + 76, + "yeast", + "taxonomy_domain" + ], + [ + 77, + 79, + "\u03b21", + "protein" + ], + [ + 80, + 90, + "propeptide", + "structure_element" + ], + [ + 91, + 102, + "replaced by", + "experimental_method" + ], + [ + 107, + 109, + "\u03b25", + "protein" + ], + [ + 110, + 121, + "counterpart", + "structure_element" + ] + ] + }, + { + "sid": 69, + "sent": "Although we observed fragments of 2FO\u2013FC electron density in the \u03b21 active site, the data were not interpretable.", + "section": "RESULTS", + "ner": [ + [ + 34, + 57, + "2FO\u2013FC electron density", + "evidence" + ], + [ + 65, + 67, + "\u03b21", + "protein" + ], + [ + 68, + 79, + "active site", + "site" + ] + ] + }, + { + "sid": 70, + "sent": "Bearing in mind that in contrast to Thr(-2) in \u03b22, Leu(-2) in subunit \u03b21 is not conserved among species (Supplementary Fig. 3a), we created a \u03b22-T(-2)V proteasome mutant.", + "section": "RESULTS", + "ner": [ + [ + 36, + 43, + "Thr(-2)", + "residue_name_number" + ], + [ + 47, + 49, + "\u03b22", + "protein" + ], + [ + 51, + 58, + "Leu(-2)", + "residue_name_number" + ], + [ + 70, + 72, + "\u03b21", + "protein" + ], + [ + 76, + 89, + "not conserved", + "protein_state" + ], + [ + 132, + 139, + "created", + "experimental_method" + ], + [ + 142, + 151, + "\u03b22-T(-2)V", + "mutant" + ], + [ + 152, + 162, + "proteasome", + "complex_assembly" + ], + [ + 163, + 169, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 71, + "sent": "As proven by the \u03b22-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the \u03b25-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in \u03b22, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j).", + "section": "RESULTS", + "ner": [ + [ + 17, + 23, + "\u03b22-T1A", + "mutant" + ], + [ + 24, + 42, + "crystal structures", + "evidence" + ], + [ + 44, + 51, + "Thr(-2)", + "residue_name_number" + ], + [ + 52, + 66, + "hydrogen bonds", + "bond_interaction" + ], + [ + 70, + 77, + "Gly(-1)", + "residue_name_number" + ], + [ + 131, + 144, + "\u03b25-H(-2)T-T1A", + "mutant" + ], + [ + 145, + 151, + "mutant", + "protein_state" + ], + [ + 191, + 199, + "exchange", + "experimental_method" + ], + [ + 203, + 210, + "Thr(-2)", + "residue_name_number" + ], + [ + 214, + 217, + "Val", + "residue_name" + ], + [ + 221, + 223, + "\u03b22", + "protein" + ] + ] + }, + { + "sid": 72, + "sent": "Notably, the 2FO\u2013FC electron-density map displays a different orientation for the \u03b22 propeptide than has been observed for the \u03b22-T1A proteasome.", + "section": "RESULTS", + "ner": [ + [ + 13, + 40, + "2FO\u2013FC electron-density map", + "evidence" + ], + [ + 82, + 84, + "\u03b22", + "protein" + ], + [ + 85, + 95, + "propeptide", + "structure_element" + ], + [ + 127, + 133, + "\u03b22-T1A", + "mutant" + ], + [ + 134, + 144, + "proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 73, + "sent": "In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8\u2009\u00c5), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k).", + "section": "RESULTS", + "ner": [ + [ + 15, + 22, + "Val(-2)", + "residue_name_number" + ], + [ + 45, + 52, + "S1 site", + "site" + ], + [ + 57, + 64, + "Gly(-1)", + "residue_name_number" + ], + [ + 176, + 180, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 74, + "sent": "These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome.", + "section": "RESULTS", + "ner": [ + [ + 62, + 82, + "active-site residues", + "site" + ], + [ + 87, + 94, + "Gly(-1)", + "residue_name_number" + ], + [ + 133, + 143, + "proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 75, + "sent": "The active site of the proteasome", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "active site", + "site" + ], + [ + 23, + 33, + "proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 76, + "sent": "Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis.", + "section": "RESULTS", + "ner": [ + [ + 38, + 49, + "active site", + "site" + ], + [ + 50, + 54, + "Thr1", + "residue_name_number" + ], + [ + 60, + 64, + "Thr1", + "residue_name_number" + ], + [ + 87, + 92, + "water", + "chemical" + ] + ] + }, + { + "sid": 77, + "sent": "However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1O\u03b3.", + "section": "RESULTS", + "ner": [ + [ + 16, + 24, + "immature", + "protein_state" + ], + [ + 25, + 33, + "particle", + "complex_assembly" + ], + [ + 34, + 38, + "Thr1", + "residue_name_number" + ], + [ + 60, + 70, + "propeptide", + "structure_element" + ], + [ + 91, + 95, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 78, + "sent": "Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1O\u03b3 (2.7\u2009\u00c5) in all catalytically active \u03b2 subunits (Fig. 3a,b), was proposed to serve as the proton acceptor.", + "section": "RESULTS", + "ner": [ + [ + 9, + 14, + "Lys33", + "residue_name_number" + ], + [ + 31, + 47, + "hydrogen-bonding", + "bond_interaction" + ], + [ + 60, + 64, + "Thr1", + "residue_name_number" + ], + [ + 82, + 102, + "catalytically active", + "protein_state" + ], + [ + 103, + 113, + "\u03b2 subunits", + "protein" + ] + ] + }, + { + "sid": 79, + "sent": "A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17O\u03b4, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site.", + "section": "RESULTS", + "ner": [ + [ + 11, + 27, + "catalytic tetrad", + "site" + ], + [ + 44, + 48, + "Thr1", + "residue_name_number" + ], + [ + 52, + 56, + "Thr1", + "residue_name_number" + ], + [ + 61, + 66, + "Lys33", + "residue_name_number" + ], + [ + 74, + 79, + "Asp17", + "residue_name_number" + ], + [ + 109, + 114, + "water", + "chemical" + ], + [ + 208, + 219, + "active site", + "site" + ] + ] + }, + { + "sid": 80, + "sent": "Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base.", + "section": "RESULTS", + "ner": [ + [ + 39, + 42, + "yCP", + "complex_assembly" + ], + [ + 43, + 59, + "X-ray structures", + "evidence" + ], + [ + 98, + 109, + "active site", + "site" + ], + [ + 117, + 127, + "proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 81, + "sent": "Mutation of \u03b25-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1).", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Mutation", + "experimental_method" + ], + [ + 12, + 14, + "\u03b25", + "protein" + ], + [ + 15, + 20, + "Lys33", + "residue_name_number" + ], + [ + 24, + 27, + "Ala", + "residue_name" + ], + [ + 82, + 117, + "structural and biochemical analyses", + "experimental_method" + ], + [ + 162, + 181, + "propeptide cleavage", + "ptm" + ] + ] + }, + { + "sid": 82, + "sent": "The phenotype of the \u03b25-K33A mutant was however less pronounced than for the \u03b25-T1A-K81R yeast (Fig. 4a).", + "section": "RESULTS", + "ner": [ + [ + 21, + 28, + "\u03b25-K33A", + "mutant" + ], + [ + 29, + 35, + "mutant", + "protein_state" + ], + [ + 77, + 88, + "\u03b25-T1A-K81R", + "mutant" + ], + [ + 89, + 94, + "yeast", + "taxonomy_domain" + ] + ] + }, + { + "sid": 83, + "sent": "This discrepancy in growth was traced to an additional point mutation L(-49)S in the \u03b25-propeptide of the \u03b25-K33A mutant (see also Supplementary Note 1).", + "section": "RESULTS", + "ner": [ + [ + 70, + 77, + "L(-49)S", + "mutant" + ], + [ + 85, + 87, + "\u03b25", + "protein" + ], + [ + 88, + 98, + "propeptide", + "structure_element" + ], + [ + 106, + 113, + "\u03b25-K33A", + "mutant" + ], + [ + 114, + 120, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 84, + "sent": "Structural comparison of the \u03b25-L(-49)S-K33A and \u03b25-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide.", + "section": "RESULTS", + "ner": [ + [ + 0, + 21, + "Structural comparison", + "experimental_method" + ], + [ + 29, + 44, + "\u03b25-L(-49)S-K33A", + "mutant" + ], + [ + 49, + 60, + "\u03b25-T1A-K81R", + "mutant" + ], + [ + 61, + 73, + "active sites", + "site" + ], + [ + 88, + 96, + "mutation", + "experimental_method" + ], + [ + 100, + 105, + "Lys33", + "residue_name_number" + ], + [ + 109, + 112, + "Ala", + "residue_name" + ], + [ + 150, + 154, + "Thr1", + "residue_name_number" + ], + [ + 171, + 181, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 85, + "sent": "This structural alteration destroys active-site integrity and abolishes catalytic activity of the \u03b25 active site (Supplementary Fig. 5a).", + "section": "RESULTS", + "ner": [ + [ + 36, + 47, + "active-site", + "site" + ], + [ + 98, + 100, + "\u03b25", + "protein" + ], + [ + 101, + 112, + "active site", + "site" + ] + ] + }, + { + "sid": 86, + "sent": "Additional proof for the key function of Lys33 was obtained from the \u03b25-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans).", + "section": "RESULTS", + "ner": [ + [ + 41, + 46, + "Lys33", + "residue_name_number" + ], + [ + 69, + 76, + "\u03b25-K33A", + "mutant" + ], + [ + 77, + 83, + "mutant", + "protein_state" + ], + [ + 94, + 104, + "propeptide", + "structure_element" + ], + [ + 105, + 125, + "expressed separately", + "experimental_method" + ], + [ + 149, + 151, + "pp", + "chemical" + ], + [ + 152, + 157, + "trans", + "protein_state" + ] + ] + }, + { + "sid": 87, + "sent": "The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by \u223c83% (Supplementary Fig. 6b).", + "section": "RESULTS", + "ner": [ + [ + 4, + 8, + "Thr1", + "residue_name_number" + ], + [ + 28, + 34, + "mutant", + "protein_state" + ], + [ + 57, + 67, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 88, + "sent": "Consistent with this, the crystal structure of the \u03b25-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the \u03b25 active sites (Supplementary Fig. 5b and Supplementary Table 1).", + "section": "RESULTS", + "ner": [ + [ + 26, + 43, + "crystal structure", + "evidence" + ], + [ + 51, + 58, + "\u03b25-K33A", + "mutant" + ], + [ + 59, + 61, + "pp", + "chemical" + ], + [ + 62, + 67, + "trans", + "protein_state" + ], + [ + 68, + 74, + "mutant", + "protein_state" + ], + [ + 75, + 90, + "in complex with", + "protein_state" + ], + [ + 91, + 102, + "carfilzomib", + "chemical" + ], + [ + 154, + 156, + "\u03b25", + "protein" + ], + [ + 157, + 169, + "active sites", + "site" + ] + ] + }, + { + "sid": 89, + "sent": "Since no acetylation of the Thr1 N terminus was observed for the \u03b25-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH.", + "section": "RESULTS", + "ner": [ + [ + 9, + 20, + "acetylation", + "ptm" + ], + [ + 28, + 32, + "Thr1", + "residue_name_number" + ], + [ + 65, + 72, + "\u03b25-K33A", + "mutant" + ], + [ + 73, + 75, + "pp", + "chemical" + ], + [ + 76, + 81, + "trans", + "protein_state" + ], + [ + 82, + 85, + "apo", + "protein_state" + ], + [ + 86, + 103, + "crystal structure", + "evidence" + ], + [ + 177, + 182, + "Lys33", + "residue_name_number" + ], + [ + 199, + 203, + "Thr1", + "residue_name_number" + ], + [ + 235, + 239, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 90, + "sent": "Furthermore, the crystal structure of the \u03b25-K33A pp trans mutant without inhibitor revealed that Thr1O\u03b3 strongly coordinates a well-defined water molecule (\u223c2\u2009\u00c5; Fig. 3c and Supplementary Fig. 5c,d).", + "section": "RESULTS", + "ner": [ + [ + 17, + 34, + "crystal structure", + "evidence" + ], + [ + 42, + 49, + "\u03b25-K33A", + "mutant" + ], + [ + 50, + 52, + "pp", + "chemical" + ], + [ + 53, + 58, + "trans", + "protein_state" + ], + [ + 59, + 65, + "mutant", + "protein_state" + ], + [ + 66, + 83, + "without inhibitor", + "protein_state" + ], + [ + 98, + 102, + "Thr1", + "residue_name_number" + ], + [ + 114, + 125, + "coordinates", + "bond_interaction" + ], + [ + 141, + 146, + "water", + "chemical" + ] + ] + }, + { + "sid": 91, + "sent": "This water hydrogen bonds also to Arg19O (\u223c3.0\u2009\u00c5) and Asp17O\u03b4 (\u223c3.0\u2009\u00c5), and thereby presumably enables residual activity of the mutant.", + "section": "RESULTS", + "ner": [ + [ + 5, + 10, + "water", + "chemical" + ], + [ + 11, + 25, + "hydrogen bonds", + "bond_interaction" + ], + [ + 34, + 39, + "Arg19", + "residue_name_number" + ], + [ + 54, + 59, + "Asp17", + "residue_name_number" + ], + [ + 128, + 134, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 92, + "sent": "Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis.", + "section": "RESULTS", + "ner": [ + [ + 73, + 78, + "Lys33", + "residue_name_number" + ], + [ + 89, + 91, + "WT", + "protein_state" + ], + [ + 92, + 102, + "proteasome", + "complex_assembly" + ], + [ + 103, + 112, + "structure", + "evidence" + ], + [ + 168, + 173, + "Lys33", + "residue_name_number" + ], + [ + 198, + 207, + "autolysis", + "ptm" + ] + ] + }, + { + "sid": 93, + "sent": "Conservative substitution of Lys33 by Arg delays autolysis of the \u03b25 precursor and impairs yeast growth (for details see Supplementary Note 1).", + "section": "RESULTS", + "ner": [ + [ + 0, + 25, + "Conservative substitution", + "experimental_method" + ], + [ + 29, + 34, + "Lys33", + "residue_name_number" + ], + [ + 38, + 41, + "Arg", + "residue_name" + ], + [ + 49, + 58, + "autolysis", + "ptm" + ], + [ + 66, + 68, + "\u03b25", + "protein" + ], + [ + 91, + 96, + "yeast", + "taxonomy_domain" + ] + ] + }, + { + "sid": 94, + "sent": "While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the \u03b25 active site (Supplementary Fig. 5e).", + "section": "RESULTS", + "ner": [ + [ + 6, + 10, + "Thr1", + "residue_name_number" + ], + [ + 44, + 46, + "WT", + "protein_state" + ], + [ + 47, + 51, + "yCPs", + "complex_assembly" + ], + [ + 53, + 58, + "Arg33", + "residue_name_number" + ], + [ + 72, + 85, + "hydrogen bond", + "bond_interaction" + ], + [ + 89, + 94, + "Asp17", + "residue_name_number" + ], + [ + 121, + 123, + "\u03b25", + "protein" + ], + [ + 124, + 135, + "active site", + "site" + ] + ] + }, + { + "sid": 95, + "sent": "The conservative mutation of Asp17 to Asn in subunit \u03b25 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 25, + "conservative mutation", + "experimental_method" + ], + [ + 29, + 34, + "Asp17", + "residue_name_number" + ], + [ + 38, + 41, + "Asn", + "residue_name" + ], + [ + 53, + 55, + "\u03b25", + "protein" + ], + [ + 63, + 66, + "yCP", + "complex_assembly" + ] + ] + }, + { + "sid": 96, + "sent": "Notably, only with the additional point mutation L(-49)S present in the \u03b25 propeptide could we purify a small amount of the \u03b25-D17N mutant yCP.", + "section": "RESULTS", + "ner": [ + [ + 49, + 56, + "L(-49)S", + "mutant" + ], + [ + 72, + 74, + "\u03b25", + "protein" + ], + [ + 75, + 85, + "propeptide", + "structure_element" + ], + [ + 124, + 131, + "\u03b25-D17N", + "mutant" + ], + [ + 132, + 138, + "mutant", + "protein_state" + ], + [ + 139, + 142, + "yCP", + "complex_assembly" + ] + ] + }, + { + "sid": 97, + "sent": "As determined by crystallographic analysis, this mutant \u03b25 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits \u03b21 and \u03b22, and with WT \u03b25 (Supplementary Fig. 7a).", + "section": "RESULTS", + "ner": [ + [ + 17, + 42, + "crystallographic analysis", + "experimental_method" + ], + [ + 49, + 55, + "mutant", + "protein_state" + ], + [ + 56, + 58, + "\u03b25", + "protein" + ], + [ + 71, + 90, + "partially processed", + "protein_state" + ], + [ + 147, + 157, + "proteasome", + "complex_assembly" + ], + [ + 168, + 179, + "carfilzomib", + "chemical" + ], + [ + 207, + 209, + "\u03b21", + "protein" + ], + [ + 214, + 216, + "\u03b22", + "protein" + ], + [ + 227, + 229, + "WT", + "protein_state" + ], + [ + 230, + 232, + "\u03b25", + "protein" + ] + ] + }, + { + "sid": 98, + "sent": "In contrast to the cis-construct, expression of the \u03b25 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome.", + "section": "RESULTS", + "ner": [ + [ + 19, + 22, + "cis", + "protein_state" + ], + [ + 34, + 44, + "expression", + "experimental_method" + ], + [ + 52, + 54, + "\u03b25", + "protein" + ], + [ + 55, + 65, + "propeptide", + "structure_element" + ], + [ + 69, + 74, + "trans", + "protein_state" + ], + [ + 99, + 108, + "isolation", + "experimental_method" + ], + [ + 113, + 128, + "crystallization", + "experimental_method" + ], + [ + 136, + 140, + "D17N", + "mutant" + ], + [ + 141, + 147, + "mutant", + "protein_state" + ], + [ + 148, + 158, + "proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 99, + "sent": "The ChT-L activity of the \u03b25-D17N pp in trans CP towards the canonical \u03b25 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.", + "section": "RESULTS", + "ner": [ + [ + 26, + 33, + "\u03b25-D17N", + "mutant" + ], + [ + 34, + 36, + "pp", + "chemical" + ], + [ + 40, + 45, + "trans", + "protein_state" + ], + [ + 46, + 48, + "CP", + "complex_assembly" + ], + [ + 71, + 73, + "\u03b25", + "protein" + ], + [ + 91, + 142, + "N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin", + "chemical" + ], + [ + 144, + 156, + "Suc-LLVY-AMC", + "chemical" + ], + [ + 162, + 205, + "carboxybenzyl-Gly-Gly-Leu-para-nitroanilide", + "chemical" + ], + [ + 207, + 216, + "Z-GGL-pNA", + "chemical" + ], + [ + 280, + 285, + "Asp17", + "residue_name_number" + ], + [ + 349, + 355, + "mature", + "protein_state" + ], + [ + 356, + 366, + "proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 100, + "sent": "Even though the \u03b25-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the \u03b1\u2032, \u03b2\u2032 epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the \u03b25 active site (Supplementary Fig. 7a).", + "section": "RESULTS", + "ner": [ + [ + 16, + 23, + "\u03b25-D17N", + "mutant" + ], + [ + 24, + 26, + "pp", + "chemical" + ], + [ + 27, + 32, + "trans", + "protein_state" + ], + [ + 33, + 36, + "yCP", + "complex_assembly" + ], + [ + 37, + 54, + "crystal structure", + "evidence" + ], + [ + 81, + 83, + "WT", + "protein_state" + ], + [ + 84, + 87, + "yCP", + "complex_assembly" + ], + [ + 117, + 137, + "co-crystal structure", + "evidence" + ], + [ + 147, + 165, + "\u03b1\u2032, \u03b2\u2032 epoxyketone", + "chemical" + ], + [ + 176, + 187, + "carfilzomib", + "chemical" + ], + [ + 243, + 245, + "\u03b25", + "protein" + ], + [ + 246, + 257, + "active site", + "site" + ] + ] + }, + { + "sid": 101, + "sent": "This observation is consistent with a strongly reduced reactivity of \u03b25-Thr1 and the crystal structure of the \u03b25-D17N pp cis mutant in complex with carfilzomib.", + "section": "RESULTS", + "ner": [ + [ + 69, + 71, + "\u03b25", + "protein" + ], + [ + 72, + 76, + "Thr1", + "residue_name_number" + ], + [ + 85, + 102, + "crystal structure", + "evidence" + ], + [ + 110, + 117, + "\u03b25-D17N", + "mutant" + ], + [ + 118, + 120, + "pp", + "chemical" + ], + [ + 121, + 124, + "cis", + "protein_state" + ], + [ + 125, + 131, + "mutant", + "protein_state" + ], + [ + 132, + 147, + "in complex with", + "protein_state" + ], + [ + 148, + 159, + "carfilzomib", + "chemical" + ] + ] + }, + { + "sid": 102, + "sent": "Autolysis and residual catalytic activity of the \u03b25-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1.", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "Autolysis", + "ptm" + ], + [ + 49, + 56, + "\u03b25-D17N", + "mutant" + ], + [ + 106, + 111, + "Asn17", + "residue_name_number" + ], + [ + 163, + 168, + "Lys33", + "residue_name_number" + ], + [ + 191, + 195, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 103, + "sent": "In agreement, an E17A mutant in the proteasomal \u03b2-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis.", + "section": "RESULTS", + "ner": [ + [ + 17, + 21, + "E17A", + "mutant" + ], + [ + 22, + 28, + "mutant", + "protein_state" + ], + [ + 48, + 57, + "\u03b2-subunit", + "protein" + ], + [ + 65, + 73, + "archaeon", + "taxonomy_domain" + ], + [ + 74, + 98, + "Thermoplasma acidophilum", + "species" + ], + [ + 108, + 117, + "autolysis", + "ptm" + ] + ] + }, + { + "sid": 104, + "sent": "Strikingly, although the X-ray data on the \u03b25-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b).", + "section": "RESULTS", + "ner": [ + [ + 25, + 35, + "X-ray data", + "evidence" + ], + [ + 43, + 50, + "\u03b25-D17N", + "mutant" + ], + [ + 51, + 57, + "mutant", + "protein_state" + ], + [ + 67, + 77, + "propeptide", + "structure_element" + ], + [ + 78, + 87, + "expressed", + "experimental_method" + ], + [ + 91, + 94, + "cis", + "protein_state" + ], + [ + 102, + 107, + "trans", + "protein_state" + ] + ] + }, + { + "sid": 105, + "sent": "On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d).", + "section": "RESULTS", + "ner": [ + [ + 47, + 50, + "CPs", + "complex_assembly" + ], + [ + 82, + 97, + "catalytic triad", + "site" + ], + [ + 112, + 116, + "Thr1", + "residue_name_number" + ], + [ + 118, + 123, + "Lys33", + "residue_name_number" + ], + [ + 128, + 131, + "Asp", + "residue_name" + ], + [ + 132, + 137, + "Glu17", + "residue_name_number" + ], + [ + 147, + 181, + "autocatalytic precursor processing", + "ptm" + ] + ] + }, + { + "sid": 106, + "sent": "This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1O\u03b3 to Thr1NH2.", + "section": "RESULTS", + "ner": [ + [ + 60, + 65, + "water", + "chemical" + ], + [ + 94, + 100, + "mature", + "protein_state" + ], + [ + 101, + 103, + "WT", + "protein_state" + ], + [ + 116, + 127, + "active site", + "site" + ], + [ + 163, + 167, + "Thr1", + "residue_name_number" + ], + [ + 173, + 177, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 107, + "sent": "To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast \u03b25 subunit.", + "section": "RESULTS", + "ner": [ + [ + 16, + 27, + "active-site", + "site" + ], + [ + 46, + 69, + "exchanged the conserved", + "experimental_method" + ], + [ + 70, + 76, + "Asp166", + "residue_name_number" + ], + [ + 89, + 92, + "Asn", + "residue_name" + ], + [ + 100, + 105, + "yeast", + "taxonomy_domain" + ], + [ + 106, + 108, + "\u03b25", + "protein" + ] + ] + }, + { + "sid": 108, + "sent": "Asp166O\u03b4 is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "Asp166", + "residue_name_number" + ], + [ + 12, + 27, + "hydrogen-bonded", + "bond_interaction" + ], + [ + 31, + 35, + "Thr1", + "residue_name_number" + ], + [ + 43, + 49, + "Ser129", + "residue_name_number" + ], + [ + 56, + 62, + "Ser169", + "residue_name_number" + ] + ] + }, + { + "sid": 109, + "sent": "The \u03b25-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 12, + "\u03b25-D166N", + "mutant" + ], + [ + 13, + 15, + "pp", + "chemical" + ], + [ + 16, + 19, + "cis", + "protein_state" + ], + [ + 20, + 25, + "yeast", + "taxonomy_domain" + ], + [ + 26, + 32, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 110, + "sent": "X-ray data on the \u03b25-D166N mutant indicate that the \u03b25 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166O\u03b4 is disrupted (Supplementary Fig. 8a).", + "section": "RESULTS", + "ner": [ + [ + 0, + 10, + "X-ray data", + "evidence" + ], + [ + 18, + 26, + "\u03b25-D166N", + "mutant" + ], + [ + 27, + 33, + "mutant", + "protein_state" + ], + [ + 52, + 54, + "\u03b25", + "protein" + ], + [ + 55, + 65, + "propeptide", + "structure_element" + ], + [ + 109, + 115, + "Ser129", + "residue_name_number" + ], + [ + 140, + 146, + "Asn166", + "residue_name_number" + ] + ] + }, + { + "sid": 111, + "sent": "Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing.", + "section": "RESULTS", + "ner": [ + [ + 11, + 16, + "water", + "chemical" + ], + [ + 29, + 37, + "bound to", + "protein_state" + ], + [ + 38, + 44, + "Ser129", + "residue_name_number" + ], + [ + 51, + 55, + "Thr1", + "residue_name_number" + ], + [ + 101, + 121, + "precursor processing", + "ptm" + ] + ] + }, + { + "sid": 112, + "sent": "The hydrogen bonds involving Ser169OH are intact and may account for residual substrate turnover.", + "section": "RESULTS", + "ner": [ + [ + 4, + 18, + "hydrogen bonds", + "bond_interaction" + ], + [ + 29, + 35, + "Ser169", + "residue_name_number" + ] + ] + }, + { + "sid": 113, + "sent": "Soaking the \u03b25-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c).", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "Soaking", + "experimental_method" + ], + [ + 12, + 20, + "\u03b25-D166N", + "mutant" + ], + [ + 21, + 29, + "crystals", + "experimental_method" + ], + [ + 35, + 46, + "carfilzomib", + "chemical" + ], + [ + 51, + 56, + "MG132", + "chemical" + ], + [ + 94, + 98, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 114, + "sent": "In the carfilzomib complex structure, Thr1O\u03b3 and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation.", + "section": "RESULTS", + "ner": [ + [ + 7, + 26, + "carfilzomib complex", + "complex_assembly" + ], + [ + 27, + 36, + "structure", + "evidence" + ], + [ + 38, + 42, + "Thr1", + "residue_name_number" + ], + [ + 49, + 53, + "Thr1", + "residue_name_number" + ], + [ + 104, + 110, + "Ser129", + "residue_name_number" + ], + [ + 122, + 124, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 115, + "sent": "In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166.", + "section": "RESULTS", + "ner": [ + [ + 7, + 24, + "MG132-bound state", + "protein_state" + ], + [ + 26, + 30, + "Thr1", + "residue_name_number" + ], + [ + 35, + 45, + "unmodified", + "protein_state" + ], + [ + 73, + 79, + "Ser129", + "residue_name_number" + ], + [ + 83, + 98, + "hydrogen-bonded", + "bond_interaction" + ], + [ + 104, + 109, + "water", + "chemical" + ], + [ + 130, + 136, + "Asn166", + "residue_name_number" + ] + ] + }, + { + "sid": 116, + "sent": "Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis.", + "section": "RESULTS", + "ner": [ + [ + 8, + 11, + "Asn", + "residue_name" + ], + [ + 39, + 45, + "Asp166", + "residue_name_number" + ], + [ + 91, + 94, + "Ala", + "residue_name" + ], + [ + 138, + 147, + "autolysis", + "ptm" + ] + ] + }, + { + "sid": 117, + "sent": "These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis.", + "section": "RESULTS", + "ner": [ + [ + 27, + 33, + "Asp166", + "residue_name_number" + ], + [ + 38, + 44, + "Ser129", + "residue_name_number" + ], + [ + 110, + 114, + "Thr1", + "residue_name_number" + ], + [ + 123, + 132, + "autolysis", + "ptm" + ] + ] + }, + { + "sid": 118, + "sent": "Substitution of the active-site Thr1 by Cys", + "section": "RESULTS", + "ner": [ + [ + 0, + 12, + "Substitution", + "experimental_method" + ], + [ + 20, + 31, + "active-site", + "site" + ], + [ + 32, + 36, + "Thr1", + "residue_name_number" + ], + [ + 40, + 43, + "Cys", + "residue_name" + ] + ] + }, + { + "sid": 119, + "sent": "Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum.", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Mutation", + "experimental_method" + ], + [ + 12, + 16, + "Thr1", + "residue_name_number" + ], + [ + 20, + 23, + "Cys", + "residue_name" + ], + [ + 40, + 54, + "20S proteasome", + "complex_assembly" + ], + [ + 64, + 72, + "archaeon", + "taxonomy_domain" + ], + [ + 73, + 87, + "T. acidophilum", + "species" + ] + ] + }, + { + "sid": 120, + "sent": "In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure.", + "section": "RESULTS", + "ner": [ + [ + 3, + 8, + "yeast", + "taxonomy_domain" + ], + [ + 15, + 23, + "mutation", + "experimental_method" + ], + [ + 90, + 100, + "propeptide", + "structure_element" + ], + [ + 137, + 152, + "X-ray structure", + "evidence" + ] + ] + }, + { + "sid": 121, + "sent": "In one of the two \u03b25 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c).", + "section": "RESULTS", + "ner": [ + [ + 18, + 20, + "\u03b25", + "protein" + ], + [ + 53, + 60, + "cleaved", + "protein_state" + ], + [ + 61, + 71, + "propeptide", + "structure_element" + ], + [ + 72, + 83, + "still bound", + "protein_state" + ], + [ + 91, + 116, + "substrate-binding channel", + "site" + ] + ] + }, + { + "sid": 122, + "sent": "His(-2) occupies the S2 pocket like observed for the \u03b25-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel \u03b2-sheet conformation as known from inhibitors like MG132 (Fig. 4c\u2013e and Supplementary Fig. 9b).", + "section": "RESULTS", + "ner": [ + [ + 0, + 7, + "His(-2)", + "residue_name_number" + ], + [ + 21, + 30, + "S2 pocket", + "site" + ], + [ + 53, + 64, + "\u03b25-T1A-K81R", + "mutant" + ], + [ + 65, + 71, + "mutant", + "protein_state" + ], + [ + 108, + 118, + "propeptide", + "structure_element" + ], + [ + 126, + 129, + "T1C", + "mutant" + ], + [ + 130, + 136, + "mutant", + "protein_state" + ], + [ + 147, + 167, + "antiparallel \u03b2-sheet", + "structure_element" + ], + [ + 211, + 216, + "MG132", + "chemical" + ] + ] + }, + { + "sid": 123, + "sent": "On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization.", + "section": "RESULTS", + "ner": [ + [ + 37, + 40, + "T1C", + "mutant" + ], + [ + 41, + 47, + "mutant", + "protein_state" + ], + [ + 56, + 66, + "propeptide", + "structure_element" + ], + [ + 93, + 104, + "active site", + "site" + ], + [ + 122, + 131, + "autolysis", + "ptm" + ], + [ + 183, + 198, + "crystallization", + "experimental_method" + ] + ] + }, + { + "sid": 124, + "sent": "Owing to the unequal positions of the two \u03b25 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits.", + "section": "RESULTS", + "ner": [ + [ + 42, + 44, + "\u03b25", + "protein" + ], + [ + 65, + 67, + "CP", + "complex_assembly" + ], + [ + 107, + 117, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 125, + "sent": "Despite propeptide hydrolysis, the \u03b25-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a).", + "section": "RESULTS", + "ner": [ + [ + 8, + 29, + "propeptide hydrolysis", + "ptm" + ], + [ + 35, + 41, + "\u03b25-T1C", + "mutant" + ], + [ + 42, + 53, + "active site", + "site" + ], + [ + 57, + 79, + "catalytically inactive", + "protein_state" + ] + ] + }, + { + "sid": 126, + "sent": "In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the \u03b21 and \u03b22 active sites, while leaving the \u03b25-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.", + "section": "RESULTS", + "ner": [ + [ + 14, + 30, + "soaking crystals", + "experimental_method" + ], + [ + 40, + 42, + "CP", + "complex_assembly" + ], + [ + 54, + 64, + "bortezomib", + "chemical" + ], + [ + 68, + 79, + "carfilzomib", + "chemical" + ], + [ + 98, + 100, + "\u03b21", + "protein" + ], + [ + 105, + 107, + "\u03b22", + "protein" + ], + [ + 108, + 120, + "active sites", + "site" + ], + [ + 140, + 146, + "\u03b25-T1C", + "mutant" + ], + [ + 147, + 166, + "proteolytic centres", + "site" + ], + [ + 167, + 177, + "unmodified", + "protein_state" + ], + [ + 230, + 237, + "cleaved", + "protein_state" + ], + [ + 238, + 248, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 127, + "sent": "Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74\u00b0 with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b).", + "section": "RESULTS", + "ner": [ + [ + 14, + 29, + "structural data", + "evidence" + ], + [ + 61, + 65, + "Cys1", + "residue_name_number" + ], + [ + 127, + 131, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 128, + "sent": "Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1.", + "section": "RESULTS", + "ner": [ + [ + 18, + 31, + "hydrogen bond", + "bond_interaction" + ], + [ + 73, + 78, + "Lys33", + "residue_name_number" + ], + [ + 82, + 84, + "WT", + "protein_state" + ], + [ + 85, + 88, + "CPs", + "complex_assembly" + ], + [ + 104, + 108, + "Cys1", + "residue_name_number" + ] + ] + }, + { + "sid": 129, + "sent": "Notably, the 2FO\u2013FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered.", + "section": "RESULTS", + "ner": [ + [ + 13, + 40, + "2FO\u2013FC electron-density map", + "evidence" + ], + [ + 48, + 51, + "T1C", + "mutant" + ], + [ + 52, + 58, + "mutant", + "protein_state" + ], + [ + 79, + 84, + "Lys33", + "residue_name_number" + ], + [ + 91, + 101, + "disordered", + "protein_state" + ] + ] + }, + { + "sid": 130, + "sent": "Together, these observations suggest that efficient peptide-bond hydrolysis requires that Lys33NH2 hydrogen bonds to the active site nucleophile.", + "section": "RESULTS", + "ner": [ + [ + 90, + 95, + "Lys33", + "residue_name_number" + ], + [ + 99, + 113, + "hydrogen bonds", + "bond_interaction" + ] + ] + }, + { + "sid": 131, + "sent": "The benefit of Thr over Ser as the active-site nucleophile", + "section": "RESULTS", + "ner": [ + [ + 15, + 18, + "Thr", + "residue_name" + ], + [ + 24, + 27, + "Ser", + "residue_name" + ] + ] + }, + { + "sid": 132, + "sent": "All proteasomes strictly employ threonine as the active-site residue instead of serine.", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "proteasomes", + "complex_assembly" + ], + [ + 16, + 31, + "strictly employ", + "protein_state" + ], + [ + 32, + 41, + "threonine", + "residue_name" + ], + [ + 49, + 68, + "active-site residue", + "site" + ], + [ + 80, + 86, + "serine", + "residue_name" + ] + ] + }, + { + "sid": 133, + "sent": "To investigate the reason for this singularity, we analysed a \u03b25-T1S mutant, which is viable but suffers from growth defects (Fig. 4a and Table 1).", + "section": "RESULTS", + "ner": [ + [ + 62, + 68, + "\u03b25-T1S", + "mutant" + ], + [ + 69, + 75, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 134, + "sent": "Activity assays with the \u03b25-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40\u201345% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c).", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Activity assays", + "experimental_method" + ], + [ + 25, + 27, + "\u03b25", + "protein" + ], + [ + 47, + 59, + "Suc-LLVY-AMC", + "chemical" + ], + [ + 104, + 107, + "T1S", + "mutant" + ], + [ + 108, + 114, + "mutant", + "protein_state" + ], + [ + 150, + 152, + "WT", + "protein_state" + ], + [ + 153, + 164, + "proteasomes", + "complex_assembly" + ] + ] + }, + { + "sid": 135, + "sent": "By contrast, turnover of the substrate Z-GGL-pNA, used to monitor ChT-L activity in situ but in a less quantitative fashion, is not detectably impaired (Supplementary Fig. 9a).", + "section": "RESULTS", + "ner": [ + [ + 39, + 48, + "Z-GGL-pNA", + "chemical" + ] + ] + }, + { + "sid": 136, + "sent": "Crystal structure analysis of the \u03b25-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5).", + "section": "RESULTS", + "ner": [ + [ + 0, + 17, + "Crystal structure", + "evidence" + ], + [ + 34, + 40, + "\u03b25-T1S", + "mutant" + ], + [ + 41, + 47, + "mutant", + "protein_state" + ], + [ + 58, + 78, + "precursor processing", + "ptm" + ], + [ + 94, + 108, + "ligand-complex", + "complex_assembly" + ], + [ + 109, + 119, + "structures", + "evidence" + ], + [ + 125, + 135, + "bortezomib", + "chemical" + ], + [ + 140, + 151, + "carfilzomib", + "chemical" + ], + [ + 197, + 201, + "Ser1", + "residue_name_number" + ] + ] + }, + { + "sid": 137, + "sent": "However, the apo crystal structure revealed that Ser1O\u03b3 is turned away from the substrate-binding channel (Fig. 4g).", + "section": "RESULTS", + "ner": [ + [ + 13, + 16, + "apo", + "protein_state" + ], + [ + 17, + 34, + "crystal structure", + "evidence" + ], + [ + 49, + 53, + "Ser1", + "residue_name_number" + ], + [ + 80, + 105, + "substrate-binding channel", + "site" + ] + ] + }, + { + "sid": 138, + "sent": "Compared with Thr1O\u03b3 in WT CP structures, Ser1O\u03b3 is rotated by 60\u00b0.", + "section": "RESULTS", + "ner": [ + [ + 14, + 18, + "Thr1", + "residue_name_number" + ], + [ + 24, + 26, + "WT", + "protein_state" + ], + [ + 27, + 29, + "CP", + "complex_assembly" + ], + [ + 30, + 40, + "structures", + "evidence" + ], + [ + 42, + 46, + "Ser1", + "residue_name_number" + ] + ] + }, + { + "sid": 139, + "sent": "Because both conformations of Ser1O\u03b3 are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1.", + "section": "RESULTS", + "ner": [ + [ + 30, + 34, + "Ser1", + "residue_name_number" + ], + [ + 41, + 56, + "hydrogen-bonded", + "bond_interaction" + ], + [ + 60, + 65, + "Lys33", + "residue_name_number" + ], + [ + 179, + 183, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 140, + "sent": "The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h).", + "section": "RESULTS", + "ner": [ + [ + 4, + 23, + "active-site residue", + "site" + ], + [ + 24, + 28, + "Thr1", + "residue_name_number" + ], + [ + 89, + 113, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 119, + 123, + "Thr3", + "residue_name_number" + ], + [ + 128, + 133, + "Ala46", + "residue_name_number" + ] + ] + }, + { + "sid": 141, + "sent": "Consequently, the hydroxyl group of Thr1 requires no reorientation before substrate cleavage and is thus more catalytically efficient than Ser1.", + "section": "RESULTS", + "ner": [ + [ + 36, + 40, + "Thr1", + "residue_name_number" + ], + [ + 139, + 143, + "Ser1", + "residue_name_number" + ] + ] + }, + { + "sid": 142, + "sent": "In agreement, at an elevated growing temperature of 37\u2009\u00b0C the T1S mutant is unable to grow (Fig. 4a).", + "section": "RESULTS", + "ner": [ + [ + 62, + 65, + "T1S", + "mutant" + ], + [ + 66, + 72, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 143, + "sent": "In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5).", + "section": "RESULTS", + "ner": [ + [ + 14, + 20, + "mutant", + "protein_state" + ], + [ + 21, + 31, + "proteasome", + "complex_assembly" + ], + [ + 55, + 65, + "proteasome", + "complex_assembly" + ], + [ + 80, + 90, + "bortezomib", + "chemical" + ], + [ + 106, + 117, + "carfilzomib", + "chemical" + ] + ] + }, + { + "sid": 144, + "sent": "Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors.", + "section": "RESULTS", + "ner": [ + [ + 14, + 31, + "inhibitor complex", + "complex_assembly" + ], + [ + 32, + 42, + "structures", + "evidence" + ], + [ + 94, + 96, + "WT", + "protein_state" + ], + [ + 97, + 100, + "yCP", + "complex_assembly" + ], + [ + 101, + 111, + "structures", + "evidence" + ], + [ + 113, + 137, + "with the same inhibitors", + "protein_state" + ] + ] + }, + { + "sid": 145, + "sent": "Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein.", + "section": "RESULTS", + "ner": [ + [ + 13, + 21, + "affinity", + "evidence" + ], + [ + 42, + 53, + "carfilzomib", + "chemical" + ], + [ + 106, + 131, + "substrate-binding channel", + "site" + ], + [ + 151, + 161, + "bortezomib", + "chemical" + ] + ] + }, + { + "sid": 146, + "sent": "Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased.", + "section": "RESULTS", + "ner": [ + [ + 11, + 30, + "mean residence time", + "evidence" + ], + [ + 34, + 45, + "carfilzomib", + "chemical" + ], + [ + 53, + 64, + "active site", + "site" + ], + [ + 123, + 127, + "Ser1", + "residue_name_number" + ] + ] + }, + { + "sid": 147, + "sent": "Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.).", + "section": "RESULTS", + "ner": [ + [ + 89, + 98, + "threonine", + "residue_name" + ], + [ + 133, + 144, + "proteasomes", + "complex_assembly" + ], + [ + 189, + 214, + "proteasome-like proteases", + "protein_type" + ], + [ + 223, + 227, + "HslV", + "protein" + ] + ] + }, + { + "sid": 148, + "sent": "The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 18, + "20S proteasome", + "complex_assembly" + ], + [ + 19, + 21, + "CP", + "complex_assembly" + ], + [ + 35, + 57, + "non-lysosomal protease", + "protein_type" + ], + [ + 61, + 71, + "eukaryotic", + "taxonomy_domain" + ] + ] + }, + { + "sid": 149, + "sent": "The \u03b2-subunit propeptides, particularly that of \u03b25, are key factors that help drive proper assembly of the CP complex.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 13, + "\u03b2-subunit", + "protein" + ], + [ + 14, + 25, + "propeptides", + "structure_element" + ], + [ + 48, + 50, + "\u03b25", + "protein" + ], + [ + 107, + 109, + "CP", + "complex_assembly" + ] + ] + }, + { + "sid": 150, + "sent": "In addition, they prevent irreversible inactivation of the Thr1 N terminus by N-acetylation.", + "section": "DISCUSS", + "ner": [ + [ + 59, + 63, + "Thr1", + "residue_name_number" + ], + [ + 78, + 91, + "N-acetylation", + "ptm" + ] + ] + }, + { + "sid": 151, + "sent": "By contrast, the prosegments of \u03b2 subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 28, + "prosegments", + "structure_element" + ], + [ + 32, + 42, + "\u03b2 subunits", + "protein" + ], + [ + 63, + 71, + "archaeal", + "taxonomy_domain" + ], + [ + 72, + 82, + "proteasome", + "complex_assembly" + ], + [ + 107, + 131, + "heterologously expressed", + "experimental_method" + ], + [ + 135, + 151, + "Escherichia coli", + "species" + ] + ] + }, + { + "sid": 152, + "sent": "In eukaryotes, deletion of or failure to cleave the \u03b21 and \u03b22 propeptides is well tolerated.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 13, + "eukaryotes", + "taxonomy_domain" + ], + [ + 52, + 54, + "\u03b21", + "protein" + ], + [ + 59, + 61, + "\u03b22", + "protein" + ], + [ + 62, + 73, + "propeptides", + "structure_element" + ] + ] + }, + { + "sid": 153, + "sent": "However, removal of the \u03b25 prosegment or any interference with its cleavage causes severe phenotypic defects.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 19, + "removal of", + "experimental_method" + ], + [ + 24, + 26, + "\u03b25", + "protein" + ], + [ + 27, + 37, + "prosegment", + "structure_element" + ] + ] + }, + { + "sid": 154, + "sent": "These observations highlight the unique function and importance of the \u03b25 propeptide as well as the \u03b25 active site for maturation and function of the eukaryotic CP.", + "section": "DISCUSS", + "ner": [ + [ + 71, + 73, + "\u03b25", + "protein" + ], + [ + 74, + 84, + "propeptide", + "structure_element" + ], + [ + 100, + 102, + "\u03b25", + "protein" + ], + [ + 103, + 114, + "active site", + "site" + ], + [ + 150, + 160, + "eukaryotic", + "taxonomy_domain" + ], + [ + 161, + 163, + "CP", + "complex_assembly" + ] + ] + }, + { + "sid": 155, + "sent": "Here we have described the atomic structures of various \u03b25-T1A mutants, which allowed for the first time visualization of the residual \u03b25 propeptide.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 44, + "atomic structures", + "evidence" + ], + [ + 56, + 62, + "\u03b25-T1A", + "mutant" + ], + [ + 135, + 137, + "\u03b25", + "protein" + ], + [ + 138, + 148, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 156, + "sent": "Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1O\u03b3, although it does not adopt the tight turn observed for the prosegment of subunit \u03b21.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 21, + "(-2)", + "residue_number" + ], + [ + 50, + 60, + "propeptide", + "structure_element" + ], + [ + 80, + 87, + "Gly(-1)", + "residue_name_number" + ], + [ + 98, + 108, + "structures", + "evidence" + ], + [ + 158, + 162, + "Thr1", + "residue_name_number" + ], + [ + 197, + 207, + "tight turn", + "structure_element" + ], + [ + 225, + 235, + "prosegment", + "structure_element" + ], + [ + 247, + 249, + "\u03b21", + "protein" + ] + ] + }, + { + "sid": 157, + "sent": "From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis.", + "section": "DISCUSS", + "ner": [ + [ + 57, + 64, + "Gly(-1)", + "residue_name_number" + ], + [ + 69, + 73, + "Thr1", + "residue_name_number" + ], + [ + 118, + 129, + "active site", + "site" + ], + [ + 147, + 156, + "autolysis", + "ptm" + ] + ] + }, + { + "sid": 158, + "sent": "In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1O\u03b3 due to the rotational freedom and flexibility of the acetyl group.", + "section": "DISCUSS", + "ner": [ + [ + 30, + 43, + "N-acetylation", + "ptm" + ], + [ + 51, + 55, + "Thr1", + "residue_name_number" + ], + [ + 88, + 92, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 159, + "sent": "The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 14, + "propeptide", + "structure_element" + ], + [ + 43, + 68, + "substrate-binding channel", + "site" + ], + [ + 90, + 97, + "Gly(-1)", + "residue_name_number" + ], + [ + 162, + 166, + "(-2)", + "residue_number" + ] + ] + }, + { + "sid": 160, + "sent": "Autolytic activation of the CP constitutes one of the final steps of proteasome biogenesis, but the trigger for propeptide cleavage had remained enigmatic.", + "section": "DISCUSS", + "ner": [ + [ + 28, + 30, + "CP", + "complex_assembly" + ], + [ + 112, + 131, + "propeptide cleavage", + "ptm" + ] + ] + }, + { + "sid": 161, + "sent": "On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture.", + "section": "DISCUSS", + "ner": [ + [ + 29, + 38, + "CP:ligand", + "complex_assembly" + ], + [ + 146, + 156, + "proteasome", + "complex_assembly" + ], + [ + 157, + 181, + "active-site architecture", + "site" + ] + ] + }, + { + "sid": 162, + "sent": "We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 28, + "catalytic triad", + "site" + ], + [ + 37, + 48, + "active site", + "site" + ], + [ + 56, + 58, + "CP", + "complex_assembly" + ], + [ + 82, + 86, + "Thr1", + "residue_name_number" + ], + [ + 88, + 93, + "Lys33", + "residue_name_number" + ], + [ + 98, + 101, + "Asp", + "residue_name" + ], + [ + 102, + 107, + "Glu17", + "residue_name_number" + ], + [ + 162, + 172, + "eukaryotic", + "taxonomy_domain" + ], + [ + 174, + 183, + "bacterial", + "taxonomy_domain" + ], + [ + 188, + 196, + "archaeal", + "taxonomy_domain" + ], + [ + 197, + 207, + "proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 163, + "sent": "Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17O\u03b4 orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+\u2013Asp17O\u03b4: 2.9\u2009\u00c5).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "Lys33", + "residue_name_number" + ], + [ + 58, + 79, + "autocatalytic removal", + "ptm" + ], + [ + 87, + 98, + "propeptides", + "structure_element" + ], + [ + 147, + 152, + "Asp17", + "residue_name_number" + ], + [ + 163, + 168, + "Lys33", + "residue_name_number" + ], + [ + 231, + 244, + "hydrogen bond", + "bond_interaction" + ], + [ + 255, + 260, + "Lys33", + "residue_name_number" + ], + [ + 265, + 270, + "Asp17", + "residue_name_number" + ] + ] + }, + { + "sid": 164, + "sent": "Analogously to the proteasome, a Thr\u2013Lys\u2013Asp triad is also found in L-asparaginase.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 29, + "proteasome", + "complex_assembly" + ], + [ + 33, + 50, + "Thr\u2013Lys\u2013Asp triad", + "site" + ], + [ + 68, + 82, + "L-asparaginase", + "protein_type" + ] + ] + }, + { + "sid": 165, + "sent": "Thus, specific protein surroundings can significantly alter the chemical properties of amino acids such as Lys to function as an acid\u2013base catalyst.", + "section": "DISCUSS", + "ner": [ + [ + 107, + 110, + "Lys", + "residue_name" + ] + ] + }, + { + "sid": 166, + "sent": "In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1O\u03b3 (Fig. 3d).", + "section": "DISCUSS", + "ner": [ + [ + 36, + 47, + "active site", + "site" + ], + [ + 72, + 76, + "Thr1", + "residue_name_number" + ], + [ + 90, + 104, + "hydrogen bonds", + "bond_interaction" + ], + [ + 207, + 231, + "endoproteolytic cleavage", + "ptm" + ], + [ + 235, + 239, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 167, + "sent": "Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), \u03b1-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.).", + "section": "DISCUSS", + "ner": [ + [ + 51, + 55, + "Thr1", + "residue_name_number" + ], + [ + 81, + 95, + "hydrogen bonds", + "bond_interaction" + ], + [ + 127, + 140, + "fellutamide B", + "chemical" + ], + [ + 149, + 161, + "\u03b1-ketoamides", + "chemical" + ], + [ + 163, + 179, + "homobelactosin C", + "chemical" + ], + [ + 191, + 208, + "salinosporamide A", + "chemical" + ] + ] + }, + { + "sid": 168, + "sent": "Furthermore, opening of the \u03b2-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+.", + "section": "DISCUSS", + "ner": [ + [ + 47, + 56, + "omuralide", + "chemical" + ], + [ + 60, + 64, + "Thr1", + "residue_name_number" + ], + [ + 123, + 127, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 169, + "sent": "The resulting uncharged Thr1NH2 is hydrogen-bridged to the C3-OH group.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 28, + "Thr1", + "residue_name_number" + ], + [ + 35, + 51, + "hydrogen-bridged", + "bond_interaction" + ] + ] + }, + { + "sid": 170, + "sent": "In agreement, acetylation of the Thr1 N terminus irreversibly blocks hydrolytic activity, and binding of substrates is prevented for steric reasons.", + "section": "DISCUSS", + "ner": [ + [ + 14, + 25, + "acetylation", + "ptm" + ], + [ + 33, + 37, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 171, + "sent": "By acting as a proton donor during catalysis, the Thr1 N terminus may also favour cleavage of substrate peptide bonds (Fig. 3d).", + "section": "DISCUSS", + "ner": [ + [ + 50, + 54, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 172, + "sent": "Cleavage of the scissile peptide bond requires protonation of the emerging free amine, and in the proteasome, the Thr1 amine group is likely to assume this function.", + "section": "DISCUSS", + "ner": [ + [ + 98, + 108, + "proteasome", + "complex_assembly" + ], + [ + 114, + 118, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 173, + "sent": "Analogously, Thr1NH3+ might promote the bivalent reaction mode of epoxyketone inhibitors by protonating the epoxide moiety to create a positively charged trivalent oxygen atom that is subsequently nucleophilically attacked by Thr1NH2.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 17, + "Thr1", + "residue_name_number" + ], + [ + 226, + 230, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 174, + "sent": "During autolysis the Thr1 N terminus is engaged in a hydroxyoxazolidine ring intermediate (Fig. 3d), which is unstable and short-lived.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 16, + "autolysis", + "ptm" + ], + [ + 21, + 25, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 175, + "sent": "Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+.", + "section": "DISCUSS", + "ner": [ + [ + 60, + 64, + "Thr1", + "residue_name_number" + ], + [ + 98, + 115, + "aspartic acid 166", + "residue_name_number" + ], + [ + 120, + 126, + "Ser129", + "residue_name_number" + ], + [ + 138, + 142, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 176, + "sent": "The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 19, + "Ser129", + "residue_name_number" + ], + [ + 24, + 30, + "Asp166", + "residue_name_number" + ], + [ + 73, + 77, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 177, + "sent": "Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%.", + "section": "DISCUSS", + "ner": [ + [ + 67, + 75, + "mutation", + "experimental_method" + ], + [ + 76, + 81, + "D166A", + "mutant" + ], + [ + 91, + 100, + "autolysis", + "ptm" + ], + [ + 108, + 116, + "archaeal", + "taxonomy_domain" + ], + [ + 117, + 119, + "CP", + "complex_assembly" + ], + [ + 128, + 136, + "exchange", + "experimental_method" + ], + [ + 137, + 142, + "D166N", + "mutant" + ], + [ + 177, + 182, + "yeast", + "taxonomy_domain" + ], + [ + 183, + 185, + "CP", + "complex_assembly" + ] + ] + }, + { + "sid": 178, + "sent": "The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2).", + "section": "DISCUSS", + "ner": [ + [ + 4, + 12, + "mutation", + "experimental_method" + ], + [ + 13, + 18, + "D166N", + "mutant" + ], + [ + 37, + 41, + "Thr1", + "residue_name_number" + ], + [ + 116, + 120, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 179, + "sent": "This interpretation agrees with the strongly reduced catalytic activity of the \u03b25-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other.", + "section": "DISCUSS", + "ner": [ + [ + 79, + 87, + "\u03b25-D166N", + "mutant" + ], + [ + 88, + 94, + "mutant", + "protein_state" + ], + [ + 150, + 161, + "carfilzomib", + "chemical" + ] + ] + }, + { + "sid": 180, + "sent": "Hence, the proteasome can be viewed as having a second triad that is essential for efficient proteolysis.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 21, + "proteasome", + "complex_assembly" + ], + [ + 48, + 60, + "second triad", + "site" + ] + ] + }, + { + "sid": 181, + "sent": "While Lys33NH2 and Asp17O\u03b4 are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1.", + "section": "DISCUSS", + "ner": [ + [ + 6, + 11, + "Lys33", + "residue_name_number" + ], + [ + 19, + 24, + "Asp17", + "residue_name_number" + ], + [ + 59, + 63, + "Thr1", + "residue_name_number" + ], + [ + 85, + 91, + "Ser129", + "residue_name_number" + ], + [ + 98, + 104, + "Asp166", + "residue_name_number" + ], + [ + 156, + 160, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 182, + "sent": "In accord with the proposed Thr1\u2013Lys33\u2013Asp17 catalytic triad, crystallographic data on the proteolytically inactive \u03b25-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken.", + "section": "DISCUSS", + "ner": [ + [ + 28, + 32, + "Thr1", + "residue_name_number" + ], + [ + 33, + 38, + "Lys33", + "residue_name_number" + ], + [ + 39, + 44, + "Asp17", + "residue_name_number" + ], + [ + 45, + 60, + "catalytic triad", + "site" + ], + [ + 62, + 83, + "crystallographic data", + "evidence" + ], + [ + 91, + 115, + "proteolytically inactive", + "protein_state" + ], + [ + 116, + 122, + "\u03b25-T1C", + "mutant" + ], + [ + 123, + 129, + "mutant", + "protein_state" + ], + [ + 166, + 171, + "Lys33", + "residue_name_number" + ], + [ + 179, + 183, + "Cys1", + "residue_name_number" + ] + ] + }, + { + "sid": 183, + "sent": "However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 21, + "Cys", + "residue_name" + ], + [ + 54, + 64, + "propeptide", + "structure_element" + ], + [ + 78, + 85, + "cleaved", + "protein_state" + ] + ] + }, + { + "sid": 184, + "sent": "While only one single turnover is necessary for autolysis, continuous enzymatic activity is required for significant and detectable substrate hydrolysis.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 57, + "autolysis", + "ptm" + ] + ] + }, + { + "sid": 185, + "sent": "Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 29, + "Ntn hydrolase", + "protein_type" + ], + [ + 30, + 48, + "penicillin acylase", + "protein_type" + ], + [ + 50, + 62, + "substitution", + "experimental_method" + ], + [ + 70, + 79, + "catalytic", + "protein_state" + ], + [ + 91, + 94, + "Ser", + "residue_name" + ], + [ + 106, + 109, + "Cys", + "residue_name" + ], + [ + 115, + 126, + "inactivates", + "protein_state" + ], + [ + 131, + 137, + "enzyme", + "protein_type" + ], + [ + 156, + 176, + "precursor processing", + "ptm" + ] + ] + }, + { + "sid": 186, + "sent": "To investigate why the CP specifically employs threonine as its active-site residue, we used a \u03b25-T1S mutant of the yCP and characterized it biochemically and structurally.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 25, + "CP", + "complex_assembly" + ], + [ + 47, + 56, + "threonine", + "residue_name" + ], + [ + 64, + 83, + "active-site residue", + "site" + ], + [ + 95, + 101, + "\u03b25-T1S", + "mutant" + ], + [ + 102, + 108, + "mutant", + "protein_state" + ], + [ + 116, + 119, + "yCP", + "complex_assembly" + ], + [ + 141, + 171, + "biochemically and structurally", + "experimental_method" + ] + ] + }, + { + "sid": 187, + "sent": "Activity assays with the \u03b25-T1S mutant revealed reduced turnover of Suc-LLVY-AMC.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 15, + "Activity assays", + "experimental_method" + ], + [ + 25, + 31, + "\u03b25-T1S", + "mutant" + ], + [ + 32, + 38, + "mutant", + "protein_state" + ], + [ + 68, + 80, + "Suc-LLVY-AMC", + "chemical" + ] + ] + }, + { + "sid": 188, + "sent": "We also observed slightly lower affinity of the \u03b25-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 54, + "\u03b25-T1S", + "mutant" + ], + [ + 55, + 61, + "mutant", + "protein_state" + ], + [ + 62, + 65, + "yCP", + "complex_assembly" + ], + [ + 112, + 122, + "proteasome", + "complex_assembly" + ], + [ + 134, + 144, + "bortezomib", + "chemical" + ], + [ + 149, + 160, + "carfilzomib", + "chemical" + ] + ] + }, + { + "sid": 189, + "sent": "Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 19, + "Structural analyses", + "evidence" + ], + [ + 52, + 55, + "T1S", + "mutant" + ], + [ + 56, + 62, + "mutant", + "protein_state" + ], + [ + 98, + 111, + "strict use of", + "protein_state" + ], + [ + 112, + 115, + "Thr", + "residue_name" + ], + [ + 128, + 139, + "proteasomes", + "complex_assembly" + ] + ] + }, + { + "sid": 190, + "sent": "Thr1 is well anchored in the active site by hydrophobic interactions of its C\u03b3 methyl group with Ala46 (C\u03b2), Lys33 (carbon side chain) and Thr3 (C\u03b3).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 4, + "Thr1", + "residue_name_number" + ], + [ + 29, + 40, + "active site", + "site" + ], + [ + 44, + 68, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 97, + 102, + "Ala46", + "residue_name_number" + ], + [ + 109, + 114, + "Lys33", + "residue_name_number" + ], + [ + 139, + 143, + "Thr3", + "residue_name_number" + ] + ] + }, + { + "sid": 191, + "sent": "Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the C\u03b3 for fixing the position of the nucleophilic Thr1.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 31, + "proteolytically active", + "protein_state" + ], + [ + 32, + 42, + "proteasome", + "complex_assembly" + ], + [ + 57, + 64, + "archaea", + "taxonomy_domain" + ], + [ + 66, + 71, + "yeast", + "taxonomy_domain" + ], + [ + 76, + 83, + "mammals", + "taxonomy_domain" + ], + [ + 161, + 164, + "Thr", + "residue_name" + ], + [ + 168, + 171, + "Ile", + "residue_name" + ], + [ + 184, + 185, + "3", + "residue_number" + ], + [ + 267, + 271, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 192, + "sent": "In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h).", + "section": "DISCUSS", + "ner": [ + [ + 15, + 19, + "Thr1", + "residue_name_number" + ], + [ + 43, + 47, + "Ser1", + "residue_name_number" + ], + [ + 77, + 81, + "Thr1", + "residue_name_number" + ], + [ + 107, + 109, + "WT", + "protein_state" + ], + [ + 110, + 116, + "enzyme", + "complex_assembly" + ] + ] + }, + { + "sid": 193, + "sent": "Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity.", + "section": "DISCUSS", + "ner": [ + [ + 16, + 35, + "threonine aspartase", + "protein_type" + ], + [ + 36, + 44, + "Taspase1", + "protein" + ], + [ + 46, + 54, + "mutation", + "experimental_method" + ], + [ + 62, + 73, + "active-site", + "site" + ], + [ + 74, + 80, + "Thr234", + "residue_name_number" + ], + [ + 84, + 87, + "Ser", + "residue_name" + ], + [ + 154, + 160, + "Thr234", + "residue_name_number" + ], + [ + 168, + 170, + "WT", + "protein_state" + ] + ] + }, + { + "sid": 194, + "sent": "Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site.", + "section": "DISCUSS", + "ner": [ + [ + 24, + 30, + "serine", + "residue_name" + ], + [ + 31, + 37, + "mutant", + "protein_state" + ], + [ + 41, + 47, + "active", + "protein_state" + ], + [ + 49, + 58, + "threonine", + "residue_name" + ], + [ + 99, + 109, + "proteasome", + "complex_assembly" + ], + [ + 110, + 121, + "active site", + "site" + ] + ] + }, + { + "sid": 195, + "sent": "The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 36, + "threonine", + "residue_name" + ], + [ + 45, + 55, + "proteasome", + "complex_assembly" + ], + [ + 56, + 67, + "active site", + "site" + ], + [ + 172, + 184, + "conservation", + "protein_state" + ], + [ + 192, + 196, + "Thr1", + "residue_name_number" + ], + [ + 212, + 223, + "proteasomes", + "complex_assembly" + ], + [ + 229, + 237, + "bacteria", + "taxonomy_domain" + ], + [ + 241, + 251, + "eukaryotes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 196, + "sent": "Conformation of proteasomal propeptides.", + "section": "FIG", + "ner": [ + [ + 28, + 39, + "propeptides", + "structure_element" + ] + ] + }, + { + "sid": 197, + "sent": "(a) Structural superposition of the \u03b21-T1A propeptide and the matured WT \u03b21 active-site Thr1.", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "Structural superposition", + "experimental_method" + ], + [ + 36, + 42, + "\u03b21-T1A", + "mutant" + ], + [ + 43, + 53, + "propeptide", + "structure_element" + ], + [ + 62, + 69, + "matured", + "protein_state" + ], + [ + 70, + 72, + "WT", + "protein_state" + ], + [ + 73, + 75, + "\u03b21", + "protein" + ], + [ + 76, + 87, + "active-site", + "site" + ], + [ + 88, + 92, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 198, + "sent": "Only the residues (-5) to (-1) of the \u03b21-T1A propeptide are displayed.", + "section": "FIG", + "ner": [ + [ + 18, + 30, + "(-5) to (-1)", + "residue_range" + ], + [ + 38, + 44, + "\u03b21-T1A", + "mutant" + ], + [ + 45, + 55, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 199, + "sent": "The major determinant of the S1 specificity pocket, residue 45, is depicted.", + "section": "FIG", + "ner": [ + [ + 29, + 50, + "S1 specificity pocket", + "site" + ], + [ + 60, + 62, + "45", + "residue_number" + ], + [ + 29, + 50, + "S1 specificity pocket", + "site" + ], + [ + 60, + 62, + "45", + "residue_number" + ] + ] + }, + { + "sid": 200, + "sent": "Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow).", + "section": "FIG", + "ner": [ + [ + 31, + 38, + "Gly(-1)", + "residue_name_number" + ], + [ + 43, + 47, + "Ala1", + "residue_name_number" + ], + [ + 55, + 65, + "propeptide", + "structure_element" + ], + [ + 75, + 80, + "G(-1)", + "residue_name_number" + ], + [ + 133, + 142, + "processed", + "protein_state" + ], + [ + 143, + 145, + "WT", + "protein_state" + ], + [ + 146, + 157, + "active-site", + "site" + ], + [ + 158, + 162, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 201, + "sent": "The black arrow indicates the attack of Thr1O\u03b3 onto the carbonyl carbon atom of Gly(-1).", + "section": "FIG", + "ner": [ + [ + 40, + 44, + "Thr1", + "residue_name_number" + ], + [ + 80, + 87, + "Gly(-1)", + "residue_name_number" + ] + ] + }, + { + "sid": 202, + "sent": "(b) Structural superposition of the \u03b21-T1A propeptide and the \u03b22-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well.", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "Structural superposition", + "experimental_method" + ], + [ + 36, + 42, + "\u03b21-T1A", + "mutant" + ], + [ + 43, + 53, + "propeptide", + "structure_element" + ], + [ + 62, + 68, + "\u03b22-T1A", + "mutant" + ], + [ + 69, + 79, + "propeptide", + "structure_element" + ], + [ + 155, + 159, + "Ala1", + "residue_name_number" + ], + [ + 164, + 171, + "Gly(-1)", + "residue_name_number" + ] + ] + }, + { + "sid": 203, + "sent": "Thr(-2)OH is hydrogen-bonded to Gly(-1)O (\u223c2.8\u2009\u00c5; black dashed line).", + "section": "FIG", + "ner": [ + [ + 0, + 7, + "Thr(-2)", + "residue_name_number" + ], + [ + 13, + 28, + "hydrogen-bonded", + "bond_interaction" + ], + [ + 32, + 39, + "Gly(-1)", + "residue_name_number" + ] + ] + }, + { + "sid": 204, + "sent": "(c) Structural superposition of the \u03b21-T1A, the \u03b22-T1A and the \u03b25-T1A-K81R propeptide remnants depict their differences in conformation.", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "Structural superposition", + "experimental_method" + ], + [ + 36, + 42, + "\u03b21-T1A", + "mutant" + ], + [ + 48, + 54, + "\u03b22-T1A", + "mutant" + ], + [ + 63, + 74, + "\u03b25-T1A-K81R", + "mutant" + ], + [ + 75, + 85, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 205, + "sent": "While residue (-2) of the \u03b21 and \u03b22 prosegments fit the S1 pocket, His(-2) of the \u03b25 propeptide occupies the S2 pocket.", + "section": "FIG", + "ner": [ + [ + 14, + 18, + "(-2)", + "residue_number" + ], + [ + 26, + 28, + "\u03b21", + "protein" + ], + [ + 33, + 35, + "\u03b22", + "protein" + ], + [ + 36, + 47, + "prosegments", + "structure_element" + ], + [ + 56, + 65, + "S1 pocket", + "site" + ], + [ + 67, + 74, + "His(-2)", + "residue_name_number" + ], + [ + 82, + 84, + "\u03b25", + "protein" + ], + [ + 85, + 95, + "propeptide", + "structure_element" + ], + [ + 109, + 118, + "S2 pocket", + "site" + ] + ] + }, + { + "sid": 206, + "sent": "Nonetheless, in all mutants the carbonyl carbon atom of Gly(-1) is ideally placed for the nucleophilic attack by Thr1O\u03b3.", + "section": "FIG", + "ner": [ + [ + 56, + 63, + "Gly(-1)", + "residue_name_number" + ], + [ + 113, + 117, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 207, + "sent": "The hydrogen bond between Thr(-2)OH and Gly(-1)O (\u223c2.8\u2009\u00c5) is indicated by a black dashed line.", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "hydrogen bond", + "bond_interaction" + ], + [ + 26, + 33, + "Thr(-2)", + "residue_name_number" + ], + [ + 40, + 47, + "Gly(-1)", + "residue_name_number" + ] + ] + }, + { + "sid": 208, + "sent": "Mutations of residue (-2) and their influence on propeptide conformation and autolysis.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Mutations", + "experimental_method" + ], + [ + 21, + 25, + "(-2)", + "residue_number" + ], + [ + 49, + 59, + "propeptide", + "structure_element" + ], + [ + 77, + 86, + "autolysis", + "ptm" + ] + ] + }, + { + "sid": 209, + "sent": "(a) Structural superposition of the \u03b21-T1A propeptide and the \u03b25-H(-2)L-T1A mutant propeptide.", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "Structural superposition", + "experimental_method" + ], + [ + 36, + 42, + "\u03b21-T1A", + "mutant" + ], + [ + 43, + 53, + "propeptide", + "structure_element" + ], + [ + 62, + 75, + "\u03b25-H(-2)L-T1A", + "mutant" + ], + [ + 76, + 82, + "mutant", + "protein_state" + ], + [ + 83, + 93, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 210, + "sent": "The (-2) residues of both prosegments point into the S1 pocket.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "(-2)", + "residue_number" + ], + [ + 26, + 37, + "prosegments", + "structure_element" + ], + [ + 53, + 62, + "S1 pocket", + "site" + ] + ] + }, + { + "sid": 211, + "sent": "(b) Structural superposition of the \u03b25 propeptides in the \u03b25-H(-2)L-T1A, \u03b25-H(-2)T-T1A, \u03b25-(H-2)A-T1A-K81R and \u03b25-T1A-K81R mutant proteasomes.", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "Structural superposition", + "experimental_method" + ], + [ + 36, + 38, + "\u03b25", + "protein" + ], + [ + 39, + 50, + "propeptides", + "structure_element" + ], + [ + 58, + 71, + "\u03b25-H(-2)L-T1A", + "mutant" + ], + [ + 73, + 86, + "\u03b25-H(-2)T-T1A", + "mutant" + ], + [ + 88, + 106, + "\u03b25-(H-2)A-T1A-K81R", + "mutant" + ], + [ + 111, + 122, + "\u03b25-T1A-K81R", + "mutant" + ], + [ + 123, + 129, + "mutant", + "protein_state" + ], + [ + 130, + 141, + "proteasomes", + "complex_assembly" + ] + ] + }, + { + "sid": 212, + "sent": "While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions.", + "section": "FIG", + "ner": [ + [ + 19, + 31, + "(-2) to (-4)", + "residue_range" + ], + [ + 60, + 67, + "Gly(-1)", + "residue_name_number" + ], + [ + 72, + 76, + "Ala1", + "residue_name_number" + ], + [ + 96, + 106, + "structures", + "evidence" + ] + ] + }, + { + "sid": 213, + "sent": "(c) Structural superposition of the \u03b22-T1A propeptide and the \u03b25-H(-2)T-T1A mutant propeptide.", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "Structural superposition", + "experimental_method" + ], + [ + 36, + 42, + "\u03b22-T1A", + "mutant" + ], + [ + 43, + 53, + "propeptide", + "structure_element" + ], + [ + 62, + 75, + "\u03b25-H(-2)T-T1A", + "mutant" + ], + [ + 76, + 82, + "mutant", + "protein_state" + ], + [ + 83, + 93, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 214, + "sent": "The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of \u03b22 forms a hydrogen bridge to Gly(-1)O (black dashed line).", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "(-2)", + "residue_number" + ], + [ + 26, + 37, + "prosegments", + "structure_element" + ], + [ + 53, + 62, + "S1 pocket", + "site" + ], + [ + 73, + 80, + "Thr(-2)", + "residue_name_number" + ], + [ + 86, + 88, + "\u03b22", + "protein" + ], + [ + 97, + 112, + "hydrogen bridge", + "bond_interaction" + ], + [ + 116, + 123, + "Gly(-1)", + "residue_name_number" + ] + ] + }, + { + "sid": 215, + "sent": "(d) Structural superposition of the matured \u03b22 active site, the WT \u03b22-T1A propeptide and the \u03b22-T(-2)V mutant propeptide.", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "Structural superposition", + "experimental_method" + ], + [ + 36, + 43, + "matured", + "protein_state" + ], + [ + 44, + 46, + "\u03b22", + "protein" + ], + [ + 47, + 58, + "active site", + "site" + ], + [ + 64, + 66, + "WT", + "protein_state" + ], + [ + 67, + 73, + "\u03b22-T1A", + "mutant" + ], + [ + 74, + 84, + "propeptide", + "structure_element" + ], + [ + 93, + 102, + "\u03b22-T(-2)V", + "mutant" + ], + [ + 103, + 109, + "mutant", + "protein_state" + ], + [ + 110, + 120, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 216, + "sent": "Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1O\u03b3 on the carbonyl carbon atom of Gly(-1).", + "section": "FIG", + "ner": [ + [ + 9, + 16, + "Val(-2)", + "residue_name_number" + ], + [ + 51, + 60, + "S1 pocket", + "site" + ], + [ + 98, + 105, + "Gly(-1)", + "residue_name_number" + ], + [ + 144, + 148, + "Thr1", + "residue_name_number" + ], + [ + 182, + 189, + "Gly(-1)", + "residue_name_number" + ] + ] + }, + { + "sid": 217, + "sent": "Architecture and proposed reaction mechanism of the proteasomal active site.", + "section": "FIG", + "ner": [ + [ + 64, + 75, + "active site", + "site" + ] + ] + }, + { + "sid": 218, + "sent": "(a) Hydrogen-bonding network at the mature WT \u03b25 proteasomal active site (dotted lines).", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "Hydrogen-bonding network", + "site" + ], + [ + 36, + 42, + "mature", + "protein_state" + ], + [ + 43, + 45, + "WT", + "protein_state" + ], + [ + 46, + 48, + "\u03b25", + "protein" + ], + [ + 61, + 72, + "active site", + "site" + ] + ] + }, + { + "sid": 219, + "sent": "Thr1OH is hydrogen-bonded to Lys33NH2 (2.7\u2009\u00c5), which in turn interacts with Asp17O\u03b4.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "Thr1", + "residue_name_number" + ], + [ + 10, + 25, + "hydrogen-bonded", + "bond_interaction" + ], + [ + 29, + 34, + "Lys33", + "residue_name_number" + ], + [ + 76, + 81, + "Asp17", + "residue_name_number" + ] + ] + }, + { + "sid": 220, + "sent": "The Thr1 N terminus is engaged in hydrogen bonds with Ser129O\u03b3, the carbonyl oxygen of residue 168, Ser169O\u03b3 and Asp166O\u03b4. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the \u03b2 subunits.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "Thr1", + "residue_name_number" + ], + [ + 34, + 48, + "hydrogen bonds", + "bond_interaction" + ], + [ + 54, + 60, + "Ser129", + "residue_name_number" + ], + [ + 95, + 98, + "168", + "residue_number" + ], + [ + 100, + 106, + "Ser169", + "residue_name_number" + ], + [ + 113, + 119, + "Asp166", + "residue_name_number" + ], + [ + 151, + 171, + "active-site residues", + "site" + ], + [ + 184, + 200, + "hydrogen bonding", + "bond_interaction" + ], + [ + 205, + 223, + "strictly conserved", + "protein_state" + ], + [ + 232, + 250, + "proteolytic centre", + "site" + ], + [ + 264, + 277, + "superposition", + "experimental_method" + ], + [ + 285, + 295, + "\u03b2 subunits", + "protein" + ] + ] + }, + { + "sid": 221, + "sent": "(c) Structural superposition of the WT \u03b25 and the \u03b25-K33A pp trans mutant active site.", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "Structural superposition", + "experimental_method" + ], + [ + 36, + 38, + "WT", + "protein_state" + ], + [ + 39, + 41, + "\u03b25", + "protein" + ], + [ + 50, + 57, + "\u03b25-K33A", + "mutant" + ], + [ + 58, + 60, + "pp", + "chemical" + ], + [ + 61, + 66, + "trans", + "protein_state" + ], + [ + 67, + 73, + "mutant", + "protein_state" + ], + [ + 74, + 85, + "active site", + "site" + ] + ] + }, + { + "sid": 222, + "sent": "In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located.", + "section": "FIG", + "ner": [ + [ + 17, + 22, + "water", + "chemical" + ], + [ + 83, + 85, + "WT", + "protein_state" + ], + [ + 126, + 131, + "Lys33", + "residue_name_number" + ] + ] + }, + { + "sid": 223, + "sent": "Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17O\u03b4 and Thr1OH.", + "section": "FIG", + "ner": [ + [ + 13, + 18, + "Lys33", + "residue_name_number" + ], + [ + 24, + 29, + "water", + "chemical" + ], + [ + 39, + 53, + "hydrogen bonds", + "bond_interaction" + ], + [ + 57, + 62, + "Arg19", + "residue_name_number" + ], + [ + 65, + 70, + "Asp17", + "residue_name_number" + ], + [ + 77, + 81, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 224, + "sent": "Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place.", + "section": "FIG", + "ner": [ + [ + 38, + 43, + "water", + "chemical" + ], + [ + 77, + 81, + "Thr1", + "residue_name_number" + ], + [ + 99, + 119, + "active-site residues", + "site" + ] + ] + }, + { + "sid": 225, + "sent": "(d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome.", + "section": "FIG", + "ner": [ + [ + 45, + 79, + "autocatalytic precursor processing", + "ptm" + ], + [ + 103, + 113, + "proteasome", + "complex_assembly" + ] + ] + }, + { + "sid": 226, + "sent": "The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red.", + "section": "FIG", + "ner": [ + [ + 4, + 15, + "active-site", + "site" + ], + [ + 16, + 20, + "Thr1", + "residue_name_number" + ], + [ + 46, + 56, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 227, + "sent": "Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Autolysis", + "ptm" + ], + [ + 68, + 72, + "Thr1", + "residue_name_number" + ], + [ + 79, + 84, + "Lys33", + "residue_name_number" + ] + ] + }, + { + "sid": 228, + "sent": "The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle.", + "section": "FIG", + "ner": [ + [ + 4, + 22, + "strictly conserved", + "protein_state" + ], + [ + 37, + 42, + "Gly47", + "residue_name_number" + ] + ] + }, + { + "sid": 229, + "sent": "Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH.", + "section": "FIG", + "ner": [ + [ + 43, + 47, + "Thr1", + "residue_name_number" + ], + [ + 102, + 112, + "propeptide", + "structure_element" + ], + [ + 151, + 157, + "Asp166", + "residue_name_number" + ], + [ + 164, + 170, + "Ser129", + "residue_name_number" + ] + ] + }, + { + "sid": 230, + "sent": "Next, Thr1NH2 polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate.", + "section": "FIG", + "ner": [ + [ + 6, + 10, + "Thr1", + "residue_name_number" + ], + [ + 26, + 31, + "water", + "chemical" + ] + ] + }, + { + "sid": 231, + "sent": "On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures).", + "section": "FIG", + "ner": [ + [ + 33, + 44, + "active-site", + "site" + ], + [ + 45, + 49, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 232, + "sent": "The charged Thr1 N terminus may engage in the orientation of the amide moiety and donate a proton to the emerging N terminus of the C-terminal cleavage product.", + "section": "FIG", + "ner": [ + [ + 12, + 16, + "Thr1", + "residue_name_number" + ] + ] + }, + { + "sid": 233, + "sent": "The resulting deprotonated Thr1NH2 finally activates a water molecule for hydrolysis of the acyl-enzyme.", + "section": "FIG", + "ner": [ + [ + 27, + 31, + "Thr1", + "residue_name_number" + ], + [ + 55, + 60, + "water", + "chemical" + ] + ] + }, + { + "sid": 234, + "sent": "The proteasome favours threonine as the active-site nucleophile.", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "proteasome", + "complex_assembly" + ], + [ + 23, + 32, + "threonine", + "residue_name" + ] + ] + }, + { + "sid": 235, + "sent": "(a) Growth tests by serial dilution of WT and pre2 (\u03b25) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation.", + "section": "FIG", + "ner": [ + [ + 4, + 35, + "Growth tests by serial dilution", + "experimental_method" + ], + [ + 39, + 41, + "WT", + "protein_state" + ], + [ + 52, + 54, + "\u03b25", + "protein" + ], + [ + 56, + 62, + "mutant", + "protein_state" + ], + [ + 63, + 68, + "yeast", + "taxonomy_domain" + ], + [ + 107, + 118, + "active-site", + "site" + ], + [ + 119, + 126, + "mutants", + "experimental_method" + ] + ] + }, + { + "sid": 236, + "sent": "(b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (\u03b25) using the substrate Suc-LLVY-AMC.", + "section": "FIG", + "ner": [ + [ + 13, + 15, + "WT", + "protein_state" + ], + [ + 20, + 26, + "mutant", + "protein_state" + ], + [ + 27, + 38, + "proteasomes", + "complex_assembly" + ], + [ + 89, + 91, + "\u03b25", + "protein" + ], + [ + 113, + 125, + "Suc-LLVY-AMC", + "chemical" + ] + ] + }, + { + "sid": 237, + "sent": "(c) Illustration of the 2FO\u2013FC electron-density map (blue mesh contoured at 1\u03c3) for the \u03b25-T1C propeptide fragment.", + "section": "FIG", + "ner": [ + [ + 24, + 51, + "2FO\u2013FC electron-density map", + "evidence" + ], + [ + 88, + 94, + "\u03b25-T1C", + "mutant" + ], + [ + 95, + 105, + "propeptide", + "structure_element" + ] + ] + }, + { + "sid": 238, + "sent": "The prosegment is cleaved but still bound in the substrate-binding channel.", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "prosegment", + "structure_element" + ], + [ + 18, + 25, + "cleaved", + "protein_state" + ], + [ + 30, + 41, + "still bound", + "protein_state" + ], + [ + 49, + 74, + "substrate-binding channel", + "site" + ] + ] + }, + { + "sid": 239, + "sent": "Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the \u03b25-T1A-K81R mutant.", + "section": "FIG", + "ner": [ + [ + 9, + 16, + "His(-2)", + "residue_name_number" + ], + [ + 37, + 46, + "S1 pocket", + "site" + ], + [ + 57, + 62, + "Met45", + "residue_name_number" + ], + [ + 101, + 112, + "\u03b25-T1A-K81R", + "mutant" + ], + [ + 113, + 119, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 240, + "sent": "(d) Structural superposition of the \u03b25-T1A-K81R and the \u03b25-T1C mutant subunits onto the WT \u03b25 subunit. (e) Structural superposition of the \u03b25-T1C propeptide onto the \u03b21-T1A active site (blue) and the WT \u03b25 active site in complex with the proteasome inhibitor MG132 (ref.).", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "Structural superposition", + "experimental_method" + ], + [ + 36, + 47, + "\u03b25-T1A-K81R", + "mutant" + ], + [ + 56, + 62, + "\u03b25-T1C", + "mutant" + ], + [ + 63, + 69, + "mutant", + "protein_state" + ], + [ + 88, + 90, + "WT", + "protein_state" + ], + [ + 91, + 93, + "\u03b25", + "protein" + ], + [ + 107, + 131, + "Structural superposition", + "experimental_method" + ], + [ + 139, + 145, + "\u03b25-T1C", + "mutant" + ], + [ + 146, + 156, + "propeptide", + "structure_element" + ], + [ + 166, + 172, + "\u03b21-T1A", + "mutant" + ], + [ + 173, + 184, + "active site", + "site" + ], + [ + 200, + 202, + "WT", + "protein_state" + ], + [ + 203, + 205, + "\u03b25", + "protein" + ], + [ + 206, + 217, + "active site", + "site" + ], + [ + 218, + 233, + "in complex with", + "protein_state" + ], + [ + 238, + 248, + "proteasome", + "complex_assembly" + ], + [ + 259, + 264, + "MG132", + "chemical" + ] + ] + }, + { + "sid": 241, + "sent": "The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "inhibitor", + "chemical" + ], + [ + 29, + 40, + "propeptides", + "structure_element" + ], + [ + 76, + 101, + "substrate-binding channel", + "site" + ] + ] + }, + { + "sid": 242, + "sent": "(f) Structural superposition of the WT \u03b25 and \u03b25-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1.", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "Structural superposition", + "experimental_method" + ], + [ + 36, + 38, + "WT", + "protein_state" + ], + [ + 39, + 41, + "\u03b25", + "protein" + ], + [ + 46, + 52, + "\u03b25-T1C", + "mutant" + ], + [ + 53, + 59, + "mutant", + "protein_state" + ], + [ + 60, + 72, + "active sites", + "site" + ], + [ + 137, + 141, + "Thr1", + "residue_name_number" + ], + [ + 170, + 174, + "Cys1", + "residue_name_number" + ] + ] + }, + { + "sid": 243, + "sent": "(g) Structural superposition of the WT \u03b25 and \u03b25-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively.", + "section": "FIG", + "ner": [ + [ + 4, + 28, + "Structural superposition", + "experimental_method" + ], + [ + 36, + 38, + "WT", + "protein_state" + ], + [ + 39, + 41, + "\u03b25", + "protein" + ], + [ + 46, + 52, + "\u03b25-T1S", + "mutant" + ], + [ + 53, + 59, + "mutant", + "protein_state" + ], + [ + 60, + 72, + "active sites", + "site" + ], + [ + 130, + 134, + "Thr1", + "residue_name_number" + ], + [ + 139, + 143, + "Ser1", + "residue_name_number" + ] + ] + }, + { + "sid": 244, + "sent": "The 2FO\u2013FC electron-density map for Ser1 (blue mesh contoured at 1\u03c3) is illustrated.", + "section": "FIG", + "ner": [ + [ + 4, + 31, + "2FO\u2013FC electron-density map", + "evidence" + ], + [ + 36, + 40, + "Ser1", + "residue_name_number" + ] + ] + }, + { + "sid": 245, + "sent": "(h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46C\u03b2 and Thr3C\u03b3.", + "section": "FIG", + "ner": [ + [ + 24, + 28, + "Thr1", + "residue_name_number" + ], + [ + 44, + 68, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 74, + 79, + "Ala46", + "residue_name_number" + ], + [ + 86, + 90, + "Thr3", + "residue_name_number" + ] + ] + }, + { + "sid": 246, + "sent": "Ser1 lacks this stabilization and is therefore rotated by 60\u00b0.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "Ser1", + "residue_name_number" + ], + [ + 5, + 10, + "lacks", + "protein_state" + ] + ] + }, + { + "sid": 247, + "sent": "Inhibition of WT and mutant \u03b25-T1S proteasomes by bortezomib and carfilzomib.", + "section": "FIG", + "ner": [ + [ + 14, + 16, + "WT", + "protein_state" + ], + [ + 21, + 27, + "mutant", + "protein_state" + ], + [ + 28, + 34, + "\u03b25-T1S", + "mutant" + ], + [ + 35, + 46, + "proteasomes", + "complex_assembly" + ], + [ + 50, + 60, + "bortezomib", + "chemical" + ], + [ + 65, + 76, + "carfilzomib", + "chemical" + ] + ] + }, + { + "sid": 248, + "sent": "Inhibition assays (left panel).", + "section": "FIG", + "ner": [ + [ + 0, + 17, + "Inhibition assays", + "experimental_method" + ] + ] + }, + { + "sid": 249, + "sent": "Purified yeast proteasomes were tested for the susceptibility of their ChT-L (\u03b25) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC.", + "section": "FIG", + "ner": [ + [ + 9, + 14, + "yeast", + "taxonomy_domain" + ], + [ + 15, + 26, + "proteasomes", + "complex_assembly" + ], + [ + 78, + 80, + "\u03b25", + "protein" + ], + [ + 108, + 118, + "bortezomib", + "chemical" + ], + [ + 123, + 134, + "carfilzomib", + "chemical" + ], + [ + 155, + 167, + "Suc-LLVY-AMC", + "chemical" + ] + ] + }, + { + "sid": 250, + "sent": "IC50 values were determined in triplicate; s.d.'s are indicated by error bars.", + "section": "FIG", + "ner": [ + [ + 0, + 11, + "IC50 values", + "evidence" + ] + ] + }, + { + "sid": 251, + "sent": "Note that IC50 values depend on time and enzyme concentration.", + "section": "FIG", + "ner": [ + [ + 10, + 21, + "IC50 values", + "evidence" + ] + ] + }, + { + "sid": 252, + "sent": "Proteasomes (final concentration: 66\u2009nM) were incubated with inhibitor for 45\u2009min before substrate addition (final concentration: 200\u2009\u03bcM).", + "section": "FIG", + "ner": [ + [ + 0, + 11, + "Proteasomes", + "complex_assembly" + ] + ] + }, + { + "sid": 253, + "sent": "Structures of the \u03b25-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel).", + "section": "FIG", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 18, + 24, + "\u03b25-T1S", + "mutant" + ], + [ + 25, + 31, + "mutant", + "protein_state" + ], + [ + 35, + 60, + "complex with both ligands", + "complex_assembly" + ], + [ + 93, + 97, + "Ser1", + "residue_name_number" + ] + ] + }, + { + "sid": 254, + "sent": "The 2FO\u2013FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1\u03c3.", + "section": "FIG", + "ner": [ + [ + 4, + 32, + "2FO\u2013FC electron-density maps", + "evidence" + ], + [ + 49, + 53, + "Ser1", + "residue_name_number" + ], + [ + 112, + 119, + "P1 site", + "site" + ], + [ + 121, + 125, + "Leu1", + "residue_name_number" + ] + ] + }, + { + "sid": 255, + "sent": "The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.", + "section": "FIG", + "ner": [ + [ + 4, + 6, + "WT", + "protein_state" + ], + [ + 7, + 35, + "proteasome:inhibitor complex", + "complex_assembly" + ], + [ + 36, + 46, + "structures", + "evidence" + ], + [ + 67, + 71, + "Thr1", + "residue_name_number" + ], + [ + 86, + 98, + "superimposed", + "experimental_method" + ], + [ + 120, + 128, + "mutation", + "experimental_method" + ], + [ + 132, + 136, + "Thr1", + "residue_name_number" + ], + [ + 140, + 143, + "Ser", + "residue_name" + ], + [ + 180, + 190, + "bortezomib", + "chemical" + ], + [ + 194, + 205, + "carfilzomib", + "chemical" + ] + ] + } + ] + }, + "PMC4871749": { + "annotations": [ + { + "sid": 0, + "sent": "The Taf14 YEATS domain is a reader of histone crotonylation", + "section": "TITLE", + "ner": [ + [ + 4, + 9, + "Taf14", + "protein" + ], + [ + 10, + 22, + "YEATS domain", + "structure_element" + ], + [ + 38, + 45, + "histone", + "protein_type" + ], + [ + 46, + 59, + "crotonylation", + "ptm" + ] + ] + }, + { + "sid": 1, + "sent": "The discovery of new histone modifications is unfolding at startling rates, however, the identification of effectors capable of interpreting these modifications has lagged behind.", + "section": "ABSTRACT", + "ner": [ + [ + 21, + 28, + "histone", + "protein_type" + ] + ] + }, + { + "sid": 2, + "sent": "Here we report the YEATS domain as an effective reader of histone lysine crotonylation \u2013 an epigenetic signature associated with active transcription.", + "section": "ABSTRACT", + "ner": [ + [ + 19, + 31, + "YEATS domain", + "structure_element" + ], + [ + 58, + 65, + "histone", + "protein_type" + ], + [ + 66, + 72, + "lysine", + "residue_name" + ], + [ + 73, + 86, + "crotonylation", + "ptm" + ] + ] + }, + { + "sid": 3, + "sent": "We show that the Taf14 YEATS domain engages crotonyllysine via a unique \u03c0-\u03c0-\u03c0-stacking mechanism and that other YEATS domains have crotonyllysine binding activity.", + "section": "ABSTRACT", + "ner": [ + [ + 17, + 22, + "Taf14", + "protein" + ], + [ + 23, + 35, + "YEATS domain", + "structure_element" + ], + [ + 44, + 58, + "crotonyllysine", + "residue_name" + ], + [ + 72, + 86, + "\u03c0-\u03c0-\u03c0-stacking", + "bond_interaction" + ], + [ + 112, + 125, + "YEATS domains", + "structure_element" + ], + [ + 131, + 145, + "crotonyllysine", + "residue_name" + ] + ] + }, + { + "sid": 4, + "sent": "Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin.", + "section": "INTRO", + "ner": [ + [ + 0, + 13, + "Crotonylation", + "ptm" + ], + [ + 17, + 23, + "lysine", + "residue_name" + ], + [ + 34, + 48, + "crotonyllysine", + "residue_name" + ], + [ + 50, + 53, + "Kcr", + "residue_name" + ], + [ + 93, + 100, + "histone", + "protein_type" + ], + [ + 150, + 159, + "mammalian", + "taxonomy_domain" + ] + ] + }, + { + "sid": 5, + "sent": "The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones.", + "section": "INTRO", + "ner": [ + [ + 4, + 18, + "crotonyllysine", + "residue_name" + ], + [ + 27, + 34, + "histone", + "protein_type" + ], + [ + 35, + 37, + "H3", + "protein_type" + ], + [ + 37, + 40, + "K18", + "residue_name_number" + ], + [ + 56, + 60, + "p300", + "protein" + ], + [ + 64, + 89, + "histone acetyltransferase", + "protein_type" + ], + [ + 111, + 122, + "acetylation", + "ptm" + ] + ] + }, + { + "sid": 6, + "sent": "Owing to some differences in their genomic distribution, the crotonyllysine and acetyllysine (Kac) modifications have been linked to distinct functional outcomes.", + "section": "INTRO", + "ner": [ + [ + 61, + 75, + "crotonyllysine", + "residue_name" + ], + [ + 80, + 92, + "acetyllysine", + "residue_name" + ], + [ + 94, + 97, + "Kac", + "residue_name" + ] + ] + }, + { + "sid": 7, + "sent": "p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation.", + "section": "INTRO", + "ner": [ + [ + 0, + 4, + "p300", + "protein" + ], + [ + 15, + 22, + "histone", + "protein_type" + ], + [ + 23, + 36, + "crotonylation", + "ptm" + ], + [ + 129, + 133, + "p300", + "protein" + ], + [ + 144, + 155, + "acetylation", + "ptm" + ] + ] + }, + { + "sid": 8, + "sent": "The discovery of individual biological roles for the crotonyllysine and acetyllysine marks suggests that these PTMs can be read by distinct readers.", + "section": "INTRO", + "ner": [ + [ + 53, + 67, + "crotonyllysine", + "residue_name" + ], + [ + 72, + 84, + "acetyllysine", + "residue_name" + ] + ] + }, + { + "sid": 9, + "sent": "While a number of acetyllysine readers have been identified and characterized, a specific reader of the crotonyllysine mark remains unknown (reviewed in).", + "section": "INTRO", + "ner": [ + [ + 18, + 30, + "acetyllysine", + "residue_name" + ], + [ + 104, + 118, + "crotonyllysine", + "residue_name" + ] + ] + }, + { + "sid": 10, + "sent": "A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity.", + "section": "INTRO", + "ner": [ + [ + 19, + 31, + "bromodomains", + "structure_element" + ], + [ + 33, + 36, + "BDs", + "structure_element" + ], + [ + 65, + 67, + "BD", + "structure_element" + ], + [ + 98, + 110, + "crotonylated", + "protein_state" + ], + [ + 153, + 163, + "acetylated", + "protein_state" + ], + [ + 189, + 201, + "bromodomains", + "structure_element" + ], + [ + 242, + 256, + "crotonyllysine", + "residue_name" + ] + ] + }, + { + "sid": 11, + "sent": "The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac.", + "section": "INTRO", + "ner": [ + [ + 14, + 26, + "acetyllysine", + "residue_name" + ], + [ + 81, + 86, + "YEATS", + "structure_element" + ], + [ + 88, + 92, + "Yaf9", + "protein" + ], + [ + 94, + 97, + "ENL", + "protein" + ], + [ + 99, + 102, + "AF9", + "protein" + ], + [ + 104, + 109, + "Taf14", + "protein" + ], + [ + 111, + 115, + "Sas5", + "protein" + ], + [ + 128, + 133, + "human", + "species" + ], + [ + 134, + 137, + "AF9", + "protein" + ], + [ + 142, + 147, + "yeast", + "taxonomy_domain" + ], + [ + 148, + 153, + "Taf14", + "protein" + ], + [ + 185, + 192, + "histone", + "protein_type" + ], + [ + 198, + 200, + "H3", + "protein_type" + ], + [ + 200, + 204, + "K9ac", + "ptm" + ] + ] + }, + { + "sid": 12, + "sent": "The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription.", + "section": "INTRO", + "ner": [ + [ + 4, + 16, + "acetyllysine", + "residue_name" + ], + [ + 41, + 44, + "AF9", + "protein" + ], + [ + 45, + 57, + "YEATS domain", + "structure_element" + ], + [ + 98, + 123, + "histone methyltransferase", + "protein_type" + ], + [ + 124, + 129, + "DOT1L", + "protein" + ], + [ + 133, + 135, + "H3", + "protein_type" + ], + [ + 135, + 139, + "K9ac", + "ptm" + ], + [ + 169, + 174, + "DOT1L", + "protein" + ], + [ + 184, + 186, + "H3", + "protein_type" + ], + [ + 186, + 189, + "K79", + "residue_name_number" + ], + [ + 190, + 201, + "methylation", + "ptm" + ] + ] + }, + { + "sid": 13, + "sent": "Similarly, activation of a subset of genes and DNA damage repair in yeast require the acetyllysine binding activity of the Taf14 YEATS domain.", + "section": "INTRO", + "ner": [ + [ + 68, + 73, + "yeast", + "taxonomy_domain" + ], + [ + 86, + 98, + "acetyllysine", + "residue_name" + ], + [ + 123, + 128, + "Taf14", + "protein" + ], + [ + 129, + 141, + "YEATS domain", + "structure_element" + ] + ] + }, + { + "sid": 14, + "sent": "Consistent with its role in gene regulation, Taf14 was identified as a core component of the transcription factor complexes TFIID and TFIIF.", + "section": "INTRO", + "ner": [ + [ + 45, + 50, + "Taf14", + "protein" + ], + [ + 124, + 129, + "TFIID", + "complex_assembly" + ], + [ + 134, + 139, + "TFIIF", + "complex_assembly" + ] + ] + }, + { + "sid": 15, + "sent": "However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology.", + "section": "INTRO", + "ner": [ + [ + 9, + 14, + "Taf14", + "protein" + ], + [ + 82, + 87, + "INO80", + "complex_assembly" + ], + [ + 89, + 96, + "SWI/SNF", + "complex_assembly" + ], + [ + 101, + 104, + "RSC", + "complex_assembly" + ], + [ + 114, + 139, + "histone acetyltransferase", + "protein_type" + ], + [ + 148, + 152, + "NuA3", + "complex_assembly" + ], + [ + 188, + 193, + "Taf14", + "protein" + ] + ] + }, + { + "sid": 16, + "sent": "In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism.", + "section": "INTRO", + "ner": [ + [ + 33, + 38, + "Taf14", + "protein" + ], + [ + 39, + 51, + "YEATS domain", + "structure_element" + ], + [ + 67, + 81, + "crotonyllysine", + "residue_name" + ], + [ + 96, + 103, + "histone", + "protein_type" + ], + [ + 104, + 106, + "H3", + "protein_type" + ], + [ + 107, + 119, + "crotonylated", + "protein_state" + ], + [ + 123, + 131, + "lysine 9", + "residue_name_number" + ], + [ + 133, + 135, + "H3", + "protein_type" + ], + [ + 135, + 139, + "K9cr", + "ptm" + ] + ] + }, + { + "sid": 17, + "sent": "We found that H3K9cr is present in yeast and is dynamically regulated.", + "section": "INTRO", + "ner": [ + [ + 14, + 16, + "H3", + "protein_type" + ], + [ + 16, + 20, + "K9cr", + "ptm" + ], + [ + 35, + 40, + "yeast", + "taxonomy_domain" + ] + ] + }, + { + "sid": 18, + "sent": "To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5\u201313 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1).", + "section": "INTRO", + "ner": [ + [ + 56, + 58, + "H3", + "protein_type" + ], + [ + 58, + 62, + "K9cr", + "ptm" + ], + [ + 83, + 100, + "crystal structure", + "evidence" + ], + [ + 108, + 113, + "Taf14", + "protein" + ], + [ + 114, + 126, + "YEATS domain", + "structure_element" + ], + [ + 127, + 142, + "in complex with", + "protein_state" + ], + [ + 143, + 153, + "H3K9cr5-13", + "chemical" + ], + [ + 164, + 168, + "5\u201313", + "residue_range" + ], + [ + 172, + 174, + "H3", + "protein_type" + ] + ] + }, + { + "sid": 19, + "sent": "The Taf14 YEATS domain adopts an immunoglobin-like \u03b2 sandwich fold containing eight anti-parallel \u03b2 strands linked by short loops that form a binding site for H3K9cr (Fig. 1b).", + "section": "INTRO", + "ner": [ + [ + 4, + 9, + "Taf14", + "protein" + ], + [ + 10, + 22, + "YEATS domain", + "structure_element" + ], + [ + 33, + 66, + "immunoglobin-like \u03b2 sandwich fold", + "structure_element" + ], + [ + 84, + 107, + "anti-parallel \u03b2 strands", + "structure_element" + ], + [ + 124, + 129, + "loops", + "structure_element" + ], + [ + 142, + 154, + "binding site", + "site" + ], + [ + 159, + 161, + "H3", + "protein_type" + ], + [ + 161, + 165, + "K9cr", + "ptm" + ] + ] + }, + { + "sid": 20, + "sent": "The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the \u03b2 strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c).", + "section": "INTRO", + "ner": [ + [ + 4, + 6, + "H3", + "protein_type" + ], + [ + 6, + 10, + "K9cr", + "ptm" + ], + [ + 30, + 51, + "extended conformation", + "protein_state" + ], + [ + 88, + 97, + "\u03b2 strands", + "structure_element" + ], + [ + 159, + 164, + "water", + "chemical" + ], + [ + 174, + 188, + "hydrogen bonds", + "bond_interaction" + ], + [ + 195, + 206, + "salt bridge", + "bond_interaction" + ] + ] + }, + { + "sid": 21, + "sent": "The most striking feature of the crotonyllysine recognition mechanism is the unique coordination of crotonylated lysine residue.", + "section": "INTRO", + "ner": [ + [ + 33, + 47, + "crotonyllysine", + "residue_name" + ], + [ + 100, + 112, + "crotonylated", + "protein_state" + ], + [ + 113, + 119, + "lysine", + "residue_name" + ] + ] + }, + { + "sid": 22, + "sent": "The fully extended side chain of K9cr transverses the narrow tunnel, crossing the \u03b2 sandwich at right angle in a corkscrew-like manner (Fig. 1b and Supplementary Figure 1b).", + "section": "INTRO", + "ner": [ + [ + 33, + 37, + "K9cr", + "ptm" + ], + [ + 82, + 92, + "\u03b2 sandwich", + "structure_element" + ] + ] + }, + { + "sid": 23, + "sent": "The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic \u03c0-\u03c0-\u03c0-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c).", + "section": "INTRO", + "ner": [ + [ + 11, + 19, + "crotonyl", + "chemical" + ], + [ + 46, + 51, + "Trp81", + "residue_name_number" + ], + [ + 56, + 61, + "Phe62", + "residue_name_number" + ], + [ + 184, + 192, + "crotonyl", + "chemical" + ], + [ + 251, + 265, + "\u03c0-\u03c0-\u03c0-stacking", + "bond_interaction" + ] + ] + }, + { + "sid": 24, + "sent": "The side chain of Trp81 appears to adopt two conformations, one of which provides maximum \u03c0-stacking with the alkene functional group while the other rotamer affords maximum \u03c0-stacking with the amide \u03c0 electrons (Supplementary Fig. 1c).", + "section": "INTRO", + "ner": [ + [ + 18, + 23, + "Trp81", + "residue_name_number" + ], + [ + 90, + 100, + "\u03c0-stacking", + "bond_interaction" + ], + [ + 174, + 184, + "\u03c0-stacking", + "bond_interaction" + ] + ] + }, + { + "sid": 25, + "sent": "The dual conformation of Trp81 is likely due to the conjugated nature of the C=C and C=O \u03c0-orbitals within the crotonyl functional group.", + "section": "INTRO", + "ner": [ + [ + 25, + 30, + "Trp81", + "residue_name_number" + ], + [ + 111, + 119, + "crotonyl", + "chemical" + ] + ] + }, + { + "sid": 26, + "sent": "In addition to \u03c0-\u03c0-\u03c0 stacking, the crotonyl group is stabilized by a set of hydrogen bonds and electrostatic interactions.", + "section": "INTRO", + "ner": [ + [ + 15, + 29, + "\u03c0-\u03c0-\u03c0 stacking", + "bond_interaction" + ], + [ + 35, + 43, + "crotonyl", + "chemical" + ], + [ + 76, + 90, + "hydrogen bonds", + "bond_interaction" + ], + [ + 95, + 121, + "electrostatic interactions", + "bond_interaction" + ] + ] + }, + { + "sid": 27, + "sent": "The \u03c0 bond conjugation of the crotonyl group gives rise to a dipole moment of the alkene moiety, resulting in a partial positive charge on the \u03b2-carbon (C\u03b2) and a partial negative charge on the \u03b1-carbon (C\u03b1).", + "section": "INTRO", + "ner": [ + [ + 4, + 10, + "\u03c0 bond", + "bond_interaction" + ], + [ + 30, + 38, + "crotonyl", + "chemical" + ] + ] + }, + { + "sid": 28, + "sent": "This provides the capability for the alkene moiety to form electrostatic contacts, as C\u03b1 and C\u03b2 lay within electrostatic interaction distances of the carbonyl oxygen of Gln79 and of the hydroxyl group of Thr61, respectively.", + "section": "INTRO", + "ner": [ + [ + 59, + 81, + "electrostatic contacts", + "bond_interaction" + ], + [ + 107, + 132, + "electrostatic interaction", + "bond_interaction" + ], + [ + 169, + 174, + "Gln79", + "residue_name_number" + ], + [ + 204, + 209, + "Thr61", + "residue_name_number" + ] + ] + }, + { + "sid": 29, + "sent": "The hydroxyl group of Thr61 also participates in a hydrogen bond with the amide nitrogen of the K9cr side chain (Fig. 1d).", + "section": "INTRO", + "ner": [ + [ + 22, + 27, + "Thr61", + "residue_name_number" + ], + [ + 51, + 64, + "hydrogen bond", + "bond_interaction" + ], + [ + 96, + 100, + "K9cr", + "ptm" + ] + ] + }, + { + "sid": 30, + "sent": "The fixed position of the Thr61 hydroxyl group, which facilitates interactions with both the amide and C\u03b1 of K9cr, is achieved through a hydrogen bond with imidazole ring of His59.", + "section": "INTRO", + "ner": [ + [ + 26, + 31, + "Thr61", + "residue_name_number" + ], + [ + 109, + 113, + "K9cr", + "ptm" + ], + [ + 137, + 150, + "hydrogen bond", + "bond_interaction" + ], + [ + 174, + 179, + "His59", + "residue_name_number" + ] + ] + }, + { + "sid": 31, + "sent": "Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d).", + "section": "INTRO", + "ner": [ + [ + 23, + 27, + "K9cr", + "ptm" + ], + [ + 45, + 58, + "hydrogen bond", + "bond_interaction" + ], + [ + 123, + 128, + "Trp81", + "residue_name_number" + ], + [ + 143, + 148, + "water", + "chemical" + ], + [ + 158, + 171, + "hydrogen bond", + "bond_interaction" + ], + [ + 208, + 213, + "Gly82", + "residue_name_number" + ] + ] + }, + { + "sid": 32, + "sent": "This distinctive mechanism was corroborated through mapping the Taf14 YEATS-H3K9cr binding interface in solution using NMR chemical shift perturbation analysis (Supplementary Fig. 2a, b).", + "section": "INTRO", + "ner": [ + [ + 64, + 69, + "Taf14", + "protein" + ], + [ + 70, + 100, + "YEATS-H3K9cr binding interface", + "site" + ], + [ + 119, + 159, + "NMR chemical shift perturbation analysis", + "experimental_method" + ] + ] + }, + { + "sid": 33, + "sent": "Binding of the Taf14 YEATS domain to H3K9cr is robust.", + "section": "INTRO", + "ner": [ + [ + 15, + 20, + "Taf14", + "protein" + ], + [ + 21, + 33, + "YEATS domain", + "structure_element" + ], + [ + 37, + 39, + "H3", + "protein_type" + ], + [ + 39, + 43, + "K9cr", + "ptm" + ] + ] + }, + { + "sid": 34, + "sent": "The dissociation constant (Kd) for the Taf14 YEATS-H3K9cr5-13 complex was found to be 9.5 \u03bcM, as measured by fluorescence spectroscopy (Supplementary Fig. 2c).", + "section": "INTRO", + "ner": [ + [ + 4, + 25, + "dissociation constant", + "evidence" + ], + [ + 27, + 29, + "Kd", + "evidence" + ], + [ + 39, + 61, + "Taf14 YEATS-H3K9cr5-13", + "complex_assembly" + ], + [ + 109, + 134, + "fluorescence spectroscopy", + "experimental_method" + ] + ] + }, + { + "sid": 35, + "sent": "This value is in the range of binding affinities exhibited by the majority of histone readers, thus attesting to the physiological relevance of the H3K9cr recognition by Taf14.", + "section": "INTRO", + "ner": [ + [ + 30, + 48, + "binding affinities", + "evidence" + ], + [ + 148, + 150, + "H3", + "protein_type" + ], + [ + 150, + 154, + "K9cr", + "ptm" + ], + [ + 170, + 175, + "Taf14", + "protein" + ] + ] + }, + { + "sid": 36, + "sent": "To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2).", + "section": "INTRO", + "ner": [ + [ + 21, + 23, + "H3", + "protein_type" + ], + [ + 23, + 27, + "K9cr", + "ptm" + ], + [ + 42, + 47, + "yeast", + "taxonomy_domain" + ], + [ + 62, + 81, + "whole cell extracts", + "experimental_method" + ], + [ + 111, + 116, + "yeast", + "taxonomy_domain" + ], + [ + 145, + 166, + "Western blot analysis", + "experimental_method" + ], + [ + 201, + 203, + "H3", + "protein_type" + ], + [ + 203, + 207, + "K9cr", + "ptm" + ], + [ + 209, + 211, + "H3", + "protein_type" + ], + [ + 211, + 215, + "K9ac", + "ptm" + ], + [ + 220, + 222, + "H3", + "protein_type" + ] + ] + }, + { + "sid": 37, + "sent": "Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast.", + "section": "INTRO", + "ner": [ + [ + 5, + 7, + "H3", + "protein_type" + ], + [ + 7, + 11, + "K9cr", + "ptm" + ], + [ + 16, + 18, + "H3", + "protein_type" + ], + [ + 18, + 22, + "K9ac", + "ptm" + ], + [ + 40, + 45, + "yeast", + "taxonomy_domain" + ], + [ + 46, + 54, + "histones", + "protein_type" + ], + [ + 102, + 104, + "H3", + "protein_type" + ], + [ + 104, + 108, + "K9cr", + "ptm" + ], + [ + 122, + 127, + "yeast", + "taxonomy_domain" + ] + ] + }, + { + "sid": 38, + "sent": "We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs).", + "section": "INTRO", + "ner": [ + [ + 17, + 19, + "H3", + "protein_type" + ], + [ + 19, + 23, + "K9cr", + "ptm" + ], + [ + 55, + 81, + "histone acetyltransferases", + "protein_type" + ], + [ + 83, + 87, + "HATs", + "protein_type" + ], + [ + 93, + 113, + "histone deacetylases", + "protein_type" + ], + [ + 115, + 120, + "HDACs", + "protein_type" + ] + ] + }, + { + "sid": 39, + "sent": "Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted.", + "section": "INTRO", + "ner": [ + [ + 50, + 55, + "yeast", + "taxonomy_domain" + ], + [ + 77, + 82, + "yeast", + "taxonomy_domain" + ], + [ + 83, + 87, + "HATs", + "protein_type" + ], + [ + 89, + 93, + "HAT1", + "protein" + ], + [ + 95, + 99, + "Gcn5", + "protein" + ], + [ + 105, + 111, + "Rtt109", + "protein" + ], + [ + 116, + 121, + "HDACs", + "protein_type" + ], + [ + 123, + 127, + "Rpd3", + "protein" + ], + [ + 129, + 133, + "Hos1", + "protein" + ], + [ + 139, + 143, + "Hos2", + "protein" + ], + [ + 150, + 157, + "deleted", + "experimental_method" + ] + ] + }, + { + "sid": 40, + "sent": "As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains.", + "section": "INTRO", + "ner": [ + [ + 52, + 54, + "H3", + "protein_type" + ], + [ + 54, + 58, + "K9cr", + "ptm" + ], + [ + 112, + 115, + "HAT", + "protein_type" + ], + [ + 116, + 124, + "deletion", + "experimental_method" + ], + [ + 182, + 186, + "HDAC", + "protein_type" + ], + [ + 187, + 195, + "deletion", + "experimental_method" + ] + ] + }, + { + "sid": 41, + "sent": "Furthermore, fluctuations in the H3K9cr levels were more substantial than fluctuations in the corresponding H3K9ac levels.", + "section": "INTRO", + "ner": [ + [ + 33, + 35, + "H3", + "protein_type" + ], + [ + 35, + 39, + "K9cr", + "ptm" + ], + [ + 108, + 110, + "H3", + "protein_type" + ], + [ + 110, + 114, + "K9ac", + "ptm" + ] + ] + }, + { + "sid": 42, + "sent": "Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs.", + "section": "INTRO", + "ner": [ + [ + 36, + 38, + "H3", + "protein_type" + ], + [ + 38, + 42, + "K9cr", + "ptm" + ], + [ + 77, + 82, + "yeast", + "taxonomy_domain" + ], + [ + 174, + 178, + "HATs", + "protein_type" + ], + [ + 183, + 188, + "HDACs", + "protein_type" + ] + ] + }, + { + "sid": 43, + "sent": "We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter.", + "section": "INTRO", + "ner": [ + [ + 36, + 46, + "acetylated", + "protein_state" + ], + [ + 47, + 54, + "histone", + "protein_type" + ], + [ + 66, + 71, + "Taf14", + "protein" + ], + [ + 72, + 84, + "YEATS domain", + "structure_element" + ], + [ + 93, + 103, + "acetylated", + "protein_state" + ], + [ + 104, + 106, + "H3", + "protein_type" + ], + [ + 106, + 108, + "K9", + "residue_name_number" + ], + [ + 163, + 165, + "H3", + "protein_type" + ], + [ + 165, + 169, + "K9cr", + "ptm" + ] + ] + }, + { + "sid": 44, + "sent": "The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b).", + "section": "INTRO", + "ner": [ + [ + 19, + 24, + "Taf14", + "protein" + ], + [ + 33, + 47, + "crotonyllysine", + "residue_name" + ], + [ + 69, + 80, + "1H,15N HSQC", + "experimental_method" + ], + [ + 110, + 120, + "H3K9cr5-13", + "chemical" + ], + [ + 124, + 134, + "H3K9ac5-13", + "chemical" + ], + [ + 147, + 155, + "titrated", + "experimental_method" + ], + [ + 165, + 176, + "15N-labeled", + "protein_state" + ], + [ + 177, + 182, + "Taf14", + "protein" + ], + [ + 183, + 195, + "YEATS domain", + "structure_element" + ] + ] + }, + { + "sid": 45, + "sent": "Binding of H3K9cr induced resonance changes in slow exchange regime on the NMR time scale, indicative of strong interaction.", + "section": "INTRO", + "ner": [ + [ + 11, + 13, + "H3", + "protein_type" + ], + [ + 13, + 17, + "K9cr", + "ptm" + ], + [ + 26, + 43, + "resonance changes", + "evidence" + ], + [ + 75, + 78, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 46, + "sent": "In contrast, binding of H3K9ac resulted in an intermediate exchange, which is characteristic of a weaker association.", + "section": "INTRO", + "ner": [ + [ + 24, + 26, + "H3", + "protein_type" + ], + [ + 26, + 30, + "K9ac", + "ptm" + ] + ] + }, + { + "sid": 47, + "sent": "Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a).", + "section": "INTRO", + "ner": [ + [ + 13, + 23, + "crosspeaks", + "evidence" + ], + [ + 27, + 32, + "Gly80", + "residue_name_number" + ], + [ + 37, + 42, + "Trp81", + "residue_name_number" + ], + [ + 50, + 62, + "YEATS domain", + "structure_element" + ], + [ + 90, + 92, + "H3", + "protein_type" + ], + [ + 92, + 96, + "K9cr", + "ptm" + ], + [ + 101, + 103, + "H3", + "protein_type" + ], + [ + 103, + 107, + "K9ac", + "ptm" + ], + [ + 171, + 218, + "crotonyllysine and acetyllysine binding pockets", + "site" + ] + ] + }, + { + "sid": 48, + "sent": "These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c).", + "section": "INTRO", + "ner": [ + [ + 41, + 46, + "Trp81", + "residue_name_number" + ], + [ + 104, + 106, + "H3", + "protein_type" + ], + [ + 106, + 110, + "K9cr", + "ptm" + ], + [ + 131, + 133, + "H3", + "protein_type" + ], + [ + 133, + 137, + "K9ac", + "ptm" + ] + ] + }, + { + "sid": 49, + "sent": "One of the conformations, characterized by the \u03c0 stacking involving two aromatic residues and the alkene group, is observed only in the YEATS-H3K9cr complex.", + "section": "INTRO", + "ner": [ + [ + 136, + 148, + "YEATS-H3K9cr", + "complex_assembly" + ] + ] + }, + { + "sid": 50, + "sent": "To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1\u201320 of H3).", + "section": "INTRO", + "ner": [ + [ + 25, + 30, + "Taf14", + "protein" + ], + [ + 31, + 43, + "YEATS domain", + "structure_element" + ], + [ + 91, + 101, + "acyllysine", + "residue_name" + ], + [ + 122, + 147, + "solution pull-down assays", + "experimental_method" + ], + [ + 154, + 156, + "H3", + "protein_type" + ], + [ + 166, + 176, + "acetylated", + "protein_state" + ], + [ + 178, + 191, + "propionylated", + "protein_state" + ], + [ + 193, + 204, + "butyrylated", + "protein_state" + ], + [ + 210, + 222, + "crotonylated", + "protein_state" + ], + [ + 226, + 234, + "lysine 9", + "residue_name_number" + ], + [ + 245, + 249, + "1\u201320", + "residue_range" + ], + [ + 253, + 255, + "H3", + "protein_type" + ] + ] + }, + { + "sid": 51, + "sent": "As shown in Figure 2d and Supplementary Fig. 5a, the Taf14 YEATS domain binds more strongly to H3K9cr1-20, as compared to other acylated histone peptides.", + "section": "INTRO", + "ner": [ + [ + 53, + 58, + "Taf14", + "protein" + ], + [ + 59, + 71, + "YEATS domain", + "structure_element" + ], + [ + 95, + 105, + "H3K9cr1-20", + "chemical" + ], + [ + 128, + 136, + "acylated", + "protein_state" + ] + ] + }, + { + "sid": 52, + "sent": "The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments.", + "section": "INTRO", + "ner": [ + [ + 19, + 21, + "H3", + "protein_type" + ], + [ + 21, + 25, + "K9cr", + "ptm" + ], + [ + 31, + 33, + "H3", + "protein_type" + ], + [ + 33, + 37, + "K9ac", + "ptm" + ], + [ + 39, + 41, + "H3", + "protein_type" + ], + [ + 41, + 45, + "K9pr", + "ptm" + ], + [ + 50, + 52, + "H3", + "protein_type" + ], + [ + 52, + 56, + "K9bu", + "ptm" + ], + [ + 74, + 107, + "1H,15N HSQC titration experiments", + "experimental_method" + ] + ] + }, + { + "sid": 53, + "sent": "Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b).", + "section": "INTRO", + "ner": [ + [ + 12, + 22, + "H3K9ac1-20", + "chemical" + ], + [ + 24, + 34, + "H3K9pr1-20", + "chemical" + ], + [ + 40, + 50, + "H3K9bu1-20", + "chemical" + ], + [ + 67, + 95, + "chemical shift perturbations", + "evidence" + ], + [ + 103, + 108, + "Taf14", + "protein" + ], + [ + 109, + 121, + "YEATS domain", + "structure_element" + ], + [ + 236, + 246, + "H3K9cr1-20", + "chemical" + ] + ] + }, + { + "sid": 54, + "sent": "We concluded that H3K9cr is the preferred target of this domain.", + "section": "INTRO", + "ner": [ + [ + 18, + 20, + "H3", + "protein_type" + ], + [ + 20, + 24, + "K9cr", + "ptm" + ] + ] + }, + { + "sid": 55, + "sent": "From comparative structural analysis of the YEATS complexes, Gly80 emerged as candidate residue potentially responsible for the preference for crotonyllysine.", + "section": "INTRO", + "ner": [ + [ + 5, + 36, + "comparative structural analysis", + "experimental_method" + ], + [ + 61, + 66, + "Gly80", + "residue_name_number" + ], + [ + 143, + 157, + "crotonyllysine", + "residue_name" + ] + ] + }, + { + "sid": 56, + "sent": "In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction.", + "section": "INTRO", + "ner": [ + [ + 123, + 131, + "crotonyl", + "chemical" + ], + [ + 143, + 150, + "mutated", + "protein_state" + ], + [ + 151, + 156, + "Gly80", + "residue_name_number" + ], + [ + 193, + 203, + "mutants of", + "protein_state" + ], + [ + 204, + 209, + "Gly80", + "residue_name_number" + ], + [ + 255, + 263, + "acylated", + "protein_state" + ], + [ + 289, + 294, + "Gly80", + "residue_name_number" + ] + ] + }, + { + "sid": 57, + "sent": "In contrast, mutation of Val24, a residue located on another side of Trp81, had no effect on binding (Fig. 2d and Supplementary Fig. 5a, c).", + "section": "INTRO", + "ner": [ + [ + 13, + 21, + "mutation", + "experimental_method" + ], + [ + 25, + 30, + "Val24", + "residue_name_number" + ], + [ + 69, + 74, + "Trp81", + "residue_name_number" + ] + ] + }, + { + "sid": 58, + "sent": "To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6).", + "section": "INTRO", + "ner": [ + [ + 31, + 45, + "crotonyllysine", + "residue_name" + ], + [ + 49, + 58, + "conserved", + "protein_state" + ], + [ + 70, + 75, + "human", + "species" + ], + [ + 76, + 89, + "YEATS domains", + "structure_element" + ], + [ + 93, + 114, + "pull-down experiments", + "experimental_method" + ], + [ + 141, + 151, + "acetylated", + "protein_state" + ], + [ + 153, + 166, + "propionylated", + "protein_state" + ], + [ + 168, + 179, + "butyrylated", + "protein_state" + ], + [ + 185, + 197, + "crotonylated", + "protein_state" + ], + [ + 198, + 205, + "histone", + "protein_type" + ] + ] + }, + { + "sid": 59, + "sent": "We found that all YEATS domains tested are capable of binding to crotonyllysine peptides, though they display variable preferences for the acyl moieties.", + "section": "INTRO", + "ner": [ + [ + 18, + 31, + "YEATS domains", + "structure_element" + ], + [ + 65, + 79, + "crotonyllysine", + "residue_name" + ] + ] + }, + { + "sid": 60, + "sent": "While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well.", + "section": "INTRO", + "ner": [ + [ + 6, + 12, + "YEATS2", + "protein" + ], + [ + 17, + 20, + "ENL", + "protein" + ], + [ + 48, + 60, + "crotonylated", + "protein_state" + ], + [ + 71, + 76, + "GAS41", + "protein" + ], + [ + 81, + 84, + "AF9", + "protein" + ], + [ + 91, + 99, + "acylated", + "protein_state" + ] + ] + }, + { + "sid": 61, + "sent": "Unlike the YEATS domain, a known acetyllysine reader, bromodomain, does not recognize crotonyllysine.", + "section": "INTRO", + "ner": [ + [ + 11, + 23, + "YEATS domain", + "structure_element" + ], + [ + 33, + 52, + "acetyllysine reader", + "protein_type" + ], + [ + 54, + 65, + "bromodomain", + "structure_element" + ], + [ + 86, + 100, + "crotonyllysine", + "residue_name" + ] + ] + }, + { + "sid": 62, + "sent": "We assayed a large set of BDs in pull-down experiments and found that this module is highly specific for acetyllysine and propionyllysine containing peptides (Supplementary Fig. 7).", + "section": "INTRO", + "ner": [ + [ + 26, + 29, + "BDs", + "structure_element" + ], + [ + 33, + 54, + "pull-down experiments", + "experimental_method" + ], + [ + 105, + 117, + "acetyllysine", + "residue_name" + ], + [ + 122, + 137, + "propionyllysine", + "residue_name" + ] + ] + }, + { + "sid": 63, + "sent": "However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports.", + "section": "INTRO", + "ner": [ + [ + 9, + 21, + "bromodomains", + "structure_element" + ], + [ + 109, + 123, + "crotonyllysine", + "residue_name" + ], + [ + 143, + 146, + "BDs", + "structure_element" + ], + [ + 150, + 154, + "TAF1", + "protein" + ], + [ + 159, + 163, + "BRD2", + "protein" + ] + ] + }, + { + "sid": 64, + "sent": "These results demonstrate that the YEATS domain is currently the sole reader of crotonyllysine.", + "section": "INTRO", + "ner": [ + [ + 35, + 47, + "YEATS domain", + "structure_element" + ], + [ + 80, + 94, + "crotonyllysine", + "residue_name" + ] + ] + }, + { + "sid": 65, + "sent": "In conclusion, we have identified the YEATS domain of Taf14 as the first reader of histone crotonylation.", + "section": "INTRO", + "ner": [ + [ + 38, + 50, + "YEATS domain", + "structure_element" + ], + [ + 54, + 59, + "Taf14", + "protein" + ], + [ + 83, + 90, + "histone", + "protein_type" + ], + [ + 91, + 104, + "crotonylation", + "ptm" + ] + ] + }, + { + "sid": 66, + "sent": "The unique and previously unobserved aromatic-amide/aliphatic-aromatic \u03c0-\u03c0-\u03c0-stacking mechanism facilitates the specific recognition of the crotonyl moiety.", + "section": "INTRO", + "ner": [ + [ + 71, + 85, + "\u03c0-\u03c0-\u03c0-stacking", + "bond_interaction" + ], + [ + 140, + 148, + "crotonyl", + "chemical" + ] + ] + }, + { + "sid": 67, + "sent": "We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs.", + "section": "INTRO", + "ner": [ + [ + 28, + 30, + "H3", + "protein_type" + ], + [ + 30, + 34, + "K9cr", + "ptm" + ], + [ + 45, + 50, + "yeast", + "taxonomy_domain" + ], + [ + 83, + 87, + "HATs", + "protein_type" + ], + [ + 92, + 97, + "HDACs", + "protein_type" + ] + ] + }, + { + "sid": 68, + "sent": "As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications.", + "section": "INTRO", + "ner": [ + [ + 42, + 52, + "acyllysine", + "residue_name" + ], + [ + 68, + 73, + "Taf14", + "protein" + ], + [ + 74, + 86, + "YEATS domain", + "structure_element" + ], + [ + 210, + 224, + "crotonyllysine", + "residue_name" + ], + [ + 276, + 281, + "Taf14", + "protein" + ], + [ + 309, + 323, + "crotonyllysine", + "residue_name" + ], + [ + 328, + 340, + "acetyllysine", + "residue_name" + ] + ] + }, + { + "sid": 69, + "sent": "Furthermore, the functional significance of crotonyllysine recognition by other YEATS proteins will be of great importance to elucidate and compare.", + "section": "INTRO", + "ner": [ + [ + 44, + 58, + "crotonyllysine", + "residue_name" + ], + [ + 80, + 85, + "YEATS", + "protein_type" + ] + ] + }, + { + "sid": 70, + "sent": "The structural mechanism for the recognition of H3K9cr", + "section": "FIG", + "ner": [ + [ + 48, + 50, + "H3", + "protein_type" + ], + [ + 50, + 54, + "K9cr", + "ptm" + ] + ] + }, + { + "sid": 71, + "sent": "(a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain.", + "section": "FIG", + "ner": [ + [ + 26, + 40, + "crotonyllysine", + "residue_name" + ], + [ + 50, + 67, + "crystal structure", + "evidence" + ], + [ + 75, + 80, + "Taf14", + "protein" + ], + [ + 81, + 93, + "YEATS domain", + "structure_element" + ], + [ + 102, + 117, + "in complex with", + "protein_state" + ], + [ + 122, + 132, + "H3K9cr5-13", + "chemical" + ], + [ + 154, + 156, + "H3", + "protein_type" + ], + [ + 156, + 160, + "K9cr", + "ptm" + ], + [ + 218, + 254, + "electrostatic and polar interactions", + "bond_interaction" + ], + [ + 264, + 269, + "Taf14", + "protein" + ], + [ + 270, + 282, + "YEATS domain", + "structure_element" + ] + ] + }, + { + "sid": 72, + "sent": "(d) The \u03c0-\u03c0-\u03c0 stacking mechanism involving the alkene moiety of crotonyllysine.", + "section": "FIG", + "ner": [ + [ + 8, + 22, + "\u03c0-\u03c0-\u03c0 stacking", + "bond_interaction" + ], + [ + 64, + 78, + "crotonyllysine", + "residue_name" + ] + ] + }, + { + "sid": 73, + "sent": "H3K9cr is a selective target of the Taf14 YEATS domain", + "section": "FIG", + "ner": [ + [ + 0, + 2, + "H3", + "protein_type" + ], + [ + 2, + 6, + "K9cr", + "ptm" + ], + [ + 36, + 41, + "Taf14", + "protein" + ], + [ + 42, + 54, + "YEATS domain", + "structure_element" + ] + ] + }, + { + "sid": 74, + "sent": "(a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains.", + "section": "FIG", + "ner": [ + [ + 7, + 19, + "Western blot", + "experimental_method" + ], + [ + 53, + 55, + "H3", + "protein_type" + ], + [ + 55, + 59, + "K9cr", + "ptm" + ], + [ + 64, + 66, + "H3", + "protein_type" + ], + [ + 66, + 70, + "K9ac", + "ptm" + ], + [ + 74, + 83, + "wild type", + "protein_state" + ], + [ + 85, + 87, + "WT", + "protein_state" + ], + [ + 90, + 93, + "HAT", + "protein_type" + ], + [ + 107, + 111, + "HDAC", + "protein_type" + ], + [ + 112, + 120, + "deletion", + "experimental_method" + ], + [ + 121, + 126, + "yeast", + "taxonomy_domain" + ] + ] + }, + { + "sid": 75, + "sent": "Total H3 was used as a loading control.", + "section": "FIG", + "ner": [ + [ + 6, + 8, + "H3", + "protein_type" + ] + ] + }, + { + "sid": 76, + "sent": "(c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in.", + "section": "FIG", + "ner": [ + [ + 17, + 28, + "1H,15N HSQC", + "experimental_method" + ], + [ + 29, + 36, + "spectra", + "evidence" + ], + [ + 40, + 45, + "Taf14", + "protein" + ], + [ + 46, + 51, + "YEATS", + "structure_element" + ], + [ + 64, + 74, + "H3K9cr5-13", + "chemical" + ], + [ + 79, + 89, + "H3K9ac5-13", + "chemical" + ], + [ + 104, + 112, + "titrated", + "experimental_method" + ] + ] + }, + { + "sid": 77, + "sent": "Spectra are color coded according to the protein:peptide molar ratio.", + "section": "FIG", + "ner": [ + [ + 0, + 7, + "Spectra", + "evidence" + ] + ] + }, + { + "sid": 78, + "sent": "(d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides.", + "section": "FIG", + "ner": [ + [ + 4, + 16, + "Western blot", + "experimental_method" + ], + [ + 29, + 53, + "peptide pull-down assays", + "experimental_method" + ], + [ + 60, + 69, + "wild-type", + "protein_state" + ], + [ + 74, + 81, + "mutated", + "protein_state" + ], + [ + 82, + 87, + "Taf14", + "protein" + ], + [ + 88, + 101, + "YEATS domains", + "structure_element" + ] + ] + } + ] + }, + "PMC4781976": { + "annotations": [ + { + "sid": 0, + "sent": "Structure of the GAT domain of the endosomal adapter protein Tom1", + "section": "TITLE", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 17, + 20, + "GAT", + "structure_element" + ], + [ + 45, + 60, + "adapter protein", + "protein_type" + ], + [ + 61, + 65, + "Tom1", + "protein" + ] + ] + }, + { + "sid": 1, + "sent": "Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments.", + "section": "ABSTRACT", + "ner": [ + [ + 50, + 71, + "cell-surface receptor", + "protein_type" + ] + ] + }, + { + "sid": 2, + "sent": "The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 20, + "adapter proteins", + "protein_type" + ], + [ + 21, + 25, + "Tom1", + "protein" + ], + [ + 30, + 36, + "Tollip", + "protein" + ], + [ + 64, + 77, + "ubiquitinated", + "ptm" + ] + ] + }, + { + "sid": 3, + "sent": "Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain\u2019s association to Tollip\u2019s Tom1-binding domain (TBD).", + "section": "ABSTRACT", + "ner": [ + [ + 15, + 19, + "Tom1", + "protein" + ], + [ + 69, + 72, + "GAT", + "structure_element" + ], + [ + 97, + 103, + "Tollip", + "protein" + ], + [ + 106, + 125, + "Tom1-binding domain", + "structure_element" + ], + [ + 127, + 130, + "TBD", + "structure_element" + ] + ] + }, + { + "sid": 4, + "sent": "In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain.", + "section": "ABSTRACT", + "ner": [ + [ + 36, + 48, + "solution NMR", + "experimental_method" + ], + [ + 57, + 66, + "structure", + "evidence" + ], + [ + 74, + 78, + "Tom1", + "protein" + ], + [ + 79, + 82, + "GAT", + "structure_element" + ] + ] + }, + { + "sid": 5, + "sent": "The estimated protein structure exhibits a bundle of three helical elements.", + "section": "ABSTRACT", + "ner": [ + [ + 22, + 31, + "structure", + "evidence" + ] + ] + }, + { + "sid": 6, + "sent": "We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states.", + "section": "ABSTRACT", + "ner": [ + [ + 3, + 10, + "compare", + "experimental_method" + ], + [ + 15, + 19, + "Tom1", + "protein" + ], + [ + 20, + 23, + "GAT", + "structure_element" + ], + [ + 24, + 33, + "structure", + "evidence" + ], + [ + 45, + 55, + "structures", + "evidence" + ], + [ + 77, + 83, + "Tollip", + "protein" + ], + [ + 84, + 88, + "TBD-", + "protein_state" + ], + [ + 93, + 108, + "ubiquitin-bound", + "protein_state" + ] + ] + }, + { + "sid": 7, + "sent": "Subject area\tBiology\t \tMore specific subject area\tStructural biology\t \tType of data\tTable, text file, graph, figures\t \tHow data was acquired\tCircular dichroism and NMR.", + "section": "TABLE", + "ner": [ + [ + 141, + 159, + "Circular dichroism", + "experimental_method" + ], + [ + 164, + 167, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 8, + "sent": "NMR data was recorded using a Bruker 800\u00a0MHz\t \tData format\tPDB format text file.", + "section": "TABLE", + "ner": [ + [ + 0, + 3, + "NMR", + "experimental_method" + ] + ] + }, + { + "sid": 9, + "sent": "Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol\t \tExperimental factors\tRecombinant human Tom1 GAT domain was purified to homogeneity before use\t \tExperimental features\tSolution structure of Tom1 GAT was determined from NMR chemical shift data\t \tData source location\tVirginia and Colorado, United States.", + "section": "TABLE", + "ner": [ + [ + 12, + 22, + "CS-Rosetta", + "experimental_method" + ], + [ + 24, + 59, + "Protein Structure Validation Server", + "experimental_method" + ], + [ + 61, + 65, + "PSVS", + "experimental_method" + ], + [ + 68, + 75, + "NMRPipe", + "experimental_method" + ], + [ + 77, + 84, + "NMRDraw", + "experimental_method" + ], + [ + 131, + 136, + "human", + "species" + ], + [ + 137, + 141, + "Tom1", + "protein" + ], + [ + 142, + 145, + "GAT", + "structure_element" + ], + [ + 216, + 234, + "Solution structure", + "evidence" + ], + [ + 238, + 242, + "Tom1", + "protein" + ], + [ + 243, + 246, + "GAT", + "structure_element" + ], + [ + 267, + 270, + "NMR", + "experimental_method" + ], + [ + 271, + 285, + "chemical shift", + "evidence" + ] + ] + }, + { + "sid": 10, + "sent": "Tom1 GAT structural data is publicly available in the RCSB Protein Data Bank (http://www.rscb.org/) under the accession number PDB: 2n9d\t \t", + "section": "TABLE", + "ner": [ + [ + 5, + 8, + "GAT", + "structure_element" + ] + ] + }, + { + "sid": 11, + "sent": "The Tom1 GAT domain solution structure will provide additional tools for modulating its biological function.", + "section": "TABLE", + "ner": [ + [ + 4, + 8, + "Tom1", + "protein" + ], + [ + 9, + 12, + "GAT", + "structure_element" + ], + [ + 20, + 38, + "solution structure", + "evidence" + ] + ] + }, + { + "sid": 12, + "sent": "Tom1 GAT can adopt distinct conformations upon ligand binding.", + "section": "TABLE", + "ner": [ + [ + 0, + 4, + "Tom1", + "protein" + ], + [ + 5, + 8, + "GAT", + "structure_element" + ] + ] + }, + { + "sid": 13, + "sent": "A conformational response of the Tom1 GAT domain upon Tollip TBD binding can serve as an example to explain mutually exclusive ligand binding events.", + "section": "TABLE", + "ner": [ + [ + 33, + 37, + "Tom1", + "protein" + ], + [ + 38, + 41, + "GAT", + "structure_element" + ], + [ + 54, + 60, + "Tollip", + "protein" + ], + [ + 61, + 64, + "TBD", + "structure_element" + ] + ] + }, + { + "sid": 14, + "sent": "Analysis of the far-UV circular dichroism (CD) spectrum of the Tom 1 GAT domain (Fig. 1) predicts 58.7% \u03b1-helix, 3% \u03b2-strand, 15.5% turn, and 22.8% disordered regions.", + "section": "TABLE", + "ner": [ + [ + 16, + 41, + "far-UV circular dichroism", + "experimental_method" + ], + [ + 43, + 45, + "CD", + "experimental_method" + ], + [ + 47, + 55, + "spectrum", + "evidence" + ], + [ + 63, + 68, + "Tom 1", + "protein" + ], + [ + 69, + 72, + "GAT", + "structure_element" + ], + [ + 104, + 111, + "\u03b1-helix", + "structure_element" + ], + [ + 116, + 124, + "\u03b2-strand", + "structure_element" + ] + ] + }, + { + "sid": 15, + "sent": "The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9\u00a0\u00c5 for backbone and 1.3\u00a0\u00c5 for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (\u03b1-helix 1), P248-Q274 (\u03b1-helix 2), and E278-T306 (\u03b1-helix 3).", + "section": "TABLE", + "ner": [ + [ + 4, + 8, + "Tom1", + "protein" + ], + [ + 9, + 12, + "GAT", + "structure_element" + ], + [ + 13, + 34, + "structural restraints", + "evidence" + ], + [ + 55, + 65, + "structures", + "evidence" + ], + [ + 85, + 111, + "root mean square deviation", + "evidence" + ], + [ + 113, + 117, + "RMSD", + "evidence" + ], + [ + 243, + 252, + "Q216-E240", + "residue_range" + ], + [ + 254, + 263, + "\u03b1-helix 1", + "structure_element" + ], + [ + 266, + 275, + "P248-Q274", + "residue_range" + ], + [ + 277, + 286, + "\u03b1-helix 2", + "structure_element" + ], + [ + 293, + 302, + "E278-T306", + "residue_range" + ], + [ + 304, + 313, + "\u03b1-helix 3", + "structure_element" + ] + ] + }, + { + "sid": 16, + "sent": "Unlike ubiquitin binding, data suggest that conformational changes of the Tom1 GAT \u03b1-helices 1 and 2 occur upon Tollip TBD binding (Fig. 3A,B).", + "section": "TABLE", + "ner": [ + [ + 7, + 16, + "ubiquitin", + "chemical" + ], + [ + 74, + 78, + "Tom1", + "protein" + ], + [ + 79, + 82, + "GAT", + "structure_element" + ], + [ + 83, + 100, + "\u03b1-helices 1 and 2", + "structure_element" + ], + [ + 112, + 118, + "Tollip", + "protein" + ], + [ + 119, + 122, + "TBD", + "structure_element" + ] + ] + }, + { + "sid": 17, + "sent": "Representative far-UV CD spectrum of the His-Tom1 GAT domain.", + "section": "FIG", + "ner": [ + [ + 15, + 24, + "far-UV CD", + "experimental_method" + ], + [ + 25, + 33, + "spectrum", + "evidence" + ], + [ + 41, + 45, + "His-", + "experimental_method" + ], + [ + 45, + 49, + "Tom1", + "protein" + ], + [ + 50, + 53, + "GAT", + "structure_element" + ] + ] + }, + { + "sid": 18, + "sent": "(A) Stereo view displaying the best-fit backbone superposition of the refined structures for the Tom1 GAT domain.", + "section": "FIG", + "ner": [ + [ + 40, + 62, + "backbone superposition", + "experimental_method" + ], + [ + 78, + 88, + "structures", + "evidence" + ], + [ + 97, + 101, + "Tom1", + "protein" + ], + [ + 102, + 105, + "GAT", + "structure_element" + ] + ] + }, + { + "sid": 19, + "sent": "Helices are shown in orange, whereas loops are colored in green. (B) Ribbon illustration of the Tom1 GAT domain.", + "section": "FIG", + "ner": [ + [ + 96, + 100, + "Tom1", + "protein" + ], + [ + 101, + 104, + "GAT", + "structure_element" + ] + ] + }, + { + "sid": 20, + "sent": "(A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green).", + "section": "FIG", + "ner": [ + [ + 21, + 44, + "superimposed structures", + "experimental_method" + ], + [ + 52, + 56, + "Tom1", + "protein" + ], + [ + 57, + 60, + "GAT", + "structure_element" + ], + [ + 75, + 79, + "free", + "protein_state" + ], + [ + 110, + 116, + "Tollip", + "protein" + ], + [ + 117, + 126, + "TBD-bound", + "protein_state" + ], + [ + 161, + 184, + "superimposed structures", + "experimental_method" + ], + [ + 192, + 196, + "Tom1", + "protein" + ], + [ + 197, + 200, + "GAT", + "structure_element" + ], + [ + 232, + 240, + "Ub-bound", + "protein_state" + ] + ] + }, + { + "sid": 21, + "sent": "NMR and refinement statistics for the Tom1 GAT domain.", + "section": "TABLE", + "ner": [ + [ + 0, + 3, + "NMR", + "experimental_method" + ], + [ + 8, + 29, + "refinement statistics", + "evidence" + ], + [ + 38, + 42, + "Tom1", + "protein" + ], + [ + 43, + 46, + "GAT", + "structure_element" + ] + ] + }, + { + "sid": 22, + "sent": "NMR structural statistics for lowest energy conformers of Tom1 GAT using PSVS.", + "section": "TABLE", + "ner": [ + [ + 0, + 3, + "NMR", + "experimental_method" + ], + [ + 4, + 25, + "structural statistics", + "evidence" + ], + [ + 58, + 62, + "Tom1", + "protein" + ], + [ + 63, + 66, + "GAT", + "structure_element" + ], + [ + 73, + 77, + "PSVS", + "experimental_method" + ] + ] + }, + { + "sid": 23, + "sent": "deviations were obtained by superimposing residues 215\u2013309 of Tom1 GAT among 10 lowest energy refined structures.", + "section": "TABLE", + "ner": [ + [ + 28, + 41, + "superimposing", + "experimental_method" + ], + [ + 51, + 58, + "215\u2013309", + "residue_range" + ], + [ + 62, + 66, + "Tom1", + "protein" + ], + [ + 67, + 70, + "GAT", + "structure_element" + ], + [ + 102, + 112, + "structures", + "evidence" + ] + ] + } + ] + }, + "PMC4802085": { + "annotations": [ + { + "sid": 0, + "sent": "Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance", + "section": "TITLE", + "ner": [ + [ + 0, + 4, + "Haem", + "chemical" + ], + [ + 15, + 27, + "dimerization", + "oligomeric_state" + ], + [ + 31, + 37, + "PGRMC1", + "protein" + ], + [ + 38, + 45, + "Sigma-2", + "protein" + ] + ] + }, + { + "sid": 1, + "sent": "Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 42, + "Progesterone-receptor membrane component 1", + "protein" + ], + [ + 44, + 50, + "PGRMC1", + "protein" + ], + [ + 51, + 67, + "Sigma-2 receptor", + "protein" + ], + [ + 74, + 97, + "haem-containing protein", + "protein_type" + ], + [ + 118, + 150, + "epidermal growth factor receptor", + "protein_type" + ], + [ + 152, + 156, + "EGFR", + "protein_type" + ], + [ + 162, + 178, + "cytochromes P450", + "protein_type" + ] + ] + }, + { + "sid": 2, + "sent": "Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95\u2009\u00c5 resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules.", + "section": "ABSTRACT", + "ner": [ + [ + 5, + 30, + "crystallographic analyses", + "experimental_method" + ], + [ + 38, + 44, + "PGRMC1", + "protein" + ], + [ + 45, + 61, + "cytosolic domain", + "structure_element" + ], + [ + 106, + 112, + "stable", + "protein_state" + ], + [ + 113, + 118, + "dimer", + "oligomeric_state" + ], + [ + 127, + 148, + "stacking interactions", + "bond_interaction" + ], + [ + 167, + 171, + "haem", + "chemical" + ] + ] + }, + { + "sid": 3, + "sent": "The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 8, + "haem", + "chemical" + ], + [ + 9, + 13, + "iron", + "chemical" + ], + [ + 17, + 36, + "five-coordinated by", + "bond_interaction" + ], + [ + 37, + 43, + "Tyr113", + "residue_name_number" + ], + [ + 58, + 65, + "surface", + "site" + ], + [ + 73, + 77, + "haem", + "chemical" + ], + [ + 87, + 99, + "dimerization", + "oligomeric_state" + ] + ] + }, + { + "sid": 4, + "sent": "Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 15, + "Carbon monoxide", + "chemical" + ], + [ + 17, + 19, + "CO", + "chemical" + ], + [ + 37, + 43, + "PGRMC1", + "protein" + ], + [ + 44, + 56, + "dimerization", + "oligomeric_state" + ], + [ + 75, + 98, + "sixth coordination site", + "site" + ], + [ + 106, + 110, + "haem", + "chemical" + ] + ] + }, + { + "sid": 5, + "sent": "Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 4, + "Haem", + "chemical" + ], + [ + 14, + 20, + "PGRMC1", + "protein" + ], + [ + 21, + 33, + "dimerization", + "oligomeric_state" + ], + [ + 68, + 72, + "EGFR", + "protein_type" + ], + [ + 77, + 93, + "cytochromes P450", + "protein_type" + ], + [ + 201, + 203, + "CO", + "chemical" + ], + [ + 207, + 211, + "haem", + "chemical" + ] + ] + }, + { + "sid": 6, + "sent": "This study demonstrates protein dimerization via haem\u2013haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer.", + "section": "ABSTRACT", + "ner": [ + [ + 32, + 44, + "dimerization", + "oligomeric_state" + ], + [ + 49, + 67, + "haem\u2013haem stacking", + "bond_interaction" + ], + [ + 96, + 106, + "eukaryotes", + "taxonomy_domain" + ] + ] + }, + { + "sid": 7, + "sent": " PGRMC1 binds to EGFR and cytochromes P450, and is known to be involved in cancer proliferation and in drug resistance.", + "section": "ABSTRACT", + "ner": [ + [ + 1, + 7, + "PGRMC1", + "protein" + ], + [ + 17, + 21, + "EGFR", + "protein_type" + ], + [ + 26, + 42, + "cytochromes P450", + "protein_type" + ] + ] + }, + { + "sid": 8, + "sent": "Here, the authors determine the structure of the cytosolic domain of PGRMC1, which forms a dimer via haem\u2013haem stacking, and propose how this interaction could be involved in its function.", + "section": "ABSTRACT", + "ner": [ + [ + 32, + 41, + "structure", + "evidence" + ], + [ + 49, + 65, + "cytosolic domain", + "structure_element" + ], + [ + 69, + 75, + "PGRMC1", + "protein" + ], + [ + 91, + 96, + "dimer", + "oligomeric_state" + ], + [ + 101, + 119, + "haem\u2013haem stacking", + "bond_interaction" + ] + ] + }, + { + "sid": 9, + "sent": "Much attention has been paid to the roles of haem-iron in cancer development.", + "section": "INTRO", + "ner": [ + [ + 45, + 49, + "haem", + "chemical" + ], + [ + 50, + 54, + "iron", + "chemical" + ] + ] + }, + { + "sid": 10, + "sent": "Increased dietary intake of haem is a risk factor for several types of cancer.", + "section": "INTRO", + "ner": [ + [ + 28, + 32, + "haem", + "chemical" + ] + ] + }, + { + "sid": 11, + "sent": "Previous studies showed that deprivation of iron or haem suppresses tumourigenesis.", + "section": "INTRO", + "ner": [ + [ + 29, + 43, + "deprivation of", + "protein_state" + ], + [ + 44, + 48, + "iron", + "chemical" + ], + [ + 52, + 56, + "haem", + "chemical" + ] + ] + }, + { + "sid": 12, + "sent": "On the other hand, carbon monoxide (CO), the gaseous mediator generated by oxidative degradation of haem via haem oxygenase (HO), inhibits tumour growth.", + "section": "INTRO", + "ner": [ + [ + 19, + 34, + "carbon monoxide", + "chemical" + ], + [ + 36, + 38, + "CO", + "chemical" + ], + [ + 100, + 104, + "haem", + "chemical" + ], + [ + 109, + 123, + "haem oxygenase", + "protein_type" + ], + [ + 125, + 127, + "HO", + "protein_type" + ] + ] + }, + { + "sid": 13, + "sent": "Thus, a tenuous balance between free haem and CO plays key roles in cancer development and chemoresistance, although the underlying mechanisms are not fully understood.", + "section": "INTRO", + "ner": [ + [ + 37, + 41, + "haem", + "chemical" + ], + [ + 46, + 48, + "CO", + "chemical" + ] + ] + }, + { + "sid": 14, + "sent": "To gain insight into the underlying mechanisms, we took chemical biological approaches using affinity nanobeads carrying haem and identified progesterone-receptor membrane component 1 (PGRMC1) as a haem-binding protein from mouse liver extracts (Supplementary Fig. 1).", + "section": "INTRO", + "ner": [ + [ + 93, + 111, + "affinity nanobeads", + "experimental_method" + ], + [ + 121, + 125, + "haem", + "chemical" + ], + [ + 141, + 183, + "progesterone-receptor membrane component 1", + "protein" + ], + [ + 185, + 191, + "PGRMC1", + "protein" + ], + [ + 198, + 202, + "haem", + "chemical" + ], + [ + 224, + 229, + "mouse", + "taxonomy_domain" + ] + ] + }, + { + "sid": 15, + "sent": "PGRMC1 is a member of the membrane-associated progesterone receptor (MAPR) family with a cytochrome b5-like haem-binding region, and is known to be highly expressed in various types of cancers.", + "section": "INTRO", + "ner": [ + [ + 0, + 6, + "PGRMC1", + "protein" + ], + [ + 26, + 67, + "membrane-associated progesterone receptor", + "protein_type" + ], + [ + 69, + 73, + "MAPR", + "protein_type" + ], + [ + 89, + 107, + "cytochrome b5-like", + "structure_element" + ], + [ + 108, + 127, + "haem-binding region", + "site" + ], + [ + 148, + 164, + "highly expressed", + "protein_state" + ] + ] + }, + { + "sid": 16, + "sent": "PGRMC1 is anchored to the cell membrane through the N-terminal transmembrane helix and interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 (ref).", + "section": "INTRO", + "ner": [ + [ + 0, + 6, + "PGRMC1", + "protein" + ], + [ + 63, + 82, + "transmembrane helix", + "structure_element" + ], + [ + 102, + 134, + "epidermal growth factor receptor", + "protein_type" + ], + [ + 136, + 140, + "EGFR", + "protein_type" + ], + [ + 146, + 162, + "cytochromes P450", + "protein_type" + ] + ] + }, + { + "sid": 17, + "sent": "While PGRMC1 is implicated in cell proliferation and cholesterol biosynthesis, the structural basis on which PGRMC1 exerts its function remains largely unknown.", + "section": "INTRO", + "ner": [ + [ + 6, + 12, + "PGRMC1", + "protein" + ], + [ + 109, + 115, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 18, + "sent": "Here we show that PGRMC1 exhibits a unique haem-dependent dimerization.", + "section": "INTRO", + "ner": [ + [ + 18, + 24, + "PGRMC1", + "protein" + ], + [ + 43, + 47, + "haem", + "chemical" + ], + [ + 58, + 70, + "dimerization", + "oligomeric_state" + ] + ] + }, + { + "sid": 19, + "sent": "The dimer binds to EGFR and cytochromes P450 to enhance tumour cell proliferation and chemoresistance.", + "section": "INTRO", + "ner": [ + [ + 4, + 9, + "dimer", + "oligomeric_state" + ], + [ + 19, + 23, + "EGFR", + "protein_type" + ], + [ + 28, + 44, + "cytochromes P450", + "protein_type" + ] + ] + }, + { + "sid": 20, + "sent": "The dimer is dissociated to monomers by physiological levels of CO, suggesting that PGRMC1 serves as a CO-sensitive molecular switch regulating cancer cell proliferation.", + "section": "INTRO", + "ner": [ + [ + 4, + 9, + "dimer", + "oligomeric_state" + ], + [ + 28, + 36, + "monomers", + "oligomeric_state" + ], + [ + 64, + 66, + "CO", + "chemical" + ], + [ + 84, + 90, + "PGRMC1", + "protein" + ], + [ + 103, + 105, + "CO", + "chemical" + ] + ] + }, + { + "sid": 21, + "sent": "X-ray crystal structure of PGRMC1", + "section": "RESULTS", + "ner": [ + [ + 0, + 23, + "X-ray crystal structure", + "evidence" + ], + [ + 27, + 33, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 22, + "sent": "We solved the crystal structure of the haem-bound PGRMC1 cytosolic domain (a.a.72\u2013195) at 1.95\u2009\u00c5 resolution (Supplementary Fig. 2).", + "section": "RESULTS", + "ner": [ + [ + 3, + 9, + "solved", + "experimental_method" + ], + [ + 14, + 31, + "crystal structure", + "evidence" + ], + [ + 39, + 49, + "haem-bound", + "protein_state" + ], + [ + 50, + 56, + "PGRMC1", + "protein" + ], + [ + 57, + 73, + "cytosolic domain", + "structure_element" + ], + [ + 79, + 85, + "72\u2013195", + "residue_range" + ] + ] + }, + { + "sid": 23, + "sent": "In the presence of haem, PGRMC1 forms a dimeric structure largely through hydrophobic interactions between the haem moieties of two monomers (Fig. 1a, Table 1 and Supplementary Fig. 3; a stereo-structural image is shown in Supplementary Fig 4).", + "section": "RESULTS", + "ner": [ + [ + 7, + 18, + "presence of", + "protein_state" + ], + [ + 19, + 23, + "haem", + "chemical" + ], + [ + 25, + 31, + "PGRMC1", + "protein" + ], + [ + 40, + 47, + "dimeric", + "oligomeric_state" + ], + [ + 74, + 98, + "hydrophobic interactions", + "bond_interaction" + ], + [ + 111, + 115, + "haem", + "chemical" + ], + [ + 132, + 140, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 24, + "sent": "While the overall fold of PGRMC1 is similar to that of canonical cytochrome b5, their haem irons are coordinated differently.", + "section": "RESULTS", + "ner": [ + [ + 26, + 32, + "PGRMC1", + "protein" + ], + [ + 65, + 78, + "cytochrome b5", + "protein_type" + ], + [ + 86, + 90, + "haem", + "chemical" + ] + ] + }, + { + "sid": 25, + "sent": "In cytochrome b5, the haem iron is six-coordinated by two axial histidine residues.", + "section": "RESULTS", + "ner": [ + [ + 3, + 16, + "cytochrome b5", + "protein_type" + ], + [ + 22, + 26, + "haem", + "chemical" + ], + [ + 27, + 31, + "iron", + "chemical" + ], + [ + 35, + 53, + "six-coordinated by", + "bond_interaction" + ], + [ + 64, + 73, + "histidine", + "residue_name" + ] + ] + }, + { + "sid": 26, + "sent": "These histidines are missing in PGRMC1, and the haem iron is five-coordinated by Tyr113 (Y113) alone (Fig. 1b and Supplementary Fig. 3).", + "section": "RESULTS", + "ner": [ + [ + 6, + 16, + "histidines", + "residue_name" + ], + [ + 21, + 28, + "missing", + "protein_state" + ], + [ + 32, + 38, + "PGRMC1", + "protein" + ], + [ + 48, + 52, + "haem", + "chemical" + ], + [ + 53, + 57, + "iron", + "chemical" + ], + [ + 61, + 80, + "five-coordinated by", + "bond_interaction" + ], + [ + 81, + 87, + "Tyr113", + "residue_name_number" + ], + [ + 89, + 93, + "Y113", + "residue_name_number" + ], + [ + 95, + 100, + "alone", + "protein_state" + ] + ] + }, + { + "sid": 27, + "sent": "A homologous helix that holds haem in cytochrome b5 is longer, shifts away from haem, and does not form a coordinate bond in PGRMC1 (Fig. 1c).", + "section": "RESULTS", + "ner": [ + [ + 2, + 18, + "homologous helix", + "structure_element" + ], + [ + 30, + 34, + "haem", + "chemical" + ], + [ + 38, + 51, + "cytochrome b5", + "protein_type" + ], + [ + 80, + 84, + "haem", + "chemical" + ], + [ + 125, + 131, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 28, + "sent": "Consequently, the five-coordinated haem of PGRMC1 has an open surface that allows its dimerization through hydrophobic haem\u2013haem stacking.", + "section": "RESULTS", + "ner": [ + [ + 35, + 39, + "haem", + "chemical" + ], + [ + 43, + 49, + "PGRMC1", + "protein" + ], + [ + 62, + 69, + "surface", + "site" + ], + [ + 86, + 98, + "dimerization", + "oligomeric_state" + ], + [ + 107, + 137, + "hydrophobic haem\u2013haem stacking", + "bond_interaction" + ] + ] + }, + { + "sid": 29, + "sent": "Contrary to our finding, Kaluka et al. recently reported that Tyr164 of PGRMC1 is the axial ligand of haem because mutation of this residue impairs haem binding.", + "section": "RESULTS", + "ner": [ + [ + 62, + 68, + "Tyr164", + "residue_name_number" + ], + [ + 72, + 78, + "PGRMC1", + "protein" + ], + [ + 102, + 106, + "haem", + "chemical" + ], + [ + 115, + 123, + "mutation", + "experimental_method" + ], + [ + 148, + 152, + "haem", + "chemical" + ] + ] + }, + { + "sid": 30, + "sent": "Our structural data revealed that Tyr164 and a few other residues such as Tyr107 and Lys163 are in fact hydrogen-bonded to haem propionates.", + "section": "RESULTS", + "ner": [ + [ + 4, + 19, + "structural data", + "evidence" + ], + [ + 34, + 40, + "Tyr164", + "residue_name_number" + ], + [ + 74, + 80, + "Tyr107", + "residue_name_number" + ], + [ + 85, + 91, + "Lys163", + "residue_name_number" + ], + [ + 104, + 119, + "hydrogen-bonded", + "bond_interaction" + ], + [ + 123, + 127, + "haem", + "chemical" + ] + ] + }, + { + "sid": 31, + "sent": "This is consistent with observations by Min et al. that Tyr 107 and Tyr113 of PGRMC1 are involved in binding with haem.", + "section": "RESULTS", + "ner": [ + [ + 56, + 63, + "Tyr 107", + "residue_name_number" + ], + [ + 68, + 74, + "Tyr113", + "residue_name_number" + ], + [ + 78, + 84, + "PGRMC1", + "protein" + ], + [ + 114, + 118, + "haem", + "chemical" + ] + ] + }, + { + "sid": 32, + "sent": "These amino acid residues are conserved among MAPR family members (Supplementary Fig. 5a), suggesting that these proteins share the ability to exhibit haem-dependent dimerization.", + "section": "RESULTS", + "ner": [ + [ + 30, + 39, + "conserved", + "protein_state" + ], + [ + 46, + 50, + "MAPR", + "protein_type" + ], + [ + 151, + 155, + "haem", + "chemical" + ], + [ + 166, + 178, + "dimerization", + "oligomeric_state" + ] + ] + }, + { + "sid": 33, + "sent": "PGRMC1 exhibits haem-dependent dimerization in solution", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "PGRMC1", + "protein" + ], + [ + 16, + 20, + "haem", + "chemical" + ], + [ + 31, + 43, + "dimerization", + "oligomeric_state" + ] + ] + }, + { + "sid": 34, + "sent": "In the PGRMC1 crystal, two different types of crystal contacts (chain A\u2013A\u2033 and A\u2013B) were observed in addition to the haem-mediated dimer (chain A\u2013A\u2032) (Supplementary Figs 3 and 6a).", + "section": "RESULTS", + "ner": [ + [ + 7, + 13, + "PGRMC1", + "protein" + ], + [ + 14, + 21, + "crystal", + "evidence" + ], + [ + 117, + 121, + "haem", + "chemical" + ], + [ + 131, + 136, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 35, + "sent": "To confirm that haem-assisted dimerization of PGRMC1 occurs in solution, we analysed the structure of apo- and haem-bound PGMRC1 by two-dimensional nuclear magnetic resonance (NMR) using heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy (Supplementary Figs 6b and 7).", + "section": "RESULTS", + "ner": [ + [ + 16, + 20, + "haem", + "chemical" + ], + [ + 30, + 42, + "dimerization", + "oligomeric_state" + ], + [ + 46, + 52, + "PGRMC1", + "protein" + ], + [ + 89, + 98, + "structure", + "evidence" + ], + [ + 102, + 105, + "apo", + "protein_state" + ], + [ + 111, + 121, + "haem-bound", + "protein_state" + ], + [ + 122, + 128, + "PGMRC1", + "protein" + ], + [ + 132, + 174, + "two-dimensional nuclear magnetic resonance", + "experimental_method" + ], + [ + 176, + 179, + "NMR", + "experimental_method" + ], + [ + 187, + 274, + "heteronuclear single-quantum coherence and transverse relaxation-optimized spectroscopy", + "experimental_method" + ] + ] + }, + { + "sid": 36, + "sent": "NMR signals from some amino acid residues of PGRMC1 disappeared due to the paramagnetic relaxation effect of haem (Supplementary Figs 6b); these residues were located in the haem-binding region.", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "NMR", + "experimental_method" + ], + [ + 45, + 51, + "PGRMC1", + "protein" + ], + [ + 109, + 113, + "haem", + "chemical" + ], + [ + 174, + 193, + "haem-binding region", + "site" + ] + ] + }, + { + "sid": 37, + "sent": "When chemical shifts of apo- and haem-bound forms of PGMRC1 were compared, some amino acid residues close to those which disappeared because of the paramagnetic relaxation effect of haem exhibit notable chemical shifts (Supplementary Fig. 6a,b; dark yellow).", + "section": "RESULTS", + "ner": [ + [ + 5, + 20, + "chemical shifts", + "evidence" + ], + [ + 24, + 27, + "apo", + "protein_state" + ], + [ + 33, + 43, + "haem-bound", + "protein_state" + ], + [ + 53, + 59, + "PGMRC1", + "protein" + ], + [ + 182, + 186, + "haem", + "chemical" + ] + ] + }, + { + "sid": 38, + "sent": "However, at the interfaces of the other possible dimeric structures (Supplementary Fig. 6a, chain A\u2013A\u2033; cyan and chain A\u2013B; violet), no significant difference was observed.", + "section": "RESULTS", + "ner": [ + [ + 16, + 26, + "interfaces", + "site" + ], + [ + 49, + 56, + "dimeric", + "oligomeric_state" + ], + [ + 57, + 67, + "structures", + "evidence" + ] + ] + }, + { + "sid": 39, + "sent": "Furthermore, free energy of dissociation predicted by PISA suggested that the haem-mediated dimer is stable in solution while the other potential interactions are not.", + "section": "RESULTS", + "ner": [ + [ + 13, + 40, + "free energy of dissociation", + "evidence" + ], + [ + 54, + 58, + "PISA", + "experimental_method" + ], + [ + 78, + 82, + "haem", + "chemical" + ], + [ + 92, + 97, + "dimer", + "oligomeric_state" + ], + [ + 101, + 107, + "stable", + "protein_state" + ] + ] + }, + { + "sid": 40, + "sent": "We also attempted to predict the secondary structure of PGRMC1 through NMR data by calculating with TALOS+ program (Supplementary Fig. 8); the prediction suggested that the overall secondary structure is comparable between apo- and haem-bound forms of PGRMC1 in solution.", + "section": "RESULTS", + "ner": [ + [ + 56, + 62, + "PGRMC1", + "protein" + ], + [ + 71, + 74, + "NMR", + "experimental_method" + ], + [ + 100, + 114, + "TALOS+ program", + "experimental_method" + ], + [ + 223, + 226, + "apo", + "protein_state" + ], + [ + 232, + 242, + "haem-bound", + "protein_state" + ], + [ + 252, + 258, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 41, + "sent": "We analysed the haem-dependent dimerization of the PGRMC1 cytosolic domain (a.a.44\u2013195) in solution (Fig. 2 and Table 2).", + "section": "RESULTS", + "ner": [ + [ + 16, + 20, + "haem", + "chemical" + ], + [ + 31, + 43, + "dimerization", + "oligomeric_state" + ], + [ + 51, + 57, + "PGRMC1", + "protein" + ], + [ + 58, + 74, + "cytosolic domain", + "structure_element" + ], + [ + 80, + 86, + "44\u2013195", + "residue_range" + ] + ] + }, + { + "sid": 42, + "sent": "Mass spectrometry (MS) analyses under non-denaturing condition demonstrated that the apo-monomer PGRMC1 resulted in dimerization by binding with haem (Fig. 2a).", + "section": "RESULTS", + "ner": [ + [ + 0, + 17, + "Mass spectrometry", + "experimental_method" + ], + [ + 19, + 21, + "MS", + "experimental_method" + ], + [ + 38, + 62, + "non-denaturing condition", + "experimental_method" + ], + [ + 85, + 88, + "apo", + "protein_state" + ], + [ + 89, + 96, + "monomer", + "oligomeric_state" + ], + [ + 97, + 103, + "PGRMC1", + "protein" + ], + [ + 116, + 128, + "dimerization", + "oligomeric_state" + ], + [ + 145, + 149, + "haem", + "chemical" + ] + ] + }, + { + "sid": 43, + "sent": "It should be noted that a disulfide bond between two Cys129 residues is observed in the crystal of PGRMC1 (Fig. 1a), while Cys129 is not conserved among the MAPR family proteins (Supplementary Fig. 5a).", + "section": "RESULTS", + "ner": [ + [ + 26, + 40, + "disulfide bond", + "ptm" + ], + [ + 53, + 59, + "Cys129", + "residue_name_number" + ], + [ + 88, + 95, + "crystal", + "evidence" + ], + [ + 99, + 105, + "PGRMC1", + "protein" + ], + [ + 123, + 129, + "Cys129", + "residue_name_number" + ], + [ + 133, + 146, + "not conserved", + "protein_state" + ], + [ + 157, + 161, + "MAPR", + "protein_type" + ] + ] + }, + { + "sid": 44, + "sent": "This observation led us to examine whether or not the disulfide bond contributes to PGRMC1 dimerization.", + "section": "RESULTS", + "ner": [ + [ + 54, + 68, + "disulfide bond", + "ptm" + ], + [ + 84, + 90, + "PGRMC1", + "protein" + ], + [ + 91, + 103, + "dimerization", + "oligomeric_state" + ] + ] + }, + { + "sid": 45, + "sent": "MS analyses under non-denaturing conditions clearly showed that the Cys129Ser (C129S) mutant is dimerized in the presence of haem, indicating that the haem-mediated dimerization of PGRMC1 occurs independently of the disulfide bond formation via Cys129 (Fig. 2a).", + "section": "RESULTS", + "ner": [ + [ + 0, + 2, + "MS", + "experimental_method" + ], + [ + 18, + 43, + "non-denaturing conditions", + "experimental_method" + ], + [ + 68, + 77, + "Cys129Ser", + "mutant" + ], + [ + 79, + 84, + "C129S", + "mutant" + ], + [ + 86, + 92, + "mutant", + "protein_state" + ], + [ + 96, + 105, + "dimerized", + "protein_state" + ], + [ + 113, + 124, + "presence of", + "protein_state" + ], + [ + 125, + 129, + "haem", + "chemical" + ], + [ + 151, + 155, + "haem", + "chemical" + ], + [ + 165, + 177, + "dimerization", + "oligomeric_state" + ], + [ + 181, + 187, + "PGRMC1", + "protein" + ], + [ + 216, + 230, + "disulfide bond", + "ptm" + ], + [ + 245, + 251, + "Cys129", + "residue_name_number" + ] + ] + }, + { + "sid": 46, + "sent": "Supporting this, MS analyses under denaturing conditions showed that haem-mediated PGRMC1 dimer is completely dissociated into monomer, indicating that dimerization of this kind is not mediated by any covalent bond such as disulfide bond (Supplementary Fig. 9).", + "section": "RESULTS", + "ner": [ + [ + 17, + 19, + "MS", + "experimental_method" + ], + [ + 35, + 56, + "denaturing conditions", + "experimental_method" + ], + [ + 69, + 73, + "haem", + "chemical" + ], + [ + 83, + 89, + "PGRMC1", + "protein" + ], + [ + 90, + 95, + "dimer", + "oligomeric_state" + ], + [ + 127, + 134, + "monomer", + "oligomeric_state" + ], + [ + 152, + 164, + "dimerization", + "oligomeric_state" + ], + [ + 223, + 237, + "disulfide bond", + "ptm" + ] + ] + }, + { + "sid": 47, + "sent": "We also analysed the haem-dependent dimerization of PGRMC1 by diffusion-ordered NMR spectroscopy (DOSY) analyses (Table 2, Supplementary Fig. 10).", + "section": "RESULTS", + "ner": [ + [ + 21, + 25, + "haem", + "chemical" + ], + [ + 36, + 48, + "dimerization", + "oligomeric_state" + ], + [ + 52, + 58, + "PGRMC1", + "protein" + ], + [ + 62, + 96, + "diffusion-ordered NMR spectroscopy", + "experimental_method" + ], + [ + 98, + 102, + "DOSY", + "experimental_method" + ] + ] + }, + { + "sid": 48, + "sent": "The results suggested that the hydrodynamic radius of haem-bound PGRMC1 is larger than that of apo-PGRMC1.", + "section": "RESULTS", + "ner": [ + [ + 31, + 50, + "hydrodynamic radius", + "evidence" + ], + [ + 54, + 64, + "haem-bound", + "protein_state" + ], + [ + 65, + 71, + "PGRMC1", + "protein" + ], + [ + 95, + 98, + "apo", + "protein_state" + ], + [ + 99, + 105, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 49, + "sent": "To further evaluate changes in molecular weights in dimerization of PGRMC1, sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis was carried out.", + "section": "RESULTS", + "ner": [ + [ + 52, + 64, + "dimerization", + "oligomeric_state" + ], + [ + 68, + 74, + "PGRMC1", + "protein" + ], + [ + 76, + 129, + "sedimentation velocity analytical ultracentrifugation", + "experimental_method" + ], + [ + 131, + 137, + "SV-AUC", + "experimental_method" + ] + ] + }, + { + "sid": 50, + "sent": "Whereas the wild-type (wt) apo-PGRMC1 appeared at a 1.9 S peak as monomer, the haem-binding PGRMC1 was converted into dimer at a 3.1 S peak (Fig. 2b).", + "section": "RESULTS", + "ner": [ + [ + 12, + 21, + "wild-type", + "protein_state" + ], + [ + 23, + 25, + "wt", + "protein_state" + ], + [ + 27, + 30, + "apo", + "protein_state" + ], + [ + 31, + 37, + "PGRMC1", + "protein" + ], + [ + 66, + 73, + "monomer", + "oligomeric_state" + ], + [ + 79, + 83, + "haem", + "chemical" + ], + [ + 92, + 98, + "PGRMC1", + "protein" + ], + [ + 118, + 123, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 51, + "sent": "Similarly, the C129S mutant of PGRMC1 converted from monomer to dimer by binding to haem (Fig. 2b).", + "section": "RESULTS", + "ner": [ + [ + 15, + 20, + "C129S", + "mutant" + ], + [ + 21, + 27, + "mutant", + "protein_state" + ], + [ + 31, + 37, + "PGRMC1", + "protein" + ], + [ + 53, + 60, + "monomer", + "oligomeric_state" + ], + [ + 64, + 69, + "dimer", + "oligomeric_state" + ], + [ + 84, + 88, + "haem", + "chemical" + ] + ] + }, + { + "sid": 52, + "sent": "SV-AUC analyses also allowed us to examine the stability of haem/PGRMC1 dimer.", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "SV-AUC", + "experimental_method" + ], + [ + 60, + 64, + "haem", + "chemical" + ], + [ + 65, + 71, + "PGRMC1", + "protein" + ], + [ + 72, + 77, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 53, + "sent": "To this end, we used different concentrations (3.5\u2013147\u2009\u03bcmol\u2009l\u22121) of haem-bound PGRMC1 protein (a.a. 72\u2013195), which were identical to that used in the crystallographic analysis.", + "section": "RESULTS", + "ner": [ + [ + 68, + 78, + "haem-bound", + "protein_state" + ], + [ + 79, + 85, + "PGRMC1", + "protein" + ], + [ + 100, + 106, + "72\u2013195", + "residue_range" + ], + [ + 150, + 175, + "crystallographic analysis", + "experimental_method" + ] + ] + }, + { + "sid": 54, + "sent": "The sedimentation coefficients calculated on the basis of the crystal structure were 1.71 S for monomer and 2.56 S for dimer (Supplementary Fig. 11, upper panel).", + "section": "RESULTS", + "ner": [ + [ + 4, + 30, + "sedimentation coefficients", + "evidence" + ], + [ + 62, + 79, + "crystal structure", + "evidence" + ], + [ + 96, + 103, + "monomer", + "oligomeric_state" + ], + [ + 119, + 124, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 55, + "sent": "The results showed that the PGRMC1 dimer is not dissociated into monomer at all concentrations examined (Supplementary Fig. 11, lower panel), suggesting that the Kd value of haem-mediated dimer of PGRMC1 is under 3.5\u2009\u03bcmol\u2009l\u22121.", + "section": "RESULTS", + "ner": [ + [ + 28, + 34, + "PGRMC1", + "protein" + ], + [ + 35, + 40, + "dimer", + "oligomeric_state" + ], + [ + 65, + 72, + "monomer", + "oligomeric_state" + ], + [ + 162, + 164, + "Kd", + "evidence" + ], + [ + 174, + 178, + "haem", + "chemical" + ], + [ + 188, + 193, + "dimer", + "oligomeric_state" + ], + [ + 197, + 203, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 56, + "sent": "A value of this kind implies that the PGRMC1 dimer is more stable than other dimers of extracellular domain of membrane proteins such as Toll like receptor 9 (dimerization Kd of 20\u2009\u03bcmol\u2009l\u22121) (ref.) and plexin A2 receptor (dimerization Kd higher than 300\u2009\u03bcmol\u2009l\u22121) (ref.).", + "section": "RESULTS", + "ner": [ + [ + 38, + 44, + "PGRMC1", + "protein" + ], + [ + 45, + 50, + "dimer", + "oligomeric_state" + ], + [ + 77, + 83, + "dimers", + "oligomeric_state" + ], + [ + 87, + 107, + "extracellular domain", + "structure_element" + ], + [ + 111, + 128, + "membrane proteins", + "protein_type" + ], + [ + 137, + 157, + "Toll like receptor 9", + "protein" + ], + [ + 159, + 171, + "dimerization", + "oligomeric_state" + ], + [ + 172, + 174, + "Kd", + "evidence" + ], + [ + 202, + 220, + "plexin A2 receptor", + "protein" + ], + [ + 222, + 234, + "dimerization", + "oligomeric_state" + ], + [ + 235, + 237, + "Kd", + "evidence" + ] + ] + }, + { + "sid": 57, + "sent": "The current analytical data confirmed that apo-PGRMC1 monomer converts into dimer by binding to haem in solution (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 43, + 46, + "apo", + "protein_state" + ], + [ + 47, + 53, + "PGRMC1", + "protein" + ], + [ + 54, + 61, + "monomer", + "oligomeric_state" + ], + [ + 76, + 81, + "dimer", + "oligomeric_state" + ], + [ + 96, + 100, + "haem", + "chemical" + ] + ] + }, + { + "sid": 58, + "sent": "We also showed by haem titration experiments that haem binding to PGRMC1 was of low affinity with a Kd value of 50\u2009nmol\u2009l\u22121; this is comparable with that of iron regulatory protein 2, which is known to be regulated by intracellular levels of haem (Fig. 2c and Supplementary Table 1).", + "section": "RESULTS", + "ner": [ + [ + 18, + 44, + "haem titration experiments", + "experimental_method" + ], + [ + 50, + 54, + "haem", + "chemical" + ], + [ + 66, + 72, + "PGRMC1", + "protein" + ], + [ + 100, + 102, + "Kd", + "evidence" + ], + [ + 157, + 182, + "iron regulatory protein 2", + "protein" + ], + [ + 242, + 246, + "haem", + "chemical" + ] + ] + }, + { + "sid": 59, + "sent": "These results raised the possibility that the function of PGRMC1 is regulated by intracellular haem concentrations.", + "section": "RESULTS", + "ner": [ + [ + 58, + 64, + "PGRMC1", + "protein" + ], + [ + 95, + 99, + "haem", + "chemical" + ] + ] + }, + { + "sid": 60, + "sent": "CO inhibits haem-dependent dimerization of PGRMC1", + "section": "RESULTS", + "ner": [ + [ + 0, + 2, + "CO", + "chemical" + ], + [ + 12, + 16, + "haem", + "chemical" + ], + [ + 27, + 39, + "dimerization", + "oligomeric_state" + ], + [ + 43, + 49, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 61, + "sent": "Crystallographic analyses revealed that Tyr113 of PGRMC1 is an axial ligand for haem and contributes to haem-dependent dimerization (Fig. 1a).", + "section": "RESULTS", + "ner": [ + [ + 0, + 25, + "Crystallographic analyses", + "experimental_method" + ], + [ + 40, + 46, + "Tyr113", + "residue_name_number" + ], + [ + 50, + 56, + "PGRMC1", + "protein" + ], + [ + 80, + 84, + "haem", + "chemical" + ], + [ + 104, + 108, + "haem", + "chemical" + ], + [ + 119, + 131, + "dimerization", + "oligomeric_state" + ] + ] + }, + { + "sid": 62, + "sent": "Analysis of UV-visible spectra revealed that the heme of PGRMC1 is reducible from ferric to ferrous state, thus allowing CO binding (Fig. 3a).", + "section": "RESULTS", + "ner": [ + [ + 12, + 30, + "UV-visible spectra", + "evidence" + ], + [ + 49, + 53, + "heme", + "chemical" + ], + [ + 57, + 63, + "PGRMC1", + "protein" + ], + [ + 82, + 88, + "ferric", + "protein_state" + ], + [ + 92, + 99, + "ferrous", + "protein_state" + ], + [ + 121, + 123, + "CO", + "chemical" + ] + ] + }, + { + "sid": 63, + "sent": "Furthermore, the UV-visible spectrum of the wild type PGRMC1 was the same as that of the C129S mutant of PGRMC1, and the R/Z ratio determined by the intensities between the Soret band (394\u2009nm) peak and the 274-nm peak showed that these proteins were fully loaded with haem (Supplementary Fig. 12).", + "section": "RESULTS", + "ner": [ + [ + 17, + 36, + "UV-visible spectrum", + "evidence" + ], + [ + 44, + 53, + "wild type", + "protein_state" + ], + [ + 54, + 60, + "PGRMC1", + "protein" + ], + [ + 89, + 94, + "C129S", + "mutant" + ], + [ + 95, + 101, + "mutant", + "protein_state" + ], + [ + 105, + 111, + "PGRMC1", + "protein" + ], + [ + 121, + 130, + "R/Z ratio", + "evidence" + ], + [ + 250, + 267, + "fully loaded with", + "protein_state" + ], + [ + 268, + 272, + "haem", + "chemical" + ] + ] + }, + { + "sid": 64, + "sent": "Analysis of the ferric form of PGRMC1 using resonance Raman spectroscopy (Supplementary Fig. 13) showed that the relative intensity of oxidation and spin state marker bands (\u03bd4 and \u03bd3) is close to 1.0, which is consistent with it being a haem protein with a proximal Tyr coordination.", + "section": "RESULTS", + "ner": [ + [ + 16, + 22, + "ferric", + "protein_state" + ], + [ + 31, + 37, + "PGRMC1", + "protein" + ], + [ + 44, + 72, + "resonance Raman spectroscopy", + "experimental_method" + ], + [ + 238, + 242, + "haem", + "chemical" + ], + [ + 267, + 270, + "Tyr", + "residue_name" + ] + ] + }, + { + "sid": 65, + "sent": "A specific Raman shift peaking at vFe\u2013CO=500\u2009cm\u22121 demonstrated that the CO-bound haem of PGRMC1 is six-coordinated (Supplementary Fig. 13).", + "section": "RESULTS", + "ner": [ + [ + 11, + 22, + "Raman shift", + "evidence" + ], + [ + 72, + 80, + "CO-bound", + "protein_state" + ], + [ + 81, + 85, + "haem", + "chemical" + ], + [ + 89, + 95, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 66, + "sent": "Since PGRMC1 dimerization involves the open surface of haem on the opposite side of the axial Tyr113, no space for CO binding is available in the dimeric structure (Fig. 3b).", + "section": "RESULTS", + "ner": [ + [ + 6, + 12, + "PGRMC1", + "protein" + ], + [ + 13, + 25, + "dimerization", + "oligomeric_state" + ], + [ + 44, + 51, + "surface", + "site" + ], + [ + 55, + 59, + "haem", + "chemical" + ], + [ + 94, + 100, + "Tyr113", + "residue_name_number" + ], + [ + 115, + 117, + "CO", + "chemical" + ], + [ + 146, + 153, + "dimeric", + "oligomeric_state" + ], + [ + 154, + 163, + "structure", + "evidence" + ] + ] + }, + { + "sid": 67, + "sent": "This prompted us to ask if CO binding to haem causes dissociation of the PGRMC1 dimer.", + "section": "RESULTS", + "ner": [ + [ + 27, + 29, + "CO", + "chemical" + ], + [ + 41, + 45, + "haem", + "chemical" + ], + [ + 73, + 79, + "PGRMC1", + "protein" + ], + [ + 80, + 85, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 68, + "sent": "Analysis by gel filtration chromatography revealed that the relative molecular sizes of the wild-type and the C129S mutant of PGRMC1 are increased by adding haem to apo-PGRMC1 regardless of the oxidation state of the iron (Fig. 3c), which is in agreement with the results in Table 1.", + "section": "RESULTS", + "ner": [ + [ + 12, + 41, + "gel filtration chromatography", + "experimental_method" + ], + [ + 92, + 101, + "wild-type", + "protein_state" + ], + [ + 110, + 115, + "C129S", + "mutant" + ], + [ + 116, + 122, + "mutant", + "protein_state" + ], + [ + 126, + 132, + "PGRMC1", + "protein" + ], + [ + 157, + 161, + "haem", + "chemical" + ], + [ + 165, + 168, + "apo", + "protein_state" + ], + [ + 169, + 175, + "PGRMC1", + "protein" + ], + [ + 217, + 221, + "iron", + "chemical" + ] + ] + }, + { + "sid": 69, + "sent": "CO application to ferrous PGRMC1 abolished the haem-dependent increase in its molecular size.", + "section": "RESULTS", + "ner": [ + [ + 0, + 2, + "CO", + "chemical" + ], + [ + 18, + 25, + "ferrous", + "protein_state" + ], + [ + 26, + 32, + "PGRMC1", + "protein" + ], + [ + 47, + 51, + "haem", + "chemical" + ] + ] + }, + { + "sid": 70, + "sent": "Under this reducing condition in the presence of dithionite, analyses of UV-visible spectra indicated that CO-binding with haem-PGRMC1 is stable, showing only 20% reduction of the absorbance at 412\u2009nm within 2\u2009h (Supplementary Fig. 14).", + "section": "RESULTS", + "ner": [ + [ + 37, + 48, + "presence of", + "protein_state" + ], + [ + 49, + 59, + "dithionite", + "chemical" + ], + [ + 73, + 91, + "UV-visible spectra", + "evidence" + ], + [ + 107, + 109, + "CO", + "chemical" + ], + [ + 123, + 134, + "haem-PGRMC1", + "complex_assembly" + ], + [ + 138, + 144, + "stable", + "protein_state" + ] + ] + }, + { + "sid": 71, + "sent": "Furthermore, the Tyr113Phe (Y113F) mutant of PGRMC1 was not responsive to haem.", + "section": "RESULTS", + "ner": [ + [ + 17, + 26, + "Tyr113Phe", + "mutant" + ], + [ + 28, + 33, + "Y113F", + "mutant" + ], + [ + 35, + 41, + "mutant", + "protein_state" + ], + [ + 45, + 51, + "PGRMC1", + "protein" + ], + [ + 74, + 78, + "haem", + "chemical" + ] + ] + }, + { + "sid": 72, + "sent": "These results suggest that CO favours the six-coordinate form of haem and interferes with the haem-mediated dimerization of PGRMC1.", + "section": "RESULTS", + "ner": [ + [ + 27, + 29, + "CO", + "chemical" + ], + [ + 65, + 69, + "haem", + "chemical" + ], + [ + 94, + 98, + "haem", + "chemical" + ], + [ + 108, + 120, + "dimerization", + "oligomeric_state" + ], + [ + 124, + 130, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 73, + "sent": "To examine the inhibitory effects of CO on haem-mediated PGRMC1 dimerization, SV-AUC analysis was carried out.", + "section": "RESULTS", + "ner": [ + [ + 37, + 39, + "CO", + "chemical" + ], + [ + 43, + 47, + "haem", + "chemical" + ], + [ + 57, + 63, + "PGRMC1", + "protein" + ], + [ + 64, + 76, + "dimerization", + "oligomeric_state" + ], + [ + 78, + 84, + "SV-AUC", + "experimental_method" + ] + ] + }, + { + "sid": 74, + "sent": "The peak corresponding to the haem/PGRMC1 dimer was detected under reducing conditions in the presence of dithionite (Supplementary Fig. 15, middle panel).", + "section": "RESULTS", + "ner": [ + [ + 30, + 34, + "haem", + "chemical" + ], + [ + 35, + 41, + "PGRMC1", + "protein" + ], + [ + 42, + 47, + "dimer", + "oligomeric_state" + ], + [ + 94, + 105, + "presence of", + "protein_state" + ], + [ + 106, + 116, + "dithionite", + "chemical" + ] + ] + }, + { + "sid": 75, + "sent": "Under these circumstances, CO application induced dissociation of the haem-mediated dimers of PGRMC1 to generate a peak of monomers (Supplementary Fig. 15, lower panel).", + "section": "RESULTS", + "ner": [ + [ + 27, + 29, + "CO", + "chemical" + ], + [ + 70, + 74, + "haem", + "chemical" + ], + [ + 84, + 90, + "dimers", + "oligomeric_state" + ], + [ + 94, + 100, + "PGRMC1", + "protein" + ], + [ + 123, + 131, + "monomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 76, + "sent": "These observations raised the transition model for structural regulation of PGRMC1 in response to haem (Fig. 3d).", + "section": "RESULTS", + "ner": [ + [ + 76, + 82, + "PGRMC1", + "protein" + ], + [ + 98, + 102, + "haem", + "chemical" + ] + ] + }, + { + "sid": 77, + "sent": "As mentioned above, apo-PGRMC1 exists as monomer.", + "section": "RESULTS", + "ner": [ + [ + 20, + 23, + "apo", + "protein_state" + ], + [ + 24, + 30, + "PGRMC1", + "protein" + ], + [ + 41, + 48, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 78, + "sent": "By binding with haem (binding Kd=50\u2009nmol\u2009l\u22121), PGRMC1 forms a stable dimer (dimerization Kd<<3.5\u2009\u03bcmol\u2009l\u22121) through stacking of the two open surfaces of the five-coordinated haem molecules in each monomer.", + "section": "RESULTS", + "ner": [ + [ + 16, + 20, + "haem", + "chemical" + ], + [ + 30, + 32, + "Kd", + "evidence" + ], + [ + 47, + 53, + "PGRMC1", + "protein" + ], + [ + 62, + 68, + "stable", + "protein_state" + ], + [ + 69, + 74, + "dimer", + "oligomeric_state" + ], + [ + 76, + 88, + "dimerization", + "oligomeric_state" + ], + [ + 89, + 91, + "Kd", + "evidence" + ], + [ + 115, + 123, + "stacking", + "bond_interaction" + ], + [ + 140, + 148, + "surfaces", + "site" + ], + [ + 173, + 177, + "haem", + "chemical" + ], + [ + 196, + 203, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 79, + "sent": "CO induces the dissociation of the haem-mediated dimer of PGRMC1 by interfering with the haem-stacking interface via formation of the six-coordinated CO-haem-PGRMC1 complex.", + "section": "RESULTS", + "ner": [ + [ + 0, + 2, + "CO", + "chemical" + ], + [ + 35, + 39, + "haem", + "chemical" + ], + [ + 49, + 54, + "dimer", + "oligomeric_state" + ], + [ + 58, + 64, + "PGRMC1", + "protein" + ], + [ + 89, + 112, + "haem-stacking interface", + "site" + ], + [ + 150, + 164, + "CO-haem-PGRMC1", + "complex_assembly" + ] + ] + }, + { + "sid": 80, + "sent": "Such a dynamic structural regulation led us to further examine the regulation of PGRMC1 functions in cancer cells.", + "section": "RESULTS", + "ner": [ + [ + 81, + 87, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 81, + "sent": "PGRMC1 dimerization is required for binding to EGFR", + "section": "RESULTS", + "ner": [ + [ + 0, + 6, + "PGRMC1", + "protein" + ], + [ + 7, + 19, + "dimerization", + "oligomeric_state" + ], + [ + 47, + 51, + "EGFR", + "protein_type" + ] + ] + }, + { + "sid": 82, + "sent": "Because PGRMC1 is known to interact with EGFR and to accelerate tumour progression, we examined the effect of haem-dependent dimerization of PGRMC1 on its interaction with EGFR by using purified proteins.", + "section": "RESULTS", + "ner": [ + [ + 8, + 14, + "PGRMC1", + "protein" + ], + [ + 41, + 45, + "EGFR", + "protein_type" + ], + [ + 110, + 114, + "haem", + "chemical" + ], + [ + 125, + 137, + "dimerization", + "oligomeric_state" + ], + [ + 141, + 147, + "PGRMC1", + "protein" + ], + [ + 172, + 176, + "EGFR", + "protein_type" + ] + ] + }, + { + "sid": 83, + "sent": "As shown in Fig. 4a, the cytosolic domain of wild-type PGRMC1, but not the Y113F mutant, interacted with purified EGFR in a haem-dependent manner.", + "section": "RESULTS", + "ner": [ + [ + 25, + 41, + "cytosolic domain", + "structure_element" + ], + [ + 45, + 54, + "wild-type", + "protein_state" + ], + [ + 55, + 61, + "PGRMC1", + "protein" + ], + [ + 75, + 80, + "Y113F", + "mutant" + ], + [ + 81, + 87, + "mutant", + "protein_state" + ], + [ + 114, + 118, + "EGFR", + "protein_type" + ], + [ + 124, + 128, + "haem", + "chemical" + ] + ] + }, + { + "sid": 84, + "sent": "This interaction was disrupted by the ruthenium-based CO-releasing molecule, CORM3, but not by RuCl3 as a control reagent (Fig. 4b).", + "section": "RESULTS", + "ner": [ + [ + 38, + 47, + "ruthenium", + "chemical" + ], + [ + 54, + 56, + "CO", + "chemical" + ], + [ + 77, + 82, + "CORM3", + "chemical" + ], + [ + 95, + 100, + "RuCl3", + "chemical" + ] + ] + }, + { + "sid": 85, + "sent": "We further analysed the intracellular interaction between PGRMC1 and EGFR.", + "section": "RESULTS", + "ner": [ + [ + 58, + 64, + "PGRMC1", + "protein" + ], + [ + 69, + 73, + "EGFR", + "protein_type" + ] + ] + }, + { + "sid": 86, + "sent": "FLAG-tagged PGRMC1 ectopically expressed in human colon cancer HCT116 cells was immunoprecipitated with anti-FLAG antibody, and co-immunoprecipitated EGFR and endogenous PGRMC1 binding to FLAG-PGRMC1 were detected by Western blotting (Fig. 4c).", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "FLAG-tagged", + "protein_state" + ], + [ + 12, + 18, + "PGRMC1", + "protein" + ], + [ + 19, + 40, + "ectopically expressed", + "experimental_method" + ], + [ + 44, + 49, + "human", + "species" + ], + [ + 80, + 98, + "immunoprecipitated", + "experimental_method" + ], + [ + 128, + 149, + "co-immunoprecipitated", + "experimental_method" + ], + [ + 150, + 154, + "EGFR", + "protein_type" + ], + [ + 159, + 169, + "endogenous", + "protein_state" + ], + [ + 170, + 176, + "PGRMC1", + "protein" + ], + [ + 193, + 199, + "PGRMC1", + "protein" + ], + [ + 217, + 233, + "Western blotting", + "experimental_method" + ] + ] + }, + { + "sid": 87, + "sent": "The C129S mutant of PGRMC1 also interacted with endogenous PGRMC1 and EGFR (Supplementary Fig. 16).", + "section": "RESULTS", + "ner": [ + [ + 4, + 9, + "C129S", + "mutant" + ], + [ + 10, + 16, + "mutant", + "protein_state" + ], + [ + 20, + 26, + "PGRMC1", + "protein" + ], + [ + 48, + 58, + "endogenous", + "protein_state" + ], + [ + 59, + 65, + "PGRMC1", + "protein" + ], + [ + 70, + 74, + "EGFR", + "protein_type" + ] + ] + }, + { + "sid": 88, + "sent": "Whereas FLAG-tagged wild-type PGRMC1 interacted with endogenous PGRMC1 and EGFR, the Y113F mutant did not.", + "section": "RESULTS", + "ner": [ + [ + 8, + 19, + "FLAG-tagged", + "protein_state" + ], + [ + 20, + 29, + "wild-type", + "protein_state" + ], + [ + 30, + 36, + "PGRMC1", + "protein" + ], + [ + 53, + 63, + "endogenous", + "protein_state" + ], + [ + 64, + 70, + "PGRMC1", + "protein" + ], + [ + 75, + 79, + "EGFR", + "protein_type" + ], + [ + 85, + 90, + "Y113F", + "mutant" + ], + [ + 91, + 97, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 89, + "sent": "We also examined the effect of succinylacetone (SA), an inhibitor of haem biosynthesis (Fig. 4d).", + "section": "RESULTS", + "ner": [ + [ + 31, + 46, + "succinylacetone", + "chemical" + ], + [ + 48, + 50, + "SA", + "chemical" + ], + [ + 69, + 73, + "haem", + "chemical" + ] + ] + }, + { + "sid": 90, + "sent": "As expected, SA significantly reduced PGRMC1 dimerization and its interaction with EGFR (Fig. 4e), indicating that haem-mediated dimerization of PGMRC1 is critical for its binding to EGFR.", + "section": "RESULTS", + "ner": [ + [ + 13, + 15, + "SA", + "chemical" + ], + [ + 30, + 37, + "reduced", + "protein_state" + ], + [ + 38, + 44, + "PGRMC1", + "protein" + ], + [ + 45, + 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were decreased by PGRMC1 knockdown (PGRMC1-KD) (Fig. 4f).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "EGF", + "protein_type" + ], + [ + 12, + 28, + "phosphorylations", + "ptm" + ], + [ + 32, + 36, + "EGFR", + "protein_type" + ], + [ + 64, + 67, + "AKT", + "protein_type" + ], + [ + 72, + 75, + "ERK", + "protein_type" + ], + [ + 94, + 100, + "PGRMC1", + "protein" + ], + [ + 101, + 110, + "knockdown", + "protein_state" + ], + [ + 112, + 121, + "PGRMC1-KD", + "mutant" + ] + ] + }, + { + "sid": 94, + "sent": "Similarly, EGFR signaling was suppressed by treatment of HCT116 cells with SA (Fig. 4g) or CORM3 (Fig. 4h).", + "section": "RESULTS", + "ner": [ + [ + 11, + 15, + "EGFR", + "protein_type" + ], + [ + 75, + 77, + "SA", + "chemical" + ], + [ + 91, + 96, + "CORM3", + "chemical" + ] + ] + }, + { + "sid": 95, + "sent": "These results suggested that haem-mediated dimerization of PGRMC1 is critical for EGFR signaling.", + "section": "RESULTS", + "ner": [ + [ + 29, + 33, + "haem", + "chemical" + ], + [ + 43, + 55, + "dimerization", + "oligomeric_state" + ], + [ + 59, + 65, + "PGRMC1", + "protein" + ], + [ + 82, + 86, + "EGFR", + "protein_type" + ] + ] + }, + { + "sid": 96, + "sent": "To further investigate the role of the dimerized form of PGRMC1 in cancer proliferation, we performed PGRMC1 knockdown-rescue experiments using FLAG-tagged wild-type and Y113F PGRMC1 expression vectors, in which silent mutations were introduced into the nucleotide sequence targeted by shRNA (Fig. 5a).", + "section": "RESULTS", + "ner": [ + [ + 39, + 48, + "dimerized", + "protein_state" + ], + [ + 57, + 63, + "PGRMC1", + "protein" + ], + [ + 102, + 108, + "PGRMC1", + "protein" + ], + [ + 109, + 137, + "knockdown-rescue experiments", + "experimental_method" + ], + [ + 144, + 155, + "FLAG-tagged", + "protein_state" + ], + [ + 156, + 165, + "wild-type", + "protein_state" + ], + [ + 170, + 175, + "Y113F", + "mutant" + ], + [ + 176, + 182, + "PGRMC1", + "protein" + ], + [ + 183, + 201, + "expression vectors", + "experimental_method" + ], + [ + 212, + 228, + "silent mutations", + "experimental_method" + ], + [ + 234, + 244, + "introduced", + "experimental_method" + ], + [ + 286, + 291, + "shRNA", + "chemical" + ] + ] + }, + { + "sid": 97, + "sent": "While proliferation of HCT116 cells was not affected by knocking down PGRMC1, PGRMC1-KD cells were more sensitive to the EGFR inhibitor erlotinib than control HCT116 cells, and the knockdown effect was reversed by co-expression of shRNA-resistant wild-type PGRMC1 but not of the Y113F mutant (Fig. 5b).", + "section": "RESULTS", + "ner": [ + [ + 56, + 69, + "knocking down", + "experimental_method" + ], + [ + 70, + 76, + "PGRMC1", + "protein" + ], + [ + 78, + 87, + "PGRMC1-KD", + "mutant" + ], + [ + 121, + 125, + "EGFR", + "protein_type" + ], + [ + 136, + 145, + "erlotinib", + "chemical" + ], + [ + 214, + 227, + "co-expression", + "experimental_method" + ], + [ + 231, + 246, + "shRNA-resistant", + "protein_state" + ], + [ + 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"protein_state" + ], + [ + 143, + 149, + "PGRMC1", + "protein" + ], + [ + 165, + 170, + "Y113F", + "mutant" + ], + [ + 171, + 177, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 100, + "sent": "Thus, PGRMC1 dimerization is important for cancer cell proliferation and chemoresistance.", + "section": "RESULTS", + "ner": [ + [ + 6, + 12, + "PGRMC1", + "protein" + ], + [ + 13, + 25, + "dimerization", + "oligomeric_state" + ] + ] + }, + { + "sid": 101, + "sent": "We examined the role of PGRMC1 in metastatic progression by xenograft transplantation assays using super-immunodeficient NOD/scid/\u03b3null (NOG) mice.", + "section": "RESULTS", + "ner": [ + [ + 24, + 30, + "PGRMC1", + "protein" + ], + [ + 60, + 92, + "xenograft transplantation assays", + "experimental_method" + ] + ] + }, + { + "sid": 102, + "sent": "Ten days after intra-splenic implantation of HCT116 cells that were genetically tagged with a fluorescent protein Venus, the group implanted with PGRMC1-KD cells showed a significant decrease of liver metastasis in comparison with the control group (Fig. 5d).", + "section": "RESULTS", + "ner": [ + [ + 15, + 41, + "intra-splenic implantation", + "experimental_method" + ], + [ + 146, + 155, + "PGRMC1-KD", + "mutant" + ] + ] + }, + { + "sid": 103, + "sent": "Interaction of PGRMC1 dimer with cytochromes P450", + "section": "RESULTS", + "ner": [ + [ + 15, + 21, + "PGRMC1", + "protein" + ], + [ + 22, + 27, + "dimer", + "oligomeric_state" + ], + [ + 33, + 49, + "cytochromes P450", + "protein_type" + ] + ] + }, + { + "sid": 104, + "sent": "Since PGRMC1 has been shown to interact with cytochromes P450 (ref), we investigated whether the haem-mediated dimerization of PGRMC1 is necessary for their interactions.", + "section": "RESULTS", + "ner": [ + [ + 6, + 12, + "PGRMC1", + "protein" + ], + [ + 45, + 61, + "cytochromes P450", + "protein_type" + ], + [ + 97, + 101, + "haem", + "chemical" + ], + [ + 111, + 123, + "dimerization", + "oligomeric_state" + ], + [ + 127, + 133, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 105, + "sent": "Recombinant CYP1A2 and CYP3A4 including a microsomal formulation containing cytochrome b5 and cytochrome P450 reductase, drug-metabolizing cytochromes P450, interacted with wild-type PGRMC1, but not with the Y113F mutant, in a haem-dependent manner (Fig. 6a,b).", + "section": "RESULTS", + "ner": [ + [ + 12, + 18, + "CYP1A2", + "protein" + ], + [ + 23, + 29, + "CYP3A4", + "protein" + ], + [ + 76, + 89, + "cytochrome b5", + "protein_type" + ], + [ + 94, + 119, + "cytochrome P450 reductase", + "protein" + ], + [ + 139, + 155, + "cytochromes P450", + "protein_type" + ], + [ + 173, + 182, + "wild-type", + "protein_state" + ], + [ + 183, + 189, + "PGRMC1", + "protein" + ], + [ + 208, + 213, + "Y113F", + "mutant" + ], + [ + 214, + 220, + "mutant", + "protein_state" + ], + [ + 227, + 231, + "haem", + "chemical" + ] + ] + }, + { + "sid": 106, + "sent": "Moreover, the interaction of PGRMC1 with CYP1A2 was blocked by CORM3 under reducing conditions (Fig. 6c), indicating that PGRMC1 dimerization is necessary for its interaction with cytochromes P450.", + "section": "RESULTS", + "ner": [ + [ + 29, + 35, + "PGRMC1", + "protein" + ], + [ + 41, + 47, + "CYP1A2", + "protein" + ], + [ + 63, + 68, + "CORM3", + "chemical" + ], + [ + 122, + 128, + "PGRMC1", + "protein" + ], + [ + 129, + 141, + "dimerization", + "oligomeric_state" + ], + [ + 180, + 196, + "cytochromes P450", + "protein_type" + ] + ] + }, + { + "sid": 107, + "sent": "Doxorubicin is an anti-cancer reagent that is metabolized into inactive doxorubicinol by CYP2D6 and CYP3A4 (Fig. 6d).", + "section": "RESULTS", + "ner": [ + [ + 0, + 11, + "Doxorubicin", + "chemical" + ], + [ + 72, + 85, + "doxorubicinol", + "chemical" + ], + [ + 89, + 95, + "CYP2D6", + "protein" + ], + [ + 100, + 106, + "CYP3A4", + "protein" + ] + ] + }, + { + "sid": 108, + "sent": "PGRMC1-KD significantly suppressed the conversion of doxorubicin to doxorubicinol (Fig. 6d) and increased sensitivity to doxorubicin (Fig. 6e).", + "section": "RESULTS", + "ner": [ + [ + 0, + 9, + "PGRMC1-KD", + "mutant" + ], + [ + 53, + 64, + "doxorubicin", + "chemical" + ], + [ + 68, + 81, + "doxorubicinol", + "chemical" + ], + [ + 121, + 132, + "doxorubicin", + "chemical" + ] + ] + }, + { + "sid": 109, + "sent": "Enhanced doxorubicin sensitivity was modestly but significantly induced by PGRMC1-KD.", + "section": "RESULTS", + "ner": [ + [ + 9, + 20, + "doxorubicin", + "chemical" + ], + [ + 75, + 84, + "PGRMC1-KD", + "mutant" + ] + ] + }, + { + "sid": 110, + "sent": "This effect was reversed by co-expression of the wild-type PGRMC1 but not of the Y113F mutant, suggesting that PGRMC1 enhances doxorubicin resistance of cancer cells by facilitating its degradation via cytochromes P450.", + "section": "RESULTS", + "ner": [ + [ + 28, + 41, + "co-expression", + "experimental_method" + ], + [ + 49, + 58, + "wild-type", + "protein_state" + ], + [ + 59, + 65, + "PGRMC1", + "protein" + ], + [ + 81, + 86, + "Y113F", + "mutant" + ], + [ + 87, + 93, + "mutant", + "protein_state" + ], + [ + 111, + 117, + "PGRMC1", + "protein" + ], + [ + 127, + 138, + "doxorubicin", + "chemical" + ], + [ + 202, + 218, + "cytochromes P450", + "protein_type" + ] + ] + }, + { + "sid": 111, + "sent": "To gain further insight into the interaction between PGRMC1 and cytochromes P450, surface plasmon resonance analyses were conducted using recombinant CYP51 and PGRMC1.", + "section": "RESULTS", + "ner": [ + [ + 53, + 59, + "PGRMC1", + "protein" + ], + [ + 64, + 80, + "cytochromes P450", + "protein_type" + ], + [ + 82, + 116, + "surface plasmon resonance analyses", + "experimental_method" + ], + [ + 150, + 155, + "CYP51", + "protein" + ], + [ + 160, + 166, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 112, + "sent": "This was based on a previous study showing that PGRMC1 binds to CYP51 and enhances cholesterol biosynthesis by CYP51 (refs).", + "section": "RESULTS", + "ner": [ + [ + 48, + 54, + "PGRMC1", + "protein" + ], + [ + 64, + 69, + "CYP51", + "protein" + ], + [ + 111, + 116, + "CYP51", + "protein" + ] + ] + }, + { + "sid": 113, + "sent": "CYP51 interacted with PGRMC1 in a concentration-dependent manner in the presence of haem, but not in its absence (Supplementary Fig. 19), suggesting the requirement for the haem-dependent dimerization of PGRMC1.", + "section": "RESULTS", + "ner": [ + [ + 0, + 5, + "CYP51", + "protein" + ], + [ + 22, + 28, + "PGRMC1", + "protein" + ], + [ + 72, + 83, + "presence of", + "protein_state" + ], + [ + 84, + 88, + "haem", + "chemical" + ], + [ + 105, + 112, + "absence", + "protein_state" + ], + [ + 173, + 177, + "haem", + "chemical" + ], + [ + 188, + 200, + "dimerization", + "oligomeric_state" + ], + [ + 204, + 210, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 114, + "sent": "The Kd value of PGRMC1 binding to CYP51 was in a micromolar range and comparable with those of other haem proteins, such as cytochrome P450 reductase and neuroglobin/G\u03b1i1 (ref.), suggesting that haem-dependent PGRMC1 interaction with CYP51 is biologically relevant.", + "section": "RESULTS", + "ner": [ + [ + 4, + 6, + "Kd", + "evidence" + ], + [ + 16, + 22, + "PGRMC1", + "protein" + ], + [ + 34, + 39, + "CYP51", + "protein" + ], + [ + 101, + 105, + "haem", + "chemical" + ], + [ + 124, + 149, + "cytochrome P450 reductase", + "protein" + ], + [ + 154, + 165, + "neuroglobin", + "protein" + ], + [ + 166, + 170, + "G\u03b1i1", + "protein" + ], + [ + 195, + 199, + "haem", + "chemical" + ], + [ + 210, + 216, + "PGRMC1", + "protein" + ], + [ + 234, + 239, + "CYP51", + "protein" + ] + ] + }, + { + "sid": 115, + "sent": "In this study, we showed that PGRMC1 dimerizes by stacking interactions of haem molecules from each monomer.", + "section": "DISCUSS", + "ner": [ + [ + 30, + 36, + "PGRMC1", + "protein" + ], + [ + 37, + 46, + "dimerizes", + "oligomeric_state" + ], + [ + 50, + 71, + "stacking interactions", + "bond_interaction" + ], + [ + 75, + 79, + "haem", + "chemical" + ], + [ + 100, + 107, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 116, + "sent": "Recently, Lucas et al. reported that translationally-controlled tumour protein was dimerized by binding with haem, but its structural basis remains unclear.", + "section": "DISCUSS", + "ner": [ + [ + 37, + 78, + "translationally-controlled tumour protein", + "protein_type" + ], + [ + 83, + 92, + "dimerized", + "protein_state" + ], + [ + 109, + 113, + "haem", + "chemical" + ] + ] + }, + { + "sid": 117, + "sent": "This is the report showing crystallographic evidence that indicates roles of the direct haem\u2013haem stacking in haem-mediated dimerization in eukaryotes, although a few examples are known in bacteria.", + "section": "DISCUSS", + "ner": [ + [ + 88, + 106, + "haem\u2013haem stacking", + "bond_interaction" + ], + [ + 110, + 114, + "haem", + "chemical" + ], + [ + 124, + 136, + "dimerization", + "oligomeric_state" + ], + [ + 140, + 150, + "eukaryotes", + "taxonomy_domain" + ], + [ + 189, + 197, + "bacteria", + "taxonomy_domain" + ] + ] + }, + { + "sid": 118, + "sent": "Sequence alignments show that haem-binding residues (Tyr113, Tyr107, Lys163 and Tyr164) in PGRMC1 are conserved among MAPR proteins (Supplementary Fig. 5).", + "section": "DISCUSS", + "ner": [ + [ + 0, + 19, + "Sequence alignments", + "experimental_method" + ], + [ + 30, + 51, + "haem-binding residues", + "site" + ], + [ + 53, + 59, + "Tyr113", + "residue_name_number" + ], + [ + 61, + 67, + "Tyr107", + "residue_name_number" + ], + [ + 69, + 75, + "Lys163", + "residue_name_number" + ], + [ + 80, + 86, + "Tyr164", + "residue_name_number" + ], + [ + 91, + 97, + "PGRMC1", + "protein" + ], + [ + 102, + 111, + "conserved", + "protein_state" + ], + [ + 118, + 122, + "MAPR", + "protein_type" + ] + ] + }, + { + "sid": 119, + "sent": "In the current study, the Y113 residue plays a crucial role for the haem-dependent dimerization of PGRMC1 and resultant regulation of cancer proliferation and chemoresistance (Figs 5c and 6e).", + "section": "DISCUSS", + "ner": [ + [ + 26, + 30, + "Y113", + "residue_name_number" + ], + [ + 68, + 72, + "haem", + "chemical" + ], + [ + 83, + 95, + "dimerization", + "oligomeric_state" + ], + [ + 99, + 105, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 120, + "sent": "Since the Y113 residue is involved in the putative consensus motif of phosphorylation by tyrosine kinases such as Abl and Lck, we investigated whether phosphorylated Y113 is present in HCT116 cells by ESI-MS analysis.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 14, + "Y113", + "residue_name_number" + ], + [ + 51, + 66, + "consensus motif", + "structure_element" + ], + [ + 70, + 85, + "phosphorylation", + "ptm" + ], + [ + 89, + 105, + "tyrosine kinases", + "protein_type" + ], + [ + 114, + 117, + "Abl", + "protein_type" + ], + [ + 122, + 125, + "Lck", + "protein_type" + ], + [ + 151, + 165, + "phosphorylated", + "protein_state" + ], + [ + 166, + 170, + "Y113", + "residue_name_number" + ], + [ + 201, + 207, + "ESI-MS", + "experimental_method" + ] + ] + }, + { + "sid": 121, + "sent": "Recently, Peluso et al. reported that PGRMC1 binds to PGRMC2, suggesting that MAPR family members may also undergo haem-mediated heterodimerization.", + "section": "DISCUSS", + "ner": [ + [ + 38, + 44, + "PGRMC1", + "protein" + ], + [ + 54, + 60, + "PGRMC2", + "protein" + ], + [ + 78, + 82, + "MAPR", + "protein_type" + ], + [ + 115, + 119, + "haem", + "chemical" + ] + ] + }, + { + "sid": 122, + "sent": "We showed that the haem-mediated dimer of PGRMC1 enables interaction with different subclasses of cytochromes P450 (CYP) (Fig. 6).", + "section": "DISCUSS", + "ner": [ + [ + 19, + 23, + "haem", + "chemical" + ], + [ + 33, + 38, + "dimer", + "oligomeric_state" + ], + [ + 42, + 48, + "PGRMC1", + "protein" + ], + [ + 98, + 114, + "cytochromes P450", + "protein_type" + ], + [ + 116, + 119, + "CYP", + "protein_type" + ] + ] + }, + { + "sid": 123, + "sent": "While the effects of PGRMC1 on cholesterol synthesis mediated by CYP51 have been well documented in yeast and human cells, it has not been clear whether drug-metabolizing CYP activities are regulated by PGRMC1.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 27, + "PGRMC1", + "protein" + ], + [ + 65, + 70, + "CYP51", + "protein" + ], + [ + 100, + 105, + "yeast", + "taxonomy_domain" + ], + [ + 110, + 115, + "human", + "species" + ], + [ + 171, + 174, + "CYP", + "protein_type" + ], + [ + 203, + 209, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 124, + "sent": "Szczesna-Skorupa and Kemper reported that PGRMC1 exhibited an inhibitory effect on CYP3A4 drug metabolizing activity by competitively binding with cytochrome P450 reductase (CPR) in HEK293 or HepG2 cells.", + "section": "DISCUSS", + "ner": [ + [ + 42, + 48, + "PGRMC1", + "protein" + ], + [ + 83, + 89, + "CYP3A4", + "protein" + ], + [ + 147, + 172, + "cytochrome P450 reductase", + "protein" + ], + [ + 174, + 177, + "CPR", + "protein" + ] + ] + }, + { + "sid": 125, + "sent": "On the other hand, Oda et al. reported that PGRMC1 had no effect to CYP2E1 and CYP3A4 activities in HepG2 cell.", + "section": "DISCUSS", + "ner": [ + [ + 44, + 50, + "PGRMC1", + "protein" + ], + [ + 68, + 74, + "CYP2E1", + "protein" + ], + [ + 79, + 85, + "CYP3A4", + "protein" + ] + ] + }, + { + "sid": 126, + "sent": "Several other groups showed that PGRMC1 enhanced chemoresistance in several cancer cells such as uterine sarcoma, breast cancer, endometrial tumour and ovarian cancer; however, no evidence of PGRMC1-dependent regulation of CYP activity was provided.", + "section": "DISCUSS", + "ner": [ + [ + 33, + 39, + "PGRMC1", + "protein" + ], + [ + 192, + 198, + "PGRMC1", + "protein" + ], + [ + 223, + 226, + "CYP", + "protein_type" + ] + ] + }, + { + "sid": 127, + "sent": "Our results showed that PGRMC1 contributes to enhancement of the doxorubicin metabolism, which is mediated by CYP2D6 or CYP3A4 in human colon cancer HCT116 cells (Fig. 6d).", + "section": "DISCUSS", + "ner": [ + [ + 24, + 30, + "PGRMC1", + "protein" + ], + [ + 65, + 76, + "doxorubicin", + "chemical" + ], + [ + 110, + 116, + "CYP2D6", + "protein" + ], + [ + 120, + 126, + "CYP3A4", + "protein" + ], + [ + 130, + 135, + "human", + "species" + ] + ] + }, + { + "sid": 128, + "sent": "While the effects of structural diversity of CYP family proteins and interactions with different xenobiotic substrates should further be examined, the current results suggest that the interaction of drug-metabolizing CYPs with the haem-mediated dimer of PGRMC1 plays a crucial role in regulating their activities.", + "section": "DISCUSS", + "ner": [ + [ + 45, + 48, + "CYP", + "protein_type" + ], + [ + 217, + 221, + "CYPs", + "protein_type" + ], + [ + 231, + 235, + "haem", + "chemical" + ], + [ + 245, + 250, + "dimer", + "oligomeric_state" + ], + [ + 254, + 260, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 129, + "sent": "We showed that haem-mediated dimerization of PGRMC1 enhances proliferation and chemoresistance of cancer cells through binding to and regulating EGFR and cytochromes P450 (illustrated in Fig. 7).", + "section": "DISCUSS", + "ner": [ + [ + 15, + 19, + "haem", + "chemical" + ], + [ + 29, + 41, + "dimerization", + "oligomeric_state" + ], + [ + 45, + 51, + "PGRMC1", + "protein" + ], + [ + 145, + 149, + "EGFR", + "protein_type" + ], + [ + 154, + 170, + "cytochromes P450", + "protein_type" + ] + ] + }, + { + "sid": 130, + "sent": "Since the haem-binding affinity of PGRMC1 is lower than those of constitutive haem-binding proteins such as myoglobin, PGMRC1 is probably interconverted between apo-monomer and haem-bound dimer forms in response to changes in the intracellular haem concentration.", + "section": "DISCUSS", + "ner": [ + [ + 10, + 31, + "haem-binding affinity", + "evidence" + ], + [ + 35, + 41, + "PGRMC1", + "protein" + ], + [ + 65, + 77, + "constitutive", + "protein_state" + ], + [ + 78, + 99, + "haem-binding proteins", + "protein_type" + ], + [ + 108, + 117, + "myoglobin", + "protein" + ], + [ + 119, + 125, + "PGMRC1", + "protein" + ], + [ + 161, + 164, + "apo", + "protein_state" + ], + [ + 165, + 172, + "monomer", + "oligomeric_state" + ], + [ + 177, + 187, + "haem-bound", + "protein_state" + ], + [ + 188, + 193, + "dimer", + "oligomeric_state" + ], + [ + 244, + 248, + "haem", + "chemical" + ] + ] + }, + { + "sid": 131, + "sent": "Considering microenvironments in and around malignant tumours, the haem concentration in cancer cells is likely to be elevated through multiple mechanisms, such as (i) an increased intake of haem, (ii) mutation of enzymes in TCA cycle (for example, fumarate hydratase) that increases the level of succinyl CoA, a substrate for haem biosynthesis and (iii) metastasis to haem-rich organs such as liver, brain and bone marrow.", + "section": "DISCUSS", + "ner": [ + [ + 67, + 71, + "haem", + "chemical" + ], + [ + 191, + 195, + "haem", + "chemical" + ], + [ + 249, + 267, + "fumarate 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dimerization of PGRMC1 through the production of CO and thereby suppress tumour progression.", + "section": "DISCUSS", + "ner": [ + [ + 6, + 10, + "HO-1", + "protein" + ], + [ + 53, + 57, + "haem", + "chemical" + ], + [ + 67, + 79, + "dimerization", + "oligomeric_state" + ], + [ + 83, + 89, + "PGRMC1", + "protein" + ], + [ + 116, + 118, + "CO", + "chemical" + ] + ] + }, + { + "sid": 135, + "sent": "This idea is consistent with the observation that HO-1 induction or CO inhibits tumour growth.", + "section": "DISCUSS", + "ner": [ + [ + 50, + 54, + "HO-1", + "protein" + ], + [ + 68, + 70, + "CO", + "chemical" + ] + ] + }, + { + "sid": 136, + "sent": "Besides the regulatory roles of PGRMC1/Sigma-2 receptor in proliferation and chemoresistance in cancer cells (ref.), recent reports show that PGRMC1 is able to bind to amyloid beta oligomer to enhance its neurotoxicity.", + "section": "DISCUSS", + "ner": [ + [ + 32, + 38, + "PGRMC1", + "protein" + ], + [ + 39, + 46, + "Sigma-2", + "protein" + ], + [ + 142, + 148, + "PGRMC1", + "protein" + ], + [ + 168, + 180, + "amyloid beta", + "protein" + ], + [ + 181, + 189, + "oligomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 137, + "sent": "Furthermore, Sigma-2 ligand-binding is decreased in transgenic amyloid beta deposition model APP/PS1 female mice.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 20, + "Sigma-2", + "protein" + ] + ] + }, + { + "sid": 138, + "sent": "These results suggest a possible involvement of PGRMC1 in Alzheimer's disease.", + "section": "DISCUSS", + "ner": [ + [ + 48, + 54, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 139, + "sent": "The roles of haem-dependent dimerization of PGRMC1 in the functional regulation of its target proteins deserve further studies to find evidence that therapeutic interventions to interfere with the function of the dimer may control varied disease conditions.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 17, + "haem", + "chemical" + ], + [ + 28, + 40, + "dimerization", + "oligomeric_state" + ], + [ + 44, + 50, + "PGRMC1", + "protein" + ], + [ + 213, + 218, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 140, + "sent": "Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers II: Sigma-2/PGRMC1 receptors mediate Abeta 42 oligomer binding and synaptotoxicity", + "section": "REF", + "ner": [ + [ + 109, + 117, + "oligomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 141, + "sent": "X-ray crystal structure of PGRMC1.", + "section": "FIG", + "ner": [ + [ + 0, + 23, + "X-ray crystal structure", + "evidence" + ], + [ + 27, + 33, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 142, + "sent": "(a) Structure of the PGRMC1 dimer formed through stacked haems.", + "section": "FIG", + "ner": [ + [ + 21, + 27, + "PGRMC1", + "protein" + ], + [ + 28, + 33, + "dimer", + "oligomeric_state" + ], + [ + 57, + 62, + "haems", + "chemical" + ] + ] + }, + { + "sid": 143, + "sent": "Two PGRMC1 subunits (blue and green ribbons) dimerize via stacking of the haem molecules.", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "PGRMC1", + "protein" + ], + [ + 11, + 19, + "subunits", + "structure_element" + ], + [ + 45, + 53, + "dimerize", + "oligomeric_state" + ], + [ + 58, + 66, + "stacking", + "bond_interaction" + ], + [ + 74, + 78, + "haem", + "chemical" + ] + ] + }, + { + "sid": 144, + "sent": "(b) Haem coordination of PGRMC1 with Tyr113.", + "section": "FIG", + "ner": [ + [ + 4, + 8, + "Haem", + "chemical" + ], + [ + 9, + 21, + "coordination", + "bond_interaction" + ], + [ + 25, + 31, + "PGRMC1", + "protein" + ], + [ + 37, + 43, + "Tyr113", + "residue_name_number" + ] + ] + }, + { + "sid": 145, + "sent": "Comparison of PGRMC1 (blue) and cytochrome b5 (yellow, ID: 3NER). (c) PGRMC1 has a longer helix (a.a.147\u2013163), which is shifted away from the haem (arrow).", + "section": "FIG", + "ner": [ + [ + 14, + 20, + "PGRMC1", + "protein" + ], + [ + 32, + 45, + "cytochrome b5", + "protein_type" + ], + [ + 70, + 76, + "PGRMC1", + "protein" + ], + [ + 90, + 95, + "helix", + "structure_element" + ], + [ + 101, + 108, + "147\u2013163", + "residue_range" + ], + [ + 142, + 146, + "haem", + "chemical" + ] + ] + }, + { + "sid": 146, + "sent": "PGRCM1 is dimerized by binding with haem.", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "PGRCM1", + "protein" + ], + [ + 10, + 19, + "dimerized", + "protein_state" + ], + [ + 36, + 40, + "haem", + "chemical" + ] + ] + }, + { + "sid": 147, + "sent": "(a) Mass spectrometric analyses of the wild-type (wt) PGRMC1 or the C129S mutant in the presence or absence of haem under non-denaturing condition.", + "section": "FIG", + "ner": [ + [ + 4, + 22, + "Mass spectrometric", + "experimental_method" + ], + [ + 39, + 48, + "wild-type", + "protein_state" + ], + [ + 50, + 52, + "wt", + "protein_state" + ], + [ + 54, + 60, + "PGRMC1", + "protein" + ], + [ + 68, + 73, + "C129S", + "mutant" + ], + [ + 74, + 80, + "mutant", + "protein_state" + ], + [ + 88, + 96, + "presence", + "protein_state" + ], + [ + 100, + 110, + "absence of", + "protein_state" + ], + [ + 111, + 115, + "haem", + "chemical" + ] + ] + }, + { + "sid": 148, + "sent": "Both proteins had identical lengths (a.a.44\u2013195).", + "section": "FIG", + "ner": [ + [ + 41, + 47, + "44\u2013195", + "residue_range" + ] + ] + }, + { + "sid": 149, + "sent": "(b) SV-AUC analyses of the wt-PGRMC1 and the C129S mutant (a.a.44\u2013195) in the presence or absence of haem.", + "section": "FIG", + "ner": [ + [ + 4, + 10, + "SV-AUC", + "experimental_method" + ], + [ + 27, + 29, + "wt", + "protein_state" + ], + [ + 30, + 36, + "PGRMC1", + "protein" + ], + [ + 45, + 50, + "C129S", + "mutant" + ], + [ + 51, + 57, + "mutant", + "protein_state" + ], + [ + 63, + 69, + "44\u2013195", + "residue_range" + ], + [ + 78, + 86, + "presence", + "protein_state" + ], + [ + 90, + 100, + "absence of", + "protein_state" + ], + [ + 101, + 105, + "haem", + "chemical" + ] + ] + }, + { + "sid": 150, + "sent": "SV-AUC experiments were performed with 1.5\u2009mg\u2009ml\u22121 of PGRMC1 proteins.", + "section": "FIG", + "ner": [ + [ + 0, + 6, + "SV-AUC", + "experimental_method" + ], + [ + 54, + 60, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 151, + "sent": "The major peak with sedimentation coefficient S20,w of 1.9\u223c2.0 S (monomer) or 3.1 S (dimer) was detected.", + "section": "FIG", + "ner": [ + [ + 20, + 45, + "sedimentation coefficient", + "evidence" + ], + [ + 46, + 51, + "S20,w", + "evidence" + ], + [ + 66, + 73, + "monomer", + "oligomeric_state" + ], + [ + 85, + 90, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 152, + "sent": "(c) Difference absorption spectra of PGRMC1 (a.a.44\u2013195) titrated with haem (left panel).", + "section": "FIG", + "ner": [ + [ + 4, + 33, + "Difference absorption spectra", + "evidence" + ], + [ + 37, + 43, + "PGRMC1", + "protein" + ], + [ + 49, + 55, + "44\u2013195", + "residue_range" + ], + [ + 57, + 70, + "titrated with", + "experimental_method" + ], + [ + 71, + 75, + "haem", + "chemical" + ] + ] + }, + { + "sid": 153, + "sent": "The titration curve of haem to PGRMC1 (right panel).", + "section": "FIG", + "ner": [ + [ + 4, + 19, + "titration curve", + "evidence" + ], + [ + 23, + 27, + "haem", + "chemical" + ], + [ + 31, + 37, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 154, + "sent": "The absorbance difference at 400\u2009nm is plotted against the haem concentration.", + "section": "FIG", + "ner": [ + [ + 4, + 25, + "absorbance difference", + "evidence" + ], + [ + 59, + 63, + "haem", + "chemical" + ] + ] + }, + { + "sid": 155, + "sent": "Carbon monoxide inhibits haem-dependent PGRMC1 dimerization.", + "section": "FIG", + "ner": [ + [ + 0, + 15, + "Carbon monoxide", + "chemical" + ], + [ + 25, + 29, + "haem", + "chemical" + ], + [ + 40, + 46, + "PGRMC1", + "protein" + ], + [ + 47, + 59, + "dimerization", + "oligomeric_state" + ] + ] + }, + { + "sid": 156, + "sent": "(a) UV-visible absorption spectra of PGRMC1 (a.a.44\u2013195).", + "section": "FIG", + "ner": [ + [ + 4, + 33, + "UV-visible absorption spectra", + "evidence" + ], + [ + 37, + 43, + "PGRMC1", + "protein" + ], + [ + 49, + 55, + "44\u2013195", + "residue_range" + ] + ] + }, + { + "sid": 157, + "sent": "Measurements were performed in the presence of the oxidized form of haem (ferric), the reduced form of haem (ferrous) and the reduced form of haem plus CO gas (ferrous+CO).", + "section": "FIG", + "ner": [ + [ + 35, + 46, + "presence of", + "protein_state" + ], + [ + 51, + 59, + "oxidized", + "protein_state" + ], + [ + 68, + 72, + "haem", + "chemical" + ], + [ + 74, + 80, + "ferric", + "protein_state" + ], + [ + 87, + 94, + "reduced", + "protein_state" + ], + [ + 103, + 107, + "haem", + "chemical" + ], + [ + 109, + 116, + "ferrous", + "protein_state" + ], + [ + 126, + 133, + "reduced", + "protein_state" + ], + [ + 142, + 146, + "haem", + "chemical" + ], + [ + 152, + 154, + "CO", + "chemical" + ], + [ + 160, + 167, + "ferrous", + "protein_state" + ], + [ + 168, + 170, + "CO", + "chemical" + ] + ] + }, + { + "sid": 158, + "sent": "(b) Close-up view of haem stacking.", + "section": "FIG", + "ner": [ + [ + 21, + 34, + "haem stacking", + "bond_interaction" + ] + ] + }, + { + "sid": 159, + "sent": "(c) Gel-filtration chromatography analyses of PGRMC1 (a.a.44\u2013195) wild-type (wt) and the Y113F or C129S mutant in the presence or absence of haem, dithionite and/or CO. (d) Transition model for structural regulation of PGRMC1 in response to haem and CO.", + "section": "FIG", + "ner": [ + [ + 4, + 33, + "Gel-filtration chromatography", + "experimental_method" + ], + [ + 46, + 52, + "PGRMC1", + "protein" + ], + [ + 58, + 64, + "44\u2013195", + "residue_range" + ], + [ + 66, + 75, + "wild-type", + "protein_state" + ], + [ + 77, + 79, + "wt", + "protein_state" + ], + [ + 89, + 94, + "Y113F", + "mutant" + ], + [ + 98, + 103, + "C129S", + "mutant" + ], + [ + 104, + 110, + "mutant", + "protein_state" + ], + [ + 118, + 126, + "presence", + "protein_state" + ], + [ + 130, + 140, + "absence of", + "protein_state" + ], + [ + 141, + 145, + "haem", + "chemical" + ], + [ + 147, + 157, + "dithionite", + "chemical" + ], + [ + 165, + 167, + "CO", + "chemical" + ], + [ + 219, + 225, + "PGRMC1", + "protein" + ], + [ + 241, + 245, + "haem", + "chemical" + ], + [ + 250, + 252, + "CO", + "chemical" + ] + ] + }, + { + "sid": 160, + "sent": "Haem-dependent dimerization of PGRMC1 is necessary for tumour proliferation mediated by EGFR signalling.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "Haem", + "chemical" + ], + [ + 15, + 27, + "dimerization", + "oligomeric_state" + ], + [ + 31, + 37, + "PGRMC1", + "protein" + ], + [ + 88, + 92, + "EGFR", + "protein_type" + ] + ] + }, + { + "sid": 161, + "sent": "(a) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44\u2013195), in either apo- or haem-bound form, were incubated with purified EGFR and co-immunoprecipitated with anti-FLAG antibody-conjugated beads.", + "section": "FIG", + "ner": [ + [ + 9, + 15, + "PGRMC1", + "protein" + ], + [ + 16, + 25, + "wild-type", + "protein_state" + ], + [ + 27, + 29, + "wt", + "protein_state" + ], + [ + 35, + 40, + "Y113F", + "mutant" + ], + [ + 41, + 47, + "mutant", + "protein_state" + ], + [ + 62, + 68, + "44\u2013195", + "residue_range" + ], + [ + 81, + 84, + "apo", + "protein_state" + ], + [ + 89, + 99, + "haem-bound", + "protein_state" + ], + [ + 111, + 120, + "incubated", + "experimental_method" + ], + [ + 135, + 139, + "EGFR", + "protein_type" + ], + [ + 144, + 165, + "co-immunoprecipitated", + "experimental_method" + ] + ] + }, + { + "sid": 162, + "sent": "Input and bound proteins were detected by Western blotting.", + "section": "FIG", + "ner": [ + [ + 42, + 58, + "Western blotting", + "experimental_method" + ], + [ + 42, + 58, + "Western blotting", + "experimental_method" + ] + ] + }, + { + "sid": 163, + "sent": "(b) In vitro binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt (a.a.44\u2013195) and purified EGFR with or without treatment of RuCl3 and CORM3.", + "section": "FIG", + "ner": [ + [ + 4, + 26, + "In vitro binding assay", + "experimental_method" + ], + [ + 57, + 67, + "haem-bound", + "protein_state" + ], + [ + 73, + 79, + "PGRMC1", + "protein" + ], + [ + 80, + 82, + "wt", + "protein_state" + ], + [ + 88, + 94, + "44\u2013195", + "residue_range" + ], + [ + 109, + 113, + "EGFR", + "protein_type" + ], + [ + 143, + 148, + "RuCl3", + "chemical" + ], + [ + 153, + 158, + "CORM3", + "chemical" + ] + ] + }, + { + "sid": 164, + "sent": "(c) FLAG-PGRMC1 wt or Y113F (full length) was over-expressed in HCT116 cells and immunoprecipitated with anti-FLAG antibody-conjugated beads.", + "section": "FIG", + "ner": [ + [ + 9, + 15, + "PGRMC1", + "protein" + ], + [ + 16, + 18, + "wt", + "protein_state" + ], + [ + 22, + 27, + "Y113F", + "mutant" + ], + [ + 29, + 40, + "full length", + "protein_state" + ], + [ + 46, + 60, + "over-expressed", + "experimental_method" + ], + [ + 81, + 99, + "immunoprecipitated", + "experimental_method" + ] + ] + }, + { + "sid": 165, + "sent": "Co-immunoprecipitated proteins (FLAG-PGRMC1, endogenous PGRMC1 and EGFR) were detected with Western blotting by using anti-PGRMC1 or anti-EGFR antibody.", + "section": "FIG", + "ner": [ + [ + 0, + 21, + "Co-immunoprecipitated", + "experimental_method" + ], + [ + 37, + 43, + "PGRMC1", + "protein" + ], + [ + 45, + 55, + "endogenous", + "protein_state" + ], + [ + 56, + 62, + "PGRMC1", + "protein" + ], + [ + 67, + 71, + "EGFR", + "protein_type" + ], + [ + 92, + 108, + "Western blotting", + "experimental_method" + ], + [ + 123, + 129, + "PGRMC1", + "protein" + ], + [ + 138, + 142, + "EGFR", + "protein_type" + ] + ] + }, + { + "sid": 166, + "sent": "(d) HCT116 cells were treated with or without 250\u2009\u03bcmol\u2009l\u22121 of succinylacetone (SA) for 48\u2009h. The intracellular haem was extracted and quantified by reverse-phase HPLC.", + "section": "FIG", + "ner": [ + [ + 62, + 77, + "succinylacetone", + "chemical" + ], + [ + 79, + 81, + "SA", + "chemical" + ], + [ + 111, + 115, + "haem", + "chemical" + ], + [ + 148, + 166, + "reverse-phase HPLC", + "experimental_method" + ] + ] + }, + { + "sid": 167, + "sent": "of four separate experiments. **P<0.01 using unpaired Student's t-test. (e) Co-immunoprecipitation assay was performed as in (c) with or without SA treatment in HCT116 cells.", + "section": "FIG", + "ner": [ + [ + 54, + 70, + "Student's t-test", + "experimental_method" + ], + [ + 76, + 104, + "Co-immunoprecipitation assay", + "experimental_method" + ], + [ + 145, + 147, + "SA", + "chemical" + ] + ] + }, + { + "sid": 168, + "sent": "(f) HCT116 cells expressing control shRNA or those knocking down PGRMC1 (PGRMC1-KD) were treated with EGF or left untreated, and components of the EGFR signaling pathway were detected by Western blotting.", + "section": "FIG", + "ner": [ + [ + 36, + 41, + "shRNA", + "chemical" + ], + [ + 51, + 64, + "knocking down", + "experimental_method" + ], + [ + 65, + 71, + "PGRMC1", + "protein" + ], + [ + 73, + 82, + "PGRMC1-KD", + "mutant" + ], + [ + 102, + 105, + "EGF", + "protein_type" + ], + [ + 147, + 151, + "EGFR", + "protein_type" + ], + [ + 187, + 203, + "Western blotting", + "experimental_method" + ] + ] + }, + { + "sid": 169, + "sent": "(g,h) HCT116 cells were treated with or without EGF, SA, RuCl3 and CORM3 as indicated, and components of the EGFR signaling pathway were detected by Western blotting.", + "section": "FIG", + "ner": [ + [ + 48, + 51, + "EGF", + "protein_type" + ], + [ + 53, + 55, + "SA", + "chemical" + ], + [ + 57, + 62, + "RuCl3", + "chemical" + ], + [ + 67, + 72, + "CORM3", + "chemical" + ], + [ + 109, + 113, + "EGFR", + "protein_type" + ], + [ + 149, + 165, + "Western blotting", + "experimental_method" + ] + ] + }, + { + "sid": 170, + "sent": "Haem-dependent dimerization of PGRMC1 accelerates tumour growth through the EGFR signaling pathway.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "Haem", + "chemical" + ], + [ + 15, + 27, + "dimerization", + "oligomeric_state" + ], + [ + 31, + 37, + "PGRMC1", + "protein" + ], + [ + 76, + 80, + "EGFR", + "protein_type" + ] + ] + }, + { + "sid": 171, + "sent": "(a) Nucleotide sequences of PGRMC1 targeted by shRNA and of the shRNA-resistant full length PGRMC1 expression vector.", + "section": "FIG", + "ner": [ + [ + 28, + 34, + "PGRMC1", + "protein" + ], + [ + 47, + 52, + "shRNA", + "chemical" + ], + [ + 64, + 79, + "shRNA-resistant", + "protein_state" + ], + [ + 80, + 91, + "full length", + "protein_state" + ], + [ + 92, + 98, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 172, + "sent": "Stable PGRMC1-knockdown (PGRMC1-KD) HCT116 cells were transiently transfected with the shRNA-resistant expression vector of wild-type PGRMC1 (wt) or the Y113F mutant (Y113F).", + "section": "FIG", + "ner": [ + [ + 7, + 23, + "PGRMC1-knockdown", + "mutant" + ], + [ + 25, + 34, + "PGRMC1-KD", + "mutant" + ], + [ + 54, + 77, + "transiently transfected", + "experimental_method" + ], + [ + 87, + 102, + "shRNA-resistant", + "protein_state" + ], + [ + 103, + 120, + "expression vector", + "experimental_method" + ], + [ + 124, + 133, + "wild-type", + "protein_state" + ], + [ + 134, + 140, + "PGRMC1", + "protein" + ], + [ + 142, + 144, + "wt", + "protein_state" + ], + [ + 153, + 158, + "Y113F", + "mutant" + ], + [ + 159, + 165, + "mutant", + "protein_state" + ], + [ + 167, + 172, + "Y113F", + "mutant" + ] + ] + }, + { + "sid": 173, + "sent": "(b) Erlotinib was added to HCT116 (control) cells, PGRMC1-KD cells or PGRMC1-KD cells expressing shRNA-resistant PGRMC1 wt or Y113F, and cell viability was examined by MTT assay.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "Erlotinib", + "chemical" + ], + [ + 51, + 60, + "PGRMC1-KD", + "mutant" + ], + [ + 70, + 79, + "PGRMC1-KD", + "mutant" + ], + [ + 97, + 112, + "shRNA-resistant", + "protein_state" + ], + [ + 113, + 119, + "PGRMC1", + "protein" + ], + [ + 120, + 122, + "wt", + "protein_state" + ], + [ + 126, + 131, + "Y113F", + "mutant" + ], + [ + 168, + 177, + "MTT assay", + "experimental_method" + ] + ] + }, + { + "sid": 174, + "sent": "of four separate experiments. *P<0.01 using ANOVA with Fischer's LSD test.", + "section": "FIG", + "ner": [ + [ + 30, + 32, + "*P", + "evidence" + ], + [ + 44, + 49, + "ANOVA", + "experimental_method" + ], + [ + 55, + 73, + "Fischer's LSD test", + "experimental_method" + ], + [ + 30, + 32, + "*P", + "evidence" + ], + [ + 44, + 49, + "ANOVA", + "experimental_method" + ], + [ + 55, + 73, + "Fischer's LSD test", + "experimental_method" + ] + ] + }, + { + "sid": 175, + "sent": "(c) Spheroid formation in control and PGRMC1-KD HCT116 cells.", + "section": "FIG", + "ner": [ + [ + 38, + 47, + "PGRMC1-KD", + "mutant" + ] + ] + }, + { + "sid": 176, + "sent": "The graph represents mean\u00b1s.e. of each spheroid size. *P<0.01 using ANOVA with Fischer's LSD test.", + "section": "FIG", + "ner": [ + [ + 54, + 56, + "*P", + "evidence" + ], + [ + 68, + 73, + "ANOVA", + "experimental_method" + ], + [ + 79, + 97, + "Fischer's LSD test", + "experimental_method" + ] + ] + }, + { + "sid": 177, + "sent": "Scale bar: 0.1\u2009mm. (d) Tumour-bearing livers of NOG mice at 10 days after intrasplenic injection of HCT116 (control) or PGRMC1-KD cells.", + "section": "FIG", + "ner": [ + [ + 74, + 96, + "intrasplenic injection", + "experimental_method" + ], + [ + 120, + 129, + "PGRMC1-KD", + "mutant" + ] + ] + }, + { + "sid": 178, + "sent": "of 10 separate experiments. *P<0.05 using unpaired Student's t-test.", + "section": "FIG", + "ner": [ + [ + 28, + 30, + "*P", + "evidence" + ], + [ + 51, + 67, + "Student's t-test", + "experimental_method" + ] + ] + }, + { + "sid": 179, + "sent": "Haem-dependent PGRMC1 dimerization enhances tumour chemoresistance through interaction with cytochromes P450.", + "section": "FIG", + "ner": [ + [ + 0, + 4, + "Haem", + "chemical" + ], + [ + 15, + 21, + "PGRMC1", + "protein" + ], + [ + 22, + 34, + "dimerization", + "oligomeric_state" + ], + [ + 92, + 108, + "cytochromes P450", + "protein_type" + ] + ] + }, + { + "sid": 180, + "sent": "(a,b) FLAG-PGRMC1 wild-type (wt) and Y113F mutant proteins (a.a.44\u2013195), in either apo or haem-bound form, were incubated with CYP1A2 (a) or CYP3A4 (b) and immunoprecipitated with anti-FLAG antibody-conjugated beads.", + "section": "FIG", + "ner": [ + [ + 11, + 17, + "PGRMC1", + "protein" + ], + [ + 18, + 27, + "wild-type", + "protein_state" + ], + [ + 29, + 31, + "wt", + "protein_state" + ], + [ + 37, + 42, + "Y113F", + "mutant" + ], + [ + 43, + 49, + "mutant", + "protein_state" + ], + [ + 64, + 70, + "44\u2013195", + "residue_range" + ], + [ + 83, + 86, + "apo", + "protein_state" + ], + [ + 90, + 100, + "haem-bound", + "protein_state" + ], + [ + 112, + 121, + "incubated", + "experimental_method" + ], + [ + 127, + 133, + "CYP1A2", + "protein" + ], + [ + 141, + 147, + "CYP3A4", + "protein" + ], + [ + 156, + 174, + "immunoprecipitated", + "experimental_method" + ] + ] + }, + { + "sid": 181, + "sent": "(c) Binding assay was performed as in (a) using haem-bound FLAG-PGRMC1 wt and CYP1A2 with or without RuCl3 and CORM3.", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "Binding assay", + "experimental_method" + ], + [ + 48, + 58, + "haem-bound", + "protein_state" + ], + [ + 64, + 70, + "PGRMC1", + "protein" + ], + [ + 71, + 73, + "wt", + "protein_state" + ], + [ + 78, + 84, + "CYP1A2", + "protein" + ], + [ + 101, + 106, + "RuCl3", + "chemical" + ], + [ + 111, + 116, + "CORM3", + "chemical" + ] + ] + }, + { + "sid": 182, + "sent": "(d) Schematic illustration of doxorubicin metabolism is shown on the left.", + "section": "FIG", + "ner": [ + [ + 30, + 41, + "doxorubicin", + "chemical" + ] + ] + }, + { + "sid": 183, + "sent": "Doxorubicin was incubated with HCT116 cells expressing control shRNA or shPGRMC1 (PGRMC1-KD), and the doxorubicinol/doxorubicin ratios in cell pellets were determined using LC-MS.", + "section": "FIG", + "ner": [ + [ + 0, + 11, + "Doxorubicin", + "chemical" + ], + [ + 16, + 25, + "incubated", + "experimental_method" + ], + [ + 63, + 68, + "shRNA", + "chemical" + ], + [ + 72, + 80, + "shPGRMC1", + "chemical" + ], + [ + 82, + 91, + "PGRMC1-KD", + "mutant" + ], + [ + 102, + 115, + "doxorubicinol", + "chemical" + ], + [ + 116, + 127, + "doxorubicin", + "chemical" + ], + [ + 173, + 178, + "LC-MS", + "experimental_method" + ] + ] + }, + { + "sid": 184, + "sent": "of four separate experiments. **P<0.01 versus control using unpaired Student's t-test. (e) Indicated amounts of doxorubicin were added to HCT116 (control) cells, PGRMC1-KD cells, or PGRMC1-KD cells expressing shRNA-resistant full-length PGRMC1 wt or Y113F, and cell viability was examined by MTT assay.", + "section": "FIG", + "ner": [ + [ + 31, + 33, + "*P", + "evidence" + ], + [ + 69, + 85, + "Student's t-test", + "experimental_method" + ], + [ + 112, + 123, + "doxorubicin", + "chemical" + ], + [ + 162, + 171, + "PGRMC1-KD", + "mutant" + ], + [ + 182, + 191, + "PGRMC1-KD", + "mutant" + ], + [ + 209, + 224, + "shRNA-resistant", + "protein_state" + ], + [ + 225, + 236, + "full-length", + "protein_state" + ], + [ + 237, + 243, + "PGRMC1", + "protein" + ], + [ + 244, + 246, + "wt", + "protein_state" + ], + [ + 250, + 255, + "Y113F", + "mutant" + ], + [ + 292, + 301, + "MTT assay", + "experimental_method" + ] + ] + }, + { + "sid": 185, + "sent": "Schematic diagram for the regulation of PGRMC1 functions.", + "section": "FIG", + "ner": [ + [ + 40, + 46, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 186, + "sent": "Apo-PGRMC1 exists as an inactive monomer.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "Apo", + "protein_state" + ], + [ + 4, + 10, + "PGRMC1", + "protein" + ], + [ + 24, + 32, + "inactive", + "protein_state" + ], + [ + 33, + 40, + "monomer", + "oligomeric_state" + ] + ] + }, + { + "sid": 187, + "sent": "On binding to haem, PGRMC1 forms a dimer through stacking interactions between the haem moieties, which enables PGRMC1 to interact with EGFR and cytochromes P450, leading to an enhanced proliferation and chemoresistance of cancer cells.", + "section": "FIG", + "ner": [ + [ + 3, + 13, + "binding to", + "protein_state" + ], + [ + 14, + 18, + "haem", + "chemical" + ], + [ + 20, + 26, + "PGRMC1", + "protein" + ], + [ + 35, + 40, + "dimer", + "oligomeric_state" + ], + [ + 49, + 70, + "stacking interactions", + "bond_interaction" + ], + [ + 83, + 87, + "haem", + "chemical" + ], + [ + 112, + 118, + "PGRMC1", + "protein" + ], + [ + 136, + 140, + "EGFR", + "protein_type" + ], + [ + 145, + 161, + "cytochromes P450", + "protein_type" + ] + ] + }, + { + "sid": 188, + "sent": "CO interferes with the stacking interactions of the haems and thereby inhibits PGRMC1 functions.", + "section": "FIG", + "ner": [ + [ + 0, + 2, + "CO", + "chemical" + ], + [ + 23, + 44, + "stacking interactions", + "bond_interaction" + ], + [ + 52, + 57, + "haems", + "chemical" + ], + [ + 79, + 85, + "PGRMC1", + "protein" + ] + ] + }, + { + "sid": 189, + "sent": "PGRMC1 proteins exhibit haem-dependent dimerization in solution.", + "section": "TABLE", + "ner": [ + [ + 39, + 51, + "dimerization", + "oligomeric_state" + ] + ] + }, + { + "sid": 190, + "sent": "\u00a0\tApo form\tHaem-bound form\t \t\u00a0\t\u00a0\tMass (Da)\t\u00a0\tMass (Da)\t \taPGRMC1 wt (a.a.44\u2013195)\t \t\u2003ESI-MS\t\u2014\t17,844.14\t\u2014\t36,920.19\t \t\u2003Theoretical\t\u00a0\t17,843.65\t\u00a0\t36,918.06\t \t\u00a0\tHydrodynamic radius 10\u22129 (m)\tMW (kDa)\tHydrodynamic radius 10\u22129 (m)\tMW (kDa)\t \t\u2003DOSY\t2.04\u20132.15\t20\t2.94\u20133.02\t42\t \t\u00a0\tS20,w (S)\tMW (kDa)\tS20,w (S)\tMW (kDa)\t \t\u2003SV-AUC\t1.9\t17.6\t3.1\t35.5\t \t\u00a0\t\u00a0\t\u00a0\t\u00a0\t\u00a0\t \tbPGRMC1 C129S (a.a.44\u2013195)\t \t\u2003ESI-MS\t\u2014\t17,827.91\t\u2014\t36,887.07\t \t\u2003Theoretical\t\u00a0\t17,827.59\t\u00a0\t36,885.6\t \t\u00a0\tS20,w (S)\tMW (kDa)\tS20,w (S)\tMW (kDa)\t \t\u2003SV-AUC\t2.0\t18.1\t3.1\t35.8\t \t", + "section": "TABLE", + "ner": [ + [ + 360, + 365, + "C129S", + "mutant" + ] + ] + }, + { + "sid": 191, + "sent": "Differences in molecular weights of the wild-type (wt; a) and the C129S mutant (b) PGRMC1 proteins in the absence (apo form) or the presence of haem (haem-bound form).", + "section": "TABLE", + "ner": [ + [ + 66, + 71, + "C129S", + "mutant" + ] + ] + }, + { + "sid": 192, + "sent": "The protein sizes of the wt and C129S PGRMC1 cytosolic domains (a.a.44\u2013195) in the presence or absence of haem were estimated by ESI-MS, DOSY and SV-AUC.", + "section": "TABLE", + "ner": [ + [ + 32, + 37, + "C129S", + "mutant" + ] + ] + } + ] + }, + "PMC4887163": { + "annotations": [ + { + "sid": 0, + "sent": "Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity", + "section": "TITLE", + "ner": [ + [ + 19, + 34, + "T cell receptor", + "protein_type" + ], + [ + 66, + 73, + "insulin", + "chemical" + ] + ] + }, + { + "sid": 1, + "sent": "However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide\u2013major histocompatibility complexes (pMHC) are unclear.", + "section": "ABSTRACT", + "ner": [ + [ + 46, + 69, + "T cell antigen receptor", + "complex_assembly" + ], + [ + 71, + 74, + "TCR", + "complex_assembly" + ], + [ + 108, + 150, + "peptide\u2013major histocompatibility complexes", + "complex_assembly" + ], + [ + 152, + 156, + "pMHC", + "complex_assembly" + ] + ] + }, + { + "sid": 2, + "sent": "Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I\u2013restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides.", + "section": "ABSTRACT", + "ner": [ + [ + 49, + 54, + "human", + "species" + ], + [ + 56, + 69, + "preproinsulin", + "protein" + ], + [ + 80, + 83, + "MHC", + "complex_assembly" + ] + ] + }, + { + "sid": 3, + "sent": "We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide.", + "section": "ABSTRACT", + "ner": [ + [ + 29, + 39, + "structures", + "evidence" + ], + [ + 47, + 54, + "1E6 TCR", + "complex_assembly" + ], + [ + 55, + 63, + "bound to", + "protein_state" + ], + [ + 66, + 89, + "altered peptide ligands", + "chemical" + ] + ] + }, + { + "sid": 4, + "sent": "Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key\u2013like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot.", + "section": "ABSTRACT", + "ner": [ + [ + 20, + 30, + "structures", + "evidence" + ], + [ + 149, + 156, + "1E6 TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 5, + "sent": "Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand.", + "section": "ABSTRACT", + "ner": [ + [ + 30, + 37, + "1E6 TCR", + "complex_assembly" + ], + [ + 60, + 94, + "antipathogen-like binding affinity", + "evidence" + ] + ] + }, + { + "sid": 6, + "sent": "T cells perform an essential role in adaptive immunity by interrogating the host proteome for anomalies, classically by recognizing peptides bound in major histocompatibility (MHC) molecules at the cell surface.", + "section": "INTRO", + "ner": [ + [ + 150, + 174, + "major histocompatibility", + "complex_assembly" + ], + [ + 176, + 179, + "MHC", + "complex_assembly" + ] + ] + }, + { + "sid": 7, + "sent": "Recent data supports the notion that, to perform this role, the highly variable \u03b1\u03b2 T cell antigen receptor (TCR) must be able to recognize thousands, if not millions, of different peptide ligands.", + "section": "INTRO", + "ner": [ + [ + 64, + 79, + "highly variable", + "protein_state" + ], + [ + 80, + 106, + "\u03b1\u03b2 T cell antigen receptor", + "complex_assembly" + ], + [ + 108, + 111, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 8, + "sent": "This ability is required to enable the estimated 25 million distinct TCRs expressed in humans to provide effective immune coverage against all possible foreign peptide antigens.", + "section": "INTRO", + "ner": [ + [ + 69, + 73, + "TCRs", + "complex_assembly" + ], + [ + 87, + 93, + "humans", + "species" + ] + ] + }, + { + "sid": 9, + "sent": "Several mechanisms, by which TCRs could bind to a large number of different peptide-MHC (pMHC), have been proposed.", + "section": "INTRO", + "ner": [ + [ + 29, + 33, + "TCRs", + "complex_assembly" + ], + [ + 76, + 87, + "peptide-MHC", + "complex_assembly" + ], + [ + 89, + 93, + "pMHC", + "complex_assembly" + ] + ] + }, + { + "sid": 10, + "sent": "Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations.", + "section": "INTRO", + "ner": [ + [ + 0, + 10, + "Structures", + "evidence" + ], + [ + 14, + 23, + "unligated", + "protein_state" + ], + [ + 28, + 35, + "ligated", + "protein_state" + ], + [ + 36, + 40, + "TCRs", + "complex_assembly" + ], + [ + 61, + 64, + "TCR", + "complex_assembly" + ], + [ + 65, + 99, + "complementarity determining region", + "structure_element" + ], + [ + 101, + 104, + "CDR", + "structure_element" + ], + [ + 106, + 111, + "loops", + "structure_element" + ], + [ + 178, + 182, + "loop", + "structure_element" + ] + ] + }, + { + "sid": 11, + "sent": "Both MHC and peptide have also been shown to undergo structural changes upon TCR binding, mediating an induced fit between the TCR and pMHC.", + "section": "INTRO", + "ner": [ + [ + 5, + 8, + "MHC", + "complex_assembly" + ], + [ + 13, + 20, + "peptide", + "chemical" + ], + [ + 77, + 80, + "TCR", + "complex_assembly" + ], + [ + 127, + 130, + "TCR", + "complex_assembly" + ], + [ + 135, + 139, + "pMHC", + "complex_assembly" + ] + ] + }, + { + "sid": 12, + "sent": "Other studies, mainly in the murine system, have demonstrated that the same TCR can interact with different pMHCs using a common or divergent modality.", + "section": "INTRO", + "ner": [ + [ + 29, + 35, + "murine", + "taxonomy_domain" + ], + [ + 76, + 79, + "TCR", + "complex_assembly" + ], + [ + 108, + 113, + "pMHCs", + "complex_assembly" + ] + ] + }, + { + "sid": 13, + "sent": "Recent studies in model murine systems demonstrate that TCR cross-reactivity can be governed by recognition of a conserved region in the peptide that allows tolerance of peptide sequence variation outside of this hotspot.", + "section": "INTRO", + "ner": [ + [ + 24, + 30, + "murine", + "taxonomy_domain" + ], + [ + 56, + 59, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 14, + "sent": "We recently reported that the 1E6 human CD8+ T cell clone \u2014 which mediates the destruction of \u03b2 cells through the recognition of a major, HLA-A*0201\u2013restricted, preproinsulin signal peptide (ALWGPDPAAA15\u201324) \u2014 can recognize upwards of 1 million different peptides.", + "section": "INTRO", + "ner": [ + [ + 34, + 39, + "human", + "species" + ], + [ + 138, + 148, + "HLA-A*0201", + "protein" + ], + [ + 161, + 174, + "preproinsulin", + "protein" + ], + [ + 175, + 189, + "signal peptide", + "structure_element" + ], + [ + 191, + 206, + "ALWGPDPAAA15\u201324", + "chemical" + ] + ] + }, + { + "sid": 15, + "sent": "CD8+ T cells that recognize HLA-A*0201\u2013ALWGPDPAAA have been shown to populate insulitic lesions in patients with type 1 diabetes (T1D).", + "section": "INTRO", + "ner": [ + [ + 28, + 49, + "HLA-A*0201\u2013ALWGPDPAAA", + "complex_assembly" + ] + ] + }, + { + "sid": 16, + "sent": "We demonstrated that the TCR from the 1E6 T cell clone bound to HLA-A*0201\u2013ALWGPDPAAA using a limited footprint and very weak binding affinity.", + "section": "INTRO", + "ner": [ + [ + 25, + 28, + "TCR", + "complex_assembly" + ], + [ + 55, + 63, + "bound to", + "protein_state" + ], + [ + 64, + 85, + "HLA-A*0201\u2013ALWGPDPAAA", + "complex_assembly" + ], + [ + 126, + 142, + "binding affinity", + "evidence" + ] + ] + }, + { + "sid": 17, + "sent": "This first experimental evidence of a high level of CD8+ T cell cross-reactivity in a human autoimmune disease system hinted toward molecular mimicry by a more potent pathogenic peptide as a potential mechanism leading to \u03b2 cell destruction.", + "section": "INTRO", + "ner": [ + [ + 86, + 91, + "human", + "species" + ] + ] + }, + { + "sid": 18, + "sent": "Here, we solved the structure of the 1E6 TCR with 7 altered peptide ligands (APLs) determined by our previously published combinatorial peptide library (CPL) screening, 2 of which mapped within human pathogens.", + "section": "INTRO", + "ner": [ + [ + 9, + 15, + "solved", + "experimental_method" + ], + [ + 20, + 29, + "structure", + "evidence" + ], + [ + 37, + 44, + "1E6 TCR", + "complex_assembly" + ], + [ + 52, + 75, + "altered peptide ligands", + "chemical" + ], + [ + 77, + 81, + "APLs", + "chemical" + ], + [ + 122, + 167, + "combinatorial peptide library (CPL) screening", + "experimental_method" + ], + [ + 194, + 199, + "human", + "species" + ] + ] + }, + { + "sid": 19, + "sent": "These APLs differed from the natural preproinsulin peptide by up to 7 of 10 residues.", + "section": "INTRO", + "ner": [ + [ + 6, + 10, + "APLs", + "chemical" + ], + [ + 37, + 50, + "preproinsulin", + "protein" + ] + ] + }, + { + "sid": 20, + "sent": "We also solved the structure of each unligated APL to investigate whether structural changes occurred before or after binding \u2014 which, combined with an in-depth cellular and biophysical analysis of the 1E6 interaction with each APL, demonstrated the molecular mechanism mediating the high level of cross-reactivity exhibited by this preproinsulin-reactive human CD8+ T cell clone.", + "section": "INTRO", + "ner": [ + [ + 8, + 14, + "solved", + "experimental_method" + ], + [ + 19, + 28, + "structure", + "evidence" + ], + [ + 37, + 46, + "unligated", + "protein_state" + ], + [ + 47, + 50, + "APL", + "chemical" + ], + [ + 161, + 194, + "cellular and biophysical analysis", + "experimental_method" + ], + [ + 228, + 231, + "APL", + "chemical" + ], + [ + 333, + 346, + "preproinsulin", + "protein" + ], + [ + 356, + 361, + "human", + "species" + ] + ] + }, + { + "sid": 21, + "sent": "The 1E6 T cell clone recognizes APLs across a large dynamic range.", + "section": "RESULTS", + "ner": [ + [ + 32, + 36, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 22, + "sent": "We have previously demonstrated that the 1E6 T cell clone can recognize over 1 million different peptides with a potency comparable with, or better than, the cognate preproinsulin peptide ALWGPDPAAA.", + "section": "RESULTS", + "ner": [ + [ + 166, + 179, + "preproinsulin", + "protein" + ], + [ + 188, + 198, + "ALWGPDPAAA", + "chemical" + ] + ] + }, + { + "sid": 23, + "sent": "From this large functional scan, we selected 7 different APLs that activated the 1E6 T cell clone across a wide (4-log) functional range (Table 1).", + "section": "RESULTS", + "ner": [ + [ + 57, + 61, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "Two of these peptides, MVWGPDPLYV and RQFGPDWIVA (bold text signifies amino acids that are different from the index preproinsulin\u2013derived sequence), are contained within the proteomes of the human pathogens Bacteroides fragilis/thetaiotaomicron and Clostridium asparagiforme, respectively.", + "section": "RESULTS", + "ner": [ + [ + 23, + 33, + "MVWGPDPLYV", + "chemical" + ], + [ + 38, + 48, + "RQFGPDWIVA", + "chemical" + ], + [ + 116, + 129, + "preproinsulin", + "protein" + ], + [ + 191, + 196, + "human", + "species" + ], + [ + 207, + 244, + "Bacteroides fragilis/thetaiotaomicron", + "species" + ], + [ + 249, + 274, + "Clostridium asparagiforme", + "species" + ] + ] + }, + { + "sid": 25, + "sent": "Competitive functional testing revealed that the preproinsulin-derived sequence ALWGPDPAAA was one of the least potent targets for 1E6, with only the MVWGPDPLYV and YLGGPDFPTI demonstrating a similar low-activity profile in MIP-1\u03b2 secretion and target killing assays (Figure 1, A and B).", + "section": "RESULTS", + "ner": [ + [ + 0, + 30, + "Competitive functional testing", + "experimental_method" + ], + [ + 49, + 62, + "preproinsulin", + "protein" + ], + [ + 80, + 90, + "ALWGPDPAAA", + "chemical" + ], + [ + 150, + 160, + "MVWGPDPLYV", + "chemical" + ], + [ + 165, + 175, + "YLGGPDFPTI", + "chemical" + ], + [ + 224, + 230, + "MIP-1\u03b2", + "protein" + ] + ] + }, + { + "sid": 26, + "sent": "The RQFGPDWIVA sequence (present in C. asparagiforme) activated the 1E6 T cell with around 1 log\u2013greater potency compared with ALWGPDPAAA.", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "RQFGPDWIVA", + "chemical" + ], + [ + 36, + 52, + "C. asparagiforme", + "species" + ], + [ + 127, + 137, + "ALWGPDPAAA", + "chemical" + ] + ] + }, + { + "sid": 27, + "sent": "At the other end of the spectrum, the RQFGPDFPTI peptide stimulated MIP-1\u03b2 release and killing by 1E6 at an exogenous peptide concentration 2\u20133 logs lower compared with ALWGPDPAAA.", + "section": "RESULTS", + "ner": [ + [ + 38, + 48, + "RQFGPDFPTI", + "chemical" + ], + [ + 68, + 74, + "MIP-1\u03b2", + "protein" + ], + [ + 169, + 179, + "ALWGPDPAAA", + "chemical" + ] + ] + }, + { + "sid": 28, + "sent": "The pattern of peptide potency was closely mirrored by pMHC tetramer staining experiments (Figure 1C and plots shown in Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI85679DS1).", + "section": "RESULTS", + "ner": [ + [ + 55, + 59, + "pMHC", + "complex_assembly" + ], + [ + 60, + 77, + "tetramer staining", + "experimental_method" + ] + ] + }, + { + "sid": 29, + "sent": "Here, the A2-RQFGPDFPTI tetramer stained 1E6 with the greatest MFI, gradually decreasing to the weakest tetramers: A2-MVWGPDPLYV and -YLGGPDFPTI.", + "section": "RESULTS", + "ner": [ + [ + 10, + 23, + "A2-RQFGPDFPTI", + "chemical" + ], + [ + 24, + 32, + "tetramer", + "oligomeric_state" + ], + [ + 104, + 113, + "tetramers", + "oligomeric_state" + ], + [ + 115, + 128, + "A2-MVWGPDPLYV", + "chemical" + ], + [ + 134, + 144, + "YLGGPDFPTI", + "chemical" + ] + ] + }, + { + "sid": 30, + "sent": "To parallel the functional analysis, we also performed thermal melt (Tm) experiments using synchrotron radiation circular dichroism (SRCD) to investigate the stability of each APL (Figure 1D).", + "section": "RESULTS", + "ner": [ + [ + 55, + 67, + "thermal melt", + "experimental_method" + ], + [ + 69, + 71, + "Tm", + "evidence" + ], + [ + 91, + 131, + "synchrotron radiation circular dichroism", + "experimental_method" + ], + [ + 133, + 137, + "SRCD", + "experimental_method" + ], + [ + 176, + 179, + "APL", + "chemical" + ] + ] + }, + { + "sid": 31, + "sent": "The range of Tm was between 49.4\u00b0C (RQFGPDWIVA) and 60.3\u00b0C (YQFGPDFPIA), with an average approximately 55\u00b0C, similar to our previous findings.", + "section": "RESULTS", + "ner": [ + [ + 13, + 15, + "Tm", + "evidence" + ], + [ + 36, + 46, + "RQFGPDWIVA", + "chemical" + ], + [ + 60, + 70, + "YQFGPDFPIA", + "chemical" + ] + ] + }, + { + "sid": 32, + "sent": "This pattern of stability did not correlate with the T cell activation or tetramer staining experiments, indicating that peptide binding to the MHC do not explain ligand potency.", + "section": "RESULTS", + "ner": [ + [ + 74, + 91, + "tetramer staining", + "experimental_method" + ], + [ + 144, + 147, + "MHC", + "complex_assembly" + ] + ] + }, + { + "sid": 33, + "sent": "The 1E6 TCR can bind peptides with strong antipathogen-like affinities.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "1E6 TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 34, + "sent": "We, and others, have previously demonstrated that antipathogenic TCRs tend to bind with stronger affinity compared with self-reactive TCRs, likely a consequence of the deletion of T cells with high-affinity self-reactive TCR during thymic selection.", + "section": "RESULTS", + "ner": [ + [ + 65, + 69, + "TCRs", + "complex_assembly" + ], + [ + 97, + 105, + "affinity", + "evidence" + ], + [ + 134, + 138, + "TCRs", + "complex_assembly" + ], + [ + 198, + 206, + "affinity", + "evidence" + ], + [ + 221, + 224, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 35, + "sent": "In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell\u2013derived TCR with a biologically relevant ligand (KD > 200 \u03bcM; KD, equilibrium binding constant).", + "section": "RESULTS", + "ner": [ + [ + 35, + 42, + "1E6 TCR", + "complex_assembly" + ], + [ + 43, + 48, + "bound", + "protein_state" + ], + [ + 61, + 74, + "preproinsulin", + "protein" + ], + [ + 84, + 94, + "ALWGPDPAAA", + "chemical" + ], + [ + 113, + 121, + "affinity", + "evidence" + ], + [ + 148, + 153, + "human", + "species" + ], + [ + 174, + 177, + "TCR", + "complex_assembly" + ], + [ + 215, + 217, + "KD", + "evidence" + ], + [ + 228, + 230, + "KD", + "evidence" + ], + [ + 232, + 260, + "equilibrium binding constant", + "evidence" + ] + ] + }, + { + "sid": 36, + "sent": "Surface plasmon resonance (SPR) analysis of the 1E6 TCR\u2013pMHC interaction for all 7 APLs (Figure 2, A\u2013H) demonstrated that stronger binding affinity (represented as \u0394G\u00b0, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson\u2019s correlation analysis value of 0.8 (P = 0.01) (Figure 2I).", + "section": "RESULTS", + "ner": [ + [ + 0, + 25, + "Surface plasmon resonance", + "experimental_method" + ], + [ + 27, + 30, + "SPR", + "experimental_method" + ], + [ + 48, + 55, + "1E6 TCR", + "complex_assembly" + ], + [ + 56, + 60, + "pMHC", + "complex_assembly" + ], + [ + 83, + 87, + "APLs", + "chemical" + ], + [ + 131, + 147, + "binding affinity", + "evidence" + ], + [ + 164, + 167, + "\u0394G\u00b0", + "evidence" + ], + [ + 204, + 208, + "EC50", + "evidence" + ], + [ + 325, + 355, + "Pearson\u2019s correlation analysis", + "experimental_method" + ] + ] + }, + { + "sid": 37, + "sent": "It should be noted that this correlation, although consistent with the T cell killing experiments, uses only approximate affinities calculated for the 2 weakest ligands.", + "section": "RESULTS", + "ner": [ + [ + 121, + 131, + "affinities", + "evidence" + ] + ] + }, + { + "sid": 38, + "sent": "First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 \u03bcM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 \u03bcM, equivalent to the binding affinity of the very strongest antipathogen TCRs.", + "section": "RESULTS", + "ner": [ + [ + 75, + 95, + "TCR binding affinity", + "evidence" + ], + [ + 126, + 150, + "1E6 TCR binding affinity", + "evidence" + ], + [ + 159, + 172, + "A2-MVWGPDPLYV", + "chemical" + ], + [ + 185, + 187, + "KD", + "evidence" + ], + [ + 211, + 218, + "1E6 TCR", + "complex_assembly" + ], + [ + 219, + 227, + "bound to", + "protein_state" + ], + [ + 228, + 241, + "A2-RQFGPDFPTI", + "chemical" + ], + [ + 247, + 249, + "KD", + "evidence" + ], + [ + 278, + 294, + "binding affinity", + "evidence" + ], + [ + 330, + 334, + "TCRs", + "complex_assembly" + ] + ] + }, + { + "sid": 39, + "sent": "Third, the 1E6 TCR bound to A2-RQFGPDWIVA peptide, within the C. asparagiforme proteome, with approximately 4-fold stronger affinity than A2-ALWGPDPAAA, demonstrating the potential for a pathogen-derived antigen to initiate a response to the self-derived sequence.", + "section": "RESULTS", + "ner": [ + [ + 11, + 18, + "1E6 TCR", + "complex_assembly" + ], + [ + 19, + 27, + "bound to", + "protein_state" + ], + [ + 28, + 41, + "A2-RQFGPDWIVA", + "chemical" + ], + [ + 62, + 78, + "C. asparagiforme", + "species" + ], + [ + 124, + 132, + "affinity", + "evidence" + ], + [ + 138, + 151, + "A2-ALWGPDPAAA", + "chemical" + ] + ] + }, + { + "sid": 40, + "sent": "Finally, these data demonstrate the largest range of binding affinities reported for a natural, endogenous human TCR of more than 3 logs of magnitude (A2-MVWGPDPLYV vs. A2-RQFGPDFPTI).", + "section": "RESULTS", + "ner": [ + [ + 53, + 71, + "binding affinities", + "evidence" + ], + [ + 96, + 106, + "endogenous", + "protein_state" + ], + [ + 107, + 112, + "human", + "species" + ], + [ + 113, + 116, + "TCR", + "complex_assembly" + ], + [ + 151, + 164, + "A2-MVWGPDPLYV", + "chemical" + ], + [ + 169, + 182, + "A2-RQFGPDFPTI", + "chemical" + ] + ] + }, + { + "sid": 41, + "sent": "To confirm the affinity spread detected by SPR, and to evaluate whether experiments performed using soluble molecules were biologically relevant to events at the T cell surface, we determined the effective 2D affinity of each APL using an adhesion frequency assay in which a human rbc coated in pMHC acted as an adhesion sensor.", + "section": "RESULTS", + "ner": [ + [ + 15, + 23, + "affinity", + "evidence" + ], + [ + 43, + 46, + "SPR", + "experimental_method" + ], + [ + 206, + 217, + "2D affinity", + "evidence" + ], + [ + 226, + 229, + "APL", + "chemical" + ], + [ + 239, + 263, + "adhesion frequency assay", + "experimental_method" + ], + [ + 275, + 280, + "human", + "species" + ], + [ + 295, + 299, + "pMHC", + "complex_assembly" + ] + ] + }, + { + "sid": 42, + "sent": "In agreement with SPR experiments, the range of 2D affinities we detected differed by around 3 logs, with the A2-MVWGPDPLYV generating the weakest 2D affinity (2.6 \u00d7 10\u20135 AcKa \u03bcm4) and A2-RQFGPDFPTI the strongest (4.5 \u00d7 10\u20132 AcKa \u03bcm4) (Figure 2J).", + "section": "RESULTS", + "ner": [ + [ + 18, + 21, + "SPR", + "experimental_method" + ], + [ + 48, + 61, + "2D affinities", + "evidence" + ], + [ + 110, + 123, + "A2-MVWGPDPLYV", + "chemical" + ], + [ + 147, + 158, + "2D affinity", + "evidence" + ], + [ + 171, + 175, + "AcKa", + "evidence" + ], + [ + 185, + 198, + "A2-RQFGPDFPTI", + "chemical" + ], + [ + 225, + 229, + "AcKa", + "evidence" + ] + ] + }, + { + "sid": 43, + "sent": "As with the 3D affinity measurements, the 2D affinity measurements correlated well with the EC50 values for each ligand (Figure 2K) demonstrating a strong correlation (Pearson\u2019s correlation = 0.8, P = 0.01) between T cell antigen sensitivity and TCR binding affinity.", + "section": "RESULTS", + "ner": [ + [ + 12, + 23, + "3D affinity", + "evidence" + ], + [ + 42, + 53, + "2D affinity", + "evidence" + ], + [ + 92, + 96, + "EC50", + "evidence" + ], + [ + 168, + 189, + "Pearson\u2019s correlation", + "evidence" + ], + [ + 197, + 198, + "P", + "evidence" + ], + [ + 246, + 266, + "TCR binding affinity", + "evidence" + ] + ] + }, + { + "sid": 44, + "sent": "Of note, these data demonstrate a close agreement between the 3D affinity values generated using SPR and 2D affinity values generated using adhesion frequency assays.", + "section": "RESULTS", + "ner": [ + [ + 62, + 73, + "3D affinity", + "evidence" + ], + [ + 97, + 100, + "SPR", + "experimental_method" + ], + [ + 105, + 116, + "2D affinity", + "evidence" + ] + ] + }, + { + "sid": 45, + "sent": "The 1E6 TCR uses a consensus binding mode to engage multiple APLs.", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "1E6 TCR", + "complex_assembly" + ], + [ + 61, + 65, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 46, + "sent": "Our previous structure of the 1E6-A2-ALWGPDPAAA complex demonstrated a limited binding footprint between the TCR and pMHC.", + "section": "RESULTS", + "ner": [ + [ + 13, + 22, + "structure", + "evidence" + ], + [ + 30, + 47, + "1E6-A2-ALWGPDPAAA", + "complex_assembly" + ], + [ + 79, + 96, + "binding footprint", + "site" + ], + [ + 109, + 112, + "TCR", + "complex_assembly" + ], + [ + 117, + 121, + "pMHC", + "complex_assembly" + ] + ] + }, + { + "sid": 47, + "sent": "The low number of contacts between the 2 molecules most likely contributed to the weak binding affinity of the interaction.", + "section": "RESULTS", + "ner": [ + [ + 87, + 103, + "binding affinity", + "evidence" + ] + ] + }, + { + "sid": 48, + "sent": "In order to examine the mechanism by which the 1E6 TCR engaged a wide range of peptides with divergent binding affinities, we solved the structure of the 1E6 TCR in complex with all 7 APLs used in Figure 2.", + "section": "RESULTS", + "ner": [ + [ + 47, + 54, + "1E6 TCR", + "complex_assembly" + ], + [ + 103, + 121, + "binding affinities", + "evidence" + ], + [ + 126, + 132, + "solved", + "experimental_method" + ], + [ + 137, + 146, + "structure", + "evidence" + ], + [ + 154, + 161, + "1E6 TCR", + "complex_assembly" + ], + [ + 162, + 177, + "in complex with", + "protein_state" + ], + [ + 184, + 188, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 49, + "sent": "All structures were solved in space group P1 to 2\u20133 \u00c5 resolution with crystallographic Rwork/Rfree ratios within accepted limits as shown in the theoretically expected distribution (ref. and Supplemental Table 1).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 20, + 26, + "solved", + "experimental_method" + ], + [ + 87, + 105, + "Rwork/Rfree ratios", + "evidence" + ] + ] + }, + { + "sid": 50, + "sent": "The 1E6 TCR used a very similar overall binding modality to engage all of the APLs, with root mean square deviation ranging between 0.81 and 1.12 \u00c52 (compared with 1E6-A2-ALWGPDPAAA).", + "section": "RESULTS", + "ner": [ + [ + 4, + 11, + "1E6 TCR", + "complex_assembly" + ], + [ + 78, + 82, + "APLs", + "chemical" + ], + [ + 89, + 115, + "root mean square deviation", + "evidence" + ], + [ + 164, + 181, + "1E6-A2-ALWGPDPAAA", + "complex_assembly" + ] + ] + }, + { + "sid": 51, + "sent": "The relatively broad range of buried surface areas (1,670\u20131,920 \u00c52) did not correlate well with TCR binding affinity (Pearson\u2019s correlation = 0.45, P = 0.2).", + "section": "RESULTS", + "ner": [ + [ + 96, + 116, + "TCR binding affinity", + "evidence" + ], + [ + 118, + 139, + "Pearson\u2019s correlation", + "evidence" + ], + [ + 148, + 149, + "P", + "evidence" + ] + ] + }, + { + "sid": 52, + "sent": "The surface complementarity values (0.52\u20130.7) correlated slightly with affinity (Pearson\u2019s correlation = 0.7, P = 0.05) but could not explain all differences in binding (Figure 3A and Table 2).", + "section": "RESULTS", + "ner": [ + [ + 4, + 34, + "surface complementarity values", + "evidence" + ], + [ + 71, + 79, + "affinity", + "evidence" + ], + [ + 81, + 102, + "Pearson\u2019s correlation", + "evidence" + ], + [ + 110, + 111, + "P", + "evidence" + ] + ] + }, + { + "sid": 53, + "sent": "The TCR CDR loops were in a very similar position in all complexes, apart from some slight deviations in the TCR \u03b2-chain (Figure 3B); the peptides were all presented in a similar conformation (Figure 3C); and there was minimal variation in crossing angles of the TCR (42.3\u00b0\u201345.6\u00b0) (Figure 3D).", + "section": "RESULTS", + "ner": [ + [ + 4, + 7, + "TCR", + "complex_assembly" + ], + [ + 8, + 17, + "CDR loops", + "structure_element" + ], + [ + 109, + 112, + "TCR", + "complex_assembly" + ], + [ + 113, + 120, + "\u03b2-chain", + "structure_element" + ], + [ + 263, + 266, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 54, + "sent": "Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR \u03b1-chain positioned over the MHC class I (MHCI) \u03b12-helix and the TCR \u03b2-chain over the MHCI \u03b1-1 helix, straddling the peptide cargo.", + "section": "RESULTS", + "ner": [ + [ + 13, + 20, + "1E6 TCR", + "complex_assembly" + ], + [ + 66, + 69, + "APL", + "chemical" + ], + [ + 79, + 82, + "TCR", + "complex_assembly" + ], + [ + 83, + 90, + "\u03b1-chain", + "structure_element" + ], + [ + 111, + 122, + "MHC class I", + "complex_assembly" + ], + [ + 124, + 128, + "MHCI", + "complex_assembly" + ], + [ + 130, + 138, + "\u03b12-helix", + "structure_element" + ], + [ + 147, + 150, + "TCR", + "complex_assembly" + ], + [ + 151, + 158, + "\u03b2-chain", + "structure_element" + ], + [ + 168, + 172, + "MHCI", + "complex_assembly" + ], + [ + 173, + 182, + "\u03b1-1 helix", + "structure_element" + ] + ] + }, + { + "sid": 55, + "sent": "However, subtle differences in the respective interfaces were apparent (discussed below) and resulted in altered binding affinities of the respective complexes.", + "section": "RESULTS", + "ner": [ + [ + 46, + 56, + "interfaces", + "site" + ], + [ + 113, + 131, + "binding affinities", + "evidence" + ] + ] + }, + { + "sid": 56, + "sent": "Interactions between the 1E6 TCR and different APLs are focused around a conserved GPD peptide motif.", + "section": "RESULTS", + "ner": [ + [ + 25, + 32, + "1E6 TCR", + "complex_assembly" + ], + [ + 47, + 51, + "APLs", + "chemical" + ], + [ + 73, + 82, + "conserved", + "protein_state" + ], + [ + 83, + 100, + "GPD peptide motif", + "structure_element" + ] + ] + }, + { + "sid": 57, + "sent": "We next performed an in-depth atomic analysis of the contacts between the 1E6 TCR and each APL to determine the structural basis for the altered T cell peptide sensitivities and TCR binding affinities (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 30, + 45, + "atomic analysis", + "experimental_method" + ], + [ + 74, + 81, + "1E6 TCR", + "complex_assembly" + ], + [ + 91, + 94, + "APL", + "chemical" + ], + [ + 178, + 200, + "TCR binding affinities", + "evidence" + ] + ] + }, + { + "sid": 58, + "sent": "Concomitant with our global analysis of 1E6 TCR binding to the APLs, we observed a common interaction element, consistent with our previous findings, that utilized TCR residues Tyr97\u03b1 and Trp97\u03b2, forming an aromatic cap over a central GPD motif that was present in all of the APLs (Figure 4).", + "section": "RESULTS", + "ner": [ + [ + 40, + 47, + "1E6 TCR", + "complex_assembly" + ], + [ + 63, + 67, + "APLs", + "chemical" + ], + [ + 164, + 167, + "TCR", + "complex_assembly" + ], + [ + 177, + 183, + "Tyr97\u03b1", + "residue_name_number" + ], + [ + 188, + 194, + "Trp97\u03b2", + "residue_name_number" + ], + [ + 207, + 219, + "aromatic cap", + "structure_element" + ], + [ + 235, + 244, + "GPD motif", + "structure_element" + ], + [ + 276, + 280, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 59, + "sent": "Interactions between these 2 TCR and 3 peptide residues accounted for 41%\u201350% of the total contacts across all complexes (Table 2), demonstrating the conserved peptide centric binding mode utilized by the 1E6 TCR.", + "section": "RESULTS", + "ner": [ + [ + 29, + 32, + "TCR", + "complex_assembly" + ], + [ + 150, + 159, + "conserved", + "protein_state" + ], + [ + 205, + 212, + "1E6 TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 60, + "sent": "This fixed anchoring between the 2 molecules was important for stabilization of the TCR-pMHC complex, as \u2014 although other peptides without the \u2018GDP\u2019 motif were tested and shown to activate the 1E6 T cell clone \u2014 we were unable to measure robust affinities using SPR (data not shown).", + "section": "RESULTS", + "ner": [ + [ + 84, + 92, + "TCR-pMHC", + "complex_assembly" + ], + [ + 143, + 154, + "\u2018GDP\u2019 motif", + "structure_element" + ], + [ + 245, + 255, + "affinities", + "evidence" + ], + [ + 262, + 265, + "SPR", + "experimental_method" + ] + ] + }, + { + "sid": 61, + "sent": "These data support the requirement for a conserved interaction between the 1E6 TCR and the GPD motif, as we observed in our previously published 1E6-A2-ALWGPDPAAA structure.", + "section": "RESULTS", + "ner": [ + [ + 41, + 50, + "conserved", + "protein_state" + ], + [ + 75, + 82, + "1E6 TCR", + "complex_assembly" + ], + [ + 91, + 100, + "GPD motif", + "structure_element" + ], + [ + 145, + 162, + "1E6-A2-ALWGPDPAAA", + "complex_assembly" + ], + [ + 163, + 172, + "structure", + "evidence" + ] + ] + }, + { + "sid": 62, + "sent": "Focused hotspot binding around a conserved GPD motif enables the 1E6 TCR to tolerate peptide degeneracy.", + "section": "RESULTS", + "ner": [ + [ + 33, + 42, + "conserved", + "protein_state" + ], + [ + 43, + 52, + "GPD motif", + "structure_element" + ], + [ + 65, + 72, + "1E6 TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 63, + "sent": "Although the 1E6 TCR formed a similar overall interaction with each APL, the stabilization between the TCR and the GPD motif enabled fine differences in the contact network with both the peptide and MHC surface that allowed discrimination between each ligand (Figure 5).", + "section": "RESULTS", + "ner": [ + [ + 13, + 20, + "1E6 TCR", + "complex_assembly" + ], + [ + 68, + 71, + "APL", + "chemical" + ], + [ + 103, + 106, + "TCR", + "complex_assembly" + ], + [ + 115, + 124, + "GPD motif", + "structure_element" + ], + [ + 157, + 172, + "contact network", + "site" + ], + [ + 187, + 194, + "peptide", + "chemical" + ], + [ + 199, + 210, + "MHC surface", + "site" + ] + ] + }, + { + "sid": 64, + "sent": "For example, the 1E6 TCR made only 47 peptide contacts with A2-MVWGPDPLYV (KD = ~600 \u03bcM) compared with 63 and 57 contacts with A2-YQFGPDFPIA (KD = 7.4 \u03bcM) and A2-RQFGPDFPTI (KD = 0.5 \u03bcM), respectively.", + "section": "RESULTS", + "ner": [ + [ + 17, + 24, + "1E6 TCR", + "complex_assembly" + ], + [ + 60, + 73, + "A2-MVWGPDPLYV", + "chemical" + ], + [ + 75, + 77, + "KD", + "evidence" + ], + [ + 127, + 140, + "A2-YQFGPDFPIA", + "chemical" + ], + [ + 142, + 144, + "KD", + "evidence" + ], + [ + 159, + 172, + "A2-RQFGPDFPTI", + "chemical" + ], + [ + 174, + 176, + "KD", + "evidence" + ] + ] + }, + { + "sid": 65, + "sent": "Although the number of peptide contacts was a good predictor of TCR binding affinity for some of the APLs, for others, the correlation was poor (Pearson\u2019s correlation = 0.045, P = 0.92), possibly because of different resolutions for each complex structure.", + "section": "RESULTS", + "ner": [ + [ + 64, + 84, + "TCR binding affinity", + "evidence" + ], + [ + 101, + 105, + "APLs", + "chemical" + ], + [ + 145, + 166, + "Pearson\u2019s correlation", + "evidence" + ], + [ + 246, + 255, + "structure", + "evidence" + ] + ] + }, + { + "sid": 66, + "sent": "For example, the 1E6 TCR made 64 peptide contacts with A2-YLGGPDFPTI (KD = ~400 \u03bcM) compared with 43 contacts with A2-RQWGPDPAAV (KD = 7.8 \u03bcM).", + "section": "RESULTS", + "ner": [ + [ + 17, + 24, + "1E6 TCR", + "complex_assembly" + ], + [ + 55, + 68, + "A2-YLGGPDFPTI", + "chemical" + ], + [ + 70, + 72, + "KD", + "evidence" + ], + [ + 115, + 128, + "A2-RQWGPDPAAV", + "chemical" + ], + [ + 130, + 132, + "KD", + "evidence" + ] + ] + }, + { + "sid": 67, + "sent": "The most important peptide modification in terms of generating new contacts was peptide position 1.", + "section": "RESULTS", + "ner": [ + [ + 97, + 98, + "1", + "residue_number" + ] + ] + }, + { + "sid": 68, + "sent": "The stronger ligands all encoded larger side chains (Arg or Tyr) at peptide position 1 (Figure 5, E\u2013H), enabling interactions with 1E6 that were not present in the weaker APLs that lacked large side chains in this position (Figure 5, A, C, and D).", + "section": "RESULTS", + "ner": [ + [ + 53, + 56, + "Arg", + "residue_name" + ], + [ + 60, + 63, + "Tyr", + "residue_name" + ], + [ + 85, + 86, + "1", + "residue_number" + ], + [ + 171, + 175, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 69, + "sent": "We have previously shown that the 1E6 TCR uses a rigid lock-and-key mechanism during binding to A2-ALWGPDPAAA.", + "section": "RESULTS", + "ner": [ + [ + 34, + 41, + "1E6 TCR", + "complex_assembly" + ], + [ + 96, + 109, + "A2-ALWGPDPAAA", + "chemical" + ] + ] + }, + { + "sid": 70, + "sent": "These data demonstrated that the unligated structure of the 1E6 TCR was virtually identical to its ligated counterparts.", + "section": "RESULTS", + "ner": [ + [ + 33, + 42, + "unligated", + "protein_state" + ], + [ + 43, + 52, + "structure", + "evidence" + ], + [ + 60, + 67, + "1E6 TCR", + "complex_assembly" + ], + [ + 99, + 106, + "ligated", + "protein_state" + ] + ] + }, + { + "sid": 71, + "sent": "In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (\u0394G) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2).", + "section": "RESULTS", + "ner": [ + [ + 41, + 45, + "APLs", + "chemical" + ], + [ + 132, + 151, + "free binding energy", + "evidence" + ], + [ + 153, + 155, + "\u0394G", + "evidence" + ], + [ + 192, + 198, + "solved", + "experimental_method" + ], + [ + 203, + 212, + "unligated", + "protein_state" + ], + [ + 213, + 223, + "structures", + "evidence" + ], + [ + 233, + 237, + "APLs", + "chemical" + ], + [ + 243, + 256, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 257, + 266, + "structure", + "evidence" + ] + ] + }, + { + "sid": 72, + "sent": "The unligated A2-MVWGPDPLYV (KD = ~600 \u03bcM) structure revealed that the side chain Tyr9 swung around 8 \u00c5 in the complex structure, subsequently making contacts with TCR residues Asp30\u03b2 and Asn51\u03b2 (Figure 6A and Figure 5A, respectively).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "unligated", + "protein_state" + ], + [ + 14, + 27, + "A2-MVWGPDPLYV", + "chemical" + ], + [ + 29, + 31, + "KD", + "evidence" + ], + [ + 43, + 52, + "structure", + "evidence" + ], + [ + 82, + 86, + "Tyr9", + "residue_name_number" + ], + [ + 119, + 128, + "structure", + "evidence" + ], + [ + 164, + 167, + "TCR", + "complex_assembly" + ], + [ + 177, + 183, + "Asp30\u03b2", + "residue_name_number" + ], + [ + 188, + 194, + "Asn51\u03b2", + "residue_name_number" + ] + ] + }, + { + "sid": 73, + "sent": "This movement could result in an entropic penalty contributing to the weak TCR binding affinity we observed for this ligand.", + "section": "RESULTS", + "ner": [ + [ + 75, + 95, + "TCR binding affinity", + "evidence" + ] + ] + }, + { + "sid": 74, + "sent": "Additional small movements in the C\u03b1 backbone of the peptide around peptide residue Asp6 were apparent in the A2-YLGGPDFPTI (KD = ~400 \u03bcM), A2-ALWGPDPAAA (KD = ~208 \u03bcM), and A2-RQFGPDWIVA (KD = 44.4 \u03bcM) structures (Figure 6, B, C, and E).", + "section": "RESULTS", + "ner": [ + [ + 84, + 88, + "Asp6", + "residue_name_number" + ], + [ + 110, + 123, + "A2-YLGGPDFPTI", + "chemical" + ], + [ + 125, + 127, + "KD", + "evidence" + ], + [ + 140, + 153, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 155, + 157, + "KD", + "evidence" + ], + [ + 174, + 187, + "A2-RQFGPDWIVA", + "chemical" + ], + [ + 189, + 191, + "KD", + "evidence" + ], + [ + 203, + 213, + "structures", + "evidence" + ] + ] + }, + { + "sid": 75, + "sent": "The unligated structures of A2-AQWGPDAAA, A2-RQWGPDPAAV, A2-YQFGPDFPIA, and A2-RQFGPDFPTI were virtually identical when in complex with 1E6 (Figure 6, D and F\u2013H).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "unligated", + "protein_state" + ], + [ + 14, + 24, + "structures", + "evidence" + ], + [ + 28, + 40, + "A2-AQWGPDAAA", + "chemical" + ], + [ + 42, + 55, + "A2-RQWGPDPAAV", + "chemical" + ], + [ + 57, + 70, + "A2-YQFGPDFPIA", + "chemical" + ], + [ + 76, + 89, + "A2-RQFGPDFPTI", + "chemical" + ], + [ + 120, + 135, + "in complex with", + "protein_state" + ] + ] + }, + { + "sid": 76, + "sent": "Apart from the case of A2-AQWGPDAAA (KD = 61.9 \u03bcM), these observations support the conclusion that the higher-affinity ligands required less conformational melding during binding, which could be energetically beneficial (lower entopic cost) during ligation with the 1E6 TCR.", + "section": "RESULTS", + "ner": [ + [ + 23, + 35, + "A2-AQWGPDAAA", + "chemical" + ], + [ + 37, + 39, + "KD", + "evidence" + ], + [ + 110, + 118, + "affinity", + "evidence" + ], + [ + 266, + 273, + "1E6 TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 77, + "sent": "Peptide modifications alter the interaction between the 1E6 TCR and the MHC surface.", + "section": "RESULTS", + "ner": [ + [ + 56, + 63, + "1E6 TCR", + "complex_assembly" + ], + [ + 72, + 83, + "MHC surface", + "site" + ] + ] + }, + { + "sid": 78, + "sent": "In addition to changes between the TCR and peptide component, we also observed that different APLs had different knock-on effects between the TCR and MHC.", + "section": "RESULTS", + "ner": [ + [ + 35, + 38, + "TCR", + "complex_assembly" + ], + [ + 94, + 98, + "APLs", + "chemical" + ], + [ + 142, + 145, + "TCR", + "complex_assembly" + ], + [ + 150, + 153, + "MHC", + "complex_assembly" + ] + ] + }, + { + "sid": 79, + "sent": "MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7).", + "section": "RESULTS", + "ner": [ + [ + 0, + 3, + "MHC", + "complex_assembly" + ], + [ + 12, + 17, + "Arg65", + "residue_name_number" + ], + [ + 41, + 62, + "MHC restriction triad", + "site" + ], + [ + 64, + 69, + "Arg65", + "residue_name_number" + ], + [ + 71, + 76, + "Ala69", + "residue_name_number" + ], + [ + 82, + 88, + "Gln155", + "residue_name_number" + ], + [ + 115, + 118, + "TCR", + "complex_assembly" + ], + [ + 119, + 122, + "MHC", + "complex_assembly" + ], + [ + 138, + 144, + "Gln155", + "residue_name_number" + ], + [ + 179, + 184, + "Ala69", + "residue_name_number" + ], + [ + 219, + 228, + "interface", + "site" + ] + ] + }, + { + "sid": 80, + "sent": "Generally, the weaker-affinity APLs made fewer contacts with the MHC surface (27\u201329 interactions) compared with the stronger-affinity APLs (29\u201335 contacts), consistent with a better Pearson\u2019s correlation value (0.55) compared with TCR-peptide interactions versus affinity (0.045).", + "section": "RESULTS", + "ner": [ + [ + 22, + 30, + "affinity", + "evidence" + ], + [ + 31, + 35, + "APLs", + "chemical" + ], + [ + 65, + 68, + "MHC", + "complex_assembly" + ], + [ + 125, + 133, + "affinity", + "evidence" + ], + [ + 134, + 138, + "APLs", + "chemical" + ], + [ + 182, + 209, + "Pearson\u2019s correlation value", + "evidence" + ], + [ + 231, + 234, + "TCR", + "complex_assembly" + ], + [ + 263, + 271, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 81, + "sent": "For instance, contacts were made between TCR residue Val53\u03b2 and MHC residue Gln72 in all APLs except for in the weakest affinity ligand pair, 1E6-A2-MVWGPDPLYV, in which a subtle change in TCR conformation \u2014 probably mediated by different peptide contacts \u2014 abrogated this interaction (Figure 7A).", + "section": "RESULTS", + "ner": [ + [ + 41, + 44, + "TCR", + "complex_assembly" + ], + [ + 53, + 59, + "Val53\u03b2", + "residue_name_number" + ], + [ + 64, + 67, + "MHC", + "complex_assembly" + ], + [ + 76, + 81, + "Gln72", + "residue_name_number" + ], + [ + 89, + 93, + "APLs", + "chemical" + ], + [ + 120, + 128, + "affinity", + "evidence" + ], + [ + 142, + 159, + "1E6-A2-MVWGPDPLYV", + "complex_assembly" + ], + [ + 189, + 192, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 82, + "sent": "An energetic switch from unfavorable to favorable entropy (order-to-disorder) correlates with antigen potency.", + "section": "RESULTS", + "ner": [ + [ + 50, + 57, + "entropy", + "evidence" + ] + ] + }, + { + "sid": 83, + "sent": "Our analysis of the contact network provided some clues that could explain the different antigen potencies and binding affinities between the 1E6 TCR and the different APLs.", + "section": "RESULTS", + "ner": [ + [ + 20, + 35, + "contact network", + "site" + ], + [ + 111, + 129, + "binding affinities", + "evidence" + ], + [ + 142, + 149, + "1E6 TCR", + "complex_assembly" + ], + [ + 168, + 172, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 84, + "sent": "For example, the 1E6 TCR bound to A2-RQWGPDPAAV with the third strongest affinity (KD = 7.8 \u03bcM) but made fewer contacts than with A2-ALWGPDPAAA (KD = ~208 \u03bcM) (Table 2).", + "section": "RESULTS", + "ner": [ + [ + 17, + 24, + "1E6 TCR", + "complex_assembly" + ], + [ + 25, + 33, + "bound to", + "protein_state" + ], + [ + 34, + 47, + "A2-RQWGPDPAAV", + "chemical" + ], + [ + 73, + 81, + "affinity", + "evidence" + ], + [ + 83, + 85, + "KD", + "evidence" + ], + [ + 130, + 143, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 145, + 147, + "KD", + "evidence" + ] + ] + }, + { + "sid": 85, + "sent": "Thus, we performed an in-depth thermodynamic analysis of 6 of the ligands under investigation (Figure 8 and Supplemental Table 3).", + "section": "RESULTS", + "ner": [ + [ + 31, + 53, + "thermodynamic analysis", + "experimental_method" + ] + ] + }, + { + "sid": 86, + "sent": "The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (\u0394H\u00b0) and entropic (T\u0394S\u00b0) changes that contributed to the different binding affinities/potencies for each APL.", + "section": "RESULTS", + "ner": [ + [ + 9, + 25, + "binding affinity", + "evidence" + ], + [ + 42, + 55, + "A2-MVWGPDPLYV", + "chemical" + ], + [ + 60, + 73, + "A2-YLGGPDFPTI", + "chemical" + ], + [ + 156, + 165, + "enthalpic", + "evidence" + ], + [ + 167, + 170, + "\u0394H\u00b0", + "evidence" + ], + [ + 176, + 184, + "entropic", + "evidence" + ], + [ + 186, + 190, + "T\u0394S\u00b0", + "evidence" + ], + [ + 234, + 252, + "binding affinities", + "evidence" + ], + [ + 272, + 275, + "APL", + "chemical" + ] + ] + }, + { + "sid": 87, + "sent": "The overall free binding energies (\u0394G\u00b0) were between \u20134.4 and \u20138.6 kcal/mol, reflecting the wide range of TCR binding affinities we observed for the different APLs.", + "section": "RESULTS", + "ner": [ + [ + 12, + 33, + "free binding energies", + "evidence" + ], + [ + 35, + 38, + "\u0394G\u00b0", + "evidence" + ], + [ + 106, + 128, + "TCR binding affinities", + "evidence" + ], + [ + 159, + 163, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 88, + "sent": "The enthalpic contribution in each complex did not follow a clear trend with affinity, with all but the 1E6-A2-RQFGPDFPTI interaction (\u0394H\u00b0 = 6.3 kcal/mol) generating an energetically favorable enthalpy value (\u0394H\u00b0 = \u20133.7 to \u201311.4 kcal/mol); this indicated a net gain in electrostatic interactions during complex formation.", + "section": "RESULTS", + "ner": [ + [ + 77, + 85, + "affinity", + "evidence" + ], + [ + 104, + 121, + "1E6-A2-RQFGPDFPTI", + "complex_assembly" + ], + [ + 135, + 138, + "\u0394H\u00b0", + "evidence" + ], + [ + 193, + 201, + "enthalpy", + "evidence" + ], + [ + 209, + 212, + "\u0394H\u00b0", + "evidence" + ] + ] + }, + { + "sid": 89, + "sent": "However, there was a clear switch in entropy between the weaker-affinity and stronger-affinity ligands, indicated by a strong Pearson\u2019s correlation value between entropy and affinity (Pearson\u2019s correlation value 0.93, P =0.007).", + "section": "RESULTS", + "ner": [ + [ + 37, + 44, + "entropy", + "evidence" + ], + [ + 64, + 72, + "affinity", + "evidence" + ], + [ + 86, + 94, + "affinity", + "evidence" + ], + [ + 126, + 153, + "Pearson\u2019s correlation value", + "evidence" + ], + [ + 162, + 169, + "entropy", + "evidence" + ], + [ + 174, + 182, + "affinity", + "evidence" + ], + [ + 184, + 211, + "Pearson\u2019s correlation value", + "evidence" + ], + [ + 218, + 219, + "P", + "evidence" + ] + ] + }, + { + "sid": 90, + "sent": "For instance, the A2-ALWGPDPAAA, A2-AQWGPDAAA, and A2-RQFGPDWIVA (KD = ~208 \u03bcM, KD = 61.9 \u03bcM, and KD = 44.4 \u03bcM, respectively) were all entropically unfavorable (T\u0394S\u00b0 = \u20132.9 to \u20135.6 kcal/mol), indicating a net change from disorder to order.", + "section": "RESULTS", + "ner": [ + [ + 18, + 31, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 33, + 45, + "A2-AQWGPDAAA", + "chemical" + ], + [ + 51, + 64, + "A2-RQFGPDWIVA", + "chemical" + ], + [ + 66, + 68, + "KD", + "evidence" + ], + [ + 80, + 82, + "KD", + "evidence" + ], + [ + 98, + 100, + "KD", + "evidence" + ], + [ + 161, + 165, + "T\u0394S\u00b0", + "evidence" + ] + ] + }, + { + "sid": 91, + "sent": "Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 \u03bcM), A2-YQFGPDFPIA (KD = 7.4 \u03bcM), and A2-RQFGPDFPTI (KD = 0.5 \u03bcM) exhibited favorable entropy (T\u0394S\u00b0 = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules.", + "section": "RESULTS", + "ner": [ + [ + 25, + 33, + "affinity", + "evidence" + ], + [ + 42, + 55, + "A2-RQWGPDPAAV", + "chemical" + ], + [ + 57, + 59, + "KD", + "evidence" + ], + [ + 71, + 84, + "A2-YQFGPDFPIA", + "chemical" + ], + [ + 86, + 88, + "KD", + "evidence" + ], + [ + 104, + 117, + "A2-RQFGPDFPTI", + "chemical" + ], + [ + 119, + 121, + "KD", + "evidence" + ], + [ + 152, + 159, + "entropy", + "evidence" + ], + [ + 161, + 165, + "T\u0394S\u00b0", + "evidence" + ] + ] + }, + { + "sid": 92, + "sent": "Furthermore, the structures of the unligated pMHCs demonstrated that, for these stronger-affinity ligands, there was less conformational difference between the TCR ligated pMHCs compared with the weaker-affinity ligands (Figure 6).", + "section": "RESULTS", + "ner": [ + [ + 35, + 44, + "unligated", + "protein_state" + ], + [ + 45, + 50, + "pMHCs", + "complex_assembly" + ], + [ + 89, + 97, + "affinity", + "evidence" + ], + [ + 160, + 163, + "TCR", + "complex_assembly" + ], + [ + 164, + 171, + "ligated", + "protein_state" + ], + [ + 172, + 177, + "pMHCs", + "complex_assembly" + ], + [ + 203, + 211, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 93, + "sent": "The potential requirement for a larger degree of induced fit during binding to these weaker-affinity ligands is consistent with the larger entropic penalties observed for these interactions.", + "section": "RESULTS", + "ner": [ + [ + 92, + 100, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 94, + "sent": "Potential epitopes for 1E6 TCR occur commonly in the viral proteome.", + "section": "RESULTS", + "ner": [ + [ + 23, + 30, + "1E6 TCR", + "complex_assembly" + ], + [ + 53, + 58, + "viral", + "taxonomy_domain" + ] + ] + }, + { + "sid": 95, + "sent": "We searched a database of over 1,924,572 unique decamer peptides from the proteome of viral pathogens that are known, or strongly suspected, to infect humans.", + "section": "RESULTS", + "ner": [ + [ + 56, + 64, + "peptides", + "chemical" + ], + [ + 86, + 91, + "viral", + "taxonomy_domain" + ], + [ + 151, + 157, + "humans", + "species" + ] + ] + }, + { + "sid": 96, + "sent": "Three hundred forty-two of these decamers conformed to the motif xxxGPDxxxx.", + "section": "RESULTS", + "ner": [ + [ + 65, + 75, + "xxxGPDxxxx", + "structure_element" + ] + ] + }, + { + "sid": 97, + "sent": "Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4).", + "section": "RESULTS", + "ner": [ + [ + 42, + 52, + "xOxGPDxxxO", + "structure_element" + ], + [ + 108, + 109, + "A", + "residue_name" + ], + [ + 110, + 111, + "V", + "residue_name" + ], + [ + 113, + 114, + "I", + "residue_name" + ], + [ + 116, + 117, + "L", + "residue_name" + ], + [ + 119, + 120, + "M", + "residue_name" + ], + [ + 122, + 123, + "Y", + "residue_name" + ], + [ + 125, + 126, + "F", + "residue_name" + ], + [ + 132, + 133, + "W", + "residue_name" + ], + [ + 162, + 172, + "HLA-A*0201", + "protein" + ] + ] + }, + { + "sid": 98, + "sent": "Thus, there are many pathogen-encoded peptides that could act as agonists for the 1E6 T cell beyond the MVWGPDPLYV and RQFGPDWIVA sequences studied here.", + "section": "RESULTS", + "ner": [ + [ + 104, + 114, + "MVWGPDPLYV", + "chemical" + ], + [ + 119, + 129, + "RQFGPDWIVA", + "chemical" + ] + ] + }, + { + "sid": 99, + "sent": "Extension of these analyses to include the larger genomes of bacterial pathogens would be expected to considerably increase these numbers.", + "section": "RESULTS", + "ner": [ + [ + 61, + 70, + "bacterial", + "taxonomy_domain" + ] + ] + }, + { + "sid": 100, + "sent": "The binding affinity of the 1E6 TCR interaction with A2-RQFGPDWIVA is considerably higher than with the disease-implicated A2-ALWGPDPAAA sequence (KD = 44.4 \u03bcM and KD > 200 \u03bcM, respectively), highlighting how a pathogen-derived sequence might be capable of priming a 1E6-like T cell.", + "section": "RESULTS", + "ner": [ + [ + 4, + 20, + "binding affinity", + "evidence" + ], + [ + 28, + 35, + "1E6 TCR", + "complex_assembly" + ], + [ + 53, + 66, + "A2-RQFGPDWIVA", + "chemical" + ], + [ + 123, + 136, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 147, + 149, + "KD", + "evidence" + ], + [ + 164, + 166, + "KD", + "evidence" + ] + ] + }, + { + "sid": 101, + "sent": "T cell antigen discrimination is governed by an interaction between the clonally expressed TCR and pMHC, mediated by the chemical characteristics of the interacting molecules.", + "section": "DISCUSS", + "ner": [ + [ + 91, + 94, + "TCR", + "complex_assembly" + ], + [ + 99, + 103, + "pMHC", + "complex_assembly" + ] + ] + }, + { + "sid": 102, + "sent": "It has recently become clear that TCR cross-reactivity with large numbers of different pMHC ligands is essential to plug holes in T cell immune coverage that pathogens could exploit.", + "section": "DISCUSS", + "ner": [ + [ + 34, + 37, + "TCR", + "complex_assembly" + ], + [ + 87, + 91, + "pMHC", + "complex_assembly" + ] + ] + }, + { + "sid": 103, + "sent": "Flexibility at the interface between the TCR and pMHC, demonstrated in various studies, has been suggested as a mechanism mediating T cell cross-reactivity with multiple distinct epitopes.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 28, + "interface", + "site" + ], + [ + 41, + 44, + "TCR", + "complex_assembly" + ], + [ + 49, + 53, + "pMHC", + "complex_assembly" + ] + ] + }, + { + "sid": 104, + "sent": "This notion is attractive because the CDR loops, which form the TCR antigen-binding site, are usually the most flexible part of the TCR and have the ability to mold around differently shaped ligands.", + "section": "DISCUSS", + "ner": [ + [ + 38, + 47, + "CDR loops", + "structure_element" + ], + [ + 64, + 88, + "TCR antigen-binding site", + "site" + ], + [ + 132, + 135, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 105, + "sent": "Focused binding around a minimal peptide motif has also been implicated as an alternative mechanism enabling TCR cross-reactivity.", + "section": "DISCUSS", + "ner": [ + [ + 109, + 112, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 106, + "sent": "Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues.", + "section": "DISCUSS", + "ner": [ + [ + 69, + 81, + "alloreactive", + "protein_state" + ], + [ + 82, + 88, + "murine", + "taxonomy_domain" + ], + [ + 89, + 92, + "TCR", + "complex_assembly" + ], + [ + 93, + 96, + "MHC", + "complex_assembly" + ], + [ + 109, + 113, + "42F3", + "protein" + ], + [ + 114, + 117, + "TCR", + "complex_assembly" + ], + [ + 122, + 127, + "H2-Ld", + "protein" + ], + [ + 199, + 208, + "conserved", + "protein_state" + ], + [ + 209, + 216, + "hotspot", + "site" + ] + ] + }, + { + "sid": 107, + "sent": "Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85\u201399 bound to HLA-DQ1).", + "section": "DISCUSS", + "ner": [ + [ + 52, + 57, + "MHCII", + "protein_type" + ], + [ + 69, + 76, + "Hy.1B11", + "protein" + ], + [ + 77, + 80, + "TCR", + "complex_assembly" + ], + [ + 161, + 172, + "deep pocket", + "site" + ], + [ + 202, + 203, + "2", + "residue_number" + ], + [ + 205, + 206, + "3", + "residue_number" + ], + [ + 212, + 213, + "5", + "residue_number" + ], + [ + 220, + 228, + "MBP85\u201399", + "protein" + ], + [ + 229, + 237, + "bound to", + "protein_state" + ], + [ + 238, + 245, + "HLA-DQ1", + "protein" + ] + ] + }, + { + "sid": 108, + "sent": "This motif was conserved in at least 2 potential foreign peptides, originating from Herpes simplex virus and Pseudomonas aeruginosa, enabling TCR recognition of foreign epitopes.", + "section": "DISCUSS", + "ner": [ + [ + 15, + 24, + "conserved", + "protein_state" + ], + [ + 84, + 104, + "Herpes simplex virus", + "species" + ], + [ + 109, + 131, + "Pseudomonas aeruginosa", + "species" + ], + [ + 142, + 145, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 109, + "sent": "First, we currently know nothing about how human MHCI\u2013restricted TCRs mediate cross-reactivity in the context of a clinically relevant model of autoimmunity, thought to be a major pathway of disease initiation in several autoimmune diseases.", + "section": "DISCUSS", + "ner": [ + [ + 43, + 48, + "human", + "species" + ], + [ + 49, + 53, + "MHCI", + "complex_assembly" + ], + [ + 65, + 69, + "TCRs", + "complex_assembly" + ] + ] + }, + { + "sid": 110, + "sent": "Second, molecular studies have not yet revealed a broad set of rules that determine TCR cross-reactivity because, with the exception of the allo\u2013TCR-MHC pair of the 42F3 TCR and H2-Ld that did not encounter each other during T cell development, studies have been limited to structures of a TCR with only 2 or 3 different ligands.", + "section": "DISCUSS", + "ner": [ + [ + 84, + 87, + "TCR", + "complex_assembly" + ], + [ + 140, + 144, + "allo", + "protein_state" + ], + [ + 145, + 152, + "TCR-MHC", + "complex_assembly" + ], + [ + 165, + 169, + "42F3", + "protein" + ], + [ + 170, + 173, + "TCR", + "complex_assembly" + ], + [ + 178, + 183, + "H2-Ld", + "protein" + ], + [ + 274, + 284, + "structures", + "evidence" + ], + [ + 290, + 293, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 111, + "sent": "Here, we investigated a highly cross-reactive MHCI-restricted TCR isolated from a patient with T1D that recognizes an HLA-A*0201\u2013restricted preproinsulin signal peptide (ALWGPDPAAA15\u201324).", + "section": "DISCUSS", + "ner": [ + [ + 46, + 50, + "MHCI", + "complex_assembly" + ], + [ + 62, + 65, + "TCR", + "complex_assembly" + ], + [ + 118, + 128, + "HLA-A*0201", + "protein" + ], + [ + 140, + 153, + "preproinsulin", + "protein" + ], + [ + 154, + 168, + "signal peptide", + "structure_element" + ], + [ + 170, + 185, + "ALWGPDPAAA15\u201324", + "chemical" + ] + ] + }, + { + "sid": 112, + "sent": "Human CD8+ T cell clones expressing TCRs with this specificity mediate the destruction of \u03b2 cells, have been found in islets early in infection, and are proposed to be a major driver of disease.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "Human", + "species" + ], + [ + 36, + 40, + "TCRs", + "complex_assembly" + ] + ] + }, + { + "sid": 113, + "sent": "We solved the structure of the 1E6 TCR with 7 APLs to enable a comprehensive analysis of the molecular basis of TCR degeneracy.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 9, + "solved", + "experimental_method" + ], + [ + 14, + 23, + "structure", + "evidence" + ], + [ + 31, + 38, + "1E6 TCR", + "complex_assembly" + ], + [ + 46, + 50, + "APLs", + "chemical" + ], + [ + 112, + 115, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 114, + "sent": "Overall, the difference in antigen potency correlated well with the binding energy (\u0394G\u00b0 kcal/mol) of the 1E6 TCR for the different epitopes, which ranged from values of \u0394G\u00b0 = ~\u20134.4 to \u20138.6 kcal/mol (calculated from 3D affinity data) or 2D affinity values of AcKa = 2.5 \u00d7 10\u20135 to 4.4 \u00d7 10\u20132 \u03bcm4.", + "section": "DISCUSS", + "ner": [ + [ + 68, + 82, + "binding energy", + "evidence" + ], + [ + 84, + 87, + "\u0394G\u00b0", + "evidence" + ], + [ + 105, + 112, + "1E6 TCR", + "complex_assembly" + ], + [ + 169, + 172, + "\u0394G\u00b0", + "evidence" + ], + [ + 215, + 226, + "3D affinity", + "evidence" + ], + [ + 236, + 247, + "2D affinity", + "evidence" + ], + [ + 258, + 262, + "AcKa", + "evidence" + ] + ] + }, + { + "sid": 115, + "sent": "The weaker end of this spectrum extends our understanding of the limits in which T cells can functionally operate in terms of TCR 3D binding affinity and is in line with the types of very low affinity, yet fully functional self-reactive CD8+ T cells we have observed in tumor-infiltrating lymphocytes.", + "section": "DISCUSS", + "ner": [ + [ + 126, + 149, + "TCR 3D binding affinity", + "evidence" + ], + [ + 192, + 200, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 116, + "sent": "Previous studies of autoreactive TCRs have shown that their binding mode is generally atypical, either due to an unusual binding manner, weak TCR binding affinity, an unstable pMHC, or a combination of these factors.", + "section": "DISCUSS", + "ner": [ + [ + 33, + 37, + "TCRs", + "complex_assembly" + ], + [ + 142, + 162, + "TCR binding affinity", + "evidence" + ], + [ + 167, + 175, + "unstable", + "protein_state" + ], + [ + 176, + 180, + "pMHC", + "complex_assembly" + ] + ] + }, + { + "sid": 117, + "sent": "Our data demonstrate the potential for an autoreactive TCR to bind with a conventional binding mode to a stable pMHC with antipathogen-like affinity (KD = 0.5 \u03bcM) depending on the peptide sequence.", + "section": "DISCUSS", + "ner": [ + [ + 55, + 58, + "TCR", + "complex_assembly" + ], + [ + 105, + 111, + "stable", + "protein_state" + ], + [ + 112, + 116, + "pMHC", + "complex_assembly" + ], + [ + 140, + 148, + "affinity", + "evidence" + ], + [ + 150, + 152, + "KD", + "evidence" + ] + ] + }, + { + "sid": 118, + "sent": "Our structural analysis revealed that the 1E6 TCR bound with a conserved conformation across all APLs investigated.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 23, + "structural analysis", + "experimental_method" + ], + [ + 42, + 49, + "1E6 TCR", + "complex_assembly" + ], + [ + 50, + 55, + "bound", + "protein_state" + ], + [ + 63, + 85, + "conserved conformation", + "protein_state" + ], + [ + 97, + 101, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 119, + "sent": "This binding orientation was mediated through a focused interaction with TCR residues Tyr97\u03b1 and Trp97\u03b2 that formed an aromatic cap over a central \u2018GDP\u2019 motif that was common to all APLs.", + "section": "DISCUSS", + "ner": [ + [ + 73, + 76, + "TCR", + "complex_assembly" + ], + [ + 86, + 92, + "Tyr97\u03b1", + "residue_name_number" + ], + [ + 97, + 103, + "Trp97\u03b2", + "residue_name_number" + ], + [ + 119, + 131, + "aromatic cap", + "structure_element" + ], + [ + 147, + 158, + "\u2018GDP\u2019 motif", + "structure_element" + ], + [ + 182, + 186, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 120, + "sent": "We have previously demonstrated the importance of the GPD motif using a peptide library scan, as well as a CPL scan approach.", + "section": "DISCUSS", + "ner": [ + [ + 54, + 63, + "GPD motif", + "structure_element" + ], + [ + 72, + 92, + "peptide library scan", + "experimental_method" + ], + [ + 107, + 115, + "CPL scan", + "experimental_method" + ] + ] + }, + { + "sid": 121, + "sent": "Although the 1E6 T cell was able to activate weakly with peptides that lacked this motif, we were unable to robustly measure binding affinities or generate complex structures with these ligands, highlighting the central role of this interaction during 1E6 T cell antigen recognition.", + "section": "DISCUSS", + "ner": [ + [ + 71, + 77, + "lacked", + "protein_state" + ], + [ + 125, + 143, + "binding affinities", + "evidence" + ], + [ + 164, + 174, + "structures", + "evidence" + ] + ] + }, + { + "sid": 122, + "sent": "This hotspot binding, defined as a localized cluster of interactions that dominate binding energy during protein-protein interactions, has been previously shown to contribute to TCR recognition of MHC as a mechanism that tunes T cell cross-reactivity by providing fixed anchor points that enable TCRs to tolerate a variable peptide cargo.", + "section": "DISCUSS", + "ner": [ + [ + 178, + 181, + "TCR", + "complex_assembly" + ], + [ + 197, + 200, + "MHC", + "complex_assembly" + ], + [ + 296, + 300, + "TCRs", + "complex_assembly" + ] + ] + }, + { + "sid": 123, + "sent": "Alternatively, interactions between the TCR and peptide have been shown to dominate the energetic landscape during ligand engagement, ensuring that T cells retain peptide specificity.", + "section": "DISCUSS", + "ner": [ + [ + 40, + 43, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 124, + "sent": "The binding mechanism utilized by the 1E6 TCR during pMHC recognition is consistent with both of these models.", + "section": "DISCUSS", + "ner": [ + [ + 38, + 45, + "1E6 TCR", + "complex_assembly" + ], + [ + 53, + 57, + "pMHC", + "complex_assembly" + ] + ] + }, + { + "sid": 125, + "sent": "Ligand engagement is dominated by peptide interactions, but hotspot-like interactions with the central GPD motif enable the 1E6 TCR to tolerate peptide residues that vary outside of this region, explaining how T cells expressing this TCR may cross-react with a large number of different peptides.", + "section": "DISCUSS", + "ner": [ + [ + 103, + 112, + "GPD motif", + "structure_element" + ], + [ + 124, + 131, + "1E6 TCR", + "complex_assembly" + ], + [ + 234, + 237, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 126, + "sent": "These findings are also analogous to the observed binding mode of the Hy.1B11 TCR, in which one aromatic residue of the TCR CDR3\u03b1 loop anchored into a pocket created by a conserved peptide motif.", + "section": "DISCUSS", + "ner": [ + [ + 70, + 77, + "Hy.1B11", + "protein" + ], + [ + 78, + 81, + "TCR", + "complex_assembly" + ], + [ + 120, + 123, + "TCR", + "complex_assembly" + ], + [ + 124, + 134, + "CDR3\u03b1 loop", + "structure_element" + ] + ] + }, + { + "sid": 127, + "sent": "In both of these examples, self-recognition is mediated by TCR residues with aromatic side chains.", + "section": "DISCUSS", + "ner": [ + [ + 59, + 62, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 128, + "sent": "Combined with evidence demonstrating that aromatic side chains are conserved in the CDR2 loops of TCRs from many species, we speculate that these aromatic residues could impart a level of \u201cstickiness\u201d to TCRs, which might be enriched in an autoimmune setting when the TCR often binds in a nonoptimal fashion.", + "section": "DISCUSS", + "ner": [ + [ + 84, + 94, + "CDR2 loops", + "structure_element" + ], + [ + 98, + 102, + "TCRs", + "complex_assembly" + ], + [ + 204, + 208, + "TCRs", + "complex_assembly" + ], + [ + 268, + 271, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 129, + "sent": "Despite some weak statistical correlation between the surface complementarity (SC) and affinity, closer inspection of the interface revealed no obvious structural signature that could definitively explain the differences in antigen potency and TCR binding strength between the different ligands.", + "section": "DISCUSS", + "ner": [ + [ + 54, + 77, + "surface complementarity", + "evidence" + ], + [ + 79, + 81, + "SC", + "evidence" + ], + [ + 87, + 95, + "affinity", + "evidence" + ], + [ + 122, + 131, + "interface", + "site" + ], + [ + 224, + 239, + "antigen potency", + "evidence" + ], + [ + 244, + 264, + "TCR binding strength", + "evidence" + ] + ] + }, + { + "sid": 130, + "sent": "However, similar to our findings in other systems, modifications to residues outside of the canonical central peptide bulge were important for generating new interactions.", + "section": "DISCUSS", + "ner": [ + [ + 102, + 123, + "central peptide bulge", + "structure_element" + ] + ] + }, + { + "sid": 131, + "sent": "For example, all of the stronger ligands encoded larger side chains (Arg or Tyr) at peptide position 1 that enabled new interactions with 1E6 not present with the Ala at this position in the natural preproinsulin peptide.", + "section": "DISCUSS", + "ner": [ + [ + 69, + 72, + "Arg", + "residue_name" + ], + [ + 76, + 79, + "Tyr", + "residue_name" + ], + [ + 101, + 102, + "1", + "residue_number" + ], + [ + 163, + 166, + "Ala", + "residue_name" + ], + [ + 199, + 212, + "preproinsulin", + "protein" + ], + [ + 213, + 220, + "peptide", + "chemical" + ] + ] + }, + { + "sid": 132, + "sent": "These data also explain our previous findings that alteration of the anchor residue at peptide position 2 (Leu-Gln) has a direct effect on 1E6 TCR binding affinity because our structural analysis demonstrated that 1E6 made 3 additional bonds with A2-AQWGPDPAAA compared with A2-ALWGPDPAAA, consistent with the >3-fold stronger binding affinity.", + "section": "DISCUSS", + "ner": [ + [ + 69, + 83, + "anchor residue", + "structure_element" + ], + [ + 104, + 105, + "2", + "residue_number" + ], + [ + 107, + 114, + "Leu-Gln", + "mutant" + ], + [ + 139, + 163, + "1E6 TCR binding affinity", + "evidence" + ], + [ + 176, + 195, + "structural analysis", + "experimental_method" + ], + [ + 247, + 260, + "A2-AQWGPDPAAA", + "chemical" + ], + [ + 275, + 288, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 327, + 343, + "binding affinity", + "evidence" + ] + ] + }, + { + "sid": 133, + "sent": "We have recently demonstrated how a suboptimal position 2 anchor in a melanoma-derived antigen can improve TCR binding through a similar mechanism.", + "section": "DISCUSS", + "ner": [ + [ + 56, + 57, + "2", + "residue_number" + ], + [ + 58, + 64, + "anchor", + "structure_element" + ], + [ + 107, + 110, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 134, + "sent": "These results challenge the notion that the most potent peptide antigens exhibit the greatest pMHC stability and have implications for the design of anchor residue\u2013modified heteroclitic peptides for vaccination.", + "section": "DISCUSS", + "ner": [ + [ + 94, + 98, + "pMHC", + "complex_assembly" + ] + ] + }, + { + "sid": 135, + "sent": "Early thermodynamic analysis of TCR-pMHC interactions suggested a common energetic signature, driven by favorable enthalpy (generally mediated through an increase in electrostatic interactions) and unfavorable entropy (changes from disorder to order).", + "section": "DISCUSS", + "ner": [ + [ + 6, + 28, + "thermodynamic analysis", + "experimental_method" + ], + [ + 32, + 40, + "TCR-pMHC", + "complex_assembly" + ], + [ + 114, + 122, + "enthalpy", + "evidence" + ], + [ + 210, + 217, + "entropy", + "evidence" + ] + ] + }, + { + "sid": 136, + "sent": "These parameters aligned well with structural data, demonstrating that TCRs engaged pMHC using an induced fit binding mode.", + "section": "DISCUSS", + "ner": [ + [ + 35, + 50, + "structural data", + "evidence" + ], + [ + 71, + 75, + "TCRs", + "complex_assembly" + ], + [ + 84, + 88, + "pMHC", + "complex_assembly" + ] + ] + }, + { + "sid": 137, + "sent": "However, more recent data have shown that TCRs can utilize a range of energetic strategies during pMHC binding, currently with no obvious pattern in terms of TCR affinity, binding mechanism, or specificity (pathogen, cancer, or self-ligands).", + "section": "DISCUSS", + "ner": [ + [ + 42, + 46, + "TCRs", + "complex_assembly" + ], + [ + 98, + 102, + "pMHC", + "complex_assembly" + ], + [ + 158, + 170, + "TCR affinity", + "evidence" + ] + ] + }, + { + "sid": 138, + "sent": "Although no energetic signature appears to exist for different TCRs, we used thermodynamic analysis here to explore whether changes in energetics could help explain ligand discrimination by a single TCR.", + "section": "DISCUSS", + "ner": [ + [ + 63, + 67, + "TCRs", + "complex_assembly" + ], + [ + 77, + 99, + "thermodynamic analysis", + "experimental_method" + ], + [ + 199, + 202, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 139, + "sent": "This analysis demonstrated a strong relationship (according to the Pearson\u2019s correlation analysis) between the energetic signature used by the 1E6 TCR and the sensitivity of the 1E6 T cell clone to different APLs.", + "section": "DISCUSS", + "ner": [ + [ + 67, + 97, + "Pearson\u2019s correlation analysis", + "experimental_method" + ], + [ + 143, + 150, + "1E6 TCR", + "complex_assembly" + ], + [ + 208, + 212, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 140, + "sent": "The weaker APL ligands were characterized by favorable enthalpy and unfavorable entropy, whereas the stronger ligands progressively shifted to favorable entropy.", + "section": "DISCUSS", + "ner": [ + [ + 11, + 14, + "APL", + "chemical" + ], + [ + 55, + 63, + "enthalpy", + "evidence" + ], + [ + 80, + 87, + "entropy", + "evidence" + ], + [ + 153, + 160, + "entropy", + "evidence" + ] + ] + }, + { + "sid": 141, + "sent": "These differences were consistent with a greater degree of movement between the unligated and ligated pMHCs for the weaker ligands, suggesting a greater requirement for disorder-to-order changes during TCR binding.", + "section": "DISCUSS", + "ner": [ + [ + 80, + 89, + "unligated", + "protein_state" + ], + [ + 94, + 101, + "ligated", + "protein_state" + ], + [ + 102, + 107, + "pMHCs", + "complex_assembly" + ], + [ + 202, + 205, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 142, + "sent": "Thus, the enhanced antigen potency was probably mediated through a shift from an induced fit to a lock-and-key interaction between the stronger ligands (less requirement for energetically unfavorable disorder-to-order changes), resulting in a more energetically favorable \u0394G value.", + "section": "DISCUSS", + "ner": [ + [ + 272, + 280, + "\u0394G value", + "evidence" + ] + ] + }, + { + "sid": 143, + "sent": "Importantly, the preproinsulin-derived epitope was one of the least potent peptides, demonstrating that the 1E6 T cell clone had the ability to respond to different peptide sequences with far greater potency.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 30, + "preproinsulin", + "protein" + ] + ] + }, + { + "sid": 144, + "sent": "The RQFGPDWIVA peptide, which was substantially more potent than the preproinsulin peptide, is within the proteome of a common human pathogen (C. asparagiforme), demonstrating the potential for an encounter between a naive 1E6-like T cell and a foreign peptide with a more potent ligand that might then break self-tolerance.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 14, + "RQFGPDWIVA", + "chemical" + ], + [ + 69, + 82, + "preproinsulin", + "protein" + ], + [ + 127, + 132, + "human", + "species" + ], + [ + 143, + 159, + "C. asparagiforme", + "species" + ] + ] + }, + { + "sid": 145, + "sent": "Indeed, we found over 50 decamer peptides from the proteome of likely, or known, human viral pathogens alone that contained both the conserved central GPD motif and anchor residues at positions 2 and 10 that would enable binding to HLA-A*02:01.", + "section": "DISCUSS", + "ner": [ + [ + 81, + 86, + "human", + "species" + ], + [ + 87, + 92, + "viral", + "taxonomy_domain" + ], + [ + 133, + 142, + "conserved", + "protein_state" + ], + [ + 151, + 160, + "GPD motif", + "structure_element" + ], + [ + 165, + 180, + "anchor residues", + "structure_element" + ], + [ + 194, + 195, + "2", + "residue_number" + ], + [ + 200, + 202, + "10", + "residue_number" + ], + [ + 232, + 243, + "HLA-A*02:01", + "protein" + ] + ] + }, + { + "sid": 146, + "sent": "Further experiments will be required to determine whether any naturally presented, human pathogen\u2013derived peptides act as active ligands for 1E6, but our work presented here demonstrates that it is at least feasible for an autoimmune TCR to bind to a different peptide sequence that could be present in a pathogen proteome with substantially higher affinity and potency than the interaction it might use to attack self-tissue.", + "section": "DISCUSS", + "ner": [ + [ + 83, + 88, + "human", + "species" + ], + [ + 234, + 237, + "TCR", + "complex_assembly" + ], + [ + 349, + 357, + "affinity", + "evidence" + ] + ] + }, + { + "sid": 147, + "sent": "In summary, this investigation into the molecular basis of T cell cross-reactivity using a clinically relevant cytotoxic CD8+ T cell clone that kills human pancreatic \u03b2 cells provides answers to a number of previously outstanding questions.", + "section": "DISCUSS", + "ner": [ + [ + 150, + 155, + "human", + "species" + ] + ] + }, + { + "sid": 148, + "sent": "First, our data shows that a single TCR has the potential to functionally (assessed through T cell activation) bind to different ligands with affinities ranging across 3 orders of magnitude.", + "section": "DISCUSS", + "ner": [ + [ + 36, + 39, + "TCR", + "complex_assembly" + ], + [ + 142, + 152, + "affinities", + "evidence" + ] + ] + }, + { + "sid": 149, + "sent": "Second, this is the first example in which ligands have been identified and characterized for a human autoreactive TCR that are substantially more potent than the natural self-ligand, demonstrating the potential for a pathogenic ligand to break self-tolerance and prime self-reactive T cells.", + "section": "DISCUSS", + "ner": [ + [ + 96, + 101, + "human", + "species" + ], + [ + 115, + 118, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 150, + "sent": "Third, this first structural analysis of a cross-reactive human MHCI\u2013restricted autoimmune TCR showed that degeneracy was mediated through TCR-pMHC anchoring by a conserved minimal binding peptide motif.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 37, + "structural analysis", + "experimental_method" + ], + [ + 58, + 63, + "human", + "species" + ], + [ + 64, + 68, + "MHCI", + "complex_assembly" + ], + [ + 91, + 94, + "TCR", + "complex_assembly" + ], + [ + 139, + 147, + "TCR-pMHC", + "complex_assembly" + ], + [ + 163, + 172, + "conserved", + "protein_state" + ], + [ + 173, + 202, + "minimal binding peptide motif", + "structure_element" + ] + ] + }, + { + "sid": 151, + "sent": "Finally, TCR ligand discrimination was characterized by an energetic shift from an enthalpically to entropically driven interaction.", + "section": "DISCUSS", + "ner": [ + [ + 9, + 12, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 152, + "sent": "Our demonstration of the molecular mechanism governing cross-reactivity by this preproinsulin reactive human CD8+ T cell clone supports the notion first put forward by Wucherpfennig and Strominger that molecular mimicry could mediate autoimmunity and has far-reaching implications for the complex nature of T cell antigen discrimination.", + "section": "DISCUSS", + "ner": [ + [ + 80, + 93, + "preproinsulin", + "protein" + ], + [ + 103, + 108, + "human", + "species" + ] + ] + }, + { + "sid": 153, + "sent": "The 1E6 T cell clone reacts with a broad sensitivity range to APLs.", + "section": "FIG", + "ner": [ + [ + 62, + 66, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 154, + "sent": "(A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201\u2013expressing C1R cells for release of MIP-1\u03b2 (A) and killing (B).", + "section": "FIG", + "ner": [ + [ + 47, + 69, + "peptide dilution assay", + "experimental_method" + ], + [ + 91, + 101, + "MVWGPDPLYV", + "chemical" + ], + [ + 110, + 120, + "YLGGPDFPTI", + "chemical" + ], + [ + 128, + 138, + "ALWGPDPAAA", + "chemical" + ], + [ + 147, + 157, + "AQWGPDPAAA", + "chemical" + ], + [ + 167, + 177, + "RQFGPDWIVA", + "chemical" + ], + [ + 191, + 201, + "RQWGPDPAAV", + "chemical" + ], + [ + 212, + 222, + "YQFGPDFPTA", + "chemical" + ], + [ + 237, + 247, + "RQFGPDFPTI", + "chemical" + ], + [ + 277, + 287, + "HLA-A*0201", + "protein" + ], + [ + 324, + 330, + "MIP-1\u03b2", + "protein" + ] + ] + }, + { + "sid": 155, + "sent": "(C) The 1E6 T cell clone was stained, in duplicate, with tetramers composed of each APL (colored as above) presented by HLA-A*0201. (D) The stability of each APL (colored as above) was tested, in duplicate, using CD by recording the peak at 218 nm absorbance from 5\u00b0C\u201390\u00b0C.", + "section": "FIG", + "ner": [ + [ + 57, + 66, + "tetramers", + "oligomeric_state" + ], + [ + 84, + 87, + "APL", + "chemical" + ], + [ + 120, + 130, + "HLA-A*0201", + "protein" + ], + [ + 158, + 161, + "APL", + "chemical" + ], + [ + 213, + 215, + "CD", + "experimental_method" + ] + ] + }, + { + "sid": 156, + "sent": "Tm values were calculated using a Boltzmann fit to each set of data.", + "section": "FIG", + "ner": [ + [ + 0, + 2, + "Tm", + "evidence" + ], + [ + 34, + 67, + "Boltzmann fit to each set of data", + "experimental_method" + ] + ] + }, + { + "sid": 157, + "sent": "3D and 2D binding analysis of the 1E6 TCR with A2-ALW and the APLs.", + "section": "FIG", + "ner": [ + [ + 0, + 26, + "3D and 2D binding analysis", + "experimental_method" + ], + [ + 34, + 41, + "1E6 TCR", + "complex_assembly" + ], + [ + 47, + 53, + "A2-ALW", + "chemical" + ], + [ + 62, + 66, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 158, + "sent": "(A\u2013H) Binding affinity of the 1E6 TCR interaction at 25\u00b0C using SPR.", + "section": "FIG", + "ner": [ + [ + 6, + 22, + "Binding affinity", + "evidence" + ], + [ + 30, + 37, + "1E6 TCR", + "complex_assembly" + ], + [ + 64, + 67, + "SPR", + "experimental_method" + ] + ] + }, + { + "sid": 159, + "sent": "Eight serial dilutions of the 1E6 TCR were measured (shown in the inset); representative data from 3 independent experiments are plotted.", + "section": "FIG", + "ner": [ + [ + 30, + 37, + "1E6 TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 160, + "sent": "The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y= [P1x]/[P2 + X]).", + "section": "FIG", + "ner": [ + [ + 4, + 32, + "equilibrium binding constant", + "evidence" + ], + [ + 34, + 36, + "KD", + "evidence" + ], + [ + 69, + 88, + "nonlinear curve fit", + "experimental_method" + ] + ] + }, + { + "sid": 161, + "sent": "In order to calculate each response, the 1E6 TCR was also injected over a control sample (HLA-A*0201\u2013ILAKFLHWL) that was deducted from the experimental data.", + "section": "FIG", + "ner": [ + [ + 41, + 48, + "1E6 TCR", + "complex_assembly" + ], + [ + 90, + 110, + "HLA-A*0201\u2013ILAKFLHWL", + "complex_assembly" + ] + ] + }, + { + "sid": 162, + "sent": "(A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) \u0394G values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson\u2019s coefficient analysis (r) and P value (including approximate values from A and B).", + "section": "FIG", + "ner": [ + [ + 4, + 21, + "1E6-A2-MVWGPDPLYV", + "complex_assembly" + ], + [ + 47, + 64, + "1E6-A2-YLGGPDFPTI", + "complex_assembly" + ], + [ + 90, + 107, + "1E6-A2-ALWGPDPAAA", + "complex_assembly" + ], + [ + 113, + 130, + "1E6-A2-AQWGPDPAAA", + "complex_assembly" + ], + [ + 136, + 153, + "1E6-A2-RQFGPDWIVA", + "complex_assembly" + ], + [ + 159, + 176, + "1E6-A2-RQWGPDPAAV", + "complex_assembly" + ], + [ + 182, + 199, + "1E6-A2-YQFGPDFPTA", + "complex_assembly" + ], + [ + 209, + 226, + "1E6-A2-RQFGPDFPTI", + "complex_assembly" + ], + [ + 232, + 241, + "\u0394G values", + "evidence" + ], + [ + 259, + 262, + "SPR", + "experimental_method" + ], + [ + 294, + 298, + "EC50", + "evidence" + ], + [ + 396, + 426, + "Pearson\u2019s coefficient analysis", + "experimental_method" + ], + [ + 428, + 429, + "r", + "evidence" + ], + [ + 435, + 436, + "P", + "evidence" + ] + ] + }, + { + "sid": 163, + "sent": "(J) Effective 2D affinity (AcKa) calculated using adhesion frequency assays, using at least 5 cell pairs, and calculated as an average of 100 cell cell contacts.", + "section": "FIG", + "ner": [ + [ + 4, + 25, + "Effective 2D affinity", + "evidence" + ], + [ + 27, + 31, + "AcKa", + "evidence" + ], + [ + 50, + 75, + "adhesion frequency assays", + "experimental_method" + ] + ] + }, + { + "sid": 164, + "sent": "(K) Effective 2D affinity plotted against 1/EC50 showing Pearson\u2019s coefficient analysis (r) and P value.", + "section": "FIG", + "ner": [ + [ + 4, + 25, + "Effective 2D affinity", + "evidence" + ], + [ + 44, + 48, + "EC50", + "evidence" + ], + [ + 57, + 87, + "Pearson\u2019s coefficient analysis", + "experimental_method" + ], + [ + 89, + 90, + "r", + "evidence" + ], + [ + 96, + 103, + "P value", + "evidence" + ] + ] + }, + { + "sid": 165, + "sent": "The 1E6 TCR uses a conserved binding mode to engage A2-ALWGPDPAAA and the APLs.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "1E6 TCR", + "complex_assembly" + ], + [ + 52, + 65, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 74, + 78, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 166, + "sent": "(A) Superposition of the 1E6 TCR (multicolored illustration) in complex with all 7 APLs (multicolored sticks) and the A2-ALWGPDPAAA ligand using the HLA-A*0201 (gray illustration) molecule to align all of the structures.", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "Superposition", + "experimental_method" + ], + [ + 25, + 32, + "1E6 TCR", + "complex_assembly" + ], + [ + 61, + 76, + "in complex with", + "protein_state" + ], + [ + 83, + 87, + "APLs", + "chemical" + ], + [ + 118, + 131, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 149, + 159, + "HLA-A*0201", + "protein" + ], + [ + 192, + 197, + "align", + "experimental_method" + ], + [ + 209, + 219, + "structures", + "evidence" + ] + ] + }, + { + "sid": 167, + "sent": "The 1E6 TCR and each peptide are colored according to the APL used in the complex as in Figure 1. (B) Position of the 1E6 TCR CDR loops (multicolored lines) in each complex.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "1E6 TCR", + "complex_assembly" + ], + [ + 58, + 61, + "APL", + "chemical" + ], + [ + 118, + 125, + "1E6 TCR", + "complex_assembly" + ], + [ + 126, + 135, + "CDR loops", + "structure_element" + ] + ] + }, + { + "sid": 168, + "sent": "The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The C\u03b1 backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 \u03b11 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface).", + "section": "FIG", + "ner": [ + [ + 4, + 14, + "ALWGPDPAAA", + "chemical" + ], + [ + 54, + 79, + "HLA-A*0201 binding groove", + "site" + ], + [ + 137, + 140, + "APL", + "chemical" + ], + [ + 191, + 201, + "HLA-A*0201", + "protein" + ], + [ + 202, + 212, + "\u03b11 helices", + "structure_element" + ], + [ + 238, + 252, + "Crossing angle", + "evidence" + ], + [ + 260, + 267, + "1E6 TCR", + "complex_assembly" + ], + [ + 360, + 370, + "ALWGPDPAAA", + "chemical" + ], + [ + 394, + 402, + "bound in", + "protein_state" + ], + [ + 407, + 432, + "HLA-A*0201 binding groove", + "site" + ] + ] + }, + { + "sid": 169, + "sent": "A conserved interaction with a GPD motif underpins the 1E6 TCR interaction with the APLs.", + "section": "FIG", + "ner": [ + [ + 31, + 40, + "GPD motif", + "structure_element" + ], + [ + 55, + 62, + "1E6 TCR", + "complex_assembly" + ], + [ + 84, + 88, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 170, + "sent": "Interaction between 1E6 TCR (gray illustration) residues Tyr97\u03b1 and Tyr97\u03b2 (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks).", + "section": "FIG", + "ner": [ + [ + 20, + 27, + "1E6 TCR", + "complex_assembly" + ], + [ + 57, + 63, + "Tyr97\u03b1", + "residue_name_number" + ], + [ + 68, + 74, + "Tyr97\u03b2", + "residue_name_number" + ], + [ + 117, + 120, + "TCR", + "complex_assembly" + ], + [ + 121, + 136, + "in complex with", + "protein_state" + ], + [ + 143, + 147, + "APLs", + "chemical" + ], + [ + 177, + 190, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 247, + 264, + "GPD peptide motif", + "structure_element" + ], + [ + 309, + 313, + "APLs", + "chemical" + ], + [ + 318, + 331, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 332, + 347, + "in complex with", + "protein_state" + ], + [ + 352, + 359, + "1E6 TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 171, + "sent": "The rest of the peptide, and the MHC\u03b11 helix, are shown as a gray illustration.", + "section": "FIG", + "ner": [ + [ + 33, + 36, + "MHC", + "complex_assembly" + ], + [ + 36, + 44, + "\u03b11 helix", + "structure_element" + ] + ] + }, + { + "sid": 172, + "sent": "The 1E6 TCR makes distinct peptide contacts with peripheral APL residues.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "1E6 TCR", + "complex_assembly" + ], + [ + 60, + 63, + "APL", + "chemical" + ] + ] + }, + { + "sid": 173, + "sent": "Interactions between the 1E6 TCR and peptide residues outside of the conserved GPD motif.", + "section": "FIG", + "ner": [ + [ + 25, + 32, + "1E6 TCR", + "complex_assembly" + ], + [ + 69, + 78, + "conserved", + "protein_state" + ], + [ + 79, + 88, + "GPD motif", + "structure_element" + ] + ] + }, + { + "sid": 174, + "sent": "The MHC\u03b11 helix is shown in gray illustrations.", + "section": "FIG", + "ner": [ + [ + 4, + 7, + "MHC", + "complex_assembly" + ], + [ + 7, + 15, + "\u03b11 helix", + "structure_element" + ] + ] + }, + { + "sid": 175, + "sent": "Hydrogen bonds are shown as red dotted lines; van der Waals (vdW) contacts are shown as black dotted lines.", + "section": "FIG", + "ner": [ + [ + 0, + 14, + "Hydrogen bonds", + "bond_interaction" + ], + [ + 46, + 74, + "van der Waals (vdW) contacts", + "bond_interaction" + ] + ] + }, + { + "sid": 176, + "sent": "Boxes show total contacts between the 1E6 TCR and each peptide ligand.", + "section": "FIG", + "ner": [ + [ + 38, + 45, + "1E6 TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 177, + "sent": "(A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data.", + "section": "FIG", + "ner": [ + [ + 28, + 35, + "1E6 TCR", + "complex_assembly" + ], + [ + 72, + 85, + "A2-MVWGPDPLYV", + "chemical" + ], + [ + 147, + 154, + "1E6 TCR", + "complex_assembly" + ], + [ + 189, + 202, + "A2-YLGGPDFPTI", + "chemical" + ], + [ + 262, + 269, + "1E6 TCR", + "complex_assembly" + ], + [ + 305, + 318, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 28, + 35, + "1E6 TCR", + "complex_assembly" + ], + [ + 72, + 85, + "A2-MVWGPDPLYV", + "chemical" + ], + [ + 147, + 154, + "1E6 TCR", + "complex_assembly" + ], + [ + 189, + 202, + "A2-YLGGPDFPTI", + "chemical" + ], + [ + 262, + 269, + "1E6 TCR", + "complex_assembly" + ], + [ + 305, + 318, + "A2-ALWGPDPAAA", + "chemical" + ] + ] + }, + { + "sid": 178, + "sent": "(D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks).", + "section": "FIG", + "ner": [ + [ + 28, + 35, + "1E6 TCR", + "complex_assembly" + ], + [ + 72, + 85, + "A2-AQWGPDPAAA", + "chemical" + ], + [ + 147, + 154, + "1E6 TCR", + "complex_assembly" + ], + [ + 195, + 208, + "A2-RQFGPDWIVA", + "chemical" + ], + [ + 274, + 281, + "1E6 TCR", + "complex_assembly" + ], + [ + 319, + 332, + "A2-RQWGPDPAAV", + "chemical" + ], + [ + 395, + 402, + "1E6 TCR", + "complex_assembly" + ], + [ + 440, + 453, + "A2-YQFGPDFPTA", + "chemical" + ], + [ + 516, + 523, + "1E6 TCR", + "complex_assembly" + ], + [ + 559, + 572, + "A2-RQFGPDFPTI", + "chemical" + ], + [ + 28, + 35, + "1E6 TCR", + "complex_assembly" + ], + [ + 72, + 85, + "A2-AQWGPDPAAA", + "chemical" + ], + [ + 147, + 154, + "1E6 TCR", + "complex_assembly" + ], + [ + 195, + 208, + "A2-RQFGPDWIVA", + "chemical" + ], + [ + 274, + 281, + "1E6 TCR", + "complex_assembly" + ], + [ + 319, + 332, + "A2-RQWGPDPAAV", + "chemical" + ], + [ + 395, + 402, + "1E6 TCR", + "complex_assembly" + ], + [ + 440, + 453, + "A2-YQFGPDFPTA", + "chemical" + ], + [ + 516, + 523, + "1E6 TCR", + "complex_assembly" + ], + [ + 559, + 572, + "A2-RQFGPDFPTI", + "chemical" + ] + ] + }, + { + "sid": 179, + "sent": "Comparison of ligated and unligated APLs.", + "section": "FIG", + "ner": [ + [ + 14, + 21, + "ligated", + "protein_state" + ], + [ + 26, + 35, + "unligated", + "protein_state" + ], + [ + 36, + 40, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 180, + "sent": "Superposition of each APL in unligated form and ligated to the 1E6 TCR.", + "section": "FIG", + "ner": [ + [ + 0, + 13, + "Superposition", + "experimental_method" + ], + [ + 22, + 25, + "APL", + "chemical" + ], + [ + 29, + 38, + "unligated", + "protein_state" + ], + [ + 48, + 55, + "ligated", + "protein_state" + ], + [ + 63, + 70, + "1E6 TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 181, + "sent": "All unligated pMHCs are shown as light green illustrations.", + "section": "FIG", + "ner": [ + [ + 4, + 13, + "unligated", + "protein_state" + ], + [ + 14, + 19, + "pMHCs", + "complex_assembly" + ] + ] + }, + { + "sid": 182, + "sent": "Peptide sequences are shown underneath each structure aligned with the peptide structure.", + "section": "FIG", + "ner": [ + [ + 44, + 53, + "structure", + "evidence" + ], + [ + 79, + 88, + "structure", + "evidence" + ] + ] + }, + { + "sid": 183, + "sent": "(A) A2-MVWGPDPLYV (black sticks).", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "A2-MVWGPDPLYV", + "chemical" + ] + ] + }, + { + "sid": 184, + "sent": "A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks).", + "section": "FIG", + "ner": [ + [ + 46, + 50, + "Tyr8", + "residue_name_number" + ], + [ + 58, + 65, + "ligated", + "protein_state" + ], + [ + 73, + 82, + "unligated", + "protein_state" + ], + [ + 110, + 123, + "A2-YLGGPDFPTI", + "chemical" + ], + [ + 142, + 155, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 215, + 228, + "A2-AQWGPDPAAA", + "chemical" + ], + [ + 249, + 262, + "A2-RQFGPDWIVA", + "chemical" + ], + [ + 287, + 300, + "A2-RQWGPDPAAV", + "chemical" + ], + [ + 322, + 335, + "A2-YQFGPDFPTA", + "chemical" + ], + [ + 357, + 370, + "A2-RQFGPDFPTI", + "chemical" + ] + ] + }, + { + "sid": 185, + "sent": "The 1E6 TCR makes distinct peptide contacts with the MHC surface depending on the peptide cargo.", + "section": "FIG", + "ner": [ + [ + 4, + 11, + "1E6 TCR", + "complex_assembly" + ], + [ + 53, + 64, + "MHC surface", + "site" + ] + ] + }, + { + "sid": 186, + "sent": "Interactions between the 1E6 TCR and the MHC \u03b11 helix residues Arg65, Lys66, and Gln72.", + "section": "FIG", + "ner": [ + [ + 25, + 32, + "1E6 TCR", + "complex_assembly" + ], + [ + 41, + 44, + "MHC", + "complex_assembly" + ], + [ + 45, + 53, + "\u03b11 helix", + "structure_element" + ], + [ + 63, + 68, + "Arg65", + "residue_name_number" + ], + [ + 70, + 75, + "Lys66", + "residue_name_number" + ], + [ + 81, + 86, + "Gln72", + "residue_name_number" + ] + ] + }, + { + "sid": 187, + "sent": "Hydrogen bonds are shown as red dotted lines; vdW contacts are shown as black dotted lines.", + "section": "FIG", + "ner": [ + [ + 0, + 14, + "Hydrogen bonds", + "bond_interaction" + ], + [ + 46, + 49, + "vdW", + "bond_interaction" + ] + ] + }, + { + "sid": 188, + "sent": "MHC\u03b11 helix are shown in gray illustrations.", + "section": "FIG", + "ner": [ + [ + 0, + 3, + "MHC", + "complex_assembly" + ], + [ + 3, + 11, + "\u03b11 helix", + "structure_element" + ] + ] + }, + { + "sid": 189, + "sent": "Boxes show total contacts between the 1E6 TCR and these key residues (green boxes are MHC residues; white boxes are TCR residues).", + "section": "FIG", + "ner": [ + [ + 38, + 45, + "1E6 TCR", + "complex_assembly" + ], + [ + 86, + 89, + "MHC", + "complex_assembly" + ], + [ + 116, + 119, + "TCR", + "complex_assembly" + ] + ] + }, + { + "sid": 190, + "sent": "Thermodynamic analysis of the 1E6 TCR with A2-ALWGPDPAAA and the APLs.", + "section": "FIG", + "ner": [ + [ + 0, + 22, + "Thermodynamic analysis", + "experimental_method" + ], + [ + 30, + 37, + "1E6 TCR", + "complex_assembly" + ], + [ + 43, + 56, + "A2-ALWGPDPAAA", + "chemical" + ], + [ + 65, + 69, + "APLs", + "chemical" + ] + ] + }, + { + "sid": 191, + "sent": "Eight serial dilutions of the 1E6 TCR were injected, in duplicate, over each immobilized APL and A2-ALW at 5\u00b0C, 13\u00b0C, 18\u00b0C, 25\u00b0C, 30\u00b0C, and 37\u00b0C.", + "section": "FIG", + "ner": [ + [ + 30, + 37, + "1E6 TCR", + "complex_assembly" + ], + [ + 89, + 92, + "APL", + "chemical" + ], + [ + 97, + 103, + "A2-ALW", + "chemical" + ] + ] + }, + { + "sid": 192, + "sent": "The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = [P1x]/[P2 + X]), and thermodynamic parameters were calculated according to the Gibbs-Helmholtz equation (\u0394G\u00b0 = \u0394H \u2212 T\u0394S\u00b0).", + "section": "FIG", + "ner": [ + [ + 4, + 32, + "equilibrium binding constant", + "evidence" + ], + [ + 34, + 36, + "KD", + "evidence" + ], + [ + 69, + 88, + "nonlinear curve fit", + "experimental_method" + ], + [ + 173, + 197, + "Gibbs-Helmholtz equation", + "experimental_method" + ], + [ + 199, + 202, + "\u0394G\u00b0", + "evidence" + ], + [ + 205, + 207, + "\u0394H", + "evidence" + ], + [ + 210, + 214, + "T\u0394S\u00b0", + "evidence" + ] + ] + }, + { + "sid": 193, + "sent": "The binding free energies, \u0394G\u00b0 (\u0394G\u00b0 = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van\u2019t Hoff equation (RT ln KD = \u0394H\u00b0 \u2013 T\u0394S\u00b0 + \u0394Cp\u00b0[T-T0] \u2013 T\u0394Cp\u00b0 ln [T/T0] with T0 = 298 K).", + "section": "FIG", + "ner": [ + [ + 4, + 25, + "binding free energies", + "evidence" + ], + [ + 27, + 30, + "\u0394G\u00b0", + "evidence" + ], + [ + 32, + 35, + "\u0394G\u00b0", + "evidence" + ], + [ + 90, + 110, + "nonlinear regression", + "experimental_method" + ], + [ + 135, + 154, + "van\u2019t Hoff equation", + "experimental_method" + ], + [ + 156, + 164, + "RT ln KD", + "evidence" + ], + [ + 167, + 170, + "\u0394H\u00b0", + "evidence" + ], + [ + 173, + 177, + "T\u0394S\u00b0", + "evidence" + ], + [ + 180, + 184, + "\u0394Cp\u00b0", + "evidence" + ], + [ + 193, + 198, + "T\u0394Cp\u00b0", + "evidence" + ] + ] + }, + { + "sid": 194, + "sent": "(A) 1E6-A2-ALWGPDPAAA; (B) 1E6-A2-AQWGPDPAAA; (C) 1E6-A2-RQFGPDWIVA; (D) 1E6-A2-RQWGPDPAAV, (E) 1E6-A2-YQFGPDFPTA; and (F) 1E6-A2-RQFGPDFPTI.", + "section": "FIG", + "ner": [ + [ + 4, + 21, + "1E6-A2-ALWGPDPAAA", + "complex_assembly" + ], + [ + 27, + 44, + "1E6-A2-AQWGPDPAAA", + "complex_assembly" + ], + [ + 50, + 67, + "1E6-A2-RQFGPDWIVA", + "complex_assembly" + ], + [ + 73, + 90, + "1E6-A2-RQWGPDPAAV", + "complex_assembly" + ], + [ + 96, + 113, + "1E6-A2-YQFGPDFPTA", + "complex_assembly" + ], + [ + 123, + 140, + "1E6-A2-RQFGPDFPTI", + "complex_assembly" + ] + ] + }, + { + "sid": 195, + "sent": "1E6 TCR-pMHC contacts, affinity measurements and thermodynamics", + "section": "TABLE", + "ner": [ + [ + 0, + 12, + "1E6 TCR-pMHC", + "complex_assembly" + ], + [ + 23, + 44, + "affinity measurements", + "experimental_method" + ], + [ + 49, + 63, + "thermodynamics", + "experimental_method" + ] + ] + } + ] + }, + "PMC4774019": { + "annotations": [ + { + "sid": 0, + "sent": "The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion", + "section": "TITLE", + "ner": [ + [ + 4, + 27, + "immunity-related GTPase", + "protein_type" + ], + [ + 28, + 33, + "Irga6", + "protein" + ], + [ + 34, + 43, + "dimerizes", + "oligomeric_state" + ], + [ + 49, + 70, + "parallel head-to-head", + "protein_state" + ] + ] + }, + { + "sid": 1, + "sent": "The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 28, + "immunity-related GTPases", + "protein_type" + ], + [ + 30, + 34, + "IRGs", + "protein_type" + ] + ] + }, + { + "sid": 2, + "sent": "Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation.", + "section": "ABSTRACT", + "ner": [ + [ + 0, + 5, + "Irga6", + "protein" + ], + [ + 11, + 31, + "dynamin-like protein", + "protein_type" + ], + [ + 100, + 117, + "Toxoplasma gondii", + "species" + ] + ] + }, + { + "sid": 3, + "sent": "Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization.", + "section": "ABSTRACT", + "ner": [ + [ + 20, + 40, + "biochemical analysis", + "experimental_method" + ], + [ + 72, + 86, + "GTPase domains", + "structure_element" + ], + [ + 90, + 95, + "Irga6", + "protein" + ], + [ + 96, + 104, + "dimerize", + "oligomeric_state" + ], + [ + 111, + 123, + "antiparallel", + "protein_state" + ] + ] + }, + { + "sid": 4, + "sent": "We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog.", + "section": "ABSTRACT", + "ner": [ + [ + 3, + 13, + "determined", + "experimental_method" + ], + [ + 18, + 35, + "crystal structure", + "evidence" + ], + [ + 42, + 66, + "oligomerization-impaired", + "protein_state" + ], + [ + 67, + 72, + "Irga6", + "protein" + ], + [ + 73, + 79, + "mutant", + "protein_state" + ], + [ + 80, + 88, + "bound to", + "protein_state" + ], + [ + 108, + 111, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 5, + "sent": "Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion.", + "section": "ABSTRACT", + "ner": [ + [ + 36, + 45, + "structure", + "evidence" + ], + [ + 61, + 75, + "GTPase domains", + "structure_element" + ], + [ + 76, + 84, + "dimerize", + "oligomeric_state" + ], + [ + 90, + 98, + "parallel", + "protein_state" + ] + ] + }, + { + "sid": 6, + "sent": "The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 15, + "nucleotides", + "chemical" + ], + [ + 37, + 46, + "interface", + "site" + ], + [ + 134, + 142, + "switch I", + "site" + ], + [ + 166, + 171, + "Irga6", + "protein" + ] + ] + }, + { + "sid": 7, + "sent": "The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site.", + "section": "ABSTRACT", + "ner": [ + [ + 39, + 42, + "GTP", + "chemical" + ], + [ + 89, + 98, + "catalytic", + "protein_state" + ], + [ + 99, + 112, + "glutamate 106", + "residue_name_number" + ], + [ + 116, + 124, + "switch I", + "site" + ], + [ + 138, + 149, + "active site", + "site" + ] + ] + }, + { + "sid": 8, + "sent": "Further dimerization contacts involve switch II, the G4 helix and the trans stabilizing loop.", + "section": "ABSTRACT", + "ner": [ + [ + 38, + 47, + "switch II", + "site" + ], + [ + 53, + 61, + "G4 helix", + "structure_element" + ], + [ + 70, + 92, + "trans stabilizing loop", + "structure_element" + ] + ] + }, + { + "sid": 9, + "sent": "The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members.", + "section": "ABSTRACT", + "ner": [ + [ + 4, + 9, + "Irga6", + "protein" + ], + [ + 10, + 19, + "structure", + "evidence" + ], + [ + 31, + 39, + "parallel", + "protein_state" + ], + [ + 40, + 53, + "GTPase domain", + "structure_element" + ], + [ + 54, + 59, + "dimer", + "oligomeric_state" + ], + [ + 107, + 114, + "dynamin", + "protein_type" + ], + [ + 119, + 125, + "septin", + "protein_type" + ] + ] + }, + { + "sid": 10, + "sent": "This study contributes important insights into the assembly and catalytic mechanisms of IRG proteins as prerequisite to understand their anti-microbial action.", + "section": "ABSTRACT", + "ner": [ + [ + 88, + 91, + "IRG", + "protein_type" + ] + ] + }, + { + "sid": 11, + "sent": "Immunity-related GTPases (IRGs) comprise a family of dynamin-related cell-autonomous resistance proteins targeting intracellular pathogens, such as Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Trypanosoma cruzi, and Toxoplasma gondii.", + "section": "INTRO", + "ner": [ + [ + 0, + 24, + "Immunity-related GTPases", + "protein_type" + ], + [ + 26, + 30, + "IRGs", + "protein_type" + ], + [ + 53, + 104, + "dynamin-related cell-autonomous resistance proteins", + "protein_type" + ], + [ + 148, + 174, + "Mycobacterium tuberculosis", + "species" + ], + [ + 176, + 195, + "Mycobacterium avium", + "species" + ], + [ + 197, + 219, + "Listeria monocytogenes", + "species" + ], + [ + 221, + 238, + "Trypanosoma cruzi", + "species" + ], + [ + 244, + 261, + "Toxoplasma gondii", + "species" + ] + ] + }, + { + "sid": 12, + "sent": "In mice, the 23 IRG members are induced by interferons, whereas the single human homologue is constitutively expressed in some tissues, especially in testis.", + "section": "INTRO", + "ner": [ + [ + 3, + 7, + "mice", + "taxonomy_domain" + ], + [ + 16, + 19, + "IRG", + "protein_type" + ], + [ + 43, + 54, + "interferons", + "protein_type" + ], + [ + 75, + 80, + "human", + "species" + ] + ] + }, + { + "sid": 13, + "sent": "In non-infected cells, most IRGs are largely cytosolic.", + "section": "INTRO", + "ner": [ + [ + 28, + 32, + "IRGs", + "protein_type" + ] + ] + }, + { + "sid": 14, + "sent": "In this way, IRGs contribute to the release of the pathogen into the cytoplasm and its subsequent destruction.", + "section": "INTRO", + "ner": [ + [ + 13, + 17, + "IRGs", + "protein_type" + ] + ] + }, + { + "sid": 15, + "sent": "Irga6, one of the effector IRG proteins, localizes to the intact parasitophorous vacuole membrane (PVM) and, after disruption of the PVM, is found associated with vesicular accumulations, presumably derived from the PVM.", + "section": "INTRO", + "ner": [ + [ + 0, + 5, + "Irga6", + "protein" + ], + [ + 27, + 30, + "IRG", + "protein_type" + ] + ] + }, + { + "sid": 16, + "sent": "A myristoylation site at Gly2 is necessary for the recruitment to the PVM but not for the weak constitutive binding to the ER membrane.", + "section": "INTRO", + "ner": [ + [ + 2, + 21, + "myristoylation site", + "site" + ], + [ + 25, + 29, + "Gly2", + "residue_name_number" + ] + ] + }, + { + "sid": 17, + "sent": "An internally oriented antibody epitope on helix A between positions 20 and 24 was demonstrated to be accessible in the GTP-, but not in the GDP-bound state.", + "section": "INTRO", + "ner": [ + [ + 43, + 50, + "helix A", + "structure_element" + ], + [ + 69, + 78, + "20 and 24", + "residue_range" + ], + [ + 120, + 125, + "GTP-,", + "protein_state" + ], + [ + 141, + 150, + "GDP-bound", + "protein_state" + ] + ] + }, + { + "sid": 18, + "sent": "This indicates large-scale structural changes upon GTP binding that probably include exposure of the myristoyl group, enhancing binding to the PVM.", + "section": "INTRO", + "ner": [ + [ + 51, + 54, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 19, + "sent": "Biochemical studies indicated that Irga6 hydrolyses GTP in a cooperative manner and forms GTP-dependent oligomers in vitro and in vivo.", + "section": "INTRO", + "ner": [ + [ + 0, + 19, + "Biochemical studies", + "experimental_method" + ], + [ + 35, + 40, + "Irga6", + "protein" + ], + [ + 52, + 55, + "GTP", + "chemical" + ], + [ + 90, + 103, + "GTP-dependent", + "protein_state" + ], + [ + 104, + 113, + "oligomers", + "oligomeric_state" + ] + ] + }, + { + "sid": 20, + "sent": "Crystal structures of Irga6 in various nucleotide-loaded states revealed the basic architecture of IRG proteins, including a GTPase domain and a composite helical domain.", + "section": "INTRO", + "ner": [ + [ + 0, + 18, + "Crystal structures", + "evidence" + ], + [ + 22, + 27, + "Irga6", + "protein" + ], + [ + 39, + 56, + "nucleotide-loaded", + "protein_state" + ], + [ + 99, + 102, + "IRG", + "protein_type" + ], + [ + 125, + 138, + "GTPase domain", + "structure_element" + ], + [ + 145, + 169, + "composite helical domain", + "structure_element" + ] + ] + }, + { + "sid": 21, + "sent": "These studies additionally showed a dimerization interface in the nucleotide-free protein as well as in all nucleotide-bound states.", + "section": "INTRO", + "ner": [ + [ + 36, + 58, + "dimerization interface", + "site" + ], + [ + 66, + 81, + "nucleotide-free", + "protein_state" + ], + [ + 108, + 124, + "nucleotide-bound", + "protein_state" + ] + ] + }, + { + "sid": 22, + "sent": "It involves a GTPase domain surface, which is located at the opposite side of the nucleotide, and an interface in the helical domain, with a water-filled gap between the two contact surfaces.", + "section": "INTRO", + "ner": [ + [ + 14, + 35, + "GTPase domain surface", + "site" + ], + [ + 101, + 110, + "interface", + "site" + ], + [ + 118, + 132, + "helical domain", + "structure_element" + ], + [ + 141, + 146, + "water", + "chemical" + ], + [ + 174, + 190, + "contact surfaces", + "site" + ] + ] + }, + { + "sid": 23, + "sent": "Mutagenesis of the contact surfaces suggests that this \"backside\" interface is not required for GTP-dependent oligomerization or cooperative hydrolysis, despite an earlier suggestion to the contrary.", + "section": "INTRO", + "ner": [ + [ + 0, + 11, + "Mutagenesis", + "experimental_method" + ], + [ + 19, + 35, + "contact surfaces", + "site" + ], + [ + 56, + 64, + "backside", + "site" + ], + [ + 66, + 75, + "interface", + "site" + ], + [ + 96, + 99, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 24, + "sent": "Extensive biochemical studies suggested that GTP-induced oligomerization of Irga6 requires an interface in the GTPase domain across the nucleotide-binding site.", + "section": "INTRO", + "ner": [ + [ + 10, + 29, + "biochemical studies", + "experimental_method" + ], + [ + 45, + 48, + "GTP", + "chemical" + ], + [ + 76, + 81, + "Irga6", + "protein" + ], + [ + 94, + 103, + "interface", + "site" + ], + [ + 111, + 124, + "GTPase domain", + "structure_element" + ], + [ + 136, + 159, + "nucleotide-binding site", + "site" + ] + ] + }, + { + "sid": 25, + "sent": "Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP).", + "section": "INTRO", + "ner": [ + [ + 7, + 25, + "structural studies", + "experimental_method" + ], + [ + 44, + 55, + "G interface", + "site" + ], + [ + 71, + 78, + "dynamin", + "protein_type" + ], + [ + 108, + 115, + "dynamin", + "protein_type" + ], + [ + 117, + 120, + "MxA", + "protein" + ], + [ + 126, + 153, + "guanylate binding protein-1", + "protein" + ], + [ + 155, + 160, + "GBP-1", + "protein" + ], + [ + 163, + 171, + "atlastin", + "protein_type" + ], + [ + 180, + 189, + "bacterial", + "taxonomy_domain" + ], + [ + 190, + 211, + "dynamin-like proteins", + "protein_type" + ], + [ + 213, + 217, + "BDLP", + "protein_type" + ] + ] + }, + { + "sid": 26, + "sent": "For several of these proteins, formation of the G interface was shown to trigger GTP hydrolysis by inducing rearrangements of catalytic residues in cis.", + "section": "INTRO", + "ner": [ + [ + 48, + 59, + "G interface", + "site" + ], + [ + 81, + 84, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 27, + "sent": "In dynamin, the G interface includes residues in the phosphate binding loop, the two switch regions, the 'trans stabilizing loop' and the 'G4 loop'.", + "section": "INTRO", + "ner": [ + [ + 3, + 10, + "dynamin", + "protein_type" + ], + [ + 16, + 27, + "G interface", + "site" + ], + [ + 53, + 75, + "phosphate binding loop", + "structure_element" + ], + [ + 85, + 99, + "switch regions", + "site" + ], + [ + 106, + 128, + "trans stabilizing loop", + "structure_element" + ], + [ + 139, + 146, + "G4 loop", + "structure_element" + ] + ] + }, + { + "sid": 28, + "sent": "For Irga6, it was demonstrated that besides residues in the switch I and switch II regions, the 3'-OH group of the ribose participates in this interface.", + "section": "INTRO", + "ner": [ + [ + 4, + 9, + "Irga6", + "protein" + ], + [ + 60, + 68, + "switch I", + "site" + ], + [ + 73, + 82, + "switch II", + "site" + ], + [ + 143, + 152, + "interface", + "site" + ] + ] + }, + { + "sid": 29, + "sent": "Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6.", + "section": "INTRO", + "ner": [ + [ + 10, + 44, + "signal recognition particle GTPase", + "protein_type" + ], + [ + 64, + 72, + "receptor", + "protein_type" + ], + [ + 81, + 84, + "FfH", + "protein" + ], + [ + 89, + 93, + "FtsY", + "protein" + ], + [ + 97, + 105, + "bacteria", + "taxonomy_domain" + ], + [ + 145, + 153, + "dimerize", + "oligomeric_state" + ], + [ + 160, + 173, + "anti-parallel", + "protein_state" + ], + [ + 211, + 217, + "GTPase", + "protein_type" + ], + [ + 268, + 273, + "Irga6", + "protein" + ] + ] + }, + { + "sid": 30, + "sent": "However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain.", + "section": "INTRO", + "ner": [ + [ + 13, + 30, + "crystal structure", + "evidence" + ], + [ + 34, + 39, + "Irga6", + "protein" + ], + [ + 47, + 58, + "presence of", + "protein_state" + ], + [ + 80, + 83, + "GTP", + "chemical" + ], + [ + 93, + 121, + "5'-guanylyl imidodiphosphate", + "chemical" + ], + [ + 123, + 129, + "GMPPNP", + "chemical" + ], + [ + 178, + 181, + "apo", + "protein_state" + ], + [ + 185, + 194, + "GDP-bound", + "protein_state" + ], + [ + 228, + 243, + "dimer interface", + "site" + ], + [ + 264, + 277, + "GTPase domain", + "structure_element" + ] + ] + }, + { + "sid": 31, + "sent": "This structure was obtained by soaking GMPPNP in nucleotide-free crystals of Irga6, an approach which may have interfered with nucleotide-induced domain rearrangements.", + "section": "INTRO", + "ner": [ + [ + 5, + 14, + "structure", + "evidence" + ], + [ + 31, + 38, + "soaking", + "experimental_method" + ], + [ + 39, + 45, + "GMPPNP", + "chemical" + ], + [ + 49, + 64, + "nucleotide-free", + "protein_state" + ], + [ + 65, + 73, + "crystals", + "evidence" + ], + [ + 77, + 82, + "Irga6", + "protein" + ] + ] + }, + { + "sid": 32, + "sent": "To clarify the dimerization mode via the G interface, we determined the GMPPNP-bound crystal structure of a non-oligomerizing Irga6 variant.", + "section": "INTRO", + "ner": [ + [ + 41, + 52, + "G interface", + "site" + ], + [ + 57, + 67, + "determined", + "experimental_method" + ], + [ + 72, + 84, + "GMPPNP-bound", + "protein_state" + ], + [ + 85, + 102, + "crystal structure", + "evidence" + ], + [ + 108, + 125, + "non-oligomerizing", + "protein_state" + ], + [ + 126, + 131, + "Irga6", + "protein" + ], + [ + 132, + 139, + "variant", + "protein_state" + ] + ] + }, + { + "sid": 33, + "sent": "The structure revealed that Irga6 can dimerize via the G interface in a parallel head-to-head fashion.", + "section": "INTRO", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 28, + 33, + "Irga6", + "protein" + ], + [ + 38, + 46, + "dimerize", + "oligomeric_state" + ], + [ + 55, + 66, + "G interface", + "site" + ], + [ + 72, + 93, + "parallel head-to-head", + "protein_state" + ] + ] + }, + { + "sid": 34, + "sent": "Our data suggest that a parallel dimerization mode may be a unifying feature in all dynamin and septin superfamily proteins.", + "section": "INTRO", + "ner": [ + [ + 24, + 32, + "parallel", + "protein_state" + ], + [ + 84, + 91, + "dynamin", + "protein_type" + ], + [ + 96, + 102, + "septin", + "protein_type" + ] + ] + }, + { + "sid": 35, + "sent": "Previous results indicated that Irga6 mutations in a loosely defined surface region (the \"secondary patch\"), which is distant from the G-interface and only slightly overlapping with the backside interface (see below), individually reduced GTP-dependent oligomerization.", + "section": "RESULTS", + "ner": [ + [ + 32, + 37, + "Irga6", + "protein" + ], + [ + 38, + 47, + "mutations", + "experimental_method" + ], + [ + 69, + 83, + "surface region", + "site" + ], + [ + 90, + 105, + "secondary patch", + "site" + ], + [ + 135, + 146, + "G-interface", + "site" + ], + [ + 186, + 204, + "backside interface", + "site" + ], + [ + 239, + 242, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 36, + "sent": "A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP.", + "section": "RESULTS", + "ner": [ + [ + 2, + 40, + "combination of four of these mutations", + "experimental_method" + ], + [ + 42, + 46, + "R31E", + "mutant" + ], + [ + 48, + 52, + "K32E", + "mutant" + ], + [ + 54, + 59, + "K176E", + "mutant" + ], + [ + 65, + 70, + "K246E", + "mutant" + ], + [ + 84, + 94, + "eliminated", + "protein_state" + ], + [ + 95, + 98, + "GTP", + "chemical" + ], + [ + 161, + 176, + "crystallization", + "experimental_method" + ], + [ + 180, + 185, + "Irga6", + "protein" + ], + [ + 193, + 204, + "presence of", + "protein_state" + ], + [ + 205, + 211, + "GMPPNP", + "chemical" + ] + ] + }, + { + "sid": 37, + "sent": "Crystals diffracted to 3.2 \u00c5 resolution and displayed one exceptionally long unit cell axis of 1289 \u00c5 (Additional file 1: Table S1).", + "section": "RESULTS", + "ner": [ + [ + 0, + 8, + "Crystals", + "evidence" + ] + ] + }, + { + "sid": 38, + "sent": "The structure was solved by molecular replacement and refined to Rwork/Rfree of 29.7 %/31.7 % (Additional file 1: Table S2).", + "section": "RESULTS", + "ner": [ + [ + 4, + 13, + "structure", + "evidence" + ], + [ + 28, + 49, + "molecular replacement", + "experimental_method" + ], + [ + 65, + 70, + "Rwork", + "evidence" + ], + [ + 71, + 76, + "Rfree", + "evidence" + ] + ] + }, + { + "sid": 39, + "sent": "The asymmetric unit contained seven Irga6 molecules that were arranged in a helical pattern along the long cell axis (Additional file 1: Figure S2).", + "section": "RESULTS", + "ner": [ + [ + 36, + 41, + "Irga6", + "protein" + ] + ] + }, + { + "sid": 40, + "sent": "Structure of the Irga6 dimer.", + "section": "FIG", + "ner": [ + [ + 0, + 9, + "Structure", + "evidence" + ], + [ + 17, + 22, + "Irga6", + "protein" + ], + [ + 23, + 28, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 41, + "sent": "a Schematic view of the domain architecture of mouse Irga6.", + "section": "FIG", + "ner": [ + [ + 47, + 52, + "mouse", + "taxonomy_domain" + ], + [ + 53, + 58, + "Irga6", + "protein" + ] + ] + }, + { + "sid": 42, + "sent": "b Ribbon-type representation of the Irga6 dimer.", + "section": "FIG", + "ner": [ + [ + 36, + 41, + "Irga6", + "protein" + ], + [ + 42, + 47, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 43, + "sent": "The nucleotide and Mg2+ ion (green) are shown in sphere representation.", + "section": "FIG", + "ner": [ + [ + 19, + 23, + "Mg2+", + "chemical" + ] + ] + }, + { + "sid": 44, + "sent": "The GTPase domain dimer is boxed.", + "section": "FIG", + "ner": [ + [ + 4, + 17, + "GTPase domain", + "structure_element" + ], + [ + 18, + 23, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 45, + "sent": "Secondary structure was numbered according to ref.. c Top view on the GTPase domain dimer.", + "section": "FIG", + "ner": [ + [ + 70, + 83, + "GTPase domain", + "structure_element" + ], + [ + 84, + 89, + "dimer", + "oligomeric_state" + ] + ] + }, + { + "sid": 46, + "sent": "d Magnification of the contact sites.", + "section": "FIG", + "ner": [ + [ + 23, + 36, + "contact sites", + "site" + ] + ] + }, + { + "sid": 47, + "sent": "e Superposition of different switch I conformations in the asymmetric unit; the same colors as in Additional file 1: Figure S2 are used for the switch I regions of the individual subunits.", + "section": "FIG", + "ner": [ + [ + 2, + 15, + "Superposition", + "experimental_method" + ], + [ + 29, + 37, + "switch I", + "site" + ], + [ + 144, + 152, + "switch I", + "site" + ] + ] + }, + { + "sid": 48, + "sent": "Switch I residues of subunit A (yellow) involved in ribose binding are labelled and shown in stick representation.", + "section": "FIG", + "ner": [ + [ + 0, + 8, + "Switch I", + "site" + ], + [ + 29, + 30, + "A", + "structure_element" + ] + ] + }, + { + "sid": 49, + "sent": "Irga6 immunity-related GTPase 6", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "Irga6", + "protein" + ], + [ + 6, + 31, + "immunity-related GTPase 6", + "protein" + ] + ] + }, + { + "sid": 50, + "sent": "Like other dynamin superfamily members, the GTPase domain of Irga6 comprises a canonical GTPase domain fold, with a central \u03b2-sheet surrounded by helices on both sides (Fig.\u00a01a-c).", + "section": "RESULTS", + "ner": [ + [ + 11, + 18, + "dynamin", + "protein_type" + ], + [ + 44, + 57, + "GTPase domain", + "structure_element" + ], + [ + 61, + 66, + "Irga6", + "protein" + ], + [ + 89, + 102, + "GTPase domain", + "structure_element" + ], + [ + 124, + 131, + "\u03b2-sheet", + "structure_element" + ], + [ + 146, + 153, + "helices", + "structure_element" + ] + ] + }, + { + "sid": 51, + "sent": "The helical domain is a bipartite structure composed of helices \u03b1A-C at the N-terminus and helix \u03b1F-L at the C-terminus of the GTPase domain.", + "section": "RESULTS", + "ner": [ + [ + 4, + 18, + "helical domain", + "structure_element" + ], + [ + 56, + 63, + "helices", + "structure_element" + ], + [ + 64, + 68, + "\u03b1A-C", + "structure_element" + ], + [ + 91, + 96, + "helix", + "structure_element" + ], + [ + 97, + 101, + "\u03b1F-L", + "structure_element" + ], + [ + 127, + 140, + "GTPase domain", + "structure_element" + ] + ] + }, + { + "sid": 52, + "sent": "Overall, the seven molecules in the asymmetric unit are very similar to each other, with root mean square deviations (rmsd) ranging from 0.32 \u2013 0.45 \u00c5 over all C\u03b1 atoms.", + "section": "RESULTS", + "ner": [ + [ + 89, + 116, + "root mean square deviations", + "evidence" + ], + [ + 118, + 122, + "rmsd", + "evidence" + ] + ] + }, + { + "sid": 53, + "sent": "The structures of the seven molecules also agree well with the previously determined structure of native GMPPNP-bound Irga6 (PDB: 1TQ6; rmsd of 1.00-1.13 \u00c5 over all C\u03b1 atoms).", + "section": "RESULTS", + "ner": [ + [ + 4, + 14, + "structures", + "evidence" + ], + [ + 85, + 94, + "structure", + "evidence" + ], + [ + 98, + 104, + "native", + "protein_state" + ], + [ + 105, + 117, + "GMPPNP-bound", + "protein_state" + ], + [ + 118, + 123, + "Irga6", + "protein" + ], + [ + 136, + 140, + "rmsd", + "evidence" + ] + ] + }, + { + "sid": 54, + "sent": "The seven Irga6 molecules in the asymmetric unit form various higher order contacts in the crystals.", + "section": "RESULTS", + "ner": [ + [ + 10, + 15, + "Irga6", + "protein" + ], + [ + 91, + 99, + "crystals", + "evidence" + ] + ] + }, + { + "sid": 55, + "sent": "Within the asymmetric unit, six molecules dimerize via the symmetric backside dimer interface (buried surface area 930 \u00c52), and the remaining seventh molecule forms the same type of interaction with its symmetry mate of the adjacent asymmetric unit (Additional file 1: Figure S2a, b, Figure S3).", + "section": "RESULTS", + "ner": [ + [ + 42, + 50, + "dimerize", + "oligomeric_state" + ], + [ + 69, + 93, + "backside dimer interface", + "site" + ] + ] + }, + { + "sid": 56, + "sent": "This indicates that the introduced mutations in the secondary patch, from which only Lys176 is part of the backside interface, do, in fact, not prevent this interaction.", + "section": "RESULTS", + "ner": [ + [ + 35, + 44, + "mutations", + "experimental_method" + ], + [ + 52, + 67, + "secondary patch", + "site" + ], + [ + 85, + 91, + "Lys176", + "residue_name_number" + ], + [ + 107, + 125, + "backside interface", + "site" + ] + ] + }, + { + "sid": 57, + "sent": "Another assembly interface with a buried surface area of 450 \u00c52, which we call the \u201ctertiary patch\u201d, was formed via two interaction sites in the helical domains (Additional file 1: Figure S2c, d, S3).", + "section": "RESULTS", + "ner": [ + [ + 8, + 26, + "assembly interface", + "site" + ], + [ + 84, + 98, + "tertiary patch", + "site" + ], + [ + 120, + 137, + "interaction sites", + "site" + ], + [ + 145, + 160, + "helical domains", + "structure_element" + ] + ] + }, + { + "sid": 58, + "sent": "In this interface, helices \u03b1K from two adjacent molecules form a hydrogen bonding network involving residues 373-376.", + "section": "RESULTS", + "ner": [ + [ + 8, + 17, + "interface", + "site" + ], + [ + 19, + 26, + "helices", + "structure_element" + ], + [ + 27, + 29, + "\u03b1K", + "structure_element" + ], + [ + 65, + 89, + "hydrogen bonding network", + "bond_interaction" + ], + [ + 109, + 116, + "373-376", + "residue_range" + ] + ] + }, + { + "sid": 59, + "sent": "Furthermore, two adjacent helices \u03b1A form hydrophobic contacts.", + "section": "RESULTS", + "ner": [ + [ + 26, + 33, + "helices", + "structure_element" + ], + [ + 34, + 36, + "\u03b1A", + "structure_element" + ], + [ + 42, + 62, + "hydrophobic contacts", + "bond_interaction" + ] + ] + }, + { + "sid": 60, + "sent": "It was previously shown that the double mutation L372R/A373R did not prevent GTP-induced assembly, so there is currently no evidence supporting an involvement of this interface in higher-order oligomerization.", + "section": "RESULTS", + "ner": [ + [ + 33, + 48, + "double mutation", + "protein_state" + ], + [ + 49, + 54, + "L372R", + "mutant" + ], + [ + 55, + 60, + "A373R", + "mutant" + ], + [ + 77, + 80, + "GTP", + "chemical" + ], + [ + 167, + 176, + "interface", + "site" + ] + ] + }, + { + "sid": 61, + "sent": "Strikingly, molecule A of one asymmetric unit assembled with an equivalent molecule of the adjacent asymmetric unit via the G-interface in a symmetric parallel fashion via a 470 \u00c52 interface.", + "section": "RESULTS", + "ner": [ + [ + 21, + 22, + "A", + "structure_element" + ], + [ + 124, + 135, + "G-interface", + "site" + ], + [ + 151, + 159, + "parallel", + "protein_state" + ] + ] + }, + { + "sid": 62, + "sent": "This assembly results in a butterfly-shaped Irga6 dimer in which the helical domains protrude in parallel orientations (Fig.\u00a01b, Additional file 1: Figure S3).", + "section": "RESULTS", + "ner": [ + [ + 27, + 43, + "butterfly-shaped", + "protein_state" + ], + [ + 44, + 49, + "Irga6", + "protein" + ], + [ + 50, + 55, + "dimer", + "oligomeric_state" + ], + [ + 69, + 84, + "helical domains", + "structure_element" + ], + [ + 97, + 105, + "parallel", + "protein_state" + ] + ] + }, + { + "sid": 63, + "sent": "In contrast, the other six molecules in the asymmetric unit do not assemble via the G interface.", + "section": "RESULTS", + "ner": [ + [ + 84, + 95, + "G interface", + "site" + ] + ] + }, + { + "sid": 64, + "sent": "The G interface in molecule A can be subdivided into three distinct contact sites (Fig.\u00a01c, d).", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "G interface", + "site" + ], + [ + 68, + 81, + "contact sites", + "site" + ] + ] + }, + { + "sid": 65, + "sent": "Contact site I is formed between R159 and K161 in the trans stabilizing loops, and S132 in the switch II regions of the opposing molecules.", + "section": "RESULTS", + "ner": [ + [ + 0, + 14, + "Contact site I", + "site" + ], + [ + 33, + 37, + "R159", + "residue_name_number" + ], + [ + 42, + 46, + "K161", + "residue_name_number" + ], + [ + 54, + 77, + "trans stabilizing loops", + "structure_element" + ], + [ + 83, + 87, + "S132", + "residue_name_number" + ], + [ + 95, + 104, + "switch II", + "site" + ] + ] + }, + { + "sid": 66, + "sent": "Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain.", + "section": "RESULTS", + "ner": [ + [ + 0, + 15, + "Contact site II", + "site" + ], + [ + 25, + 59, + "polar and hydrophobic interactions", + "bond_interaction" + ], + [ + 70, + 78, + "switch I", + "site" + ], + [ + 80, + 84, + "V104", + "residue_name_number" + ], + [ + 86, + 90, + "V107", + "residue_name_number" + ], + [ + 99, + 104, + "helix", + "structure_element" + ], + [ + 119, + 144, + "guanine specificity motif", + "structure_element" + ], + [ + 146, + 154, + "G4 helix", + "structure_element" + ], + [ + 156, + 160, + "K184", + "residue_name_number" + ], + [ + 165, + 169, + "S187", + "residue_name_number" + ], + [ + 179, + 201, + "trans stabilizing loop", + "structure_element" + ], + [ + 203, + 207, + "T158", + "residue_name_number" + ], + [ + 225, + 238, + "GTPase domain", + "structure_element" + ] + ] + }, + { + "sid": 67, + "sent": "In contact site III, G103 of switch I interacts via its main chain nitrogen with the exocyclic 2\u2019-OH and 3\u2019-OH groups of the opposing ribose in trans, whereas the two opposing exocyclic 3\u2019-OH group of the ribose form hydrogen bonds with each other.", + "section": "RESULTS", + "ner": [ + [ + 3, + 19, + "contact site III", + "site" + ], + [ + 21, + 25, + "G103", + "residue_name_number" + ], + [ + 29, + 37, + "switch I", + "site" + ], + [ + 134, + 140, + "ribose", + "chemical" + ], + [ + 205, + 211, + "ribose", + "chemical" + ], + [ + 217, + 231, + "hydrogen bonds", + "bond_interaction" + ] + ] + }, + { + "sid": 68, + "sent": "Via the ribose contact, switch I is pulled towards the opposing nucleotide (Fig.\u00a01e).", + "section": "RESULTS", + "ner": [ + [ + 8, + 14, + "ribose", + "chemical" + ], + [ + 24, + 32, + "switch I", + "site" + ], + [ + 64, + 74, + "nucleotide", + "chemical" + ] + ] + }, + { + "sid": 69, + "sent": "In turn, E106 of switch I reorients towards the nucleotide and now participates in the coordination of the Mg2+ ion (Fig.\u00a01e, Additional file 1: Figure S4).", + "section": "RESULTS", + "ner": [ + [ + 9, + 13, + "E106", + "residue_name_number" + ], + [ + 17, + 25, + "switch I", + "site" + ], + [ + 48, + 58, + "nucleotide", + "chemical" + ], + [ + 87, + 102, + "coordination of", + "bond_interaction" + ], + [ + 107, + 111, + "Mg2+", + "chemical" + ] + ] + }, + { + "sid": 70, + "sent": "E106 was previously shown to be essential for catalysis, and the observed interactions in contact site III explain how dimerization via the ribose is directly coupled to the activation of GTP hydrolysis.", + "section": "RESULTS", + "ner": [ + [ + 0, + 4, + "E106", + "residue_name_number" + ], + [ + 90, + 106, + "contact site III", + "site" + ], + [ + 140, + 146, + "ribose", + "chemical" + ], + [ + 188, + 191, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 71, + "sent": "The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis.", + "section": "RESULTS", + "ner": [ + [ + 4, + 15, + "G interface", + "site" + ], + [ + 111, + 114, + "E77", + "residue_name_number" + ], + [ + 116, + 120, + "G103", + "residue_name_number" + ], + [ + 122, + 126, + "E106", + "residue_name_number" + ], + [ + 128, + 132, + "S132", + "residue_name_number" + ], + [ + 134, + 138, + "R159", + "residue_name_number" + ], + [ + 140, + 144, + "K161", + "residue_name_number" + ], + [ + 146, + 150, + "K162", + "residue_name_number" + ], + [ + 152, + 156, + "D164", + "residue_name_number" + ], + [ + 158, + 162, + "N191", + "residue_name_number" + ], + [ + 168, + 172, + "K196", + "residue_name_number" + ], + [ + 221, + 224, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 72, + "sent": "All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191).", + "section": "RESULTS", + "ner": [ + [ + 56, + 60, + "G103", + "residue_name_number" + ], + [ + 62, + 66, + "S132", + "residue_name_number" + ], + [ + 68, + 72, + "R159", + "residue_name_number" + ], + [ + 78, + 82, + "K161", + "residue_name_number" + ], + [ + 117, + 126, + "interface", + "site" + ], + [ + 128, + 131, + "E77", + "residue_name_number" + ], + [ + 133, + 137, + "E106", + "residue_name_number" + ], + [ + 139, + 143, + "K162", + "residue_name_number" + ], + [ + 145, + 149, + "D164", + "residue_name_number" + ], + [ + 155, + 159, + "N191", + "residue_name_number" + ] + ] + }, + { + "sid": 73, + "sent": "Residues E77, K162, and D164 appear to orient the trans stabilizing loop which is involved in interface formation in contact site II.", + "section": "RESULTS", + "ner": [ + [ + 9, + 12, + "E77", + "residue_name_number" + ], + [ + 14, + 18, + "K162", + "residue_name_number" + ], + [ + 24, + 28, + "D164", + "residue_name_number" + ], + [ + 50, + 72, + "trans stabilizing loop", + "structure_element" + ], + [ + 94, + 103, + "interface", + "site" + ], + [ + 117, + 132, + "contact site II", + "site" + ] + ] + }, + { + "sid": 74, + "sent": "In the earlier model of an anti-parallel G interface, it was not possible to position the side chain of R159 to avoid steric conflict.", + "section": "RESULTS", + "ner": [ + [ + 27, + 40, + "anti-parallel", + "protein_state" + ], + [ + 41, + 52, + "G interface", + "site" + ], + [ + 104, + 108, + "R159", + "residue_name_number" + ] + ] + }, + { + "sid": 75, + "sent": "In the present structure, the side-chain of R159 projects laterally along the G interface and, therefore, does not cause a steric conflict.", + "section": "RESULTS", + "ner": [ + [ + 15, + 24, + "structure", + "evidence" + ], + [ + 44, + 48, + "R159", + "residue_name_number" + ], + [ + 78, + 89, + "G interface", + "site" + ] + ] + }, + { + "sid": 76, + "sent": "A conserved dimerization mode via the G interface in dynamin and septin GTPases.", + "section": "FIG", + "ner": [ + [ + 38, + 49, + "G interface", + "site" + ], + [ + 53, + 60, + "dynamin", + "protein_type" + ], + [ + 65, + 79, + "septin GTPases", + "protein_type" + ] + ] + }, + { + "sid": 77, + "sent": "The overall architecture of the parallel GTPase domain dimer of Irga6 is related to that of other dynamin and septin superfamily proteins.", + "section": "FIG", + "ner": [ + [ + 32, + 40, + "parallel", + "protein_state" + ], + [ + 41, + 54, + "GTPase domain", + "structure_element" + ], + [ + 55, + 60, + "dimer", + "oligomeric_state" + ], + [ + 64, + 69, + "Irga6", + "protein" + ], + [ + 98, + 105, + "dynamin", + "protein_type" + ], + [ + 110, + 116, + "septin", + "protein_type" + ] + ] + }, + { + "sid": 78, + "sent": "The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4 --bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN].", + "section": "FIG", + "ner": [ + [ + 14, + 24, + "structures", + "evidence" + ], + [ + 97, + 111, + "GTPase domains", + "structure_element" + ], + [ + 119, + 131, + "GMPPNP-bound", + 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+ "sid": 79, + "sent": "The GTPase domains of the left molecules are shown in orange, helical domains or extensions in blue.", + "section": "FIG", + "ner": [ + [ + 4, + 18, + "GTPase domains", + "structure_element" + ], + [ + 62, + 77, + "helical domains", + "structure_element" + ] + ] + }, + { + "sid": 80, + "sent": "Nucleotide, Mg2+ (green) and AlF4 - are shown in sphere representation, the buried interface sizes per molecule are indicated on the right.", + "section": "FIG", + "ner": [ + [ + 12, + 16, + "Mg2+", + "chemical" + ], + [ + 83, + 92, + "interface", + "site" + ] + ] + }, + { + "sid": 81, + "sent": "Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2", + "section": "FIG", + "ner": [ + [ + 0, + 5, + "Irga6", + "protein" + ], + [ + 6, + 31, + "immunity-related GTPase 6", + "protein" + ], + [ + 33, + 39, + "GMPPNP", + "chemical" + ], + [ + 40, + 68, 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], + [ + 126, + 133, + "dynamin", + "protein_type" + ], + [ + 163, + 170, + "dynamin", + "protein_type" + ], + [ + 187, + 195, + "atlastin", + "protein_type" + ], + [ + 211, + 216, + "GBP-1", + "protein" + ], + [ + 233, + 237, + "BDLP", + "protein_type" + ], + [ + 260, + 314, + "septin-related GTPase of immunity associated protein 2", + "protein" + ], + [ + 316, + 322, + "GIMAP2", + "protein" + ] + ] + }, + { + "sid": 83, + "sent": "However, the relative orientations of the GTPase domains in these dimers are strikingly similar, and the same elements, such as switch I, switch II, the trans activating and G4 loops are involved in the parallel dimerization mode in all of these GTPase families.", + "section": "RESULTS", + "ner": [ + [ + 42, + 56, + "GTPase domains", + "structure_element" + ], + [ + 66, + 72, + "dimers", + "oligomeric_state" + ], + [ + 128, + 136, + "switch I", + "site" + ], + [ + 138, + 147, + "switch II", + "site" + ], + [ + 153, + 182, + "trans activating and G4 loops", + "structure_element" + ], + [ + 203, + 211, + "parallel", + "protein_state" + ], + [ + 246, + 252, + "GTPase", + "protein_type" + ] + ] + }, + { + "sid": 84, + "sent": "IRG proteins are crucial mediators of the innate immune response in mice against a specific subset of intracellular pathogens, all of which enter the cell to form a membrane-bounded vacuole without engagement of the phagocytic machinery.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 3, + "IRG", + "protein_type" + ], + [ + 68, + 72, + "mice", + "taxonomy_domain" + ] + ] + }, + { + "sid": 85, + "sent": "As members of the dynamin superfamily, IRGs oligomerize at cellular membranes in response to GTP binding.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 25, + "dynamin", + "protein_type" + ], + [ + 39, + 43, + "IRGs", + "protein_type" + ], + [ + 93, + 96, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 86, + "sent": "Oligomerization and oligomerization-induced GTP hydrolysis are thought to induce membrane remodeling events ultimately leading to disruption of the PVM.", + "section": "DISCUSS", + "ner": [ + [ + 44, + 47, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 87, + "sent": "Recent structural and mechanistic analyses have begun to unravel the molecular basis for the membrane-remodeling activity and mechano-chemical function of some members (reviewed in).", + "section": "DISCUSS", + "ner": [ + [ + 7, + 42, + "structural and mechanistic analyses", + "experimental_method" + ] + ] + }, + { + "sid": 88, + "sent": "For example, for dynamin and atlastin, it was shown that GTP binding and/or hydrolysis leads to dimerization of the GTPase domains and to the reorientation of the adjacent helical domains.", + "section": "DISCUSS", + "ner": [ + [ + 17, + 24, + "dynamin", + "protein_type" + ], + [ + 29, + 37, + "atlastin", + "protein_type" + ], + [ + 57, + 60, + "GTP", + "chemical" + ], + [ + 116, + 130, + "GTPase domains", + "structure_element" + ], + [ + 172, + 187, + "helical domains", + "structure_element" + ] + ] + }, + { + "sid": 89, + "sent": "However, for other dynamin superfamily members such as IRGs, the molecular basis for GTP hydrolysis and the exact role of the mechano-chemical function are still unclear.", + "section": "DISCUSS", + "ner": [ + [ + 19, + 26, + "dynamin", + "protein_type" + ], + [ + 55, + 59, + "IRGs", + "protein_type" + ], + [ + 85, + 88, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 90, + "sent": "Our structural analysis of an oligomerization- and GTPase-defective Irga6 mutant indicates that Irga6 dimerizes via the G interface in a parallel orientation.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 23, + "structural analysis", + "experimental_method" + ], + [ + 30, + 67, + "oligomerization- and GTPase-defective", + "protein_state" + ], + [ + 68, + 73, + "Irga6", + "protein" + ], + [ + 74, + 80, + "mutant", + "protein_state" + ], + [ + 96, + 101, + "Irga6", + "protein" + ], + [ + 102, + 111, + "dimerizes", + "oligomeric_state" + ], + [ + 120, + 131, + "G interface", + "site" + ], + [ + 137, + 145, + "parallel", + "protein_state" + ] + ] + }, + { + "sid": 91, + "sent": "Only one of the seven Irga6 molecules in the asymmetric unit formed this contact pointing to a low affinity interaction via the G interface, which is in agreement with its small size.", + "section": "DISCUSS", + "ner": [ + [ + 22, + 27, + "Irga6", + "protein" + ], + [ + 128, + 139, + "G interface", + "site" + ] + ] + }, + { + "sid": 92, + "sent": "In the crystals, dimerization via the G interface is promoted by the high protein concentrations which may mimic a situation when Irga6 oligomerizes on a membrane surface.", + "section": "DISCUSS", + "ner": [ + [ + 7, + 15, + "crystals", + "evidence" + ], + [ + 38, + 49, + "G interface", + "site" + ], + [ + 130, + 135, + "Irga6", + "protein" + ] + ] + }, + { + "sid": 93, + "sent": "Such a low affinity interaction mode may allow reversibility of oligomerization following GTP hydrolysis.", + "section": "DISCUSS", + "ner": [ + [ + 90, + 93, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 94, + "sent": "Similar low affinity G interface interactions were reported for dynamin and MxA.", + "section": "DISCUSS", + "ner": [ + [ + 21, + 32, + "G interface", + "site" + ], + [ + 64, + 71, + "dynamin", + "protein_type" + ], + [ + 76, + 79, + "MxA", + "protein" + ] + ] + }, + { + "sid": 95, + "sent": "The dimerization mode is strikingly different from the previously proposed anti-parallel model that was based on the crystal structure of the signal recognition particle GTPase, SRP54 and its homologous receptor.", + "section": "DISCUSS", + "ner": [ + [ + 75, + 88, + "anti-parallel", + "protein_state" + ], + [ + 117, + 134, + "crystal structure", + "evidence" + ], + [ + 142, + 176, + "signal recognition particle GTPase", + "protein_type" + ], + [ + 178, + 183, + "SRP54", + "protein" + ] + ] + }, + { + "sid": 96, + "sent": "However, the G dimer interface is reminiscent of the GTPase domain dimers observed for several other dynamin superfamily members, such as dynamin, GBP1, atlastin, and BDLP.", + "section": "DISCUSS", + "ner": [ + [ + 13, + 30, + "G dimer interface", + "site" + ], + [ + 53, + 66, + "GTPase domain", + "structure_element" + ], + [ + 67, + 73, + "dimers", + "oligomeric_state" + ], + [ + 101, + 108, + "dynamin", + "protein_type" + ], + [ + 138, + 145, + "dynamin", + "protein_type" + ], + [ + 147, + 151, + "GBP1", + "protein" + ], + [ + 153, + 161, + "atlastin", + "protein_type" + ], + [ + 167, + 171, + "BDLP", + "protein_type" + ] + ] + }, + { + "sid": 97, + "sent": "It was recently shown that septin and septin-related GTPases, such as the Tocs GTPases or GTPases of immunity related proteins (GIMAPs), also employ a GTP-dependent parallel dimerization mode.", + "section": "DISCUSS", + "ner": [ + [ + 27, + 33, + "septin", + "protein_type" + ], + [ + 38, + 60, + "septin-related GTPases", + "protein_type" + ], + [ + 74, + 86, + "Tocs GTPases", + "protein_type" + ], + [ + 90, + 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representatives of the dynamin superfamily.", + "section": "DISCUSS", + "ner": [ + [ + 41, + 45, + "IRGs", + "protein_type" + ], + [ + 101, + 108, + "dynamin", + "protein_type" + ] + ] + }, + { + "sid": 100, + "sent": "Whereas the overall dimerization mode is similar in septin and dynamin GTPases, family-specific differences in the G interface and the oligomerization interfaces exist.", + "section": "DISCUSS", + "ner": [ + [ + 52, + 58, + "septin", + "protein_type" + ], + [ + 63, + 78, + "dynamin GTPases", + "protein_type" + ], + [ + 115, + 126, + "G interface", + "site" + ], + [ + 135, + 161, + "oligomerization interfaces", + "site" + ] + ] + }, + { + "sid": 101, + "sent": "For example, the involvement of the 2\u2019 and 3\u2019-OH groups of the ribose in the dimerization interface of Irga6 has not been observed for other dynamin and septin superfamily members.", + "section": "DISCUSS", + "ner": [ + [ + 63, + 69, + "ribose", + "chemical" + ], + [ + 77, + 99, + "dimerization 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+ 133, + "catalytic", + "protein_state" + ], + [ + 134, + 138, + "E106", + "residue_name_number" + ] + ] + }, + { + "sid": 104, + "sent": "During dimerization of GBP1, an arginine finger from the P loop reorients towards the nucleotide in cis to trigger GTP hydrolysis.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 27, + "GBP1", + "protein" + ], + [ + 32, + 47, + "arginine finger", + "structure_element" + ], + [ + 57, + 63, + "P loop", + "structure_element" + ], + [ + 86, + 96, + "nucleotide", + "chemical" + ], + [ + 115, + 118, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 105, + "sent": "In dynamin, the corresponding serine residue coordinates a sodium ion that is crucial for GTP hydrolysis.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 10, + "dynamin", + "protein_type" + ], + [ + 30, + 36, + "serine", + "residue_name" + ], + [ + 59, + 65, + "sodium", + "chemical" + ], + [ + 90, + 93, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 106, + "sent": "Irga6 bears Gly79 at this position, which in the dimerizing molecule A appears to approach the bridging imido group of GMPPNP via a main chain hydrogen bond.", + "section": "DISCUSS", + "ner": [ + [ + 0, + 5, + "Irga6", + "protein" + ], + [ + 12, + 17, + "Gly79", + "residue_name_number" + ], + [ + 49, + 59, + "dimerizing", + "oligomeric_state" + ], + [ + 69, + 70, + "A", + "structure_element" + ], + [ + 119, + 125, + "GMPPNP", + "chemical" + ], + [ + 143, + 156, + "hydrogen bond", + "bond_interaction" + ] + ] + }, + { + "sid": 107, + "sent": "Higher resolution structures of the Irga6 dimer in the presence of a transition state analogue are required to show whether Gly79 directly participates in GTP hydrolysis or whether it may also position a catalytic cation.", + "section": "DISCUSS", + "ner": [ + [ + 18, + 28, + "structures", + "evidence" + ], + [ + 36, + 41, + "Irga6", + "protein" + ], + [ + 42, + 47, + "dimer", + "oligomeric_state" + ], + [ + 55, + 66, + "presence of", + "protein_state" + ], + [ + 124, + 129, + "Gly79", + "residue_name_number" + ], + [ + 155, + 158, + "GTP", + "chemical" + ] + ] + }, + { + "sid": 108, + "sent": "In dynamin, further assembly sites are provided by the helical domains which assemble in a criss-cross fashion to form a helical filament.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 10, + "dynamin", + "protein_type" + ], + [ + 20, + 34, + "assembly sites", + "site" + ], + [ + 55, + 70, + "helical domains", + "structure_element" + ], + [ + 121, + 137, + "helical filament", + "structure_element" + ] + ] + }, + { + "sid": 109, + "sent": "In dynamin-related Eps15 homology domain containing proteins (EHDs), a second assembly interface is present in the GTPase domain.", + "section": "DISCUSS", + "ner": [ + [ + 3, + 60, + "dynamin-related Eps15 homology domain containing proteins", + "protein_type" + ], + [ + 62, + 66, + "EHDs", + "protein_type" + ], + [ + 71, + 96, + "second assembly interface", + "site" + ], + [ + 115, + 128, + "GTPase domain", + "structure_element" + ] + ] + }, + { + "sid": 110, + "sent": "For Irga6, additional interfaces in the helical domain are presumably involved in oligomerization, such as the secondary patch residues whose mutation prevented oligomerization in the crystallized mutant.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 9, + "Irga6", + "protein" + ], + [ + 22, + 32, + "interfaces", + "site" + ], + [ + 40, + 54, + "helical domain", + "structure_element" + ], + [ + 111, + 126, + "secondary patch", + "site" + ], + [ + 142, + 150, + "mutation", + "experimental_method" + ], + [ + 184, + 196, + "crystallized", + "evidence" + ], + [ + 197, + 203, + "mutant", + "protein_state" + ] + ] + }, + { + "sid": 111, + "sent": "Further structural studies, especially electron microscopy analysis of the Irga6 oligomers, are required to clarify the assembly mode via the helical domains and to show how these interfaces cooperate with the G interface to mediate the regulated assembly on a membrane surface.", + "section": "DISCUSS", + "ner": [ + [ + 8, + 26, + "structural studies", + "experimental_method" + ], + [ + 39, + 67, + "electron microscopy analysis", + "experimental_method" + ], + [ + 75, + 80, + "Irga6", + "protein" + ], + [ + 81, + 90, + "oligomers", + "oligomeric_state" + ], + [ + 142, + 157, + "helical domains", + "structure_element" + ], + [ + 180, + 190, + "interfaces", + "site" + ], + [ + 210, + 221, + "G interface", + "site" + ] + ] + }, + { + "sid": 112, + "sent": "Notably, we did not observe major rearrangements of the helical domain versus the GTPase domain in the Irga6 molecules that dimerized via the G interface.", + "section": "DISCUSS", + "ner": [ + [ + 56, + 70, + "helical domain", + "structure_element" + ], + [ + 82, + 95, + "GTPase domain", + "structure_element" + ], + [ + 103, + 108, + "Irga6", + "protein" + ], + [ + 124, + 133, + "dimerized", + "protein_state" + ], + [ + 142, + 153, + "G interface", + "site" + ] + ] + }, + { + "sid": 113, + "sent": "In a manner similar to BDLP, such large-scale conformational changes may be induced by membrane binding.", + "section": "DISCUSS", + "ner": [ + [ + 23, + 27, + "BDLP", + "protein_type" + ] + ] + }, + { + "sid": 114, + "sent": "Our structural analysis and the identification of the G-interface paves the way for determining the specific assembly of Irga6 into a membrane-associated scaffold as the prerequisite to understand its action as an anti-parasitic machine.", + "section": "DISCUSS", + "ner": [ + [ + 4, + 23, + "structural analysis", + "experimental_method" + ], + [ + 54, + 65, + "G-interface", + "site" + ], + [ + 121, + 126, + "Irga6", + "protein" + ] + ] + }, + { + "sid": 115, + "sent": "Our study indicates that Irg proteins dimerize via the G interface in a parallel head-to-head fashion thereby facilitating GTPase activation.", + "section": "CONCL", + "ner": [ + [ + 25, + 28, + "Irg", + "protein_type" + ], + [ + 38, + 46, + "dimerize", + "oligomeric_state" + ], + [ + 55, + 66, + "G interface", + "site" + ], + [ + 72, + 93, + "parallel head-to-head", + "protein_state" + ], + [ + 123, + 129, + "GTPase", + "protein_type" + ] + ] + }, + { + "sid": 116, + "sent": "These findings contribute to a molecular understanding of the anti-parasitic action of the Irg protein family and suggest that Irgs are bona-fide members of the dynamin superfamily.", + "section": "CONCL", + "ner": [ + [ + 91, + 94, + "Irg", + "protein_type" + ], + [ + 127, + 131, + "Irgs", + "protein_type" + ], + [ + 161, + 168, + "dynamin", + "protein_type" + ] + ] + } + ] + } +} \ No newline at end of file